key: cord- -yump qu authors: wu, chunxing; zhang, zhiqiang; mo, yueqiang; wang, dahui; ning, bo; xu, ping; song, jun; pei, xinhong; ni, xiaoyan; zheng, yiming; xia, tian; jing, yanhui; sun, wanlin; meng, junrong; qian, chuang title: recommendations for control and prevention of infections for pediatric orthopedics during the epidemic period of covid- date: - - journal: nan doi: . /wjps- - sha: doc_id: cord_uid: yump qu the outbreak of coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) emerged and spread rapidly throughout the world. as of february , , cases of covid- have been reported, and the outbreak is linked to deaths. the population is generally susceptible to the disease, and differences in incubation periods after infection exist among individuals. these two aspects of covid- pose significant challenges to pediatric orthopedic diagnosis and treatment. as a dedicated center for managing pediatric cases of sars-cov- in shanghai, our hospital has mobilized all branches and departments to undertake joint actions for scientific prevention and control, precise countermeasure and comprehensive anti-epidemic efforts. combined with our experience, we have consulted the relevant national regulations and the latest research advances and have formulated the prevention and control measures of sars-cov- infection, including outpatient, emergency, inpatient and surgical cares, for clinical practices of pediatric orthopedics according to the physicochemical properties of sars-cov- . it may serve as practical references and recommendations for managing sars-cov- infection in other pediatric specialties and in other hospitals. what are the new findings? ► in our hospital, a recommendation working group "recommendation formulating team for pediatric orthopedic infection controls during the epidemic's period of covid- " was formed, to focus on relevant issues for protection of medical staff in pediatric orthopedics other departments. combined with our experience, we have consulted the relevant national regulations and the latest research advances and have formulated the prevention and control measures of sars-cov- infection, including outpatient, emergency, inpatient and surgical cares for pediatric orthopedics, pediatric surgery and others. we have formulated "requirements for protection supplies in different grade (table )" and "control and surgical handling flows of pediatric orthopedics at outpatient and emergency clinics in responses to covid- infection (figure )". how might it impact on clinical practice in the foreseeable future? ► these recommendations, which are formulated to protect medical staff in pediatric orthopedics and patients, are also expected to serve as a reference and recommendations for other pediatric specialties and/or other hospitals. the outbreak of coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) emerged and spread rapidly throughout the world. as of february , , cases of covid- have been reported, and the outbreak is linked to deaths. the population is generally susceptible to the disease, and differences in incubation periods after infection exist among individuals. these two aspects of covid- pose significant challenges to pediatric orthopedic diagnosis and treatment. as a dedicated center for managing pediatric cases of sars-cov- in shanghai, our hospital has mobilized all branches and departments to undertake joint actions for scientific prevention and control, precise countermeasure and comprehensive antiepidemic efforts. is transmitted between humans mainly via aerial droplets and direct contact. thus far, no proven and specific antiviral treatments open access for covid- are available. therefore, the most important goal is to control infectious sources. protection of medical staff is obviously a crucial component of epidemic prevention and control. by consulting relevant national regulations, the latest research progress and guidance from the advisory group, our department developed these recommendations, which were also expected to serve as a reference and recommendations for other pediatric specialties and/or other hospitals. medical staff of pediatric orthopedic outpatient and emergency department, hospital administrator and staff of auxiliary department. pediatric orthopedic patients and accompanying personnel. recommendation formation process given the high demand of patients for medical treatment and the need to protect medical staff from infectious diseases, a recommendation working group "recommendation formulating team for pediatric orthopedic infection controls during the epidemic's period of covid- " (including all authors) was formed to focus on relevant issues for protection of medical staff in pediatric surgery, pediatric orthopedics, infectious diseases department, anesthesiology department, and nursing department to hospital administrators. . building and refining the first level catalog: applied population, applicable personnel, general principles, physicochemical properties of sars-cov- and clinical features of covid- . prevention and control procedures in outpatient clinic, emergency department, inpatient and surgical cares of pediatric orthopedics. "ordinary patient", "risky patient" and "suspected patient" protection. . establishing the second level catalog and retrieving the current evidence of covid- for the first and second level directory problem. . writing draft proposals. . synthesizing suggestions at all levels to form discussion papers. . discussing the proposal face to face in the proposal working group to form the applicable draft. . trying out the recommendations, gathering feedback and forming a formal version of the recommendations. general principles covid- has a general susceptibility in the population. there were many reports of nosocomial infection in literature and media, including medical staffs and hospitalized patients. therefore, who has published a recommendation for both patients and doctors, based on current knowledge of the situation in china and other countries. the strategies to prevent or limit transmission in healthcare settings include the following. . ensuring triage, early recognition, and source control (isolating patients with suspected sars-cov- infection). . applying standard precautions for all patients. . implementing empiric additional precautions (droplet and contact and, whenever applicable, airborne precautions) for suspected cases of sars-cov- infection. . implementing administrative controls. . using environmental and engineering controls. based on that, in order to reduce the iatrogenic infection and exposure infection, combined with our experience, we developed a set of recommendations for the department of pediatric surgeries, orthopedics and other relevant department. patients, families and medical staffs should adhere to the following recommendations. . control the number of accompanying family members. of the children and their close contacts in the first days before the visit. . hospitalization and surgical indications should be strictly controlled; selective operation is not allowed. . pay attention to the protection of medical staff during perioperative period. . decrease hospitalization days. . set up the transition ward, one room for one patient and continuous evaluation. do the best to avoid sars-cov- spread and cross infection. clinical features of covid- sars-cov- is the seventh coronavirus that has been found to infect humans. it is a β category coronavirus, in the same category as the coronavirus that caused middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars). spike glycoprotein (s) on virus surface is the main antigen of coronavirus and can be used for typing. the nuclear protein of the virus can be used for antigen diagnosis. most understanding of the physical and chemical properties of sars-cov- comes from studies on sars-cov and mers-cov. the virus is sensitive to ultraviolet and heat, which can be inactivated by heating at °c for minutes and by using lipid solvents such as % ethanol, chlorine-containing disinfectant, peroxyacetic acid and chloroform, but not by chlorhexidine. the transmission characteristics of covid- are as follows. . infection sources. the main sources of the infection are patients infected by covid- with or without clinical symptoms. the incubation period is between and days, with a median incubation period of days and a maximum of days. . transmission route. the novel virus is spread through respiratory droplets when patients cough, talk loudly or sneeze. close contact is also a source of transmission. aerosol and fecal-oral transmission of digestive tract are also possible, while it needs to be further clarified. . susceptible population. the age range of the reported infected population was hours old to years old, with no significant gender difference. the elderly and people with basic diseases are more likely to become severe cases. children may have mild clinical symptoms after infection. nevertheless, medical personnel still need to pay more attention, closely monitor, and strive for early identification and timely treatment. "diagnosis and treatment program of new coronavirus pneumonia (trial sixth version)" released by the national health commission of china on february , has defined as follow. suspected cases who meet any one of the following criteria. . respiratory tract or blood samples tested positive for covid- nucleic acid using reverse transcription polymerase chain reaction (rt-pcr). . genetic sequencing of respiratory tract or blood samples is highly homologous with the known covid- . on this basis, our hospital also issued clinical practice guidelines for children. screening of children at risk was enhanced through epidemiological history and clinical manifestations. although the clinical manifestations of the confirmed infection in children are mild, the clinical manifestations vary. among the confirmed cases of children with clinical data collected, fever and cough are more common, as well as fatigue, myalgia, nasal congestion, runny nose, sneezing, pharyngeal pain, headache, dizziness, nausea, vomiting, abdominal pain and diarrhea. this means that some patients with coronavirus infection are not first diagnosed with respiratory symptoms such as cough, fever and chest pain. a small number of patients will have muscle pain, abdominal pain and diarrhea as the first main complaint; pediatric orthopedic surgeons and pediatric surgeons should pay attention to these conditions during the outbreak, pay attention to screening for covid- and take measures to prevent the spread of sars-cov- infection in the hospital. control and surgical handling flow of pediatric orthopedics at outpatient and emergency clinics in responses to sars-cov- infection according to the characteristics of sars-cov- infection, national and hospital requirements for prevention and control, combined with clinical practice in pediatric orthopedics, we specially formulated the outpatient, emergency, inpatient and surgical procedures for prevention and control of sars-cov- infection in pediatric orthopedics (figure ). nurses in outpatient or emergency service will measure patients' temperature and ask epidemiological history, fever, respiratory symptoms at first, fill the epidemiological survey, and make a record about children and their accompanying guardians' identity card information, containing contact number, address and possible close contact with sars-cov- carriers after they passing the incubation period. pediatric orthopedist in the outpatient and emergency department has to check and confirm the patient's epidemiological survey again, collect the survey and turn back to preexamination nurses for centralized preservation. we divided the children into the following three categories. . "ordinary patient": without history of epidemiology or related symptoms. . "risky patient": a history of epidemiology without related symptoms. . "suspected patient": with a history of epidemiology and/or related symptoms, the patient should be transferred to the "fever clinic" in the isolation building for screening and diagnosis. for children with different categories, we clarify different levels of protection and specific requirements for different conditions (see table ). the protection of "ordinary patient'" . it is recommended that if the children are not in the emergency department, they should not seek medical treatment at the hospital to prevent the occurrence of cross-infection due to dense staff. at the peak of the epidemic, selective surgery was temporarily suspended, and then resumed gradually after the epidemic stabilized. with good precautions, pediatric orthopedists only carry out emergency operation and limited operation. if hospitalization is required, patients will be admitted to the general ward. . general protection for outpatient and emergency orthopedists: at least level protection (+ ): wearing medical overalls, medical surgical masks, medical caps and latex gloves. . ask each patient and attendant to wear masks. this cannot only prevent cross-infection between patients, but also effectively protect medical staffs. . improve the medical environment, set up waiting areas, maintain medical order and prevent crossinfection of patients. . the consultation area should be well ventilated and regularly cleaned and disinfected. . during the epidemic, hand hygiene should be strictly implemented, in accordance with the who five hand hygiene moments, following the "seven-step hand washing method". . protection for high-risk operations, such as debridement and suture, is the same as "risky patients" (see below). if hospitalization is needed, the hospitalization process for patient from other cities is the same as for "risky patients" (see below). patient needs to stay in the transition ward at first (for single person and single room admission management, the location of the transition ward should be avoided as far as possible from the entrance of the ward passage. the transition open access epidemic period ( st edition)" is warranted for the inpatients. if patients are suspected as "suspected patients" during hospitalization, he/she will be transferred to isolation wards. . note that "ordinary patients" may stay in the sars-cov- infection incubation, so close follow-up is also necessary. the protection of "risky patient" for patients with epidemic history of sars-cov- but no symptoms, we take the following preventive measures (the carriers are in the incubation period but still at risk of spreading the virus). . the orthopedist, both in outpatient and emergency department, will be informed of the situation of the patients by nurses, as the pre-warning. . the orthopedist shall adopt at least level protection (+ ): wearing medical overalls, medical caps, medical protective masks, goggles, medical isolation gowns and double latex gloves. . inquiring clinical history and performing physical examination merely in outpatient or emergency department with full protection. . patients who need auxiliary examination should wear medical surgical masks, accompanied by special personnel of the hospital. relevant auxiliary departments should be informed in advance to take preventive measures, and return to the orthopedic clinic in time after the examination. . after the treatment, the surfaces in the clinic are wiped with mg/l chlorine-containing disinfectant for minutes. then, ultraviolet radiation is used to sterilize the air for minutes. based on the results of physical and radiological examination, the orthopedic surgeon shall make a relevant decision following numbers principles. ( ) if the condition is mild, let the patient leave the hospital after treatment in the orthopedic clinic. ( ) closed fractures needing a plaster immobilization should be performed in debridement department. the orthopedist should be defended at least level protection (+ ): wearing medical overalls, medical surgical masks, medical caps and latex gloves. ( ) if the patient needs to observe the change of illness, the patient should not be left in the arbitrary observation room. the sars-cov- infection should be excluded by "clinic fever" physicians before transferring to the orthopedic transition ward. try to provide single rooms for isolated children, and reduce the chance of contact with the co-residents. after hospitalization, please refer to "diagnosis, treatment, and prevention of novel coronavirus infection in children: experts' consensus statement". ( ) if the patient needs to be hospitalized for surgery, ask physician to rule out the possibility of a suspected infection. if the infection is suspected or confirmed, follow the "suspected child" protection procedure (see below). if covid- infection is excluded, such patients will be admitted to the orthopedic transition ward with separate treatment management. ( ) if the patient needs to undergo debridement and suture operations without hospitalization, the following debridement and suture procedures should be followed: orthopedist and other personnel should have at open access least level protection (+ ) in a separate debridement room: medical overalls, medical caps, medical protective masks, face masks/goggles, medical isolation gowns and bilateral latex gloves. if tetanus vaccination is needed, human immunoglobulin (non-skin test type) should be selected and injected in the debridement room by the injector who has the same protection with orthopedist. dedusting garbage treatment: the garbage is placed in an airtight bucket with two layers of yellow garbage bags. the garbage is sprayed with mg/l chlorine-containing disinfectant and the bag is tightly sealed. after debridement, debridement room should be disinfected: the object surface is wiped and disinfected with mg/l chlorinecontaining disinfectant, acting on for > minutes. the air was sterilized by ultraviolet irradiation for minutes. the protection of "suspected patient" (including confirmed patients) . if the patient is considered to be a suspected case, the patient should first be transferred to the 'fever clinic' for screening of sars-cov- infection. if confirmed as a suspected case, the patient should be isolated immediately and reported to the hospital. nucleic acid tests are also performed twice to determine the presence of sars-cov- infection. . the following orthopedic surgeons will bring their own equipment and wear protective equipment in the infectious disease isolation building according to the prevention and control requirements. generally, level protection is provided for closed injuries and level protection is provided for high-risk operations (table ). . after experts' discussion, it is clear that if emergency or limited operation is needed, the patient will be transferred to the negative pressure operating room through the special channel by special transport vehicle. special attention should be paid to the isolation and the following protection during the perioperative period of suspected or confirmed children. ( ) reduce surgical participants. ( ) the operation should avoid rush hour and personnel flow. ( ) prevent cross infection in the operating room. for example, disposable equipment should be used as much as possible in the operating room. equipment unrelated to the operation should be moved out of the operating room, and those that cannot be moved out should be covered with protective film. during the operation, surgeons, nurses, anesthesiologists and other relevant personnel need take at least level protection: wearing double medical caps, medical protective masks (inner) and medical surgical masks (outer), protective face shield, medical protective clothing (inner) and medical isolation gowns (outer), double latex gloves and shoe covers. after the operation, the surgeons take off the medial isolation gowns (outer), the outer medical surgical mask, the outer medical cap and the outer latex gloves in the operating room. after hand disinfection by hydrogen peroxide, the surgeons take off the medical protective clothing (inner), remove the inner latex gloves, then carry out hand disinfection again with the disinfectant containing hydrogen peroxide. the operating room shall be sterilized according to the principle of disinfection of epidemic sources of class a infectious disease. the object surface of the operating room is wiped and disinfected with mg/l chlorine-containing disinfectant, acting on for > minutes. the air is atomized with hydrogen peroxide or hypochlorite for one hour. medical waste and medical fabric waste bags containing mg/l chlorine-containing preparations are sprayed and disinfected and treated as infectious medical waste and fabric. postoperative medical waste is placed in an airtight bucket with two layers of yellow garbage bags, and is sprayed with mg/l chlorine-containing disinfectant to sterilize it. occupational exposure during surgery, such as surgical instrument damage to protective gloves or protective clothing, can cause contact with body fluids to medical personnel. if this occurs, stop the operation immediately, remove the protection according to the above-mentioned principles, then deal with it according to the "emergency treatment procedure after sharp instrument injury" issued by the infection quality control center. report the incidence to the relevant departments of the hospital and fill in the "occupational exposure registration form". the relevant departments will conduct further isolation or clear diagnosis. all medical personnel involved in the operation need to be isolated for observation after the operation. if the suspected patient is excluded from sars-cov- infection, the isolation should be lifted. if the patient is diagnosed with the sars-cov- infection, all medical personnel involved in the operation are quarantined for days. when presenting with suspicious symptoms, he/she should seek medical treatment in time. during the isolation period, medical staff communicated with other staff in the department through mobile phones to avoid contact with colleagues who were not involved in contact with such patient. in conclusion, those are the clinical protective measures summarized in pediatric orthopedics, hoping to provide reference for pediatric orthopedics and other related departments. for suspected cases, we need to provide timely contact to professional doctors for judgment and treatment. at the same time, according to the changes of national epidemic prevention and control measures, to optimize protection process and measures are needed. in view of the changing epidemic situation, when there is a conflict between the treatment open access of pediatric orthopedic diseases and the prevention and control of the epidemic situation, it is necessary to make overall consideration and focus on the prevention and control of the epidemic situation. china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china emerging coronaviruses; genome structure, replication, and pathogenesis first case of novel coronavirus infection in children in shanghai national children's medical center & children's hospital of fudan university's guideline formulating team of rapid screening & clinical practices for suspected and confirmed cases of pediatric novel coronavirus infection or pneumonia clinical characteristics of novel coronavirus infection in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia what to do next to control the -ncov epidemic? world health organization. infection prevention and control during health care when novel coronavirus (ncov) infection is suspected national health commission of people's republic of china. guidelines for transmission and prevention of novel coronaviruses clinical characteristics of novel coronavirus in china diagnosis, treatment, and prevention of novel coronavirus infection in children: experts' consensus statement contributors wcx, zzq and myq contributed equally to this article. they contributed to the design, reporting, describing and interpretation of the study. wdh and nb were responsible for the overall content as guarantors. they planed, proposed and executed the study. xp, sj, pxh, nxy, zym, xt,jyh, swl, mjr and qc drafted part of the manuscript and approved the final version.funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. competing interests none declared.patient consent for publication not required. ethics approval not required for this review article.data availability statement all data relevant to the study are included in the article or uploaded as supplementary information. all text and data relevant to this study are included in this article, data are available in public and will be published in public.open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc . ) license, which permits others to distribute, remix, adapt, build upon this work noncommercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ . /. orcid ids chunxing wu http:// orcid. org/ - - - dahui wang http:// orcid. org/ - - - x references key: cord- - qfdqlpw authors: rinnert, k. j.; wigginton, j. g.; pepe, p. e. title: catastrophic anachronisms: the past, present and future of disaster medicine date: journal: yearbook of intensive care and emergency medicine doi: . / - - - _ sha: doc_id: cord_uid: qfdqlpw there is a worldwide spiraling risk for more frequent catastrophic events involving multiple casualties, not only in terms of acute injury and illness, but also subsequent psychological and public health concerns. today, such events will likely be multinational in nature, even when localized to a particular venue and this require international cooperation in terms of prevention, mitigation and relief. the best approach to preparing for disasters is to expand, modify and enhance current local ∖jobname: s batch number: infrastructures and capabilities for managing the multiple types of disaster scenarios and create a number of inter-facility cooperative agreements in advance. aside from safer internal locations for icus and surgical theaters, certain structural changes will need to be installed such as modified ventilation systems, protected water supplies, decontamination mechanisms and security renovations. a key strategy will be to proliferate interoperable, multi-disciplinary, all-hazards training initiatives such as the ama national disaster life support courses. purchases of cadres of antidotes, antibiotics and hemoglobin-based oxygen carriers should be coordinated regionally, stored in secure locations and made readily-available for the applicable disaster scenario. pointed out the concepts of modern vulnerability. not only were the victims highly concentrated in one building making it easier to target and cause many casualties, but modern technology facilitated the incident. a large modern transportation device laden with explosive fuel made such a dramatic disaster possible, one that would not have occurred to such an extent before . while such technology makes a local disaster global in nature, the same technology can bring a global disaster to each locality. the threat of worldwide spread of contagious disease, both naturally-occurring and malicious (bioterrorist) promulgation [ , ] is accelerated not only by exponential population growths, but the air ship as well. again, with more people on the planet who have the potential to become an infected vector and with more global concentrations of highly-mobile populations moving around in jet aircraft (and thus more opportunities for exposure), the risk for pandemics has clearly become greater than ever before. although the overt threats of cold war nuclear holocaust have seemingly dissipated, the underlying devices of mass destruction still exist, particularly in a world where terrorists are further spurred on by another form of technology, the mass media and internet. such threats are unimaginable, even for those who must anticipate how to deal with the aftermath [ , ] . as inferred previously, we are no longer a self-sufficient agrarian culture. major populations are now fully dependent on day-to-day energy and water supplies, sewage, garbage removal, and food products from others. therefore, in addition, to the initial impact of injury and illness are the subsequent public health sequelae such as insufficient food supplies, contaminated water, lack of shelter and the subsequent threat of associated diseases. likewise, the psychological impact of disasters on populations is only just now beginning to be better appreciated, not just for those directly affected, but for the population as a whole [ ] . for example, the economic downturns in the united states, europe and elsewhere after the events of fall are often considered one of the casualties of the terrorist attacks. it emphasizes the under-recognized widespread affects that disasters can have on the international public psyche. even when loss of life and infrastructure are relatively minimal in the grand scheme of the particular nation involved, disasters can have significant and farreaching psychological impact (e.g., the usa anthrax postal system attacks). the mass media, internet and other modern technology not only race information to all of us worldwide, but often do so in a graphic and emotionally-stimulating manner. even though the death and long-term morbidity toll was not as high as the staggering consequences of the indian ocean tsunamis, the public health risk management issues associated with the usa anthrax attacks and other identified potential threats were still enormous [ , , ] . facilitated by obsessive mass media mania, the potential threat of other public health crises (involving large populations) as a result of either terrorism or natural disease (e.g., small pox and avian flu pandemic scenarios) became more of a reality in terms of public perception. as a result, tremendous political pressure has developed for clinicians and public health officers alike to become better prepared to protect the public from all disasters, let alone doomsday scenarios. such psychological affects on the public play out in political venues as evidenced in the usa with the development of the department of homeland security and improved worldwide intelligence-gathering cooperation both domestically and internationally. nevertheless, while some improvements in surveillance and pharmacological resource allocations have evolved, the medical aspects of homeland security and catastrophic anachronisms: the past, present and future of disaster medicine public health emergency preparedness remain worrisome and clearly fall short of public expectations, appropriate expectations or not. sociologically, it is logical that wealthier countries might stand a better chance of mitigating mortality and morbidity with their robust health care systems, solid public infrastructures, substantial community resources and early detection-warning systems. this would occur not only through forewarned prevention of injury and illness, but also through rapid access to sustenance, medical and rescue assets. it could be emphasized that much of the loss of life and morbidity from the indian ocean tsunamis and their subsequent sequelae actually resulted from relative limitations in terms of such characteristics. nevertheless, the events of the fall of in the usa, the subsequent toronto experience with severe acute respiratory syndrome (sars) and the recent aftermath of hurricane katrina in the gulf coast, also exposed the vulnerability of even relatively healthy and well-resourced nations. in fact, these recent events emphasized our vulnerability in surge capacity. regardless of disaster events, on a daily basis, the great majority of healthcare facilities and emergency medical services (ems) systems, even in prosperous western nations, are overwhelmed in terms of emergency care capacity, be they government-based or private entities. many of the world's key trauma centers and ems crews are deluged on a day-to-day basis, with in-patient facilities brimming with fully-occupied beds, sub-optimal nurse and ancillary personnel staffing, despite increasing demands for service and a higher acuity of illness and injury. one could argue that these existing health care services, including ambulances, emergency centers, operating rooms, and intensive care units (icus) are facing a disaster each day as available medical resources outstrip the daily demands for urgent and critical care. this tenuous situation causes the looming threat of additional surges from disasters, be they technological or natural, to become truly deep concerns for the future of disaster medicine. table . some key reasons for increases in the frequency, magnitude and impact of disasters (natural, unintentional and intentional) since the mid- th century z exponential growth of human populations, concentrated in high-risk venues (e.g., shorelines, earthquake zones and prior uninhabited regions), particularly those externally dependent upon on others for sustenance (food supply chains), power resources and public health hygiene, making human populations increasingly vulnerable when infrastructures disrupted. z increasing exposures to zoonoses; more mutations of microbes (more human vectors); potential effects of global warming on storm formation; more sites (e.g., large buildings, subways) that are larger destructive traps. z technology that facilitate disasters (e.g., jet aircraft allowing more rapid spread of disease globally or terrorist use as a massive explosive device). z growth of hazardous industries and transportational systems (e.g., chemicals in plants, trucks, boats); genetic engineering/technology that facilitates bio-terrorism and weapons of mass effect across populations. z mass media (television, internet) that provide instantaneous imagery and disaster threat concerns, affecting populations psychologically worldwide, and, in turn, affecting markets/economies (also a stimulus for terrorists). z the global, multinational, multi-jurisdictional nature of disasters because of international-global industries, businesses, and tourist activities, and the associated global vulnerability through computer/internet disruption in essence, there is a spiraling risk for catastrophic events involving multiple casualties and population-based medical morbidity, including proximal injury and illness and subsequent psychological and public health concerns (table ) . such events will likely be multinational in nature with global ramifications, even when localized to a particular venue. therefore, this will require international cooperation in terms of prevention, mitigation and relief. however, the medical care infrastructure, even in wealthy countries already seems to operate at capacity, making a major multiple injury event or even an influenza pandemic a true challenge. z key problems in disaster medicine today just as the threats of disasters have increased and become multi-faceted, so have the medical sequelae and complications [ , , ] . explosions carry the triple threat of thermal, penetrating and blunt trauma [ , ] . associated building collapses not only cause crush injury syndromes, but a higher risk of being trapped, and modern fires induce chemical inhalation injuries as well as carbon monoxide poisoning and traditional respiratory tract impairment [ , ] . earthquakes cause all of the above, but killer tsunamis as well because of concentrated population centers. hurricanes, floods and tidal waves result in drowning, snake bites, and contaminated water supplies and subsequent mosquito-borne/zoonotic illnesses [ ] . chemical releases can result in pulmonary injury, burns, nerve system dysfunction, liver damage and cellular dissolution [ , ] . severe radiation exposure causes burns, immunological suppression, and diffuse epithelial damage, internally and externally. biological agents result in a myriad of physiological insults. from pneumonia, coagulopathies, and central nervous system compromise to cardiac suppression and liver failure, the viral and bacterial agents provide often-insidious challenges to clinicians and public health officers alike, regardless whether the root source is a natural epidemic or a malicious dissemination. while using existing medical personnel would be best, developing coordinated plans to incorporate them into a disaster plan (and provide them with timely respite and staggered shifts) is the challenge. while additional personnel theoretically could be imported from nearby (unaffected) medical facilities or from other regions or countries, the local personnel work best in their own environments and still would need to provide coordinating leadership roles. this even plays out politically as was seen in the aftermath of hurricane katrina where local medical personnel still wanted control despite the loss of resources (hospitals, supplies), both out of sense of loyalty to`their' patients, and their own sense of duty. in some respects, using the local personnel is in keeping with the number one rule of multiple casualty incident management, namely to follow day-to-day routines as closely as possible or modify day-to-day routines as much as possible to meet the unique demands of a disaster [ ± ]. the logic here is that unfamiliar activities or settings result in logistical and procedural learning curves for clinicians and that such medical care obstacles can be amplified in a strange venue with overwhelming patient care demands. learning how the laboratory or pharmacy works or how to operate less familiar equipment or communications systems, can delay and impair the true focus of patient care. in the hurricane katrina scenario or the pakistani earthquake circumstance, however, much of that infrastructure was wiped out, diluting such arguments. if, then, outside teams are to be brought in, one solution is to bring in a team that al-catastrophic anachronisms: the past, present and future of disaster medicine ready works together well. in the aforementioned dallas field hospital built to handle the surge from katrina evacuees, the medical team was used to working together. likewise, the arrival into new orleans of the uss comfort, a floating naval hospital replete with extensive operative capacity, neurosurgeons, multiple icus and airlift capacity, worked well because the team had been in place and worked well together. the question remains, however, how long could such`borrowed teams' last in the disaster zone. what is an appropriate tour of service? therefore, if they are still standing and operational, the utilization of highly-experienced trauma centers and icus would optimize the medical care skills needed and the efficiency of the delivery of care assuming that they could be off-loaded from some of their day-to-day activities. for example, a busy emergency department could off-load sore throats, urinary tract infections, and broken arms to clinics and field infirmaries (places where surge and delays in care would have less concerning consequences). through prospective, government-moderated agreements, icus at major receiving centers for disaster victims could transfer certain critical care patients to other community hospitals if necessary. this kind of arrangement has been worked out to significant degree in venues like miami, usa, a frequent target for major hurricanes in which certain hospitals themselves are at risk of damage. in any case, there will be a need for external surge capacities outside the day-to-day facilities. contrary to popular perception, most disastrous events do not create an immediate influx of critical patients [ ± ] . victims are either killed outright or they have generally survivable injuries. in most traditional situations, experience has shown that less than % of patients (those not killed outright) will require critical care or critical care monitoring [ ± ] . the main threat is actually displaced persons who need their day-to-day medical care, prescription, optometrist, dentist, and, in some cases, their dialysis or chemotherapy. in the aftermath of hurricane katrina, hundreds of thousands needed to be evacuated for extended periods of time. even if most were healthy, some of these healthy evacuees were bound to have their unanticipated appendicitis or their typical sore throats. more to the point, tens of thousands, especially the elderly nursing home patients, needed so much more. and how does one address the situation of a woman who says she is due for her chemotherapy when she has no medical records or physician contact? this becomes the real challenge, not necessarily the threat from the actual disaster event. most conventional disasters can be handled by existing resources when those resources are not involved or currently overwhelmed [ ] . if the major problem becomes the destruction of the local facilities and these concerns become further confounded by legal issues such as licensure and credentialing of medical professionals coming to volunteer at other alternative hospitals or the disaster zone itself. a physician coming to provide aid at another locale or hospital may not be authorized to do so if they are not licensed to practice medicine in that jurisdiction (i.e., country, state, province). moreover, even if licensed, hospital accreditation, in most venues, requires prospective scrutiny of physicians with relevant background checks and certain administrative requirements. all of these procedures take time and are, therefore, essentially impossible to provide at the time of a catastrophic event. while some communities have set up mechanisms to cross-credential physicians for a disaster, only a few have done so and this does not account for the issues of familiarity and skills utilization. furthermore, the key practitioners that might be needed to provide assistance at alternative locations in a disaster are skilled nurses, respiratory therapists, dialysis technicians, pharmacists and the like. while all of these practitioners could also bè cross-credentialed', it still does not account for motivators to have any of those persons participate. motivators are not just the dedication of a zealous volunteer or the lure of a financial incentive, but they also include care for those practitioners' families in the midst of a disaster during which time the families may be vulnerable as well (no food or water, loss of electricity, trapped by flooding, possible exposure to contagious disease, etc.). many of the persons trapped in the flooded new orleans hospitals were actually family members of the health care workers. finally, there could be concerns about liability coverage and protection from malpractice lawsuits when providing services outside of one's routine location [ ] . all of these concerns strengthen the argument that existing facilities, particularly major trauma centers and critical care hospitals, should all be fortified and better prepared for surge capacity. not only are there issues of skills, experience, familiarity, learning curves, licensure, credentialing, motivation and liability protection with which to be concerned, but, again, this paradigm follows the basic disaster tenet to follow one's day-to-day routines as closely as possible [ ± ] . it relies on the premise that one should prepare for such events by modifying day-to-day activities to accommodate requirements for a multi-casualty event. one of the major reasons why disaster scenarios, be they drills or actual events, often go awry is that they are encumbered by plans or procedures that fall outside normal routines. therefore, working in environments that facilitate familiar clinical and procedural behaviors in a disaster is the most advisable strategy. while local, reinforced facilities might be best suited for the more traditional st century disasters, a major nuclear or biological event can pose a more regional-national threat, if not a global risk [ ± ] . for example, even if only a fifth of a population becomes infected with a highly contagious disease over a several week period and only % of such patients require critical care, this can mean , critical care patients in a city of a million residents. assuming a week's stay for each patient whether they live or die, it still translates into the need for thousands of icu beds at any given time. also, under such circumstances, patients more than likely would not be transferred out to other communities because those other venues also may be experiencing similar, or even worse, surges in patient demands in such scenarios. similarly, certain catastrophes may involve those`routine facilities' and transport services themselves such as that seen in the hurricane katrina aftermath. they may be destroyed or inaccessible due to flood, earthquake, terrorist bombing or contamination. this scenario also entreats a plan for working outside one's normal routine or enormous surge at other facilities, either local or at a distance, established or makeshift. nevertheless, if current, day-to-day facilities are to be fortified (as they should), then these modifications will have significant fiscal impact. already strapped with budgetary challenges, such infrastructure changes would be unreasonable without full public support (government assistance) and commitment that all other facilities would share in similar burdens. in fact, far beyond the costs of constructing better ventilation mechanisms, improved water supply systems, well-placed generators and circumferential barricades (e.g., mitigating the effectiveness of car-bombs) as well as better control of hospital entrances, are the sociological conflicts. such security measures defeat the purpose and current philosophy of hospitals, clinics and medical facilities which should strive to provide even easier, patient-friendly access to medical care, particularly for the elderly, sick and chronically ill who need comfortable entrance. even with infrastructure changes, they will be of little avail without standardized training of the healthcare providers. during the last quarter of the th century, standardized, multi-disciplinary courses had been developed for the management of other major threats to life, namely cardiac arrest and trauma (e.g., the american heart association's advanced cardiac life support and the american college of surgeons' advanced trauma life support). such standardized training has even been demonstrated to be effective in increasing life-saving. however, unlike cardiac arrest and trauma cases that can present in certain facilities on a daily basis, disaster events are uncommon and infrequent events, even worldwide, making additional training and practice even more critical. even if the infrastructure is addressed and personnel trained, the complexity of nuclear explosion or`simply' a pandemic may be overwhelming. it is feared that with the typical current processes for producing vaccines, an entirely new influenza virus could sweep through even healthy worldwide populations in a matter of months, long before a vaccine could be developed, processed, and distributed, not to mention the time it takes for inoculated persons, particularly children, to develop adequate protective antibodies. a similar genetic jump from animal populations (e.g.,`avian flu') could proliferate with the same scenarios. in both cases, with more people to infect, more ways of rapidly transmitting the virus around the globe, and more persons with immunological suppression alive today, the risk for pandemic will become even more of a threat, not only for the population as a whole, but also for the ambulance crews, emergency department staff and icu practitioners. if they become ill as well, there will be even fewer healthcare providers available to care for the throngs of ill persons, making the disaster scenario even worse. such as seen in the sars situation, the medical facilities and healthcare workers may become a main vector themselves. more worrisome is the unknown. the concept of sars was unknown three years ago. with tremendous advances in genetic engineering, it is feared that both malicious and unintentional contagion threats will become new realities. and while threats for disaster are increasing, medical care resources are being spread thinner and thinner, from sociological factors (nursing shortages) to financial constraints (decreasing reimbursements for medical care). ironically, with increased demands for protection from terrorism and other public health threats, financial resources have been diverted from healthcare to national defense and homeland security efforts. unfortunately, most security efforts are overhead costs and not at all revenue-generating. even within hospitals, dealing with disaster management is generally administrative in nature (training, equipment, procedures, personnel) and consumes and diverts medical care professionals' time and efforts from their day-to-day patient care activities. these concerns are likely to become worse and resources further drained. moreover, the actual costs of the disasters are accelerating and public expectation for assistance stronger than ever before. z some potential solutions and improved preparations many lessons have been learned in our era of expanding and more frequent disasters. in a cyclone, tornado or hurricane belt, reinforced hospitals should have their icus and operating rooms secured within the center of the edifice, well above the level of potential storm surges and flood levels and far below the roof-top levels where spin-off tornadoes can rip off the top floors and blow out exterior windows. secondary and back-up generators should be protected in a similar fashion. all hospitals, particularly those in recognized earthquake zones, should have`earthquake-proof' designs with applicable structural integrity. when a hospital is a potential terrorist target such as any main receiving facility, trauma center, burn center, or children's hospital, security measures will dictate the need for barricades to bombs and specialized ventilation systems that detect and can control the spread of aerosolized poisons or biologicals. these hospitals should have a disproportionate number of negative pressure rooms and well-established decontamination zones around the potential entrance and receiving personnel (e.g., trained triage nurses and security personnel will be staged further out into the periphery and prepared with universal precautions and easy access to decon (decontamination) suits, including high level personal protective equipment (ppe). within the next ten years, spurred on by additional major terrorist events and further recognition of the vulnerability of the medical safety net, governmental resources will likely begin to make these changes and architects for new facilities will incorporate them into future design. but, economically intensive, this aspect of preparation will lag behind other efforts. this first consideration to enhance the structural aspects of disaster management would be of most value to handle the more likely conventional disasters. however, one might then return to entertaining the concept of a specialized facility or facilities to manage pandemics and disseminated bioterrorism incidents such as a mutated smallpox organism. in this case, to overcome the concepts of lack of familiarity with equipment and resources, credentialing, liability and medical skills utilization, a mitigating solution would be to dedicate a reserve team of medical personnel in the way a government entity deploys a fire service or army as a dedicated standing force. currently, a hybrid for this type of concept is accomplished through the mobile medical teams and tent hospitals designed by the national disaster medical system (ndms), an element of the federal emergency management agency (fema) in the united states department of homeland security. although this does not employ a round-the-clock standing team, it does utilize a`stand-by' team of medical and allied health volunteers who are trained and routinely exercised to operate mobile hospitals. currently, these teams would likely be inadequate for a mass casualty scenario with tens of thousands of victims, but still they are somewhat helpful in areas where the standing facilities have been destroyed, impaired or are inaccessible. the hurricane katrina incident demonstrated the clear value of expert, co-trained military medicine assets. mobile hospitals have reasonable value in some circumstances and should always be part of the planning including safe and secured storage of required equipment and assets, but community-wide plans to enhance existing facilities for surge capacity should still supercede all other plans. inter-hospital agreements to facilitate transfers of patients to balance out surges throughout the system should be in play in the best prepared communities. such agreements will also include specialized storage facilities for antidotes and antibiotics, perhaps in a separate, undisclosed and protected storage facility, but close enough for easy access. the regional hospitals should band together and purchase these items in bulk to leverage economies of scale (cheaper prices), but they should also coordinate receipt so that expiration dates on the drugs will be staggered and not all expiring at once. such coordination would also include the use of healthcare and ancillary personnel in case of overload at a given hospital or incapacity of another. not only will prospective cross-credentialing be accomplished ahead of time using familiar mechanisms, but the process may even be facilitated by course completion in advanced disaster life support and hospital disaster life support types of courses [ , , ] . in all likelihood, arrangements should first focus intra-murally. for example, they may prioritize the use of nurses from the same hospital system (i.e., many hospital systems operate more than one hospital) because of the similarities in policies, procedures, liability coverage and payroll considerations. such coordination of efforts would be a massive under-taking, but well-prepared communities will want to cooperate in such approaches. also, since many disasters are multinational and often occur in under-resourced countries with relatively poor infrastructures, the creation of dozens of international disaster teams by the united nations or other organizations worldwide could be considered. equipped with fast transport aircraft and mobile emergency units, these teams would best be available on any site within less than hours and will work closely with local organizations. very likely, each disaster team will be staffed and operated by a multinational force, and most likely a standing military team. as in any other military organization, teaching and training in disaster medicine will be part of military education in all countries and the teams will continuously drill and work together in coordination on a routine basis. if anything has been learned in recent disasters, it has been the clear value of the military. another lesson learned was the value of standardized training, not only for the military, but for the civilian population as well. after , the american medical association (ama), working with several major academic centers, began to help development of a family of standardized, interoperable, multi-disciplinary, all-hazards courses to deal with the medical aspects of disaster medicine and counter-terrorism [ ± ] . working in close conjunction with university-based trauma center teams as well as multiple federal and military agencies and, more recently, other professional societies such as the american college of emergency physicians (acep) and the society of critical care medicine (sccm), the ama courses are beginning to lay the groundwork for standardized training and improved personnel preparations for disasters [ ± , ] . during hurricane katrina, the louisiana state department of health attributed many of the medical evacuations that were accomplished quite successfully, to the use (and previous training in) adls as well the use of a table top disaster scenario called ªhurricane pamº. like advanced cardiac life support and advanced trauma life support, the advanced disaster life support course provides hands-on, multi-disciplinary scenarios in which participants learn to provide antidotes and other resuscitative skills in simulated austere, hazardous conditions requiring the donning and use of high-level ppe in insecure environments [ ] . in addition to the advanced disaster life support course, a prerequisite basic disaster life support course [ ] provides intensive exposure to the didactic elements of disaster preparation, be it chemical, biological, radiological, or traumatic in nature [ , ] . within the next ten years, it is predicted that the basic disaster life support course will be required for every medical student, paramedic student, nursing and other applicable allied health personnel in the usa [ ] . the advanced disaster life support course will also be provided to applicable trainees and nursing staff in critical care areas including advanced life support ambulances, emergency departments, and critical care areas. it is also predicted that specialized in-hospital spin-off courses (e.g., hospital disaster life support) will address elements of decontamination tactics by custodians, engineers and others. the sccm has already laid much of the groundwork for such training with some of their course developments [ ] . such endeavors will hopefully become similar to the efforts conducted by the international liaison committee on resuscitation (ilcor) for cardiac resuscitation medicine using evidence-based approaches and worldwide consensus. in addition to being involved in such standardized training, paramedics and emergency medical technicians should also be trained to deliver prophylaxis in a public emergency preparedness situation such as a smallpox or yersinia outbreak or even an influenza pandemic. prospective rules about who can be denied or provided vaccination or antibiotics and how one receives protection from liability should be addressed ahead of time in the most prepared systems. in addition to healthcare workers, certain societal infrastructure personnel (e.g., elected officials, water, power, communications, media personnel) may be prioritized as well. also, anticipating a major event, facilities should develop a cadre of antidotes, antivirals and antibiotics for biological threats. more importantly, they will need to ratchet up icu equipment, ventilators and respiratory care equipment as well as ppe and decontamination equipment. ambulances and emergency departments and hospitals at large should be fortified by new computerized technology that enhances detection and discrimination of abnormal gases, chemicals, aerosolized biologicals and other threats in ventilation systems and ambient air, just as a carbon monoxide detector or geiger counter would provide sentinel detection of carbon monoxide or radioactivity in one's home. medical records will be electronically stored and also duplicated and stored at distant sites in case of medical facility destruction and population displacements. new ventilators will need to be impervious to chemical and biological agents and provide protected ventilation in such environments. in addition, artificial hemoglobin-based oxygen carriers that can be stored in ambulances or in far-forward military conditions should be researched and eventually placed in massive storage places as well and ready to use for mass casualties [ ] . some products now in early test stages can be stored without refrigeration for several years and are likely to be standard equipment in all critical care settings, be it prehospital, emergency center or icu settings [ ] . again, all of this will require a new level of international cooperation particularly because many natural disasters can be superimposed upon on-going complex emergencies, including on-going famines, civil wars or rebel insurgencies [ ] . such circumstances can further complicate rescue and restoration of normalcy [ ] . there is a worldwide spiraling risk for more frequent catastrophic events involving multiple casualties, not only in terms of acute injury and illness, but also subsequent psychological and public health concerns. today, such events will likely be multinational in nature, even when localized to a particular venue and this require international cooperation in terms of prevention, mitigation and relief. the best approach to preparing for disasters is to expand, modify and enhance current local catastrophic anachronisms: the past, present and future of disaster medicine infrastructures and capabilities for managing the multiple types of disaster scenarios and create a number of inter-facility cooperative agreements in advance. aside from safer internal locations for icus and surgical theaters, certain structural changes will need to be installed such as modified ventilation systems, protected water supplies, decontamination mechanisms and security renovations. a key strategy will be to proliferate interoperable, multi-disciplinary, all-hazards training initiatives such as the ama national disaster life support courses. purchases of cadres of antidotes, antibiotics and hemoglobin-based oxygen carriers should be coordinated regionally, stored in secure locations and made readily-available for the applicable disaster scenario. advanced disaster life support¾, version . . american medical basic disaster life support¾, version . core disaster life support¾, version . medical response to terrorism: preparedness and clinical practice. lippincott bioterrorism and medical risk management bioterrorism and public health, st edition ten golden rules for urban multiple casualty incident management multiple casualty incident plans: ten golden rules for prehospital management responding to emergencies ± review of the phillips petroleum explosion, march issue. council of state governments field management and critical care in mass disaster fundamentals of disaster management, nd edition. society for critical care medicine bovine hemoglobin-based oxygen carrier (hboc- ) for resuscitation of uncontrolled, exsanguinating liver injury in swine differences in world responses to natural disasters and complex emergencies key: cord- -h fzti i authors: manning-geist, beryl; meyer, fremonta; chen, justin; pelletier, andrea; kosman, katherine; chen, xiaodong (phoenix); johnson, natasha r. title: pre-clinical stress management workshops increase medical students’ knowledge and self-awareness of coping with stress date: - - journal: med sci educ doi: . /s - - - sha: doc_id: cord_uid: h fzti i objectives: to investigate the effects of a stress management workshop on medical students’ knowledge of stress and potential coping strategies. methods: a panel discussion with small group breakouts on stress in clinical medicine, learning challenges, competition with colleagues, handling stressful events, and recognizing burnout symptoms was conducted with medical students entering clerkships. a longitudinal survey design was utilized to measure pre-, post-, and long-term ( -month) changes in knowledge (impact of stress on personal health, learning, and patient care), confidence, perceived skills, and attitude (towards utilizing adaptive coping strategies) among participating students (n = ). paired t test and multivariate analyses were performed to assess the differences between survey responses on a -point likert scale. results: survey response rates were pre- . %, post- %, and long-term post- . %. compared to pre-workshop, students reported significant improvement in all four domains immediately post-workshop: knowledge ( . vs. . , p < . ), confidence ( . vs. . , p < . ), perceived skills ( . vs. . , p < . ), and attitude ( . vs. . , p < . ). compared to immediate post-workshop, students’ scores slightly decreased at months but were overall significantly higher than the pre-workshop scores. conclusions: a stress management workshop can improve medical students’ knowledge of the impact of stress as well as the use of adaptive stress coping strategies. stress and burnout have been recognized as significant factors that influence medical trainees' aptitude [ ] , learning [ ] [ ] [ ] [ ] , professionalism [ ] [ ] [ ] [ ] , wellness [ ] [ ] [ ] [ ] , and care of patients [ , , ] . stress is a psychological and physical process that occurs when a person is faced with a demand that is perceived to exceed the resources available to respond to it effectively [ , ] . burnout is a state of mental and/or physical exhaustion caused by excessive and prolonged stress, and can be measured through the validated maslach burnout inventory [ ] [ ] [ ] [ ] . a recent study reported that at least half of all medical students might be affected by stress and burnout during medical school [ ] . dyrbye and colleagues revealed that the learning environment was significantly associated with medical student burnout; specifically, students rotating on hospital wards and those required to stay for overnight call were more likely to experience burnout [ ] . psychological distress can lead to depression and thus medical students are at high risk [ ] [ ] [ ] [ ] . compared with us college graduates age - , medical students were more likely to exhibit symptoms of depression and had higher levels of fatigue [ ] . even more alarming is the recent finding that the prevalence of depression or depressive symptoms among medical students was . % and that of suicidal ideation was . % [ ] . thus, more attention needs to be paid to the stress phenomenon during undergraduate medical education, including students' awareness of stress and how to improve students' stress coping skills. this is particularly important during the challenging transition from the mostly didactic-filled pre-clerkship curriculum to patient-care focused clerkships which requires medical students to adjust to new learning environments, unpredictable schedules, and different expectations. studies have investigated interventions for students to minimize the stress inherent to medical education and training and to improve wellness [ , ] . specifically, the effects of mindfulness-based stress reduction (mbsr) training on preclinical and clinical medical students have been shown to reduce depression [ , ] , anxiety [ ] , psychological distress [ , , ] , perceived stress [ , , ] , and emotional exhaustion [ ] and to increase empathy [ ] , selfcompassion [ , ] , mindfulness skills [ , ] , positive mental health [ ] , life satisfaction [ ] , and spirituality [ ] . medical students, however, use various coping mechanisms that vary by year in training and source of the stress [ ] . the specific coping strategies that students use may determine the effect of stress on their academic performance and well-being [ ] . however, little is understood regarding medical students' knowledge of the impact of stress on their own health, learning, and patient care. in addition, there is a paucity of literature on medical students' knowledge of coping strategies available and self-awareness of the need to utilize adapting coping strategies to prevent burnout and depression [ ] . given the significant prevalence of psychological distress, burnout, and depression among medical students, it is critical for medical schools to equip students with the knowledge necessary to recognize stress, ability to determine when to seek assistance, and capacity to manage stress effectively to promote well-being and prevent burnout [ , , ] . therefore, we conducted a longitudinal survey study in medical students poised to initiate clinical clerkships in order to investigate the impact of a stress management workshop on knowledge and self-awareness of stress symptoms, impact of stress, and potential adaptive positive coping strategies. a total of medical students (representing one class year) were scheduled to participate in a stress management workshop at four academic hospitals affiliated with a single medical school prior to starting their clinical rotations as part of a larger transition to the principal clinical experience course (transition to clinical clerkships course). the goals of the -h workshop were for students to ( ) demonstrate increased knowledge of the negative impact of stress on their learning, personal health, and patient care; ( ) recognize symptoms of stress; ( ) demonstrate increased selfawareness of adaptive coping mechanisms to reduce stress and reduce the risk of burnout; and ( ) identify the value of seeking help and available resources. the standardized workshop format consisted of a h didactic on stress and burnout with specific emphasis on manifestations in clinical medicine including definitions, epidemiology, time trends, hypothesized contributors, recognition in self and others, management, and approaches. in addition, the didactic highlighted the importance of seeking help and resources available to students. the second hour consisted of a panel discussion and small group breakouts on topics of role confusion, power in the hospital, benefits/risks of empathy, competition with colleagues, conflicts with other medical professionals/patients, dealing with disappointment, pimping, challenges of learning, self-care, handling stressful events, facade of perfection, and work/life balance, as well as recognizing and managing symptoms of burnout. the curriculum content was derived from the literature, a previously conducted needs assessment, and expert opinion. total population sampling was used to recruit student participants. a total of medical students, representing one class year, were invited to attend a stress management workshop at their previously assigned academic hospital affiliated with a single medical school prior to starting clinical rotations. participation in the study associated with the workshop was voluntary. three surveys, including pre-workshop, immediate post-workshop, and a long-term (conducted months after starting clinical rotations) post-workshop follow-up survey, were administered to the students. irb approval was obtained from partners and harvard medical school. we utilized paper and pencil surveys for the pre-and immediate post-workshop surveys. the long-term post-workshop survey was distributed with the qualtrics online survey tool, as students had started their clinical rotations in different hospitals. all surveys were anonymous and derived from validated instruments to measure students' knowledge of the negative impact of stress, symptoms of stress, utilization of positive coping strategies (derived from the coping strategies inventory [ ] ), resources available, likelihood of utilizing resources, and current level of burnout, as assessed using the validated maslach burnout inventory [ , ] . the maslach burnout inventory evaluates three aspects of perceived burnout including emotional exhaustion, depersonalization, and personal accomplishment [ ] . the maslach burnout inventory is widely used to assess burnout in a variety of settings, and has been extensively validated as a tool to assess perceived burnout in medical professionals [ ] . research team members reviewed and reached agreement on the final questions and construct of the survey instruments. cognitive pretesting was conducted with four medical students to ensure that students would interpret the survey questions in the intended manner. all survey instruments included identical questions to measure students' perceived changes in stress management in four domains: knowledge (impact of stress on their own health, learning, and patient care), confidence, perceived skills, and attitude (towards utilizing adaptive coping strategies to prevent burnout), which used a -point likert-type scale ( = not at all, = extremely). self-reported level of burnout ( point likert-type scale; = completely burned out, = no symptoms of burnout) was assessed in the pre-and long-term post-workshop surveys. ten questions ( -point likert-type scale; = not at all, = a lot) about workshop satisfaction and helpfulness were also included in the postworkshop survey. data were analyzed to determine the effects of the workshop on the study goals. statistical analyses were conducted using jmp pro statistical software (version ) as well as stata (version . ). differences between pre-and immediate postworkshop responses were tested using paired t tests to assess differences between continuous variables. a p value of < . was considered significant. correlation coefficients were performed to measure the relationship between students' workshop satisfaction and their self-awareness of using positive stress coping techniques. we also calculated odds ratios to examine the association between students' reported level of burnout and other variables. power analysis was not applicable as total population sampling was used and the survey response rate was reported. the overall survey response rate was . % (pre- . %, immediate post- %, -month post-workshop . %). in comparing combined ratings of and on the likert scale ("quite a lot" and "extremely") on pre-to immediate post-workshop responses, students' knowledge of the impact of stress on their own health, learning, and patient care increased. however, this was not completely sustained by the -month post-workshop survey (conducted months after starting clinical clerkships). students' confidence and perceived skills towards utilizing adaptive coping strategies to handle stress (e.g., selfawareness) also improved from the pre-to immediate postworkshop, but also was not sustained by -month post-workshop. there was no change in attitude towards stress (seeking help is not a sign of weakness) over the surveys. the trends examining impact of the workshop estimates over time (including pre-workshop, post-workshop, -month post-workshop) on the domains of knowledge, confidence, skills, and attitude are illustrated in fig. . overall, students' mean survey responses demonstrated statistically significant improvement for knowledge about the impact of stress on their overall long-term success, health, learning and patient care, and confidence and skills in recognizing and utilizing adaptive strategies to handle stress and attitude towards seeking help increased from pre-to immediate post-workshop (t to t ) (see table ). the difference between survey responses within the four domains from preworkshop and -month post-workshop (t to t ) was not statistically significant, except for scores on recognizing symptoms of burnout and stress within the confidence domain (see table ). however, the workshop was effective in increasing students' knowledge of where to find supporting resources if they feel stressed during their clinical rotations both at the medical school ( . % pre-workshop vs. . % immediate post-workshop) and at their hospital site ( . % pre-workshop vs. % post-workshop). the immediate post-workshop responses indicated that the majority of students enjoyed the workshop, learned the symptoms and impact of stress, and planned to use the knowledge of coping strategies gained from the workshop ( table ). in addition, students found specific elements of the workshop to be overall helpful (improve wellness, know effects of stress, coping strategies, talking with peers). hearing classmates' concerns about the clinical year was reported to be the most helpful element of the workshop (mean score . , sd ± . ) which occurred during the small group breakout section of the workshop. on long-term post-workshop surveying, students assigned a mean score of . (scale - , sd ± . ) to the question "did the stress workshop help to improve selfawareness of using positive coping strategies during your clinical rotations?" we conducted spearman correlation coefficient analysis and found a moderate positive correlation (r ranged from . to . , p < . ) between the following items: satisfaction with the workshop, knowledge of stress symptoms, understanding of impact of stress, knowledge of coping strategies, and students' self-awareness of using adaptive stress coping strategies during clinical rotations in the future. confidence towards utilizing adaptive coping strategies in handling stress (e.g., self-awareness), perceived skills in addressing this stress, and attitude towards stress and seeking help (seeking help is not a sign of weakness) sd standard deviation; all with - scale; data have been re-coded; the higher rating, the better. the mean differences between pre-workshop and immediate post-workshop were compared using paired t tests. the mean differences between pre-workshop and -month post-workshop were also compared using paired t tests. a p value < . was considered statistically significant students were asked to rate their current level of burnout on the pre-workshop and -month post-workshop surveys ( months after starting their clinical clerkships, scale - ; = completely burned out, = enjoy my work). there was a higher level of self-reported burnout at months (mean . sd ± . ) compared to pre-workshop (mean . sd ± . ). we found no statistically significant relationship between students' self-reported burnout at months and knowledge, confidence, perceived skill, or attitude scores. students who disagreed that asking for help was a sign of weakness were statistically more likely to have self-reported burnout at -month post-workshop (or . , % ci . - . ) ( table ). our study findings suggest that the -h stress management workshop had a positive impact on medical students' knowledge towards the symptoms and impact of stress as well as on their confidence, perceived skills, and attitude of using adaptive stress coping skills during their clinical rotations. students' self-reported knowledge, confidence, and perceived skills increased immediately after the workshop; however, this improvement was largely not durable at the -month postworkshop time point. it is possible that students' recall of the knowledge learned in the workshop was compromised by the high learning demands of clinical rotations. to prevent the knowledge decay, programs could incorporate a series of mindfulness-based stress training sessions [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and accessible support in addition to a one-time stress management workshop to enhance students' knowledge retention and skill acquisition. results of the pre-workshop survey illustrate that students generally demonstrated a high level of knowledge about stress symptoms and their impact, followed by confidence and perceived skills to cope with stress, and attitude towards seeking help. these are consistent with the immediate post-workshop survey results about workshop satisfaction and helpfulness: students rated hearing their peers' concerns and learning useful stress coping strategies as the two most helpful educational contents of the workshop. these findings suggest that future stress management workshops should focus on introducing stress coping skills and provide a safe environment for students to share concerns and experiences. students' self-reported burnout increased by months compared to pre-workshop levels ( months after starting clinical clerkships) and did not correlate with their knowledge about q questions, sd standard deviation; all with - scale, = "not at all," "slightly," = "moderately," = "very" or odds ratio; or = , exposure does not affect odds of outcome; or > , exposure associated with higher odds of outcome; or < , exposure associated with lower odds of outcome. sign of weakness was re-coded ( = strongly disagree asking for help is a sign of weakness); *only compared students who were matched across surveys (n = ) stress, confidence, or perceived skill in coping with stress. burnout was correlated with students' attitude towards seeking help: students who were more open to seeking help were twice as likely report burnout at months. this finding may be identifying premorbid depression, anxiety, or other mental health concerns; perhaps students who have already sought help in the past and are not opposed to reporting difficulty or seeking help in general. this highlights that it is critically important for students to know where to find resources should they need to seek help. further, this finding may also reveal students who are empathetic and thus more likely to talk about their feelings and seek help. one theory is that a high level of empathy may cause "compassion fatigue" which then leads to emotional exhaustion and burnout [ ] . future studies are needed to further investigate the relationship between empathy and burnout among medical students, including whether increased screening for burnout should be used in students who are more likely to ask for help. our study has limitations, as previously described [ ] . first, all participants are from a single medical school; results may therefore not be generalizable. second, all survey results represent students' self-reported knowledge, confidence, perceived skills, and attitude, and do not measure actual performance or changes in behaviors. as these data are self-reported, there is an unavoidable potential for social desirability bias. in conclusion, the stress management workshop had a positive impact on medical students' knowledge, confidence, perceived skills, and attitude of using adaptive stress coping strategies. programs are encouraged to empower students to effectively self-manage stress and prevent burnout during clinical training by providing additional interventions after a stress management workshop to increase students' 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inventory manual validity of the maslach burnout inventory for family practice physicians burnout and empathy in primary care: three hypotheses learning in a high-stress clinical environment: stressors associated with medical students' clerkship training on labor and delivery publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that there is no conflict of interest. key: cord- -bco nloe authors: fu, xiao-bing title: military medicine in china: old topic, new concept date: - - journal: mil med res doi: . / - - - sha: doc_id: cord_uid: bco nloe military medicine is important in both war and peace. in china, military medicine plays a key role in supporting and maintaining health, in preventing injuries and diseases in military staff and in enhancing the military armed forces during war. additionally, military medicine participates in actions such as emergency public health crises, natural disasters, emerging conflicts and anti-terrorist campaigns during peacetime. in this paper, we summary the current condition and achievements in military medicine in china and provide our perspective for its future. military medicine is an important field in biological and medical sciences. military medicine plays a key role in supporting and maintaining health, in preventing injuries and diseases in military staff and in enhancing the military armed forces during war [ ] . in the new millennium, military activities also involve other actions such as emergency public health crises, natural disasters, emerging conflicts and anti-terrorist campaigns during peace. thus, military medicine has faced new challenges and requirements with the military transformation. these requirements include medical services in natural disasters, accidents, terrorist attacks, and in epidemiological diseases. thus, the topic is old but has a new concept in a rapidly developing society. accompanying the new military activities, such as the chinese naval forces on escort duties in the gulf of aden, international peacekeeping activities and humanitarian relief, the military medical mission will have more responsibility in important areas. the armed forces are affected by many elements, such as military equipment, technology, organization and human resources; however, the most important element is "people". maintaining physical and mental health is the fundamental element of reinforcing the armed forces, rapidly recovering from injury, and of regenerating the fighting capacity. the aim of military medical research is to serve the people and to manage people-related health problems. traditionally, injury from weapons is called "trauma" but can be associated with many serious complications and can cause systematic reactions and internal organ damage. these damages are the central part of war injuries but has similarities with diseases in usual healthcare; sometimes these damages are called disease trauma, injury and illness, as well as injury complicated with disease. modern war can not only create somatic trauma and associated diseases but also involve psychological trauma. therefore, military medicine refers to traditional military field surgery or internal medicine as well as the overall health of military staff, particularly how to manage mental stress. currently, military surgery and internal medicine research emphasize the reform of first aid skills and equipment, regenerative medicine, etc. in march , the people's liberation army (pla) military medical institute (mmi) established the military stress research department, which began many research projects in this area and has had many accomplishments. under the current "only child" policy in china, this area has become an important issue in military medicine, covering issues such as how to consult for mental health and how to establish psychological-stress standard measurements and management guidelines [ ] . military medicine can be called translational medicine or a branch of translational medicine [ ] . using the methods of biology, pathophysiology, targeting pharmacology, pharmacological genetic phenotyping, and genetic phenotyping, military medicine can introduce new theories, concepts, and technology into basic research areas, such as epidemiology, pathogenesis and pathological response, mechanisms of complications after injury, regeneration after severe organ damage, mental stress, and psychological responses to somatic and mental trauma. the ultimate goal is to provide services in detection, prevention, diagnosis, and treatment. military activities, such as guarding, training, fighting, and surviving, are usually performed in special environments. the environment plays an important role in the health of military staff. the environment can mean the natural environment (such as remote mountain areas, tropical areas, highlands, oceans, deserts, epidemic or space areas); human-made environments (such as closed, high-pressure, weightless environments); information environments (space fields with information or information-related launching, transmitting, utilizing media carriers); and mental environments (mental stress and mental health-related). special operations should also include tasks in space, aviation, navigation, and underwater diving. the damage to humans in these areas is usually multi-factorial and more complicated and serious than single-factor damage. for historical reasons, high-altitude-related military medical research is the strong point in chinese military medicine, with many accomplished projects, such as the study of the effect and recovery of systemic or organic hypoxia and the mechanism of diseases in highlands. more than diagnostic criteria and management guidelines for high-altitude reactions and hypoxia coma have been developed, as well as a high-altitude standard sanitary system [ ] . in addition, military aviation medicine was established in . many types of aviation medical equipment have been developed by the military service in china in relation to much basic and applied research into mental health, aviation physiological training, pilot selection and unhealthy effects from hazardous aviation environments [ ] . because the space program in china has been established for more than years, aviation medical-engineering technology has rapidly developed and now has more than branches, including aviation environmental medicine, cell biology, and psychiatry [ ] . a few branches, such as cardiology, have remained in a leading position in the world; however, some branches, such as aviation environment and psychiatry, remain behind the international level. for military operations under a full-dimension environment, military medicine in china is now expanding its role in the field, reinforcing research into the military environment and into human-machine cooperation. military medical research will potentiate the capability of human effectiveness and endurance under special situations and operations, including physical fitness, skillfulness, intelligence, human-machine cooperation, emotional perception and overall military operational capability, as well as survival in extreme environments and the enhancement of initiatives. preventing and managing injuries due to high-technology and new-concept weapons because of the rapid development of high-technology, new-concept, and special weapons, the prevention and management of injuries from these weapons are essential for military medicine. new-concept weapons have new injury mechanisms beyond traditional injury mechanisms. revolutionized weapons can also increase the range of human post-traumatic response, creating more difficult and complicated situations for treatment. in the research areas of damage from nuclear, chemical, and biological weapons and of secondary injury from these weapons, china has established standard "triple-prevention" rescue guidelines, including equipment and basic rescue techniques. in the area of detecting, preventing and managing microbiological agents, military medical research in china has reached the international level but should continue developing specialized military supply systems with minimal side effects. however, because of the restrictions in international law and arms control, the possibility of using nuclear, chemical, and biological weapons is small. sub-nuclear weapons (uranium bombs, powerful fuel air explosives, electromagnetic pulse weapons, deep penetrator weapons, and metal hydrogen weapons) or fourth-generation sub-nuclear, sub-chemical, and sub-biological weapons (such as incapacitating agents and detonation inhibitors) will bring more healthcare problems. during the shanghai apec meeting, the beijing olympics, the world expo in shanghai, china and the guangzhou asian games, the chinese military "triple-prevention" medical rescue team played a key role in safeguarding and had great contributions to these conventions [ ] . military medicine in china will continue to research the prevention and management of injuries caused by high-technology, new-concept and newly developed nuclear and chemical weapons. injuries from these weapons include pathophysiological, serious, multi-organ, multi-location, combined, direct and indirect injuries; diseases during wartime; secondary psychiatric trauma and somatic trauma complicated with psychiatric trauma; and long-term effects. simultaneously, military medicine must accelerate research into medical rescue, the production of presentational drugs, storage, equipment, and fast-detecting equipment and build up the basic laboratory research platform. epidemiologic prevention is an important field in military medicine. military staff must be treated as a special group, with a close association with not only diseases in the entire country and society but also characteristics of confined living conditions, a rapidly changing population and extreme living conditions, such as remote mountains and oceans. the pathologies of such staff greatly differ from that in the general population, particularly in epidemiologic incidence and spreading. in terms of biological and microbiological attacks, epidemiologic diseases will spread quickly. in addition to working hard in regular healthcare, researchers must reinforce research into epidemiological prevention to ensure no major outbreaks of disease occur during or after war. coartem, which is a drug for malaria that was invented by the mmi, is one of three patented drugs listed by the world health organization (who) in years. this drug has saved the lives of more than million malaria patients. during the sars epidemic, researchers in the mmi first isolated and genetically analyzed the sars virus. in , severe freezing occurred because of cold weather in southern china; many people were saved by a lotion invented by the mmi and were airdropped a manual to prevent freezing. in march , the epidemic of bird flu h n occurred worldwide, and the military health organization rapidly adopted emergency actions, such as inventing a fast pathogen-detection system, epidemic surveillance, provision of preventive drugs and equipment. diagnostic kits for h n were invented, as were other management systems, such as diagnostic, prevention and treatment guidelines. the military disease-control organization joined the national public health system in , which increased the opportunities for military preventive medicine [ ] . recently, with the help of the development of the military health care system, cooperation, wide application of new material, new technologies, and of new standards, military medicine has developed research into new equipment and reforming tasks. much large health equipment, such as sanitary trains, airplanes and war-zone hospitals, appeared in the healthcare and supply competition in to connect highways, railroads, and air medical rescue transportation and to establish a full-dimension new supply system, further improving the supply and support system. six healthcare support systems were created: emergency rescue, full-dimension transportation, the prevention of nuclear-biological weapons, rapid response, and healthcare information technology and environmental protection under special situations. the largest navy hospital warship, "peace ark," which was equipped with an ambulance boat, escort helicopter, and shipboard medical modular, established a modern medical treatment and rescue platform on the ocean for systemic search and rescue, management, and transportation. by connecting information technology between the land, sea, and air, a full-dimension healthcare system has been established with high-level technology and information and improvements from single to multiple elements, from loose-constructed to integrated construction, and from equipped mobile to mobile-equipped. currently, the efficiency and quality of the epidemiological surveillance system still must be improved. simultaneously, scientists should accumulate experience in preventing epidemiological diseases adapted to preventive medicine's theory and technology system to help the public and to form the basis for future military medical activities. from the requirements for healthcare equipment from modern war, scientists must upgrade the capacity of the supply system. in the future, the multi-functional and modular system must focus on improving the emergencyresponse mobile capacity, with the function of improved intellectualization, information, and mimicking war zones. with fast-developing internet technology, newly developed practical, high-efficiency, and full-dimension information technology will be the future direction of the military medical information technology system. digitalized medical information technology has been popularized at each level of the military system. from the overseas developed technology and experiences, combined with the actual military situation in china, military medicine has begun to build its own medical information system, with military characteristics, serving its modern national defense system and with the ability to win any regional war. in , the general hospital of pla held its first clinical-case telephone conference with a hospital in germany. in , the health department of the general logistics department proposed the project of "army , , ", with denoting the military medical information technology network and tele-medical consulting system. currently, the number of satellite telecommunication systems has reached more than , including telecommunications in tibei, xinjiang, and xisha, which are remote areas. each large military district has built a telecommunications center in the central and regional district hospitals and in basic medical clinics, as well as a telecommunications working station on ocean research vessels, which have completed many tasks including medical service in the first manned flight of shenzhou in . future war will have multiple levels and mutual exchanges and will bring more difficulties to the military supply system. how to immediately transport injured military staff to the hospital behind the frontline, which has life-saving capabilities and many military medical experts, is an important issue. because of the variety of military tasks, many army forces have to live in remote areas, such as mountains, oceans, and islands with poor live conditions. with the development of digitalized information technology, many medical resources can be shared with those remote areas, thus diminishing the differences between the remote areas and large cities and helping doctors and medical services with high-level medical equipment and experts. on april , , during the strong earthquake in yushu, qinghai province, the army rescue team was sent, and the rescue response was rapid and organized, acting as the main force and lifesaving "green ark". via mutual or multiple channels, china has provided humanitarian assistance to many countries such as in asia, africa, and the caribbean, in an emergency crisis. in recent years, the chinese army has sent second-degree peacekeeping and medical teams to the congo, libya, lebanon, and sudan. china and gabon hosted a joint medical rescue action, called "peace angel ", which was the first example of a joint medical training exercise. the chinese army also sent a medical assistant team to joint international humanitarian exercises in indonesia table" . in june , the chinese military medical assistant team joined the humanitarian assistance and disaster relief/military medicine combined exercise of the asean defense ministers meeting plus expert working groups and received praise due to its excellent performance. because china is playing roles that are more important in international activities, china has routinely sent international peacekeeping forces or military medical assistant teams to disaster areas. china will keep joining these international activities, which can provide precious practical and training opportunities for military medical assistant forces [ ] . military medicine is responsible for homeland security and national safety, such as nuclear crisis rescue, emergency public accidents, biologic weapons control, international peacekeeping and medical assistance, and anti-terrorist medical assistance. military medicine must be brought to general guidelines and projects of homeland security, national safety, economy and strategy. to gain support for the protection of law and funding or for future development, military medicine should be legalized [ ] . in , the us army proposed the concept of force health protection and full-dimension protection, emphasizing improving health, protection and treatment of diseases and war-zone medical rescue or full protection, regeneration, and improvement of armed forces capability [ ] . full-dimension medical assistance has both general and special aspects. the general aspect involves fulldimension threats or injury (from natural, information, psychiatric, or social environments) and full-dimension health maintenance (before, during, and after serving the army, preventing emergency injury and its later effects, maintaining armed forces, the work force and living ability). medical service will be under the direction of military, political, and logistics leaders cooperating with local government and with local medical or related resources. the abovementioned issues involve theories and practical applications, which require further investigation to improve and to establish a new medical assistance service theory and organization system to keep pace with the new military revolution. current research in military medical service is thorough, and its design is complete. however, non-war military medical service concerning anti-terror, peacekeeping, dealing with crisis, maintaining public safety, and disaster rescue is not systemic or is absent. our plan of dealing with crises is too principled, conceptive, and general and lacks targeted, practical, applicable, or doable guidelines. our military medical research still lacks a good design and is underdeveloped without strong associated research. new developments, such as genetic, cell, enzyme, and protein engineering, as well as genetic-chip, space-information, and nanometer technology, will bring rapid developments for medical research. developments in neuron injury, space medicine, biology, radiology and radiation injury will provide important tools for military medicine research. these new development can help to develop new foundations for military medicine, to establish new research models, and to accomplish many special tasks under the new situation. these advanced and new technologies will change military medicine toward diagnosis and treatment as well as toward detection and prevention. the establishment of mimicked human technology and military medical biological engineering provide practical methods for researching medical rescue, medical assistance, the maintenance of armed forces health, and the enhancement of the effectiveness of the army supply system and for increasing the military capacity of armed forces [ ] . biotechnology can create new improvements in life information resources, micro-space and bio-control technology, involving the most important military information, such as ethnic genetic coding, functional control key, and creating new battling territory, with the ability of direct application to military defense attack systems and having the characteristics of "micro, defined, not lethal, and reversible". biotechnology can target changes in micro-structures and further define the control of special physiological functions to limit the injury of biological targets without lethal effects. medical research into these issues can help develop military medicine. under the current requirements for military drug research and national medical strategy, scientists and doctors should disseminate the knowledge of traditional medicine among every level of the military forces. scientists and doctors should find and develop military drugs from traditional medicine and must improve the survival ability of soldiers and officers, maintaining their mental health and combat force and improving military medical storage strategies. using modern pharmacological research methods, such as molecular biology, molecular pharmacology, bio-neurology, bio-physics, cell biology, natural medical chemistry and computer science, scientists should increase the intensity of research into traditional medicine, focusing on selecting traditional drugs that fit the requirements of modern war. establishing a policy of military medicine is the guarantee of the new military medicine environment and revolutionized system. establishing and completing military medical policy will move military medicine in the correct direction of scientific development. scientists should base their policy on the requirements of future war, through appropriate cooperation with civilian research resources, emphasizing the selection and education of a special military medical branch, with deepening and widening directions. in , a proposal concerning the further deepening of militarycivilian cooperation was published by the ministry of health; the general logistics department of pla; education, health, and national traditional medicine departments; the national food and drug control administration; the china science institute; and by the china academy of engineering. scientists must rely on national resources in developed and advantaged areas to widen the range of military medical research with full-dimension development. to form a solid base for future medical research, scientists also must attract military or civilian researchers and experts. abbreviations pla: people's liberation army; mmi: military medical institute; who: world health organization. the authors declare that they have no competing interests. the characteristics of military transformation and its effect on military medicine research progress on psychological pressure source of soldiers editorial: translational medicine, a new topic area of clinical and health importance research status and development of chinese altitude military medicine several development trends of frontier research and reflection on military aviation medicine. the academy of military medical sciences of the chinese pla a new discipline arising from china's manned space projectspace medico-engineering chinese military health service in non-war military operations discipline progress of chinese military preventive medicine capability development for the peacekeeping operation medical support of chinese medical teams on policy construction of military medicine interpretation of the u.s army medicine strategy map discipline construction of military biomedical engineering cite this article as: fu: military medicine in china: old topic, new concept this project was supported in part by the national nature science key: cord- -mup tzkv authors: aljohani, majed; kennedy, katharyn e. title: mass gatherings date: - - journal: ciottone's disaster medicine doi: . /b - - - - . - sha: doc_id: cord_uid: mup tzkv nan majed aljohani and katharyn e. kennedy thousands of mass gathering events take place worldwide each year. in the united states, as many as million attend national basketball association (nba), national football league (nfl), and/or national collegiate athletic association (ncaa) events. the inauguration of president obama on january , , attracted a crowd of . million to the national mall in washington, d.c.; a state of emergency was declared to allow the release of federal money and resources. protests during the arab spring in drew millions of largely peaceful protesters to central locations of tunis, tunisia, and cairo, egypt. more than million were present when the departure of egypt's president hosni mubarak was announced in february, . millions of people celebrated the world cup in brazil without significant problems; it seems that lessons have been learned from previous soccer disasters (table - ) . there is no consensus on a definition of a "mass gathering." the world health organization (who) defines it as "an organized or unplanned event where the number of people attending is sufficient to strain the planning and response resources of the community, state, or nation hosting the event." the national association of emergency medical services physicians (naemsp) defines it as "organized emergency health services provided for spectators and participants at events in which at least persons are gathered at a specific location for a defined period of time." there are two types of mass gatherings: the traditional, such as religious festivals, sporting events, music concerts, fairs, and parades; and the nontraditional, which have not been researched very well, including metropolitan subway systems, large shopping complexes, airports, cruise ships, public demonstrations, and refugee camps. medical care of some sort has been provided at such gatherings for the last years in both the united states and europe. event organizers everywhere need to take responsibility for the safety and well-being of the participants at an event. considerable variation exists in the type of medical care provided to both participants and spectators at these gatherings. general standards have been proposed for the provision of primary care, emergency and disaster care, and means of evacuation. both the american college of emergency physicians (acep) and naemsp have addressed the previous lack of guidelines and standardized care. a survey in showed that only six u.s. states provided regulatory guidance for the provision of care at mass gatherings, although many are now starting to address this omission. terrorist threats have become an unfortunate reality, as shown by the bombing at centennial park during the olympics in atlanta, georgia, and the targeting of nontraditional mass gatherings, such as the madrid train bombings in , the kenyan shopping mall mass shootings in , and the boston marathon bombing that same year. such events pose unique issues regarding the care and evacuation of patients, particularly in unsafe or unsecure scenes. at organized mass gatherings, predictable medical problems and an unpredictable wide variation in medical care exist worldwide. preplanning and prediction of resource requirements, based on careful needs assessment of anticipated medical care usage and public health risks, may lead to a standardized optimal provision of medical care (box - ). provision of medical care is the responsibility of the event planners. public health officials need to be involved early in the planning process, especially for large events, such as the olympics, world fairs, and pilgrimages. the local health department should be involved in overseeing the preparation, storage, and serving of food and sanitation requirements. once identified, those providing medical care at mass gatherings need to liaise with local emergency medical services (ems), fire, and law enforcement officials. ground and building plans, close estimates of possible attendance, and identification of any specific hazards should be shared among these providers. multiple variables interact to make planning for a mass gathering event challenging. an understanding of these may allow for a more efficient and effective planning process. some of these variables will be discussed below. estimates of attendance may be gleaned from advanced ticket sales or from attendance at previous similar events. however, previous history is notoriously unreliable, as demonstrated by the papal mass in denver ( ) where , people were predicted but , turned up. an adequate ticketing system is essential, as well as public address measures to inform crowds of no further access to an event once capacity is reached. mass gathering venues should have multiple access points for entering and exiting the site. during the hajj, people lost their lives due to a stampede as all the pilgrims were trying to leave the city of mecca at the same time through a tunnel. planners should promote a unidirectional flow of the crowd, which would significantly reduce the risk of crowd convergence. training of security staff in crowd control should be implemented before an event to improve crowd safety and avoid panic should overcrowding occur. during a football match in ellis park, south africa, tear gas was thrown into a crowd by event security in an effort to disperse intense overcrowding. this, unfortunately, served to incite panic and caused a stampede (table - ). keeping the crowd comfortable and relaxed is also essential, therefore awnings, cooling fans (in hot weather), and entertainment are sometimes helpful and should be part of the preplanning. it is crucial to establish goals for medical services. these include the following: • establishing rapid access to injured or ill patients and providing triage • stabilizing and transporting seriously injured or acutely ill patients in a timely manner • providing on-site care for injured or ill patients the type of medical care to be provided at the event needs to be considered beforehand (table - ). [ ] [ ] [ ] [ ] [ ] primary medical care, such as first aid, emergency care, and preparation for a possible disaster, should be addressed. staffing levels and type of staffing also need to be anticipated. recommended ratios include one to two physicians per , attendees, two paramedics or one paramedic and one emergency medical technician per , attendees, and one basic first aid provider per participants at the event. on-site physicians have been shown to reduce ambulance transfers to local hospitals by as much as %, significantly lessening the impact of an event on local ems services and hospitals. the majority of nondisaster injuries and medical complaints at a mass gathering can be effectively treated at the scene, which reduces the number of hospital referrals and patient presentation rates to the hospital. physician presence should be strongly encouraged at events where significant trauma may occur or where there is a long distance to definitive care. cardiac arrest cases are rare in mass gatherings, with reports of . to per , , though on-site resuscitation and early defibrillation are important and can improve patient survival rates. a prediction tool for expected medical needs considering five different variables (attendance, heat index, crowd age, crowd mood, and availability of alcohol) was developed and tested in mass gathering events (tables - and - ). after the anticipated usage rates and staffing levels have been addressed, the positioning, number, and type of aid stations should be considered. fixed events at stadiums may have areas specially designed and designated for medical care. for other events, aid station locations should be no more than a -minute walk for attendees. stations should be clearly visible, and the locations should be known to participants and other event personnel. these areas need to be adequately and appropriately staffed before the anticipated start of the event, and remain so until the event is completed. consideration needs to be given to providing medical care in the crowd for occurrences such as cardiac arrest or lower extremity fractures, including how to transport these patients to aid stations. thought needs to be given regarding provision of ambulances for hospital transport and access and egress for these vehicles. the organizers of medical care need to know the capabilities of local hospitals and should liaise with hospital personnel before the event. all patient encounters should be documented. use of noncarbon record (ncr) paper will facilitate a copy accompanying a patient who needs transport to a medical facility. records are needed for medical and legal reasons and may also be useful for research purposes. there is a positive correlation between temperature and humidity and patient presentation rates. heat indexes, which take into account the temperature and the humidity, are probably a better indicator of medical need, as it has been shown that with every °rise in the heat index, the number of patients per , spectators (pptt) tripled. thirtyone percent of physician encounters at the california aids ride were for heat-related problems. the denver papal visit resulted in an unanticipated , patient encounters at a mainly youthful gathering, due in part to the -mile walk and high temperatures. preplanning for the atlanta olympics in , in the form of education about preventing heat-related illnesses given to those who purchased advance tickets, might have led to a decrease in the number of patient encounters, despite the high heat and humidity. cold weather events generally lead to lower medical usage rates by participants and spectators. use of weather surveillance, early warning systems, and evacuation to shelter protocols is important in cases of storms and lightning. an increase in the number of patient medical encounters may be related to alcohol and illicit drug usage. historically the consumption rate of alcohol and drugs may be higher at music festivals, rock concerts, and raves. open-air music events in the united kingdom have resulted in a primary diagnosis of alcohol intoxication in % of patient encounters. banning the consumption of alcohol at wembley stadium led to a % reduction in alcohol-related problems. an increase in crowd size does not necessarily mean an increase in medical usage. one study has shown that the overall medical usage rate decreases with overall crowd size. patient encounters at events with more than million participants average per , participants. events with fewer participants average per , spectators. one interpretation of this is that it is easier to seek medical care at a less crowded venue. the medical usage rate can vary even within an event itself. at the los angeles olympics, soccer events had usage rates of per , and rowing and canoeing, . per spectators. anticipated crowd demographics may be useful in the preplanning stage. older groups may be expected at papal visits, classical music concerts, and large sporting events. younger groups frequently attend rock concerts and auto-racing events. the needs of children also must be considered. most children present with minor injuries, but medical teams need to be prepared to deal with serious medical emergencies and trauma. overall medical usage at a children's fair was . per , participants, and half of those who presented were younger than years old. protocols need to be in place for the provision of care to minors who present without an accompanying adult. event type is an unpredictable variable. certain types of music, known team rivalries, and religious furor may lead to disruptive behavior and an increase in medical usage rates. crowded events may lead to a "too-close-for-comfort" feel among attendees. environmental conditions, such as inclement weather, squalid conditions, poor sanitation, and lack of access to drinking water, may lead to ugly crowd dynamics. despite the many variables to be considered, it has been shown that event type and temperatures are the ones that best predict medical usage rates. examples of event types will be discussed later in this chapter. other variables to consider include event duration and time of occurrence. attention to the location, staffing, and communication needs of a medical command center should be addressed. communication needs should be considered for event medical providers, other event planners, and local ems, police, and fire personnel. backup communication in the form of handheld devices or cell phones should be decided on, and medical personnel must be able to connect with local dispatch centers. an incident command structure for medical personnel may be used. the event medical officer oversees all aspects of medical care provided at the site. the event triage officer conducts and directs medical assessment of casualties at designated treatment areas or while roving through the crowds and transporting patients to a central area. the event treatment officer oversees treatment to the sick and injured. the event transport officer directs transport to other facilities, and the logistics officer provides the necessary support for ems at the event. consideration may be given to the need for hazardous material teams, decontamination, wilderness medicine, or use of amateur radio groups. the naemsp addressed components on which event planners must focus (box - ). for a detailed review of these elements, the naemsp produced a comprehensive document entitled "mass gathering-medical care; the medical director's checklist." the medical usage rate may vary by type of music, with rhythm and blues having rates of . per , and gospel or christian . per , attendees. the overall median usage for concerts is . per , attendees. rock concerts typically have rates . times that of other concerts. the anticipated audience participation in "moshing," crowd surfing, and stage diving may lead to a dramatic increase in medical incidents. other problems encountered include minor trauma and alcohol or illicit drug intoxication. surgical problems may be caused by falls, assaults, being crushed against barriers, and assorted "missiles" causing head injuries. severe trauma may occur in up to . % of attendees at rock concerts. medical issues include headache, syncope, asthma, and hypoglycemia. cardiac arrest is uncommon, with a rate of . to . per , attendees. asthma may be very common at rodeos. the ultimate sporting event is probably the olympics. planning for the medical care of both spectators and participants begins as soon as the host city has been announced. around million spectators attended the london olympics. apart from routine medical care, there exists a potential for transmission of infectious diseases, risk of injury from crowd crushing, and now the very real risk of terrorist activity or political protests. extensive planning at local, state, and federal levels is vital to ensure the health and safety of all concerned. [ ] [ ] [ ] at the atlanta games, specialized incident assessment teams were set up to analyze terrorist risks and to address issues such as stockpiling of antibiotics and antidotes. medical providers of all levels received training in awareness of chemical, biological, and radiological weapons. local hospitals were updated to include mass decontamination units. local ems providers were given uniform operational plans and procedures, enhanced communications were agreed on, protocols were developed for the management of heat-related illnesses, and guidelines for response to mass casualties were issued. public health initiatives to address heat-related illnesses included a media campaign; packets sent to ticket purchasers; shelters; and provision of water, wide-brimmed hats, sunscreen, and water misters at the most crowded sites. these, plus the cooler-than-normal temperatures, may have led to a decrease in the expected number of hyperthermia victims. organizers of the world cup in south africa developed an infectious disease surveillance system for the event, which reported to a national health operations center. despite taking place over a month during the peak of the influenza season in south africa (in anticipation of which who donated . million doses of pandemic h n vaccine), only communicable disease events were reported. more than marathons are staged each year, along with countless half marathons, triathlons, and k and k events. pre-event considerations include course layout, number of runners, climate, and medical team experience. earlier start times and the addition of half marathons have led to a decreased risk of injury. , encouraging runners to seek help early has also reduced serious medical problems. educating runners before the event on issues such as dehydration, hypoglycemia, exhaustion, blisters, the importance of good preparation and training, and the use of energy drinks may lessen the need for medical intervention. runners with a history of asthma are encouraged to carry their inhalers and not to run if they feel ill. accessible, visible first aid stations along the route, use of mobile paramedic teams, and a medical control center should provide adequate medical coverage. radio communication is essential between medical providers, and a treatment tent at the finish line should include paramedic and triage teams, massage therapists, and podiatrists. the use of computer tracking chips may be used to identify how many runners use medical treatment and help in the planning of the provision of care and supplies in the future. on april , , while thousands of spectators were celebrating the boston marathon, two pressure cooker bombs exploded, killing three people and injuring an estimated others. the bombs exploded about seconds and m ( yards) apart, near the finish line. many of the victims suffered serious injuries, and at least people lost limbs as a result of the blast and its debris. many lives were saved that day because of good planning. the perfectly located medical treatment areas were well staffed and were immediately converted into a mass casualty triage unit, serving as a direct egress point for the ambulances. only lifesaving procedures were done, without delaying the transfer of the seriously injured patients to hospitals. victims were distributed equally to six level trauma centers surrounding the area, which avoided overwhelming a single center. pre-event coordination and planning among all agencies and hospitals, along with regular joined training and drills, were huge factors in the success of patient care. millions of people perform pilgrimages every year. the muslim pilgrimage, the hajj, to the holy land of makkah (mecca) in saudi arabia, may have up to . million participants from countries for a period of to days. the pilgrimage involves a -mile round trip from makkah through the plains of arafat. many of the pilgrims are elderly, come from poor countries, live in tents in extreme conditions, and perform physically exhausting religious rituals. the hajj season varies every year because it follows the lunar islamic calendar. therefore likely diseases also vary, depending on the temperature, which is a huge challenge for those involved in planning. in india, millions of pilgrims visit lord ayyappa at sabarimala each year. this occurs over a -day period and involves a -minute trek uphill to the temple. kumbh mela is a mass hindu pilgrimage of faith in which people gather to bathe in sacred rivers. it is considered the largest peaceful gathering in the world; more than million people visited during the maha kumbh mela in , a -day event. thirty-six people died in a stampede at the allahabad railway station the same year. a massive rush of passengers, returning from a dip in the waters of the ganga and yamuna rivers at the maha kumbh, contributed to the tragic event. many other pilgrimages on a smaller scale take place throughout the world. several of the problems encountered may be anticipated. many of the participants are not in good health and may have chronic medical problems. heat exhaustion is common during the hot cycle of the hajj. cases of heat stroke doubled from to . this could be overcome by educating pilgrims before and during the event. infectious disease outbreaks are also common. these include meningococcal meningitis; gastroenteritis; hepatitis a, b, and c; and various zoonotic diseases. the implementation of vaccination policies, infection control policies, and public health initiatives have proven successful in addressing these problems. pilgrims need proof of appropriate vaccinations to obtain a visa for travel to saudi arabia. face mask use is encouraged to reduce the spread of respiratory infections. some of the mitigation actions taken at the hajj are discussed in table free medical care is provided at the holy site in makkah. in and a "treat and release" program commenced, leading to a % reduction in ambulance transports. in india at sabarimala, a medical center is provided at the site. typically pilgrims receive medical care over the -day period. in the wake of various disasters at mass gatherings, it is vital to learn from previous mistakes. the european convention on spectator violence and misbehavior met in to address issues rising largely from the heysel stadium disaster. it identified the need for police and sports authorities to cooperate in ensuring segregation of rival supporters, controlling access to stadiums, and banning the consumption of alcohol. after the hillsborough disaster in england, the gibson report recommended medical care at stadiums for the first time. all-seat stadiums were to be phased in, leading to safer stadiums with greater attendance. the boston marathon has instituted a post-event clinic that is open for days after the event to meet the delayed medical needs of runners. the need for a mass gathering triage system is clear, because these events, by their very nature, have the potential to turn into mass casualty incidents (mcis). also, the current prehospital disaster triage systems are biased toward traumatic victims without considering medical complaints, drugs and alcohol, and environmental exposures, making these systems imperfect instruments for use in mass gatherings. having a unified triage system that is agreed upon among all local agencies and hospitals is essential. frequent training on triage is important for the success of any response plan. in general, most participants require minor medical interventions that may be addressed by trained first responders or paramedics. paramedics may use triage protocols to identify casualties who should be transported to a hospital after initial stabilization rather than waiting for treatment by an on-site physician. at rock concerts, . % of attendees may experience severe trauma. major and minor trauma results from falls, assaults, being crushed against barriers, and head injuries from assorted "missiles." anticipated medical problems include headaches, syncope, hyperventilation, asthma, epilepsy, and hypoglycemia. at the atlanta olympics, most of the injuries were sprains or strains ( %) and contusion abrasions ( %); bronchitis was common ( %), and heat cramps and dehydration accounted for % of those seeking medical care. in addition, three cardiac arrests were reported; the provision of defibrillators at mass gatherings is an important consideration. hyponatremia is increasingly prevalent among marathon runners. it is defined as a serum sodium level of less than mmol/l and is commonly caused by overhydration. mild cases may be treated by fluid restriction and consumption of salty foods until urination resumes. in moderate cases the patient's sodium level may need to be checked hourly and, in critical cases, intravenous access will be required and diuretics and hypertonic saline may be administered. complications such as seizures, pulmonary edema, and coma should be treated appropriately. runners should be encouraged to replace only ounces of fluid along with salt for every pound of weight lost. exercise-associated collapse may occur at the finish due to venous pooling. this may be treated by laying the patient supine with the legs elevated and rehydrating with oral electrolyte or carbohydrate solution. dehydration should be assessed clinically and treated with oral fluids. heat-related illnesses are addressed in the usual fashion. hypoglycemia is treated with oral or intravenous glucose replacement. many pilgrims are in poor health before the event and may need more than minor first aid. many suffer from heat-related illnesses or infectious diseases. provision of free medical care and on-site medical facilities with capabilities of providing even an intensive care unit level of care may meet these needs. disasters do occur at mass gatherings, and appropriate medical care needs to be available. frequently, the cause of death is traumatic asphyxia, for which rapid interventions may make a difference. every mass gathering should be considered as a unique event. careful and exhaustive preplanning may reap many benefits. it is important for those providing medical care at the event not to do so in a vacuum. local ems providers may well be needed and certainly will be required if a mass casualty event occurs. mass gatherings occurring in urban settings will have different characteristics and requirements from those in more rural or remote settings. even though it may be difficult to predict all the medical needs of the crowd, prior studies have started to use a more scientific approach to addressing these needs. event type, duration, expected attendance, and weather conditions need to be taken into careful consideration in the planning process. • lack of legislation regulating minimum standards for provision of medical care at mass gatherings • lack of a coordinated, integrated preplanning process • lack of funding to provide needed public health initiatives and medical resources • failure to identify a medical director for the event • failure to learn from previous experiences • underestimating expected attendance • failure to consider all variables, such as crowd size, demographics, event duration, and environmental factors • failure to consider and prepare for a terrorist event or mci • inability to allow capacity crowd ingress to a stadium in a -hour period • failure to consider ambulance access and egress at an event • lack of training of security personnel, leading to failure to recognize and control potentially dangerous situations mass gathering medicine: a review world health organisation. communicable disease alert and response for mass gatherings: key considerations mass gathering medicine: a review of the evidence and future directions for research provision for emergency medical care for crowds mass gathering medical care characteristics of state legislation governing medical care at mass gatherings mass gathering medical care: the medical director's checklist for the naemsp standards and clinical practice committee. lenexa, kan: national association of disasters at mass gatherings: lessons from history health risks at the hajj critical illness at mass gatherings is uncommon mass gathering medicine: a predictive model for patient presentation and transport rates mass gathering events: retrospective analysis of patient presentations over seven years at an agricultural and horticultural show medical support for children's mass gatherings emergence of medicine for mass gatherings: lessons from the hajj london: the stationery office on-site physicians reduce ambulance transports at mass gatherings mass gathering medicine: the melbourne commonwealth games experience predicting resource utilisation at mass gatherings using a simplified stratification scoring model the impact of warm weather on mass event medical need: a review of the literature association of heat index and patient volume at a mass gathering event medical care at the california aids ride : experiences in event medicine mass gathering medicine: a review mmwe: prevention and management of heat-related illness in many spectators and staff during the olympic games-atlanta health and safety at the winter olympics emergency medicine at a large rock festival concert medicine: spectrum of problems encountered at major concerts emergency medical services in mass gatherings: the experience of the formula grand prix 'san marino' in imola the mosh pit experience: emergency medical care for concert injuries prehospital severity scoring at major rock concert events frequency of asthma exacerbations at mass gatherings. chest public health response for the olympic games medical care delivery at the olympic games more than a sprint to the finish: planning health support for the sydney medical preparedness for a terrorist incident involving chemical or biological agents during the atlanta olympic games report on who support to the fifa world cup south africa™ pretoria, south africa marathon medical services: strategies to reduce runner morbidity a -year profile of medical injury and illness for the twin cities marathon glasgow marathons - . a review of medical problems infection control in saudi arabia: meeting the challenge treat and release: a new approach to the emergency medical needs of the oldest mass gatherings-the pilgrimage use and analysis of field triage criteria for mass gatherings mathematical determination of external defibrillators needed at mass gatherings mass casualty in a pop music concert instead of being a programmed event: home fair key: cord- - ksss authors: harendza, sigrid title: improvisation – a new strategy in medical education? date: - - journal: gms j med educ doi: . /zma sha: doc_id: cord_uid: ksss nan suddenly everything is different. a virus keeps the world in suspense and medical education as well. yet the older teachers among us know quite well what viral diseases are and how they can change undergraduate and postgraduate medical training. during my own undergraduate training the hiv epidemic began, which even after well over years still requires further learning [ ] , i.e. learning about contents that could not be taught at all the during the time of my studies. the situation is similar with the viral disease hepatitis c. while it was still called hepatitis non-a non-b during my studies [ ] , it has since been given its current name and everyone, including the teachers, had to learn to understand over the next decades that no vaccination was made possible, but that in the meantime drugs had been developed which can eliminate the hepatitis c virus [ ] . such influences of pathogens on the content of medical studies could be relatively easily dealt with by changing the learning objectives adapted to medical progress. in case of structural changes, which may become necessary in teaching during epidemics, adaptation processes are not quite so easily implemented. during the ehec epidemic in , our nephrological-internal ward and several other wards of the university medical center hamburg-eppendorf accommodated almost exclusively patients suffering from ehec-induced haemolytic uremic syndrome (hus) [ ] . this is a rare disease of which students only had to have heard of at that time according to the hamburg catalogue of learning objectives [ ] . within two weeks it was almost impossible for final-year students in internal medicine to see patients with other diseases. hence, we, as teachers, hat to improvise teaching -in addition to caring for the patients -and maybe the final-year students at that time learned a little less about different diseases but a lot instead about medical behaviour in unknown clinical situations. in the national competence based catalogue of learning objectives (nklm) of , hus is still listed as a rare disease under point . . [http://www.nklm.de, accessed: . . ], but the labelling with competence level a requires in any case more extensive knowledge than just knowing the name of the disease. so now, in , it is again a pathogen that influences medical education. but this time the change affects all teachers and all students and almost all teaching and examination structures in medical education and in all other healthcare professions studies as well as in all other courses of study in general -and this worldwide [university world news: https://www.universityworldnews.com/post.php?story= , accessed: . . ]. for the study of medicine, dentistry, and veterinary medicine, but also for the study of other healthcare professions, we are currently confronted with a rather small-scale structuring of teaching instruction, which prescribes content and form of teaching at most universities right down to the individual lesson. however, in the current situation, which will probably keep us busy for the next few months or even years, the ability to improvise and flexibility are required while keeping the main educational goal in mind. for postgraduate medical education, van loon and scheele recently demanded to renounce from detailed regulations and to move towards enabling teachers to engage in curricular innovation that is "only" oriented towards the educational goal [ ]. confidence in the creative design of the curriculum by teachers and their empowerment for free design opportunities [ ] should also enable universities to act quickly in times of a pandemic-related lockdown. this kind of action, i.e. the development of one's own strategies within the frame of one's own current possibilities paying attention to the global goal, but without prescription of all individual steps in detail, is called mission tactics or command and control with mission in the military. this leadership tactic has proven to be particularly effective during confusing situations to achieve a global goal [ ] . the acquisition of improvisation skills is, for example, explicitly required for students of teacher training and is already being practiced in class in some cases [ ] . for medical educators and medical students, such techniques of improvisational theatre seem to be useful as wellboth for medical activities and for teaching medical students or for designing lessons, respectively [ ] . medical work is unpredictable by its very nature. medical students must learn to deal adequately with uncertainty inherent in medical problems. this is already being implemented didactically in problem-based learning and leads to a better handling of uncertainty in everyday medical practice [ ] . furthermore, there are frameworks that use techniques of improvisational theatre to enable medical students to learn how to deal with unknown medical situations [ ] . these techniques of improvisation could also be appropriate to enable teachers to teach adequately in uncertain times [ ] . they appear to be particularly useful for learning communication skills and professional behaviour [ ] . but other teaching techniques also help to improvise appropriate medical lessons in times of a lockdown, especially e-learning, of course [ ] , because it is particularly easy to keep one's distance. this issue also contains some interesting approaches in scientific work and projects which encourage creative thinking for medical teaching and testing in the current situation, although at the time of their creation there was no mention of sars-cov- at all. rauch et al. report on the development of an instructional video for dental students to examine patients with suspected craniomandibular dysfunction [ ] . perhaps a way can be found to allow dental students to practice clinical examination techniques with people in their own homes during a lockdown, guided by such videos. the project could perhaps also be adapted to a -step video format based on the so-called peyton method, as is already used at the ludwigs-maximilians-university (lmu) in munich [https://www.med.moodle. elearning.lmu.de/mod/book/view.php?id= & chapterid= , accessed: . . ]. möltner et al. were able to show that the assessments of student raters in a formative osce in general medicine correlated highly with the assessments of medical experts [ ] . this scientific finding may also lead to the development of further training and assessment options for medical students as peers for practical and communicative skills. findyartini et al. were able to show in their study that the motivation profile of medical students is associated with the empathy they express [ ] . thus, this project also offers interesting starting points for teaching and learning empathy. zimmermann and kadmon used standardized examinees who had received training for different levels of proficiency in osce stations that were filmed and can be used both for quality assurance of osce stations and for rater training [ ] . this concept can probably be easily and contactlessly used for rater training at other universities. these examples show the essential contribution that projects in medical education research make to enabling teachers to draw on evidence even in times when they have to improvise. so let's stay tuned to improvised teaching -scientifically sound and creative. [ ] . der erwerb von kenntnissen im improvisieren wird beispielsweise für lehramtsstudierende explizit gefordert und zum teil auch im unterricht schon praktiziert [ ] . für lehrende der medizin und medizinstudierende scheinen solche techniken des improvisationstheaters ebenfalls nützlich zu sein -und zwar sowohl für die ärztliche tätigkeit als auch für das unterrichten von medizinstudierenden bzw. für die gestaltung des unterrichts [ ] . das medizinische arbeiten ist von der natur der sache her unvorhersehbar. medizinstudierende müssen lernen, der medizinischen problemen innewohnenden unsicherheit adäquat zu begegnen. dies wird bereits didaktisch im problemorientierten lernen umgesetzt und führt zu besserem umgang mit unsicherheit im ärztlichen alltag [ ] . außerdem gibt es rahmenwerke, die techniken des improvisationstheaters aufgreifen, um medizinstudierende zu befähigen, mit unbekannten medizinischen situationen umgehen zu lernen [ ] . diese techniken der improvisation könnten ebenfalls geeignet sein, um lehrenden in unsicheren zeiten adäquates lehren zu ermöglichen [ ] . besonders nützlich scheinen sie zum erlernen von kommunikationsfähigkeiten und professionellem verhalten zu sein [ ] . [ ] . dieses konzept lässt sich vermutlich leicht und kontaktarm an anderen hochschulstandorten zur prüferschulung einsetzen. diese beispiele zeigen, welchen wesentlichen beitrag projekte der medizinischen ausbildungsforschung leisten, damit lehrende auch in zeiten, in denen sie improvisieren müssen, auf evidenz zurückgreifen können. bleiben wir also dran an improvisierter lehre -wissenschaftlich fundiert und kreativ. die autorin erklärt, dass sie keine interessenkonflikte im zusammenhang mit diesem artikel hat. the fourth hiv epidemic non-a, non-b viral hepatitis from non-a, non-b hepatitis to hepatitis c cure the fourth hiv epidemic non-a, non-b viral hepatitis from non-a, non-b hepatitis to hepatitis c cure hus diary" of a german nephrologist during the current ehec outbreak in europe improving graduate medical education through faculty empowerment instead of detailed guidelines auftragstaktik -geschichte und gegenwart einer führungskonzeption. frankfurt/m: report-verlag improvisation in teaching and education -roots and applications medical education empowered by theater (meet) the effects of problem-based learning during medical school on physican competency: a systematic review common ground: frameworks for teaching improvisational ability in medical education twelve tips for using applied improvisation in medical education medial improv: a novel approach to teaching communication and professionalism skills learning under lockdown: navigating the best way to study online development of an instruction movie illustrating a standardized clinical examination of patients with tmd symptoms formative assessment of practical skills with peerassessors: quality features of an osce in general medicine at the medical faculty of heidelberg relationship between empathy and motivation in undergraduate medical students standardized examinees: development of a new tool to evaluate factors influencing osce scores and to train examiners improvisation -a new strategy in medical education? the author declares that she has no competing interests. key: cord- - hn wsv authors: bruce, lori title: a pot ignored boils on: sustained calls for explicit consent of intimate medical exams date: - - journal: hec forum doi: . /s - - - sha: doc_id: cord_uid: hn wsv unconsented intimate exams (uies) on men and women are known to occur for training purposes and diagnostic reasons, mostly during gynecological surgeries but also during prostate examinations and abdominal surgeries. uies most often occur on anesthetized patients but have also been reported on conscious patients. over the last years, several parties—both within and external to medicine—have increasingly voiced opposition to these exams. arguments from medical associations, legal scholars, ethicists, nurses, and some physicians have not compelled meaningful institutional change. opposition is escalating in the form of legislative bans and whistleblower reports. aspiring to professional and scientific detachment, institutional consent policies make no distinction between intimate exams and exams on any other body part, but patients do not think of their intimate regions in a detached or neutral way and believe intimate exams call for special protections. uies are found to contribute to moral erosion and moral distress of medical students and compromise the sacred trust between the medical community and the general public. this paper refutes the main arguments in favor of the status quo, identifies a series of harms related to continuing the current practice, and proposes an explicit consent policy for intimate exams along with specific changes to medical school curriculum and institutional culture. because patients are the rights-holders of their bodies, consent practices should reflect and uphold patient values which call for explicit consent for intimate exams. from hippocrates to percival, classic medical scholars demonstrated "virtually no appreciation of a patient's right to consent" (beauchamp , p. ) . they avoided disclosures perceived to "harm or upset patients" and viewed patients' bodies as vessels for research and physician education; a view not to be superseded by a patient's preferences (beauchamp ) . prioritizing research and education over a patient's preferences implies utilitarianism, when the goodness of an action is rooted in creating the most positive outcome for the greatest number of people. with the advent of bioethics, clinical medicine moved to a deontological frame in which the goodness of an action is based on whether that action in itself is right or wrong. bioethics also phased out the idea that physicians' perceptions of a patient's best interests should supersede the patient's own values. informed consent allows the patient to decide what happens to their body based on an understanding of the proposed procedure, and this concept was "imposed on medicine through nonmedical forms of authority such as judges in courts and government officials in regulatory agencies" (beauchamp , p. ) , underscoring the dichotomy between physicians' and nonphysicians' perceptions of rights. consent is now a well-established tenet of medical practice. physicians must request a patient's permission before conducting research or performing procedures on them. consent "must be rooted in autonomous choice by patients" (beauchamp , p. ) and requires an explanation of the procedure and disclosure of the risks and benefits. medicine is comprised of committed, talented individuals who dedicate their lives to healing the wounded and comforting the dying; however, certain practices still demonstrate concerning perceptions. recent surveys and reports reveal that some physicians and medical students still conduct unconsented intimate exams (uies) on male and female patients. unconsented rectal exams on men and women have been reported for training purposes (reese and monrouxe ; coldicott et al. a, b) and diagnostic reasons (chan ) . unconsented vaginal exams have also been reported, mostly during gynecological surgeries (schniederjan and donovan ; barnes ; picard ) but also during abdominal surgeries (graham ; kelly ) and in efforts to rule out sexually transmitted disease in a patient with uncontrolled vomiting (laird ) . the referenced studies surveyed hundreds of medical students in the u.s., england, wales, and australia. in one study, nearly three-quarters of students believed that patients did not consent to their training exams (schniederjan and donovan ) . in another study, patient consent for intimate exams was not acquired by half of the third-year students or one-third of patients examined by second-year students (coldicott et al. a, b) . medical students are eager to participate in procedures and quickly realize the delicate balance of gaining new skills while respecting patient autonomy. students are also careful to navigate complex organizational dynamics inherent to the academic culture of their medical schools. a student refusal to participate in an unconsented intimate exam (uie) may be viewed as an act of personal embarrassment or unwarranted disobedience. because students worry about jeopardizing their budding careers, they sometimes participate in clinical experiences that conflict with their personal moral compasses. fraught dynamics between student and supervisor are central to the controversy of intimate exams on patients. a pelvic exam is a standard precursor to gynecologic surgery to ascertain the position and mobility of the reproductive organs. the exam requires the insertion of one or two fingers into the vagina to palpate the ovaries and uterus. rectal exams similarly require digital penetration with the aim of detecting abnormalities in the rectum, prostate, and abdomen. medical schools consider these digital exams as standard training procedures and typically require all students to practice them during their third-year clerkships. some institutions have policies in place requiring explicit consent for intimate exams, but they are the exception; most institutions have no such policies. physicians often assume consent for these training exams, believing it is covered under general admissions clauses-or vague language on consent forms-relating to student participation in teaching hospitals. consent forms often require the attending physician to hand-write the major risks of a specific procedure but otherwise follow a standard template. patients and members of the general public are often shocked to learn that physicians and medical students may perform intimate exams on them without explicit consent and believe such exams meet the criteria for battery, malpractice, or sexual assault. whether these exams meet any such criteria is contested, but this tension underscores the dichotomy between societal and medical views of the intimate regions of the body. while the medical professional may aspire to a professional, scientific detachment, patients simply do not think of their intimate regions in a detached or neutral way and tend to have an inherent conviction that consent practices should mark this distinction. for several decades, ethicists and some number of physicians have expressed concern about uies. in , cohen et al. emphasized the uniqueness of the pelvic exam and how patients have a right to refuse participation in these training exams. since then, uies continue while opposition increases. singer considers uies "definitely among the most egregious of… ethical issues" in medical schools (boyles ) ; caplan ( b) recently called for "an abrupt and immediate halt" to these exams; and friesen ( a) refers to uies as "immoral and indefensible" (p. ). adashi, a former dean at the warren alpert medical school of brown university, states, "viewed in hindsight, it is difficult to see how the conduct of unapproved pelvic examinations by medical students could have been rationalized, let alone condoned" ( ). medical students are increasingly raising concerns to their ethics advisors (friesen b; caplan a) , researchers (reese and monrouxe ), the media (tsai ), and state legislators (barnes ) . when medical students feel they must warn the public about uies in their medical institution, their actions meet the criteria for whistleblowing which: results from a malfunction of the ethical environment of the organization to focus on its accountability for the safety and welfare of the patients. individuals [who engage in whistleblowing] believe they must take a stand for the wrongdoing in the organization. (lachman , p. ) some of these whistleblower reports may have contributed to changes in association guidelines. medical associations have developed consensus statements recommending bans on unconsented pelvic exams (association of american medical colleges ; american college of obstetricians and gynecologists ); however, these statements are advisory and incomplete. associations simply do not have the capacity to compel systemic change, as evidenced by institutions' inaction. in response, nine states recently passed legislation related to unconsented intimate exams, and other states are considering similar measures. this paper describes current laws and association guidelines related to uies then explores the main arguments in favor of continuing the status quo. next, the paper examines the implications of continuing the status quo and closes with a series of practical recommendations to curtail the occurrence of uies. the association of american medical colleges (aamc), american college of obstetricians and gynecologists (acog), and the american medical association (ama) have called for explicit, mandatory consent before pelvic exams. the aamc's statement ( ) declared, "medical students and residents not only must acquire clinical skills, they must also learn to always treat their patients with respect and dignity. recent reports have suggested that medical students are performing pelvic examinations on women under anesthesia, without their knowledge and approval. aamc believes that such practice is unethical and unacceptable" ( ). acog's opinion includes, "pelvic examinations on an anesthetized woman that offer her no personal benefit and are performed solely for teaching purposes should be performed only with her specific informed consent obtained before her surgery" (p. ). the ama's code of medical ethics opinions on patient-physician relationships states that patients' "refusal of care by a trainee should be respected in keeping with ethics guidance" ( , p. ) , and a statement by the ama council on ethical and judicial affairs calls for explicit disclosure of student involvement in exams on anesthetized patients ( ) . nurses have also expressed concern: fourteen nursing organizations banded together to urge the american academy of medical colleges to require explicit consent (wilson ) . the nine states who passed laws banning some form of intimate exams are iowa, illinois, utah, oregon, maryland, virginia, new york, california, and hawaii. other states are also considering or have proposed legislative action. utah and maryland's laws are gender-neutral but most other states explicitly specify unconsented pelvic exams on women. uies are also under scrutiny in other countries. new zealand has a national consensus statement requiring written consent (bagg et al. ) , and france has taken steps to ban both unconsented rectal and pelvic exams (the local ) . associations in other countries, including canada (liu et al. ) and the united kingdom (royal college of obstetricians and gynaecologists ), have explicit consent guidelines but they are limited to women's intimate exams. as mentioned, medical associations, legislators, ethicists, and nurses-along with some physicians and medical students-have argued in favor of explicit consent of intimate exams. arguments in favor of the status quo (which we define as failing to explicitly consent for intimate exams) are limited to those made by some physicians, institutions, and medical students. this section defines and discusses arguments in favor of the status quo. some medical students voice disinterest in the importance of consent (cohen et al. ). one medical student called explicit consent an "attempt to justify the obsession with political correctness," opining that explicit consent is an inappropriate overreaction to a necessary training procedure (bhangu (bhangu , p. . some physicians also find that explicit consent is simply not a priority. citing his inclination as a "policy minimalist," george washington university's ob-gyn chairman, john larsen, expressed disinterest in codifying explicit consent (goldstein ) . interestingly, ubel, a physician who examined uie practices in teaching hospitals in a survey of medical students, found that completion of the obstetrics/gynecology clerkship is associated with decreased interest in informed consent (ubel ) . after this clerkship, students were also "less likely to think consent for rectal examinations was important" (p. ). in a follow-up interview, ubel stated that the experience of participating in "exams without detailed consent on anesthetized patients desensitized doctors about the need for patients to grant consent" (goldstein ). this view is in contrast with women who "almost universally [feel] that pelvic examinations that are conducted by medical students while the woman is anesthetized should occur only after the woman has given her permission to the students' supervisors" (ubel , p. ) . to claim that explicit consent is unnecessary is in defiance of the fundamental principles of autonomy and self-determination. adult patients have the right to self-determination: "the right to make the ultimate decision concerning what will or will not be done to their bodies" (annas et al. ) . along with this right comes the right to refuse procedures. "courts have… declared that both the common law and the united states constitution protect an individual's right to refuse medical treatment" (annas et al. , p. ) . similarly, the ama's ethical and policy guidelines voice respect for the patient's refusal of an exam by a trainee ( ). the idea that explicit consent for intimate exams is optional therefore fails to meet the most basic tenets outlined in law, bioethics principles, and association guidelines. another argument against explicit consent relates to discomfort felt by novice medical students when performing intimate exams. such exams are "anxiety-or fear-provoking for the novice learner, [and they] often feel embarrassed or even uncomfortable" (nelson , p. ) . some therefore say there is a benefit to uies because it is easier on the medical student to learn the exam while they do not have to consider student-patient dynamics. if the medical student makes a mistake or does something that would be physically or emotionally uncomfortable for the patient, the student will not be embarrassed by a conscious patient's reaction. however, there are several points against this argument. first, teaching institutions have a duty to train their medical students, but training is secondary to their primary responsibility of the patient's health and well-being. second, part of medical school is learning to exhibit professionalism with patients even when the student may be uncomfortable. third, there are many ways of learning intimate exams to help students overcome their nerves before direct patient contact. pressure-sensitive pelvic mannequins, training videos, and gynecologic teaching assistants (gtas) contribute to comprehensive training. gtas are women "trained on how to instruct the pelvic exam and allow students to perform the exam on them" (nelson , p. ) . gtas are used in the u.s. as well as the netherlands, sweden, belgium, australia, and the uk (walsh ) . women with a history of sexual violence are at risk for pronounced fears, anxiety, pain, and discomfort during intimate exams (qaseem et al. ) , and it is reasonable to assume men with similar histories would have similar reactions. other populations who experience pronounced levels of distress during intimate exams include people with disabilities, those who meet the criteria for obesity, and members of sexual and gender minorities (potter ) . in the u.s., the prevalence for sexual violence is % for women and % for men (national sexual violence resource center ); the prevalence for disabilities is approximately % (center for disease control and prevention ); the prevalence of obesity is nearly % (centers for disease control and prevention ); and those who identify as lgbt + in the u.s. account for approximately % of the population (family equality council ). medical students ought to develop sensitivity and understanding for these vulnerable populations which comprise a notable portion of the patient population. gtas, sexual assault counselors, and experts in lgbt + ethics, and other experts can and should be consulted to help students learn to communicate sensitively with vulnerable patients during intimate exams. for instance, gtas could expand their teaching to role-play as anxious patients so that students can practice their communication skills in a forgiving environment. sexual assault counselors uniquely understand the trauma felt by survivors of sexual assault and can offer guidance in terms of vocabulary and alternative positioning of the patient body to more fully empower these patients. experts in lgbt + ethics can similarly be consulted to help medical students more fully understand how current medical practice may marginalize these communities and how to more fully provide empathic care. medical schools can carefully plan their teaching modules so that students can participate in these lower-stress learning experiences-and learn basic patient interaction skills and consent procedures-before they perform the exam on a patient. in short, there are obvious, uncomplicated means to help students overcome their nerves and increase competency, and student nervousness should not take priority over the right of a patient to decide what will happen to their body during a medical procedure. some argue that students must perform extensive numbers of intimate exams because the exams are a necessary component of regular patient care. however, in their final recommendation statement: screening for prostate cancer, the u.s. preventive services task force (uspstf) recommends that men can either forgo a prostate exam or "make an individual decision about whether to be screened after a conversation with their clinician about the potential benefits and harms" ( ). similarly, physicians are now questioning the "very utility of the traditional pelvic examination" (adashi ). a recent study reviewed data from . million women aged - years old and found that . % of their pelvic exams "were potentially unnecessary… and may cause harms such as false-positive test results, overdiagnosis, anxiety, and unnecessary costs" (qin et al. , p. e ) . the american college of obstetricians and gynecologists advocates "against performing screening pelvic examinations in asymptomatic, nonpregnant, adult women" ( , p. e ). similarly, the uspstf's final recommendation statement for gynecological conditions states, "it is unclear whether performing screening pelvic examinations in asymptomatic women reduces morbidity and mortality" ( ). as medicine has evolved, the need for frequent, across-the-board digital intimate exams has lessened. this development especially decreases the need for frequent training exams by students in their clerkships. some say that uies should continue because an explicit consent policy would substantially reduce the number of training exams, resulting in poorly trained physicians. in other words, if patients are asked, the belief is that they would likely refuse, so physicians may avoid the consent conversation so as not to jeopardize training opportunities. however, a recent survey of over women indicated that % of women would consent if asked, and only % were sure they would refuse (wainberg et al. ) . other surveys have similar positive feedback from patients related to their willingness to participate (friesen a) , and students also have additional training opportunities in clinics and during training sessions with gtas. in sum, explicit consent is an unlikely barrier to physician training. some physicians argue that explicit consent for intimate exams is unnecessary because intimate regions of the body are no different than any other body part. in response to coldicott's landmark study on intimate exams ( a, b), kaushik, an ophthalmic surgeon, argues in favor of the status quo, believing it is "dangerous" to consider intimate exams differently, and that "examination of the fundus of the uterus is just as intimate for a gynaecologist [emphasis added] as examining the fundus of the eye is to an ophthalmologist" ( a, b, p. ) . this thinking contributes to physician preference for a standard, genericized surgical consent form for all surgeries. yet other physicians understand that the frame of reference should not be about whether the physician finds the exam to be intimate. rather, the decades-long conflict relating to uies relates to how these exams are perceived by the patients themselves. women's pelvic exams are "particularly threatening" to the patient (cohen et al. ) and "in the area of gynecologic care [patients' right to refuse student exams] takes on heightened sensitivity" (cohen et al. ) . and unlike eye exams, some women may find the standard gynecologic exam to be "disempowering, abusive, and humiliating" (bates et al. , p. ) . the american college of physician's clinical practice guideline also acknowledges the uniqueness of pelvic exams, noting how women may experience "fear, anxiety, embarrassment, pain, and discomfort" (qaseem et al. , p. ) during a pelvic exam. men may also express embarrassment (myers et al. ) , shame (consedine et al. ) , and mistrust (robinson ) during rectal exams, and there is no literature relating to women's or men's shame or embarrassment during eye exams. these responses to intimate exams contribute to lower rates of doctor visits which may predispose patients to poorer health in the long term. as discussed, women with a history of sexual violence (along with patients with sexual and gender minority status and other groups described earlier) are even more likely to experience distress during intimate exams. ubel stated, "we don't see a pelvic exam as having any sexual content at all, but that's not how other people perceive it" (goldstein ) . "there's no way a physician would ever equate a pelvic exam with rape-there is no rape content to it. but the fact that someone else perceives it that way makes it important" (goldstein ). an unconsented intimate exam may feel like a sexual violation to patients. when medical school administrations consider the values they wish to promote in the next generation of physicians, they must consider patient perception. even if some physicians and institutions have ambivalence over explicit consent for intimate procedures, patients, the general public, and others have repeatedly issued calls for explicit consent. surveys indicate that women are unanimous in their stance against unconsented intimate exams by students when anesthetized (bibby et al. ) , and this is another signal that the medical community cannot gloss over this issue by, as an example, comparing the uterus to the eye. even though a patient's body may be "naked on a brightly lit table for all to see" (friesen a, p. ) during a procedure, the patient should still have rights to decide what happens to their body. in a recent discussion forum about uies, a student wrote: as a female student not yet in the medical field, i am disturbed to hear that by consenting to surgery, i risk having someone literally in my vagina without consent for purposes that benefit only [the students], and not me. are patients really viewed as a teaching tool rather than a human being? that i will be splayed and sliced during the procedure doesn't mean that additional indignities are acceptable. it's still my vagina, even if i am naked and unconscious. i didn't lend it to anyone to practice techniques (friesen a, p. ) . when people repeatedly express a fervent and heartfelt preference for how their bodies are treated, and this preference differs from physicians' perceptions, physicians should carefully examine why their perceptions should prevail. clearly, standard consent forms fail to give patients the granularity they need to properly consent. some argue that explicit consent is unnecessary because uies rarely occur. however, greger, a physician who has researched unconsented exams, stated, "if they have five medical students on an ob-gyn rotation, they aren't going to let one do it and not the other four… i never heard of anyone out of some kind of respect for the patient just limiting it to a few" (goldstein ) . and, following recent discussions with concerned medical students, caplan agreed: "sometimes, more than one student [on their gynecology rotation] will practice the exam, with many sets of gloved fingers in the patient's vagina without their knowledge" (caplan a) . even if physicians rarely practice or condone uies, some do, and when institutions fail to enact explicit consent on the grounds that uies are infrequent, they send a message to the public that the affected patients' experiences are unimportant. institutions should not fear that explicit consent will look badly on them. instead they should view them as necessary protections for patients and know that patients are then more likely to relax in a clinical setting because of the policy's existence. in sum, the most common arguments in favor of the status quo generally lack substance and should not pose barriers to explicit consent. critics of explicit consent point to perceived harms of intimate exams but fail to mention harms of continuing the status quo. critics may argue that patients are not harmed if they are anesthetized (and therefore unable to remember the procedure), and perhaps that is true. however, some patients have recently woken during an uie under anesthesia. ashley weitz, a young woman in utah, sought emergency care for uncontrolled vomiting and when the physician suggested a pelvic exam to rule out a sexually transmitted disease, she explicitly declined (laird ) . weitz passed out from the sedative effects of phenergan then "woke up screaming" (laird ) because the physician was performing a pelvic exam. a survivor of childhood sexual assault, weitz was so traumatized by the unconsented exam that she became an advocate for a law banning the procedure in her home state. weitz is not the only patient to wake during an intimate exam (mcdermott and johnson ), and the idea that patients are unaware under anesthesia is also increasingly refuted: a large u.s. multicenter study found that intra-anesthetic awareness occurs in nearly cases every year (sebel et al. ) . patients with awareness during surgeries most commonly report "hearing conversations… but there are many cases where they report pain, paralysis and anxiety because of the fact that they feel helpless [and some] patients describe this situation as the worst experience they ever had in a hospital" (kotsovolis and komninos ). one compelling report is from an anesthesiologist who, as a patient, was aware of aspects of his urogenital surgical experience and found his experience "terrifying" (peduto et al. ) . therefore, the assumption that patients are unaware of their unconsented exams may be questionable. other patients have been informed of an uie after they woke from anesthesia. one such patient, a nurse, woke from her laparoscopic stomach surgery in to be informed by a medical student that her cervix seemed abnormal (graham ) . she, too, is a survivor of sexual assault and was so distraught by the procedure that she tried to bring charges but was told by local attorneys that state laws would not allow her to prosecute the hospital. definitions of medical malpractice vary by state, but medical malpractice occurs when a physician fails to disclose the risks of a surgery, provides substandard medical care, and subsequently injures the patient (bal ). this nurse-patient's sense of violation and trauma could be considered injurious to her and may therefore meet the definition of medical malpractice. unconsented intimate exams typically occur on unconscious patients. after all, few would ever perform an unconsented intimate exam on a conscious patient because such an act is far outside of ethical standards. and yet there are recent cases of unconsented intimate exams on conscious patients. in some of these cases, the patients denied consent before the procedure but felt too overwhelmed to advocate for themselves during the exam. one patient, a physician from maryland, was at his oncologist's office for a manual rectal exam related to his recovery from prostate cancer. '…the doctor turned to a med student and said, 'why don't you go ahead.' the student dug right in, caving to the intense pressure med students are placed under during training. stern, himself a doctor, was flabbergasted-not only because the doctor hadn't asked permission, but because stern had expressly denied the student's own request to perform the exam not min prior. "it was terrible," says stern now. "i was awake. i'd said no. but the trainee went ahead anyway, and neither of them spent any time telling me why they thought it was useful. "a patient shouldn't have to meet the needs of the provider," stern goes on. "if he decides it's appropriate to help, great. but he needs to be asked" (redfearn and cao ) . while some may say that the patient could have spoken up, consent "must be given freely [and] validity is easily threatened if the patient… is asked for consent at an inappropriate time or is coerced" (uk department of health ). in stern's case, the consent process was poorly executed and failed to protect the patient. it is interesting to note that the patient was a physician, someone who would seemingly feel most empowered to refuse a student exam, and yet even he felt constrained by hierarchical dynamics. other cases of uies on conscious patients may occur when the patient is unaware of the exams. in one such case (rees and monrouxe ), a male patient was conscious but unaware of receiving sequential digital rectal exams by several students since a sheet divided him from the students. two of the students discussed this case as part of a recent multicenter study on intimate exams. one of the students stated: …they just had a queue of medical students doing a rectal examination he wasn't consented but… you don't have the confidence to say 'no' you just do it…i think there was [sic] about three or four medical students… [the patient] had no idea that we were there and… none of the theatre staff spoke to him about what was happening (rees and monrouxe , p. ) . these cases find their way into public dialogue. as cases of conscious and unconscious uies become more widely known, patients may suspect their physicians are failing to fully disclose what will happen to their bodies during an exam. the fear that physicians may not share a sense of primary responsibility for their patients' health and well-being can damage the relationship between the general public and the medical community and lead to legal protections. medical students can also experience harm when the status quo goes unchallenged. as discussed, ubel's study found that medical students were less supportive of explicit consent after their obstetrics and gynecology clerkship. no other rotations were reported to have this effect on medical students. …this decline [in valuing the importance of informed consent] is not gradual and inexorable, but instead… it is associated with specific experiences. students who have completed obstetrics/gynecology clerkships place significantly less importance on seeking permission from women… (ubel et al., , p. ). this shift in perceptions of consent is an example of ethical erosion, an attrition of values, leading to decreased respect for the patient's autonomy and emotional detachment. this shift may be attributable to the hidden curriculum, described by mahood ( ) as: a socialization process [by which] norms and values transmitted to future physicians often undermine the formal messages of the declared curriculum… [it] consists of what is implicitly taught by example day to day, not the explicit teaching of lectures, grand rounds, and seminars. students move from being open-minded to being closed-minded; from being intellectually curious to narrowly focusing on facts; from empathy to emotional detachment; from idealism to cynicism; and often from civility and caring to arrogance and irritability. this erosion of empathy and "vanquishing of virtue" is repeatedly documented in studies of physicians in training (p. ). feudtner et al. ( ) have examined ethical erosion of medical students during their clerkships. nearly all of the affected students in their study overheard physicians disparage patients, and over half observed members of the clinical team engage in unethical behavior and felt that their own ethical principles suffered during their clerkship. when students are faced with ethical dilemmas, feudtner et al. find that "for at least a sizable minority, exposures to… [ethical] dilemmas coincide with deterioration of the students' ethical self-identities" ( , p. ) , and those who saw others participate in unethical behaviors were increasingly likely to do so themselves. ethical erosion is not the only ill effect on medical students when they perform uies on patients. the experience of some students may be more accurately described in terms of moral distress, a "response to a perceived conflict between what one is expected to do and what morality requires" (weber , p. ) . moral distress may arise "when one knows the right thing to do, but institutional constraints make it nearly impossible to pursue the right course of action" (jameton , p. ). moral distress is a "uniquely painful phenomenon… frequently associated with feelings of being powerless" (tigard , p. ) , and it correlates with compassion fatigue and high turnover rates. when shawn barnes ( ) was still a medical student, he felt coerced by institutional constraints to perform unconsented pelvic exams. he stated: for weeks, four to five times a day, i was asked to, and did, perform pelvic examinations on anesthetized women, without specific consent, solely for the purpose of my education. [when] the patient was asleep, the attending or resident would ask me to perform a pelvic examination on the patient for educational purposes. to my shame, i obeyed… my medical education experience has reinforced the notion that the medical student should not question the practices of those above him or her. i was very conflicted about performing an act that i felt was unethical, but owing to both the culture of medicine and my own lack of courage, i did not immediately speak out against what i was asked to do by residents and attendings" (barnes , p. ). barnes felt powerless at the time but later chose to take action and helped to pass hawaii's law banning unconsented pelvic exams. symptoms of moral distress can ensue after direct or indirect participation in ethically questionable behavior, so nurses and other members of the clinical team may also experience moral distress when they witness or facilitate uies. it is not unreasonable to assume that indirect participants of uies may also experience ethical erosion. thus, a move to an explicit consent process is not only good for the patient; it is also beneficial for the health and well-being of students and practitioners because explicit consent safeguards them from ethical erosion and symptoms of moral distress. when institutions decline to make consent explicit, they are a complicit party to ethical erosion and moral distress. and when institutions embrace an explicit consent process, they signal to their students and practitioners that they care about their well-being and are willing to make a small policy change to protect students' and practitioners' mental health and well-being. in sum, the continuation of the status quo results in a series of harms. harms may occur to patients who are aware of having received an uie, to medical students who are directed to conduct these exams, and to other members of the clinical team who watch or facilitate the procedure. harms may also occur to the fragile relationship between the public and the medical community. while the process of explicit consent may seem to be a trivial, burdensome, and perhaps uncomfortable administrative task, failure to acquire explicit consent inevitably harms some number of students, clinicians, and patients-and tarnishes the medical community's relationship with the general public. when medical schools enact an explicit consent process, they demonstrate the institution's respect for the patient as the gatekeeper to their body, recognize medical students' worth, and bolster their relationship with the general public. as discussed, most unconsented intimate exams are for training purposes on anesthetized patients during urological and obstetrical/ gynecological clerkships, and some uies have occurred for diagnostic purposes, on conscious patients, or during abdominal procedures. a sufficient resolution therefore requires explicit, institution-wide policies and also requires time, a precious commodity in medicine. mercurio ( ) reflects on the physician's daily challenges, stating: we know what we ought to do. the right to informed consent or permission, for example, has important ethical underpinnings… most often, we fall short of our ethical ideals not from lack of deliberation, poor analysis, or inadequate understanding of ethical principles. we fall short because of time (p. ). these words should be taken to heart. even when time is constrained (and it always seems to be), physicians should not overlook or gloss over the consent process with the patient. indeed, the aspect of time is a fundamental determinant of resolving the decades-long conflict of uies. we spend our time on what we value. spending time on explicit consent sends a signal to all parties that a patient's permission is sacred and valued. the resolution to uies requires a multi-tiered response. first, institutions should develop an explicit consent policy for all intimate exams. second, medical school curriculum should support the explicit consent policy. third, institutions should consider top-down changes to bolster ethics across the organization. institutions should create an explicit consent policy for all intimate exams regardless of the patient's gender, whether the patient shall be conscious or anesthetized during the procedure, and whether the exam is diagnostic or for training purposes. if the patient is unconscious and in a medical crisis, physicians can presume consent if and only if the patient's condition is so dire that the intimate exam cannot wait until after the patient regains consciousness. finally, a patient should not receive an intimate exam unless it is indicated for the patient's underlying medical condition; a patient should never fear having an unconsented intimate exam when they are, for example, undergoing a cardiac procedure. additionally, some consent forms may include a vague reference to student participation but may not specify whether a student intends to conduct an extra exam explicitly for training purposes. the presence of students-and the intent of their exams-should be made explicit to the patient. the design of the consent form matters. as discussed, some hospitals use a standard surgical consent form. these forms should be modified to document explicit consent for intimate exams. consent forms are also sometimes in electronic form. while some patients may prefer this method (winter et al. ) , electronic consent should not replace the physician-patient conversation. policy logistics also matter. the explicit consent policy should be publicly available and easily accessible so that all interested parties, including legislators and members of the general public, can locate it and have confidence in its existence. also, when institutions care about the intent of a policy, they invest in training and prioritize periodic evaluations. periodic reviews which thoughtfully examine the successes and failures of the policy could be employed to gauge progress. communications between physician and medical student also matter. students rarely witness the consent conversation between the attending and the patient, so they do not always know whether explicit consent was acquired. efforts should be made to allow students to observe (and learn) the consent process when possible. when students are unaware of the consent conversation, and are subsequently told to perform a student exam, they worry they are on shaky ethical ground. students may not feel empowered to directly ask their supervisor whether explicit consent was obtained; they do not wish to challenge their superior's actions. to clear the air, once the physician has completed the explicit consent process with the patient, the physician should unambiguously inform students that explicit consent was obtained. this brief conversation between physician and medical student seems like a banal administrative task but it will ameliorate students' misplaced guilt, shame, and distress and provide enormous relief to them. spending time on these conversationsand including students in the conversation whenever possible-underscores the importance of consent to students and the rest of the clinical team. enhancements to the medical school curriculum will better prepare students to perform intimate exams. as discussed, some medical schools already employ complementary methods to learn intimate exams. these methods should become standard in all medical schools to help students learn intimate exams before patient contact, and the gta position could be expanded to include role-playing. and as mentioned earlier, experts such as sexual assault counselors should be consulted to learn how to best support the sizeable patient population with risk factors predisposing them to anxiety or discomfort during intimate exams. the inclusion of these expert voices honors the psychosocial complexities inherent to the patient population. as discussed, ubel's survey ( ) found that the completion of the obstetrics/ gynecology clerkship is associated with students' decreased interest in informed consent-a trend not found after any other clerkship. medical schools should examine the dynamics of this rotation to determine root causes and take action to reverse this trend. medical training should also teach students how to have a consent discussion with patients. informed consent is a learned skill that can improve with practice and (as discussed) should be incorporated in students' training experiences with gtas. mercurio ( ) wrote a thoughtful reflection to help physicians request access to the bodies of newly deceased children for training exams. his reflection demonstrated how the use of sensitive, respectful language can build rapport with patients and families and provided guidelines which give structure and confidence to physicians also wishing to have these conversations with families. similarly, guidelines could be developed to help physicians request consent for intimate exams and give them confidence during these sensitive discussions. medical school curriculum should provide bioethics education to students before and during clerkship rotations. discussing cases in advance-and paths to resolution-will bolster students' understanding of ethical principles, help them understand how to navigate ethical conflicts before they arise, and may build their confidence. medical schools should also tell students where to turn when they experience an ethical conflict. medical schools should offer multiple channels for students to seek counsel; these delicate conversations require rapport between student and mentor. during their clerkships, students should have avenues to report an ethically challenging encounter. some medical schools have anonymous upward feedback mechanisms. medical schools could also consider allowing a student to opt out of a procedure on ethical grounds. physicians may elect a conscientious objection when they face an ethical challenge, and medical students should also have the means to refrain from an ethically fraught procedure without repercussions. student access to conscientious objection is not widely available but is offered in some u.s. institutions (card ). even with enhanced medical school education, students may not feel at liberty to refrain from conducting an unconsented intimate exam for training purposes. in coldicott's study ( a, b) , many students felt unable to refuse their supervisor's call to perform an uie. similar concerns were voiced by medical students in rees and monrouxe's open-ended survey about professionalism in medicine ( ). this study collected reflections from medical students and when asked generally about ethically charged circumstances, many students elected to discuss their experience with unconsented prostate and vaginal exams. one student who performed an uie remembers feeling "almost star struck that i was in theatre for the first time" (p. ). another student felt bowled over by their supervisor, stating, "he was quite an aggressive surgeon anyway so i just went and did [the unconsented intimate exam]" (p. ). these examples highlight medical students' inability to voice their concerns within the institution. it makes sense then, that some students approach outsiders to talk about their conflicts. it is insufficient to make medical students solely responsible for halting their participation in uies. therefore, directors of clerkships, the dean of students, attending physicians, and residents should all make clear that they support explicit consent, that explicit consent is mandatory, and that they support a student's right to exercise and voice an ethical concern. institutions could also declare that violations to explicit consent will be investigated and offenders will be held accountable. physicians must act impeccably to preserve their trust with patients; when they do not, they damage the good name of their profession. physicians should remember that their actions impact how patients view other physicians, and even small incidences of mistrust create fissures that are hard-if not impossible-to heal. thus, physicians and medical schools should be supportive of policies that protect students and patients from indiscriminate physician behavior and help restore trust between patients and physicians. ethics can also be integrated across all levels of the medical institution. a notable example of integrated ethics includes the model developed for the veterans health administration (fox et al. ). fox's model consists of three functions which target ethics at "the level of decisions and actions;… the level of systems and processes; and…the level of environment and culture" (p. ). work by callahan et al. ( ) can also be considered: they promote "three trust-building tactics: 'accountability, reliance, and aspiration'… these principles provide people and mechanisms to reach the outcome of an ethical organizational culture" (p. ). for instance, they suggest an ombudsperson to build employees' trust in the institution's policies and standards and recommend principles to increase empathy and trust and thereby foster organizational integrity. similarly, lachman ( ) suggests an implementation of committees, forums, and procedures to amplify integrity, and greene and latting ( ) recommend the formation of an organizational ethics committee. while these scholars propose robust approaches to institutional ethics, even small organizational changes can have lasting and far-reaching effects. to bolster a sound ethical culture, institutions should review these studies to identify methods most suitable to their institution. in summary, recommendations include an explicit consent policy and enhancements to both medical school curriculum and institutional culture. conflicts between physicians and families are a part of the public conversation. the case of ms. jahi mcmath, an example of a family's refusal to accept a brain death diagnosis, caught national attention (truog et al. ) . the mcmath family did not feel heard by their practitioners when their repeated calls to curb jahi's postsurgical loss of blood were disregarded-neglect believed to have caused her brain death-and the case is emblematic of widespread racial bias in medicine (goodwin ) . other families may also experience treatment denial, such as vaccine-hesitant families who are denied pediatric care (flanagan-klygis et al. ) . these fissures reflect the challenge of conveying complex medical information to families who may already mistrust the medical system and the fissures seem to be so far-reaching that some fear there is little hope for resolution. other recent news stories have covered larry nassar's rampant abuse of his pediatric patients (mountjoy ) and the atlanta journal-constitution's investigation uncovering "more than cases of sexual misconduct by physicians across the nation since " (teegardin and norder , p. ) . given these recent headlines, it comes as no surprise that news coverage on unconsented intimate exams could cause further discord between physicians and the general public. ethicists, legal scholars, nurses, and professional associations-along with some physicians and medical students-have voiced persistent and prolonged calls for change, and the dichotomy between societal and traditional medical views of intimate regions shows no signs of abatement. uies continue to receive attention in the media, and legislators continue to propose statutes-underscoring the sustainability of these concerns. however, most institutions still decline to implement an explicit consent policy. we are reminded that the bodies in question belong to the patients, and patients are the rights-holders of their bodies. asking for permission is a fundamental tenet of modern medicine. without permission, touching intimate areas can feel like an assault, and may even meet the criteria for battery or malpractice. this paper demonstrates how the status quo leads to ethical erosion, moral distress, patient distress, and distrust by the general public. these ill effects are surely not intended by physicians whose goals are to heal and comfort patients. when rhodes urges physicians to "seek trust and deserve it as their moral law, as their creed," she notes that physicians should "pay serious attention to the patient's view of what is good" ( , p. ) . formalizing explicit consent can therefore be an act of trust-building between patients and physicians and call attention to physicians' honorable commitment to patients' safety and well-being. we encourage medical institutions to reexamine their stance towards intimate exams to restore trust, protect clinicians, medical students, and patients, and underscore the sacred right of a patient to decide what happens to their body. finally, while institutional change can minimize future harms, consideration should be paid to healing the wounds of medical students, physicians, and patients who have already suffered from past harms related to their roles in unconsented intimate exams. jama forum: teaching pelvic examination under anesthesia without patient consent medical student involvement in patient care professional responsibilities in obstetric-gynecologic education the utility of and indications for routine pelvic examination opinions on patient-physician relationships & the legal advisors committee, concern for dying informed consent to human experimentation: the subject's dilemma medical students and informed consent: a consensus statement prepared by the faculties of medical and health science of the universities of auckland and otago, chief medical officers of district health boards, new zealand 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speaking out can we create lasting change': what can we learn from the dr larry nassar tragedy? factors associated with intention to undergo annual prostate cancer screening among african american men in philadelphia get statistics teaching the female breast and pelvic exam: a student-and patient-centered approach an anesthetized anesthesiologist tells his experience of waking up accidentally during the operation time to end pelvic exams done without consent challenging pelvic exam screening pelvic examination in adult women: a clinical practice guideline from the american college of physicians prevalence of potentially unnecessary bimanual pelvic examinations and papanicolaou tests among adolescent girls and young women aged - years in the united states are med students practicing on you? medical students learning intimate examinations without valid consent: a multicentre study understanding the trusted doctor and constructing a theory of bioethics attitudes of african americans regarding screening for prostate cancer obtaining valid consent ethics versus education: pelvic exams on anesthetized women the incidence of awareness during anesthesia: a multicenter united states study abusive doctors: how the atlanta newspaper exposed a system that tolerates sexual misconduct by physicians france to ban vaginal exams without consent the positive value of moral distress brain death at fifty: exploring consensus, controversy, and contexts medical students regularly practice pelvic exams on unconscious patients don't ask, don't tell: a change in medical student attitudes after obstetrics/gynecology clerkships toward seeking consent for pelvic examinations on an anesthetized patient final recommendation statement. gynecological conditions: periodic screening with the pelvic examination teaching pelvic examinations under anaesthesia: what do women think? oxford textbook of medical education moral distress, workplace health, and intrinsic harm autonomy suspended: using female patients to teach intimate exams without their knowledge or consent the use of portable video media vs standard verbal communication in the urological consent process: a multicentre, randomised controlled, crossover trial publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the author wishes to thank jason eiseman and jordan jefferson at the lillian goldman law library at yale law school, and patty martin at the bioethics research library, kennedy institute of ethics, georgetown university for their valuable support during the research process. key: cord- -lvw h w authors: atzema, clare; poirier, vincent title: career options in aerospace and aviation medicine() date: - - journal: ann emerg med doi: . /j.annemergmed. . . sha: doc_id: cord_uid: lvw h w nan underlying all these issues is a thorough understanding of human physiology, basic physics, toxicology, infectious disease, and engineering principles. , in general, physicians trained in aerospace medicine practice health care in populations exposed to flight and space, consult on the physical and engineering aspects of the flight environment, and manage public safety issues at a variety of regulatory agencies. they may choose to specialize in civil aviation, military aviation, or space flight. they might work for the national aeronautics and space administration, the federal aviation administration, a commercial or corporate airline, the department of transportation, an aerospace manufacturer, the occupational safety and health administration, the centers for disease control and prevention, or in private practice. practitioners may choose to focus on medical research, they may teach, or they may consult on a variety of preventive, workplace safety, and environmental programs , or on aerospace health care system development. aerospace medicine and emergency medicine are complementary specialties, for several reasons. each requires the evaluation and stabilization of patients in a less than perfectly controlled environment. training for mass casualty incidents serves as preparation for airline crashes, and disaster medicine training, along with out-of-hospital care and toxicology, contributes to the preparation for manned space flight. the variety of clinical problems encountered in aerospace medicine overlaps with emergency medicine, ranging from treatment of multiple trauma patients after an airline accident to running a medical clinic for aircrew, which may mirror some of what is seen in the minor area of an emergency department. in addition, the problem-solving and teamwork skills that are inherent to emergency medicine are also basic tenets of astronautics. the daily practice of a physician in aerospace medicine depends on his or her area of expertise. a medical consultant for a major airline, for example, will oversee the health of the pilots, flight attendants, and ground staff. he or she is likely to run daily clinics to manage the medical problems of the airline crew, such as barotrauma, occupational injuries (back pain is a common complaint), and return to work assignment. the consultant is usually a trained aviation medical examiner (ame) and, as such, gives out the pilot medical certificates from the federal aviation administration, the canadian civil aviation, or the united kingdom civil perhaps the plane will be unable to land because of weather conditions. there is little routine, and by flying full-time, there is a risk of becoming physically exhausted. , in addition, teamwork is ubiquitous in aerospace medicine: one is likely to take part in several committees, and if one is a flight physician, one works in a confined environment with a medical team (ie, a respiratory therapist, a nurse, paramedics), often for many hours at a time. if you like to work alone, this may be a drawback. there are many topical issues in aerospace medicine, which you are likely to find yourself debating if you choose this career. traveler's thrombosis is being addressed by the wright project (world health organization research into global hazards of travel), which is a collaboration of an international research project and the world health organization. severe acute respiratory syndrome and aeromedical transport will likely remain a serious issue for anyone in this field, and the effects of cosmic radiation are a continuing concern. other issues include the use of selective serotonin reuptake inhibitors by pilots, refractive eye surgery in pilots, long-haul flight and aircrew fatigue, and long stay in the international space station. if you have a predisposition toward research, there are a plethora of opportunities in this field. the routes to becoming a specialist in aerospace medicine are through either civilian training (table ) or the military (table ) . after graduation as a medical doctor at an accredited medical school, one may: ( ) enter an internship of choice, and, after completion of the internship, enter a residency in aerospace medicine lasting an additional years; or ( ) enter the united states military to complete a military residency for to years. however, there are many other options available (tables and ) . the final pertinent issue to consider is that military programs require their trainees to fly, and in civilian programs, although it is not a requirement, flight training is encouraged through several affiliated flight schools. thus, the strong desire to fly is an important prerequisite. aviation authority. in addition, he or she will ensure that protocols are followed, including medical clearance of passengers, medical kit onboard the aircrafts, emergency procedures, and air quality control, to name a few. another example is that of a flight physician, who usually works for an aeromedical transport company. this individual is in charge of flight planning, in conjunction with the medical team. in particular, the flight physician will determine whether the patient is fit to fly, assess the patient's oxygen requirements and the aircraft's cabin pressure, and determine if any special equipment is required. the physician escorts patients onboard different aircraft types (ie, small fixed wing, commercial, helicopter) and will work to minimize the risk of barotrauma and problems resulting from the hypoxic inflight environment. often, this physician is attached to a university center and will be in charge of medical education for residents and medical staff. a third example is that of a director for a specialized aviation authority, such as the civil aerospace medical institute or the international civil aviation organization. the director will lead a professional, technical, and clerical team that is engaged in policy development, evaluation, and administration. this individual will develop aeromedical education programs for flight crews and civil aviation pilots. he or she will supervise the creation of the didactic materials used in these programs, as well as general aeromedical publications such as aviation safety brochures and research technical reports. these jobs have some distinct advantages. if one works for an airline or an aviation authority as a medical consultant, a typical workday extends from am until pm. despite additional on-call duties, one is likely to be called in only a few times a year (for emergencies such as crash investigations, emergency landings, etc). a flight physician who retrieves injured or ill passengers is afforded the opportunity to discover the world, particularly because some travelers have the desire to visit some fairly unusual places. some aerospace medicine physicians will maintain a part-time position in an emergency department or another ambulatory setting, providing both variety and the opportunity to maintain clinical skills. a career in aerospace medicine has disadvantages as well. the daily medical practice of an airline consultant is usually limited to the health care issues of a healthy population. flight physicians are on call many times a month and may be required to leave within to minutes of a telephone call. although these physicians often know when they are going to leave, they may not know when they will return: the patient may be more unstable than predicted (potentially requiring an emergency landing) or table . fundamentals of aerospace medicine aerospace medicine residency at university of texas medical branch-galveston and nasa/jsc emergency medical preparedness and response to a singapore airliner crash space shuttle operations at the nasa kennedy space center: the role of emergency medicine aviation medicine space doctor similarities of medicine and astronautics the preventative medicine physician: a national study a review of human physiological and performance changes associated with desynchronosis of biological rhythms fatigue in operational settings: examples from the aviation environment key: cord- -xw i k authors: dederichs, melina; weber, jeannette; muth, thomas; angerer, peter; loerbroks, adrian title: students’ perspectives on interventions to reduce stress in medical school: a qualitative study date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: xw i k the mental health of medical students remains to be a matter of concern. numerous setting-based and individual-based interventions for student mental health have been proposed in the literature. however, the student perspective on those interventions has been largely neglected. this study aims to explore how medical students perceive different interventions and if they desire any additional changes with regard to their studies. eight focus groups with participants were conducted at a large german medical school. focus groups were recorded, transcribed and content-analyzed using maxqda . we found that medical students prefer setting-based interventions. most proposed interventions were on a setting-based level. for instance, students asked for more information on the university’s psychosocial counseling services and for better information management regarding contact persons. interventions proposed in the literature received mixed reactions: several participants did not favour a pass/fail grading system. students considered a peer-to-peer mentoring program for freshmen very helpful. students had diverse attitudes towards balint groups. they approved of several self-management courses, most of them being related to time or stress management. interestingly, the most urgently wanted interventions appear to be rather easy to implement (e.g. a mentoring program). this study explored the medical student perspective on student mental health interventions. additionally, our study illustrates the benefit and feasibility of involving students early on in the conception of interventions. further research with a representative sample is needed to obtain broader information on the acceptance of the suggested interventions. medical students show a high prevalence of mental illnesses worldwide, such as high levels of depression and depressive symptoms [ ] .to address medical students' poor mental health, setting-based and individual-based interventions have been proposed [ ] [ ] [ ] [ ] . setting-based interventions aim to improve health by modifying environmental factors, e.g. curricular changes in medical schools [ , , ] . individual-based approaches, by contrast, seek a change in the individual, e.g., by providing skills that help to cope with stress [ , ] . although some interventions different perspectives. we therefore conducted a qualitative study with focus groups to gather insights and specific explanations for why students deemed specific interventions useful or not. our study aims to answer the following questions: . what interventions do medical students themselves suggest and wish for? . how do medical students perceive interventions that have been described in the literature (pass/fail grading, a peer-to-peer mentoring program, balint groups, and self-management courses)? participants were recruited through social media or advertisement on campus of heinrich-heine-university of düsseldorf in germany. the only criteria for participation were to be currently enrolled as medical student and not having participated at our previous focus groups [ ] . participants of one focus group were recruited through a stress-management seminar. participation was explicitly on a voluntary basis. all participants gave their informed consent. students were compensated for their participation with two cinema tickets and one cinema discount card. focus groups were conducted until data saturation was reached. students were grouped into focus groups according to their period of study. this maximised the likelihood that participants could relate to each other in terms of experience regarding classes and exams. four groups included students in the preclinical part of their studies (year to ) and four groups included students both from the clinical part (year to ) as well as students in their clinical internship year (after the second state examination). the focus groups took place in june and july at the medical faculty of heinrich-heine-university düsseldorf. in , the medical faculty introduced a new curriculum. traditionally, medical students take one final exam organised by the state (state-examination) for each of the three periods of study: the first after the preclinical phase (after year ), the second after the clinical phase (after year ) and the last one after one year of practical internship at a hospital. with the new curriculum, the first state examination takes place after the first two years. permission to conduct this study was given by the ethics committee of the medical faculty of heinrich-heine-university of düsseldorf. after provision of written informed consent, participants were asked to fill out a questionnaire covering demographic data. the focus groups were conducted by tm, a faculty member and psychologist by training, while md, a junior researcher and trained psychologist, took notes. the focus groups followed a topic guide developed by md and tm (s file). both facilitators provide lectures for medical students and are involved in research on medical students' wellbeing. participants were informed about the facilitators' research focus. first, participants were asked about their experience in medical school so far and in which situations they had encountered obstacles or experienced stress. secondly, students were asked about potential changes and interventions that could contribute to stress reduction in medical school. previous research at our faculty already pointed out high stress levels and specific stressors of our medical students [ , ] . to answer the second research question (how the students perceived literature-based interventions), we discussed interventions proposed in the literature with medical students. the topic guide contained a list of the interventions (see introduction section), which were explained by the facilitator and then discussed by the students one after the other. if necessary, the facilitator asked follow-up questions to further explore students' views. every single participant was encouraged to contribute to discussions at an early stage. all focus groups were audio-recorded, transcribed and content analyzed by md and jw according to mayring [ ] using maxqda . jw is experienced with occupational health and qualitative research [ ] . research questions were transformed into deductive main categories (students' suggestions, interventions from literature). during analysis these categories were further broken down into sub-categories by inductive category formation. after md completed the first coding round of % of the data, al, an experienced qualitative researcher [ , , , ] , reviewed the coding scheme. the coding scheme was then adapted and a second coding round was performed by md. jw coded the data of four groups independently. both versions were compared and discrepancies resolved. after that, md recoded the remaining four groups and transferred changes made to the coding system in the previous discussion with jw. finally, jw and md discussed the focus groups once more. there was no need for further recoding since there were only minor differences between the analyses of md and jw. due to logistic constraints, corrections and feedback on transcripts and research findings were not obtained from study participants. the completed checklist of consolidated criteria for reporting qualitative research (coreq; [ ] ) can be found in s file. eight focus groups consisting of to medical students were conducted. focus groups lasted between and minutes. in total, medical students ( women) participated. with n = medical students being enrolled at the medical faculty in the summer semester of , we included . % of the population in our focus groups. participant age ranged between and years (mean = . ; sd = . ). all participants engaged in a comprehensive discussion about what they would like to change in medical school to improve student wellbeing. in the following, we will present their proposed starting points for interventions. regulations for absence of lecturers. students reported that especially in the clinical study phase, many lecturers did not show up for teaching. students asked for a substitution or at least a designated contact person. attendance. attendance rules were perceived to be too strict. according to the study regulations, an attendance of at least % presence per subject is required. however, if one subject consists of three appointments for example, all three seminars have to be attended. by attending only two of them, the presence would be %, thus below %. students therefore requested that if one misses class due to sickness or death of a family member, an exception should be granted. according to the participating students, there should also be the option to complete a compensatory task, for instance, holding a presentation on the subject that was missed. as another solution for not missing a mandatory event, participants suggested switching groups with their peers for the required event only. due to the high number of mandatory classes during the preclinical phase, students wished for less mandatory attendance during this phase. coordination of practical and theoretical phases. the düsseldorf medical curriculum combines phases of theoretical studying (lectures) and practical experience (clinical traineeship). students wished that the curriculum is adapted so that theoretical and practical lectures as well as corresponding tutorials are better aligned and deal with the same subjects. this was believed to increase their learning effect. students noted that the workload between theoretical and practical phases fluctuates significantly with high workload during theoretical phases and low workload during practical phases. therefore, they wished for shorter practical phases to distribute the workload more evenly. often, the lecture-free week takes place after the clinical traineeship. however, students would rather have a week off for studying after completing the theoretical lectures when they find to have more content to repeat and memorize. counseling. participants pointed out that they would benefit from a broader psychological support system. regarding the on-campus counseling service, students wished for longer opening hours as well as opening hours that are compatible with their schedules. free of cost confidential counseling services were perceived as useful for reaching out to students to address mental health issues. overall, students wished for more information regarding mental health problems and interventions. possible solutions students brought up were for instance a mandatory lecture about stress related to medical school, coping strategies and support contacts. additionally, students suggested that a newsletter should be emailed close to the first state examination to present information on emergency contacts and counseling services. registration processes for elective subjects. students explained that when they want to register for a subject, they have to log in to an online portal. there they would have to wait until midnight until the registration process opens. they further explained that registrations are allocated on a first come, first serve basis. therefore, students stated that without access to a fast and reliable internet connection at that time they would not be able to take the class they prefer. students would like this procedure be replaced by a selection based on preference. in this procedure, students could indicate their preference within a certain period. then they would be matched according to their choice. if there is no personnel to do this task, some students suggested that at least the first come-first serve procedure was opened during daytime. information management. students reported a lack of information management in medical school. they requested a comprehensive online portal containing information on classes and exams. moreover, students expressed that they would benefit from an introductory lecture for each new topic block that contains all relevant administrative information, e.g. on how to register for an exam. a faq that addresses most urgent questions concerning the first state examination was considered useful. online lectures. many students raised the topic of online lectures. they perceived online lectures as an efficient measure to reduce stress and improve flexibility. especially commuters were thought to profit significantly from it. online lectures were thought to be a great learning tool and aid with the preparation of future seminars. one student requested a contact person in case any questions arise during a session. "i think it would be great to have this opportunity. i personally learn at night. in the morning, i don't pick up much, but at night i am really diligent. and it were a lot easier to combine family, job and university. that would be a huge advantage." refresher courses. students stated that they would benefit from refresher courses before the second state examination. "i believe it would be pretty fair, for instance, to have courses that prepare you for the state examination in practical terms during the semester. that would be, i think, not bad." examinations. students reported that their exams cover several subjects at once. students asked for a restricted number of subjects within the same exam. they reported that this could be achieved if exams included more questions on the same subject per exam instead of the same subject posing only a few questions per exam over several exams. "now for instance [ they also suggested grades acquired in seminars should be added to the exam score and act as a buffer. it was discussed whether more open-ended questions instead of multiple-choice questions would be beneficial. some students stated that they could explain their knowledge better in open-ended questions. moreover, it would motivate them to learn more details. others preferred the existing multiple-choice format. teaching content. participants made a range of suggestions to improve the quality of teaching. for instance, students wished for a contact person to report verbal harassment of students like depreciative comments by teaching staff. exact teaching guidelines containing learning goals for every subject were considered potentially helpful. they also wished for more covering of content relevant for the second state examination. a perceived lack of interest and skill of teaching staff was thought to be solved through employing senior students or external referents. they asked for more interactive and clinically orientated teaching. elective subjects. in total, elective subjects have to be completed over the course of five years at the medical school in düsseldorf. some of the subjects are graded. participants raised the idea to reduce the total number of required courses to reduce stress and provide more time for state exam preparation. "it is good that we have elective subjects. but there are too many. and it is stressful, to have eight elective subjects here and another six elective subjects there. it would be great if we had less." alternatively, additional credit for scientific projects, clinical lectures or extracurricular activities was suggested. clinical traineeship. students asked to move the clinical traineeships towards the end of the medical curriculum, because students felt that they are often confronted with medical conditions for which they are not prepared yet and often clinicians presuppose content they have not dealt with yet. students reported frequent difficulties in their clinical traineeships. for instance, clinicians that are supposed to teach them are often not available and, if they are present, students felt that they do not take time to teach them adequately. more available contact persons or a central complaints office that assists them were mentioned as a possible solution to this issue. preparation for the second state examination. students wished for lecture free and exam free time for preparation before the second state examination. they requested to have at least days to dedicate completely to studying. this way they stated they could follow the learning schedule they wanted to use (so called " -day study plan" which is well-known and widely used by medical students in germany). "it is a question of planning. then, if you have these one hundred days, i think that would help a lot. and it would be nice if it wasn't exactly one hundred days but a couple of days more because maybe you don't study seven days a week." participants had many ideas on how to provide more time to study. participants proposed shortening clinical trainings, or to move them towards the end of the medical curriculum to avoid that students have to pass other exams right before the second state examination. students were willing to complete their clinical training during semester break or to begin the semester early to provide more preparation time for the second state examination. measures proposed in the literature pass-fail system. there was no consensus among the students in our focus groups related to the usefulness of pass-fail systems. some were in favour of eliminating grades because it would reduce pressure and stop demotivation through bad grades. others in favour of the pass-fail system argued grades did not have any informative value regarding their ability as a physician anyway and were thus of no importance. students opposing the pass-fail system reported to appreciate the incentive they received from good grades and to appreciate the feedback to better understand whether they had learned enough. students depending on a scholarship or on receiving particularly good grades raised concerns regarding their ability to compete and prove their performance. scholarship holders among the students reported that in order to keep their scholarship, they had to prove to belong to the top students in their semester. without grades, this might be difficult to prove and students from other universities might have an advantage. in düsseldorf, the final grade for the first state examination is calculated as a cumulative sum score of grades from the first to the third year and of the performance on the day of the state examination. students argued that this way they did not solely depend on one's day performance and therefore the stress on the day of the first state examination was reduced. some preferred keeping grades to maintain this system. however, students suggested alterations of the pass-fail system. they were in favour of eliminating grades from some subjects to reduce stress but maintain the current system of collecting grades for the first state examination. mentoring. almost all participants were in favour of a mentoring program. they appreciated it for its easy access and voluntary nature. the idea of having a permanent contact person that provides not only informational and emotional support but also further networking opportunities to higher semesters was welcomed. the personal experience and organizational knowledge of a senior student was thought to reduce stress significantly. "i would have liked having someone who told me 'everything is fine, this is normal. i experienced it too.' especially at the beginning of medical school. . . i had just moved here. . . and everything was new. having someone who can answer questions related to studying or simply help you." many students in senior semesters expressed the desire to become a mentor themselves. those who did not want a mentoring program stated they already established such a contact to a senior student by themselves. there were many suggestions regarding the design of a mentoring program. a pool with volunteers, possibly on an online-based platform was proposed. students further proposed that mentors should receive some form of compensation, either monetary or in a non-monetary form, like an honorary position. students argued that their willingness to participate in a mentoring program also depended on the mentor's personality. they feared that overly performance-driven students might be a bad role model in terms of stress management. students who would not want to participate in a balint group stated that they preferred being mentored by their peers or supervising physicians. they reported to rely on their own social networks for emotional support. some students expressed fear of stigmatisation due to participation in a balint group. they worried about talking freely about unpleasant experiences and being stigmatized by their peers. therefore, they would not want to participate. self-management courses. students were interested in learning self-management strategies or competences to strengthen their resilience. in this context, they wished for subjects on stress-management and relaxation techniques. moreover, students wished for a time-management course and more capacities in an existing elective subject deals with mind-body-medicine. participants stated they would benefit from courses that covered efficient learning strategies and presentation skills. there was little consensus among participants regarding the question when and how those classes should be implemented in their curriculum. in general, students perceived lack of time as a barrier to participate in a non-obligatory class. some students suggested that it should be an ongoing offer over a long period of time where students could decide for themselves every week whether they wanted to participate or not. students preferred to schedule such courses at the beginning of medical school and shortly before the first state examination. general dissatisfaction with already existing self-management courses on campus was expressed. students either perceived lack of information about those classes, lack of quality of available classes or limited capacity to enroll all interested students. the aim of the present study was to explore which interventions students suggest to improve their mental health and to discuss interventions suggested in the literature. in our eight focus groups, students suggested specific solutions to their perceived obstacles in medical school. interventions that students proposed most frequently pertained to setting-based aspects such as curricular changes, improved information management and new regulations concerning absence of teaching staff and students. many of the suggested interventions, such as online lectures, appear rather easy to implement. due to the current covid- pandemic (time of publication; [ ] ), our faculty addressed the wish for more online lectures by creating new digital learning opportunities and structures. eventually these structures will persist after the university switches to conventional lectures once again. it is also striking that many of the suggested interventions might not only reduce stress, but also improve several aspects of teaching (e.g. the coordination of practical and theoretical phases). this suggests that improvement of well-being and improvement of academic outcomes go into the same direction. in contrast to students' wish for setting-based interventions, most interventions being proposed in the literature focus on the individual [ ] . however, such interventions are believed to not tackle the root of the problem [ ] . it is argued that instead of teaching students how to cope with stress, the causes for stress need to be addressed [ ] . some individual-based interventions (e.g. stress-management courses) might even have the opposite effect by being an addition to existing classes and workload. it is further of interest that we observed that a significant number of our participants did not favor the interventions suggested by the literature. for instance, several students did not prefer a switch to a pass/fail grading system. these students perceived grades as helpful, were concerned that the absence of grades would have a negative impact on their academic performance, or would cause increased stress at the first state examination. however, this latter concern is not supported by the data [ , ] . it is possible that the idea of a pass/fail grading system elicits discomfort among our students because it is rather uncommon in germany. when the pass/fail system was first introduced in the us similar concerns were raised [ ] . a shift to a pass/fail system was perceived as a disadvantage for students and students coming from a university with pass/fail grading were thought to experience difficulties finding a residency placement. therefore, the authors did not recommend such a transition [ ] . however, a recent study showed that a pass/fail grading does not curtail career prospects [ ] . specifically, there were no differences in residency placement (receiving a placement at all and percentage of students that receive top specialty choices) and overall academic performance between pass/fail and tiered grading system cohorts [ ] . besides pass/fail grading being uncommon in germany, it would also require a change of the persisting score calculation for the first state examination. additionally, it would require a cultural shift towards a non-competitive environment among all students in order to be successful. therefore, instead of fully switching to pass/fail grading only, a mixed approach might be more feasible in which pass/fail grading could be adopted in some subjects while other subject are still graded. in this approach, grades still contribute to the result of the state examination. this would address medical students' preference to accumulate marks and thereby reduce stress during the first state examination without a prompt change that requires time for adaptation. possible mentoring programs by senior students were well received by the participants in our study. more advanced students were believed to provide invaluable informational and emotional support. since such a program has relatively low costs and was also favored by potential mentors, we propose starting a peer-to-peer mentoring program at our university as recommended by others [ ] . balint groups received mixed reactions. this is in line with previous research suggesting indifferent to mildly positive student attitudes towards balint groups [ , ] . a qualitative study from finland reported a more positive evaluation of balint groups [ ] . here, students reported being satisfied with their participation and benefiting from the groups [ ] . in our present study, especially those students who had already encountered a difficult situation with a patient stated that they would be grateful for this kind of opportunity and support. therefore, the implementation of a balint group with voluntary participation on request as suggested by students could be considered. it could build on already established peer-support-programs such as the programs at brigham and women's hospital in boston, which has repeatedly served as a model for peer-support-programs [ ] . students themselves requested a variety of self-management courses. most were interested in learning relaxation techniques and how to deal with stress. they stated that existing offers, like a time management class, are less attractive because their study curriculum does not allow for the implementation of taught strategies in this class. overall, they expressed the wish that classes to improve resilience should be accessible to everyone. some stress management courses (e.g. "stress management" or "mind-body-medicine") are only offered as elective subjects and are therefore only available for a fraction of the students. we suggest that all students receive a basic stress-management training and psychoeducation, preferably at the beginning of the curriculum. at some universities, such as the monash university medical school in australia, mindfulness and stress management have been part of the curriculum for many years [ ] . since some studies found reliable short-term effects [ , ] , students could benefit from it in most stressful times without the concern of losing time to study. for the latter reason we also suggest that more classes that address mental health are offered. in addition to face-toface training, e-mental-health-solutions could be made accessible for students for quick support. students' wish for e-learning opportunities suggests that they are open to digital formats that grant them more independence in terms of time and location of use. furthermore, information on specific counseling opportunities should be made more accessible. it is possible that raising awareness of medical students' mental health will reduce fear of stigmatization by peers which was mentioned as a concern in the context of balint groups. overall, we find that students proposed more setting-based interventions than individualbased interventions. this is in sharp contrast with the persistent emphasis on individual-based interventions in medical schools [ ] [ ] [ ] ] . importantly, while setting-based interventions are sometimes considered expensive or difficult to implement [ ] , most ideas in the focus groups (e.g. attendance rules, a new course selection procedure, teaching guidelines) seem easily feasible and resource-friendly and will not only improve wellbeing, but also academic performance. a strength of this study is its rich data, which were collected among as much as students from a broad range of semesters in eight focus groups until data saturation was reached. however, the generalizability of our findings may be limited since only students from the medical school at the university of düsseldorf were included in this study. our findings might be specific to issues and obstacles encountered at universities in germany and therefore only somewhat transferable to universities in other countries. when we asked the participants about their opinions on a pass/fail grading system, we did not clarify in detail whether they would still like feedback on their performance if a pass/fail grading system was implemented. thus, we do not know to what extent students perceive a pass/fail grading system and feedback on their performances mutually exclusive. further, we cannot rule out selection bias. one might argue that only those students suffering from stress or those who are especially dissatisfied with medical school attended the focus groups. on the other hand, one might assume that those students experiencing a high level of stress choose not to participate because of the additional workload. the fact that focus group facilitators were members of the teaching staff and that one focus group was held within a class on stress management could also have affected the observations. md's and tm's position as a teaching staff member might have elicited a social desirability bias and reduced willingness to share sensitive topics. however, also in this seminar, we felt that students spoke very openly about their issues. the contents of this focus group did not differ thematically from the other focus groups. this study explored which type of interventions students consider acceptable and useful. based on our data we are however unable to evaluate the feasibility and effectiveness of proposed interventions. however, we believe that only interventions that are favored by students and address their specific needs will be successful. in contrast to interventions to improve medical students' mental health that are proposed in the literature, we find that medical students mostly proposed interventions on a setting level rather than on an individual level. importantly, many interventions suggested by the students are low-cost and easy to implement. we believe that considering the student perspective is a key factor in designing mental health interventions. further research with a representative sample is needed to obtain more generalizable information on the acceptance of the proposed interventions and to test them in terms of feasibility and effectiveness by using both qualitative and quantitative approaches. prevalence of depression, depressive symptoms, and suicidal ideation among medical students: a systematic review and meta-analysis medical student mental health: culture, environment, and the need for change medical student mental health . : improving student wellness through curricular changes. academic medicine: journal of the association of american medical colleges medical school strategies to address student well-being: a national survey. academic medicine: journal of the association of academic psychiatry: the journal of the american association of directors of psychiatric residency training and the association for academic psychiatry holistic and sustainable health improvement: the contribution of the settings-based approach to health promotion setting-based interventions to promote mental health at the university: a systematic review effectiveness of universal programmes for the prevention of suicidal ideation, behaviour and mental ill health in medical students: a systematic review and meta-analysis. evidence-based mental health mindfulness interventions in medical education: a systematic review of their impact on medical student stress, depression, fatigue and burnout association between learning environment interventions and medical student well-being: a systematic review the benefits of pass-fail grading on stress, mood, and group cohesion in medical students impact of pass/fail grading on medical students' well-being and academic outcomes differences in medical students' academic performance between a pass/fail and tiered grading system a systematic review of the literature describing the outcomes of nearpeer mentoring programs for first year medical students introducing first-year students to medical school: experiences at the faculty of medicine of erasmus university students helping students: vertical peer mentoring to enhance the medical school experience the doctor, his patient and the illness research on balint groups: a literature review the impact of a physician awareness group and the first year of training on hematology-oncology fellows stressors perceived by the para-clinical undergraduate medical students teaching the clinical encounter in psychiatry: a trial of balint groups for medical students reflecting on our practice: an evaluation of balint groups for medical students in psychiatry a qualitative analysis of student balint groups in medical education: contexts and triggers of case presentations and discussion themes. patient education and counseling self-management education: history, definition, outcomes, and mechanisms. annals of behavioral medicine user involvement: a review of the benefits and challenges. behaviour & information technology acceptability of healthcare interventions: an overview of reviews and development of a theoretical framework. bmc health services research helping medical students develop lifelong strategies to cope with stress a comprehensive medical student wellness program-design and implementation at vanderbilt school of medicine. academic medicine: journal of the association of american medical colleges stressors and resources related to academic studies and improvements suggested by medical students: a qualitative study medical students' perceptions of stress due to academic studies and its interrelationships with other domains of life: a qualitative study qualitative content analysis: theoretical foundation, basic procedures and software solution what are the perceived influences on asthma self-management at the workplace? a qualitative study psychosocial working conditions and diabetes self-management at work: a qualitative study consolidated criteria for reporting qualitative research (coreq): a -item checklist for interviews and focus groups meeting between federal chancellor merkel and the minister-presidents of the lä nder to discuss the coronavirus-press release medical student perspective on stress: tackling the problem at the root evaluating a grading change at ucsd school of medicine: pass/fail grading is associated with decreased performance on preclinical exams but unchanged performance on usmle step scores pass fail grading-a disadvantage for students applying for residency stressors and starting points for health-promoting interventions in medical school from the students' perspective: a qualitative study peer support for clinicians: a programmatic approach the health enhancement program at monash university medical school enhancing the health of medical students: outcomes of an integrated mindfulness and lifestyle program academic psychiatry: the journal of the american association of directors of psychiatric residency training and the association for academic psychiatry we would like to thank johanna schwerdt for her invaluable help understanding the medical school curriculum and putting the students' perspectives into the right context. loerbroks. key: cord- -t w cdr authors: nan title: royal academy of medicine in ireland date: - - journal: ir j med sci doi: . /s - - - sha: doc_id: cord_uid: t w cdr nan the tuberculin-skin-test is the most commonly used test to screen for tuberculosis worldwide. in most cases it is administered by the most junior member of the medical team. there is some anecdotal evidence to suggest that junior doctors have limited knowledge of how to administer and interpret this test correctly. the aim of this audit was to assess the proficiency of interns and senior-house-officers in st. vincent's university hospital at performing the tuberculin-skin-test and improve standards. a multiple choice questionaire was used to assess doctors' knowledge of tuberculin-skin-test administration, interpretation, alternatives and the availability and awareness of information regarding the tuberculin-skin-test within the hospital. interns and senior-house-officers were assessed. of those questioned . % correctly identified intradermal as the method of administration. . % knew to correctly assess the induration at - h, but only % knew that the induration should be measured across the forearm. only . % were aware of the information leaflet within the hospital. . % of senior-house-officers correctly identified intradermal injection as the method of administration. it is apparent that the tuberculin-skin-test is often administered and/or interpreeted incorrectly. we recommend formal teaching for junior doctors in this area, coupled with improved availability of the information leaflet. mucinous tubular and spindle cell carcinoma (mtscc) is an extremely rare type of kidney tumour that has only recently been described, with less than eighty cases in the literature. this was only recognized as a specific entity in the world health organization classification of renal cell carcinoma (rcc). mtsccs are polymorphic renal neoplasms characterized by small, elongated tubules lined by cuboidal cells with cords of spindled cells separated by pale mucinous stroma. we report the case of a -year old lady who had an incidental finding of a mass in her right kidney. the radiological features were consistent with a rcc and following a multidisciplinary team discussion she underwent a laparoscopic radical nephrectomy. macroscopic examination revealed a well circumscribed . . right lower pole mass. histologically it was composed of elongated tubules, small tubules and papillary structures with a necrotic centre. the cells demonstrated cuboidal and spindle cell morphology. histological grade was fuhrman grade . subsequent ct thorax abdomen and pelvis staged the tumour as pt b. the majority of mtsccs are indolent, and there is only one report of a distant metastasis which responded favourably to adjuvant sunitinib. to date there is no international consensus on long term surveillance of these patients. due of the favourable prognosis with this type of tumour, mtscc must be differentiated from papillary renal cell carcinoma to avoid administration of excessive adjuvant treatment to patients. this is the first recorded case of this recently classified, rare tumour in ireland. this incidental finding of solid pseudopapillary neoplasm (spn) was discovered when a -year-old female underwent a chest x-ray to investigate a wheeze. a subsequent ct abdomen revealed a cm well circumscribed mass adjacent to the tail of the pancreas. this neoplasm had reached a significant size of cm appreciable on radiological imaging and yet was asymptomatic and not palpable on physical examination. laparatomy revealed a highly haemorrhagic and calcified mass emanating from the pancreas. this was adherent to the omentum, distal pancreas and splenic vessels. distal pancreatectomy was performed with en bloc resection of the mass. repeated ct scans at , and months failed to demonstrate recurrence. solid pseudopapillary neoplasms are rare entities accounting for between . and . percent of pancreatic tumours. this neoplasm has a predilection for females under the age of . these tumours are indolent and usually reach a large size before detection. diagnosis is confirmed on histology and complete surgical excision of localised tumours is curative. we aimed to assess the prevalence of smoking among patients with vascular disease and the role of the health care profession in encouraging smoking cessation. patients who attended the vascular outpatient department were surveyed over a month period in . patients gave verbal consent to partake in the audit and the surveyor entered the responses into a standardised questionnaire response sheet. % of patients were current smokers, % ex-smokers and % had no history of smoking. % smoke over cigarettes per day and % had a smoking history spanning over years. just % of smokers who are under the care of the vascular service have been advised to give up smoking in the past by a healthcare professional. smoking has long been established as a major modifiable risk factor for the development of atherosclerosis however % of patients attending the vascular service continue to smoke. just half of patients who were offered smoking cessation advice found it was effective. therefore a system needs to be put in place where all vascular patients are advised of the benefits of smoking cessation and the manner in which information is dispensed needs to be revised. to investigate the optimum location for the teaching of procedural skills to medical students english n, o'flynn s introduction: procedural skill training is a vital component of medical education. traditionally it has been teaching hospital based however general practice rotations may provide greater opportunities than previously thought. aims: this study aimed to ascertain whether a general practice setting or a teaching hospital setting provided a better environment for acquiring procedural skills in terms of opportunity to practice and the variety of skills performed. the correlation between end of year osce results and the amount of procedural skill exposure was also looked at. methods: a cross-sectional quantitative study which included all rd year medical students at ucc was conducted. a log book listing procedural skills was made available to all students before beginning both general practice and teaching hospital rotations. students were instructed to indicate on the log when they performed any of these skills and in which location. logs were returned to medical school. data was obtained and analysis performed using spss . results: a response rate of % was achieved. . % of students performed more skills at the gp setting. . % (n = ) did not perform any skills while in a teaching hospital . skills were performed significantly more frequently in a gp setting while were performed more frequently in a teaching hospital. students who performed a high number of skills in one location were no more likely to perform a high number in the other. conclusions: students were able to take greater advantage of procedural skills opportunities in a gp setting. as this was the students first clinical year it is likely that the one-to-one teaching scenario provided them with a more suitable location to practice skills for the first time. this study also highlighted the diverse nature of procedural skills which a general practice setting can provide. accuracy of sentinel node biopsy in determining the requirement for second axillary surgeries in t -t breast cancer with retrospective application of z criteria background: lymph node status is the most important prognostic marker in breast cancer management. in tandem with breast conser-vative surgery, surgical approaches to the axilla have also become less invasive thus decreasing the morbidity associated with axillary clearance. the acosog z trial reported no difference in survival in patients undergoing sentinel lymph node biopsy (slnb) alone versus axillary lymph node dissection (alnd) in t -t tumours. our aims were to establish whether sentinel lymph node biopsy was a true representative of axillary burden. we also analysed whether retrospective application of criteria from z trial would have prevented patients undergoing second axillary surgery. methods: all patients with t -t tumours undergoing sentinel node biopsy were included in our study (n = ). analysis of our prospectively updated breast cancer database was performed. minitab version . was used to carry out statistical analysis of the data results: slnb procedures for t & t tumours were performed over a year period. patients were reported as histologically negative and were positive. of the lymph node positive group, patients progressed to axillary clearance. staging of patients remained unchanged with only patients having [ axillary lymph nodes reported as positive. patients from the slnb negative group also had an axillary clearance. of these patients had further axillary disease with patient being upstaged having [ axillary lymph nodes positive. with retrospective application of z criteria % of patients would have avoided second axillary surgery. conclusions: sentinel node biopsy is a strong indicator of axillary tumour burden. this study highlights the accuracy of sentinel lymph node biopsy in staging disease and representing overall tumour burden. flaherty ra, kelly bd, coyle d, quinlan mr, d'arcy ft, rogers e, jaffry sq we report the first case of a spontaneous right nephrocutaneous fistula (ncf) with an accompanying fistula limb communicating with the right ureter. a -year-old man presented with a groin mass, which was initially diagnosed as a hernia. he was scheduled for an inguinal hernia repair. upon incision there was extravasation of urine from the wound and the procedure was abandoned. a ct urogram identified a ncf running from the right lower pole calyx, anterior to the psoas muscle and emerging on the right groin skin with an accompanying fistula limb communicating with the right ureter. during the course of investigation it was discovered that the patient was suffering from chronic indolent calculus pyelonephritis which led to the formation of both aberrant pathways from the kidney and the ureter and that both had calculi located at their origins. the patient was first treated with a nephrostomy and ureteric stenting to relieve urinary obstruction and after this failed to resolve the fistula, was successfully treated with percutaneous nephrolithotomy for removal of the calculi and fibrin glue injection into the fistula. this case is one of only a few reported cases of spontaneous nephrocutaneous fistula and the anatomy of the fistulous tract in this case is very unusual and posed a particular challenge for surgical management. this case report further advocates the use of fibrin glue in the management of complicated ncf. this is a retrospective case study. there were six cases of ocular tuberculosis over the year period, one annually, four of whom are women, with ages ranging from to years old. two were foreign-born. all patients presented with reduced visual acuity. four developed posterior uveitis, one anterior uveitis and one panuveitis. this was also complicated by vitritis, retinal detachment and retinal vasculitis in four. the median duration of symptoms until commencement of treatment was months. all cases had a positive mantoux and one case had evidence of pulmonary tuberculosis on chest x-ray. tuberculosis was isolated in two cases. the intended duration of anti-tuberculous therapy for all patients was months. vision improved in all cases. ocular tuberculosis is rare in developed countries, with prevalence ranging from \ to %. however, it is important to be considered in all cases of uveitis. despite the use of pcr, most cases are presumptive. this leads to delayed commencement of therapy causing further complications. a high index of suspicion is required. we describe the case of a -year-old gentleman who presented to our emergency department (ed) with a very unusual complication of central venous catheterisation. this resulted in spontaneous extrusion of a retained intravenous guide wire from the base of the occiput. this has been described only once previously in the literature, but not at such a delayed time interval from insertion [ ] . this -year-old gentleman presented to the ed reporting that he felt the point of a sharp object irritate his finger in his midline occipital area. he had successfully retrieved approximately cm of a thin metal wire. he had a history of rheumatic fever and had undergone an elective aortic valve replacement years previously, necessitating central venous cannulation. he had remained asymptomatic up to this time. plain radiography of his neck revealed a short segment of wire in the posterior spinal musculature. this segment of wire (approximately cm) was removed manually with minimal force and minor manipulation. the procedure was uncomplicated and the patient was discharged shortly afterwards. retained foreign bodies may migrate slowly over many years eventually extruding from the body, without any serious complications. events such as retained or lost guide-wires are rare. this phenomenon may become more frequent with increasing complexity of medical care and with increasing use of cv catheters in the treatment of sepsis and other emergent critical conditions. physicians should be aware of the possibility of retained foreign bodies and should be somewhat re-assured by reports of simple uncomplicated removal. we present the case of a -year-old gentleman who was recently admitted with symptomatic right heart failure and new onset atrial fibrillation. our patient had been treated in the community for symptoms suggestive of ccf but had not previously been investigated. of note, he has no history of a chronic inflammatory condition and no symptoms suggestive of an underlying neoplastic process. on presentation he was also noted to have evidence of an arthropathy affecting his knees and ankles and bipedal oedema. renal function was abnormal with a urea of . and a creatinine of . urinalysis was positive for protein and h urine collection for protein is ongoing. liver enzymes were also elevated and revealed a cholestatic picture. echocardiogram showed a reduced ejection fraction of % and findings consistent with amyloidosis. biopsy of abdominal fat pad at time of writing is pending. amyloidosis refers to an uncommon group of disorders characterised by extracellular tissue deposition of a variety of proteins in an abnormal fibrillar pattern which are resistant to degradation. it can occur alone (primary) or can complicate many chronic inflammatory conditions (secondary). the major sites for clinically reported amyloid deposition are the kidneys, heart and liver. clinically patients present more often with right heart failure; pulmonary oedema is rare. amyloid infiltration results in increased echogenicity on echocardiogram and gives a ''sparkling'' appearance to the myocardium. biopsy is diagnostic. this was achieved using a retrospective review of all children receiving gh therapy (n = ) over a -year period (october -october ). of patients on gh therapy had ghd. of these, had ighd ( male) and had cphd ( male). all had appropriate work-up and follow-up. age at presentation to endocrinology was older in the ighd group (mean . years) than in those with cphd (mean . years). / children with ighd presented with short stature, compared to only with cphd; the remainder presenting with clinical features of other pituitary hormone deficiencies. the mean height centile at diagnosis was lower in the ighd group ( . th) versus the cphd group ( th). mri brain/pituitary was abnormal in the majority of patients ( / ) with cphd, compared with / with ighd. both groups responded well to treatment and height increased by one centile on average at months. all patients diagnosed with ghd at temple st had appropriate work-up and follow-up. children with ighd presented later than those with cphd, and had shorter height centiles at diagnosis. there was a strong male predominance in children presenting with ighd, which may reflect psychosocial factors. structural pituitary abnormalities were more common in those with cphd, and their clinical presentation was more varied. response to therapy was similar in both groups. background: out of hospital cardiac arrests have poor survival rates approx - %. improving outcomes in ireland have been seen in the past decade. better outcomes are seen if arrest is witnessed and when bystander basic life support was initiated. worse prognosis is seen in a rural setting due to delay in paramedic response times and in administration of advanced cardiac life support. case report: a -year-old donegal male experienced chest pain in his rural home and subsequently cardiac arrested. his spouse, whom months prior had trained in basic life support as part of a fas course contacted the 'out of hours' gp and ambulance service and commenced cpr. the gp failed to reach the house and the first ambulance broke down. on arrival of second ambulance, one person cpr had been administered for [ min. paramedics delivered dc shocks and intubated the patient. in the regional hospital pc was admitted to the intensive care unit for days being managed with acute respiratory distress syndrome (ards). transoesophageal echocardiogram on day of admission showed ef %. ct brain carried out showed no acute pathology. once stable, angiography was carried out showing multivessel disease. discussion at st james's hospital (sjh) cardio-thoracic conference resulted in plan for transfer and pci. in sjh pressure wire study of left anterior descending (lad) coronary artery was positive and stenting (drug-eluting) commenced. lad stents, left circumflex stents and right coronary artery (rca) stents. patient is currently well with no overt signs of hypoxic brain injury and is enrolled in cardiac rehabilitation programme. discussion: this is an incredible case of an out of hospital cardiac arrest. elapsed time in the chain of survival events would predict a negative outcome. however, adequate cpr was administered preventing long term brain injury and certain death. this highlights the need for a greater community-based cpr skill base. recently citalopram and escitalopram have been reported to cause dose dependent qtc prolongation. prescribing guidelines have since changed including contraindication of co-prescription with other qtc prolonging agents. domperidone is a dopamine antagonist widely used as an anti emetic. qtc prolongation and ventricular arrhythmias have also recently been highlighted with domperidone and, since november , caution advised when prescribing domperidone, particularly in patients [ years of age, or at doses [ mg/day. in this audit, we aimed to study whether information on qtc prolongation affects prescribing practice by looking at the prescription of a commonly used medical drug, with recently highlighted qtc effects, and its co-prescription with psychotropics. a list of drugs with substantial evidence for qtc prolonging effects was obtained. a kardex review was completed from acute medical and surgical; long stay and rehabilitation wards. kardexes with domperidone were reviewed for dose, age, gender and co-prescription of other qtc prolonging agents. of surveyed kardexes, % (n = ) were prescribed domperidone. % were[ years. % were on[ mg/day. coprescription with another qtc prolonging agent seen in % of cases; of these % were psychotropics, most commonly citalopram (n = ). four patients were co-prescribed [ qtc prolonging agent. qtc prolonging agents were commonly co-prescribed with domperidone, which continues to be used even in at-risk groups. psychotropics were the most likely class to be concurrently prescribed. further work in this area is necessary to inform clinical psychiatric practice and encourage responsiveness to new evidence regarding cardiac risk. the development of a mathematical model to predict the time to osteoporosis (tto) using dexa scanning background: dual-energy x-ray absorptiometry (dexa) is the gold standard used for measuring bone mineral density and such readings are currently used to predict osteoporosis and osteoporotic fractures. however, no similar prediction model has been developed to identify the time it will take to become osteoporotic based on dexa scanning. objective: the aim of this study was to develop a mathematical model to determine the tto based on two or more dexa scans with tto defined as the age at which the patient will enter the osteoporotic t-score range. methods: fifty patients who had previously undertaken five dexa scans were identified from the dexa database. t-scores were graphed against patient age using graphpad prism software. straight line curves for the most recent scans and cumulative scans were generated with the age at which the curve intersects t = - . being classed as tto. results: the mathematical model developed successfully predicted the time to osteoporosis for each patient, as well as creating a cumulative osteoporotic trend based on total dexa scans performed. additionally, if the patient was classified as osteoporotic following dexa scanning, the model also successfully predicted the time out of osteoporosis. implication: the tto provides a simple and informative parameter of dexa scanning that a patient can immediately comprehend and understand, while also providing a more simple measure to monitor response to therapy. based on the results presented tto can be incorporated into future dexa scans result summaries. further research will involve validation of this tool. an audit of clinical outcomes in transcervical resection of the endometrium compared to outpatient balloon thermablation anglim bc, von bunau g department of gynaecology, adelaide and meath children's hospital, tallaght, dublin thermablation was introduced to the coombe in november and thus far it has provided a quick and effective means of treating women with menorrhagia refractive to medical treatment. a retrospective audit was carried out over a year period in tallaght hospital from november to october . the aim of the study was to compare the efficacy of balloon thermablation compared to transcervical resection of the endometrium (tcre) with or without mirena coil insertion, in the treatment of menorrhagia. patients in total were studied, of which underwent a tcre, and of which underwent balloon thermablation. out of those who underwent a tcre had successful treatment of the menorrhagia and and weekly follow up, had continued menorrhagia which may require a future hysterectomy, however one of which was due to a large fibroid, and one patient described a reduction in menorrhagia however an increase in dysmenorrheoa. out of those who underwent thermablation were treated successfully, had continued menorrhagia to be considered for hysterectomy, had reduced bleeding but increased dysmenorrhoea and one patients symptoms had resolved however she then developed idiopathic thrombocytopenia purpura which led to a recommencement of symptoms. one can therefore conclude that there are both pros and cons to both procedures, tcre being less expensive, however it requires general anaesthesia and may require mirena insertion. thermablation is more expensive however it is a quick outpatient procedure ( min, s) and is done under local anaesthetic. akinmoluwa s, tormey s department of breast surgery, mid-western regional hospital, limerick breast pain is a common problem especially among women of reproductive age. it accounts for a great percentage of gp visits by young women. it represents a huge proportion of gp referrals to the breast clinic. the palpable effects of this include, among others, an increase in waiting time, increase in healthcare cost, stress on the limited resources and ultimately a decrease in quality of care. in this era of unfavorable economic climate, it is prudent to sanitise our healthcare systems by way of identifying and eliminating practices that have not been proven to alter the course of care. in this study, i reviewed the number of breast pain cases referred to ms tormey's breast clinic in the month of march. the objective of this study is to determine whether or not all breast pain complaints should be referred for specialist review. to achieve this objective, i reviewed all the cases of breast pain referred to the breast clinic in march. the table represents my findings. it is evident from the study that hormonal mastalgia accounts for majority of breast pain complaints in women of reproductive age while a few other cases are attributed to musculoskeletal and other benign disorders. these women, with no risk factors, only need reassurance and pain relief. they do not require specialist intervention. alrashed d introduction: anaemia is a common finding in the elderly population. it may be a sign of chronic disease, underlying malignancy, nutritional status, or blood loss. depending on the classification of anaemia, further investigations such as haematinics and endoscopy may be warranted, as replacing the haemoglobin deficit is never a definitive treatment. objective: to determine the prevalence of anaemia in a population of elderly in-patients and whether further screening was performed. methods: this was a cross-sectional review of all patients years and older under a gastroenterology, a rheumatology, and three geriatrics services at a large teaching hospital. patients' full blood counts were reviewed during their current admission. anaemic patients were then categorised based on anaemia subtype and whether haematinics were investigated. results: out of patients under the five teams, were years and older. out of of these elderly patients were anaemic. none of these subjects had microcytic anaemia during their current admission. out of of these patients had normocytic anaemia. out of of anaemic patients had macrocytic anaemia. haematinics were investigated in out of , including out of patients with normocytic anaemia and out of patients with macrocytic anaemia. one patient had abnormal haematinics after being investigated for macrocytic anaemia. conclusion: anaemia was very prevalent in the patients selected for this audit, with the normocytic subtype being the commonest. haematinics were investigated in half those patients. anglim b, murphy c aims: to determine the nature of surgical management of ovarian cysts in the adolescent and paediatric population over a year period. methods: a retrospective audit was carried out over a year period in tallaght hospital from january to december . this audit reviewed cases of ovarian cystectomy, oopherectomy and salpingooopherectomy using both a hospital online database and records of theatre procedures to identify these patients. results: a total of cases were identified. the commonest presentation was due to pelvic pain. there was a total of ovarian cystectomies, fimbrial cystectomies, oopherectomies, bilateral oopherectomy, and salpingo-oopherectomies. a total of appendicectomies were performed in conjunction with these. histology varied from functional and non functional cysts to dermoids and cystadenomas. there were a total of functional cysts, of which were hemorrhagic. there were follicular cysts, fimbrial cysts, paratubal cysts, dermoid cysts, endometrial cysts, cystadenomas, ovarian torsions and fimbrial torsions. of the total amount of procedures performed were done by a paediatric surgeon, and by a gynaecologist. notably there were fewer cases of benign histology in those procedure performed by gynaecologists. conclusions: adnexal surgery is commonly performed in adolescents and children. pathology is frequently benign. there may be a role for more conservative management. we suggest that imaging of the pelvis and tumour markers should be used more frequently in the pre-operative period. protocols may be developed for future implementation. anglim bc, crowley p day surgery is an efficient way of using hospital beds, provided patients are discharged as planned on the day of surgery. unplanned overnight stay following day surgery places an extra burden on a hospital with the busiest accident and emergency department in ireland. a retrospective audit was carried out of one years day case admissions to determine the incidence and causes of unintended or unplanned overnight stay. women were admitted as day cases over the period of st july to june th . a total of diagnostic laparoscopies, operative laparoscopies, diagnostic hysteroscopies, ninety-three operative hysteroscopies, tension free vaginal tapes (tvt) and miscellaneous minor procedure were carried out during this time period. women ( . %) were retained overnight. the main reason for overnight stay was excessive post-operative pain. additional reasons included voiding difficulties, reactions to spinal anaesthetic, asymptomatic tachycardia and the need for intravenous antibiotics. there was no evidence of inappropriate selection amongst the laparoscopies and hysteroscopies, however % of the patients undergoing tvt required admission. one can conclude from this study that most patients were appropriately selected for day case admission. patients undergoing tvt surgery should be scheduled for a h hospital stay. a vulval clinic is an ideal and efficient way of detecting patients with vulval cancer. once potential patients have been flagged by general practice clinicians or other specialities within the hospital, immediate steps can be taken to rule out malignancy. a retrospective audit was carried over a month period on a new vulval clinic which commenced in tallaght hospital on / / . the aim of the study was to determine the need for a specialised vulval clinic for detection of vulval cancer. a total of patients were referred to the four clinics which took place over this time frame. the majority of referrals were from general practice, other referrals were from dermatology, gynaecology and colposcopy clinics. the main reason for referral was vulval pruritis and pain. nine patients were referred with suspicious lesions on clinical examination. a total of biopsies were taken, two of which showed vulval intraepithelial neoplasia (vin). amongst the other biopsies were cases of lichen sclerosis and the remaining biopsies showed non specific dermatitis. one can conclude from this study that a combined dermatological-gynaecological clinic would be of benefit. in addition a . % detection rate of vin was achieved and therefore highlights the necessity of this clinic. the prevalence of renal disease in patients aged above with normal serum creatinine balasubramanian i, peters c, lyons d and o'connor m department of ageing and therapeutics, mid-western regional hospital, limerick background: the prevalence of chronic kidney disease (ckd) increases with age. older patients have lower lean muscle mass and therefore using serum creatinine alone as marker of renal function can lead to underdiagnosis of ckd. objective: the aim of this study was to review the prevalence of ckd amongst a cohort of elderly patients with normal serum creatinine. methods: doctot application on the smartphone was used to calculate egfr in a cohort of patients over years with a normal serum creatinine on admission. patients were included. this application is based on the mdrd formula (includes age, sex, ethnicity and serum creatinine). patients were then classed into the various stages of ckd. results: of the patients reviewed, had renal disease. interestingly, only had a diagnosis of renal impairment recorded in the medical notes. of the patients had stage ckd and the other had stage ckd. of the patients with renal impairment especially stage , were found to be frail females over years. this group also had a number of co-morbidities including diabetes and hypertension. conclusion: egfr is better than serum creatinine alone for assessment of renal function in the elderly. it is important not only for diagnosis but also for appropriate medical investigation and drug prescribing. as the mdrd formula excludes bmi, further research is warranted to compare measurement of egfr using mdrd formula with the cockcroft and gault equation in this older population. chronic obstructive pulmonary disease (copd) is increasingly prevalent worldwide and the main responsibility for it's prevention and management lies with general practitioners. the aim of this audit was to analyse current standards of care of copd patients in a suburbanrural general practice by examining icgp criteria and comparing results with best practice guidelines. the existing coded population of active patients with copd were telephoned and consent was obtained to ask a set of questions designed to examine certain criteria chosen from the icgp copd quick reference guide [ ] . . of the patients included in the audit (n = ), % of patients were male, the mean age was years (sd = . ) and % were general medical service (gms) patients. there was poor recording of smoking status, high uptake of influenza vaccines compared to international figures, a lower uptake of pneumococcal vaccinations and an increased need for osteoporosis prophylaxis. vaccination reminders, smoking cessation advice and information leaflets have been posted to these patients. development of protocols for coding and management have been implemented. in conclusion, general practitioners must focus on ensuring optimum managment of copd in the community. clinical audit is a useful tool to initiate change. we assessed the accuracy of continuous non-invasive haemoglobin measurement using the sphb pulse co-oximeter Ò when compared to traditional laboratory haemoglobin assessment in an outpatient antenatal population. a total of women were recruited. traditional laboratory haemoglobin samples were taken and quantified in the hospital laboratory. the sphb pulse co-oximeter Ò was calibrated and the mean of three non-invasive measurements of haemoglobin were recorded prior to venipuncture. bland-altman plots were used to determine acceptability of the new non-invasive test as a replacement for invasive testing in a clinical setting. the mean gestation at haemoglobin estimation was . ( . ) weeks. laboratory haemoglobin values ranged from . to . g/dl with a mean of . ( . ) g/dl. the range for the sphb pulse co-oximeter Ò assessment was . to . g/dl with a mean of . ( . ) g/dl. non-invasive haemoglobin measurement provides a clinically acceptable accuracy compared to traditional haemoglobin testing. pressure wound therapy (npwt). in this review we examine the role of npwt in wound healing, compare the products available to clinicians in irish hospitals and explore cost implications today. we achieved this through review of online data, peer reviewed articles regarding efficacy, collection and assessment of data from suppliers of npwt and examining the use and cost of npwt in the mater misericordiae university hospital. we summarise the mechanism of action of npwt, patient selection and indications for its use. the products available on the irish market are compared. through examination of these elements we clarify a role of npwt in management of complex wounds and identify flaws in the management of this service that are both wasteful of money and hospital services and create barriers to discharge. potential strategies to correct the issues identified are detailed, for example, funding of the product by the treating hospital rather than by local authorities in the community or selection of less costly devices in negotiation with suppliers by local health authorities. the solutions we outline will potentially have a financial benefit to the hospital, will lead to the more efficacious running of the hospital system and as such will benefit the patient. we conclude that this is a fundamental service and that there are alternative approaches to implementing use of the product in a more efficacious manner. poster q fever: questions to be answered? brandon l, bannon c, fleming c department of infectious diseases, university hospital galway q fever, an aptly named condition, describes infection with gramnegative bacteria coxiella burnetti. q denotes a question, and there are many to be answered in this rare, but not unknown, condition. take mr. m.c, a -year-old farm worker, who had an aortic valve replacement in , for congenital aortic valvular disease. he next presented to medical services in , with fevers, sweats, fatigue and weight loss. investigations at the time diagnosed autoimmune hepatitis, following liver biopsy. he commenced prednisolone and azathioprine. in , again symptomatic, he had another aortic valve replacement. post-operatively, he required weeks of antibiotics for a culture negative valvular infection. in , still on immunosuppression, he developed culture negative meningitis, requiring weeks of antibiotics. azathioprine was discontinued. a renal biopsy revealed proliferative glomerular nephritis in , carried out for macroscopic haematuria. he commenced high dose prednisolone and cyclophosphamide. throughout this time, he regularly presented to medical personnel with high fevers, up to c, present since . they responded to steroids but relapsed on doses below mg. in , the fevers were investigated with a toe, and vegetations seen on the aortic graft. he was diagnosed with culture negative bacterial endocarditis, and subsequently tested positive for q fever. this case highlights the q behind q fever, and raises important issues for medical personnel. when should we remember it? when should we test for it? and what can we do to ensure high risk populations dont slip through the cracks, as this gentleman did? previous point prevalence studies of antimicrobial use in sch have consistently produced the same conclusions and recommendations pertaining to prescribing habits, highlighting doctors' failure to meet ideal standards of antimicrobial prescription. the aim of this study was to assess antimicrobial prescribing habits from the doctors' point of view, to compare this to available prescription data and to raise awareness of the principles of prudent antimicrobial prescribing. a multiple choice questionnaire was used to examine antimicrobial prescribing habits with regard to documentation of indication, documentation of a stop/review date, awareness of local empiric guidelines and other principles of prudent antimicrobial prescribing. trainee and consultant doctors were surveyed. of those questioned, % claimed they always ensure that an indication for commencing antimicrobial treatment is documented in the patient's healthcare record. only % always document a stop/ review date when prescribing antimicrobials, while % indicated that they had failed to do this at least once in the preceding month. % of those surveyed sometimes or never consult local guidelines. when switching patients from intravenous to oral therapy, % believed oral bioavailability to be an important factor, with only % citing cost as being relevant. identifying doctors self-reporting of their deficits allows us to target appropriate interventions to these deficits. our survey identifies areas where awareness of diverted resources and safety issues could be used as a fulcrum for changing prescribing practices. we recommend formal teaching for doctors in this area, with particular emphasis on prudent prescribing and the correct use of empiric guidelines. venous thromboembolism (vte) is a cause of inpatient morbidity and mortality which may be reduced by appropriate thromboprophylaxis. it is well established that vte risk assessment and thromboprophylaxis prescribing may often be inadequate. recently it has been estimated that as many as , deaths per year due to hospital-acquired vte in england may have been prevented with appropriate prophylaxis [ ] . in the current study, a cross section of inpatients was examined to establish concordance with current evidence-based guidelines for vte prophylaxis. data was collected from inpatient charts and drug kardexes relating to patients on three medical wards. laboratory data was also obtained from the hospital it system. data relating to patient mobility was obtained from medical charts, nursing staff, observation, and the patient themselves. sixty-three medical patients and surgical patients (including one patient under obstetrics and gynaecology) were included in the study. ( . %) out of at-risk medical patients who were suitable candidates for thromboprophylaxis had sub-cutaneous heparin prescribed, whereas out of of the suitable at-risk surgical patients were prescribed thromboprophylaxis. medical patients ( . %) and surgical patients ( %) were prescribed anti-embolism compression stockings. prescribing of thromboprophylaxis is relatively thorough in this patient population although it remains less than optimal. there exists some evidence of disagreement amongst clinicians regarding the optimum vte prophylaxis strategy [ ] . implementation of hospitalspecific guidelines regarding thromboprophylaxis is recommended in keeping with recognised guidelines [ ] . ali sheikh a, chandra r, gardezi a, o'hare j mid-western regional hospital, limerick background: good documentation represents good medical practice. objectives: to assess our current standard of documentation of allergy in admission notes, synchronicity with risk alert bands, information given by the patient, documentation on the front allergy alert section of medical notes and drug kardex. methodology: we assessed five parameters i.e. drug kardex, alert band, medical and admission notes and gathered information from each patient staying in medical and surgical services. there were patients in hospital. results: % of the patients admitted under medical and surgical teams had allergy or allergies to different drugs. % had single allergy, whereas % had multiple allergies. penicillin allergy was the commonest . % followed by opioids . %. furthermore, it is found that recording of allergies was under par as % was on front page and % appeared in medical notes. more than half of allergy information was found on drug kardex %, patient knowledge % and allergy bands %. it is well documented that use of elastic compression stockings (ecs) prevents post thrombotic syndrome in patients with prior deep venous thrombosis (dvt). a % reduction in these complications has been noted, with year duration of therapy suggested [ ] . the advice given to patients, their understanding of the benefits of this therapy and adherence issues has not been documented at sligo general hospital (sgh), this research aimed to address this. a short patient questionnaire was undertaken. this consisted of demographic information, and questions regarding the advice and use of ecs. the population consisted of patients with prior dvt attending the warfarin clinic at sgh. this data collection took place from october -february . the questionnaires were collated and results identified using microsoft excel with simple statistical analysis. eleven patients were included in the study, % were not advised to wear ecs, and only % wore the ecs daily. reasons for nonadherence include; difficulty fitting, discomfort and no benefit noted. improvement in adherence could be achieved if advice was given promoting use, the benefits explained, optimal frequency/duration of use advised and correct measurement. as research strongly supports use of ecs, it is essential adherence is encouraged to reduce the risk of post-thrombotic syndrome and future dvt. opiate injecting drug use is a well-established phenomenon in inner city dublin. the complications arising from this practice affect a predominantly young cohort of patients, who under different circumstances would be expected to enjoy good health. acute infections, acute vascular issues such as pseudoaneurysm, and chronic medical conditions such as hepatitis c and hiv are well recognised and frequently encountered by medical physicians who care for these patients. we present the case of a lady in her thirties with a long history of opiate injecting drug use. approximately months prior to presentation, she underwent left sided pseudoaneurysm repair. she presented to the emergency department in a drowsy opiate induced state. on waking, she stated that she had lost some needles while injecting into her groins. plain radiology of pelvis revealed the two ''lost'' needles ( fig. ) . on closer questioning she admitted to significant manipulation of the needle injecting path and angle in the weeks prior to presentation. she had attributed this to her previous surgery and the duration of her injecting drug use. follow-up duplex sonography revealed bilateral intact femoral arteries. further surgical management was non-operative with the focus on addiction counselling and further attempts at facilitating cessation of heroin use. bilateral ''lost'' needles is an unusual complication of injecting drug use and certainly would not rank as one of the protean manifestations of such practices. the aim of the study was to investigate patients' recall of their surgery and influencing factors. a questionnaire was given to patients at outpatient follow up and surgical details were recorded. patients completed the questionnaire. the median age was years (range - ) and the median follow up was months (range - ). the extent of surgery did influence patients' recall with those having an alnd (n = , %) having significantly more accurate recall of their surgery as opposed to those who had slnb (n = , %), p = . . the presence of ongoing postoperative symptoms also significantly improved recall, p = . . almost half the patients who had slnb ( . %) could not accurately remember the extent of the surgery they had but . % were more careful of their arm or would not allow cannulation. the patient's consent process influenced patient accuracy. patients who filled the consent at both the outpatient consultation and in the hospital were significantly more accurate than those who had signed the consent at the clinic or hospital alone, p = . . patients who have minimally invasive surgery, such as slnb are not accurate at recalling their surgery. this misinformation results in confusion over the subsequent vigilance of their upper limb. the consent process may have a role in improving patient recall. introduction: waiting times can exceed days for general surgical clinics and can reach up to months in different surgical specialities. many outpatient slots are lost by patients who do not attend (dna) to their scheduled appointment. we sought to ascertain whether a reminder text message (rtm) could decrease the number of patients who dna to surgical outpatients. methods: a single text message was sent to patients days before their scheduled appointment, reminding them of the date and time of their upcoming surgical outpatient visit. this incentive was initiated in january . outpatient appointment scheduling and attendances for a single surgical team were analysed over a year period, encompassing two month periods before and after implementation of the rtm service. data was exported to spss v for statistical analysis with p \ . considered statistically significant. results: over the month period there were , scheduled outpatient appointments for the surgical service, with attending prior to the implementation of the reminder text message service and the remaining attending in the months following its implementation. the percentage of dna patients did not differ significantly ( . classically, focussed assessment with sonography in trauma (fast) addresses a yes/no binary question as to whether fluid is present in the context of trauma. fast generally concentrates on four areas: perihepatic, peri-splenic, pelvic and a sub-xiphoid view of the pericardium. we report on two patients who were the victims of trauma. both patients had normal haemodynamic parameters. in both patients, the initial fast ultrasound scan was technically negative but it exhibited other signs of intraperitoneal injury. in the first case, a young gentleman sustained a penetrating injury to his right upper quadrant area. morison's pouch (the interface between the liver and the right kidney) did not exhibit any fluid. there was, however, a thin anechoic strip around the gallbladder. ct confirmed the suspicion of peri-cholecystic fluid and this patient required urgent laparotomy and repair of his hepatobiliary injury. in the second case, a gentleman in his thirties sustained a blunt injury to his left upper quadrant. ultrasonography exhibited heterogeneous echogenicity of the spleen. this patient proceeded to have urgent laparotomy and splenectomy for this shatter-type injury. as experience with fast techniques grows, the binary question of whether intra-peritoneal fluid is present becomes more nuanced. the objective of this audit was to review the hospitals compliance with hospital guidelines, to get an overview of how fluids are being prescribed in the hospital and to produce quality improvement plans. thirty drug kardexs were chosen randomly from wards around the hospital, both medical and surgical. if a kardex was found to have no fluid prescription, an alternative kardex was chosen in its place. note was taken on whether the prescription had the patient name and hospital number, the date, name, dosage and strength of the prescription, the route of administration and the frequency and rate of administration. the main areas of non-compliance were found to be: name: only ( . %)orders out of had the name on the order medical record number: only ( . %)orders out of had the mrn on the order, and the route of administration was not present on any of the orders checked. in conclusion, this audit would suggest that there is a lack of compliance with detailing the patients name and mrn on fluid orders, that the route of administration was not written on any kardex, however the back page of each is exclusively dedicated to iv fluid prescription and also that non-approved abbreviations are being used when prescribing fluid orders. spontaneous hip fractures, or fractures without a fall have been described in up to % ( , ) of cases of hip fracture. an upsurge in such cases was recently observed in our emergency department. we present these in the form of a retrospective case series. patient is a -year-old ex intravenous drug user who presented with non-traumatic right-sided hip pain over a period of weeks. initial plain films did not reveal fracture. over week her symptoms deteriorated to the extent that she became unable to weight-bear. patient is a -year-old gentleman with increasing left sided hip pain following a seemingly innocuous fall months prior to index presentation. again initial radiographs did not reveal an abnormality. patient is an -year-old bed-bound nursing home resident with end-stage alzheimer's disease. she was noted by nursing staff to have bilateral hip symptoms post seizure. the patient was unable to mobilise independently and had not fallen out of bed at any stage. patient is a -year-old lady who presented with unilateral sacroiliac pain following a recent intensive exercise program including kickboxing week previously. in each of these cases, subsequent review and plain films demonstrated fracture and in one case bilateral fractures secondary to seizure were demonstrated. our cases highlight the need for diagnostic vigilance and a structured approach in dealing with possible radiologically occult hip fractures, even in patients with no proximate antecedent history of trauma. delerium, or acute confusional state, is a common presentation to our emergency departments, and occurs in up to % of hospitalised patients. we describe the case of acute deterioration in mental status, on a background of alzheimer's disease, with an interesting aetiology. mr k's family sought emergency medical review of days deterioration; withdrawal, somnolence and general disorientation. he is a -year old with moderate alzheimer's disease. history and initial investigations were unremarkable. he was mildly dehydrated and physical exam showed only mild truncal ataxia. further investigations to elucidate cause included lumbar puncture, mri brain and immunological and vasculitic parameters. serology revealed human immunodeficiency virus (hiv) infection with acute seroconversion pattern. a history obtained with help of his family identified several casual heterosexual partners within past year. this included a contact who may be an intravenous drug user, with involvement in commercial sex work. symptoms abated within a week of admission, following pattern of hiv viral load. he has subsequently commenced antiretroviral therapy. this case highlights several areas of interest. sexual history is often overlooked in the older patient, which can be deleterious to outcomes. trends of hiv infection in ireland include primary infection in the older person, in addition to greater longevity of people infected in earlier adult life. we would advocate opt-out testing within the emergency department, and this is currently under study in our tertiary emergency department. comparison of comorbidities in patients with pre-diabetes to those with diabetes mellitus type the management of type diabetes and its complications are well researched. the prevalence of these complications in pre-diabetes has not been researched to the same extent. there has been no research comparing the prevalence of complications in pre-diabetes and type diabetes in ireland. a cross sectional study performed on pre-diabetes and type diabetes patients, selected from the diabetes interest group database (a database of the diabetic patients in general practices in cork region) using stratified sampling for age and gender. a questionnaire was designed and completed in each practice assessing the presence of diabetes related complications in pre-diabetes and type diabetes patients. data was analyzed on spss. the prevalence of complications was determined and the chi square test performed to see is there a statistically significant difference in the prevalence of these complications between pre-diabetic and type diabetic patients. the prevalence of ischaemic heart disease and autonomic neuropathy is actually higher in pre-diabetes but the prevalence of renal disease and cerebrovascular disease is higher in type diabetes. none of these differences in prevalence are statistically significant. the prevalence of peripheral vascular disease, eye disease and peripheral neuropathy is higher in type diabetes, this difference being statistically significant. the prevalence of many of the complications in pre-diabetes is as high as in type diabetes which may have implications for the screening and management of these conditions and the related comorbidities. the both groups were evenly matched. the median age was and median homocysteine level was (range - . ). results: in group b, immediate clinical improvement was equivalent between the normal homocysteine group and treated hyhc group. median time to binary restenosis in hyhc was months and in normal homocysteine was months. p = . . secondary endpoints and all cause survival showed no significant difference. pre-treatment multivariate logistic regression for group a; depicts that hyhc is the main culprit of graft occlusion and limb loss p \ . . multivariate logistic regression for treatment group reports that corrected hyhc is no longer a significant factor of operative outcome. conclusion: patients with treated hyhc have similar outcomes compared to those with normal homocysteine. it is therefore crucial to measure homocysteine in all patients with cli and correct aggressively prior to intervention to improve outcomes. the efficacy of clinical guidelines in promoting co-prescription of bone protection with glucocorticoids among hospital doctors treating inpatients background: therapeutic glucocorticoids (gc) rapidly decrease bone mineral density, inducing a catabolic shift by promoting osteoclast differentiation and activation and by inhibiting osteocytes. current guidelines ( ) direct that bisphosphonates (bp's) and calcium carbonate , mg (ca ++ co -) with vitamin d (vit. d ) should be given at initiation of gc therapy as it is known that bone catabolism occurs early with steroid usage. we circulated these guidelines within our hospital after auditing the existing practice of the hospitals doctors and year later we sought to measure the efficacy of our intervention by completing an audit loop. methods: a cross sectional audit was performed of all adult medical and surgical inpatients in a tertiary referral centre teaching hospital. it was noted if inpatients had been prescribed gc and if concurrent anti osteoporotic medication had been prescribed. subsequent to the initial audit, guidelines promoting the use of bp's, ca ++ co and vit. d when prescribing gc's were advertised on hospital notice boards, in hospital bulletins, hospital prescribing guidelines and on the hospital website. one year after publishing the new guidelines the audit loop was completed by performing a similar cross sectional audit. results: all inpatient medical records (n = ) were reviewed in jan of whom % were female and % were older than . / ( %) inpatients were prescribed gc's. ca ++ co with vit. d was prescribed for % of patients on gc's with % also receiving bp therapy. % of patients were also receiving-post menopausal hormone replacement therapy. in nov year after guideline publication all inpatient medical records (n = ) were reviewed of whom % were female and % were older than . / ( %) inpatients were prescribed gc's. ca ++ co with vit. d was prescribed for % of patients on systemic steroids with % also receiving bp therapy. creation and circulation of hospital guidelines resulted in an improvement in the co-prescription of ca ++ co and vit. d and bp's with gc's by the order of . and respectively. however % of patients on systemic steroids received no bone protection and % received suboptimal bone protection from steroid induced osteoporosis. conclusion: publication and advertisement of current bone protection guidelines when prescribing systemic steroids resulted in a substantial but suboptimal improvement by hospital doctors in our hospital in the co-prescription of bone protecting drugs to prevent steroid induced osteoporosis. in this audit it appears that the majority of prescribers do recognise the necessity to protect bone health when a patient requires steroids. however a substantial number of patients did not receive any bone protection. it is our perception that most physicians are not aware that short courses of steroids reduce bone mineral density and therefore greater efforts must be made to enhance doctor awareness of the necessity for bone protection to be prescribed at initiation of systemic steroids. there is a trend towards longer total survival for jetflow tcvcs. these results suggest a potential advantage from using this line type, however, further study and formal cost analysis needs to be undertaken prior to changing our practice. with increasing resource restrictions, appropriate ordering of blood tests is vital for medical economic viability. this study evaluated the pattern and cost of thyroid function test (tft) requests and aimed to determine if tsh alone identifies thyroid abnormalities. a retrospective review of tfts performed on in-and-out-patients at a -bedded regional hospital was undertaken in january , evaluating the number, results and costs of tsh, t and t levels. tsh, t and t were ordered. / patients ( . %) were euthyroid. tsh abnormalities occurred in / ( . %) ( table ) only / ( . %) patients had a normal tsh despite an abnormal t or t level. / ( . %) of these patients had known thyroid disease, undergoing treatment with thyroxine or thyroidblocking medications. / ( . %) had t levels \ nmol/l outside the normal range and asymptomatic so were considered to be euthyroid. / ( . %) had a variety of diagnoses, for example, pituitary disease. tft reagents alone cost € , . ir j med sci ( ) (suppl ):s -s this study has identified that non-selective requests for t and t add little diagnostic value, except in certain circumstances like treatment of thyroid disease, in pregnancy or if pituitary disease is suspected. optimising tfts requests could save in the region of € , /per annum. tsh alone would appear to be adequate for the majority of patients. case study: neurodegenerative disorders we present a case with an unusual combination of neurodegenerative disorders. a fit and healthy -year-old man, with no history of medical or psychiatric illness deteriorated progressively over a year span, presenting initially with speech and language difficulties, followed by development of extra-pyramidal signs non responsive to levodopa. neurological permacol Ò mesh is an acellular porcine-derived dermal collagen surgical implant used in a wide variety of surgical reconstructions and repairs. we describe two cases where permacol Ò mesh was used to anchor the contents of the femoral triangle in patients undergoing radical block nodal dissection as part of the surgical management for metastatic penile squamous cell carcinoma, one of whom had an atrophied sartorius muscle due to previous infection with poliomyelitis. both patients underwent successful inguinal node dissections and femoral triangle repairs, with permacol Ò proving to be an effective means of protecting the femoral vessels in both patients despite complications related to wound healing secondary to a fixed flexion deformity in one patient. a -year-old gentleman, with a past history of vestibular schwannoma requiring a ventriculoperitoneal shunt (vps) was admitted with acute diverticulitis. his condition worsened and required a laparotomy for bowel perforation and faecal peritonitis. this case reports the successful perioperative management of the patient with a vps in situ in the setting of an emergency abdominal surgery. vps placement is an effective treatment of hydrocephalus, diverting cerebrospinal fluid (csf) into the peritoneal cavity. unfortunately, the shunt devices have a high incidence of malfunction mainly due to catheter obstruction or infection and are associated with various complications, % of which are abdominal [ ] . incidental pathology unrelated to the vp shunt can also occur such as appendicitis [ ] , endometriosis [ ] and diverticulitis as in this case. no standard current set of guidelines for perioperative management of vps exists for patients undergoing general gastrointestinal or urologic procedures with varying degrees of contamination [ ] . this case reports successful and conservative management of a patient with a vp shunt that underwent contaminated abdominal surgery. there is no consensus on the management of vps in patients undergoing elective or emergent abdominal surgery and further studies are required in this area. the use of antithrombotic therapy on management of atrial fibrillation in an irish general practice malomo k , breen n , dunne l , farrell g , bryne p ucd (university college dublin), ireland, now intern, mid-western regional hospital, limerick; general practice, dublin, ireland; pottersway medical centre, bunclody, ireland background and objective: atrial fibrillation (af) is a common cardiac arrhythmia associated with increased risk of stroke events [ ] . to assess the use of antithrombotic therapy in patients with known af attending an irish general practice (igp) and use of stratification schemes to assess their suitability for oral anticoagulant therapy. methods and subject: permission to carry out the study was sort from university-college-dublin ethics committee. there were patients with af attending the igp identified using the computerized disease coding system who international classification of disease (icd- ). thirty patients were diagnosed between / / and / / and their data from the computerized medical notes was used to calculate chads , cha ds -vasc, has-bled scores and identify antithrombotic therapy they were using. results: there were af patients. sixty-three percent (n = ) were males and % (n = ) were females (ratio . : ). twentythree percent (n = ) of patients were aged \ years, % (n = ) - years inclusive and % (n = ) =/[ years. two patients with chads score zero were on warfarin although one of them had cha ds vasc score of one. sixty-percent (n = ) were on warfarin alone, % (n = ) aspirin alone, % (n = ) warfarin plus aspirin, % (n = ) aspirin plus clopidogrel and % (n = ) on warfarin plus clopidogrel. seven patients were not on warfarin for various reasons. the has-bled score revealed patients at low risk, moderate risk and at high risk of bleeding. implications: ninety-three percent of patients were correctly managed and two patients were on warfarin with chads scores of zero. the use of evidence based management guidelines is necessary to manage patients. keywords: atrial fibrillation, chads score, cha ds vasc score, has-bled score meckel's diverticulum is the most common congenital abnormality of the gastrointestinal tract. only % of meckel's diverticulum are symptomatic [ ] . it can cause complications such as ulceration, obstruction, intussusception, haemorrhage and perforation and these complications are more common in the paediatric age group. a -year-old has a lifetime risk of . of developing a complication, this falls to zero over time [ ] . adults most commonly present with bleeding [ ] . we have a case of a -year-old male who presented with a day history of abdominal pain, constipation and anorexia. on examination he had rif tenderness, but no signs of peritonism. a provisional diagnosis of appendicitis was made. the patient was taken to theatre the next morning for laparoscopy and appendicectomy. the appendix was normal and surgery proceeded to laparotomy. an inflamed and perforated meckel's diverticulum was found. a terminal ileum resection with side to side anastomosis was performed. the patient made an uneventful recovery and was discharged to opd follow up. this case illustrates the importance of further evaluation following normal laparoscopy in the case of the ill patient. references: neonatal graves disease is a rare condition, caused by transplacental transfer of thyroid stimulating antibodies from mother to fetus. . % of pregnant women have graves disease and . % of their offspring will have overt hyperthyroidism. a further % will have biochemical thyrotoxicosis without symptoms. this is the case of a baby girl with neonatal graves disease. her antenatal course was uncomplicated until weeks gestation. at this point, her mother became clinically thyrotoxic. maternal blood tests showed an elevated free thyroxine level ( pmol/l) and positive thyroid receptor antibodies. a diagnosis of graves disease was made. she was commenced on treatment but remained thyrotoxic at the time of delivery. the baby was healthy at birth. however, thyroid function tests on day of life showed an elevated free thyroxine ( pmol/l) and thyroid receptor antibodies were positive. clinically, she remained asymptomatic and examination was normal. treatment with carbimazole was commenced and the dose titrated to maintain her euthyroid. most neonates affected by neonatal graves disease will have biochemical thyrotoxicosis but are clinically asymptomatic. the minority will be severely affected with goitre, eye signs, weight loss, tachycardia, arrhythmias and heart failure. it is a transient disorder, limited by clearance of maternal thyroid receptor antibodies and is usually self-limiting over - weeks. mortality rates of up to % are reported in untreated cases, usually from arrhythmias and heart failure. this case emphasises the importance of close monitoring of pregnant women with a history of thyroid disorders, before and during their pregnancy, as well as monitoring their babies in the neonatal period. fibreoptic bronchoscopy is considered a safe diagnostic tool [ ] . it is suggested however that post-bronchoscopy complication rate increases with age [ ] . we decided to study the complication rate and the outcomes of bronchoscopy in patients over the age of years in our institution. a retrospective review of the case notes of patients aged greater than years who underwent bronchoscopy between september and november was carried out. data on complications experienced during and after bronchoscopy and the influence of the results on subsequent management of patients were collated and analysed. ninety-six patients were included. the mean age was . years (sd . ). thirty subjects ( . %) had a documented lung disease. fifty-nine patients ( . %) were current or ex-smokers. indications for bronchoscopy were; to evaluate for malignancy ( . %) and to evaluate for tb ( . %). post bronchoscopy complications were noted in eight ( . %) cases including hypoxia ( . %), infection ( . %), tachycardia ( %) haemoptysis ( %) and pneumothorax ( %). six patients required treatment including nebulised bronchodilators ( . %), antibiotics ( . %), and oxygen therapy ( . %). malignancy was diagnosed in twenty cases ( . %). clinically significant pathogens were detected in six cases ( . %). as a result of bronchoscopy fourteen patients ( . %) had alterations to their drug therapy, three ( . %) received lung cancer treatment with curative intent, eighteen ( . %) had palliative care input, seventeen ( . %) were referred for further investigation and thirty-seven ( . %) had no change to their management. in conclusion, bronchoscopy is relatively safe and has good diagnostic utility in patients aged more than years. patient records were identified from a database of patients who underwent a spinal mri to investigate spinal metastatic disease between november and april . an analysis of the management of those diagnosed with mscc, specifically radiotherapy and/or surgical intervention was performed. three hundred and sixtyone patient records were identified with one hundred and seventy-one patients having metastatic spinal column disease. of these, thirty-four had mri evidence of metastatic spinal cord compression. radiotherapy alone was the most common therapy employed for patients with mscc. a multidisciplinary team approach was not taken in the majority of cases. a surgical opinion was sought in the minority of cases. this is not congruous with nice guidelines as a management protocol. the complexity of management decisions for metastatic spinal cord compression demands a multi-disciplinary approach. current practise in this major supra-regional cancer centre does not routinely employ this approach. a surgical opinion is sought in the minority of cases. this reflects the national trend with some centres having no spinal surgeons as staff. we recommend the establishment of a care pathway in order to comply with best evidence based practise as outlined by the nice guidelines. pet ct as a staging modality in primary cervical cancer; to establish the correlation between histological subtype and fdg- avidity of the primary lesion purpose: pet ct has become one of the mainstays of diagnostic imaging both in staging and prognosis of cervical cancer. we wanted to establish the link between fdg- uptake in the primary lesion and correlation with specific histological subtypes of cervical cancer including squamous cell carcinoma, adenocarcinoma and other rarer subtypes such as clear cell and adeno-squamous carcinoma. methods and materials: the main audit involved working out the fdg uptake in the primary lesions from the cervical cancer database of patients. the patient list was derived from a database of patients collated by the gynaecological services at sjh of all patients who received workup and treatment for cervical cancer from - . the computer system at sjh was employed for analysing pet-ct reports and histology reports. microsoft excel was used to store this information parameters and complete statistics on the data. results: the results of this study are to follow. conclusion: there is a correlation between fdg avidity and histological subtype of cervical cancer and this provides valuable information on the reliability of pet-ct findings in a specific cohort of patients with cervical cancer. we present the case of a -year-old male with a primary piriform fossa squamous cell carcinoma (scc) who attended for staging positron emission tomography/computerised tomography (pet/ ct) scan. distant to the primary lesion, focused f fluorodeoxyglucose (fdg) uptake was noted in the left iliac bone, without underlying abnormality on the accompanying ct scan. low grade uptake was also noted in subcentimetre upper mediastinal nodes, without any underlying lung parenchymal abnormality. these nodes were felt to be inflammatory or reactive in origin. though an unusual pattern for metastatic head and neck scc, the left iliac bone lesion was concerning for malignancy. thus, a percutaneous biopsy of this region was performed under image guidance. histology revealed non caseating epithelioid granulomata consistent with sarcoidosis. the patient was subsequently able to have potentially curative treatment of his head and neck primary. discussion: sarcoidosis is a chronic inflammatory multisystem condition characterised by the presence of non-caseating granulomas in affected organ tissues. it commonly affects young and middle aged adults with a slightly higher prevalence in women. the disease shows a predilection for adults under , peaking between and , with a second peak in women over [ ] . despite its unknown aetiology, it is felt that t lymphocytes play a central role in the development of sarcoidosis, as they likely propagate an excessive cellular immune reaction. it has been shown that abnormalities with the cd /cd ratio and production of t helper and (th /th ) cytokines such as interferon and tumour necrosis factor (tnf) are found in sites of disease activity [ ] . the importance of tnf in sarcoidosis is demonstrated by the efficacy of anti-tnf medications such as pentoxifylline and infliximab [ ] . it is estimated that bone lesions occur in - % of sarcoidosis patients [ ] . these figures are however based on radiographic data and are likely an underestimate as the majority of bone lesions would be asymptomatic [ ] . varying osseous manifestations of sarcoid have been described; punched out lytic lesions, lace-like destruction and subperiosteal resorption mimicking hyperparathyroidism. commonly, the small bones of the hands and feet (predominantly the middle and distal phalanges) are involved often bilaterally and symmetrically. while pulmonary involvement occurs in % of patients with sarcoidosis [ ] , bony involvement is rare without other clinical manifestations of the disorder [ ] . indeed our patient had low grade subcentimetre mediastinal nodes. the fdg avidity of sarcoid is a well documented phenomenon. indistinguishable from metastatic disease on f fdg pet scan alone it can lead to false-positive appearance of metastatic disease on pet/ ct. furthermore one-third of pet/ct positive sarcoidosis have osseous abnormalities on pet/ct the majority of which will not be evident on low dose ct [ ] . this case serves to remind us of the diagnostic limitations of f fdg pet in the differentiation of inflammatory and metastatic processes. in a patient with an unusual pattern of 'metastatic' disease tissue diagnosis is a necessity. distinct islet auto antibodies against antigens insulin, gad , ia and znt have been identified. the presence of autoantibodies has been shown to be predictive of reduced beta cell mass. international data suggests that - % of patients with newly diagnosed t dm are positive for at least one of the above antibodies. our aim is to study the prevalence of autoantibody positivity in our population of children with newly diagnosed t dm over a year period ( ) ( ) ( ) ( ) ( ) . details of all children newly diagnosed with t dm were collected using the endocrinology department database and chart review was undertaken. children diagnosed elsewhere whose care was transferred to our centre and children who had non type diabetes were excluded. one hundred and thirty-six children were diagnosed with t dm in our centre, of which ( %) presented in diabetic ketoacidosis. age at diagnosis ranged between months and . years. the male to female ratio was : . other autoimmune conditions (coeliac disease, hypothyroidism, addison's) were present in %. ninety-six percent (n = ) were tested for one of the three antibodies. % were positive for at least one antibody, % positive for two, % positive for all three antibodies. the most common antibody found was anti gad ( %). positive autoantibodies are helpful in confirming the presence of t dm and their absence in raising the possibility of monogenic diabetes. the absence of pancreatic islet autoantibodies at diagnosis can be predictive for maintained beta cell function during the years after diagnosis. maternal obesity, based on a body mass index (bmi)[ . kg/m , is associated with increased pregnancy complications. moderate exercise during pregnancy is associated with decreased complications such as pre-eclampsia [ ] and gestational diabetes mellitus [ ] and has a beneficial effect on mood with those who exercise experiencing fewer symptoms of depression and anxiety both during and after pregnancy [ ] . the purpose of this study was to determine if obese women exercise less during pregnancy. we recruited women at their convenience after a routine scan confirmed an early ongoing pregnancy. maternal height and weight were measured accurately and bmi calculated. women completed the international physical activity questionnaire. of the studied in early pregnancy, . % took no exercise, . % walked only, . % undertook moderate exercise and . % undertook vigorous exercise. of the obese women (n = ), only % reported moderate-vigorous exercise in early pregnancy compared with . % in women from the normal bmi category (n = ). also % of the obese group reported doing no exercise compared with % of those with a normal bmi. women with a bmi of or more were found to sit for an average of min per day whereas those with a normal bmi sit for min per day on average. although bmi increases with age and parity, these variables were not found to influence exercise levels in early pregnancy. exercise may be physically challenging in obese women, particularly if morbidly obese, but due to its beneficial effects it should be encouraged antenatally in all pregnant women irrespective of their bmi category. references: angiogram showed an absence of coronary artery disease and echocardiogram ruled out structural abnormality. exercise stress test showed short runs of vt in recovery. further tests included ajmaline and adrenaline challenges. cardiac mri showed right ventricular outflow tract scarring consistent with either a primary diagnosis of arvc or secondary with that of myocarditis. sarcoidosis was outruled by further laboratory and radiological means. non-sustained runs of vt on telemetry were noted and a dual chamber implantable cardiac defibrillator was placed. on discharge, medication included atenolol mg daily and patient will undergo genetic screening. follow up for the siblings included phenotyping and mri. discussion: history, presentation and pathology uncovered are consistent with a diagnosis of arvc. suspected paternal inheritance of an autosomal dominant genetic defect predisposed to the ventricular arrhythmias which at first, manifested as self-limiting palpitations however, later caused a near fatal event. long term management may include cardiac transplantation. prevalence of diagnosed atrial fibrillation in adults: national implications of rhythm management and stroke prevention: the anticoagulation and risk factors in atrial fibrillation (atria) study complications of fiberoptic bronchoscopy at a university hospital the relationship between age and process of care and patient tolerance of bronchoscopy central skeletal sarcoidosis mimicking metastatic disease sarcoidosis is a th /th multisystem disorder osseous sarcoidosis treated with tumour necrosis factor-inhibitors: case report and review of literature. spine (phila pa ) musculoskeletal manifestations of sarcoidosis multiple atypical bone involvement in sarcoidosis imaging in sarcoidosis. semin respir crit carre med f- fdg pet/ct for detecting bane and bone marrow involvement in sarcoidosis patients poster diaphragmatic rupture: delayed diagnosis and its consequences-a case report diaphragmatic rupture: a frequently missed injury in blunt thoracoabdominal trauma patients diaphragmatic rupture due to blunt trauma: sensitivity of plain chest radiographs the introduction: lymphoscintigraphy has been shown to be accurate in identifying sites of potential nodal metastases in melanoma patients. recent guidelines published by the eortc-eanm have defined specific criteria with relation to performing lymphoscintigraphy in melanoma patients. methods: the aim of this study was to audit all patients with malignant melanoma who underwent sentinel lymph node biopsy (slnbx) and lymphoscintigraphy in university college hospital galway between - . results were compared with eortc-eanm recommendations. results: melanoma patients underwent slnbx during the study period. patients had preoperative lymphoscintigraphy using intradermal injections of technetium m. sentinel nodes were identified in of patients ( . %) on lymphoscintigraphy. . % of lymphoscintigrams were reported on the same day as the procedure, . % after day and . % greater than day postop. obligatory imaging, as defined in the eortc-eanm guidelines, was obtained in % of all patients undergoing lymphoscintigraphy. no nodal uptake was reported in patients, of whom received imaging in accordance with the guidelines. the location of those melanomas with no nodal uptake was . % on the head and neck, and . % on the trunk. the overall rate of false-negative lymphatic mapping and sentinel node biopsy was . %. in patients receiving lymphoscintigraphy the false negative rate was . versus . % in patients who did not have lymphoscintigraphy. conclusion: preoperative lymphoscintigraphy is an essential adjunct in identifying the sentinel lymph node in clinically node negative melanoma patients and should adhere to eortc-eanm guidelines. conflict of interest: none. on examination, she was alert, hr , bp / . she was tachypnoeic, but reported this to be her baseline. there was a palpable, non-reducible mass in the left upper quadrant. a chest x-ray showed loops of bowel above the diaphragm. ultrasound showed an abscess in the rectus sheath, which drained mucopurulent fluid.mb opted not to have the diaphragm repaired, despite medical advice. she was readmitted weeks later with a recurrence of the abscess. her clinical condition deteriorated, with severe abdominal pain, and oxygen saturations of %. an emergency laparotomy was performed, which showed an obstructing lesion in the descending colon, with large and small bowel above the diaphragm. she had an extended right hemicolectomy, with restoration of bowel to the abdominal cavity and mesh repair of the diaphragm. histology showed an descending colon adenocarcinoma, t n m .traumatic diaphragmatic rupture is a rare problem, occurring in - % of blunt and penetrating traumas. ( ) plain films and ct scans are not always diagnostic in the acute phase, due to concomitant injuries. ( ) repair is essential once diagnosis has been reached to avoid herniation of abdominal viscera. patients with ongoing dyspnoea after blunt trauma may benefit from a repeat chest x-ray. a -year-old retired veterinary surgeon was referred to tertiary referral with a months history of a painless enlarging neck mass. clinical examination showed a right side neck mass approximately cm cm in size which extended through both anterior and posterior triangles. cervical lymphadenopathy was not appreciated and the patient was clinically euthyroid. patient was admitted under the care of the maxillofacial service, where he underwent a needle core biopsy of the neck mass. this was returned showing poorly differentiated spindle cell tumour with large pleomorphic nuclei and abundant abnormal mitoses. the immunoprofile was consistent with metastatic poorly differentiated sarcomatoid carcinoma and the differential diagnosis included origin fro the kidney, lung or thyroid.the case was discussed at the head and neck mdm and a consensus was reached that the patient as developed a sarcoma of the neck, with a level neck dissection the most appropriated intervention.intraoperatively, following the removal of the neck mass it was noted that the right lobe of the thyroid was enlarged. an intra-op fna was performed on the mass in the right lobe of the thyroid. the fna was returned showing bizarre giant cells, suggestive of malignancy. ultimately the patient underwent a total thyroidectomy but, despite surgery the patient died weeks post-operatively. using feedback from the pilot study and analysis of the preexamination consultant and registrar-led teaching schedule for students a further 'intern-led' tutorial timetable was structured. it allowed for a weekly maximum of h of teaching dependent on demand and intern availability. programme duration was weeks, january to march . group sizes were a maximum of students. tutorials were all at the patient bedside. feedback forms were distributed at the end of the programme.sixty-four tutorials were given in total. seventy feedback forms were returned. mean number of tutorials attended per student was . . students rated statements - ( -strongly disagree, -disagree, -neutral, -agree, -strongly agree). median scores were used. scoring showed improvements were made from last year in terms of level of intern preparation for tutorials and importantly, the students own subjective view of their level of preparation for forthcoming examinations. most importantly, students agreed that tutorials improved their history taking skills and strongly agreed that their examination skills improved. matching feedback from the pilot study, students strongly agreed that intern-led teaching is an appropriate adjunct to the final year programme.of the intern working in st james's hospital, participated. seventeen of these had received tutorials on the pilot programme. of the that did not participate, many had never received formal intern teaching.the feedback obtained from the pilot study was invaluable in organising and delivering this teaching programme. ongoing improvements will be made for next year based on this audit. this also highlights that the intern-teaching tool is extremely beneficial, yet largely underused. key: cord- - uqy bnd authors: taylor, robert b. title: now and future practice date: - - journal: white coat tales doi: . / - - - - _ sha: doc_id: cord_uid: uqy bnd in the past chapters, we have visited some heroes, diseases, and treatments that are part of the heritage of the healing professions, we have discussed some of the language of medicine and some thoughts of our most erudite predecessors, and we have examined some instances of clinical practice, including a few times when what happened was not what we today would have wished. i hope that the pages that we have traveled together so far have convinced you of the rich and (usually) honorable traditions of medicine. the tales are sometimes epic, at times tragic, sporadically triumphant, and occasionally lamentable. this last chapter suggests some ways that you, as a health professional or informed consumer, will now—i hope—learn more about medicine’s history and lore. when seeking to learn more about anything, and especially what has happened in the past, books are a great place to start. of course, you may be thinking, "the computer and the internet are replacing books." perhaps this will be true some day; certainly e-mail has replaced handwritten letters as a means of personal correspondence. we should all grieve this loss of civility and of what should become of valuable historical records. imagine what would have happened if the correspondence of thomas jefferson and john and abigail adams had been via e-mail, read by the recipients and then deleted forever. as i wrote this book, i did a great deal of research on computer, as well as in books and scientific journals. the computer can provide fast facts: the internet was invaluable in allowing me to tell dates of birth and death for persons mentioned in the various chapters; it permitted me to do a lot of important fact-checking. however, the internet could seldom provide the context of facts presented and the linguistic richness of books. the world wide web lacks the thoughtful, often lyrical prose found in the best-written books. also, with a book or article, i know who to blame if facts are wrong; the internet is much more impersonal. and so with that introduction, let us look at selected books that may help you learn more. sometime around , sir william osler ( - ) collected a number of his addresses "delivered at sundry times and diverse places in the course of a busy life." the book's title is aequanirnitas with other addresses to medical students, nurses and practitioners of medicine. on the last (and curiously unnumbered) page of the book, the one item in the book (other than the preface) that is not a record of an address is osler's recommendations for a "bedside library for medical students" (osler, last page): "a liberal education may be had at a very slight cost of time and money. well filled though the day be with appointed tasks, to make the best possible use of your one or of your ten talents, rest not satisfied with this professional training, but try to get the education, if not of a scholar, at least of a gentleman. before going to sleep read for half an hour, and in the morning have a book open on the dressing table. you will be surprised to find how much can be accomplished in the course of a year. i have put down a list of ten books which you may make close friends. there are many others; studied carefully in your student days, these will help in the inner education of which i speak. i. old a good friend once told me, "there are two kinds of people in the worldthose who travel and those who don't." sometimes it's good to get out of the office and travel. when traveling in your own country or abroad, take some time to visit medical landmarks-historical sites and museums that recall medical history. sometimes they are a little hard to find; the typical tourist is probably not likely to seek out the semmelweis museum in budapest or the museum of questionable medical devices at the science museum in st. paul, minnesota. however, visiting the site of an important medical event or a collection of medical artifacts can become the highlight of a trip. here is a list of some suggested medical landmarks to visit. some merit an afternoon excursion if you happen to be visiting a city; others, such as the greek island of kos, represent worthwhile travel destinations. kos from the balcony of our hotel in kos, we could see turkey across the waters of the karpathian sea. my wife and i visited kos in with a group of fellow physicians. kos, of course, was the home of hippocrates (ca. - bce), and here we visited the two chief medical history attractions of the island. the first, in a quiet city square, is the plane tree-or at least a descendent of the original tree-under whose branches hippocrates taught his pupils. the other awe-inspiring medical site is the aesculapion, the temple of healing built in the fourth century bce. here, the physicians in our group recited in unison the oath of hippocrates on a bright, sunny morning in october. on the northeast coast of the peloponnesus lies the ancient city of epidaurus. here was built the aesculapion, a center of healing well known throughout greece (mentioned in chapter ). sick persons came to spend the night in the sleeping hall, the enkoimitiria, where during the night aesculapius might help them find their way back to health. the pilgrims to the aesculapion brought riches to the city and allowed the construction of the nearby amphitheater, famous for its remarkable acoustics. some may hold that the baths of caracalla in rome are not truly a medical attraction, but i include them to highlight that public health measuresincluding providing clean water and other advances in sanitation-were the chief medical contributions of the ancient romans. built in the early third century ce during the reign of emperor caracalla, the baths are worth a visit during the day or perhaps on a summer evening when the ruins are the backdrop for the teatro del opera di roma. the oldest anatomy theater in the world is in padua, italy, and it was here that andreas vesalius ( - ) conducted his anatomic dissections. the remarkably well-preserved auditorium is part of the university of padua. among the other noteworthy persons who taught at the university were copernicus, galileo, and giovanni battista morgagni. you will probably need a guide to find this well-preserved architectural treasure among the many halls and rooms of the university. in , our own ubiquitous john hunter, recalled for making surgery a scientific discipline as well as for his syphilitic misadventure and his eventually fatal temper, began what is now the hunterian collection of the royal college of surgeons in london. the collection holds thousands of specimens, including , of hunter's original preparations, such as a specimen showing his successful ligation of the femoral artery for popliteal aneurysm. the home of ignaz semmelweis ( - ) in budapest, hungary, is now the site of a small museum showing artifacts from his life and a selection of other antique medical items. we recall that, in , john snow ( - ) helped stop a cholera epidemic in the broad street neighborhood of london (see chapter ). a short walk from leicester square in soho is broadwick street; in , the suffix "wick" was added to the name of historic broad street-in my opinion, a sad distortion of historical nomenclature. you willnot find a formal museum. what is there may be even better: there is a handleless pump, a commemorative plaque, and the john snow pub, with some framed memorabilia of snow's work. ironically, snow did not drink alcohol, and yet the only building honoring his memory is a tavern. nestled in the pasteur institute in paris is the pasteur museum, opened in . here you will find mementos of the life and work of french microbiologist louis pasteur ( - ), whose work and name give us pasteurization. you can view the apartment where pasteur and his wife lived, and you can visit pasteur's tomb. national museum of medical science history of the islamic republic of iran this museum, opened just a few years ago in tehran, presents iran's historical contributions to medical science. the exhibits draw on an inventory of "more than , objects, tools, pictures, documents, and books related to medical sciences" ( ). although most medical schools in the people's republic of china lean toward an allopathic, "western" approach to medicine, there are still some traditional medical colleges in china. in , i visited one such medical college and hospital in beijing, where i witnessed acupuncture, moxibus-tion, cupping using short pieces of bamboo, and cauterization of enlarged tonsils in children. (the children, wide awake during the procedure, felt no pain. the tonsils have no nerve fibers that conduct pain.) shanghai, for example, has the museum of medical history of shanghai college of traditional chinese medicine. if the chinese city you are visiting has a traditional medical college, a tour of the facilities will be memorable. this section is a personal indulgence, and it is risky for two reasons. the first is that i am stating-in print-some of my opinions, and a few of them have prognostic overtones, thus exposing myself to scorn if i am proven wrong in the future. the second risk is that opinions can be challenged, although such questioning should be considered an invitation for dialogue. after reading the readable books and consulting the reference sources listed in the bibliography, after spending hundreds of hours searching pubmed and other internet sites, and after following the trail of scores of clues that might explain curious tales, i have formulated some thoughts. here they are: john hunter, louis pasteur, pierre curie, william t.g. morton, joseph goldberger, and others all used themselves as experimental subjects. wilhelm roentgen took the first x-ray film-of his wife's hand. jonas salk injected his whole family, as well as himself, with his new polio vaccine. the days of heroically putting one's health, and that of one's family, at risk are gone and should not be lamented. today, human experimentation is performed under tight control. we in academic medicine sometimes grumble about the administrative hurdles placed in our path by institutional review boards and government agencies, but they help to ensure that episodes such as the tuskegee syphilis study will not be repeated. as i worked on this book and looked for the name of the single pioneering scientist responsible for breakthroughs in the mapping of the human genome, the control of the hemorrhagic fevers, and the new advances in treating acquired immunodeficiency syndrome (aids), i found lists of team members, but no pasteur, ehrlich, or salk. in future books of medical tales, the great advances will be attributed to institutes, consortia, and teams, with the names of the individual scientists often ignored. happily, nobel prizes still recognize individual scientists and physicians. the ascendance of research teams and the twilight of the lone investigator do not mean that the single, stubborn innovator has vanished entirely from the scene. furthermore, the traditional bias against novel advances that challenge current dogma is alive and well. more than years ago, portland, oregon otolaryngologist john epley, md developed what he believed was an effective and non-invasive treatment for benign paroxysmal positional vertigo. it was a simple maneuver involving moving the body to reposition particles in the inner ear canals. no surgery and no medication were needed. the technique came to be called the epley maneuver and eventually involved a rotating, computer-controlled chair used to standardize movement of the patient. a investigative report in the portland newspaper, the oregonian, tells the reaction of epley's colleagues: "inexplicably, they rejected him, heaved accusations that threatened his license to practice medicine." today, however, the epley chair is gaining acceptance, has garnered several million dollars in federal grants, and may be on its way to a medical clinic near you ( ). pasteur's words still ring true, but many discoveries are made differently today than in the past. instead of happy accidents and eureka moments, investigators today may study hundreds of compounds hoping that one will prove clinically useful (and commercially successful). nevertheless, dr. john eng must have had an "aha" experience when he first realized that a drug for diabetes, now marketed as exenatide (byetta), might possibly be derived from the venom of the gila monster. because he was a veteran's administration (va) employee and because the va was not interested in seeking a patent, eng paid for the patent himself and subsequently licensed the patent to a pharmaceutical company ( ) . eng shows that there is still a place in medical science for the persistent, intuitive entrepreneur. the language of medicine is far from dead. every year, we create new medical words, phases, syndromes, abbreviations, acronyms, euphemisms, jargon, and slang. during my lifetime, and perhaps yours, we have had the following additions to medicalese: aids, human immunodeficiency virus, prion, erectile dysfunction (well known to all who view television today), nutraceutical, telemedicine, and ehealth. in prior chapters, i described jersey finger, runner's knee, legionnaires disease, fred astaire legs, and the salk vaccine. a few paragraphs ago, i told of the epley maneuver; this technique is not in my current edition of stedman's electronic medical dictionary but probably will be at some point in the future. we have developed a host of new abbreviations, acronyms, and slang expressions: wasp (wait-and-see prescription), pet (positron emission tomography), sars (severe acute respiratory syndrome), and emr (electronic medical record). we also have learned the meanings of commonly used abbreviations such as nsaid (non-steroidal anti-inflammatory drug) and some that are more specialized, such as uval (ultraviolet argon laser). medicine can take less pride in the creation of squash (brain), fascinoma (an interesting and unexpected clinical finding), and positive gown sign (painting a somewhat graphic image of a patient who exits the hospital with neither permission nor street clothes). how rapidly is medicine's vocabulary growing? the first edition of what we know as dorland's medical dictionary was published in , with the title american illustrated medical dictionary (see chapter ); it had pages. (for temporal orientation, osler's text the principles and practice of medicine was first published in .) by , when the th edition of dorland's was published, the book had ballooned to , pages describing , items. in , the th edition discussed , items in , pages. in four short years, we added , new medical words, phrases, and abbreviations. yes, staying current in medicine today involves knowing not only new tests, drugs, and procedures, but new medical words and their permutations. for example, i told above about dr. john eng paying for the patent for what is now the drug exenatide with his own money. although the internet has several sites telling about eng and his discovery, the only source i could find telling about his personal payment for the patent was http://www. mendosa.com/monster.htm ( ) . this web site is entitled mendosa.com, with the subtitle: your on-line diabetes resource. the author isdavid mendosa. i like mendosa's story about dr. eng and his patent. is the story accurate? because mendosa's piece provides no reference citations, i am not quite sure. in online research for this book, i tended to trust web sites that are sponsored by government agencies, specialty organizations, and peerreviewed publications. with appropriate skepticism, i pondered facts presented by web sites maintained by advocacy groups, such as organizations concerned with single diseases (e.g., morgellon disease), and i skipped by web sites whose url (uniform resource locator) contains words such as aquarius, underground, and celestial. these include alexander fleming (died in ), abraham flexner (died in ), and jonas salk (died in ). i wish that i had met these men. while i was in medical school, i heard a lecture by famed pediatrician and book editor waldo e. nelson. while in private practice in upstate new york, i met cardiologist paul dudley white when he spoke at a county medical society meeting; he struck me as a kind, humble man. if you get the chance to hear or meet a medical giant-or a rising star-be sure to take advantage of the opportunity. by no means have all the colorful tales of medicine been lived and written. every scientific advance has the potential to set the stage for several more. i won't try to be nostradamus and predict specific events in the future, but during the lifetimes of today's young physicians, we can hope to see effective vaccines for the infectious diseases to which we are currently vulnerable (e.g., aids), some means to prevent cancers of various types, and interventions to help patients avoid chronic diseases such as diabetes mellitus and hypertension. from time to time, i mention an event in medical history to a medical student or resident. every month, when i teach a headache seminar for third-year students, i tell the story of ergot and st. anthony's fire. i describe how lewis carroll might have visualized some characters in alice's adventures in wonderland as part of a migraine aura visual distortion. i note too many blank stares. only a few synaptic connections. fortunately, when i attended temple medical school long ago, we had some excellent lectures in medical history. i recall the image of joseph auenbrugger ( - ), inventor of percussion as a diagnostic maneuver, tapping on wine barrels filled to various levels. could anyone in our class forget the story of how rene laennec ( - ) invented the stethoscope in ? embarrassed to place his ear directly on the chest of a young woman he was examining, laennec rolled several sheets of paper to make a tube that could carry sound from her chest to his ear. wonder how many of today's young physicians learn these stories. suspect not many. until we senior physicians once again assume responsibility for passing on the oral history of our discipline, books like this will be needed. i will share one example, humorously encapsulated by woody allen: "eighty percent of life is showing up." remember one day that-counter to all logic-i drove miles over snowy roads to make a very routine hospital visit to an elderly patient. as entered the room, she smiled and said, "i knew you would come." that day felt especially proud to be a physician, and the snowy drive home didn't seem as long as the trip to the hospital. a less happy lesson was learned in my very first year in practice. my patient, an older man, quite overweight and severely diabetic, was dying. he and his wife lived in a trailer about minutes from our medical group's office. one day, not unexpectedly, his wife awoke to find that her husband had died at home during the night. as was local practice at the time, a physician was needed to go to the home to pronounce him dead. our group had a designated "house call" physician each day, and this was not my day. my office schedule was full. what should i do? i kept seeing my scheduled patients and dispatched the "house call" doctor to my patient's home. he pronounced the patient dead, doing the job that needed to be done. the next time i saw the widow, she began to weep. "why didn't you come when he died? he was your patient." if there are ever times when persons remember every detail of what happens, it is at times of major life events; death is one of these. you really can't learn lessons such as these from books. medical students and young physicians often do not understand this simple truth and seem to assume that the faith and respect that patients accord them is something that they somehow merit. with few exceptions, our patients do trust us. for the experienced physician, the trust comes from past actions, but it comes also from patients' subconscious memories of their childhood physicians, the legends of hippocrates and maimonides, the discoveries of pasteur and fleming, and the clinical skills of freud and osler. today's physician trust and respect have been earned, but largely by those who have gone before. we, today's and tomorrow's physicians, must aspire to be like the best of our professional forebears. we must be hardworking, resourceful, and patient-centered. as phillips and haynes wrote (see chapter ), we must "be there" when our patients need us. we must always act with integrity and do what is right for the patient, striving to be the physician our patients believe us to be. some of the tales we will tell young physicians in the future will be about what happens today and tomorrow-perhaps in your examination room or hospital. some of these stories will concern aids, which oldstone calls "a plague as bad as any ever known" (p. ). other viruses also threaten us, including ebola, marburg, and ranta viruses. could one of them mutate in a way that matches or even exceeds the devastation caused by the influenza pandemic? then there is the concern that avian influenza (r n ) will become a pandemic with human-to-human transmission. on the more positive side, we can expect many new developments in genetic screening and gene therapy. electronic technology promises to revolutionize day-by-day patient care, with electronic medical records serving as the platform. in years to come, we will see innovations in messaging between patients and physicians, virtual office visits and home visits, online group therapy, and much more. robotic surgery is already a reality, and we will see innovations that are difficult to imagine today. in the end, perhaps we will even begin to conquer some of america's actual causes of death, which include tobacco use, poor diet and physical inactivity, excessive alcohol consumption, motor vehicle accidents, careless sexual behavior, and illicit drug use ( ) . in the meantime, while the dramas that will generate tomorrow's tales are unfolding, what should you and i do? first of all, we should take good care of our families and ourselves and enjoy the practice of medicine-a rare privilege for which we should give thanks every day. famed investor warren buffet has been quoted as saying, "when i go to my office every morning, i feel like i'm going to the sistine chapel to paint" ( ) . my wish for you is that you share this feeling with buffet and with me. there is more. medicine is much more than seeing your patients in the office tomorrow, more than performing three operations or passing an endoscope four times in the morning, more than making hospital rounds or visiting a patient in the nursing home or whatever else your specialty calls for you to do in your workday. what is more is doing your part in regard to tomorrow's white coat tales. how can you or i do this? how can we-in some small way-help pass on the history of medicine to the next generation? to accomplish this, i suggest five tasks: • enhance your personal knowledge about medicine's past. begin a program of reading about medical giants, language, and culture. choose among the books in the bibliography or perhaps even osler's reading list (see page ). become, in osler's words, "close friends" with them. keep an enrichment book at your bedside and read a little each night. • make the extra effort to learn about newly encountered medical terms and syndromes. look up clinical word origins and search out the life stories of persons for whom diseases and syndromes are named. then keep a notebook, entering new findings as you learn them. • become a medically oriented traveler. use your personal tourism to expand your knowledge of medical history, visiting some of the sites listed above or by discovering some of your own. involve your family in the exploration and learning. • add to the medical literature. you may find an intriguing connection between a current clinical finding and medical history. perhaps you will have an insight involving a medical hero or one of the historic plagues. you may have noted a previously unreported cluster of diseases or an unusual manifestation of an illness. when you have a thought worth sharing, consider writing a short piece and submitting it for publication. if you do so, you have added a tale to medicine's anthology. along the way, you will discover that writing for publication is a journey of education, self-discovery, and personal growth ( ) . • seek to develop a sense of how the medical information you use in daily practice has come not only from scientific "evidence-based" sources. your medical knowledge and mine also has its roots in the lore of medicine. it began with the incense-burners, amulet-makers, and quasi-religious ritualists and evolved from those days through the centuries of discoveries to the knowledge we now take for granted. i urge you to stop often and reflect that every diagnostic maneuver, every therapeutic intervention, and every word and phrase in medicine come from some source, some person, or some event in history. the stories of these origins are medicine's white coat tales. thank you for reading my book. i will end, as i began chapters ago, with a quote from felix marti-ibanez (p. ): in your future work, you will be in good company. the great physicians of history, the glorious figures of the past, will always be near you. when you perform a dissection, a red-bearded young man with flashing eyes, andreas vesalius, will be peering over your shoulder; when you conduct a physiological experiment, the melancholy, pensive eyes of william harvey will be watching you; when you teach medicine, the venerable figure of william osler with his apollonian head will come and sit like a medical goethe beside you; and when you approach the sickbed, the shades of hippocrates, sydenham, and fleming will gather round to counsel you, the young princes [and princesses] of our profession. persian history: the national museum of medical science medical landmarks usa: a travel guide to historic sites, architectural gems, remarkable museums and libraries, and other places of health-related interest doctor and intervention outlast jeers and threats. the oregonian foege who actual causes of death in the united states the change makers the clinician's guide to medical writing key: cord- - zkhz p authors: pattisapu, nikhil; anand, vivek; patil, sangameshwar; palshikar, girish; varma, vasudeva title: distant supervision for medical concept normalization date: - - journal: j biomed inform doi: . /j.jbi. . sha: doc_id: cord_uid: zkhz p we consider the task of medical concept normalization (mcn) which aims to map informal medical phrases such as “loosing weight” to formal medical concepts, such as “weight loss”. deep learning models have shown high performance across various mcn datasets containing small number of target concepts along with adequate number of training examples per concept. however, scaling these models to millions of medical concepts entails the creation of much larger datasets which is cost and effort intensive. recent works have shown that training mcn models using automatically labeled examples extracted from medical knowledge bases partially alleviates this problem. we extend this idea by computationally creating a distant dataset from patient discussion forums. we extract informal medical phrases and medical concepts from these forums using a synthetically trained classifier and an off-the-shelf medical entity linker respectively. we use pretrained sentence encoding models to find the k-nearest phrases corresponding to each medical concept. these mappings are used in combination with the examples obtained from medical knowledge bases to train an mcn model. our approach outperforms the previous state-of-the-art by . % and . % classification accuracy across two datasets while avoiding manual labeling. medical social media is a subset of social media focusing on medical topics. it primarily includes medical forums, blogs, and tweets. it draws participation from a variety of cohorts such as patients, caretakers, consultants, doctors, pharmacists, researchers, and journalists [ ] . recent studies have shown that medical social media can be leveraged to find the side effects of a particular drug [ ] , detect the spread of infectious diseases, monitor public health [ ] and understand a patient's experience in healthcare [ ] [ ] . however, automatically identifying such insights is challenging due to the lexical and grammatical variability of the language used in social media which contains informal language, non-standard grammar, and typographic errors. due to the varied backgrounds and expertise levels of its users, medical social media also contains non-standard medical terminology, jargon and abbreviations [ ] . the task of medical concept normalization (mcn) aims to map a variable length phrase to a medical concept in some external coding system. table provides a few examples of social media phrases mapped to medical concepts. mcn has several applications in improving patient care, such as the understanding and answering of patients' questions, early detection of patients requiring immediate attention, and digital disease surveillance [ ] . the same gassy feeling bloating (snomed id: ) table : examples of mappings between social media phrases and medical concepts. snomed ct is a medical knowledge base. we use italics to denote medical phrases and typewriter to denote medical concepts. medical entity linking, which is closely related to mcn, links medical entity mentions in the text with their corresponding entities in a medical knowledge base (kb) such as snomed ct [ ] . while both tasks share the primary goal of disambiguation of a sequence of words, there exist a few substantial differences. medical entity linking operates on medical entity mentions which have an entity type such as disease, drug, symptom, treatment, and test whereas mcn operates on phrases which may or may not have an entity type. for instance, the phrase cant shut up for the whole day is not recognized as a medical entity by any medical entity recognizer and yet is mapped to hyperactive behavior, snomed id: by mcn models [ ] . furthermore, mcn, unlike medical entity linking, allows mapping between the source text and target concept to be loosely defined. for instance, no way i 'm gettin any sleep nite could be mapped to insomnia, snomed id: . medical entity linking uses the context of an entity mention along with the information from the medical kb, to decide which entity is being referred to in the text [ ] . lack of such information makes mcn a relatively more challenging task than medical entity linking. most of the current deep learning models (section ) formulate mcn as a supervised text classification problem. this formulation has several major shortcomings. existing approaches for mcn can be divided into three major categories. the first category formulates mcn as a monolingual translation problem, where the task is to map informal phrases (l ) to formal medical concepts (l ). limsopatham et al. [ ] adapt phrase-based machine translation [ ] to translate medical phrases from twitter language to formal medical language. during inference, output of the machine translation model is mapped to one of the concepts in medical lexicons based on the ranked similarity of their word vector representations. similar to [ ] , statistical machine translation based techniques have also been used to normalize non-medical phrases [ ] [ ] [ ] . the second category poses mcn as a supervised text categorization problem, in which an informal medical phrase is categorized into one of the predefined categories wherein each category represents a unique concept in a medical lexicon. limsopatham et al. [ ] use convolutional neural networks (cnns) and recurrent neural networks (rnns) along with the pretrained word embeddings for normalizing medical concepts. their approach outperforms lexical matching and machine translation based approaches by up to %. lee [ ] propose the use of multilayered bidirectional transformer encoder bert [ ] to extract the vector representation of input phrases which are subsequently used to train a softmax classifier. the third type of mcn methods project the informal phrases and formal medical concepts into a common embedding space. during inference, cosine similarity based ranking is used to retrieve the most similar concept to a given input phrase. metke-jimenez et. al. [ ] use tf-idf representation to retrieve relevant medical concepts corresponding to paraphrased concept mentions. in our recent work [ ] we propose a roberta [ ] based neural model which maps the input phrase and medical concepts into a common embedding space. we leverage existing medical knowledge bases (such as snomed ct) to first obtain embeddings for each medical concept using various text and graph based embeddings. subsequently, we train a neural model to map informal phrases into the target embedding space. we have shown that this approach outperforms all the existing approaches and achieves an improvement of up to . % compared to the previous state-of-the-art [ ] . for the extent of this work, we regard [ ] as the state-of-the-art for this task. most of the recent works [ , , , , , ] use cadec dataset for experimentation, while some of these [ , ] also use psytar dataset. both cadec and psytar datasets were created from the patient discussion forum askapatient.com. few works [ , , , ] , use twadr-l dataset for experiments which consisted of medical phrases extracted from twitter mapped to the medical concepts in the sider medical lexicon . almost all recent works [ , , , , , , , , , , , ] use classification accuracy as the primary metric for measuring the performance of mcn models. main drawback of previous approaches such as [ ] , [ ] and [ ] is that they rely on the availability of pairs of medical phrases and concepts for training mcn models. creating such a training dataset entails the painstaking task of manually identifying social media phrases and mapping them to one of the concepts in a medical lexicon. in our prior work [ ] we used two techniques to overcome this challenge. first, we encoded all target medical concepts into a common embedding space using a variety of text and graph embeddings methods and thereafter transformed an input phrase into a vector in the target embedding space. this allowed our model to map phrases to even those medical concepts which were not present in the training set. second, we generated labeled examples for mcn using snomed ct synonyms by treating each synonym as a medical phrase. we have shown that our model trained exclusively on snomed ct synonyms demonstrates reasonable performance when compared to other approaches. architecture as proposed in our prior work (section . ). our main contribution in this work is the automatic creation of distantly supervised mcn dataset which is discussed in section . . for the extent of this work, we use our prior work [ ] as a baseline. in this section, we describe our previous approach for mcn. we used a two staged approach for normalizing medical concept mentions. in the first stage, all medical concepts from a target lexicon (such as snomed ct) are encoded into fixed sized embeddings using a variety of text and graph based embedding methods such that similar medical concepts are closer in the target embedding space. in the second stage, each input phrase is transformed into a vector m i using pretrained roberta model [ ] which is then transformed into a vector in the target embedding space r i using the two layered feed forward neural network shown in equations , , where w w , b w , w r , b r and the weight matrices of the roberta model are trainable parameters. all parameters are trained using adamw [ ] stochastic optimizer which aims to maximize the cosine similarity between the transformed vector r i and the corresponding target embedding. during inference, a new input phrase m j is mapped to a vector r j in the target concept embedding space which is then classified to a concept using the -nn (nearest neighbour) method. we now describe the process of obtaining target embeddings for medical concepts using text and graph based embedding methods. for text embedding methods we extract the concept description of each target concept id by doing a lookup in snomed ct knowledge base. figure . this graph is given as input to graph embedding algorithms such as deepwalk [ ] , node vec [ ] , harp [ ] and line [ ] , each of which return an embedding for every vertex in the graph. both text and graph label embedding methods aim to map medical concepts into vectors, such that similar concepts are closer in the target embedding space. these embeddings are made publicly accessible at https://zenodo.org/record/ . figure select pairs with cosine similarity greater than a pre-designated threshold and discard the remaining pairs. although this approach seems reasonable, we do not use it as it might introduce label imbalance in our dataset. for instance, common concepts such as pain might have a high representation in our dataset when compared to rare concepts such as aarskog syndrome, which is undesirable. tracting noun phrases from a news corpora [ ] (non-medical corpora) using stanford corenlp library [ ] . we use binary support vector machine (svm) with radial basis function kernel to categorize the social media phrases, where each phrase is represented by the averaged word vector of the words present in it. the embeddings for each word were obtained from the pretrained word vec model [ ] . we use metamap [ ] as an off-the-shelf medical entity linker. it identifies concepts from text and links them to entries of kbs such as snomed ct and mesh [ ] . metamap assigns one or more semantic types such as, sign or symptom and disease or syndrome to each identified concept. existing study shows that metamap exhibits high precision and poor recall when used to extract concepts from medical social media posts [ ] . in this work, precision is of paramount importance for generating high quality distant data. therefore, we further improve the precision using the following two heuristics. first, medical entities which correspond to one of the major semantic types described by [ ] are retained while others are discarded. second, the most frequent incorrect mappings are pruned using a manually created list. for instance, the generic word hi, which is highly frequent, is wrongly identified as a disease dataset from these mappings which were made publicly accessible. . psytar: psychiatric treatment adverse reactions (psytar) corpus [ ] is an mcn dataset created from askapatient.com. in this dataset, all the posts which mention the medications cymbalta, effexor, lexapro and zoloft were obtained. similar to the cadec dataset, human experts were asked to manually discover medical phrases and map to snomed ct concepts which resulted in , medical phrases mapped to concepts. miftahutdinov et al. [ ] created a five fold dataset from these mappings which were made publicly accessible. . in our prior work [ ] we used the publicly accessible folds of the cadec and psytar datasets to evaluate the performance of our mcn model. for a fair comparison, we use the same folds to evaluate the performance of our current approach. the snomed ct lexicon consists of a collection of medical concepts wherein each medical concept has a unique snomed id. every concept in this lexicon is associated with its fully specified name and its synonyms. currently, snomed ct contains over , medical concepts. table shows few examples of medical concepts obtained from snomed ct. in our prior work [ ] we leveraged this resource for training our mcn model. we extracted the synonyms of medical concepts present in psytar and cadec datasets and created an automatically labeled dataset by treating each synonym as a medical phrase. that it also contains some noisy labels, for instance, the phrase heart burn is wrongly associated with the medical concept abdominal discomfort with a cosine similarity of . . we observed that compared to the snomed ct synonyms dataset, this dataset has a better coverage of phrases containing slang words, non-standard terminology, acronyms and spelling errors. our main objective in this work is to build an mcn model which does not use man- ually labeled examples. we therefore use the snomed ct synonyms dataset and the distantly supervised dataset described in section to train our model. we do not use the training folds of cadec and psytar datasets. our baseline is the mcn model proposed in our prior work [ ] which is trained using snomed ct synonyms dataset. to train our mcn model, we initialize its parameters (section . evaluating the performance of both the models we compute the average classification accuracy across the test folds. table shows the comparison of our approach with the baseline [ ] across multiple target embedding methods. our model outperforms the baseline by a significant margin. our model achieves the best classification accuracy of . % on psytar and . % on cadec datasets, whereas the best accuracy using the baseline is . % and . % respectively. the best performance across both datasets and approaches was achieved using graph embedding methods (deepwalk and harp). across all text embedding methods, we find that universal sentence encoder (use) consistently gives better performance . . analysis and discussion we find that the performance improvement across target embedding methods is inconsistent. we attribute this to the suitability of the target embedding for this task. we observe that the improvement is higher for low performing embeddings (such as elmo) as compared to high performing embeddings (such as harp). additionally, we observe that the proposed approach improves the performance while reducing the standard deviation across multiple target embedding methods. in a way, distant supervision reduces the impact of the choice of target embedding method. we also find that the performance improvement is not consistent across datasets. we also observe that the performance improvement is not consistent across all target embedding methods. we attribute this to the quality of target embeddings and the nature of evaluation dataset. an input phrase to a correct target concept which does not match with the corresponding human labeled concept. we find this to be the most common error type, table lists although our model shows relatively better performance across lengthy phrases, there is still a large scope for improvement. in general, we observed that distant supervision boosts the performance of our model in most cases. figure shows the t-sne representation of medical concepts and phrase embeddings obtained using the baseline and the proposed approach. we observe that the mcn model trained using distant supervision maps the medical phrases closer to their corresponding concepts. in this work, we address the problem of medical concept normalization (mcn) in social media which aims to map a variable length social media phrase to a medical concept in some external coding system. most of the current approaches pose mcn as a supervised text classification task which requires a large number of medical phrase, concept pairs for training. creating such a training dataset entails the painstaking task of manually identifying social media phrases and mapping them to one of the concepts in a medical lexicon. in our prior work [ ] , we have leveraged medical knowledge bases (such as snomed ct) to extract synonyms of medical concepts and use the synonym, concept pairs as training data. our model trained on snomed ct synonyms dataset alone shows a reasonable performance accuracy across multiple datasets. in this work, we extend this idea further by augmenting the snomed ct dataset with an automatically constructed distantly supervised dataset created from patient discussion forum posts. experimental results across multiple datasets show that the mcn model trained using the proposed approach outperforms the baseline by a significant margin. in future, we would like to increase the coverage of medical concepts in our distant data by obtaining multiple posts from a variety of medical social media forums. in our current work, we use generic pretrained models such as universal sentence encoders to measure the similarity between medical phrases and concepts. in future, we would like to use the model trained on snomed ct synonym dataset to select medical phrase, concept pairs. our current work explores the use of several text and graph based embeddings. it would be interesting to experiment with heterogeneous label embeddings. we would also want to experiment with graph convolutional networks [ ] to obtain target embeddings for medical concepts. in our current work, we use avgemb based approach to extract medical phrases (section . . ) from patient discussion forums. it would be interesting to see if we can further improve the performance of our medical phrase extractor (and thereby the performance of our mcn model) by using other sentence encoders discussed in section . . medical persona classification in social media pharmacovigilance from social media: mining adverse drug reaction mentions using sequence labeling with word embedding cluster features social media mining for public health monitoring and surveillance twitter social media is an effective tool for breast cancer patient education and support: patient-reported outcomes by survey take two aspirin and tweet me in the morning: how twitter, facebook, and other social media are reshaping health care medical concept normalization by encoding target knowledge medical concept normalization for online user-generated texts snomed-ct: the advanced terminology and coding system for ehealth normalising medical concepts in social media texts by learning semantic representation entity linking with a knowledge base: issues, techniques, and solutions adapting phrase-based machine translation to normalise medical terms in social media messages statistical phrase-based translation a character-level machine translation approach for normalization of sms abbreviations text normalization based on statistical machine translation and internet user support statistical machine translation based text normalization with crowdsourcing using an ensemble of generalised linear and deep learning models in the smm h medical concept normalisation task medical concept normalization in social media posts with recurrent neural networks multi-task character-level attentional networks for medical concept normalization deep neural models for medical concept normalization in user-generated texts pre-training of deep bidirectional transformers for language understanding concept extraction to identify adverse drug reactions in medical forums: a comparison of algorithms a robustly optimized bert pretraining approach decoupled weight decay regularization deep contextualized word representations efficient estimation of word representations in vector space deepwalk: online learning of social representations proceedings of the nd acm sigkdd international conference on knowledge discovery and data mining harp: hierarchical representation learning for networks line: large-scale information network embedding visualizing data using t-sne the stanford corenlp natural language processing toolkit the reuters corpus volume -from yesterday's news to tomorrow's language resources effective mapping of biomedical text to the umls metathesaurus: the metamap program medical subject headings (mesh) extracting medical concepts from medical social media with clinical nlp tools: a qualitative study cadec: a corpus of adverse drug event annotations a systematic approach for developing a corpus of patient reported adverse drug events: a case study for ssri and snri medications semi-supervised classification with graph convolutional networks nikhil pattisapu: conceptualization, writing -original draft preparation, methodology, software. vivek anand : data curation, resources, visualization. sangameshwar patil : validation, formal analysis, writing -reviewing and editing. girish palshikar : investigation, funding acquisition. vasudeva varma : supervision • medical concept normalization maps informal phrases to formal medical concepts.• training mcn models requires creating labeled datasets, which is effort intensive.• we overcome this problem by creating automatically labeled dataset from social media.• we augment our distant dataset with examples extracted from the snomed ct lexicon.• our approach significantly outperforms the previous state-of-the-art on two datasets. ☐ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests:we have no conflicts of interest to disclose. key: cord- -klewqw u authors: zhang, yan; xie, simiao; wang, pu; wang, guixiang; zhang, li; cao, xiaochen; wu, wenzhi; bian, yueran; huang, fei; luo, na; luo, mingyan; xiao, qiang title: factors influencing mental health of medical workers during the covid- outbreak date: - - journal: front public health doi: . /fpubh. . sha: doc_id: cord_uid: klewqw u background: since the outbreak of covid- , physical and psychological harm has been spreading across the global population alongside the spread of the virus. currently, the novel coronavirus has spread to most countries in the world, and its impact on the public is also increasing. as a high-risk group in direct contact with the virus, medical workers should be monitored, and their mental health deserves extensive attention. the aim of this study was to explore the mental health of medical workers facing the novel coronavirus and the main factors affecting it. methods: the present cross-sectional study including , eligible individuals from , hospitals in china was conducted through the network platform powered by www.wjx.cn, a platform providing functions equivalent to amazon mechanical turk. we used a self-designed questionnaire to collect demographic information and data on mental states, including gender, age (years), educational level, job rank, body and mind reaction, cognition of risk, and the judgment of the epidemic situation. independent samples t-tests and one-way (anova) analysis were carried out to compare the differences in the mental reactions according to the demographic and psychological states of the participants. results: there were males ( . %) and , females ( . %). the participants reported feeling calm ( . %), tense ( . %), scared ( . %), angry ( . %), sad ( . %), afraid ( . %), optimistic ( . %), impressed ( . %), and confident ( . %) during the epidemic. at the same time, the psychological stress responses of medical staff were significantly different according to the levels of exposure in their environments, duration and personal experience. conclusions: prolonged exposure to the virus and intense work are detrimental to the mental health of medical care personnel. it is necessary to adjust work conditions and intensity according to workers' mental state flexibly and systematically. at the end of , a large outbreak of disease that was widespread with a high speed and a large number of infected people broke out in wuhan ( , ) , hubei province, china. it spread quickly over a short period of time ( , ) , and it has been a serious threat not only to physical health ( ) but also to mental health issues throughout the population ( ) . since april, there have been no additional diagnoses for many days outside of hubei, china ( ) , and the number of additional infections in hubei has been largely in the double-digits, as if the chinese epidemic were about to end. however, since the outbreak of the global epidemic ( ) ( ) ( ) , the number of imported cases has been increasing continuously, making the slightly calmer mood tense once again. if the control of imported potential patients is not adequately strong to prevent the epidemic from spreading again, previous efforts could be in vain. according to the latest real-time statistics of johns hopkins university, as of : beijing time on march th, the cumulative number of confirmed cases of coronary pneumonia worldwide was more than , , and the cumulative number of confirmed cases outside china exceeded , . studying the novel coronavirus is not only a matter of fighting covid- in china but also an international public health crisis that needs to be fought by the whole world. since the outbreak of the epidemic, tension, anxiety and other negative emotions have spread throughout china on a large scale, so much so that people have fallen into a series of psychological crises ( ) . medical care personnel, as the backbone of the front line of epidemic prevention and control, have been taking on heavy work tasks with a high risk of infection and great work pressure ( ) . health-care workers, especially those in hospitals who take care of confirmed or suspected patients, are more vulnerable than the general population, experience a high risk of infection and negative emotional stress, and further risk spreading the virus to their family, friends or colleagues ( ) . moreover, dangerous and susceptible environments as well as traumatic experiences caused by high exposure can all have a certain impact on an individual's emotional state and induce emotional stress responses ( ) as well as severe anxiety and depressive disorders and posttraumatic stress disorder (ptsd, posttraumatic stress disorder) ( , ) . a psychological survey published in the lancet· psychiatry showed that the prevalence of depression, anxiety, insomnia and stress among medical staff involved in the prevention and control of the epidemic were as high as . , . , . , and . %, respectively ( ) . until now, despite the rudimentary principal notice issued by the china national health commission in january regarding the emergency psychological crisis intervention measures for covid- pneumonia, no one has been able to provide timely and effective psychological intervention measures for medical staff. therefore, it is urgent and important for psychological researchers to focus on the mental health problems of medical workers during the epidemic, explore the main factors affecting their psychological stability and health, and try to prevent longterm irreversible psychological trauma to medical workers. some scholars ( , ) in environmental psychology have studied the effects of the environment on the individual, especially in the face of danger. according to ecological theory, the individual behavior and environment are part of an interactive ecosystem, and individual behavior has a temporal and spatial background; that is to say, there is an integrated behavioral situation ( ) . for the same environmental phenomenon, arousal theory argues that the influence of the same spatial and temporal background on individuals is determined by the degree of arousal experienced by any particular individual ( ) . the level of arousal experienced by individuals is closely related to personal experience. inspired by this theory, this study attempted to investigate whether differences in the exposure environment, personal experience, and exposure duration of medical care personnel would lead to differences in their psychological responses, and advice and assistance were provided to personnel to prevent the development of mental health issues. the questionnaire was designed for medical workers from all provinces in china. in the formal test, , medical workers were selected from , hospitals in provinces to fill out the questionnaire, including in wuhan and , outside of wuhan; males and , females were included. among them, . % were under years of age, . % were aged - , . % were aged - , . % were aged - and . % were over years of age. the study was designed in accordance with the tenets of the declaration of helsinki. approval from the ethical authority of the school of educational science, huazhong university of science and technology, was granted. confidentiality and the statement confirming informed consent were managed by anonymous coding of the self-report questionnaires. this survey used wechat, online questionnaires and other online surveys to investigate the emotional and psychological stress states of medical staff. we used a selfdesigned questionnaire to collect demographics and mental state data including factors such as gender, age (years), educational level, job rank, body and mind reaction, cognition of risk, and the judgment of the epidemic situation, which was started in the third week after the outbreak, and the specific time is from february to february , . our team sent out questionnaires through the internet platform powered by www. wjx.cn, a platform providing functions equivalent to amazon mechanical turk. participants filled in the questionnaire on the web page through mobile phone or computer. first, information was collected through small-scale interviews; next, we compiled a stress response questionnaire and determined the questionnaire topics and factors through exploratory factor analysis (efa). data from subjects were collected as preliminary test through a web questionnaire with items, including in wuhan and outside wuhan, males and females. before the exploratory factor analysis, the results showed that the kmo (kaiser-meyer-olkin) measure of sampling adequacy was . (chi-square = . , df = , p < . ), and the bartlett's test of sphericity indicated that the correlation matrices on which the pca was based were suitable for analysis. according to the factor load matrix after the rotation axis, the analysis process of the items was as follows. first, delete three items with insufficient load and which are difficult to name on each factor; next, compare the load of each item on each factor, and delete three items with small load and similar load on different factors; third, analyze each factor, and delete the items with poor division and which are difficult to explain. as per the above principles, all nine items were retained and three factors were confirmed as the result, and the total variance was . %. the factors, which were named in turn, were cognition of danger (cd), reflecting the evaluation of the environmental risk of the subjects; judgment of the situation (js), reflecting the confidence in successfully combating the epidemic and the psychology of the anti-epidemic work; and the stress reaction (sr), reflecting the physical and mental stress response produced by the subjects' current environment. see table . after constructing a stress reaction questionnaire with good reliability and validity, we used confirmatory factors analysis (cfa) to confirm the validity of the questionnaire to provide a questionnaire that reflected the ideal standard. data from subjects were collected as cfa, including in wuhan and outside wuhan, males and females, and the fitting index tables and model diagrams drawn through amos software of cfa are shown in table and figure ; finally, we conducted a wide range of formal tests. the internal consistency reliability (cronbach α coefficient), partial reliability and the correlation between each factor score and the total score of the questionnaire were calculated by spss . , and the results showed that the overall internal consistency and reliability and the overall parity factor for both was . . see table . all data analysis was carried out using spss . (spss inc, chicago, illinois), and a two-tailed probability value of < . was considered to be statistically significant. descriptive statistics for the demographic and psychological states of the medical staff were shown as the mean, standard deviation (sd), number (n), and percentage. independent samples t-tests and one-way (anova) analysis were carried out to compare the differences in the mental reactions according to the demographic and psychological states of the participants. among the , subjects who filled in the questionnaire, the distribution was not uniform, and . % were doctors ( , in this study, the differences in the health care workers' environmental exposure were demonstrated mainly by whether they participated in the covid- resistance front and had direct contact with confirmed patients. there were significant table . the results show that those involved in the first-line response believed they were at greater risk of exposure to infection in the workplace(t = . , p < . ), and they had more anxiety about infection (t = . , p = . ), thought they were more likely to get sick (t = . , p < . ), worried more about family infection (t = . , p = . ), had lower confidence in obtaining victory over the epidemic (t = . , p = . ), had poor sleep quality (t = . , p < . ) and had a higher demand for psychological counseling (t = . , p < . ). however, there were no significant differences for the cognition of the current epidemic severity and the fear of epidemic prevention. the differences in health care workers' personal experiences were affected mainly by whether they had experienced sars or another epidemic. there were significant differences in the levels of cognition of danger, judgment of their situation and stress reactions to personal experiences. specific statistical results for the medical care personnel and group comparisons are displayed in table , which shows that medical staff involved in sars prevention believed they had a greater risk of exposure to infection in the workplace (t = . , p = . ), were more likely to be infected (t = . , p = . ), had more confidence in the success in epidemic prevention and control (t = − . , p = . ), less fear of fighting the epidemic (t = − . , p = . ), and poor sleep quality (t = . , p = . ). however, there were no significant differences for the items regarding being worried about getting infected, the cognition of the current epidemic severity and the need for psychological counseling. since the outbreak of the epidemic, medical workers have been stressed and made to work for long periods of time, with little time for rest. the difference in exposure duration was reflected mainly by the number of continuous working days. this study compared the differences in the duration of the participants' operational time in medical care work and divided the working hours into four levels for horizontal comparison, which found that the longer the working hours were, the more likely the participants believed they would be infected (f = . , p < . ), the more worried they were about family members being infected (f = . , p < . ), and the poorer their sleep-quality was (f = . , p < . ). however, the fear of epidemic prevention was lower (f = . , p < . ). furthermore, there were significant fluctuations in two dimensions, cognition of the current epidemic severity (f = . , p = . ) and confidence in anti-epidemic measures (f = . , p < . ), caused by the increase in working hours, which at first declined a certain degree, then increased significantly. see table . since the emergence of the new coronavirus pneumonia in wuhan at the end of december , numerous medical staff have been working intensively for nearly months and will continue to do so in the future. the results showed that the current mental health status of health care workers was not stable, with a general mean of more than . in terms of the cognition of danger, and most of the mean values were above (according to richter's five-point score, which gradually declined from to ). regarding the dimensions of the judgment of the situation and the stress reaction, the medical staff were optimistic, and there was no obvious negative somatization phenomenon. it was found that the exposure environment, personal experience, and exposure duration had significant effects on the psychological stress and emotional responses of medical staff. medical workers involved in the front-line of prevention were affected to different degrees in these three dimensions, and the statistical level was significantly different. this may be due to direct exposure to close contact with the virus and negative tension in their environment as well as the fear of threats to their own lives. additionally, the medical work environment is infested with patients' senses of grief and panic, resulting in a constant psychological burden for front-line medical workers. at the same time, there is no clear and targeted cure for the novel coronavirus infection. doctors and nurses are not in a position to cope with the suffering of infected patients, which is further increasing their psychological burden. the influence of medical workers who have experienced sars and other epidemic diseases was not synchronized in these three dimensions. in the dimension of the cognition of danger, employees with experience of sars and other epidemic prevention situations felt more serious psychological pressure, while for the dimension of the judgment of the situation, they had more confidence about overcoming this epidemic. this may be explained by the success of the prevention and control of infectious diseases like sars, which has enhanced the collective sense of the efficacy of health care groups in the face of similar diseases, thus enhancing their confidence. however, the difficulties of living through that process and the negative emotions experienced are difficult to describe, and the impact has not gradually disappeared over time. the outbreak of the epidemic quickly awakened the former unhappy memory, so the iteration and development of risk cognition were derived from a certain preexisting foundation. this is also a wake-up call for psychological workers to remind us to do a good job of psychological intervention and health care even after illness. as the time of exposure to the virus increases, the mental state of the medical staff deteriorates. regarding the factor of risk cognition, the negative psychological state of the medical staff gradually intensifies with the passage of time, whereas the optimistic hope dimension presents the inverted u curve change. in the physical and mental response dimension, the sleep-quality of the medical staff is generally poor, but the difference in the level of demand for psychological counseling is not significant. this may be because, in the early days of the outbreak, a large number of patients poured into hospital emergency rooms and fever outpatient departments, increasing the already heavy workload and responsibility of all medical staff. meanwhile, the high intensity of work continued without rest, there were inadequate protective supplies and protective isolation measures, the outpatient procedure organization became cluttered, and other phenomena have continually aggravated the psychological burden of medical staff, reducing the confidence of medical workers in prevention and control. as the epidemic situation gradually comes under control, medical work tends to stabilize, so the confidence in prevention and control has been steadily recovered. however, the negative feelings of health care workers have not been effectively vented, such as the grievances, fears, and powerlessness of medical staff in the face of dissatisfaction from patients and their families because of the lack of timely treatment. the inner suffering cannot slowly dissipate over time. by contrast, it is highly likely that the backlog of negative emotions causes some mental health issues, especially ptsd, requiring the attention of psychological workers. ptsd usually occurs within a few weeks of traumatic events but can also appear after a few months or even a few years, and the duration is usually half a year or more ( ) ( ) ( ) , depending on the severity of the event and the individual state of mind ( , ) . the current trend of the epidemic situation in china has been obviously controlled, and the tension of the medical staff can be relaxed in stages, which is the best time for online psychological guidance. moreover, the outbreak of foreign epidemics is rapid, and many countries lack the experience of prevention and control. china plans to send some supportive medical workers to countries where the epidemic is ongoing. the relief of tension is about to face new challenges, and it is essential to effectively perform psychological intervention and regulation for medical staff. both chinese and international mental health workers must pay attention to this problem and stabilize psychological security ( , ) . this study found that the psychological state of medical workers was significantly affected by the high-risk environment of direct contact infection, long working hours, and personal experiences. however, the only factors that can be controlled are the working environment and working hours. the authors suggests the establishment of a matching system between the psychological state and the working intensity of medical staff; after all, only upon a foundation of psychological security can the work be completed efficiently. the psychological security work needs to be carried out in a systematic and hierarchical manner from the local level to a more general investigation by utilizing close attention to ensure that every corner of the mental health of medical staff is explored. first, based on the overall comprehensive investigation, a medical staff psychological state tracking system should be established. second, all mental state files should be classified into attention levels, such as core, focus, general attention, etc. meanwhile, each health worker will be assigned a psychologist who is responsible for paying regular attention to their mental health problems. psychological workers need to evaluate whether the medical staff 's work schedule matches their psychological status and periodically review their appropriate work intensity level. finally, specific psychological interventions need to be carried out for all health workers who are marked as working at a certain level of focus and above by recording any incidents in their mental state file. by investigating the emotional and psychological stress responses of medical staff during the prevention and control of the new coronary pneumonia, it was found that the high intensity of medical work had a variety of negative effects on their risk cognition, confidence in overcoming the epidemic situation and physical and mental reactions, all of which are detrimental to the mental health of medical staff. in addition, the exposed environment, personal experiences and differences in the length of their work hours played important roles. to maintain the mental health and stability of medical staff and avoid the influence of mental health issues like ptsd, psychological workers need to take targeted measures to systematically solve the mental health problems of medical workers in the face of major infectious disease crises. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. approval from the ethical authority of the school of educational science, huazhong university of science and technology, was granted. confidentiality and the statement confirming informed consent were managed by anonymous coding of the self-report questionnaires. the patients/participants provided their written informed consent to participate in this study. yz, pw, and lz conceived and designed the questionnaire. lz recruitment and payment of participants. sx, gw, xc, yb, fh, nl, ml, and qx analyzed the data. sx wrote and revised the paper. all the authors have approved the manuscript and agreed with submission to your esteemed journal. a novel coronavirus outbreak of global health concern clinical features of patients infected with novel coronavirus in wuhan travelers give wings to novel coronavirus ( -ncov) incubation period of novel coronavirus -ncov infections among travellers from wuhan, china -novel coronavirus outbreak: a new challenge timely mental health care for the novel coronavirus outbreak is urgently needed will novel virus go pandemic or be contained? journey of a thai taxi driver and novel coronavirus importation and human-to-human transmission of a novel coronavirus in vietnam community pharmacist in public health emergencies: quick to action against the coronavirus -ncov outbreak vicarious traumatization in the general public, members, and non-members of medical teams aiding in covid- control theoretical perspectives of traumatic stress and debriefings long-term psychiatric morbidities among sars survivors online mental health services in china during the covid- outbreak environmental psychology empirical research in environmental psychology: past, present, and future an ecological perspective on theory, methods, and analysis in environmental psychology: advances and challenges a problem for cognitive load theory-the distinctively human lifeform a systematic review of ptsd prevalence and trajectories in dsm- defined trauma exposed populations: intentional and non-intentional traumatic events perception counts: the relationships of inner perceptions of trauma and ptsd symptoms across time post-traumatic stress disorder and somatization symptoms: a prospective study emotional distress and positive and negative memories from military deployment: the influence of ptsd symptoms and time does acute stress disorder predict post-traumatic stress disorder in traffic accident victims? analysis of a self-report inventory mental health and psychosocial support in crisis and conflict: report of the mental health working group crisis exploitation: political and policy impacts of framing contests we thank all participants for their time and interest as well as the editor and reviewers for their valuable feedback. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © zhang, xie, wang, wang, zhang, cao, wu, bian, huang, luo, luo and xiao. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -de o q authors: bagshaw, michael; nicolls, deborah j. title: aircraft cabin environment date: - - journal: travel medicine doi: . /b - - - - . -x sha: doc_id: cord_uid: de o q nan the physiology of the human being is optimized for existence at sea level. most individuals, however, can ascend to around - ft ( - m) above sea level before hypoxia begins to have ill effects and reduces performance. with increasing altitude, there is a fall in the atmospheric pressure, together with a decrease in density and temperature. the pressure at sea level in the standard atmosphere is mmhg and this falls to half at ft ( m), where the ambient temperature is about − °c. the composition of the atmosphere remains constant up to the tropopause ( ft or m), the most abundant gases being nitrogen ( %) and oxygen ( %), with the remaining % being argon, carbon dioxide, neon, hydrogen and ozone. the relationship between the oxygen saturation of hemoglobin and oxygen tension minimizes the effect on the human of the reduction in partial pressure of oxygen. ascent to an altitude of ft ( m) produces a fall in the partial pressure of oxygen in the alveoli, but only a slight fall in the percentage saturation of hemoglobin with oxygen. once altitude exceeds ft ( m), however, the percentage saturation of hemoglobin falls quickly, resulting in hypoxia. indeed, above ft ( m), the effects of lack of oxygen will begin to appear and a decrease in an individual's ability to perform complex tasks and a reduction in night vision can be measured. figure . shows the oxygen dissociation curve of blood. the concentrations of physically dissolved and chemically combined oxygen are shown separately and the curve illustrated is the average for a fit young adult. the actual shape of the curve will be influenced by factors such as age, state of health, tobacco use and ambient temperature. healthy individuals can tolerate altitudes of up to - ft ( - m) with no harmful effects. however, in the case of the elderly or of individuals suffering from some diseases of the respiratory or circulatory system, there is less tolerance of the mild hypoxia at even this altitude. in an ideal world, the cabin would be pressurized to maintain sea level conditions. to achieve this would require an extremely strong and heavy aircraft structure with severe implications on load carrying capacity, fuel consumption and resulting effects on the external environment. as a result, a compromise has to be struck, and airworthiness regulations (us federal aviation regulations and european joint aviation requirements) state that 'pressurized cabins and compartments to be occupied must be equipped to provide a cabin pressure altitude of not more than ft ( m) at the maximum operating altitude of the aeroplane under normal operating conditions.' pressurization is achieved by tapping bleed air from the engine compressors and passing this flow of air through the air-conditioning packs into the cabin. the outside air is very dry and cold, and the temperature is controlled via the air-conditioning packs. the cabin pressure is maintained at the desired level by regulating the flow of air overboard. figure . illustrates ambient and cabin altitudes for a typical flight. section as a result of the required cabin pressure change during climb and descent, it is possible for individuals to suffer discomfort as a result of expansion of gas trapped within the body. in particular, gas can be trapped within the gut and within the middle ear and sinuses. normally, this trapped gas is able to escape without any problem, but there may be occasions when this is not so, such as when the individual is suffering from upper respiratory tract congestion due to infection or allergy. in particular, the human ear is very sensitive to rates of pressure change, the threshold for detection being . psi ( . kpa). this is equivalent to a change in cabin altitude of ft ( m) at sea level. in the human ear, the cavity of the middle ear is separated from the outer ear by the tympanic membrane. it communicates with the nasopharynx and, in turn, the atmosphere by way of the eustachian tube, the proximal two-thirds of which has soft walls that are normally collapsed. during ascent to altitude, the gas in the middle ear cavity expands and escapes along the eustachian tube into the nasopharynx, equalizing the pressure across the tympanic membrane. the pharyngeal portion of the eustachian tube acts as a one-way valve, thus allowing expanding air to escape easily to the atmosphere. this can be sometimes felt as a 'popping' sensation as air escapes from the tube during ascent. during descent, air from the nasopharynx must enter the middle ear to maintain equilibrium. in some individuals, the one-way valve mechanism of the eustachian tube can prevent passive flow of air back into the middle ear cavity. this causes a relative increase of pressure on the outside of the tympanic membrane, pushing it into the middle ear cavity and causing a sensation of fullness, a decrease in hearing acuity and eventually pain. it is possible to perform active maneuvers to open the eustachian tube, such as swallowing, yawning, and jaw movements. in some people, these simple maneuvers are not effective and it may be necessary to occlude the nostrils and raise the pressure in the mouth and nose to force air into the middle ear cavities. this increase in pressure can usually simply be achieved by raising the floor of the mouth with the glottis shut, while other individuals raise the pressure in the lungs and the respiratory tract by contracting the expiratory muscles while forcibly exhaling (valsalva's maneuver). in addition to regulating the airflow rate required to pressurize the aircraft, the environmental control system controls the flow rate of outside air required to remove contaminants and controls the temperature in the cabin. this requirement is facilitated by the practice of recirculation of approximately % of the cabin air. this is achieved by extracting air from the cabin and mixing it with conditioned outside air. recirculation provides two benefits: one, it allows the total airflow rate to be higher than the flow rate of the outside air, so good circulation in the cabin can be maintained independently of the outside airflow; and two, the conditioned air is mixed with comparatively warm recirculated air before being introduced into the cabin. as a result, the conditioned air is supplied at a much lower temperature without causing discomfort from cold drafts. the recirculated air will also have picked up moisture from the cabin occupants and the cabin activities, improving the humidity level. in older generation jet aircraft, all the air supplied to the cabin came from outside air, without the benefits of recirculation (improved humidity, reduction in perceived drafts). this practice was inefficient with a substantial energy, and hence environmental, cost. in pressurized jet aircraft manufactured since the beginning of the s, the recirculated air is passed through filters. these are high efficiency particulate (hepa) filters, which have an efficiency of . % for . μm particles. they are effective in removing bacteria and viruses from the recirculated air, so preventing their spread through the cabin by this route. air filters are changed during routine aircraft maintenance, as specified by the manufacturer in the servicing schedule. recirculated air is obtained from the area above the cabin or under the floor; air from the cargo bay, lavatories and galleys is not recirculated. the flow rate of outside air per seat ranges from . to . l/s ( . - . cubic ft/min) with the percentage of recirculated air distributed to the passenger cabin being of the order - % of the total air supply. the result of filtering the recirculated air is a significant improvement in cabin air quality by the removal of particles and biological microorganisms. it is not necessary to pass the compressed air from outside through hepa filters, because the ambient air at high altitude is free of microorganisms and particulates. the use of recirculation has been common in the design of building environmental control systems for many years. building environmental systems are commonly designed and operated with up to % of recirculated air, which compares with the maximum recirculated air flow in aircraft of %. the air supply to the flight deck (or cockpit) is derived from the same source, but is delivered at a slightly higher pressure than the air supplied to the cabin. this ensures a positive pressure differential to prevent the ingress of smoke or fumes to the flight deck in the event of a fire or similar in-flight emergency. the flow rate of the flight deck air supply is also slightly higher than that to the cabin because this supply is used for cooling the avionics and other electronic equipment. humidity is the concentration of water vapor in the air. relative humidity is the ratio of the actual amount of vapor in the air to the amount that would be present if the air was saturated at the same temperature, expressed as a percentage. saturated air at high temperatures holds more water vapor than at low temperatures, and if unsaturated air is cooled, it becomes saturated. high humidity can lead to passenger and crew discomfort when it is accompanied by high temperature. high humidity can cause condensation, dripping and freezing of moisture on the inside of the aircraft shell, which may lead to safety problems such as corrosion. condensation can also give rise to biological growth thus causing adverse effects on cabin air quality. at a typical aircraft cruising altitude of ft ( m), the outside air temperature is in the region of − °c and is extremely dry, typically containing about . g/kg of moisture. for pressurized aircraft flying at these levels, the conditioned air entering the cabin has a relative humidity of < %. exhaled moisture from passengers and crew, together with moisture from galleys and toilet areas, increases the humidity to an average level of - %, which is below the % normally accepted as comfort level. research has shown that the maximum additional water lost from an individual during an -h period in % humidity, compared with normal day-to-day loss, is around ml. the sensation of thirst experienced by healthy individuals in the low humidity environment is due to local drying of the pharyngeal membranes, and this itself may lead to the spurious sensation of thirst. there is no evidence that exposure to a low-humidity environment itself leads to dehydration, although local humidity can cause mild subjective symptoms, such as dryness of the eyes and mucous membranes. , no significant effect has been shown on reaction time or other measures of psychomotor performance, although there can be some changes in the fluid regulatory hormones. , it is unlikely that low humidity has any long-or short-term ill-effects, provided overall hydration is maintained by drinking adequate amounts of fluid. the body's homeostatic mechanisms ensure that central hydration is maintained, although the peripheral physical effects can lead to discomfort. dry skin can be alleviated by using moisturizing aqueous creams, particularly just before flight, and dry eye irritation can be alleviated by the use of moisturizing eye drops. individuals prone to develop dry eyes are advised not to wear contact lenses during long flights in pressurized aircraft. a national academy of sciences report identified humidity as one of the areas deserving more attention in future research concerning aircraft cabin environment. the aircraft cabin is similar to many other indoor environments, such as homes and offices, in that people are exposed to a mixture of external and recirculated air. the cabin environment is different in many respects (e.g. the high occupant density, the inability of the occupants to leave at will and the need for pressurization). in flight, there is a combination of environmental factors including low air pressure and low humidity, as well as low frequency vibration and constant background noise. although the noise and vibration can contribute to fatigue, the levels are all below those which are accepted as potentially harmful to hearing. , ozone ozone is a highly reactive form of oxygen found naturally in the upper atmosphere. it is formed primarily above the tropopause as a result of the action of uv light on oxygen molecules. the amount and distribution of natural ozone in the atmosphere varies with latitude, altitude, season and weather conditions. the highest concentrations in the northern hemisphere are generally found at high altitude over high latitude locations during the winter and spring. the effects of high ozone concentration on human beings can include eye irritation, coughing due to irritation of the upper respiratory system, nose irritation and chest pains. as a result of this, the airworthiness regulatory authorities (e.g. us federal aviation authority, european joint aviation authorities) require that transport category aircraft operating above ft ( m) must show that the concentration of ozone inside the cabin will not exceed . parts/million by volume (sea level equivalent) at any time, and a time weighted value of . parts/million by volume (sea level) for scheduled segments of > h. for this reason, long haul transport jet aircraft are now equipped with ozone catalytic converters that break down or 'crack' the ozone before it enters the cabin air circulation. natural radiation consists of cosmic rays from outer space (galactic radiation) and the gamma rays from rocks, earth, and building materials. cosmic radiation is produced when primary photons and alpha particles from outside the solar system (galactic cosmic radiation) interact with components of the earth's atmosphere. a second source of cosmic radiation is the release of charged particles from the sun, which becomes significant during periods of solar flare ('sun storm'). cosmic radiation is an ionizing radiation; ionizing radiation also includes x-rays and that from radioactive materials. ionizing radiation is a natural part of the environment in which we live and is present in the earth, buildings, food we eat, and even in the bones of our bodies. the other type of radiation is known as non-ionizing radiation and this includes uv light, radio waves and microwaves. humans, animals and plants have all evolved in an environment with a background of natural radiation and, with few exceptions, it is not a significant risk to health. the amount of cosmic radiation that reaches the earth from the sun and outer space varies and depends on the latitude and height above sea level. the amount of cosmic radiation entering the atmosphere follows an -year cycle, with the intensity of galactic radiation being lowest when solar activity is at its highest. this is because during high levels of solar activity, the resulting magnetic flux between the sun and the earth deflects much of the galactic cosmic radiation. cosmic radiation is effectively absorbed by the atmosphere and is also affected by the earth's magnetic field. the effect on the body will depend on the latitude and altitude at which the individual is flying and also on the length of time in the air. cosmic radiation may be measured directly using sophisticated instruments, as was done in the concorde supersonic transport and subsonic long range aircraft, or can be estimated using a computer software program. these programs look at the route, time at each altitude and the phase of the solar cycle, and calculate the radiation dose received by the aircraft occupant for a particular flight. a number of airlines and research organizations have compared actual measurements taken on board an aircraft with the computer estimations, and the two are very similar. the effect of ionizing radiation depends not only on the dose absorbed, but also on the type and energy of the radiation and the tissues involved. these factors are taken into account in arriving at the dose equivalent measured in sieverts (sv). however, doses of cosmic radiation are so low that figures are usually quoted in microsieverts (μsv, millionths of a sievert) or millisieverts (msv, thousandths of a sievert). when ionizing radiation passes through the body, energy is transmitted to the tissues, which affects the atoms within the individual cells. very high levels of radiation, such as that from a nuclear explosion, will cause severe cell damage in a human being, particularly to the bone marrow cells and the reproductive cells, which cannot be repaired by the body. low-level doses of radiation, such as cosmic radiation or medical x-rays, do not cause such severe damage to the cells and in most cases, any such damage is repaired satisfactorily by the body's own mechanism. it is not possible to predict a maximum safe threshold of exposure to low levels of radiation, because individuals vary in their biological response. , the international commission for radiological protection (icrp) recommends maximum mean body effective dose limits of msv/ year (averaged over years) for workers exposed to radiation as part of their occupation (including flight crew), and msv/year for the general population, with an additional recommendation that the equivalent dose to the fetus should not exceed msv during the declared term of pregnancy. when the concorde supersonic transport aircraft was flying, the effective dose rate for the occupants at cruising altitude was measured to be in the range of - μsv/h. on ultra long-haul flights at high latitudes, such as a boeing - flying between london and tokyo, the effective dose rate at cruising altitude is around μsv/h. on short-haul commercial operations, the effective dose rate in europe is in the region of - μsv/h. for typical annual flight schedules, crew members accumulate around or msv/year operations, and between and msv/year on european short-haul operations from cosmic radiation. for airline passengers, the icrp recommended limit for the general public of msv/year equates to about flying hours/year on the trans-equatorial routes. there are essentially two types of airline passenger: the occasional social traveler and the frequent business traveler. the public limit ( msv/year) will be of no consequence to the social traveler but could be of significance to the frequent business traveler. the msv annual limit would be exceeded if the business traveler were flying more than eight transatlantic or five antipodean return journeys per year. business travelers are exposed as an essential part of the occupation, and it is entirely logical to apply the occupational limit of msv/year to this group. cosmic radiation is of no significance at altitudes below about ft ( m) because of the attenuating properties of the earth's atmosphere. there is no evidence from epidemiological studies of flight crew of any increase in incidence of cancers linked to ionizing radiation exposure, such as leukemia. in general terms, as far as the risk of developing cancer induced by radiation exposure is concerned, it has been calculated that an accumulated dose of msv/year for years increases the risk of developing cancer (in the general population) from - . %, i.e. an increase of risk of . % over years. compared with all the other risks encountered during a working life, this is very low. cosmic radiation is both a complex and emotive subject. it cannot be seen, touched, smelled or tasted and yet it is present all around us. while it is known that there is no level of radiation exposure below which effects do not occur, all the evidence indicates that there is an extremely low probability of airline passengers or crew suffering any abnormality or disease as a result of exposure to cosmic radiation. the world health organization and the international civil aviation organization recommend that aircraft arriving through countries reporting certain indigenous infectious diseases, be treated with pesticides. this remains a controversial issue. while it is understandable that countries such as new zealand would not want to risk the entry of certain vector-borne diseases transmitted by mosquitoes, there remains concern about the safety and efficacy of various pesticides used, particularly while passengers are in the cabin (top of descent spraying). there appears to be less concern about using residual pesticides during routine aircraft maintenance. at this time, however, the us environmental protection agency has not promoted the registry of any pesticides in the usa for use in aircraft disinsection on american carriers. as vector-borne diseases re-emerge, however, these policies may need re-evaluation in order to prevent the transmission of disease. further education of air crew and passengers will be needed, and these issues are being reviewed. there are five main routes by which microorganisms may be transmitted to humans: contact (direct and indirect), droplet, air-borne, common-vehicle, and vector-borne. all of these modes have been implicated, at least anecdotally, in the transmission of disease aboard an aircraft; [ ] [ ] [ ] [ ] however, there has been increasing concern over the respiratory spread of infections during air travel, via droplet and air-borne transmission. droplet transmission may occur whenever a person coughs, sneezes, or talks. droplets are relatively large particles (> μm) that can travel only a short distance through the air; they do not remain suspended. infection occurs when microorganisms within the droplets come into contact with the conjunctivae, nasal mucosa, or mouth of a susceptible person. in air-borne transmission, smaller respiratory particles (≤ μm), called droplet nuclei, are inhaled by a susceptible host. these particles remain suspended in air indefinitely and may spread over long distances, depending on environmental factors. humans are the most important reservoirs of infectious agents on aircraft. most microorganisms that have been isolated from occupied spaces, including aircraft cabins, are human in source, including bacteria that have been shed from exposed skin and scalp, as well as from the nose and mouth. these microorganisms are typically part of the normal human flora and very rarely cause infections. studies have shown no statistically significant differences in the concentrations of bacteria and fungi sampled from the aircraft cabin: ▪ among different aircraft, airlines or flight durations ▪ between aircraft cabins and other types of public transport vehicles ▪ between aircraft cabins and typical indoor and outdoor urban environments. when microorganisms are spread by droplet transmission, the risk of acquiring infection is highest for the passengers seated closest to a source person, typically within feet. in contrast, microorganisms that are spread by air-borne transmission potentially could distribute throughout the entire aircraft cabin. in reality, however, this does not occur. microorganisms suspended in cabin air are removed by the high efficiency particulate (hepa) filters during the air recirculation process; unfortunately, these filters provide no protection from the cough or sneeze emitted by an infected neighbor. the natural or acquired immunity of most individuals prevents the development of infectious disease. limited data are available on the true risk of disease transmission during air travel. studies of potential infectious disease transmission on aircraft have considered tuberculosis, influenza, measles, meningococcal disease, sars, and acute respiratory infections, such as the common cold. , tuberculosis the transmission of mycobacterium tuberculosis (tb) during air travel has been most extensively studied. tb is transmitted by the inhalation of droplet nuclei (air-borne transmission). two of six investigations conducted by the centers for disease control and prevention (cdc) between and have indicated that the transmission of tb from a symptomatic person to other passengers or crew members does occur during air travel. , in one investigation, a flight attendant was diagnosed with cavitary pulmonary tuberculosis in november, . of the flight crew who worked with this flight attendant between august and october, , . % had a positive tuberculin skin test (tst) result. two of these contacts had documented conversion of tst status. tst positivity and conversion was associated with a cumulative flight-time exposure of h or more. in the second investigation, a foreign-born passenger developed symptomatic pulmonary tuberculosis while visiting friends in the usa. she traveled from baltimore, maryland to chicago, illinois ( h flight) and then from chicago to honolulu, hawaii ( h and min flight). three of the passengers on the baltimore to chicago flight had a positive tst, two were foreign-born. fifteen of the passengers on the flight from chicago to honolulu had a positive tst. four of these passengers converted their tst. in both investigations, all of the new infections were latent tb infection; none developed active tuberculosis. the hepa filters used in newer commercial aircraft are able to filter out tb bacteria from the recycled air and are also used in hospital respiratory isolation rooms to prevent the spread of tb within the hospital setting. the number of air exchanges per hour in aircraft exceeds the number recommended for hospital isolation rooms. furthermore, the prevalence of transmissible tuberculosis among air travelers is estimated to be - / passengers, depending on the route of the plane. the risk of tb transmission on a commercial aircraft, therefore, remains low. mathematical models estimate that the chance of acquiring tb during air travel while sitting near a highly infectious source is approximately in . human influenza virus is spread by droplet and air-borne transmission. one of the first well-documented outbreaks of influenza on a commercial airline was published by moser and colleagues in . thirty-eight of passengers and crew members became ill after exposure to a passenger with symptomatic influenza infection. another two passengers showed serological evidence of infection without becoming ill. the outbreak was attributed to an engine malfunction, which required the aircraft to remain on the runway for approximately . h without adequate ventilation. most of the passengers remained on the plane during the delay, and the clinical attack rate varied with the amount of time spent aboard the aircraft. another outbreak of presumed influenza that was associated with air travel occurred among workers traveling from a remote mine in northwestern australia. one worker became symptomatic with an influenza-like illness prior to the flight, which lasted h and min. over the - days that followed the flight, workers presented with similar complaints and were unable to work. an additional five workers reported upper respiratory tract symptoms when questioned but were able to continue working. there was no ventilation system malfunction reported for the aircraft associated with this outbreak. a significant limitation of this report is the lack of etiologic confirmation. since , a new strain of avian influenza virus (h n ) has been responsible for a number of outbreaks involving a large number of birds, and rarely, humans. from december -february , laboratory-confirmed human infections were identified in asia and se asia, and have primarily been due to direct contact with poultry. , limited, non-sustained person-to-person spread has also been described in a few cases. as with sars, the airline industry will look to public health authorities for guidance in the event that an (avian) influenza pandemic should occur. the severe acute respiratory syndrome (sars) was first identified in southern china in november, , and recognized as a global threat by march, . it was caused by a novel coronavirus, the sars-associated coronavirus, and was spread primarily via droplet transmission, although spread by air-borne and contact transmission could not be ruled out. despite the clear role of international travel in the spread of sars during the outbreak, , there had been only one case of likely in-flight transmission. limited transmission probably occurred aboard aircraft, but the risk was felt to be low. fortunately, no suspected cases of sars have been reported since may, . neisseria meningitidis is a leading cause of meningitis and sepsis in children and young adults and is spread by droplet transmission. meningococcal disease has been documented in travelers, particularly those traveling for the hajj; however, transmission due to exposure while aboard an aircraft has not been definitively documented. guidelines for the management of airline passengers who have been exposed to meningococcal disease are based on information available from investigations of tb transmission aboard aircraft. us guidelines are online and available at: http://www.cdc.gov/travel/menin-guidelines.htm measles is a highly contagious viral disease that is spread by air-borne transmission. a person infected with measles is contagious from the first onset of vague symptoms (up to days before rash appears) to approximately days after the development of rash; therefore the potential for disease transmission during air travel exists. from to , of the cases of imported measles into the usa developed a rash directly before or on the day of air travel. from these imported cases, only one documented case of in-flight transmission was reported. despite the risk, few cases of measles occur as a direct result of inflight exposure. , amornkul and colleagues investigated the extent of measles transmission aboard an international commercial flight after exposure to an infected passenger. of the potentially exposed passengers, % completed a follow-up questionnaire, including passengers who reported close contact with the ill passenger before, during, or after the flight. none of the contacted passengers reported symptoms consistent with measles. available data indicate that infectious agents can be transmitted from person-to-person aboard an aircraft on the ground and during flight, just as they can in any other situation where people find themselves in close proximity. from investigations of disease outbreaks associated with air travel, two main risk factors for the spread of communicable diseases have been identified: flight duration (> h, including ground time) and seating proximity to the source. there is no evidence that the pressurized cabin itself makes transmission of disease any more likely. once the aircraft doors are closed, airconditioning is provided from the auxiliary power unit until it can be supplied from the aircraft engines, thus ensuring the filtration benefits of air recirculation. there does appear to be increased risk of disease spread when the aircraft ventilation system is inoperable. guidelines are in place to assist flight crew in the management of ill passengers and can be accessed from www.who.org, www.cdc.gov, or www.iata.org. in general, good respiratory and hand hygiene practices should always be encouraged, and people with febrile illnesses should postpone air travel whenever possible. aircrews, particularly of international fights, are at risk for the same illnesses to which other travelers are exposed. because of their very short-term stays, and stays that are generally in major cities, they may not be as intensely exposed to some illnesses as others. however, their lifestyle may predispose them to other risks. because of their frequent international travel, flight crews should receive health recommendations that are specifically geared to their travel patterns, itineraries, and lifestyles. for example, an international pilot flying frequently to sub-saharan africa would do well not to take routine anti-malarial medication, but should take precautions to avoid insect bites, may carry a self-treatment regimen if desired and if the layovers are extended, and should be instructed to seek medical attention immediately if a fever occurs. the medical department of international carriers should either be able to provide good information and appropriate immunizations to aircrew, or should have an arrangement with a travel clinic for appropriate education of flight crews. other than transmission of infectious agents, flight crews have other health concerns that though briefly mentioned here, are best reviewed in an occupational health setting. issues that have surfaced with regard to pilot health have included the safety of refractive surgery, the use of serotonin re-uptake inhibitors, safety of pilots to fly with insulin-dependent diabetes mellitus or even with the human immunodeficiency virus, as well as issues related to pilot fatigue. flight attendants' concerns have ranged from reproductive health problems, menstrual disorders, chronic back pain, to fears of an increased risk of breast cancer. epidemiological studies have shown an increased prevalence of malignant melanoma among flight crew members. this is thought to be a result of a lifestyle, allowing exposure to sunlight that is perhaps increased during layover periods. flying as a passenger should be no problem for the fit, healthy, and mobile individual. but for the passenger with certain pre-existing conditions, the cabin environment may exacerbate their underlying problems. although many problems relate to the physiological effects of hypoxia and expansion of trapped gases, it should be remembered that the complex airport environment can be stressful and challenging to the passenger, leading to problems before even becoming air-borne. although passengers with medical needs require medical clearance from the airline, passengers with disabilities do not. disabled passengers do need to notify the requirement for special needs, such as wheelchair assistance or assignment of seats with lifting armrests, and this should be done at the time of booking. an area of little exploration has been air travel stress and anxiety. whereas typical issues such as fear of flying have been addressed by the airlines in the past, problems that have surfaced related to terrorism and airline security have increased. passenger lines, terminal crowding, lesser personal services due to cost-cutting, etc. have taken a toll on the ease in which air travel is currently perceived. addressing these potential problems with the air traveler, particularly with those who have any underlying illness or general anxiety, is helpful. the objectives of medical clearance are to provide advice to passengers and their medical attendants on fitness to fly, and to prevent delays and diversions of the flight as a result of deterioration in the passenger's well-being. it depends upon self-declaration by the passenger, and upon the attending physician having an awareness of the flight environment and how this may affect the patient's condition. most major airlines provide services for those passengers who require extra help, and most have a medical advisor to assess the fitness for travel of those with medical needs. individual airlines work with their own guidelines, but these are generally based on those published by the aerospace medical association on fitness for travel. (copies of the asma guidelines may be obtained from the association online at: www.asma.org or tel: .) the international air transport association (iata) publishes a recommended medical information form (medif) for use by member airlines (fig. . ) . the medif should be completed by the passenger's medical attendant and passed to the airline, or travel agent, at the time of booking to ensure timely medical clearance. medical clearance is required when: ▪ fitness to travel is in doubt as a result of recent illness, hospitalization, injury, surgery or instability of an acute or chronic medical condition ▪ special services are required (e.g. oxygen, stretcher or authority to carry or use accompanying medical equipment such as a ventilator or a nebulizer). medical clearance is not required for carriage of an invalid passenger outside these categories, although special needs (such as a wheelchair) must be reported to the airline at the time of booking. cabin crew members are unable to provide individual special assistance to invalid passengers beyond the provision of normal in-flight service. passengers who are unable to look after their own personal needs during flight (such as toileting or feeding) will be asked to travel with an accompanying adult who can assist with these needs. it is vital that passengers remember to carry with them any essential medication, and not pack it in their checked baggage. deterioration on holiday or on a business trip of a previously stable condition -such as asthma, diabetes, or seizure disorder -can often give rise to the need for medical clearance for the return journey. a stretcher may be required, together with medical support, and this can incur considerable cost. it is important for all travelers to consider supplemental travel insurance, which includes provision for the use of a specialist repatriation company to provide the necessary medical support. if yes, describe and indicate for each item, (a) segment(s) on which required (b) airline arranged or arranging third party, and (c) at whose expense. provision of special equipment such as oxygen etc. always requires completion of part overleaf. if yes, add below fremec data to your reservation requests. if no, (or additional data needed by carrying airline(s)), have physician in attendance complete part overleaf. if yes, specify below and indicate for each item, (a) the arranging airline or other organisation, (b) at whose expense, and (c) contact addresses/phones where appropriate, or whenever specific persons are designated to meet/assist the passenger this form is intended to provide confidential information to enable the airlines' medical departments to provide for the passenger's special needs. to be completed by attending physician • when fitness to travel is in doubt as evidenced by recent illness, hospitalisation, injury, surgery or instability. • where special services are required, i.e. oxygen, stretcher, authority to carry accompanying medical equipment. completion of the form in block letters or by typewriter will be appreciated. specify if any** in determining the passenger's fitness to fly, a basic knowledge of aviation physiology and physics can be applied. any trapped gas will expand in volume by up to % during flight, and consideration must be given to the effects of the relative hypoxia encountered at a cabin altitude of up to ft above mean sea level. the altitude of the destination airport may also need to be taken into account in deciding the fitness of an individual to undertake a particular journey. the passenger's exercise tolerance can provide a useful guide on fitness to fly; if unable to walk a distance > m without developing dyspnea, there is a risk that the passenger will be unable to tolerate the relative hypoxia of the pressurized cabin. more specific guidance can be gained from knowledge of the passenger's baseline sea level blood gas levels and hemoglobin value. a good source of guidance is provided by the british thoracic society website. table . shows the guidelines recommended by one international carrier. this list is not exhaustive, and it should be remembered that individual cases might require individual assessment by the attending physician. the prolonged period of immobility associated with long-haul flying can be a risk for those individuals predisposed to develop deep venous thrombosis (dvt). pre-existing risk factors include: ▪ blood disorders and clotting factor abnormalities ▪ cardiovascular disease ▪ malignancy ▪ major surgery ▪ lower limb/abdominal trauma ▪ dvt history ▪ pregnancy ▪ estrogen therapy (including oral contraception and hormone replacement therapy) ▪ > years of age ▪ immobilization ▪ pathological body fluid depletion ▪ smoking ▪ obesity ▪ varicose veins. although many airlines promote lower limb exercise via the in-flight magazine or videos, and encourage mobility within the cabin, those passengers known to be vulnerable to dvt should seek guidance from their attending physician on the use of compression stockings and/or anti-coagulants. there is currently no evidence that flying, per se, is a risk factor for the development of dvt, but those at high risk should avoid any form of prolonged immobilization such as is associated with travel. in addition to the reduction in ambient pressure and the relative hypoxia, it is important to consider the physical constraints of the passenger cabin. a passenger with a disability must not impede the free egress of the cabin occupants in case of emergency evacuation. there is limited leg space in an economy class seat and a passenger with an above-knee leg plaster or an ankylosed knee or hip may simply not fit in the available space. the long period of immobility in an uncomfortable position must be taken into account, and it is imperative to ensure adequate pain control for the duration of the journey, particularly following surgery or trauma. even in the premier class cabins with more available legroom, there are limits to space. to avoid impeding emergency egress, immobilized or disabled passengers cannot be seated adjacent to emergency exits, despite the availability of increased leg room at many of these positions. similarly, a plastered leg cannot be stretched into the aisle because of the conflict with safety regulations. there is limited space in aircraft toilet compartments and if assistance is necessary, a traveling companion is required. the complexities of the airport environment should not be underestimated, and must be considered during the assessment of fitness to fly. the formalities of check-in and departure procedures are demanding and can be stressful, and this can be compounded by illness and disability, as well as by language difficulties or jet lag. the operational effect of the use of equipment such as wheelchairs, ambulances, and stretchers must be taken into account, and the possibility of aircraft delays or diversion to another airport must be considered. it may be necessary to change aircraft and transit between terminals during the course of a long journey, and landside medical facilities will not be available to a transiting passenger. there is often a long distance between the check-in desk and the boarding gate. not all flights depart from or arrive to jetties, and it may be necessary to climb up or down stairs and board transfer coaches. passengers should specify the level of assistance required when booking facilities such as wheelchairs. in addition to the main gaseous system, all commercial aircraft carry an emergency oxygen supply for use in the event of failure of the pressurization system or during emergencies such as fire or smoke in the cabin. the passenger supply is delivered through drop-down masks from chemical generators or an emergency reservoir, and the crew supply is from oxygen bottles strategically located within the cabin. the drop-down masks are automatically released en masse (the socalled 'rubber jungle') in the event of the cabin altitude exceeding a pre-determined level of between and ft. this passenger emergency supply has a limited duration if provided by chemical generators, usually in the region of min. the flow rate is between and l (ntp)/min, and is continuous once the supply is triggered by the passenger pulling on the connecting tube. oxygen supplied from an emergency reservoir is delivered to the cabin via a 'ring main', and in some aircraft it is possible to plug a mask into this ring main to provide supplementary oxygen for a passenger. sufficient first-aid oxygen bottles are carried to allow the delivery of oxygen to a passenger in case of a medical emergency in-flight, at a rate of or l (ntp)/min. this cannot be used to provide a pre-meditated supply for a passenger requiring it continuously throughout a journey, however, since it would then not be available for emergency use. if a passenger has a condition requiring continuous ('scheduled') oxygen for a journey, this needs pre-notification to the airline at the time of booking the ticket. most airlines charge a fee: one major british international airline charges gb£ (us$ , euro€ ) per sector, whether the supply is derived from gaseous bottles or via a mask plugged into the ring main. for us carriers, a fee of approximately us$ (€ ) is charged for providing oxygen during flight. some airlines also allow passengers to use their own portable oxygen concentrators, provided the unit meets certain specifications. an additional fee may be charged for medical clearance by an airline representative. passengers who require continuous oxygen during travel should be made aware that airlines provide oxygen service only during the flight, and not during the time spent in the airport terminal. passengers need to make arrangements for oxygen to be supplied at their destination upon arrival. oxygen bottles, regulators and masks must meet minimum safety standards set by the regulatory authorities, and the oxygen must be of 'aviation' quality, which is a higher specification than 'medical' quality. an in-flight medical emergency is defined as a medical occurrence requiring the assistance of the cabin crew. it may or may not involve the use of medical equipment or drugs, and may or may not involve a request for assistance from a medical professional traveling as a passenger on the flight. it can be something as simple as a headache, or a vaso-vagal episode, or something major, such as a myocardial infarction or impending childbirth. the incidence is comparatively low, although the media impact of an event can be significant. one major international airline recently reported incidents occurring in something over million passengers carried in year. the breakdown of these incidents into generalized causes is shown in table . . , the top six in-flight emergency medical conditions reported by the same airline are shown in table . . , any acute medical condition occurring during the course of a flight can be alarming for the passenger and crew because of the remoteness of the environment. cabin crew members receive training in advanced first aid and basic life support and the use of the emergency medical equipment carried on board the aircraft. many airlines give training in excess of the regulatory requirement, particularly when an extended range of medical equipment is carried. although the crew are trained to handle common medical emergencies, in serious cases they may request assistance from a medical professional traveling as a passenger. such assisting professionals are referred to as 'good samaritans'. cabin crew members attempt to establish the bona fide of medical professionals offering to assist, but much has to be taken on trust. the international nature of air travel can lead to complications in terms of professional qualification and certification, specialist knowledge, and professional liability. an aircraft in flight is subject to the laws of the state in which it is registered, although when not moving under its own power (i.e. stationary at the airport) it is subject to the local law. in some countries, it is a statutory requirement for a medical professional to offer assistance to a sick or injured person (e.g. france), whereas in other states no such law exists (e.g. uk or usa). some countries (e.g. usa) have enacted a good samaritan law, whereby an assisting professional delivering emergency medical care within the bounds of his or her competence, is not liable for prosecution for negligence. in the uk, the major medical defense insurance companies provide indemnity for their members acting as good samaritans. some airlines provide full indemnity for medical professionals assisting in response to a request from the crew, whereas other airlines take the view that a professional relationship is established between the sick passenger and the good samaritan and any liability lies within that relationship. at the time of writing, there has been no case of successful action against a good samaritan providing assistance on board an aircraft. recognition by the airline of the assistance given by the good samaritan is complicated by the special nature of the relationship between the professional, the patient, and the airline. indemnity, whether provided by the airline or the professional's defense organization, depends upon the fact that a good samaritan act is performed. if a professional fee is claimed or offered, the relationship moves away from being that of a good samaritan act to one of a professional interaction with an acceptance of clinical responsibility. this implies that the professional is suitably trained, qualified, and experienced to diagnose, treat and follow-up the particular case, and the good samaritan indemnity provision no longer applies. follow-up of the outcome for the passenger after disembarkation is frequently difficult, because the sick passenger is no longer in the care of the airline and becomes the responsibility of the receiving hospital or medical practitioner. responsibility for the conduct of the flight rests with the aircraft captain who makes the final decision as to whether or not an immediate unscheduled landing or diversion is required for the well-being of a sick passenger. the captain has to take into account operational factors as well as the medical condition of the sick passenger. in practice, it is rarely possible to land immediately, even if a suitable airport is in the immediate vicinity. the aircraft has to descend from cruising altitude, possibly jettison fuel to reduce to landing weight, and then fly the approach procedure to land. consideration has to be given to the availability of appropriate medical facilities, and in many cases, it is of greater benefit for the sick passenger to continue to the scheduled destination where the advantage of appropriate facilities will outweigh the risks of continuing the flight. operational factors to be considered include the suitability of an airport to receive the particular aircraft type. the runway must be of sufficient length and load-bearing capacity, the terminal must be able to accommodate the number of passengers on the flight, and if the crew go out of duty time, there must be sufficient hotel accommodation to allow an overnight stay of crew and passengers. the cost to the airline may be substantial, including the effects of aircraft and crew unavailability for the next scheduled sector, as well as the direct airport and fuel costs of the diversion. in making the decision whether or not to divert, the captain will take advice from all sources. if a good samaritan is assisting, he or she has an important role to play, perhaps in radio consultation with the airline medical advisor. many airlines use an air-to-ground link, which allows the captain and/or the good samaritan to confer with the airline medical adviser regarding the diagnosis, treatment, and prognosis of the sick passenger. the airline operations department is also involved in the decision-making process. some airlines maintain a worldwide database of medical facilities available at or near the major airports; others subscribe to a third party provider giving access to immediate medical advice and assistance with arranging emergency medical care for the sick passenger at the diversion airport. the link from the aircraft is made using either radio-telephone voice or datalink (vhf or acars), high-frequency radio communication (hf) or a satellite communication system (satcom). satcom is installed in newer, long-range aircraft, and is gradually replacing hf as the industry norm for long-range communication. the advantage is that satcom is unaffected by terrain, topography or atmospheric conditions, and allows good transmission of voice and data from over any point on the globe. digitization and telephone transmission of physiological parameters is a well established practice, particularly in remote areas of the world. an aircraft cabin at ft ( m) can be considered a remote location in terms of availability of medical support, and the digital technology used in satcom is similar to that used in modern ground-to-ground communication. the advent of satcom has enabled the development of air-to-ground transmission to assist in diagnosis. pulse oximetry and ecg are examples of data that can assist the medical advisor to give appropriate advice to the aircraft captain, although the cost/benefit analysis has to be weighed very carefully. national regulatory authorities stipulate the minimum scale and standard of all equipment to be carried on aircraft operating under their jurisdiction, which includes emergency medical equipment. these standards stipulate the minimum requirement, although in practice many airlines carry considerably more equipment. tables . and . give the minimum standard of equipment mandated by the federal aviation administration (faa) to be carried by aircraft registered in the usa, while table . gives the standard determined by the joint aviation authorities (jaa) for aircraft registered in european states. in determining the type and quantity of equipment and drugs to include in the medical kits, the airline must fulfill the statutory requirements laid down by the regulatory authority. other factors to be considered are: ▪ the route structure and stage lengths flown. different countries of the world vary in their regulations on what might be imported and exported, particularly in terms of drugs. for example, it is illegal to import morphine derivatives into the usa, even if securely locked in a medical kit approved first aid kits required by β . must meet the following specifications and requirements: the approved emergency medical kit required by β . for passenger flights must meet the following specifications and requirements: . each first aid kit must be dust and moisture proof, and contain only materials that either meet federal specification gg-k- a, as revised, or are approved. . approved emergency medical equipment shall be stored securely so as to keep it free from dust, moisture, and damaging temperatures. . required first aid kits must be distributed as evenly as practicable throughout the aircraft and be readily accessible to the cabin flight attendants. . one approved emergency medical kit shall be provided for each aircraft during each passenger flight and shall be located so as to be readily accessible to crew members. . the minimum number of first aid kits required is set forth in the following table: . the approved emergency medical kit must contain, as a minimum, the following appropriately maintained contents in the specified quantities. it is important to audit the incidence and outcome of in-flight medical emergencies and maintain a review of the kit content. this review should also take into account changes in medical practice ▪ space and weight. the medical equipment must be accessible, but securely stowed. some airlines divide the equipment and drugs between basic first-aid kits, which are readily accessible on the catering trolleys, and a more comprehensive emergency medical kit that is sealed and stowed with other emergency equipment. space and weight are always at a premium within the cabin, and the medical kits must be as light and compact as possible ▪ shelf life and replenishment. a tracking system for each kit must be in place to ensure that contents have not exceeded their designated shelf life. similarly, after use of a kit, there has to be a procedure for replenishment. in practice, the aircraft can depart if the kit contents meet the statutory minimum, even though drugs or equipment have been used from the non-statutory part of the kit. many airlines subcontract the tracking and replenishment to a specialist medical supply company. although basic cardiopulmonary resuscitation (cpr) techniques are an essential part of cabin crew training, the outcome of an in-flight cardiac event may be improved if appropriate resuscitation equipment is available. this can range from a simple mouth-to-mouth faceguard, to a resuscitation bag and mask and airway, to an all crew members will receive initial training on the emk and on the location, function, and intended operation of an aed. flight attendants will receive initial and recurrent training in cpr and on the use of aeds. medical personnel are frequently onboard and can assist fellow passengers during an in-flight medical event. in addition, a 'good samaritan' provision in the aviation medical assistance act of limits the liability of air carriers and non-employee passengers unless the assistance is grossly negligent or willful misconduct is evident. the following should be included in the first aid kits: note: an eye irrigator, while not required to be carried in the first aid kit should, where possible, be available for use on the ground. in addition, for aeroplanes with more than nine passenger seats installed, an emergency medical kit must be carried. the following should be included in the emergency medical kit: sphygmomanometer -non-mercury stethoscope syringes and needles oropharyngeal airways (two sizes) tourniquet coronary vasodilator (e.g. nitro-glycerine) anti-spasmodic (e.g. hyoscine) epinephrine : adrenocortical steroid (e.g. hydrocortisone) major analgesic (e.g. nalbuphine) diuretic (e.g. furosemide) antihistamine (e.g. diphenhydramine hydrochloride) sedative/anticonvulsant (e.g. diazepam) medication for hypoglycemia (e.g. hypertonic glucose) antiemetic (e.g. metoclopramide) atropine digoxin uterine contractant (e.g. ergometrine/oxytocin) disposable gloves bronchial dilator, including an injectable form needle disposal box anti-spasmodic drug catheter a list of contents in at least two languages (english and one other). this should include information on the effects and side-effects of drugs carried. endotracheal tube and laryngoscope, to an automatic external defibrillator (aed). the decision on the scale of equipment to be carried has to take account of the same parameters used in determining the content of the emergency medical kits (table . ). in addition, a cost/benefit analysis has to balance the cost of acquisition, maintenance and training against the probability of need and the expectation of the traveling public. the european resuscitation committee and the american heart association endorse the concept of early defibrillation as the standard of care for a cardiac event both in and out of the hospital setting. however, the protocol includes early transfer to an intensive care facility for continuing monitoring and treatment, which is not always possible in the flight environment. despite this inability to complete the resuscitation chain, it is becoming increasingly common for commercial aircraft to be equipped with aeds and for the cabin crew to be trained in their use. this has been mandated in the usa by the faa (table . ). experience of those airlines which carry aeds indicates that there may be benefits to the airline operation as well as to the passenger. some types of aed have a cardiac monitoring facility, and this can be of benefit in reaching the decision on whether or not to divert. for example, there is no point in initiating a diversion if the monitor shows asystole, or if it suggests that the chest pain is unlikely to be cardiac in origin. lives have been saved by the use of aeds on aircraft and diversions have been avoided, so it could be argued that the cost/benefit analysis is weighted in favor of carrying aeds as part of the aircraft medical equipment. nonetheless, it is important that unrealistic expectations are not raised. an aircraft cabin is not an intensive care unit and the aed forms only a part of the first aid and resuscitation equipment. many airlines have in place a procedure for the follow-up of crew members involved in a distressing event, such as a serious medical emergency. this can be valuable in avoiding long-term posttraumatic stress disorder, and also in reinforcing the training that the crew member has already undergone. the pressurized aircraft cabin provides protection against the hostile environment encountered at cruising altitudes. ▪ although the partial pressure of oxygen is less than at sea level, it is more than adequate in a pressurized aircraft cabin for normal healthy individuals ▪ the cabin air, although dry, does not cause systemic dehydration and harm to health. dry skin and eyes can lead to discomfort, which can be alleviated by the use of moisturizing creams and eye drops ▪ although up to half of the air in modern pressurized aircraft is recirculated, the amount of fresh air available to each occupant exceeds that available in air-conditioned buildings. recirculating the air has the advantage of reducing cold draughts and increasing the humidity ▪ in modern aircraft, all the recirculated air is passed through high-efficiency particulate filters which remove > % of particles, including bacteria and viruses ▪ there is an extremely low probability of airline passengers or crew suffering any abnormality or disease as a result of exposure to cosmic radiation. the passenger cabin of a commercial airliner is designed to carry the maximum number of passengers in safety and comfort, within the constraints of cost-effectiveness. it is incompatible with providing the facilities of an ambulance, an emergency room, an intensive care unit, a delivery suite, or a mortuary. the ease and accessibility of air travel to a population of changing demographics inevitably means that there are those who wish to fly, who may not cope with the hostile physical environment of the airport, or the hostile physiological environment of the pressurized passenger cabin. it is important for medical professionals to be aware of the relevant factors, and for unrealistic public expectations to be avoided. transmission aboard aircraft of illnesses, such as tuberculosis, sars and other respiratory diseases, has been reported, but is infrequently documented and probably rare. there is no evidence that a properly functioning environmental control system contributes to increased disease transmission aboard an aircraft. most airlines have a medical advisor who may be consulted before flight to discuss the implications for a particular passenger. such pre-flight notification can prevent the development of an in-flight medical emergency that is hazardous to the passenger concerned, inconvenient to fellow passengers, and expensive for the airline. for those with disability, but not an acute medical problem, preflight notification of special needs and assistance will reduce the stress of the journey and enhance the standard of service delivered by the airline. the importance of adequate medical insurance coverage for all travelers cannot be over-emphasized. finally, as is the case in commercial aviation, there is a continuing 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republic of ireland in response to the covid‐ pandemic date: - - journal: anat sci educ doi: . /ase. sha: doc_id: cord_uid: kab f the covid‐ pandemic has driven the fastest changes to higher education across the globe, necessitated by social distancing measures preventing face‐to‐face teaching. this has led to an almost immediate switch to distance learning by higher education institutions. anatomy faces some unique challenges. intrinsically, anatomy is a three‐dimensional subject that requires a sound understanding of the relationships between structures, often achieved by the study of human cadaveric material, models, and virtual resources. this study sought to identify the approaches taken in the united kingdom and republic of ireland to deliver anatomical education through online means. data were collected from different universities in the united kingdom and republic of ireland and compared adopting a thematic analysis approach. once themes were generated, they were collectively brought together using a strength, weakness, opportunity, threat (swot) analysis. key themes included the opportunity to develop new online resources and the chance to engage in new academic collaborations. academics frequently mentioned the challenge that time constrains could place on the quality and effectiveness of these resources; especially as in many cases the aim of these resources was to compensate for a lack of exposure to cadaveric exposure. comparisons of the actions taken by multiple higher education institutions reveal the ways that academics have tried to balance this demand. discussions will facilitate decisions being made by higher education institutions regarding adapting the curriculum and assessment methods in anatomy. the coronavirus pandemic is an unprecedented emergency that has affected all global industries, including education (ayittey et al., ) . with the widespread implementation of social distancing and self-isolation policies, it is not feasible for educators and students to attend lessons or assessments as they have previously. the covid- pandemic has disrupted our long-standing educational practices and has precipitated an urgent need for many institutions to rapidly implement alternative educational and assessment strategies. on st december , the world health organization (who) was informed of a small cluster of pneumonia cases with unknown etiology in the wuhan city, hubei province, china (who, a: zhu et al., ) . the novel coronavirus was isolated on th january and genetically sequenced on th january (who, a; wong et al., ) . the virus was confirmed to have subsequently spread to thailand, japan, and the republic of korea, on th , th and th january, respectively (who, a). on january rd , the government of china imposed a modern form of quarantine, shutting down all transportation in and out of wuhan. the lockdown expanded to twelve other cities in the hubei province (rubin and wessely, ) . covid- was first confirmed in the united kingdom (uk) and republic of ireland (roi) on th january and th february , respectively (holmes, ; hpsc, ) . on th march , the who officially declared the outbreak a pandemic (who, b) . to control the outbreak of the virus, public health england (phe, ) and the department of health and department of the taoiseach in the roi (dh&dt, ) introduced increasingly restrictive policies governing the movement and gathering of people. social distancing was implemented in the uk on th march (mahase, ) and even greater restrictions to movement were implemented on rd march (phe, ). historic pandemics, such as the severe acute respiratory syndrome (sars) also saw changes to educational practices and important lessons were learned. during the sars epidemic, chinese and canadian medical schools were forced to cease their clinical clerkships and electives (ahmed et al., ) . chinese medical schools supplemented their students learning experiences with the introduction of online problem-based learning (ahmed et al., ) . at the same time in hong kong, university policies decreed that the external examiner must be present for final-year medical distinction examinations (patil and yan, ) . these examinations traditionally took place as viva voce examinations; however, because the external examiner could not be present, these examinations took place by phone call. due to concern regarding the stability of the phone connection, the first part of the examination was conducted by an external examiner and no interruptions were reported (patil and yan, ) . these solutions could similarly be utilized by anatomists in addressing the challenges that have arisen due to the covid- pandemic. education institutes across all levels (pre-primary, primary, secondary and tertiary) have closed in countries across the globe, impacting over % of the world's student population (unesco, ) . universities in the uk started to close from the th of march. universities in the roi were formally closed on th march (rtÉ, ) and face-to-face teaching was suspended from th march, following government advice. universities have moved all teaching activities online and set up communication platforms, such as webpages and email streams to inform students of the ever-changing circumstance. the majority of higher education institutions in the uk and roi begin their academic term in august or september, and this cessation of face-to-face teaching occurred during the second or third semesters. educators were forced to rapidly find solutions to many challenges; for some this meant moving to online delivery within days. governmental education departments released guidance for students and educators in response to this (des, ; gov.uk, a, b) . there are over , medical students in the uk and roi (gmc, ; heffron and socha-dietrich, ) . approximately half of these students are completing formal anatomical education during the early years of their program (heylings, ) . therefore, alternative educational and assessment strategies will impact a large cohort of approximately , students with immediate effect. for final years, the general medical council (gmc) urged medical schools to fast track the graduation and registrations of these students to supplement the healthcare workforce (msc, ). the google hangouts (google meet) video conferencing application (google inc., mountain view, ca) has successfully been used to deliver lessons to medical students assigned to surgical specialties and it has been speculated that these could similarly be implemented to supplement anatomical education (moszkowicz et al., ) . at imperial college london, teleteaching and telemedicine modalities have been implemented to supplement the clinical needs of final-year medical students, and these students are currently perfecting their clinical judgment using an online repository of patient interviews and clinical cases (mian and khan, ) . in related fields, such as dentistry, there have also been recommendations to shift education toward online platforms (meng et al., ) . there are medical schools in the uk and seven in roi. recently, five new medical schools in the uk were created but have not been included in the sample as they are either not currently taking students or are under the umbrella affiliation of another medical school and are hence using their curriculum. the number of hours dedicated to anatomy teaching and the number of anatomical staff members significantly varies throughout the uk. anatomy is taught through systems-based curricula, problem-based curricula, or in the traditional regional format. lancaster university medical school, peninsula college of medicine and dentistry, and university of limerick medical school are the only institutions that do not use human cadavers to teach anatomy (heylings, ) . educators across the globe have been forced to replace traditional learning modalities with distance and blended learning approaches. distance learning has been described as an information delivery mechanism where the educator and learner are separated in both time and space (billings, ) , whereas blended learning combines traditional classroom methods of learning with online learning modalities (green and whitburn, ) . neither approach is novel, and both have been used successfully as part of anatomy education (pereira et al., ; ferrer-torregrosa et al., ) . however, these approaches are usually implemented after strategic planning, collaboration with other academics, and careful consideration of the pedagogical evidence. in order to utilize online approaches, educators must invest considerable time up-front to learn how to create online learning material. in many cases, academics are required to develop these new skills and create or adapt resources in parallel with a time frame that reflects the normal progression of student learning. the storage, maintenance, and embalming of cadaveric specimens poses a further challenge to anatomy departments with limited on-site access. with the body donation program largely halted in the uk and roi (hta, a), there may be a reduced number of available specimens for teaching in the following academic year. guidance from the international committee of the red cross (icrc) has also suggested that medical schools may provide body storage facilities as a contingency for mortuary overflow (finegan et al., ) , with updated guidance on licensing of emergency mortuaries concurrently released by the human tissue authority (hta) to support this (hta, b). this may result in existing cadaveric material being compromised, placing further strain on availability of specimens going forward. due to the unprecedented demand that the covid- pandemic has placed on public health services, clinically trained anatomists have been encouraged to return to the clinical workforce (willan et al., ) . in addition, non-clinical academic staff may contract covid- , or have caring responsibilities placing additional demands on the remaining members within a team of anatomists, disproportionately affecting universities with smaller academic teams, or in locations where only one anatomist is employed placing considerable burden on them. the negative impact on the mental wellbeing of healthcare workers due to anxieties and pressure in relation to covid- has already been documented (shaw, ) . this research aimed to understand the variety of solutions being utilized across institutions in the uk and roi and to facilitate easier decision-making surrounding changes to curriculum and assessment strategy. the two research questions were: ( ) what strategies have been adopted by uk and roi anatomy departments in response to what strengths, weaknesses, opportunities, and threats does the instant move to online learning bring? the questionnaire was conceptualized by the researchers for this study (g.j.l., d.m.s., k.d., d.s., and t.c.) in order to obtain information from anatomy departments across the uk and roi on their institutional and departmental responses to the covid- pandemic. all questions proposed by the researchers were selected for review. only the questions that were agreed by all contributors were selected. the seven questions that were selected focused around three themes: ( ) location of university and size of anatomy team, ( ) adaptations to teaching and resources used in lectures and practical sessions and ( ) major challenges and opportunities (see supporting information ). all feedback was solicited by way of free-text responses. a pilot study was completed in the respective institutions of the researchers (g.j.l., d.m.s., k.d., d.s., and t.c), and on review there were no postpilot modifications made. the questionnaire was tabulated in a microsoft word document (microsoft corp., redmond, wa) and emailed to anatomists in higher education institutions in the uk and roi. the list of anatomists and institutions selected for this study was created using a convenience sample of known contacts. a total of responses were received from the medical schools that were contacted ( % response rate). the adaptations to lectures, practical, and assessment were collated and summarized and a thematic analysis was carried out on the opportunities and challenges. all responses were analyzed using thematic analysis by the researchers (g.j.l., d.m.s., k.d., d.s., and t.c.) (glaser and straus, ; braun and clarke, ) . to achieve this, the six-phase process described by braun and clarke ( ) was followed. first, all authors read through the entire data set to familiarize themselves with the data. initial codes were then manually generated using an open-coding approach by highlighting interesting and relevant aspects of the responses. this was done individually and then any discrepancies discussed in order to minimize bias. codes were then collated into more general themes. these themes were reviewed by all authors to ensure they fully reflected the responses. due to the small sample size, themes were generated without digital programs. the most commonly reoccurring themes were then used to inform the next stage of the analysis, as well as other essential themes as identified by the authors. the authors assigned these themes to either strengths, weaknesses, opportunities, or threats for the purpose of swot analysis, an analytical approach commonly used to inform strategic planning and decision making (helms and nixon, ; teoli and an, ) . a swot analysis was originally developed as a business tool to aid decision making, but has since been used to analyze strategies in higher education, including medical education (sharma, ; burr, ; liu, ; hazzan et al., ; leiber et al., ) . due to the urgent and rapidly evolving nature of covid , it was not possible to secure ethical approval. to mitigate this, it was made clear that respondents were providing data and by doing so were consenting for the data to be used. the authors are planning a follow-up study from different stakeholders' perspectives and this will go through full ethical approval and the data presented here will be considered as pilot data. fourteen universities provided information on the departmental response to covid- . twelve universities were uk based and two were based in the roi. the results presented here represent % and % of medical schools in the uk and roi, respectively. ten of the uk-based universities are located in england, one is located in wales and another one in northern ireland. the two irish universities are located in dublin. lectures. universities opted to replace lectures with recorded presentations and accompanying audio that was uploaded to the virtual learning environment. the most common lecture recording platform used was "panopto" (panopto inc., seattle, wa), with % of universities citing its use. thirty-six percent of universities also provided live sessions and tutorials via platforms such as "zoom" (zoom voice communications inc., san jose, ca), "collaborate ultra" (blackboard inc., new york, ny), and "big blue button" (big blue button inc., ottawa, canada). practical sessions. all bar two universities in the sample group used cadaveric material to teach anatomy prior to the pandemic. universities replaced practical sessions by supplementing virtual learning environments with additional resources. twenty-nine percent of universities used digitized cadaveric resources only, seven percent used d virtual resources only, and % used a combination of cadaveric and d virtual resources (table ). digitized cadaveric resources included "acland's video atlas of human anatomy" (acland, ) , high-quality cadaveric images, bespoke videos of prosected/ plastinated specimens, youtube (tm) videos (youtube, san bruno, ca), and the visible human project (u.s national library of medicine, bethesda, md) ( table ) . three-dimensional virtual resources included "visible body" (argosy publishing, inc., newton, ma), "complete anatomy" ( d medical/elsevier, dublin, republic of ireland), d models using "sketchfab" (sketchfab, new york, ny), "anatomy tv" (primal pictures ltd., colchester, uk), and "sectra" (a virtual dissection table; sectra ab, linköping, sweden) ( table ) . forty-three percent of universities provided further support for practical sessions by hosting live tutorials and sessions using platforms such as "zoom" (zoom voice communications inc., san jose, ca) and "microsoft teams" (microsoft corp., redmond, wa). assessment. fourteen percent of universities did not have a summative practical assessment in their curriculum, a further % of universities canceled their assessment and % completed online digital spotter examinations (table ) . written examinations were canceled in seven percent of universities (table ) . thirty-six percent opted for online multiple-choice questions (mcqs)/extending matching questions (emqs)/ single-answer questions (saqs)/single-best-answer (sbas) examinations and % opted for open-book examinations (table ). there were no regional differences noted with regard to assessment. opportunities and challenges. despite chronic disruption to curricula, the covid- pandemic has presented many opportunities to universities. the opportunity to develop new online resources was highlighted by % of universities. fifty percent of universities cited academic collaboration as an important opportunity, and % highlighted the importance of working from home. universities identified other opportunities such as upskilling in new technologies ( %), incorporation of blended learning ( %), development of alternative examination methods ( %), and free access to online resources ( %) ( table ). the most commonly expressed concern was the time investment associated with the development of new resources to replace lectures and practical classes. this challenge was highlighted by % of universities. fifty percent of universities were concerned about lack of practical sessions/cadaveric exposure, and % of universities identified reductions in student engagement. universities also highlighted concerns regarding the teacher-student relationship ( %), assessment ( %), working from home ( %), suspension of body donor programs ( %), and lack of technical support ( %) ( table ). many universities documented similar pedagogical opportunities and challenges. themes from the responses to top opportunities and challenges are sorted into a swot analysis and used to form the basis of this discussion. reoccurring strengths included the development of new resources and skills. time constraints, lack of exposure to cadaveric material, and changes to assessment were identified as weaknesses. opportunities identified included academic collaboration and working remotely, as well as with the opportunity to implement blended learning in future curriculum development. the reduction in quality of resources, reduced student engagement, and diminished student-teacher relationship were considered threats (fig. ). the approaches taken by higher education institutions and the effect these have on the strengths, weaknesses, opportunities, and threats currently faced are also discussed to elicit which approaches could prove most effective. development of new online resources. in response to the covid- lockdown, both digitized cadaveric resources and d virtual anatomy platforms were used by % of universities, in an attempt to emulate canceled practical sessions. twenty one percent of universities created unique videos. bespoke resources have been shown to lead to a significant increase in student satisfaction (mandernach, ) . new resources created and developed by academics include videos, virtual dissections, formative quizzes, and updating practical dissection notes that will be available to students through virtual learning environments. previous studies have elucidated that students appreciate anatomical videos and they have the potential to improve test scores (pereira et al., ; dilullo et al., ; topping, ) . to date, there are limited data on the effectiveness of virtual dissections; however, the results have been positive, when used in conjunction with cadaveric material (yammine and violato, ; darras et al., ) . a significant number of medical students have reported that they find online resources helpful (smith et al., ) this echoes a statement by one academic who stated gaining "free access to high quality resources from publishers" was a positive aspect of the pandemic. interestingly, the usefulness of online resources for individual students has been linked to personality preference, and thus the switch to distant learning may prove harder for certain groups of students (mcnulty et al., ) . the context of the anatomical learning environment has undergone a paradigm shift since the lockdown, and this may result in students adopting different strategic approaches to their learning (smith et al., ) . thus, it is imperative to ensure that these new resources are based upon sound pedagogical theory in order to foster deep learning strategies (kreber, ) . in addition, the number of new resources has the potential to overwhelm the learner, having negative implications for their extraneous cognitive load. extraneous cognitive load is affected by how information is presented by the instructor (leppink and van den heuvel, ) . upskilling in new technologies and resources. three institutions ( %) noted that this is an opportunity to develop technological skills. one academic stated this was a "chance to upskill and incorporate alternative software and novel resources into our teaching." this sudden leap in upskilling and innovation by a large proportion of academics has the potential to transform medical education by incorporating online learning to all aspects of the curricula (skochelak and stack, ; rose, ) . one academic stated that that this was an "opportunity to improve staff awareness of online teaching methods and their confidence in using them." for anatomists, this is a unique opportunity to assess the educational benefits of this software, encouraged by free licenses offered by many companies during this time, such as "visible body" (argosy publishing, inc., newton, ma) and "human biodigital" (biodigital inc., seoul, south korea). furthermore, it has triggered the development of new skills in assessment modalities. as all universities have adopted an online approach to written examinations, new skills in utilizing the full capabilities of virtual learning environments are being explored. virtual learning environments allow academics to easily build question banks and design assessments that can be marked automatically or manually. virtual learning environments also facilitate randomized presentation of questions, time constraints, and prohibit backtracking in order to prevent collaborative answering. for example, "brightspace" (d l corp., kitchener, canada) facilitates precise keystroke recording-an important feature to prevent cheating during online spotters. the move to online assessment has identified an opportunity to improve examination conditions (dennick et al., ) . specifically, this is true for summative continuous assessments that often involve image-based spotter examinations as there is a high workload associated with designing and implementing station-based spotter exams. schubert et al., ( ) reported that designing, implementing, and invigilating a station-based gross anatomy examination encompassed . days' work (shubert et al., ). dennick et al., ( ) showed that student performance on gross anatomical examination is no different between traditional spotter examinations compared to digital online modes. the development of skills in online assessment modalities may be of benefit for future academic practice without endangering academic integrity or student performance (meyer et al., ) . time constraints. time is ultimately of the essence when it comes to creating additional anatomical resources in response to covid- , with universities identifying it as the top challenge in the delivery of anatomical education. one academic noted that the "time needed to learn how to use technologies" was an issue. creating new resources can take at least three times as much work compared to a traditional format (gewin, ) . this may be attributed to the steep learning curve required to learn how to use technologies effectively. the added pressure of producing "high-quality resources," in addition to fulfilling other academic duties in a limited time period may put undue pressure on academics. therefore, it is imperative to weigh-up the cost-benefit implications of the time needed to create new resources against the educational benefit for the students. indeed, it was observed that instructor-made videos lead to no measurable improvements in student achievement (mandernach, ); however, it may have an impact in student engagement as discussed in the section outlining potential threats. furthermore, well-renowned anatomical educational resources already exist, so one must decide if it is worth 're-inventing the wheel', so to speak. for example, "acland's video atlas of human anatomy" (acland, ), recurrent themes in the context of the swot paradigm. the frequency of recurring themes was calculated based on respondent survey data (a total of responses from the contacted medical schools in the united kingdom and republic of ireland). the authors subsequently assigned these themes to the most appropriate heading of the swot paradigm. created in , is still used by medical schools today along with other useful online resources as outlined by participating universities ( table ) . lack of practical sessions and cadaveric exposure. the results show that a major concern for academics is the loss of cadaveric-based teaching, as expressed by % of participating universities. cadavers are utilized as the pillar pedagogical tool in many medical schools across the globe (habicht et al., ) . indeed, one academic stated that "not physically attending practical sessions will limit haptic understanding and reduce discussions concerning variations and pathology." the benefits of cadaveric-based teaching have been eluded in a number of studies (aziz et al., ; estai and bunt, ) . cadavericbased teaching has been shown to lead to a deep understanding of the three-dimensional relationships of the human body and allows students to appreciate anatomical variations and pathologies (azer and eizenberg, ; smith and mathaias, ; fruhstorfer et al., ; hafferty and o'donnell, ) . cadaveric dissection can be a sensory explosion, arguably one that cannot be stimulated by technology (korf et al., ) . in addition, it allows students to develop competencies of medical profession and gain attributes through the hidden anatomical curriculum, for example, encountering death, empathy, and professionalism, in addition to communication and teamwork skills (estai and bunt, ; kumar ghosh and kumar, ) . additionally, students themselves believe that working with cadaveric material helps them learn anatomy and is an important component of becoming a healthcare professional (smith et al., ; flack and nicholson, ) . in the uk and roi, fortunately the lockdown occurred nearer the end of the academic term, thus students will have been exposed to cadaveric material for approximately six or seventh months. conversely, this is not the case for other countries, e.g., in australia and new zealand, where the academic term begins in february/march. all medical students may be disproportionately affected by the lack of cadaveric-based classes. although further studies will elucidate how the current covid- crisis will affect students' understanding of anatomy, a previous report revealed that students participating in gross anatomy laboratories received significantly higher grades, compared to those taking an online course (mathiowetz et al., ) . with many institutions looking to produce new online content to replace cadaveric dissection the hta have released a new statement regarding the use of cadaveric images, reiterating the importance of protecting the dignity of the deceased (hta, a). where cadaveric images are to be used to supplement online teaching, it is imperative to check if the donor provided consent for their remains to be used in images and videos. (hta, ; hennessy et al., ) . some institutions have also put in measures to limit sharing of cadaveric material now available on virtual learning environments by ensuring students are able to view but not download this content. issues with assessment. twenty-nine percent of the institutions surveyed did not have pre-existing summative assessments based on practical content. of those that did, % canceled the summative element, and % switched to online assessments to maintain normal student progression (table ) . however, online assessments pose their own unique challenges. one key principle that needs to be considered is the preservation of constructive alignment of the assessment task to the intended learning outcomes and teaching/learning activities (biggs and tang, ) . many higher education institutions teach through cadaveric dissection and assess the practical element of learnings through a traditional spotter style format. this will encourage students to construct their knowledge of anatomy through learning activities with a focus on cadaveric material. however, online assessments cannot replicate the wealth of information available in a cadaveric specimen that can act as a retrieval cue for learning that took place during practical classes (ali et al., ) . a switch to online assessment could cause a breakdown of constructive alignment between the teaching learning activity and assessment task. the results highlighted that % of universities identified compromised assessment standards as a significant concern, with "questions around validity and practicality of delivering exams online." one way of mitigating this would be to ensure that images used in online assessments reflect those used during teaching and learning activities. research has suggested that assessment modality does not significantly influence student achievement (attardi and rogers, ) and highlights the need for carefully chosen images with clear orientation (meyer et al., ) . in order to maintain constructive alignment, it is important that new assessment modalities are chosen that best assess the skills students are expected to achieve through their learning. academic collaboration and working remotely. the covid- pandemic presented opportunities for online collaboration between academic peers, both within and between institutions to gain "insight and inspiration" from others that "are facing similar challenges." these were ranked as the main opportunities by % of the universities in this study. additionally, four institutes commented on the opportunity to create new and effective online working environments which particularly highlights the opportunity for remote working in the future. while there were two responses regarding the negative aspects of working from home, they both reflected the impact of the current covid- crisis (i.e., lack of textbooks and childcare responsibilities). already, there has been a wealth of support and resources offered by numerous academics on twitter (tm) (twitter inc., san francisco, ca), facebook (tm) (facebook inc., menlo park, ca), and other social media platforms. in addition, the anatomical society has a collaborative database containing online resources (as, ). an online network, particularly in circumstances such as these can have an impact on academic success (maican et al., ) . external collaborations can act as a source of support and are particularly important in institutions with small teams of anatomists. nurturing these collaborations and developing an online community of practice in the future is a huge opportunity for the anatomical community. this is especially important for the next academic year as the impact of covid- on the body donor program and anatomical education is yet to be fully appreciated. additionally, as many anatomy departments are developing new technological skills in teleconferencing, there is a potential to pool expertise among institutions. it gives departments the opportunity to have "online connections made between peers," to share expertise in anatomical sub-specialties and to showcase cutting-edge anatomical research across satellite campuses, and indeed across institutions worldwide. incorporation of blended learning in future curriculum development. academics reported being excited at the chance to develop new resources as it was the most frequently cited opportunity among the cohort ( % of responders). the decline in the priority of cadavers, and indeed anatomy as a subject, within medical curricula is well documented (drake et al., ; drake et al., ) . with diminished resources including curricula time, budgets, and staffing, in conjunction with increased student numbers, innovative and novel approaches are needed to solve these pressing issues. perhaps covid- could act as a catalyst to do so. fourteen percent of academics suggested that this crisis presented them with an opportunity to develop resources that can be used in the upcoming academic year, allowing them to incorporate more blended learning techniques and review the curriculum. therefore, it is imperative that enough time can be dedicated in order to create high-quality references. reduced student engagement. a frequently expressed concern was diminished student engagement as a function of the rapid implementation of distance learning ( % of responders). on consideration, this may be compounded by the fact that many universities are no longer pursuing typical mandatory attendance policies for teaching sessions. moreover, the current covid- crisis may cause an increased risk of isolation, anxiety, and boredom among the population (rubin and wessely, ) . this was also highlighted by one institution as a major concern among the student population where they were worried about the levels of "anxiety" that students face on "the future of their education." previous studies have shown a decrease in quality of life and stress negatively impacts student motivation and academic results (artino et al., ; lyndon et al., ) . specifically, the responses from this study included comments that highlighted their concerns with "creating resources that students will actively engage with" as well as how to "encourage and maintain student engagement." thus, academics adapting their approach to anatomical education should consider the need for support, interactivity, and social engagement with and between the students. a reduction in these elements may ultimately impede academic progress and student satisfaction. ultimately, one academic expressed they wanted to ensure that students "were getting value for money" from their course. additionally, technical issues, for example, unstable internet connections or lack of suitable electronic devices, will also impact student engagement (wimpenny and savin-baden, ; ilgaz and gülbahar, ) . the unexpected shift to distance learning means that some students may not be technically prepared for distant learning and assessment. therefore, due consideration to accessibility must also be considered as a potential threat to delivery of distance learning and assessment. in conjunction with potential issues in relation to the student population, staff may not have proficient knowledge of pedagogical techniques specific to the delivery of online teaching. some staff members may not have been able to fully evaluate the theoretical and practical implications of distance learning prior to the cessation of face-to-face teaching. this may also impact on their ability to boost student engagement (wimpenny and savin-baden, ) . teacher-student relationship. maintaining the teacherstudent relationship was considered a threat ( % of responders) as online resources lead to a perceived improvement in the online instructor social presence (draus et al., ) . the online instructor presence effectively reduces the transactional distance between student and instructor (attardi et al., ; stone and barry, ) by increasing the likelihood of interaction. one way to address this issue is to host synchronous (live) classes (mcbrien et al., ; o'flaherty and laws, ) . this approach was taken by % of universities that hosted live lectures and % hosted live tutorials to support practical sessions. many synchronous software packages contain interactive polls that can be used to assess knowledge, encourage engagement, and provide instant feedback to students. studies have shown that timely feedback from instructors is hugely important in maintaining engagement (martin and bolliger, ; ragusa and crampton, ) . interactive polling can therefore be utilized in a flipped-classroom approach, shown to increase student interactivity as all levels of bloom's taxonomy can be assessed if correctly designed (gilroy et al., ) . providing clear guided instructions for problem solving and linking content to clinical practice and prior knowledge are other methods that are proven to improve engagement (wimpenny and savin-baden, ; boton and gregory, ; ilgaz and gülbahar, ; buelow et al., ; martin and bolliger, ) . thus, within the synchronous classes, staff can potentially utilize anatomical education software programs to incorporate applied anatomy and clinical scenarios into their sessions. in this regard, there is evidence that virtual synchronous conferencing methods may increase peer-peer and peer-teacher interactions (mcbrien et al., ; o'flaherty and laws, ) . however, in the covid- crisis, synchronous teaching may be deemed unsuitable for a proportion of the student population. students living in different time zones or those with parental and caregiving responsibilities may be unable to attend scheduled synchronous sessions. as synchronous sessions offer potential for interactivity, students unable to attend may feel particularly isolated if departments solely relied on synchronous teaching methods. to address this, interactive asynchronous teaching methods have been employed, for example, twitter (tm) and discussion boards or padlet (tm) (padlet, inc., san francisco, ca) (table ) . a strong teacher-student relationship results in students feeling more comfortable engaging with online communication platforms such as discussion boards (griffiths and graham, ; rose, ). the use of social media has been shown to diminish anxiety and foster a sense of community (hennessy et al., ) . however, if these options are unavailable, adapting resources that are already used across ten institutions in our study can include interactive elements within asynchronous teaching. this includes creating a short video content of problem-solving tasks with recorded audiovisual input from the tutor. such an approach permits students to consistently see and hear their tutor, with the potential to aid in student engagement (wimpenny and savin-baden, ; boton and gregory, ; ilgaz and gülbahar, ; buelow et al., ; martin and bolliger, ) . it is also important to consider the limitations of the methodologies employed and the data presented. first, the presented data are representative of a subset of the total number of medical schools in the uk and roi. it is conceivable that adaptions utilized by an individual institution or minority of universities may have been missed due to inadequate sampling. second, during the data collection process only a single individual was contacted from each institution. it is our presumption that respondents' answers accurately reflect the most commonly utilized adaptations at their respective institutions-though our methodology did not establish this. third, the authors did not sample any universities in scotland. this is important to consider as differing national practices could affect the various adaptations utilized by universities in response to the pandemic. fourth, our survey sought to establish information pertaining to the education of medical students only and did not consider the allied health disciplines. it would be interesting to also identify the adaptations utilized by universities to manage this cohort's education as any niche solution employed could also be translated to the medical cohort. fifth, the survey did not explicitly state that respondents should describe the strengths and weaknesses of the various adaptations implemented by their respective university. the authors believed that incomplete responses would be returned if respondents were required to provide more information. in hindsight, the utility of these adaptations would have been better contextualized if the authors had formally sought for respondents to comment on the strengths and weaknesses of the adaptations implemented at their university. sixth, due to the small sample size, statistics for interval validity were not possible. everyone is currently amid an unprecedented global event. the effects of covid- upon anatomical education, and medical education, are not yet fully understood. changes to curriculum normally take years to research, enforce, and evaluate. however, the current crisis has forced academics to make radial adjustments in a short period of time. the swot analysis and embedded pedagogical theory of these changes has been presented in the hope that anatomists feel more confident in their decision-making. additionally, as the change in anatomical education has been so sudden over the course of the next few months, academics' responses may change and new threats and opportunities may arise. currently, negative effects, such as the time pressure, changes to assessment, and implications in students' engagements and relationship, are somewhat balanced by, positive consequences, such as the potential to create new resources and foster academic collaborations, also arise. indeed, this crisis may be a catalyst for the integration of novel technological resources and the development of stronger ties and collaborations between anatomy departments. it is evident that anatomy departments across the uk and roi are putting tremendous effort into the delivery of high-quality education in the midst of the pandemic. it is hoped that the adaptations utilized by universities will ultimately translate to a lasting positive change in the delivery of anatomical education; and as such there are plans to assess the sustainability of these modalities with a follow-up survey. the authors would like to express their sincerest gratitude to the fellow anatomists who contributed to this study. it is our hope that this crisis leads to the beginning of many new collaborations. georga j. longhurst, b.sc., m.sc. s.p. cert., is a lecturer in anatomical sciences at st george's university of london in london, uk. she teaches anatomy to medical and allied health students. she has a research interest in anatomical education. danya m. stone, b.sc., m.sc., f.h.e.a., is a lecturer in anatomy brighton and sussex medical school (brighton), falmer, uk. she teaches anatomy to medical students and contributes to teaching on multiple external courses. her research interest is in medical education. kate dulohery, b.sc., m.sc., ph.d., f.h.e.a., is a lecturer in anatomy in school of medical education at newcastle university, newcastle upon tyne, uk. she teaches anatomy to medical, dentistry and allied health students and her research interests include incorporation of technology in anatomical education. deidre scully, b.sc., m.sc., ph.d., is a teaching fellow in the department of anatomy, school of medicine at trinity college dublin, the university of dublin, dublin, republic of ireland. she teaches medical and allied health students and her research interests include reproductive biology and anatomical education. thomas acland's video atlas of human anatomy ® covid- and medical education lancet inf dis the perception of anatomy teaching among uk medical students second-year medical students' motivational beliefs, emotions, and achievement improving online interactions. lessons from an online anatomy course with a 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(social distancing) sense of connection, identity and academic success in distance education: sociologically exploring online learning environments student perceptions of the use of instructor-made videos in online and face-to-face classes medical student education in the time of covid- rtÉ. . raidió teilifís Éireann. coronavirus in ireland -a timeline the psychological effects of quarantining a city assessment of spatial anatomical knowledge with a 'three-dimensional multiple choice test' ( d-mc) health education in india: a strengths, weaknesses, opportunities, and threats (swot) analysis hopelessness, helplessness and resilience: the importance of safeguarding our trainees' mental wellbeing during the covid- pandemic key: cord- -l heb or authors: nagarajan, kiran koushik; bali, atul; malayala, srikrishna varun; adhikari, ramesh title: prevalence of us-trained international medical graduates (img) physicians awaiting permanent residency: a quantitative analysis date: - - journal: nan doi: . / . . sha: doc_id: cord_uid: l heb or background: international medical graduates (imgs) contribute to about % of the physician workforce in the usa. certain us-trained imgs face long wait times for transitioning to a permanent resident status, which limits their ability to work to fullest capacity, especially during a public health emergency. objectives: to estimate the number of us-trained imgs awaiting permanent residency. study design: data were obtained from national residency matching program (nrmp) to quantify the number of imgs who secured residency training in the us from to . estimates of physician demographics were based on nrmp/ecfmg match data and federation of state medical boards (fsmb) physician census data. results: between and , a total of , non-us imgs who were not us citizens successfully matched to residency training programs. applicants from india and china were noted to be impacted by delays in adjustment to permanent resident status. per our estimate, there are between , and , us-trained physicians from china currently awaiting permanent residency, with applicants waiting since october , and between , and , us-trained physicians from india currently awaiting permanent residency, with applicants waiting since june . conclusions: the total number of us-trained immigrant physicians in active practice awaiting permanent residency to the usa is estimated to be ranging between , and , . each year, multiple physicians from all over the world attempt to secure residency training in the usa [ ] . physicians who graduate from international medical schools are called international medical graduates (imgs) [ ] . many of these physicians require visa sponsorship for legal entry into the usa (us) to initiate their residency training [ ] . after completion of their training, they become eligible to apply for a permanent resident status in the usa, often colloquially referred to as a 'green card' [ ] . however, due to the limited annual quota of green cards, which are allocated based on the country of birth of the applicant, the wait times can vary widely among applicants. due to the sheer number of applicants from countries like india and china, expectedly higher from the two most populous countries in the world, these applicants have the longest wait times before they become eligible to adjust their status to that of a permanent resident [ ] . while every applicant seeking an employment-based immigrant visa must demonstrate that they possess specific skills, advanced degrees or extraordinary abilities for their eligibility, physicians constitute a small but particularly highly trained subset of these applicants. notably, these physicians are perhaps the few immigrants where federal tax dollars have already been utilized for their training, as they navigate through years of grueling residency training, often followed by even more specialized fellowship training. given the resource-intensive process of training a physician, there are several legislative programs in places such as the appalachian regional commission (arc) program and the conrad program, with the primary purpose of retaining these assets to serve areas of the usa with inadequate physician access [ ] . furthermore, if these highly trained physicians are unencumbered by restrictions imposed by their visa status, they could be mobilized to areas of higher need. this flexibility could prove crucial amidst a public health crisis, such as the covid- pandemic [ ] . therefore, it is imperative to quantify the number of immigrant physicians who are awaiting permanent residency status due to the reasons mentioned above. to the best of our knowledge, the usa citizenship and immigration services (uscis) does not publish or track profession-specific data pertaining to applicants with an approved i- (immigrant petition for alien workers), who have not yet become eligible for applying for an adjustment of status [ ] . in the absence of this information, one can only estimate the number of immigrant physicians currently awaiting permanent residency status. for this reason, we have attempted to quantify the prevalence of ustrained immigrant physicians awaiting permanent residency. we formulated a stepwise approach to the problem, by first quantifying the number of non-us imgs (international medical graduates who were not us citizens), who had sought residency training in the usa, from until . residency training in the usa has a minimum duration of years, with some physicians pursuing subsequent fellowship training of minimum years duration, only after completion of which would the physician start working in the capacity of an attending physician and become eligible to apply for permanent residency [ ] . most physicians qualify for the 'eb- ' immigrant visa category, which specifies that the applicants must be 'professionals holding advanced degrees and persons of exceptional ability' [ ] . as of may , based on data from the us department of state, the subset of eb- applicants most impacted by delays in transition to permanent residency had applied in july and onwards [ ] . to account for years of training as elucidated above, we chose as the earliest data point for the sake of our analysis. the national residency matching program (nrmp) is a private, non-profit organization that utilizes a sophisticated algorithm to allow medical students to be matched with their preferred residency training programs[ ]. with rare exceptions, the overwhelming majority of applicants utilize the nrmp match to secure a residency training position. we collected data from the nrmp residency match data and reports to determine the cumulative number of non-us imgs who had successfully matched for each year since [ ] [ ] [ ] [ ] [ ] . determining the country of birth of practicing physicians posed a unique challenge, as this demographic information is not evaluated by agencies tracking data pertaining to the us physician workforce. while there are exceptions, most individuals complete their medical education prior to residency in their countries of birth. therefore, we used the country where the physician completed their medical school graduation as a surrogate for their country of birth. based on data from the biennial census of physicians with active licenses conducted by the federation of state medical boards (fsmb), we determined the percentage of practicing physicians who completed their medical school graduation from various countries [ ] . these numbers were used to get an estimate of physicians who had likely applied for immigrant petitions currently practicing in the usa but were awaiting permanent residency. the cumulative number of non-us imgs who had successfully matched for residency training from to was , [ ] [ ] [ ] [ ] [ ] . based on the may [ ] visa bulletin released by the us department of state, all eb- applicants with an approved i- were eligible to adjust their status, other than those who were born in india and china [ ] . thus it was important to determine the percentage of non-us imgs from these countries who had secured residency training since . the fsmb census data suggested that while the proportion of physicians from china was relatively small and was not quantified, the majority of licensed imgs that were currently in practice in the us had graduated from medical schools in india, with their numbers being estimated at , ( %) (figure ) [ ] . upon review of prior biennial census data from the fsmb, this percentage of imgs from india has remained remarkably stable over several years. furthermore, match data obtained by a joint effort from the nrmp and the educational council for foreign medical graduates (ecfmg) in , also obtained every applicant's citizenship at birth. review of these demographic data suggested that . % ( / ) of matched applicants were indian citizens at birth. therefore, given the stability of this percentage despite the influx of medical graduates each year, we operated under the assumption that the composition of imgs matching to us residency programs was similar to fsmb census data. we thereby estimate approximately between , and , us-trained physicians from india currently awaiting permanent residency, with some applicants waiting since june (figure ). since the fsmb census data did not assign a definitive number of us-trained chinese physicians, we revisited the nrmp-ecfmg match data and noted that . % ( / ) were chinese citizens at birth (figure ) [ ] . additionally, we utilized data from a study published by duvivier et al. in april [ ] . the authors procured data from the american medical association physician masterfile, noting that the total number of chinese-educated physicians had risen modestly from , in , to , in . additionally, they noted that as compared to data where physicians were in residency training, only physicians were in residency training positions in . they inferred that there had been a relative decline in the influx of physicians who completed their medical school graduation in china. according to data from the may visa bulletin, eb- applicants from china with an approved i- from october are eligible for adjustment of status to permanent residency [ ] . assuming the modest rate of influx of chinese physicians as noted in the duvivier study, with a relatively higher rate noted in the nrmp-ecfmg data, there would be approximately between , and , us-trained physicians from china currently awaiting permanent residency, with some applicants waiting since october . combining the estimates listed above, the total number of us-trained foreign physicians in active practice awaiting permanent residency to the usa is estimated to be ranging between , and , . the robust clinical and research experience offered during residency and fellowship training attracts thousands of foreign physicians to the usa each year. this has resulted in nearly a quarter of practicing physicians in the us to be imgs. american medical association (ama) suggests that about . % of the licensed us physicians are imgs. owing to the ever-expanding population and the retirement of the existing physician workforce, the [ ] number of imgs in practice has grown by nearly , since , a . % rise and that figure is bigger than the % rise in us. medical graduates over that same time period [ ] . in a report published by the ama in october , it was noted that % of imgs speak two or more languages fluently, helping patients overcome linguistic and cultural barriers that can impede care. imgs tend to work mostly in the primary care specialties (about %) and that is about double the % of all us. physicians who work in a primary care specialty, helping to meet a critical workforce need. this influx of highly trained immigrants has positively impacted the physician shortages across the country. due to programs redirecting these us-trained foreign physicians such as the arc program and conrad program, these physicians serve in higher proportions in rural and underserved areas, where there are greater african american, hispanic and non-white populations [ ] . this suggests that these actively licensed physicians are caring for the most medically vulnerable americans. in this manner, physician immigration into the usa is mutually beneficial. however, the inability of us-trained immigrant physicians to secure permanent resident status for prolonged periods, in comparison to their peers who trained in other countries, increases physician dissatisfaction and may serve as a disincentive to continue to practice in the usa. furthermore, their visa restrictions prevent them from pursuing additional opportunities such as offering new medical services or hiring additional staff members, which would benefit the communities they serve from both healthcare and economic standpoint. for this reason, it is important to be able to quantify this untapped resource and introduce means to expedite the permanent residency status of these highly skilled immigrants working towards the nation's best interests. there are nearly , us-trained foreign physicians in the usa who are awaiting permanent resident status and are consequently limited in their scope due to visa restrictions. this cohort represents a large pool of us-trained, licensed physicians who are not being utilized to their fullest potential. devising means to expedite their legal immigration would bolster the healthcare workforce and prevent growing physician shortages in the usa, during and even after the ongoing global public health emergency. there are several limitations and potential sources of error that we would like to acknowledge, which limit the precision of our estimates. while the eb- immigrant visa is the pathway that most physicians are eligible for, a few others may have obtained permanent residency via alternative means which greatly reduced wait times, such as the eb- immigrant visa (alien of extraordinary ability) or the eb- immigrant visa (immigrant investor) category. there are personal circumstances which may alter our estimate, such as dual foreign physician households (only one family immigrant petition would be required), foreign physicians married to us citizens, foreign physicians who accepted a residency position outside of the nrmp match, and us-trained foreign physicians who may no longer be in practice (left the us, became disabled, or deceased). these factors are difficult to quantify based on publicly available databases. however, our effort appears to be the only publication attempting a quantitative analysis of this unique problem afflicting the us physician workforce. no potential conflict of interest was reported by the authors. http://orcid.org/ - - - x srikrishna varun malayala http://orcid.org/ - - - ramesh adhikari http://orcid.org/ - - - x about . % of the nrmp match applicants are from india, followed by china, . %. rest of the world constituted about . %. international medical graduates in the us physician workforce international medical graduate (imgs) immigration information for international medical graduates -year wait for indian immigrants with advanced degrees foreign medical graduates: a brief overview of the j- visa waiver program events as they happen, world health organization requirements for foreign medical graduates green card eligibility criteria ] national residency matching program. results and data: main residency match® results and data: main residency match® national resident matching program and educational commission for foreign medical graduates. charting outcomes in the match for international medical graduates main residency match® advance data tables for the main residency match® fsmb census of licensed physicians in the usa the contribution of chinese-educated physicians to health care in the usa how imgs have changed the face of american medicine american immigration council. foreign-trained doctors are critical to serving many u.s. communities key: cord- -pnjhi cu authors: foreman, stephen; kilsdonk, joseph; boggs, kelly; mouradian, wendy e.; boulter, suzanne; casamassimo, paul; powell, valerie j. h.; piraino, beth; shoemaker, wells; kovarik, jessica; waxman, evan(jake); cheriyan, biju; hood, henry; farman, allan g.; holder, matthew; torres-urquidy, miguel humberto; walji, muhammad f.; acharya, amit; mahnke, andrea; chyou, po-huang; din, franklin m.; schrodi, steven j. title: broader considerations of medical and dental data integration date: - - journal: integration of medical and dental care and patient data doi: . / - - - - _ sha: doc_id: cord_uid: pnjhi cu dental health insurance coverage in the united states is either nonexistent (medicare and the uninsured), spotty (medicaid) and limited (most employer-based private benefit plans). perhaps as a result, dental health in the united states is not good. what public policy makers may not appreciate is that this may well be impacting medical care costs in a way that improved dental benefits would produce a substantial return to investment in expanded dental insurance coverage. have been rising at double digit rates. most employers have been dropping health care coverage rather than expanding it ( kaiser family foundation ) . medicare trust funds are bankrupt (social security and medicare boards of trustees ). adding coverage would exacerbate an already alarming problem. medicaid funding is a major source of state government defi cits. many states are slashing medicaid coverage during this time of crisis (wolf ). improving medicaid dental coverage during times of budget crisis would meet substantial political resistance. strikingly, strong and increasing evidence suggests relationships between oral health and a range of chronic illnesses. for example, recent fi ndings show relationships between periodontal infl ammatory conditions and diabetes, myocardial infarction, coronary artery disease, stroke, preeclampsia and rheumatoid arthritis. this suggests that improved oral health may well have the potential to reduce the incidence of chronic diseases as well as their complications. if chronic disease incidence is reduced it may be possible to avoid medical care costs related to treating them. it would be important to know more about the extent to which improved oral health could reduce health care costs and improve lives. there are few, if any, studies of the costs of providing medicare dental benefi ts, the costs of improving the medicaid dental benefi t or the cost of providing dental insurance to the uninsured. there are a few studies that indicate that periodontitis increases medical care costs, perhaps by as much as % (ide et al. ; albert et al. ) . ideally there should be a controlled study to assess the benefi t of providing dental coverage through a government payer system. for a preliminary inquiry we can consider work already done and using some cost and benefi t estimates, determine whether it is possible that benefi ts of extending dental coverage may outweigh costs. the failure of medicare to cover dental care has engendered some (albeit not much) public debate. in , congress enacted the medicare prescription drug, improvement, and modernization act (medicare part d). by medicare provided $ . billion in benefi t payments for outpatient prescription drugs and medicaid paid . billion for outpatient prescription drugs (center for medicare and medicaid services ) . benefi ciaries provided billions more in the form of monthly part d premiums. the expense of the medicare prescription drug program and the controversy surrounding its enactment may well have eroded public support for increased medicare coverage. so while there has been no shortage of effort paid to improving medicare, the one common theme in all of the recent initiatives is that dental care has been conspicuously a new study by hedlund, jeffcoat, genco and tanna funded by cigna of patients with type ii diabetes and periodontal disease found that medical costs of patients who received maintenance therapy were $ . per year lower than patients who did not. cigna, research from cigna supports potential association between treated gum disease and reduced medical costs for people with diabetes, http://newsroom.cigna.com/newsreleases/research-from-cigna-supports-potential-association-between-treated-gum-disease-and-reduced-medical-costs-for-people-with-diabetes. omitted. as a result, million medicare recipients in (us census bureau ) continue to have no dental insurance coverage through medicare. medicaid dental coverage is an optional benefi t that states may or may not elect to provide. in medicaid, both the state and the federal government provide funds to cover healthcare services to eligible patients. the bulk of the money comes from the federal government. because the medicaid dollars are limited and coverage for systemic diseases has precedence, medicaid coverage of dental care has been spotty. even where it has been provided, payments to dental providers have been so low as to make it diffi cult or impossible for medicaid benefi ciaries to obtain adequate dental care (broadwater ) . the recession increased the number of medicaid eligible individuals nationwide. further, the federal budget defi cits of the past few years have reduced the federal contribution to state medicaid programs. the combination of increases in the number of benefi ciaries and diminished revenues has caused a number of states to eliminate or curtail medicaid dental coverage (ehow ; mullins et al. ) . the result, million medicaid benefi ciaries in the us (us census bureau ) in either had no dental insurance coverage or inadequate coverage. approximately million people in the united states do not have health insurance (kaiser family foundation ) . presumably, they have no dental insurance either. further, not every employer provides dental insurance. a cdc survey found that . % of adults do not have dental insurance coverage (centers for disease control ) . a montana survey found that % of employers who offer health insurance do not offer dental insurance coverage (montana business journal ) . in there were approximately million people enrolled in health insurance plans (us census bureau ) . if half (a rough combination of the cdc and montana percentages) of them do not have dental insurance it is likely that an additional million (nonelderly, non-poor) people in the us do not have dental insurance coverage. finally, the term "dental insurance" is actually a misnomer. dental policies cover routine treatments, offer discounts for more complex treatment and impose a low yearly on total payments. in fact, it has been called "part insurance, part prepayment and part large volume discount" (manski ) . effectively, many (if not most) people who have dental insurance fi nd it coverage to be quite restrictive. for example, many impose a small yearly cap ($ , is common) or large coinsurance amounts ( % for orthodontia, for example) (rubenstein ) . even with discounts it is easy for many people to exceed the annual limit. given the lack of dental insurance coverage it is not surprising that the status of oral health in the us is not particularly good. in approximately . % of adults between the ages of and had untreated caries, % had decayed, missing and fi lled tooth surfaces and more than one-half of adults had gingival bleeding (dental, oral and craniofacial data resource center of the national institute of dental and craniofacial research ) . three fourths of adults in the us have gingivitis and % have periodontitis (mealey and rose ) . if these levels of untreated disease were applied to most systemic diseases, there would be public outcry. over the past decade evidence has been building that there is a relationship between dental disease, particularly periodontal disease, and chronic illnesses. mealey and rose note that there is strong evidence that "diabetes is a risk factor for gingivitis and periodontitis and that the level of glycemic control appears to be an important determinant in this relationship" (mealey and rose ) . moreover, diabetics have a six times greater risk for worsening of glycemic control over time compared to those without periodontitis and, periodontitis is associated with an increased risk for diabetic complications. for example, in one study more than % of diabetics with periodontitis experienced one or more major cardiovascular, cerebrovascular or peripheral vascular events compared to % of the diabetic subjects without periodontitis (thorstensson et al. ) . also, a longitudinal study of type diabetics found that the death rate from ischemic heart disease was . times higher in subjects with severe periodontitis and the death rate from diabetic nephropathy was . times higher (saremi et al. ) . clinical trials have demonstrated that treatment of periodontal disease improved glycemic control in diabetics (miller et al. ) . moreover, investigations have found an association between periodontal disease and the development of glucose intolerance in non-diabetics (saito et al. ) . while it is diffi cult to establish causality and it is possible that other factors infl uence periodontal disease and medical complications, these studies suggest that treatment of periodontitis substantially improves health and greatly reduces medical complications related to diabetes. similarly, periodontitis is associated with cardiovascular disease and its complications including ischemia, atherosclerosis, myocardial infarction and stroke. a study by slade and colleagues found both a relationship between periodontitis and elevated serum c-reactive protein levels (systemic marker of infl ammation and documented risk factor for cardiovascular disease) as well as a relationship among body mass index, periodontitis and crp concentrations (slade et al. ) . hung and colleagues evaluated the association between baseline number of teeth and incident tooth loss and peripheral arterial disease. they determined that incident tooth loss was signifi cantly associated with pad, particularly among men with periodontal disease potentially implying an oral infection-infl ammation pathway (hund et al. ) . the same group of researchers used the population enrolled in the health professionals' follow-up study ( , men free of cardiovascular disease and diabetes at baseline) to assess the relationship between tooth loss and periodontal disease and ischemic stroke. controlling for a wide range of factors including smoking, obesity, and dietary factors, the researchers found a "modest" association between baseline periodontal disease history and ischemic stroke . as early as destefano and colleagues found that among subjects, those with periodontitis had a % increased risk of coronary heart disease relative to those without. the association was particularly high among young men. the authors questioned whether the association was causal or not, suggesting that it might be a more general indicator of personal hygiene and possibly health care practices (destefano et al. ) . in wu and colleagues used data from the first national health and nutrition examination survey and its epidemiologic follow-up study to examine the association between periodontal disease and cerebrovascular accidents. the study found that periodontitis was a signifi cant risk factor for total cva, in particular, for non-hemorrhagic stroke (wu et al. ) . in addition to diabetes and coronary artery disease, associations have been found between periodontal disease and rheumatoid arthritis and respiratory disease. this is not surprising given the role of periodontal disease in the production of infl ammation related proteins. dissick and colleagues conducted a pilot study of the associate ion between periodontitis and rheumatoid arthritis using multivariate regression and chi square tests. they found that periodontitis was more prevalent in patients with rheumatoid arthritis than in the control group and that patients who were seropositive for rheumatoid factor were more likely to have moderate to severe periodontitis than patients who were rf negative and also that patients who were positive for anti-cyclic citrullinated peptide antibodies were more likely to have moderate to severe periodontitis (redman et al. ) . paju and scannapeico investigated the association among oral biofi lms, periodontitis and pulmonary infections. they noted that periodontitis seems to infl uence the incidence of pulmonary infections, particularly nosocomial pneumonia in high-risk subjects and that improved oral hygiene has been shown to reduce the occurrence of nosocomial pneumonia. they found that oral colonization by potential respiratory pathogens, for possibly fostered by periodontitis and possibly by bacteria specifi c to the oral cavity contribute to pulmonary infections (paju and scannapeico ) . the implications for these fi ndings are profound. professionally, they suggest that managing patients with chronic illness and periodontal disease will require teamwork and a deeper knowledge base for dentists and for physicians (mealey and rose ) . dentists will need to be alert for early signs of chronic illness among their patients and physicians will need to be alert for signs of dental disease. both will need to consider wider treatment options than their specialty indicates. dentistry and medicine have operated as professional silos in the past. the relationship between dental disease and chronic medical conditions suggests that continued separation is detrimental to patient centered care. beyond treatment implications, there are extremely important health policy concerns. if treatment of periodontitis and other dental problems leads to reduced incidence of chronic illness, fewer complications from chronic diseases and reduced morbidity among chronically ill patients, increased access to dental services could signifi cantly reduce health care costs. the diseases associated with periodontitis are among the most common illnesses, the fastest growing and the most expensive diseases that we treat. a recent robert wood johnson report notes that approximately million americans have one or more chronic conditions, that the number of people with chronic conditions is expected to increase by % per year for the foreseeable future and that the most common chronic conditions include hypertension, disorders of lipid metabolism, upper respiratory disease, joint disorders, heart disease, diabetes, cardiovascular disorders, asthma and chronic respiratory infections (anderson ) (see fig . . ). one in four americans has multiple chronic conditions. ninety-one percent of adults aged and older have at least one chronic condition and % have two or more of them (anderson ) . people with chronic conditions account for % of all healthcare spending. seventy eight percent of private health insurance spending is attributable to the % of privately insured persons with chronic conditions. seventy three percent of healthcare spending for the uninsured is for care received by the one third of uninsured people who have chronic conditions. seventy nine percent of medicaid spending goes to care for the % of non-institutionalized benefi ciaries who have chronic conditions (anderson ) (see fig. . ). further, health care spending increases with the number of chronic conditions (anderson ) (see fig. . ). more than three fi fths of healthcare spending (two thirds of medicare spending) goes to care for people with multiple chronic conditions. those with multiple chronic conditions are more likely to be hospitalized, fi ll more prescriptions, and have more physician visits (anderson ) . in the american diabetes association estimated direct medical expenditures for diabetes at $ . billion: $ . billion for diabetes care, $ . billion for chronic complications and $ . billion for excess prevalence of general medical conditions. approximately % of direct medical expenditures were incurred by people over . indirect expenditures included lost workdays, restricted productivity mortality and permanent disability -a total of $ . billion. all told, diabetes was found to be responsible for $ billion of $ billion in total expenditures. per capita medical expenditures totaled $ , annually for people with diabetes and $ for people without diabetes (hogan et al. ) . more recently, dall and colleagues estimated that the us national economic burden of prediabetes and diabetes had reached $ billion in , $ million in higher medical costs and $ billion in reduced productivity. annual cost per case was estimated at $ , for undiagnosed diabetes and , for type diabetes (dall et al. ) . the costs of caring for people with diabetes have risen both because the numbers of diabetics has been increasing and because the per capita costs of care have increased. the number of diabetics increased from . million on to . million in (ashkenazy and abrahamson ) . a recent report by the unitedhealth group center for health reform & modernization provides a dire estimation -that more than % of adult americans could have diabetes ( %) or prediabetes ( %) by at a cost of $ . trillion over the decade. this compares with current estimates of % of the population with diabetes and % with prediabetes, or %. these estimates conclude that diabetes and prediabetes will account for % of total healthcare spending in at an annual cost of $ billion, up from an estimated $ billion in (unitedhealth center for health reform and modernization ) . average annual spending over the next decade by payer type is $ billion for private health insurance, $ billion for medicare, $ billion for medicaid and $ . billion for the uninsured. what about cardiovascular disease and rheumatoid arthritis? among the top ten health conditions requiring treatment for medicare benefi ciaries in approximately % of benefi ciaries suffered from hypertension, % from heart conditions, % had hyperlipidemia % had copd, % had osteoarthritis and % had diabetes (thorpe et al. ) . the american heart association estimates the cost of cardiovascular disease and stroke to be $ billion in direct expenditures and $ . billion for productivity losses due to morbidity and $ . billion in lost productivity due to mortality (present value of lost wages at %) (lloyd- ) . the centers for disease control estimates that during - million americans had selfreported doctor diagnosed arthritis, million of them with activity limitations (cheng et al. ) . cisternas and colleagues estimated that total expenditures by us adults with arthritis increased from $ billion in to $ billion in . most of the increase was attributable to people who had co-occurring chronic conditions (cisternas et al. ) . the cisternas study appears to aggregate all medical care expenditures by people with arthritis (which would include expenditures to treat diabetes and cardiovascular disease). an earlier cdc study focused on the direct and indirect costs in attributable to arthritis that estimated $ . billion in direct costs (medical expenditures) and $ billion in indirect costs (lost earnings) (yelin et al. ) . in short, current cost estimates for direct health care expenditures (excluding productivity losses) related to diabetes are approximately $ billion, for cardiovascular treatment, $ billion, and for rheumatoid arthritis, approximately $ billion (estimating that the $ . billion in costs have grown approximately % per year), a total of $ billion of the $ . trillion that will be spent in the us in . moreover, given current growth in the prevalence of diabetes, the unitedhealth estimate of $ million in spending for diabetes alone is not unreasonable. if health care costs attributable to diabetes, cardiovascular disease and rheumatoid arthritis only increase by % over the next decade (even given added demand produced by the aging baby boomer population), annual costs of these chronic diseases will exceed $ . trillion in . if we use the unitedhealth estimates for the proportions of diabetes costs paid by private insurance ( %), medicare ( %), medicaid ( %) and the uninsured ( %) and estimate total costs based on the studies projecting a % increase in years and a % increase in years we can obtain an estimate of future costs for treating diabetes, cardiovascular disease and arthritis. table . set forth below, summarizes these cost estimates. by medicare costs for these chronic illnesses would be approximately $ billion. the estimated costs to medicaid will be approximately $ billion. the costs for the uninsured will be approximately $ billion. any intervention that has the potential to substantially reduce these costs will produce meaningful results. unfortunately, even though there had been a substantial numbers of studies that show relationships between dental disease and chronic illness that are have been very few studies that actually test whether improved dental treatment reduces the incidence of chronic illness and complications due to chronic illness. the potential for large health care cost savings through an active and aggressive program of dental care is so large that such studies are clearly indicated. suppose, for example, that % of all medical care costs required to treat diabetes, cardiovascular disease and arthritis could be avoided through an active aggressive program of dental care. what this would mean is that in private health insurers could see a $ billion reduction in healthcare costs, medicare would see a $ . billion reduction and medicaid pay $ . billion reduction. recent health reform has provided for the issuance of health insurance to the uninsured by state exchanges. aggressive dental care that saved % of costs attributable to diabetes, cardiovascular disease and arthritis could save the exchanges $ billion per year. and, if greater proportions of costs can be saved or if the estimates of costs are low, potential benefi ts will be even larger. once again, it would be important to know whether aggressive dental care could produce such savings and how much. ide and colleagues found that people who were treated for periodontitis incurred % higher health care costs than those who were free of periodontal disease (ide et al. ) . similarly, albert, et al., found medical costs associated with diabetes, cardiovascular disease and cerebrovascular disease were signifi cantly higher for enrollees who were treated for periodontitis than for other dental conditions (albert et al. ) . additional studies of this nature would be important to support a measured approach to expanding dental coverage. so what do we mean by an aggressive dental treatment plan? suppose we were to provide dental insurance to all medicare benefi ciaries at the level of current private dental insurance coverage and strongly encourage benefi ciaries to receive dental treatment. suppose we were to provide for medicaid payment for all benefi ciaries at the level of current private dental insurance coverage. suppose health care insurers provided dental coverage in order to reduce their costs and that such coverage was consistent with current private dental insurance coverage. suppose health insurance companies, understanding the benefi ts from dental care, were to require their private employer customers to cover the costs of dental care. how much would all of this cost? how would it compare to the benefi ts that may be available? in order to estimate the potential costs of providing enhanced coverage for dental care we start use the cms estimates of national health care spending for dental services and statistical abstract of the us estimates for medicare enrollment, medicaid enrollment, private health insurance enrollment and uninsured persons. based on the estimate that half of private employers with health insurance provided dental insurance coverage we estimate that of the private health insurance enrollment one half would have dental insurance coverage and one half would not. table . sets forth the national health care expenditures for dental services in millions and enrollment in private dental plans, medicare, medicaid, the uninsured without health insurance and dental insurance, the uninsured with health insurance and dual eligibles. from this we derive a cost per enrollee for private dental insurance, medicare dental benefi ts and medicaid dental benefi ts. in order to estimate the annual cost of providing full dental coverage to medicare benefi ciaries we subtracted dual eligibles (who receive some dental insurance) from total medicare enrollees to determine the number of persons who would need coverage. in our example there were million medicare benefi ciaries including million dual eligibles. accordingly, the estimates would cover the million medicare benefi ciaries that are not dual eligible at a cost equal to the per capita cost of private dental insurance ($ . ) less amounts that medicare is already paying for dental services ($ . per person). the result provides an estimate of the cost of covering all medicare benefi ciaries for dental services at a level equivalent to private health insurance. using the example the cost of providing full dental insurance coverage to medicare benefi ciaries would have been $ . billion. in addition, we used the cms national health expenditure fi gures to determine administrative costs for private health insurance, medicare and medicaid as a percentage of program expenditures for medical care. we found that the administrative costs of the medicare program were . % on average for - . in order to fully estimate the cost of medicare dental coverage we added . % to the cost health insurers will be in the same position as medicare and medicaid regarding dental coverage. if quality dental coverage saves health care costs attributable to diabetes, cardiovascular disease and rheumatoid arthritis then the exchanges will have an incentive to provide quality dental coverage to reduce costs. accordingly, we estimated the cost of providing dental coverage equivalent to private dental insurance coverage through the exchanges. again we assume that the costs of such coverage will be equivalent to the number of uninsured persons multiplied by the annual per capita cost of coverage. for the example, this would refl ect coverage for million people at $ . per person, a total of $ . billion. with administrative costs, the cost of providing dental insurance coverage to the uninsured at a level equivalent to private dental coverage would be $ . billion. finally, given the evidence that improved dental care has the potential to reduce health care costs private health insurers may wish to expand health insurance to cover dental care. here, we estimate the cost of providing dental insurance to the % of the workforce whose employers currently do not provide dental insurance benefi ts. once again, we multiply the number of covered lives by the estimated annual per capita cost. for the example we estimate million adults will receive dental coverage at $ per person: $ billion for dental services and $ . billion for administrative costs or a total of $ . billion. of course, as noted a number of times above, these estimates are based on providing full "universal" dental insurance coverage at levels equivalent to current benefi t levels for private dental insurance. it may be that an appropriate package of dental services that deals specifi cally with periodontitis can be provided for less than the full cost of private dental insurance. once again, further research should provide better information. the health reform law does not attempt to provide coverage to all million people without health insurance. estimates are that only million people will be covered by the bill. even though this is the case we prepare our estimates using all million uninsured americans. indeed, the failure of % of employers to cover dental services may well constitute a classic externality in the market for health insurance. internalizing this externality may well provide better effi ciency. it is also possible that dental care for persons with greater incidence of chronic illness as is the case with medicare benefi ciaries may require even higher levels of spending per benefi ciary. again, it would be good to know scientifi cally if this is the case. as noted in sect. above, costs for diabetes, cardiovascular disease and arthritis will be $ billion for private health insurance, $ billion for medicare, $ billion for medicaid and $ billion for the uninsured. costs of providing "full" dental coverage will be $ . billion for medicare, $ . billion for medicaid, $ . billion for the uninsured and $ . billion for private health insurance. given this, if . % or more of the medicare costs can be "saved" through improved dental care, medicaid dental insurance will pay for itself and will provide a positive return on investment. see table . . similarly, private health insurers could justify providing dental insurance coverage to employees who do not have it so long as they spend . % or more of their chronic care costs for diabetes, cardiovascular disease and arthritis. on the other hand, it would appear that medicaid expansion would require cost savings of approximately % and that health care insurance coverage of the uninsured would require savings of approximately % in order to justify coverage. while it is possible, it may not be likely that full dental coverage would be justifi ed for these programs. of course, these estimates do not consider indirect costs in the form of lost wages or premature death. these costs are externalities to the health insurance programs. to the extent that they represent a social benefi t that a national dental insurance program might internalize, it would be appropriate to consider their impact in the cost-benefi t analysis. in any event, better understanding of the potential for deriving savings in health insurance costs related to chronic diseases like diabetes, cardiovascular disease and arthritis would be crucial to any determination whether to expand insurance coverage for dental care. heretofore the case for expanding medicare coverage to include dental care has taken the form of "benefi t" to patients rather than benefi t to health insurance programs and society and has been cast in emotional and political terms. for example, oral health america grades "america's commitment to providing oral health access to the elderly" (oral health america ) . in truth, there is no american commitment to providing oral health access to any age group, much less the elderly. rubenstein notes that "at least one commentator has suggested that the dental profession should join with senior citizen groups when the time is right to ask congress to expand medicare to cover oral health" (rubenstein ) . rubenstein emphasizes that "calls for action" are "mere words" unless they are accompanied by political actions that health policy professionals and the dental profession must help promote (rubenstein ) . another commentator has suggested that "as soon as the debate over medicare prescription drug coverage and, the debate to provide dental care coverage for the elderly may soon begin" (manski ) . rubenstein, again suggests that "the dental community must convince americans, and particularly aging boomers, that oral health is integral to all health, and for that reason, retiree dental benefi ts are an important issue". in truth, a decade of defi cit spending and public distaste for out of control program costs in the medicare and medicaid programs as well as the unpopularity of the process that was used to provide medicare prescription drug coverage (with perceived abuses by the health insurance and drug lobbies) and national health reform makes it unlikely that the public would be willing to approve expansions in insurance coverage for dental care "for its own sake" or "as the right thing" or to "benefi t seniors." what this political climate has produced is an arena in which a good idea that could provide appropriate return on investment for society might well be rejected out of hand based on political history of health insurance coverage. as a result, it is incumbent on policymakers, medical and dental research scientists and health economists to investigate and confi rm the potential savings that expansion of dental insurance coverage has the potential to produce and to develop hard evidence regarding potential costs of the expansion prior to, not as a part of, political efforts aimed at dental coverage expansion. a responsible, well informed effort to expand dental coverage may well go far to restore public confi dence in the health policy process. joseph kilsdonk and kelly boggs the adage of "putting your money where your mouth is" is often referenced when being challenged about public statements or claims. in this instance, we use it literally. in health care costs in us were $ . trillion. there have been numerous reports on health disparities, the burden of chronic diseases, increasing healthcare costs and the need for change. long-term economic benefi ts associated with the cost of care are dependent upon integrating oral health with medicine. this is particularly true as it relates to the management of those conditions which impact the economics of healthcare the most. as examples, % of medicare costs and % of medicaid costs are in managing chronic health conditions (partnership for solutions national program offi ce ) . more than % of the u.s. population has one or more chronic condition (cartwright-smith ) and in , % of medicare spending was on patients with fi ve or more chronic diseases (swartz ) . effective management of health care resources and information are critical to the economic well-being of our healthcare system. we can no longer afford to manage care in isolation. integration of care between medicine and dentistry holds much promise in terms of reducing the cost of care and an integrated medical-dental electronic healthcare record (iehr) is the vehicle that will lead to downstream cost savings. in the united states the center for medicare & medicaid services (cms) has conducted demonstration projects around chronic disease management. section of the benefi ts improvement and protection act of mandated cms to conduct a disease management demonstration project. april , , as an effort to reduce the cost of care and improve quality associated with chronic diseases, cms partnered with ten premier health systems to effectively manage chronic diseases in a medicare physician group practice demonstration (pgp). it was the fi rst pay-for-performance initiative for physicians under the medicare program (center for medicare and medicaid services ) . it involved giving additional payments to providers based on practice effi ciency and improved management of chronically ill patients. participants included ten multispecialty group practices nationwide, with a total of more than , physicians, who care for more than , medicare benefi ciaries (frieden ) . the chronic diseases that were targeted were based on occurrence in the population and included diabetes, heart failure, coronary artery disease, and hypertension (frieden under the pgp, physician groups continued to be paid under regular medicare fee schedules and had the opportunity to share in savings from enhancements in patient care management. physician groups could earn performance payments which were divided between cost effi ciency for generating savings and performance on quality measures phased in during the demonstration as follows: year , measures, year , measures and years and having quality measures. for each of the years only the university of michigan faculty group practice and marshfi eld clinic, earned performance payments for improving the quality and cost effi ciency of care. a large part of the success of this project was attributed to being able to extract, evaluate, and monitor key clinical data associated with the specifi c disease and to manage that data through an electronic health record (table . ). during the third year of the demonstration project marshfi eld clinic, using a robust electronic health record succeeded in saving cms $ million dollars; that's one clinic system in year. as a result of such demonstration projects and as of this writing, cms is looking to establish accountable care organization's as the medical front runners to new care delivery methods for quality and cost control. accountable care organization (aco) is a term used to describe partnerships between healthcare providers to establish accountability and improved outcomes for the patients. in a cms workshop on october , , don berwick, the administrator of cms, stated "an aco will put the patient and family at the center of all its activities…" an emerging model of an aco is the patient-centered medical home (pcmh). pcmh is at the center of many demonstration projects. acos were derived from studies piloted by cms. since funds provided by cms, do not cover routine dental care as part of the patient management or quality and cost objectives cms aco studies are limited if they become models for the pcmh, due to the exclusion of dental. more recently, organizations representing the major primary care specialtiesthe american academy of family practice, the american academy of pediatrics, the american osteopathic association, and the american college of physicianshave worked together to develop and endorse the concept of the "patient-centered medical home," a practice model that would more effectively support the core functions of primary care and the management of chronic disease (fisher ) . in geisinger health system, kaiser permanente, mayo clinic, intermountain healthcare and group health cooperative announced they will be creating a project called the care connectivity consortium. this project is intended to exchange patient information. although progressive in their approach their project does not include dental. these benefi ts however, are yet to be adapted in the arena of oral health. as of this writing, dentistry remains largely separate from medical reimbursement mechanisms such as shared billing, integrated consults, diagnosis, shared problem lists, and government coverage. for example, cms does not cover routine dental care. dentistry is also working to establish its own "dental home" with patients. however to reap the economic benefi ts of integrated care, a primary care "medical-dental" home is what needs to be created. according to an institute of oral health report ( ) it is widely accepted across the dental profession that oral health has a direct impact on systemic health, and increasingly, medical and dental care providers are building to bridge relationships that create treatment solutions. the case for medical and dental professionals' comanaging patients has been suggested for almost the past century, in william gies reported that "the frequency of periodic examination gives dentists exceptional opportunity to note early signs of many types of illnesses outside the domain of dentistry" (gies ) . as described by dr. richard nagelberg, dds "the convergence of dental and medical care is underway. our patients will be the benefi ciaries of this trend. for too long, we have provided dental care in a bubble, practicing -to a large degree -apart from other health-care providers. even when we consulted with our medical colleagues, it was to fi nd out if premedication was necessary, get clearance for treatment of a medically compromised patient, or fi nd out the hba c level of a diabetic individual, rather than providing true patient co-management. we have made diagnoses and provided treatments without the benefi t of tests, reports, metrics, and other information that predict the likelihood of disease development and progression, as well as favorable treatment outcomes. we have practiced in this manner not due to negligence, but because of the limitations of tools that were available to us" (nagelberg ) . integrated medical/dental records need to be a tool in a providers' toolbox. in the case of marshfi eld clinic, dental was not included in their past cms demonstration project as dental is not a cms covered benefi t, and thus not part of the demonstration. however, as a leader in healthcare, the marshfi eld clinic recognizes the importance of data integration for both increased quality and cost savings. "marshfi eld clinic believes the best health care comes from an integrated dental/medical approach," said michael murphy, director, business development for cattails software. integration enhances communication between providers and can ultimately lead to better management of complex diseases with oral-systemic connection, avoidance of medical errors, and improved public health. while the cms pgp and other demonstration projects along with independent studies have shown to improve quality and reduce costs through integration, greater results may be afforded if studies are not done in isolation from dental data. in fact, if healthcare does not fi nd a way to manage the systemic nature of the pathogens known to the oral cavity the economic impact and cost savings around chronic disease management will hit a ceiling. the economic opportunity of having clinical data for integrated decision making is readily identifi ed by the insurance industry. the effective management of clinical data around chronic and systemic oral and medical disease as part of an iehr is the greatest healthcare cost savings opportunity associated with such a tool. the insurance industry sustains itself through risk management [obtaining best outcomes] using actuarial analysis [data] and controlling costs [reduction of costs] in order to ensure coverage [profi tability]. as such they have pursued the economic and outcome benefi ts of integrated medical -dental clinical decision making. as an example, in there was a study conducted by the university of michigan, commissioned by the blue cross blue shield of michigan foundation ( ) , the study included , blue cross blue shield of michigan members diagnosed with diabetes who had access to dental care, and had continuous coverage for at least year. with regular periodontal care, it was observed diabetes related medical costs were reduced by %. when compounding chronic health complications were also examined, the study showed a % reduction in cost related to the treatment of cardiovascular disease in patients with diabetes and heart disease. a % reduction in cost related to treatment of kidney disease for patients with diabetes and kidney disease. and a % reduction in costs related to treating congestive heart failure for patients with diabetes and congestive heart failure. according to a joint statement by lead researchers, and blue cross blue shield of michigan executives, "our results are consistent with an emerging body of evidence that periodontal disease…it addresses quality of care and health care costs for all michigan residents." also, at the institute for oral health conference in november joseph errante, d.d.s., vice president, blue cross blue shield of ma reported that blue cross blue shield of massachusetts claims data showed medical costs for diabetics who accessed dental care for prevention and periodontal services averaged $ /month, while medical costs for diabetics who didn't get dental care were about $ /month (errante ) . similarly insured individuals with cardiovascular diseases who accessed dental care had lower medical costs, $ /month lower than people who did not seek dental treatment (errante ) . the cost is $ less per visit for those diabetics who accessed prevention and periodontal services. those savings could be translated into access to care or additional benefi ts for more individuals. in the case of neonatal health there is similar research. over % of all births in the u.s. are delivered preterm, with many infants at risk of birth defects ( martin et al. ) . according to a january statement issued by cigna, announcing their cigna oral health maternity program, "the program was launched in response to mounting research indicating an increased probability of preterm birth for those with gum disease. these research-based, value-added programs are designed to help improve outcomes and reduce expense" (cigna ) . the program was initially designed to offer extended dental benefi ts free of charge to members who were expecting mothers, citing "research supporting the negative and costly impact periodontal disease has on both mother and baby." according to research cited by cigna, expecting mothers with chronic periodontal disease during the second trimester are seven times more likely to deliver preterm (before th week), and the costs associated with treating premature newborns is an average of times more during their fi rst year, and premature newborns have dramatically more healthcare challenges throughout their life. cigna also cited the correlation between periodontal disease and low birth weight, pre-eclampsia, gestational diabetes as additional rationale to support extended dental benefi ts to expecting mothers. six months later cigna initiated well aware for better health, an extended benefi ts free of charge program for diabetic and cardiovascular disease patients aimed at "turning evidence into action by enhancing dental benefi ts for participants in disease management" programs. it is interesting to note, not only does cigna offer extended dental benefi t to targeted groups, they also reimburse members for any out-of-pocket expenses associated to their dental care (co-pays, etc.) in , columbia university researchers conducted a -year retrospective study of , aetna ppo members with continuous medical and dental insurance, exhibiting one of three chronic conditions (diabetes mellitus, coronary artery disease, and cerebrovascular disease) (aetna ) . researchers found members who received periodontal treatments incurred higher initial per member per month medical costs, but ultimately achieved signifi cantly lower health screening (episode risk group/erg) risk scores than peers receiving little or no dental care. convinced by the data and understanding lower risk scores ultimately leads to healthier people and cost savings, aetna initiated the dental/medical integration (dmi) program in . aetna's dmi program offers enhanced benefi ts in the form of free-of-charge extended benefi t dental care to aetna's . million indemnity, ppo and managed choice medical plan members, specifi cally targeting members deemed at-risk, including those who are pregnant, diabetic, and/or have cardiovascular disease and have not been to a dentist in year as a result of various outreach methods during the pilot, % of at-risk members who had not been to a dentist in the previous months, sought dental care (aetna ) . "the fi ndings from this latest study we conducted continue to show that members with certain conditions who are engaged in seeking preventive care, such as regular dental visits, can improve their overall health and quality of life," said alan hirschberg, head of aetna dental (aetna ) . delta dental of wisconsin understands the connection between oral and systemic health and has created a program that is designed to offer members with certain chronic health conditions the opportunity to gain additional benefi ts. more than , groups now offer delta dental of wisconsin's evidence-based integrated care plan (ebicp) option (delta dental of wisconsin ) . ebicp provides expanded benefi ts for persons with diseases and medical conditions that have oral health implications. these benefi ts include increased frequency of cleanings and/or applications of topical fl uoride. they address the unique oral health challenges faced by persons with these conditions, and can also play an important role in the management of an individual's medical condition. ebicp offers additional cleanings and topical fl uoride application for persons who are undergoing cancer treatment involving radiation and/or chemotherapy, persons with prior surgical or nonsurgical treatment of periodontal disease and persons with suppressed immune systems. the ebic offers additional cleanings for persons with diabetes and those with risk factors for ie, persons with kidney failure or who are on dialysis and for women who are pregnant. the iehr provides the insurance industry in partnership with the healthcare industry an integrated tool to facilitate these health and subsequently economic outcomes across medicine and dentistry. in addition to the anticipated savings through better outcomes using integrated clinical data, an example of a positive economic outcome associated with an integrated record as related to increased effi ciency and patient safety is found in the united states veterans administration (va) hospitals and clinics. the va is one of the few institutions that have implemented the shared electronic medical-dental record successfully. the va has the ability to be the "one stop shop" for their patients. an april press release published on the department of veterans affairs website highlighted the success of va's health information technology in terms of cost reductions and "improvements in quality, safety, and patient satisfaction" (department of veterans affairs ) . the press release spotlighted a recent study conducted by the public health journal, health affairs, which focused on va's health it investment from to . the study confi rmed that while va has spent $ billion on their technology initiative, a conservative estimate of cost savings was more than $ billion. after subtracting the expense of the it investment, there was a net savings of $ billion for the va during the years covered by the study (mcbride ) . furthermore, the study estimated that "more than percent of the savings were due to eliminating duplicated tests and reducing medical errors. the rest of the savings came from lower operating expenses and reduced workload." independent studies show that the va system does better on many measures, especially preventive services and chronic care, than the private sector and medicare. va offi cials say "its [integrated] technology has helped cut down hospitalizations and helped patients live longer" (zhang ) . recently, the journal of obstetrics and gynecology reported on a tragic loss of life due to the systemic nature of oral health. a study found oral bacteria called fusobacterium nucleatum was the likely culprit in infecting a -year-old woman's fetus through her bloodstream (carroll ) . the doctors determined that the same strain of oral bacteria found in the woman's mouth was in the deceased baby's stomach and lungs. integrated records would provide critical data to the obstetrician including oral health issues and when the patient had her last dental exam. how does one measure the economic impact of a life not lived and another derailed by such tragedy? in a randomized controlled study, lopez et al. ( ) determined that periodontal therapy provided during pregnancy to women with periodontitis or gingivitis reduced the incidence of preterm and of low birth weight. the institute of medicine and national academies estimate that preterm births cost society at least $ billion annually . data integration of the iehr enables the effective management between the dentist and obstetrician to ensure proper periodontal therapy has been provided during pregnancy. such management based on the lopez et al. study, will have direct impact in reducing the prevalence per preterm births leading to reduced health care costs. there have also been studies indicating a correlation between poorer oral hygiene or defi cient denture hygiene and pneumonia or respiratory tract infection among elderly people in nursing homes or hospitals (rosenblum ; ghezzi and ship ; scannapieco ) . one such study of elderly persons in two nursing homes in japan (adachi et al. ) concluded that "the number of bacteria silently aspirating into the lower respiratory tract was lower in the group who received professional oral care, which resulted in less fatal aspiration pneumonia in that group." over the month period of the study, of the patients receiving professional oral care, % died of pneumonia versus . % of the patients that died of the same cause who maintained their own oral hygiene. lack of access is certainly a key factor to consider. however, lack of available data respective to the interrelationship between oral health and systemic health also contributed to the apathy in these cases. as identifi ed above, complications are correlated to cost. as conditions compound, costs go up. marshfi eld clinic, as part of their iehr is creating a shared problem list that identifi es both oral and medical conditions and history to recent visits and medication lists for monitoring at point of care [be it a medical or dental visit], such cross access to clinical data and care management milestones serves as a tool to prevent conditions from compounding and escalating costs such as those described above. several other areas of economic impact will be seen as iehr's become broadly deployed. some of these are listed as follows: medication management. a great deal of provider and allied support time is • spent obtaining medication information between dentistry and medicine [and vice versa] including current medications, contraindications, tolerances, etc. marshfi eld clinic cattails software has created a dashboard that readily identifi es this for both the medical and dental providers. not is time saved but chances for complications or escalation of conditions is reduced [both of which impact cost]. for example an integrated record allows medical providers treating respiratory infections to include or exclude oral fl ora as the possible source of the infection which would lead to more knowledgeable prescribing decision on the antibiotic used. coordination of care has a direct impact on cost for the system and the patient. • for example, in . % of the us population aged years and older that was diagnosed diabetes had been to the dentist in the past year (healthy people ( )). the us government's program healthy people includes an initiative to increase the proportion of people with diagnosed diabetes who have at least an annual dental examination. the american diabetes association recommends that diabetic patients be seen semi-annually and more if bleeding gums or other oral issues are present. the american diabetes association also recommends the consultation between the dentist and doctor to decide about possible adjustments to diabetes medicines, or to decide if an antibiotic is needed before surgery to prevent infection. the target from the healthy people is a % improvement at . %. integrated medical/dental records could allow for the coordination of efforts between providers to include communication of treatment plan and services leading to quicker resolution, increased patient compliance, and less patient time away from work or home and potentially less travel. similarly, integrated records also create a platform to integrate clinical appointing • between medicine and dentistry. as such, combative patients or severely disabled patients needing anesthesia in order for care to be delivered can be treated with one hospital sedation vs. multiple sedations. family health center of marshfi eld, inc. (fhc) dental clinics shares an iehr with marshfi eld clinic and uses it integrated scheduling feature to complete dental care, lab work, ent care, woman's health, preventive studies, all in one visit. follow up care management can be more focused and coordinated. for example, • without the knowledge or dental conditions, medical providers could spend months attempting to control diabetes with periodontal disease. however, with access to an iehr, the practitioner or allied care manager can determine patient's oral health status immediately to determine possible infl uence of periodontal disease. similarly an iehr with a shared patient data dashboard brings to light history • and physical examination data without having to have patients be the historian to their physician on their last dental visit or for the dentist to have to rely on the patient's recall of medications or medical diagnosis. for example, if an integrated record saved providers min per hour of patient care, that would be min per day. imagine giving a physician or dentist min more a day. in a capitated system, this allows for more patients to be seen in a day for roughly the same amount of expenditure. in a production based clinic this allows more patients to be seen and more charges per day. in either case, the investment into informatics is covered. in an underserved area, more patients get care quicker, which creates the opportunity for quicker resolution, which can lead to a healthier society, which in turn may lead them back to a productive livelihood sooner. an iehr results in one system for acquisition, orientation, training and support. • pc based owners who also own a mac and mac owners who also have to operate a pc can relate. need we say more? imagine if your pc function just like a mac [or your mac function just a pc]. no cross learning of software quirks. not having to purchase two separate units to begin with. reduced costs, increased space. not having to jump from one computer to the other computer to get data from one data from another to create a report. not having to call two separate computer companies for service or updates. third party coordination. having an iehr creates a platform for interfacing • with third party payers. a common system and language for timely reimbursement. in part, the result of an iehr is driving the diagnostic coding for dentistry. such an integrated interface provides a tool to bridge with healthcare payors that historically kept payment as segregated as the oral and medical health professions. the iehr overcomes that limitation. timely payment, consolidation of payment, expansion of covered patient and provider benefi ts based on clinical integration, and a viable system for interfacing are all potential economic benefi ts of iehr clinical data. the iehr creates new horizons for research that will lead to cost saving discov-• eries. as example, knowing the benefi ts of research, marshfi eld clinic research foundation (mcrf) has created an oral and systemic health research project (oshrp). the creation of oshrp, led by dr. murray brilliant, will allow mcrf to capitalize on its existing and growing strengths in the areas of complex disease interactions and personalized health care (phc) to advance oral health and the health of the rest of the body. the oshrp has three specifi c goals: understand the connections between oral and systemic health (diabetes, heart disease, pre-term births) understand the causes of oral diseases and determine the effect of genetics, diet, water source (well/city + fl uoridation) and microbiome. understand how improving oral health aids systemic health (comparative effectiveness) and bring personalized health care (phc) to the dental arena. the oshrp research resource will be unique in the nation. as mcrf has done • with other projects, it will share this resource with qualifi ed investigators at other academic institutions both within and outside of wisconsin. oshrp will advance scientifi c knowledge, improve healthcare and prevention, reduce the cost of oral healthcare, and create new economic opportunities. such knowledge will have a direct economic impact on the cost of care and care management. the iehr creates an ability to have an integrated patient portal to comprehen-• sively maintain their health. portals are becoming more and more popular in the healthcare industry as a means to helping maintain compliance with care management recommendations and preventative procedures. portals provide patents a tool to stay up to date on their care and recommendations. portals can take iehr clinical data, adapt it through programming, and provide creative visual reinforcement for patients as they monitor their health status. the more patients engage in owning their health status, the more preventative services are followed through with. the more medicine and dentistry can leverage the prevention potential [which insurance companies have come to realize] the more likely costly conditions can be avoided. the link between oral health and systemic health is well documented. the separation of dental and medical is not a sustainable model in modern healthcare delivery. a new model of integrated care is necessary. aristotle said, "the whole is greater than the sum of its parts." increased access to combined medical and dental histories and diagnosis at the providers' fi ngertips makes vital information available. shared diagnosis between physicians and dentists could aid in formulating interventions and to accelerate decision making abilities by allowing for prioritizing of medical/ dental procedures. clinical management and treatment of the patient would be expedited with immediate access to both records. quality could be improved through a complete picture of the patient through the dashboard. all of which have a direct or indirect economic benefi t. the iehr will be the tool that facilitates such delivery and the studies and scenarios described in these pages point to signifi cant economic benefi ts to patients, payors, and providers. if increased access, multi-provider monitoring, shared problems lists with enhanced decision making abilities from iehr could reduce healthcare costs. the greatest cost reduction will be with using the iehr to manage chronic disease. a combined dental-medical electronic record with a shared data informatics platform is most likely to yield the best long-term economic solution while maintaining or enhancing positive patient outcomes. this section reveals viewpoints from a variety of medical and dental providers. one section focuses on optimal use of ophthalmic imaging, which should show how that the challenges of clinical data integration go beyond those encountered in the effort to bring oral health and systemic health together. wendy e. mouradian , suzanne boulter , paul casamassimo , and valerie j. harvey powell oral health is an important but often neglected part of overall health. historically separate systems of education, financing and practice in medicine and dentistry fuel this neglect, contributing to poorer health outcomes for vulnerable populations such as children, while increasing costs and chances for medical error for all patients. advances in understanding the impact of oral health on children's overall health, changing disease patterns and demographic trends strengthen the mandate for greater integration of oral and overall healthcare, as reviewed in two recent institute of medicine reports (iom a, b ) . the pediatric population could realize substantial benefit from oral disease prevention strategies under a coordinated system of care enhanced by integrated electronic health records (ehr). this approach would benefit all children but especially young children and those from low socioeconomic, minority and other disadvantaged groups who are at higher risk for oral disease and difficulties accessing dental care. this section focuses on the pediatric population and the need for close collaboration of pediatric medical and dental providers. first we consider how a child's developmental position and their parents' level of understanding might affect oral health outcomes. next we address the importance of children's oral health and the urgency of seizing missed opportunities to prevent disease. we then briefl y outlines some steps to preventing early childhood oral disease utilizing some of the many health providers that interact with families. finally we examine one pediatric hospital's approach to choosing an integrated ehr technology. children have unique characteristics which distinguish their needs from those of adults. children's developmental immaturities may increase their risks for poor oral health outcomes ( fig. all children, but especially young children, are limited in their ability to care for their own health and must depend upon adults. a child's parent/caregiver may also lack basic oral health knowledge and an awareness of their child's oral health needs, and/or suffer from poor oral health themselves. low oral health literacy is prevalent among patients and health professionals alike in america; individuals of low socioeconomic status or from ethnically diverse backgrounds may be at particular risk for low oral health literacy (iom a ) . without appropriate education, a parent…. may not correctly interpret a child's symptoms or signs of oral disease • may not know that caries is an infectious disease that can be spread to a child by • sharing spoons, for example, may not know the potential value of chewing gum with xylitol, • may not fully grasp the importance of good oral health hygiene habits, • may not grasp the consequences of a child consuming quantities of sugared • foods or beverages, may have diffi culty controlling the child's consumption of sugared foods or bev-• erages in or out of the home, may not realize the consequences of chronic use of sugared medications, • may not know the potential for systemic spread of disease from a toothache, or • for liver damage due to overuse of acetaminophen or other analgesics, may not grasp the long-term consequences of early childhood caries, • may live in a community without fl uoride in the tap water and not know about • alternative sources of fl uoride, may overlook oral health due to the stress of living in poverty, • may be fearful of dentists or oral health care due to their own experiences, • may have diffi culty locating a dental provider accepting public insurance, or • have other problems navigating the health care system. parents in turn depend on access to medical and dental providers with current understanding of the most effective ways to prevent caries and promote the child's oral and overall health. an important element in helping families is the provision of culturally-sensitive care to a diverse population. children are the most diverse segment of the population with % from minority backgrounds compared with % of the overall population (us census bureau ) . the separation of medical and dental systems and the lack of shared information can create additional barriers for families, especially for those with low health literacy or facing linguistic or cultural barriers. all pediatric health professionals have increased ethical and legal responsibilities to promote children's health, including advocacy for them at the system level (mouradian ) . although many factors can infl uence children's oral health outcomes, caries is largely a preventable disease. despite this, national trends and other data on broader considerations of medical and dental data integration children's oral health attest to this persistent national problem ) . some important facts include the following…. caries is the most prevalent chronic disease of childhood, • caries is a preventable disease unlike many chronic diseases of childhood, • yet according to (nicdr ) % of children - have had dental caries in • their primary teeth; % of children - have untreated dental caries. further, " % of children - have had dental caries in their permanent teeth; % of children - have untreated decay." overall "[c]hildren - have an average of . decayed primary teeth and . decayed primary surfaces," the latest epidemiologic evidence shows increasing rates of caries for young-• est children, reverse from the healthy people goal of decreasing caries. according to (nicdr ), overall "dental caries in the baby teeth of children - declined from the early s until the mid s. from the mid s until the most recent ( ) ( ) ( ) ( ) ( ) ( ) ) national health and nutrition examination survey, this trend has reversed: a small but signifi cant increase in primary decay was found. this trend reversal was more severe in younger children." disparities in children's oral health and access to care persist by age, income • level, race and ethnicity, and parental education level (edelstein and chinn ) . of concern, the latest increase was actually in a traditionally low-risk group of young children (dye and thornton-evans ) . the human and economic costs of early childhood caries are substantial • (casamassimo et al. ) . according to catalanotto ( ) , health consequences include… extreme pain, spread of infection/facial cellulitis, even death (otto - ) diffi culty chewing, poor weight gain falling off the growth curve (acs et al. - ) risk of dental decay in adult teeth (broadbent et al. - ; li and wang ) crooked bite (malocclusion) -children with special health care needs (cshcn) may be at higher risk for oral • disease and diffi culties accessing care. analyzing data from the national survey of children with special health care needs, (lewis ) found that "cshcn are more likely to be insured and to receive preventive dental care at equal or higher rates than children without special health care needs. nevertheless, cshcn, particularly lower income and severely affected, are more likely to report unmet dental care need compared with unaffected children." children who were both low-income and severely affected had . times the likelihood of unmet dental care needs, dental care is the highest unmet health care need of children; . million children • had unmet dental care needs because families could not afford care compared with . million with unmet medical needs for the same reasons (cdc ) , according to the national survey of children's health, children are . times as • likely to lack dental as medical insurance (lewis et al. ) , there is evidence that children who get referred to a dentist early may have lower • costs of care and disease. savage et al. ( ) reported that children "who had their fi rst preventive visit by age were more likely to have subsequent preventive visits but were not more likely to have subsequent restorative or emergency visits" and concluded that preschool "children who used early preventive dental care incurred fewer dentally related costs," ramos- gomez and shepherd ( • ) , in their "cost-effectiveness model for prevention of early childhood caries," conclude that preventive ecc interventions could reduce ecc by - % for a particularly vulnerable population of children, and that part of the costs of interventions will be offset by savings in treatment costs. as these facts convey, and the deaths of more than one child from consequences of untreated caries make painfully clear, there is an urgent need for more attention to the oral health needs of children. a more coordinated system for oral health care including integrated ehr would be an important advance. a glance at table . , an ideal model, reveals that intervention should begin before birth and that a range of medical and oral health professionals can contribute to the child's oral health. early intervention is necessary because of the transmissibility of cariogenic bacteria from mother/caregiver to infant, and importance of oral health practice in preventing disease. the following professionals may be involved: • pediatric medical provider family physician pediatrician pediatric nurse nurse practitioner in pediatric/family practice physician assistant in pediatric /family practice - other appropriate allied health professionals • the availability of some of these professionals can be affected socioeconomic status, health insurance, place of residence, or by a child's special health care need. one obvious limitation on developing a "relay" as in table . , with a "hand-off" from family care to obstetric care to pediatric care is the education of the medical providers. as part of pre-conception and perinatal healthcare, providers should address oral health, but may lack the knowledge to do so. additionally, as noted by ressler-maerlaender et al. ( ) , "some women may believe that they or their table . timeline of some oral health interventions to prevent early childhood caries (ecc) -birth to years age (marrs et al. ; lannon et al. ; han et al. ; ezer et al. ; aap ; mouradian et al. ) child's age intervening professional(s) planning conception, prenatal and perinatal family physician the physician and/or obstetric provider educates mother-to-be about good maternal oral hygiene and infant oral health issues, including transmissibility of caries. mother's dentist assesses and treats caries, gingivitis or other oral health problems and educates the mother-to-be obstetrician/nurse midwife obstetric nurse general dentist obstetric nurse obstetric nurse advises new mother to chew xylitol gum, limit salivary contact between mother and infant, and help child avoid sugar intake (exposure) while asleep and from common sugar sources (medicines, sugared water, bottle feeding on demand at night with fl uid other than water -following tooth eruption, certain foods), and to schedule dental exam at year age months pediatric medical provider first dental examination recommended by aapd when the fi rst tooth comes in, usually between to months pediatric/general dentist educate mother about optimal fl uoride levels . assess the woman's oral health status, oral health practices, and access to a dental home; . discuss with the woman how oral health affects general health; . offer referrals to oral health professionals for treatment; . educate the woman about oral health during pregnancy, including expected physiological changes in the mouth and interventions to prevent and relieve discomfort; and . educate the woman about diet and oral hygiene for infants and children and encourage breastfeeding a combination of anticipatory guidance, with continuity from prenatal and perinatal care to pediatric care, can help move infant oral health from "missed opportunities" to "seized opportunities." others who may be of assistance to families in closing these gaps are professionals at the women, infants and children's (wic) supplemental nutrition program, early head start/head start and neurodevelopmental/birth to three programs. together medical, dental and community professionals can help create a system of care to improve maternal and child oral health. for the envisioned model in table . to be realized, the mother requires access to a general dentist with accurate information on her oral health during pregnancy and on her infant's oral health, including the need for an early dental visit. the mother and child then need access to a pediatric medical provider who will provide oral health screening/counseling, and who will guide the family to establishing the child's dental home by age . success in dental referral requires access to a pediatric or general dentist willing and able to provide infant oral health. (dela cruz et al. ) , in a discussion of the referral process mentioned that among the factors in assessing the likelihood of a dental referral were the medical providers' "level of oral health knowledge, and their opinions about the importance of oral health and preventive dental care." since young children are much more likely to access medical than dental care, the medical provider plays an important role in promoting children's oral health. (catalanotto ) recommends, as part of a pediatric well child checkup: an oral screening examination, • a risk assessment, including assessment of the mother's/caregiver's oral health, • application of fl uoride varnish • anticipatory guidance (parental education) including dietary and oral hygiene • information, attempted referral to a dental home. • the aap recommends that child healthcare providers be trained to perform an oral health risk assessment and triage all infants and children beginning by months of age to identify known risk factors for early childhood caries (ecc). the oral health component of pediatric care is integrated into the aap's "recommendations for preventive pediatric health care (periodicity schedule)" (aap ) . to what extent are medical and dental and providers aware of recommendations for a fi rst dental visit for a child by age one, as recommended by the aap, the american academy of pediatric dentistry (aapd), and the american dental association? (wolfe et al. ) reported that % of licensed general dentists in iowa were familiar with the aapd age dental visit recommendation and that most obtained the information through continuing education; % believed that the fi rst dental visit should occur between and months of age. however, according to (caspary et al. ) , when pediatric medical residents were asked the age for the fi rst dental visit, the average response was . years, while % reported received no oral health training during residency. in a national survey of pediatricians ) reported that less than % of had received oral health education in medical school, residency, or continuing education. finally (ferullo et al. ) surveyed allopathic and osteopathic schools of medicine and found that . % reported offering less than h of oral health curriculum, while . % offered no curriculum at all. other workforce considerations relevant to preventing early childhood caries include the training of dentists in pediatric oral health (seale et al. ) , the number and diversity of the dental workforce, the number of pediatric dentists, and the use of alternative providers such as dental therapists, expanded function dental assistants and dental hygienists (mertz and mouradian ; nash ) . examples of integrated care models do exist, such as that presented by (heuer ) involving school-linked and school-based clinics with an "innovative health infrastructure." according to heuer, "neighborhood outreach action for health (noah)" is staffed by two nurse practitioners and a part-time physician to provide "primary medical services to more than , uninsured patients each year" in scottsdale, arizona. heuer counts caries among the "top ten" diagnoses every year. mabry and mosca ( ) described community public health training of dental hygiene students for children with neurodevelopmental/intellectual disabilities. they mentioned that the dental hygiene students had worked together with school nurses and "felt they had impacted the school nurses' knowledge of oral disease and care." the decision to acquire an integrated ehr as pediatric clinicians (both medical and dental) work more closely together, they require appropriate ehr systems that integrate a patient's medical and dental records. following is a set of local "best practices" from nationwide children's hospital in columbus, ohio, which may help other children's hospitals in planning acquisition of an integrated pediatric ehr system. integrated (medical-dental) ehr technologies are becoming more widely available outside the federal government sector (see integrated models e and e in fig. . ). nationwide children's 'drivers' for the acquisition process were, in : . minimize registration and dual databases . patient registration takes time and requiring both a stand-alone dental and a medical patient registration inhibits cost-effective fl ow of services. integration allows for the use of single demographics information for all clinics in the comprehensive care system serving the patient. clinicians always have an updated health history on patients, if they have been a patient of record. if not, and for a dental clinic that sees walk-ins, a brief "critical" dental health history can be completed on paper by a parent and scanned into the emr. in designing an integrated medical-dental record for patients of record, the system can sort essential health history elements into a brief focused dental history without the detail needed by other medical specialty clinics. kioskdriven electronic health histories for those children who are new to clinic similar to those used in airline travel could be considered if feasible in busy clinics. . for charting, no more key/mouse strokes than with paper . some commercial dental record products try to accomplish too much. moving from paper to electronics should be driven in part by effi ciencies. the tooth chart, which is an essential part of any dental record, must be such that examination fi ndings can be transferred quickly and accurately to either paper or electronic capture. a helpful exercise is visualization of the functionality of the charting process, including both the different types of entries (caries, existing restorations and pathology) and how these are entered in the paper world. if charting will be able to be used for research the system should be able to translate pictures to numerical values, often a complex programming function. dental practitioners and faculty may want to use drawings of teeth or graphics of surfaces because that is their current comfort level. a true digital charting is possible with no images of teeth, but some habits are hard to change. . maximizing drop downs with drop down building possible . duplication of paper chart entries using drop downs which can be upgraded as more clinical entities are found is a staple of an emr. the paper process usually relies on a clinician's wealth of medical-dental terms since inclusion of every possible, or even the most common fi ndings, is prohibitive on a paper chart. the emr drop down requires front-end loading of the most common clinical fi ndings with opportunity for free-hand additions. being able to add terms to any drop down is a needed capability. . don't design a system for uncommon contingencies, but for your bulk of work . a pediatric dental record should be primarily designed around dental caries, with secondary emphases on oral-facial development (orthodontics) and a lesser capability to record traumatic injuries and periodontal fi ndings. these second and third level characteristics can be hot-buttoned and should not drive the design of the basic system which is caries charting for % of our patients. sadly in most dental schools, the chart is slave to every teaching form, few of which ever exit with the dds into practice! these forms may have little relationship to patient care and only create "signature black holes" that need to be addressed, usually after treatment is completed. . progress notes should be designed for the routine entries with free-hand modification possible . student learners tend to write too much and a carefully crafted progress note format with standard entries in required fi elds helps patient fl ow and record completion. in federally funded clinics and residencies, attending reconciliation of student/resident service delivery is a compliance requirement. a well-designed emr system can "stack" required co-signing tasks on a computer screen, offer standard entries as well as free-hand options, and create a process far faster than paper records for an attending's validation (same as reconciliation?). . tie examination results to treatment planning and treatment planning into billing . a good system allows easy transfer of clinical fi ndings needing treatment into some problem "basket" and ideally in a tabulated format. an alternative is a split screen that allows a clinician to visualize clinical fi ndings, radiographic fi ndings while compiling a treatment plan. again, in clinical settings where compliance to medicaid/medicare regulations is required, the design of the record should give attention to auditing principles and security. a good emr system allows portals of entry for billing and compliance personnel. . plan for users of different skill levels and different periods of exposure . the teaching hospital or dental school environment often involves learners and attendings with varying skill levels and computer experience who may be there for brief periods of time. this reality adds signifi cant security and userfriendliness issues. some medical record systems are far too complex for shortterm or casual users. a well-integrated medical-dental emr allows navigation of the depths of the medical side should a user want to explore, but should focus on the dental portion. some suggestions in design: initial opening or logging into the dental portion for dental users, rather than • opening into the medical portion, clearly indicated options for exploration of medical portions, • orientation of major dental component (examination, radiographs, treatment • plan) in a logical dental treatment fl ow to replicate the way dentistry works rather than trying to reshape dentistry's normal fl ow to the record, minimization of seldom-used functions on the main dental screen, such as • specialty medical clinics, old laboratory tests and hyperfunctionalities like letter writing, clear identifi cation of existing non-caries dental portions like orthodontics or • trauma, so a novice user need not randomly search to see if a patient has any of these records. unfortunately, many pediatric hospitals do not yet have an ehr system that supports convenient communication among a pediatric patient's medical and dental providers. evidence of this state of affairs was provided unintentionally by (fiks et al. ) . some pediatric hospitals may have an awkward mix of systems serving physicians, dentists, and orthodontists and their shared patients. this section demonstrates how closely medical and dental professionals must collaborate to deliver appropriate oral health care for infants and children. such collaboration is especially important given the developmental vulnerabilities of children and the urgency of the oral health needs of many children, especially those from underserved populations. collaboration is made more diffi cult by the long-standing separation of medical and dental systems and poor oral health literacy of parents and medical professionals alike. teamwork in the delivery of pediatric care requires appropriate electronic patient record technology to facilitate sharing of patient information, to avoid patient record discrepancies between systems, and to create effi ciencies by maintaining only a single repository for patient demographics. only comparatively recently have appropriate integrated systems become available to support a range of clinical sites from pediatric special needs clinics to the largest children's hospitals. nationwide children's has given practical examples of effi cient decision-making in identifying an integrated system to acquire. much more work will be needed to develop the means to move towards integrating offi ce and community-based care for children through the sharing of electronic health records. oral health is an oft neglected area in the care of patients who have chronic kidney disease. furthermore, the provision of care by dentists and physicians to the same patient is fragmented as communication between the two health care providers is scant. emerging data suggesting the periodontal disease is closely linked to chronic kidney disease highlights the importance of proper oral health and the importance of communication between dentists and physicians in the care of the patient. investigators used data from nhanes iii, including information on , adults who had an oral examination by a dentist who categorized each patient as having no periodontal disease, periodontal disease or edentulous to examine the relationship between numerous risk factors for moderate to severe chronic kidney disease, as determined by calculation of estimated gfr through use of the mdrd formula (fisher et al. ) . no chronic kidney disease was defi ned as an estimated gfr of ml per min per . m . three percent of the patients had ckd, . % were hypertension and . % had diabetes ( . % with glycated hemoglobin of % or higher). four models were constructed to examine the potential relationship between periodontal disease and ckd. in model one adults with either periodontal disease or edentulous had an adjusted odds ratio of . (with % confi dence intervals of . - . ) of having ckd, independent of the other risk factors for ckd including of age above years, ethnicity, hypertension, smoking status, female gender and c-reactive protein elevation. the fourth model contained potential risk factors including the periodontal disease score and for every -unit increase in the score, the risk of having ckd increased by % controlling for the other risk factors. the authors hypothesized from their results that the relationship between periodontal disease and ckd was bidirectional in that ckd may increase the risk of periodontal disease which in turn increases the risk of ckd. grubbs et al. ( ) also used nhanes data to look more closely at the relationship between periodontal disease and ckd, using dental examinations obtained from to (n = , adults, - years) (grubbs v, et al. ) . in this analysis edentulous subjects were excluded and those with albuminuria were included in the defi nition of ckd. in the entire population ckd was present in . %, but in those with moderate to severe periodontal disease this increased to . %. other associations with moderate to severe periodontal disease were being older, male, nonwhite, less educated and poor. there was a strong relationship between periodontal disease and ckd ( . unadjusted odds ratio). when adjusted for age, gender, tobacco use, hypertension, diabetes, ethnicity, poverty and educational attainment, the odds ratio for the association of periodontal disease and ckd was still signifi cant ( . ). in some groups (mexican american, poor, and poorly educated) dental care was not received on an annual basis in the majority of this segment of the population. periodontal disease has been associated with an increased risk of death in hemodialysis patients (kshirsagar et al. ). this relationship has been poorly studied in peritoneal dialysis patients. this requires further study but it appears possible that periodontal disease might hasten loss of residual kidney function and perhaps contribute to atherosclerosis in dialysis patients and therefore, contribute to the high mortality in this population. patients who desire a kidney transplant are required to undergo a thorough evaluation beforehand including an oral examination by a dentist. some patients on dialysis have inadequate insurance which does not cover dental care, leading to a situation in which a kidney transplant is denied because the patient cannot afford the dental examination. communications between dentists and physicians in the care of the patient is scant. if oral surgery is required in a dialysis patient, the surgeon generally requires a brief summary from the nephrologist with recommendations. these might include suggestions for prophylactic antibiotics, avoidance of vasoconstrictor agents to an excess locally (which can elevate blood pressure) and the increased risk of bleeding of a dialysis patient. for more routine dental examinations no information is requested which could potentially lead to drug interactions or a dangerous situation. most nephrologists and health care providers in the dialysis unit do not inquire of the patient concerning dental health and examination of the mouth is quite uncommon. although the dialysis patient is seen monthly at a minimum, there is little conversation or documentation of oral health. connecting the electronic health records of in-patient care, the out-patient dialysis unit and the dentists' offi ce could potentially have a large impact in improving the care of those with end stage kidney disease. integrating medical and dental records in ehr's may or may not be the "golden ring." first, we need to integrate the clinical thinking…something we both realize is important, but not likely to be solved by an inert computer. i also think that integrated records will be very cumbersome, given the fact that the language used by the separate disciplines is so different, and the kind of detail required to support good decisions and good work is so different. it could be done…but for many professionals on either "side," they would never open the other module. to me, a more sensible solution may be to have a condensed "nugget" of information that could cross populate. "moderate periodontal disease" may be what the medical doctor needs to know, plus know what a treatment plan may include. she won't need to know the number of the teeth with the deepest pockets and erosions but will need to support the patient's determination to follow through. on the other hand, if the patient has shown remarkable initiative in gum care and has successfully migrated to a lower severity index, that would be important for congratulation and reinforcement…and also to encourage similar diligence in managing, let's say, the hypertension that is not optimally controlled. in the other direction, the dentist should know that a patient has been erratic in clinical follow up, does not self-test blood glucose, uses hypoglycemic drugs only intermittently, and has failed several appointments for eye exams. this would lead to a rather different set of approaches from a highly motivated grandmother who is enrolled in a community cultural center's senior exercise club, and is learning to become a lay community teacher for diabetes. right now, i don't think even this superfi cial degree of information is exchanged. we need to support each other's efforts, but we probably do not need to share minute details. the benefi ts of an electronic health record are well described. ehrs allow for legible standardized documentation and easier sharing of patient data between providers at multiple locations. they are less prone to loss and require much less space to store. they have the potential to result in a reduction in the cost of health care. a distinct disadvantage of the ehr, in its current confi guration, is the problem of information overload. simply put, there is often too much information presented in a way that is diffi cult to review and digest. the ehr equivalent of thumbing through a chart quickly is not yet available. as a result we frequently see practitioners look only at the last note or two as they review a patient's history. we require a way to communicate information directly relevant to patient diagnosis, treatment and prognosis among subspecialists and primary care providers. we require a way to identify subclinical cerebrovascular disease in a patient, independent of blood pressure and other traditional risk factors. we require a way to recognize which patients with cerebrovascular disease are two to four times more likely than average to develop a stroke in the next years. we have a way -retinal imaging. the eye is the one place in the body we can directly observe arteries, veins and a cranial nerve in a noninvasive manner. routine imaging of the retina and optic nerve could allow primary care providers to assess retinal, and by proxy systemic, end organ damage from atherosclerosis in an effi cient manner. the key to optimal use of the medical record and effi cient yet effective communication among providers may lie with the familiar adage; a picture is worth a , words. traditionally, when ophthalmologists communicate with primary care providers they send brief letters regarding the fi ndings seen during a yearly dilated examination and the presence, absence or progression of diabetic retinopathy. these letters end by exhorting the virtues of improved blood sugar, blood pressure and lipid control, a sentiment that the primary care provider likely shares. this system of communication does not provide particularly useful information for the primary care provider, except to serve as a notice that the standard of care screening guidelines have been met. the box has been checked. if primary care providers, cardiologists, nephrologists had access to routine ophthalmic imaging, they would be able to directly visualize the effect that suboptimal blood sugar control is having on their diabetic patients. as importantly, they would be equipped with information directly predictive of congestive heart failure, stroke, and cardiovascular mortality for their patient with hypertension, hyperlipidemia and for those who smoke. large clinical studies have shown that assessment of retinal vascular changes such as retinal hemorrhages, microaneurysms and cotton wool spots provides important information for vasculopathy risk stratifi cation. as an example, wong et al. showed that the presence of retinopathy indicates susceptibility to and onset of preclinical systemic vascular disease, independent of and qualitatively different from measuring blood pressure or lipids (wong and mcintosh ) . in the atherosclerosis risk in communities (aric) study, individuals with hypertensive retinopathy signs such as cotton wool spots, retinal hemorrhages and microaneurysms were two to four times more likely to develop a stroke within years, even when controlling for the effects of blood pressure, hyperlipidemia, cigarette smoking and other risk factors (wong et al. ) . in a recent study by werther et al., patients with retinal vein occlusions were found to have a two-fold increased risk of stroke compared to controls (werther et al. ) . in addition, the aric study group reported that individuals with retinopathy were twice as likely to develop congestive heart failure as individuals without retinopathy, even after controlling for pre-existing risk factors (wong et al. a ) . interestingly, even among individuals without pre-existing coronary artery disease, diabetes or hypertension, the presence of hypertensive retinopathy was associated with a three-fold increased risk of congestive heart failure events (wong et al. a ) . in the beaver dam eye study, cardiovascular mortality was almost twice as high among individuals with retinal microaneurysms and retinal hemorrhages as those without these signs ( wong et al. a, b ) . the aric and beaver dam eye studies have also shown that, independent of other risk factors, generalized retinal arteriolar narrowing predicts the incidence of type ii diabetes among individuals initially free of the disease (wong et al. a (wong et al. , b . a primary care provider with access to patients' retinal photographs may therefore have the evidence needed to suggest which patient with either established systemic vascular disease or preclinical systemic vascular disease requires a more aggressive treatment and risk factor modifi cation. they could do this without wading through the electronic equivalent of piles of records. one photograph could refl ect both acute changes in blood pressure (retinal hemorrhages, microaneurysms and cotton wool spots) and chronic changes resulting from cumulative damage from hypertension (av nicking and generalized arteriolar narrowing) (sharrett et al. ; wong et al. a ; leung et al. ) . in brown et al. out of patients, excluding those with known diabetes, that presented with a single cotton wool spot or a predominance of cotton wool spots on examination of the retina were found to have underlying systemic disease (brown et al. ) . systemic work-up revealed diagnoses including previously undiagnosed diabetes, hypertension, cardiac valvular disease, severe carotid artery obstruction, leukemia, metastatic carcinoma, systemic lupus erythematosus, aids and giant cell arteritis (brown et al. ) . these fi ndings illustrate the importance of retinal fi ndings on a systemic level. the utilization and integration of ophthalmic imaging may serve to achieve more effective communication among subspecialists and primary care providers and ultimately to provide improved diagnosis and treatment for delivery of optimal quality of patient care. moreover, the improved integration and maximal use of resources may serve to reduce overall health care cost and perhaps decrease provider frustration with the electronic health record (fig. . ). there are cotton wool spots, exudates, intraretinal dot-blot hemorrhages and microaneurysms. av nicking is also present especially along the superior arcade just as the vessel leaves the optic nerve ( fig. . ) . av nicking, tortuosity of vessels, intraretinal hemorrhages and dry exudates are seen ( fig. . ) . there is edema of the optic nerve head, with cotton wool spots and fl ame shaped hemorrhage along the disc margin. there are several cotton wool spots along the vascular arcades and scattered dot hemorrhages throughout the posterior pole and periphery ( fig. . ) . notice the cholesterol plaque in the vessel just as it exits the optic nerve head and the pallor in the superior macula corresponding to retinal ischemia and edema ( fig. . ). the cholesterol embolus has resulted in lack of blood fl ow to the superior arcade ( fig. . ) . there is pooling of subretinal blood just superior to the optic disc with a central fi brin clot and associated vitreous hemorrhage (fig. . ) . optic disc edema, fl ame hemorrhages and venous congestion are seen in a patient with severe hypertension. biju cheriyan in clinical practice, an otolaryngologist often needs a dental consult not only because of the topographically adjacent nature of the structures but also because most structures are supplied by the same neurovascular bundle and therefore there is overlapping of symptoms. the converse scenario can also apply. apart from this, there are many systemic medical conditions (for example: bleeding diatheses, diabetes) a hypertensive optic neuropathy dentist encounters throughout his or her practice which can determine the outcome of a successful treatment. sometimes, providers may observe a cluster of diagnostic criteria which may have to a single source. in the sections below, i will explore a few of these scenarios and conditions, and indicate where and how an integrated electronic health record (ehr) could optimize delivery of health care by dentists and otolaryngologists. cleft palate/cleft lip : cleft lip and cleft palate (cl/cp) are congenital conditions that require multidisciplinary management by dentists, oral and maxillofacial surgeons, orthodontists, otolaryngologists, speech pathologists and plastic surgeons a number of studies report that a multidisciplinary approach is essential for better treatment outcomes (wangsrimongkol and jansawang ) and for post operative rehabilitation (furr et al. ). these multidisciplinary approaches may lead to new ways to manage and treat cl/cp patients (salyer et al. ). hutchinson's teeth : notching of the upper two incisors is typically seen in individuals infl icted with congenital syphilis. macroglossia refers to enlarged tongue in relation to oral cavity. macroglossia is an important sign. it can indicate important systemic diseases like systemic amyloidosis, congenital hypothyroidism, acromegaly, or down syndrome. a common complaint that dentists and otolaryngologists encounter in their practice is the common headache. because of the special nature of the neurovascular bundle of the head and neck this symptom can be presented to both dentists and otolaryngologists (ram et al. ). any sinus pathology can present as a headache to an otolaryngology practice. since the maxillary sinus fl oor is in close proximity to the maxillary premolars and molars, it is imperative to obtain a dental evaluation in persistent cases of headache. there are a number of causes for headache from the dental and otolaryngology perspective. a mal-aligned denture patient with chronic headache, whom i saw in my practice was shuttled between departments and an array of investigations only to fi nd at the end that an ill-fi tting denture caused the intractable headache. in these cases, an integration of fi ndings is extremely important in providing quality treatment to the patient and also saves money and time for the whole health care system. hence it is important to have an integrated patient record for this particular symptom alone. trigeminal neuralgia is facial pain of neurogenic origin experienced along the distribution of the trigeminal nerve(fi fth cranial nerve). it can present as a dental pain and can also be triggered by brushing teeth among other trigger factors. as a result, patients with dental pain without obvious causes are required to have a physicians' consultation to rule out this obscure condition. sometimes it is diagnosed by omission (aggarwal et al. ; rodriguez-lozano et al. ; spencer et al. ). any tumor of the nasal sinuses (specifi cally maxillary and ethmoids) can erode the lower bony wall and present in the oral cavity (usually the maxillary arch) as dental pain, loose tooth, etc. therefore, these are areas of interest to both dentists and otolaryngologists. such tumors most commonly present fi rst to a dentist or could also be an accidental fi nding. cancers of the naso/oro/laryngo pharynx can also present as toothache to a dentist as these structures have a common nerve supply from cranial nerves , and . therefore, an integration of the patient record may even help in early diagnosis of the tumor. the same principle applies to all oral tumors, tumors of the nasopharynx, the oropharynx etc. this is especially true of malignant lesions of the oral cavity as these may help in early detection and treatment of cancer. in these cases, an early biopsy and histopathology can save the life of the patient. therefore, it is imperative to say that a collaborative patient record can save patients' lives. ulcers of the oral cavity from aphthous ulcers to carcinomas can present both to a dentist and an otolaryngologist. oral ulcers can be of dental origin. contact ulcers from sharp edges of a mal-aligned tooth can result in intractable ulcers, where a simple smoothing of sharp edges may eradicate the ulcer and terminate it as a chronic condition and can even prevent the ulcer turning into a malignancy. if you have an integrated electronic health record (ehr) these problems are immediately addressed and managed. otherwise, the condition will consume valuable time of both the patient and the physician concerned. in addition to this, there are a few conditions which require special attention: aphthous stomatitis (canker sore), which may indicate oral manifestation of defi ciencies of iron, vitamin b , folate deficiency and oral candidiasis, which can be a sign of diabetes mellitus or of an immunocompromised patient (e.g. aids). temperomandibular joint (tmj) disorders can present in a variety of symptoms to both dentists and otolaryngologists. they can present as a headache, earache, toothache, or as facial pain. there can be a number of causes for this including osteoarthritis of the tmj, recurrent dislocation, bruxism, or even an ill fi tting denture. there have been cases where patients have been subjected to removal of teeth for chronic toothache only to discover at the end that the symptom was a referred pain from tmj! therefore, an integrated ehr can prevent misdiagnoses and resulting impairment or disability to patients. trismus (lock jaw) can indicate important diagnoses such as tetanus and rabies.it is due to a spasm of muscles of mastication, which is an important oral manifestation of widespread muscle spasm. apart from these conditions, other causes of trismus are peritonsillar abscesses, and scleroderma. other problems dentists and otolaryngologists encounter in clinical practice are concurrent systemic diseases (patients with multiple problems): patients with bleeding diatheses, diabetes mellitus and a hidden primary malignancy. a non-healing ulcer in the oral cavity may hide a primary malignancy behind it. in these cases, you have to look for it specifi cally. similarly, one has to be aware of oral manifestations of internal pathology. some of them are crohn's disease, ulcerative colitis and gastro-intestinal tract malignancies. often dentists see patients after a tooth extraction with intractable bleeding to fi nd that they have a bleeding diathesis. so, this may be the fi rst presentation of these patients' bleeding disorder. when this patient undergoes any elective procedure in future, it will be a great help to surgeons to be aware of this information to prevent any inadvertent complications. therefore an integrated ehr can prevent unwanted complications where a patient's life may be in jeopardy. the source of otalgia or earache can be from a number of sites other than ear itself. technically ear lobe and ear canal are supplied by four different cranial nerve branches ( th, th, th, th). therefore, an area with a common nerve supply can present as earache. common dental problems which present as referred otalgia are ( ) dental caries ( ) oro-dental diseases or abscesses ( ) an impacted molar tooth (which is a common cause) ( ) malocclusion ( ) benign and malignant lesions of oral cavity and tongue (kim et al. ) . therefore, it is essential these two departments collaborate with each other in diagnosing and treating these diseases, and one way of facilitating it is through an integrated ehr system. there is a lot of overlap between dentists and otolaryngologists in the diagnosis and treatment of patients with halitosis (delanghe et al. ; bollen et al. ) . poor oral hygiene is the most common cause for this common complaint. oral causes include tooth caries, oral ulcers, periodontal diseases, unhealthy mucosa of the oral cavity. it is interesting to note that a simple oral ulcer can form an abcess eroding the fl oor of mouth and becoming a life-threatening oral cellulitis (ludwig angina). once the cellulitis has developed, it becomes a medical emergency. therefore, it is essential to prevent it before it can progress into a life-threatening condition, which of course is possible. causes pertaining to otolaryngologists include: chronic sinusitis or mucociliary disorder, chronic laryngitis or pharyngitis, pharyngeal pouches-related pathology, tumors or ulcers of naso/oro/laryngopharynx, diseases or conditions that impair normal fl ow of saliva such as salivary gland diseases or stones preventing fl ow of saliva, medications which cause dryness of mouth: antihistamines, antidepressants; local manifestation of systemic disorders: auto immune disorders, sjögren syndrome, dehydration from any cause, diabetes mellitus and gastro esophageal refl ux disorder (gerd). gerd is caused by improper neuro-autonomy of the lower esophageal sphincter (les). the les does not close tightly after food intake which causes gastric content to enter the esophagus. over time this can erode mucosa and cause various diseases even becoming cancerous (friedenberg et al. ). this disorder is attributed to life style. fast food consumption habits (oily fried foods) and eating habits (swallowing food without properly chewing) are partly responsible for this disorder (lukic et al. ; al-humayed et al. ) . here again an early diagnosis can manage the disease process before it is fully developed. at present there are no integrated ehr systems serving these specialties (dentistry and otolaryngology). an integrated ehr would facilitate effi cient communication between a dentist and an otolaryngologist who are providing care to the same patient and addressing a problem with a shared focus between the two disciplines. such integrated communication, may only require consulting the available medical or dental record of the patient, based on the particular circumstance. even enabling this simple communication would avoid duplication of effort, clarify the context of certain symptoms and reduce stress endured by the patient. it also has the potential to reduce healthcare delivery costs, and in some cases, even contribute to saving the patient's life. henry hood, allan g. farman, and matthew holder in this chapter, the authors attempt to put forth a justifi cation for precisely this kind of collaborative approach through a summary and discussion of a series of actual clinical cases. the protocols discussed in the management of each of these clinical cases illustrate the value in providing whole-person, interdisciplinary health care to this complex patient population. there is arguably no single patient population for whom the provision of collaborative, interdisciplinary health care is more challenging than for patients with neurodevelopmental disorders and intellectual disabilities (nd/id). in planning and delivering the generally-accepted standard of health care to this unique population, myriad biomedical, psychosocial and sociopolitical realities converge to create a landscape that is, at best, daunting for patients with these disorders, and for the clinicians who are charged with their care. anecdotal and scientifi c evidence suggest that this landscape has produced a paucity of physicians and dentists who are willing and able to provide care to patients with nd/id, and that american medical and dental schools are providing little training focused on their care (holder et al. ; wolff et al. ) . in february of , th surgeon general david satcher issued a report, which documented that americans with nd/id experience great diffi culty accessing quality health care (thompson ) . in that same report, former health and human services secretary tommy thompson said, "americans with mental retardation and their families face enormous obstacles in seeking the kind of basic health care that many of us take for granted." (thompson ) the disparities identifi ed by dr. satcher and secretary thompson require that physicians and dentists approach this population in a spirit of collaboration, compassion, and teamwork in order to produce positive health outcomes for them. perhaps, an even greater imperative driving the need for collaboration between medicine and dentistry in this arena is the fact that many patients with intellectual disabilities have developed this cognitive impairment as the result of an underlying neurodevelopmental disorder that is often undiagnosed. and it is this neurodevelopmental illness and the constellation of potentially devastating complications associated with that illness that create a biomedical fragility and a vulnerability that neither begins nor ends at the oral cavity, and that leaves these patients at risk in almost every aspect of their daily lives. when, for example, patients with nd/id are dependent upon publicly-funded programs for their health care, and when these systems fail to provide the health services that biomedically complex cases require because they fail to account for and accommodate the link between medical and dental pathologies, the risk of a negative outcome is greatly enhanced. such was the case for an intellectually disabled woman in michigan who, in october of , was unable to access dental services through the state's public medical assistance program, and who fatally succumbed to a systemic bacteremia resulting from an untreated periodontal disease (mich. dent. assoc. ). the american academy of developmental medicine and dentistry (aadmd) defi nes a neurodevelopmental disorder as a disorder involving injury to the brain that occurs at some point between the time of conception and neurological maturationapproximately age or (zelenski et al. ). examples of frequently-encountered neurodevelopmental disorders would include fragile x syndrome, a genetically acquired neurodevelopmental disorder caused by a mutation at the distal end of the long arm of the x chromosome (see fig. . ), trisomy , another genetic disorder, which features extra genetic material at the chromosome site (see fig. . ), and cerebral palsy, a prenatal or perinatal, acquired neurodevelopmental disorder (see fig. . ). patients with neurodevelopmental disorders tend to present clinically with one or more of fi ve frequently-encountered, objective symptom complexes or primary complications. these fi ve, classic primary complications include intellectual disability (aka: mental retardation), neuromotor impairment, seizure disorders, behavioral disturbances, and sensory impairment (aadmd). additionally, multiple secondary health consequences can derive from the fi ve primary complications; and any one of these secondary health consequences, or a combination of them, can produce profound morbidity. an example of a common secondary health consequence seen in patients with nd/id, which is derived from intellectual disability and / or neuromotor impairment, is the patient who is unable to care for his or her own mouth, and who develops ubiquitous caries and advanced periodontal disease as a result (see: fig. . ). another example would be the patient who suffers from the secondary health consequence of gastroesophageal refl ux disease (gerd) as a result of the neuromotor impairment associated with multiple neurodevelopmental disorders; and whose tooth enamel and dentinal tissues become chemically eroded as a result of the chronic intraoral acidity produced by gerd (see: fig. . ) . the diagnosis and management of these secondary health consequences provide dentists and physicians with a unique opportunity to work together to improve the quality of health and quality of life for their patients by implementing a team approach, which crosses the traditional interdisciplinary lines of communication, and which expands each clinician's ability to make meaningful treatment options available. indeed, it is often the case that quality primary care provided in one discipline will provide potentially valuable information to an attending clinician from another discipline. such is the case with the patients featured in figs. . and . . the patient whose intraoral photograph is featured in fig. . is a year-old male patient who presented to a special needs dental clinic accompanied by his mother. the mother indicated that her son was exhibiting hand-mouthing behaviors that she believed suggested he was experiencing mouth pain. a comprehensive radiographic and intraoral exam revealed, among other maladies, notched incisors, multiple diastemas, grossly decayed mulberry molars, and advanced periodontal disease. the patient also exhibited moderate to severe intellectual disability. these fi ndings were all consistent with a diagnosis of congenital syphilis. however, in developing the medical history with the mother, it was learned that no previous diagnosis of syphilis had been discussed with the mother, nor was it included in the health history. in cases like this, a comprehensive dental treatment plan should always include consultation with the primary care physician for purposes of moving forward with confi rmation of the clinical diagnosis by serologic testing, and consultation with a cardiologist to assist in the management of potential cardiovascular sequelae. as the dental treatment plan is being developed, consideration should also be given to human immunodefi ciency virus (hiv) testing for this patient, as coinfection is a common fi nding . this issue could easily be attended to by a primary care physician, an internist or an infectious disease specialist. in the absence of any of these team members, the dentist should feel entirely comfortable ordering hiv testing. the primary care physician and the developmental dentist should continue to advise each other and their respective consultant specialists of any signifi cant developments or new information, which could in any way impact either the medical or the dental treatment plan. as treatment progresses, both the physician and the dentist should expect improvement in the patient's periodontal status, which will likely be refl ected in a decrease in the frequency of immune-related illnesses, and in the maladaptive behaviors produced by chronic oral pain. it is quite often the case in this patient population that, with a reduction in maladaptive behaviors, comes a reduction of the use of psychotropic medications prescribed in a frequently futile attempt to manage behaviors that were born of an undiagnosed medical or dental illness. gerd is defi ned as the refl ux of gastric contents into the esophagus. gerd is primarily associated with incompetence of the lower esophageal sphincter; however there are numerous co-contributors, which may predispose a patient to gerd or exacerbate an existing refl ux problem. these co-contributors include a diet high in fat, neuromotor impairment associated with functional abnormalities such as dysphagia, neuromotor impairment associated with impaired ambulation and prolonged periods of recumbence, and the use of multiple medications including anxiolytics, calcium channel blockers, and anticholinergics. gerd is thought to affect approximately - % of the general us population. it has been established in the literature that the incidence of gerd in patients with intellectual disabilities is signifi cantly higher than in the neurotypical population, and that the relative number of unreported cases of gerd is much higher in patients with a neurodevelopmental diagnosis, as well. patients who have gastric refl ux as a function of a neurodevelopmentally-derived neuromotor impairment and a coexisting intellectual disability are impaired in their ability to voice the complaint that would, in the neurotypical patient, commonly lead to an encounter with either a family physician or a gastroenterologist and, ultimately, to a diagnosis. this inability to voice a complaint can be problematic in that, left untreated, gerd can produce maladaptive and sometimes aggressive behaviors in this population. and, of even greater concern, is the fact that undiagnosed esophageal refl ux can lead to more complex conditions that can produce signifi cant morbidity or even mortality -maladies such as barrett's esophagus or adenocarcinoma of the esophagus. chronic gerd can also produce an acidic intraoral environment, which can lead to the chemical erosion of the enamel and dentinal tissues of the teeth. ali et al. have established a link between erosion of the enamel and dentinal tissues of the teeth and gerd. there is additional anecdotal evidence suggesting a link between tooth enamel erosion and gerd, and related maladies. a special needs dental clinic in the eastern united states serving , patients with nd/id, has reported that, of nine patients referred to gastroenterology who presented for dental exam with a fi nding of either tooth enamel erosion or ubiquitous caries, two cases were diagnosed with gerd, two with barrett's esophagus, three with gastritis, and one with duodenitis. in all cases, medical treatment was required. in light of all that is known about the incidence of gerd and of the gerdrelated risks unique to this patient population; and in light of the link between tooth enamel erosion and gerd, it is incumbent upon any dentist encountering tooth enamel erosion in a patient with an intellectual disability to immediately refer that patient to gastroenterology for a work up, which should include esophagogastroduodenoscopy (egd) and ph monitoring. a dentist encountering gerd in a patient with an intellectual disability must be aware that he or she may be the fi rst and only link between that patient and the diagnosis of a potentially life-threatening illness. phenytoin-induced gingival enlargement can appear as either an infl ammatory lesion or a more dense, fi brotic hyperplastic lesion. the infl ammatory lesion is one in which the gingival tissues are swollen and bleeding, and in which pain is often a component. this type of gingival enlargement is the more acute lesion, frequently seen in patients who are currently taking phenytoin. in advanced cases of infl ammatory gingival enlargement, the tissues can appear botryoid, with a characteristic grape-cluster appearance. in advanced cases of phenytoin-induced gingival enlargement, the lesion can sometimes shroud entire sections of the dentition. phenytoin has long been a common medication used to treat seizure disorders in patients with neurodevelopmental disorders and intellectual disabilities. however, the gingival enlargement it produces, and the obstacle this lesion can pose to effective oral hygiene -especially in a population in which oral hygiene is typically compromised -can, over time, lead to periodontal disease, edentulism, and in advanced cases, systemic bacteremias. gingivectomy performed to reduce phenytoin-induced gingival enlargement will typically fail unless the patient is weaned off the offending medication, and another anti-seizure medication is titrated to effect. multiple alternative anti-seizure medications are currently available, which do not have the side effect profi le of phenytoin, and most patients who are weaned off phenytoin will demonstrate a virtual % resolution of the infl ammatory lesion within a matter of or months. the image in fig. . is of a year-old, microcephalic african-american male with intellectual disability, neuromotor impairment, and a seizure disorder. figure . illustrates the appearance of this patient's gingival tissues while he was currently on phenytoin. figure . features the same patient months after being weaned off phenytoin and placed on topiramate. these images illustrate the dramatic result that can be achieved when a dentist and a physician work in collaboration in the best interests of the patient. it is worth noting that this particular collaboration required only one intervention to achieve this result: the patient was weaned off phenytoin and was placed on a safer alternate anti-seizure medication. any dentist caring for a patient with an intellectual disability who presents with phenytoin induced gingival enlargement should immediately contact either the primary care physician or neurologist managing the patient's seizure disorder, and strongly urge that the patient be weaned off phenytoin and placed on a safer alternative anti-seizure medication. edentulism and bacteremia need not be a side-effect of a seizure management protocol. the patient seen in fig. . is a year old male patient with idiopathic intellectual disability who presented to an outpatient dental clinic for comprehensive dental evaluation and treatment. he was accompanied by his father. his father was referred to the clinic by the staff at his son's day program workshop. the day program staff had observed hand-mouthing behaviors, and they had voiced concern that the patient may be in pain. in the waiting room, the patient exhibited behaviors consistent with neurodevelopmental dysfunction. he was non-communicative, and his gaze aversion and tactile defensiveness were suggestive of autism. he was resistant and somewhat combative when directed to the dental chair, and effective behavior management in both the waiting room and operatory required the combined efforts of his father and two staff fig. . the adult patient suspected of having fragile x syndrome members. the patient's health history was positive for attention defi cit hyperactivity disorder (adhd), and there was no history of seizure or neuromotor impairment. the father indicated that, at age ten, the patient was admitted to an inpatient psychiatric unit for evaluation of his uncontrollable behavior. the following day, the parents were told that managing the patient's behavior was beyond the ability of the psychiatric unit staff, and the parents were asked to take the child home. the father also indicated that the psychiatric unit staff described the child's behavior as overwhelming. the patient was last seen by a dentist years prior to presentation; examination and treatment at that time were carried out in the operating room under general anesthesia. effective oral examination of this patient required utilization of papoose board and molt mouth prop. multiple options for behavior management, including utilization of general anesthesia in the operating room, were discussed with the father, and informed consent to utilize medical immobilization techniques for purposes of this examination was obtained and documented prior to taking the patient into the operatory. in the operatory a dental examination was performed, and a baseline panel of digital radiographs was obtained. the head and facial features of this patient were suggestive of fragile x syndrome (see: fig. . ) . the body of the mandible was somewhat elongated; the nose was prominent; the head had somewhat of a triangular shape, and the patient readily averted his gaze. upon further inquiry, the father reported that the patient also exhibited macroorchidism, although he indicated that no physician or dentist had ever suggested a work up for fragile x. fragile x syndrome is a disorder with which many clinicians are unfamiliar. yet it is the second leading genetic cause of intellectual disability in the united states, and it is the leading known cause of autism in the u.s. in addition to the phenotypic fi ndings noted in this case, there are other frequently-encountered physical characteristics consistent with fragile x that may move a clinician toward this diagnosis. they include pectus excavatum or funnel chest (see fig. . ) and joint laxity (see fig. . ) . gaze aversion, as previously mentioned, is a typical fi nding in autism and in fragile x syndrome. indeed, in conjunction with non-verbal behaviors, gaze aversion is often the fi nding that initially alerts the clinician to the possibility of a neurodevelopmental diagnosis featuring autism as a complication. figure . features a photograph of fi ve children at a school for children with special needs. four of the children have been diagnosed with autism, and a fi fth child is a neurotypical child who was visiting his brother on the day the photograph was taken. the reader is left to decide which child is the neurotypical child. any physician or dentist who encounters a patient with an obvious intellectual disability, who does not have an established underlying neurodevelopmental diagnosis, and who presents with additional fi ndings, which may include gaze aversion, shyness, a prominent chin, pectus excavatum, a large nose or large ears, should suspect a possible fragile x diagnosis. the primary care clinician -physician or dentist -should discuss with the guardian or family member the importance of establishing a neurodevelopmental diagnosis. the family member or guardian should be informed that genetic counseling should be made available to all members of the extended family, since fragile x syndrome is a genetic disorder that can be passed from parents to offspring. once this discussion has taken place, a referral to a geneticist for a complete genetic work up is indicated. both the dentist and physician should feel entirely comfortable making this referral. in remote areas where the services of a geneticist may not be available, the attending physician or dentist may order a high resolution chromosomal analysis and a fragile x dna test, and have those results sent to a remote location for interpretation by a geneticist. consultation with a psychiatrist or a clinical psychologist may also be advisable, as patients with fragile x can sometimes experience enhanced social integration as a benefi t of behavioral therapy. the healthcare access problem for americans with neurodevelopmental disorders and intellectual disabilities is, at its core, a healthcare education problem -an education problem resulting from a long-standing defi ciency in professional training focused on the care of this patient population. and it is clear that the medical and dental professions share equally in responsibility for these defi ciencies. eighty-one percent of america's medical students will graduate without ever having rendered clinical care to a single patient with a neurodevelopmental disorder or intellectual disability; and the graduates of % of america's medical residency programs will graduate from those residencies having had no formal training whatsoever -didactic or clinical -in the care of this patient population. additionally, % of graduating dentists have never treated a single patient with a disability. it is no wonder that patients like those whose cases were discussed in earlier sections of this chapter have such diffi culty accessing quality health care. as robert uchin, dean of nova southeastern university college of dental medicine observed in a speech in to his faculty, "not only do we not have enough doctors to care for these patients; we don't have enough teachers to teach them how to care for them." as a result of these defi ciencies in professional education, few clinicians with any expertise in developmental medicine or developmental dentistry are to be found in communities across america. the experts in developmental medicine and dentistry, for the most part, tend to be physicians and dentists who work at the few remaining intermediate care facilities, and at special needs outpatient clinics, psychiatric hospitals, and nursing homes. these physicians and dentists possess the knowledge and expertise in these disciplines because they are the physicians and dentists with the clinical experience. unfortunately for the patients with neurodevelopmental disorders who are clamoring for quality care, there are too few of these clinicians. national experts in developmental medicine and dentistry, however, have begun to collaborate in the creation of patient care protocols; and they have produced multidisciplinary curricula in both dvd and online format. the aadmd has made available hours of online curriculum in developmental medicine, developmental dentistry, and developmental psychiatry (see: list of urls). the curriculum program is entitled, the continuum of quality care , and it teaches collaborative patient care in three disciplines through an interdisciplinary format. the aadmd, through a grant from the wal mart foundation and the north carolina developmental disabilities council, and in collaboration with the north carolina mountain area health education center and the family medicine education consortium, has also established the national curriculum initiative in developmental medicine. this initiative, which is scheduled for completion in , will develop curriculum standards for physicians in the primary care of adults with nd/id. the curriculum stresses the importance of a collaborative approach, which includes medicine, dentistry, podiatry, optometry, and multiple ancillary health professions. if the disparities in access to healthcare for americans with nd/id are to be resolved, physicians and dentists must be willing to cross professional boundaries and work together to plan and deliver whole-person healthcare to their patients with nd/id. interdisciplinary protocols in the diagnosis of neurodevelopmental disorders and in the management of the secondary health consequences associated with these disorders must be established. additionally, clinicians with expertise in these arenas must be willing to work and teach in our nation's medical and dental schools. the clinicians with expertise must be willing to develop predoctoral and postdoctoral curricula, and the deans of america's professional schools must be willing to include these curricula as part of their larger programs in primary and specialized care. the clinicians with expertise in developmental medicine and dentistry must also be willing to conduct patient-focused, interdisciplinary, clinical research in an effort to solve the myriad problems that create obstacles to the delivery of the standard of care for patients with nd/id. they must be willing to obtain institutional review board approval for this research, and they must be willing to make this research available to their colleagues through publication in peer-reviewed journals and text books, and in professional lecture forums. the patient featured in figs. . and . is a man named james. he is a year old patient with idiopathic intellectual disability who presented to a dental clinic for evaluation of a painful facial swelling. a comprehensive intraoral exam revealed a cellulitis resulting from multiple grossly decayed teeth, and a generalized advanced periodontitis. no fewer than fi ve clinicians became involved in this patient's care. they included a general dentist, two oral surgeons, a family practice physician, and a geneticist. over the course of several months, as the treatment plan was completed, and as the chronic dental and periodontal infections were eliminated, james experienced signifi cant improvement in his overall state of health. a comparison of these two photographs reveals not only signifi cant improvement in his aesthetic appearance, but also in his skin turgor and color. these improvements in the patient's health translated to improvements in his daily life. he found gainful employment, and his caregivers now report that he smiles constantly -at work and at home. these photographs were entered into evidence in before a congressional subcommittee investigating the death of a young african-american boy who died as a result of an untreated dental abscess. the photographs were intended to make the point that patients with intellectual disabilities need not die as a result of medical illnesses derived from untreated dental disease. this patient's case illustrates that, when physicians and dentists are willing to work together toward a common goal of whole-person health for their patients, profoundly positive outcomes can be achieved. in a larger context, if our nation's medical and dental professions are willing to commit to a shared agenda, one which promotes the idea of collaborative, interdisciplinary care as a foundational concept, signifi cant improvements in quality of health and quality of life can be realized, not just for americans with neurodevelopmental disorders, but for every patient seeking quality care. in light of the events of , bioterrorism has become subject of increased attention from all members of society. government agencies, professional associations, academia, etc. have expressed their determination to wage war on such threats by all means available. dentists can also participate in this effort by providing assistance at interested groups and the general public (flores et al. ) . in this chapter we will examine the elements and components that may play a role in the establishment of an electronic network for the dental profession for supporting the fi ght against bioterrorism. in this section we review the threats, the public health system, current electronic surveillance systems, regulations and ethical issues, the computerization of dentistry, and how dentistry can serve in improving biosurveillance efforts. the aftermath of september and the anthrax incidents in october ( lane and fauci ) , made the us government reorganize its priorities and reform its current structure (white house offi ce of the press secretary ) . in response to these incidents, president bush proposed the "health security initiative" (white house letter ) in february nd of . this effort labeled the "bioshield initiative," (white house letter ) has the purpose to stimulate research and development of medical countermeasures against bioterrorism attacks. however, despite all these efforts, terrorist attacks are likely to happen in the future and even the best work from intelligence and security agencies will be unable to prevent such events (betts and richard ; council on foreign relations ; baker and koplan ) . to cope with this threat, a report published by an independent task force sponsored by the council on foreign relations "america-still unprepared, still in danger" (council on foreign relations ) , suggested a series of steps to assist the government in preparing to better protect the country. one of these suggestions is the bolstering of the "public health systems". baker et al. defi ne the u.s. public health system as a system that consists of a broad range of organizations and partnerships needed to carry out the essential public health services, such as hospitals, voluntary health organizations, other non-governmental organizations and the business community (baker and koplan ) which can collaborate with local, state and federal public health entities. after the unfortunate incidents in the public health system was revisited and the realization that "the nation's public health infrastructure is not fully prepared to meet this growing challenge" (frist ) became clear. to address this need, congress and president bush enacted the public law (p.l.) - titled "public health security and bioterrorism preparedness and response act of " (frist ; th congress ) . the main purpose of this law was to improve the public health capacity by means of increasing funding and fostering other measures. frist ( ) , described the law as a "good start" and that "to be prepared for bioterrorism, it is imperative that we develop a cohesive and comprehensive system of ongoing surveillance and case investigations for early detection". in this way, several early detection systems have been implemented with different levels of success among different geographic regions in the us. one of the most important initiatives over the years has been the establishment of the national electronic disease surveillance system (nedss) (baker and koplan ; nedss ) . the national electronic disease surveillance working group establishes that the "nedss is a broad initiative focused on the use of data and information systems standards to advance the development of effi cient, integrated, and interoperable surveillance systems at the state and local levels. the long-term objectives for nedss are the ongoing automatic capture and analyses of data needed for public health surveillance". the purpose of this system is to take into consideration and integrate the information of current public health systems implemented at different health department levels: county, state and fi nally at the centers for disease control and prevention (cdc). another initiative spearheaded by the cdc is biosense (looks ) . the purpose of this program is to develop advance detection capabilities of health related events including disease outbreaks. in addition, its emphasis is to improve situational awareness by integrating advanced analytics to process data generated by different health providers and other entities in the us. now that we have examined the general aspects, we will continue our background review focusing on the aspects that pertain to the specifi cs of the dental profession. this section will provide some perspective of the structure of the dental profession in comparison with its medical counterpart. "there are approximately , active dentists in the united states" (mertz and o'neil ) . in the dentistto-population ratio was of - , . and it is expected that by the year the ratio will be . , which translates into one dentist for every , people. " in contrast, the physician-to-population ratio has been increasing for the past years and now stands at per , , about one physician for every people." eighty percent of the dentists are in general practice. during march and of , the american dental association and the us public health service sponsored the conference "dentistry's role in responding to bioterrorism and other catastrophic events" (palmer ; national institute of dental and craniofacial research ) . this meeting reviewed several aspects of bioterrorism and the dental profession: the nature of biological pathogens and its oral manifestations, what needed to be communicated, how dentists should participate, etc. dr. michael c. alfano described the diffi culties that biological pathogens create for clinicians because "they are so insidious." while discussing the anthrax mailings after september th he pointed out that: "… early symptoms appeared so they resembled the aches, fever, and malaise of fl u so those affected delayed seeking treatment, a delay that has proven fatal in some cases". lieutenant colonel ross h. pastel of the us army medical research institute of infectious disease (usamriid) listed the "category a" pathogens as defi ned by the centers for disease control and prevention, and those are: smallpox, anthrax, plague, botulinum toxin, tularemia and viral hemorrhagic fever. he also described an outbreak of smallpox in yugoslavia in and the measure that had to be taken to control it. dr. michael glick described the oral manifestations of smallpox showing "signs hours before skin rash. these oral signs include tongue swelling, multiple mucosa vesicles, ulceration, and mucosal hemorrhaging. oral signs are also evident in inhalation and gastro-intestinal anthrax. in oropharyngeal anthrax the mucosa appears edematous and congested; there may be neck swelling, fever, and sore throat" . dr. ed thompson, deputy director of the centers for disease control and prevention mentioned that "none of the new counter-bioterrorism measures can be effective unless local health practitioners are vigilant in observing and reporting a possible disease outbreak. such surveillance-knowing what to look for and whom to report to-is critical and applies not only to suspected bioterrorist agents, but to a list of reportable diseases which has grown to include such entities as west nile virus and sever acute respiratory syndrome (sars)." dr. sigurs o. krolls presented the response at the local level and he "stressed the importance of communication and the need for redundant systems", "to keep all the parties informed". he also posed the question "can dentists recognize signs and systems of contagious diseases?", and emphasized that education can be essential. dr. louis depaola made several connotations that can be key in the scope of this paper by saying "dentists can contribute to bioterrorism surveillance by being alert to clues that might indicate a bioterrorism attack. such surveillance would note if there is an infl ux of people seeking medical attention with non-traumatic conditions and fl ulike or possibly neurological or paralytic symptoms… or even specifi c signs of a bioterrorist agent. patterns of school of work absence, appointment cancellations or failures to appear, could also be indicators." dr. depaola made clear that in cases of limited release of bioterrorist agents, dentists "have little to offer" but "a widespread attack can certainly tap into dental professional skills in recognition, isolation and management". in addition, dr. guay ( ) lists all the possible roles in which dentists can participate including "education, risk communication, diagnosis, surveillance and notifi cation, treatment, distribution of medications, decontamination, sample collection and forensic dentistry." dental informatics must pay attention to these and other recommendations, in order to develop integrated systems that take these recommendations into consideration. it is also important to understand that informatics has to work with technologies already in place like the computer-based oral health record and current standards. the fi nal recommendation from the meeting stated that to play an important role in biodefense, a serious amount of coordination and preparation will be required, not only from dentists but from other groups, most likely requiring medical and dental data integration. the cohr as described by rhodes ( ) "can provide a structure for documentation that goes beyond the concept of a blank form on a page, it includes a glossary of dental terminology for the entire content of the form as well as knowledge bases and expert systems that can enhance the practitioner's diagnostic and treatment planning decisions". he also acknowledges that one of the advantages of this type of documentation is that it "is much more transportable". he also recognizes the need for standardized methods for collecting information from dentists. schleyer and eisner ( ) defi ned several scenarios where the cohr is used in a "shared" environment where several healthcare providers interact and information is seamless communicated, improving the decisions made by clinicians. delrose and steinberg ( ) discuss how the "digital patient record" enhances clinical practice by providing "better quality information" to the clinician. although all of these benefi ts sound promising and encouraging some still express concern of the lack of standards among different information systems, which translates in communication breakdowns (schleyer ) . on the other hand, heid and colleagues ( ) mention a list all the steps that are currently being taken by different organizations such as the ada in order to produce a standardized cohr. other examples of standardization can be found in a paper presented by narcisi ( ) where ada's participation as a voting member in the american national standards institute has allowed edi or the cohr to be discussed and improved at a national level. additional infl uences in the standardization of the cohr are the security regulations mandated by hipaa, the health insurance portability and accountability act of . dentists are required to "adopt practices necessary for compliance" (sfi kas ; chasteen et al. ) . these and other regulations (szekely et al. ) will encourage the homogeny among different system vendors. computer ownership, on the other hand, has increased steadily during the last years. according to schleyer et al. ( ) in only % of dental professionals used computers in their practices compared to % in the year . additionally similar trends in internet connectivity where described. the issues mentioned above describe the issues that have to be considered in order to create surveillance system against bioterrorism for the dental profession. this review has tried to be inclusive by covering different aspects starting with the current state of affairs and environment, treats, technology, law, etc. next we present a blueprint for developing a biosurveillance system. the purpose of developing an electronic health surveillance system is to gather information from patients directly ( wagner et al. ) by detecting signs and/or symptoms, or indirectly by obtaining other types of information such as over the counter medication sales, patients' no-shows, usage of internet search engines keywords, etc. in this particular case, the proximity of contact between the dentist and the patient is equivalent to a medical inspection in terms of immediacy and/or closeness. such signs and symptoms can be easily detected if the dentist is properly prompted to search for them. this is just one example of ways how a system could provide assistance in the detection of a bioterrorism incident. but, before describing our proposed system, it would be important to address the fact that current syndromic surveillance systems have certain advantages in terms of its particular technological implementation . the rods laboratory obtains data directly from chief complains in the emergency departments from hospitals. the advantage of this surveillance system is that the implementation has to be made with only a limited number of parties (hospitals, clinics, health systems, etc.). on the other hand, our system would have to deal with thousands of different implementations (one in each dental offi ce). this and other challenges have to be considered when designing the proposed system: the proposed system should work at multiple levels: the system would have to provide a mechanism to alert the dentist if there is • suspicion that a bioterrorist attack may be happening. the mechanism would increase the dentist's awareness in case of fi nding suspicious signs or symptoms in a patient. this can be triggered by the patient's characteristics such as geographic location of residence, etc. automated collection of information from the patient's oral health record. the • system would report to a central database signs or symptoms of interest. the aggregation of this data could generate information that would eventually identify the presence of patterns that may lead to the early detection of such events. collection of additional information, which combined with other sources, can be • useful in terms of detecting or tracing some incident. patients' "no-shows" is the primary example, that, if combined with others such as work or school absenteeism can provide a relevant pattern for public health offi cials. dr. x, who practices in a community min away from capitol city, installed a new clinical management system months ago. among the features that were included in this new clinical management system (cms), a bioterrorism detection module was added. she felt curious because of recent news she read in the newspaper about possible attacks against the us and decided to install such feature. he read about how the module would work in combination with the cms she just bought. the educational information provided with the software instructed dr. x, that in case that a patient victim of a bioterrorism attack happens to be seen in her practice, the software would collect information and would send it to public health offi cials. when installing the software, dr. x was asked if she agreed to share such information with authorities. she was provided the option to receive notifi cation in case some information was sent but she decided not to enforce it. during the last week a patient walked into dr. x's dental offi ce. the patient presented some signs that indicated the presence of a disease; still its origin was not clear. an epidemiologic study later would show that the patient was present at the football stadium when an infectious agent was released (fig. . ) . although, at that time his medical history showed no indication of a systemic disease, the presence of multiple oral vesicles prompted the dentist to make an annotation into the cohr. the system, by using a natural language processing engine, detected such sign and sent this information to a central database. the patient was discharged and instructed to take some support medication to treat the oral ulcers. the next day, the central database pinpointed the presence of an out of the ordinary increase in the number of cases with the same signs and symptoms around that region. when the presence of this peak was detected, the central server sent a request to the dentist computer for additional information. one of the requested elements was if there was any use of medication for treating oral ulcers. fortunately this information was available. the central database crossed this with the information of other surveillance systems together with the information from other patients that happen to have similar clinical signs and/or symptoms. dr. x received an email from a public health offi cial asking her to communicate to the local health department to discuss information about one her patients. the case depicted above simulates the release of smallpox during a football game. in the case of smallpox oral symptoms include tongue swelling, multiple oral mucosal vesicles, ulceration, and mucosal hemorrhaging (national institute of dental and craniofacial research ) . dentists could be alerted by an electronic system to search for such signs or they can be detected automatically. in case of a high incidence within a group of patients, in a confi ned area, public health offi cials get to be notifi ed. in our hypothetical case there are issues that need to be addressed in order to make such detection system feasible: as described by schleyer et al. ( ) , % of dentists in the us use a computer in their practices. this fi gure would generate an estimate of , computers in dental practices. this prevalence of computers represents an opportunity for public health data collection. the creation of a software application for surveillance purposes must rely on existing technology. currently there are approximately major clinical management software packages in the market (dentistry today ) . out of these , clearly permit direct database manipulation. this characteristic can easily allow the creation of a "querying" application that would look for specifi c information within the data stored by those packages. additionally, a natural language processing engine could be embedded into the application in order to detect variations in data input on the computer oral health record. nevertheless, it is necessary to obtain a detailed list of the oral manifestations of diseases that are likely to be found on patients. successful implementations of similar systems have been shown to work successfully (chapman et al. ; ivanov et al. ) and using the same approach for our system seems technically feasible. this collected information later would be send to a central server in order to be analyzed and interpreted. the components of our system would be as follows (fig. . ) : thin client: a software application distributed for data collection. it would be • conformed of a "querying" mechanism, combined with a natural language processing engine and a communication module. this software client should be as thin as possible to reduce the work load on the dentist's equipment and should be embedded as a plug-in for current clinical management systems. vendors should be contacted to ask for their collaboration in the development of such application to ensure maximum compatibility and integrity of data collection. central servers: server software in charge of integrating all the data collected • from dental offi ces. it has to be capable of handling simultaneous requests from multiple users. this server would integrate all the data and would perform an analysis with the intention of detecting anomalies. it would be recommended that redundant servers should be located in different data centers with mirroring capabilities to guarantee their survivability in case of technical diffi culties. communication network: the transmission of information should be done using • the internet. this, of course, would essentially depend on the practitioner's current connectivity. if that is not available, backup connection to the central servers should be established. dentistry uses several standards for transmission of health related information. clinical management systems use standard-based technology to transmit information (narcisi ; chasteen et al. ; szekely et al. ; dentrix dental systems ) . dentists are aware of these standards and use them in a day-to-day basis to transmit information to insurers. additionally, in order to interact with other surveillance systems such as the nedss, our application should rely on the same standards. the software both client and server should be thoroughly verifi ed to be secure in terms of being safe against hacker attacks. on the server side, redundancy should be provided so downtime is reduced from design. the system should be developed so mirrored servers are always up and running. data integrity mechanism should also be considered. privacy and confi dentiality are important issues that need to be incorporated as part of a robust biosurveillance system and distinct regulations such as hipaa require protecting patient information (frist ; chasteen et al. ; bayer and colgrove ; etzioni ; ivanov et al. ) . in our hypothetical case we describe the use of several sources of information for detecting a bioterrorist attack. we described how syndromic information is transmitted to a central database which initially should be de-identifi ed. later, after the suspicion a bioterrorist attack more information is requested (medications) and more inferences are made. this, although technically possible, would require changing our processes and also the will to share clinical information. this leads to the discussion mentioned in the background section about "individual rights" vs. "common good". although hipaa addresses public health , some other implications may arise and the health professionals including dentists, physicians, public health offi cials and patients should discuss and address such issues. as discussed earlier, legislators face a diffi cult task in terms of determining what is best on behalf of the individuals they were asked to represent. legislation may have to be passed in order to guarantee the functioning of such a system. individual freedom and privacy are important values which may pose a confl ict when collecting individuals' information even for their own good. in any case, careful consideration has to be given to which information is required to detect a bioterrorist attack and also, by keeping in mind that it is always important to reduce, as much as possible, the collection and transmission of patients' information over the internet or any other network. a detection algorithm has to be created or adapted in order to determine the presence of a bioterrorist attack. some algorithms have proven their effectiveness (wong et al. a, b ) and it is likely that from these, a new analysis should be done in order to select or create one that addresses the particular needs of our system. a study was conducted to assess the feasibility of using oral manifestations in order to detect disease outbreaks (torres-urquidy et al. ) . it was found that for diseases such as botulism and smallpox it would be feasible to gather data that contains oral manifestations that would allow creating a detection signal using natural language processing followed by the use of statistical methods such as moving average to serve as part of a detection algorithm. the system should also be thoroughly evaluated, before and after implementation. to perform the evaluation before the system implementation computer simulation can be used to assess the effectiveness and likelihood of detection. simulation and modeling techniques (reshetin and regens ) have been used to estimate the effects of a bioterrorist attack. the same techniques can be used to evaluate our system. in case of the study by torres-urquidy ( ) , the investigators utilized synthetic outbreaks to test the performance of different signals. from their evaluation process, they learned, for instance, how many cases would be necessary to occur for the system to reach certain detection thresholds. several dental organizations have shown publicly their support of measures against bioterrorism. the american dental association and the national institute of dental and craniofacial research are two organizations who could play an important role in the development, deployment and ongoing support for our system. local dental societies also would also play an important role in the deployment of the proposed system. similarly, local, state and federal public health agencies should engage in activities that could make these mechanisms for health surveillance feasible. if dentists want to play an active role in the fi ght against bioterrorism, they should commit to collaborate with public health entities as well as to seek a way to integrate their information with the rest of electronic biosurveillance systems. professional organizations such as the american dental association can also participate by endorsing such efforts and by collaborating in the educational process of the dental professionals and their patients. as mentioned by dr. depaola (national institute of dental and craniofacial research ) dentists "have little to offer" in the current biosurveillance state of affairs. however, the integration of different technologies can change this perception. goldenberg et al. ( ) described over-the-counter medication sales as a technique for discovering disease outbreaks and stated that their approach may be "more timely" than traditional medical or public health approaches. medical cases that result from bioterrorism attacks do not produce symptoms until they have fully developed, so it is likely that different patterns can be detected before the patients start reaching the emergency department. as stated earlier (torres-urquidy et al. ) , it may be possible to have dentists participating of biosurveillance efforts, if we solve the proper organizational and technical challenges. dr. john r. lumpkin ( ) states that "hippocrates noted the health of the community was dependent on characteristics of a community and the habits of the people who lived there." dr. krolls (nidcr ) in his fi nal remarks during his presentation at the dentistry's role conference against bioterrorism, said, "dentists may pick up telltale information about what is happening in the community. after all, dentists spend more time with their patients than any other health specialty". kass-hout t, zhang x. biosurveillance: methods and case studies. muhammad f. walji maintaining patient records are essential for both clinical care and research. clinical research often occurs in the context of also providing patient care, yet the systems that are used for each are different and often cannot exchange data. the lack of data exchange between systems pose signifi cant barriers to effi ciently treating patient and conducting clinical research in dentistry. the purpose of this section is to review the benefi ts and challenges of integrating electronic health record (ehr) used for patient care and electronic data capture (edc) which is used for clinical research such as clinical trials. an increasing number of dentists routinely use ehrs (schleyer et al. ) . most dental schools in north america also use ehrs. benefi ts of using ehrs include increased legibility, portability, and improved patient safety (buntin et al. ) . recent federal incentives, although not directly benefi cial to dentists, will also likely spur the adoption of ehr (blumenthal and tavenner ) . clinical researchers, especially those conducting clinical trials, are also discovering benefi ts of using electronic data capture compared to paper. a clinical trial is a process in which new treatments, medications and other innovations are tested to evaluate safety and effi cacy. a standard part of health care, clinical trials are often lengthy and costly due to myriads of regulatory oversight. recent estimates set the cost of drug development in excess of $ million (grabowski et al. ) . accurately documenting data with suffi cient detail is critical for providing patient care and conducting clinical research. while the medical record is the foundation for patient care, the case report form is the foundation in a clinical trial. not all clinical research is clinical trials. clinical trials whose data will be submitted to the fda as a new therapy or device have additional requirements relating to the collection and transmission of the data. similarly for patient care data, ehrs need to meet the privacy and security requirements of hipaa. case report forms (crf) are a medium in which research study sites collect subject data in pre-defi ned formats for communication with clinical trial sponsors (rondel and webb ) many clinical trials data are collected on paper (rondel and webb ) . data measurement, collection, and recording are considered the "most crucial stage" in the data management process (hosking et al. ) . traditionally, study coordinators often record information in a case report form and subsequently mail or fax the crf to the centralized coordinating center. there, data entry staff, sometimes with the aid of optical character recognition systems, input crf data into a computer. errors made during this second data entry process are diffi cult to detect and correct (hosking et al. ) . lengthy guidelines in literature discuss methods for developing paper case report forms to reduce data entry mistakes (hosking ) . a well-designed crf may allow a user to effi ciently collect and record pertinent data. however, forms are often revised and redesigned during a clinical trial due to changes in protocol, unforeseen outcomes, or oversight (singer and meinert ) . there has been a recent drive to use electronic case report forms (ecrf). direct data entry at a study site shortens time to analysis and provides opportunities to audit data at time of entry. this could reduce data errors that might otherwise be caught weeks after submission. for quality control purposes, some studies require double data entry using computers and paper (day et al. ) , though alternative solutions have been explored including the use of data sampling (king and lashley ) and probability statistics to select only those forms likely to contain errors (kleinman ) . ecrfs may also facilitate data collection from existing electronic information systems such as lab systems. however, ecrfs are almost always reside in a separate system that is not linked to a patients record. although many clinical research studies are still being conducted using paper, an increasing number of studies are using ecrfs and electronic data capture (edc). for example, a review of canadian clinical trials found that % use edc (el emam et al. ). studies that are sponsored by a pharmaceutical company and are multicenter appear to use edc at a higher rate than those sponsored by government or a university. the cost of a commercial edc is substantial. recently a freely available edc has become popular amongst universities called redcap. a tool originally developed at vanderbilt university, it is now being used at over a institutions worldwide (harris et al. ). however, such tools are generally not integrated with the institutions ehr. although moving from paper to electronic will afford benefi ts there is a great need to allow data exchange between the patient care and clinical research components of information systems. although ehr and edc are similar, several challenges remain unresolved that prevent integration. one of the major barriers is likely to be different workfl ows for patient care purposes and to collect data for research. research is needed in defi ning an optimal workfl ow that can streamline the tasks associated with patient care and research, while at the same time providing a unifi ed information system that support these activities. also, the data that are collected for care and research are likely to differ. a researcher may require far more granularity of an oral health measurement than a clinician seeking to provide care. in cases when conducting a double blind placebo controlled clinical trial, the investigator may not even know the type of treatment that has been delivered to the patient. due to complexities of each domain, and large differences in goals, to date mutually exclusive workfl ows have arisen. a clinician investigator who sees a patient for both care and research, will likely need to enter data on this same patient twice; once in the ehr and once in the edc system. despite the availability of electronic systems, a major barrier is the integration and compatibility of disparate health information systems to converse with one another. the languages are important because they can help data sharing. clinical trials are not usually conducted in isolation, but are part of conventional medical care. therefore sharing data by clinical trials, patient care and laboratory systems becomes especially important with the adoption of ehrs in dentistry. in biomedical informatics, standardized terminologies are recognized as a critically important area to help better represent and share data for use in electronic systems (cimino ) . the systematized nomenclature of medicine clinical terms (snomed-ct), developed by the college of american pathologists, is the most comprehensive medical terminology (strang et al. ; chute et al. ) and is used in a number of health informatics applications. the us department of health and human services ( ) has also licensed snomed-ct, allowing access throughout the us at no charge. therefore snomed-ct is even more likely to be the vocabulary used in electronic formats of patient records in the future. the medical dictionary for regulatory activities (meddra) is terminology used by the fda and drug development industry to classify, retrieve, present, and communicate medical information throughout the medical product regulatory cycle (brown et al. ) . in particular it is used to record and report adverse drug event data. therefore standard languages are essential in sharing clinical trials data between sites, and also with regulatory agencies. no one single terminology is suited for all tasks. snomed-ct is likely to be more comprehensive to code clinical encounters, while meddra is more suited to help adverse event reporting. however, it is important that terminologies are widely adopted and used for similar purposes. even when standard terminologies are agreed upon, such information needs to be interchanged in standard formats. health level (hl ) is an important organization whose standards are widely adopted in healthcare to exchange information between computer systems. the clinical data interchange standards consortium (cdisc) is also an important group that helps to defi ne different data standards specifi cally for clinical trials research, such as clinical trials or regulatory submissions. one particular challenge in oral health has been the lack of a standardized terminology to describe diagnoses. although icd contains oral health concepts, they are often not granular enough to be useful for some patient care or research purposes. recently a dental diagnostic terminology has been developed by a group of dental schools, and has already been adopted by several institutions and used within dental ehrs (kalenderian et al. ) . the american dental association (ada) has also been developing snodent, but is not yet publically available for clinical use (goldberg et al. ). another link between ehr data and clinical research is the potential to fi nd human subjects. recruiting suffi cient numbers of patients that meet eligibility requirements within an allotted time frame for clinical trials is challenging. as ehrs contain detailed information about patients, they can be used to fi nd patients that meet specifi c inclusion and exclusion criteria. informatics for integrating biology and the bedside (i b ), an open source data warehousing platform, has been found to be a useful tool for cohort selection especially if the source data from an ehr is represented in a structured format (deshmukh et al. ). further, with health information increasingly available to patients through the internet, it is possible interested patients will be more effective in fi nding clinical trials than investigators looking for patients. many clinical trial registers are now available online. the national institutes of health (nih) have made available their database of nih funded research (mccray ) . there is currently no single repository for patients to fi nd all trials studying a health condition. a recent study assessed the comprehensiveness of online trial databases concerning prostate and colon cancer and found that online trial registries are incomplete, especially for industry-sponsored trials (manheimer and anderson ) . a more collaborative effort between government and industry-sponsored research groups to compile and standardize information may be a mutually benefi cial effort. it is not clear how many patients now enroll in clinical trials through online discovery. ehr data originally collected for patient purposes can be potentially used for research. aggregating data from multiple sources can provide a large dataset that could otherwise not be available. electronic health records (ehr) contain a wealth of information and are a promising source to conduct research. data extracted from ehrs differ from other sources such as population surveys or data obtained from payers, as they provide a more detailed and longitudinal view of patients, symptoms, diseases, treatments, outcomes, and differences among providers. therefore ehr data in dentistry can potentially provide valuable insight into oral health diseases, and treatments performed on a large cohort of subjects. ehrs also play an important role in enhancing evidence-based decision-making in dentistry (ebd) and improving clinical effectiveness through decision support (atkinson et al. ; walji et al. ; valenza and walji ; taylor et al. ; spence et al. ; chambers et al. ; langabeer nd et al. ; walji mf et al. ). the consortium of oral health related informatics (cohri) provides an example of how dental ehrs are used for research purposes (schleyer et al. ; stark et al. ) . cohri was formed in by a group of dental schools who used the same ehr platform and who are interested in sharing clinical and education data. through funding from the national library of medicine, four dental schools are participating in a pilot project to develop an inter-university oral health research database by extracting and integrating data from ehrs. one promising area where data repositories derived from ehr data can be used for new discoveries is in the area of comparative effectiveness research. comparative effectiveness research is defi ned as "a rigorous evaluation of the impact of different options that are available for treating a given medical condition for a particular set of patients." (congressional budget offi ce ) further, such research includes focusing on the clinical benefi ts and risks of each option (clinical effectiveness), and an analysis on the costs and benefi ts (cost effectiveness analysis). comparative effectiveness research (cer) is also likely to reduce costs of dental care and increase access to the majority of the population who currently receive no dental care. unfortunately many recent systematic reviews focusing on cer questions in dentistry have been inconclusive due to the lack of existing evidence in the scientifi c literature. secondary analysis of the data that reside in dental electronic health records (ehr) is a particularly appealing approach to facilitate cer and generate new knowledge. ehr data has the potential to provide a comprehensive picture of patients' histories, treatments, and outcomes, and if integrated with similar data from other dental clinics can include a large and diverse set of patients. however, numerous challenges must be solved before ehrs can be used for cer. first, data suitable for cer must actually be collected from ehr systems. second, this data, which often resides in proprietary systems, must be accessible and retrievable. and lastly, this data should be structured in a format that can be integrated with data from other sources or institutions. practice-based research networks (pbrn) are groups of primary care clinicians and practices working together to answer community-based health care questions and translate research fi ndings into practice. pbrns engage clinicians in quality improvement activities and an evidence-based culture in primary care practice to improve the health of all americans. in , the national institute of dental craniofacial research funded three such research networks. the dental pbrn's to date have been conducting both prospective and retrospective research. for example, barasch et al. conducted a case controlled study to investigate risk factors associated with osteonecrosis of the jaws . many prospective studies conducted as part of pbrns still require separate data collection systems for the research data. ehr data contained in practices as part of pbrns are beginning to be used for secondary purposes. for example fellows et al. conducted a retrospective analysis of data contained in electronic health records to estimate incidence rates of osteonecrosis of the jaws ( fellows et al. ) . pbrns provide great promise of how ehr and clinical research data can be used effectively to promote both patient care and new discoveries. another area that intersects both the patient care and research realm are patient registries. patient registries are ways to track groups of patients who have had specifi c diseases or have had certain treatments. while ehr data would contain information on all types of patients, their diseases, and treatments, registries would allow focus on specifi c diseases or treatments of interest. registries would not be as costly in terms of resource requirements like a traditional clinical trial, but would require specifi c eligibility criteria, informed consents, and collection addition to that collected as part of routing care. dentistry has lagged far behind in forming data registries, primarily because dentistry is practiced in small offi ces and not in large hospitals making the process of integrating data very diffi culty. however, dental schools which themselves house large clinical operations are ideally positioned to create disease specifi c registries that can potentially use data collected for patient care and extend for research purposes. there is great potential for providing new insight in oral health by the integration of patient records and clinical research from both a workfl ow and information systems perspective. the technology challenges of developing systems that can exchange data, and use standardized terminologies appear solvable. however, the socio-technical issues such as determining how to incorporate optimal workfl ows for conducing both patient care and research with minimal additional overhead appear to be the greatest challenge before widespread adoption. similarly, there appears to be great potential in using ehr data originally collected for patient care for the secondary use of research and discovery. this will require collaboration between patients, providers and researchers from all healthcare disciples, and institutions with friendly policies for sharing data to improve both patient care and drive new discoveries. amit acharya , andrea mahnke , po-huang chyou , and franklin m. din more recently there has been a strong push from the united states federal government for the adoption of the electronic health record (ehr) within the healthcare industry. as a result, $ . billion is made available to incentivize the physicians, dentists and hospitals for the adoption of the ehr through the health information technology for economic and clinical health (hitech) act. as the nation head towards adoption of the ehrs, there has also been a growing interest with the majority of the u.s. dental schools to implement ehrs within the educational setting. fifty of the fi fty-six u.s. dental schools, as well as dental schools in canada and europe, are either using or in the process of adopting some aspects of a common dental ehr framework (white et al. ) . a group of dental schools known as consortium for oral health-related informatics (cohri) was formed in which used this common dental ehr framework -axium (stark et al. ) . currently there are about dental schools within cohri. the ehr will not only support clinical care, but will also result in training the next generation dental students and to conduct innovative research that was not possible earlier. however, not much is known about how many of these dental schools' electronic dental records are integrated with their respective university's electronic medical record. a common medical-dental ehr model at healthcare universities would enable a holistic approach to providing patient care and provide the much needed electronic infrastructure to study interrelationship between the various oral-systemic diseases. recently a group of researchers from marshfi eld clinic in wisconsin, us conducted a survey to investigate the current states of health information technology and informatics within the dental school in the us. list of us dental schools were identifi ed through the american dental education association (adea) web site. dental schools were contacted to determine who the most appropriate person to take the survey would be. once the list of contact was developed from each dental school, an email was sent to us dental schools with a link to a survey created in surveymonkey. the survey was administered on tuesday march , . reminder survey emails were sent to all recipients on march and march . the survey was closed on march . the anonymous survey was at most questions, depending on how questions were answered. the survey focused on topics such as presence of dental informaticians within the dental schools, use of fi nancial and clinical information systems, interest in federal stimulus support for ehr adoption provided through american recovery and reinvestment act and meaningful use of ehr, relationships with health care entities and bidirectional nature of the dental and medical ehrs. the study was approved as exempt from the marshfi eld clinic institutional review board under section cfr . (b) and waived requirement for an authorization. thirty out of the fi fty fi ve dental schools responded to the survey (response rate of %). however, fi ve of the thirty dental schools representative did not complete the survey and hence their response was not included in the analysis. regarding the question about the presence of a dedicated department or a center for information technology (it) or informatics within the dental school in us, % (n = ) of the responding dental schools had a dedicated it/informatics department or center (p-value of . ). the it or the informatics department size (in terms of the number of personnel) at the dental schools is illustrated in fig. . . thirty fi ve percent (n = ) of the us dental schools that housed an it / informatics departments had personnel with not only it training but also dental informatics training. while % (n = ) of the dental schools were considering integration of dental informatics personnel within their department or center. twenty fi ve percent (n = ) of the dental schools did not have any plans of integrating personnel with dental informatics personnel within their department or center (see fig. . ). partial responses to additional questions in the section of the survey is provided under table . . the majority of the responding dental schools were currently using financial electronic systems (fes) (p-value of < . ) and electronic dental records (edr) (p-value of . ). the use of fes outnumbered the use of edrs in the dental schools (see fig . ) . about % of the dental schools that were currently utilizing the edrs used it in all the clinical modules (p-value of . ), while % of the dental schools used the edrs in some of the clinical modules. when asked about the commercial edr system that the dental schools were using, axium (exan group, canada) was by far the most implemented edr system. two dental schools had salud (two-ten health limited, ireland) implemented and two dental schools had gsd academic (general systems design group, iowa, us) implemented. combinations of two ehr systems (home grown and dentrix) were implemented at two dental schools. one school had a dentrix only implementation, while another had developed its own edr system (home grown) (see fig. . ). there were dental schools which had implemented an edr fi ve or more years ago, dental schools - years ago, dental schools - years ago and dental schools less than a year ago (see fig. . ) (p value of . ). when the dental schools were asked the question as to whether they were expecting to apply for the medicaid meaningful use incentive program, majority ( %) of the dental schools did not know and only % of the dental schools were expecting to apply within the next years (fig. . ) (p-value of . ). challenges or barriers identifi ed by some of the dental schools in complying with the meaningful use objectives were (a). lack of certifi ed edr and information regarding it, (b). issues with getting auxium certifi ed and (c). qualifi cations of the edr as many of the meaningful use objectives do not apply to dentistry and lack of specifi c information about it. only % of the responded dental schools were part of a health system. fifty two percent (n = ) of the responded dental schools had a formal relationship with other health care delivery entities in terms of sharing facilities, patient transfer, training programs. some of the types of relationship mentioned by the dental schools that had a formal relationship with other health care delivery entities included: (a). a gpr program and an emergency dental unit in the hospital, (b). affi liated hospital, (c). affi liation agreements, (d). oral and maxillofacial surgery (omfs), anesthesia and pedodontics all have some portion of education in medical health center, (e). omfs residents are also residents of medical health center, (f). residents providing care under contract with area hospitals, (g). sharing patients wand facilities with the health center, (h). students rotating in the community health centers and (i) collaborative grand programs. eighty fi ve percent of the dental schools that had a formal relationship with the health care delivery entities had routine interaction with them because of their existing relationship (p-value of . ). their usual method for exchanging information was through informal medium such as phones, emails and faxes and formal medium such as memorandums, letters and contracts. when the dental schools were asked about the communication between the health systems' emr and the school's edr, majority of the dental schools did not have any communication ( %) or did not know is such a communication existed ( %) (p-value of < . ) (see fig. . ). out of the % (n = ) of the responded dental schools who's edr did not communicate with the health system's emr, % (n = ) of the dental schools stated that they did not need to exchange patient information electronically as a reason for the non-communication, while % (n = ) dental schools states that they would like to exchange patient information electronically but there were barriers that prevent them from doing so. some of the barriers identifi ed by these dental schools were (a). the hospitals and the dental school are not part of the same medial system and hipaa concerns prevent sharing data, (b). the dental school currently neither did have an edr nor the infrastructure to support one and (c). hospital is not interested and has high and perhaps unrealistic security standards. the remaining % (n = ) of the dental schools expected to exchange patient information electronically in the near future (next years). some of the information categories that were shared between the edr and emr in the small number of dental schools are illustrated in fig . . finally when asked about any research projects under way in their dental school to investigate discrepancies between medical and dental records for the same patient, only ( %) dental school was currently undertaking such project. in all common diseases, including those that affect the oral cavity, both the environment and genetics are pathogenic conspirators. unfortunately, we currently know little about the specifi c mechanisms underlying any common disease; and oral diseases are among the least understood. elucidating the etiology of chronic oral diseases will involve a synthesis of results from careful experiments of environmental exposures such as diet and tobacco use, the oral microbiome, co-morbidities, largescale, well-designed genetic studies, and the various interaction effects. with regard to genetics, the past few decades have witnessed transformative developments in our ability to interrogate the entire genome for genes that contribute to disease. while dramatic advances in experimental designs, statistical approaches, and clinical insights have greatly aided this scientifi c campaign, the central driver of this progress has been the development of high-throughput, inexpensive genetic technologies. following initial molecular studies using variant forms of enzymes, or allozymes, a major breakthrough was the use of highly informative dna-based markers throughout the genome (botstein et al. ) . this idea of directly assaying existing dna variation to conduct linkage and association studies in genetics began a revolution in disease gene mapping. recent interest from commercial entities has produced a feverish pace of technological innovation, markedly reducing cost and expanding the depth of inquiry. previously unfathomable, the testing of over one million single nucleotide polymorphisms (snps) in thousands of patients and controls is now commonplace (wellcome trust case control consortium ; schaefer et al. ) ; and very recently, next generation sequencing technologies have progressed to the point where sequencing of the entire protein-coding portion of the genome (exome) or even the entire genome is a costeffective method to examine disease traits across the entire spectrum of genetic variants in small numbers of affected individuals (ng et al. ) . there is little doubt that soon whole genome sequencing will be applied to nuclear family-based designs, studies among distantly-related affected individuals in extended pedigrees, and case/control studies involving thousands of individuals. this unprecedented scope of inquiry made possible by large-scale genetics, has begun to yield fascinating resulting into predisposition to oral cancers, caries, and periodontal disease that will molecularly redefi ne these pathologies, explicate unique biological connections with related diseases, give impetus to the development of directed therapeutics, and indeed personalize medicine. still, much more genetic focus on oral disease phenotypes is required if we are to realize this medical impact in a timely fashion. as genetic technologies have allowed the progression of interrogating single protein variants to single dna markers to entire genes to markers across the genome, and now to the entire genome sequence, the promises of these large-scale genetic studies have understandably undergone monumental expansion. it may be reasonable to expect the results from whole genome sequencing to decidedly revolutionize medicine within the next two decades. however, this new scientifi c capacity comes at a cost. as genetics, and biology in general, transitions to a data-rich science, practitioners have found themselves woefully unprepared to store and analyze the volume of data generated. once analyzed, interpretation and integration of these abundant and multifaceted results into medical practice will also be an appreciable challenge. insuffi cient assimilation of genetic fi ndings into merged dental and medical records will severely limit the ability of clinicians to appropriately treat patients. inadequately addressing these informatics issues will severely derail efforts in the basic sciences efforts as well as the translational and clinical sciences. this chapter explores the current state of genomics studies, what we have learned from genetic investigations into oral diseases, and where we may be headed. genetic studies have much to offer investigations of disease etiology. why do some acquire diseases and others do not? for those affected, why do some progress more rapidly than others? what causes some patients to respond to therapies, while others suffer from adverse reactions? these are all fundamental questions in both biology and medicine, whether the focus is on the gastrointestinal tract, the hippocampus, the lymphatic system, metabolic disorders, or oral diseases. speaking generally across disease areas, a portion of the answers to these questions often lies in described environmental effects. in numerous chronic diseases, infectious agents are likely contributors to the disease process -periodontitis, for example, is initiated by gram negative anaerobes in susceptible individuals (holt and ebersole ) . surely, unique and latent environmental exposures provide a random component to common disease susceptibility and progression. through twin studies, studies of risk in close relatives, and quantitative traits experiments, it is well-understood that heritable factors, including but not limited to dna variation, are typically responsible for - % of the phenotype variability for common diseases. this section will attempt to cover, at least at a cursory level, the major salient developments affecting genetic insights into chronic and aggressive periodontitis, with some comment on genetic factors infl uencing susceptibility to caries and oral cancers. while it would be extremely naïve to view genetic studies as an immediate panacea for our ills, the discovery of disease-causing genes does illuminate hitherto unknown biological pathways and molecular mechanisms, draws unforeseen connections with other traits, may improve prognostic models applicable for individuals, and suggests specifi c therapeutics. industrialization has brought forth increased lifespan and wellness through vaccination, modern sanitation practices, public health policies, and advances in medical science translated into practice. however, the accompanying physical inactivity coupled with a high calorie diet are probable contributors to an extremely common, chronically infl amed metabolic syndrome (hotamisligil ) that is thought to give rise to a multitude of intimately related disease traits: insulin resistance, compromised insulin signaling, hyperglycemia, obesity, dyslipidemia, hypertension, impaired kidney function, elevated liver enzymes and steatohepatitis, poor wound healing, neurodegeneration, vascular disease, pregnancy complications, accelerated immunosenescence, and periodontal disease (ford et al. ; ferrannini et al. ; eaton et al. ; holvoet et al. ; speliotes et al. ; eckel et al. ; d'aiuto et al. ) . these diseases often co-occur within the same patient and could be considered variable expression complications arising from a state of aberrant caloric fl ux that induces metabolic dysfunction and chronic, systemic infl ammation. these features constitute a disruption in a fundamental homeostatic mechanism with intensifying pathogenic consequences. the rapidly increasing incidence and decreasing age of onset for this pathophysiological state have generated a major source of mortality and morbidity in modern cultures (ford et al. ; ferrannini et al. ; weiss et al. ) . it is becoming increasing clear that many chronic diseases have an infectious component. there is relatively convincing evidence that many systemic, t-cell mediated autoimmune disorders may be initiated by infections. for example, from archaeological data, it is believed that an infectious agent -currently unknown -is necessary for rheumatoid arthritis (firestein ) , and both guillain-barre syndrome and rheumatic fever have well-described pathogeneses triggered by specifi c infections in susceptible individuals (bach ) . in many instances, oncogenesis and tumor progression can be traced to pro-infl ammatory responses at the site of chronic infection (coussens and werb ) , although it is not known whether these effects are mediated through the actions of the immune system, the infectious agents, or a combination thereof. several cancers fall into this category including gastric adenocarcinoma (uemura et al. ) , cervical cancer (walboomers et al. ) , hepatocellular carcinoma (saito et al. ) , and kaposi's sarcoma (dictor ) , all having unequivocal infectious agent etiologies. recent fi ndings of antiinfl ammatory pharmaceuticals, particularly those that inhibit cox- and cox- , reduce the incidence of certain classes of cancers are consistent with this view (dannenberg and subbaramaiah , rothwell rothwell et al. ) . in addition, there is moderate evidence that several bacteria -the most studied is chlamydia pneumoniae -play a role in atherosclerosis and myocardial infarction (saikku et al. ; watson and alp ) , however the studies are not conclusive and antibiotic treatment does not appear to be effective (andraws et al. ) . chronic periodontal disease is fi rmly footed at the intersection of infection, chronic infl ammation, and metabolic dysfunction. chronic periodontitis is characterized by infl ammation of the periodontal membrane, slowly causing gingival recession and eventual bone loss. the proximate cause of periodontitis is the virulent oral microbiome. the involvement of gram negative anaerobes has been fi rmly established for the disease. aside from the known oral pathogenic species p. gingivalis , t. denticola , and t. forsythensis , the so-called "red complex" (holt and ebersole ) , new bacterial species associated with chronic periodontitis have also been described (kumar et al. ) . the advent of an extensive database covering the oral microbiome will surely propel such investigations . numerous studies have shown that periodontal disease covaries with many diseases, presumably due to overlapping molecular etiologies. compelling meta-analyses demonstrate a highly signifi cant synchronicity of obesity and periodontal disease (chaffee and weston ) . in addition, the correlation between periodontal diseases/ alveolar bone loss and frank metabolic syndrome is repetitively observed (nesbitt et al. ; andriankaja et al. ) . extensive work has also shown a strong role for both infl ammation-related genes and circulating infl ammatory markers in periodontal disease (nikolopoulos et al. ; bretz et al. a, b ) . treatment studies further support the link between periodontal disease and immuno-metabolic syndrome. these experiments have demonstrated a signifi cant improvement in intermediate molecular markers of infl ammation when chronic periodontitis in the presence of metabolic syndrome (acharya et al. ) or type diabetes (iwamoto et al. ) was treated. conversely, treatment of periodontal disease with reduction of bacterial load leads to greater glycemic control among diabetic patients (simpson et al. ; stewart et al. ) . given the high prevalence of periodontitis and the co-morbidity of metabolic syndrome with periodontal disease, these treatment experiments appear to suggest that the virulent oral microbiome could play an important role in the pathogenesis of systemic infl ammatory metabolic syndrome, and is exacerbated by the syndrome. certainly, further studies are needed to defi nitively answer this question. as chronic periodontal disease seems to be a critical feature of sustained, systemic dysfunction of both metabolic and infl ammatory networks, uncovering the genetic variants carried by susceptible individuals would not only provide much needed insight into the molecular pathogenesis of chronic periodontal disease, but would also markedly aid our understanding of the infl ammatory metabolic syndrome and how it drives related co-morbidities. such genetic studies may also shed light on the specifi c mechanisms that appear to improve cardiovascular, infl ammatory, and diabetic outcomes when periodontal disease is treated, potentially leading to therapies and medical/dental intervention with greater effectiveness. such studies may also provide clues to which subsets of individuals respond more effectively than others and why they do so. periodontal disease can also present in a rapid manner with aggressive bone loss and early-onset. this is termed aggressive periodontitis (lang et al. ) . in contrast to chronic periodontitis, there is often a greater degree of familial aggregation with aggressive periodontitis, and it is hypothesized that most aggressive cases may affl ict individuals with one or more defective immune genes (zhang et al. ; amer et al. ; machulla et al. ; carvalho et al. ; toomes et al. ; hart et al. ; hewitt et al. ) . mutations in the lysosomal protease, cathepsin c, have been shown to be responsible for some forms of aggressive periodontitis, along with complications associated with other infl ammatory diseases (laine and busch-petersen ) . the specifi c hla variants thought to play a role in aggressive periodontitis, are also involved in infectious disease susceptibility and autoimmunity; and, interestingly, two of the non-mhc-linked regions, fam c and a locus on chromosome p , have been implicated in myocardial infarction (connelly et al. ) and may have action as a tumor suppressor in tongue squamous cell carcinoma (kuroiwa et al. ) . as with chronic periodontal disease, an infectious microbiome is heavily involved. however, in general, microbiome differences could not explain the presence of chronic versus aggressive forms of the disease, although in some aggressive periodontitis patients, a highly leukotoxic a. actinomycetemcomitans strain may contribute to the disease process (mombelli et al. ) . we currently do not fully know the differences between the genetic susceptibility factors for the chronic and aggressive forms of the disease. the most prevalent chronic disease in both children and adults is dental caries (national institute of dental and craniofacial research) . caries formation is a complex disease with several interacting components form the environment and host genetics. similar to gingivitis and periodontitis, caries have an infection-initiating etiology with acidifi cation leading to localized demineralization. epidemiological studies have long shown that diet is a strong predictor of caries formation; and the reduction in ph is exacerbated by high consumption of carbohydrates. the principal pathobacterial species are streptococcus mutans and lactobacillus ( van houte ) . there are also several reports of positive correlations of caries with infl ammatory diseases, although the association is not always repeatable. it is also not clear what proportion of the putative association with infl ammatory disease is due to innate upregulation of immune networks in contrast to the immuno-modulating pharmaceuticals prescribed to those with infl ammatory disease (steinbacher and glick ) . much of the effect is reported to result from lack of saliva volume (steinbacher and glick ) . interestingly, the presence of epilepsy may be associated with higher caries rates (anjomshoaa et al. ) . fluoride is an effective antimicrobial agent that interferes with bacterial growth and metabolism (wiegand et al. ) . hence, topical fl uoride administration as well as ingestion of fl uoridated water inhibits cariogenesis and caries progression (ripa ) . amelogenesis is a key process involved in modifying the rate of caries formation. both common variation and rare mutations in enamel formation genes such as amelogenin and enamelin are involved in caries rates (patir et al. ; kim et al. ; crawford et al. ) , the molecular actions of which are beginning to be revealed (lakshminarayanan et al. ) . over , new cases of cancers affecting the oral cavity and pharynx were expected in the united states for , with deaths numbering , (jemal et al. ) . the majority of these malignancies involved solid tumors originating from cancerous changes in squamous cells of the mouth. again, oral cancers have a complex etiology existing of entangled genetic, epigenetic, infectious, and dietary causes, further modifi ed by tobacco, alcohol and other environmental exposures. as with most cancers, it is reasonable to expect that both germline and somatic genetic changes will be involved in carcinogenesis, tumor growth, and metastasis. promoter hypermethylation of genes central to cellular growth, differentiation, dna fi delity, apoptosis, and metabolic stability is an important facet of these cancers (poage et al. ) . indeed, methylation-mediated silencing of genes involved in tumor suppression (e.g. the cyclin-dependent kinase inhibitor a), detoxifi cation (e.g. mgmt ), and apoptosis (e.g. the death-associated protein kinase- ) are commonly found in oral squamous cell carcinoma samples (ha and califano ) . to quantify the proportion of the variance in a phenotypic trait that is due to variance in genetic factors, population geneticists defi ned the concept of heritability (visscher et al. ; falconer and mackay ) . researchers subsequently developed several methods for estimating heritabilities using the measure of a trait (e.g. occurrence of disease/not-disease) in combinations of relatives (e.g. parentoffspring, or monozygotic-dizygotic twins). in general, the higher the measured heritability of a variable phenotype, the larger the contribution of genetic factors is in comparison to environmental effects. it is fallacious to assume that the heritable variation is composed entirely of alleles residing in the dna sequence, for heritability studies simply examine the covariance between relatives without comment on specifi c molecular mechanisms. hence, any heritable variation such as methylation patterns, vertically-transmitted infectious agents, as well as dna variation can contribute to the heritability measure. heritability results are important because they not only give a rough estimate of the collective effects of heritable factors, but also can provide a measure to quantify how much of the total genetic effect is accounted for by specifi c loci examined. for periodontal disease, four twin-based studies of heritability have been performed (michalowicz et al. ; corey et al. ; michalowicz et al. ; mucci et al. ) . although varying in sample size and methodological details, all four arrived at consistent results, with - % of the variance in periodontal disease being attributed to genetic variability for chronic periodontitis. given the segregation patterns described in the literature, it is reasonable to assume that aggressive periodontal disease exhibits a higher heritability. therefore, given the prevalence of periodontal disease, heritable factors within the population at large are likely appreciable. using twin pairs, bretz and colleagues reported substantial heritability values for multiple traits related to caries ranging from % to % (bretz et al. a, b ) . lastly, mutagen sensitivity studies of head and neck cancer patients suggest a signifi cant effect of genetic factors for the carcinogenesis of oral cancers (cloos et al. ) . hence, there is every reason to believe that a sizable pool of genetic and/or epigenetic factors await discovery for oral diseases. once the development of pcr (saiki et al. ) was applied to the idea of using naturally-occurring dna variation (botstein et al. ) , large-scale dna-based studies of disease underwent a substantial acceleration (schlotterer ) . genotyping of short, tandem repeated sequences (weber and may ) -microsatellites -spurred on a wave of genome-wide linkage studies, which evaluate the co-segregation of disease state with microsatellite markers, for both rare mendelian disorders as well as more common diseases with complex inheritance patterns. while the rarer traits with more coherent transmission patterns generally relinquished their genetic secrets to linkage analysis, more common diseases did not. in the mid-to late s, several theoretical studies had shown that the power to detect disease-causing alleles is higher with association-based designs such as a case/control experiment or association in the presence of a linkage signal as in the transmission/disequilibrium test if the frequency of those alleles is high and the effects are moderate (kaplan et al. ; risch and merikangas ; jones ; long and langley ) . however, to conduct genomewide association studies presented an ominous obstacle for the genetic technologies at the time. the number of markers required to effectively cover the genome was prohibitively large as the chromosomal blocks in population-based samples used in association designs were expected to be small. even within large extended families, the limited number of recombination events generates substantial chromosomal blocks passed through the pedigree, but researchers had both theoretical and empirical evidence that the blocks in population-based samples were on the order of k base pairs for most large human populations. as the reader can imagine, the mean length of blocks that are shared by descent is inversely related to the product of recombination rate, the number of affected individuals and the number of meioses separating the affected individuals. in practice, even very large extended families segregate regions shared by affected members on the order of several million base pairs in length. however, once geneticists seriously considered large-scale studies using a case/control design where individuals are separated by say , meioses, it became clear that to adequately cover the much smaller shared regions across the entire genome, hundreds of thousands of markers would be required (kruglyak ) . utilizing the human genome sequence (venter et al. ; lander et al. ) , a number of studies at celera diagnostics provided an intermediate solution, where approximately , putative functional snps primarily located in genes were assayed through allelespecifi c pcr in a number of common diseases using a staged case/control design. these studies were successful in identifying several gene-centric polymorphisms associated with common diseases (begovich et al. ; cargill et al. ) (fig. . ) . concurrently, several groups had performed extensive sequencing and genotyping across the genome to produce a genome-wide map of haplotype structure (hinds et al. ) , useful in linkage disequilibrium mapping. within years, technology for snp hybridization arrays had advanced so as to enable genome-wide association studies capable of capturing most of the common genetic variation in the genome either through direct genotyping or indirect interrogation using linkage disequilibrium -the term linkage disequilibrium is a measure of the correlation of alleles at closely-linked sites (see fig. . ). these investigations were met with numerous successes (klein et al. ; kathiresan et al. ; graham et al. ; gudmundsson et al. ) . inexpensive genotyping platforms and urging from theoreticians ensured that these genome-wide association studies were, in general, highly powered to detect all but very mild effects from high frequency alleles. these efforts, led by large academic consortia such as the wellcome trust, the international multiple sclerosis genetics consortium, and the broad institute and commercial entities such as decode genetics and perlegen have greatly expanded our understanding of the basic biology of common diseases: we now know, for example, that (i) autophagy-related genes are involved in crohn's disease (rioux et al. ) , (ii) there are a number of genes such as the protein tyrosine phosphatase, ptpn and the interleukin- receptor, il r , that exhibit ample pleiotropic effects among autoimmune conditions (lopez-escamez ; safrany and melegh ) , (iii) in the case of age-related macular degeneration, predictive models using the genetic results enable fairly accurate prognosis of individuals who are at high risk of disease (seddon et al. transcription factor tcf l plays a role in type diabetes (grant et al. ) , and (v) aberrant il- signaling likely contributes to multiple sclerosis susceptibility (gregory et al. ) . the plot shows the tremendous progress in genotyping technology where, a decade ago, very little of the genome was accessible for disease studies using association designs through the current wave of viable sequencing-based whole exome studies ( ) ( ) and whole genome studies ( ) ( ) . in fig. . , the average distance between adjacent genetic markers is plotted as a function of year of introduction to the disease mapping community. impressively, the total number of genetic markers has increased a million-fold over the past decade. although successful in uncovering numerous pathogenic pathways for common diseases, results from the current wave of genome-wide association studies, with a few exceptions, explain little of existing disease heritability. the reasons for this are cryptic and the subject of heavy debate (manolio et al. ) . multiple rare sequence variants generating high levels of allelic heterogeneity, functional de novo mutations, structural mutations such as copy number variants and large deletions, and epigenetic effects constitute four of several possible disease models that could account for the heritability discrepancy. the answer will almost certainly consist of a conglomeration of these and other effects. bringing forth the new genome-wide technologies that illuminate these previously non-or under-interrogated properties of the genome to bear on this enigma is a reasonable next step for all complex traits including oral diseases. the most commonly used measure of ld in a sample of chromosomes is linkage disequilibrium (ld) is a measure of the correlation between alleles at two sites in a sample of chromosomes. for two biallelic sites, if the a allele is always paired with the b allele, and the a allele is always on the same haplotype as the b allele, then the two sites are said to be in perfect ld. successive recombination diminishes ld. interrogating one site for disease association allows investigators to indirectly interrogate other sites in sufficiently high ld with the interrogated site. a key feature explicitly studied in molecular population genetics and implicitly used in disease gene mapping studies is the site frequency spectrum; that is, the distribution of allele frequencies at single sites in the genome that vary in the human population studied. from both diffusion models (kimura ) and coalescent theory (hudson ) in theoretical population genetics, we know that the vast majority of realistic models generate many more rare variants compared to common polymorphisms. this is particularly true for expanding populations. are these rare variants the source of much of the missing heritability? recently, with the application of high-throughput sequencing technology to human studies over the past decade, empirical studies have clearly verifi ed these predictions -the large majority of variants have low frequencies (the international hapmap consortium ) . the distribution of deletions appears to be skewed toward more rare frequencies, presumably due to the deleterious effects of such variants. individual mutations appearing de novo typically are extremely rare events per locus, but collectively are numerous. other types of genetic variability, such as copy number repeats, span both ends of the frequency spectrum with the preponderance of the markers being rare. thus, there is a sizable pool of low-frequency variants in human populations that have yet to be thoroughly investigated. over the past few years it has become increasingly clear that structural variants exist in the human genome at a far higher rate than previously thought. structural variants can exist in a multitude of forms including deletions, copy number variants, and inversions among others. due to the nature of these genetic changes, many are considered to be highly disruptive of molecular function if they lie in functional motifs. indeed, there are several mendelian diseases are caused by fully-penetrant structural variants impacting a chromosomal region (lupski ) . numerous structural variants have recently been reported to be associated with common diseases, particularly in the neurological fi eld (sebat et al. ; stefansson et al. ; elia et al. ) , infectious disease susceptibility (gonzalez et al. ) , and drug metabolism (zackrisson et al. ) . although they have improved dramatically over the past few years, algorithms using snp-based data from hybridization arrays to infer copy number variants have had high error rates, perhaps explaining the rather low rates of replication of structural variation association results for common diseases. nevertheless, given the high frequency of structural variants, their pathogenic potential, and that we are on the precipice of a sequencing revolution in genome-wide studies, examination of these variants should be a high priority for new sequencing-based studies in oral disease susceptibility, progression, and related pharmacogenetic applications. as different technologies examine different portions of the site frequency spectrum (i.e. genome-wide snp scans interrogate variation that is common in the hapmap populations, whereas sequencing-based studies typically interrogate the entire frequency spectrum), where one believes genetic causation is harbored should infl uence the selection of genotyping technology. if common genetic variation contains the vast majority of heritable effects on disease phenotypes, then an investigator would be wise to employ a snp-based experimental design. if, however, there is reason to believe that a signifi cant portion of the genetic load of the disease studied exists in the highly populated portion of the distribution -the rare variants -then a sequencing-based study may be better suited to unravel causative alleles. the studies of heritability discussed previously show that there is heritable variation underlying a substantial portion of the variance observed in oral diseases. as discussed above, sequencing technologies may address many aspects of dna variation including copy number loci, rare haplotypes, inversions, and insertions/deletions, but it is also worthwhile to repeat that the molecular mechanisms for disease heritability are not necessarily limited to variation at the dna level. for a disease state, the covariance between relatives could be driven by co-inherited chromosomal regions or other phenomena. chief alternative heritable mechanisms include dna methylation (hammoud et al. ) , modifi cations to the histones (bestor ) , complex rna zygotic transfer (rassoulzadegan et al. ) , and vertical transmission of infectious agents. additionally, transgenerational effects offer an intriguing class of epigenetic mechanisms (nadeau ) . in a thorough review on epigenetics and periodontitis, gomez et al. make a strong argument for consideration of both cpg dinucleotide methylation and deacetylation actions on cytokine expression as a credible avenue for further investigation in periodontal disease etiology (gomez et al. ) . genome-wide epigenetic studies have been successfully conducted for oral cancers (poage et al. ) . the scale of this study on head and neck squamous cell carcinomas allowed these researchers to show a global pattern of tumor copy number changes signifi cantly correlated with methylation profi les that was not detectable at the individual gene promoter level. with advanced chromatin immunoprecipitation and new methods to study dna methylation, efforts to apply highthroughput epigenetic methods to oral diseases should be accelerated. numerous studies have been conducted in oral disease traits using a candidate gene approach. there are two large reviews of the existing candidate gene results (nikolopoulos et al. ; ) . laine and colleagues have recently put together a comprehensive review article covering gene polymorphisms. there are some suggestive fi ndings for cyclooxygenase- gene, cox- , the cytokineencoding genes, il and il b , the vitamin d receptor, vdr , a polymorphism immediately upstream of cd , and the matrix metalloproteinase- gene, mmp . however, these initial results will require further confi rmation, for the association patterns are inconsistent across independent studies, the statistical signifi cance is moderate, and the posterior probability of disease is decidedly bland. the striking pattern that emerges from the laine et al. summary data is the lack of coherent replication of genetic association for the vast majority of polymorphisms examined. the situation is reminiscent of genetic association studies prior to large-scale snp studies where poor repeatability of results plagued the fi eld. in a pivotal study from , hirschhorn and colleagues (hirschhorn et al. ) examined the state of genetic association studies, fi nding that "of the putative associations that had been studied three or more times, only six have been consistently replicated." the dearth of robust results was largely remedied when large-scale genetic studies were applied to very substantial numbers of well-characterized patients and geneticallymatched controls and stringent statistical criteria enforced. one can only suspect that a similar state of affairs is operating in genetic studies of chronic periodontitis. perhaps efforts to ( ) reduce the heterogeneity of the disease state through detailed clinical and laboratory assessments, ( ) drastically increase sample sizes, and ( ) expand the scope of inquiry to larger numbers of genes/regions, and examine a more comprehensive set of variants/epigenetic effects will improve the current situation. the second large study is a meta-analysis of studies, where nikolopoulos and colleagues analyzed six cytokine polymorphisms linked to il a , il b , il , and tnf-alpha (nikolopoulos et al. ) . two of these, an upstream snp in il a and a snp in il b , exhibited signifi cant association with chronic periodontal disease risk. although the results were not particularly strong, as is typical with complex diseases, the results do suggest the importance of infl ammation-response variability in chronic periodontitis predisposition. perhaps the strongest, most replicable genetic association fi nding with coronary heart disease and myocardial infarction is centered on the short arm of chromosome ( p . ) (mcpherson et al. ; helgadottir et al. ) . two studies of periodontal disease showed that the same alleles at the p . locus confer risk for aggressive periodontitis (schaefer et al. ; ernst et al. ) . the discovery of such a pleiotropic locus may explain a portion of the aggregation of periodontal disease with other co-morbid conditions. further studies investigating overlapping genetic susceptibility factors between periodontitis and cardiovascular disease, diabetes mellitus, metabolic syndrome, rheumatoid arthritis, and other related diseases may be a fruitful strategy for honing in on shared genes affecting these immuno-metabolic disorders. using patients from families from the philippines, the fi rst genome-wide linkage study for caries was completed in (vieira et al. ) . the study identifi ed fi ve loci which exhibit suggestive statistical evidence (lod scores exceeding . ): q . , q . , xq . , q . , and q . . the latter of which overlapped with a quantitative trait locus discovered from mapping work in the mouse. further work is necessary to refi ne these signals and localize the variants that may be driving these linkage signals. aggressive periodontal disease and rarer dental diseases have also been subjected to linkage analysis. results from linkage studies for dentinogenesis imperfecta type i, for example, have gone on to produce the novel gene fi ndings of the dentin sialophosphoprotein-encoding gene on q . being responsible (song et al. ; crosby et al. ) . a linkage study in african american families examining localized aggressive periodontitis found a strong linkage signal in a region covering approximately megabases on chromosome (li et al. ) . several interesting genes are in this region. in a study earlier this year further mapping from carvalho et al. in brazilian families identifi ed haplotypes in this region on q in fam c which were associated with aggressive periodontitis (carvalho et al. ) . the function of the fam c protein is not fully understood. fam c is localized in the mitochondria and it appears to play a role in vascular plaque dynamics and risk of myocardial infarction (laass et al. ) . it should also be noted here that other types of mapping analyses such as homozygosity mapping to identify have yielded gene discoveries. for example, the lysosomal protease cathepsin c gene for the recessively-inherited papillon-lefevre syndrome which is characterized by aggressive and progressive periodontitis was effectively mapped using homozygosity mapping (fischer et al. ; connelly et al. ) . cathepsin c is highly expressed in leukocytes and macrophages and is a key coordinating molecule in natural killer cells (rao et al. ; meade et al. ) . although sparse, these linkage results are undoubtedly encouraging. employing very large extended families subjected to genome-wide genotyping or sequencing will surely shed much needed light on chromosomal regions and genes relevant to oral disease research (fig. . ). for periodontitis, a single study has employed a genome-wide association design in an effort to uncover aggressive periodontal variants (schaefer et al. ) . this study by schaefer and colleagues discovered and replicated an intronic snp, rs , in the glycosyltransferase glt d which is signifi cantly correlated with aggressive periodontal disease in both german and dutch samples. often, seemingly signifi cant results from large studies are due to the effect of reporting the top result from a great many statistical tests -this is called the multiple testing problem. in this situation, the strength of the fi nding, along with the replication across three case/control studies, argues for true association with aggressive periodontal susceptibility. the snp may modulate the binding affi nity of gata- . the association with glt d is currently one of strongest genetic associations for aggressive periodontal disease, testifying to the power of genome-wide studies to generate novel, relevant molecular pathophysiology for complex diseases. it seems unlikely that glt d would be extremely high on a candidate gene list, and it was only through a genome-wide scan that it appeared. like many excellent studies, the fi nding by schaefer et al. raises more questions than it answers and will undoubtedly provide fertile ground for ensuing molecular work. after a somewhat sluggish start, due to a lack of critical mass of investigators aiming to collect large numbers of patient samples and bring high throughput genetic technologies to caries susceptibility, gingivitis, and periodontal disease traits, the future of genetic studies in oral health is bright. scientifi c progress in revealing the molecular pathogenesis of oral diseases is dependent on genome-wide genetic studies; and i have argued that progress in related immuno-metabolic diseases is also dependent on these large-scale genetic studies in periodontal disease. to study sporadic disease, substantial patient collection efforts are required for the application of these technologies. this may involve a combination of new recruitment and consortiumrelationships with existing collections. the beginning of such a collection for sporadic aggressive periodontitis in europe has shown extremely intriguing initial results, but more patients are needed to examine rare variants of moderate effect. both the german/dutch collection of aggressive periodontitis and the brazilian collection have begun to revolutionize the study of periodontal disease susceptibility with the discovery of glt d snps and fam c -linked haplotypes. there is little doubt that subsequent molecular work on these two genes will uncover novel mechanisms for the predisposition to aggressive periodontal disease. focus should also be placed on the collection of extended families segregating these diseases. applying sequencing technologies to large pedigrees can be an effective method of identifying rare variants and structural variants in a highly-refi ned phenotype. furthermore, applying these methods to the entire genome would make for a comprehensive genetic study. several trends in large-scale genomics science hold promise to signifi cantly advance our understanding of oral disease pathogenesis: the sociology of biological sciences has changed over the past years so as to • become more collaborative. essential for association-based designs, consortiumbased genetic research has blossomed over that time period, increasing sample sizes and therefore the power to detect disease-causing variants. there currently is consortium-based research in periodontal disease and oral cancer. further expanding these efforts will enhance subsequent studies, particularly those investigating rare alleles and/or rare epigenetic effects. through over a century of laboratory work, the collective knowledge of bio-• chemical pathways, signal transduction, cell physiology, regulatory mechanisms, and structural biochemistry is weighty. incorporation of this information into etiological models may substantially advance oral disease work as well as the fi eld of complex disease genetics in general. sophisticated analysis techniques are needed to perform this task. recent advances merging results from network science with probability theory within the context of computer science have produced the fi eld of machine learning. this rigorous framework can be used to identify those factors responsible for disease status and can also be used to develop robust predictive models using known biological networks and genetic data. the output from such models, typically the probability of disease, an estimate of disease progression rate, or a probability of adverse reaction, can be used by physicians and dentists to personalize medical care. until relatively recently, population genetics did not contribute a great deal to • human genetics research. that has changed in the past decade where effort spent on association studies surpassed that spent on family-based studies. those investigating disease gene mapping began to collaborate with population geneticists and population geneticists took up a wide-spread interest in fi nding disease alleles. incorporation of population genetics theory into such studies markedly improved association studies on several levels: confounding by population stratifi cation was effectively treated using population genetics, linkage disequilibrium patterns. use of population genetics theory in large-scale oral disease mapping studies may accelerate discoveries. sequencing technology has rapidly progressed over the past decade. currently, • sequencing studies across the exome can be accomplished at reasonable cost and yield data for all known genes in the genome. within the next few years, sequencing costs will depreciate to a point where whole-genome sequencing studies will be commonplace, using both family-based and population designs. application of these technologies to oral disease studies is imperative for comprehensive studies of etiology. high-throughput dna methylation and chromatin immunoprecipitation studies • will enable large-scale epigenetic studies in oral diseases (meade et al. ; ehrich et al. ; bibikova et al. ; ren et al. ; pokholok et al. ) . these have already started to play an important role in delineating mechanisms responsible for oral cancers (poage et al. ) . additional application of these techniques to studies of gingivitis, caries, and periodontal diseases may generate novel fi ndings. molecular biologists and pharmacologists have increasingly become able to • develop and evaluate highly targeted pharmaceuticals based on genetic discoveries. the use of such genetic information may improve the chances of developing effi cacious therapies. geneticists and disease researchers are beginning to realize that oral diseases • both impact and are intrinsically tied to susceptibility and progression of other common diseases. a synthesis of genetic fi ndings from immuno-metaboliclinked disorders would seem to greatly increase the knowledge of these diseases and better pinpoint their respective etiologies. as the new high-throughput genomics and epigenomics technologies become • implemented in oral disease research, the storage, management, analysis, and interpretation of the ensuing colossal amounts of data will be critical to enable clinicians to use these results in daily practice. advances in dental and medical informatics will facilitate these steps. we are in exciting times where advances in genetic technologies will uncover the genetic causes of diseases, including those that affect the oral cavity. with more focus in the area of oral disease genomics and the harnessing of new high-throughput sequencing and epigenetic technologies, novel insights into the pathways driving these diseases are imminent. these discoveries will, in turn, motivate directed therapies, aid in illuminating the molecular etiology of related disorders such as diabetes, and increase the level of personalized medicine. joseph kilsdonk the title of this section reinforces a institute of medicine (iom) report titled "dental education: at the crossroads." to quote yogi berra, a baseball sage: "when you come to a fork in the road, take it." the implication being that dental education must take action and move beyond its crossroads. these crossroads are described in the fi rst third of the section. it includes a summary and recommendations of the iom report and three transitional reports that followed: the surgeon general's report identifying oral health as a silent epidemic, the josiah macy foundation report, and a "pipeline" study funded by both the robert wood johnson and the california foundations. having been at the crossroads for a decade or so, the middle portion of the section highlights educational models that may lead to a more promising future. the later third of this section describes an alternative path of action for dental education which emphasizes the central roles of clinic-based education and dental informatics in dental education curriculum. it is unknown how traditional dental educators may view this model; however, it is effectively a logical conclusion and responsive to the reports. in the institute of medicine (iom) published "dental education at the crossroads" (field ) . the title was apropos as the authors' analysis concluded: ( ) economics surrounding dental education were unsustainable ; ( ) student service learning opportunities and access to care for patients were limited; and ( ) new dental schools were not replacing those forced to close due to the economic climate. the iom report additionally proposed key recommendations to reform dental education and service delivery. fifteen years later, we remain at "the crossroads" as these issues remain largely unresolved. furthermore, these recommendations have retained their validity. their implementation would directly impact structures and services for contemporary models of dental education in the future. the following iom recommendations (field ) are intrinsic to the proposed dental education reform: recommendation : to increase access to care and improve the oral health status of underserved populations… recommendation : to improve the availability of dental care in underserved areas and to limit the negative effects of high student debt… recommendation : to prepare future practitioners for more medically based modes of oral health care and more medically complicated patients, dental educators should work with their colleagues in medical schools and academic health centers to: move toward integrated basic science education for dental and medical • students; require and provide for dental students at least one rotation, clerkship or • equivalent experience in relevant areas of medicine and offer opportunities for additional elective experience in hospitals, nursing homes, ambulatory care clinics and other settings; continue and expand experiments with combined md-dds programs and • similar programs for interested students and residents; increase the experience of dental faculty in clinical medicine so that they, • and not just physicians, can impart medical knowledge to dental students and serve as role models for them. recommendation : to prepare students and faculty for an environment that will demand increasing effi ciency, accountability, and evidence of effectiveness, the committee recommends that dental students and faculty participate in effi ciently managed clinics and faculty practices in which the following occurs: patient-centered, comprehensive care is the norm; • patients' preferences and their social, economic, and emotional circumstances • are sensitively considered; teamwork and cost-effective use of well-trained allied dental personnel are • stressed; evaluations of practice patterns and of the outcomes of care guide actions to • improve both the quality and the effi ciency of such care; general dentists serve as role models in the appropriate treatment and referral • of patients needing advanced therapies; larger numbers of patients, including those with more diverse characteristics • and clinical problems, are served. recommendation : because no single fi nancing strategy exists, the committee recommends that dental schools individually and, when appropriate collectively evaluate and implement a mix of actions to reduce costs and increase revenues. potential strategies, each of which needs to be guided by solid fi nancial information and projections as well as educational and other considerations, include the following: increasing the productivity, quality, effi ciency, and profi tability of faculty • practice plans, student clinics, and other patient care activities; pursuing fi nancial support at the federal, state, and local levels for patient-• centered predoctoral and postdoctoral dental education, including adequate reimbursement of services for medicaid and indigent populations and contractual or other arrangements for states without dental schools to support the education of some of their students in states with dental schools; rethinking basic models of dental education and experimenting with less • costly alternatives; raising tuition for in or out-of-state students if current tuition and fees are low • compared to similar schools; developing high-quality, competitive research and continuing education • programs; consolidating or merging courses, departments, programs, and even entire • schools. in summary, the iom report identifi ed that: ( ) an outdated curriculum continues to be retained which refl ects past dental practice rather than current and emerging practice and knowledge; ( ) clinical education does not suffi ciently incorporate the goal of comprehensive care, with instruction focusing too heavily on procedures; ( ) medical care and dentistry are not integrated; and ( ) the curriculum is crowded with redundant material, often taught in disciplinary silos. the iom's report was followed by the surgeon general's report on oral health in and a subsequent supplement by the surgeon general in called "the national call to action" (u.s. department of health and human services ) . five signifi cant fi ndings and recommendations from the surgeon general's report(s) that have implications pertaining to the envisioned structure and services of new models for dental education include: changing the perception of oral health so that it will no longer be considered • separate from general health; improving oral health care delivery by reducing disparities associated with popu-• lations whose access to dental treatment is compromised by poverty, limited education or language skills, geographic isolation, age, gender, disability, or an existing medical condition; encouraging oral health research, expanding preventive and early detection pro-• grams, and facilitating the transfer of knowledge about them to the general population; increasing oral health workforce diversity, capacity, and fl exibility to overcome • the underrepresentation of specifi c racial and ethnic groups in the dental profession. in this regard, the national call to action urged the development of dental school recruitment programs to correct these disparities and to encourage parttime dental service in community clinics in areas of oral health shortage; increasing collaboration between the private sector and the public sector to cre-• ate the kind of cross-disciplinary, culturally sensitive, community-based, and community-wide efforts to expand initiatives for oral health promotion and dental disease prevention. spurred by the iom report and the surgeon general's report, the josiah macy foundation ( ) conducted a study entitled "new models of dental education." the study was prompted by concerns about declines in dental school budgets and the diffi culties experienced by schools in meeting their educational, research, and service missions. the macy study concluded that: financial problems of dental schools are real and certain to increase. • current responses of schools to these economic challenges are not adequate. • most promising solutions require new models of clinical dental education. • macy study lead researcher dr howard bailit, and his team recently concluded in reference to points one and two above, that: "if current trends (to aforementioned) continue for the next years, there is little doubt that the term crisis will describe the situation faced by dental schools. further, assuming that it will take at least ten or even more years to address and resolve these fi nancial problems, now is the time for dental educators, practitioners, and other interested parties from the private and public sectors to come to a consensus on how to deal with the coming crisis. clearly, these fi nancial problems will not be solved by minor adjustments to the curriculum, modest improvements in the clinical productivity of students or faculty, or even signifi cant increases in contributions from alumni. the solutions 'must involve basic structural changes in the way dental education is fi nanced and organized' (bailit et al. ) ." this statement is supported by the fact that in the past years more dental schools have closed than opened. specifi cally eight schools have closed, whereas to date a couple has opened and a handful is pending. curriculum relevance was also a focus of the study. findings concluded that "changing the curriculums in dental schools to allow students to spend more time in community venues would be highly benefi cial to both society and student. society benefi ted from having underserved patients cared for while students were assessed as being fi ve to ten times more productive, more profi cient, more confi dent, more technically skilled and more competent in treating and interacting with minority patients" (brodeur ) . macy study (formicola et al. ) outcomes represented signifi cant and foundational guideposts for assessing and planning any future models for dental education. their report led to the robert wood johnson foundation pipeline study ( ) , a major research study funded by the robert wood johnson foundation and the california endowment (tce). the goal of the dental pipeline program was to reduce disparities in access to dental care. the pipeline study provided over $ million for the start up or expansion of schools and student clinical programs that incorporated services to underserved extramural clinical settings (primarily community health centers). the following recommendations from the surgeon general's report structured • the goals of the pipeline's initiative: increase the number of under-represented minority and low-income students enrolled in the dental schools participating in the pipeline program so that there would be a voice of minority and low-income students at all the funded schools. provide dental students with courses and clinical experience that would prepare • them for treating disadvantaged patients in community sites. have senior dental students spend an average of days in community clinics • and practices treating underserved patients. increasing the community experience of dental students was expected to have an immediate impact on increasing care to underserved patients (brodeur ) . this third point is pivotal to future success of dental curricula and dental education economics. recently published in a supplemental volume to the journal of dental education , february, , the pipeline study reported the following outcomes: minority recruitment of low-income students increased by %; • the rate of recruitment for under-represented populations was almost twice that • of non-pipeline schools; the length of time dental students spent in extramural rotations increased from a • mean of days to a mean of days over a period of years. procedural profi ciency increased compared to that of their non-extramural peers. of the pipeline-funded programs, only four schools achieved the goal of • days of extramural rotations; through extra funding from tce, the four schools extended extramural rotations to an average of days; based on this publication, it appears that only a handful of pipeline schools defi -• nitely plan to sustain their extended extramural rotations. financial concerns were highlighted as the major problem in sustaining future recruitment and placement of students beyond the timeframe of the study; a survey of program seniors indicated a mean of % [range of - % by • school] were planning to devote ³ % of their practice to serving minority patients. only % [range of - % by school] were planning to practice at community clinics. in the context of these outcomes, discussion indicated that the unwillingness of students to practice in underserved settings was based on several factors: students that participated were already enrolled in traditional programs and were • not necessarily seeking a pipeline experience or a future in community service. concern over future reimbursement as a provider in a community setting; • limited time spent in underserved settings; • limited loan forgiveness scholarship opportunities. • the fact that the large majority of pipelines were unsustainable was attributed to lack of productivity in the school clinics while the students were on rotation at community based clinics. schools generate meager, yet necessary revenue streams on intramural student clinical activity to support the costly clinical and faculty infrastructure. currently, similar economic constraints involved with outsourcing students to serving rural and underserved populations impacts the ability of tradition dental schools to participate in sustained outreach programs. most recently, the pew center on the states national academy for health policy ( ) released "help wanted: a policy makers guide to new dental providers". this report provided an excellent summary outlining workforce needs, access issues, and strategies for dental-related services to help states and institutions develop creative ways to solve oral health access and care issues. the guide proposes the following relevant components and trends for consideration in development of future sustainable school models: dental colleges are willing to bear a large and disproportionate share of the burden • in terms of access to care, particularly during a time of incredibly scarce resources. expanded, extensive, and/or creative extramural rotations have been developed • in recent years under the conceptual umbrella of service-learning. these often involve clinics providing direct or indirect payment to dental schools or clinics managed in some way by dental schools. dental education has certain obligations. first, education must adhere to accreditation standards with the goal of producing competent practitioners. second, education must remain responsive and impact the societal need for care. lastly, the delivery of dental education must be economically sustainable. the macy, rwjf, and iom reports note that improved oral health, sustainable dental education economic models, and competent workforce pipelines converge around community health centers (chc). university of michigan researchers fitzgerald and piskorowski ( ) reaffi rm this conclusion in an evaluation of an ongoing -year program, stating that: (the chc model) is self-sustaining and can be used to increase service to the underserved and increase the value of students' clinical educational experiences without requiring grant or school funding, thus improving the value of dental education without increased cost. self-sustaining contracts with seven federally qualifi ed health centers (fqhcs) have resulted in win-win-win-win outcomes: win for the underserved communities, which experienced increased access to care; win for the fqhcs, which experienced increased and more consistent productivity; win for the students, who increased their clinical skills and broadened their experience base; and win for the school in the form of predictable and continuing full coverage of all program costs (fitzgerald and piskorowski ) (fig. . ). however, unlike medicine that outsources their students to clinical sites, dental education programs retain the majority of the student time within their own "clinical laboratories" as documented by the aforementioned studies, this limits students' exposure to extramural experiences. costs to operate such intramural clinical programs are ever increasing and many schools' clinical operations run defi cits. if that component can be outsourced to community-based resources such as a chc, then the burden of cost is shifted away from the school. an example would be a.t. still university's arizona school of dentistry and oral health (asdoh) which matriculated its fi rst class in . at the prompting of the state's community based clinics, asdoh designed a program that placed students into community-based settings for up to months, an unprecedented length of time for an extramural rotation. they also saw this as an opportunity to use an adjunct centric faculty that signifi cantly reduced traditional education overhead. through this innovation, the school was able to develop a program that was sustained by "fair market" value tuition and trained students where community needs were greatest for up to months (which was then unprecedented). conversely, if the chc can rely on student service-learning to care for patients, the cost of care is reduced. other schools are also advancing with innovative education and care delivery. adea's charting progress (valachovic ) fig. . the synergy between access to care, student competency, and fi nancially sustainable dental education converge around chc/fqhcs little rock, arkansas; and the university of southern nevada in south jordan, utah. western university is planning placement of % of their fourth year class in community health centers, while east carolina is seeking to set up rural clinical campuses as well as clinical partnerships with the state's fqhc. at the time of this publication, several existing schools are expanding or looking to expand including the university of north carolina, marquette university, midwestern university in downer's grove, il. such expansions will contribute to solving the existent access supply and demand issues. however, it was observed even with all the start ups and expansions, graduation numbers will not approach the output of schools in the late s and early s. these creative models establish the foundation for a sustainable clinic structure by generating self-sustaining revenue through student service-learning, which, unlike medical student services, are billable. simultaneously these new models provide access to care for the needy while student exposure to clinical experiences that are often not available in academic patient pools. these models also shift some of the cost of providing clinical education from the dental college to community-based clinics. however, this innovation is not without criticism. schools are dependent on the success of their clinics and clinic partnerships. one author cautions: "however, these creative models also may present potential political strategic risk or confl ict: private practitioners may organize and protest higher than normal reimbursement schemes. potentially, such protests could even jeopardize the very existence of such models (dunning et al. ) ." notably, community health centers have historically received strong bipartisan support. for example, during the bush administration, fqhc funding was doubled and most recently expanded through health reform legislation by the obama administration. according to the institute for oral health, "the group practice of the future is the dentist working with the physician" (ryan ) . the ada reported "multidisciplinary education must become the norm and represent the meaning and purposes of primary care as it applies to dentistry. educational sequences should include rotation strategies across discipline specialties in medicine and dentistry, clerkships and hospital rotations, and experience in faculty and residency clinics." (barnett and brown ) the models alluded to, were school-based attempts at improving educational outcomes. perhaps the proverbial fork in the road regarding the future of dental education leaves two paths for consideration. is it better to travel down a road that leads a school to develop and operate a clinic? or is the road less traveled, where a clinic becomes a school, the better of the two options? the answer, perhaps, is that a combination of both will accomplish the desired outcome. for example, didactic knowledge is measured by examination whereas competency as a practitioner is measured by clinical demonstration. at a minimum, the result must achieve learner competency, quality, and sustainability. however, the road less traveled has not been taken yet. william gies, in his revered report written years ago on the state of american dental education, wrote "dental faculties should show the need…. for integrated instruction in the general principles of clinical dentistry and in its correlations with clinical medicine" (gies ) . basic sciences aside, could a clinicalbased educational training center have an advantage over a school-based clinical center? soon-to-be-implemented new commission on dental accreditation (coda) standards will require schools to demonstrate competency in patient-centered care (valachovic ) . might an enterprise profi cient at running a successful clinical business model have an advantage running a professional, patient-centered clinical training program as compared to a pedagogical business model attempting to run a clinical training model? these questions should challenge us to reexamine why our thinking about educational models should be limited to schools being the starting point for the development of a profession that demands clinical competency, patientcenteredness, and integration as outcomes. the clinic based model may serve as an equivalent starting point and, have some distinct advantages for achieving responsiveness to recommendations and directions cited in this section. beginning in november through august , the family health center (fhc) of marshfi eld, inc, marshfi eld, wisconsin, launched of a broad network of developing dental clinics, targeting dental professional shortage areas with the provision of dental services to the underserved communities whose dental needs were not being adequately met by the existing infrastructure. fhc-marshfi eld is a federally qualifi ed health center (fqhc). as an fqhc, fhc receives cost-based reimbursement for its dental services to medicaid populations. along with the cost-based reimbursement, fqhcs are obligated to provide care to anyone regardless of their ability to pay. presently, fhc is the nation's largest federally qualifi ed dental health center. to date, this network of dental clinics has served over , unique patients, % of whom were under % of poverty. notably, service was provided to a signifi cant number of cognitively and developmentally disabled patients in special stations developed for serving patients with special needs. these patients frequently travel the furthest to get to our dental centers for care. beginning in , fhc stepped up the pace of dental clinic expansion, constructing two new dental centers in , two in , and two more are slated to open in . when fully operational, this will establish capacity to serve , patients annually. each site has proactively included dedicated clinical and classroom training space for dental residents or students, thus laying the framework for clinic-based training of new dental professionals. the plan is to continue to stand up new dental centers until they have the capacity to serve , patients annually or approximately % of the , underserved patients in the rural service area. in addition to the capacity for training residents and students, a dental post-baccalaureate program is being considered in partnership with regional year under graduate campuses. the post-baccalaureate program is aimed at preparing students from rural and underserved areas who desire to practice in rural and underserved areas for acceptance and success in dental schools. presently fhc in partnership with marshfi eld clinic is moving forward with plans to develop dental residencies at these sites and a dental post baccalaureate training program to better prepare pre-doctoral students from rural and/or underserved backgrounds to be successful in dental school as a means to create a dental academic infrastructure responsive to rural environments which have been classically underserved. marshfi eld clinic has a long-standing history in medical student education and multiple medical residency programs. creating access for the underserved population was the major motivational force driving the establishment of the dental clinic network back in . the fi ndings of the iom, macy, and rwjf reports became the foundational framework for developing the vision of a dental education model that would realize the major recommendations found in the reports. by establishing clinical campuses in regional underserved dental health professional shortage areas, access to care where care is needed most was provided. sustainment of a work force for provision of care across the dental clinic network is accomplished by schools contracting with fqhc's for service learning, thus circumventing challenges associated with releasing dental students at traditional dental schools to distant extramural training sites as discussed previously. this model is however not without its own set of challenges including calibration of faculty, supervision and evaluation of students in training, and achieving accreditation acceptance. however, through video connectivity and iehr technology curriculum, learning plans, competency assessment, progression, performance, faculty development, and learner evaluations can be centrally calibrated. additionally, this dental service-learning model based in a community health center setting offers students unique state-of-the-art exposures to alternative access models, cutting-edge informatics (including access to a combined dental-medical record) and a quality-based outcomes-driven practice. given the novelty of such an extended extramural dental clinical training model, there is limited data on the success of rural placement leading to retention to practice in a rural setting. the pipeline study piloted a model for getting students into underserved communities. however, that experiment was limited to -day rotations. outcome driven programs may provide a predictive surrogate for purposes of comparative analysis. for example, the rural medical education "rmed" program of the university of illinois medical school at rockford, has sustained a longstanding program in illinois. over years in duration with over student participants of whom % have been retained as primary care medicine practitioners in rural illinois. rabinowitz et al. ( a ) further reinforced that medical school rural programs have been highly successful in increasing the supply of rural physicians, with an average of - % of graduates choosing to practice in rural areas. they also noted rural retention rates of - % among the programs (rabinowitz et al. a) . recently, the university of wisconsin school of medicine and public health (uwsmph) launched the wisconsin academy for rural medicine (warm program). the warm program places medical students in rural academic medical centers during their third and fourth years in medical school. marshfi eld clinic is one of those sites. warm students affi liating with marshfi eld clinic's system would ultimately share learning experiences with dental students, clinical rotations, team-based rounding, lectures, and exposure to a combined medical-dental patient record. in an analogous manner, the marshfi eld clinic dental education model will incorporate a curriculum that embeds students in rural clinical practice for up to years. a secondary but not insignifi cant outcome of placing residents and students in clinical campuses focused on developing competency and providing care where needs are often greatest is the cost savings to taxpayers associated with the public care of patients. these savings are accomplished through the "service-learning" of the student. for example, in the model described where clinical training is embedded within the fhc clinics, the stipend resident or unpaid student learner provides the patient care as part of their service learning training while requiring oversight from one paid faculty per four to six learners. as a result, an academic based clinical partnership creates a model that reduces the cost for care provided to underserved patients. an additional benefi t to the community based clinic might be realized through tuition assistance by the academic program to help support patient procedures that develop learner competencies. in educational quality and infl uence, dental schools should equal medical schools, for their responsibilities are similar and their tasks analogous (william gies ) . the commission on dental accreditation (coda) notes that one of the learning objectives of an advanced education general dentistry (aegd) residency is to have the graduate function as a "primary care provider". to function competently in this role, the graduate needs to have a strong academic linkage to primary care medicine. at a dental deans forum, years after the gies report, dr polverini made the statement "dentistry has never been linked to the medical network but unless dentistry becomes part of the solution to the challenge of providing comprehensive patient care, it will be looked on as part of the problem, and ultimately, all dental schools will be called into question." (polverini ) the use of dental informatics and an integrated record are elements essential to this competency. on april , , fhc and marshfi eld clinic successfully transitioned all of their dental centers to a new practice management and electronic health record system that fully integrates medical and dental; one of the fi rst such systems in the nation. along with the benefi ts derived in fig. . , chc placement also exposes students to an integrated medical-dental care setting where learners can develop skills in system-based practice to include the interdependence of health professionals, systems, and the coordination of care. on the administrative side, dental and medical appointments can be coordinated to enhance convenience for patients and improve compliance with preventive dental visits. in , marshfi eld clinic's research foundation biomedical informatics research center hired their fi rst dental informatician, dr. amit acharya, bds, ms, phd. with dedicated biomedical informatics and research resource centers, the marshfi eld clinic has laid the groundwork for true medical/dental integration with appropriate electronic health record decision support and is positioned to develop a dental education curriculum capable of implementing the iom recommendations. downstream benefi ts of using such a curriculum are the ability of future practitioners to use informatics to improve quality of care and reduce the burden of disease. according to an institute of oral health report ( ) it is widely accepted across the dental profession that oral health has a direct impact on systemic health, and increasingly, medical and dental care providers are building to bridge relationships to create treatment solutions. as early as , william gies recognized that "the frequency of periodic examination gives dentists exceptional opportunity to note early signs of many types of illnesses outside the domain of dentistry" (gies ) . the following examples show how integration of dental and medical care can impact patient outcomes, underlining the importance of this concept in dental curriculum design. a study of , blue cross blue shield of michigan (bcbs) members with diabetes, who had access to dental care lead researchers, and bcbs executives to conclude that treatment of periodontal disease signifi cantly impacts outcomes related to diabetes care and related costs (blue cross blue shield of michigan ) . another example is found in the context of preterm delivery and miscarriage. according to research cited by cigna ( ) , expecting mothers with chronic periodontal disease during the second trimester are seven times more likely to deliver preterm (before th week), and have dramatically more healthcare challenges throughout their life. cigna also cites the correlation between periodontal disease and low birth weights, pre-eclampsia, gestational diabetes. equally important is the opportunity to develop and implement the team-based curriculum that trains future dentists and physicians in the management of chronic disease as an accountable care organization (aco) in a patient-centered environment. as an example, joseph errante, d.d.s., vice president, blue cross blue shield of ma, reported that medical costs for diabetics who accessed dental care for prevention and periodontal services were signifi cantly lower than those who didn't get dental care (errante ) . these data suggest that team based case management of prevalent chronic health conditions have considerable cost savings opportunities for government payers, third party payers, employers and employees (errante ) . these economic benefi ts to integration as it relates to the iehr are discussed elsewhere in this book, but begin with the ability of providers to function in a team based environment and as such, underscore the importance of training in such an environment. dentists trained in a fqhc iehr integrated educational model will be well positioned to function successfully within an aco model. an aco is a system where providers are accountable for the outcomes and expenditures of the insured population of patients they serve. the providers within the system are charged with collectively improving care around cost and quality targets set by the payor. within this system, care must be delivered in a patient-centered environment. the patient-centered environment according to the national committee for quality assurance (ncqa), is a health care setting that cultivates partnerships between individual patients and their personal physicians and, when appropriate, the patient's family. care is facilitated by registries, information technology, health information exchange and other means to assure that patients receive defi ned, timely and appropriate care while remaining cognizant of cultural, linguistic and literacy needs of the patient being served. the model includes the opportunity to deliver patient care that is patient-centric, incorporates the patient in the care planning, considers the patient's beliefs and views, and incorporates the patient's families as needed. the model allows providers to deliver care that is inclusive of needs, attentive, and accessible. the model equips payers to purchase high quality and coordinated care among teams of providers across healthcare settings. while this describes the medical home, most dental practices also follow this process. many dental practices function in this regard with insured populations and refl ect elements of the model that medicine is creating. william gies would be proud. training in the delivery of accountable and patient-centered medical-dental care must be done purposefully. commenting on the inadequate training relative to the integration of medical and dental education, baum ( ) stated that "we need to design new curricula with meaningful core competencies for the next generation of dentists rather than apply patches to our existing ones." while this statement was made in reference to the basic sciences, the same holds true for patient-centered system-based practice competencies. utilizing state-of-the-art electronic medical records as a tool and the fhc infrastructure as the service venue, meaningful patient-centered system-based practice core competencies achievement becomes possible in a manner highly responsive to societal needs. by defi nition, fqhcs must provide primary medical care, dental care, and behavioral health. fqhc have also historically been utilized as healthcare workforce training centers and the affordable care act of reinforced their role as healthcare training centers. specifi cally, this legislation serves to promote fqhcs as the entity through which the primary care workforce (including dental) will be developed and expanded. in combination, fqhcs and primary care centers are positioned to be the front runners in a medical/-dental home training model which will be essential to preparing future practitioners for practice in an aco. critical to this success is the ability to train these practitioners on an integrated medical-dental record and informatics platform. use of this platform imprints most strongly during the learner's formative years of training; instructing and guiding disease management, decision making, patient care coordination, prevention, and both outcome-based and comprehensive care. training in this hybrid academically orientated clinically integrated setting moves dental education off its crossroads and creates the highway to its future. concerns with the new models extend to their ability to integrate medical and dental disciplines at the clinical and informatics level. while the iom report identifi ed the need to integrate medical and dental curriculum, success at the curricular and technological level within schools, has been limited. three major factors have contributed to the limited progress: access priorities. creating access to care has outranked the need to integrate • care. in part, this refl ects societal need for care and public demand to reduce the burden of the "silent epidemic." schools play an important role as a safety net to care for the uninsured and underinsured through intramural clinical service learning. even though "dental colleges seem to be willing to bearing a large and disproportionate share of the burden in terms of access to care" (dunning et al. ) , schools were challenged as part of iom, surgeon general, and macy reports, to expand that role. while these reports have prompted creative educational solutions to increase access, the reports understate the tremendous opportunity, quality and cost benefi ts that could result from an integration of medicine and dentistry. it is diffi cult to change the culture and structure of existing schools. this is not • unique to dentistry. however, the iom report specifi cally recommended that schools "eliminate marginally useful and redundant courses and design an integrated basic and clinical science curriculum". the challenges with this are many. examples include: some schools may not have other disciplines to draw from to create an inte-grated curriculum; a number of schools use a faculty senate to determine curriculum. this can result in curriculum that preserves the current faculty structure; changing curriculum is associated with expense and can be fi nancially pro-hibitive to some schools physical changes may be needed and represent an expense and/or may, in some instances, may not be practicable based on structure of existing facilities. public school programs may direct the fi nal curriculum, as boards or regent's one or two steps removed from the curriculum often have fi nal authority conversely, private schools may specify business or mission objectives that determine fi nal design. perhaps most germane to this text is the lack of a common technology plat-• form between disciplines in a learning environment. an integrated curriculum requires an integrated platform to accomplish delivery and evaluation. this is particularly essential to clinical management of the patient by professionals in training as part of a healthcare delivery team. some progress in establishing shared basic sciences curricula has been documented in the literature. to date, no single integrated electronic health (medical-dental) record has been meaningfully adapted for educational purposes, including incorporation of assessment of the learner relative to integrated competencies, integrated case-based and problem-based curriculum, and integrated evaluation and assessment. another concern with new educational programs emerging in response to these reports and relative to creating a transformational integrated curriculum is that some of the programs are focusing primarily on creating clinicians with no value or emphasis on integrating training with research and/or scholarly activity. integrated training models counter such concerns. research will be fundamental to measuring the relative benefi ts and outcomes associated with treatment of patients in a shared curriculum setting and will be the catalyst for the development of integrated medical-dental informatics incorporating educational capabilities. additionally, accreditation will also need to evaluate its response to such models. presently it is unclear how accrediting bodies will view an integrated crossdisciplinary curriculum. further, due to its integrated nature, such a curriculum would lie outside of the expertise of a single traditional accrediting body focused on one particular discipline. it has yet to be determined how accrediting bodies will review and appraise such cross-disciplinary competencies. lastly, it is important to recognize that a successful education model with innovative informatics is only successful if its focus is patient care. graduating learners with competency only in the use of informatics will be limited unless adapted to training and delivery programs that result in patient centric care. research and reports over the past years support the need to reform dental education. first steps have been taken and lead the way for continued innovation around clinic-based education and integrated curriculum. the models identifi ed point to a strong partnership and interrelationship with chcs for creative, cost saving, effective and sustainable delivery methods. moreover, chc's must be more involved in a training curriculum integrated with informatics. chcs, in turn, benefi t from residents and students through service-learning to help meet a societal and workforce need, while the learners benefi t from increased competency. in order to train an evidence-based, patient-centered, medical-dental workforce, it is imperative that medical and dental data and record accessibility be incorporated into these training and care delivery initiatives. in order to keep moving away from the crossroads, such integration must become the pathway on which curriculum is developed and implemented. public law - 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doi: . /j.banm. . . sha: doc_id: cord_uid: gy lc jz nan the author declares no conflict of interest related to this publication. in france, enforcing clinical leadership within hospitals has once again run into its « glass ceiling »: the lack of academic and professional recognition of management as a part of medical practice, alongside clinical expertise. a look at other countries provides insights into how to achieve such an endorsement. in the wake of the covid- epidemic, several medical unions or interest groups in france have stressed the need to increase medical involvement in hospital decision making. the general public mostly welcomes the idea of having medical doctors "run" hospitals; yet it remains an exception in france. upon request of the ministry of health, pr olivier claris [ ] recently published a report on hospital governance, which duly recommends to give greater emphasis on management issues in medical training; it also points out the need to strengthen management skills among medical doctors, thereby helping them assume clinical leadership. such recommendations, however, seem to have been expressed time and again. special requirements for medical department heads have existed since the beginning of the s, when the current standards of medical practice were being defined for france's public sector hospitals. ten years later, a major law on hospital reform expressed concern over the "lack of involvement of medical staff in the responsibilities of management". as public-sector hospital budgeting switched to drg-based payments in , another law was passed in order to promote medical intrapreneurship and therefore increase the self-determination of clinical departments ("pôles") ; since , head physicians are required to follow at least hours of initial training when newly promoted. today, most university or large-scaled hospitals provide leadership programmes to medical doctors assigned to new responsibilities as department heads. pr claris' report specifies that gateways do exist for medical doctors wishing to become executive officers, but scarcely attract candidates. increasing doctors' initial training and adding skills will not solve the underlying problem: healthcare management needs academic and professional endorsement, alongside the other clinical and public health specialties. a quick international survey shows that france is lagging behind on this aspect [ ] [ ] [ ] [ ] [ ] [ ] . in germany, for instance, the association of medical hospital executives has been active since , and is hosted by the federal medical association. it provides continuing professional development in healthcare management with the same level of requirements as expected for clinical cpd . in the uk, the faculty of medical leadership and management was created in with similar assignments; it is endorsed by all other royal academic colleges and faculties. in portugal, the national medical association sets standards for qualifications and training within health services management. the european association of senior hospital physicians [ ] published a statement on clinical leadership in and has assigned a permanent working group to promoting it towards national medical associations. outside europe, the japanese association of medical sciences includes a specific society for healthcare administration. the royal australasian college of medical administrators (racma) was created in and extends beyond australia to new zealand and the asia pacific region; it considers medical administration as a specialty. in the us, the american medical association includes a section called « organized medical staff » which started issuing a code of conduct in , after the ama adopted an ethical opinion stating that health professionals have a "responsibility to address situations in which individual physicians behave disruptively.". the ama considers that such behaviour can impede high-quality care and may be a manifestation of physician burnout, which top management must address. given its greater share of senior executive positions held by medical doctors, norway [ ] provides some other insights. within their national medical association, a section is dedicated to management since : its official policy states that "all medical activities imply responsibilities as a leader" [ , ] . the section publishes guidelines on medical conduct when engaging reorganizations and best practices on clinical leadership. moreover, the medical association insists on the importance of anticipating the switch back to clinical practice through training packages; and of providing adequate compensation, as a prerequisite of sufficient authority among peers. as these examples show, the way clinical leadership is acknowledged may vary according to national settings: as a section of the medical association in some countries, as an academic society in others. however, they all share two aspects in common : firstly, peer group recognition ; secondly, using cpd as their major lever for improvement. pondering this fact, it seems that french institutions could resort to cpd, rather than focusing on initial training, in order to promote clinical leadership among its medical staff. with regards to clinical practice, cpd has successfully led medical communities to engage in lifelong learning, with a strong focus on three-yearly, peer-based evaluation of professional practice. french authorities have been cautious to avoid the pitfalls of "top-down" guidelines : through its accreditation process for training programs, cpd is gradually enforcing a framework, in which every specialty identifies "good practice" based on medical evidence. all healthcare professionals are involved regardless of age. the dynamics of change that cpd has harnessed among medical specialties could be applied to clinical leadership ; in particular, evaluating managerial practice based on the self-assessment methods used in cpd (called "epp" in france) could prove very insightful. such methods would have to be transferred with specific attention on crossdisciplinarity and on patient safety. evaluation as applied to management would encourage the production of guidelines and promote evidence-based approaches to clinical leadership. medical simulation should have a great part to play ; in the long run, it would benefit entire healthcare teams. pr claris' report recommends that the centre national de gestion, an authority in charge of coordinating the careers of public sector physicians nation-wide, be entrusted with the task of defining the training requirements for new department heads. based on the insights above, it seems the cng may only partially succeed if left on its own. greater change is at hand, if clinical leadership is granted the academic recognition it requires. how to achieve such an endorsement remains to discuss, and would imply several french institutions such as the medical association, the academy of medecine, the federation of medical specialties (fsm) and the higher health authority (has). it could serve as a landmark for further change, as technology and innovation frees medical doctors from front-end delivery of care, and sets a new focus on patient journey and health services organization. le professeur olivier claris a remis son rapport de recommandations sur la gouvernance de l'hôpital à olivier véran royal australasian college of medical administrators set clear rules to stop bad behavior that worsens morale agence nationale du développement professionnel continu association européenne des médecins des hôpitaux (aemh) engaging doctors in leadership key: cord- -ds y ks authors: schiller, marcus; pilette, marijatta; rahlf, björn; von see, constantin; gellrich, n.-c. title: management of pandemic or large-scale emergencies in germany with a focus on the current and potential role of university schools of dentistry: can it help in covid- time? date: - - journal: bull natl res cent doi: . /s - - - sha: doc_id: cord_uid: ds y ks background: the study presented here systematically examines the potential involvement of dental, oral and maxillofacial centres (zmk) in the management of pandemia or in large-scale emergencies. it looks at available material and infrastructural resources and how they can be brought to bear in such incidents or situations. the aim was to gain an initial scientific overview of how zmk can potentially contribute to the handling of a pandemia or mass casualty (mascal) situation in terms of available resources as well as their location within the hospital as a whole and their integration into the existing infrastructure. the study was conducted on the basis of a questionnaire consisting of individual questions, which was sent to all universities in germany that offer a course of study in dental medicine. the responses were then statistically evaluated. results: the study outlines the current status of zmk and discusses what could be an important component of emergency medical care in the overall hospital context. conclusion: the involvement of zmk—with their own resources and existing infrastructural links to the hospital as a whole—could lead to faster and more effective patient treatment in the event of a pandemic or mascal situation. the increasing threat of international terrorism has shifted public focus in germany onto the question of how large-scale emergencies can be managed. after the / terrorist attacks in new york, the standing conference of federal and state ministers and senators of the interior decided to critically review civil protection and disaster control policies (simon and tepermann ; cook ) . as a result, hospitals were also forced to take a closer look at this issue and to reevaluate existing emergency response plans, especially after the recent terrorist attack on a christmas market in berlin. one question that needs to be answered is how to sensibly involve dental medical centres in existing emergency concepts. so far, there have been widely differing views on this subject at the various hospitals, although there already page of schiller et al. bull natl res cent ( ) : are several german and international studies available on the potential integration of such centres (pahor ; sakr ; mitchell ) . that is because in general, each dental treatment unit (i.e. dentist's chair) could be considered a small operating table in its own right. after all, it provides an opportunity for surgical hand disinfection, a small surgical light, a suction device, monopolar electrocoagulation and, above all, various ways to position the patient, including the shock position. examinations and surgical treatments such as wound care, splinting or other emergency treatments are possible. taking into account previous studies, the aim of this study was thus to systematically investigate the current rate and potential increase in integration of dental medical centres at university hospitals in the emergency response plan of the hospital as a whole. for this purpose, a specifically developed questionnaire was used to survey the structures and resources of the dental medical centres at german university hospitals. their potential involvement in providing emergency medical treatment in case of large-scale emergencies is illustrated using hannover medical school (mhh) as an example. previous contingency plans for a mass casualty scenario had foreseen the fire service, germany's federal disaster relief agency (thw) and the red cross setting up and operating treatment stations at the outpatient clinic of the mhh to support the hospital. the option of falling back on the dental treatment stations of the dental medical centre is currently not included. emergency medicine as we know it today is a relatively new medical discipline. its history can be traced back to lessons learned in military campaigns that were translated into principles for the rescue and evacuation of casualties. it was not until after world war ii that civilian emergency medicine became truly established (ambulances, triage, etc.). härtel stated in that physicians who worked in other specialties during times of peace had to get used to thinking and acting as surgeons in times of war (robertis et al. ( )). the way ambulance services in germany are organised today is a result of the adaptation of military principles, the further development of medical knowledge and increasing regulation (skandalakis et al. ) . during world war i, emergency rescue was the responsibility of the red cross and the fire services. after world war ii, the emergency rescue services were shaped by the occupying powers and the different occupation zones. rutherford suggested that the order of evacuation should depend on the pattern of injury in the different triage categories. on the federal level, civil disaster control tasks fall into the remit of the federal ministry of the interior and are allocated to the federal office of civil protection and disaster assistance. this office is a higher federal authority and supports the supreme federal authorities in uniform civil defence planning. in case of a hazard or emergency situation, crisis staffs at the federal ministry of the interior assume coordination tasks (niska and shimizu ) . in germany, % of medical assistance delivered in an emergency situation is provided by nongovernmental organisations. , a system of fast response units has been established, which are mainly employed for preclinical tasks (sakr ) . , the länder (german federal states) disaster control in germany reflects the german federal system as federal law assigns certain tasks to the länder [article ( ) of the basic law]. however, the federation also makes recommendations and cooperates with the länder [article ( ) of the civil protection and disaster management law]. this is especially the case in largescale emergency situations or emergency situations of national significance (niska and shimizu ) . in order to improve joint coordination and practice, the federal minister of the interior and the ministers of the interior of the länder decided in to conduct a national crisis management exercise. over hospitals in lower saxony provide medical care for the state's population. in the event of an emergency, the number of patients they will treat will exceed normal capacity. in this context, a contingency plan may be important to facilitate an appropriate response. according to sefrin et al., a working emergency response plan is a prerequisite for extending the treatment capacities of every hospital in an emergency (schenk ) . page of schiller et al. bull natl res cent ( ) : allocating patients in successive waves may counter clinical overload (adams and tecklenburg ) . this plan of admitting patients in "waves" makes it possible to maintain the hospitals' ability to act, even though requests for treatment capacity can no longer be accommodated. the tool used by the länder for adapting to actual needs is the so-called hospital plan. this plan provides the basis for ensuring requirement-oriented support of the population with respect to the hospitals needed according to their location, specialties, number of beds and functional units (niska and shimizu ; mistovich et al. ) . the state of lower saxony has such an emergency response plan in place. the hannover medical school (mhh) is a well-established university hospital and a supramaximal care hospital. with a capacity of about beds (as of ), the mhh is one of the largest hospitals in lower saxony. together with the university medical centre göttingen, the mhh is one of two hospitals in lower saxony to feature a university dental medical centre and offer a course of study in dental medicine. the hospital plan of lower saxony states that the number of beds assigned to oral and maxillofacial surgery is equivalent to . inpatient beds per , inhabitants (adams and tecklenburg ) . a credo of emergency medicine is that each patient should be provided with individual care as quickly as possible, but not past the point where, in the case of a large number of casualties, the treatment of that individual patient would have a disproportionate negative effect on the prognosis of others. forecasts about the type of patients admitted to hospital as well as their patterns of injury and time of arrival are mostly based on the nominal analysis of patient numbers. the population of the study consisted of hospitals. questionnaires were sent out to the following university hospitals with dental medical centres and/or dental student training (fig. in the run-up to the study, ethics commission at the hanover medical school was asked. this study does not require an ethics vote, as the study is purely anonymous. of the university hospitals-based dental medical centres that were invited to participate in the study, . % returned the questionnaire. most dental medical centres feature instruction rooms ( . %). in ( . %) of these centres, such rooms have separate entrances with direct access to the outside. only . % of them lack access to the outside area. sterile processing is carried out in three different places: at the central sterile processing department of the entire hospital, at the central sterile processing unit of the dental medical centre or locally, i.e. at the individual departments of the dental medical centre. if only the central sterile processing department (n = ) or a combination of that department and the central sterile processing unit at the dental medical centre (n = ) is used, transportation/supply of sterile items is possible h a day. if the hospital provides transportation services for patients on a -h basis all year round, sterile material will also always be transported/supplied h a day (n = ). of the hospitals that do not provide -h transportation/supply of sterile items, % did not provide a patient transportation service either. on average, the dental medical centres feature a total of . dental treatment units and . surgical rooms, which fall into the categories of minor surgery rooms, emergency operating theatres and operating theatres. in dental treatment centres equipped with dental treatment units, the numbers are: (fig. ) . the number of minor surgery rooms, operating theatres and emergency operating theatres was also determined. the graph illustrates the results (fig. ) . four dental medical centres are equipped with intensive care capacities. a maximum of three intensive care beds are available in . % of all dental medical centres. two such beds are available in . % of the centres. this results in an average capacity of . intensive care beds at dental medical centres. the ratio of surgical room capacities to bed capacities is : (one operating theatre per seven ward beds). the ratio between operating theatre capacity ( . ) and recovery room places ( . ) is approximately : . schiller et al. bull natl res cent ( ) : digital networking upon investigating the dependency on information technology (it), we found that a digital record exists in . % of all hospitals, including dental medical centres. the number of patients to be treated in a mass casualty event is always difficult to estimate (mistovich et al. ; rutherford ) . it depends not only on the total number of people affected by the event but particularly on the number of people with injuries that see them classified as triage categories i-iii. a comparison of the german eschede train disaster with the terrorist attacks in madrid in terms of victims shows that the number of injured who reach a hospital does not have a linear correlation with the overall number of people affected. in eschede, only % of casualties were alive upon arriving at a hospital for treatment, compared with % of victims in madrid (turégano-fuentes et al. ) . insufflation anaesthesia is required for almost all patients of triage categories i and ii. of the total number of patients to be expected, % will fall into these two categories. at least % of trauma patients classified as "category red" are in need of life-saving emergency surgery. for critically injured patients, surgical capacity is one of the decisive bottlenecks. analyses of the surgeries performed after the madrid bombings have shown that out of the operations carried out within the first h, were maxillofacial surgeries. about % of these injuries were pure fractures of the jaw and facial bones, while around % affected the face, head and neck area. of all seriously injured category i patients, approximately % require surgical interventions in the region of the head and neck. patients with minor injuries can be expected to make up a proportion of %. adams has in different publications already suggested having these patients treated at a dental, oral and maxillofacial clinic by staff of the dental medical centre. triage category iii patients can usually be treated under local anaesthetic. treatment under local anaesthetic is part of the standard treatments routinely performed by dentists (pahor ; daubländer ) . they have the required facilities and equipment at their disposal, and treating their patients in a dentist's chair is common practice for them. this resource, which is available in relatively large numbers, should thus be considered for use in major emergency situations. in that case, such facilities and the dental treatment units would mainly be used for the treatment of triage category iii patients. this would involve the organisational integration of the staff and in some cases the students at dental medical centres, who would cooperate with doctors and assistant personnel in a multidisciplinary approach. a dentist's chair can fig. numbers of available minor surgery rooms, operating theatres, emergency operating theatres page of schiller et al. bull natl res cent ( ) : generally also be used for the treatment of regions other than the head and neck area. an average of dental treatment units are available, which means that treatment stations are available for triage category iii patients. these units are spread all over germany according to the distribution of universities for dental, oral and maxillofacial medicine. in addition, dental medical centres lend themselves to providing rooms for crisis staffs or families as well as for pastoral care, etc. this workload sharing within hospitals equipped with a dental medical centre would make it possible to more efficiently use the resources for triage categories i and ii at the main hospital. if the condition of a triage category iii patient deteriorates during treatment and a more severe triage category needs to be assigned, the traditional surgical and monitoring facilities within the dental medical centres can be incorporated across the specialties. the objective of this study was to investigate the existing and future potential integration of dental, oral and maxillofacial clinics into the emergency concepts of hospitals as a whole. the data obtained were statistically evaluated and analysed. the majority of dental medical centres in the area surveyed were found to not be included in the emergency concepts of university hospitals. of the dental medical centres that are in fact included in emergency concepts, only two-thirds were able to provide details on the exact nature and extent of this involvement. we thus conclude that resources for patient examination, treatment and admission are generally available but not used to their full extent. in terms of these resources, we were able to establish the numbers of dental treatment units (average . ), minor surgery rooms (average . ), operating theatres (average . ) and ward beds (average . ). we further conclude that dental medical capabilities in germany are insufficiently used as a potential resource in case of an emergency. there are doubts about the integration of dental (assistant) personnel. however, the geographical distribution of existing dental medical centres is an additional positive aspect in terms of their potential involvement in case of large-scale emergencies. in the current situation, hospitals are flooded with a large number of patients. the focus of the hospitals is on the treatment of emergencies and especially on the treatment of covid- patients. additional places are being created for triage, either by setting up additional treatment places or by putting up tents. the use of the resources of dental medical centres to relieve the main clinics should be included in the considerations. dental medical centres offer a large number of possibilities for the initial treatment and/or treatment of patients with mild to severe diseases. the integration of dental medical centres, with their own resources and existing infrastructure connections to the hospital compound, could facilitate quicker and more efficient treatment in a mass casualty event. in such emergency situations, physicians and other non-dental medical personnel could take up work at the dental medical centres, and their dentist colleagues who work in hospitals and in outpatients setting could also be involved. the necessary statutory provisions would first have to be established, however. the usa sets a positive example in this regard. constant further development of the task spectrum of dentists as part of mass casualty planning and the creation of a clear statutory framework ensure that all capabilities are exploited to their full potential. through extended training in the field of emergency medicine as students, dentists are thoroughly prepared for their future tasks and thus able to provide a real contribution to casualty care in large-scale emergencies. der notfallplan des krankenhauses the world trade center attack. the paramedic response: an insider's view neue notfallfolge trainieren für den tag x. zahnärztl mitteil the monopolisation of emergency medicine in europe: the flipside of the medal prehospital emergency care a brief history of triage hospital preparedness for emergency response: united states historical article: ear, nose and throat in ancient egypt triage for simple compensated disasters casualty, accident and emergency, or emergency medicine, the evolution krankenhaus-alarm-und -einsatzplan (kaep) -niedersächsisches muster the world trade center attack. lessons for disaster management to afford the wounded speedy assistance": dominique jean larrey and napoleon injury patterns from major urban terrorist bombings in trains : the madrid experience publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions mp, ms, br, cs and ncg conceived the study and participated in its design and coordination. mp and ms made substantial contributions to data acquisition and conception of manuscript. ms, br and cs drafted and designed the manuscript and contributed equally to this work. ms and ncg were involved in revising the manuscript. all authors read and approved the final manuscript. not applicable. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. not applicable. yes. the authors declare that they have no competing interests. key: cord- -qd fht y authors: bucciol, alessandro; camboni, riccardo; valbonesi, paola title: purchasing medical devices: the role of buyer competence and discretion date: - - journal: j health econ doi: . /j.jhealeco. . sha: doc_id: cord_uid: qd fht y this paper investigates the price variability of standardized medical devices purchased by italian public buyers (pbs). a semiparametric approach is used to recover the marginal cost of each device. average prices vary substantially between pbs; we show that most of the difference between the purchase prices and estimated costs is associated with a pb fixed effect, which, in turn, is related to the institutional characteristics and size of the pb. repeating the main estimation using device fixed effects yields similar results. finally, an exogenous policy change, i.e. the termination of the mandatory reference price regime, is used to assess how discretion affects medical device procurement given the skills of each pb. our results show that less pb discretion — i.e. when mandatory reference prices apply – determines efficiency gains and losses for low- and high-skilled pbs, respectively. the european medical technology market -comprising mainly medical devices and in vitro diagnostics -was valued at roughly e billion in . at % of the world market, it is the second largest in the world, after the united states. medical technology is characterised by a continuous innovation, and the short life-cycle of products (on average, - months) . in , in this sector, more than , patent applications were filed with the european patent office (epo), twice the number from the pharmaceutical sector. medical technologies provide value in various ways, saving and improving lives by promptly detecting disease and providing effective treatment options for patients and healthcare systems. they can also deliver significant savings (i.e. via efficiency) to the health system over time. this paper empirically investigates public procurement for medical devices in italy, decomposing price variation into components explained by buyer fixed effects and buyer characteristics. the study period overlaps with a time when reference pricing was temporarily imposed at a national level, allowing examination of the heterogeneous effects of the policy on different buyers. in the us healthcare system the purchase of medical technologies is usually by direct trade negotiated by private hospital managers and suppliers. the resulting contracts are characterised by strategic discretion and flexibility (grennan ; ) . by contrast, in eu countries, purchasing is heavily regulated: in particular, public buyer (pb) discretion, awarding mechanisms and contract management are restricted and determined by law. accordingly, pb competence involves coping with regulated business-to-government procedures, often with open tender mechanisms (lian and laing, ; spagnolo, ) . in italy, as in many other national health systems in europe, the procurement of medical technologies is managed at the local level. occasionally, the national press has reported prices differences for the same standard medical device (e.g. a simple syringe) paid by different pbs (public hospitals and healthcare units). in a period of tight public budgets, this evidence has fuelled an extensive public debate and -in -led to the introduction of reference prices, a policy imposing a cap on the unit price of each standard entry regulations and quality information requirements for new medical devices play a key role in this process. grennan and town ( ) exploit differences between us and eu regulations on testing new medical devices to address the trade-offs arising from more frequent testing, which can overcome consumer uncertainty about product efficacy but increases entry costs and delays product launches. their empirical results for stents show that us regulation is close to the optimal policy in terms of trading off testing with access to innovation, while eu regulation is too lax. estimation of market values and information on patents from med tech europe ( ). see, amongst others, p. russo 'garze e siringhe d'oro: le spese pazze delle asl' (bandages and syringes of gold: the berserk expenditure of italian local health agencies) in la stampa, july , . greater attention to price differences (and corruption) for the purchase of medical devices has been paid during the recent coronavirus pandemic. indeed, the public healthcare system in italy has faced severe challenges regarding procurement strategies for masks, hand sanitizers, ecmos, etc., speed having been prioritised over transparency, with competitive bidding and other safeguards dropped to keep pace with the pandemic. as reported by the international press, the same has occurred in many other countries affected by the coronavirus (see: a. faiola and a.v. herrero, 'a pandemic of corruption', in washington post, april , ). medical device procured by tender. the aim of this policy was to limit pb discretion in an attempt to reduce public procurement expenditure. reference prices for medical devices were applied from july , to may , and were then scrapped after a ruling of the administrative court of rome. this provides a quasi-natural experimental setting for a clean test on how pb discretion affects the procurement of medical devices. based on procurement data, this paper initially estimates the net contribution of pb competence to price variability and then assesses how reference prices, which reduce pb discretion, interact with that competence. our analysis was run on an original italian dataset including classes of standard medical devices sold to local pbs between january and december . the empirical approach is based on two important features of the medical devices investigated: i) they are standardised and relatively cheap, so renegotiations are rare; ii) they are grouped into classes of functionally homogeneous products (i.e. in each class, quality differentiation is not an issue). given these features, a semiparametric approach is adopted, inspired by guerre et al. ( ) , to recover the marginal cost of each device. using the official classification provided by the italian technical advisor for health policies (agenas), we group functionally homogeneous medical devices into classes and, for each class, establish its benchmark marginal cost. we then show empirically that most of the discrepancy between purchasing prices and estimated costs is related to the pb fixed effect. this way, we infer a proxy for pb competence in managing each purchase. the main estimate is repeated, in the spirit of best et al. ( ) , using device fixed effects rather than marginal costs, obtaining similar results. hence, the findings are not specific to the structural approach used to infer the marginal cost of medical devices. as a further step in the analysis, the determining factors of pb competence are investigated based on balance sheet data from local public hospitals and healthcare units. finally, the exogenous termination of the reference price policy is used to identify its impact on the competence of pbs. our main findings are summarised below. first, the average prices of standard medical devices paid by italian local public hospitals and healthcare units vary substantially. second, the differences between pb purchase prices can be explained by pb fixed effects, which, in turn, are related to the size and characteristics of the pb. specifically, size (measured either via overall personnel costs or by the procurement of health-related materials) has a generally positive and significant effect on competence in managing procurement efficiently. furthermore, the ratio of non-health personnel over total personnel costs drives the overall positive and significant effect of size on pb competence. by contrast, once pb size has been accounted for, overall procurement expenses for health-related goods push competence down, in line with the definition of pb competence we adopted in this paper. regarding organizational characteristics, our results indicate significant differences in purchasing medical technology between local public healthcare units and hospitals, with higher prices paid for standard medical devices by the former. comparing the period of reference pricing during which pb discretion was restricted with the period after the removal of reference pricing suggests that this policy reduced price dispersion, but had a non-linear effect on pb competence to purchase efficiently. indeed, reference pricing increased the average prices paid by highly competent pbs and reduced the prices paid by pbs with lower competence. when reference prices were in force, the main determinants of pb competence decreased in magnitude or lost their overall significance, reducing the disparity between pbs, with observations moving towards an average value. in a period in which, in many countries, regulatory policies for medical device are often set to improve procurement efficiency, such a non-linear effect calls for the careful adoption of measures that evenly affect pbs in the healthcare system and their discretion in managing purchasing procedures. our study contributes mainly to three strands of economic literature. the first is the empirical literature on the effect of pb discretion on purchasing activities. di tella and schargrodsky ( ) investigate the prices of standard medical devices following the introduction of a strict monitoring policy for hospital purchases in buenos aires. they estimate a % reduction in the average prices paid by hospitals because of the crackdown. similarly to these authors, we investigate the effects of a policy to reduce public procurement expenditure for standard medical devices. by exploiting exogenous changes in the size (i.e. threshold value) of the public works tenders in which pbs are granted larger degrees of discretion in managing procedures, recent studies have focussed on the effect of pb discretion on procurement performance (palguta and pertold, ; baltrunaite et al. ; ). our work differs from these as our empirical strategy is able to isolate the net effect of reference prices on pb discretion and, in turn, on public expenditure. second, we add to the literature on the role played by pb competence to purchase goods or services and on the related regulation policies. by investigating procurement performance as related to the competence of the public workforce, a recent work by decarolis et al. ( ) empirically assesses the causal effect on us bureaus. using an instrumental variable strategy and combining data on office-level competencies and procurement performance (i.e. cost and time overruns), the authors find that cooperation within the office matters the most to improve outcomes. considering the price paid for standardised goods and services by different classes of italian pbs, bandiera et al. ( ) find that the expenditure would be reduced by % -corresponding to a saving of between . % and . % of the italian gdp -if all pbs paid the same prices as the pbs at the tenth percentile. these authors also found that at least % of the estimated waste is related to bureaucratic inefficiency. using a large dataset for russian procurement in - , best et al. ( ) estimated that % of within-product purchase price variation over million purchases was due to bureaucratic (in)competence. moreover, investigating a specific procurement policy which sets preferences for domestic firms, these authors show that its optimal design depends on how effective the purchasers are in implementing the policy itself. to these studies, we add the novel approach of measuring pb competence and its effects in managing the procurement of standard devices in the healthcare sector. finally, we contribute to the literature on medical technology procurement (laing and lian, ; sorenson and kanavos, ; grennan grennan , kastanioti et al. ; grennan and swanson, ; den ambtman et al. ) by focusing on standard medical devices and the determinants of purchasers' competence in managing a highly regulated process, partly by investigating the effect of a reference price regime. the remainder of the paper is organised as follows: section describes the institutional setting ( . ) and our dataset ( . ), presenting some preliminary evidence ( . ). section illustrates the theoretical structural framework by introducing the definition of pb competence ( . ) and showing the marginal cost estimate for our medical devices ( . ) . section estimates pb competence ( . ) and its determinants ( . ). section replicates the analysis, exploiting the event of reference price termination as a quasinatural experiment. pb competence ( . ) and its determinants ( . ) are compared before and after this event. finally, section concludes by summarising our findings and providing policy implications. the appendix sets out details estimation and further robustness checks. context, data and preliminary evidence . institutional setting the italian healthcare system is a regionally based national health service that provides universal coverage mostly free of charge. the main sources of its funding are national and regional taxes, supplemented by co-payments for pharmaceuticals and outpatient care. the system comprises three levels of action: national, regional and local. the highest level is responsible for ensuring the general goals and fundamental principles of the national health system. regional governments are responsible for ensuring the delivery of services through a network of population-based local public health units (aziende sanitarie locali, asl) and local public hospitals. in italy, the purchasing of medical technologies is decentralised at the local level. in , the year covered in our dataset, approximately local public buyers (pbs) had procurement responsibilities for these items. according to italian public procurement law, these items are purchased through public tenders (first price auctions and scoring rule auctions, fpas and sras respectively), with direct negotiation only in certain special cases. to take part in a public procurement tender for medical devices, potential suppliers must meet a minimum set of common requirements (e.g. submit standard tender documents and meet certain financial and technical prerequisites). each pb uses its discretion when establishing procedures or adding further requirements. hence, each pb responsible for purchasing medical devices, within the finite set of mechanisms established by law, chooses the award method and sets sometimes costly requirements. in , the italian authority for public contracts (avcp) was asked to set reference prices for classes of functionally equivalent medical devices purchased by public hospitals and local healthcare units. each reference price consists of a cap on unit prices for a class of medical devices. the aim of this policy was to help standardise the prices paid for very similar items by different pbs. reference prices were mandatory for the public procurement of medical devices from july , to may , . on the latter date, responding to an appeal jointly submitted by some suppliers, the administrative tribunal of lazio (tar), outlawed the reference price regime. this decision was taken because the listed devices in some classes were too heterogeneous both functionally and technically to come under a single price. the differences were mainly related to complex devices such as stents and prostheses. note that heterogeneity within classes of medical devices is not a problem for the present empirical analysis, because our investigation is carried out using the database including only simple medical devices, as confirmed in a subsequent, more detailed classification precisely dealing with this issue. to investigate unit prices paid by pbs when purchasing standard medical devices, we base our empirical analysis on a database with four sources of information. the first and main source of information is an original dataset consisting of unit prices paid by italian pbs to purchase simple medical devices from january , to december , . all these devices were subject to reference prices until may , , and the related information on awarding tenders was collected by the avcp. pbs usually adopt procedures under which they aggregate the purchase of similar the previous contract has expired and no participants bid in the following tender or (iv) unpredictable events. in , the responsibilities of the avcp were transferred to the italian anticorruption authority (anac). the policy on reference prices included a safeguard clause. if a tender applying reference prices was null, the pb could then proceed with a new tender where reference prices were no longer applied. anecdotal evidence suggests the clause was rarely implemented. in line with the date and with reference to the date recorded in each tender transcript, in our database we divided unit prices for medical devices into two groups, when the reference price regime was applicable or not, i.e. before/after may , . the italian national agency for regional health services (agenas) produced two lists for classes of homogeneous products. the first, published in , was used to set the reference prices, ruled out on may , . the second, published in , is a more detailed list created to address the tribunal's concerns about excessive intra-class product heterogeneity (mainly stents and prostheses). our empirical analysis uses the latter. medical devices into lots, with a separate tender for each lot. each procedure corresponds to a framework with common rules for supplier requirements, bank guarantees, etc.. as an example, let us consider a public buyer, p b a , awarding a lot, lot , through a tender in the form of a first price auction, fpa. let us assume lot includes different devices, e.g. three bandages of different lengths (say, , and cm), and that only the first two are subject to the reference price regime. in this tender for lot , all participating suppliers offer a price for each of the three bandages. the winner of lot is the supplier who offers the lowest price for the whole lot , corresponding to the sum of each device's unit price multiplied by the quantity requested. accordingly, our database records the unit price offered by the winner for each of the two devices subject to the reference price regime, as well as the quantity and the classes of functionally equivalent medical devices they refer to. however, no information is available for the third device in lot which is not subject to reference prices (or for lots including only these devices). our database also has information regarding the id of the pb organising the tender, the awarding mechanism and, for a subset of observations, the number of bidders for each lot. second, from the financial statements of each pb in our database, we collected information on the total value of services supplied, total costs, costs for personnel split into health-related personnel (doctors, nurses, healthcare assistants) and non-health related personnel (clerks), and procurement costs for health-related goods and services. summary statistics on the financial statements are set out in table . pbs were also classified according to rural or metropolitan location. third, we gathered information from the national institute of statistics (istat) on the size of the regional population and total annual regional spending on healthcare. this information is relevant, given the decentralised nature of the italian health system, because political decisions at the regional level may impact on pb competence. note that the ratio between these two variables, i.e. per-capita health expenditure, is a dimensionally invariant measure of the resources each region devotes to healthcare every year. on average, in , the per-capita health expenditure was e , . finally, from assobiomedica, the main italian association of medical device pro- via the cnd code (national device classification, a top-down digit structure from general to narrow descriptions of devices), we investigated the extent of heterogeneity within each lot in our database. for the first level of classification, which gives the general description of the device, we found that only . % of the lots included heterogeneous items, and this percentage becomes . at the third level of classification. summary statistics at the lot level are presented in appendix a. . . according to italian law, the financial statements of each pb, which include the balance sheet and profit/loss account, are disclosed following a standard format. financial statements were downloaded from official pb websites. for two pbs in our database, the financial statements were not available on their official websites. a pb is located in a metropolitan area if its headquarters are in a municipality which is part of a metropolitan city as defined by italian law. data are available from the national institute of statistics (istat), at the following link: https://www.istat.it/it/archivio/ , accessed on / / . to check if the regional per-capita health expenditure is driven by economies of scale, we compare the per-capita health expenditure for regions above and below the average population size by kolmogorov-smirnov test. we find no significant difference. note. data in million euros. j o u r n a l p r e -p r o o f ducers, we gathered data on average days of delay in payment at the pb level. this information is of interest as delays in payment affect pb competence in obtaining a better deal. indeed, suppliers may discount expected late payments by initially offering a higher price. in - , we observed such delays varying from to , days, recording a median of days. note that overdue payments are a lagging indicator, so delays in are used to study pb competence in . the unit of observation in our dataset is the price paid by each pb for the purchase of medical devices subject to the reference price regime. starting from the avcp original dataset of , observations in the period from january to december , we discarded observations relative to classes of medical devices where fewer than observations were available; we discarded further observations for which the awarding mechanism was unspecified. according to italian statutory requirements for public procurement, each pb can choose the awarding mechanism to adopt in the form of first price auctions (fpas), direct negotiations or scoring rules auctions (sras). our original database includes unit prices resulting from all these awarding mechanisms (see appendix a. for summary statistics on sras, compared to fpas). given our research question and the empirical strategy of focusing on simple items in order to avoid confusing effects relating to pb competence and their discretion when purchasing medical devices, we exclude sras from our analysis. indeed, unlike fpas, the sra format includes competition between suppliers on quality elements. as a result, we end up with , observations, split almost equally between fpas ( observations) and direct negotiations ( observations). this dataset records observations on classes of functionally homogeneous medical devices subject to reference prices. the median unit price for each device is e . . within each class, d = , . . . , d, we observe the price paid by each pb for the purchase of medical devices subject to the reference price regime. for example, the class of syringes with three-piece eccentric cone, luer tip; capacity ml, graduated, with a triple-sharpened needle, mounted gauge g -g and a length of mm has unit prices ranging between e . and e . . information is missing for % of our observations. in these cases, we use the regional average as a proxy. the regional average delay in payments is highly correlated with local one (correlation . ). for data on overdue payments, see also guglieri and carbone ( ) . inspecting quality components in sra tender documents, we found no significant supplementary services, but frequent references to subjective characteristics as suggested by operators (e.g. the ease of slipping the needle, the force required to press the plunger, etc.), unrelated to the direct choice of the pb in the purchasing process. for the fpas for which information is available, the average number of bidders is . appendix a. . reports, for each device class, the average, minimum and maximum price observed. j o u r n a l p r e -p r o o f this sub-section presents preliminary findings on the unit price. first, we investigate whether prices vary according to the pb and the identity of the supplier. second, we compare some common reduced-form estimates, to find the most appropriate to describe unit prices. for the first task, we ran a set of one-way anova tests to see if unit prices, on average, change according to medical device, pb or the supplier (one at a time). the three tests, shown in column ( ), table , (with p-values in square brackets), always reject the null hypothesis, indicating that prices indeed vary for all three dimensions, especially for the device categories (as suggested by the higher value of the test). we then checked if unit prices change for each dimension, after controlling for the other two. hence, the same test was carried out, with prices now cleaned of their average in two dimensions. for example, in one case we considered the difference between prices and average prices by pb plus average prices by supplier, and looked to see if this difference changes according to medical device. this way, after removing pb-and supplier-specific linear fixed effects, we can see if something nonetheless varies according to the device. column ( ) shows that the tests always reject the null hypothesis, indicating that prices still vary for each dimension, once the fixed effects of the other two are removed. in another case, shown in column ( ), we repeated the anova exercise using the ratio instead of the difference between the price and the average price paid. the purpose here was to see if, after removing pb-and supplier-specific multiplicative fixed effects, there is still something that varies according to the device. this evidence suggests that prices are determined by all the three dimensions and that identifying the contribution of each is possible. our results are also confirmed using a non-parametric kruskal-wallis test in place of the anova test (output available upon request). note. "price -avg. price" and "price / avg. price" respectively subtract and divide the price by its average over the two remaining dimensions (e.g. the average by pb and the average by supplier when running the test on the device dimension); p-values in squared parentheses. for the second task, a standard approximation requires unit prices to be explained by costs, quantities purchased and measures of market power. as we run our analysis on medical devices grouped into classes of functionally homogeneous products, a vector of device dummies is a good proxy for their costs. quantities purchased are used to control for the presence of economies of scale. to consider market power, we take two variables: the number of different suppliers recorded in our dataset for each category of medical devices (to account for potential competition), and the number of bidders in the auction (to account for actual competition). we also incorporate a dummy for fpas, which generally have a larger number of bidders than is the case for direct negotiations. using a linear regression model of prices on device dummies, the number of suppliers and of bidders, we find that % of the medical device dummies are significant at the % significance level, with r = . . with the use of a log-log model, the fit increases to r = . , with % of the medical device dummies being significant (see columns ( ) and ( ) of table ). this suggests that the log transformation is better suited to describe prices. moreover, f-tests strongly reject the hypothesis that all device dummy coefficients are equal. moving from a fixed-effect (fe) to a random-effect (re) model has no relevant impact on these results. variables on the number of bidders and the awarding mechanism may be affected by endogeneity. columns ( ) and ( ) replicate the two previous analyses without including these variables in the specification. the log-log model is still largely preferable to the linear model. in what follows, we stick to fe regressions with log prices as a dependent variable. finally, in column ( ), we use a log-log model of prices on quantities, device dummies and device-quantity interactions to control for potential economies of scale, allowing device dummies to interact with the quantities purchased. the fit is high (r = . ) and we would find almost no variation (r = . ) with the same specification, removing quantity and quantity-device interactions. furthermore, % of the log quantity and device-dummy interactions are not significant at the % significance level. similar results are obtained using a linear regression model. hence, the analysis suggests that in our dataset, no economies of scale are present in the levels of quantity purchased by pbs. theoretical framework . definition of the pb's competence consider a market in which − on the demand side − a pb, h ∈ { , h}, is in charge of managing the purchase of medical devices − such as hypodermic needles for syringes − belonging to class d ∈ { , d}. on the supply side, there are s suppliers, and each supplier s ∈ { , s} is willing to sell the requested quantity q dh . we assume that, for a medical device of class d, each supplier's profit function, π ds , with constant return to scale, is given by ( ) according to italian law, requests to procure medical devices cannot refer to a specific brand existing in the market, so as not to favour a specific supplier. requests are limited to a detailed technical description of the medical device required. note. robust standard errors in columns ( )-( ); clustered standard errors using pb id in column ( ); ***p < . , **< . , *p < . . where p is the awarding price of the medical device, c d (·) is the cost function to produce the medical device d, and θ s ∈ θ, θ is the supplier type, known only by the supplier. we assume that θ s is distributed according to a cumulative distribution function f (θ), which is common knowledge amongst suppliers and not observed by the econometrician. assuming a cost function with unidimensional private information θ s and no economies of scale, means it is possible to use unit prices in the presence of lots. in other words, no cross-subsidisation between different medical devices in the same lot is allowed. finally, some suppliers may not be active for a specific tender. we define n dh ≤ s as the number of active suppliers in a specific tender managed by a local pb h, for class d of medical devices. the observed unit price paid, p dhs , can be written as the sum of the supplier's marginal cost c ds = c d (θ s ) and a mark-up µ dhs , as follows: when standard devices are purchased, the aim of the pb is to buy them at the lowest possible price. under full information, the pb utility is then is the marginal cost of the most efficient supplier. to maximise its utility, a pb needs to both award the contract to the most efficient supplier (with the lowest marginal cost) and obtain a price as close as possible to that supplier's marginal cost. however, in a realistic framework, several elements might prevent a pb from obtaining this price: some of them are exogenous to pb choices, whereas others can be totally or partially controlled by the pb. in order to investigate pb competence in the purchasing of different classes of medical devices, we need to set a benchmark supplier s = with marginal costs c d = c d (θ ). defining Ψ dhs = µ dhs + (c ds − c d ), equation ( ) can then be rewritten as follows: we define pb competence as a persistent effect on Ψ dhs recorded for all the tenders (i.e. fpas and direct negotiations) to procure medical devices. this effect refers to the pb choice of awarding mechanism, the definition of the reserve price, collecting information on supplier cost structures and inviting the best suppliers to take part in the tender, among other factors. according to equation ( ), the higher the persistent pb effect, the higher the price paid on average by these pbs (the lower their utility), and the lower the pb competence in managing the procurement process. to estimate this effect, we assume that Ψ dhs can be broken down into a pb-specific effect γ h and a residual component γ ds . assuming linear separability (i.e. Ψ dhs = γ h + γ ds ), means that γ h can be estimated consistently from equation ( ) by using a regression of prices on medical devices' and pb fes. in this case, the choice of the benchmark supplier is irrelevant, as its effect is captured by the medical devices' fes. however, our preliminary analysis in sub-section . suggests that a log-log structure and hence a multiplicative separability (i.e. Ψ dhs = γ h γ ds ), better fits our data. accordingly, equation ( ) can be rewritten as follows: ln(Ψ dhs ) = ln(p dhs − c d ) = ln(γ h ) + ln(γ ds ) ( ) thus requiring a structural estimation of marginal costs and a careful choice of c d . in sub-section . below, we focus on how to derive the benchmark marginal cost for each class of medical device and, in section , we estimate each pb fe γ h . we then explore the correlation between pb competence and pb balance sheet data. following the methodology of the seminal work of guerre et al. ( ; henceforth gpv) , we use only fpa observations to estimate the marginal cost for each class of awarded medical device. in so doing, we implement the gpv approach with three main changes. first, we account for heterogeneous devices in our dataset (see sub-section . . ). second, we adapt the gpv methodology developed for direct auctions -where the highest price wins -to procurement auctions, where the lowest price wins (see sub-section . . ). finally, we extend gpv to consider sealed bid auctions in which bidders do not directly observe their competitors, i.e. they may receive a noisy signal on the level of competition (see appendix a. ). medical devices include different goods -bandages, syringes, etc. -which are categorised by class. unfortunately, the number of observations in our dataset is too small to compute the conditional distribution of bids for each class d. to address this issue, in line with the preliminary evidence of sub-section . suggesting that a log-transformed model is well-suited to describing our data, we assume that a bidder's private evaluation (i.e. its marginal cost) is multiplicatively separable in the supplier type θ s and in a technological parameter α d specific for each class of medical device. this separability is preserved by equilibrium bidding (haile et al. ) . for example, suppose the marginal cost of a medical device of class d is twice the marginal cost of a medical device of class d , then the same ratio between the marginal costs of d and d applies to all suppliers. in this case, in equilibrium and for each supplier, the price of d will be twice the price of d . accordingly, we assume that in an auction for medical device d, marginal costs (i.e. the bidders' private values) are given by the following equation: where the bidder-specific private information θ s is independent of the device-category parameter α d . the assumption of multiplicative separability in the cost function has already been used in the literature (e.g. to model adaptation costs in bajari et al. ) and is in line with the preliminary results of sub-section . . let a category d = be such that α d = . then, the equilibrium price has the same separable structure as the marginal costs: where p d (·) is the equilibrium bidding function for device d. given this functional form, the technological parameter α d can be obtained using a regression of the observed log bids on medical device fes (the dummy variable d d ) and on the number of bidders in each fpa (n dh ), as follows (output available on request): appendix a. . collects all device-category parameters α d . as a robustness check and to exclude the fact that devices with few observations lead to biased estimates, we repeated the estimation of equation ( ) in two subsamples of the medical device classes, i.e. for all medical devices where we have at least five observations (roughly half of the classes) or eight observations (roughly half of the observations used to estimate the α parameter). then, in both cases, we test whether the estimated α d (for the considered devices) are equal to the same α d estimated in the entire sample. in both cases and for all the devices considered, we find no statistical difference with a % confidence interval. all observed unit prices p dhs paid by pbs, hence the winning bids, are then normalised by dividing by α d . we define homogeneous price p hs as follows: this price p hs is used from now on to make all observations of our dataset comparable and to obtain a consistent estimate of the bid that each supplier would have submitted in an fpa for the provision of a medical device of class , with α = with the level of competition n h . the distribution of p hs derived from the data is presented in figure . in a fpa, procurement framework, the lowest bid wins. the resulting nash equilibrium bid p (θ i ) of the i-th bidder of type θ i is given by the following: following gpv, equation ( ) can be inverted to express the unobserved marginal cost θ i as a function of the observed prices and price distribution observed through kernel estimation. in our dataset, for each auction, we observe the winning price rather than all the bids. for standard fpas, athey and haile ( ) propose using the winning prices of multiple auctions to identify private values because the winning price is the maximum order statistic of bid distribution for a given level of participation. in a procurement framework, winning prices can be considered as the first (i.e. minimum) order statistic of bid distribution. the structural equation that states unobserved marginal costs as a non-parametric function of observed winning prices, winning price distribution and the level of competition is as follows: where n h = { , } is the noisy signal about the level of competition that bidders receive for the given auction, g ( ) (p hs |n h ) is the cumulative density function of all transaction prices, conditional on n h , evaluated at p hs , and g ( ) (p hs |n h ) is its relative probability density function. the derivation of equation ( ) is presented in appendix a. . the resulting distribution of θ s based on our sample is plotted in figure . since we impose no constraint on equation ( ), estimates of the private value θ s can be negative. indeed, the distribution in figure displays negative values in % of cases. the distribution of private values is based on our benchmark sample in order to use as many observations as possible. however, we find a similar distribution if we restrict our attention to several sub-samples of the data, that is, if we concentrate on bidders who manufacture the devices (ignoring bidders who are merely sellers), if we separate auctions with reference prices from auctions without reference prices, and if we look at devices or pbs with more observations. details of these robustness checks are discussed in subsection . . below. the fact that the distribution of private values includes negative values is not a problem for subsequent analysis, because we need only one representative value from the distribution. indeed, our analysis requires us to choose a benchmark supplier, the same for all medical devices. the prices paid by different pbs are then compared to the marginal costs of that supplier. we choose the median value θ of the distribution and, accordingly, use equation ( ) to obtain the benchmark marginal cost c d for each class d, as follows: we use the median marginal cost mainly for two reasons: (i) deviations from a median value provide a simple interpretation of the price-cost difference (p dhs − c d ) used to derive pb competence, as it measures how much the winning supplier differs from a representative, median value; (ii) the median value is a robust choice, as the distribution of the private values is structurally estimated and not directly observed by the econometrician. while reasonable, using the median private value is an arbitrary decision. the choice of the benchmark value has consequences for equation ( ) and in particular for the pbspecific effect γ h . in our case, γ h describes the pb effect compared to the median supplier. however, changing the benchmark value does not alter the subsequent analysis: since it is constant with respect to the pb, the pb-specific effect γ h may change in size, but it maintains the same ranking. in appendix a. . we replicate the benchmark results of section when taking the rd and th percentiles from the distribution of private values. the key results are unaltered. to investigate pb competence in relation to the procurement of medical devices, we consider c d , along with the price paid by the pb. in our dataset, benchmark marginal costs are always above zero and, except in . % of our observations, below the actual prices paid by pbs. this sub-section replicates the marginal cost estimate to control for different issues which may arise from our structural model. we are particularly concerned about the median value θ used to define the benchmark marginal cost c d because the prices paid by pbs are compared to this cost. to further strengthen our results, for each robustness check listed below, we also compare the distribution of private values θ s with the baseline estimate shown in figure . only producers -one concern with our analysis might be the use of a private value model to structurally estimate the auction game. this model is consistent with a setting where bidders are, simultaneously, producers and sellers , that is, are endowed with a privately observed cost function. differently, the presence of bidders who are, simultaneously, distributors and sellers may introduce a common-value component into the information structure, thus determining biased estimates. non-parametric tests to control for the presence of a stochastic common value component exist in the literature, but they require the observation either of all bids (haile, hong and shum, ) or, at least, the winning and second lowest bids (athey and haile, ) . decarolis ( ) suggests using the reserve price in place of the second lowest bid to control for the presence of a deterministic common cost component (unobservable by the econometrician, but observed by all bidders). unfortunately, such information is not recorded in our dataset, which has information on winning bids only. to address the issue, we collect additional data on bidders to identify them as producers and sellers or distributors and sellers . the marginal cost estimate is then repeated using only bidders identified as producers and sellers . reference price -understanding the conditions under which the reference price may affect bidding decisions is relevant. note that inconsistencies in estimation of θ arise only when the following conditions are both met: (i) the reference price is higher than the winning firm's marginal cost, but lower than its equilibrium bid when the reference price policy is not adopted, and (ii) at least a second firm with marginal costs below the reference price took part in the auction. as a robustness check, we consider observations before and after the termination of the reference price policy separately. device and public buyers' restrictions -to prevent devices with few observations leading to biased estimates, we repeat the estimation excluding device classes with fewer than five observations. we carry out a similar exercise for pbs, and in line with the next section, exclude pbs with fewer than observations. table . the median θ in table shows that the largest deviation from the baseline estimate of θ arises when the marginal cost estimate is repeated using only auctions when the reference price policy was applicable. however, the estimate of θ is only . % larger than the baseline estimate. the sample is the smallest of the robustness checks considered, and kolmogorov-smirnov finds no difference between the two distributions. remarkably, the differences in θ from the remaining robustness checks are tiny. for example, the deviation in θ from the baseline, considering solely producers, is equal to only %. this, with the use of the kolmogorov-smirnov test, is the only distribution of θ s found to be significantly different from the baseline estimate. however, the suppliers included also differ, with producers and distributors in the baseline distribution, but we now investigate each pb-specific fixed effect in purchasing standard medical devices. considering the price paid, after estimating the benchmark marginal cost c d of each medical device, we obtain Ψ dhs = p dhs −c d . we then estimate the pb-specific component γ h by using the following ols regression: the specification in equation ( ) includes the pb dummies a h and the dummy variable r equal to when the reference price regulation was in force and otherwise . this variable interacts with the pb dummies to capture any change in the pb fixed effect attributable to the reference price. excluded from this estimate are pbs with fewer than observations, i.e. pbs that managed under auctions in the period considered. we obtained pbs and , observations for awarded medical devices. in the analysis, we use standard errors clustered at the level of medical devices to control for potential serial correlation. our aim is to provide an estimate for pb parameters γ h = ln (γ h ), where γ h is the pb-specific fixed effect in purchasing medical devices, as set out in equation ( ) . the higher the coefficient, the lower the competence of the pb. in the regression, almost all dummies are significant, suggesting that each pb is endowed with its own specific procurement competence. the r of the regression is . , which means that about two-thirds of the difference between prices and marginal costs can be explained by pb fixed effects. estimates are available on request. pb competence may affect various decisions in purchasing procedures such as the choice of awarding mechanism and of the reserve price, or how to attract more or better suppliers, among others. below we study pb competence in managing the purchasing of medical devices to see if it is associated with any observable characteristics. hence, we run the following regressions of a proxy for each pb fixed effect on a set of explanatory variables: in these regressions, the unit of analysis is a single pb. we consider weighted regressions, in which the weight is proportional to the number of auctions the pb managed in our sample period. this way, we attribute more importance to the pbs that more frequently organised tenders to award medical devices. we consider two different measures for the dependent variable. first, we take pb competence derived earlier from equation ( ) with the structural estimation of the marginal costs. second, we take a measure of pb competence originating from the following equation: equation ( ) differs from equation ( ) in two ways. first, the dependent variable comprises prices only and therefore excludes marginal costs which, in our approach, are recovered from prices. second, the specification now includes medical device dummies. the purpose is to obtain estimates of the pb fixed effects unaffected by our structural approach to infer the marginal cost of medical devices. indeed, the heterogeneity of the costs of the devices is now captured through the assumption-free medical device dummies, similarly to best et al. ( ) . the resulting estimates of γ h are generally smaller but highly correlated ( . ) with those obtained from our benchmark analysis. the r of the regression in equation ( ) is equal to . , but falls to . when the regression is repeated with only pb fixed effects. even after excluding the structural analysis, almost half of the price disparities is explained by pb fixed effects (estimates are available on request). note that in equation ( ) , the sign of the dependent variable is inverted to ease interpretation. in so doing, higher coefficients indicate higher competence in managing procurement. as the dependent variable is itself an estimate, we make use of bootstrapped standard errors based on , iterations. the specification includes four groups of variables: m h refers to the auction mechanism applied (the fraction of direct negotiations), h h refers to potential scale economies in purchasing (the logarithm of total personnel cost or the logarithm of healthcare material purchases), p h refers to the distribution of costs (the fraction of non-health personnel over total personnel costs and the fraction of healthcare material purchases over total health costs) and the average number of days the pb takes to pay its suppliers (the logarithm of days of payable outstanding in ), and c h refers to control variables on the nature of the pb (the dummy asl, identifying local healthcare units, other than hospitals), its location in a metropolitan/rural area, in the north/center-south of the country, and per-capita health expenditure in the region of the pb. the latter variable interacts with the center-south dummy because of countrywide disparity, with northern outspending southern regions. table shows the output of our regressions by using the proxy of pb competence obtained from equation ( ) in columns ( ) and ( ), using the proxy obtained from equation ( ) in columns ( ) and ( ). for each measure, we consider two variants of the specification, depending on which variable is used for h h (either total personnel costs or healthcare material purchases). we do not include the two variables in the same specification because they both proxy for the size of the pb, and are highly correlated (the correlation is . ). a regression equation using both variables would not precisely identify the contribution of each. since, a priori we have no preference for either variable, we look at them in two separate models. table shows the output of iv rather than standard ols regressions (shown in appendix a. . ). this is due to concern that there may be simultaneity for the mechanism variable m h . the pb decision about which auction mechanism to implement may influence and, at the same time, be influenced by pb competence itself. this could create endogeneity, yielding inconsistent estimates. in all columns, we therefore instrument the mechanism variable (the fraction of direct negotiations) with two variables, the fraction of multi-device auctions and the pb-specific average quantity of devices auctioned. both instruments inform on the size of the auction. this is important, as smaller auctions face fewer legislative constraints in using direct negotiation. the two instruments should be directly correlated with the procurement mechanism (i.e. they should be relevant) but not with pb competence (i.e. they should be exogenous). this set of instruments is found to be relevant and exogenous according to the standard tests, as it rejects the null hypothesis of the kleibergen-paap test of relevance, and accepts the null hypothesis of the sargan test for over-identifying restrictions (p-values at the bottom of the table; see also the output of first-stage and reduced-form regressions in appendix a. . ). moreover, the hausman-wu test suggests that endogeneity is indeed present, at least in columns ( ) and ( ) , and iv models (p-values at the bottom of table ) are therefore advisable. below, we comment only on coefficients that are significant at least at a % level. importantly, iv estimates in table and ols estimates in appendix a. . produce similar results, with the main exception of the fraction of direct negotiations, that is the endogeneous explanatory variable. the key findings from all the models in table are qualitatively the same. our analysis shows that direct negotiations have a negative impact on pb competence. according to column ( ), a % increase in the fraction of direct negotiations decreases pb fixed effects by . or . % (- . divided by the average of the dependent variable, . ); according to column ( ), the same change has an effect of - . or - . % (- . / . ). the reason could be that, in line with italian public procurement law, direct negotiations are used when the purchased item has specific characteristics a competition would neglect. this explains the higher prices paid by the pb, and thus a negative impact on pb competence. we also find that the pb size effect, measured using either total personnel cost or healthcare purchases, is positive and significant. considering the variables on costs, we find a positive and highly significant effect for the ratio of non-health personnel to total personnel costs. when two pbs of the same size are compared, the pb with higher costs for non-healthcare personnel is more competent in procuring medical devices. we also find a generally negative effect of healthcare purchases over total healthcare expenditure, in line with the definition of pb competence. once we focus on the type of pb, we find that local healthcare units (measured by the coefficient on the asl dummy) are less competent than hospitals. these two types of pb differ in their form of territorial organization: local healthcare units provide services -typically, in a county -with several offices, outpatient clinics and services spread over the territory. hospitals generally comprise numerous departments all in one location and, accordingly, are more sensitive to size-related economies of scale. hence, we repeat the analysis adding to the specification the interaction of the asl dummy with pb size (measured either by personnel costs or healthcare purchases). the results (available on request) show that the negative effect of local healthcare units disappears for small pbs, but remains negative (increasingly by size) for all the others. pb type does not only influence the effect of size on competence. the negative effect of location in a metropolitan area disappears when the interaction between metropolitan area and asl is added to the specification, and only that interaction is negative and highly significant (results available on request). moving on to the control variables, there is a negative overall effect for the number of outstanding days for payment. the expectation of delayed payments from a pb is built into supplier offers, i.e. they submit higher prices because they expect to be paid late. column ( ) shows a significant and negative effect for pbs located in the center-south. this geographical effect is in line with the well-known north-south divide in italy (federico et al. ). with a focus on local public institutions and procurement in italy, show that court and public works inefficiencies are more severe note. bootstrapped standard errors ( , repetitions) in parentheses; *** p < . , **< . , *p < . . in southern regions. similarly, our analysis provides further evidence of institutional inefficiency -for pbs in the healthcare sector -located in the center-south of italy as compared to pbs in the north. significant effects can also be seen, in columns ( ) and ( ), for the size of per capita health expenditures (positive) and the interaction between the latter and the geographical variable (positive). no other variable in the specification turns out to be significant. appendix a. . shows, among others, the output of columns ( )-( ) in table using, for the estimate of pb fixed effects, the rd and th percentiles (rather than the median) of the private values. our main findings are unaltered, with the exception of the fraction of direct negotiations, which is never significant in the new estimates using the th percentile. reference prices and public buyer competence this section empirically investigates the effect of the reference price policy (for classes of medical device) on pb competence. our dataset covers the period from january , to december , . until may , , pbs were forced by law to apply the reference price established by the avcp for each class of homogeneous medical device. first, we replicated the regression in equation ( ), but only on the subset of pbs that managed awarding procedures both before and after the termination of the reference price policy. this leads to a dataset of observations, with prices paid by pbs both before and after the abolition of the reference price. figure compares the distribution of pb fes, as measured by γ h , with pb fes under the reference price regime, given by γ h + φ h . with the reference price policy, the distribution of fes is more concentrated around central values of the distribution. this is not surprising, since the reference price policy reduced pb discretion and hence removed a degree of competence. to further clarify what is going on, we divided the sample of pbs into four groups, depending on whether their competence falls below any of the quartiles of the distribution. ranking pbs h = , ..., h from the lowest to the highest competence, we can create four groups of similar size (from observation h i− to observation h i for i = , ..., ; h = and h = h). for each group i we then ran the following regression: the authors show that -in areas with inefficient local courts -public works are delivered with longer delays and the larger the contract, the longer the delay. specifically, since local courts enforce procurement contracts managed by local pbs, their inefficiency weakens the pb, making it less able in managing the overall purchasing process. one limitation of our study may be the treatment of endogenous entry in awarding procedures. such entry can be affected both by the buyer choice about which awarding procedure to implement (i.e. fpa or direct negotiation) and by the supplier decision to enter the procedure. explicitly incorporating these types of endogeneity would have required a multiple-stage model, which we leave for future research. however, it is reassuring that both the semi-parametric and parametric estimates provide similar conclusions. j o u r n a l p r e -p r o o f equation ( ) differs from equation ( ) for the inclusion of a common rather than pb-specific effect on the reference prices. the results are shown in table separately for the models with and without a structural estimation of the costs (panel a and panel b respectively), following the approach of section . although reference prices overall have a negative impact on the final price net of the marginal cost (with a magnitude of around - %), their effect varies widely depending on the initial level of competence of the pb. in fact, reference prices have a strong and negative impact on low-competence pbs (first quartile) and a strong positive impact on high-competence pbs (fourth quartile), and have no impact on average-competence pbs (second and third quartiles). with the reference price, the distance between prices and marginal costs shrinks for low-competence and increases for high-competence pbs. changes are non-negligible (- . % in the lowcompetence sample and . % in the high-competence sample). the findings are similar in panel b), except that there is no longer a significantly negative effect of the reference price in the full sample. the r statistics in panel b) are much higher than in panel a) due to the inclusion in the specification of device fes accounting alone for about % of the fit. we interpret this non-linear effect of the reference price policy on awarding prices as directly related to pb discretion in determining the reserve price, distinguishing high-competence from low-competence pbs. indeed, in the absence of mandatory reference prices, high-competence pbs can freely determine the reserve price (the maximum price the pb is willing to pay) so as to maximise savings via the most efficient supplier, while low-competence pbs are very far from obtaining a price close to the most efficient supplier's marginal cost. note. standard errors clustered by medical device in parentheses;*** p < . , **< . , *p < . . when mandatory reference prices are at work, high-competence pbs have less discretion in each awarding procedure, and this decreases their competence in obtaining the best final price. by contrast, low-competence pbs may benefit from reference prices, as they can be lower than the reserve price they would have adopted in the absence of a reference price, thus allowing pbs to pay lower final prices. we perform three robustness check on the regressions in table . first, we are aware that our results originate from a before-after setting with no control groups, which does not allow the effect of the reference price to be distinguished from the effect of time. in order to disentangle the two effects we would need a setting with both treatment and control groups, where we could compare our medical devices with other goods for which the reference price remained in force throughout . hence, in appendix a. we comment on an exercise obtained from a dataset including homogeneous drugs no longer covered by patent. unfortunately, the new data can be used to replicate only the analysis without structural estimation of the costs. the results of the diff-in-diff analysis are consistent with those in table panel b). second, from panel a) we take the median in the distribution of private values. in appendix a. . we repeat the analysis using the rd and th percentiles rather than the median of the private values. although the estimates for each column may differ from table , the general pattern is clear and shows that the effect of reference prices is negative (positive) for low-(high-) competence pbs. third, some results might be driven by devices for which we have only a few observations. removing sparsely observed devices is challenging, as we want to preserve the threshold of at least observations for each pb. we repeated the analysis excluding devices with only or fewer observations. the results are set out in appendix a. . . we found that the reference price significantly reduces the difference between prices and marginal costs for pbs with lower competence. additionally, the effect of reference price on pbs endowed with high competence (in the fourth quartile) is significantly different from the effect on pbs endowed with low competence (in the first quartile). we conclude our analysis by repeating the iv regression in equation ( ) where the unit of analysis is an individual pb but now using, as a dependent variable, a proxy for pb competence in the reference price regime: our aim here is to determine if pb competence correlates with different variables when the reference price is in play. table shows the relevant outputs, comparing estimates of competence under the reference price (− ( γ h + φ h )) with those without (− γ h ). the latter scenario stems from equation ( ), but the output differs from table because here, we only consider pbs facing at least one auction with a reference price and one auction without a reference price. appendix a. . shows the corresponding estimates based on ols regressions. the comparison of column ( ) with column ( ), and column ( ) with column ( ) provides systematic evidence that under the reference price policy, key effects (of the fraction of direct negotiations, healthcare personnel cost, healthcare purchases and non-healthcare personnel over the total personnel cost) are generally smaller. these coefficients generally become closer to zero. our explanation is that the reference price policy limits the discretion of pbs in designing the awarding process, so each pb-specific competence (or incompetence) no longer significantly affects the outcome. the only exception is the dummy variable related to the metropolitan area, which switches from a negative and significant to a positive value, suggesting that pbs located in those areas benefited most from reference prices. focusing on the geographic dimensions in table , an interesting pattern emerges. without the reference price, pb competence was much lower in the center-south of the confidence intervals of the reference price estimated in the first and the fourth quartiles (at % with costs, at % without costs) do not overlap. italy; with reference price, pb competence was still lower in the center-south, but not as low as without. one possible explanation for the negative effect is the inefficiency of institutions in the center-south of italy, as mentioned in the comments on table results. based on our findings, inefficiency is mitigated under mandatory reference prices, possibly because pb discretion is more limited. by using the dependent variable obtained without structural estimation of the costs, we obtain similar findings. however, this estimation does not pass the sargan test for the exogeneity of the over-identifying restrictions. the output is available on request. in european countries, the procurement of medical devices by hospitals and local health units (pbs) takes up a large share of national budgets. in the us, healthcare expenditure is rising. anecdotal and empirical evidence of huge variations in the prices of medical devices -among and within countries -makes procurement efficiency a relevant issue. this study empirically investigates the price differences in the purchasing of standard medical devices by italian pbs, with a focus on pb competence in managing procurement during the period from january to december , . it also studies the effects of the mandatory reference price on the prices paid by pbs, a nationwide policy adopted until may , to increase public spending efficiency. for each purchase, we measured the difference between the price of a medical device (resulting from the procurement procedure) and its benchmark marginal production cost (resulting from our structural estimation). we defined pb competence as pb fixed effects on this difference for each item purchased. our results show that italian pbs pay substantially different prices for standard medical devices. in particular, the quartile-based coefficient of variation of the prices paid is . . this difference between purchasing prices can be explained by pb fixed effects, which we investigated as related to institutional characteristics, geography and size. we found that pb size (measured by overall personnel costs, corresponding to the sum of healthcare personnel and non-healthcare personnel costs or by the size of healthcare-related procurement) has a generally positive and significant effect on pb competence in relation to the procurement of medical devices. our empirical analysis showed that non-healthcare personnel costs drive the overall positive and significant effect on pbs' competence. this result supports the centralisation of purchasing for medical devices, i.e. a few large pbs in the country with non-healthcare personnel addressing (possibly skilled) efforts in the procurement activities. a centralised for variations in these prices, see medtech europe ( ); henschke, c. and redberg ( ); den ambtman et al. ( ). analysing hospital surveys carried out by the millennium research group, wenzl and mossialos ( ) provide evidence of large price differences between the us and germany for cardiovascular technologies. for example, in , prices for bare-metal stents and pacemakers were five times higher in the us compared to germany and significant price differences were recorded even within eu countries. to make all observations comparable, the quartile-based coefficient of variation is calculated using homogeneous prices, as defined by equation ( ) , applied to the entire dataset. note. bootstrapped standard errors ( , repetitions) in parentheses; *** p < . , **< . , *p < . . system for procurement -whatever the design of the national health sector considered -would also better enable policymakers to implement measures to control prices while accounting for the value of the medical devices procured. we then investigated the effect of mandatory reference prices as a cap on the winning price. this policy seems to have a weak effect in fostering the efficiency of purchasing medical devices. considering an average price of e . , our back-of-the-envelope calculation shows that the average price decreased by e . , or %. this translates into a reduction of e in a median auction with a total cost of e , . particularly noteworthy, this overall result masks a non-linear effect of the reference price on pbs with different abilities: we found that reference prices have a significant negative effect on high-competence pb purchasing and a significant positive effect on low-competence pb purchasing. according to our back-of-the-envelope calculation, the effect of the reference price ranges from a e . ( . %) average price decrease for low-competence pbs to a e . ( . %) average price increase for high-competence pbs. this finding regarding the effect of reference prices is not free of limitations. indeed, it relies on a before-andafter analysis which compares medical device purchases in two periods, with and without reference prices. hence bias may arise if events not controlled for in the regression analysis take place in one only of the two periods. to remove any bias, in appendix a. we compare our findings for medical devices with those from a control group of drugs (no longer covered by patent) for which reference prices were not abolished in the period considered. our benchmark finding was supported by these further results. in a period in which, both in europe and the us, policymakers are increasingly relying on regulatory policies for medical devices to improve procurement efficiency, our results show that pb discretion should be considered along with each pb competence in managing the process. specifically referring to the impacts of mandatory reference prices on pb competence, our results suggest a move towards a discriminatory approach -implementing mandatory requirements only for pbs performing below a given benchmark. note that our results were obtained in a procurement setting with standardised and simple items. considerations of pb competence and discretion would be even more relevant regarding policies designed for the procurement of more complex items (kelmann, ). note that the centralisation of purchasing for medical devices might be life-saving in some countries during crises such as the recent covid- pandemic. espitia et al. ( ) estimate that surges in demand for critical covid- products (e.g. masks, gloves, aprons, suits, ventilators, etc.) and export restrictions on leading producers will drive up prices by % on average (aprons and masks recording % and % increase, respectively), with critical effects (very limited or no access to medical devices) in developing countries. there, the centralisation of procurement for medical devices could also address -in addition to price control -the issue of coordinating international trade policy for these products. this calculation is based on predictions of the price/cost difference from column ( ), table . we obtain: (exp (− . − . ) − exp (− . )) / . = − . . these calculations are based on predictions of the price/cost difference from column ( ) and ( ), table . low-and high-competence pbs are defined in − respectively − as the st and the st quartile of the overall pb competence. we are grateful to the editor, prof. owen o'donnell, for suggesting this test. note that drug data do not give rise to the structural estimation of marginal costs. future research may replicate our benchmark analysis with a richer dataset. given the high value of european public procurement in the healthcare sector (both for standard and non-standard items) and the core relevance of the sector in the europe strategy and forthcoming policies, new empirical investigations are expected to shed light on further improvements in spending efficiencies in the sector. they are also expected to address the effect of the recent policy by the trump administration regarding the transparency of "price and quality information" in the healthcare industry. the search for spending efficiency in this industry seems -both in the us and in europeto encourage the spread of information on prices, but more empirical work is necessary to explore the costs and benefits of the mechanisms adopted and to assess their policy implications. relational contracts and the theory of the firm identification of standard auction models bidding for incomplete contracts: an empirical analysis of adaptation costs discretion and supplier selection in public procurement law enforcement, malfeasance, and the compensation of enforcers privatization and quality: evidence from elderly care in sweden individuals and organizations as sources of state effectiveness, and consequences for policy relational contract and competitive screening the effects of discretion in procurement performance public requirements in public procurement: evidence from a regression discontinuity design court efficiency and procurement performance buyer competence in procurement: evidence on cooperation, incentives and skills comparing public procurement auctions analysing actual prices of medical products: a cross-sectional survey of dutch hospitals the role of wages and auditing during a crackdown on corruption in the city of buenos aires trade and the covid- crisis in developing countries the origins of the italian regional divide: evidence from real wages self-enforcing agreements and relational contracting: evidence from california highway procurement price discrimination and bargaining: empirical evidence from medical devices bargaining ability and competitive advantage: empirical evidence from medical devices transparency and negotiated prices: the value of information in hospital-supplier bargaining regulating innovation with uncertain quality: information, risk, and access in medical devices optimal nonparametric estimation of first-price auctions average payment time for private and public health organizations nonparametric tests for common values in first-price sealed bid auctions medical device price differentials in the u.s. and europe -rethinking price regulation? competitive bidding for contracts under alternative auction procedures public procurement of health technologies in greece in an era of economic crisis procurement and public management: the fear of discretion and the quality of public performance bid takers or market makers? the effect of auctioneers on auction outcome relational incentive contracts public sector purchasing of health services: a comparison with private sector purchasing the european medical technology industryin figures health at a glance: europe : state of health in the eu cycle manipulation of procurement contracts: evidence from the introduction of discretionary thresholds price transparency for medical devices corruption and government: causes, consequences and reform medical technology procurement in europe: a cross-country comparison of current practice and policy reputation, competition and entry in procurement prices for cardiac implant devices may be up to six times higher in the us than in some european countries key: cord- -bhzctz authors: ngan, olivia miu yung; sim, joong hiong title: evolution of bioethics education in the medical programme: a tale of two medical schools date: - - journal: international journal of ethics education doi: . /s - - - sha: doc_id: cord_uid: bhzctz bioethics education in the anglo-european context developed since and was incorporated into the undergraduate and postgraduate education, residency training, and continuous education. in the asia-pacific region, bioethics education is less structured and often dependent on contextual constraints. this paper provides a cross-sectional analysis, describing institutional experiences in developing bioethics curriculum at two medical schools in malaysia and hong kong. the medical programmes of the two institutions are distinctive in terms curriculum framework, teaching approach, and topic selection, and common challenges include implementation of bioethics courses, students’ resistance to bioethics, and limited teaching capacity, emerged as they evolve. the reported experiences revealed that there is room for improvement regarding how medical schools integrate bioethics education in regions where curriculum development remains at an early stage. at least, a bioethics education requires both top-down support from the faculty to improve teaching and educational quality, as well as from the bottom-up approach to empower students to raise awareness and concerns toward bioethics, and helps students developing reasoning through challenging issues. moral conduct in the clinical practice demonstrated through attitudes and behaviours is a transcendental element in medical education. entering medical school as students and later becoming clinicians involves a transformative process emphasising knowledge competencies, codes of practice compliance, and moral development (holden et al. ) . clinical knowledge acquisition has been the core in the medical education and establishment of virtues values through bioethics training have been underexplored in medical education. bioethics in the anglo-european context has developed since and has been incorporated into the undergraduate and postgraduate medical curriculum, residency training, and continuous professional education. in the asia-pacific region, bioethics education did not gain attention until the s, and teaching deliveries remained inconsistent and less structured depending on contextual factors (miyasaka et al. ). one of the common barriers in the implementation of a curriculum in a resource-constrained context is a lack of teaching capacity (ten have ) . teaching bioethics requires expertise both in ethics and medicine. nonetheless, many teachers who are involved in bioethics curriculum in medical programmes were either philosophers, ethicists, or scientists with limited experience in medicine, or clinicians with inadequate formal training in ethics (ekmekci ; sim et al. ) . universities have a limited capacity to provide professional training of ethics and moral philosophy to sustain teaching quality. another common challenge is limited curriculum time to include ethics in the packed medical teaching timetable (ravindran ) . successful curriculum development implementation requires continuous and multifaceted consideration. there is a four-stage framework describing the action plan for progressive changes in the curriculum development, specific to bioethics curriculum in medical education over time, in terms of the implementation, content, teaching capacity, and instruction method (sherer et al. ) . in stage , there is no bioethics course in the institution. in stage , bioethics is taught from a philosophy course with a limited clinical perspective, emphasising on theories and knowledge acquisition. the capacity in supporting teaching is little and didactic lecture is the primary teaching delivery. in stage , bioethics is taught as an independent course, covering ethical issues arise from clinical practice, communication skills, and empathy. in addition to didactic teaching, other teaching methods, including small-group discussion and research project, are adopted to encourage intellectual exchange. in stage , the bioethics curriculum is taught by a multidisciplinary faculty team, including physicians, philosophers, and attorneys. the framework is a good indicator, particularly for developing countries, in determining the current status of bioethics education at medical schools. bioethics is taught in almost all medical schools in malaysia (sim et al. ) and hong kong (becker ) , and the teaching framework and deliveries vary to a great extent depending on the contextual factors. this paper provides a narration describing institutional experiences in developing bioethics curriculum at two medical schools the university of malaya and the chinese university of hong kong. malaya gained independence from british colonial rule in . in , malaysia, comprising the peninsula (west) and sabah and sarawak (east) was formed. the country is run based on parliamentary democracy. the legal system incorporates civil law, derived from british elements, and aspects of syariah laws for muslims. malaysia, with a population of . million, is a prime example of a plural society. the largest ethnic groups in malaysia are the malays ( %), chinese ( %), indians ( %), and others ( %) (department of information ). in sabah and sarawak, there is a myriad of indigenous ethnic groups with their own unique culture and heritage. due to the multi-racial characteristic of the population, it also has varieties of culture, religion, social norms and values. all these cultures have influenced each other, creating a genuinely diversified malaysian culture. the malaysian medical council (mmc) operates under the medical act of and regulates the registration and practice of medical practitioners (malaysian medical council ). over the years the mmc has undertaken the role of granting recognition to other medical schools in the country and overseas and added the name of these schools to the existing register of recognised medical degrees in the second schedule of the act. it is recommended that the mmc establishes an active functional 'education committee' and that the role of mmc in medical education should be clearly and explicitly stated in the act. currently, there are malaysian medical schools, including public and private institutions (wong & kadir ) . the university of malaya (um) is the oldest public tertiary institution in malaysia, providing a five-year undergraduate medical programme -bachelor of medicine and bachelor of surgery (mbbs). at present, um adopts a system-based and problembased approach that integrates the teaching around the major body system, covering both basic scientific foundations and clinical reasoning skills development. before the medical curriculum reform took place in , the teaching and assessment focused on the acquisition of medical knowledge and clinical skills, and the humane aspect of medicine was largely neglected. the course content is divided into three main components that run vertically through the course, namely: (i) the scientific basis of medicine, (ii) the doctor, patient, health and society, and (iii) personal and professional development. the scientific basis of medicine component includes the study of the normal human body and its function (anatomy, biochemistry, physiology); the body's reaction to injury (medical microbiology, pathology, parasitology pharmacology) and practice-based clinical medicine (anaesthesiology, biomedical imaging, emergency medicine, internal medicine, obstetrics and gynaecology, orthopaedic surgery, opthalmology, otorhinolaryngology, paediatrics, primary care medicine, psychological medicine, social and preventive medicine surgery). the doctor, patient, health and society (dphss) module includes the study of preventive, promotive, environmental and occupational health in the community; medical statistics, epidemiology, two field projects (community residency program, crp; organisation and management of health services, omhs); community family case studies, cfcs and family health. the personal and professional development (ppd) module includes improvement of learning, analytical, critical thinking and communication skills, nursing skills, medical ethics, research methodology and exposure to optional fields of interest via elective programs. under the past curriculum, bioethics was appended under personal, and professional development theme and lecture was the primary mode of delivery. there was no formal assessment on the subject. bioethics was not a popular subject among medical students. this was reflected by poor attendance, in particular, among pre-clinical students. some faculty members also do not consider bioethics a 'real' topic to be taught. at that time, the development of bioethics education was slow and insignificant. starting from , the medical education and research development unit (merdu) has been actively involved in the curriculum reform and is now responsible for overseeing the bioethics curriculum. the current medical programme comprises of four themes, namely basic and clinical sciences, patient and doctor, population medicine, and personal and professional development. compared with the past curriculum, the most significant change in the revised curriculum is that personal and personal development module has been upgraded to a theme. while only ethics and professionalism were emphasised previously, bioethics, professionalism, humanities, and health law are given emphasis in the revised curriculum. this is to ensure that students not only grow professionally into good doctors but also receive a holistic experience in humanities other than science teaching in the medical curriculum. a unique feature is the 'language in medicine' component, which provides an early introduction to the humanities in medicine (sim et al. ) . students learn about icons in medicine, historical events in medicine, death and dying, end-oflife issues, and organ transplantation. (figure ). today, bioethics is not taught as an independent course or a stand-alone course but as part of an integrated curriculum. moral theories and related application are oriented vertically and horizontally along with relevant clinical topics. for example, core topics such as informed consent and confidentiality are introduced in year and revisited throughout the programme (vertical integration). other contentspecific topics such as ethical issues in human fertility are embedded into relevant learning block such as endocrine and reproductive health block in year (horizontal integration) and revisit in later years during clinical postings such as obstetrics and gynaecology (vertical integration). in terms of content, a wide range of topics is being covered. topics more commonly taught in pre-clinical years include principles of bioethics, history of medicine, origins and philosophy in ethics, and medical humanities. it is believed that these topics need an early introduction to help lay the foundation for bioethics education. some cases, such as the refusal of treatment, reproductive issues, paediatrics and neonatal issues, are very clinical and could be addressed at a later stage, e.g. during clinical postings. however, certain topics need to be emphasised throughout the medical programme. some examples are informed consent, patient-doctor relationship, privacy and confidentiality, health law, truth-telling, cultural issues and diversity awareness, religious and spiritual issues, conflicts of interest. these topics aim to prepare medical students for work readiness in the healthcare setting of a plural society like malaysia. bioethics was not a popular subject among medical students, in particular, among pre-clinical students. some faculty members also do not consider bioethics a 'real' topic to be taught. before the medical curriculum reform, the development of bioethics education was slow and insignificant. nonetheless, since embarking on the change via curriculum transformation in our undergraduate medical programme at the beginning of , our medical school has taken a giant step forward. murals related to bioethics on the walls of faculty buildings (figure ) , initiated by faculty members and completed by medical students, is testimony there is increased awareness and acceptance of the importance of bioethics education among faculty members and students. local setting hong kong, now a territory of china, was a former british colony from to and the british common law system continued after . british influence on the local medical practice and law also endures after the sovereignty handover to china. the current population of hong kong is about . million composed of chinese ( %). among the non-chinese ethnic groups, the majority are filipino ( %), followed by indonesia ( %), caucasian ( %), and others ( %) (the census and statistics department, ). compared with malaysia, hong kong is mostly a homogenous society. the medical council of hong kong is established under the medical registration ordinance handling the registration, professional conduct and discipline of medical practitioners in hong kong. the council also review the standard and structure of undergraduate medical education and training in the accreditation processes. in , the council updated the document "hong kong doctors", listing a set of essential attributes and skills in addressing the medical dilemmas and moral difficulties among local medical practitioners (the medical council of hong kong ). the chinese university of hong kong (cuhk) is one of the two local universities, providing a six-year undergraduate medical degree programme -bachelor of medicine and bachelor of surgery (mbchb). it offers comprehensive longitudinal doctoring curriculum to train physicians. at present, cuhk adopts conventional, studentcentred and subject-based curricula offered by the preclinical-clinical division. overview of the current medical curriculum the first three years of the mbchb curriculum encourage breadth and depth learning, offering pre-clinical studies focusing on interdisciplinary education in the field of humanities, social science, natural science. in addition to courses teaching fundamental biomedical courses relevant to medicine (e.g. public health, anatomy, physiology), students are required to courses humanities and language. before transiting to the clinical year, a six-week bridging course is in place to equip student pathology and clinical anatomy and clinical communications skills. the latter three years of studies focus on learning advanced biology and clinical medicine subjects, such as anatomical and chemical pathology, anaesthesia and intensive care, diagnostic imaging, clinical microbiology, medicine and surgery. students receive clinical exposure through a rich mixture of bedside teaching, simulation training, and community or laboratory research opportunities. courses for communication skills and professionalism are introduced throughout the entire six-year programme. the overarching objective of the medical curriculum is to provide an allrounded training of science and humanities (figure ). before the structured bioethics teaching rolled out, ethics was not yet implemented vertically and horizontally that sporadic ethics-related teaching took place on an ad hoc basis in the clinical years only. in , a workshop "integrated professionalism teaching session" was introduced for final year medical students as part of the induction towards independent clinical practice (joynt et al. ). it is a -h session comprises of lectures, small group discussions, and group presentations. the teaching topics broadly include the principles guiding professionalism, medical regulations and government ordinances. starting from , the quarterly clinical ethics grand round was also introduced in parallel in discussions about professional misbehaviours or ethical dilemmas in fig. coursework during the six-year mbchb curriculum at the chinese university of hong kong healthcare and life science. during the symposium, students and staff members are required to vote using a green-red card that encourages active and interactive participation. in this -h event, students identify key ethical issues that arise in the context of the case, discuss possible conflicting principles, and provide reasonable resolutions based on the four principles approach that underpins much of our teaching of ethics. with this framework, the teaching of bioethics in the medical schools was the focus in the clinical years only. the acquaintance of ethics knowledge in the pre-clinical year were less emphasised. starting from the mid- , the new bioethics curriculum is integrated into the curriculum, covering a wide range of ethical issues in biomedical sciences, clinical practice, public health, law, and policy. the teaching goal of bioethics education along three dimensions encompasses acquiring cognitive knowledge and behavioural skills and encouraging positive character development. besides, teaching modules are designed to raise ethical sensitivity and develop critical thinking skills while encouraging robust communication and interpersonal skills. in the clinical years, students can then link the ethical theory, knowledge, and examples taught in the preceding years for an application to the clinical context. table summarises the major themes of the curriculum. the topic-oriented approach in the pre-clinical year entails a teaching goal to ( ) observe inter-dependence between medicines and ethics, ( ) acquire bioethics knowledge and language, ( ) recognise vital ethical issues in medical practice, and ( ) apply reasoning skills when present in a dilemma. in the clinical years, students can then link the ethical theory, knowledge, and examples taught in the preceding years for an application to the clinical context. the section discusses barriers in the implementation of bioethics teaching from our experience, corroborating with literature. students' resistance to bioethics learning is a source of challenge. students, in general, believed that ethics is a 'soft' or 'fluffy' subject that lacks scientific rigour that could be reasoned based on common sensesocial and cultural normsthat does not require formal training (leo & eagen ) . malaysian and hong kong medical students, similarly, also regarded learning of ethics and medical humanities are of lower priority and prefer to spend more time learning clinical knowledge. ethics in medical education is sometimes regarded as a diversion from the core study of medical knowledge. at worst, ethics may be viewed as a tedious repetition of the obvious with an emphasis on procedural requirements for informed consent, protection of patient confidentiality, etc. less acknowledged is the unique role of bioethics in the development of core clinical skills such as critical reasoning, familial conflict management, navigation of discourse among professional peers, and understanding healthcare policy. the contemporary bioethics stems moral philosophy applied in research and healthcare contexts and the field has grown ever-present in medical curricula around the globe in an effort to produce doctors capable of navigating complex patient circumstances. however, given that most medical students gravitate towards hard science with its clear and testable answers, the theoretical-and humanities-based roots of bioethics reduce its importance in the eyes of pupils. another speculative reason is that students were uncertain about the need for bioethics in pre-clinical years, although they observe the vital role of bioethics for developing the professional identity. young medical students often neglected the importance of developing moral awareness for a future career during the time of learning 'skills' in early university years. interestingly, practising physicians observed the importance of bioethics when recognising their identity as primary caregivers as opposed to students (chan et al. ) . as young medical student progress to advanced years, they became interested in learning topics related to contemporary ethical issues and moral dilemmas that physicians faced in daily practice (aldughaither et al. ). the discrepant perceived view of bioethics education between young medical students and experienced practising doctors point out the need of a formal orientation about the instrumentality of bioethics training in early medical education years among students whose curricula in high school were predominately focused on mathematics, engineering and natural sciences. in other words, the influence of bioethics on professional identity construction has been largely ignored in reality. it is imperative to connect students with bioethics teaching such that relevance to the future career as a doctor is obvious rather than dismissed. there are studies in the united states, netherland, indonesia, or nigeria, reported that medical students witnessed unethical decision, unprofessional behaviour, inappropriate conduct by superiors or co-workers in early clerkship (muhaimin et al. ; okoye & nwachukwu ; seiden et al. ; yamey & roach ) . they, however, were uncertain about the resolution in preventing ethical lapses. importantly, implementing ethics training in the clinical years may be late, since students learn by being told what to do by senior doctors with limited opportunity to think and reflect critically (hren et al. ). bioethics training is not a one-time, but a longitudinal and reflective learning process in the career trajectory shaping a professional identity and cultivate a culture of humanism (fernandes ) . it needs to be implemented throughout medical practice years to help reconstruct correct values in medicine in the shifting role from 'layman or students' to 'physician' identity. when combined with traditional scientific learning, bioethics forms a critical component of comprehensive medical education, which has developed as the new international standard. to achieve parity with global education norms, bioethics teaching must adapt both to evolving learning styles and geographic realities. in the asia-pacific context, the euro-american dominance of teaching materials causes students to dismiss bioethics curricula as impractical. a lack of local contexts in the curriculum affects the long-term effect of learning. the framework should consider the deliberation of not only locally specific systems and events but also cultural belief systems and philosophical foundation of the society. modern bioethics which is developed in western philosophies might not adequately provide tools to address ethical complexity in clinical practices of ethical and religious diversity (chattopadhyay et al. ; fuscaldo et al. ) . the contested notion of cultural relativism in pluralistic societies as a response to the conventional belief of universality of morals needs to be discussed in bioethics education (sokol ) . the topic of public health is a great example illustrating the need to respect cultural difference from our experience. for instance, in the us context, the use of restrictive measures (i.e. quarantine) in response to infectious disease epidemics was disputable, as it is perceived to violate individual freedom for population good. this contrasts with hong kong, where citizens experienced the sars epidemic in , and using quarantine to monitor individuals for the 'greater good' was found reasonable from the local perspective (blendon et al. ) . in malaysia, the recent examples of covid- challenge the right in a religious gathering while implementing individual movement's restriction (yezli & khan ) . our recent transcultural experience introducing local topics cases has resulted in more lively discussions, with students more willing to express their observations and opinions on topics of bioethical relevance. for example, the discussion of imposing a mandatory influenza vaccination program for healthcare workers is somewhat less challenged in the us (babcock et al. ) . still, it remains unsettled in malaysia and hong kong, which results in a lively debate in the class (hudu et al. ; so et al. ) . these examples demonstrated that there is no unified approach to ethical problems, and responses are the result of a complex mix of moral and value-related issues turner ) . a study also discussed that incorporating cases that are relevant to the local medical systems helps cultivate bioethical responsiveness among the students . our learning experience is that placing local contexts as equally as western bioethics enhance the quality of a participatory bioethics teaching in our institutions and it should be done clearly at the design phase of the curriculum. one of the common challenges in the implementation of a bioethics curriculum in a resource-constrained context is a lack of teaching capacity (sim et al. ; ten have ) . teaching bioethics requires expertise both in ethics and medicine, and universities, nonetheless, have a limited ability to provide professional training of ethics and moral philosophy to sustain teaching quality. in the university of malaya, there is only a retired o&g specialist with a law degree, who is well-qualified to teach bioethics. other faculty members involved in bioethics teaching are mainly clinicians with some exposure to ethical issues in clinical practice but not trained specially in bioethics. in other malaysian medical schools, bioethics teaching is majorly lead by healthcare professionals or a multidisciplinary team of scientists, lawyers, and ethicists (sim et al. , bilgin et al. . in hong kong, many teachers who are involved in bioethics curriculum in medical programmes were either philosophers, ethicists, or scientists with limited experience in medicine, or clinicians with some training in bioethics (rothchild & holmquist ) . to address limited teaching capacity limitation, the faculty development team shall progressively organise workshops to provide training to faulty with little ethics background on areas such as how to integrate bioethics teaching into clinical postings. placing local contexts as equally as western bioethics will enhance the quality of a participatory bioethics teaching in asia, and it should be done clearly at the design phase of the curriculum. to overcome limited teaching resources, the success of the collaborative learning activities depended upon the training of local faculty who were responsible for leading the teaching. indeed, the concept of "training of trainers (tot)" has been widely implemented globally in the fields of health education by international institutions (e.g., un agencies) and major universities in developed countries in resource-limited settings, mostly developing countries (ewert et al. ; world health organisation, ) . unesco offers "ethics teachers' training courses (ettc)" in developing countries as part of its bioethics professional capacity building programme (ten have ; langlois ) . while tot can be effective to introduce the change in the place of intervention by teaching individuals, providing basic resources and implementing an initiative programme, another level of local resources is necessary to achieve sustainability (mormina & pinder ) . thus, the long-term effect of the bioethics curriculum requires the local commitment and leadership as resource-limited academic settings need more collaborative, participatory learning through interaction with teachers and peers in class (avci ). this paper described two evolving experiences in implementing bioethics teaching within existing institutional capacity. both medical schools are unique and very different in terms of the curriculum, and common challenges include curriculum design, students' resistance to bioethics teaching, and limited teaching capacity, were observed as the curriculum evolve. the reported experiences revealed that there 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the face of political, economic, social and religious challenges publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions jhs conceived the study. omyn and jhs drafted the manuscript. omyn made substantial edits on the writing. omyn and jhs approved the final manuscript.data availability not applicable. competing interests all authors declare no conflict of interests.code availability not applicable. key: cord- -ez ke of authors: moxon, nathaniel r.; goyal, anju; giaconi, joann a.; rosenberg, jamie b.; graubart, emily b.; waxman, evan l.; knoch, daniel; forster, susan h.; sankar, privthi s.; mirza, rukhsana g. title: the state of ophthalmology medical student education in the united states: an update date: - - journal: ophthalmology doi: . /j.ophtha. . . sha: doc_id: cord_uid: ez ke of nan the state of medical student education in ophthalmology eye disease and vision loss worsen quality of life, independence, mental health, social function, and mortality. [ ] [ ] [ ] visual impairment affects . % of the u.s. population and requires frontline providers to diagnose and manage eye conditions. , however, the time dedicated to teaching medical students about these conditions has been on the decline for half a century. ninety-five responses were collected on google forms ( % response rate). our results revealed four main themes. first, the decline of ophthalmology's role in medical student curricula has slowed, if not plateaued. second, ophthalmology teaching has shifted to the preclinical years. third, ophthalmology has compensated for its decline in the traditional curriculum by teaching knowledge and skills outside the regular curriculum. finally, programs with dmses have higher rates of both required and extracurricular exposure, and higher rates of faculty engagement with medical students. each of these findings raises further points of discussion regarding how to shape ophthalmology curriculum, within models of medical education that are constantly changing. the first trend seen in the new data demonstrates how the decreasing curricular time for ophthalmology has slowed, if not plateaued, particularly since the last survey in . we found that required exposure to ophthalmology during medical school in was very similar to where it was - years earlier, despite the rapid decline of the required clinical ophthalmology clerkship noted in decades prior to the study. in , required clerkships existed at % of responding institutions while required preclinical coursework existed at % of institutions compared to % and % respectively in . these statistics indicate a much slower decrease compared to that over the decade prior. of note, this plateau has occurred after competence of graduating medical students was already compromised. by the time medical students graduate, an estimated % are not able to properly use an ophthalmoscope. further, primary care residency program directors report that the state of medical student education in ophthalmology most of their residents do not meet aupo standards for ophthalmic skills. , while % of schools did have some required ophthalmology training, medical students in % of medical schools have no required exposure to ophthalmology training in skills or clinical ophthalmology exposure unless it is sought by individual students. the question that remains is whether the current number of ophthalmology hours and teaching methods during medical school are sufficient to produce competence in graduating medical students. the second theme we identified is that ophthalmology teaching has shifted to the preclinical years. the vast majority ( . %) of institutions included some form of required exposure to ophthalmology during medical school. most institutions ( . %) required preclinical coursework only, . % required both preclinical and clinical exposure, and . % had clinical exposure only. preclinical coursework was . hours on average (median= ; n= ) but varied from . to , with one outlier at . lecture was the most common way to teach ophthalmology, followed by skills training and problem-based learning. in terms of clinical exposure, only % of schools required clinical rotations, half of which were embedded within another course. on the other hand, clinical electives, typically four weeks in length, were ubiquitous, and % of institutions also offered ophthalmology as an option during the surgery rotation (see table online). the third theme relates to non-traditional teaching, which has allowed schools to maintain exposure to ophthalmology. as an increasing number of institutions transition to shorter preclinical time, ophthalmology will have to continue to creatively reinvent itself in new curricula alongside all other specialties condensed into the same period. we have already seen this occur, as ophthalmology has compensated for loss in the traditional curriculum by teaching knowledge and skills in other ways. many institutions report activities outside of the curriculum the state of medical student education in ophthalmology for medical students, such as interest groups ( %), medical student advisors ( %), career fairs ( %), and outreach or community service experiences ( %). more recently, our experience with covid- shows that virtual electives can be developed to teach content to medical students. efforts creating shared resources such as those on the american academy of ophthalmology medical student portal are essential, especially when circumstances demand remote learning. a final trend observed in the data highlights a novel discussion related to designated medical student educators. programs with dmses have higher rates of both required and extracurricular exposure, and higher rates of faculty engagement. most aupo institutions had a dmse ( %), under half ( %) of whom had funding for such a position. institutions with a dmse offered more preclinical coursework ( . versus . hours), had more students opting into electives ( versus ), and had higher rates of faculty engagement and extracurricular exposure. there was a significantly higher likelihood that institutions with a dmse actively engaged medical students outside of the curriculum (p= . ), had an ophthalmology interest group (p= . ), or had a formal student advisor in the department (p= . ) (see table online) . the dmse designation may be a marker for preserved ophthalmology education efforts. the pipeline of students entering ophthalmology is also germane to this discussion. research has uncovered that racial and ethnic disparities in eye care exist. underrepresented minorities and women continue to make up a minority of the ophthalmology workforce despite an increasingly diverse pool of medical students. medical student exposure to ophthalmology during medical school are the first steps to integrating potentially interested students into this subspecialty. the state of medical student education in ophthalmology the survey results showing that ophthalmology is still required at a large percentage of schools are encouraging. medical student exposure to inspiring ophthalmologists is the best way to teach the vital ophthalmology skills required of most physicians. medical education is changing rapidly, and ophthalmology must be an active participant in this change. eye health needs to be a population health longitudinal relationships among visual acuity, daily functional status, and mortality: the salisbury eye evaluation study committee on public health approaches to reduce making eye health a population health imperative: vision for tomorrow age-related eye diseases and visual impairment among u.s. adults ophthalmology teaching in medical schools medical student education in ophthalmology: crisis and opportunity the state of ophthalmology medical student education in the united states and canada ny: carnegie foundation for the advancement of teaching, . the state of medical student education in ophthalmology assessing the status of ophthalmic education years after the unpublished survey of us ophthalmology departments. oral presentation at: association of university professors of ophthalmology annual meeting evaluating medical students' proficiency with a handheld ophthalmoscope: a pilot study stern ga. teaching ophthalmology to primary care physicians. the association of university professors of ophthalmology education committee nonmydriatic ocular fundus photography in the emergency department vision health disparities in the united states by race/ethnicity, education, and economic status: findings from two nationally representative surveys current and future status of diversity in ophthalmologist workforce a proposal to improve ophthalmic education in medical schools ophthalmology and specialty education for medical students teaching doctors about the eye: trends in the education of medical students and primary care residents is there more than one type of elective? how many weeks is it? how much time is spent in clinic? how much time is spent in the or? how many students take it each year? do you have a research elective?how long is it? how many students take it each year? are there other opportunities for medical students to get involved in research? do you have any equipment that aids in teaching? if so, what equipment? do you use the aupo guidelines in assisting the writing of your ophthalmology curriculum? is anyone in the department a member of the schools curriculum, retention, or other committees?is anyone on specifically the curriculum committee? are you doing anything outside of the curriculum for medical students? do you have a freecare/outreach/community service experience for students? is there an ophthalmology interest group? do you have career fairs? do you have student advisers in the department? are there additional opportunities (non-committee, non-curriculum) that can provide faculty opportunities to work with medical students (i.e. medical student mentoring programs, cv workshops, etc.)? if yes, please specify: key: cord- - vpu xq authors: wu, anette; leask, betty; choi, edward; unangst, lisa; de wit, hans title: internationalization of medical education—a scoping review of the current status in the united states date: - - journal: med sci educ doi: . /s - - - sha: doc_id: cord_uid: vpu xq this article explores the current status of internationalization of medical education in the united states. dominant themes of articles published from to indicate that common formats are institutional partnerships, international learning at home, and student mobility programs. critical analysis on the basis of internationalization of higher education, recommendations, and future perspective is given. in the last years, the internationalization of higher education (iohe), which includes internationalization of the curriculum, has emerged as a dynamic field of study supported by a rich body of scholarly literature and research. the consensus is that internationalization of the curriculum, referring to the incorporation of international, intercultural, and global dimensions into the curriculum in ways that are relevant to graduates' professional practice [ ] , is important for all students. while motivations for internationalization of individual higher education institutions are varied, including quality improvement, provision of access, competitiveness, growth, and financial profits [ ] , the provision of a professionally relevant education that prepares all students to be interculturally proficient professionals and citizens is the responsibility of the disciplines [ ] [ ] [ ] . in medical education, this might mean, for example, developing students' ability to access and evaluate new ideas and practices from diverse national and cultural sources, using advanced intercultural skills in their professional practice, and practicing medicine in a global context. recent global health events, namely the ebola virus, sars, or covid- pandemic, have demonstrated the need for efficient international collaboration and communication in biomedical research, education, and patient care. such global health emergencies require efficiency in international communication, culturally competent and expert healthcare leadership (locally, nationally, and internationally), rapid international public health action, and collaborative international biotechnology and medical science research. today, more than ever before, these elements are not optional choices but rather represent essential components that should be included in medical education curricula in all parts of the world. in this context, it is important to take stock of where we are and where we might want to go next in the internationalization of medical education (iome), using research that has been undertaken into iohe curriculum, teaching, and learning, tailored to the needs of the medical profession. the authors argue that this offers a useful way forward because it focuses attention on preparing all medical graduates to meaningfully contribute to society as professionals who practice locally but are also global citizens. to date, iome is a broad term with no formally agreed upon definition. for the purpose of this paper, we utilize a definition commonly used in research on iohe and apply it to medical education. thus, we define iome as the process of purposefully integrating international, intercultural, or global dimensions into medical education in order to enhance its quality and prepare graduates for professional practice in a globalized world. this definition is consistent with research-based and frequently cited definitions of higher education internationalization [ ] , internationalization of the curriculum, and internationalization at home frequently cited in the literature on higher education [ ] [ ] [ ] . it constructs iome as a concept that includes intentional, systematic, and evidence-based activities designed to ensure students achieve specific learning outcomes through engaging in high-quality learning experiences. it positions iome as a means to a specific international educational goal, not a goal in and of itself. educational goals and competencies for iome overlap with other subject matter taught in medical school (i.e., cultural competency). in recent years, public health (ph) and global health (gh) have become an important part of medical curricula, incorporating elements of social equity, diversity, inclusivity, and cultural competence into medical education. in order to differentiate these different terms, short definitions are provided below. ph is regarded as "…the science and art of preventing disease, prolonging life, and promoting health through the organized efforts and informed choices of society, organizations, public and private communities, and individuals…" [ ] . gh as its global counterpart historically evolved from international health-an area that addresses local, national, and international health concerns on all levels [ ] . while both terms still exist, gh was defined by koplan in as "…an area for study, research, and practice that places a priority on improving health and achieving health equity for all people worldwide…" [ ] . based on koplan's initial definition, others have proposed a shorter definition-"collaborative trans-national research and action for promoting health for all" [ ] . there is still ongoing debate on a commonly agreed upon definition [ ] and there is inconsistency in the way medical educators use the term gh. gh is frequently conceived as a multidisciplinary field of service, research, and education undertaken as part of an altruistic "global north to global south" collaboration, and/or as part of a national/local health equity program. its primary goals-as it oftentimes appears in published work-are to improve the health of underserved individuals and populations, and to achieve health equity for all people worldwide, with a primary focus on the resource-poor abroad [ ] . for medical education, gh education programs in the in low-and middle-income countries (lmics) are frequently used to report on the internationalization of the medical curriculum-mostly to describe extracurricular activities or projects in lmics or the underserved populations. thus, the enactment of gh in medical education programs appears to be narrower and more limiting than the definition of gh itself suggests. while there is overlap with the broader definition of gh, iome should not be equated with gh. gh-as the broader term suggests-includes the vision of improvement of health for all people worldwide. in contrast, iome is an area of educational science, an educational concept, a framework, and a means to develop international and intercultural learning outcomes in all students. despite their value as a means of internationalizing the learning outcomes of some students, gh programs-particularly its narrower term-should not be a proxy for the modern concept of iome. goals and outcomes set by gh (i.e., the improvement of health for all people worldwide) are considered the ultimate goal for iome. thus, iome is a means to achieving this ultimate goal, and includes the acquisition of cultural competency and knowledge about health issues worldwide. iome encompasses the international collaborative goals and dimensions between nations and does not solely focus on cultural differences. iome can play an important role by preparing future physicians to practice medicine in a global context and laying the framework for international collaboration and understanding of differences among nations. iome includes, but is not limited to, learning about and understanding differences in international healthcare education, healthcare delivery systems, health economics, health ethics, and health laws; building international peer networks; and providing future physicians with skills in intercultural competencies, collaboration, and international leadership. iome can therefore improve the practice of future physicians by purposefully developing students' understanding of international social, cultural, economic, and ethical differences in healthcare, and by supporting better communication, fostering international leadership, collaboration, and understanding. iome creates awareness building of the importance of these differences regarding patients and healthcare, and creates physician feelings of being part of a global medical world. one can argue that iome highlights meaningful differences between individual nations with the goal of international understanding and improvement in healthcare, whereas gh issues transcend individual nations. iome is a means to improve gh, leading to a globalized and better healthcare world. hanson [ ] describes three different models of iome drawing on literature from to . the "market model" focuses on competition for students internationally and positioning of academic institutions in the global market. hanson argues that this approach, focused on revenue growth, market consolidation, and fiscal returns rather than educative outcomes, typifies the us approach to internationalization. in contrast, the "liberal model" focuses on the promotion of international and intercultural understanding and cooperationintroduced for international higher education in the post-wwii era. curricula in this model might prepare students to work in international or diverse cultural settings, or to be future global collaborators through exchange. the third "social transformation model" emphasizes cross-cultural understanding operationalized "in a spirit of mutuality and reciprocity, through networks or partnerships." hanson argues that this model is the one most closely aligned with approaches to current international practices in medical education. iome necessarily involves multiple players-students, faculty, medical professionals, medical educators, administrators, institutions, and governments [ ] -and includes curricular components such as content, pedagogy, assessment, and learning outcomes. the key players include students, for whom iome may involve didactic lectures in the classroom ("iome at home"), peer-to-peer connections and networking, intercultural experiences in clinical settings, outbound mobility activities, and inbound mobility including international student recruitment. other key players are faculty, for whom iome involves research, teaching, and service. and finally, the academic institutions as major stakeholders, who play a significant role in initiating and orchestrating these international endeavors. stutz et al. [ ] highlighted the paucity of contextualized national studies on the topic of internationalizing the curriculum in medicine. this is concerning, given the myriad varieties of professional practice, and the impact of our globalized world on local medical practice. the overall goal of this study was to evaluate the current status of iome in the united states (us) by capturing scholarly work on iome in the published literature. the aims were to distill what is being reported and how, and to assess whether more educational research in this area is needed. identifying gaps and limitations in reporting can help iome moving forward as an area of investigational research for the medical educator community. the authors addressed the following question: "what is the current status of scholarly work on the internationalization of medical education in the united states?" intentionally, the authors avoided searching non-scholarly work such as websites or surveyed institutions for international educational curriculum elements or programs. in order to find a way forward, this article summarizes usbased reports on programs, including gh programs at medical schools, discussed over the last years in peer-reviewed journals. the authors focus their discussion on didactics and formats of programs and reasons for gaps in the literature. it provides a picture of the evolving conceptualization and practice of the internationalization of medical education as it is commonly understood and compares this with contemporary approaches to iohe curriculum that have evolved over a similar period of time. the comparison informs suggestions for future approaches to iome. a scoping review was undertaken to identify what has been published on iome in us medical schools. the purpose of this scoping review was to provide an overview of current approaches. the review was conducted in several steps [ ] : identify the research question, identify relevant studies, study selection, chart the data, and summary of results. the research was undertaken to answer the question "what is the current status of scholarly work on the internationalization of medical education in the united states?" a scoping review was conducted using a literature search in medline, pubmed, google scholar, and ebscohost. searches targeted the date range from january , , to august , . this task was performed by an interdisciplinary team consisting of a medical educator, researchers in internationalization, and research assistants in international higher education, from diverse linguistic and cultural backgrounds, working at two major us institutions. the search included keywords, titles, and abstracts of studies from the databases listed. the search terms/keywords or phrases used for this review were as follows: "international," "medical," "education," "united states," the boolean operators "or" and "and" were used to expand and narrow the searches to include all the pertinent publications within the period indicated above. the following limitation was encountered when using a combination of these keywords. for example, a search in pubmed exceeded , articles. to narrow down the results, an advanced search included the term "curriculum," resulting in over , . since this did not represent a significant reduction of results, the keywords "medical school" and "training" were added. albeit not a term the authors were intending to include initially, "global" was added, resulting in about , results. adding "usa" or "united states" resulted in about , hits. combining with "internationalization" and "internationalisation," the search resulted in or articles respectively. none of the articles was deemed appropriate for the intended search as they included other health professions or were not relevant for medical education. given the paucity of the initial search pairing, the authors decided to focus on scanning titles and abstracts. overall, searches using the keyword combinations below were effective in returning a moderate quantity of articles with topic relevance. the authors avoided the term "global health" in the search ( ) to not limit the searches to global health education, and ( ) to not mix key terms that have different meanings. when hand-searching individual articles, crossreferencing articles that were not found in the initial search were added to supplement the list. pubmed-the keywords "international and medical and education and united states" resulted in a total of articles using a filter for journal articles inclusive of reviews and systematic reviews. a title and abstract search was then conducted. similar to the pubmed searches, the queries in google scholar, ebscohost, medline, and web of science combining the keywords returned too many results. this limitation was addressed by narrowing the search parameters to scanning article titles, which provided published works with a primary focus on iome. the purpose of our scoping review was to provide an overview of current approaches to iome. as iome as a research field itself is not formally defined and still evolving, modification and adjustment of keywords used in the searches were necessary in order to capture all relevant articles. based on a title and abstract scan, the relevant studies were compiled into a master spreadsheet of non-duplicate articles. this spreadsheet including title and abstracts was reviewed independently by the authors (a.w., e.c., l.u.) and as a group using a set of inclusion and exclusion criteria. criteria for inclusion comprised any articles that reported on international activities and/or programmatic efforts for us medical students. those included, but were not limited to, journal articles of ( ) original research, ( ) case reports, ( ) reviews and systematic reviews, ( ) descriptive articles, and ( ) program overviews. exclusion criteria were as follows: ( ) articles written in non-english language, ( ) articles about programs in non-us institutions (unless collaborating with us institution on iome, ( ) articles describing international postgraduate (residency) training, ( ) reports on international students at us medical schools or foreign medical graduates, ( ) international comparisons of medical curricula, ( ) opinions/oped on iome, ( ) work reporting on offshore us academic institutions or us institutions supporting other nations in building medical curricula/schools, ( ) studies from other allied health professions (nursing, dentistry, public health, pharmacy, social work, emergency medicine technician, biomedical sciences, or veterinary medicine), and ( ) reflective pieces, book reviews, or commentaries. this narrow screening was deemed important to identify relevant articles on iome and to answer the research question on the current status of, and approaches to, iome in the us. cross-references in identified review articles were only included when deemed appropriate for the purpose of the study. based on this screening, the authors indexed each article according to the extent to which it met the inclusion/exclusion criteria, resulting in articles selected for intensive study. a total of articles over a span of years were identifiedsorted by author, publication year, location, sample, method, major findings, discussion points, and limitations ( table ) . despite this relatively low count, we found a diversity of internationalization efforts described, including narratives of individual international experiences [ ] , research on student initiatives [ ] , description of single school programs [ , , , ] , institutional partnerships [ , , ] , cultural learning at home [ , ] , reviews on school websites [ ] , and international gh programs [ , ] . as the goal of the study was to determine the current status of iome in the us via viewing of published literature, the authors identified three overarching themes that summarized the articles, to help in identifying current practices in iome-institutional partnerships, international learning at home, and student mobility programs. definition of each category below is based on descriptions found in iohe [ ] : institutional partnerships articles that described programs in which us academic institutions and medical schools partnered with one or more schools or programs outside of the country to provide elements of iome to medical students were listed in this category. in the majority of the articles, these partnerships were formed with low-and middleincome countries (lmics). internationalization at home studies that contained information on international learning and exposure of medical students to either global content or international courses or formats within their school curriculum (at home) fell into this category. studies that included students' pre-departure training for site visits were included. student mobility programs programs describing study abroad, international medical service missions, clinical electives, or short-term rotations were included in this category. many, if not the majority, of the programs described student outbound mobility. while often these studies were part of activities organized via institutional partnerships, some that report trips that were self-organized by students and individual faculty members are included. with few exceptions, most described reports focused on partnerships between us schools and institutions in the lmics, the majority in south america and africa [ - , , , , , , ] , and some in south east asia [ , ] . rybak described a partnership with the ukraine [ ] , and there were two reports of the same partnership with israel [ , ] . the described partnerships with lmics originated from only nine us medical schools, with some schools reporting on multiple occasions, such as brown university or suny [ , , , , , ] . the report by finkel et al. in was the only article found that reported on partnerships solely with european schools [ ] . ip et al. do not mention formal partnerships with japan and germany, but student exchanges at brown university were administered via an official exchange program; so there is an assumption that a partnership was in place [ ] . international institutional partnerships appear to be a common activity associated with the iome, although partnership strength and impact varied greatly. at one end of the scale; formal institutional partnerships were reported "for show" [ , , , ] , while at the other end of the scale, they are mentioned as part of other reports on iome activities without further details [ , , ] . overall, there was a full range of number of partners reported, including schools partnering with multiple partners [ , , , ] , whereas others report on one specific partnership only [ , , , , ] . while many more international partnerships may exist, they appear not to description of a global health program for medical education be utilized or recognized as vehicles for the iome or are simply not reported in the literature. several articles discussed international learning at home including descriptions of on-campus didactics. the articles were wide-ranging, including descriptions of full gh programs, individual courses, local student involvement with refugees, or preparation for travel [ - , , , , - , , , - , , , ] . two articles were reviews on international gh programs and one an overview of international school activities. drain [ ] suggested including gh education (e.g., learning about tropical disease, different cultures) in medical school curricula and/or creating a gh track for interested students. in seven articles, pre-departure training and activities were mentioned [ , , , , , , ] . in one article by vora [ ] , the international student travels were part of a requirement of an at home international elective. furthermore, we found nine articles addressing or mentioning cultural competencies and language education as part of their international efforts at home [ - , , , , , , ] . of these, two are review/summary articles [ , ] . griswold described in two reports the involvement of medical students working at home with international refugees [ , ] -obtaining cultural competency through this experience. we found a much narrower interpretation of the iome at home than that found in the broader scholarship. most articles focused on preparatory training for students prior to travel to a lmic as part of a gh program and failed to report on international or intercultural learning objectives or outcomes of these programs, which medical educators agree should be specific and measurable [ ] . thus, while we found evidence for internationalization at home in the peer-reviewed articles we reviewed, it was clearly connected to gh-related student mobility programs and did not engage the majority of students. student mobility, with travel duration ranging from week to several months, involved in almost all cases a low-and middle-income country (lmic). although it is often advocated from work in other countries [ ] , we found limited reporting on pre-departure training in the us [ , , , , , , ] . ramsey describes pre-departure training but only as a cross-reference for a program description [ ] . furthermore, we identified case reports [ ] , summaries [ ] , reviews [ , , , ] , and follow-up studies or reviews that study long-term outcomes of these international experiences [ , , , , , , , , ] . the followup studies focused on effect on career choices and postgraduate international work of the graduates. at least two articles summarized reviews of gh programs [ , ] . one article reported on the iome not specific to gh programs [ ] . finkel [ ] described the only article that addressed a focused comparative component of international healthcare systems as an educational goal for students to study while aboard. with very few exceptions, our research demonstrated that us-focused literature regarding international experience during medical school appears limited to work contexts in the lmics. we found limited evidence in the literature of us medical programs engaging with high-income countries (hics) for the purpose of internationalization of the curriculum, teaching, and learning. although historically outbound student exchanges in medical education involved the european countries [ ] , only five reports involved student mobility to european or japanese hics [ , , , , ] according to our search. vora [ ] includes european countries such as armenia and the czech republic. in one case, where international placements included australia, students worked specifically with indigenous populations in remote areas [ ] . in general terms and overall, our review found a range of literature on the theme of student mobility, from individual small program reports to comprehensive reviews of large gh programs. this suggests attention to both micro and macro detail in considering the impact of gh programs, and a strong emphasis on mobility programs for a minority of students. the articles were published in a wide range of medical journals of relevance to various health professionals, suggesting broad interest. some groups and institutions were represented multiple times in our pool of literature, perhaps reflecting uneven interest in these themes across us institutions. there was a general consensus that these activities have a positive impact for student learning [ , ] . furthermore, most articles addressing student mobility did not go beyond students' personal perceptions of the impact of their experience on their learning, which is necessarily subjective. this may be a result of no scientific metric of international/intercultural competency being recognized by medical educators [ ] . the purpose of this review was to assess the current state of iome in the us by looking at its presence in published scholarly work. in today's world, and for the foreseeable future, graduates of medical programs will require international and intercultural perspectives to perform both their professional and civic duties. while scholarly research on the development of all students' international understanding and intercultural skills in professional programs of higher education has increased dramatically in the last years, the present review of the literature on iome in the us did not find a commensurate rise in published peer-reviewed articles or research on this topic in medical education. a number of themes were identified characterizing the current state of scholarship regarding iome in the us. some of them deserve more attention going forward. overall, we found a focus on program descriptions in the majority of articles with few discussing research into the learning outcomes and goals achieved. the reports were classified into three main categories found in iohe (i.e., institutional partnerships, the internationalization of medical education at home, and student mobility). the significance of the findings of the studies reported was limited because they considered outputs from only a few medical schools and were lacking in comparative data from related professions, and/or for not having a focus on standardized goal settings and outcome research. there was little evidence of structured learning regarding differences in healthcare delivery systems between nations. a number of missing elements and gaps in the three areas of reported activity were identified. these and some possible improvements that could be made are discussed below. first, in relation to institutional partnerships, the authors found no descriptions of how institutional partnerships were used to facilitate peer connections between students and between faculty respectively, or how technology was used to facilitate students at home interacting with students, researchers, or professionals abroad. only one paper reported that students connected via videoconferencing [ ] . this is consistent with approaches to international partnership engagement in professional programs outside of medicine, and is recognized in the literature on internationalization of the curriculum as a lost opportunity [ , ] . reports on how to build a larger global medical community using modern technology and social media led by institutional partnerships will be of utmost importance in the time to come-particularly given recent global health events. as with other areas of online education, existing technology needs to further be embedded and expanded in medical school curricula given the limited reports the study shows. second, the commonly described partnerships with lmics limit students' ability to appreciate the full spectrum of healthcare systems and collaborative opportunities worldwide, and restricts them from acquainting themselves with other hics-thus preventing them from including the "best of each world" in their future practice. a more balanced combination of partners from different areas of the world is suggested. however, the authors also advise against connecting with too many partners, given experiences shared from iohe [ ] . third, according to the published reports reviewed, internationalization at home has not been enacted to reach its full potential in medical schools in the us. by comparison, in other disciplines and professional programs (particularly, in the social sciences), there is a stronger focus on identifying international and intercultural learning outcomes for all students, and on how to teach and assess these outcomes at home [ , , ] . given that iome is an emerging concept in medical education, it may be that there is more evidence of internationalization at home in conference papers and institutional reports. however, scholarly work on this topic is of high importance so that educators can standardize goals, research outcomes, agree on best practices, and share ideas and work. the authors suggest that medical educational societies (e.g., the american association of medical colleges, aamc) take the lead and set the framework to encourage sharing this work. finally, student outbound mobility in us medical schools-representing the main theme for reports on iome-appears to be reported when it involves the lmics. in contrast, other industrialized countries, mainly european, more commonly report outbound student mobility and related activities in other hics [ ] . this may be due to entrenched european medical student exchange programs which have been in existence for several decades, stimulated and supported by the erasmus program of the european commission [ ] and others. the authors advocate increasing bilateral exchange between the us and other hic to be more inclusive for building a global medical world. while mobility programs are an important aspect of internationalization, they are on their own insufficient. global estimates reflect that at most % of students will engage in mobility programs. furthermore, the international literature recognizes that students from marginalized backgrounds are less likely to participate in these programs. approaches to internationalization that focus primarily on mobility are likely to exacerbate rather than address recognized inequalities in medical education and the practice of medicine [ ] . another area that is not well researched is medical students (often from the aforementioned marginalized backgrounds) who obtain part or all of their medical training in the caribbean and move to the us for medical practice. these students and their experiences in the lmics provide diversity, an important component for iome at home [ ] [ ] [ ] [ ] . however, these students are not captured within reports about iome. likewise, in this report, studies on international students were not included, to focus solely on reports about offerings from us medical schools. the authors argue that the reported approaches for student mobility as an element for iome are insufficient and call for an internationally informed approach to iome that focuses on all students and medical educators. furthermore, during a time of discussion about climate change, increasing ethical sensitivity, and sustainability of global resources, it is important to take a step back and rethink current existing formats for student mobility as a means for iome. particularly in the wake of the pandemic, medical educators need to evaluate if medical students' outbound short-term missions to the lmics is ethical, safe, and justified, and whether goals and outcomes achieved from these missions can be acquired via other alternative routes. the authors argue that optimizing internationalization at home may be a suitable alternative and should be the focus of further research. the very limited number of published articles on comparative international health systems requires further investigation. while us schools of public health offer these fields of study, most medical students apparently have no access to acquiring knowledge about this topic. for future collaborative international work, this information is deemed important by the authors. considering that medical school curricula generally leave no space for the inclusion of additional coursework, medical educators need to come to a decision regarding what is deemed necessary in the future. furthermore, in the us, medical educators are mostly practicing clinician-researchers with little or no international professional experience and little time for curriculum review and innovation. unless medical educators, as a community, identify and articulate a strong need for the internationalization of medical education for all students, it is likely that it will continue to be regarded as an extracurricular, optional activity for an elite minority. we found little connection between the iome in the us and the broader literature on iohe and the curriculum. educational research is an acknowledged field of study in its own right, as is internationalization. although % of us medical schools have established internationalization programs, we identified a relatively small number of peerreviewed articles on the topic over a span of nearly years [ , ] . lack of research and publication has a significant impact on exchange of experience, programming efforts, and quality improvement. increased communication among us and international peers via publications and conferences can have a positive impact on medical education and is important for the future of the iome. the limited publication of peer-reviewed articles on the internationalization of medical education may not be a reflection of the iome in the us occupying a low priority within the medical educator community, but the consequence of unreported programs and experiences. while clearly international options are available at most us medical schools, the lack of reporting found in this study warrants further investigation and research [ ] . considering that international programs and activities involve a significant amount of resources, an analysis of financial impact or studies on cost efficiency would appear to be important but were not easily found [ ] . at a time of growing globalization of healthcare, the internationalization of medical education can help in improving the global healthcare world. despite a high proportion of international offerings in us medical schools, this study demonstrates that very limited sharing of experiences via published work in this field exists. the reported literature details three areas of medical educational research consistent with research in international higher education: institutional partnerships, internationalization at home education, and student mobility. all three areas will benefit from further investigation and expansion to promote internationalization of medical education for all students. these improvements should be investigated in an interdisciplinary manner in collaboration with the longstanding field of international higher education, thus leading 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cultural exchanges in europe medical education in the caribbean: a longitudinal study of united states medical licensing examination performance medical education in the caribbean: quantifying the contribution of caribbean-educated physicians to the primary care workforce in the united states the medical education of united states citizens who train abroad -citizen international medical graduates-a boon for the workforce? publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements the authors would like to thank laura rumbley, phd, at the european association for international education (eaie) for her helpful discussions. many thanks to michael fortgang, md, for helpful review of the manuscript. conflict of interest the authors declare that they have no conflict of interest. key: cord- -mtl x gz authors: leung, t. i.; pendharkar, s. s.; chen, c.-y. a.; snyder, r. title: physician suicide: a scoping review to highlight opportunities for prevention date: - - journal: nan doi: . / sha: doc_id: cord_uid: mtl x gz objective: the aim of this scoping review is to map the current landscape of published research and perspectives on physician suicide. findings could serve as a roadmap for further investigations and potentially inform efforts to prevent physician suicide. methods: ovid medline, psycinfo, and scopus were searched for english-language publications from august , through april , . inclusion criteria were a primary outcome or thesis focused on suicide (including suicide completion, attempts, and thoughts or ideation) among medical students, postgraduate trainees, or attending physicians. opinion articles were included. studies that were non-english, or those that only mentioned physician burnout, mental health or substance use disorders were excluded. data extraction was performed by two authors. results: the search yielded , articles, of which articles passed to the full-text review round. the oldest article was an editorial from ; ( . %) articles were published from to present. authors originated from countries and ( . %) were opinion articles. most discussed were suicide risk factors and culture of practice issues, while least discussed themes included public health and postvention. conclusions: consistency and reliability of data and information about physician suicides could be improved. data limitations partly contribute to these issues. also, various suicide risk factors for physicians have been explored, and several remain poorly understood. based on this scoping review, a public health approach, including surveillance and early warning systems, investigations of sentinel cases, and postvention may be impactful next steps in preventing physician deaths by suicide. physician suicide is a significant problem for the medical community and general public and is poorly understood, suggesting that important knowledge and implementation gaps towards prevention remain. to address this gap, this literature review aims to describe the state of current knowledge and research on physician suicidal behaviors among medical students, postgraduate trainees, including residents and fellows, and physicians. suicide is estimated to occur at a higher rate among physicians than the general population and perhaps even other professions ( , ), however, estimates by profession vary ( ). several medical organizations have begun launching various initiatives to address physician well-being, yet efforts to address physician suicide remain organization-or institution-specific. the aim of this scoping literature review is to map the current landscape of published research and perspectives on physician suicide. findings could serve as a roadmap for informing further study, evidence-based policy, and interventions to prevent physician suicide. differs from other reviews such as a systematic review which gathers and assesses the quality of quantitative evidence to report on the effectiveness of a particular intervention in achieving a certain outcome ( ). the research question was broadly designed to gather and analyze articles that mention physician suicide. no date range for the search was specified. the initial searches were performed on august , in ovid medline, and october , in ovid psycinfo. authors contributed seed articles that they had previously identified as relevant to physician suicide ( , - ), which were analyzed by the medical librarian co-author. search terms and databases were then selected and tested based on this analysis. the search strategy also underwent a peer review process with two additional medical librarians. an updated search was run again on april , in ovid medline, psycinfo, and scopus. detailed search terms are available in appendix . inclusion criteria were english-language papers with a primary outcome, measure, or thesis focused on death by suicide or suicidal behaviors among physicians, including suicide attempts, and suicidal thoughts and ideation. physicians included medical students, postgraduate trainees (residents and fellows), and physicians at any career stage. opinion articles were also included, if they otherwise met inclusion criteria; these included perspectives, letters to the editor or their replies, essays, and viewpoints with a focus specifically on physician suicide. exclusion criteria were non-english publications and those only pertaining to physician burnout, mental health, substance use disorders, or other media, such as newspaper and magazine articles. the aim was to inductively identify key themes across the published literature included in this scoping review. during each subsequent round of review, tags could be added to articles. after all articles were reviewed and tagged by two authors (tl,sp,cyac), one of the authors (tl) re- reviewed all articles to add tags until a point of saturation was reached and no further topic tags could be added. tags were then condensed into a core set of themes. these themes were assembled into a framework based on their frequency of occurrence, resulting in a map of the most published themes about physician suicide. findings are summarized in narrative form. results the articles that met inclusion criteria ( figure ) covered a broad range of publication types over time and from countries worldwide. the earliest publications were editorials ( primarily sought to estimate epidemiology of suicidal behaviors among physician populations. in other words, death by suicide was most often studied among attending physicians because of availability of data on occupation and death, particularly in vital statistics or death certificates. however, the tools used to perform such estimates varied. deaths by suicide were most frequently estimated based on th or th revisions of the international classification of diseases (icd- or icd- ) codes on death certificates. other commonly used sources of data included membership masterfiles from physician organizations or associations ( ), published obituaries, and charts from medical records or forensic reports ( records, and any other information available, such as suicide notes ( ). such an approach can elucidate potential contributors to an individual's death, providing detailed contextual information, all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint their life circumstances, and how they managed such circumstances up until their death. however, psychological autopsies are not systematically applied in the setting of deaths by suicide. case reports also may describe a psychological profile, in addition to medical and clinical aspects of the case ( , , ), but this was also infrequently published. in , gold et al examined data from the u.s. national violent death reporting system (nvdrs), which is a combination of information and data from death certificates, coroner and medical examiner reports, toxicology reports, and law enforcement reports ( ), the only study to triangulate multiple data sources to estimate suicide incidence. the wide variety of data sources and methods of estimation suggest that there is no standard. suicide clusters are suicides that occur near each other, typically with respect to time and geography. suicide contagion refers to spread of information about a suicide via media and other channels, which can increase suicide risk in a community. only three studies acknowledged these topics. two opinion papers described suicide clusters that occurred in a short time span and in a focused geographic region, one in winnipeg, canada ( ) and another cited suicides in australia that had been reported in news media ( ). both articles focused on intense or distressing working conditions as important factors in the suicide deaths but did not elaborate further on suicide clustering. in fact, the canadian article, along with one other case series of physicians who died by suicide while on probation in oregon state ( ), used the term "epidemic" rather than "clusters." nonetheless, all three papers referred to the concepts of suicide clusters and suggest contagion, even though they do not use these terms explicitly. these were articles were tagged with the public health theme. suicide clusters also appeared to follow physician subpopulations, although numbers were small in such case series and opinion articles. jewish dermatologists during the holocaust: some emigrated, some died during nazi rule, and others survived ( ). three of these dermatologists died by suicide, which was "very common among the doctors [under the nazi ordeal], especially those physicians of the older generation" ( ). another unexpected analysis described the confusing ethical circumstances of physicians as terrorists ( ) . overall, suicidal ideation was the second most studied thesis among articles included in this literature review, especially suicidal ideation among medical students. to assess suicidal ideation and attempts, surveys were most commonly used; overall, cross-sectional survey studies were performed (table ). validated surveys are available to assess suicidal ideation ( suicide attempts in general were poorly studied in any physician subpopulation, but suicidal behaviors among postgraduate trainees were the least studied of all, with resident physicians' behaviors studied more than fellows. among articles reviewed, only two validated tools, paykel's instrument and the beck hopelessness scale, were identified that were specifically designed to assess all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint suicide attempts; otherwise, investigators developed survey items to assess respondents' self-reported attempts. physicians' risk factors for suicidal behaviors, especially mental health disorders, are among the most common topics of study. gold et al examined nvdrs data and identified certain risks unique to physicians compared to non-physicians: physicians were more likely to have a job problem preceding death by suicide, and a greater likelihood of known mental health disorders, yet no accompanying increased likelihood of antidepressant therapy ( ). additional risk factors potentially include professional setbacks prior to suicide death, such as facing complaints ( , ), feeling overloaded, working long hours, or being unable to cope with job responsibilities ( ), or experiencing disability as a result of medical illness ( , , ). related to these conditions, physicians are also at high risk of burnout, which has been found to be associated with suicidal ideation in u.s. medical students ( ) and dutch residents ( ), although no direct relationship between burnout and death by suicide has been established. to assess these risk factors, a variety of validated questionnaires were used to inquire about general health, burnout and stress, mental health (e.g. depression, anxiety or insomnia), substance use including alcohol use, and other measures of career, work, personality, and other life experiences (table ) . additionally, investigator-developed survey questions were also used, for example, to assess attitudes towards suicidal behaviors of a peer. because the earliest publications about physician suicide suggested an increased incidence compared to the general population, certain theories have been applied to attempt to explain the increased suicide risk among physicians. or provocative stimuli, including events triggering second victimization, such as patients' poor outcomes, death, and suffering. this can then lead to desensitization when exposed to death in general. role strain describes physician risks as a result of their direct work environment or professional norms; a mismatch between social and institutional norms and the physician's roles can manifest as an unrealistic expectation of perfect function at a maximum level of competence ( ). professional self-image and identity, along with self-stigma, are also considered relevant mediating themes ( , , ) . early studies of suicide rates by specialty suggested that psychiatry and anesthesiology had the highest suicide rates ( ). indeed, these two specialties occurred most frequently in this scoping literature review, followed by surgery or general surgery. however, the findings remain mixed due to data limitations in ascertaining suicide rates as noted previously ( - ). furthermore, no explanations for the association have been offered other than a proffered but unstudied hypothesis that some medical specialties may possess more acquired capability than others ( , ). the access and knowledge hypothesis is a commonly discussed risk factor for suicide death among physicians. physicians acquire specialized medical knowledge of the human body and have access to the means (e.g. prescription drugs) that can cause lethal self-harm. observational studies of means of all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint suicide death suggest that firearm, prescription drug overdose, and hanging are among the most common methods used by physicians ( , , ), although this varies by country. psychological autopsies are consistent with the access and knowledge hypothesis. one set of psychological autopsies in the united kingdom examined physician suicides, finding that of the physicians died by self-poisoning and by self-injury (other than poisoning), as one physician used both methods ( ). in a finnish psychological autopsy of physician suicides, only had previous suicide attempts, suggesting that physicians may be more likely to cause self-harm leading to death by suicide; in other words, physicians who die by suicide may not have had any prior suicide attempt, which is risk factor in the general population ( ). self-treatment or self-prescription is also a concerning contributor due to specialized access to controlled substances, as "medical doctors exploit their profession for purpose of self-treatment" ( ). personality and life experience prior to medical training might also contribute to physician suicide risk, although are poorly studied. in a prospective study of norwegian medical students, the control personality trait, or the degree of compulsiveness, was independently linked to suicidal ideation; and more neuroticism, or the vulnerability personality trait, as a medical student independently predicted more serious suicidal ideations and planning in the first two postgraduate years ( , , ). reality weakness, more commonly associated with serious psychiatric pathology, predicted a transition from suicide ideation to planning ( ). neuroticism and tendency towards perfection may even be traits sought in potential medical trainees. one editorial speculates about the influence of adverse childhood experiences on risk for physician suicide ( ). one south korean study found a more than threefold risk of lifetime suicidal ideation, planning, and attempts among medical students who experienced emotional abuse early in life, characterized by "a continuously cold and uncaring parental attitude" ( ). in china, medical students were surveyed about parental relationships and parenting communication styles, hypothesizing that this could be a highly influential aspect of the student's character ( ). the study found that for chinese medical students, a good relationship with parents was statistically significantly associated with less suicidal ideation, plans and attempts. some studies sought to identify risk factors unique to their cultural context. researchers in the united arab emirates collaborated with eskin ( ) to again assess medical students' religiosity and their attitudes towards suicide ( ). in this study, investigators suggested that the islamic faith may serve as a protective factor against suicidal thoughts and death by suicide among their medical students. in pakistan, a study of suicidal ideation among medical students included religion as a demographic characteristic but did not otherwise include religion in further analyses ( ). among opinion articles, certain conceptualizations of suicide also varied. for example, in one commentary from japan, two concepts directly link overwork with death: karoshi (death due to overwork) and karojisatsu (suicide due to overwork) are considered causes of death in the japanese culture ( ) but nowhere else in the world. early studies in the s and earlier explicitly excluded female and minority physicians ( , , ). results published in from the women physicians' health study, which surveyed a sample of women physicians from the american medical association masterfile ( ), described prior studies that reported an odds ratio as high as for women physician suicides compared to other categories of women but that such studies were based on small numbers of suicide deaths. in , schernhammer performed a systematic review and meta-analysis, concluding that the suicide rate ratio for women physicians was . compared to the general women population, and . for male physicians all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint gender difference in terms of rates of suicidal ideation among medical students ( ). little data is known on suicide among physicians of gender minorities, as only one perspective piece written by a nurse who wrote about her transgender child who died by suicide ( ) . health initiatives for surveillance and prevention. this imbalance quickly identifies potential areas for further work. themes such as suicide as a public health issue, postvention, and legal issues were far less studied than others. in the second level of the framework are epidemiologic and demographic focuses for study, which were previously described in section . . along with these subjects are a relatively lesser focus on substance use, compared to other risk factors such as mental health, and on developing educational curricula to address knowledge gaps about suicide and its prevention. wellness is an increasingly popular topic for publication on this level of the framework. in the third level of the framework are prevention approaches. thirteen articles described interventions with a primary aim of preventing physician suicides. frequently, programs were implemented as an organizational response to a physician suicide. five interventions implemented the afsp's interactive screening program for suicide prevention ( , ) as a component of a larger initiative to promote physician well-being; three of the five came from the same institution ( - ). only one of was a randomized controlled trial of a web-based screening program ( ). treatments and responses to physician suicide were discussed as frequently as prevention; treatments and responses differed because treatments tended to focus on individual physician treatment for suicidal behaviors or related mental health disorders, and responses tended to focus on small group, physician health program, or medical board responses to individual circumstances. for example, young et al described the difficult grieving process for a healthcare team after the death a physician ( ). patients were told that the physician who had died "was not available," trust between staff and administrators eroded, morale decreased, and grieving, guilt and other negative consequences remained unaddressed until six weeks after the physicians' death ( ). the process of facilitating a normal grieving process after a suicide death has occurred requires active planning, called postvention. in another study by kaltreider et al, medical students were interviewed after the death of a classmate by suicide ( ). this qualitative study found that students experienced intense, all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint intrusive, and persistent thoughts and emotions relating to their classmate's death, sometimes lasting for months. proximity to the site of the death, prior exposure to another suicide previously, or familiarity with depression in themselves or family seemed to increase these effects. legal and policy issues are increasingly recognized as affecting physician health and well-being, and appear in the third and fourth levels of figure . physicians may avoid seeking mental health treatment, avoid self-report, and self-treat mental health and other health issues ( , ) , out of fear of reputational damage or concern about perceived weakness or impairment ( ). licensure application questions could be improved ( ) and compassionate, confidential assistance programs, such as one implemented in the united kingdom in , could offer bespoke services to physicians who might be considered at risk of suicide ( ). in , the uk's general medical council was recommended to fund a pilot program that would include developing a national support service for doctors ( ). however, no literature was found to describe such policies in other countries. only three articles describe suicide clustering and suicide contagion, which are unique public health concepts pertaining to suicide ( - ). however, no further follow-up or other literature regarding these suicide clusters were found in this scoping review. as a result, the themes of postvention and public health approaches to physician suicide both were at the vertex (bottom) of the inverted pyramid. discussion as a scoping literature review, this review did not include a quality assessment of quantitative evidence from included papers nor did it perform a meta-analysis or any statistical comparisons of published evidence, which would be characteristic of a systematic review ( ). more papers on physician suicide have been published over the last two decades than before. this follows an overall trend in published literature on physician well-being and may also reflect growing public recognition of and decreasing stigma relating to suicide. furthermore, the proportion of opinion articles published is lower in the recent past compared to previously, indicating growing application of contemporary statistical and scientific methods to the study of physician suicide. nonetheless, themes identified reveal opportunities for further work. the inverted pyramid framework ( figure ) suggests that there is a dearth of work on examining physician suicide using typical public health approaches to investigate suicide clusters. figure offers a proposed framework that highlights priorities for continued work on physician suicide. this framework highlights the potential for developing an organized public health approach to preventing physician suicide. the pyramid's base identifies areas for immediate, targeted actions, forming the foundation for the following recommendations for action: • mandatory reporting, an approach used for workplace injury reporting, which could improve surveillance of physician deaths by suicide (prevention); • identification of minimum acceptable community standards of validated instruments to assess suicidal ideation and attempts (prevention, public health); • development and implementation of evidence-based screening and service provision for suicide risk factors ( - ) (prevention); • development of early warning systems, like those used to monitor infectious disease outbreaks, to signal suicide clusters and contagion (public health); • implementation and dissemination of best practices, especially regarding postvention or additional directed interventions (postvention); all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint • increase education in the healthcare community about suicide warning signs and prevention (education, wellness); • standardized education in the medical community about appropriate reporting on suicides to prevent suicide contagion (education); • reduction of barriers to help-seeking behavior, reduce systematic stigma in licensing and investigative processes (policy issues, legal issues). higher levels in figure interpreted the data. til drafted the manuscript, except for the methods section which was drafted by all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint rs, and the entire manuscript was produced with critical revisions and important intellectual content from all authors. acknowledgments the authors wish to acknowledge dr. christine moutier - - - - - - - - - no. 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key: cord- -ulvqm g authors: hammond, david; louca, christina; leeves, laura; rampes, sanketh title: undergraduate medical education and covid- : engaged but abstract date: - - journal: medical education online doi: . / . . sha: doc_id: cord_uid: ulvqm g nan we were interested to read the letter of our colleagues regarding the impact covid- has had on medical education [ ] . that engagement levels with the online content have been high certainly seems to be the case, with virtual attendances closer to capacity than faceto-face teaching, despite some students currently being in other time-zones. the move to open-book examinations may yet be a practice that lives on, especially as one of the biggest concerns of students was finding a quiet place to sit the exam [ ] -yet in a post-lockdown world, quiet -at home, or in a library, will be easier to obtain. however, we feel it is important not to get carried away with the apparent success of online medical education. whilst the thoroughness of online teaching may surpass other mediums, being a good doctor requires patient contact and time on the wards. nothing else can adequately prepare someone for the realities of working life. focusing on paediatrics as an example, whilst a webinar can tell you how to perform a paediatric examination, only experiencing it can teach you to, sometimes, just sit on the floor and play with a child, improvising the examination as opportunities arise. additionally, nothing can truly replace the pain and anger of parents concerned for their child -only by actually speaking to them can communication skills be honed for the situation. as noble argued, 'although knowledge is important, it is not what makes the difference between a good doctor and a less good doctor [ ] . ' whilst the pandemic was rife and hospitals were quickly reconfigured to cope with the surge in covid- patients [ ] , medical students on the wards may have been left unsupervised, or else diverted the attention of doctors away from patients -so education, correctly, had to temporarily take a back seat. as things begin to return to normal, however, it is vital that medical schools around the country do not think that online teaching is an acceptable substitute for clinical placements. perhaps in the early years of medical school, mixing online lectures and webinars with face-to-face teaching may be worthwhile, in order to maintain the benefits accrued from increased real time engagement. when it comes to clinical time, however, no amount of exposure can be too great for a student and this must be protected at all costs, in order to ensure that future graduates are both well prepared and safe doctors. the impact of covid- on the undergraduate medical curriculum medical students take final exams online for first time, despite student concern are national qualifying examinations a fair way to rank medical students? london begins major covid- reconfiguration key: cord- -b dk sl authors: cheng, wenhong; zhang, fang; liu, zhen; zhang, hao; lyu, yifan; xu, hao; hua, yingqi; gu, jiarong; yang, zhi; liu, jun title: a psychological health support scheme for medical teams in covid- outbreak and its effectiveness date: - - journal: gen psychiatr doi: . /gpsych- - sha: doc_id: cord_uid: b dk sl background: medical staff fighting the covid- pandemic are experiencing stress from high occupational risk, panic in the community and the extreme workload. maintaining the psychological health of a medical team is essential for efficient functioning, but psychological intervention models for emergency medical teams are rare. aims: to design a systematic, full-coverage psychological health support scheme for medical teams serving large-scale emergent situations, and demonstrate its effectiveness in a real-world study in leishenshan hospital during the covid- epidemic in wuhan, china. methods: the scheme integrates onsite and online mental health resources and features team-based psychosocial support and evidence-based interventions. it contained five modules, including a daily measurement of mood, a daily mood broadcast that promotes positive affirmation, a daily online peer-group activity with themes based on the challenges reported by the team, balint groups and an after-work support team. the daily mood measurement provides information to the other modules. the scheme also respects the special psychological characteristics of medical staff by promoting their strengths. results: the scheme economically supported a special medical team of members with only one onsite psychiatrist. our data reflected that the entire medical team maintained an overall positive outlook ( – out of in a daily mood index, dmi) for nearly weeks of continuous working. since the scheme promoted self-strengths and positive self-affirmation, the number of self-reports of life-related gains were high and played a significant effect on the dmi. our follow-up investigations also revealed that multiple modules of the scheme received high attention and evaluation levels. conclusion: our quantitative data from leishenshan hospital, wuhan, china, show that the programme is adequate to support the continuous high workload of medical teams. this scheme could be applied to medical teams dealing with emergent situations. to reinforce the shortage of medical services during the outbreak of covid- in wuhan, china, more than medical staff from all over the country served in hubei province. they worked in hospitals including temporary infectious disease hospitals and in medical isolation units. due to factors such as the lockdown of wuhan, prolonged isolation, family infections, lack of medical resources, and witnessing death, these physicians generally experienced stress symptoms or bereavement-related depression. faced with patient's infectiousness and psychological needs, as well as locked wards, separation from home, and continuous work, medical staff experienced emotional pain, insomnia, stress reactions and exhaustion. this increased the risk of illness in the staff themselves. [ ] [ ] [ ] [ ] [ ] [ ] in addition, the medical workers became easily dissatisfied with their job. due to the emergent nature of the epidemic, many doctors and nurses were not from the departments of infectious diseases, respiratory or intensive care units (icu), so they lacked confidence in treating covid- , which also increased their stress. therefore, to maintain efficient work, it was essential to alleviate the psychological stress of the team. there has been a considerable shortage of professional mental health workers in china. [ ] [ ] [ ] in this epidemic, there were only about professional mental health workers among the medical workers dispatched from outside to hubei ( . %). in our case, a -member medical team from shanghai general hospital arrived at leishenshan hospital on february and took over beds in two general wards and beds in the icu. there was only one psychiatrist among the medical team. in addition to maintaining the mental health of medical team members, this psychiatrist also provided psychological support for patients under the jurisdiction of this medical team. therefore, onsite mental health resources were minimal. to overcome these difficulties, a psychological health support programme was designed and implemented to reinforce the medical team sent to leishenshan hospital in wuhan, china. the programme was based on strength-oriented peer support groups and positive self-feedback coaching. previous studies have demonstrated the effectiveness of peer support group approaches, such as balint groups, on alleviating the exhaustion of healthcare workers. [ ] [ ] [ ] [ ] these activities have been validated both in face-to-face and online forms. besides being financially economical and resource efficient, it adapts easily to the special characteristics of healthcare workers. doctors and nurses tend not to accept the role of help-seeker, and they are more inclined to learn and solve problems by themselves, bringing challenges to classical psychological interventions. our programme thus emphasised motivating self-strengths by promoting positive self-feedback and coaching selfregulation exercises everyday. this paper introduces the details of the psychological health support programme and presents data that reflect the effects of this programme. we examined whether this programme can effectively promote positive emotions, maintain team work efficiency and prevent burnout. there were medical personnel in the medical team, including doctors, nurses and administrative staff. mean age of the team was years ( - years), with men ( . %). figure shows the details of the enrolment and follow-up of subjects in the study. figure presents the architecture of our psychological health support programme developed in response to the specific needs of the medical team. the programme consists of five modules, including a daily measurement of mood (referred to as a psychometer), positive selffeedback training (in the form of a daily mood broadcast, an online means to tell staff about positive happenings), peer-group psychological support and education (in the form of a daily online themed chat), a balint group and an after-work support team that provides necessary life skills and organises after-work activities. as shown in figure , medical team members reported their emotional state, gains and issues in the psychometer module. the information then inspires the daily mood broadcasts and the topics of the daily online themed chat. the after-work support team also provides assistance to medical team members based on the needs reflected in the psychometer module (blue lines in figure ). medical team members receive positive self-feedback from the daily broadcast. they also obtained timely support that meets their needs, such as physical and cultural activities, from the after-work support team. at the same time, they participated in daily online themed chats and weekly balint groups based on interests and needs during their after-work time. details of each module are described below. in the psychometer module, we created an online, anonymously completed daily mood questionnaire, which includes an age-and-gender form, a subjective units of feeling (suf) scale and a gain-and-issue scale. the suf is a - scale ( for no pleasure and for most intense pleasure) to measure the subjective intensity of pleasure that an individual is currently experiencing. the scale was adapted from the subjective units of distress scale (wolf, ) which changes the distress rating to a positive emotional rating. the gain-and-issue scale consists of two semi-open-ended questions that ask for answers to the question, 'what was your biggest gain today?'; 'what is the challenge you are experiencing today?'. the results were used to capture individual impressions of gains and challenging issues. the entire questionnaire was made into a web page that could be accessed via smartphone, and a qr code link was sent to the medical team's online general psychiatry themed chat group daily. the information collected in this questionnaire was summarised daily and used as valuable reference information for other intervention modules. at the same time, these data are one of the primary sources for later data analysis. positive self-affirmation training (in the form of a daily mood broadcast) based on the level of positive emotions and positive self-reporting provided by the psychometer module, we composed a daily mood broadcast to report the mood index and convey positive feedback. we sent the daily broadcast to the medical team's online themed chat group every evening. in this way, we reinforced the selfaffirmation of the entire staff. peer-group psychological support, and education (in the form of a daily online themed chat) we conducted a daily hour online themed chat group to invite people to share their experiences and thoughts. the topics were selected according to the most representative issues based on the information collected daily in the psychometer module. the chat topics covered mood regulation, cognitive restructuring, problem-solving, pleasurable activities, relaxation meditation and positive mindfulness exercises (see online supplementary table for specific daily chat topics). the moderator (a psychologist) also posted relevant mental health support tips in the forms of text, video and audio. we ran a weekly balint group activity, namely leaded by the psychiatrist and to discuss with health care professionals. which recruiting group members on a sign-up basis, up to - members at one time. participants attended experiential learning from each other, tried to understand difficult emotions at work and shared solutions. as an essential part of the psychological intervention programme, the after-work support team provided targeted materials based on the issues collected by the psychometer module and organised after-work activities, such as sports, cooking and photography. this team also took care of unexpected out-of-work events that occurred to the medical team members. these efforts promoted relaxation and social supports among the medical team members. we conducted a follow-up study of the effectiveness of our psychological health support programme after the medical team had finished their work assignment within week after leaving wuhan. an online questionnaire was sent to the entire medical team as they were in a rest-and-quarantine phase. the subjective unit of feeling questionnaires asked the following information: ( ) level of attention to the daily mood broadcast ( - scale; : never attention, : special attention); ( ) subjective effectiveness rating of the balint group ( - scale; : no effect, : a very significant effect), and ( ) the most impressive topics in the daily online themed chat. the data collected in this study came partly from the follow-up study and partly from the psychometer data collected during the implementation of the psychological health support programme. for the follow-up questionnaire data, we depicted the distribution of the medical team members' attention to the daily broadcast and their effectiveness rating scores for the balint group. we also calculated the percentage of 'impressive' topics in the daily online chat activity, that is, the number of individuals who were impressed with the daily topic divided by the sample size. through these analyses, we preliminarily examined the subjective effects of the intervention programme on the medical team members. the psychometer data collected during the implementation of the intervention programme included data from the emotional suf and the gain-and-issue scale. we first averaged the reports of the suf across individuals to calculate a daily mood index (dmi). due to the intensive nature of emergency medical work, there were a few dates when these data could not be collected. in data analysis, therefore, we ignored these missing values. for the results of the gain-and issue open scales, we split the report into events. we averaged the number of daily reported gain events and the number of challenging events, respectively. further, we coded the reported gain events and issues as work-related, life-related and physiological. we calculated the daily average of gain-work, gain-life, gain-physiological, issue-work, issue-life and issue-physiological, respectively. at the same time, we also obtained the total number of patients under the responsibility of the daily medical team and the number of cases in critical condition. to further investigate the effectiveness of the intervention programme, a stepwise regression analysis was used to examine how the dmi was affected by the total number of patients, the number of critical cases, the number of gains and the number of issues. further, we examined how the three different aspects of gains and issues affected the dmi. since the main thrust of the intervention programme is positive self-feedback and regulation, testing the effect of the number of reports of gains and issues on the dmi would reflect the effectiveness of the intervention programme. one hundred and twenty-four members of the medical team responded to the follow-up investigation, including doctors, nurses and administrative staff. as shown in table , this sample does not differ significantly from the entire medical team in terms of role composition. there was also no significant difference between the general psychiatry sex ratio of the sample and the entire medical team (see table ). according to the survey, . % of the participants followed the daily mood broadcast, . % participated in daily themed chats and . % participated in the balint group. figure a shows the distribution of respondents' attention to the daily mood broadcast. respondents rated this using an evaluation scale of - . the distribution shows a significant leftward bias, with a median of , indicating that the daily mood broadcast received widespread attention from the medical staff. figure b shows the distribution of the evaluation scores of respondents who had participated in the balint group. similar to figure a , the distribution also showed a significant leftward bias, with a median of . among the respondents, . % rated or higher. the results showed that the team members who participated in the balint group were satisfied with the benefits. figure c shows the percentage of respondents who were impressed by each daily themed chat activity. the highest impression rate was . %, the lowest . % and the median . %, suggesting that daily themed chat activities have some influence on medical team members. the above data show that several essential components of our programme received the attention and made the medical team members feel positive. the number of people who filled out a daily suf scale while working at leishenshan hospital ranged from to , depending on the days, with a median of . some data were missing (eg, [ ] [ ] [ ] [ ] [ ] [ ] [ ] because of interruptions, such as the only psychiatrist had to deal with emergency of the patients or the medical team being busy with other things. figure a general psychiatry patients, including those in critical condition, were transferred to the wards, which broke the hope of going home for the team members ( figure b ). these changes in the dmi, which are strongly associated with external events, suggest that the dmi can sensitively portray mood states of the medical team. the dmi remained high (> ) most of the time compared with its initial level and the lowest point ( march) and showed a gradual upward trend over time between february and march. after experiencing a valley with a sudden spike in the number of patients, the dmi quickly returned to higher levels. perceiving gains and building positive attitudes are essential goals of our intervention programme. as shown in figure c , the average number of gains reported by medical team members increased rapidly from a starting point of . to . and subsequently above . with the implementation of the intervention programme, which means the members participating in the psychometer reported more than one gain per day on average. conversely, the average number of issues reported fell rapidly from about . at the starting point to around . . despite an increase between march and march, the ratio of issues to the number of gains remained mostly in a range of . - . , after february. this fact suggests that the intervention programme may help to increase awareness of the gains and to promote positive attitudes. we further classified three aspects of work, life and physiological factors according to the specific content of medical team members' reporting. figure d shows the daily values of three types of gains over time. similarly, figure e shows the daily values of three types of issues over time. the amount of reports on issues regarding work slightly decreased over time, while the amount of gains regarding life showed an upward trend. we used stepwise regression analysis to examine how the daily average of gains/issues, the number of patients and the number of severe cases affected the dmi. we applied two linear models (table ). model used the total number of patients, the number of severe cases, the daily average of gains and the daily average of issues as regressors to predict the variation of dmi. model further broke down the gains and issues into three types, namely the total number of patients and the number of severe cases, gain-work, gain-life, gain-physiology, issuework, issue-life, issue-physiology were used as regressors to predict the variation of dmi. the initial diagnosis of both models suggested that the data on march and march affected the normality of the residuals, so they were eliminated. in the two-way stepwise regression analysis, the fitting of model was significant. the model showed that both the number of severe cases and the daily average of gains significantly explained the variation in dmi. the total number of patients and the daily average of issues did not reach significance. model also significantly explained the variance of dmi. the model showed that both the number of severe cases and the gain-life significantly explained the variation in dmi. the total number of patients, gain-work, gain-physiology, issue-work, issue-life and issue-physiology did not show significant effects. the above results indicate that in addition to the significant impact of the number of severe cases on the dmi, the subjective gains reported by the medical team members, especially the daily average of life-related subjective gains, can also significantly explain the variation in dmi. the subjective sense of gain is precisely the aim of the psychological health support programme that we intended to achieve. therefore, our programme may improve dmi by improving the subjective sense of gain. the main finding of our study was that the entire medical team maintained an overall positive outlook with a dmi mainly between and out of for nearly weeks of continuous working. the number of severe cases had a significant effect on the dmi of the medical team. further analysis of the reported gains showed a significant effect of life-related gains on dmi. since the primary feature of this psychological support programme was to promote self-strengths and positive self-affirmation, these findings support the effectiveness of this psychological support programme. meanwhile, the follow-up investigations also revealed that the modules of the programme, such as positive self-affirmation training (daily broadcast), peer psychological support and education (daily themed chat) and the balint group, received high levels of attention and evaluation from staff, supporting the effectiveness of this programme. a highlight of our psychological health support programme is that it reconciles the mental health needs of medical team members in extreme settings with the minimal resources available for mental health support. research has shown that exposure to a working environment where patients' conditions changed rapidly, were severe, urgent or even fatal could increase occupational exhaustion and trigger psychological trauma, thus adversely affecting the emotion and behaviour of healthcare workers. impairment of mental health and quality of life could affect the quality of medical care, increase medical errors and reduce job satisfaction. furthermore, emotional burden, excessive workload and frustrations due to lack of control are potential risks for stress and burn-out. additionally, being away from home, inability to connect with family and lack of family support due to work are also significant risk factors for occupational exhaustion in healthcare workers. given that these risk factors coincided together with medical teams in the midst of a disaster (the covid- pandemic), it was necessary to apply a psychological support programme. indeed, as shown in this study, the dmi of medical team members was adversely impacted when the workload increased, as reflected in the number of severe cases. on the other hand, positive events especially positive life experiences could enhance the dmi of medical team members. therefore, in the midst of major disasters, it is necessary and crucial to maintain the mental state of front-line healthcare workers to ensure their adequate working status and health, despite minimal resources for mental health support. the psychological support programme presented here is an effective and promising solution. the core ideas of this programme are resource-based learning, peer support and positive self-affirmation training. the daily mood broadcasts, online themed chat groups and balint group activities in the programme were all designed around these aims. studies have shown that positive self-affirmation promotes healthy behaviours, better academic performance and more prosocial behaviours, [ ] [ ] [ ] as well as improving psychological and physical well-being. self-affirmation acts as a defence mechanism to help mobilise the individual's internal resources and enhance psychological openness and flexibility when facing external threatening events. when psychologically threatened, well-functioning selfaffirmation becomes a protective factor that enhances internal resources, broadens the perspective on events and uncouples the self and threat, thus protecting selfesteem. research on healthcare workers has also found a significant relationship between self-efficacy and burn-out. healthcare workers with better self-efficacy reported higher job satisfaction. they were more likely to mobilise new resources when faced with difficulties and obstacles, such as seeking help from colleagues and supervisors. they were also more motivated, persistent and engaged in their work. [ ] [ ] [ ] [ ] social support is another powerful buffer against the adverse effects of emergency stressors and traumatic events, with better social support preventing and reducing the incidence and severity of post-traumatic stress disorder. moreover, both self-efficacy and social support play an essential role in reducing and ameliorating the adverse effects of traumatic events. the above arguments explain the effectiveness of the core ideas in this psychological health programme. previous studies on psychological interventions for medical workers have mainly focused on long-term programmes to reduce burn-out. while a study has reported psychological intervention efforts, such as educational lectures and face-to-face psychotherapies for medical staff treating patients infected with severe acute respiratory syndrome, the literature on psychological general psychiatry intervention programmes for medical staff under highrisk continuous emergency work is sparse. in recent qualitative research, jonker et al proposed a framework for psychological trauma management in high-risk occupations that highlights three levels of interventions for acute stress. the primary interventions are a job-related resource, management support and immediate needs; the secondary intervention is psychoeducation and a team-based approach; and the tertiary interventions are onsite psychological support, psychological assessment and connection with social support. the five modules in our psychological health support programme fit this framework. the support team tried to satisfy the needs of staff from food to life events (primary intervention), the balint group and daily chat group provided psychoeducation and team-based psychological social support (secondary intervention), the daily broadcast promoted positive self-affirmation (secondary intervention) and the psychometer collected data on daily mood and needs of team members (tertiary intervention). therefore, our programme provided comprehensive coverage for the various aspects of preventing psychological issues. considering the high working pressure of medical team members in wuhan, we were worried that filling in standardised scales for evaluation of emotions and other conditions would increase their burden, which led to the lack of standardised scales for evaluation of intervention efficacy in this study. in addition this research lacks a control group to further demonstrate the effectiveness of interventions. as the number of victims of global disasters continues to increase, the damage caused by disasters to humans will also increase. it is necessary to formulate defence strategies, related intervention teams and working mechanisms. the psychological support programme we designed in the practice of maintaining the mental health of medical staff can be used as part of the disaster prevention strategy. investigation of the mental health of patients with novel coronavirus pneumonia factors associated with 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survivors - years after / take care! the evaluation of a team-based burnout intervention program for oncology care providers can skill-development training alleviate burnout in hospital social workers? comparison of psychological status and sleep disorder between earlier stage and final stage of psychological intervention in medical staff of sars wards an intervention framework to facilitate psychological trauma management in high-risk occupations key: cord- -z wuajpx authors: martin-fumadó, carles; gómez-durán, esperanza l.; morlans-molina, màrius title: medico-legal and ethical considerations on resource limitation and clinical decisions during the covid- pandemic date: - - journal: nan doi: . /j.remle. . . sha: doc_id: cord_uid: z wuajpx abstract the covid- pandemic has raised ethical and medico-legal problems, which include the equitable allocation of health resources, especially in relation to the prioritization of patients and the rationing of resources. priority setting is always present in healthcare systems and depends on the theory of justice applicable in each society. resource rationing has been necessary in the covid- pandemic, and therefore consensus documents have been published for decision-making based on four fundamental ethical values: maximization of benefits, treating people equally, contributing to creating social value and giving priority to the worst off, from which specific recommendations derive: maximize benefits; prioritize health workers; do not prioritize attendance on a first-come, first-served basis; be sensitive to scientific evidence; recognize participation in research and apply the same principles to covid- patients as to non-covid- patients. march the who declared that covid- had become a pandemic. since then, as medical professionals around the world have confronted their professional responsibilities in the fight against this pandemic, a range of ethical problems have emerged. they are hard to resolve, and include the fair assignation of limited available medical resources. , it must firstly be remembered that all medical actions, and therefore clinical-medical decision-making on the fair assignation of medical resources, must be governed by the ethical principles of medicine established by beauchamp and childress : the principle of autonomy, beneficence, absence of malice, and justice. the principle of autonomy refers to the individual capacity of everyone to deliberate and freely decide, i.e., to take decisions about themselves in connection with aspects that concern them. the principle of beneficence refers to the moral obligation to act in a way that benefits others, promoting their well-being and legitimate interests. the principle of absence of malice, which is also known as primum non nocere, refers to the obligation not to harm or prejudice patients, while also preventing any possible harm. finally, the principle of justice refers to the obligation to treat each individual as corresponds to them, understood essentially as a principle of medical policy and the fair distribution of medical resources. the aspects associated with the principle of justice expressed by beauchamp and childress will therefore be of key importance in resolving the ethical and medical-legal problems arising from the fair assignation of resources during the covid- pandemic. the approach to the said ethical and medical-legal problems makes it necessary to differentiate between three different concepts. these arise within the context of medical care, and they centre on clinical-treatment decision-making. they are the concepts of therapeutic futil-ity, leading to limited therapeutic effort, the prioritisation of patients and the rationing of resources. the limitation or suitability of therapeutic effort, which includes the limitation of life support treatments, is a measure that is often applied in the context of critical patients. it refers to the decision for a specific patient not to commence a life support treatment or to withdraw it if one has commenced in the belief that it offers no significant benefit apart from prolonging the process of dying. in this case, there is no doubt that there is no moral obligation to supply futile treatment. the problem lies in determining whether or not a specific therapy is in fact futile. the factors that determine whether a medical act is futile refer to three different aspects: the strictly technical aspects, the ethical questions and the social implications. in any case, futility refers to a medical procedure that it is not worth administering as it is of very little use, or one with a remote probability of achieving an effect, or when the patient believes that it will not produce any benefit, or, simply, when its excessive cost compared to the negligible probability of its achieving the desired effect means that it would be unwise to use it. in this case, the principle of the absence of malice demands that we strive at all times not to cause harm to the patient. this takes the form of mitigating and palliating suffering, even if this accelerates the process of dying. the patient may decide how their end of life should be managed. if it is medically indicated then palliative sedation may be selected, accepting the consequences of this. thus even though it may sometimes seem confusing, it is clear that although there are cases where therapeutic effort is limited in patients affected by covid- , the covid- pandemic has not altered medical decision-making in connection with suppositions of futility. prioritisation involves establishing an order in medical care, while rationing involves assigning a resource to one patient rather than another one. it is not about resources that would be futile for the patient in question, but rather about accepting the loss of opportunity arising from this decision in terms of the evolution of a pathology. triage is habitually used in emergency services, with the aim of prioritising patients. triage is a structured form of working which make it possible to swiftly evaluate the severity of a patient in a way that is rule-governed, valid and reproducible. it aims to prioritise the order of medical care, determining the place and measures which a patient needs. however, triage is also a process that manages clinical risk and patient flows appropriately and safely when demand and clinical needs overwhelm resources. the severity of disease, the speed of its progression, waiting time and pain are considered to be the most important and widely used criteria for prioritising patient care in emergency departments. a good prioritisation system also has to include the utility of healthcare, to guarantee equity, so that the need for the selected treatment must also be taken into account. over and above specific scenarios, such as the emergency department, healthcare systems always set priorities in medical care, and this involves macroassignation processes as well as the microassignation of medical resources. resource macroassignation refers to the resources assigned by the administration. resource microassignation refers to the way in which within clinical contexts it is decided who, how, when and for how long patients will receive care. thus two of the most controversial aspects in the decision-making process when setting priorities are: firstly, defining how this is to be decided, i.e., the type of methodology to be used to determine the ranking of priorities and ensure that it is socially valid. they have to fair according to aristotle's definition of distributive justice, treating equals as equals. secondly, the role of experts in the decisionmaking process regarding priorities must be defined, as well as the degree to which the population will take part in the decisions about what to select. respecting the first question, the response will be determined by the theory of justice in which we start by delimiting the principles that have to govern the distribution of medical resources. determining the scopes and limits of what it is fair to demand depends on the way in which the right to medical care is defined and specified, together with the creation of criteria for the distribution of medical resources. there are different attitudes to this question, and they basically consist of the libertarian, egalitarian, utilitarian and community viewpoints: the libertarian perspective is governed by the principle of commutative justice (everyone receives according to what they have given). this legitimises the segmentation of medical care and charity as means of attending to the health problems of the population. access to medical ser-vices depends on spending power and the capacity to take out medical insurance, governed by market laws. the egalitarian perspective is based on the equality of persons and the need for social institutions to counterbalance the lack of opportunities caused by the fact of having become sick. this theory considers equality of access to the medical system to be the premise for achieving a fair medical system. within this context of egalitarianism, some versions make the right to healthcare conditional on individual responsibility for the cause of disease, i.e., the healthcare system recognises that it must only compensate individuals for unexpected risks, not for those they run because of their lifestyle decisions. the utilitarian viewpoint defines the value of the right to healthcare and clinical interventions solely in terms of their measurable outcomes, ''results'' or efficiency. nevertheless, it is not clear, even from different religious perspectives, who should measure these results or how this should be done. the number of years lived adjusted for quality of life, for example, gives units to measure health results that unit survival with quality of life. from this point of view, distributive justice in healthcare must always be governed by its cost-benefit ratio, so that the moral obligation to do something that is not economically optimum never arises. finally, the community viewpoint considers that each good or set of goods is a sphere of distribution, and that each one of these spheres has its own appropriate criteria and guidelines. on the one hand, this implies considering the ''social meaning of healthcare'', which is determined culturally and is therefore dynamic and subject to constant interpretation; on the other hand, it is necessary to accept that there is no single principle of distributive justice that governs all social goods, but rather that there are a series of constructed principles that constitute different ''spheres of justice''. in our social context, where the theory of egalitarian justice predominates, the socially valid scheme of priorities for assigning medical resources includes aspects such as patient age or quality of life, and these criteria are strongly supported socially. respecting the second aspect, the role of experts in the process of deciding on priorities has to be defined, together with the degree to which the population takes part in decisions on the microassignation of resources. rationing resources to certain patients rather than others while accepting the consequences of this decision must take place on the basis of standardised medical criteria, using decision-making algorithms agreed in clinical practice guides. , in many cases decisions on establishing priorities involves tragic choices, so that society has to set moral limits. these decisions therefore have to be based on democratically informed deliberations, in which all of the agents involved take part. bioethical professionals should therefore emerge as the figure able to promote dialogue between the world of medicine and the community, confronting ethical dilemmas even in emerging clinical situations. nevertheless, we must admit the need for an immediate response in a situation such as the covid- pandemic. this is especially complex due to a demand that surpasses the available resources, so that experts have to decide on priorities. this decision-making process has to be based on medical criteria which must be socially validated, and this is hindered by the lack of an immediate social debate on this question. during the covid- pandemic, medical professionals have had to deal with scenarios in which sufficient resources were not available (beds in intensive care units, ventilators, ecmo (extracorporeal membrane oxygenation . . .) for the number of severely ill patients, so that the decision on resource assignation depends on who has the best chance of living or dying. medical resource rationing is fortunately very rarely necessary outside catastrophe medicine, where there are far fewer available resources than necessary and they have to be rationed. however, even in these circumstances the question is not free of ethical debates and clinical arguments. , therefore, and in a prudentially similar way to what happens during care in the case of major catastrophes, the covid- pandemic has brought about a global state of emergency. for example, in italy there was insufficient time to test or verify plans or protocols for covid- infection, at least at large scale. the speed with which the scenario evolved made it necessary to adopt easy and pragmatic solutions, even for critical and delicate matters given the lack of resources for everyone. , medical resources rationing has since then been the case in many countries. resources have had to be rationed even in countries such as the united states, which theoretically had longer to prepare, due to the devastating effects of the pandemic. , foresight makes it possible to plan for future scenarios, including ethical debate on the assignation of resources. the implementation of algorithms for critical decision-making should be a part of the preparation for a pandemic in any part of the world, and these algorithms should include more than the most evident prognostic factors: fragility, comorbidity and functional state. the fact is that an additional hindrance to implementing decision-making algorithms in the covid- pandemic has been the urgency with which decisions about clinical practice and organisational protocols are being reconfigured. this was necessary due to gradually increasing knowledge about the infection, giving rise to uncertainty in patient care and leading to a considerable degree of variation in treatments. in italy, it should be underlined that in the initial phase of the medical crisis the italian society of anaesthesia, analgesia and intensive care (siaarti) drew up a series of ethical recommendations for treatment assignation in exceptional situations with limited resources. this document aroused strong reactions in the medical-scientific community, in the academic world and in the media. nevertheless, it was also supported by a large part of the medical community. , doctors were not surprised that the patient age criterion, which is socially accepted, started to be adopted implicitly in the decision-making algorithm for the assignment of scant medical resources due to the growing number of severe patients. , in fact, the siaarti document mentions patient age in two critical recommendations: ) ''it may necessary to set an age limit for admission to intensive care. this is not simply a selection associated with values, but rather the reservation of extremely limited resources for those who have the greatest possibility of survival and those who may live for more years once saved, to maximise the benefits for the highest number of people'', and ) ''the presence of comorbidity and functional state must be carefully evaluated as well as age. it is possible that short admission in healthy individuals may be prolonged, and that therefore resources should not be consumed in the case of persons who are elderly, with fragility or severe comorbidity''. in spite of the implicit acceptance of italian medical professionals to use the age criterion, disagreements arose in the academic world about this situation, and more specifically about whether it is incorrect to prioritise younger patients with covid- . in fact, in spain the general board of official spanish doctors' associations (the consejo general de colegios oficiales de médicos de españa) (cgcom) and other associations drew up different protocols in reaction to the crisis in care caused by covid- , giving rise to a certain degree of discomfort due to their references to age. currently, the documents by organisations such as the bioethics committee of spain, the central professional ethics commission of the cgcom or the spanish society of intensive and critical medicine and coronary units use a text that confirms the age criterion as being of undeniable importance in prioritisation, although it is not exclusive, and they underline the difference between prioritisation and refusing or restricting care. lastly, it should be remembered that the rationing of medical resources must be exceptional, and it is only justifiable if all of the parties involved (hospitals and institutions. . .) have attempted to increase resource availability and have also evaluated the feasibility and safety of transferring patients to other hospitals. in ethical terms, the novelty of the current emergency due to the covid- pandemic lies in the extraordinarily high number of people who are personally affected by the results of the assignation of scarce resources. they have suddenly realised that the principle of ''equals should be treated equally'' (the aristotelian definition of distributive justice) may no longer be applicable. given this dilemma, and based on the fact that by definition ethical dilemmas have no correct answer, we have to design a protocol-governed strategy for action that makes it possible to assign resources as equitably as possible. the assignation of resources and therefore also their prioritisation and rationing in the covid- pandemic should thus be based on strictly medical criteria. these have to be supported by clinical decision scores and they have to be socially validated even when we know that in these circumstances, the principle of patient autonomy may be restricted. thus in spain , --- and at an international level , --- many consensus documents have been published on ethical recommendations for decision-making in this exceptional crisis situation. they are revised at regular intervals, given that knowledge of the disease is evolving very quickly. all of the said recommendations have the aim of supporting medical professionals when making difficult clinical decisions. they supply criteria that are approved by professional associations which make it possible share responsibility in situations that involve a strong emotional charge, specifying criteria for the assignation of resources in an exceptional situation of scarcity. there is agreement on the need to prioritise the assignation of life support measures for those individuals with the highest probability of surviving their critical situation and recovering their project for the future. this decision is based on biological parameters that are guidelines, although others should also be taken into consideration. the capacity of a patient to tolerate the high level of physical, metabolic and cognitive demand involved in intensive therapy over the short to medium terms must be evaluated. this aspect is highly important, and it is not usually taken so much into consideration, and it may lead to therapeutic obstinacy. lastly, guideline documents may be prepared at local level, and experts underline the importance of a higher level centralised committee regularly revising clinical decisionmaking documents prepared by different committees or workgroups to ensure that inequalities do not arise. proposals for resource assignation in pandemics and other scenarios involving a total scarcity of resources agree on four basic ethical values: the maximisation of the benefits produced by the scant resources in question, treating everyone equally, contributing to the creation of social value and prioritising the most severe situation. , , , it is obvious that a single basic value is not enough to determine the rationing of a resource for one patient rather than another, as an ethical framework containing multiple values is needed. this framework has to be adapted, depending on the resource in question and the context in which the medical intervention takes place. it has been suggested that six specific recommendations can be derived from the four basic values described above for the equitable assignation of medical resources during the covid- pandemic : maximise benefits; give priority to healthcare workers; do not prioritise care in order of arrival; be sensitive to scientific evidence; recognise participation in research; and apply the same principles to patients with covid- and those without covid- . it is primordial to maximise benefits in a pandemic. for example, this involves the possibility of withdrawing a ventilator or icu bed from a patient when they would give another patient a greater therapeutic benefit. patients and their families must be aware of this possibility. , the clinical suitability of admission to the icu is a recurring aspect in the debate during the current pandemic, and decisions on this involve detailed analysis of the probability of survival and discharge from hospital. this analysis must be based on clinical judgment of the physiological reserve of the patient and their capacity to recover. patients are therefore classified in one of three priority groupings: high, moderate or low, and this will determine which patients are admitted first. the second recommendation involves prioritising healthcare professionals for the interventions intrinsic to covid- (testing, personal protective equipment, icu beds, ventilators, treatments and vaccines). all of these facilities must be available for first line medical care workers and those who maintain critical infrastructures, particularly those who are at high risk of infection and whose training makes them hard to replace. , these elements are essential parts of how our society responds to the pandemic, and prioritising them benefits the population as a whole. the third recommendation is not to prioritise medical care according to the order in which patients arrive. however, due to the pressure in reaching a decision, in circumstances when information is lacking, random selection is preferable to attempting to make more precise prognostic judgements within a group of more or less similar patients. being sensitive to scientific evidence involves not only prioritising resources in a restrictive way for those patients with the worst prognosis, as it also has a positive meaning. for example, when prioritising access to future vaccines for the most vulnerable individuals (after healthcare workers), with the aim of saving as many lives as possible, given that covid- evolution is significantly worse in older people and those with chronic diseases. lastly, recognising participation in research means prioritising resources as well for those people who are performing research into therapies for covid- . their acceptance of risk during their research work will help future patients, so that they should be rewarded. the difficult role of professionals in the microassignation of medical resources was described above, and this creates an additional emotional charge over and above the current complex care situation. some experts recommend distributing functions in teams, separating clinical professionals who provide care for the sick from those who, with the help of an expert team, work on the triage of resource allocation. , , , the latter will then report on their decision to the clinical teams, the patient and their family. in any case, as well as the information on the ethical basis and recommendation that documents such as the current review may make, or the protocols drawn up by the different bodies mentioned in this paper, as is the habitual practice in decision-making for complex questions at the level of care, we recommend team discussions to support decisions and to share the enormous emotional burden arising from more complex ethical decisions, and perhaps even urgently consulting the ethics assistance committee. medical professional responsibility consists of the obligation of doctors to remedy and satisfy the consequences of their actions, omissions and voluntary and involuntary errors, within certain limits, committed while doing their job. analysis of medical praxis in each specific case requires study of the specific circumstances in each situation (lex artis ad hoc). medical care now faces a praxis scenario that is not usual, and professionals act within the framework of a medical organisation conditioned by the crisis in care due to the pandemic and new regulations. this may give rise to claims against the action of professionals being transferred to the system itself or public authorities. in the current situation movements such as the one promoted by the humanising justice association have arisen. these underline that during this crisis professionals have been working without sufficient resources for the conventional exercise of their profession, under conditions contrary to conventional health at work. they have done so in a crisis and in unsuitable conditions in connection with the demandsatisfaction equation. many professionals have intervened in ways usually reserved for specifically qualified professionals, and that they have worked in installations that cannot strictly be considered medical. due to these considerations, they suggest forms of institutional rather than individual or professional responsiblity. it is within this scenario that the prioritisation of resources emerges. this question may have arisen beforehand in emergency situations or major accidents, when urgency is the core concern. the difference now is that it is not urgency but rather the lack of medical material or instruments for treatment that makes it necessary to prioritise and apply rationing, in a close association with the legal concept of loss of opportunity. another question will be to determine the degree of responsibility of the public authorities, or to apply grading, proportionality and responsibility criteria to the working of the administration. although it may be that there is no way to prevent a covid- pandemic in a globalised world, verified information is the most effective way to prevent panic caused by the infection. in the same way, faced with ethical problems that may arise in care during extraordinary situations such as those deriving from covid- , as well as to mitigate disinformation and the resulting uncertainty, exemplary transparency is needed regarding the criteria for allocating medical resources and especially sensitive clinical decisions. it is necessary to clearly explain the circumstances that give rise to rationing, if it exists, to patients, family members, medical professionals, the community and the media, as well as the form it takes. the current crisis in care leads to an especially complex scenario that means doctors have to work in a different way in comparison with their usual practice. this has to give rise to an active and intense ethi-cal debate, the results of which should be included in plans for care during future emergencies. who director-general's opening remarks at the media briefing on covid- - healthcare ethics during a pandemic ethics in the time of coronavirus: recommendations in the covid- pandemic principles of biomedical ethics evolución en las decisiones de limitación de los tratamientos de soporte vital en una unidad de cuidados intensivos durante una década limitación del tratamiento en una unidad de cuidados intensivos limitación de 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recommendations for the allocation of intensive care treatments, in exceptional, resource-limited circumstances clinical ethics recommendations for the allocation of intensive care treatments in exceptional, resource-limited circumstances: the italian perspective during the covid- epidemic is it wrong to prioritise younger patients with covid- ? informe del comité de bioética de españa sobre los aspectos bioéticos de la priorización de recursos sanitarios en el contexto de la crisis del coronavirus informe de la comisión central de deontología en relación a la priorización de las decisiones sobre los enfermos en estado crítico en una catástrofe sanitaria recomendaciones éticas para la toma de decisiones en la situación excepcional de crisis por pandemia covid- en las unidades de cuidados intensivos facing covid- in italy -ethics, logistics, and therapeutics on the epidemic's front line the choices in covid- 's time manejo clínico del covid- : unidades de cuidados intensivos manejo en urgencias del covid- consideracions sobre la limitació de recursos i decisions clíniques en la pandemia covid comite bioetica catalunya covid triage of scarce critical care resources in covid- an implementation guide for regional allocation: an expert panel report of the task force for mass critical care and the american college of chest physicians swiss society of intensive care medicine. recommendations for the admission of patients with covid- to intensive care and intermediate care units (icus and imcus) for the covid-task force of palliative ch. covid- : decision making and palliative care anzics guiding principles for complex decision making during the covid- pandemic principles for allocation of scarce medical interventions ethical considerations of the duty to care and physician safety in the covid- pandemic toma de decisiones en materia de triaje en cuidados intensivos en la emergencia del covid- . guía práctica para médicos y directivos de hospitales ethical rationing of personal protective equipment to minimize moral residue during the covid- pandemic whose life to save? scarce resources allocation in the covid- outbreak aspectos médico-legales de la responsabilidad profesional médica criterios medicolegales en la valoración de la responsabilidad profesional médica comunicado asociación humanizando la justicia en defensa del personal sanitario humanizando la justicia cuantificación de la pérdida de oportunidad en responsabilidad profesional médica covid- : fighting panic with information covid- : we need to be open about rationing ventilators key: cord- -i n mu b authors: callaghan, chris title: pseudoscience in medicine: cautionary recommendations date: - - journal: afr health sci doi: . /ahs.v i . sha: doc_id: cord_uid: i n mu b introduction: certain real life applications of scientific and social science ideas that knowingly reject accumulated empirical biomedical evidence have been termed ‘pseudoscience,’ or empirical rejectionism. an uncritical acceptance of empiricism, or even of evidence-based medicine, however, can also be problematic. objectives: with reference to a specific type of medical denialism associated with moral failure, justified by dissident aids and anti-vaccine scientific publications, this paper seeks to make the argument that this type of denialism meets certain longstanding definitions for classification as pseudoscience. methods: this paper uses a conceptual framework to make certain arguments and to juxtapose arguments for evidence-based approaches to medicine against literature that highlights certain limitations of an unquestioning approach to empiricism. results: discussions of certain real life examples are used to derive the important insight that, under certain conditions, moral failure can result in the violation both type i and type ii scientific error types, with catastrophic consequences. conclusion: it is argued that the validity of all theory should not be assumed before sufficient empirical evidence has accumulated to support its validity across contexts. however, caution is required, to avoid the consequences of an unquestioning approach to empiricism. certain emergent problems with potentially catastrophic consequences require medical responses in a matter of hours, or days. examples of these include outbreaks of zika , ebola , lassa , middle east respiratory syndrome (mers-cov) , swine flu/h n , or rapidly increasing global antibiotic resistance . to meet these challenges, healthcare professionals require up to date scientific knowledge. it is not enough that this knowledge is up to date. it also needs to be scientifically rigorous. a reliance on empirical evidence and rigor, however, should not be confounded with a dogmatic faith in empiricism itself, which can also be problematic [ ] [ ] [ ] . in light of this tension, between a need for a pragmatic approach to evidence-based medicine (ebm), and the dangers of an uncritical acceptance of ebm, amidst uncertainties asso-ciated with its shortcomings, this paper seeks to provide a discussion of these issues, and to derive cautionary recommendations for those exposed to these same issues. in doing so,dissident examples of aids , and vaccine denialism [ ] [ ] [ ] [ ] [ ] [ ] [ ] are employed as an interpretive schema, or lens, through which to view events that link dissident perspectives in scientific publication to harmful outcomes. such an approach offers a useful heuristic in order to highlight a certain type of harmful use of science, which, according to established literature , has previously been categorised as 'pseudoscience.' these examples are taken to represent pseudoscience in medicine, which is defined here as scientific denialism associated with moral failure, justifying itself in terms of dissident scientific publication in order to advance agendas with the potential to do medical harm to human populations. according to kuhn , [with the exception of extraordinary problems] the "three classes of problems-determination of significant fact, matching of facts with theory, and articulation of theory-exhaust, i think, the literature of normal science, both empirical and theoretical (p. ) ." medical pseudoscience, as discussed here, falls outside of kuhn's classes of problem. this definition of medical pseudoscience is derived here,specifically, from two examples. the first relates to the moral failure on the part of a government, associated with aids denialism, which justified its medically harmfulactions at the time on the basis of certain dissident scientific publications . the policy stance in this case persisted, for years, after the emergence of evidence of the large scale loss of human life caused by these actions. the second example relates to the emergence of the anti-vaccination movement, based on the publication of two scientific papers in particular , , which mooted a causal relationship between measles-mumps-rubella (mmr) vaccination and autism. this movement has also been linked to an increase in fatalities due to non-vaccination . through the use of a heuristic, in the form of the interpretive schema offered by these two examples, a textured discussion of the dangers of medical pseudoscience is provided here. the problems of dogmatic empiricism are also, however, highlighted, to demonstrate the pervasiveness of medical pseudoscience as a problem that cannot be simplistically argued away with reference to notions of scientific evidence. in light of the problem of medical pseudoscience, the objective of this paper is to inform those potentially affected by the phenomena discussed here, and to provide cautionary insights on the basis of a considered engagement with the literature. given recent literature that highlights the vulnerability of the scientific publication process to political and ideological agendas , it is important to note, up front, that science itself is also subject to agendas of power , , , and that an empirical approach to solving these problems of medical denialism cannot also be considered to be value free or more just than other, non-empirical approaches. indeed, although some have argued that political conservatives in the us have been prone to deny scientific evidence related to climate change, recent evidence finds that scientific denialism may characterise those both on the left and the right of the political spectrum . if no political denomination at this time has a monopoly on dissident, or anti-science, discourse, the arguments made in this paper are perhaps timely, and serve to provide cautionary insights, particularly for those at the nexus of ideological and political forces, where scientific work, and particularly empirical findings, might be vulnerable to immoral misrepresentation. it is argued here that the use of the term pseudoscience, albeit uncomfortable, might therefore be useful (or even necessary), in that such uncomfortable debates may be to the benefit of those most vulnerable and powerless in the face of medical need. the paper proceeds as follows. the need for a cautionary perspective on empiricism is first introduced. to further contextualise the discussions, the topic of medical nihilism is then also introduced. certain challenges associated with the notion of consensus are then considered, and discrepancies between published recommendations and clinical practice are then discussed. finally, consideration of the topics of medical denialism and academic pseudoscience round off the paper, and the conclusion section summarises its objective and its key arguments. unwarranted faith in empiricism can amount to little more than dogma. it must be acknowledged that here is little in the way of evidence to date that demonstrates that an empirical approach can eliminate the dangers inherent in human interpretation and subjectivity. the pervasiveness of medical denialism [ ] [ ] [ ] [ ] [ ] , and well-known historical cases, such as the rejection of semmelweiss's empirical evidence of how to reduce infections in sur-gicalprocedures , seem to highlight this problem. the discussions of medical pseudoscience undertaken here therefore need to be grounded in an acknowledgement of the limitations of empiricism itself. the problem of medical nihilism, according to poland , highlights seemingly insoluble problems associated withhuman choices to believe or disbelieve medical evidence. an important debatein the literature relates to discussions of medical nihilism. the mckeown thesis derives from mckeown's argument that in certain contexts the fall in the crude mortality rate over time was largely "due not to life-saving advancements in the field of medicine or public health, but instead to improvements in overall standards of living (p. )," and especially those related to advances in nutrition that resulted from improved economic conditions . debates about mckeown's thesis have persisted in light of the question, are "public health ends better served by targeted interventions or by broadbased efforts to redistribute the social, political, and economic resources that determine the health of populations? (p. )" . the mckeown thesis has, however, led to what some have termed medical health nihilism , whereby some (perhaps "an entire generation of social scientists, historians, and policymakers") have considered the contributions of economics and nutrition to overshadow the role of public health interventions (p. ) . while some, for example, have questioned the provision of treatment for multi-drug resistant tuberculosis,in certain contexts, in that it may not be cost effective, farmer and nardell stress that it is important to avoid the trap of medical health nihilism, in that even though poverty and inequality persist, it is necessary to, nevertheless, "move forward with focused interventions and insist on universal access to high-quality tb care (p )." consensus about medical phenomena, can, however, be problematic under certain conditions. according to social empiricism, consensus is typically epistemologically undesirable (solomon, ) . dissent is valuable, even when there is no discussion (because deliberation can sometimes "make things worse rather than better"), as it does not require discussion to make it valuable, as originally argued by the likes of mill, popper and longino (p. ) . an over-reliance on the dogma of empiricism might therefore open scientific endeavour up to the shortcomings of problematic forms of consensus. ebm, based on the work of clinical epidemiologists in the s and s, emerged as a "new paradigm" at the start of the s, associated with methods such as randomised controlled trials, systematic reviews, and meta-analyses, which produced "an extensive and powerful body of research (p. )" . relying heavily on statistics, probability theory and utility theory provide ebm with its conceptual underpinnings. with its hierarchy of evidence, ebm takes its epistemic techniques as superior to traditional methods such as expert opinion, clinical experience, and physiological reasoning. this stance has not gone unchallenged, however. indeed, solomon stresses that ebm has been described as a kuhnian paradigm, and that it has been criticised in terms of its procedural aspects , , in terms of its fallibility, or its lack of replicability , and its incompleteness as a philosophy of science. in terms of the latter, there has been a persistent criticism of ebm that it ignores the basic sciences that inform research and clinical practice, whereby it is "scientifically superficial: it measures correlations," failing to "theorize about the complete organism, still less the complete organism in its social and environmental context (p. )" . further, according to solomon , literature suggests that "publication bias, time to publication bias and pharmaceutical funding bias (which subtly affects trial design and evaluation) are responsible for the worse-than-expected track record" of random controlled trials, systematic reviews, and meta-analyses (p. ). considering these criticisms, solomon suggests an instrumental, or pragmatic approach, whereby evidence should be ranked with reference to actual (and not theoretically expected) reliability of results. according to solomon , with the "recent emphasis on translational medicine, we are seeing a restoration of the recognition that clinical research requires an engagement with basic theory (e.g. physiological, genetic, biochemical) and a range of empirical techniques," in that ebm "works best when used in this context (p. )." translational medicine seeks to develop interdisciplinary synergies between researchers, and to create interactive linkages between basic science research and research in clinical medicine. translational medicine might ultimately hold the key to reducing problems associated with discrepancies between published recommendations and clinical practices. much work in other fields draws on precedent in medical research, in order to follow a systematic process, so as to develop evidence-informed knowledge . thus medical science is acknowledged to lead the way in evidence-based research, notwithstanding the problems associated with dogmatic approaches to empiricism. research on medical practice patterns has revealed a discrepancy between "published recommendations and clinical practices" . according to bryg and johns : the emotional appeal of interventional therapy is often so strong that rational thought is denied…physicians and patients often feel better trying "something" rather than waiting; interventional procedures are powerful and seductive for those seeking action….many different factors can be given to explain differences between clinical practices and recommendations based on controlled trials. the current efforts to address the medicolegal system and remuneration may not be enough. to change the "art' of medicine through the "science' of randomized trials, attention to all of these issues is needed to change practice patterns and help us "practice what we preach (p. )." seminal theory also points to how discrepancies between published recommendations and clinical practices. the seminal work of kuhn predicts that paradigmatic change can occur in scientific research, but that researchers typically reject novel facts that do not conform to the assumptions and values associated with a particular paradigm. in light of the potential consequences of pseudoscience, this paper seeks to explore the central tenet of kuhn's work, that advances in science are subject to changes in human values systems and not only to the objective advances of science itself, in relation to examples of how evidence can be ignored, at great human cost. by making these relationships explicit, we might be less likely to repeat the mistakes of the past. kuhn's theory suggests that advances in science are essentially a function of how the values and beliefs of scientists in fields change, whereby in 'normal science' fields will typically resist paradigmatic change in the face of contrary evidence, until eventually a tipping point is reached, and sets of values and beliefs then change to accommodate the new paradigm. kuhn's theory can be used to draw useful inferences, particularly when applied to the phenomenon of 'pseudoscience' and its potentially harmful influence. as discussed, a powerful example of kuhn's theory in the medical context is the work of semmelweiss. the case of semmelweiss illustrates how professionals can reject innovative ideas even in the face of evidence of the catastrophic human costs associated with this rejection. semmelweiss, a doctor in the s, demonstrated dramatically lower surgical mortality rates due to handwashing using a chloride of lime solution. even after providing evidence of this life saving process, his ideas were rejected. it was only two decades later that his work was revisited by the medical profession , and the 'new paradigm' of sterile hand washing was embraced. other seminal work, for example by lakatos also stresses the subjectivity of the research process, as fields like newtonian physics were shown to have rejected novel facts that challenged the 'hard core,' or fundamental tenets of the field. this work can also be taken to support kuhn's arguments that it cannot always be assumed that objective evidence will be the basis for how scientific decisions are made. there are other examples of kuhn's theory at work in the social sciences. events such as the sokal affair, orsokal hoax have highlighted the vulnerability of the academy to empirical rejectionism, or rejection of evidence-based approaches in academic or scientific research, or pseudoscience. still and dryden suggest two types of pseudoscience, one related to academic fields entailing deep engagement with some kind of academic process, termed 'big' pseudoscience, and one related to 'erroneous' public beliefs, with less of a deep engagement in academia, but often with a tenacious grip on the beliefs of many within populations. a core argument presented in this paper is that, as predicted by kuhn's theory, professional medical work, including policymaking, requires constant vigilance on the part of medical professionals so as to avoid the influence of pseudoscience. the examples of aids and vaccine denialism might be a useful interpretive schema, or lens, through which to view events that link dissident perspectives in scientific publication to harmful outcomes. as stressed previously, science is also an agenda of power , , , and an empirical approach to solving these problems of medical denialism therefore cannot also be considered to be value free or more just than other, non-empirical approaches. nattrass summarises certain catastrophic events at the nexus of pseudoscience, political power, and moral failure on a national scale: aids policy in post-apartheid south africa has been shaped by persistent antipathy towards antiretroviral drugs (arvs). this hostility was framed initially by [the president's] questioning of aids science and subsequently by direct resistance to implementing prevention and treatment programmes using arvs. once that battle was lost in the courts and in the political arena, the health minister [at the time] continued to portray arvs as 'poison' and to support alternative untested therapies. demographic modelling suggests that if the national government had used arvs for prevention and treatment at the same rate as the western cape (which defined national policy on arvs), then about , hiv infections and , deaths could have been prevented between and . two key scientific bodies, the medicines control council (mcc) and the medical research council (mrc) fall under the ambit of the national department of health. although notionally independent, both have experienced political interference as a consequence of their scientific approach towards aids (p. ). this example is illustrative, offering useful insights into a paradoxical situation of power misuse that led to the deaths of many who were the most vulnerable and powerless. this paradox seems to mirror the anti-science discourse driven by politicians in current political discourse, whether relating to the denial of climate change, or the selective use of science in support of political agendas (for a useful summary of these current issues, see washburn and skitka ). given the hundreds of thousands that may have died due to the amoral implementation of political ideology in the case of south africa, describing this using the term 'medical pseudoscience' is perhaps necessary (uncomfortable as the term may be to the academic ear), so as to not do disservice to those that have perished due to the application of this particular type of scientific denialism. indeed, it would seem that such events pass out of consideration relatively quickly, both by the public and by academic discussions. those involved in this perpetration live on, with little in the way of accountability. this example seems to illustrate a unique form of pseudoscience, which bears classification as such, and the development of its own stream of literature.exploring this paradox is useful, as it can offer insights into tensions between science and agendas of power. further exploration of this example offers more detailed insights into the harm that pseudoscience can cause. the use of this particular example might also serve to highlight the differences between pseudoscience and discourse that highlights legitimate criticisms of evidence-based approaches to medicine. this particular form of pseudoscience is taken here to relate to the power relationships of political actors and the prioritisation of ideology, in such a way as to actively engage in empirical rejectionism, at the expense of the most vulnerable and powerless in a society. an important lesson can be learned from these events. this large scale loss of human life was found to be due to scientific denialism on the part of certain respected academics. according to nattrass the cause can be traced back to the publication of scientific research, as accomplished (highly respected at the time) academics argued that azt itself caused aids rather than treating or preventing it. an example of the link between these events and scientific publication is duesberg's . denialist members of the presidential aids advisory panel (half orthodox scientists, half aids denialists) asserted that "aids would disappear instantaneously if all hiv testing was outlawed and the use of antiretroviral drugs was ter-minated (p. ) . another example of this type of pseudoscience, in the form of medical denialism, or empirical rejectionism, is the spread of the anti-vaccine movement. the link between dissident academic publication and the rise of the pseudoscientific anti-vaccine movement is also seen in the emergence of the populist anti-vaccine movement, which advocated halting mmr immunisation . this example of vaccine nihilism, according to poland , highlights the problem that "there are no magical solutions to the deep divide between those who accept the scientific method and evidence and those who do not and simply choose to disbelieve the evidence (p. )." this movement emerged on account of scientific publications that put forth the thesis that mmr vaccine was linked with the development of autism , . this example also serves to highlight the dangers of denialism, and its potential human costs [ ] [ ] [ ] . policy applications of pseudoscientific ideas and ideologies, including economic ideas, and the human costs of these are beyond the scope of this work, but further study of examples like this by further research might be instructive. this example clearly highlights kuhn's predictions, challenging assumptions that empirical rejectionist behaviour is necessarily benign. resistance to scientific thought itself has a long history. contestations between empirical rejectionist belief systems and science, and the catastrophic costs in human life caused by belief-system resistance to science throughout history is well documented . following kuhn , a condition for scientific advancement seems to be its coincidental alignment or convenient instrumental value for powerful agendas. in contrast to south african aids denialism in the context of a developing nation, the anti-vaccine movement serves as a cautionary tale about the pervasiveness of pseudoscientific beliefs in developed nations. academic literature on pseudoscience may offer further insights into these discussions. seeking to define prevalence of pseudoscience in the psychology discipline, lilienfeld suggests that academics follow bunge's seven 'indicators of pseudoscience' for guidance. these include: (i) overuse of ad hoc hypotheses to avoid refutation, (ii) emphasising confirmation, above refutation, (iii) lack of self-correction, (iv) reversed burden of proof, (v) excessive reliance on testimonial and anecdotal evidence, (vi) use of obscurantist language, and ( ) lack of connectivity with other disciplines. the use of science to justify the objectives of the aids denialist state, and the objectives of the anti-vaccine movement are considered to warrant the term 'pseudoscience' as they seem to meet these criteria. more than this, these examples offer examples of moral failure that seem to require differentiation from other instances of conflict with empiricism. thus, it is arguably useful to differentiate these examples by using the term pseudoscience, so as not to confound their discussion with broader debates on empiricism itself. it is therefore important to provide an informed discussion that embraces these broader debates, and which locates these two examples of a specific form of pseudoscience in relation to debates on empiricism, and the weaknesses of dogmatic empiricism as a potential remedy for the problem of denialist pseudoscience. to understand the real threats of pseudoscience it is perhaps necessary to return to first principles. an example of this kind of 'first principle' can perhaps be found in the doctrine 'do no harm.' with reference to the field of psychology, which has no equivalent of the food and drug administration's (fda's) oversight function, lilienfeld stresses the importance of the medical and mental health professions' credo primum non nocere, or 'first, do no harm.' the harm associated with pseudoscience is well illustrated in light of the examples considered above. the tensions between unlimited thought in academic contexts (which are good) and opportunistic use of ideas for political or ideological reasons that are largely inseparable from issues of power use and abuse, which can lead to loss of human life (which are bad) are not independent of real life contexts informed by medical science. a conceptual framework is needed in order to better understand these tensions. according to solomon , from "the work of historians of science, sociologists of science, anthropologists of science, feminist critics, social psychologists and decision analysts, we now know much more about the variety and pervasiveness of bias," whereby no one "has designed a group (or individual) scientific practice in which bias is eliminated, or even reduced to insignificant levels (p. )." solomon's work has sought to offer scientists a heuristic based on decision vectors, or the decisions that are made to accept one theory over another, particularly when both offer overlapping predictions about phenomena, or where both seem to fit the available data. these decision vectors can be based on empirical data or conceptual rationales. arguably, the wellspring for most pseudoscientific ideas has been academia. as well it is expected to be, given academic freedom and the role of the academy in generating all types of ideas, without limit. how do we then differentiate theory from recommendations for healthcare practice? figure offers a model, with the hope that it might be a useful heuristic for understanding the tensions between the two scientific error states, namely between making a type error of rejecting a true novel finding, versus making a type ii error of failing to reject false novel findings. these representations acknowledge the criticisms of empiricism highlighted in previous sections, and relate only to discussions of the tensions between theorising and empirically testing theory. in health sciences research, the centre line (in bold) shown in figure represents the optimal state, where the types of errors are balanced. this, in many studies, is put at the % confidence threshold, where statistical testing is at the five percent level of significance. as we know, however, this value is arbitrary, as increasing the size of a sample can increase the chances of obtaining a significant effect. a health science practitioner or researcher, however, needs to navigate between modes in different roles. as a theorist, one might need to be open to all ideas, and to consider all potentialities. to theorise innovatively, one would need to sacrifice validity temporarily, to develop models that can then be tested for validity. dissident theory that predicted that hiv was caused by arvs, for example, is an example falling into the low validity portion of the continuum shown in figure . however, so should all health theory that has not been empirically tested, in some way. this example is useful as a special case, however, in that it represents the extreme of type error, whereby these scientists rejected accumulated evidence that clearly supported the use of arvs. a type ii error, however, is also evident, in that there was a failure to reject theory that was patently at odds with the accumulated evidence to date. the model shown in figure is only useful in that it identifies problems related to validity versus problems related to conservatism and lack of innovation, but it does not show that at the extremes, both the violation of type and type ii errors can converge. the example of aids denialism illustrates that it is important for a healthcare practitioner to understand that there is not simply one continuum of error that can be made, but that both kinds of errors can mirror each other at the extreme. whereas many have discussed the example of semmelweiss , this instance of a type i error has seemingly not to date been considered in relation to an example of the coincidence of type and ii. this is however evident in a government's refusal to allow treatment of hiv victims with arvs on the basis of theory developed by respected academics. similarly, while seminal theory bylakatos ( ) also stresses how core tenets of fields can reject new knowledge, this theory also relates primarily to type errors, as does kuhn's seminal work. the sokal hoax highlights the potential for theory to be published by respected journals, but which was specifically formulated on the basis of confounding logics. further research should differentiate between extreme forms of type i and type ii error violation. the two forms of pseudoscience discussed here might be taken to exhibit characteristics of these extremes, and that such a characterisation might be useful in differentiating them from other forms of medical denialism. a contribution of this paperis arguably in the way it identifies and highlights dangers associated with the way that both type i and type ii errors can actually be committed under certain circumstances. at these extremes, a phenomenon is identified, termed medical pseudoscience, a relatively unique form of pseudoscience that takes the form of extreme denialism, but which is vulnerable to those with political agendas or other motives associated with moral failure. knowledge of this categorisation is arguably important, particularly when healthcare providers and policy makers can be lulled into a false sense of security in deriving policy or practice from dogmatic perspectives of empiricism that do not acknowledge the shortcomings of empiricism, or ebm itself. certain conclusions and recommendations that derive from this analysis are now presented. the objective of this paper was to provide a discussion of the dangers of medical pseudoscience, which might be particularly relevant in a global context wherein anti-science, or dissident and denialist discourse, seems to alsobe driven by political forces, making such discourse vulnerable to agendas associated with moral failure. it is concluded that taking recourse to unquestioning approaches of empiricism may however also be wrongheaded. instead, the approach of solomon - is recommended, one of cautious pragmatism. more specifically, evidence should be ranked with reference to actual (and not theo-retically expected) reliability of results . thus, threats associated with medical nihilism might be mitigated, pragmatically, to reduce harm. in extending debates about dissident and denialist medical discourse, this work sought to build on other, related, work. such work includes research that highlighting the dangers of inflexible and slow responses to epidemics , a lack of an adequate bioethical response to novel discoveries , constraints posed by human values to scientific progress itself , and changes to the scientific discovery process on account of emergent technologies , . this work also sought to extend discussions of empiricism to embrace issues associated with medical pseudoscience. it is also concluded that these examples of medical pseudoscience might benefit from further research that conceptualises them as extreme forms of the violation of type and type ii errors, whereby at the extremes, such phenomena share epistemic similarities. in conclusion, much hope rests on the promise of translational medicine , and its potential to address certain of the shortcomings of empiricism through a more interdisciplinary and pluralistic approach to both the methodological and theory-development processes of medical science. going forward, however, epistemic caution may be an important watchword in a world in which 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management knowledge by means of systematic review falsification and the methodology of scientific research programmes a physicist experiments with cultural studies the social psychology of 'pseudoscience': a brief history disaster management, crowdsourced r&d and probabilistic innovation theory: toward real time disaster response capability ethics preparedness for public health emergencies: recommendations from the presidential bioethics commission citizen science and biomedical research: implications for bioethics theory and practice values infections and the epidemiology of values: implications for management reinventing discovery surviving a technological future: technological proliferation and modes of discovery anonymous inputs and insights resulting from the review process are gratefully acknowledged. key: cord- -d s akd authors: akabayashi, akira title: a brief history of bioethics in japan date: - - journal: bioethics across the globe doi: . / - - - - _ sha: doc_id: cord_uid: d s akd in this chapter i look back at the history of bioethics in japan, which can be divided into three phases: phase i, introduction ( – ); phase ii, development ( – ); and phase iii, the recent past ( –present). phase i marks the period when the concept of bioethics that originated in the west came to japan. it was also when japanese society faced its first difficult bioethical issues: namely brain-death and organ transplantation. other issues emerged during this period, particularly pertaining to death, such as end-of-life medical care and euthanasia. in phase ii, the problems shifted to those pertaining to the beginning of life, such as the moral status of the human embryo. as well, during this period the government implemented ethical guidelines for research ethics. during this period, social awareness of bioethics increased, and bioethics education began to appear not only medical education, but also within high school curricula. in phase iii, japan began to tackle its own ethical issues, such as enhancement, regenerative medicine, neuroethics, public health ethics, and precision medicine. some of my thoughts concerning projections for the future are discussed at the end of this chapter. in this introductory chapter, i consider the current period (ca. ), look back on the history of bioethics in japan over the past years, and finally, look briefly toward the future. first, i present an overview of the types of problems that have been handled in the fields of bioethics and medical ethics in japan since the s. i begin my discussion in this era because modern bioethics began its development in japan in the early s. i divide this time period (beginning in the early s until around ) into three parts, corresponding to phase i, introduction: - ; phase ii, development: - ; and phase iii: the recent past ( -present) . no significant distinction is intended between use of the terms "bioethics" and "medical ethics." for full description, refer to references [ ] [ ] [ ] . this chapter is a brief summary of those three papers and some further considerations. in japan, the issue of brain-death and organ transplantation was highly influential to the development of bioethics (see chap. ) . beginning in the s, nationwide debates began as to whether brain-death constituted human death, and whether organ transplantation from a brain-dead body should be permissible. these issues were discussed in medical circles, religious groups, political groups, the media, and among the general population. with the establishment of a commission on braindeath, and the enactment of the organ transplantation law, organ transplantation from a brain-dead body was finally deemed permissible, with strict conditions. specifically, the clear and written expression of the intent to donate from the organ donor ( years or older) as well as the family's consent were both required. politically, this marked a major milestone. several positive outcomes were achieved, including discussion on the definition of death and japanese views on life and death; this set a healthy tone for how bioethical arguments should be handled in japan from that point forward. at about the same time, the idea of informed consent began to emerge as an issue in clinical settings (see chap. ) . informed consent in the clinical context entails an act in which "medical caregivers provide sufficient explanation to those with sound judgment capacity and ensure that the patient understands, while the patient then offers consent of their own volition." today this is considered common protocol, but such was not always the case in japan. there was even extensive debate on how to translate the english term "informed consent" into japanese; today, the term "infomudo konsento" is used, but is presented in katakana, the phonetic alphabet used for foreign terms and names. in the course of this discussion, those arguing to protect patient autonomy, basing their arguments on self-determination and the right to know, were in a dominant position. others countered that argument, stating that prioritizing individual autonomy so heavily is unsuited for japan. however, an explanation of the patient's condition has now been systematically added to the treatment plan at the time of hospital admission and discharge, such that it is covered by health insurance [ ] . informed consent is therefore a common theme that has been integrated into medical care. this flow of events necessitated a change in paternalism among physicians. it also led to the medical record disclosure system based on the private information protection law in . one other major event during phase i was the tokai university euthanasia incident. in this case, a physician, in response to a clear request from the patient's family, administered potassium chloride (kcl) to a terminal cancer patient [ ] . he was found guilty of murder and given a suspended sentence by the yokohama district court in . the trial was an unprecedented case in which a doctor performed euthanasia. as the court decision presented the conditions that would allow for active euthanasia in the obiter dictum, it was erroneously relayed to other countries that active euthanasia was legally permissible in japan. reports of physicianassisted suicide emerged from abroad, and terms such as euthanasia, mercy killing, and dying with dignity were used without clarity. currently in japan, the act of a medical caregiver administering muscle relaxants or kcl to a patient, leading to the latter's death, carries with it a high probability that the medical caregiver will be placed on trial for murder. notably, unlike several other countries, japan has not legalized active voluntary euthanasia. in addition, with the increased popularity of hospice or palliative care, fewer cases of "withholding treatment" requested ahead of time by patients are legally problematic. however, the issue of "treatment withdrawal" (removal of an artificial respirator) from a terminal patient has yet to be subject to legal resolution (see chap. ). there are three characteristics of phase ii. first, the topics of discussion shifted from issues pertaining to the end of life (brain-death, end-of-life medical care, and euthanasia) to those dealing with the beginning of life, particularly the moral status of the embryo and problems related to reproductive medicine. second, numerous policy guidelines and legislation were produced in the fields of life sciences and . phase ii: development ( - medical care, paving the way for the establishment of a framework for policy decisions. third, bioethics came to be socially acknowledged in the fields of medical education and research. in the second phase, topics in medical ethics shifted to issues pertaining to the beginning of life. among them, the heightened discussion surrounding the moral status of the human embryo became particularly significant. at this time, research on embryonic stem cells (hereafter, escs) had become widespread. human escs have the capacity to differentiate into all kind of tissues and all cell types, and there was great hope for these cells in the field of regenerative medicine. however, in order to create human escs, one must destroy a fertilized egg (embryo) that has the capacity to become a human individual in the future. if one perceives that human life is present in an embryo, then this type of use of embryos cannot be approved. in , the council for science and technology policy and the experts panel on bioethics compiled the document "basic principles concerning the handling of human embryos." this document stated that the human embryo is not a person per se, but it is also not an object. the document instead uses the phrase, "sprout of human life." the writers conclude that a human embryo is worthy of respect and requires careful handling (see chap. ). fundamentally, "the guidelines for derivation and utilization of human embryonic stem cells" and "the act on regulation of human cloning techniques" take the same stance as "the basic principles concerning the handling of human embryos." given that the moral status of the human embryo is a topic of deep religious and political debate in other countries, it seems that japan has actually handled this issue with relative ease. however, it remains a mystery as to why abortion-another debate that should be begging many questions about the moral status of an individual prior to birth-did not gain as much momentum as that surrounding the human embryo. at the end of the twentieth century, numerous governmental ethical guidelines (ethical guidelines for human genome/gene analysis research, ethical guidelines for epidemiological research, and ethical guidelines for clinical research) as well as a number of laws (the organ transplant law, the act on regulation of human cloning techniques) came into the fields of life science and medical care. many of these were drafted by governmental review boards or committees. these drafting sessions came to include medical and legal specialists as well as ethicists, and representatives from the general public such as journalists. by this time, a new framework had been put in place for policy-making in the fields of life sciences and medical care. this was a linear process that began with forming a public committee comprising specialists from many disciplines, drafting the guidelines, implementing the public comment system, and publishing the final results on the internet, followed by media reporting and revisions over several years. this framework reflected the nature and process of the debate surrounding organ transplantation from brain-dead donors. by the year , "medical ethics" had been added to the curriculum of medical schools nationwide, and faculty members were tasked with teaching these classes. in the early stages, these classes were taught by faculty in forensic medicine, public health, and philosophy/ethics departments as part of general education courses. however, in , the first graduate level department of biomedical ethics was established at the kyoto university graduate school of medicine, and several full-time faculty members were hired. in , the university of tokyo graduate school of medicine also created a department of biomedical ethics. as this process continued to unfold, the "ethics of medicine" education for students in healthcare-related areas became standard. meanwhile, the elsi (ethical, legal, and social implications) programs in the life sciences and medicine were offered a substantial amount of public and private research funding. by the end of phase ii, bioethics had earned a well-respected place in education and research. in what follows i introduce several topics that have emerged or are just emerging in japan. these are not unique to japan, but i would like to emphasize those which have become prominent in the japanese context. "enhancement" refers to a specific use of medical technology, that is not been used to treat or prevent disease. it has been defined as an "intervention aimed to improve the human form and function more than is necessary for maintaining health or recovery" [ ] . a more straightforward definition is set forth by kato, a japanese philosopher, who defines enhancement as "employing medical technology for a purpose other than treatment." examples of enhancement include the administration of growth hormones for children who are not suffering from growth restriction, "doping" or the use of steroids to enhance muscle strength in sports, and the use of mind-altering drugs such as ritalin (methylphenidate) and prozac (fluoxetine hydrochloride) by healthy individuals to enhance their attention (learning capacity) or mood ("smart pills" or "happy pills"). in the future, we may be dealing with designer babies due to genome editing, increased longevity, and enhancement using a brain-machine interface that can operate machinery by connecting a brain to a computer. ideas to improve humans are not new, but developments in medical technology now present the possibility of altering the human body, which has led to many debates surrounding the ethical issues involved. those opposed to enhancement are concerned about its widespread use, advocating that it is unnatural for human beings to "play god," that there are unknown dangers, and that we would lose sacred values such as the importance of weakness and being interlinked with one another. meanwhile, those whose stance is one of the passive promotion, that is "so long as each individual's freedom to choose is respected we cannot go so far as to prohibit this," are in favor of enhancement, as are those who actively promote it based on utilitarian principles, arguing that enhancement promotes the happiness and enjoyment of human beings, and thus we are obligated to pursue it. in japan, rather than either approving or prohibiting all forms of enhancement, each issue is considered individually. some also feel that as long as society respects individual self-determination and the multidimensionality of values, blanket prohibition of enhancement cannot be enforced, but some restrictions may be warranted. these discussions will help us to grapple with some important issues related to the nature of human beings and society. in recent years, a new and rapidly developing field of study has emerged that deals with ethical problems related to neurology or neuroscience and applicable technologies. this field has come to be known as "neuroethics." safire defines neuroethics as "the examination of what is right and wrong, good and bad about the treatment of, perfection of, or unwelcome invasion of and worrisome manipulation of the human brain" [ ] . two major developments in neuroscience aided in creating momentum for neuroethics. first, the development of brain functional imaging technology such as pet and fmri made it possible to observe the brain functions of a living human being. this new technology presents the possibility of obtaining an even more diverse array of information, such as lie detection and clarifying an individual's level of consciousness. however, it also opens up possibilities for mind-reading, or the ability to read the condition of another person's spirit. the second major technological development involves selective pharmacological and anatomical intervention/manipulation of neurological processes. for example, it is now possible to control tremors caused by parkinson's disease with deep brain stimulation. this technology could potentially be applied to patients with other diseases. moreover, it could also be used for brain enhancement in healthy people. brainwashing and mind control for military purposes have also come into the debate. the thought of a brain-machine interface and chimeras of humans and animals being created evokes science fiction-like images of cyborg production, generating fear among people. some attempts have been made to resolve this fear through twoway communication between scientists and non-scientists, for example, and by improving scientific literacy among the general population and performing risk assessments and encouraging participation from ordinary citizens. the japanese government is proactive about neuroscience research and has a national brain science project; the strategic research program for brain science. while there are no specific topics unique to japan, elsi studies of techniques developed in japan such as decoded neurofeedback are ongoing. in , dr. shinya yamanaka created the nobel prize-winning human induced pluripotent (ips) cells at kyoto university, using technology that allows for the creation of cells with the same differentiation function as human escs by incorporating multiple genes into an adult somatic cell. this technology did not require the destruction of a human embryo, and thus generated much hope for cell transplantation medicine that does not involve ethical issues or rejection responses. it was a breakthrough in the field of life sciences as well as the ethical arena. currently, ips cell research has moved from the basic research stage to drug development and even clinical applications such as cell transplantation. the japanese government is actively promoting ips cell research. however, were the ethical problems truly and completely eliminated by ips cell technology? here, i discuss some of the remaining issues. the first of these is safety. the fact that these cells can differentiate into many cell types means that the possibility of cancerization cannot be ruled out. in this regard, attempts are being made to create ips cells (using drugs, for example) that lower the number of, if not eliminate the need for, gene recombinants. this is something that future technological advancements will likely be able to overcome. next, whether or not it is permissible to create reproductive cells (sperm or egg cells) from ips cells is an important ethical issue. in research using human cloned embryos, there was some concern that if the cloned embryo was returned to the uterus, then an individual (a cloned human being) would develop. as such, returning a cloned embryo to the uterus was prohibited by the act on regulation of human cloning techniques. for ips cell research, at least in theory, if it is possible to induce differentiation into sperm and egg cells, then it may also become possible to fertilize these. as was the case with the argument surrounding cloning, problems arise concerning the uniqueness of a human individual. another developing discussion in this field concerns differentiation into neurons. for example, if a human neuron is grown for research in an animal brain, there is some concern that this animal will develop a human personality. this may present to us a problem related to fusion between humans and animals at the cellular or genomic level. another challenge is that of stem cell research, for which various uses can be imagined (for example, controlling the direction of differentiation, genetic recombination, and fusion with cells from other species). the problem for researchers then becomes how much consent to obtain from cell donors, and for cell donors, how much they should understand about the future destinations of their cells. the course of medical ethics in the second half of the twentieth century can be summed up by the phrase "from paternalism to individual self-determination." part of the background for this was the change in disease patterns. namely, from infectious diseases to lifestyle diseases, which led to a change in thinking about disease as something that should be prevented at a population level, to that which should be prevented and treated individually. however, in recent years, the dangers of new and re-emerging infectious diseases such as hiv/aids, malaria, sars, and novel forms of influenza have increased. in addition, concern has mounted for bioterrorismled smallpox outbreaks, or a recurrence of polio, leading us to refocus our attention on the importance of mass prevention. in japan, the worldwide spread of the h n influenza virus in is a recent collective memory. this engendered social debate, especially with regard to who would be the first to receive the vaccine. it is clear that situations are emerging that cannot be well addressed by the kind of bioethics that prioritizes individual self-determination. the opposition and tension between public welfare and individual freedom can be seen in the debates about quarantines and vaccinations for infectious diseases, discussion on handling vaccine distribution, and individual lives being affected by governmental interventions aimed to promote health. meanwhile, they also create more difficulties than ever before for equal distribution (justice) of benefit and cost. simply stated, the central arguments concerning these issues pose the following questions: in what situations should individual autonomy and self-determination be restricted, and what degree of intervention for the sake of the common good and paternalistic interventions should be permitted? a new discussion framework for medical ethicsone that is not limited to the conventional patient-caregiver relationship but rather forms the basis for healthcare policy for the various issues plaguing modern-day public health-is in the process of development. discussion concerning this topic is just beginning in japan. due to groundbreaking developments in genomic medicine, the time will come when at the time of our birth (or indeed at conception), we will know our genetic destiny. this raises many serious ethical issues. for example, if it is discovered at birth that a child would develop a severe disease by the age of , what sort of care should that child receive? what would we do if we found out that we would develop dementia by the age of ? would we be tested early on and take preventive drugs? adults may have the capacity to make these sorts of decisions, but the ethics of administering these tests to underage individuals is not as clear. is testing justifiable in children if the intent is to prevent disease or slow its progression? should genome editing be conducted, ensuring that the genes causing disease are eliminated prior to birth? alternatively, is this an area into which humans should not intervene? this would change the nature of medicine entirely. similar issues are raised by the development of artificial intelligence, as implementation of this technology may change the physician's role markedly. currently, in an age in which progress has been realized in genomic medicine, precision medicine will likely become one of the most difficult ethical dilemmas for mid-twentyfirst century medical care. having touched on the history of bioethics in japan, i will turn to some issues for the future. first, i anticipate that the life sciences and medical technology will continue to progress further, giving hope to many suffering from currently incurable diseases. some of the developments, such as immune-checkpoint inhibitors, ips cells, and escs, are technological innovations that may help to overcome conventional ethical problems. however, as is evident in issues related to enhancement and neuroethics, forward progress in medical technology that hides within itself great possibilities can, on the one hand, give us great hope for the development of new treatments, while also creating friction between conventional values and ways of living, causing anxiety in society. moreover, when technology is first introduced, it is difficult to envision sufficiently the influence it will have. how should society handle the "uncertainties" that inevitably and constantly accompany these new technologies? constructing a framework to ensure that these are addressed effectively is one major challenge for the future of medical ethics in the japanese context. in addition, even if the arguments in medical ethics shift over to those concerning the beginning of life against the backdrop of new technological developments, issues with terminal medical care and end of life will continue to cause worry among patients and their families, as well as among medical caregivers involved in treatment. with regard to the many problems that will inevitably emerge, as well as for those that already exist, attempts to address these will likely occur by establishing guidelines or legislation through japanese governmental committees, as described above. thus, the framework required to address these issues, while befitting the japanese context, also comprises an effective method that listens to external voices and adjusts to create harmony in society in order to resolve ethical issues in life sciences and medical care. yet, i cannot help but assume that the excessive use of . the future of bioethics in japan guidelines and laws creates a dependency among medical caregivers and researchers, such that they lose the motivation to face problems head-on and think deeply about them, or when necessary, communicate to the outside world about issues. in questioning whether a hospital ethics consultation (a system in which an ethics committee or its equivalent would offer advice for individual cases) would even function well in this particular climate, i can imagine numerous medical caregivers who would be baffled by having to find answers to difficult ethical issues in the clinical setting. indeed, the demand not only to accomplish all they do in their busy workday but also to tackle these ethical issues is a tall order. however, we cannot assume simply that because laws and guidelines are in place, all problems will be resolved quickly. if we hope to resolve these ethical issues, we must take a multifaceted approach that is both policy-based (guidelines are issued, ethics committees are established, and healthcare systems are improved) and education-based (awareness and problem-solving capacities are increased among medical caregivers and researchers in those fields as a result of medical ethics education). this is what i envision for bioethics in japan. in the chapters that follow, there are many passages in which i question the japanese government's policies in regard to bioethics and research ethics. however, my intent is not to criticize the government, but rather to bring the issues to light and describe them in their rawness through the lens of japanese culture. if i do not do this, my concern is that international trust and confidence in japan's research and medical care will deteriorate over time, and the value quality of japan as a nation will decrease. this is not a lack of patriotism. as noted in the introduction, the present book was written to achieve a global scale of bioethical dialogue; to this end, i cannot shy away from criticism of my own country's policies or cultures in this process when this is necessary. the late japanese scholar donald keene, in dialogue with jakucho setouchi in the book "nihon no bitoku [the virtues of japan]," said, "since obtaining japanese citizenship, i have come to think that i should …express my opinions as a japanese citizen," and, "now that i have become a japanese citizen, i intend to freely speak out against japan in ways that i have refrained from in the past." there can be no deep-rooted democracy in a society where the act of criticism itself comes under critique even before validation of the substance of the criticism. in japanese society today, however, many people internalize an obedient spirit of servility toward current systems, without so much as questioning authority. i too have been quite hesitant to criticize japan up to this point, but like keene, i must say what should be said before i reach the final years of my life. in this book, i speak of what i call "the dark side" of japan, as seen through the lens of bioethics, so that non-japanese readers can truly understand japan. there are already enough publications which describe japan's advantages. open access this chapter is licensed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ): importation, development, and the future biomedical ethics in japan: the second stage nurturing the future of medical ethics in japan paying for informed consent euthanasia, assisted suicide, and cessation of life support: japan's policy, law, and an analysis of whistle blowing in two recent mercy killing cases enhancement uses of medical technology neuroethics: mapping the field. new york: dana foundation key: cord- -dxrrpxl authors: marchiori, chiara; dykeman, douglas; girardi, ivan; ivankay, adam; thandiackal, kevin; zusag, mario; giovannini, andrea; karpati, daniel; saenz, henri title: artificial intelligence decision support for medical triage date: - - journal: nan doi: nan sha: doc_id: cord_uid: dxrrpxl applying state-of-the-art machine learning and natural language processing on approximately one million of teleconsultation records, we developed a triage system, now certified and in use at the largest european telemedicine provider. the system evaluates care alternatives through interactions with patients via a mobile application. reasoning on an initial set of provided symptoms, the triage application generates ai-powered, personalized questions to better characterize the problem and recommends the most appropriate point of care and time frame for a consultation. the underlying technology was developed to meet the needs for performance, transparency, user acceptance and ease of use, central aspects to the adoption of ai-based decision support systems. providing such remote guidance at the beginning of the chain of care has significant potential for improving cost efficiency, patient experience and outcomes. being remote, always available and highly scalable, this service is fundamental in high demand situations, such as the current covid- outbreak. shortage of physicians and increasing healthcare costs have created a need for digital solutions to better optimize medical resources. in addition, patient expectations for mobile, fast and easy / access to doctors and health services drive the development of patient-centered solutions. the need to be triaged, diagnosed and treated remotely or at home without having to wait in crowded rooms has never been more relevant than in outbreak periods as the covid- one [ ] [ ] [ ] . symptom checkers and differential diagnosis generators (ddx) developed for the public in form of web and mobile apps are at the crossroad of these needs. already several of these developed over the past few years are established on the market. isabel healthcare is mostly designed for medical doctors (mds). it allows the user to enter some demographic information and symptoms and simply suggests a list of all potential diagnoses, ranking them by likelihood and red flagging the potentially dangerous ones. it also displays many literature and web resources for a further exploration of the potential diagnosis (from popular wikipedia to specialized medical sources such as uptodate). ada is developed for the patient. it has a similar starting point, but then engages in a long dialog with the patient to collect additional information on the initially entered symptom and the presence of additional ones. at the end of the dialog, ada provides a generic and high-level summary on the urgency of the condition and returns a list of the most probable diagnoses (such as "n out of people with the same symptoms had this condition"). for each displayed diagnosis there is an associated recommendation, such as "this condition can usually be managed at home", "seek medical advice", "seek emergency care". the telemedicine triage that we developed, the artificial intelligence triage engine (aite), is an integral part of a rich ecosystem of telemedicine services and is fundamentally different from symptom checkers in several aspects. starting with few input symptoms (see figure ), aite engages in short and focused dialogues to finally provide clear and simple recommendations to patients. patients receive suggestions on which medical service provider to go to depending on the characteristics of their condition. in critical cases, patients are recommended to call the telecare provider immediately for a teleconsultation. if the health condition of the patient is assessed as being less urgent, they can select an appointment of their choice via a phone or video consultation with a trained telecare specialist (see figure ). should patients require physical intervention, they will be referred to an appropriate care provider for a faceto-face consultation. the advantages are clear. by using the mobile application, emergency cases can be treated faster than in standard call center settings. moreover by automatically filtering out patient requiring physical intervention, telemedical resources can invest more time in treating telecare eligible patients. furthermore, to recommend the most appropriate course of action, the app draws from the patient's personal medical history as well as from similar cases handled by the telecare provider in the past. built on case records and guidelines using ai-based methods, the system consists of the following building blocks: ) an engine for the automated ingestion of unstructured clinical notes, the extraction of relevant medical entities and their organization into a knowledge graph (kg); ) a data-driven dialog system that allows a conversation with such medical knowledge base and drives the patient interactions; ) an inference engine able to suggest the most appropriate recommendation in terms of point of care and time frame for treatment. all these components are depicted in figure and will be discussed in detail in the following sections. to build the triage application described here, we used more than k case records written in german and collected over more than years. this is only a fraction of the available data, since only records generated by top-ranked doctors (based on years of experience and internal audits) were selected. the records are notes that call center agents and doctors took while talking to the patients over the phone. they are structured in sections that contain demographic data such as age and gender, previous illnesses, and free text descriptions of the patient's current medical condition. it should be noted that in this kind of records, the free text contains a shallow and subjective description of the patient's problem. patient narrative can contain mention of recent surgery or treatments and whether the patient can perform or not a specific test/movement, but no physical exams nor lab tests. potential diagnoses consistent with the symptom description are listed. the descriptions in the records are expressed in formal medical language as well as in layman's terminology. typical for these settings, sentences are not always complete (e.g., subjects or verbs may be missing) and include misspellings, dialect vocabulary, non-standard medical abbreviations and inconsistent punctuation. this is a challenge for the linguistic processing of the case records. a natural language processing (nlp) pipeline extracted medically relevant concepts from each case record. the pipeline consisted of the following stages: ( ) data preprocessing for misspelling correction and abbreviation expansion, ( ) named entity recognition (ner) and ( ) concept clustering for the dynamic creation of an ontology of medical concepts from the corpus. acronyms and abbreviations used unambiguously were linked to the corresponding entities directly in the medical dictionaries. ambiguous acronyms and abbreviations were resolved, when possible, using algorithms that include context for disambiguation. although we were able to detect many entity types, i.e. from anatomy and physiology to medical procedures and medicines, the main focus was on the extraction of current, non-current and negative mentions of symptoms and diagnosis in simple and complex expressions. special attention was devoted to symptom and disease characterization in terms of body part location (e.g. pain in the leg, abscess in the arm), intensity (strong, light), time duration (chronic, acute), activity by which the symptom manifests or changes in character (e.g. dyspnea at rest, exertional dyspnea). for medical entity extraction we used a system based on the combination of dictionary look-up, advanced rules and deep learning. the dictionaries used in the ner were built partly using existing german-language medical dictionaries and ontologies (umls mapped german terms, icd , meddra, etc.) and partly using the list of words contained in the case records. they therefore contain a mapping of technical and layman's terms, for a total of more than k unique words (including declination, capitalization). negated mentions of medical entities are very frequent in this type of records and were detected using german language-specific negation particles or expressions. for relations that associate a medical entity to a specific body part, advanced parsing rules were used to detect short distance relations with high precision, while a deep learning (dl) stage was used to detect distant relations, improving therefore the overall recall. we trained a dl binary classifier with positive and negative examples. the annotated data consisted of k triples (e , r, e ) manually generated on the raw texts; where e is a symptom, disease, or operation, e is an anatomical location, and r is the positive or negative relation between the two entities. to obtain a good balance of training examples, % triplets had r = "located in" and % triplets had r = "not located in". this approach can also be used in case of multi-relation extraction by building a binary classifier for every relation, where the use of correctly balanced negated examples helps improve the training. the raw text data was processed by (a) removing stop words, (b) transforming to lower case, (c) normalizing to ascii characters, (d) transforming delimiters (e.g. ";", ",", ".") to word embeddings by adding white spaces, and (e) excluding any non-alphanumerical characters and characters not in the delimiter set. for each word, we concatenated word-, positional-and part-ofspeech-features (pos-features) creating the input feature vector. word features were constructed using pre-trained word embeddings on the raw text data, using either word vec or elmo embeddings. positional features were defined as the combination of the relative distances of the current word from the entities e and e . we explored three different architectures convolutional neural network (cnn) and bidirectional gated recurrent unit network (bi-gru), bidirectional long short term memory network (bi-lstm), combining the models proposed in [ ] [ ] [ ] [ ] . in the cnn architecture, the input feature vector with dimension k was fed to convolutional layers. these layers encoded the word sequence into n-gram representations, to capture the contextual information. for a given sentence, a weight matrix w ∈ r m×k was applied to generate the new features c i from the window of words x i:i+m− where: with m convolutional window. this filter was applied to each possible window of words in the sentence to produce a feature map: with c ∈ r n−m+ . by applying multiple filters (denoted f ) on x i:i+m− , we obtained a new representation of the sentence. by setting different values for m, we obtained different n-gram representations of the sentence which was input to a max pooling layer, similarly to , , . the last layers of the cnn architecture comprised several fully connected layers with dropout and a softmax for the classification. the architecture schema of the cnn model is shown in the left panel of figure . the dimensions of the word embeddings w, the positional embeddings p, the number and size of the convolutional windows, the number of filters f , the number of fully connected layers and their dimensions, the number of epochs and the dropout rate were tuned during training. in the bi-gru and bi-lstm architectures, the input feature vector with dimension k was fed into bi-gru or bi-lstm layers, followed by an attention and a softmax layer for the classification. the architecture schema of the bi-lstm is given in the right panel figure . for all architectures, we used grid search to find the optimal values of the model-specific hyperparameters. we used a randomly constructed cross-entropy loss function with mini-batch updating and adam optimizer for five epochs. in our experiment, we found optimal values for w = and p = . the annotated data was split in % training, % validation and % testing. precision, recall and f-score on the test set on the different architectures are shown in table . notably, the described nlp pipeline is able to ingest one million patient records at run time and extract the relevant medical entities and their relations in about two hours. therefore, if needed, new, improved versions of the system could be produced almost on a daily basis, by ingesting new case records whenever available. a highly performant pipeline is also important for efficient debugging of the nlp algorithms. several approaches have been developed for ontology learning , , which is defined as the extraction of terms, concepts, taxonomic relations and non-taxonomic relations from data. an ontology can be built from scratch for example table : prediction results of the different architectures on relation extraction (i.e., predicting whether the relation is r or r ), where p(r k ), r(r k ), f(r k ) are precision, recall and f-score and r = "not located in", r = "located in". similar values were obtained by conducting several experiments and averaging the results. using clustering algorithms or from an existing ontology performing classification tasks. the first step in the creation of the ontology in our application was the grouping of the annotations gathered through the nlp pipeline that describe the same medical concept. for this process we used a hierarchical procedure, named concept clustering. this is important to improve performance of the inference engine to ensure high recall for systems based on patient similarity and reduce feature correlation for machine learning classifiers. it is especially laborious in german, due to the frequent use of complex compound names that can be also expressed by several permutations of the corresponding simple entities. the created medical concepts are then organized in an ontology, where the following relations among medical concepts were considered: ) child of (taxonomic relations) and ) located in, negation of, characterization of, specification of (non-taxonomic relations). the relations child-of were automatically built using as a reference the relations contained in two manually curated elementary ontologies, "anatomy" (e.g. augenlid is child of auge -eyelid is child of eye) and "general symptom" (e.g. druckschmerz is child of schmerz), which were derived from standard medical resources(e.g., ). to summarize, more than one million medical expressions were merged into k medical concepts specific to medical triage, related to a very broad range of problems (e.g. urinary, digestive, respiratory, locomotive, skin, eye). these medical concepts were organized in a large ontology containing k nodes ( k symptoms, k diseases) and k edges. in addition to a name and its synonyms, each medical concept was also characterized by metadata, used, for example, to assign the semantic type (symptoms, disease), distinguish between red flags and common symptoms, female or male specific conditions, psychological or other types of symptoms. metadata are consumed by the system which drives the dialogue with the end users (q&a system). the diagram of the stages used to construct the ontology is summarized in figure . it should be noted, that this solution allowed for the creation of an ontology of medical concepts directly and automatically from the ingested data, whereby layman's and technical terminology as well as many synonym expressions are mapped to the same concept. in addition, this solution is useful for languages that are not yet well covered by standard ontologies such as umls/snomed. after ontology creation, the input case records together with the extracted medical concepts and metadata were automatically ingested and organized in a language agnostic knowledge graph (kg). kgs are structured knowledge bases (kbs) that store information in form of relationships (edges) between entities (nodes or vertices) [ ] [ ] [ ] . the graph is represented by its sparse adjacency matrices, indicating which vertices are connected with a given relation. the created kg consisted in more than m of nodes of the type case record, age, gender, symptom, disease, red flags, historical recommendation and m edges linking these entities, such as, for example symptom-to-patient, diseaseto-patient, patient-to-point-of-care, age-group-to-patient. the language agnostic kg was built for multilingual triage applications by first using data in one specific language (german) as a reference and then mapping the medical en- tities (kg nodes) to numerical codes. to obtain language interoperability, these numerical codes should correspond to international standards, such as the umls concept unique identifier, or icd and snomed codes. however, the coverage of these coding systems for the specific languages can vary considerably and therefore a complete mapping was not possible. an essential part of the triage process is the q&a session, during which additional, carefully chosen symptoms are asked to the patient. as the patients do not have the necessary knowledge for proper triage, this process ensures that they enter all relevant symptoms, even the ones that they might consider irrelevant or unnecessary to input. users might not know correlations between symptoms that are important. the question generation algorithm drives the interaction with the patient. its goal is to identify which medical concepts need to be asked as the most relevant to the initial input provided by the patient, emulating the human expert decision process. in our system, the question generation algorithm is fully learnt from the data: the questions are determined dynamically based on the patient's input and the system's knowledge on the training data. we explored two different approaches. one based on information retrieval algorithms, i.e., pseudo-relevance feedback based query expansion techniques (such as entropy, mutual information) and the second one on neural network techniques. in the first approach question generation and patient risk assessment are jointly optimised so that the collection of additional relevant information from the patient aims to improve the classification task. while in the second approach the two tasks are optimised (i.e., trained) separately. for the neural network based model we constructed a training corpus masking one or multiple medical concepts from each patient case and optimised the network to predict the obscured features. we split the data in % for training, % for validation and % for testing. due to the high number of word features that may be obscured, the prediction task becomes very hard. a random classifier would only achieve an accuracy of . × − (inverse of the number of word features) for the top one (i.e., highest probability) prediction. in table we compared the performance of a neural network based query expansion model with the following rankers: i) frequency-based (f), ii) binary independence model (bim ), iii) χ value (chi ), iv) robertson selection value (rsv ), v) kullback-leibler divergence (kld , relative entropy). for a comprehensive review of those methods see also . the frequency-based ranking of the terms (f) ranks the most often occurring symptoms the highest, while low-occurrent ones lower as in the formula f (s, r) = r∈r i s∈r , where r is the set of relevant cases retrieved, s is the token to be ranked and i the identity if the token s belongs to the set r, zero otherwise. a drawback of this method is that it ranks potentially irrelevant symptoms high, that are only common because they are frequent in the data, hence also in the relevant subset of cases. the binary independence model (bim) ranks symptoms based on the equation below: where s denotes the symptom to be ranked, r is the set of relevant retrieved cases and n is the case base. p(s|r) and p(s|n ) stand for the probabilities of the symptom occurring in the relevant set of cases and the whole case base respectively. the exact probabilities are unknown but can be approximated with the counts. the χ value is used to determine whether there is significant difference in expected values of two events. in our case, these two events are the query occurrence in the relevant set of cases and the occurrence in the whole set of training data. the definition is given in the equation below: the kullback-leibler divergence (kld, relative entropy) of the aforementioned probabilities is also used as a term ranking metric. it is defined as written in the equation below: kld is a measure of how two probability distributions differ from each other. the robertson selection value (rsv) ranks the symptoms based on the product of their differences in occurrence probabilities and their term weight for each document in the relevant records. this is intuitively promising as certain tokens might be low-occurrent compared to others, yet of major importance for the downstream classification task, which is captured by a higher term weight. rsv is described in the equation below: where w(s, r) denotes the weight of term s in the given relevant record r. nn based question generation systems show very promising results in order to generate questions that are medically relevant and consistent with the initially provided symptoms, hence fundamental for the construction of a q&a system. the described experiment has been used to benchmark different algorithms. in real settings, the question generation system is only one component of the dialogue system and only a subset of medical conditions is used. a key aspects in the dialogue is the usage of the medical ontology. central to the development and digitization of ai-based decision support system are user acceptance and user friendliness. in aite, for example, patients can state their symptoms in a natural way since many different forms of expressing the same symptom have been learned from the training data provided by the telemedicine provider. an elastic search engine provides the mapping from the user input to the medical concepts using all variety of symptom expressions extracted from the data and stored in the ontology. the q&a part plays an important role in defining the overall user experience, hence speed and usability are crucial. in order to achieve the best user experience, the q&a was customized according to patient age, gender, and medical status, and special attention was paid to a simple and understandable formulation of the questions. questions should be relevant to the input symptoms and conversations should be short. this required that a final recommendation could be reached in only few interactions, with a short waiting time between these, i.e., less than one second. especially difficult was to design a system to avoid asking "similar questions" that are interpreted by a human as repetitive and redundant. this was partially solved by using the hierarchical structure of the medical ontology, by excluding all the children of a medical concept as possible question candidates, if the patient has denied having their parent. for instance, the system will not ask any symptoms related to a specific part of the abdomen (upper, lower, right, left, center) if the patient denies having an abdomen problem. these key performance indicators (kpis) were explicitly evaluated during several testing campaigns in different settings which led to continuous refinement of the q&a model and logic. the inference engine is the part of the system that will reason on the collected information and give a final recommendation. the are several approaches to build a recommendation engine for patient risk assessment. in one type of approach, patient similarity is calculated on the kg using node and edge weights, to identify the sub-graph region containing the patient data most similar to the user. this region might also be used to retrieve the possible key missing medical conditions, in systems where question generation and recommendation inference are coupled. node and edge weights in the kg are learned from the data and medical guidelines with cross entropy cost minimisation. in a second type of approach, the kg is used as a knowledge base to extract feature vectors, (e.g. by learning embeddings from the graph [ ] [ ] [ ] [ ] , combining embeddings learned from the original text with graph features) to build a machine learning classification engine. while it is not possible to report final accuracy levels due to confidentiality, we tested an implementation of this second approach which achieved f-scores as high as . %, . % and . % on high-, medium-and low-risk classes respectively, with a confidence threshold of . . based on cnn methods , it is extensively described in . finally, patient risk assessment can be performed with high classification performance also with advanced dl techniques. in this latter case, however, the methods are perceived as a 'black box" in terms of how they generate the predictions from the input data and the addition of an explanation method is needed for user acceptance . in implementing the first approach, we modeled the retrieval of similar patients on the kg with a sequence of graph operations. the technology was developed on proprietary libraries on sparse linear algebra , . the final implementation was extremely efficient and allowed for very fast graph operations. the computation of graph traversals with arbitrary depth for sparse adjacency matrices was of the order of milliseconds (o(ms)) for single graph traversal. high performance was obtained by keeping the kg in memory. the response time for any single query was measured to be well below the four seconds requirement with a single worker, as the system architecture enables scaling, and thus efficient large-scale patient support. the kg was also used for traceability and transparency in the recommendation rationale. the retrieval of similar patients to the users allows to understand why a certain recommendation was given from historical cases. a key requirement for usability and user-friendliness was speed and scalability. patient requests can be spread over the available servers, and speedup from concurrency is linear with the available hardware resources as shown in figure . all patient interactions can be handled on average in less than one second with enough computational resources. clinical testing and validation were fundamental for the certification of the solution. in such a complex system, testing and validation were performed at different levels, encompassing: ) semi-automated validation of single building blocks (e.g. nlp, ontology, q&a); ) automated validation of the end-to-end output on a set of clinically validated ground truth cases (automated recommendation testing); ( ) validation of the system in real-life and production settings. whenever possible automated testing procedures were streamlined to the execution of the pipeline for the creation of the system, so that they could be run at any update or modification of training corpus or pipeline components. physicians support was fundamental in all testing steps as depicted in the right column of figure . automated recommendation testing was performed by first creating a ground truth set in which patient demographic, symptoms and the corresponding recommendations were known and validated by clinicians. recall, sensitivity, precision and fscore were then calculated by comparing those recommendations with the ones given by the system on the same input. although metric values can not be disclosed, it has to be noted that all had to be above required thresholds (with a special attention to emergency recall) for system release . validation of the system in real life was performed by physicians and other subject matter experts, by evaluating defined kpis on a statistically significant sample during the usage of the tool in different settings. to reduce the time and effort requested to the physicians in these activities, special methods and tools were developed to enable a continuous loop between clinician review and quick implementation. we have presented results, solutions and approaches used to build an interactive, ai-based system for automated medical triage from a large corpus of case records. we have considered various aspects in developing such an endto-end system, from precision on the recommendation, transparency for trust, adoption and end-user friendliness, to the system scalability in terms of number of users served. a key aspect of our research work was the development of a pipeline capable of automatically generating a very rich ontology of medical concepts and a knowledge base, directly from a large corpus of ingested records. this included a highly efficient dl-augmented nlp stage with high precision and recall for the extraction of medical entities and their relations, and a language agnostic implementation of a medical knowledge graph for multilingual applications. a second key aspect was the implementation of reasoning methods on such a knowledge base, comprising a user-friendly, fully data-driven question generation technology and an inference engine for the final recommendation. a third key aspect was that several testing procedures were automated and streamlined to the development activities, for a continuous and consistent feedback implementation. the abstraction and modularity of the underlined solutions are crucial for the reusability of the components in general settings such as automated knowledge ingestion and organisation, development of dialogue systems and decision support applications. providing such automated triage services will help healthcare providers satisfy a larger number of patients and focus their valuable medical resources on telecare eligible patients at the same time. the optimisation of the medical resources will improve patient experience overall. a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a novel coronavirus from patients with pneumonia in china distributed representations of words and phrases and their compositionality relation classification via convolutional deep neural network convolutional neural networks for sentence classification bidirectional recurrent neural networks long short-term memory finding structure in time learning phrase representations using rnn encoder-decoder for statistical machine translation natural language processing (almost) from scratch ontology learning from text: a survey of methods ontology learning from text: a look back and into the future a reference ontology for biomedical informatics: the foundational model of anatomy semiempirical molecular dynamics (semd) i: midpoint-based parallel sparse matrix-matrix multiplication algorithm for matrices with decay method for performing sparse matrix-matrix multiplication. us a , united states patent and trademark office a review of relational machine learning for knowledge graphs relevance weighting of search terms aid, an associative interactive dictionary for online searching on term selection for query expansion an information-theoretic approach to automatic query expansion query expansion techniques for information retrieval: a survey. information processing & management deepwalk: online learning of social repreentations line: large-scale information network embedding node vec: scalable feature learning for networks revisiting semi-supervised learning with graph embeddings patient risk assessment and warning symptom detection using deep attention-based neural networks key: cord- -xj i fgd authors: camhi, stephanie s.; herweck, alexandra; perone, hanna title: telehealth training is essential to care for underserved populations: a medical student perspective date: - - journal: med sci educ doi: . /s - - -w sha: doc_id: cord_uid: xj i fgd telehealth is an area of medicine which has magnified the ability to treat patients remotely. presently the education of medical professionals pertaining to the value, use, and implementation of telehealth is not adequate to harness the potential of available technologies. patients engaging in telehealth experience time and cost savings, improved disease management through remote monitoring programs, and high-quality care regardless of geographic location. despite this, medical education has been slow to evolve. it is therefore imperative that medical curricula incorporate training for this rapidly advancing mode of healthcare delivery to enable students to best care for their future patient population. telehealth (telemedicine, e-health) refers to utilization of technology to deliver healthcare services outside of traditional healthcare facilities such as through remote monitoring, video-conferencing, or electronic consultations [ ] . surveys from the american hospital association support that use of telehealth has grown dramatically in the past decade, with % of united states (us) hospitals offering some form of telehealth services [ ] . while thirty-five states in the us currently have enacted parity laws requiring health insurers to reimburse telehealth services, medicare reimbursement is slow to provide adequate compensation to all involved parties [ ] . the advent of the coronavirus disease (covid- ) global pandemic propelled telehealth to the forefront with virtual care visits predicted to surpass billion in this calendar year [ , ] . while the agility of the healthcare system to adapt to this unprecedented challenge is admirable, the retroactive application of telehealth approaches highlights how unprepared our health system is to embrace widespread telehealth implementation. in part, this may be related to the fact that medical schools are similarly slow to adopt the growing movement of telehealth despite its promotion by the american medical association (ama). telemedicine has significant potential to combat health disparities, improve access to healthcare across all specialties, including the care of stigmatized populations, and improve patient outcomes through remote monitoring programs; however, effective implementation by healthcare providers requires early exposure throughout both allopathic and osteopathic education to adequately harness this potential. as of , less than half of allopathic medical programs in new york and texas reported implementing preclinical telemedicine education. in the same time frame, states with predominantly rural populations, namely north dakota, kansas, and oklahoma, had yet to implement preclinical telemedicine in any of their allopathic schools [ ] . according to the association of american medical colleges (aamc), the number of medical schools offering clerkship electives in telemedicine has increased from in to in [ ] . while this increase is promising, the elective nature of telehealth training remains concerning. the ama seems to share this sentiment and recently launched the "accelerating change in medical education consortium" which granted $ . million to medical schools for telehealth curricular development [ ] . it is worth noting that medical education already aims to deliver a surplus of crucial biomedical information on a rather accelerated timeline, which appears to push telemedicine to the hierarchical backburner. one study from australia highlighted that telehealth training is not a priority for medical students given that accrediting bodies do not expect competency in this domain from medical graduates [ ] . however, with the advent of the covid- pandemic, and despite the fact that mandatory telemedicine education is currently lacking in most allopathic schools, the united states medical licensing examination (usmle) has suggested that a telemedicine-style clinical skills examination may be forthcoming. this presents additional challenges including the complexity of technical work required for initiation of telehealth education, which to this point has been lacking, as well as the concerns of standardization and validation of new assessments over electronic video platforms [ ] . despite these challenges, a few preliminary studies suggest that curricular reform to introduce and instruct regarding telemedicine is likely to be promising. one study from the university of iowa implemented a -module introductory educational program for second-year students and improved students' telemedicine knowledge and confidence [ ] . a study from the uniformed services university of the health sciences similarly demonstrated an increase in telehealth knowledge following student completion of their pilot program consisting of telemedicine training, faculty-supervised patient encounters, and an introduction to telehealth surgical equipment [ ] . telehealth applications stand to benefit patients given that medical appointments cause hourly workers to lose valuable income through loss of time at work, personal transportation fees, and copayments or other associated visit costs. recent work by ray et al. found the total time invested in travel to and from, wait time associated with, and clinical interaction comprising a single appointment to be minutes [ ] . after this substantial time and monetary investment, the patient may not even be visiting a center that is specialized in treatment of their specific complaint, leading to receipt of suboptimal medical care. another study at vermont veterans hospital showed an average travel and cost savings of miles and minutes per visit with the use of telemedicine clinic appointments [ ] . in addition to permitting time and cost savings, telemedicine is essential to bridge structural barriers and provide access to vital emergency and/or specialty care to individuals in under-resourced settings. utilization of telehealth for triage has shown to be acceptable to patients and to potentially reduce the number of emergency department (ed) visits [ ] . telehealth approaches also show promise for providing crucial urgent care services to rural populations [ ] . this reduction in structural barriers does not come at a cost to patient satisfaction. a study in oncology patients suggested equivalent satisfaction ratings regardless of the care delivery method including telephone versus in-person appointments [ ] . furthermore, telemedicine has improved access to specialists in resource-poor communities. one study of an interactive video colposcopy initiative in rural arkansas allowed health department nurses to conduct colposcopies and cervical biopsies through real-time inclusion of an obstetric-gynecologic specialist [ ] . similarly, dermatology and gastroenterology telehealth pilot programs have decreased time to initiation of patient care [ ] . during the covid- pandemic, telemedicine has been harnessed to continue regular non-urgent outpatient care and to provide covid-related care such as symptomatic monitoring and post-discharge follow-up [ ] . widespread use and availability of mobile technologies allow for telemedicine utilization throughout the country. as of , % of americans own a smartphone, nearly % of adults own desktop or laptop computers, and approximately % own tablet computers [ ] . more than ever, individuals from all racial, ethnic, and income backgrounds can afford these technologies and therefore already possess the necessary supplies to engage in telehealth services. in the last years, remote patient monitoring (rpm) via noninvasive devices has gained popularity and increased in availability for collection of immediate medical information and thereby monitoring of chronic health conditions. the most common rpm devices used include smartphones ( %), wearable devices ( %), biosensor devices ( %), and computerized systems ( %), with most rpm programs including multiple methodologies of monitoring patients ( %) [ ] . rpm programs have improved self-management, which has been shown to reduce clinic visits and hospitalizations, for patients with chronic disease and reduced the severity of symptoms related to chronic respiratory, metabolic, and cardiovascular conditions [ ] . patients with type diabetes mellitus in an rpm program lost weight and had positive a c outcomes [ ] . in a study utilizing rpm for blood pressure control, systolic blood pressure outcomes were similar in patients with rpm alone compared with those who received both rpm and regular office visits, suggesting that both approaches were as efficacious as usual office care [ ] . telehealth is also an important avenue for providing care to populations who may otherwise be stigmatized. men who have sex with men (msm) face many structural and cultural barriers, and studies have shown worse health outcomes in this population, particularly in those with hiv or aids. a recent study of msm in oklahoma suggested that geographical isolation significantly limits access to quality sexual healthcare and resources including hiv pre-exposure prophylaxis (prep) [ ] . a pilot study in the rural us showed that electronic prescribing of prep resulted in many individuals initiating treatment, and additionally was preferred by patients over standard in-person care [ ] . in addition to prep availability, msm have also indicated acceptability of telemedicine efforts to increase sexually transmitted infection (sti) screening. in a pilot study of home sampling for sti screening, forty-nine of fifty enrolled msm couples completed testing, indicating high acceptance among the msm population for this model [ ] . telehealth further stands to improve ease of access to female reproductive health services including oral contraception. as of february , nine platforms were electronically prescribing oral contraception with a greater adherence to cdc prescribing guidelines compared with in-person office visits [ , ] . tele-contraception efforts have not only increased access to oral contraception but also empowered women with adequate knowledge pertaining to their contraceptive options [ ] . these are merely a handful of studies highlighting the immense power for telemedicine to improve healthcare accessibility in stigmatized populations and promote safe sexual health practices. delivery of the highest quality telehealth care relies on physicians from numerous specialties, and therefore, all osteopathic and allopathic students would benefit from early exposure and training. there is much conversation in the medical education community pertaining to curricular reform and training "physicians of the future." while these groups discuss shortening the preclinical experiences, integrating basic sciences into clinical studies, and transitioning to symptombased education, an emphasis on telehealth is generally lacking. greater urgency should be exhibited to incorporate telemedicine into the training of young physicians and thereby harness the full potential of our rapidly advancing medical technologies. introduction specifically at the medical student level will provide students with a framework for developing further telehealth knowledge and experience as they progress through training. early and repeated exposure across varying institutions (i.e., medical school, residency training, fellowship training) will provide a more thorough "tour" of available telehealth approaches and technologies. in addition, the initial clerkship year, or just prior, is an ideal time frame for introduction given that students will then feel as though telemedicine is engrained into their clinical workflow. medical students in germany identified the growing prevalence of telehealth applications as an important learning goal [ ] . a mixed-methods review of telehealth applications in us allopathic schools similarly details that most medical students find telemedicine-based training to be valuable [ ] . telehealth applications are a main reason that the covid- pandemic failed to bring our medical education to a startling halt. objective structured clinical examinations (osces) were adjusted to take place as telemedicine encounters over video conference for core clerkships including pediatrics and psychiatry. in addition, a medical student-driven initiative to offer telehealth education to clerkship students, consisting of an introductory lecture and participation in telehealth encounters, was mandated for all third-year students at our institution. following the lecture, students were paired with a provider in their specialty area of interest to participate in telehealth encounters. these experiences provide just a small taste of what telehealth applications stand to contribute to our future toolkit as physicians, and similar activities should be built into the curriculum rather than mandated in the context of pandemic response. some of the barriers to widespread implementation of telemedicine include uncertainty pertaining to reimbursement and licensure requirements. recent governmental actions have lessened these concerns, leaving the lack of training of medical professionals as a key barrier to implementation [ ] . without expedited implementation of training within undergraduate medical education, the likelihood of provider knowledge keeping up with rapid technological advances is slim. how will we become public health physicians of the future without this vital training? how will we bridge the inequities that structural barriers have enacted? how will we offer the best care, to all patients, regardless of gender, race, religion, stigmatization of their medical condition, or physical location? and perhaps most pressingly, how can we ensure that our pandemic response is not dampened by the learning curve associated with retroactive telemedicine implementation? the answer is that we, as current physicians-in-training, will not succeed in these endeavors if our medical education does not prepare us to do so. our inability, as care providers, to implement telemedicine is a disservice to the rural, structurally disadvantaged, impoverished, and stigmatized communities which we, as physicians, cannot morally afford to underserve. telehealth transformation: covid- and the rise of virtual care telemedicine in the era of covid- telemedicine training in undergraduate medical education: mixed-methods review required courses and elective courses. aamc ama encourages telemedicine training for medical students, residents it's important, but not important enough: ehealth as a curriculum priority in medical education in australia united states medical licensing examination | announcements model for medical student introductory telemedicine education an interdisciplinary, multi-institution telehealth course for third-year medical students opportunity costs of ambulatory medical care in the united states va telemedicine: an analysis of cost and time savings potential of mobile health technology to reduce health disparities in underserved communities advancing health equity and access using telemedicine: a geospatial assessment telemedicine versus clinic visit: a pilot study of patient satisfaction and recall of diet and exercise recommendations from survivorship care plans distributing medical expertise: the evolution and impact of telemedicine in arkansas teledermatology consultation can optimize treatment of cutaneous disease by nondermatologists in under-resourced clinics remote patient monitoring via non-invasive digital technologies: a systematic review association between weight loss and glycemic outcomes: a post hoc analysis of a remote patient monitoring program for diabetes management randomized clinical trial to assess the effectiveness of remote patient monitoring and physician care in reducing office blood pressure barriers to access and adoption of pre-exposure prophylaxis for the prevention of hiv among men who have sex with men (msm) in a relatively rural state acceptability and feasibility of a telehealth intervention for sexually transmitted infection testing among male couples: protocol for a pilot study breaking down barriers to birth control access: an assessment of online platforms prescribing birth control in the usa a study of telecontraception | nejm the closer, the better:" the role of telehealth in increasing contraceptive access among women in rural south carolina the future of practical skills in undergraduate medical educationan explorative delphi-study acknowledgments the authors are thankful to dr. daniel lichtstein for his thoughtful review and support of our work.code availability not applicable.authors' contributions all authors made substantial contributions to the conception of the work, drafted the work and revised it critically for important intellectual content, and approved the version to be published. conflict of interest the authors declare that they have no conflict of interest. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -or zzjs authors: zhou, liang; zhang, ping; zhang, zhigang; fan, lidong; tang, shuo; hu, kunpeng; xiao, nan; li, shuguang title: a bibliometric profile of disaster medicine research from to : a scientometric analysis date: - - journal: disaster med public health prep doi: . /dmp. . sha: doc_id: cord_uid: or zzjs this study analyzed and assessed publication trends in articles on “disaster medicine,” using scientometric analysis. data were obtained from the web of science core collection (woscc) of thomson reuters on march , . a total of publications on disaster medicine were identified. there was a mild increase in the number of articles on disaster medicine from (n= ) to (n= ). disaster medicine and public health preparedness published the most articles, the majority of articles were published in the united states, and the leading institute was tohoku university. f. della corte, m. d. christian, and p. l. ingrassia were the top authors on the topic, and the field of public health generated the most publications. terms analysis indicated that emergency medicine, public health, disaster preparedness, natural disasters, medicine, and management were the research hotspots, whereas hurricane katrina, mechanical ventilation, occupational medicine, intensive care, and european journals represented the frontiers of disaster medicine research. overall, our analysis revealed that disaster medicine studies are closely related to other medical fields and provides researchers and policy-makers in this area with new insight into the hotspots and dynamic directions. (disaster med public health preparedness. ; : – ) n atural disasters, biological terrorism, nuclear leakage, public health emergencies, epidemic diseases, and other disasters directly threaten the survival and development of mankind. currently, a major disaster occurs almost daily in some part of the world. most population centers are concentrated in high-risk locales like metropolitan cities, which feature very frequent and multiple person-to-person contacts. high-risk occupations, international trade, and housing construction all increase the possibility of human exposure to disasters, leading to increased casualties after each disaster. the ever-increasing spiral of human populations, the rapid growth of technology, swift world-wide travel by millions of persons, and the exponential expansion of at-risk industries and residences combine to increase human exposure to disasters. in particular, the major casualties caused by the wenchuan earthquake, the nepal earthquake, and the indian ocean tsunami pose great challenges to disaster medicine. the wenchuan earthquake was one of the most devastating disasters in the past years and caused more than , casualties; the main causes of death were trauma and crush syndrome. in addition, there was a significant increase in the number of respiratory infections, enteritis, and skin diseases in the week after the earthquake. even a full year after the earthquake, some survivors began to suffer from posttraumatic stress disorder. disaster medicine has attracted global attention gradually by implementing emergency medical treatment, disease prevention, and health care science under the conditions of disastrous damage. after the september attacks, the united states made two major adjustments to the national disaster medical system (ndms) to form a high-efficiency operating mechanism called the national disaster medical rescue system. japan has also established a national rescue medical center in tachikawa city, tokyo. as a data transmission and command center for disaster medical care, it features a disaster medical information system used to determine damage for medical institutions. in addition, japan has enhanced its disaster emergency medical rescues by launching civil and community organizations. after the outbreak of severe acute respiratory syndrome (sars) in , china likewise began to attach importance to the establishment of an emergency medical system. then, after experiencing the wenchuan, lushan, and yushu earthquakes, and observing the actual rescue experience, it built an emergency medical rescue system, including a rescue command center and a medical rescue scene and rescue information platform. disaster medicine scholars have published a substantial amount of original research based on care during disaster rescues, emergency medical treatment, and disease prevention. however, the bibliometric profile of disaster medicine in the literature is still unknown. therefore, in this study, a scientometric analysis was conducted on disaster medicine to estimate the productivity of specific journals, countries, institutions, authors, and research areas, and to identify research hotspots and trends in this field. all of the data for this study were obtained from the web of science core collection (woscc) of thomson reuters on march , (incomplete data existed in ). the woscc, which includes the social sciences citation index, current chemical reactions, and index chemicus, is the most frequently used source of scientific information. the search term "disaster medicine" was used to retrieve titles, keywords, author information, abstracts, and references published from to . the following search string was used: (ts = (disaster medicine)) and languages: (english) and document type: (article or review). the impact factor of each journal was obtained from the journal citation reports science edition, accessed on march , . citespace iii ( bits) was used to analyze publication outputs and construct knowledge maps, to analyze the extracted records for citation characteristics, and to visualize the patterns and trends in disaster medicine. [ ] [ ] [ ] [ ] [ ] a total of studies on disaster medicine, published from to , were retrieved from the woscc (incomplete data existed in ). of these, ( . %) were original articles. although the number of publications increased only mildly from (n = ) to (n = ), the number of citations increased substantially from (n = ) to (n = ) ( figure ). of studies, ( . %) were cited at least once, with an average of . citations per article for , total citations. table shows the most frequently cited articles. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] among these articles, the most common topic was disaster rescue and post-disaster health effects ( of [ %]). the top-ranking paper ( citations) was published in chest and involved critical care treatment: "definitive care for the critically ill during a disaster: a framework for allocation of scarce resources in mass critical care -from a task force for mass critical care summit meeting, january - , , chicago, il." the article provided suggestions to perform triage while allocating scarce critical care resources during a disaster. the studies were published in different journals. the top-ranked journal, which published papers, was disaster medicine and public health preparedness, followed by two other journals with more than papers each: academic emergency medicine (n = ) and american journal of preventive medicine (n = ). disaster medicine and public health preparedness also had the greatest number of total citations (n = ), again followed by academic emergency medicine (n = ) and american journal of preventive medicine (n = ) ( table in the online data supplement). these research studies were published by countries. the top countries published of the studies, accounting for . % of the total number of publications. the country with the greatest number of publications was the united states (n = ), followed by japan (n = ) and china (n = ) ( table in the online data supplement). similarly, among the sponsoring institutions, the top institutions published literatures, accounting for . % of the total number of publications. new york university had the most publications (n = ), followed by the university of washington (n = ) and johns hopkins university (n = ) (table s ). a total of authors contributed to these studies; the top authors accounted for studies, . % of the total. three authors tied for first place, each with publications: m. d. christian, f. della corte, and p. l. ingrassia ( table in the online data supplement). a total of research areas were represented, with the majority of articles focusing on public environmental/occupational health (n = ), general internal medicine (n = ), and emergency medicine (n = ) (table s ). the visualization analysis for reference citations was conducted by citespace iii. the parameters in citespace were as follows: time span = years ( - ); time slicing = ; term type = burst terms; selection criteria (c, cc, ccv) = ( , , ) ( , , ) ( , , ) . the top most cited or occurring items from each slice were selected. the pathfinder network method was used to streamline the network and to map the visualization analysis. the network revealed nodes and lines. in figure , the thicker circle indicates a higher level of between-study centrality. in general, a study with a centrality value equal to or greater than . can be considered a key study; therefore, the key studies were [subbarao i, ] ( . ), followed by [einav s, ] ( . ) and [gillett b, ] ( . ). in addition, the red circles represent burst studies which represent the frontier of a period ; among them, the key burst studies were [subbarao i, ] and [walsh l, ] , which had the highest citation rates between and . figure presents the timeline view for hot keywords. the results show that the hotspots of disaster medicine during this period were spinal cord injury, conceptual framework, health professional, occupational medicine, medical surge capacity, oleic acid, lifesaving intervention, terrorist bombing, developing country, workforce, professionalization and west africa (figure ). in addition, the research papers published in journals represent the frontiers of certain subjects, and the references cited in these papers provide the knowledge base of the papers. in figure , the nodes of clusters # , # , # , # , and # were mainly distributed before (the knowledge base), while the nodes of clusters # , # , # , # , # , # , and # were mainly distributed between and (the frontiers). a total of papers on disaster medicine research were included in this analysis. the visualization was generated by the carrot system based on the first results of a search on regenerative medicine. there were results from the lingo clustering algorithm; the first ranked cluster was practices in disaster (n = ), followed by hospital disaster (n = ), disaster events (n = ), disaster setting (n = ), and earthquake disaster (n = ) (figure ). in addition, the time interval is depicted as a blue line, whereas the time period that represents a burst cited journal is depicted as a red line, indicating the beginning and the end of the time interval of each burst. the top burst references were public health preparedness ( . ), hurricane katrina ( . ), and european journal ( . ) ( table ) . to the best of our knowledge, this is the first scientometric analysis on the topic of disaster medicine. the results indicated a significant increase in the number of publications ( and ) . this increase may be due to several major disasters. the year was the first year after the haiti earthquake, and much of the related research focused on the implementation and development of international medical rescue. [ ] [ ] it was also the third year after the wenchuan earthquake, research on which mainly focused on disease classification and patient management of the patients. [ ] [ ] finally, was the th anniversary of the / attacks; the relevant research mainly focused on post-disaster effects, including respiratory and mental health problems, among survivors and rescuers. [ ] [ ] the united states, japan, and china published the most research on disaster medicine. this may be because the frequent occurrence of disasters in these countries has caused serious casualties and property losses. , [ ] [ ] [ ] likewise, these countries have participated in international humanitarian relief efforts many times, so they have a wealth of medical rescue experience. [ ] [ ] [ ] the top studies (by number of citations) all emphasized a lightweight map of major terms on disaster medicine. burst strength: representing the intensity of the frequency of a key word suddenly increasing over a short period of time. the burst-detection algorithm can be adapted for detecting sharp increases of interest in a specialty. in citespace iii, a current research front is identified based on such burst terms extracted from titles, abstracts, descriptors, and identifiers of bibliographic records. burst-detection algorithms can identify emergent terms; centrality: evaluating the parameter of the number of lines on a certain node, the larger the value, the more the number of lines, that is, the importance of the node in the whole network. disaster medicine and public health preparedness categories of disaster medicine, except for [leaning j, ] , which summarizes the challenges and pressures posed by natural disasters to public health. these categories are disaster rescue , [ ] [ ] [ ] [ ] and post-disaster health effects. [ ] [ ] [ ] [ ] overall, the most cited article was "definitive care for the critically ill during a disaster: a framework for allocation of scarce resources in mass critical care -from a task force for mass critical care summit meeting, january - , , chicago, il," which offers guidance for allocating scarce critical care resources, drawn from a task force on mass critical care. this task force provided several suggestions for managing the triage process when medical systems are overwhelmed. in addition, [devereaux av, ] was sponsored by new york university, which sponsored the greatest number of disaster medicine publications from to . among the authors, f. della corte, m. d. christian, and p. l. ingrassia were the most productive. in their papers, the most cited articles were all associated with the "task force for mass critical care." , [ ] [ ] among the studies of post-disaster health effects, the most cited article was "trends in respiratory symptoms of firefighters exposed to the world trade center disaster: - ," which described trends in post- respiratory and gastroesophageal reflux disease symptoms in wtc-exposed fire fighters. this study also contributed to the literature on public environmental/occupational health, the discipline that produced the most disaster medicine studies from to . based on the co-citation analysis, "a consensus-based educational framework and competency set for the discipline of disaster medicine and public health preparedness" had the highest centrality; it developed a new educational framework for disaster medicine and public health preparedness, based on consensus identification from an expert working group in the american medical association. a time-view map of the co-citation activities appears to the left of the column with the clusters' labels. new clusters include cluster # on spinal cord injury, # on conceptual framework, and # on health professionals; the landmark publications include [subbarao i, ] and [walsh l, ] . they were also the newest burst strength publications, which suggests that "public health preparedness" will become a hot topic in disaster medicine. terms analysis provides a reasonable description of research hotspots (areas of focused attention by a number of scientific researchers, addressing a set of related research problems and concepts), whereas burst words represent new research frontiers (emerging trends and abrupt changes that occur in a timely manner). as shown in figure , the top hotspots of disaster medicine research were: ( ) "practices in disasters." these papers focus on the practical elements of disaster medicine, including the treatment of wounded, effect evaluation, first aid, and disaster medical education. [ ] [ ] [ ] [ ] [ ] ( ) "hospital disasters." these papers also focus on practical elements of disaster medicine, including modular management, humanitarian relief, first aid management processes, and disaster emergency departments. [ ] [ ] [ ] ( ) "disaster events." these papers summarize the casualties of disasters and their impact on public health. - ( ) "disaster settings." these papers focus on the medical needs of the disaster, including medical personnel, medical equipment, and medical technology. - ( ) "earthquake disasters." these papers focus on medical rescues during an earthquake, including the medical decision-making, the rescue process, and the treatment of the special population. [ ] [ ] [ ] [ ] in addition, several burst terms were detected by cite space iii and are considered indicators of research frontiers over time. in the results, the time interval is shown as a blue line, and the time period that represents a burst term category is shown as a red line, indicating the beginning and the end of the time interval of each burst. therefore, the three newest frontiers were: ( ) "impact." these papers focus on post-disaster effects on the physiology and mental health of survivors, including their daily behaviors, physiological indicators, and mental states. [ ] [ ] ( ) "public health preparedness." these papers focus on the establishment, evaluation, and management of medical rescue systems. - ( ) "public health emergencies." these papers focus on the training of emergency personnel, the promotion of emergency technology, and the management of emergency procedures. , [ ] [ ] limitation in this study, although the noise data can be reduced by setting up the requisite statistical parameters in citespace iii, the source of data is limited by a generic search term strategy, which is likely to lead to some noise in the selection of articles. the major findings of the present scientometric study are helpful for all those involved in worldwide disaster medicine research. indeed, this study can help researchers better understand disaster medicine research worldwide and be useful, for example, in choosing appropriate journals for publication and collaborations. fellows choosing an institution for advanced work may also be interested in such an analysis. journals can determine where they stand in relation to other journals in publishing articles related to disaster medicine. governments and policy-makers can also ascertain the most effective countries and institutions in the world in this field, and this analysis may assist them to apprehend and predict the hotspots and dynamic directions of disaster developing health information documentation in disaster update in intensive care and emergency medicine medical action and reflections on china's rescue in nepal emergency medical rescue efforts after a major earthquake: lessons from the wenchuan earthquake the pandemic and all-hazards preparedness act: its contributions and new potential to increase public health preparedness development of the japanese national disaster medical system and experiences during the great east japan earthquake emergency medical rescue major earthquakes: lessons from the wenchuan earthquake emerging trends in regenerative medicine: a scientometric analysis in citespace bibliometric analysis of atmospheric simulation trends in meteorology and atmospheric science journals bibliometric analysis of obstructive sleep apnea research trends global regulatory t-cell research from to : a bibliometric analysis visualizing the knowledge domain of nanoparticle drug delivery technologies: a scientometric review exploring the gis knowledge domain using citespace detecting and visualizing emerging trends and transient patterns in scientific literature rete-netzwerk-red: analyzing and visualizing scholarly networks using the network workbench tool definitive care for the critically ill during a disaster: current capabilities and limitations -from a task force for mass critical care summit meeting natural disasters, armed conflict, and public health trends in respiratory symptoms of firefighters exposed to the mental health response to community disasters: a systematic review the use of simulation for pediatric training and assessment profile of injuries arising from the kashmir earthquake: the first h simulation for team training and assessment: case studies of online training with virtual worlds summary of suggestions from the task force for mass critical care summit occupational toxicant inhalation injury: the world trade center (wtc) experience. intern archive occup environ health treatment of posttraumatic stress disorder in postwar kosovar adolescents using mind-body skills groups: a randomized controlled trial a consensus-based educational framework and competency set for the discipline of disaster medicine and public health preparedness in-hospital resource utilization during multiple casualty incidents simulation in a disaster drill: comparison of high-fidelity simulators versus trained actors global liposome research in the period of - : a bibliometric analysis core competencies for disaster medicine and public health knowledge mapping of hospitality research -a visual analysis using citespace disaster relief in post-earthquake haiti: unintended consequences of humanitarian volunteerism haiti earthquake : a field hospital pediatric perspective triage during the week of the sichuan earthquake: a review of utilized patient triage, care, and disposition procedures rescue efforts management and characteristics of casualties of the wenchuan earthquake in china trends in respiratory diagnoses and symptoms of firefighters exposed to the role of academic institutions in community disaster response since physician-diagnosed respiratory conditions and mental health symptoms seven to nine years following the world trade center disaster occupational toxicant inhalation injury: the world trade center (wtc) experience. intern archive occup environ health the great east japan earthquake and devastating tsunami: an update and lessons from the past great earthquakes in japan since the academic health center in complex humanitarian emergencies: lessons learned from the haiti earthquake awareness of disaster reduction frameworks and risk perception of natural disaster: a questionnaire survey among philippine and indonesian health care personnel and public health students medical emergency rescue in disaster: the international emergency response to the haiyan typhoon in philippines summary of suggestions from the task force for mass critical care summit definitive care for the critically ill during a disaster: a framework for optimizing critical care surge capacity -from a task force for mass critical care summit meeting trends in respiratory symptoms of firefighters exposed to the detecting and visualizing emerging trends and transient patterns in scientific literature trends in respiratory symptoms of firefighters exposed to the regional anesthesia for painful injuries after disasters (rapid): study protocol for a randomized controlled trial assessment of the scope and practice of evaluation among medical donation programs. globa health are belgian military students in medical sciences better educated in disaster medicine than their civilian colleagues? evaluation of a new communitybased curriculum in disaster medicine for undergraduates practical "modular design" research of emergency drug supplies in hospitals extending a helping hand: a comparison of israel defense forces medical corps humanitarian aid field hospitals capabilities for clinical management of radiation injuries of the nikiforov russian center of emergency and radiation medicine (emercom of russia) observation services linked with an urgent care center in the absence of an emergency department: an innovative mechanism to initiate efficient health care delivery in the aftermath of a natural disaster mass disasters observed at the sapienza university of rome: a retrospective study between ebola, quarantine, and the scale of ethics lessons from the earthquake(s) in nepal: implication for rehabilitation a community-led medical response effort in the wake of hurricane sandy education in disaster management: what do we offer and what do we need? proposing a new global program portable ultrasound in disaster triage: a focused review coping behavior and risk of posttraumatic stress disorder among federal disaster responders prepared for what? addressing the disaster readiness gap beyond preparedness for survival equilibrium decision method for earthquake first-aid medicine allocation based on demand information updating transfusion under triple threat: lessons from japan's earthquake, tsunami, and nuclear crisis nepal earthquake: analysis of child rescue and treatment by a field hospital the tohoku medical megabank project: design and mission predictors of decline in iadl functioning among older survivors following the great east japan earthquake: a prospective study effect of evacuation on body weight after the great east japan earthquake what are the most common domains of the core competencies of disaster nursing? a scoping review knowledge levels and training needs of disaster medicine among health professionals, medical students, and local residents in building community disaster resilience: perspectives from a large urban county department of public health assessing disaster preparedness among latino migrant and seasonal farm workers in eastern north carolina association of community health nursing educators: disaster preparedness white paper for community/ public health nursing educators characteristics of effective disaster responders and leaders: a survey of disaster medical practitioners national differences in regional emergency department boarding times: are us emergency departments prepared for a public health emergency this study was supported by the third military medical university military doctoral program (no. jskt ).lz and pz conceived and designed the paper. lz, nx, and sl wrote the article. zz, lf, st, kh, lz, and pz collected and analyzed the data. sl provided critical revisions. all authors approved the final version of the paper. to view supplementary material for this article, please visit https://doi.org/ . /dmp. . key: cord- -nypkr authors: drexler, richard; hambrecht, jan m; oldhafer, karl j title: involvement of medical students during the coronavirus disease pandemic: a cross-sectional survey study date: - - journal: cureus doi: . /cureus. sha: doc_id: cord_uid: nypkr background: the coronavirus disease (covid- ) pandemic affects the education of medical students around the world and countries have had differing responses in dealing with this dynamic situation. the role of medical students in fighting this pandemic is controversial and it is yet to be elucidated how they can best be of service. the aim of this study is to evaluate the working fields of volunteering students and the impact of the pandemic on final year students from a student's perspective. methods: an anonymous online survey was conducted amongst medical students from hamburg (germany), using an institutional online data collection program. results: a total of questionnaires ( . %) were completed. of these, participants were students from academic year three to five ( . %) and students were in the final year of medical school ( . %). of the students from academic year three to five, students ( . %) signed up for voluntary duties during the pandemic. interestingly, only . % of the students were called for voluntary work in hospitals or health authorities. final year students had already been working in hospitals since before the outbreak, with . % of them assisting doctors in the treatment of covid- positive patients during their placements. using a -point likert scale, the students who volunteered self-assessed their work as more useful and received more gratitude than final year students (p< . ). conclusions: the majority of medical students are willing to make a significant contribution in the response to covid- and do not wish to be overlooked. furthermore, the current pandemic offers novel educational opportunities for medical students. the coronavirus disease (covid- ) pandemic has spread globally with major outbreaks in the usa, italy and spain. although the end is not yet in sight and the final lasting impact of covid- is difficult to gauge, it is clear that the current situation is overwhelming for health care systems and economies around the world. as the pandemic progresses, staff shortages will likely occur, and this raises the question of who will step up to the plate in this ongoing crisis. in this context, the role of medical students is unclear and contentious [ ] [ ] . due to legal requirements, social distancing was implemented and in-person medial classes have been cancelled and replaced with online lectures and virtual teaching for most medical students [ ] . however, the educational situation is crucial for final year students, as they are completing clinical clerkships. these students are normally integrated into medical teams as well as clinical routines and gaining clinical experience is a crucial aspect for success in future residencies. nevertheless, student participation in clinical care varies across medical schools and different countries. some countries, such as italy, china and the united kingdom, integrated the medical students into their health care systems or graduated students early [ ] [ ] [ ] . on the contrary, other countries cancelled clerkships and restricted patient contact for medical students [ ] [ ] . in germany the treatment of final year students varied across hospitals, but non-final year students were encouraged by the german minister of health to volunteer in healthcare facilities [ ] . it is clear that there is a dissonance between medical students' roles during the covid- pandemic and the progression of patient-centered medical education. in this study, we present medical students' involvement during the covid- pandemic. we evaluate the usefulness of volunteering students as a response to the pandemic as well as the assessment from a student's perspective during volunteering. in addition, we describe the impact of the pandemic on final year students and their clinical education. an online survey was designed and sent to medical students enrolled at the campus hamburg (germany) of the semmelweis university budapest (hungary) via personalised institutional email. the survey was conducted using an institutional online data collection program. two surveys were performed, one survey for medical students from academic year three to year five and a separate survey for final sixth-year students. the surveys consisted of single, multiple-choice, and -point likert scale questions and recruitment began on may , . the survey was open from may , , to may , , and available in german. inclusion criteria were students currently active from academic year three to six at the medical faculty hamburg of the semmelweis university budapest. ten external students piloted the survey prior to publication, and minor amendments to wording to improve clarity were made. after a period of data collection, three researchers checked the database for errors and false data independently from each other. four main topics were addressed: ( ) reasons for volunteering or rejection of volunteering; ( ) work circumstances of volunteering students; ( ) critical self-reflection of usefulness of volunteering and ( ) impact of covid- pandemic on planned clerkships and future plans after graduation. students from academic year three to five were initially asked if they had registered as volunteers. individuals who volunteered until may , , then answered a more detailed questionnaire about reasons, working field, type of activity, working hours, and contact to covid- -positive patients. furthermore, they selfreflected their volunteer work answering a -point likert scale ("strongly disagree", "disagree", "neutral", "agree", "strongly agree"). the final year students were asked about the changing workload since the beginning of the covid- pandemic, contact to covid- positive patients, possible infection, and the impact on future residencies. in closing, final year students self-reflected their clerkship answering the -point likert scale as described above. all study procedures were reviewed and approved by the ethics committee hamburg, germany (wf- / ). no patient data was included. we informed participants that their answers would be anonymously used for statistical analyses and that they would not be transmitted to third parties. variables were processed and analysed using ibm spss statistics for mac version (ibm corp., armonk, ny, usa). data were reported as number with percentage. differences in proportions were analysed with the chi-square test or fisher exact test. differences in -point likert scale between third to fifth year and final year students were compared, using the two-sample t-test as stated by norman [ ] . a two-sided p-value less than . was considered as statistically significant. the survey was sent to medical students including third-year students ( . %), fourth-year students ( . %), and fifth-year students ( . %). the remaining students ( . %) were in the final year of medical school. in total, questionnaires ( . %) were completed. of these, participants were non-final year students ( . %) and students were in final year ( . %). we enrolled students from academic year three to five with an equal distribution within the years (p= . , table ). these students were affected by the cancellation of in-person medical classes, which were consequently replaced by online lectures. the students were therefore enabled to volunteer in hospitals or aid health authorities during the covid- pandemic. of the students, the majority ( . %) registered as volunteers, while students ( . %) did not ( table ) . depending on their first answer, students were asked what motivated them to sign up as volunteers or to decline the opportunity. the volunteering students had quite similar answers with the majority indicating a sense of duty to society ( . %), interest in medical activity ( . %), and social commitment ( . %) as their incentives. the main reason given for not signing up was a lack of time due to studying commitments or part-time jobs ( . %). however, six students ( . %) were either part of a high-risk group or had a first-degree family member at risk. finally, students ( . %) received notification that their clinical clerkships were cancelled due to the on-going pandemic. multiple answers were possible, in total: answers. multiple answers were possible, in total: answers. as previously stated, students signed up for voluntary commitment but only students ( . %) were called for work. these students were asked further questions regarding their work circumstances in the survey ( table ). the majority ( . %) were called within a week after signing up. almost half of the students ( . %) were assisting health authority bodies, which involved telephone consultations or data administration ( . %). five students ( . %) volunteered in the hospital, either on the ward, in icu, or in the emergency department. the remaining volunteers worked in the ambulance service ( . %) or worked in general practices ( . %). during their voluntary work, six students ( . %) had physical contact with patients who tested positive for covid- . however, none of these students were suspected of having or tested positive for covid- . to evaluate the usefulness and gratitude of the voluntary work during covid- pandemic, the volunteering students were confronted with several statements that were answered using a -point likert scale (figure ) . when self-reflecting on their work, % of the students felt helpful and % were under the impression that other medical staff valued their work. approximately half of the volunteers acquired new skills through their work and only % felt overburdened. academic year three to five. the final year students have a special status as they were already working in hospitals at the beginning of the covid- pandemic. therefore, a second survey was specifically designed to evaluate the impact of covid- on their workload and clinical education. the response rate was . % and consisted of final year students ( table ) . when comparing the students' workload before and during the covid- pandemic, most students ( . %) experienced a reduced workload. of note, students ( . %) were directly involved in treating covid- positive patients. during their rotations students ( . %) were suspected to be covid- positive, but ultimately tested negative. as previously described for the first survey, the final year students were confronted with different statements regarding their role during the covid- pandemic ( figure ). only . % of the students evaluated their work as helpful and felt their contribution was valued by other medical staff. in addition, . % agreed that they developed new skills due to the covid- pandemic. most importantly, students felt the pandemic had a negative impact on education; especially bedside teaching for example, was experienced by . % of students. focussing on future perspectives, the majority ( . %) felt the pandemic had a negative impact on their planned residencies or research activities after graduation. we described the perspectives of two student groups during the covid- pandemic. on one hand, the volunteering students from academic year three to year five, and on the other, the final years in their practical placements. both groups were equally involved in covid- patient care ( . % vs. . %, p> . ). however, final year students were more often suspected of being covid- positive (p< . ). when evaluating the role of both groups using a -point likert scale, significant differences were observed. the volunteering students evaluated their work as more helpful and more of them felt they had acquired new skills (p< . ). in addition, volunteers' work was appreciated and valued more by the medical staff in comparison to final year students conducting their clinical placements (p< . ). the covid- pandemic has spread around the world and poses a significant challenge ubiquitously for all health care systems. in the wake of finding solutions for possible staff shortages, the role of medical students is contentious as they could significantly contribute to the ongoing crisis. to address this eminent topic, we performed an online survey amongst medical students from hamburg, one of germany's epicentres during the pandemic. we evaluated the willingness to volunteer among third to fifth-year medical students. the actual demand for students was also evaluated as well as the practicality for volunteers in hospitals and health authorities. in addition, our study focussed on the ever-changing clinical education experienced by final year students. these students were already in the final months of their practical placements when the pandemic broke out. as the traditional didactic, in-person teaching shifted towards online lectures and virtual seminars since the beginning of the pandemic, most students wanted to contribute to the fight against the covid- pandemic [ , [ ] [ ] [ ] [ ] . the results of our survey underline the willingness of medical students to contribute to the handling of the crisis as . % of the students signed up for voluntary commitment. of the remaining students who were unwilling to volunteer, nearly % were either already employed in a part-time job in hospitals or unable to sign up due to a higher risk of severe illness from covid- . however, it was remarkable that only . % of the students had been called for voluntary work, mainly in the realm of hospital care or health authorities. this low number begs the question if hospitals in germany really need student volunteers and gives the impression that hospitals are capable of solving staff shortages without the helping hands of medical students. one explanation might be that lower numbers of infections were experienced here than had been anticipated. however, when looking at current reports from several countries it becomes clear, that this abrupt conclusion cannot simply be drawn and medical students are indeed able to have a positive impact during the covid- pandemic [ , ] . this is reiterated by the fact that % of the final year students in this study were already involved in the treatment of covid- patients and the role of medical students in general could prove to be indispensible in the on-going fight against the pandemic. furthermore, it is noteworthy that staff shortages and the demand for medical students correlates with the extent of the pandemic outbreak and the strain on the respective country's healthcare system. as major outbreaks were experienced in italy and the uk, medical students played a central role in dealing with the crisis in comparison to germany [ ] [ ] [ ] . however, bearing in mind the unequivocal will of medical students to participate in the fight against covid- , the role of medical students must be considered. focussing on our results, volunteering students supported doctors in patient care or worked at a covid- hotline service. students who took over these tasks unburdened doctors from administrative duties so that they could entirely shift their focus to essential care of critically ill patients. in our study, volunteering students were encouraged to self-reflect on the usefulness of their tasks via a -point likert scale. the majority of students considered their work as useful and felt it was valued by medical staff. this emphasizes that students could undertake certain tasks with a symbiotic advantage for both doctors and students, and is consistent with ideas by miller and colleagues [ ] . however, it is doubtful if these students are sufficiently trained to undertake roles with more responsibility, such as assisting with invasive ventilation of patients [ ] . the student group most affected by the covid- pandemic is undoubtedly students in their final year, due to restricted patient contact, reduced bed-side teaching and even cancelled clerkships [ , [ ] [ ] [ ] [ ] . therefore, it is unsurprising that . % of the final year students in this study reported a negative influence on their clinical education. in addition, the dissonance in suitable roles for final year students during the pandemic has lead to huge dissatisfaction in our study cohort. final year students have gone through years of rigorous training, are nearing graduation, and therefore could have a decisive role and make a significant contribution during this pandemic. even though clinical education is currently not the main focus, the integration of final year students into critical care of covid- patients could be of indispensable value for teaching certain skills, such as critical care or ventilation therapy. furthermore, it can prove useful for crisis management preparation with regard to possible future pandemics. the ongoing pandemic is a challenging time for maintaining clinical education for students on the cusp of graduation, but could be regarded as a worthwhile opportunity to educate them beyond the traditional curriculum. innovative ideas are urgently needed in order to implement these novel-learning opportunities and simultaneously integrate final year students as full members into critical care or emergency teams. there have been a few promising approaches introduced in recent literature [ , , ] . rasmussen and colleagues initiated fast-track courses in ventilation therapy and nursing assistance for medical students attending aalborg university and successfully integrated the majority of these students into medical care teams [ ] . another promising approach came from klasen and colleagues who developed a training curriculum including all necessary aspects for working in a triage-test-center for diagnostic swab-testing. after educating medical students according to this new curriculum, they were assigned to emergency teams that evaluated hundreds of patients daily and provided over swabs during a five-week period [ ] . these concepts combine the contribution of medical students to the pandemic with the utilization of these novel learning opportunities. these newly acquired unique clinical skills further the student's opportunities in their future career as a physician. more importantly, rasmussen and klasen demonstrate how medical students' role was crucial to the fight against the covid- pandemic. the involvement of medical students is entirely dependent on the gravity of the situation in the respective country. our study showed the current situation in one of the worst-affected cities in germany and evaluated the medical students' involvement in the covid- response from the students' perspective. in addition, ramussen and klasen et al. introduced concepts functioning in denmark and switzerland [ ] [ ] . it would be favourable to share further experiences from various other countries, which would show the integration of medical students into clinical settings. this could prove invaluable in the ongoing fight against covid- and much can be learned from different countries' approaches. nevertheless, the students' point of view must be taken into consideration in order to benefit future generations of medical students and patient care. it is our hope that the covid- pandemic leads to the development of alternative plans in how medical students could be positively utilised in future pandemics and in other exceptional circumstances, so that hospitals and medical schools are better prepared for future crises. we are convinced that pandemics, especially the current one, could offer various educational opportunities for medical students and should not lead to educational disadvantage and feelings of overall dissatisfaction. the majority of medical students are willing to make a significant contribution in the response to covid- and do not wish to be overlooked. furthermore, the current pandemic offers novel educational opportunities for medical students. however, it is mandatory that medical schools and hospitals implement functioning concepts in which students are integrated into clinical settings. disclosures medical students are not essential workers: examining institutional responsibility during the covid- pandemic the role of medical students during the covid- pandemic virtual bedside teaching rounds on patients covid- : medical students and fy doctors to be given early registration to help combat covid- covid- : medical schools are urged to fast-track final year students guidance-on-medical-students-participation-in-direct-patient consequences of covid- on international medical graduates and students applying to residencies in the united states likert scales, levels of measurement and the "laws" of statistics let us help'-why senior medical students are the next step in battling the covid- pandemic we signed up for this! -student and trainee responses to the covid- pandemic coalition for medical education-a call to action: a proposition to adapt clinical medical education to meet the needs of students and other healthcare learners during covid- senior medical students in the covid- response: an opportunity to be proactive. acad emerg med. medical students for healthcare staff shortages during the covid- pandemic wab team instead of swat team -students as front-line force during the covid- pandemic covid- : medical students should not work outside their competency, says bma the impact of covid- on medical education medical student involvement in the covid- response letter: covid- impact on the medical student path to neurosurgery a bold response to the covid- pandemic: medical students, national service, and public health students supporting critical care -a contention plan to prevent the decompensation of icus in the covid- pandemic: translating bjorn ibsens' polio-lessons to modern times human subjects: consent was obtained by all participants in this study. ethics committee hamburg, germany issued approval wf- / . all study procedures were reviewed and approved by the ethics committee hamburg, germany (wf- / ). no patient data was included. we informed participants that their answers would be anonymously used for statistical analyses and that they would not be transmitted to third parties. . animal subjects: all authors have confirmed that this study did not involve animal subjects or tissue. conflicts of interest: in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. key: cord- -tcuv v authors: hood, ernie; jenkins, kristin p. title: evolutionary medicine: a powerful tool for improving human health date: - - journal: evolution (n y) doi: . /s - - - sha: doc_id: cord_uid: tcuv v modern evolutionary research has much to contribute to medical research and health care practices. conversely, evolutionary biologists are tapping into the rapidly expanding databases of medical genomic information to further their research. these two fields, which have historically functioned in almost complete isolation, are finding mutual benefit in the exchange of information. the long-term benefits of this synthesis of two major areas of research include improved health care. recently, efforts to catalyze this relationship have brought together evolutionary biologists, medical practitioners, anthropologists, and ethicists to lay the groundwork for further collaboration and exploration. the range of overlap is surprisingly broad and potentially invaluable. evolution has not traditionally been considered to be an important aspect of medicine, and medical practitioners and researchers have not traditionally approached their work from the perspective offered by evolutionary biology. medical practice tends to focus on the immediate, or proximate, causes of disease, including genetics and lifestyle, to identify a potential treatment. an evolutionary viewpoint pushes the focus out farther to look at long-term ecological relationships, including symbiotic bacteria, parasites and pathogens, historical lifestyles, and the genetics of populations. conversely, evolutionary biologists have not traditionally paid much attention to the medical implications or applications of their findings, with a few exceptions such as the study of emerging infectious diseases. other evolutionary studies have seemed far removed from contemporary human health issues. recently, however, these traditions have been giving way to a new approach, as researchers and medical practitioners discover connections based on evolutionary biology that are leading them to new conclusions about their respective fields. evolution is providing clues about puzzling medical results, and studies of human health are giving us new information about the rate and driving forces of evolution. initiatives such as the human genome project and the haplotype map have provided new tools for studying evolution and human health. genes are important, environment is important, the interaction between the two is important-but so is human evolution, as it exerts an ongoing influence on characteristics of the human population. in may , evolutionary and medical researchers, doctors, ethicists, and anthropologists met at the national evolutionary synthesis center to catalyze the emerging scientific discipline known as evolutionary medicine. although the participants work on different aspects of human health, they all realize the value of evolution in their work. as the title of the meeting-evolution in contemporary human populations: medical, genetic and behavioral implications (govindaraju )-suggests, this group of scientists clearly share the belief that evolution is going on in contemporary human populations. in fact, a recent study presents evidence that evolution may have been occurring even faster in human populations than expected over the past , years (hawks et al. ) . the field of medicine has developed to address the wide array of environmental and lifestyle-induced diseases. over time, doctors have learned more about the relationship between environment and disease, including factors such as hygiene and lifestyle. more recently, genomic studies have opened the door to studying genetic differences between populations, bringing us the possibility of more effective individualized medicine. all in all, medicine seems to be making great progress in preventing, treating, and curing disease without including evolutionary information. so what does understanding our evolutionary history add to our ability to improve human health? it has been shown that human evolution, in the form of natural selection for particular traits, can occur over the short term-just a few generations, "the blink of an eye to evolutionary biologists," according to yale university evolutionary biologist stephen stearns, one of the nescent meeting organizers. continued lactose tolerance in adults is an example of a trait that has evolved in certain populations within the last , years, since the domestication of milkproducing livestock (tishkoff et al. ; swallow ) . "if a doctor can become aware of how all these different traits actually interact with each other, then you begin to see your individual patient as something that isn't just the symptom sitting in front of you in the office that day," stearns says. "if you treat that symptom, then the perspective of evolutionary biology will tell you what all of the possible associated reactions are. the point is, they're a bit broader, and they may contain more surprises than a strictly medical orientation would give you. evolution gives you a broader view of the whole way an organism reacts, and why it does sobecause it's been shaped by evolution in certain ways." psychiatrist randolph nesse of the university of michigan, a strong advocate of evolutionary medicine, agrees: "once a doctor starts understanding how the body came to be, instead of seeing the body as a machine-which it's not-he or she starts seeing the body as a product of natural selection, where everything in the body is pretty good, because it's got to be, and nothing is perfect….this perspective is needed for literally every aspect of the body that's vulnerable to disease. why hasn't natural selection made it better? there are always good reasons." among those reasons is the idea that evolution involves an array of trade-offs, compromises in selection that favor one trait at the inevitable expense of another. stearns, nesse, and david haig of harvard university eloquently explain the importance of evolutionary trade-offs in the initial chapter of evolution in health and disease (stearns and koella ): no trait is perfect. every trait could be better, but making it better would make something else worse. our vision could be as acute as that of an eagle, but the price would be a decreased capacity to detect color, depth, and movement in a wide field of vision. if the bones in our wrists were thicker they would not break so readily, but we would not be able to rotate our wrists in the wonderful motion that makes throwing efficient. it the stomach made less acid we would be less prone to ulcers, but more prone to gi infections. every trait requires analysis of the trade-offs that limit its perfection. consideration of these compromises that have evolved over time can guide medical decisions about altering our physiology and behaviors. the evolutionary trade-off between survival and reproductive success of the individual is an interesting example. reproduction is expensive, on many fronts. just looking at mating success, high testosterone levels may provide a man with a competitive advantage in securing a mate, but will also decrease his resistance to pathogens and parasites (muehlenbein and bribiescas ) . a woman who has regular and frequent menstrual cycles has more opportunities to become pregnant, but is also at increased risk of cancer (strassmann ) . exposure to higher hormone levels, either incidentally or by design, can have unexpected side effects because of this evolutionary trade-off. reproductive success is ultimately the more important factor in evolution, as one's evolutionary fitness is measured by the number of offspring that survive to reproductive age themselves. the compromise results from the fact that we produce only one or two offspring at a time, and human offspring require extended parental care. humans who threw everything into mating but did not survive long enough to raise the few resulting offspring would be unsuccessful because their offspring would not survive either. radically changing traits that have been shaped over time runs the very real risk of tipping the balance in unexpected, and potentially unpleasant, directions. homo sapiens has been around for about , years, and both fossils and genomic studies present evidence of ongoing evolution in h. sapiens. as a species, h. sapiens has experienced many lifestyle changes-some generated by culture, others by new or changing environments. anthropology can provide valuable insights into our past, and this information can improve our application of evolutionary medicine (trevathan ) . we learn from studies of ancient societies, but also from the few remaining traditional hunter-gatherer societies and by comparisons between different societies. a major shift in lifestyle was the development of agriculture. agriculture led to many changes, including dietary alterations and exposure to zoonoses from domesticated animals. agriculture required groups to abandon a mobile hunter-gatherer lifestyle and live in sedentary populations, such as farming villages. ultimately, agriculture provided the ability to support large urban populations. sedentary groups encounter issues such as accumulation of wastes and transmission of epidemic diseases (armelagos et al. ) , issues that increase with population size. even more recently, increases in expected life span and change in diets have led to a rise in the frequency of chronic diseases (armelagos et al. ; leonard ). these changes in the environment and the increase in population size have the potential to create a surge in the rate of evolutionary change (hawks et al. ). clinicians and biomedical researchers need to incorporate an understanding of this mismatch to modernity into their explorations of pathophysiology. in simple terms, many aspects of our modern environment and lifestyle, including diet, exercise, exposure to chemicals, hygienic practices and circumstances, and a variety of other elements, are mismatched to our bodies' evolutionary state. for example, if-thanks to our prehistoric ancestors-our physiology is evolutionarily hardwired for low caloric intake and intense physical activity, our rich modern diets and sedentary lifestyles mismatch our innate metabolism (neel ; wang and mariman ) . evolutionary medicine tells us that many of today's so-called "diseases of civilization" that are so highly prevalent and growing rapidly in incidence, such as diabetes, obesity, and cardiovascular disease, may well be fueled by a metabolic mismatch (leonard ) . this phenomenon can be readily observed in contemporary populations that have undergone a "nutrition transition" in which rapid socioeconomic, demographic, and technological changes have brought profound changes in diet and activity patterns, often resulting in sudden and widespread increases in metabolic disorders. for example, a recent paper examined the changes in diet and health in the rapidly urbanizing black south african population. the study found that the change in lifestyle was accompanied not only by an improvement in micronutrients but also by an increase in obesity and chronic diseases (vorster et al. ). characterizing such mismatches through the use of today's analytical tools promises to shed important new light on the etiology of these and many other common, complex disorders (e.g., autoimmune, inflammatory, and mental/behavioral conditions) and offers an innovative approach to the identification of novel interventions and therapies. continuing to look at mismatches between what we are prepared for evolutionarily and what we experience with a modern lifestyle, we find puzzling results that require an understanding of our evolutionary history to solve. for example, recent studies have demonstrated a connection between decreased intestinal parasite load and increased incidence of autoimmune diseases such as asthma (lau and matricardi ) . in one study, children who had been dewormed showed an increase in the occurrence of allergic reactions to house mites. why? what was the link between removing worms and developing allergies? one proposed explanation is the "hygiene hypothesis" (strachan ) , which takes an evolutionary view and considers that the historically "normal" condition for humans is a constant barrage of parasites and that the immune response evolved in response to these conditions. an over-reactive immune system is a recognized cause of allergies, and it turns out that the presence of helminthes, common intestinal parasites, actually increases regulation and control of the immune response. when these children were dewormed, regulation of their immune response decreased, allowing an allergic response to house mites. an interesting application of this research is the experimental use of intestinal parasites in the treatment of autoimmune diseases such as irritable bowl syndrome, crohn's disease, and multiple sclerosis (wickelgren ) . evolution can also shed light on normal physiological responses such as vomiting and fever and guide medical interventions (nesse and williams ) . in terms of evolution, a trait will be selected for if it provides some advantage. an example is "morning sickness"-the nausea and vomiting suffered by % of pregnant women (badell et al. ) . nausea and vomiting are commonly experienced in the first and second trimesters, and generally, foods such as meat, fish, poultry, and eggs elicit the strongest responses. clearly, feeling nauseated and vomiting frequently are unpleasant experiences at the very least, and hospitalization for rehydration is frequently required. occasionally, more serious complications arise. however, there is significant evidence that this physiological process is associated with successful pregnancies (weigel and weigel ) . one tested hypothesis posits that this response is beneficial to both mother and embryo because it reduces the mother's intake of, and embryo's exposure to, potentially toxic chemicals and pathogens during key developmental stages (flaxman and sherman ) . in addition, the foods most commonly avoided are animal products which tend to harbor more pathogens than plant foods. the immune system of pregnant women is depressed to avoid rejection of the fetus, so both mother and embryo are at higher risk from pathogens. the benefits of this response may well justify the discomfort and risks-or at least provide some psychological comfort to suffering women. evolution is not something that happened long ago and then stopped. it remains an active force in the modern world not only in human populations but also in the pathogens and parasites with which we live. one need look no farther than the local news or health clinic for proof. the evolution of antibiotic-resistant strains of tuberculosis made the news in spring when a man with extremely drugresistant (xdr) tb made multiple international flights (cnn ) . fortunately, no one else was infected in this instance, but the recent evolution of drug-resistant tb has revived old fears, especially in places where the complicated medical treatment required for these strains is not available. evolutionary medicine can help physicians take into account the effects of people's changing environments and lifestyles. human behaviors such as conflict, poverty, and urbanization provide opportunities for infectious diseases to spread (halstead ) . today, more people live in areas of high population density-a change from the more traditional small community. high population density urban centers are ideal for transmission of pathogens, particularly when coupled with high rates of poverty (armelagos et al. ) . old antagonists are not the only concern. behaviors that bring humans into contact with pathogen reservoirs in wildlife, such as logging and the exotic animal and bush meat trades, also create opportunities for the transmission of "zoonoses"-pathogens normally found in animal hosts but capable of infecting humans (patz et al. ) . for example, logging brings roads to new areas, providing easy access for local inhabitants who come to hunt for food. in the process, hunters either become prey themselves as they are infected by novel pathogens or bring the pathogens back to populated areas in the form of bush meat (chomel et al. ). sometimes, the infection is relatively benignfor example, children often pick up worms from household pets-but sometimes, the transfer is deadly. the ancestor of the human immunodeficiency virus (hiv) is the simian immunodeficiency virus, which is found in a wide range of primate species and rarely causes disease in the primate host (hahn et al. ) despite the fact that hiv is devastating in humans. taking an evolutionary approach to medicine can help us deal with novel pathogens by increasing our understanding of the pathogen and facilitating the development of appropriate responses. when the mysterious severe acute respiratory syndrome (sars) virus emerged on the world scene, researchers quickly gathered information about its physical structure, life cycle, and genetics. by comparing its characteristics with those of known viruses, researchers were able to quickly identify the novel sars virus as a coronavirus (who ) . that crucial piece of information allowed health care professionals to develop appropriate diagnostic tools, preventive measures, and treatments based on knowledge of how other coronaviruses behave. additionally, the information about viral type allowed field researchers to identify the source of the infection based on known hosts of coronaviruses. civet cats, sold live in meat markets, were frequently found to be infected with a sars virus. further research indicated that horseshoe bats, which were also sold in these markets, had a more ancestral variant of the virus (britigan ; lau et al. ; wendong et al. ) . the virus was also found to be more widespread in bats. the civet cats probably act as an intermediate host, or "amplifier." understanding the lifestyle and environment in which the disease arose helped to rapidly control it, preventing a feared worldwide epidemic. recently, new technologies such as improved genome sequencing and microarrays are generating huge quantities of genomic information. bioinformatic tools for analysis of this ever-expanding volume of raw data are developing apace, and genomics, the study of whole genomes, has become an independent biological discipline. the medically relevant value of this data is the potential to identify the genes underlying disease for the purpose of developing treatment or even prevention. evolutionary medicine plays a critical role in the interpretation and application of this information, since human evolutionary history has shaped the genome. humans as a species are relatively young with a mere , -year history. the pattern of our relatively recent global dispersal from our home in africa is reflected in our genes. different populations demonstrate our common ancestry and recent isolation during dispersal (kidd and kidd ) . interpretation of genomic data must take our evolutionary history into account to yield useful applications. one of the most informative analytical approaches is comparison between sequences, either of small genomic regions or entire genomes. by examining differences and similarities between the genomes of two organisms, researchers learn about ancient genes that have deep evolutionary roots and about genetic differences between organisms that make them unique. in comparing the human genome with those of closely related primates, we learn what minor differences may be important keys to our human condition. comparisons between the human genome and more distantly related organisms provide information about ancient genes that play important roles in fundamental processes such as development and physiology. this can be done on various levels. microarrays allow comparisons between sets of genes or two entire genomes. bioinformatic tools allow comparisons of multiple genes, sections of genomes, or entire genomes. a new method that exploits both microarray technology and genome sequencing is the genome-wide association (gwa) study. in a gwa, a microarray of all of the known human single nucleotide polymorphisms (which are simply a change in one nucleotide) is screened for differences between diseased and healthy individuals. to give the study statistical significance, samples from thousands of individuals are used. in a recent gwa, researchers identified genomic polymorphisms potentially associated with seven different diseases (wellcome ). this information can be used to guide future research on the genetics of these diseases. the gwa is yet another method based on enormous data sets that may lead to significant advances in medicine. resources to promote this kind of megaanalysis include the human genome project and the hapmap, which uses collections of single-nucleotide differences as markers for larger genetic regions. the hapmap is important because genomes of any two humans differ only by . %. researchers can use the general markers from the hapmap to narrow down their searches to areas of variation. an important key to drawing useful information from genomic data is the ability to link a genotype to a phenotype. this is a difficult problem for several reasons: genomic data are not usually collected from a specific individual with an extensive health history; family medical histories are rarely complete; environmental conditions vary between populations, etc. one approach to deal with this issue is the use of large, long-term studies of specific populations. the framingham heart study (fhs) is the quintessential large cohort, long-term study. initiated in to investigate the causes of heart disease and stroke, the project collected data on more than , people living in framingham, massachusetts. in addition to birth, marriage, reproduction, and death data, the participants were given physical exams and lifestyle interviews. followup physicals were given every years. adult children of the original cohort and the children's spouses, as well as the original cohort's grandchildren, also participated in the study. this study allowed researchers to identify the major environmental risk factors for cardiovascular disease, such as smoking and high blood pressure. because of the extensive data collected for the project and the virtually unprecedented length of the study, the fhs is incredibly valuable to medical researchers. the data are now being mined by groups researching other diseases such as obesity and eye and lung diseases. other studies of large or multigenerational cohorts are also under way using different populations. diddahally govindaraju is the director of the fhs genetics laboratory at the boston university school of medicine and was one of the organizers of the nescent meeting. he is enthusiastic about the concept of applying evolutionary principles to cohort data such as that generated over nearly six decades in the fhs. "i was essentially sitting on a wealth of medical data that has been collected for the last years," he says. "having been raised on evolutionary biological thought in the first part of my professional life, i looked at this and realized that the doctors were not looking at these data from an evolutionary biology perspective." dr. govindaraju and his colleagues have organized a working group to explore the fhs from an evolutionary viewpoint. the group proposes to look for microevolutionary changes in the study population and to use the extensive medical data to correlate differences in genomic inheritance and phenotypic outcomes. given the vast quantities of data available because of the genomics revolution and multi-generational, longitudinal clinical cohorts such as the framingham heart study, it has become increasingly clear that the analysis of new and existing data from an evolutionary perspective promises to yield important insights into long-standing questions about human physiology and pathophysiology. combined with the development of new technologies that have given us entire genomes and the tools with which to study them, these vast data sets have the potential to launch an evolutionary medicine revolution. it should come as no surprise that evolutionary medicine comes with a unique set of ethical issues. a field that encompasses so many aspects of who we are as individuals and has the power to provide better health to so many people also has the potential to be abused in a variety of ways. positive applications such as personalized medicine, in which an individual would receive the most beneficial medical treatment based on his or her genomic makeup, hold great promise for improving the efficacy of medical care. however, it is no great stretch to imagine that genetic information about individuals could be misused outside of the medical realm. communication of complex evolutionary medicine concepts to the general public will be fraught with cultural perspectives, biases, and historical antagonism, and miscommunication could have serious consequences for individuals and society. privacy concerns are a major issue. when so much data, including genetic information, are being gathered on so many people, how can a participating individual's privacy be protected while simultaneously making the information as widely available as possible (nhgri ) ? also, the use of particular populations in studies is a double-edged sword. in some cases, researchers can learn a great deal by studying a particular group whose members share a lifestyle or ancestry. but how can that group be certain that the information will not be used against them, by insurance companies, for example? on the other hand, if beneficial information is gleaned from one group, how is that benefit made available to other groups who were not studied? another important issue to consider is the concept of race (kittles and weiss ) . particularly in the usa, the sensitive nature of this issue cannot be overstated. the genetic concept of race and the social concept of race are not the same. in colloquial use, the term race designates a group of people based on their appearance and culture. among the research community the definition of race has not been consistent or well defined. scientifically, race correlates roughly with shared genetic ancestry usually based on geographic proximity, but even these parameters fail to clearly delineate groups. there is some debate as to whether race is a legitimate biological concept at all. social concepts of race also influence how research is done by biasing selection of populations to be sampled, methodologies, and other aspects of research. paradoxically, some diseases are associated with socially defined races, such as tay-sachs disease in the ashkenazi jews, making it necessary to take into account these social groupings in conducting research. the exciting potential of evolutionary medicine to yield important new insights into long-standing questions about human physiology and pathophysiology was summed up by stearns: "when you see how evolution illuminates those questions, you see there's the potential for a new way of looking at them to save literally hundreds of millions of lives. that is an enormous payoff, and it's the kind of thing that people in medical research have felt all of their lives, but it's a new motivation for evolutionary biologists, and it's a revelation to them to realize that they could bring that to the table." evolutionary, historical, and political economic perspectives on health and disease treatment options for nausea and vomiting during pregnancy journal watch infectious diseases exotic pets, and emerging zoonoses man knew he had tb before flying to europe morning sickness: a mechanism for protecting mother and embryo evolution in contemporary human populations: medical aids as a zoonosis: scientific and public health implications human factors in emerging infectious diseases recent acceleration of human adaptive evolution human genetic variation of medical significance race, ancestry, and genes: implications for defining disease risk worms, asthma, and the hygiene hypothesis severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats lifestyle, diet and disease: comparative perspectives on the determinants of chronic health risks testosterone-mediated immune functions and male life histories genetic discrimination diabete mellitus: a "thrifty" genotype rendered detrimental by "progress evolution and the origins of disease members of the working group on land use change disease emergence. unhealthy landscapes: policy recommendations on land use change and infectious disease emergence (eds) evolution in health and disease hay fever, hygiene, and household size menstrual cycling and breast cancer: an evolutionary perspective genetics of lactase persistence and lactose intolerance convergent adaptation of human lactase persistence in africa and europe evolutionary medicine the nutrition and health transition in the north west province of south africa: a review of the thusa (transition and health during urbanization of south africans) study insulin resistance in an energy-centered perspective nausea and vomiting of early pregnancy and pregnancy outcome. a meta-analytical review wellcome trust case control consortium. genome-wide association study of , cases of seven common disease and , shared controls bats are natural reservoirs of sars-like coronaviruses can worms tame the immune system coronavirus never before seen in humans is the cause of sars acknowledgments the authors would like to thank the organizers and participants of the evolution in contemporary human populations: medical, genetic and behavioral implications meeting at nescent for providing access to the meeting and supporting information, and reviewers for their helpful comments. key: cord- - am l ms authors: nan title: spr date: - - journal: pediatr radiol doi: . /s - - - sha: doc_id: cord_uid: am l ms nan dear colleagues, i confess i haven't read many "welcome letters" at the beginning of the spr program book over the years. perhaps the only defensible benefit of this is that there is no preconception about the content of this message…or the length. i will be brief. this meeting is about building bridges…bridges from our past to the future and bridges between all of us who believe fundamentally in maintaining or improving the health of our children. the content, which is detailed on subsequent pages, speaks for itself. this material will be presented during the sessions with an appreciative look back at past accomplishments-the legacy of our subspecialty-with a vision to the future of pediatric imaging. we can only measure how broad and deep our successes have been by connecting with these beginnings. looking beyond the titles (and the speakers), i think you will see that the material is not only about techniques and tactics but about ideas, insights, energy, all conspiring in the creative process … an aggregate for excellence in pediatric imaging. the content is also punctuated by a strong presence of our clinical colleagues. again, this builds bridges. how can we maintain and expand these relationships? moreover, the connections between science and clinical practice are evident in the structured blending of scientific papers and topical presentations by both imaging and clinical experts. this blending is also "fraternal" in that there will sometimes be disagreement and critical commentary, but this is essential in the advancement of medicine. support and criticism make a stronger mortar. in the end, this gathering is about fostering a connected community, including technologists, nurses, physicists and other allied health experts including industry experts. finally, the emblem of pediatric radiology has always been embossed by cooperation, passion, commitment, and humanistic care. i believe the program content, the presenters and you, the participants, all embrace this. i hope that you will feel the spirit and the passion of the meeting and all of us will in many ways be better able to care for children because of this-even if you never read this message! donald p. frush the gold medal of the society for pediatric radiology is our most distinguished honor. the spr medal is awarded to pediatric radiologists who have contributed greatly to the spr and our subspecialty of pediatric radiology as a scientist, teacher, personal mentor and leader. marilyn goske has always wanted to make a differenceand what a difference she has made! her role as an educator, and her lifelong commitment to improving training for residents, fellows, faculty, medical staff and radiologic technologists has resulted in many wonderful initiatives that have benefited all in pediatric radiology. the work she is most proud of-the cleveland clinic web based curriculum, working with the leadership of spr's philanthropic campaign for children, launching the image gently campaign and the pediatric research component within the american college of radiology's dose index registry share a common theme: educating others in providing the best care possible for children. born in berea, ohio, marilyn's father, george, was a chemical engineer. her mother, cornelia aka "corky", loved writing as one of the first women journalists for the associated press and later teaching, passions she passed on to her daughter. while marilyn was blessed with a strong female role model in her mother, it was her brother, james, who was her cheerleader, always pushing her to dream big. he encouraged her to follow in his footsteps first at ohio university, then on to the ohio state college of medicine to pursue an md degree during an era when nursing would have been a more conventional goal. marilyn met her husband rick on a double date in college-unfortunately, they were with different dates! luckily, they were able to get together for an actual date with each other months later. they quickly became engaged and married within a year of that first true date. when rick started his residency in internal medicine, marilyn transferred to the university of connecticut school of medicine in farmington. it was here that she met her first pediatric radiologist-and what a giant-mike ozonoff! when rick moved on to a neurology residency in rochester, new york, marilyn followed and met another pediatric radiology giant: beverly wood, at strong memorial hospital. beverly proved to be a wonderful teacher, mentor, co-researcher and lifelong friend. marilyn describes beverly as inspirational and "fearless" in trying new technologies. it was during her time in rochester that marilyn went to her first spr meeting and, not surprisingly, won the caffey award for her work on "experimental neonatal intraventricular hemorrhage: clinical, radiographic and pathologic features." by then marilyn had two young children and moved on to the private sector, practicing part-time for several years first in rochester, then in cleveland, ohio. her years in private practice were particularly helpful in learning the importance of patient oriented service-and paved the way for her intuitive public relations strategies when designing the image gently campaign in later years. dr. goske was asked to join the cleveland clinic in , as the first full-time section head of pediatric radiology. it was here that she built a new section and spearheaded the web based education program for pediatric radiology residents with co-founder janet reid. this important free web site with modules is used widely by over radiology residencies nationally and internationally. her passion for education continued, inspiring her to complete a medical education fellowship focused on professionalism within the cleveland clinic lerner college of medicine. her work towards this fellowship has led to many creative educational initiatives including yearly educational summits at the spr. she was named chair of the professionalism committee of the rsna where she along with her committee have sponsored interactive workshops on this topic dear to her heart. dr. goske's energy and effective leadership skills brought her to become involved in the society for pediatric radiology, first as the coordinator for spr's first video-taped course in . mentors diane babcock and carol rumack proposed her for the nominating committee. this was followed by chair of the membership committee, where she organized the first formal survey of the society, then as a board member, then as secretary, and finally as president and chair of the board of directors, completing years on the spr board. working together with stuart royal, she successfully energized the campaign for children raising funds for the research and education foundation of the spr and expanded the work of prior presidents in further organizing the corporate support committee. marilyn's years as president and chairman of the board of the spr were highly successful with many unique strategic goals. she was instrumental in the founding of the junior spr. she led the wonderful spr national meeting in miami which included the first educational summit to enhance knowledge in adult learning and resident competencies. most people would rest after completing their arduous year as president but as chairman of the board, marilyn was just beginning! she moved to cincinnati children's hospital, joining the radiology department and was named the dr. corning benton endowed chair for radiology education where she got to work with dr. janet strife, another influential mentor and friend. acknowledging spr's long focus on reducing radiation doses in the imaging of children but concerned about the lack of change in practice by a majority of radiologists despite increasing reports of possible side effects, marilyn developed a public relations and awareness campaign. her goal was to inspire all to work towards decreasing radiation exposure to children when possible. with the help of many, she founded the alliance for radiation safety in pediatric imaging and the image gently campaign, initially focusing on ct. her ability to encourage numerous experts and societies to work together and get involved in "child sizing the amount of radiation used" has resulted in a groundswell of research and activity in this area. currently organizations with over , members have joined the alliance including international societies. the web site, www.imagegently.org, has been immensely successful filled with free information pamphlets in over languages, pqi projects, and modules for parents, physicians, and technologists. the image gently campaign has received several awards including the associations advance america honor roll, rt image magazine group with the "most influence in radiology" and the most effective philanthropy program from aunt minnie. image gently has spawned the creation of the adult-focused image wisely campaign. the alliance has been named by the joint commission, u.s. food and drug administration, and the american medical association in their influential statements on radiation dose as providing much needed guidance and information. marilyn's exceptional talent is inspiring and coordinating experts in multiple fields to work together towards common goals. she continues to work hard on the image gently campaign with more safety and quality messages planned for the coming years. she is also proud of her work with the acr dose index registry and quality improvement registry in ct scans in children in working toward developing diagnostic reference levels with a talented consortium of pediatric radiologists, medical physicists and technologists. she has acted as a national and international expert in her work with the international atomic energy agency, the world health organization and the national council on radiation protection in medicine and the fda. dr. goske's multiple committee appointments are taken seriously, and her work is always meticulous, well thought out, and brought to successful completion. she has been an active member of numerous national and international societies including the john caffey honorary society, acr, rsna, espr, aawr, and scorch. it is important to remember that dr. goske is also a successful researcher with numerous grants obtained through the spr ref including the thorne griscom education award and the rsna scholar grant. she has published over peer-reviewed articles, electronic publications, chapters, and presented scientific exhibits as well as given numerous scientific presentations. an articulate and engaging speaker, she has been invited to give over lectures locally, nationally and internationally. while marilyn has been very focused on her work with the spr, she believes that it is her amazing family and their love that really fuels her life. her husband rick is an internationally known neurologist and researcher in multiple sclerosis. her adult children jamie and brian, both in manhattan, remain close, and spending quality time together as a family remains the joy of her life. whether it is relaxing together in florida cooking or fishing, or taking an exotic vacation to india, being with rick, jamie and brian makes her the happiest. marilyn's genius is partly refusing to take "no" as an answer. along the way, at every turn there were those who believed that what she wanted to do couldn't be done. her approach was to draft the nay-sayers to the team and charge ahead with their willing and enthusiastic help. daniel burnham might have been talking about marilyn and not about his plan for the city of chicago when he said: make no little plans; they have no magic to stir men's blood and probably themselves will not be realized. make big plans; aim high in hope and work, remembering that a noble, logical diagram once recorded will not die, but long after we are gone be a living thing, asserting itself with evergrowing insistence. as an amazing change agent, inspirational leader, and wonderful role model, the spr is proud to honor marilyn goske with the gold medal. she made big plans! dorothy i. bulas, md pioneer honorees were first acknowledged in as a means to honor certain physicians who made special contributions to the early development of our specialty. it is time to reevaluate the meaning of the pioneer honoree. the subspecialty of pediatric radiology has been in existence now for more than years. we are beyond "the early development"; we must recognize other pioneering paths and should consider contributions to the subspecialty beyond the bounds of a modality, a technique, an observation or a change in practice. whatever this advancement is, it must be forged with vision, innovative ideas, and the ability to enable and sustain science and application. george s. bisset, iii, m.d. why george bisset? has he been part of the pediatric radiology landscape these last ten years? been part of the dialogue that has been increasingly influential across all of radiology, a conversation steeped in a deep tradition of excellence in diagnosis and treatment, and the safety and welfare of our children? been a leader in science and application? part of the landscape? no. but he has been beyond that and has worked tirelessly within the horizon, surveying…a step before, but guiding us on towards our destiny. a conversant? part of the dialogue? maybe. but he has been defining thought and concept upon which such conversation is born and nurtured. part of the science and application? yes, as much as anyone who promotes, who facilitates and sustains discovery, then here we are. horizons, innovation, and the gift of en-abling…what else is needed to define a true pioneer? how was this done? simply stated, george bisset has devoted at least the last decade to the advancement of our specialty in truly novel ways through his leadership, especially in rsna and the abr. in the rsna, as the scientific program committee chair several years ago he was instrumental in the conception, development and implementation of the integration of scientific papers and refresher course topics. this has been a resounding success, is currently used in other categories during rsna and is a model for other meetings, including the annual meeting for the society for pediatric radiology over the past few years. pediatric radiology was first in this effort. george continued to endorse topics that were marquees for pediatric radiology over the year in his education role on the rsna board of directors. he endorsed and implemented the pediatric campus concept at the rsna. early returns are that this was an extremely successful model to consolidate experts in pediatric radiology (and those interested in this subspecialty), pertinent science, education and administration. george is now the president of rsna, perhaps the most widely respected scientific and educational organization for our profession across the globe…and i would argue, with more promise for our future success in pediatric radiology than has ever existed. and george bisset, who through two terms as the pediatric trustee for the board of trustees for the abr, again, was on the horizon of a critical, sometimes perilous, and complete transformation of our certification examination process, always mindful of his constituency and colleagues, his duty as a physician, and the public and patients. this required delicate diplomacy, forward thinking, professionalism, and enlistment of a cadre of experts from within our subspecialty to assure excellence in pediatric radiology through abr certification. he was also a leader in the development, validation and implementation of the imagerich computer based examination model (the pediatric caq) now the standard for the new abr examinations. with these successes in mind, who better to embody the concept of bridging horizons that is the theme for this entire meeting? if you were looking for more numbers and accolades, i apologize. here are some: more than contributions to medical and scientific literature, advancing care through pediatric body ct and mr imaging research, a litany of presentations and invited lectures, vice chairs, chairs, chiefs, boards of directors, committee member and committee leader, clinical excellence including as a pediatric cardiologist and interventional radiologist, a superb speaker and author.… all are on his cv but i believe serve really as signposts for his gifts, some of those mentioned above, that a cv simply cannot convey. he could have played it safe with all of these successes on his cv. but pioneers don't play it safe. they are on the horizon, too busy defining thought and enabling (our) advancement-building bridges. i believe it is time to reevaluate the meaning of the pioneer honoree and i have the greatest honor and pleasure of introducing george s. bisset, iii for the pioneer award for . linked with past awardees, he continues an exceptional legacy and i don't believe his explorations and discoveries are finished… donald p. frush, md the society bestows presidential recognition awards on members or other individuals whose energy and creativity have made a significant impact on the work of the society and its service to its members. in , david kushner was recognized by the spr with its first presidential recognition award for his vision and foresight in working with both the american college of radiology (acr) and the society for pediatric radiology (spr) in developing an important new relationship and for his service to the spr. in summarizing his considerable efforts for that award, i noted that he "contributed substantively to the increased visibility of the spr within the acr. his tenure as our treasurer placed our organization on a firm financial foundation." with the current award, the society recognizes his indefatigable continuing efforts on our behalf including: his work with the acr: . establishing a pediatric radiology caucus at the annual acr meeting, . convincing the acr of the value of managing specialty societies by making the spr its first successful new model for imaging society management, . advocating tirelessly for pediatrics and children's health within the acr by serving on the council steering committee and then as acr council vice speaker and speaker, . helping establish the first pediatric commission, assuring that pediatric issues will receive support of the college and its resources while serving on the board of chancellors of the acr for the past five years. the spr's "image gently" campaign was a beneficiary of this pediatric commission of the board of chancellors, . continuing to shepherd and contribute to the pediatric component of the acr practice guideline process. his work with the spr: since his earlier award, david has served as: foundation from to , including the launch of the formal fundraising effort, "the campaign for children," . spr president - , organizing and running a very successful meeting in savannah, . chair of the board of directors of the spr from to , including leading a strategic planning process that resulted in a new, more focused division of labor amongst board members and defined board responsibilities. david was born in fargo, north dakota, received a ba from the university of minnesota, and received his medical education at the university of pennsylvania. this was followed by two years of training in pediatrics at children's hospital, boston. he then did a two-year fellowship at the national institute of health in bethesda, performing research in embryology and teratology. he returned to massachusetts general hospital for training in diagnostic radiology. this was followed by a year of residency in pediatric radiology at children's hospital boston, followed by a one-year fellowship. he then became director of the pediatric radiology section at massachusetts general hospital, a position he held from to . from to , david was chief of the division of diagnostic imaging and radiology at children's national medical center in washington, dc attracting a strong faculty, training many fellows and promoting research. during that time, he served as a volunteer radiologist and pediatrician to inner city healthcare systems aiding the indigent and homeless, and developing telemedicine capabilities linking free clinics with radiology experts. in , our man inside the beltway moved a bit outside by accepting the medical directorship of radiology at the children's hospital of the king's daughters in norfolk, virginia, and professor of radiology and pediatrics at the eastern virginia medical school. he assures me that life there is good, being a bit more "laid back" with fishing and sailing just outside the door. he also finds time for italian cooking and practicing jazz on his several guitars. fortunately for all of us in the spr, david is close enough to our central office and the acr that he will be able to continue work on our behalf for many years to come. the society bestows presidential recognition awards on members or other individuals whose energy and creativity have made a significant impact on the work of the society and its service to its members. the spr presidential award is given in recognition of stuart's numerous significant and outstanding contributions to the spr over many years of service. the awardee is selected by the honors committee, a committee comprised of the three most recent past presidents of the society. dr. royal is a proud native of birmingham, alabama. he is a second generation physician who came naturally to his desire to care for children as the son of a pediatrician, arnold royal, who took care of children in the birmingham community until he was years old. dr. royal attended rice university in houston, texas followed by md and ms degrees from the university of alabama at birmingham. he subsequently moved to san francisco, where he completed a pediatric internship followed by a diagnostic radiology residency at the university of california, san francisco. dr. royal credits dr. charles gooding at ucsf for influencing his decision to pursue a career in pediatric radiology. during his internship stuart observed dr. gooding make a plain film diagnosis of tapvr, type on a severely ill and perplexing newborn, and he was immediately hooked into radiology. while at ucsf dr. royal was also appointed as a national institute of health research fellow in the department of radiology. following residency, stuart completed a fellowship in pediatric radiology at the children's hospital medical center in boston. from boston, stuart returned to his roots in birmingham, alabama in , where he was appointed as a pediatric radiologist at the university of alabama and subsequently the children's hospital in birmingham. in recognition of his outstanding leadership skills and accomplishments at the children's hospital, dr. royal was appointed as the radiologist-in-chief in , and subsequently the harry m. burns endowed chair of pediatric radiology. he also holds appointments as clinical professor of radiology and pediatrics at the university of alabama at birmingham and serves on the children's hospital board of trustees. at alabama dr. royal has earned the high esteem of his colleagues, referring physicians, and staff for his outstanding clinical acumen as a diagnostic radiologist and for his undaunting commitment to excellent care of children. colleagues describe stuart as one who fosters a strong work ethic, high commitment to teaching, and sincere compassion for children. in , stuart was the recipient of the children's advocate award by childcare resources for improving the quality of care and access to radiological services for underserved children in birmingham. stuart has been married to the love of his life, barbara royal, for the past years. stuart and barbara are the proud parents of two very accomplished children, jeremy a budding radiologist in training at the university of alabama, and rachael, who has an mba and works as a vice president for moody's in new york. stuart and barbara are also the proud grandparents of three grandchildren. in conversation, stuart is quick to pull out his iphone and share the latest pictures of family members while recounting their latest activities and milestones. throughout his professional career, dr. royal has worked tirelessly to advance the mission of the society for pediatric radiology. he is past president and chairman of the board of the spr and has served on numerous spr committees. he ran a highly successful spr meeting in new orleans in . those in attendance will recall the jubilant parade stuart led through the streets of new orleans to culminate the meeting. as president and then chair of the spr board, stuart played a critical and instrumental role in bringing the spr management contract under the umbrella of the acr. the synergy achieved by the spr-acr relationship has yielded results well beyond a simple management contract. pediatric radiology and spr now have a voice at the "radiology table." stuart has also been a strong advocate for supporting translational research to advance the care of children via imaging. to help achieve this goal, he has worked aggressively to secure increased funding for the society of pediatric radiology research and education foundation. following the launch of the ref's campaign for children in , stuart made it his personal mission to work with the leadership of the society, both past and present, to discuss major gifts to the foundation. through stuart's personal effort, the foundation received pledges for many significant leadership gifts, including from spr pioneers drs. hooshang taybi and ed singleton and from himself and barbara. the spr is highly fortunate to have benefited from stuart's numerous contributions and dedication to the care of children. the society is very proud to bestow the president's award on dr. stuart a. royal. the society extends honorary membership to individuals outside of pediatric radiology who have made outstanding contributions to the care of children. this evening, dr. harvey l. neiman, whose leadership of the american college of radiology is resoundingly praised, is the recipient of the honorary member award. for , as in when his contributions were similarly recognized, dr. neiman's selection by the society for pediatric radiology honors committee was made in appreciation for the strength of his efforts to further the spr's philosophy, goals, and programs for responsible diagnosis and treatment of the young patient as embodied in the acr and spr's "image gently" campaign. image gently has succeeded not only in raising awareness of the great diagnostic benefits we can offer to pediatric patients but also directs us to acknowledge the downside of overzealous diagnostic efforts where excessive radiation becomes a risk. importantly, the "image gently" campaign, an upbeat, positive program rather than a punitive one, a smile rather than a frown, makes pediatric and all radiologists aware that their best practice reflects balanced, educated, up-to-date utilization of state-of-the art technology with exercise of responsible leadership in protecting the pediatric patient. for adults, awareness of the need for patient protection is communicated in image wisely. dr. neiman's vision and successful achievements are evident on every page of his curriculum vitae. a consummate strategist in assembling teams to make forward-looking goals a reality, harvey now stands at the top of our specialty as the first physician executive director of the american college of radiology. at this time in big-business medicine, as we see the physician, leader of the patient care team, being diminished to one of many "providers," it is so important for our patients' well-being for us to recognize the obligations commanded by our training, clinical experience and commitment. dr. neiman's recognition of the need for physicians' leadership in improving the quality of patient services and his development of programs in all areas of the college's activities have been just short of miraculous-image wisely for adults, quality and safety including the performance guidelines and accreditations, education, government relations, economics, imaging metrix, acrin, and the new radiology leadership institute-to name only a few. all have contributed significantly to the care of our patients and the stature of our specialty. dr. neiman was born in detroit and attended mumford high school. from wayne state university, he received his b.s. in and his md in . harvey's postgraduate training was at the university of michigan, where he was a resident in radiology ( - ), chief resident ( , and a - fellow in angiography (cardiovascular radiology), receiving abr certification in and a caq in vascular and interventional radiology in february . dr. neiman often expresses his gratitude to and profound respect for his mentor and beloved chief at the university of michigan, dr. william martel. dr. neiman was chief of cardiovascular radiology at walter reed army hospital and a lecturer in cardiovascular radiology at the afip from - . in , he joined the northwestern radiology faculty, rising to professor in , and for ten years he headed up the section of angiography and sectional imaging, advancing its technology and honing the skills of northwestern's radiology residents. harvey also offered a highly sought-after fellowship in interventional radiology, us, and ct. in , dr. neiman left northwestern to assume the chair in medical imaging at the western pennsylvania in pittsburgh. i was the first woman to have completed his fellowship in us, ct, and interventional radiology at northwestern and accompanied him to pittsburgh. his tenure at west penn attests to his talent in making his visions a reality: the department became a highly respected, successful academic private practice notable in many areas including ultrasound, breast and women's imaging, and interventional radiology. harvey instituted an excellent radiology residency program in as well as fellowship programs in in the areas of excellence noted above. during the years since harvey received his md, he has been awarded honors from many national, international, and specialty societies, has been an invited lecturer over times on ultrasound, interventional radiologic, radiologic educational, management, turf issues, disruptive and new technology topics to name just a few. dr. neiman, who was a founder of the sru (society of radiologists in ultrasound), has to his credit peer-reviewed articles, scientific presentations and exhibits, a text co-authored with dr. james yao, angiography of vascular disease ( ) , and book chapters. he has received many honors including fellowship in the american college of radiology, american institute of ultrasound in medicine, society of radiologists in ultrasound and the society of cardiovascular and interventional radiology (now sir). as part of his strong commitment to the future leaders of radiology, for diagnostic radiology he has served as a member of the residency review committee of the accreditation council for graduate medical education. he has been a member of the american college of radiology and its committees and commissions for many years including the commissions on education, ultrasound, and economics. he also served as chair of the commissions on ultrasound and economics. from to , he was a member on the acr board of chancellors, serving as its chairman - . he was president of the radiology advocacy alliance from to . in , nine years ago, dr. neiman became the acr's executive director. he currently serves in this position, where his excellent business skills, knowledge of health policy and economic issues, and strong administrative background have furthered our specialty. his goal, to ensure that the acr's resources benefit all radiologists and patients across all economic strata, is evident in his actions at the college. harvey has a devoted, wonderful family that often included me and my youngest daughter on many pittsburgh occasions. his beautiful, elegant wife of many years, ellie neiman, is here tonight to celebrate with him the spr's recognition of his many achievements. dr. neiman has two accomplished, lovely daughters, jennifer, extremely successful in her marketing career, and hilary, an attorney. jennie's husband, dr. seth kligerman, one of many young radiologists whom harvey has mentored, is on the radiology staff at the university of maryland. how harvey has had time between, through, and among all of these achievements to have become mentor, colleague, and friend to me and to so many others who have been inspired by his ability to see into the future and to shape it in a positive way is remarkable. now that dr. neiman has taken all of radiology under his wing, not just its component parts, the future of our specialty, one of the best, can be assured but also recognized for its centrality to all of medicine. it is my honor and privilege to introduce to you harvey l. neiman md, facr as this year's society for pediatric radiology honorary member. the singleton-taybi award is given in honor of edward singleton and hooshang taybi, in recognition of their personal commitment to the educational goals of the spr. initiated in , the award is presented annually to a senior member of the spr whose professional lifetime dedication to the education of medical students, residents, fellows, and colleagues has brought honor to him/her and to the discipline of pediatric radiology. it comes as no surprise to those who know him that dr. daneman, "dr. d" as some of us call him, has been named the recipient of the singleton-taybi award in recognition of his many years of dedication to the education of residents, fellows, and colleagues. born in south africa in , he received his medical degree at the university of the witwatersrand, johannesburg, receiving the harwood-nash award for the most successful student in surgery. initially, dr. d thought he would become a pediatric surgeon; but after passing the part i examination offered by the royal australasian college of surgeons, he changed his mind and began his training in diagnostic radiology. he chose a radiology residency at the royal prince alfred hospital, in sydney, australia. this included a year in pediatric radiology at the royal alexandra hospital for children in sydney where his interest and love of pediatric radiology began. dr. d then had the foresight to pursue pediatric radiology fellowship training at the hospital for sick children in toronto, canada. after completing the fellowship, he was immediately offered a position as staff radiologist at "sick kids." he became director of body imaging in and radiologist-in-chief in serving in that capacity for years. his management style was simple but effective. he chose staff that were young, but smart and innovative. he nurtured them and provided them with all the tools they needed to become successful professionals, like him. but contributing to his own department was not enough for him. he also found the time and strength to contribute, teach, train, and help pediatric radiologists in the most remote portions of the globe in every continent, which resulted in recognition from prestigious organizations in places such as south america, israel, europe, taiwan and australia: he is an honorary member of the european society for pediatric radiology and the sociedad latinoamericana de radiologia pediatrica as well as other national societies. dr. d is an "institution" inside the great institution that is sick kids. his teaching is unique and praiseworthy in being enthusiastic, provocative, and fun at the same time. his lectures have been regarded as both instructive and practical by his students and trainees due to his special gift of making the most complicated things look as simple as possible. in sharing his diagnostic knowledge and know-how, he passes his own, innate teaching spirit on to his apprentices. he has earned several awards for this, including the outstanding teacher award granted by the university of toronto fellows at sick kids for the past consecutive years. dr. d receives numerous invitations to present at national and international meetings and symposia and has been invited as a visiting professor to more than a hundred institutions across the globe. he does not only teach us the ins and outs of pediatric radiology, but he makes sure that we learn to love it and understand the importance not only of good practice but also the imperative to pass knowledge on by teaching and publishing. dr. d is someone who inspires us to reach beyond our limits, someone we want to emulate. he shares his knowledge, his wisdom, and his advice freely. he shares with us the most incredible secrets of his own career, so we understand from his personal experience. dr. d never tells you what to do, he suggests to you, in an incredible articulate fashion, what you want to do yourself. dr. d has been and is for many of us, more than an educator, more than a mentor, he is our "coach." well before this concept was introduced into medicine by a. gawande , dr. d intuitively had the vision to "coach" his trainees, trying to get the best out of them, without pressure, but with love and passion, and especially emphasizing the importance of achieving a worklife balance in order to prevent the now so common "stress and burnout" affecting the radiology community . he warned us that many high achievers reach their goals only at the expense of their personal lives, but dr. d has been as successful personally as he is professionally. his wonderful wife of years, louise, his two daughters and his recently newborn granddaughter serve as sources of strength and pride. he is a truthful and generous friend to many, both in and out of radiology. it is not uncommon for many of us, who came through sick kids, to come back and visit and be invited to his house to share a wonderful dinner with other invitees, who may be radiologists from north america or from other parts of the globe visiting sick kids to learn from him. dr. daneman's research has widely influenced the field of pediatric radiology. examples include the work of dr. daneman and his colleagues on intussusception, which has promulgated the use of ultrasound for diagnosis, and the use of air enema for reduction. this approach has been adopted as standard practice at many institutions in north america and across the globe. to share his research with others in the field, dr. daneman has authored or co-authored more than publications, including peer reviewed articles and book chapters on a wide range of topics related to the imaging of children. dr. d is one of those rare people who are irreplaceable. he is a superb teacher, a gifted academician, a capable administrator, and a person called "friend" by so many of us. we are thrilled and proud to present our society's singleton-taybi award to dr alan daneman in recognition of his lifelong accomplishments and personal commitment to the educational goals of the spr. we cannot imagine anyone more deserving of this award than dr. d. thank you "coach"! monica epelman, md and oscar navarro, md john caffey, md - dr. caffey was regarded throughout the world as the father of pediatric radiology. his classic textbook, pediatric x-ray diagnosis, which was first published in , has become the recognized bible and authority in its field. the seventh edition of this book was completed several months before his death in . it has been among the most successful books of its kind in the medical field. dr. caffey was born in castle gate, utah on march , . it is interesting that he was born in the same year that roentgen discovered the x-ray. dr. caffey was graduated from university of michigan medical school in , following which he served an internship in internal medicine at barnes hospital in st. louis. he spent three years in eastern europe with the american red cross and the american relief administration, and returned to the united states for additional training in medicine and in pediatrics at the universities of michigan and columbia, respectively. while in the private practice of pediatrics in new york city at the old babies hospital of columbia university college of physicians and surgeons, he become interested in radiology and was charged with developing a department of pediatric radiology in . he frequently expressed appreciation and admiration for the late ross golden, chairman of radiology at columbia presbyterian hospital, who allowed him to develop a separate department of diagnostic radiology without undue interference, and who was always available to help and advise him. dr. caffey's keen intelligence and inquiring mind quickly established him as the leader in the fields of pediatric x-ray diagnosis, which recognition became worldwide almost instantaneously with the publication of his book in . dr. caffey received many awards in recognition of his achievements. outstanding among these were the mackenzie davidson medical of the british institute of radiology in , the distinguished service award of the columbia presbyterian medical center in , the outstanding achievement award of the university of michigan in , the howland award of the american pediatric society in , the jacobi award of the american medical association in , and the gold medal award of the american college of radiology in . he had been a member of the american journal of roentgenology. he was a counselor of the society for pediatric radiology and was an honorary member of the european society of pediatric radiology. dr. caffey's contributions to the pediatric radiologic literature were many. he was instrumental in directing attention to the fact that a prominent thymic shadow was a sign of good health and not of disease, an observation that literally spelled the end to the practice of thymic irradiation in infancy. infantile cortical hyperostosis was described by him and is called "caffey's disease." dr. caffey in first recognized the telltale radiographic changes that characterize the battered child, and his students helped disseminate his teachings about these findings. it was dr. caffey who first recognized and descried the characteristic bony changes in vitamin a poisoning. he recognized and described the findings associated with prenatal bowing of the skeleton. in , three years after his retirement from babies hospital, he joined the staff of the children's hospital of pittsburgh as associate radiologist and as visiting professor of radiology and pediatrics at the university of pittsburgh school of medicine. although dr. caffey came to children's hospital and the university of pittsburgh in an emeritus position, he worked daily and on weekends throughout the years he was there. in pittsburgh, he made four major new contributions to the medical literature. he described the entity, "idiopathic familial hyperphosphatasemia." he recognized and described the earliest radiological changes in perthes' disease. he called attention to the potentially serious effects of shaking children, and used this as a subject of his jacobi award lecture. he described, with the late dr. kenny, a hitherto unrecognized form of dwarfism that is now known as the caffey-kenny dwarf. the john caffey society, which includes as its members pediatric radiologists who have been intimately associated with dr. caffey, or who have been trained by his students, was established in . this society is now among the most prestigious in the field of radiology. his book and the society named in his honor will live on as important memorials to this great man. his greatness was obvious to all who worked with him. he was warm, kind, stimulating, argumentative, and above all, honest in his approach to medicine and to x-ray diagnoses. his dedication to the truth was expressed in his abiding interest in the limitations of x-ray signs in pediatric diagnosis and in his interest in normal variation in the growing skeleton. he was concerned with the written and spoken word and was a skilled semanticist. his book and his articles are masterpieces of language and construction. he stimulated and was stimulated and loved by all who had the privilege of working with him. radiology and pediatrics have lost a great man, but they shall ever have been enriched by his presence. interstitial lung disease, which is more common in infants than older children, is defined as a rare heterogeneous group of parenchymal lung conditions primarily due to underlying developmental or genetic disorders. affected infants typically present with clinical syndromes characterized by dyspnea, tachypnea, crackles, and hypoxemia. mainly due to a lack of evidence based information regarding underlying pathogenesis, natural history, imaging findings, and histopathologic features of interstitial lung disease, the understanding of interstitial lung disease in infants has been limited in the past. however, in recent years, the understanding of interstitial lung disease in infants has been substantially improved primarily due to: ) advances in imaging technology for better detection; ) improvement of thoracoscopic techniques for lung biopsy; ) established pathologic criteria for consistent diagnosis; and ) development of new classification system based on underlying etiology of the interstitial lung disease. in fact, several forms of interstitial lung disease in infants that exhibit distinct clinical, radiological, and pathological patterns are currently emerging. the overarching goal of this article is to review a new classification system, imaging findings, and pathological correlation of interstitial lung disease in infants. improved understanding of this often challenging disorder can aid in early and accurate diagnosis, which in turn, will result in improved patient care. large airway disease in pediatric patients: impact of advanced post-processing techniques catherine m. owens, bsc mbbs mrcp frcr the introduction of multidetector row computed tomography (mdct) scanners has altered the approach to imaging the pediatric thorax. in an environment where the rapid acquisition of ct data allows general hospitals to image children instead of referring them to specialist pediatric centers, it is vital that general radiologists have access to protocols appropriate for pediatric applications. this lecture will focus on the main principles of volumetric ct imaging that apply generically to all mdct scanners and in particular we describe the reconstruction techniques for imaging the pediatric thorax and the low-dose protocols used in our institution on a -slice dual source ct scanner. examples of important clinical applications with the impact and added value of post processing are also given. neoplasms, by definition, comprise an abnormal uncoordinated proliferation of cells that persists even after the inciting stimulus as ceased. the resulting mass may be benign or malignant and arise from any tissue that is normally found in the location where the mass develops. thus, tumors of the chest may arise from bone, lung, pleura, lymphatics, muscle, etc. whether benign or malignant, chest masses may be incidental findings on imaging obtained for other reasons. this presentation will focus on malignant tumors of the chest, address the imaging characteristics and staging of the most common chest malignancies and discuss characteristics that may aid in distinguishing these lesions from their corresponding benign or infectious counterparts. included in this presentation will be the most common chest wall malignancies (ewing family of tumors and rhabdomyosarcoma), mediastinal malignancies (lymphoma, germ cell tumors, and neurogenic malignancies) and pulmonary primary malignancies (pleuropulmonary blastoma and carcinoid). the changing appearance of selected tumors in patients treated with new targeted therapies will be introduced. lung disease is the most common chronic disease of childhood, but young children cannot perform the breathing maneuvers required for the most commonly used method for assessing lung function, spirometry. ct provides exquisite structure information about the lung but concerns regarding the long-term consequences of the relatively high radiation dose limit its use particularly in the pediatric population. magnetic resonance imaging (mri) has the potential to provide regional information about the lung without the use of ionizing radiation. while conventional proton mri has found widespread clinical application in most organs of the body, mri of the lung lags behind because the lung is intrinsically difficult to image with mri. the strength of the mr signal depends on the physical density of protons in the tissue being imaged and the local environment of the protons. the lung has a low physical density and thus a low proton density so little mr signal is generated by the lung. furthermore, the magnetic susceptibility effects from its many air-tissue interfaces cause what little signal is generated to rapidly decay so that the lung typically appears dark on conventional proton mr images. a variety of strategies have been developed to overcome the inherent difficulties of mri of the lung, resulting in recent substantial improvements in image quality. additionally by administering an inhaled gaseous contrast agent, such as the hyperpolarized noble gases helium- or xenon- , direct visualization of lung airspaces in an mr image is possible. a number of unique strategies for evaluating the structure and function of the human lung using hyperpolarized gas mri have been developed. although the level of structure detail possible with lung mri may never equal that of ct, mri may nonetheless has the potential to provide clinically useful information and be a sensitive, effort independent test of pediatric lung disease. for a matter of time, we will focus in this presentation only on the following: intestinal malrotation a normal visceral situs can be inferred sonographically in relation to the right-sidedness of the superior mesenteric vein, to the retromesenteric location of d and to the right iliac position of the ileocecal valve. conversely, intestinal malrotation is likely when the aforementioned features are reversed. in addition, cdu can display the whirlpool pattern in case of midgut volvulus or internal hernia, alleviating the need for preoperative opacification. the reliability of us in diagnosing intussusception is well documented since the early s. the value of us in predicting the success or failure of pneumatic reduction and/or bowel necrosis is more debatable, based upon a coexisting bowel occlusion, the presence of interloop fluid, bowel wall changes (intramural air, dilated mural vascular channels), absent blood flow at cdu. the continuous down-grading of us in comparison to ct, and the opposite conclusions of various series regarding imaging of pediatric appendicitis are based upon different prerequisites and definitions. historically and in most usa institutions, sonography reports are either negative (entire normal appendix), positive (abnormal inflamed appendix), or equivocal (non-visualization or partial visualization of appendix). the equivocal group is then logically investigated by a subsequent abdominal ct. in europe, some usa centers, and in our practice, us reporting include groups and take into account ancillary findings: . normal appendix (blind-ended, lamellated, compressible, < mm in diameter, without peristalsis; . appendix not depicted, no secondary signs; . appendix not depicted, with one of the following: hyperchoic mesenteric fat, fluid collection, local dilated small bowel loop; . appendix inflamed. group represents most cases of perforated appendicitis, groups and the negative sonogram. ct is then indicated only in obese patients and to assess the feasibility of percutaneous interventions. inflammatory bowel disease in the recent literature, mr enterography is often preferred to ct enterography. small bowel series look prehistoric and us is rarely mentioned. sonography however is very valuable both for screening children presenting with abdominal pain, diarrhea, weight loss, or gi bleeding and for following the course of the disease and searching for complication. hypervascularization has been proved to parallel the disease activity. initially mentioned by dr. rita teele, the interest of us for differentiating high-intermediate/low varieties of imperforated anus has been re-emphasized more recently. a perineal rectal cul de sac distance of mm is quoted as the significant cut-off value. us can also display rectourinary fistulae outlined by air. update on mdct and mri of hepatobiliary disease in children: what's new lisa h. lowe, md a variety of disorders may affect the pediatric liver. recent advances in histopathological knowledge and imaging techniques have led to important changes that radiologists must be aware of in order to allow for an accurate limited differential, and in some cases, specific, diagnosis. this presentation will focus on recent developments that have lead to a better understanding of the embryopathogenesis for fibropolycystic liver diseases (including choledochal cysts and caroli disease), histopathological findings that have led to new classification systems for of pediatric vascular anomalies, technological advances and contrast agents in magnetic resonance imaging that are useful to characterize and limit the differential diagnosis of hepatic masses. diagnostic errors in pediatric abdominal imaging: diagnostic pearls and pitfalls george a. taylor, md this presentation reviews the types of diagnostic errors in abdominal imaging occurring over a -year period in an academic pediatric radiology practice. radiologists engage in two interrelated processes when interpreting imaging studies: perception and analysis. failures in perception (failure to identify an important finding) are a common source of diagnostic error in pediatric imaging, while failures in the analytic portion of the process (over-or faulty interpretation of a finding) are not as common. under-interpretation of findings can be related to a number of perceptual and visual phenomena including visual isolation where attention is selectively focused on a main area of the image while less or no attention is given to secondary areas, and satisfaction of search which occurs when additional lesions remain undetected after detection of an initial lesion. many analytic errors are the result of commonly used heuristics or shortcuts in reasoning. these include the availability heuristic in which likelihoods are based on memory of a similar case, the framing effect in which a different diagnosis is reached based on how the information is presented, and the anchoring heuristic in which the initial impression is difficult to change, despite conflicting new information. another recognized pitfall is blind obedience, in which a diagnostician stops thinking when confronted by authority. this authority can be human or technical (reliance on a laboratory value). finally, diagnostic errors can result from an attitude of overconfidence. examples of these heuristics and strategies to minimize cognitive errors will be discussed. marta hernanz-schulman, md, faap, facr this session will consider abdominal masses that present in the neonatal period, spanning developmental, inflammatory and neoplastic conditions. time constraints do not allow an exhaustive list or description, but the more important or frequent lesions are discussed. the presentation is subdivided by systems. the renal section discusses various conditions presenting with hydronephrosis, such as ureteropelvic junction obstruction and duplication anomalies, followed by autosomal recessive polycystic kidney disease and multicystic dysplastic kidney, cystic entities commonly presenting in the perinatal period. neoplastic renal entities include lesions with benign behavior, such as ossifying renal tumor of infancy, with the discussion extending to entities with very poor prognosis such as clear cell sarcoma and rhabdoid tumor, while discussing the congenital mesoblastic nephroma, its histologic subtypes and the differences in their presentation, imaging findings and clinical behavior. suprarenal lesions include the adrenal hemorrhage, congenital neuroblastoma and subdiaphragmatic sequestration. hepatic lesions include developmental anomalies that present as mass lesions, such as choledochal cysts, vascular lesions such as congenital and infantile hemangiomas, and neoplastic lesions such as the mesenchymal hamartoma and hepatoblastoma. differences in clinical presentation, imaging characteristics and behavior of the lesions are discussed. the section on pancreatic lesions discusses pancreatic cysts and pancreaticoblastoma. gi tract and mesenteric lesions include duplication cysts, lymphangioma, and meconium pseudocyst, and their relationship to bowel obstruction and persistent perforation. ovarian cysts can present as large masses in neonatal girls, and should be high in the differential diagnosis of large masses encountered in female infants; the imaging characteristics of simple and complicated cysts are described, as well as their course and potential complications. pediatric procedures: from imaging to intervention the spectrum of vascular anomalies in pediatric patients: multimodality imaging evaluation and current treatment patricia e. burrows, md vascular anomalies are categorized into two main groups, vascular tumors and vascular malformations. genetic and molecular regulation of vascular genesis of angiogenesis, and mutations responsible for some of the vascular malformations, have been delineated. in order to implement future targeted treatment of vascular lesions, accurate diagnosis is important. imaging modalities that are effective in distinguishing the various types of vascular anomalies and demonstrating the extent include ultrasonography with doppler interrogation, mri and various forms of mr vascular flow imaging, conventional angiography and venography. techniques used to image lymphatic channel anomalies, conventional lymphangiography, lymphoscintigraphy and infrared fluorescent lymphangiography. in this presentation, common forms of vascular anomalies will be described and rare or recently recognized anomalies will be mentioned. current treatment of the different forms of vascular anomalies will also be discussed, including pharmacotherapy using beta blockers, angiogenesis inhibitors and mtor inhibitors. endovascular techniques used in treating vascular malformations, including embolization and sclerotherapy will be presented. pediatric vascular disease is extremely varied, with a wide range of conditions requiring diagnostic or therapeutic intervention. technological improvements in non-invasive imaging modalities such as mri and ct have reduced the need for diagnostic angiography; however, with advances in interventional techniques, arteriography in the pediatric patient is now often performed for therapeutic reasons. pediatric arteriography presents unique issues and challenges. tremendous variability in patient size and physical maturity limits the ability to standardize technical aspects of performing arteriography. in addition, radiation protection, sedation/anesthetic support, monitoring of fluid balance, and maintaining patient warmth must be considered. a regimented protocol for assessment of the pediatric patient must be followed, with review of the indications for the study requested, and review of patient-specific issues such as coagulation profile, concurrent medical disease, patient weight, and anesthetic concerns. appropriate patient monitoring is imperative to ensure patient safety. vascular access can be quite challenging. ultrasound and micropuncture access techniques have tremendously improved successful access while reducing associated complications. the smallest catheter that can accomplish procedure objectives should be used. for most diagnostic cases, french systems can be used for children> kilograms, while french catheters are preferred in those < kg. intraprocedural heparinization ( - iu/kg) is also more often used, especially in children weighing less than - kg. rates and volumes of contrast injected for pediatric arteriography are not standardized, as in adult patients. in general, contrast dose should be limited to - ml/kg, and - ml/kg in premature infants and neonates. all these new technique are less invasive, improve patients' outcomes and reduce morbidity. they are also cost-effective as patients are discharged home earlier and recover faster from the intervention. the future holds promising new technologies such as high-intensity focused ultrasound (non-invasive method of thermal ablation) and nanoparticles for drug delivery. pediatric interventional radiology will continue to be an essential part of these minimally invasive therapies. musculoskeletal imaging: from planning to performance kirsten ecklund, md the purpose of this talk is to review advanced mr imaging techniques currently being used in the evaluation of pediatric musculoskeletal tumors. the goals of these techniques include improved image resolution and quality, lesion tissue characterization, and increased acquisition speed. diffusionweighted (dw) and perfusion imaging will be emphasized; however, whole body, metallic artifact mitigation, and volumetric sequences will also be discussed. dw mri is based upon the brownian motion of water within extra and intra-cellular spaces which depends upon tissue cellularity. dwi can aid in the differentiation of benign from malignant lesions, which generally have restricted diffusion. there is even greater potential for dwi in the assessment of tumor response to therapy. the apparent diffusion coefficient (adc) maps are critical to accurate interpretation of diffusion sequences. adc maps distinguish between restricted diffusion and t effect, both of which appear bright on dwi. both qualitative and quantitative tissue assessments can be made with dwi. challenges for dwi in the pediatric musculoskeleton include susceptibility artifacts from bone, motion vulnerability, and geometric distortion at larger fields of view. our current protocols and parameters for dwi will be presented. contrast-enhanced (dce) mr using one of a variety of vendor specific sequences. qualitative and quantitative assessments of inflow and distribution of contrast have been shown to help differentiate between benign and malignant lesions and to evaluate drug efficacy during therapy. this technique is especially promising in those patients undergoing antivascular and antiangiogenic therapy. tal laor, md congenital abnormalities of the musculoskeletal system can result in alterations of limb size, configuration, and/or segmentation. these disorders often affect both the osteocartilaginous skeleton as well as the surrounding soft tissues and can be localized or diffuse. in this session, we will focus on the imaging features of several congenital abnormalities that result in a small or short limb, in altered configuration of a limb, or in abnormal segmentation. deformities of both upper and lower limbs will be examined. like congenital abnormalities, developmental disorders of the pediatric musculoskeletal system can be limited to a single area or can affect numerous sites within the body. for example, neonatal brachial plexopathy is a localized disorder that produces characteristic musculoskeletal alterations about the shoulder girdle and elbow of affected children. the alterations of morphology and function of the shoulder develop over time with growth of the child and change in response to a variety of therapies. we will review the features of developmental anomalies of the pediatric musculoskeletal system and evaluate the role that imaging plays in the initial evaluation and in the subsequent assessment of these children during treatment. multimodality imaging of skeletal trauma in children: using all of the tools peter j. strouse, md skeletal trauma is a common indication for imaging throughout the pediatric age range. newborns may suffer birth trauma. infants and toddlers may be subject to abusive injury. children of all ages may suffer accidental injury. older children and adolescents are increasing hurt in sporting activity and vehicular accidents. fracture patterns vary with maturation of the child. interference with normal growth is a potential complication. imaging of skeletal trauma begins with radiography. proper anatomic and age specific radiographic technique assures optimal diagnostic yield. radiography suffices in most cases to diagnose fracture or confirm normalcy. "clinical correlation" aids in diagnosis. ultrasound, ct, mri and nuclear medicine may play a role in specific instances where plain radiographs are non-diagnostic or to better delineate certain fractures. arthrography and conventional tomography have occasionally been used in the past and tomosynthesis may prove useful. follow-up radiographs may be useful for diagnosis or confirmation of some fractures. this presentation will focus on the imaging of acute skeletal injury. technique and approach for plain radiography will be emphasized. specific indications and roles for ancillary imaging techniques will be defined and illustrated with representative cases. although classically thought of as a disease of adulthood, stroke is much more common in the pediatric population than was once appreciated. this may be due to many factors, not the least of which is increased awareness due to the presence of subspecialty stroke teams now fairly commonplace in many children's hospitals, and the fairly recent advent of more advanced imaging technique such as diffusion-weighted imaging (dwi) and its routine use in imaging the central nervous system (cns) in the child and adolescent. causes of stroke in children can be protean, and range from idiopathic on one end of the spectrum, to traumatic on the other, with many causes in between, many of which may not be intuitive to the clinician without further research. moyamoya disease and its many causes, such as sickle cell disease (scd), trisomy and neurofibromatosis type i (nf i) can all lead to stroke in children, as can congenital clotting deficiencies such as factor v leiden deficiency and congenital cardiac lesions with their resultant shunting of blood between the left and right cardiac circulations. although usually arterial in nature, strokes may arise from the venous system in clinical scenario of venous thrombosis with resultant venous infarctions. factors contributing to venous thrombosis in children and adolescents can be due to dehydration (especially in the very young), severe iron deficiency anemia, inflammatory bowel disease and exogenous hormone ingestion such as is seen with oral contraceptives (ocp) in young women. advanced imaging techniques for neuroimaging in pediatric patients: where are we now? blaise v. jones, md the past decade has seen a large number of advanced imaging techniques introduced to the clinical armamentarium of the pediatric radiologist. from the development of multidetector ct scanners that can obtain whole head diagnostic studies in less than s to the routine use of t mr imaging, technical advances have dramatically changed our ability to diagnose and manage neurological disorders in children. however, all of these advances are not of equal clinical utility, and it is imperative that the pediatric radiologist be well versed in their judicious and appropriate application. this presentation will discuss the effective use of volume ct scanning, cta, swi, asl, fmr, pmr, and other advanced imaging techniques in the diagnosis of neurological disorders presenting in childhood. at the conclusion of the presentation the attendee will have a better understanding of how to ideally apply these technologies in practice. a spectrum of abnormality in pediatric neck: practical imaging choices and interpretation caroline d. robson, mbchb learning objectives: . become familiar with an optimized imaging approach for head and neck infections . recognize the complications of head and neck infections . recognize the utility and interpretation of imaging for neck masses this talk will cover the imaging approach and interpretation of findings in head and neck infection and neck masses. infection includes acute complicated sinusitis, coalescent mastoiditis, neck infection and local and intracranial complications. optimized imaging protocols and image interpretation for neck masses will also be discussed and illustrated. acute complicated sinusitis is diagnosed when acute sinusitis is accompanied by orbital symptoms (e.g. proptosis) and/or mental status changes, seizures or other neurological findings. coalescent mastoiditis is diagnosed when otomastoiditis is accompanied by tenderness and/or swelling over the mastoid process. ct and mr provide complementary information. ct is obtained with contrast. mr sequences include fatsuppressed t , t , diffusion, and fat-suppressed contrastenhanced t weighted images with mr venography. intracranial complications include epidural abscess, subdural empyema, meningitis, cerebritis, brain abscess, venous thrombosis and venous infarction. the limitations and usefulness of ct in the diagnosis of neck abscess will be illustrated. the imaging approach to masses depends on patient age, and the size and location of the mass. us, ct, mr, and nuclear medicine studies provide complementary information. as for infection, optimized imaging approaches and key imaging features for various masses will be discussed. embryology and diagnostic approach in spinal dysraphism l. santiago medina, md, mph and esperanza pacheco-jacome, md congenital anomalies of the spine are malformations that can be confusing due to the complexity of their embryology, and to the sometimes unclear classifications and terminology. the purpose of this review is to give a clear and basic understanding of the different stages of the embryological development of the spinal cord, starting with the bilaminar disc in the first week of gestation. during the second week, the formation of a trilaminar disc (gastrulation), the notochord, and the formation of the neural tube or neurulation. also, a review of the development of the distal cord: conus medullaris, filum terminale, ventriculus terminalis, by a different mechanism, canalization and retrogressive differentiation. beside the embryological review, a case correlation will be presented using mr imaging to demonstrate these malformations. open spina bifida entities include meningocele and myelomeningicele. closed or occult spinal dysraphism (osd) is characterized by a spinal anomaly covered with skin and hence with no exposed neural tissue. osd spectrum includes dorsal dermal sinus, thickened filum terminale, diastematomyelia, caudal regression syndrome, intradural lipoma, lipomyelocele, lipomyelomeningocele, anterior spinal meningocele and other forms of myelodysplasia. several studies have shown that mri and ultrasound have better overall diagnostic performances (i.e., sensitivity and specificity) than plain radiographs for detection of occult spinal dysraphism. for h n , most patients had mild illness but a small percentage required mechanical ventilation and icu admission. the high risk groups include children < years old and those with chronic medical conditions in particular neurodevelopmental impairment. pediatric mortality was . % of all deaths associated with the pandemic reported in the u.s. in both conditions, the most prominent radiographic and ct features were airspace disease including ground glass opacities (ggo) and consolidation, commonly with multi-focal and bilateral involvement. pleural effusion, adenopathy and cavities were absent. in some patients with viral infection, respiratory symptoms may be mild but are complicated by neurological manifestations. a brief review of mri features in h n related encephalopathy including acute necrotizing encephalopathy (ane) will be given. bernard f. laya, do tuberculosis (tb) is a worldwide major public health problem with one-third of the world's population being infected. it is a leading cause of death and disability from infection worldwide. children are amongst the most vulnerable group because of their immature immune status. a child usually gets tb infection after being exposed to a sputum-positive adult. depending on many factors, the infection can lead to latency or tb disease. it can affect virtually any organ in the body and can be devastating if left untreated. tb in children remains a diagnostic challenge. in addition to history of tb exposure, signs and symptoms, laboratory and microbiologic tests, medical imaging remains a valuable tool in its diagnosis. although findings are nonspecific, the radiograph is the most commonly ordered initial imaging tool for screening and diagnosis of pulmonary and musculoskeletal involvement. computed tomography and magnetic resonance imaging offer more detailed assessment especially in cranial and abdominal involvement. medical imaging is also utilized to follow up patients during or after anti-tb treatment. knowledge of the common imaging patterns, pitfalls and dilemma are very important in establishing the diagnosis of tb in children. the pathophysiology of pediatric tb will be discussed as it correlates with imaging findings. the wide spectrum of imaging manifestations in various modalities will be presented. imaging updates along with pitfalls and dilemma in the interpretation will also be discussed. tb can affect almost every organ system but the author will present cases that are more commonly encountered. and concurrent ct/ pet-ct (k . ) panels. there were more indeterminate nodule predictions by pet-ct (n of ; %) and concurrent ct/pet-ct (n ; %) than by ct alone (n ; %). the overall accuracy of ct alone was %, pet-ct alone % and concurrent review %. worst case sensitivity and specificity were % and % for ct alone, % and % for pet-ct alone, and % and % for concurrent ct/pet-ct. conclusions: pet-ct assessment of pulmonary nodules in children is feasible but limited by non-diagnostic quality ct images and atelectasis caused by sedation. subjective assessment of nodules by pet-ct does not appear to improve the ability to distinguish benign from malignant histology in children with solid malignancies. semi-quantitative nodule assessment using the standardized uptake value may improve the performance of pet-ct in this setting and will be investigated in the future. purpose or case report: nec is the most common lifethreatening medical/surgical emergency of the gastrointestinal (gi) system in neonates, with an incidence up to % in infants weighing < g. with advances in treatment of nec, increased survival rates result in rise in post-nec gi complications such as feeding intolerance. development of post-nec bowel strictures results from healing of involved bowel and can result in bowel obstruction. it has been routine to study the bowel of infants after medical treatment for nec by contrast enema and small bowel follow-through prior to initiating feeding. however, in order to "image gently" we are attempting to decrease the radiation exposure to these patients. we postulate that in patients with no abnormal bowel dilation prior to initiation of feeds, the incidence of colonic stricture would be so low that routine enemas would be unnecessary and could be eliminated from the workup. recorded as present or absent in anatomic abdominal regions defined as: and -from the dome of the diaphragm to top of l to the right and left of midline, respectively; and -from top of l to the iliac crest to the right and left of midline, respectively; -from the iliac crest to the top of the sacrosciatic notch; -below the top of the sacrosciatic notch. we assessed the frequency of findings in each region and how often findings in regions and were associated with findings in regions - . % confidence intervals were calculated. results: the fewest pertinent findings were present in region in . % ( / ) ( % ci: . - . %) of radiographs. findings included: abnormal bowel % (n ), bowel gas paucity . % (n ), pneumatosis . % (n ), inguinal hernia . % (n ) and osseous abnormalities . % (n ). pertinent findings were present in region in . % ( / ) ( % ci: . - . %). findings included: abnormal bowel . % (n ), bowel gas paucity gas . % (n ), pneumatosis . % (n ), free air . % (n ), and abnormal bowel with pneumatosis . % (n ). among patients with an abnormality in region , ( . %) also had an abnormality within at least one of regions through . among the patients with an abnormality in region or , ( . %) also had an abnormality within at least one of regions through . catheter/tube tips were located in region in . % (n ) and region in . % (n ) of radiographs, respectively. pneumatosis was present most frequently in regions ( . %), ( . %), and ( . %). free air was present most frequently in regions ( . %), regions and ( . % each). conclusions: our preliminary data suggest that pertinent findings on neonatal portable abdominal radiographs are rarely isolated to the pelvis, implying that gonadal shielding of regions and should not compromise diagnostic accuracy. purpose or case report: the purpose of this study was to determine the sensitivity, specificity, and positive and negative predictive value of ultrasound in diagnosing appendicitis when the appendix is visualized, using three diagnostic categories: positive, negative, and equivocal. the -category diagnostic accuracies for appendiceal diameter and radiologist impression were compared. methods & materials: a retrospective study was performed evaluating all right lower quadrant ultrasound reports dictated over a -month period. included studies were interpreted as positive, negative, or equivocal for appendicitis. report impressions that did not specify one of these categories and studies where the appendix was not seen were excluded. the pathologic diagnosis of appendicitis was considered the gold standard for a positive diagnosis. because virtually all pediatric surgical cases in the region are referred to our hospital, it was assumed that the patient did not have appendicitis if surgery was not performed. logistic modeling using appendiceal diameter as the independent variable established cutoff diameters of ≤ mm negative, > mm positive, and <x≤ mm equivocal. sensitivity, specificity, and positive and negative predictive values were calculated for both methods. results: of the patients imaged during the study period, ( %) met the inclusion criteria; ( %) reports did not categorize the impression and in ( %) studies the appendix was not seen. surgery was performed on ( %) subjects, of which ( %) had a pathologic diagnosis of acute appendicitis. using the radiologist impression, there were true positives (tp), false positives (fp), false negatives (fn), and true negatives (tn), for a sensitivity of % ( % ci - %), specificity of % (ci - %), ppv of % (ci - %), and npv of % (ci - %). there were ( %) cases reported as equivocal; of these, ( %) had a pathologic diagnosis of appendicitis. using appendiceal diameter, there were tp, fp, fn, and tn, for a sensitivity of % (ci - %), specificity of % (ci - %), ppv of % (ci - %), and npv of % (ci - %). there were ( %) cases categorized as equivocal; of these, ( %) had a pathologic diagnosis of appendicitis. conclusions: the use of diagnostic categories enables high ppv and npv with a relatively low number of equivocal cases. using appendiceal diameter alone results in higher ppv than that of the radiologist impression, but also yields a higher number of equivocal cases. purpose or case report: intussusception is an obstructive condition that a segment of the bowel invaginates into adjacent bowel and is an urgency that needs to be reduced as soon as possible to prevent bowel ischemia or necrosis. when it occurs in the infants and young child, it usually develops idiopathically and reduced by the technique such as air enema under the fluoroscopic guidance or saline enema under the ultrasound guidance. the success rate of the non-surgical reduction has reported over %. the pneumatic reduction is to introduce air into the patients' rectum via a ballooned foley catheter by hand pump. a mercury manometer connecting with the foley catheter calibrates the pressure of insufflation, and the recommended maximum pressure is mmhg. the hydrostatic reduction is to drip saline from a bag at the height of ft that may be different according to the institute's guidelines. until recently, in both techniques, the precise measurement of the intraluminal pressure was not possible. with the purpose of intraluminal pressure monitoring, we introduced a specific digital pressure gauge that can measure both pneumatic and hydrostatic pressure in various units and can give the collected data to our workstation. with the data, we purpose to make the curve of intraluminal pressure during the procedure and to compare the pattern and difference in both techniques of reduction, which permits suggesting the better technique for reducing the intussusception. from february , we performed the intraluminal pressure monitoring in children; cases of pneumatic reduction and cases hydrostatic reduction. the results indicated that the curves were similar in both techniques. however, the hydrostatic reduction tended to need longer time and higher pressure difference than the pneumatic reduction. we assumed that a higher flow rate and more fluctuating introduction of pressure in the pneumatic reduction made the difference between both methods. purpose or case report: anecdotal evidence suggests that some pediatric brain tumors, particularly pilocytic astrocytomas, demonstrate greater enhancement on delayed post-contrast t weighted mr imaging compared to early post-contrast imaging. our goal was to compare immediate post-contrast imaging with delayed post-contrast imaging in pediatric brain tumors. methods & materials: the research protocol was granted exempt status by the institutional review board (comirb# - ). between november and march , all patients for whom an mri of the brain was ordered to follow-up known brain tumor or to evaluate new brain tumor underwent a modified protocol that included both immediate and min delay post-gadolinium images. the modified protocol sequence order was dwi, t mprage, contrast injection, immediate post-contrast t mprage, t , flair, min delayed post-contrast t mprage. this sequence allowed both immediate and delayed post-contrast imaging to be performed without scanner downtime. a total of unique patients met inclusion criteria of an enhancing primary brain tumor with the delayed imaging performed within a window of - min. these studies were evaluated independently by pediatric neuroradiologists (ns and lf) and a radiology resident (jm) to compare the degree of lesion conspicuity and edge discrimination, and to determine an overall preference. in cases of discrepancy between reviewers, final grading was determined by group consensus. results: delayed post-contrast images were preferred in / tumors ( %), immediate post-contrast images were not preferred in any case, and / cases were graded as equivalent ( %). notably, delayed imaging was preferred for / pilocytic astrocytomas ( %). in / cases ( %), the improvement in conspicuity and edge discrimination on delayed imaging was felt to have a potential impact on clinical management, e.g. by extending resectable or treatable tumor margin. conclusions: delayed post-contrast imaging can be performed easily by reordering existing mri protocols, and no scanner downtime is required for this protocol change. our results indicate that pediatric brain tumors (particularly pilocytic astrocytomas) often enhance more conspicuously on delayed imaging, that no diagnostic confidence is lost on delayed imaging, and that clinical management may be more accurately guided. -directions) were retrospectively reviewed, after irb approval. both sporadic (n ) and nf- associated optic gliomas (n ) were included. tumors confined to intraconal optic nerve were excluded due to technical limitation of epi dwi within the bony orbit. apparent diffusion coefficient (adc) of the solid portions of the tumor was measured using the region of interest method both at baseline and on follow-up mri. results: for the patients, required treatment (surgery and/or chemotherapy) for symptomatic tumor progression ( sporadic opg and cases of nf- ). median follow-up interval was . years (range: months to . years, sd . years). there was no difference in adc values between sporadic and nf- associated opg. opg that required therapy had significantly higher baseline mean adc ( x - mm /s, sd . ), whereas opg with low mean adc ( x - mm /s, sd . ) remained clinically asymptomatic or stable (stratified mann-whitney test, p . ). an increase in mean adc of x - mm /s doubled the odds the patient will require treatment (or . , % ci: . - . , p< . ). enhancement patterns did not predict tumor behavior or nf- status (fisher's exact test, p . ). conclusions: significantly higher mean adc was seen in opg that required therapy for tumor progression. mri may be a useful prognostic tool in further defining a subset clinically significant sporadic or nf- associated opg. purpose or case report: fast brain mri imaging has recently been introduced for assessing brain ventricles in patients with ventriculo-peritoneal shunts (vps). however, there is minimal literature on how this method could be beneficial for other indications. compared to ct, this method reduces lifetime cumulative radiation dose and has multiplanar capabilities. it is much faster than full brain mri, reducing exam costs and potentially reducing the need for sedation. however, fast brain mri has lower resolution than a standard brain mri and cannot detect hemorrhage. the goal of our study is to retrospectively assess additional clinical applications for fast brain mri. methods & materials: patients who had undergone fast brain mri were found in xenobase, a database which references deidentified electronic medical records at our institution. xenobase was then used to identify subgroups by diagnosis. one of the most common diagnoses was arachnoid cyst. after irb approval, patients with fast brain mri and arachnoid cyst were identified so that their fast brain mri could be compared to any full brain mri and to clinical outcomes. fast brain mri findings were excluded if there was not a full brain mri within year. agreement with full brain mri, and documented effect on clinical management were coded for statistical review. results: between and , there were arachnoid cyst patients with fast brain mri exams. in members of this group, fast brain mri was used alone or in comparison with a full brain mri in the decision to follow or discharge the patient. exams failed to report a pituitary cyst and mass effect which were reported on prior exams. patients had no complications in the year following their fast brain mri. the most common complication was headache ( cases). conclusions: fast brain mri reduces radiation exposure, time, cost, and the need for sedation in vps patients. these benefits may cautiously be extended to other patient groups also requiring routine imaging, such as those with arachnoid cysts. disclosure: the authors disclose that they will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: susceptibility-weighted imaging (swi) is a new technique that exploits susceptibility differences in different tissues, to provide a different type of tissue contrast. swi is a gradient echo sequence utilizing both phase and magnitude data to achieve exquisite sensitivity to tissue magnetic susceptibility effects. it is extremely sensitive to even minute amounts of paramagnetic substances and therefore for detection of blood products (hemosiderin, ferritin), deoxygenated blood, calcium, iron, and small vein depiction. it is particularly suited for vascular imaging, especially in cerebral ischemia. swi has been shown to be more sensitive in detecting cerebral microbleeds than is conventional t *-weighted gradient-recalled echo imaging (gre) partly because of its inherent sensitivity, increased spatial resolution and the thinner slices acquired. recent studies have shown that swi detected more microbleeds in more patients, irrespective of their location and that swi may be more sensitive in detecting traumatic lesions than ct or mri. methods & materials: between january and october we have performed swi on pediatric patients (from day to yrs old) affected with different pathologies. mri was performed using either a -or . -t magnet (achieva ; philips healthcare, best, the netherlands). among these patients also received a classic gre sequence. results: our experience confirms a greater sensibility of swi than gre in detecting microbleedings and larger bleedings. swi is much more helpful in patients younger than year since it can distinguish hemorrhagic and non hemorrhagic punctuate white matter lesions detected as hyperintensities on t -w images. it is more accurate in distinguishing petechial hemorrhages and ischemic foci in patients with germinal matrix hemorrhages. swi can provide important diagnostic information not only in vascular pathologies but also in tumors showing the altered tumor microvascularity, the degree of intratumoral necrosis and the presence of subtle defects of the bbb within the surrounding parenchymal tissue. the technique also allows the detection of possible bleedings in infectious lesions. conclusions: swi is a valuable technique in the evaluation of a wide variety of intracranial pathologies in the pediatric population and its use is advisable on a routine basis. purpose or case report: a common problem with pediatric mr imaging is motion. diffusion-weighted imaging (dwi) is highly sensitive to the effects of both rigid-body motion and brain pulsation. the resulting motion artifacts can manifest in several ways, and include blurring (motion between volumes), slice 'drop-outs' (motion during the diffusion encoding), and aliasing (motion during the parallel imaging/ghost calibration scan). in order to improve image quality in pediatric dwi and avoid the potential for repeat scans, we have implemented our grappa-accelerated diffusion tensor (dt)-epi sequence on over , pediatric patients at our institution, and have developed tailored reconstruction software to correct for large motion and reduce the need for general anesthesia (ga). methods & materials: grappa-accelerated epi dti data was collected on patients at . t and , patients at t between the ages of month and years old. with these data, we investigate the use of a method to select the best grappa and ghost calibration weights; d rigid-body realignment with importance weighting; a new volume rejection criterion for large motion; together with other procedures to lower image noise and reduce phase artifacts. all image reconstruction was performed using compiled threaded matlab code on the vendor's (ge) multiprocessor reconstruction hardware. results: using the maximum motion detected from the d realignment procedure on the , dti exams, % of patients did not move (either through co-operation or ga, denoted here by < mm of motion); % of patients moved between - mm; % between - mm; % between - mm; and % between - mm. except for patients in total, the motion correction steps employed here were extremely robust in correcting for motion. in addition, the motion correction steps employed here do not blur or negatively affect the data on non-moving patients. conclusions: our proposed integrated reconstruction scheme suggests that correcting for different types of motion at different stages of the dti acquisition is critical to provide scans of diagnostic quality. apart from the very rare instance ( . %) where the patient moved continuously an extensively throughout the scan, our motion correction procedures employed here are extremely robust. using this scheme, we show that highly diagnostic motion-corrected dti data can routinely be read on our patient database within min. . participating sites followed mutually agreed upon vendor-specific dti protocols used for neonates in clinical practice: ss-epi; b ;~ mm voxel; - directions; te~ msec; acquisition; parallel imaging; multi-channel ( - ) head coil. tensor data was analyzed at one site using internally developed software in idl. b and eddy current distortion were assessed by analyzing grid line distortion on acr slice ; deviations from linearity were measured in the phase encoding direction. acr slice was used to assess snr of the center and periphery of the b image, and fractional anisotropy (fa) of the phantom, which should approach . deviations from specified sequence parameters were recorded. results: both siemens scanners had some image degradation due to vibration susequently corrected by the vendor. nonlinear gridline distortion was highest on the ge followed by philips but was corrected with the use of nd order shimming. eddy current distortion was consistently smaller than acquisition voxel size after image registration. te varied from - msec; acquisition bandwidth range was - hz/pixel. the mm slice thickness was problematic on clinical ge scanners resulting in non-isotropic voxels. across vendors, snr was consistently higher in the image periphery than center by a factor of . - . ). the greatest difference was showed in patients with fhc. no association was found between the value of ldl-c and imt. the . % received drug treatment at the time of evaluation. in dbt- a no association was found between the imt and the levels of hba c and lipids. and in the ob group found no association between imt and bmi z score. fhc %, % of dbt- and % of ob showed atherosclerotic plaques and intima irregularities. conclusions: the imt ultrasonography was able to demonstrate that patients with chronic diseases, with increased cardiovascular risk in adulthood, showed early alterations of carotid intima media in children. this allows clinical applications of preventive and therapeutic guidance. a benchmark for precision of flow assessment was determined from differences between aortic and pulmonary arterial flows, which should agree. the precision of flow when utilizing a slow flow structure (vein) and a fast flow structure (artery) was then determined, serving as an indicator of the precision of slow flow measurements. this was done in pairs for the upper body (svc flow vs difference between ascending and descending aortic flow), lower body (ivc flow vs descending aortic flow) & total body (svc flow+ivc flow vs ascending aortic flow). paired t-tests were used to identify significant differences in the respective arterial and venous flow rates. with an f-test, the relative precision of the arterial flow (determined from aortic and pulmonary arterial flow) was compared against the precision of agreement between upper, lower, and total body flows, each of which has a slow flow venous component, thus assessing precision of venous flow measurement. results: arterial & venous flow measurements were strongly correlated for the upper body (r . ), lower body (r . ) and total body flow (r . ); net aortic and net pulmonary trunk flow rates were also tightly correlated (r . ). moreover, there was no significant difference in the precision of arterial & venous flow measurements (t-test, p . ). lower venc ( cm/s or lower) improves the accuracy and precision associated with low velocity venous flow, a finding that tended to but it did not reach statistical significance. conclusions: with -d pc mri, the accuracy and precision of venous flow quantification are comparable to arterial flow quantification, though venc settings may improve venous flow assessment. disclosure: dr. tariq has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. the spatial resolution requirements for imaging tiny target vessels in neonates have so far precluded its use of cemra in this patient population. the purpose of our study, therefore, was to explore the potential of high resolution cemra at . t in neonates with suspected congenital vascular anomalies methods & materials: neonates (mean age . days, range - days; mean weight . kg, range . kg- . kg) underwent cemra at . t. cemra was performed under controlled apnea on a -channel . t system using either a -channel head-neck coil ( pts) or a -channel adult knee coil ( pts). gd contrast delivery was calculated based on a total dose of . mmol /kg. parallel imaging factors of - were used, generating . x . x . mm voxels in an acquisition time of - s. images were scored for quality and the presence of artifacts independently by two observers. the cemra series were evaluated to identify pulmonary arterial and venous anatomy. extra-pulmonary vascular anatomy was divided into sections for image quality assessment on mpr. results: the mra data sets had high mean image quality and low mean artifact grading scores. no significant difference was evident between the two observers for scoring image quality and artifact (p . ), and there was good interobserver agreement (k . , % ci: . - . ). the pulmonary arteries were confidently visualized to at least the nd order branches in all patients, at least the rd order branches in ( %). out of evaluated segments, ( %) were graded as the vessel "very well visualized with excellent definition of vessel" pa abnormalities were diagnosed in patients and pv anomalies in patients. correlation was made with open surgical findings in cases, confirming the cemra diagnoses with no discrepancy between the operative and imaging conclusions. the results of our study suggest that high resolution cemra at . t is extremely promising in neonatal cchd and, in combination with echocardiography, may often provide comprehensive information for treatment planning. with appropriate use of physiological motion compensation, parallel imaging and coil configuration, voxel volumes less than . mm are routinely achievable. purpose or case report: it is not well understood whether transfusion-dependent children (mainly those who have hereditary anemias subject to excessive iron levels from regular blood transfusions) with no or mild cardiac iron overload and without signs of heart failure exhibit early changes in cardiac dysfunction. this study aims to evaluate intramyocardial deformation and subsequent cardiac dysfunction in such patients using the cardiovascular magnetic resonance technique of myocardial tagging as well as assess iron overload by another cardiovascular magnetic resonance technique of t star (t *). we hypothesize that transfusion dependent children undergoing chelation therapy will be at high-risk for cardiomyopathy despite normal or slightly elevated iron levels and no signs of heart failure due to progressive myocardial deformation as assessed by myocardial tagging; that is, we expect abnormal mechanical parameters to exist despite t * values which correspond to normal or slightly elevated myocardial iron levels. methods & materials: patients (mean age yrs) requiring regular blood transfusions for various reasons (e.g. thalassemia, sickle cell disease, other anemias) were investigated using harmonic phase magnetic resonance tagging and t * imaging. various myocardial mechanical parameters were quantified and correlated to cardiac iron levels as measured by t *. results: children with no overt signs of heart failure (as measured by ejection fraction and volume indices) exhibited normal t * iron levels in combination with global circumferential abnormal myocardial strain and children exhibited regional strain. conclusions: this preliminary data suggests that transfusiondependent children without overt signs of heart failure exhibit early signs of cardiac dysfunction (an effect of strain as assessed by myocardial tagging) in direct relation to normal or slightly elevated myocardial iron levels (as measured by t *). a larger sample size of children could support the idea of abnormal myocardial mechanics being a diagnostic imaging marker for the development of myocardial iron overload and eventual iron-mediated cardiomyopathy. the choice of coil and field of view varied according to the age and size of the patient. imaging was performed on a . t magnet (philips mri system). after the cine gradient echo time of flight sequences were acquired, the dynamic keyhole mr angiographic sequence was applied in the coronal orientation. this sequence commenced - s after initiation of the first bolus administration of - ml of undiluted gadopentetate dimeglumine ( . mmol/ml, magnevist) at a rate of - ml/sec followed by - ml of saline at a rate of ml/sec. image acquisition was repeated multiple times without delay for s during shallow breathing. contrast material was administered through a peripheral or central venous line, and sometimes via simultaneous bilateral extremity injections. rarely manual hand injection was performed. the parameters were as follows: centra k-space filling, acquired voxel size - . x - . x - , reconstructed voxel size . - x . - x - . , number of dynamics - , keyhole percentage - %, sense (parallel imaging) factor x - x , dynamic times - . s, and injection rate - cc/second. image analysis was based on original contrast-enhanced data sets, mip and volume rendering. the quality of the mrv was graded by a subjective scale from - ( excellent, no limitations; nondiagnostic). comparison with unenhanced mr venographic sequences and doppler ultrasound were available in all cases. results: d mrv provided diagnostic information on patency of venous segments in all cases, including the presence of venous occlusion or stenosis, the segments involved, and collateral venous pathways. conclusions: d mr venography using the method outlined is a reliable means of estimating central venous thrombosis in children, using short dynamic scan times with high spatial and temporal resolution, creating an effect similar to conventional venography. paper #: pa- analysis of optimal phase interval for prospective ekg-triggered cardiac ct in children prakash masand, md, texas children's hospital, savivek bogale, lorna browne, rajesh krishnamurthy purpose or case report: evaluate high temporal resolution cine mri of the heart in children to determine relationship of heart rate to the optimal motion free interval during the cardiac cycle, and to derive a chart depicting optimal reconstruction intervals across a range of heart rates which could be applied for prospective ekg triggered ct imaging of the heart. methods & materials: pediatric patients who had high temporal resolution cine steady state free precession sequence ( frames acquired across a single cardiac cycle) performed as part of a mri/mra of the heart from - were included. a single reader analyzed the high temporal resolution cine data to determine the location and duration of the motion-free phase interval in systole and diastole for heart rates ranging from - bpm. the following information was collected: age, heart rate, duration of systole and diastole, ekg phase and phase percentage of motion-free interval, and duration of motion-free interval. heart rates were grouped in ranges of ( - , - , - etc.), with at least patients in each group. wilcoxon sum rank test was performed to compare the static durations of systolic and diastolic phases for each heart rate group. two-tailed p-values< . were considered to indicate statistical significance. a chart matching heart rate range to the optimal phase of image acquisition and reconstruction was created. results: optimal reconstruction intervals for motion-free imaging of the heart in systole and diastole, and single best phase for a given heart rate are as follows: (please see table ) mean duration of the motionless phase in systole and diastole for a given heart rate is as follows: (please see table ) conclusions: this is the first time that variability in location of the motion-free interval within the cardiac cycle with respect to heart rate in children has been studied in rigorous fashion. this chart will have immediate application in the so-called 'target scan', which uses prospective ekg triggered acquisition of data in combination with half-scan reconstruction to minimize radiation dose with respect to retrospective ekg gated imaging. results: a total of patients were found to have aberrant pda by ccta. nine patients had a pda emanating from the brachiocephalic artery to the pulmonary arteries and one patient had pda emanating from the carotid or the subclavian artery according to the operative report. all ten patients had echocardiography in addition to ccta. nine of the ten patients underwent surgery. one patient was lost to follow up. comparison between ccta and echocardiography to operative report showed the following: out of the echocardiograms agreed completely with ccta and operative report. out of the echocardiograms did not report aberrant pdas which were seen on ccta and confirmed during surgery. in one patient, the echocardiogram reported a double aortic arch which was found to be an aberrant pda from the left brachiocephalic artery; a finding that changed the surgical management and pre-surgical planning. the results were statistically significant with a p value of < . . conclusions: diagnostic accuracy of ductus arteriosus anatomy is important in pre-surgical planning and management of patients with ductal dependent pulmonary circulation. cardiac ct angiography is more accurate as compared to echocardiography in diagnosis of aberrant patent ductus arteriosus. ) in patients younger than year of age. the prospective ecg-gated protocol scanned % of the cardiac cycle with msec padding in patients with heart rates greater than bpm. two experienced radiologists evaluated the image quality and course of the coronary arteries. image quality was assessed based on the degree of homogeneity of vascular enhancement, presence of stair-step artifacts, image signal and noise as well as the overall subjective image quality based on a point scale. the course of the left and right coronary arteries was also evaluated based on a point scale, focusing on how well the evaluator was able to visually follow the coronary artery from its origin to its termination. radiation dose was recorded and compared. results: the mean score of the homogeneity of vascular enhancement, stair step artifacts, image noise and signal, overall image quality, and score given to visualization of the course of the coronary arteries showed no significant difference (p> . ) in the retrospective and prospective ecg gated groups. the mean estimated effective dose was significantly lower for the prospective ecg gated group compared to the retrospective group, ( . msv vs . msv) respectively (p< . ). conclusions: prospective ecg-gated cardiac mdct provides comparable assessment of coronary anatomy, image quality with significantly less radiation dose when compared to the retrospective ecg-gated mdct. prospective ecg gated cardiac mdct is a powerful adjunct to the treatment and surgical planning of pediatric patients with congenital heart disease less than yr of age with lower radiation dose. methods & materials: pediatric neuroradiology lectures were recorded and made available to radiology residents at a university program through on-line streaming video viewed through an internet link. topics included brain tumors, phakomatosis, and congenital brain malformations. one lecture per week was recommended prior to case conferences that reviewed the same topic. pre-and post-tests and a feedback survey were administered. nonparametric paired sign test and analysis of covariance were used to evaluate changes in test scores overall and according to feedback responses. spearman's partial rank correlation coefficient was used to evaluate the relationship between the number of viewed videos and test scores. results: twenty-nine residents completed the pre-test and the post-test. the means (sd) scores were . % ( . %) and . % ( . %) respectively. there was a significant improvement in test scores (p . ). residents that agreed/strongly agreed that the streaming technology lectures were convenient had greater improvement than those who did not ( . vs. - . %, p . ). similarly, those who agreed/strongly agreed that being able to replay a lecture was helpful had greater improvement than those who did not ( . vs. - . %, p . ). finally, those who agreed/ strongly agreed that the streaming technology lecture format was a better teaching tool had greater improvement than those who did not ( . vs. - . %, p . ). significant positive correlation between number of videos watched postconference and improvement was present (spearman's rho . , p . ). conclusions: on-line streaming video with live case conferences enhances radiology resident learning of pediatric neuroradiology. step (mpps) software provided an accurate measurement of scan time. visual charting made gaps in utilization apparent. technologists and nursing notes correlated to gaps identified barriers and opportunities for improvement. nursing and anesthesiology reduced redundancies. standardized protocols lead to more consistent scan times. appointment access for sedated mri was measured by the third available appointment. results: manually entered data points were time consuming, inconsistent and unreliable. the process improvement was most effective when fewer more reliable data points were used to evaluate the effect of change. the program resulted in a reduction of appointment access for sedated mri from days to days with no change in the hours of operation. magnet utilization was increased from % to %. induction outside the scan room provided the most efficient process tested. we ranked first in utilization in a children's healthcare cooperation of america (chca) survey as measured by exams per scanner. patient preference for a.m. scheduling was shown by survey and corroborated by scheduling data. consistent scan times were achieved by protocol standardization augmented by indication driven decision support. conclusions: a consise definition of mr utilization established a metric that was used in the process cycle of analyze-optimize-measure. anesthesia induction outside the magnet was the most efficient practice but required collaboration between nursing, mr technologists and anesthesiology. protocol standardization were valuable aspects of process improvement essential to optimizing parallel sedation. these adjustments reduced appointment access from days to days, increased utilization from % to % and produced a number one rank in utilization by chca survey. we exploited the synonym option in epic order entry to translate indications into procedures mapped to specific protocols for mri neuroimaging. the order screen allows the provider to enter an indication. that indication is linked through a synonym option to a specific exam and protocol. the recommendation is based upon institutionally created clinical care guidelines, and can be accepted with a single click to complete the order. the requesting provider retains the option to override the recommendation. an step in process development utilized an order queue established within the epic inbasket. a pediatric radiologist monitored the queue and communicated with referring providers to obtain additional history and educate toward best imaging practice. these interactions facilitated development of a robust index of clinical indications used to create the synonym pool. results: mri neuroimaging indications were expanded into a robust data set linked to specific mri exams aligned with specific protocols. the synonym option within epic created the opportunity for the requesting provider to simply enter an indication which drives the procedure and recommended protocol. provider satisfaction has been high and concurrence with recommendations nearly universal. conclusions: indication driven order entry was achieved through the synonym option in order entry within the epic emr. imaging recommendations are based upon institutional clinical care guidelines developed through consensus. a robust compilation of pediatric mri neuroimaging indications has been created and linked to specific exams and protocols. compliance with the indication driven recommendation has been high. modifications of the current system are currently under development for all cross sectional modalities and organ systems. paper #: pa- cost-effectiveness of routine neonatal renal ultrasound in non-syndromic complex congenital heart disease elfrides traipe, tch, extraipe@texaschildrens.org; jill v. hunter, marthe munden purpose or case report: to assess the prevalence of abnormal renal ultrasound in non-syndromic complex congenital heart disease (cchd) and assess the cost-effectiveness of routine renal ultrasound in this population. we restrospectively reviewed the initial neonatal renal ultrasound and any subsequent renal imaging in patients with non-syndromic cchd. etiologies included hypoplastic left heart syndrome (hlhs), transposition of the great vessels (tga), coarctation of the aorta (coa), truncus arteriosos (ta), double outlet right ventricle (dorv) with or without patent ductus arteriosus. patients were recruited consecutively as part of a prospective trial for pre-and post-operative magnetic resonance imaging of the brain. results: the neonatal pre-operative renal ultrasounds were analyzed in female and male patients. only of the patients showed any congenital renal anomaly. this patient was born with hypospadias, that would have routinely stimulated neonatal follow up. conclusions: knowledge of embryology would not lead us to anticipate a high co-incidence of congenital renal and cardiac pathology. based on this statement and our findings, our recommendation to improve cost-effectiveness is not to perform routine neonatal renal ultrasound in non-syndromic cchd, but only if otherwise clinically indicated. purpose or case report: to conduct a meta-analysis of the diagnostic performance of contrast enhanced voiding urosonography (cevus) in comparison to voiding cystourethrography (vcug) or direct radionuclide cystography (drnc). a literature search was conducted for studies published on cevus in the pediatric age group. studies were included if the ultrasound contrast agents (usca) levovist® (bayer-schering pharma, germany) or sonovue® (bracco, italy) were used and enough data was available to extract x tables. if the cevus was compared to both vcug and drnc in the same patients the results for each were analyzed separately. a bivariate hierarchical model that takes into account the heterogeneity in cevus sensitivity and specificity in different studies was used for the assessment of the summary diagnostic metrics. the summary roc curve was derived and presented graphically from the parameters of the model. additionally, the % confidence intervals (ci) and the positive (lr+) and negative (lr-) likelihood ratios were calculated. results: out of publications only comparative studies fulfilled the inclusion criteria. these encompassed cevus studies in comparison with vcug and with drnc with regards to detection of vesicoureteric reflux. in studies the usca levovist® and in sonovue® were used. a total of children with pelvi-ureteral-units (puus) were included in the meta-analysis. cevus compared to vcug and drnc had a sensitivity of % (ci: - ) and a specificity of % (ci: - ) with lr+and lr-of . and . , respectively. the performance of cevus was better when compared to drnc than to vcug (sensitivity %, specificity % versus % and %, respectively. the meta-analysis of the diagnostic performance of cevus regarding the urethra included patients ( boys). excellent imaging of urethral anatomy was reported in over % of the patients. however, currently there is only one comparative study with patients available. in this study % sensitivity and % specificity were reported. conclusions: sufficient evidence is available clearly demonstrating the high diagnostic performance of cevus compared to vcug or drnc regarding the detection or exclusion of vur in children. these findings combined with the absence of radiation should be convincing reasons for promoting the widespread use of cevus in children. disclosure: dr. darge has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: prenatal ultrasound (us) has increased identification of infants with asymptomatic renal pelvic dilatation. society for fetal urology (sfu) grading is used in the sonographic evaluation of pediatric hydronephrosis. based on us findings, a nuclear medicine diuretic renogram may assess renal function, which could result in operative intervention. standardized protocols for diuresis renography, the "welltempered renogram," already exist; however, no current study has assessed effect of intravenous hydration (iv) status with us in the evaluation of childhood hydronephrosis. our study assesses the effect of hydration on sfu grading. in this prospective irb approved study, pediatric patients diagnosed with pelvicaliectasis requiring a diuretic renogram were recruited to undergo pre and post hydration renal us. a urinary catheter was placed followed by renal us pre and post iv hydration ( ml/kg normal saline bolus). imaging was performed by the same sonographer on the same us machine. a well-tempered renogram was then performed. all images were reviewed by two blinded radiologists, one pediatric radiologist who assigned sfu grades to each kidney. results: data were collected from studies, with ages ranging from weeks- years, with an average age of months. there were unique patients. of these, underwent a single renogram, underwent two renograms, and underwent renograms. one patient had a solitary kidney due to mcdk. thus, there were usable paired sonograms ( kidneys) for analysis. sfu grades were compared in the pre-and post-hydration us for each kidney. two-sided statistical tests were done to assess whether sfu grades changed significantly after hydration (sign test). of ( %) kidneys remained the same grade post hydration. when there was a difference, most demonstrated an increase ( of kidneys and p< . ). no change in sfu grade pre-and post-hydration differed by more than . only kidney went from grade to grade . sfu above grade is considered clinically significant. no kidney that was grade - pre-hydration became grade - post-hydration. when sfu is dichotomized grade - vs. - , there was no significant change in grade from pre to post hydration (p ). conclusions: hydration does not appear to have a clinically significant effect on sfu grade. therefore, performance of a "well-tempered" us is unnecessary. disclosure: dr. lee has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. study type was significantly associated with both total and weighted score (both p < . ). rus was better and mag was worse than vcug, rnc, and dmsa, which did not differ from each other. other factors associated with worse total scores included patient age - years (p< . ) and non-white race (p . ). gender, prior testing history, wait time, and parent education were not associated with total scores. in the multivariate model, rus remained the best, mag the worst, and dmsa, vcug, and rnc in the middle (p< . ). compared directly, dmsa and vcug total score did not differ (p . ). conclusions: this study documents significant differences among gui studies with respect to the patient and family experience, but there was no overall difference between dmsa and vcug. these findings may be useful to aid decision-making when considering gui for pediatric patients. we retrospectively reviewed the imaging findings of consecutive patients with histologic diagnosis of cd ( males, females; mean age . years; age range - years) who underwent mre between / / and / / . the mre was performed in a siemens avanto . tesla scanner. standard departmental volume of polyethylene glycol and fluid were administered for bowel distention. the imaging protocol included dwi with eight b values ranging between sec/mm and sec/ mm and gadolinium enhanced dynamic d vibe (volume interpolated breath hold exam) in the coronal plane. the studies were qualitatively evaluated in a blinded fashion by two board certified radiologists. disease activity was defined as bowel wall thickening and enhancement in the gadolinium enhanced images. dwi abnormality was defined as bowel thickening, increase signal on dwi images and decrease signal on adc maps of the ileum. intra voxel incoherent motion (ivim) dwi parameters were used as quantitative biomarkers for the analysis of slow diffusion (d) and fast diffusion fraction (f). results: gadolinium enhanced vibe images identified abnormal thickening and enhancement of the ileum in / ( %) patients. dwi identified abnormal signal in / ( %) patients. the sensitivity and specificity of the qualitative dwi for identifying ileitis, as shown by gadolinium enhanced imaging, were % and %, respectively. quantitative analysis showed statistically significant difference in ivim maximal values for f (fast diffusion fraction) between abnormal (mean . , std . ) and normal (mean . , std . ) ileum segments (p . ). there was statistically significant difference in ivim maximal values for d (slow diffusion) between abnormal (mean . μm /ms, std . μm /ms) and normal (mean . μm /ms, std . μm /ms) ileum segments (p . ). abnormal loops of bowel had decreased slow and fast diffusion parameters. conclusions: diffusion weighted imaging has excellent sensitivity and specificity for the detection of active ileitis in pediatric cd. furthermore, quantitative ivim model parameters provide effective biomarkers for this condition. ivim dwi has the potential to assess bowel inflammation without intravenous contrast enhancement and further increase our understanding of cd. methods & materials: forty pediatric patients (median age . years, range . - . ) with suspected (n ) or confirmed ibd (n ) were included and underwent gastroileocolonoscopy with biopsies followed by mre (median interval days, range - ). the mre results were compared with macroscopic and microscopic assessment of the ileum. the clinical importance of the mre results was registered. results: crohn disease (cd) was diagnosed in cases, ulcerative colitis (uc) in , and ibd unclassified (ibdu) in three. macroscopic ileitis was detected in / ( %) of cd cases and in / ( %) of uc (backwash ileitis). microscopic inflammation was found in another four cd cases and one ibdu patient. in total, discrepancy between macroscopic and microscopic inflammation was found in cd, uc and one ibdu patients. the sensitivity of mri was % (against macroscopy and/or microscopy) to % (against macroscopy alone), while the specificity was % and %, respectivley. mre findings was decisive for diagnosis in / ( %) and led to treatment adjustments in / ( %) in the following six months. conclusions: mre is a reliable method for imaging of intestinal inflammation in pediatric ibd, and can be supportive or essential for clinical treatment decisions. results: of children with ec, had ct imaging of the abdomen and pelvis. these children ranged in age from months to years (mean . years +/− . ) with a male predominance (n , %). the most common presenting symptoms were abdominal pain (n ), bloody diarrhea (n ), and rectal bleeding (n ). ec was characterized as a dense and predominant eosinophilic inflammatory infiltrate in the lamina propria and/or epithelium without granulomas. ct scans were abnormal in ( %). no colonic luminal contrast was present in patients, and in one of these, the colon appeared normal. abnormal ct findings included cecal wall thickening (n , %), mucosal enhancement without colonic wall thickening, (n , %), mesenteric lymph node enlargement (n , %), terminal ileal thickening (n , %), jejunal and ileal thickening (n , %), and pneumatosis (n , %). of the patients with cecal involvement, primarily involved the cecum with less severe or no ileal or downstream colonic involvement. pneumatosis extended along the length of the colon with rectal predominance. conclusions: the predominant ct finding in our ec series was wall thickening, most severe in the cecum with variable extent downstream with mild or no involvement of the terminal ileum. although there is overlap, these findings are different from the most common patterns encountered with ulcerative colitis or crohn disease and should raise the possibility of ec in children presenting with abdominal pain and bloody diarrhea. purpose or case report: timely identification of childhood arterial ischemic stroke (ais) is critical to development of acute treatment strategies. we present our experience prior to and following development of a pediatric stroke alert system (sas). through multi-disciplinary collaboration in a tertiary care setting, a pediatric sas was established in . we describe the system, imaging protocol evolution, and impact upon the time between admission and mri initiation (time-to-mri) in patients with childhood ais. of patients in our stroke database (comirb # - ), % met inclusion criteria for stroke alert initiation (acute focal neurological deficit within h). eleven pre- and nine post- patients met criteria. we compared the time-to-mri between these two groups, utilizing a two-tailed t-test. results: the pediatric sas has two phases: i-neurological evaluation and ii-imaging and treatment consideration. phase i stroke alert is initiated when a child presents with an acute focal neurologic deficit. if neurology confirms stroke symptoms and ct head is negative for an alternative etiology, a stroke alert is called prompting an emergent brain mri. if mri confirms an acute stroke, hyperacute therapies are considered. initial mri protocol included dwi, t , flair, d tof cow mra, d tof neck mra and fat saturated t neck imaging. after internal quality review, t mprage brain and contrast enhanced d neck mra were added. the sequence order was also altered so diagnostic sequences were scanned first (dwi and cow mra). there was a trend towards decreased time-to-mri in the post- group (mean min, sd +/− ) as compared to the pre- group (mean min, sd +/− ; p . ). conclusions: institution of a pediatric sas improved urgent neurologic evaluation and demonstrated a trend towards shorter time-to-mri. ongoing quality review has enhanced imaging quality and decreased time-to-mri. continued refinement of pediatric sas's will be critical to the success of recently funded phase i clinical trials in the evaluation of hyperacute therapies. results: there were female and male infants. the mean post-gestational age at presentation was days (range - days), while the corrected age was less than days for all patients. patients presented with seizures and signs of infection; presented with lethargy and later proved to have protein c deficiency. mri was performed - days from presentation in these patients. another patient with known protein c deficiency underwent mri at d for followup of screening us abnormalities. there were a total of deep cerebral white matter lesions: frontal, parietal, temporal lobe. lesions were fluid signal cavities with restricted diffusion. larger lesions had dependent debris. all lesions had associated hemorrhage and most lesions had evidence of adjacent small vessel venous thrombosis. lesions imaged after gad showed peripheral enhancement. three lesions were seen to increase in size on follow-up imaging. three patients, with meningitis confirmed via microbiology and with presumed meningitis by csf counts, underwent surgical aspiration of a total of lesions. all specimens were sent for pathology and culture and were negative for microorganisms. conclusions: recognizing the mr appearance of necrosis and liquefaction after deep white matter cerebral venous infarction in neonates can distinguish this entity from cerebral abscess and potentially avoid an unnecessary neurosurgical aspiration procedure. all four children were initially treated with aspirin but experienced recurrent events on therapy. all four were subsequently anticoagulated. two children have remained on warfarin for - years without recurrent events, while the other two had recurrent events despite adequate anticoagulation. these two children underwent uncomplicated coil embolization of the affected vertebral artery segment, and they have remained symptom-free for five and months since then. conclusions: dava was diagnosed by ca in / patients. all four children with dava in our series suffered recurrent strokes despite aspirin therapy. two of the four experienced further strokes on anticoagulation, necessitating endovascular therapy. these findings suggest that dava in children may require ca to diagnose, and that it may be refractory to standard adult therapies. ongoing multicenter efforts in childhood ais should further evaluate the diagnostic approach and recurrence risk of childhood dava. ) and cerebral gray matter abnormalities were present in ( %). posterior fossa lesions were seen on us in . % , but mastoid views were included in only % of the centrally read us. conclusions: in the largest extreme preterm cohort to date with near-term mri and serial us, % had mod-severe wma on brain mri, similar to previous reports. cerebellar abnormalities were detected more frequently by mri than by us. neurodevelopmental outcomes at - months and school age will assess the relative and combined values of mri and us as outcome predictors. , an analysis technique based on task-free resting state fmri recording, can be useful in assessing disruption of connectivity in certain disease states, including epilepsy. in healthy control subjects, functional connectivity reveals strong bilateral interhemispheric connectivity in such system as sensory-motor, visual, auditory as well as dorsal attention and default mode networks. in patients with epilepsy associated with unilateral diffuse hemispheric disease such data is limited. differences in the pattern of activation would suggest alteration in connectivity in these entities. this finding would impact the typical interpretation of this data that is becoming routinely collected for epilepsy pre-surgical evaluation. methods & materials: siemens (erlangen, germany) system, -tesla (trio) scanner was used for imaging (epibold sequence, te ms, flip angle °). resting state fmri scan were performed in both awake and anesthetized patient. awake patients were instructed to relax and rest while keeping their eyes open. analysis was performed using functional connectomes project scripts based on afni and fsl software packages. resting state data were analyzed for connectivity with the following seeds: somatomotor, visual, auditory, and default mode (posterior cingulated cortex (pcc)). results: we applied this technique to evaluate patients with hemispheric seizure disorders, including rasmussen's, neonatal infarct and migration disorders. all the subjects demonstrated some deviation from typical interhemispheric connectivity with a spectrum of findings. the figure below shows connectivity patterns in a patient with cortical dysplasia. while some interhemispheric connectivity remained in somatomotor (sm) and auditory (a ) systems, it was disrupted in visual (v ) and default mode (pcc) networks. variable patterns were found across the cases that corresponded to lesion side, supportive of disruption in interhemispheric connectivity as measured by fmri. conclusions: resting state functional connectivity patterns are well documented in healthy subjects. these results suggest that interhemispheric connectivity disruption is a typical feature of unilateral diffuse hemispheric disease though variable in presentation, either being limited to select systems or demonstrating broad disconnect between the two hemispheres. these results should be carefully considered when evaluating data for pre-surgical epilepsy evaluation. purpose or case report: premature birth is associated with white matter injury leading to a wide ventricular system. however,normative standards for ventricular size are lacking for this particular group.aims: we aimed to, in a controlled, population based norwegian cohort of ex-prematures without major handicaps, and for men and women separately,to ) create standards for radiological indices of ventricular dilatation, )investigate associations of these measurements with subjectively assessed ventricular size, ) examine differences in ventricular size between ex-prematures and healthy controls methods & materials: the initial birth cohort included neonates, birth weight below g (low birth weight)born within hordaland county, norway, between april st and august th . of eligible survivors (without major handicaps)underwent mr examination during the period january to may . of these were expremature (born before gestational age weeks) and were included in this sub-study. based on t weighted images, the ventricular size was subjectively judged as being normal, mildly, moderately or severely dilated by an experienced paediatric neuroradiologist, while objective measurements were performed in a blinded fashion, by a second observer (sma) using an imaging software program (nordic ice®). results: the normative standards for the ventricular system in ex-premature young adults showed wide variations, in particular for the occipital horns. the agreement between subjective and objective assessment of ventricular size was good. ex-prematures had smaller heads than those born term (control group). there was no difference in ventricular size between the two groups, even after adjusting for head size. ex-premature males had larger ventricles than females; however, the difference disappeared after adjusting for head size. conclusions: young adults born prematurely with a birth weight below g do not have larger lateral ventricles than healthy controls born term, even after correcting for a smaller head size. paper #: pa- best practice for reproducibility when measuring t *: implications for liver and cardiac iron assessment mark ferguson, md, radiology, seattle children's hospital, markferg@uw.edu; randolph otto, seth d. friedman purpose or case report: patients with red blood cell transfusion-dependent conditions receive high amounts of iron that can lead to abnormal iron accumulation in tissues resulting in organ damage. while the liver is the dominant excess iron storage organ, iron related cardiotoxicity is a leading cause of morbidity and mortality in patients with transfusion-dependent thalassemia. therefore, accurate determination and tracking of tissue iron levels in both the liver and the myocardium is important for patient prognostication as well as monitoring treatment changes. while multi-echo gradient echo mri (t *) is widely used and validated method employed for iron assessment, less attention has been given to derived metrics. specifically, the literature almost exclusively reports and uses the mean value for t * from a pixel-wise (pw) map. infrequently used is the median. the median is a potentially superior metric than the mean because it is insensitive to outliers. outliers will always occur in data because of either noise or imperfect vessel exclusion. to compare mean versus median on reproducibility of t * measurement, subjects who had paired heart/liver measurements were examined. the entire liver (excluding vessels) and the interventricular septum myocardium were traced on representative images from each series. mean and median t * values were generated from the pixel maps. r * ( / t *) and coefficient of variation (cv) were computed on a patient-by-patient basis. these measures were then summarized for the group. results: markedly higher r * values were observed in both heart and liver using median summary measures (liver: t − . , p . , heart: t − . , p . ). these findings were accompanied by lower cv's (better reproducibility) for the median approach (liver: t . , p . ; heart: t . , p . ). conclusions: the consistent difference in derived t * values between the methods (median>mean) should be considered when comparing derived r * values to established normal ranges. cv data support that using the median as the final summary metric will always outperform mean metrics for measuring change in r *. this finding has immediate implications for the scientific literature and for guiding therapeutic management over time. results: twelve children (age mo- yo; m:f : ) with gsds ( abca mutations, sp-c mutations, undefined mutation) and children (age wk- yo; m:f : ) with other dlds (including pulmonary interstitial glycogenosis, neuroendocrine cell hyperplasia of infancy, lymphocytic interstitial pneumonia, lipoid pneumonia, diffuse alveolar damage, granulomatous infection, capillaritis and other pulmonary hemorrhage syndromes) were identified. ct findings with highest sensitivity for gsds were ground glass attenuation ( %), parenchymal cysts ( %), and interstitial thickening ( %). parenchymal cysts, honeycombing, and pectus excavatum were more specific for gsds compared to other dlds (p< . ). the combination of either parenchymal cysts and honeycombing or ground glass attenuation and pectus excavatum provided the highest specificity ( %) but low sensitivity ( %). the combination of parenchymal cysts and ground glass attenuation provided good specificity ( %) and modest sensitivity ( %). no combination of findings provided both high sensitivity and specificity. conclusions: ground glass attenuation is the most sensitive finding for gsds, while parenchymal cysts, honeycombing, and pectus excavatum are more specific findings for gsds than other chronic dlds of childhood. however, no single finding or combination of findings on chest ct is both highly sensitive and specific for gsds, and chest ct cannot substitute for genetic testing or lung biopsy for the differentiation of gsds from other dlds. pes were observed in scans of patients without a history of congenital heart disease (chd), and pes in scans of patients with a history of chd. z-axis scan lengths for the chest ct exams ranged from . - . cm. a z-axis scan length of cm centered . cm below the carina captured all pes in all patients, and a length of cm centered . - cm below the carina in patients with chd. a z-axis scan length of cm centered cm below the carina was sufficient to capture at least one pe in all patients, and a length of cm centered - cm below the carina in patients with chd. the radiation effective dose of the chest ctpa exams ranged from - msv. limiting the z-axis scan length on ctpa exams to cm or cm would have resulted in a % or % decrease in z-axis coverage, respectively, and estimated radiation effective dose reduction of - % due to less radiation exposure to the intrathoracic structures, thyroid gland and upper abdominal viscera. conclusions: limiting the z-axis scan length coverage for ctpa exams based on a model of the typical anatomic distribution of pes relative to the reference level of the carina permits a substantial reduction of radiation dose in children without reducing the sensitivity for detection of pulmonary emboli. purpose or case report: to determine whether the addition of multiplanar reformation mdct images affects reader performance parameters and provides added diagnostic value compared to the use of axial ct mdct images alone for diagnosing pe in children. this was an institutional review board-approved retrospective study of consecutive pediatric patients who underwent ctpa for clinically suspected pe. two faculty pediatric radiologists and two radiology residents independently reviewed each study initially using only axial mdct images and later using mpr mdct images in any x-, y-, or z-axis for detecting pe. diagnostic accuracy, confidence level, and interpretation time of mpr mdct images were compared to axial mdct images using mcnemar's test and paired t-tests. the kappa coefficient was calculated to assess interobserver agreement. diagnostic accuracy was compared between faculty pediatric radiologists and radiology residents by logistic regression whereas confidence level, interpretation time, and added diagnostic value were evaluated with analysis of variance (anova). results: the final study cohort consisted of ctpa studies from children ( m/ f; mean age . years). nine ( %) of ctpa studies were found to have pe. diagnostic accuracy in correctly detecting pe ranged from . to % (mean . %), with no significant differences between the use of axial and mpr mdct images. logistic regression indicated no significant difference in diagnostic accuracy of detecting pe between faculty pediatric radiologists and radiology residents for axial mdct images (p . ) or mpr mdct images (p . ). confidence level and interobserver agreement were significantly higher and average interpretation time was longer in evaluating pe with mpr mdct images compared to axial mdct images for all reviewers (p<. ). compared to faculty pediatric radiologists, significantly greater increases in confidence level, interobserver agreement, interpretation time, and added diagnostic value using mpr mdct images compared to axial mdct images to diagnose pe were found for radiology residents (p< . ). conclusions: use of mpr mdct images in diagnosing pe on ctpa in children significantly increases confidence, interobserver agreement, and interpretation time among faculty pediatric radiologists and radiology residents. because mpr mdct images provide significantly greater improvements in reading parameters for residents than for faculty members, their routine use should be encouraged for trainees. paper #: pa- chest ct in children, anesthesia and atelectasis beverley newman, md, radiology, stanford university, bev.newman@stanford.edu; elliot krane, terry e. robinson purpose or case report: in spite of advances in ct equipment and speed, sedation/ anesthesia is required in many young children for optimal quality ct for detailed parenchymal evaluation; resultant atelectasis is a common and important quality issue. our purpose was to evaluate the safety and effectiveness of a standardized lung recruitment technique. methods & materials: with irb approval and parental informed consent, controlled ventilation, low dose, chest ct's (cooperative effort between anesthesia, pulmonology and radiology) were performed in children ( had - cts) ( f, m; ages . - . yrs, mean . yrs). indications included cystic fibrosis ; ciliary dyskinesia ; chronic or interstitial lung disease ; evaluate pulmonary metastases . ct parameters were - kvp, - mas, iv contrast . various prior methods employed by the pediatric anesthesiologists to maintain lung inflation had unpredictable results (a brief survey showed / nonintubated anesthetized cases had problematic atelectasis). a standardized intubation technique was therefore adopted: .use of a tight fitting face mask during induction and iv placement, inspiratory pressures of - and peep of . . introduction as early as possible using an appropriately sized cuffed endotracheal tube. . alveolar recruitment maneuvers- - s breaths to cm h o/ ( - in st cases). . three breaths at / , inspiratory breathold followed by - cm on th breath for scout and inspiratory scan, and complete ventilator disconnection for expiratory scan. recruitment breaths repeated before each scan. two experienced readers reviewed and scored the images on a point scale for overall quality and atelectasis. results: all studies were completed safely with no procedural complications. one child had propofol-related postoperative emergence delirium. all ct scans were diagnostically good to excellent with small subsegmental atelectasis in ( / were the initial cases with lower recruitment pressures) and segmental atelectasis in . cases had prior cts, without this technique, that were suboptimal due to moderate procedural atelectasis, in spite of tracheal intubation in the majority of cases. conclusions: an intubation lung recruitment technique can be performed safely and consistently by different individuals using a standardized protocol. procedural atelectasis that affects quality is reliably absent and repeat sequences are not needed. obtaining a high-quality dynamic airway imaging study is critical for accurate interpretation and subsequent medical decision-making. the ideal mri sleep study is one that allows successful completion while maintaining spontaneous breathing without artificial airway, which can be an anesthesia challenge. dexmedetomidine has been shown to have sedative properties paralleling natural sleep with minimal respiratory depression. we hypothesized that dexmedetomidine compared to propofol would have less effect on upper airway tone and airway collapsibility and provide favorable conditions with less airway interventions required during dynamic mri airway imaging in children with osa. in this prospective study, we examined the requirement for airway intervention for propofol ( - mcg/kg/m) and dexmedetomidine( - mcg/kg/h) in children and adolescents with osa. severity of osa was analyzed by overnight polysomnography. for children with history of mild osa there was no intervention unless oxygen saturation decreases below %; while for children with history of moderate/severe osa, an artificial airway was placed when oxygen saturation decreased below %. results: demographics and osa severity by polysomnography were comparable. requirement for artificial airway by severity of osa as documented by polysomnography will be shown. mri sleep studies required airway intervention in / ( %) children in the dexmedetomidine group versus / ( %) children in the propofol group. mri sleep studies were successfully completed without the use of artificial airways in children ( %) in the dexmedetomidine group versus children ( %) in the propofol group. conclusions: safe and effective anesthetic management is a key factor in obtaining good quality mr images of the airway. although there was no statistical significant difference in the need for airway intervention between drugs, dexmedetomidine provided acceptable sedation for mri sleep studies with less airway intervention in children with osa. dexmedetomidine may be the preferred agent for sedation during mri sleep studies in children, and may offer benefits to children with sleep disordered breathing requiring anesthesia or sedation for other diagnostic imaging studies. an open mouth and administration of cpap resulted in smaller ap diameter of the retroglossal airway compared to images without cpap due to cpap pressure pushing the tongue posteriorly. in patient volume of oral cavity anterior to the tongue increased from . ml to . ml. meanwhile, the ap diameter of the retroglossal airway decreased from . to . mm ( % decrease). in patient the mouth was initially closed but parted when the pressure of cpap was added with the oral volume increasing from . ml to . ml. the ap measure of the retroglossal airway decreased from . mm to . mm ( % decrease). in patient the mouth was then closed and cpap reapplied resulting in an ap measurement of . mm ( % increase). the ap diameter difference between cpap and no cpap were tested with paired t-test, but were not statistically significant (p . ). conclusions: positive airway pressure on a patient by full facemask and an open mouth can have an adverse effect on the retroglossal airway. this adverse effect is an important consideration in the use of positive airway pressure to support airways for osa, or during emergency resuscitation when a full facemask is used. paper #: pa- the purpose or case report: a nanoparticle blood pool iodinated contrast agent (nctx) has been designed and tested in preclinical animal models. we report data in animal models exemplifying its advantages over conventional contrast in the setting of ct pulmonary angiography methods & materials: nctx blood pool nanoparticles of nm diameter with an encapsulated total iodine concentration of~ mgi/ml were administered by intravenous injection to mice, rabbits, dogs, pigs and sheep. (these studies were actually conducted for other purposes and a review of the data revealed the similarities that motivated this paper.) total injected volumes were~ ml/kg in large animals, and as high as ml/kg in small animals to provide satisfactory vessel enhancement. iohexol or iopamidol was administered for comparative studies with conventional contrast. in a subset of pigs, iatrogenic pulmonary arterial emboli were introduced prior to contrast administration. toxicity studies were conducted in mice and monkeys. results: the visualization of pulmonary vessels using nctx blood pool nanoparticles was generally at least equivalent to using conventional contrast, and superior in several cases, particularly in small veins and when bolus timing of the conventional contrast was suboptimal. in all cases, satisfactory vessel enhancement was achieved for a duration of several hours following a single infusion of nctx blood pool nanoparticles. there was no evidence of renal toxicity, and only transient elevation of hepatic enzymes at relevant dose levels. conclusions: nctx nanoparticle blood pool agents demonstrate several advantages over conventional glomerularfiltered iodinated contrast agents for ct pulmonary angiography in animal models, including no nephrotoxicity, no dependence on bolus injection technique, superior depiction of small veins, and capability of re-imaging for follow-up studies without needing contrast re-injection. potential applications in human pediatric subjects include the diagnostic and post-therapeutic evaluation of cardiopulmonary anomalies and pulmonary embolism, especially in patients with renal insufficiency or tenuous vascular access. disclosure: dr. annapragada has indicated that he is a stock holder and consultant for marval biosciences inc. paper #: pa- cardiovascular image quality using a nanoparticle ct contrast agent: preliminary studies in a pig model rajesh krishnamurthy, radiology, texas children's hospital, rxkrishn@texaschildrens.org; ketan ghaghada, prakash masand, abhay divekar, eric hoffman, ananth annapragada purpose or case report: image quality in a separate study using a long circulating, liposomal-based nanoscale blood pool iodinated contrast agent (nctx) suggests clinical utility in pediatrics, potentially reducing difficulties in contrast-ct of children with congenital heart disease (chd) including the size of intravenous cannula, need for accurate timing, inability to simultaneously opacify multiple targets of interest (requiring repeated contrast administration and/or repeated imaging). methods & materials: six pigs (average weight kg) were imaged after slow intravenous infusion of nctx ( mg i/ml) at an iodine dose of approximately mg i/ kg ( . ml/kg). retrospective ekg gated ct imaging was performed h later using a -slice dual-source ct scanner at and kvp. two radiologists analyzed and graded (on a -point scale with : unreadable, : excellent) images aimed at anatomic structures relevant to chd. quality of images obtained at and kvp were compared. uniformity of contrast opacification was measured using a roi-based ctnumber method at various intracardiac and extracardiac sites and mean non-uniformity was calculated. results: there was excellent agreement between the two readers on all counts at kvp. kvp images received lower scores for coronary morphology ( / ), and aortic valve visualization ( . / ), but were comparable in other aspects. pulmonary artery and pulmonary vein branch visualization extended up to the th generation in all cases. visualization of coronary artery branches was possible up to the second generation, with good arteriovenous separation. subtle morphologic features including crista terminalis, thebesian valve, foramen ovale, membranous septum, and chordae of the mitral valve were demonstrated in all cases. automated functional analysis and myocardial mass quantitation was feasible in all cases. there was no significant difference in blood pool attenuation between the atria, ventricles, and extracardiac vasculature on quantitative assessment. no image artifacts were visible on the reconstructed images. conclusions: these findings suggest that nctx promises to be superior to conventional contrast agents for ct imaging of complex congenital heart disease, due to the absence of nephrotoxicity, avoidance of repeated contrast administration, and reduced number of scans performed. avoiding the need for accurately timed scans precludes the need for large bore intravenous access. these attributes make it a promising agent that warrants further studies. disclosure: dr. annapragada has indicated that he is a stock holder and consultant for marval biosciences inc. paper #: pa- theoretical cost and x-ray dose reduction in pediatric congenital heart disease imaging by the use of a nanoparticle contrast agent robert bell, the university of texas-houston; rajesh krishnamurthy, gabriela espinosa, christopher petit, ananth annapragada purpose or case report: the purpose of this study is to determine the effective, population averaged reduction in costs and radiation dose that can be achieved in the diagnosis of congenital heart disease by use of a nanoparticle long circulating blood pool contrast agent. methods & materials: a markov model of the decision tree followed at the texas children's hospital in the image based diagnosis of congenital heart disease was constructed in treeage software. the model included ct angiography, mr angiography, cardiac catheterization, and echocardiography diagnostic modalities. patient records, accumulated between and were examined to inform the model. the radiation dose and cost for each step were encoded as penalty functions. markov simulations were run for two decision trees: ( ) utilizing ct angiography and ( ) replacing conventional ct angiography with blood-pool agent based ct angiography. the overall population x-ray dose and accrued cost was calculated for each pass through the model. results: x-ray dose distributions for the example populations showed substantial reductions per ct study, as much as %. averaged over the population, since a sizeable fraction of patients are diagnosed without ever being exposed to any x-ray based modality, reductions were more modest, but still substantial. costs per ct study were slightly higher when the blood pool contrast agent was used. when the diagnostic probability using the blood pool agent increased, it led to an automatic overall cost reduction. conversely when the diagnostic probability remained unchanged, costs rose, commensurate with the increased cost of the contrast agent. conclusions: the use of a blood pool contrast agent for ct angiography leads to substantial reduction in radiation dose in the setting of congenital heart disease. cost reductions are more modest, and are driven almost completely by the reduction in the number of mr and invasive angiography procedures resulting from increased diagnostic success using blood pool based ct angiography. the model as constructed does not account for potential workflow changes that might result from the use of a new contrast agent. actual reductions realized may therefore be higher. disclosure: dr. annapragada has indicated that he is a stock holder and consultant for marval biosciences inc. paper #: pa- frequencies and patterns of situs discordance in chest and abdomen justin boe, stanford, justinj.boe@gmail.com; beverley newman, shreyas vasanawala, frandics chan purpose or case report: incidence of situs anomalies, including heterotaxy and situs inversus, is estimated at . % of population. as the first step in the segmental analysis of structural heart disease, the determination situs position is of fundamental importance. abdominal situs, as defined by splenic position and morphology, and cardiac situs, as defined by atrial morphology, are usually but not always in agreement. echocardiographers also employ the relative position of the great arteries and vein at the hiatus to determine cardiac situs. we evaluate the frequencies of discordances among abdominal, hiatal and cardiac situses. methods & materials: with retrospective irb approval, imaging records from to were reviewed for the diagnosis of cardiac situs inversus and heterotaxy. patients who had cardiac ct or mri were included. images were evaluated on a d-processing station by a cardiac radiologist. cardiac situs was determined by the morphology of the atrial appendages. when an atrial appendage was not adequately visualized, cardiac situs was assessed by the relative position of the main pulmonary artery and bronchi. hiatal situs was determined by the relative position of the aorta and the systemic venous return, and abdominal situs by the position and morphology of the spleen. results: thirty-five cases were identified, with cardiac ct and mri. patients' age ranged from day to years old. in the abdomen, the numbers of situs inversus, asplenia, and polysplenia were ( %), ( %), and ( %). for the heart, the numbers of situs solitus, inversus, rightisomerism, and left-isomerism were ( %), ( %), ( %) and ( %). the abdominal and cardiac situses were discordant in ( %) cases. polysplenia had the highest number of discordance with the heart. hiatal situs was discordant with the abdomen in cases ( %) and with the heart in ( %) cases. conclusions: situs disagreement between the abdomen and the heart is not uncommon and they should be documented separately in radiology reports. hiatal situs, as used by echocardiographer, disagrees with the cardiac situs in a quarter of the cases. it should be used with caution in the segmental analysis. paper #: pa- diminished asl intracranial perfusion in children with neurofibromatosis type kristen yeom, md, stanford university, kyeom@stanford. edu; cynthia campen, patrick barnes purpose or case report: neurofibromatosis type (nf ), a neuro-cutaneous syndrome affecting / children is associated with moyamoya syndrome (mms). however, no comparisons of cerebral perfusion in patients with nf and nf -associated mms to healthy controls exist. we hypothesize cerebral blood flow (cbf), as measured by magnetic resonance imaging (mri) arterial-spin-labeled (asl), is diminished in children with nf compared to healthy controls, with the lowest levels seen in patients with nf -associated mms. methods & materials: twenty children aged - years with nf , four with mms, and age-matched controls underwent asl cbf on a t magnet. pseudocontinuousspin-echo-asl technique was used. measurements were taken bilaterally in cerebral cortical-subcortical regions, and the deep gray nuclei. trends in measurements as a function of disease severity were tested with the jonckheere-terpstra test for ordered alternatives. a bonferroni-adjusted p-value less than . was considered significant. results: we identified / areas with significantly diminished asl cbf (ml/ g/min) in patients with nf (midrange), and nf -associated mms (lowest) compared to healthy controls (highest). these included the: thalami (left: p . , right: p . ); superior/middle temporal lobes (left: p . , right: p . ); temporooccipital lobes (left: p . , right: p . ); occipital poles (left: p . , right: p . ); centrum semiovale (left: p . , right: p . ); and left parietal lobe (p . ). conclusions: cerebral perfusion diminishes in a graded fashion in children with nf and nf -associated mms, particularly in the posterior circulation and the mca-pca posterior watershed zones. future studies may demonstrate an important role for asl in the presymptomatic diagnosis of cerebral vasculopathy, and the definition of nf -related vasculopathy patterns. paper #: pa- cingulate gyrus mri sign in pediatric nf patients: a novel imaging marker nadja kadom, md, radiology, children's national medical center, nkadom@childrensnational.org; nabila hai, rhea udyavar , amir noor, gilbert l. vezina, maria t. acosta purpose or case report: we observed a magnetic resonance imaging (mri) signal abnormality in the anterior cingulate gyrus of pediatric patients with neurofibromatosis type (nf ). the cingulate gyrus could play a role in cognitive deficits of nf patients. the first objective here is to document inter-rater reliability scores for visual detection of this sign. the second objective is comparing adc values of the cingulate gyrus in areas of visually abnormal mri signal in nf patients to matched normal mris to confirm a pathophysiological basis of the visual mri sign. methods & materials: retrospective analysis, irb approved, nf patients and matched controls. in the visual assessment part, two blinded neuroradiologists rated presence or absence of mri signal abnormality in the cingulate gyrus in three different age groups of nf patients mixed with normal controls. cohen's kappa inter-rater reliability coefficients were calculated. the same blinded neuroradiologists evaluated the cohort one year later, this time by agreement at the workstation. in the adc measurements part, two researchers, one blinded, manually placed roi's in the anterior and posterior cingulate regions of nf patients and their matched controls, and student t-test was used to assess for significance of differences in measured values. results: cohen's kappa for the three age groups showed very good agreement (kappa coefficients were either . or . ). rater agreement at the workstation was %. all subjects with a positive finding also had nf and the sign was not seen in any of the normal controls. the prevalence of the sign was %. adc measurements showed significantly higher adc values in the anterior cingulate gyrus of nf patients when compared to normal controls and also when compared to the posterior cingulate gyrus in nf patients. conclusions: our results show that visual t /flair mri abnormalities in the anterior cingulate gyrus are present in % of patients with nf from ages to years. adc measurements confirm a pathophysiological basis for this finding. future correlation with clinical manifestations, such as learning and behavioral manifestations in patients with nf , are under way to further evaluate the clinical importance of this finding. tract-based spatial statistical analysis of diffusion tensor imaging in pediatric patients with mitochondrial disease seth friedman, phd, seattle children's, seth.friedman@ seattlechildrens.org; andrew v. poliakov, sandra l. poliachik, dennis w. shaw purpose or case report: often diagnosed at birth or in early childhood, mitochondrial disease presents with a variety of clinical symptoms, particularly in organs and tissues that require high energetic demand such as brain, heart, liver, and skeletal muscles. in a group of pediatric patients identified to have complex i or i/iii deficits, but with white matter tissue appearing qualitatively normal for age, we hypothesized that quantitative dti analyses might unmask deficits in microstructural integrity. methods & materials: dti and structural mr brain imaging data were collected in pediatric patients with confirmed mitochondrial disease and clinical control subjects matched for age, gender, scanning parameters, and date of exam. paired tract-based spatial statistics (tbss) were performed to evaluate differences in fractional anisotropy (fa) and mean diffusivity (md). results: in patients with mitochondrial disease, significant widespread reductions in fa values were shown in white matter tracts. md values were significantly increased in patients, having a sparser distribution of affected regions compared to fa. results of tbss statistical analysis will be shown. to be shown in green is the mean fa skeleton which represents the centers of main white matter tracts. all results p<. . red and yellow represent a significant increase, blue and light blue represent a significant decrease. conclusions: despite qualitatively normal appearing white matter tissues, patients with confirmed mitochondrial disease have widespread microstructural changes measurable with quantitative dti. this supports the evaluation of such metrics in other populations where gross imaging features may be normal. to extend our studies to patients with other plp mutations, we analyzed the brains of male pmd patients (ranging in age from to ) and female carriers for whom the plp genotype had been determined and analyzed by mri. for each patient we measured, white matter volume (wmv) and the intercaudate distance (icd). the mri data were correlated with functional disability scores (fds) using a system we developed for clinical evaluation of pmd patients and which was validated by assessments of pmd patients. brain volume and segmentation were measured using nih image . . the average number of coronal slices analyzed from each patients mri was slices. when graywhite contrast was not adequate, then the intercaudate distance (icd) and intercaudate ratio were measured as described in caon et. al., ( ) . results: comparison of the mr measurements and the fds demonstrated that white matter volume inversely correlates with functional disability, suggesting that the initial disability does correlate with the extent of myelination. the intercaudate distance also correlated with the fds, and may usefully substitute when gray-white matter segmentation is not possible. conclusions: pmd is a clinically and genetically heterogeneous disease caused by mutations in the gene encoding the major cns myelin protein, proteolipid protein (plp). myelin is a major target of disease pathogenesis in most cases of pmd, but how the various mutations cause clinical disability is not fully understood. our data demonstrate that the extent of brain white matter atrophy, measured directly by volumetric fractionation, or indirectly by analyzing the intercaudate ratio, is significantly correlated with the patient's functional disability. white matter atrophy is thus the main cause of clinical disability in patients with pmd of all ages and mutation type. paper #: pa- maturational effects on language localization in children demonstrated by fmri susan palasis, md, children's healthcare of atlanta at scottish rite, spalasis@yahoo.com; binjian sun, laura l. hayes, richard a. jones purpose or case report: language localization is of paramount importance when contemplating surgery in children with intractable epilepsy or brain tumors. the potential risk of injury to language centers in the developing pediatric brain needs to be weighed against the potential benefits of surgery. in the past, language localization was crudely and invasively determined using the wada test. most institutions are now transitioning to non invasive localization using functional mri (fmri). the purpose of our study was to analyze language localization relative to age in children using age appropriate language paradigms and fmri. methods & materials: forty three healthy, english speaking, right handed children underwent fmri evaluation for language localization. the studies were performed on a t system. three novel age appropriate language block paradigms were utilized, targeted both to expressive and receptive language processing. these paradigms were the auditory category decision task (audcat), the auditory description decision task (addt), and the listening task. the spatial statistical maps generated by the fmri data were fused to the d anatomical mri dataset. language areas were localized and statistical analysis was performed with age as the variable in a general linear model. results: our results demonstrate a distinct trend in language localization and lateralization with brain maturation. in the young age groups (less than years) the localization tended to be less focused and bilateral in the frontal and temporal regions of the brain. in the older age groups (greater than years), language became more localized and lateralized to the expected left sided pattern. the findings were more robustly demonstrated with the addt task and were statistically significant (p< . ). conclusions: our study clearly demonstrates the plasticity of language centers in the maturing pediatric brain. this observation is significant for neurosurgical planning and rehabilitation in the pediatric population. ( ) no slc a mutations were found in , and subjects, respectively. significantly higher association with slc a mutations was found in bilateral eva+v/c dysplasia ( / ). double mutations of slc a is more often associated with combined eva+ v/c dysplasia, while a single mutation with eva only. cochlear aplasia without eva ( / ) and snhl with normal imaging ( / ) are less likely associated with slc a mutation. conclusions: slc a mutation is highly associated with eva and v/c dysplasia. once eva with or without v/c dysplasia are found at imaging, genetic investigation is recommended for slc a mutation because of possible thyroid involvement. moderate-severe hie who were randomized to cooling ( . °c for h). there were in the hypothermia group and in the control group. all mris were reviewed by a cental reader masked to the clinical findings, groupings, and outcomes. the mri findings were scored according to pattern and extent of injury, including involvement of the cerebral hemispheres, basal ganglia, thalami, internal capsules, and other structures. brain injury scores were correlated with death or disability at months postnatal age. results: no mri abnormalities were observed in of infants ( %) in the hypothermia group and in of infants ( %) in the control group (p . ). infants in the hypothermia group had fewer areas of injury ( %) as compared with the control group ( %, p . ). there were of the infants with death or disability at months. the brain injury score correlated with outcome of death or disability (p . ) and disability among survivors (p . ). conclusions: fewer areas of brain injury on mri were observed following whole-body hypothermia. the mri brain injury score is a marker of death or disability at months following hypothermia for term hie. . presence or absence of the "red dot" on fa color maps was correlated to clinical (ataxia, oculomotor abnormalities etc.) and morphological data, and to fa and md measurements. results: the "red dot" was absent in js and hgpps (genetic cross wiring impairment diseases) and present in coma and wvs (no reported gene abnormalities so far) as in normal controls. js and coma presented on mri molar tooth appearance. hgpps presented "split pons" appearance. js and coma patients presented oculomotor apraxia, wvs and hgpps palsy of the horizontal gaze. mirror movements were found in js and in wvs. ipsilateral responses are present in hgpps. wvs presented multiple cranial nerves impairment. in js, fa and md values of scp, pt and pc were significantly lower than in normal controls (p > . ). in hgpps high fa and low md were found in pc and pt (p > . ) and normal in scp. conclusions: the "red dot" absence is unrelated to morphological or clinical abnormalities. absence of the "red dot" is associated to abnormal measurements of fa and md in pc and pt( low in js and high in hgpps). these findings indicate a pivotal role for the pc in the physiopathology of these diseases. the "red dot" absence seems to be a marker of genetic cross wiring diseases. in this view, coma and wvs should not be considered as part of these diseases. sonographic predictors of intermittant testicular torsion in the pediatric patient jennifer williams, md, pediatric radiology, texas children's hospital, jlwilli @texaschildrens.org; marthe munden purpose or case report: intermittent testicular torsion (itt), defined as sudden onset unilateral scrotal pain with spontaneous resolution, is difficult to confirm both clinically and sonographically. the purpose of this study was to determine if sonographic predictors exist for diagnosing itt in the pediatric patient. methods & materials: a search of the pacs data system for patients presenting with suspected intermittent testicular torsion was performed. fifteen patients with a total of episodes presenting over a year period were found. a retrospective review of the medical records for clinical presentation, surgical outcome, and comorbidities was performed. scrotal ultrasound images and reports were reviewed for testicular size and echotexture, testicular flow, epididymal appearance, vascular bundle appearance, and presence of hydrocele. results: an abnormal appearance of the vascular bundle was found in % of episodes ( / ). initial absence of testicular flow followed by reperfusion during the scan was seen in % of episodes ( / ); % had increased flow ( / ), % had decreased flow ( / ), and % had normal flow ( / ) . nine of the patients had surgery; of these were found to have evidence of itt and was found to have acute testicular torsion. of patients with itt, % ( / ) had an abnormal vascular bundle. testicular flow was not initially visualized but returned during the exam in % of patients ( / ), was increased in % of patients ( / ) and was decreased in % patients ( / ). conclusions: itt is a difficult diagnosis. the most reliable sonographic indicator is an abnormal spermatic cord, found in % of episodes and % of surgically proven itt. dedicated views of the spermatic cord must be obtained in order to differentiate an abnormal epididymis from an engorged vascular bundle (the so-called pseudomass). attention to testicular flow is of particular importance. while visualization of a transition from no or decreased testicular flow to normal flow during the sonogram is certainly diagnostic of itt, increased testicular flow should not lead to false reassurance. purpose or case report: testicular torsion is a common acute condition in boys requiring prompt and accurate diagnosis. the objective was to evaluate ultrasound accuracy and findings, and clinical predictors in testicular torsion in boys presenting to the stollery pediatric emergency department (ed) with acute scrotal pain. methods & materials: retrospective review of us, surgical and ed records for boys aged month to years, presenting with acute scrotum from to , was performed. age, demographics, clinical symptoms, physical findings, us and surgical techniques, findings and diagnoses were recorded. surgical results and follow-up were used as the gold standard as all pediatric urology in our region is performed at our centre. results: patients presented to ed with acute scrotum with the following diagnoses: testicular torsion, possible torsion-detorsion, torsion of appendix testes, epididymo-orchitis, and other. of us performed, boys had torsion confirmed by surgery. there were inconclusive us reports, none of which had torsion at surgery or follow-up. the false positive rate of us was . % ( patients), and there were no false negatives. six torsion patients had no us. median time from ed to us and surgery for torsion patients was and min. six patients had non-salvageable testes. diagnostic accuracy of us compared to surgery was % for torsion and % for other. sonographic heterogeneity was seen in % of patients with testes that the surgeon felt were non-viable at surgery and % of patients with viable testes (p . ). sudden-onset scrotal pain ( %), abnormal position ( %) and absent cremasteric reflex ( %,) were most prevalent in torsion patients. conclusions: color doppler us is accurate and sensitive for diagnosis of torsion in the setting of acute scrotum. despite heterogeneity on pre-operative us, many testes were felt to be salvageable at surgery. rate of salvage of torsion was high. common symptoms and findings of torsion were sudden onset of pain, abnormal testicular position and absent cremasteric reflex. paper #: pa- diagnostic twists of tubal torsion srikala narayanan, md, children's national medical center, snarayan@childrensnational.org; anjum n. bandarkar, dorothy bulas purpose or case report: fallopian tube torsion is a rare cause of acute pelvic pain in a young female and requires prompt diagnosis for immediate surgical intervention. our purpose is to review varied imaging findings of surgically proven cases of tubal torsion. methods & materials: retrospective review of our data base from to revealed cases of surgically proven fallopian tube torsion. ages ranged from to years of age. all had pelvic ultrasound performed, cases had additional ct performed for acute pelvic pain. results: us findings included thickened dilated tubular hypoechoic structure ( ), cystic mass ( ); adnexal ( ), midline ( ). five cases had normal ovaries bilaterally ( with paratubal cysts). ct imaging findings include dilated, fluid filled, thickwalled tube with internal hyperdensity ( hu) likely debris/ hemorrhage in case. additional findings included cystic adnexal mass ( cases), beak sign ( case) and increased vascularity ( case). secondary signs included free fluid ( ), peritubular fat stranding ( ), vascular congestion and thickening of the broad ligament ( ) and enlarged draining vein ( ). laparoscopic salphingectomy was performed in cases (including cases with isolated tubal torsion). laparoscopic detorsion was performed in a total of cases. in addition, laparoscopic cyst drainage was performed in out of these cases. detorsion with paratubal cystectomy and hemorrhagic ovarian cystectomy was performed in of the cases. conclusions: imaging diagnosis of tubal torsion can be difficult. it can occur in isolation with a dilated thickened tubular structure adjacent to a normal ovary or potentially mimic appendicitis, pyosalpinx, complex adnexal cyst or cystic adnexal neoplasm. presence of normal ovaries, beaked tapered tubular structure with intratubal fluid level and hemorrhage may help in making the diagnosis. it is important to recognize this entity in a patient with acute pelvic pain to facilitate prompt tubal sparing surgery. paper #: pa- adjusted renal length in pediatric bone marrow transplant recipients nicholas bodmer, md, university of washington, nbodmer@gmail.com; teresa chapman, sangeeta hingorani, marguerite parisi purpose or case report: bilateral nephromegaly has been observed in the bone marrow transplant (bmt) patients at our institution. this study aims to quantify this observation, thereby providing radiologists with an adjusted baseline agedetermined renal growth curve for bmt patients. methods & materials: a retrospective clinical chart and imaging review was performed on patients who underwent bmt between and and who had abdominal imaging including the kidneys. ultrasound, ct, and mri exams were used for renal length measurement. renal lengths were assessed for each age group, first as an average length of all the patients within that age group overall, and subsequently as an average renal length by age group divided into the following time frames after transplantation: - days, - days, - days, and + days. clinic chart information collected included bun, creatinine, weight, and medication use. results: renal length was measured using imaging cases, distributed across each age group as follows: - months, n ; months- . years, n ; . - . years, n ; . - . years, n ; . - . years, n ; . years and higher, n . renal lengths were greater, on average, within every age group, compared with previously established normative age-related renal lengths (rosenbaum et al.) . the augmented renal lengths universally were observed in the - day post-transplantation timeframe. return to normal renal lengths typically occurred by months post transplant. clinic chart review revealed that the majority ( %) of patients received nephrotoxic medication within two weeks of imaging. conclusions: pediatric bmt patients have larger kidneys in the absence of known renal disease than age-matched peers. a revised, age-based renal length chart for post-bmt patients has been generated which should help prevent the misdiagnosis of nephromegaly in this population, eliminating unnecessary diagnostic evaluations. multiple etiologies to explain renal enlargement in these patients are possible, including fluid overload, nephrotoxic medication, or direct effect of the transplant. purpose or case report: mr urography can be a comprehensive exam for anatomical and functional pediatric renal evaluation. quantification of renal function may benefit when dynamic contrast enhanced images can be obtained at high spatiotemporal resolution and with minimal respiratory motion artifacts. though respiratory triggering may decrease motion artifacts, it results in loss of temporal resolution by a factor of about three. a two-echo gradient echo sequence with segmented outer k-space sampling and view-sharing/dixon image reconstruction (disco, differential subsampling with cartesian ordering) was chosen as a starting point due to its high temporal resolution. it was then modified to enable respiratory triggering while maintaining temporal resolution of one temporal frame every one to two respirations, with segments of k-space only acquired in the expiratory phase of respiration. imaging parameters were: °flip angle, ± khz bandwidth, tr~ . , matrix x , fov - cm, slice thickness mm, and x spatial acceleration. with irb approval and informed patient consent consecutive patients referred for mri renal function evaluation were recruited (age range; . to . years, mean±sd: . ± . years; males % females %), and scanned on a ge t mr using a -channel torso array with the respiratory-triggered high spatiotemporal resolution technique to extract regional gfr maps. two readers by consensus assessed image qualitative snr, motion artifacts and volumetric fat-water suppression performance. results: data acquisition was obtained to completion in all subjects without triggering failure. temporal resolution was approximately s for two respiratory cycles. no case had major fat suppression failure, whereas minor fat suppression failure was seen in % ( % c.i. to %). all cases had diagnostically acceptable snr. no motion artifacts were noted in / cases, while some artifacts with ghosting in / cases. regional gfr maps could be successfully extracted for each patient without the need for image registration. attached figure shows image quality. conclusions: view-sharing offsets loss of temporal resolution from respiratory triggering. thus, high spatiotemporal resolution renal dynamic contrast enhanced respiratory triggered images can be obtained with minimal motion artifacts in a pediatric clinical setting to evaluate renal function. disclosure: dr. chowdhury has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. methods & materials: the study cohort was selected from the irb approved children's oncology group aren b study. cases are evaluated for pre-operative wt rupture based on central review of surgical/pathology findings. wt cases with rupture were matched to wt controls by age and tumor weight (within months and g). ct scans were independently reviewed by blinded radiologists, for presence/absence of rupture and the following ct signs: poorly circumscribed mass, perinephric fat stranding, peritumoral fat planes obscured, retroperitoneal fluid, ascites beyond cul-de-sac, peritoneal implants, ipsilateral pleural effusion, intratumor hemorrhage. sensitivity, specificity of ct for assessing pre-operative wt rupture was determined. the relationship between ct signs and rupture was assessed by mcnemar's test, and the most predictive ct signs determined by backward selection multivariate logistic regression. results: sensitivity, specificity for detecting wt rupture were: reviewer - . %, . %, reviewer - . %, . %. kappa coefficient for interobserver agreement was substantial: . (p< . ). all ct signs tested, except peritoneal implants and intratumoral hemorrhage, had significant association with tumor rupture (p< . ). for reviewer , ascites and fat stranding around tumor were most predictive (odds ratio . and . , p< . ). for reviewer , ascites and retroperitoneal fluid were most predictive (or . and . , p< . ). conclusions: ct has high specificity but relatively low sensitivity for detecting preoperative wt rupture. the presence of ascites beyond cul-de-sac is the best indicator of preoperative rupture, followed by fat stranding and retroperitoneal fluid. paper #: pa- the failed pyeloplasty: evaluation with mr urography damien grattan-smith, children's healthcare of atlanta, damien.grattansmith@mac.com; ricahrd jones, stephen little, wolfgang cerwinka, hal scherz, andrew kirsch purpose or case report: to identify imaging characteristics associated with failed pyeloplasty seen with mr urography. we have performed mr urography in children following pyeloplasty. from this group, children had follow-up surgical intervention with repeat pyeloplasty or balloon dilatation of the upj. imaging features reviewed included degree of hydronephrosis, calyceal transit times, renal transit times, signal intensity versus time curves, as well as functional analysis based on volumetric and patlak differential function and change in the asymmetry index. results: all children who underwent a second surgical procedure had delayed calyceal transit times. the degree of hydronephrosis and renal transit times were either stable or worse when compared to pre-operative evaluation. functional derangement could show stability, slight improvement or deterioration. the asymmetry index estimated the severity of the obstruction. conclusions: mr urography is valuable in the evaluation of children who have undergone pyeloplasty. the calyceal transit time appears to be the most reliable discriminator when comparing successful and failed pyeloplasty. calyceal transit times may be prolonged before the hydronephrosis becomes progressive. disclosure: dr. grattan-smith has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: initial attempts at interpreting functional mr urography (fmru) can be challenging. a time intensive navigation through a multitude of both subjective and objective functional results is necessary to render a useful interpretation. this is a guided review of fmru, noting the important functional findings in high-grade unilateral pelvicalyceal dilatation (pcd), in the absence of ureterectasis, with a contralateral normal kidney allowing for an optimal functional comparison. methods & materials: a retrospective functional evaluation of cases with unilateral pelvicalyceal dilatation (pcd), without prior pyeloplasty, was conducted. the fmru studies were carried out according to a standard protocol and post-processing using the chop-fmru software. this included iv hydration, bladder catheterization and iv furosemide administration. fifteen minutes after diuretic administration, a dynamic coronal d fat saturated t sequence was performed in a supine position over min. a sagittal d t and delayed single coronal t , both fat saturated, followed in a supine and/or prone position. the following functional features were evaluated: visualization of the ureter, the presence of a contrast-urine level and swirling of contrast in the dilated renal pelvis. the functional results included in the analysis were calyceal transit time (ctt), renal transit time (rtt), time-to-peak (ttp), parenchymal volume (pv), differential renal functions (volumetric-vdrf, patlak-pdrf and volumetric patlak-vpdrf) and the difference between vdrf and pdrf. results: patients were comprised of males and females with an age range of . - . years (median . yrs). of the kidneys with pcd, the ureter was visualized in , during the dynamic sequence, / during supine delay and / only in prone position. a contrast-urine level was present in of the dilated systems, and swirling in . the ureter was visualized during dynamic sequence in all contralateral normal kidneys and at no time was swirling or a contrast-urine level identified. the average functional parameters are seen in table . a statistically significant (p< . ) difference between the normal and dilated pelvicalyceal systems was achieved in ttp, pdrf and vpdrf for this small sample size. conclusions: awareness of multiple functional features and the range of calculated results may aid in subsequent combined interpretation of the fmru with the morphologic analysis. disclosure: dr. lecompte has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. and . demographics, clinical presentation , diagnostic studies and treatment outcomes were evaluated. post-procedure imaging was evaluated for clot burden reduction (patency) and residual venous stenosis by two-reader consensus. results: ten patients ( male; female, mean age years, range - ) presenting with acute upper extremity swelling and pre-procedure imaging revealing % occlusion of the axillary and subclavian veins received successful endovascular therapy. all patients underwent infusion catheter placement for thrombolysis with tissue plasminogen activator or urokinase. patients received additional pharmacomechanical treatment. angioplasty was also performed in all patients. the mean treatment duration was h (range - ). post-procedural imaging revealed that of patients achieved - % patency (clot burden reduction) and patient achieved - % patency. the residual venous stenosis was graded: patients had - % stenosis, patients had - % stenosis and patients had - % stenosis. all patients were discharged on full anticoagulation therapy with low molecular weight heparin. patients had surgical rib resection postthrombolysis with an average length of time from thrombolytic therapy to surgery being days (range - ). patients had re-thrombosis events during the follow-up period (mean months; range - ), with one re-thrombosis event occurring within one week of thrombolytic therapy, prior to surgery and the other two occurring - weeks post-rib resection. there were no procedure related complications. one patient was lost to follow-up after initial successful catheter directed therapy. conclusions: percutaneous endovascular techniques such as pharmacomechanical thrombolysis and angioplasty appear to be feasible and safe options for paget schroetter syndrome in otherwise healthy adolescent patients. in attempt to prevent rethrombosis and chronic symptoms, we refer all patients for adjunctive surgical decompression. future larger studies are needed to address optimal strategies for these patients. combined d fluoroscopy image guided percutaneous intervention with real-time optical sensing at the tip of a needle for tissue characterization rami nachabe, philips, rami.nachabe@philips.com; john m. racadio, drazenko babic, ross schierling, jasmine hales, benno hendriks purpose or case report: to investigate the feasibility and potential of real-time tissue characterization at the tip of a needle with diffuse optical spectroscopy (dos) sensing capabilities during d fluoroscopy guidance using cone beam ct and dedicated needle path planning software. methods & materials: a c-arm x-ray system that combines fluoroscopy and d imaging from a cone beam ct was used to image a woodchuck with hepatocellular carcinoma (hcc). the imaging system enabled needle path planning, which was used to perform insertion and navigation of a needle toward the liver tumor. the needle was integrated with optical fibers for real-time tissue spectral sensing at its tip. optical spectra measurements were obtained continuously as the needle passed through healthy liver tissue and then into the tumor. from the diffuse optical spectra measurements, the following clinical parameters were extracted for tissue characterization: blood volume fraction, blood oxygenation, lipid volume fraction and tissue light scattering (related to tissue density). the tissue parameters were compared for healthy liver and tumor using the kruskal-wallis test. results: the tissue density of the healthy liver was lower than that of the tumor. higher blood and lipid volume fractions as well as oxygenation levels were observed in the healthy liver as compared to the tumor. all differences were statistically significant (p< . ). additionally, a much wider heterogeneity in tissue density was observed in the tumor as opposed to the healthy liver. conclusions: differences in tissue properties between tumor and healthy liver enable discrimination between these two types of tissues. adding real-time optical sensing at the tip of a needle to d fluoroscopy image guidance is a feasible technique that complements the imaging information with relevant physiological parameters; it facilitates more precise definition of tumor boundaries despite any target motion during needle insertion. disclosure: dr. racadio has disclosed that he is a consultant for philips healthcare and receives travel reimbursement. rami nachabe, drazenko babic and benno hendriks are employees of philips healthcare. methods & materials: two children aged months and months were treated at this institution for liver failure resulting from urea cycle disorders, with a hepatocyte transplant procedure. the recipient liver was irradiated prior to transplant to facilitate engraftment. the procedure involves the injection of prepared hepatocytes from a suitably screened, compatible donor, via a main portal vein branch into the recipient liver. in both procedures access to the umbilical vein was achieved by the surgery service and a french arterial sheath was placed. a french angled catheter was used for diagnostic runs and to access the right and left main portal vein. a french fogarty catheter (edwards lifesciences, irvine, ca) was placed to isolate each portal vein branch in turn and hepatocytes injected using hand injections. pressures in the main, right and left portal veins were measured and hand injections of contrast made at regular intervals. careful attention must be paid for evidence of pruning of portal branches, indicating occlusion of small portal branches, or portal to hepatic vein shunting. if shunting is seen, infusion must be stopped as embolism of hepatocytes into pulmonary arteries may result with serious clinical sequelae results: in both patients, the desired number of hepatocytes were successfully delivered into the recipient liver. in both cases, mild pruning of the portal vein branches was evident at the end of the procedure. portal vein presssures remained steady. there was no venographic or clinical evidence of pulmonary arterial embolization. conclusions: the interventional radiologist plays a central role in the hepatocyte transplant procedure. familiarity with catheterizing portal branches from an umbilical vein approach, measuring venous pressures, using small occlusion catheters and recognizing venographic end points such as portal vein pruning and portal to hepatic vein shunting are necessary to the safe and successful completion of this new technique. purpose or case report: the aim of the study was to evaluate the trends in term of type of tube placed, number of procedures per year, number and age of the patients as well as the number of procedures per patient and the interval of time between two placements, and finally the irradiation burden borne by the patients. methods & materials: after reb approval the radiologic files of the patients who underwent naso-duodenal-jejunal (ndj) or gastro-jejunal (gj) or jejunal (j) tube placement under fluoroscopy over the past five years ( to ) were extracted from the ris and reviewed. the results were tabulated as a single batch and stratified by year. results: eighty-nine patients representing procedures ( ndj, , gj, j) were included. only patients underwent a single procedure. the average number of procedures per patient was . with a maximum of during the study period. the average patient's age was . months (sd . , median . ). the average fluoro time per procedure was . min (sd . , median . ). the average interval between two procedures was days (sd , , median ). the average fluoroscopy time per patient combining those having a single procedure and those having multiple ones, was . min (range . to . , sd . , median . ). conclusions: fluoroscopic placement of enteric tubes delivers a significant amount of irradiation. our data led to two interventions with respect to insertion and management of the tubes. on one hand, when the attempt pursued by a radiologist is not successful after min of fluoroscopy other strategies should be considered including another operator or an alternative technique for tube positioning. on the other hand, information will be distributed toward the clinicians and nurses in order to improve the management of these tubes and avoid fortuitous displacement which was responsible of a significant amount of repeated procedures leading to undue irradiation. purpose or case report: to evaluate drug elution pharmacodynamics of doxycycline in an albumin-based solution, as used in percutaneous imaging-directed therapy of aneurysmal bone cyst (abc) and microcystic lymphatic malformation (lm) methods & materials: doxycycline mixed with % human serum albumin (hsa), and doxycycline mixed with saline solution (both mg/ml) were evaluated using a fluid diffusion chamber system over h, recording ph and doxycycline concentration. static ph and doxycycline concentrations were recorded every min for the first min, then every min for a total of h, averaged over trials in each of the hsa and saline systems. statistical analysis evaluated standard deviation and rate of change for the trials in each system. drug elution dynamics data were correlated with clinical experience in the doxycycline/albumin treatment of patients ( treatments) with aneurysmal bone cyst (abc) and patients with lymphatic malformation microcysts. results: drug elution was linear in both the hsa and saline systems, with statistically significant (p<. ) slower elution drug release from the albumin system as compared with the doxycycline and saline solution, both over and h. purpose or case report: to describe a successful interventional radiologic approach to the management of paget schroetter syndrome presenting as acute arm swelling in adolecent athletes. methods & materials: institutional review board approval was obtained for this retrospective study. five patients aged to years (mean . years) were treated at this institution over a year period all presenting with acute arm swelling (july -july ). ultrasound confirmed subclavian vein thrombosis in all cases. all were treated with placement of an infusion catheter (ev , plymouth, mn), infusion of tissue plasminogen activator (tpa) at a rate of mg/hour overnight and aspiration of remaining clot with a "trellis" (bacchus vascular, santa clara, ca, usa) thrombectomy device. results: clot was successfully removed in all five patients. complete clearance of clot was confirmed by contrarst venography in all cases. in four patients balloon angioplasty of a narrrowing at the junction of the subclavian and brachiocephalic veins was carried out. in one, the thrombus recurred within h. the patient was retreated the next day with aspiration of clot using the "trellis" device and an infusion catheter placed with low dose ( . mgs/hour) tpa commenced until surgical review; this patient was operated on within h of final thrombolysis. all patients were seen by a vascular surgeon with an interest in this condition. all underwent surgical decompression; at end of the study period all patients were asymptomatic. conclusions: interventional radiologic management of acute axillo-subclavian thrombosis due to paget schroetter syndrome is safe and highly successful in the adolescent population. early recurrence of thrombus is not uncommon and prompt surgical consultation with a view to early surgical decompression is recommended. purpose or case report: diagnostic reference levels (drl) or target radiation dose ranges for pediatric ct scans are needed in the u.s. the first u.s. pediatric ct dose index registry (quircc) within the american college of radiology recorded estimates of patient radiation dose using a new method (ssde) based on body width(bw) for the purpose of developing diagnostic reference levels (drl). in addition to developing drl at the th percentile, the purpose of this study was to determine the ssdes associated with the lower range of acceptable image quality through subjective image quality evaluation. methods & materials: six children's hospitals participated in a retrospective review of abdominal ct with iv contrast on patients < yrs of age. from exams, each site submitted de-identified images for selected cases based on ssde and patient width. a total of cases were selected from the lowest, first quartile and median ssde. six investigators reviewed images from each case under identical viewing conditions and rated them for subjective quality according to a score sheet and reference scale of images with known quantum mottle. cases were considered non-diagnostic if at least of reviewers ranked them as such. results: first, second, and third quartile ssde and ctdi-vol values from sites for each bw will be shown. / cases were ranked non-diagnostic by the reviewers. / non-diagnostic cases were below the th percentile based on ssde. / of "non-diagnostic" cases had ssde less than the th percentile. the unacceptable case with ssde above the th percentile ( cm, ssde . mgy) was due to subcutaneous metal implant with artifact. the quircc th percentile using ctdivol for a yr old is . mgy which is % lower than the acr ct accreditation data's published th percentile. conclusions: this consortium developed target dose ranges (drls) for ct of the abdomen with iv contrast for routine exam indications based on evaluation of image quality that establish lower and upper ranges ( - percentile) of patient dose(using ssde) associated with clinically acceptable images. this study demonstrates that pediatric radiologists in this consortium are comfortable interpreting images at or above the percentile ssde and judged all but one image within this target range as diagnostically acceptable. table ). with the exception of neonate chest, most used age-based techniques; only two centers reported using thickness. no survey used grids for wrist images, while / of the surveys used grids for chest and abdomen exams in -year-olds. at the most common sid there was up to a kvp variation ( year-old chest ap) and up to -fold variation in mas ( year old scoli lat). only two surveys used equipment that displayed the new iec exposure index. conclusions: participants report variability in the techniques and methods used to acquire common radiographic studies, reflecting differences between detector types and users. radiologists, technologists, medical physicists, manufacturers, and the fda have an opportunity to work together to standardize the techniques based on detector type to optimize radiation exposure for pediatric radiographic exams. disclosure: dr. don has indicated that he performs contract research for carestream and that he is on the speaker's bureau for siemens and receives an honoraria. purpose or case report: this study assesses community adoption of ct radiation dose guidelines after a -year international initiative to reduce medical radiation exposure in children. size-specific dose estimates (ssde) from community pediatric body ct scans are compared to ssde from matched scans obtained at a children's hospital that adheres to image gently campaign principles. we reviewed pediatric ct scans ( chest (c), abdomen/pelvis (ap), chest/abdomen/ pelvis (cap)) transferred from community imaging centers to our university children's hospital between july and february . community scans were acquired with variable parameters and reconstructed with traditional filtered back projection (fbp). comparison was made to children's hospital ct scans, performed in accordance with principles of the image gently campaign. because iterative reconstruction (ir) software was added to our scanner during the study, enabling us to reduce ctdivol by %, children's hospital scans were divided into two groups: a) scans obtained with standard weightbased pediatric protocols and fbp (october -october ; c, ap, cap) and b) scans obtained with reduced-dose weight-based pediatric protocols and blended ir/ fbp (october -april ; c, ap, cap). ctdivol and greatest lateral dimension were recorded from each scan and were used to calculate ssde. mean ssde from community scans was compared to mean ssde from children's hospital groups a and b. statistical analysis was performed with student's t-test. results: patient age range was - years in both community and children's hospital groups. mean ssde for community c, ap, and cap scans was . , . , and . times higher than mean ssde for matched scans in control group a (p< . ) and . , . , and . times higher than mean ssde for matched scans in control group b (p< . ). conclusions: ssde was significantly higher for community pediatric body ct scans than for matched scans performed at a children's hospital that adheres to image gently campaign principles. results suggest that more community outreach and education are required in implementation of low-dose ct protocols outside of children's hospitals. concurrent use of ir provides a means of achieving even greater ssde reduction than is possible with fbp alone and should be encouraged. paper #: pa- optimization of tube voltage and current in size-based pediatric ct imaging: a phantom study boaz karmazyn, md, radiology, riley hospital for children, bkarmazy@iupui.edu; yun liang, keith kaser, peter johnson, mervyn cohen purpose or case report: determine the change in ct dose index (ctdivol) required to maintain the same quantum mottle noise when using lower tube voltages ( and kvp) relative to kvp in different sized cylinder water phantoms (cwp) representing a wide range of pediatric body sizes. we performed mdct scans of , , , and cm cwp. thirty scans were performed for each phantom. the tube currents ranged from to mas with increments of mas, and the tube voltage levels were , , and kvp. the noise (standard deviation in hu) was measured using center region of interest (roi) that was % of phantom's area. two other rois (each % of the area) were placed at the center and periphery of the phantom images to measure noise gradient. results: in the smallest ( cm) cwp, approximately the same noise level was maintained with all three tube voltages without a significant change in ctdivol. for the , , and cm phantoms, the average ctdivol needed to be increased by %, %, and %, respectively, to maintain same noise level when the voltage was decreased from to kvp. the average ctdivol needed to be increased by %, % and % to maintain the same noise level in the , , and cm cwp when the tube voltage was decreased from to kvp. the difference between central and peripheral noise increased on average by . %, . %, . %, and . % in the cwp of , , , and cm, respectively. in each cwp, the central to peripheral noise difference was more pronounced (up to . % more) with decrease in kvp from to or . conclusions: noise measurements in the water phantom model indicate that tube voltage could be decreased from to in cwp of cm without significant change in ctdivol. it is also possible to decrease the voltage from to kvp with a minimal (< average %) increase in dose in cwps of , , and cm. the noise gradient increases with larger cwp and smaller kvp. paper #: pa- comparison of radiation dose estimates, image noise, and scan duration in pediatric body imaging using -row and -row ct jennifer johnston, md, radiology, cincinnati children's hospital medical center, jhtai@yahoo.com; daniel j. podberesky, erin angels, terry t. yoshizumi, greta toncheva, donald p. frush purpose or case report: to compare effective dose (ed) estimates, image noise, and scan duration for pediatric chest, abdomen and pelvis protocols using -row and -row ct scanners in various acquisition modes. methods & materials: organ doses were measured using mosfet dosimeters. dose, scan duration, and noise measurements were made in a -year-old anthropomorphic phantom for conventional helical, -detector helical and volume acquisition modes for chest, abdomen and pelvis protocols on a -row ct, and for helical mode on a row ct (aquilion one and aquilion , toshiba medical systems, otawara, japan) using similar scan parameters representing currently used clinical protocols. mean organ doses from three runs for each protocol, in combination with icrp tissue weighting factors, were used to obtain ed for each protocol. noise was measured as the standard deviation of hounsfield units in equivalent locations at levels for each protocol with an roi tool. ed and noise were compared with a paired t-test or sign test. results: compared to helical acquisitions on the -row ct, ed of all tested acquisition modes on the -row volume ct were significantly lower for chest, abdomen/pelvis (ap) and chest/abdomen/pelvis (cap) protocols (table) . scan durations were lower across the board on the -row volume ct. compared to acquisitions on the -row ct, noise was in general similar to those on -row ct protocols, but some acquisition protocols on the -row ct produced greater noise (table) , specifically volume acquisition for chest ct and -detector helical and volume modes for ap and cap protocols. conclusions: dose savings can be achieved for chest, ap and cap ct examinations on a -row ct scanner compared to helical acquisition on a -row ct, with shorter scan durations. image noise was in general comparable between protocols. although noise differences between some modes did reach statistical significance, the impact on overall image quality will need to be studied further. paper #: pa- the observed to expected total fetal lung volume as a predictor of short-and long-term morbidity in surviving infants with congenital diaphragmatic hernia emily stenhouse, the royal hospital for sick children, emilysten@doctors.org.uk; neil patel, judith simpson, watt andrew, gregor walker, carl davis purpose or case report: observed-to-expected total fetal lung volume (o:e tflv) is a validated mr measure which we have previously demonstrated to be significantly reduced in non-surviving infants with congenital diaphragmatic hernia (cdh). our aim was to investigate the relationship between o:e tflv and short-and long-term morbidity outcomes in surviving infants with cdh. methods & materials: a retrospective analysis of cases of isolated left-side cdh referred to our institution for fetal mr evaluation between - weeks. mr imaging studies were performed on a . t philips system using a phased array body coil. the observed tflv was calculated by multiplying the summed area of the region of interest by the section thickness. the expected tflv was calculated with a formula previously described in the literature using the gestational age of the fetus. the observed tflv was expressed as a percentage of the expected tflv at a given gestation. morbidity outcome data was obtained from the case records of all surviving infants. specific measures of illness severity relating to short-term intensive care management and long-term outpatient management were recorded. differences in o:e tflv between outcome groups were assessed by t-test. results: liveborn infants with isolated left-side cdh and antenatal mr scans were identified. scans were performed at - weeks gestation. infants survived to discharge; gestation . ( . - ) weeks, birth weight . ( . - . ) kg. median length of admission was ( - ) days, median duration of follow-up was . ( . - . ) years. o:e tflv was significantly lower in non-surviving infants; vs. %, p . . o:e tflv was significantly lower in infants who received high frequency oscillation ventilation (hfov) versus those who were conventionally ventilated ( % vs %, p . ). o:e tflv was also significantly lower in those infants who had a length of admission greater than the median of days ( % vs. %, p . ). o:e tflv trended lower with other measures of increased morbidity; inhaled nitric oxide use, patch repair of diaphragm, rehospitalisation within year, supplemental feeding at discharge, gastro-oesophageal reflux, and developmental delay. conclusions: as well as predicting survival, lung volume measurement by o:e tflv is a promising predictor of outcome and morbidity in surviving infants with cdh. further studies in larger populations are required to provide quantitative predictive risk data. characterization of the inherent acoustic noise of a dedicated nicu mri system jean tkach, phd, cincinnati children's hospital medical center, jean.tkach@cchmc.org; yu li, ron g. pratt, christopher villa, beth m. kline-fath, charles dumoulin purpose or case report: we have developed a small foot print . t mri scanner specifically for neonatal imaging that can be easily installed in a neonatal intensive care unit (nicu). the scanner has a maximum patient bore diameter of . cm (without rf coil), and roughly twice the gradient performance of the best conventional adult whole-body . t mr systems. it is known that sensory stimulation such as acoustic noise can elicit autonomic instability in both term and preterm neonates. the inherent noise properties of the nicu mri system were measured as part of the initial safety evaluation of the system and compared against that of a conventional . t mri system. to evaluate the inherent acoustic noise characteristics of the nicu mri scanner, sound pressure level (spl) measurements were performed on it and on a conventional adult sized whole body . t hdx ge mri system (ge healthcare, waukesha, wi). a brüel & kjaer model sound level meter (brüel & kjaer sound & vibration measurement a/s, denmark) was used to perform the spl measurements for several different mr acquisitions (spin echo, gradient echo, fast rf spoiled gradient echo, fully balanced steady state free precession, gradient echo echo planar, and diffusion weighted) using acquisition parameters consistent with clinical protocols. the mr sequences, acquisition parameters, noise measurement equipment and methodology were identical for the two mr systems. the maximum spl in units of a weighted decibels (dba) was recorded for each of the mr acquisition/mr system combinations evaluated. results: the maximum spl values measured during each of the mr acquisitions were lower for all sequences (average . dba (range - dba)) for the nicu mri unit as compared to the conventional mri scanner ( table ). the average measured maximum spl value, reported in dba, across all acquisitions was . ± . for the nicu scanner, and . ± . for the conventional mri scanner. the highest spl values were measured for the diffusion-weighted sequence: and dba, for the nicu and conventional mri scanner respectively. conclusions: because of the smaller dimensions of the gradient coils in the nicu mri system, acoustic noise is less than that of conventional mri scanners despite the superior gradient performance of the smaller coils. the lower inherent acoustic noise level of the nicu system provides improved safety for the neonate, and facilitates siting of the unit in the nicu. disclosure: dr. tkach has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. paper #: pa- late neurologic events in extremely premature infants carlos guevara, md, radiology, duke university, cjg @duke.edu; brett bartz, caroline l. hollingsworth, caroline w. carrico, michael c. cotten, charles m. maxfield purpose or case report: germinal matrix hemorrhage (gmh) is a major complication of prematurity. persistence of germinal matrix and immature neurovascular autonomic regulation in the premature neonate is thought to predispose to gmh. most gmh in premature population occurs during the first days of life, and yet the persistence of the germinal matrix to weeks gestation may allow for post-natal gmh outside of the immediate perinatal period. to our knowledge, this is the first systematic review of late gmh (after the first week of life) in a large population of extremely preterm neonates (less than weeks of gestation). this irb approved retrospective review included patients weighing less than g or born at less than weeks of gestation from through . the study population included infants who had a head ultrasound (hus) within the first week of life and at least one follow hus after the first week of life. all hus were reviewed by three experienced pediatric radiologists for the presence and grade of ich or late developing hemorrhagelike lesions (hll). infants with and without hll were evaluated for several clinical variables, including neurodevelopmental outcomes (bayley scales). results: average gestational age of study population was . weeks. the incidence of gmh in the first week of life was % grade , . % grade , . % grade / , and . % posterior fossa. new echogenic foci (hll) at the caudothalamic groove were seen in . % after the first week of life. % of these lesions were bilateral. a four-fold increase in incidence of hll was seen in infants < g compared to those> g. higher grade hemorrhages were not seen in this patient population, although % of infants had late posterior fossa hemorrhages. the clinical course of infants with hll trended towards a higher incidence of stressors, but this was not statistically significant. the psychomotor development index scores were lower than those infants without hemorrhage. conclusions: small hll at the caudothalamic groove are common in extremely preterm infants after the first week of life. higher grade ( - ) hemorrhages were not seen. there were no cases of intraventricular extension and no direct complications. if isolated, this finding necessitates no follow-up imaging, but may be associated with poor neurodevelopmental outcome. disclosure: dr. guevara has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: tof/apv is a rare congenital heart lesion in which pulmonary arteries may become aneurysmally dilated and compress adjacent airways. pulmonary arterioplasty is often required to relieve tracheobronchial compression in addition to intracardiac repair. the purpose of this study was to review pre and postnatal imaging findings and their impact on patient management and clinical course. methods & materials: a retrospective database search identified infants with tof/apv between - ( fetal diagnosed cases and diagnosed postnatally). for fdc, prenatal ultrasound (us) and fetal mri were correlated with postnatal ct for the size of the central pulmonary arteries, airway compression, and presence / distribution of air trapping/atelectasis. for all cases postnatal ct findings (between - days of age) were correlated with clinical management and outcome. results: prenatal diagnosis of tof/apv was suggested sonographically, based on dilated central pas, between - weeks gestational age (ga). fetal mri, performed between - weeks ga confirmed the diagnosis and aneurysmal central pas and demonstrated air trapping &/or atelectasis in / with normal appearing lungs in fetus. size of the pas ( / ) and presence and distribution of lung abnormality ( / ) correlated closely between fetal mri and postnatal ct, although detailed visualization of the central airway/ vascular relationships were better defined on ct. fetal mri identified an unexpected diaphragmatic hernia (dh) not seen on us. for the pnd cases, ct showed aneurysmal pas and airway compression with air trapping &/or atelectasis in / infants. seven infants with airway obstruction on ct required pulmonary arterioplasty; infant with no air trapping did not have arterioplasty. / operative patients survived, one with concomitant dh died at age days due to hemorrhagic shock. one fdc was inoperable due to poor cardiac function and died at age days. conclusions: prenatal mri correlates well with postnatal ct for assessing pulmonary artery size and location and severity of lung abnormality in patients with tof/apv, this allows for appropriate management planning and may negate the need for an immediate postnatal ct. ct accurately depicts the location and extent of airway compression and resultant air trapping or atelectasis, serving to guide the need for and extent of the arterioplasty procedure. paper #: pa- craniosynostosis syndromes: prenatal findings by us and mri eva rubio, md, cnmc, rubioeva@yahoo.com; anna blask, alexia egloff, dorothy bulas purpose or case report: craniosynostosis with associated malformations is a feature of several related syndromes resulting from a fgfr or twist genetic mutation. syndromes include apert, crouzon, pfeiffer, and carpenter syndromes. our purpose was to review imaging findings which aid in suggesting the diagnosis prenatally. we retrospectively reviewed prenatal us and mri findings in cases with prenatal ( with postnatal/molecular) diagnosis of a craniosynostosis syndrome: cases of apert, case of carpenter, and cases of pfeiffer syndrome. results: / cases were correctly diagnosed prenatally. in the second trimester findings may be subtle, with mild calvarial changes; digit abnormalities, in particular, may elude the imager in unsuspected cases. although the diagnosis could be made with either modality, the full spectrum of abnormalities was best appreciated using a combined imaging approach of mri and us. by us many salient features were depicted: turribrachycephaly/trigonocephaly/cloverleaf ( / ); syndactyly ( / ); polydactyly ( / ). agenesis of the corpus callosum was identified by us in ( / ) cases. conversely, mri, performed in all cases, contributed additional observations not well seen by us: the fetal airway was well delineated in all cases ( / ); a low lying spinal cord was noted ( / ), midface hypoplasia ( / ) and migrational/sulcation abnormality ( / ). additional findings of absent ductus venosus with biliary atresia ( / ), abdominal wall defect ( / ) and renal anomalies ( / ) were seen with both modalities. reimaging in later pregnancy depicted important changes ( / ), including worsening hydrocephalus and resolution of suspected airway occlusion. conclusions: us and mri are complementary modalities in evaluating fetuses with craniosynostosis. airway patency, midface hypoplasia, spinal cord abnormalities and intracranial abnormalities are often better seen with mri. fetal activity, digits, bone detail, and cardiac anomalies are better appreciated by us. findings may be subtle in the second trimester. repeat imaging in later pregnancy may reveal specific information affecting delivery planning. paper #: pa- pcpra best paper hyperpolarized carbon- mrsi for pediatric disease john mackenzie, md, department of radiology and biomedical imaging, ucsf, john.mackenzie@ucsf.edu; yi-fen yen, linda nguyen, jeffrey gu, john kurhanewicz purpose or case report: to study the potential of carbon- mr spectroscopic imaging ( c-mrsi)-a radiation free molecular imaging strategy-for the detection and treatment monitoring of pediatric disease. methods & materials: the potential of c-mrsi to detect pediatric disease was tested in rodent models of pediatric arthritis. animals were induced with arthritis and subsequently given intravenous hyperpolarized c-pyruvate, and imaged. the amount of c-lactate produced from pyruvate in normal and arthritic joints was measured both at single points in time and dynamically at either or tesla. the c-mrsi data were compared with clinical measures of arthritis, cell stimulation studies, and joint changes on conventional anatomic mri and histology. results: alterations in lactate production as measured by c-mrsi appear to depict sites of arthritis and correlate with other more established but potentially less reliable or more invasive measures of disease status. imaging robust mouse models of pediatric disease may be feasible at telsa. this method may also be translated from high-field to clinical equipment with reasonable hardware and software modifications that allow detection of hyperpolarized c compounds. c-mrsi depicts increased lactate production at specific regions of inflammation within arthritic joints and is confirmed by histological inspection and anatomic mri. on average, lactate production is increased by % in areas affected by inflammation. conclusions: the intravenous injection of hyperpolarized carbon- compounds and subsequent imaging with c-mrsi provides a unique molecular imaging strategy to noninvasively monitor pediatric disease. this non-invasive imaging strategy may eventually provide clinical utility for several pediatric diseases involving inflammation, infection and tumor. disclosure: dr. mackenzie has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. methods & materials: using the hemangioma-vascular malformation clinic registry at cincinnati children's hospital, we searched for patients diagnosed with khe whose evaluation included mri. twenty such patients were found, although three of the patients had no pre-therapy mris. the imaging studies were reviewed by the authors with assessment of the following characteristics: location, margin definition, soft tissue involvement, and pre and post contrast signal intensity. results: location: lesion location was as follows: trunk ( ), extremity ( ), extremity plus trunk ( ), and head/neck ( ). signal: all lesions were dark on t weighted sequences with diffuse enhancement after contrast administration. the majority of the lesions were bright on t weighted sequences, but there were cases that had heterogenous to low t signal (with all involving the retroperitoneum). of the cases, only one had both high arterial and venous flow by mri. margin definition: four of the lesions had well defined borders (greater than % well circumscribed) with minimal to no adjacent infiltration/edema. two of those four cases were exophytic masses. the remaining cases were poorly defined lesions with adjacent infiltrative fluid signal intensity and enhancement. tissue/organ involvement: tissue/organ involvement was counted if abnormal fluid-signal intensity or enhancement was identified at that site. review of these cases showed fifteen patients with muscular involvement. dermal and subcutaneous involvement was observed in all but cases, with the uninvolved lesions being isolated and deep. additional sites of suspected involvement included bone ( ), pleura ( ), penis ( ), and pancreas ( ). conclusions: khe is a rare neoplasm of infancy with a spectrum of features by mri. poorly defined lesions are much more frequent than well-circumscribed masses. however, pathologic correlation of such infiltrative margins is usually not available as treatments after biopsy are primarily medical rather than surgical. common additional mri features include predominant involvement of muscle, subcutaneous fat, and skin over viscera and bone with lesions generally showing increased t signal and enhancement. is dedicated chest ct needed in addition to pet ct for evaluation of pediatric oncology patients? ibrahim tuna, montefiore medical center, dristuna@yahoo. com; jeffrey levsky, jeremy rosenblum, rosanna ricafort, benjamin taragin purpose or case report: to evaluate the diagnostic accuracy of low dose ct performed during pet-ct as compared to dedicated chest ct in the assessment of pulmonary findings in children with malignancy. the institutional review board approved this hipaa compliant research. pediatric oncology patients, ages between - , with known solid malignant tumors who were referred to pet-ct and standard chest ct within days for staging or assessment of treatment response between - and - were eligible for this retrospective study. radiology reports were reviewed for potential discrepancies. two radiologists re-evaluated the standard chest ct and low dose chest ct portion of the pet ct of the discordant cases, while comparing with the most recent prior studies. studies were scored for pulmonary nodules, bony metastasis, adenopathy, and pleural effusions. true discrepancies were assessed by a panel of pediatric oncologists to judge whether the differences in reports might lead to a significant change in management. results: ( female, male) patients were identified. radiologic reports of different patients ( female, male) had potential discrepancies based on review of the reports. the primary tumors were rhabdomyosarcoma (n ), hodgkin's lymphoma (n ) and others (n ). reevaluation of the original images showed true discrepancies in . % ( / total ). in studies, the discrepancy had no clinical significance. in studies, a pulmonary nodule was identified on standard chest ct which was not described on the pet-ct. both of these patients had rhabdomyosarcoma. one of these patients had findings that pediatric oncologists considered significant enough to alter patient management. conclusions: we found a low false negative rate for clinically significant findings on the low dose portion of pet-ct as compared to standard chest ct. in the future, improvements in acquisition technique and post processing of the ct portion of the pet-ct may further improve its diagnostic utility, obviating the need for a routine separate diagnostic ct, thereby minimizing radiation exposure in these young patients. methods & materials: low-dose cta examinations were performed in pediatric patients over a three year period to evaluate suspected vascular traumatic injury with some patients receiving scans of more than one area of the body. areas scanned in this include the head and/or neck (n ), chest (n ), abdomen and/or pelvis (n ), upper extremity (n ) and lower extremity (n ). in of these patients, suspected vascular injury was due to a history of either blunt (n ) or penetrating (n ) trauma. patients were referred directly from the emergency department, while were inpatients and the remaining were referred from an outpatient setting. patients ( f: m) ranged in age from to years old (mean age ). studies were performed on a -channel mdct scanner with or kv, to mas, . to . mm section thickness, reconstructed with % overlap, and . to . pitch. contrast medium was power-injected using weight-based protocols to optimize iodine delivery. exams were interpreted on a workstation using advanced imaging techniques. patient radiation dose was calculated in all cases. clinical outcome was assessed through a month follow-up when possible. results: all studies were technically adequate. . % (n ) of studies revealed no vascular injury, while . % (n ) revealed acute vascular pathology. vascular injuries included vascular occlusion (n ), vasospasm (n ), narrowing/dissection (n ), pseudoaneurysm (n ), and transection (n ). extravascular traumatic findings were demonstrated in . % (n ), including fractures, lung injury, soft tissue hematomas, and a ruptured baker's cyst. of the patients with acute vascular findings, . % (n ) underwent surgical management (including for vascular injury), while . % (n ) were managed conservatively. one patient with active extravasation was managed with angiographically-guided embolization. in no case was catheter angiography required to confirm cta findings. conclusions: low dose cta is a reliable means to screen pediatric patients emergently for acute vascular injury. vascular and non-vascular pathology can be diagnosed noninvasively for efficient patient management. paper #: pa- elasticity measurement by acoustic radiation force impulse (arfi) technique of normal liver, kidney and spleen in healthy children mi-jung lee, radiology, severance children's hospital, mjl @yumc.yonsei.ac.kr; myung-joon kim purpose or case report: there are many previous studies about using acoustic radiation foce impulse (arfi) value to measure the elasticity of tissue, mainly the liver in adult patients. however, there was limited study about arfi measurement in the children. the purpose of this study is to evaluate the arfi value in the normal liver, kidney and spleen in healthy children and to evaluate the effect of sex, age, and body mass index (bmi). the study prospectively enrolled healthy pediatric volunteers who are under years old, and underwent abdominal ultrasonography and arfi between july and august . arfi velocity measuring was performed by - mhz linear probe for children under years old and - mhz convex probe for older children. arfi velocity was measured three times at each organ. however this measurement was stopped if the child cannot tolerate. results: two hundred two children (m:f : ; mean age, ± . years) were enrolled. and arfi measurement was performed only two time for some organs in three children. the mean arfi value was . ± . m/s in liver, . ± . m/s in right kidney, . ± . m/s in left kidney, and . ± . m/s in spleen. arfi velocity was not different between boys and girls. however, arfi velocity was different between right and left kidneys (p . ). the arfi value of right kidney, left kidney and spleen was correlated with age, height, weight and bmi (p< . ). however, the arfi value of liver was not correlated with these parameters. conclusions: arfi measurement is feasible in children with only three times acquisition for each abdominal organ. the mean arfi velocity was increased according to the age, height, weight and bmi in kidney and spleen, but it was constant in liver. disclosure: dr. lee has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: diagnostic image quality can be achieved over a wide range of radiation exposure in digital radiography. "exposure factor creep" or "dose creep'" in which technologists tend to increase dose to avoid the appearance of noise has been well described. using the alara principle, acceptable images can be achieved while minimizing dose. at our institution "dose creep" has been observed in bedside pediatric chest radiography. to address this we coupled a data mining tool with a continuous quality improvement (cqi) initiative which educates individual technologists on appropriate technique. methods & materials: radiation dose in digital radiography is estimated from an exposure index, a proprietary format that varies among manufacturers. our institution uses a fuji computed radiography system which calculates an s, or sensitivity value, that provides an approximation of the radiation dose to the imaging plate, using an inverse scale. overexposed bedside chest radiographs were defined by a s value less than . a data-mining program was developed to extract from the dicom header the s value and other relevant information, on a monthly basis. these data were used to provide training and feedback on a one-on-one-basis. results: with ad hoc feedback and group training initiatives prior to implementation of this new system, approximately . % ( / ) of bedside chest radiographs were overexposed over a four month period. after one-on-one intervention with the technologists, preliminary findings reveal a trend towards fewer overexposed radiographs with approximately . % ( / ) with s< . conclusions: our tool provides a simple method for systematically identifying overexposed radiographs and the corresponding responsible technologists. we anticipate that this personalized educational program will continue to reduce the proportion of overexposed radiographs and thus the radiation dose to our pediatric patients. purpose or case report: ensuring radiation protection for children undergoing ct scans is challenging due to rapidly changing technology, differences in ct equipment and potential lack of understanding of unique aspects of scanning children. the joint commission has named technologists' training as an "action" item. we developed online training modules to fill potential gaps in ct technologists' education. methods & materials: four modules were created by pediatric radiologists, radiologic technologists and medical physicists; were developed by education/training experts from major ct vendors (ge, philips, toshiba, siemens) through the medical imaging technology alliance. modules were created as microsoft word documents containing de-identified images and edited by education specialists at the american society of radiologic technologists and the alliance for radiation safety in pediatric imaging. they were converted to audio/video format using question/answer narration. vendor modules were created in microsoft powerpoint format and edited. all modules were converted into adobe captivate learning program to achieve uniformity of appearance. modules are hosted on the asrt server and linked to the image gently website. a certificate may be printed as documentation of completion. results: all modules are available at www.imagegently.org. two introductory modules discuss basics of ct equipment and medical physics related to radiation dose in children. the third and fourth modules discuss dose-saving strategies for neu-roct and body ct. four vendor-produced modules address unique aspects of equipment design such as automatic exposure control and dose saving strategies for children. conclusions: through collaborative efforts with medical imaging professionals and vendors, we have developed free online modules addressing radiation protection for children. ct technologist training in specific dose saving strategies for children is variable and limited. these modules have the potential to improve ct technologists' understanding of equipment. end confusion which focused attention on improving communication with patients and families. there is little research regarding health literacy (hl) in radiology. the purpose of our study was to determine if an educational intervention (brochure) improves hl for parents whose child will undergo a fluoroscopic study. methods & materials: an education exemption was obtained from the irb. a multidisciplinary team developed brochures for fluoroscopic procedures. participants were randomly selected and asked to complete a survey to assess their knowledge of the procedure and use of radiation both before and after reading a brochure. a final survey to rate and gain feedback about the brochure was completed. results: median age of children whose parents participated (n ) was years. vcug was most commonly performed ( %). prior to the brochure, % of participants knew the name of the test their child was having. after the brochure, % knew the name (p < . ). prior to the brochure, % felt informed about the test, whereas % felt informed after (p<. ). test scores showed an improvement in parent knowledge about the procedure with a median increase of points after the brochure (scale of - ; p<. ). even after reading the brochure, % of parents wanted more information. prior to the brochure, % of parents knew the test involved radiation compared to % afterwards (p<. ). parents improved their understanding of the relative amount of radiation compared to background from % before to % after the brochure (p<. ). overall, % rated the brochure > on a -point scale with % rating the brochure (p<. ). written feedback was uniformly excellent. conclusions: improving hl for parents is part of the mission of radiology medical professionals. our study demonstrates that there is room for improvement in communicating with parents about fluoroscopy. straightforward information for parents provided as a brochure improves their understanding of radiologic fluoroscopic procedures. paper #: pa- compendium of resources for radiation safety in medical imaging anum minhas, duke university, anum.minhas@duke.edu; donald frush purpose or case report: diagnostic imaging, including ionizing radiation modalities, maintains a foremost role in evaluation of medical disorders. there is increasing awareness and need for information across varied sectors about low level radiation and potential risks. many medical/scientific organizations have resources discussing radiation risk and management. however, there is no one resource compiling the same available information. methods & materials: websites, including those of national and international medical organizations (e.g., acr, "image gently" alliance, iaea) were reviewed for information on radiation dose, risk, justification, optimization, guidelines (which included general information about improvement in quality and dose reduction without specific mention of optimization techniques), appropriateness criteria, and general principles of radiation safety for radiography, fluoroscopy/angiography, and ct. this information was divided by modalities and separated into adult and pediatric populations. information from organizations that were not arbitrarily considered to be national (e.g., subspecialty society, regional organization, individual institution/practice) was not reviewed. the resources were then organized into tables, organized by modality. websites with training modules were noted as well. results: websites were explored. overall, less information is available about medical radiation safety in children compared to adults. across both, most information is available on ct, then fluoroscopy, and finally radiography. across all groups and modalities, there is no information available for patients/parents on optimization, appropriateness, or guidelines, with the exception of adult radiography where there were some guidelines. conclusions: this compendium on medical imaging radiation serves as a collective resource for communities including the public and regulatory organizations. additionally, the compendium can be used to determine redundant or deficient areas, providing opportunities for more comprehensive and efficient efforts in medical radiation protection for patients. inappropriate and cloned histories in children: how big a problem is it? leann linam, md, radiology, uams/ach, llinam@uams. edu; chetan c. shah, s bruce greenberg purpose or case report: acr standards require appropriate clinical history for obtaining imaging examinations. cloning clinical histories is a federal violation. our purpose is to determine the frequency of inappropriate histories (ih) and/or cloning histories (ch) at a tertiary children's hospital. methods & materials: three pediatric moc radiologists reviewed clinical histories for radiographs obtained at a tertiary children's hospital on randomly selected dates ( weekdays and weekend day) for appropriateness and cloning. appropriate histories have associated icd- codes. cloning is defined by identical clinical histories occurring on consecutive days and could be clinically appropriate or inappropriate. only the first patient radiograph on a day was included. χ testing was performed to determine significant differences. results: % ( / ) of exams had ih. ih were significantly more common in inpatients than outpatients (p< . ). nicu examinations accounted for % of all ih and were significantly more frequent than other inpatient locations (p . ). the cvicu examinations accounted for % of all ih and was the second most common patient location for ih, but not significantly different from other inpatient locations (p . ). the increased frequency in ih on the weekend reflects a change in patient mix with fewer outpatient examinations performed than on weekdays and was not significant (p . ). the most common ih included: evaluate ett or evaluate lungs ( each). cloning only occurs in inpatients and was combined with ih in % of patients with ch. the nicu accounted % of ch which was significantly greater than other inpatient locations (p . ). conclusions: in radiographs had ih which can lead to misdiagnoses or nonpayment by insurance companies. inpatients, especially the nicu were the most common patient locations. cloning was also a common problem and was frequently combined with ih. identifying the extent of ih allows for corrective educational measures to be instituted which should improve compliance with existing medical and legal standards for ordering radiographs. paper #: in vivo validation of size-specific dose estimates (ssde) through breast entrance skin dosimetry (esd) during pediatric chest ct angiography sjirk westra, md, radiology, massachusetts general hospital, swestra@partners.org; xinhua li, mannudeep kalra, bob liu, suhny abbara purpose or case report: ssde is a new ct dose measure that corrects scanner console ct dose index (ctdi) for cross-sectional body diameter, being a better estimate of absorbed dose in individual patients of varying body size. ssde has been developed through phantom studies and computer simulations of ct dose, but has not yet been validated in vivo. the purpose of our study was to determine correlation between ssde and measured breast entrance skin dose (esd) for pediatric chest cta across a variety of scanning techniques, scanner models and patient sizes. methods & materials: our study was irb-approved, with waiver of written informed consent. during consecutive chest cta exams done on different scanners over a period of years, we measured mid-sternal esd as an approximation of breast dose with skin dosimeters, which was also expressed as mammogram equivalents. for each scan, we recorded patient age, weight, effective ma, kvp, console ctdivol- cm and dlp- cm (from which we calculated age-adjusted effective dose (ed)). we measured effective chest diameter Ø to convert ctdi to ssde, and we correlated ssde with measured breast esd, using linear regression. we evaluated image quality with regard to answering the clinical question. (table) , due to systematic introduction of automatic exposure control, low kv and high pitch scanning techniques. all studies were of diagnostic image quality to address the clinical question. conclusions: ssde is a valid measure of ct dose in pediatric patients undergoing chest cta over a wide range of scanner platforms, techniques, and patient sizes, and may be used to model breast and other organ dose, and to document results of dose reduction strategies over time. purpose or case report: the purpose of this project was to create an automated system capable of quantifying slice-byslice ct image quality and radiation dose data based on patient size. the information generated from this system should enable size-specific optimization of ct scan parameters in order to obtain images of diagnostic quality at the lowest possible radiation doses. methods & materials: a mathematical model was developed to predict ct image noise based on kvp, effective mas, and water-equivalent diameter of the patient. a conical water phantom was used to calibrate the model on multiple scanners and accounting for different operational modes and scan parameters, including tube voltage (kvp), tube current (effective mas), bowtie filter, and focal spot size. a software application was created to process image data from the scout topogram and incorporate dicom metadata from the axial images. a database and data viewing application were developed to display individual and aggregate study data. all of these systems were integrated and automated to enable real-time monitoring of image quality and radiation dose as a function of patient size. results: since the completion of the automated system, ct exams have been processed. a search application allows the user to find an individual study or a collection of studies based on parameters such as body part imaged or study protocol. the viewing application displays slice-by-slice patient diameter, radiation dose, and image quality for each study. radiation dose estimates are adjusted for patient size, yielding size-specific dose estimates. the application also graphs individual study data compared to those of comparative studies that are included in the search. conclusions: we have successfully developed an automated system that monitors ct image quality and radiation dose data based on patient size. the system enables simultaneous real-time monitoring of all studies performed on all ct scanners at our institution. specifically, the system enables size-specific radiation dose estimates at every scan level. this system will be used to guide protocol adjustments in order to optimize ct image quality and thus optimize radiation dose. disclosure: dr. larson has disclosed that he has a patent application in process through cchmc for ct radiation dose reduction. purpose or case report: at many institutions, ct scan parameters for children are determined by patient age or weight. aapm task group recommends cross sectional body dimension, such as patient width to determine size specific dose estimates. the purpose of our study was to develop prediction models of body width based on patient age and weight and compare these models with actual measured body widths for children undergoing body ct. methods & materials: children's hospitals participated in a -month retrospective review of abdominal ct scans on patients < years of age after local irb approval. recorded values included patient width(cm) from an axial image at the level of the splenic vein, patient age (yrs) and patient weight (lbs). a regression model for predicting patient width as a function of age and weight was determined. results: exams, had all measurements. both age and weight were significant predictors of patient width (p<. ). there was also a significant interaction between weight and age (p<. ), indicating that the relationship between patient width and weight depended on the age of the patient. the r for the regression model for predicting patient width from age and weight individually were . and . respectively. the r for the model including both age and weight and their interaction was . leaving % of the variation unexplained. the regression equation for this model is: patient width . + . x age(yrs)+ . x weight(lbs)- . x age x weight. despite the r of . for the model using both age and weight, the average error (rmse) for predicting patient width compared to a direct measurement of width was . cm. the plot of observed minus predicted values (residuals) versus predicted values indicates that the best model (combination of weight and age) results in measurable errors of predicted patient width relative to direct measurement. conclusions: a combination of both patient age and weight results in a more accurate patient width prediction than using age or weight alone. while age and weight can be used to predict body width, this is not sufficiently accurate for generating ct protocols. therefore, direct measurement of body width form either physical measurement on the patient or from the scout view or an axial image is preferred to select appropriate scan parameters for pediatric abdominal ct. paper #: pa- automated size-adjusted dose monitoring for pediatric ct dosimetry olav christianson, clinical imaging physics group, olav. christianson@duke.edu; ehsan samei, donald frush purpose or case report: the potential health risks associated with low levels of ionizing radiation have created a movement in the radiology community to minimize radiation dose during ct imaging; this is especially important for pediatric patients due to their increased sensitivity to radiation. it is thus essential to accurately assess the risks to pediatric patients undergoing ct imaging. current efforts to monitor radiation dose, however, are limited because they do not account for differences in risk from ionizing radiation due to variability in patient size, age, and gender. in this context, we developed an automated size-adjusted dose monitoring program capable of performing patient-specific risk estimation to facilitate protocol optimization. methods & materials: dicom routing software was used to send dose reports and scout images to an image repository on a dosimetry server. optical character recognition was used to extract dose-relevant data from dose reports; patient size was determined from corresponding scout images. based on anatomical location, risk estimation conversion coefficients (qfactors) were determined for each series in the dose reports. the q-factors were adjusted according to patient size, age, and gender and then multiplied by the dlp to estimate the risk to each patient. this process was applied to the cohort of pediatric patients undergoing ct examination at our institution. to evaluate the impact of including patient size, age, and gender, risk estimates were obtained excluding and including the dependencies on size, age, and gender. the results were computed in units of cancer incidence per cases exposed (cpt). results: the average patient-generic risk estimate for a pilot group of patients undergoing body ct was . ± . cpt. by including patient size, the risk estimate was increased to . cpt± . cpt. by including patient age and gender, the average risk estimate was further increased to . cpt± . cpt. conclusions: we developed a new size-adjusted dose monitoring program for pediatric ct dosimetry. comparisons between patient-generic and our new patient-specific risk estimates show that failure to consider patient size, age, and gender resulted in risk estimates that were too low by a factor of seven. additionally, the increase in standard deviation we observed demonstrates that our method of including patient size, age, and gender is sensitive to the inherent variability in the patient population. disclosure: dr. christianson has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: treatment of prenatally diagnosed lung masses is controversial, with many specialists recommending elective surgical removal in the first year of life because of a reported or perceived increased risk of infection and malignancy, while other centers recommend a conservative approach to management. the natural history of unresected lung masses is not clear. in our center, our standard recommendation is prophylactic resection of asymptomatic lesions, although not all families choose this option. we asked whether respiratory morbidity increased during the time prior to elective resection of prenatally diagnosed lung masses. methods & materials: ninety-eight pregnant women carrying fetuses with chest masses were imaged by ultrasound (us) and magnetic resonance imaging (mri). medical records of the liveborn infants were retrospectively reviewed. results: fetal diagnosis of a lung mass was made at a mean of weeks gestation (range - wks). intrauterine fetal demise was documented in pregnancies. there was one elective termination of pregnancy. three infants were lost to follow up. thus, outcomes were available for children ( % m, % f) with prenatally diagnosed lung masses. significant respiratory morbidity (rm) was defined as the occurrence of pneumonia, asthma, chronic coughing or wheezing, or respiratory symptoms severe enough to require an emergency room visit or hospitalization. of the children who had surgical removal of their lung mass, ( %) had rm prior to surgery. fifteen out of children did not have surgery but have been followed expectantly, and of ( %) developed some form of rm. fifteen of ( %) infants had immediate and significant rm (tachypnea, grunting, increased work of breathing, increased oxygen requirements or need for intubation) in the newborn period leading to urgent surgery (range of age at surgery: - d; mean . d). of the initially asymptomatic infants, ( %) developed rm prior to elective removal of the mass (range - weeks, mean weeks). of the lesions removed, histology revealed: cystic adenomatoid malformation (ccam) %, ccam + sequestration %, sequestration %, congenital lobar emphysema (cle) %, ccam+cle %, other %. conclusions: the risk of respiratory morbidity appears to be increased during the time prior to elective resection of prenatally diagnosed lung masses, which may be important for parents and pediatric specialists to consider when deciding whether to remove an initially asymptomatic lung mass. purpose or case report: it is now accepted that fetal mri with its superior tissue resolution can be very helpful in clarifying anomalies detected during obstetrical ultrasound. this is particularly the case with intracranial abnormalities, although indications are expanding. the current english medical literature, though, appears to be focused on evolving mri techniques and how mri compares to ultrasound with regards to image quality and detection of additional findings which may alter the diagnosis. however, we found no study specifically evaluating the clinical relevance and impact of the information obtained by fetal mri to the specialists who counsel and treat these patients. a "satisfaction and clinical impact" survey was created and sent to all the members of our fetal diagnosis and treatment group, asking specifically how the clinicians rated their satisfaction with this type of imaging, its influence on their counseling and on various clinical decisions, both prenatal and postnatal. results: we received responses from specialists in different clinical disciplines. the greatest number of respondents came from our obstetricians ( %), many of whom perform their own ultrasounds, and from members of our medical geneticists/genetic counselors ( %), although % of respondents were from various other clinical disciplines, both medical and surgical. there was a surprisingly high degree of satisfaction overall with the quality of the images and with the type and amount of information provided. most respondents indicated they felt fetal mri was "moderately" or "extremely" useful for their particular clinical decisions, and most respondents agreed that fetal mri impacted "moderately" or "significantly" on counseling and management of these pregnancies. impact appeared greatest on the counseling of the parents and their decision to terminate/pursue the pregnancy, and the least impact was on issues around delivery. conclusions: fetal mri, in addition to providing images of better quality, particularly in certain conditions, has clinical value in that it directly impacts on the counseling of parents and on clinical decisions. - . ultrasound reports were reviewed to determine sonographic diagnoses. selected patients from this cohort underwent mri using ge . tesla magnet without contrast (sequences included ssfse, fiesta, fgre or dual echo in planes). the images were reviewed and multiple characteristics were assessed for specifiying the area of obstruction. the features included: presence of normal fluid-filled bowel, small rectum for gestational age, signal of meconium in the rectum, and meconium filled dilated bowel. results: cases of sonographically suspected bowel obstruction were identified during the study period; of these underwent fetal mri. of these cases, had normal mri and postnatal outcomes, cases did not have postnatal findings available, and had postnatal meconium peritonitis but no obstruction. one case of congenital chloride diarrhea was diagnosed by fetal mri. a variety of bowel abnormalities were observed amongst the remaining cases. proximal obstruction was diagnosed in cases: jejunal atresia (n ) and multiple atresia (n ). distal obstruction was diagnosed in cases: ileal atresia (n ), meconium plugging (n ), closed gastroschisis (n ), enteral duplication cyst (n ), and imperforate anus (n ). characteristic patterns of features were identified amongst these cases that specified the location of obstruction. these patterns of findings allowed accurate localization of the level of obstruction in all cases when compared to postnatal findings. distal obstruction was characterized by normal fluid-filled small bowel and high t signal in distended loops. jejunal atresia was characterized by multiple loops of dilated bowel with high t signal primarily in the left upper quadrant. small rectum for gestational age was not consistently associated with proximal or distal atresia. conclusions: evaluation of fetal mri with attention to specific features allows localization of bowel obstruction. this may aid in counseling and postnatal management, including the need and type of postnatal imaging study. early diagnosis and treatment of ph may prevent clinical deterioration. pvt may produce a spectrum of imaging appearances, which has not been fully recorded in the literature. the goal of this paper is to review the spectrum of imaging appearances of neonates and survivors of neonatal pvt with special emphasis on the role of us and to correlate these findings with the clinical findings including outcome. methods & materials: a retrospective review of consecutive neonates admitted between - and diagnosed with pvt was conducted. diagnosis was established by us at a mean age of days (range: - ). health records, initial and follow-up (f/u) imaging were reviewed. findings were classified as non occlusive, single branch, pvt (grade ); occlusive pvt (grade ) and pvt with extensive parenchymal ischemia (grade ). results: pvt was diagnosed in patients, of whom were followed up to for years or longer. twelve patients were excluded due to liver disease, expired and were lost to f/u. of the in whom f/u was available, at the time of initial diagnosis, grade pvt was present in , all were on the left. grade pvt was diagnosed in and grade pvt in . on f/u physical exam, findings were unremarkable in / patients. liver function tests (lft) and thrombophilia assessment were available in children, mild lft abnormalities were noted in and children had evidence of thrombophilia. us exams were available in / children. among the survivors of neonatal pvt, us was regarded as normal in only children; showed left lobar atrophy (lla), had slowly progressive splenomegaly without other signs of ph, and developed clinically significant ph requiring shunting. conclusions: pvt has a wide spectrum of imaging appearances, it is possibly underdiagnosed and clinically unsuspected. varying degrees of lla are likely a sequela of clinically silent left pvt. us is a sensitive method for the detection of disease and assessment of progression. paper #: pa- fetal mri in arthrogryposis hedieh eslamy, md, radiology, lucile packard children's hospital, hkeslamy@gmail.com; erika rubesova, louanne hudgins, britton rink, richard a. barth purpose or case report: to present the fetal mri findings in fetuses with a prenatal diagnosis of arthrogryposis and correlate with postnatal outcome or autopsy results. arthrogryposis refers to contractures involving more than one joint which often represent deformational changes secondary to decreased or absent fetal movement. prognosis varies widely dependent on diagnosis, ranging from isolated contractures in amyoplasia to lethality in some cases. we hypothesized that fetal mri may demonstrate central nervous system (cns) pathology and muscle abnormalities which are important for predicting postnatal outcome. methods & materials: we identified fetuses with a diagnosis of arthrogryposis between january and october . all had fetal mri which was performed on a ge . tesla magnet, with ssfse, fiesta and fgre sequences in planes. the fetal mri's were evaluated for cns and muscle abnormalities. the extremities were evaluated for: muscle mass, increase in subcutaneous fat (indicative of muscle atrophy), and extremity joint positioning. these findings were subsequently correlated with the clinical exam of the neonates, pathology in the abortus and karyotype when available. results: results of fetal us, amniocentesis, fetal mri and post-natal or post-termination outcomes will be summarized. five fetuses had ≥ limb joint contractures. a sixth case had neck hyperextension and lateral flexion associated with akinesia and hydrops. on mri, no structural brain or spine abnormalities were identified. the abnormalities detected in the extremities were: severe decrease in muscle mass associated with increased subcutaneous fat ( cases); normal muscle mass ( cases); moderate decreased muscle mass associated with increased subcutaneous fat ( case). in the cases that delivered, the diagnoses were amyoplasia ( ) and distal arthrogryposis ( ). in a fourth case that underwent elective termination, autopsy was consistent with amyoplasia. two cases are pending delivery. conclusions: while fetal mri can be useful to rule out cns anomalies, it may also provide important information on decreased muscle mass as an important prognostic sign in a fetus with arthrogryposis. in our series, severely decreased muscle mass was predictive of amyoplasia, and joint contractures limited to hands and feet with preserved proximal muscle mass was predictive of distal arthrogryposis. both diagnoses are associated with relatively good prognosis and usually normal intelligence. purpose or case report: the purpose of this study is to assess the effects of iterative reconstruction technique (irt) on image quality metrics measured in child-sized anthropomorphic phantoms as kvp is changed. methods & materials: ct scans were performed on anthropomorphic phantoms with sizes of , & years (atom phantoms, cirs, norfolk virginia) using low dose pediatric chest protocols ( . , & msv) to determine baseline noise and dose levels. subsequently three voltage levels ( , & kvp) were used while adjusting mas to maintain baseline ctdivol and without mas adjustment which allowed varied ctdivol. images were reconstructed using % filtered back projection (fbp) and blends fbp: ir ( : , : , & : ) . parameters including ctdivol, dose length product, scan length, kvp, and mas, were recorded for each scan. image noise, contrast:noise (cnr), and signal:noise (snr) data were recorded from rois in phantoms and dilute iodine contrast filled syringes ( , , . %). results: as kv is lowered from to , image noise is doubled if mas is not increased to maintain ctdivol, and cnr is increased but snr is decreased due to the increased image noise. as kvp is lowered from to , image noise is increased nominally ( - %) if mas is increased to maintain ctdivol; therefore the increase in cnr and decrease in snr is negligible. ctdivol is reduced > % in all phantom scans as kv is reduced from to . irt reduces image noise by up to % [range - %] in all phantom sizes and in clinical images. as ctdivol is maintained in patient scans, image noise, cnr, and snr are reduced in patients (p< . ), resulting in improved image quality. conclusions: when lowering kvp, compensation with increases in mas is necessary to maintain ctdivol. however, lower target ctdivol can be achieved when adding irt as image noise can be decreased. for these phantoms, cnr and snr improved using all [selected] levels of ir, even when kv was reduced, resulting in lower ctdivol in phantoms. at all kvp settings when irt is applied, image noise is reduced, resulting in improved cnr and snr for all phantoms. disclosure: dr. bardo has indicated she is in the speaker's bureau and receives an honorarium from koninklijke philips. paper #: pa- adaptive iterative dose reduction in evaluation of the pediatric abdomen with ultra-helical -channel mdct jeffrey hellinger, md, stony brook university, jeffrey. hellinger@yahoo.com; bernice hoppel, richard mather, monica epelman purpose or case report: radiation reduction is paramount for pediatric patients. ultra-helical -channel mdct allows for rapid acquisitions at low dose. we evaluated the ability of a new adaptive iterative dose reduction algorithm (aidr) to reduce noise in low-dose ultra-helical pediatric abdominal ct scans. aidr is an iterative algorithm that adaptively reduces noise in the raw and image domains while preserving image structure. the raw data from consecutive low-dose pediatric abdomen exams was gathered. a dose simulation tool which adds noise to raw projection data was employed to simulate tube current at / of baseline ma. data were reconstructed with both standard filtered back projection and with aidr. regions of interest were drawn in the liver and lumbar musculature to determine the signalto-noise (snr), contrast-to-noise (cnr) and overall diagnostic quality of each data set. statistical significance was determined using a student's t-test. subjective image quality was evaluated by two reader blind review using a five point scale ( excellent, unacceptable). results: the snr and cnr were significantly lower for the % dose reduction datasets compared to the original filtered back projection reconstructions (snr: . vs . , p< . ; cnr: . vs . , p . ). when aidr was applied to the % dose reduction data, the snr and cnr improved to be superior to the native case (snr: . vs . , p< . ; cnr: . vs . , p . ). the average image quality score for the low dose datasets with aidr was . compared to . with standard filtered back projection at the baseline ma conclusions: aidr significantly improves the image quality of pediatric abdominal ct images. with a simulated % reduction in dose, aidr produces images with significantly greater snr and cnr. the subjective image quality scores for aidr showed dramatic improvement over standard filtered back projection. aidr processing algorithms with ultra-helical mdct will allow % reduction in radiation exposure while achieving the same diagnostic quality as compared to routine pediatric abdomen mdct radiation protocols with filtered back projection processing algorithms. purpose or case report: to explore incorporating asir into pediatric head ct protocols, to reduce patient radiation dose while maintaining image quality. methods & materials: an alderson rando head phantom was estimated to approximate the size of a -year-old child's head, and was scanned at decreasing % ma intervals ( to %, to ma) relative to this institution's age-based head ct protocols. each of these studies was then was reconstructed at % asir intervals ( % to %), and a mm roi was obtained in a consistent location behind the frontal bone to estimate noise (sd). using this phantom data, our ventriculoperitoneal (vp) shunt follow-up ct protocol was modified, and patients were scanned at % asir with approximately % ma reductions relative to our normal age-based mas. these asir studies were then anonymously compared to older non-asir head ct studies from the same patients (with identical kvp/slice thickness) by two blinded attending pediatric neuroradiologists. all studies were evaluated subjectively for diagnostic utility ( - ), sharpness ( - ), noise ( - ), and artifacts ( - ). - mm rois were drawn in consistent locations to estimate noise in air, bone, csf, and white matter (wm). results: the phantom study suggested similar same noise levels at % ma/ % asir ( . ) and % ma/ % asir ( . ). patients (average , range to years) were then scanned at approximately % ma reductions, with an average of days (range to days) between the asir study and prior non-asir study. the average ctdivol and dlp values of the % asir studies were . mgy and . mgy-cm, and for the non-asir studies were . mgy and . mgy-cm, representing statistically significant decreases in the ctdivol ( . %, p . ) and dlp ( . %, p . ) values. there were no significant differences between the asir studies and non-asir studies in respect to diagnostic acceptability (p . ), sharpness (p . ), or noise (p . ). there was a non-significant trend that the asir studies had a lower artifact score ( . vs . , p . ). there was good to perfect (kappa . to . ) agreement. the asir studies had statistically significant decreased csf noise ( . vs . , p . ), but no noise differences were seen in air (p . ), bone (p . ), or wm (p . ). conclusions: our findings suggest that asir can provide dose reductions in pediatric head ct without affecting image quality. purpose or case report: to validate the t map as a noninvasive quantitative biomarker of fatty infiltration of muscles and to determine whether the t map can differentiate between boys with dmd and healthy boys. methods & materials: two groups of boys with similar ages (range - years) were evaluated: boys with dmd (mean age . years) and healthy boys (mean age . years). mr images were performed at t. fatty infiltration of the pelvic and thigh muscles on t -weighted images (wi) was graded from to . on t maps with and without fat suppression, the muscle with the greatest fatty infiltration on t -wi was selected, and a region of interest was placed to obtain t values. t values from t maps with fat suppression were subtracted from values of t maps without fat suppression and designated as the "t fat value." t fat values were obtained from the same muscles in all boys. comparison was made between the t fat values of the two groups. the upper reference limit of the reference interval (ri) of t fat values was obtained from the control group to establish the normal range and applied to both groups to determine the accuracy of the t map. results: the gluteus maximus muscle had the greatest fatty infiltration on t -wi. median t fat value was . msec for dmd ( % ri . , range . - . ) and . msec for the control group ( % ri . , range . - . ). when applied to the two groups, the upper reference limit of the ri for control patients yielded % sensitivity, % specificity, % positive predictive value, and % negative predictive value. conclusions: utilization of t maps for the quantitative measurement of fatty infiltration of muscles can clearly differentiate between dmd and normal control boys with a high degree of accuracy and precision. this advanced noninvasive technique may potentially replace invasive muscle biopsies currently used for diagnosis. purpose or case report: prior work has shown that the gluteus maximus muscle has the greatest t relaxation time on mr imaging using t mapping in boys with duchenne muscular dystrophy (dmd). however, an increased t value on t relaxation time mapping may reflect both fatty infiltration and inflammation of the muscle. fatty infiltration characteristically follows inflammation in this disease process. therefore, the purpose of this study was to determine the contribution of each component (fat and inflammation) within gluteus maximus muscles and to correlate each component to clinical assessments. methods & materials: forty-six boys with dmd (ages: - years) were recruited. mr imaging of the pelvis using t maps with and without fat suppression were performed. the t map "fat values" (t value calculated from the t map without fat suppression [fs] minus t map with fs) and the t map "inflammation value" (t value from the t map with fs) were obtained. clinical assessments typically used to evaluate dmd patients (including clinical functional score, ft run, gower score, and step-up time) were also performed. spearman correlation coefficients between fat and inflammation values and the clinical assessments were calculated. results: there was a statistically significant correlation between the fat value of the gluteus maximus muscle and each clinical assessment test (p< . ). however, the inflammation value of the gluteus maximus muscle did not correlate with any clinical assessment. conclusions: in dmd, the amount of fatty infiltration of the gluteus maximus muscle has excellent correlation with clinical assessment. the amount of inflammation of the gluteus maximus muscle, however, does not correlate with clinical function. therefore, further study is needed to determine whether components (fatty infiltration or muscle inflammation) of the single most involved muscle reflect the components of all the muscles of the pelvis and thighs and whether the cumulative muscle involvement of each component represents clinical disease severity. utility of contrast-enhanced mr imaging in children with osteonecrosis: does gadolinium help? lamya atweh, md, radiology, texas children's hospital, laatweh@texaschildrens.org; robert c. orth, wei zhang, r. paul guillerman, herman kan purpose or case report: at our institution, gadolinium contrast-enhanced mr sequences are often obtained to assess epiphyseal and non-epiphyseal osteonecrosis in children. several studies have shown that dynamic contrast-enhanced sequences may provide prognostic information about long-term complications and healing of osteonecrosis. to our knowledge, no studies have determined the added value of routine post-contrast mr imaging in assessing acute complications related to chronic osteonecrosis. the purpose of this study was to evaluate the utility of intravenous gadolinium contrast in the mri identification of complications in children with an established diagnosis of osteonecrosis. methods & materials: patients were restrospectively identified (age range: . years to . year; m:f : ) with an imaging diagnosis of chronic osteonecrosis who underwent contrast-enhanced mr studies between / and / . the pre-and post-contrast mr images were consensus reviewed by two caq pediatric radiologists. pre-and post-contrast images were reviewed at separate times. the pre-contrast images were available during the review of post contrast images. studies were assessed for: osteonecrosis location (epiphyseal, non-epiphyseal osteonecrosis, or both), joint effusion, marrow edema, and epiphyseal collapse. % confidence interval (ci) and cohen's kappa coefficient(κ) was calculated to assess observed agreement. results: the diagnosis of osteonecrosis without complicating features was made in . % (ci: . - . %) ( / ) of pre-contrast studies and . % (ci: . %- . %) ( / ) of post-contrast studies. when chronic osteonecrosis with complicating features was identified,pre-and postcontrast images idenfied joint effusion in . % ( / ) and . % ( / ) (κ . , p< . ); marrow edema in . % ( / ) and . % ( / ) (κ . , p< . ); and epiphyseal collapse in . % ( / ) and . % ( / ) (κ . , p< . ), respectively. myositis or muscle strain was incidentally diagnosed in . % ( / ) pre-contrast and . % ( / ) post-contrast (κ . , p < . ) studies. conclusions: the high observed agreement between the pre-and post-contrast mr images shows that the addition of intravenous gadolinium may not be necessary in the majority of children with chronic osteonecrosis. paper #: pa- systematic protocol for assessment of the validity of bold mri in a rabbit model of inflammatory arthritis at . tesla michael chan, bhsc, university of toronto, mw.chan@ utoronto.ca; afsaneh amirabadi, anguo zhong, antonella kis, rahim moineddin, andrea s. doria purpose or case report: blood oxygen level-dependent (bold) mri has the potential to identify regions of early hypoxic and vascular joint changes in inflammatory arthritis. at this point, there is no standard protocol for data analysis of bold mri measurements in musculoskeletal disorders. standardization of the technique is paramount to compare results between studies and assess the validity of this technique in tissues outside the blood-brain barrier. our objective is to optimize bold mri reading parameters in a rabbit model of inflammatory arthritis by determining the diagnostic accuracy of ( ) statistical threshold values (r> . vs r> . ), ( ) summary measures of bold mri contrast [ the mean of the % bold signal differences within the region of interest (roi) (diff_on_off) and the percentage of suprathreshold voxels within the roi (pt%)], and ( ) voxel activation algorithm (positive, negative, and positive_negative). methods & materials: using bold mri protocols with a carbogen stimulus on a . t magnet, we imaged injected and contralateral knee joints of juvenile rabbits at baseline, and days , and after a unilateral intra-articular injection of carrageenin. nine non-injected rabbits served as controls. receiver operating characteristic (roc) curves were plotted to determine the diagnostic accuracy of the reading parameters. the bold measures from [(injected knee-control knees)/control knees] were counted as positive cases, while the bold measures from [(contralateral knees-control knees)/control knees] were regarded as negative cases. areas under the curve (aucs) were calculated to determine the most accurate parameters. results: using diff_on_off and positive_negative activations as constants, r> . was found to be more accurate than r> . (p . at day ). comparison of diff_on_off and pt% yielded no statistically significant difference (p> . ). finally, positive_negative activations for diff_on_off and negative activations for pt% using r> . were the most diagnostically accurate (auc . , p< . at day , and auc . , p< . ). conclusions: from the results of this study, the most diagnostically accurate and clinically relevant reading parameters included the use of a more lenient threshold of r> . , a diff_on_off measure of bold contrast, and a positive_negative voxel activation algorithm. pt% may used as an ancillary measure of bold contrast. quantitative versus semi-quantitative mr imaging of cartilage in blood-induced arthritic ankles andrea doria, md, phd, diagnostic imaging, the hospital for sick children, andrea.doria@sickkids.ca; ningning zhang, carina man, pamela hilliard, ann marie stain, victor blanchette purpose or case report: to cross-sectionally compare the ability of a scoring system (semi-quantitative method) with a manual segmentation technique (quantitative method) to evaluate the status of the articular cartilage of growing ankles of children with blood-induced arthritis. methods & materials: boys, with hemophilia (a, n ; b, n ) and with von willebrand disease, median age (range, - ) underwent a high resolution mri protocol at . tesla, x-rays, and physical examination using the hemophilia joint health score (hjhs) system. two blinded radiologists scored the mri examinations for cartilage items (horizontal component: surface erosions, scores - and vertical component: cartilage degradation, scores - ) according to the semiquantitative method (international prophylaxis study group mri scale). an experienced operator applied a validated quantitative d-mri method (horizontal components: ac, vc, vctab, thctab; vertical component: thccab) to corresponding high resolution mr images of ankles. results: internal correlation of the semi-quantitative method components was substantial (r . , p< . , tibia) to high (r . , p< . , talus) in any site of investigation, but it was site-specific with the quantitative method, being significant only in the talar trochlea (r . , p< . ). external correlation of corresponding components of the semiquantitative and quantitative methods was moderate (r . , p . ) to poor (r . , p . ) for horizontal components, and non-existent for vertical components. components of the semi-quantitative method highly correlated with lifetime number of previous ankle bleeds (r . - . , p< . ), pettersson x-ray (r . - . , p< . ), and hjhs scores (r . , p< . ). this correlation was poor (r . , p . ) to moderate (r − . , p . ) for horizontal components of the quantitative method. conclusions: the biologic concepts of the semi-quantitative and quantitative mri methods are distinct for assessment of ankles. the semi-quantitative method is valid for assessing cartilage changes in cross-sectional studies of blood-induced arthropathy, however the quantitative method is suboptimal or less powerful for this purpose. paper #: pa- shoulder mr arthrography in skeletally immature patients nancy chauvin, md, department of radiology, the children's hospital of philadelphia, chauvinn@email. chop.edu; camilo jaimes, victor ho-fung, diego jaramillo purpose or case report: there has been a well documented increase in sports participation in children which has lead to an increase in sports-related injuries. to date, there are no studies describing the value of shoulder mr arthrography compared with the gold standard, arthroscopy. we retrospectively reviewed mr shoulder arthrograms obtained in pediatric patients between and who underwent subsequent shoulder arthroscopy. interpretation of the images was performed by three pediatric radiologists who were blinded to the arthroscopy findings. images were evaluated in consensus and independently. assessment included evaluation of the osseous structures, labral-ligamentous complex, joint space and the rotator cuff interval. the mr results were compared with reported surgical findings. sensitivity and specificity were calculated. results: nine patients were excluded due to technical reasons. of the remaining patients, were boys ( . - . years, mean . years) and were girls ( . - . years, mean . years). at arthroscopy, patients ( %) had injury to the anterior inferior glenoid labrum. mr sensitivity was % for depiction of bankart-type injuries with a specificity of %. patients ( %) had hill sach lesions and mr had sensitivity of % with specificity of %. superior labrum anterior posterior (slap) tears ( %) were identified at arthroscopy with mr sensitivity of % and specificity of %. overall, mr arthrography had a positive predictive value of % for identification of a surgical lesion. agreement between the observers was high. interobserver reliability was calculated with an intraclass correlation coefficient (icc)of . with a cronbach's alpha of . . conclusions: mr shoulder arthrography can accurately depict labral and osseous injury and provides pertinent preoperative information. a novel multi-channel mr coil for improved pediatric elbow coil imaging suraj serai, phd, cchmc, suraj.serai@cchmc.org; randy giaquinto, kathleen emery, charles dumoulin purpose or case report: single flex coils or adult size coils are currently used for imaging the pediatric elbow. this frequently results in uncomfortable patient positioning, motion, poor fat suppression, low snr and there is currently lack of a dedicated pediatric elbow coil in the commercial market. our goal was to explore the usefulness of a new coil array dedicated for pediatric elbow imaging and to compare quantitative & qualitative imaging findings to commercially available coils. methods & materials: an eight channel elbow coil was designed. the coil frame was designed to be rigid and lightweight. seven identical loop coils were built into a polycarbonate frame and an eighth coil built into a paddle that fits into the top frame. the coil elements were constructed with heavy copper to provide a high q-factor and increased snr. the complete coil including electronics & covering, weighs only . kg. mr imaging under irb approval was performed on a ge . t scanner using a routine clinical elbow protocol including t w, pdw, t w, fat-sat, non-fat-sat, d & d sequences. subjects were positioned feet-first with the elbow on the side & were subjectively assessed for comfort level. images obtained from the new coil & from the current commercial coils were compared for snr. results: scan positioning was reported to be comfortable. snr was between - % higher as compared to the routine coils. fat saturation was uniform, indicating that the magnetic susceptibility of the coil is well-matched to human anatomy. anatomical detail depiction was subjectively better for anatomic features such as trochlea. detection & diagnostic confidence of elbow disorders were improved with the new coil & greatly decreased motion artifacts were observed. conclusions: the new pediatric elbow coil provided excellent image quality, patient acceptance and clinical performance improvements over existing coils. the open coil design also allows for imaging of the elbow in a partially flexed position or in a cast. the advantages provided by the new coil are expected to include shortened image acquisition times (via parallel imaging) & increased snr. disclosure: dr. serai has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. incremental value of knee radiography in the interpretation of pediatric knee mri yen-ying wu, texas children's hospital, yxwu@ texaschildrens.org; robert c. orth, wei zhang, r. p. guillerman, herman kan purpose or case report: the acr appropriateness criteria recommendation for the imaging work-up of knee pain is radiography followed by mri. in many cases, mri is performed prior to review of radiographs or the referring subspecialist does not feel radiographs add value, particularly when ligamentous injury is suspected. the purpose of this study is to determine if radiography adds incremental value in the interpretation of knee mr studies electively referred by pediatric sports medicine and orthopedic subspecialists. knee mri studies referred from pediatric sports medicine physicians or pediatric orthopedic surgeons between / and / (n , ages - years, m:f : ) with accompanying radiographs were identified. patients were separated into groups based on mri findings: normal, ligamentous injury, or osteochondral injury (osteochondral lesions, bone contusions/fracture, and avulsion injury). knee radiographs were consensus reviewed by two caq pediatric radiologists blinded to mri findings and categorized into the same groups. radiograph and mri findings were compared and categorized into groups: neutral if radiograph and mri findings were the same, misleading if findings were discordant, or helpful if radiographs improved mr interpretation. the latter group was analyzed for impact on mr diagnosis. results: for knee radiographs, were normal, showed ligamentous injuries, and showed osteochondral injuries. when radiographs were interpreted as normal (n ), by mr % were normal, % had ligamentous injury, % had osteochondral injury, and % had both ligamentous and osteochondral injury. when radiographs were interpreted as ligamentous injury (n ), by mr % were normal and % had ligamentous injury. when radiographs were interpreted as osteochondral injuries, by mr % had ligamentous injury, % had osteochondral injury, and % had both ligamentous and osteochondral injury. subset analysis of true positive radiographs (n ) found % to be helpful and % to be neutral in mr diagnosis. for radiographs considered helpful, % resulted in a change in mr diagnosis. in regards to the influence of radiographs on mr interpretation, % ( / ) were misleading, % ( / ) were neutral, and % ( / ) were helpful. conclusions: a minority of pediatric knee radiographs aided mr diagnosis, and none resulted in a change in diagnosis. pediatric knee mri and interpretation should not be predicated on radiologist review of knee radiographs in this subset of patients. paper #: pa- sonographic evaluation of pediatric skeletal lesions: is it worthwhile? henrietta rosenberg, md, radiology, the mt. sinai medical school, henrietta.rosenberg@mountsinai.org; amish patel, neil lester purpose or case report: the purpose of this paper is to demonstrate how ultrasound(us) may serve as a readily available, cost-effective, non-invasive, non-ionizing, practical tool for the evaluation of a variety of skeletal abnormalities in the pediatric age range. we reviewed the clinical and imaging findings in patients seen during the past years in whom us demonstrated abnormalities related to the skeletal system, excluding patients with hip joint effusions or ddh. results: us proved useful in the following situations: evaluation hard superficial immobile mass (osteoma shin) ( ), absent medial end clavicle on x-ray in region of neck mass (us showed abc medial end clavicle)( ), to determine if soft tissue mass involves adjacent bone nodular fasciitis surrounding clavicular head ( ), for diagnosis and followup fracture (displaced/non-displaced) in infants ( ), diagnosis osteomyelitis in patients with cellulitis ( ), question of fracture underlying cephalohematoma or subgaleal hematoma ( ), rib mass (osteochondroma) ( ) or mass costochondral junctions (contour deformities costochondral cartilage) ( ), firm posterior knee mass (baker's cyst) ( ), firm anterior knee mass (septated cystic mass suprapatella region due to rheumatoid disease) ( ), immobile hard scalp mass due to epidermoid cranial vault ( ), painful mass occipital bone with soft tissue components extending through the skull externally and internally due to langerhan's histiocytosis ( ), indeterminate mass clavicle clinically thought to be post-traumatic sequellae, resolved on follow-up ( ), assessment craniosynostosis ( ), for differentiation of pathological entity from normal anatomic structure (lump on back of slender baby proved to be normal posterior spinous process) ( ). conclusions: us is worthwhile for evaluation of wide range of pediatric skeletal abnormalities and helps to determine if the a lesion is one that is "touch" or "don't touch". to maximize diagnostic accuracy, the imager should have thorough knowledge of the clinical history, physical findings, laboratory and other imaging findings. in equivocal cases or in those patients in whom the field of view (fov) is insufficient for complete visualization of an obvious lesion or if malignancy is suspected, us serves to triage those patients in whom further imaging is necessary. high incidence of vertebral fractures in children with acute lymphoblastic leukemia months after the initiation of therapy mary ann matzinger, md frcp(c), university of ottawa, matzinger@cheo.on.ca; nazih shenouda , brian lentle, josée dubois, helen r. nadel purpose or case report: vertebral fractures due to osteoporosis are a potential complication of childhood acute lymphoblastic leukemia (all). to date, the incidence of vertebral fractures during all treatment has not been reported methods & materials: we prospectively evaluated children with all during the first months of leukemia therapy. lateral thoracolumbar spine radiographs were obtained at diagnosis and months. vertebral bodies were assessed for incident vertebral fractures using the genant semi-quantitative method, and relevant clinical indices such as spine bone mineral density (bmd), back pain and the presence of vertebral fractures at diagnosis were analyzed for association with incident vertebral fractures. results: of the children, ( %, % confidence interval [ci] % to %) had a total of incident vertebral fractures, of which ( %) were moderate or severe. thirteen of the children with incident vertebral fractures ( %) also had fractures at the time of diagnosis. vertebral fractures at diagnosis increased the odds of an incident fracture at months by an odds ratio of . ( % ci . to . , p . ). in addition, for every . standard deviation reduction in spine bmd z-score at diagnosis, there was . -fold increased odds for incident vertebral fracture at months ( % ci . to . %, p . ). conclusions: children with all have a high incidence of vertebral fractures months after diagnosis, and the presence of vertebral fractures and reductions in spine bmd zscores at diagnosis are highly associated clinical features. purpose or case report: to provide objective measures of acetabular morphology utilizing volume-rendered ct and to better characterize normal acetabular development in adolescents. implications for the diagnosis of femoroacetabular impingement (fai) will be discussed. methods & materials: hips in consecutive patients ( female, male; ages - years) who underwent abdominal and pelvic ct for non-hip related complaints were retrospectively examined. examinations were performed for a variety of complaints, including abdominal pain, nephrolithiasis, vomiting etc. patients with obvious hip pathology were excluded. pelvic rotation was eliminated, and pelvic inclination was measured and corrected to °u tilizing a volume rendered ct model. measurements of femoral head diameter (fhd), anterior femoral head coverage (fhca), and posterior femoral head coverage (fhcp) were obtained. femoral head area (fha) was defined as π(fhd/ ) . percent anterior femoral head coverage (%fhca) was defined as (fhca/fha)* . percent posterior femoral head coverage (%fhcp) was defined as (fhcp/fha)* . acetabular version by volume-rendered ct (avvr) was defined as (fhcp/fhca). results: average pelvic inclination angle (sd) was . ( . ) for females and . ( . ) for males. average (sd) %fhca was . ( . ) for males and . ( . ) for females. average (sd) avvr was . ( . ) for males and . ( . ) for females. among males, average avvr decreased with subject age. on the other hand, there was little change in average avvr with age among females. conclusions: average avvr is greater for females than males, and this difference becomes more striking with increasing subject age. this represents an unexpected finding given the reported increased incidence of "pincer" type fai among females. characterization of acetabular morphology among adolescents with clinical fai should consider subject age and gender. in this regard, volumerendered ct is capable of providing an objective measure of acetabular morphology. mistakes in musculoskeletal plain film interpretation james crowe, pediatric radiology, texas children's hospital, jecrowe@texaschildrens.org; george s. bisset purpose or case report: to evaluate the mistakes made by trained pediatric radiologists when interpreting radiographs of the extremities obtained for the evaluation of outpatient acute pain (mostly post-traumatic). we retrospectively evaluated all radiographs and associated interpretations obtained during a month period from april , , to october , , of the elbows, wrists, knees and ankles in pediatric outpatients who presented with acute pain in the affected area. all radiographs were previously interpreted by a caq-certified pediatric radiologist varying in experience from year to years. abnormals were identified, including elbows, wrists, knees and ankles. all radiographs were determined to be "as dictated", missed significant finding, or overcall. attention was focused on the missed findings and overcalls. results: findings were as follows: elbow radiographs- missed findings and overcalls, wrist radiographs- missed findings and overcalls, knee radiographs- missed findings and overcalls, ankle radiographs- missed findings and overcalls. this resulted in a total of missed findings ( . % of abnormals) and overcalls ( . % of abnormals). of the misses, % were fractures. the highest mistake percentage occured in the ankles where the combined misses and overcalls approached %. this was also the location where we found the highest percentage of missed fractures ( . %) conclusions: when just abnormal cases were considered, fully trained pediatric radiologists have a mistake rate of approximately . %, if misses and overcalls are included. from a quality improvement perspective, we will review all of the types of misses and overcalls to expose common themes. longitudinal assessment of osteoporosis in a blood-induced hemophilia rabbit model using quantitative ultrasound kuan-chieh wang, university of toronto, kc.wang@ utoronto.ca; afsaneh amirabadi, anguo zhong, christopher tomlinson, andrea s. doria purpose or case report: the reduction of physical activities in hemophilic patients may lead to bone demineralization and consequent osteoporosis. quantitative ultrasound (qus) is free of ionizing-radiation, relatively inexpensive, and easy to use that making this technique suitable for follow-up of hemophilic children with clinical suspicion of osteoporosis. to our knowledge, no previous study has investigated the value of qus for longitudinal assessment of growing bones in an animal model which is paramount for clinical translation of the technique once change in measurements could relate to either the baseline pathology or physiologic bone growth variability. the objective of this study is to investigate the intra-and inter-operator reliability of qus over time, and its ability to discriminate bone loss in pathologic vs control knees of a rabbit model of blood induced arthritis. methods & materials: sixteen juvenile white new zealand rabbits distributed into two groups: received intraarticular blood injections over weeks (n pathologic and contralateral knees), and noninjected rabbits were used as controls (n knees). midshaft tibia speed-of-sound (sos) was measured at baseline, and weeks and of the experiment. two operators scanned each site twice at each time point. qus measurements were compared to microct (reference standard) on week to validate the study results. results: the sos measured in the control group increased significantly (p< . ) over the week period. there was not such an increase in the arthritis sos value (p> . ). in both groups the overall intra-operator coefficient of variation of sos measurements was % at baseline and decreased to % at week likely due to increased tibia size. the inter-operator reliability was % at baseline and % at week . with regard to the effect of bone growth on qus measurements for the control group (n ), sos values increased by . m/s, whereas for the pathologic group (n ), they only increased by m/s. statistically significant differences in ratios of sos between final/baseline results were noted (p . ) between the pathologic and control groups. conclusions: the longitudinal use of qus has an acceptable intra-and inter-operator reliability. even accounting for the significant impact that bone growth has on qus measurements over time, qus can differentiate pathologic from control knees in the proposed animal model and holds potential for clinical use in the assessment of osteoporosis in hemophilic children. methods & materials: the study was approved by the institutional review board. pediatric patients with abdominal tumors ( malignant and benign lesions) underwent diffusion-weighted mr imaging (dwi) on clinical . t (n ) and t (n ) mri scanners. adc maps were generated from b dwi and adc values were retrospectively and independently measured by two radiologists. adc values of benign and malignant tumors were compared with the welch two sample t-test. a p value of . was considered to indicate statistical significant differences. in addition, a receiver operating curve analysis (roc) was performed to determine the optimal cut-off adc value for differentiating benign and malignant tumors. results: the mean adc value (mm /sec) of benign tumors was . x - for the first reader and . x - for the second reader. the mean adc value (mm /sec) of malignant abdominal tumors was . x - for the first reader and . x - for the second reader. the differences between benign and malignant tumors were statistically significant (p< . for both readers). roc analysis revealed an optimal cut-off adc value for differentiating malignant and solid tumors as . x - mm /sec. conclusions: diffusion-weighted imaging with adc maps can be used to differentiate between benign and malignant pediatric abdominal tumors. creation of a database to evaluate imaging findings in long-term survivors of pediatric malignancy alexander towbin, md, radiology, cincinnati children's hospital medical center, alexander.towbin@cchmc.org; seth hall purpose or case report: over the past years, there have been significant improvements in the treatment of pediatric malignancies. improved therapy has led to an increase in the number of long-term survivors. many of these survivors are now experiencing late effects as a result of the original disease process or its treatment. these late effects are frequently identified on imaging. the purpose of this study is to create a database of the imaging findings of long-term survivors of pediatric malignancy in an attempt to begin to classify the findings and identify associations. methods & materials: after irb approval, the institutional cancer registry was searched to identify all patients younger than years of age who were diagnosed with a solid tumor between and . patients were included in the database if they survived for more than years from the date of their initial diagnosis. the electronic medical record system was then used to obtain demographic and treatment information for each included patient. the dictated reports from all cross-sectional imaging studies evaluating the chest, abdomen, or pelvis performed more than two years from the date of diagnosis were then reviewed. each positive imaging finding was classified by the involved organ. results: after querying the institutional cancer registry, patients were identified who met the inclusion criteria for this database. the most common neoplasms were neuroblastoma, wilms tumor, and astrocytoma. of the included subjects, had imaging of the chest, abdomen, or pelvis. overall, reports were evaluated and classified. findings were most commonly identified in the lungs, musculoskeletal system, kidneys, liver, and lymph nodes. conclusions: a database examining the late effects in longterm survivors of pediatric malignancies was created. this database has the potential to help identify the radiologic manifestations of the complications of cancer therapy and thus help guide rationally determined long-term risk-benefit ratios in the treatment of pediatric malignancies. imaging followup of lymphoma in pediatric patients: is pelvic ct necessary? javier lopez bueno, md, children's hospital of eastern ontario, jlopezbueno@cheo.on.ca; nishard abdeen purpose or case report: pelvic ct is often included in the imaging followup of patient with lymphoma before, during and after treatment to assess response to treatment and monitoring for relapses. while such followup is expected to improve detection of relapse, there is little objective evidence of its effectiveness in lymphoma. anecdotally, there are few pelvic relapses in pediatric patients with lymphoma regardless of primary site. we hypothesize that pelvic ct could be avoided as part of the followup without adverse impact on survival or in the detection rate of relapses, and with subsequent significant reduction in the radiation dose, particularly to the gonads. methods & materials: research ethics board approval was obtained. patients diagnosed with lymphoma and with at least one year of followup at our tertiary care pediatric hospital were included. sex, age, type of lymphoma, stage, primary site, site of relapse if any as well as the number of ct scans of the head, neck, chest, abdomen and pelvis were recorded. results: a total of patients met study criteria. there were males and females, with an average age of . years (range - years). eighteen patients had hodgkin disease ( %) and eleven had non-hodgkin lymphoma ( %). mean length of followup was . years (range - years). an average of . pelvic scans per patient were performed for surveillance (range - ). three relapses were detected. of these only one was in the pelvis, in a patient whose initial t cell non-hodgkin lymphoma was extensive and involved the neck, chest, abdomen and pelvis. conclusions: this study suggests a low incidence of pelvic relapse in pediatric patients with lymphoma. the routine use of pelvic ct in surveillance protocols may therefore be of little benefit while imposing a significant radiation burden. our study is limited by small sample size and short length of followup. further large scale studies are required. (esft) is performed by measuring the size of the tumors before and after chemotherapy. the proposed method of measuring tumor size, however, differs amongst recist . (response evaluation criteria in solid tumors), who (world health organization) and cog (children's oncology group) response criteria. in our project, we assessed whether response classification differs between the three different methods. methods & materials: after irb approval, we retrospectively analyzed mri studies of patients with ewing sarcoma who were treated at stanford and ucsf medical centers. tumor size was assessed before and after therapy. tumor measurements were obtained using recist . (longest single diameter), who (longest diameter and perpendicular diameter), and cog criteria (three measurements to calculate tumor volume). tumor response was assessed by the differences in sizes of the tumors before and after treatment using four response categories: progressive disease (pd), stable disease (sd), partial response (pr), and complete response (cr). concordance between the three response classification systems was assessed using cohen's kappa (k) coefficient and percentage of disagreement per response category. results: the k statistic for concordance in cog/who, cog/ recist and recist/who were . , . and . respectively. disagreement rates for recist/who, cog/ who, and cog/recist were . , . , and . % respectively. using tumor volume, twenty-six patients were reclassified: twenty-four cases of stable disease coded by recist were reclassified as progressive disease by cog and two cases of partial response coded by recist were reclassified as complete response by cog. conclusions: this study demonstrates poor agreement between the recist . and cog response criteria in esft. given the degree of discordance between response criteria in esft, evaluation of the prognostic impact of each of these classification systems may guide selection of the optimal system for future use in this disease. imaging recognition of chylous ascites following surgery for abdominal neuroblastoma zeyad metwalli, md, baylor college of medicine, metwalli@bcm.edu; r. p. guillerman, heidi v. russell, eugene s. kim purpose or case report: surgical resection is a standard part of multimodality treatment of neuroblastoma, the most common abdominal malignancy of infancy and early childhood. chylous ascites is a rarely reported complication of surgery for abdominal neuroblastoma, and is likely underrecognized, posing the risk of nutritional deterioration and sepsis. to facilitate early diagnosis and institution of appropriate therapy, we present the salient imaging findings of the largest known series of chylous ascites following surgery for abdominal neuroblastoma. methods & materials: all patients with abdominal neuroblastoma complicated by post-operative chylous ascites over a five-year period at a large children's hospital were identified by a database search. a retrospective review of the imaging studies and clinical charts was conducted. results: chylous ascites developed following surgical resection of abdominal neuroblastoma in of patients, with the diagnosis made between postoperative days and . four cases were high-risk neuroblastoma and one was intermediaterisk neuroblastoma. all cases involved resection of an adrenal mass and dissection around the abdominal great vessels. all cases manifested with abdominal distention on physical exam, and ascites was suspected clinically in cases. computed tomography (ct) in all cases revealed a large volume of ascites of near-water attenuation (range of − to . hounsfield units). the cases imaged with ultrasound (us) showed hypoechoic or anechoic ascites without septations. the chylous ascites resolved after - months of treatment with dietary fat restriction, medium chain triglycerides, intravenous octreotide, or peritoneal catheter drainage. conclusions: chylous ascites is an under-recognized complication of surgical resection for abdominal neuroblastoma, occurring in % of patients in this series. the diagnosis is supported by the demonstration on ct or us of a large volume of ascites causing abdominal distention - weeks post-operatively. the ascites is typically near-water in attenuation rather than fatty in attenuation and should not be misattributed to peritonitis, hemorrhage, bowel leak, or early tumor recurrence. cervical spine injuries in patients with suspected physical abuse nadja kadom, md, radiology, children's national medical center, nkadom@childrensnational.org; zarir p. khademian, tanya hinds, katherine deye, allison m. jackson, eglal shalaby-rana purpose or case report: to evaluate the incidence and nature of cervical spine injuries and relationship to posterior fossa abnormalities in children who underwent brain and cervical spine mri as part of the clinical workup for suspected physical abuse. methods & materials: authors retrospectively analyzed records of eighty-five children less than three years of age who were documented by the child protective services at a level one pediatric trauma center over a period of four years ( ) ( ) ( ) ( ) ( ) . only patients who underwent both mri imaging of the cervical spines (c-spine) in addition to brain imaging as part of the clinical workup were included. cspine and posterior fossa of brain mris were independently reviewed by two pediatric neuroradiologists, both blinded to clinical details. c-spine abnormalities (bone marrow edema, cord edema, intrathecal blood, disc pathology, soft tissue/ ligamentous injury, vascular injury) were documented and correlated with abnormalities seen in the posterior fossa (blood, brainstem edema, cerebellar edema). results: at this time, / patients have been reviewed. twenty patients ( %) had both cervical spine injuries and posterior fossa abnormalities. there were no patients with isolated cervical spine injuries without posterior fossa abnormalities, but there were five patients ( . %) that had posterior fossa abnormalities in the absence of c-spine injuries. fifteen patients ( . %) did not have any spinal or posterior fossa imaging abnormality. none of the patients had bone marrow edema, disc pathology, or intrathecal blood. one patient had vascular neck injury and cord edema. conclusions: our results show that the incidence of cervical spine injury in children under investigation for abusive head trauma is as high as %. our data show further that cervical spine injury predicted posterior fossa injury in all patients, while presence of posterior fossa injury predicted concomitant c-spine injury in only %. the incidence of c-spine trauma we found in these patients is higher than reported elsewhere in the literature and may impact whether or not routine c-spine mri will be included in national imaging guidelines for children under investigation of abusive head trauma. pediatric skull fracture andre loyd, phd, biomedical engineering, duke university, aml @duke.edu purpose or case report: skull fractures are often seen in the setting of non accidental trauma (abuse) abuse, and are usually attributed to falls from heights above m. part of the difficulty in assessing height is due to uncertainties in actual distance. objective: to determine what types of skull fractures can occur in pediatric and adult post-mortem human specimens during controlled impacts on hard surfaces from various heights. methods & materials: skull fracture patterns in postmortem human specimens from a unique bank of pediatric specimens ( -week gestation to -years-old, n ) were subjected to controlled drops from both arbitrarily low heights ( and cm) and high heights ( m) onto an aluminum platen. the specimens were dissected from the neck at the occipital condyles and intracranial were sealed inside the head using pmma. the heads were dropped on to five different impact locations. fractures were identified using palpation and high resolution mdct. results: no specimens between -weeks-gestation and days-old sustained fractures from the - cm drops. three out of four ( %) specimens ages between -and -months old fractured due to the or cm drops. the -and -year-old specimens and all adult specimens survived the - cm drops. all specimens subjected to the m drop fractured. the specimen between -months and -months sustained either a linear fractures or diastatic fractures from the cm and cm drops. the results indicate that some aged infants and young children can sustain skull fractures by being dropped or falling from relatively low heights. drops, as low as cm, can cause linear and diastatic fractures in pediatric skulls. the presences of compliant sutures and fontanelles in neonatal heads allow the head to deform during impact. these data add very important information to mechanisms of skull fractures across ages, including ages in which child abuse is a consideration. evaluation of a new classification system for temporal bone fractures in children aimed at increasing prognostic value badriya al-qassabi, md, mcgill university, albahlania @ yahoo.com; lucia carpineta, rania ywakim, bahar torabi, andrew m. zakhari, lily h p. nguyen purpose or case report: to compare a new classification of temporal bone fractures which specifically evaluates involvement of the otic capsule against the traditional classification system (transverse versus oblique versus longitudinal), to evaluate whether this new classification is able to better identify patients at risk of adverse otologic outcome and neurologic complications in the pediatric population. methods & materials: a retrospective hospital chart review was performed by ent colleagues searching for all patients with temporal bone fractures seen at our center over the past years. this was followed by a blinded review of the ct heads by a resident and a trained pediatric radiologist with neuro expertise. these cts were evaluated for petrous involvement, otic capsule involvement and any associated intracranial lesions. this information was then correlated with clinical outcome measures including post-traumatic hearing deficit, facial nerve palsy, persisting csf leak and global neurologic sequelae. the new classification was compared to the traditional one, and specifically analysed for the ability to better predict the clinical outcomes. results: expectedly, pediatric temporal bone fractures were infrequent and otic involvement even more rare. fractures with involvement of the otic capsule (versus otic sparing) were found more frequently in boys. they were also more likely to be associated with immediate otologic signs and neurologic findings on presentation. these fractures also had the highest association with conductive hearing deficit (> %) and were twice as likely as otic sparing fractures to be associated with immediate facial nerve palsy and with more important concomitant intracranial injuries such as midline shift. conclusions: while our numbers are small, our results suggest a trend that when temporal bone fractures show involvement of the otic capsule, there is higher risk of adverse otologic outcome and neurologic complications even in the pediatric population. absence of a causal relationship between mr detected subdural hematomas (sdh) in neonates with hypoxic-ischemic encephalopathy (hie) deniz altinok, children's hospital of michigan; jay shah, harut haroyan, gulcin altinok, nitin chouthai purpose or case report: the existing controversy regarding subdural hemorrhages noted in patients with hie is an important discussion in the medical, legal and child-welfare realms. it is our goal to provide additional information to this critical debate through mr findings on patients with clinically diagnosed hie. methods & materials: all patients born with clinically diagnosed hie, and treated at children's hospital of michigan in the past years were examined; those with head mri taken within days of life were selected. in total, patients fit the criteria, this included: males and females, and an age range of - days at scan (average age of days at scan). all traumatic births, coagulopathies, and other pertinent clinical findings were noted. mr imaging was reviewed and reported by a blinded pediatric neuroradiologist, these reports were then compared to the "original read". results: all patients were confirmed radiologically to have hie. the causes of hie in all cases examined were either intrauterine/delivery asphyxia, aspiration, or congenital disease. of these cases, the findings were: sdh, parenchymal hemorrhages, intraventricular hemorrhages, cephalohematomas, subarachnoids, large subcutaneous hemorrhage and instance of mca stroke. all patients with mr detectable sdh had or more confounding factors ( meningitis, coagulopathies, chest compressions, cardiac malformation, pph, severe pulmonary hemorrhage requiring transfusion of plasma and prbc). conclusions: it has been hypothesized that sdh is often found incidentally in children diagnosed with hie, this is however a dubious conclusion considering our results. in fact, the presence of sdh and hie concomitantly is low even when including a population with traumatic births such as ours. sports-related concussion in children: an mri and mrs study kim cecil, phd, cincinnati children's hospital medical center, kim.cecil@cchmc.org; todd a. maugans, james l. leach, mekibib altaye purpose or case report: the pathophysiology of sportsrelated concussion (src) is poorly understood, especially for children. following src and mild traumatic brain injury in adults, a few mri and proton mrs studies have identified axonal injury with declines in the neurometabolite n-acetyl aspartate (naa). we wanted to examine a src adolescent population with proton mrs, diffusion tensor imaging (dti) and other mri methods within h of concussion and with short term followup to determine if there were differences in imaging metrics with age and sex matched healthy control participants. methods & materials: twelve children, ages - years, who experienced src were evaluated with impact neurocognitive testing, t -weighted mri, susceptibility weighted imaging (swi), dti, proton mrs, and phase contrast angiography (pca) at less than h, days and days or greater post-concussion. healthy, age and sex matched controls for each src participant were recruited and evaluated at a single time point. quantitative imaging metrics included fractional anisotropy, metabolite concentrations, and global cerebral blood flow (cbf). group comparisons were examined by paired t-test or wilcoxon signed rank test. correlational data employed spearman rank correlation. results: impact results revealed significant differences in initial total symptom score (tss), and reaction time (rt) for the src group compared with the control group, with tss resolving by a mean of days and rt at days. no evidence of structural injury was observed qualitatively for either group. analyses between groups or over time within the src group found no decreases in naa or elevation of lactic acid upon mrs, and no changes in fractional anisotropy upon dti. within the src group, significant changes in the global cbf were observed. improvement towards control values occurred by days for % and by days for % of src group participants. conclusions: pediatric src affects global cbf without evidence of structural or metabolic injury. predictive value of high resolution mr imaging of brain and sella in children with clinical optic nerve hypoplasia for hypopituitarism charles glasier, radiology, arkansas childrens hospital, glasiercharlesm@uams.edu; raghu h. ramakrishnaiah, julie shelton, chetan c. shah, paul h. philips purpose or case report: to review the spectrum of cns abnormalities and their incidence in children with optic nerve hypoplasia and to calculate the sensitivity and specificity of magnetic resonance imaging in predicting endocrine abnormalities. methods & materials: this is an irb approved retrospective study of children with clinical optic nerve hypoplasia who underwent mri of the brain and orbits as part of the clinical workup in a tertiary care pediatric hospital. high resolution mri studies were performed on . tesla scanners. mri studies were reviewed for optic nerve hypoplasia, absent or ectopic posterior pituitary, absent pituitary infundibulum, absent septum pellucidum, migration anomalies and hemispheric injury.radiologists were blinded to patients endocrinologic status.all patients had clinical evaluation by a pediatric neuro-ophtalmologist and endocrinologist. a standardized panel of serologic testing that included serum cortisol, acth, tsh, and free t levels were performed on all patients. statistical analysis was performed to determine the sensitivity and specificity of mr findings in predicting endocrinologic deficiency. results: study included children( males and females) who had clinical optic nerve hypoplasia. the mean age of the study population was yr (sd: . yr). children had unilateral and children had bilateral optic nerve hypoplasia by mri. children had absent posterior pituitary bright spot and had ectopic posterior pituitary, had absent infundibulum, had complete callosal agenesis, partial callosal agenesis and had callosal thinning. had absent septum pellucidum. had hypopituitarism. of the patients with hypopituitarism had abnormal abnormal pituitary on mri, had absent septum pellucidum, and child had migration abnormality. none had corpus callosal abnormality. the sensitivity and specificity of mri in predicting hypopituitarism by demonstration of abnormal pituitary is % and % respectively. the positive predictive value and the negative predictive value is % and % respectively. among the patients with normal endocrinologic function, none had pituitary abnormalities on mri. conclusions: pituitary abnormalities are the most common intracranial abnormality in patients with optic nerve hypoplasia followed by absent septum pellucidum. detection of pituitary abnormalities by the mri has high specificity and high negative predictive value for endocrine abnormality. paper #: pa- ct imaging pearls for shunted pediatric brains srikala narayanan, md, children's national medical center, snarayan@childrensnational.org; nadja kadom purpose or case report: shunted pediatric patients frequently present emergently with symptoms that could indicate shunt malfunction, such as headache and vomiting. here, we present imaging pearls on non-contrast head ct in shunted children. methods & materials: illustration of each of the following: . shunt tip and volume averaging-consider location of side holes and use of multiplanar reformatted images. . shunt at burr hole-consider radiolucent shunt parts. . shunt rupture in the neck-remember to investigate the lower extracranial shunt parts. . shunt in cyst/subdural shunt (vs dislocation)-consider primary shunt location in a cyst rather than shunt dislocation. . enlarged temporal horns-look for it. in infants occipital horns may dilate first. . enlarged rd ventricle-look for bulging of lateral walls. . sulcal effacement-use the "three shades of gray" rule. . small cisterns-detecting shape distortion can help. . periventricular edema-easily overlooked because of similar low density compared to ventricular fluid. . slit-ventricle -requires cautious reporting. conclusions: careful evaluation of ct images in shunted pediatric patients can reveal important clues for making an accurate diagnosis, even when prior images are not available. successful treatment of mice with creatine transporter deficiency kim cecil, phd, cincinnati children's hospital medical center, kim.cecil@cchmc.org; diana m. lindquist, matthew r. skelton, gail j. pyne-geithman, joseph f. clark purpose or case report: creatine transporter deficiency (ctd) is an untreatable x-linked mental retardation syndrome with severe cognitive and speech impairment. patients are identified by an absence of creatine in the brain on mr spectroscopy (mrs) and distinguished from two creatine synthesis deficiency syndromes with genetic testing. for ctd, the absence of the transporter (slc a ) prevents creatine from crossing the blood brain barrier and entering brain cells. a brain specific ctd knockout mouse was developed replicating key features of the human disease and establishing an animal model for treatment of ctd. we report the successful treatment of the ctd knockout mouse and present confirmation by mrs. methods & materials: brain specific knockout and littermate control mice were randomly assigned and treated with on one of three supplements: agentx (confidential), creatine or maltodextrine as placebo. h and p mrs data were collected on a t mr system (bruker). mice (n ) were studied with mrs after weeks of supplementation. single voxel h data were acquired on a ul voxel covering the cerebrum using a double spin echo sequence. p data were acquired with an isis sequence from the same voxel. metabolite quantification was performed with jmrui and compared between groups and over time with statistical tests for significance (t-tests, anova). results: creatine and phosphocreatine levels in the brain were all significantly higher after weeks supplementation of agentx in knockout mice, compared to creatine and placebo fed knockout mice (phosphorus mrs [ ul brain voxel] with phosphocreatine (pcr) ( ppm) observed only in agentx treated knockout mice. adenosine triphospate (atp) gamma (− . ppm), alpha (− . ppm) and beta (− ppm) peaks are noted in all three knockouts. conclusions: successful treatment was achieved in a slc a brain specific knockout mouse for the second largest known cause of x-linked mental retardation in humans, ctd. disclosure: dr. cecil has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. prevalence of abusive injuries in siblings and contacts of abused children kenneth feldman, md, general pediatrics/children's protection program, university of washington/seattle children's, kfeldman@u.washington.edu purpose or case report: siblings and children who share a home with a physically abused child are thought to be at high risk for abuse. however, rates of injury in these children are unknown. disagreements between medical and cps professionals are common and screening is highly variable. our objective was to measure the rates of occult abusive injuries detected in contacts of abused children using a common screening protocol. this was a multi-center, observational cohort study of child abuse teams who shared a common screening protocol. data were collected for all children < years undergoing evaluation for physical abuse and their contacts. for contacts of abused children, the protocol recommended physical examination for all children < years, skeletal survey and physical exam for children < months, and physical exam, skeletal survey and neuroimaging for children < months old. results: among , children evaluated for abuse, met criteria as "physically abused" and these had contacts. for each screening modality, screening was completed as recommended by the protocol in approximately % of cases. of contacts who met criteria for skeletal survey, new injuries were identified in ( . %). none of these fractures had associated findings on physical examination. physical examination identified new injuries in . % of eligible, examined contacts. neuroimaging failed to identify new injuries among imaged, eligible contacts less than months old. twins were at significantly increased risk of fracture relative to other non-twin contacts ( . % vs . %, or . ). conclusions: these results support physical examinations and skeletal survey, regardless of physical examination results, for contacts of abused children < months of age. too few children had cranial imaging to change recommendations to image contact children less than months old. even for children where no injuries are identified, these results demonstrate that abuse is common among children who share a home with an abused child. they support including contacts in evaluations and interventions (foster care, safety planning, social support) designed to protect physically abused children. the project was supported by the health resources and services administration/maternal shown that pediatric rib fractures may be a marker for significant intrathoracic injury. this information has been used to suggest that children with rib fractures and no underlying intrathoracic injury may have sustained them due to insufficient bony mineralization and minor trauma rather than inflicted injury. methods & materials: irb approval was obtained for a retrospective review of all children under years of age with imaging diagnosis of rib fracture over a -year period at two university hospitals. children with prior thoracotomy, previously recognized metabolic bone disease, and prematurity < weeks were excluded. medical records were reviewed and children with documented abuse or accidental trauma were evaluated. children with indeterminate injury mechanisms were excluded. sixty-six patients with rib fractures were included in analysis, due to abusive injury and due to accidental trauma. children were analyzed for associated intrathoracic, abdominal or intracranial injury, additional fractures and retinal hemorrhage. results: abused children were younger ( . +/− . months) than accidentally injured children ( . +/− . months, p< . ). children with rib fractures due to accidental trauma had a higher incidence of intrathoracic injury compared to those due to abusive injury ( % vs %,p< . ). there was no difference in the incidence of abdominal or intracranial injury between groups. mortality and icu admission rates were similar. abused children had a higher total number of rib fractures (mean . vs . , p< . ) and were more likely to sustain additional fractures outside of the thoracic cavity ( % vs %, p< . ). conclusions: abuse is a more common cause of rib fractures in young children than accidents. children with rib fractures due to abusive trauma are less likely to have intrathoracic injury compared to those sustaining rib fractures due to accidental trauma. this suggests differences in mechanism of injury between groups. pediatric elbow fractures: a different angle on an old topic shannon zingula, md, pediatric radiology, cincinnati children's hospital medical center; kathleen emery, christopher g. anton purpose or case report: the most common elbow fractures classically reported in pediatric orthopedic texts are supracondylar (sc) ( - %), lateral condylar (lc) ( %), and medial epicondylar (me) fractures ( %) with fractures of the proximal radius (including but not limited to fractures of the radial neck) being relatively uncommon ( - %). our experience at a large children's hospital suggests a different distribution. purpose: ) to describe the frequency of different elbow fracture types in a large pediatric population, and ) to determine the fracture types that were occult on initial radiographs but detected on follow-up. methods & materials: review of medical records identified children, median age years and interquartile range for age of - years (range, . - years) diagnosed with elbow fractures at our institution from october through july . initial and follow-up radiographs were reviewed in blinded fashion independently by two experienced pediatric musculoskeletal radiologists to identify fracture type(s) on initial and follow up radiographs. note was made of fractures identified on follow up only. results: the most common fractures included sc (n , %), radial neck (rn) (n , %), and lc fractures (n , %). as compared to classically referenced incidences, rn fractures were seen significantly more (p< . ) and me fractures (n , %) significantly less (p . ) than would be predicted. in patients without fracture seen on initial films, occult fractures were seen on follow up; sc (n , %) and rn fractures (n , %) were most common. the frequency of rn fractures compared to the overall group ( % vs. %) approached but did not reach statistical significance (p . ). patients with one fracture had additional fractures seen on follow-up not seen initially with olecranon fractures most frequent (n , %.) this was significantly more common than the number identified on initial radiographs (n , %) (p< . ). conclusions: sc fractures are the most frequent elbow fracture seen initially and in follow up followed by rn and lc fractures in a distribution different than classically described. the relatively high frequency of rn and olecranon fractures detected on follow up speaks to their potentially occult nature. careful attention to these areas is warranted in patients with initially normal radiographs. purpose or case report: previous studies have found that fractures involving the spine, hands and feet are rare on skeletal surveys for suspected child abuse, leading some authors to suggest eliminating views of these regions from the initial skeletal survey protocol. the purpose of this study was to assess this recommendation by performing a historical review of these injuries in a population undergoing screen-film based skeletal surveys for suspected abuse. this cross-sectional, retrospective irb approved study reviewed the reports of the initial skeletal surveys of all children < years of age with suspected abuse imaged between april, and december, . infants underwent skeletal surveys according to acr standards acquired on a mammographic type screen-film imaging system with at least line pairs per millimeter resolution. studies in toddlers were performed using a par speed screenfilm system. results: % ( / ) of all skeletal surveys demonstrated positive findings, and % ( / ) had > fracture. . % ( / ) of all studies had fractures involving the spine, hands or feet. of all positive skeletal surveys, . % ( / ) had fractures involving the spine, hands or feet, and . % ( / ) of all patients with > fracture on skeletal survey had fractures involving these regions. conclusions: these data, acquired in the screen-film era, suggest that fractures of the spine, hands and feet may not be rare in infants and toddlers in cases of suspected child abuse. the benefits of eliminating views of these regions from the initial skeletal survey should be carefully weighed against the cost of missing these potentially important injuries in at-risk pediatric populations. purpose or case report: dating fractures is critical in cases of suspected infant abuse, but there are little scientific data to guide radiologists, and dating is generally based on personal experience and conventional wisdom. we previously reported a scientific scheme for dating fractures in infants based on an analysis of subperiosteal new bone and callus formation in birth-related clavicular fractures. we hypothesize that when used as a guide this system can significantly improve the ability of radiologists to accurately date fractures in young infants. methods & materials: radiographs of presumed birthrelated clavicular fractures in infants - months were reviewed by pediatric radiologists with (reader a) and (reader b) years experience in two reading sessions separated by one year. for the first read, no guidelines were provided. training was carried out prior to the second session, and readers were given the dating scheme as a guide during fracture analysis. readers were asked to provide an estimate of the minimum and maximum fracture age in both sessions. the primary outcome was whether or not the reader's estimated range for fracture age included the actual fracture age. a secondary outcome was the width of the estimate of fracture age. these outcomes were compared across the two reading sessions. results: the rate of correct response significantly increased after training for each reader (reader a: % to %, p<. ; reader b: % to %, p . ). the width of estimated fracture age after training was significantly smaller for each reader (reader a: mean width days to days, p<. ; reader b: days to days, p . ). conclusions: our results suggest that the ability of a radiologist to accurately date fractures can improve significantly when provided with a scientifically based system outlining patterns of fracture healing. this scheme can be applied in radiologic practice and may prove particularly useful in cases of suspected abuse, where fracture dating often has forensic implications. purpose or case report: to demonstrate the acute and subacute features of proximal femoral physeal fractures in the abused child. also to demonstrate how to recognize this injury in patients with unossified femoral heads. the database of patients with suspected non-accidental trauma, accumulated over years, was reviewed. out of a total of patients ( %) were proven to be cases of non-accidental trauma, as determined by the child abuse pediatrician. from these patients, the cases of proximal femur growth plate fractures were identified. results: patients with proximal femur growth plate fractures were identified for a prevalence of . %. one patient had bilateral proximal femoral fractures, for a total of fractures in patients. were boys, were girls with ages ranging from . mos to yrs mos. in patients, the fracture was revealed on imaging performed because of refusal to bear weight; in the other patients, the fracture was found during imaging for the skeletal survey. the fracture was on the left side in cases and on the right side in (the patient with bilateral fractures). in all of the fractures, there was lateral displacement of the femoral shaft. in fractures, the femoral head was not yet ossified simulating the appearance of a dislocation. location of the femoral head in the hip joint was verified by ultrasound or ct (ct abdomen had already been done in patient) thus delineating the presence of a physeal fracture. / fractures were salter-harris i and the other were salter-harris ii fractures. the fracture was acute in cases and subacute in cases. in these subacute cases, periosteal reaction and/or calcifying subperiosteal hemorrhage was present in , and irregularity and scalloping of the metaphysis was present in the other . conclusions: proximal femoral growth plate fractures are quite uncommon in non-accidental trauma. the injuries are typically salter-harris i or ii fractures, seen more often in the healing phase. in the presence of an unossified femoral head, the laterally displaced femoral shaft can simulate hip dislocation; this can be clarified with hip sonogram. purpose or case report: in recent years, metal stents have been used to overcome airway obstruction in children for whom no better surgical option is available. these devices are not designed for use in the airway, however, and may cause significant complications. bioabsorbable airway stents may avoid some of the problems associated with metal stents. methods & materials: this is a retrospective review of all endoluminal insertions of bioabsorbable airway stents at a single institution from april to september . custom-made polydioxanone stents of various sizes (ella dv, ella, czech republic) were used. results: twelve stents were inserted in the airways of seven children. indications were: recurrent obstruction after slide tracheoplasty ( ), persistent airway compression after correction of a congenital cardiac lesion ( ), collapse of stem cell supported tracheal homograft, tracheomegaly following fetal balloon insertion, and syndromic tracheobronchomalacia (tbm). eleven stents (diameters to mm) were placed in the trachea and one in the left main bronchus. two stents had to be removed and replaced for technical reasons (one was too long and the other too narrow). the child with syndromic tbm died when treatment was withdrawn because she could not be weaned from the ventilator. the remaining children are alive at a median follow-up of nine months (range to months). the granulation tissue response was similar to that seen after placement of metal stents. the stents were observed to absorb gradually over a period of approximately three months, requiring serial stenting in two children. conclusions: bioabsorbable airway stents are more difficult to insert than metal stents. they cause similar early complications, especially granulation tissue formation, but appear to avoid potential long-term complications of metal stents, including vascular erosion and growth limitation. disclosure: dr. mcclaren has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: renal angiomyolipomas (amls) in tuberous sclerosis complex (tsc) grow at a faster rate, exhibit a wider and more problematic range of symptoms, and hemorrhage more frequently than sporadic amls. we examined the efficacy of prophylactic embolization of renal amls in tsc in decreasing tumor size, alleviating symptoms, and preventing hemorrhage while preserving renal function. we retrospectively reviewed the charts and imaging studies of consecutive patients who underwent transarterial, transcatheter embolization of amls. tumor volume was measured from available ct or mri imaging before and after embolization. pre-and postembolization symptoms and creatinine levels were documented. results: patients had available follow-up imaging at a mean of months post-embolization. the mean preembolization tumor volume was ml and postembolization was ml; median decrease in volume was %. using the schwartz method, the mean glomerular filtration rate before embolization was calculated to be . ml/min/ . m . after embolization the mean value was statistically unchanged at . ml/min/ . m . none of the patients experienced renal hemorrhage or symptom recurrence during the follow-up period. conclusions: selective embolization of renal amls in patients with tsc decreases tumor volume, relieves symptoms and reduces the risk of future hemorrhage while preserving renal function. etoh was injected percutaneously with g needle; transductal ablation performed through a f micropuncture sheath. drug volumes, technical difficulties, percentage reduction in saliva production, family reported clinical significance, and complications were recorded. results: salivary gland ablation (sga) included bilateral smg and slg ablation without parotid gland ablation in cases, and with unilateral parotid gland ablation in cases. one case of bilateral parotid gland ablation following surgical resection of bilateral smgs. mean etoh dose for smg . ml, and . ml for slg. one case of focal skin necrosis was noted; no other complications. patient families reported response to sga in / cases ( %) with mean saliva production of %. greatest health and family impact was reported with elimination of hospitalizations for recurring aspiration pneumonia ( cases), elimination of choking in bed ( cases) , and improved patient sense of self-hygiene in cases. one complication occurred with temporary marginal mandibular nerve paralysis (resolution in months). conclusions: percutaneous and transductal sga is feasible, safe, and effective in this small patient series, offering an alternative to surgical salivary gland resection, or treatment option following failed surgical intervention. paper #: pa- mr-guided procedures in children: initial experience joao amaral, md, diagnostic imaging, the hospital for sick children, joao.amaral@sickkids.ca; michael temple, dimitri parra, philip john, bairbre connolly purpose or case report: the primary purpose of this study was to review our initial experience with mr-guided procedures in children. our secondary objective was to share some aspects on how to start an mr-guided program in a tertiary pediatric center. patients with lesions identified only on magnetic resonance (mr) imaging were selected to undergo an mr-guided procedure. patients' demographic data, primary diagnosis, referring team's clinical suspicion, lesion's anatomical location, tissue adequacy for pathology, final diagnosis and clinical follow up were reviewed. aspects of starting a program of mr-guided procedures, safety concerns, imaging and technical challenges, and mr compatible materials were also addressed. results: to date, procedures ( bone biopsies, soft tissue biopsy and pre-surgical needle localization) were performed in patients during months. there were girls and boys with a mean age of . years ( y mo- yrs). one patient had a nasopharyngeal carcinoma, cardiofacial syndrome, wilm's tumor and had no previous medical issues. the clinical suspicion for procedures in patients was metastatic disease and for procedures in patients was primary malignancy or infection. lesions were located in the tibia ( -metaphysis and diaphysis), femur ( -metaphysis and epiphysis), thigh ( -soft tissues), sacrum ( ) and retroperitoneum ( ). all biopsies provided adequate tissue for diagnosis. needle localization and hook deployment was also accurate. malignancy was excluded in all patients. final diagnosis included chronic recurrent multifocal osteomyelitis (crmo), osteomyelytis, fibrous tissue, osteoid osteoma, and scar tissue. mean follow up was . months. no patient required a second procedure to confirm the diagnosis. conclusions: mr with its unique soft tissue resolution and lack of ionizing radiation is an excellent method to guide interventions in children. one of the greatest advantages of this method is the precise target localization especially in lesions located in the bone marrow or lesions better identified on mr. special safety measures, specific mr compatible material (needles, surgical instruments), dedicated imaging techniques to reduce or increase material/needle artifact and careful technique are paramount. - . m, f; . - months, mean . months, median months, mode months. sonographic approach expanded as our experience grew over months. studies performed by a single pediatric radiologist. bilateral sonography included: interscalene and supraclavicular neck, nerve roots at neural foramina, cervical spinal canal, diaphragm during spontaneous respiration, rhomboid muscle, serratus anterior muscle, posterior shoulder, all performed and interpreted blind to other imaging. results: interscalene and supraclavicular neck evaluated in all patients. all exhibited echogenic interscalene portion of brachial plexus.size and extent of traction neuroma varied. nerve roots at foramina noted in axial and coronal planes. in cases enlarged root(s)noted. cervical spinal canal studied in patients: cord oscillated normally, no syrinx, cord concentric in canal. intracanalicular traction pseudomeningoceles on concurrent ct myelography or mri were not apparent on us. in cases a "clumped" retracted nerve root on the cervical cord was later found to correspond to a pseudo-meningocele on ct myelogram. otherwise, cervical spinal canal us was unremarkable in cases. diaphragm motion was evaluated in patients during spontaneous respiration; no phrenic nerve palsy. rhomboid muscle was evaluated for atrophy in patients; had atrophy. the rhomboids are innervated by the dorsal scapular nerve which arises solely from c , prior to c joining the brachial plexus. intact rhomboid indicates that the central c root is intact. serratus anterior muscle, innervated by the long thoracic nerve (c ,c ,c ), was evaluated for atrophy in patients; had atrophy. dynamic evaluation of the posterior shoulder looking for posterior laxity was evaluated in patients; had laxity. posterior shoulder dislocation or subluxation is a known sequela of brachioplexopathy which sometimes requires muscle transfer when the child is older. conclusions: comprehensive us evaluation of perinatal brachioplexopathy detects: extent of traction neuromafibroma from the interscalene region peripherally toward clavicles (important for neurosurgeon), thick nerve roots, phrenic nerve diaphragm palsy, muscle atrophy from denervation, and posterior shoulder subluxation. us misses: intracanalicular traction pseudomeningoceles. paper #: alt- impact of the image gently campaigns in adult-focused hospitals: a survey of practice leaders brett bartz, duke university medical center; donald frush, kimberly applegate, michael callahan, laura coombs, marilyn goske purpose or case report: the alliance for radiation safety in pediatric imaging is an organization that uses social marketing to promote radiation protection for children and effect change across radiology practices. the impact of the alliance's image gently campaigns on practice patterns in radiology practices has yet to be assessed, especially outside of freestanding children's hospitals. the purpose of this investigation was to assess the impact of the image gently campaigns on academic and private practices/institutions that treat children but primarily serve adults. a web-based survey was emailed to leaders in radiology practices (n ) who do not practice at freestanding children's hospitals utilizing the acr's pred database. the survey consisted of questions designed to measure the recognition and impact of the image gently campaigns, including the impact on practice patterns. results: a total of practice leaders in u.s. states and territories responded for a response rate of . %. the majority ( %) of sites image pediatric patients in their practices. respondents consisted of department chairs ( %), group presidents/ceos ( %), and division chiefs ( %). the majority ( %) of respondents described their practice as a hospital-based private practice without a dedicated pediatric radiology division. the vast majority ( %) of respondents was familiar with the image gently campaigns; % of respondents reported that image gently had effected a change on how they imaged children. specifically, respondents (%) reported that the campaign caused a modification to lower dose protocols for head ct ( %), chest ct ( %), and abdominal/pelvic ct ( %). slightly more than half of respondents ( %), however, estimated that the image gently campaign resulted in no modification of pediatric fluoroscopy exposure. conclusions: to our knowledge, this is the first survey evaluating the impact of the image gently campaigns. there is near universal recognition of the campaigns, which have impacted practice patterns beyond the freestanding children's hospital in ct, but not in fluoroscopy. reliability of shear-wave velocity using different frequencies in acoustic radiation force impulse (arfi) elastography mi-jung lee, radiology, severance children's hospital, mjl @yumc.yonsei.ac.kr; suyon chang, myung-joon kim purpose or case report: although there are many studies about acoustic radiation force impulse (arfi) measurement, standard protocol has not been established. and a new probe with high frequency has been developed which can be applied for pediatric patients. the purpose of this study was to assess the reliability of shear-wave velocity (swv) at various depths using different frequencies to suggest standard measurement in arfi elastography. methods & materials: arfi elastography of both the elasticity phantom and normal liver was performed at different depths ( - cm) with convex ( - mhz) and linear ( - mhz) probes. ten valid swv measurements at each depth were performed. it was repeated ten times with the phantom and it was done in healthy volunteers (m:f : , age - years; mean . ). the mean value and standard deviation of swv were calculated. results: in both the elasticity phantom and the liver, variability of swv was different between the depths in both probes. the depth with lower variability in the phantom was and cm with the convex probe and cm with the linear probe. in the liver, the depth with lower variability was cm with the convex probe and and cm with the linear probe. in comparison of two probes, the linear probe showed lower variability at and cm depth in the phantom and at cm depth in the liver whereas the convex probe showed it at cm depth in both the phantom and the liver. conclusions: in arfi elastography, measurement of depth shows different variability in both low and high frequency probes. to obtain the most reliable measurement of swv, using high frequency probe is recommended for - cm depth and using low frequency probe is recommended for - cm depth. disclosure: dr. lee has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. imaging -year-old fetuses sabah servaes, children's hospital of philadelphia; teresa victoria, ann johnson, sandra kramer, richard markowitz, diego jaramillo purpose or case report: to demonstrate normal anatomy and pathology of medical museum specimens without disturbing the specimens. methods & materials: nine fetal specimens from a medical museum were imaged with ct and mri ( . t and . t) when possible with the specimens in their preserving fluid and containers. results: the fetal specimens are estimated to be approximately years old. one specimen is from the first trimester, seven are from the second trimester, and one is from the third trimester. normal anatomical structures at various stages of development including the brain (and varied sulcation pattern), lungs (lobar anatomy), and skeletal structures (several developmental features such as the ossification centers, perichondrial structures, and marrow cavitation) can be evaluated using imaging without causing harm to the specimens. pathologic entities including anencephaly and sirenomelia are also evaluated demonstrating features of these entities. conclusions: imaging historical fetal specimens provide an opportunity to evaluate normal developmental changes and pathological entities and also to gain a better understanding of the museum pieces without damaging the museum specimens. pediatric ct interpretations: does a tertiary care radiologist make a difference? wendy d. ellis, monroe carell jr. children's hospital at vanderbilt university; sumit pruthi, david johnson, christopher eakins, chang yu, marta hernanz-schulman purpose or case report: to determine whether a substantive difference exists between the pediatric imaging reports of community radiologists and reinterpretations by tertiary care radiologists at a free-standing children's hospital; and how those interpretations were related to the final diagnosis. methods & materials: this retrospective review examined the computed tomography (ct) reports of all pediatric patients referred to our tertiary care children's hospital over a month period ( / / - / / ). the outside reports and the requested second interpretation reports were compared and their content categorized as "agreement" vs. "disagreement: major or minor". a representative sample of major disagreements in which there was reliable followup information was correlated with the final diagnosis to determine if there was added value provided by the reinterpretation. results: ct scans from patients were submitted for reinterpretation. disagreements were found in / cases ( . %); with . % ( / ) classified as major disagreements. among the neurologic cases, major disagreements occurred in patients ( . %) and minor disagreements in patients ( . %). among the body scans, major disagreements occurred in cases ( . %) and minor disagreements in cases ( . %). in the cohort of cases reviewed for final diagnosis, the second read interpretation was more accurate in . % of cases with a p-value of < . (neurologic . %, p < . ; body . %, p < . ). conclusions: in our review, discrepancy rates between community and tertiary care radiologists in interpretation of pediatric ct scans were substantial, with discrepancies occurring in more than % of cases. further review of the cases for final diagnosis, showed that a significant number of the tertiary care interpretations were more accurate. possibilities that may account for this discrepancy include subspecialty training and elapsed time since performance of the study, which might provide additional clinical data in some cases. diagnostic ct scans performed at outside institutions should not be repeated considering added radiation burden to the child and additional expense. our data indicates there is added value to the reinterpretation which impacts the accuracy of the report (as assessed by the final diagnosis), and should be recognized by payors as integral to optimal patient care. ionizing radiation exposure from radiography in the neonatal intensive care unit-per-patient cumulative effective doses amaya basta, radiology and biomedical imaging, ucsf; jesse courtier, john mackenzie purpose or case report: to better understand the levels of exposure to ionizing radiation for infants in the neonatal intensive care unit (nicu). we retrospectively collected the number and types of radiographs performed per infant in our nicu by searching our radiology information system database over a five-year period. we focused on the most common examinations ( % of all radiographs) and assigned each an estimated equivalent dose based on published literature: chest and abdomen . micro sieverts (μsv), one-view chest . μsv, abdomen . μsv, twoview chest . μsv, two-view abdomen μsv. we then calculated a cumulative equivalent dose (ced) for each infant based on the number of each type of examination they received. descriptive statistics were generated to depict the distribution of number of examinations and ced. results: over five years, , infants cared for in our nicu received at least one radiograph of the chest and/or abdomen. the number of examinations obtained on these infants was . , , , (mean, median, minimum, maximum). the st quartile was and the rd quartile was examinations. the cumulative equivalent dose these infants received was . , . , . , , . μsv (mean, median, minimum, maximum) . the st quartile was . and the rd quartile was . μsv. two hundred infants ( . % of the study population) received a ced of over μsv. conclusions: descriptive statistics provide a valuable assessment for the broad range of radiation that infants receive in the nicu. although the distribution is skewed towards a low level of exposure, a subset of patients ( . %) received a ced of over μsv. identification of factors that cause infants to enter this group will be important for future dose reduction strategies. poster #: cr- congenital cardiac fibroma: a case report earic bonner, meharry medical college, ebonner @email. mmc.edu; seth crapp, david parra purpose or case report: a -week-old male presented to his pediatrician with a ii/vi systolic ejection murmur along the left sternal border. he had mild tachypnea without cyanosis. his oral intake was adequate with no evidence of failure to thrive. he was referred to a pediatric cardiologist who performed an ecg and a transthoracic echocardiogram. the ecg showed normal sinus rhythm at beats per minute with no abnormalities. the transthoracic echocardiogram showed a x x mm homogeneous mass originating from the anterior free wall of the right ventricle, and mild dilation of the right ventricle. mild dynamic subpulmonary stenosis and a secundum atrial septal defect were also noted. although the murmur was significantly louder at one month follow-up, a repeat echocardiogram did not reveal any increase in the size of the mass. at months of age, a cardiovascular magnetic resonance imaging (cmri) study under general anesthesia was performed. cmri revealed a x x mm cardiac tumor that was causing narrowing of the right ventricular outflow tract. the tumor was hypointense on t -weighted imaging and hyperintense on t weighted imaging, with positive delayed enhancement. these findings, along with the size and location of the mass, are consistent with a diagnosis of a cardiac fibroma. chest mra, that was also performed, showed normal extracardiac vascular anatomy with no evidence of peripheral branch pulmonary stenosis. cardiac fibromas do not usually increase in size; however, the concern is the child's risk of arrhythmias. frequent holter monitoring was recommended for this patient. considerations were also made for an electrophysiology study in the next - years to determine the risk of ventricular ectopy. at that point, the patient can be assessed for the possibility of resection of the fibroma. purpose or case report: treatment of pulmonary atresia is complex and demands intricate solutions. one solution is the creation of a conduit between the right ventricle and the main pulmonary artery. the lifespan of these conduits is limited by progressive occlusion over time, which can be treated with endovascular stent placement in lieu of surgical re-intervention. however, these stents are at high ( %) risk for fracture, typically at the stent waist. the radiologist should be aware of this complication, as they may be the first to identify it on chest radiograph. the purpose of this electronic poster is to familiarize radiologists with this entity by presenting cases of stent fracture and migration. methods & materials: over a month period, we identified three children with rv-pa stent fractures and associated stent migrations on chest radiography. imaging analysis was focused on the appearances of these fractured stents. patient management and outcomes were reviewed. results: three children, males, female (ages , , and years) were found to have asymptomatic rv-pa conduit stent fractures with fragment migration. one chest xray was performed in the er for fever and cough; one was pre-op for gi surgery; one was done to confirm abnormal findings seen on a routine cardiac echo. the time between stent placement and fracture detection ranged from to months. two patients had stent fractures and embolizations to the right ventricle that required open surgery to remove stent fragments. the third patient had embolization to both pulmonary arteries, but did not require treatment. all patients did well. conclusions: stent fractures and migrations are a relatively common complication of rv-pa conduit stent placement. pediatric radiologists need to be aware of this complication in order to provide value-added interpretations. purpose or case report: we describe the case of a week stillborn fetus with a . cm diameter craniopharyngioma detected by ultrasonography. a g p woman in her third decade had ultrasonographic examination showing hydrocephalus, polyhydramnios and an intracerebral mass. the nature of the mass was uncertain and intracerebral hemorrhage was considered. the pregnancy was terminated at weeks gestation. at postmortem examination the decedent was a g male fetus with a head circumference of . cm and a crown-rump length of . cm. anterior and posterior fontanelles appeared large. no other external abnormality was found. the placenta was unremarkable and cytogenetics on placental tissue showed a normal male karyotype. examination of fetal viscera was remarkable for mildly underweight adrenal glands ( . g, expected . g) and hepatomegaly ( . g, expected . g). intracranial csf was increased in volume. there was a suprasellar . cm diameter somewhat gritty, but smooth-surfaced tumor. the brain and tumor together weighed g. the floor of the cranium and sella turcica were grossly normal. histologic examination of the tumor showed an adamantinomatous type craniopharyngioma with characteristic epithelium, stellate reticulum, focal keratinizing squamous epithelium and calcification. pre-and postnatal mri of caudal regression syndrome claire b. beaumont, md, university of arkansas for medical sciences, cbbeaumont@uams.edu; nafisa k. dajani, leann e. linam purpose or case report: caudal regression syndrome is a rare form of caudal dysplasia characterized by a spectrum of findings including agenesis of the lumbosacral vertebra, multiple orthopedic deformities in the lower limbs, as well as anomalies of the gastrointestinal and genitourinary tracts. the mechansim of caudal regression syndrome is not completely understood but is believed to be secondary to a defect in the induction of caudal elements. mri is a valuable tool for identifying the specific anomalies involved with caudal regression syndrome on a case-by-case basis. the following is a case from our institution which includes both pre-and postnatal mri. unsuspecting tuberous sclerosis diagnosed on neonatal cranial ultrasound vikas menghani, md, pediatric radiology, women's and children's hospital, drvikasmenghani@gmail.com; puneet gupta, richard thomas, vaseem iqbal, jan najdzionek. purpose or case report: tuberous sclerosis (ts) is a rare autosomal dominant genetic disorder causing hamartomatous proliferation in number of organ systems. because the classical triad of epilepsy, mental retardation and adenoma sebaceum is not commonly seen on clinical examination, imaging plays a central role in the diagnosis and treatment of tuberous sclerosis. central nervous system features of ts include subependymal nodules, cortical tubers, subependymal giant cell astrocytoma, white matter bands and cysts. in patients with ts, cerebral involvement in the form of subependymal nodules is seen in % to % and white matter abnormalities are noted in % to % of cases. knowledge of expected radiological features is thus important in making the correct diagnosis. recent studies have indicated that earlier appearance of brain lesions indicate a greater risk of mental retardation and a more severe clinical course. we present a case of a -day-old neonate who was referred to us with concerns for hydrocephalus. the cranial ultrasound demonstrated multiple echogenic subependymal nodules of varying sizes and mild asymmetry of the ventricles. the differential diagnosis included ts, torch infections, and x-linked subependymal heterotropia. areas of increased echogenicity were noted within the white matter of the left frontal lobe, which favored ts. subsequently, an mri was performed to validate these findings and assess for additional white matter lesions. the mri showed classic manifestations of ts that included periventricular lesions and streaky, linear, wedge-shaped hyperintensities on flair imaging. a noncontrast ct scan was also performed which revealed classic calcified subependymal nodules. cardiac rhabdomyoma and renal angiomyolipoma are the other recognized manifestations of ts and were respectively excluded by subsequent echocardiogram and renal ultrasound. pyloric atresia with epidermolysis bullosa: fetal mri diagnosis with postnatal correlation arnold c. merrow, md, radiology, cincinnati children's hospital medical center, carl.merrow@cchmc.org; jason s. frischer, anne w. lucky purpose or case report: pyloric atresia (pa) is an uncommon disorder, accounting for % of congenital gastrointestinal atresias. up to % of cases have associated anomalies, the most common of which is epidermolysis bullosa (eb). prenatal findings have been reported sonographically for each of these anomalies, both in isolation and in the rare case of association. a case of isolated pa has been reported by fetal mri. we present the first reported case of pa with eb diagnosed by fetal mri with corroborative postnatal imaging and surgical findings. the mother of this child was initially referred to the fetal care center of cincinnati at weeks gestation for a possible myelomeningocele diagnosed by prenatal ultrasound at an outside facility. these ultrasound images were not available for review at the time of our workup. a fetal mri was the first study to be obtained at our institution. the mri showed no myelomeningocele or brain anomalies. the stomach was moderately enlarged throughout the exam and did not empty. subjective polyhydramnios was also noted. no duodenal dilation was seen, and there was minimal fluid in the distal bowel loops. this constellation of findings raised concern for pyloric atresia, resulting in a careful search for any sign of epidermolysis bullosa due to a known association of these disorders. prominent debris was seen layering dependently in the amniotic fluid and in the dilated fetal stomach, and the external ears were abnormally small and misshapen. the pa-eb association was proposed as the underlying diagnosis based on our mri findings. it was also postulated that skin blistering over the lumbosacral spine at the time of the prior outside ultrasound could have mimicked a myelomeningocele, thus prompting the referral to our center. at delivery, the baby had numerous skin defects, and the ears were malformed. an abdominal radiograph obtained after nasogastric tube placement and air injection showed no gas beyond the stomach. a pyloric ultrasound showed a distended stomach without a patent pyloric channel to the duodenal bulb, consistent with pyloric atresia. a skin biopsy confirmed epidermolysis bullosa, and the patient underwent a resection of the pa with gastroduodenostomy. the baby subsequently expired less than two weeks later, most likely due to sepsis based on wound cultures and autopsy results. our case demonstrates the ability of fetal mri to diagnose this rare condition and highlights the key imaging manifestations of the pa-eb association. disclosure: dr. merrow has indicated that he is an author for amirsys and receives a royalty accordingly. purpose or case report: we demonstrate a case where the changing position of the contrast filled appendix lead to the diagnosis of malrotation, with review of the embriology of intestinal rotation. a newborn preterm female presented with a golf ball sized umbilical mass, that reduced by itself, thought to represent an umbilical hernia vs omphalocele. she was unstable to undergo an upper gi exam under fluoroscopy, therefore a limited contrast study was performed at bedside and was inconclusive for malrotation. subsequent nicu radiographs showed changing position of the appendix filled with residual contrast, visiting all quadrants of the abdomen in a random pattern over a few days period. this confirmed our suspicion for malrotation. it is well know that in malrotation the position of the cecum can be variable, most commonly located in the right upper quadrant or left lower quadrant. to our knowledge it has not been described yet that the changing position of the appendix can lead to the diagnosis of malrotation. through this case we display the embriology of the intestinal rotation and the radiologic signs of malrotation. poster #: cr- mr imaging patters of liver transplant complications in the pediatric population edward richer, md, emory university, richerej@gmail. com; adina alazraki, jonathan loewen purpose or case report: pediatric liver transplantation is a relatively common surgery, with more than transplants in the united status annually. the spectrum of post transplant complications has been previously described, primarily utilizing ultrasound. as mri has become a more widely used technique in pediatric imaging, and ultrasound findings may be non-specific, knowledge of mr imaging patterns is an important adjunct in the post-transplant evaluation. we present a spectrum of complications, including vascular, biliary, hepatic parenchymal, and systemic complications. methods & materials: using an electronic record system, we identified pediatric patients with prior liver transplantation who subsequently underwent abdominal mri at our institution and were found to have a post transplant complication. patient management and outcomes were reviewed. results: our review of a subset of the available patients shows vascular complications to be the most commonly encountered abnormality at our institution, including hepatic artery stenosis/thrombosis, and portal vein stenosis/ thrombosis, cavernous transformation of the portal vein. biliary complications were relatively common, including bilary stenoses and bilomas. hepatic parenchymal and systemic complications, such as ptld, were less common. we demonstrate the mr imaging patterns of these complications. conclusions: pediatric liver transplantation is a relatively common surgery, and the mri appeance of post transplant complications warrants illustration as abdominal mri becomes more widely used in pediatric imaging. we present a pictorial review of common patterns of complication. imaging of progressive familial intrahepatic cholestasis (pfic) matthew d. dobbs, md , radiology, vanderbilt university medical center, matthew.dobbs@vanderbilt.edu; sumit pruthi, stephanie e. spottswood purpose or case report: progressive familial intrahepatic cholestasis (pfic) is a relatively rare pediatric liver disease due to a genetic mutation (abcb gene on chromosome q - ) in a bile salt export protein causing cholestasis leading to chronic inflammation within the biliary system. the diagnosis is made clinically with detection of a low ggt in the face of an elevated bilirubin and alkaline phosphatase. genetic testing confirms the diagnosis. one of the subtypes, type , was shown in to be highly related to the development of hepatocellular carcinoma. the vast majority in children in this study developed hcc at less than years of age. radiological contribution to the management of these chronic liver disease patients is to perform surveillance imaging to detect hcc. due to the rarity of this condition, almost no reports exist in the radiological literature describing the imaging features or management of this condition. our presentation will review the imaging findings in our small population of pfic type patients on us, ct, and mri. we will also review suggested surveillance imaging techniques and imaging algorithms. renal rhabdoid mimics wilms tumor vikas menghani, md, pediatric radiology, women's and children's hospital, drvikasmenghani@gmail.com; paul montgomery, jan najdzionek, vaseem iqbal purpose or case report: in the past most pediatric renal tumors have been classified together under the umbrella of wilms tumor. however, over the last decade with advancement in imaging, several distinctive imaging features specific to renal tumors have been recognized which aid in their classification as being distinct pathologically. we present a case of rhabdoid tumor where in the primary tumor arose from the kidney. it had classical imaging features of wilms tumor. we want to highlight that even with the most sophisticated imaging techniques, specific renal tumors cannot always be diagnosed with preoperative imaging and how this alters the management and prognosis for child with a renal mass. in our case, the postoperative findings, pathology and immunohistochemical techniques confirmed a rhabdoid tumor. differentiation of these two tumors is essential since in patients with rhabdoid tumor survival is poor with -year overall survival rates of % for stages i and ii and % for stages iii, iv, and v. on imaging, there are several features that suggest the diagnosis of rhaboid tumor. these include subcapsular fluid collections, linear calcifications outlining tumor lobules, and vascular invasion. also, a pertinent feature of rhabdoid tumor due to its aggressive nature is the presence of lung metastasis ( %) and synchronous malignant brain lesions ( %). these findings were not present on our case, which led us in formulating a diagnosis of wilms. our patient is unusual in the fact that the local renal findings and absence of metastasis, synchronous malignant lesions, and vascular invasion led us to an incorrect diagnosis of wilms tumor. in conclusion, we would like to stress that diagnosis of rhabdoid tumor of the kidney on imaging presents a challenge because of its imaging similarity to wilms tumor. ectopic ureters in young infants: mru findings shin-lin shih, md, department of radiology, mackay memorial hospital; yi-fang chen, chun-chao huang, fei-shih yang purpose or case report: to localize the terminations of ectopic ureters by mri methods & materials: mr urography (mru) was conducted in four female patients with hydroureter and a suspected ectopic orifice. mr imaging was performed with a t mr scanner (achieva; philips). the imaging protocol mainly consisted of a single-shot t -weighted turbo spin echo sequence with a slice thickness of mm and multiplanar reformations. the ages of the four patients were day, days and months (for two). the latter two patients presented with urinary tract infection. the newborn patients presented with abnormal prenatal examination. the pertinent findings and descriptions of a variety of renal anomalies were described. results: the locations of the ectopic ureters were two in the vagina, one in the uterus and one in the bladder neck. the associated renal anomalies were a right duplex kidney in four, a left duplex kidney in one, a left ectopic dysplastic kidney in one and vesicoureteral reflux in one (confirmed by vcug). conclusions: mru may demonstrate the exact point of termination of an ectopic ureter and also the associated renal anomalies. poster #: cr- acquired polycystic kidneys in neuroblastoma survivors richard bellah, , radiology, the children's hospital of philadelphia, bellah@email.chop.edu; bernard kaplan, camilo jaimes, yael p. mosse, jill p. ginsberg, kevin e. meyers purpose or case report: neuroblastoma (nbl) is the most common extracranial solid malignancy of childhood. with current therapy, the prognosis and long term survival of patients affected by this condition has dramatically improved. nevertheless, the treatment for nbl may account for some complications further in life. in patients with neuroblastoma, acute renal failure can occur usually as a result of a thrombotic microangiopathy associated with bone marrow transplantation. in addition, end-stage renal disease has been reported in long-term survivors of nbl. this exhibit describes and illustrates the first case series of five patients with treated nbl in whom the imaging features of polycystic kidney disease (pkd) developed over time, and in some cases, as progressive renal failure ensued. methods & materials: medical and imaging records were reviewed (irb approved) of patients with treated nbl in whom pkd became apparent during the course of followup imaging. results: five patients displayed findings of pkd on us and/or ct. three of the five patients (where images were available) had normal renal imaging at time of nbl diagnosis. the mean age at nbl diagnosis was . years (range . - . yr). the mean age at time pkd was detected was . years (range - yrs). none of the patients had a family history of pkd, or had previously undergone dialysis. all patients received chemotherapy and total body irradiation prior to bone marrow transplantation. four patients survived nbl therapy but eventually developed end-stage renal disease. conclusions: an association between acquired pkd and nbl has not been previously reported. the etiology of this observation is still unclear, but a toxic insult is likely to account for the renal changes. further research is needed to establish the epidemiology, prognosis, and etiology of this association. abnormal migration of the retention anchor suture in a case following gastrostomy tube insertion surendra narayanam, mbbs, dmrd, dnb, division of image guided therapy, department of diagnostic imaging, the hospital for sick children, nrssbabu@gmail.com; joao amaral, luke toh, bairbre connolly, vicente deoliveira, dimitri parra purpose or case report: during percutaneous gastrostomy tube placement, retention anchor suture(s) are deployed into the stomach to tack the anterior gastric wall to the abdominal wall. in our practice the thread of the retention anchor suture is cut at days and the metallic portion passes pre rectum. we report an interesting and very rare migration of the metallic portion of the retention anchor suture in post-primary gastrostomy tube insertion. an -month-old girl, with a mitochondrial disease and severe hypotonia underwent percutaneous gastrostomy placement. during the procedure the retention anchor suture thread snapped and the metallic portion of the suture remained within the stomach. day post procedure, the child became uncomfortable, so a gastrostomy tube check was performed. the suture was not visible in the abdomen on abdominal x-ray or fluoroscopically. on close review of the images, the suture was found projected over the distal esophagus. initial impression was the anchor suture had refluxed into the esopahgeal lumen. careful attempts were made to remove it along with the nasogastric tube, from above under fluoroscopic control. however on withdrawal of the nastogastric tube, the retention anchor suture moved enbloc with the nasogastric tube. once removed the retention anchor suture was confirmed to be within the nasogastric tube. this case illustrates the importance of examining the chest x-ray carefully before assuming a retention anchor suture has passed. to understand the appropriate post procedural radiographic workup and its technique for timely diagnosis. . to learn the potential complications of delayed diagnosis. pediatric retroperitoneal synovial sarcoma ahmad aouthmany, university of toledo medical center, ahmad.aouthmany@utoledo.edu; asif abdullah purpose or case report: pediatric synovial sarcoma most commonly affects the extremities, especially the lower thigh and knee region; other primary sites such as the retroperitoneum have been only infrequently reported. we report an extremely rare case of a retroperitoneal synovial sarcoma masquerading as retroperitoneal hematoma in a -year-old white female with non-traumatic back pain and non-contrast enhanced ct findings of right quadratus lumborum and psoas region presumed hematoma. coagulation studies revealed factor xi deficiency also known as hemophilia c. however, on follow-up imaging, the presumed retroperitoneal bleed persisted and a subsequent mr examination revealed a solid enhancing mass. ct, mr, and fdg-pet findings as well as a brief histopathology are discussed. our case is rare in the regards that the tumor occurred in an uncommon retroperitoneal location in a pediatric patient and was mimicking a retroperitoneal hematoma which posed a significant diagnostic challenge. despite a rare entity, synovial sarcoma among other sarcomatous lesions maybe considered in the differential consideration of a spontaneous retroperitoneal hematoma even in hemophiliac patients. longitudinal bracket epiphysis michael jubang, geisinger, mjjubang@geisinger.edu; farzad sedaghat, william j. malone, george wu, william mirenda purpose or case report: longitudinal bracket epiphysis is a rare anomaly with multiple synonyms such as delta bone, triangular bone, and congenital angular deformity. the purpose of this case report poster is to discuss an -month-old male born with an adducted right great toe with a broad nail and a notch in the center of the distal phalanx. the review will discuss radiographic findings, the natural progression of the disease, the treatment options, the mri findings used for pre-surgical planning, and associated pathology. whole body mri in pediatric non oncologic diseases: pictorial review ramy el jalbout, md, radiology, chu sainte justine, ramy.jalbout@yahoo.com; vijay moorjani purpose or case report: with the advances in scanning techniques and the scanning sequences, the role of wbmri is expanding. mri has a great role in the pediatric population owing to its inherent advantages namely lack of radiation, high tissue specificity, and high diagnostic yield at the level of the entire body under a single sedation. unlike the application of wbmri in the assessment of metastasis and bone marrow involvement in leukemia, its role in systemic diseases is yet to be further investigated. certain diseases such as crmo are very often multifocal. the extent of osteonecrosis in patients on steroids, dermatomyositis and the lesions related to child abuse are very often wide spread in the skeleton. we intend to present some of the findings of these pediatric systemic and multifocal diseases on wbmri. chronic relapsing multifocal osteomyelitis (crmo): crmo can be acute or chronic and is multifocal. the abnormality manifests as high signal intensity. wbmri can guide for the best site for biopsy and provides monitoring for response to treatment. osteonecrosis: only few small studies evaluated the usefulness of wbmri in the diagnosis of both the symptomatic and asymptomatic sites of osteonecrosis in all patients on steroid therapy. wbmri is more sensitive than conventional radiographs. the abnormalities are typically geographical areas of high stir signal intensity. myopathies: wbmri has also the role of detecting the extent of idiopathic inflammatory myopathies such as dermatomyositis in the entire skeleton. child abuse: wbmri has a low sensitivity for the highly specific fractures that are pathognomonic for child abuse. conclusions: wbmri is a useful examination in the pediatric patient that is radiation free, quick and allows imaging of the entire body. it is an adjunct to dedicated mris to look for multifocality and extent of systemic diseases such as crmo, osteonecrosis in patients on steroids and dermatomyositis. it has a great potential as a screening examination but at the same time can detect both the symptomatic and the asymptomatic lesions in the bone marrow and muscles that are otherwise not seen on conventional radiography. it also allows guidance for biopsy and monitors response to treatment. mobile "cerebroliths" in hemihydranencephaly: a case report usha d. nagaraj, md, the ohio state university medial center, usha.nagaraj@osumc.edu; brent adler purpose or case report: hydranencephaly is a congenital central nervous system disorder manifested by the replacement of the cerebral hemispheres with a thin membranous sac filled with cerebrospinal fluid and necrotic debris. hemihydranencephaly is an extremely rare brain condition in which the vascular anomaly is unilateral, with fewer than cases previously reported in the literature. this is a case of a -month-old male who presented to the ophthalmologist for evaluation of possible leukocoria of the right eye. the patient had a history of a difficult vaginal delivery that required forceps delivery with possible associated trauma to the right eye. dilated fundoscopic exam revealed retinal calcifications. this caused a clinical concern for retinoblastoma and ct and mri of the orbits were obtained. ct demonstrated profound dilatation of the left lateral ventricle with only a thin rim of cortex surrounding it. there was some midline shift to the right with mild dilatation of the right lateral ventricle. the thalami and brainstem were spared. there were multiple soft tissue bodies that layered in the dependent portion of the left lateral ventricle, which were isodense to grey matter. mri revealed similar findings consistent with hemihydranencephaly involving the left cerebral hemisphere. there were multiple round soft tissue masses that measured up to cm in size that layered posteriorly in the left lateral ventricle. these masses were isointense to grey matter on t and hyperintense on t . when the patient was placed with his head turned to the left, these masses moved to the dependent portion of the left lateral ventricle. the orbits were normal on both ct and mr. these soft tissue collections are presumed to be mobile collections of infarcted brain tissue. this unusual appearance has not been described in the radiology literature. we review the ct and mr findings and review the relevant literature. purpose or case report: citrullinemia type i is a rare inborn error of urea cycle metabolism resulting in hyperammonemia. in the classic form, the newborn presents with poor feeding, vomiting, progressive lethargy and signs of increasing intracranial pressure - days after birth, rapidly progressing to apnea, coma and death if left untreated. we present a case of a term infant who presented to the hospital on the th day of life with a typical history of poor feeding and profound hypotonia. upon admission he had multiple episodes of apnea and hemodynamic instability prompting intubation and intensive support. laboratory evaluation revealed multiple abnormalities, most notably, hyperammonemia ( umol/l) and elevated citrulline (> umol/l). mri of the brain performed on the th day of life showed findings consistent with term hypoxic ischemic encephalopathy with restricted diffusion in bilateral rolandic cortex and subcortical white matter, bilateral caudate heads and lenticular nuclei, bilateral insular cortex, and bilateral cerebral peduncles. the genu of the corpus callosum, bilateral deep frontal white matter, and the left parietal white matter also demonstrated restricted diffusion suggesting infarction secondary to thrombosis of deep intramedullary veins. an area of restricted diffusion in the right parietal cortex was suspicious for superficial venous infarct. review of the literature reveals that this case of neonatal citrillunemia has unique mri findings. while our patient had diffusion changes with some shared similarities to the previous two cases in the literature, there are also findings consistent with deep intramedullary venous thrombosis and infarction. poster #: cr- duplicated internal auditory canal: a rare anomaly of the temporal bone ahmad aouthmany, university of toledo medical center, ahmad.aouthmany@utoledo.edu; asif abdullah purpose or case report: duplicated internal auditory canal (iac) is a rare anomaly of the temporal bone, which is usually associated with sensorineural hearing loss. only a few cases have been previously described in literature. we describe an extremely rare case of duplicated right internal auditory canal in a six month-old patient with a history of down syndrome. a six month-old male with trisomy presented with profound bilateral sensorineural hearing loss. the patient failed the newborn hearing screening tests. past medical history was unremarkable for recurrent ear infections. on focused physical examination, the auricles were normal appearing. external auditory canals were patent bilaterally revealing clear and translucent tympanic membranes. patient did not reveal a facial palsy. subsequently, a high resolution computed tomography (hrct) of the temporal bone was performed. duplicated appearance of the right internal auditory canal with separation of facial and vestibulocochlear segments was noted. the facial nerve canal demonstrated normal caliber while there was significant narrowing of the cochlear canal near the fundus. significant stenosis of the vestibulocochlear segment of the duplicated iac was identified at the porus acousticus. dehiscent right posterior semicircular canal was also seen. an enlarged right vestibule was also noted. a single iac was identified on the contralateral side with significant stenosis at the porus acousticus. high-resolution magnetic resonance imaging of iac was recommended which revealed normal appearance of the bilateral cochlear and vestibular nerves. duplication of the iac is an extremely rare anomaly involving a redundant osseous canal extending from the cerebellopontine angle through the otic capsule bone toward the labyrinth or cochlea. a duplicated iac may or may not be associated with congenital sensorineural hearing loss secondary to aplasia or hypoplasia of the vestibulocochlear nerve. to evaluate for structural abnormalities that may preclude cochlear implantation, it is important to evaluate pediatric patients with sensorineural hearing loss radiologically. although hrct is the best imaging modality for evaluation of osseous iac, the iac contents are best viewed on mri in oblique sagittal planes of the iac using a -d volumetric steady state sequence. neuroimaging in hemiplegic migraine: cases and review of the literature nicholas v. stence, md, children's hospital colorado-radiology, nicholas.stence@childrenscolorado.org; sita kedia, john a. maloney, jennifer armstrong-wells, timothy bernard purpose or case report: hemiplegic migraine (hm) is a rare variant of migraine with aura. it is characterized by a motor deficit lasting up to h that is fully reversible. little neuroimaging data for hm exists in the literature. we report our experience with two pediatric cases of hemiplegic migraine. we also review published cases of pediatric hm with abnormal findings on neuroimaging. methods & materials: cases and presented to our institution with severe headache (ha), acute right-side weakness, aphasia, and altered mental status (ams), which did not resolve after h. magnetic resonance imaging (mri) and genetic testing are reviewed for these cases. the literature was reviewed for pediatric cases with neuroimaging changes during hm attacks. results: initial mri, including diffusion-weighted imaging (dwi), was negative in both patients within h of onset. repeat mris at h (case ) and h (case ) were both positive for mild hyperintensity on trace diffusion images, and corresponding reduced diffusion on adc maps, involving regions of the cortex and juxtacortical white matter in left middle cerebral artery distributions. these findings completely resolved at months in both cases. mr angiograms (mra) were negative in both cases. case had a family history of migraines and was found to have an unreported mutation in atp a gene at a highly conserved location in vertebrates. case had a family history of hm and was found to have an indeterminant mutation in the cacna a gene. infectious, metabolic and hypercoagubility work up was negative. case required inpatient rehabilitation and at year follow up was requiring speech therapy. case resolved completely. in the literature, cases of hemiplegic migraine with neuroimaging changes were reported. all cases had prolonged hemiplegic migraines (symptoms> h) and showed cerebral edema with or without restricted diffusion. conclusions: all eight hm cases in the literature with abnormal findings on neuroimaging had prolonged attacks. mris for our two cases and two cases reported in the literature were initially normal at admission. mild swelling and restricted diffusion developed in our two cases after h, and resolved on follow up mris. subtle findings on diffusion and t imaging may lag behind the clinical picture in hm, therefore serial neuroimaging may be useful in individuals with prolonged symptoms. most cases eventually show resolution clinically and on mri. correlation of neurosonographic anatomy with matching mr scan planes denise castro, hospital for sick children, denisecastro @ gmail.com; pam rasalingham, omar islam, don soboleski purpose or case report: new high-resolution mr sequences have allowed for exquisive anatomic detail and enables reconstruction of images in any scan plane desired. this ability allows for precise matching of mr image planes with the standard oblique coronal, sagittal and axial images obtained during routine neurosonography. the purpose of this poster is to correlate the morphology demonstrated on neurosonography with the mr image, utilizing this ability in order to enhance our understanding of the neuroanatomy distinguishable on sonographic imaging. we believe this will allow a better appreciation of the subtle differences in echotexture of neuroanatomic structures which are often ignored or overlooked on neurosonography and help improve our detection of subtle sonographic abnormalies. ectopic cerebellum in the posterior cranial fossa: report of a case and review of the literature usha d. nagaraj, md, the ohio state university medical center, usha.nagaraj@osumc.edu; daniel boue, lisa martin purpose or case report: cerebellar heterotopia is a common congenital anomaly frequently encountered in the form of cell rests around the fourth ventricle. however, isolated well-differentiated cerebellar ectopia is extremely rare. of the previously reported cases in the literature, only have presented as a discrete, extraaxial mass and none have been described in the posterior cranial fossa. we present a case of a -year-old male who initially presented with persistent daily headaches. physical exam including a detailed neurologic exam was within normal limits. non-contrast computed tomography (ct) of the brain was initially performed, demonstrating no abnormalities. further work-up with magnetic resonance imaging (mri) was performed, which revealed a well-defined, extra-axial mass superior to the cerebellum and inferior to the tentorium, immediately beneath the vein of galen. the mass was isointense to grey matter on t and t sequences and there was no significant enhancement on post-contrast images. there was mass effect on the vermis and the cerebellar tonsils were displaced mm below the foramen magnum. neurosurgery was consulted and the mass was removed for diagnosis and treatment of the patient's symptoms. the mass was easily identified intra-operatively and gross total resection was accomplished successfully. pathologic analysis of the mass revealed well-formed cerebellar tissue without evidence of neoplasia. to the best of our knowledge this is the only case of ectopic cerebellum presenting as a discrete extra-axial mass in the posterior cranial fossa. our case shows that an extra-axial mass that parallels grey matter on all sequences can be a presentation of ectopic cerebellum. we describe the ct and mri findings, surgical and histopatholgical results and review the relevant literature. pediatric isodense acute subdural hemorrhage jeffrey s. kao, md, msee, university of kansas-wichita, run boston@gmail.com; debbie desilet-dobbs purpose or case report: the density (attenuation coefficient) of subdural hemorrhage (sdh) in computed tomography (ct) is important in assessing the acuity of sdhs. an acute sdh is traditionally described as hyperdense and then becoming isodense in approximately weeks when entering the subacute phase. in this report, we document the case of a pediatric patient with the new appearance of an acute sdh within h of the prior ct that was isodense. greater than % of the collection was isodense, with a small focus of hyperdensity. acute sdhs are known to be isodense to gray matter in patients with anemia (wp smith, am j neurorad ). however, the hemoglobin and hematocrit was within normal limits. in addition, acute sdhs that are only a few hours old can have a mixed hyperdense and hypodense appearance because of uncoagulated blood before clotting takes place (j provenzale, ajr ) . thus, an acute sdh can have an isodense appearance in a non-anemic patient. radiologists should consider the possibility of an acute sdh with an isodense appearance, especially in case of possible non-accidental trauma where timing of an injury is important. undifferentiated sarcoma of the esophagus in an year-old male: case report and radiologic/pathologic correlation michael e. daniel, md, ut southwestern / children's medical center dallas, michael.daniel@utsouthwestern. edu; lisa sutton, sandy cope-yokoyama, neil j. fernandes purpose or case report: mesenchymal neoplasms of the gastrointestinal (gi) tract occur infrequently in the adult and are extremely rare in the pediatric population. the occurrence of these lesions in the esophagus is limited to a collection of case reports in the available literature. most esophageal mesenchymal tumors in the pediatric gi tract are benign leiomyomas. the vast majority of malignant mesenchymal tumors in children are categorized as either sarcomas or gastrointestinal stromal tumors (gist). we report a case of a high grade undifferentiated sarcoma of the distal esophagus in an year-old male. while this tumor most closely resembles a gist, the immunohistochemical profile of the lesion is not typical of any distinct mesenchymal neoplasm. a review of the literature demonstrates a single case report of a likely benign undifferentiated mesenchymal neoplasm of the distal esophagus in an adolescent. to our knowledge, this is the first reported case of an undifferentiated esophageal sarcoma in a pediatric patient. we provide radiologic and pathologic features of the above lesion, and review the typical imaging and pathologic characteristics of mesenchymal gi neoplasms. potential airway management issues in sedated children kimberly fagen, md, ms, children's national medical center, kfagen@childrensnational.org; nadja kadom, ira cohen purpose or case report: many pediatric imaging studies require sedation. it has been shown that a variety of health care professionals other than anethesiologists may provide sedation, including advanced practice registered nurses, nurse practitioners, physician assistants, fellow level trainees, emergency medicine physicians, intensivists, pediatricians and, last but not least, radiologists. moderate sedation, also called "conscious sedation", does generally not require an anesthesiologist as there is usually adequate spontaneous ventilation and no airway intervention required. however, in case of a complication during the imaging study intubation may become necessary. for patients with certain congenital or acquired conditions emergent intubation may be very difficult and should be brought to the attention of an anesthesiologist prior to inducing moderate sedation. the four "d's" is a quick way to assess potentially difficult airways that necessitate consultation with anesthesia prior to moderate sedation: dentition (incisor/tooth size, dental alignment, and macroglossia), distortion (swelling from infection, tumor, or trauma), disproportion (hyoid-chin ratio, such as with micrognathia), and dysmobility (jaw or cervical spine movement issues, i.e. trauma or atlanto-occipital instability). presence of some of these features may be an indication to consider general anesthesia for sedation; at the very least, anesthesiologist's awareness of a potentially difficult intubation adds to patient safety during moderate sedation. purpose or case report: lymphangiomatosis describes the presence of multiple lymphangiomas often with multiorgan involvement; typically bones, spleen, mediastinum and lungs. although lymphangiomatosis has been described in patients ranging from birth up to years, it most frequently presents in childhood. the lesions can occur in any tissue in which lymphatics are normally found, with a predilection for neck and chest involvement. the clinical presentation is variable including pleural or pericardial effusion, hemoptysis, protein wasting enteropathy, peripheral edema, hemihypertrophy and disseminated intravascular coagulopathy. the coexistence of lytic bone lesions and chylothorax serves as an important diagnostic clue. we describe typical radiographic, ct and mri findings in the appropritate clinical setting that narrow the differential diagnosis and raise concern for this rare entity as the etiology for the patient's symptoms. we report a -year-old girl and year-old boy with pulmonary lymphangiomatosis with typical presentation and imaging findings. results: bilateral interstitial infiltrates, pericardial and pleural effusions are evident on chest radiograph. sampling of the pleural fluid demonstrates chylous effusion. ct scans of the thorax reveal diffuse smooth thickening of interlobular septa and bronchovascular bundles with extensive infiltrative involvement of mediastinal fat. osseous and splenic lesions are demonstrated both on ct and mr. differential diagnosis includes interstitial edema, lymphoma and sarcoidosis. conclusions: the natural history of pulmonary lymphangiomatosis is characterized by progressive growth and compression of adjacent structures. therapy should aim to decrease the compressive effects, to control chylous effusions, and to maintain cosmesis. the success of surgical resection is limited by inability to separate lymph collections from normal structures. characteristic clinical and radiographic presentation, chylothorax, and extrathoracic lymphatic dysfunction should prompt a consideration of lymphangiomatosis and prevent delay in diagnosis. aortic arch congenital anomalies: what the radiologist needs to know luana stanescu, radiology, seattle children's hospital, stanescu@u.washington.edu; stephen done purpose or case report: . review classic imaging findings in congenital aortic arch anomalies which can improve detection on radiographs and barium esophagogram . describe pertinent embryologic basis of the radiologic findings . describe correlative imaging findings on ct and/or mri in dedicated cases . describe common diagnostic pitfalls methods & materials: after obtaining institutional irb approval we reviewed various patients presentations with this condition and analyzed images to characterize this particular entity and it's manifestations for better definition of diagnostic criteria. results: radiographs and barium esophagogram: algorithmic approach in reviewing chest radiographs in order to improve detection of aortic arch anomalies; classic findings and common pitfalls. cross-sectional imaging (ct and mri): what the surgeons need to know before surgical repair; detection of associated cardiac anomalies. sample cases: double aortic arch, double aortic arch with complete or partial atresia of one of the arches. conclusions: major teaching points of this exhibit are: . review of classic features of congenital aortic anomalies on radiographs, esophagogram, ct and mri with pertinent embryologic basis . describe the utility of various imaging modalities in congenital aortic anomalies, emphasizing common pitfalls. cardiovascular and mediastinal imaging in children with unexpected clinical presentation shunsuke nosaka, md, radiology, national center for child health and development, nosaka-s@ncchd.go.jp purpose or case report: children with cardiovascular and mediastinal diseases can be congenital or acquired in etiology. they usually present with straightforward clinical course. in certain situation, however, some of the children show unexpected clinical presentation predominantly with those of neighboring organs such as respiratory tract, hepatobiliary system, and gastrointestinal tract. these unexpected presentations can be the cause of delay in proper diagnosis and treatment. the purpose of this exhibit is demonstrate a variety of imaging findings of cardiovascular and mediastinal diseases in children with unexpected clinical presentation. this exhibit is case based presentation of cardiovascular and mediastinal imaging in children including tips, pitfalls and lessons learned among patients presented with unexpected clinical presentation. diagnostic imaging modalities for cardiovascular disease usually consist of various combinations of plain radiography, ultrasound, ct, mr imaging, fluoroscopy, nuclear medicine, and angiography. the general concept of alara-as low as reasonably achievable-should always be utilized when radiation-producing modalities are indicated in children. the diseases included will be double aortic arch found during workup for the cause of aspiration pneumonia, unilateral pulmonary vein atresia presented with recurrent episodes of pneumonia, severe mitral regurgitation secondary to chordal rupture mimicking fluminant hepatic failure, myocarditis initially present as acute abdomen, cardiomyopathy as unusual initial presentation of neuroblastoma, and thymolipoma mimicking gradual development of cardiomegaly. conclusions: it is important for radiologist to be familiar with imaging findings of cardiovascular and mediastinal diseases in children with unexpected clinical presentation. cardiac embryology made easy: a novel teaching approach using claymation andrew phelps, children's hospital boston, aphelpsmd@ gmail.com; purpose or case report: congenital heart disease can be an intimidating subject for radiology residents, and cardiac embryology is key to its understanding. however, this can be an equally intimidating topic to teach! various diagrams and animations are available in textbooks and online, but much like advanced origami, many of these resources suffer from being visually too complex for the first-time learner. to overcome this teaching obstacle, i created my own cardiac embryology animations using modeling clay and incorporated them into a comprehensive didactic lecture on congenital heart disease. methods & materials: cardiac embryology animations were created using modeling clay, a digital camera, and microsoft powerpoint. surface and cross-sectional views were generated, depicting the key events in cardiac embryology: heart tube formation, cardiac looping, chamber division, truncus arteriosus division, and pulmonary venous connection. example models are shown in figure . results: in this lecture, the animations are presented alongside actual embryonic heart photographs. the lecture then uses the embryology knowledge as a basis to explain the common congenital heart diseases and their mri appearances. examples of septal defects, ventricular hypoplasia, and transposition of the great arteries are presented, among others. conclusions: understanding cardiac embryology is required in order to approach congenital heart disease in a logical fashion. modeling clay animations are a cheap and easy way to simplify this complex topic. arterial tortuosity syndrome: an introduction to the clinical and radiologic manifestations in the pediatric population neal desai, umkc som, neal @gmail.com; suchit patel, ayushi gupta, marius hubbel, doug rivard purpose or case report: . to describe the clinical findings of arterial tortuosity syndrome and give a brief discussion of the disease process. . to describe the radiologic manifestations of arterial tortuosity syndrome. . to give a brief discussion of loeys-dietz syndrome-a disease with similar arterial findings, but with unique molecular characteristics from arterial tortuosity syndrome. . to use this knowledge to help establish the diagnosis and reduce mortality. methods & materials: arterial tortuosity syndrome overview • epidemiology • molecular basis • pathophysiology • review of signs, symptoms and presentation • brief discussion of treatment differential diagnosis of arterial tortuosity syndrome • loeys-dietz syndrome-similarities and differences radiologic findings and discussion • chest radiograph • computed tomographic angiography • magnetic resonance angiography-neck • magnetic resonance angiography-head • conventional angiography making a diagnosis • sample case report • review questions conclusions: arterial tortuosity syndrome is a rare disease whose chief manifestation is severe cardiovascular connective tissue defects. due to the nature of these defects and the significance of rapid intervention, it is important to be aware of and recognize the radiologic manifestations associated with arterial tortuosity syndrome in the presence of appropriate clinical history to help offer a better prognosis to the patient. dynamic pulmonary computed tomography for evaluation of cardiopulmonary disease shilpa v. hegde, md, arkansas childrens hospital, university of arkansas, shilpavhegde@gmail.com; s. bruce greenberg purpose or case report: dynamic pulmonary computed tomography (dpct) is a wide-detector ct technique that allows for continuous chest imaging during respiration. when combined with intravenous contrast, the technique is a unique tool for evaluation of cardiopulmonary abnormalities in children with cardiopulmonary abnormalities. the purpose of this poster is to illustrate the technique of dpct for evaluation of cardiopulmonary disease in children with congenital heart disease and persistent respiratory distress. methods & materials: methods and materials: dpct exams with intravenous contrast were performed on infants with a history of congenital heart disease and palliative surgery. four continuous msec gantry rotations were obtained with respiratory rates set at /minute. the imaging was accomplished during the time of a single respiratory cycle. kvp and low ma resulted in effective dose of≈ . msv. eight respiratory phases were reconstructed to create d and mpr cine loops for evaluation of cardiopulmonary abnormalities. results: cardiopulmonary abnormalities were detected in all patients. patency of sano shunt, blalock tausig shunt or patent ductus arteriosus stent was established. intimal thickening was identified in one sano shunt. hypoplastic branch pulmonary arteries were present in infants and pulmonary vein thrombosis in infant. left bronchomalacia was identified in four of five infants and best or only identified on the expiratory phase of respiration. left lung air trapping was present in two patients. conclusions: dpct with intravenous contrast is the ideal study for evaluation of the post-operative infant with congenital heart disease and persistent respiratory distress. the role of low-dose ct angiography in the evaluation of renovascular hypertension in children jessica kurian, md, chop, kurianj@email.chop.edu; monica epelman, kassa darge, els nijs, jeffrey hellinger purpose or case report: historically, the evaluation of renovascular hypertension has been accomplished via us and conventional angiography. based on the reported adult experience we introduced renal ct angiography (cta) for the evaluation of renovascular hypertension in mid- . our institution has a robust, well-established protocol, which results in reproducible, high quality images. we aim to present our imaging strategies for the evaluation of these patients and to discuss and illustrate the role of low-dose cta with -d imaging as a noninvasive alternative in the evaluation of pediatric renovascular hypertension. methods & materials: we used our department information system to identify pediatric patients (< years of age) who had documented renovascular hypertension confirmed either by conventional angiography and/or surgery during a -year period. we present our protocol and discuss the indications, limitations and benefits of renal cta. ct thin slice data, obtained employing dose reduction strategies, was reviewed and reconstructed in d and d renderings. pertinent us and mr studies as well as demographic and clinical data were reviewed and recorded. several causes for renovascular hypertension were documented and relevant ct angiographic findings were selected for presentation. results: radiation dose ranged . - msv. fibromuscular dysplasia was the most common diagnosis followed by neurofibromatosis type . vascular pathology included stenoses, beading, occlusions, and aneurysms. disease was noted in the extraparenchymal renal arteries in approximately % of the cases. the choice of the imaging modality for the investigation of renovascular hypertension in pediatric patients remains controversial. in the authors' experience, cta with -d imaging is a valuable, non-invasive diagnostic tool for the evaluation of pediatric renovascular hypertension. low dose protocols can reduce the radiation exposure associated with ct. this method can spare patients the complications associated with conventional angiography. fetal mri: brain, head and neck malformations-a pictorial essay sumit singh, md, children's hospital of wisconsin, sumitsingh @yahoo.com; mohit maheshwari, teresa c. gross kelly, tushar chandra, ibrahim s. tuna, craig johnson purpose or case report: the purpose of the exhibit is to illustrate various brain, head and neck massses/vascular anomalies on fetal mri. we will also briefly discuss the normal fetal brain anatomy as seen on fetal mri. methods & materials: major indications for fetal mri include evaluation of inconclusive sonographic findings in cases of cns malformations. in our institute patients are scanned on . t mr scanner. a body surface six channel phased array coil is used to maximize signal to noise. all the scans are checked by a neuroradiologist to make sure adequate plane imaging of the brain or other lesion in question were performed. plane scanning of the fetal body is also performed for the laterality determination of the lesion and also screen for other congenital anomalies. results: prenatal usg is frequently inconclusive for evaluation of complex fetal brain and head and neck anomalies. most studies suggest that mri after first trimester is safe. in addition, advent of rapid mri sequences like single shot fast spin echo (ssfse) have helped in reducing scan time and motion artifacts leading to availability of diagnostic quality images. these have led to increasing use of mri as supplemental tool to further investigate inconclusive fetal sonographic findings. mri provides better anatomical delineation of these complex abnormalities. it helps in making appropriate diagnosis with high confidence and aids in appropriate obstetric and prenatal/neonatal surgical planning or intervention. this educational exhibit will illustrate few common fetal anomalies. these will include agenesis of corpus callosum, malformation of cortical development, posterior fossa malformations, ventriculomegaly, in-utero stroke, orbital abnormalities and some fetal neck masses/ vascular malformation. correlation and confirmation with the postnatal mri will also be provided for some cases. conclusions: technical and therapeutic advances have driven the development of fetal mri. it is an important adjunctive tool for prenatal imaging in those instances in which a complex anomaly is suspected by sonography, when fetal surgery is contemplated, or when a definitive diagnosis cannot be determined. it has prognostic implications and may help in optimal and timely obstetric and neonatal management. purpose or case report: this educational report will provide a review of the imaging appearance of intradiaphragmatic and subdiaphragmatic pulmonary sequestrations on fetal mri. the proposed pathophysiology, review of sequestration subtypes, and surgical management options will also be described. case examples will be provided to illustrate the fetal mr imaging findings of these variants of pulmonary sequestration that help support the diagnosis. specifically a "triangle sign" of t hyperintense tissue directed toward the diaphragm will be demonstrated. illustrative case examples will be placed in the context of a differential diagnosis for subdiaphragmatic masses seen on prenatal imaging. imaging signs that help make a diagnosis of these pulmonary sequestration variants and separate this entity from other lesions will be emphasized. poster #: edu- mri of the fetal head and neck masses alok jaju, md, mallinckrodt institute of radiology, alokjaju@gmail.com; joshua shimony, per amundson purpose or case report: fetal magnetic resonance imaging (mri) is a useful problem solving tool for abnormalities detected by prenatal ultrasound (us). masses of the head and neck region can vary from benign incidental lesions to devastating neurological lesions and life threatening tumors. we share our experience in characterizing these lesions by prenatal mri, that can have a bearing on follow up imaging, perinatal management and overall prognosis. we did a retrospective review of all fetal mri studies performed at our tertiary care children's hospital between / and / , to identify fetuses with head and neck masses. we reviewed the maternal demographic and clinical data, prenatal ultrasound, fetal outcomes and post natal imaging (when available). results: out of the fetal mri studies, had dominant head and neck masses. majority were encephaloceles ( occipital, parietal). the remaining included variety of masses such as nasal glioma, teratoma ( ), epidermoid cyst, hemangioma and lymphatic malformation ( ) . mri played a useful role in distinguishing encephaloceles from other masses based on underlying bone defect and intracranial extension. it also helped in characterizing other masses based on location and signal characteristics. the presence and degree of airway compromise was determined. intracranial anomalies associated with encephaloceles including callosal dysgenesis, cerebral and cerebellar hypoplasias, migrational disorders and spinal anomalies were also correctly identified. conclusions: we present the prenatal mr imaging findings of a spectrum of head and neck lesions, correlating with prenatal ultrasound, postnatal imaging and clinical or pathological outcomes. purpose or case report: the immaturity of the cns in neonatal infants makes neurologic assessment difficult. neuroimaging plays an essential role in the assessment of brain injury by helping to indentify the injury and expected neurologic outcome. cranial ultrasound (us) is usually the first neuroimaging modality used since the technique is portable, does not involve radiation and can be used sequentially. magnetic resonance imaging (mri), however, is the most sensitive imaging modality for the detection of hypoxic brain injury. the goal of this presentation is to compare the us and mri performed within a -hour interval, and evaluate these findings to improve the interpretation of the us which is usually the first methodology used to evaluated these patients. we performed a retrospective review of the neonatal imaging studies with us and mri performed within -hour interval on preterm and term newborns with clinical history of hypoxia-ischemia. the imaging findings of the two modalities, mri and us, were correlated with the pattern and severity of the injury and brain maturity. results: diffuse white matter abnormalities were observed in % of the patients by us or mri. the ultrasound identified diffuse increased echogenicity which did not show correlation with mri in % of patients. focal white matter abnormalities were better identified by mri on non-cavitary leukomalacia which is the most common pvl observed in premature neonates with low birth weight and the most difficult to identified using us. cavitary leukomalacia showed strong agreement in both methodologies. the mri identified % more cases of intraventricular hemorrhage, however, the corresponding increase in hemorrhage was of minimal clinical significance. in most cases extra axial hemorrhage was better identified by mri. conclusions: after viewing this exhibit, the viewer will gain a better appreciation and understanding of the neuroimaging characteristics of hypoxia-ischemia in us and mri, and thus improving the interpretation of the us which is usually the first imaging modality used to evaluate this patient population. purpose or case report: the most common thoracic lesions found on prenatal imaging, congenital pulmonary airway malformation (cpam), bronchopulmonary sequestration (bps), and congenital diaphragmatic hernia (cdh), usually have characteristic imaging findings previously described in detail. however, common entities presenting with atypical findings and rarer thoracic entities do occur and can be characterized by fetal magnetic resonance (mr) imaging. the purpose of this educational exhibit is to show examples of atypical presentations of common thoracic lesions and more unusual thoracic entities on fetal mr. when applicable, prenatal mr is compared with prenatal ultrasound, postnatal imaging, operative findings, or pathology. methods & materials: using a radiology information system database, the reports of all fetal mr exams at our institution from january through january were reviewed. when unusual thoracic findings were described in the report, all prenatal and postnatal images (when available) were evaluated. in the cases selected, medical charts were reviewed for operative findings and pathologic reports. results: the cases to be described, both pulmonary and extrapulmonary in location, include: hybrid lesion in a horseshoe lung, cpam extending across the midline, bilateral bps, bps located within the mediastinum, bps located within the leaves of the diaphragm, ectopia cordis and cdh as components in pentalogy of cantrell, cdh with herniation of liver into the pericardium, elongated esophageal duplication cyst, chest wall lymphatic malformation, and tight double aortic arch causing congenital high airway obstruction syndrome (chaos). conclusions: after studying this educational exhibit, the reader will be acquainted with a variety of unusual fetal pulmonary and extrapulmonary lesions, with emphasis on fetal mr. prenatal and postnatal imaging findings in megacystis-microcolon-intestinal hypoperistalsis syndrome (mmihs) mary kitazono, chop, mkitazono@gmail.com; richard bellah purpose or case report: to review the classic constellation of findings seen in prenatal and postnatal imaging of megacystis-microcolon-intestinal-hypoperistalsis syndrome (mmihs), as well as to illustrate additional imaging features that are variably seen in this syndrome. the imaging database at our children's hospital was searched for all cases of mmihs diagnosed since . all available prenatal and postnatal imaging studies were reviewed in patients with a diagnosis of mmihs, and representative images are provided with a description of the findings. results: since , patients ( girls, boy) have been diagnosed with mmihs at our institution, including on prenatal mri and us. the characteristic prenatal imaging findings include marked urinary bladder distension, bilateral pelvicaliectasis, and dilated, tortuous ureters, as well as a diminutive colon containing no or minimal t w-hyperintense meconium on mri. postnatal imaging studies also characteristically demonstrate a massively distended urinary bladder (with no apparent mechanical cause of obstruction) as well as a small, unused colon with dilated, hypoperistaltic small bowel seen proximal to the microcolon. additional findings which are variably seen include intestinal malrotation, stomach and esophageal hypoperistalsis or aperistalsis, gastroesophageal reflux, and biliary stasis. conclusions: although a rare syndrome, the constellation of imaging findings in mmihs is pathognomonic, and recognition of the classic pattern of findings can allow the radiologist to make a diagnosis of mmihs in both the in-utero and postnatal setting. early diagnosis is essential for allowing prenatal counseling regarding this generally fatal disorder, as well as to optimize early management options. purpose or case report: gastric mass lesion are uncommon. this presentation is an educational review of pediatric gastric mass lesions including gastro-intestinal stromal tumor (gist), inflammatory myofibroblastic tumor (pseudotumor). burkitt's lymphoma, squamous cell carcinoma, gastric teratoma, gastric varices, gastric hamartoma, gastric polyp and hypertrophic pyloric stenosis (hps). clinical presentation is varied with upper gi bleeding, feeding intolerance, pain, weight loss and fatigue manifesting. the imaging work-up might initially have been endoscopy or ultrasound. cross section imaging (ct mr) can be invaluable. the role and impact of fdg pet on the management, staging and follow up of the oncologic pathology will be emphasized. imaging findings in megacystic microcolon intestinal hypoperistalsis syndrome, a rare disease kiery braithwaite, pediatric radiology, emory-egleston, kieryb@yahoo.com; kiery braithwaite, paula dickson, marianne m. ballisty purpose or case report: megacystis microcolon intestinal hypoperistalsis (mmih) syndrome is a rare congenital form of severe functional intestinal obstruction which is more commonly found in females. the presenting clinical and imaging features of this disease can often mimic other causes of proximal bowel obstruction in the neonate. in combination with its common association with intestinal malrotation, the clinical picture of mmih syndrome may be confusing at times. awareness of additional imaging features characteristic of mmih syndrome may help the radiologist suggest this diagnosis. the purpose of this study is to enhance the ability of the pediatric radiologist to suggest this rare diagnosis by recognizing this unusual constellation of imaging features. we retrospectively reviewed the clinical data and imaging studies of four patients with mmih syndrome at our institution. imaging studies included plain radiography, ultrasonography, fluoroscopy, and cross sectional imaging. the initial presentation and clinical outcome was also reviewed. results: the clinical presentations of our patients, who were all female, were somewhat varied but typically included symptoms of intestinal obstruction. the diagnosis of mmih syndrome was made in our patients from the first few weeks of life through early childhood. the four patients demonstrated imaging features characteristic of this disease including a very large dilated bladder, severe bilateral hydroureteronephrosis, gaseous distention of the stomach and proximal small bowel, intestinal hypoperistalsis, and a very small colon. the clinical course of these patients that we observed was also quite variable, with some patients dying in neonatal period while another patient continues to do reasonably well at years old after a multi-organ transplant. conclusions: mmih syndrome is a rare and frequently lethal disease. the ability of the pediatric radiologist to recognize this constellation of imaging findings can help the clinical team arrive at a diagnosis of mmih syndrome. more prompt diagnosis can aid in the development of a long term management plan for the patient and in counseling the family regarding the prognostic implications of this disorder. pathologies of omphalomesenteric duct remnant: radiologic-surgical correlation swapnil bagade, md, pediatric radiology, mallinckrodt institute of radiology, bagades@mir.wustl.edu; geetika khanna, rebecca hulett purpose or case report: . to facilitate understanding of embryology of the omphalomesenteric(vitelline) duct and normal anatomy of the umbilicus. . review the spectrum of omphalomesenteric duct malformations and diversity of clinical presentations of these remnants. . illustrate the imaging findings of omphalomesenteric remnants, from the common such as meckel's diverticulum to the uncommon such as the omphalomesenteric duct cyst, with surgical correlation. methods & materials: cases with complications of persistent omphalomesenteric duct were collected from the joint surgery/radiology conferences at a tertiary level children's hospital. imaging features were correlated with intraoperative findings. conclusions: preoperative diagnosis of complications related to the omphalomesenteric duct remnants can be challenging because clinical and imaging features overlap with other etiologies of acute abdomen. knowledge of the embryologic, clinical, radiologic, and surgical characteristics of omphalomesenteric duct remnants will aid in early and accurate diagnosis. neonatal bowel obstruction-a pictorial essay tanmay patel, university of kentucky; harigovinda challa purpose or case report: bowel obstruction is the most common abdominal emergency in the newborn period and in most cases is secondary to a congenital anomaly requiring early surgical intervention. however not every case of abdominal distension or dilated bowel is secondary to mechanical bowel obstruction or underlying surgical condition. radiologic imaging forms a central role in the work up of newborns with suspected intestinal obstruction. the role of the radiologist is to identify whether or not mechanical obstruction is present; if obstruction is identified on initial radiographs, to determine the level of obstruction, and finally to identify the etiology of obstruction. initial plain radiographic evaluation also helps to determine the subsequent diagnostic or therapeutic approach. methods & materials: a retrospective review of multiple radiographic and fluroscopic examinations in patients with diagnosis of neonatal bowel obstruction was performed at kentucky children's hospital. multiple examples of classical imaging findings were compiled and placed into a pictorial review. results: neonatal intestinal obstruction generally presents with nonspecific symptoms such as abdominal distention, vomiting, or failure to pass meconium depending on the level of obstruction and time of occurrence of underlying congenital lesion/atresia in the intrauterine life. initial plain radiographs of the abdomen reveal dilated bowel loops when obstruction is present. high intestinal obstruction is suspected when only few dilated loops are identified, while multiple dilated bowel loops are seen in low obstruction. most cases of high obstruction may not need another diagnostic imaging test. all cases of distal intestinal obstruction require water soluble enema to identify the etiology of obstruction. in conditions like functional immaturity of the colon, and meconium ileus water soluble enema is therapeutic and thus surgery can be avoided in most cases. the objective of this presentation is to present an educational exhibit of classical imaging findings of various types of neonatal bowel obstructions, and how to differentiate between them. conclusions: bowel obstruction is the most common abdominal emergency in the new born period. most cases are secondary to a congenital surgical condition and early diagnosis and treatment significantly reduces mortality and morbidity. radiographic evaluation plays a central role in the diagnosis and treatment of these conditions. poster #: edu- d t -weighted mrcp in the pediatric population-a pictorial review nathan egbert, mbbs mph, university of michigan, nathaneg@med.umich.edu; jonathan r. dillman, peter j. strouse purpose or case report: to demonstrate the utility of d t -weighted magnetic resonance cholangiopancreatography (mrcp) in the pediatric population, and to illustrate the mrcp findings of various conditions affecting in the pediatric pancreaticobiliary system. we identified all mrcp exams performed on pediatric patients (< years of age) from january , through august , by searching institutional electronic medical records. we then identified representative d t -weighted mrcp images of various conditions affecting the pediatric pancreaticobiliary system. results: representative d t -weighted mrcp images (including source, maximum intensity projection, and volume rendered images) from the following conditions will be presented: abnormal biliary narrowing/stricture (including sclerosing cholangitis, anastomotic strictures following kasai procedure & liver transplantation, and "pseudostricture"), biliary atresia, choledochal cyst (including various subtypes, based on todani classification), choledocholithiasis & cholelithiasis, congenital anomalies of the pancreaticobiliary system (including pancreas divisum and anomalous pancreaticobiliary junction), pancreatobiliary system trauma (including main pancreatic duct transection), and other rare conditions affecting the pancreaticobiliary system (including rhabdomyosarcoma of the biliary tree). conclusions: d t -weighted mrcp has become an extremely useful tool in the evaluation of children with suspected disorders of the pancreaticobiliary system. since mrcp has distinct advantages over alternative diagnostic techniques, such as endoscopic retrograde cholangiopancreatography (ercp) or percutaneous cholangiography, including lack of ionizing radiation and noninvasiveness, mrcp is a much preferred initial study for pediatric pancreaticobiliary imaging. this pictorial review is intended to highlight the d t weighted mrcp appearances of various pancreaticobiliary conditions occurring in the pediatric population. purpose or case report: magnetic resonance enterography (mre) is rapidly emerging as an important imaging tool for the diagnosis and follow-up of inflammatory bowel disease (ibd). its lack of ionizing radiation makes this imaging modality especially vital to the pediatric population. using a casebased approach, we will demonstrate the usefulness of diffusion-weighted imaging (dwi) as part of a comprehensive mre protocol for the assessment of ibd in children. the basics of dwi will be discussed with particular attention to abdominopelvic techniques. the role of mre dwi for the evaluation of pediatric crohn disease (cd) and ulcerative colitis (uc) will be reviewed using a case-based approach. key images from pertinent imaging studies will be identified by searching institutional electronic medical records and presented with relevant clinical data. results: a review of pediatric mre examinations suggests dwi can be used to detect the following: ) small and large bowel segments affected by ibd (both cd and uc) ) abdominopelvic abscesses (including within the mesentery, body wall, iliopsoas muscle, and liver) ) abnormal lymph nodes ) sacroiliitis ) perianal disease (including abscesses and other penetrating complications). conclusions: dwi has the potential to play a very important role in the diagnosis and follow-up of pediatric ibd. this mre technique is particularly useful for detecting a variety of disease-related complications. as the exact meaning of bowel wall restricted diffusion is poorly understood to date, continued investigation will be necessary to determine the clinical and histologic significance of this finding. cases of cf involving the gi tract were collected from clinical workflow encounters of the authors and from the main hospital medical records database. relevant imaging studies were reviewed for known gi manifestations of cf. these imaging studies were correlated with clinical histories and available intraoperative and pathologic findings. results: cf involvement of the gi tract presents over a wide range of ages, organs involved, and associated symptoms. these manifestations can generally be divided anatomically into those involving the alimentary tract, hepatobiliary system, and pancreas. alimentary tract manifestations consist of meconium ileus in uncomplicated and complicated forms (with the latter including secondary intestinal atresia, volvulus, and perforation with meconium peritonitisdistal intestinal obstruction syndrome, constipation, rectal prolapse, duodenal fold thickening, and appendiceal dilation. hepatobiliary disorders secondary to cf include microgallbladder, cholelithiasis, biliary ductal abnormalities, neonatal hepatitis, and cirrhosis (including complications such as portal vein thrombosis and ascites). pancreatic expressions of cf include fatty infiltration, calcifications, and cysts/ cystosis, frequently in the setting of malnutrition and/or stooling abnormalities. this exhibit will demonstrate the spectrum of clinical and radiologic gi findings in this disease from the fetal and neonatal period through adolescence across a range of imaging modalities. conclusions: gastrointestinal manifestations of cystic fibrosis occur frequently in the pediatric population and may be the earliest clinical expression of the disease. familiarity with the variety of gastrointestinal imaging findings of cystic fibrosis can expedite appropriate diagnosis and therapy, particularly in those children in whom the primary disease is not clinically suspected. beyond acute appendicitis: imaging of additional pathologies of the pediatric appendix kelly dietz, md, cincinnati children's hospital; arnold c. merrow, daniel j. podberesky, alexander j. towbin purpose or case report: primary acute appendicitis (or appendiceal inflammation caused by a superimposed bacterial infection in the setting of appendiceal obstruction) is by far the most common pathology of the appendix, and imaging evaluations to exclude this diagnosis occur daily in the pediatric radiology setting. the clinical and imaging differential diagnosis in a patient with right lower quadrant pain and suspected appendicitis is a broad but well-recognized list that predominantly involves structures adjacent to the appendix including the ovaries, small and large bowel, and ureters. there are, however, less common pathologies primarily involving the appendix which can create an imaging diagnostic dilemma in the setting of right lower quadrant symptoms. our goal is to review the imaging and clinical manifestations of these less commonly encountered appendiceal abnormalities. methods & materials: cases of appendices that were abnormal by imaging but ultimately determined not to be due to primary acute appendicitis were collected from clinical encounters by the authors as well as through a search of the radiology and pathology report databases. clinical course, surgical findings, and pathology reports (if available) were subsequently reviewed through the main hospital medical records system. results: the collected cases demonstrate a wide range of additional pathologies of the appendix outside of primary acute appendicitis. a variety of imaging modalities were employed in the workup of these cases. examples reviewed in this exhibit include crohn's disease, ulcerative colitis, cystic fibrosis, carcinoid tumor, inguinal hernia with incarceration, retained foreign body, pinworm infestation, and ileocolic intussusception. conclusions: despite the frequency of primary acute appendicitis, there is a differential diagnosis when an abnormal appendix is found by imaging. familiarity with these alternative diagnoses may be particularly helpful in guiding management of the patient whose clinical presentation is not typical for primary acute appendicitis. methods & materials: a hospital pacs database search from the past years for patients with bws. selected cases, with multimodality imaging, were cross-referenced with pathology reports from patient records database. results: intricate abdominal pathologies are depicted utilizing multimodality imaging, such as plain films, us, ct, mri and pet/ct, and with pathologic correlation. cases with highlight the following: liver: hepatoblastoma, nonspecific hepatobiliary cysts, multiple hemangiomas mimicking metastatic disease; adrenal: dysplastic organomegaly mimicking neoplasm; pancreas: diffuse and focal hyperplasia in the setting of hyperinsulinism, organomegaly; renal: neprocalcinosis, including medullary sponge kidney, nephroblastomatosis, organomegaly; adnexal: ectopic paraovarian adrenal tissue mimicking metastatic lymph node; urinary bladder: benign fibro-uroepithelial polyp. conclusions: diagnosis of bws can be difficult when the classic clinical and radiological findings are not present. these few cases highlight the unusual abdominal pathologies, so when detected, a radiologist can aid in the appropriate diagnosis and help guide therapy for these young patients. this poster will discuss pharmaceuticals the fda considers investigational for their intended use. disclosure: dr. lecompte has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. radiologic-pathologic review of pancreatic masses encountered at a tertiary pediatric hospital over a -year period no kwak, md, radiology, long island jewish medical center, kwak_nb@yahoo.com; karen naar, jeanne choi-rosen, lee collins, sukhjinder singh, anna thomas purpose or case report: review of pathologically proven pancreatic masses in pediatric patients encountered at a tertiary pediatric hospital over a -year period. describe the key morphologic features and other pertinent findings using various imaging modalities. correlate pathologic and radiologic findings. methods & materials: illustrate the various imaging characteristics of pathologically proven pancreatic masses including pseudocyst, pancreatoblastoma, solid pseudopapillary tumor, acinar cell carcinoma, ductal adenocarcinoma, lymphoma, pancreatic neuroblastoma, and inflammatory myofibroblastic tumor. correlate pathologic and radiologic findings. identify the key imaging features that allow narrower differential diagnosis. results: pancreatoblastoma and solid pseudopapillary tumor are the more commonly encountered pediatric primary pancreatic tumors. both are bulky and heterogeneously enhancing tumors with solid and cystic elements. pancreatoblastoma occurs more commonly in young children. internal hemorrhage and fibrous capsule favor solid pseudopapillary tumor which more commonly occurs in adolescent girls. ductal adenocarcinoma, acinar cell carcinoma and an inflammatory myofibroblastic tumor, which were pathologically proven in our pediatric patients, are exceedingly rare entities. the imaging findings of these cases and their pathology when available will be presented, as well as a quick literature review of these rare tumors. illustration and correlation of the pathologic and radiologic findings. conclusions: pancreatic masses in children are rare but in general have a better prognosis than in adults. salient imaging findings for the various tumors encountered at a tertiary care center with pathologic and radiologic correlation. evaluation of hepatoblastoma with gadoxetate disodium-typical, atypical, pre and post treatment evaluation arthur b. meyers, radiology, cincinnati children's hospital, arthurbmeyers@yahoo.com; alexander j. towbin, daniel j. podberesky purpose or case report: gadoxetate disodium (gd-eob-dtpa) is a hepatobilliary mri contrast agent that is widely used in adults for characterization of liver tumors and is being increasingly used in pediatric patients. hepatoblastoma is the most common primary hepatic malignancy of childhood. the purpose of this presentation is to describe our experience with the use of this agent in the mri evaluation both before and after initiating therapy in patients with hepatoblastoma. methods & materials: the radiology report system at our institution was queried for all patients with pathology proven hepatoblastomas who underwent a liver mr with administration of gadoxetate disodium between / / and / / . the mr imaging characteristics of the patient's primary hepatoblastoma pre-and post-therapy (when available) and post treatment findings (when available) were reviewed. results: mri studies in different patients were reviewed. the patients ranged in age from months to years. patients had pre and post treatment evaluation with gd-eob-dtpa enhanced mri, patient had only pretreatment evaluation and patients had only post treatment evaluation. of the hepatoblastomas did not take up gd-eob-dtpa during the hepatocyte phase and were therefore low signal intensity during the hepatocyte phase of imaging. this was useful in the pretreatment evaluation of hepatoblastoma, particularly in defining the relationship of the tumor to hepatic and portal veins. post treatment gd-eob-dtpa imaging allowed characterization of the biliary anatomy and demonstrated the communication of a postoperative fluid collection with the biliary tree, consistent with biloma. atypical hepatoblastoma showed uptake of gd-eob-dtpa on hepatocyte phase imaging, similar to what has been described in adults with atypical hepatocellular carcinoma. conclusions: gadoxetate disodium enhanced mri is useful in the imaging evaluation of hepatoblastoma, particularly in defining the relationship of tumor to vascular and biliary anatomy and in characterizing post-treatment complications. disclosure: dr. meyers has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. imaging of the gallbladder and biliary tree in pediatric age group ihsan mamoun, md, cleveland clinic, ihsanmamoun@ yahoo.com; s. pinar karakas, unni udayasankar, neil vachhani, ellen park purpose or case report: interactive educational exhibit to illustrate the embryology, anatomical variants as well as congenital and acquired diseases of the bile ducts and gallbladder in pediatric patients. methods & materials: a)the embryology of the gallbladder and biliary tree will be demonstrated with diagrams. b) imaging techniques for gallbladder and biliary tree including us, ct, mri, ercp and intraoperative cholangiogram will be discussed. c)imaging findings of various lesions with special emphasis on key findings that can lead to accurate diagnosis will be discussed. d)an appropriate list of differential diagnosis will be provided. e)an algorithm for the assessment of suspected biliary pathology will be presented. f)the exhibit will be interactive and the reader will answer questions about the discussed entity, related imaging algorithm and management. results: a)discuss congenital anomalies including duplicated and septated gallbladder, choledochal cyst, caroli disease, situs abnormalities and biliary atresia. b)discuss infectious and inflammatory conditions including cholecystitis, kawasaki's disease, sclerosing cholangitis and hepatitis. c)discuss iatrogenic complications including post transplant biliary stricture and leak. d)discuss benign and malignant neoplasms invoving the gallbladder including polyps, ptld and rhabdomyosarcoma. conclusions: this exhibit will demonstrate a logical approach to imaging of the congenital and acquired diseases of the gallbladder and biliary tree based on the embryology and underlying pathology. postnatal work up of congenital uronephropathies-a pictorial essay harigovinda r. challa, radiology, university of kentucky, hch @uky.edu purpose or case report: the use of obstetric ultrasound routinely in the prenatal care has lead to the discovery of many fetal anomalies. uronephropathies in the newborn represent one of the largest groups of anomalies amenable to neonatal management. since these uropathies are detected mostly in asymptomatic patients the treatment is mainly preventive. the pediatric radiologist has a key role in the post natal work up and management of these patients with prenatally diagnosed neprhouropathies and familiarity with the congenital urinary tract abnormalities is necessary. methods & materials: a retrospective review of multiple radiographic, sonographic and fluroscopic examinations performed in the newborn babies and infants with prenatal diagnosis of urinary tract abnormalities was performed at kentucky children's hospital. multiple examples of classical imaging findings were compiled and placed into a pictorial review. results: numerous anomalies can be detected in utero, including anomalies of renal number, position, morphology, collecting system dilation and bladder, urethral abnormalities. of these postnatal work of congenital hydronephrosis is the most common routinely encountered clinical entity. renal ultrasound is the initial examination in the evaluation in all cases of prenatal hydronephrosis, which is best performed around postnatal day . if collecting system dilatation persists on postnatal ultrasound, further imaging work up with vcug, radionuclide imaging may be required depending on degree of dilatation. conclusions: uroneprhopathies are increasingly detected in the prenatal life with increasing use of obstetric ultrasound. the objective of this presentation is to demonstrate in a pictorial essay of different neprhouropathies and their workup in newborns. isolated fallopian tubal torsion: causes, imaging findings, and how to suggest the diagnosis jesse courtier, md, ucsf dept of radiology, jesse. courtier@ucsf.edu; amaya m. basta, rebecca maine, pierre-alain cohen, shinjiro hirose, john d. mackenzie purpose or case report: the purpose of this educational report is to describe the rare entity of isolated fallopian tubal torsion in the pediatric population and depict the cross sectional imaging findings that help make a diagnosis and guide management. the proposed pathophysiology, predisposing factors, and surgical management will be described. an illustrative case example of -year-old female patient will be provided with surgical correlation. the exhibit will review imaging findings on us, ct and mri that help support the diagnosis including, dilated tubular structure in the pelvis, normal ovaries, and corkscrewing and beaking of the proximal fallopian tube. isolated fallopian tubal torsion will be placed in the context of a differential diagnosis for girls presenting with pelvic pain and the imaging signs that help make a diagnosis of isolated tubal torsion and separate this entity from other causes of pediatric pelvic pain will be emphasized. multimodality imaging characteristics of genitourinary rhabdomyosarcoma rhea udyavar, md, george washington university medical center, rudyavar@gwmail.gwu.edu; amir noor, pranav k. vyas purpose or case report: in this pictorial essay, we will demonstrate salient imaging features of mr, us, and ct modalities for the diagnosis of genitourinary rhabdomyosarcoma in male (n ) and female (n ) children ages - years, evaluated at our institution over the past years. background information, including tumor biology, staging, and treatment will also be discussed. the swollen scrotum: ultrasound technique and differential diagnosis kelli r. schmitz, md, oregon health & science university, schmitzk@ohsu.edu; roya sohaey purpose or case report: to review the ultrasound protocol for the performance of scrotal ultrasound and illustrate the ultrasound appearance of conditions resulting in scrotal swelling in pediatric patients. a retrospective review of the imaging database at a tertiary pediatric referral center was performed to identify pediatric patients who presented with scrotal swelling and underwent diagnostic ultrasound. when available, surgical/pathologic correlation was obtained. results: a variety of pathologic processes result in scrotal swelling. causes illustrated include: testicular torsion, epididymitis/orchitis, hydrocele, varicocele, inguinal hernia, trauma, adrenal rest, and testicular or paratesticular neoplasm. conclusions: the causes of scrotal swelling are myriad, including infectious/inflammatory, developmental, traumatic, and neoplastic etiologies. in children, the clinical presentation of a swollen scrotum is nonspecific, and ultrasound plays a key role in making the correct diagnosis. experiences of starting a functional mr urography program at a university hospital: trials and tribulations steven l. blumer, bsc, montefiore medical center/albert einstein college of medicine, sblumer@montefiore.org; ibrahim tuna, amanda north, benjamin taragin, netta blitman, terry l. levin purpose or case report: starting a functional mru program can be challenging as there are numerous potential hurdles to overcome. this presentation describes the process of starting a functional mr urography (fmru) program at a university hospital and discusses the difficulties encountered starting such a program. selecting a sufficient patient referral base, resolving common and uncommon technological issues, and education of clinicians, patients and technical staff are some of the challenges that will be discussed. conclusions: awareness of the common pitfalls in fmru imaging and close partnering with referring physicians can make establishing a functional mru program easier. despite many potential obstacles, the benefit of exquisite anatomical and functional information provided by fmru in children, without exposure to ionizing radiation, greatly outweighs any challenges. disclosure: dr. blumer has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. pictorial review of ultrasound findings in boys presenting to emergency department/urology with acute scrotum teresa liang, faculty of medicine, university of british columbia, teresaliang @gmail.com; peter metcalfe, william sevcik, michelle noga purpose or case report: testicular torsion is a common acute condition in adolescent boys. rapid and accurate diagnosis is critical. diagnosis is currently based on history, physical findings, and ultrasound (u/s) with doppler. the objective of this poster is to demonstrate ultrasound findings from a retrospective review of acute scrotum over years, and to demonstrate some pitfalls of the technique with regard to testicular torsion diagnosis. we reviewed the u/s, surgical and ed records at the stollery children's hospital for boys aged month to years, presenting with acute scrotum from july , to . age, demographics, clinical symptoms, and physical findings, u/s and surgical techniques, findings, diagnoses and follow-up were also recorded. results: patients presented to uah stollery ed with acute scrotum: were diagnosed with testicular torsion ( inguinal torsion), were suspected of a torsion-detorsion, torsion of appendix testes, epididymitis/orchitis, and other diagnoses including hydroceles, varicoceles, epididymal cysts, abscesses, cellulitis and hematomas. for the patients who had ultrasound, % sensitivity and % specificity for testicular torsion. the ultrasound findings including size, vascularity and echogenicity associated with both salvageable and necrotic testicles including use of color and pulse doppler will be reviewed. the sonographic findings and pictorial examples associated with the more common acute scrotum etiologies will be presented. sonographic findings from problematic cases (those with inconclusive ultrasound reports or false positive reports) will also be addressed. conclusions: ultrasound imaging problem case examples and characteristic findings of common acute scrotum presentations at stollery hospital at the university of alberta are reviewed in this poster. primary and secondary amenorrhea in pediatric patients: from the beginning to the end cesar cortes, md, miami children's hospital, n c @ hotmail.com; yanerys ramos, ricardo restrepo, alejandro diaz, lorena sequeira, edward lee purpose or case report: to describe the role of imaging in evaluating patients with primary and secondary amenorrhea and to illustrate the normal imaging findings of the reproductive organs in the pediatric population as well as the imaging findings of the different etiologies causing amenorrhea. a search of the literature is done to determine the different etiologies of amenorrhea and the role of imaging in their evaluation. first, we will focus on the normal physiologic hormonal influence and changes of the girl's reproductive organs since birth until adolescence on ultrasound and mri. images of the normal appearance of the female reproductive organs as well as imaging findings of the different common and uncommon etiologies of amenorrhea will be shown. then, specific reference will be made to crucial related concepts such as minipuberty of infancy, latest criteria for polycystic ovarian disease and ovarian failure syndrome among others. finally, the treatment, either medical or surgical will be briefly discussed. results: causes of amenorrhea in children range from disorders affecting the hypothalamus, pituitary gland, adrenal glands, and ovaries, as well as uterine and vaginal structural abnormalities. even though history and clinical exam are essential in evaluating a patient with amenorrhea, the pediatric radiologist plays a pivotal role helping guide the area to be imaged and thus the modality that should be used. mri and ultrasound are the main modalities in the evaluation of amenorrhea. conclusions: ultrasound and mri are the main imaging modalities used in the evaluation of amenorrhea in children and are usually part of the work up. amenorrhea in children can have implications in girl's fertility allowing pediatric radiologists to play an important role in helping not only the patient but also their offspring. imaging of mullerian duct anomalies in children kelly k. horst, md, radiology, university of michigan, khorst@med.umich.edu; maryam ghadimi mahani, deepa pai, jonathan r. dillman, peter j. strouse purpose or case report: the purpose of this educational exhibit is to provide an up-to-date appraisal of mullerian duct anomalies presenting in the pediatric population. the appearances of anatomic variants on ultrasound and mri will be used to illustrate the strengths and potential pitfalls of these imaging modalities. methods & materials: patients who have previously undergone ultrasound and/or mri in the course of their clinical workup within the university of michigan health system (umhs) were identified using electronic medical records. imaging reports were reviewed by a single author in order to identify relevant imaging findings (interesting anatomic variations, associated anomalies, etc.). pertinent images from these imaging examinations were de-identified and saved to a secure hard drive. the medical record was accessed by a single researcher to obtain relevant information regarding the patients' clinical presentations. in cases of corrective surgery, pathology reports were reviewed, if available, for correlation with the imaging findings. results: cases of mullerian duct anomalies were reviewed within the pediatric population. clinical manifestations were correlated with imaging appearances. conclusions: mullerian duct anomalies represent a range of developmental variants. although functioning ovaries and age-appropriate external genitalia are characteristic, there may be anomalies ranging from uterine and vaginal agenesis, to duplication of the uterus and vagina, to minor uterine cavity abnormalities. müllerian malformations are frequently associated with abnormalities of the renal and axial skeletal systems, and pediatric patients in particular may present with these associated anomalies. menstrual abnormalities may represent a more typical presentation in the adolescent age group. this is in contrast to the adult population, which may be more likely to present with infertility. the variation in clinical presentations make mullerian duct anomalies difficult to diagnose and, because surgical techniques for correction and treatment depend on the underlying anatomy, understanding these variants in the context of imaging studies is important to their diagnosis and management. patient had radiographs which showed an irregular left humeral metaphysis with an associated fracture. patient had a phase bone scan that showed slightly increased uptake on the angiographic and blood pool phases and increased activity on the delayed phase in the right femur. radiographs showed a moth eaten appearance of the right femur with soft tissue swelling. patient had radiographs that showed periosteal reaction in the right tibia with an associated fracture. patient , in addition to radiographs, had an mri that showed osteomyelitis of the left humerus and scapula with an associated subperiosteal abscess. patient had multi focal osteomyelitis that was demonstrated on radiographs by irregular cortices and periosteal reaction involving the upper and lower extremities. conclusions: neonatal osteomyelitis is an uncommon entity that can have severe complications if not diagnosis and treated promptly. it is important to review cases and to review the appearance of neonatal osteomyelitis on multiple modalities. radiographs will usually demonstrate periosteal reaction and possibly soft tissue swelling. additional studies may be obtained to evaluate for complications, such as abscesses or involvement of the joint space. purpose or case report: review the epidemiology of ddh. describe the critical diagnostic imaging findings of ddh. understand the role of imaging accompanying treatment. methods & materials: images including radiographs, ultrasound, ct and mri will be used to demonstrate the current and historical role of imaging in caring for patients with ddh. discussion of the importance of reducing radiation exposure when choosing imaging studies will be included. results: radiographs and ultrasound are used primarily in making the diagnosis of ddh. ultrasound and mri are most often used during the course of treatment to assess its effectiveness. mri is increasingly utilized without sedation for patients in spica cast. conclusions: imaging is critical in the care of patients with ddh. pediatric musculoskeletal ultrasound of the proximal lower extremity (pelvis to thigh) julia rissmiller, md, dept of radiology, children's hospital boston, julia.rissmiller@childrens.harvard.edu; howard christianson, michael j. callahan purpose or case report: to review indications for ultrasound of the proximal lower extremity (pelvis, hip and thigh), and to illustrate the practical use of ultrasound in evaluation of the proximal lower extremity, emphasizing the sonographic appearance of various musculoskeletal disorders. ultrasound is a well-established modality for the evaluation of painful hip, developmental hip dysplasia, soft tissue infection, palpable masses, and foreign bodies in children. in general, ultrasound has a more limited role for the primary evaluation of other pediatric musculoskeletal disorders including trauma, articular and periarticular diseases and tumors or tumor-like processes. advantages of ultrasound, a relatively non-invasive technique, include excellent spatial resolution, low cost, lack of ionizing radiation, lack of need for sedation, and the ability to image the patient in real-time. the major disadvantage of ultrasound is operator dependency, which is particularly evident in musculoskeletal applications. we present ultrasound examples of pathology involving the proximal lower extremity (pelvis, hip and thigh). cases include developmental hip dysplasia, hip effusion, osseous metastasis to the iliac bone, osteomyelitis of the hip, femoral acetabular impingement, rectus femoris hernia, vascular malformation, ewing's sarcoma and myositis ossificans. results: a range of images from pediatric diagnostic ultrasounds performed of the proximal lower extremity (pelvis to thigh) will be presented emphasizing the sonographic appearance of various musculoskeletal disorders. conclusions: ultrasound is an excellent modality for evaluating the proximal lower extremity in children, beyond the current indications of painful hip, developmental hip dysplasia, soft tissue infection, palpable masses, and foreign bodies in children. a multi-modality pictorial review of lesions of the epiphysis in infants and children ernesto i. blanco, md, st. christopher's hospital for children, eiblanco @gmail.com; jacqueline urbine, evan geller, peter pizzutillo purpose or case report: to review the imaging spectrum of epiphyseal lesions in infants and children. a retrospective review of our imaging database was performed to identify studies with either primary lesions of the epiphysis or processes that affect the epiphysis. results: multiple epiphyseal lesions were elucidated primarily by radiography, with cross-sectional imaging included where clinically necessary. congenital lesions include the epiphyseal dysplasias represented here by chondrodysplasia punctata. epiphyseal infarction may due to multiple etiologies including slipped capitol femoral epiphysis, developmental dysplasia of the hip, sickle cell disease, or idiopathic reasons. neoplasms may occur in the epiphysis, including chondroblastoma and histiocytosis. traumatic lesions include fracture and avulsion. osteomyelitis can occur in the epiphysis as well. pseudolesions that mimic pathology will also be reviewed. other pathologies that can affect the epiphysis include juvenile idiopathic arthritis and hemophilia. conclusions: a wide spectrum of congenital and acquired pathologies may affect the epiphysis in the infant and child. plain radiography, computed tomography, and magnetic resonance imaging all contribute to the diagnosis of these varied lesions. purpose or case report: we aim to present the spectrum of common and uncommon hip disorders in pediatric population. we will formulate a systematic approach and present a flowchart to workup and characterize hip diseases. methods & materials: relevant imaging appearances of normal as well as pathological hip will be presented. normal hip anatomy will be discussed through anatomic drawings and radiological images (plain radiographs, ct, usg, and mri). we will illustrate the various anatomic landmarks, measurements and lines on plain radiographs and ultrasound of hip. results: evaluation of limp and hip pain in the pediatric population has undergone rapid evolution. surgical treatment for these disorders continues to be refined, and our ability to identify patients along the spectrum of disease continues to improve. yet, despite our advances, obtaining an accurate diagnosis can remain challenging, especially in the setting of mild structural abnormalities. many imaging studies can be used to evaluate the bones and soft tissues, but conventional radiography is the primary imaging modality for most clinical conditions. plain radiographs usually are obtained first because they are sensitive and specific for a wide range of bone pathology. more sophisticated imaging modalities including radionuclide scintigraphy (bone scan), ultrasonography (usg), computed tomography (ct) and magnetic resonance imaging (mri) are reserved for specific clinical situations. each of these imaging modalities has specific advantages and disadvantages. it is the aim of this review to guide in selecting and interpreting the appropriate imaging modality for a variety of common disorders. this exhibit will illustrate imaging features of developmental dysplasia of hip, perthes disease, slipped capital femoral epiphyses, hip malformations in syndromes, femoral acetabural impingement, labral disorders, septic arthrits and other disorders. the role of various imaging modalities in evaluation of these disorders will be discussed, along with common imaging pearls and pitfalls. conclusions: a systematic approach is necessary for evaluation of pediatric hip disorders. familiarity with normal appearances, pitfalls and specific imaging of these entities is essential for proper diagnosis and management. osteoid osteomas: a pain in the "night" diagnosis nancy k. laurence, md, the children's hospital of philadelphia, nkang @gmail.com; monica epelman, richard markowitz, camilo jaimes, diego jaramillo, nancy chauvin purpose or case report: a common benign bone-forming lesion, osteoid osteoma comprises approximately % of all benign bone tumors. the tumor is composed of a nidus of vascular osteoid tissue and woven bone lined by osteoblasts, frequently with considerable surrounding inflammation. the radiolucent nidus surrounded by variable degrees of reactive sclerosis usually leads to a straightforward diagnosis; however, sometimes the diagnosis of osteoid osteoma can be challenging, as it may have a non-specific and misleading appearance on different imaging modalities, particularly on mri. the purpose of this exhibit is to review the typical and atypical features of osteoid osteomas on different imaging modalities. we present diagnostic dilemmas of osteoid osteomas from our institution and how imaging characteristics can aid in diagnosis. we performed a retrospective review of our imaging database to identify cases of typical and atypical osteoid osteomas, with special emphasis on cases which posed a diagnostic dilemma on imaging. results: when osteoid osteomas occur in atypical locations the diagnosis can be elusive. when located in the intraarticular space there is often minimal or absent cortical thickening and there may be a joint effusion with synovial hypertrophy. phalangeal lesions may cause extensive bone marrow edema and surrounding soft tissue swelling. both of these types of osteoid osteomas can be mistaken for infection. the recently described "ct vessel" or "vascular groove" sign, a low density vascular groove adjacent to the nidus, is highly specific for osteoid osteoma. in the authors' experience, a rim of sclerosis surrounding the nidus may aid in diagnosis on mri and can be identified as an outer hypointense halo on all sequences. we illustrate the findings in cases of atypical osteoid osteomas which may be difficult to diagnose including intraarticular, phalangeal, and vertebral osteoid osteomas. we also show examples of the newly described sign which has high specificity for osteoid osteoma. conclusions: imaging findings in osteoid osteomas can be misleading and cause misdiagnosis, especially in atypical cases. knowledge of their appearance in atypical locations and specific findings can aid in the correct diagnosis. ultrasound of normal entheses in the growing skeleton nancy chauvin, md, department of radiology, the children's hospital of philadelphia, chauvinn@email.chop. edu; pamela f. weiss, monica epelman, diego jaramillo purpose or case report: ultrasound is an underutilized modality in the evaluation of the pediatric musculoskeletal system. evaluation of tendon insertions about the elbow, knee and foot can be easily performed with ultrasonography. a good knowledge of the age dependent normal ultrasound appearance of the entheses is crucial in order to evaluate for pathology, such as trauma or ethesitis-related arthritis. this exhibit will serve to provide the reader with a practical approach to imaging when assessing tendon insertions. optimal patient positioning and transducer selection will be discussed. in addition, important anatomic landmarks will be described to allow for reproducibility and avoiding pitfalls. methods & materials: transverse and longitudinal ultrasound images of entheseal insertion sites were performed on healthy girls and boys between the ages of and years. ultrasound of the elbow was performed while in full extension and the insertions of the common flexor and common extensor tendons were evaluated. the quadriceps and patellar insertions were imaged with patients in the supine position, with the knees flexed at degrees. the achilles tendon and plantar fascia insertion were evaluated with the patient prone, with the feet hanging off the edge of the table. results: tendons demonstrated the expected fibrillar pattern with parallel echogenic lines. the appearance of the entheses changed as the insertion matured from sonolucent cartilage to echogenic bone. conclusions: using a systematic approach and knowledge of the normal anatomy, sonography of the tendons of the elbow, knee and foot can easily be performed in children. pediatric musculoskeletal ultrasound of the distal lower extremity (knee to ankle) howard christianson, md, radiology, children's hospital boston, howard.christianson@childrens.harvard.edu; julia rissmiller, michael j. callahan purpose or case report: ultrasound is a well-established technique in children for evaluation of the painful hip, developmental dysplasia of the hip, soft tissue infection, palpable masses and foreign bodies. in general, ultrasound has a somewhat more limited role for the primary evaluation of several other pediatric musculoskeletal disorders in the setting of trauma, articular and periarticular diseases and tumors and tumor-like conditions. inherent advantages of ultrasound include excellent spatial resolution, a lack of ionizing radiation, a relatively non-invasive technique and lack of a need for sedation. real-time imaging allows problem solving not available with other modalities which is well suited for musculoskeletal applications, particularly in the setting of trauma. the major disadvantage of ultrasound is operator dependency, which is particularly evident in musculoskeletal applications. the purpose of this study is to illustrate the practical use of ultrasound in the evaluation of the distal lower extremity (knee to ankle) emphasizing the sonographic appearance of various musculoskeletal disorders. examples include: ) cystic lesions around the joints: baker's cyst, synovial cyst, ganglion cyst and suprapatellar bursitis; ) infectious processes: pretibial, subperiosteal and intramuscular abscess; ) tumor and tumor like lesions: nerve sheath tumor, tumoral calcinosis; ) trauma related injuries: sinding larsen johansson, tibialis anterior muscle herniation, hematoma. methods & materials: cases selected for presentation from a series of diagnostic musculoskeletal ultrasounds performed at our institution. results: a range of images from diagnostic ultrasounds performed of the distal lower extremity (knee to ankle) will be presented emphasizing the sonographic appearance of various musculoskeletal disorders. conclusions: selected musculoskeletal ultrasounds of the distal lower extremity are presented to familiarize the audience with the sonographic appearance of various musculoskeletal disorders and to highlight the tremendous potential of ultrasound in evaluating musculoskeletal disease in children and adolescents. role of conventional and dynamic contrast enhanced magnetic resonance imaging in diagnosis of hemihypertrophy syndromes in children shrey k. thawait, md, phd , radiology, yale university-bridgeport hospital, sthawai @jhmi.edu; gaurav k. thawait, sally e. mitchell, laura m. fayad, john a. carrino, kate puttgen purpose or case report: hemihypertrophy syndromes in children are complex and there is some overlap among these conditions. hence, establishing a diagnosis can be challenging. identification of the correct vascular anomaly associated with these overgrowth disorders helps to correctly classify the disease into one of the several syndromes, which in turn guides management. in this educational poster, we will review the definition, clinical presentation, conventional magnetic resonance imaging (mri) and contrast enhanced magnetic resonance angiography and venography (mra / mrv) features of hemihypertrophy syndromes in children. methods & materials: . learn the diagnostic criteria for overgrowth syndromes in children such as klippel-trenaunay syndrome (kts) and parkes weber syndrome (pws) with special emphasis on associated vascular anomalies. . gain knowledge of high resolution mri technique for evaluation of vascular anomalies associated with the hemihypertrophy syndromes. . understand the additional value of dynamic contrast enhanced mra / mrv in the differentiation of the hemihypertrophy syndromes in the pediatric age group. results: . mri technique for a dedicated "vascular anomaly protocol" consisting of fat saturated t weighted, pre contrast axial t weighted, and post contrast triplanar t weighted fat saturated imaging will be described. . special emphasis will be provided on dynamic contrast enhanced mra/mrv. . conventional and dynamic mri features of clinically proven cases of hemihypertrophy syndromes will be demonstrated. conclusions: systematic mri interpretation utilizing a dedicated vascular anomaly protocol enables the radiologist to correctly identify the hemihypertrophy syndrome, and provide detailed extent of disease. correlative ultrasound, mri imaging and physical examination of elbows in hemophilic children andrea s. doria, md, the hospital for sick children-diagnostic imaging, andrea.doria@sickkids.ca; frederico xavier, arun mohanta, carina man, ningning zhang, pamela hilliard purpose or case report: .to report a systematic ultrasound (us) protocol for assessment of hemophilic elbows. . to discuss advantages and disadvantages of us and mri for evaluating hemophilic elbows in comparison with physical examination. .to illustrate us and mri findings and associated pitfalls in hemophilic joints. background: the value of physical examination for assessment of early arthropathic changes in hemophilic joints is unknown. us does not require sedation in young children, but involves operator training and standardized technique. mri is the reference standard imaging modality for assessment of pathology in hemophilic joints. standardization of a systematic protocol for data acquisition and interpretation of us findings and understanding of the correlation of findings between physical examination, us and mri in hemophilic elbows is essential for the use us as an outcome measure both in clinical practice and research. so far such information is not available for growing elbow joints. methods & materials: eight hemophilic boys (age range/ median, - / years) with a history of prior elbow bleeds underwent us and mr imaging, and physical examination on the same day. corresponding images on us and mri were highlighted to illustrate abnormalities and pitfalls. soft tissues (effusion/hemarthrosis,synovial hypertrophy,hemosiderin deposition) changes were characterized as small, moderate, or large. erosions, cartilage and subchondral abnormalities were graded based on depth or extent of articular changes. results: . us is helpful for discriminating synovial hypertrophy, joint effusion/hemarthrosis, and large hemosiderin deposition which otherwise generates susceptibility artifacts on gradient-echo mri obscuring adjacent tissues. . us can visualize erosions, cartilage and subchondral abnormalities at the joint periphery. however,differentiation between subchondral cysts and erosions is usually unfeasible by us. . prior knowledge of the degree of joint maturation is essential for an accurate evaluation of cartilage loss by us. . physical examination has limitations for assessment of early joint changes in contrast to us. conclusions: us can be useful for assessing hemophilic elbows, with advantages over mri in the evaluation of soft tissues. further development of an us-mri atlas on normal cartilage in growing joints is needed for definition of the value of us in the assessment of minimal osteochondral abnormalities. digital atlas of skeletal surveys of common skeletal dysplasias shawn parnell, mbbs, md, dnb, radiology, seattle children's hospital, shawn.parnell@seattlechildrens.org; corey wall, edward weinberger purpose or case report: skeletal dysplasias are conditions of abnormal bone and cartilage growth which result in short stature. developing expertise in the radiographic evaluation of skeletal dysplasias can be difficult, as more than dysplasias exist. exhaustive description of individual dysplasias can be found in hard copy textbooks, without the ability to compare individual dysplasias side by side. by providing radiographic images and descriptive text of thirteen common skeletal dysplasias and two comparative normal skeletal surveys, we aim to facilitate understanding of the terminology and highlight the differences in imaging appearances one may commonly encounter in interpreting skeletal dysplasias. methods & materials: initial skeletal surveys and/or follow up radiographs obtained for evaluation of skeletal dysplasias at our institution from to were compiled and reviewed for best quality images. selected images for each case were labeled according to body part and view, to include ap and lateral views of the spine and skull and ap views of the extremities and pelvis. for neonates, ap and lateral babygram images were used. the software program used for viewing the atlas, written in c#, may be freely downloaded. it permits linked scrolling and resizing of the images, and simultaneous comparison of different cases is available. cases may be viewed as unknowns or in a selfteaching mode. results: radiographic images for thirteen common skeletal dysplasias and two comparative normal skeletons (neonate and child) are provided within an interactive digital atlas. cases include achondroplasia, pseudoachondroplasia, cleidocranial dysplasia, thanatophoric dysplasia, diaphyseal dysplasia, multiple epiphyseal dysplasia, osteopetrosis, osteogenesis imperfecta, multiple hereditary exostoses, dysostosis multiplex, fibrous dysplasia, asphyxiating thoracic dysplasia (jeune syndrome), and spondyloepiphyseal dysplasia. conclusions: by displaying radiographic images of several common skeletal dysplasias in an interactive and comparative format with descriptive text, understanding of basic radiographic terminology and appearances will be facilitated. purpose or case report: . to classify various pediatric msk soft tissue masses . to describe pathogenesis, imaging appearances and differential diagnosis of these lesions methods & materials: radiology and clinical medical records were reviewed and pediatric patients with musculoskeletal soft tissue masses were identified. representative images were collected as examples of each lesion. the lesions were then classified into different groups based on the similar pathology and etiology. brief discussion is done for each of these masses with their multimodality imaging appearances. results: the search yielded pediatric soft tissue masses of multiple different etiologies, including post-traumatic (hematoma, fat necrosis, fibromatosis coli, myositis ossificans), inflammatory or infectious (cellulitis, abscess, granuloma annulare, retained foreign bodies), pseudotumors (synovial cysts, ganglion cysts, vascular malformations) and neoplastic lesions (fatty, vascular, neural, fibrous, muscular). multiple different imaging modalities were used to evaluate these masses, including ultrasound, ct and mri. representative examples of different lesions and their appearances on different imaging modalities will be presented and an organized approach to the diagnosis of these lesions will be discussed. conclusions: musculoskeletal soft tissue masses are relatively common in children. majority of these are benign; however, up to % of these lesions can be malignant "sarcomas". multiple different imaging modalities often provide complimentary information in the work-up of these lesions. despite multimodality imaging approach, tissue diagnosis or short interval follow-up is still often required when the mass does not show typical features of a benign etiology. pediatric radiologists should be familiar with various pediatric msk soft tissue masses and their imaging appearances, and should be able to guide appropriate management. results: elbow mri examinations were reviewed on children aged months to years with ( %) of these investigating clinical instability in children. mechanism of injuries included congenital dislocation ( %), traumatic dislocation ( %), fracture or avulsion ( %) and other injuries ( %). the patient's with congenital elbow dislocations most commonly presented with radial head dislocation and associated dysplasia or flattening, effusion and less frequently dysplasia of the olecranon or capitellum. patient's with traumatic dislocations were frequently associated with ligamentous or capsular disruption, bone oedema and epicondylar avulsion with effusion, loose osseous bodies and fractures less often. the epicondylar avulsions and ligamentous or tendon injuries occurred equally often in those few patients with unspecified injury mechanism. conclusions: a number of the bony, ligamentous, articular and developmental anomalies evident on elbow mri have been illustrated highlighting the importance of careful and systematic review of all elbow structures when presented with a child with elbow instability. accurate identification of these abnormalities is vital to facilitate their appropriate management. methods & materials: from our computerized radiology information system, we retrieved all patients that have foot ultrasound for evaluation of vertical or oblique talus deformities in the last years ( / - / ). the us was performed by a pediatric radiologist using a high resolution linear and tight convex curve probes with foot in neutral, plantar flexion and dorsiflexion. all medical charts, ultrasound scans and foot radiographs were reviewed by a pediatric radiologist. results: we identified nine patients' with foot deformities who were suspected of vertical or oblique talus and were evaluated by ultrasound. seven patients are male; two of them had initial foot radiographs that were not diagnostic. two female patients had unilateral oblique talus deformity. there were patients with vertical talus deformity; three of them had bilateral deformities. conclusions: us can directly visualize the unossified navicular, the talar cartilage and their alignment. dynamic us can. ultrasound evaluation of costal chrondral pathologies in children presented as anterior chest wall mass or pain nucharin supakul, md, radiology, riley hospital for children, tanyasupakul@yahoo.com; boaz karmazyn purpose or case report: to summarize our experience with the use of ultrasonography (us) for evaluation of costal cartilage pathology presented as anterior chest wall mass. methods & materials: from our computerized radiology information system, we retrieved all patients that have chest wall ultrasound for evaluation of a mass in the last . years ( / - / ) . the us was performed by a pediatric radiologist using a localized scan with high resolution linear probe. all medical charts, pathology results, ultrasound scans and other imaging studies were reviewed by a pediatric radiologist. results: ten patients were found with costal chrondral pathologies. nine patients presented with anterior chest wall mass and one with chest wall pain. eight patients had angular deformity of a single costal cartilage and one patient had biopsy proven osteochrondroma, presented with anterior chest wall mass. one patient had a non-union fracture after motor vehicle accident, presented with anterior chest wall pain. in patients with rib deformity, the mass was non-tender. nine patients had prior imaging study including chest x-rays (n ), ct scan (n ), breast mr (n ). all these studies were negative. conclusions: us optimally demonstrated costal cartilage abnormities. chest radiographs and cross sectional studies were negative. we therefore recommend using high resolution chest wall us in children with negative chest radiograph and anterior hard chest wall mass. challenges in pediatric marrow imaging-boning up on current mr techniques srikala narayanan, md, division of radiology, children's national medical center, snarayan@childrensnational.org; neha kwatra, nabile safdar purpose or case report: a wide range of pathologies demonstrate similar findings when imaged using conventional mr sequences. however, pediatric musculoskeletal imagers are increasingly leveraging newer techniques to add specificity to their diagnoses when abnormal marrow signal is detected. the purpose of this educational exhibit is to review the application of current mr techniques to pediatric marrow imaging across the spectrum of normal, variant, and pathologic processes. methods & materials: cases with potentially overlapping imaging appearances on conventional mr sequences, including hematopoetic marrow, sickle cell disease, osteomyelitis, chronic recurrent multifocal osteomyelitis, and infiltrative neoplasms, will be presented. the basis of various mr techniques including chemical shift imaging, "whole body" marrow imaging, diffusion weighted imaging, and fat-water separation techniques such as dixon or ideal (ge) will be reviewed. the strengths and weaknesses of such techniques in differentiating between infection, neoplasm, and normal variation will be emphasized through the case examples. challenges and pitfalls in the imaging of these pathologies using such techniques will be discussed. results: current mr imaging techniques add specificity to diagnoses of marrow pathology which are otherwise difficult to differentiate using traditional sequences alone. the use of opposed phase imaging can be helpful in differentiate hematopoietic marrow or infection from infiltrative and neoplastic conditions. "whole body" marrow imaging may serve as an alternative to other modalities which involve significant radiation exposure. the use of diffusion weighted imaging is a promising, but developing, technique being applied to marrow pathology. conclusions: pediatric bone marrow mr imaging is a challenging area for a vast majority of the radiologists. understanding normal developmental bone marrow changes and being aware of the pitfalls is crucial to render accurate diagnosis. current techniques such as ideal, chemical shift imaging, and "whole body" mri have a potentially important role in further characterization of marrow abnormalities. radiologists beware: unusual imaging manifestations in child abuse eglal shalaby-rana, mbbs (hons), children's national medical center, erana@childrensnational.org; allison m. jackson, tanya hinds, katherine deye purpose or case report: to present less common imaging manifestations of injuries in child abuse that may not be readily recognized as possibly abusive injury. methods & materials: through bi-monthly review of cases with the child protection team over a period of years, the imaging studies of patients with suspected non-accidental trauma were recorded. of the pts with suspected nonaccidental trauma, outcomes were available in patients. the child protection team concluded ( %) were cases of non-accidental trauma with reasonable medical certainty. this data base was reviewed for less common injuries that were found in these medically confirmed cases of child abuse. results: less common manifestations of abuse identified by radiographs included salter-harris injuries in the proximal humerus, and proximal femur. pelvic fractures were rare and when present were associated with sexual abuse. severe chest wall injury, with associated rib fractures, causing complete or near-complete white-out of the chest was occasionally encountered. soft tissue injures, such as hematomas were found in various locations in the body including the buttocks and anterior abdominal wall, were imaged on ultrasound and ct. paraor prevertebral injuries, with or without associated bone injury were identified; one infant presented with retropharyngeal soft tissue swelling. mri identified cervical spine injuries which included ligamentous injury and intrathecal hematomas. conclusions: while classic metaphyseal lesions and rib fractures are the most common, specific injuries documented by radiologic work up of suspected non-accidental trauma, less common injuries to the soft tissue and skeletal system may occur as a result of child abuse. the ability of the radiologist to recognize these uncommon manifestations of demonstrated in axial, coronal, and sagittal planes. the ligament of interest will be denoted by arrows. at the conclusion of the anatomy section, there will be a self assessment exam. the participants then will be asked to identify the ligament. if answered correctly, a summary slide will be displayed and, if common, images of the relevant pathology will be demonstrated. if an incorrect answer is indicated, a slide will appear denoting the incorrect answer with explanation. conclusions: hopefully, with review of this educational exhibit, the participant will have a better understanding of the relevant ligamentous anatomy of the ankle and hindfoot. purpose or case report: the purpose of this educational exhibit is to demonstrate the pathologic sonographic findings, one might encounter in the pediatric ankle. a systematic methodological approach including patient positioning, transducer orientation and sonographic technique are vital for ideal sonographic assessment of the pediatric ankle. using a data search program from a large academic institution, pediatric ankle ultrasounds performed in the last years were reviewed. pathologies include trauma, inflammation/infection, masses and congenital abnormalities. examples of normal anatomy will be included particularly when demonstrating ligament and tendon pathology. the normal side was often assessed for comparison purposes. results: ankle sonography is a useful modality to evaluate commonly encountered pathologies in the pediatric ankle. radiographically occult fractures may be discovered. ligament and tendon pathology, such as tears of the anterior talofibular ligament, high ankle sprain and peroneus longus tendon tears, can be easily detected. signs of infection that can be radiographically occult such as subtle periosteal reaction or fluid collections can be identified. finally, "lumps and bumps" can be characterized. for example, one of the most commonly encountered masses in the pediatric ankle is a ganglion cyst which can be well characterized sonographically. awareness of imaging pitfalls is also critical to avoid misdiagnosis and to guide appropriate management. conclusions: with basic ultrasound skills and knowledge of normal anatomy, sonography of the pediatric ankle is a useful modality to evaluate soft tissue structures and other pathologies. it is comparable to mri and allows for dynamic evaluation without need for anesthesia. resonance angiography (mra) using time resolved imaging is a relatively new technique that has become increasingly utilized in the diagnosis of vascular anomalies. we will describe the technique used at our institution, time resolved imaging of contrast kinetics (tricks, ge healthcare, milwaukee, wi), and the parameters that can be adjusted to optimize the exam. we will review key imaging features of hemangiomas and vascular malformations in various modalities, with a special emphasis on the tricks appearance. we performed a retrospective review of all the tricks studies performed at our institution for suspected vascular anomalies. in addition to the mr imaging features, we specifically analyzed the t weighted with fat saturation post tricks enhancement and the temporal tricks enhancement pattern. we reviewed all additional imaging including plain film, ultrasound, and ct and correlated the radiographic imaging with the available clinical and histopathologic features. results: we present illustrative cases of hemangiomas, kaposiform hemangioendothelioma, venous malformations, arteriovenous malformations, lymphatic malformations, and other pitfall lesions. we propose a diagnostic algorithm that relies heavily on the post contrast t weighted with fat saturation post tricks enhancement pattern and the temporal tricks enhancement pattern. conclusions: time resolved contrast-enhanced mra has become an increasingly important adjunct in the diagnosis of vascular anomalies. optimization of the exam technique and familiarity of the tricks imaging appearance is essential and can often assist in accurate lesion characterization. purpose or case report: vertical expandable prosthetic titanium rib (veptr) is increasingly used in the treatment of thoracic insufficiency, scoliosis, and chest wall defects in children. in contrast to spinal fusion surgery, veptr allows for growth while stabilizing the deformity. we review the indications, pre-operative imaging, normal radiographic appearance, and complications of this device. methods & materials: on review of the literature, the indications for veptr have expanded in the past several years to include thoracic insufficiency, idiopathic and neuromuscular scoliosis, and chest wall defects. we illustrate the normal radiographic appearance of the three common configurations of veptr (cradle-to-cradle assembly, cradle with lumbar extension assembly; cradle-to-ala hook assembly). we discuss the potential complications of veptr, including infection, rib fracture, dislodged hardware, and neurological injury, with an emphasis on imaging diagnosis. results: there is a relatively high rate of reported complications with veptr in the literature. therefore, awareness of the growing number of indications, as well as the expected and unexpected appearance of this device, aids in radiographic diagnosis of complications. conclusions: vertical expandable prosthetic titanium rib (veptr) is gaining acceptance in the treatment of thoracic insufficiency, scoliosis, and chest wall defects in children. recognition of the indications, normal radiographic appearance, and complications of this device will facilitate timely and accurate diagnosis. disclosure: dr. philips has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. spectrum of pediatric spinal neoplasms: an interactive tutorial benjamin t. haverkamp, md, radiology, university of missouri-kansas city, haverkampbt@umkc.edu; salvador f. iloreta, maha jarmakani, lisa lowe, seth gibson purpose or case report: the objective of this educational electronic exhibit is to provide the radiologist with an approach to pediatric spinal neoplasms. emphasis will be placed on narrowing the differential diagnosis using a combination of lesion location, characteristic imaging findings, relevant history, and associations. the exhibit format will include a casebased review of various pediatric spinal neoplasms, radiologicpathologic correlation, and a brief discussion of imaging findings useful in guiding surgical management. an interactive selfassessment exam will be presented at the end of the exhibit. results: a review of the specific etiologies will be presented with classification as intra-and extra-medullary and extradural lesions. radiologic images will be related to gross and microscopic pathology. conclusions: after viewing this exhibit, the learner will be able to: . recognize the clinical, imaging, and pathologic characteristics of pediatric spinal neoplasms. . understand relevant imaging findings useful in surgical management. . test their understanding of the presented material through an interactive exam. poster #: edu- craniosynostosis jason tsai, mbchb, children's hospital boston, jason. tsai@childrens.harvard.edu; diana p. rodriguez purpose or case report: to review the normal developmental appearance of the cranial sutures with computed tomography (ct) and to describe ct findings of the various forms of craniosynostoses. in this irb-approved retrospective study we reviewed ct images of subjects diagnosed with craniosynostosis between and september . we included patients with single-suture synostosis, isolated bilateral coronal synostosis, pansynostosis, and combined craniosynotoses. additionally, we identified individuals with normal appearing sutures from to years of age imaged with head ct to describe the pattern of normal development of the cranial sutures. results: a description of the normal developmental ct appearance of the cranial sutures using computed tomography has been provided. of the group of patients with craniosynostosis the following variables were recorded: age at presentation, the pattern of sutural fusion, skull shape, presence of hydrocephalus, genetic testing, and types of surgical correction. conclusions: we have demonstrated the normal developmental ct appearance of the cranial sutures and the ct patterns of the various forms of craniosynostoses, with clinical, genetic and surgical correlation. posterior fossa tumours: a pictorial review sam byott, md, manchester children's hospital, sambyott@ hotmail.com; neville wright, vivian tang, abdu shabani, stavros stivaros purpose or case report: posterior fossa tumours account for - % of childhood brain tumours. the most common differentials include pilocytic astrocytoma, medulloblastoma and ependymoma. mr imaging is crucial to diagnosis, staging and identification of complications such as hydrocephalus and haemorrhage. soft tissue characteristics alongside tumour location, invasion and clinical history facilitate radiological discrimination prior to surgery. however, there is significant clinical equipoise with regards to the imaging appearances in a significant proportion of cases making definite diagnosis difficult. the aim of this study is to evaluate the radiological findings and correlate with histological data. this will allow identification of the key morphological features that discriminate different tumours. these can then be presented to educate fellow radiologists. methods & materials: radiology pacs and patient notes were used to collate radiological, histological and clinical data. results: there were patients presenting at our institution with posterior fossa tumours. had pilocytic astrocytomas, had medulloblastomas and had ependymomas. one patient had an atypical teratoid rhabdoid tumour (atrt). traditional features alongside more advanced mr characteristics were correlated with histology, and the features allowing for discrimination of tumour types are presented in this pictorial review. conclusions: posterior fossa tumours have a highly variable radiological appearance. we present a range of appearances and describe the important morphological features that allow radiological discrimination of tumour type. poster #: edu- dt imaging of the pediatric spine teresa c. gross kelly, children's hospital of wisconsin, tkelly@chw.org; ibrahim s. tuna, mia s. kelly, tushar chandra, sumit singh, mohit maheshwari, hervey d. segall purpose or case report: some abnormalities of the pediatric spine can be challenging. we have discovered that in many such cases, diagnosis of spinal lesions can be faciliated by using the dt weighted sequence. the purpose of this educational poster is to demonstrate the remarkable usefulness of dt weighted images for delineating pathology of the pediatric spine. methods & materials: lesions of the spine that will be reivewed in this educational exhibit will be categorized as: ( ) vascular ( ) due to infection/inflammation ( ) neoplastic/ neurogenic ( ) congenital ( ) traumatic/iatrogenic ( ) endocrine/metabolic. the imaging characteristics of lesions found in the pediatric spine will be described and the utility of d t -weighted mr sequences for the evaluation of these lesions will be discussed. finally the role of imaging in the treatment planning of abnormalities of the pediatric spine will be addressed. results: this educational exhibit will provide numerous examples of how d t -weighted imaging can elucidate diagnosis of lesions involving the spine. examples include enhancement of the cauda equina in guillain barre syndrome, lipomatous malformations, spondylolysis in children with low back pain, thecal cysts, filar cysts, metastasis, hydromyelia and ventriculus terminalis. conclusions: d t -weighted images of the spine performed in the sagittal plane with coronal and axial reformations, as well sagittal oblique reformations (scotty dog reformations) for evaluation of spondylolysis, can facilitate the evaluation of lesions involving the pediatric spine. the normal pediatric spine: a pictorial review of mr anatomy and development in the infant, child and adolescent ibrahim s. tuna, md, radiology, children's hospital of wisconsin, dristuna@yahoo.com; teresa c. gross kelly, tushar chandra, mohit maheshwari, sumit singh, hervey d. segall purpose or case report: radiological evaluation of the pediatric spine can be more challenging in child than in the adult patient due to the wide range of normal anatomic variants and synchondroses, combined with the unique effects of trauma in children. mri is an excellent imaging modality for the evaluation of the pediatric spine. however, in order to provide an accurate interpretation of acute posttraumatic changes in the pediatric spine, particularly in the setting of abusive head trauma, a fundamental knowledge of normal anatomy, variants and pathology of the pediatric spine is required. the aim of this educational exhibit is to illustrate normal mri anatomy of the spine in the infant, child and adolescent. methods & materials: this exhibit will first describe basic spinal embryology and development of the vertebra and spinal cord, followed by mri depiction of the developmental anatomy of the spine from infancy through adolescence. the changing appearance of the spinal canal, spinal cord and vertebral bodies with age will be illustrated using normal cases from the radiology database. sagittal and transverse diameter of vertebral bodies, thickness of the dural thecal sac, dimensions of the spinal canal, normal bone marrow signal changes, vertebral body heights, level of conus medullaris, prevertebral and paraspinous soft tissues and epidural fat thickness will be described and changes according to age will be pointed out. results: in early life, the spinal cord extends to the inferior aspect of the bony spinal column. because the vertebral bodies grow longitudinally faster than the spinal cord does, the conus medullaris may change. ossification of the vertebral bodies and posterior elements is nearly complete by age , with a resultant decrease in the spinal canal diameter. the nucleus pulposus becomes smaller after years and spans approximately half the disk space in the sagittal plane. the spinal cord is elliptical in cross section in the cervical spine and demonstrates a difference in signal between the normal gray and white matter of the spinal cord which should not be mistaken for intramedullary pathology. conclusions: a solid understanding of normal spine anatomy and embryological development is essential in evaluation of pediatric spine, mainly in the setting of trauma. familiarity with normal anatomic variants is essential to provide an accurate interpretation of pathology in the pediatric spine. spectrum of intracranial cystic lesions in infants and children ernesto i. blanco, md, st. christopher's hospital for children, eiblanco @gmail.com; eric faerber purpose or case report: to review the imaging spectrum of intracranial cystic lesions in the pediatric population. methods & materials: a retrospective review of our imaging database was performed to identify studies obtained in which the findings included intracranial cystic lesions. results: multiple cystic lesions were elucidated primarily by computed tomography or magnetic resonance imaging. these lesions can be divided into nonneoplastic and nonneoplastic tumor-associated cysts. the nonneoplastic cysts, which is the largest group, include: cavum septi pellucidi and cavum veli interpositi, choroid plexus cyst, enlarged peri-vascular spaces, pineal cyst, the large spectrum of arachnoid cysts, colloid cyst, epidermoid cyst, rathke cleft cyst, and porencephalic cyst. nonneoplastic tumor-associated cysts include: craniopharyngioma, optic glioma, pilocytic astrocytoma, hemangioblastoma, and ganglioglioma. conclusions: intracranial cystic lesions are relatively common entities in the pediatric population. a wide spectrum of nonneoplastic and nonneoplastic tumor associated pathologies are presented using both computed tomography and magnetic resonance imaging. kelly, sumit singh, ibrahim s. tuna, hervey d. segall purpose or case report: the aim of this educational exhibit is to provide a comprehensive review of imaging features, classification and management of pediatric spinal cord tumors. we also aim to elicit the differences between pediatric spinal cord tumors and their adult counterparts. we will summarize the differences between the individual tumors based on histological cell types and the pertinent implications on management and outcome methods & materials: this exhibit will provide an overview of the common as well as uncommon tumors of the pediatric spinal cord. various classification systems for these tumors-anatomical as well as histological will be discussed. we will illustrate the relevant imaging findings that can help in differentiating these tumors. results: pediatric spinal cord tumors account for % to % of all pediatric central nervous system tumors. mri is the mainstay for the initial diagnosis as well as the post surgical evaluation and surveillance of these tumors. pediatric and adult spinal cord tumours differ both in terms of anatomical location as well as histology. the disease and treatment related morbidities are also different in children as compared to adults. astrocytomas, ependymomas, glioneural tumors and csf metastasis represent the vast majority of cord neoplasms in the pediatric age group. some of cord tumors may also be associated with inherited syndromes (like neurofibromatosis type ) or may have genetic predisposition. these would also be discussed. we will also illustrate and discuss common non neoplastic spinal masses that may mimic tumors. conclusions: pediatric spinal cord tumors have varied clinical presentations, imaging appearance and outcome. this review would improve the understanding of these tumors thereby helping in diagnosis, management and follow up of these uncommon neoplasms. multi-modality imaging of pediatric head and neck lesions jason au, md, oklahoma university health sciences center, jasonmau@gmail.com; anthony alleman, mahmoud elkaissi, roy jacob purpose or case report: the purpose of this study is to present a side by side comparison of the multi-modality imaging features of pediatric masses. using cases that have been imaged with multiple modalities, the exhibit will delineate the sonographic, mr, and ct appearance of congenital, infectious, and neoplastic head and neck lesions in the pediatric population. methods & materials: a restrospective search of pacs was performed on studies completed at the oklahoma university medical center on the oklahoma university health science center campus from january to the present. ultrasound, ct, and mr examinations were selected that depicted relevant pediatric head and neck pathology. all studies were de-identified prior to image export. results: over twenty representative cases of pediatric infections, fibrous tumors, cystic neoplasms, vascular malformation, bony tumors, developmental anomalies, and other neoplasms were selected for inclusion. results: pictorial review of cases including the following representative cases: myelonmeningocele associated with arnold chiari malformation, lipomyelomeningocele, tethered cord with spinal lipoma/fibrofatty filum, tethered cord and dermal sinus tract, and chiari i with syringohydromyelia. several unique cases including the following will be presented as well: thoracic meningocele with arnold chiari malformation, terminal myelocystocele, diastematomyelia, and myelomeningocele without arnold chiari malformation. while mri demonstrates the cranio-cervical junction and the cervicothoracic spinal cord better than ultrasound, ultrasound often allows for improved resolution of the distal spinal cord, lumbosacral spinal canal, and spinal dysraphism structures near the skin surface in the neonate. conclusions: congenital spinal malformations are complex and variable in imaging appearance. it is important to understand the classification in order to determine the appropriate management and prognosis. in the neonatal period imaging should be performed with ultrasound and mri studies, as they may provide different and complementary information. conclusions: hypoxic ischemic injury is a common condition resulting in a wide spectrum of severe neurological defects. while in the past treatment only consisted of supportive care for hii, recent advances have yielded promising treatment options if initiated within a limited time window. thus due to the severity of the disease and the need for rapid intervention, it is important to recognize radiological manifestations of hii along with its clinical signs and symptoms to offer a better prognosis to the patient. craniosynostosis: looking beyond the sutures tushar chandra, md, children's hospital of wisconsin, drtusharchandra@gmail.com; teresa c. gross kelly, mohit maheshwari, sumit singh, ibrahim s. tuna, hervey d. segall purpose or case report: the aim of this educational exhibit is to provide a framework upon which the diagnosis of the various types of craniosynostosis can be facilitated. our goal is to provide an efficient way to evaluate craniosynostosis for the radiologist in clinical practice. we plan to accomplish this goal by providing a succinct review of the sutures, an overview of the various classification schemes for craniosynostosis and potential complications associated with premature sutural closure. the role of imaging in the evaluation of craniosynostosis will be described and the features of craniosynostosis that are most important to the craniofacial surgeon will be elucidated. finally, surgical strategies for the repair of craniosynostosis and postoperative findings will be described. results: some of the forms of craniosynostosis may have a genetic basis, but many are spontaneous in nature. untreated progressive craniosynostosis can lead to inhibition of brain growth, and an increase in intracranial pressure. mdct with mip and d surface reformations is the preferred modality for diagnostic evaluation of craniosynostosis. it is also a robust modality for post operative assessment and long-term follow up. mri is a useful adjunct for assessment of associated intracranial anomalies and complications. timely and appropriate imaging is essential to assess for potential complications of craniosynostosis which may include intracranial hypertension, anomalies of external and middle ear, hydrocephalus, chronic tonsillar herniation, cranial base deformity, impaired venous drainage, enlarged emissary foramina and veins and optic atrophy. on the other hand, positional plagiocephaly should not be misinterpreted as craniosynostosis. surgical management is typical for nonsyndromic craniosynostosis, which involves correction of craniosynostosis between three to six months of age. conservative management is the mainstay for syndromic craniosynostosis. postoperative follow up imaging for surveillance for ventricular size and signs of raised intracranial pressure are necessary. conclusions: craniosynostosis is a challenging area of pediatric neuroimaging. knowledge of the sutural anatomy, an understanding of the potential intracranial complications caused by premature sutural closure, as well as the role that imaging plays in presurgical planning, can provide a practical way for the radiologist to evaluate craniosynostosis in a fast-paced clinical setting. poster #: edu- the perinatal brain and spinal cord-imaging across a life border: a case-based approach anand dorai raju, md, radiology university of tennessee, araju@uthsc.edu; harris l. cohen, matthew whitehead, asim choudhri purpose or case report: to review normal and abnormal perinatal ultrasound (us) and magnetic resonance (mr) imaging findings and note their significance for the analysis of the fetal and neonatal brain as well as spinal cord and vertebral column using a case based approach. to highlight us and mr capabilities in allowing correct perinatal diagnosis of congenital and acquired central nervous system abnormalities. methods & materials: cases will be shown of normal and abnormal anatomic findings in fetal and neonatal brain and spinal cord imaging. key teaching points necessary for the diagnosis of such brain abnormalities as ventriculomegaly, chiari malformations, holoprosencephaly, and agenesis of the corpus callosum as well as dandy walker malformations and avms will be discussed. intraventricular hemorrhage, periventricular leukomalacia, anoxic injuries and infectious abnormalities will be reviewed. abnormal anatomic findings in fetal and neonatal spine evaluations for congenital and acquired abnormalities and key teaching points necessary for the accurate diagnosis of tethered cord, myelomeninocele, caudal regression syndrome, hydromyelia, diastomatomyelia and sacrococcygeal teratoma will be reviewed. some diagnostic difficulties and controversies will be addressed. conclusions: ultrasound aided by mri can provide ready diagnosis to many central nervous system abnormalities involving fetuses and neonates. ever improving perinatal imaging experience and technique allow for better prenatal as well as postnatal diagnosis. cases showing such imaging and key points helping such imaging diagnoses will be reviewed. overview of imaging of pediatric extraocular orbital tumors srikala narayanan, md, division of radiology, children's national medical center, snarayan@childrensnational.org; nadja kadom, gilbert l. vezina purpose or case report: to show the spectrum of benign and malignant extraocular orbital tumors in children. methods & materials: we reviewed the cross-sectional imaging of orbit (ct and mr) done in the last years. specific imaging signs of extraocular tumors including benign and malignant tumors such as hemangiomas, lymphangiomas, optic nerve glioma, optic nerve sheath meningioma, pseudotumors, rhabdomyosarcoma, orbital myofibroma, eosinophilic granuloma and neuroblastoma metastases will be shown. important imaging features that should be considered when formulating a differential diagnosis will be described. conclusions: the spectrum of diseases affecting pediatric orbit is substantially different from what we see in the adults. it is not easy always to differentiate between different tumors. important imaging characteristics will help us towards better differential diagnosis. in this exhibit, we will illustrate ultrasound anatomy of the neonatal spinal cord. discussion of the normal anatomic variants and pathological conditions of the spinal cord will be provided. representative images of a variety of common and uncommon pathological conditions of the spine will be presented to illustrate teaching points. in abnormal cases, follow up mri images will also be illustrated for comparison. results: ultrasound is a robust screening modality for evaluation of the lumbosacral spine in neonates. it is cheaper, readily available, safer first line imaging modality in neonates suspected to have spinal malformations. under able and well trained operator, diagnostic accuracy of spinal ultrasound approaches mri. however, mri remains the gold standard for imaging evaluation of spine. normal variants that simulate disease processes like ventriculus terminalis, prominent filum terminale and central echo complex will be presented. congenital malformations of the cord such as tethered cord, hydromyelia, lipoma, diastematomyelia, myelomeningocele, lateral meningocele and presacral masses will also be discussed. conclusions: ultrasound is a very useful screening technique for evaluation of pathological conditions of lumbosacral spine in neonates. this review would improve the understanding of utility and limitations of ultrasound in evaluation of neonatal spinal malformations. purpose or case report: although mri is the standard for detecting epilepsy and brain tumor abnormalities, pet-ct is performed to ascertain metabolism related to epileptogenic regions or characterize tumor metabolic activity. asymmetric metabolism often correlates to structural abnormalities like cortical dysplasia. metabolic activity often correlates with tumor aggressiveness or grade. fdg pet is commonly used to assess seizure and tumor metabolism. the lesser utilized amino acid pet tracers (c methionine, fdopa) show increasing value with lower grade tumors due to high tumor to normal tissue contrast. literature is accumulating regarding c methionine (cmet) in the detection of lesions like cortical dysplasia and its ability to delineate low grade seizure related tumor lesions. despite the established fdg and accumulating cmet literature, little information exists about the imaging seen with both in pediatrics. as these studies are increasingly viewed as part of fusion mri images, there is more scrutiny of focal metabolism correlating with mri findings and less interpretative reliance on abnormality based solely on asymmetry. methods & materials: review of patients who underwent cmet and fdg brain pet-ct was performed. each was imaged on a philips scanner and had prior mri. studies demonstrating a variety of tumors, postoperative findings of residual or recurrent tumor, and pseudoprogression were selected. epilepsy cases with structural cortical abnormalities or seizure-associated tumors were also selected. cmet and fdg studies were analyzed by pediatric neuroradiologists and the imaging findings correlated with prior mri and any pathology or follow-up imaging. pictorial galleries of the cmet and fdg imaging patterns were created. results: pathologically proven low-grade glial tumors showed increased cmet uptake and no hypermetabolism on fdg. high-grade tumors showed increased uptake on cmet and hypermetabolism on fdg. patients with residual or recurrent tumors showed uptake similar to their original tumor. granulation tissue and pseudoprogression changes showed increased uptake on cmet and no hypermetabolism on fdg. epilepsy surgery patients with cortical dysplasia or low grade glial tumors showed increased uptake on cmet and fdg hypometabolism. conclusions: this study illustrates the variety of findings on cmet and fdg pet-ct in pediatric patients clinically evaluated for brain tumor and epilepsy. this atlas provides readers with a guide to the appearance of these findings on an emerging imaging technique. pediatric head and neck neoplasms: a multimodality pictorial review alok jaju, md, mallinckrodt institute of radiology, alokjaju@gmail.com; marilyn j. siegel purpose or case report: neck masses are common in children and most occur in the suprahyoid region. knowledge of the fascial spaces involved in conjunction with imaging features can help in diagnosis. in this pictorial review, we present a multimodality imaging approach based on anatomy of the suprahyoid fascial spaces for evaluation of pediatric neck tumors. methods & materials: radiology information system (ris) at our tertiary care children's hospital was queried to identify patients with suprahyoid neck masses who had imaging performed between july and present. a variety of conditions having congenital, inflammatory, neoplastic, or vascular origin were identified and the anatomic location in the neck as well as imaging and clinical findings were retrospectively reviewed. results: the imaging evaluation included ultrasound, ct and mri. lesions arose within the following fascial spaces of the suprahyoid neck: superficial, carotid, masticator, submandibular, sublingual, parotid, parapharyngeal, visceral, retropharyngeal and prevertebral. key imaging features important in diagnosis included lesion vascularity, calcification, necrosis and bone invasion. we discuss and illustrate these imaging findings and relate them to specific suprahyoid fascial spaces. specific lesions include vascular and lymphatic malformations, teratoma, nerve sheath tumors, thyroglossal duct and branchial cleft cysts, pleomorphic adenoma, dermoid cyst, ranula, lymphadenopathy, abscess, lymphoma, rhabdomyosarcoma, neuroblastoma and nasopharyngeal carcinoma. conclusions: knowledge of fascial spaces of the suprahyoid compartment and key imaging features on multiple modalities can aid in the diagnosis of pediatric neck masses. pediatric sinusitis: spectrum of imaging findings with clinicopathologic correlation roy jacob, md, university of oklahoma, drjacobr@gmail. com; paul digoy, robert s. glade, anthony alleman purpose or case report: the clinical spectrum of sinusitis in children can range from uncomplicated bacterial sinusitis to invasive fungal sinusitis. most cases respond favorably to medical management. however, complications occasionally occur due to the spread to adjacent structures. imaging plays an important role in characterizing the disease and guiding the clinical and surgical planning and treatment. this electronic presentation outlines the following- . review radiologic anatomy and unique characteristics of pediatric sinuses. . review the clinical features, pathophysiology, and microbiology of sinusitis. . review of ct and mri imaging characteristics of sinusitis with representative cases such as complicated sinusitis and invasive fungal sinusitis. . review the treatment approaches of sinusitis. methods & materials: a retrospective search of pacs was performed on studies completed at the ou children's hospital in oklahoma city for the last three years. ct and mr examinations were selected that depicted relevant disease processes. corresponding nasal endoscopic pictures were obtained from cases which required surgical management. all studies were de-identified prior to image export. results: over fifteen representative cases of the clinical spectrum of sinusitis and its complications were selected for inclusion. conclusions: this educational exhibit provides a concise review of imaging, clinical features, and treatment of pediatric sinusitis. findings will be richly illustrated with radiological and clinical images. microcephaly or hydrocephalus. knowing the embryology of the cerebellum and th ventricle is important to perceive the development of posterior fossa malformations and to further understand the imaging findings. several classifications schemes have been proposed from a pure embryologic to an imaging-based approach using some essential findings such as the size of the posterior fossa, the presence of csf collection or expansion of csf space, and the size and morphology of the cerebellum. mr is the gold-standard for adequately access and characterize the posterior fossa structures. this pictorial essay will review the mr findings of some of the most common posterior fossa malformations including dandy-walker malformation, persistent blakes pouch, mega cisterna magna, arachnoid cyst, paleocerebellar hypoplasia, cerebellar agenesis, cerebellar and pontocerebellar hypoplasia, cerebelar cortical malformations, isolated brainstem hypoplasia/dysplasia and chiari malformations. we will provide a practical approach to the mr findings of posterior fossa malformations in children. conclusions: mr plays a crucial role in identifying and characterizing malformations of the posterior fossa structures. it should give a logical approach to these complex malformations thus guiding the refereeing physician into the clinical approach and in determining further investigations. results: neuroimaging features of abnormal thalami as encountered in the pediatric population were detailed, and wherever applicable, the relevance of additional mr imaging sequences and techniques to determine etiology was described. while there was considerable overlap in imaging appearances, making a precise diagnosis was found to be challenging in difficult cases, and by and large, a stepwise approach was successfully formulated and used to: . diagnose the more emergent conditions and to . devise a management algorithm for the less acute abnormalities. conclusions: bilateral thalamic lesions are occasionally encountered in pediatric neuroimaging and have a limited differential; a good knowledge base and adequate technique are imperative to tease out the precise diagnosis and institute appropriate management. cortical developmental abnormalities in pediatric seizure patients ibrahim s. tuna, md, radiology, children's hospital of wisconsin, dristuna@yahoo.com; mohit maheshwari, teresa c. gross kelly, sumit singh, tushar chandra, hervey d. segall purpose or case report: to describe various cortical malformations with illustrative examples. we will also briefly discuss the embryology, genetic basis, classification schemes and characteristic imaging findings . methods & materials: this exhibit will illustrate three main categories of cortical malformations: neuronal proliferation, migration and organization. understanding of this complex topic would be facilitated by brief discussion on the embryological basis and proposed genetic causes of some of these cortical malformations. classification schemes on embryology and imaging will be discussed. characteristic imaging findings of these malformations will be discussed and examples from the authors database will be shown. results: neuroimaging in pediatric seizures is challenging. mri is considered the imaging modality of choice because of superior soft tissue contrast and better ability to characterize the pathologic process. we will also discuss the dedicated seizure protocol which is used in our institute. pet-ct imaging can also provides additional information in cases where mri is negative, inconclusive or does not correlate with eeg/clinical findings. brief discussion on advanced imaging techniques will also be presented. malformations are frequently detected in infancy. however, if the initial mri scan performed in infancy is negative, a repeat scan after years of age may be helpful. conclusions: evaluation of cortical malformation in seizure patients still remains a challenging area of pediatric neuroimaging. reviewing of the embryological basis, classification schemes and characteristic imaging findings would improve the understanding the cortical malformations and interpreting the images. poster #: edu- sprs best poster cystic neonatal lesions associated with the spinal cord: discussion and differential diagnosis for these uncommon lesions jacob pirkle, md, jpirkle@mc.utmck.edu, james boyd, brian dupree purpose or case report: to review intradural cystic neonatal spine lesions and discuss the various causes and appearance of these lesions. this poster presentation provides a brief review of neonatal cystic spine lesions, including their etiologies, and presents the targeted audience (radiology resdients, fellows, and practicing radiologists) a helpful differential diagnosis of these lesions based upon their imaging appearance. methods & materials: a brief overview of neonatal cystic spine lesions, their etiology, and imaging appearance is presented in poster format utilizing both literature search and printed reference material. images from several cases of cystic neonatal spine lesions are presented. results: a brief overview of neonatal cystic spine lesions, their etiology, and imaging appearance is presented in poster format utilizing both literature search and printed reference material. images from several cases of cystic neonatal spine lesions are presented. conclusions: neonatal spine ultrasound is often performed to evaluate for abnormalities related to the presence of sacral dimples, cutaneous stigmata, skin tags, hairy tufts, during the evaluation of other congenital anomalies, or when prenatal ultrasound/mri demonstrates an abnormality warranting postnatal follow-up. the identification of cystic spinal cord lesions is relatively rare in the neonate. however, the etiology of these lesions can often be deduced or surmised based upon the location and the imaging appearance of the lesion. the most common cause of a cystic intramedullary spinal lesion is ventriculus terminalis, with a reported incidence of . %. additional lesions include transient dilatation of the central canal, filar cyst, syringohydromyelia, intramedullary arachnoid cyst, and myelomalacia related to in utero/birth trauma. extremely rare etiologies in the neonate include epidermoid/dermoid, cavernous malformation, intranatal cystic infections etiologies, neuroepithelial cysts, and cystic neoplasms. mimics include diastematamyelia, spinal lipomas, and intramedullary hematomas. numerous imaging examples of these lesions are provided in the accompanying poster. brain mri in peroxisomal disorders: a pictorial essay bruno p. soares, md, radiology and biomedical imaging, university of california at san francisco, bruno.soares@ucsf. edu; leonardo vedolin, guido gonzalez purpose or case report: our presentation aims to illustrate the brain mri patterns in peroxisomal disorders. peroxisomes are intracellular organelles involved in important cellular processes including beta-oxidation of very-longchain fatty acids and plasmalogen production. peroxisomal disorders can be categorized into disorders of peroxisomal biogenesis, in which the peroxisomes are abnormally formed and several peroxisomal functions are deficient, and in defects involving a single peroxisomal function, in which the structure of the peroxisome is intact. disorders of peroxisomal biogenesis include zellweger syndrome, neonatal adrenoleukodystrophy, infantile refsum disease and rhizomelic chondrodysplasia punctata. numerous disorders are caused by loss of a single peroxisomal function including x-linked adrenoleukodystrophy and acyl-coa oxidase deficiency. clinical findings in peroxisomal disorders include dysmorphic features, hepatic dysfunction, neurodevelopmental delay, retinopathy and hearing impairment. methods & materials: pictorial essay illustrating brain mri patterns in peroxisomal disorders, including disorders of peroxisomal biogenesis and disorders with loss of a single peroxisomal function. results: brain abnormalities in peroxisomal disorders have a wide spectrum of patterns. neuronal migration disorders with abnormal myelination are typically seen in zellweger disease and neonatal adrenoleukodystrophy. specifically, the association of abnormal myelination with germinolytic cysts is suggestive of zellweger syndrome. classic x-linked adrenoleukodystrophy typically shows posterior central white matter involvement and symmetric demyelination also involving the corticospinal tracts and corpus callosum. a similar pattern of white matter involvement is seen in acyl-coa oxidase deficiency and infantile refsum disease. conclusions: brain mri helps narrow the differential diagnosis and guides subsequent evaluation in infants presenting with clinical features concerning for peroxisomal disorders. therefore, knowledge of the brain mri patterns in peroxisomal disorders is important for the radiologist interpreting neuroimaging studies. clots in tots: role of imaging in diagnosis of acute stroke and its causes in children asif abdullah, c.s. mott children's hospital of the university of michigan, asifa@med.umich.edu; ellen hoeffner, augusto elias purpose or case report: stroke is a major cause of morbidity and mortality in children. long-term neurologic deficits occur in % to % of infants and children after arterial ischemic stroke. limited awareness regarding pediatric stroke among physicians and in general community is a major concern. imaging plays crucial role in the diagnosis of pediatric stroke. the goal of this presentation is to provide awareness to the reader about the role of imaging in childhood stroke and its myriad causes in children. we will provide a case based approach to imaging diagnosis of acute pediatric stroke based on three categories: ( ) arterial ischemic stroke, ( ) cerebral venous thrombosis, and ( ) hemorrhagic. arterial ischemic stroke (ais) is classified according to the pediatric stroke classification (psc). psc includes eight subtypes of ais: ( ) sickle cell disease, ( ) cardioembolic disease, ( ) moyamoya syndrome, ( ) cervical arterial dissection, ( ) stenoocclusive cerebral arteriopathy, ( ) other determined etiology, ( ) multiple probable etiologies, and ( ) undetermined etiology. we will describe the role of computed tomography (ct) and magnetic resonance imaging including angiography (mri/mra) in identifying these causes in relation to available clinical data. the etiologies of cerebral venous thrombosis related infarction would be discussed from an imaging perspective with a case-based approach with emphasis on mrv and swi techniques. finally, we will focus on hemorrhagic causes of childhood stroke such as vascular malformation, aneurysm, neurocutaneous disorders, coagulopathy, and a variety of other causes from an imaging standpoint. perfusion imaging in pediatric stroke demonstrates flow within the brain and can detect areas that are at risk of ischemia; however, further studies in the pediatric population need to be validated for the role of this technique in pediatric stroke. results: the most important factors in the diagnosis of childhood stroke are causal investigation, appropriate laboratory tests, and imaging studies. imaging is frequently the first step in the evaluation of an acutely ill child. conclusions: pediatric stroke is a debilitating disease that requires urgent multidisciplinary approach for diagnosis and treatment. in cases of both ischemic and hemorrhagic origin, the radiological approach to be obtained in emergency setting leads to the initial screening and the first therapeutic possibility. methods & materials: this exhibit will illustrate the characteristic imaging findings of vascular anomalies in the head and neck region. vascular anomalies are divided into vascular tumors and vascular malformations which include slow flow malformations (capillary malformations, venous malformations, lymphatic malformations and their combinations) and high flow malformations (arteriovenous fistula and arteriovenous malformations). complex malformations are also seen in several syndromes including klippel-trenaunay syndrome, phace syndrome, etc. cases from author's database will be used for illustration. results: a review of clinical manifestations, characteristic imaging findings and interventional treatment strategies in cases of head and neck vascular anomalies will be presented with pre and post treatment imaging features. ultrasonography and mri are the mainstay in diagnosis of these malformations. ct scan and catheter angiography may occasionally be needed for diagnosis and treatment planning. various imaging findings and main treatment options will be listed. conclusions: head and neck vascular malformations are common in pediatric population. understanding the characteristic imaging findings and clinical presentation is essential in evaluating the vascular malformations. interventional procedures are generally the preferred treatment modality, either alone or in association with surgery in majority of these cases. isolated cortical diffusion restriction in pediatric brain mri ihsan mamoun, md, cleveland clinic, ihsanmamoun@ yahoo.com; sarah stock, s. pinar karakas, unni udayasankar, janet r. reid purpose or case report: diffusion-weighted imaging continues to emerge as a powerful neuroimaging tool. isolated cortical restricted diffusion is a particularly striking pattern with specific differential in the pediatric population. we aim to review this specific imaging pattern supplemented by case examples and key physiologic and imaging concepts. methods & materials: review the concept of diffusion restriction a) pathophysiology b) specific imaging appearances pictorial review of pediatric conditions that lead to cortical restricted diffusion: a) post ictal change b) infection-i. meningoencephaliitis ii. herpes c) hypoxic ischemic injury d) infarct: venous and arterial e) posterior reversible leukoencephalopathy f) mitrochondrial cytopathy g) metabolic: hypoglycemia. discuss certain artifacts. summary table and differential clues conclusions: the pattern of isolated cortical restricted diffusion has specific differential diagnosis in the pediatric population. the radiologist should be aware of this as use of dwi continues to grow. this exhibit with familiarize the reader with common conditions that specifically affect the cortex and produces true restricted diffusion. methods & materials: high resolution ct scan and mri are mainstay of diagnosis and assessment in patients with sensorineural hearing loss. in this exhibit we will present a pictorial review of ct scan and mri images of various causes of sensorineural hearing loss (snhl) that are seen on imaging. reviewing the embryologic basis of these anomalies would enable better understanding of this complex subject. results: the new system classifies these malformations according to descending order of severity into complete labyrinthine aplasia, cochlear aplasia, common cavity, cystic cochleovestibular malformation or incomplete partition-i (ip-i), cochleovestibular hypoplasia, and incomplete partition-ii (ip-ii). there is a lot of confusion in literature pertaining to mondini deformity. the new classification divides incomplete partition into ip-i representing cystic cochleovestibular malformation and ip-ii representing the classic mondini deformity with three components (cystic cochlear apex, dilated vestibule, and large vestibular aqeduct). recently a subclassification of ip-i and ip-ii has been proposed (subdividing into typical and atypical subtypes)[ ]. this will be discussed briefly. isolated large vestibular aqueduct without associated cochlear abnormalities will also be discussed. we will discuss the relevant embryology with correlations of malformations to the timing of embryologic insult. conclusions: the new classification system provides precision in description of inner ear malformation. this also helps in providing a uniform scale for comparison of effectiveness of cochlear implant for different malformations. purpose or case report: congenital cranial nerve anomalies often present as sensory and/or motor deficits of unknown etiology in the pediatric age group. the early recognition of a definitive cranial nerve abnormality using high-resolution imaging can focus further clinical investigation and shorten the time to diagnosis. methods & materials: to promote appropriate recognition of cranial nerve anomalies, we present the imaging findings of the most commonly affected cranial nerves and provide correlation with clinical presentation. all studies were performed on a . t magnet with dedicated high resolution imaging of cranial nerve exit zones. results: ours is a tertiary care pediatric hospital with an extensive neuroimaging database. we intend to review all known cases of cranial nerve anomalies from the prior years and present interesting and representative images including optic nerve hypoplasia as part of septo-optic dysplasia, kallman syndrome, duane retraction syndrome, and mobius syndrome. conclusions: congenital cranial nerve anomalies present with varied symptomatology including anosmia, impaired vision, occulomotor deficits, and hearing loss. additionally, clinical manifestations of cranial nerve anomalies can be difficult to recognize in the pediatric age group. effective imaging and prompt diagnosis is crucial to initiate appropriate clinical management. purpose or case report: mr is the standard for evaluation of tumors or epilepsy. pet-ct imaging is often performed to ascertain metabolic asymmetries related to epileptogenic regions or to better characterize the metabolic activity of tumors. a baseline for normals with pet-ct fdg- and c methionine does not exist. methods & materials: retrospective review was performed of the pediatric patients who underwent pet-ct with c methionine and fdg. representative studies were selected for patients imaged during infancy (< yr), early childhood ( - ), childhood ( - ), late childhood ( - ), teenage ( - ). c methionine and fdg studies were analyzed for normal patterns of uptake and any trends identified across the stratified age groups. representative pictorial image galleries of the c methionine and fdg imaging patterns through development were created. results: the pattern of radiotracer uptake on c methionine differed from that of fdg. the c uptake remained low level throughout development compared to fdg uptake, which was robust in much of the cortex. the cortical fdg uptake within the frontal lobes progressively increased with age. the c uptake within the brainstem and thalamus was equal to cortex throughout development. the fdg uptake within the basal ganglia was equal to cortex while the brainstem and thalamic uptake was generally less than cortex. several anatomic structures showed robust c uptake not seen on fdg. these included the lacrimal, submandibular and parotid glands. incidentally, the pituitary gland and hippocampus consistently showed c uptake equal to cortex contrary to their appearance on fdg. our institutional protocol regarding the performance of combination c methionine and fdg brain pet-ct studies is presented. conclusions: this study illustrated the normal appearance of brain pet-ct imaging performed with c methionine and fdg in a representative cohort of the pediatric patients through development. normal variance imaging patterns and developmental trends seen with each radiotracer was demonstrated. the pediatric cerebellum: a pictorial review of normal anatomy using mri and diffusion tensor imaging ibrahim s. tuna, md, radiology, children's hospital of wisconsin, dristuna@yahoo.com; sumit singh, teresa c. gross kelly, mohit maheshwari, tushar chandra, hervey d. segall purpose or case report: the aim of this educational exhibit is to illustrate normal anatomical and functional anatomy of the cerebellum in the pediatric patient. the cerebellum receives sensory input from the brain and spinal cord and integrates this information to coordinate motor control. in addition, the cerebellum also plays a role in some cognitive functions such as attention and language. the first step toward understanding how cerebellar abnormalities can lead to neurological dysfunction, is to provide a solid understanding of the neuroanatomy and functional pathways of the cerebellum. we will describe basic cerebellar embryology, the various cell types and gross anatomy using mr images as well as dti fiber tractography. methods & materials: this exhibit will describe the microstructure, gross anatomy and functional pathways of the cerebellum through illustrations, mr images, diffusion tensor imaging (dti) and pathological correlation. first embryology of the cerebellum will be described, followed by mri depiction of the developmental anatomy of the cerebellum from infancy through adolescence. finally dti tractography images will be used to delineate functional pathways to and from, as well as within, the cerebellum. pathological specimens will be photographed to further illustrate gross anatomy. results: afferent white matter pathways travel mainly via the inferior and middle cerebellar peduncles. the main efferent cerebellar white matter pathway is through the superior cerebellar peduncle. transverse fiber tracts are present in the vermis. there are mainly two main systems of cerebellar white matter fibers which are easily visualized with dti color mapping; however more anterior components of dti tracts are intermixed with afferent white matter projections following the middle cerebellar peduncle. conclusions: knowledge of the precise neuroanatomy and white matter tracts of cerebellum may elucidate our ability to comprehend the clinical manifestations of cerebellar diseases in children. a solid understanding of normal cerebellar anatomy, development and functional fiber tracts in the pediatric patient can provide a baseline that may help predict the clinical outcome of various diseases or interventional procedures. gastroesophageal reflux scintigraphy: a low radiation alternative to gerd evaluation in children vikas menghani, md, pediatric radiology, women's and children's hospital, drvikasmenghani@gmail.com; feraas jabi, jan najdzionek, vaseem iqbal purpose or case report: gastroesophageal reflux disease (gerd) is among the common causes for failure to thrive, recurrent cough and aspiration in children. early diagnosis of gerd is essential in avoiding long-term sequelae such as growth delay, chronic lung disease, esophageal stricture, and esophagitis. gastroesophageal reflux scintigraphy, a noninvasive imaging modality, has been applied for detection of gerd and gastric emptying in children over the past few decades. the radiation burden is considerably small given that a very low dose of radioactivity via a short half-life radioisotope like technetium- m tagged to oral sulfur colloid is administered to a patient. this feature makes reflux scintigraphy especially attractive as the patient can be scanned for prolonged and delayed periods without increasing radiation dose permitting not only identification but also assessment of severity of gerd. characterizing gerd severity is essential in determining how aggressive the pediatrician should be with therapy. gastroesophageal reflux scintigraphy also allows a child to be fed their regular meal tagged to radiopharmaceutical without altering food taste. qualitative and quantitative parameters like gastrointestinal transit and gastric emptying time can be measured, respectively. scintigraphy is highly sensitive to low grade reflux making it very desirable for monitoring response to therapy. while scintigraphy like all other imaging modalities,has limitations, it continues to be an excellent technique for gerd identification and characterization as well as in monitoring response to gerd therapy. the pediatric kidney-a review of common and uncommon renal anomalies ruby lukse, staten island university hospital, drjosemorey@gmail.com; josé morey, jeremy neuman, arnold brenner, oren herman, adam bernheim purpose or case report: renal parenchymal imaging in nuclear medicine has long been performed with mtcdimercaptosuccinic acid (dmsa) due to its sufficient binding to the renal tubules to permit renal cortical imaging. dmsa is of particular value when high-resolution images of the renal cortex are needed. this poster will be a pictorial review of common and uncommon congenital anomalies evaluated on dmsa imaging, such as horseshoe kidney, pelvic kidney, sshaped kidney and crossed-fused ectopia. the poster will also correlate planar imaging findingss with appropriate additional imaging including computed tomography (ct), magnetic resonance imaging (mri), fluoroscopic imaging and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (alara) principle set forth by the american college of radiology (acr). purpose or case report: in this poster we will review the differential diagnoses of congenital anomalies that give the appearance of hydronephrosis on renal imaging of the pediatric patient. we will show a pictorial review of both common and uncommon congenital anomalies such as congenital megaureter, ureterocele, uretero-pelvic junction (upj) obstruction, uretero-vesicular junction (uvj) obstruction and posterior urethral valves (puv). we will also review common mimickers of hydronephrosis such as multicystic dysplastic kidney (mcdk) and pseudo-obstruction secondary to bladder overdistention. the poster will also correlate planar imaging finds with appropriate additional imaging including computed tomography (ct), magnetic resonance imaging (mri), fluoroscopic imaging and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (alara) principle set forth by the american college of radiology (acr). the pediatric bone scan-a review of neoplastic pathology shrita smith, staten island university hospital, drjosemorey @gmail.com; josé morey, jeremy neuman, arnold brenner, daniel klein, purpose or case report: bone imaging continues to be the second greatest-volume of nuclear imaging procedure performed today, offering the advantage of total body examination, low cost, and high sensitivity. the diagnostic utility, sensitivity, specificity and predictive value of m-tc bone imaging of malignant conditions have long been established. in fact, more than , , bone scans were performed in the united states in . in this poster we will review the current indications for planar bone imaging for the evaluation of malignant and benign neoplasms in the pediatric population, such as osteoid osteoma, langerhan cell histiocytosis (lch), osteoblastoma, ewing's sarcoma, lymphoma, osteosarcoma and osseous/hepatic metastatic disease from neuroblastoma. the poster will also correlate planar and single-photon emission computed tomography (spect) imaging findings with appropriate additional imaging including computed tomography (ct), magnetic resonance imaging (mri), positron emission tomography (pet), and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (alara) principle set forth by the american college of radiology (acr). the many faces of duplex kidneys on dmsa scans-a pictorial essay neha kwatra, children's national medical center, nskwatra@childrensnational.org; massoud majd purpose or case report: renal duplication is the most common malformation of the urinary tract and is often seen in children with urinary tract infections (uti). the purpose of this study is to learn to recognize duplex kidneys on dimercaptosuccinic acid (dmsa) scintigraphy, review their entire spectrum of findings and correlate with other imaging modalities. methods & materials: dmsa scintigraphy is routinely performed in the nuclear medicine department with a single-head gamma camera (siemens e.cam, schaumberg, illinois). about . h after injection of dmsa, posterior and posterior oblique images are obtained using parallel and pin hole collimators. differential renal function is also calculated. dmsa scan reports containing the words "duplex" or "duplicated" from - were populated using a radiology search engine (montage health care solutions inc.). the images were then reviewed in pacs and representative examples were selected for the poster. the scans were evaluated for renal position, size, contour, any evidence of duplication and parenchymal damage. results: patterns of duplication included non complicated duplex kidney recognized by asymmetric renal size and a prominent cortical bar separating the two moieties, complicated duplex systems with hydronephrosis, scarring or pyelonephritis of one or both moieties. a small nonfunctioning upper moiety was sometimes evidenced by just an indentation along the superomedial aspect of the larger lower moiety. cases with bilateral duplex kidneys were also seen. illustrative examples of each will be provided. correlating findings on other imaging modalities will also be included. conclusions: establishing the diagnosis of duplex kidney on a dmsa scan requires a careful systematic review of the images. the findings can be subtle and it is important for the radiologist to recognize them. correlation with other modalities such as ultrasound or voiding cystogram can be complementary. the assessment of parenchymal function of the upper and lower moieties separately on dmsa scintigraphy can be of immense value in patient management and in choosing surgical options. poster #: edu- f-fdg pet/ct imaging of pediatric brain tumors, neurofibromatosis (nf ) and non-lymphomatous head and neck tumors. lisa states, md, radiology, chop, states@email.chop.edu; purpose or case report: this educational poster will review the current literature and summarize the value of f-fdg pet/ct in standard clinical practice in the evaluation of pediatric brain tumors, nf plexiform neurofibromas and malignant peripheral nerve sheath tumors, and nonlymphomatous head and neck tumors. normal variants and pitfalls will be reviewed. comparison with other pet tracers will be briefly discussed. case examples will be used to illustrate the value of f-fdg pet/ct in grading, staging, assessment of therapeutic response and detection of residual or recurrent disease in various pathologic entities. results: examples of cases will include: benign brain tumor, residual brain tumor in the post-operative bed, brain metastasis, malignant peripheral nerve sheath tumor in nf , head and neck rhabdomyosarcoma, mandibular osteosarcoma, and infection. conclusions: an understanding of the value of pet molecular imaging is essential to the success of the next phase of hybrid imaging with pet/mri which has the potential to play an important role in the development of new diagnostic and therapeutic approaches for the treatment of pediatric brain tumors, nf , and pediatric head and neck tumors. disclosure: dr. states has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. the pediatric bone scan-a review of non-malignant pathology josé morey, staten island university hospital, drjosemorey@ gmail.com; jeremy neuman, arnold brenner, vinh phan, cheryl lin purpose or case report: bone imaging continues to be the second most performed nuclear imaging procedure, offering the advantage of total body examination, low cost, and high sensitivity. the diagnostic utility, sensitivity, specificity and predictive value of m-tc bone imaging of benign conditions have long been established. in fact, more than , , bone scans were performed in the united states in . in this poster we will review the current indications for planar bone imaging for the evaluation of non-malignant diseases in the pediatric population, such as acute osteomyelitis secondary to salmonella enterobacteriaceae and tubercle bacillus (tb), chronic osteomyelitis, reflex sympathetic dystrophy, spondylolysis, bone infarcts in the setting of sickle cell disease, fractures (occult/stress), ankylosing spondylitis, dermatomyositis and non-accidental trauma. the poster will also correlate planar and single-photon emission computed tomography (spect) imaging findings with appropriate additional imaging including computed tomography (ct), magnetic resonance imaging (mri), positron emission tomography (pet), and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (alara) principle set forth by the american college of radiology (acr). purpose or case report: we review the radiologic features of pathologic conditions linked to diesel exposure. the hydraulic fracturing ("fracking") technique is increasingly used in many areas of the country to extract natural gas from rock formations. diesel fuel, or fluids containing diesel, are one component of fracking fluid and create a potential for ground water contamination and risk to air quality. the toxic effects of diesel exhaust are described in the literature, and include asthma, hydrocarbon pneumonitis, and leukemia. there are no scientific data currently available on the effects of chronic diesel ingestion. methods & materials: multi-modality examples of pathology were obtained from a radiology database at a tertiary care pediatric hospital. the specific cases displayed are not known to have diesel exposure, but are intended to serve as representative examples of the type of pathology that may be encountered in the setting of chronic diesel exposure. results: imaging findings of asthma include hyperexpansion, atelectasis, peribronchial thickening, and air-trapping. hydrocarbon pneumonitis may demonstrate low attenuation consolidation and subsequent pneumoatocoeles with ct. leukemia may present on plain radiographs with lucent metaphyseal bands and with marrow infiltration on mri. conclusions: in conjunction with other symptoms not necessarily evaluated in the radiology department, including rhinitis, laryngitis, acute coronary syndrome, and dementia, the radiologist may suggest the diagnosis of diesel toxicity, particularly in populations that may be at high risk of exposure. pediatric radiology in the philadelphia region: a historical review* richard markowitz, md, children's hospital of philadelphia, markowitz@email.chop.edu purpose or case report: the specialty of pediatric radiology in the philadelphia region has grown and evolved over the past eight decades originating from early "visiting" radiologists to drs. hope and kirkpatrick, the "giants" of the s and ' s, to over fifty practicing pediatric radiologists today. clinical excellence, commitment to teaching, and advancement of knowledge through research remain the goals and ideals, much as they were many years ago. philadelphia has been a fertile home and environment for this evolution, mostly because of outstanding leaders and role models who have trained and influenced generations of pediatric radiologists. developments and leadership at the children's hospital of philadelphia, st. christopher's hospital for children, and a.i. dupont institute are highlighted. the purpose of this poster is to tell the story of the growth and development of pediatric radiology in this area and to explore the intellectual origins, professional "genealogy," and legacies left by those who created and those who have carried on this tradition. *note: this material is based on a previously published article: pediatric radiology ( ) : - and "addendum" (pediatric radiology : - ), but never presented at spr. superficial lumps and bumps henrietta k. rosenberg, md, radiology, the mt. sinai medical school, henrietta.rosenberg@mountsinai.org; diane belvin, neil lester purpose or case report: superficial soft tissue masses in the pediatric age range can be quite challenging to the pediatrician and the imager. the purpose of this presentation is to demonstrate the efficacy of duplex/color doppler ultrasound for the diagnosis and follow up of a large gamut of superficial lumps and bumps. methods & materials: we reviewed our experience during the past years using ultrasound to evaluate superficial soft tissue masses that had been encountered in many parts of the body, from the skull to the soles of the feet, in a large group of patients ranging in age from newborn to years. all sonograms were performed after obtaining pertinent clinical information as well information regarding the clinical characteristics of each of the masses, e.g. location, consistency (firm [solid], compressible [cystic]), fixed or easily movable, smooth or irregular surface, tenderness. the masses were palpated by the imaging team and duplex/color doppler ultrasound was performed. comparison sonographic views of the opposite side were obtained as needed. clinical followup and surgical/pathological correlation was obtained in most of the patients. results: most of the masses were benign and included a wide variety of etiologies. most often, us was sufficient for assessment of soft tissue masses if the entire mass was included in the field of view. if the lesion was too large for the field of view or malignancy was suspected, ct/mri were required preoperatively. nuclear medicine studies are reserved for midline masses likely due to ectopic thyroid and pet was used for more complete evaluation of a lesion that was likely malignant. conclusions: duplex/color doppler ultrasound (us) is the modality of choice for evaluation of superficial lumps and bumps! this modality allows for rapid acquisition of information without the use of ionizing radiation, intravenous contrast material, or sedation/anesthesia. reliable information can be rapidly acquired regarding the size, shape, borders, location, internal consistency, vascularity, vascular encasement/displacement. correlation of the ultrasound and clinical findings helps narrow differential diagnosis. sonography helps to determine what is the next best step: watchful waiting (clinical observation, follow-up us), surgical resection, or us guided interventional procedure. present day imaging of down syndrome rupa radhakrishnan, md, radiology, university of cincinnati college of medicine, radhakrp@ucmail.uc.edu; alexander j. towbin purpose or case report: down syndrome is a common genetic condition characterized by unique physical traits and multisystem anomalies. the purpose of this exhibit is to portray the imaging findings of down syndrome and discuss with illustrative examples, the use of imaging in multidisciplinary management. methods & materials: published literature was reviewed to identify the multisystem imaging findings in down syndrome. the electronic medical record system was then searched to find illustrative case examples from our institution. results: in patients with down syndrome, abnormalities can be found in the musculoskeletal, cardiovascular, respiratory, gastrointestinal, and central nervous systems. abnormalities can range from emergent, life threatening conditions such as malrotation with midgut volvulus to chronic conditions such as scoliosis. examples of abnormalities from each organ system and the modalities used for diagnosis and management are described. cardiovascular system: echocardiogram and cardiac mri and ct are useful in evaluating congenital heart disease associated with down syndrome. respiratory system: micrognathia with macroglossia and hypotonia predisposes patients to sleep apnea which can be evaluated with dynamic mri. chest ct demonstrates subpleural cysts which are characteristic of this syndrome. gastrointestinal system: fluoroscopy and/or radiographs are the mainstay in diagnosing many gastrointestinal disorders including duodenal atresia, malrotation, annular pancreas, imperforate anus, and hirschsprung disease. central nervous system: choroid plexus cysts may be identified on prenatal ultrasound in a fetus with down syndrome. imaging is used in the evaluation of epilepsy, hearing loss and alzheimer disease that is more common in these individuals. musculoskeletal system: multiple skeletal anomalies can be present in patients with down syndrome. radiographs are often used as the method of identifying and, if needed, following the anomalies. prenatal imaging: increased nuchal translucency is the earliest imaging finding. other features of down syndrome can be identified on prenatal ultrasound or mri. prenatal imaging is helpful in determining the prognosis of the fetus and in guiding management. conclusions: modern day multidisciplinary management has improved quality of life and survival in individuals with down syndrome. imaging plays a critical role in guiding management in these individuals. imaging the spectrum of lymphatic malformations in the pediatric patient andrew schapiro, md, radiology, university of wisconsin, aschapiro@uwhealth.org; kara gill, bradley maxfield purpose or case report: lymphatic malformations (lm) occur as a result of abnormal development of the lymphatic system during embryogenesis. as % of lm present by years of age, these lesions represent an important pediatric entity. lm can often be suspected clinically in an infant with the classic presentation of an asymptomatic, soft mass in the head, neck, or axilla. however, myriad presentations are possible as lm occur in numerous other anatomic locations, can be multiple, and can be a component of mixed vascular malformations. in addition, the true extent of lm is often not apparent clinically. given these considerations and the implications for proper management, imaging plays an important role in the assessment of lm. the purpose of this exhibit is to review the spectrum of radiographic, ct, sonographic, and mr imaging findings of a variety of lm presentations. methods & materials: cases of lymphatic malformation in pediatric patients identified at a single institution over the past ten years with available imaging were reviewed utilizing pacs. results: images of lm involving the head and neck, chest, abdomen, retroperitoneum, extremities, and skeletal system were identified. in addition, cases of lymphangiomatosis and mixed venolymphatic malformation were identified. various imaging modalities including radiography, ct, sonography, and mr were represented. conclusions: adequate knowledge of the imaging characteristics of lm across multiple modalities enables proper diagnosis, assessment of disease extent, and guidance of appropriate therapy in pediatric patients. results: ct and mr imaging findings in nine cases will be presented. they include ) congenital absence of the inferior vena cava with thrombosis of the external iliac vein secondary to venous stasis ) pyelophlebitis complicating ruptured appendicitis ) left iliac vein thrombosis in a patient with may-thurner syndrome ) splenic vein thrombosis complicating pancreatitis ) splenic vein thrombosis following splenectomy ) renal vein thrombosis in an infant of a diabetic mother ) adrenal vein thrombosis as the presenting sign of antiphospholipid syndrome ) budd-chiari syndrome associated with underlying myeloproliferative disease ) iliac vein thrombosis as a manifestation of behcet's syndrome (hughes-stovin syndrome, a variant of behcet's syndrome, which presents with systemic venous thrombosis and pulmonary artery aneurysms will also be discussed). conclusions: thrombosis of large abdominal and pelvic veins in children and adolescents is uncommon. certain conditions, both congenital and acquired, predispose to the development of venous thrombosis. ct/mr imaging defines the extent of thrombosis, and demonstrates additional findings that may elucidate the nature of the underlying condition leading to clot formation. purpose or case report: because abnormal gait in a young child has a wide range of causes, imaging plays a critical role in establishing the definitive diagnosis. the purpose of this exhibit is to review the clinical clues (age, duration, laboratory markers) and imaging findings of the causes of abnormal gait in a toddler and to assess the strengths and limitations of radiographs, ultrasound, magnetic resonance imaging (mri), and computed tomography (ct). methods & materials: cases, from a single institution experience with various causes for abnormal gait in a toddler, are reviewed and categorized into congenital, traumatic, inflammatory, neoplastic, or neuromuscular etiologies. results: there are various causes of abnormal gait in a toddler. the congenital causes include spinal dysraphism, proximal and distal skeletal deformities and dysplasias. the traumatic causes include non-accidental trauma, toddler's fracture, foreign body, and soft tissue injuries. the inflammatory causes include juvenile idiopathic arthritis, transient synovitis, and infection, including osteomyelitis, septic arthritis, discitis, cellulitis, and abscess. the neoplastic causes include various neurogenic, bone, and soft tissue tumors. the neuromuscular causes include cerebral palsy and spinal bifida. the combination of clinical presentation, supporting laboratory findings, and classic imaging findings help to distinguish the possibilities and often allows confident diagnosis. conclusions: knowledge of imaging findings and clinical factors can demystify the diagnosis of abnormal gait in a toddler. familiarity with the clinical presentation can ensure the performance of the appropriate diagnostic studies, timely diagnosis, and effective treatment. nonaccidental causes should never be overlooked. ultrasonography has become an important tool in the radiologist's armamentarium, augmenting radiography, mri, and ct. approximately different contrast agents for mri and ct are now commercially available for use. although most of them are fda approved in adults, information on usage and safety in children is not readily available. the most important reason is lack of controlled studies in children, especially for the age of - years. however, the lack of fda approval has not limited the use of these promising agents in children. in fact, there is widespread off-label use of these agents in most major pediatric hospitals in the country. based on a review of relevant literature in children, and based on a survey of radiology faculty at major pediatric hospitals, this poster will address the gap between approved use and reality in the setting of pediatrics. results: using a tabular format, this poster will provide a list of mr and ct contrast agents that are available for clinical use, their relevant clinical properties (ionic or nonionic, viscosity, linear or macrocyclic, degree of relaxivity for mri, iodine concentration for ct, cost, dosage, halftime, incidence of allergic reactions, nephrogenic systemic fibrosis and other adverse reactions), fda approval status (for ages - days, days- years, and - years), common pediatric applications, and contrast injection protocols for common applications. conclusions: to enlighten imaging personnel about usage and safety of contrast agents in children. disclosure: dr. krishnamurthy has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. a pictorial essay and literature review of the spleen in sickle cell disease david hindson, md, boston medical center, david. hindson@bmc.org; heather imsande, philippa sprinz, ilse castro-aragon purpose or case report: the morbidity and mortality of sickle cell disease (scd) results from acute and chronic infarction events that affect almost every organ. repeated infarction has some of its greatest visual and physiologic impact within the spleen. continuous hemolysis, sequestration and vaso-occlusion within the spleen result in loss of splenic function early in life and frequently autosplenectomy thereafter. by years of age, approximately % of children with hemoglobin ss disease will have diminished splenic function, putting them at increased risk for infections. treatments for scd have evolved over the last years, and among others include penicillin prophylaxis and immunizations, hydroxyurea and transfusion therapy (or hypertransfusion program). imaging findings are a reflection of the different treatments and their efficacy. methods & materials: our institution cares for a large group of patients with sickle cell disease, from birth to adulthood. this offers an unprecedented opportunity to document the imaging findings of the spleen with different treatment regimens, and over many years. the splenic size and morphology can be followed, by ultrasound, in a very straightforward way. we have compiled a pictorial essay of the various imaging characteristics of spleens from infants to adults. we also performed a literature review to compare and supplement the findings of our images. results: there is a spectrum of imaging findings in the spleen of patients with scd that changes from birth to childhood. the findings range from the normal appearance of a spleen to a calcified spleen, and include regenerative nodules, fibrosis, altered parenchymal echotexture, increased echogenicity, and changes in size, including enlargement secondary to sequestration. the ultrasound characteristics not only change with advancing age, but also appear to depend on whether or not the patient has received specific treatments, and at what age treatment was initiated. conclusions: the ultrasound appearance of the spleen in patients with scd is variable. treatments such as blood transfusions and hydroxyurea, patient compliance with therapy and type and severity of the disease are some of the factors that affect imaging characteristics. cystic fibrosis: not just for children cindy miller, md, radiology, yale-new haven hospital, cindy.miller@yale.edu purpose or case report: cystic fibrosis has been recognized for hundreds of years with the first descriptions of it including such anecdotes as mothers licking the foreheads of their children and knowing that if it tasted salty, an early death could be predicted. it was not until that the disease was first named by dr. dorothy andersen, and for the following years, treatment was largely supportive, and imaging was essentially done with plain films alone. in with the elucidation of the cftr gene, there was an explosion of knowledge which included the range of increased awareness and understanding of the suspected etiology, imaging findings and significance of the ductus bump. the contribution of d imaging for evaluation of the pediatric central airways jessica kurian, md, the children's hospital of philadelphia, kurianj@email.chop.edu; monica epelman, david a. mong purpose or case report: evaluation of the central airways in children has historically been accomplished by flexible bronchoscopy, an invasive technique associated with inherent risks and complications. multidetector ct (mdct) with volume rendering offers a noninvasive alternative for airway evaluation. in this educational exhibit, we will review imaging techniques and clinical applications of mdct for the assessment of large airway maladies in children. methods & materials: mdct imaging in children with a variety of tracheobronchial disorders is reviewed. for each entity, the characteristic clinical features are described, and key imaging features are illustrated. emphasis is placed on the contribution of d techniques for characterizing complex airway anomalies. dose reduction strategies are also highlighted. results: the entities reviewed in this exhibit include, but are not limited to, congenital anomalies of tracheobronchial branching, airway malformations associated with situs, and congenital or acquired airway compression and/or obstruction. conclusions: mdct with volume visualization is a useful adjunct for evaluation of the pediatric central airways in a variety of pathologies. as a noninvasive technique, it avoids sedation risks and spare patients from complications associated with conventional flexible bronchoscopy. low dose protocols should be used to minimize radiation exposure. bronchopulmonary foregut malformation that result from abnormal budding of the primitive foregut. currently, many such anomalies are initially detected by prenatal ultrasound and are further delineated by fetal magnetic resonance imaging (mri), while others may be incidentally detected on postnatal radiologic examinations or later in life in the setting recurrent pulmonary infection. imaging plays a very important role in the diagnosis and characterization of these lesions and assists surgical planning. the purpose of our educational exhibit is to illustrate the common and uncommon radiologic appearances of cpams using various imaging modalities, including radiography, computed tomography, prenatal and postnatal ultrasound, and prenatal and postnatal mri. methods & materials: all pediatric and adult cpam (including both sequestration and ccam) patients were identified using electronic medical records. pertinent imaging reports (including radiography, prenatal and postnatal ultrasound, ct, and prenatal and postnatal mri) were reviewed by a single author in order to identify relevant imaging findings. relevant images from these imaging examinations were de-identified and saved to a secure hard drive. medical records were accessed by a single researcher to obtain relevant demographic information as well as data regarding the patients' clinical presentations. in cases of corrective surgery, operative and pathology reports were reviewed, if available, for correlation with the imaging findings. results: cases of pediatric and adult cpam were identified and presented in a variety of clinical contexts. their appearances were reviewed through multiple imaging modalities. conclusions: congenital pulmonary airway malformations are varied in their clinical presentation and imaging appearance. the purpose of this pictorial essay is to enhance understanding of their diagnosis and to use a multidisciplinary approach in order to highlight imaging aspects that may alter clinical management. disclosure: dr. horst has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. the imaging evaluation of cystic lung disease in children: an evidence-based approach jordan caplan, md, pediatric radiology, lucile packard children's hospital, stanford university, caplan@stanford. edu; beverley newman purpose or case report: the goal of the poster is to provide a framework for use when confronted with cystic lung disease in a child. methods & materials: the differential diagnosis for the types and causes of cystic lung disease in children will be presented using an evidence-based, age appropriate approach. categories of disease discussed and illustrated with case examples will include: a. congenital cystic bronchopulmonary malformations b. infectious cysts c. autoimmune/inflammatory/vasculitic disease with cavitating lesions d. neoplastic conditions e. collagen/soft tissue abnormalities f. mimics of cystic lung disease results: the pathophysiology, imaging appearance, and demographics of the above entities will be reviewed with attention to relevant recent literature. important educational points include the differentiation of bronchopulmonary malformations from neoplasm, notably pleuropulmonary blastoma (ppb), the relationship between lung cysts and ppb, and the management and surveillance of lung cysts in children. conclusions: an evidence-based approach to the broad spectrum of causes of cystic lung disease in children is a useful starting point in forming a concise and pertinent differential diagnosis. an understanding of the pathophysiology, imaging appearance, and demographics of these entities is essential in guiding patient management. pediatric interstitial lung disease (ild): a pictorial review with radiologic and pathologic correlation hollie west, md, diagnostic radiology, vanderbilt university, hollie.c.west@vanderbilt.edu; melissa a. hilmes, sudha p. singh, jennifer soares, lisa young purpose or case report: while adult interstitial lung disease is a well-described and fairly well understood group of disease processes, pediatric interstitial lung disease (ild) remains a subject of uncertainty and misunderstanding for many clinicians and radiologists. confusion surrounding the phenomenon of pediatric ild stems not only from the rarity of the disease, but also from the extensive list of disease entities that can produce ild, the existence of certain patterns that are restricted to infants and children and the fact that patterns of ild manifest differently in a child's developing lung than in an already developed adult lung. imaging plays an important role in diagnostic work-up of this disease and can guide lung biopsy in specific patient populations. methods & materials: the irb approved retrospective study will show patients at our institution over a year period diagnosed with various types of ild, including pulmonary insterstitial glycogenolysis (pig), diffuse neuroendocrine cell hyperplasia (nehi), surfactant deficiency diseases, and lung diseases associated with other systemic processes such as downs syndrome and inflammatory bowel disease. we will include patients with biopsy proven ild and will provide examples of the major ilds, including clinical, radiologic and pathologic correlation. our pictorial review will describe the radiologic patterns associated with the different forms of ild, emphasizing what the radiologist needs to know and how to be helpful to a multidisciplinary team in the diagnosis and treatment of these diseases. results: the study will report the frequency of ild at our institution, including a breakdown of the various subtypes of ild. we will show examples of the subtypes with correlative chest radiography, computed tomography, and pathology. we plan to highlight specific differentiating factors between the different diseases and demonstrate how a radiologist can be helpful in collborating with clinicians in diagnosing and treating these diseases. conclusions: pediatric ild can be a confusing topic for radiologists. increasing knowledge and awareness of these diseases, their clinincal presentation, work up, and treatment is important for pediatric radiologists who work as part of of a multidiciplinary team. poster #: sci- ct radiation dose delivered by community hospitals and imaging centers stephen little, children's healthcare of atlanta, stephen. little@choa.org; damien grattan-smith, bonnie johnson purpose or case report: to evaluate and compare ct radiation dose for pediatric abdominal and cranial ct examinations performed by community hospitals and imaging centers. methods & materials: consecutive ct examinations ( cranial, abdominal) from community hospitals and imaging centers were reviewed following transfer of care. the examinations were performed between january and july . consecutive ct examinations ( cranial, abdominal) performed at our own institution were also reviewed. ctdivol and dlp were obtained from the dose report for each examination ( cm-phantom for abdominal exams, cm-phantom for cranial exams). patient age and weight were obtained from the medical record. results: average ctdivol for abdominal ct performed by local community hospitals and imaging centers was . mgy, while average ctdivol was . mgy for abdominal ct performed at choa. there was a wide variation in ct radiation dose delivered. while some sites delivered a ct radiation dose comparable to our own, others delivered a substantially greater dose. in fact, % of pediatric abdominal ct exams performed by local community hospitals and imaging centers exceeded the notification value recommended by the aapm ( mgy using the cm phantom). low kvp technique for imaging small children was infrequent. multi-phase examinations were more often performed, resulting in additional elevation in ct radiation dose when dlp is considered. average ctdivol delivered by local community hospitals and imaging centers for cranial ct was mgy compared to a ctdivol of mgy for cranial ct performed at choa. % of pediatric cranial ct exams performed by local community hospitals and imaging centers exceeded the notification value recommended by the aapm ( mgy for - years, mgy for > years). conclusions: despite ongoing efforts at education, there is wide variation in ct radiation dose delivered for pediatric abdominal and cranial ct examinations performed by local community hospitals and imaging centers. appropriate use of dose check software on newer scanners may help reduce the number of children subjected to excessive ct radiation dose. ultimately, each site performing pediatric ct must take responsibility for minimizing radiation dose while producing diagnostic quality exams. the impact of adaptive statistical iterative reconstruction on ct image quality parameters -a phantom study karen thomas, md, radiology, hospital for sick children, karen.thomas@sickkids.ca; nancy ford, angjelina protik, paul babyn purpose or case report: to quantify the effect of adaptive statistical iterative reconstruction (asir) on ct image quality parameters. methods & materials: phantom (catphan ) studies were performed on a ge hd -slice scanner to investigate the impact of a) % asir compared to routine filtered back projection using variable kvp ( - ) and mas ( - ), and b) incremental asir % ( , , , , %), scanning at mas and variable kvp ( - ). pitch, acquisition fov and detector width were kept constant. image noise, spatial and contrast resolution, contrast noise ratio (cnr) and wiener spectrum analysis were performed on . mm ax, mm ax mpr and mm cor mpr series. results: % asir resulted in a mean decrease in noise of % ( . mm ax), % (ax mpr) and % (cor mpr) and improvement in cnr of - %. incremental advantage was seen with stepwise increase in asir %. however, application of asir was associated with a small reduction in spatial resolution ( - % at % asir). low contrast detectability (lcd) improved except at the smallest target lesion size. image quality effects at very low mas and at high asir % will be presented. conclusions: image noise reduction and improvements in cnr and lcd with asir hold considerable potential for dose reduction in pediatric ct. this study provides quantitative data that may be used to design asir-enhanced protocols with consideration of diagnostic task, balancing image quality benefits and potential pitfalls. pictorial essay on cardiac mr for congenital heart disease on t mr scanner with rf multi-transmit technology (tx) taylor chung, md, diagnostic imaging, children's hospital & research center oakland, taylorchung @gmail.com purpose or case report: this is a pictorial essay (e-poster) to show artifacts on cine ssfp images pre-tx and post-tx upgrade on congenital heart disease cardiac mr; to illustrate methods prior to tx-upgrade to minimize artifacts. disclosure: dr. chung has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. revisiting the relationship between anthropometric parameters and left ventricular mass abdullahi adamu, md, phd, ahmadu bello university, scorpion kd@yahoo.com purpose or case report: the purpose of this study was to find the correlation between anthropometric parameters and left ventricular mass in normal adolescents and young adults. methods & materials: healthy individuals in the age range to years ( males and females) were included in this study. anthropometry was performed with standard anthropometry kit and measurements of height, weight, body surface area (bsa), upper arm circumference and upper hip circumference were taken. echocardiography was performed and the american society of echocardiography (ase)-recommended method was employed for calculation of left ventricular mass (lvm). statistical analysis was performed using statistica . (stat soft, usa). results: the mean value of lvm for all our subjects was found to be . ± . g. there was significant correlation between lvm and height (r . , p< . ), weight (r . , p< . ) and bsa (r . , p< . ). correlation with upper arm circumference was moderate (r . , p< . ), while it was found to be weak with upper hip circumference (r . , p< . ). diagnostic. both field strengths can be used successfully for cardiac and vascular imaging. the decision as to which to use is weighted by local availability and the relative requirement for detailed vascular vs intra-cardiac imaging. disclosure: dr. nguyen has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. poster #: sci- color coded d cardiac cta of congenital heart disease: a five year experience nhi huynh, md, radiology, st. joseph hospital and medical center, e.nhihuynh@gmail.com; randy richardson purpose or case report: post-processing of cardiac computed tomography angiograms can be performed on a commercially available workstation to create color coded d volume rendered images of the segmented heart and great vessel anatomy in patients with congenital heart disease. these studies optimally demonstrate complex anatomy, streamlining communication between members of the healthcare team and providing a tool for communicating complex anatomy and treatment options with families. these studies have been ordered with more frequency over the past five years. we retrospectively reviewed the types of congenital heart disease demonstrated by cardiac d cta over the past five years at a congenital heart center. methods & materials: color coded cardiac cta postprocessing was performed from ecg gated prospective and retrospective cta data on a commercially available workstation for / patients over the past three years. the anatomy was initially segmented and colored into individual parts of the anatomy of the heart and great vessels as follows rv purple, lv light red, aorta red, pulmonary arteries blue, systemic veins and right atrium aqua, pulmonary veins and left atrium pink, pda or collaterals green, airway yellow, coronary arteries neutral. the anatomy was then reassembled and images obtained every °in a °rotation for display. results: d color coded cta images were used in the treatment and care of congenital heart patients for the following types of congenital heart diseases: cases of complex anatomy (tga, truncus arteriosus, hlhs, tricuspid atresia, tof…), coronary artery anomalies, cases of pulmonary atresia or stenosis, cases of systemic and venous anomalies, cases of coarctation or interruption of the aortic arch, and tracheobronchial tree anomalies. conclusions: color coded cardiac cta post-processing is an effective and viable method for demonstrating anatomy in complex congenital heart patients. it is an excellent tool for demonstrating anatomy which is difficult to see by echocardiography such as: coronary artery anomalies, pulmonic atresia, aortic arch coarctation or interruption, and tracheobronchial anomalies and/or stenosis. neuroimaging in the evaluation of hie in term neonates post hypothermia therapy julio m. araque, md, radiology, medical college of georgia, jaraque@georgiahealth.edu; jatinder bhatia, leann vanlandingham purpose or case report: to illustrate and review the potential utility of brain mri, ct and ultrasound in hypoxic ischemic encephalopathy in newborns treated with hypothermia. neuroimaging studies including brain ultrasound, ct and mri of fifteen term newborns treated in our institution with therapeutic hypothermia, since april were evaluated retrospectively. more relevant lesions are depicted and the diagnostic and prognostic value of the findings is discussed and compared with a review of the literature. results: recent studies showed that patients treated with cooling had a more favorable prognosis than was suggested by the clinical grade of encephalopathy compared with infants treated with standard care. our institutional protocol includes the performance of mri, and ultrasound. ct is performed when is a clinical impossibility of perform mri. brain ultrasound was performed in all the patients. mri scans were obtained in neonates. ct was obtained in patients. all mri studies included dwi. the utility of dwi and adc maps as an aid in diagnosis of non-ischemic lesions is becoming increasingly established. mri evidence of brain injury was visible on basal ganglia in cases with negative ultrasound. abnormal signal intensity in the posterior limb of the internal capsule coexists with lesions in the basal ganglia and thalami have been associated with abnormal motor outcome. the remaining newborns did not develop significant mri evidence of brain injury. it has been suggested that the ability of mri to predict subsequent neurological impairment is unaltered by therapeutic hypothermia. further research is needed for defining the relation between mri findings and cooling. it is possible that imaging findings might be delayed in cooled infants. conclusions: mri offers the highest sensitivity in detecting anoxic injury of the neonatal brain. mr biomarkers in combination with clinical markers may identify patients with adverse outcome with therapeutic implications. disclosure: dr. araque has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: to correlate bowel wall diffusionweighted imaging (dwi) apparent diffusion coefficient (adc) values with multiple mr enterography (mre) and clinical findings in pediatric small bowel crohn disease. methods & materials: pediatric crohn disease patients with mre exams containing diffusion-weighted imaging and demonstrating terminal ileitis were identified. minimum bowel wall adc values were tested for correlation/association with other mri findings and clinical parameters (including laboratory values). results: there is negative correlation between adc value and degree of bowel wall thickening (r (−) . ; p . ). lower adc values were significantly associated with striated pattern of arterial phase postcontrast enhancement (p . ), greater degree of arterial phase postcontrast enhancement (p . ), and presence of stricture (p . ). adc values were not associated with diseased bowel length, degree/pattern of delayed postcontrast enhancement, degree of mesenteric inflammation or fibrofatty proliferation, or clinical markers of inflammation. conclusions: restricted diffusion in pediatric small bowel crohn disease is associated with other mri findings of that are suggestive of active disease, including degree of bowel wall thickening and degree and pattern of arterial phase postcontrast enhancement. our data also suggests that dwi may be useful when attempting to characterize small bowel strictures as either predominantly inflammatory or fibrotic, although further investigation is needed. quantification of blood flow into and out of the liver with d phase contrast mri in the pediatric patient binh huynh, md, radiology, stanford, bhuynh@stanford. edu; shreyas vasanawala, albert hsiao purpose or case report: the ability to probe blood flow dynamics in the liver may aid management of children with liver disease, including shunt fractions in portal hypertension and arterial flow fraction in diffuse liver disease. the purpose of this study is to evaluate the ability to measure blood flow into and out of the liver with time resolved volumetric ( d) phase contrast mri in the pediatric patient. methods & materials: nineteen consecutive patients were retrospectively identified who underwent d flow imaging through the level of the hepatic vessels on . t and t magnets. a software enabling d flow program was utilized to first assess for the feasibility of measurement of flow in the hepatic artery (ha), portal vein (pv), splenic vein (spv), superior mesenteric vein (smv), supra (sivc) and infrahepatic (iivc) inferior vena cava. if measurable, calculations were performed to evaluate for internal consistency by comparing the sum of smv and spv flow to pv flow. calculations were then performed to compare hepatic inflow (pv+ha) to hepatic outflow (sivc-iivc) and for the percentage of pv and ha contribution to hepatic inflow. results: of the nineteen patients, all of the above mentioned six vessels were visualized and measurable in two patients, both of which were imaged on the . t magnet. in the remaining patients, flow measurements were limited by respiratory motion artifacts obscuring the smaller vessels, and severe eddy currents, particularly in patients imaged with the t magnet. the evaluation for internal consistency demonstrated an average of . % ( . % & - . %) difference between smv+spv and pv flow. hepatic inflow was found to closely match the measured hepatic outflow with an average difference of . % ( . % & . %). the portal vein was found to contribute . % and . % to hepatic inflow, while the hepatic artery contributed . % and . %. conclusions: measurement of hepatic flow with phase contrast mri is more challenging than assessment of thoracic flow. when respiratory artifacts are minimal, vessels can be identified and measurements have internal consistency and good agreement between hepatic inflow and outflow at . t. conversely, flow measurements were limited at t by eddy currents. thus, ongoing efforts are aimed at mitigating respiratory motion artifacts at . t. poster #: sci- mri findings in post-fontan hepatopathy adina alazraki, md, radiology, emory university/children's healthcare of atlanta, adina.alazraki@choa.org; pinar bulut, kiery braithwaite, miriam vos, rene romero, nitika a. gupta purpose or case report: as advances in congenital heart disease continue to improve both mortality and quality of life, associated complications are becoming more prevalent. amongst patients who have had fontan repair for hypoplastic left heart syndrome, tricuspic atresia, or other right heart dysfunction, it is well known that liver disease is a complication. we describe the mri findings in post-fontan patients and propose mri as a useful tool to the hepatologist's evaluation of these patients. methods & materials: irb approval was obtained for a retrospective review of patients who underwent fontan repair and were subsequently referred for hepatology evaluation between - . all but one patient was scanned on a siemenstriotrim t magnet; one patient was scanned on a ge twinspeed . t magnet with an equivalent protocol due to orthodontics. a standardized departmental protocol was utilized. mri findings were correlated with age at surgery and years since surgery. mr images were reviewed independently by pediatric radiologists and compared with the dictated report in the patients record. results: patients underwent mri of the abdomen. patients had mri incompatible hardware and patients were not scanned secondary to insurance denial. patients were divided into groups based on elapsed time since surgery: less than years, - years, - years, and greater than years.(table ) mr images were evaluated for the presence of fibrosis, congestion and any other hepatic abnormalities. fibrosis was determined based on a specific pattern of delayed reticular enhancement in combination with liver morphology. congestion was deemed present if there was increased t signal in the liver parenchyma or periportal regions in combination with cloud-like enhancement on dynamic post-contrast images. all patients demonstrated morphologic changes in the liver with varying degrees of hepatic fibrosis and hepatic congestion. fibrotic changes were often non-uniform, and thus could be underdiagnosed by biopsy. interestingly, patients, %, had focal arterially enhancing lesions speculated to represent vascular proliferative lesions, however, none warranted biopsy. conclusions: it is established that patients who undergo fontan develop hepatic abnormalities. mri is a reliable, non invasive technique that accurately demonstrates these findings. mri may be a more sensitive method to evaluate the etiology and full extent of hepatic disease. poster #: sci- complications within the interventional radiology division of a tertiary care children's hospital: initiatives for ongoing quality and practice improvement brian dillon, children's hospital boston, brian. dillon@childrens.harvard.edu; pamela sanborn, yolanda milliman-richard, darren orbach, stephan voss purpose or case report: between and , procedure-related complications occurring within the division of interventional radiology at our institution were recorded and classified according to level of severity. the goals of this study were to determine rates of procedurebased complications based on severity, to establish thresholds for complications, and to determine whether measurable trends in complications over time were evident. methods & materials: between and , , interventional procedures were performed within the division of interventional radiology at our institution. adverse events were characterized both according to level of severity (using an institutional point severity scale), and with brief descriptions of individual events. adverse events were reviewed monthly at the division's morbidity and mortality conference, with respect to procedure type and operator. based on review of our interventional radiology data and benchmarks rates used for diagnostic errors, threshold complication rates were established by consensus between the department quality improvement committee and the division of interventional radiology. for severe events (level and ) there is no allowable threshold; all such events were subjected to both internal and institutional review. results: the overall complication rate was less than % for all procedures performed. the complication rates for the respective severity levels were: level ( . ), level ( . ), level ( . ), level ( . ), and level ( . ). the severity of a given complication was not associated with procedural complexity. no operator-specific trends were identified. conclusions: since , the society of interventional radiology has offered guidelines and strategies for improving safety and quality in interventional radiology. however, no specific benchmark data or procedural recommendations are available for pediatric interventional procedures. our results demonstrate rates of complications well below published overall complication rates for interventional radiologic procedures. this database of procedure-based complications serves as a foundation for a quality improvement program that allows review of complications with respect to specific procedure types, individual operators, and procedural complexity, in an effort to institute an ongoing and continuous process of quality improvement within interventional radiology. purpose or case report: dysosteosclerosis (dss), an extremely rare dense bone disease, features short stature and fractures and sometimes optic atrophy, cranial nerve palsy, developmental delay, and failure of tooth eruption in infancy or early childhood consistent with osteopetrosis (opt). bone histology during childhood shows unresorbed primary spongiosa from deficient osteoclast action. additionally, there is remarkable progressive flattening of all vertebrae mimic ppi blocking mineralization. during ehdp treatment for gaci, in our patient prolonged high dose ehdp resulted in severe skeletal deformity resembling hypophosphatasia which was reversable with drug stoppage. methods & materials: a -year-old boy with gaci referred for profound, acquired, progressive skeletal deformity. he was receiving mg/day of ehdp and was wheelchair bound. we studied him and his response to stopping ehdp. results: skeletal radiographic findings resembled pediatric hypophosphatasia with pancranial synostosis, widened physes with metaphyseal osteosclerosis, "tongues" of radiolucency, along with cupping and fraying, and long-bone bowing. in addition there were large intra and extraarticular calcifications. radiographic features of bp-induced opt included femoral erlenmeyer flask deformity and osteosclerosis (lumbar sine dxa z-score + . ). biochemical parameters of mineral homeostasis were essentially normal although serum osteocalcin was low and he had markedly elevated serum levels of creatine kinase and trap- b consistent with osteopetrosis (opt). after stopping ehdp, he improved quickly with remarkable healing of his rachitic appearing skeleton and decreased joint calcifications. conclusions: our patient with gaci had profound skeletal deformities from high-dose ehdp therapy that significatly improved with drug stoppage. magnetic resonance imaging in the evaluation of infants with hypoxic ischemic encephalopathy julio m. araque, md, radiology, medical college of georgia, jaraque@georgiahealth.edu purpose or case report: to illustrate and review a spectrum of brain abnormalities of infants with hie. defining the most useful approaches and mri sequences, to facilitate identification and early diagnosis of lesions with the potential to predict outcome and abnormal neurodevelopment. methods & materials: reviewed available evidence on mri strategies for evaluating infants with hypoxic ischemic encephalopathy. different cases illustrating lesions are presented and discussed for proper diagnosis correlating physiopathology and imaging appearance. more relevant findings are depicted with didactic illustrations. identifying studies where new techniques such as dwi, adc, dti, swi, or mrs adds significant diagnostic value to the overall interpretation. results: mri is routinely performed as a very sensitive method for detection of hie lesions. advanced mr techniques, such as dti, dwi, adc, mrs, swi offer the possibility of detecting injuries at a time when intervention is theoretically possible. the understanding of the physiopathology allows for prediction of the location and extent of lesions, facilitating identification and appropriate classification. the identification of infants with potentially abnormal neurodevelopment, offers the opportunity to provide therapeutic neurodevelopmental interventions in early childhood. mrs is the best mr biomarker to predict neurodevelopmental outcome in asphyxiated full-term neonates. brain metabolite ratios and regional adc values may vary between mr systems and coils. development of normal values for each institution is required, and support of physicists is mandatory. conclusions: mri continues to evolve as a valuable adjunctive tool routinely obtained in nearly all cases of hie. advanced mri techniques increase sensitivity of conventional t and t -w images and outperform computer tomography and ultrasound for confirming the diagnosis of hypoxic-ischemic brain injury or providing prognostic information for the care of patient with hie. disclosure: dr. araque has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. posterior fossa abnormalities in children amit gupta, mbbs, radiodiagnosis, r.n.t. medical college, udaipur, rajasthan, india, amitsensation@yahoo.co.in purpose or case report: the aim of this exhibit is to demonstrate various conditions involving the posterior fossa in children with emphasis on importance of embryologic development of cerebellum in reaching a correct diagnosis. methods & materials: this pictographic presentation displays the imaging features of cases encountered in our clinical practice on . tesla magnetic resonance (mr) imaging. results: with the advent of mr imaging, there has been a revolution in identification and characterization of malformations of the brain this is especially true in posterior fossa, where the sensitivity and specificity of mr imaging with its multidimensional imaging capability are far superior to those of computed tomography (ct) in the detection of subtle morphologic abnormalities. however, there is still a great deal of confusion regarding their classification, terminology, and spectrum of expression and this is where neuroembryology is of great help. this exhibit demonstrates : ) review of embryology and normal anatomy of cerebellum. ) mr appearance of spectrum of conditions involving posterior fossa in children which includes developmental abnormalities (dandy-walker complex, arnold chiari malformations, cerebellar dysplasia/ hypoplasia, joubert's syndrome, etc.), cysts (arachnoid cyst, giant cisterna magna etc.), tumours (medulloblastoma, ependymoma, hemangioblastoma etc.) and miscellaneous conditions. significantly reduces dose ( / of other gadolinium based contrast agents), and doesn't require trigger imaging. conventional mri provides important information regarding the anatomical extent, size, and relation to critical anatomical structures thus when combined with twist, mri provides the best information without use of radiation in children. functional connectivity mri in pediatric brain tumor patients with and without epilepsy andrew v. poliakov, phd, radiology, seattle children's hospital; david bauer, edward novotny, seth d. friedman, dennis shaw, jeff ojemann purpose or case report: functional connectivity mri (fcmri) is a way to evaluate cortical networks across different modalities such as motor, sensory, vision, and the default mode network using functional magnetic resonance imaging. fcmri relies on correlation in fmri image intensity that occurs between functionally connected regions. this effect can be seen in awake as well as anesthetized patients. we evaluated these pathways in pediatric patients with brain tumors. methods & materials: patients were randomly selected from our tumor database. inclusion criteria included age less than , history of brain tumor resection, and complete fcmri data. imaging was performed on a t siemens trio system. functional mri data were acquired as part of a clinical imaging protocol over . - min using a gradient echo, echo-planar sequence. preprocessing of fmri data followed by independent component analysis (ica) was performed using fsl software. functional connectivity analysis was performed using software provided by functional connectomes project, based on afni and fsl software packages. correlation maps were produced by extracting the bold time course from a seed region, computing the correlation coefficient between that time course and the time course from all other brain voxels, correcting for multiple sampling and degrees of freedom and thresholded at a z value of . . results: fourteen patients were included in the study, eight female and six male. tumor types include ganglioglioma ( ), pleomorphic xanthoastrocytoma ( ), juvenile pilocytic astrocytoma ( ), ependymoma ( ), anaplastic astrocytoma ( ), glioblastoma multiforme ( ), and primitive neuroectodermal tumor ( ). seven patients had tumor-associated epilepsy, and seven patients did not. the figure shows connectivity patterns in the motor network in patients without (a) and with (b) epilepsy. in the patients without epilepsy, functional connectivity was often displaced but not decreased or absent. in the patients with epilepsy, we observed decreased or absent functional connectivity. similar results were found for default mode network: connectivity was diminished or absent in the patients affected by epilepsy. conclusions: fcmri is a novel technique that may prove useful for evaluation and presurgical planning by giving us insight into how tumors disrupt function. functional connectivity was often displaced but relatively preserved in the patients without epilepsy. it was disrupted or absent in the patients with epilepsy. poster #: sci- corpus callosum dti measurements in neurofibromatosis type and normal controls nadja kadom, md, radiology, children's national medical center, nkadom@childrensnational.org; amir noor, rhea udyavar, marine bouyssi-kobar, iordanis evangelou, maria t. acosta purpose or case report: many patients with neurofibromatosis type (nf ) have corpus callosum enlargement; pathogenesis and underlying pathophysiology are unclear. the goal of our study is to investigate the pathophysiological basis of corpus callosum enlargement in nf patients through mri diffusion tensor (dti) measurements. methods & materials: retrospective study, irb approved. patients consecutively selected from institutional data base; inclusion criteria: established diagnosis of nf , brain imaging with dti sequence, abnormally high corpus callosum to skull ratio; excluded were patients with complications of nf that could affect size of the corpus callosum. age and gender matched normal controls were randomly selected from the radiology data base. roi were placed manually over the corpus callosum for dti measurements using dti-studio by two independent researchers, one blinded to diagnosis. results: fifteen nf patients and matched controls were analyzed. the corpus callosum to skull ratio was found to be significantly different between the experimental and control group (p . ). for nf patients we found: a trend to lower apparent diffusion coefficient (adc, p . ), significantly higher radial diffusivity (p . ), significantly lower axial diffusivity (p . ), and significantly lower fractional anisotropy (fa, p . ). conclusions: the significantly lower axial diffusivity in nf can indicate that there are more crossing fibers in the corpus callosum of nf patients than in normal controls. further studies using comparative dti tractography may be helpful in further investigating this stipulation. the significant increase in radial diffusivity can be explained by a variety of factors, including thinner myelin sheaths, increased interstitial fluid, smaller axons, or a combination thereof. the trend of lower adc may indicate low axonal diameter, as adc has been shown to more strongly correlate with axonal diameter without the myelin sheath. in future studies we will correlate abnormal corpus callosum dti markers with cognitive functions in nf patients to see if relationships exist that can be used as predictors of cognitive deficits in nf patients. screening for vitamin d deficiency in children with suspected non-accidental fracture conor kain, md, tripler army medical center; veronica rooks, laura keller, jordan pinsker, allyson cordoni, sarah frioux purpose or case report: determine if routine screening of vitamin d levels after suspected non-accidental fracture detects vitamin d deficiency and changes clinical outcomes. methods & materials: after irb approval we reviewed all skeletal surveys performed at tripler army medical center (tamc) in the last years and selected the children who were evaluated for suspected non-accidental fracture. we determined if -hydroxyvitamin d [ (oh)d] level was requested for these patients and characterized the provider's clinical suspicion of vitamin d deficiency as high or low. per the institute of medicine report and endocrine society guidelines we defined vitamin d deficiency as a (oh)d level of less than ng/ml. we calculated the prevalence of children with low (oh)d levels whose providers had low clinical suspicion for vitamin d deficiency. results: skeletal surveys were done at tamc from november to july . were performed after identifying a suspected non-accidental fracture. of these patients children from ages to months had (oh)d levels requested. for children whose providers had a low pre-test suspicion for vitamin d deficiency, the prevalence of vitamin d deficiency was . % ( % binomial ci . - . , of cases. these results indicate that at least one out of every three hundred children evaluated for nonaccidental fracture could have vitamin d deficiency despite a low clinical suspicion by their provider, although the actual rate is likely much higher given that we found one in eight cases. the child we identified with a low vitamin d level whose provider had no suspicion for rickets was treated with ergocalciferol and continued to be evaluated for abuse. conclusions: routine vitamin d level screening after nonaccidental fracture may detect vitamin d deficiency in children for whom there is low clinical suspicion. as our population resides at a low latitude and receives greater than average sun exposure, the rate of deficiency in children with suspected non-accidental fracture may be much greater in other areas. comet tails and dirty shadows: the secrets behind artifacts in pediatric ultrasound adam edelstein, pediatric radiology, massachusetts general hospital; anuradha shenoy-bhangle, katherine nimkin purpose or case report: to review common ultrasonographic artifacts, explain what causes them, and show how they can be used to aid in diagnosis in a variety of pediatric conditions, including less common entities. methods & materials: ultrasonographic images in patients less than years of age were reviewed. cases were selected that showed classic artifacts which helped with the diagnosis of a variety of entities. results: ultrasound artifacts include comet tail, reverberation, ring down and "dirty" shadowing. these can be used to help characterize a variety of pediatric conditions including gossypiboma, bezoar, subcutaneous foreign body, complications of nec, and staghorn calculus. artifacts can also be used to confirm the presence of stool or bowel gas. conclusions: familiarity with ultrasonographic artifacts is critical for tissue characterization and can help narrow the differential diagnosis in difficult pediatric cases. cardiac cta: non-vascular ring tracheobronchial compression secondary to enlarged patent ductus arteriosus in infants with congenital heart disease. nhi huynh, md, radiology, st. joseph hospital and medical center, e.nhihuynh@gmail.com; todd chapman, randy richardson purpose or case report: tracheobronchial compression or narrowing secondary to a vascular ring has been well documented. the purpose of this study is to describe the frequency of airway compression secondary to an enlarged patent ductus arteriosus detected by ccta without the presence of a vascular ring. methods & materials: a retrospective study of ccta exams in infants was performed over the period between / / and / / . ccta was performed with a -slice mdct, with ekg gating, followed by three-dimensional reformations. results: of the congenital heart disease infant patients, there are patients with tracheobronchial compression or narrowing. of these patients, patients reported to have patent ductus arteriosus as the primary cause of tracheobronchial compression or narrowing. approximately % of patients with airway compression in patients with congenital heart disease are secondary to an enlarged and/or tortuous patent ductus arteriosus. none of these cases were due to a vascular ring. of these patients, , , and patients demonstrated to have mild, moderate, and severe airway compression respectively. conclusions: tracheobronchial compression or narrowing secondary to vascular ring with a patent ductus arteriosus has been well documented. in this study, we demonstrate that a significant percentage of airway compression in patients with congenital heart disease without a vascular ring is due to a tortuous enlarged patent ductus arteriosus. cardiac cta is uniquely equipped to evaluate airway compression due to an enlarged patent ductus arteriosus and can help improve patient care in congenital heart disease patients with respiratory symptomatology. pediatric liver mr elastography: a primer suraj serai, phd, cchmc, suraj.serai@cchmc.org; daniel j. podberesky, alexander j. towbin purpose or case report: a wide variety of pediatric liver disorders may be complicated by the development of liver fibrosis and ultimately cirrhosis. with early interventions, the progression to hepatic fibrosis can be slowed, halted, and in some cases reversed. liver biopsy has long been considered the gold standard for assessing the presence and degree of liver fibrosis. however, liver biopsy has disadvantages, due to its potential sampling error, risk of complications, relatively high cost, intra-and inter-observer variability, and, in general, poor acceptance by pediatric patients and their parents. mr elastography (mre) is a relatively new, non-invasive technique that provides a safe, rapid and cost-effective method for objectively evaluating of a wide variety of hepatic diseases by quantitative stiffness evaluation of the liver-parenchyma. the purpose of this exhibit is to review our clinical experience with this technique and illustrate the application of liver mre in the pediatric population at our medical center. methods & materials: a review of pathogenesis and staging of liver fibrosis in children and current methods available for assessing liver fibrosis will be provided. a review of mre physics and technique, including the specific liver mre protocol used at our institution will be illustrated. we will review widely-used and emerging clinical indications for liver mre, as well as benefits and limitations to the technique, supported by brief literature review. results: in addition to sharing our liver mre technique, we will illustrate clinical case examples from our institution of a variety of liver disorders including non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, storage disorders, cardiac disease, and idiopathic elevated liver enzymes. conclusions: this educational exhibit will review our experience with liver mre, a safe, newly available technique which will play an increasingly important role in the noninvasive evaluation of pediatric liver disease. poster #: sci- spectrum of tuberculosis in children amit gupta, mbbs, radiodiagnosis, r.n.t. medical college, udaipur, rajasthan, india, amitsensation@yahoo.co.in purpose or case report: the aim of this exhibit is to present a spectrum of tuberculosis (tb) in the human body which commonly involves pulmonary, nervous, musculoskeletal, gastrointestinal and genitourinary systems. this pictographic presentation displays the imaging features of tb cases encountered in our clinical practice with reference to plain x-rays, ct and mri as appropriate. results: with the advent of the newer modalities, the utility of the plain skaigram has been largely limited a initial screening tool only. whereas ct scores over mri in pulmonary tb (parenchymal disease, lymphadenopathy, pleural effusion, empyema, miliary disease) and abdominal tb (spectrum from mesenteric lymphadenitis to visceral involvement), the magnetic resonance (mr) imaging is much better in diagnosing cns tb (tuberculoma, abscess, meningitis, subdural empyema and myelitis). in musculoskeletal and genitourinary tb, ct and mr imaging may be preferred based on the stage of disease and the character of the lesion. cardiac involvement (pericarditis) is among the less common affections of tb. conclusions: tuberculosis is a multisystem disease that can affect virtually any part of the body from head to toe. tb demonstrates a variety of clinical and radiologic findings and has a known propensity for dissemination from its primary site and therefore can mimic numerous other disease entities. hence it is imperative for radiologists to understand the typical disease distribution, patterns and imaging manifestations of tb. janet l. strife, md carol m. rumack md ole a. eklof, md clement c. faure, md andres giedion, md denis lallemand, md arnold lassrich md donald r. kirks, md beverly p. wood, md hooshang taybi md marta hernanz-schulman, md, facr m. ines boechat, md, facr neil d. johnson, mbbs dorothy i. bulas, md *deceased singleton-taybi award investigator award this award is given to the author of the best paper presented by a resident or fellow at the spr meeting md ricardo faingold, md andrea doria, md nina m. menezes, phd anthropometric parameters are a strong determinant of lvm in healthy individuals kiyarash mohajer, pierangelo renella, paul j. finn purpose or case report: despite theoretical advantages of higher field strength ssfp cine imaging, time-resolved magnetic resonance angiography (tr-mra), and high resolution contrast-enhanced mra (ce-mra) were performed. two readers independently evaluated the data for image quality, vessel and cardiac chamber definition, and presence of artifacts. snr and cnr were calculated. results: % of ssfp cine images at t were rated as good or excellent quality with % having mild and % having moderate artifacts (k . ) % of arterial and venous phase ce-mra images were considered good or excellent cardiac chamber definition was considered good or excellent in % of arterial and venous phase ce-mra images (k . ). % ce-mra images showed good or excellent definition of the thoraco-abdominal vessels on average, both readers scored cine ssfp images higher at . t and cemra images higher at . t. overall diagnostic performance was high at both field strengths. conclusions: mri of pediatric patients with chd and vascular abnormalities at . t is feasible. relative to . t, snr and cnr are both improved at higher field strength and higher resolution cemra is achievable t are more prevalent, they rarely render cine imaging non-poster # exclusion criteria were lack of correlating us or follow up information. two pediatric radiologists blinded to us findings reviewed the mr images and analyzed the contents of abdominal wall defect, organ location and attachment; spine anomalies; umbilical cord and limb anomalies. results: our search yielded patients. all fetuses had ventral wall defects, small thorax and eviscerated liver and bowel. in two cases kidneys were in extracorporeal location. in / there was no membrane covering extruded organs. in five mr showed organs attached to the placenta or uterine wall (mainly bowel and liver) mahmoud al-hawary and, by adolescence, paradoxical metaphyseal osteopenia with thin cortical bone. reports of consanguinity indicate autosomal recessive inheritance our studies, spanning ages - mo, showed weight %, but length diminishing from~ % to − . sd. head circumference was + sd. she had frontal bossing, blue sclera, normal teeth, genu valgum, and unremarkable joints. radiographs showed orbital and facial sclerosis, basilar thickening, "bone-in-bone" appearance in the pelvis, sclerotic long bone ends, and fractures of ribs and extremities. progressive metaphyseal widening occurred as vertebrae changed from ovoid to flattened and became beaked anteriorly. consistent with opt, serum pth concentrations reflected dietary calcium levels. serum bone alkaline phosphatase, osteocalcin, and trap b were sub-normal. iliac crest contained excessive primary spongiosa and no osteoclasts. splice sites and exons were intact for the genes encoding cholride channel , t-cell immune regulator , opt-associated transmembrane protein the hallmarks include stippled epiphyses, nasal hypoplasia, and hypoplastic distal phalanges and developmental delay. punctate calcifications are seen not only in the epiphyses but also in the paravertebral regions. paravertebral puncta are commonly associated with defective ossifications in the cervical spine. the malformation of the cervical spine causes spinal canal stenosis and instability, which occasionally necessitate surgical intervention none of the cases had brain infarction. conclusions: tortuousity and luminal narrowing of the cervical arteries is a common finding in cdp-bt. this previously unknown malformation is an important factor to discriminate patients at increased risk of cerebral ischemia, particularly in patients undergoing surgical intervention. disclosure: dr. okabe has indicated that she will discuss or describe severe skeletal toxicity from protracted etidronate therapy for generalized arterial calcification of infancy william h. mcalister, md, mallinckrodt institute of radiology campbell sheen purpose or case report: generalized arterial calcification of infancy (gaci) is an autosomal recessive disorder caused by deactivating mutations within the gene for ectonucleotide pyrophosphatase phosphodiesterase- (enpp ). enpp on osteoblasts, chondrocytes, and vascular smooth muscle cells hydrolyzes nucleotide triphosphates to nucleotide monophosphates and inorganic pyrophosphate (ppi) can time-resolved contrast-enhanced mra (twist) classify soft tissue vascular anomalies in the head and neck in children accurately? aylin tekes, md children from - years of age were enrolled. twist and conventional mri was performed (triplanar t -weighted [t -w] imaging with fat saturation, pre-contrast axial t -weighted [t -w] imaging, and post contrast triplanar fat-suppressed t -w imaging). twist was performed in coronal plane using blood-pool mr contrast agent (ablavar-lantheus) to enhance image quality and spatial resolution of mra. two pediatric neuroradiologists evaluated all patients in two different sessions, days apart: one session conventional mri with contrast was evaluated, in the second session twist was evaluated. clinical evaluation and/or percutaneous venogram/lymphogram data were the gold standard. results: our patients had diagnosis of infantile hemangioma (n ), venous malformation (n ), and lymphatic malformation (n ). twist alone could accurately classify / , conventional mri with contrast could accurately classify / . conventional mri with contrast combined with twist could accurately classify all cases. conclusions: twist offers high temporal resolution in the order of seconds, and provides functional data about the dynamics of contrast enhancement comprising the arterial, venous and delayed venous phases kiery cr- , sci- edu- , edu- edu- , edu- , pa- edu- , pa- a- , pa- , pa- , pa- , pa- edu- , edu- , edu- , edu- , edu- , edu- , edu- edu- , edu- , edu- , edu- , edu- , edu- suraj sci- , pa- , pa- edu- pa- , edu- , pa- the society for pediatric radiology gratefully acknowledges the support of the following companies in presenting the th annual meeting and postgraduate course: cme committee reviewers for this activity have disclosed any relevant financial relationships. no conflicts of interest exist.abuse and offer potential mechanisms of injury may help make the diagnosis of child abuse. the pediatric elbow-mri findings with multimodality correlation michael guandalini, md, royal children's hospital; murray bartlett purpose or case report: to describe and illustrate elbow abnormalities identified by mri performed in a cohort of pediatric patients with multimodality correlation. methods & materials: retrospective review of mri elbow studies performed at the royal children's hospital, melbourne between and . the studies were reviewed by a pediatric musculoskeletal radiologist and pediatric radiology fellow with patient demographics, clinical indication, findings and selected images recorded. results: elbow mri examinations were reviewed on children aged months to years ( boys, girls) with equal numbers of left and right sides examined. clinical indications included previous trauma in cases ( %) and nontraumatic conditions in ( %). the most common traumatic indication was suspected or confirmed fractures or avulsions ( %) followed by osteochondral or cartilage injuries ( %), growth arrest ( %), loose bodies ( %) and ligament injuries ( %). hemophilia ( %) was the most frequent nontraumatic indication followed by neoplasm ( %). mild to severe arthropathy, fractures, physeal growth arrest, subluxations, osteochondral lesions and loose bodies were the most frequently demonstrated abnormalities. ligament strains and tears, bone oedema, neuromuscular abnormalities, infections and several neoplasms including lipomas, vascular/lymphatic malformations and bone tumors also featured. conclusions: this pictorial review illustrates the broad range of abnormalities one might expect to encounter on pediatric elbow mri studies, highlighting the major features and corresponding appearances on ct and plain x-ray. spectrum of patellar tendon avulsive injury on mri in children: differentiation between acute and chronic avulsive injuries of the inferior patellar pole and tibial tuberosity zeyad metwalli, md, baylor college of medicine, metwalli@ bcm.edu; herman kan, scott rosenfeld, r. p. guillerman purpose or case report: the extensor mechanism of the knee is an intricate component of the joint and is frequently injured in pediatric athletes. due to the strength of the patella tendon, trauma to the anterior knee is often manifested by avulsive injuries, which may occur on an acute or chronic repetitive basis. purpose: this pictorial review will illustrate differentiating radiographic and mri features of acute and chronic avulsive injuries of the pediatric knee. outline: . anatomy and physiology a. discuss the anatomic differences of the pediatric and adult knee extensor mechanism b. pathophysiology and biomechanical basis for chondro-osseous avulsion injuries versus tendon tears in the skeletally immature. purpose or case report: the purpose of this educational exhibit is to demonstrate the magnetic resonance imaging (mri) appearance of the ankle and hindfoot ligaments using an interactive approach. methods & materials: a tesla siemens mri scanner with a multichannel ankle coil was utilized in the acquisition of images of ankle and hindfoot. three dimensional volume acquisition proton density images will be used to demonstrate the ligamentous anatomy of the ankle and hindfoot in axial, axial oblique, coronal, and sagittal planes. results: the exhibit will begin with an interactive review of the ankle and hindfoot ligamentous anatomy with each ligament poster #: edu- cns imaging findings in hemophagocytic lymphohistiocytic syndrome rupa radhakrishnan, mbbs, md, dnb, radiology, university of cincinnati college of medicine, radhakrp@ucmail. uc.edu; marcia k. kukreja, alexandra filipovich, alexander j. towbin purpose or case report: hemophagocytic lymphohistiocytosis (hlh) is a rare, life threatening condition caused by an uncontrolled proliferation of activated lymphocytes and histiocytes with high levels of inflammatory cytokines. the organs most commonly involved in this disorder include the liver, spleen, lymph nodes, bone marrow and central nervous system (cns). the purpose of this exhibit is to review the cns imaging findings associated with hlh, its complications, and its management. the published literature was reviewed to identify the potential imaging findings hlh. the electronic medical record system was then searched to find illustrative case examples from our institution. cases demonstrating the primary imaging findings as well cases highlighting complications of the disease or its therapy were selected. results: cns involvement is common in hlh with approximately % of patients demonstrating neurological symptoms. ct findings of cns involvement include diffuse parenchymal atrophy, low attenuation lesions in the white matter and calcifications. mr findings include diffuse leptomeningeal and perivascular enhancement, t hyperintense lesions with nodular or rim enhancement as well as confluent white matter lesions, and diffuse parenchymal volume loss of the cerebrum and cerebellum. restricted diffusion has been demonstrated in some lesions. ring enhancing parenchymal lesions have been described representing active demyelination. intracranial hemorrhage may occur as a result of thrombocytopenia and coagulation abnormalities. sepsis with opportunistic organisms can involve the cns and produce intracranial findings such as parenchymal abscesses. cns changes, such as posterior reversible encephalopathy syndrome, are also seen with the commonly used immunomodulatory regimen used in the treatment of hlh. conclusions: this exhibit will aid the viewer in identifying the cns imaging findings of hlh as well as the complications of the disease and its therapy. while the cns imaging findings are not specific, they may help the radiologist formulate a diagnosis in association with the other clinical and imaging findings; furthermore, imaging can help the clinical team in managing the disease and its complications. methods & materials: medical records of our pediatric patients with palpable head masses over the last years, were reviewed and images were collected. correlation of us of these lesions with other imaging modalities and/or pathologic diagnosis was done. results: us appearances of various head masses including congenital/developmental (encephalocele, meningocele, dermoid, occipital protuberance), traumatic (cephalhematoma, subgaleal hematoma, calvarial fracture), inflammatory/infectious (sebaceous cyst, histiocytosis, dermatitis), vascular (malformations, pseudoaneurysm) and neoplastic (benign and malignant lesions including metastases) etiologies, will be illustrated with case based approach. mri and/or ct or tissue diagnosis can be problem solving. role of ultrasound guidance for percutaneous procedures (biopsy, sclerotherapy) will also be described. conclusions: ultrasound can play an important role in the delineation, diagnosis and guiding further management of pediatric palpable head masses. us can differentiate various scalp lesions and suggest the underlying calvarial defect or involvement to some extent, helping to narrow the differential diagnosis for such lesions. color doppler us can be useful to detect vascularity within the lesion or vascular lesions. given that us is often requested for the evaluation of palpable head masses, pediatric radiologists should be familiar with their sonographic features. posterior fossa malformations-a pictorial review rui santos, md, bc children's hospital, ruiradiologia@gmail. com; khalid khashoggi, angela t. byrne purpose or case report: posterior fossa malformations are a group of central nervous system anomalies that may be detected during pregnancy or present early infancy with features that include hypotonia, developmental delay, mutations responsible for the disease and proposals as to mechanism of action of the mutation with respect to disease manifestations. this preceded the development of hypotheses regarding the relationship between genotype and phenotype and the attempt to utilize imaging modalities that could better assess disease activity as it related to functional status. the purpose of this exhibit is to briefly review the history pre- and to focus on the numerous ways in which the understanding has improved since that time. conclusions: . there are over different mutations of the cftr gene responsible for cystic fibrosis with varying prevalence throughout the world. . the class of mutation often dictates its particular mechanism of action. . there is some relationship between genotype and phenotype-particularly with respect to pancreatic involvement. . newer imaging modalities including ct and mri with or without hyperpolarized helium are better predictors of disease severity than is plain film. imaging pulmonary tuberculosis in infants: what are the most useful diagnostic radiological findings? handan cakmakci, pediatric radiology, dokuz eylul university hospital, handancakmakci@gmail.com; nevin uzuner, filiz tetik purpose or case report: early diagnosis and treatment are very important for infants with tuberculosis. infantile pulmonary tuberculosis is more symptomatic, and the risk of severe and life-threatening complications such as tuberculous meningitis or miliary tuberculosis is higher. bacteriologic confirmation of the disease in children is difficult and in younger infants (< months), the tuberculin skin test is frequently negative. therefore, radiological findings play important role in diagnosing tuberculosis in infants. the purposes of this study are to identify chest x-ray and lung ct findings in pulmonary tuberculosis of infants and consider the most useful diagnostic findings of these age group patients.methods & materials: chest radiographs and chest ct images of infants who were diagnosed in our hospital from to were retrospectively reviewed. the study group included boys and girls ranging in age from to months (mean age, months). chest x-ray and computed tomography images were analyzed considering air space consolidation, nodular lesions, cavitating lesions, mediastinal enlargement, hyperinflation, bronchial narrowing, atelectasis pleural effusion on plain radiography and additional mediastinal calcific or caseating lymph nodes on ct images.results: air space consolidation was seen on out of chest x-ray and computed tomography images. nodular lesions were seen out of chest x-ray and computed tomography images. cavitating lesion was seen on out of chest x-ray and computed tomography images. mediastinal enlargement suggesting lymph node was seen out of chest x-ray and computed tomography images. hyperinflation, bronchial narrowing was seen out of chest x-ray and computed tomography images. atelectasis, pleural effusion was seen out of chest x-ray and out of computed tomography images. mediastinal caseating lymph nodes, mediastinal calcific lymph nodes were seen out of computed tomography images. conclusions: frequent and the most useful diagnostic radiological findings of pulmonary tuberculosis in infants are mediastinal or hilar lymphadenopathy with central necrosis and air space consolidations. disseminated nodules including miliary lesions and airway complications are also detected in this age group. ct can show detailed parenchymal lesions and tuberculous lymphnodes especially calcified ones. the ductus bump: radiographic findings of this normal variant and differential diagnoses anusuya mokashi, staten island university hospital, anusuya.mokashi@gmail.com; jeremy neuman, cheryl lin purpose or case report: the ductus bump: review of radiographic findings, differential diagnoses and current controversies. the ductus bump was first described in by berdon et al as a transient physiologic mass in the chest in newborn infants. some controversy remains as to the exact etiology and clinical significance. although initially thought to represent a dilated ductus arteriosus, recently it has been suggested that it actually represents a ductus arteriosus aneurysm that spontaneously resolves. others contend it represents dilation of the infundibulum of the closing ductus. regardless of etiology, the time of discovery, location, and rapid resolution are characteristic of this entity. in this presentation we will review the radiographic and echocardiogram findings of the ductus bump, as well as discuss the differential diagnosis. the frontal radiographic findings are a round mass to the left of the vertebral spine projecting from the mediastinum near the aortic arch. this mass does not indent the esophagus and it cannot be seen on the lateral view. it is classically said to resolve within the first few days of life. the controversy regarding the etiology has also led to some disagreement involving the clinical significance and appropriate follow up, which will also be discussed. after reviewing this educational poster, the reader will have conclusions: abnormalities of the posterior fossa are often difficult to differentiate solely on the basis of their radiologic appearances alone. however, an accurate diagnosis is essential for proper treatment planning and genetic counselling. therefore it is imperative for radiologists to be well versed with the normal anatomy and development of cerebellum so as to correctly diagnose the various posterior fossa abnormalities.poster #: sci- imaging of oculoauriculofrontonasal syndrome with low-dose -dimensional computed tomography paritosh c. khanna, md, radiology, seattle children's hospital, pkhanna@uw.edu; kelly evans, gisele ishak, joseph gruss, michael cunningham, anne hing purpose or case report: oculoauriculofrontonasal syndrome (oafns) combines elements of abnormal morphology of the frontonasal and maxillary processes of the face. the aim of our exhibit is to demonstrate the low-dose computed tomography (ct) features of this syndrome, in seven patients who have been followed at seattle children's hospital (sch) over years. we underscore the imaging features of this condition, and describe additional features including bony nasal abnormalities not previously described in the literature, to improve imaging recognition of this spectrum. we present d ct imaging features of a series of eight patients with oafns. in keeping with the alara (as low as reasonably achievable) concept and the image gently recommendations (www.imagegently. org), ct head and face studies were obtained on six of eight patients at sch, while two had prior exams at outside institutions. using a -slice multidetector ct scanner (ge lightspeed vct, waukesha wi), low-dose ct ( kv, mas or lower depending on age) of the head and face was obtained. planar bone window and d surface rendered images were analyzed. results: our series of patients demonstrated bifid nasal bones, uni-or bilateral mandibular hypoplasia, temporomandibular and zygomatic dysplasia and bony external auditory canal abnormalities. one patient had an interfrontal bone with a frontal bony defect that was contiguous with the metopic suture. we describe additional previously unidentified ct anomalies of the nasal bones, anterior nasal spine and nasal septum. these structures are involved in all patients who had ct imaging available, although unique features are present in each case. conclusions: ct is the mainstay of imaging of craniofacial anomalies in the post-natal period, both pre-and postoperatively. in addition to our low-dose ct imaging findings of oafns, novel nasal bone anomalies identified by our group serve to identify a new subset of patients with this syndrome and may help refine the phenotype of the oafns spectrum. key: cord- - wdwrttn authors: li, taisheng title: diagnosis and clinical management of severe acute respiratory syndrome coronavirus (sars-cov- ) infection: an operational recommendation of peking union medical college hospital (v . ): working group of novel coronavirus, peking union medical college hospital date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: wdwrttn since december , china has been experiencing an outbreak of a new infectious disease caused by severe acute respiratory syndrome coronavirus (sars-cov- ). the clinical features include fever, coughing, shortness of breath, and inflammatory lung infiltration. china rapidly listed sars-cov- -related pneumonia as a statutory infectious disease. to standardize the diagnosis and treatment of this new infectious disease, an operational recommendation for the diagnosis and management of sars-cov- infection is developed by peking union medical college hospital. severe acute respiratory syndrome coronavirus (sars-cov- ) is a novel type of coronavirus of β genus that leads to an emerging infectious disease with remarkable pulmonary involvement in china since december . the clinical features include fever, dry cough, shortness of breath, normal or low levels of peripheral white blood cells, and inflammatory changes on chest x-ray. china has designated sars-cov- infected pneumonia as a statutory infectious disease. to standardize the clinical diagnosis and treatment, peking union medical college hospital (pumch) has established a working group and formulated the following operational recommendation regarding "diagnosis and clinical management of severe acute respiratory syndrome coronavirus infection" (v . ). selection of front-line personnel front-line medical personnel are qualified only after passing the physical examinations and professional training of sars-cov- . staff with the following conditions are exempt from sars-cov- related clinical/ laboratory work, including pregnancy, age over years old, a past history of chronic diseases such as chronic hepatitis, renal diseases, diabetes mellitus, autoimmune diseases and tumours. individuals affected with acute fever should also be excluded from sars-cov- related work. baseline tests should be arranged including the complete blood count, urine analysis, biochemical tests, creatine kinase, and chest x-ray. see "the guidelines for the diagnosis and treatment of severe acute respiratory syndrome coronavirus (sars-cov- )infection (pilot rd version)" published by the national health commission of the people's republic of china [ ] . isolation and observation of medical personnel after close contact with sars-cov- ( ) medical personnel in close contact with sars-cov- infected pneumonia patients should be relatively isolated, avoiding walking around and extensive contact with others. ( ) medical personnel should be isolated immediately and receive relevant examinations upon onset of fever, cough, shortness of breath and other symptoms. ( ) when work in the sars-cov- infection ward is finished, nasopharyngeal or oropharyngeal swabs for sars-cov- and a complete blood count should be carried out. those who have abnormal test results should undergo strict isolation and observation; while others will be generally isolated for observation and resume work after one week. screening criteria [ ] [ ] ( ) epidemiological history: history of travel or residence in hubei province within weeks prior to the onset of illness, or contact with patients from hubei province with fever and respiratory symptoms within days prior to onset, or presented with clustering onset. ( ) acute onset of fever within h without influenzalike symptoms, which could not attribute to other confirmed etiology. ( ) supportive epidemiological history ( ) clinical manifestation: fever; normal or low levels of white blood cells or decreased lymphocyte counts at onset. chest radiology at early stage is characteristic of multiple small patchy shadows and interstitial changes, more prominent in the extrapulmonary bands. multiple ground-glass opacities and infiltrations may develop bilaterally with disease progression, with possible consolidation in severe cases. hours) and normal chest imaging, if the absolute count of peripheral lymphocytes is less than . × /l, or the count of cd + and cd + t cells decreases significantly, isolation and close observation should be conducted at home even if the first sars-cov- nucleic acid test is negative. repeat of rrt-pcr should be considered after h, and a chest ct scan should be performed when necessary. screening cases on the day of visit nucleic acid examination of sputum or naso-/oropharyngeal swabs, complete blood count, urine analysis, arterial blood gas analysis, liver and kidney function, c-reactive protein (crp), procalcitonin, creatine kinase plus myoglobin, coagulation, and chest ct should be performed. inflammatory cytokines [such as interleukin (il)- , il- , and tumour necrosis factor (tnf)-α], tb lymphocyte subsets, and complement can be tested as appropriate [ ] [ ] [ ] . ( ) complete blood count, liver and kidney function, creatine kinase and myoglobin, coagulation function and crp can be checked on the rd, th and th days after admission and on discharge according to the disease status. pct and tb lymphocyte subsets can be repeated on days - if feasible [ ] [ ] [ ] . ( ) the chest x-ray or ct scan should be re-examined - days after the admission, and the time for subsequent re-examination depends on the disease status, no longer than days. ( ) complete blood count, chest x-ray, liver and kidney function, and all abnormal examinations on admission should be re-examined before discharge except for referrals. place of treatment according to the severity of the disease all cases with screening indications are subject to onsite medical isolation (single-room isolation), and once diagnosed, should be transferred to a designated hospital. according to the definition of the national health commission [ ] , patients in accordance with one of the following standards should be hospitalized and transferred to beijing designated medical institution as soon as possible; ( ) respiratory rate increased (≥ per min) or dyspnoea; ( ) oxygen saturation ≤ % when breathing ambient air, or arterial oxygen tension (pao₂) over inspiratory oxygen fraction (fio₂) of less than mm hg ( mm hg equals to . kpa); ( ) lung imaging indicating multilobular lesions or progression of lesions over % within h; ( ) quick sequential organ failure assessment (qsofa) score ≥ ; ( ) community-acquired pneumonia- (curb- ) score ≥ ; ( ) combined pneumothorax; ( ) other clinical conditions that require hospitalization. according to the definition of the national health commission [ ] , patients in accordance with respiratory failure, septic shock or other organ failure should be transferred to intensive care unit immediately and to designated medical institution as soon as possible when feasible. patients should be kept in bed and closely monitored for vital signs and levels of oxygen saturation. supportive treatment should be ensured, including enough supply of energy and fluid, maintenance of electrolyte and acid-base homeostasis. oxygen therapy patients with hypoxemia should be given oxygen therapy immediately and maintain a blood oxygen saturation level to no less than % in man and non-pregnant women, and between % and % in pregnant women. choice of oxygen therapy. patients with mild hypoxemia should be put on nasal cannula, l/min. if the patient is getting worse, high flow nasal cannula should be considered, starting with l/min and increasing to - l/min gradually. fraction of oxygen should be adjusted according to oxygen saturation. the way of respiratory support. non-invasive ventilation is only considered for patients who can tolerate. for the patients requiring invasive ventilation, endotracheal intubation should be performed by experienced physician with personal protective equipment. we recommend protective ventilation strategy for acute respiratory distress syndrome (ards). for those patients with most severe ards, extracorporeal membrane oxygenation or prone position is recommended. interventions should be implemented to prevent complications associated with critical illness. standard precautions should always be routinely applied in all areas of healthcare facilities. currently, there is no evidence to support the effectiveness of existing antiviral drugs against sars-cov- . lopinavir/ritonavir can be used when appropriate, tablets, twice daily for days. severe patients could receive glucocorticoid at early stage, e.g. intravenous methylprednisolone - mg, once daily for days, and the course of treatment can be prolonged according to the clinical condition and radiological manifestations. early intravenous infusion of human immunoglobulin is recommended for critically ill patients, based on their clinical condition, at . - . g/(kg·d), for - days [ ] [ ] . if bacterial infection is suspected according to the patient's clinical and imaging findings, patients of mild type can take oral antibiotics for communityacquired pneumonia, such as second-generation cephalosporins or fluoroquinolones. as regards patients of severe type, all possible pathogens should be covered when necessary. ( ) once the critically ill patients are diagnosed and endotracheal intubation is anticipated, they should be immediately transferred to the icu ward with negative pressure. all the procedures should be carried out in accordance with the requirements of comprehensive protection. ( ) oxygen storage mask is used to supply oxygen above l/min during transport, during which complete filling of the oxygen storage airbag should be ensured. ( ) endotracheal intubation should be induced by standard rapid procedure, and muscle relaxants should be used as much as possible to avoid droplet transmission caused by choking. ( ) reusable items such as goggles should be disinfected after intubation before taken out of the negative pressure ward. ( ) patients with intubation should use closed endotracheal suction to avoid airborne transmission caused by ventilator airflow. ( ) under certain circumstances when the ventilator must be disconnected for operation, the standby function of the ventilator should be set to avoid airborne transmission caused by ventilator airflow. once the standby function is not available, the y-tube port of the ventilator should be blocked to avoid air spread. criteria of isolation release and discharge [ ] patients could be discharged or transferred to the other departments for comorbidities, when the body temperature returned to normal for more than days, the respiratory symptoms significantly improved, the pulmonary lesions markedly absorbed, and respiratory nucleic acid is tested negative for sars-cov- for two consecutive times at least day apart. wrote by department of infectious diseases, peking union medical college hospital) department of infectious diseases, peking union medical college hospital) department of internal medicine icu, peking union medical college hospital) hongwei fan (department of infectious diseases, peking union medical college hospital) experts participating in the discussion (sorted by family name of chinese phonetic alphabet) wenzhao chai national health commission. the guidelines for the diagnosis and treatment of severe acute respiratory syndrome coronavirus (sars-cov- )infection (pilot rd version clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance rapid loss of both cd + and cd + t lymphocyte subsets during the acute phase of severe acute respiratory syndrome the alterations of t cell subsets of severe acute respiratory syndrome during acute phase characteristics and prognostic value of peripheral blood t lymphocyte subsets in patients with severe influenza prominent plasmacytosis following intravenous immunoglobulin correlates with clinical improvement in guillain-barré syndrome use of intravenous immunoglobulins for prophylaxis or treatment of infectious diseases no potential conflict of interest was reported by the author(s). key: cord- - r gh authors: tabatabai, shima title: covid- impact and virtual medical education date: - - journal: j adv med educ prof doi: . /jamp. . . sha: doc_id: cord_uid: r gh the corona-virus disease (covid- ) pandemic has had a tremendous effect on medical education. it is also challenging the medical educationists' ability to adapt to this whole unique situation. considering the hospital-based education, clinical mentors, and students in all health professions are potential carriers. however, the current crisis is revitalizing the necessity for online learning opportunities and virtual education. most medical schools are following reacting to lockdown with a shift to live online or video-based learning. maintaining standard in medical education, keeping the clinical learning on stream, and minimizing the assessment disruption are unprecedented challenges under pandemic conditions. adaptation to this new situation is necessary to prepare future clinicians for practice. this commentary discusses how this pandemic may affect medical education. in this commentary, the author highlights the importance of virtual education and the potential implications of integrating virtual simulation technologies into medical education for the future of clinical competency learning and assessment. outbreak, as a rapidly evolving situation, has influenced all members of the community and has caused tremendous disruption in all life aspects, not least in the health professions who provide front-line care for patients. currently, the ongoing pandemic has a tremendous effect on medical education. it is also challenging medical educationists' ability to adapt to this whole unique situation. considering the hospital-based education, clinical mentors, and students in all health professions, frequently working in the most difficult circumstances are potential victims and carriers for the virus ( ) . in the state of emergency, medical educators as role models are facing major challenges to train as the next generation of doctors ( ) . we have to respond to the immediate concerns among medical students, to channel our student's energy from panic to resolve this problem and to apply our energy to what we know we can do about medical education based on the lessons learned during pandemics. universities across the world have reacted quickly to the crisis, having announced immediate closures. medical schools are enacting emergency contingencies that allow their student populations to complete the semester remotely ( ). in addition, the current ongoing crisis is revitalizing the necessity for virtual learning opportunities. most medical schools are preparing to transform the medical education from real classes to the virtual education to minimize the teaching and assessment disruption ( ) . the transformative drivers are associated with the pandemic length of time. they are likely to interact with other factors such as types of available resources and emergent technology. virtual education refers to instruction in a learning environment where educators and students are separated by time or space, or both, and the instructors provide course content through course management applications, multimedia resources, the internet, videoconferencing, etc. students receive the content and communicate with the teacher via the same technologies ( ) . since the perspective of medical education after the covid- pandemic will transform ( ), it is necessary to transform the medical education through the following suggested steps: . determining medical education priorities during the pandemic: as the covid- outbreak has rapidly transitioned, following the social distancing practices, keeping learners safe, supported and engaged, and helping keep education programs moving forward with high quality, are the medical education systems' priorities. . assessing available resources: digital learning management systems and massive open online courses offer free online courses and classes aimed at the professional development of educators. in addition, there are also many free tools enabling video communications, video and audio conferencing, chats, and webinars for medical educators such as zoom, skype, and google hangouts meet ( , ) . these online communication tools support collaboration and the sharing of expertise between universities and providers. in iran, most universities of medical sciences have created a platform for the provision of virtual education. regarding authentic medical education, iran's virtual university of medical sciences (vums) has provided resources and educational content to help medical schools all around the country. at vums, the national learning management system was established to provide an electronic platform for the medical sciences education. this learning management system (lms), like comparable lmss, was intended to help instructors reflect on medical sciences students' processes of learning as well as meeting the needs of individual medical sciences students. some of its major facilities include course content repository, management of users, courses, instructors, and facilities, and generation of reports, course calendar, learning path, and discussion forum where members could provide their own contributions, prepare exercises, assign deadlines to and correct them, display scores and transcripts, etc. besides, the national virtual university of medical sciences is providing what are known as moocs -massive open online courses-in partnership with leading universities of medical sciences in iran. under the current circumstances, the educational contents are produced by the professors of different universities to provide integrated authentic learning experiences for iranian medical students. . determining challenges and related solutions: this outbreak widens the gap between those able to access online learning opportunities and those who are not. many students suffer a type of unequal access whereby they lack the digital connections ( ). maintaining performance standards and quality assurance are unprecedented challenges under pandemic conditions. as the covid- pandemic continues to develop, it could decrease the medical students' and residents' exposure to specific clinical conditions, and causes a significant effect on their clinical clerkship evaluation and competency assessment exams ( , ) . keeping the students engaged and on track with curriculum-driven coursework, even though they're learning remotely, are unprecedented challenges under pandemic conditions. hd-quality mobile or fixedcamera live streaming to establish a standardized, classroom-like experience in real-time improves interaction and ensures that the remote learners can participate at the same baseline ( , ). the latest innovations in flexible educational technologies will change the future of medical education ( ) . providing engaged and high-quality medical education will be guaranteed through virtual medical universities and simulation-based solutions for healthcare training environments ranging from clinical simulation management software and hardware to counselor education, case development, and virtual patient training ( , ) . virtual learning and education require careful thinking about how educators are equipped for the shift. the long-term impact of the current pandemic on medical education could be considerable. to maintain high-quality medical education, administrators and educators forced to look for innovative technologies. medical educationists will need to use emergent technologies which impact on the future way that their institutions will provide medical education ( ) . virtual reality is the use of software to create a simulated environment where educators can engage in virtual situations in a way that feels real ( ) . simulation is a vital part of medical education and students just don't get to do it enough. virtual reality simulation (vrs) has the unique ability to make learners believe they are in a different environment. this allows medical students to learn from virtual simulated clinical experiences as they would do in hospital-based real experiences. virtual simulation-based medical education has been found to be 'superior to traditional clinical education ( , ) . integrating vrs into medical education has enabled medical educationists in providing the simulated clinical experiences to a greater number of medical students in a shorter time. vrs is used in postgraduate medical education by the university of northampton and oxford university hospitals. vrss have also been used across england's health education and healthcare systems since ( , ) . the future of medical education lies in technological developments and the ongoing integration of virtual clinical simulation experiences into medical curriculum. as pressure to increase delivery of simulation continues, vr simulation will continue to expand ( ) ( ) ( ) . a number of institutions are also investigating vrs from the standpoint of objective structured clinical examinations (osces), as a method of decreasing the cost and increasing the objectivity of their assessment processes ( ) . it is essential to ensure medical students' safety and on-time evaluation during this pandemic crisis. the universities of medical sciences need to invest in individualized learning for competency-based education and in the technologies necessary for virtual assessments of student clinical competency ( , ) . virtual simulation-based education products prepare the necessary tools to provide benchmarking and best practice insights to medical students, better preparing them for realworld practice and these tools are set to expand over the coming years ( , , ) . simulationbased educational technologies can be effective for the transformation of clinical education, even the most complicate traditional-to-virtual universities, in the following ways: the simulation-based educational technologies allows medical education to quickly move to a virtual environment ( , ) . these technologies schedule simulated meetings, facilitate virtual education with video conferencing and reviewing clinical decision making through simulated clinical experiences. the vrs changes traditionally timeconsuming manual processes to create an integrated approach that improves clinical outcomes, and saves time. the vrs platforms allow learners to access the clinical scenarios via the computer or the mobile device regardless of their location, improve educational task flows, and customizes remote learning parameters specific to the program requirements. by simulation-based platform, clinical educators could conduct high-impact, casebased simulated training scenarios online and run live, virtual objective structured clinical examinations (osces) from anywhere ( , ) . simulation-based virtual osce could offer the perfect cloud-based platform to lead standardized patient confronts in a completely virtual, videoenabled environment to address all clinical education needs ( , ) . therefore, the virtual simulation platform helps medical educationists to maximize the learning potential of osces. in addition, simulation-based virtual osce facilitates fostering core competency for clinical decision making to transform clinical practice into telemedicine forms in the context of virtual medical education. we have to adapt to the new situation to prepare future clinicians for practice under pandemic conditions. educational methods are changing and new domains of medical sciences and technological innovations, as well as educational technologies, are growing. today medical sciences students are digital natives and rely more and more on virtual education and virtual reality simulated experiences. virtual reality simulation products are the bases for simulation-based virtual education that are urgently needed by many medical education programs. simulationbased virtual education has the potential to ensure that transformative developments continue to benefit medical education across the world. we need new innovative strategies for utilizing the medical education system to support uninterrupted education and assessment. considering the benefits of virtual reality and simulation-based technologies, medical university administrators should invest in simulation-based virtual educational products to keep the clinical competency education and assessment on stream. conflict of interests: none declared. covid- and medical education. the lancet infectious diseases medical student education in the time of covid- educause horizon report teaching and learning edition covid- reviving need explore online teaching and learning opportunities encyclopedia of e-leadership creating the medical schools of the future effects of physician-patient electronic communications on the quality of care applications and challenges of implementing artificial intelligence in medical education: integrative review necessity of designing a national model of foresight-based policy-making in medical education virtual reality and the transformation of medical education a critical review of simulation-based medical education research extended reality in medical education: driving adoption through provider-centered design ophthalmic education and ophthalmologists growth trends in iran simulated patient methodology: theory, evidence, and practice conducting a high-stakes osce in a covid- environment key: cord- -t quwfva authors: loh, tiffany y.; hsiao, jennifer l.; shi, vivian y. title: covid- and its impact on medical student education in dermatology date: - - journal: j am acad dermatol doi: . /j.jaad. . . sha: doc_id: cord_uid: t quwfva nan dermatology is a visual field, and repetitive patient encounters are required in order for clinicians to develop the skills necessary for diagnosing and managing dermatologic conditions. teledermatology offers a potential solution to medical student education during the covid- pandemic. herein, we discuss methods of implementing teledermatology in order to optimize medical student learning. optimizing teledermatology for medical student education although many dermatology appointments have been transitioned to virtual visits, medical students can still participate in these encounters. medical students may join video conferencing patient care encounters at the patient's and attending's discretion (table ). this allows medical students to learn fundamental dermatologic concepts while participating in patient care, which can help optimize their learning in the absence of in-person visits. other online resources can also be helpful for supplementing medical education (table ) . many dermatology residency programs hold online lectures, kodachrome sessions, and journal clubs, and residents nationwide are often invited to join. virtual dermatology society conferences, webinars, and podcasts are also available to most residencies. although it is commendable that these resources are often available free of charge to residents, it may also be beneficial to extend the invitation to medical students. although telemedicine is useful, it is also important to recognize that there are elements of dermatology education that cannot be replaced virtually, such as the ability to assess texture, perform biopsies, or use tools such as dermoscopy, wood's lamp, and koh scraping. ultimately, in-person visits are still needed. for the time being however, it is important to optimize tele-education and to involve medical students as much as possible. conclusions medical education has changed drastically during the covid- pandemic, and teledermatology has become essential for educational continuity. dermatology education is important for all medical students, as the majority will likely continue to encounter dermatologic problems throughout their careers, and it is our responsibility to include them in our educational endeavors to the best of our ability. optimizing teledermatology visits for dermatology resident education during the covid- pandemic important guidance for medical students on clinical rotations during the coronavirus (covid- ) outbreak medical student core curriculum in dermatology survey assessment of medical students' proficiency in dermatology: are medical students adequately prepared to diagnose and treat common dermatologic conditions in the united states? dermatology in primary care: prevalence and patient disposition key: cord- -jrsg pdk authors: hu, na; li, ying; he, su-shuang; wang, lei-lei; wei, yan-yan; yin, lu; chen, jing-xu title: impact of the family environment on the emotional state of medical staff during the covid- outbreak: the mediating effect of self-efficacy date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: jrsg pdk during the outbreak of the coronavirus disease (covid- ), the medical staff was facing severe work pressure, which led to a negative emotional state. the purpose of this study was to explore the relationship between the family environment and the emotional state of the medical staff members during the covid- outbreak. due to the importance of self-efficacy in regulating mental health, the mediating role of self-efficacy in the association between family environment and emotional state was also explored. a cross-sectional survey was performed, using an online questionnaire, on medical staff who participated in the epidemic prevention and control tasks during the covid- outbreak in beijing. family environment, self-efficacy, anxiety, and depressive symptoms were measured by the family environment scale-chinese version (fes-cv), the general self-efficacy scale (gses), the generalized anxiety disorder scale- (gad- ), and the patient health questionnaire- (phq- ), respectively. correlation analysis and mediating effect analysis were used to explore the relationships between them. first, a higher prevalence of anxiety ( %) and depressive ( %) symptoms were confirmed among the medical staff. second, the symptoms of anxiety and depression were negatively correlated with the dimensions of cohesion and expressiveness and positively correlated with the dimensions of conflict in the fes-cv scale. third, self-efficacy significantly mediated the association between the family environment and anxiety symptoms (p < . ) as well as the family environment and depressive symptoms (p < . ). these findings show that a negative family environment was the main predictor of symptoms of anxiety and depression in the medical staff during the covid- outbreak. furthermore, we found that self-efficacy played a critical mediating role between the family environment and the symptoms of anxiety and depression. our study also indicates that improvements in the family environment benefit the mental health care of the medical staff, and high self-efficacy enhances this effect. the outbreak of coronavirus disease continues to attract worldwide attention . to date, covid- cases have been confirmed in more than countries around the world, and it has become a public health emergency of international concern. many people who have directly faced this large-scale public crisis, especially the medical staff involved in the prevention and control of this epidemic, showed anxiety symptoms (huang et al., ) . they faced a high risk of getting infected at their workplace and the possibility of their family members at home getting infected through them (xiang et al., ) . medical staff working in a high-pressure environment suffer from psychological problems, especially anxiety and depression (kang et al., ; wang et al., ) . although guidelines on the care of the mental health issues in medical staff have been issued in china (kang et al., ) , some of the staff refused to receive mental health care . it has been reported that the incidence of anxiety and depression was high among the medical staff during the outbreak of covid- , with prevalence rates of anxiety and depressive symptoms being . % and . %, respectively (lai et al., ) . due to the stigma around mental health problems in china (bai et al., ) , many members of the medical staff community were reluctant to accept professional psychological help . however, they preferred to seek help from their family members. some medical staff was far away from their families because of the fear of infection, and they were reluctant to have close contact with them. others were isolated and cannot return home for a long time (raven et al., ) . it has shown that family support is very important for medical staff involved in epidemic prevention (mohindra et al., ) . however, there has been no research on how family members can help medical professionals suffering from mental health issues and what kind of family environment can be useful in relieving negative emotions in these individuals during an epidemic. the family, which is the basic social unit, may affect the mental health of family members (cheng et al., ) . in chinese culture, family relations are highly valued, and family is a very important support system (poulin et al., ) . it means that the importance of family is the core feature for most chinese people. previous studies have shown that the family environment can directly affect the emotions in family members, especially the dimension of cohesion (harris and zakowski, ; burnett et al., ) , expressiveness (luebbe and bell, ) , and conflict (yap et al., ; yap and jorm, ; fosco et al., ) . however, whether the family environment has an effect on the mental health of medical staff during the epidemic is still not clear. self-efficacy refers to an individual's judgment about his or her ability to complete a certain task successfully, and it reflects the self-confidence of an individual to cope with various difficulties and setbacks in life (tang et al., ) . previous research has shown a positive correlation between the family environment and self-efficacy (mahmoudi, ) . individuals with a better family environment were shown to be more likely to have higher levels of self-efficacy (hemati et al., ) . for example, individuals can freely communicate with family members, express more about themselves, and have a frequent high contact of intimacy with other family members, which will lead to a high level of selfefficacy (hemati et al., ) . studies show that increasing selfefficacy is an essential aspect of the psychological intervention to protect the mental health of individuals (caldwell et al., ) . it seems that the higher the self-efficacy in an individual, the better the mental health (sebastian, ; jiang et al., ) . high self-efficacy can improve an individual's mental health (bandura, ). however, a low level of self-efficacy harms a person's psychological well-being (cieslak et al., ; sachs-ericsson et al., ) . importantly, studies have shown that self-efficacy is one of the critical factors that modulate an individual's emotions, especially feelings of anxiety and depression (bandura et al., ; kanfer and zeiss, ; cybulski et al., ; schönfeld et al., ) . these studies suggested that higher levels of selfefficacy are associated with lower levels of feelings of anxiety and depression. during the outbreak of sars, medical staff with low self-efficacy often had a higher fear of the epidemic, which was positively correlated with their poor mental health status (ho et al., ) . self-efficacy can predict the significant difference in mental health during the epidemic. the lower the self-efficacy, the worse the mental health status (yıldırım and güler, ) . it has been found that lower psychological stress among dentists during the covid- epidemic is associated with being in a stable relationship and having a higher sense of self-efficacy (shacham et al., ) . it suggests that family relationships and self-efficacy during the epidemic may both affect the mental health of medical staff. the partial mediating role of self-efficacy in some psychological trait relationships has been supported by relevant research (haj-yahia et al., ) . indeed, it has been found that self-efficacy was a mediator for the association of daily stress and mental health (schönfeld et al., ) . however, there has been no study focusing on the relationship between family environment, self-efficacy, and the emotional state of medical professionals during an epidemic. the relationship between family environment, self-efficacy, and depression and anxiety, and whether the influence of family environment on anxiety and depression is regulated by selfefficacy needs to be further studied. therefore, the purpose of this study was to further explore the direct and indirect impact of the family environment on symptoms of anxiety and depression among the medical staff involved in controlling the epidemic. when examining the indirect effects, we took self-efficacy as an intermediary variable. mediating effect analysis was performed to explore the role of self-efficacy in the relationship between family environment and symptoms of anxiety and depression. based on our findings, we provide viable strategies for the family based psychological intervention of the medical staff during an epidemic that will help to improve our psychological crisis intervention system. this research was a cross-sectional study using the convenience sampling method to collect survey results through an online questionnaire. the online survey was conducted in beijing from february , to march , . inclusion criteria for the study were as follows: ( ) chinese, working in beijing; ( ) - years old; ( ) medical staff including doctor or nurse involved in covid- epidemic prevention and control; and ( ) has read and agreed to the online informed consent. the protocol of this study was approved by the ethics committee of the beijing huilongguan hospital. the family environment scale-chinese version (fes-cv) this scale was based on the family environment scale (fes) developed by moss (moos and moos, ) , which was translated into chinese by wang et al. (wang et al., ) . the scale has entries in total, including dimensions (cohesion, expressiveness, conflict, independence, achievement orientation, intellectual-cultural orientation, active-recreational orientation, moral-religious emphasis, organization, and control), with nine entries for each dimension. each entry has a true and false option. the higher the score of a particular dimension, the more prominent the characteristics of the family in that aspect. the scores of conflict and control were negatively correlated with the family environment, whereas others positively correlated with the family environment. this scale has been shown to have good structural, content, and external validity when applied to the chinese population (phillips et al., ) . in this study, we selected three dimensions of this scale that were closely related to emotions investigated in previous studies: cohesion, expressiveness, and conflict (harris and zakowski, ; burnett et al., ) . this scale was developed by german psychologist schwarzer (schwarzer, ) , which was translated and revised for the chinese version by wang et al. (wang et al., ) . the scale comprises of items with four answer options, and the answer options range from (not at all true) to (completely true). higher scores mean higher levels of self-efficacy; a score of . - . means low level, . - . means medium level, and . - . means a high level of self-efficacy. the revised scale has been shown to have excellent reliability and validity in the chinese population (wang et al., ) . the cronbach's alpha of this scale is . , the retest reliability is . , and the correlation coefficient between the items and the total scale score is . - . . this scale was developed by spitzer et al. (spitzer et al., ) . previous research demonstrated that the chinese version of gad- has good reliability and validity, and the sensitivity and specificity of this version were . and . , respectively (huang et al., ) . this scale is composed of seven items, and each item has a - points scale. the total score range is - points; - for no anxiety, - for mild anxiety, - for moderate anxiety, and more than for severe anxiety. the patient health questionnaire- (phq- ) the scale was developed based on the fourth edition of the diagnostic and statistical manual of mental disorders (kroenke et al., ) . we used the chinese version of this scale. this scale is composed of nine items, and each item has a - points scale. the symptom severity is determined by the total score, with - being mild, - being moderate, - being moderately severe, and - being severe. cronbach's alpha of the phq- in the chinese population is . , and the retest reliability is . , which indicates that this test has excellent reliability and validity (wang et al., ) . all of the analyses were performed using spss for windows . . we reported means and standard deviations for continuous variables showing normal distribution and frequencies and proportions for categorical variables. the original scores of the dimensions of conflict in fes-cv were not normally distributed and were presented as medians and quartiles. a chisquared test was used to test the relationship between the demographic data of the participants and their anxiety and depressive symptoms. the study participants were grouped based on whether they showed/did not show anxiety or depressive symptoms. the inter-group comparison of the scores of fes-cv and gses was carried out using the independent sample t-test and the independent sample kruskal-wallis test. spearman correlations were calculated to determine the relationships between the scores of the various scales. we categorized the family environment (cohesion, expressiveness, and conflict) as an independent variable, anxiety and depression symptoms as dependent variables, and self-efficacy as an intermediary variable. after controlling for demographic variables, the direct, indirect, and total effects of the family environment on the symptoms of anxiety and depression were examined. the mediation analysis was run on the process macro for spss (preacher and hayes, ), using , bootstrap samples for bias correction and to establish % confidence intervals. all of the tests were twotailed, and the significance level was set at p < . . the medical staff involved in this study mainly includes two groups. one is the staff of the hospital involved in treating patients infected with covid- ; the other is the staff at the isolation point, whose main task is nucleic acid testing and medical services for the quarantined personnel. a total of medical staff members completed the online questionnaire, out of which six individuals did not complete the basic information, and two took more than min to answer the questionnaire. thus, medical staff members participated in the study, of which ( %) were women, and ( %) were men, aged - , with an average age of . ± . . most of the participants were under ( %), had a bachelor's degree ( %), were married ( %), and were living with their families ( %). among these participants, ( %) had anxiety symptoms, and ( %) had depressive symptoms. the symptoms of anxiety and depression were closely related to the gender of the medical staff and whether they lived with their family members or not. the prevalence rate of anxiety and depression symptoms was higher in women (x = . , p = . ; x = . , p = . ) and in those who did not live with their families (x = . , p = . ; x = . , p = . ) ( table ) . we divided the participants into two groups: one group with anxiety symptoms (gad- score > ) and the other group without anxiety symptoms (gad- score ≤ ). we then investigated the significance of the differences in the scores of the two groups for the fes-cv and gses scales. next, the participants were divided into two groups according to whether they had depressive symptoms or not (depressive symptoms: phq- score > ; no depressive symptoms: phq- score ≤ ). like the anxiety symptoms, we investigated the significance of the difference between the scores of fes-cv and gses scales in the two groups. the results, which show the significant differences among groups, are shown in table . next, we used correlation analysis to determine the correlation between the scores of each scale. the results showed that there were statistically significant relationships between the anxiety and depressive symptoms of medical staff, their family environment, and their sense of self-efficacy. anxiety and depressive symptoms showed a significant positive correlation with the dimension of conflict in fes-cv scale (r = . , p < . ; r = . , p < . ), and a significant negative correlation with the dimension of cohesion (r = − . , p < . ; r = − . , p < . ), expressiveness (r = − . , p < . ; r = − . , p < . ), and self-efficacy (r = − . , p < . ). more specifically, individuals with bad family environments and low self-efficacy were more likely to show symptoms of anxiety and depression. besides, selfefficacy positively correlated with the dimension of cohesion and expressiveness and negatively associated with the dimension of conflict (table ) . after controlling for demographic variables, we examined the mediating effects of self-efficacy (figure ) . self-efficacy was significantly associated with the symptoms of anxiety and depression. it significantly mediated the association between the family environment and anxiety symptoms (β = − . ; % ci, − . to − . ; β = − . ; % ci, − . to − . ; and β = . ; % ci, . to . ). similarly, it mediated the association between family environment and the depressive symptoms (β = − . ; % ci, − . to − . ; β = − . ; % ci, − . to − . ; and β = . ; % ci, . to . ). when controlling for self-efficacy, the association between the family environment and anxiety symptoms were still significant (β = − . , p < . ; β = − . , p < . ; and β = . , p < . ) and similarly for family environment and depressive symptoms (β = − . , p < . ; β = − . , p < . ; β = . , p < . ). thus, self-efficacy partly mediated the relationship between the family environment and the symptoms of anxiety and depression. in the current study, we conducted an online questionnaire survey of some medical staff involved in covid- prevention and control in hospitals and isolation sites in beijing. we found that a considerable proportion of medical professionals had anxiety ( %) and depressive symptoms ( %), as noted in previous studies (huang et al., ; lai et al., ) . moreover, our results showed that the family environment of medical staff and their symptoms of anxiety and depression during the epidemic were closely related to self-efficacy, and self-efficacy partly mediated the relationship between the family environment and the symptoms of anxiety and depression. in this present study, we found that the self-efficacy of male medical staff was significantly higher than that of female medical staff (t = . , p = . ). however, the symptoms of anxiety (p < . ) and depression (p = . ) during covid- were significantly lower than that of female medical staff, which was consistent with previous research results. for example, it showed that female medical workers experience higher levels of anxiety, depression, and distress during covid- (lai et al., ) . it also reported that the self-efficacy of male medical staff is significantly higher than that of female medical staff (tang et al., ) . it should also be noted that medical staff who did not live with their families were more likely to have symptoms of anxiety and depression during the epidemic. thus, our study suggests that we should pay more attention to such medical staff and provide them psychological intervention. this observation also indicates that the family plays a certain role in regulating negative emotions. furthermore, our study showed a close relationship between the family environment of the medical staff and their symptoms of anxiety and depression. the medical staff members with low cohesion and expressiveness, as well as high conflict in the family environment, were more likely to show anxiety and depressive symptoms during the epidemic. notably, in this present study, the relationship between the family environment and the symptoms of anxiety and depression reveals that the family environment can, directly and indirectly, affect the emotions of medical staff during an epidemic. the family environment can significantly predict the emergence of anxiety and depressive symptoms directly, which is consistent with previous results. for example, some studies found that there was a correlation between cohesion in the family environment and depression in family members (burnett et al., ) ; families with high cohesion, which have high levels of family support and ties, likely reduce depression (park et al., ; cano et al., ) . in contrast, low family cohesion and conflict between parents increased the risk of depression and anxiety in family members (park et al., ; cano et al., ) . in families with a high degree of cohesion, individuals can get more psychological help and emotional support within the family (birgisdóttir et al., ) , so that the psychological pressure can be appropriately relieved. positive emotional expression within the family can prevent suppression of inner feelings and buffer internal conflicts, especially in the face of stressful events. in contrast, negative emotional expression and low emotional expression within the family are associated with higher anxiety and depression (luebbe and bell, ; park et al., ) . in a high-conflict family, family members are prone to conflict between each other, leading to anxiety. therefore, the results in this study support the hypothesis that the family environment can influence the emotional state of the family members and that a negative family environment is a psychological risk factor for the rising emotional distress of the medical staff during an epidemic. additionally, the influence of the medical staff 's family environment on their symptoms of anxiety and depression during the covid- epidemic is partly through the role of self-efficacy, which means self-efficacy plays a critical role in mediating the effect of family environment on symptoms of anxiety and depression. indeed, previous studies supported that self-efficacy had a protective effect on mental health (bandura, ) and played a vital role in the regulation of stress (bandura et al., ) . high self-efficacy was related to better psychological adjustment (bandura, ) and lower emotional distress (benight and harper, ) . individuals with high selfefficacy had positive expectations and beliefs, had successful experiences, generated positive emotions, and were more likely to seek psychological support to modulate their emotions when facing stressful situations (tsang et al., ) . a bad family environment can reduce an individual's self-confidence and ability (hemati et al., ) . self-efficacy is the embodiment of such confidence and ability (tang et al., ) . that is to say, the family environment affects self-efficacy by affecting people's self-confidence and ability, thus affecting individuals' behavioral patterns and emotional responses to stress (tsang et al., ) . for example, a medical worker with a good family environment has confidence in the success of the fight against the epidemic and also believes that he is capable of doing his job, which will ease his fear of the epidemic and anxiety about the high-risk work of infection. self-efficacy played a partial mediating role between the family environment and symptoms of anxiety and depression, indicating the existence of other variables between them. future studies should, therefore, include other relevant variables that are likely involved in the relationship between the family environment and negative emotions. this study indicates that adjusting self-efficacy is a meaningful way to regulate the anxiety and depressive symptoms of medical staff during an epidemic. because of the close correlation between the family environment and the symptoms of anxiety and depression of medical staff during an epidemic, we need to pay more attention to psychological assistance for medical staff from the perspective of their family situation. when providing psychological assistance to medical staff during the epidemic, we should not only focus on the medical staff but also care about their family members and family relations. by improving the family environment and increasing the active support of the family, their emotional problems can be effectively alleviated (mohindra et al., ) . the focus of the medical staff 's treatment of family relations should be to enhance the intimacy between family members, increase their interaction, encourage them to talk to each other, resolve the family conflicts in time, and create a good family atmosphere. based on the results of this study, we propose the following suggestions for medical staff. first, we suggest that medical staff should have time to communicate with their families and that they should be encouraged to share their feelings with family members and get their support and encouragement. for example, they should be encouraged to record their routines in the hospital and share them with their families . the hospital or isolation point shall provide relevant communication conditions and equipment for this purpose. second, during the epidemic period, the staff of the relevant departments of the hospital should be aware of the difficulties existing in the family of medical staff, and they should guide these staff members and help them solve those problems to avoid family conflicts. third, the family members of medical staff should be aware of the mental health issues of the staff member. family safety plays the most important role in reducing the pressure of medical staff during the epidemic . therefore, the staff members should stay connected with their families through wechat, sms, and other apps to understand their health status, which will help lessen the negative mental state of the medical staff during the covid- pandemic outbreak. these suggestions can bring medical staff closer to their families, have more emotional communication, and reduce family conflicts. with the implementation of these measures, the medical staff 's sense of self-efficacy will also be improved. besides, our results suggest that improving self-efficacy will help to alleviate the anxiety and depressive symptoms of medical staff during the covid- outbreak. manipulating self-efficacy is an important way to prevent mental health problems when dealing with stress (schönfeld et al., ) . previous studies have focused on the effects of self-efficacy on the mental health and work quality of medical staff (amiri et al., ; tang et al., ) , and it suggested that necessary interventions should be implemented to improve the self-efficacy of medical staff. in the prevention and control of covid- , medical staff is faced with two main difficulties. on the one hand, medical staff has heavy work tasks, great pressure, high risk of infection, and lack of support (spoorthy et al., ) . on the other hand, most of the medical staff are required to be isolated in hospitals or isolation points. their families will face more prominent problems (mohindra et al., ) . some positive motivation factors can boost morale and improve the self-efficacy of medical staff, such as family and social support, positive example, recognition, and appreciation from others, successful experience, self-identity (spoorthy et al., ) . positive feedback and encouragement from others could also effectively improve self-efficacy (bandura, ; zinken et al., ; brown et al., ) . the pre-job training, encouragement from colleagues and family, affirmation from patients and society, and sufficient material support were all helpful ways to improve the self-efficacy of medical staff during the covid- outbreak. medical staff in a good family environment can get better family support. the support reduces the sense of uneasiness caused by isolation, and improve self-efficacy, increase work confidence, improve work efficiency and quality, and reduce the negative emotions caused by epidemic infection. it has been reported that the mental health status of chinese medical staff is poor (zhou et al., ) , and they are exposed to immense workplace pressure and face complex doctor-patient relationships. the reason lies in the contradictions in the current medical system reform in china, such as the uneven distribution of medical resources (lu et al., ) , the disequilibrium between health care needs and medical development (zhou et al., ) , and the imperfection of the medical system (ta et al., ) . during an epidemic period, protecting the mental health of the medical staff would benefit their health as well as the control of the epidemic worldwide (kang et al., ) . the national health commission of china has published a national guideline of psychological crisis intervention for covid- , which is guided for the protection of the mental health of the medical staff (kang et al., ) . however, the family environment is particularly important to the mental health of the medical staff, and self-efficacy plays an important role in regulating the relationship between them. appropriate guidelines should be issued nationally to improve the family environment of the medical staff and for the improvement of their selfefficacy. there are some limitations to the current study that need to be addressed. first, there are limitations to the method of sampling. sampling bias may have occurred by using a convenient sampling method. second, we have a small sample size, and all participants are from beijing, so the research participants in this study may not be sufficiently representative of the population we are interested in studying, which may limit the conclusion of research results. third, online questionnaire surveys cannot observe the participants' answering process, there is the possibility of random answer and perfunctory answer, cannot guarantee the complete authenticity of data. fourth, we did not measure other potential confounding variables that may exist between the family environment and the emotional state of medical staff during the covid- outbreak. finally, the researchers are all medical staff, and the design of the survey may be more based on clinical observation. in the future, the research design can be combined with clinical observation and the existing theoretical framework. in the current study, we found that the anxiety and depressive symptoms of medical staff during the covid- outbreak was closely related to their family environment, and their self-efficacy regulated the relationship between them. this study provides a new direction for the psychological intervention in medical staff during the epidemic that mainly focuses on improving their family environment and their self-efficacy. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. nh contributed to the manuscript writing. j-xc, yl, and s-sh contributed to the conception and designed the work. yl, l-lw, y-yw, and ly contributed to the critical revision of the article. all authors contributed to the article and approved the submitted version. study of the relationship between self-efficacy, general health and burnout among iranian health workers survey of stress reactions among health care workers involved with the sars outbreak self-efficacy in changing societies on the functional properties of perceived self-efficacy revisited 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a systematic review and metaanalysis parental factors associated with depression and anxiety in young people: a systematic review and meta-analysis covid- severity, self-efficacy, knowledge, preventive behaviors, and mental health in turkey determinate factors of mental health status in chinese medical staff: a cross-sectional study analysis system for selfefficacy training (asset). assessing treatment fidelity of self-management interventions key: cord- -uuaa ta authors: schaffir, jonathan; strafford, katherine; worly, brett; traugott, amber title: challenges to medical education on surgical services during the covid- pandemic date: - - journal: med sci educ doi: . /s - - - sha: doc_id: cord_uid: uuaa ta nan the arrival of the covid- pandemic to the usa in march of had a profound and unforeseen effect on education of american medical students. students and faculty in the middle of academic programs were given little advance notice that learning would be disrupted. as the epidemic spread, concerns grew for medical systems becoming overwhelmed with new cases and needing to conserve personal protective equipment (ppe). medical schools moved to cancel classes and restrict students from participating in clinical activities or even setting foot in their affiliated medical centers. with recommendations for social distancing and state-ordered mandates to stay at home, students were additionally forbidden from getting together in groups and had to remain physically separated from anyone other than immediate family. previous disruptions in medical education have been described in the literature, generally in the forms of natural disasters such as floods and earthquakes [ ] [ ] [ ] . unlike these events, several factors made the covid- pandemic unprecedented. the disruption was on a global scale-not just affecting one medical school or one community, but institutions across the country and around the world. solutions that helped to circumvent the effects of financial failures and natural disasters, such as the distribution of students to other institutions or programs would not apply in this case [ ] [ ] [ ] [ ] . the length of the disruption is likely to be greater than that for other examples as well. floods recede and earthquakes cease, with recovery linked to the time it takes to rebuild and redevelop infrastructure. in the case of the current pandemic, effects on student participation will likely last until the threat of infection abates, which may not occur until a reliable and effective vaccination program is in place, an intervention predicted to require a year or more. for other disasters, there was little or no risk to students beyond the initial insult; students suffered from emotional and financial trauma following natural disasters but did not remain at risk of physical harm. in those cases, students were often enlisted to help with recovery efforts and participate in clinical care, giving them a sense of purpose and a useful hands-on education. in the pandemic scenario, students are isolated and restricted from participation due to health concerns and the need to preserve resources for more extensively trained providers. nevertheless, some lessons learned from responses to natural disasters have been applicable to the pandemic situation. following hurricane katrina, the interruption in medical education necessitated some acceleration of the educational process, with a reevaluation of what requirements were necessary to graduate with an md degree [ ] . the pandemic situation is forcing educators to take a hard look at what the minimum requirements should be to complete a field of medical study. medical educators and administrators are asked to make a difficult ethical decision, where the education and safety of trainees are being weighed against the increasing demands of medical personnel needed in high-risk environments. these situations also demonstrate the potential psychological trauma on students and emphasize the need for adequate student counseling and mental health resources [ ] . regarding educational content, all of these situations have prompted educators to look for alternative means of instruction outside of the classroom. introduction of remote learning with televised lectures, online learning modules, and interactive online discussions have become important solutions to the lack of available time spent within institutional walls. televised and electronic learning is most appropriate when replacing didactic content, whether related to basic sciences or clinical teaching. however, for rotations that involve large numbers of procedures and hands-on learning, such solutions may prove inadequate. clinical clerkships in surgery, obstetrics and gynecology, and anesthesiology require students to participate in surgeries and procedures to learn technical skills, and then demonstrate such skills to assess competence. in addition to learning the skills required for competence as a physician, students also learn vital lessons about communication and function in a surgical team within these arenas. exposure to the surgical or obstetrical atmosphere is often what guides a student into or away from choosing these specialties as a medical career [ ] . unfortunately, these experiences are not as easily replaced by electronic learning as are other clinical arenas in medical education. the remainder of this paper focuses on the potential solutions and barriers to providing adequate undergraduate medical education in procedure-oriented fields in the setting of the covid- pandemic. many of the challenges of teaching surgery and obstetrics/ gynecology content have been successfully met with online educational platforms. a central learning objective for students in these fields is gaining the clinical judgment to weigh the relative merits of medical and surgical interventions. working through clinical scenarios of patients with potentially surgical conditions is a tried and true method for mastering this objective [ , ] . in the absence of having such patients physically present, it is useful to work through constructed cases that describe such scenarios. several online services are available that offer these cases and give students a chance to interactively work through their management. onlinemeded, geeky medics, oxford medical simulation, and aquifer are platforms that walk students through clinical scenarios that require them to decide when surgery is necessary, for example, in the setting of appendicitis or ectopic pregnancy (table ) . other available resources include case vignettes prepared by professional organizations like the association of professors of gynecology and obstetrics (apgo) [ ] and the association for surgical education (ase) [ ] (table ). these can be utilized via remote faculty-led sessions to guide students through surgical decision-making similar to case conferences that more typically happen on clinical rounds. a greater educational challenge lies in reproducing the experience of assisting in surgical procedures. part of the experience lies in witnessing the procedures themselves and learning the relevant techniques and anatomy. there are many excellent examples of online surgeries on internet video platforms such as youtube and vimeo that display recordings of surgical cases. they range from demonstrations of classical open approaches to cutting-edge techniques that employ the newest technologies available. these latter examples are often geared towards practicing physicians seeking to expand their repertoire of surgical skills and are not applicable to students learning basic surgical concepts. educators looking to provide examples of surgical videos to their learners need to curate these collections with the knowledge base of the learner in mind [ , ] . although many videos are narrated to edify their audience, the content and expertise of narration varies greatly. for some, the intent is to point out impressive pathological findings or to highlight potential surgical complications. others, more relevant to the undergraduate medical experience, concentrate on basic anatomy and rationale for surgical techniques. even when the perfect video is found that exposes learners to an appropriate procedure with narration at an appropriate learning level, asking a student to watch it alone can still be isolating. excluding the ability to interact with the surgeon and ask questions diverges substantially from the usual experience of being in the operating room. a potential solution is to enlist an interactive online platform to create an electronic classroom where the experience can be shared. a surgical faculty member can share his/her screen with a group of students as he/she describes the video being viewed. the video may be one found online or one recorded by the surgeon. providing real-time narration ensures that the content matches the faculty's objectives and is given at the correct level of complexity. it also provides a point of contact with a faculty member who may be a resource for future questions about the material or about career exploration. the situation still falls short in the instruction of how to assist at surgery. the importance of providing helpful and knowledgeable assistance is a skill that medical students often fail to appreciate. providing instruction through surgical videos about what role the assistant plays is a viable option, though it fails to reproduce the experience of retracting and suctioning. very few resources are available online that are not designated for professional surgical assistants, whose goals may be quite different from the undergraduate learner. creating educational tools that instruct students in surgical assisting may fill an important need. another established technique used in remote surgical education is the use of simulation. in general, simulation is used to demonstrate procedural techniques on a simulated patient (whether physical or virtual) in order to reduce patient risk and increase available opportunities for practice [ , ] . telesimulation, which provides the benefits of simulation instruction to learners at an off-site location, has been described in settings where a single faculty may teach students in several disparate locations [ ] . this technology may be useful in settings such as developing nations where local faculty lack the expertise to train their learners, and the challenge is in finding a reliable electronic infrastructure to communicate the simulations being explained [ ] . in the setting of medical students being physically isolated from their institution, the challenge is not with the equipment used in communication but rather with the equipment used for the simulation itself. without being able to gather centrally at a simulation center, students lack the opportunity to use resources such as manikins and surgical instruments. a newer technology that has been proposed to authentically reproduce live experiences remote from the operating room is virtual reality. devices that incorporate virtual reality vary from partially immersive (where a surgical environment that can be manipulated by user controls appears on a screen) to totally immersive (with the use of a headpiece that places the user within a -degree environment). such devices have been used in surgical training, particularly in training for minimally invasive surgery and robotics [ , ] . virtual reality has been proven to be an effective educational tool even for learners at the early stage of medical training [ ] . while virtual reality devices may someday be affordable and available outside of the institutional setting, they are currently too expensive to put in the hands of every student learning from home. other less expensive resources are more realistic. providing each student with a prefabricated kit of surgical tools may be cost-effective, depending on the nature of the materials and the size of the class. at minimum, a hemostat, a suture removal kit, and a pack of suture could be provided to train students in suturing and knot-tying on the surface of his or her choice. alternatively, a student could create simulation models using implements at home. several do-it-yourself simulation kits have been proposed to educate students on topics ranging from cervical exams in labor [ ] to performing hemorrhoidectomy [ ] using household items. students choosing to pursue such instructional methods would be remarkable for high levels of motivation. objectives for surgical education that are more abstract are difficult to teach remotely. a key element of training in surgery is teaching trainees the importance of professionalism and communication. much of the education provided in the clinical arena is by modeling appropriate behaviors and encouraging students to reproduce positive examples of communication that they witness. being physically separated from their instructors, students at home may lack opportunities to observe professional behaviors. sharing web-based video examples of appropriate communication techniques has been shown to be effective in teaching surgical trainees [ ] . use of an immersive virtual reality device to teach communication skills has also been proposed [ ] . choosing what routes of instruction to pursue may be influenced by students' perceptions of each teaching method's value. students at our institution who were asked which remote teaching methods they considered the most helpful gave similar ratings to interactive online cases, remote lectures, and faculty-guided surgical videos. several students commented that the latter were superior to teaching they received live in the operating room, since the faculty spent more time teaching and the views of the surgical field were clearer. another challenge in having students forced to stay at home during the pandemic is finding optimal means to assess their clinical performance. assessment of student performance is crucial to ensure that students graduate with adequate knowledge to address clinical conditions they will encounter and adequate skills to safely and effectively treat them. assessment is also important to identify struggling students who may require remediation and exemplary students worthy of commendations or honor programs. assessment usually includes written, detailed performance feedback from supervising residents or faculty, and objective evaluation of knowledge and critical thinking based on oral and/or written examination. this tiered system has been used for decades, but recent evidence suggests it may be subject to implicit bias [ ] . assessment of medical knowledge is carried out with standardized testing that requires little change in either preparation or execution. students may study using a variety of online and textual resources, and the test can be administered remotely, although significant trust is needed for ensuring adherence to an honor code to prevent cheating. modern medical education has embraced the addition of structured patient encounters and note writing assessments into many course grades [ , ] . these encounters can be accomplished in an online format where patient, student, and grader may be physically remote from one another. it is more difficult to assess clinical performance in a rotation where a student has never set foot in the hospital. the ability to distinguish one student from another and provide the individual assessments so important to the progression on to residency is further challenged. grading performance on surgical rotations also includes proficiency at specific procedural and examination skills. direct observation when students are not present is limited by both the lack of resources to perform such procedures and the time requirement for faculty to set aside time for observation. it is unreasonable to expect assessment of complex procedures done remotely, but a video assessment of basic skills such as suturing and knot-tying is feasible if students are provided with a basic, prefabricated kit as previously mentioned. video assessment of surgical skills has been previously described using monitors as handy as a smartphone or gopro [ ] . because the limited interaction with students has made it difficult for faculty to objectively discriminate degrees of mastery, many institutions elected to transition affected courses to a pass-fail format. with students confined at home, it is difficult to judge when they have either motivation or skills that exceed those of peers. professional behaviors such as attention to required attendance and appropriate interactions with standardized patients may be evaluated remotely, but in this setting, it is fairer to define a single standard of competency than to establish gradations of excellence. the use of pass-fail in medical education and its effect on ability of students to make a successful residency match without the distinctions that tiered grading provides was an additional stressor for students and will continue to be debated [ ] . while everyone would like to see a return to the usual format of patient care and student education, it has become increasingly apparent that things may never return to what has previously been considered normal. as educators plan for a gradual reentry of students into the clinical workplace, they must carefully balance the need for education with the safety of patients and the students themselves. students participating in surgical and obstetrical procedures require access to personal protective equipment (ppe) to safeguard against covid- as well as blood-borne infections. with rationing of ppe to ensure an adequate supply for health care workers, institutional administrators need to judge the importance of student education against the needs of the community. depending on limitations in supply, students may be forced to assume an increasingly distant role from the surgical bedside. the covid- crisis has forced training programs, patient care, and workplaces to incorporate tools for simulation and remote engagement in ways that have been unprecedented. many of these tools will prove to be useful, even desirable, for our learning environments once the crisis has passed. remote learning will play an expanded role in our training programs for the foreseeable future. but which experiential activities and assessments are essential to retain, and which could be effectively replaced or supplemented by simulation or by remote activities? we now have an opportunity as educators to test our existing tools, develop new strategies, and collect a body of evidence surrounding remote learning interventions for clinical education. it is incumbent on us as a profession to develop evidence-based best practices to guide curricular development as we move forward in this rapidly changing environment. authors' contributions all authors contributed substantively to the 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the context of the coronavirus disease (covid- ) pandemic. existing health system deficiencies are at the heart and core-el corazón-of the health inequities being magnified during the current public health crisis for racial, ethnic, and linguistic minorities [ , ] . among the most important factors that influence whether health information is understood and whether individuals can access care is language. individuals with limited english proficiency (lep) have improved health outcomes when they experience languageconcordant health care-that is, when their clinician speaks their language [ ] . the lack of medical interpreter availability and increased difficulties with in-person access to care during the covid- health crisis have further complicated health care for linguistic minorities [ ] . cities such as new york, boston, and chicago are increasingly reporting large clusters of infection rates and disproportionately high death rates in hispanic/ latinx patients [ ] [ ] [ ] . rural hispanic/latinx groups are also at elevated risk given their significant representation in meat and poultry packing plants throughout the midwest [ ] . some of the health system deficiencies that may result in a disproportionate impact of covid- on language minority communities include a paucity of data regarding patient language and ethnicity, limited availability of languageappropriate services, and the lack of language-concordant health-care professionals [ ] . medical spanish education has been proposed as a strategy to increase the language-concordant physician workforce [ ] [ ] [ ] . although many medical schools provide opportunities for students to enhance language skills such as medical spanish [ ] , the covid- pandemic also has affected the landscape of medical education, causing medical schools to seek virtual educational options for students [ ] . existing experiences in which students might have gained medical spanish skills or clinical exposure to minority communities, such as clinical clerkships, away electives, study-abroad opportunities, and service-learning experiences, have in many cases been canceled or postponed in light of pandemic safety restrictions. despite these restrictions, virtual learning platforms may present a new opportunity to effectively teach communication skills to care for linguistic minority populations. building a professionally multilingual physician workforce through medical spanish courses can address the languageconcordant physician deficit that affects a growing number of communities in the usa [ ] . according to the most recent population estimates, the usa is home to . million individuals who identify as hispanic/latinx, representing . % of the population and constituting the largest and fastestgrowing racial or ethnic minority in the usa [ ] . moreover, million us residents ( . % of the population) report speaking spanish at home, and . % of hispanics who speak spanish at home report speaking english less than very well [ ] . the number of spanish speakers in the usa represents a . % increase between and [ ] , making the usa the country with the second most spanish speakers in the world (after mexico) [ ] . this trend is expected to continue, exacerbating unmet challenges to health systems and clinicians who, despite legal requirements [ ] and guidelines to provide linguistically and culturally appropriate services to all patients [ ] , may be unprepared to do so, particularly during patient surges such as the covid- pandemic. existing data shows that a majority of medical students- . % according to electronic residency application system data-have some pre-existing spanish skills [ ] , but many are being asked to use skills in patient care without appropriate training or confirmation of skills [ ] . moreover, a recent analysis of the alignment of non-english languagespeaking resident physicians and the lep patient population geographic distribution demonstrates that spanish is the us language with the lowest ratio of language-concordant resident physicians to patients [ ] . despite the public health need to increase the number of competent spanish-speaking health professionals, the intrinsic complexities of medical spanish education have resulted in barriers to medical spanish course implementation. for example, lack of qualified educators and lack of time in the curriculum are two of the main reported barriers to offering medical spanish education in medical schools [ ] . additionally, many schools' medical spanish educational opportunities are student-led [ ] , a model that challenges long-term sustainability as well as curricular standardization. importantly, concerns about unintentionally promoting medical student "false fluency" by imparting partial knowledge of medical spanish without appropriately teaching limitations and self-assessment skills may also be a reason why schools are reticent to provide formal medical spanish education [ ] . virtual access to medical spanish programs can contribute to the democratization of knowledge by bridging previously identified physical and geographic barriers to medical spanish education since educators need not be locally situated. scholars in spanish language pedagogy have made a compelling case that the appropriate use of technology can increase contact with the target language, address the variety of content interests that learners bring to language learning, and augment exposure to comprehensible input [ ] . a thoughtful approach to virtual medical spanish education may serve as an effective strategy to teach patient-centered communication skills for a linguistically vulnerable and growing patient population, particularly in schools in which it was not previously accessible or available. for example, some areas of the usa with the highest growth of the hispanic/latinx population are rural areas. north dakota, west virginia, and south dakota are the three states with the highest immigrant population growth between and with %, %, and % immigrant population growth, respectively, the majority of whom identify as hispanic/latinx [ ] . at least half of the us immigrant population has lep [ ] , although this may represent an underestimate due to difficulties in data capture for populations with language needs besides english [ , ] . medical school programs located in rural environments may experience an influx of patients with lep due to immigrant population growth in rural communities, but their curricula and onsite faculty may not be prepared to provide language skills education such as medical spanish courses. additionally, considering pandemic restrictions suspending or postponing student in-person clinical experiences, opportunities to learn clinical and interpersonal skills through virtual strategies now are being sought widely and actively by medical schools both in rural and urban locations. medical schools should take advantage of the current need to adjust curricula to virtual environments as an opportunity to also ensure that the educational content equitably addresses the linguistic and cultural communication needs of the us patient population. given population trends, this would require intentional inclusion of communication skills with linguistic minority communities, particularly spanish speakers. furthermore, it is critical to acknowledge that the challenge of communication with spanish-speaking patients and other communities with lep should not be addressed by medical students and physicians alone but also should involve other health professions and incorporate the perspectives of language experts. the more flexible online environment may facilitate medical spanish collaboration among institutions and disciplines, and across health professions programs. for example, language educators and clinician experts have been called upon to collaborate in the development of medical spanish educational standards, but their work is often unintentionally siloed in their respective departments [ ] . additionally, a virtual classroom in which students from various health programs, such as medicine, pharmacy, nursing, physical therapy, dentistry, and others, could collaborate to learn medical spanish has never been published but could enable medical spanish programs to more widely address the need for a linguistically and culturally prepared healthcare workforce. to understand how a medical spanish course can be transformed into a high-quality virtual educational opportunity, the overall purpose, specific learning objectives, and assessment methods of such a course must be clearly defined. historically, medical spanish courses have lacked consistent curricular structure or objectives, resulting in many variations of course content, teaching activities, student spanish proficiency pre-requisites, and learner objectives [ , , ] . medical spanish in medical school settings has been defined by expert consensus as "the use of spanish in the practice of medicine for communication with patients" [ ] . a standardized list of five core learner competencies for medical spanish students in medical school have been proposed and include ( ) medical interviewing skills, ( ) comprehension of the patient story, ( ) patient-centered discussion of the assessment or diagnoses, ( ) patient-centered discussion of the plan of care, and ( ) self-assessment of confidence and limitations [ ] . the fifth competency is a progressive lifelong learning goal for students to continually reassess strengths and weaknesses in communication skills with linguistic minority patients and has also been referred to as global linguistic competency [ ] . within the patient-centered learning objectives, medical spanish courses are tasked not only to teach the linguistic aspects of communication but also to address the learner's cultural humility and cultural competency in order to understand the spanish-speaking patient's sociocultural context and health communication needs [ , ] . teaching these communication skills goes far beyond teaching a vocabulary or terminology list or correcting grammar; in fact, while these activities form part of the background content necessary to teaching medical spanish, experts recommend that medical spanish courses focus on the patientcentered communication rather than on the technical medical vocabulary [ ] . synchronous, asynchronous, and blended synchronous/asynchronous strategies can be applied to virtual teaching and learning of medical spanish skills. for example, elements of grammar and vocabulary-focused education may be effectively taught through asynchronous learning methods such as vocabulary and grammar exercises or games that aid memorization and support other cognitive processes that reinforce learning in general and language learning in particular [ ] . patient-centered communication skills, however, must be taught, observed, and practiced using more interactive modalities. for instance, a flipped-classroom approach is a type of blended learning in which students are introduced to content through pre-recorded videos and texts (individual learning space), and face-to-face classroom time using video communication solutions is dedicated to interactive activities that require personal engagement such as dialogues, debates, or presentations (group learning space). this approach can be an effective way for students to review and learn material in preparation for class, which would be dedicated to applied language use and faculty-guided reinforcement. a digital voiceboard, through which students can record themselves speaking in response to prompts, is an asynchronous strategy that has been shown to provide students an opportunity to both practice and reflect on responses to patient queries during the medical interview [ ] . table summarizes our recommended pedagogical practices and examples for implementation in virtual medical spanish courses, as well as how these practices align with medical spanish course competencies, learning objectives, and expected performance outcomes [ ] . transforming medical spanish courses from in-person to virtual learning platforms need not require recreating the curriculum for schools with pre-existing formal medical spanish courses but rather adapting the teaching methods and practices to the virtual environment. adapting courses for virtual education may present a useful opportunity to revisit the course objectives, to maximize the productivity of face-to-face sessions through high-yield clinically relevant interactive methods, and to address some potentially deficient components of the program. for example, the assessment of medical spanish linguistically and culturally appropriate communication skills is a critical element of medical spanish education, yet one that has been lacking in a majority of reported medical spanish courses [ ] . for example, arranging virtual role-play pairings by level of proficiency, providing real-time constructive feedback on performance, fostering peer-to-peer reviews and assessments, and guiding students on how to effectively self-reflect on their strengths and limitations can be important ways to continually assess students' performance, confidence, and skill levels. this learner-centered assessment method should ideally not only evaluate but also lead to the progressive improvement of the students' language skills and cultural competence, and to the gradual growth in student confidence commensurate with skills acquisition. a continuous assessment through role-play activities and other interactive classroom experiences may better contribute to this goal than a single final exam as the only learner assessment methodology. moreover, standardized patient (sp) encounters, which are the recommended approach to assessing student skills at the end of a medical spanish course [ ] , can be conducted through virtual solutions. the simulated telehealth encounter can be useful for assessing medical student performance in a setting that reflects the realities of current access to care under pandemic circumstances. telehealth communication presents particular challenges to clinicians and patients with lep, since it may be more difficult to interpret gestures, display empathy, or address limited health literacy using video technology [ ] . during the covid- crisis, health systems are encountering barriers to the technological application of their prior language assistance staff, such as interpreters or patient advocates, to telehealth encounters [ , ] . further, patients with lep have increased difficulty and decreased comfort with using technology to access health services [ ] . as a result, using telehealth sp encounters as part of medical spanish courses may not only be useful for assessing student performance of medical spanish skills but may also serve to educate learners on strategies to communicate empathically and effectively pre-class assignments: students receive a video lecture, podcast, or reading assignment to complete prior to class. conversation sessions: between class virtual sessions, students are assigned to participate in pre-arranged small group virtual meetings to foster conversation focused on specific health topics in spanish. self-study exercises: students independently complete vocabulary and grammar exercises or games to cognitively reinforce new vocabulary or grammar through workbooks or online software (e.g., quizlet or anki). student-led role play: a student must prepare a case related to the class topic and will play the role of the patient while another student is tasked as the clinician interviewing the patient. digital recordings: students are assigned to prepare a video or audio recording in response to a video/audio prompt or a patient scenario as oral/aural practice using digital voiceboards. the faculty reviews and provides constructive feedback. competency : patient-centered discussion of the assessment or diagnoses competency : patient-centered discussion of the plan of care demonstrate patient-centered explanation of medical diagnoses/assessment. demonstrate patient-centered explanation of treatment/evaluation plan. orally explain findings of the medical evaluation and plan to the patient, adjusted for cultural, emotional, and literacy needs assess patient comprehension of the information provided and address knowledge gaps or concerns guided self-assessment at key intervals: students are guided on how to self-reflect on their personal strengths and limitations in medical spanish at various points throughout the course, such as pre-course, mid-course, and end-course. post-activity guided self-reflection: students respond to a self-reflection prompt following a role play or performance assessment activity, such as a standardized patient encounter. competency : self-assessment of confidence and limitations accurately and progressively self-assess confidence and limitations in medical spanish identify when and how to work with a medical interpreter seek a medical interpreter if/when limitations are reached effectively work with a medical interpreter with spanish-speaking patients using video technology. such an educational and assessment strategy for medical students may enhance their preparedness to provide competent language-concordant telehealth care for vulnerable populations. language is an important factor in the health inequities experienced by linguistic minority groups and currently magnified by the covid- pandemic. addressing language needs is at the core of health equity for linguistic minorities, and improving virtual access to high-quality medical spanish education is an important strategy to prepare medical students and other health professionals to care for vulnerable communities during the current pandemic and beyond. next steps in enhancing virtual medical spanish education and assessment should include an evaluation of medical school strategies and methodologies in transforming current medical spanish courses into virtual learning environments, collaboration among medical spanish faculty to evaluate and share virtual learning resources, and the development of a national repository of standardized virtual sp encounters and assessment methodology for evaluation and eventual certification of medical student and physician medical spanish skills. code availability not applicable. author contributions the first draft of the manuscript was written by pilar ortega, md, and all of the authors commented on previous versions of the manuscript. all of the authors read and approved the final manuscript. data availability not applicable. conflict of interest p. ortega receives author royalties from saunders (an imprint of elsevier) for a textbook. real-time feedback is provided during synchronous classroom interactive activities such as role plays; feedback may be provided by the faculty member as well as peer-to-peer feedback guided by faculty. simulated case post-assessments: a simulated telehealth encounter is conducted on a virtual platform to assess student skills, and student receives feedback from standardized patient and faculty member. competencies - evaluate the extent to which the listed course learning objectives and performance objectives have been met for each learner. improve the learner's ability to successfully communicate with spanish-speaking patients better serve spanish-speaking patients by identifying and addressing communicative gaps and limitations the disproportionate burden of covid- for immigrants in the covid- and racial/ethnic disparities a systematic review of the impact of patient-physician non-english language concordance on quality of care and outcomes when coronavirus care gets lost in translation latinos disproportionately dying, losing jobs because of the coronavirus: 'something has to change coronavirus patients who don't speak english could end up 'unable to communicate in their 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services in health and health care: a blueprint for advancing and sustaining clas-policy and practice non-english-language proficiency of applicants to us residency programs medical students' experiences and perspectives on interpreting for lep patients at two us medical schools supply and demand: association between non-english languagespeaking first year resident physicians and areas of need in the usa let's not contribute to disparities: the best methods for teaching clinicians how to overcome language barriers to health care brave new digital classroom: technology and foreign language learning immigrants in the u.s. states with the fastestgrowing foreign-born populations the limited english proficient population in the united states. migration policy institute medical spanish programs in the united states: a critical review of published studies and a proposal of best practices medical spanish standardization in u.s. medical schools: consensus statement from a multidisciplinary expert panel strategies for teaching linguistic preparedness for physicians: medical spanish and global linguistic competence in undergraduate medical education beginning medical spanish: oral proficiency and cultural humility cultural humility versus cultural competence: a critical distinction in defining physician training outcomes in multicultural education words matter: translanguaging in medical communication skills training minds online: teaching effectively with technology spanish in health care: policy, practice and pedagogy in latino health addressing equity in telemedicine for chronic disease management during the covid- pandemic hospitals have left many covid- patients who don't speak english alone, confused, and without proper care the digital divide in health-related technology use: the significance of race/ethnicity publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -nnh i k authors: jumreornvong, oranicha; yang, emmy; race, jasmine; appel, jacob title: telemedicine and medical education in the age of covid- date: - - journal: acad med doi: . /acm. sha: doc_id: cord_uid: nnh i k the covid- pandemic has offered medical schools an opportunity to incorporate telemedicine training into the curricula in a timely and practical manner. telemedicine has grown exponentially in the united states, and the shift toward remote care to align with social distancing guidelines is fueling this growth. training medical students to deliver high-quality, secure, and personalized health care through telemedicine will prepare the next generation of physicians to conscientiously use these technologies and meet a growing need for telehealth services. telemedicine-specific educational goals can be incorporated into curricula and integrated with existing clinical experiences to provide students with core telemedicine and clinical skills to prepare them for current and future pandemics. medical educators could explore major telemedicine domains: ( ) access to care, ( ) cost, ( ) cost-effectiveness, ( ) patient experience, and ( ) clinician experience. schools could use the following learning vehicles to help medical students explore these domains: ( ) asynchronous lectures covering telehealth history; ( ) discussions on applications, ethics, safety, etiquette, and patient considerations; ( ) faculty-supervised standardized patient telehealth encounters; and ( ) hands-on diagnostic or therapeutic procedures using telehealth equipment. incorporating telemedicine into the medical school curriculum exposes students to the application of telemedicine across specialties as well as its limitations. medical education has been affected by the rapid outbreak of the coronavirus . in line with social distancing guidelines and recommendations from the association of american medical colleges, in march of this year, medical schools transitioned the preclerkship curriculum to online, and many paused clinical rotations. , lectures and small-group learning activities continued virtually. clinical skills sessions either moved online or were deferred. many examinations are now administered online. in other times of crisis, such as after the september , , terrorist attacks, medical students were able to continue their education and help in the crisis response. however, given virus transmission risk and personal protective equipment (ppe) shortages, educators have been cautious in allowing students to be involved in patient care during the pandemic. fourth-year medical students were given the option to graduate early. medical students also volunteered to support frontline workers remotely by triaging patients, assessing patients using telehealth platforms, transferring patient calls, conducting covid- research, and providing both peer and patient mental health support. additionally, they have assisted onsite with ppe donation and pharmacy logistics. thus, lectures and exams are not the only aspects of the medical student experience that can be done remotely. these students also witnessed and took part in remote versions of vital medical tasks such as scribing, telemedicine encounters, medication reconciliation, and social work coordination. with research models predicting that intermittent social distancing may be recommended through , this pandemic has allowed medical schools the opportunity to incorporate telemedicine into their curricula. here, we examine the state of telemedicine in the united states and propose a longitudinal telemedicine curriculum in undergraduate medical education so that future doctors may be prepared to smoothly transition to telemedicine during future pandemics. telemedicine is defined as "the provision of health care services over a spatial distance through the use of telecommunication technology with the aim of benefitting a patient or population." telemedicine can improve both the care experience and patients' health, and reduce per capita costs of health care. in , the telemedicine market was worth over $ . billion. the world health organization also highlighted telemedicine as an essential service in response to the covid- emergency. the health care landscape in the age of covid- presents ideal conditions to accelerate this growth. a global physical workforce shortage, geographic maldistribution of primary and specialist care, and high national health care expenditure incentivize the deployment of telemedicine in rural, urban, military, and veterans affairs settings. , in a survey, about % of consumers were interested in receiving virtual health care, but only % had access to it. in response to covid- , electronic health record providers such as cerner and epic have evolved to support more telemedicine capabilities. for example, the mobile epic medical records system now includes infection tracing, communication with isolated patients, and remote monitoring. it also allows for the coordination of patient transfers between facilities, ppe tracking, handheld charting, and guidance for the covid- pandemic has offered medical schools an opportunity to incorporate telemedicine training into the curricula in a timely and practical manner. telemedicine has grown exponentially in the united states, and the shift toward remote care to align with social distancing guidelines is fueling this growth. training medical students to deliver high-quality, secure, and personalized health care through telemedicine will prepare the next generation of physicians to conscientiously use these technologies and meet a growing need for telehealth services. telemedicine-specific educational goals can be incorporated into curricula and integrated with existing clinical experiences to provide students with core telemedicine and clinical skills to prepare them for current and future pandemics. medical educators could explore major telemedicine domains: ( ) access to care, ( ) cost, ( ) cost-effectiveness, ( ) patient experience, and ( ) clinician experience. schools could use the following learning vehicles to help medical students explore these domains: ( ) asynchronous lectures covering telehealth history; ( ) discussions on applications, ethics, safety, etiquette, and patient considerations; ( ) faculty-supervised standardized patient telehealth encounters; and ( ) hands-on diagnostic or therapeutic procedures using telehealth equipment. incorporating telemedicine into the medical school curriculum exposes students to the application of telemedicine across specialties as well as its limitations. telemedicine and medical education in the age of covid- nurses on remote patient support. these technologies have enabled continuity of care during the pandemic and can continue to bolster care beyond the immediate crisis. providers' concerns about telemedicine include compromised quality of care as compared with in-person visits, reduced privacy and security of patients' health information, and the potential lack of personal connection between providers and patients during telemedicine visits. , in addition, there is evidence that direct-to-consumer telehealth may increase medical malpractice. , health care providers may also resist innovations in telemedicine because they perceive technology to be in competition with their services. however, training physicians during medical school to deliver high-quality, secure, and personable health care through telemedicine can alleviate concerns and promote population-wide adoption of the technology. through a curriculum that incorporates telemedicine training, medical students could learn how to maintain a strong patient-doctor relationship, protect patient privacy, promote equity in access and treatment, and seek the best possible outcomes while using telemedicine platforms. store-and-forward telemedicine education-involving the acquisition of clinical information transferred to multiple clinical sites and appropriate specialists, such as radiology, dermatology, pathology, and ophthalmology-has also been shown to be effective. medical trainees who completed this curriculum reported that it helped them develop core competencies in patient care, medical knowledge, practice-based learning and improvement, and systems-based practices. concerns about telemedicine at a system-wide level include legal and liability uncertainties, licensure requirements, and nascent reimbursement mechanisms. a longitudinal telemedicine curriculum could equip future providers with a more comprehensive understanding of the legal and regulatory resources in the states in which they practice. the american medical association has encouraged telemedicine training for medical education, and the liaison committee on medical education's survey shows that over a quarter of md degree-granting medical schools have implemented telemedicine training components into the preclinical phase of their curriculum and nearly half have implemented it into the clerkship phase. , therefore, we hope to advocate for more institutions to rapidly incorporate telemedicine education into their curricula and recommend how the process could occur. the basic goals of using telemedicine platforms in medical education include: facilitating basic knowledge acquisition, improving decision making, enhancement of perceptual variation in anatomy lessons or -dimensional simulations, improving skill coordination, practicing for rare or critical events, learning team training, and improving psychomotor skills. these goals can be incorporated into curricula and integrated with existing clinical experiences to provide students with core telemedicine and clinical skills to prepare them for current and future pandemics. through meaningful and sustained remote patient care in a wide variety of clinical settings, supervised by a diverse, interprofessional faculty body, students will learn how to incorporate telemedicine in a patient-centered, compassionate way while practicing history taking, guiding patients through self-examination, and acquiring clinical reasoning skills remotely. historically, the clerkship curriculum is where most medical schools incorporated their telemedicine education. however, preclinical education is also an opportunity for telemedicine training and exposure during didactic lectures or small-group discussions. videoconferencing technologies, such as project echo (extension for community healthcare outcomes), can aid in the incorporation of case-based discussions, short didactic presentations, and real-time evaluations to educate future providers in underserved areas or global settings on remote care. although medical students may not directly be involved in patient care at such an early stage of their training, it is important to expose them to different technologies and teach them how to incorporate telemedicine into their own careers. medical educators could explore major telemedicine domains: ( ) access to care, ( ) cost, ( ) cost-effectiveness, ( ) patient experience, and ( ) clinician experience. they could use the following learning vehicles to help medical students explore these domains: ( ) asynchronous lectures covering telehealth history; ( ) discussions on applications, ethics, safety, etiquette, and patient considerations; ( ) faculty-supervised standardized patient telehealth encounters; and ( ) hands-on diagnostic or therapeutic procedures using telehealth equipment such as live video, the store-and-forward method, remote patient monitoring (rpm), and mobile health. , telemedicine's applications are abundant and we will focus on how medical schools can consider formalizing the medical student exposure in key areas: ( ) telesurgery, ( ) telerehabilitation, ( ) tele-intensive care units (tele-icus), and ( ) chronic disease management and rpm. during telesurgery, surgeons use wireless networking and robotic technologies to operate on patients who are distantly located. it facilitates surgical education in medically underserved locations, especially in rural areas where health care capacity may be lower. virtual interactive presence allows for real-time, long-distance surgical collaboration during complex microsurgical procedures using shared -dimensional displays via high-definition binoculars. this technology could enable remotely located experts to mentor medical students or residents at the surgical site with applications in surgical training programs, remote proctoring for proficiency, and expert support for rural and global health settings. haptic sensation feedback technology also enables the transmission of tactile information to teleoperators, which allows the operators to feel tissue consistency and tension within the sutures, preventing damage to fragile tissues or tearing of sutures during the operation. , therefore, telesurgery can improve surgical patient care, eliminate the need for long-distance travel and costs, and increase surgical collaboration and education among academic medical centers around the world. with telesurgery, medical students on virtual surgical rotations and those interested in surgical specialties would gain observational experience of core procedures and become familiar with technologies they may use in the future to instruct other trainees globally. telerehabilitation allows for the transfer of inpatients to their homes after an acute phase of disease and reduces hospitalization costs for both patients and health care providers. after recovering from covid- , patients are at high risk of developing postintensive care unit syndrome-which could include cognitive, psychiatric, or physical disability after treatment. telerehabilitation may promote social distancing and more effective integration of exercise routines into daily life. individualized exercise routines at home on a treadmill, telemonitoring by a physiotherapist via videoconferencing using a tablet computer, and selfmanagement via a customized website have been shown to be effective. rehabilitation specialists and medical students can use technology to observe patients as they execute movements and monitor their improvement. covid- has also been reported to increase stroke incidence in younger patients. telerehabilitation has proven to be effective in helping poststroke patients recover motor and sensory function of affected limbs and may be adapted to help a surge of younger poststroke patients. individuals with underlying comorbidities, such as cardiovascular and pulmonary diseases, are at higher risk of developing severe covid- disease. medical students may want to become skilled in cardiac telerehabilitation because it is as effective in decreasing morbidity and mortality as facility-based cardiac rehabilitation programs. medical students can employ motivational interviewing skills, a core competency of clinical skills curriculum, to promote step counts, measure walking activity, and increase adherence to a schedule of regular physical activity. supervising members of the rehabilitation team could provide feedback on students' communication skills and efficacy of counseling after receiving quantitative data from step counters. students can serve as active partners with rehabilitation teams and form longitudinal relationships with rehabilitation patients while acquiring core competencies at the same time. a tele-icu uses technology to assist in providing care for critically ill patients with offsite clinical resources. two types of tele-icus have been described in the literature. the decentralized tele-icu is one or more medical facility that can be accessed from remote sites such as the office, home, or mobile clinic. in the centralized system, a single icu provides intensive care via telemedicine and remote monitoring to several satellite icus. physicians working in these systems might be able to more effectively treat icu patients, providing better clinical outcomes for patients at lower costs compared with hospitals without an icu. patients and health care workers have reported that the streamlining of workflows for rapid diagnosis and isolation, clinical management, and infection prevention are important during the covid- pandemic. there may also be a shortage in icu resources such as dialysis machines and respiratory support equipment, so it is important that future providers learn how to adapt tele-icu care to their hospital's resource status. research also indicates that a structured tele-education critical care program using case-based learning and icu management principles can facilitate knowledge translation and quality improvement in the critical care setting. medical students rotating through a remote critical care elective would still be expected to learn the systems-based approach to the critically ill patient, report on patient statuses during daily rounds, and identify a plan for each systems-based issue. a shortcoming of such an elective, particularly for students interested in specializing in critical care, would be a lack of hands-on experience with standard icu interventions such as central catheter and line placements, intubation, and extubation, among others. such a deficiency could be countered by remote video monitoring capabilities that allow providers to be virtually present in patient rooms at all times, such that learners could continue to observe the procedures. remote monitoring of arterial lines, mechanical ventilators, dialysis machines, and infusion pumps would also allow for more accurate reporting of clinical status and assessment for next steps in management. emerging technologies such as inpatient telemedicine and online file sharing applications can enable clinical programs to continue to function while protecting medical staff and patients from the spread of covid- . rpm programs have been rapidly deployed to safely monitor the physical and mental well-being of patients who are at high risk of contracting or have contracted covid- . rpm has previously been shown to vastly reduce the rates of visits to the emergency department and re-hospitalizations in individuals managing multiple complex conditions. emotional trauma instigated by largescale human disasters, such as covid- , may require use of tele-psychiatry to mitigate costs and increase access to global psychological counseling. rpm can also be used for continuous glucose monitoring, home monitoring of cardiovascular implantable devices, and remote consultations and diagnosis. the food and drug administration also authorized a covid- test using saliva for home collection under the guidance of telehealth professionals. such technologies invite students to learn and actively contribute in troubleshooting and refining interventions to be user friendly through community-based participatory research. forward triage-sorting patients before they arrive in the emergency department-has been a central strategy for health care surge control during covid- . telemedicine allows efficient screening of patients while protecting patients, clinicians, and the community from exposure. it has also been deployed in the primary care setting and is valuable for patients in need of chronic or preventative care who are reluctant to visit the clinic because they're afraid of viral transmission. medical students could be directly involved in testing, screening, and triage using history taking and clinical reasoning skills to report findings to their preceptors. the current pandemic has rapidly accelerated the move toward telemedicine and has provided an opportunity for medical schools to prepare students to participate in and develop the competencies for this transition. over half of u.s. medical schools included telemedicine in required or elective courses during the - academic year. we believe that a swifter transition toward telemedicine in medical education will help future doctors prepare for the present age of covid- and future pandemics. with the recent relaxation of restrictions on video visits, more practitioners are able to perform virtual visits across state lines and on non-health insurance portability and accountability act compliant platforms. covid- has challenged providers to deliver health care remotely and has demonstrated which kinds of care are more readily offered virtually. incorporating telemedicine into medical school curricula will not only expose medical students to relevant telemedicine technologies but also increase their understanding of the complex ethical, regulatory, and legal issues related to such cases. importantly, patients should give their consent to student involvement in their telemedicine care. threats to patient privacy in a telemedicine education program, where information is shared on different devices and accessed by multiple students and providers, may make patients reluctant to participate. it is essential that a robust privacy and security plan is well implemented and disclosed to patients. medical students should also be exposed to telehealth visit disclosures in which providers discuss the risks and benefits of phone or video visits. as informed consent is a core competency for providersin-training, these disclosures must be conveyed to learners, and in turn, to their patients. in addition, research indicates an inequity in geographical distributions of telemedicine training, especially in rural areas where hospitals may benefit most from telemedicine. the covid- pandemic has also exposed inequities in access to educational technologies. thus, hospitals and/or state governments should consider supporting these initiatives to establish the technological infrastructure needed for providers and patients. to address the potential lack of educator training in teaching telemedicine, communities of collaboration, professional credentialing standards, and automated guidance systems based on learner feedback are warranted. telemedicine curricula should include the limitations of telemedicine platforms, especially in emergency or urgent care. patients using on-demand telemedicine may encounter reduced care continuity since the same provider may not be available at different unscheduled visits. insufficient or disjointed efforts in establishing high-quality telehealth training may lead to inconsistent quality of care and risks to patient safety. it could also place additional burdens on medical students who are already overwhelmed with existing coursework. rapid implementation of telemedicine based on profit-driven motives may lead future providers to ignore critical research on quality and cost of care and focus on efficient, rather than evidence-based, technology implementation. in addition, the lack of implicit cues, such as body language, during telehealth consultations increases the risk of misunderstandings between patients and providers. new sensitivities are also required when discussing new diagnoses and end-of-life care across lines of technology. some clinicians have stepped in to provide guidelines for video and telephone visits. learners should be briefed on these limitations and be provided with tools, such as the teach-back method, to ensure mutual understanding between clinicians and patients. in the teachback method, providers invite patients to explain their understanding of the information or action plans discussed. teach-back may be useful not only for patient communication but also among telehealth providers to prevent medical documentation errors. by integrating telemedicine training into existing curricular structures such as clinical rotations, lectures, ethics programming, rural care exposures, electives, and research opportunities, medical schools can expose students to telemedicine without significant additional burden. existing online resources for students and providers include the american telemedicine association, center for telemedicine and ehealth law, health resource and services website, and the american academy of pediatrics' section on telehealth care and council on clinical information technologies. , - as telemedicine training is incorporated into more medical schools, systemic evaluation of these training methods may also allow for more effective implementation of programs. more research on the efficacy of existing telemedicine curricula and practice implementation should be explored. covid- has highlighted possibilities for technological advancement within medicine and medical education. with greater knowledge about which aspects of medicine work best with telehealth, it is important to train future providers to use these technologies and provide these modes of care. telemedicine curricula should train future providers to deal with the ethical, legal, and regulatory implications of telemedicine this training is especially important in light of the imminent care needs during the covid- pandemic. physicians must not only be trained to use telemedicine but also learn how to do so professionally, safely, and in an evidence-based manner. news-insights/press-releases/ important-guidance-medical-studentsclinical-rotations-during-coronavirus-covid- -outbreak medical student education in the time of covid- mount sinai takes on mental health impact of covid- crisis among health workers projecting the transmission dynamics of sars-cov- through the postpandemic period telemedicine training in undergraduate medical education: mixed-methods review the triple aim: care, health, and cost telemedicine market size, share, and trends analysis report analysis of third global survey on ehealth based on the reported data by countries utilization of telemedicine in the u.s. military in a deployed setting the use of deployable telehealth centers by military beneficiaries to access behavioral healthcare: an exploratory evaluation in american samoa survey: % of consumers are interested in receiving 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collection using saliva building a primary care/research partnership: lessons learned from a telehealth intervention for diabetes and depression virtually perfect? telemedicine for covid- from bedside to webside: future doctors learn how to practice remotely medicare telemedicine health care provider fact sheet telemedicine and telehealth: the potential to improve rural access to care time to fix american education with race-for-space resolve telehealth innovations in health education and training acknowledgments: the authors would like to acknowledge the students and student leaders at the icahn school of medicine at mount sinai who contributed their time to the covid- response in new york, new york. they are the inspiration for this article. key: cord- -o biwypo authors: asai, atsushi; okita, taketoshi; ohnishi, motoki; bito, seiji title: should we aim to create a perfect healthy utopia? discussions of ethical issues surrounding the world of project itoh’s harmony date: - - journal: sci eng ethics doi: . /s - - - sha: doc_id: cord_uid: o biwypo to consider whether or not we should aim to create a perfect healthy utopia on earth, we focus on the sf novel harmony ( ), written by japanese writer project ito, and analyze various issues in the world established in the novel from a bioethical standpoint. in the world depicted in harmony, preserving health and life is a top priority. super-medicine is realized through highly advanced medical technologies. citizens in harmony are required to strictly control themselves to achieve perfect health and must always disclose their health information to the public and continuously prove their health. from a bioethical standpoint, the world in harmony is governed by a “healthy longevity supremacy” principle, with being healthy equated to being good and right. privacy no longer exists, as it is perceived ethical for citizens to openly communicate health-related information to establish one’s credibility. moreover, there is no room for self-determination concerning healthcare because medical interventions and care are completely routinized, automated, centralized, and instantly provided. this is a situation where the community exhibits extremely powerful and effective paternalism. one can argue that healthy longevity is highly preferred. but is it right to aim for a perfectly healthy society at all costs? should we sacrifice freedom, privacy, vivid feelings, and personal dignity to achieve such a world? in our view, the answer is no, as this would require the loss of many essential values. we conclude by proposing an alternative governing principle for future healthcare, and refer to it as the “do-everything-in-moderation” principle. tuan kirie, a -year-old woman, is the main character of harmony. this work is told in the first person of tuan. it is the year , and tuan is a world health organization (who) monitoring officer who protects people's right to life. her job is to ensure that society guarantees healthy and humane lives for its members and to intervene when necessary. tuan describes her mission as follows: we are basically a flag-waving troop of diplomats-cum-peacekeepers charged with the protection of life everywhere… [t] o check whether they were ensuring their populace a lifestyle that was sufficiently healthy and human. (p. ). she was ordered to temporarily return to japan by her boss after a certain incident. shortly afterwards, more than people around the world attempted suicide simultaneously, and people, including one of tuan's best friends from high school, were successful in the attempt. an unidentified criminal declares to all citizens in the world: within the next week, i want you each to kill at least one other person… those who are unable or unwilling to perform this small task will die. (p. ). the main story in harmony concerns tuan kirie's investigation into the cause of the multiple suicides and identification of the person who issued the one-man-one-kill order. harmony's world is governed by an ideology referred to as lifeism (project itoh's coinage). lifeism is explained as being a politically enacted policy or tendency to view the preservation of health as the community's highest responsibility (p. ). harmony's world consists of communities which include people who have reached a consensus on a particular medical system and these medical conclaves are referred to as administrations (project itoh's coinage) (p. ). the medical conclaves (or administrations) have successfully realized super-medicine that uses advanced medical technologies including ai, ict, and nanotechnology. based on the welfare societies of the twenty-first century, this means the inclusion at adulthood of a constant internal monitoring network through a device called watchme, the establishment of a high-volume medical consumer system with affordable medicine and medical activities, and the provision of proper nutrition and lifestyle advice designed to mitigate predicted lifestyle-related illnesses. these activities are seen as the basic minimum conditions for human dignity (p. ). community groups cover % of the world (p. ), but some areas do not belong to a medical consensus community. for these %, super-medicine has nothing to do with citizens' lives. those who live outside medical conclaves are just like usthey catch colds, get headaches, get cancer, and die around the age of sixty or seventy (p. ). on the other hand, in medical conclaves, all people install watchme by injection into their bodies when the body finishes growing, i.e., when they become adults. watchme constantly monitors the level of rna transcription errors and immunological consistency in human blood at the molecular level. based on data sent from watchme, a little pharmaceutical factory in every household called medicare (personal medical drug purification system) synthesizes substances called medicule (project itoh's coinage) needed to eliminate pathogenic substances in the blood, and sends medicule, in a matter of milliseconds, to the area where the abnormality occurred at pinpoint and eliminates substances harmful to the human body. medicule can also issue a warning via measuring homeostasis in the body (p. ). as a result, swarms of medicines have essentially eliminated most diseases off the face of the planet (p. ). medicare performs autonomous, automatic, and instant medical interventions. medical treatments are nearly perfect and citizens' health management is almost completely realized. interestingly, healthcare professionals do not appear in the novel at all. a healthy longevity world has been established where no one is ill, the elderly are extremely active and healthy, and no one dies except in accidents or due to senility. in addition, each person's life is designed together with a health consultant. it is the extreme health-conscious society (p. ). in harmony's world, there are no interventions based on scientific technologies such as nanotechnology, let alone devices for monitoring the brains of community members. watchme is not designed to directly monitor mental activities, nor is medicule able to pass through the blood-brain barrier. behavior modification is attempted by request for self-restraint via traditional educational methods and social pressure. promotion of healthy behavior, and improvement of the environment by eliminating factors that could adversely affect mental and physical health, also lead people to live a healthy life. good health and health efforts are awarded high social assessment points, further motivating people to make efforts toward self-restraint. watchme warns people when a disturbance in the autonomic nervous system is detected and blocks harmful information, which also helps maintain peace of mind. children and young adults who cannot tolerate health education or the demand to live a public life attempt suicide or self-harm, as discussed later in sect. , unrelated to the aforementioned incident by the unidentified criminal. suicide that occurs frequently in this society is interpreted as a rebellion against controlled mental health by individuals who suffer from feelings of disillusionment and despair toward society, as well as hopeless, disconsolate feelings and a sense of entrapment. the above-mentioned medical conclaves were born after reflecting on a frightening history. in , a riot in the united states led to ethnic slaughter, and nuclear warheads spread around the world during the ensuing turmoil. this led to frequent nuclear terrorism, and radiation led to the development of cancer in many people. in addition, an unknown virus was generated due to the radiation, and many people succumbed to it and fell ill. this event is referred to as "maelstrom," meaning "the great disaster." the world in the face of an imminent health crisis established medical conclaves overnight. now, most parts of the world are free from chaos, savage, and slaughter, and a utopian world of peace, healthy longevity, and love has been realized (p. ). as it becomes apparent during the course of the story, harmony's world adopts an oligarchy political system; % of humans are basically under the watchful eye of a select group of people (p. ). this group refers to itself as the next-generation human behavior monitoring group. group members (i.e., leading senior citizens of medical conclaves, highest authorities of the medical industrial complex, and some scholars and scientists) have no intention of exploiting members of medical conclaves and created the healthy longevity society purely based on good intentions. they are solely dedicated to preventing the return of "maelstrom," and have the noble intentions of ensuring healthy longevity and world peace. no malice exits and the rulers remain concerned that all it would take is one mishap for cancers and viruses to return (p. ). we present here a brief overview of the history of medicine and medical development which are relevant to harmony's world. progress in medicine has provided solutions to various diseases including infectious diseases, genetic disorders, malignant tumors, and cerebrovascular disorders, and brought about mental stability to many people. all of these medical advances are cornerstones of the medical technologies used in harmony's world. in fact, a comparison between modern-day medical care that has evolved over the past years to that of harmony's world reveals a number of similarities. for example, wearable wireless devices that measure and display vital signs, steps, calorie consumption, and other variables have been in widespread use, with smartphones providing health guidance. continuous blood glucose monitoring devices have also been put into practical use. the use of "digital pills" started, in which a patient's medication status and physical condition are monitored by a sensor patch and ingestible sensor and sent to a central server and physicians, and improvement in medication adherence is attempted via guidance feedback. digital medicine includes a wide range of devices, such as temperature-monitoring foot mats capable of automatically detecting diabetic foot ulcers or clinically validated smartphone apps for smoking cessation combined with video tutorials and nicotine replacement therapy (ohta ; martani et al. ; biesen et al. ) . behaviors of members of the public are monitored, recorded, and aggregated for the purpose of preventing the spread of infectious diseases using smartphone location information and surveillance cameras. anti-cancer therapy with a drug delivery system using nanomolecules created by nanotechnology, micro-sample diagnosis, medical treatment by nano-robots, diagnosis/drug selection/prediction of disease onset based on genome analysis, gene manipulation by crispr/cas genome editing in fertilized eggs, and organ regeneration by regenerative medicine technology have also begun (sekai-cyoukan ; kanaya and ichikawa ; kobayashi ; johnston et al. ; sunshine and paller ; king and bishop ; itai ); medicule and genetic-level repairs of abnormalities described in harmony' world are beginning to be realized. the development of diagnostic systems using ai deep learning which had made a substantial breakthrough in the past decade is another active area of research. medical ai is used to carry out pathological diagnosis, diagnostic imaging, endoscopic diagnosis, diagnosis of and critical care for rare and intractable diseases, prediction of patient prognosis, and big data management. ai robots also provide nursing care. direct-to-consumer medicine using a digital healthcare application has also been put into motion (susskind and susskind ; kirinuki-sokuho ) . many countries guarantee affordable healthcare and good access to medical care through universal health care systems. in japan, the health promotion act ( ) states that citizens are responsible for health promotion, as well as local governments and the state, thereby setting the stage to support healthy lifestyles and promote behavior modifications. simultaneously, the act prescribes environmental maintenance to prevent passive smoking in society ). thus, we would argue that harmony's world is an sf story depicting a "brave healthy world" and can also be regarded as a speculative fiction about a health promoting society. as mentioned above, medical conclaves in harmony's world covers % of the world population and exclusively aim to promote healthy longevity and life retention. harmony's world devotes itself to realizing the health-supremacy principle and healthism, and this leads to various problematic issues, which are discussed in detail below (table ) . we can gain insight into important lessons about ethical, social, and psychological issues of our future world with strong health orientation by analyzing harmony's world in detail. first, the extreme healthoriented and health-conscious society has given rise to health worship. in this world, healthy longevity is considered more important than anything else. health the value we call health is trampling us over everything else. it means that the flood is coming. we are about to drown. (p. ). another one of tuan's colleagues, who is critical of the excessive health-oriented society, mentions: since we are both here on business, we don't have to worry about oil or cholesterol or any moral concerns. let's eat! (p. ). here, we note that cholesterol and ethical concerns are referred to in the same dimension. taking cholesterol would be considered unethical in the context of a non-work situation. also, a goody-goody woman in the community who claims that coffee addiction is a serious problem and should be banned mentions: i was just wondering if there isn't a moral problem with the taking of caffeine. (p. ). tuan describes harmony's world as follows: plague, booze, and smokes -loot too good to pass up. you could not find any of these things in japan, a nation obsessed with health, or anywhere else under admedistration rule, for that matter. all these vices, things which had gone more or less ignored in the past, had been carved in a list of sins by the all-powerful hand of medicine, and one by one, they had been purged form a society. (p. ). she also mentions: the invention of medicines had put the human body and moral precept side by side on the same lab table… the program took care of the signal sent form the body and transmitted morals in return. the moral code over percent of the people in the world had taken for their own. (p. ). substance intake that leads to poor health is a sin in harmony's world. issues surrounding alcohol, smoking, and coffee suggest that health-related perfectionism is an endless slippery slope. in harmony's world, medicine is the subject of people's faith. in this regard, tuan states: our gods, asklepios and hippocrates, watched closely over us, the 'medicine people' and in their name we built temples to clinical medicine and shock down nearly every disease ever known. our faith was such that we would continue striking them down, and so the medicine people who would never be abandoned by their gods. (p. ). the propensity to avoid caffeine and animal fats, and taking only healthy food, can be considered orthorexia nervosa, an obsession with eating only healthily or an obsessional adherence to a clean, pure, and healthy diet (mccartney ). one commentator argues that individuals with orthorexia consider their eating styles to be virtuous and wholesome, citing ethical reasons for their eating practices, and they may even proudly discuss their dietary practices because they believe their eating behaviors symbolize moral superiority (donini et al. ) . the healthy life is the good life and striving for health is one of the most important goals in life. therefore, a healthy life equates to a good life (komduur et al. ). medical conclaves do their best to help members eliminate health risks. for example, life pattern designers instill in people the notion that they should do as the medical doctor says, i.e., a type of health counseling. life pattern designers determine a lifestyle pattern to optimize both their client's health and their social assessment score by looking at information supplied by watchme (pp. - ). interestingly, emergency rooms in harmony's world are referred to as emergency morality centers (pp. - ). we argue that all aspects of life protection, medical care, and ethics are inseparable, and that healthy longevity equates to goodness in this world. beyond maintaining complete health and extending life to the extent possible, there exists a desire for immortality. in harmony's world, citizens are required to live as resources to the community. tuan explains this as follows: resource awareness. that was how people defined their obligation to society. that and the concept of a communal body. always be aware that you are an irreplaceable resource, they would tell us. (p. ). with the population so dramatically reduced, our bodies are considered public property, valuable resources to society, and as such they were something to be protected, or so went the publicly correct thinking. (p. ). medical conclaves obligate their members to live healthily as long as possible in order to maintain the community. this may be an inevitable consequence of the major disaster which happened half a century ago. but to live for society rather than oneself renders people merely a means to an end-they are objectified. nonetheless, the majority of citizens are willing to fulfill their duty of being healthy and live as long as possible for the sake of their medical conclaves. in harmony's world, one's body no longer belongs to oneself. some people, especially children and adolescents, disagree with this awareness. for example, miach as a high school student wanted a world where one's body was one's own. tuan recalled that this was what miach wanted. miach wanted a body that was hers, not beholden to a society or its rules (p. ). given that citizens are a public existence in harmony's world, individual privacy no longer exists. members of medical conclaves are required not only to keep themselves healthy at all costs, but also to continuously prove their own health to other members of the community. private information such as name, age, occupation, and social assessment score, as well as current health status, must be discussed with other members of society (p. ). in this lifeist society, where it was considered a moral obligation to reveal personal information, especially that concerning one's health, the very word "private" had the illicit stench of secrecy to it. the regional ethics committee ("administration moral consortium") tracks members' assessment scores and discloses them to the public. citizens are constantly evaluated and ranked based on their health. according to tuan: the augmented reality (ar) in my contacts kicks in. ar public metadata: everything in our world had a user review attached to it. even people had little social assessment stars stuck on them. (p. ). it can be argued that a healthy person is a person that you can trust and those who are not healthy seem to be the person who cannot be trusted. however, miach suggests her friends that people are getting tired of this telling-everyone-whothey-are-all-the-time business. what a drag it is to have you to show you are healthy and you are taking care of yourself all the time (p. ). finally, by fully pursuing health and longevity, as well as a medically optimal body, physical diversity is lost in society and the delusion of a "standardized" human body has become reality. the myth of a normalized human body was elevated to a high public standard (p. ). under the constant monitoring of watchme and advice from health consultants, obesity and emaciation were both driven out of the human experience (p. ). all citizens live within the prescribed margins of a healthy adult. regarding this, tuan states: they were all the same. everyone. i realized how bizarre a sight the medically standardized japanese populace presented. (p. ). deviations from standard physique really stood out when everyone was listening so attentively to their health consultant's advice and following their perfectly designed lifestyle plans to the letter. the range of acceptable body types grew narrower every year. (p. ). the atmosphere of conformity that the society generated was too hard to break free of. (p. ). it is clear to us that harmony's world would be intolerant of those who deviate from social standards in any respect. in addition, because of the similarity of harmony's world we would like to refer to, one of the famous english utopian novels, samuel butler's erewhon or over the range. this novel presents a society that values health above all else and punishes citizens who fall sick, blaming them for a moral failing. it seems that erewhon's society is much more unsparing against unhealthy people than harmony world (butler ) . in erewhon, sick people are treated like criminals and they are stigmatized, fined, and imprisoned for a condition they could not help, whereas criminals are treated like sick people and they are helped, attended, and given decent treatments (butler ; osborn ; jenkins ) . in other words, the law of erewhon outlaws physical rather than moral deviations, punishing disease and physical disability while treating acts of dishonesty and theft in the way that we would treat illnesses (parrinder ) . for an example a man in the last stage of tuberculosis being sentenced to life imprisonment. the rationale is that if harsh measures were not taken against disease, a time of universal dephysicalisation would ensue (parrinder ; bulter ). the judge who assumes that the sick are responsible for their acts said to the patient in chronic respiratory failure with severe tuberculosis, "you may say that it is not your fault…if you tell me that you had no hand in your parentage and education and that it is therefore unjust to lay these things to your charge. you may say that it is your misfortune to be criminal; i answer that it is your crime to be unfortunate… infliction of pain upon the weak and sicky was the only means of preventing weakness and sickness from spreading… you are a terrible and perilous character, and stand branded in the eyes of your fellow-countrymen with one of the maximum heinous recognized offences." the underlying rationale for self-responsibility for sickness seems to be based on the erewhonians strange belief that an unborn child in erewhon voluntarily choose to be born according to self-determination, even knowing very bad things could happen in their life. therefore, individuals in the society are completely responsible for their health regardless of its causes or circumstances. many ill individuals attempt to hide their disease in the society (bulter ). in the previous sections, we introduced harmony's world and brought to light various issues pertaining to health worship and the healthy longevity supreme principle, including the strong faith in medicine and healthcare, moralization of health issues, identification of health and good, people's obligation to be healthy, objectification of citizens, loss of privacy and diversity, and one's life designed by others ( table ) . all of these would be problematic from an ethical standpoint. this is because individual freedom would be deprived on many fronts. moreover, harmony's world is value-monistic, perfectionistic, and intolerant of lifestyles that are not perfectly healthy and diverse ways of thinking about healthcare. there are also other issues in this world. below we discuss some of the ethically dubious means and policies that are used in harmony's world to achieve health and longevity. the first means by which harmony's world achieves a perfect healthy utopia is the routinization and automation of healthcare delivery. the combination of watchme, medicare, and medicule have enabled the provision of instant, matter-ofmilliseconds, diagnosis and therapy, as well as prevention. naturally there exists no room for self-determination and the concept of treatment refusal or informed refusal has disappeared completely. every medical intervention has become a matter of course. all medical care procedures are quick and completely routinized. regarding issues with routinization, japanese philosopher kenji hattori rightfully noted that routine examinations are often conducted implicitly without a sufficient explanation or with little expectation that they will be refused, and everyday medical care is being provided with such routine examinations as a matter of course. he also argued that the word "routine" or routinized healthcare system would deprive patients of their initiative in medical decision making by suggesting that all other people or patients would usually do the same thing. he concluded his criticism against routinization by noting that, whenever an action or intervention is intended to be routinized, we should ethically scrutinize potential problems caused by the routinization (hattori ) . in harmony's world, which effortlessly provides citizens with adequate and affordable medical care, the pressure to use it is stronger because no financial barrier exists. in our opinion, there likely is no option not to install watchme, not to set medicare in one's house, or not to receive a diagnosis, prevention, and interventions through medicule in medical conclaves. harmony tells us that the medical conclaves are a gathering of people who have reached a consensus on a particular medical system (p. ), but it is unclear to us the extent to which the consensus is voluntary or what members have actually consented to. the second means by which harmony's world achieves a perfect healthy utopia is through cryptic and clever techniques and covert interventions, including educating individuals to voluntarily internalize social norms, incentives tied to social assessment scores, domination by the atmosphere of society, and kind watching supported by affirmative paternalism. in this world, there is no unilateral and forced order to become healthy-no enforcement exists. rather, people are skillfully driven toward the direction of health promotion. from childhood, they are repeatedly instructed and guided to suppress human instincts and impulsive desires which could result in an unhealthy state through polite education. the world has made them think that they themselves should voluntarily try to be and stay healthy. society requires citizens to voluntarily internalize morality consistent with social norms (p. ). such a framework creates a contradiction within the individual; since it is not simple or high-pressure coercion, face-to-face rebellion and rejection are psychologically difficult. miach notes: i knew how barbaric people could be. and i knew how broken they could become when they tried to repress that nature. i thought that this society, admedistrative society, this lifeist system was all wrong. a society that wanted me to regulate myself internally, even while people were killing themselves all around me. it was just bizarre. (p. ). in our view, if you failed to internalize social norms which are long taught to be self-evidently good and right, you may feel guilty or have reduced self-esteem. you may also feel alienated from others who have naturally accepted these social norms. freedom inside would be lost because you are expected to monitor yourself on your own initiative. to this, miach further notes: a society where rigid self-monitoring was the only path to peace and harmony. (p. ). one commentator states: the everyday practices involved in improving or maintaining our own health -eating, exercising, feeling emotions, managing time, sleep, and so on -do not merely constitute our health; they also constitute our identities. the combination of obligations to internalize and act in accordance with shared moral norms and a focus on individual achievement seems likely to limit human freedoms. (carter et al. ). not only educating people about social norms, but social assessment would also be an incentive to become healthy in harmony's world. careers and medical records are used to award social assessment points. in a world where you must always keep others informed of your health efforts and health condition, the majority of people may want to get as high a score as possible. families and supervisors would also pressure people to do so. there is also psychological control by "the air" or atmosphere, rather than written laws or rules. the atmosphere of harmony's world decides almost everything. invisible rules that reflect common sense and "the air" are conceptually very japanese. regarding impressions about buildings in japanese cities, tuan mentions: there weren't any laws against painting a building something more exciting, and yet here they were, an endless line of houses, all cast in bland, nondescript shades. none of them stood out against others. (p. ). in fact, there are no laws that prohibit drinking in most medical conclaves. one of tian's who colleagues noted: funny thing is, it turns out that out of all the thousands of admedistrations in the world, only twenty-six have laws on the books actually prohibiting alcohol. just twenty-six that forbid their members to imbibe. in all the rest, it's just not done. i am sure that that social assessment analysis has something to do with that. that is how the social assessment points work. as long as enough people agree about something, it starts being reflected in your points, and before you know it, you had better behave or else. and enforcement is built in. (pp. - ). good intentions and kindness make it difficult for people to counter or refute the world's health interventions. harmony's world is not malicious. it is not a cold and cruel surveillance society, but rather a warm and kind watching society. it attempts to connect all people to its server from good will and kindness. importantly, some citizens, like miach's adoptive mother, are thankful. she mentions the following to tuan: my watchme (the public correctness monitoring module in it) just warned me my emotional state was beyond acceptance parameters for interfacing with others. it is a real lifesaver, having another pair of eyes inside me to help me through these things. (p. ). but tuan felt that mankind was trapped in an endless hospital. (p. ). informational regulation as an additional kindness is also notable. it serves as a paternalistic block on harmful information. tuan notes that parts of their history have been censored, images in particular, such as horribly disfigured corpses, and that people need special clearance to see those. even what had probably been considered tame content by the standards of yesteryear was teeming with violence by the peaceful, elegant standards of society (p. ). novels are no exception. tuan indicates that any novel or essay people are about to read would be scanned in advance and cross-referenced with their therapy records (p. ). in the middle of the story, the perpetrator of a massive simultaneous suicide attempt sent a tv station a one-man-one-kill order. the newscaster who read the statement was killed on the air by the mysterious perpetrator. this killing scene was instantly censored by the ai censor. the image cut out and was replaced by other thing (p. ). but how is the ai programmed? perhaps information other than that which protects the individual is also censored, for example, the problems of medical conclaves. information can be fabricated and falsified. delivered contents could be biased in favor of the community. information critical against authorities will probably be cut off. totalitarian control of information is a substantial barrier to autonomous thought and independent decision-making. the third means by which harmony's world achieves a perfect healthy utopia is through inappropriate responses to issues. in harmony's world, there are many suicide attempts and acts of self-harm, particularly among children and young people. yuki keita, an -year-old university professor who does brain research, told tuan that the statistical rise in suicides is troubling and that pharmaceuticals and novel therapeutic treatments, as well as legal support for such treatments, would likely eventually bring these under control. he also noted that those who would want to destroy the safe and stable cycle of life are anathema to the rest of us (pp. - ) . tuan also pointed out that even without the mass simultaneous suicides, administration reports showed an increasing trend in suicide rates among youngsters. more kids were cutting their wrists, hanging themselves, and jumping off buildings (p. ). however, the cause of suicides among youngsters resides exactly in the very system of medical conclaves. in harmony's world, many cannot bear to fit into the molds society stamps for them and, according to tuan, the breakpoint of an over-considerate society is nearing (pp. - ). souls in danger of being crushed by society were, in turn, gnawing away at its underbelly. there are souls that just did not fit, soul of children yearning for disease, for damage, for pain are predominant (p. ). with wickedness in their hearts, they tried to ruin their own precious lives, and they knew what they were doing. tuan thought that something had to be wrong with this picture and even in her brainwashed society people had begun to realize it (pp. - ). a boy who committed suicide said he hated this world and did not belong to the world (p. ). we argue that the high suicide rate in harmony's world is clearly a serious side effect brought about by its social institutions, healthism, super-medicine, and lack of freedom and diversity. one could argue that it is in fact medical conclaves that require emergent intervention and correction, rather than the young wouldbe-suicidal. nevertheless, the leaders of the next-generation human behavior monitoring group carried out the "harmony project" without resolving the true causes. tuan's father, who is a brain scientist, explains the "harmony project" as follows (p. ): in controlling the feedback web in the midbrain with medicules, we found we were able to influence human decisions, emotions, and thoughts. the control of human will was a hot topic with upper leader at who and some of the admedistrations. (p. ). in those days, we picked up a lot of kids like her (miach) and put them into treatment. we gathered the ones that wanted to kill themselves, especially the ones who overate or refuses to eat, the ones who wanted to watch themselves grow weak and die. our goal was to create a harmonized will inside the human brain. we call it the "harmony program." there were plenty of kids back then who had attempted suicide more than once, just as there were plenty of them now. (p. ). because their system of values is fashioned to be in perfect harmony with society, there are far fewer suicides, and the kinds of stress we find in our admedistrative society disappear completely. (p. ). to tuan, it appeared as though her father and his colleagues of the next-generation human behavior monitoring group were trying to create a self-evident person, perfectly adapted to the stresses of the administrative society (p. ). because the correctness of society is self-evident to the leaders, they attempted to alter the values of young people by manipulating their brains. in our view, this is unilateral brainwashing and cannot be ethically justified-the "harmony project" is a typical example of an unethical human experiment. as mentioned above, harmony's world makes it self-evident that health longevity has the highest value and that all members have an ethical obligation to maintain healthy longevity and world peace. the rightness of the object of strong faith would of course be self-evident and an absolute right to believers. those who believe there is self-evident truth would rarely reflect on the justification of the very truth. there is no room for doubt or reflection on the truth. for the sake of respecting self-evident truth, self-evident persons may consider it justified to physically manipulate the brain and alter the consciousness and thoughts of young individuals who cannot accept social norms. we argue that the "harmony program" is a future-world lobotomy. instead of dealing with the real cause that is disturbing and confusing the mind, lobotomy destroys the brains of the mentally ill. before concluding our paper, we present four sf works that are deeply relevant to our discussion of harmony's world, and touch on their contemporary significance in comparison with that of harmony's world. since it is beyond the scope of this paper to introduce in detail the contents of these works, we will mainly focus on backgrounds and events described in each work. these include mental therapy by means of nanotechnology, environmental manipulation, and ultimate harm to japanese society. the first two of the four is a duology, queen of angels ( ) and slant ( ) , written by american sf writer greg bear (bear (bear , . in the world of queen of angels and slant, nanotechnology has been perfected, and humans are given the ability to change their environment and themselves at a cellular level, as well as to achieve perfect mental health. in los angels, the setting of queen of angels, % of people are receiving psychiatric treatment by 'nano therapy,' a literal brain reconstruction in which tiny surgical prochines are used to alter neural pathways. nano therapy is performed to repair genetic defects and improve work, sociality, and human relationships, and to correct the behavior of criminals. in other words, the society has reached a point where effective therapy is a necessity. people are even subjected to permanent mood adjustment. the world depicted in these books is fundamentally different from harmony's world, in that no direct interventions to the mind or brain are generally performed in the latter. nanotechnology is used not only in psychotherapy but also to transform, heal, and strengthen the body. with this technology, police officers can regenerate their severely damaged bodies many times. at the same time, their wounds and diseases are instantly fixed from inside the body using medical monitors and stabilizer infusions. indeed, nanotechnology has realized an 'in-body hospital,' and in this aspect, similarities can be found with harmony's world. murderers are given 'enforced deep therapy,' a therapy that fixes and changes them. personality correction is mandatory for all criminals, resulting in a sharp decline in crime with almost zero murder cases. this therapy is similar to the harmony project in harmony's world. the latter, however, is only used as a last resort for social survival due to its side effects (i.e., deprivation of consciousness). in the world of queen of angels and slant, discrimination and economic disparities emerge between those who receive nano therapy and those who do not. contrarily, harmony's world is free of conflict and disparities. in slant, it becomes apparent that mental therapy based on nanotechnology is ineffective, and difficult-to-treat cases in which individuals repeat anti-social behaviors ("core therapy reject," often with a long criminal history) or experience relapse and worsening of conditions ("fall-out") start to appear. the harmony project in harmony's world would have been implemented to treat suicidal people as planned, had it been successful, and perhaps, its use would have been extended to people with mild mental ailments and mood disorders. if no one followed the one-man-one-kill order in harmony's world, and had the harmony project been successful as mental therapy with no side effects, a peaceful, equal 'slant' might have been realized. as described above, nanotechnology depicted in the duology has been used in the real world for treating various physical illnesses. in the near future, it may be applied to the brain, leading to the establishment of nextgeneration psychiatric surgery. the third story we introduce, from the perspective of quasi-forced environmental manipulation for the purpose of health promotion, is "ai no seikatsu (life of love) (hayashi ) ." it is a part of a collection of short stories on the theme of "creating the future," published by the japanese society for artificial intelligence with the cooperation of science fiction and fantasy writers of japan. the main character is an obese, diabetic male writer. his cohabitating girlfriend left him, as she ran out of patience due to his sloppy and irregular lifestyle. he ended up renting a furnished apartment with home appliances in a brand-new building. however, strange things start to happen in this apartment: in the morning, the curtains open automatically and coffee is made; at pm, room lights automatically turn off. when he tries to have a bowl of rice with curry and pork cutlet for breakfast at a restaurant, his smartphone all of a sudden sends a message, "stop!" he can only buy healthy food with his smartphone payment app. the writer loses weight without even knowing it, and his physical condition improves. he begins to lead a regular life. the story ends with his illness getting better, and his work being a success. as it turns out, a woman had built a self-learning system that combines ai, iot, and ict in order to improve her former roommate's lifestyle habits. this system was manipulating the furniture and household electronics, as well as the writer's smartphone. the main character of this story loses weight and becomes successful and happy. the theme here is how ai embedded in our living environment can change our lives and values without us knowing. it is a world in which people are manipulated into selecting a healthy choice unknowingly. people are constantly monitored, warned if their health condition is poor, and are restricted in terms of access to unhealthy food and products. it is a miniature version of harmony's world, in which the healthier people are, the higher their social assessment score. all is well and good, but we are left with a complex feeling about the clever manipulation, as in the case of harmony's world. the manipulation toward a healthy lifestyle depicted in this story has already begun around the world, as with the above-mentioned japanese health promotion act ( ) (okita, enzo, asai ) . lastly, "ai houkai (ai collapse)" by rintaro hamaguchi depicts the collapse of japanese society, i.e., a crisis of ultimate harm, caused by the malfunctioning of medical ai (hamaguchi ). in , japanese society is completely reliant on a medical ai called nozomi ('hope' in japanese), which has deep learning and self-learning abilities, to carry out medical examination, vital sign monitoring, data management, and physical condition management. roughly % of all japanese medical institutions have introduced nozomi in inpatient practice. patients who visit these institutions send their physical data to nozomi from their wearable devices on a regular basis, and nozomi administers insulin, provides instructions on medication, or controls cardiac pacemakers. moreover, infrastructures related to everyday life such as finance, security, and transportation are also single-handedly managed by this ai. one day, a terrorist hacks nozomi, and nozomi suddenly starts killing patients. the medical ai with the power to decide life and death, now running out of control, brings about a crisis of collapse in japanese society. the terrorist attempts to kill all incompetent people, those who cannot work, and those with low productivity-i.e., the sick, disabled, and elderly-with the aim of rejuvenating japan's super-aged society and creating a society with high economic power and no poverty via the selection of life. this story portrays the horror of ai malfunctioning, and the crisis of ultimate harm brought about by one individual. it would not be strange for something similar to happen in the future. whatever the cause, if watchme's sensor, its network, or medicare which synthesizes medicule falls out of order, a similar crisis would easily fall upon harmony's world. harmony's world aimed to establish a perfect healthy utopia and nearly completely realized this goal. we believe the creation of the world depicted in harmony to be a great work when considered solely from the standpoints of medical science and healthcare. intuitively, the healthier we are and the longer we live, the better. however, from an ethical standpoint, it is clear that there are serious issues with the medical conclave system and unacceptable measures taken to maintain it (table ) . we argue that the existence of harmony's world cannot be accepted as is. we oppose the basic idea of creating a super-healthy society at the expense of all other worthy things, in particular, our freedom, diversity, and privacy. we need to remain a free and diverse existence with the ability to make decisions about one's own life without interference. we do not want to be monitored by a watchful society, even if the intentions are good and ultimately would make us healthier. in harmony's world, even decisions about lifestyle are outsourced. any decision must always be made together with others, and thus it is no longer possible to live according to one's idiosyncratic ways. tuan says, we are expected to always keep personal information on display, to participate in admedistration discussions on morality sessions, and to make decisions only after receiving advice from the appropriate expert. (p. ). she feels that people in administrations prefer that other people decide things for them (p. ). however, in the real world, there are things which we cannot help but decide by ourselves. in harmony, a perfect example of this is the response to the one-man-one-kill declaration. regarding this order, tuan was disgusted with the society that calls for people to gather and talk about the situation. most admedistrations had called immediate sessions to discuss the declaration, but hardly anyone had shown up. what was there to discuss? this was not something you could discuss in public. this was a decision everyone had to make on their own. (p. ). thinking alone and deciding alone is sometimes important, and we should not lose the ability and will to make decisions on our own without support from others, even if it is difficult to do so. we need not be perfect. there is no end to the pursuit of perfection concerning health and longevity in our life because there is no such thing as an objectively "long enough" life-span, and because the occurrence of diseases and disorders is best kept as close to zero as possible. the higher the quality of life, the better. the lower the individual out-of-pocket payments, the better. healthcare is the subject of infinite craving. health needs and desires are virtually limitless beauchamp and childress ) . thus, the pursuit of medical perfection will inevitably end in vain and ruin our satisfaction, acceptance, and peace of mind. social solidarity is important and good, but we argue it is not something we should aim for from the beginning. on the contrary, it is something we achieve naturally and unintentionally as a result of our activities and relations with others. medicine and ethics are different, and health and good are not identical. there is no self-evident truth in our lives. although being healthy is obviously good, health should not be regarded as the only good in our lives. the monistic claim that health has the sole highest value is unacceptable to us pluralists. we consider the infinite craving for health and healthcare problematic, but survival is a more powerful craving by human nature. naturally, some people think they are better off staying alive even in poor health rather than having to face ultimate harm, i.e., the extinction of mankind. however, the validity of this claim depends on the context of poor health. for example, persson & savulescu argued the following in their discussion of ultimate harm: "something could be ultimately harmful by forever extinguishing sentient life, or by damaging its conditions so drastically that, in general, life will not henceforth be worth living (persson and savulescu ) ." therefore, we need to consider what an acceptable condition would be in which we would want to keep living. would people want to live even if in a vegetative state, or as perhaps even a robot or zombie? the answer will be different for each individual. some may not want to keep living if their condition is such that it meets the requirements for euthanasia (e.g., unbearable pain with no prospect to recover), which is practiced in the netherlands and elsewhere. however, others might not feel the same way. with the presence of a loving family, a purpose in life, and valuable memories, some people may wish to live under any physical condition. yet, for those of us who live outside the religious culture of a monotheistic personal god, it is somewhat unrealistic to think that life in any condition is a gift of god and should thus be better lived than lost. we do not understand, frankly, the meaning of "reason for being" in a state of the eternal absence of "self," self-consciousness, and free will, which is realized in the ending of harmony. it can be argued that quality of life studies, by shifting away from ends-oriented criteria such as survival rate and life expectancy and towards subjective psychometrics including our purpose and meaning of life, they propose that a patient's quality of life must be determined in relation to his or her affective preferences and would agree with our position. they translate both the medical condition affecting survival and the affective response to that condition into a common register of temporal progression: qalys. however, the body's physical well-being, i.e. survival or length of life, is taken into account only to the extent that it impacts the patient's affective experience of the time period under consideration (eatough ) . we would argue that our perception concerning our life strongly rely on the purpose and meaning of our lives to provide us with a sense of self-worth and that our sense of self-worth would naturally have a tremendous impact on our subjective estimation of quality of life, which is sometime more important than the length of life. we do not deny the great value of a healthy longevity society. naturally, we do not want to live in a world ridden with disease, disability, pain, premature death, and separation. what is needed is a world where people are adequately healthy, while avoiding harmful health worship and healthism. how can we achieve this? in our view, the answer is provided early on in harmony. the answer is "do everything in moderation." a warrior of the tuareg, one of the characters in the tribe that does not belong to any medical conclave, suggests this. he referred to tuan and her colleagues as "medicine people." below is a conversation between the warrior and tuan. you think we bow too deeply to our gods, then? warrior in a word, yes. 'all things in moderation' you say, but you do not practice it. you are so filled with your faith that you must push it upon us as well. tuan we moderates are in the minority. (pp. - ). finally, in harmony, the top leaders of the next-gen human behavior monitoring group faced a chaotic situation in which murders and suicides among citizens became frequent after the perpetrator's declaration. they were frightened of chaos, frightened of people losing their rational minds, and frightened of riots that would lead to genocide and nukes going off all over the world again. (p. ) as a consequence, they destroyed the consciousness of all members of medical conclaves, including themselves and tuan, in order to conserve the medical conclaves. in a nutshell, harmony tells us a story in which individual consciousness was deprived forever for the sake of preserving the healthy longevity society. to us, the ending suggests that we human beings should not have individual consciousness, self-consciousness, or "i" if we hope to maintain a healthy and peaceful society. we are ambivalent about the story's ending. certainly, if unhealthy foods have disappeared and a medically ideal life plan is presented and exactly followed, then we would be healthier as a group and live longer. those who are self-aware and think freely with different interests and value systems are likely to have conflicts with others and disobey medically appropriate health advice. we are also willing to admit that there are many unhealthy people who are "prematurely dead" from inappropriate lifestyles and addictions to smoking or alcohol. if this is the case, we should not simply dismiss harmony's message as completely nonsensical or fictional. nonetheless, we cannot accept harmony's ending, because a free and autonomous agent must not be used solely for any purpose. harmony's world wrongly defines good, or being ethical, as being healthy in a very monistic way. we argue that it is ethically sufficient for us to live moderately healthily and happily, so long as we remain self-aware, self-conscious, and unique. even if society needs to preserve its values, it is wrong to destroy individual consciousness. a commentator who discusses aldous huxley's two novels argued the danger of forgetting that, in our world, complete control is impossible and that it is even more dangerous to forget why we should be happy that this is so. he added that our world is pluralistic and people hold vastly different views on what a good life entails and that a peaceful pluralism is impossible (schermer ) . in conclusion, we argue that we can lead a reasonably healthy and long life in a peaceful world, so long as everything is done in moderation, we pay some attention to our health, and we are considerate to others in accordance with the no harm principle. however, our self-awareness and consciousness could lead to an unhealthy life and premature death, which otherwise could be preventable. we would rather accept the consequences than lose our free will and self-aware consciousness. another point to note is that the extent to which social control over individuals is considered acceptable changes depending on whether or not the actions of individuals have the potential to harm others. naturally, enhanced social interventions are justified if an act causes direct harm to others, or relates to murder or terrorism. but as argued above, if people have compassion for others and act in accordance with the no harm principle, the potential for ultimate harm could be reduced. our argument is not directed at nuclear weapons or bioterrorism; it is primarily a criticism of excessive health management. just because a certain number of people are unhealthy, it does not mean the whole world would collapse (although healthcare costs would be high). those who follow their own life philosophy and avoid strict health management would have to take responsibility for the consequences of their behavior. healthcare systems could be modified in such a way that, if those individuals get sick due to their own carelessness, they can get treated at their own expense. however, it should be noted that to take a stance that all diseases are to be considered an individual's responsibility overlooks one's upbringing, peer pressure, genetic predisposition, and luck as significant influencing factors. on the other hand, people with highly pathogenic infectious diseases, such as covid- , have the potential to harm others. thus, their isolation is warranted, and the restriction and monitoring of their behaviors as long as they remain infectious cannot be avoided. yet, such requirements must be kept to the necessary minimum, and human rights and social defense must always be balanced. that said, we are not advocating that each person is free to do anythig in any way they desire. we maintain that everything should be done in moderation, and that we should pay attention to our health and be considerate to others in accordance with the no harm principle. we value 'moderation,' which we consider a moral virtue. if many people embodied this virtue, our world would be a better place. we anticipate also that the likelihood of ultimate harm would decrease. according to the analects of confucius, a classic of eastern philosophy, "there is little to choose between overshooting the mark and falling short," and "supreme indeed is the mean as a moral virtue. it has been rare among the common people for quite a long time." these ideas are the basis of the 'everything-in-moderation' principle we advocate (confucius ) . some argue that human beings lack sufficient altruism, empathy for the suffering of others, and a sense of justice, and that it is difficult to cultivate those with conventional methods of education (persson and savulescu ) . unfortunately, we cannot completely deny that assertion. perhaps humans are morally imperfect and our brains cannot stop us from harming others. that said, is it permissible to perform a lobotomy on a person's brain or perform medical interventions aimed at improving morals? or, as in the case of harmony's world, to take away self-consciousness permanently from people? at this time, we cannot agree with these. furthermore, we think that the rights and wrongs of permanently depriving consciousness from people should be determined at least by democratic means, and not by a handful of people. hope for the best and prepare for the worst: ethical concerns related to the introduction of healthcare 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jurisdictional claims in published maps and institutional affiliations acknowledgements our paper has been writing under the support of human information technology ecosystem (hite). hite is a research and development (r&d) focus area delivered by the research institute of science and technology for society (ristex), japan science and technology agency (jst). conflict of interest the authors declare that they have no conflict of interest. key: cord- - dmv ep authors: fu, xiao-wei; wu, li-na; shan, ling title: review of possible psychological impacts of covid- on frontline medical staff and reduction strategies date: - - journal: world j clin cases doi: . /wjcc.v .i . sha: doc_id: cord_uid: dmv ep like soldiers, frontline medical staff provide a first line of defense and have played a critical role in responses to the outbreak of coronavirus disease- in december . it is important to acknowledge the considerable pressure placed on frontline medical staff in the face of a new type of coronavirus that is highly infectious and for which no specific treatment is available. here, we review the various kinds of psychological problems afflicting frontline medical staff who are combatting the severe acute respiratory syndrome epidemic. these include anxiety, insomnia, depression, interpersonal difficulties, and post-traumatic stress disorder syndrome. we further present a summary of countermeasures for alleviating these problems based on our findings. these countermeasures include ensuring the provision of adequate protective gear for frontline medical staff, developing timely and clear guidelines, strengthening social support, and providing clear criteria and additional training, focusing on the choice of frontline medical staff. an understanding of the psychological impacts of an epidemic situation and of relevant countermeasures will contribute to reducing the psychological pressures on frontline medical staff. consequently, they will be able to cope better with outbreaks of infectious diseases in the future, to reduce the psychological pressure of the front-line medical staff, and to improve the treatment level. according to statistics released by the national health protection committee in china, as of march , , the total number of individuals infected with coronavirus disease- (covid- ) in the country was , and the total number of successfully treated patients was . increasing cumulative number of cured patients can be attributed to the meticulous treatment and care provided by the medical staff throughout hubei, more generally, and specifically by the frontline medical support staff in this province. as of february , , medical staff had been afflicted by pneumonia caused by covid- . more than % of the infected medical staff were from hubei, and medical workers succumbed to covid- according to statistics provided by the national health commission. an effective new treatment method for the highly infectious covid- is still lacking, and the number of infected medical and nursing staff has gradually increased. consequently, frontline medical personnel are under tremendous psychological pressure. the intense war waged against the epidemic has been ongoing for over mo. this situation raises the following critical questions. what kinds of psychological pressure are being experienced by frontline medical staff? what are the impacts of the considerable psychological pressure on frontline medical staff? how can psychological pressure on frontline medical personnel arising from the epidemic situation be mitigated or avoided? currently, pneumonia infections caused by covid- continue to occur, and there is a paucity of research on the psychological pressure on frontline medical staff. the purpose of this review is to provide guiding inputs for solving these problems. a large number of studies have reported that infectious epidemic diseases, such as severe acute respiratory syndrome (sars), induce considerable psychological pressure that continues to impact frontline medical personnel a full year after such incidences [ ] . therefore, effective and timely psychological interventions are critical for reducing the psychological pressure inflicted by the epidemic on frontline medical staff, improving the work outcomes of these staff, and reducing the long-term impacts of the epidemic on them. at an early stage of the epidemic disease, there was a lack of comprehensive awareness of the disease and corresponding protective measures. consequently, frontline medical staff experienced psychological conflicts, as being torn between their responsibilities and ensuring their own safety. the resulting psychological pressure on these individuals was extensive given the increasing numbers of infections among their colleagues. a positive development has been the government's increasing awareness and attention to the psychological stress endured by medical staff under current conditions of the covid- epidemic. on march , , the national health commission dispatched mental health professionals to hubei to provide professional psychological consultations and crisis intervention services for patients and medical staff. however, these interventions are woefully inadequate. an understanding of the sources of the psychological pressure experienced by frontline medical staff is critically required to aid the formulation of targeted policies and efforts that fundamentally reduce this pressure. in this paper, we present the findings of a review of studies that have investigated the psychological pressure and causes of stress associated with previous outbreaks of infectious diseases, such as sars and influenza a (h n ). the characteristics of studies that met our inclusion criteria are presented in table . we further examine potential solutions for reducing the impacts of stress on frontline medical staff following the current outbreak of covid- pneumonia so that they can resume their normal lives as soon as possible. these measures also provide the basis for - to measure perceived stress; : pss- , dass- to measure depression and anxiety, and ies-r to measure posttraumatic stress tam et al [ ] hong kong questionnaires the general health questionnaire was used to identify psychological distress su et al [ ] taiwan prospective and periodic follow-up design study bdi to identify cases of symptomatic depression; stai to identify anxiety; insomnia and psqi to identify sleep disturbance chan et al [ ] singapore questionnaires ghq- to detect psychiatric cases; ies to assess ptsd symptoms wu et al [ ] china questionnaires ies-r to assess pts symptoms goulia et al [ ] greece questionnaires questionnaire (part-i) and the ghq- to assess hcws psychological distress chong et al [ ] taiwan questionnaires ies to assess the frequency of intrusive and avoidant phenomena in response to a specific stressful life event; chq- to assess psychiatric morbidity wu et al [ ] china questionnaires ies-r to assess pts symptoms; ces-d to measure depression; questionnaires hong et al [ ] china follow-up interviews ies to assess symptoms of intrusive thoughts and avoidance resulting from traumatic life events. sas and sds to assess symptoms of depression and anxiety; scl- to screen for psychopathological symptoms; sf- to assess health function and sdss to access the function in a variety of occupational, social, marital, and family roles mok et al [ ] hong kong face-to-face or telephone interviews a semistructured interview, qualitative approach phua et al [ ] singapore questionnaires cope to assess coping strategies, ies to measure psychological reactions and ghq- to measure psychiatric morbidity styra et al [ ] toronto questionnaires event scale-revised to screen for ptss χ statistics to examine the association between nurses' perceived stress from sars and their corresponding chan et al [ ] hong kong questionnaires a total of of the hong kong public hospitals perceived health status during the sars epidemic kim et al [ ] seoul and in kyung-gi province psychological interventions targeting frontline medical staff prior to the outbreak of epidemic infectious diseases in the future to reduce or avoid the pressures and impacts of these epidemics on them. according to findings reported in the literature, the psychological impacts of epidemic diseases on frontline medical staff are universal. different researchers working in diverse regions on different diseases have all reached the same conclusion: epidemic diseases extensively impact frontline medical staff, with typical symptoms and a long duration. one such study entailed an investigation of frontline medical workers combatting sars in three hospitals in hong kong. a total of % of these medical workers reported that they were under great pressure, with about % of them experiencing psychological pain [ ] . the findings of a second study conducted on nurses in sars hospital wards in taiwan in found that % of the nurses suffered from post-traumatic stress disorder (ptsd) [ ] . in a third study, frontline medical staff at a singapore middle hospital were interviewed. approximately % of the respondents had ptsd [ ] . a fourth study conducted in china revealed that since the outbreak of sars in this country, about % of the medical staff have experienced symptoms indicating high levels of post-traumatic stress (pts) [ ] . moreover, more than half ( . %) of the researchers investigated in another study suffered from high levels of anxiety during the outbreak of h n [ ] . most of the specific psychological symptoms experienced by frontline medical staff in relation to epidemic diseases are relatively common and include anxiety, insomnia, depression, interpersonal difficulties, and ptsd. in addition, they experienced physical problems caused directly or indirectly by these psychological problems. during the early stages of an outbreak of an infectious disease when its spread is rampant, frontline medical staff perceive it as life threatening and experience feelings of extreme vulnerability and uncertainty, which mainly manifest as physical and cognitive anxiety symptoms. during the recovery period after the infection has been brought under control, symptoms of depression and a desire to escape are evident [ ] . mcalonan et al [ ] investigated the psychological stress experienced by frontline medical staff during the sars outbreaks in and , respectively, using the perceived stress scale, the depression anxiety stress scale, and a revised version of the events scale. the results of the study revealed that frontline medical personnel evidenced mental problems, such as fatigue, poor sleep quality, worry about health status, and august , volume issue fear of social interactions in . moreover, in , the stress had been transformed into chronic depression, anxiety, pts, and other symptoms [ ] . the findings of another study showed that exposure to severe infectious diseases could not only lead to ptsd but it could also lead to other mental diseases, such as alcoholism or alcohol dependence [ ] . chronic stress can weaken the immune system and increase the risk of disease; in addition, stress can adversely impact cognitive functions and task performance. further research is, however, needed to determine whether this kind of psychological pressure will affect the work of frontline medical staff in future outbreaks of epidemic diseases. therefore, it is very important for hospital managers to reduce or avoid the short-term and long-term psychological impacts of an epidemic on frontline medical staff. the impacts of epidemic diseases on frontline medical personnel are not limited to the duration of their outbreak, and they may last a long time. the results of a survey indicated that about % of the respondents still experienced high-level pts symptoms years after the sars outbreak during the interview [ ] . moreover, another study found that . % of sars patients still had ptsd years after the epidemic [ ] . these findings raise the following question: how long do the impacts of an epidemic endure among frontline medical staff? currently, there are no reported research results. therefore, continued investigations are required to obtain solid scientific research data and to make comprehensive preparations for future outbreaks of epidemic diseases. although sudden outbreaks of epidemic diseases have significant physiological and psychological impacts on medical personnel, they also have certain positive effects [ , ] . according to the investigated medical staff, sudden outbreaks of epidemic diseases induce a new understanding of life and interpersonal relationships, leading to positive changes relating to the setting of priorities, such as greater relaxation and having more fun in life. we hope that more frontline medical staff will experience such positive impacts of the epidemic through our efforts. the findings of our review revealed that during the outbreak of an epidemic disease, frontline medical staff experience considerable pressure emanating from different sources. to reduce the psychological pressure on frontline medical staff, it is necessary to know the sources of this pressure. the following main sources of pressure were derived from the literature. a number of studies have reported that the most disturbing aspect of disease outbreaks that creates psychological pressure is the infection of medical personnel [ , ] . such infections raise concerns among individual staff members about their vulnerability, as they share similar personal characteristics and are emotionally identified with their sick colleagues. the findings of a survey revealed that the personal risk level of frontline medical staff was an undeniable source of stress. in addition to worrying about themselves, some of the interviewees expressed more concern about the health of their relatives [ ] . they deliberately avoided close social contact to reduce the risk of transmission to relatives. consequently, their family lives and social relations were seriously damaged. in addition, the repeated emphasis on disease incidence and mortality rates during this period within the media may have been an added source of trauma for many medical personnel, either directly or indirectly [ ] . role-related pressure mainly stems from the conflict between professional responsibility and the helplessness of medical personnel. members of the public place high hopes on medical personnel and highly value their professionalism. the public perception is that professionals should be selfless and brave in battle. however, many staff members experience doubts relating to their self-efficacy and even their medical knowledge. any delay or misdiagnosis incurs a risk of becoming infected and of infecting colleagues, other medical staff, and family members, and such individuals will also be admonished by their superiors [ ] . maintaining this state of high alert over a long stretch of time leads to extreme stress for frontline medical staff resulting in a high incidence rate of anxiety disorders. august most respondents in studies that examined work-related factors complained about unclear policies, inadequate information dissemination, and a lack of feedback mechanisms. during the sars outbreak, heavy personal protective equipment (ppe), strict infection control measures, and the news of colleagues' illness or death caused tension and strained atmosphere in emergency rooms and hospitals [ , ] . one study reported that frontline medical staff feared being isolated from members of their families, their spouses, children, and society during an outbreak [ ] . during the sars outbreak, some families refused to support nurses, and some spouses of medical staff were forced to take temporary leave, while their children were asked to stay away from school. these social phenomena have significant impacts on frontline medical staff. some nurses feel very depressed because they have no place to go except the hospital, which is their workplace [ , ] . this issue raises the question of what can be done to mitigate the impact of the epidemic on medical staff. evidently, effective measures are required that target the source of the pressure, with the aim of effectively reducing the psychological pressure experienced by frontline medical personnel as much as possible. below, we present psychological measures targeting frontline medical personnel reported in the literature. practical measures for ensuring that frontline medical staff are free from infection during the epidemic are a primary concern. controlled case studies conducted in five hospitals in hong kong revealed that medical staff wearing ppe (masks, goggles, gloves, and robes) and practicing hand washing were less likely to develop sars than those who did not take such measures [ ] . ideally, full protection for frontline medical staff can be provided, preventing their exposure to the risk of infection. hospitals should therefore provide frontline medical staff with the best and most effective protective gear, such as protective clothing, n masks, goggles, boots, and surgical gloves. to meet the safety needs of medical staff more effectively, ppe of different types and sizes can be provided that cater to the needs of different people [ ] . adequate protective measures can greatly alleviate the anxiety and worry of frontline medical staff regarding the possibility of infection. in addition, taking a bath after each shift in the hospital minimizes the possibility of transmission of the sars-coronavirus- from the hospital to the families and communities of medical staff, thereby reducing the worry of medical personnel about infecting their families and friends with the virus. many medical staff were infected with covid- because the characteristic features of human transmission of covid- during the outbreak were unknown. this knowledge gap undoubtedly increased the panic and anxiety of frontline medical staff. it was impossible to provide appropriate guidelines and policies for infection management because of the lack of understanding regarding the pathogen during the early stage of the outbreak. nurses have been instructed to use ppe every hour, leading to their confusion and depression [ ] . when individuals are constantly exposed to uncontrollable events, they feel helpless, experiencing a reduced sense of control over outcomes along with depression and a lack of motivation to solve new problems [ ] . uncertainty is reduced through the possession of knowledge. during the period of the epidemic, medical personnel on the front line should be regularly provided with the latest available information on the disease. access to such information not only promotes their timely responses but it also contributes to patients' education and a reduction of their psychological problems and worries. clearly, it takes time to develop an understanding of the pathogen during the early stages of an outbreak. however, valuable experiences elicited from previous outbreaks can be compiled to form a set of centralized and integrated mechanisms for effectively disseminating information to frontline medical staff. with a gradual deepening of understanding of the disease, the corresponding process for dealing with it will also gradually improve. hospitals should apply state guidelines on the epidemic in a timely manner to impart immediate learning to frontline medical staff. accordingly, august , volume issue they will grasp the latest information and treatment methods so as to formulate possible treatment plans, infection control policies, and personal protection measures. consequently, the confidence of frontline medical staff in the face of the epidemic will be enhanced and their concerns will be reduced. having the support of their family members, friends, colleagues, and society greatly influences the willingness of medical staff to care for patients during the outbreak of an epidemic [ , , ] . a study found that korean nurses were reluctant to take care of sars patients due to lack of support from family and friends [ ] . frontline medical staff are afraid to tell others what they do because they are afraid that they and their family members will become outcasts. medical staff were not allowed to go home because they were engaged in frontline work during the covid- outbreak, which undoubtedly added to the heavy psychological pressure that they were already experiencing. on the one hand, societal resonance and support for frontline medical staff can be strengthened through advocacy and publicizing of their achievements, thereby enhancing their psychological well-being. on the other hand, the professional beliefs of frontline medical staff can be enhanced to strengthen their conviction that taking care of patients is the right choice and to convince people around them that as medical staff, they know how to protect themselves and their families from infection. the research findings indicate that the contents of training programs conducted during the epidemic entail many aspects. the imparting of different themes has varying but significant effects on frontline medical staff. for example, to increase frontline medical staff's knowledge of epidemic diseases and infection control, the speaker, who understood the needs of the trainees, designed an education plan and training program. the main content of the training program comprises information on epidemic diseases, the correct use of ppe, risks for medical staff and their families, and infection control policies and procedures. one study found that the proportion of participants who felt confident about coping with influenza after participating in the training program ( %) was significantly higher than the proportion of those with such confidence before the training session ( %) [ ] . another study emphasized the importance of providing training and education to all hospital staff members on all aspects of sars during the outbreak period to enable them to cope better with future outbreaks of infectious diseases [ ] . for example, frontline medical staff could receive training in which stress management is the core component to guide them toward mobilizing their self-efficacy. such training can be helpful for the early detection of high-risk groups that cannot effectively adjust themselves during the epidemic. there is evidence that mental health resilience, which is the ability of individuals to adjust and optimize their psychological and physical needs, is particularly important during the outbreak of epidemic diseases [ ] . in addition, training is required that focuses on strengthening the professional dignity of frontline nursing staff and alleviating job burnout. during the outbreak of epidemic diseases, job burnout is considered to be one of the most serious health-related problems faced by nurses [ ] . enhanced support and training can reduce burnout and pts. one study found that a pilot training workshop held for primary healthcare workers to manage acquired immunodeficiency syndrome patients greatly improved the participants' attitudes [ ] . such training programs lead to the development of skills and confidence among medical staff that enable them to improve their personal efficiency, enhance their professional dignity, and reduce job burnout. the selection of frontline medical staff should be done cautiously to minimize the impacts of epidemic diseases on them in the face of heightened tensions relating to future biological disasters. one study found that when working in a high-risk ward, frontline medical staff who had more contact with sars patients experienced less pain [ ] . this finding suggests that if conditions permit, medical staff who have previous experience relating to the treatment and care of patients afflicted by epidemic diseases should be selected preferentially in the future. this is because choosing such individuals would contribute to reducing the impacts of the epidemic on the frontline medical staff. a history of mood disorders and an age below years may be risk factors predicting depression, pts symptoms, and sleep disorders [ ] . in addition to the above described measures, professional psychological consultants should be included in crisis working groups at an early stage of the outbreak in view of the long-term and august , volume issue deep impacts of the epidemic on frontline medical staff [ ] . their inclusion would enable frontline medical workers to get timely and effective psychological consultations, thereby reducing the impacts of the epidemic on them. some studies have also suggested other ways of relieving the psychological stress experienced by frontline medical staff, such as humor [ ] . one such study showed that nurses and emergency workers used humor to deal with crises [ ] . other studies have found that humor has a positive meaning because it can shift perspectives, disperse tension, improve energy levels, and build cohesion within the team, in addition to strengthening an individual's physical, psychological, and spiritual abilities [ , ] . some studies have found that sars patients can also alleviate the trauma caused by the epidemic through tai chi [ ] . however, further studies are needed to assess whether tai chi can relieve psychological pressure experienced by frontline medical staff. during the initial outbreak of covid- , it was reported that frontline medical staff and patients in some hospitals in wuhan danced rhythmically in the square to relieve their inner tension and discomfort. when faced with another epidemic brought about by covid- , there was a sudden realization of how valuable the experience of beating sars was. the valuable experiences brought by each epidemic disease outbreak should be applied in planning for future pandemics. with the gradual increase of relevant research, people are becoming increasingly aware of the psychological problems endured by frontline personnel during an epidemic. therefore, one of the main goals for improving the ability of medical staff to deal with future outbreaks must be to improve their capacities to deal with psychological stress. this paper has presented the findings of a review of studies that assessed the impacts of the sars epidemic on frontline medical staff and potentially effective solutions, with the aim of reducing the impacts of future epidemic diseases on frontline medical staff and providing guiding inputs for comprehensive preparatory work. immediate and sustained psychological impact of an emerging infectious disease outbreak on health care workers severe acute respiratory syndrome (sars) in hong kong in : stress and psychological impact among frontline healthcare workers prevalence of psychiatric morbidity and psychological adaptation of the nurses in a structured sars caring unit during outbreak: a prospective and periodic assessment study in taiwan psychological impact of the severe acute respiratory syndrome outbreak on health care workers in a medium size regional general hospital in singapore the psychological impact of the sars epidemic on hospital employees in china: exposure, risk perception, and altruistic acceptance of risk general hospital staff worries, perceived sufficiency of information and associated psychological distress during the a/h n influenza pandemic psychological impact of severe acute respiratory syndrome on health workers in a tertiary hospital alcohol abuse/dependence symptoms among hospital employees exposed to a sars outbreak posttraumatic stress disorder in convalescent severe acute respiratory syndrome patients: a -year follow-up study an exploratory study of nurses suffering from severe acute respiratory syndrome (sars) coping responses of emergency physicians and nurses to the severe acute respiratory syndrome outbreak impact on health care workers employed in high-risk areas during the toronto sars outbreak uniformed service nurses' experiences with the severe acute respiratory syndrome outbreak and response in taiwan nurses fighting against severe acute respiratory syndrome (sars) in hong kong the impact of work-related risk on nurses during the sars outbreak in hong kong advisors of expert sars group of hospital authority. effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) sars (severe acute respiratory syndrome): reflective practice of a nurse manager attitude, beliefs, and intentions to care for sars patients among korean clinical nurses: an application of theory of planned behavior resilience training for hospital workers in anticipation of an influenza pandemic hospital preparedness and sars long-term psychological and occupational effects of providing hospital healthcare during sars outbreak new strategies for monitoring the health of canadian nurses: results of collaborations with key stakeholders integrated hiv prevention and care strengthens primary health care: lessons from rural haiti facing sars: psychological impacts on sars team nurses and psychiatric services in a taiwan general hospital the effect of mirthful laughter on stress and natural killer cell activity the use of gallows humor and dark humor during crisis situations an antidote for terrorism coping with future epidemics: tai chi practice as an overcoming strategy used by survivors of severe acute respiratory syndrome (sars) in post-sars hong kong key: cord- -y whb u authors: roberts, chris title: how medical education can help in a covid‐ crisis date: - - journal: clin teach doi: . /tct. sha: doc_id: cord_uid: y whb u nan i am recovering from a state of shock at what is happening with the covid- crisis and how clinical education is undergoing rapid change. here, i reflect on how medical educators can help in this crisis. as a clinical academic (teaching and research), i have responsibility for faculty development and interprofessional learning at a medical school in a researchintensive university in australia. i am one of the leadership group delivering the first year of a new graduate-entry doctor of medicine (md) curriculum, whilst also continuing with the previous md programme. the university has recently completed a major organisational restructure, bringing together medicine, dentistry, nursing, pharmacy and allied health to form one comprehensive faculty. in the medical school, we are currently grappling with: (i) preparing volunteer final-year medical students to contribute usefully to clinical service during the pandemic as 'assistants in medicine' (preinternships), as well as providing educational supervision and assessment while dovetailing with the requirements of the state-wide health education and training agency responsible for managing internships; (ii) delivering my own and supporting colleagues' bedside clinical teaching via zoom, as clinical placements have largely been suspended for the safety of students, patients and hospital staff; (iii) delivering large-scale online interprofessional activities that we have delivered face to face for years; and (iv) providing additional feedback and support to promote well-being via online learning advisors, for students who are studying remotely. clinicians are being asked whether they are prepared to work outside their usual scope of practice if they are called to be on the covid- frontline. academic clinician-educators have never worked harder. there are lessons for medical educators from previous epidemics, but there is no blueprint for a pandemic. prompted by the challenge of covid- , leading medical educators have questioned the value of the international medical education research community. i had wondered whether we already had enough research evidence and the real problem was that it was not being translated into practice and enacted into policy. our collective history of responding to change suggests that the fundamental issue of improving the methods of teaching medicine to produce doctors who are well prepared to provide highquality care has changed only incrementally in the century since the flexner reforms. contemporary competency-based medical education frameworks have improved fitness to practise worldwide; however, there was a lost opportunity on the flexner report's centennial anniversary to find collective ways to tackle the fundamental restructuring of medical education required for the st century. the covid- crisis has led to wholesale restructuring towards online teaching based on necessity rather than a settled evidence base of what works and for whom. in this time of disruption, our accreditation bodies must rapidly adapt to approaches that work best and must become more agile in order to shorten the decade-long cycle of introducing sustainable curriculum reforms. in assessment, the preponderance of rote learning in the ever-expanding discipline-based basic sciences needs to be addressed and decoupled from the dominance of the multiplechoice question in written assessment. we need to develop online forms of active learning methods, such as team-based learning, to ensure the integration of basic science into clinical competence. in clinical assessment, do we really want to see the objective structured clinical examination, a beast of its time, but increasingly flawed in its objectivity, injected into online testing or delivered under the duress of infection control? we need to agree to assessments based on the longitudinal collection and triangulation of information about a student or trainee's performance and progress, now and when the pandemic is over. such assessments have been shown to lead to higher levels of competence when compared with single-event examinations and are a better fit for work-integrated learning. none of us individually has a magic solution as to what medical educators should do in this crisis. the right groups of people can develop the evidence base by rapidly putting together reviews that address the major problems facing us in in our disrupted training programmes. it is particularly challenging for educators to think at a systemic level and include broad societal considerations, especially when taking an interdisciplinary approach to addressing concerns about the outcomes of medical schools. an interdisciplinary approach can build on disciplinary perspectives to offer creative solutions for those who grapple with the preparedness for practice of medical students. experts in the use of technology need to deliver learning environments built on what is pedagogically sound, not what is in vogue. health professional educators need to re-engage with the fundamental issues of learning and teaching that can be adapted to a time of disruption. these include how we learn, learner engagement and agency, ethical practice and a wider lens through which to view assessment. medical educators can address the modest uptake of research evidence in clinical education. knowledge translation strategies suggest that educators can increase that uptake by becoming knowledge brokers and sharing decision making with accreditation bodies, university leaders, vocational training organisations and professional colleagues. this is a tall order, yet the need has never been greater to reframe health professional education and to translate the research base into practice. the focus is not on transferring innovation, but on adapting longstanding good practice to new contexts and disseminating scholarship in a way that balances efficiency and critical review. this is a global conversation that will continue for the foreseeable future and i am keen to contribute. principles to support medical students' safe and useful roles in the covid- health workforce sars and its effect on medical education in hong kong strange days learning and teaching in clinical contexts: a practical guide medical education in the united states and canada bulletin number four (the flexner report) american medical education years after the flexner report team-based learning (tbl) in the medical curriculum: better than pbl? conducting a high-stakes osce in a covid- environment joining the dots: conditional pass and programmatic assessment enhances recognition of problems with professionalism and factors hampering student progress the intersection of assessment, selection and professionalism in the service of patient care ethical approval: not required. doi: . /tct. key: cord- - xjkv m authors: martin, anastasia; blom, iris martine; whyatt, gemma; shaunak, raghav; viva, maria inês francisco; banerjee, lopamudra title: a rapid systematic review exploring the involvement of medical students in pandemics and other global health emergencies date: - - journal: disaster medicine and public health preparedness doi: . /dmp. . sha: doc_id: cord_uid: xjkv m objectives: the role of medical students in the current coronavirus disease (covid- ) pandemic is rapidly evolving. the aim of this review is to explore the involvement of medical students in past global health emergencies, to help inform current and future scenarios. methods: a rapid systematic review was undertaken, including articles from online databases discussing the roles, willingness and appropriateness of medical student involvement in global health emergencies. data were extracted, appraised and written up as a narrative synthesis. this study was registered with prospero (crd ). results: twenty-eight articles were included. medical students played a wide variety of clinical and nonclinical roles including education and logistics, although medical assistance was the most commonly reported role. challenges included a lack of preparedness and negative mental health impacts. a total of . % of included articles about willingness found medical students were more willing to be involved than not. conclusions: this review shows medical students are capable and willing to be involved in global health emergencies. however, there should be clear protocols for the roles that they play, taking into account the appropriateness. as a rapid review, there were study limitations and more research is required regarding the impact of these roles on medical students and the system. m edical students represent a largely untapped reservoir of potential in global health issues. this group can provide a youth perspective to global health issues, contribute to research in a manner in which busy seniors cannot, and be an addition to the global health workforce in times of need. , this last point is the focus of our research. at the time of writing (april ), the world was experiencing coronavirus disease (covid- ); a pandemic with unprecedented social and financial impacts that was placing pressure on healthcare systems around the world. one solution to alleviating this pressure was by asking medical students to help. [ ] [ ] [ ] many medical schools have been suspended or moved to online platforms, leaving students with extra time on their hands; time that can potentially be spent helping. , medical students worldwide have already started moving to the frontline. in the united kingdom and brazil, for example, final year medical students have been fast-tracked to join the workforce early. , guidance toward medical students helping on the frontline has been published by several organizing bodies, but due to the dynamic situation, this guidance continues to change. , therefore, it is important to ascertain what exactly would be the best way for medical students to help. the primary role of the medical student is to learn to be a doctor, and deviations from this may have consequences both for medical students and the healthcare system around them. however, having students in the healthcare environment during a pandemic can be an increased burden to clinicians who need to invest time to teach them. consequently, students' education and patients' care may be compromised. currently, there are multiple articles describing the role of medical students in past similar scenarios, their willingness to assist, as well as opinions on what roles medical students should play during covid- . , however, to our knowledge, a systematic review that combines and assesses this information does not currently exist. filling this gap in the evidence base will help better inform what role medical students can play in future global health emergencies, including covid- . the aim of this review was to systematically assess how medical students can be involved in pandemics and global health emergencies. by outlining the roles medical students have already undertaken or potentially can undertake, the appropriateness of these roles, as well as how willing medical students are to be involved, this review can be used to guide decision-makers to design safe and effective roles for medical students in current and future global health emergencies. a rapid systematic review was used to collate, critically appraise, and synthesize the information, because it answered our question in a timely manner so our research could potentially inform the covid- pandemic. prisma guidelines were followed throughout the process. the review protocol submitted on march , , accepted april , and can be found in the prospero database [number: crd ]. four independent reviewers (a.m., r.s., i.b., i.v.) conducted a systematic search on march , . pubmed, medline, embase, and the global health database were searched for eligible studies with no date restrictions. studies were available from to march . the full search strategy can be found in appendix . bibliographies from eligible articles were screened (ie, snowballing) and the "journal of disaster medicine and public health preparedness" was hand searched through all volumes for further articles by reviewer l.b. papers were initially screened by title and abstract and shortlisted articles were screened for full-text analysis against eligibility criteria by all reviewers and any disputes were discussed. data from eligible articles were inputted into a tailored data collection form which was trialed by reviewers before use. all steps were recorded using the prisma diagram. all studies that discussed roles medical students have played or can play, their willingness to do so, or whether they should do so in an acute global health emergency, including hypothetical studies, were included. "global health emergency" was defined as any event that significantly and acutely affected the capacity or functioning of a health system in or more countries, including but not limited to: infectious disease outbreaks, natural or man-made disasters, and armed conflict. only english language articles were included. the population of interest was medical students, defined as anyone from any country enrolled in a university course (undergraduate or postgraduate) training to be a doctor who has not yet finished their medical education. articles that involved other participants (eg, nursing students) but also mentioned medical students, were included. qualitative research, quantitative research, and mixed methods studies, including systematic, scoping and literature reviews, published editorials, commentaries, and conference abstracts were included. we included gray literature to add a wider perspective. gray literature included unpublished or nonpeer reviewed papers, reports, theses, and technical documentation. studies involving nonacute events and diseases that were deemed nongeneralizable to the covid- outbreak, eg, the opioid epidemic and obesity crisis, were excluded. furthermore, studies that did not discuss medical students, eg, studies that exclusively discussed other populations (eg, veterinary, dental, or nursing students) were excluded because they did not include our population of interest. non-english language papers were also excluded due to time constraints of translating these papers. our primary outcome was to collate descriptions of the roles medical students can play in a pandemic. these were predefined in the data extraction proforma as: clinical assistance; testing; helplines; triage; raising awareness; or "other," if a different role was described, which the reviewer then specified. our secondary outcomes included the level of willingness of medical students to help, factors affecting willingness, and appropriateness of the role. data were extracted using the predefined standardized form and included: article, author, year, journal, country, article type, article design, aim, area studied (role/willingness/appropriateness/preparedness), article population, setting/context, the number of participants, methodology, outcomes, key findings, relation to past global health emergency (if applicable), comments on the role; comments on willingness, comments on appropriateness, and critical appraisal. articles were critically appraised globally and briefly judged for risk of bias; however, the full use of quality assessment tools was not feasible due to time constraints. all included papers were critically appraised using the reviewers' judgment, expertise, and by means of discussion among the researchers. the design, outcome measure, and whether the study was peerreviewed or a gray literature study played a role in guiding whether articles are of high or low quality and are reported on in the discussion section. data were analyzed thematically and written up as a narrative synthesis. our search identified articles as well as from extra sources. a total of articles were screened for title and abstract, and of those were excluded. sixty-six articles were screened for the full manuscript, and of those met the inclusion criteria ( figure ). most of the included articles had good quality of evidence but unpublished gray literature (eg, expert opinion pieces) had poorer evidence quality. table shows the baseline characteristics of all included articles. seventeen articles discussed roles, discussed willingness, and directly discussed appropriateness (chen e, goodman kw, fiore rn. involving medical students in disaster response: ethics, education and opportunity [unpublished pdf]. ; - ). as well as articles purely discussing medical students, articles also examined nursing, pharmacy, and dental students. the views of medical students across multiple years were examined with an even spread. the context of the global health emergencies is detailed in table , with articles reporting on past situations that happened and articles discussing hypothetical scenarios (table ). seventeen of the included articles discussed the roles of medical students during global health emergencies ( table ). the most commonly reported role was providing clinical assistance (n = ). in of these articles, medical students acted as junior doctors. , in , during the outbreak of the spanish flu, medical students acted as nurses and interns because the usual medical workforce were away assisting the war troops. during the kashmir earthquake in pakistan, medical students were sent to less accessible places compared with senior doctors due to their young age. in , after an earthquake, a tsunami hit chile and medical students provided medical care in remote areas. recently, it has also been hypothesized that medical students can provide clinical care by distributing medication to hospital staff and providing manual ventilation. , the role of medical students as educators through raising awareness was also highlighted (n = ). in , during the hiv epidemic, medical students developed a peer-to-peer teaching system to raise awareness and contribute to better patient care. during the severe acute respiratory syndrome (sars) and ebola outbreaks, students held (online) campaigns to promote healthier behavior. , other reported roles entailed nonclinical assistance. in , in the immediate liberation of belsen concentration camp, medical students set up an acute care hospital for emergency treatment of the inmates waiting to be transferred and a pharmaceutical dispensary. during the wtc terrorist attack, medical students participated as "runners" to carry information between triage stations, prepared food for rescue workers, worked in emergency hotlines to provide information to families of victims, fundraised, donated blood, and assisted in psychiatric disaster services (chen e, goodman kw, fiore rn. involving medical students in disaster response: ethics, education and opportunity [unpublished pdf]. ; - ). medical students who were further in their medical education were more likely to provide medical assistance (n = ). , , , , , furthermore, the challenges faced by medical students in their roles during the global health emergency was discussed (n = ). these challenges included: a lack of supervision, treating children, and prioritizing medical need. , the impact on the mental health of medical students was also highlighted, both during and after the emergency event (n = ). , , for example, during the provision of clinical care during the influenza epidemic, medical students experienced psychological distress during their fieldwork, such as fear, anxiety, depressive symptoms, despair, and panic. an article on the wtc terrorist attack suggested that the type of role played appeared to correlate with how much the students were affected. it was found that students working in fundraisers and hotlines had significantly higher symptoms. medical students who assisted in medical care at hospitals had the fewest symptoms and a greater sense of empowerment. the same article also concluded that involvement in the disaster was associated with a reinforced desire to become a physician. twelve of the included articles evaluated the willingness of medical students to help in disaster situations (table ) . most (n = ) reported quantitative data on the level of willingness of medical students to help in a disaster as shown in table . the percentage of medical students willing to help ranged between % and . %, with of the articles reporting a willingness percentage greater than or equal to %. one article reported the willingness of medical students to be involved on a different scale but equated it as "moderate willingness." one article did not provide quantitative data but reported that "the majority of students responding to the survey were willing to respond to disaster events." seven of the included articles provided reasons to explain the medical students' level of willingness. obligation or social responsibility was stated most commonly, along with altruism, as a reason why medical students were willing to be involved. concerns for personal health and safety, as well as concerns for family health and safety, were the most commonly stated reasons for students being unwilling to help (table ). some (n = ) also discussed factors that affected the willingness, which are described in table . when comparing willingness levels with medical students' confidence and knowledge in the final y of medical school, willingness was seen to be proportionally higher than knowledge. , , [ ] [ ] [ ] one article reported over % of students willing to help, but only . % of students believed they have the skills to help. thirteen articles discussed appropriateness of the roles. this included looking at medical students' confidence and preparedness for specific roles. five studies measured students' skill levels; confidence to deal with emergency outbreaks ranged between . and . % (table ). , , [ ] [ ] [ ] three articles found that students' willingness was high, but when compared with their self-perceived knowledge, they did not feel prepared. [ ] [ ] [ ] the perspective study by starr describes "for me and my classmates, knowledge of the disease we were to face so soon was limited to the contents of that lecture." the need for disaster management training was also highlighted (n = ), due to the lack of preparation of students. , only articles reported training their students before carrying out their role, and reyes described volunteers as being "quickly trained." . . there are ethical issues of involving medical students in global health emergencies, including students' safety, medico-legal issues, and health insurance of students. , , eastwood et al. specifically highlighted the importance of students to be well informed and be able to make the decision of being involved themselves. the review outlines the past involvement of medical students in global health emergencies. this can help to guide decisionmakers in choosing appropriate roles that medical students are willing to do and are prepared to carry out in a global health emergency. historically, medical students have been involved in the response to global health emergencies and pandemics in a variety of ways. , , , , , our results identified a range of roles that students have played in the past, thus highlighting the potential roles medical students are able to play in the current covid- pandemic or future global health emergencies. unlike members of the general public, medical students have attained several relevant clinical skills during their years of training, which can be useful in a situation where the healthcare system is under pressure. for example, in study, students assisted the resuscitation team, a skill students learn during medical school. the current data suggest that the most common roles played were, in fact, clinical roles. however, nonclinical roles, such as runners to carry information, assisting in hotlines, and psychiatric services were also described (chen e, goodman kw, fiore rn. involving medical students in disaster response: ethics, education and opportunity [unpublished pdf]. ; - ). a role that should not be neglected, however, is the normal role of a medical student-to learn. it is important to consider whether these alternative roles are appropriate in a specific situation. of interest, study concluded that being involved in such situations helps strengthen students' desires to become physicians, suggesting that involvement does not only help to serve healthcare systems but the students themselves. other studies have also found clinical volunteering to be perceived positively by medical students, helping them develop clinical skills and collaborate with other healthcare professionals. the range of roles described in the included articles highlights the versatility of medical student involvement. for example, articles also described that medical students further in their medical education were more likely to provide medical assistance. , , , , , one study found younger students were involved in more educational roles as first year students helped by raising awareness for hiv. this demonstrates that involvement in the response to global health emergencies is not limited to advanced medical students and that younger students, even though they may have less clinical knowledge, can still offer a valuable contribution when given an appropriate role. furthermore, the variety of roles can allow certain challenges to be avoided. for instance, if a hospital is deemed to have a lack of supervisors, a commonly faced challenge even during nonemergency times, then medical students can get involved in a nondirect clinical role. several challenges were described as students undertook these roles, including a lack of supervision, a lack of experience, and a negative impact on their mental health. , , , pro-actively addressing these challenges will not only enable any involvement medical students have in the current or any global health emergency to be efficient and effective, but will also reduce any detrimental effect on the medical students' mental health and medical education. our results demonstrate that such involvement in highly stressful situations can lead to anxiety, depressive symptoms, and emotional distress. , , consequently, stress can lead increased severity (very dangerous and contagious compared to very infectious) , greater level of knowledge , , pediatric patient care history of a severe illness to them or family member respiratory transmission compared to contact less than y old medical role (compared to admin role) more than h of previous volunteering natural disaster (compared to an infectious disease) ppe availability training before work travel compensation well run organization table overview of the students' confidence in their skills, knowledge, and education to deal with global health emergencies article id context (self-perceived) percentage of students gouda ( ) sufficiently skilled to respond to an emergency outbreak . kaiser ( ) sufficiently skilled to respond to natural disasters . sufficiently skilled to respond to pandemic influenza . mortelmans ( ) sufficiently educated to help in h n pandemic mortelmans ( ) sufficient knowledge on disease management in disaster situations mortelmans ( ) sufficient skills to deal with infectious outbreak . academic commitments cv improvement and gaining future contacts family health and safety concerns increase self-confidence in sim situations family/social commitments obligation/social responsibility , financial implications professional and skills development inefficiency reduce guilt about less fortunate lack of confidence in skills sense of ethics lack of information needless sacrifice personal health and safety concerns work commitments disaster medicine and public health preparedness to a higher prevalence of university student dropout. to avoid negative mental health impacts, adequate support must be provided to medical students if they are to be involved. this also highlights the need for further research that explores the short-and long-term effects on the students involved, and more specifically, the impact on their mental health and their future careers. ultimately, the roles of medical students might be very context-specific. a limitation of this review is the lack of data providing specific recommendations on the most suitable role of medical students in the differing types of global health emergencies. a further limitation is the paucity of data evaluating the roles medical students have previously undertaken. it is important to ascertain how effective medical students were in the emergency response and whether they made a real impact. therefore, further research into the effectiveness of medical student involvement would be beneficial, and perhaps comparing this to global health emergencies that have not involved medical students. eleven of the articles ( . %) discussing willingness reported a greater willingness than unwillingness among medical students to be involved in the response to pandemics and global health emergencies. only hwang et al. found the opposite pattern of only % of students willing to be involved. this article focused specifically on whether medical students would enter a hypothetical closed area with a highly infectious disease and high fatality rate. of interest, only % of those not wanting to enter stated fear of safety as their reason, and in contrast, % were unwilling to enter due to inefficiency. this highlights the importance of organization when adding medical students to the workforce. obligation to help and social responsibility were the most commonly stated reasons for wanting to help in such situations (chen e, goodman kw, fiore rn. involving medical students in disaster response: ethics, education and opportunity [unpublished pdf]. ; - ). [ ] [ ] [ ] this suggests that medical students believe they "should" volunteer, rather than "want" to. in turn, this can lead to students putting pressure on themselves to help, when they may not feel ready to do so, which will potentially hinder any positive effect they may have on the disaster situation, and could be dangerous for patients. making students aware of both their mental and physical capabilities is essential to ensure that they only volunteer if they are prepared. certain factors encouraged student involvement, including a greater level of knowledge and being trained and receiving travel compensation. the most common discouraging factor was the severity of the event or outbreak. , , , , it is important to consider these factors, specifically by emphasizing encouraging factors, such as travel compensation and training before work. this, along with minimizing any discouraging factors where possible, will ensure the maximum number of medical students will be prepared to volunteer and, thus, increase the size of the workforce. it is vital to support medical students to gain the extra help that is needed, as medical students would be going beyond their role of simply learning and being a student in an already stressful situation. considering ways to decrease stress can help reduce negative mental health effects both during and after the event. factors such as age and previous volunteering experience can be used to help target a specific cohort of medical students to volunteer with specific roles. for instance, initially approaching medical students who are already experienced with volunteering, before approaching the remainder of the medical student cohort if required, may be a more beneficial way of organizing a volunteering scheme. several studies found students reported that their willingness was higher than their knowledge and readiness. , , [ ] [ ] [ ] this illustrates the need to better prepare medical students, who can indeed be a great addition to the workforce in disastrous situations. knowledge about a specific disease is vital to students undertaking both clinical and nonclinical roles, such as raising awareness. this preparation could be spread throughout their medical school education, or delivered immediately before a specific role. , the latter, however, would require further resources acutely during a disease outbreak, which may not be available. the studies discussing willingness were limited by their methodology as they only measured medical students' responses and did not compare with the general population. furthermore, most studies were hypothetical situations and, therefore, students may answer differently if the real situation arose. furthermore, there was no standardized survey of measuring willingness between all studies and this, along with the studies not comparing to a general cohort, limited the ability to carry out a meta-analysis. three articles reported that, although medical students may have been willing to help, they may not have necessarily felt prepared or felt that they knew enough. [ ] [ ] [ ] the ethical considerations of the roles of medical students must be thoroughly explored before students are invited to help, especially where patient care may be compromised. during times of nonglobal health emergencies, the primary role of medical students is education and to learn to be a doctor, which can take between and y. the need to help the workforce in times of crisis must be balanced with the educational and wellbeing needs of the medical student to complete their training. whereas "learning on the job" can be an invaluable experience, the safety of both medical students and their patients must be considered. however, there was considerably less data on this aspect in the evidence base. this rapid systematic review provides an overview of the previous involvement of medical students in global health outbreaks, which, to the authors' knowledge, no other systematic review has previously discussed. the inclusion criteria were kept broad, which allows the results to be generalizable for other future global-health emergencies, as well as the current covid- pandemic. the predefined aims and objectives were answered and previous roles and their appropriateness were summarized. although completing a rapid review may help inform the current covid- pandemic, this study design also has limitations, especially due to time constraints. first, although each included article was discussed and data were extracted by authors, no full formal critical appraisal or risk of bias tool was used. second, due to the data not being sufficiently homogenous, a meta-analysis was not possible. third, gray literature was included, which may yield lower quality evidence; however, on balance, it provided an invaluable insight into previous roles that had taken. the fact that medical students are the authors of this review is both a strength and a limitation. the authors are themselves experiencing the possibility of being involved in the current covid- pandemic and, therefore, have insights into the difficulties and lack of data about this process. much has been written on previous roles students have taken in previous situations, and this can be used to inform future policy regarding covid- and future global health emergencies. when designing the role itself, the willingness and preparedness of the medical students should be strongly taken into account. this will ensure specific roles are safe and within appropriate student competencies. medical students should be given clinical roles within reason, and educational and social media roles may be given for the less clinically confident. this highlights the importance of co-production and including students themselves when planning these roles. the authors of this review experienced firsthand the difficulty in defining roles for medical students in the current covid- pandemic, within their own universities and hospital trusts. this highlights the lack of robust policy and knowledge surrounding this topic, and the consequent unnecessary delay in the use of a skillful resource. we thoroughly encourage governments to have predefined policies for medical schools if such events arise again. an infographic was made to summarize the findings of this review (figure ). future research to both describe and evaluate the effectiveness of medical students' roles during the current pandemic should be carried out to help guide future pandemics. this research should also encompass the safety of these roles and the shortand long-term effects on the medical students themselves. furthermore, there is a dearth of literature on the ethical aspects of medical student involvement and duty during such situations. this has been discussed when looking at residents/ junior doctors participating in such situations, but not for the medical student cohort. finally, this area of research can be expanded to provide information on the role of other healthcare students in global health emergencies, namely nursing, pharmacy, dental, and veterinary students. medical students are a willing and resourceful potential addition to the healthcare workforce during global health emergencies. their involvement is vast and many roles have been identified; however, adequate and proactive support must be provided to help them overcome any challenges they may face. choosing the perfect role is very subjective to each emergency. therefore, it is vital to consider available resources, students' opinions, and the nature of the emergency itself when planning roles. future research should be targeted at filling important gaps in the literature discussed above, including evaluating the effectiveness of different roles undertaken by medical students in global health emergencies and the ethical issues regarding the appropriateness of the medical students' involvement. the international federation of medical students' associations. ifmsa policy proposal meaningful youth participation medical student research: 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trajectory of university dropout: investigating the cumulative effect of academic vulnerability and proximity to family support are saudi medical students aware of middle east respiratory syndrome coronavirus during an outbreak? planning and incorporating public health preparedness into the medical curriculum using an h n vaccination drive-through to introduce healthcare students and their faculty to disaster medicine it is time to prioritize education and well-being over workforce needs in residency training or student doctor[title/abstract] or medical student*[title/abstract])) and (pandemic[title/abstract] or disease outbreak[title/abstract] or disaster medicine[title/abstract] or epidemic[title/abstract] or acute respiratory infection[title/abstract] or severe acute respiratory syndrome[title/abstract] or global health emergency ovid: ((medical student or student doctor or medical student*) and (pandemic or disease outbreak or disaster medicine or epidemic or acute respiratory infection or severe acute respiratory syndrome or global health emergency or public health emergency)).ab,ti all authors have completed the icmje uniform disclosure form at http://www. icmje.org/coi_disclosure.pdf and declare: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous y, no other relationships or activities that could appear to have influenced the submitted work. the lead author affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained. key: cord- -t zvz zg authors: liu, yin; wang, li; chen, long; zhang, xianhong; bao, lei; shi, yuan title: mental health status of paediatric medical workers in china during the covid- outbreak date: - - journal: front psychiatry doi: . /fpsyt. . sha: doc_id: cord_uid: t zvz zg background: a novel coronavirus (covid- ) outbreak has occurred in china, and national medical workers have been thrown into this silent battle. paediatric medical workers have been an important part of this battle and under enormous pressure. this paper evaluates the depression, anxiety, and stress of paediatric medical staff during the epidemic and examines related impact factors. methods: we conducted this study using online questionnaires via social networking software during the week of feb. to feb. , . the -item depression anxiety stress scale (dass), which is a revised, simplified version of the original dass developed by lovibond et al., was used in this study. results: among all , respondents, . %, . %, and . % had depression, anxiety and stress symptoms, respectively. males, doctors, individuals aged between ‑ years, those with senior job titles, those who had contact with patients with confirmed or suspected cases of covid- , those who worked on the clinical frontlines fighting the epidemic and those who had experience combating similar outbreaks were more likely to have depression, anxiety or stress symptoms. respondents in beijing and chongqing had lower negative psychological symptom scores than the national average. conclusion: during the covid- outbreak, depression, anxiety and stress are present to varying degrees among paediatric medical workers across the country. psychological crisis interventions should be implemented to protect the mental health of paediatric medical workers during and after the epidemic. in december , a novel coronavirus outbreak occurred in wuhan, hubei, china. the virus has rapidly spread across china and to at least countries ( ) . by march , , , confirmed cases (nearly child cases) and suspected cases, among which , cases were cured and , were fatal, were reported in all provinces of china. since the outbreak, china's health authorities have taken a series of actions to control the spread of infectious diseases. however, during this dangerous epidemic situations, medical treatment resources were in short supply. medical staff were exhausted, and facing enormous pressure ( ) . previous studies have shown that during and after the outbreak of sars in , medical workers suffered from different degrees of psychological distress, such as stress, anxiety, and depressive symptoms, due to a lack of mental health care. these mental health problems not only affect medical workers' attention, understanding, and decisionmaking ability, which might hinder the fight against covid- , but also could have a lasting effect on their overall wellbeing. therefore, psychological crisis intervention is another important task in the fight against covid- . the chinese government has incorporated psychological crisis intervention into the overall efforts for epidemic prevention and control ( ) , but because of the relatively low incidence of covid- infection in children, psychological intervention for paediatric medical staff has not attracted the attention of the government and relevant departments. thus far, there is no relevant report on protective measures for the mental health of paediatric medical workers. according to the "white paper on the current situation of paediatric resources in china" issued by the paediatric branch of the chinese medical association in , the number of paediatric medical staff in china is seriously insufficient. this deficit is associated with high occupational risk, conflict between doctors and patients, heavy workload, long working hours, and low pay, among other factors. because of this medical environment, paediatric medical staff experience greater long-term psychological pressure than other chinese medical workers. in addition, respiratory tract infection is a common clinical disease in children, and more than half of paediatric outpatients have fever and cough symptoms, which are also common symptoms of covid- . after the outbreak of the epidemic, due to fear of the disease, a large number of children with fever and cough symptoms poured into paediatric clinics, making paediatric medical resources more scarce and placing paediatric medical staff under great pressure. protecting the mental health of paediatric medical workers during and after the epidemic is thus important for the control of the epidemic and for workers' own long-term health. an investigation of the mental health status of paediatric medical staff is the first step. in this study, paediatric medical workers in china were surveyed to understand the severity of depression, anxiety, and stress among such workers during the epidemic in order to provide a scientific basis for psychological protection and intervention. to collect data, this study used a web-based cross-sectional survey based on the internet survey of chinese medical doctor association (cmda), an ongoing, online mental health-related survey of chinese paediatric medical workers. this web-based survey was sent to several wechat working groups of paediatric medical staff involved in the cmda. paediatric medical workers answered the questionnaire by scanning the quick response code (qr code) of the questionnaire address or clicking a link. two members of the research team answered participants' questions online during the survey completion process. to encourage participation, all participants could receive reports about their mental health after completing the survey. this survey was completely voluntary and non-commercial. the doctors and nurses who were interviewed were told that the results of the survey would be used for scientific research, but they were not informed of the specific research plan. all respondents were anonymous. participants answered the questionnaires anonymously on the internet from feb. to feb. , . the questionnaire included questions on sociodemographic characteristics, anxiety symptomology, depressive symptomology, and stress symptomology. sociodemographic characteristics included gender, age, identity (doctor/nurse), professional title, education, health condition, area of work during the coronavirus outbreak, experience with similar public health events and frontline anti-epidemic personnel status (exposure to patients with confirmed or suspected cases of covid- ). a total of , respondents (doctors or nurses) completed the questionnaire. to ensure the quality of the survey, we set the following exclusion criteria: over years old, questionnaires completed in less than min or more than min, invalid content and incomplete data. finally, a total of , valid questionnaires were included in this research ( figure shows the case selection algorithm). the -item depression anxiety stress scale (dass), which is a revised, simplified version of the original dass developed by lovibond et al., was used in this study ( ) . the dass- has the same reliability and validity as the full version ( ) . the chinese version of the dass- was used in this study ( , ) . the dass- includes three subscales with a total of items that investigate the degree of depression, anxiety and stress. items are scored on a point scale ranging from - , where is never, is sometimes, is often, and is almost always or always. the sum of the item scores for each subscale multiplied by is the subscale score, which ranges from - points. for the depression subscale, a score of ≤ points is normal, while a score of -l points indicates mild depression, - points moderate depression, - points severe depression, and ≥ points very serious depression. for the anxiety subscale, a score of ≤ points is normal, while - points indicates mild anxiety, - points moderate anxiety, -l points severe anxiety, and ≥ points very serious anxiety. for the stress subscale, a score of ≤ points is normal, while -l points indicates mild stress, - points moderate stress, - points severe stress, and ≥ points very serious stress. the higher the score is, the more serious the degree of depression, anxiety or stress ( ). cronbach's alpha coefficient of this questionnaire is . . this study was approved by the ethics committee of children's hospital of chongqing medical university. electronic informed consent was obtained from each participant prior to starting the investigation. participants could withdraw from the survey at any moment without providing any justification. wps software was used for data entry in this study. after the data were checked, they were entered into spss . for statistical analysis. quantitative variables were described by the mean and standard deviation, while qualitative variables were described by the frequency distribution. t tests and variance homogeneity tests were used to compare the mental health status scores by participants' demographic characteristics. the distribution of different mental statuses by demographic characteristics was determined by chi-square tests. the results were corrected for multiple comparisons. p < . was statistically significant. a total of , valid questionnaires from provinces or political areas across china (except hong kong and taiwan) were included in this research. among the respondents, . % were female and . % were aged - years. a total of . % of the respondents were doctors, and . % were nurses; . % had experience combating similar outbreaks. a total of . % had been exposed to patients with confirmed or suspected cases of covid- (table ) . one respondent was confirmed to be infected with covid- , two respondents had relatives who were suspected or confirmed to be infected with covid- , and five respondents were under isolation observation because of suspected exposure. the regional distribution of the survey population is shown in table . descriptive statistics of anxiety, depression, and stress as shown in table , the total possible questionnaire score ranges from - . the higher the score is, the more serious the degree of depression, anxiety, or stress. the results indicated that the mental states of most participants were normal. the average depression score in the whole cohort was . ± . , the average anxiety score was . ± . , and the average stress score was . ± . (table ) . however, there were some differences among participants in terms of the degree of mental health problems. a total of . %, . %, and . % of the whole cohort had symptoms of depression, anxiety and stress, respectively, and the rates of depression, anxiety and stress varied by demographic characteristics ( table ) . we found that men's average stress scores were higher than women's (p= . , table ). there was no significant difference in the average score of depression, anxiety or stress among medical staff with different educational levels, but trends towards an increased score of depression and stress were observed with increasing educational levels ( table ) . compared with nurses, doctors had higher average scores of perceived stress (p= . ) and depression (p= . ) ( table ) , as well as a higher prevalence of anxiety (p= . ) and stress (p= . ) ( table ) . compared with junior and intermediate workers, workers with senior job titles had higher average scores and probabilities of perceived stress (p= . in tables and ) and depression (p= . in table ; p= . in table ) (multiple comparisons were conducted). respondents between the ages of and years were more likely to report stress than those < years old (p= . in tables and ) (multiple comparisons were conducted); respondents over years old showed normal levels for these three mental states (tables and ) . respondents who had contact with patients with confirmed or suspected cases of covid- working on the clinical frontlines were more likely to have higher perceived stress, anxiety, and depression (p= . in tables and ). in addition, respondents who had previous experience combating similar outbreaks showed higher perceived stress (p= . in table ; p= . in table ). there was no significant difference between the scores of respondents from four provinces (chongqing, shandong, guizhou, and beijing) and the national average, while respondents from other provinces, especially hubei province, had higher scores than the national average ( table ) . with the outbreak and spread of the novel coronavirus epidemic in , the number of confirmed cases is rising rapidly daily. to quickly control the epidemic and save the lives of infected patients, chinese medical workers have been extremely busy doing hard work in the past months and have made great sacrifices. as of feb. , according to the report of the joint inspection expert group of china and the who, , medical staff from medical institutions in china have been infected with covid- ( , confirmed cases, , clinically diagnosed cases, and suspected cases); more than % of infected medical staff ( , cases) came from hubei province, and of them have died. as was previously known, paediatric medical staff in china face several hurdles, including high occupational risk, multiple conflicts, heavy workload and long working hours. since the outbreak, due to overwork and the potential risk of infection, paediatric medical staff may be under great psychological pressure that may not disappear immediately with the end of the epidemic. some of them are at risk of experiencing posttraumatic stress disorder symptoms. to understand psychological problems among paediatric medical workers during this epidemic and to explore effective psychological intervention measures, we surveyed , paediatric medical workers from provinces or political areas in china by using an internet application. we found that . % of the respondents had depressive symptoms, . % had anxiety symptoms, and . % had stress symptoms. the rate of depressive symptoms is higher than that found in a previous study on the general chinese population prevalence of depressive symptoms ( . %) ( ) . a comparison of the dass- scores of paediatric medical staff in different regions revealed that among the provinces with a sample size of more than respondents, there was no significant difference between the scores of respondents from four provinces (chongqing, shangdong, guizhou and beijing, respectively) and the national average. among these provinces, respondents from chongqing and beijing scored slightly lower than the national average, while those from shangdong and guizhouo scored slightly higher than the national average. in particular, the scores of respondents from hubei were significantly higher than the national level, likely due to the serious situation in hubei ( ) . chongqing and beijing are both municipalities that are directly under the central government of china. the numbers of confirmed cases in these two cities were among the highest in the country, but the perceived stress of paediatric doctors and nurses was generally low, which may be related to the professional medical backgrounds of medical staff and their more objective cognition of disease risk ( ) . this finding may also be attributed to the early awareness of the particularity and severity of the epidemic by local governments as well as the timely and detailed working guidelines they developed. great differences in the occurrence of psychological problems among different medical workers have been documented in previous studies ( ) ( ) ( ) . our study also found that frontline paediatric medical workers who were fighting against the epidemic had higher stress, anxiety, and depression because ( ) ( . ) ( . ) ( . ) ( . ) ( ) ( . ) ( . ) ( . ) ( . ) ( ) ( . ) ( . ) ( . ) ( . ) they were the highest-risk group for covid- infection, and medical staff who had experienced similar events showed higher levels of perceived stress due to their higher awareness of the risk of disease. in addition, men's perceived stress perception was higher than women's, and doctors had higher perceived stress and depression levels than nurses. by comparing the emotional states of paediatric medical staff in different age groups, we found that stress were most likely to occur among -to -year-olds. we also found that the more senior the job title was, the higher the depression and stress scores. an analysis of the reasons for these findings showed that most of the paediatric medical staff between - years old, with senior job titles, especially in men and doctors, were the main persons in charge of clinical work, and they needed to take more responsibility and bear greater pressure. these meaningful social and demographic characteristics analysed above can guide us to develop psychological intervention programmes that are tailored to address the different emotional symptoms and needs of paediatric medical workers. our study had several limitations. first, although a national sample was obtained, the sample of respondents affected by covid- as of the time of the survey was not large enough. second, we used online questionnaires through social network software and could not fully explain the questionnaire to the respondents. third, we did not have mental health data from non-epidemic periods for comparison; conducting such a comparison will be our next step. these factors may have biased the results of the study. during the covid- outbreak, medical staff have taken on high-intensity work every day. in addition to suffering physical exhaustion, they have suffered psychologically. similar to everyone else, they feel depressed, anxious, and stressed. paediatric medical staff are a group that experiences high psychological pressure on chinese medical teams, which may have further increased their psychological burden since the outbreak. depression, anxiety, and stress symptoms due to natural disasters can be prevented from becoming chronic problems through appropriate mental health management, prompt responses, efforts to meet individualized needs, and efforts to provide appropriate interventions after the identification of symptoms of anxiety, depression, or stress ( ) . understanding the emotional problems that different groups may face and implementing corresponding psychological intervention programmes to protect the psychological health of paediatric anti-epidemic personnel can have a profound impact on the fight against the epidemic and workers' own longterm health ( ) . the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. the studies involving human participants were reviewed and approved by ethics committee of the children's hospital of chongqing medical university. written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. lb and ys conceptualized and designed the study, drafted the manuscript, and approved the final manuscript as submitted. lw and yl designed the data collection instruments, coordinated and supervised the data collection, carried out the initial analyses, drafted the manuscript, and approved the final manuscript as submitted. lc and xz coordinated and supervised the data collection and approved the final 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original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -qctfl r authors: monday, lea m; gaynier, anthony; berschback, madeline; gelovani, david; kwon, henry y; ilyas, sahrish; shaik, asra n; levine, diane l title: outcomes of an online virtual boot camp to prepare fourth-year medical students for a successful transition to internship date: - - journal: cureus doi: . /cureus. sha: doc_id: cord_uid: qctfl r introduction changes in medical education and health care delivery have limited the ability of fourth-year medical students to perform the role of an intern prior to graduating from medical school. to address this issue, many schools have instituted residency preparation courses (sometimes referred to as boot camps) particularly for students entering surgical fields. courses for students entering nonprocedural fields are less common and most assess increases in self-reported confidence without providing objective evidence of a gain in knowledge or skills improvement. materials and methods we used a plan, do, study, act (pdsa) model to develop and pilot cycle of a nonprocedural internship preparation elective in . feedback was used to refine the course and map sessions to core competencies outlined by the accreditation council of graduate medical education (acgme) for pdsa cycle . the curriculum was adapted for remote synchronous delivery due to the coronavirus pandemic in spring using a combination of didactic lectures containing embedded polls and case-based role play responses using a chat box. students completed anonymous surveys assessing self-perceived levels of confidence, as well as an objective comprehensive assessment after course completion. results a total of students participated in the course. pre-session confidence was lowest for transfusion medicine, handling pages from nursing while on call, and knowledge of the role of a chief resident. a statistically significant increase in median scores for self-reported knowledge or confidence was seen in all sessions. the percentage of students reporting that they were either confident or extremely confident also increased significantly after each session (p< . for all). all sessions analyzed were rated as useful or extremely useful by more than half of the students, and % of the students scored % or higher on the comprehensive course assessment. conclusions an online virtual synchronous boot camp increased students’ confidence in handling common topics encountered during residency and demonstrated an appropriate gain in knowledge using a comprehensive assessment. we were able to adapt our curriculum to a remote model and will likely keep several sessions in an online format in the future. the goal of medical schools is to prepare students to be doctors ready for postgraduate training in their field of choice. since the publication of flexner's x model in , undergraduate medical education (ume) curriculum has been divided into two phases: the preclinical phase, which is focused on pathophysiology and medical theory, and the clinical phase, which is focused on training in surgical and non-surgical fields. in the united states, the fourth year of medical school varies in content and structure to maximize student freedom to choose elective courses and non-clinical pursuits [ ] [ ] [ ] . ideally, students will have opportunities during clerkship to care for patients from admission until discharge and immersed in patient care as if they are already interns. however, the degree of student immersion and experience deviates from this ideal due to several factors. patient length of stay has shortened, procedures are increasingly outsourced to interventional subspecialists, patient handoffs are frequent, and care is generally more fragmented than decades ago [ ] . in addition, changes in medical education such as duty hour regulation and limited privileges within the electronic medical record (emr) reduce student autonomy. the degree of student experience with the emr varies. in one recent survey, fewer than % of students on their obstetrics clerkship had entered orders and less than half ( %) had written a history and physical note in the patient chart [ ] . in another large survey of around , graduating medical students, % had entered information of some type into the emr on their internal medicine clerkship or subinternship; however, % had never entered admission orders [ ] . yet students are expected to step into residency on july and care for patients, including night shifts and with varying degrees of supervision and support. these challenges result in increased stress for interns, educational difficulties for program directors, and, most importantly, potentially unsafe care for patients. in order to address the difficulties in the transition to internship, some medical schools have created capstone courses focused on communication, common problems, procedural skills, or some combination of topics for students entering internship. elective courses for students entering dentistry [ ] , surgery [ ] [ ] [ ] [ ] [ ] , and obstetrics and gynecology [ ] [ ] [ ] have been shown to increase confidence in procedural skills and communication. similar courses in non-surgical specialties are not as common; however, their numbers are increasing. boot camp experiences have been replicated in family medicine [ ] , pediatrics [ , ] , emergency medicine [ ] , and with students entering a mix of fields including internal medicine [ ] [ ] [ ] . a common result is an increase in the level of confidence or preparedness reported among students. in a meta-analysis, boot camps were shown to be an effective educational strategy to improve learners' clinical skills, knowledge, and confidence [ ] . medical students graduating in were removed from all direct patient care and in-person educational activities due to the coronavirus disease (covid- ) pandemic. unfortunately, this drastically altered the content and delivery of courses at medical schools throughout the united states, including internship preparation courses. remote delivery of one boot camp course has been briefly reported for students entering pediatric residency [ ] . as of may , there is no published work describing the logistics and results of executing a nonsurgical internship preparation course open to all medical students regardless of match specialty. we describe a one-month intensive course entitled, "internship boot camp", which was designed after a previous pilot and adapted to be delivered remotely during the covid- pandemic. this online virtual synchronous interactive elective was taught by faculty, residents, and near peers, with the goal of assisting fourth-year medical students with the transition to internship. the goals of this study were to determine the effectiveness of the internship boot camp on three measures: ( ) level of confidence about entering their internship, ( ) ability to develop a framework for responding to common challenges, and ( ) ability to demonstrate an appropriate gain in clinical knowledge using a comprehensive post-course assessment. here, we report the outcomes of a non-surgical intern preparatory course. faculty from the department of surgery at our institution had been offering a procedural boot camp focused on laparoscopic skills and knot tying since ; however, no such course existed for students entering nonprocedural fields. starting in spring , we developed and piloted a nonprocedural preparation elective utilizing pdsa (plan, do, study, act) methodology ( figure ). fifty-three students from the wayne state university school of medicine who matched into a variety of non-surgical residencies were offered an ungraded elective entitled, "intern boot camp." before developing the boot camp curriculum, a needs assessment (a systematic process for determining and addressing needs or "gaps" existing in the knowledge and skills of medical students) was conducted through a voluntary online survey. input was provided by clerkship directors, residency program directors, residents, recent graduates, and our graduating medical students. next, a targeted needs assessment was performed to determine specific deficiencies, and course sessions were created with specific goals and objectives to meet the deficiencies identified. for example, if program directors answered that new interns did not know how to write admission orders, the specific deficiencies were asked on the targeted needs assessment (such as not understanding order sets, or diet and activity orders). this course was approved by the curriculum management committee and added to the course catalog for april and may of . faculty, chief medical residents, and residents taught each session. oversight was provided by the course director. course content was delivered using a variety of modalities including a combination of didactic powerpoint lectures, procedures on lightly preserved cadavers, a session on how to teach and give effective feedback, and case-based role play for handling difficult conversations, medical emergencies, and simulated pages from nursing. lastly, students completed symptom-based modules (which covered common clinical scenarios encountered on call) through the wise-on-call program distributed by aquifer® on behalf of new york university (nyu) school of medicine [ ] . after the pilot, a post-course survey was conducted to evaluate students' self-assessed learning achieved through the course and to learn where improvement was needed. these data were taken into consideration while adjusting and creating the schedule for april . institutional review board approval was not obtained as students participated voluntarily, topics covered constituted routine education, and the assessment of the learners was part of the normal course evaluation process. in early , planning began to scale this activity up to approximately one half of the class for april , ( figure ), in preparation for delivery to all students by spring . student feedback was used to adjust session topics, and the curriculum was redeveloped so that content was divided into six interrelated domains based on the accreditation council for graduate medical education (acgme) core competencies: patient care, medical knowledge, practicebased learning and improvement, interpersonal communication skills, professionalism, and systems-based practice ( table ) [ ] . residents must be able to provide patient care that is compassionate, appropriate, and effective for the treatment of health problems and the promotion of health. residents must be able to demonstrate knowledge about established and evolving biomedical, clinical, and cognate (e.g. epidemiological and social-behavioral) sciences, and the application of this knowledge to patient care. practice-based learning and improvement residents must be able to investigate and evaluate their patient care practices, appraise and assimilate scientific evidence, and improve their patient care practices. interpersonal communication skills residents must be able to demonstrate interpersonal and communication skills that result in effective information exchange and teaming with patients, patients' families, and professional associates. residents must be able to demonstrate a commitment to carrying out professional responsibilities, adherence to ethical principles, and sensitivity to a diverse patient population. systems-based practice residents must be able to demonstrate an awareness of and responsiveness to the larger context and system of health care and the ability to effectively call on system resources to provide care that is of optimal value. the curriculum was also designed to reinforce the american academy of medical colleges (aamc) core entrustable professional activity's (epas) whenever possible [ ] . content was delivered in sessions ( mandatory and optional). detailed mapping of each boot camp session topic with a description and the corresponding acgme core competency is provided in table . with the onset of the covid- pandemic, all student face-to-face activities were furloughed on march , . the course was rapidly transformed in days to an entirely online curriculum. we adapted content for synchronous remote delivery using the canvas online learning management system (instructure®, salt lake city, ut, usa). synchronous sessions used a combination of didactic powerpoint presentations that were made interactive by adding facilitator polling, multiple-choice questions, and small group or individual writing exercises that were shared with the group through incorporation into the presentation or chat board. for example, students were given a mock patient sign out electronically and had to use this to respond to virtual pages from a nurse for a simulated night-on-call. students were asked to write a note about some of the overnight events, which were later de-identified and shared with the group for feedback during the "sign out" lecture. procedural training on cadavers was adapted to include an independent review of online procedure videos by students. training in pronouncing death on a recently deceased cadaver and calling his/her family was adapted to include an interactive session on pronouncing patient death and a reflective reading assignment. an "ask the interns" panel discussion was adapted by requesting students to email questions for review by three high functioning interns who then took turns answering them through video chat and the live message board. pertinent hot topics were added, including a session on telehealth and a covid- patient case discussion that included a rich discussion of end-of-life care and the ethics surrounding decision-making with limited resources. in addition to the synchronous remote sessions, students were required to submit written exercises with a focus on compassionate care, wellness, and self-reflection. students wrote a letter to members of the incoming class of medical students. additional independent study requirements included aquifer cases and online naloxone training. sessions were grouped so that students had a monday or friday free each week to facilitate their end-of-year personal tasks such as finalizing housing plans or pre-employment requirements for their upcoming residencies, which may be in different cities or states. four additional optional sessions were held, one about preparing a professional powerpoint and three focused on topics pertinent to the care of pediatric patients. for out of sessions, the facilitators created a voluntary pre-session survey through a secure online data collection tool. medical students were asked to rate their confidence and understanding of requisite knowledge points on the topic before these sessions. items were graded on a -point likert scale ( meaning confidence or knowledge was very poor, meaning neutral, and meaning very high). after delivery of each session, students were given a postsurvey with one additional question about whether they found the activity useful. a competency-based exam was administered at the conclusion of the course to assess the knowledge gained. two to three multiple-choice questions were written for each lecture session assessing student competency of objectives identified by the educator. the questions were each mapped to acgme core competencies and written with varying degrees of difficulty addressing knowledge, application, and problem-solving [ , ] . the resulting -question comprehensive assessment was completed by all students except for one who contracted covid- . after completing the comprehensive assessment, students were given a link to the correct responses. pre-/post-survey median scores were analyzed using the wilcoxon rank-sum test (mann-whitney u test) for unpaired non-parametric data. to categorically compare the percentages of students who answered positively about each session, responses in the likert scale were combined into a x contingency table (positive, neutral, or negative responses), and data were analyzed using the chi-square test. for example, the likert scale response categories of extremely confident and confident were combined into one, "positive" category. usefulness of sessions and post-test grades were analyzed with descriptive statistics. all tests were twotailed, with p < . as the significance level. statistical analysis was performed using ibm spss version (ibm corp., armonk, ny, usa). a total of students and instructors participated remotely. student learners had matched into a mix of non-surgical residency programs, with internal medicine, emergency medicine, family medicine, being most common. instructors included faculty, chief residents, and current interns. pre-surveys were completed by - students ( - %), and post-surveys were submitted by - students ( - %). pre-test confidence scores were lowest for the sessions on transfusion medicine, the nursing pager on call activity, and chief medical resident expectations, with less than % of students feeling confident or extremely confident in those topics. pre-session confidence scores were highest in teaching medical students and providing feedback ( % and % of students felt at least confident and comfortable, respectively). the percentages of students who answered that they were confident or extremely confident in the subject matter before and after each session are summarized in table . all synchronous sessions resulted in a measurable increase in the portion of students reporting confidence or extreme confidence in the subject matter. the largest increases in confidence (over % increase) were seen in the session about when go to your program director, chief medical resident expectations, and what do after a patient dies, followed by the session on using blood products ( % increase). all values are rounded to the nearest %. to compare the percentages of students who answered positively for each question, the response categories in the likert scale were combined into a x contingency table (positive, neutral, and negative), and data were analyzed using the chi-square test. median confidence scores before and after the sessions are presented in table . before the session, median scores were lowest regarding knowledge about the role of a chief resident, confidence writing admission orders independently, using blood products, and how to fill out a death certificate. baseline median confidence scores were highest for teaching medical students and knowledge about how to give students feedback. a measurable increase in numerical confidence or knowledge scores was demonstrated for all sessions, even if the median score did not change. figure . overall, all sessions analyzed were rated as useful or extremely useful by over half of the students ( - %). the top five most useful sessions were the sessions on clinical documentation, common clinical scenarios, the night-on-call nursing paging activity, and clinical reasoning, which were rated as useful or extremely useful by %, %, %, and %, respectively. only of the sessions received any feedback of "not useful at all", and in all cases it was fewer than % of students. the cumulative post-test was taken by of students. of the students, ( %) achieved a score of % or higher, ( . %) scored in the - % range, and scored %. students were asked to provide feedback comments and state whether they believed certain sessions could remain virtual; comments are summarized in table . positive comments i enjoyed that this course was online. this is a big transition time for us, and it was very convenient to be able to have this online. i am glad that this was online because it allowed me to go home and be with my family for the month. the interactive nature of being able to type comments at any point and have your comments be addressed and interacted with by other students was helpful. i felt much more engaged than i normally do during in-person lectures. mandatory attendance and the chat box made it very interactive. virtual boot camp made it easier and less tiring to attend lecture since i could do it from home, and there was still great participation from the students. chat allowed other students to clarify things while the instructor was speaking, so the instructor didn't need to answer more questions. excellent resource that i plan on reviewing again the days prior to starting residency. constructive comments i think it would have been helpful to have more clinically oriented courses in-person. anything that's super participation heavy should be done in person if possible, but lectures that are just lectures (with perhaps a poll here and there) are great to have online. if you kept these lectures online, i would suggest shortening/separating them if possible. as a student, it is hard to pay full attention online with multiple hour-long lectures. summarizing handouts would be helpful to carry forward into residency. positive feedback was related to the flexibility of an online course and utility of the chat box, which, as some students reported, allowed them to be more engaged than they had been in live lectures. constructive comments included the suggestion that sessions with a focus on patient care may be better achieved by a live session in coming years. positive feedback was related to the flexibility of an online course and utility of the chat box, which, as some students reported, allowed them to be more engaged than they had been in live lectures. constructive comments included the suggestion that sessions with a focus on patient care may be better achieved by a live session in coming years. student preferences for which sessions could continue remotely versus which would be better facilitated in person are shown in figure . for a majority of sessions, the percentage of students preferring a virtual format outnumbered those preferring an in-person session. the sessions most heavily focused on medical knowledge and patient care such as the clinical scenarios, sign-out activity, and night-on-call had the highest percentage of students preferring an in-person session. this study demonstrates that students who participated in this elective non-surgical boot camp felt more confident and knowledgeable about handling situations they may encounter as interns and demonstrated an objective gain in knowledge beyond self-reported levels. most of the teaching sessions were adapted to an online virtual synchronous format without difficulty, and feedback from students was overwhelmingly positive despite the change in format. had there been more time to prepare, pagers could have been given to students to make the night-on-call activity more realistic; however, despite the absence of a pager, this was one of the highestrated sessions. the course was well received with significant increases in student's perceptions of readiness and comfort in multiple domains, which were linked to acgme core competencies. facilitators volunteered their time to construct and deliver the course content, and students used their personal computers; therefore, no high-fidelity simulation manikins or costly equipment was required. although several studies on internship preparation courses have been published demonstrating increases in self-reported confidence, very few have included objective assessments such as the cumulative post-test included in our study. all students except for one achieved a score of above %, which would correlate with the score required to pass a national shelf examination. limitations of this remote activity include the fact it was a single-school study and that student's level of interest and engagement may have been related to not having any other residency preparation options given the covid- pandemic. the number of students who signed up for the course increased significantly in the seven days prior to the start of the course, which may reflect that. other limitations include those of analyzing participation in pre-and post-surveys when some of the students did not complete both for each session. lastly, it is a possibility that students who filled out their surveys were more engaged, and thus results were more likely to be positive. covid- has presented many challenges to students and educators in undergraduate medicine. now more than ever students need all the help they can and to feel more confident and prepared when they start internship on july . despite the challenges we encountered, synchronous remote delivery of this boot camp was successful, and we will likely continue to hold certain sessions remotely in the future. this may be more useful for students traveling to arrange for new living arrangements during residency. future work includes include scaling up to all students and obtaining more volunteer facilitators that would enable small group breakout sessions which may increase opportunities to interact. we plan to re-survey students after their first month of internship to re-evaluate their perceived usefulness of this course. overall, an online synchronous boot camp was rated as useful by students, helped them feel more confident about common topics encountered during residency, and demonstrated an appropriate gain in knowledge using a comprehensive post-course assessment. we were able to adapt our curriculum to a remote model and will likely keep several sessions in an online format in coming years based on positive feedback from students. human subjects: all authors have confirmed that this study did not involve human participants or tissue. animal subjects: all authors have confirmed that this study did not involve animal subjects or tissue. conflicts of interest: in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. course offerings in the fourth year of medical school: how u.s. medical schools are preparing students for internship what training is needed in the fourth year of medical school? views of residency program directors the fourth year of medical education: a literature review the impact of fragmentation of hospitalist care on length of stay medical student experiences with accessing and entering patient information in 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milestones guidebook. acgme core entrustable professional activities for entering residency: toolkits for the core epas education techniques for lifelong learning: writing multiple-choice questions for continuing medical education activities and self-assessment modules drs. monday and levine had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. key: cord- -z ucelh authors: franchi, thomas; magudia, adit; rasheed, adib title: appropriate use of self-directed learning at medical school prepares students for future clinical practice date: - - journal: med educ online doi: . / . . sha: doc_id: cord_uid: z ucelh nan we would firstly like to credit hill et al. [ ] for providing insightful research into the evaluation of self-directed learning for first-year medical students. as fourth-year medical students at the university of sheffield medical school, we believe that self-directed learning is an integral aspect of the medical curriculum. as postgraduate examinations are an essential part of career progression, it is paramount for healthcare professionals to be proficient in self-directed learning whilst working in a clinical setting. therefore, we agree with the sentiments discussed by the authors, and would like to present some additional points with regard to the value of self-directed learning. the integrated curriculum at our medical school accounts for self-directed learning throughout a students' progression from the preclinical to clinical years. from the beginning of our medical education, we were introduced to self-directed learning in small groups via integrated learning activities (ilas). ilas build on the principles of problem-based learning and are designed to allow students to explore the core clinical situations in collaboration with other medical students that newly qualified doctors often experience. thus, ilas help to develop adeptness in communication, problem solving and personal responsibility for learning; all key components of a doctor [ ] . furthermore, our ability to study independently is tested on numerous occasions via written examinations. a significant component of our examinations is formed by the ilas and a core list of diseases, with a selection of these specifically not taught by the medical school. the examinations, therefore, provide an insight into our ability to carry out self-directed study, as a strong element of independent learning is required by students to progress through medical school. we feel that an additional point to consider following the contributions made by the hill et al. is students' perceived attitudes towards the quality of knowledge obtained as a result of the self-directed learning activity. as medical students, we feel that there is significant difficulty in creating an environment in which we can adequately learn content indepth under tight time constraints. as a consequence, self-directed study can be undermined, with medical students often lacking confidence in the relevance of information obtained through independent study. this has previously been observed where the preparedness of medical students from different curricula (traditional lecture based versus problem based versus integrated) was assessed. students in heavily selfdirected learning curriculums had a significantly lower self-rating of knowledge compared to students from a traditional curriculum [ ] . therefore, we suggest that medical educators look to address and minimise ambiguity over depth and duration spent on self-learning activities as a way of increasing confidence in the knowledge gained. from our experiences in medical school, we believe there is a distinct difference between students benefiting from selfdirected learning versus feeling unsure about whether they have met the intended learning outcomes. with the ongoing covid- pandemic, and the subsequent temporary but indefinite suspension of the majority of uk medical school teaching and clinical placements [ ] , it is pertinent that medical students make effective use of their time. by engaging effectively with self-directed learning, students will be able to ensure that they remain informed and competent whilst continuing to develop their knowledge of the standards required to graduate medical school. whilst learning opportunities will be missed, we believe this can serve as an example to the importance and critical value of self-directed learning in medical education. no potential conflict of interest was reported by the authors. implementation and evaluation of a self-directed learning activity for first-year medical students problem based learning medical graduates' preparedness to practice: a comparison of undergraduate medical school training advice from medical schools council to uk medical schools on actions surrounding covid- . medical schools council http://orcid.org/ - - - key: cord- -bnbinwnl authors: rallis, kathrine s.; allen tejerina, andrea m. title: tele-oncology in the covid- era: are medical students left behind? date: - - journal: trends cancer doi: . /j.trecan. . . sha: doc_id: cord_uid: bnbinwnl nan we read, with interest, the recent article by shirke et al. [ ] 'tele-oncology in the covid- era: the way forward?', describing the benefits and limitations of tele-oncology and its role during the coronavirus disease (covid- ) pandemic. as medical students experiencing the disruptions caused by covid- , this article resonates with us. we appreciate the value of telemedicine in cancer services and agree that tele-oncology offers excellent prospects to help patients with cancer during covid- and in the future. yet, we are confronted with the fact that there is little mentioned by the authors of the opportunities and challenges associated with integrating telemedicine education and training (et) in tele-oncology. in response to this statement, here, we set out ways in which telemedicine et can be integrated in tele-oncology services for medical students. in addition, we discuss challenges of telemedicine et and propose ways to overcome these. emergence of the covid- pandemic has exerted an unprecedented strain on medical education. as medical students interested in specialising in oncology, we are disheartened by the missed learning opportunities caused by the suspension of clinical placements and cancellation of summer electives and research programmes. although there has been an accelerated transition to telemedicine et through virtual conferences, webinars, online platforms, and free open-access medical education (foamed), these predominantly offer theoretical education, with little hospital training and medical simulation [ ] . we are concerned that reductions in clinical teaching will hinder our clinical skills performance and selfconfidence. even though all aspects of medical education have been significantly disrupted, we believe that oncology is disproportionately affected because it is already underrepresented [ ] [ ] [ ] . furthermore, early specialty exposure through academic opportunities and mentorships significantly increases the likelihood of medical students pursuing oncology-related careers [ ] [ ] [ ] . the decline in such experiences amid covid- may impede oncology specialty selection, causing a shortage of trainees to join a workforce that is already overstretched [ , ] . increased physician burnout and poorer quality oncology services will likely result. to prevent these outcomes, we believe a revitalised medical education effort is crucial. if telemedicine is indeed the way forward, it is paramount to incorporate medical student et. first, streaming tele-oncology clinics is an easy method to offer clinical exposure to medical students who are restricted from attending clinical placements. live participation in outpatient clinics through videocall and webchat offers real-time patient access, replacing the need for students, doctors, and patients to be in physical proximity and, thus, risking covid- contraction. allowing students to interview and clerk patients online can offer invaluable clinical experience. students can assist in taking notes during consultations, supporting clinicians' undivided attention to patients. additionally, medical students should be invited to join virtual multidisciplinary team (mdt) meetings, which are easily accessible and a treasured source of learning. most importantly, we strongly advocate for the provision of remote access to electronic patient records (epr) for all clinical year students. we believe that such access will cause a paradigm shift, enabling students to familiarise themselves with patient cases and bolstering student research initiatives by allowing them to conduct audits, retrospective patient studies, and case-series remotely. indeed, restricted access to eprs is a main obstacle in students' preparation for clinical practice [ ] . telemedicine et has its limitations, including the fact that medical students cannot practice physical examinations. moreover, face-to-face patient exposure offers a better chance of cultivating empathy, practicing motivational interviewing, observing body language, and engaging in more sensitive patient discussions. additionally, the presence of medical students in virtual clinics may reinforce uncomfortable feelings from patients who already report increased nervousness, emotional distance and difficulty, or reluctance, associated with communicating with providers using television-based systems [ ] . lastly, although remote access to eprs will exponentially increase research output and aid clinical learning, it also poses a risk to patient confidentiality by information miss-sharing. undergraduate medical curricula must be revised to prepare future doctors for a telemedicine-dominant era. training students on tele-oncology tools and increasing awareness of their limitations should form part of undergraduate education. medical schools must teach students how to decide when telemedicine is appropriate by triaging patients who require in-clinic assessment. emphasis should be placed on developing online communication skills and reassuring patients who are reluctant to use televisionbased systems. lastly, students must recognise that tele-oncology needs require modification on a case-by-case basis. given that the duration of healthcare disruptions remains unknown, with a significant trends in cancer, month , vol. xx, no. xx trecan no. of pages backlog in services anticipated even after the pandemic has subsided, medical et cannot be neglected. the pandemic has accelerated the transition to tele-oncology, unmasking a new era in telemedicine that is expected to remain in the long term. as medical schools adapt to the new face of medicine, students' transition to telemedicine should be prioritised, given that they have an important future role. further efforts should focus on integrating telemedicine et in tele-oncology services and educating students on using telemedicine systems. k.r. wrote the letter, and a.a. contributed to comments and editing. tele-oncology in the covid- era: the way forward? knowledge, attitudes and practices of telemedicine education and training of french medical students and residents discordance between cancer prevalence and training: a need for an increase in oncology education medical student teaching in the uk: how well are newly qualified doctors prepared for their role caring for patients with cancer in hospital? a nationwide medical student assessment of oncology education medical student exposure to radiation oncology through the pre-clerkship residency exploration program (prep): effect on career interest and understanding of radiation oncology preclinical medical student training in radiology: the effect of early exposure medical student career choice: a qualitative study of fourth-year medical students at memorial university future supply and demand for oncologists : challenges to assuring access to oncology services barriers to medical students' electronic health record access can impede their preparedness for practice how effective is video consultation in clinical oncology? a systematic review key: cord- - l ulis authors: cleland, j. a.; foo, j.; ilic, d.; maloney, s.; you, y. title: “you can’t always get what you want…”: economic thinking, constrained optimization and health professions education date: - - journal: adv health sci educ theory pract doi: . /s - - -w sha: doc_id: cord_uid: l ulis every choice we make in health professions education has a cost, whether it be financial or otherwise; by choosing one action (e.g., integrating more simulation, studying more for a summative examination) we lose the opportunity to take an alternative action (e.g., freeing up time for other teaching, leisure time). economics significantly shapes the way we behave and think as educators and learners and so there is increasing interest in using economic ways of thinking and approaches to examine and understand how choices are made, the influence of constraints and boundaries in educational decision making, and how costs are felt. thus, in this article, we provide a brief historical overview of modern economics, to illustrate how the core concepts of economics—scarcity (and desirability), rationality, and optimization—developed over time. we explain the important concept of bounded rationality, which explains how individual, meso-factors and contextual factors influence decision making. we then consider the opportunities that these concepts afford for health professions education and research. we conclude by proposing that embracing economic thinking opens up new questions and new ways of approaching old questions which can add knowledge about how choice is enacted in contemporary health professions education. consider some of the choices facing health professions educators. should we give our students more feedback? more or less simulation? expand the number of stations in an objective structured clinical examination (osce)? giving more feedback probably requires more faculty time and training, which might mean less faculty time for something else. more simulation may be desirable in terms of mastering skills but which activity should it replace, and can the simulated patients, rooms and other resources be feasibly sourced? more osce stations might increase reliability and curriculum coverage but will require more examiners, more questions and more preparatory and examination time. not only must we make choices as faculty, so too do students and the greater society. a student may choose not to study for a formative assessment to focus their time on preparing for summative examinations. as a society, we may want fair access to medicine, so that medical students represent all groups in society as well as excelling in terms of academic and personal qualities. both aspects may be desirable, but attaining excellent pre-entry qualifications may conflict with those aspects and therefore require trade-off with expanding the diversity of the medical student body (see later for further discussion). in short, every choice we make has a cost, whether they be financial or otherwise; by choosing one action we lose the opportunity to take an alternative action; and these opportunity costs may be felt in different ways by different individuals, groups and society. choice is a focus of study in many different fields. in this article, we introduce thinking about choice from the economic perspective (johnes ) . although there is no single definition of economics, this description is helpful in understanding its nature and scope: "economists study how individuals, organizations, and societies employ time, money, and effort. economics provides a framework for understanding the behavior of individuals and organizations as they generate and allocate human, material, and financial resources." (brewer et al. , p. ) . in short, economic thinking allows us to examine, understand and reflect on how individuals, groups and societies choose among the alternatives available to them. it is timely to step back and consider some of the key concepts in economics and how these can be applied to health professions education (hpe). first, there seems an appetite in the literature to rigorously evaluate and optimise allocation of medical education resources maloney et al. ) , and to apply economic methodologies which could be used to address hpe questions (e.g., cleland et al. b) . second, in a recent commentary about strategic planning during covid- , tolsgaard et al. ( ) stated that the "choices that we make now for how to use our workforce, which education and training activities to stop, continue or modify, will [also] have profound workforce consequences and implications for quality of non-covid- areas of clinical care". this commentary alluded to the role of constraints and boundaries in decision making but did not expand on their nature. without this granularity, medical education decision makers do not have the necessary building blocks to consider their decision making, the ramifications of decisions or insight into creative ways to solve everyday hpe problems. third, longstanding increases in medical education costs and student debt (aamc ), combined with more recent covid- related pressures on institutional budgets (burki ) , has led to pressures to optimise medical education while continuing to generate value for all involved. the concept of value is also of interest in respect to value to whom? economics provides language and frameworks for considering this and other tensions-opening up new and creative ways of thinking and illuminating the implications of actions at the level of individuals, groups and institutions in hpe. to set the scene, we provide a brief historical overview of modern economics. in table we give a very brief overview of key time periods, figures, theories and concepts in economics. for more details we direct readers to key economics primers (e.g., backhouse and tribe ) . we then explain the core concepts of economics: scarcity (and desirability), rationality, and optimization (table ) . we then consider the opportunities that these concepts afford for health professions education and research. economic ideas have been present since antiquity. however, modern economic thought was laid down during the scientific revolution ( th- th century) (backhouse ) . adam smith, with debts to his predecessors and contemporaries, is regarded as the father of classical/classical political economics via his book, the wealth of nations, which was first published in (note that smith was also a philosopher but in this article we focus on his contribution to economics). classical economists such as adam smith and karl marx regarded economy as a part of broader social, political, historical and institutional domain and discussed economic process within this context (e.g., meek ; skinner ) . within this framework, classical economic doctrine asserts that the value of a good (how much of another good or service it exchanges for in the market) is proportional to how much labour was required to produce it, including the labour required to produce the raw materials and machinery used in the process. a second core assertion is that the power of the market system, if left alone (i.e., without government intervention), will ensure full employment and usage of economic resources. within this natural order (as proposed by john locke in his second treatise of government in ), there may be occasional deviation, but individual self-interest based on the information required for rationale decision making, supply and demand, preferences and constraints (e.g., laws, religion, customs), and social order (what smith referred to as the "invisible hand": smith , p. ) will underpin economic self-regulation. in the years following smith's wealth of nations, the world changed, and economic theories also evolved as generations of economists tried to understand and explain economic phenomenon in changing times and societies. classical or political economics as a field then made a transition to neoclassical economics which still assumed that the market and the "invisible hand" will ensure the optimal allocation of resources. however, the basic determinant of economic decisions became individual choice and maximizing behavior, the optimal utilization of scarce resources to satisfy the needs and desires of individual agents, not social processes within a specific social system (e.g., de vroey ). the term "marginalist revolution" is commonly used to indicate this change of direction in economic science, with its shift to a subjective theory of value (roncaglia , p. ) . however, as per smith's classical model, neoclassical economics assumed that everyone has free access to the information they require for decision making, enabling rational decision making in terms of choosing between identifiable outcomes and associated with values (e.g., firms will make choices which will maximize profits, individuals will make choices which maximize personal benefit or satisfaction). moving forwards in time, in s, in the time of the great depression, john maynard keynes was highly critical of classical economic arguments that natural economic forces and incentives would be sufficient to help the economy recover. instead, keynes advocated for increased government expenditures and lower taxes to stimulate demand and pull the global economy out of the depression. subsequently, keynesian economics was used to refer to the concept that optimal economic performance could be achieved by influencing choice through economic intervention policies by the government. it was at this point in time that the separate disciplines of micro-and macro-economics first emerged, with the recognition that supply and demand at the level of individual markets was not sufficient to explain (macro-) factors such as inflation, market failures such as the great depression and unemployment. in macro-economics, the government and, increasingly, global markets are the object of analysis. as with those before him, keynes ( ) argued that people make economic decisions on essentially rational grounds, using all the information available to them to form accurate probabilities about the world. however, as time progressed keynes' position shifted to apparently question this central tenet of economics, that actors are fundamentally rational. his view was that people often lack sufficient information and face too much uncertainty to calculate probabilities and make rationale choices in any meaningful way. instead, they filter information using less than reliable practices, including assuming that the present is the best available predictor of the future and looking to those around them/social convention table core economics principles-bounded rationality, scarcity and optimization/satisficing bounded rationality rationality is limited when individuals make decisions: by the tractability of the decision problem, the cognitive limitations of the mind, and the time available to make the decision, and so on bounded rationality is the idea that we make decisions that are rational, but within the limits of the information available to us and our mental capabilities scarcity due to limited resources and infinite demands, society needs to determine how to produce and distribute these relatively scarce resources. as a result of scarcity of resources, economic thinking considers opportunity cost-having to make choices between two or more options. for example, investing in donor dissection for anatomy education, leaving less time and finances to invest in virtual reality anatomy education optimization/satisficing optimization refers to getting the best deal/outcome possible within resource constraints. satisficing is a decision-making strategy that aims for a satisfactory or adequate result, rather than the optimal solution. it refers to taking a "good enough" decision which requires lesser resources, compared to committing greater resources (effort, costs and time) to find the best possible decision ( ) . neither of these practices are efficient, optimal or stable but they are not entirely unreasonable if there is no other information that instructs otherwise, and one is short of time and/or analytical/cognitive capability. keynes states "human decisions… cannot depend on strict mathematical expectations, since the basis for making such calculations does not exist… "our rational selves [choose] between the alternatives as best we are able, calculating where we can." failing those instances, however, decisions are necessarily left open to "whim or sentiment or chance" (keynes , p. ) . this is an important shift and led to the concept of bounded rationality-rationality taking place within operational constraints (simon ) . bounded rationality is not the same as irrationality: decision-makers are still attempting to make as rational a decision as possible given the information, time and capability to store and utilize information available to them. bounded rationality explains situations where people do not take the action that would maximize results (from an economic point of view), because they do not have all the information needed to make a truly rational choice. bounded rationality also acknowledges that individual and meso-factors-such as embedded values and personal preferences, limited feedback and tendencies to avoid discomfort, as well as exogenous factors such as the environment in which the individual inhabits-influence decision making (e.g., dawnay and shah ; thaler and sunstein ; dolan et al. furubotn and richter ) . eagle-eyed readers and those with an interest in cognition will have noted the references to cognitive capacity and heuristics above. although originating in economics, bounded rationality is now a very multi-disciplinary concept, and the basis of theories and studies in cognitive-and neuropsychology, biology, computer science, and philosophy. these perspectives all add to current understanding (for further information, we direct readers to https ://plato .stanf ord.edu/entri es/bound ed-ratio nalit y/#bettb oun). as stated earlier, selecting among alternatives involves considering scarcity (including supply and demand) and optimization, as well as (bounded) rationality (mankiw ) . in almost every situation, resources are limited, or perceived to be limited, and you have to give up something to get something else (opportunity cost again). for example, a 'free lunch' is not really free, as to consume the meal, a person has to forgo opportunities that could be taken to engage in other activities (e.g., work, nap, exercise), even if he/she doesn't have to pay the bill for the lunch. in hpe, an example of scarcity might be investing in dissection for anatomy education, leaving less time and finances to invest in virtual reality anatomy education. other examples might be employing/allocating staff for one part of the curriculum meaning no, or less resource for another part of the curriculum, or removing sessional teachers and replacing them with tenured research staff who are contractually obliged to teach, thus reducing costs but possibly also compromising quality and the student learning experience. economists view decision-making behaviour according to the basic principle that people make decisions by comparing perceived benefits with costs, to get the best deal/ outcome possible within resource constraints (optimization). an example of this at the individual level might be the best use of time (a limited resource) in the run up to examinations. for a student, the benefit of gaining an extra couple of hours of study might be the best use of time and worth the cost of missing rugby training. in medical education, faculty may ask whether greater benefits can be found by incurring greater costs (e.g., shifting from large-group to smaller group teaching with the resultant costs in terms of more educators and more time but potential gains in terms of student learning and thus potential savings from less remediation and attrition); whether the gains from substituting group feedback for individual feedback would be worth the cost of changing systems and a potential drop in student satisfaction. because bounded rationality thinking is limited by factors such as the available information, the nature of the problem, cognitive and time limits, the goal posts and language have shifted to acknowledging that economic decision making is usually about doing the best you can with what you have. this formulation of boundedly rational decision making as a constrained optimization problem is commonplace in economics (arrow ) , and is labelled "satisficing," (a combination of satisfy and suffice, coined by simon ) . economic thinking, with its core principles of (bounded) rationality, scarcity and optimization/satisficing (see table ), provides a novel lens to understand the behaviours (choices) of organisations, groups and individuals in hpe, one which can encourage different ways of thinking, and open new avenues for thought and exploration. note however, that while borrowing from other disciplines is not new in hpe (e.g., varpio et al. ) , it is critical to carefully consider theories and concepts, and check their assumptions are congruent with the specific research question (murray and evers ) . we suggest some outstanding research questions and topics that could be addressed using economic concepts and thinking below. our unit of analysis is micro-economicsthe level of the individual, group and organisation. we acknowledge that there are macroeconomic factors that are very relevant to hpe (e.g., walsh ), such as the number of medical and other healthcare students trained and government policy on different aspects of hpe. however, our focus and examples reflect our own interests and observations, and mostly relate to bounded rationality and optimization. they are by no means exhaustive but refer to day-to-day activities and tensions which will be familiar to educators across the globe. we also direct readers to two recent papers, one which sets out how to read economics studies (maloney et al. ) and the second which provides guidance on how to conduct cost and value analyses in hpe (foo et al., in press) . our first example is that of bounded rationality. in many contexts and institutions students are asked to rate their satisfaction with their course/aspects of their course. aggregate responses or ratings of this data are used as quality "signals" or performance indicators and used by different people for different purposes. for example, potential applicants may use these satisfaction rankings to help them decide where to study (e.g., horstchraer ), governments may use them as indicators of institutional performance and management [and link them to funding; e.g., mccormack et al. ) ] and institutions may use them for performance review. however, student responses to such surveys are bounded by their lack of experience of other university education, scheduling and time available to them to fill in feedback forms, ease of completing such evaluations, the importance they give to filling in such forms, how interested they are in their course, current standing in the course (e.g. first year or final year), academic performance in the course and whether they "like" the teachers or not. they may also be influenced by the norms of student feedback in their institution. is this mandatory? do students take it seriously? do they believe their views make any difference? moreover, econometric analysis suggests that "teaching quality" factors such as student-staff ratios are associated with responses, but so too are extraneous factors such as whether or not students pay fees or not (raising expectations linked to education as needing to deliver value for money, or the commodification of education; e.g., bacher-hicks, chin, kane and staiger, ; staddon and standish ), or how likely they are to get a job after graduating (e.g., lenton ) . or indeed whether they are offered cookies in class or not (hassler et al. )! student satisfaction may be quantifiable, but it should not be relied upon given bounded rationality (e.g., dawnay and shah ; thaler and sunstein ; dolan et al. ). yet many institutions continue to use student satisfaction for different purposes. the concept of optimization is useful here. student satisfaction is a simple and cheap outcome assessment (given the complexity of establishing a simple, valid multi-factorial evaluation strategy) which may be a "good enough" (satisficing) indicator, aligning with an organisational reference point in respect of performance/ quality management. in other words, organisations also are boundedly rational, situated within their own economic, social and historical contexts and norms (nielsen and moynihan ) , and need to make judgements within resource constraints. a second example of bounded rationality is that of medical careers decision making. many studies report on the importance of exposure to specialities and different clinical environments for medical student career decision making. not considering certain career options appears to relate to the lack of information or knowledge needed in terms of the various options, understanding one's self, motivation and "dysfunctional myths": all factors which reflect bounded rationality (e.g., stamovlasis and vaiopoulou ). an example of a dysfunctional myth, linked to lack of information and faulty heuristics, may be the assumption that rural training programs provide poorer educational outcomes than urban ones, or similarly placement at large teaching hospitals opens future employment opportunities compared to placement at smaller teaching hospitals. a well-advertised league table comparing successful completions or licencing exam scores across training programs, or any other credible source of information (self ), may correct this assumption and support accurate decision-making on educational outcomes. medical careers decision making leads us into health workforce planning. the societallevel challenge for workforce planners is to ensure the right mix of health professions, specialities within professions, and geographical distribution to ensure the right capabilities are in the right place to meet the health needs of local populations. this is optimization of relatively scarce resources, particularly in countries with insufficient training capabilities. economic theories can help to explain how medical students and residents/trainees make career choices (see earlier), as well as provide testable hypotheses and methodological approaches to inform interventions designed to encourage choice towards areas of need. healthcare organisations seeking to attract residents/trainees can look to strengthen those aspects of the workplace for which evidence using economic approaches, such as discrete choice experiments, suggest are highly valued by the target group (e.g., good working conditions: e.g., scanlan et al. ; cleland et al. cleland et al. , . at the same time, and as above, organisations must also make sure that information about these positive changes are readily available to potential applicants so it can factor into their decision-making process. admissions is fundamentally an optimization process for medical schools: identifying and implementing processes which select the best applicants from the available pool at the same time as balancing political, social and academic drivers for admissions (e.g., fielding et al. ; cleland et al. b) . a recent review highlighted the need to consider not only the reliability and validity of admissions tools and processes, but also to bring fairness and cost-effectiveness into admissions research and decision making (e.g., patterson et al. ). several studies have done so (e.g., hissbach et al. ; rosenfeld et al. ; schreurs et al. ; tiller et al. ; ziv et al. ) but this remains an underdeveloped area of admissions research. admissions costs can be considered from different angles [e.g., of the institution or the individual applicant (e.g., ]. outcomes measures must assess value as well as cost (cheapest is not necessary best: see scheurs et al. ) , and look at individual-level outcomes for both distal (e.g., location and type of position postqualification) as well as proximal (e.g., performance in medical school) indicators. admissions can also be considered an optimization/satisficing process from a different angle. places are scarce and admissions committees must consider the balance between the expected relative effort and benefit of using different selection criteria or processes to shift through more applicants than places. an admissions committee which takes the position that academic attainment is the most important factor in selection will likely invest relatively less resource into selecting for personal qualities than would an institution that places high value on the latter . looking at the mission statements of different medical schools can give some indication of what they value and their admissions priorities, and may explain differences in admissions processes, including how different components within an admissions process are weighed (see table ). our last point in respect to admissions is that of bounded rationality. medical school selection committees are faced with an abundance of "traditional" applicants who, experience suggests, will be likely to progress and graduate in a satisfactory manner. "nontraditional" applicants, while valued at a macro-level in respect of government policies to increase diversity, are more of an unknown quality and hence challenge the bounded rational decision-maker (better the devil you know), potentially jeopardising institutional reputation and metrics if they do not perform adequately. that medical schools and medicine are slow to change is not news (e.g., whitehead ). however, economic concepts provide a novel lens by which to consider admissions decision making, and can inform the design and evaluation of change interventions so admissions aligns more with macrodrivers (e.g., governmental drivers to increase the diversity of the medical profession to meet changing societal needs). (van der vleuten ) . however, years on this remains the most under-researched component of the utility framework, possibly because of anxiety about balancing change with maintaining standards. only a few authors have reported on direct assessment costs (e.g., brown et al. ) while others have looked directly at assessment approaches which reduce costs but maintain robust psychometric properties (e.g., currie, sivasubramaniam and cleland ) . drawing on healthcare research practices (e.g., halpern et al. ) , economic modelling may help the hpe community measure and compare assessment activities with the necessary impact, scalability, and sustainability to optimize educational outcomes. economic thinking also opens up assessment questions at the individual and organisational levels of resource allocation (michaelis and schwaneback, ) . in respect to the former, how much time do students take to prepare for different types of assessment (e.g., formative vs. summative)? what is the optimal strategy in allocating time between formative and summative assessments preparation? how do institutional processes (e.g., re-sits, repeating a year) influence student assessment behaviour? how is innovation in assessment developed and implemented, for example the use of computerised adaptive testing as either a formative or summative activity? in respect to organisational level of resource allocation, how many osce stations are optimal given each station will have a cost (e.g., examiner time, an actor or simulated patient, consumables, refreshments, room hire)? is the investment of staff training and quality assurance processes required to score short-answer questions (saqs) considered worthwhile? or do medical schools take a more satisficing approach, appreciating that some aspects of their assessment are not optimal but the best they can do within resource limits. there are many other aspects of education we could consider through an economic lens. the relatively new phenomena of micro-credentialing, for example, can be considered in terms of optimization from both the learner perspective (just enough learning for professional recognition, achievable in terms of motivation, cost and time) and that of the provider (a new pool of consumers, efficient use of existing resources, extending brand recognition and an alternative pathway for study in award or other courses) (e.g., young, west and nylin ) . another relevant area is curricular innovation and reform. using an economic lens, the ultimate goal of curriculum reform can be considered as optimizing the curriculum in some way (e.g., removing lectures and investing in online resources to free up previous didactic time for more interactive face to face teaching and learning). drawing on evidence from healthcare, this optimization is likely to be linked to abundance or scarcity of resources. scarcity promotes adoption of innovations that enhance efficiency, whereas availability of resources promotes advanced, expensive innovations (e.g., lambooij, engelfriet and westert ) . the context (societal structures and characteristics, including the macroeconomic environment, level of regulation, expectations and norms, and so on) in which hpe operates and, at an individual level, faculty and student perceptions and attitudes, including self-interest (e.g., wishing to protect curriculum time for one's own subject), will also act as bounds in terms of both the problems faced and the decisions that are made in terms of change. consider educational responses to the covid- pandemic in . social distancing and infection control measures limited access to clinical and campus environments overnight (scarcity) and hpe providers rapidly shifted to online and digital learning (efficiency), where they had the systems and resources to do so ). in conclusion, as per jagger and richards, you can't always get what you want. but if you try sometimes, well, you just might find you get what you need. aspects of individual, group and institutional decision making in hpe may sometimes seem less than rational on the surface but make sense when considered using the concepts of scarcity, optimization and bounded rationality. embracing economic thinking opens up new questions and new ways of approaching old questions which can add knowledge about how choice is enacted in contemporary health professions education. an experimental evaluation of three teacher quality measures: value-added, classroom observations and student surveys the penguin history of economics. london: the penguin group the history of economics: a course for students and teachers theoretical concepts in the economics of education money makes the (medical assessment) world go round: the cost of components of a summative final year objective structured clinical examination (osce) covid- : consequences for higher education thinking of selection and widening access as complex and wicked problems what do uk doctors-intraining value in 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florence; dubrowski, adam; patocskai, erica title: repercussions of the covid- pandemic on the well-being and training of medical clerks: a pan-canadian survey date: - - journal: bmc med educ doi: . /s - - - sha: doc_id: cord_uid: ykxvaqcz background: the covid- pandemic has been an unprecedented and potentially stressful event that inserted itself into the – canadian medical curriculum. however, its impact on stress and subsequent professional pathways is not well understood. this study aims to assess the impact of the covid- pandemic on the mental well-being, training, and career choices of canadian medical clerks within the first three months of the pandemic. it also aims to assess their use of university support systems and their appreciation of potential solutions to common academic stressors. methods: an electronic survey composed of four sections: demographics, stressors experienced during the pandemic, world health organization (who) well-being index, and stress management and resources was distributed to canadian clerks. results: clerks from of the canadian medical faculties participated in this study (n = ). forty-five percent of clerks reported higher levels of stress than usual; % reconsidered their residency choice; and % reconsidered medicine as a career. the factors that were most stressful among clerks were: the means of return to rotations; decreased opportunities to be productive in view of residency match; and taking the national licensing exam after the beginning of residency. the mean who well-being index was . / ± . , indicating a poor level of well-being among a considerable proportion of students. clerks who reconsidered their residency choice or medicine as a career had lower mean who well-being indices. most clerks agreed with the following suggested solutions: training sessions on the clinical management of covid- cases; being allowed to submit fewer reference letters when applying to residency; and having protected time to study for their licensing exam during residency. overall, clerks were less concerned with being infected during their rotations than with the impact of the pandemic on their future career and residency match. conclusion: the covid- pandemic had a considerable impact on the medical curriculum and well-being of clerks. a number of student-identified solutions were proposed to reduce stress. the implementation of these solutions throughout the canadian medical training system should be considered. the well-being of university students has been a subject of particular concern in recent times. a national study conducted in canada found that % of undergraduate university students reported elevated psychological distress, which was significantly higher than the general population [ ] . medical trainees are no exception to these statistics as they report a high prevalence of anxiety and stress [ , ] . evidence suggests that these trends among medical students tend to worsen during their medical curriculum, with a significant increase of perceived stress among third-year medical students when compared to their first-year of medical school [ ] . high levels of stress and anxiety were correlated with depression and burnout [ , ] . one study found that % of third year and % of fourth year medical students experienced moderate or high degree of burnout [ ] . the deleterious impact of this psychological distress on clerks' lives has been well documented, such as a detrimental effect on cognitive functioning and academic performance, as well as intention of dropping out [ ] [ ] [ ] . an unprecedented and potentially stressful event that inserted itself into the - medical curriculum is the covid- pandemic. the impact of this pandemic on the wellbeing of health-care workers has already been well documented [ ] [ ] [ ] . yet, its impact on stress and subsequent professional pathways for medical trainees is still not well understood. during the pandemic, although the academic curriculum of preclinical students continued online, the situation was different for most medical clerks. in many countries, senior clerks had to graduate prematurely, [ ] while, in other parts of the world, clerks were pulled from their respective rotations. in canada, most junior and senior clerks saw their medical rotations be suspended. the canadian medical curriculum follows a traditional format where students in third-year transition to clerkship in a teaching hospital. the impact of this pandemic may be particularly pronounced for students during clerkship who are preparing for their future careers. in fact, in canada, it is during clerkship that third-year medical students, also known as junior clerks, choose electives and obtain reference letters from attendings in order to apply to residency. on the other hand, fourthyear medical students, known as senior clerks, are tasked with completing the national licensing exam. however, due to the pandemic, the specific requirements and dates of the canadian resident matching service (carms) and the medical council of canada qualifying examination (mccqe) are being reconsidered. given their known predisposition to decreased mental well-being, [ , , ] this study aims to assess the impact of the covid- pandemic on the mental well-being, training, and career choices of canadian medical clerks within the first three months of the pandemic. it also aims to assess the use of mental well-being support systems, as well as their appreciation of potential solutions to common academic stressors. an electronic survey composed of four sections: demographics, stressors and their impact on residency and career choice, world health organization (who) wellbeing index and stress management and resources was distributed to clerks (third-and fourth-year medical students) in all canadian medical schools, with of the faculties participating and forwarding the survey to their clerks. the survey was developed and delivered in french and english. the study protocol, consent form and recruitment documents were approved by the institutional review board of the université de montréal (cerses- - -d). eligible participants were clerks attending one of the canadian medical faculties, and whose rotations were suspended due to the covid- pandemic. clerks whose clerkship had been suspended for any other reason were excluded. the survey (additional file ) contained four sections: ( ) demographics, ( ) stressors ( ) who well-being index, and ( ) stress management and resources. these four sections of the survey were developed by either adapting existing sources [ ] or by conducting short focus group sessions with a small sample of junior and senior clerks from a single canadian medical school. the first section of the survey consisted of questions on demographics; age, gender, level of education, university, implications and occupations, covid- infection status and preferred residency. senior clerks were asked about the specialty to which they had already matched. stressors included in the survey were developed with a focus group of canadian medical clerks. this focus group was composed of four third year medical students and two fourth year medical students whose clinical rotations were suspended due to the covid- pandemic. a meeting was held in order to select the main stressors by reflecting on their own experience as clerks and the experience of their classmates. this section included an initial question allowing an assessment of the respondent's stress level in the context of the covid- pandemic. the three subsequent questions assessed the impact of the pandemic on their choice of residency and their decision to pursue a medical career. if the respondent reconsidered his residency choice, an additional question was asked to see the type of residency change they considered. this section also contained a question allowing the respondent to rate, on a likert scale, the level of stress associated with potential stressors they may have experienced. most of these stressors were directly linked to future educational and professional pathways. the third section of the survey assessed the respondent's state of well-being by using the who well-being index [ ] . this measurement tool consists of five statements that respondents apply to their own lives. it has high clinical validity and is among the most widely used questionnaires assessing subjective psychological well-being [ ] . it has been translated into over languages, including french [ ] . the maximum score of this index is and a score of or lower indicates a poor state of well-being [ ] . the fourth section of the survey contained five questions evaluating whether the respondent used university psychosocial resources during the covid- pandemic, as well as the perceived usefulness of the resource. this section also included a question exploring the level of agreement regarding certain academic solutions. the level of agreement with each solution was assessed using a -point likert scale ranging from "strongly agree" to "strongly disagree". a final open-ended question allowed respondents to offer other potential solutions. in order to ensure validity of the survey, its contents were reviewed by a group of experts composed of members of the medical stimulation and education research group at the université of montréal and the ontario tech university, consisting of physicians, researchers in medical education, residents, and medical students. the survey was developed and distributed using the qualtrics xm platform [ ] . the request to participate was sent to the deans and curriculum directors of all canadian medical faculties. they were asked to review the summary of the study and the survey and decide if the faculty would participate. because the aim of this study was to capture the well-being, stressors, and management within the acute stage of the covid- pandemic, we requested a response within a month. for the faculties that agreed to participate, the clerkship directors were asked to send the survey to all eligible clerks by distributing an email containing a digital link. for faculties that chose not to participate, the clerkship directors were asked for a short explanation and reasoning for not participating in the study. participation in this study was voluntary and consent was obtained with a consent form at the beginning of the survey. the survey was anonymous and took to min to complete. the study was conducted between april th, and june rd, . data collected for each of the four sections of the survey were analyzed separately. the data was first cleaned and analyzed in microsoft excel. except for participants' characteristics, missing data from participants who elected not to answer certain questions were excluded from all calculations. categorical variables (age, level of study, home university, preferred residency match, covid status, and family covid status) were described as proportions of the study population. descriptive statistics were performed. proportions were used to assess the number of clerks who reconsidered medicine as a career and their residency choice, and to assess levels of stress during the pandemic. first, we assessed the frequency distribution of the index and performed descriptive statistics for all participants with the mean and standard deviation. second, we compared the mean who well-being index between two groups of participants: participants who reconsidered medicine as a career and participants who did not. this data was obtained from part of the survey (stressors). we used unpaired t-tests to compare these two means. for each analysis, confidence intervals were computed for a %-degree confidence. p-values less than . were interpreted as indicating a statistical difference. all inferential statistics were performed in graphpad prism [ ] . descriptive statistics were performed using proportions. for the final open-ended question, responses were grouped by theme. the themes that came up more than ten times by different students were considered valid solutions. of the canadian medical faculties, accepted to participate and were able to send the survey to their students within an acceptable timeframe. of the seven faculties that chose not to participate, one did not reply, two chose not to distribute the survey without providing a reason, and four chose not participate because their institutional research ethics board reviewing process could not meet the deadline of the study. there were responses, of which ( . %) were excluded because their clerkship rotations had not been suspended due to covid- , and two did not provide informed consent. therefore, a total of participants were included in the final survey. table shows the major characteristics of the participants. the majority of the included participants were female (n = , . %). during the confinement period, most clerks resided at home with their parents (n = , . %) or with a partner (n = , . %). a minority of respondents were taking care of a dependent, including a child and/or an elderly person (n = , . %). the vast majority of students reported partaking in hobbies and/or self-care activities during the pandemic (n = , . %) and most were involved in volunteering (n = , . %). only a minority of students participated in remunerated work outside of clerkship (n = , . %). many students had already had online medical classes at some point during clerkship suspension (n = , . %). figure reports that clerks ( %) reconsidered medicine as a career, and clerks ( %) reconsidered their residency choice. figure shows the clerks' level of stress during the pandemic with clerks ( %) reporting higher levels of stress than usual. figure describes participants' self-reported level of stress for various stress factors. the factor that was the most stressful for all clerks was the means of return to rotations (e.g., lack of supervision, lack of learning opportunities). the most stressful factors for junior clerks were feeling the need to stay productive in view of carms, and stress regarding all aspects of carms application. for senior clerks, having to take the mccqe after the beginning of residency was the most stressful factor. the overall mean who well-being index of the included participants was . ± . , indicating a poor level of well-being among a considerable proportion of students. figure shows the proportion of participants who reconsidered their medical career (n = ) and those who reconsidered their choice of residency (n = ) during the pandemic. participants who reconsidered medicine as a career had a mean who well-being index of . ± . . this value was significantly different when compared to participants who did not reconsider medicine ( . ± . ; t ( ) = . , p = . ). the mean who well-being index of participants who reconsidered of the included participants, ( . %) reported using various university mental health support resources to assist them during the pandemic. of these students, ( . %) did not find the resource they used helpful. with the recent covid- pandemic sweeping across canada, medical clerks were removed from clinical rotations and had their clerkship suspended, missing-out on valuable clinical experience [ ] . our study aimed to assess the impact of the covid- pandemic on the mental well-being, training, and career choices of canadian medical clerks. it also aims to assess the use of mental well-being support systems, as well as their appreciation of potential solutions to common academic stressors. the need to adapt to new environments is one of the challenging elements of medical students' educational journey [ , ] . the relative lack of tolerance for uncertainty and ambiguity has been found to be an important predictor of psychological distress among medical students [ ] . our study showed that approximately % of surveyed students had higher stress levels than usual during the pandemic (fig. ) . this may be due to the challenge of facing such a unique event. historically, there has been very few dropouts from canadian medical schools. according to the association of faculties of medicine of canada, only . % of students had dropped out of a medical school in [ ] . as shown in fig. , our study found that % of surveyors reconsidered medicine as a career during this pandemic, with % reconsidering their choice of residency. this could be due to limited clinical exposure, or perhaps the fear of not being competitive for selective residencies [ ] . however, this change in perspective may also be due to their mental state during the pandemic. in fact, the results of the third part of our survey showed that a considerable amount of respondents had a who wellbeing index below , which suggests poor well-being and would require psychological support [ ] . furthermore, clerks who reconsidered medicine as a career or reconsidered their residency choice had significantly lower mean who well-being indices. yet, our results showed that only a minority of students used a university support system, and of those who used a resource, half regarded it as unhelpful. this begs the question on whether the resources currently available to medical clerks are both accessible and well-tailored to their needs. our study also identified several factors that could explain this increase in stress levels during the pandemic (fig. ) . both junior and senior clerks were found to have increased stress with respect to means of clerkship resumption. as shown in fig. , the majority of clerks were not in favour of the resumption of rotations only at the end of the pandemic. instead, returning when cases cease to increase was deemed more favorable to them. interestingly, clerks were less anxious about contracting covid- during their rotations despite the inherent risk, which challenges the pertinence of suspending clerkship altogether. indeed, as evidenced by fig. , clerks were more concerned with their eventual return to rotations and the impact of the pandemic on their education and future careers than with contracting the disease or contaminating others. according to the demographics part of our survey, clerks were also more willing to stay active during clerkship suspension as more than half decided to volunteer during the crisis. it could therefore be argued that suspending rotations may not have been in the clerk's best interest. that being said, faculties made such a decision with more than just the interest of their students' in mind, as reducing the amount of vectors in clinical settings was pivotal to flattening the curve [ ] . among junior clerks, the need to stay productive during the pandemic in view of carms was a major source of anxiety (fig. ) . literature has shown that medical students utilize clerkship to make career decisions [ ] . it also provides students with a chance to undertake projects and obtain reference letters from senior staff members to help support their residency applications. with clerkship suspension, clerks were impeded from participating in patient care and working with attendings or residents [ ] . junior clerks were also in favour of flexibility so as not to be penalized for a lack of electives in their specialty of choice (fig. ) . this is particularly important as many faculties have decided to prioritize core rotations, and the possibility of doing electives outside one's home university has been suspended for an undetermined amount of time across the country [ ] . regarding senior clerks, a major stress factor was writing the mccqe part i during residency (fig. ) . traditionally, senior clerks were able to write the exam prior to starting their residency. however, due to social distancing rules, many test centers were shut down causing postponement of the exam [ ] . at the time of the survey, the majority of clerks were in favour of having multiple time periods to take the mccqe part i examination and having protected time during residency to study for it (fig. ) . surprisingly, as shown in fig. , graduating prematurely to actively help during the crisis was not a major source of stress. this is in keeping with the notion that most students were not afraid of contracting the disease or contaminating others. as part of the solutions that were explored in our study, the majority of clerks were highly in favor of novel learning methods (fig. ) . for many years, medical faculties have been aiming to incorporate technology into their teaching methods, and with the pandemic many were able to quickly transition their teaching sessions into online formats [ ] . this can provide a solid foundation to permanently incorporate remote learning in medicine. a limitation in our study is that medical schools in alberta, british colombia and saskatchewan are not represented. also, participating in the survey was optional and may have introduced a selection bias. furthermore, some elements of the survey were designed to be as specific as possible to the reality of canadian medical clerks. as such, not all means of measuring outcomes utilized validated questionnaires and may have introduced response bias. overall, the results of this survey should be viewed as a qualitative and descriptive assessment of the general impact of the pandemic on canadian clerks rather than an analytical assessment. the covid- pandemic has forced medical students to adapt to a unique situation in order to achieve their educational goals. the pandemic resulted in considerable stress among clerks which medical faculties should address. various solutions regarding the medical curriculum were viewed more favourably than others. this study can serve as a reference for faculty leaders when taking important decisions on behalf of their students. centre for addiction and mental health., canadian electronic library (firm) systematic review of depression, anxiety, and other indicators of psychological distress among u.s. and canadian medical students levels and sources of stress in medical students depression and stress amongst undergraduate medical students stress, coping, and well-being among third-year medical students burnout in medical students: examining the prevalence and associated factors perceived stress during undergraduate medical training: a qualitative study sources of stress for residents and recommendations for programs to assist them burnout and suicidal ideation among u.s. medical students mental health problems faced by healthcare workers due to the covid- pandemic-a review factors associated with mental health outcomes among health care workers exposed to coronavirus disease when health professionals look death in the eye: the mental health of professionals who deal daily with the coronavirus outbreak covid- and early medical school graduation: a primer for m s accessed supporting the well-being of medical students provision of mental health support for medical students the world health organisation-five well-being index (who- ) the who- well-being index: a systematic review of the literature who (five) well-being index (who- ) graphpad prism. . . for mac ed the association of faculties of medicine in canada emotional challenges of medical students generate feelings of uncertainty uncertainty and ambiguity and their association with psychological distress in medical students why do students quit medical school far less often in canada than in other countries? a systematic review of the factors affecting choice of surgery as a career medical students are not essential workers: examining institutional responsibility during the covid- pandemic free choice and career choice: clerkship electives in medical education medical student education in the time of covid- notice of suspension of application to the mccqe part i due to covid- springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to acknowledge every member of clerkship direction who helped with the distribution of the survey. received: july accepted: october the online version contains supplementary material available at https://doi. org/ . /s - - - . this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. the study protocol, consent form and recruitment documents of this study were approved by the institutional review board of the université de montréal (cerses- - -d). participation in this study was voluntary and consent to participate was obtained with a consent form at the beginning of the survey. participation in this study was voluntary and consent to participate was obtained with a consent form at the beginning of the survey. the consent form explained that the data obtained from the survey would be used for the purpose of publication. there is no conflict of interest to declare. key: cord- - apdn authors: hamlin, matilda; steingrimsson, steinn; cohen, itzhak; bero, victor; bar-tl, avishay; adini, bruria title: attitudes of the public to receiving medical care during emergencies through remote physician–patient communications date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: apdn providing health services through remote communications for sub-acute health issues during emergencies may help reduce the burden of the health care system and increase availability of care. this study aimed to investigate the attitudes of the public towards receiving medical services and providing medical information through remote communication in times of emergencies. during the pandemic outbreak of the novel coronavirus (covid- ), pandemic outbreak, participants answered a structured online survey, rating their mean willingness to receive medical care and provide medical information, on a four-point likert scale. furthermore, demographic characteristics, social media use, and trust in data protection was collected. the mean willingness to receive medical services was . ± . and the mean willingness to provide medical information was . ± . , with a strong significant correlation between the two (r = . ). the multiple regression model identified higher trust in data protection, level of education, and social media use as statistically significant predictors for a higher willingness to receive medical information while the first two predicted willingness to provide information. the findings suggest an overall positive attitude to receive medical care through remote communications. major disasters and emergency events pose continual threats to the public health. the current ongoing pandemic outbreak of the novel coronavirus (covid- ) constitutes a grave risk to the health of the global community and presents complex challenges in striving to contain the spread of the virus and mobilizing responses to maintain public health [ , ] . at the late end of december , the first human cases of covid- were observed in wuhan, china [ ] [ ] [ ] . the outbreak is currently ongoing, and as of the beginning of may , over . million confirmed cases were identified globally [ ] . to control a pandemic spread, strategies to minimize contact between infected and uninfected, must be utilized [ , ] . for example, in south korea, during , one single infected patient that visited a crowded emergency department (ed) caused additional cases of the novel middle east respiratory syndrome coronavirus (mers-cov), with a % attack rate of those who were in close the study was conducted among a population insured by meuhedet health services (mhs), which is one of four health funds operating in israel, with over million insured. this health fund was chosen for the study as it has advanced computerized systems for managing emergencies and was a leading actor in integrating telemedicine into the response plans for emergency scenarios. the sample size was calculated to respondents, using the open source software for epidemiological statistics, openepi at openepi.com. to encompass the two main populations that characterize the israeli society, both hebrew-and arabic-speaking individuals were included. as the division of the two groups in the israeli population is : , responses from the hebrew-speaking population and responses from the arabic-speaking population were collected. only individuals over years of age were included in the study. a cross-sectional study was performed in the end of january through february , in the midst of the covid- outbreak, following the publication that several israelis may have contracted the virus while visiting wuhan, china. the study was planned by the researchers prior to covid- , resulting from the need to prepare for varied types of emergencies, including security scenarios and potential natural disasters. as covid- erupted prior to the dissemination of the questionnaire to the respondents, it is not possible to disregard the impact of the pandemic on the answers of the participants. a supplementary figure illustrates a timeline for the different study procedures (supplementary material figure s ). the research tool used to collect data was a quantitative questionnaire developed specifically for this study, checked for content validity by content experts and pilot tested among participants, prior to its distribution. the questionnaire was conceptualized in hebrew, and translated to arabic (and re-translated to ensure validity) to enable both the majority and minority groups of israel to partake in the study. the first part of the questionnaire charted attitudes of the respondents concerning receiving medical services through remote communications during emergencies. six statements focused on the attitudes towards receiving medical care from the health fund's providers through remote communication (for example, receiving information about available medical facilities, instructions concerning needed medical treatment, distant medical examination through video-conference, etc.). six other statements concentrated on attitudes towards providing the health fund with information or requesting medical/mental health services through remote communication (for example, updates concerning your current medical condition, transmit photos or video of your medical condition, request a home visit, etc.). a -point likert scale was used ranging from = "not ready at all" to = "very ready" for each of the different statements. the second part of the questionnaire included demographic questions on gender, age, place of residence, marital status, number of children under the age of /adult offspring living with the respondents, education, religion, religiosity, and socioeconomic status. the third part of the questionnaire collected information on social media use, such as the frequency of activity and through which types of social media the respondents are willing to be contacted through in times of emergency. furthermore, the third part contained a question concerning the trust of the data protection in transferring medical information through remote communication. the questionnaire was distributed to members of mhs through an internet panel that employs a panel of over , individuals (http:www.ipanel.co.il). one of the exclusion criteria was members of other health funds, so that only members of this specific health fund were sampled. further, through the panel, a representation of all demographic and geographic components of the population was conducted using stratified sampling methodology, based on quotas of age, gender, and geographic classification. the representation was based on data from the central bureau of statistics. the likert scale for statements - was converted into two different indices, one index for the willingness to receive medical services from a health fund through remote communications during emergencies and another index for the willingness to provide information to a health fund through remote communications during emergencies. the reliability of the indices was tested by cronbach's alpha and found to be very high ( . for willingness to receive medical services and . for willingness to provide information through remote communication). to investigate differences in mean willingness between groups of characteristics, an independent samples t-test was calculated for dichotomous variables and an analysis of variance (one-way anova) was calculated for variables with more than two values. when statistically significant values in the one-way anova were presented, it was followed by bonferroni's post hoc test. the pearson's correlation coefficient (pearson's r) was calculated to examine a correlation between the willingness to receive medical services and the willingness to provide medical information through remote communication during emergencies. spearman's rank correlation coefficient (spearman's r s ) was calculated to examine monotonic correlations between the willingness to either receive medical services or provide medical information and the ordinal variables social media use and trust of the data protection in transferring medical information through remote communication. the correlation was considered weak when coefficients r or r s ranged between − . and . ; moderate between >− . and − . or > . to . ; and strong between >− . and − . or > . and . . finally, a prediction analysis was conducted using a multiple linear regression model. all statistical analysis was conducted using ibm spss statistics version (ibm corp., armonk, ny, usa). p value below . was considered significant. the study protocol was approved by ethical committees at the tel aviv university (# - ) and at meuhedet health services (# p- - - - ) according to local ethical guidelines. each participant gave an informed consent after receiving information of the study before any data was collected. furthermore, the study followed the declaration of helsinki principles in research ethics where participation was voluntary without a risk for breach of personal integrity. the characteristics and descriptive statistics of the respondents to the questionnaire are presented in table . the mean age was . (standard deviation (sd) = . ) years and ranged between and years. the majority of the respondents had an income level near or above the average monthly salary ( . %) and more than one child ( %). self-reported frequency in use of social media was ≤ a week ( . %), several times a week ( . %), and several times a day ( . %). when rating the trust of data protection, . % answered they "do not trust", . % answered they "trust", and . % "highly trust". the mean willingness to receive medical services from the health fund through remote communications, in times of emergencies, was . (sd = . ). the mean willingness of the respondents to provide medical information to the health fund through remote communications, in times of emergencies, was . (sd = . ). figure shows the statistically significant differences that were found in mean willingness to receive medical services through remote communications during emergencies. according to age, the mean willingness to receive was significantly higher among those above years of age compared to those under years (t( ) = . , p = . ). similarly, the mean willingness to provide medical information through remote communications was found to be numerically higher in the age group above years (mean . , sd = . and mean . , sd = . , respectively); however, this was not statistically significant (t( ) = . , p = . ). figure presents the statistically significant differences in the mean willingness to provide medical information through remote communications during emergencies. socioeconomic-related differences were statistically significant in the mean willingness to both receive medical services (f( ) = . , p = . ) and provide medical information through remote communication (f( ) = . , p = . ). post hoc analysis using bonferroni's multiple comparison showed higher levels of mean willingness to receive medical services among respondents with an above-average salary compared to respondents with a salary near average (p = . ); however, the multiple comparison was not statistically significant in willingness to provide. significant differences were seen between the respondents according to their level of trust of the data protection when transferring medical information through remote communications during emergencies, in both mean willingness to provide medical information (f( ) = . , p < . ) and to receive medical services (f( ) = . , p < . ). further post hoc analysis, using the bonferroni multiple comparison, showed that the mean willingness of both providing and receiving was higher between all groups that reported higher levels of trust of data protection (p < . ). there were no significant differences between different religions, which is equivalent to the two language categories, hebrew and arabic. furthermore, gender, living area, religiosity, marital status, education, and social media use were not associated with a higher mean willingness to receive or provide medical care/information. there was a significantly strong positive linear correlation between willingness to provide medical information and willingness to receive medical services through remote communication during emergencies (r = . , p < . ). significant moderate positive monotonic correlations were found between trust in data protection in transferring medical information through remote communication and both the willingness to provide (rs = . , p < . ) as well as the willingness to receive (rs = . , p < . ), i.e., higher willingness was associated with higher trust. a linear regression model was conducted in order to examine which variables can predict the willingness of the respondent to receive medical services through remote communications. the anova test was significant (f( ) = . , p < . ) and showed a good fit of the model (r = . ). there was a significantly strong positive linear correlation between willingness to provide medical information and willingness to receive medical services through remote communication during emergencies (r = . , p < . ). significant moderate positive monotonic correlations were found between trust in data protection in transferring medical information through remote communication and both the willingness to provide (rs = . , p < . ) as well as the willingness to receive (rs = . , p < . ), i.e., higher willingness was associated with higher trust. a linear regression model was conducted in order to examine which variables can predict the willingness of the respondent to receive medical services through remote communications. the anova test was significant (f( ) = . , p < . ) and showed a good fit of the model (r = . ). there was a significantly strong positive linear correlation between willingness to provide medical information and willingness to receive medical services through remote communication during emergencies (r = . , p < . ). significant moderate positive monotonic correlations were found between trust in data protection in transferring medical information through remote communication and both the willingness to provide (r s = . , p < . ) as well as the willingness to receive (r s = . , p < . ), i.e., higher willingness was associated with higher trust. a linear regression model was conducted in order to examine which variables can predict the willingness of the respondent to receive medical services through remote communications. the anova test was significant (f( ) = . , p < . ) and showed a good fit of the model (r = . ). this means that in total, % of the variance of a respondent's willingness to receive medical services through remote communications can be explained by using three variables: the respondent's level of trust in data protection (p < . ), respondent's level of education (p = . ), and respondent's level of activity on social media (p = . ). among these three variables, the trust of data protection has the highest effect on respondents' willingness to receive medical services (b-coefficient = . ). the linear regression model is presented in table . gender, age, marital status, religion, religiosity, socio-economic status, number of children under the age of , or other dependent adults living with the respondents were not statistically significant in the multiple analysis and therefore were not included in the model. similarly, a linear regression model was conducted to examine the predicting variables in the willingness to provide medical information through remote communications. the anova test was significant (f( ) = . , p < . ), with r = . . in total, % of the variance of a respondent's willingness to provide medical information through remote communications can be explained by using two variables: the respondent's level of trust in data protection (p < . ) and the level of education (p = . ). among the two variables, the trust of data protection has the highest effect on respondents' willingness to provide medical information (b-coefficient = . ). the linear regression model is presented in table . gender, age, marital status, religion, religiosity, socioeconomic status, number of children under the age of , or other dependent adults living with the respondents and level activity on social media were not statistically significant in the multiple analysis and therefore were not included in the model. this study, examining attitudes to receive health services through remote communications in times of emergency, was conducted during an emergency situation-the covid- crisis. though the actual cases that were infected in the israeli communities were only confirmed at a later date, the study was conducted during a period in which the population was notified of an evolving epidemic that though (at that time) it impacted other countries, it was expected to also impact the local society. therefore, it can be assumed that the results of this study realistically reflect the attitudes and perceptions of the public concerning consumption of services through remote communication, in a way which could not have been achieved otherwise [ ] . in this situation, when mobility in society is restricted and medical facilities are primarily dedicated to treating the moderate and severe patients, it is more likely that respondents can relate and prefer health services through remote communications as an alternative way to receive needed medical care for sub-acute illnesses and monitoring of chronic illnesses [ ] . furthermore, it may reflect their concern from being exposed to the risk, such as contracting the covid- if they were to physically go to the medical facilities [ ] , and thus consent to utilizing the services through telemedical means. the willingness of the general public to provide medical information and to receive medical services from a health fund through remote communications in times of emergency was found to be high. by testing each factor's effect on patient's willingness to receive medical services and provide medical information, the factors associated with a higher willingness to receive medical services were age, salary in relation to average income, and trust of the data protection while for willingness to provide medical information, the latter two were statistically significant. the respondents scored very similar in willingness to receive medical services and provide medical information to a health fund through remote communications in times of emergency. not surprisingly, there was a significant strong positive correlation between the two variables. the predictors of willingness to receive medical services by using a multiple model were trust of data protection transferring medical information through remote communications, level of education, and level of activity on social media. similarly, the predictors of willingness to provide medical information were trust of data protection and level of education. the r value represents the percent of the variance of the dependent variable that can be explained by using the regression model including the independent variables. the prediction values were relatively low, which could be explained as there was a low variance in the willingness reported by the participants. further, including other independent variables in order to predict the dependent variables could have increased the r value. according to the conducted literature review, no previous studies concerning attitudes towards health services through remote communications in times of emergency were identified. however, the willingness to use telemedical means among patients with specific medical conditions, such as chronic diseases with increased risk of cardiovascular disease or depression, has been studied [ ] [ ] [ ] . in one of them, an american study investigating attitude towards telemedical means when monitoring diabetic retinopathy, valikodath et al. reported ( ) that % of their respondents had never been in contact with the word telemedicine before [ ] . this indicates that there are barriers to overcome when implementing telemedical means in general; however, patient satisfaction rates are reported as very high in the majority of studies when it is implemented [ ] [ ] [ ] . the trust of data protection transferring medical information through remote communications is the one factor that remained statistically significantly associated with willingness to use remote communications in all our analyses. these results are in accordance with previous studies [ ] [ ] [ ] . in a dutch study, trust of the data protection in transferring medical information through remote communications is shown to be the one sub-factor that affects the overall trust in using telemedicine the most, with a higher impact than both trust in health care providers, health care workers, and offered treatment [ ] . furthermore, a german study showed how the trust of data protection is of great importance, especially among healthy respondents, when considering the use of health services through remote communications [ ] . this study indicates that socioeconomic differences might affect the attitude towards medical services through remote communications. an american study [ ] found that the level of education was associated with increased use of health services through remote communications. similarly, green and colleagues found that a lower education level was associated with a higher likelihood in refusing to participate in a trial to monitor and optimize blood pressure through remote communications [ ] . level of activity on social media was a predictor in the willingness to receive medical services through remote communications. in a recent study, it was shown that in an ed-visiting population with a low socioeconomic status, % had access through a smart phone [ ] , indicating that in high-income regions, the access to social media is high regardless of socioeconomic status. although age was not found to be an independent predictor, it is surprising that younger age was not a strong predictor. even though technological use is increasing in the older generation [ ] , it is common that the younger generation uses technology to a greater extent. it is reasonable to believe that more frequent use and convenience in technology would correlate with a higher willingness towards telemedicine. in accordance with this consideration, a polish study examining the willingness to use telemedicine among those years and older showed that % of the respondents were reluctant to conduct a video consultation with a physician [ ] . one possible explanation to the counterintuitive age-related results found in our study can be that in times of emergency, the older generation are more likely to trust the authorities in emergency management compared to the younger generation. it may also reflect a higher degree of concern with health-related issues among older individuals compared to younger people, who tend to be generally healthier. more so, as the elderly were defined as highly vulnerable to covid- , their willingness to access services through remote communication may signify their perceived concern of leaving their homes and thus a higher preference for maintaining their safety by not being potentially exposed to the virus. the younger populations, perceiving themselves as less vulnerable, were most probably less concerned with the need to strictly maintain social distancing [ ] . the cross-sectional design cannot determine causality nor capture changes in attitudes over time. given this, it is difficult to assess the extent of the impact that covid- has had on the results. future research is needed to evaluate the differences in usage and patient satisfaction of telemedical means, actually implemented in clinical practice during the covid- pandemic outbreak. furthermore, a strength of the study is how the internet panel, with over , individuals, made it possible to stratify the sampling, based on quotas of age, gender, and geographic classification, enabling a comparison to the israeli population and also to collect the exact calculated sample size. however, distributing the questionnaire exclusively through an internet panel when the aim was to investigate willingness to access health services using electronic tools could exclude groups in the society who do not have access or are reluctant to use electronic devices. if the questionnaire were to be conducted also by post or phone-call, respondents with less access to electronic tools may have been more fully represented. in this cross-sectional study, conducted during an ongoing worldwide pandemic outbreak, the willingness to receive medical services and to provide medical information through remote communications in times of emergency was found to be high. there are factors associated with statistically significant differences in the attitude towards health services through remote communications, where the trust of data protection in transferring medical information through remote communications was the factor with the highest impact. the results are important in future implementation of these solutions in clinical practice. funding: this research received no external funding. steinn steingrimssons' research time was financed by grants from the swedish state under the agreement between the swedish government and the country councils, the alf-agreement (alfgbg- ). will ebola change the game? ten essential reforms before the next pandemic. the report of the harvard-lshtm independent panel on the global response to ebola characteristics of and public health responses to the coronavirus disease outbreak in china three emerging coronaviruses in two decades epidemiological and clinical characteristics of cases 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accommodate in times of emergency) risk of influenza a (h n ) infection among health care workers exposed to patients with influenza a (h n ), hong kong local public health workers' perceptions toward responding to an influenza pandemic virtual first responders: the role of direct-to-consumer telemedicine in caring for people impacted by natural disasters experts' views on the gaps in public health emergency preparedness in israel: a qualitative case study the effectiveness of video-telemedicine for screening of patients requesting emergency air medical transport (eamt) evaluation of an accident and emergency teleconsultation service for north-east scotland emergency department telemedicine shortens rural time-to-provider and emergency department transfer times israeli, a. health and health care in israel: an introduction validating human behavior representation model of general personnel during offshore emergency situations covid- pandemic: what consequences for cardiac rehabilitation? monaldi arch anxiety regarding contracting covid- related to interoceptive anxiety sensations: the moderating role of disgust propensity and sensitivity are people with chronic diseases interested in using telehealth? a cross-sectional postal survey patient attitudes toward telemedicine for diabetic retinopathy potential of mobile health technology to reduce health disparities in underserved communities patients' satisfaction with and preference for telehealth visits patterns of use and correlates of patient satisfaction with a large nationwide direct to consumer telemedicine service systematic review of patient and caregivers' satisfaction with telehealth videoconferencing as a mode of service delivery in managing patients' health measuring patient trust in telemedicine services: development of a survey instrument and its validation for an anticoagulation web-service trust in telemedicine portals for rehabilitation care: an exploratory focus group study with patients and healthcare professionals privacy and data security in e-health: requirements from the user's perspective. health inform predictors of ehealth usage: insights on the digital divide from the health information national trends survey patient ability and willingness to participate in a web-based intervention to improve hypertension control factors associated with change in internet usage of swedish older adults health in old age, and patients' approaches to telemedicine in poland awareness, attitudes, and actions related to covid- among adults with chronic conditions at the onset of the u.s. outbreak: a cross-sectional survey the authors would like to thank the respondents to the questionnaire. the authors declare no conflict of interest; funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- - naeuo authors: zhou, yongjie; wang, wenjuan; sun, yanping; qian, wei; liu, zhengkui; wang, ruoxi; qi, ling; yang, jiezhi; song, xiuli; zhou, xin; zeng, lingyun; liu, tiebang; li, zezhi; zhang, xiangyang title: the prevalence and risk factors of psychological disturbances of frontline medical staff in china under the covid- epidemic: workload should be concerned date: - - journal: j affect disord doi: . /j.jad. . . sha: doc_id: cord_uid: naeuo background: to our best knowledge, this was the first time to investigate the prevalence and risk factors of psychological disturbances, including depression, anxiety, somatization symptoms, insomnia and suicide, among frontline medical staff, who were working with the covid- infected patients directly. methods: patient health questionnaire depression (phq- ), generalized anxiety disorder questionnaire scale (gad- ), symptom check list- (scl- ) somatization, insomnia severity index (isi), and the suicidal module of the mini international neuropsychiatric interview were used for online survey. results: a total of frontline hospital staff and general population were recruited. the prevalence of depression, anxiety, somatization symptoms, insomnia, and suicide risk in frontline medical staffs were . %, . %, . %, . % and . %, respectively. except for suicide risk, the prevalence of other psychological disorders in frontline medical staff were higher than those in general population (all p< . ). among the frontline medical staff, the daily working hours were associated with all psychological disturbance (all p< . ), women with anxiety (p= . ), body mass index (bmi) with anxiety and insomnia (p= . , p= . ). age was negatively associated with depression, anxiety, and insomnia (all p< . ). finally, years of working and family income were negatively associated with suicide risk (p= . , p< . ). conclusion: our study demonstrates that during the outbreak of covid- , the frontline medical staff are more likely to suffer from psychological disturbances than general population. it is noticeable that daily working hours are a risk factor for all measured psychological disturbances, and some other variables may be involved in certain psychological disturbances of frontline medical staff. in december , a novel coronavirus epidemic broke out in wuhan, the capital city of hubei province, china paules et al., ) . epidemiological studies have confirmed its spread from person to person and spreads rapidly around the world. it is named as corona virus disease by the world health organization (who) (world health organization, ) . covid- is a highly contagious disease with asymptomatic infection during the incubation period, and can be transmitted through respiratory droplets, contact and aerosols (world health organization, ) . as a result, covid- quickly spread throughout the country and many other countries. covid- is highly contagious and has a high mortality rate in some populations, but there is even no special treatment zhou et al., ) , which seriously threatens the physical and mental health of the public (gao et al., ) . in particular, frontline medical staff are vulnerable to infection and spread the virus to their colleagues or family members. since january , , china has sent more than , medical staff to provide medical support to hubei province. to date, more than , medical personnel across the country have been infected with covid- (guangming online, ) . previous studies have shown that in high-stress and high-risk disease pandemics, medical staff often encounter huge mental burdens and various psychological barriers (lehmann et al., ; lin et al., ; kang et al., ) . although attention has been paid to the mental health of medical staff in the fight against covid- , few studies have investigated the psychological disturbances and risk factors of frontline medical staff, especially considering the impact of the work conditions, such as workload. therefore, the purpose of this study was to investigate the prevalence and risk factors (including work overload) of psychological disorders among frontline medical staffs in china, including depression, anxiety, and somatization symptoms, insomnia and suicide risk. to the best of our knowledge, this was the first study to investigate the prevalence and risk factors of psychological disorders of frontline medical staff. since this study was conducted during the covid- pandemic, it was difficult to conduct face-to-face interviews on quarantine policy. therefore, online questionnaire was constructed using a mobile application. this online questionnaire was designed to investigate psychological disorders during the epidemic by using ranxing technology "surveystar". the questionnaire was anonymous, and the information of all respondents was confidential. the data was collected from february to march , . responders aged years or older were included in this study. a total of first-line hospital staff and ordinary people were recruited from cities in china. the survey was approved by the institute of psychology, chinese academy of sciences. each participant signed an electronic informed consent form before starting the survey. demographic and working status information were collected, including age, sex, body mass index (bmi), ethnicity, marriage status, education, annual family income, infected status of relatives or friends, years of working, daily working hours, and whether experienced sars outbreak in . depressive symptoms were assessed using the patient health questionnaire depression (phq- ), which is a convenient and effective scale for detecting depressive symptoms (kroenke et al., ) . phq- includes questions, and each question has a score of to . the total score ranges from to , and a score≥ is identified as having depressive symptoms kroenke et al., ) . anxiety symptoms were assessed using the generalized anxiety disorder questionnaire scale (gad- ) (spitzer et al., ) . gad- includes questions and each question has a score of to . the total score ranges from to , and a score ≥ is identified as having anxiety symptoms spitzer et al., ) . somatization symptoms were assessed by symptom check list- (scl- ) somatization factor (som). there are questions, and each question has a score of to (fau et al., ) . the total score ranges from to , and a score≥ is identified as having somatization symptoms. insomnia was assessed by using the insomnia severity index (isi). there are questions, and each question has a score of to . the total score ranges from to , and a score≥ is identified as having insomnia. neuropsychiatric interview (mini), which includes questions. the total score ranges from to . a score≥ and < is classified as mild suicide risk, ≥ and < as moderate suicide risk, and ≥ as severe suicide risk. kolmogorov-smirnov one-sample test was conducted to test the normality of the distribution. an analysis of variance (anova) of continuous variables and a chisquare test of categorical variables were conducted. then analysis of covariance (ancova) was applied to adjust for the confounding factors to compare scores on each assessment scale. in order to explore the risk factors for each psychological disorders of front-line medical staff, a univariate analysis was conducted. then the variables with significant differences were included in the multivariate logistic regression. bonferroni correction was applied for multiple tests. the data were considered statistically significant when p < . . analyses were performed by using spss version . (ibm co. ltd, chicago, il, usa). as shown in table , except bmi, there were significant differences in age, sex, education, marital status, ethnicity, family income, physical diseases and the covid- infection of relatives and friends between frontline medical staff and the general population (all p< . ), which were adjusted for in the following statistical analyses. after adjusting for age, sex, education, marital status, ethnicity, family income, physical diseases and infection of relatives and friends, ancova showed that except suicide risk score (f= . , p= . ), frontline medical staff had significantly higher scores of phq- (f= . , p< . ), gad- (f= . , p< . ), scl- somatization (f= . , p< . ), and isi than general population (f= . , p< . ). after adjusting for age, sex, education, marital status, ethnicity, family income, physical diseases and infection of relatives or friends, , the prevalence rates of depressive symptoms, anxiety symptoms, somatization symptoms, and insomnia were higher in frontline medical staff than those in general population ( . % vs. as shown in table , univariate analysis found that there was a significant difference in some variables between frontline medical staff with and without psychological disorders. then these significant variables were included in each logistic regression to investigate factors related to each psychological disorder of frontline medical staff. in terms of depressive symptoms, logistic regression showed that age was negatively associated with depressive symptoms (b=- . , wald's covid- is a stressor that has a great impact on both individuals and the public, especially those at the core of the event. in the face of the covid- patients, frontline hospital staff are under unprecedented tremendous mental distress, thus it is becoming more and more urgent to research and solve the needs of these health care workers. our study mainly found that that ( ) except for the risk of suicide, the symptoms of depression, anxiety, somatization and insomnia in frontline medical staff were more serious than those in the general population; ( ) in frontline medical staff, daily working hours were positively associated with all psychological disorders, including depression , anxiety, somatization, insomnia and suicide risk. ( ) female gender was positively associated with anxiety symptoms in frontline medical staff. ( ) bmi was positively associated with anxiety symptoms and insomnia in frontline medical staff; ( ) age was negatively associated with the symptoms of depression, anxiety and insomnia, while the years of working and family income were negatively associated with suicide risk in frontline medical staff. we found that the prevalence of depressive symptoms, anxiety symptoms, somatization symptoms, insomnia and suicide risk in frontline medical staff were . %, . %, . %, . % and . %, respectively. lai et al., recently reported that depression, anxiety symptoms and insomnia accounted for . % . %, and . % of the total medical staff, which was very similar to our results (lai et al., ) . however, zhang et al. recently reported that depression, anxiety symptoms, insomnia and somatization symptoms were . %, . %, . % and . % in medical workers, respectively, most of which were lower than our results (zhang et al., ) . the reason may be that they did not distinguish between frontline and non-frontline medical staff, because frontline medical staff who were in face of infected patients were reported more severe mental health symptoms than other health care workers (lai et al., ) . compared with the general population, we found that frontline medical staff experienced higher symptoms of depression, anxiety, somatization, and insomnia. zhang et al. also showed that medical staff reported much more depression, anxiety, somatization symptoms and insomnia than nonmedical workers (zhang et al., ) , suggesting that the medical staff, especially frontline medical staff had greater psychological distress than the general population. most medical staff in hospitals usually had not received mental health training (lima et al., ) . therefore, during this covid- epidemic, china also sent a large number of psychiatrists and psychologists to hubei province to provide mental health support to both patients and frontline medical staff. in this study, we also investigated the risk factors of psychological disorders in frontline medical staff. the main causes of psychological distress for frontline medical staff may include long working hours, fatigue and the risk of infection, as well as putting their families at life-threatening risk. however, no studies have investigated the impact of these factors on the psychological disorders under the covid- epidemic. we first reported that daily working hours were a risk factor for all measured psychological disorders including depression and anxiety, somatization, insomnia and suicide risk. in the covid- pandemic, frontline medical staff were facing overwhelming workload pressure, which may lead to job burnout (moazzami et al., ) . previous evidence indicates that work overload was a critical cause of exhaustion, which led to burnout of health care workers (portoghese et al., ) . in addition, cole et al., found that medical staff with the largest workload were more likely to receive mental health treatment (cole et al., ) . we found that women were a risk factor for anxiety symptoms in frontline medical staff, which was partly consistent with recent study. lai et al. showed that women had more severe anxiety symptoms than male medical staff (lai et al., ) . interestingly, we also found that bmi was positively associated with anxiety symptoms and insomnia. there is growing evidence that overweight/obesity was associated with anxiety (simon et al., ) and poor sleep quality in adults (jennings et al., ) . huang et al., indicated that less slow-wave sleep, or deep sleep, in patients with insomnia was associated with higher bmi (huang et al., ) . shochat et al., found that high bmi was associated with shorter sleep time and lower sleep efficiency than normal and low bmi individuals (shochat et al., ) . in this study, several protective factors for psychological disorders were observed in the frontline medical staff. we found that the advanced age was a protective factor for depression symptoms, anxiety symptoms and insomnia, while years of working and family income were protective factors for suicide risk. it is worth noting that since the covid- epidemic, there have been cases of medical staff suicides worldwide (goyal et al., ; nicola, ) . recently, the new epicenter of the covid- pandemic, the national suicide prevention lifeline, has been launched to prevent suicide in the united states due to psychological distress related to covid- . there were several limitations in this study. first, due to the special situation of the covid- epidemic, the online questionnaire was designed by using psychological scales, and the diagnosis cannot be determined by face to face interviews. we only detected psychological symptoms such as depression, and anxiety but did not make a diagnose. further, the information of the participants were not obtained if they did not submitted the online questionnaire. therefore, we could not obtain the data of the participants who did not submit the questionnaire, which could not eliminate the selection bias. second, the cross-sectional design prevented investigation of the causal relationship between related factors and psychological disorders. the psychological disorders of medical staff may become more serious when they encounter the covid- epidemic. further prospective cohort studies should be conducted to verify our results and hypotheses. in summary, our study shows that compared with the general population, the frontline medical staff are more likely to develop depression, anxiety, somatization and insomnia. among frontline medical staff, daily working hours are a risk factor for all measured psychological disturbances. further, women and bmi are risk factors, while age, years of working and family income are protective factors for certain psychological disorders of frontline medical staff. however, considering the limitations of our cross-sectional study, our findings should be validated using a prospective cohort design in future studies. all participants provided their online informed consent. the study was approved by the institute of psychology, chinese academy of sciences. the authors declare no conflicts of interest. y.z., z.l., and x.z. designed the study. y.z. and z.l. wrote the paper. z.l. and r. w. analyzed the data; w. q., z. l., l. q., j. y., x. s., x. z. provided administrative, technical, or material support; z.l., and x.z. revised the manuscript. this study was supported by the national natural science foundation of china regions, hospitals and health outcomes over time: a multi-level analysis of repeat prevalence among a cohort of health-care workers scl- : an outpatient psychiatric rating scale--preliminary report / / ). mental health problems and social media exposure during covid- outbreak fear of covid : first suicidal case in india! china medical treatment expert group for covid- . clinical characteristics of coronavirus disease in china beijing: central steering group: over , medical staff in hubei were infected in the early stage of the epidemic, currently no infection reports among medical aid staff polysomnographically determined sleep and body mass index in patients with insomnia selfreported sleep quality is associated with the metabolic syndrome the mental health of medical workers in wuhan, china dealing with the novel coronavirus the benefits of indirect exposure to trauma: the relationships among vicarious posttraumatic growth, social support, and resilience in ambulance personnel in china the phq- validity of a brief depression severity measure factors associated with mental health outcomes among health care workers exposed to coronavirus disease ebola and psychological stress of health care professionals early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia prevalence and associated physical symptoms of depressive and anxiety symptoms in neurology outpatient clinic the emotional impact of coronavirus -ncov (new coronavirus disease) the psychological effect of severe acute respiratory syndrome on emergency department staff covid- and telemedicine: immediate action required for maintaining healthcare providers well-being the emotional impact of covid- : from medical staff to common people short sleep duration and weight gain: a systematic review coronavirus infections-more than just the common cold burnout and workload among health care workers: the moderating role of job control relationships between body mass index and sleep quality and duration in adults years and older association between obesity and psychiatric disorders in the us adult population a brief measure for assessing generalized anxiety disorder: the gad- geneva: who characterizes covid- as a pandemic mental health and psychosocial problems of medical health workers during the covid- epidemic in china a pneumonia outbreak associated with a new coronavirus of probable bat origin we appreciate dr. meihong xiu, lei zhao and yong fan for their kindly help. the data that support the findings of this study are available from the corresponding author x.z. upon reasonable request. key: cord- -dddf u authors: patil, poorvaprabha; chakraborty, stuti title: where does indian medical education stand amidst a pandemic? date: - - journal: j med educ curric dev doi: . / sha: doc_id: cord_uid: dddf u the recent outbreak of covid- , declared a pandemic, has affected medical education globally. the scenario is no different for medical students in india as they find themselves at a crossroads in their careers, with clinical and elective postings called off. missing out on the opportunity to learn from “first-hand” clinical observation stands to threaten the quality of medical education and learning procured by indian medical students which is extremely essential to deal with the vast patient load that awaits them in their impending future as healthcare professionals. is the indian medical education system being able to cope with the challenges imposed by the increasing burden of covid- ? the authors propose few administrative and on-ground interventions that must seek to work collectively with all government and private medical institutions in order to help students/interns and residents in coping with stress, anxiety or academic losses incurred due to the pandemic. health outcomes are immensely affected by the structure of health systems and policies and also play a pivotal role in determining how various healthcare services are delivered. due to the presence of a federal government in india, shouldering the responsibility of running health care systems' operations and governance have been divided amongst the union (central) and individual state governments. medical education in india is governed by the union and state governments in unison. india's healthcare system consists of a combination of private and public healthcare services and facilities-both providing medical education either under directives of the state/union governments or through private institutional affiliation. the formal training for indian medical education spans a long time and was initiated before india's independence from colonization in . over the years, medical education has evolved significantly to where it stands todaybeing one of the most competitive fields of education to secure a future in, for indian students. a total of medical colleges recognized by the mci (medical council of india) provide approximately seats for more than . million medical aspirants (as per ) who take the national eligibility cum entrance test (neet). once an aspiring student secures a place amidst the grinding competition, she/he is expected to undergo five and a half years of medical training and education, including a period of year of compulsory internship. in addition to studying various clinical subjects, medical students are expected to complete compulsory clinical postings with moderated number of hours in each clinical subject, are subjected to continuous clinical and paper-based evaluation/assessments, need to fulfil clerkships, elective postings-all while dealing with the anxiety and stress that comes with course load completion before final exams. [ ] [ ] [ ] [ ] the recent sars cov- (also known as covid- or novel coronavirus) disease outbreak was declared as a pandemic by the world health organization (who). the rapid spread of the infection has disrupted daily life and put even the best healthcare systems across the world to a test. this has put an insurmountable amount of pressure on all medical services, including medical education. medical students across the world stand at a difficult juncture amidst this unanticipated situation. currently, several governments and medical schools around the world have taken measures to curb electives and clinical teaching in its entirety while some have chosen to maintain a few. nevertheless, medical students find themselves at a painful crossroad of being the future of healthcare in their respective countries and at the same time being unable to provide services during this crisis as a result of not being fullfledged, licensed professionals. [ ] [ ] [ ] [ ] scenario in india covid- has impacted the journey of medical students in proportions far beyond what could possibly be fully mitigated. most medical schools in the country have cancelled face-toface lectures, clinical postings, practical classes, demonstrations, and have moved to online lectures. impact on medical students. in india, students preparing to be medical doctors study anatomy, physiology and biochemistry in the first year of their five and a half years coursework. during the first year, in addition to lectures, students visit the dissection hall almost on a daily basis where they learn about the human anatomy. dissection hall sessions help them in building an understanding of how the intricate structure of the human body functions and lays the foundation for a better understanding of more advanced subjects like orthopedics and surgery. this is further supplemented by practical classes, demonstrations, seminars, small group tutorials and hospital visits. in light of the lockdown, medical students in their first year have been walloped, with most of these lessons now being attempted to be delivered online, which makes it challenging to understand the relationship of different human anatomical structures in the body and eliminates the "learning by doing" component of education by performing dissections. hospital postings/clinical rounds are undoubtedly the most crucial component of medical education that follows after the first year. budding doctors learn how to take a case history, identify signs and symptoms, interact with patients, and observe interesting cases while they also shadow doctors on rounds. everything that they spend hours and days learning from books finally starts to fall into perspective on witnessing findings in patients. a lot of medical students have at least once heard during the course of their education, "what you read, you might forget. what you see (in clinics), you will always remember". covid- has taken away the chance for medical students to learn through clinical rounds; thousands of them are engulfed in uncertainty and fear of how they will be able to make up for this academic loss. with the rapidly evolving nature of the current situation, most medical schools across the country have not been able to decide on how they will make changes to the existing curricula and annual plan to be able to do justice to their students. several colleges have resorted to assigning humongous portions as "self-study" modules failing to take into account familial conditions and the possible disruptive environment in homes while mental health issues and domestic violence are at a steep rise during the nationwide lockdown. this is an even bigger concern, especially for many indian students, residing in joint families, consisting of a large number of family members, where the environment encountered might not always be conducive for academic pursuits. final year students, just a year away from finally being doctors, are anxious about how they will make up for the academic loss and cope with the added burden of an already extremely intense course year. impact on interns and residents. the condition of medical students is not particularly promising but interns and residents undoubtedly have it far more difficult. while residents learn specialty-specific skills during their post-graduation, a -year-long internship is the last component of undergraduate medical education where interns apply and develop their clinical skills, under the guidance of senior doctors. at some health facilities, senior doctors and specialists are not attending to patients, leaving the entire responsibility to interns and resident doctors. several interns and residents have been working extra duty shifts in resource-deprived conditions, with a gross lack of personal protective equipments (ppes)-away from their families and homes. at a few centers, ppes strangely seem to be following a hierarchy-the senior doctors get the best of what is available, residents follow, and interns are left with barely any quality equipment to protect themselves adequately. in a study conducted in may , around % of all respondents also perceived discrimination based on hierarchy (eg, senior doctors vs junior doctors, nurses vs interns) or departments, in the distribution of ppe. a large number of doctors have been reusing masks, gloves, and scrubs. moreover, faced with a shortage of personal protection equipment (ppe), india's healthcare workers have been reduced to using raincoats and garbage bags instead. while stories are surfacing all over social media and internet globally, calling doctors and health professionals covid heroes and sentinels, there have been far too many instances of doctors being assaulted in india over the past weeks. while having put themselves and the lives of their families at risk to protect their communities from the pandemic, there are reports of health professionals facing discrimination and stigmatization, being removed from homes, fighting for proper ppes, being beaten and ill-treated. such incidents doing rounds on social media and news broadcasts could have a long-lasting and indelible impact on aggravating the anxiety and panic of the impending future on the minds of medical students and young doctors. , how this will impact the current generation of budding doctors, in the long run, remains a mystery. amidst the long-standing anarchy that presently shrouds india which has been on lockdown since march th, , the future of medical education for millions of indian medical students is at stake, and the stakes are high. impact on career paths. it is not just the impact of the pandemic on the mindsets of budding doctors that is worrisome; it is also the uncertainty that looms over the future prospects of these medical students. with international licensing exams being called off, admission processes halted within and outside the country, examinations for many postgraduate medical courses postponed, there have been numerous qualms, making the mental impacts of the pandemic on some medical students worse than the others. postponement of national level competitive entrance exams proposes another threat to continuity of the supply of trained doctors and health workforce, which might result in a crunch in trained workforce in a few years. hence, the impact is being experienced not just by existing medical students, but future medical aspirants as well. patil and chakraborty conclusion covid has put healthcare education and health systems around the world to a stress test. the future of healthcare in india, and the world at large, depends on how our institutions collectively mitigate the damage to healthcare education caused as a result of this pandemic. there ought to be administrative interventions into the functioning of medical institutes across the nation to ensure that concrete efforts are made to help students to cope with stress, anxiety and the academic losses they incurred due to the pandemic. examples of which could be: ( ) extension of semesters in institutions across the country to provide adequate time for medical students enabling completion of recommended hours of coursework and hospital rounds at an appropriate pace. ( ) postponement of exams on the horizon enabling students adequate time for preparation. ( ) allowing students with more opportunities to retake/ reappear in written or oral examinations that may have been conducted during the lockdown period which might have warranted for poor performance for students unable to cope with the psychosocial and emotional stressors of dealing with the pandemic (especially students who might have a family member with positive diagnosis). ( ) "self-study/homework" and online lectures are widely being used as a way out, but it is essential to reconsider and reassess the complete reliance and effectiveness of these methods in indian medical education during these times of disquiet; and take into thorough consideration the academic disadvantage a number of future doctors will suffer from, compared to their peers, due to veiled hindrances like distressing familial conditions and unpleasant environments within homes preventing them from reaching their full academic potential. health systems in india ministry of human resource development number of neet aspirants increases by lakh to a record lakh. the times of india depression, anxiety, and stress among final-year medical students covid- and medical education sars and its effect on medical education in hong kong the impact of covid- on medical education medical student involvement in the covid- response competency based undergraduate curriculum for the indian medical graduate risks: the impact of online learning and technology on student physical, mental, emotional, and social health. international technology, education and development conference prevalence and correlates of depression among college going students of district amritsar senior doctors do not attend to us, say covid- patients at victoria. the hindu coronavirus in india: lack of equipment forces doctors to fight covid- with raincoats, helmets. india today india covid- : online survey shows not all is well on ppe availability front. the wire science ppe shortage throws doctors out of gear madhya pradesh: lady doctors hurt in stone pelting in indore. national herald indian doctors fighting coronavirus now face social stigma. quartz india all stages of neet pg counselling postponed due to covid- lockdown. the times of india all authors understand and meet the icmje authorship criteria. both authors contributed to conceptualisation, evidence, literature search, drafting and editing of the manuscript. https://orcid.org/ - - - key: cord- - vn ozqj authors: wayne, diane b.; green, marianne; neilson, eric g. title: medical education in the time of covid- date: - - journal: sci adv doi: . /sciadv.abc sha: doc_id: cord_uid: vn ozqj nan i n current circumstances, one rightfully wonders if persistence of sars-cov- will fundamentally alter the landscape of medical education and hospital training. because of the absence of a vaccine, the prevalence of this virus adds to annual respiratory illnesses caused by seasonal influenza, respiratory syncytial virus, rhinoviruses, and other coronaviruses. faced with a looming new normal, many educators are ruminating on how best to ensure rigorous medical training that produces a steady stream of competent physicians. by way of background, the umbrella of medical education covers a highly structured curriculum in a variety of preclinical and clinical environments whose architecture and requirements are set by the liaison committee for medical education (lcme) ( ) and the accreditation council for graduate medical education (acgme) ( ) . these requirements reflect established habits for producing quality outcomes. capricious changes to these requirements can alter the carat of each uncut gem matriculating to medical school. for this reason, students follow inviolate course work to their doctorate. graduates can apply for state licensure to become physicians after receiving accredited training as interns and residents. only later when seen serving a public good are physicians fully vested professionals. such training can last to years. modern training encompasses a well-thought out system of educational milestones that are highly interactive, increasingly team-based, and guided by educators who have mastered their craft. competent physicians are not born; they are made-taught to integrate the language of science with recent concepts of disease, diagnosis, treatment, and empathy. a gradual move to some outcome measures now provides a few entrustable professional activities and procedures that students must master before residency training ( ) . technology also plays an increasingly important role in teaching core clinical skills as simulation centers and computerized anatomy laboratories become more prevalent ( ). patient histories and physical examinations, how hospitals and medical records work, and the art of detective conversations can only be honed, however, in supervised settings that engage real patients. this spring, medical education in many institutions experienced abrupt disruptions in the face of the coronavirus disease (covid- ) pandemic. teaching hospitals encountered a rapidly expanding illness, shortages of personal protective equipment (ppe), and growing concern for exposures to asymptomatic carriers. at northwestern medicine, our least experienced clinicians, the medical students, were removed temporarily from direct patient care. the preclinical curriculum was migrated online with evidence of similar or improved learning compared to prior years. students on hospital rotations completed virtual clerkships and clinical skill assessments and participated in newly created online electives. interdisciplinary faculty similarly launched covid- courses focusing on the pathophysiology, diagnosis, and treatment of the infection, as well as the health disparities and ethical considerations associated with pandemics. while marching along this curriculum, medical students also channeled their energy and concern through community service. they led donation efforts for ppe, set up food drives, participated in patient education at community sites, worked with clinicians to contact patients diagnosed with covid- to assess their health status, and made food and prescription deliveries to senior citizens and others needing help across chicago. through these experiences, students learned about health inequities and social determinants of disease in a manner not easily addressed in the classroom. several months into this disruption, our focus has shifted toward creating a new normal. while a return to contact patient care and teaching remains highly desirable, we are carefully evaluating various formats for delivering other parts of the curriculum. what seems certain is that a return to a typical pre-covid- teaching platform is unlikely, and that many creative changes are here to stay. large-scale adoption of online education during the pandemic shows that it is possible to achieve a number of teaching objectives virtually. faculty previously resistant to technology-enhanced learning now have evidence of its ability to meet the needs of preclinical students who value adaptive and self-directed study; some may need to review specific content several times, whereas others will proceed more rapidly. augmented intelligence and machine learning will support this model by achieving the goal of a more tailored outcomes-based education. undoubtedly, this undertaking will result in additional innovations, flexibility, and experimentation in areas such as anatomy, problem-based learning, clinical skills education, assessment of student well-being, and mentorship or career advising. many improvements to medical education are a natural consequence of disruptive moments. as we reflect on the covid- pandemic, changes to the medical curriculum that ensure more focus on infection control, pandemic modeling, population and public health, telemedicine, and health equity are desirable. new learners need modern tools to prepare for a response to unexpected medical events in the future. in addition, we have witnessed the spreading value of resilience, grit, and tolerance for uncertainty on the front lines of patient care. we must continue to select for these qualities in future matriculants. despite the temptation for unfettered innovation, we also know that some elements of the curriculum cannot change. the heroic actions of health care workers currently treating patients with covid- reaffirm professionalism and community service as core attributes of a well-taught student. clinical competency also depends on reliable assessment tools that ensure that our graduates are prepared to enter residency training with the knowledge and skills to provide safe and effective patient care. sinek reminds us that "working hard for something we don't care about is called stress, while working hard for something we love is called passion" ( ) . the latter is achieved by witnessing selflessness and a desire to make a difference, core traits that inspired our students to pursue careers in medicine in the first place. as we look toward the future, medical education may never be the same again, and our accrediting agencies will have to join in the adaptation. we now have an opportunity to create a better medical school experience with improved flexibility and outcomes that still ensures competence from this increasingly complex effort. accreditation council for graduate medical education aamc core epas for entering residency pilot team, implementing an entrustable professional activities framework in undergraduate medical education: early lessons from the aamc core entrustable professional activities for entering residency pilot approaches to teaching the physical exam to preclerkship medical students: results of a national survey top simon sinek quotes that reveal the hard truths about success key: cord- - uhhqasl authors: dimitriu, mihai c.t.; pantea-stoian, anca; smaranda, alexandru c.; nica, anca a.; carap, alexandru c.; constantin, vlad d.; davitoiu, ana m.; cirstoveanu, catalin; bacalbasa, nicolae; bratu, ovidiu g.; jacota-alexe, florentina; badiu, cristinel d.; smarandache, catalin g.; socea, bogdan title: burnout syndrome in romanian medical residents in time of the covid- pandemic date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: uhhqasl burnout is a state of physical or mental collapse caused by overwork or stress. burnout during residency training has gained significant attention secondary to concerns regarding job performance and patient care. the new covid- pandemic has raised public health problems around the world and required a reorganization of health services. in this context, burnout syndrome and physical exhaustion have become even more pronounced. resident doctors, and especially those in certain specialties, seem even more exposed due to the higher workload, prolonged exposure and first contact with patients. this article is a short review of the literature and a presentation of some considerations regarding the activity of the medical residents in a non-covid emergency hospital in romania, based on the responses obtained via a questionnaire. burnout prevalence is not equal in different specialties. we studied its impact and imagine the potential steps that can be taken in order to reduce the increasing rate of burnout syndrome in the pandemics. the burnout syndrome in health care professionals has gained significant attention over the last several years. given the intense emotional demands of the work environment, clinicians are particularly susceptible to developing this syndrome more than in other jobs. residency can cause a significant degree of burnout, leading to individuals' ability to establish correct communication with the patient, solve diagnostic dilemmas, and have a good overview of the correct treatment. burnout is associated with a variety of negative consequences including depression, risk of medical errors, and negative effects on patient safety. the goal of this review is to provide an overview of the prevalence in different specialties of the burnout syndrome, even more pronounced in covid context, provide team leaders with options to minimize the risks and prevalence and recognize its potential hazards towards the medical act and its medical caregiver [ ] . although reported and discussed long before the pandemic, after its outbreak, burnout syndrome became even more pregnant [ ] . the coronavirus epidemic broke out in wuhan, china, in a metropolis of million people in december [ ] . free movement of people led to the spread of the virus in europe (italy), the united kingdom and the united states in january-february , becoming soon a pandemic that affected almost all countries [ ] . resident physicians often represent the communication interface between the attending physician and the patient, so that the time spent with the patient is often longer. in the context of the pandemic, physical fatigue is added to the mental stress associated with a possible infection. resident physicians have the most prolonged contact with patients, including in the time window from the testing moment to the arrival of the result for sars-cov- , therefore the stress is even more pronounced. some physical factors are added to the equation. prolonged wearing of personal protective equipment, excessive heat provided by them, lack of hydration, alimentation, sleep deprivation, all together accentuate fatigue and the burnout syndrome. from this point of view, the most affected doctors are from the emergency units, radiology, intensive care units, but also from the specialties that ensure surgical, obstetrical, orthopedic and neurosurgery emergencies. the modification of the shifts and of the work schedule, of the type of the current activity, the time and the technique of dressing and undressing the protective equipment, all constitute the novelty that adds an additional stress factor. last, but not the least, wearing facial protection equipment leads to a depersonalization of the activity, both in contact with patients and medical staff. the impossibility of reading facial expressions, the lack of interpersonal interactions and facial expressions decreases the possibility of socialization and mutual encouragement. in this sense, the idea related to wearing a photo of the holder on the protective suit appeared. there are studies that paradoxically indicate a higher level of stress in regular, non-covid wards of hospitals, compared to front-line ones [ ] . a possible explanation would be that better organization and a sense of control reduce the stress level of those in the front line, while the fear of being exposed when the protocols do not seem perfect is higher in non-covid wards. here, however, there is a permanent threat of a positive patient, so no one seems to be safe. some studies in china and uk have also revealed depression and anxiety due to the covid- outbreak that medical staff experienced [ , , ] . the pandemic has led to numerous cases of depression and anxiety, as well as worsening preexisting mental illnesses [ ] . the term "burnout" was described by psychologist herbert freudenberger [ ] in in an article entitled "staff burnout" in which he discussed job dissatisfaction precipitated by workrelated stress. a broadly applicable description defines burnout as a state of mental and physical exhaustion related to work or care-giving activities. a long-standing conceptual and operational definition characterized burnout as a triad of emotional exhaustion (emotional overextension and exhaustion), depersonalization (negative, callous, and detached responses to others), and reduced personal accomplishment (feelings of competence and achievement in one's work) [ ] . the maslach burnout inventory (mbi) [ ] is the most used questionnaire to measure burnout in research studies. the mbi human services survey is a self-administered, item questionnaire that was developed to measure burnout in human services workers and is the "gold standard" of measuring burnout [ ] . the mbi items are rated on a likert scale from to ( = never, = a few times per year, = once a month, = a few times per month, = once a week, = a few times per week, and = every day) and score sample items such as: "i feel emotionally drained from my work.". it is designed to assess the primary dimensions of burnout: emotional exhaustion, depersonalization, and personal accomplishment. burnout is detected using cutoff scores of high emotional exhaustion (≥ ), high depersonalization (≥ ), and low personal accomplishment (≤ ), based on normative data from medical professionals [ ] . a study in china, comparing burnout of front line workers with that of normal ward staff, in covid context, used maslach questionnaire for medical workers and the results were interesting and quite unexpected [ ] . in , martini et al [ ] did a unique study that compared burnout rates among the different specialties using the mbi. the overall burnout rate was % and ranged from % to % among different specialties. this variation among specialties was not statistically significant; however, burnout rates were as follows: % in obstetrics-gynecology followed by % in internal medicine, % in neurology, % in ophthalmology, % in dermatology, % in general surgery, % in psychiatry, and % in family medicine. however, this variation among specialties was not statistically significant. being in one's first year in residency, mood fluctuation, dissatisfaction with clinical faculty, recent family stress, and being unmarried were all associated with increased likelihood to meet burnout criteria [ ] . psychiatry residents were noted to have additional stressors including fear and exposure to patient violence and suicide [ ] [ ] [ ] [ ] [ ] in a study by fahrenkopf et al [ ] , no actual correlation was found between burnout and the number of medical errors seen in collected data. one plausible explanation may be that residents reporting symptoms of burnout may be more likely to over-report their errors. in this pandemics, there is a need for practical methods to assess medical stuff burnout. some researchers have even proposed the continuous wearing of sensors to quantify fatigue [ ] . the aim of our study was to compare the frequency of burnout syndrome between medical residents considered to work on the front line (emergency unit, radiology and intensive care unit) and those working in normal hospital wards (surgery, obstetrics and gynecology, obstetrics). our hypothesis that we wanted to prove is that there is higher prevalence of burnout syndrome in regular, non-covid wards of hospitals, compared to front-line ones. the study was conducted in a hospital with an emergency clinical profile, which is not in the frontline in the fight against coronavirus, ensuring non-covid emergencies or suspects until the result of the real time polymerase chain reaction test is obtained. during the pandemic, the teaching processes in the hospital were stopped, both for medical students and residents, opting for online teaching. students' access to the hospital was stopped by a university decision to limit the spread of the infection. the medical residents continued to carry out medical activity, restricted to the activity in this only hospital where they have an employment contract. we distributed a survey of questions to medical residents, for resident doctors in the emergency department, radiology and intensive care unit (considered first-contact with the questionnaires for first-line medical resident were distributed as follows: for residents in emergency unit, for residents in intensive care unit and for residents in radiology department (lot a). the other questionnaires were distributed in that we considered normal wards. they were allocated for residents in general surgery ( questionnaires), gynecology ( ) and orthopedics ( ) -lot b in our study. all the invited participants anonymously completed the survey and the responses were valid to be analyzed. demographics characteristics can be found in table . demographic characteristics showed that the two groups were relatively homogeneous in terms of distribution by age and gender. the extreme ages were and years, respectively, given that the target was represented by resident doctors. burnout was defined as a high level of emotional exhaustion (≥ ), and/or high level of depersonalization (≥ ), and/or low personal accomplishment (≤ ). according to our results, we obtained an average burnout for medical residents of %, about two months after the outbreak of the pandemics in our country, which is superior to studies conducted in normal periods. the global prevalence of burnout syndrome among medical residents is high, proving that the threat posed by sars-cov- is a major stressor for medical staff. the results are all the more worrying as the target group was represented by resident doctors, of young age (maximum years), who, at least theoretically, should have a better adaptability to the new condition represented by this pandemics, compared to senior doctors. the burnout was significantly more frequent in normal wards workers (lot b) (prevalence %) compared to medical residents working in places that we considered front-line departments: emergency unit, radiology, including ct/mri department and intensive care unit -lot a in our study, that showed a prevalence of burnout of only % (p< . , from chi-square statistic test) (table ). we considered emergency unit, radiology, including ct/mri department and intensive care unit as front-line departments as all the patients, at presentation time, are now considered potentially infected ones till invalidation by a negative real time polymerase chain reaction test that usually takes hours in our hospital. the prolonged time is due to the fact that this test is analyzed in an external laboratory. there can be an array of methods that can be used to fight against the burnout sydrome. in a sample of professionals, maslach [ ] showed that venting, laughing, and discussing care with colleagues decreased personal anxiety. in conditions of social distancing, even between colleagues, all these mechanisms are annulled. the place of direct socialization is partially replaced by social media groups and net socialization. there are no studies on suggested interventions for reducing the prevalence or how it works on each individual. each person is different genetically, racially, different sex and culture, different family environment and so on. with these many factors comes the problem of pinpointing the trigger factor for each individual. that is why with these many stress factors comes a multitude of anti-stress factors which can be used: from physical activity to meditation. mentoring programs in residency training can also be helpful in this regard [ , ] . the negative impact of burnout on patient care includes risk of medical errors, patient safety risks, and potential compromise of quality of care. burnout and fatigue can affect the caution of medical staff, lead to negligence on self-protection measures and increase the risk of infection. negative consequences of burnout on physicians in training include depression, suicidal tendencies, and medical illnesses. the problems of medical errors related to fatigue and burn-out syndrome seem to be more serious for the surgical specialties [ ] . effective interventions to address burnout should be developed at both the individual and institutional levels [ ] . in a previous study conducted in our hospital, in non-pandemic conditions, the conclusion was that surgeons' fatigue seems to be a more subjective self-perception of surgeons than an objective fact and that surgeons tend to attribute their mistakes to burn-out syndrome, this being more acceptable for their conscience. in normal times, the rates of complications were not statistically higher on call-days and the days immediately after, when exhaustion should be maximum [ ] . maslach [ ] summarized effective working through burnout by stating: "if all of the knowledge and advice about how to beat burnout could be summed up in word, that word would be balance-balance between giving and getting, balance between stress and calm, balance between work and home." pandemics of this magnitude have appeared in humanity about once every years [ ] . so you cannot talk about personal experience in managing a crisis like this. it is also difficult to assume that the competent centralized structures, such as governments, public health organizations, could fully manage the situation at individual level. in certain critical situations, it even turned out that local authorities, along with the population, had more competent organization and involvement (the example of hurricanes). in our opinion, the local organization at the hospital level is much more important for decreasing the stress level and the prevalence of the burnout syndrome. the existence of clear protocols for any possible situation, the practical trainings with the personnel regarding the protection measures, the adequate use of the protection equipment are all measures that ensure a state of confidence and control, which obviously decreases the stress level. this could explain the higher prevalence of burnout syndrome in staff in regular wards, compared to employees in the emergency department. medical residents in emergency unit had more training hours about the protective equipment and the wearing of the personal protection equipment was continuous, throughout the working time, that gave them the feeling of being safe, reducing the stress. all the successes of medical teams must be promoted by all means, being a source of positive emotions. the shift program must be organized in such a way as to respect the epidemiological timing (incubation period or quarantine time). periods of rest and relaxation are important and must be observed to prevent burnout, even if, often, they cannot take place in the privacy of families. the burnout-syndrome is a real phenomenon and may manifest in many forms. each resident and other higher level caregiver is susceptible to it. being aware of this, the new physician generations are shown to have an attraction towards balancing activities. all in all medical leaders and mentors should be aware of their colleagues and residents, thus allowing themselves to partake in the well-being of the team and making the work environment less stressful. the research in individual stress-factors and its many ways in which to actively fight them it is a gateway to making a whole medical environment better by concentrating upon the individual and giving a successful education to the next generation of physicians. in the context of covid- , the best way to combat burnout seems to be, in our opinion, the precise local organization within the hospital and practical training sessions on the use of personal protective equipment, source of a mental comfort feeling. mean age (standard deviation) low personal accomplishment total (burnout frequency) ( %) ( %) p= . table . characteristics of burnout syndrome elements of the two lots burnout during residency training: a literature review coronavirus disease (covid- ) and beyond: micropractices for burnout prevention and emotional wellness emergence of a novel coronavirus causing respiratory illness from wuhan the outbreak of coronavirus disease (covid- )-an emerging global health threat a comparison of burnout frequency among oncology physicians and nurses working on the frontline and usual wards during the covid- mental health care for medical staff in china during the covid- outbreak occupational risks for covid- infection covid- : give nhs staff rest spaces and free parking not thank yous, says doctor factors associated with mental health outcomes among health care workers exposed to coronavirus disease staff burnout burnout: a multidimensional perspective professional burnout: recent developments in theory and research maslach burnout inventory manual stress in health professionals: psychological and organizational causes and interventions evaluating stress: a book of resources burnout comparison among residents in different medical specialties general psychiatry in no-man's land hidden ethical dilemmas in psychiatric residency training: the psychiatry resident as a dual agent observations on burnout in family medicine and psychiatry residents burnout among dutch medical residents burnout and internal medicine resident work-hour restrictions rates of medication errors among depressed and burnt out residents: prospective cohort study continuous monitoring and detection of post-traumatic stress disorder (ptsd) triggers among veterans: a supervised machine learning approach burned out an exploratory study of resident burnout and wellness mentoring matters: mentoring and career preparation in internal medicine residency training burnout and medical errors among american surgeons surgical malpractice in relation to long calls burnout: the cost of caring the mother of all pandemics is years old (and going strong)! maria sklodowska curie carol davila sos. pantelimon, , bogdan.socea@umfcd.ro, + , kindly ask you to take in consideration our submission for your journal, having the title all authors have been read and approved the final manuscript. the study was performed according to the world medical association declaration of helsinki and according to national legislation, using a protocol approved by the local bioethics committee. all subjects have previously signed an informed written consent about future publication of data all authors have significant scientific contribution to the manuscript. all authors have been read and approved the final manuscript. the study had no funding. there are no conflicts of interest. key: cord- -en xkotn authors: fourtassi, maryam; hjiej, ghita; touissi, youness; hajjioui, abderrazak; abda, naima title: how has the covid- pandemic improved evidence-based-medicine awareness among undergraduate medical students? date: - - journal: medical education online doi: . / . . sha: doc_id: cord_uid: en xkotn nan evidence-based medicine (ebm) is a key component of current medical practice, as it induces effective decision-making regarding patients care, based on the best available evidence [ ] . however, delivering ebm in the undergraduate curriculum is a real challenge, especially in settings where undergraduate medical education is mainly based on textbooks-like information that is usually presented to students as solid and verified knowledge with less room to doubt and uncertainty. medical information is usually taught to undergraduates as a set of general truths they can build their analytic thinking around in order to reach a diagnosis or an intervention decision. hence, the transition from theory to 'bedside' practice becomes difficult and might cause a real shock to some learners when they see that a patient could actually have the condition without it fitting the exact textbook description, or when they discover that an antibiotic is not always % efficient as they might have thought. while early introducing undergraduate medical students to the basics of ebm is important to improve their critical thinking [ ] , and raise their awareness about how medical knowledge is actually built over years of translational research, it might seem difficult to make them understand and accept its common frustrating pitfalls, and to explain how these limitations are part of the whole process effectiveness. interestingly, this current ongoing covid- pandemic represents a comprehensive and easily perceptible teaching example. despite its worldwide devastating health and socio-economical consequences, the covid- pandemic has brought a great opportunity to promote the vital importance of ebm among undergraduate medical students, who have found themselves in the middle of an international research race to face the virus. each of the phases this global health crisis has crossed comprises a valuable basic ebm lesson we can teach our undergraduate students, with higher chances for them to understand and integrate it, even if they have not been used to it before. moreover, this pandemic has brought to students' attention the vital need of often overlooked disciplines in their learning such as 'epidemiology', 'biostatistics', 'critical reading' or even 'medical english' for those studying in non-english-speaking countries like morocco [ ] , to be able to understand scientific resources. thanks to the covid- pandemic, our undergraduate students understand better how mastering the exact modes of the virus transmission can impact the choice of prevention measures and how strictly they should be implemented to contain the viral contamination spread. they are also aware of how critical physiopathology knowledge is to defining the best treatment approaches, and choosing the most effective drugs to prescribe. they better understand how symptoms can be misleading, and how atypical clinical presentations should always be considered especially when the patient's life is endangered. on the other hand, the global multi-centred clinical trials to identify the best therapy against the disease and the basic-science-laboratories race to find a vaccine are even richer of key messages regarding the inseparable vulnerability and strength of fundamental and clinical research. indeed, the scientific evidence provided by research results can be affected by some limitations, mainly related to the lack of follow up and insufficient observation time and materials regarding a recent and unknown phenomenon, which makes decisions and recommendations evolve and change with every new piece of information that can be a source of new evidence. hence, this commonly used sentence: 'more research is needed to conclusively establish … ' that is usually added in every research paper conclusion has never been as meaningful as today with this unlimited coronavirusrelated research papers production, providing complementary and sometimes paradoxical information to be carefully examined and interpreted. today, our students are aware that published evidence still needs to be confirmed or infirmed by further investigations, and that publishing in a high impact journal does not make your results or conclusions beyond scientific criticism, leading to possible paper retraction [ , ] . they are also aware of the multiple levels of evidence every kind of research can provide, and that research can be biased by conflicts of interests that should be controlled and fully declared. today, our undergraduate students can see that despite its limitations, the scientific research and publishing system is still strong by implementing a double security process, with the prior publishing peer-review and post publishing possibility for the scientific community to openly discuss and criticise peer-productions through scientific correspondence. these security and quality guarantors are the main source of research strength and a good reason to convince our students of its importance. one could argue that those key lessons about research and ebm can still be depicted by other older pandemic examples and yet, only when you are personally involved in the experience, that you really get it deeply integrated. this pandemic experience has thought to medical students over the world about ebm and research more than they could ever learn through their medical schools' theoretical settings. and we should all take this opportunity to draw lessons and teachings that we can share with our students. no potential conflict of interest was reported by the authors. contemporary global perspectives of medical students on research during undergraduate medical education: a systematic literature review teaching evidence-based medicine to medical students using wikipedia as a platform medical education in morocco: current situation and future challenges retraction-hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis retraction: cardiovascular disease, drug therapy, and mortality in covid- key: cord- -rxhoe cw authors: hay-david, a.g.c.; herron, j.b.t.; gilling, p.; miller, a.; brennan, p.a. title: reducing medical error during a pandemic date: - - journal: br j oral maxillofac surg doi: . /j.bjoms. . . sha: doc_id: cord_uid: rxhoe cw on january , the who declared the coronavirus disease (covid- ) a public health emergency of international concern. by march , it was designated a pandemic owing to its rapid worldwide spread. in this short article we provide some information that might be useful and help equip colleagues to reduce medical error during a pandemic. we advocate a systems-based approach, rather than an individual’s sole responsibility, and, look at ways to provide safer healthcare. at time of writing ( april ), we are in the midst of a pandemic: covid- currently affects countries, with , cases and , confirmed deaths. [ ] [ ] [ ] the annual estimated cost of avoidable medical errors in the uk has been quoted as £ bn and is the third leading cause of death in the us. , the potential risk for medical errors in the current climate is potentially even more likely. prior to covid- , medical error was seen as a global priority that prompted the first "world patient safety day" by the who on september th, . the intention is to make healthcare safer, to raise global awareness of patient safety, and, to encourage people to show their commitment (to "speak up for patient safety"). during emergency situations, several inter-connecting themes have been identified as impacting on the quality of care and its timely delivery. these include organisational sys- * corresponding author. e-mail address: a.hay-david@nhs.net (a.g.c. hay-david). tems, workload, time pressure, teamwork, individual human factors, and case complexity. in the remainder of this article, we provide an overview of specific individual human factors (hf) and working within the hierarchy during a pandemic. simulation training is an established training tool used in the military, aviation, and civilian settings (fig. ). it facilitates kinaesthetic learning but is also a highly effective way to learn from mistakes and failures in a safe environment. there is evidence to support that simulators are effective in improving clinical skills as well as non-technical skills. (live training during an exercise period) pilot in found it a suitable means to ensure operational preparedness was maintained. this is particularly pertinent in the military medical setting when a hospex (hospital exercise simu-lation) is employed ( fig. a and b) . this can be adapted to a particular threat, such as the ebola crisis seen in op gritrock, and, many of the lessons are relevant to covid- . the royal college of surgeons in ireland offers a human factors msc and phd programmes are increasingly being offered across the uk. the royal college of surgeons of england is sharing educational material with its members online such as the start course that includes a hf section. the intensive care society has set up a webpage dedicated to the pandemic with up to date guidance/resources. there is an opportunity to learn from the lessons of other countries via podcasts and webinars. social media platforms such as twitter, facebook, and instagram have been used to good effect in the past; for example, providing information for disaster response between authorities and the public during the haitian earthquake. it has been suggested, that social media may have a useful role over the more traditional means of reporting to track emergence of disease trends and outbreaks. [ ] [ ] [ ] analysis of social media use over a week period during the ebola outbreak identified it as a potential source of surveillance to enhance detection, preparedness, and, the public health response during a crisis (used in addition to traditional surveillance approach). on march the uk's information commissioner's office (ico) provided advice to the government that it can use anonymised mobile phone data in the fight against coro- navirus. similar measures have been taken by other nations to help track the likely spread of the virus. the gmc and the bma provide guidance on social media use by the medical profession and respect and confidentiality for patients as well as colleagues remains of paramount importance. , the authors have recently written an article (in press) on the potential advantages and perils of using social media in medicine. optimisation and empowering a team can be reinforced at a brief: encouraging situational awareness; asking others to look out for you; taking regular breaks to rest and to eat; staying well hydrated; and, to stop if anything doesn't seem quite right. , assertive followership although a systems-based approach is advocated, to have the best effect still requires good hf integration. a key element of flight simulators is not just how the crew interacts with the system, but also with each other. the need for subordinate crew members to challenge authority by asking questions, expressing concerns, or airing vulnerabilities without the fear of retribution or humiliation is encouraged and taught to enhance flight safety. consequently, to enable effective systems-based medical training, a challenge culture of assertive followership is a vital element, not only to enhance learning but to reduce medical error. however, for a challenge culture to be effective requires the whole organisation to adopt and encourage this culture, with those in positions of authority actively flattening the hierarchy by championing and adopting such open practices. there were significant lessons learnt from the medical response request for military aid to civilian authorities (maca) seen in the terrorist attacks. there was a clear need for command and control (c ), with a senior decision maker working in teams with the application of an ethos of mutual support. while there is a requirement to have this c approach, clinical teams should strive for a flattened hierarchy to allow junior team members to give their opinion without fear of retribution and this should be included in any team brief. the c element was of particular relevance when a single point of contact (spoc) is established, which in turn reduces the bandwidth of other team members, regular situation reports (sitreps) are briefed to ensure that every team member is informed and can add value during this update. in order to ensure absolute trust between the care deliverers and the policy makers at this time, there must be transparency and also an opportunity for open discussion. , there are multiple anecdotal incidents from around the world of frontline care givers having different views to the system managers and politicians as to how well systems are preparing or coping. consequently, both views should be balanced so that decisions are based on reality, and, where there is doubt, an abundance of caution is advisable from a risk reduction perspective, given the poor outcomes for healthcare workers in many countries thus far. there are several factors that are under our control: self-care (hydration, eating regular healthy meals, exercise), staying in touch with loved ones, and, sleep. [ ] [ ] [ ] [ ] the military employ a buddy-buddy system to ensure that self-care is maintained and correct application of personal protective equipment. this was one of the lessons learnt from operation gritrock that ensured if someone was tired or under the influence of other hf performance influencing factors, a safe environment was maintained. the buddy-buddy system was not just to monitor discrete tasks but also to allow each clinician to maintain a good standard of self-care and highlight potential errors and allow mitigation. the debrief session is an excellent opportunity to recognise or address the psychological or emotional needs of the clinical team, and, in dealing with terrorism or pandemics, this burden can be quite significant. this increases significantly if there is an expectant death category added to triage, particularly when services are overwhelmed, as seen in the recent covid- response in italy. it is especially important, during these highly stressful times, that all medical professionals are aware of the importance of how they treat their team. for high-functioning teams to work effectively members need to feel safe to speak up. team members report that they know when the atmosphere is right for 'psychological safety': when the team feels like a family. to enable this, leaders must constantly self-monitor and ask for open feedback. are they empowering others, encouraging questions or, just as importantly, apologising for any overbearing behaviour? leaders must also challenge colleagues whom they feel are not adopting appropriate behaviours and coach a more appropriate response. the current situation has presented challenges to the workforce in terms of adapting to a new way of working, clinical redeployment, and concern for our own and our loved ones' health. many frontline colleagues are being separated from family members for fear of virus transmission. all formal assessments such as mrcs and frcs examinations have been suspended. there is also an intense daily exposure to politics and politicians. given previous responses, for example to the junior doctors' contract, there is a degree of scepticism and mistrust about the handling of the situation. covid- is affecting all us, our extended families and all those we serve. we must continue to adapt to these new ways of working: within our levels of competency, to deliver safe patient care, and, to minimise the potential for error. not required. second-meeting-of-the-international-health-regulations-( )-emergency-committee-regarding-the-outbreak-of-novel-coronavirus-( -ncov) world health organisation. who director-general's opening remarks at the media briefing on covid- - march world health organisation. coronavirus disease (covid- ) outbreak situation nhs wastes at least £ bn a year on avoidable errors, claims health secretary medical error-the third leading cause of death in the us decision-making under pressure: medical errors in uncertain and dynamic environments does simulation-based medical education with deliberate practice yield better results than traditional clinical education? a meta-analytic comparative review of the evidence a pilot study to evaluate the utility of live training (livex) in the operational preparedness of uk military trauma teams hospex and concepts of simulation operation gritrock: the defence medical services' story and emerging lessons from supporting the uk response to the ebola crisis covid- podcast from italy with roberto cosentini european centre for disease prevention and control. covid webinars emergency knowledge management and social media technologies: a case study of the haitian earthquake monitoring influenza trends through mining social media you are what you tweet: analyzing twitter for public health social and news media enable estimation of epidemiological patterns early in the haitian cholera outbreak social media in ebola outbreak uk information commissioner oks use of phone data to track coronavirus spread. the register use of social media bma. social media guidance for doctors. london. . available from url social media (#some): a life support or a life drain leading article: what can we do to improve individual and team situational awareness to benefit patient safety? impact of hydration and nutrition on personal performance in the clinical workplace manchester and london terrorism incidents. part : introduction and the prehospital phase end the paternalism and top-down orders, to move forward in fighting the pandemic improving patient safety: we need to reduce hierarchy and empower junior doctors to speak up challenging hierarchy in healthcare teams -ways to flatten gradients to improve teamwork and patient care the impact and effect of emotional resilience on performance: an overview for surgeons and other healthcare professionals sleep: its importance and the effects of deprivation on surgeons and other healthcare professionals review: avoid, trap, and mitigate -an overview of threat and error management personal protective equipment solution for uk military medical personnel working in an ebola virus disease treatment unit in sierra leone the toughest triage -allocating ventilators in a pandemic we have no conflicts of interest. key: cord- -tnvp n g authors: hauer, karen e.; lockspeiser, tai m.; chen, h. carrie title: the covid- pandemic as an imperative to advance medical student assessment: areas for change date: - - journal: acad med doi: . /acm. sha: doc_id: cord_uid: tnvp n g conditions caused by the covid- pandemic have disrupted clinical practice and all aspects of medical education. yet the need to continue to train physicians to care for patients and communities is greater than ever. medical educators are responding by adapting curricula in response to requirements for social distancing, changing availability of clinical learning opportunities, and limitations on testing center availability for examinations. these disruptions require a systems approach to ensure that learners achieve competence in preparation for advancement in training toward unsupervised practice. in this article, the authors assert that medical educators, obligated by current conditions to adapt educational experiences, should seize the opportunity presented by the pandemic to make needed changes in areas aligned with competency-based medical education: focusing on outcomes, broadening the assessment toolbox, and improving the undergraduate medical education (ume)-to-graduate medical education (gme) transition. defined outcomes, as exemplified by entrustable professional activities, will enable curricular designers to shorten and adapt learning experiences by focusing on students’ achievement of prespecified learning outcomes. broadening the assessment toolbox entails capturing more and different assessment information about learners to provide a well-rounded view of their strengths and areas for growth in both traditional and novel settings, such as telehealth. limitations on available data, such as licensing examination scores and clerkship grades, heighten the urgency to revise the system for the ume-to-gme transition by enhancing the quality and usability of information available to residency program directors. educators should capitalize on the opportunity presented by altered conditions due to the covid- pandemic to make these needed changes to the educational system, to prepare physicians to provide health care and lead the health care system into the future. the covid- pandemic has upended clinical practice and all aspects of medical education. despite major challenges to classroom and clinical learning, however, the need to continue to train physicians to meet the health care needs of patients and communities is greater than ever. from the educational community, early responses to the disruption to medical education have been swift. discussion boards and listservs are replete with ideas of how to convert curricula to online learning, administer exams remotely, adjust clinical rotations for students and residents, and engage learners in telehealth. because this disruptive crisis is unprecedented and was largely unanticipated, medical schools, residency programs, and clinical care delivery systems have not had their usual timeframes to plan educational changes. the aim has been "speed over elegance" and, understandably, many solutions are not perfect. unfortunately, as education programs continue to debate how to respond to the crisis situation, discussions tend to focus on adapting the inputs, or the delivery of an acceptable curriculum, rather than on ensuring outputs, or desired learner outcomes. questions are arising regarding the boundaries of acceptable adaptations. how much or should any clinical workplace learning be replaced by virtual experiences? residents and fellows who are assigned to help cover other services may be asked to practice new skills, but at what point are they practicing beyond their qualifications? redeployment of clinical teachers to meet care needs prompts questions about the adequacy of trainee supervision by faculty who are practicing outside of their usual scope of practice. students eager to contribute to the response to covid- and medical centers seeking to reinforce their workforce have asked whether students can enter the workforce early. the core concern underlying these questions is the educational community's ability to assure readiness for practice, with heightened urgency due to pandemic conditions. the current disruption to medical training and clinical practice thus presents a unique obligation to focus on outcomes and catalyze desirable change in competency assessment. evidence that the pandemic may be an ongoing challenge for months or years to come should compel educators to take a longrange view and a systems approach when addressing immediate needs, to identify lessons learned that can extend beyond the pandemic. , this strategic thinking entails innovating in the direction toward which the medical education community already has wanted to go, and implementing approaches that are better aligned with current educational theory and evidence. conditions created by the pandemic invite us to tackle barriers that have rooted us to traditional practices that exist because "that is the way things have always been done," and emerge better and with more robust educational systems. in this article, we discuss opportunities for innovation abstract conditions caused by the covid- pandemic have disrupted clinical practice and all aspects of medical education. yet the need to continue to train physicians to care for patients and communities is greater than ever. medical educators are responding by adapting curricula in response to requirements for social distancing, changing availability of clinical learning opportunities, and limitations on testing center availability for examinations. these disruptions require a systems approach to ensure that learners achieve competence in preparation for advancement in training toward unsupervised practice. in this article, the authors assert that medical educators, obligated by current conditions to adapt educational experiences, should seize the opportunity presented by the pandemic to make needed changes in areas aligned with competency-based medical education: focusing on outcomes, broadening the assessment toolbox, and improving the undergraduate medical education (ume)-to-graduate medical education (gme) transition. defined outcomes, as exemplified by entrustable professional activities, will enable curricular designers to shorten and adapt learning experiences by focusing on students' achievement of prespecified learning outcomes. broadening the assessment toolbox entails capturing more and different assessment information about learners to provide a well-rounded view of their strengths and areas for growth in both traditional and novel settings, such as telehealth. limitations on available data, such as licensing examination scores and clerkship grades, heighten the urgency to revise the system for the ume-to-gme transition by enhancing the quality and usability of information available to residency program directors. educators should capitalize on the opportunity presented by altered conditions due to the covid- pandemic to make these needed changes to the educational system, to prepare physicians to provide health care and lead the health care system into the future. the covid- pandemic as an imperative to advance medical student assessment: areas for change and improvement in learner assessment that are aligned with competency-based medical education (cbme) that we believe can make enduring contributions: focusing on outcomes, broadening the assessment toolbox, and improving the undergraduate medical education (ume)-to-graduate medical education (gme) transition. first, current experience with the effects of the covid- pandemic highlights the need to accelerate the transformation of medical education from a purely time-based model to one that operationalizes outcomes and the principles of cbme. these principles demand clearly stated, standardized outcomes that all learners must achieve, along with tailored learning experiences based on individual learner needs as well as programmatic assessment. , despite the common endorsement and adoption of competency frameworks, competency language, and learning objectives, almost all medical schools and residency programs in the united states currently base achievement on time rather than measurement and documentation of expected learner outcomes. before and in the midst of the pandemic, a few medical schools have taken the bold step to graduate students early to strengthen the workforce, and medical schools in the united kingdom (uk) have received permission to do so for graduates who had achieved competence. these decisions for early graduation must be supported by robust longitudinal data collection to ensure attainment of competence. some educators have asked themselves what evidence they would use to know that their students have achieved readiness for gme. by viewing time as the proxy rather than the outcome, educators can be persuaded that many students should have achieved the skills they need by a few months before the usual graduation time. the question then arises: why not graduate students early next year as well? creating more clear medical school outcomes, and robust assessment methods that provide evidence of achievement of those outcomes, can shift the emphasis to graduating students when they are ready for residency, rather than at a specified time point. to enable this shift, students and faculty must have a shared understanding of the essential facets of competence and the purposes of assessments. the association of american medical college's (aamc's) core entrustable professional activities for entering residency (epas) represent one example of a step toward clearly defined minimal outcomes for medical school that have been variably adopted. whether outcomes are shared or individually defined by a particular school, schools must be able to gather the data necessary to make the determination of achievement. educators must then hold students, their teachers, and school leaders accountable for student achievement of those outcomes through robust performance data collection and ongoing progress monitoring, and by only graduating students for whom there is evidence of outcome achievement. similarly, with restrictions on student placements in clinical settings due to the covid- pandemic, educators now debate how much or little time is needed to ensure that students have met the requirements for each core clerkship. learning experiences in core clerkships are mostly time based, with a certain number of weeks in each specialty. this focus on time creates challenges for modifying clerkships in response to conditions created by the pandemic. in contrast, clear outcomes required of each clerkship, with robust assessment systems, would enable determination of whether individual students have achieved those outcomes or whether additional time or adjusted experiences are needed to meet those goals. defining the types of patients students need to see and the core knowledge/skills they need to demonstrate for each core clerkship, as well as a robust system of assessing and tracking completion, would yield more opportunity for flexibility in learning experiences. one example of the benefits of more individualized learning experiences commonly arises within longitudinal integrated clerkships (lics); for example, students receiving more individualized clerkship experiences at harvard and university of british columbia achieved the same or better outcomes than students in traditional block clerkships. [ ] [ ] [ ] the experiences of students in lics, and potentially all students, could be adapted to the changing environment of the pandemic through opportunities to meet certain requirements for one field with experiences in a different field (i.e., seeing a pediatric patient with a family medicine preceptor), as recently endorsed by the liaison committee on medical education. coupled with flexibility to alter schedules, this approach can meet students' individual learning needs. by defining core learning experiences based on competencies and focusing on specific learning outcomes for each clerkship, educators can optimize the time students have in medical school even if that time is different from before covid- . second is the responsibility to broaden our toolbox of assessment methods. for example, medical educators have traditionally relied heavily on certain standardized assessments such as united states medical licensing examination (usmle) step and ck and clerkship subject examinations to assess students' medical knowledge achievement and readiness for the next phase of medical training. these medical knowledge metrics are less available, or available on a different schedule, due to testing center closures that limit learners' access to computer-based testing. a way forward is illustrated by the decisions by some undergraduate colleges to make standardized testing optional rather than required due to the pandemic, and to emphasize other aspects of college applications. the situation is further complicated in medical training by licensing requirements; as such, any adjustments to the licensing schedule require the buy-in of not only education programs but also state licensing boards. though not currently available for licensing examinations, remote administration of medical school examinations including the national board of medical examiner (nbme) subject ("shelf ") examinations has met with mixed success. remote video proctoring is flexible, with students able to take examinations at home. however, technical requirements and lack of distraction-free environments may present barriers for some learners and exacerbate inequities. in addition, questions persist about exam security with remote proctoring. information about students' medical knowledge achievement will still be needed and could be captured more flexibly through confirmation of minimum competence on clerkship examinations or one but not necessarily both of the step examinations before residency, given the high correlation among scores on these examinations. ultimately, the ability to individualize learning experiences and advance students based on achievement of outcomes necessitates assessments that can provide a well-rounded picture of learner strengths and weaknesses, and evidence of learner readiness. to meet these goals, medical educators need to rely on assessments beyond existing standardized or "objective" knowledge exams. objective structured clinical examinations (osces), now the norm in ume, can be further developed and emphasized to enable competency-based assessment to inform progression and provide competency-based feedback to learners. [ ] [ ] [ ] as schools temporarily change grading schemes to adjust for shortened or altered clinical placements, a variety of new tools that enable students to demonstrate their learning within and across disciplines may emerge. innovative examples such as workplacebased assessment tools to capture narrative assessment of how a learner participated in telehealth patient visits or documentation of their volunteerism to support communities at risk of infection should be embraced. educators can identify, implement, and adapt assessment tools as needed to measure achievement of different outcomes. for example, virtual technology can enable remote assessment through oral examinations, standardized patient televisits, or simulations. however, students may justifiably perceive remote administration of osces that simulate telehealth encounters as assessing telehealth skills that may not have been taught through the curriculum as well as failing to assess important physical exam skills. workplace-based assessments using epas can provide multiple measures of performance that could be aggregated by advancement committees to make judgments about overall learner achievement. online portfolios or dashboards can facilitate gathering varied and sufficient assessment data to assure that all outcomes have been met. accelerated attention to the incorporation of artificial intelligence and learning analytics in medical education can support adaptive learning that is responsive to each student's learning edge. lastly, with supportive faculty coaching, students can and must be active partners both in learning and in gathering assessment data about their own performance. in the uk, increased reliance on learner self-assessment contributes to competence determination. as medical educators in the united states adapt, clear communications to learners about expectations and empowerment of students to be proactive in self-assessing their learning needs and seeking feedback will be critical. third, the disruption to medical education caused by the pandemic highlights multiple issues in the transition from ume to gme; most pertinent to this article is the poor trust between ume and gme programs, with lack of confidence in evidence of learner readiness by gme programs. improved precision of communication from ume about learner competence can encourage trust in programbased performance data from medical schools. gme overreliance on specific performance measures will be upended due to the disrupted availability of usmle scores, "audition" rotations that provide firsthand knowledge of students' performance, and proxy measures such as number of specialty rotations completed. medical schools have asked for, and the aamc is exploring, the addition of template medical student performance evaluation language for explanations of curricular adjustments due to covid- to help gme programs understand the unique circumstances at each school and better interpret changes in student assessments and grading. the increased transparency and uniformity of reporting should be embraced not just during the pandemic but as an ongoing expectation, and expanded to include more details of program-specific assessments of student performance and documented achievement of competence. in addition, schools that invest in examination of students' achievement of graduation milestones signaling readiness to provide care at the gme level can use these individualized data in partnership with gme programs to enable early transition to residency training. , ume and gme must not see each other as opposing sides, but rather as relay partners in the continuum of medical training, especially in this time in which collaboration is more necessary than ever. gme programs already know they must reconsider what data to use for selection. earlier this year, the nbme announced plans to change step score reporting from -digit numeric scores to pass/fail outcomes only, to take effect sometime on or after january . changes wrought by covid- has accelerated this push to think beyond current practices. for instance, the council of residency directors in emergency medicine (em) has encouraged their gme programs to relax the - application cycle requirements for standardized letters of evaluation from away rotations and the number of em rotations completed. accordingly, gme programs may need to seek new types of information and place trust in information from a broader range of sources. the experience in the - application cycle will challenge previous assumptions and processes, requiring reexamination of what student data residency programs should use moving forward and how these data are used and prioritized. rather than depend primarily on who has the highest ranking or usmle scores to predict student success in residency, gme programs could use a constellation of outcomes data including clinical performance ratings, osce performance, and portfolio or dashboard records. together, these data constitute evidence for student demonstration of growth over time (rather than the unrealistic expectation that students will achieve "top %" status early in training), readiness for transition, and capacity for success in the residency program regardless of differences in the students' curricular contexts. the covid- pandemic will continue to present an ongoing challenge to health, health care, and medical education. when location, time, and familiar assessment benchmarks are not available as organizing principles for education, and the usual, comfortable sequence of learning and assessment events is not possible, educators are forced (or freed up) to use other methods to determine whether learners have achieved competence. the scale of this crisis requires all education programs as well as larger national and international educational organizations to rethink systems and modify usual procedures. the variable impact of the pandemic by region and its uncertain duration will continue to require flexible responses that can address local conditions and adapt over time. in this context, the medical educational system faces increased accountability and an imperative to use best evidence and medical education frameworks to optimize assessment of learner competence. by focusing on the outcomes of training, defined through the competencies essential for practice, and by rethinking what constitutes useful, meaningful assessment data, educators can design flexible educational systems while ensuring outcomes can be achieved and measured using an adaptable set of tools. rapidly implemented responses from educators during this crisis can constitute a series of pilot experiments or pdsa (plando-study-act) cycles on a path forward to produce enduring improvements to medical education. innovation in response to the covid- pandemic crisis the heart of resilient leadership: responding to covid- . deloitte insights a vision of the use of technology in medical education after the covid- pandemic preparedness of medical education in china: lessons from the covid- outbreak competencybased medical education: theory to practice international competency-based medical education collaborators. a core components framework for evaluating implementation of competency-based medical education programs early graduation at nyu grossman school of medicine sends new doctors to join covid- fight. nyu langone news. https:// nyulangone.org/news/early-graduationnyu-grossman-school-medicine-sends-newdoctors-join-covid- -fight covid- : medical schools given powers to graduate final year students early to help nhs enhanced requirements for assessment in a competency-based, time-variable medical education system core epas for entering residency pilot program. toolkits for the core entrustable professional activities for entering residency outcomes of longitudinal integrated clinical placements for students, clinicians and society why wait until residency? competency-based education in longitudinal integrated clerkships lcme update on medical students, patients, and covid- : approaches to the clinical curriculum uc's response on admissions to covid- residents-students/medicalschool-life/medical-school-assessmentduring-covid- -shelf-exams-go predicting united states medical licensure examination step clinical knowledge scores from previous academic indicators international competency-based medical education collaborators. advancing competency-based medical education: a charter for clinician-educators impact of the united states medical licensing examination step clinical skills exam on medical school clinical skills assessment curriculum reports. assessment methods at us and canadian medical schools is the osce a feasible tool to assess competencies in undergraduate medical education? medical and surgical education challenges and innovations in the covid- era: a systematic review remote assessment of clinical skills during covid- : a virtual, high-stakes, summative pediatric objective structured clinical examination the accountability of clinical education: its definition and assessment covid- : health education england shares advice for trainees epac study group. education in pediatrics across the continuum (epac): first steps toward realizing the dream of competency-based education accelerated graduation and the deployment of new physicians during the covid- pandemic united states medical licensing examination. invitational conference on usmle scoring consensus statement regarding sloes and away rotations from the advising students committee in emergency medicine the authors wish to thank key: cord- -nbkvd le authors: ashcroft, james; byrne, matthew h v; brennan, peter a; davies, richard justin title: preparing medical students for a pandemic: a systematic review of student disaster training programmes date: - - journal: postgrad med j doi: . /postgradmedj- - sha: doc_id: cord_uid: nbkvd le objective: to identify pandemic and disaster medicine-themed training programmes aimed at medical students and to assess whether these interventions had an effect on objective measures of disaster preparedness and clinical outcomes. to suggest a training approach that can be used to train medical students for the current covid- pandemic. results: studies met inclusion criteria assessing knowledge (n= , . %), attitude (n= , . %) or skill (n= , . %) following medical student disaster training. no studies assessed clinical improvement. the length of studies ranged from day to days, and the median length of training was days (iqr= – ). overall, medical student disaster training programmes improved student disaster and pandemic preparedness and resulted in improved attitude, knowledge and skills. studies used pretest and post-test measures which demonstrated an improvement in all outcomes from all studies. conclusions: implementing disaster training programmes for medical students improves preparedness, knowledge and skills that are important for medical students during times of pandemic. if medical students are recruited to assist in the covid- pandemic, there needs to be a specific training programme for them. this review demonstrates that medical students undergoing appropriate training could play an essential role in pandemic management and suggests a course and assessment structure for medical student covid- training. registration: the search strategy was not registered on prospero—the international prospective register of systematic reviews—to prevent unnecessary delay. global disasters, such as a pandemics or warfare, are events that cause a major disruption to health and social care, industry and economy, and community and education. disasters on this scale result in substantial loss of life, and an immeasurable burden is placed on healthcare services to deliver core medical care. disaster healthcare provision requires a collaborative approach that uses the expertise and skills of as many people as possible. much of what is formally taught in medical school is around the knowledge, skills and behaviours required of a physician for patients at the bedside. however, the broad training medical students receive could be applied to disaster scenarios especially if supported with adjunct specialist training. the current medical student curriculum already covers a wide range of specialties, and some may argue it is stretched. however, the rising incidence of worldwide disasters and the impact of the current coronavirus (covid- ) pandemic has justified the need for disaster preparation training in medical students. in some respects, students with disaster training may be better suited to assist in both clinical and non-clinical roles in disaster scenarios than redeployment of senior physicians with super-specialist skills and knowledge. curricula using multidisciplinary methods of simulation and human factors training have been proposed for implementation by the usa (association of american medical colleges ) and europe (government of the federal republic of germany computer science applied to medical practice, italy ). however, at present, it is recognised that there is a brief or non-existent exposure to disaster training within current medical training curricula across the world, which may leave students unprepared for an intimidating and unfamiliar setting if assisting in the healthcare workforce. the current covid- pandemic is rapidly driving the need for healthcare workers in the uk. on march , the uk health secretary, matt hancock announced plans to introduce medical students as volunteers to the nhs in order to assist in this pandemic. in response, the british medical association and medical schools council issued clear advice regarding medical students joining the uk healthcare workforce including ensuring correct induction, training and supervision. the aim of this study was to systematically review disaster training courses for medical students. we describe the educational structure and methodology employed, and evaluate both preparedness for disaster medicine and learning outcomes to inform the development of covid- -specific training programmes. we adhered to prisma (preferred reporting items for systematic reviews and meta-analyses) guidelines and recommendations for systematic reviews of observational studies. we searched embase, medline and cochrane central from for all articles published until march evaluating training that medical students receive to prepare them for pandemics and disasters, with no language restriction. we identified articles, which we then screened for inclusion. the search was conducted using the following medical search headings: 'coronavirus', 'covid- ', 'sars virus', 'disasters', 'natural disaster', 'major catastrophe', 'mass casualties', 'crisis event', 'extreme weather', 'disease outbreaks', 'infectious disease transmission', 'epidemics', 'pandemics', 'mass drug administration', 'warfare', 'biohazard release', 'chemical hazard release', 'radioactive hazard release', 'radiation exposure', 'radiation injuries', 'hazardous', 'waste', 'chemical water pollution', 'radioactive water pollution', 'medical students', 'medical schools', 'education' with terms exploded as appropriate. the search strategy was not registered on prospero-the international prospective register of systematic reviews-to prevent unnecessary delay. we selected randomised controlled trials, case-control studies and cohort studies that measured medical student training outcomes in the context of pandemics and disasters. studies were selected only if they contained a detailed report of the training implementation and used objective precourse and/or postcourse assessments related to medical student knowledge, attitude, skills or clinical care outcomes. importantly, if medical student outcomes were grouped with other healthcare students or professionals and not reported separately, the study was excluded. we excluded non-english language articles in order to ensure data quality, logistical training process evaluations, literature reviews, case reports, clinical trial proposals, conference abstracts, editorials, letters and articles evaluating non-medical student populations only. pandemic infections that may be secondary issues to a disaster, but were not the primary cause, were also excluded, for example, hiv, dengue, malaria; situations where medical students were unlikely to be required to volunteer en masse, for example, active shooter situations; and interventions that were not in a disaster setting, for example, basic life support and routine clinical infection control procedures were excluded. duplicates were removed and two reviewers (ja and mhvb) independently screened titles and abstracts using rayyan, an online software to aid blinded abstract screening. any discrepancies were resolved by consensus. of the citations screened, we identified articles for full text and reference review, of which final studies met the inclusion criteria for data synthesis (figure ). two reviewers (ja and mhvb) independently extracted relevant information from each training report using standardised data extraction proforma in keeping with best evidence medical education recommendations and one author with medical training expertise (rjd) reviewed all extracted data. we recorded administrative information including authorship, institution and year of publication; training-related data including details and duration of intervention, participants and teaching methods; and quantitative and qualitative outcome measures. the quality of training intervention and risk of bias in reporting of results was assessed using the robins- for non-randomised controlled trials. meta-analysis was not performed on the training outcomes assessed due to the wide heterogeneity in training interventions and reporting of results. descriptive analysis was performed instead. interventions were assessed against kirkpatrick criteria and kirkpatrick's levels were assigned: impact on learners' satisfaction (level ), changes in learners' attitudes (level a), measures of learners' knowledge and skills (level b), change in learners' behaviour (level ), changes to clinical processes/organisational practice (level a) and benefits to patients (level b). twenty-three studies met the inclusion criteria, and their characteristics are displayed in table . the majority of studies (n= , . %) were from the usa, and other countries were germany (n= ), israel (n= ), italy (n= ), saudi arabia (n= ) and south korea (n= ). five studies ( . %) involved a multidisciplinary cohort and reported outcomes for other healthcare students and professionals, as well as outcomes for medical students individually without pooling of results. the course structures and learning objectives were grouped into three categories: broad concepts in disaster medicine (n= , . %), trauma or haemorrhage mass casualty management (n= , . %), or influenza pandemic management, airborne viral management or personal protection (n= , %). the length of studies ranged from single day teaching to -week boot camps, and the median length of training was days (iqr= - ). the majority of training interventions used traditional didactic lectures with simulative or experiential teaching methods, with courses ( . %) containing lectures and simulation. of the simulation experiences, four courses ( . %) contained outdoor actor-based mass casualty simulation. multimedia approaches were used in eight courses ( . %) as an adjunct to training, often precourse, in order to efficiently provide material to attendees. problem-based learning or casebased learning was used as a predominant feature in five courses ( . %) in a classroom setting with or without other teaching methods. no courses involved only didactic teaching methods, all included studies were prospective cohort studies measuring the impact of their training intervention postcourse evaluation, with using precourse evaluation for comparison as displayed in table . the majority used subjective assessments of knowledge or preparedness in disaster medicine (n= , . %) with nine studies ( . %) using objective measures. there was a wide range in number of medical students attending the courses, with the median number of participants being (iqr - ). however, five studies did not clearly describe their medical student population, either omitting total number of participants or seniority of students. only two studies reported data that assessed longitudinal learning beyond the year of course implementation. common limitations of study design included training being limited to a single institution, studies which were excluded due to being randomised controlled trials evaluating the efficacy of different teaching methods or simulation technology, or studies excluded due to reporting medical student outcomes pooled with outcomes of other healthcare student or professionals. of the studies included in this review, studies in total measured precourse and postcourse outcomes. of these studies, knowledge was measured in ( . %), of which undertook objective knowledge measurements, making this the most measured outcome. however, none of the subjective or objective measures of knowledge were previously described or undertaken with validated measures. a total of four studies measured attitude-either preparedness for disaster (n= ) or confidence in approaching a disaster (n= ) by precourse and postcourse assessment. disaster medicine skills were subjectively measured in four studies and objectively measured in two, one being pass rates in personal protective equipment fitting and the second being accuracy of disaster triage. in these studies, validated measures were used to create scores or pass rates. of the five studies inviting a multi-disciplinary participant group, none trainee satisfaction was assessed in ( . %) studies and most commonly assessed using likert type scales. medical students were asked to rate the overall quality of the courses in addition to whether they would recommend courses to colleagues for disaster preparedness. course satisfaction was generally very high and appeared to be enhanced by multimodal approaches to curriculum design including the incorporation of simulation and technology. however, one group discussion and interactive activity-based study did report mixed reviews with postcourse overall ratings of positive ( %), undecided ( %) and negative ( %). this is reflective of overburdening medical students with work, with one student stating, "this was way more work than it should have been. i would rather have an hour lecture on the flu than do all that group stuff. this was just frustrating to have at the end of the year when finals are right around the corner". attitudes and perceptions of knowledge in medical students were assessed in studies ( . %). attitudes were broadly assessed as level a following courses measuring either a simple change such as interest in disaster medicine, a measurement of a medical student's willingness to volunteer in or preparedness to practice disaster medicine, or by mapping trainee responses to learning objectives. level b was measured by a total of studies ( . %) which assessed medical student knowledge or skill acquisition, with assessing knowledge and assessing skills, either alone or in combination. in courses training students in mass casualty scenarios, discrete and measurable skills were easily assessed including tourniquet application and triage skills. as behavioural change is a difficult area to measure in nonpracticing medical students, only one study was deemed to adequately assess behavioural change. this study assessed confidence and perceived stress handling emergencies once the medical students had graduated . years following the course. no studies investigated the impact of disaster training on clinical performance (level a) or organisational delivery of care (level b). all included studies were cohort studies and risk of bias was assessed using robins- (figure ). risk of bias was low to goolsby, , university of the health sciences, usa postcourse questionnaire assessing perceived confidence at assessment and procedures in a combat casualty situation and perceived preparedness at managing combat casualties. the majority of students feel more confident and better prepared to assess and perform procedures in a combat casualty situation after course. the majority of students preferred the high-fidelity simulation to their normal learning environment. attitude, skill to a ingrassia, crimedim, italy precourse and postcourse examination assessing disaster medicine knowledge. triage accuracy was measured in a disaster simulation assessed by an examiner. there was a significant improvement in knowledge of disaster medicine after course from % to % (p< . ), and a significant improvement in triage accuracy in the disaster medicine simulation after course from % to % (p< . ). the majority of students felt that disaster medicine should be part of their curriculum and evaluated the course highly. knowledge and assess learning of the didactic material immediately before the min case-based lecture, and the posttest immediately after the lectures. the class could take their pretest via an e-learning tool up to several days before the class, and the post-test was available online for weeks after completion of the course. over a -year assessment period the first year cohort's post-test knowledge scored improved from . / (below average to average) compared with . / (average to above average) and the second year's post-test scores improved from . / (average) before and . / (above average). in the first year cohort he average overall rating for the experience was . / , and % of the respondents recommended the class for next year's students allowing it to continue. knowledge, attitude to b scott, , medical university of south carolina, usa participants undertook precourse and postcourse assessment developed to meet learning objectives of the course. selfassessment of personal capability and comfort to handle a disaster and multiple choice questions of knowledge and subjective skill were undertaken. most ( %) of the trainees considered their emergency preparedness knowledge and skill as average or below average before the training experience. after the curriculum, % of trainees considered their emergency preparedness knowledge and skill above average, and % would recommend the course to other healthcare workers. knowledge, skill to b scott, , medical university of south carolina, usa participants undertook an online precourse and postcourse assessment developed to meet the learning objectives and competencies of the course in addition to giving post-test feedback on the implementation of the course. in discrete knowledge, subjective knowledge and skills all participants demonstrated significant improvements in their postcourse test results when compared with pre-test. course evaluation was performed, and it was found that students would recommend this course (median . %), whether the course was feasible (median . %) and overall evaluation ( . %). knowledge, skill to b silenas, , the texas a&m college of medicine, usa students answered likert type scales to assess the extent to which the objectives and understanding of key concepts had been accomplished. written and verbal comments from the students and facilitators about their experience were gathered. sixty-six medical students completed the knowledge test before and again days after the avian influenza exercise. the lowest scores for knowledge were best and all tested knowledge areas except one (endemic influenza as a public health issue) decreased postcourse. the course received mixed ratings which overall were positive ( %), undecided ( %) and negative ( %). knowledge. knowledge, skill b table continued figure individual risk of bias for non-randomised control trials determined by robins- . overall risk of bias for non-randomised control trials determined by robins- . critical ( figure ). confounding bias was serious overall as many studies did not present pretest control data. there was critical overall bias in the selection of participants as courses were often not open to all students, for example, self-selected recruitment from student emergency medicine interest groups. classification bias and bias due to deviation from intended interventions was low. a single study had a classification bias because they did not adequately describe their educational intervention-as all other studies had low bias, this domain was classified as having low overall bias. overall missing data bias was moderate as three studies had some form of missing data and were not able to adjust for this in their analysis. measurement of outcomes had serious bias overall, as many questionnaire evaluations were subjective without any objective measures. selection and report of results had serious bias overall. while some studies did note ethical approval there was no priori registration of the results and some studies had limited reporting of results. this systematic review identified approaches used to train medical students in disaster medicine in order to suggest training approaches for medical students in the current covid- pandemic. we identified studies published between and march . overall, medical student disaster training programmes improved student disaster and pandemic preparedness and resulted in improved attitude, knowledge and skills. there was an improvement in all studies that measured precourse and postcourse outcomes. we found that all interventions ranging from simple classroombased interactive discussion to complex multimodal simulative experiences resulted in improved knowledge, skill and attitudes towards participation in disaster medicine. the main outcomes of the courses reviewed were subjective; however, there was evidence to suggest that disaster medicine training does improve objective knowledge and can teach skills which can be used by medical students, relevant to a pandemic. the majority of courses were just day in duration, indicating that short courses can still be impactful. the courses identified in this review required expert faculty or high-fidelity equipment and were implemented alongside an already busy medical school curriculum. these barriers prevented the majority of courses in this review from reaching longitudinal integration into medical school training. however, this may be overcome in the current covid- pandemic by collaboration and coordination, particularly when many medical students have had their studies either postponed or converted to telemedicine/online teaching. the main limitations of this review are related to study design, as the majority of studies were single centre and often focused on very specific aspects of disaster medicine. the overall reporting of both participant factors and outcome factors was generally poor, and the educational methodology was very heterogeneous-this was represented by critical risk of bias in selection of participants, and serious risk of bias in measurement of outcomes. this bias inevitably weakens the strength of the conclusions drawn, but given that all studies demonstrated a positive benefit, it can still be concluded that there will be benefit to students who undertake disaster preparedness courses. another limitation was the kirkpatrick levels that were evaluated. only one study evaluated change in behaviour (level ) and no studies evaluated change in clinical performance (level a) or organisational patient benefit (level b). furthermore, only three studies focused solely on pandemic influenza, airborne viral management or personal protective equipment (n= , %), and only a single study assessed resuscitation in a disaster setting. this is of particular importance for the covid- pandemic, where respiratory personal protective equipment is a necessity and there are specific resuscitation guidelines. clinical impact and clinical utility must be taken into account when making suggestions for training during the covid- pandemic. although medical students working during the covid- pandemic will likely be deployed to non-infectious areas of work, there is no guarantee that medical students will not be exposed to the virus. furthermore, a strain will be placed on healthcare services and contingency care may need to be provided in place of a traditional care service. here, students may be essential in ► medical students could play a crucial role in the sars-cov- healthcare response. ► disaster medicine programmes using multimodal techniques improve knowledge, skills and attitudes which are imperative for medical practice in a pandemic. ► training programmes incorporating previously successful techniques could ensure the successful integration of disaster training into global medical school curricula. ► do disaster training programmes aimed at medical students demonstrate direct patient benefit? ► can disaster training programmes improve the integration of medical students into the healthcare workforce during the sars-cov- pandemic? ► how can disaster training programmes be adapted to manage future pandemics? preserving the resilience of hospitals and community healthcare systems. there will ultimately be more pressure on medical students to work than previous cohorts and this review suggests that disaster medicine training as a part of medical school's curriculum is not common practice. therefore, medical students may require a very different set of competencies than those acquired during medical school. unsurprisingly, some final year medical students do not feel ready to start as a newly qualified doctor, due to worries they are not well prepared for clinical placements, or feeling under prepared for covid- . moreover, the medical schools council have advised that medical students from any year should not take on roles that will impact on their studies. this review suggests that early mobilisation of medical students into the workforce could be accompanied by disaster medicine training. all courses reviewed in this study were positively evaluated by medical students, and if a similar programme was offered to current medical students, it would likely be well received improving willingness and preparedness to work in the healthcare service. this is of particular importance as medical students are already being asked to join the workforce as volunteers, or to graduate early in order to join healthcare systems as physicians. there is great concern that students who give assistance during a disaster without training are at an increased risk of both harm to themselves and psychological consequences. there is therefore a need to create novel courses to teach medical students pandemic skills in these unprecedented circumstances. this review suggests that the most beneficial medical student disaster medicine courses should consist of mixed modalities of didactic sessions, case-studies, practical hands on training and simulation experiences. these training methods could be used to train medical students in covid- specific knowledge and skills and prepare them for clinical practice. table shows a proposed covid- course and assessment based on the findings of this systematic review. the course structure includes the variety of elements found in other studies. didactic lectures on covid- could be delivered in a lecture hall with social distancing measures in place, or perhaps more appropriately as a distance learning component consisting of video, podcast and computer activities. practical activities could include fitting of respiratory personal equipment as well as donning and doffing. the simulated element could consist of a patient with covid- who requires cardiopulmonary resuscitation. in resource-limited scenarios, this could be undertaken using computer-based tutorials or video tutorials. as new doctors and medical students may have a substantial volume of information to learn in addition to this course, handouts and online refresher courses should be offered. the proposed assessment aims to cover all kirkpatrick levels and criteria. it is also important to teach and train human factors awareness, particularly in relation to team dynamics, lowering authority gradients and empowering anyone to speak up if concerned. maintaining both individual and team situational awareness is also important during any clinical duty, and even more so during a crisis setting. it may be useful to incorporate a credentialing process for medical students undergoing disaster training, thereby allowing students to demonstrate a background of competency and separating this cohort from unskilled volunteers when aiding a disaster medicine response. the successful implementation of these suggested disaster training techniques will require the encouragement of people-centred training, the development of peer-learning, coordination and funding of training systems, and regular disaster preparedness exercises of multimodality format. the covid- pandemic has caused unprecedented disruption to healthcare services in peacetime. medical students may play a crucial role in the healthcare response. there is an imminent demand for educational interventions to train medical students to better assist in this response. the disaster medicine courses reviewed in this article improved knowledge, skills and attitudes through multimodal techniques and were well received original research by learners. although no studies in this review demonstrated direct patient benefit, the courses increased student preparedness and similar courses should be implemented prior to medical students joining the healthcare workforce during the covid- pandemic. future courses should note the methodological and longitudinal flaws demonstrated in previous studies so that direct patient benefit can be demonstrated in the covid- pandemic. future work should be undertaken to ensure the successful integration of disaster training into global medical school curricula. twitter james ashcroft @jamesashcroftmd, matthew h v byrne @mhvbyrne, peter a brennan @brennansurgeon and richard justin davies @jdcamcolorectal contributors ja and rjd undertook conceptualisation of this article. ja and mhvb undertook data collection, analysed the data, and drafted and revised the manuscript. rjd and pb undertook critical review of all data collection and analysis and guided critical revisions. rjd approved final manuscript for submission. funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. the importance of evidence-based disaster planning achieving good medical practice 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protective equipment simulation training for in-flight medical emergencies improves provider knowledge and confidence a short medical school course on responding to bioterrorism and other disasters disaster preparedness medical school elective combating terror: a new paradigm in student trauma education disaster : a novel approach to disaster medicine training for health professionals high-fidelity multiactor emergency preparedness training for patient care providers teaching mass casualty triage skills using immersive three-dimensional virtual reality teaching mass casualty triage skills using iterative multimanikin simulations disaster training in hours: evaluation of a novel medical student curriculum in disaster medicine competing interests none declared. provenance and peer review not commissioned; externally peer reviewed. this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid- pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. james ashcroft http:// orcid. org/ - - - matthew h v byrne http:// orcid. org/ - - - x key: cord- -w m ci i authors: yamin, mohammad title: it applications in healthcare management: a survey date: - - journal: int j inf technol doi: . /s - - - sha: doc_id: cord_uid: w m ci i healthcare management is currently undergoing substantial changes, and reshaping our perception of the medical field. one spectrum is that of the considerable changes that we see in surgical machines and equipment, and the way the procedures are performed. computing power, internet and associated technologies are transforming surgical operations into model based procedures. the other spectrum is the management side of healthcare, which is equally critical to the medical profession. in particular, recent advances in the field of information technology (it) is assisting in better management of health appointments and record management. with the proliferation of it and management, data is now playing a vital role in diagnostics, drug administration and management of healthcare services. with the advancement in data processing, large amounts of medical data collected by medical centres and providers, can now be mined and analysed to assist in planning and making appropriate decisions. in this article, we shall provide an overview of the role of it that have been reshaping the healthcare management, hospital, health profession and industry. internet and web . have been instrumental in evolving most of the information technology (it) applications that we are now accustomed to. barely years ago, we had not imagined the technological advancements, which are now implemented into our lives. technological advancement, riding on the wave of disruptive technologies of the last quarter of a century, is reshaping and redefining the world's social order and norms. the medical field is part and parcel of our lives and is perhaps one that we care the most about. in the recent times, this field has undergone vast changes. the medical profession today has evolved greatly since the beginning of this century. not only the medical procedures, the medical data flow and management has also undergone considerable improvement. advance data transfer and management techniques have made improvements in disease diagnostic and have been a critical role in national health planning and efficient record keeping. in particular, the medical profession has undergone substantial changes through the capabilities of database management, which has given rise to the healthcare information systems (his). medical data collected from different institutions can now be mined and analysed in time using big data analytics tools. this article shall provide an overview of the involvement, importance and benefits of it and how data management is contributing to the medical science field. in particular, the following will be discussed: . patient and data management . big medical data analytics . privacy and security of healthcare data artificial intelligence and robotics in healthcare artificial intelligence (ai) is a field of computer science which has its roots in logic (mathematics), psychology, philosophy, linguistics, arts, science and management among others. many ai-dominant tools and applications can be found in games, auto spare parts, heavy machinery and various medical instrumentations. according to [ ] , many programs are developed with the help of ai to perform specific tasks which make use of many activities including medical diagnostic, time sharing, interactive interpreters, graphical user interfaces and the computer mouse, rapid development environments, the linked listdata structure, automatic storage management, symbolic, functional, dynamic, and object-oriented programming. here are some other examples. pharmaceutical developments involve extensive clinical trials in real environment and may take many years, sometimes more than a decade. this process requires a considerable amount of resources and can cost billions of dollars. in order to save lives and money, it is desirable to expedite this time consuming process to make healthcare cheaper. an urgency of this nature was witnessed during the ebola crisis in some west african nations [ ] . an urgency of this nature was witnessed during the ebola crisis in some west african nations [ ] , where a program powered by ai was used to scan existing medicines that could be redesigned to fight the disease. nowadays medical consultation, with the help of an online illness database, can take place without the direct interaction of a medical practitioner. an example of such a practice can be found in [ ] . advantages of such consultations lies in the fact that the symptoms presented online by the patients are matched with those of about ten thousand known diseases [ ] , whereas doctors can only match the symptoms against a fraction of the known diseases which they are familiar without the intervention of an it database. likewise virtual nursing to support patients online is also available from [ ] . yet another app [ ] developed by boston children hospital provides basic health information and advice for parents of ill children. since the introduction in s, robots have been used in many scientific and social fields. their use in fields like nuclear arsenal, production of automobiles, and manipulations in space has been widespread. in comparison, the medical science field has somewhat been gradual to take advantage of this technology. nevertheless, robots are now days being utilised to assist in or perform many complex medical procedures, which has given rise to the term robotic surgery. a comprehensive discussion of robotic surgery is available in [ ] . indeed there are many advantages of using robots in healthcare management. for example, developing pharmaceuticals through clinical trials can take many years and cost may rise to billions of dollars. making this process faster and more affordable could change the world. robots can play a critical role in such trials. robots are also very effective in medico training where simulations are achieved. use of robots in abdominal surgery [ ] is innovative in comparison to the conventional laparoscopy procedures, and has the potential to eliminate the existing shortcomings and drawbacks. according to [ ] , the most promising procedures are those in which the robot enables a laparoscopic approach where open surgery is usually required. likewise robotic surgery for gynaecology related ailments [ ] can also be very effective. the conventional laparoscopy, the surgeon would have limited degree of freedom with a d vision, whereas the robotic system would provide a d view with intuitive motion and enable additional degrees of freedom. prostatic surgery, being complex in the way of the degree of freedom, is another area where robots are very effective [ ] . nosocomial infections pose a major problem hospitals and clinics around the world. this becomes worst in case of large and intense gatherings of people like in hajj [ ] . to keep the environment free of these viruses and bacteria, the environments needs to be cleaned efficiently and regularly. however some viruses like ebola [ ] and mers coronavirus [ ] are highly contagious and cannot be cleaned by humans without having protective gear, which can be costly and may or may not be effective. cleaning the hospital with chlorine is an effective way. however, there are many drawbacks of this method including a high risk for cleaners to be infected themselves. a sophisticated approach was implemented in us hospitals by using robots [ ] to purify the space. however, some of the robots used are not motorized and may require a human to place them in the infected room or area, posing the risk of infection to humans. it is not known as to which substance or matter can effectively eliminate deadly viruses like ebola and mers coronavirus. however, according to [ ] , beta-coronaviruses, which is one of the four strands of mers coronavirus, can be effectively eliminated by uv light. for such a deadly and highly contagious virus clearing, a robot that can be remote controlled is needed, which can move forward, backward and sideways, and is able to clean itself. concept and related technology of virtual reality (vr), augmented reality (ar) and mixed reality (mr), which originate from image processing and make extensive use of ai, have been in use for decades in games and movies. for some time now, the vr, ar and mr tools and technology are assisting the medical field in a significant way. virtual reality has been used in robotic surgery to develop simulators to train surgeons in a stress free environment using realistic models and surgical simulation, without adversely affecting operating time or patient safety [ ] . an account of ar and vr technologies, including the advantages and disadvantages can be found in [ ] . the ar and vr technology can be very useful in medical and healthcare education, an account of which can be found in [ ] . applications of vr, ar and mr can also be very helpful in urology [ ] . healthcare management information systems (hmis) is the generic name for many kinds of information systems used in healthcare industry. the use of hmis is widespread in various units of hospitals, clinics, and insurance companies. a doctor records patient data (habits, complaints, prescriptions, vital signs, etc.) in one such system they use. a number of hospital/clinic units like radiology, pathology, and pharmacy store patient data in these systems for later retrieval and usage by different stakeholders as shown in fig. . appointment system itself can also be part of a hmis or a standalone system. modern healthcare without hmis cannot be comprehended. an information system or electronic system dealing includes a database system, sometimes known as the backend of the main system. a database is a collection of data, tailored to the information needs of an organisation for a specific purpose. it can grow bigger or shrink to a smaller size depending on the needs of different times. a database in itself would be of no use unless it was coupled with a software system known as the database management system (dbms), which facilitates various functions and processes like data entry and storage, retrieval, and update, data redundancy, data sharing, data consistency, data integrity, concurrency, and data privacy & security. more information about database systems can be found in [ ] . data in an information system can be organised in several different ways. classical representation of data [ ] is tabular. most tables can be regarded as mathematical relations, for this reason, a dbms with tabular data organisation is known as relational. relational databases have by far been dominant in the business organisations until recently. in the eighties and nineties of the last century, object-oriented and semantic data models were also introduced. in particular, object-oriented databases [ ] became very popular for managing audio-visual data. semantic database technology [ ] , although very efficient for search queries, did not reach its expected peak, perhaps because of its complex data model dealing with ontologies. a comparative study of relational, object oriented and semantic database systems is carried out in [ ] . although the evolution of information technologies began much earlier, however, its implementation by businesses and corporations occurred in the second half of the last century. the healthcare industry a bit slow to take the advantage of the technology. earlier, medical records and systems were paper based. thus the first phase of the usage of information technology and systems in hospital and healthcare management was to transform paper based records to database systems. not only did it change paper based systems to electronic ones but also allowed data manipulation. as a result, more data could be processed in a very short time, which was critical in making timely decisions. a discussion of early stages of this transformation can be found in [ ] . consolidation of this technology resulted in healthcare management information systems (hmis). timely decision is critical in all walks of life but it assumes a much greater urgency in some healthcare cases. hmis is capable to transferring data from one location or application to another within split of a second, as opposed to days without the help of electronic systems. this allows users to access, use and manipulate data instantly and concurrently. as the technology continues to evolve and refine, the performance of information systems operations, in particular those of hmis, is bound to improve. ubiquitous health system is meant to provide healthcare services remotely. according to [ ] , ubiquitous healthcare system means the environment that users can receive the medical treatment regardless of the location and time. essentially, a ubiquitous healthcare system would monitor daily or periodic activities of patients and alert the patients or health workers of problems, if any. a discussion of national healthcare systems in korea, including the concept of u-hospitals, is discussed in [ ] . a rich picture of a ubiquitous healthcare system is shown in fig. . internet of things (iot) is an emerging technology. data in organisations, including hospitals, clinics, and insurance companies, is increasing every day. to process this large data, advanced data processing techniques are combined into what is now known as the big data analytics. internet of things (iot) is a general paradigm. when dealing with medical environment, it is known as internet of medical of things (iomt), which is same as the medical internet of things. a discussion of iomt can be found in [ ] . the fundamental objective of iot is to provide ubiquitous access to numerous devices and machines on service providers (sps) [ ] , covering many areas such as location based services (lbs), smart home, smart city, e-health [ , ] . these tools, devices and machines cover service providers in many areas of location based services. a few other could be smart home, street or city, elements of ubiquitous health systems, tools for electronic learning and electronic business, the number which is increasing day by day and it may reach fifty billions in [ , ] . we have conceptualised this in fig. . the iot applications invariably use cloud storage coupled with fog computing [ ] . data in organisations is growing at a rapid rate. some organisations have collected huge pile from their operations. organisational data needs to be mined and analysed on a regular basis to uncover hidden patterns, unknown correlations, market trends, customers, patients and treatment records to help organizations to plan and make more informed decisions. mining and analysis of big data captures the immense wealth of intelligence hidden in organisational data, makes information transparent and usable at much higher frequency, and enhance performance. all of these contribute to providing higher value in efficient decision making. when data grows, it cannot be handled manually, often big data organisations use robotic arm to perform data loading and unloading operations. medical data is also growing at a rapid rate in many hospitals, clinics and insurance companies. mining and analysis of healthcare data is even more critical as it relates to the wellbeing of the society. data centric research is highly desirable to find answers to questions on many chronical diseases, and to plan and manage national and international health programs. potential befits of big data analytics in healthcare are discussed in [ ] . in particular, authors have provided five strategies for success with big data. these are ( ) implementing big data governance, ( ) developing an information sharing culture, ( ) training key personnel to use big data analytics, ( ) incorporating cloud computing into the organization's big data analytics, and ( ) generating new business ideas from big data analytics. a survey of big data analytics in healthcare and government is conducted in [ ] , where the need for the analytics is linked to the provision and improvement of patient centric services, detection of diseases before they spread, monitoring the quality of services in hospitals, improving methods of treatment, and provision of quality medical education. authors in [ ] identify medical signals as a major source of big data, discuss analytical techniques of such data. some of the well known tools used for big data analytics are apache tm hadoop Ò is highly scalable storage platform. it provides cost-effective storage solution for large data volumes. it doesn't require any particular format. apache spark is an open source, in-memory processing machine. its performance is much faster than that of hadoop. mapreduce is used for iterative algorithms or interactive data mining. there are many other big data analytics tools, a list of them can be found at http://bigdata-madesimple.com/top- -big-data-tools-data-analysis/. there are always security and privacy concerns associated with data. protection of privacy and security is a responsibility of data holding organisations. with medical data these concerns assumes higher pronounce and priority. medical data can be very sensitive, and may be linked to life and death of the patients. often socio political issues are linked with medical data. it is well known that india was divided into two countries giving rise to pakistan in . mohammad ali jinnah, very clever and shrewd, was the leader of pakistan campaign, larry collins and dominique lapierre [ ] argue that the partition of india could have been avoided if ''the most closely guarded secret in india'' had become known. jinnah was suffering from tuberculosis which was slowly but surely killing him. in [ , , ] , privacy issues of data are discussed. healthcare procedures and management are now greatly relying on it applications, which are providing realtime access to data and utilities. without these applications, healthcare will be limited, compromised and prone to major problems. many of the medical equipments, which are used in procedures, are highly dependent on technology. ai, robots, vr, ar, mr, iomt, ubiquitous medical services, and big data analytics are all directly or indirectly related to it. hmis is critical for efficient management of records, appointment systems, diagnostics and their needs of medical centres. as the technology advances, the healthcare is likely to further improve. artificial intelligence: a modern approach perspectives on west africa ebola virus disease outbreak intelligent machines, the artificially intelligent doctor will hear you now meet molly-the virtual nurse boston children's hospital launches cloud-based education on amazon alexa-enabled devices robotic surgery-a personal view of the past, present and future robotic abdominal surgery status of robotic assistance-a less traumatic and more accurate minimally invasive surgery? robotic surgery in gynaecology a randomised controlled trial of robotic vs. open radical prostatectomy: early outcomes health management in crowded events: hajj and kumbh. bvicam's middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir the iowa disinfection cleaning project: opportunities, successes, and challenges of a structured intervention program in hospitals inactivation of middle east respiratory syndrome-coronavirus in human plasma using amotosalen and ultraviolet a light impact of virtual reality simulators in training of robotic surgery augmented and virtual reality in surgery-the digital surgical environment: applications, limitations and legal pitfalls preliminary study of vr and ar applications in medical and healthcare education application of virtual, augmented, and mixed reality to urology database systems. a practical approach to design, implementation and management, th edn a relational model of data for large shared data banks semantic, relational and object oriented systems: a comparative study health care in the information society: a prognosis for the year unified ubiquitous healthcare system architecture with collaborative model national health information system in korea medical internet of things and big data in healthcare information analytics for healthcare service discovery intelligent human activity recognition scheme for e-health applications preserving privacy in internet of things-a survey internet of things: converging. technologies for smart environments and integrated ecosystems security of the internet of things: perspectives and challenges a survey on fog computing: properties, roles and challenges big data analytics: understanding its capabilities and potential benefits for healthcare organizations a survey of big data analytics in healthcare and government. nd international symposium on big data and cloud computing (isbcc' ) big data analytics in healthcare freedom at midnight: the epic drama of india's stuggle for independence improving privacy and security of user data in location based services big data security and privacy in healthcare: a review key: cord- -fw qt authors: belhouideg, soufiane title: impact of d printed medical equipment on the management of the covid pandemic date: - - journal: int j health plann manage doi: . /hpm. sha: doc_id: cord_uid: fw qt very high mortality rates of coronavirus pandemic (covid‐ ) are observed around the world due to lack of medical equipment. the increased need for medical devices and personal protective equipment (ppe) has kept several healthcare professionals at risk. fortunately, d printing technology allows to overcome the lack of medical supplies. this study highlights the impact of d printing on the combat against covid , and its importance in the medical product supply chain. indeed, the existing medical equipment fabricated by d printing technology and its role in the management of covid pandemic is presented. moreover, the last works are examined to know whether the models of the medical equipment are free of use and whether useful informations are presented (eg, available design data and setup guidelines). very high mortality rates of coronavirus pandemic are observed around the world due to lack of medical equipment. the increased need for medical devices and personal protective equipment (ppe) has kept several healthcare professionals at risk. fortunately, d printing technology allows to overcome the lack of medical supplies. this study highlights the impact of d printing on the combat against covid , and its importance in the medical product supply chain. indeed, the existing medical equipment fabricated by d printing technology and its role in the management of covid pandemic is presented. moreover, the last works are examined to know whether the models of the medical equipment are free of use and whether useful informations are presented (eg, available design data and setup guidelines). ) becomes a danger to the public at the international level. [ ] [ ] [ ] the covid- is carried mainly through connection with an infected person, through droplets that are made when the individual coughs or sneezes, or through tiny droplets from the saliva or even the nasal cavity. the human respiratory system is targeted by the coronavirus. simple actions such as individual hand cleaning and social distancing are important. however, based on world health organization guidelines, a protective material is necessary for all medical services. with the number of cases confirmed each day, the available medical equipment cannot support the volume of patients. then, the medical product supply chain is disrupted. thus, people die because of lack of some equipment such as ventilators and protective masks. [ ] [ ] [ ] the existing medical system is based on private equipment from suppliers. generally, most of the medical equipment is patented by medical firms. , in the case of a sudden surge in demand, suppliers are unable to prepare the necessary quantity of equipment: innovation and new manufacturing strategies are needed. moreover, transport and trade restrictions, quarantines, border controls, and production disruptions greatly affect the global supply chain of critical medical devices. fortunately, d printing technology can be an effective solution to overcome this problem. , using this technology, the developed open-source designs of medical equipment are shared freely. [ ] [ ] [ ] then, other people around the world can reproduce the medical equipment on their d printers. [ ] [ ] [ ] the most commonly used d printers are desktop d printers based on fused deposition modeling (fdm) technology. generally, these types of d printers are not fast when creating parts. but, by using all the existing d printers, the production capacity increases significantly. in recent weeks, the national governments around the world have called industry, scientific, and makers to develop and produce more personal protective equipment. with a d printer and the necessary material, everybody can be a part of the "citizen supply chain". the designs of personal protective equipment (ppe) are shared for free by the maker community. the goal of this study is to present the role of d printing to fight against the covid- pandemic. also, the impact of d printed medical equipment on the medical product supply chain is analyzed. the existing designs for medical equipment are summarized. it analyzes previous works to determine whether the designs are open source and whether practical details are provided, which include accessible design source files (eg, computer-aided design, cad), production files (eg, surface tessellation language, stl) and assembly instructions. quarantine procedures in the particular situation of this specific pandemic have started to give stress and worry between people. a regrettable result of this is the unnecessary anxiety buying, causing those individuals who need ppe products, such as healthcare workers, within restricted supply. then, people from the d printing community have developed a variety of reusable ppe products using low-cost desktop d printers, such as n masks, surgical masks, face shields, and controlled air purifying respirator (capr) system. in a local context, the user must employ adequate sterilization techniques for the ppe devices. the protective mask is a medical device aimed to filter bacteria and to avoid contracting a virus. these masks must be worn by the general public to protect themselves from microorganisms in the pandemic context. since the beginning of covid , the problem of supplying a protective mask has become one of the main challenges to overcome. several initiatives are taking place to remedy these deficiencies around the world. in all situations, the end customers must plainly recognize that only prototypes are obtainable at this stage and local test techniques are important to evaluate the quality of the protective mask. swennen et al have presented a procedure to produce d printed personalized masks using available methods and materials. the authors have shared the design of this mask for free of charge. a d printed reusable n comparable respirator was developed by provenzano et al at the george washington university. these masks can be used with multiple filtration units, easy to produce and easy to clean. the d model is available upon request. chagas et al summarize several open source projects to fabricate personal protective equipment and the developed approaches in covid- diagnostics. students at cedarville university have printed d masks for healthcare workers. the project nanohack was launched by the manufacturer, copper d, to produce the model n of protective mask using d printing technology. a new version of the project nanohack . was published. it is an improved version that protects against the covid- infection. the production files (stl) are provided for free of charge. polylactic acid (pla) filament can be used to fabricate this mask as a flat part. therefore, it is designed to be manually assembled into the final d configuration after heating to a temperature of c to c (figure ). another design of the protective mask was developed by the thingiverse user kvatthro. moreover, this mask can be produced using a desktop d printer and pla filament. it is designed with a box for hepa filter insertion ( figure ). the chinese company, creality, suggested a similar style of the hepa mask with different arrangements of the filter container, that allows to insert folded fabric or filters (figure ). during the covid pandemic, a face shield was designed and fabricated using d printing. it contains a d printable headpiece and an attachable transparent sheet of plastic. this device protects the user's eyes and mouth (figure ) . table summarizes the different ppe that has been fabricated using d printing technology during the covid pandemic. it is noticed that the d printed ppe must be approved by a regulatory body. most ppe is difficult to sterilize. however, several studies were conducted to investigate the insertion of drug molecules in the ppe during fabrication to offer an antimicrobial and antiviral protection. - a non-exhaustive list of open-source medical devices for covid- is given in table . the link to download the stl files is also given. the ventilation system helps patients to maintain an adequate level of oxygen (> %) in the arterial blood. ventilation system is the last hope for the majority of patients most affected by the new coronavirus "covid- ." most of the national health systems do not have the needed ventilation systems that the covid- pandemic is believed to require. this situation forced the doctors to make a difficult decision regarding the patients: who should be connected to these devices and who will be disconnected from them. since the appearance of covid- pandemic, local and international communities have been working together to develop an open-source ventilator. their documentation and information are freely available on their slack team. for example, a d printed ventilator system was designed and tested. the design files and stls are freely available. also, some makers are working on the development of pandemic ventilators. despite the many promising approaches, it is found that there is not enough information on the performance of ventilator designs to recommend them for medical use. also, it is noticed that a basic test was carried on the designed ventilators and some ventilators were not tested at all. it is reported that the makers can use the free inactive patent that contains useful information f i g u r e creality mask to improve the open-source ventilator design. these designed ventilators must be validated by a medical team. the untested ventilators can harm the lungs of the patients. [ ] [ ] [ ] [ ] for those who design open-source ventilators, there is abundant literature. for example, the essentials of mechanical ventilation can be found in "the ventilator book" by owens and "principles and practice of mechanical ventilation" by tobin. pham et al as a guide to build a similar device. a minimal oxygen consumption pneumatic ventilator during a respiratory malfunction pandemic was developed by williams et al. for different lung compliances, three designs were tested. the main schematics are given, but insufficient for a complete open hardware design. it is noticed that most of the low-cost ventilators use the bag approach; however, the contemporary professional ventilators, commonly, are not produced with bags. the current effect of covid- in italy has caused local crisis due to the lack of medical equipment such as masks and venturi valves. during this crisis, venturi valves ( figure ), important elements of respiratory support equipment, demonstrated a hard problem to reproduce. the design of venturi valve is protected under patent law. but particular disasters leading to life-or-death choices may explain the total usage of intellectual property. this specific need led the d printing community to develop a solution for producing these valves to reinforce regional supply. another method of reinforcement is the use of a single ventilator for multiple patients with a d printed ventilator splitter. in the above, a subset of projects that are aiming to produce protective equipment against covid- using d printing are summarized. for a disaster situation, open-source systems allow rapid development based on the contributions of many people who work remotely. the "citizen supply chain" based on d printing has confirmed to be a powerful solution during covid . this solution can be used in many industrial sectors. in fact, many of the d printed ppe products need several hours to print it on conventional desktop d printers. during covid pandemic, organized communication between d printing community and local hospital supply chain is recommended. despite the urgency of the growing covid- crisis, healthcare devices must be very controlled for safety. indeed, the d printed medical equipment must follow the standard safety and quality measures. moreover, the development of antimicrobial polymers for d printing provides the possibility to fabricate several critical medical products. in the future, a d printed ppe manufacturing protocol may be developed. this protocol contains the following information: cao model, d printing technology to use, material, and assembly instructions. sometimes, it is important to develop an open-source program for disasters before the disaster strikes, because some regions of the world suffer from supply disruptions and shipping challenges. moreover, it is necessary to develop policies and funding mechanisms for such projects. an additional challenge is keeping a suitable level of sterility of the equipment manufactured. covid- infection: origin, transmission, and characteristics of human coronaviruses coronavirus disease (covid- ): emerging and future challenges for dental and oral medicine world health organization. critical preparedness, readiness and response actions for covid- : interim guidance the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak covid- ) advice for the public: when and how to use masks flusurge-a tool to estimate demand for hospital services during the next pandemic influenza italy rush to buy life-saving ventilators as manufacturers warn of shortages preparing for the most critically ill patients with covid- : the potential role of extracorporeal membrane oxygenation estimates of the demand for mechanical ventilation in the united states during an influenza pandemic against intellectual monopoly the crisis of intellectual monopoly capitalism experimental d printed -port ventilator manifold for potential use in disaster surges d 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print -d masks for healthcare workers. news releases makers guide additive manufacturing can assist in the fight against covid- and other pandemics and impact on the global supply chain. d print addit manuf food and drug administration (fda). d printing of medical devices antimicrobial polymers with metal nanoparticles d printed antibacterial prostheses aerosol and surface stability of sars-cov- as compared with sars-cov- a novel anti-influenza copper oxide containing respiratory face mask the role of additive manufacturing and antimicrobial polymers in the covid- pandemic a review of open source ventilators for covid- and future pandemics pandemic ventilator' could offer solution in potential 'worst case' coronavirus scenario open source database and website to provide free and open access to inactive u.s. patents in the public domain ventilator-induced lung injury ventilator-induced lung injury ventilator-induced lung injury: the anatomical and physiological framework mechanical power and development of ventilator-induced lung injury the ventilator book principles and practice of mechanical ventilation mechanical ventilation: state of the art development of field portable ventilator systems for domestic and military emergency medical response a new compact and low-cost respirator concept for one way usage prototyping of artificial respiration machine using ambu bag compression design and prototyping of a low-cost portable mechanical ventilator a low oxygen consumption pneumatic ventilator for emergency construction during a respiratory failure pandemic how to cite this article: belhouideg s. impact of d printed medical equipment on the management of the covid pandemic the authors have no competing interests. https://orcid.org/ - - - key: cord- -mcee wr authors: tempski, p.; arantes-costa, f. m.; kobayasi, r.; siqueira, m. a.; torsani, m. m.; amaro, b. q.; nascimento, m. e. f.; siqueira, s. l.; santos, i. s.; martins, m. a. title: medical students perceptions and motivations in time of covid- pandemic date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: mcee wr background: there has been a rapid increase in the number of cases of covid in latin america, africa and asia, in many countries that have an insufficient number of physicians and other health care personnel, and the need for the inclusion of medical students as part of the health teams is a very important issue. it has been recommended that medical students work as volunteers, have appropriate training, do not undertake any activity beyond their level of competence, have continuous supervision and adequate personal protective equipment. however, motivation of medical students must be evaluated in order to make volunteering a more evidence-based initiative. the aim of our study was to evaluate motivation of medical students to be part of the health team to help in the covid pandemic. methods and findings: we developed a questionnaire specifically to evaluate medical students perceptions about participating in care of patients with suspected infection due to coronavirus during the covid pandemic. the questionnaire had two parts: a) individual characteristics, year and geographic location of medical school; b) twenty eight statements responded on a point likert scale (totally agree, agree, neither agree nor disagree, disagree and totally disagree). to develop the questionnaire, we performed consensus meetings of a group of faculty and medical students. the questionnaire was sent to student organizations of medical schools in brazil and answered by , students. we used multinomial logistic regression models to analyse the data. statements with greater odds ratios for participation of medical students in covid pandemic were related to sense of purpose or duty (it is the duty of the medical student to put himself at the service of the population in the pandemic), altruism (i am willing to take risks by participating in practical in the context of pandemic), perception of good performance and professional identity (i will be a better health professional for having experienced the pandemic). males had higher odds ratios than females ( . [ % ci: . to . ] to . [ % ci: . to . ]). conclusions: medical students are motivated by sense of purpose or duty, altruism, perception of good performance and values of professionalism more than their interest in learning. these results have implications in the developing of programs of volunteering and in the design of health force policies in the present pandemic and in future health emergencies. the coronavirus covid- pandemic is the most important global health crisis of our time and the greatest challenge the health system has faced since world war two. since its emergence in asia in , the virus has spread to every continent, except antarctica. cases are rising daily in europe, north america, and, in the last weeks, also in latin america and africa. [ , ] covid- pandemic resulted in a disruption of undergraduate medical education. in many countries, medical education faculty have quickly transitioned the first-years curriculum to on-line activities in response to the need of social isolation to flatten the curve of new cases of covid- . [ ] in addition, in the final years of medical schools, in many countries, clerkships were severely affected by the rapid changes in hospitals due to the need of care of an increasing number of covid- patients and medical students were advised to stay at home, considering the potential risk of medical students spreading covid- infection in health care settings and the shortage of personal protective equipment (ppe). [ , ] however, the role of medical students in the covid- pandemic is changing rapidly, due to the shortage of health professionals in many cities, even in developed countries. association of medical colleges (aamc) of the united states have published guidelines for the participation of medical students in the global effort to give the best care to patients with covid- . [ , ] both associations recommend that medical students work as volunteers, have appropriate training, do not undertake any activity beyond their level of competence, have continuous supervision and adequate ppe. [ , ] in the last weeks, there has been a huge increase in the number of cases in countries that have an insufficient number of physicians and other health care personnel, and it is possible to anticipate the need for the inclusion of medical students as part of the health teams. [ , ] in many countries thousands of medical students have volunteered their services to support the fight against the coronavirus pandemic. [ , ] motivation is pivotal to volunteering and must be evaluated in order to make volunteering a more evidence-based initiative. the aim of our study was to evaluate motivation of medical students to be part of the heath team to help in the covid- pandemic. it was performed in a developing country, brazil, in the first week of the increase in cases of covid- in brazil, including , medical students. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint we developed a questionnaire specifically to evaluate medical student's perceptions about participating in care of patients with suspected infection due to coronavirus during the covid- pandemic. the questionnaire had two parts: a) individual characteristics, year and geographic location of medical school; b) twenty-eight statements responded on a -point likert scale (totally agree, agree, neither agree nor disagree, disagree and totally disagree). to develop the questionnaire, we performed consensus meetings of a group of faculty and medical students. the questionnaire was developed on a google form survey administration app. with the help of the brazilian section of the international federation of medical students associations (ifmsa), the questionnaire was sent to medical student organizations of medical schools of all regions of brazil, using instagram and whatsapp social media. in the first page of the questionnaire, the purpose of the study was explained, and the student had to agree with the objectives of the study before filling the questionnaire. the survey was performed between th and th of march, since after these three days of survey we had received more than , answers. in this period, there were only a small number of patients with the diagnosis of covid- disease in brazil; , confirmed cases and deaths due to according to the ministry of health of brazil. [ ] . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . [ ] the questionnaire was sent to the student organizations of medical schools ( . % of brazilian medical schools). participants informed their agreement or not with two statements regarding their view about the role medical students should have during the covid- pandemic. these questions were (s ) medical internship students must participate in health care assistance during pandemic; and (s ) all students regardless their year during the medical school, must participate in health care assistance during pandemic. participants were classified according to their agreement with statements s and s as: ( ) all students should participate (agree with s ); ( ) only students in internship should participate (not agree nor disagree / disagree with s and agree with s ); and ( ) no students should participate (not agree nor disagree / disagree with s , not agree nor disagree / disagree with s ). for the analysis, we combined the responses "completely agree" with "agree" for each statement. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint table shows the number of medical students from the five geographic regions of the country that answered the questionnaire, and the relationship between the number of participants and the number of vacancies in each region of the country. there was a similar proportion of respondents/vacancies, showing that the sample was homogenously distributed across the country. table shows the number (and percentage) of medical students that answered "completely agree" or "agree" for each one of the statements of the questionnaire. students were divided considering year of medical course and sex. supplemental table shows the answers to the questionnaire for the hole group of medical students. of the , students who completed the survey, , ( . %) were females, and , ( . %) were in internship years (interns). the mean age was . ± . years. all students should participate in the response to the covid- pandemic according to . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . table shows the participants' characteristics and perceptions according to their view about the role of medical students during the covid- pandemic. the statements which most participants agreed to were: s "i know how to guide for preventive measures" ( . %), s "i know how to use personal protection equipment (pfe)" ( . %), and s "medical schools must suspend their academic activities during the first to fourth year" ( . %). . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint table shows the adjusted odds ratios (and % confidence intervals), from multinomial models, for the association between students' characteristics and perceptions and their view about the role of medical students during the covid- pandemic (crude models results are presented in supplemental table ). compared to the participation of no medical students, odds ratios [or] for the participation of students in internship or all medical students were, respectively, . ( % confidence interval: . - . ) and . ( % ci: . - . ). men were more prone to support the participation of medical students in the fight against the pandemic (or for the participation of students in internship or all medical students respectively . [ %ci: . - . ] and . [ %ci: beliefs towards a participation of medical students in covid- pandemic healthcare with highest odds ratios were: s "it is the duty of the medical student to put himself at the service of the population in the pandemic", s "i am willing to take risks by participating in practical in the context of pandemic", s "i am able to participate in the care of patients who seek health care", s "i feel able to communicate the diagnosis for covid- infection", and s "i will be a better health professional for having experienced the pandemic". on the other hand, beliefs against a participation of medical students in covid- pandemic healthcare with lowest odds ratios were statements s /s (suspension of academic activities), s "i feel insecure regarding the future", s "my emotional state during the pandemic affects my learning", and s "the role of the medical students during the pandemics time is irrelevant". . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint analysis with only the students in the last years (interns) was also performed (table ). in this subgroup, male sex was also associated with the support of a participation of medical students. similar to the findings of the entire sample, statements s , s , s , s , s and s were the beliefs towards a participation of medical students with highest odds ratios in this subgroup. on the other hand, agreement with the statements s /s (suspension of academic activities), s "i feel stressed in the hospital at the moment", s "i am afraid of contaminating myself" and s "the role of the medical students during the pandemics time is irrelevant" were more relevant beliefs associated with a perception against the participation of medical students in the actions during the pandemic. crude models for this subgroup are presented in supplemental table . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint beliefs towards a participation of medical students in covid- pandemic healthcare s . it is the duty of the medical student to put himself at the service of the population in the pandemic beliefs not / poorly related with the participation of medical students in covid- pandemic healthcare s . i prefer to study theoretical content using distance learning methods beliefs towards a participation of medical students in covid- pandemic healthcare s . it is the duty of the medical student to put himself at the service of the population in the pandemic beliefs against a participation of medical students in covid- pandemic healthcare s . medical schools must suspend their academic activities during the first to fourth year. in this study we aimed to evaluate the factors that motivate a medical student to participate in the care of patients in the context of covid- pandemic. we also aimed to understand the students' desires related to continuing medical training or to have online education during the pandemic. to our knowledge, there is no previous study that evaluated a large sample of medical students with these purposes. we believe that this study can contribute to the planning of medical education and work force organization in the covid- pandemic and in other future health emergencies. although our sample was a convenience sample, we evaluated a large group of medical students ( , ) and there was a uniform distribution of subjects across the country (table ) . when we performed this study, the number of covid- cases in brazil was low, and only · % of participants (table ) had a family member or a friend with a diagnosis of covid- or had a personal diagnosis of this disease. [ ] we were able to study the factors that influence the desire to participate in the care of covid- without a strong emotional influence of sick relatives or friends. when we compared the answers to the statements of the questionnaire provided by students in the first two years of medical course to students of the last two years (interns) ( table , univariate analysis) we observed many differences. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint the largest differences in percentages of agreement with the statements were related to feeling of competency to take care of patients (differences from . % to . %, statements s , s , s , s and s ). as expected, interns felt more confident concerning identification of a suspected case, identification of signs of severity, guidance of therapeutic measures, participation in the care of patients and communication of diagnosis. in addition, interns were more prone to accept on-line learning and the interruption of both classes and internship activities (differences from . % to . %). in contrast, students from the first years were more insecure about the substitution of classes and practical activities by on-line learning (statements s and s , differences . % and . %). more students preferred to delay their training and fully restore the academic activities. although interns felt more secure concerning competencies to take care of covid- patients, many interns probably needed psychological support. in fact, more interns agreed to the statement s ("i feel stressed in the hospital at the moment", difference . % compared to first year medical students) and less interns agreed with statement s ("i have access to psychological support", difference . %). in addition, many students were afraid of contaminating themselves (statement s , percentages of agreement from . to . %) and considered that their emotional state affected learning (statement s , percentages of agreement from . to . %). to perform the multivariate analysis, we decided to divide the medical students into three groups, concerning their responses about participation of medical students in the covid-. cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint pandemic (no participation, only interns participate or all students participate, table ), to better understand the factors that were most important in the student's decision to participate. since we observed many differences between the answers of students in the first years of medical program and interns, we performed two analysis, one including all medical students and the second including only interns (tables and ). interestingly, we did not observe important differences when we compared the two analysis, suggesting that individual factors are more important than professional identity developed during medical course. maybe the decision to be a volunteer in a health emergency such as covid- pandemic is more linked to an emotional or attitudinal decision than beliefs of self-efficacy or performance. the statements that had greater odds ratios when the groups of "all students should participate" and "only students in internship should participate" were compared to "no students should participate" group (reference, odds . ) were, for all students, statements s , s , s , s and s , and for interns only, statements s , s , s , s , s and s . we observed that sense of purpose or duty (moral values linked to medical profession) (s "it is the duty of the medical student to put himself at the service of the population in the pandemic") was the most important factor that influenced the desire to work in the pandemic, followed by the willingness to take risk (altruism) (s "i am willing to take risks by participating in practical in the context of pandemic") and the perception of good performance (self-efficacy) (s "i am able to participate in the care of patients who seek health care" and s "i feel able to communicate the diagnosis for covid- infection", . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint for all medical students and s , s "i can identify signs of severity in a patient" and s "i know how to guide for therapeutic measures") for interns. in addition to statements that suggest moral values, altruism and confidence in professional competence, in both analysis, statement s ("i will be a better health professional for having experienced the pandemic"), that is related to building of professional identity, had odds ratios above . in all comparisons. in other words, the desire to help was stronger among the students that believed that they should work in the pandemic than their interest in learning during this health emergency. allowing students to participate can reinforce important values, such as altruism, service in times of crisis, and solidarity with the profession, contributing to the building of professional identity. [ ] why people act or decide to serve others is important to better organize volunteerism in social and career. [ ] they concluded that self-determined motivation results in more volunteer's satisfaction. [ ] . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint in our study we observed that the student with higher sense of duty (s ) and altruism (s ) were more prone to engage in health care activities during covid- , and it could be interpreted as self-determined motivation or values function of motivation. we also observed that the self-perception of competence (s and s ) was the third factor influencing motivation and the fourth was the desire of learn with the experience of working in the pandemic. desire to learn demonstrated for student can be interpreted as understanding function by clary et al., and it is also a self-determined motivation defined by guntert et al. [ , ] the controlled motivation was not important in our data. we also evaluated factors that can influence the decision to not participate in the covid- health effort. we observed that predictors of do not be a volunteer were beliefs that all educational activities should be suspended (s and s ), fear of contamination (s "i am afraid of contaminating myself") and emotional factors (s "i feel stressed in the hospital at the moment"). the differences we observed between male and female medical students were smaller than the differences among students of different years of medical program ( table , univariate analysis). differences between males and females concerning the percentage of agreement with the statements of the questionnaire were greater than % in only statements (females vs males): s "i feel insecure regarding the future" ( . %), s "i feel able to communicate the diagnosis for covid- infection" (- . %), s "my emotional state during the pandemic affects my learning" ( . %), s "i am willing to take risks by participating in practical activities in the context of pandemic" (- . %) and . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint s "i am afraid of contaminating myself" ( . %). all these statements are related to emotional competencies. results from multinomial regressions performed with all medical students (table ) and with only interns ( table ) also showed that males were more prone to believe that only interns should participate (odds rations . and . , respectively, for all students and interns) and that all students should participate (odds ratios . and . , respectively). we argue that this difference is possibly due males' higher propensity to take risks in health/safety domain. [ ] also, females are more prone to develop anxiety and stress disorders, be more affected by human suffering and have worse perceptions about their own quality of life, health and skills. [ ] [ ] [ ] these factors can influence their intentions to act in pandemic. nonetheless, we cannot disregard gender bias as a possible limitation of our instrument. [ , ] covid- pandemic had a substantial impact on medical education across the world. [ , ] there is uncertainty and disagreement about the appropriate roles for medical students during this pandemic, and student participation in clinical care has varied across institutions and countries. [ , ] many medical schools forbid any patient interaction, others included medical students in patient care and others decided to graduate medical students early in order to make them frontline clinicians. the american association of medical colleges recommended that "unless there is a critical health care workforce need locally, we strongly suggest that medical students not be involved in any direct patient care activities". is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint medical students in a local or global health emergency. some educators chose the position to ensure student security and to avoid exposition, and this perspective can send a message (hidden curriculum) that students have a passive role without a significant social responsibility. on the other hand, medical educators that defend the inclusion of the medical student in health teams send to medical students a message that social responsibility is pivotal to professional identity. interestingly, in our study, . % of medical students agreed with statement s ("i will be a better health professional for having experienced the pandemic"). a shortage of health professionals has been reached even in some cities of developed countries. [ , ] this risk will be even greater as the pandemic is reaching developing countries in south america, asia and africa. the participation of medical students in the care of people with suspected or confirmed covid- increases personal risk of acquiring this disease. however, the risks of severe disease are probably lower than to retired clinician volunteers, more susceptible to complications of covid- owing to their age. [ ] since the personal risks cannot be eliminated, there is predominant agreement that involvement of medical students in the care of patients should be voluntary. [ , ] medical students that work as volunteers, must have appropriate training, do not undertake any activity beyond their level of competence, have continuous supervision and adequate personal protective equipment. [ , ] one implication of our study for medical education is that allowing students to participate in pandemic efforts reinforces important values, such as altruism, service in times of . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint crisis, and solidarity with the profession and disposition to serve society. [ ] it will probably influence the development of professional values and identity. our study showed that medical students who believe that they must participate in the fight against covid- pandemic are motivated by sense of purpose or duty, altruism, perception of good performance and values of professionalism more than their interest in learning. these results have implications in the developing of programs of volunteering and in the design of health force policies in the present pandemic and in future health emergencies. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint the authors declare no conflicts of interest related to this study. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . beliefs towards a participation of medical students in covid- pandemic healthcare s . it is the duty of the medical student to put himself at the service of the population in the pandemic beliefs against a participation of medical students in covid- pandemic healthcare s . distance learning must be implemented in the suspension of academic activities . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint supplemental table : crude odds ratios ( % confidence intervals) for the association between internship students' characteristics and perceptions and their view about the role of medical students during the covid- pandemic. only students in internship should participate all students should participate clinical characteristics of coronavirus disease in china medical student education in time of covid- guidance on medical students' clinical participation: effective immediately. association of american medical colleges medical schools council of united kingdom. statement of expectation. medical student volunteers in the nhs the role of medical students during the covid- pandemic understanding and assessing the motivations of volunteers: a functional approach the quality of volunteers' motives: integrating the functional approach and self-determination theory gender differences in risk assessment: why do women take fewer risks than men? in their own words: stressors facing medical students in the millennial generation gender differences in the perception of quality of life during internal medicine training: a qualitative and quantitative analysis assessing gender bias in key: cord- -cqk c ie authors: plachouri, kerasia‐maria; georgiou, sophia title: how well prepared are dermatologists redeployed to covid‐ wards? date: - - journal: int j dermatol doi: . /ijd. sha: doc_id: cord_uid: cqk c ie nan a serious challenge for the national health systems of numerous countries, such as italy or the united states (us). the massive patient influx has led to shortages not only in vital medical supplies but also in medical personnel, resulting in health authorities taking emergency initiatives so as to face the consequences of this public health crisis. among these initiatives is the recruitment of doctors of specialties other than internal medicine or anesthesiology and, in some cases, even of medical students. - such actions, although necessary and evident in critical times, can be a cause of concern for numerous physicians who are suddenly redeployed to covid- wards yet lack even the basic intensive care training. as far as the structure of the dermatologic medical specialist training is concerned, there are certainly differences among the different countries and even among residency programs in the same country: some residency programs include a general medicine common trunk of variable duration, while others are constituted solely of a full-time dermatology training. it is therefore a reasonable concern up to which extent a skin physician senior medical students in the covid- response: an opportunity to be proactive with virus surge, dermatologists and orthopedists are drafted for the e.r [www document training requirements and recommendation for the specialty of dermatology and venereology european standards of postgraduate medical specialist training medical dermatology: a view to the future taking responsibility for complex medical dermatology patient management funding source: none. doi: . /ijd. key: cord- -pltz viz authors: lieberman, joshua a; nester, theresa; emrich, brooke; staley, elizabeth m; bourassa, lori a; tsang, hamilton c title: coping with covid- : emerging medical student clinical pathology education in the pacific northwest in the face of a global pandemic date: - - journal: am j clin pathol doi: . /ajcp/aqaa sha: doc_id: cord_uid: pltz viz objectives: the first coronavirus disease (covid- ) case in the united states was reported in washington state. the pandemic caused drastic disruptions to medical institutions, including medical education. the department of laboratory medicine at the university of washington responded by rapidly implementing substantial changes to medical student clerkships. methods: in real time, we converted one ongoing case- and didactic-based course, labm , to remote learning. results: fifteen of scheduled sessions proceeded as planned, including two sessions for student presentations. two didactics were canceled as the functions of the teleconferencing platform were not sufficient to proceed. one grand rounds speaker canceled due to covid- precautions. elements of an immersive clinical laboratory clerkship, labm , were repurposed to accommodate medical students per class via remote learning, highlighting clinical laboratory activities that continue throughout the outbreak. a new remote clerkship, medsci c, was developed incorporating distance learning and guided small-group sessions. this coincided with parallel efforts to make resident and fellow service work, conferences, and didactics available remotely to comply with social distancing. conclusions: the changes in medical education described reflect the dynamic interplay of current events affecting the world of clinical pathology. throughout this, technology—while with some limitations—has provided the platform for innovative learning. on maintaining optimal patient care while protecting the well-being of the workforce and trainees. in march , as cases of covid- increased throughout the state, the som halted all clinical clerkships and advised students to return to their home states. a parallel gubernatorial proclamation required all classes and meetings at the university to be held remotely, prohibiting in-person classroom instruction. simultaneously, the us federal government declared the pandemic a national emergency. these developments required the rapid implementation of significant changes to medical education in clinical pathology at our institution. the department of laboratory medicine routinely offers two -week clerkships for medical students: laboratory medicine : "laboratory case studies for clinical diagnosis" (labm ) and laboratory medicine : "clinical laboratory testing-methods and interpretation" (labm ). labm is a casebased course offered once per academic year (ay) using lectures and guided discussion to teach senior medical students the efficient selection and rational interpretation of laboratory tests. labm pedagogy relies on classroom discussions, laboratory shadowing, and student presentations. enrollment is an average of students per ay. two days prior to the start of labm , the viral genome analysis from two regional patients diagnosed with covid- suggested weeks of cryptic transmission in the local community, a finding broadcast through social media and that set the tone for the session. labm is a -week, modular clinical laboratory experience offered throughout the ay in which one to three students participate at an introductory level in division-specific and interdisciplinary conferences, didactics, and a weekly review of calls handled by the laboratory medicine residents ("call rounds") and tour clinical laboratories at the university-based medical center and the general trauma hospital. division-specific activities include sign-out of coagulation and hemoglobinopathy testing, identification of alloantibodies, review of transfusion reactions, and other clinical consultation encountered in resident service work. in addition, three interdisciplinary conferences occur weekly between clinical microbiology faculty and infectious diseases clinical services ("plate rounds"). both courses are graded with a pass or fail format. the clerkships are not available simultaneously because each clerkship draws from the same pool of instructors. medical student instruction in laboratory medicine is distinct from other specialties in part because while critical patient care activities are performed, relatively little direct patient contact takes place. this feature greatly facilitates the transition to remote learning. however, being provider-facing means clinical laboratory activities are often less recognizable to nonpathologists as patient care. nonpathologists are often unaware of the regulatory environment in which clinical laboratories operate, a topic that has been a source of increased discussion recently in the context of developing diagnostic testing for sars-cov- . these features make medical education in laboratory medicine a valuable resource, especially during the covid- pandemic, which serves as a unique realtime case study highlighting multiple central aspects of laboratory medicine for learners at all levels. sars-cov- remained a key topic of discussion throughout the course. specific questions from or interests expressed by students prompted additional discussions and supplemental reading/journal articles related to both the new pathogen and vaccine development in other outbreak situations (ie, ebola, severe acute respiratory syndrome virus, zika virus). impromptu and extemporaneous discussions or modifications were made to existing scheduled lectures to incorporate sars-cov- biology, epidemiology, and laboratory responses. the rapidly changing som policies for medical students and employee health guidelines were also discussed with students. the zoom teleconferencing platform (zoom video communications) was used to conduct all remote learning. the professional version of the teleconferencing application was made available for university students, faculty, and staff on march , , including a health insurance portability and accountability act (hipaa)compliant version for protected health information (phi). support for the application was available through administrative staff and course directors. the new online-only course, medical science c: "clinical laboratory testing distance learning-methods and interpretation" (medsci c), relied on the cloud-based canvas learning management system (canvas gfx) for distribution of course materials and submission of assignments. the zoom "breakout room" feature was used for small-group discussions in medsci c. changes to course structures made due to covid- are summarized in ❚table ❚. briefly, the second week of labm was conducted online. scheduled sessions of labm (march to june ) were canceled by the uw som along with all in-person medical student rotations. labm was replaced with medsci c, which provided most regularly scheduled labm curricula in an online format. in addition, we identified previously recorded and upcoming lectures that could be recorded, as well as the clinical services with remote-access capabilities for trainees, and created supplemental activities not typically part of labm , such as guided discussions, self-study resources, and reflective writing assignments. after assessing capacity, we planned to accommodate up to students. to maintain low trainee/faculty ratios, we also planned twice-weekly small-group discussions facilitated by at least one faculty plus a second faculty or senior fellow. no changes were made to an additional clerkship with rare enrollment, labm , which covered individualized independent study or research projects with a faculty mentor. no students were enrolled at the time, and this course likely would have been canceled under som policy. no changes were made to previously planned labm feedback collection through an anonymous survey and discussion between course directors and students. for medsci c, student feedback was gathered through ( ) a final wrap-up session with the whole class, ( ) an assigned reflection piece, and ( ) a course survey. the final wrap-up session invited students to provide verbal feedback and closing remarks. the reflection piece, due at the end of the course, was a one-to two-page reply to the following prompt: "describe three key aspects of lab medicine you've learned and how they apply to the covid- pandemic." the anonymous course survey contained quantitative and freetext elements for students to give feedback on small groups, lectures, and the overall course (supplemental table ; all supplemental materials can be found at american journal of clinical pathology online). survey questions were designed to assess aspects of the course such as interactivity, clinical casework integration, achievement of course objectives, and overall impression. students graded the course on a scale of to , with indicating the strongest level of agreement or most positive assessment. when the centers for disease control and prevention began issuing guidance to curb the spread of covid- , students were enrolled in course-based learning via labm ❚table ❚. adjustments to labm (table ) were implemented in both week and week , with significant structural changes in week ❚table ❚. the most significant alteration implemented in week was the real-time incorporation of the covid- pandemic as an overarching theme or case study to highlight the role of laboratory medicine. course directors led either impromptu or rapidly prepared discussions focused on multiple aspects of the growing pandemic. these covered a diverse array of topics, including clinical features of covid- , understanding outbreak dynamics through representative graphs, specific infection control precautions in clinical laboratories, molecular epidemiology from viral genome sequence data, and sharing emerging data and reports. other discussions covered sars-cov- test development and regulatory requirements for clinical laboratories. these discussions were facilitated by the start of clinical testing for sars-cov- by the clinical virology laboratory on the first day of labm . one clinicalhematopathology conference was canceled. during week , the faculty and administrative staff responsible for the course anticipated and planned to convert to remote-only instruction using the university teleconferencing platform. in-person instruction was suspended at the midpoint of the course in concordance with university official policy. week of labm was conducted entirely online. two of the scheduled presentations were canceled as they were formatted to be given as "chalk talks." the "whiteboard" feature of the teleconferencing platform did not meet the needs of the two presenters, and the lectures could not be rewritten as slide-based presentations in the time available. in addition, one grand rounds presentation was canceled by the invited speaker, an expert in tropical medicine, who declined to travel given the risks of sars-cov- transmission. all other pedagogic activities proceeded, including the student presentations, which were conducted in two sessions at the end of the course. the success of these changes informed modifications to the upcoming labm course. before the next section of labm began, the som canceled all upcoming clerkships to allow a window of opportunity for the development and implementation of alternate educational plans. the course directors and clerkship administrator for labm collaborated to develop a new course, medsci c, based on labm activities but conducted as remote learning. many activities in which labm students participated were concurrently being transitioned online to allow residents and fellows to work remotely. these included clinical microbiology-infectious diseases plate rounds, resident didactics, call rounds, and coagulation/hemoglobinopathy testing sign-out. the course directors effectively leveraged these parallel changes in clinical activity and resident education to provide medical students remote access to these activities, which had been components of labm ❚table ❚. the online format allowed a greater capacity of up to students with the first course scheduled to begin late april. small-group sessions and flipped classroom teaching sessions were incorporated. the new course, medsci c, provided third-and fourth-year medical students an opportunity to develop their diagnostic and patient management skills through directed distance learning in the hospital-based clinical laboratory. fourteen students completed each session of the new course, medsci c. in the first session, five of thirty-four students were registered for labm , but nine were dropped from the rotation due to covid- . students were located in states outside of washington (one in alaska, two in idaho, one in iowa, and one in wyoming) and four of students in the second session (one in alaska, one in idaho, two in wyoming). three additional students in the second session were more than a -hour drive from seattle. as a required assignment, all students completed the reflection piece. students most frequently commented on phases of testing (n = ), test utilization (n = ), regulatory agencies (n = ), and the experience of teaching reverse lectures (n = ). all students completed the course evaluation survey in the first session, and of students completed the course evaluation in the second session. average rating of the course overall for each session was . and . out of ❚table ❚, corresponding to good to excellent (supplemental table ). the response to "this course helped me appreciate the significance of the subject matter" was consistently in the top two highest scores ( . of ). for both sessions, the lowest average score to a targeted question was in response to whether there was adequate protected study time (table ). in response to whether the class length was appropriate for the subject matter, respondents answered it was "just right," five "too short," and four "too long." critical remarks about the course most frequently included teleconferencing fatigue (n = ) and the density of complex subject matter in sign-out and conferences (n = ), with the majority of these comments directed toward the first session. only free text or verbal feedback on the adaptation to labm was available. during the covid- pandemic, the clinical laboratory response has been dynamic. as new developments have arisen on a global and national scale, these disruptions to the community at large have greatly affected academic practice. , , other groups in dermatology, surgery, , nursing, and anatomic pathology , have reported on rapid and drastic changes in medical education and ways to proceed. in laboratory medicine, most divisions-except for therapeutic apheresis-conduct clinical service work without direct patient contact. in compliance with covid- distancing measures, laboratory medicine service work, sign-out, and didactics have shifted to a predominately remote-access paradigm for trainees and faculty alike. this represents a unique opportunity for medical students to participate in similar activities with additional guidance to interpret the clinical experience. therefore, the course structure involved elements of direct sign-out observation, in addition to both guided and independent study. additional activities involve small-group sessions, journal article reading, and live or prerecorded didactics. the lack of a vaccine against sars-cov- has placed diagnostic tests for the virus at the leading edge of the pandemic response. the result has been enormous medical and popular interest in laboratory diagnostics with the recognition that issues pertaining to the laboratory have vital repercussions for society. this was apparent from the student response during labm . we have leveraged this interest by creating a remote course, medsci c, so future clinicians have a better understanding of the laboratory's role. as in the pre-covid rotation, the goal was to provide exposure to multiple laboratories and the breadth of testing performed at our institution. special focus included how the clinical laboratories have mobilized in response to an urgent pandemic and whether this response is generalizable or unique to this institution. more quotidian goals included introducing students to common activities of the laboratory; exploring concepts of preclinical variables, including labeling/patient identification and patient safety in the clinical laboratory; and encouraging consultation with laboratorian/pathologist colleagues in future clinical practice. the change of the course from a two-to four-student rotation conducted in person to a potentially -student rotation conducted entirely online necessitated changes in structure to preserve interactivity, engagement, and participation in sign-out. to facilitate discussions, we structured portions of the clerkship as small groups of up to students co-led by laboratory medicine faculty and fellows who meet twice a week for an hour. the first two sessions consisted of guided discussion and question-and-answer sessions, while the last two sessions consist of flipped classroom teaching sessions moderated by small-group instructors ( table ). the benefits of small-group and reverse teaching methods in the classroom in promoting interaction and collaboration have been reported on previously, [ ] [ ] [ ] and students commented positively on their experience in evaluations. for the first spring quarter that this clerkship was offered, students rotated through the clerkship. this brings the total students rotating through uw laboratory medicine clerkships in ay to students-roughly double the number of students who normally would have been exposed to laboratory medicine ( table ). together with the distance learning clerkship offered by our anatomic pathology colleagues, pathology accounted for two of courses offered to third-and fourth-year medical students at this institution during covid- . the changes in the clerkship increased the visibility of and access to laboratory medicine education, including nine ( %) medical students who were out of state. however, serving students in different regions revealed two unanticipated challenges: accommodating a -hour span of time zones and coping with limited or disrupted internet access, particularly in rural areas. to meet these needs, we minimized activities at the extremes of the workday and encouraged early submission of student presentations when weather threatened to disrupt infrastructure, with the option to have students narrate their presentations by phone. additional trade-offs between traditional and online format are presented in table . technology such as teleconferencing, didactic recording, and learning management software was already partially used in the som, but the global pandemic quickly forced universal adoption of these tools to facilitate education in this period of social distancing. , , an unprecedented amount of online resources, media, and readings have been made available through both online commercial modules purchased by the university and free online learning materials. , , , stored content has the main advantage of flexibility-students and trainees can access high-quality content at any time-but lacks the immersion of a real case and an experienced mentor to navigate the clinical thinking around each case. hence, a curriculum that would bring students into the environment of clinical pathology, combined with small-group activities to navigate cases and increase learning engagement, was used. we identified two additional challenges unique to the remote format: teleconferencing fatigue and hipaa compliance. students in the first session reported teleconferencing fatigue on days with back-to-back conferences. this was partially mitigated in the second session by increasing the number of breaks between live sessions and reliance on several previously recorded lectures that were viewable at any time. concerns over the security of teleconferencing applications are not unique to this setting, but because teleconferencing was used extensively for didactics and sign-out of clinical cases that could involve sensitive phi, hipaa compliance was critical. this required that teleconferencing applications could ( ) ensure the confidentiality of electronic phi, ( ) protect against reasonably anticipated threats to such information, ( ) protect against reasonably anticipated nonpermitted uses or disclosures of such information, and ( ) ensure compliance by its workforce. , we relied on the hipaa-compliant accounts made available by our institution, required students include their role in their screen name (eg, "medical student," "ms- "), and encouraged teleconference hosts of phi-sensitive meetings to require a passcode for access as recommended. our efforts reflect one of several ways in which medical educators have adapted to covid- . on the basis of this experience and the positive student feedback (table ), we will continue to offer a remote learning course for up to medical students per session-including students in quarantine, those at wwami/rural sites, and visiting students-for the rest of calendar year . even though our som is returning to in-person clerkships with some limitations in the summer and fall of , this remote learning approach has the benefits of providing in-depth instruction in laboratory medicine, exposing students to the critical role of the clinical laboratory in response to emerging infections, and affords flexibility for educators to respond to the continuing pandemic. we note that other solutions have been employed for medical and nursing students. these have ranged from graduating senior medical students early , to training medical students to perform critical public health activities, particularly contact tracing, conducting telehealth visits, and staffing covid- call centers. [ ] [ ] [ ] we encourage such innovative solutions and believe that our approach to rapidly adapt to distance learning dovetails with engaging medical students in a variety of alternative learning activities. the changes in medical education afford a new opportunity for medical student exposure to pathology and laboratory medicine. as the united states faces a prolonged first wave of covid- infections, such changes in medical education appear to reflect a new normal and may require further evolution. corresponding author: hamilton c. tsang, md; hamiltn@ uw.edu. first case of novel coronavirus in the united states novel coronavirus outbreak (covid- ) covid- united states cases by county prepare to adapt: blood supply and transfusion support during the first weeks of the novel coronavirus (covid- ) pandemic affecting washington state three uw students in seattle screened for novel coronavirus medical student education in the time of covid- proclamation by the governor amending proclamations proclamation on declaring a national emergency concerning the novel coronavirus disease (covid- ) outbreak. white house proclamations the team at the @seattleflustudy have sequenced the genome the #covid community case reported yesterday from snohomish county sars-cov- testing comparative performance of sars-cov- detection assays using seven different primer/probe sets and one assay kit moving classes online and concluding the winter quarter covid- and medical education strange days medical education and care in dermatology during the sars-cov pandemia: challenges and chances the effects of covid- on academic activities and surgical education in italy residency and fellowship program accreditation: effects of the novel coronavirus (covid- ) pandemic [published blended learning via distance in pre-registration nursing education: a scoping review implementing a structured digitalbased online pathology curriculum for trainees at the time of covid- leveraging technology for remote learning in the era of covid- and social distancing: tips and resources for pathology educators and trainees self-regulated learning: the effect on medical student learning outcomes in a flipped classroom environment analysis of satisfaction and academic achievement of medical students in a flipped class informatics training for pathology practice and research in the digital era daily medical education for confined students during covid- pandemic: a simple videoconference solution blood bank guy transfusion medicine questions the coronavirus pandemic made zoom the most popular app, and that came with consequences voip for telerehabilitation: a risk analysis for privacy, security, and hipaa compliance covid- : states call on early medical school grads to bolster workforce itching to get back in": medical students graduate early to join the fight. association of american medical colleges medical students: how to keep learning as covid- volunteers alberta medical students help with covid- contact tracing. global news uw nursing students join frontline efforts to battle covid- , meet public health needs key: cord- -z x stl authors: wilkening, dean a. title: combatting bioterrorism date: - - journal: encyclopedia of violence, peace, & conflict doi: . /b - - . - sha: doc_id: cord_uid: z x stl concern that a terrorist group might attack civilian populations or agriculture by releasing deadly pathogens has grown in the past decade. failed attempts by the japanese cult aum shinrikyo to release botulinum toxin and anthrax in tokyo on several occasions in the early s, and the anthrax letter attacks in the united states seem to confirm these fears. however, there were only five fatalities in the us case and none in the japanese case. the question naturally arises: how serious is this threat and, if it is serious, what strategy should states take to combat it? this article draws on us experience to outline a strategy for combating bioterrorism that is general enough to apply to most states, especially ones with well developed public health and medical infrastructures. concern that a terrorist group might attack civilian populations or agriculture by releasing deadly pathogens has grown in the past decade. failed attempts by the japanese cult aum shinrikyo to release botulinum toxin and anthrax in tokyo on several occasions in the early s, and the anthrax letter attacks in the united states seem to confirm these fears. however, there were only five fatalities in the us case and none in the japanese case. the question naturally arises: how serious is this threat and, if it is serious, what strategy should states take to combat it? this article draws on us experience to outline a strategy for combating bioterrorism that is general enough to apply to most states, especially ones with well developed public health and medical infrastructures. the nature of the threat historically, attacks using biological agents are rare. this is not surprising given the relative ineffectiveness of biological weapons as a military weapon due to the difficulty of infecting opposing forces, the availability of protective clothing (a simple mask will do in most cases), prompt medical treatment for troops, and the risk that the attacker's troops may also become infected. the centuries-old revulsion to killing people with poison or disease codified in the hague convention, the geneva protocol, and the biological and toxin weapons convention (btwc) is perhaps the best explanation for why we have not witnessed more deaths due to biological attack. norms have a powerful, albeit imperfect, influence over human behavior. finally, with respect to terrorist use, the traditional view has been that ''terrorists want lots of people watching, not lots of people dead.'' to the extent this is true, terrorists have little incentive to attack civilians indiscriminately because they would loose political support within their community and the attack would likely provoke a draconian response by the state to eliminate the group. the question is whether this trend will continue. there is reason for concern regarding bioterrorism because the knowledge, materials, and equipment to make biological weapons are spreading worldwide; the incentives for states or terrorists to acquire and use such weapons may be increasing; and civilians, as well as agricultural sectors, remain quite vulnerable to such attacks. the knowledge, materials, and equipment to manufacture biological weapons are spreading due to advances in biomedical technology, the dual-use character of this technology, the global nature of the biotechnology and pharmaceutical industries, and the pervasive access to knowledge through rapid global information sharing. unlike nuclear weapons, where - kg of fissile material is required to build a rudimentary fission bomb, no such barrier exists for biological weapons. in fact, biological weapon proliferation is governed more by the spread of knowledge than the spread of material and equipment. traditional biological agents can be found in the environment or in numerous unprotected strain collections around the world and large batches of bacteria or virus can be grown in simple fermenters. former state biological programs represent another potential source of materials, equipment and, especially, knowledge. the remnants of the former soviet biological weapon program, estimated by some accounts to have once employed over scientists and technicians, represents an avenue by which states or terrorists might acquire biological weapons without the painstaking research and development required to create them ab initio. president yeltsin declared in that the former soviet biological weapon program had been dismantled and that biopreparat, the civilian biomedical research organization that conducted much of the biological weapons research, would be converted solely to peaceful pursuits. due to a lack of transparency, concerns remain that a covert biological weapons program may still exist in russia. cuba, iran, north korea, and syria are also believed by the us government to have biological weapon programs, at various levels of development, and south africa and iraq formerly had programs from which materials or expertise could leak. in addition, numerous countries have biological weapon defense programs, which produce small quantities of pathogens for peaceful purposes (e.g., testing prophylaxis efficacy), which is allowed under the btwc, from which knowledge or materials could also leak. biological weapons of varying degrees of sophistication clearly are within a state's means to acquire covertly. the dual-use nature of the equipment and supplies make biological weapon programs easy to hide under the guise of legitimate biomedical activities. only small quantities of pathogens are required for seed stocks, and biological agents emit no detectable signal, making them virtually impossible to detect remotely. the fact that biological weapon facilities can be small and have no distinct physical features makes their identification difficult even with intrusive on-site inspections, as the un special commission charged with locating and destroying iraq's weapons of mass destruction discovered after the iraq war. it took the commission years to locate most of iraq's biological weapon facilities and then only after kamel hussein divulged the scope of the secret iraqi program after defecting to jordan in . finally, legitimate peaceful activities such as vaccine and biopesticide production can be converted to biological weapon production within weeks to months. it is less clear whether terrorists can acquire effective biological weapons without state support. terrorists operating rudimentary laboratories face challenges obtaining lethal pathogen strains, extending pathogen shelf life and, in particular, mastering the 'weaponization' hurdles -agent drying, stabilization, and aerosolization (i.e., creating a respirable aerosol of viable agent less than mm in diameter so it does not settle out of the atmosphere close to the release point and so it can penetrate the alveolar region of the lungs where pathogens are most infectious). wet pathogen slurries are relatively easy to produce but difficult to disseminate in a -mm aerosol. dry powders can be ground to less than mm prior to dispersal (although clumping and electrostatic charge can be a problem), but dry agent is more difficult to produce and handle in a safe manner. thus, each path has its hurdles. most terrorists lack the practical knowledge required to circumvent these hurdles, even if they have trained microbiologists in their ranks, unless they receive outside help. (aum shinrikyo failed to kill anyone with anthrax because they used a nonlethal vaccine (stern) strain in and attempts to aerosolize the spores failed. this led the cult to carry out its more infamous sarin gas attack on the tokyo subway in .) of course, terrorists may not strive for highly efficient weapons. causing panic, if not terror, is possible even with a rudimentary biological weapon. not only are the means for acquiring biological weapons spreading but the incentives to acquire, and possibly use, them are increasing as well. the united states emerged from the cold war as the world's unrivaled conventional military power, while the collapse of the former soviet union left its allies to fend for themselves. consequently, opponents of the united states must search for 'asymmetric' means -including, possibly, biological weapons -to counter us military might. the suicide bombings in iraq and afghanistan are a current example of this approach. terrorists' incentives to use such weapons may be changing as well. the september attack in the united states and subsequent indiscriminant attacks in bali, madrid, beslan, and london suggest that terrorists may wish to inflict mass casualties. biological attacks can also devastate sectors of the us or world economy -an attractive goal to some terrorists. still, some constraints may exist. for example, terrorists may be reluctant to use contagious agents because the subsequent contagion might spread to their home country or social group, which may have less access to public health. they may also eschew biological attacks because the operation is more likely to fail, preferring conventional explosives instead. to divinely inspired perpetrators, failure can be a deterrent because it suggests that god does not support their actions. the vulnerability of civilian populations and agriculture may encourage bioterrorism. while modern military forces are relatively invulnerable to biological attack, civilians are quite vulnerable because they do not have protective clothing, would not know when to put it on if they had it, and they do not routinely receive prophylaxis against common biological warfare agents. the agricultural sector in most countries is also vulnerable because farms, animal pens, animal feed, and even finished agricultural products (e.g., milk) typically are not very secure against malevolent actors. the sudden appearance of diseases such as foot and mouth disease, bovine spongiform encephalitis, wheat rust, or similar plant or animal diseases can shut down an agricultural sector very quickly, preventing exports if not domestic consumption, as demonstrated by natural outbreaks of these diseases in the past. the bioterrorism threat is complex and diverse. biological weapons may be toxins or living pathogens. they may target humans, livestock, or crops and, hence, be aimed at mass murder or economic impact. pathogens may be lethal or nonlethal, contagious or noncontagious, and they may infect the host via contact with openings in the skin, animal or insect vectors, ingestion of contaminated food or water, or inhalation, giving rise to a wide range of delivery mechanisms and attack outcomes that vary by many orders of magnitude in terms of their consequences. the current threat largely involves naturally occurring pathogens and toxins. in many ways, biological attacks are similar to the scourge of disease that has wrought havoc on human, animal, and plant populations for millennia, the main difference being that the consequences are greatly compressed in time if not scope. in the future, genetically altered or synthetic pathogens may be possible because the science of genetic manipulation and dna synthesis is evolving rapidly. this diversity of threats makes it difficult to comprehend bioterrorism as a singular phenomenon requiring a singular strategy. the complexity of the bioterrorism threat suggests a multifaceted approach. to simplify the remaining discussion, this article focuses on the elements of a strategy for combating bioterrorism aimed at humans, especially airborne releases because they have the potential for creating the greatest number of casualties. the strategy for protecting the agricultural sector will have similar elements but requires a separate analysis. the strategy for coping with any proliferation problem involves four complementary elements: diplomacy, deterrence, preemption, and defense. diplomatic initiatives may help prevent the spread of proscribed weapons, thereby eliminating the problem at its source. if weapons proliferate, deterrence may dissuade their use. if deterrence is about to fail, preemptive attacks may destroy the weapons before they can launch and, if preemption is not a viable option, 'active' defenses may interdict weapons before they arrive and 'passive' defenses may protect people from their effects after detonation. this framework applies to any proliferation problem -nuclear, biological, chemical, or ballistic missile. all four elements are important, with different emphasis depending on the nature of the proscribed weapon. for example, the cold war nuclear threat was principally addressed by deterrence, complemented by diplomatic (i.e., arms control) efforts to circumscribe the threat and limited efforts at preemption and defense. for biological weapons, the main emphasis should be on defense, complemented by diplomatic efforts, preemption, and deterrence. diplomatic efforts to prevent the proliferation of biological weapons include the geneva protocol, which bans the first use of biological (and chemical) weapons; the btwc, which bans the development, production, stockpiling, acquisition, and transfer of biological agents ''of types and in quantities that have no justification for prophylactic, protective or other peaceful purposes'' and their means of delivery; the australia group, which coordinates the export control policies relating to chemical and biological weapons materials and equipment among the or so member states; and un security council resolution , which calls upon states to ''refrain from providing any form of support to non-state actors that attempt to develop, acquire, manufacture, possess, transport, transfer, or use nuclear, chemical or biological weapons and their means of delivery,'' to adopt and enforce domestic legislation to prevent non-state actors from engaging in these activities, and to establish domestic controls to prevent such proliferation. the fundamental problem with monitoring biological weapon proliferation is that biological agents, material and equipment are quintessential dual use items, making it difficult to separate benevolent from malevolent applications, and covert biological weapon facilities have few unique signatures that would allow for identification without intrusive inspections. even with intrusive inspections, it can be difficult to identify covert facilities. for this reason, the btwc did not include any monitoring or verification provisions, common to most arms control treaties, because member states could not agree on inspection provisions. this difficulty is compounded by the fact that the btwc allows states to acquire small quantities of pathogens for prophylactic and other peaceful purposes. the difference between a small pathogen sample used to test antibiotics or vaccines and one used as seed stock in a biological weapon production facility is solely one of intent. attempts during the s to strengthen the btwc with an inspection protocol ended in when the bush administration withdrew its support; arguing, with some justification, that covert biological weapon programs could easily be hidden even if this protocol entered into force; that legitimate facilities could be converted to produce biological weapons in a short period of time (weeks to months); and that any inspection regime that was intrusive enough to detect covert programs could compromise proprietary information crucial to commercial companies. similarly, the australia group faces an enormous challenge distinguishing between legitimate and illegitimate end uses for exported biological material and equipment. at best, export controls may impede large-scale acquisition or activities, but it probably cannot thwart small-scale operations. future diplomatic efforts should extend beyond traditional arms control treaties and export control regimes. for example, the us-russian cooperative threat reduction program, which has focused principally on securing nuclear weapons, materials, and scientists in the former soviet union, should be expanded to cover a wider range of former soviet biological weapon facilities and activities. material protection, accounting, and control would help prevent the spread of pathogens, equipment, and materials; and funding to employ key former soviet personnel on peaceful biomedical research would discourage them from selling their expertise to foreign bidders. the latter is particularly important because biological weapon acquisition is largely an issue of acquiring the tacit knowledge, as opposed to the equipment and materials, to build such weapons. an international code of conduct for biomedical researchers could help reinforce the norm against biological weapons development. the promulgation of international standards and shared best practices for safety and security at pathogen collections or biocontainment facilities that work with deadly pathogens, for example, could reduce the risks associated with accidents or diversion and would help promote interaction among biomedical practitioners engaged in potentially dangerous research. international association and collaboration among biologists, medical professionals, and public health practitioners would help address emerging infectious diseases and the transparency produced through such collaborations would have, as a collateral benefit, the potential to detect covert activities. the soviet biological weapons program would have been more difficult to conceal had there been international collaboration with soviet biologists, medical and public health practitioners during the cold war. detecting covert biological weapon activities increasingly will be a matter of detecting the people involved, not the weapons. improving international disease surveillance, specifically improving public health laboratories in less-developed countries and their connectivity to the international health community, is a worthy objective that also has security benefits. if an attack with a contagious agent occurs in a foreign country, the sooner this is detected, the better governments will be able to limit its entry into their state by monitoring borders and ports of entry, and restricting travel. improved international disease surveillance might also detect the presence of covert biological weapon programs in the event of an accident that infects the local population. the accidental anthrax release in sverdlovsk, russia would have been readily detected if such a surveillance system were in place at that time. of course, hiding covert programs, as well as avoiding political embarrassment from natural disease outbreaks, is precisely why some states will resist disease surveillance that is not under their control. still, efforts by the world health organization (who) to implement the global outbreak alert and response network are well placed and the recently revised who international health regulations, which require reporting of any disease of international public health concern within hours, when fully implemented, will have public health and security benefits for all nations. these efforts need sustained diplomatic and financial backing. ultimately governments must recognize that the spread of disease does not respect international boundaries. hence, public health is not solely a sovereign issue, especially in an age of rapid international travel and commodity transport. however, diplomacy alone ultimately cannot prevent the spread of biological weapons. revelations about the size and scope of the covert soviet biological weapons program during the cold war, much of which existed under the guise of legitimate biomedical research, demonstrates the limited utility of diplomatic means. diplomacy's greatest benefit may be to reinforce the widely held norm against the use of disease as a weapon of war or terror. reinforcing this norm is important not because one hopes to convince malevolent actors to abide by the norms to which the status quo powers adhere, but rather for deterrence; it convinces malevolent actors of the sincerity with which the united states and other likeminded states abhor biological weapons and, hence, the resolve with which they will respond if attacked. despite the best diplomatic efforts, biological weapons may still spread. the question then becomes whether states can dissuade other states or terrorists from using them. the efficacy of deterrence against 'rogue' states or terrorists has been questioned because their leaders are believed to be irrational and, hence, cannot be dissuaded by retaliatory threats. this argument distorts the character of regional leaders. authoritarian leaders may be ruthless, unsavory characters with little regard for their civilian population; however, generally, they are not suicidal. similarly, terrorist groups often have clear strategic and tactical goals, with an infrastructure that supports their operations logistically and financially. while those who carry out acts of terror may be suicidal, the top-and mid-level leadership frequently is not. therefore, in principle, one may be able to identify targets against which retribution will dissuade some terrorist groups from acting. in practice, this often is more difficult, especially if one wants to avoid civilian casualties. effective deterrence depends upon the ability to identify the perpetrator of an attack. therefore, attribution, especially against terrorists or state-sponsored terrorists, is crucial for effective deterrence. by holding states responsible for terrorists who acquire material from them, states will have greater incentive to secure any biological agents they might possess and they will be more reluctant to provide them to terrorist groups with whom they sympathize. unauthorized acquisition is a problem and will be the obvious cover for any state that contributes to a terrorist's biological weapon capability, un security council resolution notwithstanding. deterrence relies upon clearly communicated, credible retaliatory threats whose consequences outweigh any benefits the attacker might hope to gain. credibility, in turn, depends on a state's capability and resolve to retaliate. public commitment to the geneva protocol, the bwtc, and un resolution helps convey resolve, in conjunction with statements by top government officials. however, states often lack the capability to respond, in part, because they lack biological weapons themselves with which to make tit for tat retaliatory threats. nuclear threats, for those states that can make them, are less credible because they require nuclear first use -a difficult political/strategic decision under any circumstances. the united states may be alone in having sufficient conventional military power with which to threaten retaliation, for example, to topple the regime that aided or carried out the attack. although nuclear response options cannot be ruled out, the united states clearly should emphasize conventional military capabilities to deter biological attack. such threats may help deter biological attacks by states, but they are bound to be less effective than deterrence of nuclear attacks during the cold war. deterring terrorists may have common elements with deterring states, assuming one can locate enough of the top leadership and infrastructure that supports their operations. however, terrorists have less to lose so the cost-benefit calculus is more difficult to shift in the deterring state's favor. perhaps the best way to dissuade terrorists from attacking is to deny the success of the attack. terrorists often are risk averse when it comes to the success of their mission, preferring tactics, techniques, and targets that assure success. dissuading an opponent by convincing him that his chances for success are slim is referred to as 'deterrence by denial'. however, this terminology confounds deterrence with defense. true, defenses may divert an attack to less well defended targets or dissuade a terrorist group from attacking altogether because of the reduced chance of success. however, the objective of a defense is not to influence terrorist calculations, but to protect the defender regardless of their calculations. preemptive attacks, that is, thwarting an opponent's ability to strike first when the threat is imminent by destroying his weapons before they can be launched, is frequently practiced in conventional war and was considered by both the united states and the former soviet union with respect to nuclear war. preemption will play less of a role against biological attacks because it is impractical -biological facilities and weapons are easy to conceal and, even if located, they are not easy to destroy without the risk of collateral damage, whether this is from the radioactive fallout from a nuclear blast or the dispersal of pathogens due to their incomplete destruction in a conventional strike. against terrorist attacks, preemption certainly will be attempted if a state knows where biological weapons are located but, again, this is unlikely unless intelligence or law enforcement agencies get lucky. efforts to improve intelligence on suspect groups or individuals are useful; however, there are no technical fixes in the offing that will allow intelligence agencies to improve their ability to detect covert biological weapon programs in the future. better human intelligence is imperative. therefore, while states should be alert to the opportunity to preempt state or terrorist attacks, it is impractical to rely upon preemption for coping with biological attacks because of the demands it places on accurate, timely intelligence. active defense/interdiction 'active' defenses interdict weapons before they reach their targets. interdiction frequently is cited as a preferred strategy against terrorists. however, interdiction is difficult against covert biological delivery because pathogens have no signature that allows one to detect them in transit on a person, in luggage, or in any other container. moreover, biological agents can be released in a myriad of ways, complicating surveillance efforts. again, there are no clear fixes that will allow intelligence agencies to improve their ability to determine who, when, where, and how a biological attack might occur. therefore, interdiction programs like the proliferation security initiative -a us effort to create international agreements and partnerships with other countries to allow the united states and its allies to board airplanes or ships suspected of carrying weapons of mass destruction or their components -may have some deterrent role but without accurate and timely intelligence, it will likely be of limited effectiveness against biological threats, unlike chemical, nuclear, or ballistic missile threats where the cargo is easier to detect. examples of efforts to improve border and transportation security in the united states either focus on identifying potential terrorists (e.g., the united states visitor and immigrant status indicator technology (us-visit) program) or dangerous cargo (e.g., advance electronic cargo manifests, the container security initiative, and the customs-trade partnership against terrorism program). screening travelers at ports of entry is useful because it potentially detects malevolent actors, not their weapons. screening cargo is much less useful for biological threats because, again, biological agents emit no detectable signature. not surprisingly, most cargo screening efforts focus on detecting nuclear or radiological materials. traditional forms of defense such as air and ballistic missile defense will be of limited use against bioterrorism attacks. air defenses can be effective provided air defense networks are alerted to the attack, but covert air delivery using a commercial or private airplane will be very difficult to detect without prior intelligence. ballistic missile defenses are of limited use because terrorists are unlikely to have ballistic missiles at their disposal, except possibly very short-range missiles or rockets. against the latter, defenses such as patriot advanced capability- (pac- ) interceptors or the mobile tactical high energy laser (mthel) may have some utility, assuming they can prove their effectiveness on the test range. against biological weapon attacks by a state, ballistic missile defenses also will be of limited value because biological submunitions released early in flight, a technology the united states and the former soviet union mastered in the s, can easily overwhelm missile defenses. 'passive' defenses protect a population from the effects of weapons after they detonate. against biological weapons, passive defenses can be quite effective. inhalation is the most infectious method of exposure for biological agents. therefore, a simple mask can provide considerable protection if one knows when to don it. standard, inexpensive n or n masks filter out % or %, respectively, of the submicron particles from inhaled air, thereby substantially reducing the inhaled dose. their use, for example, would substantially reduce the scale of an epidemic if donned immediately after a contagious disease outbreak is detected, thus increasing the effectiveness of any medical response because it could focus on a smaller infected population. in principle, homes could be outfitted with high-efficiency particle (hepa) filters, although this would require substantial modifications to most home heating, ventilation, and air conditioning (hvac) systems and would require positive overpressure systems to prevent infiltration through cracks. however, hermetically sealed office buildings frequently have hepa filters and positive overpressure hvac systems, making it easier to 'harden' such buildings if they are likely targets of attack or if they perform critical functions in the midst of an emergency. despite the simplicity and relatively low cost associated with most physical protection schemes, they all suffer from the fact that, to be effective, protection must either be in place at all times (e.g., hermetically sealed office buildings) or individuals must know when to seek shelter or don masks. the latter relies upon adequate warning and the ready availability of shelters and masks, both of which currently are not available in most countries. during the iraq war, most israeli citizens carried gas masks which they donned each time their ballistic missile surveillance system warned of an incoming iraqi scud missile attack. however, most countries do not have such plans or provisions and, in any case, at best they work only in war and not against covert bioterror attacks in peacetime. preattack vaccination conceptually is the simplest approach to preventing disease from a biological attack. this is the approach taken for most infectious diseases of public health concern. for example, almost all children in the united states are vaccinated against polio, measles, mumps, rubella, pertusses, and varicella; and large segments of the population who may be at risk are vaccinated against pneumococcal infection, hepatitus a, hepatitus b, tetanus, and influenza. the reason this approach has not been widely adopted as a defense against bioterrorism is twofold. first, unlike mother nature, terrorists are strategic opponents. when it becomes known that a population has been vaccinated against specific pathogens, terrorists will choose an alternate pathogen or, if sufficiently sophisticated, they may design the pathogen to circumvent the vaccine. second, some vaccines have serious medical side effects in a very small percentage of cases. vaccinating the entire population prior to an attack could produce several hundred severe reactions, possibly including death, and hence is an option of which political leaders will be chary unless the threat of attack is imminent, which as noted above will be difficult to determine. postattack medical prophylaxis, on the other hand, does not suffer from this political liability and, hence, is the preferred strategy, assuming it can be effective. if, however, postattack medical intervention is not effective against some pathogen or if the threat of attack from a particular pathogen is a clear and present danger, then a case can be made for preattack vaccination against this particular agent, assuming the vaccine exists. otherwise, preattack vaccination is contraindicated until safer vaccines become available. all biological agents, with the exception of toxins, incubate in their hosts for a period of days to weeks, depending on the pathogen and the dose to which the individual is exposed. moreover, medical interventionantibiotics and vaccines in the case of bacterial agents, and antiviral drugs and vaccines in the case of viral agentscan be very effective if administered prior to the development of symptoms in the host, or shortly thereafter under some circumstances. therefore, a window exists inside of which medical intervention can be very effective at saving lives. this is the basis for the most important element of a biodefense strategy, namely, rapid postattack medical response. postattack medical response involves three elements: biological agent detection and identification, medical logistics to deliver supplies to the exposed population, and a prophylaxis campaign that can dispense the appropriate medications to the entire exposed population in a short period of time, including to a potentially large number of people who believe they have been exposed but are not. all three steps must occur before too many people become symptomatic if this strategy is to provide a high degree of protection. the incubation period for a given disease sets the timescale on which these activities must occur. the medical efficacy of postattack antibiotic treatment against inhalation anthrax, for example, is illustrated in figure , which plots the percentage of hypothetical victims that can be saved as a function of the time at which medical intervention begins. the airborne anthrax release upon which this calculation is based is one for which the exposed population is approximately people, of which would become infected in the absence of medical treatment. the medical intervention posited here consists of antibiotic distribution to % of the exposed population over a period of hours (possibly followed by vaccination), that antibiotic treatment prior to the onset of symptoms is % effective, and that postsymptomatic antibiotic treatment is approximately % effective if delivered within days of symptom onset, assuming intensive medical care is available of the sort provided to the victims of the fall us anthrax letter attacks (i.e., multidrug regimens and pleural fluid drainage). (different curves are required for different attack sizes and different diseases, and, in the case of contagious diseases, one must account for secondary transmission.) from figure , one observes that over % of this exposed population can be saved if treatment begins within days after exposure. the maximum medical efficacy asymptotes at % due to assumptions embedded in the model. clearly, the sooner one can provide prophylaxis to the exposed population, the better. as the detection and identification time is reduced, more time is available to implement the medical response. and, some functions can overlap in time; for example, the logistics associated with activating the strategic national stockpile (warehouses in the united states that currently store medical supplies for a massive infectious outbreak) and setting up and staffing the points of distribution (pods) where people will eventually queue up to receive treatment can occur simultaneously and immediately after an attack is detected but before the agent has been identified. this figure should be interpreted with care because it is based on optimistic assumptions, given current us preparedness, regarding the detection time, the speed with which medical logistics can deliver antibiotics to the pods within the affected area, the speed with which queues can be processed at these pods, and the effectiveness of the prophylaxis regime (e.g., certain medications may be contraindicated for some subpopulations such as children, pregnant women, and immunocompromised individuals and other victims may not comply with the entire prophylaxis regimen). but, figure does represent the level of protection that, in principle, can be achieved. moreover, a % effective medical response against an attack that hypothetically infects people, still leaves people infected, which in the case of inhalation anthrax implies close to deaths, a horrific outcome compared to any bioterrorism witnessed to date. this will likely be viewed as an unacceptable outcome. thus, the tendency will be to strive for medical effectiveness well above %. to achieve levels of protection above %, it will be very important to implement policies that reduce the percentage of people who do not receive prophylaxis (assumed to be % of the exposed population in figure ) or that do not adhere to the full antibiotic regimen over timeboth of which are important social, as opposed to technical, challenges for a prophylaxis campaign. finally, it is important to note that this strategy overlaps with efforts to improve public health. consequently, some of the cost for biodefense will have benefits even if an attack never occurs. given the low, albeit uncertain, likelihood of a biological attack, emphasizing those biodefense programs and activities that have substantial public health benefits is a prudent way to proceed, although some important biodefense programs will not meet this criterion (e.g., developing an improved smallpox vaccine). moreover, given that infectious diseases are the leading cause of death in many developing countries, a strategy for coping with bioterrorism that does not address the legitimate public health concerns of the developing world will gain little sympathy, support, or cooperation. contrariwise, the developed world stands to gain tremendous good will if it helps the governments of developing countries solve their public health problems -a commodity not irrelevant in the global struggle against terrorism. two issues are important with respect to detecting a bioterrorism event: low false alarm rate and speed. false alarms quickly erode confidence in any detection system, not to mention their economic costs, and consequently should be very infrequent (e.g., on the order of one per decade for a given facility or urban area being monitored). the false positive rate depends on the detection technology, the detection threshold (lower thresholds give more false positives), and background biological aerosol levels in the environment that cannot readily be discriminated from the agents one wishes to detect. as a rule, using two or more different detection technologies, with uncorrelated noise and background signals, reduces the false alarm rate considerably. these detection methods can be sequenced in time to reduce cost, with the first detector cueing the second; however, this increases the detection time. rapid agent detection and identification is important because medical treatment is most effective if delivered prior to a victim becoming symptomatic. prophylaxis efficacy drops rapidly after symptoms appear and intensive supportive care is required to ward off death, making medical intervention more burdensome and costly and, hence, less likely to reach as many people. real-time detection is not required, unless physical protectiondonning masks or entering protected buildings -is part of the defensive strategy. detection within hours is a reasonable goal for rapidly incubating diseases such as anthrax, thus leaving - days to conduct an effective medical response. for diseases that incubate more slowly (e.g., smallpox) or for contagious diseases where preventing secondary infections is an important part of preventing a widespread epidemic, slower detection speeds are acceptable. finally, detailed dna analysis and trace element detection is important for forensic evidence; however, this can be collected and analyzed within days or weeks after an attack. the us government has funded the development of a wide range of biological agent detectors. they fall into two categories: environmental sampling and detecting the host response to infection. environmental sampling involves collecting air, water, food, or swab samples and analyzing them for the presence of pathogens using antibody tests, or matching dna sequences to known pathogens. environmental sampling has the virtue that it can, in principle, be rapid (on the order of a fraction of a day, depending on how frequently samples are taken) and it can be used to identify the pathogen, though not necessarily whether the pathogen is virulent. the us biowatch program, which currently monitors the air in approximately american cities for a range of pathogens, is an example of this approach. if a pathogen plume passes one of the biowatch air sampling stations in sufficient concentration, it would be detected within approximately day. the disadvantage of environmental sampling is that a large number of sensors, or air collection stations, must be deployed to ensure that small releases are detected with high probability. if intelligence is available, mobile sensors can be deployed to the area of concern. however, without reliable intelligence, the system costs become prohibitive if one wishes to monitor the air for small releases in hundreds of cities all the time. finally, such a sensor network provides little benefit to public health because it cannot detect contagious diseases of public health concern, for example, severe acute respiratory syndrome (sars) or influenza, because the concentration of these microorganisms in the open environment is well below any detection threshold. methods to detect the human response to infection currently involve clinical diagnosis and syndromic surveillance, the data from which would be sent over a nationwide alerting network. clinical diagnosis relies upon symptomatic patients visiting a physician or hospital emergency room. not all symptomatic victims would seek medical care immediately because the early symptoms of diseases caused by biological agents are frequently similar to those for influenza -fever, nonspecific cough, congestion, etc. physician aids can improve differential diagnosis of the relatively uncommon diseases caused by biological warfare agents, thus reducing the time for detection and identification; however, one must still wait for the first few victims to present which may take on the order of - days. moreover, laboratory cultures commonly used to confirm the infectious agent take an additional - days, although antibody tests such as enzyme linked immunosorbent assays (elisa) can reduce this to a fraction of a day if the test is conducted immediately. therefore, clinical diagnosis currently cannot warn of an attack within hours. syndromic surveillance systems monitor clinical reports, pharmacy sales, school absentee rates, and other data to detect an above-normal rate of symptoms in a geographic area. these systems, for example, biosense in the united states, have detected unusual increases in local infection levels from natural outbreaks. however, they detect events only when the number of symptomatic cases rises above the background level which, by definition, is late for effective medial intervention in the event of a biological attack. hence, while syndromic surveillance systems may have public health benefits, they cannot provide sufficient warning (i.e., within hours) of a biological attack to implement a highly effective medical response. veterinary and wild animal disease surveillance may detect disease among animal and bird populations before they become apparent in the human population, as was the case with the west nile virus natural outbreak between and in the united states. however, monitoring animal disease outbreaks benefits public health more than defense against intentional attacks, where exposure of animal and human populations would be simultaneous, because the incubation period in animals and birds frequently is comparable to that in humans, implying that animal disease detection is unlikely to precede the detection of zoonotic diseases in humans. in any case, rapid veterinary and wild animal disease detection, and its integration with human disease surveillance systems, has not occurred in the united states. future research and development in the area of attack warning should emphasize improving the detection time for the host's response to infection, as opposed to environmental sampling, because the latter will be expensive and have very limited benefits for public health. automated laboratory testing, for example, using elisa techniques or dna chips, can reduce detection and identification times to a few hours (from the time samples enter the laboratory); however, clinical diagnosis still requires the presentation of symptomatic patients. presymptomatic diagnostic techniques that detect the early host immune response to infection, for example, mrna transcription of the genes involved in the host's immune response which begins within approximately hours of exposure, would be more useful, assuming further research demonstrates that gene expression patterns are reliable fingerprints for the presence of a given pathogen or small class of pathogens. such techniques could reduce the warning time to approximately day, assuming it is used routinely in hospitals and medical clinics, by detecting asymptomatic victims who visit for other reasons. presymptomatic diagnostic methods would have tremendous benefits for routine medical diagnosis of common infectious diseases and the resulting economies of scale will reduce their cost. the united states currently can probably detect and correctly identify a bioterrorism event within approximately days of the initial release by clinical diagnosis. note, however, that it took - days to diagnose cases from the anthrax letter attacks, including the time for blood culture confirmation. diagnosis information would then be conveyed to the us center for disease control (cdc) via the public health information network in a matter of hours from most major urban areas, although this network has not been fully implemented. in the future, the us national biosurveillance integration system is being designed to provide warning of disease outbreaks of natural or terrorist origin, integrating food, agriculture, public health (clinical diagnosis and syndromic surveillance), and environmental sampling data. the speed with which this system will be able to detect an outbreak will be constrained by the abovementioned limits associated with the different detection methodologies. again, real-time detection is not required. twenty-four hour warning should be sufficient to mount an effective medical response. since medical prophylaxis should begin within approximately hours of an atmospheric release (in the case of anthrax), a -hour detection capability would leave at least one day to implement medical logistics and to begin providing prophylaxis to a large number of exposed and worried citizens. providing prophylaxis to a large number of people requires the delivery of large quantities of medical supplies to the exposed population. the suggestion that people keep supplies of the necessary medications at home, thus obviating the need for rapid distribution, have been rejected because some people will take the medications inappropriately (e.g., antibiotics when they have the flu), they may take inappropriate doses, a large selection of medications would be needed to protect against all possible biological agents, and self-vaccination would not be possible. consequently, current us plans call for stockpiling the necessary medical supplies (antibiotics, antiviral drugs, vaccines, syringes, intravenous supplies, ventilators, etc.) in central warehouses referred to collectively as the strategic national stockpile, with the intent to rapidly dispense these supplies after an attack has been detected. palletized 'push packs' of medical supplies can be delivered to any local staging area in the united states by aircraft or truck within hours of a decision to deploy them. the greater challenge is to distribute these supplies from the local staging area to the pods where people queue up to receive medications. these pods could be local schools, fire stations, or shopping malls, but not hospitals. hospitals should remain free from congestion to handle acute cases of victims who have passed into the symptomatic disease phase. the united states has little hospital surge capacity due to managed healthcare. however, hospital surge capability is important only for those victims who need intensive care. the principle challenge for effective medical response is to provide prophylaxis prior to the appearance of a large number of symptomatic cases. if hospitals become overwhelmed with acute cases, surge capacity is not the answer but rather more rapid and effective presymptomatic prophylaxis. therefore, pod surge capacity is more important, in particular, augmenting the personnel capable of servicing queues to increase pod throughput. diagnostic techniques to triage noninfected individuals will greatly reduce the number of people requiring prophylaxis because the number of people concerned about exposure will exceed the actual number of people who become infected by a factor of to , if not more. the time it takes to transfer medical supplies from the local staging area to the pods depends on the time required to repackage supplies into smaller quantities and to transport them to individual pods, most likely via small trucks. efforts clearly should be made to minimize the extent to which repackaging is necessary. helicopter backup may be required if roads are congested with people fleeing an exposed area. depending on the size of the urban area, several tens to several hundred pods will be required to minimize the time required to treat the exposed population. local officials must identify suitable pod locations, transportation, and staff for each pod, and exercise these logistics plans so they go smoothly in an emergency. this is beginning to occur in the united states. the prophylaxis campaign requires medically trained personnel to triage individuals according to their medical status and prescribe the appropriate prophylaxis regimen. paperwork is required to track individuals and the mediations they receive, and to provide information about the medications and possible side effects. security personnel will be required to ensure order. if detection occurs within hours, and it takes hours to distribute supplies from the strategic national stockpile to local staging areas, hours to distribute supplies from there to the pods, and hours to set up the pods (this can occur concurrently with stockpile dispersal), then prophylaxis can begin within hours of a release. if urban pods can process people each per hour around the clock, then such a rapid medical response can, in principle, provide prophylaxis to approximately million people within days, thereby saving over % of a population exposed to anthrax according to figure . again, these numbers do not reflect current capability but rather the level of protection that is possible with sufficient effort. effective medical treatment depends on stockpiling the appropriate medications in sufficient quantity. while antibiotics have an efficacy of approximately % for healthy individuals, they may be contraindicated for certain subpopulations (e.g., children, pregnant women, and immunosuppressed individuals) . in the united states, the immunosuppressed population is growing due to cancer treatments, human immunodeficiency virus (hiv), organ transplants, and other medical interventions, which is cause for some concern if a highly effective medical response is desired for all significant subpopulations. vaccination is an effective defense against many infectious diseases. however, vaccines often take - weeks for primary seroconversion, and may require one or more booster shots thereafter to achieve full protection. consequently, they are generally less effective for postattack prophylaxis unless antibiotic or antiviral drugs are available to control the disease until vaccination takes effect. moreover, vaccines do not exist for some biological agents and they do not exist in sufficient quantity for others because they are not routinely stockpiled for diseases that are not current public health concerns. therefore, research and development should focus on new broad spectrum antibiotics, antiviral drugs, and safe, effective vaccines against known biological agents that can be administered to most segments of the population. medical research in these areas will also have important benefits for public health as new treatments are discovered for emerging infectious diseases. concern with antibiotic or antiviral resistant pathogens is best addressed by limiting the overuse of these drugs and by having multiple medications on hand that are effective against a given pathogen strain, again highlighting the importance of medical research and development. finally, genetically engineered pathogens that have enhanced effects, circumvent detection systems, or circumvent medical countermeasures may become more widespread in the future. however, increasing the virulence of pathogens through genetic manipulation is not trivial, notwithstanding the australian mousepox experiment. nor would terrorists need to go to this trouble since natural pathogens are terrifying enough. at the current time, bioterrorism countermeasures should focus on naturally occurring pathogens. however, biotechnology is in its infancy and powerful discoveries lie ahead. hence, any defensive policy must strike a balance between developing countermeasures to current versus possible future pathogens. research on new prophylactic drugs and vaccines should be carried out largely by private biotechnology and pharmaceutical companies because they have the resources and the expertise to create new drugs, with government sponsored financial incentives to encourage them to develop countermeasures that otherwise would be unprofitable. the us bioshield act of and follow-on bioshield ii legislation currently under congressional review illustrate the kinds of incentives that may be effective, for example, tax credits, patent extension, and liability limitations. debates have also surfaced about the wisdom of censorship in biomedical publications. the world has a lot more to gain from improved public health than it stands to lose from bioterrorism by allowing unfettered access to scientific advances in biology and medicine. thus, censorship or classification schemes to keep certain information from malevolent actors should be carefully scrutinized, with open access being the norm unless a clear and present danger exists. this is the best hope for having the medical countermeasures available if and when the need arises while at the same time providing benefits to public health. the us government has established the national advisory board for biosecurity, a group of biologists, physicians, and security experts from outside the government, to help maintain the balance between scientific openness and preventing bioterrorism. in addition to developing guidelines for research and publications in the life sciences, they have helped draft a code of conduct for life science professionals and fostered international cooperation to help define these issues. decontamination is required to prevent bioterrorism from becoming a threat to physical infrastructure by rendering buildings unusable for months or years because public officials cannot certify that they are safe for occupancy. an effective decontamination policy must determine safe public exposure levels, which depend on site use and individual susceptibility to infection. little data exists on environmental background levels for common pathogens. moreover, the scientific debate regarding the effects of low-level exposure to pathogens may be as contentious as the low-level radiation debate. therefore, the seemingly simple question of how clean a site must be to ensure public safety will, in fact, be difficult to answer with existing scientific data. answering this question should be the first priority for decontamination research. pathogen levels will have to be monitored for months to years after an event, which is both expensive and complicated by the fact that some detection techniques (e.g., dna sequence matching) do not distinguish living from dead pathogens. in addition, communicating the risks associated with residual contamination to the public in a credible way is vital to allay public anxiety and the economic consequences that flow from these fears. this will be a nontrivial challenge for federal, state, and local authorities working in conjunction with the media. postattack vaccination of the local population is an important adjunct to any decontamination strategy because vaccinated individuals can live safely with much higher residual contamination. however, not everyone can be vaccinated due to health risks and vaccinating all visitors to a contaminated area will be inconvenient. decontamination strategies differ for outdoor and indoor contamination. outdoor contamination can be partially removed by washing surfaces with water and, in any case, will decrease with time due to environmental degradation of the agent (e.g., ultraviolet light reduces anthrax spore viability by -to -fold each day). locating 'hot spots' will be important but costly because all possible outdoor locations where pathogens might collect in quantity must be sampled initially and monitored thereafter. outdoor chemical decontamination is expensive and, therefore, will be feasible only for areas on the order of a few square kilometers. indoor contamination is a more serious problem because of the absence of ultraviolet light, thus slowing environmental degradation, and because of the amount of time people spend indoors, thus increasing exposure. indoor remediation can be very expensive, as demonstrated by the experience with the hart senate office building and the brentwood postal facility after the us anthrax letter attacks, the latter of which took year at a cost of approximately $ million to clean up. moreover, current decontamination chemicals (e.g., chlorine dioxide, methyl-formaldehyde, para-formaldehyde, methyl-bromide, hydrogen peroxide, and household bleach) are corrosive, carcinogenic, and/or toxic. safer, more effective decontaminants are an important area for research and development, for example, gaseous germination agents that cause anthrax spores to germinate whereupon the vegetative bacillus becomes much more vulnerable to environmental degradation. combining advanced decontamination agents with high-efficiency particulate air (hepa) vacuuming could reduce the indoor decontamination problem to a manageable level, especially when combined with vaccination of the local population. ultimately, a mixed decontamination strategy that takes advantage of environmental degradation, washing, chemical decontamination, and vacuuming where appropriate should be able to render public areas usable, but the cost may be quite high depending on the extent to which future decontamination technologies can avoid a repeat of the us anthrax decontamination experience. the logic behind the belief that bioterrorism is a serious emerging threat is sound, although reasonable people can disagree about its urgency. moreover, this threat is multifaceted and complex, owing to the range of pathogens, delivery modes, and targets for attack. substantial uncertainties exist in predicting the outcome of any hypothetical biological attack, which implies that attack outcomes can look bleak or relatively benign depending on one's assumptions. the tendency to focus on worst case scenarios, which leads one to the conclusion that biological weapons are the poor man's nuclear weapon, originate, in part, from exaggerations by those trying to move governments to action, which may have the unintended consequence of convincing some that defense against bioterrorism is too hard and others that pathogens are an ideal weapon of terror. hence, states must develop a coherent strategy for combating bioterrorism at reasonable cost. such a strategy involves diplomacy, deterrence, preemption, and defense, with the emphasis on defense. arms control and export controls may constrain largescale biological weapon programs. more importantly, they reinforce the norm against the acquisition or use of pathogens in war or as weapons of terror. this helps reinforce deterrence, which may be effective against states but is less likely to be effective against terrorist groups. attribution will be crucial to deter states from aiding terrorist groups. however, neither diplomacy nor deterrence is sufficient alone, or in combination, to reduce the biological weapon threat to a satisfactory level. in terms of limiting damage from such threats, preemption, attractive as the concept might be, will be impractical because it relies on accurate, timely intelligence. interdiction of covert biological attacks also will be very difficult because one must detect the malevolent actors since one cannot detect the biological weapons themselves. consequently, the emphasis should be placed on passive defense, which involves detecting the release of pathogens in a timely manner, rapid postattack medical intervention, and effective decontamination to restore contaminated areas to a usable state. in principle, postattack medical response can protect over % of an exposed population if pathogens can be detected within day of their release, medical logistics can deliver appropriate medical supplies to the exposed population within day, and a prophylaxis campaign can be mounted to treat the exposed population, potentially numbering into the millions, within days. while no state currently can claim to have such an effective defense in place, except possibly against small outbreaks, such a defense is possible. moreover, to the extent biodefense overlaps with efforts to improve public health, the expenditures may be justified because resources are not wasted even if a biological attack never occurs. chemical and biological warfare; health services, effects of war and political violence on; health consequences of war and political violence biohazard. new york: random house biotechnology and the challenge to arms control biological weapons threat reduction committee on research standards and practices to prevent the destructive application of biotechnology biowarrior: inside the soviet/russian biological war machine biosecurity: a comprehensive action plan biological weapons: from invention of statesponsored programs to contemporary bioterrorism responding to terrorism across the technological spectrum expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox will terrorists go nuclear, p- unconquerable nation: knowing our enemy, strengthening ourselves, mg- -rc investigation of bioterrorism-related anthrax confronting zoonoses, linking human and veterinary medicine the cult at the end of the world effectiveness of nuclear weapons against buried biological agents animals as sentinels of bioterrorism federal agencies face challenges in implementing initiatives to improve public health infrastructure adherence to and compliance with arms control, nonproliferation, and disarmament agreements and commitments us regional deterrence strategies, mr- -a/af hepa/vaccine plan for indoor anthrax remediation bwc protocol talks in geneva collapse following us rejection sheltering effects of buildings from biological weapons -national science advisory board for biosecurity key: cord- -bwuag ek authors: matloubieh, jubin e.; eghbali, manizheh; abraham, nitya title: strategies to encourage medical student interest in urology date: - - journal: curr urol rep doi: . /s - - - sha: doc_id: cord_uid: bwuag ek purpose of review: urology residency positions have steadily increased but applications have remained stagnant. this is an alarming trend given the aging general population and thus increased need for urologists. the purpose of this review is to describe barriers and suggest strategies to encourage medical students to pursue urology. recent findings: barriers to interest in urology include educational factors, such as timing of exposure to urology in medical school, usmle scores, research experience, and deciding in time for an early match, as well as socioeconomic barriers, such as cost, being underrepresented in medicine, and gender. steps the urological community can take include increasing involvement in medical school curricula, increasing faculty mentor availability, and broadening students’ range of urological experiences. summary: strategies to encourage interest in urology fall into three categories: creating interest, supporting interest, and removing barriers for students considering urology. ultimately, the goal is to garner excellent residents in a field that must expand to meet the needs of a growing and aging population. in , the american urological association (aua) match had a record urology residency positions, an increase from in . however, the number of applicants over the same period has remained about the same, with applications and an overall match rate of % in (table ) [ ] . given the increased number of residency positions, the growing need for urological care due to the aging us population, and the relatively high happiness outside of work reported by urologists relative to their peers in other specialties, it is surprising that the number of applicants has not increased in tandem with the increase in residency positions [ ] . to further compound the issue of recruiting medical students into urology, urologists as a group are aging and approaching retirement age, which is projected to lead to a larger shortage of urologists by [ •] . a survey of the literature reveals barriers that deter students from urology include academic factors, the application process, and certain socioeconomic factors. on the other hand, factors that motivate students to enter urology include clinical exposure, educational tools, and mentorship. by better understanding barriers that medical students face, as well as factors that motivate medical students to enter urology, the urological community can devise new or revise current strategies to encourage medical students' interest in urology and fill the increasing need for urological care (table ). barriers medical students face in pursuing urology include educational factors, such as audition rotations and research, as well as socioeconomic factors, such as cost, underrepresentation in medicine, and gender. another element worthy of mention is the burnout rate among urologists, despite their high career satisfaction. without addressing these often intertwined barriers, medical students often face a high wall to entering urology that may deter even highly qualified students from applying in the first place. educational barriers that medical students face in choosing urology include medical school curricular obstacles, a common feeling among applicants that a research background is necessary, and the perceived difficulty of applying and matching to urology. curricular barriers result from wellintentioned medical school curriculum committees that seek diversify medical student experiences as well as the inherant structure of medical education in the usa. some schools, for example, only allow students to take a certain number of rotations within a given field. in other schools, students may take their surgical clerkship later in their third year, which compounds the already shortened deadline imposed by the aua's relatively early match. subsequently, many students have little to no exposure to urology as a field until their third or even fourth year, resulting in less time to decide on urology as a specialty to participate in multiple audition rotations, which are a strong predictor for matching into a given program [ ] [ ] [ ] . the problem of limited time during medical school also arises in research. anecdotally, many medical students are told that research is important for applying to surgical specialties. a large survey of us medical students found that one quarter had no research interest, and that a majority of medical students found research experiences to not be meaningful. barriers to involvement in research included the time necessary to complete research, lack of research mentors availability, and the perception that the student may not be appropriately acknowledged for their contributions [ ] . both curriculum design and the research conundrum factor into a larger barrier for medical students applying to urology: the perceived difficulty of matching into urology. while urology remains one of the more competitive specialties, this perceived difficulty likely further deters many qualified applicants. the first barrier that medical students must overcome in this sense is the united states medical licensing examination (usmle) step exam, where various "score cutoffs" are circulated among medical students. a study looking at the association of step score and specialty at their institution from to found that the mean usmle step score was for those matching in urology [ ] . a large survey of program directors found that a lower step score was one of the most deleterious criteria for selection for interviews [ ] . while this has historically been an important factor in applying to urology, the sponsors of the usmle recently announced a change in step score reporting to make it purely a pass-fail examination [ •] . for students considering applying to competitive specialties, this is a noteworthy change in one of the most significant selection criteria, and it raises the question of what will fill the role that the step score had as an "objective" measure for applicants. it remains to be seen whether usmle step will become a requirement so that programs will have a new "objective" measure, adding further pressure to applicants who are attempting to balance multiple audition rotations and applying to urology, or whether programs devise more holistic ways of evaluating applicants. gender and underrepresentation in urology are oft-discussed topics, as urology has remained a white male-dominated field even as other medical and surgical fields have diversified. although the percentage of female applicants in urology has been increasing, male applicants still outnumber their female counterparts by double, while the match rate for male and female applicants is about equal [ ] . the gender disparity has been studied from a number of angles, including mentorship and from trainees' perspectives. for example, the disproportionate underrepresentation of female urology faculty manifests as a lack of female mentors, impeding female students from considering urology-a finding corroborated in a study that found having more female faculty predicted having a higher number of female applicants [ ••] . further studies have found that having pediatric, trauma, reconstructive, and/or female pelvic medicine programs predicted a higher number of female applicants as they may be more interested in those subfields [ •] . urology departments throughout the country have attempted to address the gender gap by hiring more female faculty and diversifying their clinical offerings, but the disparity remains [ ] . another unique barrier for females is misogyny within a male-centered field. a study of female urology residents found that over half of those surveyed had experienced negative behaviors from both male patients and colleagues [ ] . students from communities underrepresented in medicine also face similar barriers from lack of mentorship and faculty they can identify with. one study of residents in urology, surgery, and in general found that urology as a field had a lower proportion of student underrepresented in medicine compared to both surgerical residents and residents in all fields, similar to the representation of women in urology [ ] . steps the urological community can take to mitigate these issues and improve female and underrepresented medical student engagement in urology include hiring diverse faculty, involving diverse faculty in direct medical education, providing sensitivity and implicit bias training for all attendings and residents, and fostering healthy work cultures where people feel comfortable discussing issues involving sexism or other hostile elements. another large barrier to pursuing a career in urology is the enormous time and money cost seemingly required by the application process. for urology applicants, the basic residency process is stretched out and inflated: students start considering where to do sub-internships midway through their third year, go through a competitive sub-internship application process with a financial and time cost, travel and pay to participate in month-long audition rotations in hopes of obtaining a letter of recommendation where they must secure stable housing remotely, and then apply to residency programs. in , the average number of programs applied to was , an increase from prior years, yielding a further cost since the application service has financial penalties for a larger number of applications [ , ] . once interview invitations arrive, students then arrange travel and lodging, often at the last minute and with little flexibility. studies of urology applicants have found that the financial cost of applying to residency for subspecialties can be upward of $ since almost % of applicants undertake away rotations [ •, •] . given the rising number of applications students submit, it follows that the financial barrier to applying for urology will continue rising. because of this alarming picture, institutions and larger governing bodies have taken some steps to attempt to overcome the enormous financial burden that may deter students from considering urology. multiple institutions offer scholarships for students from disadvantaged or underrepresented backgrounds that can be applied toward room, board, and transportation for audition rotations. other institutions have low or no charge for visiting student rotators. in perhaps the most dramatic attempt to address this issue, all of the canadian programs joined together to create the canadian urology fair starting in . after surveying students and programs in the pilot year of this program, the overseeing committee found that the cost to students was significantly less than traveling to interviews, the costs to programs were not significantly different than from hosting interviews, and that students missed fewer days of school compared to their counterparts in other surgical specialties who had to travel to interviews [ ] . the main drawbacks to this process are the lack of the social element, for applicants are also interviewing the programs, and the fact that there are ten times as many urology residency programs in the usa than in canada. one way to reconcile the canadian model in the sprawling usa and thus reduce the cost barrier could be to adopt a similar model to music and dance school audition committees that host auditions at sites around the country, perhaps at regional aua sectional meetings. in addition, the changes to the application process in response to the covid- pandemic have resulted in residency programs creating new ways to connect with interested medical students. anectdotally, these changes include virtual open houses, sub-internships, and interviews, as well as increased use of social media. it remains to be seen whether these changes will remain permanent if the pandemic subsides. urology ranked highest ( %) in a medscape national survey of , physicians on burnout and suicide with the leading cause of burnout being too many bureaucratic tasks ( %), like charting and paperwork. critics of these findings point to the limited sample size of urologists surveyed in the past (n = ) [ ] . the aua census data surveying a matrix sample of practicing urologists found a burnout rate of . %, which is comparable to other specialties [ • ]. nevertheless, a systematic review of factors influencing medical students' choice of subspecialty found that the third most important factor was controllable lifestyles or flexible work schedules ( %), following academic interests ( )% and competencies ( ) [ ] . high reported burnout rates in urology could deter student interest, especially in those concerned about work-life balance. anectodally, urologists report a high work-life balance satisfaction, so perhaps increasing medical student mentorship could combat the perception that urologists have a higher burnout rate. early clinical exposure while medical students may have some exposure to urology prior to medical school, the initial exposure to most topics in medicine, including urology, occurs during the preclinical years of medical schooling. a number of studies have found that there is a paucity of exposure to urology in these formative years of medical school. unfortunately, these studies have also found a decline in urology exposure. a study found that % of medical schools had no exposure to urology in the preclinical years [ ] . a follow-up to this study found that this number had increased to % of medical schools not having urology exposure in the preclinical years [ ] . this decline in urology exposure can be to the detriment of future patients, for as one study noted, knowledge of basic urology was low for both medical students and primary care faculty taking care of the bulk of communities [ ] . as exposure is critical to fostering medical student interest in urology, it is imperative that the urological community devise strategies to increase and maintain exposure to the field of urology. one such strategy was studied in england where early sustained exposure to urology was correlated with considering a career in urology [ ] . despite this encouraging finding, the state of medical education in the usa seems to slant students away from pursuing urology, for a study found a decline in medical schools requiring urology rotations from % in the s to % in [ ] . further compounding this is another discouraging report that % of american medical schools surveyed reported that it was possible to graduate from their school without any clinical exposure to urology [ ] . fortunately, a number of methods have been studied that increase students' knowledge of and exposure to urology, and thus hopefully increase their interest. while the number of studies and data obtained is limited, the variety of approaches examined is encouraging in the way of urological educational innovation. educational strategies that have specifically been studied in the urological literature include dedicated clinical rotations in urology, the use of simulators, the use of technology in education, the presence of a urology interest group, and mentorship. multiple studies have found that having a clinical rotation in urology increases students' comfort in managing urological problems and potentially increases consideration of a career in urology. clinical exposure to medical problems and treatments is crucial in cultivating, expanding, and reinforcing knowledge learned in the preclinical years. a study of outpatient versus inpatient settings for learning urology found that students in the outpatient clinical setting had a larger exposure to urological problems, better knowledge of urological physical examination, and were more likely to perceive that they learned more. further, students in the outpatient setting may have greater exposure to urology faculty in the clinic compared to the floors, encouraging the mentorship needed to motivate students into a career in urology [ ] . a study conducted years later found similar results and expanded into acquiring skills such as foley catheter insertion [ ] . by including students more in clinical management, such as in placement of foley catheters, a sense of inclusion in the team is fostered, further reinforcing the social bonds that encourage students to pursue urology. the social aspect to medical education and garnering student interest was reported in an earlier study where third year medical students felt that clinical learning followed by resident teaching to be the most helpful, whereas watching surgery, especially endoscopic surgery, to be less helpful [ ] . this calls into question surgical clerkship structure where maximal operative exposure is encouraged, even in endoscopic or robotic cases, perhaps at the expense of the potentially more educational and social clinical setting. the use of various simulations in urological education has had mixed results in increasing interest in urology. a study of a clinical clerkship in urologic laparoscopy had a high level of interest and satisfaction among medical students, but did not find a change in the desire to pursue a career in urology [ ] . a different study found that students' confidence in performing catheterization and digital rectal examination, especially female students' confidence, was improved postsimulation; while this study did not comment on the change in interest in pursuing urology, it did address an issue urology faces in recruiting female trainees [ ] . the conclusion to be drawn from these experiences may be that simulations have an adjunctive role in urological education, but may be less helpful in increasing interest in urology when used alone. similarly, the use of media and technology in urological education to allow for broader exposure to urology is inconclusive in the ability to attract students to urology. two methods described in the literature include the use of a massive open online course (mooc) and the use of a "learning package for medical students in a busy urology department." the former method was innovative in that it was the first mooc that expressly taught urology; while over participants completed this mooc and % of countries were represented, it neither commented on its utility in attracting students to urology nor was it specifically geared toward medical students [ ] . in another study, the use of online "learning package for medical students" was helpful for learning core clinical problems. again, this study did not comment on the utility of the online material in helping attract students to urology, and even its title emphasized important barriers for students considering a career in urology, namely lack of access to faculty mentors in a "busy urology department" [ ] . the educational strategy that has been most uniformly effective in recruiting students to a career in urology has been mentorship. a study of why certain medical schools have more students pursuing urology found that mentorship was the single most important factor between schools [ ] . this broader finding was confirmed in a study within a single school where having positive role models in urology led to a positive perception of urology and thus increased interest in urology [ ] . mentorship may be a helpful factor in helping bridge the gender gap in urology, as having a higher percentage of female urology faculty is correlated with a higher percentage of female students pursuing urology [ ••] . a similar conclusion can be extrapolated to students underrepresented in medicine. one specific method that has been studied to connect medical students to mentors is the presence of a urology interest group, which has also been found to have a positive impact on medical student interest in urology [ ••] . other opportunities through which urology faculty can serve as mentors include offering research projects in urology during the summer between first and second year of medical school, leading small group or one-on-one sessions in first and second year clinical courses, and serving as teachers in preclinical classes like anatomy or male reproductive pathophysiology. creating a more formal mentorship program is a further step that the urological community can take to encourage students to enter urology. this idea was tested in germany, where a national mentoring program for urology was started in and has continued since then due to an overwhelmingly positive response [ ] . while this may seemingly be more difficult in the more populous usa, the urological community remains small and well-connected, so a national, or at least regional, mentorship program may not be difficult to implement. the early match requires that applicants decide before the end of third year if they are interested in urology. switching to the national residency matching program (nrmp) match, which occurs in march, may afford more time to students exploring urology late in third year and may increase the number of applicants. in otolaryngology, another competitive surgical subspecialty switched from an early match to the regular nrmp match. however, a comparison of the applicants and match rate before and after the change found no difference in the number of applicants or competitiveness of the specialty, suggesting a switch to the nrmp match may not necessarily lead to increased interest in urology [ ] . it remains to be seen whether changes in the application cycle in response to the covid- pandemic will have an impact on students applying to urology. a recent viewpoint piece in jama suggests another alternative to the current early match and regular match: an optional early application and acceptance program. this strategy is akin to the early acceptance process used by colleges and universities and may suit several groups of students including highly competitive students with clear program choice or the seemingly less competitive students who may be hesitant in applying to a competitive field such as urology. another advantage of this program would be a substantial reduction in time and cost expenditure as each applicant pursues fewer programs [ ••] . broadly speaking, strategies to encourage medical student interest in urology fall into three categories: creating student interest, supporting student interest, and removing barriers to students considering urology. to address the issue of encouraging medical student interest in urology requires a threepronged approach and increased effort on the part of urology residency programs. future steps that the urological community can take include increasing involvement in the medical school curriculum during the preclinical years, increasing faculty availability as mentors, broadening the range of urological experiences that students have, lowering academic and socioeconomic barriers, and finding ways to continue vetting quality applicants despite changes in board exam scoring and possible restrictions in the number of programs students can apply to. ultimately, the goal is to continue garnering excellent residents in a field that needs to expand to meet the demands of a growing and aging population. conflict of interest the authors declare that they have no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. medscape physician lifestyle & happiness report : the generational divide projects an increased deficit in urologists by due to both the aging us population as well as the aging urologist population who will be retiring the afmc electives diversification policy: potential drawbacks and benefits for medical students applying to urology the current status of medical student urological education in the united states an update on the current status of medical student urology education in the united states a survey on the attitudes towards research in medical school the association of usmle step and step ck scores with residency match specialty and location program directors criteria for selection into urology residency usmle step scoring will be changing from a numerical score to purely pass/fail, eliminating one of the filters that urology residencies use for residency applications having more female urology faculty is positively correlated with more female medical students applying to urology. having a urology residency program and having a urology having a broad array of urological subspecialties is correlated with more female medical students applying to urology gender and subspecialty of urology faculty in department-based leadership roles a survey of women urology residents regarding career choice and practice challenges examining trends in underrepresented minorities in urology residency fees for eras residency applications survey of applicant experience and cost in the urology match: opportunities for reform corroborates the value of medical students undertaking audition rotations with respect to matching into a residency program the canadian urology fair: a model for minimizing the financial and academic costs of the residency selection process burnout statistics match those of other specialties, yet there is still a growing deficit in the number of students applying factors influencing subspecialty choice among medical students: a systematic review and meta-analysis deficits in urological knowledge among medical students and primary care providers: potential for impact on urological care factors affecting uk medical students' decision to train in urology: a national survey does the outpatient setting provide the best environment for medical student learning of urology? the value of a core clinical rotation in urology for medical students simulators help improve student confidence to acquire skills in urology an innovative medical student clinical clerkship in advanced urologic laparoscopy: a preliminary experience kiurologyx: urology as you like it-a massive open online course for medical students, professionals, patients, and laypeople alike a learning package for medical students in a busy urology department: design, implementation, and evaluation the gatekeeper disparity-why do some medical schools send more medical students into urology? survey on the perception of urology as a specialty by medical students bringing excellence into urology: how to improve the future training of residents? state of otolaryngology match: has competition increased since the "early" match? otolaryngol head neck surg improving the residency application and selection process: an optional early result acceptance program describes a novel method for decreasing the cost of applying into urology and lowering potential barriers for students applying into urology publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - m l t authors: xing, jun; sun, ning; xu, jun; geng, shuling; li, yuqian title: study of the mental health status of medical personnel dealing with new coronavirus pneumonia date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: m l t this paper studied the relationship between personality traits and mental health conditions of medical personnel to provide a basis and reference for the implementation of targeted education on mental health. a self-report inventory, the symptom checklist- (scl- ), was used to investigate the mental health status of medical personnel dealing with the new coronavirus pneumonia in eight provinces and cities of china. the overall mean scl- score and mean values of factors (somatization, obsessive-compulsive, anxiety, phobic anxiety, and psychoticism) of the medical personnel were significantly higher than in the norm group (p < . ), while their average interpersonal sensitivity score was significantly lower (p < . ). in addition, personal factors affecting the mental health status of medical personnel were identified (all p < . ). the overall mental health status of medical personnel responding to new coronavirus pneumonia is generally higher than that of the norm group in china. the results of this study should contribute to measures to alleviate the psychological pressures on medical personnel dealing with the new coronavirus epidemic in china. novel coronavirus pneumonia (ncp) is a pathogenic coronavirus often referred to as the novel coronavirus. on january , , who officially named the disease coronavirus disease (covid- ). the first case of covid- was reported in wuhan in china on december , [ ] [ ] as causing severe acute respiratory infection (sari). coronaviruses are a large family of viruses known to cause illnesses such as cold and more serious diseases such as middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars). covid- is a new strain of coronavirus that has never been found in the human body before [ ] [ ] . covid- patients typically show symptoms such as fever, coughing, shortness of breath, and difficulty in breathing. in more severe cases, the disease can lead to pneumonia, severe acute respiratory failure, kidney failure, and even death [ ] . there is as yet no specific treatment for covid- . patients mainly receive symptomatic treatment and care for the prevention of complications, while supplementary medical services also appear to be very effective for infected people. the rate of covid- infections has increased rapidly in a short period of time. as of february , , the number of confirmed cases exceeded , , with , deaths [ ] . in addition, the disease can cause secondary infections, which has created a huge burden and pressure for the prevention and treatment of the disease in various places. at present, more than , medical personnel from various medical teams across the country have provided support in wuhan [ ] . though the help from these medical personnel has relieved the local pressure for medical care to save critically ill covid- patients, there have been serious infections among the medical staff in wuhan and other places in hubei province. at present, over medical personnel have been infected, which has greatly increased the psychological pressure they experience. in the face of the catastrophic health emergency of covid- , medical personnel have been affected by different kinds of subjective and objective factors and confront several mental health problems. mental illness is a form of human stress response, an explanatory, emotional, and defensive response within the human body, and a physiological response of the human body to the impact of needs or injuries. therefore, this study aims to analyze the psychological state of medical personnel dealing with covid- and its influencing factors in order to provide an objective basis for the prevention of further transmission, interventions, and countermeasures for covid- . this study adopted convenience sampling to recruit research subjects. from january to february , , medical personnel from hospitals in eight provinces and cities across the country were enrolled as research subjects. ① survey of demographic characteristics of medical personnel, including questions related to the following aspects: province, hospital, department, occupation, gender, age, highest education level, work experience, level of expertise, marital status, children, living status, whether you have participated in training for handling of public health emergencies, whether family members support your working on the front line against coronavirus, whether you have supported in affected areas in hubei, designated hospitals, department of infectious diseases, fever clinics or emergency department, level of concern whether you and your family have been infected, degree of suspicion that you were infected when coronavirus-related symptoms occurred, and whether you have received medical observation recently. ② the scl- self-report inventory: the scl- inventory, compiled by derogatis in , includes factors and a total of items. it had been translated into chinese version and used in the study [ ] . each factor reflects the symptoms and pain of a patient in a certain aspect, and the distribution of symptoms can be understood through the factor scores. the factors include somatization, obsessive-compulsive, depression, anxiety, hostility, phobic anxiety, paranoid ideation, psychoticism, sleep, and diet. each item was scored using a -point likert scale, ranging none, mild, moderate, moderate to severe, to severe. the total score is the sum of the scores of the items. in previous studies, the homogeneity reliability of the total scl- scale was . , and the homogeneity reliability of each sub-scale was also greater than . . the test-retest reliability was greater than . , and the content validity was above . , which suggest sound reliability and validity [ ] . . . survey methods and medical ethics. the study used online questionnaires for data collection. researchers conducted surveys upon the completion of general training. all procedures were approved by the ethics committee of harbin medical university (hrbykd-a ). the research purpose and methods were explained to subjects to seek their cooperation. online informed consent forms were signed by participants. they were informed that their participation was completely voluntary, and they could withdraw from the study at any time. the link of the online questionnaire was then sent to a total of medical personnel via the internet. . in this study, data analysis was performed after logical checks using the statistical software spss . . a p-value less than . (p < . ) was considered statistically significant. . statistical description: the mean value, standard deviation, and frequency were used to describe the demographic data of medical personnel, while the mean value and standard deviation were used to describe the scores for the mental health status of medical personnel responding to covid- . . statistical inference: multivariate linear regression was adopted to analyze the impact of the demographic data of medical personnel on their mental health status. a total of questionnaires were distributed, and valid questionnaires were recovered, for an effective recovery rate of . %. these questionnaires were completed by medical personnel from eight provinces and cities in china, namely heilongjiang, liaoning, jilin, inner mongolia, tianjin, sichuan, shanxi, and shandong. details of the respondents' personal information are given in table . the overall average of scl- and mean values of factors (somatization, obsessive-compulsive, anxiety, phobic anxiety, and psychoticism) of medical personnel were significantly higher than that of the norm group (p < . or p < . ), while the average score of the interpersonal sensitivity factor of medical personnel was significantly lower than that of the norm group (p < . ). details are provided in table . stepwise linear regression was performed using the total score of mental health status as the dependent variable and items of personal information as independent variables. the items include: hospital, department, occupation, gender, age, highest education level, work experience, level of expertise, marital status, any children, living status, whether you have participated in training for handling of public health emergencies, whether family members support your working on the front line against coronavirus, whether you have supported in affected areas in hubei, designated hospitals, department of infectious diseases, fever clinics or emergency department, level of concern whether you and your family have been infected, degree of suspicion that you were infected when coronavirus-related symptoms occurred, and whether you have received medical observation recently. the α-values for importing and exporting a variable in the regression equation were set to . and . , respectively. factors affecting the mental health and status of medical personnel based on their significance from high to low are: the degree of suspicion that they were infected when the novel coronavirusrelated symptoms occurred, the level of concern whether they and their family members have been infected, age, whether they have supported in affected areas in hubei province, designated hospitals, and other places for the novel coronavirus, and whether family members support them working on the front line (p < . ). details of the regression results are listed in table . covid- is a fulminant infectious disease. as it is highly contagious, many people are frightened by it and even talk fearfully about coronavirus, which can also be observed in front-line medical staff. li et al. reported how much people and medical staff suffer from vicarious traumatization and how this vicarious traumatization of non-front-line medical staff is more serious than that of front-line medical staff [ ] . as in south and southeast asia countries, also in italy, there are similar problems in medical staff due to high workload and intermittent lack of protective devices. in addition, some slight form of racism is demonstrated against health care professionals who potentially have a higher risk of being infected and between non-front-line medical staff towards front-line medical staff [ ] . the results of the study have shown that the overall mean of the scl- and the mean values of the factors (somatization, obsessive- compulsive, anxiety, phobic anxiety, and psychoticism) of medical personnel were significantly higher than that of the norm group (p < . ), while the average score of the interpersonal sensitivity factor of medical personnel was significantly lower than that of the norm group (p < . ). the results were partly similar with some research in wuhan city [ ] . more specifically, medical personnel are most of the people closest to covid- patients, so they are at high risk of exposure to the disease. moreover, they have a deep understanding of the dangers of covid- , so they are prone to anxiety and fear. the infection protection procedures for covid- are highly complex and medical staff need to constantly change clothes and replace protective equipment, so they are more likely to establish obsessive-compulsive behaviors. medical personnel, especially young medical staff, have less experience in the field and in dealing with difficulties and hardships in life. therefore, when they suddenly encounter such sudden public health events, they tend to suffer anxiety and phobic anxiety, leading to physical and mental problems. that is why scores of the factors somatization, obsessive-compulsive, anxiety, phobic anxiety, and psychoticism were significantly higher than in the norm group. this result suggests that psychologists and team leaders should pay more attention to the anxiety, phobic anxiety, and psychoticism issues of the medical personnel in a team. the average score for the interpersonal sensitivity factor of medical personnel was significantly lower than that of the norm group. this shows that in the event of an infectious disease epidemic, the majority of medical personnel are united and have good professional strengths and qualities for self-regulation and self-protection. the results of this study show partial consistency with the studies of the mental health status of front-line medical personnel for sars in [ ] . the results of this study have shown that the factors affecting the mental health status of medical personnel based on the significance from high to low are: the degree of suspicion that they were infected when the novel coronavirus-related symptoms occurred, the level of concern whether they and their family members have been infected, age, whether they have supported in affected areas in hubei province, designated hospitals, and other places for the novel coronavirus, and whether their family members support them working on the front line. specific reasons are given in the following sections. degree of suspicion that they were infected when the novel coronavirus-related symptoms occurred, the level of concern whether they and their family members have been infected, whether they have supported in affected areas in hubei province, designated hospitals, and other places for the novel coronavirus, and whether family members support them working on the front line. covid- patients are the main source of transmission of the disease. patients with latent infection (i.e., asymptomatic infection) may also constitute a source of infection [ ] . medical personnel are in frequent close contact with patients during their treatment and care, hence the high risk of infection [ ] . among patients admitted consecutively from january to , , to zhongnan hospital of wuhan university, the proportion of medical personnel was as high as % [ ] . a retrospective analysis of confirmed covid- patients from hospitals in provinces (diagnosis as of january ) found that the proportion of medical staff was . % [ ] . therefore, medical staff are at high risk of infection and are under great psychological pressure. suicidal cases were reported in india but also in other countries, italy included, where two infected italian nurses committed suicide in a period of a few days probably due to fear of spreading covid- to patients. it is possible that fear and anxiety of falling sick or dying, helplessness will drive an increase in the suicide rates [ ] . if they become infected as a result of supporting affected areas in hubei and covid- designated hospitals, it will not only affect their physical and mental health but also that of their families. therefore, with the emergence of symptoms and the increase in the level of concern, the mental health status of clinical medical staff may deteriorate. furthermore, if their families do not support them working on the front line against the disease, then the psychological burden of the medical staff will also increase due to the resulting sense of conflict with professional ethics, resulting in further impact on their physical and mental health. we found that the higher the age, the higher the mental health score and the more psychological problems. based on the age distribution of patients across the country, all ages have no resistance to the novel coronavirus, and a person of any age can be infected as long as virus transmission conditions are met [ ] . an analysis of confirmed patients nationwide (diagnosis date as of january ) also shows that people of all ages are generally vulnerable to the disease, of whom . % are aged to years [ ] . as people get older, the risk of exposure to the disease may increase in people with underlying illness such as asthma, diabetes, and heart diseases [ ] . therefore, older medical personnel have more psychological stress when dealing with covid- patients. it is advised that older medical personnel receive psychological counseling before and during work to help them adjust their status as soon as possible. despite all the above, this study believes that after such a "smokeless" war against the novel coronavirus, the psychological quality of medical personnel can be improved to a certain extent. in this study, the applicability of the results is limited by the nature of cross-sectional studies, and because of its use of convenience sampling from hospitals in eight provinces and cities of china. in subsequent research in this project, a longitudinal study should be conducted that uses a wider sample and measures the mental health status of medical personnel from multiple dimensions, which can help better identify the mutual influence between demographic data and mental health status. in the face of the catastrophic health emergency caused by covid- , medical staff have been affected by different kinds of subjective and objective factors. their mental health problems are a form of human stress response, an explanatory, emotional, and defensive response within the human body, and a physiological response of the human body to the invasion of needs or injuries. in this special environment, their work, life, and emotions tend to be regularly abnormal. due to the requirements for isolation and disinfection, medical personnel need to wear several layers of protection clothing. this increases the intensity of their work and requires great physical energy, causing severe hypoxia and physical symptoms such as headache and muscle soreness. other symptoms such as obsessive-compulsive symptoms, interpersonal sensitivity, depression, anxiety, phobic anxiety, hostility, and paranoid ideation are all normal psychological reactions in the handling of emergencies and environmental stimuli. in face of a disaster, persons with good mental health will tend to actively take measures such as catharsis, transference, compensation, relaxation, humor, self-consolation, and rational response. the results of this study show that the overall mental health of medical staff is generally poor when dealing with covid- . psychological tests show that people have a process of adaptation to catastrophic emergencies, from initial rejection, shock, and fear, to habituation, acceptance, and calm, to co-existence and living together, which is a regular process. in the face of such a sudden disaster as covid- , these psychological symptoms have manifested in both doctors and patients. for medical personnel, it is particularly important to pay attention to mental health conditions while fulfilling their responsibilities. in future research, it is worth exploring how to strengthen the monitoring of mental health conditions of medical personnel and establish an active, systematic, and scientific psychological defense system under such special circumstances. report of clustering pneumonia of unknown etiology in wuhan city chinese center for disease control and prevention. wuhan municipal health committee's report on the status of viral pneumonia of unknown cause world health organization. novel coronavirus-china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia epub ahead of print clinical features of patients infected with novel coronavirus in wuhan chinese center for disease control and prevention. epidemic distribution of new coronavirus pneumonia the authoritative release of the joint prevention and control mechanism of the state council mental health status of medical staff during the outbreak of coronavirus disease preliminary analysis of scl- evaluation results of chinese normal people vicarious traumatization in the general public, members, and non-members of medical teams aiding in covid- control the emotional impact of covid- : from medical staff to common people published online first care for the psychological status of frontline medical staff fighting against covid- mental health survey of front-line medical personnel for sars. beijing genomic characterisation and epidemiolooy of novel coronavirus: implications for virus origins and receptor binding a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china the continuing -ncov epidemic threat of novel coronaviruses to global health: the latest novel coronavirus outbreak in wuhan fear of covid : first suicidal case in india national health commission of the people's republic of china novel coronavirus ( -ncov) advice for the public: myth busters the authors would like to thank the medical personnel who participated in the study. conceptualization: ning sun. key: cord- -n ljjdhu authors: sandhu, preeti; de wolf, maisie title: the impact of covid- on the undergraduate medical curriculum date: - - journal: med educ online doi: . / . . sha: doc_id: cord_uid: n ljjdhu the coronavirus pandemic has impacted medical education globally. as universities seek to deliver medical education through new methods of modalities, this continuing of education ensures the learning of the future workforce of the nhs. novel ways of online teaching should be considered in new medical curricula development, as well as methods of delivering practical skills for medical students online. the coronavirus disease (covid- ) pandemic has had a worldwide impact on the population, global economy and heath care systems. whilst the spread of the virus has resulted in far-reaching consequences, the closure of schools and universities has led to innovative methods of delivering education, ensuring that students continue to receive teaching, albeit different methods of modality. a national effort by uk medical schools in graduating early nearly , final-year students will allow for these interim foundation year doctors to support the nhs during the pandemic [ ] . as medical students in our penultimate clinical year of undergraduate education, we have experienced cancellations of all clinical placements into the beginning of final year, with online learning presently proving essential in the continuation of medical education. the coronavirus pandemic has seen the introduction of novel methods of delivering education to medical students. lectures have rapidly been developed to be delivered online as webinars using various platforms such as zoom, with such technologically enhanced approaches already being proven to have high levels of engagement with medical students [ ] . with international students making up . % of the total student population [ ] , and many having returned to their native homes during the coronavirus outbreak, online teaching platforms are beneficial due to their worldwide accessibility, ensuring that all medical students regardless of their current location are able to access webinars as they happen or can be recorded for later use. thus far, our experience of online webinars that have included key clinical conditions, case studies and examination questions have been well received, with a regular number of medical student attendees engaging in these lessons throughout these unprecedented times. the transition to online medical education has also seen a change in examination methods. following on from the recent success of imperial college london's first ever online exam for final years [ ] , other medical schools are also adopting a similar approach to ensure students remain engaged with their studies, with many universities adopting an open-book examination (obe) approach. the introduction of obes will be a sudden change to nearly all medical students from previous exam-hall settings. however, obes have been shown to reduce student anxiety [ ] and with a global level of heightened fear and apprehension during the current covid- pandemic, an approach to examining students during this difficult time that can minimise symptoms of stress is welcomed. to ensure the future workforce of the nhs are qualified, continuing education is vital and this can be achieved by medical school staff continuing to engage regularly with medical students using online teaching platforms. the current success of online teaching and obes provides an initial insight into new and innovative ways of teaching for medical education. consideration is encouraged as to how such online methods may be adapted to deliver teaching on clinical and practical skills remotely that would otherwise have been developed during clinical placements. no potential conflict of interest was reported by the authors. covid- : early provisional registration for final year students using technology to increase student (and faculty satisfaction with engagement in medical education international facts and figures medical students take final exams online for first time, despite student concern effects of online testing on student exam performance and test anxiety key: cord- -wrc dzvl authors: novintan, shonnelly; mann, simran; hazemi-jebelli, yasmin title: simulations and virtual learning supporting clinical education during the covid pandemic [letter] date: - - journal: adv med educ pract doi: . /amep.s sha: doc_id: cord_uid: wrc dzvl nan we read with great interest the article by tabatabai regarding virtual learning supporting clinical education and examination during the covid- pandemic. as final year medical students, we have experienced months of remote teaching followed by online exams. it is interesting that we share same similar benefits of virtual learning in the uk. however, we believe that virtual platforms fail to meet the practical needs of students and could leave significant gaps in our clinical competency. in iran, there has been a national partnership between medical schools to provide standardised massive online open courses. conversely, in the uk, universities have differed in each decision, from date of closure, to the commencement of online teaching and from the format of exams to the return of students to placement: it has not been a unified approach. the controversy of some universities returning their students in july, but others waiting to , raises questions on the impact of prolonged virtual learning on the clinical competency of a student. tabatabai correctly emphasises the need for universities to have the right tools to keep the education of medical students on track. additionally, we would like to bring the reader's attention to a more current, revolutionary example: virtual reality ward rounds. in this example, education goes beyond simulation and provides a livestream of the consultant-patient interaction on the ward, whilst the remaining clinical team observes from an isolated area, allowing students to take histories from patients. this allows for dynamic interaction that is superior to the one-way dialogue of live lectures and better prepares students for clinical scenarios and exams. the author states that simulation-based, virtual objective-structured clinical exam (osce) provides the perfect platform to address all clinical education needs. however, tabatabai fails to explain how this extends to the practical element of the osce. unlike other exams, the osce is unique in its assessment of bedside manner and practical skills, ranging from demonstrating venepuncture skills to diagnosing heart murmurs. students need to develop and be assessed on practical skills in a competent and, more importantly, safe manner. this is evident as, during the pandemic, medical schools have chosen to repeat uncompleted practical assessments to ensure that students demonstrate the skills required by the general medical council to graduate (eg, catheterisation or intramuscular injection). at present, technology cannot replicate the assessment of these skills, which is a fundamental flaw in the simulationbased virtual osce. for this reason, we disagree with tabatabai, as a virtual osce would not increase objectivity, but instead make the exam more incomprehensive and provide an inaccurate score of the student's capabilities with the potential of graduating unsafe doctors. this is highlighted in the mistakenly interpreted cited article that actually expresses the importance of in-person, as opposed to virtual, osce conducted by a singaporean medical school. this commentary highlights an important issue that faces all universities: the need to continue medical education and assessment, whilst maintaining patient and student safety. however, we must acknowledge the limitations of virtual education and adapt accordingly. dove medical press encourages responsible, free and frank academic debate. the content of the advances in medical education and practice 'letters to the editor' section does not necessarily represent the views of dove medical press, its officers, agents, employees, related entities or the advances in medical education and practice editors. while all reasonable steps have been taken to confirm the content of each letter, dove medical press accepts no liability in respect of the content of any letter, nor is it responsible for the content and accuracy of any letter to the editor. advances in medical education and practice is an international, peerreviewed, open access journal that aims to present and publish research on medical education covering medical, dental, nursing and allied health care professional education. the journal covers undergraduate education, postgraduate training and continuing medical education including emerging trends and innovative models linking education, research, and health care services. the manuscript management system is completely online and includes a very quick and fair peer-review system. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. virtual learning supporting clinical education during the covid pandemic london hospital starts virtual ward rounds for medical students. guardian; conducting a high-stakes osce in a covid- environment the authors report no conflicts of interest in this communication. key: cord- -itdorszt authors: farha, loulwa; abi jaoude, joseph title: lebanese healthcare system: how will the aftermath look? date: - - journal: cureus doi: . /cureus. sha: doc_id: cord_uid: itdorszt the recent lebanese port explosion came as a continuation of a series of socioeconomic disasters the country has been facing during the past year. in addition, the massive impact of the coronavirus disease- (covid- ) pandemic further hastened the collapse of the lebanese healthcare system. in light of all those events, the lebanese healthcare sector has faced major blows that will be difficult to recuperate from. in the aftermath of the beirut port explosion, lebanon received immense financial and medical support from the international community in a timely fashion, which secured first level care to victims of the explosion. nevertheless, this forced lebanon, which was considered a prominent tertiary medical hub in the middle east, to slowly regress into an exclusive primary care provider. as such, it is crucial for local and regional stakeholders to build strong collaborations, and shape a unified vision of lebanon’s future healthcare system. on august , beirut, the capital of lebanon, witnessed one of the biggest explosions in the history of mankind [ ] . although not really a nuclear explosion, the blast was strong enough for people from around the world to question a potential nuclear nature to the event, with some going to the extent of dubbing the explosion "beirutshima", owing to its similarity with the tragic events of the hiroshima bombing [ ] . the explosion led to the deaths of hundreds, wounded thousands, and resulted in a colossal physical and financial burden on the country [ ] . while this particular day was truly incomparable, it merely comes as a continuation of a wave of disasters the country has faced during the past year. as a matter of fact, the recent political and economic situation has driven lebanon into one of the most serious financial breakdowns in its modern history, and has pushed a large proportion of its population below the poverty line. in addition, the massive impact of the coronavirus disease- (covid- ) pandemic further compounded the effects of the lebanese financial collapse [ ] . in light of all those events, it becomes quite evident that the major blows sustained by the lebanese healthcare system have elicited a negative chain reaction that will be difficult to recuperate from. from the beginning of the lebanese economic crisis which began around a year ago, many of the small-scale hospitals and medical centers faced threats of potential bankruptcy. however, with the continuous financial strain, even the bigger tertiary medical centers had to succumb to those dire circumstances and adopt harsh measures to stay afloat [ ] . the most noteworthy measures included laying-off a considerable number of hospital staff, and closing some of the hospital wards that were operating at suboptimal capacity [ ] . moreover, many highly qualified physicians and nurses opted to leave lebanon for better opportunities abroad as a result of pay cuts that were later accompanied by a depreciation of the national currency. as the lebanese pound lost approximately % of its value, the importation of medications and medical equipment became exceptionally difficult and restricted. as such, the pressure on the healthcare system began to build up early on, sending shock waves into the foundations of what was once considered one of the top healthcare systems in the region. as the number of covid- cases drastically increased around the world, lebanon was no exception. the covid- pandemic began to pick up pace in the country just weeks prior to the explosion with infections reaching the range of a couple hundred new cases registered daily [ ] . although the absolute number may seem small, the number of covid- infections is considered to be relatively high after accounting for the country's small population size (with rates reaching as high as cases per million persons as of july ) [ ] . hospitals and medical centers have been suffering for months to keep up with the increase in demand for intensive care unit beds, medical equipment, and medications necessary to cater to patients with severe covid- infections. it would thus come as no surprise that the events of august were the final nail in the coffin of an already toppling healthcare system. the consequences of the explosion were directly evident as the number of injured people was estimated at approximately , patients [ ] . healthcare workers who have already been suffering from psychological burnout due to the covid- pandemic were also among those who lost their lives or were severely injured. to make things worse, the capital was faced with yet another repercussion that served as both a direct obstacle, and will serve as a future impediment, and that is the destruction of several hospital facilities. three of the major tertiary medical centers serving the beirut area were severely damaged rendering them completely non-operational, and forcing them to evacuate their patients immediately. furthermore, most of the hospitals and medical centers in beirut and the neighboring areas were also affected, albeit to a lesser extent. those facilities sustained damages to their infrastructure and medical equipment which forced them to operate under harsh conditions. as such, in a matter of hours, lebanon was faced with an acute increase in demand for healthcare, only to be met by a massive and abrupt decrease in supply. within hours, lebanon transformed from a major tertiary medical hub, to the equivalent of a huge emergency room, providing nothing but the most basic medical services to its patients. in the aftermath of all this chaos, lebanon received and is still receiving immense financial and medical support from the international community. a significant fraction of this aid came in the form of financial funding to trusted non-governmental organizations (ngos). other notable support was offered in the form of basic nutrition and medical supplies, such as field hospitals, medical equipment, and medications. this remarkable aid was offered in a timely fashion, and allowed the situation in lebanon to slightly stabilize. although far from optimal, this secured first level care to victims of the explosion, which was of prime significance in such an exceptional situation. nevertheless, this rapid and chaotic surge in hospital admissions further increased the rate of covid- infections, which posed yet another problematic issue to an already struggling sector. lebanon's healthcare system has been on the receiving end of repeated blows that have already started to change the nature and quality of its healthcare delivery. a once prominent tertiary medical hub in the middle east is slowly turning into an exclusive primary care provider. for that reason, it is now crucial for local and regional stakeholders to start planning out the aftermath of these unfortunate events. despite vital support from the international community, funding is currently being distributed among several entities that lack common management that is capable of assessing the gaps in the entire sector, and allocating resources so as to reestablish a coherent system. the prevailing circumstances also necessitate the immediate resumption of functions at tertiary medical care centers that offer specialized treatments, disease screening, and disease prevention. similarly, tackling the psychological and financial stress of healthcare personnel is essential to prevent further losses of valuable human resources. finally, given the unstable political and socioeconomic condition in the country, it becomes of utmost importance to build a solid collaboration among the key contributors who will help shape a unified vision of lebanon's future healthcare system. in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. why beirut's ammonium nitrate blast was so devastating coronavirus infections-more than just the common cold major beirut medical centre lays off hundreds as crisis bites covid- response in lebanon: current experience and challenges in a lowresource setting key: cord- -t bedot authors: byrnes, yasmeen m.; civantos, alyssa m.; go, beatrice c.; mcwilliams, tara l.; rajasekaran, karthik title: effect of the covid- pandemic on medical student career perceptions: a national survey study date: - - journal: medical education online doi: . / . . sha: doc_id: cord_uid: t bedot background & objective: the covid- pandemic and resulting cancellation of medical student clinical rotations pose unique challenges to students’ educations, the impact of which has not yet been explored. design: this cross-sectional survey study collected responses from april until april . students at us allopathic medical schools completed the survey online. results: , responses were analyzed. a total of ( . %) respondents thought the pandemic would affect their choice of specialty, with differences across class years: . % ( ) of first-years (ms s), . % ( ) of second-years (ms s), . % ( ) of third-years (ms s), and . % ( ) of fourth-years (ms s) (p < . ). among all classes, the most common reason chosen was inability to explore specialties of interest ( , . %), and the second was inability to bolster their residency application ( , . %). out of the ms s who chose the latter, the majority were concerned about recommendation letters ( , . %) and away rotations ( , . %). as high as . % ( ) of ms s said they were more likely to take an extra year during medical school as a result of the pandemic. region of the us, number of local covid cases, and number of local covid deaths had no effect on whether respondents thought the pandemic would affect their specialty choice. conclusions: our study found that about one-fifth of surveyed medical students currently believe that the covid- pandemic will affect their choice of specialty, with many of these citing concerns that they cannot explore specialties or obtain recommendation letters. with prolonged suspension of clinical rotations, targeted efforts by medical schools to address these concerns through enhanced virtual curriculum development and advising strategies will become increasingly important. further study is needed to explore whether these cross-sectional student perspectives will manifest as changes in upcoming national residency matching program data. the global pandemic caused by sars-cov- has caused unprecedented changes to almost every aspect of society, with social distancing orders taking effect across the usa (us) beginning in march [ , ] . medical student education has been uniquely impacted by these changes. for the vast majority of medical students across the country, all clinical learning has been indefinitely suspended in accordance with the american association of medical colleges (aamc) recommendations on march [ ], including visiting student learning opportunities (away rotations) [ ] . many medical schools have now developed virtual curricula to continue medical education until students are able to return to hospitals and clinics [ ] [ ] [ ] [ ] [ ] . however, lack of in-person clinical exposure may pose a challenge to medical students, particularly those attempting to choose a specialty or apply to residency programs. published commentaries by medical students have indicated a simultaneous sense of duty to do their part by staying home, as well as concern about how their education will proceed [ , ] . several previous disruptions to medical school clinical curriculum and their effects have been described in the literature. during part of the - severe acute respiratory syndrome (sars) pandemic, medical students in affected countries were barred from direct in-person patient care [ ] [ ] [ ] . at these institutions, alternative virtual coursework was adopted, such as e-learning modules, video vignettes, and virtual reality simulators [ ] . similar measures were adopted in certain regions during the middle eastern respiratory syndrome (mers) pandemic [ ] . when hurricane katrina struck new orleans, tulane university medical school relocated medical student education, including clinical rotations, to four medical schools in houston, texas [ ] . while to our knowledge there are no studies exploring sars or mers impact on medical student specialty choice, one post-katrina new orleans study found that medical students were significantly more likely to specialize in emergency medicine (em) and less likely to specialize in psychiatry in the years after the hurricane than in the years before [ ] . changes in medical education in response to these disasters, while locally disruptive, were not as widespread as the changes brought on by the covid- pandemic. the current changes to us medical education are unprecedented. although there are some existing anecdotal commentaries describing effects of the covid- pandemic on medical education [ , [ ] [ ] [ ] [ ] , studies describing virtual curriculum development [ ] [ ] [ ] [ ] , and reports of medical student mobilization in response to the pandemic [ ] [ ] [ ] [ ] [ ] , to our knowledge no data yet exists describing the current impact on medical students' academic lives. it is not yet known how long the suspension of clinical rotations will continue [ ] . as such, early identification of concerns and potential issues may help guide career advising, virtual curriculum development, and general support for medical students during this unique time. this study aimed to provide a crosssectional view into the impact of the covid- crisis on us medical students' perception of their education and career development. this cross-sectional online survey study collected response data from medical students across the usa from april through april . the survey was designed by three investigators (yb, ac, and kr) and contained multiple choice items with up to possible follow-up multiple choice items depending on the participant's answers (see appendix ) . it contained no identifying information. respondent demographics included us state in which the respondent attended medical school and class year. the survey gave students the option of selecting up to two specialties of interest. a variable for certainty of specialty was created using these responses. those who selected only one specialty were considered certain. those who selected either two specialties or chose the option 'undecided' were considered uncertain. the survey also asked students which electives they had completed if any. using their response to this in conjunction with choice of specialties, a variable was created to indicate whether students had taken an elective in their specialty of interest. those whose specialty was listed as 'undecided', those who had not taken any electives, and those who had taken electives that did not match their specialty of interest were considered as not having taken an elective in their specialty of interest. prior to national distribution, feedback on survey design and content was solicited and incorporated from home-institution medical students with a variety of specialty interests via open-ended verbal discussions (one -person group discussion and four -on- discussions). responses to the survey were collected and managed using redcap electronic data capture tools [ , ] . the survey was selfadministered and accessed by participants via an electronic link. participation was voluntary, and participants were allowed to terminate the survey at any time. this project was reviewed and determined to qualify as quality improvement by the university of pennsylvania institutional review board. the survey was distributed nationally using a multipronged approach. a list of all american association of medical colleges (aamc) medical schools in the continental usa, hawaii, and alaska was generated [ ] . canadian, caribbean, and puerto rican aamc medical schools were excluded from this list. for each school on the list, deans or directors of student affairs were contacted via email. they were given a description of the study and asked to distribute the survey link to their institution's entire medical student body. we did not have a method by which to confirm which deans or directors actually distributed it. in addition to this approach, based on investigators' knowledge of existing communication structures among students, additional medical student-led distribution was also achieved at many schools through email listservs and social media groups. all analyses were conducted using sas version . (sas institute, cary, nc). cross-tabulated frequencies and percentages were calculated, and all associations were quantified using chi-squared and fisher's exact tests, as applicable. fisher's exact tests were used in cases where a combination of two variables resulted in any cell counts of five or less. a p-value equal to or less than . was considered to indicate a statistically significant relationship between two variables. state of medical school location was categorized as one of four usa regions (northeast, south, midwest, and west) using guidelines from the us census bureau [ ] . publicly available covid- data were used as proxy measures for pandemic severity in each us state. covid- confirmed cumulative positive case counts and cumulative death counts by state as of april at pm est were acquired from the covid tracking project [ ] . this date was chosen because it was halfway through our -day study period. the number of positive cases in each state was originally a continuous variable with median , . using this, number of cases was dichotomized into roughly two equal groups, at least , cases and under , cases. similarly, the median number of deaths was and was dichotomized into at least deaths and under deaths. responses were analyzed, from us states [ table ]. the majority of responses were from the northeast ( , . %), south ( , . %), and midwest ( , . %), with a minority ( , . %) from the west. a plurality of respondents were ms s ( , . %), with ( . %) ms s, ( . %) ms s, and ( . %) ms s. a small minority of respondents ( , . %) were md-phds within the research part of their program, students taking a year out during medical school for research, dual degree, or other reasons, or 'other'. for simplicity of analysis, these respondents were collapsed into one category titled 'other'. respondents' level of clinical exposure (as measured by whether they had completed, not started, or partially completed core rotations) differed across class years (p < . ) [ table ]. a majority of ms s had not yet begun clerkship year ( , . %), while the majority of ms s had begun the year but not completed it ( , . %). in total there were ( . %) students who were part-way through clerkship year when the pandemic started. out of these, roughly half of the ms s had not completed either surgery or internal medicine ( , . %), while about the same proportion of ms s had completed both ( , . %). the percentage of respondents who were certain about their specialty choice differed significantly across classes; . % ( ) of ms s were certain, as opposed to . % ( ) of ms s and . % ( ) of ms s (p < . ). when asked to indicate up to two specialties they were 'seriously considering', ms s were considering a broad range of options, with internal medicine (im), pediatrics, and emergency medicine (em) being the most commonly chosen [ table ]. as high as . % ( ) of medical students surveyed thought that the pandemic would affect their choice of specialty. there was a significant difference across class years, with . % ( ) ms s, . % ( ) ms s, . % ( ) ms s, and . % ( ) of ms s saying their specialty choice would be affected (p < . ). across all class years, the most common reason chosen was 'i may not have the opportunity to explore my specialty or specialties of interest' ( , . %). the majority of ms s had not done an elective in their specialty of choice ( , . %). however, on further analysis of ms s, there was no significant relationship between completion of an elective of interest and certainty in specialty choice. out of those who thought the pandemic would affect their choice of specialty, the second most common reason given was, across all classes, 'i no longer have the ability to bolster my application' ( , . %). when asked what they meant, ms s who chose this reason were most commonly concerned about 'letters of recommendation' ( , . %) and 'away rotations' ( , . %), whereas ms s most often chose 'taking board exams' ( , . %), and ms s most often chose 'research' ( , . %). when ms s were asked whether the covid- pandemic has made them more likely to take an extra year in medical school before applying to residency, ( . %) said yes. out of these, the most common reason given was 'it would make me more likely to match to my satisfaction' ( , . %). three hundred and twenty-seven ( . %) of ms s and ( . %) of ms s were concerned about fulfilling graduation requirements in time. respondents who thought the pandemic would affect their choice of specialty were asked, as a follow-up, what specialties they were 'more likely' and 'less likely' to apply into, respectively. no clearly favored or disfavored specialties emerged. im was the most common answer for 'more likely', while em was the most commonly chosen as 'less likely'. however, em was also the second most common choice put for 'more likely', and im was the second most common choice put for 'less likely'. an analysis using proxy measures for covid- severity level near where respondents attended medical school was done. region of the us, number of statewide covid cases, number of statewide covid deaths all had no correlation with the proportion of respondents who thought the pandemic would affect their specialty choice (p = . , . , and . , respectively). when asked how they were spending their time away from the clinics [ table ], 'research' and 'virtual classes through medical school' were the only academic-related options selected by greater than half of respondents. five hundred and nine ( . %) and ( . %) indicated involvement in service work or community engagement/organizing, respectively. in the non-academic category, 'hobbies', 'exercise', and 'self care/relaxing' were each selected by greater than half of respondents. our survey also included an optional comment box. relevant comments will be summarized in the following section as qualitative information to help lend nuance to our discussion of the results. this study serves as an early snapshot into medical students' perspectives on their education and careers at a time when the covid- pandemic is in full force and clinical rotations remain cancelled. despite substantial curriculum upheaval, only about one-fifth of our respondents indicated that their specialty choice would be affected. most of these students cited concerns about not having time to explore their specialties of interest. medical student specialty choice has been written about prior to the covid- pandemic, and studies have suggested many factors can play a role, such as exposure to role models and core rotation experiences [ ] . jones et al. found that, as one might expect, the positive predictive value of a medical student's top specialty choice increases from end of first year (ranging from % to % depending on specialty) through end of third year (ranging from % to %), suggesting that more exposure to fields of potential interest throughout medical school may result in increased accuracy of selfpredictions. they also found that students' self-reported level of certainty was not correlated with the positive predictive value of their specialty choice [ ] . on the other hand, manuel found that there was a significant relationship between first-semester medical students' predictions of whether they would apply into a technique-oriented or person-oriented specialty and their eventual choice [ ] . at baseline (pre-pandemic), though they might have an idea of the broad category of career they prefer, ms s, ms s, and even ms s can be inaccurate at predicting their future specialty choice regardless of whether they feel certain. our results found that . % ( ) of ms s thought the pandemic would affect their specialty choice, an odd finding considering that by april, when our survey was distributed, ms s have generally already matched or not matched into a single specialty of their choice. there are a couple possible explanations for this finding. some ms s may have chosen not to apply to residency. also, students taking a year out during medical school between rd and th year may have identified themselves as ms s rather than year out students. in our survey, students who indicated that they did not think their specialty choice would be affected were not given follow-up questions asking about their specific concerns. however, many of them independently brought up some of the same concepts in the optional comment box at the end of the survey. though these respondents did not think their choice would be affected, many were still concerned about their ability to be as competitive in their chosen specialty as they otherwise would have been, and submitted comments about away rotations, letters of recommendation, completing board exams in time, and inadequate clinical preparedness due to virtual coursework being a poor substitute for rotations. literature on virtual learning in medical students prior to the covid- pandemic have found that though students feel it augments their education, they do not view it as an adequate replacement for in-person learning [ , ] . by now, many medical schools have created virtual coursework so that students can continue learning from home [ ] [ ] [ ] [ ] [ ] ] . literature on the precise structure and content of coursework being created at different institutions is limited thus far, but given that course development is not centralized on a national level, one can only imagine it varies greatly from school to school in terms of fidelity to a true clinical rotation, level of interactivity, presence of virtual patient contact, or quality of contact with residents and faculty. as such, these curricula likely also vary in terms of how well they address the concerns described in our results, particularly obtaining letters of recommendation, gaining clinical preparedness, and accurately evaluating specialty fit. it is still unclear when medical students across the us will be able to return to clinical rotations [ ] . the longer that rotations remain canceled, the more crucial it will become to carefully examine which types of virtual curricula, if any, are effective at addressing medical students' career-related concerns. our results show that students are currently filling their time with a variety of personal and professional endeavors, most notably, research and the previously mentioned virtual coursework. although medical student volunteering and community-based efforts to combat covid- have been frequently featured in the news and described in the literature [ , , , , ] , our data indicate that only about half of students are involved with these endeavors, which may be less than the common perception. the severity of the covid- pandemic in the us state in which medical students attended school did not have an effect on their perception of the pandemic's effect on their careers. this is a logical finding; local climate may certainly affect other aspects of students' lives such as magnitude of student-led volunteer efforts [ , ] , and concern for safety of self and loved ones. however, if medical students have concerns about their education and career choices, these are most likely to stem from the sudden and drastic disruption to their curriculum [ , , ] . furthermore, our analysis of measures of pandemic severity was at the state level. it is possible that a city-level analysis would produce different results, as it is becoming increasingly clear that covid pandemic severity often varies across different cities within states [ , ] . strengths of this study include a large sample size, a wide geographical distribution, and timely development and distribution. however, several limitations must be discussed as well, particularly the possibility of response bias. students who are willing to take surveys may be a group with different attitudes toward the pandemic and toward career choices than those who are not willing to take surveys. since our survey was also distributed via medical student social media groups, it is also possible that students who regularly engage with these groups on social media may have different traits and perceptions as well. in addition, to maximize anonymity, our survey did not prompt respondents to include their medical school name (merely the state in which they attended medical school); thus, we were not able to track which institutions out of those we contacted actually distributed the survey. our early cross-sectional study did not identify any specialties that were clearly favored or disfavored as a result of the pandemic. future study will be necessary to determine whether the thought processes and concerns revealed in our results manifest as real changes in national residency matching program (nrmp/the match) data, or whether they are transient issues that resolve as students are allowed to resume rotations. in the aforementioned study on medical student specialty choice before and after hurricane katrina (another public health event that disrupted medical education), there were significant changes in specialty choice before and after the event [ ] . however, this change was evident over a -year study period. a similar long-term analysis of medical student specialty choices before and after the covid- pandemic would certainly be a worthwhile study in the future. the covid- pandemic has disrupted clinical education at medical schools across the us. our results indicate that though the majority of medical students do not think this disruption will affect their specialty choice, about one in five do. it is impossible to tell at this early stage whether the careerrelated concerns revealed by our findings will manifest as changes in residency match data. what we can conclude is that the longer the covid- pandemic prevents medical students from completing clinical rotations, the more important it will become for medical schools to address these concerns in a targeted manner through enhanced virtual coursework and advising support. ethics approval and consent to participate: this project was reviewed and determined to qualify as quality improvement by the university of pennsylvania's institutional review board. no potential conflict of interest was reported by the authors. beatrice c. go http://orcid.org/ - - - trump extends social distancing guidelines through end of april -the new york times social distancing. quarantine, and isolation covid- ) and the vslo program the impact of covid- on medical student surgical education: implementing extreme pandemic response measures in a widely distributed surgical clerkship experience leveraging technology for remote learning in the era of covid- and social distancing: tips and resources for pathology educators and trainees going virtual to support anatomy education: a stop gap in the midst of the covid- pandemic medical students' perceptions and an anatomy teacher's personal experience using an e-learning platform for tutorials during the covid- crisis outpatient clinical student rotation without direct patient contact: leveraging care transitions to benefit students and patients. aamc icollaborative medical students are not essential workers: examining institutional responsibility during the covid- pandemic our education mx. our concerns: medical student education impact due to covid- the challenges of "continuing medical education" in a pandemic era medical school on bypass during the sars outbreak sars and its effect on medical education in hong kong avoiding student infection during a middle east respiratory syndrome (mers) outbreak: a single medical school experience baylor college of medicine's support of tulane university school of medicine following hurricane katrina snap shots: the effect of hurricane katrina on medical student career choice. acad psychiatry medical student education in the time of covid- the change to pass/fail scoring for step in the context of covid- no classrooms, no clinics: medical education during a pandemic | aamc the impact of covid- on medical education bright lights in a dark time: medical students step up to help out | aamc itching to get back in": medical students graduate early to join the fight | aamc medical student mobilization during a crisis: lessons from a covid- medical student response team medical student involvement in the covid- response let us help'-why senior medical students are the next step in battling the covid- pandemic guidance on medical students' participation in direct patient contact activities research electronic data capture (redcap) -a metadata-driven methodology and workflow process for providing translational research informatics support the redcap consortium: building an international community of software partners the impact of clerkships on students' specialty preferences: what do undergraduates learn for their profession? positive predictive value of medical student specialty choices person-oriented versus technique-oriented specialties: early preferences and eventual choice the impact of e-learning in medical education the role of e-learning in medical education senior medical students in the covid- response: an opportunity to be proactive fathy r coronavirus is keeping philly medical students out of hospitals, but we are still contributing l opinion. the inquirer pennsylvania department of health key: cord- -y xma authors: mathieson, georgia; sutthakorn, roxanne; thomas, olivia title: could the future of medical school examinations be open-book - a medical student’s perspective? date: - - journal: medical education online doi: . / . . sha: doc_id: cord_uid: y xma nan the stress and uncertainty of undertaking a medical degree and summative open-book examinations (obe) amidst the covid- pandemic is undeniable. however, we wanted to propose the question of a shift in medical written examinations becoming purely online obes in the future. we see this as a positive change for the future of medical education in contradiction to the negative stance taken by others around the open-book nature as well as negativity about continuing with medical education through a pandemic [ ] , something we see as crucial at this time. having completed our summative obes, overall, we believe the process was a fair assessment of our knowledge that was not drastically different due to the open-book nature, supported by analysis of data from imperial college london [ ] . several steps were taken by our institution to overcome the anxieties and challenges highlighted by the authors. the unfamiliar format obes were tackled with a trial run of questions using the assessment interface, the medical schools council assessment alliance assessment suite (mscaa). we found the mscaa to be extremely user friendly with options to highlight and annotate questions, flag answers to come back to later and unanswered questions were clearly identifiable so that they were not missed. in comparison to traditional paper examinations, it was much easier to identify questions that had been flagged or left unanswered, as well as eliminating the transfer of answers to paper computer-marked answer sheets where errors commonly occur. additionally, we were allocated minutes extra per hour of the exam due to unfamiliarity using the mscaa. concerns about internet connection problems were mitigated as the mscaa saved our answers automatically as we progressed through the examination; therefore, if the connection was lost, we could pick up where we left off. furthermore, immediately after the examination, we were informed that if we had experienced any issues, we should file for mitigating circumstances with clear instructions on how to do this. after completing our obe, in hindsight, the use of google and personal notes facilitated the answer to some of the simpler questions. however, these had mostly been committed to memory during revision and through clinical exposure due to their simple nature. yet, more complicated problem-solvingbased questions, often accompanied by investigation results or a long patient prologue, were much more difficult to answer using google or notes. we appreciate that in clinical practice, resources are commonly used by the clinical team to aid treatment in the form of guidelines or referencing the british national formulary (bnf) and we feel that obes are a fantastic way to learn to utilise these in a timepressured environment. the purpose of progression through medical school is to ensure we are safe doctors, even in emergency situations guidelines are followed and advice over management is sought, safe junior doctors do not jump into treatment without consulting a senior if there is doubt. overall, it is undeniable that students require a strong core knowledge, and this remained unchanged by the new obes. however, the style of our revision may have changed from memorising facts to learning how to utilise guidelines, but we feel this is a positive change that corresponds more closely to working life. despite the steps taken by the medical school, academic misconduct remained the main concern for our cohort. students were most concerned about others 'cheating' rather than focusing on their own learning. we feel that it is easy to get side-tracked during exam season about learning purely for exams, and unfortunately, the competitive nature about exam results between medical students is reinforced by the ranking system that contributes towards the allocation of foundation posts once qualified. however, we found comfort from remembering that we are learning to become safe doctors, well equipped with a good set of clinical knowledge rather than purely for exam grades. regardless, cheating was addressed by the medical school and we were informed there would be strict formal disciplinary action for students found to be sharing answers or asking colleagues for help. additionally, the randomised question order in a time-pressure examination meaning there simply was not time to be consulting with others. we have recently been informed that all of our written examinations in our final year will take place as obes, and this is something we see as a positive change for the future. overall, the obe was not more anxiety-provoking, being able to complete it in a familiar and comfortable environment with simple home comforts. as medical education and the curriculum evolves, we envisage that obes could form more frequent and regular 'knowledge checks' as the logistical organisation is greatly reduced compared to traditional paper examinations. undertaking more frequent obes also has the power to reduce overall anxiety around examinations [ ] . finally, we feel that the continued learning of medical students is vitally important and disagree with the view that 'our time as medical students could be spent more productively' [ ] . the role of medical students, especially those entering their final year of study needs to be fully engaged in continued learning to ensure clinical competency to enter the national health service (nhs) as safe and effective junior doctors [ ] . although we recognise the ethical and moral dilemmas we face as medical students wanting to help out, ultimately to have the greatest impact we must be ready to join the nhs when it is our time. cancelling examinations and teaching is not the answer to this. continued learning in such uncertain times gives us structure to our days, which otherwise may be empty, gives us purpose and a goal to work towards, as well as a sense of accomplishment as we progress through. no potential conflict of interest was reported by the authors. the prospects of sitting 'end of year' open book exams in the light of covid- : a medical student's perspective medical students take final exams online for first time, despite student concern effects of online testing on student exam performance and test anxiety statement of expectationmedical student volunteers in the nhs key: cord- -zkm mph authors: wang, jessie j.; deng, aaron; tsui, ban c.h. title: covid- : novel pandemic, novel generation of medical students date: - - journal: br j anaesth doi: . /j.bja. . . sha: doc_id: cord_uid: zkm mph nan united kingdom (uk) and worldwide, and while healthcare professionals fight frontline battles and systems leaders negotiate public health measures, the needs of medical students at various stages of training cannot be forgotten. preclinical students, removed from in-person sessions, have transitioned to rapidly adapted online curricula and assessments. final-year students' experiences differ widely by jurisdiction, ranging from early graduation and provisional registration to entering residency despite postponed board examinations. clinical students not in their final year face perhaps the greatest uncertainty, with limitations to and/or suspension of inpatient and outpatient rotations. as the pandemic evolves, so too must strategies to address the issue it raises for medical education. internationally, displacing medical trainees from clinical settings amidst an infectious disease outbreak has precedent in the severe acute respiratory syndrome (sars) outbreak of . medical programmes had variable restrictions depending on the local extent of the pandemic, including interruptions to licensing examinations, human subject research, and clinical rotations. in canada, clerkship placements saw a significant reduction in quantity and scope, and adaptations to the canadian residency application process were required even following containment of the crisis. nevertheless, even in impacted areas around the world, medical school cohorts were able to receive accreditation as usual. with mounting caseloads and increasing strains on healthcare capacities, some jurisdictions have released final-year medical students into the workforce. the general medical council (gmc) is provisionally registering early graduates as foundation year doctors. association of american medical colleges (aamc) allows student involvement in patient care in locales with critical healthcare workforce shortage, and medical schools in several states have offered students the option to graduate early and join the frontlines. harnessing medical students in the fight against covid- has the advantage, while addressing public health needs, of providing a learning environment with authentic patient experiences for students. many medical students deem it an obligation to contribute to the healthcare response during a pandemic and are eager to do so. considering that today's students will be tomorrow's doctors, it is arguably counterproductive to artificially shield them from the realities of medicine in crisis times. indeed, even off the wards, students are far from immune to the reverberations of the recently evolving medical landscape, as a profession so soughtafter for reasons of security and prestige is suddenly cast as a dangerous occupation. by being proactively incorporated into the healthcare team and allowed to step into the altruistic role that inspired them to pursue medicine, students are able to experience firsthand the role their specialty of interest plays in a medical crisis and make more informed decisions for their future. however, bringing students into hospitals at this time does have significant implications. although final-year medical students are mere months from the change in status to residents and interns, they are not yet bound by contractual obligation as residents are, thus requiring official policies from regulatory bodies. the uk has organised a nationwide, unified approach to deploying early graduates on an "opt in" basis, whereas the aamc has provided guidance on students' participation in direct patient contact activities but left decisions to individual institutions. in the early weeks of the pandemic, personal protective equipment (ppe) shortage, imperfect infection control curricula and adherence, and limited availability of covid- testing posed major safety concerns. students on the wards also increase supervisory demands on physicians, adding strain to already overwhelmed staff capacities. students themselves face unprecedented pressures of high-stakes rapid training, high-acuity hospital environments, and potentially emotionally taxing patient encounters. there is no easy replacement for real-life patient interactions, however. clinical students are understandably concerned about their ability to achieve the competencies tested in clinical exit examinations and, more critically, their future finesse in the postgraduate environment. one solution for keeping students involved with patients is to engage them in the telemedicine systems already in place for physician providers. having been shown to improve equitable access to care, reduce costs, and increase patient satisfaction, telemedicine is sure to become a growing part of the futures of current medical students. while % of us medical schools included telemedicine as a component of required or elective courses in , formal telemedicine training has yet to become mainstream. , there is no better time than now to bridge the gap between medical education and the demands for telemedicine services, both to support virtual patient care during the pandemic, as one us institution is already doing, and looking beyond to an increasingly technology-driven healthcare landscape. as the global medical community addresses and mitigates the immediate effects of the covid- pandemic, indirect impacts on medical education cannot be overlooked. whether or not jurisdictions harness students in the healthcare workforce to care for patients directly, their clinical training should not simply be put on hold. telemedicine can maintain a clinical learning environment through virtual interactions with patients and should be explored fully both for the present crisis and for meeting mounting demands in years ahead. now is the time to instate novel educational experiences to equip this generation of medical students for their future as physicians, beyond the pandemic. collateral damage: the effect of sars on medical education joint statement from the uk health departments, the general medical council, health education england, nhs education for scotland, health education and improvement wales, the northern ireland medical and dental training agency, and the medical schools cou guidance on medical students' participation in direct patient contact activities medical student education in the time of covid- statement of expectation: medical student volunteers in the nhs simulation-based learning: no longer a novelty in undergraduate education lack of telemedicine training in academic medicine: are we preparing the next generation from bedside to webside: future doctors learn how to practice remotely medical students train to support telehealth visits with patients the authors have no conflicts to declare. key: cord- -c oc authors: hui, jin; qing-xin, kong; hui-min, wang title: covid- prevention and control strategy: management of close contacts in hangzhou city, china date: - - journal: j infect public health doi: . /j.jiph. . . sha: doc_id: cord_uid: c oc nan coronavirus disease (covid- ) has been spreading rapidly across the country since its discovery, causing the epidemic in communities, families and hospitals. on february , world health organization announced that it would raise the level of global communication and impact risk to "very high". how to effectively block the transmission of covid- has become an urgent public health problem. in the whole stage of prevention and control, how to effectively manage close contacts is very important [ ] . in hangzhou, what we have done was an intensive medical observation of close contacts according to prevention and control project established by national health commission of the people's republic of china [ ] . close contacts are defined as those individuals who have not taken effective protection and come into close contact with suspected and confirmed cases within meter. the definition of time is from the days before the onset of symptoms of suspected and confirmed cases, or days before the sampling day of asymptomatic infections. management measures include: ) the body temperature and health status of close contacts shall be monitored twice a day; ) close contacts once have any symptoms, including fever, chills, cough, runny nose, sore throat, headache, muscle pain, shortness of breath, vomiting, diarrhea and so on, immediately report to the local health department and sent to designated medical institutions for diagnosis and treatment. ) at the end of the medical observation period, throat swab specimens will be sampled and tested for -ncov nucleic acid detection by real-time pcr. we supervised sixteen medical observation sites in seven districts and counties (table ). all sites have a medical team, a daily life supply team, and a vehicle for transferring the patients under observation to the designated treatment hospitals in case of manifestation fever, cough, diarrhea and other related gastrointestinal and respiratory symptoms . during the supervision, the following issues were recognized. due to the centralized medical observation sites were temporarily requisitioned, there was no possibility to perform a completely separate decontamination of passageway, so it iswas recommended to concurrently disinfect the passageway after use. the waste generated in the sites wa defined as medical waste, which should be collected by a designated person who was only responsible for collecting and transporting medical wasteand stored in a temporary storage room conforming to the requirements medical waste management. during the supervision, it was found that some temporary storage room had potential leakage just like that the medical waste would be taken away or residents living nearby would enter the room, which could not be locked, or was not convenient for routine disinfection. at one observation site, the medical staff entered the room of close contacts to measure body temperature twice a day. however, this practice was considered as increasing the risk of infection among medical staff as well as the increasing the consumption of personal protective equipment. therefore, in most of the centralized medical observation sites, the body temperature monitoring was performed by the observed individuals themselves, then text the body temperature data and their health status to the medical staff by point-to-point wechat and recorded. another objective monitoring method was to take a throat swab for detecting -ncov nucleic acid three times. one was within three days after entered in the medical observation room , the other two was just before be dismissed and the interval time needed to more than hours. covid- can be effectively controlled by setting up the centralized medical observation points, controlling infection sources and cutting off the route of transmission. early containment strategies and core measures for prevention and control of novel coronaviruspneumonia in china national health commission of the people's republic of china. prevention and control project for corona virus disease general office of national health commission key: cord- -u q yxhh authors: miller, anne-marie; behan, robert; smith, ian; griffin, matthew; keane, fionnuala; langan, james; o'rourke, colm; mcaleenan, niall; pandit, abhay; watson, mark title: a multidisciplinary approach to online support for device research translation: regulatory change and clinical engagement date: - - journal: health policy technol doi: . /j.hlpt. . . sha: doc_id: cord_uid: u q yxhh objectives: to promote medical device eu regulatory understanding in the biomedical research community and encourage greater levels of clinical engagement to further medical device research innovation, translation and effective clinical trials. methods: an interdisciplinary, iterative, needs-based design approach was used to develop medical device regulatory training, information and clinical expertise resources. results: a multimedia based self-paced e-learning course focusing on the ‘fundamentals of medical device design and regulation’ was produced in tandem with an interactive online web portal: medtech translate. conclusions: health research translation relies on both clinical input and regulation to drive progress and to ensure quality and safety standards from concept development to clinical investigation. a lack of regulatory awareness and access to clinical expertise has the potential to significantly impact on health research translation and ambition for market. our interdisciplinary academic-regulator-clinical-industry led approach meets the need for a coordinated stakeholder response to support innovation and promote growth in the medical technology sector. regulation functions to protect public health and to provide stability in the marketplace by ensuring delivery of safe, high quality medical devices for clinical use across the european union (eu). in europe the medtech industry employs over , people directly and accounts for some , + companies . ireland figures prominently in this sector, with of the world's top medtech companies based there. ireland has the highest number of personnel per capita employed in healthcare technology in the eu, and is ranked the second largest exporter of medical devices in the same jurisdiction with national output accounting for as much as % of global orthopaedic knee joint production and % of cardiovascular stent production . with the eu medical device market comprising some half million devices, their variation in complexity, risk profile and application makes the implementation and enforcement of harmonised regulation across eu member states an important and challenging task. the eu medical device and in vitro diagnostic regulations (eudr) , were first published in with a view to addressing the many regulatory challenges observed in such a diverse market where safety, consistency of performance and risk control are of paramount importance. on full application the eudr will supersede the existing medical device directives; being broader in scope with greater numbers of products (including devices manufactured within hospitals) subject to their requirements and placing greater emphasis on quality management system implementation. they also involve device classification changes (ivd devices in particular) and increase requirements for in-house regulatory expertise and pre-and post-market clinical data generation. the official date of application of the medical device regulations is may . this is a year later than originally planned with the delay being primarily to allow regulatory agencies, authorities and manufacturers to attend to covid- related concerns. the date of application for the in vitro diagnostic regulations to date remains unchanged. the main objectives of the eudr are to strengthen and develop the existing regulatory system for medical devices, to increase consistency within the system, increase the oversight on the highest risk medical devices and increase transparency. however, the eudr also have enormous potential to impact on device development processes and commercial competitiveness in the sector by significantly changing device innovation and business practices . at all stages of the device development process the implementation of the eudr will demand greater clinical input to meet the increased requirement for proof of clinical safety and performance. in contrast to the directives, the regulations require, for certification purposes, the generation of greater amounts of clinical data for the specific device rather than relying on data from equivalent devices . in addition, the obligations for more robust post-market surveillance systems, post-market clinical follow up in particular, are described within the new regulations. this is particularly true for ivds for which there is a requirement to show, not only that the test is valid, but also that it is necessary for making a treatment decision that will affect patient outcomes . therefore, effective, informed navigation of the regulatory framework by health researchers is vital to facilitate the future translation of innovative medical devices from concept to clinical application. innovation of new technologies affords patients and health systems access to new diagnostic and therapeutic options. significantly, however, in its horizon coordination and support action, the eu identifies a lack of regulatory science in medical teaching and training programmes as . the reason for diminished capacity amongst researchers to incorporate a regulatory approach to research translation, and . as a significant barrier to patient access to state-of-the-art healthcare . from an economic viewpoint continuous innovation and translation of biomedical research is vital for the ongoing success of the medtech industry in europe and to the irish economy. research led innovation and academic partnership has been recognised as key to improving rates of health technology innovation. consequently, a growing need for industry to be part of an integrated ecosystem comprising academia supported by national enterprise strategies focusing on successful commercialisation and reduced times to market has been identified . by increasing clinical involvement from the earliest stages of academic research, a more practical approach to 'disruptive' device development can be driven from a basic research level to advance healthcare. such an approach can make best use of limited resources and produce targeted, effective devices . importantly, from this perspective, in ireland the sector spans some companies with % of those involved in r&d . key to ireland's success has been the level of active collaboration in industry with some % of companies having a shared services mandate and with many of them fostering close linkages with national academic centres of excellence. it is a stated objective of the irish government that 'education and training providers place a stronger focus on providing skills development opportunities that are relevant to the needs of learners, society and the economy' , . in this context, and being particularly mindful of the changing regulatory infrastructure in europe and the recognised need for greater levels of clinical involvement in device innovation, clinical research development ireland, a research partnership supporting biomedical research translation in leading irish universities, in collaboration with cÚram, the science foundation ireland (sfi) research centre for medical devices have sought to firstly, address the lack of medical device regulatory knowledge and training amongst our target audience (clinician scientists, biomedical researchers and small / medium enterprises (with early stage innovation projects)); and secondly, facilitate more active clinical engagement to support innovation and clinical trials. in collaboration with strategically chosen stakeholders to include the national regulatory agency, national standards authority, representation from industry and the national clinical research infrastructure our primary objective was to develop online regulatory training tools focused towards our target audience coupled with a web based regulatory information portal underpinned by a device development clinical support function. greater understanding as to how to navigate the regulatory system and its requirements in the biomedical research community is key to advancing healthcare innovation nationally and europe-wide. a coordinated approach to the gathering and provision of such expertise and knowledge is central to serving this need. the primary objectives of this work were twofold: . to make positive changes to the state of regulatory knowledge in the field; and . to support clinical involvement throughout the life cycle of device development. to realise these aims a joint needs-based design and development approach, supported by strategic stakeholder engagement, was taken to support project resource development regulatory expertise is provided by the irish national competent authority, the health products regulatory authority (hpra) and the national standards authority of ireland (nsai). the nsai, a designated notified body, is responsible for the assessment of conformity of medical devices to the applicable eu medical device regulatory legislation as part of the pre-market approvals, or ce marking process. the hpra, as the national designating authority, is responsible for the surveillance and control of the performance and activities of notified bodies operating within its remit as well as the market surveillance of approved devices to ensure that they are safe, perform as intended and do not pose an unnecessary risk to public health. the hpra is also responsible for reviewing and approving applications to conduct clinical investigations (research) of medical devices in ireland. both agencies support the goals of this partnership by providing oversight of training content and by contributing to the development of project resources, including case study videos describing the conformance assessment process for ce marking from the perspective of the notified body. industry engagement aerogen ltd., a world leading aerosol drug delivery company develops nebulisers for use in diverse clinical settings. in its business and innovation practices aerogen focuses strongly on research translation to ensure that its technology remains state-of-the-art. for this reason we partnered with aerogen ltd. to generate educational video case studies aimed at explaining to our target audiences, from the manufacturer's perspective, how the medical device regulatory framework influences research translation and is applied to manufacturing and business processes. these case studies also reflect on the potential for the eudr to impact on existing and future product generation. results e-learning e-learning is a useful tool to provide a subject matter-focused, flexible and easily accessed knowledge base to support clinical research and career development , . in collaboration with partnership stakeholders, we produced a bespoke online e-learning course, comprising modules, focusing on the 'fundamentals of medical device design and regulation (fmddr)'. fmddr was designed to deliver regulatory knowledge in the context of medical device design and development. it comprises two modules, each devised using different methodological approaches yet both delivering multimedia based, self-paced blended learning supported by web-accessible resources. the purpose of using differing delivery methodologies was three-fold: . to reflect and compliment the divergent subject matter; . to utilise access to contributor expertise as effectively and efficiently as possible; and , the differing styles of both modules will act as methodological test cases to assess user learning preferences to inform future e-learning development and existing module updates. the first module, 'principles of medical device design (pmdd)' describes how the disciplines of engineering and life sciences combine during medical device design and development. content generation was academic led and aimed to convey the multi-and inter-disciplinary nature of the field as well as speaking to key concepts such as biocompatibility and biomaterial / biomechanical processes. pmdd content generation involved identifying and gaining support from internationally recognised medical device researchers, based in cÚram and elsewhere in europe. in all experts contributed to the course content; industry based, from national university of ireland, galway with from universities in italy, the united kingdom and switzerland. contributor competencies include tissue engineering, therapeutic angiogenesis, regenerative medicine, design by self-assembly, the neural-device interface (nanotubes) and orthopaedic implantation, each contributor developed course content based on their area of expertise to include face-to-camera pieces to support the production of video lectures. they also provided course resources to include relevant reading lists and examination material. in addition to core design and development content pmdd also covers more holistic aspects of medical device design including future design and development strategies, the device translation process (how devices are tested and transformed for therapeutic effectiveness and market), the process of assessment of clinical need/market identification, the selection of optimal treatment strategies and the identification of design factors needed for particular clinical applications. the second fmddr module 'eu regulation of medical devices (eurmd)' was formulated using the 'addie' instructional design model (figure ) which supports a systematic iterative needs-focused method of knowledge delivery for specified audiences year and fmddr participants ( % response rate) were surveyed as to their impressions of the course and were asked to rate their responses from (totally disagree) - (totally agree). respondents were very positive regarding the approach to content delivery and indicated strongly that the 'eu regulation of medical devices' module made a positive contribution to their understanding of the field (table ) . also, importantly, % of total respondents ( our work to date, in recognition of the importance of incorporating regulatory concepts and the clinical perspective from the earliest stages of the device development journey, aims to address the recognised paucity of regulatory knowledge/training in our target audience whilst supporting access to clinical expertise in the device innovation space. the particular challenge in respect to this was how to optimise participation and meet the learning needs of diverse audiences. to this end we focused on harnessing a collaborative multifaceted (academic, regulatory and industry) approach to the origination of high-quality, sector focused, digital learning and information platforms. moreover, in reaching out to academia and clinicians our work aligns closely with research and innovation priorities identified at eu level which seek to address regulatory impediments to, and to increase the impact of, medtech innovation through the strengthening of regulatory sciences and supporting regulatory scientific advice in these cohorts , . our method of e-learning design and delivery serves to juxtapose core principles of device design and development with regulatory concepts central to the successful translation of medical device research for the clinic. this approach is important in that it addresses directly a need for the development of skills relevant to industry and academic entrepreneurship, whilst promoting an appreciation of: . the importance of incorporating regulatory principles and concepts from the earliest stages of device research and . the potential of regulation to impact upon device development and innovation processes. the novelty of this work stems from the fact that, in addition to e-learning, we are looking beyond a formal style of knowledge delivery. the information delivered as part of fmddr was designed to align with, and will be delivered in formats complimentary to, the regulatory information and clinical expertise being delivered through the medtech translate portal. furthermore, this work is also unique in that output delivery is not commercially driven and has been specifically developed in support of medical device research and development in clinical academic and early innovation settings, nor is it, unlike a large number of regulatory training offerings available online at present, targeted at those seeking a formal professional regulatory qualification. when discussing device design and development it is important to remember that it is not a unidirectional, one-task-at-a-time process. indeed, many of its facets, to include the manufacturer's regulatory considerations are complex, frequently run in parallel, overlap and often benefit from an iterative approach. a cyclical or iterative approach from concept development through to commercialisation can benefit the manufacturer in that it serves to keep their development team focused on several critical project-related issues (to include regulatory compliance) at any given time. from a strategic perspective this work is also of value to our project partners in that it promotes entrepreneurship and serves to further their respective outreach and education objectives by enhancing regulatory awareness in the field; promoting quality in regulatory approval submissions; enhancing clinical academia-industry collaboration and supporting medical device clinical trials activity. the hpra, for example, in its - strategic programme, identifies supporting life sciences and the leveraging of new technologies for patient benefit amongst its strategic objectives . equally the nsai, is committed to 'actively engaging with key stakeholders to disseminate technical knowledge' and 'to targeting and promoting nsai activities to research and innovation communities and third level institutions' . correspondingly, hrb crci, mandated in its role as national clinical research network coordinator to support trials feasibility, and through its association with the national enterprise agency, 'enterprise ireland', is strategically placed to facilitate access for clinical academics, small-medium enterprises and high potential start-ups to our e-learning and medtech translate portal with a view to supporting device innovation processes and enhancing device investigation capacity in the sector. however, despite the success of the work to date, some limitations and difficulties need to be acknowledged. in the first instance the cost of development of each resource is considerable and may be prohibitive for those considering a similar approach in other projects. equally, the subsequent maintenance of these outputs in terms of personnel hours, expertise and technical support is not inconsequential. as a result we relied greatly, throughout the process, on 'in kind' access to much of the expertise and opinion required to make the ideation and development of this work viable. also, importantly, an ongoing concern throughout was that the material and information made available via the e-learning, and through medtech translate in particular (as a public facing resource), could be considered by end users as being both definitive and prescriptive in terms of the legislation and/or construed as a checklist resource for achieving a ce mark and access to market. as such, every effort was made in the generation of these outputs to ensure that only primary sources were referred to in the text and that users are continually reminded that the platforms are solely designed for education and information purposes and should not be considered as a categorical interpretation of the directives, the regulations or indeed their supporting materials. moreover, having successfully integrated the e-learning into academic streams we must now look to growing, in parallel with the initial strong uptake from clinical research and academia, our external innovation audience. it will be important for sustained growth in the sector to ensure that this cohort of device developers are aware of the regulatory requirements for market access and, as such, have easy open access to training and information tools in this regard. correspondingly, we would envisage medtech translate acting for this cohort as a conduit to the clinical expertise necessary for basic research development and clinical trial support. we also anticipate that medtech translate will, through its linkages to the national clinical research and innovation support infrastructure, serve the increasing need for industry to be part of an integrated ecosystem comprising academia supported by national enterprise strategies . in conclusion, the introduction of the eudr has the potential to significantly impact clinical research and commercialisation activities eu wide. effective navigation of regulatory processes is key to the delivery of new devices to patients and to the eu market. as such, training and access to pertinent legislative guidance and clinical expertise are vital to ensure sufficient levels of regulatory proficiency among health research audiences to support medical device research translation and continued buoyancy in the medtech market. strong, positive sector relationships and clinical engagement are required to harness the appropriate expertise to meet knowledge gaps in the area. our work benefits from an interdisciplinary approach to regulatory knowledge delivery and was borne of an identified need for a coordinated stakeholder response to government and eu policy by supporting clinicalacademic led growth in the sector. our researcher training and support resources benefit from the involvement of clinical, academic, industry and regulatory stakeholders as well as the support of the national clinical research infrastructure. this joint approach to regulatory knowledge delivery and clinical engagement strategically positions the partnership to support the expansion of ireland's, and europe's, position as a global leader in healthcare technology innovation. competing interests: none declared ethical approval: not required about the industry facts and figures irish medical devices association. statement of strategy - . the global medtech hub regulation (eu) / of the european parliament and of the council regulation (eu) / of the european parliament and of the council new eu medical device regulations: impact on the medtech sector big changes for eu medical and in vitro diagnostic device regulations. applied clinical trials strengthening regulatory sciences and supporting regulatory scientific advice european commission succeeding in delivery of value based care. collaborative data-driven and customer-centric innovation for sustainable healthcare multidisciplinary collaboration as a sustainable research model for device development irish medtech association position on the european commission proposal for a regulation on ie) medical technology sector in ireland ireland's national skills strategy -ireland's future development of an online smartphone-based e-learning nutrition education program for low-income individuals e-learning in clinical teaching the effectiveness of blended learning in health professions: systematic review and meta-analysis e-learning methodologies: a guide for designing and developing e-learning courses council directive / /eec of june concerning medical devices directive / /ec of the european parliament and of the council of october on in vitro diagnostics medical devices regulatory framework health products regulatory authority. medical devices engine solutions association (ie) irish medtech association position on ensuring ireland's medtech sector is prepared for the date of application of medical device regulation / and in vitro diagnostic medical device regulation (eu) / new medical technology regulations proceedings of the icde world conference on online learning. zenodo sport and culture (european commission). ects users guide new crdi initiative: open badges awarded for e-learning course completion stage-gate process for the development of medical devices national feasibility study programme a comparison of medical records and patient questionnaires as sources for the estimation of costs within research studies and the implications for economic evaluation feasibility of a physical activity programme embedded into the daily lives of older adults living in nursing homes: protocol for a randomised controlled feasibility study joint incorporation of randomised and observational evidence in estimating treatment effects health products regulatory authority [ie]. our vision, mission and values (health products regulatory authority strategic plan engine solutions policy documents (nsai strategic plan key: cord- - htcturz authors: bleakley, alan title: embracing the collective through medical education date: - - journal: adv health sci educ theory pract doi: . /s - - -y sha: doc_id: cord_uid: htcturz the journal advances in health sciences education: theory and practice has, under geoff norman’s leadership, promoted a collaborative approach to investigating educationally-savvy and innovative health care practices, where academic medical educators can work closely with healthcare practitioners to improve patient care and safety. but in medical practice in particular this networked approach is often compromised by a lingering, historically conditioned pattern of heroic individualism (under the banner ‘self help’). in an era promising patient-centredness and inter-professional practices, we must ask: ‘when will medicine, and its informing agent medical education, embrace democratic habits and collectivism?’ the symptom of lingering heroic individualism is particularly prominent in north american medical education. this is echoed in widespread resistance to a government-controlled public health, where the usa remains the only advanced economy that fails to provide universal health care. i track a resistance to collectivist medical-educational reform historically from a mid-nineteenth century nexus of influential thinkers who came, some unwittingly, to shape north american medical education within a protestant-capitalist individualist tradition. this tradition still lingers, where some doctors recall a fictional ‘golden age’ of medical practice and education, actually long since eclipsed by fluid inter-professional health care team practices. i cast this tension between conservative traditions of individualism and progressive collectivism as a political issue. in this article, i engage the reader with a slice of north american medical and cultural history to better understand the present. in particular, i ask why conservative heroic individualism in medicine still lingers in a global, connected age in which medical education's main political role is to democratize medicine. it is only through flattening hierarchies a crude metaphor, but the point is made-that we can authentically realise the twin promises of patient-centredness and inter-professionalism (bleakley ) . these sociallyresponsible practices, seeded some decades ago, are often now termed "coordinated care" (langberg, dyhr and davidsen ) . i prefer the term 'collaborative care', progressing surface co-ordination of tasks to deeper emotional and values commitment to collaboration as democratic "habits of the heart" (bellah et al. ). at the point of acceleration of the novel coronavirus pandemic in america, with the death toll nearing , and rising (at the time of writing it is over , ), dr anthony fauci, a distinguished public health expert and the primary medical and scientific advisor in the white house, said of his work: "you stay completely apolitical and non-ideological, and you stick to what you do i'm a scientist and i'm a physician. and that's it" mccarthy, ) . in contrast in the uk, also at the height of the coronavirus crisis, richard horton also a doctor, and longstanding editor of the prestigious medical journal the lancetvociferously challenged the uk government's response to the pandemic as too little too late. in an interview with the financial times horton ( ) said, contra fauci: "the idea you can strip out politics from medicine or health is historically ignorant. the medical establishment should be much more politicised, not less, in attacking issues like health inequalities and poor access to care." in an opinion piece in the wall street journal-take two aspirin and call me by my pronouns-stanley goldfarb ( ) , an experienced physician and retired associate dean of curriculum at the university of pennsylvania's perelman school of medicine, complains about 'woke' medical schools where "curricula are increasingly focused on social justice rather than treating illness". 'woke' is invoked here in a disparaging way. it is an african-american term referring to vigilance to acts of discrimination (staying awake, or 'woke') goldfarb's nostalgia for a golden age of medical education supposedly lost to 'woke' interests is badly misguided, as the state of american medicine shows. there is a serious faultline running through american medicine that medical education fails to address. this is the systematic refusal to address upstream community health issues, particularly structural health disparities (inequities and inequalities), focusing instead upon downstream hospitalism unhooked from political concerns. a glaring upstream political issue is that of health care insurance, another is structural racism. stanley goldfarb is not a lone wolf -he represents a reactionary movement in health professions education (hpe) that hankers after a supposed golden age. it is difficult to gauge just how pervasive this movement is, and it may be a generational effect, but my point here is that there is a wider phenomenon at work. prejudice against medical education's potential interest in social justice issues is grounded in a long-standing historical divide between health in the community setting (focused on prevention of illness) and hospital-based medicine (focused on treatment). the latter particularly privileges the doctor as authority figure. william hsiao ( , pp. - ) , a harvard economist, notes: "there are many statistics that illustrate the flaws of the u.s. health-care system", where "the united states is the only advanced economy that does not offer universal health care coverage". where americans spend nearly twice as much per capita on health care than other advanced post-industrial economies, in comparison with european, japanese, australian and canadian societies, americans have "lower life expectancy, higher infant mortality rates, and a higher prevalence of heart disease, lung disease, and sexually transmitted diseases", where income inequality feeds health disparities grounded in poverty and structural racism. this is fed by a dominant values system that resists conservation and celebrates consumption. hsiao notes that around % of the capital spent on health care per annum in america is wasted just as vast amounts of food and energy are wasted. this amounts to $ trillion a year bleeding out from the health care budget. fraud and abuse in insurance claims, high administrative expenses, and duplication of services all add to this waste, yet, again, many americans still do not have any or adequate health insurance. hsiao (ibid, p. ) notes: "health care still remains primarily a private-sector activity driven by the profit motive". here, i am reminded of an acid remark of the celebrated socialist economist john maynard keynes ( ) : "the love of money as a possession -as distinguished from the love of money as means to the enjoyment and realities of life will be recognised for what it is, a somewhat disgusting morbidity". daniel e. dawes ( ) meticulously tracks the development of president barack obama's "comprehensive health care reform" initiated in march , as a model for "the political determinants of health". 'obamacare' was an attempt to address the dilemma of health insurance for all. by july , under pressure from opponents of reform, the president referred to "health care reform" dropping the descriptor "comprehensive", and by august , this had mutated to "health insurance reform". dawes shows how political machinations practically wrecked the plan for health care for all, and how, after donald j. trump's election as president in , efforts have been made to sabotage and dismantle the proposal. but dawes does not get at the root reason why universal health care is so disliked in america, where it is perceived as big government meddling in an individual's business. many individuals saw compulsory obamacare as a 'soviet-like' intrusion on choice. as we shall see, this rabid individualism-a key identity position in particular for wider american culture (bellah )-also permeates american medical education. in the late ′s, president harry truman tried to introduce a universal health care insurance, but this was vigorously blocked by the american medical association (ama), which stirred up a bitter campaign to disparage truman's plan, labelling it "socialized medicine" and even calling it "un-american" and supporting "the moscow party line" (hsiao ) . during the same postwar period, uk doctors responded in the same reactionary way to initial proposals for a national health service, fearing they would lose private work. this stubbornness against government intrusion is part of a frankly hysterical element in the american psyche that so treasures individualism that it favours citizens carrying guns, despite the consequences for high levels of gun crimes. true to type, stanley goldfarb complains about the american college of physicians making statements supporting gun control, suggesting that here medicine has "stepped out of its lane", or is meddling in politics. there are , deaths in the usa annually from gun use, and treating injuries from firearms costs $ billion annually. guns are the leading cause of death among children and adolescents and african american youth, and are implicated in % of over suicides per annum. it was the powerful national rifle association (nra) and the pro-gun lobby who recently advised doctors to "stay in their lane" when issuing statements on gun control and safety. in the usa, while physician political action committees (pacs) are common, doctors seem reluctant to upset the nra and wider gun lobby. this echoes an earlier era where doctors were not outspoken enough about tobacco use as they remained in some cases financially supported by the tobacco industry. but change may be afoot. rebecca cunningham and colleagues (cunningham et al. ) report the "beginning of the end of the medical community's silence on the issue of firearm research and safety". goldfarb also notes disapprovingly: "during my term as associate dean of curriculum at the university of pennsylvania's medical school, i was chastised by a faculty member for not including a program on climate change in the course of study", where "such programs are spreading across medical schools nationwide" as if this were an unwelcome infection. indeed, the ama now welcomes such programmes. out-of-touch sceptics such as goldfarb, meanwhile, might take heed of analyses such as that of alice hill and leonardo martinez-diaz ( , p. ) who suggest that "lost productivity due to climate-related illness are projected to consume an estimated $ billion per year by the time a child born today has settled into retirement". goldfarb chastises medical schools for "inculcating social policy" when they should be teaching students to "cure patients", and blames "a new wave of educational specialists" who are "increasingly influencing medical education", to emphasise "social justice" topics "at the expense of rigorous training in medical science". this in turn, claims goldfarb (and here is the rub), comes of a mindset that "abhors hierarchy of any kind and the social elitism associated with the medical profession in particular". further, "the prospect of this 'new,' politicised medical education should worry all americans". then goldfarb lays the blame at the doorstep of socio-cultural learning theorists supposedly infecting medical education: "theories of learning with virtually no experimental basis for their impact on society and professions now prevail. students are taught in the tradition of educational theorist Étienne wenger, who emphasized 'communal learning' rather than individual mastery of crucial information". what is wrong with "communal learning"? surely this is in any case unavoidable, where no person is an island. indeed, contemporary psychology points to 'self' as a delusion and a philosophical category error, in a networked world in which both skin-to-skin social connections and technology enhanced virtual connections are the norm (oliver ). well before our virtual age, i know of no better description of collaboration than the celebrated passage in herman melville's moby dick, where ishmael and queequeg are roped together for efficiency as they attend to a whale's carcass, dangling over the edge of the ship. if one goes, then the other goes too. melville describes this "as the precise situation of every mortal that breathes". goldfarb and others' collective prejudice for individualism in any case runs counter to the evidence base for the effectiveness of collectivism advertised by inter-professional clinical teams. for example, changes in operating theatre practices that embrace the mandatory global surgical safety checklist must include collaborative briefing and debriefing of lists, found to improve surgical outcomes (gawande ; haynes et al ) . further, more democratic team structures in healthcare lead to better patient outcomes, also improving patient safety and worker satisfaction (borrill & west ) . the source of prejudice against collectivism may rest with a values system grounded in an unexamined emphasis upon the moral status of the heroic individual, the heart of protestant-capitalist ideology (as argued by max weber ( ) in the protestant ethic and the spirit of capitalism, first published in german in and not translated into english until ). subscription to such a values perspective leads to a prejudice against nationalised and centralised healthcare, and collaborative rather than individualistic practices of learning. democracy is translated back to the freedom of the individual (such as the right to bear arms) rather than progressed forward to collective responsibility, however tough to achieve as compromise is always built in (participatory and democratic clinical team process). goldfarb's doomsday polemic properly attracted a largely hostile online response. one hundred and fifty plus alumni of the pennsylvania's school of medicine bothered to strongly refute his claims. on medscape (zheng ), penn med graduate doctors said: "we are compassionate, socially responsible, and grounded in the deep-rooted belief that doctors are vehicles for social justice. we believe that social justice should not only have a place, but a central place, in the medical school curriculum". what then are the historical conditions of possibility for the emergence of such rabid individualism (expressed as 'selfhelp') and demonizing of collectivity? traditions of self-sufficiency permeate mainstream medical education. self-help's centre of gravity for over a century has been north america, but its birthplace is scotland. the scottish doctor, author and reformer samuel smiles , who trained in medicine at edinburgh, published self-help in . smiles was critical of excessive wealth and materialism, but he also thought that poverty was a product of irresponsibility, and avoidable. ironically smiles, one of eleven children, was supported through medical school by finances provided by his mother, after his father had died from cholera. in the ′s, smiles engaged deeply with political reform, arguing for democratic principles including the rights of women. but by the ′s, he had stopped campaigning for general political reform as he vigorously promoted the idea of self-sufficiency. self-help sold , copies in the first year of publication, and by the time of smiles' death in the book had sold over , copies. orison swett marden , an american physician and polymath, had degrees in law, science, and arts as well as medicine. orphaned at the age of seven, as a teenager marden fortuitously came across a copy of self-help and was smitten with smiles' ideas. marden wrote his own self-help book pushing to the front. published in , it was the first and most influential popular psychology book in america. by it had run to editions and became a global bestseller. spurred on by the initial success of his book, in marden founded a magazine called 'success', with a circulation of , . it was indeed a runaway success as the first motivational self-help journal. by then, marden had left medicine, turning his back on hospitals to enter the hospitality industry. he ran several hotels and a holiday resort, and eventually employed over people to produce and deliver his periodical. in , he became the first president of 'the league for the larger life' in new york, an organisation whose mission statement was "to spread a knowledge of the fundamental principles that underlie healthy and harmonious living" and "to assist the individual in the solution of personal problems". the 'pop' psychology, personal development culture was established, grounded in the wider values of the 'lone frontiersman' mentality of heroic-individualism, self-sufficiency, strong work ethic (protestantism's main secular value) and opportunistic capitalism. here, private feelings and emotional life become commodities for capitalist enterprises of self-help an therapy, as arlie russell hochschild ( ) in particular details in the managed heart: commercialization of human feeling. the uk's observer newspaper recently reported a boom in sales of self-help books, particularly pertaining to mindfulness, with sales of million in (walker ). in france, nearly million self-help or health and wellbeing improvement books were sold in , compared with million cookery books and million books on gardening, animals and nature (staista ) . the self-help or personal growth market in the usa is now turning to 'life coaching' and is worth $ billion, predicted to rise to $ billion by (larosa ). while more women than men read self-help books, more men than women write them (zhou ) . stressing the 'frontiersman' virtues of resilience and persistence (core to self sufficiency) and the protestant work ethic, orison marden recounted how his first manuscript copy of pushing to the front had been destroyed in a fire when one of his hotels burned down. he immediately wrote three new versions and sent them simultaneously to three different publishers each wanted to publish the book. again, marden, inspired by samuel smiles, had created the self-realisation movement that today we know by descriptors such as 'personal growth' and 'humanistic psychology', and that has exploded through you tube and social media, affording a significant psychological public health intervention. marden himself was almost certainly influenced by a th century american movement called 'new thought' formed initially by the ideas of phineas quimby. drawing on ideas from a number of religious denominations, the new thought movement's doctrine was that health is a product of 'right thinking', and conversely, sickness a product of 'wrong thinking'. in short, the individual is in charge of his or her fate, and responsible for his or her actions, where psyche precedes and shapes soma. the scottish-american industrialist and philanthropist andrew carnegie ( - ) made a fortune from producing steel. he was a staunch believer in independence, the protestant work ethic and self-help, and admired marden's work in particular. carnegie set up a charitable foundation to re-distribute around % of his considerable fortune (around $ - billion in today's money). there was, however, a dark side to carnegie's beliefs that also characterised samuel smiles' philosophy: those who could not help themselves were seen as either weak or lazy and should be allowed to perish. this twisted reading of darwinian 'survival of the fittest' offered a cruel injunction to the physically or mentally challenged, or to those stuck in a poverty trap. marden's and carnegie's shared value system would come to describe a cultural style and trait among north americans that would shape educational systems and pedagogical practices, focused on self 'improvement'. its main proponent would be john dewey, born in the year that samuel smiles' self-help was first published ( ) and a contemporary of both marden and carnegie. dewey was a firm believer in democracy, but more, in autonomy. democracy advertised individual freedom of expression rather than compromise for the common good. this has now become the credo for neoliberal (free-market and competitive) capitalism, and has proved to be illusory. 'self help' readily becomes 'every man for himself'. should ishmael or queequeg encounter trouble, one would cut the tie and clamber to safety, leaving the other to plunge onto the carcass of the whale into a mess of trouble. the darker competitive side of samuel smiles' legacy has dominated north american pedagogy and this has bled into medical education, including western european versions. the individual, and the cult of individualism expressed competitively (prizes! awards! leadership! mastery!), has been a primary driver for medical pedagogies. (this is not to ignore the major influence of liberatory and explicitly politicised and activist educationalists such as paulo freire). where success stems from self-reliance, so failure is described as reliance on others. recall stanley goldfarb's invective recounted earlier, where in medical education: "theories of learning with virtually no experimental basis for their impact on society and professions now prevail. students are taught 'communal learning' rather than individual mastery of crucial information". such 'mastery' carries the sinister overtones of a master/slave relationship refusing reciprocity and productive social bonds. the self-help philosophy of marden, the pedagogy of dewey, and the philanthropy of carnegie converge in the work of abraham flexner, an ambitious educationalist who ran his own innovative school. for the origins of modern medical pedagogy, we must go back over a century to the politics of abraham flexner and his hugely influential reports on medical education in north america and canada (flexner ) ; and, two years later, in europe. these have been picked over many times by contemporary medical educators. importantly, brett schrewe ( ) argues that flexner has now become mythologised through a "metanarrative" that surely clouds rather than illuminates our understanding of his contribution to hpe. the flexerian myth suggests that the shape of modern medical education should be ascribed to the work of one man again, an heroic endeavour. a classics graduate turned educationalist, abraham flexner was a contemporary of orison marden, his educational inspiration was john dewey, and the funding that allowed his vision of medical education to be realised was gained originally from the legacy of andrew carnegie in the form of the carnegie foundation for the advancement of teaching. this was not explicitly a cabal, but the interconnections between the members of this male group are surely as interesting as the individual figures. flexner's ( ) medical education in the united states and canada: a report to the carnegie foundation for the advancement of teaching is in fact a text of immense political and ethical interest. the carnegie foundation did not want a doctor to carry out the on-the-ground research and subsequent writing of the report, but somebody who would be dispassionate about medical education. many considered flexner ( flexner ( - to be the most important educationalist of his era, even more so than his contemporary john dewey ( - ) whose educational methods flexner greatly admired. on his death, the new york times front page obituary said of flexner: "no other american of his time contributed more to the welfare of his country and of humanity in general". not everybody agreed. an american doctor, lester king ( ) called flexner's report "probably the most grossly overrated document in american medical history", pointing out that recent medical historical scholarship has placed flexner among a network of many equally important figures and factors influencing medical education of his time. the celebrated medical historian kenneth ludmerer ( ) agrees, as we shall see. flexner was the sixth of nine children born to german jewish immigrant parents in louisville, kentucky. his father moritz was a hat seller and his mother esther a seamstress. his eldest brother jacob supported flexner through his first degree at johns hopkins university in baltimore. flexner completed his bachelor degree in years. jacob, a pharmacist, later trained as a doctor, while flexner's older brother simon became a renowned pathologist and bacteriologist. flexner taught greek and latin at high school, and on the back of private education tuition from wealthy donors set up his own experimental school in which lessons were not compulsory and students did not enter for exams. yet many went on to attend prestigious universities and colleges, and the school gained a reputation for educational innovation. flexner married one of his former students, anne crawford, who had become a teacher and playwright. she had a broadway success and the profits financed flexner to close his school and study full time for a master's degree in psychology from harvard, and then to spend a yearlong sabbatical in germany at berlin and heidelberg universities, something of a homecoming, where he decided that the german educational system was the finest in the world. the wealthy carnegie foundation asked flexner in to conduct the planned survey of the quality of medical education in north america and canada. the president, henry s. pritchett, had read flexner's recently published ( ) critique of higher education, the american college: a criticism, in which flexner attacked in particular large lecture teaching as a 'cheap' and 'wholesale' way of turning education into cruder management of learning. flexner was taken by surprise at the invitation as he knew nothing about medical education and had never set foot inside a medical school. when he did, what he saw shocked him. from january to april , flexner visited all medical schools in north america and canada, some only briefly but some of them twice, clocking up visits. most visits revealed available medical education to be largely a totally unacceptable way to prepare doctors for practice. within the dominant flexner narrative, in the majority of schools, admissions policies were poor, haphazard or non-existent. curricula had no formal shape. pedagogies went unexamined. resources were lacking of the schools had no library; schools were poorly equipped and had no link with nearby hospitals. yet the certificates received upon completion of studies licensed graduates to practice medicine. most importantly, there was an over-production of doctors (flexner claimed four to five times as many doctors were being trained in north america as in germany per head of population). only one school-johns hopkins in baltimore, flexner's alma mater-required entrants to possess a prior degree. flexner took this institution as his standard for future development of medical schools. he did not think along lines of equity and equality, such as how such poorly performing schools could be better resourced or helped a 'seeding' approach. rather, he set a standard and then employed the scythe, and brutally. as fee-paying private institutions, medical schools were more interested in profits than standards, often recruiting their students from industrial occupations. flexner had apparently uncovered a scandal. fifteen thousand copies of his report were printed and distributed free of charge causing widespread alarm. by , of the schools only remained. flexner himself had called for a maximum of . he initiated four major changes: medical schools would be university based; faculty would be involved in research, both scientific and educational; students would be recruited only after obtaining an undergraduate degree in sciences; and would learn through a standardised curriculum of two years of anatomy (through cadaver dissection) and bench sciences, followed by two years of clinical study through the university's attachment to hospital settings. in a comprehensive history of north american medical education kenneth ludmerer ( ) points out that flexner's extreme foregrounding from a background of multiple medical educational activities provides a distorted picture. flexner did not suddenly initiate modern medical education singlehandedly, while his observations of medical schools often suffered from a lack of appreciation of how far many schools had come since inauguration especially those that catered for women and minority groups. appalled by the laxity of home-based medical education, some more thoughtful, inquisitive and morally sensitive -american doctors had gained experience in germany where medical students underwent a rigorous education, first socialised as anatomists (through cadaver dissection) and then bench scientists before engaging in clinical medicine. ludmerer (ibid, p. xxii) shows that the flexnerian revolution had antecedents since the mid-nineteenth century "when a revolution occurred regarding how medicine should be taught". the revolution was not confined to medicine, but was one of ideas and adventures in pedagogy based on a social contract. capitalist society, driven by entrepreneurs, would provide the climate for the generation of schools and universities, including medical schools. in turn, doctors educated in these schools would engage in a social contract in which they committed not only to serving communities, but also to developing the highest possible standards of research and professionalism within their field. as ludmerer (ibid.) says, this was a "financial, political, and moral" exchange. financial capital flowed in to medical school development not in dribs and drabs, but in huge quantities, reflecting flexner's new influential role at the carnegie foundation and then the rockefeller foundation. ludmerer (ibid, p. xxiii) notes that in a leading medical school may have a budget of around $ , , by that would have swelled tenfold, to $ million. more sinister is whether or not flexner had consciously decided to come down heavily on those schools that catered for women and minorities (hodges ) . they were the most vulnerable, short of funding and then equipment and expertise. nowadays, as noted above, we would see this as good reason to support them and invest in them to counter both inequity and inequality. it did not seem to matter to flexner that women would be dispossessed of the opportunity to study medicine -a condition that persisted until relatively recently. however, there were certainly open motives for discouraging people of colour to study medicine. flexner suggested that black doctors should only work with black patients using the spurious argument that such doctors might infect white patients with illnesses carried only by people of colour (nevins ). flexner's view about race issues was complex. in a letter from concerning recruitment of staff at princeton university, flexner's belief in offering opportunities to all individuals is clear: "it is fundamental in our purpose, and our express desire, that in the appointments to the staff and faculty as well as in the admission of workers and students, no account shall be taken, directly or indirectly, of race, religion, or sex". further, while flexner was jewish, he never openly spoke out against anti-semitism and was strangely quiet when hitler came to power. in the s, when high-profile jewish intellectuals and scientists emigrated to america, flexner was often involved in employing them through his role as founding director of the institute for advanced study at princeton, where his biggest 'catch' was albert einstein. as michael nevins (ibid.) argues, flexner's achievements in pedagogy and medical education have been lauded, while his infamous character flaws of irascibility and narcissism have been noted, yet his values and beliefs, despite his autobiography and an update in , remain opaque or cloudy, complex, contradictory and difficult to decipher. he must have been conflicted over his love for the german educational system, his jewish ancestry, and what he was seeing in germany as hitler came to power, but he never made this plain. nevins asks why flexner did not openly come out against institutionalized anti-semitism that was reflected in the popularity of the eugenics movement in america during the first half of the th century. the german sociologist max weber ( - was a contemporary of marden, carnegie, dewey and flexner. first published in , but not translated into english until , and then probably unknown to the spearhead figures in american self-help and self-sufficiency thinking, weber put forward a radical idea in the protestant ethic and the spirit of capitalism. goethe's novel elective affinities took the idea-prevalent at the time-that certain chemicals were attracted to other chemicals and would react with these and not with others. goethe took this as a metaphor for human passions and relationships. we still use the term 'chemistry' to describe such affinities. weber poached goethe's idea to explore social and intellectual bonds. he was puzzled as to why market-driven capitalism was so successful in the western world, and suggested that this can be explained by capitalism's elective affinity with the central ethic of protestant belief: 'getting ahead' through self-help and independence, or what we now call a 'work ethic'. protestant calvinism in particular, popular in scotland, england, germany and the netherlands, mapped on to the rapid development of capitalist economies in these european countries when compared to catholic-dominated countries such as spain, france and italy. calvinism encouraged hard work in this life with reinvestment of profits (rather than what was seen as frivolous spending) to set up salvation in an afterlife. more, buying in to this predestination eased any conscience about social and economic inequality in this life -that could be put down to others' laziness or indulgence. we have seen that flexner's fieldwork inquiry and subsequent report uncovered a common model amongst north american and canadian medical schools. whatever their quality and standing, they were all profit-seeking private institutions or businesses. paradoxically, drawing more on john dewey's idea of independent learning than on democratic engagement as the primary pedagogy for medical education, flexner's purging and reconceptualising of medical education drove curricula deeper into capitalist ideology, where knowledge and skills were obtained through individual effort and retained as personal capital. with flexner's initiative, the political body of north american (and then western european) medical education is laid bare. in short, the dominant model of learning remained individualistic and not social, as free-market neoliberal capitalistic and competitive, certainly up to the dawn of the twenty-first century. collaborative or socio-cultural learning theories were still on the horizon. marxist dialectical materialism, where physical objects and artefacts (including languages and symbol systems) played an equal role in learning along with the humans who drew on them, was until recently for most medical educators a foreign language. medical educators whose pedagogies celebrated individual achievement ignored the work of american scholars who had spent time in russia studying collectivist and dialectical-materialist learning theory, such as the psychologist michael cole (cole et al. ). so, for example, peer assessment methods were never properly explored, while the collectivity possible in problem-based learning was consistently displaced by focus on individuals' efforts and how these could be isolated and assessed. stanley goldfarb, above, speaking for an entrenched conservative view, complained of both political activism and pedagogical collectivism tainting contemporary medical education, where "the prospect of this 'new,' politicized medical education should worry all americans". as noted earlier, he lays the blame at the feet of social (and socialist) learning theorists: "students are taught in the tradition of educational theorist Étienne wenger, who emphasized 'communal learning' rather than individual mastery of crucial information". poor Étienne wenger, who came from a medical family but decided to study psychology, and never wrote a thing about medical education until late in his career. wenger's work, with his colleague the anthropologist jean lave, was focused on craft apprenticeships in areas such as the work of butchers. better that he target the psychologist michael cole, mentioned above, who on the advice of jerome bruner went to study learning theory in moscow in for a year, under the direction of alexandr luria. or yrjö engeström, the leading finnish educator, who has done more than anybody globally to promote and develop cultural-historical activity theory (chat) (a descriptor coined by cole) first developed by the russian psychologist lev vygotsky after the revolution. engeström too has worked in america on and off since the ′s, at the university of california, san diego in a department founded by cole, where engeström ( ) says: "i had to learn about multiculturalism and to appreciate ethnic, religious, and other differences between people". here then is a glaring paradox -engeström, seeped in collectivist tradition in helsinki, learns about multiculturalism in san diego, but north american hpe fails largely to learn from collectivist chat. social learning theories did not register in the medical education literature until the early s (bleakley ) . michael cole later became a translator and editor of luria's writings and, for over three decades, was editor of the journal soviet psychology. cole's political interests came partly from the influence of his father, lester cole, a hollywood film screenwriter who held leftwing views and was under surveillance during the height of the mccarthy era. he refused to answer questions when interviewed about his possible communist affiliations, along with a group of hollywood directors and writers who became known as the 'hollywood ten'. in conclusion, stanley goldfarb and others should know better -all medical students and junior doctors learn in social or communal settings such as inter-professional teams on ward rounds, crash teams in resuscitation scenarios, or surgical teams in the operating theatre, and the evidence base shows -as noted earlier-that the more collaborative the team, the better the outcomes not only for patients' health and safety, but also for team members' work satisfaction. goldfarb and others, i guess, would not welcome medical students and junior doctors challenging the system to afford innovation, but would wish neat and quiet absorption into traditions of individualism without a hint of revolution. you would think that current generations of medical students and junior doctors would surely change that, after #metoo and black lives matter, but lingering traditions of heroic individualism are so entrenched that they will surely persist (sabin ) . such entrenchment would mirror what yrjö engeström ( ) calls a "will-to-stability", as opposed to the horizon of "possibility knowledge" achieved through questioning unproductive historical habits. social learning theories follow a model of dialectical learning, in which challenge from below is invited and contradiction is the engine of change. medicine's major contradiction is that, within multi-professional healthcare provision, it remains a stubbornly hierarchical culture while advertising patient-centredness and interprofessionalism, or "co-ordinated care". medicine must democratise, and quickly. john dewey's dilemma was how to fuse rabid autonomy and unbridled capitalism (the american way) with democracy, to give equal weight to 'mind' and 'culture'. how should the individual mind and "habits of the heart" adapt in commitment to collaborative democratic progress? dewey's answer was to integrate culture into individual mind, as a commitment to collective endeavour without losing individual rights and freedoms. this is the american way (sabin ) . but this path morphed into what kenneth galbraith ( ) derides as a "culture of contentment", one of smug self-satisfaction in the face of growing structural inequities and inequalities. rather, as michael cole insisted, we should integrate mind into culture, as extended cognition and affect. in this 'ecological perception' view, the 'mind', or thinking and feeling, is not in the skull, but is afforded by interactions between environmental context and human intentions. context includes artefacts such as technologies, languages and semiotics (signs and symbols). human intentions include 'predictive processing'-the ability to link improvised activity with changing environmental cues (clark ) . if we take this model of cognition seriously, then 'individualism' is already a mis-description, as we are all embedded (and then embodied) in a wider ecological context of shared technologies and languages. in the neoliberal capitalist model, as individuals gain more money, power and prestige, they typically abandon collectivism and drift away from the common good. the less successful are cast adrift, because now we know that as the economic tide rises, it does not bring all boats with it (piketty ) . the socialist way in contrast is to sacrifice individualism for what the collective can afford the individual. this honours principles of equity and equality. while equality provides the same measures for all, equity provides differing measures depending on needs. following alexandr luria's maxim that "the determining factor in the psychological development of the child is the social development of the child", if goldfarb had, for example, been born in cuba, or even a scandinavian country where individualism is subsumed in the collective ideal, he probably would have followed a quite different set of values as a doctor and medical educator. i have -often polemically -argued for the value of a collectivist hpe over an individualistic model and have provided a rationale for this. ahse is a journal that has been at the cutting edge of thinking and practice in the arena of hpe, recognising that major advances in healthcare have been achieved through genuine patient-centred and inter-professional collective care team practices. again, i applaud geoff norman's pivotal role in advancing work in the field. habits of the heart: individualism and commitment in american life broadening conceptions of learning in medical education: the message from teamworking patient-centred medicine in transition: the heart of the matter team working and effectiveness in health care: findings from the health care team effectiveness project surfing uncertainty: prediction, action, and the embodied mind mind, culture and activity money, politics, and firearm safety: physician political action committees in the era of the political determinants of health learning by expanding: years after expertise in transition: expansive learning in medical work medical education in the united states and canada: a report to the carnegie foundation for the advancement of teaching the culture of contentment the checklst manifesto: how to get things right take two aspirin and call me by my pronouns a surgical safety checklist to reduce morbidity and mortality in a global population adapt or perish: preparing for the inescapable effects of climate change the managed heart: commercialization of human feeling the many and conflicting histories of medical education in canada and the usa: an introduction to the paradigm wars it's the biggest science policy failure in a generation how to fix american health care: what other countries can-and can't-teach the united states economic possibilities for our grandchildren xx the flexner report of development of the concept of patient-centredness -a systematic review the $ billion self-improvement market adjusts to a new generation time to heal: american medical education from the turn of the century to the era of managed care abraham flexner: a flawed american icon the self delusion: the surprising science of how we are connected and why that matters capital in the twenty-first century medicine and society from history to myth: productive engagement with the flexnerian metanarrative in medical education stressed brits buy record number of self-help books observer sat the protestant ethic and the "spirit" of capitalism and other writings an open letter to our former dean, dr stanley goldfarb, from + alumni of the university of pennsylvania school of medicine goodreads data show that women reading self-help books are getting advice from men. quartz key: cord- -z ayolzb authors: gaur, uma; majumder, md anwarul azim; sa, bidyadhar; sarkar, sankalan; williams, arlene; singh, keerti title: challenges and opportunities of preclinical medical education: covid- crisis and beyond date: - - journal: sn compr clin med doi: . /s - - - sha: doc_id: cord_uid: z ayolzb covid- pandemic has disrupted face-to-face teaching in medical schools globally. the use of remote learning as an emergency measure has affected students, faculty, support staff, and administrators. the aim of this narrative review paper is to examine the challenges and opportunities faced by medical schools in implementing remote learning for basic science teaching in response to the covid- crisis. we searched relevant literature in pubmed, scopus, and google scholar using specific keywords, e.g., “covid- pandemic,” “preclinical medical education,” “online learning,” “remote learning,” “challenges,” and “opportunities.” the pandemic has posed several challenges to premedical education (e.g., suspension of face-to-face teaching, lack of cadaveric dissections, and practical/laboratory sessions) but has provided many opportunities as well, such as the incorporation of online learning in the curriculum and upskilling and reskilling in new technologies. to date, many medical schools have successfully transitioned their educational environment to emergency remote teaching and assessments. during covid- crisis, the preclinical phase of medical curricula has successfully introduced the novel culture of “online home learning” using technology-oriented innovations, which may extend to post-covid era to maintain teaching and learning in medical education. however, the lack of hands-on training in the preclinical years may have serious implications on the training of the current cohort of students, and they may struggle later in the clinical years. the use of emergent technology (e.g., artificial intelligence for adaptive learning, virtual simulation, and telehealth) for education is most likely to be indispensable components of the transformative change and post-covid medical education. the covid- pandemic required a massive and rapid change in the way we deliver medical education, particularly to the junior years of the medical program. [ ] the global covid- pandemic impacted every sphere of human life including medical education, resulting in the suspension of face-to-face teaching in medical schools across the world [ , ] . medical schools have adopted several innovative strategies in response to the crisis, with shift to online learning and assessment [ ] . however, medical schools are currently facing the biggest challenge, "training medical students within limitations of social distancing" especially in the preclinical phase due to suspension of practical/lab sessions. the british model of mbbs curriculum is spilt into preclinical and clinical phases; the initial years are preclinical years dealing with basic science subjects, i.e., anatomy, physiology, biochemistry, pharmacology, microbiology, and pathology. preclinical phase plays a very crucial role in shaping up scientifically grounded and clinically competent physicians [ , ] . before the outbreak of covid- , blended learning was highly recommended for preclinical years using active learning strategies which were found to be more effective learning this article is part of the topical collection on covid- experiences [ , , ] . online learning emerged as a sole option to continue the semester during lockdown and social distancing era. implementation of a well-structured "blended" preclinical curriculum with integration of knowledge and skills to prepare a better trained doctor is highly recommended. we searched relevant literature in pubmed, scopus, and google scholar using specific keywords, e.g., "covid- pandemic," "preclinical medical education," "online learning," "remote learning," "challenges," and "opportunities." original studies, reviews, editorials, commentaries, perspectives, short or special communications, and policy papers on covid- pandemic were reviewed. information from websites of different medical schools, universities, professional associations, and international or national organizations were extracted. reference lists from retrieved articles were also examined manually for relevant information. preclinical curriculum is considered the "foundation" and "core component" for clinical education and allows students to learn basic sciences. learn in-depth mastery of the preclinical knowledge necessary to prepare tomorrow's doctors for the scientific advances that are rapidly changing the perspective of medical practice. preclinical curriculum utilizes didactic lectures with practical-oriented, laboratory-based hands-on training [ ] [ ] [ ] . however, in recent years, preclinical faculty has been facing the reduction of contact hours with the introduction of integrated curriculum and limited resources [ ] [ ] [ ] [ ] . to accommodate the transition from regional to systemic integration in anatomy, preclinical education in the last few decades observed a slow transition from didactic, lecture-based classroom teaching to small group sessions, problem-based learning, teambased learning, and flipped lectures [ ] [ ] [ ] [ ] [ ] . introduction of these new teaching strategies makes learning more active, studentcentered, engaging, creative, effective, and enjoyable [ ] . new strategies are emerging in the preclinical phase that integrates technology to make learning effective and appealing to students, [ ] [ ] [ ] , making them more relevant and applicable for remote learning during the current covid- pandemic. these strategies proved very useful in the current covid- crisis, and medical students appreciated synchronized online learning, especially preclinical students [ ] . medical education in the last two decades has seen the extensive implementation of evidence-based teaching with the adaptation of new learning strategies supported by digital technology [ ] [ ] [ ] [ ] [ ] . technology-based medical curriculum was found to be more effective than the traditional approach and was well-received by medical fraternities [ ] [ ] [ ] [ ] [ ] [ ] . digital approaches transformed medical education to achieve higher levels of knowledge and skills through competency-based education with the application of adult learning theory [ ] [ ] [ ] ] . in this unprecedented and unforeseen circumstance raised by covid- , online learning may be the only way to continue teaching basic sciences during post-covid- . we will need to develop new and enduring ways to support medical students who may be scarred by this pandemic. [ ] the effect of current pandemic has an enduring transformation on medical education and will likely to have long-lasting repercussions on student learning. the academic leadership has been placed under tremendous pressure in terms of delivery, access, and assessment of courses; [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] while simultaneously preserving the principles of integrity, equity and inclusiveness, fairness, ethics, and safety [ ] . the unprecedented use of online pedagogy as an emergency measure during covid- period, with an adaptation to the "new normal" to deliver preclinical medical education, has brought both challenges and opportunities to medical education [ , [ ] [ ] [ ] [ ] . the main impediments to providing online learning include lack of reliable network infrastructure (hardware and software, network bandwidth) and compatible online platforms that work with existing learning management system (lms) [ , , , [ ] [ ] [ ] [ ] . other challenges included shortage of competent it personnel to extend technical support, zoombombing/cyberattacks on online platforms, increased cost (schools, students, and faculty), time constraints, poor computer technical skills of faculty and students, inadequate infrastructure and resources, and an absence of institutional policies, which will pose real challenges for low-and middleincome countries [ , , , [ ] [ ] [ ] [ ] . the transition from on-campus medical school settings to home environments results in isolation, stress, lack of concentration, reduced interaction with peers, and disturbance caused by family affairs [ , , , , ] . sudden transition from classroom to home learning affected the performance of students and faculty. adaptability to home learning would be a challenging task [ , , , ] . moreover, students would lose access to dissection rooms and practical labs, prosections, models, pathology specimens, skeletons, and other lab-or practical-based materials and equipment [ , , , [ ] [ ] [ ] . this is worrying because the medical curriculum has already shortened the basic medical sciences teaching time and limited students' exposure to anatomy and other important foundation subjects of the medical program [ ] [ ] [ ] . very short notice transitioning of the teaching community from face-to-face to online teaching with limited and in some cases no prior exposure has been a real challenge for preclinical faculty. this resulted in a struggle to find "a best-suited medium" for teaching anatomy and other basic sciences [ , , , [ ] [ ] [ ] [ ] [ ] . the current situation also raised concerns regarding unlimited amount of time spent in front of the computer screen, lack of social interaction, and lack of real-life verbal and nonverbal communication which could result in a passive form of learning [ , , , [ ] [ ] [ ] [ ] . moreover, facial expression and voice would be compromised in online teaching to some extent. online learning will minimize the chances of building a better mentoring relationship between faculty and students as well. the lack of hands-on training in the preclinical years may have serious implications on the training of the current cohort of preclinical students, and they may struggle later in the clinical years [ , [ ] [ ] [ ] [ ] . it is well establish that clerking patients cannot be replace by online learning as "clinical experience and human interaction are extremely important for the practice of medicine" and online learning cannot completely replace in-person live sessions [ ] . clinical students can be benefited when teaching is supplemented by virtual simulations and computer-based models [ , ] . to create a conducive learning environment (internet access, dedicated space) to deliver a "forced home learning" has also been a challenge for both staff and students [ , , [ ] [ ] [ ] . physical (e.g., lack of social interaction and outdoor activity) and psychological wellbeing of both students and staff is of great concern [ ] [ ] [ ] ] . the preparation of online assessments and training for various online platforms has been a struggle for faculty as well as students [ , , , ] . furthermore, unproctored online assessments may encourage students towards academic misconduct or dishonesty, i.e., plagiarism and cheating, [ , ] which may "severely affect the knowledge and conduct of future doctors and therefore the quality of healthcare" [ ] . while covid- has thrown many challenges for preclinical medical education, it has also created opportunities-academic leaders with servant leadership, distribution of leadership responsibilities, and clear lines of communication dealt with the situation aptly [ , [ ] [ ] [ ] [ ] . during this challenging time, the academic leadership presented guidelines/protocols to conduct course delivery and assessment in a remote environment which were found to be helpful for students and staff [ ] [ ] [ ] [ ] . medical schools and faculty utilized novel methods of delivering education, which need to be adopted much earlier to teach "adult learners" [ , ] . online delivery of courses has the potential to enhance student engagement, geographical accessibility, and synchronous/asynchronous learning and assessment [ , , , , ] . medical educators worldwide are trying to learn from experiences of innovative schools and developing survival packages for their medical schools. many medical schools have worked to transform pedagogy by increasing online lectures of basic medical sciences, using technology to replace and enhance basic sciences and laboratory teaching, and implementing online formative and summative assessments [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . online lectures, flipped lectures, videos, virtual simulators, webcasting, online chatrooms, and other technologies and simulation-based teaching modalities have been used to teach preclinical courses [ - , , ] . small group teaching in virtual team settings are found to be effective, even for some laboratory science and practical training contents [ , ] . for example, a recent study showed that fourteen medical schools in the uk and republic of ireland used technology to teach and assess anatomy in medical students (table ) [ ] . medical schools in australia and new zealand used diverse platforms/policies for the delivery of online lectures and active learning (asynchronous/synchronous, discussion boards, prerecorded lectures, and feedback), practical sessions (asynchronous/ synchronous), and online assessments (random q&a, modified weighting, pass/fail, and team exams) [ ] . imperial college london's first-ever online exam by open-book examination (obe) approach, for final years, has proven successful [ , ] . many medical schools have adopted similar approaches to engage students with their studies. all these positive impacts will certainly provide "an initial insight into new and innovative ways of delivering medical education," especially in preclinical phase during the post-covid era [ ] [ ] [ ] [ ] ] . the pandemic has provided opportunities for staff to acquire skills in online pedagogy and digital media production while developing alternative modes of assessments and transferring principles of learning [ - , , , ] . online learning during covid- received positive students' feedback with "an extremely high level of satisfaction and engagement with online learning activities" and brought a psychological resilience among staff and students [ , , ] . this is an opportunity for the academic sector to converge, strengthen connections across countries and continents, and collaborate. medical schools, regulatory bodies, and other stakeholders have a responsibility to ensure that tomorrow's doctors are "sufficiently trained and supported to deliver essential patient care, even in crises" [ ] . medical educators should "learn from this experience and prioritize a forward-thinking and scholarly approach" to implement best practices and flexible approaches adopted in medical education around the world for an effective transition to online learning and assessment [ ] . as pandemic exposed the "flaws" in medical education and healthcare, medical educators should review curriculum using the "bioscientific model of health" and widen the educational approach by incorporating determinants of health (behavioral, social, and environmental factors) [ ] . medical curriculum should include "surveillance for disease, investment in disease and injury prevention, and disaster planning" as core components [ ] . we must remain optimistic as the medical education fraternity has accepted that "transition is inevitable" and has already started preparing to ensure that "transition can be smooth through resilient educational systems" [ ] . we are waiting to see what ingenuities for medical education will emerge in the face of the covid- pandemic [ ] . basic or preclinical sciences are considered the foundation of medical practice. the covid- pandemic has impacted medical education globally. covid- has disrupted medical education and forced basic science teaching to rapidly adapt to changes which will pose unique challenges to the clerkship training. the preclinical medical curricula have seen the introduction of novel methods of delivering education online, and online classes received positive responses from students and faculty. the use of remote learning as an emergency measure has brought both challenges and opportunities for many medical schools worldwide affecting faculty, students, and administrative and support staff. however, the lack of hands-on training in the preclinical years may have serious implications on the training of the current cohort of students, and they may struggle later in the clinical years. medical educationists need to adopt a "forward-thinking and scholarly approach" with curricular innovations to train future medical graduates during and after the pandemic [ ] . to make an online learning package beneficial, it needs to be "suitably integrated into curriculum design" to "guide students through a learning journey, rather than simply made available as another resource" [ ] . moreover, technology-based teaching modalities must be evaluated for effectiveness and sustainability for application in the preclinical settings. appropriate attention must be paid to pedagogical requirements of online learning as well as providing face-to-face clinical training so that students can develop required competencies to practice medicine. if the higher education sector believes that digital education should replace contact teaching and learning, it runs the risk of producing highly qualified people with severely underdeveloped human or social skills. they will just be robots [ ] . (dr. sizwe mabizela, vicechancellor of rhodes university in south africa) terry hartle, vice-president of the american council on education, mentioned the pandemic as an "epochal event" which will "leave a huge impression on the education sector" [ ] . mccullough et al. [ ] also warned: "the covid- pandemic will not be the last major infectious disease that puts learners at risk." however, many medical schools have successfully transitioned their teaching and learning environment to emergency remote learning, having monitored and examined student and staff readiness, and completed online formative and summative exams both in preclinical and clinical years. while bringing closure to the turmoil academic year - , medical schools are now making final preparations for the start of the new academic year and carefully monitoring national and international covid- environments. due to the covid- pandemic, online learning has been expanding as a relatively new concept in medical education. implications for educators now include increased responsibility; a need to alter teaching style, curriculum planning, and assessment strategies; more meaningful and continuous communication; and better preparedness to not only challenge learners of the digital age [ , , , , , ] but also to obtain "deeper learning" and "apply knowledge in the clinical context" [ ] . however, due to the "immersive nature of medical education," medical educators should find innovative ways to have face-to-face teaching alongside online delivery [ ] . novel and authentic assessment strategies are required where students can develop a set of graduate attributes or "soft skills" (e.g., communication skills, reflective practice, group work, and collaboration) and demonstrate understanding and application of the relevant basic science knowledge required for clinical phase [ ] . and, further design of quality assurance strategies that will monitor the online delivery of curriculum and assessment is also crucial. during and after this pandemic, extensive research is required to examine the effectiveness of superimposed online pedagogy and indicators such as students' academic performance, their mental health, and teaching transferable graduate mcqs multiple-choice questions, emqs extending matching questions, saqs single-answer questions, sbas single-best-answers skills. the use of emergent technology for education, such as artificial intelligence for adaptive learning, virtual simulation, and telehealth, are most likely to be essential components of the transformative change and the future of medical education, including clinical training [ ] . author's contributions ug: conceptualized the review, conduct literature review, extracted relevant information, and drafted the manuscript. maam: conceptualized the review, conduct literature review, extracted relevant information, and drafted the manuscript. bs: conduct literature review, extracted relevant information, and reviewed and revised the manuscript. ss: conduct literature review, extracted relevant information, and reviewed and revised the manuscript. aw: conduct literature review, extracted relevant information, and reviewed and revised the manuscript. ks: conduct literature review, extracted relevant information, and critically reviewed the manuscript for important intellectual content. other disclosures none. ethical approval not applicable. disclaimer none. consent for publication all authors reviewed and approved the final version and have agreed to be accountable for all aspects of the work including any issues related to accuracy or integrity. abbreviations covid , coronavirus disease of ; mbbs, bachelor of medicine bachelor of surgery; lms, learning management system; it, information technology; uk, united kingdom; q&a, question and answer; mcqs, multiple-choice questions; emqs, extending matching questions; saqs, single-answer questions; sbas, single-bestanswers the impact of covid- pandemic on medical education a vision of the use of technology in medical education after the covid- pandemic we signed up for this!" student and trainee responses to the covid- pandemic pandemics and their impact on medical training: lessons from singapore medical education today: all that glitters is not gold medical education: past, present and future gamification in action: theoretical and practical considerations for medical educators teaching anatomy using an active and engaging learning strategy technology-enhanced learning in asia: new educational possibilities for the tomorrow's doctors and tomorrow's cures the sudden transition to synchronized online learning during the covid- pandemic in saudi arabia: a qualitative study exploring medical students' perspectives twelve tips for covid- friendly learning design in medical education going virtual to support anatomy education: a stopgap in the midst of the covid- pandemic covid- -the impact on our medical students will be far-reaching medical student education in the time of covid- [published online ahead of print medical education amid the covid- pandemic strength, weakness, opportunity, threat (swot) analysis of the adaptations to anatomical education in the united kingdom and republic of ireland in response to the covid- pandemic academic leadership in a time of crisis: the coronavirus and covid- guidance from the board for undergraduate studies on the waiver of faculty regulations on exams due to covid- forced disruption of anatomy education in australia and new zealand: an acute response to the covid- pandemic the impact of the covid- pandemic on current anatomy education and future careers: a student's perspective the virtual anatomy laboratory: usability testing to improve an online learning resource for anatomy education teaching anatomy at the time of covid- effective use of educational technology in medical education. in colloquium on educational technology: recommendations and guidelines for medical educators. aamc institute for improving medical education: washington covid- and online teaching in higher education: a case study of peking university medical-school-life/medical-school-assessment-during-covid- -shelf-exams-go self-reported cheating among medical students: an alarming finding in a crosssectional study from saudi lifting the carpet" on cheating in medical school exams the impact of covid- on the undergraduate medical curriculum using technology to increase student (and faculty satisfaction with) engagement in medical education medical students take final exams online for first time, despite student concern. the guardian effects of online testing on student exam performance and test anxiety institutional report for covid- impact on medical education medical and surgical education challenges and innovations in the covid- era: a systematic review medical student involvement in the covid- response covid- : lessons from the disaster that can improve health professions education covid- : the global disrupter of medical education covid- and medical education covid- : 'this is not the end of contact teaching covid- : consequences for higher education teaching professional formation in response to the covid- pandemic barriers and solutions to online learning in medical education -an integrative review publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we extend our gratitude to mrs. stella williams, former lecturer in health communication, centre for medical sciences education, faculty of medical sciences, the university of the west indies, st. augustine campus, and trinidad & tobago, for her assistance in reviewing this manuscript for english language and grammar. key: cord- -ef xg q authors: kelen, gabor d; kraus, chadd k; mccarthy, melissa l; bass, eric; hsu, edbert b; li, guohua; scheulen, james j; shahan, judy b; brill, justin d; green, gary b title: inpatient disposition classification for the creation of hospital surge capacity: a multiphase study date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: ef xg q background: the ability to provide medical care during sudden increases in patient volume during a disaster or other high-consequence event is a serious concern for health-care systems. identification of inpatients for safe early discharge (ie, reverse triage) could create additional hospital surge capacity. we sought to develop a disposition classification system that categorises inpatients according to suitability for immediate discharge on the basis of risk tolerance for a subsequent consequential medical event. methods: we did a warfare analysis laboratory exercise using evidence-based techniques, combined with a consensus process of expert panellists. these panellists were asked to define the categories of a disposition classification system, assign risk tolerance of a consequential medical event to each category, identify critical interventions, and rank each (using a scale of – ) according to the likelihood of a resultant consequential medical event if a critical intervention is withdrawn or withheld because of discharge. findings: the panellists unanimously agreed on a five-category disposition classification system. the upper limit of risk tolerance for a consequential medical event in the lowest risk group if discharged early was less than %. the next categories had upper limits of risk tolerance of about % (iqr – %), % ( – %), % ( – %) and % ( – %), respectively. the expert panellists identified critical interventions with a likelihood of association with a consequential medical event if withdrawn, ranging from to on the -point scale. interpretation: the disposition classification system allows conceptual classification of patients for suitable disposition, including those deemed safe for early discharge home during surges in demand. clinical criteria allowing real-time categorisation of patients are awaited. the ability to provide medical care during sudden increases in patient volume during a disaster or other highconsequence event is a substantial concern for health-care systems. history has shown that during disasters and epidemiological outbreaks, hospitals take the burden of caring for the sick and injured. [ ] [ ] [ ] [ ] [ ] [ ] in most major city hospitals, inpatient capacity is constrained on a daily basis. [ ] [ ] [ ] thus, hospitals are concerned about maintenance of inpatient capacity during normal operating conditions, and they are developing methods to improve resource capabilities during surges. , several measures have been suggested for the creation of hospital surge capacity. [ ] [ ] [ ] [ ] [ ] these include: cancellation of elective admissions and operations; redesign of current inpatient space to accommodate more beds when needed; opening of unlicensed or unstaff ed beds; rapidly outfi tting temporary nearby space, such as cafeterias or outpatient facilities; or deployment of temporary portable inpatient units. all these possibilities present unique operational challenges, such as imposition of unpractised routines in the context of rapid incremental demand for inpatient census. furthermore, most options need hospital staffi ng to be increased at a time when the case-mix index can also be expected to increase. however, during some publichealth emergencies, especially those of a com municable nature, hospitals are likely to have substantial reductions in available staff , as happened during the severe acute respiratory syndrome outbreak. [ ] [ ] [ ] thus, for acute-care hospitals, a means to achieve maximum availability of capacity resources to allow for the care of victims of a disaster is a priority. one novel approach to capacity management during disasters is reverse triage, which includes the safe discharge of current inpatients and refocus of hospital resources to those in even greater need. patients might be discharged home, to facilities set up to care for less acute inpatients (eg, public-health contingency stations specifi cally designed for this purpose), nursing homes, or other acute-care facilities. thus, a need exists to develop an easy method to categorise hospital patients for safe discharge to suitable venues (including early discharge home), taking into account the existence and competence (or lack thereof) of available external resources. we report the fi rst phase in the development of inpatient disposition criteria for the creation of hospital surge capacity during catastrophic or high-consequence events. the aim of this phase was to develop a disposition classifi cation system (reverse triage) based on risk tolerance of a consequential medical event as a result of discharge; assuming that critical interventions were withdrawn or withheld. we focus on the main components of the fi rst phase: ( ) conceptualisation of the disposition classifi cation system; ( ) development of operational defi nitions of consequential medical events and critical interventions; and ( ) derivation of risk estimates related to early discharge from a multidisciplinary panel of experts. subsequent phases of this project will use clinical data obtained from nearly hospital patients to examine the validity of the classifi cation system to correctly identify those who can be discharged early. we used evidence-based materials combined with an expert panel consensus that was approved by the institutional review board on human subjects research. we hosted a warfare analysis laboratory exercise, which was developed by the johns hopkins applied physics laboratory in . the exercise uses networked computers that allow anonymous participation through streaming comments entered into individual laptops by panellists. expert panellists ( clinicians or practitioners, nonclinicians, or non-practising clinicians) took part in the -h exercise. conference organisers determined the composition and the variety of desirable backgrounds of the panel members, who were then identifi ed and specifi cally invited. all panellists were involved in health care, but represented diverse perspectives, including those of nonclinicians. the composition of the expert panel is shown in panel . of the participants were from various components of the johns hopkins medical institutions, and eight were from outside agencies and institutions. almost all the local panellists had experience in other health systems in the usa, and four had experience in other countries. those from the home institution represented three distinctly diff erent establish ments: a major academic health centre, an affi liate teaching centre, and a community hospital. four of the authors participated in the panel discussions. individual panellists received a detailed manual of background information on the day's proceedings, goals, objectives, and expectations from the expert panel. need for written informed consent for participation was waived by the institutional review board on human subjects research because all written comments and risk estimates made by the panellists were anonymous and could not be linked back to an individual. the warfare analysis laboratory is a specialised facility equipped with a network of laptop computers, an integrated audio and visual infrastructure with six large-screen, high-resolution, interactive three-dimensional computer graphics display monitors, a designated data-analysis station, and a -seat observation gallery. plans, defi nitions, and assumptions can be displayed on large-screen monitors at the front of the room. one or more of the screens can be dedicated to continuous display of anonymous comments from panellists on each networked computer in real time. items that need opinions or need to be voted on can also be processed and displayed in real-time. computer comments are numbered consecutively to help with responses to specifi c statements. the exercise is a formally defi ned process and is further described elsewhere. the warfare analysis laboratory has a professional team that oversees the process. a trained facilitator guides the process, allowing for discussion of key issues, and maintaining the focus on the day's objectives. before the exercise was started, eight consensus panel organisers held two separate full-day rehearsals for the conference, to ensure that presentations, discussion, and voting would go smoothly. on the day of the exercise, panellists were shown how to use the facility and computer data-gathering techniques with presimulations unrelated or indirectly related to the planned session. panellists were then exposed to short formal presentations on disaster management, surge-capacity concepts (fi gure), notions of risk and risk tolerance in health care, an overview of the project, and rationale and specifi c objectives. evidencebased information was presented about decision-making devices (eg, patient outcomes research team [port] and acute physiology and chronic health evaluation [apache] iii), peer-reviewed publications, and data for adverse events, readmission rate of discharged patients, and iatrogenic events related to being in hospital. the tasks of the expert panel during the exercise were to: ( ) develop an evidence-based disposition classifi cation system for discharge or transfer based on tolerance for the occurrence of a consequential medical event or the need for a critical intervention during the ensuing h after discharge or transfer; ( ) develop consensus defi nitions of consequential medical event and critical intervention; ( ) assign, for each category of the system, the panel's tolerance for a subsequent consequential medical event, or need for an in-hospital critical intervention; and ( ) compile a list of ( ), psychiatry ( ), infectious diseases ( ), obstetrics/gynaecology ( ), oncology ( ), paediatrics ( ), paediatric emergency medicine ( ), general surgery, paediatric surgery ( ), critical care/anaesthesiology ( ), cardiology ( ), nephrology ( ), trauma surgery ( ), ear nose and throat ( ), military surgery ( ), military emergency medicine ( ), military psychiatry ( ) obstetrics and gynaecology ( ), psychiatry ( ), internal medicine ( ), emergency medicine ( ), surgery ( ), paediatrics ( ), military ( ), oncology ( ) disaster management ( ), disaster and military triage ( ), hospital epidemiology ( ), research methodology ( ), risk management ( ), hospital administration ( ), home health services ( ), social work ( ), medical law ( ), medical ethics ( ), patient safety ( ), data analysis ( ), military public health ( ), city and state public health ( ), public health ( ), public health preparedness and response ( ), homeland security ( ), prehospital care ( ), international health care ( ) numbers in each category are shown in parentheses. some physicians and experts are included in more than one specialist area. critical interventions that would imply the need for continued stay in an acute-care hospital. discussions were held of assumptions to be used for defi nition of the disposition classifi cation system. panellists were presented with proposed assumptions developed by the project leaders and were invited to criticise and include additional assumptions. final assumptions under which the conference proceeded are shown in panel . panellists were shown examples of classifi cation systems used for clinical decision making. [ ] [ ] [ ] [ ] the notion and number of categories were discussed by panellists both via open comment and anonymous computer input. discussion focused on dispositions that defi ne severity of illness and likelihood of treatable complications. the process also included a discussion on the language used to defi ne the categories to help with assignment of tolerance of risk for a consequential medical event. consensus was reached about the number of categories based on tolerance for a consequential medical event. to determine into which category of the disposition classifi cation system a patient falls requires the assessment of risk for a consequential medical event. assessment of risk took into account the possibility of medical deterioration, a new medical event, or the untoward eff ect of the withdrawal of a continuing treatment (ie, withdrawal of a critical intervention). the consensus defi nition of consequential medical event developed by the panellists was: unexpected death, irreversible impairment, or reduction in function within h of hospital discharge for which an in-hospital critical intervention would be initiated to stabilise or ameliorate the medical disorder or disorders. panellists reasoned that medical events were consequential only if acute in-hospital treatment or intervention was needed. thus, when the probability of a consequential medical event was determined, the panellists agreed that likelihood should be judged on the basis of need for a new or continuing in-hospital treatment, termed critical intervention. thus, failure to initiate or continue a critical intervention risked a consequential medical event. to orientate panellists with respect to tolerance of risks taken in daily medical care, panellists were presented data for: -day and -day readmission rates taken from adult discharges during year ( ) for various medical services from one of our hospitals (unpublished data), as well as data of baseline inpatient hospital errors from the institute of medicine. risk, consequential medical event, and the defi nition of critical intervention were then discussed in detail. after the defi nitions had been decided on, the panellists voted to defi ne the upper limit of acceptable risk for the occurrence of a consequential medical event (ie, need for critical intervention), for each of the categories of the disposition classifi cation system. voting was on a scale of - %, with votes given to one decimal point. results of the fi rst vote were revealed and further discussion ensued. a second (and fi nal) consensus vote was then taken. after discussion, a consensus defi nition of critical interventions was formed and became embedded in the defi nition of consequential medical event, as noted above. panellists were then presented with a list of critical interventions to consider, and asked to validate or criticise each in view of the consensus defi nition. a list of possible candidate critical interventions was initially developed by the conference organisers. all panellists were allowed to suggest and add interventions to the list. panellists with clinical experience or responsibilities were then asked to rank on a likert scale of to (whole numbers only), how likely, in their opinion, the withholding or withdrawal of • panellists should think globally and not specifi cally about their facilities • the hospital will remain functional (ie, not a target of terrorist threat or disaster) • government and other disaster response will occur • disaster plans will exist • health-care system will recover or be aided within - h • assume ability to smoothly discharge or transfer patients (specifi c logistical issues will be considered separately). • a model to compare risk profi les of competing new patients requiring hospital resources exists • hospital liability protection exists or is not an issue • inpatient beds could be redistributed across various services • quality of care will not be compromised • only basic (non-professional or family) care will be available to patients discharged into the community (relatives, shelters, or home) • hospital patients with dismal prognosis will be considered separately • other eff orts to improve surge capacity will be continuing each of the listed critical interventions would lead to a consequential medical event. a score of ten was deemed to be synonymous with certainty of a consequential medical event, whereas a score of one implied virtually no associated risk with withholding or withdrawing the specifi c critical intervention. panellists without clinical experience or responsibilities participated in discussions, but did not vote. separate votes were taken for withholding and withdrawing the critical intervention. data generated by these votes were presented. after further discussion, a fi nal vote was taken. data were recorded and stored in a database and were then tabulated. only the fi nal votes were used for analysis. data only from clinicians were used for those questions requiring clinical expertise (clinicians could be identifi ed because they logged on as "clinician" rather than "nonclinician"). because the warfare analysis laboratory exercise requires anonymity in recording participant responses, subgroup analyses by medical specialty were not possible. the study sponsor had no role in the study design, data collection, data analysis, data interpretation, or the writing of the report. the corresponding author had full access to all data in the study and had fi nal responsibility for the decision to submit the paper for publication. the panellists agreed that a fi ve category disposition classifi cation system was optimum, for the intended task (table ) . consensus was reached on the understanding and wording of the categories. the upper limit of risk tolerance for a consequential medical event for the fi rst category was less than %. this category represented patients who the panellists judged to be at low risk of a consequential medical event if discharged early; on the assumption that no skilled medical care was available outside the hospital setting. an example would be a patient admitted for intravenous antibiotics for uncomplicated cellulitis, who could readily be discharged and switched to oral medication. the next categories had upper limits of risk tolerance of about %, %, %, and % respectively. the panellists agreed that category represented those patients who, although at some risk of a consequential medical event, might nonetheless warrant discharge if surge capacity was needed for victims of a disaster; especially if incoming victims are at higher risk of consequential medical event than those patients considered for discharge. panellists also agreed that this category of patients could warrant discharge in certain biothreat or other contagion situations in which spread of disease in hospitals would present a substantial added risk. an example would be a patient with acute coronary syndrome with no evidence of high risk for adverse events. category represented patients who were potentially suitable for transfer to another medical facility. as a group, the risk was judged too high for simple discharge home. an example would be a stable elderly patient who is progressing well days after hip-fracture surgery. category patients were those at substantial risk and judged likely to need continued acute-hospital resources. examples of such patients include those in need of emergency surgery, those in need of pressors, and those in active labour. finally, category patients were those who might be too unstable or critically ill even for transfer to another appropriate facility. most of these patients would require specialised medical care. most patients in intensive-care units would be in this category. table shows the consensus list of critical interventions that if withdrawn would result in a consequential medical event. less than half the critical interventions were assessed as or higher (on a scale of - ). only two-thirds were ranked higher than . for every situation, panellists' scoring for critical interventions that were withdrawn compared with those that were not available or withheld was much the same as that for critical interventions that were withheld. we have proposed the notion of in-hospital disposition classifi cation scheme (reverse triage) of inpatients to deal with increased hospital demand during high consequence disasters. a system of patient categorisation based on risk tolerance developed by an expert panel provides a context for further work to determine which patients can be safely discharged early at the time of overwhelming need for hospital-based resources. mean upper limit of tolerance for consequential medical events (iqr) high consequent events are disasters that overwhelm local or regional infrastructure including the health system. the need to plan for and increase hospital capacity during high consequence disasters is now a standard recommendation. , many techniques have been suggested, such as cancellation of elective admissions and surgeries, opening of licensed but unstaff ed beds, use of other spaces that can be readily converted for clinical use, and triage and treat off site as many victims as possible. most of these techniques need increased staffi ng at a time when either staff are not available, staffi ng is degraded because of the disaster itself, or staff are co-opted by the public-health system for other needs. volunteer staff who are unfamiliar with hospital routine, remain an untested resource, and have yet to be shown as valuable for augmenting hospital capacity in any disaster. thus, other ideas for increasing or preserving capacity become important. much has been written about the appropriate triage of disaster victims. the focus is to concentrate resources on the most severely injured or ill who are likely to survive with defi nitive medical care. [ ] [ ] [ ] one way to create hospital surge capacity-through reverse triage-has received scant attention. in military terms, reverse triage refers to treating those who are not seriously injured fi rst to allow them to return to the battlefi eld sooner. here, we expand the notion to the civilian medical model and include consideration of early discharge of the least sick. with present constraints on hospital capacity, making the most of available resources, rather than hoping that plans to augment them can be implemented, becomes an important consideration. patients are generally not discharged from hospitals if a consequential medical event is potentially foreseeable. however, a zero risk policy, although desirable, is not feasible. forster and colleagues report that up to % of discharged patients have an adverse event in the immediate ( week) post-discharge period. despite a strong patientsafety movement today, zero risk for an out of hospital consequential medical event as a condition of discharge is not attainable, and must be balanced against risk tolerance for various medical adverse events, including iatrogenic ones. , [ ] [ ] [ ] [ ] [ ] despite the accepted risks discussed, our experienced panellists were reluctant to ascribe a substantially higher risk to the category patients. even the risk tolerance assigned for category patients can be regarded as conservative in view of the complex issues faced in major disasters. if a % risk of a consequential medical event is accepted, as was the consensus for category , the rate is still much less than the adverse event rate reported by forster. the iqr for votes on categories , , and were fairly narrow, indicating a cohesive consensus. however, the iqr for categories and were quite wide. categories and would be unlikely to be discharged. this report represents the fi rst part of a multiphase study. once the categories are established, the next step will be to derive a system that allows classifi cation of individual hospital patients in real time. to accomplish this, measurable outcomes and prognostic variables are required. the panel initially defi ned critical interventions as a means to understand the notions of risk. however, the consensus critical interventions will also serve as the outcome measures in subsequent studies. what constitutes a consequential medical event is subjective. thus, the critical interventions that are the outcome proxy measures were weighted for likely importance. future analysis will consider the accuracy of such weighting. the ultimate goal is to produce a disposition classifi cation system that guides clinical decision making during a disaster in a manner similar to the use of the apache score , or port score in everyday clinical practice. in view of the increasing trend toward electronic medical data, prognostic variables of potential interest-eg, vital signs, key laboratory values, diagnostic considerations, co-morbid conditions-could be captured and continuously updated in real-time. thus, in the event of a disaster, patients would be predesignated for the lowest risk disposition options, through the use of a system such as the one proposed here. if such a system were in place, each patient would have a continuously, updated reliable risk score to assist in disposition making. starting with those with the lowest risk, discharges would take place in an ascending order of risk. furthermore, in the future when prognostic scoring of risk for pre-hospital or emergency-department based disaster triage are developed and reliable, the entire demand for inpatient resources from all sources can be simultaneously ascertained and decisions made accordingly. the value of such a device is not only to augment surge capacity, but also to help with rapid decisions when complete or part hospital evacuations are required, such as happened during hurricanes katrina and rita, and the severe acute respiratory syndrome epidemic in toronto. a further and potentially important value of the development of a risk-based disposition classifi cation system is that it is also designed for everyday hospital use. emergency departments in many parts of the world are often overcrowded, mainly as a result of severely restricted in-patient capacity. thus, on a small, but real scale, disaster-like settings arise every day. the device being developed here could have a substantial eff ect on the safe management of hospital capacity on a routine daily basis. in fact, preliminary data from the next phase of the study, available at this time, show that such a device might prove useful for such dual purpose. simulating a disaster, we randomly sampled non-intensive-care unit adult patients from medical units in our three-hospital system during weeks. of the patients sampled, ( %) were discharged routinely on the day of the disaster. of the remaining patients, ( %) either had no critical interventions or were discharged in any event during the next h. although full analysis remains to be completed, these data are consistent with those reported by davis and colleagues. physicians and nurses in this study opined that about % of patients were dischargeable during h. when controlling for patients in intensivecare, the proportion of capacity is similar to ours. finally, a study from a tertiary-care teaching hospital in the uk, also using opinion survey of nurses showed that more than a third of inpatient beds (including those in intensive-care units) could be made available within - h of a disaster if increased care were available in the community. the fundamental problem with estimation of capacity on the basis of opinion surveys is absence of evidence that the estimates would result in safe practice. furthermore, as in the case of the uk study, which called for aug mentation of community resources, it is unclear whether these resources could reasonably be increased during catastrophic events. our approach is to establish the scientifi c basis for a practice that can be modifi able for use both daily and during catastrophic events. comments are needed on concerns that might be raised about the work presented here. the expert panellists were from a wide array of professions. although many of the panellists have varied geographic and institutional experience, we did not attempt to ensure that every hospital structure and size was represented by all disciplines. thus, inherent geographic or regional bias in the established ranges of risk tolerance could have occurred. however, as noted, about % of the panel were not from our system, and most of the panellists had other national and international experience. even so, the model described is suffi ciently robust that risk tolerance notions can be scaled to an individual hospital and even an individual service. if adopted for daily routine use, risk tolerances for categories and might change or be subject to local customs. we did not consider patient or lay perspectives, but again, the variables of the proposed device can be modifi ed in those settings that wish to consider such views. also, simulations used here might not represent reality, but most disaster planning is based on simulated exercises due to an absence of empirical data. furthermore, other systems have been developed on the basis of clinical judgment, in the absence of empirical data. one example is the abbreviated injury scale, , which was originally based on clinical-judgment ratings, and has subsequently been shown to be both valid and reliable in measuring injury severity. more recently, hick and o'laughlin, using a less structured consensus format, developed an ethical framework for the distribution of scarce intensive-care-unit resources during disasters. the - h period of concern is not arbitrary. generally local resources are highly stressed for this duration in the aftermath of a severe and sudden disaster. , in fact, the us joint commission on accreditation of healthcare organizations position on hospital preparedness states that hospitals should "ensure a - hour stand-alone capability through the appropriate stockpiling of necessary medications and supplies." other systems for augmenting surge capacity resources, including those defi ned by the national incident management system, are activated within our period of concern. thus, external validation of our approach by the general community is needed, and this report serves as an appropriate foundation for such discussions. the next step is to derive a real-time decision rule or scoring system based on clinical variables that allows accurate categorisation of individual patients, followed by prospective validation. the experience of st vincent's hospital, manhattan two new york city hospitals' surgical response to the emergency department impact of the oklahoma city terrorist bombing severe acute respiratory syndrome and critical medicine: the toronto experience tornado: assessment of the trauma system response and the resulting injuries emergency department visits for home medical device failure during the north america blackout the role of public hospitals in urban health hospital and emergency department crowding in the united states emergency department overcrowding following systematic hospital restructuring: trends at twenty hospitals over ten years us government accountability offi ce. hospital preparedness: most urban hospitals have emergency plans but lack certain capacities for bioterrorism response health care at the crossroads: strategies for creating and sustaining community-wide emergency preparedness systems health care facility and community strategies for patient care surge capacity bioterrorism preparedness i: the emergency department and hospital lessons learned from the evacuation of an urban teaching hospital impact of a major hurricane on surgical services in a university hospital lessons learned from a nightclub fi re: institutional disaster preparedness clinical course and management of sars in health care workers in toronto: a case series an outbreak of severe acute respiratory syndrome among hospital workers in a community hospital in hong kong sars in three categories of hospital workers implications of hospital evacuation after the northridge, california, earthquake us centers for disease control and prevention. fiscal year justifi cation of estimates for appropriations committee the walex process a prediction rule to identify lowrisk patients with community-acquired pneumonia modeling the severity of illness of icu patients: a systems update a new simplifi ed acute physiology score (saps ii) based on a european/north american multicenter study the apache iii prognostic system. risk prediction of hospital mortality for critically ill hospitalized adults institute of medicine committee on quality of health care in america health care system surge capacity recognition, preparedness, and response casualties treated at the closest hospital in the madrid report of the events and the prehospital emergency response can external signs of trauma guide management? lessons from suicide bombing attacks in israel the incidence and severity of adverse event aff ecting patients after discharge form the hospital incidence of adverse events and negligence in hospitalized patients: results of the harvard medical practice study i the nature of adverse events in hospitalized patients: results of the harvard medical practice study ii institute of medicine medical error fi gures are not exaggerated deaths due to medical errors are exaggerated in the institute of medicine report creation of health system surge capacity by the immediate discharge of inpatients hospital bed surge capacity in the event of a mass-casualty incident accelerated discharge of patients in the event of a major incident: observational study of a teaching hospital rating the severity of tissue damage: i. the abbreviated scale rating the severity of tissue damage: ii. the comprehensive scale concept of operations for triage of mechanical ventilation in an epidemic state and regional responses to disasters: solving the -hour problem learning from disaster: the role of federalism and the importance of grassroots response national incident management system this study was sponsored by funding from the agency for healthcare research and quality (u hs - ). data in this manuscript were presented, in part, at the th world congress of disaster and emergency medicine, may , , edinburgh, scotland. key: cord- -wacoz t authors: thirumalaikolundusubramanian, ponniah; meenakshisundaram, ramachandran; senthilkumaran, subramanian title: ethics, legality, and education in the practice of cardiology date: - - journal: heart and toxins doi: . /b - - - - . - sha: doc_id: cord_uid: wacoz t advances in diagnostics, drugs, and devices have revolutionized cardiology practice, and improved quality of life of the patients. however, awareness, achievements, and advances in cardiac health care have enhanced the demands and expectations of the community, which lacks awareness about the social, economic, administrative, professional, and technical constraints or limitations of implementation. being unfamiliar with medical ethics and legal aspects of care, and suboptimal virtues among physicians, have led to patients and the public utilizing legal remedies. in addition, sophistication of medical practice, increasing costs of health care, involvement of insurance systems, increased awareness and high expectations from the community and patients, and increased participation of the media have altered the attitude toward claiming compensation and acquisition of health care delivery among the public. in view of the changing trends of consumers and emerging areas in health care, providers must get accustomed to patient empowerment and come up to their expectations. interactive programs on these aspects in medical education will help students understand not only the intricacies of medical ethics and the laws and shortcomings of health professionals and/or health systems, but also strengthen their knowledge of ethics and laws to improve their practice. principles of ethics and legal issues have to be discussed at every stage of medical education. to improve the quality of the physician–patient encounter, we need to develop formal and informal curricula about effective communication skills. this chapter also highlights preventive measures and educational aspects of cardiology practice. cardiology is a branch of medicine characterized by state-of-the-art biomedical technology. evidence-based medicine is incorporated in the practice of cardiology. ethical dilemmas known to the specialty arise from the decisions to be made between what can be done and what should be done for varieties of cardiovascular pathologies. some of the ethical deliberations are resurrection of a patient with acute coronary syndrome or malignant arrhythmias. the practice of cardiology is increasingly constrained by guidelines, regulations, and legal considerations. all doctors, including cardiologists, have a primary duty of care to individual patients, in addition to their responsibilities to society, institutions, and colleagues. among cardiologists, the propensity to test and treat is closely associated with fear of malpractice suits. cardiology practice has gained importance as a result of the introduction of advanced diagnostics, drugs, and devices. early recognition and appropriate intervention minimizes the consequences of cardiac illnesses, which in turn reduces the cardiovascular mortality and morbidity. at the same time, awareness about cardiac care among community members has increased their expectations. when the expectations are not achieved, legal issues creep in. in an analysis of judicial sentences related to cardiology in spain, % of the decisions were made for plaintiffs and the defects were misdiagnosis including gaps in communication and informed consent. , misuse and unethical practices related to state health insurance in india was reported by a weekly magazine, and it urged medical associations to curtail such violations. factors linked to malpractice claims are patient dissatisfaction, noncompliance with patients' demands, incorrect diagnosis, complication(s) related to procedures, medical malpractice, and so forth. even though many physicians recognize impairment and incompetency among coprofessionals, they generally do not bring them to the attention of the respective authorities or report them. medical ethics is a dynamic field that plays an important role in cardiovascular medicine. codes of ethics, advanced directives, informed consent, privacy rules, and disclosure of conflicts of interest have changed the practice of cardiology. ethical issues are concerned with the ideas of right or wrong, duties or obligations, and rights or responsibilities. there are always gray areas in ethics. ethical norms derived from various laws and federal and state constitutions (fundamental laws of nations) are related to citizens' welfare, safety, and security; professional councils and statutory organizations that deal with practitioners, policies of professional organizations, professional standards of care, fiduciary obligations, institutional policies, and judgments delivered; and public health regulations and other laws related to patient care and the hospital environment. the fundamental principles and the expectations for ethical conduct are: codes of ethics provide a wealth of information on a variety of aspects such as virtue-based ethical decision making, professional responsibilities, counseling relationships, consulting, private practice, evaluation and assessment, research and publications, counselor education, training, and supervision. keith-spiegel and tabachnick offer eight general ethical principles: respect for autonomy of others, doing no harm, benefit to others, fairness and equity, fidelity and honesty, dignity, caring, and doing one's best. trust patients and the public trust doctors and health care systems, despite limitations. trust has various dimensions and determinants. developments in sociophysical scenarios, market-based diagnostics, drugs and devices, economic crises, and the needs of providers have had an affect on systems and services. in addition to blind trust, gopichandran described four more types of trust: calculated trust, trust but with verification, skeptical trust, and impersonal trust (with some overlapping features among them). information given to patients and/or caregivers should have certainty, clarity, and eliminate divergent meaning; whereas komesaroff suggested that clinical communication often requires the deliberate preservation of uncertainty. the doctorÀpatient relationship is the cornerstone in health care. this helps with diagnosis, therapy, prevention, and compliance, as well as improvement of psychosocial quality of life and patient satisfaction. medicolegal duties are essential components to maintain a physicianÀpatient relationship. web-based medical tourism facilitators are the connectors between foreign patients and host countries that identify concerns regarding the information displayed. patients or caregivers are attracted by price comparisons, perceived quality of care, additional support services, other social websites, and patient blogs. these sites do not mention patients' rights. e-health is a multidisciplinary field. the facilitators of web-based services have to be educated about the ethical aspects of patient care, be prevented from exploiting patients or clients, and be required to respect privacy and confidentiality. international organizations, such as the world health organization (who) and the united nations educational, scientific and cultural organization (unesco), have formulated standard ethical guidelines for the quality of health information available on the internet. the various organizations need to focus on four basic principles of e-health information: self-regulation, evaluation of information, regulation, and awareness of users. facilitators of medical tourism have to ensure confidentiality; disclose health regulations, ethical guidelines, and laws related to compensation; provide information on potential risks of travel, facility stays, and procedures; and divulge the correct details of hospitals and providers. modern medicine is supported by advanced technologies and devices used for the purposes of diagnosis, prevention, monitoring, alleviation of symptoms, and therapy. each hospital has a plethora of machines. ubiquitous health care is an emerging field of technology. it uses a variety of environmental and patient sensors and actuators to monitor and improve patients' physical and mental conditions. such machine-dependent health care poses challenging ethical questions related to trust, efficacy, and societal issues; longevity gaps related to economic status; and clinical problems related to failure of medical devices have led to mounting concerns in the regulatory processes. À medical, technical, and computer professionals have to collaborate to develop and deploy ubiquitous health care systems. geppert and shelton have described various aspects of consultation. it is a service and the professionals who provide consultation should follow certain principles and norms. dubois et al. identified environmental factors that enable wrongdoing in medical practice and research. to avoid wrongdoing, education and policy of ethics have to be strengthened. health care providers face ethical, moral, psychological, and medicolegal challenges while providing care for end-stage heart failure. although cardiac transplantation may have limitations, left ventricular assist devices are becoming a more prominent therapeutic option for some. while dealing with such cases, physicians find themselves in a peculiar situation of having a professional obligation to perform whatever is the best for the patient, but doing it in the context of shared decision making. at the same time, health care providers must refrain from doing anything that might conflict with the law or raise legal concerns. in some clinical cases, discussion of medical futility reflects an apparent conflict between respect for patient autonomy and the preservation of ethical integrity of the medical profession. the danger of futility-based exception to informed consent includes the emergence of paternalism and physicians' avoidance of difficult discussions with patients about dying and death. hariharan et al. have described indifference toward ethical and legal issues among doctors and nurses, and the need for appropriate training. integrating the use of complementary therapies in medical practice increases the physicians' risk for license suspension or revocation. , it is suggested that physicians should continue to provide conventional medical care. medical errors are bound to happen. even though they may be known, errors often do not come to light or go undocumented because of the inherent threatening attitude of the existing health care system and the possibility of self-harm. moreover, medical errors are not discussed as a part of medical education. in most situations, nurses and paramedical professionals do not have the liberty to write medical errors in their reports and are not trained to document such incidents because they are considered suboptimal. as a result, prevalence cannot be estimated, occurrence cannot be prevented, and root caused cannot be assessed. the complexity of the current health care system contributes to various types of medication errors, leading to legal liability. reducing such errors is an ongoing process of quality improvement, which is an essential component of clinical audits. in some cases, root cause analysis and redesigning faulty systems have reduced medication errors. medical laboratories have responsibilities to three main groups: patients, professional colleagues, and society. they are required to maintain high standards in sample collection, good laboratory practices, reporting, monitoring, maintenance of records, and confidentiality. while making decisions about resuscitation, health care providers should be guided by science; individual patient or surrogate preferences; local policy and legal requirements with reference to respect for autonomy, advanced directives, living wills, patient self-determination, do not attempt resuscitation (dnar) orders, withholding and withdrawing cardiopulmonary resuscitation (cpr), and terminating resuscitation efforts. all these should include providing emotional support to family, limitations of care and withdrawal of life-sustaining therapies, ethics of organ donation, and privacy. deliberations about ethical issues among cardiothoracic surgeons have gained prominence in recent years and is starting to become part of medical schools' curriculum. a recent review described various ethical issues related to cardiac surgery in a detailed manner. wait time for access to a cardiovascular specialist's evaluation and therapy has an impact on a vulnerable population in many ways including lawsuits. shortage of cardiologists and demands from the community have been discussed by professional societies, universities, and health ministries. if a fetus is found to have a cardiac anomaly, the ethical principle of autonomy creates a responsibility for the physician to help the pregnant woman make an informed decision based on her values and aspirations. a decision to terminate is partly a medical matter and partly a personal decision bound by legal, cultural, and religious constraints. "do no harm" is an established mantra of medical ethics and the profession. in the modern era, medical bills are escalating and are the leading cause of financial harm. physicians, while providing patient-centered care, should also consider patients' financial well-being. they should understand financial ramifications, provide appropriate care, and optimize care plans for individual patients considering their socioeconomic status. in order to achieve patient-centered care, family members and/or caregivers need unprecedented education and assistance. this will entail a fundamental shift from individual autonomy to family-and caregiver-centered care. ward rounds provide a benefit to patients that is safe, effective, and efficient. in addition, rounds can lead to staff and patient satisfaction. when various levels of health care providers see and talk to patients, they gain confidence and are more satisfied. in addition, ward rounds involve teamwork, where senior and junior doctors, medical students, nurses, and paramedical professionals share patient information and gain knowledge. standard ethical guidelines need to be practiced while disseminating information about health, disease, institutions, and physicians to the media. the media (i.e., print, television, internet) has become a powerful source for sharing information and knowledge. issues related to ethics and the media, including showing patients in news stories or taking pictures of them, have to be evaluated, and students of health sciences and practitioners/physicians should be aware of these issues. the importance of the media was revealed during the epidemics of severe acute respiratory syndrome (sars) and the h n influenza infection. it has been suggested that the media become a partner in health care delivery to carry messages quickly to the population, to enforce prevention or therapeutic strategies, and to protect communities from hazards. the medical profession is considered a noble profession. ethics is important for the correct conduct of the professionals and practitioners. there are two levels in a teacher's ethics: legal and/or administrative and personal. medical educators have various roles including teacher, manager, administrator, researcher, and physician, and all roles are governed by and require a code of ethics. in clinical teaching, patients are brought to a consultation room or students are taken to the patient for bedside instruction. in developing countries, many times ethical principles are violated or not followed when patients are involved in medical education. principles of ethics have to be discussed at every stage of clinical teaching. like the fact that human beings need oxygen to breathe, science needs research to live and progress. thus, research is an essential component in the science. publications help exchange and share knowledge for the betterment and advancement of medicine. therefore, authors are expected to follow standard ethical guidelines when submitting research for publication. hall et al. have noted that cardiac patients should be assessed by cardiologists as a matter of course. the authors suggested that health care providers listen to patients' various needs; provide access to information technology (it) for audit, clinical governance, and continuous professional development (cpd); and collect valuable outcome data. the authors, while discussing human rights, stated that cardiac cases should not be delayed when starting treatment or denied ethics for want of resources because of an unpredictable response from the illness. defensive medicine is defined as the ordering of tests, procedures, and visits and/or referrals, or avoidance of high-risk patients or procedures, primarily to reduce malpractice liability. a study from israel revealed that defensive medicine is prevalent, especially among surgical specialties, and more so among those exposed to lawsuits. studies have revealed that defensive medicine is a well-known practice in industrialized countries. emergency medicine, general surgery, neurosurgery, obstetrics, gynecology, orthopedic surgery, and radiology specialists were common in defensive medicine and were affected more by liability cases. the influencing factors for defensive medical practice are increasing demands of the patients, lack of resources, work and patient loads, deviation from standard guidelines, and increasing lawsuits and compensation amounts. measures to reduce lawsuits and complaints are improving the quality of care, clinical audits, anonymous disclosure of medical mistakes, resources, practicing standard guidelines, lawsuit immunity, and no-fault compensation. current public reports compare health care providers in terms of quality or cost to help consumers, who in turn decide where and from whom to seek care. in view of the changing trends of consumers, health care providers need to become accustomed to patient empowerment and meet their expectations. the public expects standard guidelines for treatment; however, they too have limitations, and conflict of interest (coi) is prevalent in the ones related to cardiology. sophistication of medical practice, increasing costs of health care, involvement of insurance systems, increased awareness and high expectations from the community, and increased participation of media have enhanced society's attitude toward claiming compensation and acquisition of health care delivery. this section outlines the various ways by which hospitals and health care providers can minimize their exposure to legal liability or consequences. there are numerous sources for obligation in emergencies and health care providers should identify legal issues and adopt a risk management approach, including characterizing the hazards, establishing a community profile, determining vulnerability, analyzing risk, and identifying and evaluating the management plan. an alternative way to avoid legal liability is to use the pprr (prevention, preparedness, response, and recovery) framework. laws include federal and state constitutions, statutes, and administrative and judicial decisions, which may vary from country to country and/or state to state; thus, a legal cause of action may be created by a constitutional right, by a statute, or by a common law. a cause of action consists of several distinct elements; however, the following must exist before there is legal liability: g plaintiff must have an interest that is protected by law g there must be a legal duty g a breach of duty by the defendant must be provable g an injury must be shown as damage to the protected party g it must be proved that the breach of a law caused injury negligence is the most common allegation (cause of action) in medical practice cases. it is legally defined as the omission of something that a reasonable person, guided by ordinary considerations of someone who regulates human affairs, would do; or the doing of something that a reasonable and prudent person would not do. therefore, negligence is a violation of the duty to use care. it arises when injury results from the failure of the wrongdoer ("tortfeasor") to exercise due care. the four elements required to establish a prima facie case for negligence are duty, breach, causation, and compensable injury. malpractice is defined as a tort or civil wrong committed by a professional acting in his or her professional capacity. the term malpractice refers to any misconduct that encompasses an unreasonable lack of skill or unfaithfulness in carrying out professional or fiduciary duties. malpractice law is a part of tort or personal injury law. the three goals of malpractice litigation are to deter unsafe practices, to compensate persons injured through negligence, and to exact corrective justice. plaintiffs' legal theories or causes of action against a physician are ethical negligence, wrongful death, loss of a chance of recovery or survival, battery and assault, lack of informed consent, abandonment, breach of privacy and confidentiality, product liability because of drugs and medical devices, vicarious responsibility for the acts of others, negligent referral, false imprisonment, defamation, failure to warn or control, negligent infliction of emotional distress, outrage, failure to report, fraud or misrepresentation (deceit), and loss of consortium. the issue of determining patient competence for medical decision making is often difficult. no absolute definition exists. competency is essentially the ability of a person to make a decision, and it is where understanding, appreciation, nationality, and religion begin. the prescriber exercises clinical skill in diagnosing a condition and determines which drug is indicated and in what dose. also, the prescriber has to provide sufficient information to the patient so as to make an informed decision whether or not to take the drug in the light of any possible adverse effects or risks that may be associated with the choice. illegible prescriptions have been a source of legal liability for both pharmacists and prescribers. therefore, prescribers should respond to queries from pharmacists to avoid the possiblity of injury to the patient and expensive litigation for all concerned. patients may be motivated to follow the directions on product information (pi). a consumers' movement view is that an off-label prescription indicates it as experimental; under such circumstances, the prescriber has to provide complete information to obtain the patients valid informed consent. prescribers should focus on evidence and drug quality, as opposed to commercial influences. both prescribers and pharmaceutical companies need ethical regulation. the practice of gifts from pharmaceutical companies to doctors alters the physicians' decision in prescribing the drug, which may be expensive and in turn could cause poor patient compliance. such practice should not be encouraged. pharmaceutical companies should disclose payments made to doctors above a certain value in their accounts, as is already practiced in some countries. in the united states, according to federal law, drug companies must disclose payments made over a certain value to physicians for research and other miscellaneous expenses. a health care provider is likely to be charged for civil actions against injuries resulting from lack of informed consent and standard care. medical treatment and malpractice laws are specific to each state and may vary with different countries. ethical and legal standards require a health care provider to obtain informed consent (ic) before delivering care. if the patient cannot provide it, a legally authorized surrogate decision maker may do so. in an emergency, when the patient is not legally competent to give ic and no surrogate decision maker is readily available, the law implies consent on behalf of the patient. information should be conveyed in a language and at a level that the patient can understand, and consent should be obtained for diagnostic tests, treatment's nature and character, anticipated results, and risks and benefits of treatment (or no treatment), along with other alternative options if available. the informed consent must be voluntary. the physician is responsible for obtaining ic and delineating the potential risks, benefits, and alternatives. physicians must be transparent for all potential ethical or financial conflicts concerning therapies and various other matters related to them or the devices employed in patient care. patients' reaction to confidentiality, liability, and the financial aspects of ic in cardiology research have been described by fortune-greeley et al. in a study, informed consent forms for cardiology practice were found to be suboptimal and lacking in clarity. the number of patients coming to the emergency department (ed) for diversities of toxicological emergencies is on the rise. each patient requires immediate care and yet is unable to give consent due to impaired consciousness-that is, prevents the patient from making informed decisions. some of the challenges faced by emergency physicians are acute organic impairment manifested by confusion, irrational thoughts, or dangerous behavior at times. they should familiarize themselves with relevant laws, which vary from state to state and country to country, and carry out immediate management to avoid liability for negligence and abandonment. there has been much discussion about ic and its types. medical ethicists claim that informed consent is valuable because it supports individual autonomy. in addition, it provides an assurance that patients and others are neither deceived nor coerced. in real clinical scenarios, there are variations among patients. since there are many distinct conceptions of individual autonomy, ethical importance varies. consent forms should be designed to give patients and others control over the amount of information they receive and the opportunity to rescind consent given already. limitations of informed consent are getting informed consent from the young, very ill, mentally impaired, or unconscious patients; or patients brought to the ed. other limitations are nonapplicability for public health policies, access to a patient's personal information by third parties, and duress or constraints experienced by patients. a patient has to prove the provider's failure to follow the accepted standard of care. there are various sources for standard of care. many states apply different standards to specialists and practitioners with accommodations for practice limitations such as availability of medical facilities, services, devices, and the like. common ethical issues are related to medical decision making and good patientÀphysician communication. laws and regulations on surrogate decision making are slowly evolving due to societal changes. it is highly recommended that readers become familiar with the various laws and regulations related to decision making according to their country or state. other areas of ethics with regard to treatment are advanced directives, futile resuscitation, withdrawal of treatment, missed diagnosis, medical issues, incorrect procedures or drugs, prescription-related matters, adverse effects to drugs, failure to screen family members, withholding diagnosis and information, organ transplantation or harvesting, and breach of confidentiality. hospital authorities are vicariously liable for the unlawful act of physicians or nurses, and/or others employed by them when such acts occur during the course and scope of individuals' work. doctors are often alarmed by the patient's bill of rights but should remember it is for everyone, including doctors. familiarizing oneself with the patient's bill of rights is especially important in this era of medical tourism. care providers should disclose patients' rights to their clients before entering a contract. benefits of a patient's bill of rights are: physicians have the privilege of making decisions to investigate, treat, or refer cases. decisions are influenced by clinical, psychological, economic, and other issues. during clinical practice, however, doctors may be at risk of abusing this privilege. similarly, in recommending leave on medical grounds, some doctors are likely to overuse their privilege for want of definitive guidelines. granting medical leave has an impact on productivity; thus, there is a need to develop standard guidelines for recommending a medical leave. both doctors and patients should realize that sickness, pain, and death are part of life and medicine, that diagnostics and therapy have limitations, and that predictions of clinical course and outcome may not always be certain. all these may become less or averted once personalized medicine becomes a reality. doctors and patients have to accept the risk of developing a drug reaction (i.e., drug reactions can neither be predicted nor prevented on all occasions) and treatment complications. good communication and understanding of all these aspects is a part of ethics and, if well understood, litigations are less likely to arise. on most occasions, health care professionals are unaware of the importance of ethical practice. it needs to be introduced in education curricula, and training on clinical ethics in health care settings is also beneficial. , medical schools organize clinico-pathological conferences (cpc) where students and faculty interact with each other, but discussions on judgments related to health care or liability cases and ethics are not often presented. interactive programs in medical schools and clinical practices about such issues will help students and physicians understand not only the intricacies of laws related to health care and the shortcomings of health professionals and health systems, but also strengthen their understanding of ethics and laws, improve their practice and standard of care, and minimize litigation issues. medical students are trained and exposed to cpcs regularly during their studies. they are confident about substantiating their statements with evidence. however, most students do not have the confidence to do a clinical audit, to find deficiencies in medical records, to correlate the laws related to patient care, and/or to provide legal support to patients. this is because knowledge about the legal issues is suboptimal among medical teachers, and legal and ethical aspects are not discussed during regular ward rounds or grandrounds, nor are they incorporated into lecture classes. anything that is not learned in medical school is less likely to be remembered and practiced or inculcated. the university of iowa college of medicine is among a relative few to offer separate courses in ethics and law as part of their curriculum. the european society of cardiology (esc) has made recommendations about how to minimize bias in all scientific communications and continuing medical education (cme), and how to ensure proper ethical standards and transparency between the medical profession and industry. , medical education and training do not focus on the intricacies and importance of consultation aspects and, thus, these have to be incorporated into the curriculum. suitable modules have to be prepared for training doctors on the principles of consultation. current medical curricula in india lacks ethics and medical humanities; training programs to acquire such skills were proposed by the medical council of india, but implementing them awaits permission from the indian government. law and ethics support health care professionals and services rendered. the united kingdom's national health service (nhs) has introduced programs to teach and train health care professionals about law, ethics, education, and development (leed) based on surveys. a central thrust of the program was the initiation of an "introduction to health care law and ethics" study day. in this survey, to % of participants rated the educational value as excellent. although the program has now been stopped, there is a need for all to have access to law and ethics. by and large, there is no dispute for dissemination of knowledge on ethics and law to health professionals. some of the subjects that are included in teaching and training for legal medicine and medical ethics are medical humanities, public health policies, the business aspects of medicine, forensic sciences, end-of-life issues, how to care of special patients, the liability of specialties, and medical liability. also such a curriculum should cover hospitals' reputation in medical practice, nursing facilities, managed care organizations, laws, courts and judicial processes, consumer protection acts, human rights, state medical boards, hospital ethics committees, and medical examiners. cardiovascular training should recognize and address ethical issues, the rational use of resources, and conflicts related to end-of-life care. a cardiovascular advance directive should address the deactivation of a pacemaker, use of an implantable cardioverter defibrillator (icd), or a left ventricular assist device (lvad). in addition, cardiovascular clinicians should understand ethical dynamics and equip trainees with the tools of ethical reasoning because the complexity of the specialty continues to increase. À medical students are exposed to the intricacies and interconnections between physiological systems and their influence on living organisms. there are also opportunities available to introduce and discuss ethics and ethical issues and reinforce ethical principles. introduction of ethics in physiology has led to a greater awareness of the importance of ethical issues and are likely to be more widely practiced in clinical practices too. even though doctors make appropriate efforts to treat patients, they have to be trained and retrained about proper documentation from the point of view of litigation; encouraged to record dying declarations and obtain valid consents; and trained on the preservation and confidentiality of medical records, on the avoidance of hiding facts, and on adherence to standard guidelines for therapy. the latter includes selection of the therapeutic agents, methods, and/ or procedures and substantiating written statements if there are any deviations. in addition, guidance is needed for the reporting of illnesses to public health authorities as per the regulations of the county or state and hospitals. a clinical audit is an essential component of clinical governance. it considers procedures adopted to diagnose, methods used to deliver health care and treatment, utilization of resources, and the effect of outcome along with quality of life of the patient. principles for the best practice in clinical audit, published by the national institute for health and clinical excellence (nice), defines it as a quality improvement process that seeks to improve patient care and outcomes through systemic care delivered against explicit criteria and the implementation of change. even though the terms medical audit and clinical audit are used interchangeably, clinical audit has multiple components related to patients, education and training, health care delivery, resources for health, working relationships, and so on. in addition, a clinical audit helps when listening to patients; understanding patients' expectations; deals with evidence-based practice; assists in the development of local guidelines or protocols; minimizes error or harm to patients; and reduces incidents, complaints, and claims. because a clinical audit involves patients and patient-related matters, confidentiality of information has to be adhered to strictly. for the review of case notes during a clinical audit, there is no need for informed consent from patients as long as confidentiality and anonymity are preserved. medical ethics has been incorporated into medical education and training. however, the shortfall in ethics core competencies does not preclude graduation. there is a need for greater integration of ethics in theory and practice. major concerns are how to make a medical faculty impart medical ethics to students of health care sciences. the "cardiology ethics curriculum" at the university of toronto is a useful resource. ethical components are rarely discussed in during ward rounds. teaching ethics should be incorporated into all clinically oriented teaching and training activities. students can also learn ethics through role models and interaction with patients, instead of formal lectures. because medicine and ethics are inseparable, teaching ethics at the bedside becomes essential but is seldom done. there is a need to introduce bioethics as part of the curriculum at all health science education institutions. the complexity of the technology involved in making a diagnosis and therapy requires a careful search for the right answer under some very difficult situations. training on descriptive bioethics using various modern medical education technologies and discussion with fellow doctors, hospital administrators, religious leaders, and social scientists in difficult situations, can help to make policy decisions amicable to the patient, family, and society. achievements in medical practice and health care delivery depend on the status of the medical education system, the nature of medical manpower, economic resources, the health care system, and, importantly, medicolegal law. the quality of medical manpower and medical services are determined by policy, findings, regulatory mechanisms, professional organizations, direction from judiciary, and expectations and participation of the community. a common difficulty in the current medical education system is that it fails to inculcate appropriate skills and competencies to recognize and tackle ethical issues among the learners. although developing an ethics-based medical education system is a challenging task, we have to take initiatives to deal with a variety of issues. a. knowledge in medicine and timely application in a given situation to assess the case at the bedside meticulously with empathy; analysis of clinical issues and challenges, and differentiating one from another with or without the help of laboratory data; application of appropriate specific and nonspecific measures to alleviate the symptoms or to treat cases with currently available guidelines; acceptance of limitations of knowledge and skills, resources, and constraints; arrangement for referral or consultation; provision of follow-up care, rehabilitation services, or welfare program depending on the case; proper documentation, adequate informed consent, continuous monitoring to strengthen physicianÀpatient relationship, patient satisfaction, and quality of life, thereby avoiding any conflicts or liability issues. b. the attitude of health care professionals toward the patients shall be: . receive them with care and concern . respond with empathy . realize the underlying clinical problem(s) and manage with appropriate measures . review the clinical course, therapeutic strategies, and outcome . revise the plan and management with shared decision . seek further help, if needed c. cardiologists should be informed to establish the kind of actions, communication skills, and empathy that are required to build a stronger patientÀhealth care professional relationship, which improves prognosis, treatment efficacy, and therapeutic adhesion. d. cardiac report cards should be developed that incorporate an ethical framework, which can identify points of ethical concern for practitioners, patients, policymakers, and researchers e. the integrity and behavior of an individual physician plays a major role in the prevention of liability cases. professional associations play a major role in the regular monitoring process and for initiating self-regulation of professionals. to provide a written notice of their privacy policy to all those who seek medical care for the first time. patients must be informed of how the institution may use and disclose information. the written notice must describe patient's rights, including the right to access their medical information and their right to provide feedback or complaint, if they believe their rights were violated. the notice shall be in plain language and presented in an understandable manner. an acknowledgment of receipt of the information notice must be obtained from the patient. the institution shall have a close working relationship among medical personnel, legal, and risk management team. the team, through interaction on case by case basis, can acquire a wealth of information, learn, and meet the needs of clinical dilemma, which are evolving constantly. such interactions enhance health care and ensure patient safety, and thereby establish a high standard of care. also, the institution shall ensure quality assurance, organize risk management programs, and conduct routine audits or monitoring as suggested by a specialist's team. laws generally require that therapeutic interventions be done in a reasonable and medically approved manner, preferably based on clinical evidence or standard guidelines. medical staff dealing with cases should remember that the issues raised by any one case are complex and application in a real situation is difficult. hence, staff should review the hospital counsel and the local laws and regulations pertaining to these issues. l. medical records: medical personnel should be trained and motivated for proper documentation, adherence to risk management principles, and entry of every aspect related to the case in the respective medical record. the physician is required to write a medical record that will support the basis for the appropriate medical judgment made. the medical record should reveal supporting clinical data or facts, the patient's history available at that time, hemodynamic status, physical examination, investigation reports, and treatment plans administered. one must remember that hospital records are prima facie evidence. therefore physicians who do not record supporting clinical data and history deprive themselves of a strong "medical judgment defense." moreover, inappropriate entries or markings on the medical record can weaken the defense in a liability case. if any corrections are required in the medical record, the preferable method is to draw a single line over the word or value to be changed; and at the place where such correction is made, initials or signature should be affixed. however, total obliteration of a number or word may indicate that obliteration was done intentionally. quality-assurance reviews of records of eds often reveal inadequate charting by physicians and nurses. a lapse of documentation of patient's clinical condition for hours or more after initial physician and nursing assessment will be utilized by the plaintiffs attorney in a lawsuit to develop the theory that no care was given. the uncooperative acts of the patient and the difficulties encountered in providing care to the patient because of his or her actions should be recorded. poorly written physicians' notes may become an issue in a medical malpractice action and considered a less-than-caring attitude by the doctor. descriptions of a patient's behavior and lifestyles should depict compassion and a professional manner. health care institutions and cardiovascular physicians should realize consumer rights and responsibilities such as information disclosure, choice of providers and plans, access to emergency services, participation in a treatment discussion, respect and nondiscrimination, confidentiality of health information, and complaints and appeals. m. incentives and ethics: incentives, in different forms, have been introduced for various services and are not accessible in health care delivery. incentive mechanisms are based on four types of motives: traditional, selfinterest, affective, and shared purposes. financial incentives have helped to achieve the quality or efficiency of related targets but have potential adverse consequences including conflicts of interest that threaten patientÀphysician trust. the ethical allegations, implications for effectiveness, and mechanisms of different motives were highlighted. incentive schemes based on a robust sense of shared purposes protect and promote physicians' sense of moral responsibilities and ethical standards. these also enable physicians to take ownership of it rather than make them feel it is imposed on them. n. patient-centered specialty practice: as a part of a new phase of health care delivery systems, the national committee for quality assurance (ncqa) launched patient-centered specialty practice (pcsp) in . the objectives of the committee are to reinforce care coordination, improve access to specialty care, reduce the use of unnecessary or duplicate tests, enhance communication, and assess and improve performance. delay in diagnostic investigations, the nonavailability of interventional procedures, recurrent and repeated system failures, and so on, are real aspects of all our daily lives, yet rarely are considered in mitigation. careful documentation of these failures is paramount, if we need to defend ourselves against a system that seeks criminal prosecution or compensation. a legal decision for violation of ethics may lead to loss of status, career, and livelihood. it is worth studying the patterns and prevalence of defensive medicine and the status of lawsuits in different countries to find any economical impacts and adverse effects. patterns of malpractice insurance systems adopted in various countries and the compensation paid to patients should be analyzed and the information shared among health care professionals, community members, and payers, so as to design modalities to reduce expenses. periodic analyses of physician behaviors and attitudes, along with health care utilization and lawsuits, are likely to help professional organizations and health science educators introduce teaching and training modules for reduction in malpractice lawsuits. the consequences of wait time for health care services in terms of quality of life and health care economics should be studied for the effective introduction of remedial measures. the attitude of physicians and their beliefs, preparedness, and experiences related to impaired and incompetent colleagues should be explored, as well as suggestions to overcome them. issues related to devices, drugs, and diagnoses with reference to ethical and legal aspects and alternatives for better treatment of the patient should be analyzed. periodical analyses of judgments related to health care issues, health care professionals, and health services at national and international levels, as well as identification of the causes, to find methods to avert them can be undertaken at educational and industrial levels. the capacity of medical students and practitioners to get informed consent should be evaluated and suitable measures introduced to teach and train them. analyses of curriculum and methods of teaching and training should be undertaken, and medical students should be assessed on their knowledge of ethics and laws. a common curriculum should be introduced so that medical students can be trained uniformly to deliver health care effectively and as expected. moreover, such a curriculum will help patients and practitioners, as patients start moving between countries for medical treatment (medical tourism). the current status of consultation and its impact on the part of referring doctors and their patients have to be studied to find lacunae. suitable modules should be prepared for training doctors on consultation. there are inter-and intracountry variations in one or more components of bioethics, thus the boundaries are disputable. disagreements about the relationship between ethics and law should be addressed. future directions in therapeutics should focus on the role of pluripotent stem cells in cardiotoxicity, the feasibility implications of personalizing health care, and the assessment of myocardial neuronal function. health science research explores basics, epidemiology, risk, prevention, diagnostics and management, financial burdens of disease, and behavioral aspects. each component of health science should focus on ethical aspects before asking for funding. cardiovascular research should produce guidelines on management of end-oflife care, palliative care, and support for patients' decisions to forgo treatment. cardiovascular physicians should prepare and produce modules to facilitate the patient and faculty decision-making process about cardiovascular health. managed care organizations (mco) and cardiovascular physicians should plan and develop guidelines to handle ethical issues or problems related to mco and standard of ethics for hospital administrators and all others involved directly or indirectly with patient care. protocols and algorithms need to be developed to curtail inappropriate prescribing, unnecessary testing, overuse of technologies, diversion of scarce resources, and nonrecognition of complications. the public has realized that "doctors are not always to be trusted, as they have fallen off their pedestal." doctors, therefore, have to remain up to date and demonstrate their continuing competency. currently the duties and responsibilities of doctors have been defined by medical councils belonging to the individual country where they practice. as medical tourism has developed and patients are moving from one country to another for treatment, there is a need to develop an international norm to overcome inter-and intracountry variations. cardiovascular care is increasingly complicated, device-oriented, and expensive, and it requires striking a balance for quality of life and longevity. the modern cardiovascular specialist has to explain these spectra, considering individual patients' wishes and best interests, the maleficence of side effects, and the impact of therapeutic interventions in the larger social context. to achieve these things, physicians should acquire skills in the art of medicine and interact with patients and caregivers with empathy, astute observations, acccurate interpretation of clinical and laboratory data, and application of up-to-date knowledge with evidence and ethics. this can strengthen insight into disease and suffering and provide a means to diagnose, treat, and prevent disease; if followed meticulously and documented sincerely, legal liability is less likely to develop. treatment decisions, in general, should be based on a profound respect for the patient's autonomy, balanced by an appreciation of what may be in the patient's best interest. decision making involves a wide array of personal, professional, institutional, spiritual, and social values. in routine practice, clinical management decisions should be coupled with outcome analyses and guide physicians' approach to best address the needs and expectations of patients. to improve the quality of the physicianÀpatient encounter, we need to develop formal and informal curricula at medical institutions on communication skills and ethics. key learning points g illness remains universal; however, human 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principles of ethics and legal aspects have to be incorporated into health science education and discussed at every stage of clinical teaching. g ethics and legal aspects related to emerging areas of health care, such as medical tourism, ubiquitous health care, consumer-driven health care, integration of complementary therapy, and so on, need to be addressed.g curriculum about medical ethics should be included in all aspects of medical education and training, including continuing education. such measures should reduce the possibilities of medical liability cases. key: cord- -m tkgf x authors: ravi, kumar satish title: dead body management in times of covid‐ and its potential impact on the availability of cadavers for medical education in india date: - - journal: anat sci educ doi: . /ase. sha: doc_id: cord_uid: m tkgf x technology integration in teaching is an evolving concept in modern medical education like other study disciplines in india. the domain of medical science education is presently deficient of deploying technology-based practices. currently, majority of medical schools and colleges continue to choose traditional wet laboratory dissections over new virtual dissections which are being followed in institutions such as all india institute of medical sciences (aiims) and handful of other governmental as well as private medical schools of india like aiims rishikesh, aiims jodhpur, government medical college vimsar, burla, odisha, gsl medical college rajahmundry, andhra pradesh, yennapoya university mangalore, dutta megha medical college, gmc chindwara madhya pradesh, symbiosis institute pune and apollo medical college chitoor. not to mention, as the current push for technology integration increases, it is likely that a large number of indian academic scholars will begin seeing virtual dissections as beneficial to their classroom. to the editor of anatomical sciences education: technology integration in teaching is an evolving concept in modern medical education like other study disciplines in india. the domain of medical science education is presently deficient of deploying technology-based practices. currently, the majority of medical schools and colleges continue to choose traditional wet laboratory dissections over new virtual dissections which are being followed in institutions such as all india institute of medical sciences (aiims rishikesh) and handful of other governmental as well as private medical schools of india like aiims jodhpur, government medical college vimsar, burla, odisha, gsl medical college rajahmundry, andhra pradesh, yennapoya university mangalore, dutta megha medical college, gmc chhindwara, madhya pradesh, symbiosis institute pune, and apollo medical college chitoor. not to mention, as the current push for technology integration increases, it is likely that a large number of indian academic scholars will begin seeing virtual dissections as beneficial to their classroom. obviously, it is difficult to reach any consensus and conclusion as of now and only time will tell whether this new wave of change will be incorporated within our anatomy teaching and replace the traditional methods. in times of covid- , to maintain social distancing when the whole country is locked down, the ministry of health and family welfare, government of india, and board of governors of medical council of india have issued directives to all the medical institutions to suspend classes for courses such as m.b.b.s., b.sc., nursing, and other paramedical and allied health sciences to maintain social distancing (government of india, ) . keeping this in view, most of the medical institutions are conducting online classes for various undergraduate courses. theory classes of several subjects can easily be conducted by using video software like zoom, google classroom, google hangouts, google meet, and moodle to name a few. however, anatomy dissection classes remain challenging to conduct online. anatomy lays the foundation of surgical disciplines and a logical understanding of various surgical equipment and techniques can be successfully attained through cadaveric dissection classes (sugand et al., ) . cadaveric dissection is one of the methods of training hands and fingers for future surgical procedures on living human beings (ajita and singh, ) . understanding the significance of dead body, government of india, ministry of health and family welfare, directorate general of health services (emr division) have issued guidelines of dead body management in view of covid- pandemic. the guidelines for dead body management have been released by central government on march (government of india, ) . guidelines were issued with regard to precautions, infection prevention and control measures, handling of the human body, and environmental disinfection. it is based on the current epidemiological knowledge about the covid- as there was some delay in the cremation of the second disease-related casualty of a -year-old woman in delhi (india today, ). the first covid- casualty in india was a -year-old man in karnataka who died on march , . he had recently returned from saudi arabia (india today, ) . considering the fact that novel coronavirus is a new disease and there is a knowledge gap on best practices such as how to dispose the dead body of a suspect or confirmed case of covid- , the necessary guidelines were issued. the main mode of transmission of covid- is through droplets, fomites, and close contact with possible spread through feces (who, ). hence, the augmented risk of covid- contamination from a dead body to healthcare professionals or relatives who follow standard precautions while handling the body is quite unlikely (government of india, ) . till date there is no evidence of individuals getting infected from exposure to the bodies of people who died from covid- (who, ) . it is advised that healthcare workers identified to handle dead bodies in the isolation area, mortuary, ambulance, and in the crematorium/burial ground should be trained with respect to best infection prevention and control practices. the healthcare worker attending to the dead body should practice hand hygiene and ensure proper use of personal protective equipment such as coveralls, head covers, shoe covers, n respirators, surgical masks, goggles/face shields, etc. if the exterior of the body is visibly contaminated with body fluids, excretions or secretions, it has to be ensured that the gown is waterproof. they should remove all tubes, drains and catheters from the dead body. all puncture holes or wounds should be properly disinfected with % hypochlorite solution and dressed with impermeable material. it is advisable to properly dispose and take precaution while handling intravenous catheter or any sharp devices. to avoid leakage of body fluids, all the orifices of dead body should be plugged. viewing of the dead body for the relatives to see the body for one last time may be allowed by unzipping the face end of the body bag by using standard precautions. guidelines also advise to keep the dead body in a leak-proof plastic body bag. the exterior of the bag can be decontaminated with % sodium hypochlorite (naocl) solution. mortuary personnel managing the dead body of covid- patients are advised to observe standard precautions like storing the dead body in coolers maintained at approximately degrees celsius ( °c), disinfecting environmental surfaces, instruments, transport trolleys and cleaning the doors, handles, and floor with sodium hypochlorite % solution after removing the body. as per the guidelines, embalming of such dead bodies should not be allowed (government of india, ) . similarly, autopsy may be avoided and in case if it is to be performed for special reasons, the recommended infection prevention and control practices should be followed. the government of india has directed to avoid huge congregation either at the crematorium or burial ground as a social distancing measure due to an obvious reason that close family members or relatives may be symptomatic of corona virus disease (government of india, ) . according to voluntary body donation program, the general population can will their bodies to serve the purpose of medical education and scientific research. a beautiful quote by famous indian academician, author, and psychologist dr. amit abraham makes sense here: "after i die if i am buried, i will rot. if i am burnt i will become ash but if my body is donated i will live to give life and happiness to many" (abraham, ) . body donation in india is followed as per the bombay anatomy act ( ): an act to provide for the supply of unclaimed bodies of deceased persons (or donated bodies or any part thereof of deceased persons) to hospitals and medical and teaching institutions for the purpose of anatomical examination and dissection and other similar purposes (mohan foundation, ). body donation not only is useful for understanding human body and for advancing medical science but also helps medical students in learning relations of human anatomic structures and development of psychomotor skills by cadaveric dissection (ajita and singh, ) . each human body is a new source of knowledge with anatomical variations and a medium for gaining medical knowledge, more effective than any textbook or computer. nowadays, cadaveric dissection is not only limited to medical graduates and post graduates but it also helps various surgical disciplines such as surgical oncologists, neurosurgeons, burns and plastic surgeons, radiotherapists, head and neck and orthopedic surgeons to experiment innovative surgical skills and procedures in the form of cadaver laboratories and cadaveric workshops, to explore human body in a realistic manner before performing any major and complex surgery on living patients (ajita and singh, ) . apart from these, there are other uses of body donations such as cadaver banks for brain, skin, vessel and bones for molecular research and cadaver grafting. with the growing concerns regarding the corona virus (covid- ) pandemic, many anatomists have questions about the impact on body donation. we should maximize all donation opportunities, while keeping our healthcare professionals, medical students, and community safe. the risk of a covid- infection from an infected donor is unknown at this time. factors that could impact the risk of similar coronavirus such as sars-cov- transmission include epidemiological risk factors, incubation period, degree of viremia, and viability of the virus within the blood and specific organ compartments (lai et al., ) . we should follow infection prevention guidelines which address covid- . these include the screening and testing of body donors or potential donors that could have contracted covid- . donors should be screened for potential covid- infection (ast, ) . screening for covid- includes three different methods such as ( ) epidemiologic screening for travel and potential exposures, ( ) history for symptoms suggestive of covid- , and ( ) laboratory screening like nasopharyngeal and oropharyngeal or bronchoalveolar lavage sample for testing . recent studies indicate that negative results of laboratory screening may not rule out covid- infection (winichakoon et al., ) . the optimal approach to donor screening may change over time as more data accumulate. in the present scenario, medical professionals including anatomists need to be vigilant about evolving covid- guidelines and adopt their standard operating procedures accordingly. the covid- pandemic is unpredictable; still we are hopeful for the success of social distancing as the country is locked down else it might have a severe impact not only on medical education but on healthcare infrastructure and capacity crisis as well. amit abraham > quotes > quotable quote body donation and its relevance in anatomy learning -a review faqs for organ donation and transplantation bombay act no. xi of [the bombay anatomy act covid- : guidelines on dead body management government of india, ministry of health and family welfare, directorate general of health services (emr division -year-old coronavirus victim cremated under medical supervision in delhi. india today severe acute respiratory syndrome coronavirus (sars-cov- ) and corona virus disease- (covid- ): the epidemic and the challenges asp?gclid =cj kc qjw _ vzbrc iaris aos z tsf fowo tzpz d qze wtln m wz ebopj-r eck wwu w fccb ihqaa o sea lw_wcb [accessed the anatomy of anatomy: a review for its modernization world health organization. infection prevention and control for the safe management of a dead body in the context of covid- : interim guidance negative nasopharyngeal and oropharyngeal swab does not rule out covid- key: cord- - ccsvf h authors: hickland, maria m.; gosney, eleanor r.; hare, katie l. title: medical student views on returning to clinical placement after months of online learning as a result of the covid- pandemic date: - - journal: medical education online doi: . / . . sha: doc_id: cord_uid: ccsvf h nan medical education has faced drastic changes as a result of the covid- pandemic. the suspension of clinical placements across medical schools from march resulted in online delivery of the medical curricula through the use of virtual platforms. the recent evaluation of online medical education by our colleagues highlighted key advantages to these novel methods of teaching, including increased accessibility and higher levels of engagement [ , ] . however, as senior clinicalyear students are now either preparing to, or have, returned to placements within hospital and community settings, the insurance of medical student safety whilst ensuring the preparedness of the future doctors of tomorrow is paramount. as final year medical students returning to placement, we highlight key areas for discussion amongst medical schools and hospital undergraduate administrators amid the covid- pandemic. the general medical council (gmc) states medical students as tomorrow's doctors, thus it is imperative that clinical-year medical students return to placements to ensure the continued development of core competencies, outlined in the outcomes for graduates by the gmc [ ] . as final-year medical students due for graduation in , we look forward to this return due to the limitations in patient-contact during online learning, as well as reduced opportunities in developing our communication skills and the challenges in remote non-clinical working. our enthused return to placement is accompanied by a widespread feeling of anxiety amongst ourselves and our peers. the british medical association recently released a statement highlighting some of the issues and possible solutions regarding returns to clinical placements, but we question as to whether this goes far enough in alleviating student anxieties, particularly those with pre-existing health conditions or at-risk backgrounds such as ethnic minorities [ ] . whilst we anticipate that the skills we have developed during the covid- pandemic through online teaching will ensure a fluent return to placement, we are concerned about widespread lack of confidence in communication and clinical skills upon our return to placement, especially when compared to previous cohorts. will our colleagues be understanding, or adjust their expectations if our clinical competencies differ from previous final years? much like other health care professionals working on the front line during the covid- pandemic, medical student anxiety remains high, with particular concerns surrounding their interactions with nonmedical family members and friends. medical schools have required students to undertake a risk assessment prior to returning to placement, and whilst we have been assured that appropriate personal protective equipment (ppe) will be provided, this does not negate the fear of contracting the virus. there is particular concern amongst individuals returning to placement who live with at-risk family members, thus assurances in how risk of transmission and infection are being managed is vital to help ease student fears. one study found that a third of medical students preferred not to return to the clinical setting during the pandemic, a higher proportion of which was in the lower pre-clinical years. those not wishing to return also had a statistically significantly higher level of perceived personal risk compared to those who wished to return, highlighting the concern about risk to self [ ] . the impact of covid- has been a global challenge, but as medical students we have found ourselves at an impasse -conflicted between the desire to return to a clinical environment, and the safety of online learning, for our own and others health. we hope that with the continuing improvements in safety measures will not only help negate some of these fears, coupled with the support of both medical schools and hospital staff, students will feel wellsupported upon placement return and continue to thrive as tomorrow's doctors. the impact of covid- on the undergraduate medical curriculum undergraduate medical education and covid- : engaged but abstract gmc publications position statement: implications of resuming clinical placements during the covid- pandemic medical students' preference for returning to the clinical setting during the covid- pandemic the authors report no conflict of interest. maria m. hickland http://orcid.org/ - - - key: cord- - umby l authors: cai, qi; feng, hongliang; huang, jing; wang, meiyao; wang, qunfeng; lu, xuanzhen; xie, yu; wang, xing; liu, zhenxing; hou, botong; ouyang, keni; pan, jing; li, qin; fu, beibei; deng, yongchao; liu, yumin title: the mental health of frontline and non-frontline medical workers during the coronavirus disease (covid- ) outbreak in china: a case-control study date: - - journal: j affect disord doi: . /j.jad. . . sha: doc_id: cord_uid: umby l background and objective: coronavirus disease (covid- ) is a new infectious disease with high transmissibility and morbidity. it has caused substantial mental distress to medical professionals. we aimed to compare the psychological impact of the covid- outbreak between frontline and non-frontline medical workers in china. methods: this case-control study recruited frontline and age- and sex-matched non-frontline medical workers during the covid- outbreak (february to , ). a set of online questionnaires were used to measure mental problems (i.e., anxiety, insomnia, and depressive symptoms), and help-seeking behavior and treatment for these mental problems. results: frontline medical workers had higher rates of any mental problem ( . % vs . %, adjusted or= . , % ci= . - . ), anxiety symptoms ( . % vs . %, adjusted or= . , % ci= . - . ), depressed mood (marginally insignificant; . % vs . %, adjusted or= . , % ci= . - . ) and insomnia ( . % vs . %, adjusted or= . , % ci= . - . ) than non-frontline medical workers. no significant difference was observed in terms of suicidal ideation ( . % vs . %, adjusted or= . , % ci= . - . ), help-seeking ( . % vs . %, adjusted or= . , % ci= . - . ) or treatment ( . % vs . %, adjusted or= . , % ci= . - . ) for mental problems. limitations: the case-control nature of the data precludes causal inferences, and there is a possibility of bias related to self-reports. conclusions: frontline medical workers had more mental problems but comparable help-seeking behaviors and treatment for these problems than non-frontline medical workers. these findings highlight the timely mental support and intervention for medical workers, especially for those on the frontline. coronavirus disease was first reported in wuhan, hubei province, china, in december (phan, ) , followed by an outbreak throughout the country and beyond. as of february , , a total of , medical workers were infected in china with novel coronavirus (including confirmed cases, suspected cases, clinically diagnosed cases, and asymptomatic infected persons), , medical workers have been confirmed to be infected, with ( . %) critical cases and ( . %) deaths ((epidemiology working group for ncip epidemic response, ). medical workers, especially those who are working in the frontline, may suffer huge amounts of stress during the fight against covid- . several studies have reported that individuals exposed directly or indirectly to life-threatening situations may have a high risk of psychological morbidities (bills et al., ; chan and huak, ; ji et al., ; mak et al., ; ofner-agostini et al., ; sim et al., ; wang et al., ; wu et al., ) . for example, during the outbreak of severe acute respiratory syndrome (sars) in , it was shown that health care workers had a higher rate of sars infection than other people (ofner-agostini et al., ) , and they were emotionally affected and traumatized during the outbreak (chan and huak, ; wu et al., ) . at present, the severe acute respiratory syndrome from coronavirus (sars-cov- ), which has been reported to have more severe transmissibility than sars-cov (liu et al., b) , puts medical workers, especially the frontline medical staff at very high health risk. with an increasing number of patients, long hours of intensive work and less sleep time, inadequate protection from contamination, and the risk of infection at any time, medical workers have a high risk of physical and mental exhaustion, resulting in a variety of mental health problems. the mental health of medical workers has gained tremendous attention. as this outbreak has highlighted the fragility of psychological resilience, we also need to pay attention to the psychological state of health care workers during epidemics (ho cs et al. ). the government and hospitals have formulated a series of measures to address this problem . a recent study found that nearly one-sixth of , medical staff had psychological distress and therefore needed to seek help from psychological or psychiatric professionals. the prevalences of psychological distress, anxious symptoms, and depressive symptoms were . %, . %, and . %, respectively . another study investigating the mental health status of medical staff found that . % of the medical staff that fought against covid- in wuhan had a high score of anxiety compared with medical staff in xian . however, the deficiency lies in the lack of systematic assessment of mental problems between frontline and non-frontline medical workers during the covid- outbreak. since an increasing number of newly infected cases among the medical staff have been observed to date, and since the arduous task of the fight against the covid- might be far from over, timely mental health care for medical staff during the covid- outbreak is urgently needed. the present study attempted to compare the immediate psychological impact of the covid- outbreak on frontline and non-frontline medical workers in china. this case-control study was conducted between february to , , which was approved by the clinical research ethics committee of zhongnan hospital of wuhan university (ref. no.: ) . this study was conducted through a program called questionnaire star. the sample was obtained based on a non-probability sampling design. all medical workers participated in the survey through the link or quick response (qr) code of questionnaire star, which is a bar code that can store the website link of the questionnaire used in this study. once the questionnaire was submitted, the data would be saved on the questionnaire star server, and the questionnaire creator can download or analyze the data. the requirement for written consent from subjects was waived by the research ethics committee. no identifiable information was collected. a set of questionnaires were sent to medical workers working in hospitals in china via the qr code of questionnaire star. the medical workers included physicians, nurses, and other healthcare workers (e.g., medical technicians, respiratory therapists, or emergency room attendants). medical workers dealing with covid- were considered frontline medical workers. otherwise, they were categorized into the non-frontline medical workers group. other inclusion criteria were as follows: ) age to years old and ) chinese-speaking residents of china. a total of , eligible frontline medical workers and , eligible non-frontline medical workers participated in this study. . % of the non-frontline medical workers (n = , ) were selected and matched to frontline medical workers by age and sex. the matching was performed using the "matchit" package in r (version . . ; r foundation for statistical computing, vienna, austria) (ho, d., imai, k., king, g., & stuart, e., ) . the study instrument was a structured questionnaire that comprised demographic details, three main self-reported rating scales that have broad credibility, and information about whether the participant sought help or treatment for mental problems. the detailed explanation is as follows. participants' demographic characteristics, including age, sex, education level, marital status, jobs, annual household income, living status, and geographic origin, were obtained. the epidemic parameters for the covid- of the areas where the subjects stayed were used to estimate the levels of exposure to the epidemic. the epidemic parameters, including the number of cumulative cases, number of new daily cases, prevalence, and daily incidence, were obtained from the official online platform for controlling the covid- epidemic in china (national health commission and provincial health commissions. a). the beck anxiety inventory (bai) was used to measure anxiety symptoms over the last seven days (beck et al., ; chinese version: che et al., ) . this scale has a total of self-report items with responses rated on a -point likert scale ranging from (not at all) to (severely). higher scores reveal a higher level of anxiety. the following cut-off scores were used to assess different levels of anxiety: ) scores between and denote no anxiety; ) scores between and denote mild anxiety; ) - denotes moderate anxiety; ) - denote severe anxiety (ahmed et al., ) . the presence of anxiety was defined as a bai score > in our study. the insomnia severity index (isi) was used to assess participants' perceptions of insomnia over the past two weeks (chahoud et al., ) . it has seven items targeting the subjective symptoms and daytime consequences of insomnia, as well as the degree of distress caused by these difficulties. each item of this scale is rated on a -point likert scale of - , and higher scores indicate greater insomnia severity (morin et al., ; chinese version: li et al., ) . the optimal cut-off point of isi for detecting clinical insomnia in the chinese population was a total score of (chung et al., ) . therefore, an isi score higher than nine was chosen as the cut-off for insomnia for this study. the patient health questionnaire- (phq- ) was used to measure depressive symptoms over the past two weeks (michel et al., ) . all nine items are scored from to , and the total scores range from to , with a higher score indicating more severe symptoms (kroenke et al., ; chinese version: min et al., ) . scores of to indicate no depression, scores of to indicate mild depression, scores of to indicate moderate depression, scores of to indicate moderately severe depression, and scores of to indicate severe depression (xia et al., ) . participants with a phq- > were defined as depressed in our study. the chinese versions of the three abovementioned rating scales have been proven to have satisfactory reliability and validity (chung et al., ; liang et al., ; wang et al., ) . in addition, suicidal ideations were measured by one question: "over the past two weeks, have you ever had suicidal thoughts?" the responses to this question were "once/several times" or "never" (hassan et al., ) . whether participants sought help or received treatment for mental problems, including anxiety symptoms, depressed mood, suicidal ideation, and insomnia, during the covid- outbreak was recorded. the question "have you ever sought help from psychiatrists or clinical psychologists since the outbreak of covid- began?" was used to estimate help-seeking behavior. the question "have you ever received any treatment for psychiatric or psychological problems since the outbreak of covid- began?" was used to measure treatment history for mental problems. mann-whitney u test and t test were used to compare means of two groups of non-normally and normally distributed variables, respectively. chi-square test was used to compare the inter-group differences for categorical variables. univariate and multivariate logistic regressions were performed to evaluate the relationships of frontline medical workers (vs. non-frontline medical workers) with mental problems, and help-seeking behaviors and treatment for mental problems. a value of two-tailed p < . was considered statistically significant. in table , p values for multivariate logistic regressions were further adjusted using a false discovery rate (fdr) method (bernhard, ). an fdr of % using q values would mean that % of results called significant (p < . ) are false-positives (benjamini et al., ) . all statistical tests were performed using spss version . for windows (armonk, ny: ibm corp) and r (version . . ). a total of participants were enrolled in our study, including frontline workers and age-and sex-matched non-frontline medical workers. table presents the demographic characteristics of the participants. there were no differences in education years and unmarried status between non-frontline and frontline medical workers (all p > . ). however, frontline medical workers had a higher proportion of medical staff (mainly including doctors and nurses) than non-frontline medical workers ( . % vs. . %, p < . ). frontline medical workers showed higher proportions of annual household income of < , usd per head ( . % vs. . %, p = . ) and living alone ( . % vs. . %, p < . ) than non-frontline medical workers. compared with non-frontline medical workers, frontline medical workers had higher proportions of participants from wuhan and hubei province (excluding wuhan) (p < . ). in addition, frontline medical workers had higher exposure levels of the covid- epidemic, including larger numbers of cumulative cases and daily new cases, higher prevalence, and higher daily incidence than subjects from other areas (all p < . ). the psychological impact of acute infection outbreaks on medical workers has aroused considerable concern from the government, the public, and medical professionals. the current study has shown that frontline medical workers directly dealing with patients confirmed or suspected of having covid- had a higher level of various mental problems than those non-frontline medical workers. in addition, the two groups had comparable low rates of help-seeking behaviors and treatment for their mental problems. data from the present study showed that the mental health of frontline medical workers was particularly worrying. the rate of mental problems, such as anxiety, depression, and insomnia, was significantly increased in frontline medical workers, compared with non-frontline medical workers. in this study, about . % of enrolled frontline medical workers experienced anxiety symptoms, and the prevalence is lower than the general population during the covid- outbreak ( . % experienced moderate to severe anxiety symptoms) . similarly, about . % of enrolled frontline medical workers experienced depressive symptoms, and the prevalence is slightly lower than the general population during the covid- outbreak ( . % reported moderate to severe depressive symptoms) . the higher prevalence of anxiety and depression in the general public could be due to less access to personal protection equipment as compared to frontline medical workers. a great deal of evidence demonstrates the dramatic psychological impact of the epidemic on healthcare workers, and the importance of dedicated interventions to deal with mental problems, such as stress, anxiety, depressive symptoms samantha et al., ; . our findings are consistent with those reported zhu et al., who found a significant percentage of psychiatric symptoms during the covid- pandemic, . % for stress, . % for depression, and . % for anxiety among healthcare workers (zhu, et al, ) . a recent study indicated poor sleep quality among frontline clinical nurses fighting with the covid- ( . in contrast, a recent study conducted in singapore found that there was a higher prevalence of anxiety among non-medical healthcare workers compared to medical personnel (tan et al., ) . the opposite findings in singapore could be due to the fact that covid- was a less severe problem in singapore as compared to china and frontline healthcare workers encountered lower level of anxiety and depression. compared with non-frontline medical workers, frontline medical workers might be exposed to much more physical and mental stresses, which may contribute to their higher rates of mental problems. for example, frontline medical workers have had to be extra vigilant when working in the fever clinics or infectious wards, ensuring that suspected patients were timely identified and transferred to the designated hospital to reduce exposure risk for others. in addition, the rapid increase of the infected patients and the uncertainty of transmission in the early stage of the outbreak increased the enormous workload and psychological burden of medical workers . during the covid- outbreak, many medical staff were infected , which may have increased the psychological stress of their colleagues. moreover, inadequate protective materials against the virus, negative emotions from the patients, quarantine, and lack of contact with their families also added to the psychological burden of frontline medical workers. furthermore, mental problems could interact with each other. for example, sleep disorders were reported to be related to anxiety (bélanger et al., ; papadimitriou and linkowski, ) . our results showed poor mental health among frontline medical workers, but contrary to our expectation, we did not observe significantly higher rates of seeking help or receiving treatment for mental problems among these subjects. the phenomenon that medical workers have difficulty accepting and disclosing emotions is not unique to the covid- outbreak (fridner et al., ; king et al., ; tyssen et al., ) . emotional distress is common among hospital doctors, many of whom do not seek professional help or support from their colleagues, because they either think they did not need or are embarrassed to seek help and worried about confidentiality (fridner et al., ) . these findings remind future psychological intervention providers should pay more attention to medical workers with mental health problems. the mental health status of medical workers presented in the current study prompts the need for appropriate measures and timely treatment for covid- -related psychological problems. according to previous experience with severe infectious diseases (such as sars and evd), medical workers with a good awareness of the disease developed relatively fewer psychological symptoms (chua et al., ; huang et al., ; ji et al., ) . therefore, authoritative knowledge about covid- should be disseminated among medical workers as early as possible. mental health professionals should be deployed in the medical teams to provide psychological support. furthermore, external material and spiritual support were crucial to confronting psychological symptoms, including virus prevention implementation, living and medical supplies, as well as spiritual support from colleagues, team leaders, family, and friends (nasser and overholser, ) . since the start of the covid- outbreak, the chinese government, hospitals, and psychological and psychiatric centers have taken measurements to address mental health problems (bao et al., ) . a national guideline for psychological crisis intervention during the covid- outbreak, in which psychological protection measures for medical workers were provided, was published by the national health commission of china (national health commission of the people's republic of china, b). psychological intervention teams were set up to deliver mental health services to medical staff. in addition, a variety of online mental health services through communication programs, such as wechat, weibo, and tiktok, were gradually put into practice for people in need (liu et al., a; . as the development of the epidemic, further psychological support and measurements should be provided, especially for frontline medical workers. there were several limitations to our study. first, medical workers participated in the investigation via the internet without random sampling, so the response rate was hard to estimate. however, the relatively large number of participants reduced this potential sampling bias. second, the mental health status of the participants before the covid- outbreak was not available, making it difficult to know whether their pre-existing mental health status also impacted the posttraumatic morbidity of covid- . third, this study did not address other potential confounding factors, such as personality traits, family history of mental disorders, life events, and social support. fourth, the case-control nature of the present study did not allow us to make any conclusions regarding causality. the present study highlighted the mental health problems and unmet needs of medical workers during the covid- epidemic in china, especially among frontline medical workers. further strategies should be provided urgently to alleviate the mental distress of medical workers. long-term surveillance should be provided to monitor the mental health of frontline and non-frontline medical workers. involved in study design, revising the manuscript. municipalities and special administrative regions. b the participants' exposure to the covid- epidemic was estimated by the regional epidemic parameters. would mean that % of results called significant (p values< . ) are false-positives. epidemic of covid- in china and associated psychological problems -ncov epidemic: address mental health care to empower society an inventory for measuring clinical anxiety: psychometric properties insomnia and generalized anxiety disorder controlling the false discovery rate -a practical and powerful approach to multiple testing multiple testing: correcting for alpha error inflation with false discovery rate (fdr) or family-wise error rate? mental health of workers and volunteers responding to events of / : review of the literature psychological impact of the severe acute respiratory syndrome outbreak on health care workers in a medium size regional 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mental health care for the novel coronavirus outbreak is urgently needed epidemiological and clinical features of the novel coronavirus outbreak in china recommended psychological crisis intervention response to the novel coronavirus pneumonia outbreak in china: a model of west china hospital a survey on mental health of medical staff fighting novel coronavirus diseases in wuhan covid- in wuhan: immediate psychological impacton we are grateful to professor jihui zhang (sleep assessment unit, department of psychiatry, faculty of medicine, the chinese university of hong kong, shatin, hong kong sar, china) for valuable contributions on data processing and the revision of the article. we also thank all the participants in our study. dr. hongliang feng was supported by the hong kong ph.d. fellowship scheme of the research grants council. the other authors have nothing to declare. key: cord- -kvenaqj authors: wang, xuan; wang, min-jie; jiang, xiao-bing; wang, hai-jun; zhao, hong-yang title: letter: strategies for prevention and control of novel coronavirus infection among medical staff date: - - journal: neurosurgery doi: . /neuros/nyaa sha: doc_id: cord_uid: kvenaqj nan lack of awareness and prevention measures taken at that time, the epidemic spread rapidly as people returned home for the chinese new year. despite the government's closure and traffic control of wuhan since january , , covid- had been spread into other provinces of china and even other countries. on january , the outbreak in china was announced as a public health emergency of international concern at a meeting of the who emergency committee in geneva, in an effort to suppress global spread of the disease. so far (from january , to february , ) as of this writing, there were medical professionals were infected with sars-cov- ; were diagnosed with covid- and unfortunately died. in the very early stage of the epidemic, the first of these infected medical staff were from the department of neurosurgery, which was located in the epidemic center of the covid- outbreak. in such an urgent and unexpected situation, the department took immediate and effective prevention and control strategies. these staff was now completely recovered and there were now new infected cases in the department. here, we would like to summarize and share our successful experiences in the prevention and control of sars-cov- infection in medical staff of hospital departments, especially those devoted to non-infection diseases. on december , , a patient with pituitary adenoma was hospitalized for a surgery in the department of neurosurgery. prior to the hospitalization, the patient showed no covid- symptom. but d after the neurosurgical operation, the patient showed low fever and decreased lymphocyte on january , , and was admitted to isolation ward on january . however, he was diagnosed as covid- and transferred to the designated hospital for the treatments that assigned by the government on january , . at that time, the medical community experienced an initial delay in understanding that covid- has a long incubation period prior to diagnosis, and protective measures at the onset were not at the current level iii (table ). in the department of neurosurgery, of the medical staff, including nurses and doctors, were infected after january , . fortunately, by february , , all the medical personnel had recovered and been discharged after active treatment. an immediate retrospective analysis was conducted of the differences between infected and uninfected workers in the department. the investigated factors included age, gender, duration of contact with the infected, and night shift experience ( figure ). the results of binary logistic regression showed that, after adjustment for the remaining variables, the risk of infection in the medical staff with longer exposure to an infected patient (> min) was ( . - . ) times higher than that of those with shorter exposure (< min). furthermore, the risk of infection was . ( . - . ) times higher for those who had worked a night shift, compared with those who had not. with the awareness of the high rate of infection in the medical staff, combined with the statistical results, we drew up strategies for emergency management and daily protection for medical staff in departments of non-infection disease during this severe epidemic. after the implementation of these measures in our department, there has been no further infection among patients or medical staff. the study was done after agreement from the local ethics committee and informed consent was provided by participating patients. • reduce non-urgent and non-necessary medical activities, and assist diagnosis and treatment via the internet. • effectively manage all patients at different levels and categories, that is, patients are categorized as with or without fever, and each group further stratified as emergency or nonemergency, as appropriate. • reduce direct contact between medical staff and patients, and avoid mutual contact between patients. • aim for early detection, early isolation, and early treatment of all infected individuals. • implement continuous disinfection of the departmental environment. in a scenario of epidemic and shortage of medical resources, the medical staff should triage outpatients and inpatients, to achieve early detection and warning of covid- infection ( figure ). elderly patients and those with underlying diseases of the heart, lung, liver, and renal system are susceptible to higher pathogenicity and mortality in response to covid- infection. the risk of infection varies with the clinical department. for example, patients in the neurosurgery department are at high risk of covid- infection. therefore, the hospital should be committed to a strict -level ward management system (table ) . patients suspected of infection should be partitioned from other patients, and each ward should employ different sets of medical staff to avoid cross-contamination. • patients with systemic abnormalities beyond the respiratory system. among patients with covid- there are reports of %, %, and % rates of acute cardiac impairment, acute renal impairment, and diarrhea, respectively. liver injury has also been reported. these concomitant conditions are aggravated in covid- , and even present a major threat of mortality. therefore, it is especially important during this epidemic that the basic vital signs and dysfunctional organs of such vulnerable patients are safeguarded. the effect of possible infection should be minimized, with early detection and intervention. • patients with associated mental stress. because covid- is highly contagious, can quickly progress in severe cases, and there is no specific effective treatment, hospitalized patients often experience anxiety, mania, insomnia, or other psychological problems. patients should be reassured that, although the number of infected persons has rapidly increased, the prognosis is better than for sars (severe acute respiratory syndrome), and the fatality rate is much lower. the medical staff should inform inpatients that the epidemic will subside if scientific prevention, control, and treatment measures are adhered to. for outpatients, prevention and control procedures for the new coronavirus can refer to the diagnosis and treatment plan for pneumonia of the national health and medical commission (trial sixth edition). this includes a previewing triage system ( figure ). outpatient treatment should be arranged for patients with suspected infection or fever. in addition, the management and disinfection of outpatient clinics should be given priority. patients should be instructed regarding how wear masks correctly, and to put them on before entering the waiting area. • thorough cleaning and disinfection is essential after contact with each patient. hands must be carefully washed for to min with disinfectant ( % ethanol or chlorine-containing disinfectant). • as far as practicable, the medical staff should remain . to meters away from the patient during diagnosis and treatment, and time of contact should be minimized. if an atomizer, stethoscope, or other examination instrument must be used, the medical personnel should avoid contact with patients' secretions, especially for patients with trachea intubation or tracheotomy. medical personnel should note the color of phlegm when suctioning sputum, avoid the airway opening as much as possible, and wear a protective mask. body fluids, secretions, excreta, and used articles collected from confirmed and suspected patients should be regarded as biological pollutants, and treated as clinical waste at the highest biosafety level. • testing throat swabs for nucleic acid is important for diagnosing covid- infection. however, the sampling process poses great risk to the healthcare worker. to protect the medical staff to the greatest extent, only designated, senior, and experienced medical staff should collect samples, and in accordance with a standardized protocol with level iii protection measures. • inpatients and accompanying persons should be instructed and required to wear masks in the ward. warnings should be posted at the entrance of the ward, and information regarding protection against infection should be promoted and disseminated to increase public knowledge. accompanying persons should not be permitted to take the inpatients outside the hospital without permission, or visit other wards or congregate. • all wards should institute a layered management system, with the wards strictly divided into levels according to risk: common ward, isolation ward for suspected patients, and single isolation for the diagnosed patient. free movement of persons among the ward levels should be strictly limited to avoid cross contamination. each level should deliver its own medical treatment. • wards should be ventilated at least to times daily, for not less than half an hour each time. all objects in the ward that are frequently handled, such as door knobs, bed edges, window sills, and wall switches, should be cleaned regularly and disinfected with mg/l chlorine-containing disinfectant twice per day. • as much as possible, individual items that are commonly used in departments should be designated for use in a single ward, including blood pressure meters, thermometers, and stethoscopes. if the number is insufficient or it is necessary to borrow in an emergency, the items must be thoroughly disinfected. • for patients undergoing elective neurosurgery, stepwise preoperative screening for covid- should be conducted based on the diagnostic criteria. the surgery should be performed only after no abnormality is detected. due to the long incubation period, medical staff should take level ii protection measures (table ) . • in principle, no elective surgery should be scheduled for patients with suspected or confirmed covid- . if emergency craniotomy is needed, the medical staff must take the following level iii precautions: strictly wearing disinfected first-layer work clothes (hospital scrubs) and additional gown with hood; double latex gloves; layers of cap; goggles and other facial protection tools; and medical protective masks (n ). it is necessary to avoid or reduce endotracheal intubation, tracheostomy, and other procedures that aggravate infection. if possible, during surgery adequate and proper hemostasis should be maintained to prevent the need for blood transfusion. the spatter of body fluid should be avoided to lower the risk of infection. • a one-way path should be used to enter and exit the operating room. suspected pollution areas should be avoided. the operating room should contain infrared thermometers to measure the body temperature of the medical staff and patients at any time to give early warning of acute illness. • after surgery, medical staff must use quick-drying hand disinfectant before and after removing protective clothing, after taking off googles and hat, after removing the inner gloves, and again after removing masks before donning new masks and immediately leaving the operating room. in addition to operating rooms and wards, the offices and living areas of the medical staff are important potential areas of infection improve the awareness of the medical staff about the epidemic covid- differs from any other known infectious disease, and information regarding adequate protection is lacking. in addition, a high incidence of infectious disease in a department of neurosurgery is unusual. the neurosurgery staff must be instructed regarding what is known about infections of this disease. in addition to basic hospital training, our department has improved education regarding important guidelines and procedures related to covid- infection ( table ). the great importance of early detection and isolation of suspected cases must be emphasized, and safety measures strictly implemented. the staff must receive both theoretical and practical knowledge about covid- infection and medical protection to enhance vigilance and the ability to confront emergencies. under the pressures imposed by the severe epidemic and intense clinical work, medical staff may experience negative emotions that can reduce daily work efficiency and have longterm adverse effects on the entire medical team. negative emotions may manifest as anxiety, depression, or insomnia, among others. appropriate mental health education and positive encouragement are crucial for protecting the staff against these stressful conditions. the number of infected persons and the rapid spread of covid- are unprecedented as a public health emergency. this epidemic is a serious challenge to china's medical system and its ability to cope. the nation cannot afford further misjudgments regarding the need for information, prevention, and protection against covid- infection. herein, we have summarized the experience of our neurosurgery department and the protection measures we have implemented. because of these new preventive protocols, there have been no further cases of infection among our medical staff. coping with this emergency will require better understanding of infection characteristics and route of transmission, and achieving early detection, isolation, and treatment. with adequate upgrades in protective protocols and training, medical personnel in departments not related to infectious diseases can face this outbreak in a scientific, effective, and easy manner, and ensure the normal procedures of diagnosis and treatment. according to tedros ghebreyesus, the director-general of who, "no single country or organization can stop this outbreak alone. our greatest and only hope is to work together." to this end, we share with our colleagues around the world our practical experience in dealing with the outbreak, and we hope to contribute to the world health system. this project was supported by the medjaden academy & research foundation for young scientists (grant no. ncov_mja ). the authors have no personal, financial, or institutional interest in any of the drugs, materials, or devices described in this article. clinical characteristics of novel coronavirus infection in china clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china novel coronavirus -ncov: early estimation of epidemiological parameters and epidemic predictions beware of asymptomatic transmission: study on -ncov prevention and control measures based on extended seir model clinical features of patients infected with novel coronavirus in wuhan, china the global spread of -ncov: a molecular evolutionary analysis the mental health of medical workers in wuhan, china dealing with the novel coronavirus this project was supported by the medjaden academy & research foundation for young scientists (grant no. ncov_mja ). wuhan, hubei p.r. china * xuan wang and min-jie wang contributed equally to this work. key: cord- -xj p ba authors: savage, nicholas; jain, anshini; ng, suat chin title: the impacts of the covid‐ pandemic on medical students in australia date: - - journal: anz j surg doi: . /ans. sha: doc_id: cord_uid: xj p ba nan covid- has been formally declared a pandemic by who on the th of march ( ). the effects of this on the health systems cannot be underrated. in this unprecedented situation, perhaps one of the more underestimated repercussions will be on medical students. there are several challenges common to all medical students such as the uncertainty in course requirements, the fear of exposure to covid- , the lack of guidance and the loss of learning opportunities. nonetheless, it has also affected medical students of different levels indistinctly. preclinical medical students are accessing e-learning solely while socially isolating themselves. e-learning certainly has its merits but lacks the engaging qualities of in-person learning. the study of medicine is complex, requiring a multimodal approach with specialists providing clinical experience and guiding students out of the maze of endless archaic textbooks. adjuncts to classroom teaching such as anatomy dissections, microbiology and histopathology labarotory work as well as the development of interpersonal skills in clinical interactions are paramount to these years. furthermore, e-learning may present new challenges including technical issues, adaptability-struggle and computer literacy. it may be unaccessible by some, without access to on-campus wifi. to overcome this, certain universities have provided usb modems to students to increase accessibility to internet facilities ( ) . other universities have altered term outlines and exam dates( ). these amendments in addition to technical issues that arise during exams may cause unneccesary added stress. there have also been concerns that students may be disadvantaged when exams are uninvigilated, with co-students cheating. during such times, we have little choice but to trust that students will maintain the highest standards of academic integrity. students in clinical years are experiencing different challenges. disruption to normal teaching and examination has left many students feeling uncertain and disoriented. the structure and length of clinical placement blocks are modified, along with semester breaks. traditional examinations en masse are not possible. practical assessments such as osce's require alteration given the transmission risk associated with close contact of simulated patients and examiners. most students in this group have been removed from clinical placement, leaving them without any practical teaching to rely upon. final year medical students face a more diverse spectrum of outcomes, albeit from a much stronger vantage point to both comprehend pandemic management and contribute to it directly. their core study comprises almost exclusively of clinical placement with practical teaching in preparation for internship. for these students, changes to staffing and services are challenging. in victoria, where a merit-based system is normally used for internship positions, the pmcv has now announced that references will no longer be considered due to the already significant workload on consultants( ). some final year students have been allowed to continue clinical placement but are left to determine what risk they are willing to accept in order to graduate. most clinical specialties have reduced clinic attendance in favour of telehealth, resulting in less face-to-face time with patients. attendence of students are often discouraged or limited, e.g. during ward rounds. the reduction in elective surgery has also affected learning opportunities. with the presence of students in hospital, consideration must be given to the increased risk of cross-contamination among medical teams. education on the correct use of personal protective equipement is also vital. several countries have accelerated final year medical students to the level of intern during the pandemic ( , ) . whether this occurs in australia remains to be seen. they may have the option to work in covid- or non-covid- facing roles, be it voluntary or via formal employment. this needs to be very carefully assessed at all levels by the state government in consultation with medical schools, taking into account the students' competency and scope of practice, before being executed. there has been the suggestion that students can volunteer as clinical assistants, receiving telehealth accepted article calls, performing sample collection and packing test kits in fever clinics ( ) . while this relieves workforce strain, it also places students in higher risk situations without compensation. students are in general younger, have less comorbidities and at a lower risk of becoming critically ill than the senior clinicians. it may therefore be logical to place them on the frontlines. whether students feel compelled to act in such a selfless manner for the public good is a personal decision that should be made voluntarily, with the provision of avenues for open disclosure of any safety concerns. at a global perspective, there were international students enrolled in australian medical schools in ( ) . covid- has far reaching effects on this cohort, who have invested significant financial and personal sacrifice to undertake their degree. several are met with disappointment at the abridged version of schooling provided, despite ongoing full fee payment requirements ( ) . some rely on part-time employment to fund their international living, which may also be affected with social distancing measures in place ( ) . some have their education interrupted by international travel restrictions ( ) . compounding these changes is the fear that if less students graduate in , future years may enrol a larger cohort and as such, increase competition for desired internship positions, for which they are already ranked inferior to their domestic colleagues. the covid- pandemic presents a dynamic situation, making it difficult for faculty, health services and the government to plan and deliver new strategies for education and support of medical students. communication with students is now more vital than ever for wellbeing and continuing learning. additionally, the consideration and recognition that students often contribute to the health system without any direct compensation is important during these times of increased risk. undoubtedly, there are more important issues for the public good than medical students in this time of crisis. however, medical students are also being significantly affected and many are willing to contribute to the effort against covid- . overall, the widespread ramifications of covid- are becoming more evident over time. the authors hope to highlight the effect on and potential contribution that the medical student cohort can offer in these unprecedented times. this article is protected by copyright. all rights reserved. get online with a usb modem covid- faqs for uq doctor of medicine students postgraduate medical council of victoria inc umms, three boston medical schools to accelerate graduation of class of fresh out of medical school, young italian doctors are being fasttracked to the coronavirus frontline: cnn ready for the rmh recruitment campaign: the rmh student statistics tables medical deans australia and new zealand australia's international students are anxious about experiencing covid- away from home sbs news how the coronavirus pandemic could shift the multicultural make-up of our society abc news disclosure statement: nicholas savage, anshini jain and suat chin ng declare that they have no conflict of interest. this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. key: cord- -o e na authors: nan title: scientific session of the th world congress of endoscopic surgery, jointly hosted by society of american gastrointestinal and endoscopic surgeons (sages) & canadian association of general surgeons (cags), seattle, washington, usa, – april : poster abstracts date: - - journal: surg endosc doi: . /s - - - sha: doc_id: cord_uid: o e na nan purpose: to evaluate the efficacy of single-incision laparoscopic surgery for totally extraperitoneal repair (sils-tep) of incarcerated inguinal hernia. patients and methods: clinical setting a retrospective analysis of patients undergoing sils-tep for incarcerated hernia from may to august at kinki central hospital was performed. exclusion criteria sils-tep was contraindicated for the following conditions in our hospital: a history of radical prostatectomy; a small indirect inguinal hernia in a young patient; and unsuitable for general anesthesia. surgical procedure laparoscopic abdominal exploration through a single, . -cm, intraumbilical incision was performed. the incarcerated hernia content was gently retracted from the hernia sac into the abdominal cavity. in some cases, simultaneous manual compression on the incarcerated hernia from the body surface was required. if no bowel resection was needed, a standard sils-tep using mesh was performed following laparoscopic abdominal exploration and incarcerated hernia reduction. if bowel resection was required, inguinal hernia repair using mesh was not performed to avoid postoperative mesh infection, and two-stage sils-tep was performed - months after the bowel resection. results: fourteen patients ( men, women) with irreducible inguinal hernias, including with unilateral hernias and with bilateral hernias, underwent surgery. the patients' median age was years (range, - years), and median bmi was . kg/m (range, . - . kg/m ). of the patients, had acute incarceration, and had a chronic irreducible hernia. seven patients with acute incarcerated hernias underwent emergency surgery, and two of the seven patients needed singleincision laparoscopic partial resection of the ileum, followed by two-stage sils-tep. twelve patients, excluding two patients who required single-incision laparoscopic partial resection of the ileum, underwent laparoscopic exploration with hernia reduction followed by sils-tep. one case of chronic incarceration out of the twelve patients who underwent sils-tep after hernia reduction required conversion to kugel patch repair. the median operative times were min (range - min) for unilateral hernias and min (range - min) for bilateral hernias. the median blood loss was minimal (range - ml). the median postoperative hospital stay was day (range - days). the median follow-up period was months (range - months). a seroma developed in % ( / ) of patients and was managed conservatively. no other major complications or hernia recurrence were noted during the follow-up period. conclusions: sils-tep, which offers good cosmetic results, could be safely performed for incarcerated inguinal hernia. objective: introduction of mis in pediatric age group has been proved feasible and safe. there is considerable evolution with introduction of a number of invovation in mis pediatric inguinal hernia repair. high ligation of sac is the basic premise of surgical repair in pediatric inguinal hernias. there are different mis techniques broadly grouped into intracorporeal or intracorporeal with extracorporeal component namely the suturing. every techniques has its own complications. the main objective of our study was to focus on different anatomical pointers which can lead inadverent complications mainly bleeding and recurrence. methods and procedures: prospective review of hernias ( male and female) ( months- years) performed laparoscopically between september and june . under laparoscopic guidance, the internal ring was encircled extraperitoneally using a - non-absorbable suture and knotted extraperitoneally. data analyzed included operating time, ease of procedure, occult patent processus vaginalis (ppv), contralateral inguinal hernia, complications, cosmesis and recurrence. results: sixteen right ( %), left ( %) and bilateral hernia ( %) were repaired. five unilateral hernias ( . %), all left, had a contralateral ppv that was repaired (p= . ). mean operative time for a unilateral and bilateral repair were . ( - ) and . min ( - min) respectively. one hernia repair still recurred ( . %) even with all precautions and another had a post operative hydrocoele ( . %). one case ( . %) needed an additional port placement due to inability to reduce the contents of hernia completely. because of our techinique we could not find any adverent peroperative bleeding. there were no stitch abscess/granulomas, obvious spermatic cord injuries, testicular atrophy, or nerve injuries. conclusion: the results confirm safety, efficacy and cost effectiveness of laparoscopic inguinal hernia repair. during our per-operative analysis we focus to address the anatomical landmark to minimize future recurrence and peroperative surgical complications. we identified and named a point as j. point at the tip of triangle of "doom". that is most important point to address peroperatively. there is high chance of recurrence if that point is not encircled well or inadequately circled because of fear of iliac vessels injury. we aslo concluded that 'water dissection technique' is effective techniques in un-experienced hand and in early stages of laparoscopic hernia repair to prevent inadvertent iliac vessels injury. medstar georgetown university hospital, georgetown university school of medicine, introduction: incisional hernias following abdominal surgery can be associated with significant morbidity leading to decreased quality of life, increase in health care spending and need for repeat operations. patients undergoing gastrointestinal and hepatobiliary surgery for malignant disease may be at higher risk for developing incisional hernias. identifying these risk factors for incisional hernia development can help decrease occurrence. this will be the largest multi-institutional study looking at incidence of symptomatic hernia rates for major abdominal operations including colectomy, hepatectomy, pancreatectomy, and gastrectomy. methods and procedures: an irb-approved retrospective study within the medstar hospital database was conducted, incorporating all isolated colectomy, hepatectomy, pancreatectomy, and gastrectomy procedures performed across hospitals between the years of to . all patients were identified using icd- and icd- codes for relevant procedures and then subdivided into either having benign or malignant disease. exclusion criteria comprised of patients who had concomitant organ resection, or those undergoing organ transplant. data validation was performed to verify the accuracy of the data set. the rate of symptomatic incisional hernia rates (ihrs) were determined for each cohort based on subsequent hernia procedural codes identified and repairs performed. descriptive statistics and chi squared test were used to report ihrs in each group. results: during this -year span, a total of , major abdominal operations were performed at all institutions, comprising of , colectomies, , hepatectomies, , pancreatectomies, and gastrectomies. malignancy was the indication for surgery in , ( . %) colectomies, ( . %) hepatectomies, ( . %) pancreatectomies, and ( . %) gastrectomies. ihr in each cohort for benign vs malignant etiologies, respectively, are as follows: ( . %) vs ( . %) in colectomy (p= . ), ( . %) vs ( . %) in hepatectomy (p= . ), ( . %) vs ( . %) in pancreatectomy (p= . ), and ( . %) vs ( . %) in gastrectomy (p= . ) patients. conclusion: symptomatic incisional hernia rates following major gastrointestinal and hepatobiliary surgery ranges from . to . %. there was no significant increase in hernia rates in patients undergoing surgery for malignancy. patients undergoing colectomy for benign disease had a high incidence of symptomatic ihrs. introduction: prosthetic infections, although relatively uncommon, are a major source of cost and morbidity. the study aimed to evaluate the influence of mesh structure including the polymer type and mean pore size on bacterial adherence in a mouse model. methods: three commercially available hernia meshes were included in the study. for each mesh type, a cm square was surgically placed intraabdominally in mice. one mouse served as a control while an enterotomy was made in the subsequent mice to introduce a bacterial load onto the mesh. after hours the meshes were harvested. the inoculated meshes were then plated on agar plates and bacterial counts were counted after hours. the bacterial counts were compared between the various mesh types. results: the mean bacterial adherence was increased in the large pore mesh was colonies, for the small pore mesh was colonies, and in the biologic mesh group it was colonies. conclusions: through the use of a mouse model, the influence of mesh type and pore size on bacterial adherence was evaluated. meshes that have larger pores with a lower prosthetic load and the biologic mesh interestingly had lower early bacterial colonization after hours following an enterotomy. further evaluation with a longer incubation time could be helpful to determine the effect of bacterial colonization of mesh. hrishikesh salgaonkar, raquel maia, lynette loo, wee boon tan, sujith wijerathne, davide lomanto; national university hospital, singapore laparoscopic repair of groin hernias is widely accepted approach over open due to lesser pain, faster recovery, better cosmesis and decreased morbidity. however, there is still debate on its use in large inguino-scrotal hernias, recurrent hernias and history of lower abdominal surgery anticipating adhesions and difficulty in dissecting extensive hernia sac. retrospective analysis of prospectively collected data was done of patients undergoing laparoscopic repair of large inguino-scrotal, incarcerated groin hernia, recurrent cases after open or laparoscopic repair and history of previous lower abdominal surgery. between january to july , patients with large inguino-scrotal hernias, recurrent hernia, history of lower abdominal surgery, incarcerated femoral hernia underwent laparoscopic inguinal hernia repair. patient characteristics, operating time, surgical technique, conversion rate, complications and recurrence up to months recorded. patients had large inguino-scrotal hernia, recurrent hernia ( previous open, previous lap) , history of lower abdominal surgery ( lscs, appendectomy, prostatectomy, midline laparotomy), incarcerated femoral hernia, meshoma removal. patients underwent total extraperitoneal (tep) repair, transabdominal pre-peritoneal (tapp), needed conversion to open. mean operation time was min for unilateral and min for bilateral hernia. seroma formation seen in patients, minor wound infections treated conservatively. we conclude that the laparoscopic approach can be safely employed for the treatment of complex groin hernias; surgical experience in laparoscopic hernia repair is mandatory with tailored technique in order to minimize morbidity and achieve good clinical outcomes with acceptable recurrence rates. mesh fixation in ventral incisional hernia is a topic of ongoing debate. permanent and absorbable tacks are acceptable and widely used methods for mesh fixation. the purpose of this study was to compare outcomes of permanent tack fixation versus absorbable when used alone or with suture fixation in laparoscopic incisional hernia repairs. a retrospective review of all patients undergoing laparoscopic ventral hernia using tack fixation (absorbable/permanent) alone or in conjunction with suture fixation was queried from the ahsqc database. outcome measures included hernia recurrence rate, pain, quality of life, wound related issues, and hospital length of stay. propensity match scoring was performed to compare patients undergoing tack only fixation versus tack and suture fixation with a p-value of . considered significant. a total of patients were identified after propensity match scoring with who underwent repair with permanent tacks alone or with sutures and who underwent repair with absorbable tacks alone or with sutures. following matching there were no differences in bmi, age, hernia width/length, or baseline pain/ quality of life. there were no significant differences found in outcome measures including recurrence rates, pain and quality of life outcomes at days, months, and year, surgical site infection (ssi), and postoperative length of stay (p[ . ). there was a significant increase in any post op complication in the permanent tack fixation group compared to the absorbable tack fixation group ( % vs %, p. ) which is likely due to the increase in surgical site occurrences noted in the permanent tack fixation group ( % vs. %, p. ). based on this large data set, there are no significant differences in postoperative outcomes in permanent versus absorbable fixation in laparoscopic hernia repair except in surgical site occurrences. further study is needed to evaluate but at the present time, there is no convincing evidence that one type of fixation is superior to another in laparoscopic ventral hernia repair. introduction: inguinal hernia repair is the most common procedure in general and visceral surgery worldwide. laparoscopic transabdominal preperitoneal mesh hernioplasty (tapp) has been also popular surgical method in japan. single incision laparoscopic surgery is one of the newest branches of advanced laparoscopy, and its indication has been spread to not only simple surgery such as cholecystectomy, but also complex surgery. we report our experience with single incision laparoscopic tapp (s-tapp) for japanese patients with inguinal hernia. case description: a consecutive series of patients ( male, female) who underwent s-tapp during june to september in a single institution. twenty eight of the patients had bilateral inguinal hernia. the mean follow-up was days. the average age of the patients was . ± . years. establishment of the ports: a -mm vertical intra-umbilical incision is made for port access. one -mm optical port and two -mm ports were placed side-by-side through the umbilical scar. surgical procedure: the procedure was carried out in the conventional fashion with a wide incision in the peritoneum to achieve broad and clear access to the preperitoneal space, and an appropriate placement of polypropylene mesh ( dmaxtm light, bard) with fixation using the tacking device (absorbatack®, covidien). the hernia sac is usually reduced by blunt dissection, or is ligated and transected with ultrasound activated device. the peritoneal flap is closed by one suture with - pds and the - tacks using absorbatack®. discussion: in one patient, we encountered a large sliding hernia on the right side having sigmoid colon as content of the sac, which required conversion to the conventional laparoscopic procedure. there were nine recurrence cases after surgery of laparoscopic or anterior approach, and two cases after prostatectomy. there was no intra-operative complication. the mean operative time was . ± . min, and blood loss was minimum in all cases. the average postoperative stay was . ± . days. there was one recurrence case ( . %) months after the surgery. there was no severe complication after the surgery, but there were seromas ( . %) and one hematoma ( . %). two patients had blunt tactile sense in the area of the lateral femoral cutaneous nerve ( . %), which improved in two months. conclusion: our results suggest that s-tapp is a safe and feasible method without additional risk. moreover, cosmetic benefit is clear. however, further evaluation for postoperative pain and longterm complications compared to standard laparoscopic tapp mesh hernioplasty should be required. manuel garcia, md, daniel srikureja, md, marcos j michelotti, md, facs; loma linda university health introduction: prosthetic mesh use has become standard practice during ventral hernia repair to reduce the risk of recurrence. the ideal mesh is macro-porous which favors rapid cellular ingrowth and tissue integration, has limited tissue reactivity, low profile and weight, and has high tensile strength to add resilience to the repair. additionally, the material is expected to have good handling characteristics. currently, there is a wide variety of options for mesh. biosynthetic material (poliglycolic acid/trimethylene carbonate -pga/tmc) has been shown to behave well in terms of early vascularization and ingrowth as well as adequate long term tissue generation. gore® synecor® biomaterial is a composite mesh including two layers of absorbable biosynthetic material (pga/tmc) with one tridimensional non-absorbable macro-porous knit of dense ptfe mesh. it has shown good vascularization and ingrowth at days in animal examination. however, there is still no evidence of long term behavior of this mesh in human tissue. we present the first histologic analysis of this mesh year after placement in a human. objective: to perform a histologic analysis of the gore® synecor® biomaterial one year after placement in the human body. methods: after incidentally finding incorporated gore® synecor® mesh in a patient with prior ventral hernia repair year ago, during open bilateral inguinal hernia repair, a sample of mesh was taken and sent to pathology lab for analysis. tissue healing, vascularization, and ingrowth of the composite mesh were analyzed. results: histologic findings significant for a biomaterial consistent with a knitted ptfe material surrounded by mature fibrovascular tissue and foreign body inflammation consistent with expected healing response for this time frame. no evidence of any other biomaterial (pga/tmc) or evidence of infection. conclusion: gore® synecor® biomaterial has shown to be well integrated into appropriately healed tissue, with pronounced vascularization and ingrowth. the pga/tmc layers have been seen to be completely absorbed and replaced by collagen. these findings, in a human months sample, replicate what had been shown in animal specimens. method: from to , patients came to hospital with renal paratransplant hernia. they were evaluated for this study. the following data were collected from their records: age, gender, weight, age at graft rejection, surgical complications, treatment method and the treatment results with composite ptfe mesh. results: for laparoscopic repair of incisional hernia after renal transplant, the median interval between kidney transplantation and developing of incisional hernia was (range to ) days. predisposing factors were obesity, age over fifty years, and female gender. in six patients, hernia was large, and the repair was performed with using composite ptfe mesh. one patient had developed serous collection in surgical site, which was managed successfully with multiple punctures. hernia recurrence or infection was not noted in these patients during to months follow-up periods. conclusion: incisional hernia is not a rare entity after kidney transplantation. predisposing factors, such as obesity, age over years, and female gender have a role in its development. repeated surgeries in kidney recipients can increase the risk of incisional hernia. managing this complication by laparoscopic approach is a safe and effective method. sujith wijerathne, raquel maia, hrishikesh salgaonkar, wee boon tan, lynette loo, davide lomanto; national university hospital, singapore introduction: a femoral hernia is a less common type of hernia. it is estimated to account for less than % of all abdominal wall hernias. only about in every groin hernias are femoral hernias. they are found more commonly in females due to wider shape of pelvis. laparoscopy by offering magnification and better vision provides us the opportunity for clear visualization of the myopectineal orifice. laparoscopy seems to be a safe and feasible approach for femoral hernia repair in an asian population. case description: between and , consecutive patients with femoral hernia who underwent laparoscopic hernia repair were prospectively studied. patient demographics, hernia characteristics, operating time, conversion rate, intraoperative, postoperative complications and recurrence were measured. discussion: total of femoral hernias were repaired, on right and on left groin. this included patients with bilateral and unilateral hernia. concomitant obturator hernia were found. there were male and female patient. no conversion was reported. one patient had injury to bowel at the mm port entry site, without contamination, identified and managed immediately. patients developed seroma, all were managed conservatively except one who needed aspiration. peri-port bruising was noticed in patients and patients had hematoma. one patient with hematoma underwent excision of the organised hematoma. of the hematoma patient was on aspirin pre-operatively. no wound infection, chronic groin pain or recurrence was documented during follow up till date. conclusion: laparoscopic repair offers accurate diagnosis and simultaneous treatment of both inguinal and femoral hernia with minimum morbidity and good clinical outcomes. better visualisation and magnification gives us an opportunity to identify occult hernias which can be repaired during the same setting, thereby reducing the chance of recurrence and possible need for second surgery. laparoscopic repair has become the procedure of choice for the treatment of the majority of groin hernia at our institution. introduction: totally extraperitoneal (tep) repair that does not require peritoneal incisions is a good procedure that involves minimal visceral damage. however, balloon-or camera-assisted blunt dissections that are performed in a haphazard manner do not follow precise dissection of the fascia layer. furthermore, they have a disadvantage in that they are difficult to understand anatomically. we therefore developed a novel preperitoneal approach to resolve this issue. methods: a -mm trocar is inserted into the rectus abdominis sheath cavity after a small incision is made below the umbilicus and the posterior rectus sheath is exposed. a -mm trocar is inserted cm towards the pubic bone from the umbilicus. using forceps from this position, narrow branches that enter the posterior rectus sheath from the inferior epigastric vessels are dissected, thereby broadly exposing the anterior surface of the posterior rectus sheath. the third mm-trocar is inserted near the lateral margin of the rectus abdominis. on the outside, local anesthetic is injected beneath the posterior rectus sheath and the preperitoneal cavity is separated in fluid so that the peritoneum is not injured during posterior rectus sheath incision. a small incision is made to the posterior rectus sheath or attenuated posterior rectus sheath at one finger width higher than the expected upper margin of the prosthetic mesh. due to the effects of local injection, a sharp incision to the fascia can be made with an electric scalpel. utilizing this mechanism, the posterior rectus sheath aponeurosis and the lining transverse fascia and superficial preperitoneal layer are individually identified. once the preperitoneal cavity is reached, the peritoneal margin is determined in the lateral direction, and the peritoneum that is pulled due to pneumoperitoneum is separated from the preperitoneal fascia on the outside from the cranial side towards the deep inguinal ring. on the inside, the pneumoperitoneum pressure pushes the peritoneum inferiorly, leading to enlargement and increased visibility of the posterior rectus sheath deep fascia, which is dissected one layer at a time from the outside. the umbilical prevesical fascia is dropped inferiorly, and the dissection of the preperitoneal cavity necessary for mesh deployment is performed. results: by individually dissecting each fascia using emphysema through pneumoperitoneum and enlargement through local injection, the method for reaching the preperitoneal cavity could be successfully completed by following the dissection of the fascia layer without proceeding with the operation blindly, thereby resulting in the elimination of intraoperative bleeding and postoperative hematoma. introduction: in the field of abdominal wall reconstruction, the utility of drain placement is of debatable value. we present outcomes evaluating drain placement vs no drain placement at the time of robotic transversus abdominis release (rtar) technique with placement of mesh in the retromuscular position, a currently understudied subject. methods: retrospective review of a prospectively maintained hernia patient database was conducted identifying individuals who received either drain placement or no drain placement during abdominal wall reconstruction via the rtar technique from august to june at a single high volume hernia center. perioperative data and postoperative outcomes between the two groups are presented with statistical analysis for comparison and quality of life (qol) measures assessed using the carolina comfort scale. results: thirty-five patients were identified for this study, of which had drains placed intraoperatively in the retromuscular position at the conclusion of rtar (drn) and underwent rtar without the placement of draining devices (nd). the drn cohort had a mean bmi, defect area, mesh area, and operative time of . , cm , cm and minutes, respectively, compared to . , cm , cm , and minutes in the nd group. all cases utilized medium weight macroporous polypropylene synthetic implantable mesh materials in both the drn and nd subgroups. there were no reported postoperative complications, including no development of hematoma, seroma, or surgical site infections in either group. hernia recurrence was not identified in either the drn or nd cohorts through a mean follow up of days ( . months). there were no statistically significant differences in postoperative qol outcomes. conclusion: our series review suggests that the use of intraoperative drains may not afford any benefits with the rtar technique when mesh is placed in the retromuscular position. additional postoperative management associated with drain care may be unnecessary. surg endosc ( ) :s -s background: appendectomy is one of the most common operations performed during emergency surgery. although laparoscopic appendectomy (la) has become the treatment of choice, there is still a debate regarding the use of la for treating complicated appendicitis. in this retrospective analysis, we aimed to clinically compare la and open appendectomy (oa) for treating complicated appendicitis. methods: we retrospectively identified patients who underwent an operation for complicated appendicitis at our hospital; these patients were operated on between and july . [editor ] in total, patients underwent conventional appendectomy and patients were laparoscopically treated. outcomes included operation time, blood loss, length of hospital stay, and postoperative complications. logistic regression analysis was performed to analyze the concurrent effects of various factors on the rate of postoperative complications. objective: small bowel perforation has conventionally been dealt with open exploration, which frequently leads to many wound-related complications. wound infection is the major reason for increasing morbidity in these patients and delay recovery. laparoscopic surgery has various benefits over open surgery like, smaller wound, lesser pain and faster recovery. the aim of this study was to relay the advantages of minimally invasive surgery (mis) to patients with small bowel perforation to decrease postoperative wound complications and duration of hospital stay. methods: it is a retrospective study, including patients with small bowel perforation from to . of these , had traumatic etiology, had typhoid-related perforation and the remaining had a duodenal perforation. of them were male, and the average age was . years. only patients who presented within hours of perforation were included in the study. laparoscopic exploration was done on introducing camera from -mm infraumbilical port after intraperitoneal carbon dioxide insufflation. the remaining two -mm working ports were then introduced depending on the site of perforation once identified. the perforations were then repaired using intracorporeal single-layer suturing using polydioxanone - suture. the peritoneal cavity was given thorough lavage and abdominal drain placed in the pouch of douglas. fecal contamination was found in all the patients. a total of patients underwent conversion to open surgery due to inability to find the site of perforation laparoscopically. of the operated patients, patients developed port-site infection, and there were no major postoperative complications in the -week follow up period. conclusion: we conclude from our study that laparoscopic intervention in early small bowel perforation is a safe approach with favorable outcomes, especially with regards to wound complications, that are a major factor in increasing the morbidity in such patients postoperatively. laparoscopic approach leads to early discharge and recovery postoperatively. with the emerging era of laparoscopic surgery, leading to its easy accessibility, more patients can advantage from this technique when they arrive in emergency with intestinal perforation. s surg endosc ( ) :s -s introduction: pneumatosis intestinalis (pi), or gas in the bowel wall, can be seen on various imaging modalities. the pathophysiology behind pi is unclear. one theory proposes a mechanical cause (e.g. small bowel obstruction) while another proposes a bacterial etiology. management of pi in adults is difficult as often there is a benign clinical course. however, when paired with specific clinical features such as hepatic portal venous gas (hpvg) on imaging, the course of management changes as the suspicion of bowel ischemia increases. hpvg alone has been associated with a high mortality rate and a poor prognosis. management in this case becomes surgical. case presentation: we present a case of -year-old latino male who presented to the emergency room with abdominal pain and altered mental status. focused physical examination revealed a non-rigid abdomen, no rebound tenderness, no guarding, and diffuse tenderness only to deep palpation. ct scan of the abdomen and pelvis demonstrated moderate portal venous gas in the right and left hepatic lobes, an upper midline dilated small bowel loop with pneumatosis intestinalis, and a moderately distended stomach with gas and fluid. laboratory studies revealed metabolic acidosis and a lactic acid level of . mmol/l. due to these findings, bowel ischemia was suspected, and the patient was taken to the operating room for a diagnostic laparoscopy. the laparoscopy was converted to an exploratory laparotomy due to extensive adhesions. intraoperatively, there was no small bowel compromise and no identifiable transition point. extensive lysis of adhesions and repair of iatrogenic enterotomy were performed. patient tolerated the procedure well, clinically improved, and was discharged from the hospital. discussion: this case illustrates the difficulty in management of a patient with pneumatosis intestinalis and, specifically, hepatic portal vein gas seen on ct imaging. hpvg has traditionally been a harbinger of morbidity and mortality, but exploratory laparotomy revealed only diffuse abdominal adhesions and the absence of bowel ischemia despite high clinical suspicion. background: ventral hernia repair is one of the most common surgical procedures facing the general surgeon. there is little consensus as to the best surgical technique for complex scenarios. often these patients have complicating co-morbid conditions such as radiation therapy, that has an inevitable effect in the abdominal wall structures, which can lead to non-traditional repairs. case report: we present a case of a year-old female who underwent a tah/bso and right hemicolectomy which was complicated by wound dehiscence. she underwent primary repair and adjuvant whole pelvis radiation for her squamous cell carcinoma. subsequently, the patient developed acute obstructive symptoms do to a stricture within her small bowel and a large ventral hernia measuring cm with non-reducible abdominal contents below the level of the fascia more prominent in the suprapubic area. the patient's bmi was . . various considerations are important in planning a surgical repair in a previously irradiated field with loss of domain which include, minimal dissection, and the use of an atraumatic surgical techniqueque with either external oblique release or transversus abdominis muscle release (tar). we chose a a tar, as it provides wider myofascial release and dissection below the arcuate line towards the space of retzius and bogros allowing for a larger sublay mesh placement. also it avoids the need of skin flaps reducing the risk for wound complications in under-perfused tissue. the tar was performed successfully and there were no intraoperative and postoperative complications. her follow-up at months revealed no wound complications or hernia recurrence. conclusion: for patients with compromised tissue and loss of domain a tar technique may be useful when reconstructing complex abdominal wall hernias. it provides the core principals of hernia repair such as primary fascial closure, wide mesh overlap, and finally it provides a reliable approach for the under-perfused tissue without need of skin and soft tissue flap creation. outcomes in the management of cholecystectomy patients in the setting of a new acute care surgery service model: impact on hospital course larsa al-omaishi, bs, william s richardson, md; ochsner medical clinic foundation introduction: the acute care surgery (acs) model, defined as a dedicated team of surgeons to address all emergency department, inpatient, and transfer consultations, is quickly evolving within hospitals across the united states due to demonstrated improved patient outcomes in the non-trauma setting. the traditional model of call scheduling consisted of one senior attending and one senior resident on call per -hour shift. attendings were responsible for consults, previously scheduled operations, as well as clinic time. multiple recent studies have shown statistically significant improvements in several parameters of patient care by using acs including but not limited to . time from emergency department to surgical evaluation . time from surgical evaluation to operating room . operative time . percent laparoscopic . length of hospital stay . intra-operative complications (blood loss, perforation rates) . post-operative complications (fever, infection, redo) . cost. one study demonstrated a statistically significant cost savings for the acute care surgery model with respect to appendectomies, but not cholecystectomies. study design: a retrospective analysis of patients who underwent cholecystectomy in the setting of non-traumatic emergent cholecystitis was performed to compare data from two cohorts: the traditional model and the acs between january , and dec , at ochsner medical center, a -bed acute care center in new orleans. parameters gathered included . time from emergency department to surgical evaluation . time from surgical evaluation to operating room . operative time . percent laparoscopic . length of hospital stay . intra-operative complications (blood loss, perforation rates, conversion to open) . post-operative complications (fever, infection, redo). demographics were also collected including age, weight, height, ethnicity, asa, etc. inclusion criteria included: age[ and having undergone cholecystectomy between jan , and december , . exclusion criteria included choledocholithiasis, gallstone pancreatitis, ascending cholangitis, gangrenous cholecystitis, septic complications precipitating further procedures and delays, or researcher discretion. results: patients were initially identified as having undergone cholecystectomy within the allotted time period [ - , - , - , - ] . were excluded due to one of the reasons above. median patient age was years old and the average patient encounter was . days. conclusion: the acs model is better suited to manage emergent non-traumatic cholecystectomies than the traditional call service at our institution, as evidenced by several parameters. s surg endosc ( ) :s -s he nailed it background: nail guns are powerful tools and are widely used. injuries with these devices may be devastating due to the significant force they can deploy. patients and methods: we herein report a first case of a self inflicted abdominal injury with a nail gun. results: a year old male with history of coronary artery disease, type dm and early signs of dementia attempted to refill a nail gun. he lodged the device against his right abdomen while the air hose was still attached and then accidently fired nails into his abdomen. after he unsuccessfully tried to pull the nails out he drove himself minutes to our emergency room. he was hemodynamically stable on arrival; pain control was achieved, antibiotics were given and he received tetanus immunization. ct-scan showed the two foreign bodies penetrating from the ruq with one reaching the transverse colon. on emergency laparoscopy, the nails were found to have penetrated the thick omentum and the puncture site of one nail into the colon was identified. the omentum was resected off the colon and the right colon was completely mobilized. no additional injuries were found. the entrance area of the nails was then used to create a loop colostomy. the postoperative course was initially uneventful but the patient developed a severe posttraumatic inflammatory reaction of the fat tissue in the right upper quadrant and had to be readmitted for pain control and antibiotics were again administered. he recovered and was discharged with a plan for laparoscopically assisted colostomy closure after weeks. discussion: to the best of our knowledge this is the first reported isolated colonic injury by a nail gun. given the tremendous force of the device with unknown collateral damage to the surrounding tissue it was decided to manage the accident with a laparoscopic assisted colostomy using the entrance point of the nails for fecal diversion. introduction: it is difficult to diagnose obturator hernias by routine physical examination. obturator hernias are frequently complicated by ileus and the diagnosis is often first made from abdominal ct. obturator hernias are difficult to reduce, and often necessitate emergency surgery. they are common in elderly people, and they often had bad general condition. so it was high in the death rate. at our hospital, we first attempt to reduce the hernia from the body surface under ultrasonographic guidance. after relieving the strangulation, we perform radical operation electively in patients who are for possible for surgery under the general anesthesia. we perform laparoscopic repair for obturator hernias. obturator hernias are often complicated by other types of hernia. in these cases, we perform total repair. herein, we present a review of the patients who underwent surgery for obturator hernia at our hospital. methods: we review the data of cases of obturator hernia encountered by us from february to december . we performed total repair in three of the cases. however, it is difficult to procure a mesh that would be adequate for all the defects (inner inguinal ring, femoral ring, obturator). no single mesh can fit, because the inguinal and pelvic curves present opposing curves near the obturator. therefore, we placed two pieces of mesh available at our hospital ( d max [bard] and onlay sheet of kugel patch [bard] ) together in the patientswe could successfully cover all the defects using these two pieces of mesh and could fit the mesh to the pelvic shape by devising an appropriate connection between the meshes. results: we reviewed a total of operated cases for obturator hernia. the hernia was bilateral in cases, and complicated by other hernias in cases. we first determined the appropriate approach for the repair. we performed total repair in cases. they were no complications and no cases of recurrence. conclusion: our approach to the repair of obturator hernias was very useful. we can use the exact area and shape of the mesh needed in individual patients by this method. we show the method of shaping the mesh to fit the pelvic form. demin aleksandr, do, ajit singh, do, noman khan, do; flushing hospital introduction: internal hernias are known complications that are well documented to involve peterson's defect. in bariatric patient's post gastric bypass there is a high index of suspicion for internal hernias as well as a low threshold to operate. there have been some debates around the closure of the potential peterson's space with several studies advocating closure versus some which show that there is no difference in the rate of symptomatic internal hernias. we present a case of an unusual cause of small bowel obstruction due to internal hernia caused by a cecal volvulus. it is an atypical presentation however the patient was triaged and brought to the or within hours of admission. although it is rare there have been reports of internal hernias caused by other structures like congenital bands or natural potential spaces. there have been reports of unusual presentations of the cecum herniating through the foramen of winslow. the anatomical rearrangements after bypass create potential areas where an internal hernia can occur. in this case a bowel resection was undertaken due to the anatomical variation of the cecal bascule and cecal volvulus due to high rate of recurrence of this cecal pathology. majority of internal hernias do not require bowel resection especially when detected earlier and prompt surgical exploration is undertaken. mortality as direct consequence internal hernia is extremely rare. however late diagnosis of internal hernias can lead to catastrophic gut loss and may require lifelong tpn and/or visceral transplantation or autologous reconstruction. conclusion: careful history and physical of our bariatric patient can elicit the signs and symptoms of internal hernias and prevent the morbidity and mortality that can come with the complications of this condition. unusual presentations and causes are reason for prompt diagnosis and complete exploration. shingo ishida , naotsugu yamashiro , satoshi taga , koichi yano ; shinkomonji hospital, shinmizumaki hospital symptomatic cholelithiasis is common disease performed with laparoscopic cholecystectomy (lc). we will hesitate to operate if the patient is pregnant in the third trimester. pregnant patients undergoing laparoscopic surgery have been reported increasingly. however, most case reports are confined to patients in the first and second trimester. we report a patient who underwent lc in the third trimester and review the relevant literature. a -year-old woman in the third trimester ( w d) of pregnancy was seen in the emergency department of our hospital with a history of upper abdominal pain. there was no problem in the course of pregnancy. the result of the examination proved to be attack of gallstone colic. she was hospitalized the same day and underwent lc the next day. the base of pregnancy uterus was cm above the navel. we needed to consider the surgical approach, for example inserting the first trocar under left hypochondrium. operative duration was minutes. she complained abdominal distension at postoperative day (pod) and but there was no abnormality in the fetus. she was discharged on pod . after that she gave birth to a healthy baby. lc in third trimester of pregnancy was safely performed with obstetrics back up. weekday or weekend hospital discharge: does it matter for acute care surgery? ibrahim albabtain , roaa alsuhaibani , sami almalki , hassan arishi , hatim alsulaim ; kamc, background: hospitals usually reduce staffing levels over weekend. this raises the question of whether patients discharged over a weekend may be inadequately prepared and possibly at higher risk for adverse events post-discharge. the aim of this study was to assess the outcomes of common acute care surgery procedures for patients discharged over weekend, and identify the key predictors of early readmission. methods: this retrospective cohort study was conducted at a tertiary care hospital between january and december . surgical procedures included were cholecystectomy, appendectomy, and hernia repairs. patients' demographic, co-morbidities, complications, readmission and follow-up details were collected from the electronic medical records. predictors and post-operative outcomes associated with weekend discharge were identified by multivariable analysis using univariable and multivariable logistic regression models controlling for potential confounders. results: a total of patients were included. overall median age was years (iqr: , ). the majority of patients were female (n= , . %). patients ( . %) underwent a cholecystectomy, ( . %) an appendectomy, and ( . %) hernia repairs. weekend discharge was . % vs. . % of weekday discharge. patients discharged during weekend were younger ( . vs. , p-value. , mean) . post-discharge -day follow-up visits were significantly lower in the weekend discharge subgroup ( . % vs. . %, p-value . ). overall, -day readmission rate was . % (n= ), and did not differ between those of weekend and weekday discharge (or= . , % ci . - . ). conclusions: patients discharged on weekends tended to be younger in age and less likely to have chronic diseases. patients discharged over the weekend were less likely to follow up compared to weekday discharge patients. however, the readmissions rate did not differ between the two groups. intrauterine device (iud) migration out of the uterine cavity is a serious complication. its incidence in the us has been reported to be about . % annually. previously published systematic review supports the use of laparoscopic surgery for elective removal of migrated iucds from the peritoneal cavity. we present the safety and efficacy of the laparoscopic approach to this complication in the acute care setting. depicted is an otherwise healthy year old female with no previous surgical history who presented to the ed with worsening abdominal pain for one week with no associated symptoms. on physical exam, patient was non toxic. abdomen was moderately distended with guarding and rebound tenderness to palpation, no rigid. patient had been seen shorlty prior to ed admission by her obgyn and recent work up with abdominal/pelvic x-ray and ultrasound has revealed a misplaced iud in the transverse position (side ways). pregnancy test was negative. based on patient clinical presentation and recent radiologic findings, we decided to proceed with diagnostic laparoscopy. after systematic review of cavity, the foreign body was found to be incorporated within the greater omentum. we procceded, laparoscopically with omentectomy+foreign body removal. there were no perioperative complications, patiet was discharged on the following day. the use of laparoscopy in elective iud retrieval within in the abdominal cavity has been considered standard of care in surgical management to date. this poster demonstrates its use as an effective approach for safe removal of intra-abdominal foreign bodies also in the acute setting. symptomatic inguinal and umbilical hernias in the emergency department: opportunity lost? andrew t bates, md, jie yang, phd, maria altieri, chencan zhu, bs, salvatore docimo, jr., do, konstantinos spaniolas, md, aurora pryor, md; stony brook university hospital introduction: patients with symptomatic inguinal and umbilical hernias often present to the emergency department (ed) when their symptoms change or increase, usually not requiring emergent surgery. however, little is known about how often these patients present prior to eventual repair and whether they undergo surgery at the initial presenting institution. the aim of this study was to assess the clinical flow of patients presenting in the ed for inguinal and umbilical hernia. methods: all patients presenting to eds in new york state from to with symptomatic inguinal and umbilical hernias were identified using the new york state longitudinal hospital claims database (sparcs). patients were followed for records of hernia repair and subsequent inpatient and outpatient visits up to . results: , patients presenting to the ed for symptomatic inguinal hernia were identified. . % ( , ) of ed presentations resulted in inpatient admissions. , ( . %) had repair later and their average time from ed presentation to inguinal hernia repair was (± ) days. . % of patients who did not have subsequent surgery had only one ed visit. of those that underwent interval repair, . % had only one ed visit prior to surgery. for those patients with only one ed visit before repair, . % had repair at a different hospital, as opposed to . % if multiple ed visits were made. , umbilical hernia patients presenting to the ed were identified. . % ( , ) resulted in inpatient admission. , ( . %) had interval repair, with the average time from ed presentation to umbilical hernia repair being (± . ) days. % of patients who did not record of later repair presented to the ed once. of those patients who underwent repair, . % did so after one ed visit. for those patients with only one ed visit before repair, . % had repair at a different hospital, as opposed to . % if multiple ed visits were made. conclusion: a majority of patients with symptomatic inguinal and umbilical hernias that present to the ed do so once with no subsequent follow-up or repair. for those patients that undergo interval repair, a significant portion willnopt for surgery at other hospitals. a significant proportion of patients with acutely symptomatic inguinal/umbilical hernias who undergo interval repair after a previous ed visit, will opt for definitive surgery at another hospital facility. this represents a missed opportunity for continuity of care for providers and healthcare systems. nikhil gupta, dr, himanshu agrawal, dr, arun k gupta, dr, dipankar naskar, dr, c k durga, dr; pgimer dr rml hospital, delhi introduction: peritonitis is the inflammation of the serous membrane that lines the abdominal cavity and the organ contained therein and is one of the most common infections, and an important problem that a surgeon has to face. reproducible scoring system that allows a surgeon to determine the severity of intra-abdominal infections are essential to prognosticate the patient. this study was done to compare apache ii scoring and mpi score to assess prognosis in perforation peritonitis. methods: all patients admitted with hollow viscus perforation from st november till st march was included in the study. it was a cross sectional observational study. apache ii and mannheim peritonitis index (mpi) scoring systems were calculated in all the patients in order to assess their individual risk of morbidity and mortality. the outcome variables were studied postoperatively -post-operative wound infection, wound dehiscence, anastomotic leak, respiratory complications, duration of hospital stay, need of ventilator support and mortality. the inferences were drawn with the use of appropriate tests of significance. results: the study comprised of patients. neither apache ii nor mpi could predict postoperative wound infection. the mean apache ii score of subjects included in the study was . ± . with range of to and the mean mpi score of subjects included in the study was . ± . with range of to . apache ii was able to predict postoperative respiratory complications, post-operative need for ventilatory support, hospital stay duration and mortality while mpi was able to predict post-operative wound dehiscence, post-operative respiratory complications, post-operative need for ventilatory support and mortality. neither apache ii nor mpi could predict postoperative anastomotic leak and postoperative wound infection. conclusion: mannheim peritonitis index is a useful and simple method to determine outcome in patients with peritonitis. mpi is comparable to apache ii in assessing the prognosis in perforation peritonitis and can well be used in emergency setting in place of apache ii scoring when time is a definite constraint. microrna- and the prognosis of human carcinomas: a systematic review and meta-analysis chengzhi huang, mengya yu; guangdong general hospital (guangdong academy of medical science) muhammad nadeem , julian ambrus, md , steven schwaitzberg, md , john butsch, md ; university at buffalo, introduction: mitochondria is a small energy producing structure of a cell. mitochondrial myopathy (mm) is mixed disorder clinically, which can affect various systems besides skeletal muscle. mm starts with muscle weakness or exercise weakness. mm patients have decreased skeletal muscle mitochondrial function than the healthy person, because of weakened intrinsic mitochondrial function and decreased mitochondrial volume density. no one has studied the mm role in gerd and constipation so far. this study is aimed to see effects of mm on the gastrointestinal system specifically gastroesophageal reflux disease (gerd), gall bladder issues, and constipation. methods: between may and june , mm diagnosed patients at buffalo general hospital were included in this retrospective study. we assessed their demeester score for gerd and wexner's constipation questionnaire for constipation. demeester score[ and constipation score[ were set points for gerd and constipation respectively. data was analyzed by using spss version . mitochondrial enzymes were assessed by using their muscle biopsy report. results: out of ( . % female, . % male) mitochondrial myopathy patients, . % and . % were suffering from gerd and constipation respectively. . %, . % and . % patients had gall bladder issues, obstructive sleep apnea (osa) and fatigue respectively. mm gerd patients ( . % female, . male) had mean demeester score . (sd: . ) more than normal although . % patients were on gerd medications and . % patients had nadh cytochrome c reductase, cytochrome c oxidase and citrate synthase abnormal mitochondrial enzyme in mm associated gerd but . % mm patients had abnormal cytochrome c oxidase enzyme only. mm along with constipation had mean wexner's constipation score . (sd: . ) more than the normal although . % were taking enema, medications or digital assistance. % patients had cytochrome c oxidase and nadh cytochrome c reductase enzymes were abnormal in those patients. . % mm associated gall bladder issues patients had cytochrome c oxidase abnormal. . % mm associated gerd and constipation patients had gall bladder issues. conclusion: in this present study, we found that mm had effects on gastrointestinal system causing gerd, constipation and gall bladder issues. gerd, constipation and gall bladder problems are common in mm patients even patients are taking medications for gerd and constipation. cytochrome c oxidase, citrate synthase and nadh cytochrome c reductase are the most commonly impaired mitochondrial enzyme in mm patients and mm associated gerd, constipation and gall bladder issues patients. objectives: gulf war illness (gwi) is a chronic, multisymptom illness marked by cognitive and mood dysfunction and disrupted neuroendocrine-immune homeostasis affecting % of gw veterans. after + years, useful treatments are lacking and its cause is poorly understood, although exposures to pyridostigmine bromide and pesticides are consistently identified among the strongest risk factors. previous work in our laboratory using an established rat model of gwi identified persistent elevation of microrna- (mir- ) levels in the hippocampus whose gene targets are involved in cognition-associated pathways and neuroendocrine function, suggesting that mir- inhibition is a promising therapeutic approach to improve the complex symptoms exhibited by gwi. the purpose of this study was to identify broad effects of mir- inhibition in the brain by profiling the expression of genes known to play a critical role in synaptic plasticity, glucocorticoid signaling, and neurogenesis in gwi rats administered a mir- antisense oligonucleotide (mir- inhibitor). methods and procedures: nine months after completion of a -day exposure regimen involving gw-relevant chemicals and stress, rats underwent intracerebroventricular infusion of mir- inhibitor (n= ) or scrambled negative control oligonucleotide (n= ) and were implanted with -day osmotic pumps delivering . nmol/day. intranasal delivery of oligonucleotides was performed on additional rats (n= per group; daily for days) to determine whether mir- inhibition is achievable using a noninvasive procedure. hippocampi were harvested and quantitative pcr arrays were used to profile the expression of focused panels of genes important for ) synaptic alterations during learning and memory, ) signaling initiated by the glucocorticoid receptor (known mir- target), and ) neurogenesis. hippocampi were also analyzed by quantitative pcr to examine expression levels of endogenous mir- . results: upregulation ([ . fold change, p. ) of synaptic plasticity genes, glucocorticoid signaling genes, and neurogenesis genes was observed in the hippocampus of gwi rats infused with mir- inhibitor compared to scrambled control, consistent with a significant reduction (p\ . ) in mir- levels detected in rats receiving mir- inhibitor. altered gene expression and a reduction in mir- levels were not observed in rats after intranasal delivery. conclusion: mir- antagonism in the hippocampus upregulates the expression of several downstream targets involved in synaptic plasticity, glucocorticoid signaling, and neurogenesis and is a promising therapeutic approach to improve cognition, emotion regulation, and neuroendocrine dysfunction in gwi. further testing is being pursued to discover the optimal dose for intranasal administration to test viability of this option for ill gw veterans. nikhil gupta, dr, ananya deori, dr, arun k gupta, dr, dipankar naskar, dr, c k durga, dr; pgimer dr rml hospital, delhi background: the ultrasonic dissector, commonly known as the harmonic scalpel, has been in use for achieving haemostasis in surgery for almost yrs. its advantages in breast surgery, especially in the dissection of axilla, have been a matter of debate as previous studies have shown inconsistent results. this study compares the outcomes of the ultrasonic dissector in axillary dissection with that of the conventional electrocautery. methods: patients who were undergoing mrm and bcs with axillary dissection from november till march were included in the study. patients were randomized into two groups, group a undergoing axillary dissection with ultrasonic dissector and group b with electrocautery. the operative time, intra-op bleeding, post-op pain, post op drain volume, hospital stay and any other complications were noted in the two groups. results: the numbers of patients in both groups were each. group a had a significantly shorter operative time, both for axillary dissection ( . min vs. . min, p. ) and the total duration ( . vs. . min, p= . ). the blood loss was significantly less in group a, as measured by the mop count. there was significant reduction in the total post-op drainage volume, which resulted in fewer days of drain in-situ and the total number days stayed in the hospital. there was no significant change in the post-op complications such as haematoma, seroma, flap necrosis, oedema, etc. conclusion: with the use of ultrasonic dissector, the operative time, blood loss and the axillary drainage was significantly reduced. the axillary drainage in turn, reduced the hospital stay. there was no significant difference in terms of complications like haematoma formation, seroma formation, skin flap necrosis or oedema. for the statistical analysis, χ or fisher's exact tests to compare proportions and the nonparametric mann-whitney u test for analysis of values with abnormal distribution were used. discussion: the study included patients. all preoperative laboratory indicators were elevated. the laboratory tests do not demonstrate any statistical significance between these two groups. the group of the patients without stones in the cbd diagnosed by ioc was also divided in patients with diameters. ?mm and with diameters≥ . ?mm of the cbd. also in these two groups, the statistical analysis of the laboratory tests does not demonstrate significant difference. all patients underwent ioc. ioc showed stones in / patients ( . %) . a comparison of patients with and without stones at ioc showed similar mean times from hospitalization to surgery ( . background: housed in a high volume tertiary referral center, our division receives a large amount of transfers and referrals from outside institutions for patients who require completion cholecystectomies. in this study "completion cholecystectomy" refers to patients that meet one of three criteria: . previous subtotal cholecystectomy, . previously aborted cholecystectomy, or . previous cholecystectomy with incidental finding of cancer on pathology. traditionally, exploration of a reoperative field in the right-upper quadrant mandates an open approach due to dense adhesions and inflammation. over the past few years, we have found that robotic-assisted surgery has allowed us to perform these completion cholecystectomies in a minimally invasive fashion. methods: case logs and operating room billing logs were reviewed from to to identify all robotic-assisted cholecystectomies performed at our institution. review of all reports identified completion cholecystectomies. all additional variables including demographics, operative variables, and postoperative outcomes were determined from manual chart review of all consultation notes, operative reports, anesthesia records, progress notes, discharge summaries, and postoperative office visits. results: of the identified robotic-assisted completion cholecystectomies, patients had a previous subtotal cholecystectomy, patients had an aborted cholecystectomy, and patients had an incidental finding of t gallbladder carcinoma on pathology. fifteen patients ( %) underwent preoperative ercp either for choledocolithiasis or to determine biliary anatomy. average time from original procedure was months with . % of previous procedures performed in an open approach. average or time was . minutes, average ebl was . cc, and average length of stay was . days. one patient ( . %) was readmitted within days for nausea that resolved with antiemetics. three patients ( . %) had minor postoperative complications (clavien-dindo grade or ) which resolved with pharmacologic therapy. no patients suffered a -day mortality. all cases were completed in minimally invasive fashion without a conversion to an open procedure. conclusions: although rare, completion cholecystectomies present a challenging surgical scenario. although traditionally performed in an open approach, we have had success in recent years at our institution with a robotic-assisted approach to completion cholecystectomy. we feel that the robotic approach offers certain advantages in a hostile, reoperative field which allows us to perform these procedures in a minimally invasive fashion with no conversions to an open procedure to date. previously limited to case reports, this report of procedures represents the largest case series of robot-assisted completion cholecystectomies to our knowledge. s surg endosc ( ) :s -s background: percutaneous cholecystostomy tube (pct) has been used as a bridge treatment for grade ii-iii moderate to severe acute cholecystitis (ac) to "cool" the gallbladder down over several weeks and allow the inflammation to resolve prior to performing interval cholecystectomy (ic) and removal of the pct, often laparoscopically. the aim of this study was to assess the impact of timing of ic after pct on operative success and outcomes. methods: a retrospective review of electronic medical records of patients who were treated for ac with a pct, and subsequently underwent ic at our institution between january to december was performed. the patients were divided into three groups (n= each), based on the duration of the pct prior to ic, and these groups were comparatively analyzed. a comparative sub-analysis of clinical outcomes between patients who underwent surgery within the first week vs. third week or later after pct was also performed. results: a total of patients met the study criteria. each group had patients. there were no statistically significant differences between the groups in regards to age, gender, bmi, imaging findings, and indications for cholecystostomy tube placement. overall, there was no statistically significant difference in outcomes between performing ic within the first weeks, - weeks and [ weeks after pct placement. the length of stay, overall morbidity, clavien-dindo grade of complications and mortality were similar between the time intervals. however, a sub-analysis showed that patients who underwent ic within the first week of pct placement had statistically significant higher mortality rate (p= . ) compared to those who underwent ic[ weeks of pct placement. the two patients who died in our sample had ic within a week after pct placement. even though there was a statistically significantly higher morbidity rate in those who had ic[ weeks after pct, the clavien-dindo grade of these complications was lower than. conclusion: delaying ic to [ weeks after pct placement for ac is not associated with any improvement in patient morbidity, length of stay or rate of conversion from laparoscopic to open cholecystectomy. cholecystectomy within the first week of pct placement is associated with higher mortality rate than after weeks likely due to associated sepsis. introduction: the effect of intraoperative bile spillage during laparoscopic cholecystectomy (lc) on operative time (or time), length of stay (los), postoperative complication rates, and day readmission rates was analyzed. laparoscopic cholecystectomy is the gold standard operation for gallbladder disease in the united states. number of studies have shown that same day discharge in elective laparoscopic cholecystectomy is feasible and safe. bile spillage during this procedure can be a common occurrence in teaching institutions, however, data on the effects of operative outcomes is lacking. methods: this is a retrospective study analyzing all of the laparoscopic cholecystectomies performed at the brooklyn hospital center (tbhc), both emergent and elective, from to . patient data was collected on demographics, comorbidities, bile spillage, operative findings, complications, los, and day readmission rates. statistical analysis was performed using imb spss statistics v. . covaried analysis of variance (ancova) was performed on continues variables and significance levels were calculated. pearson's chi square significance level was calculated for all binomial variables. results: of the patients who underwent lc during this time period, intraoperative bile spillage was encountered in patients. interestingly, bile spillage was significantly more likely to be seen in elective cases over acute cases ( . % vs . %, p. ). there was a statistically significant increase in or time in cases where intraoperative bile spillage was encountered vs. cases where no bile spillage was encountered ( vs. min, p= . ). there was a significant increase in rate of conversion to open procedure when bile spillage was encountered ( . % vs. . %, p. ). drain placement rates increased, not surprisingly, when bile spillage was encountered ( . % vs. . %, p. ). there was no statistically significant difference in los between cases with bile spillage and cases without ( . days vs. . days). there was no significant increase in complication rate or day readmission rates. conclusions: intraoperative bile spillage significantly increases or time, conversion to open procedure, and drain placement. however, there was no significant effect observed of intraoperative bile spillage on length of stay, complication, and day readmission rates. thus, intraoperative bile spillage appears to have little clinical significance on surgical outcomes. however it may have an impact on overall healthcare costs. larger prospective studies evaluating the effect of intraoperative bile spillage on los, or time, complication rates, and day readmission rates are needed to analyze these effects further. tariq nawaz, md; rawalpindi medical university study design: prospective and observational study. place and duration: from january, to july . surgical unit ll, holy family hospital, rawalpindi. patients and methods: thousand patients with a diagnosis of cholithiasis were included. exclusion criteria are patient younger than year and older than year. calot's triangle dissection was done meticulously. cystic artery and hepatic artery anomalies and variations were observed and analyzed on spss . results: the age varies from to years. on the basis of distributional variation the cystic artery was single in % cases, branched in % cases and absent in % cases. on positional variations the cystic artery was superomedial to the cystic duct in % cases, anterior in % cases, and posterior in % cases and low lying in % of the cases. on the basis of length variation results showed that ( %) cases had a normal cystic artery. a short cystic artery was found in ( %) cases and a long cystic artery was present in ( %) cases. other arterial variations are of hepatic artery i.e moynihan's hump ( %) and and right hepatic artery present in calots triangle in % conclusions: for the safety of laparoscopic cholecystectomy one should be well aware of the anatomical variations of the cystic and hepatic artery. keywords: cholelithiasis, cholecystitis, laparoscopic cholecystectomy. as small as it gets: micro-invasive laparoscopic cholecystectomy using only two mm trocars and a needle grasper background: the majority of surgeons use four ports including for laparoscopic cholecystectomy (lc). multiple efforts have been made to reduce number and size of ports. left upper quadrant (luq). patients and methods: of lcs performed from / - / , ( %) were done using three instruments including cases in which trocars and the teleflex needle grasper were used. in cases only two mm trocars were (left upper quadrant (luq) and umbilicus) with the minigrasper being placed between the two. the gallbladder (gb) serosa was incised on both sides and a window was created behind the gb midportion and widened towards fundus and infundibulum. cystic artery (ca) and cystic duct (cd) were dissected out obtaining the critical view and after the last fundus adhesion was cut, ca and cd were secured with clips or endoloop. results: median age of women and men was . (range . - . ) years. lc was done for acute cholecystitis (n= ), chronic cholecystitis (n= ), biliary dyskinesia (n= ), choledocholithiasis (n= ). three patients had an ercp with bile duct clearance prior to the lc. in one case a keith needle was used to suspend the gb fundus for better exposure. twelve patients had additional procedures together with their lc (wedge liver biopsy ( ), lysis of adhesions ( ) , umbilical hernia repair ( ) , mesenteric/lymphnode biopsies ( ) . median or time was (range - ) minutes. the specimen was removed through the luq port site in patients. there were no vascular or bile duct injuries in this series. % of cases were done as outpatient procedures, % of patients required hours observation only three patients were hospitalized for medical reasons. conclusion: in selected cases with either small stones or biliary dyskinesia, lc with only two mm ports and a needle grasper is possible. the teleflex minigrasper can completely replace a port based grasper. introduction: the standard treatment for lithiasic acute cholecystitis remains the laparoscopic cholecystectomy despite the timing of surgery is still controversial. the aim of this prospective study is to evaluate the advantages and limitations of early laparoscopic cholecystectomy in a district hospital. methods and procedure: all patients undergoing laparoscopic cholecystectomy at the surgical department of "carlo urbani" hospital in jesi (italy) from may to september were consecutively enrolled. clinical data such as gender, age, bmi, comorbidity, previous abdominal surgery, previous acute cholecystitis were collected. subsequently, the patients were arranged in two groups according to the timing of intervention (early versus elective surgery). for each group, we compared data concerning surgery, such as operative time, intraoperative and postoperative complications, length of hospital stay and cost analysis. results: this study is a part of an ongoing research. so far, we collected laparoscopic cholecystectomies. ten ( %) of them were admitted with acute cholecystitis and were operated during the hospital stay (group a). group b included patients scheduled for elective surgery (n= ; %). the two groups were comparable with respect to clinical data. conversion to open approach was performed in cases, all of them in group b. mean surgical time was . ± . minutes in group a and . ± . minutes in group b (p= . ). no significant differences in intraoperative and postoperative complications rates were seen in the two groups, just a few in both of them. mean overall length of hospitalization was . ± . days in group a and ± . days in group b (p= . ), whereas the difference in length of postoperative hospitalization was not statistically significant. due to the extended hospitalization for group a, the cost increase as compared to group b was statistically significant, too. conclusions: early laparoscopy is comparable to delayed laparoscopy in terms of postoperative hospitalization and complications in the management of acute cholecystitis. a longer hospital stay among patients scheduled for immediate surgery may be associated with a more time-consuming diagnostic work-up before surgery. however, in future research we expect to enhance our cost analysis with more data regarding the costs incurred in the first hospitalization reserved to nonoperative treatment of group b inpatients with acute cholecystitis. s surg endosc ( ) introduction: with improvements in healthcare access and technology, admissions of octogenarian population with acute cholangitis (ac) are increasing. octogenarians are vulnerable to inferior outcomes. there is no study to evaluate factors predicting outcomes of ac in octogenarians. the aim of our study is identify factors predicting outcomes, and to evaluate the quick sequential organ failure assessment (qsofa) score and tokyo guidelines (tg ) severity grading for octogenarian patients with ac. methods: a retrospective review of octogenarian patients admitted with ac from january to december was performed. demographic profile, clinical presentation and discharge outcomes were studied. systemic inflammatory response syndrome (sirs), qsofa and tg severity grading scores were calculated. mortality is defined as death within days of admission or in hospital mortality. statistical analysis was performed using spss version . results: there were a total of patients admitted for ac, of which ( %) were octogenarians. majority (n= , %) were female, with a mean age of (range - ) years. majority were secondary to gallstones (n= , %), and ( %) were due to malignancies. ( %) and ( %) patients fulfilled sirs and qsofa criteria of severity respectively. ( %) and ( %) of patients had a tg severity grading of moderate and severe respectively. nine ( %) patients required inotropic support in the emergency department (ed) and ( %) patients were admitted to critical care unit (ccu). ( %) patients underwent endoscopic retrograde cholangiopancreatography (ercp) and ( %) underwent percutaneous transhepatic biliary drainage (ptbd) for biliary decompression. patients underwent index cholecystectomy. length of stay was . (range - ) days and -day mortality of %. multivariate analysis performed showed that an abnormal glasgow coma score (p= . ) and malignancy (p. ) predicted -day mortality. the use of ed inotropic support predicted ccu admission (p= ). a positive blood culture (p= . ), presence of malignancy (p. ), use of ed inotropes (p= . ), and index cholecystectomy (p= . ) predicted a longer length of stay. qsofa (p. ) and tg severity grading (p= . ) were predictive of -day mortality. sirs criteria did not predict -day mortality. conclusion: reduced consciousness and malignancy predicted -day mortality in octogenarian patients with ac. qsofa and tg severity grading system is superior to sirs criteria in predicting mortality of octogenerians with ac. our group has performed needlescopic grasper assisted silc (nsilc) to overcome these problems. we evaluate the technical feasibility, safety and benefit of nsilc versus three-port laparoscopic cholecystectomy (tplc). methods and procedures: this prospective randomized control study was conducted to compare the advantages if any between the nsilc and tplc. one hundred and forty eight patient were randomized into two groups, with one group underwent n slic ( patients) and a control group underwent tplc ( patients). basic information about the patient and diagnosis was collected. the surgical outcome that was composed with critical view of safety (cvs) time, major procedure time and total operation time, and the comparison of postoperative complication was made. result: nsilc group was consisted of male ( . %) and female ( . %), and tplc group was consisted of male ( . %) and female ( . %) (p= . ). the average age of nsilc group was . ± . years old, and tplc group was . ± . years old (p= . ). cvs time of tplc group was shorter than silc group (nsilc: . ± . min, tplc: . ± . min, p= . ), major procedure time (skin incision to gb removal from liver bed) of tplc group was shorter than nsilc group (silc group: . ± . min, tplc: . ± . min, p= . ). however, there was no significant difference in postoperative complication (nsilc: , tlc: , p= . ). conclusion: although cvs time, major procedure time, and operation time of silc were longer than tplc, overall clinical results were similar. nsilc is feasible and safe surgical procedure in patient with benign gallbladder disease. introduction: management of malignant biliary obstruction not amenable to surgery is usually by means of ercp or pthc. however, on occasions, these routes are not accessible and the alternate decompressive technique of percutaneous cholecystostomy (pc) has to be adopted. the aim of this study was to evaluate the efficacy and outcomes of pc in a highly selected series at a tertiary referral center. methods: we retrospectively reviewed all patients that had undergone pc from to . data collected included baseline demographics, comorbidities, details of pc placement and management, etiology of mbo, and post-procedure outcomes. the charlson comorbidity index (cci) was calculated for all patients at the time of pc. results: four hundred and eight patients underwent pc placement of which patients including ( %) males and ( %) females, with malignant biliary obstruction. the mean age at the time of pc placement was . ± . years of age, and the mean cci was . ± . for all patients. of mbo in all patients was due to pancreatic malignancies (n= ), cholangiocarcinoma (n= ), primary hepatic malignancies (n= ), secondary hepatic tumors (n= ), and ampullary carcinoma (n= ). pc tube complications were reported in ( %) patients. mean number of tube exchanges was . ± . . mean duration from pc tube placement to death was ± . days. total deaths were recorded. conclusion: pc placement appears to be a viable option in mbo in elderly and frail patients. in this cohort, pc may be a potential definitive management to improve quality of life. melanie boyle, daivyd palencia, philip leggett; houston northwest medical center background: there are very few studies assessing the relationship between gastroesophageal reflux and biliary disease. this is surprising as they share presenting symptoms as well as risk factors, particularly obesity. our group previously produced a review of patients in our practice who had undergone some type of reflux procedure. conclusions showed that the prevalence of gallbladder disease in our severe reflux population is much higher compared to that found in the general population. our goal of this study is to expand on that data to include a larger sample size to investigate the incidence of biliary disease in our reflux population and decide if this should influence our pre-operative algorithm for anti-reflux surgery patients. methods: we expanded on our previously performed retrospective review of patients that underwent laparoscopic fundoplication for reflux disease. we previously reviewed data from to . we are now looking at data from to . our expected sample size will include approximately patients, of which have currently been reviewed. our previous study included only . the surgery preformed was either a toupet or nissen fundoplication, and one underwent a dor. demographic data, imaging studies, and pathology results were reviewed. results: we looked at whether each patient who underwent antireflux surgery had a prior cholecystectomy either remotely or recently, underwent concomitant cholecystectomy, or had no biliary disease in their workup. the groups had similar age and were predominantly women. we once again demonstrated that the prevalence of gallbladder disease in our severe reflux population is much higher than the general population. when approaching a patient with gastroesophageal reflux disease, attention should be paid to gallbladder symptomatology as well. we recommend that it may be beneficial to include gallbladder ultrasound in pre-operative workup for antireflux surgery so that concomitant cholecystectomy can be performed if indicated. steven schulberg, do, jonathan gumer, do, matt goldstein, vadim meytes, do, george ferzli, md; nyu langone hospital -brooklyn introduction: acute cholecystitis is a common surgical disease with roughly , cholecystectomies performed in the us annually. the current dogma revolves around the " hour rule" advocating early cholecystectomy if within the window, and if beyond hours, conservative treatment and interval operation. in patients beyond the hour window, as well as with multiple comorbidities, advanced age, and other complicating factors, cholecystostomy has become an acceptable treatment as a bridge to interval cholecystectomy. while this has become an appropriate treatment modality, it does not come without its own set of complications. we aim to evaluate the rate of complications in our institution. methods: this is a retrospective review of all patients at our institution who underwent cholecystostomy placement between and . we evaluate the comorbidities, readmission rate, overall rate of complication associated with cholecystostomy tubes, and eventual definitive cholecystectomy. results: our cohort includes patients, % of whom were male, with a mean age of . we had an overall complication rate of . %, including tube dislodgements, leaking tubes, and misplaced tubes. all cause readmission rate was % and only % of patients who had cholecystostomy drains underwent interval cholecystectomy. conclusion: there has been much interest in treatment of acute cholecystitis in patients with multiple comorbidities. in review of our data, a surprisingly large number of patients had mechanical complications involving the cholecystostomy drain. in an era focused on decreasing readmission rates and their associated costs, drains carry a high risk of malfunction which will in turn, lead to increases in these two metrics. while there is more work to be done in the evaluation of early cholecystectomy versus cholecystostomy in this subgroup of patients, we suspect that early cholecystectomy in the medically optimized patient will lead to reduced length of stay and hospital costs as well as increased patient satisfaction. does selective use of hepatobiliary scintigraphy (hida) scan for diagnosis of acute cholecystitis, following equivocal nondiagnostic gallbladder ultrasonography, affect outcomes fahad ali, ba, amir aryaie, md, eneko larumbe, phd, mark williams, md, edwin onkendi, md; texas tech university health sciences center introduction: acute cholecystitis (ac) is diagnosed by characteristic gallbladder ultrasonographic findings (high specificity, low sensitivity). hepatobiliary scintigraphy (hida) may be needed to confirm ac (higher sensitivity and specificity). the aim of this study was to assess the impact of the current selective use of hida scan for sonographically equivocal cases of ac on outcomes. methods: a retrospective chart review of patients treated for ac at our institution ( / to / ) was performed. patients were divided into groups: the ultrasound only group (us-only) and the ultrasound-hida group (us-hida). timing of us and hida, and intervention for ac since presentation to emergency room (er), and their impact on outcomes were analyzed. ac severity was graded per the tg -tokyo guidelines. results: a total of patients were analyzed. the groups were statistically similar with regards to age, body mass index, asa class ii, iii and iv, extent of leukocytosis at presentation and liver functions test levels at presentation. in the us-only group, diagnostic ultrasound was obtained sooner, [median of (interquartile range, iqr . - . ) hours] from presentation to the er compared to the us-hida group, ) hours], p= . . hida was obtained after a median delay of . (iqr . - ) hours from a nondiagnostic ultrasound. majority of patients ( %) in the us-only group had mild (tg grade i) to moderate (tg grade ii) ac, while % of the us-hida group had moderate (tg grade ii) to severe (tg grade iii) ac (p= . ). despite this, more patients in the us-hida group ( %) had a "normal" non-diagnostic ultrasound compared to the us-only group ( . %), p. . seven patients in the us-hida group had no intervention due to normal hida scan ( ) , ac misdiagnosis due to liver cirrhosis ( ) , and severe medical comorbidities ( ) . more patients ( %) in the us-only group underwent laparoscopic cholecystectomy, compared to % in the us-hida group (p= . ). between the two groups, there was no significant differences in -day morbidity, mortality and reoperations. however, the length of stay was longer by a median of . days in the us-hida group (p= . ). conclusion: patients with moderate to severe ac are more likely to need hida scan due to a "normal" non-diagnostic ultrasound, have a delay in diagnosis, not have intervention for ac due to severe medical comorbidities and have lower chance of laparoscopic cholecystectomy. the length of hospital stay is significantly longer for these patient by a median of . days. introduction: benign gallbladder disease is commonly treated with laparoscopic cholecystectomy (lc). gallbladder cancer (gbc) is a rare malignancy characterized by high invasiveness and poor survival. in our institution, all gallbladder specimens are routinely sent to pathology, to rule out gbc. the purpose of our study was to assess the efficacy for routine histopathology of gallbladder specimens after cholecystectomy (cly) for all gallbladder disease. methods and procedures: after obtaining approval from our institutional review board, a retrospective review was conducted on all patients who underwent cly from june of to may were included in the study. the data obtained include gender, age, american society of anesthesiologist score (asa), body mass index (bmi), comorbidities, length of stay (los), radiological imaging and pathology results. independent t and chi-square tests were performed using ibm® spss® software. results: there were cly performed at our institution, of which ( %) were lc. females composed of ( %) patients and the median age was . ( %) gallbladder specimens were found to be cancerous. ( %) gallbladder specimens were benign. majority ( %) were chronic cholecystitis, ( %) were acute cholecystitis and ( %) were gangrenous cholecystitis. ( %) were found to be acalculus cholecystitis and ( %) were cholelithiasis. ( %) were found to be adenomyositis, and other. conclusion: in our institution, less than % ( ) of all gallbladder specimens were found to be cancerous. it would decrease cost and work load if gallbladder specimens are selectively sent to pathology. emanuel a shapera, md we sought to determine clinical factors associated with recurrent cholangitis in two las vegas community hospitals to aid providers in management of this disease. methods and procedures: retrospective, multi-center study. over ercps were analyzed between and . patients were identified as having multiple ( ) admissions for cholangitis per tokyo criteria. univariate and multivariate analysis was conducted. results: patients with a significantly (p. ) higher albumin level on admission ( . ) were discharged home more often than patients discharged to a facility or hospice ( . ). on multivariate analysis, non-home discharge was associated with lower albumin level at admission (p= . ) and greater maximum temperature prior to decompression (p= . ). increased hospital stay was associated with lower albumin level at admission (p= . ). a majority ( / ) of recurrent episodes involved stent placement, exchange or removal. patients ( %) had either biliary malignancy, gallbladder or both. blood cultures were drawn in % of all episodes and positive in %, e coli being the most common pathogen isolated. all patients had low hdl levels ( - , mean ) . conclusions: high fevers and poor nutritional status was associated with increased length of hospital stay and fewer home discharges. tumors, gallbladders and malfunctioning stents contribute substantially to morbidity. close follow up for indicated gallbladder removal, stent management and nutritional optimization is critical to reduce the burden of this disease. we compared the surgical method in neonate choledochal cyst between oec and lec. the perioperative and surgical outcomes that were reviewed included age, operative time, postoperative hospital stay, time to diet, and surgical complications. the patients were followed up for months (range, - months) . results: there was no difference in range of bile duct excision and manner of roux-en-y hepaticojejunostomy between oec and lec groups. there was no intraoperative complication in both groups and no open conversion in the lec group except one case which was ruptured choledochal cyst. the median age of oec and lec groups were days (range, - ) and . days (range, and median body weight at the time of operation were . kg (range, . - . ) and . kg (range, . - . ) , respectively. the median operative time was minutes (range, - ) in oec and . minutes (range, in lec groups and there was no significant difference between oec and lec groups (p= . ). intraoperative bleeding was minimal in both groups. the postoperative hospital-stay, time to start diet, and time to return to full feeding had no significant differences in both groups. after discharge, of ( %) oec patients experienced readmission due to cholangitis and ileus, while there were none in the lec group. conclusions: this study revealed that lec had better prognosis compared to oec. lec provided an excellent cosmetic result. so we suggest lec could be the treatment of choice for neonatal choledochal cyst. this is a small series, therefore future studies will have to include a larger number of patients and evaluate long-term follow-up. keywords: choledochal cyst, laparoscopy, neonate. laparoscopic narrow band imaging for intraoperative diagnosis of tumor invasiveness in gallbladder carcinoma: a preliminary study yukio iwashita, hiroki uchida, teijiro hirashita, yuichi endo, kazuhiro tada, kunihiro saga, hiroomi takayama, masayuki ohta, masafumi inomata; oita university faculty of medicine introduction: determining tumor invasiveness before operation is one of the most important unsolved issues in the management of gallbladder cancer. we hypothesized that the assessment of irregular vessels on the gallbladder wall may be useful for detecting subserosal infiltration. we present an initial report on the clinical usefulness of laparoscopic narrow band imaging (nbi) for the intraoperative diagnosis of tumor invasiveness in gallbladder carcinoma. methods: thirteen patients with gallbladder cancer were included in this study. patients with tumors located in the liver bed and those with definitive invasion observed on computed tomography findings were excluded from this study. gallbladders were observed using nbi and the microvasculature was evaluated. according to previous reports of endoscopic nbi, we defined four findings as positive: vessel dilatation, tortuousness, interruption, and heterogeneity. the nbi findings were compared with postoperative pathological findings. the study protocol was approved by the institutional review board of the oita university. results: the serosal surface of the tumor site and its microvasculature were successfully observed in all patients. laparoscopic nbi detected at least one abnormal finding in seven patients, and postoperative pathology showed subserosal infiltration accompanied by vessel invasion. on the contrary, six patients with no positive nbi findings showed mild or no subserosal infiltration and no vessel invasion. conclusions: our study indicated that laparoscopic nbi may be useful for diagnosing subserosal infiltration accompanied by a vessel invasion. shuichi iwahashi, mitsuo shimada, satoru imura, yuji morine, tetsuya ikemoto, yu saito, hiroki teraoku; department of surgery, tokushima university introduction: laparoscopic cholecystectomy (lap-c) is the standard operation for the benign diseases. we have reported reduced port lap-c (rpl-c) was safely and comparable method to sils-c and conventional lap-c (sages ) . in this time, we examined the utility of rpl-c containing the post-operative adverse event. procedures: the adjustment is the benign illness including the cholecystolithiasis, and advanced obesity and the cases of the inflammation remaining have been excluded. the incision is put and cut open the abdomen to the umbilical region, and camera port was inserted. we used mm flexible scope. mm forceps for holding of the gallbladder bottom and left hand of operator were inserted directly with no port. methods: rpl-c has been introduced in this department since july, . we performed cases of lap-c, containing sils-c and american style conventional lap-c, and we performed rpl-c has been performed already cases. we compared the patient background and the operation factor between rpl-c, sils-c, conventional lap-c. operators were young surgeons, they were not specialists of gastroenterological surgery or endoscopic surgery. results: the difference was not admitted in the age, gender, the physique, and the disease, and the difference was not admitted in hospital stay after the operation (rpl-c:sils-c:conventional lap-c= . ± . days: . ± . days: . ± . days) and the amount of blood loss (rpl-c:sils-c:conventional lap-c= . ± . ml: . ± . ml: . ± . ml) and operation time (rpl-c:sils-c:conventional lap-c= ± min: ± min: ± min). and surgical wound after rpl-c was cosmetically acceptable. regarding as the post-operative adverse event, there were no patients of bile duct injury. conclusion: in the patients on reduced port lap-c, there were no bile duct injuries of postoperative adverse event. reduced port lap-c is safely for young surgeons and comparable method. introduction: acute cholangitis is an ascending infection of the biliary tree secondary to obstruction and can be severe if proper intervention and treatment are not performed in a timely fashion. the most common management of cholangitis with ductal obstruction due to choledocholithiasis is intravenous hydration, empiric antibiotic therapy, endoscopic retrograde cholangiopancreatogram (ercp) with sphincterotomy and stone extraction with or without stent placement, followed by a delayed laparoscopic cholecystectomy. we present the case of a patient with blood clot obstruction of a common bile duct (cbd) stent after ercp with sphincterotomy and stone extraction. case presentation: a year old male presented to the emergency department with jaundice, right upper quadrant abdominal pain, truncal pruritis, nausea, vomiting, and fever. biochemical analyses and liver profile demonstrated an elevated white blood cell count, hyperbilirubinemia, and elevated liver enzymes consistent with cholestasis. biliary ultrasound demonstrated multiple gallstones and dilation of the cbd with a distal obstructing calculus. he proceeded to ercp where biliary cannulation was achieved, sphincterotomy performed, and a large amount of sludge and pus was drained. an mm stone was removed from the cbd by balloon sweep with completion cholangiogram demonstrating no filling defects. a stent was then placed in the cbd with adequate flow. following the procedure, the patient continued to have increasing hyperbilirubinemia. a repeat ercp revealed a large blood clot and continued bleeding at the previous sphincterotomy that resolved with epinephrine injection. the former stent was visualized in the proper position, removed with a snare, and found to be fully occluded with blood clots. after retrieval of additional clots, a new stent was placed with adequate return of bile. the patient recovered with resolution of his symptoms and hyperbilirubinemia with laparoscopic cholecystectomy. discussion: cholangitis is characterized by charcot's triad of right upper quadrant abdominal pain, fever, and jaundice due to an ascending bacterial infection of the biliary tree coinciding with obstruction of biliary flow most commonly from gallstones. cholangiography via ercp with associated sphincterotomy, stone extraction, and stenting is both diagnostic and therapeutic. while debated by endoscopists, stent placement has shown to reduce recurrent biliary complications, decrease length of hospital stay, and lessen morbidity. although pancreatitis is the most common cause of hyperbilirubinemia post-ercp, stent occlusion secondary to stones or blood clots should be considered to effectively treat patients. proper hemostasis is important in any procedure and close patient follow-up should be performed to prevent further complications. sarrath sutthipong, md, panot yimcharoen, md, poschong suesat, md; bhumibol adulyadej hospital background: choledochal cyst (cc) is a rare disease, characterized by dilatations of the extra-or/ and intrahepatic bile ducts. ccs occur most frequently in asian and female populations. cc is associated with biliary lithiasis and considered at risk of malignant transformation. todani's classification dividing cc into types is the most useful in clinical practice. the current standard treatment is complete cyst excision with roux-en-y hepaticojejunostomy and cholecystectomy for the extrahepatic disease (todani type i and iv). in this report we present our experience using a total laparoscopic technique to treat adult patients with cc in -year period. methods: a retrospective review of the records of the patients above years who underwent laparoscopic cyst excision and roux-en-y hepaticojejunostomy in our hospital between january and may was carried out. the data included the clinical presentation, investigation, perioperative details and complication. the type of cc was classified according to todani's classification. results: seven cases of cc were reviewed, females and male with mean age years (range - years). these included cases of todani type ib and cases of type a. the predominant symptoms were chronic abdominal pain and jaundice. a case of both pancreatitis and cholangitis were also seen. investigations included ultrasound with mrcp in cases and ercp in case. the mean operative time was hours and minutes ( hours minutes to hours range) with mean intraoperative blood loss ml (range - ml). all the resected specimens showed chronic inflammation. malignancy was not seen in any patients. the early postoperative complications included bile leakage with intra-abdominal collection in patients, which were managed conservatively (evidenced by clinical status and imaging study), re-operation was not required. the median duration of hospital stay was days (range - days). there was no perioperative mortality. all patients were followed up at , , and months postoperatively, late complication were not detected during each visit. conclusion: in our opinion, laparoscopic cyst excision and hepaticojejunostomy could offer more feasible and safe methods of treatment for ccs in adult patients with potentially less postoperative morbidity, a shortened length of stay and a lower blood loss when compared to the preferred open approach. however, we would need to study this on a larger sample of patients to report the efficacy and safety of laparoscopic approach. endoscopic trans-papillary gallbladder drainage (etgbd) in acute cholecystitis: a single center experience arun kritsanasakul, chotirot angkurawaranon, jerasak wannapraset, thawee rattanachu-ek, kannikar laohavichitra; rajavithi hospital background: surgery is the mainstay of treatment for cholecystitis, however, it may not be safe or feasible in some circumstances such as severe cholecystitis or cholecystitis in extremely high-risk patients. gallbladder drainage may be an appropriate alternative or a bridging option prior to cholecystectomy. endoscopic trans-papillary gallbladder drainage (etgbd) has been proposed as a modality that is feasible and effective in cholecystitis. objective: the primary outcome of this study is to evaluate the effectiveness of etgbd. the secondary outcome is to evaluate the safety, early experience outcomes, and complications of this procedure. methods: retrospective medical records review between january -december from a single tertiary referral hospital center, rajavithi hospital, bangkok, thailand. a total of patients who was diagnosed with cholecystitis and underwent etgbd. the procedure was performed at the endoscopic suite under light sedation via total intravenous anesthesia. the patient demographic data and procedures were collected. the technical success of etgbd was defined as decompression of the gallbladder by successful cystic duct stent placement. the clinical success was defined as resolution of symptoms and/or improved laboratory data or ultra-sonographic findings. results: a total of patients underwent etgbd. among these patients, were high risk for surgery due to age or comorbidity, had concomitant jaundice and was failure of medical treatment. both technical and clinical success of etgbd was achieved in of cases ( %). the two patients that did not achieve technical success were due to failure to cannulate guidewire through cystic duct and the other had trans-cystic guidewire perforation that needed surgical intervention. there were two intra-operative complications ( %). one was the patient who had trans-cystic guidewire perforation and another had anesthesia-related complication (hypoventilation requiring endotracheal intubation). there were no -day mortality. conclusion: endoscopic trans-papillary gallbladder drainage is an alternative treatment modality for patients with cholecystitis who are at high-risk for surgery and or those who are unsuitable for percutaneous gallbladder drainage. the technique is feasible, however, careful case selection and high endoscopic skill is needed. julia f kohn, bs , alexander trenk, md , woody denham, md , john linn, md , stephen haggerty, md , ray joehl, md , michael ujiki, md ; university of illinois at chicago; northshore university healthsystem, northshore university healthsystem introduction: subtotal cholecystectomy, where the infundibulum of the gallbladder is transected to avoid dissecting within a heavily inflamed triangle of calot, has been suggested as a method to conclude laparoscopic cholecystectomy while avoiding common bile duct injury. however, some case reports have suggested the possibility of recurrent symptoms from the remnant gallbladder. this retrospective case series reports a minimum of two-year follow-up on patients who underwent subtotal cholecystectomy within one four-hospital system. methods: a retrospective chart review database containing randomly selected cholecystectomies, all of which occurred between and , was reviewed to identify all instances of subtotal cholecystectomy. charts for these patients were reviewed through / , including any documentation from other providers, including primary care. results: six patients who underwent subtotal cholecystectomy with a remnant of infundibulum left following surgery were identified. surgical approach and the choice to perform subtotal cholecystectomy were dependent on the attending surgeon; all decisions were made intraoperatively. there was an average of months of follow-up for these patients within our institution. discussion: this case series adds six cases to the literature surrounding long-term outcomes in patients who underwent subtotal cholecystectomy. although one patient was lost to follow-up, no patient had recurrent biliary colic or other complications arising from the remnant gallbladder. this may be encouraging to surgeons who feel that subtotal cholecystectomy with an infundibular remnant is the safest way to proceed with cholecystectomy in patients with severe inflammation. objective: this study aims to evaluate the utility and efficiency of icg as an alternative to routine intraoperative cholangiogram in patients undergoing cholecystectomy. introduction: common bile duct injury is an uncommon, but serious complication associated with laparoscopic cholecystectomy. current guidelines state that when used routinely intraoperative cholangiogram (ioc) can decrease biliary injury, however it is not routinely used due to increased time of operation, and inaccessibility of equipment. indocyanine green (icg) has been found to be effective for identification of biliary anatomy during cholecystectomy, however has not yet been widely adopted. we aim to assess if icg is able to overcome the obstacles of ioc, while still effectively assessing biliary anatomy. methods: we performed a retrospective analysis of laparoscopic cholecystectomies performed in a single institution from january to september . elective and emergent cases were included. we stratified patients into icg and non-icg groups. patients who had concomitant procedures performed were excluded. we analyzed patient demographic information, as well as bmi, asa classification and comorbidities in both groups. our primary outcome was operation time (skin to skin), and laparotomy conversion rate. secondary outcomes were effectiveness of icg in visualizing biliary anatomy, and cost. results: patients were included in our study, in the non-icg arm and in the icg arm. both groups were similar in background. there were no statistical differences in patient demographics, asa classification, bmi, or comorbidities. there was no statistical difference in operation time ( . vs . minutes; p. ) or conversion rate ( . vs %; p. ). icg was able to delineate biliary anatomy in % of the patients. the cost of a mg/vial kit of icg is approximately $ . conclusion: the use of icg does not increase operating time during laparoscopic cholecystectomy. icg is an inexpensive and effective tool used to delineate biliary anatomy without the inherent burden and limitations of ioc. benefsha mohammad, md , michele richard, md , steve brandwein, md , keith zuccala, md ; danbury hospital, danbury hospital department of gastroenterology, introduction: obesity is a prevalent issue in today's society, which has increased the number of gastric weight loss surgeries. this presents an anatomical challenge to biliary disease requiring endoscopic retrograde cholangiopancreatography (ercp). in gastric bypass patients, traditional ercp via the mouth in these patients is technically more challenging, requiring a longer endoscope with a reported success rate of less than %. a solution is laparoscopic assisted ercp (la-ercp) via gastrostomy. this minimally invasive technique has become increasingly more prevalent and safe. we present our experience with la-ercp at our teaching community hospital in a large cohort of patients. methods and procedures: retrospective chart review was performed on all patients with a history of prior laparoscopic gastric bypass surgery who underwent la-ercp from april to april . the procedure was performed by two different general surgeons and one gastroenterologist. a pursestring suture and transfacial stay sutures were used to bring the gastric remnant to the abdominal wall. a gastrostomy was then created and accessed by the duodenoscope to perform the ercp. biliary sphincterotomy, papillary or biliary dilation, lithotripsy, stent placement, and/or stone removal were performed as indicated. we observed the incidence of postoperative outcomes, including acute pancreatitis, reoperation, post-procedure infection, pain control, hospital re-admission and bile leak. results: thirty-two patients met inclusion criteria. six patients were male and twenty-six were female, with mean ages of (std dev ) and years (std dev ), respectively. indications for la-ercp included suspected choledocholithiasis ( / ), cholangitis with choledocholithiasis ( / ), acute pancreatitis ( / ), abdominal pain with abnormal lft ( / ), cholangitis with cholecystitis ( / ), and bile leak ( / ). la-ercp was successfully performed in all thirty-two patients. biliary cannulation, sphincterotomy and stone extraction were performed on / patients, and one patient underwent sphincterotomy and stent placement for bile leak after recent laparoscopic cholecystectomy. one patient developed acute pancreatitis with elevated pancreatic enzymes which resolved after conservative treatment. one patient required a second la-ercp for stent replacement due to a persistent bile leak. the median length of stay was days (range - days). conclusions: la-ercp is a safe and feasible alternative to open surgery, and can be safely implemented at community hospitals with adequately trained providers. obesity is a growing burden on society, increasing the incidence of weight loss surgery. our large study proves that in this minimally invasive era, la-ercp provides gastric bypass patients a safe alternative with less pain and increased satisfaction. ahmed elgeidie, elsayed adel; gastrointestinal surgery center background: endoscopic sphincterotomy (es) is an effective therapeutic procedure for common bile duct (cbd) stone clearance but it carries a substantial risk of recurrent stones at long-term outcome. aim of the study: to evaluate the rate of cbd stones recurrence after primary complete endoscopic clearance, and to identify the risk factors of recurrence. methods: between january and december , patients with cbd stones who underwent successful es and complete stone clearance were studied retrospectively. recurrent cbd stone, was defined by the confirmation of the presence of cbd stone at least months after previous complete cbd stone clearance by es. the risk factors for recurrent cbd stones and mean time interval between initial es and stone recurrence were analyzed. results: in total, patients we included. the median follow up period was months. recurrent cbd stones appeared in / ( . %) patients after a median time interval of ( - ) months following es. stone recurrences were observed on multiple occasions in patients ( . %). on the univariate analysis, the significant risk factors related to recurrent cbd stone were male sex (p= . ), previous history of cholecystectomy (p= . ) multiple cbd stones (p= . ), large cbd stone (p= . ) the presence of periampulary diverticulum (p= . ) and stone crushing using mechanical lithotripsy (p= . ) conclusion: recurrence of cbd stones is an identified long-term risk after es and stone clearance. background: laparoscopic cholecystectomy during advanced pregnancy is challenging due to the limited intraabdominal space. patients may be at increased risk for developing trocar site hernia. case report: a year old hispanic female in her th week of pregnancy came to the er with acute right upper quadrant pain. due to lack of accessibility she had poor prenatal care. she had mildly elevated amylase but normal lfts and ultrasound showed some gallbladder wall thickening suggestive for acute cholecystitis and no dilated biliary duct. fetal ultrasound was normal. she was admitted to the hospital and started on antibiotics, obstetrics was consulted. her amylase peaked at [ u/l but then normalized and indication for laparoscopic cholecystectomy was made. mrcp and ercp were not performed as it was assumed that the patient had passed a stone. five mm trocars were placed in the luq and the umbilicus and a teleflex minigrasper between the tow. the uterus was found at the umbilical level. the gb was pulled out and the serosa was incised on both sides and a window was created behind the gb midportion and widened towards infundibulum and fundus. there was gb wall thickening and edema. the critical view was obtained and the cystic artery and duct were clipped and divided. the common bile duct appeared normal and no ioc was done. the specimen was retrieved through the luq port site using a mm endobag after dilatation to . cm due to the presence of two large stones. the port site fascia was closed using a suture passer. the postoperative course was uneventful and both mother and baby were well at the two weeks follow up. discussion: in case of biliary pancreatitis during pregnancy, lc should be performed and if ultrasound shows a normal biliary system and amylase/lipase normalize, mrcp/ercp and ioc may be avoidable to protect the baby. lc with two ports is feasible during pregnancy. removal of the specimen through a lateral abdominal wall site may help prevent an umbilical port site hernia in this patient population. s surg endosc ( ) :s -s introduction: splenic abscess is a rare, potentially lethal condition, with autopsy studies showing incidence rates between . - . %. mortality rates ranging from to % making early diagnosis and prompt intervention vital. several case reports have documented post surgical splenic abscess, most notably after laparoscopic sleeve gastrectomy. to the best of our knowledge, there has not been any reported cases of splenic abscess arising after laparoscopic cholecystectomy. it is important to remember this disease process for expeditious targeted treatment in future cases. case presentation: a year-old female with past medical history significant for cholilithiasis, hypertension, and hyperlipidemia presented to the emergency department (ed) with a chief complaint of abdominal pain for two days. labs and imaging were obtained which confirmed the diagnosis of choledocholithiasis and pancreatitis. ercp was performed which showed a . cm stone causing obstruction, with several other smaller filling defects. the stones were removed after sphincterotomy. post procedurally, the patient underwent an uncomplicated laparoscopic cholecystectomy on hospital day (hd) # . post operatively, the patient had persistent leukocytosis peaking at . thousand on postoperative day (pod) # . a ct scan was performed which showed a rim-enhancing splenic collection measuring . . cm suggestive of an abscess. interventional radiology was consulted and aspirated ml of purulent fluid. cultures grew out klebsiella pneumoniae and enterobacter cloacae complex, and the patient was discharged home on zosyn. discussion: laparoscopic cholecystectomy has become the cornerstone in treatment of symptomatic biliary colic and acute cholecystitis. of the many recognized complications of laparoscopic cholecystectomy, splenic abscess has not yet been reported in current literature. the nonspecific signs and symptoms of splenic abscess make clinical diagnosis difficult. the classic triad of fever, palpable spleen and left upper quadrant pain are only seen in about two-thirds of patients. ct scan has been shown to be the most sensitive imaging modality for diagnosis of splenic abscess. current treatment options for splenic abscess are broken down into two subsets: percutaneous and surgical intervention. percutaneous treatment includes image guided aspiration with or without placement of drainage catheter. surgical intervention can be either laparoscopic or open and includes drainage of abscess with splenectomy or splenic conservation. the best treatment option remains unclear, and there is lacking prospective data demonstrating which modality is superior. introduction: laparoscopic subtotal cholecystectomy is widely accepted as a safe alternative to the conventional laparoscopic cholecystectomy in case of acute cholecystitis with frozen calot's triangle. the remnant stump of the gallbladder may be either sutured or looped. however, there are limited studies comparing the outcomes of the two techniques. the present study is aimed at comparing loop and suture closure of the gall bladder stump. methods: a retrospective analysis of our prospectively maintained database revealed that between january and december . patients underwent laparoscopic subtotal cholecystectomy for acute cholecystitis, chronic cholecystitis or empyema gallbladder with frozen calot's triangle. the decision to use endoloop or sutures for stump closure was made intra-operatively after dividing the gallbladder through the infundibulum. a no. sized drain was kept in all the cases. the patients were discharged with drain in situ, and were reviewed on post-operative day during which an ultrasound was done and drain removed if the progress was satisfactory. the intra-operative and post-operative data between the two groups were recorded and analyzed. results: endoloop closure was performed in patients and suture closure using . ethibond was done in patients. three patients from the sutured group had post operative bile leak among which one patient underwent endobiliary stenting. the other were managed conservatively while the drain had to be retained for weeks. two patients in the endoloop group were detected to have retained stone in the remnant gallbladder cuff among which one had recurrent cholecystitis requiring laparoscopic completion cholecystectomy. none of the patients had bile duct injury or surgical site infection. mean post operative stay was . + . days, did not significantly vary between the groups. suturing needed more surgical expertise and had prolonged operative time than endoloop ( + min versus + min, p= . ). conclusion: suture or loop closure of the remnant gallbladder after subtotal cholecystectomy are equally effective. suturing the stump may be associated with increased incidence of biliary leak while endoloop may have higher incidence of retained gallstones. the choice between the two may be made intra-operatively based on the surgeon's expertise and preference. background and aim: in recent years, due to the spread of laparoscopic cholecystectomy, bile duct injury as its complication has been reported at a certain frequency. current surgical treatments include ) suturing and closing the injured part laparoscopically during surgery, ) transitioning to laparotomy and closing the suture, ) inserting a tube such as t-tube under the laparotomy, ) bile duct-intestinal anastomosis under the laparotomy, etc. are taken into consideration. regardless of which treatment method, it is not a definite ideal treatment. we have developed a bioabsorbable material (caprolactone: lactic acid ( : ) polymer reinforced with polyglycolic acid fiber and designed to be absorbed in about weeks). at this conference, we would like to talk about the current state and problems of development of minimally invasive therapy for biliary damaged area using bioabsorbable materials we developed. method: in order to overcome the problem of the current bile duct injury cure method, we have been developed, a) a method of closing a perforation part endoscopically from the luminal side of a bile duct (a covered stent using a bioabsorbable material in the damaged part), b) develop a method of closing the biliary duct injury under the laparoscope from the outside of the bile duct (adhering the bioabsorbable sheet to the bile duct perforation using a biocompatible adhesive). results: experimental results of suturing the bioabsorbable material in the biliary duct in surgery of laparotomy were able to regenerate the bile duct without stenosis in the damaged area. however, various adhesives were tried to bond the sheet of this bioabsorbable material and the native bile duct under the endoscope, but at the moment, there is no glue that will allow the sheet to be adhered readily and reliably where there is moisture to a certain extent. a tool for delivering the sheet from the bile duct into the injured part is under development and good results are obtained at present. conclusion: it is possible to regenerate the bile duct without constriction using a bioabsorbable material. it is difficult to laparoscopically adhere to the injured part of the bile duct, but we hope that it will be possible in the near future to develop further adhesives. s surg endosc ( ) , - kg/m (c) and more than kg/m (d). we made a . -cm longitudinal skin incision within the umbilicus. a wound retractor and a surgical glove were applied at that incision. we used the three -mm ports technique. after retracting the gallbladder upward, the cystic duct and artery were divided and identified using pre-bending forceps through the flexible port and laparoscopic coagulating shears (lcs). the cystic artery was dissected using the lcs and the cystic duct was also dissected after clipping. the gallbladder was freed from the liver bed using the lcs, and the specimen was retrieved from the umbilical wound. results: there were conversions to open laparotomy in cases ( . %) and requirement of additional ports in ( . %). the mean age (years), operation time (min), blood loss (ml) and postoperative hospital stay (days) in group a, b, c and d were . , . , . and . (p= . [), . , . , . and . (p= . ), . , . , . and . (p= . ) , and . , . , . , and . (p= . ), respectively. there was a significant difference in age only. the complications were bile duct injury in one case ( . %) and pneumothorax in two ( . %). conclusion: obesity had no influence of surgical outcomes for performing silc. introduction: recent studies have reported mixed outcomes when comparing surgeon case volume and laparoscopic cholecystectomy (lc) outcomes. formal minimally invasive surgical training (mist) has been shown to be associated with shorter post-operative length of stay (los), but no difference in major adverse events such as bile leak, bile duct injury, intra-abdominal abscess formation, and death. we aim to determine -day rates of major adverse events after lc in a university hospital setting, to identify significant associated risk factors, and to determine if mist or surgeon volume are associated with differences in los and major adverse events. methods: we conducted a single-center retrospective review of , cholecystectomies performed over a seven-year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . characteristics and outcomes were compared using chi squared or rank sum tests. multivariable regression modeling was used to determine independent associations with the two main outcomes, major adverse events and los. results: we identified , adults who underwent lc during the study period, with a median age of , and % women. about % (n= ) of patients had a los[ day and . % (n= ) were re-admitted within the first days after surgery for any reason. within days of lc, . % (n= ) of patients suffered from one or more major adverse events. this includes . % (n= ) of patients with bile duct injury, . % (n= ) of patients with bile leak, . % (n= ) of patients with intra-abdominal abscess, and . % (n= ) of patients died for reasons related to their procedure or post-operative recovery. table shows the characteristics of the patients and procedures with a comparison of the patients with an adverse event versus those without one. in univariate analysis, high annual surgical volume ( + cases/year) and procedure urgency were found to be significant predictors of adverse events and los, however, mist was not. in multivariable analysis, controlling for significant univariate predictors, urgent or emergent cases were associated with a -fold increase in odds of an adverse event (or= . introduction: laparoscopic cholecystectomy is an extremely common procedure in the united states, with over , cases performed annually. despite the procedure's overall safety, there has been some evidence that tobacco use is associated with increased risk of wound infection after lc. this retrospective chart review sought to examine whether tobacco use is associated with increased complications following laparoscopic cholecystectomy within a high-volume healthcare system. methods: after irb approval, of approximately , cholecystectomies performed within one four-hospital system between and were randomly selected, and patient charts were retrospectively reviewed. pre-, intra-, and postoperative data were collected, including all complications within days. tobacco use cohorts were defined as follows: never, former (any historical tobacco use), and current (active tobacco use within year of surgery) per the acs nsqip surgical risk guidelines. following preliminary data analysis, multivariable logistic regression models were generated to identify whether tobacco use was predictive of outcomes of interest. of the cases analyzed, patients ( . %) were never smokers; . % were former smokers, and . % were current tobacco users or had quit less than months prior to surgery. there were surgical site infections, one wound dehiscence, one port site hernia, three common bile duct injuries, and medical complications requiring prolonged hospitalization or readmission within days. current tobacco users were significantly more likely to undergo urgent surgery (following emergency admission or direct admission to the hospital) than former or nonsmokers. however, there was no difference between cohorts for prolonged duration of surgery, conversion to an open procedure, surgical site infection, wound dehiscence or hernia, common bile duct injury, or other medical complication. there was no significant difference between cohorts when all postoperative complications were pooled. conclusions: there does not appear to be a significant difference in -day surgical outcomes or complications in active tobacco users vs. former or non-users. although studies in other surgical settings have indicated a possible reduction in complications if patients abstained from smoking prior to surgery, this may not be beneficial in laparoscopic cholecystectomy. moreover, as current tobacco use appears to be associated with higher rates of urgent surgery, these patients may not be able to stop smoking prior to an elective procedure. prospective studies to further clarify whether there is any benefit towards tobacco cessation prior to lc may be valuable. , [ , [ respectively ( - ) , cyfra were . , . , . respectively ( - . ) . afp and cea were negative. as for this patient, he is of high risk of hepatobiliary system diseases. introduction: thymoma is one of the rare tumor entity benign or malignant arsisng from the epithelial cells of thymus gland, frequently associated with neuromuscular disorder myasthenia gravis. so, we are presenting this rare case of thymoma with myasthenia gravis in our institute. methods: we operated a single patient of thymoma in a case of myasthenia gravis by video assissted thoracoscopic approach. results: operative time- min, intraoperative blood loss − ml, post operative analgesia requirement in form of nsaids is for days, no ventilatory support required post operatively, with follow up reduction in achr ab from nmol/l to nmol/l and reduction in symptoms in form of reduced ptosis. conclusion: thoracoscopic thymectomy is feasible and safe in terms less operative time, less post operative pain and analgesia requirement and no post operative ventilatory support requirement. carter c lebares, md, stanley j rogers, md; ucsf background: duodenal fistulas are uncommon but morbid complications of acute necrotizing pancreatitis. if percutaneous drainage fails, surgical correction via roux-en-y diversion or pancreaticoduodenectomy can be required. while self-expanding metal stents have been tried, complications like migration and perforation have limited such use. endoscopic transmural stents have successfully treated fistulas of the stomach, particularly post-sleeve gastrectomy. here we present a case of endoscopic transmural stents used to treat a non-resolving duodenal fistula following acute necrotizing pancreatitis. methods: under general anesthesia, using a standard adult gastroscope, the fistula was identified in the second portion of the duodenum (fig. ) . a flexible-tipped guide wire was used to identify the fistula tract and two fr cm double pigtail biliary stents were deployed ( fig. ) with positioning verified under fluoroscopy. two weeks later these were removed and a single stent deployed into the visibly smaller tract (fig. ). two weeks after that, the single stent was removed and contrast medium was injected under fluoroscopic visualization, demonstrating resolution of the fistula (fig. ) . case: this patient is a year old woman with hypertension and congenital hearing loss who underwent a cholecystectomy for biliary colic and subsequent ercp with sphincterotomy for retained stone. this was complicated by acute pancreatitis which progressed to severe necrotizing pancreatitis with infected retroperitoneal necrosis. percutaneous drainage yielded initial improvement but a persistent moderate collection ( cc per day) lead to the identification of a fistula in the second part of the duodenum. repositioning and exchange of percutaneous drains over weeks did not hasten resolution. endoscopic transmural pigtail stents were tried after visualization of a large ( - mm diameter) fistula tract. stents were utilized as described in methods, with a total of three endoscopic interventions, at week intervals, resulting in resolution of the fistula as evidenced by contrast injection into the duodenum under fluoroscopy and subsequent ct scan with oral contrast. the patient's symptoms resolved and she was tolerating a normal diet. she remained thus at month follow-up. conclusion: this case demonstrates the benefit of endoscopic transmural stents for the resolution of duodenal fistulas, expanding the utility of this technique to address leaks and fistulas of the upper gastrointestinal tract. further study is warranted to clarify the timing and adjuncts to optimize the use of this promising approach. totally laparoscopic alpps combined with the microwave ablation for a patient with a huge hcc hua zhang; department of hepatopancreatobiliary surgery, west china hospital, sichuan university introduction: associating liver partition and portal vein ligation for staged hepatectomy (alpps) is a novel technique for resecting hepatic tumors that were previously considered unresectable due to the insufficient future liver remnant (flr) which may result in postoperative liver failure (plf). the procedure has been accepted and modified in many medical centers worldwide. but reports about the laparoscopic alpps were rare. this study aimed to report a totally alpps combined with microwave ablation for a patient with huge hcc and confirm the feasibility of laparoscopic alpps. methods: a -year-old man had complained of -year history of right upper abdominal pain, and the syndrome was worsened in recent month. abdominal enhanced computed tomography (ct) imaging revealed a cm solid mass in right lobe of liver with non-uniform and unclear boundary, the right posterior branch of the portal vein was invaded. in addition, a small lesion was simultaneous found in left lateral lobe of liver. the tumor was evaluated as unresectable due to the flr was only ml ( %). we decided to perform the laparoscopic alpps procedure. first stage including microwave ablation of the lesion in left lobe, cholecystectomy, ligation of the portal vein and transection of liver parenchyma. the second stage was done days later and consisted of laparoscopic right hemihepatectomy. results: the two stages were underwent by laparoscopy successfully. the operation duration was and minutes, respectively. estimated blood loss was and ml. the hospitalization time in intensive care unit was and days. there was no need for transfusion in both stages. the patient was discharged days after the second stage and the total hospitalization time was days. recovery of the patient was uneventful in addition to the incision infection after the second stage which recovered with conservative management. the patient did not show any signs of liver failure. the ct scan before the second stage showed an enlargement of left lobe, the flr was ml ( . %). there was no signs of residual liver disease in the ct scan days after the operation. the patient showed no signs of recurrence or liver failure in the following up period of six months. conclusion: totally laparoscopic alpps combined with microwave ablation is safe and feasible for the multiple hcc which was not resectable. the hypertrophy of remaining liver was fast and can achieve an adequate volume in a short time. introduction: chronic pancreatitis is a benign, irreversible inflammatory disorder characterized by the conversion of the pancreatic parenchyma into fibrous tissue. initial management should be conservative, surgery is applied in case of failure of medical treatment. the development of minimally invasive techniques has made it possible to perform these highly technical procedures in a laparoscopic manner. materials and method: we have the history of patients with and years with chronic pancreatitis and pancreatic lithiasis of difficult handling but intractable pain to those who decided to surgical management. we performed the procedure under general anesthesia, epidural analgesia catheter was placed. neumoperitoneum technique of cali, at mmhg and approach using a mm umbilical port, working ports of and a of mm port,. the pancreas was exposed by a section of the gastrocolic ligament with a mm ultrasonic scalpel, with cephalic retraction of the stomach, opening of a smaller sac and approaching the transpavity of omentum. the ventral surface of the pancreas was exposed from the neck. an incision was made in a pancreas body with a monopolar hook. primary pancreatic duct lumen was identified and the incision was extended longitudinally from the neck to the tail of the pancreas ( cm). roux's y loop was prepared cm from the treitz ligament, with a jejunum section with a mm stapler, roux's loop was transmecoscopically retrocollic, closing the gap of the mesocolon with monocryl. a -cm jejunum-jejunal anastomosis was performed with endo-gia stapler and closure of enterotomy with - polypropylene intracorporeal suture. jejunal (roux) isoperistaltic loop was placed longitudinally at the opening of the main pancreatic duct, and enterotomy was performed with monopolar in antimesenteric segment. the intracorporeal pancreatico and jejunum anastomosis was performed using a lower and an upper plane, with single points of total thickness with ethnobond - . closed drains were placed towards each anastomosis. this procedure was performed in the patients reported. operative time - min complications none operative time - days minimal bleeding drains no retired in both cases at days year follow-up of patients improved pain\ conclusions: minimally invasive surgery is a fundamental tool for the approach and management of patients with biliopancreatic pathologies. the establishment of multidisciplinary groups, offer an excellent alteranativa in the integral management of the patients. surg endosc ( ) gallbladder anatomy is highly variable, and surgeons must be prepared to identify anomalies of form, number, and position. variants include gallbladder agenesis, diverticulum, duplication, bilobed, multiseptate, phrygian cap, ectopic, and hourglass gallbladder. the hourglass gallbladder has been described from the earliest days of cholecystectomy, as morton described a congenital case in , and else thoroughly described the acquired and congenital strictures leading to the hourglass deformity in . we describe a case of an hourglass gallbladder found during one-step endoscopic retrograde cholangiopancreatography (ercp) and laparoscopic cholecystectomy. this year old male presented to an outside hospital with one day of nausea, and constant, severe, epigastric pain that radiated to his back. he endorsed a history of similar pain several times in the past. his abdomen was soft, nontender, and without murphy sign. laboratory evaluation revealed total bilirubin . mg/dl, alkaline phosphatase u/l, ast u/l, alt u/l, and no leukocytosis. ct abdomen and pelvis revealed cholelithiasis, distal choledocholithiasis, intra-and extra-hepatic ductal dilation, and a . centimeter left liver hemangioma. he was transferred for management of choledocholithiasis, and an abdominal ultrasound revealed cholelithiasis, without gallbladder wall thickening or pericholecystic fluid, and a . millimeter common bile duct without choledocholithiasis. he was taken to the operating room for a one-step ercp and laparoscopic cholecystectomy. upon laparoscopy, dense adhesions to the gallbladder were found. after initially attempting to obtain the critical view of safety, we then embarked on the retrograde "top down" dissection. this isolated a spherical structure measuring . . centimeters. two very thin tubular structures were identified, clipped, and transected after we found they were too small to place a cholangiocatheter. the common bile duct appeared to be pulled anteriorly by surrounding inflammation, though this was later found to be the proximal segment of gallbladder. the intra-operative ercp identified a remnant gallbladder with cholelithiasis and no extravasation of contrast. given the unusual anatomy, we completed the operation, ordered a post-operative ct liver and mrcp, and consulted a hepatopancreatobiliary surgeon. a small remnant gallbladder was identified on ct liver, though not on mrcp. completion laparoscopic cholecystectomy with intraoperative cholangiogram and ultrasound was performed on hospital day . this hourglass gallbladder variant likely occurred secondary to chronic fibrosis from cholecystitis, leading to a proximal and distal gallbladder lumen. in anatomic uncertainty, the "top down" dissection, intraoperative cholangiography, ct liver, and expert consultation are safe methods to avoid iatrogenic injury. introduction: endoscopic entero-enteral bypass could change our approach to small bowel obstruction in patients with prohibitively high operative risk. magnetic compression anastomoses have been well-vetted in animal studies, but remain infrequent in humans. isolated cases of successful use in humans include treatment of biliary strictures and esophageal atresia. while endoscopic gastro-enteric magnetic anastomoses have been described, the associated multicenter cohort study was terminated due to serious adverse events. since then, the technology has evolved and recently our own institution reported results of the first in-human trial of magnetic compression anastomosis (magnamosis), deployed through an open approach. here we present the first case of endoscopic delivery of the magnamosis device and the successful creation of an enteroenteral anastomosis for chronic small bowel obstruction in a patient with prohibitively high operative risk. methods: the magnamosis device has previously been approved by the food and drug administration (fda) for use in clinical trial. our institutional review board approved emergency compassionate endoscopic use of the device in this patient due to a non-resolving small bowel resection and prohibitively high operative risk. case: this is a year old man with advanced liver disease, chronic obstructive pulmonary disease, and history of emergent right colectomy with end ileostomy for cecal perforation. he presented with multiple acute on chronic episodes of small bowel obstruction with a stable transition point in the distal ileum, radiographically estimated at centimeters proximal to the ileostomy. endoscopic evaluation through the ileostomy revealed a traversable obstruction with proximally dilated small bowel. the magnets were delivered via endoscopic snare under fluoroscopic guidance and positioned in adjacent loops of bowel on either side of the obstruction (image ). by days post-procedure, healthy villi were visible through the central portion of the mated magnetic rings (image ). by days the magnetic rings were mobile and the anastomosis was widely patent allowing easy passage of the gastroscope (image ), and the patient's symptoms were completely resolved. the rings passed through the ileostomy days post-procedure. at month follow up, the anastomosis was unchanged (image ). conclusion: this case demonstrates the benefit of an endoscopically created magnetic compression anastomosis in a patient with small bowel obstruction and high operative risk. further studies are indicated to evaluate the use of this technique in similar patients or those with malignant obstruct, ion. desiree raygor, md, ruchir puri, md; university of florida health jacksonville cholecystectomy is one of the commonest operations in general surgery [ ] . occasionally chronic cholecystitis can lead to a small contracted gallbladder. this diagnosis can be misleading as it may represent congenital agenesis of the gallbladder [ ] . a -year-old female with a past history of pancreatitis presented with a three day history of right upper quadrant pain associated with nausea and vomiting. upon exam she exhibited tenderness in the right upper quadrant. her leukocyte count and liver function tests were within normal limits. ultrasound revealed a poorly visualized, contracted gallbladder without stones and a dilated common bile duct (cbd). cholescintigraphy revealed non visualization of the gallbladder after two hours, which was suggestive of acute cholecystitis. decision was made to proceed with a laparoscopic cholecystectomy. the abdomen was entered by an open hasson technique and standard trocar placement for a cholecystectomy was performed. on initial inspection, the gallbladder was not readily visible. a structure appearing to be the cbd was present and was mobilized circumferentially (fig. ) . a gauge butterfly cannula was utilized and multiple cholangiographic images were obtained (fig. ). no cystic duct or gallbladder was identified which was suggestive of congenital agenesis of the gallbladder. the patient did well postoperatively, and was discharged home on postoperative day two. the patient's symptoms resolved and she continues to be pain free one month postoperatively. congenital agenesis of the gall bladder is a rare disorder. a high index of suspicion is required especially in the setting of a small contracted gall bladder. if preoperative imaging is inconclusive then diagnostic laparoscopy should be the next step. cholangiogram should be performed routinely to confirm the diagnosis and to rule out an ectopic gall bladder. conversion to open does not offer any distinct advantage, and laparotomy should be avoided if possible given its associated morbidity. there are many reports upper abdominal major arterial aneurysms. however, an aneurysm of left inferior phrenic artery had never been reported. a -year-old woman with liver cirrhosis associated with hepatitis b viral infection was referred to department of surgery for treatment of aneurysm of left inferior phrenic artery. she underwent trans-arterial chemoembolization (tace) for treatment of hepatocellular carcinoma three times, previously. on months after last tace, mm sized highly enhancing nodular lesion of gastric fundus was found on follow-up abdomenpelvis computed tomography (a-p ct). one year later, the size of this lesion increased to mm, and an aneurysm was diagnosed. she underwent angiography and attempted embolization with an aneurysm of the left inferior phrenic artery, but access failed. we performed a laparoscopic vessel ligation. she recovered with no complication and discharged on the th postoperative day. s surg endosc ( ) :s -s yousef almuhanna, vatsal trivedi, fady balaa; university of ottawa a years old female, g and weeks pregnant, was brought to the hospital by ems, after being found on the floor in her toilette surrounded by vomitus and urine. mother-inlaw, who happens to be at the house that time, have heard severe retching followed by a loud bang sound. firefighters have found no pulse and therefore started cpr. return of spontaneous circulation was achieved, yet unfortunately, she had arrested again minutes prior to arrival to er. pocus assessment showed large rvot, and therefore tpa was started on the assumption of pulmonary embolism. upon arrival of blood work, it was found that her hemoglobin had dropped from to . fast was repeated showing moderate to severe amount of free fluid in the morrison's pouch and pelvis. she was then taken to the operating theatre, had undergone laparotomy showing liver segment ii injury. pringle's maneuver and aortic clamping did not control the bleed, therefore finger fracture and venous clips were used to temporary minimize the bleed, and head to interventional radiology suite. after multiple attempts to control the bleed, and the massive transfusion, she vital signs were not maintained, and had arrested afterwards. sarrath sutthipong, md, chumpunut chuthanan, md, chinnavat sutthivana, md, petch kasetsuwan, md; bhumibol adulyadej hospital, bangkok, thailand background: mesenteric panniculitis (mp) is a rare, benign and chronic fibrosing inflammatory disease that affects the adipose tissue of the mesentery of the small bowel and colon. the specific etiology is unknown and no clear information about the incidence. the diagnosis is suggested by ct and is usually confirmed by surgical biopsy. treatment is based on some selected drugs. surgical resection is sometimes attempted for definitive therapy, although the surgical approach is often limited. we reported a case of the mp diagnosed with ct and surgical biopsy by laparoscopic approach. case report: -year-old woman with months history of chronic abdominal pain, mainly localized in the sub-epigastrium, intermittent and mild. she had anorexia but no weight loss or change in bowel habits. no history of medical illness or surgery. the physical examination was unremarkable, except for palpation of ill-defined mass about cm at mid-abdomen, firm, smooth surface with mild tenderness. the laboratory profile and tumor marker were normal. ct of the abdomen, which showed focal heterogeneous enhancement of the mesenteric fat with stranding ( . . cm) with multiple internal subcentimeter lns in the supraumbilical area, which was probably inflammatory in origin and suggestive of mp. f-fdg pet/ct showed faint fdg uptake in multiple mesenteric lns. the patient was subsequently underwent diagnostic laparoscopy with biopsy. intra-operative finding showed a fat-like surface of yellowish mass at mesentery of jejunal segment, incisional biopsy was performed laparoscopically. the histology showed adipose tissue with areas of fat necrosis, fibrosis, foamy macrophages infiltration and predominant chronic inflammation, no evidence of malignancy. ihc studies (including cd , s- , cd and cd ) were performed and the result was compatible with reactive process. treatment was started with mg prednisone once daily and planned for follow-up with repeated ct scan. discussion: mp involves the small bowel mesentery in over % of cases. the diagnosis is made by pathologic findings: fibrosis, chronic inflammation and fatty infiltration. the differential diagnosis is broad and has been associated with malignancies such as lymphoma, well-differentiated liposarcoma and melanoma. the imaging appearance varies depending on the predominant tissue component. a definitive diagnosis is biopsy but open biopsy is not always necessary. no data of laparoscopic biopsy, which has been reported previously. treatment has been reserved for symptomatic cases with a variety of drugs. our case was started on oral corticosteroid treatment and waited for responsive evaluation. background: laparoscopic appendectomy is the gold standard for treatment of acute appendicitis. stapled closure of the appendiceal stump is often performed and has been shown to have several advantages. few prior cases have been reported demonstrating complications from free staples left within the abdominal cavity after the laparoscopic stapler has been fired. case report: a previously healthy year old female initially underwent laparoscopic appendectomy for acute uncomplicated appendicitis during which the appendix and mesoappendix were divided using laparoscopic gastrointestinal anastomosis (gia) staplers. her initial postoperative recovery was uncomplicated and she was discharged home the same day. the patient returned to the emergency department on postoperative day with one day of sharp mid-abdominal pain, obstipation, and emesis. her abdomen was distended and mildly tender but not peritoneal. she was afebrile but was found to have a leukocytosis of . . ct demonstrated twisted loops of dilated small bowel in the right lower quadrant with two transition points, suggestive of internal hernia with closed loop bowel obstruction. diagnostic laparoscopy was performed through the three prior appendectomy incisions. an adhesion was noted between the veil of treves and the mesentery of a more proximal loop of ileum caused by a solitary free closed staple, remote from the staple lines, resulting in an internal hernia containing several loops of ileum ( fig. ). the hernia was reduced, and the small bowel was noted to have early ischemic discoloration. the adhesion was lysed by removing the staple from both structures to prevent recurrence. through the remainder of the procedure, the compromised loops of bowel began to peristalse and the color normalized. the procedure was concluded without resection. the patient recovered on a surgical floor and was discharged home on postoperative day one. conclusion: gastrointestinal staplers are commonly used secondary to ease of use and low complication rate. it is not uncommon to leave free staples in the abdomen during laparoscopy as retrieval can often be more difficult and time consuming. our case is only the second in the literature reporting an internal hernia with closed loop bowel obstruction as a complication of retained staple. choosing the most appropriate size staple load, to reduce the number of extra staples after the fire, and removing as many free staples as possible can prevent potentially devastating complications. video-assisted thoracoscopic pulmonary wedge resection in a patient with hemopytsis and intralobar sequestration: a case report mary k lindemuth, md, subrato j deb, md; the university of oklahoma health science center case report: a -year-old male with history of noonan's syndrome, bronchitis, and asthma presented with acute hemoptysis. while chest x-ray was unremarkable, a computed tomography angiogram of his chest was significant for intralobar pulmonary sequestration in the right lower lobe. the aberrant pulmonary artery originated from the abdominal aorta, immediately proximal to the celiac axis, and coursed through the hiatus in the retroperitoneum. flexible, fiberoptic bronchoscopy revealed blood within the right lower lobe bronchus with no appreciable source. a right video-assisted thoracoscopic approach was taken for wedge resection of the sequestration. twoportal technique was utilized with the patient on single lung ventilation. the sequestration was easily identified; the anomalous pulmonary artery coursed directly to a large, focal area of hemorrhage noted within the lower lobe pulmonary parenchyma, as seen in image [rectangle marking the aberrant artery and oval marking the sequestration]. pathologically, the specimen was noted to be benign lung parenchyma with bronchiectasis and abundant, acute hemorrhage. discussion: pulmonary sequestration (ps) is a rare, congenital bronchopulmonary foregut malformation. literature describes the incidence of ps to be only . - . % of all pulmonary malformations. as ps is most frequently diagnosed during childhood, the occurrence of diagnosis during adulthood is estimated to be less than per , adults. two types (intra-and extralobar) are described, with intralobar sequestration most common and contained within the normal visceral pleura. both types have aberrant systemic arterial blood supply, most frequently from the thoracic aorta. likewise, both types are nonfunctioning lung tissue, as there is no direct communication with the bronchopulmonary tree. the most common presentation is pneumonia, and often patients will have had recurrent symptoms before diagnosis. it is rare to present with hemoptysis, which is understood to be secondary to elevated capillary pressure within the sequestration and then communication through the pores of kohn. while endovascular embolization of the aberrant pulmonary artery has been described as a safe alterative for surgical intervention, the subjects of these studies have primarily been children and long-term outcomes are unknown. the definitive treatment of ps continues to be surgical intervention. the surgeon should strive to leave as much normal lung parenchyma as possible. video-assisted thoracoscopic resection is well tolerated by patients when compared to thoracotomy. however, it is vital for the surgeon to be aware of the potential risk of life-threatening hemorrhage secondary to the sequestration having systemic blood supply that must be controlled and ligated. case report: a years-old female patient with history of an increased mass and weight loss of kilograms in months, associated with vomiting and nausea for eight months. abdominal ultrasound showed an irregular cyst, without solid projections and without signs of flow in doppler, measuring cm. investigation continued with ct scan that showed a large homogeneous cystic lesion with no septum in the abdominopelvic region, possibly mesenteric, measuring . . cm. a laparoscopic approach for resection of the cyst was then performed. the surgery was performed with a patient in the dorsal decubitus, using three trocars: one in the umbilical region ( -mm) for the camera, and where the pneumoperitoneum was created by the hasson open technique under direct vision; and another two located in the epigastrium ( -mm) and in the right upper quadrant ( -mm) . in addition to the mesenteric cyst, a simple cyst in the right ovary and a solid nodule with a lipomatous characteristic of approximately cm in the abdominal cavity were visualized. total resection of the mesenteric cyst with periprancreatic fibrous tissue was performed. the cyst was punctured and its contents fully aspirated. resection of the right ovarian cyst was also performed. at the end of the procedure the mesenteric and ovarian cysts, the nodule, part of the omentum, and the peripancreatic tissue were removed through the -mm trocar at the umbilicus. patient had no further complications, being discharged four days after the procedure. histopathologic result showed a serous cyst in the right ovary, serous cyst in peripancreatic mesentery with chronic inflammatory process and signs of calcification; no signs of malignancy were observed in any specimen. we aimed to present the succesul therapeutic approach utilizing laparoscopy for safely removing a gastrointestinal stromal tumor. depicted is a year old jehova's witness female who presented to the emergency department for evaluation of bitemporal headache and dizziness and found with profound anemia with hemoglobin . and hematocrit . upon arrival to ed. the patient refused blood transfusion as her religious beliefs, jehovah's witness, preclude her from taking blood products. as part of her work up, endoscopy was performed and revealed a large, approximatelly cm, prolapsed, ulcerated, nodular lesion with active bleeding in the cardia of the stomach. this was temporized but the friable tissue, with no single identifiable lesion for clip placement, left the patient at high risk for re-bleeding. she was taken to the operating room and laparoscopic partial gastrectomy with intraoperative esophagogastroduodenoscopy were succefully perfomed, with minimall blood loss and no intra operative complications. patient was discharged on post op day . we present the case of a -year-old male with a history of morbid obesity with an initial bmi of . , who underwent an elective laparoscopic single anastomosis duodenal-ileal bypass with sleeve gastrectomy (sadi-s). postoperatively he developed an anastomotic leak at the duodeno-ileal anastomosis that would not resolve despite reoperation. he was then converted to a roux-en-y gastric bypass (rygb). postoperative imaging failed to reveal any signs of anastomotic leak and the patient was discharged tolerating an oral diet. he returned to the emergency department days later with a cm sub-hepatic collection arising from the duodenal stump from the surgical conversion. interventional radiology percutaneously drained the collection and found a connection between the cavity and the duodenum. using this connection, a percutaneous decompressive duodenostomy drain was successfully inserted into the duodenum using a guidewire through the abscess cavity along with an extra-enteric drain placed within this cavity. the collection was obliterated and the duodenal leak was controlled successfully with percutaneous drainage, bowel rest with parenteral nutrition and broad-spectrum intravenous (iv) antibiotics. the patient was reintroduced to a bariatric clear diet after a week of bowel rest and the abscess drain was then discontinued during the same hospital admission. the patient was discharged with the percutaneous duodenostomy tube which was removed in clinic days later, after the patient tolerated capping trials and imaging failed to reveal any further collections, oral contrast extravasation or distal obstruction. in this article we analyze notable imaging from the case and review current literature on the different management options for a duodenal stump blowout. we also discuss the basics of the sadi-s procedure and conversion of a sadi-s procedure to a rygb. keywords: anastomotic leak, duodenal stump blowout, sadi-s, duodenostomy tube. pancreatopic heterotopia is often an incidental finding on autopsy, but in some cases can lead to abdominal pain, obstruction, or intussusception. we present a case of pancreatic herterotopia mimicking an internal hernia on radiologic imaging. a year old female with seven month history of chronic abdominal pain treated for low back pain and recurrent urinary tract infections. she was found to have a computed tomography (ct) scan concerning for internal hernia and labs consistent with acidosis. she was taken for a laparotomy and did not have an internal hernia, but an exophytic mass in the proximal jejunum. the mass was resected and a stapled side to side jejunojejunostomy was created. on pathologic review, the specimen was found to be pancreatic heterotopia. her post operative course was complicated by an ileus, but was discharged post op day three. at her two week follow up she had minimal incisional pain and at one year follow-up she had resolution of her left upper quadrant abdominal pain. prior to this report, pancreatic heterotopia has never been described as presenting on ct scan as an internal hernia. although uncommon it should remain in the differential when evaluating a patient presenting with abdominal pain and radiologic evidence of obstruction or internal hernia. case report: a -year-old male patient who was diagnosed with high blood pressure at years-old and presented tetraparesis and intense asthenia for six months. blood tests showed hypokalemia, hypernatremia, and suppressed renin activity. ultrasound of the urinary tract was normal. ct scan of the abdomen showed a hypodense nodule with regular margins, measuring . . cm with a density of hu in the non-contrast phase and heterogeneous uptake after the injection of the contrast in the left adrenal gland. thus, the diagnosis of hyperaldosteronism secondary to the left adrenal nodule was confirmed, and surgical resection was indicated. the procedure was performed with the patient in the right lateral decubitus. two -mm and one -mm trocars were used on the left flank, as well as the -mm portal for the camera in the lower right quadrant under direct vision. the pneumoperitoneum was created by the hasson open technique in the transumbilical incision. the procedure consisted of the dissection, isolation and electrocautery of the left renal capsule and the left adrenal region with ultrasonic device, as well as the periadrenal vessels, adjacent lymph nodes and periadrenal and adrenal fat tissue. the surgery was uneventful and the patient had no further complications, being discharged the next day. histopathologic result showed a completely excised adrenocortical adenoma. conclusions: the hybrid minimally invasive approach proved to be safe and effective for this procedure, and the known advantages of minilaparoscopy such as less trauma, better visualization, better dexterity, better aesthetics, and reduced hospital stay were observed. s surg endosc ( ) background: coccidioidomycosis is a fungal infection endemic to the southwestern united states, central america and south america. coccidioides is ubiquitous in many of these endemic regions, with near % seroconversion in some communities. two-thirds of these mycotic infections may be asymptomatic. the most common presentation of coccidioidomycosis consists of "flu-like" symptoms or pneumonia. less than five percent of symptomatic cases progress to disseminated coccidioidomycosis which may involve any organ system. very rarely infection may include the peritoneum. we report a case of coccidioidomycosis with peritoneal involvement in an immunocompetent individual. case: a -year-old male presented to the emergency department with progressive abdominal pain. he was seen and treated for pneumonia in the emergency department one week prior. the patient worked outdoors in arizona and was otherwise healthy with a family history of malignancy and blood disorders. fever, leukocytosis and ascites on computed tomography scan prompted a diagnostic laparoscopy which revealed peritoneal granulomas positive for coccidioides. the patient was treated outpatient with fluconazole. discussion: since this is the th reported case of peritoneal coccidioidomycosis to our knowledge. the patient described in this case report was an otherwise healthy -year-old male; this is incongruent with many of the previously recorded cases which involved disseminated disease in immunocompromised patients. the patient's family history of malignancy and blood disorders suggests a potential underlying genetic predisposition that could account for this abdominal presentation. possible mutations include genes coding for the interleukin- β receptor and the signal transducer and activator of transcription which have been implicated in increased coccidioidomycosis susceptibility. peritoneal infection presents a unique challenge in diagnosis. in these cases coccidioidomycosis may not be suspected due to nonspecific symptoms and imaging, the infrequency of this extra-pulmonary manifestation and clinical characteristics that mimic the presentation of tuberculosis and malignancy. abdominal infections have been misdiagnosed as appendicular abscesses, iliopsoas abscesses, adnexal abscesses and pancreatic masses. consequently, the diagnosis of peritoneal coccidioidomycosis is often made after laparoscopic exploration of the abdomen and histopathology, as it was in this case report. conclusions: coccidioidomycosis incidence is on the rise in endemic areas and it often falls on the surgeon to make the diagnosis in extra-pulmonary cases. the peritoneal subset of coccidioidomycosis should be considered in endemic areas when a young, otherwise healthy patient presents with abdominal pain. failure to recognize the possibility of coccidioidomycosis may lead to unnecessary treatments and procedures. indocyanine green cholangiography to detect anomalous biliary anatomy steven d schwaitzberg, md, gabrielle yee, ms; university at buffalo jacobs school of medicine introduction: common bile duct injury is the most feared complication of cholecystectomy. imaging with indocyanine green (icg) is a safe and effective technique to detect biliary anatomy in open, laparoscopic and robotic surgery. several studies report detecting aberrant biliary anatomy with the use of icg in laparoscopic cholecystectomy with high success rates. by identifying the cystic duct-common hepatic duct confluence before dissecting calot's triangle, icg allows surgeons to perform "virtual" cholangiography at the start of procedures to identify either normal anatomy or possible anatomic variants. it is clear that icg use is an effective tool to achieve the critical view of safety. however, no reports have suggested icg cholangiography as the last operative step in cholecystectomy to identify hidden biliary anomalies and avoid postoperative bile leak complications. case report: we report a novel use of icg cholangiography in visualizing anomalous biliary anatomy prior to closing, thus avoiding potential bile duct leakage. in our case, icg cholangiography was used to fluoresce the common hepatic duct, common bile duct and cystic duct. the cystic duct was transected, and the gallbladder was removed using electrosurgery. at the completion of the gallbladder removal, the liver was elevated to inspect the clips on the cystic duct and artery. at this point, near infrared imaging was reinitiated, and a small mm structure was noted to fluoresce next to the cystic artery. this structure was identified using white light and subsequently clipped. discussion: the use of icg in this context after the completion of the cholecystectomy facilitated the identification of a small hepatocystic or aberrant duct, which would have likely leaked bile sometime in the postoperative period. based on our experience, we recommend one additional routine near infrared viewing to identify small structures or potential leaks at the completion of cholecystectomy. improved visualization of the extrahepatic biliary anatomy by icg has the potential to translate into improved clinical outcomes. solitary fibrous tumors (sft) are uncommon fibroblastic mesenchymal neoplasms that display a wide range of histologic behaviors. these tumors, which are estimated to account for % of all soft tissue neoplasms, typically follow a benign clinical course. however, it is estimated that - % of sfts are malignant and demonstrate aggressive behavior with local recurrence and metastasis up to several years after surgical resection. we report a case of sft arising from the stomach, which is an exceptionally rare finding and has been reported only six times in the literature. additionally, this tumor was associated with dedifferentiation into undifferentiated pleomorphic sarcoma. to our knowledge, there are no documented cases of a malignant sft arising from the stomach to demonstrate dedifferentiation into an undifferentiated pleomorphic sarcoma. a -year-old male presented to the emergency department with vague complaints of right-sided flank pain. the patient had a history of nephrolithiasis and underwent a ct abdomen. this scan revealed a large heterogeneous mass in the left upper quadrant. the patient underwent endoscopic ultrasonography with fine needle aspiration of the mass, which stained strongly for cd . gastrointestinal stromal tumor (gist) was the favored diagnosis as it is by far the most common mesenchymal neoplasm of the stomach, especially cd positive spindle cell neoplasm. accordingly, the patient began treatment with imatinib; however, after four weeks of therapy, there was no significant radiologic regression. a second biopsy was performed and the specimen was sent for stat immunohistochemistry, which revealed diffuse strong nuclear positivity. a diagnosis of solitary fibrous tumor was provided. surgical resection of the tumor was performed, which measured . cm. the patient was to undergo surveillance imaging every to months post-operatively. surveillance scan showed solitary metastatic disease in the left lateral segment of the liver. he underwent left lateral segmentectomy with an uneventful recovery. our case was complicated by diagnostic dilemma with gist, highlighting the challenges of diagnosing and characterizing sfts. dedifferentiation, or the abrupt transition from a classic sft into a high-grade sarcoma, is a particularly concerning finding in our case, as it is associated with a worse prognosis than classic malignant sft. the stat marker by immunohistochemistry is very specific for sft and may have aided in the diagnosis earlier. therefore, it is imperative to keep solitary fibrous tumor, albeit exceedingly rare, in the differential diagnosis of mesenchymal neoplasms of the stomach. appendiceal diverticulitits is an uncommon pathology that can clinically mimic acute appendicitis. some radiographic distinctions have been reported, but final pathologic examination of the surgical specimen is required to confirm the diagnosis. symptoms are often more mild, which can lead to a delayed diagnosis, and increases the risk of severe complications such as perforation. a year old female presented with a three day history of right lower quadrant pain. she described the pain as constant and radiating to the left lower quadrant. associated symptoms included nausea and vomiting, and decreased appetite; she denied fevers or diarrhea. the patient had no significant past medical history, and surgical history was significant for a total nephrectomy for living donor kidney transplant to her mother. on physical exam she was tender in the right lower quadrant with rebound and a positive rosving's sign. all laboratory results were unremarkable, and she was hemodynamically stable. ct scan was performed and demonstrated a dilated fluid filled appendix with surrounding inflammatory change without abscess or free intra-peritoneal air. she was subsequently admitted to the hospital, made npo, started on iv antibiotics, and was taken to the operating room where she underwent an uncomplicated laparoscopic appendectomy. post-operatively, her hospital course was unremarkable. pathology revealed acute suppurative appendicitis secondary to an acutely inflamed appendiceal diverticula, consistent with a final diagnosis of acute appendiceal diverticulitis. appendiceal diverticulitis should be considered in patients presenting with acute right lower quadrant abdominal pain. although some consider appendiceal diverticulitis a variant of acute appendicitis, it is important to distinguish between the two diagnoses. appendiceal diverticulitis has a higher rate of complications, including perforation, and is associated with a higher risk of neoplasm, particularly mucinous adenomas and carcinoid tumors. appendectomy should be performed in all cases in order to obtain appropriate pathological examination and rule out coexistent neoplasms. laparoscopic appendectomy is a safe and appropriate approach to treatment of appendiceal diverticulitis. upper gi endoscopy and biopsy showed a gastrointestinal stromal tumor (gist) in the stomach. a videolaparoscopic partial gastrectomy was then proposed. the surgery was performed with the patient in the right lateral decubitus. two -mm minilaparoscopic trocars, a -mm conventional trocar for an ultrasonic instrument and a -mm trocar in the umbilical region for the camera were used. pneumoperitoneum was created using the hasson open technique under direct vision. trans-operatory endoscopy was perfomed to identify the tumor easily. initially, the ultrasonic device released the large omentum, and, then, the tumor was resected in the body of the stomach. the gastric wall was manually sutured with a - vicryl, and the tumor was removed in an endobag through the -mm incision in the umbilicus. the surgery was uneventful, with a total time of minutes. the patient had no further complications, being discharged two days after the procedure with good clinical conditions. histopathological result showed a free margins gist. conclusion: the minimally invasive approach proved to be safe and effective for this procedure. the known advantages of video-surgery such as less trauma, better visualization, increased dexterity, better esthetics, and less postoperative recovery time were confirmed. the upper gi endoscopy contributed to improve the safety and efficacy of the procedure, allowing a more precise resection of the gist, as well as the intragastric review of the suture line at the end of the surgery. background: portal vein thrombosis (pvt) is a rare post-operative complication, which has been associated with a wide range of precipitating factors. most commonly described associated conditions include; cirrhosis, bacteremia, myeloproliferative disorders and hypercoagulable states. pvt most frequently occurs as a complication after hepatobiliary surgery, and although possible, very few cases have been documented occurring after laparoscopic surgery of the gastrointestinal tract. herein, we describe a case of pvt in a patient who underwent elective laparoscopic right hemicolectomy and was treated successfully at our center. case: a year-old female with past medical history of depression, migraines and endometriosis underwent an uncomplicated laparoscopic right hemicolectomy at our facility, for recurrent rightsided diverticulitis. she had suffered previous episodes of diverticulitis and desired definitive surgical treatment. her hospital course was uneventful and she was discharged to home on postoperative day . on post-operative day , she presented to the emergency department complaining of severe abdominal pain, back pain and nausea. computed tomography of abdomen and pelvis revealed pvt. she was initiated on therapeutic anticoagulation with heparin. hematology was consulted for hypercoagulable workup. further investigation revealed that she had a family history of a brother who had had a lower extremity deep venous thrombosis, with negative hypercoagulable workup. she had also previously been taking leuprolide and conjugated estrogen and medroxyprogesterone for her endometriosis. she was ultimately found to have a heterozygous prothrombin g a gene mutation. her anticoagulation was bridged to coumadin and she was discharged home. she has recovered as expected, without any further complications. discussion: although more common in patients with cirrhosis after hepatobiliary surgery, pvt is a rare complication that can occur after virtually all types laparoscopic surgeries, including elective right hemicolectomy. patients may be completely asymptomatic, or present with a broad spectrum of symptoms including; severe abdominal pain, fever, diarrhea, or gastrointestinal bleeding. physicians should be aware of this possible complication, since early diagnosis and treatment is imperative to prevent life-threatening complications, such as intestinal ischemia and perforation. a detailed medical and family history is imperative, and all patients with post-operative pvt should undergo complete hypercoagulability workup. this is a case of a year old male with a previous history of a redo-hiatal hernia years prior who presented with two episodes of upper gastrointestinal bleeding with no identifiable source noted on both endoscopy and angiography. during his second admission, initial hemoglobin was . g/dl and endoscopy performed showed massive amount of blood in the stomach. continuous oozing was seen originating in the fundus area but no clear source could be identified. empiric epinephrine was injected to the area but failed to achieve hemostasis. angiography was also negative. repeat endoscopy performed showed no active bleeding, however, distention of the wrap into the gastric cavity was observed. the patient re-bled and was taken to the operating room emergently after failed attempt at endoscopic control. the patient underwent proximal gastrectomy after intra-operative gastrostomy and exploration was unable to identify a bleeding source. the patient was left with an open abdomen and in discontinuity while resuscitation was performed in the surgical intensive care unit. he subsequently underwent a roux-en-y reconstruction and gastrostomy tube placement via the distal gastric remnant. upper gastrointestinal series performed demonstrated absence of leak, and the patient was started on a liquid diet supplemented with tube feeding. his recovery was uneventful and he was discharged home in stable condition. pathology revealed gastric ischemia at the base of the wrap making it impossible to visualize through endoscopy. on reviewing the literature, gastric ulcers and ischemia have been previously described. incidence was up to % and their onset of presentation ranged from the early post-operative period up to years. most were located in the lesser curvature. the exact pathophysiology for its occurrence is not completely understood. factors hypothesized include technical aspect of the fundoplication causing inappropriate tension, vessel disruption and ischemia, and injury to the vagus nerve affecting gastric emptying which was thought to increase gastrin secretion. treatment includes medical management with proton pump inhibitors; however, few cases describe antrectomy with inclusion of the bleeding ulcer. our case presents failed medical and endoscopic management. we recommend take down of the fundoplication in hemodynamically stable patients to completely evaluate the gastric mucosa, identify, and address the source of bleeding. otherwise emergent cases will require staged gastrectomy including the wrap followed by roux-en-y reconstruction. acalculous cholecystitis associated with a large periampullary duodenal diverticulum: a case report peng yu, md, phd, austin iovoli, aaron hoffman, md; department of surgery, suny buffalo, kaleida health system, buffalo, ny introduction: periampullary diverticulum (pad) could compress common bile duct (cbd), and consequently cause obstructive jaundice and cholangitis as few publications have documented. here we first report an acalculous cholecystitis associated with a pad-related cbd obstruction. case: the patient was a -year-old female with a past surgical history of laparoscopic sleeve gastrectomy who presented at the emergency room with upper abdominal pain and vomiting for one day, associated with leukocytosis and left shift. serum total bilirubin raised up to . mg/dl on hospital day (hd) . ct, ultrasound, and mrcp images confirmed a distended, wall-thickening gallbladder with pericholecystic fluid, and a significantly dilated cbd at . cm of diameter ( fig. ) , without cholelithiasis or choledocholithiasis. ercp was unable to be completed due to the post-gastrectomy anatomy and the failure in cannulation into the ampulla which embedded in a large foodimpacted pad (fig. ). on hd , the patient underwent a diagnostic laparoscopy and an intra-operative cholangiogram which confirmed a mildly inflamed edematous gallbladder, and a . . cm large pad with a narrow neck that was distorting the distal cbd (fig. ). since the patient's bilirubin level had been improving, we decided to only do a laparoscopic cholecystectomy. intraoperatively an anatomic variation of the cystic artery encircling the cystic duct ( fig. ) was also identified. postoperatively the patient recovered well during the thereafter inpatient course and at the postoperative -week outpatient follow-up. the pathology of the excised gallbladder confirmed cholecystitis without cholelithiasis. discussion: lemmel's syndrome is defined, in the absence of cholelithiasis or other detectable obstacle, by obstructive jaundice due to pad. since lemmel described this duodenal-diverticulum-obstructive jaundice in , there still have been very few cases reported or investigated. to date there is no report describing the association of acalculous cholecystitis with lemmel's syndrome. this patient's mild acalculous cholecystitis probably attributed to the biliary obstruction and consequent gallbladder hydrops. her symptoms could be from either acalculous cholecystitis or intermittently worsening biliary obstruction. in this case, the contribution of the anatomic variation of the cystic artery is unclear. in the future, if this patient's symptoms recur, the treatment plans for her will be sphincterotomy, removal of the impacted food in the pad, or diverticulectomy. accidental fish bone ingestion masquerading as acute abdomen aim: to report a case of fish bone ingestion masquerading as acute abdomen. case report: a years old female patient presented with complaints of severe abdominal pain since days. there was no history of associated nausea or vomiting, fever or altered in bowel habits. on examination patient had tenderness and guarding localized to the right iliac fossa. blood investigations revealed raised inflammatory markers. ultrasound whole abdomen and contrast enhanced computed tomography (cect) were normal. patient was managed conservatively but in view of persistence of symptoms a triple puncture diagnostic laparoscopy was performed on day of admission. omental inflammation with soapy appendix was found and appendicectomy was performed. on further assessment a foreign body was also found in the ileum which was removed and identified as a fish bone. patient had a satisfactory post operative recovery and was discharged in stable condition. discussion: acute abdomen due to fish bone ingestion is not a very common occurrence. unfortunately the history is often non-specific and these people can be misdiagnosed with acute appendicitis & other pathologies. ct scans can be useful to aid diagnostics. it is however not fully sensitive in detecting complications arising from fishbone ingestion. conclusion: any patient with acute abdomen, with non-specific history and normal imaging may still benefit from a diagnostic laparoscopy. discussion: this patient presented with a bowel obstruction, partial cecal necrosis and neuroendocrine carcinoma. literature suggests that cecal necrosis in the majority of cases is caused by a vascular event, occlusive or non-occlusive. the patient had atherosclerosis and an underlying malignancy which can be associated with prothrombotic states and contributes to an overall risk of thrombosis. the cecum can sustain ischemic ischemic injury in the presence of severe or prolonged hypotension. most frequent causes being decompensated heart failure, hemorrhage, arrhythmia or severe dehydration, only of which was present in this patient. the midgut neuroendocrine tumor is generally located in the terminal ileum, as a fibrotic submucosal tumor cm or less. mesenteric metastases are often larger than the primary tumor and associated with fibrosis which may entrap loops of the small intestine and cause bowel obstruction. this may eventually encase the mesenteric vessels with resulting venous stasis and ischemia in segments of the intestine as seen in this patient. conclusion: cecal necrosis is a rare entity, but its incidence increases with age. isolated cecal necrosis may manifest as a ct-negative appendicitis or a small bowel obstruction in the absence of past surgical history. s surg endosc ( ) laparoscopic transection of the falciform and triangular ligament successfully released the entrapped loop with successful reperfusion by the end of the surgery. in the absence of any prothrombotic comorbidity, the patients were discharged asymptomatic without further anticoagulation. to date only few similar cases have been reported, and most of them described in neonates and pediatric patients. to our knowledge, this cases reporteds in the elderlys. in this patients laparoscopic approach was both diagnostic and therapeutic with the transection the ligament. roberto javier rueda esteban , andres mauricio garcia sierra , felipe perdomo ; universidad de los andes, fundacion santa fe this is a patient´s rare case of spontaneous splenic rupture associated to chronic myeloid leukemia as an uncommon complication. the case report and review of the relevant literature on symptomatology and clinical management is presented. emphasis is made about the importance of including splenic rupture as differential diagnosis for acute abdominal pain, especially in a patient with neoplastic hematopathology, since early treatment increases patient survival and prognosis. esophagectomy is a complex operation associated with serious immediate complications and long term chronic complications. gastric ulcers are a common chronic complication after esophagectomy with gastric conduit reconstruction. these are rarely complicated by significant bleeding or perforation. we report a case of delayed diagnosis of a fistula forming between a gastric conduit and right bronchial tree years after esophagectomy. this was successfully treated using multiple therapeutic approaches including endoscopic localization and resection through a right thoractomy. to the best of our knowledge, our patient is the only survivor from a chronic gastric conduit bronchial fistula. a year old male with type diabetes mellitus, dyslipidemia, asthma and smoking history presented years after an ivory-lewis esophagectomy for a gastrointestinal stromal tumor (gist) with a chronic cough starting years after his esophagectomy followed by multiple episodes of hematoptysis over the next years. the patient was known to have ulcers in his gastric conduit with a massive bleed year after his esophagectomy. repeat endoscopy revealed two large chronic ulcers that had increased in size based on comparison of pictures from endoscopies to years after his esophagectomy despite maximal medical management. the patient presented to numerous specialists at tertiary care centers in canada and the united states. ultimately, in a clinic the patient was observed to cough immediately after the ingestion of water, but not solids leading to a provisional diagnosis of a gastrobronchial fistula. a barium swallow failed to show a fistula (fig. ). however at endoscopy, instillation of saline directed at an ulcer immediately induced a cough, but this was not reproduced when the saline was directed away from the ulcer. the fistula was ultimately demonstrated by placing a wire through the ulcer and visualizing it bronchoscopically in the right superior segmental bronchus . in an effort to pursue a minimally invasive approach two attempts were made to close the fistula with over-the-scope clips (otsc). unfortunately, the patient's symptoms persisted. a wire was placed through the fistula and delivered through the patient's mouth and endotracheal tube. a right thoracotomy allowed access to the conduit, which was opened and the fistula localized using the wire. the fistula was resected and the bronchus closed. at twelve month follow up the patient did not have a recurrent cough or hemoptysis while tolerating a full diet. introduction: roux en-y gastric bypass (rygb) is one of the initial and most studied weight reduction procedures and remains the gold standard for comparison in bariatric surgery clinical outcomes. although rygb is an effective procedure for weight loss, it has been less popular over last several years because of increased morbidity compared to the more utilized vertical sleeve gastrectomy (vsg). early complications of rygb include bleeding, perforation, or leakage. late complications include internal hernias, small bowel obstruction, anastomotic stenosis, marginal ulcers, and gastrogastric fistulas. case report: a -year old female with a past medical history of morbid obesity, diabetes mellitus type , hypertension, gerd, peptic ulcer disease, cholelithiasis, liver dysfunction with ascites, asthma, and a past surgical history of rygb ( years ago) presented to our institution with acute on chronic abdominal pain associated with nausea, vomiting, dysphagia, inability to eat and maintain hydration, and an additional weight loss of about lbs. over the last year. in addition, the patient was a chronic opioid and nsaid user, had an extensive smoking history, and had not followed with her surgeon for years. at the time of presentation, the patient weighed lbs (bmi: . ), had normal vital signs, and appeared cachectic. an upper gastrointestinal study followed by an upper endoscopic examination demonstrated complete obliteration of the gastrojejunal anastomosis and revealed a -cm long gastrogastric fistula originating from the distal end of the gastric pouch to the lesser curvature of the excluded stomach. after conservative measures were initiated to hydrate and metabolically stabilize the patient, the decision was made to proceed with diagnostic laparoscopy and surgical placement of a gastrostomy tube to the gastric remnant. the patient was discharged after tolerating a full liquid diet and gastrostomy tube feedings, for plan of future revision of gastrojejunostomy when optimal nutritional status is achieved. conclusions: late complications of rygb occur at a rate of - %. major risk factors for anastomotic complications include non-compliance, smoking, and opiate and nsaid abuse. though abdominal pain, anastomotic stenosis, marginal ulcers, and fistulas are relatively common late complications of rygb, complete obliteration of the gastrojejunal anastomosis has not been well described in the literature. this case demonstrates the importance of long term follow up post rygb for early diagnosis of late complications and brings attention to this rare, but possible sequele that can arise in patients after rygb. contrast radiograms and upper endoscopic photographs will be presented. introduction: retroperitoneal sarcoma represents approximately - % of all sarcomas and less than . % of all neoplasia. radiotherapy and chemotherapy still do not represent valid therapeutic alternatives; therefore complete surgical resection is the only potential curative treatment modality for retroperitoneal sarcomas. the ability of complete resection of a retroperitoneal sarcoma with tumor grading remains the most important predictor of local recurrence and disease-specific survival. in a patient with a large fibrosarcoma and associated hypoglycemia, assays for insulin-like activity (ila) were found to be high in the extract of tumor tissue, while insulin was not detected in significant concentration neither in the same extract nor in his serum. laparoscopic surgery represents an alternative technique for radical resection of such tumors as a minimally invasive rather than traditional surgery. only few cases were reported in the literature. introduction: roux-en-y gastric bypass (rygb) is a frequently performed bariatric procedure, of which internal hernia (ih) is a known complication. we discuss a rare finding of occult gastric remnant perforation as a result of an obstructed ih in a post bypass patient. methods: we present a case report of a single bariatric surgeon's experience at a tertiary care hospital. literature review of pubmed confirms the unique presentation and operative findings in our patient, as few similar cases have been published. a -year-old male s/p rygb years ago presented to the ed with right upper quadrant pain, nausea, vomiting, and a leukocytosis of , . bmi was . ; weight was lbs. workup included an abdominal ultrasound showing gallbladder distention without signs of cholecystitis. liver function tests were normal. further imaging included a ct scan, remarkable for a paraesophageal hernia (peh) containing the gastric pouch, and an elevated left hemidiaphragm. the scan showed no evidence of ih or bowel obstruction. an upper gi series was additionally obtained, which was also negative for small bowel obstruction. due to unclear etiology for this patient's symptoms or source of leukocytosis, diagnostic laparoscopy was planned. results: intraoperative findings were significant for ih containing dilated small bowel with twisted and incarcerated omentum through the jejunojenunostomy site, as well as a distended gallbladder without acute inflammation. ih was reduced and closed without bowel resection. cholecystectomy was completed. subsequent inspection of the diaphragmatic hiatus revealed uncomplicated herniation of the gastric pouch. in attempts to dissect the left diaphragmatic crus, a large pocket of purulent material was encountered below the left diaphragm in the region of the remnant stomach fundus. methylene blue test and intraoperative endoscopy did not demonstrate any connection to gastric pouch. the purulence was attributed to an occult remnant stomach perforation related to distal obstructed ih. a drain was left in the abscess and the peh was not surgically addressed. patient was discharged on postoperative day . he has not suffered any further complications or recurrent complaints. conclusion: gastric perforation following rygb is an uncommon complication resulting from ih. this diagnosis was missed by preoperative imaging and was only found after thorough laparoscopic investigation. surgeons should maintain a high clinical suspicion of ih in post rygb patients with otherwise unexplained abdominal symptoms, fever, and leukocytosis, even in the absence of confirmatory diagnostic testing. threshold for operative exploration in this clinical setting should remain low. alejandro garza, md, robert alleyn, md, jose almeda, md, ricardo martinez, md; utrgv obesity is an epidemic condition worldwide carrying significant morbidity and mortality. surgical therapy is the only proven effective method to sustain weight loss. among the different surgical procedures gastric bypass is the most effective. during this surgery, most of the stomach is excluded from the upper gastrointestinal tract which makes future evaluation of the same very challenging. this could potentially lead to delay in diagnosis of any pathology in the bypass stomach. gastric cancer is the th most common cause of cancer and cause of cancer death in the united states. we present a case report of a patient who underwent a roux-en-y gastric bypass and went on to developed adenocarcinoma in the gastric remnant year after her surgery. she underwent an exploratory laparotomy, extended antrectomy, subtotal gastrectomy including the gastro-colic ligament, and incidental appendectomy. pathology showed grade undifferentiated adenocarcinoma that penetrated the visceral peritoneum with clear margins. there was angiolymphatic invasion and perineural invasion along with metastatic carcinoma in out of lymph nodes. introduction: polyarteritis nodosa (pan) is a systemic transmural inflammatory vasculitis that affects medium-sized arteries. inflammation of the vessel wall and intimal proliferation creates luminal narrowing which can lead to stenosis and insufficiency. the same inflammatory process causes disruption of the elastic lamina leading to aneurysm formation and possible spontaneous rupture with life-threatening bleeding. multifocal segments of stenosis and aneurysm formation are characteristically identified as a "rosary sign" or "beads on a string". unlike other vasculitides, pan does not involve small arteries or veins, and is not associated with anti-neutrophil cytoplasmic antibodies. we present the case of a year old female with a significant intra-abdominal bleed that was explored and repaired primarily. she was subsequently found on angiogram and postmortem pathology to have findings consistent with pan. case presentation: year old female who presented to the emergency department with abdominal pain followed by hemorrhagic shock and found to have a ruptured left hepatic artery aneurysm during exploratory laparotomy. this aneurysm was suture ligated with a successful outcome. a mesenteric arteriogram was performed the following day and demonstrated lesions consistent with pan including aneurysms of the left gastric branches, right and left hepatic arteries, and beaded appearance of the iliac artery. however, days after hospital discharge she developed massive pulmonary embolism from which she did not recover. postmortem examination confirmed rupture of the left hepatic artery aneurysm in addition to gross anatomical and histological findings consistent with pan. discussion: polyarteritis nodosa is a systemic inflammatory vasculitis that causes intimal proliferation and elastic lamina disruption. this multifocal disruption of the vessel results in aneurysm formation alternating with stenosis creating a characteristic "rosary sign" on imaging. spontaneous rupture of these aneurysms is rare and almost always fatal due to life-threatening hemorrhage. with acutely ruptured aneurysms, prompt diagnosis, aggressive resuscitation, and hemostasis through transarterial embolization or surgery is paramount for patient survival. while acute rupture of an aneurysm as the result of pan is exceedingly rare, it must be considered as a differential diagnosis in the setting of acute abdominal pain and hemodynamic instability. in a patient known to have a medical history of pan and aneurysm formation, routine monitoring and disease progression should be followed. introduction: , surgeries are done annually in the us for small bowel obstruction, which is most commonly caused by intraabdominal adhesions, malignancy, and hernias. . to . % of small bowel obstructions are due to paraduodenal hernias. paraduodenal hernias carry a % lifetime risk of incarceration with a mortality of to %. case report: the patient is a year old male who presented with severe upper abdominal pain for one day. he was passing flatus and had had a bowel movement the previous day. on examination, the patient was tender over the upper abdomen. computed tomography (ct) scan with iv contrast showed a mesenteric swirl sign. the decision was made to perform diagnostic laparoscopy with possible small bowel resection. intraoperatively, a mesenteric defect was noted posterior and to the right of the duodenum, through which bowel was herniating. the herniated bowel and its mesentery were edematous. the defect was sutured closed, taking seromuscular and mesenteric bites through the stomach, jejunum, and mesentery. the patient had an uneventful recovery postoperatively and was discharged on postoperative day . he returned on postoperative day with periumbilical pain which resolved with conservative management. he was followed up weeks postoperatively and was doing well. discussion: paraduodenal hernias are the most common internal hernias. they are seen more often in males. they are caused by failure of the counterclockwise rotation of the prearterial segment of the embryonic midgut in weeks to of embryonic development. paraduodenal hernias usually present with chronic intermittent abdominal pain, weight loss, nausea, and vomiting. they may present acutely with symptoms of bowel obstruction. peritoneal signs are often not appreciated due to retroperitoneal position of the hernia. ct scan of the abdomen often shows clustering of bowel loops, which cannot be displaced on repositioning the patient. if imaging is equivocal, diagnostic laparoscopy may be undertaken. surgical correction consists of reducing the bowel, resecting nonviable segments, and either closing the defect or opening the sac laterally into the general peritoneal cavity. in summary, paraduodenal hernias are a rare cause of bowel obstruction and as such present a challenge in diagnosis and early intervention. diverticulosis of the appendix is a rare disease found in . - . % of appendectomies, first described in . the clinical presentation may be acute inflammatory with or without appendicitis or it may be an incidental finding in an uninflamed appendix. the congenital type is rare and it has all the bowel wall layers. it most frequently represents as pseudo diverticulum which lacks the muscularis layer. the pathogenesis of appendiceal diverticula is not completely elucidated. its symptoms are similar to and often misdiagnosed for that early acute or chronic appendicitis. while appendectomy is curative for both entities, it is important to distinguish diverticulum of the appendix from appendicitis as it is four times more likely to perforate and may be a sign of an underlying neoplasm. we reported a very rare giant pseudo diverticulum of the appendix in a -year-old male presenting with chronic abdominal discomfort for months. abdominal x-ray showed abnormal gaseous finding. physical exam was significant for a soft rubbery mass in the periumbilical region. blood work revealed slight elevation of c-reactive protein. preoperative ct and mri showed a -centimeter-large cavity composed of thin wall, located at the tip of the appendix with peri appendicular fat stranding. in the concern of pending obstructive symptom and chronic abdominal pain, we decided to perform the resection laparoscopic. the soft mass arose from the tip of the appendix. there were dense adhesions between the appendix, mesentery, and sigmoid colon. after adhesiohedlysis, laparoscopic appendectomy was performed with endogia. the specimen was extracted through a small incision without spillage. hospital course was uneventful and the patient was discharged on post-operative day . the pathological finding was consistent with a pseudo diverticulum of the appendix which lacked muscularis layer and the inner wall of the cavity was lined with a scattered cubital epithelial layer in the continuity with the appendiceal mucosal membrane. here we report a successful laparoscopic resection of an extremely rare giant chronic pseudo diverticulum of the appendix. yvette farran, ms, jorge a miranda, ms, benjamin clapp, md, elizabeth de la rosa, md; texas tech university health sciences center introduction: sigmoid colon intussusception is rarely encountered and given its vague symptomatology diagnosis and management can be difficult. the treatment of an intussusception in adults is different than in children. lipomas as the causative etiology for intussusception are encountered up to . % of the times and up to %- % of the patients require surgical resection for treatment. methods: this is a case report about a year old male that presented with two weeks of worsening abdominal pain and distention. physical exam was only pertinent for abdominal pain on light palpation, guarding and moderate distress. ct scan of abdomen and pelvis demonstrated a lipomatous mass causing complete obstruction of the sigmoid colon with intussusception. this was managed with laparoscopic sigmoidectomy. the patient had an uncomplicated post-operative period and was discharged on post-operative day . pathology of the lipomatous mass confirmed a benign lipoma. discussion: intussusception is rarely encountered in clinical practice in adults and constitutes % of all cases. lipoma induced sigmoid intussusception with complete obstruction is rare. symptoms can be non-specific as in this case. this case report highlights the importance of timely diagnosis and treatment of an intussusception in adult patients. ct scan is the gold standard for diagnosis and often shows a "target sign". other imaging techniques like ultrasound have shown adequate results but remain less effective than ct scan. the treatment in adults is not a reduction by enema like in pediatrics but rather resection of the lead point. this can be appropriately done with a laparoscopic technique in most cases. conclusion: colonic intussusception is rare. surgery is the only treatment for an intussusception in adults since the lead point needs to be removed, and can be attempted safely with a laparoscopic approach. surg endosc ( ) :s -s joshua smith, md, kern brittany, md, amie hop, md, amy banks-venegoni, md; spectrum health case report: year-old female with no significant past medical history presents with a -year history of nocturnal cough that had worsened over the past months and had associated regurgitation. she underwent esophagogastroduodenoscopy (egd) that showed a tortuous esophagus and tight lower esophageal sphincter that required dilation. she received an upper gastrointestinal (ugi) contrast study that showed a dilated, tortuous esophagus with 'bird's beak' tapering, consistent with achalasia, as well as a large epiphrenic diverticulum measuring cm. esophageal manometry confirmed "pan-esophageal pressurization" consistent with type ii achalasia. given her symptoms in the presence of these findings, she elected to proceed with surgery. she underwent laparoscopic, trans-hiatal epiphrenic diverticulectomy, heller myotomy and dorr fundoplication. extensive dissection allowed for approximately cm of retraction down from the chest and we were able to come across it with a single blue load of a mm linear cutting stapler. post-operatively, she tolerated the procedure well with immediate improvement in her symptoms. her ugi on post-operative day showed no evidence of leak, she tolerated a soft diet and was discharged home. she was seen at -week and -year follow-up appointments with complete resolution of symptoms. discussion: epiphrenic diverticula in the presence of achalasia has an occurrence rate of %. large diverticula ([ cm), are even more rare with only a handful of case reports in the literature. historically, thoracotomy or, more recently, thoracoscopic approaches are required for resection. however, thoracic approaches are associated with a % increase in morbidity, namely due to staple line leak and the resulting pulmonary complications. only a single case report exists on our review of the literature that demonstrates successful trans-hiatal laparoscopic resection without post-operative complications of a diverticulum of this size. the shortest documented length of hospital stay postoperatively for similar cases is days, while the average is - days or longer for those with complications. our patient was able to go home on post-operative day after a normal ugi and was tolerating a soft diet. not only does this case show that a large epiphrenic diverticulm can be successfully resected via the trans-abdominal laparoscopic approach, this case makes the argument that patients undergoing any minimally-invasive epiphrenic diverticulectomy and myotomy, with or without fundoplication, may be successfully managed with early post-operative contrast studies and dietary advancement, thus decreasing their length of hospitalization and overall cost of treatment. kazuma sato, shunji kinuta, koichi takiguchi, naoyuki hanari, naoki koshiishi; takeda general hospital background: situs inversus totalis (sit) is a rare congenital condition in which the abdominal and thoracic organs are located opposite to their normal positions. few cases of laparoscopic surgery for gastric cancer with sit have been reported. we report a case of laparoscopic distal gastrectomy with d lymph node dissection performed for gastric cancer in a patient with sit. case description: an -year-old woman was admitted to our hospital for treatment of gastric cancer that was diagnosed by esophagogastroduodenoscopy (egd) at a local clinic after she experienced anemia and nausea. egd identified an irregularly shaped gastric ulcer located at the anterior side of the lesser curvature of the antrum. a biopsy revealed a moderately differentiated adenocarcinoma. she was then diagnosed with sit by chest radiography and abdominal computed tomography (ct). the abdominal ct showed that all organs were inversely positioned and that the wall of the antrum had thickened; it also showed the lymph nodes in the lesser curvature of the stomach, without distant metastasis or an abnormal course of vascularity. the patient was clinically diagnosed with t n m stage iiia gastric cancer according to the japanese classification of gastric carcinoma. a laparoscopic distal gastrectomy with d lymph node dissection in accordance with the japanese gastric cancer treatment guidelines as well as a roux-en-y anastomosis due to an esophageal hiatal hernia were performed. the surgery was safely and successfully performed, although it required more time than usual because the inverted anatomic structures were repeatedly examined during the surgery. the postoperative course was positive, and the patient was discharged on postoperative day without any complications. the final stage of this case was pt bn m stage ia. currently, the patient is doing well without recurrent gastric cancer. conclusion: gastric cancer with sit is an extremely rare occurrence. we experienced a case of laparoscopic distal gastrectomy with d lymph node dissection performed for gastric cancer in a patient with sit. we simulated the operation for sit by viewing left-right reversed ordinary surgical videos. the abdominal ct angiography with a three-dimensional reconstruction helped reveal any variation and confirmed the structures and locations of vessels before the surgery. the operation could safely be performed following the standardized surgical technique by reversing the surgeon standing position and trocar position. sternum or chest wall resection is performed for a variety of conditions such as primary and secondary tumors of the chest wall or the sternum. sternum reconstruction has been a complex problem in the past due to intraoperative technical difficulties, surgical complications, and respiratory failure caused by the chest wall instability and paradoxical respiratory movements. advances in the fields of surgery and anesthesia result in more aggressive resections. nowadays neither the size nor the position of the chest wall defect limits surgical management, because resection and reconstruction are performed in a single operation that provides immediate chest wall stability. chest wall resection involves resection of the ribs, sternum, costal cartilages and the accompanying soft tissues and the reconstruction strategy depends on the site and extent of the resected chest wall defect. here i'll present, the youngest ever case reported, years old girl with rhabdomyosarcoma involving the sternum. i will present the management challenges and the reconstruction options. introduction: neuroendrocrine malignancies constitute . % of all cancers. the gastrointestinal tract is the commonest site, followed by the lung. the last decade has seen a steady increase in their incidence. this is a case series of twenty five such tumours and their clinicopathological characteristics. materials and methods: twenty five patients with neuroendocrine tumours of the gastrointestinal tract were studied with reference to their demographic and clinicopathological characteristics. apart from routine pathological examination, these tumours were also checked for e cadherin expression as an independent marker of aggressive disease. results: the age of our patients ranged from to years. we had female and male patients, contradicting a female preponderance in literature. the vast majority of the tumours we encountered were from the stomach and duodenum, with and patients, respectively. two tumours were at the gastroduodenal junction, two from the appendix, small intestine and pancreas, each, and one each from the rectum and gall bladder. this is in contrast to literature that shows that neuroendocrine tumours of the git most commonly arise from the appendix and small bowel, followed by the rectum, stomach and duodenum. two of these tumours were functional. the diagnosis was confirmed by immunohistochemistry staining for chromogranin a and synaptophysin. grading was done using who criteria that takes into account the mitotic count, ki index and necrosis. of our cases were grade i. further, immunohistochemistry for e cadherin showed that absence of expression correlated with more aggressive clinical behavior. out of twenty five patients were operable at presentation and standard resections depending on the organ of origin with adjuvant therapies were given as required. could only be given palliative care. the functional tumours were treated with radiolabelled somatostatin analogues following uptake studies. conclusion: as neuroendocrine tumours are relatively rare, information about them is not as abundant as with other malignancies. absence of e cadherin expression is associated with more aggressive disease. more studies are required that document the pathological characteristics and clinical behavior in order to offer well rounded treatment protocols that treat not only the primary, but also the generalized effects of the secretions produced by them. targeted chemotherapy is gaining prominence, but more specific drugs directed at the plethora of receptors these tumours express, could potentially revolutionize treatment. ( ) . unfortunately there are no publications from denmark. we would like to present first to our knowledge reported case of double gallbladder in denmark. double gallbladder is a rare anomaly with a prevalence of : in autopsy studies, described first by boyden in ( ) . there are several classifications of double gallbladder that are based on relation between gallbladder, cystic duct and common bile duct ( , ) . non-specific symptoms and inadequate imaging are possible causes of lack of awareness of the condition. removal of all gallbladders, preferably laparoscopic with special attention to the biliary anatomy, is recommended ( ). method: case report with review of the literature. a -year-old female patient of polish origin was hospitalized due to upper right quadrant pain. on admission clinical manifestations and paraclinical abnormalities of pancreatitis were present. ultrasound scanning of the abdomen showed bile stones, ultrasonic manifestations of acute cholecystitis and normal intra-and extrahepatic bile ducts. because of elevated liver enzymes mrcp was performed and showed double gallbladder, double cystic duct and signs of pancreas anulare. scheduled ercp confirmed bile stones in cbd, double gallbladder with double cystic duct, h-type according to harlaftis classification ( ) . because of minor retroperitoneal perforation second ercp was needed for removal of all stones. the patient was then scheduled to laparoscopic cholecystectomy with perioperativ cholangiography. conclusion: anatomical variations of the gallbladder such as double gallbladder are rare and often remain unnoticed. they are most often identified because of clinical manifestations symptoms, diverse imaging studies, during surgery or autopsy. as most of them are not expected, they can contribute to complications during surgery. careful preoperative imaging is very important to prevent accidental bile duct injury. looking at the number of case reports, double gallbladder seems to be slightly more common than expected. the interesting question is whether a gallbladder discovered during an unrelated radiological investigation in a patient that previously underwent a cholecystectomy can represent undetected case of double gallbladder. we would like to present a review of the literature as well as images from mrcp, ercp and laparoscopy. michael jaroncyzk, md, courtney e collins, md, ms, vladimir p daoud, md, ms, ibrahim daoud, md; st. francis hospital; hartford ct introduction: several decades ago, surgical training was saturated with procedures to treated peptic ulcer disease. since the introduction of histamine- blockers and proton pump inhibitors, these procedures have dwindled significantly. however, there are still instances where patients require surgical intervention for peptic ulcer disease. perforation is one of the indications for surgery. the surgical options to treat a perforated peptic ulcer are numerous. one of the most common options is a graham patch. we are presenting a case of a patient with a perforated ulcer that did not have available omentum for the repair. methods and procedures: recently, a -year-old female with a past history of an open total abdominal hysterectomy and bilateral salpingo-oophorectomy presented as an outpatient with chronic lower abdominal pain. she underwent a work-up and imaging that did not reveal any pathology. at diagnostic laparoscopy, she had diffuse lower abdominal adhesions, which were lysed. she was discharged on the same day, but presented to the emergency department two days later with severe abdominal pain and fevers. the work-up revealed tachycardia, diffuse abdominal tenderness with peritoneal signs, leukocytosis and a large amount of free air on imaging. she was emergently brought to the operating room for a diagnostic laparoscopy. during laparoscopic exploration, the lower abdominal cavity appeared normal for a recent lysis of adhesions. attention was turned to the upper cavity to find the pathology. bile-stained free fluid and peri-gastric exudates were identified, but no perforation was visualized. intra-operative endoscopy revealed the site of perforation in the antrum on the lesser curvature. a biopsy was performed and the decision was made to perform a graham patch. however, the omentum was already densely involved with the lower abdominal cavity from the enterolysis. due to the close proximity of the falciform ligament, it was mobilized laparoscopically and the pedicle was used as a graham patch. the patient recovered without any additional issues. the biopsy was reported as a chronic gastric ulcer. conclusion: surgical history has given us many options to treat peptic ulcer disease that are not nearly as common as they were decades ago. perforated ulcers can be managed laparoscopically and graham patches are a common choice for repair. however, the lack of the omentum for a proper pedicle flap can pose a problem in some patients. we have shown in this patient that a falciform pedicle flap can be successfully used as a substitution. laparoscopic management of boerhaave's syndrome after a late presentation: a case report and literature review tahir yunus, hager aref, obadah alhallaq; imc background: boerhaave's syndrome involves an abrupt elevation in the intraluminal pressure of the oesophagus, causing a transmural perforation. it is associated with high morbidity and mortality. having a nonspecific presentation may contribute to a delay in diagnosis and results in poor outcomes. treatment is challenging, yet early surgical intervention is the most important prognostic factor. case presentation: we present a case of a thirty-two-year-old male with a long medical history of dysphagia due to benign oesophagal stricture. he presented with acute onset of epigastric pain after severe emesis. based on computed tomography scan, he was diagnosed with boerhaave's syndrome. presenting with signs of shock, mandated immediate surgical exploration. for which he was taken for laparoscopic primary repair with uneventful postoperative recovery. the golden period of the first hours of insult still applies for cases of oesophagal perforation. the rarity of these cases makes a comparison between the various treatment methods difficult. our data support that the use of laparoscopic operative intervention with primary repair as the mainstay of treatment for the management of oesophageal perforation. lipomas of the gastrointestinal tract are rare benign soft tissue tumors that are often discovered incidentally. these lesions are often asymptomatic, but have occasionally been reported to have clinical significance as will be described in this case report. a year old male initially presented to his primary care physician's office with a three week history of vague intermittent abdominal pain. his pain was located in the mid epigastrium and was associated with mild nausea. past medical history was significant for hyperlipidemia and a right-sided goiter, and he denied any previous surgeries. outpatient work up revealed a microcytic anemia, intermittent melena and hemoccult positive stools. the patient was referred to hematology and gastroenterology. endoscopies revealed gastritis, and small internal and external hemorrhoids. he underwent an outpatient ct scan which demonstrated a . . cm mass within the lumen of the jejunum causing long segment non-obstucting intussusception. subsequently, the patient was referred to surgery and underwent a diagnostic laparoscopy. at the time of surgery, an approximately twelve centimeter segment of proximal jejunum was identified intussuscepting into a distal limb. this segment was attempted to be reduced laparoscopically, however there was significant mesentery within in the intussusceptum and the segment could not be safely reduced. therefore, the section of bowel was delivered through a small periumbilical incision. the intussusceptum was then able to be manually reduced from the intussusception. at this point a large mass was palpated inside the lumen of the jejunum. a small bowel side to side, functional end to end resection and anastomosis was preformed. the bowel was returned to the abdomen and the abdomen was re-insufflated. the remainder of the small bowel was run and no additional lesions were identified. final pathology revealed a . . . cm submucosal partially obstructing lipoma with ulceration at the tip. the patient recovered uneventfully and was discharged home on the second post operative day. this case report describes a submucosal jejunal lipoma that was acting as a lead point for intermittent non-obstructing small bowel intussusception, while simultaneously causing a microcytic anemia due to ulceration at the tip of the lipoma. laparoscopic assisted reduction and small bowel resection is a safe and effective treatment for gastrointestinal tract lipomas that are unable to removed endoscopically. percutaneous endoscopic gastrostomy (peg) is an alternative to laparotomy for open gastrostomy tube placement to provide enteral nutrition for those who are unable to pass nutrition orally. despite being less invasive, the procedure is not without its complications, one of which includes the formation of a gastrocolocutaneous fistula. the case describes a year old female who presented with a peg placed months prior with reports of leakage of tube feeds from the gastrostomy site. as there was concern for possible ileus or obstruction, an upper gi series was completed which seemed to indicate dislodgement of the g-tube. the g-tube was replaced and a follow-up gastrograffin study was repeated which now indicated that the g-tube was within the lumen of the colon. soon thereafter fecal matter was noted to be draining around the g-tube site; however, patient was without clinical signs of peritonitis. the patient was managed non-surgically as she was a poor surgical candidate with multiple prohibitive co-morbidities. the g-tube was removed bedside by cutting it flush at the skin level with the anticipation that the remainder of the tube would be excreted with bowel movements. the decision was then made to attempt closure of the gastric fistula endoscopically which was accomplished with hemoclips. a follow up upper gi study hours later showed no extravasation of contrast through the gastric fistula. the colocutaneous fistula had self-resolved over the next couple days as well. placement of the peg tube through the transverse colon can present with varying ill effects including diarrhea, pneumoperitoneum, peritonitis, gram negative pulmonary infection or feculent vomiting with the formation of a gastrocutaneous fistula. treatment historically for a gastrocolocutaneous fistula has been exploration and excision of the fistula tract with resection of the involved colonic segment. however, there currently is no gold standard for the management of, and really ranges from conservative management to surgical and is dependent on the presenting symptoms. if the peg becomes dislodged with resultant spillage from the colon with resultant peritonitis, surgical exploration is needed with removal of the g-tube and repair of the stomach and colon. on the other hand, non-surgical management has been suggested in management of a well-established fistula. fistula closure may be spontaneous; however, can be inhibited due to delayed gastric emptying or leakage of gastric secretions through the fistula. endoscopic clipping of the fistula tract employing the hemoclips is a treatment option. median arcuate ligament syndrome (mals) is a rare etiology of abdominal pain caused by narrowing of the celiac artery at its origin by the median arcuate ligament with relative hypoperfusion downstream. patients suffer from post-prandial abdominal pain, abdominal pain associated with exercise, nausea, and unintentional weight loss. diagnosis is historically made by demonstrating elevated celiac artery velocities and respiratory variation on dynamic vascular studies. standard of care for mals patients is laparoscopic celiac artery dissection with release of the median arcuate ligament. at our institution, we have encountered fourteen patients (eleven female, three male) diagnosed by elevated peak velocity in the celiac artery by duplex ultrasound in conjunction with ct angiogram, mr angiogram, arteriogram, or multiple modalities. all but one patient had multiple diagnostic imaging modalities, with the most common being ct angiogram; eight patients had invasive imaging. the mean age at presentation was . years in men and . years in women. on average, male patients presented with a longer duration of symptoms, . years (range - years), as compared to women, . years (range - years). symptoms were fairly consistent between genders and included nausea, emesis, abnormal bowel habits, early satiety, post-prandial pain, and weight loss. all male patients reported at least two symptoms, most commonly nausea and post-prandial pain. in female patients, % reported having three or more symptoms. notably, post-prandial pain was universal among men and women, while weight loss was exclusive to female patients as reported by %. pre-operative peak velocities were recorded in all but one patient, with mean values more elevated in female patients as opposed to male patients, cm/s versus cm/s. post-operative duplexes were obtained in seven patients; pooled data show a mean change of negative cm/s for an average of cm/s after decompression. in all cases, the celiac artery trifurcation was visualized and noted to have a distinct change in artery caliber after division of the ligament. in total, % of patients reported significant improvement with return to normal diet and healthy weight gain post-operatively. of the three without complete resolution, two were diagnosed with motility disorders and one was lost to follow-up. our experience demonstrates that laparoscopic release of the median arcuate ligament in patients with significant flow limitation of the celiac artery on dynamic and anatomic imaging can be a successful treatment option for patients with recalcitrant pain and gastrointestinal dysfunction with no alternative diagnosis. matthew a goldstein, ma, kirill zakharov, do, sharique nazir, md; nyu langone brooklyn adhesions are fibrotic bands that form between and among abdominal organs. the most common cause of abdominal adhesions is previous surgery in the area as well as radiation, infection and frequently occurring with unknown etiology. these bands occur among abdominal organs, commonly the small bowel, and can lead to obstruction or remain asymptomatic, akin to the patient discussed here. congenital abdominal adhesions are rare and have received little attention in research and field of study. the patient described in this case is a -year-old female with a past medical history of morbid obesity, bmi of , hypertension and no past abdominal surgical procedures. the patient presented in august for bariatric surgical consultation and was ultimately taken for an attempted laparoscopic sleeve gastrectomy. upon entering the abdomen, significant adhesions were encountered and an additional attending was called to assist in identifying the stomach. the splenic flexure was found to be plastered to the diaphragm and the descending and transverse colon were adhered to the anterior surface of the stomach. additionally, small bowel adhesions encased the area between the right and left hepatic lobes as well as the caudate lobe. after extensive enterolysis, the pylorus remained the only identifiable portion of the stomach. the patient also demonstrated significant hepatomegaly and a wedge resection was performed. the amount of adhesion and matting of the small and large bowel obscured the view of the stomach and the procedure was deemed too dangerous and terminated. this case represents the uncommon scenario in which an abdomen with no prior surgical history presents with extensive, obscuring adhesions. one such recent study describes the influence of cytokines and proinflammatory states as contributors to obstruction and malrotation in children, but this patient demonstrated no significant history. further investigation is needed to determine potential etiologies of symptomatic and non-symptomatic congenital adhesions among bariatric patients who fail conservative treatment. today the patient is doing well and the surgical team will attempt to complete the procedure in the coming months. laparoscopic spenulectomy: an interesting case report riva das, md , daniel a ringold, md , thai q vu, md ; orlando health, abington jefferson health introduction: spenules, or accessory spleens, are a rare disease entity. most often, they are asymptomatic, and found incidentally during radiographic workup for an unrelated problem. torsion can cause a splenule to not only become symptomatic, but also confound the results of usual diagnostic studies. case description: a -year-old female patient with history of uncomplicated hypertension, hyperlipidemia, hysterectomy, cholecystectomy, spinal surgery, and partial left nephrectomy, presented to the hospital with a two-week history of intermittent left upper quadrant abdominal pain. she denied any similar episodes in the past, or any associated symptoms. further investigation with a ct scan of the abdomen and pelvis showed an acute inflammatory process in the left upper quadrant in same location as some colonic diverticulosis, as well as a . cm soft tissue mass. this indeterminate soft tissue mass was described as having decreased attenuation compared with the spleen. differential diagnosis for this mass included malignancy, an atypical splenule, or an infectious/inflammatory mass. an mri was recommended for further evaluation, but did not reveal any additional significant findings. nuclear medicine liver/spleen scintigraphy was performed, which showed no focal activity associated with the indeterminate left upper quadrant mass, therefore making it unlikely to reflect a splenule, and making malignancy the diagnosis of exclusion. following a period of observation with analgesia, intravenous antibiotics, and bowel rest, her abdominal pain did not resolve, and the decision was made to proceed with operative exploration. diagnostic laparoscopy revealed an approximately cm spherical mass in the left upper quadrant located just below the inferior aspect of the spleen. the superior aspect of the mass gave rise to a vascular pedicle, which upon tracing, seemed to originate from the splenic hilum. this pedicle was easily ligated, and the mass removed. pathology revealed an extensive infarcted hemorrhagic nodule with organizing thrombus and attached thrombosed artery, consistent with an infarcted splenule due to torsion along its own axis. the patient had an uncomplicated postoperative course. discussion: this case report demonstrates the unusual presentation and workup of a patient that was ultimately diagnosed with an infarcted splenule, despite imaging findings that did not correlate, and may even have confused her diagnosis. scintigraphy, which is normally the gold standard for diagnosing and localizing accessory splenic tissue, was in this case unrevealing, due to inability of the tracer to traverse the torsed vascular pedicle. operative exploration was both diagnostic and therapeutic. patients which was treated with antibiotics suggested by culture and sensitivity report and local wound care. one patient died due to sepsis at presentation. conclusion: chikungunya virus was found circulating in rodents in pakistan as early as . duodenal ulcer perforation which is a common surgical emergency in our part of the world usually presents with pinpoint perforation in ant wall of first part of duodenum unlike in already diagnosed cases of chikungunya disease where a slit like duodenal perforation is noted in the anterior wall of first part of duodenum. literature and consensus relate this perforation with the excessive use of nsaids due to usual presentation of arthritis in chikungunya disease but the unusual presentation is still to be answered. introduction: bouveret's syndrome is a rare form of gallstone ileus in which an impaction of a gallstone in the duodenum results in a gastric outlet obstruction. gallstone ileus accounts for approximately - % of all cases of small bowel obstruction. the terminal ileum is the most common location for a calculus to cause obstruction followed by the proximal ileum, jejunum and duodenum/stomach respectively. open and laparoscopic surgery has previously been the mainstay of treatment for bouveret's syndrome, however with the advent of new endoscopic techniques and instruments there has been increasing success in endoscopic management. this case report looks at a patient with a gastric outlet obstruction from a gallstone, and discusses the current literature regarding diagnosis and management. case: year old male presented with several day history of epigastric abdominal pain and multiple episodes of nonbloody, nonbilious emesis. he had previously been diagnosed with cholelithiasis, however had refused surgery at that time. on admission the patient was found to have a leukocytosis of . . an ultrasound was performed in which the images were limited due to pneumobilia. a subsequent ct scan revealed pneumobilia, and a large cm gallstone impacted in the first portion of the duodenum causing a gastric outlet obstruction. the patient underwent failed endoscopic attempts at removal and ultimately required a laparotomy, enerotomy with stone extraction. discussion: bouveret's syndrome is a rare variant of gallstone ileus. with newer endoscopic techniques and electrohydraulic lithotripsy, there has been increasing success with endoscopic retrieval of the impacted gallstones. there is some controversy in regards to the need for definitive operative management. stone extraction, without cholecystectomy and fistula repair, has been shown to have less postoperative complications as well as lower mortality rates compared to when a cholecystectomy and fistula repair has been performed. total mesorectal excision (tme) with neoadjuvant chemoradiotherapy (nacrt) is standard treatment for rectal cancer, which has resulted in a decrease in local recurrence. however, nacrt has shown no significant overall survival and some adverse effects mainly caused by radiation therapy. recently, the usefulness of neoadjuvant chemotherapy (nac) has been reported. we retrospectively assessed the efficacy and safety of the neoadjuvant mfolfoxiri compared with nacrt followed by laparoscopic surgery. a total of patients undergoing laparoscopic surgery for lower rectal cancer (clinical stage: ii or iii) from july to february in our department were retrospectively evaluated. patients underwent nac, and patients underwent nacrt. the following data were collected: pathological complete response (pcr), histological grade, down staging, radial margin (rm) and postoperative complications. histological grade was defined as follows: tumor cell necrosis or degeneration is present in less than one third of the tumor area (grade a), between one and two thirds (grade b), more than two thirds but viable cells remain (grade ), and complete response (grade ). these two groups were demographically comparable. down staging did not differ between the two groups. histological grade (?grade b) and pcr were significantly higher in the nacrt than in the nac group (p. ). rm had no significant difference in both groups, but tended to be able to secure negative rm in the nac group ( % vs. . %, p= . aims: increasing evidence suggest that cme may improve overall and disease free survival in colon cancer. our aims were to investigate the safety and efficacy of single incision laparoscopic cme colectomy (silcc) compared to multiport cme laparoscopic colectomy (mpclc) providing the first meta-analytical evidence. methods: pubmed, scopus and cochrane library were searched. studies comparing the silcc to mpclc in adults with colon adenocarcinoma were included. the studies were critically appraised using the newcastle ottawa scale. statistical heterogeneity was assessed with x and i . the symmetry of funnel plots was examined for publication bias. results: one randomized and four case control trials were included ( silcc vs sl introduction: obesity has been associated with increased morbidity following total proctocolectomies with ilealpouch anal anastomosis (tpc-ipaa). however, the incremental added risk of increasing obesity class is not known. the aim of this study was to evaluate the additional morbidity of increasing obesity class for tpc-ipaa. methods: after ethics board approval, the acs-nsqip database ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) was accessed to identify patients who underwent elective tpc-ipaa. body mass index (bmi, kg/m ) was classified as normal ( . - . ) , overweight ( . - . ), obesity class-i ( - . ), obesity class-ii ( - . ) and obesity class-iii (≥ ). primary outcomes were overall surgical site infection (ssi) and organ-space infection (osi). secondary outcomes were -day major morbidity and length of hospital stay (los aim: in curatively intended resection of sigmoid and rectal cancer, many surgeons prefer to perform ligation of the root of the inferior mesenteric artery (ima), high tie, because of oncological reasons. however, ligation of the ima has been known to decrease blood flow to the anastomosis. there are few reports of patients undergoing the reduced port laparoscopic approach (rps) including single-incision laparoscopic approach (sils) even among those undergoing laparoscopic lymph node dissection around the ima with preservation of the left colic artery (lca). our objective was to evaluate the quality of this procedure regarding application of rps for the treatment of sigmoid and rectal cancer. methods: the feasibility of this procedure was evaluated in consecutive cases of rps for sigmoid and rectal cancer. a lap protector (lp) was inserted through a . cm transumbilical incision, and an ez-access was mounted to lp and three -mm ports were placed. almost all procedures were performed with standard laparoscopic instruments using a flexible scope (sils). a mm port was inserted in right lower quadrant mainly in rectal cancer surgery (sils + ). our method involves peeling off the vascular sheath from the ima and dissection of the ln around the ima together with the sheath. results: lymph nodes around the ima were dissected with preservation of the lca in cases (group a). the ima was ligated at its root in cases (high tie, group b). in group a, patients were treated with sils and patients were treated with sils+ . in group b, patients were treated with sils and patients were treated with sils+ . median operative time was . , and . min for group a, and b, respectively. the operative time was significantly longer in group a. estimated blood loss was . and . g, and mean numbers of harvested ln were . , and . . none of the other operative results of groups a and b were different statistically. in this series, there was only one anastomotic leakage in group b. conclusion: our method allows equivalent laparoscopic lymph node dissection to the high tie technique. the operative time tends to be longer, however this procedure has a possibility to reduce an anastomotic leakage. introduction: the routine mobilization of the left colonic flexure in colorectal surgery is still a matter of debate. we present our surgical approach with data. this technique may increases the surgical expertise/confidence when the surgical maneuver is necessary. up to % of all splenectomies are for surgery-related injuries; % of those splenic injuries are treated by splenectomy. the iatrogenic splenic injury rate during colorectal surgery is . %. iatrogenic splenic injuries create: increased risk of mortality/morbidity, extended operative time/patient in-hospital stay and increased healthcare costs. risk factors for iatrogenic splenic injury are: advanced age, adhesions, underlying pathology. obesity is not a risk factor. it is debated if the left colonic flexure mobilization is a risk factor for splenic injury. the ligament over-traction is the most frequent damage mechanism. the most dangerous surgical manuever is the spleno-colic ligament surgical dissection. moreover, laparoscopy descreases by almost , times the splenic injury risk. some surgeons are reluctant to routinely take down the splenic flexure. materials and procedures: robotic left colonic/rectal cases with routine splenic flexure mobilization technique have been performed: left colectomy (n= ), rectal surgery (n= ), transverse-colectomy (n= ) and pancolectomy (n = ). conversion rate , %, ebl\ ml, postop-leak ( . %) and % iatrogenic splenic injuries. results: in our approach, there are pathways that need to be mastered for the splenic flexure mobilization:a) medial to lateral dissection (underneath the inferior mesenteric vein); b) lateral to medial (from the lateral peritoneal reflection); c) access to the lesser sac with omental detachment from the transverse colon; d) access to the lesser sac with the gastrocolic opening, following the inferior border of the pancreas. the dissection should be closer to the colon rather than to the spleen. in our experience the routine mobilization of the splenic flexure may have some advantages: a) better (without tension) distal anastomosis formation; b) better perfusion of the proxiaml stump; c) wider oncological dissection; d) no need of going back to the flexure when the proximal stump is too short; e) mastering a surgical manuver useful in other procedures (e.g. distal pancreasectomy). the theoretical drawbacks of routine splenic flexure mobilization can be:a) longer operative time, which is on average increased by minutes; b) risk of splenic injuries, in our experience, no splenic injuries have been registered. conclusions: technical accuracy with cautious dissection/visualization can reduce iatrogenic splenic damages rate. laparoscopy decreases splenic injury rate. robotic surgery may have the potential to further reduce this complications. our data suggest that the routine mobilization of the splenic flexure, has more advantages than drawbacks and it can reduce the iatrogenic splenic injury rate. more trials are needed in order confirm our findings. introduction: the robotic stapler with the endowrist™ technology (intuitive surgical, inc.) includes a larger range of motion and articulation compared to the laparoscopic device, and may provide some benefits in difficult areas like the pelvis. to date, few studies have been published on the application of robotic endowristed stapling. we present our preliminary experience using the robotic stapler in low anterior rectal resection (larr) with total mesorectal excision (tme) for rectal cancer. methods and procedures: between march and september , patients underwent elective robotic larr with tme and primary colorectal anastomosis within the eras program. patient demographic, intra-operative data and post-operative outcomes were compared between the endowrist™ robotic stapler group (rs group) and the laparoscopic stapler group (ls group). results: the two groups were homogeneous in terms of demographic and clinical characteristics. thirteen ( males) and patients ( males) were included in rs and in ls group, respectively. seven patients received preoperative chemoradiation in rs group, in ls group. there was no difference in intra-operative blood loss and total operative time. the median number of stapler fires for patients in rs group and in ls group was (range, - ) and (range, - ), respectively. loop-ileostomy was fashioned in patients in rs group ( . %) and patients in ls group ( . %). the days mortality was nil. two cases of anastomotic leaks have been detected in rs group ( . %), cases ( . %), occurred in ls group, all treated conservatively. the mean length of postoperative stay was . ± . days in rs group, . ± . days in ls group. conclusions: in our preliminary experience the application of robotic stapler during larr with tme has shown to be safe and feasible with acceptable morbidity. even if our case series is pretty small, fewer stapler fires were required in the rsg compared to lsg. we believe that the robotic stapler might lead to a more precise firing during pelvic surgery: it can explain the trend toward a decreased number of fires, that has been well documented in literature to be related to a lower risk of anastomotic leak. further high quality studies are required to confirm these findings. background and objectives: the present study was aimed at investigating the safety and feasibility of laparoscopic ultra-low anterior resection (l-ular) with total mesorectal excision (tme) and transanal specimen extraction for rectal cancer located at lower one-third rectum, and specifically understanding the oncological outcome of the operation. patients and method: a prospective designed database of a consecutive series of patients undergoing laparoscopic ultra-low anterior resection for rectal malignancy with various tumornode-metastasis (tnm) classifications from to at the texas endosurgery institute was analyzed. in this study ultra-low anterior resection is defined as low anterior resection for the malignant lesion at distal / of rectum. results: ultralow anterior resections were completed laparoscopically with tme and transanal specimen extraction. the operating time for the surgery was . ± . minutes, and estimated blood loss during the procedure was . ± . ml. the length of the lesion from the anal verge measured with intraoperative colonoscopy ranged from . cm to . cm, and shortest distance of colorectal anastomosis from the anal verge is cm. since diverting ileostomy was routinely installed after l-ular, none was found to have anastomotic leakage, however patients developed anal stenosis within -month follow-up. therefore the overall rate of postoperative complication is . %. moreover patients were reported to have local recurrence in -year followup with the rate of . %. conclusions: l-ular is safe and effective procedure for the rectal cancer at distal / rectum with comparable local recurrence and postoperative complication rates, thereby suggesting l-ular can be considered as a procedure of choice for rectal cancer at very low location in the rectum. for rectal cancer, however, local full-thickness excisions are fraught with high local recurrence rates -even if limited to early and best selected lesions. this corroborated observation is likely caused by a combination of missed nodal disease and direct implantation of tumor cells into the mesorectum, which upstages even early t lesions to at least a t lesion. the treatment of choice for invasive adenocarcinoma consists of an oncological total mesorectal resection, possibly with other modalities. rectal tumors of uncertain behavior can present a treatment dilemma between over-treatment vs under-treatment. concept: if the nature of a lesion is not certain or if contradictory results have been obtained, we propose a superficial local excision as a mucosal excisional biopsy to establish the diagnosis while avoiding interference with subsequent definitive treatment modalities by preserving the integrity of the external rectal wall and mesorectum. a benign final pathology concludes the treatment, whereas a detection of invasive cancer will be managed with a subsequent oncological resection. methods: this is a case report of a -year-old woman found to have a . cm villous lesion in the mid to distal rectum without proven or disproven invasive cancer. a tems-guided mucosal resection of the rectal mass at cm above the anal verge was performed whereby the lesion was dissected off the underlying muscularis. results: with preoperative discrepant erus and mri staging ut - vs ct lesion, a technically successful mucosal resection of the large rectal mass was carried out. pathology revealed a tubulovillous adenoma without high grade dysplasia or malignancy and a complete resection. conclusion: tems mucosal excisional biopsy of rectal tumors of uncertain behavior allows for a less invasive diagnostic approach that may (a) be definitive treatment if the lesion is proven benign, or (b) confirm the need for more aggressive treatment without having burned any treatment bridges or upstaged an early tumor by violating the mesorectal plane. an oncologic resection with appropriate (neo-)adjuvant chemotherapy can be carried out while preventing the potential for tumor seeding at initial operation. background: adequate visualization of the entire lumen of the large bowel is essential in detecting pathology and establishing diagnoses during colonoscopies. patients are provided dietary instructions and medications in order to achieve adequate bowel preparation. given the extensive amount of preparation required, some patients may be unable to adhere to the prescribed routine, resulting in rescheduling or repeat procedures and misallocation of limited resources. a number of previous quality-improvement efforts have been implemented to ensure adequate preparation prior to colonoscopy. objective: the objective of this study was to develop and assess the feasibility of a novel smart phone application in the delivery of bowel preparation instructions. methods: a novel smart phone application was developed to deliver bowel preparation instructions to patients undergoing colonoscopy for the first time. patients were included in the pilot phase of this project if they were undergoing a colonoscopy for the first time. we included patients who had access to a smart phone, had not previously had a bowel preparation for any reason. we excluded patients with a previous diagnosis of inflammatory bowel disease or colorectal cancer. patient surveys were administered at the time of colonoscopy. patients were questioned regarding the completeness of bowel preparation and adherence to bowel preparation instructions. patient questionnaires were completed to ascertain the ease of use of the smart phone application and any concerns that arose. quality of bowel preparation was assessed by the colonoscopist using the validated ottawa bowel preparation score. this is the pilot study results for the "coloprep" trial (nct ). results: a total of patients were enrolled in the pilot phase of this study. patient satisfaction, adherence to instructions and ease of use of the smart phone application were ascertained. bowel preparation, as assessed by the colonoscopist, was reported. conclusions: this study assessed the feasibility of using a novel smart phone application for delivery of bowel preparation instruction. this pilot study is the initial phase of a randomized controlled trial to compare smart phone application vs. written instructions in the delivery of bowel preparation instructions. the . median follow-up was months. there were no statistically significant differences found in clinical features and laboratory findings between the two groups. no statistically significant difference was found regarding the overall success rates and the complication rates between the conservative and the surgical arms (success rates: . % and . % (p= . ) and complication rates: . % and . % (p= . ), respectively). however, surgical treatment was better than conservative treatment in preventing recurrent diverticulitis (recurrence rates: % and . % (p= . ), respectively). conclusion: conservative management with bowel rest and antibiotics is a safe and effective treatment for right-sided colonic uncomplicated diverticulitis and may be considered as the initial option. on the other hand, laparoscopic diverticulectomy is also safe, effective and adequate. surgery is advocated to decrease the recurrence rate. introduction: it has been hypothesized that the structural and functional changes that develop in the defunctioned segment of bowel may contribute to the development of postoperative ileus (poi) after loop ileostomy closure (lic). as such, longer intersurgery interval between ileostomy creation and lic may increase poi. methods and procedures: after institutional review board approval, all patients who underwent lic at a single institution between - were identified. the primary endpoint, primary poi, was defined as either a) being kept nil-per-os on or after postoperative day for symptoms of nausea/vomiting, distension, and/or obstipation or b) having a nasogastric tube (ngt) inserted, without postoperative obstruction or sepsis. secondary endpoints included length of hospital stay (los) and non-poi related morbidity. patients who left the operating room with a ngt, had a planned laparotomy with a concomitant procedure at the time of lic, had a total proctocolectomy as their index operation, or had secondary poi, were excluded. patients were then divided into two groups based on timing from the index operation to lic (\ months vs. objective: fecal incontinence can be a debilitating problem significantly diminishing productivity and quality of life. sacral neuromodulation has emerged as a first line surgical option treatment in patients with fecal incontinence. though its efficacy has been rigorously evaluated in adult populations there is scant data available for its use in the pediatric pateints with fecal incontinence. this case study discusses the management of fecal incontinence in a pediatric patient with a history of hirschsprung's disease utilizing sacral nerve stimulation. methods: our patient is a -year-old female with a history of hirshsprung's diagnosed in infancy and treated surgically with coloanal pull through at the age of who presented with complaints of fecal incontinence. the patient was wearing pads daily, noting frequent uncontrolled bowel movements as well as having frequent missed days of school due to these symptoms. despite maximal medical management and pelvic floor physical therapy the patient continued to have - episodes of fecal incontinence daily. a ct scan with rectal contrast was used to establish her postoperative anatomy. anal manometry showed low rest/squeeze pressures, absent resting anal inhibitory reflex, and abnormal sensation. furthermore, during balloon expulsion testing the patient failed to pass device. the patient was deemed a candidate for stage testing with sacral nerve neuromodulation. during follow-up, the patient was noted to have resolution of her episodes of fecal incontinence and the second stage was completed. the patient continues to note % continence and dramatic improvement in her quality of life. conclusion: in this patient with a history of severe fecal incontinence due to hirschsprung's disease, sacral neuromodulation has had a significant impact on her quality of life. post-operatively she continues to have marked improvement in her symptoms with - bowel movements a day with no recurrence of fecal incontinence. the use of sacral neuromodulation is a promising treatment for fecal incontinence in the pediatric population. future research investigating the longterm efficacy of this treatment modality in the pediatric population is needed. cases of bowel obstruction caused by colorectal cancer recurrence and progression were excluded. surgical cases ( . %) were considered to be early bowel obstruction and ( . %) were classified as late bowel obstruction. left hemicolectomy (n= , . %) was a significantly more frequent procedure in early bowel obstruction, and abdominoperineal resection (n= , . %) was significantly more common in late bowel obstruction (p. ). both early and late bowel obstruction included adhesive small bowel obstruction (n= ), internal hernia (n= ), and strangulation obstruction (n= ). internal hernia (n= ) and strangulation obstruction (n= ) occurred after left hemicolectomy and abdominoperineal resection, respectively. there is no apparent relationship between surgical procedures and adhesion regions (abdominal wall, intestinal tract, and pelvic cavity). the incidence rate of postoperative small bowel obstruction remained low, and laparoscopic colectomy had been safely performed. however, countermeasures are needed because of the high frequency of both early and late bowel obstruction which occurred after left hemicolectomy and abdominoperineal resection, respectively. improved utilization of resources as an improvement introduction: nowadays, treatment decisions about patients with rectal cancer are increasingly made within the context of a multi-disciplinary team (mdt) meeting. the outcomes of rectal cancer patients before and after the era of multi-disciplinary team was analyzed and compared in this paper. the purpose of the present study is to evaluate the value of discussing rectal cancer patients in a multi-disciplinary team. methods and procedures: in our health institute, weekly mdt conferences were initiated in january . meetings were attended by surgeons, radiologists, radiation and medical oncologists and key nursing personnel. all rectal cancer patients diagnosed and treated in - in the general surgery division of the "carlo urbani" hospital in jesi (an, italy) were included. then, the data from rectal cancer patients in were evaluated, before the adoption of mdt and in year , after the adoption of meetings. datasets regarding demographics, tumor stage, treatment, and outcomes based on pathology after operation were obtained. during an mdt discussion patient history, clinical and psychological condition, co-morbidity, modes of work-up, clinical staging, and optimal treatment strategies were discussed. a database was created to include each patient's workup, treatments to date and recommendations by each specialty. ''demographic variables'' consisted of age at diagnosis, sex, body mass index, comorbidities, american society of anesthesiologists physical status classification system, clinical stage and pathological stage. other analyzed variables included baseline carcinoembryonic antigen (cea), the type of imaging, use of neoadjuvant chemo-radiation, restaging following neoadjuvant therapy, distance from the anal verge, operation type and use of adjuvant chemo-radiation. ''outcome variables'' consisted in a comparison for each group between clinical and pathological stage. results: sixty-five patients were included in this study: thirty patients in (pre-mdt) and thirty-five patients in . demographic variables did not differ significantly between groups. preoperative clinical stages with baseline preoperative cea and postoperative pathological stage were analysed, too. thanks to the mdt and the increased use of the neoadjuvant therapy, a statistically significant difference in reduction of the stage between the clinical and pathological stage in the patients of the mdt group was verified. conclusions: the vast majority of rectal mdt decisions were implemented and when decisions changed, it mostly related to patient factors that had not been taken into account prior to the adoption of multi-disciplinary team. analysis of the implementation of team decisions is an informative process in order to monitor the quality of mdt decision-making. purpose: in japan, lateral pelvic node dissection (lpnd) is the standard treatment for locally advanced lower rectal cancer. there are few reports of patients undergoing single-incision plus one port laparoscopic (sils+ ) lpnd even among those undergoing laparoscopic lpnd. the aim of this study is to describe our initial experience and assess the feasibility and safety of sils+ lpnd for patients with advanced lower rectal cancer. methods: a lap protector (lp) was inserted through a . cm transumbilical incision, and an ezaccess was mounted to lp and three -mm ports were placed. a mm port was inserted in right lower quadrant. a single institutional experience of sils+ lplnd for rectal cancer are presented. inclusion criteria was indications for lld were lower rectal cancer with t - , or t - rectal cancer with metastasis of lateral lymph node, as described by the japanese society for cancer of the colon and rectum (jsccr) guidelines for the treatment of colorectal cancer. perioperative outcomes including operative time, operative blood loss, length of stay, postoperative complications, and histopathological data were collected prospectively. introduction: endoscopic stenting with a self-expandable metallic stent (sems) is widely accepted procedure for malignant colorectal obstruction. we assessed the safety and efficacy of insertion of a sems followed by elective surgery as 'bridge to surgery (bts)' in our institute. methods: this study was a retrospective study in our institute. the data was collected from medical charts from january to june . results: a total of consecutive patients underwent radical surgery for colorectal malignancy during this period. in this series, patients ( . %) were diagnosed malignant colorectal obstruction and intended to a bts. the stent was successfully placed in patients and all the patients were planned to undergo radical surgery. the failed patients underwent stoma creation ( patients) and hartmann's procedure. the technical success rate was % and the clinical success rate was %. the median time from sems to surgery was days ( - days) . open and laparoscopic surgery was performed in and patients, respectively, except for one patient refused radical surgery because of a great age. the tumor could be resected in patients (bts patients) with primary anastomosis. however, diverting stoma creation was needed in patients and decompression rectal tube was placed in patient. the entire patient laparoscopically was no conversion to open surgery. there was no anastomotic leakage in bts patients. the median duration of postoperative hospital stay was days ( - days). the overall postoperative complication was % ( / ) including bowel obstruction and anastomotic stricture. the median follow-up period was days. during the follow-up period, patients were relapsed peritoneal dissemination, ovarian metastasis, and liver and pulmonary metastases, respectively. former patients were diagnosed stage iva at the time of primary surgery. one patient died from sudden death. conclusions: our data suggested that routine use of sems insertion was safe and effective procedure for malignant colorectal obstruction as a bts. moreover, laparoscopic procedure was useful procedure in bts patient. the short-and long-term surgical outcomes were also acceptable. introduction: serpin e , also known as plasminogen activator inhibitor- (pai- ) is an inhibitor of urokinase type plasminogen activator (upa) and tissue-type plasminogen activators (tpa ). pai- plays a role in the regulation of angiogenesis, wound healing, and tumor cell invasion; over expression has been noted in breast, esophageal, and colorectal cancer (crc). pai- is also a potent regulator of endothelial cell (ec) proliferation and migration in vitro and of angiogenesis and tumor growth in vivo. the plasminogen/plasmin system plays a key role in cancer progression by mediating extracellular matrix degradation and tumor cell migration. surgery's impact on plasma pai- levels is unknown. this study's purpose was to measure plasma pai- levels before and during the first month after minimally invasive colorectal resection (micr) for crc. objectives: retroflexion in the rectum at the end of a colonoscopy is a requirement for a complete endoscopic evaluation. retroflexion helps to visualize and detect polyps which would be missed otherwise. currently new endoscopes are available which can do retroflexion in the caecum. aim: our study aims to compare the rate of polyp detection rate in cecum and ascending colon with and without retroflexion in cecum. methods: this is a single center, single operator, retrospective study. a total of two hundred patients were involved. a single center irb waiver was obtained. patients were divided into two groups based on the presence/absence of retroflexion in caecum during their colonoscopy. the data was obtained from records. group a (n= ) had colonoscopy without retroflexion in caecum group b (n= ) had colonoscopy with retroflexion in caecum inclusion criteria: patients undergoing screening colonoscopy between the age of and . results: group a: total of patients were screened. a total of polyps were detected in group a. number of cecal polyps were ( . % of total polyp count). number of ascending colon polyp were ( % of total polyp). on analyzing the pathology % of the cecal polyps were tubular adenoma, % hyperplastic polyps % and % lymphoid aggregate. number of ascending colon polyps were , of which % were tubular adenoma, % tubular adenoma and % tubulovillous adenoma group b: total of patients were screened. a total of polyps were detected. number of cecal polyps detected were ( . % of total polyp count). number of ascending of ascending colon polyps were ( %). on analyzing pathology, % cecal polyps were tubular adenoma and % were sessile serrated. out of the ascending colon polyps % were tubular adenoma, % sessile serrated, % tubulovillous and % hyperplastic polyp. side events: two mass lesions were noted in both group a and b. there was incomplete colonoscopy in group a and b. conclusion: this retrospective analysis reveals a small increase in polyp detection in the cecum with retroflexion, especially in detecting sessile polyps which have more malignant potential. however, a large multicenter analysis will be required to validate the above observation. background: while uncommon, rectal prolapse is a disabling condition affecting older females. in a small subset of patients, concomitant organ prolapses with or without incarceration can lead to significant morbidity. as the field of laparoscopy has evolved, minimally invasive surgical options for rectal prolapse have led to improved quality and reduced morbidity for patients suffering this debilitating disease. methods: the - acs-nsqip databases was queried for patients undergoing a traditional or minimally invasive rectopexy based on cpt codes ( , , , and ) . emergent cases and patients with preoperative infections or inflammatory states were excluded. the primary outcome of interest was a -day postoperative composite morbidity score. statistical analysis incorporated multivariate analysis and binomial logistic regression with p. holding significance. results: these inclusion and exclusion criteria identified patients undergoing traditional ( ) and minimally invasive ( ) rectopexy for prolapse between and . patients undergoing traditional rectopexy were older (p. ), had a higher body mass index (p= . ), more comorbid conditions (diabetes, copd, hypertension) and less functional independence (p= . ). patients undergoing a traditional rectopexy had a higher composite morbidity incidence of . % vs. % for minimally invasive rectopexy (p. ). specifically, minimally invasive rectopexy patients had a . % reduction in wound complications (p= . ) and a shorter hospital stay ( . days vs. . days, p . ) compared to a traditional rectopexy. readmission rates were also . % lower in the minimally invasive group (p= . ). after controlling for the differences in the cohorts, a minimally invasive approach was a significant protective factor against the incidence of -day postoperative morbidity (or . , p. ). conclusion: a minimally invasive rectopexy has improved -day postoperative morbidity compared to a traditional rectopexy and should be strongly considered for the treatment of rectal prolapse. objectives: the short-term safety and efficacy of a self-expandable metallic stent (sems) placement followed by elective surgery, "bridge to surgery (bts)", for malignant large-bowel obstruction (mlbo) have been well described. the aim of this study was to investigate the risk factors for postoperative complications and optimal interval between sems placement and surgery in patients with mlbo. methods: retrospective examination of patient records revealed that the bts strategy was attempted in patients with mlbo from january to march in our institution. two of these patients were excluded because they had undergone emergency surgery for sems migration; thus, patients with mlbo who had undergone sems placement followed by elective surgery were included. of these patients, eight had developed postoperative complications (clavien-dindo grading≥ii) (postoperative complication: poc group) whereas patients had no such complications (no poc group). results: univariate analyses showed that the factors of asa score, number of lymph nodes resected, interval between sems and surgery, and preoperative albumin concentration were associated with postoperative complications. multivariate analysis identified only the interval between sems and surgery as an independent risk factor. furthermore, a cut-off value of days for interval between sems and surgery was identified by roc curve analysis. conclusions: an interval of ≥ days from sems placement to surgery is an independent predictive factor for postoperative complications in patients undergoing elective surgery in a bts setting. thus, an interval of over days is recommended for minimizing postoperative complications. haseeb kothar, ronan cahill; mater misericordiae university hospital current clinical advances in operative near-infrared visualisation of cells, tissues and structures are predicated on the use of commercial available near-infrared cameras to excite and visualise emission energy from non-selective, approved compounds (predominantly indocyanine green (icg)). it is expected that new generation compounds wholly selective for specific cellular components are now needed for further advance and a variety of molecular targets have been proposed and are being developed primarily for oncological imaging purposes. recent publications have however suggested icg itself is retained within malignant tissue differently to its uptake and clearance from surrounding non-malignant tissue which is important for two reasons. firstly, it exploits and makes visual the increased vascular permeability and disordered clearance associated with carcinogenesis which is a common endpoint of a variety of mediators including but not limited to vegf. this raises the useful option of targeting downstream effects of cancer compounds on a metabolic basis as opposed to tagging individual cell or antigen components. this means that a single agent could be used to target a variety of cancers rather then needing a specific one for each specific sub-type as well as obviating the issue of cancer cells heterogeneity even in a single cancer deposit. second, it is very likely that some or all of the "localisation" effect of proposed selective compounds may well be due to a similar phenomenum rather then cell-specific binding and may make distinction from other areas of similar metabolic behaviour (ie inflammatory regions) difficult. the crucial step-advance for such agent development so may well relate to timing of compound delivery and "visualisation window" at the region of interest rather then highly selective oncocellular-targeting. to illustrate this in more detail, we have been examining the tissue-specific effects and actions of near-infrared excitation in patients (n= ) with localised malignant colorectal primaries receiving an aliquot of icg before such examination at the time of resection. icg can be selectively apparent in the colorectal primary minutes after its systemic administration likely due to altered vascular dynamics. additional dose-related work has shown that early administration ( - minutes before examination) does not give useful information related to tumour fluorescence. interestingly none of these patients had fluorescence seen within their regional lymphatics but none also had malignant lymph nodes associated with their large primaries on pathological examination. however, this procedure is not usually performed in laparoscopic apr for its technique difficulty, which may lead to increased rates of complications ( fig. ) . here, we compared the feasibility and peri-operative outcomes of the laparoscopic apr with and without pelvic peritoneum closure (ppc) for lower rectal cancer. introduction: there are reports of increased operative duration, blood loss and postoperative morbidity, caused by difficulties in obtaining good visualization and in controlling bleeding when laparoscopic resection is performed in obese patients with colon cancer. purpose: the aim of this study was to investigate the impact of obesity on perioperative outcomes after laparoscopic colorectal resection performed by various operative methods in our department. patients and methods: we conducted a retrospective analysis of patients with colorectal cancer who underwent laparoscopic surgery between january to december . right colectomy was performed in patients, sigmoidectomy in patients, and low anterior resection in patients. the surgical outcomes were compared between non-obese (body mass index [bmi]\ kg/m ) and obese (bmi ? kg/m ) patients. results: right colectomy cases: the amount of blood loss was significantly increased in the obese group compared with the non-obese group, but operation time did not differ significantly between the groups. there were no significant differences between the two groups in the rate of postoperative complications and duration of post-operative hospitalization. sigmoidectomy cases: there were no significant differences between the two groups in operation time and amount of blood loss. even though the preoperative asa score and the rate of postoperative complications were higher in the obese group, the mean postoperative hospital stay did not differ significantly between the two groups. low anterior resection cases: there were no significant differences between the obese group and the non-obese groups in operation time, amount of blood loss, rate of postoperative complications, and duration of post-operative hospitalization. discussion: although there are some reports of increased operative times in obese patients, the operative procedure was not extended in any of the present study patients. the amount of blood loss was significantly increased in the obese group compared with the non-obese group when right colectomy was performed. among the patients undergoing sigmoidectomy, the postoperative rate of complications was higher in the obese group; however, the preoperative asa status was also higher in the obese group than non-obese group, indicating that factors other than obesity may be involved. conclusion: we concluded that laparoscopic colorectal resection appeared to be safe and feasible in both obese patients and non-obese patients. however, bmi may not accurately reflect the amount of visceral fat present. background: for the complete rectal prolapse (basically longer than cm), we thought sling rectopexy was most reasonable to hang up and fix the rectum, which drooped down and prolapsed due to the relaxation of supporting tissue. we considered ripstein method had enough fixed power of rectum to sacrum. however, complications of rectal stenosis, constipation, mesh infection and mesh penetration were reported. therefore, we modified ripstein method to conquer such complications. aim: a prospective study beyond the randomized control trial (rct) between our modified ( introduction: the results of the japan clinical oncology group (jcog) study suggested that total mesorectal excision (tme) and lateral lymph node dissection (llnd) could become the standard treatment for lower rectal carcinoma. however, llnd must also be performed laparoscopically if surgery for lower rectal carcinoma is to be carried out as a completely laparoscopic procedure. transanal tme (tatme) is expected to provide better results than the conventional tme, both oncologically and in terms of pelvic function, and its use has recently been spreading in japan. we started performing laparoscopic tatme+llnd in our department in july and here report the short-term outcomes. subjects and methods: we used laparoscopic tatme+llnd to treat men and women with ct or deeper rectal carcinoma in whom the inferior margin of the tumor was on the anal side of the peritoneal reflection. this was a retrospective study of short-term postoperative outcomes. surgical procedure: laparoscopic surgery was started simultaneously by two teams, one working transabdominally and the other working transanally. the transabdominal team performed the standard proximal llnd and mobilization of the splenic flexure via five ports. they then dissected the bilateral lateral lymph nodes, mainly in the obturator (# ) and internal iliac (# ) groups. during this time, the transanal team performed laparoscopic tatme. finally, both dissection layers were connected and the cancer was excised. results: six patients had clinical stage ii and two had clinical stage iii lower rectal carcinoma. all the patients underwent preoperative chemotherapy with s- +l-ohp. five underwent a sphincterpreserving surgery, and three underwent rectal amputation. the mean operating time was minutes (range, - minutes), and the mean amount of hemorrhage was g ( - g). the mean number of lymph nodes dissected was , and r resection was performed in all the cases. the mean length of hospital stay was days, and a postoperative complication of clavien-dindo grade iii or higher occurred in one patient (anastomotic failure). conclusions: laparoscopic tatme+llnd performed by two teams simultaneously is an extremely useful procedure that not only reduces operating time, but also is less invasive than laparoscopic surgery. it may also be effective for improving curative nature, nerve preservation, and anal function. objective: in laparoscopic appendectomy, the base of the appendix is usually secured by applying a roeders knot. the aim of this study was to compare the advantages of using staplers and hem-olocks for securing the base of the appendix. method: the study included patients between age of to years with acute appendicitis randomly divided into two groups. in the first group, the base of the appendix was secured using roeders knot. in the second group, mesoappendix was not dissected and was included in the endostapler jaws. the primary outcome was overall morbidity. secondary outcomes were total duration of surgery, total length of stay and ease in difficult cases. result: no morbidity was recorded in any group. the time of the operative procedure was significantly longer in the cases with roeders knot than in the stapler group (p. ) as mesoappendix was not dissected in the later. cases with unhealthy base were progressed to laparoscopic quadricolectomy. apart from the ease of applying a stapler, cases of second group with gangrenous base were easily tackled using endostapler, avoiding the need of a hemicolectomy. conclusion: all forms of closure of the appendix base are acceptable, but endostapler technique apart from providing a secure base, reduces operative time and is an essential tool in cases of gangrenous base. introduction: accurate staging is essential to estimate the prognosis of patients with colorectal cancer (crc) and lymph node evaluation is key to determine it. in non-metastatic crc, the number of harvested lymph nodes is the strongest prognostic factor for outcome and survival. additionally, it is thought that a higher lymph node yield may be representative of a higher quality of surgical care. due to the importance of the association between lymph node evaluation and outcome in crc, it is necessary to evaluate factors which may affect lymph node harvest. introduction: hatmann's procedure is commonly done in treating complicated diverticulitis, negleccted rectal trauma with sepsis and sometimes malignancy. the traditional techniques to restore the intestinal continuity after hartmann's procedure were for many years the standard of care in these operations, but in fact they carry many morbidity and even mortality and failure. laparoscopic techniques is not only carry the advantage of minimal invasive surgery, but also of better visualizationn and magnification. the aim is evaluating the outcome of using the laparoscope in reversal of hartmann's procedure as regard feasibility and safety. patients and method: forty patients were subjected to laparoscopic reversal of hatmann's procedure in tanta university hospital, there ages ranged between to years, the time elapsed after the original operation ranged from months to years, excluding advanced malignany. conversin occurred in cases due to extensive adhesions and bleeding. results: no mortality, or major morbidity in our study and only single leak treated by covering ilestomy. conclusion; laparoscopic hartmann's procedure is feasible, promising tehnique with minimal morbidity. background: minimal invasive surgery has been well established in the elective colorectal surgery and it has been proven better clinical outcome compared with open surgery. in the emergent setting, laparoscope is used mostly in the colecystectomy, appendectomy but laparoscopic emergent colorectal surgery is limited for it's complexity and difficulity. the aim of this study was to envaluate the feasibility of laparoscopic emergent colorectal surgery. methods: this study is prospective collected, observational single center study of patients undergoing laparoscopic emergent colorectal surgery from to . the patient demographics, surgery indication and detail, complication, clinical outcome and hospital stay were collected and analyzed. results: there are total emergent colorectal operations and patients were managed with minimal invasive method. among these laparoscopic emergent surgery, there are male patients and female patients. mean age of the patients was . years (range - years). the main indication for operation: perforation . % ( / ), leakage after elective colorectal surgery . % ( / ), obstruction . % ( / ), ischemia colitis . % ( / ,), bleeding . % ( / ). there are cases in asa , cases in asa , cases in asa . the qsofa score for sepsis: cases was , cases was , cases was , case was . there are cases undergoing laparoscopic lavage with diverting stomy, cases were hartmann procedure, cases were anterior resection, cases were right hemicolectomy, cases were perforation repair, cases were redo anastomosis. there are cases coversion to open method including cases were due to bowel adhesion, cases were due to bowel distension, case was due to severe shock status. mean operative time is . minutes. the overall mortality rate was . % and major complication rate (clavien-dindo grade above ) was . %. re-operation rate was . %. the mean hospital stay was . days. conclusions: this study presents evidence of an initially clinical outcome in emergent laparoscopic colorectal suregry. in the absence of large case series, the benefits of a laparoscopic approach should befall to at least a minority of these patients. confocal laser endomicroscopy (cle) can provide real-time observation of the cell structure and tissue morphology. in our study, we aim to assess the situation of anastomotic perfusion using cle. method: the experimental rabbits were separated into two groups: group a (good anastomotic perfusion, n= ), group b (poor anastomotic perfusion, n= ). the partial colectomy and anastomosis was performed for group a and b. then detection for anastomotic perfusion using cle was carried out after the surgery. during the continuous scanning, we counted the number of blood cells that cross over the certain point of anastomotic stoma in the same period. results: assistant with fluorescein sodium, the blood vessels are highlighted. we can see significant difference of imaging effect between group a and group b. the average number of blood cells are . /min of group a and . /min of group b (p. ), which has significant difference. conclusion: cle can allow real-time observation of the blood flow of anastomotic stoma in vivo. therefore, it is feasible to assess the anastomotic perfusion using cle in colorectal surgery. cigdem benlice, ahmet rencuzogullari, james church, gokhan ozuner, david liska, scott steele, emre gorgun; cleveland clinic background: intraoperative colonoscopy (ioc) is an adjunct in colorectal surgery (crs) especially in patients with malignancies in order to detect location of the primary or synchronous lesions as well as assessing anastomotic integrity. however, effects of intraoperative colonoscopy on short term outcomes during crs is a concern. this study aims to evaluate safety and feasibility and post-operative outcomes of intraoperative colonoscopy in left-sided colectomy patients for colorectal cancer patients by using the nationwide database. patients and methods: patients undergoing elective left-sided colectomy with low pelvic anastomosis without any proximal diversion for colorectal cancer were reviewed from the american college of surgeons national surgical quality improvement program (acs-nsqip) proceduretargeted database ( ) ( ) ( ) according to their primary procedure current procedural terminology (cpt) code. subsequently, patients who underwent intraoperative colonoscopy were identified from concurrent cpt codes and divided into two groups based on the simultaneous intraoperative colonoscopy. demographics, comorbidities, -day postoperative complications were evaluated and compared between the groups. multivariate logistic regression was conducted adjusting for significant factors between the groups. results: a total of patients were identified and ioc was performed for ( . %) patients. objective: laparoscopic ileostomy commonly performed for the patients with colorectal obstruction due to cancer, peritonitis with perforation of colon or the other reason. reduced port surgery is a novel technique that may be performed when considering minimally invasive surgery and desiring a cosmetic benefit. the aim of this study was to evaluate safety and feasibility of reduced port laparoscopic ileostomy for the patients with advanced colorectal cancer before chemotherapy. methods: between july and august , patients who underwent reduced port laparoscopic ileostomy were included ( male and female, age: years old. the outcomes were evaluated in terms of operation time, intraoperative blood loss and perioperative complications. sugical procedures: the patients were placed in the supine position and the operator stood left side. an access device with the wound-protector (ez access, hakko, nagono, japan) was inserted on the future ileostomy site in the right lower abdomen, inserting two of -mm trocars, maintaining pneumoperitoneum at mmhg with carbon dioxide. a -mm trocar was inserted in the left lower abdomen. a -mm flexible laparoscope was inserted from access device port. after exploring abdominal cavity, ileum end was identified. then the marking using dye was put on the ileum of cm proximal from the ileum end. the ileum marked by dye was grasped, and extracted through the access devise. then a blooke ileostomy was created. results: reduced port laparoscopic ileostomy was performed for patients with colorectal obstruction due to cancer before chemotherapy. the mean operative time was minutes, the mean blood loss was . ml. three patient received one additional port. there were no intraoperative complications. five patients ( . %) experienced postoperative complications (two of deep surgical site infection, one of pneumonia, one of outlet obstruction and one of renal dysfunction). there were no other intraoperative or postoperative complications. conclusion: reduced port laparoscopic ileostomy is a safe and feasible procedure for the patients with advanced colorectal cancer before chemotherapy. methods: we performed elective lcr on patients for primary colorectal cancers between june and june . seventy-two patients were excluded in this study following reasons: patients underwent multiple organ resection, and colorectal cancer was diagnosed with stage iv in patients. accordingly, patients were eligible for comparative analysis, with in group po (post operation) and in group c (control). in group po, past operative procedures were as follows: appendectomy ( %), digestive tract ( %), hepato-billiary-pancreatic ( %), gynecologic ( %), urologic surgery ( %), and others ( %). results: there were no significant differences between two groups in asa (grade≤ : vs. %, p= . ), bmi ( introduction: the treatment of rectal cancer requires highly skilled practice by the entire multidisciplinary team. important aims of treatment are: to reduce the risk of residual disease in the pelvis, with lower morbidity and to preserve good sphincter function. the tata procedure is transanal transabdominal radical proctosigmoidectomy with coloanal anastomosis. this technique was first developed in by dr. gerald marks to avoid a permanent colostomy for low-lying rectal cancer. this study reports the long-term results of tata procedure for low rectal cancer. methods and procedures: a prospective study was on patients with low rectal cancer between april and july in a tertiary referral university-affiliated center specializing in laparoscopic surgery. all resections were carried out by a team of dedicated colorectal surgery and standard protocol was used for all pre-and-post-operative care. all the patients underwent total mesorectal excision. results: consecutive patients ( male, female, mean age ) underwent tata procedure, of them ( , %) after neoadjuvant radiochemotherapy. the mean operation time was min (range - ) and the mean estimated blood loss was ml (range - ). the overall incidence of morbidity was , % ( / ) and the mean hospital stay was , days. the mean follow-up period was , (range, - ) months with a recurrence rate of , % ( / ), overall estimated -year survival , % and the disease-free survival rate , %. conclusion: laparoscopic total mesorectal excision with tata procedure is safe with excellent local recurrence and disease-free survival rate. jacek piatkowski, md, phd, marek jackowski, prof; clinic of general, gastroenterological and oncological surgery introduction: more than years ago, laparoscopic technique was considered to be a fully accepted surgical method for treatment of rectal cancer. the following years are a further search for a new surgical method that reduces invasiveness and improves treatment outcomes. it seems that such a method is transanal total mesorectal excision. the aim of this study was to evaluate the new method of rectal cancer surgery (tatme) after years of its use. methods: radicality of treatment (r resection, local recurrence), outcome of surgical treatment and quality of life of patients after surgery were evaluated. results: in the period from . . . - . . . patients ( men, women) were operated in the clinic. in cases the indication for surgery was lower and middle rectal cancer and in cases high grade dysplasia. all patients underwent laparoscopic rectal proctectomy with transanal access (tatme). in all cases, complete oncological radicalization (resection r ) was obtained. the average operation time was minutes. we had used two teams approach (cecil approach) with laparoscopic sets -abdominal and perineal starting at the same time. in the postoperative course, patients had signs of anastomosis leak ( of them required reoperation). the follow-up period is - months. none of the patients had any recurrence of cancer. conclusions: . transanal tme for rectal cancer surgery is an alternative method to conventional laparoscopic surgery. . in a large proportion of patients with lower and middle tumors, the rectum can avoid abdomino-perineal resection with permanent colostomy. background: the double stapling technique (dst) has widely spread colorectal anastomosis especially for anastomosis after low anterior resection. as for the colorectal cancer treatment, heald reported total mesorectal excision (tme) in , and has been accepted as the standard technique for rectal resection due to the decreased local recurrence rate and improved functional results. with advent of dst, there is a background that it has become possible to preserve anus, even in the case with the lesion at lower rectum. laparoscopic surgery for colon cancer was introduced in the s, and has had promising results including long-term outcomes. according to the spread of laparoscopic surgery, laparoscopic surgery had been applied to the rectal resection, with technical difficulty. one of the reasons for the difficulty is that the high rate of anastomotic leakage, a critical adverse effect of low anterior resection (lar). thus, risk factors for anastomotic leakage were widely discussed, including technical factors such as pre-compression and number or firing. the decisive difference in conventional lar and laparoscopic lar in dst, is the stapler used for transection of the rectum. the laparoscopic staplers which are currently available are thought to be not ideal, and there is little evidence of specific specifications of stapler for laparoscopic surgery. materials and methods: all method described in this study was approved by the institutional ethical review committee. we reviewed the colon and rectal wall thickness according to histological examination using h&e staining of distal margin of resected specimen of the patients who conclusions: rstc for severe acute uc is at least as safe as the laparoscopic approach. although the robotic cohort had more comorbidities, major postoperative complications, readmissions, and reoperation rates were less when compared to lstc. rstc was also associated with an earlier return of bowel function and shorter length of stay. a prospective study with larger numbers is needed to see if the superiority of robotic versus laparoscopic approaches is reproducible. s surg endosc ( ) introduction: complete mesocolic excision (cme) has been advocated based on oncologic superiority, but is not commonly performed in north america. furthermore, many data are limited to case series with few comparative studies. therefore the objective was to systematically review studies comparing the short-and long-term outcomes between cme and non-cme colectomy for colon cancer. methods: a systematic review was performed according to prisma guidelines of medline, embase, healthstar, web of science, and cochrane library. studies were only included if they compared conventional resection (non-cme) to cme for colon cancer. quality was assessed using the methodological index for non-randomized studies (minors). the main outcome measures were short-term morbidity and oncologic outcomes. study eligibility, data extraction and quality assessment was performed by two independent reviewers, and disagreements resolved by consensus. weighted pooled means and proportions with %ci were calculated using a randomeffects model when appropriate. results: out of citations, studies underwent full-text review and met the inclusion criteria, of which were unique series. mean minors score was . (range - ). the mean sample size in the cme group was (range - ) and (range - ) in the non-cme group. in the unique studies, included only right-sided resection, and . % ( % ci . - . ) of the remaining were right-sided colectomies. of the studies that reported surgical approach, . % ( %ci . - . ) of cme were performed laparoscopically. there were papers reporting plane of dissection, with cme plane achieved in . % ( . - . ). mean or time in cme group was minutes (range - ) and in non-cme group minutes (range - ). perioperative morbidity was reported in studies, with pooled overall complications of . % ( %ci . - . ) for cme and . ( %ci . - . ) for non-cme resections. anastomotic leak occurred in . % ( %ci . - . ) of cme versus . % ( %ci . - . ) in non-cme colectomies. cme surgery consistently resulted in more lymph nodes retrieved, longer distance to high tie, and specimen length. there were studies that compared -or -year overall or disease-free survival, or local recurrence. only studies reported statistically significant higher disease-free or overall survival in favour of cme. local recurrence was lower after cme in of reported studies. conclusions: the quality of the current evidence is limited and does not consistently support the superiority of cme. more rigorous data are needed before cme can be recommended as the standard of care for colon cancer resections. gilberto lozano dubernard, md, facs, ramon gil-ortiz, md, gustavo cruz-santiago, md, bernardo rueda-torres, md, javier lopez-gutierrez, md, facs; hospital angeles del pedregal introduction: to assess the feasibility of a single-stage colorectal laparoscopic re intervention without ostomy. colonic laparoscopic interventions on patients that previously underwent a minimally invasive procedure, constitutes the current boundary in the management of the acute colorectal pathology. that includes, patients with fecal peritonitis due to diverting procedures already treated surgically. the outcome of our patients could significantly improve if the surgical procedure is performed in one time, with no stoma. method and procedures: from september to june , one hundred thirty-two patients underwent colorectal laparoscopic surgery. five of these patients developed complications: three perforations due to colonoscopy and two due to dehiscence of the anastomosis. these five patients underwent a second laparoscopic procedure that included resection and anastomosis. no stoma required. results: all five patients underwent a second laparoscopic procedure due to an anastomosis leak. no stoma was required. the procedure consisted on resection of the previous anastomosis, re anastomosis, abdominal lavage, aspiration and drains placement. all of them supported with parenteral nutrition. there were no surgical complications. only one patient developed pneumonic symptoms that were solved. conclusion: the reported results, regarding no conversion rate, nor mortality, on our series of patients, suggest that single stage laparoscopic re intervention is feasible, despite fecal peritonitis. introduction: total mesorectal excision is known to be a gold standard surgical procedure for the rectal cancer. subsequently complete mesocolic excision (cme) is recognized as an essential surgical procedure for the colon cancer. the transverse colon is relatively minor location for colon cancer. variety of vessels and mobilization of splenic flexure and dissection close to pancreas make operations for the transverse colon cancer complicated. laparoscopic transverse mesocolic excision in our hospital is presented. method: laparoscopic surgery is conducted with five trocars under the lithotomy position. inferior mesenteric vein is cut after dissection of the descending colon with medial approach. the lower edge of pancreas is exposed near the inferior mesenteric vein and is dissected along toward the tail of pancreas. the splenic flexure is mobilized with lateral approach and the dissection between transverse mesocolon and the lower edge of pancreas is continued in the direction to the pancreas head. coming to the exposure of superior mesenteric artery and vein, the origin of middle colic artery and vein are cut. the transverse mesocolon is separated from the pancreas head and the duodenum with preserving the gastrocolic trunk of henle and the right gastroepiploic vein. the hepatic flexure is mobilized and cme for the transverse colon is finished. this method, the 'tail to head of pancreas' approach, we called, was performed from september . this method is well performed with one series of surgical view, and seems to be a simple procedure as cme with central vascular ligation for the transverse colonic cancer. there were no intraoperative complications, and one postoperative pancreatitis with grade ? of clavien-dindo classification of surgical complications. conclusion: our method, the 'tail to head of pancreas' approach, with transverse mesocoloc excision is simple, safe and feasible. the introduction: anastomotic complication after stapled anastomosis in colorectal cancer surgery is a considerable problem. there are various types of anastomotic complication and they have different severity. this study was aimed to evaluate the impact of intraoperative colonoscopy on detection of anastomotic complication, and its effectiveness in treatment of anastomotic complications after anterior resection (ar) and low anterior resection (lar) for colorectal cancer intraoperatively. methods: from dec. to jul. , a total of patients who underwent anastomosis between sigmoid colon and rectum after colorectal resection were reviewed retrospectively. intraoperative colonoscopy was performed routinely since december in our hospital after anterior resection and low anterior resection. to identify effectiveness of intraoperative colonoscopy, we compared postoperative complications with non-intraoperative colonoscopy group during previous months. intraoperative colonoscopy was performed after anastomosis to visualize the anastomosis line and to perform an air leakage test. if anastomotic defect and moderate bleeding were found in intraoperative colonoscopy, it was managed by means of reinforcement suture or transanal suture repair. we used logistic regression to analyze anastomotic complication between two groups with or without intraoperative colonoscopy. results: of the patients who were performed intraoperative colonoscopy after ar (n= ) and lar (n= ), abnormal findings including bleeding and air leak were found in patients ( . %). among those, cases were observed without any procedure, additional procedures were performed in patients ( . %, transanal suture ( ), lembert suture ( )). postoperative complication was developed in patients; patients had anastomosis bleeding ( . %), patients had ileus ( . %), patient had pneumonia ( . %), patients had minor complication ( . %, acute urinary retention, chylous drainage, laparoscopic port site bleeding). among patients who had anastomosis bleeding, patients were treated by endoscopic clipping, patients were cured by conservative treatment. there was no postoperative anastomotic leakage. the cases of ar and lar were and in non-intraoperative colonoscopy group, there was no significant difference between two group (p= . ). the proportion of laparoscopic surgery was . % and . % on intraoperative colonoscopy and non-intraoperative colonoscopy group, respectively, there was significant difference statistically (p= . ). however, there was no significant difference in anastomotic complication rate between two groups. (rr= . , % ci, . - . ). conclusions: although there was no significant difference in postoperative anastomotic complication rate between two groups, intraoperative colonoscopy may be valuable method for decreasing postoperative complication by visualizing anastomosis line and performing additional procedure. conclusion: it was suggested that lymph node dissection of both middle and left colic regions is necessary for splenic flexure colon cancer, because lymph node metastasis was recognized in both region. surg endosc ( ) :s -s the aims: laparoscopic right hemicolectomy became the standard of care for treating cecum, ascending and proximal transverse colon cancer in many centers. most centers use laparoscopic colectomy with extracorporeal resection and anastomosis (lc). single-incision laparoscopic colectomy with intracorporeal resection and extracorporeal (sc) remains controversial. the aim of the present study is to compare these two techniques using propensity score matching analysis. methods: we analysed the data of patients who underwent laparoscopic right hemicolectomy with lc or sc between december and december . the propensity score was calculated from age, gender, body mass index, the american society of anesthesiologists score, previous abdominal surgery and d lymphnode dissection. short-term outcomes were recorded. postoperative pain was evaluated using a visual analogue scale (vas) and postoperative analgesic use as outcome measure. results: the length of skin incision in the sc group was significantly shorter than in the lc group: median (range) ( . - ) cm verses ( - ) cm (p= . ). the vas score on day and day after surgery was significantly less in the sc group than in the lc group: median (range) ( - ) verses ( - ) on day (p= . ) and median (range) ( - ) verses ( - ) on day (p= . ). significantly fewer the number of requiring analgesia in the sc group on day and day after surgery: median (range) ( - ) times verses ( - ) times on day (p= . ) and ( - ) times verses ( - ) times on day (p= . ). there were no significant differences in operative time, intraoperative blood loss, the number of lymph nodes removed and postoperative courses between the groups. conclusions: sc for right colon cancer is safe and technically feasible. sc reduces the length of skin incision and postoperative pain compared with conventional lc. patients were divided into the following groups: cephalo-medial-to-lateral approach group (cml group, n= ) and medial-to-lateral approach group (ml group, n= introduction: laparoscopic technique has been widely used in the treatment of colorectal cancer, while playing its minimally invasive advantages, but also achieved a good effect of radical oncology. however, t colorectal cancer is not recommended laparoscopic surgery. methods: retrospectively collected pt colorectal cancer data from to in guangdong general hospital, all cases were undergoing radical surgery. results: a total of cases were enrolled in the pt group, including cases of laparoscopic group, cases of open group, conversion rate was . %. there was no difference in baseline data (age, sex, bmi, asa, etc.)(p. ). there was a significant difference between the two groups (p. ) in blood loss, postoperative complications and postoperative recovery index. in the pathologic t a/b, combined-organ resection, postoperative recurrence, the laparotomy group had more cases, and there was a statistically significant difference between the two groups (p\ . ). the -and -year overall survival rates were . % and . % for the lap group and . % and . % for the open group (p= . ). meanwhile, the -and -years disease-(p= . ). iiic stage, lymph node status, ca - and adjuvant chemotherapy were independent prognostic factors affecting overall survival. the age, pt a/b, iiic stage, ca - and adjuvant chemotherapy were independent influencing factors of disease-free survival. conclusions: laparoscopic surgery for pt colorectal cancer surgery, it is not only in the play of its minimally invasive but also obtained with the similar long-term effect. but we need more multicenter, prospective, and large sample clinical studies to validate our findings. introduction: lymph node (ln) retrieval after surgery is important. in the present study we evaluated the efficacy of the fat dissolution technique using fluid containing collagenase and lipase to avoid staging migration after laparoscopic colorectal surgery. methods: seventeen patients who underwent laparoscopic ln dissection for colorectal cancer were evaluated. first, unfixed lns within the resected mesentery were explored by visual inspection and palpation immediately after the operation by the surgeon, which is the most common practice in japan. subsequently, the fat dissolution technique was used on remnant fat tissue, and the lns were evaluated again. the primary endpoint was whether the second assessment increased the number of lns evaluated. results: the median number of lns identified at the first and second assessments was and , respectively, resulting in a significant increase in the total number of lns evaluated ( vs. , p\ . , paired t-test). one positive node was identified among all the additional lns identified ( . %; / ). although staging was not altered in any patient, the second assessment resulted in an increase in the originally insufficient number of lns evaluated (\ for stage ii) in three patients, whose treatment may be altered. tumor cells detected after the fat dissolution technique were stained with carcinoembryonic antigen and cytokeratin- . conclusion: using the fat dissolution liquid on remnant fat tissue of the mesentery of the colon and rectum enabled identification of additional lns. this method should be considered when the number of lns identified is not sufficient after conventional ln retrieval, and may avoid stage migration. aim: the aim of this study is to evaluate the pathological resection margin after laparoscopic intersphincteric resection for low rectal cancer. method: from to , there were eight laparoscopic intersphincteric resection cases for low rectal cancer. we evaluated the clinicopathological findings and the positivity of pathological resection margin. results: the median distance from the anal verge to the tumor was mm (range, - ), and the median diameter of the tumor was mm (range, - ). there was no case with neoadjuvant therapy. the estimated tumor depth were ct in cases ( . %) and ct in cases ( . %), and the actual tumor depth were ptis in cases ( . %) and pt in cases ( . %) and pt in cases ( . %). the median distal resection margin was mm (range, - ). pathological resection margin, such as the proximal, distal and circumferential margin was negative in all cases ( %). there was no mortality, but morbidity occurred in two cases (one case of anastomotic leakage and one case of small bowel obstruction). no recurrence nor distant metastasis was observed in the follow up period. conclusion: there was no positive resection margin case in the series. our patient selection, indication and the technique were considered to be precise and appropriate. introduction: the fistulas of the intestine to the vagina or the bladder include a highly morbid entity, with several functional limitation and loss of the quality of life, its diagnosis is complex and more than its treatment, which include a wide range of possibilities that go from the simple derivative colostomy in search of the spontaneous closure of the fistula, under the complete correction of the pathology with resections, anastomosis and mini-vasive reconstructions. give to know our experience in the minimally invasive treatment of whole vaginal and whole vesicial fistules by laparoscopic via, for the last years. results: a total of patients were operated in this period, women and men, all those by laparoscopic via, with intestinal resection, in thick intestine cases, in one small intestine and in another case with the commitment of the two, everyone restriction and intestinal anastomosis and in no matter were colostomy, primary closures of the fistula in patients were required, conversion to open surgery in a case and there was no recurrence, patients had prolonged hospitalization for localized infections, a requirement reintervencion for revision. a patient suffried a umbilical eventration for the extraction site, which was corrected one year after laparoscopy. conclusion: minimally invasive surgery in patients with this type of pathology becomes an excellent strategy for the integral management of these patients. group work guarantees good results. robbie sparks, dr, ronan cahill; mater misericordiae university hospital background: precise preoperative localisation of colonic cancer is a prerequisite for correct oncological resection. effective endoscopic lesional tattoo is vital for small, radiologically unseen tumors planned for laparoscopic resection but its practice may be imperfect. methods: retrospective review of consecutive patients with preoperative endoscopic lesional tattoo who underwent laparoscopic colonic resection identified from our prospectively-maintained cancer database with supplementary clinical chart and radiological, histological, endoscopic and theatre database/logbook interrogation. results: patients ( males, mean age years, median bmi . kg/m , left sided lesions, screen detected, benign polyps, % conversion rate). in operations ( %) tattoo visibility was documented with tattoo absence noted in ( . %) although tattoo was identifiable in the pathological specimen in four. in those with "missing tattoos", six of the lesions were radiologically occult and in three the tumor was found in a different colonic segment then had been judged at colonoscopy. four patients had on-table colonoscopy and five were converted to laparotomy ( % conversion rate, p. ). mean postoperative length of stay was . (range - ) days. one patient's segmental resection contained only benign pathology requiring a second operation to remove the cancer. on univariate analysis, time between endoscopy and surgery (but not patient age, gender, bmi, endoscopist or surgeon seniority, tumor size or location) was significantly associated with absence of tattoo intraoperatively (p= . ). conclusion: recording related to tattoo is variable but definite lack of gross tattoo visualisation significantly impacts the procedure. the mechanism of tattoo absence is multifactorial needing careful consideration but solvable. the aim of the present study was to perform a systematic review of the literature to determine the role of antibiotics in the management of acute uncomplicated diverticulitis (aud). diverticular disease is the most common disease of the large bowel and poses a significant burden on healthcare resources. in the united states alone, the cost of diverticular disease has been estimated to be over $ billion making it the fifth most important gastrointestinal disease economically. the use of antibiotics in the management of aud, however, is primarily based on expert opinion as current high-quality evidence is lacking. recent studies have not only questioned the optimal type and duration of antibiotic regimens, but whether antibiotics provide any benefit in the treatment of aud. conclusions: antibiotic use in patients with acute uncomplicated diverticulitis is not associated with a reduction in major complications, readmissions, treatment failure, progression to complicated diverticulitis, or need for elective and emergent surgery. however, it increases the length of hospital stay. given the risk of selection bias in included studies, further randomized trials are needed to clarify the need for antibiotics in uncomplicated diverticulitis. laparoscopic para-aortic lymph node resection for colorectal cancer aim: we want to highlight the feasibility of a sigmoidectomy using total laparoscopic with a transanal extraction of the specimen. methods: it is a -year-old female patient, obese (bmi= kg/m ) to the antecedents of laparoscopic cholecystectomy and chronic constipation. she was treated three months ago for a sigmoidal diverticulitis complicated with a pelvic abscess. the evolution has been favorable under antibiotic therapy and percutaneous drainage of the abscess. the colonoscopy showed a multiple diverticula located between and cm from the anal verge. prophylactic sigmoidectomy was performed laparoscopically using trocars ( mm supra ombilical, mm fid and mm right flank). the specimen was extracted transanally, thus avoiding a pubic incision. the steps of the intervention were: -mobilisation of left colon -closing of distal left colon stump -rectal stump lavage -opening on the rectum -transanal introduction of the anvil -specimen transanal extraction -closing og rectal stump -colonic positioning of the anvil -coloractal anastomosis. results: the intervention was minutes. no perioperative incidents. the liquid regime was authorized on the night of the intervention. the operating procedures were favorable with an exit to j post operative. the anapath examination of the surgical specimen confirmed the presence of sigmoidal diverticula. conclusion: laparoscopic sigmoidectomy with transanal extraction of the specimen for benign desease is a seductive technique with satisfactory results. it avoids a pubic incision with its parietal and aesthetic complications. chengzhi huang; guangdong general hospital (guangdong academy of medical science) background: colorectal cancer (crc) is one of the most common malignant diseases over the world. of the causes of the death of crc, metastasis to liver or lung are the major factors. however, there is still lack of precise tumor biomarker that precisely predict the clinical outcome of crc. the salt-inducible kinase (sik ) encodes a serine kinase of amp-activated protein kinase (ampk) family, which may play critical roles in tumorigenesis and tumor progression. this study aimed the study the expression and clinical significance of sik and crc patients. methods: the expression of sik protein was measured by western-blot and analysis of immunohistochemistry. sik mrna expression in cancerous tissue was measured by rt-pcr. results: the expression level of sik was correlated with the following factors: tumor invasion (t stages), lymph node metastasis, clinical stages (tnm) and tumor location. the down-regulated sik implies poor clinical outcome measured by kaplan-meier analysis (p-value. ), and may act as an independent risk factor of crc patients. background: surgical specimens for resected colon cancer vary in quality and there remains no universally accepted technique to guide resection margins. a minimum of lymph nodes provides some quality assurance, however this remains a crude marker of optimal oncological surgery. a tool to precisely identify lymphatic drainage within the mesentery could improve the oncologic quality of resection and better guide adjuvant treatment through more optimal mesenteric lymphadenectomy. while fluorescence imaging (fi) has been described to identify nodal disease in several other cancers, feasibility and best practices have not been established in colon cancer. we describe a novel technique of fi using indocyanine green (icg) to identify lymphatic spread and potentially guide optimal mesenteric lymphadenectomy in colon cancer. methods: three consecutive patients with colon cancer undergoing a laparoscopic resection had peritumoral subserosal injection of icg for fi after extracorporealization of the mobilized specimen. three concentrations of icg were injected − mg/ ml, mg/ ml, and mg/ ml. a total of ml was given for each patient. using a modified laparoscopic camera, the icg was excited by light in the near-infrared (nir) spectrum, for real-time visualization of the lymphatic drainage. the main outcome measure was identification of lymphatic drainage. results: three patients with right-sided primary colon cancer were evaluated. all three patients had successful identification of the lymphatic drainage pattern along the mesentery. the most successful protocol was ml (concentration mg/ ml) subserosal injection at points within close proximity ( cm) of the tumor with a -gauge needle, then waiting minutes for complete mapping. no intraoperative or injection-related adverse effects occurred with -day follow-up. the median lymph node yield was . all specimens had tumor-free margins. conclusion: from this small series, fluorescence imaging with icg is a potentially safe and feasible technique for identifying mesocolic lymphatic drainage patterns. this proof of concept and protocol will lead to future studies to examine the utility of fluoresence imaging to guide more precise surgery in colon cancer. introduction: anastomotic leakage in colon/rectal surgery is a dangerous event with an occurance rate ranging from to %. the associated mortality rate is between - %. the white-light intraoperative subjective surgical assessment (the most frequently used approach) underestimates the actual anastomotic leakage rate. intraoperative tissue perfusion assessment by indocyanine green (icg)-enhanced fluorescence has been reported in multiple clinical scenarios in laparoscopic/ robotic surgery, as well as for for bowel perfusion assessment. this technology can detect microvascular impairment, potentially preventing anastomotic leakage. we reviewed the literature and present our data to evaluate the feasibility and usefulness of icg-enhanced ?uorescence in the intraoperative assessment of vascular peri-anastomotic tissue perfusion in colorectal surgery. methods and procedures: a pubmed literature narrative review has been performed. moreover, out of a total of robotic colorectal cases, we retrospectively analyzed icg-enhanced fluorescence robotic colorectal resections ( left colectomies- rectal resections- right- transverse- pancolectomy). results: after icg-technology use, the biggest (n[ ) case-series showed a rate of . - % of cases in which they changed the level of resection based on icg. icg technology may variably reduce the anastomotic leak rate from to %. however, the threshold values to define the actual sub-optimal perfusion are still under investigation. in our experience, out of icg cases performed: the conversion, intraoperative complication, dye allergic reactionand mortality rates were all %. post-op surgical complications: case of leak ( , %) and sbo for incarcerated hernia ( . %). in cases, with normal white-light assessment, the level of the anastomosis was changed after icg showed ischemic tissues. despite the application of icg, anastomotic leak has been registered. conclusions: icg-enhanced ?uorescence may intraoperatively change the white-light assessed resection/anastomotic level, potentially decreasing the anastomotic leakage rate. our data shows that this technology is safe, feasibile and may prevent anastomotic leakage. however, the decision making is still too subjective and not data driven. at this stage icg, beside being a promising technique, doesn't have high level of evidence (most of the reports are retrospective). some randomized prospective trials with an adequate statistical power are needed. a precise injection dose and timing standardization is required. the main challange is to develop a method to objectively obtain a real-time intensity assessement. this may provide objective metric tresholds for an intraoperative evidence/data-based surgical decision making. introduction: according to the world health organization, colorectal cancer is the rd most commonly diagnosed cancer in the world. one of the main risk factors for the development of colorectal cancer is obesity. obesity is seen to increase the risk of colorectal cancer by % in women per kg/m and % in men per kg/m . bariatric surgery is one of the treatments that is considered to achieve and sustain a significant amount of intentional weight loss in patients. considering that fact that bariatric surgery decreases obesity, this intentional weight loss would seem to provide a favorable outcome in terms of diagnosis and prognosis of colorectal cancer. a systemic review of the literature was conducted via pubmed to identify relevant studies from january through may . the main outcome for this study is to assess whether patients who underwent bariatric surgery (restrictive and malabsorptive procedures) had an increased or decreased risk of colorectal cancer. all studies included in this meta-analysis are retrospective cohort studies. results were expressed as standard difference in means with standard error. statistical analysis was done using fixed-effects meta-analysis to compare the mean value of the two groups between bariatric surgery and non-surgery in patients with colorectal cancer. (comprehensive meta-analysis version . . software; biostat inc., englewood, nj). results: four out of studies were quantitatively assessed and included for meta-analysis. among the four studies, , underwent bariatric surgery and , did not undergo bariatric surgery. there is a significant decrease ( . ± . ; p= . ) in the risk in patients developing colorectal cancer in patients who underwent bariatric surgery compared to those who didn't get surgery. conclusion: bariatric surgery patients appear to have a decreased risk of colorectal cancer compared to patients who did not have bariatric surgery. guh jung seo, hyung-suk cho; department of colorectal surgery, dae han surgical clinic, gwangju, south korea introduction: the incidence of rectal carcinoid tumors is increasing due to the widespread use of screening colonoscopy. endoscopic mucosal resection (emr) is a useful method for small rectal carcinoid tumors (≤ mm) because of its simplicity, quick procedure and low complication rates. we aimed to describe our experience and evaluate the outcomes of emr for rectal carcinoid tumors. the patients enrolled in this study were patients with small rectal carcinoid tumors who underwent emr using a submucosal injection technique of epinephrinesaline mixture between august and october . all medical records, including characteristics of the patients and tumors, complications, were retrospectively reviewed. results: the patients were men and women, with a mean age of . years (range, - years). en block resection was performed by emr in all cases. the endoscopic mean size of tumors was . mm (range, - mm). the pathologically measured mean size of the resected specimens was . mm (range, - mm). the mean size of resected carcinoid tumors was . mm (range, . - mm). the tumor shape was submucosal tumor in and polyp in . histological examination revealed that cases had resection margin positive of tumor and case had undetermined resection margin of tumor. of the patients, patients underwent endoscopic treatment and patients underwent transanal excision. no residual tumor was found in additionally removed tissue. there were cases with emr-related complications: early postprocedural bleeding and postpolypectomy syndrome. there was no significant bleeding requiring blood transfusion or perforations. conclusion: endoscopic mucosal resection is considered to be a relatively safe and useful method for treatment of small rectal carcinoids in selected patients. background: disturbance of sexual function after an operation for rectal cancer has often occurred. the relationship between autonomic nerves and arteries in pelvis was examined. methods: clinical studies of male patients with resected rectal cancer were performed using snap gauge method, penile-brachial index and evoked bulvo-cavernous reflex. in canine experiments, pelvic splanchnic nerve (psn) electric stimulation, arterial flow measurement, corpus cavernosum pressure measurement and muscle strip study using drugs were evaluated. results: in clinical studies of male patients, transection of the hypogastric nerve (hgn) and the sympathetic trunk did not affect the erectile function in the postoperative course. in animal experiments transection of these nerves did not affect the increase in inner pressure of the penis cavernosum. in postoperative cases in which only one side of the lower grade branches of the psn (s ) were preserved, the erectile function was preserved. in animal experiments in which the psn of one side was disturbed, the ipa flow of the same side decreased, while the flow of the other side increased. we have evaluated the role of adrenergic components in the psn on the erectile function in the dog. the effect of norepinephrine hydrochloride on canine vascular smooth muscle was examined in vitro. vascular smooth muscle strips from the ipa relaxed longitudinally. electrical stimulation of the psn increased blood flow in the ipa and also elevated the cavernous pressure. these increases were blocked in part by phentolamine, but not by propranolol or atropine. the effects of cholinergic and adrenergic agonists and antagonists on mechanical responses were also examined in muscle strips obtained from various arteries in the intra-pelvic region including the ipa. norepinephrine induced contraction in the iliac artery and relaxation in the ipa, and both the contraction and relaxation responses were blocked by phentolamine but not by propranolol. these findings suggest that in the dog, α-adrenergic components projected through the psn may contribute to penile erection. conclusion: blood flow in the ipa was controlled significantly by the same side psn, but compensatory by the other side psn. it is also conceivable that the erectile function through the psn is controlled by the sympathetic nerve, not by the parasympathetic nerve. in postoperative cases in which only one side of the lower grade branches of the psn (s ) were preserved, the erectile function was preserved. introduction: currently, neoadjuvant chemo-radiotherapy (ncrt) followed by low anterior resection or abdominoperineal resection are the standard treatments for locally advanced rectal cancer. ncrt can improve resecability, achieve better sphincter preservation and reduce local recurrence. although total mesorectal excision is the standard treatment for advanced rectal cancer, recent trends in minimally invasive treatments led to an increase in local excision or "watch and wait" in patients with an excellent response to ncrt. the purpose of this study, part of an ongoing research, is critically evaluating the feasibility of "non-operative treatment" for rectal cancer in a district hospital. methods and procedures: a total of patients with rectal cancer, who where treated with ncrt from january to august at "carlo urbani" district hospital in jesi (italy), were retrospectively reviewed. all patients had histologically-confirmed primary adenocarcinoma of the rectum located within cm from the anal verge. the involved patients completed ncrt and had no recurrence disease, distant metastasis, synchronous malignancies. they were classified according to the mandard's tumor regression grade (trg) into two clusters: group a (trg - ) and b . results: the average age of people is . and were male. five patients underwent abdominoperineal resection and % fell within group a. six patients had lymph nodes involved. four patients suffered relevant complications, such as wound complication, anastomotic leak, operative reintervention and death. univariate analysis showed that the main predictors of tumor regression were the absence of lymph-nodes involvement from initial imaging (p. ), normal initial carcinoembryonic antigen level (p. ) and tumor downstaging in imaging (p. ). in addition, most relevant complications occurred to elderly patients although they observed a good clinical response. besides, % of patients were found to be complete pathologic responders upon examination of the surgical specimen. conclusions: the oncologic feasibility of non-operative management for the patients with complete clinical response after ncrt has been growing, but some studies have suggested lack of oncologic safety in these patients. the patients with a complete clinical response expect good survival, but they may still harbor residual disease. no consensus on "watch and wait" policy in the field of rectal cancer was obtained, yet. our data did not entirely support this policy although it might be the best strategy, based on the predictors of tumor regression, to avoid the complications associated with surgery in elderly patients with significant medical comorbidities and fear of a permanent stoma. introduction: conventional incision laparoscopic surgery procedure for rectal cancer is widely accepted as a successful alternative to laparotomy now, bestowing specific advantages without causing detriment to oncological outcome. evolving from this, single-incision laparoscopic surgery (sils) has been successfully utilized for the removal of colonic tumors, but the literature lacks sufficient data analyzing the suitability of sils for rectal cancer especially for total resection mesorectal excision (tme), particularlyon oncological outcome. we report the short-term clinical and oncological outcomes from a large cases retrospective analysis of observational study of sils for tme procedure of rectal cancer. methods: rectal cancer patients who underwent transumbilical single incision laparoscopic tme surgery were recruited in the current study. short-term perioperative clinical parameters and oncological outcomes were observed and all patients were followed up after surgery. then summarize the preliminary application results. results: operations were accomplished successfully with single incision laparoscopy, patients were converted to multiport approach, and was converted to laparotomy, no diverting ileostomy was performed. the average operative time was ( . ± . ) min, with an average blood loss of ( . ± . ) ml, the median postoperative hospital stay was ( . ± . ) days. all patients received a r resection and the surgical margin were conformed negative in all cases, the median number of harvested lymph node is ( . ± . ), the specimens met the requirement of tme. there were postoperational complications, no operation-related mortality or postoperative anastomotic leakage was observed. no patient appeared recurrent in a median follow up of months. conclusions: total mesorectal excision surgery for rectal cancer can be safely performed using transumbilical single incision laparoscopic technique, with acceptable short-term clinical and oncological outcome. surg endosc ( ) background: any surgical trauma induces an inflammatory response, which is considered as a negative factor in the general immune response, specially in malignant disease. the c-reactive protein (crp) is an acute phase protein often used as a marker of surgical trauma. stent treatment has been used as a treatment option for colonic obstruction in palliative cases for many years, and also as a bridge to surgery in selected cases. in a pilot study we compared the inflammatory response after acute stent treatment or surgery for malignant colonic obstruction. method: we compared two consecutive series of treatment of acute malignant colonic obstruction, stent treatment or emergency surgery during - . all patients were admitted with acute colonic obstruction due to colorectal cancer. choice of treatment was based on attending senior colorectal surgeons' preference, patient comorbidities and disseminated disease was considered. patient age, crp, time to first defecation and length of stay was recorded. results: a total of patients were identified in a retrospective analysis. patients had acute stent treatment and had acute surgical treatment for colonic obstruction, all due to colorectal cancer. median age was y ( - ) with no difference between the groups. there was no difference in metastatic disease between the groups. median time until first defecation after treatment was significantly shorter for the stented patients ( h ( - )) compared with those operated ( h ( - )) (p, ). median hospital stay was also shorter in the stent group, days ( - ), versus days ( - ) in the surgical group (p= , ). crp did not differ between the groups before treatment. both treatments resulted in increased crp levels at postoperative days and , but the crp levels were significantly higher in the surgical group than in the stent group at both time points (pod p= , , pod p, ) conclusion: acute stent treatment in colonic malignant obstruction seems to induce a less pronounced inflammatory response compared with surgery, as shown by a significantly reduced increase in postoperative crp resulting in shorter time to first defecation and a shorter hospital stay. introduction: meckel's diverticulum is the most common congenital abnormality in newborns, present in about - % of them. diagnostic of meckel's diverticulum requires a high index of suspicion, and even with the use of modern imaging technologies, they are often diagnosed intraoperatively. what to do when an asymptomatic diverticulum is found incidentally during surgery for other causes is a matter of discussion. objective: the aim of this article is to report symptomatic and asymptomatic incidentally found cases seen in a fourth-level hospital in colombia. the reports of the histopathologic examinations carried out in the hospital in the last years were reviewed searching for those containing meckel's diverticulum in their diagnosis. patients were divided in asymptomatic and symptomatic groups. the asymptomatic group was defined as patients who were operated for a different indication and a meckel's diverticulum was found incidentally. morbidity was divided in early and late complications after the initial surgery. results: from january to june , a total of pathology reports included the diagnosis meckel's diverticulum. a total of adult patients were retrieved. all of those patients with meckel's diverticulum a total of patients were symptomatic, being sbo the most common complication and required the surgical remove incidentally. conclusion: the correct approach of the patients with diverticular pathology allows the early identification and the appropriate management of the surgical complications that can be presented. robert j czuprynski, md, grace montenegro, md; saint louis university hospital presacral masses are a rare entity, with an incidence of . % and can be classified in several categories, including inflammatory, neurogenic, congenital, osseous and miscellaneous. in this case, a neuroendocrine tumor was identified with concern for iliac chain lymphatic and gluteal metastasis. the patient underwent abdominoperineal resection, excision of presacral mass, lymph node biopsy and omental flap. final pathology returned as a grade ii neuroendocrine tumor arising from a tailgut cyst. a year old female with a ten year history of recurrent perianal, ischiorectal and deep postanal abscesses presents with a presacral mass biopsy proven well-differentiated neuroendocrine tumor. octreotide scan demonstrated avidity for presacral mas as well as left intergluteal lymph node and two internal iliac lymph nodes. chromogranin a, neuron-specific enolase and serotonin markers were all negative. the patient was taken to the operating room and underwent abdominoperineal resection, resection of presacral mass and internal iliac nodes with an omental flap. neuroendocrine tumors arising from tailgut cysts of the presacral space are rare in nature. in a retrospective study from great britain, four of thirty one tailgut cysts had malignant transformation, so it is generally recommended to resect the cysts. in this case, the patient's tumor was a moderately differentiated, grade ii with extensive lymphovascular and perineural invasion. there are no prospective studies showing neoadjuvant therapies in neuroendocrine tumors of the presacral space. according nccn guidelines, patient is currently asymptomatic with low tumor burden. recommended treatment at this time is observation with surveillance tumor markers every - months or octreotide. anastomotic leakage has been commonly regarded as one of the toughing postoperative complications in laparoscopic mid/low rectal cancer surgery, attenuating the short-term clinical benefits. the left colic artery (lca) has been routinely central-ligated in dissection process to guarantee the oncological effects, which may potentially attribute to the postoperative ischemia-induced anastomotic leakage in the patients with left-colic vessel variation, e.g. bypass or absent of riolan arch. however, no specific study focuses on the surgical benefits of lca preservation compares to conventional ones. herein, we conduct a single center randomized controlled trial, demonstrating that lca-preserving technique shows significant reduction rate of postoperative leakage as well as overall complications comparing to the traditional central-ligation group. no difference in survival rate and recurrence in short term is found between the two groups. the lca-preserving strategy is proven to be repeatedly safe and feasible, potentially reduce the risk of anastomotic leakage with comparable short-term outcomes. further investigation is required for both the oncological safety and long-term prognosis for this innovative technique. background: three-photon imaging (tpi), which was based on the field of nonlinear optics and femtosecond lasers, has been proved to be able to provide the -dimensional ( d) morphological feature of living tissues without the administration of exogenous contrast agents. the purpose of this study is to investigate whether tpi could make a real-time histological d diagnosis for colorectal cancer compared with the gold standard hematoxylin-eosin (h-e). methods: this study was conducted between january and august . a total of patients diagnosed as colon or rectum carcinoma by preoperative colonoscopy were included. all patients received radical surgery. the fresh, unfixed and unstained full-thickness cancerous and the corresponding normal specimens in the same patient, were immediately prepared to receive tpi after surgery. for d visualization, the z-stacks were reconstructed. all tissue went through routine histological procedures. tpi images were compared with h-e by the same attending pathologist. results: the schematic diagram of tpi is shown in fig. a . peak tpi signal intensity excited at nm was detected in living tissues. the field of view (fov) was µm and the imaging deep was µm in each specimen. in normal specimens, glands lined regularly and characterized as a typical foveolar, which was comparable to h-e images ( fig. b and d ). in cancerous specimens, irregular tissue architecture and shape were identified by tpi, which was also validated by corresponding h-e images ( fig. c and e ). tpi images can be acquired with a view of d visualization. based on rates of correlation with pathological diagnosis, the accuracy, sensitivity, specificity, positive predictive value, negative predictive value were %, %, %, %, . %, respectively. conclusions: it is feasible to use tpi to make a real-time d optical diagnosis for colorectal cancer. with the miniaturization and integration of colonoscopy, tpi has the potential to make a real-time histological d diagnosis for colorectal cancer in the future, especially in low rectal cancer. erica pettke , abhinit shah , vesna cekic , daniel feingold , tracey arnell , nipa gandhi , carl winkler, md , richard whelan ; mount sinai west, columbia university introduction: alvimopan (alvim) is a peripherally acting µ-opioid receptor antagonist used to accelerate gastrointestinal functional recovery postoperatively (postop) after bowel resection. the purpose of this retrospective study was to compare the time to first flatus and bowel movement (bm) as well as length of stay (los) following elective minimally invasive colorectal resection (crr) in a group of patients (pts) who received alvimopan perioperatively (periop) vs a group that did not get this agent. methods: a data review from to from irb approved databases was carried out. operative, hospital and office charts were reviewed. routine use of alvim for elective crr cases was stared in . besides gi data, preoperative comorbidities and day postop complication rates were assessed. the results with periop alvim were compared to a no-alvim group. the students t and chi-square tests were used. results: a total of pts underwent elective crr. alvim was administered periop to pts ( %). the breakdown of indications between groups were similar. alvim pts were younger ( . vs. . years old, p= . ) and, as regards comorbidities, less likely to have heart disease (cad . % vs . %, other heart disease . % vs . %) but were otherwise similar. the rate of laparoscopic-assisted (alvim, . %; no alvim, %) and hand assisted or hybrid operations (alvim, . %; no alvim, %) were similar. alvim pts had significantly earlier return of flatus ( . vs . days) and first bm ( . vs . , p. for both) than the no alvim group. there was also a trend toward a shorter los ( . vs . days, p= . ) for the alvim group. overall complication rates were similar, however, alvim pts had lower rates of post-operative ileus ( . % vs . %, p. ), sssi's ( . vs %, p= . ), and blood transfusion ( . vs . %, p= . ) than the no alvim group. conclusion: the two groups compared were largely similar (most co-morbidities, indications, crr type) with the differences in age and cardiac issues noted. the impact of the higher rates of sssi's, blood transfusion, and mi in the no alvim group on gi function is unclear. pts who received alvim periop had an accelerated return of bowel function, decreased postoperative ileus and shorter length of stay. these results suggest that alvim is effective in reducing the postoperative ileus but further study is warranted. background: laparoscopic total proctocolectomy (tpc) is selected for minimally invasive surgical treatment of familial adenomatous polyposis (fap) and ulcerative colitis (uc). our policy of tpc is no diverting ileostomy for fap and creating ileostomy for ibd because most of the patients received steroid therapy. objective: we examined the outcome of laparoscopic tpc according to disease of fap and ibd (uc and crohn's disease). methods: twenty-three consecutive patients who underwent laparoscopic tpc between april and march were examined. the patients were divided into fap group and ibd group. results: seven patients of fap and patients of ibd (uc , crohn's disease ) underwent laparoscopic tpc or total colectomy. among them, patients (fap , ibd ) were cancerassociated cases. the procedures of the fap group was tpc with iaca in patients and hals total colectomy with ira in patient. the procedures of ibd group were tpc with iaca in patients, tpc with iaa in patients, total colectomy with ira in patients, of which hals cases. the mean operative time and blood loss were minutes, . g in the fap group and minutes, . g in the ibd group, respectively. diverting ileostomy was constructed in patients of only uc group. early complications of fap group were observed in cases (postoperative ileus , anastomotic leak with conservative treatment ), and those of ibd were observed in cases (ileus , anastomotic leak with conservative treatment , abdominal abscess , wound infection ). the median postoperative hospital stay was days in the fap group and days in the ibd group. complications requiring reoperation were cases (fap : intestinal obstruction, ibd : inflammation of stoma-closure site). no cancer recurrence and mortality were observed. one case of fap underwent additional transanal mucosal resection due to new lesion of adenoma. conclusions: laparoscopic total proctocolectomy for fap and ibd was performed safely, especially less complications occurred in fap patients without diverting ileostomy. in addition, followup of remaining mucosa is important in iaca and ira patients. treatment of complex anal fistula has always been a nightmare for surgeonsby conventional means. even the lowest and simple looking fistula at times comes out to be a complex one with high incidence of recurrence above %. most of the availability diagnostic including mri is nit conclusive and many a times the surgeon remains in a state of confusion as to what is going to come at the operation table. the conventional treatment modalities also usually leave the patient wounded needing almost to weeks to heal with a risk of sphincter damage and a high risk of recurrence. we would be presenting the technical details and results of our series of cases of complex anal fistula treated by video assisted endoscopic therapy. jun higashijima, phd, mitsuo shimada, professor, kozo yoshikawa, phd, takuya tokunaga, phd, masaaki nishi, phd, hideya kashihara, phd, chie takasu, phd, daichi ishikawa, phd; department of surgery, the university of tokushima background: one of the important causes for anastomotic leakage (al) in anterior resection is an insufficient blood flow of the stump. the hems (hyper eye medical system) and spies (laparoscopic icg system) can detect the blood flow of fresh organ intraoperatively by injection of indocyanine green (icg). and thermography also can evaluate the bloodflow less invasively. the aim of this study is to evaluate the usefulness of icg system and thermography in laparoscopic anterior resection. patients and methods: this study retrospectively included patients who underwent laparoscopic anterior resection for colon cancer with double stapling anastomosis procedure. blood flow evaluation of oral stumps was performed with measurement of fluorescence time (ft) using hems and spies. and bloodflow was also evaluated by thermography. result: evaluation by icg system: in all cases, the al rate was . % ( / cases). over ft cases, the al rate was %, higher than that of under s cases and these patinets need additional management, covering stoma or additional resection. and in border cases, ft * sec, al rate is . %, higher than under s cases. in these borderline cases, if covering stoma was performed in patinets with more than three well known risk factors, the al rate reduced to . % and false positive was . %. and under s cases, they need no additional management. evaluation by thermography: in residual intestine, the temperature was siginificantly higher than resected intestine ( . vs . ?, p. ). and the temperature in ft under s cases was significantly higher than over ft over s cases ( . vs . ?). the temperatue and ft was tended to be oppositely correlated (r = . ). conclusion: both icg system and thermography may be useful to avoid anastomotic leakage. introduction: some patients who undergo neoadjuvant chemoradiation therapy (crt) for rectal cancer achieve a pathologic complete response (pcr) in which no tumor cells are discovered during pathologic analysis of the resection specimen. achievement of pcr is correlated to improved prognoses relative to non-pcr counterparts. such correlations are not well established in the context of a community-based hospital. the study sought to examine response rates, recurrences, and survivals in locally advanced rectal cancer patients and compare patient outcomes to those achieved at major academic institutions. methods and procedures: a single-center retrospective chart review was performed at a local, community-based hospital. study population consisted of patients with locally advanced rectal cancer treated with neoadjuvant crt followed by surgical resection. patients with a history of metastasis, inflammatory bowel disease (ibd), hereditary cancer syndromes, concurrent or prior malignancy, and emergent surgery were excluded. results: patients ( . %) achieved pcr in the test population. across both groups, mean age (p =. ), gender (p=. ), and ethnicity (p=. ) were found to be comparable. mean interval between crt and or (p=. ), pre-op stage (p=. ), number of nodes (p=. ), radiation dose (p=. ), tumor location (p=. ), and days of follow-up (p=. ) presented statistically insignificant differences between groups. at years, non-pcr patients ( . %) had a recurrence with zero recurrences in the pcr group. -year mortality presented non-pcr patients ( . %) compared to pcr patient ( . %). conclusion: a multidisciplinary approach to rectal cancer consisting of standardized preoperative treatment and surgical resection can achieve patient outcomes and survival similar to those of larger academic institutions, even in the context of a community-based hospital. objective: the aim of this study was to assess safety and feasibility of total mesorectum excision (tme) within the holy plane based on embryology for rectal cancer. methods: prospectively collected data of consecutive patients with rectal cancer who underwent tatme from november to august were enrolled. surgical outcomes including tme completeness, operative time for tme completion, blood loss, complications, pathological findings and length of hospital stay were assessed. surgical procedure: after performing ractal lavage, self-retaining anal retractor was set, and anal dilators were used for an atraumatic introduction of the transanal access devise (gelpoint path). three of -mm trocars and one of -mm trocar were inserted through the gelpoint path in a quadrant shape. then the gelpoint path was introduced through the anal to rectum. after rectosigmoid colon was temporally clamped using an atraumatic endo bulldog clip, pneumoperitoneum was maintained at mmhg with carbon dioxide via an air seal platform. a purse-string suture using a polypropylen with -mm rounded needle was performed clock-wise to tightly occlude the rectum with a cm margin distal to the tumor. after irrigation with saline and marking dissection line with tattooing the rectal mucosa distal to the mucosal folds, a mucosal transection of rectum was initiated. then a full-thickness rectal transection was performed circumferentially. after dissection of rectococcygeal muscle at o'clock and rectourethral muscle in the anterior wall, circumferential sharp dissection within the holy plane was performed. dissection proceeded between the endopelvic fascia and the prehypogastric nerve fascia in the posterior plane, between the denonvilliers's fascia and the anterior mesorectum in the anterior plane, and between pelvic nerve and the mesorectum with recognition of the neurovascular bandle in the lateral plane. then the dissection connected to the abdominal plane via laparoscopic team with working together until tme completed. results: tme completion performed in ( . %) patients. thirty five ( . %) patients had negative of circumferential resection margin. mean of tme completion time and blood loss were min and g, respectively. one ( . %) patient had an intraoperative complication and ( . %) patients had postoperative complications. no other complications occurred. the length of hospital stay was days. conclusions: tatme within the holy plane on based on embryology is a safe and feasible procedure for rectal cancer. abstract: acromegaly is a debilitating condition marked by excessive production of growth hormone. this leads to disfiguration, cardiopulmonary complications, and increased risk for cancer. with up to a two-fold increased risk of developing colon cancer and worse prognosis for diagnosed patients, earlier and more frequent screening has been recommended. we present a case of a -year-old hispanic male with acromegaly who presented to our hospital with hematochezia and weight loss. a near-obstructing rectal adenocarcinoma with metastasis to the liver was discovered. after completing neoadjuvant chemoradiotherapy, he underwent laparoscopic low-anterior colon resection and simultaneous open hepatic trisegmentectomy. in this case report, we review the literature and current guidelines in screening this high-risk group of patients. introduction: in this study, we discovered that in cme for laparoscopic right hemi-colectomy starting at the ileocolic vessel and proceeds along the superior mesenteric artery (sma) achieved a better oncologic outcome compared with the conventional ones proceeding along the superior mesenteric vein (smv). methods and procedures: patients admitted to a shanghai minimally invasive surgical center were included from september to january and were randomly divided into two groups: study group (n = ) and conventional group (n = ). operation time, blood loss during surgery, liquid intake time, postoperative hospital stay, postoperative complications within days after surgery, specimen length, and number of lymph nodes harvested as well as the positive lymph node rate were observed and studied. results: there was no statistical difference between the two groups with the exception of number of lymph node dissected and the positive lymph node rate for stage iii colon cancer. the study group had more lymph node retrieved and also a higher positive rate compared with the conventional group. the mean number of lymph node retrieved of study group was . ± . , while the conventional group was . ± . (p. ). and the positive lymph node rate for study group was . %, the conventional group was . %. conclusion: when performing the laparoscopic right hemi-colectomy, dissecting the lymph node along with the left side of sma could be achievable and there were no differences of surgical outcomes compared with the conventional ways, while there was a higher number of lymph nodes dissected and positive rate probably leading to a better oncologic outcome. aims: we describe laparoscopic surgery for rectal cancer using needlescopic instruments performed at our department. methods: from to , cases of rectal cancer underwent surgery using needlescopic instruments: cases at rectosigmoid colon, at upper rectum, and at lower rectum. an umbilical camera port ( -mm) and two needlescopic instruments (endorelieftm) were directly punctured into the assistant surgical site. we started with port sites. in low rectum cancer cases, we kept the good pelvic visualization to lifting the peritoneum of the bladder onto the ventral side using the lone star retractor staystm. results: the median age was years ( - years), with males and females, and body mass index was . kg/ m ( - kg/m ). anterior resection was performed in cases, low anterior resection in cases, intersphincteric resection in cases, abdominoperineal resection in cases, hartmann's procedure in cases, and lateral lymph node dissection in case. in addition, one case of t b (bladder) was converted from laparoscopic to open surgery. however, there were no cases in which needlescopic instruments were replaced with conventional forceps. moreover, intraoperative complications related to the forceps were not observed. conclusions: in rectum cancer surgery, needlescopic instruments leave a small postoperative wound; healing is rapid and the cosmetic result is excellent. surgical safety is comparable to that using conventional forceps. there is no problem with the rigidity of needlescopic instruments. however, where the shaft is curved, operative control requires attention to mobility and directionality. in low rectum surgery, use of needlescopic instruments is limited due to the curvature of the shaft during the dissection of the anterior rectum wall, but it is possible to maintain a good field of view by using auxiliary equipment. therefore, more cases could be considered for surgeries using needlescopic instruments with the help of auxiliary equipment. introduction: anastomotic leaks are devastating complications of colorectal operations that lead to significant morbidity and potential mortality. inadequate tissue perfusion is considered a key contributor to anastomotic failure following colorectal operations. currently, clinical judgment is the most commonly used method for evaluating adequate blood supply to an anastomosis. more recently intraoperative laser angiography using indocyanine green (icg) has been utilized to assess tissue viability, particularly in reconstructive plastic surgery. this technology provides a real-time evaluation of tissue perfusion and is a helpful tool for intra-operative decisions, particularly in deciding to revise an intended colorectal anastomosis. our study aimed to determine if there is a statistical significance in colorectal anastomotic leak or abscess rate using icg compared to common clinical practice. methods and procedures: patients undergoing left-sided colorectal operations, between march and february , were retrospectively reviewed. patients' colorectal anastomoses were evaluated using icg angiography (icga) to qualitatively assess tissue perfusion (icg group). peri-operative and post-operative outcomes, including anastomotic leak and abscess rates, were compared to patients who had colorectal operations without icga (control group). the primary outcomes of intra-abdominal leak rate and intra-abdominal abscess rate were compared using exact chi-square tests. the secondary outcomes of -days or return, mortality, and readmission rate were compared using chi-square tests. all statistical analyses were performed using sas software. results: two leading indications for surgery included malignancy (n = ) and diverticulitis (n = ). the majority of patients either had a low anterior resection (n = ) or sigmoidectomy (n = ). all operations were primarily minimally invasive. no statistically significant difference was seen between the two groups in regards to patient demographics, rate of proximal diversion (p = . ), and splenic flexure mobilization (p = . ). patients in the icga group were more likely to have high ima ligation than in the control group ( . % vs. . %, p-value. ). of the icga group, of the patients underwent additional colonic resection while of the did not undergo additional colonic resection. there was no statistically significant difference in primary or secondary outcomes between the two groups. conclusion: icg angiography has become a helpful adjunct in determining adequate perfusion to an intended colorectal anastomosis. this data is unable to support any difference in patient outcome utilizing this technology over surgeons' visual and clinical assessment. our results may contribute to larger studies to determine if there is a true difference in anastomotic leak or abscess rate using this technology. objective: to investigate the feasibility and surgical strategy of complete mesocolic excision (cme) with completely medial access by "page-turning" approach (cmapa) for the laparoscopic right hemi-colectomy. the cmapa is a modified medial approach of cme, which focus on the exploration of surgical plane instead of the recognition of vessels. surgical procedures: ( ) start point: the anatomy projection of ileocolic vessel; ( ) expose the whole trunk of smv to the level of inferior edge of pancreas before ligating any branches, for the purpose of high tie and verifying their location; ( ) enter the intermesenteric space (ims) and right retrocolic space (rrcs) with cranial and right extension through transverse retrocolic space (trcs); ( ) complete mobilize the mesocolon and remove the tumor en-bloc. see figure ? . clinical outcome: from september to march , there were patients underwent cmapa in shanghai ruijin hospital. the average operation time was . ± . minutes, average blood loss was . ± . ml, number of lymph node was . ± . , average specimen length was . ± . cm, flatus time was . ± . days, fluid intake time was . ± . days and average hospital stay was . ± . days. the overall complications rate was . % ( / ). compared to traditional medial approach of cme performed in our center, the blood loss, operation time and hospital stay were significantly reduced by performing cmapa for laparoscopic right hemi-colectomy. conclusion: the advantage of the cmapa ( ) to avoid the laparoscopic "leverage effect" and "tunnel effect". ( ) to make the branches of superior mesenteric vessels more easily recognized. ( ) to offer surgeons an alternative route entering the trcs, ims and rrcs. ( ) to avoid repetitive flipping of the colon complying with the "no touch" principle, and to lower the requirements of assistants. figure : anatomy and surgical planes concerning cmapa. aim: we have reported a possibility of "one-stop shop" simulation for liver surgery by mri using gadoliniumethoxybenzyl-diethylenetriamine pentaacetic acid (eob-mri) (emerging technology, sages )., which is characterized by ( ) one-time examination, ( ) no-radiation exposure, ( ) demonstration of liver vasculatures including biliary tract, ( ) diagnosis of tumors, ( ) volumetry and ( ) estimation of liver functional reserve in each segment. the aim of this study is to investigate usefulness of "one-stop shop" simulation for liver surgery using eob-mri. methods: accuracy of liver vasculatures: d-reconstruction of dynamic eob-mri imaging was done by synapse vincent software (fujifilm medical co., ltd., japan), using a manual tracing method. visualization of hepatic vessels in eob-mri was compared with that in dynamic ct in patients. assessment of liver functional reserve: the standardized signal intensity (si) of each segment was calculated by si of each segment divided by si of the right erector spine muscle. the standardized total liver functional volume (tlfv) was calculated by ∑ [k= to ] (standardized si of segment (k) volume of segment (k)) divided by body surface area. the following formula of resection limit was established using normal liver cases ( % of the liver is resectable) and unresectable cirrhotic patients such as recipients of liver transplantation ( % of the liver is resectable). the estimated resection limit (%)= % (the standardized tlfv of the patient - )/ , . this formula was validated using other patients who underwent hepatectomy. results: accuracy of liver vasculatures: the liver simulation by eob-mri succeeded in demonstrating hepatic vasculatures including biliary tract, diagnosis of hepatic tumors, and volumetry without any radiation exposure. regarding the vessel anatomy at hilar area, biliary tract was more clearly visualized in eob-mri. regarding the hepatic artery, right and left hepatic arteries were well visualized in all cases, however, small-sized middle hepatic artery was visualized in only one out of patients. assessment of liver functional reserve: as a result of validation of the patients, one patient having resection volume with over the resection limit died of liver failure, however, the other cases within their resection limits did not suffer from liver failure. conclusion: "one-stop shop" liver surgery simulation could contribute to safety of liver surgery such as laparoscopic hepatectomy, because of no radiation exposure, accurate assessment of anatomical variations especially biliary tract, and helping decision making of resection volume. showing key steps of the procedure to be viewed. the in-studio program was hosted by an education specialist from the science center and a surgical resident from our institution, with laparoscopic instruments available for manipulation by participants. participants then viewed a video highlighting the roles of all healthcare providers involved in the specialty to be featured, including nurses, physicians, dietitians, psychologists, technologists, etc. live questions and answers were then encouraged between students and surgeons during the surgery broadcast. the program also expanded from high schools to vocational-technical colleges and nursing schools. results: during the - academic year there were sessions presented to schools, with student participants. by the - year this increased to sessions presented to schools, with participants. in sum, throughout the first years of the program, there were schools attending, with a total of , participants. of polled high school participants, % of responders acknowledged considering a career in healthcare after this experience. conclusion: over years, our program has grown steadily in popularity such that schools from several counties attend and regularly return, and we have been asked to expand the program to create a surgical summer camp for students interested in science and technology. live broadcast surgery in an elective, minimally invasive format provides unique visibility and access to surgical procedures for student audiences and promotes future interest in healthcare careers. surg endosc ( ) :s -s p improving trainees' self-assessment through gaze guidance introduction: effective learning to become competent in surgery depends on a trainee's ability to accurately recognize their strengths and weaknesses. however, a surgical trainee's self-assessment is poorly correlated with expert assessment. this study aimed to improve self-assessment by the visual gaze guidance provided through telestration in laparoscopic training. we hypothesized that visual conveyance of where to look or perform actions on the laparoscopic video enhances the trainees' awareness of the gaps in their skills and knowledge. methods and procedures: a lab-developed telestration system that enables the trainer to point or draw a free hand sketch over a laparoscopic video was used in the study (fig. ). seven surgical trainees ( surgical fellow, research fellow, pyg- and pyg- ) participated in a counterbalanced, within subjects controlled experiment, comparing standard guidance with telestration-supplemented guidance. the trainees performed four laparoscopic cholecystectomy tasks -mobilizing cystic duct and artery, clipping the duct, clipping the artery, and cutting the duct and artery, on a laparoscopic simulation. performance assessment, adapted from the global rating scale (grs) instrument, was completed by the trainers and trainees at the end of each task. the mean self-assessment scores were compared with the trainers' scores by the linear mixed model, where the trainees' performance indicated by the trainers' scores was control. the assessment alignment was evaluated by spearman's rho. results: the trainers' scores were significantly lower than the self-assessment scores in the standard guidance, while the scores of the trainers and trainees were much more similar (fig. ) . the correlation between the trainers' and trainees' assessment in telestration guidance was high (r= . , p. ), compared to the standard guidance (r= . , p= . ). the correlation comparison for each grs criterion shows a significant increase (p= . ) in the assessment alignment for depth perception in telestration guidance (r= . , p. ), compared to the standard guidance (r= . , p= . ) (fig. ) . the visual gaze guidance improved the alignment of assessment between the trainer and trainees, especially for the assessment alignment in depth perception. for visual gaze guidance to become an integrated part of the training, further work needs to be conducted to understand how gaze guidance change the nature of the training process. applying to surgical residency: what makes the best candidates? yann beaulieu, beng, louis guertin, md, frcsc, ariane p smith, md, margeret henri, md, frcsc, facs; university of montreal objective: while quotas for canadian surgical residency programs are at their lowest point in ten years, the number of canadian graduating medical students is at an apogee. this year, only spots in surgical residency programs were available for students applying to carms. undergraduate medical students individually collect anecdotal information regarding what influences admission to their surgical subspecialties of interest, as scarce literature covers the topic. we thus surveyed surgeons and residents to analyze the relative importance of modifiable factors and innate attributes in the selection of new surgical residents. methods: an electronic survey was sent to all surgeons and surgical residents affiliated with the university of montreal. participants were asked to specify their surgical subspecialty, their status, their level of experience and whether they were an active member of a residency selection committee. the subjective importance of predefined application elements and candidate qualities was assessed using -point likert-type items. results: of the surgeons and residents to whom the survey was sent, ( . %) and ( . %) completed the survey. evaluations of elective rotations and evaluations of core rotations were considered very important by . % and . % of responders respectively. regarding letters of recommendation, the content was rated very important ( . %) more often than the notoriety of the author ( . %). networking with key surgeons was considered the least important element to prioritize with % of negative assessments. with regards to the fundamental qualities of surgical candidates, the extremes were "clinical judgement" with . % and "innate technical ability" with . % of responders rating them very important. no significant differences in responses were observed between staffs and residents, between members and non-members of selection committees, between different levels of surgical experience and between surgical subspecialties. conclusion: clinical judgement and performance in core and elective rotations along with strong personalized letters of recommendation should be prioritized by medical students aiming for a surgical career. kazuhiko shinohara, phd, md; school of health science, tokyo university of technology background and objective: many types of training devices had been proposed since the early days of endoscopic surgery. however, they are too expensive for daily training of novices. we developed a simple and economical training device made of frozen fruit and agar. material and methods: to make this device, g of agar powder was added to ml of boiling water and boiled for min. the solution was then poured into a stainless steel tray containing frozen blueberries and lychees and refrigerated for h. basic maneuvers required during endoscopic dissection and resection of a tumor with laparoscopic forceps and electrosurgical devices were then performed using this agar model in a conventional laparoscopic training box. results: using this model, endoscopic dissection and enucleation of a tumor with an electrosurgical device could be practiced repeatedly with minimal expense and preparation. background: situs inversus totalis (sit) is a rare congenital anatomy and a challenging condition for laparoscopic surgeries because standardized strategy to overcome such anatomical difficulties. mirror-reversed video images of laparoscopic surgeries for patients with normal anatomy could help to develop surgical strategies for patients with sit. we had a chance to evaluate this idea with a treatment of a patient of early gastric cancer, and describe the surgical results of the case. patient and methods: seventy-two-year-old women with a history of sit was referred to our department for the treatment of early gastric cancer, and laparoscopic distal gastrectomy with d + lymphadenectomy was scheduled. a video record of the same surgery for a patient with similar physical attribute performed before then was retrieved, and was edited with a computer into full length, totally mirror-reversed images of the surgery. designated operator and assistant simulated the operation using the video several times before surgery. results: laparoscopic distal gastrectomy was performed with d + lymphadenectomy while the operator was on the left side of the patient and the assistant on the other side, being opposite positions as usual. laparoscopic b- reconstruction was followed using "delta anastomosis" technique reported by kanaya et al. total laparoscopic procedures were completed with the operation time of minutes and the blood loss below measurable limits. no appreciable complications were observed after surgery and the patient was discharged on postoperative day . no recurrence of the disease was detected until years after surgery, conclusion: although further validation is unlikely because of a rare incidence of this anatomy, the same technique would be recommended for one of the preoperative preparations for similar cases. background: surgical simulation is thought to provide a basis for improvement of resident surgical skill training, in the safety of a simulation setting. it is unclear whether surgical skills learned in a simulation curriculum actually contribute to the improvement of surgical skills when transferred to the or. methods: a ten question online survey was sent to attending surgeons and residents. the questionnaire focused on domains: confidence, independence, transferable skills, improvement of skills/knowledge and time spent on the simulation curriculum. evaluation data was collected and anonymously analyzed. background: minimally invasive surgery poses a unique learning curve due to the requirement for non-intuitive psychomotor skills. programmes such as the fundamentals of laparoscopic surgery (fls) provide mandatory training and certification for many residents. however, predictors of fls performance and retention remain to be described. this single-centre observational study aimed to assess for factors predicting the acquisition and retention of fls performance amongst a surgically naïve cohort. methods: laparoscopically naïve individuals were recruited consecutively from preclinical years of a medical university. participants completed five visuospatial and psychomotor tests followed by a questionnaire surveying demographics, extracurricular experiences and personality traits. individuals completed a baseline assessment of the five fls tasks evaluated by fls standards. subsequently, participants attended a -minute training-course over week one and two on inanimate box trainers. a post-training assessment was performed in week three to evaluate skill acquisition. participants were withdrawn from laparoscopic exposure and retested at four onemonth intervals to assess skill retention. introduction: bipolar energy can cause thermal injury to adjacent organs when used improperly. sages fuse curriculum provides didactic knowledge on principles and best practices for safety, but there is no hands-on component to practice these skills. the objective of this study is to compare the effectiveness of the vest™ bipolar training module in addition to the fuse curriculum. methods and procedures: the study was a mixed design with two groups, control and simulation. after a pre-test that assessed their baseline knowledge, the subjects were randomized to two groups. both groups were given a min presentation, reading materials from the fuse manual and an online didactic module on bipolar energy. the simulation group also practiced on the simulator for one session that consisted of five trials on the effect of activation time on thermal damage and the importance of providing a margin of safety by sealing short gastric vessels. after one week the performance of both groups was assessed using a post-questionnaire. one week after the post-test both groups performed sealing of vessels on an explanted porcine mesentery with vessels perfused. their performance was videotaped and their activation times were recorded. a total safety score was calculated by assessing the proximity of the location of activation to the intestine by two independent raters. wilcoxon -signed rank and mann-whitney u tests were used to assess difference within and between groups. results: a total of residents ( in each group) participated in this irb approved study. median test scores for both groups increased (simulation, p= . and control, p= . ). no difference was found between the two groups in their pre-test (p= . ) and post-test (p= . ) scores indicating learning. the median total activation time for control group was higher ( . s) compared to simulation ( . s) but was not statistically significant (p= . ). there was a moderate agreement between two raters for margin of safety (kappa= . , p. ). total safety scores showed no difference between the two groups (p= . ). conclusions: subjects with simulation training had lower activation time compared to control. training for margin of safety requires more simulation refinement. small sample size and variations in the explanted models contributed to variability in data but even with small sample size, simulation training along with the fuse curriculum trended towards being more beneficial than the fuse curriculum alone. the general, that aims to build educational infrastructure and standardize training and education in laparoscopy throughout mexico. ilap participants engage in didactic and hands-on modules in educational theory, laparoscopic techniques, and simulation based education (sbe), and then develop and implement a -day sbe course for local trainees. the purposes of this study were to understand the existing educational environment at a single institution in mexico and measure the changes in perceptions, attitudes, and engagement in surgical education after an intensive training course. methods and procedures: all faculty and of general surgery resident participants completed a survey that contained items designed to assess the existing educational environment at a large, public hospital in mexico. using a -point likert scale, residents self-rated the quality of faculty feedback and the learning environment within their institution ( =strongly disagree, = neutral, =strongly agree). faculty rated their perceptions of the same educational themes. upon completion of a faculty-lead simulation course, residents rated the educational environment during the course. faculty provided additional qualitative feedback. descriptive analyses were performed. irb-exemption was obtained through lurie children's hospital. results: discordance existed in perceptions of the existing educational environment. the greatest disparity between resident and faculty perceptions included "faculty provide sufficient feedback in the operating room" ( % vs. %), "faculty promote an active learning environment" ( % vs. %), and "residents may ask questions without fear of negative evaluation" ( % vs. %). faculty and residents agreed with "residents are sometimes afraid to speak up in the operating room for fear of retaliation" ( % each). post-course evaluations (n= ) revealed universal improvement in all educational themes during the simulation course. qualitative feedback revealed most faculty plan to incorporate open communication and safe learning into their practice. residents were equally positive, with % optimistic that they will see changes within the educational environment. conclusions: significant discordance exists in resident and faculty perceptions of the educational environment at a large teaching hospital in guadalajara, mexico. after participation in the ilap course, residents noted demonstrable change in the faculty approach to education and feedback, and both faculty and residents expressed optimism for increased engagement in education. the immediate successes of the ilap initiative should be followed over time, as the ultimate measure of success is sustainability and scalability throughout mexico. background: laparoscopic anterior resection is technically challenging and the learning curve is long. well-designed formative assessments can provide trainees effective and constructive feedback, an important element in efficient learning. previously reported assessments for laparoscopic colorectal procedures were developed for summative assessment. we aimed to develop a formative assessment tool to evaluate competence and provide trainees with effective feedback in laparoscopic anterior resection. methods: the assessment tool was developed by an expert panel from mcgill university affiliated hospitals. the procedure was deconstructed into a series of sequential steps including general domains, surgical principles, injury prevention and technical skills specific to laparoscopic anterior resection. the tool contains discrete items with global rating scales for each step of the operation; each domain was scored using a -point likert scale, with anchors for scores of , and . each operation was assessed through direct observation in the operating-room by the attending, a trained observer, and trainees themselves. intraclass correlation coefficients (iccs) were calculated to estimate interrater reliability for ( ) attending surgeon and trained observer, ( ) attending surgeon and self-assessment, and ( ) trained observer and self-assessment. internal consistency was measured using cronbach's alpha. comparison between training levels was done using mann-whitney u-test. the global operative assessment of laparoscopic skills (goals) was also used to assess trainees' general laproscopic skills. spearman's correlation was used to determine association between goals and this procedure-specific tool. overall usefulness of this tool was evaluated using a cm visual analog scale. results: in this pilot study, fourteen operations, performed by experienced surgeons and trainees were assessed. the icc between ( ) attending surgeon and observer was . ( % ci . to . ) ( ) observer and self-assessment was . ( % ci . to . ), and ( ) attending surgeon and self-assessment was . ( % ci - . to . ). the internal consistency of the items was excellent (cronbach's α= . ). there was a significant difference in median total score between experienced surgeons and trainees ( . ± . vs. . ± . ; p= . ). there was strong correlation (r= . ) between goals and this procedure-specific score. overall usefulness of this assessment tool was rated as . ± . . all assessments were completed in about minutes. conclusions: we present a new procedure-specific formative assessment tool for laparoscopic anterior resection and provide preliminary evidence of its reliability and validity. this formative assessment tool could be used for constructive feedback and tracking performance in competencybased surgical training. cullen introduction: one of the key challenges to the proliferation of endoscopic submucosal dissection (esd) in the west has been a lack of training platforms. therefore, the virtual endoluminal surgery simulator (vess) is being developed as a training tool for esd. the aim of our study is to inform the design of vess using cognitive task analysis (cta), which is a human factors engineering framework to describe practitioners' mental models and cognitive processes and incorporate insights into the simulator's design. methods and procedures: cta-based interview questions were developed to probe the cognitive challenges and strategies employed at each stage of the esd procedure. six esd practitioners were interviewed for varying lengths of time. two of these interviews were conducted simultaneously during an observation of a training workshop where the cta participants were instructors (total observation time was five hours, and interview time was * minutes). another interview was conducted during observation of esd procedures (total observation time was hours, and interview time was * minutes). participants had varying levels of experience in esd, with of them being 'super-experts' (exclusively esd exponents), an 'expert' and a fellow. a cta of the data is currently being conducted to systematically inform design of functionalities in the simulator. results: analysis of our data highlights a few prominent themes at each stage of esd: goals, challenges (e.g., avoiding perforation of muscularis); points of decision-making (e.g., partial or full incision for boundary demarcation); skills involved (e.g., dissection); and ambiguity (e.g., unclear lesion boundaries). participants also described risks associated with each stage of esd and strategies to prevent or overcome the same. conclusions: qualitative data for a cta were collected through observations and interviews of esd practitioners. preliminary analysis has indicated prominent themes to consider in the design of the training simulator. the next step in the study is to conduct a full-scale cta of esd based on the current data. the ultimate benefit of the cta would be to incorporate the results into informing the design of vess in a way that is compatible with the mental models of esd trainees, thus enhancing the fidelity and effectiveness of the simulator. background: colonoscopy is an important diagnostic and therapeutic procedure in the management of colonic disease; achieving competence during residency is an integral part of performing high-quality colonoscopy in-practice, regardless of specialty. there is debate and controversy however, regarding what, if any, number of procedures achieves said proficiency. furthermore, there is significant heterogeneity in the current guidelines and studies published to-date on the definition of competence in colonoscopy. objective: to determine individualized learning curves as an alternative to 'number of procedures' for assessing colonoscopy competence. methods and procedures: this is a multi-institutional prospective cohort study involving eleven surgical trainees (novice endoscopists). the main outcome, colonoscopy competence, was assessed by determining the independent colonoscopy completion rate (iccr), the number of procedures required to reach % independent colonoscopy completion and polyp detection rate. individual and overall iccr were calculated using moving average analysis. conclusions: while a benchmark for a minimum number of procedures may be necessary to allow supervisors to adequately assess performance, it is difficult to determine what number is optimal. there appears to be significant heterogeneity in both overall number of colonoscopies completed by each resident, as well as the mean iccr and the number of procedures required to reach the current benchmark for competency. the use of learning curves allows real-time tracking of progress and training tailored to the individual, as we move forward in the era of competency-based medical education. background: with the growing popularity of robotic-assisted surgery, new methods for evaluation of technical skill are necessary to determine when a surgeon is qualified to perform an operation independently. current evaluation methods are limited to point likert scales which require a degree of subjective scoring. surgeons in training need an objective method of evaluation to view progress and target areas for improvement. one method of objectively evaluating surgical performance is a cumulative sum control chart (cusum). by plotting consecutive operative outcomes on a cusum chart, surgeons can view their learning curve for a given task. another method of objective evaluation is the dv logger®, or "black box," which records objective measurements directly from the da vinci® system. methods: we followed two hpb fellows during dry lab simulation of robotic-assisted hepaticojejunostomy reconstructions using biotissues to model a portion of a whipple procedure. we simultaneously recorded objective measurements of dexterity from the da vinci® system and performed cusum analyses for each procedural step. we modeled each variable using machine learning (a self-correcting and autoregressive modeling tool) to reflect the fellows' learning curves for each task. statistically significant objective variables were then combined into a single formula to create an operative robotic index (ori). results: variables that significantly improved over the course of the simulation included completion time (p= . ), economy of motion in arm (p= . ), number of times head was removed from the console (p= . ), total time left master manipulator was active (p= . ), total time right master manipulator was active (p. ), and total time that any arm was active (p\ . ). the inflection points of our cusum charts and plots of objective variables both showed improvement in technical performance beginning between trials and [ fig. and fig. ]. the operative robotic index showed a strong fit to our observed data and improved with additional trials (r = . ). [ figure ]. conclusions: in this study we identified objective variables recorded by the da vinci® system which correlated with the technical dexterity of fellows during a robotics dry lab. we broke a complex procedure down in stepwise fashion with cusum analyses to determine targets for improvement. using variables which correlated with the improved performance of the fellows, we effectively modeled the learning curve with the creation of an operative robotics index (ori). this study successfully models the learning curve of novice robotic surgeons using a novel combination of objective measures. georg wiese, md, paula veldhuis, steve eubanks, md, facs, scott w bloom, md, frcsc, facs; florida hospital institute for surgical advancement introduction: robotic surgery is a specialized skill which requires time and resources to master. in a general surgery residency program that seeks to train competent surgeons in both open, laparoscopic and endoscopic techniques it is difficult to see where adding robotic training will be of benefit and at what cost this will be to the remaining surgical skills. we therefore sought to ascertain robotic surgery's current role in the training of new general surgeons by soliciting the opinions of current general surgery program directors on the role of robotic surgery at their respective institutions. methods: an irb approved survey was created and sent to general surgery program directors across the country to assess how robotic surgery training is being integrated into current surgical training. the survey was sent via email to publicly available email addresses from the acgme website of program directors. it was voluntary in nature and consisted of questions regarding current status of robotic training in residency as well as future goals. results: overall response from our pd survey were at % of the surgical programs with addresses available via acgme, though responses continue to be submitted at the time of this abstract. approximately % of all respondents are from independent, university based programs. % felt that robotics was an emerging skillset important for residents to master versus % feeling that it was more appropriate for fellowship. all respondents noted that robotic surgeons were present at their institution, % within the core faculty, and % indicated that they were actively recruiting robotically trained surgeons. additionally, % of programs indicated that residents were exposed to robotic surgery, % of these on core general surgery rotations. % of respondents indicated that they had a formal robotic training curriculum with % of programs taking measures to integrate robotics into the future curriculum though % lacked specific milestones for such training. finally, opinion was evenly divided among respondents as to whether one could sign off on residents to perform robotic assisted cases upon completion of pgy year with % agreeing with that statement and the remainder indicating some additional training would be necessary. conclusions: our study highlights the emerging field of robotic assisted mis surgery and its increasing role in residency training. it is evident from the data, that robotic surgery is a growing part of residency experience. importantly, however, milestones were significantly lacking for determining resident progress in robotic training. introduction: in chile, medical students have the opportunity to undertake a month-long medicine elective (me) in a community hospital, primary care center or emergency department within the country at the end of their first clinical year. due to the lack of opportunities to practice suturing in the first years, students usually do not have an optimal performance in this type of medical procedure during the me. simulation training programs in suturing improve technical skills, selfconfidence and patient safety in the medical internship. the objective of this study is to evaluate the impact of implementing a simulated suture training program earlier in the medical curriculum, before the me. methods: we conducted a prospective, randomized controlled trial with medical students at the end of their first clinical year. they were randomized into two equal groups. the intervention group received an intensive suture training program consisting in one theory class, four practical sessions and effective feedback from an expert surgeon. the control group did not receive training, remaining with the classic opportunistic learning approach during the me. after the me, all students undertook an electronic survey. statistical analysis was performed on the answers of both groups. per protocol analysis was applied. results: there were no statistical differences between groups in terms of age and sex. four students did not complete the training program. one student in the control group did not reply to the survey. higher self-confidence with regards to suturing was reported in the intervention group in comparison with the control group [ / ( %) vs / ( %), p, ]. also, a greater student desire to carry out suture-related procedures was reported in the intervention group than the control group [ / ( %) vs / ( %), p, ]. in addition, a lower rate of overseeing physician intervention was reported in the intervention group [ / ( %) vs / ( %), p, ] ( table ) . a greater number of patients requiring sutures were treated by the intervention group than the control group, with a median of patients ( - ) against ( ) ( ) ( ) ( ) . the intervention group performed a higher number of sutures with a median of ( - ) vs ( - ), with a statistically significant difference (p, ) in both cases (fig. ) . conclusion: a simulated suture training program prior to the me generates a positive impact on medical students by improving self-confidence and desire to attend patients that require sutures. this leads to a higher rate of both exposure to suture techniques and suture execution. introduction: measuring performance in the operating room (or) is challenging. performance is a multifaceted construct a complex interaction of many behaviors and actions that reflect an individual's knowledge and skill. no assessment tool to date provides an expertise-based, comprehensive evaluation of the various aptitudes necessary to excel in the or, especially with respect to advanced cognitive skills. using qualitative methodologies, we previously defined behavioral themes that guide surgeons' behaviors, decisions, and actions, within a universal framework of domains that reflect intra-operative performance. the purpose of this pilot study was to use this framework to derive a comprehensive assessment tool and to obtain evidence for its validity as a measure of intra-operative performance. methods: an assessment tool was developed by a panel of surgeons and surgical trainees based on the five-domain model of intra-operative performance: ) psychomotor skills; ) declarative knowledge; ) interpersonal skills (two items); ) personal resourcefulness, and ) advanced cognitive skills (ten items). all items were rated on an ordinal scale of (inadequate) to (expert) and equally weighted. surgical residents and surgeons from a single academic center were evaluated on their performance during standard general surgery operations, for example, open inguinal hernia repair and laparoscopic cholecystectomy. for residents, there were evaluators -the attending surgeon and an observing surgeon. attending surgeons evaluated their own performances and were also assessed by observing surgeons. internal consistency, inter-rater reliability, and correlation of total scores with training level (junior residents, senior residents, staff surgeons) were calculated. likert scale questionnaires were administered to evaluate the tool's usability, feasibility, and educational value. results: fifteen subjects ( junior residents, senior residents, surgeons) participated. the total score on the assessment demonstrated significant differences between training levels ( figure) . inter-rater reliability was high (interclass correlation coefficient= . ), as were internal consistency between each domain score (cronbach's alpha= . ), internal consistency amongst items in the advanced cognitive skill domain (cronbach's alpha= . ), and internal consistency amongst items in the interpersonal skills domain (cronbach's alpha= . ). all assessments required less than five minutes to complete. overall, evaluators agreed that the assessment tool was easy to use, was comprehensive, and should be used routinely throughout training to track performance and provide formative feedback. conclusion: in this pilot study, we developed a comprehensive assessment tool for intra-operative performance and provide preliminary validity evidence for the score. surg endosc ( ) introduction: the purpose of this study was to evaluate the validity of our developed system for assessing suturing skills in laparoscopic surgery (fig. ) . we have updated numbers of participants and a comparison method compared with the last year report. methods and procedures: fig. shows our developed computerized system for objective assessment of suturing skills by using a laparoscopic intestinal suturing model, e-lap. the system includes a new artificial intestinal model that mimics living tissue and pressure-measuring and image-processing devices. each examinee performs a specific skill using the artificial model, which is linked to a suture simulator instruction evaluation unit. the model uses internal air pressure measurements and image processing to evaluate suturing skills. five criteria, scored on a five-grade scale, were used to evaluate participants' skills ( fig. ) . the volume of air pressure leak was determined by the volume of air inside the sutured artificial intestine. for example, for the criterion "air pressure leakage", the approximate midpoint of the acceptable range was grade . values lower than the minimum acceptable value received lower grades and those above the midpoint of the acceptable range higher grades. we enrolled surgeons who participated a simulator competition event at the th annual meeting of the japan society for endoscopic surgery (jses houston methodist hosptial, baylor college of medicine introduction: the sages flexible endoscopy course for minimally-invasive surgery (mis) fellows has been shown to improve confidence and skills in performing gi endoscopy. this study evaluated the long-term retention of these confidence levels and investigated how fellows have changed practices within their fellowships as a result of the course. methods: participating mis fellows completed surveys six months after the course. respondents rated their confidence to independently perform sixteen endoscopic procedures ( =not at all; =very). while the pre-and post-course surveys identified anticipated endoscopy uses and barriers to use, the -month follow-up survey evaluated actual usage and barriers to use in each fellow's practice. respondents also noted participation in additional skills courses and status of fundamentals of endoscopic surgery (fes) certification. comparison of responses from the immediate postcourse survey to the -month follow-up survey were examined. mcnemar and paired t-tests were used for analyses. results: twenty-three of ( %) course participants returned the -month survey. % had passed the fes skills examination and % had attended another flexible endoscopy course. no major barriers to endoscopy use were identified. in fact, fellows reported less competition with gi providers as a barrier to practice compared to their original post-course expectations ( % versus %, p. ). in addition, confidence was maintained in performing the majority of the endoscopic procedures, although fellows reported significant decreases in confidence in independently performing snare polypectomy (− %; p. ), control of variceal bleeding (− %; p. ), colonic stenting (− %; p. ), barrx (− %; p. ), and tif (− %; p. ). fewer fellows used the gi suite to manage surgical problems than was anticipated post course ( % versus %, p. ). fellows without fes certification reported loss in confidence to independently perform barrx (− %; p. ) and colonic stenting (− %; p. ), and also a % decrease in the use of gi suite to manage surgical problems (p. ) fellows who passed fes noted no significant loss of independence, changes in use, or barriers to use. % of fellows made additional partnerships with industry after the course. % stated flexible endoscopy has influenced their post-fellowship job choice. % would recommend the course to other fellows. the sages flexible endoscopy course for mis fellows results in long-term practice changes with participating fellows maintaining confidence to perform the majority of taught endoscopic procedures six months later, and over % reporting that flexible endoscopy influenced their career choice. additionally, fellows experienced no major barriers to implementing endoscopy into practice. the materials and methods: at our center, we formulated a laparoscopic mentorship program where a senior consultant was paired with a particular trainee resident for a period of weeks. consultants & residents were a part of the study. the or schedules were rearranged to accommodate these pairs. an evaluation of the residents' views was performed prior to the study and once at its completion, using a simple questionnaire with each parameter scored between & . results and discussion: continuous, consistent evaluation by a consultant over an extended period of time allowed them to assess their assigned resident's laparoscopic skill set. all pairs observed an increased frequency of errors being noticed & improved upon. the consultants stressed upon shedding undesirable operative habits. there was a significant improvement in residents' scores at the end of the short study. conclusion: we found that the short-term mentorship program was easy to incorporate within our or schedule and was well received by the participants. continuous short rotations under senior consultants appear to allow residents to not only fully observe and imbibe correct operative techniques, but also helps shed unfavorable habits. we are currently amid the second cycle of our study & looking forward to the results at the end of this academic year. introduction: colorectal cancer is one of the most common cancers in the united states. endoscopic submucosal dissection (esd) is an emerging minimally invasive technique that allows complete en-bloc resection and a much lower recurrence rate at long-term follow-ups. however, performing colorectal esd is technically demanding since the colorectal wall is thin and constantly moving, and potentially higher rates of complications (e.g., bleeding and perforations). hence, an adequate training for colorectal esd is needed to acquire basic proficiency with minimum complications. objectives: a virtual reality (vr)-based simulator with visual and haptic feedback for training in colorectal esd is being developed, which the aim to allow trainees to attain competence in a controlled environment with no risk to patients. in this work, a newly developed application of the virtual simulator that promotes the endoscopists to perform and assess technical skills in esd is developed. training tasks are built based on physics-based computational models of human anatomy with tumors. methods: the main modules of the vr-based simulator for colorectal esd involve: ( ) rendering; ( ) haptic interface; ( ) physics-based simulation; and ( ) performance recording and assessment metrics. the rendering engine allows surgical tasks to be performed in the three-dimensional virtual environment. haptic feedback mechanisms allow users to physically feel the interaction forces. physics-based simulation technologies are employed to enable the complicated simulation for performing virtual surgical tool-tissue interactions. the simulator can also collect learners' performance data to offer feedback based on the built-in metrics. results: four training tasks involving marking, injection solution, circumferential cutting, and submucosal dissection are designed to practice skills with different surgical tools. the marking task aims to identify the lesion. the injection solution task minimizes the risk of bleeding and perforation to protect the muscularis. in the circumferential cutting task, the objective is initial incision of the lesion with the surgical tools. the objective of the dissection task is to remove the tumor from the connective tissue of the submucosa under the lesion. conclusions: the vr-based simulator enables realistic esd tasks to provide a possibility for developing, validating and objectively evaluating the performance metrics in colorectal esd training, and offers an opportunity to rise up the learning curve before application to patients. background: the virtual translumenal endoscopic surgery trainer (vtest) simulator is a virtual reality system that was designed to train the hybrid-notes technique. transfer of skill acquired while training on the vtest was measured in a near-real cholecystectomy procedure staged in the easie-r model. methods: sixteen medical students were divided randomly and evenly into groups: control, training. all subjects performed the cholecystectomy procedure on the vtest simulator to establish a baseline (pre-test). the training group received training sessions, over a period of consecutive weeks, consisting of trials per session or as many trials as can be accomplished in one hour, whichever was achieved first. at the end of the training period, all subjects performed one trial on the vtest simulator (post-test), and again to weeks later (retention test). two months after that, subjects performed the hybrid-notes cholecystectomy procedure on an easie-r model. performance with the easie-r simulator was video-recorded, and three tasks within the cholecystectomy procedure were isolated for evaluation: clipping, cutting, and dissecting the gallbladder. objective performance measures, such as time and error, were extracted from the videos by two independent reviewers, while subjective performance was scored by four expert surgeons who were blinded to the training conditions. expert reviewers used a modified version of the operative performance rating system by the american board of surgery and the objective structured assessment of technical skills (osats) tool. results: there was no difference in task completion time between the control and training groups, (t( )= . , p =. ) in the cutting and clipping tasks. however, there was a significant difference in the number of errors, t( )=- . , p=. . there was no difference in subjective performance between the training groups for the clipping and cutting tasks. in the gallbladder dissection task, however, there was a statistical significance in "instrument handling" based on one of the surgeons' ratings (t( )= . , p=. ), and a statistical significance in "time and motion" based on another surgeon's rating (t( )= . , p=. ). conclusions: results indicate that weeks of training on the vtest simulator did not allow the subjects to transfer their learned skills equally to the near-real environment, even though they retained the skills when tested for retention. this new insight suggests that modification of the training method for different types of surgical skills may be warranted to optimize their transfer to the real environment. examining conclusions: this study provides evidence to suggest that for bariatric surgeons, experience and skills acquired in performing non-bariatric surgery may not translate to improved outcomes in bariatric surgery. as seen in this study, improvement in bariatric surgical outcomes is likely more dependent on experience specifically performing bariatric procedures. as there may be no benefit acquired from performing surrogate procedures, this may have implications in the design of subspecialty training programs and for accreditation purposes. . a universally adjustable cellphone holder was used where smartphones could be placed inside the fls box in order to capture the task from a similar angle as the onboard camera. residents were able to use their own smartphones to record their performance on each of the five fls tasks in high definition (hd) quality. after each practicing session, they would upload their videos to a designated folder on a password-protected computer in the simulation lab. this folder was linked to a cloud-based storage system that fls instructor had exclusive access. the faculty was able to review each video in the next hours and provide immediate feedback to the residents via email, over the phone or in-person. the video library of performance also allowed the instructor to track the progress of the residents and whether they reached proficiency level in all five tasks to take the fls examination. this program was offered to all surgical trainees. results: utilization of simulation lab to practice fls tasks increased significantly across all postgraduate years after implementation of this model. six residents took the fls examination. the passing rate of the residents remained the same ( % before and after) but their scores in fls manual skills improved significantly compared to the group prior to implementation. the residents evaluated this change positively and reported that the use of videos and immediate feedback by faculty was a valuable intervention in their learning experience. conclusions: the smartphone cameras are readily available and can be used for telementoring. incorporation of telementoring in standard proficiency based fls training can promote self-directed learning and improve the access to experts for immediate feedback as a crucial element of effective training in acquisition of laparoscopic skills. background: it is important that making individual procedures a language, and an objective qualitative evaluation for the laproscopic training. recently, task training and the sham operation using the virtual simulator are carried out for medical students as the basic laparoscopic maneuver training, but there are few reports of objective qualitative evaluation for the training. in this study, we investigated rubric evaluation as the qualitative evaluation for laparoscopic training. materials and methods: one hundred and six students in th grade of tokushima univ. were participated. basic laparoscopic task training (gummy band ligation, beads transfer, delivery of beads, gauze excision) with training box and sham laparoscopic cholecystectomy with virtual simulator were performed. task execution time and rubric evaluation which includes the evaluation standard that became a language for each maneuver were performed before and after basic task training and sham operation. the group who are bad at laparoscopic maneuver was decided by time exceeded in tasks more than two from before practice. relationship between the group who are bad at laparoscopic maneuver and the group which self-evaluation was higher in a rubric evaluation was investigated. results: in basic task training, average task execution time in all students was shortened after practice compared with before practice, but investigated individual, students exceeded in more than two tasks. rubric evaluation in basic task training showed no difference between self-evaluation and evaluation by tutor before and after practice. in sham laparoscopic cholecystectomy, all students and tutor showed high score by rubric evaluation after practice compared with before practice. some students showed higher score than tutor, especially in part of extension of operation field by elevation of the gall bladder, exposure of triangle of calot, and exposure of cystic duct. students who showed high score by self-evaluation in many maneuver of sham laparoscopic cholecystectomy also exceeded in more than two basic tasks. conclusions: as rubric evaluation showed the point of the maneuver is made a language definitely, it was useful for an objective qualitative evaluation for laparoscopic training. pre introduction: bariatric surgery candidates have the opportunity to research bariatric surgeons and hospitals prior to scheduling their elective surgery. pre-operative information sessions are important tools for bariatric surgeons to provide patient education while increasing their patient population. online education is becoming increasingly popular, but its utility over in-person education is uncertain. our objective was to compare patients attending the two most commonly used educational formats: online (webinars) and in-person (seminars) and determine which were more likely to undergo bariatric surgery. methods: we conducted a retrospective cohort study of , patients who attended pre-operative information sessions from january to december by reviewing data maintained by the obesity, prevention, policy and management (oppm) database from our institution. the patients were divided into two groups: those who attended an in-person session (n= ) and those who attended an online session (n= , ). the proportion of patients who went on to have bariatric surgery was compared between the two groups. to categorize the study sample, patient demographics, surgeon providing the information session, and procedure performed were compared between groups. multivariate logistic regression model was applied to compare the effectiveness of in-person session and online session. results: of , patients analyzed, % attended online information sessions ( % female, mean age ). the remaining % attended in-person information sessions ( % female, mean age ). analysis found that . % of patients who attended online information sessions went on to have a bariatric surgical procedure, while . % of patients who attended in-person sessions went on to have a bariatric surgical procedure. after controlling for differences in age and gender, results of multivariate logistic regression analysis indicate that patients who attended inperson sessions were % more likely to have a bariatric surgical procedure than patients who attended an online session ( introduction: knot security is the ability of knots to resist slippage as force is applied, and the optimal number of throws to ensure a secure knot improves efficiency and outcome. the literature on the accepted number of throws per type of suture material has been largely anecdotal, often referring to throws for silk, for polyglactin (vicryl), five for polydioxanone (pds), and six for polyproprolene (prolene). we report a pilot knot-tying study of four suture types to determine optimal numbers of throws. materials and methods: four senior general surgery residents (pgy- and above) and four attending surgeons participated. participants viewed a standardized instructional video and a one-handed knot-tying tutorial. they were instructed to tie one-handed knots, beginning each knot with two throws in the same direction, and square the third and subsequent throws in the opposite direction. each surgeon tied knots, using differenttypes of - suture material: silk, polyglactin, polydioxanone, and polyproprolene. suture types were evaluated using , , , or throws. the participants were randomized to both suture type and order of throw numbers. the knots were then tested on the f.a.s. t knot tester (sawbones, vashon island, wa) for slippage (insecure knot) or breakage (secure knot). generalized estimating equation (gee) analysis was used to determine optimal throw number. results: knots were individually tested on the knot tester for slippage and recorded as % slipped (see table) . the percentage of slipped knots varied by participant and ranged from to %. generalized estimating equation analysis suggested that the only significant variable when determining knot security was number of throws (p= . ), not suture type or participant training level. the optimal number of throws for - silk, polydioxanone, and polypropylene was five, whereas six throws was optimal for polyglactin. conclusion: knot security is dependent on the number of throws placed, and these optimal numbers were higher in our study than the commonly accepted number of throws. evaluation of take introduction: laparoscopic skills can be learned using portable simulators and these skills are transferrable to the operating room. several training regions within the uk have therefore developed and delivered home-based laparoscopic training programmes for junior surgical trainees. although performance improved in some, overall engagement has been poor. similar results have been observed in north america. the aim of our study was to uncover the reasons for poor engagement with home-based simulation with a view to developing a future, more successful, programme. methods: this was a qualitative study utilising focus groups. interviews were undertaken with key stakeholders involved in various laparoscopic home-based simulation programmes through the uk. training equipment comprised the eosim portable simulator paired with online training tasks. the tasks were similar to those used in the fundamentals of laparoscopic surgery programme (fls). basic metric feedback was provided (eg time to complete task). a total of individuals were interviewed, including surgical trainees, consultant trainers, training directors and programme faculty. this generated approximately hours of data which was coded using nvivo software. a basic thematic analysis was performed. results: trainees cited multiple competing professional commitments as a barrier to engaging with home-based simulation. they tended to focus on scoring 'points' which contributed toward career progression rather than tasks which were interesting, or associated with personal development. this approach is perpetuated by the surgical training system, which rewards trainees with points for publications and exams, but not for operative skill. this leads to conflict between trainers and trainees, the former expecting trainees to instead focus upon developing their technical abilities. trainees were unsatisfied with metric feedback and wanted individual feedback from consultant trainers (attending equivalent). trainees generally perceived consultants as lacking interest toward the programmes and training in general. however, some consultants were in fact unaware of the programmes being delivered and others felt lacking in confidence to deliver necessary training to trainees. conclusions: our findings are widely generalizable and have implications for any institution delivering a similar programme. as a means of improving engagement, the the inception of scheduled simulation study days, providing trainees with the opportunity for personalised feedback from consultants, has been suggested. equipping trainers with the necessary competencies to deliver training can be achieved by ensuring attendance at the necessary professional development courses. tackling the 'box ticking' culture is more challenging and may involve a move toward restructuring the current surgical training scheme. introduction: to provide evidence for the face and content validity of a hybrid active-shooter team training simulation and the impact of a hybrid curricular model on learner's engagement and performance. the following study was conducted because hospitals are increasingly threatened by active-shooter incidents, and no active and noticeable training is currently available to train hospital staff members. methods: thirty-five volunteers (medical students, residents and other allied health providers) from the university of minnesota affiliated medical centers were randomly selected and divided into control and experimental groups. the control group (n= ) was given a traditional lecture-style presentation. the experimental group (n= ) participated in the hybrid curriculum which included augmented reality, kinesthetic simulation, and debriefing components. following both curriculum styles, nasa task load index (tlx) surveys were completed by each group member. a final active shooter simulation experience was presented and evaluated by active-shooter trained raters using a checklist of critical actions from the department of defense. a post-simulation nasa tlx survey and post-test were provided. to assess face and content validation of a hybrid team-training simulation exercise to prepare healthcare personnel in the event of a hospital-related active-shooter crisis, a -point likert-scale survey determined the realism, utility, and applicability of this type of training while engagement and performance during the simulation were measured using a nasa-tlx survey and contrasted with the rater's evaluation. our study provided evidence to support the face and content validation of an active-shooter simulation team training curriculum as a useful adjunct to health care institutional safety planning. we demonstrated that this type of training requires an optimal level of cognitive activation to increases learner's engagement and performance. we concluded that the hybrid design of our curriculum was successful in delivering these optimal levels of cognitive stimuli by producing engaging team training simulation experience capable of motivating our learners to acquire the tactical skills and life-preserving behaviors consistent with better survival opportunities during a hospital related active-shooter crisis. the introduction: the virtual electrosurgical skill trainer (vest) provides surgeons and trainees with a hands-on approach to learning the best practices in electrosurgery. it is comprised of five modules covering tissue effects, stray currents, bipolar tools, monopolar tools and or fire safety. the module in this study teaches the origins of stray currents and shows the learner how they can cause damage to non-target tissues via direct and capacitive coupling. the aim of this study was to assess learning using the vest system. methods: the irb approved study followed a single group pretest-posttest design and was conducted at the sages learning center. thirty-eight subjects participated and out of these, % were attending surgeons while the rest were medical students, residents and fellows. % of subjects had prior fuse exposure, while the remaining had none. subjects were asked to complete a five-question multiple choice questionnaire before and after using the simulator. it assessed their knowledge in topics such as direct coupling, capacitive coupling and insulation failure. participants then used the simulator to complete three tasks. first, the subject used direct coupling to seal a vessel and observed the desired effects and potential pitfalls. in the second task the subject was immersed inside the peritoneal cavity and was directed to use the active electrode to observe how the activation of energy can cause capacitive coupling. in the third task the subject practiced evaluating the insulation of electrosurgical tools for defects. wilcoxon's signed rank test was used to differentiate between pre-and post-test scores, and the mann-whitney u test was used to differentiate between the groups of subjects as a function of fuse experience. results: the median score on the pre-simulator assessment was % and the post-simulator median score was % (p = . ). there was no statistically significant difference in pre-assessment scores between attending surgeons and the others (p= . ). subjects with prior fuse exposure scored significantly higher on the pre-module assessment compared to those that had no prior fuse exposure ( % vs %, p= . ). in the post-assessment their median scores were % and %, respectively (p= . ). conclusions: the vest simulator module successfully increased the overall participants' knowledge of coupling in electrosurgery regardless of level of surgical experience. participants with prior exposure to the fuse curriculum had increased knowledge on this topic at baseline as compared to participants without any fuse exposure. introduction: the objective of this study was to assess the reliability of a modified notechs rating scale for the evaluation of medical students' non-technical (nt) skills. the importance of physician nt skills for the safe care of patients is receiving increasing attention in the literature. tools to assess nt skills such as notechs that addresses communication, situation awareness, cooperation, leadership, and decision-making have been shown to be valid and reliable. despite its importance, the assessment of nt skills of medical students, our future physicians, has received little attention. methods and procedures: twenty-seven medical students participated in of acute care simulated scenarios, each approximately minutes long. video recordings of student performance were reviewed and assessed using a modified notechs rating tool adapted for these scenarios with input from a team of clinicians, nurses, and human factors specialists. the rating scale ranged from to , representing very problematic behavior (e.g., not vocalizing concerns or decision process) and representing model behavior (e.g., identifies future problems and remains calm to unexpected events). two reviewers rated all videos independently on the notechs domains and specific subscales. student scores in each nt skill domain and interrater reliability were assessed. results: a summary of the scores of each notechs domain is shown in table . the highest overall average score of a participant was . while the lowest was . . the intra-class correlation (icc; two-way random model) was . , and the cronbach's α coefficient was [ . . the lowest icc agreement was in the situation awareness domain ( . ) while the highest agreement was in leadership ( . ). conclusion: medical student nt skills during acute care simulated scenarios vary significantly using a modified notechs assessment. this newly developed tool provides a framework for educators to evaluate medical students' nt skills during simulation training. it further identified domains where students scored lower, such as situation awareness, and could be targeted for education. the moderate icc, between the . - . range, shows that further refinement of the tool is needed to reliably assess the constructs. future steps to obtain validity evidence include additional raters and applying the tool in non-simulated settings. introduction: a general misperception of the real concept of robotic surgery seems to be revealed in our clinical practice. despite its introduction almost years ago, robotic surgery is still related to many myths and beliefs. before designing a trial to see if these false awareness could impact on outcome, we measured this misperception by a survey. moreover we tested if medical school is able today to give to the future doctors a necessary knowledge about robotic surgery. with the same survey we explore the feelings about the introduction of the artificial intelligence in medicine and the perception of the consequences of a larger use of technology in medicine. methods and procedures: a multiple choice survey was designed and anonymously administered via the platform surveymonkey (http://www.surveymonkey.com). a total of questions were selected from the research team and included in the survey. the questionnaire was divided in three parts: the first was to get information on participants' population; the second asked specific questions about robotic surgery; the third focused on technology use in medical education. results: we received and analyzed questionnaires, of which totally filled. many undergraduates consider robotic surgery as "experimental", will prefer open surgery on themselves and see a risk for robotic surgery in damaging the patient-surgeon relationship. this situation is better for medical students, but still a great diffidence were encountered. % of ug consider robotic surgery as "experimental" vs only . % of ms (q ). most thought robotic surgery had been used for only years or less (q ). . % of ug and . % of ms gave the right answer (p=. ). almost % of ug see robotic surgery as a risk in damaging the patient-surgeon relationship. this is not seen among ms (q ) (p=. ). % of ug are fearful of robots used to operate them. this fear is significantly reduced among medical students (p=. ). ug were less familiar with the indications and uses for robotics. ms gave a correct response more frequently (q , . % vs . %, p= . ). conclusions: our results indicates that nowadays, the robotic surgery is related a lot of misperceptions and a generally low level of information. this general picture is partially mitigated during the medical school, but the level of knowledge is still low. a big effort seems mandatory in clarify every technical aspect and an ethic debate about robotics, technology and ai as part of medical curriculum is advisable. background: learning theory states that a certain level of physiological stress or cognitive activation is required to achieve optimal task engagement and performance by the learners. our study will seek to determine if a hybrid team training curriculum inclusive of a task-oriented interactive virtual environment could help achieve the optimal level of cognitive activation required to result in a higher task engagement and performance. methods: a total of thirty-five medical professionals from the university of minnesota participated in several team training simulations. participants were randomly selected to an experimental and control groups. the experimental group (n= ) was exposed to a hybrid team training module, consisting of a task-oriented augmented reality phase followed by a second and third phase consisting of a kinesthetic simulation scenario and debriefing, respectively. the augmented reality phase presented the trainees to an interactive -degree image of the same clinical room where the simulation would take place allowing for ''situated-learning'' to take place. during the learning phase, trainees were encouraged to interact and communicate with each other while completing the tasks allowing for ''social-learning'' to effect. the control group (n= ), educational component consisted of a traditional audiovisual lecture-style introductory presentation, a simulation, and debriefing. after completing their respective educational components, each group completed a nasa task load index survey to assess the cognitive load experience of the individual educational models. subjects were then exposed to a final simulation (test simulation) similar in content and structure to the initial simulation. this was followed by a second nasa tlx survey. raters evaluated both group level of engagement and performance using a validated checklist of critical actions. results: the experimental groups showed higher weighted overall nasa cognitive load index scores than the control group (p= . ) prior to the test simulation. the weighted nasa score remained elevated in the experimental participant groups following the test simulation, whereas in the control group the post-simulation nasa assessment revealed a decrease in cognitive load (p= . ). expert raters using a validated checklist determined that . ± . % of the experimental (hybrid curriculum) group and . ± . % of the control group appeared to be more engaged and performed better during the simulation. conclusions: pre-simulation task-oriented augmented reality learning environments designed to incorporate situated, and social learning virtual experiences can provide the optimal level of cognitive boost that can result in a higher participant engagement and performance during team training simulation scenarios. introduction: despite the huge importance of laparoscopy, medical students have a brief contact with this surgical specialty during medical school in brazil. usually, they get in touch with this specialty during the surgery clerkship in the last years of medical school. therefore, few students perform clinical research or develop interest for this area during graduation. objective: to awaken the interest in laparoscopy of medical students early in medical school, improving the development of clinical research projects, and to prepare new generations of minimally invasive surgeons. discussion: the academic league of videolaparoscopy was created in under the guidance of dr. gustavo carvalho from the university of pernambuco, brazil. an academic league is a group of medical students who are guided by a tutor to develop three areas: research, teaching, and clinical practice. every year new students join the league after being selected with a multiple question test and an analysis of the curriculum vitae. the students are stimulated to participate in laparoscopic procedures as observers, learning about the techniques and instruments. moreover, there are minimally invasive surgery lectures and courses during the year. general surgery residents can also be part of the program as tutors. they are encouraged to present lectures, and to assist with research projects. medical students participated of this program in years. % pursued a surgical specialty after graduation. % did minimally invasive surgery as a fellowship. conclusions: the students who participate in several activities provided by the league have an increased interest in pursuing the path to become a laparoscopic surgeon. background: surgical education is an active and adaptive process of developing knowledge, technical and non-technical skills. the rise of social media has created a paradigm shift in surgical education, with online learning platforms offering exposure to real-time content, expert instruction, and global collaboration. while these disruptive technologies evolve, their influence on surgical education has not been investigated. our goal was to evaluate the growth and impact of an online surgical education model-the advances in surgery (ais) channel. our hypothesis was that utilization and engagement with the platform continues to grow, providing novel methods of measuring successful education. methods: assessment of the platform's membership demographic, user activity, and engagement was performed from inception in to quarter . the ais channel uniquely provides free, high quality, innovative content from elite surgeons in scheduled and continuously available formats across colorectal, bariatric and endocrine surgery service lines. users login to access content, with demographics, time spent, and content accessed recorded as measures of active account utilization and engagement. the main outcome measures were overall membership trends, utilization patterns by region, content type, and surgical specialty for the platform. results: users were predominately male ( . %), surgeons ( . %), and ranged in age from to years ( . %). the main surgical subspecialty represented was colorectal ( . %). active account usage/weekly recurrence was . % ( % industry benchmark), with users engaged for a mean minutes/session (excluding live events). since inception, steady exponential growth was seen across several dimensions. registered users and unique ip addresses increased from over , and , in to over , and . million in , respectively. the number of countries represented increased to reach across continents. at present, over live surgeries and live congresses have been broadcast from countries, with over , surgical videos available on demand to facilitate surgical education. the greatest engagement is seen with live surgical broadcasts. conclusion: our analysis demonstrated proof of concept for a unique, online surgical education model to provide effective surgical education. success was validated through the increase in overall users, sustained active account usage, and global penetration. user preferences for live surgical broadcasts were seen. knowing the utilization and preference patterns, the platform can continue to evolve and enhance the learners' experience. with this growth and penetrance, there is the potential to globally improve patient outcomes and the quality of care provided. background: a realistic simulator for transabdominal preperitoneal (tapp) inguinal hernia repair would enhance the surgeons' training experience before they enter the operating theater. the purpose of this study was to evaluate the efficacy of d-printed tapp simulator in evaluating preoperative skill before entering operative theater. methods: surgeons in our institution were enrolled in this study. they performed simulation tapp and the performance score was measured using tapp check list. the tapp simulator allows for the performance of all procedures required in tapp. the correlation between post -graduate years (pgys), age, experienced a number of laparoscopic surgery (more than , less than ), experienced number of tapp and the performance score was evaluated. results: strong correlation between experienced member of tapp inguinal hernia repair and the performance score was evaluated in this study (r= . ). however, the correlation between pgy, age and score was weak ( introduction: as the field of laparoscopic surgery grows, the need for standard measures of complex laparoscopic surgical skills is apparent. fundamentals of laparoscopic skills (fls) testing is required to complete general surgery residency, but there is no standard metric to convey expertise in advanced laparoscopic procedures. in an effort to develop a standardized assessment of laparoscopic suturing expertise, a group of experts was surveyed using delphi methodology to reach consensus on observed laparoscopic suturing skills reflective of performing at an expert level. methods: expert laparoscopic surgeons participated in serial surveys via redcap (research electronic data capture). experts included surgeons who perform[ /year laparoscopic procedures that involve intra-corporeal suturing, obtained from the authors' personal and professions networks. using a point likert scale, participants were asked to agree/disagree if different observed laparoscopic suturing skills indicate performing at an expert level. these skills were chosen from prior assessment instruments in the literature and the authors' previously published work. tasks were considered to meet criteria for consensus and eliminated from the next round of the survey after reaching % consensus as "strongly agree." results of the previous round of surveys were shared with participants at the start of the next round. the predefined endpoint for the delphi was set as maximum of rounds, reaching % consensus on each skill, or if[ % of initial respondents fail to return for subsequent surveys. results: after the first round of the delphi survey, respondents met inclusion criteria. preliminary data demonstrated skills that reached consensus ([ % of respondents chose "strongly agree"): forehand suturing, avoiding tissue trauma, having a technically acceptable final product (ie. tight closure), and tying a secure knot at the end of suturing. items did not approach consensus (\ % of respondents chose "strongly agree" or "agree"): alternating hands for each throw while tying, never missing a target when grabbing needle/suture, alternating direction of throws when tying, and backhand suturing. data from all four rounds of surveys as well as the final draft of the assessment instrument will be available at time of presentation. conclusion: preliminary data of this delphi study allowed us to reach consensus amongst a group of expert laparoscopic surgeons on the characteristics of expert laparoscopic suturing, which will allow creation of a comprehensive assessment tool for this domain. validation of such a tool will help advance the surgical field towards true competency-based credentialing and promotion. the study was designed to assess the knowledge of scp among european surgeons (specialists and residents). additionally, surgeons' opinion on usefulness of each of the rules of scp was gathered. the data were analyzed in terms of differences between residents and specialists. this is to set ground for and an educational program and increase the safety of elective laparoscopic cholecystectomy by minimizing the occurrence of cbdi. methods: the data on the knowledge of scp and opinion on usefulness of its rules were gathered in form of an anonymous questionnaire distributed among participants of several surgical conferences in poland. the questionnaire then asked about the surgeon's experience in terms of cholecystectomies performed and the number of complications in form of cbdi. it then listed the scp rules and asked the surgeon about their opinion on usefulness of each of the rules on a -point scale. gathered data were subject to statistical analysis and a comparison between specialists and residents was performed. the study has been registered in the clinicaltrials.gov-nct . , although these numbers are still low. significant differences in the mean usefulness score between residents and specialists were observed in regard to two rules: rule was found more useful by residents (mean score , vs. , , p= . ), whereas rule was found more useful by specialists (mean . vs. . , p= . ). the awareness of the sages safe cholecystectomy program in poland is still low and needs to be promoted. both surgical residents and specialists consider the rules of scp to be useful during surgery, although there are slight differences in the usefulness scores between the groups. an educational program to promote and further implement the scp should be established. introduction: transanal total mesorectal excision (tatme) has attracted substantial interest amongst colorectal surgeons throughout the world. technical challenges of the technique however have been acknowledged by early adopters and this may underpin the early reports of visceral injuries which occurred during the perineal phase. evidence from previous surgical training programs suggest that a structured proctorship programme can shorten the learning curve, operative time and most importantly reduce major complications. the aim of this study was to report on the first national pilot training initiative which was developed in the uk to ensure safe introduction of this technique. methods: a pilot training programme for the uk has been established in partnership with the healthcare industry, and supported by the association of coloproctology of great britain and ireland. the programme consists of three phases: (i) development of a consensus process on the optimum training curriculum of tatme from all relevant stakeholders, including experts, early adopters, and potential learners, to guide the training of this technique (ii) piloting of this training curriculum and (iii) assessment and quality assurance mechanisms to monitor training and measure outcomes. results: a cohesive multi-modal training curriculum has been developed providing clear guidance on case selection, supporting multi-disciplinary and multimodal training including online modules, dry-lab, purse-string simulators, cadaveric training and formal clinical proctoring programme. the uk pilot programme opened for applications in may and, after a rigorous selection process, the initiative was launched in september with trainers mentoring consultant colorectal surgeons from five centres. the selection of learners was based on suitable case volume and prior experience in laparoscopic rectal surgery. objective assessment tools were applied to an unedited video of a laparoscopic rectal surgery case for each applicant. for the selected centres, access to the ilapp tatme app was provided to access educational content including operative video footage, prior to attending a bespoke cadaveric workshop. each learner will then benefit from a structured, centrally organised and funded proctorship programme at their own institutions. a global assessment score form has been specifically designed to monitor training and a formal accreditation process will be used to sign off each learner using competency assessment tool. data on the cadaveric workshop and initial outcomes of the clinical mentorship will be presented at the conference. conclusion: a competency-based pilot training programme for transanal total mesorectal excision has been launched in the uk to support safe introduction of this technique. practicing on a fls trainer box is effective but requires large amount of consumables and is scored subjectively. the purpose of this study is to evaluate the face validity of the intracorporeal suturing task on a virtual fundamentals of laparoscopic surgery simulator (virtual fls). we hypothesize that the virtual fls will demonstrate face validity. methods and procedures: after a video demonstration and a practice period, twenty-three medical students and residents completed an evaluation of the simulator. the participants were asked to perform the standard intracorporeal suturing task on each of the virtual fls and the traditional fls box trainer. the presentation order of the devices was balanced. the performance scores on each device were calculated based on time (seconds), deviations to the black dots (mm), and incision gap (mm). the participants were then asked to finish a -question questionnaire regarding the face validity of the simulator. participants answered questions with ratings from (not realistic/useful) to (very realistic/ useful). a wilcoxon signed ranks test was performed to identify differences in performance on the virtual fls compared to the traditional fls box trainer. results: responses to of the questions ( . %) averaged above a . out of . those questions that rated the highest were the degree of realism of the target objects in the virtual fls compared to the fls ( . ) presently, most training methods for thoracoscopic esophagectomy use live porcines; this presents several problems including cost, long preparation times, and ethical issues. these problems further prevent frequent training. currently, no alternative models for thoracocopic esophagectomy training. we report, for the first time, the development and use of a non-biomaterial training model for thoracoscopic esophagectomy. methods: we collaborated with sunarrow co., ltd. (tokyo, japan) to develop the training model. we created organ models for esophagus, trachea, bronchus, aorta, vagus nerve, recurrent nerve, bronchial artery, lymph node, vertebrae, azygos vein, and thoracic duct, and filled the models with a polyvinyl alcohol hydrogel. the gaps between organs were filled with a filler material mimicking connective tissue. we chose a synthetic resin that closely mimics the characteristics (rigidity or elasticity) of each organ. after each organ was fixed, the model was covered with a filler to create a pleural membrane to allow training in peeling operations. in addition, because a patient plate was attached to the rear of the training model, excision with an energy device was possible and more closely simulated surgical conditions. results: using the training model resulted in a highly satisfactory level of experience in three trainees. the trainees were able to learn anatomical positions and sequence of surgical procedures, including endoscope handling. centre for rural health, aberdeen university introduction: as doctors become expert in a complex procedure, they develop automatic nuances of performance that are difficult to explain to a peer or a trainee (so called 'unconscious competence'). traditional methods which aim to allow sharing of expertise have limitations: concurrent reporting alters the flow of the task at hand while retrospective reporting is subject to bias and often incomplete. iview expert is a technique validated in the aerospace domain which externalises an expert's cognitive processes, without disrupting the task at hand. the aim of this project is to assess the feasibility of adapting the technique to medical training. methods: this was an observational case study in which an expert endoscopist wore a head mounted camera to capture a complex procedure (colonoscopy). captured video was reviewed during a facilitated debrief which externalised the expert's cognitive processes. the debrief was recorded and formed an audio commentary. the video and accompanying audio commentary formed a learning package which was watched by a specialty trainee. the technique differs from standard procedural videos in that it provides a more detailed insight into cognitive processes of the expert. this is achieved through the debrief, which encourages reflection upon kinaesthetic (head movement) as well as auditory and visual cues, resulting in a higher level of experiential immersion. questionnaires examined acceptability and educational value of the technique using likert scales and free text answers. quantitative data were presented using basic descriptions in terms of agreement with statements. qualitative data from free text responses were coded in order to identify key themes. results: the expert agreed that wearing the camera was acceptable and did not interfere with the procedure, nor usual decision making processes. qualitative analysis revealed the debrief process to be associated with a high level of experiential immersion: "as if they were there". both the expert and the trainee strongly agreed that the process was educationally valuable and that they learned something new. qualitative analysis demonstrated that the technique revealed useful and unique nuances of the procedure. the intervention could represent a powerful adjunct to existing training methods, especially amongst more experienced practitioners. we are currently undertaking a larger study involving a greater range of procedures with more learners. introduction: endoscopy is an important skill for general surgeons to possess. however, there is lack of training within surgery residency programs. we implemented a one-day endoscopic surgery course with the aim of improving the confidence of surgical residents in performing endoscopic procedures. we also aimed to examine the effect of the exposure to this course on self-reported confidence in performing endoscopic procedures. methods and procedures: the fundamental of endoscopic surgery course at texas tech university health science center is a one-day course consisting of both didactic training and lab training. the didactic part of the course is taught by attending physicians and focuses on the basics of endoscopy, management of upper and lower gastrointestinal (gi) bleed, and techniques to perform a variety of gi endoscopic procedures on swine esophagus and stomach explant. the lab portion of the course allows residents to perform different endoscopic surgical procedures with the attending physicians providing guidance. residents from pgy- to pgy- participated in the course. a -item questionnaire that measured the self-reported confidence in performing several endoscopic procedures on a - likert scale was administered before and after the course. results: twenty-two participants successfully completed the training and the questionnaires. a significant improvement was observed in the overall confidence in performing a variety of endoscopic procedures ( . ± . , p. ). the improvements remained significant even after controlling for the years of postgraduate surgical training (p. ). conclusion: the one-day fundamental of endoscopic surgery course enabled residents to be more confident with endoscopic procedures. overall, the residents felt that the course was helpful and would like to attend more than one session per year. this course should be held, at least, annually to allow the general surgery residents to become even more confident with this important skill. by being more confident in their surgical endoscopy skills, they will ultimately be able to provide better care for patients. introduction: a course evaluation study on the effectiveness of improving laparoscopic skills of surgical residents using swine models was evaluated through a self-report questionnaire administered before and after course completion. the purpose of the training is to provide surgical residents opportunities to practice and advance their laparoscopic proficiencies. methods and procedures: participating residents in all post-graduate year levels (pgy through pgy , n= ) were provided anesthetized pigs with which to perform a variety of simple to complex laparoscopic cases. prior to training, residents were given a questionnaire composed of eleven questions requiring the subjects to rate their confidence in performing various laparoscopic procedures on a - likert scale. after completion of the course, an identical questionnaire was distributed with two additional questions relating to the overall impact of the course. all statistical analyses were conducted using r statistical software (version . conclusion: overall, one-day hands-on training using swine models improved resident's skills, confidence, and understanding of laparoscopic surgery. the information acquired through the questionnaire emphasized the importance of providing a laparoscopic training course as a standard requirement at all medical institutions. allowing opportunities for surgical residents to practice their laparoscopic skillset will not only help in their individual academic advancements, it will allow them to provide optimum care for their patients. background: learning laparoscopy is difficult and many educational tools including simulation training are required. feedback plays a crucial role for motor skill training but require expert tutors and its time consuming. e-learning increases knowledge acquisition through a more interacting multimedia experience and reduces de costs of learning. in the last decade multiple applications (apps) have been developed for mobile medical training. a new ios app was developed using specially designed educational videos that explain the main technical aspects in advanced laparoscopy through simulation training. the aim of this study is to present the first results of its incorporation in a surgical simulation lab as a complement of effective feedback. methods: twenty-five consecutive residents were trained in our simulation lab through a session validated training program for the acquisition of advanced laparoscopic skills needed for the performance of a laparoscopic hand-sewn jejuno-jejunostomy. every session had written instructions and a basic tutorial video. the app consist two main sections, the first one explains the essential techniques needed for intracorporeal suturing and the second is a complete walkthrough of the validated training program. the trainees were divided in two groups, the first was trained without using the app (napp) and the second group was trained using the app (yapp). both groups of trainees could ask for feedback anytime they needed. trainees were assessed before and after the training program using validated rating scales and the number of necessary tutor-feedback sessions were registered. finally the yapp group answered a survey about the strengths and weaknesses of the app for learning advanced laparoscopic skills. results: twenty-five residents completed the training program; yapp and napp. both groups finalized their training with no statistical significant differences in their scores (p: . ). the number of tutor-feedback needed to complete the training in the yapp vs napp was of [ ( - ) vs ( - ) (p. )] respectively. in the questionnaire all participants considered that the app was effective for learning advanced laparoscopy. over downloads have been registered since the app was published in the apple app store in . we present a novel smartphone app that guides laparoscopic training using simulation-based educational videos with very good results. the use of app guided learning reduces de need of expert tutor feedback reducing the costs of simulated training. jemin choi, young-il choi; kosin university gospel hospital purpose: laparoscopic appendectomy (la) has been widely performed for acute appendicitis. in addition, minimally invasive surgery such as la is common surgical technique to the surgical residents. however, single incision laparoscopic surgery (sils) is a challenge to inexperienced surgical residents. we described our initial experience in teaching sils procedure for appendectomy in our medical center. methods: twenty nine cases of single incision laparoscopic appendectomy (sila) were performed by single surgical resident and cases of la were performed by surgical residents and boardcertified surgeons. a study was reviewed retrospectively. ( ) clinical stressors (i.e., vitals of patient coding). we developed a stress simulator testbed by integrating an fls box trainer with a linux computer, running custom c++ code. the code generated various stressor conditions, while recording sensor data from the trainer and human operator. we tested groups of participants in an irb approved trial including: novices (non-medical students), intermediates (medical students), and experts (pgy residents and fellows). the study consisted of subjects performing the peg transfer and the pattern cut six times (baseline, four randomized stressors, posttest). after each task, the nasa-tlx survey was administered to determine the overall workload of that stressor condition. an analysis of variance was conducted to identify significant trends in terms of stressor type. results: when compared to baseline nasa-tlx scores, the intermediate group had the greatest changes in overall workload than novices and experts (p= . ). additionally, the change between baseline and post-test workload was significantly lower than for the environmental, negative evaluative, and clinical stressors (p= . ). for pattern cutting, subjects reported a significantly lower perception of failure (p= . ) in both the positive evaluative (mean= . ) and post-test conditions (mean= . ), yet, though not statistically significant (p= . ), the measured accuracy in the task during the positive evaluative condition was actually worse ( . %), second only to the pre-test accuracy ( . %). the best accuracy for pattern cutting across all expertise levels was % for the post-test followed by . % in the negative evaluative condition. these results are interesting as they show that despite perceived improvements in performance with a positive feedback condition, performance actually degrades and is better in the negative feedback condition, which is perceived to be more difficult. these results were not found in the peg transfer task, which is arguably an easier task. conclusion: from the evidence gathered in the study, it is clear that there is a correlation between distractors and performance. further analysis is needed to identify the relationship between the type of stressor, and inherent difficulty of the tasks, in terms of which type of stressor best improves learning and outcomes. surg endosc ( ) each received credentials to perform diagnostic and therapeutic ercp from their respective hospitals in nevada, minnesota, and idaho. one continues to teach ercp to general surgery residents, and another taught the skill to fellows in an advanced endoscopy fellowship. all three continue to use ercp in their practice ( to times per month), as they each specialized in a field that utilizes ercp routinely. choledocholothiaisis is the most frequent indication, though ercp is also performed for iatrogenic biliary duct leaks, traumatic biliary or pancreatic duct leaks, chronic pancreatitis, and malignancy. conclusions: training in esophagogastroduodenoscopy and colonoscopy is required for general surgery residents, but the addition of ercp to select residents' training enables them to completely manage their patients' surgical disease. the training of select general surgery residents in this skill has been successful, evidenced by the continued use of ercp in the practices of three residents who completed this training program at our institution. the decision to train residents in this skill should be left to individual program directors and department chairs. we recommend that residents selected for this additional training should plan to practice in specialties where ercp can be implemented. conclusion: same-day discharge after nissen fundoplication and hiatal hernia repair is feasible for select patients. one major challenge for same day discharge is the current insurance provisions required for hospital reimbursement. within the parameters of this study, bmi and asa score did not differ between discharged and admitted patients, while older age and increased procedure duration were associated with need for admission. premkumar anandan, ms, facs; bangalore medical college and research institute introduction: minimal access surgery is an imperative element of enhanced recovery program and has significantly improved the outcomes. enhanced recovery program (erp) synonym "fast track" surgery "was first conceived by dr henrich kelhet. largely described for colorectal surgery and reported to be feasible and useful for maintaining physiological function and smooth the progress of recovery. most of the patients who present for surgical emergency are not adequately prepared and many are not in normal physiological state. the feasibility of enhanced recovery programs protocol in such emergency minimal access surgery remains indistinct. this study was designed to validate an enhanced recovery program in patients who undergo emergency minimal access surgery. introduction: pathways for enhanced recovery after surgery (eras) have been shown to improve length of stay and postoperative complication rates across various surgical fields, however there is a relative lack of evidence-based studies in bariatric surgery. the objective of the current study was to determine if starting a bariatric full liquid diet on postoperative day (pod) zero was associated with shorter length of stay (los) for patients who underwent laparoscopic sleeve gastrectomy (lsg) or roux-en-y gastric bypass (rygb). methods: retrospective review of a prospectively collected dataset was conducted at a single institution before and after implementation of a new diet protocol for lsg and rygb. postoperative diet orders were changed from full liquid diet on pod to pod . length of stay and -day readmissions were reviewed from june to august . independent samples t-tests were used to compare continuous variables and chi-squared tests for categorical variables before and after diet change was implemented. patients were excluded if they were undergoing revision surgery, were discharged directly from pacu, or had significant intraoperative complications or required reoperation within the same admission. introduction: data suggests value in using tap (transversus abdominis plane) neural blockade in abdominal surgical procedures. we deploy tap blockade using liposomal bupivacaine via ultrasound (us) as part of a narcotic sparing pain management pathway for patients undergoing abdominal surgery in our rural community setting. our goal was to evaluate adequacy of postoperative discomfort and the success in avoiding narcotic usage. methods and procedures: records of patients undergoing abdominal surgical procedures performed by one surgeon over an month period were reviewed under irb approval. patients taking narcotics prior to the procedure (except for discomfort due to the condition being surgically treated) were excluded from analysis, as were those admitted to the hospital for postoperative treatment. us guided lateral tap blocks were performed by the surgeon using mg of liposomal bupivacaine and mg of bupivacaine in the or prior to the incision. unilateral block was performed for unilateral procedures (e.g. inguinal hernia) and bilateral for laparoscopic or midline procedures. incisional sites were treated with a field block of mg of bupivacaine. prescriptions for medications included , mg of acetaminophen qid and mg of naproxen sodium tid for days. a prescription for tramadol ( to mg prn up to times daily; tablets with no refill) was given. patients were seen in followup two weeks postoperatively. data (following standard scales/metrics) for patient-reported-outcomes e.g. pain, nausea-vomiting, & fatigue will be analyzed with the above data and the analysis with conclusions will be presented & discussed. federico sertic, md, ashwin gojanur, dr, ahmed hammad, md; guy's and st thomas' hospital introduction: the aim of this project is to assess the quality of post-operative pain relief in colorectal surgery and identify patients in whom pain management has not been effective, in order to improve the quality of post-operative care. effective management of post-operative pain has long been recognised as important in improving the post-operative experience, reducing complications and promoting early discharge from hospital. standards: all patients should be pain free at rest, % of elective patients should be told about what analgesia they will have post-operatively, % of patients should be satisfied with their pain management and % of patients should feel staff did everything they could to control their pain. methods and procedures: questionnaires were given to patients on the day prior to discharge. questions about pre-operative and post-operative pain experience were asked. data regarding post-operative analgesia were collected from medication charts and medical notes. data were collected over a period of two months (august/september ). range of procedures: elective laparoscopic abdomino-perineal-excision-of-rectum with igap flaps, elective laparoscopic right hemicholectomy, laparotomy+bowel resection/stoma formation ( elective, emergency), elective repair of parastomal hernia, appendicectomy ( laparoscopic elective, laparoscopic emergency, laparotomy emergency) and elective reversal of ileosomy. pain scores ( - ): immediately post-operative pain, day post-operative pain, post-operative pain after day and pain on moving/coughing/straining. results: mean immediate post-operative pain score was . ( % of patients with score +), mean day post-operative pain score was . , mean post-operative pain score after day was . , mean pain score on moving was . ( % of patients with score +), mean pain score on coughing/ straining was . ( % of patients with score +). % of patients were satisfied with their post-operative pain management and felt that the staff had done everything they could to manage their pain. % of patients were not aware of their post-operative analgesia regimen and % did not know how regularly they could request analgesia. conclusions: effective management of post-operative pain is a key part of post-operative care and an important component of enhanced recovery programmes. patient satisfaction with pain management has been found to correlate with received pre-operative information. increasing ward nurses' and acute pain teams' knowledge is important in improving patients' pain experiences. interestingly, those patients who had a background of long-term opioid requirements reported that they were satisfied with their pain management. methods and procedures: a patient undergoing a standard ultrasound guided ql block by an anesthesiologist established the baseline anticipated response, and procedure time. the procedure, performed under sedation preoperatively, required over minutes. for this study, patients undergoing laparoscopic colorectal surgery were administered a lateral ql block (modified ql ) under ultrasound guidance by the operating surgeon. ml of a mixture ( ml injectable liposomal bupivacaine suspension, ml . % bupivacaine hydrochloride and ml normal saline) was injected bilaterally, after induction, skin preparation, draping, and prior to the operation. postoperative narcotic use and pain vas scores were documented. results: six patients were administered a bi-lateral ql block intraoperatively. procedures were: laparoscopic sigmoid colectomies, one end ileostomy reversal, laparoscopic completion proctectomy with ileal pouch anal anastomosis, and a laparoscopic descending colectomy. of the narcotic naïve patients, mean pain vas on post op days , and were . , . and . respectively within a multimodality pain management/enhanced recovery program, where standing orders prompting narcotic administration by nursing staff is pain vas . all were discharged on pod or without narcotic prescriptions. two of the patients were chronic narcotic users, and they were discharged on their baseline narcotics, i.e. without additional narcotics. all intraoperative blocks were performed in less than minutes. conclusion: a novel, surgeon-administered lateral ql block under ultrasound guidance, is feasible and provides post-operative pain control. patients are discharged home on no/baseline narcotics. a randomized controlled trial is being constructed based on these striking findings. keywords: lc-laparoscopic cholecystectomy, ga-general anaesthesia, sa-spinal anaesthesia. nikhil gupta, rachan kathpal, dr, arun k gupta, dr, dipankar naskar, dr, c k durga, dr; pgimer dr rml hospital, delhi introduction: cholecystectomy have shown some advantages when done under spinal anaesthesia (sa) and associated with less intra operative and post -operative morbidity and mortality. laparoscopic cholecystectomy (lc) under regional anaesthesia alone included patients with coexisting pulmonary disease, who are deemed high risk for ga. the aim of the present study is to assess the efficacy and safety of laparoscopic cholecystectomy under sa. materials: this prospective, interventional study was conducted on patients with chronic calculous cholecystitis attending general surgery out-patient department of our institution. results: in our study, intraoperative complications recorded were hypotension, bradycardia, intra op shoulder tip pain, bleeding from the liver bed, bile spillage, post-op pain and vomiting. % patients had intraoperative pain, % had shoulder tip pain, . % had bradycardia, . % had hypotension, . % had bile spillage and . % had bleeding. laparoscopic cholecystectomy under spinal anaesthesia should be promoted more even in developing countries but we need to establish well evaluated safety guidelines that could be followed faithfully for minimizing the risk of complication. background: the "opioid crisis" has taken over headlines with increasing public attention brought to the drastically increasing rates of addiction to prescription narcotics. in , the american society of addiction medicine reported million americans with an addiction to prescription pain relievers and a four-fold increase in overdose related deaths. in a medical setting, increased opiate use is associated with increased rates of delirium, ileus, urinary retention, and respiratory depression. these risks are increased in the obese/bariatric population. transversus abdominis plane (tap) block is a safe and effective approach to achieve optimum pain control. it reduces the use of opiates in patients undergoing major abdominal surgery. however, there is currently no data in the literature examining its use in the bariatric population. our study examines the use of liposomal bupivacaine for tap block in patients undergoing laparoscopic sleeve gastrectomy (lsg). methods: sixteen patients undergoing lsg with tap block were compared with historical cohort of sixteen patients undergoing lsg without tap block (standard group). the primary outcome measured was post-operative in-hospital opiate use (morphine equivalents). statistical analysis was performed using student's t test for continuous variables and fisher's exact test for categorical variables. results: both groups were well matched in regards to bmi, age, and asa class. there was a significant decrease in the post-operative use of opiates with the use of the tap block ( . mg in the tap block group vs. mg in the standard group; p . ). there was no difference in the mean length of stay between the two groups. there was an increase in the mean operative time with use of the tap block ( minutes in the tap block group vs. minutes in the standard group; p. ) conclusions: the use liposomal bupivacaine for tap block provides substantial analgesia, allowing for significant reduction in post-operative opiate use in our bariatric patients. this can be an important adjunct in pain control for the bariatric population and aid in post-operative complication risk reduction. introduction: the objective of this study was to identify variation in weight and demographics in the distribution of pre-operative clinical characteristics between super obese females compared with males who were about to undergo bpd/ds surgery. as the american obesity epidemic increases, morbidly obese patients have become integral to every surgical practice; they are no longer limited to bariatric surgeons. every clinical insight helps the surgeon to optimize outcomes when operating on and managing these medically fragile individuals. in this context, however, clinically and statistically significant differences in demographics, body mass, and in the distribution of weight-related medical problems between super-obese women and men are unknown. introduction: a transversus abdominis plane (tap) block is an ultrasound-guided injection of local anesthetic in the plane between the internal oblique and transversus abdominis muscles to interrupt innervation to the abdominal skin, muscles, and parietal peritoneum. currently there are incongruent findings on the benefit of this regional anesthetic to surgical patients, particularly the obese population. we hypothesized the addition of a tap block in an enhanced recovery pathway (eras) for bariatric patients would decrease opioid use and shorten hospital length of stay. methods: a retrospective review of all patients who underwent bariatric surgery at a single institution from january to december was performed. patients were identified as: no tap block (no tap), tap blocks that were performed after induction either pre-surgery (pre-tap) or post-surgery (post-tap). the primary outcome was time to first opioid (min) and total morphine (mg) equivalents in pacu. objective: prolonged postoperative ileus increases hospital length of stay and therefore impacts healthcare costs. although many surgeons recommend ambulation in the postoperative period to hasten return of bowel function, little evidence exists to support this practice. our hypothesis is that early ambulation does reduce the time to return of bowel function after intestinal surgery. methods: a subset of patients undergoing intestinal surgery from an ongoing, prospective trial evaluating perioperative physical activity was analyzed. preoperatively, patients wore an activity tracker for a minimum of three days to establish a baseline activity level, measured by daily steps. postoperatively, steps were recorded for days. patients were included in this study if they underwent an operation on the small bowel, colon, or rectum. resolution of postoperative ileus was defined as the postoperative day when patients were noted to meet all of the following criteria on review of nursing documentation: passing flatus, stooling or having ostomy output, and tolerating a regular diet without intravenous fluids. "early" postoperative activity was defined as the average number of daily steps during the first two postoperative days. discussion: these results suggest the patients who received an intraoperative block laparoscopically were more likely to be able to spend less time in the post anesthesia care unit and be discharged home the same day. based on these results, additional process improvement ideas will be implemented in an attempt to improve outcomes. riley d stewart, md, msc, frcsc, james ellsmere, md, msc, frcsc; dalhousie university division of general surgery introduction: oropharyngeal and gastrointestinal (gi) perforations from bbq brush bristles are being reported in the literature with increasing frequency. media attention to this problem has increased awareness by the public. most commonly, bbq bristles lodged in the gi tract can be removed endoscopically or pass without complication. rarely, surgical intervention is required for removal of the bristle or drainage of an associated abscess. we report a case of gastric perforation by a bbq bristle leading to a pancreatic abscess. case report: a -year-old male presented to a regional center with epigastric pain and malaise. his medical history included: hypertension, dyslipidemia, gerd, and smoking. his surgical history included: a tonsillectomy, excision of bronchial cleft cyst, and an umbilical hernia repair. on presentation, his laboratory investigations where unremarkable aside from an elevated white blood cell count. investigations including an abdominal x-rays and an abdominal ultrasound were unremarkable. he was initially treated with a proton pump inhibitor for presumed peptic ulcer disease. he returned to the local emergency room, no better than before. a ct scan was arranged which demonstrated a foreign body at the pylorus consistent with a bbq bristle and a peripancreatic fluid collection (figs. & ) . a gastroscopy failed to identify the bristle. he was admitted, placed on iv antibiotics and referred to our center. despite several days of antibiotics prior to arrival, the collection size on repeat ct scan had increased and the patient had ongoing pain. we repeated the endoscopy with a side viewing endoscope. the perforation was identified posteriorly at the pylorus. the bristle had migrated into the peripancreatic space. the perforation was cannulated with a jagtome. fluoroscopy was used to confirm the position of a wire in the fluid collection (figs. & ) . pus was drained from the collection into the stomach by placement of a french pigtail catheter (fig. ) . the patient was discharged pain free the following day. the patient was asymptomatic at weeks' follow-up. a repeat ct scan showed resolution of the abscess and safe migration of the bristle and stent out of the gi tract (fig. ) conclusion: to our knowledge, this is the first reported transgastric endoscopic drainage of a peripancreatic abscess caused by a bbq bristle gastric perforation. this case is a demonstration of the ever-expanding role of therapeutic endoscopy in a surgical practice. andrew w white, md, carl westcott, md; wake forest baptist medical center introduction: endoscopic balloon dilation of the gastroesophageal junction (gej) is generally limited to mm in diameter. in many stenotic or spastic disorders of the gej mm is just not big enough. larger balloon sizes are available ( and mm), although these are deployed under fluoroscopy without endoscopy. thus, these larger dilations are often not feasible at the time of the diagnostic endoscopy because different facilities and/or equipment are needed. also, fluoroscopic mm balloon dilations are associated with a percent perforation rate. to address these shortcomings we present an experience with a retroflexed "against the scope" balloon dilation of the gej. in detail, the gej is visualized while retroflexed and a balloon is then placed through the scope. the gej is cannulated next to the scope and deployed. please see the attached image for example. methods and procedures: a retrospective chart review was performed for a single surgeon during the past five years. we identified those who had retrograde dilations and evaluated the indications, repeat dilations, complications and symptomatic response. results: a total of retrograde dilations were performed on patients with gej related dysphagia. the average age was . years. of dilations were with a mm balloon while other dilations used as small as a mm balloon. dilations were performed for persistent dysphagia after cardiomyotomy between and days after surgery. other indications for dilation were dysphagia after fundoplication ( / ), dysphagia after paraesophageal hernia repair ( / ) and achalasia during pregnancy ( / ). patients required a total of repeat retrograde dilations at an average time of days after previous dilation. there were instances reported where the dilation did not improve symptoms. there was mucosal breakdown noted in instances although there were no perforations. bleeding was noted in instances although this was always minimal and selfresolving. conclusions: retrograde endoscopic dilation is safe and effective in this small series. the mm balloon against a mm scope gives a mm diameter, but a different shape and a decreased total circumference. there is a possible added safety advantage given that the balloon is inflated under visualization. it can be inflated in steps or stopped if it appears too aggressive. in addition these larger dilations were provided at the time of the initial diagnostic egd without extra equipment. more studies are needed to compare retrograde endoscopic dilation to other methods of management of gej stenosis. introduction: robot-assisted surgery allows surgeons to perform many types of complex laparoscopic surgical procedures. more and more patients are treated with this sophisticated system. however, all the instruments used in the currently available surgical robot system is rigid. therefore, there exists a limitation in the extent of reach to the deeper surgical fields. in order to overcome this difficulty, we are developing a novel flexible endoscopic surgery system (fess) which has flexible single port platform of cm in diameter, independently controlled endoscope and instruments, open architecture that is compatible with existing flexible devices and a magnified d hd camera that has sensors of both rgb and infrared. furthermore, the system is smaller and would be more cost-effective than existing robotic surgical system. a preliminary experiment was performed in surgical procedures using porcine model to evaluate effectiveness and feasibility of fess. methods and procedures: experimental protocols were approved by the animal research committees of our institution. we used a female swine of kg. an assistant forcep lifted up the fundus of gallbladder to create good visualization of surgical field. the cystic duct was ligated by laparoscopic clip device from assistant port. blunt dissection was performed by pushing the forceps and sharp dissection by monopolar electrocoagulation. results: the fess accomplished the dissection of the gallbladder from the liver bed successfully. two mm forceps had enough grasping and dissecting force and dexterity. the gallbladder was removed from single port site easily. conclusions: this experiment showed that it is feasible to intuitively operate single-site cholecystectomy with fess. in order to realize a pure fess procedure, an additional novel device to create good visualization of the surgical field is necessary for the fess platform. a prototype has already been developed for evaluation in securing the surgical field. the optimal working range, or "sweet spot" of fess is not relatively large. in addressing this issue, the feature of easy setup is being improved to enable more efficient positioning and shifting of the sweet spot for the surgical field. this mechanism could enhance the expansion of procedures suitable for fess. the target procedures of fess are those specifically suitable for single port surgery, such as transanal surgeries and transcervical mediastinoscopic surgeries. intraluminal procedures and natural orifice translumenal surgery (notes), which are not considered suitable for rigid surgical robot, are also good applications of fess. regression of anal and scrotal squamous cell carcinoma (hpv related) with imiquimod index patient is a year old hiv positive homosexual man with anal-scrotal condylomas (ain) initially resected in , then treated with radiation in for recurrence. recurred in with changes severe enough to ''…consider diagnosis of invasive squamous cell carcinoma…''. patient elected trial of imiquimod % cream three times per week to defer recommendation of abdominoperineal resection. imiquimod has no antiviral effect but stimulates interferon and cytokines to suppress hpv subtypes and , among other immune effects. no data exists as to systemic effects of imiquimod. after three months of therapy, lesions had largely regressed with only one specimen showing ''…concern for squamous cell carcinoma in situ…''. patient has elected to continue treatment pending further biopsy. this report is typical of a number of other reports of small numbers of cases of neoplasia regression with imiquimod % cream to include melanoma-in-situ, basal cell cancer of skin and other cutaneous malignancies as well as vin. a second female patient, years old, hiv+ with hpv lesions (ain ) including urethral lesions, is being treated with vulvar application of imiquimod to determine if urethral lesions will regress. there is no fda-approved indication for mucosal application of imiquimod. biopsies are pending at completion of six month trial of imiquiimod. surg endosc ( ) introduction: training in flexible endoscopy remains a critical skill for surgeons, as therapeutic endoscopy procedures continue to evolve and to supplant standard surgical operations. the role of endoscopy across surgical subspecialties is shifting, as endolumenal procedures (like per-oral endoscopic myotomy and endolumenal bariatric interventions) have become commonplace. while surgical residency minimum case volumes are mandated, little is known about the volume of endoscopic procedures surgical fellows participate in. we aimed to characterize the volume of flexible endoscopy cases logged by surgical subspecialty fellows as a measure of endoscopic platform use by surgeons. methods: operative case logs for fellows enrolled in post-graduate training programs participating in the fellowship council were de-identified (no patient or program specific information) and provided for analysis. the case log is an online, mandatory, self-reported collection of all surgeries, procedures and endoscopies performed during fellowship year. all cases listed within the category of "gi endoscopy" in which the fellow designated their role as "primary" surgeon for the procedure were further sorted based on subcategory and linked to the year of fellowship graduation. rigid endoscopy, trans-anal endoscopic procedures, and those in which the fellows roll was "first assistant" were excluded. introduction: complex pancreatic and duodenal injuries due to trauma continue to present a formidable challenge to the trauma surgeon with a described mortality of - % and morbidity of - %. duodenal fistula formation subsequent to failure of attempted primary repair is associated with significant morbidity and mortality. we present the first reported series of four patients with complex trauma-related duodenal injuries who had failure of primary repair which were managed with duodenal stenting. we compared outcomes to a matched case control cohort of patients with trauma related duodenal injuries. the aim of this study is to document our experience with enteral stents in patients with complex duodenopancreatic traumatic injuries. methods: a retrospective review at a level i trauma center identified patients who underwent endoscopically placed indwelling covered metal stents after failure of primary duodenal repair in the form of high output duodenal fistulas. a matched case control cohort was identified including patients with duodenal fistulas who were not treated with stents. drainage volumes were collected and classified according to source and phase of intervention (i.e. admission to fistula diagnosis, to stent insertion, after removal, and until discharge). results: there was a decrease in the mean combined drain output of ml/day (p= . ) after stent placement. when comparing the sum of all output sources, there was a statistically significant difference across phases (p= . ) and "after removal" was significantly less when compared to the reference phase (p= . ). there was also a change in the directionality of the slope for the sum of all drain outputs with an increase of ml/day prior to stent placement compared to a decrease of ml/day (p= . ) after stent placement. the stenting group demonstrated a decrease in mean drain output ( ml/day vs ml/day, p= . ) and increase in distal gastrointestinal output ( ml/day vs ml/day, p= . ). one patient in the stent group required later operative repair. all other patients in the stenting and control group had resolution of their fistulas over time. there were late mortalities in the control group. the stent treated patients demonstrated diversion of approximately ml/day of enteral contents distally. while all patients eventually healed their fistulas, the stent treated patients demonstrated an accelerated abatement of drain outputs when compared to the control cohort, but did not reach statistical significance. indwelling enteral-coated stents appear to be an effective rescue method for an otherwise inaccessible duodenal fistula after failure of primary repair. kevin l chow, md, hassan mashbari, md, mohannad hemdi, md, eduardo smith-singares, md; university of illinois at chicago introduction: esophageal trauma represents an uncommon but potentially catastrophic injury with a reported overall mortality of up to %. the management of iatrogenic and spontaneous perforations have been previously described with well-established guidelines which have been mirrored in the trauma setting. esophageal leaks are the most feared complication after primary surgical management and present a challenge to salvage. there has been increasing reports in the literature supporting the use of removable covered metal stents to treat esophageal perforations and leaks in the non-trauma setting. we present the first reported case series of four patients presenting with external penetrating trauma induced esophageal injuries, complicated by failure of initial primary surgical repair and leak development, successfully managed with the use of esophageal stents. materials and methods: a retrospective review was performed at a level i trauma center identifying four patients who underwent endoscopically placed removable covered metal stents, either by a surgical endoscopist or an interventional gastroenterologist, after failure of primary surgical repair of esophageal traumatic injuries. demographic information, hospital stay, additional interventions, complications, imaging studies, iss scores, and outcomes were collected. results: our cohort consisted of patients with penetrating injuries to the chest and neck with esophageal injuries ( thoracic and cervical esophageal injuries) managed with esophageal stenting after leaks were diagnosed following primary surgical repair. their initial esophageal injuries included grades , and . leaks were diagnosed on average post-operative day . two patients underwent an additional attempted surgical repair and subsequent leak development. esophageal stents were placed under endoscopic and fluoroscopic guidance within days of leak diagnosis. there was resolution of their esophageal fistulas with all patients resuming oral intake (averaging days after stent placement). three patients ( %) required further endoscopic interventions to adjust the stent due to migration or for dilations due to strictures. mortality was %, all patients survived to be discharged from the hospital with average icu length of stay of days. conclusion: the use of esophageal stenting has progressed over the last few years, with successful management of both post-operative upper gastrointestinal leaks as well as benign, spontaneous, or iatrogenic esophageal perforations. while the mainstay of external penetrating traumatic esophageal injuries remains surgical exploration, debridement, and repair with perivisceral drainage; our case series illustrates that the use of esophageal stents is an attractive adjunct that can be effective in the management of post-operative leaks in the trauma patient. results of the ovesco-over-overstitch technique for managing bariatric surgical complications introduction: since , the preferred method of enteral access has been the percutaneous endoscopic gastrostomy tube (peg). accidental removal is a common complication associated with excessive cost and possible significant morbidity. removal prior to days is considered ''early removal.'' early removal has more significant risk associated with it, and can necessitate emergent operation to prevent peritonitis and sepsis. some patients, who do not exhibit signs of peritonitis, may be simply observed. for these patients, peg replacement would typically be delayed - days to ensure closure. this delay results in prolonged npo status and worsened nutritional status. presented below is a case of early accidental removal followed by endoscopic clip closure, and immediate peg replacement. case report: a -year-old male presented after a large left middle cerebral artery infarct. a peg placement was completed without complication. eleven hours after the procedure the patient had pulled the peg tube out of the abdominal wall. at this time the patient appeared to have no abdominal pain and no signs of peritonitis. twelve hours following the accidental removal of his peg tube, the patient was taken back to the endoscopy suite, and an egd was performed. the previous peg site was identified and appeared closed and ulcerated. the mucosal defect was closed with two endoscopic metallic clips. a peg tube was then placed at an adjacent site. the following day, the patient was restarted on trickle feeds and advanced to regular tube feeding over a period of hours. since that time, his peg has been functioning well. discussion: we propose that in the case of early accidental peg removal, the patient should be examined first for evidence of peritonitis. if initial physical exam and radiographic investigation do not reveal peritonitis or significant pneumoperitoneum, the patient should undergo urgent repeat endoscopy. at this time, the gastrotomy can be closed endoscopically via metallic clips and peg can be replaced immediately. tube feeds can be initiated after a - hour period of dependent drainage with serial abdominal exams. introduction: since its inception in , poem has become a viable procedure for the treatment of achalasia and esophageal dysmotility disorders. however many institutions are in the beginning stages of implementing the procedure into their programs. in view of training, we report the successful ability to dissect and identify common landmarks during a poem procedure performed by trainees under supervision in a high volume poem center. methods: posterior poem procedures performed by trainees with experienced proctor guidance during the period between february to july were evaluated for the frequency of identifying the perforating vessels, the presence of sling fibers, and position on the lesser curvature of stomach evaluated by double scoping method during the creation of the tunnel and myotomy for procedure. results: all poem procedures were successfully completed by trainees (gi and surgery fellows). the average length of procedure was minutes. indication for procedure included patients with type achalasia ( %), with type achalasia ( %) and des ( %). average length of myotomy for all procedures was . cm. during these procedures or perforator vessels were identified in ( %) of patients, sling muscle was identified in patients ( %) of patients. myotomy extended to anterior lesser curvature of stomach on double scope exam in % of patients. no patient had a serious complication requiring intervention. conclusion: trainees performing a posterior poem procedure were able to correctly dissect and identify the sling muscle and/or perforating vessels in approximately % and % respectively of procedures. however the myotomy position was correctly placed in all procedures. this indicates that while ideally the sling fibers and perforating vessels should be identified, a correctly positioned myotomy can still be successfully performed by trainees without identification of these landmarks. introduction: gastroparesis is a rapidly increasing problem with sometimes devestating patient consequenses. surgical treatments, particularly laparoscopic pyloroplasty, have recently gained popularity but require general anesthesia, advanced skills and create risk of leaks. peroral pyloromyotomy (pop) is a less invasive alternative but is technically demanding and not widely available. we propose an hybrid laparo-endoscopic collaborative approach using a novel gastric access device to allow a endoluminal stapled pyloroplasty as an alternative treatment option for functional gastric outlet obstruction. methods and procedures: under general anesthesia six female pigs (mean weight kg) had endoscopic placement of or mm intragastric ports (taggs, kansas, usa) using a technique similar to percutaneous endoscopic gastrostomy. a mm laparoscope was used for visualization. endoflip (crospon, inc., galway, ireland) was used to measure cross sectional area (csa) and compliance of the pylorus before intervention, immediately after and at week survival. pyloroplasty was performed using a mm articulating laparoscopic stapler (dextera microcutter). after removing the taggs ports, the gastrotomies were closed by either endoscopic clip, endoscopic suture or suture under laparoscopic vision. the animals were survived for week. after - days, a second laparo-endoscopic procedure was performed to verify healing of the pyloroplasty as well as intraluminal dimensions. at the end of the protocol, animals were euthanized. results: six endoluminal linear stapled pyloroplasty were performed. the mean operative time was min. in all cases, this technique was effective in achieving optimal pyloric dilatation. median pyloric diameter (d) and median cross-sectional area (csa) pre-pyloroplasty were mm ( . - . mm) and . mm ( - mm ). after the procedure, these values were increased to . mm ( . - . mm) and . mm ( - mm ) respectively (p= . the quality of endoscopic examination depends on the quality of endoscopic equipment, experience of the endoscopist and preparation of the patient. contemporarily electronic endoscopes make feasible to transfer image directly to external device which is subsequently linked to computer network and can be transferred further. dynamic image viewed in real time is more accurately interpreted by a physician than a static one. the possibility of simultaneous voice contact makes teleconsultation sterling. the aim of this study was to present our own experience regarding endoscopic teleconsultations. materials and methods: analysis enrolled examinations performed in endoscopic centers located in lesser poland district and in denmark. consultations took place in real time, consulting physicians had more than years of experience in endoscopic procedures and over colonoscopies and therapeutic procedures performed. there were teleconsultations via standard internet connection mb/s. endoscopic centers were equipped with olympus and series linked to video card. each card had its own ip address, and the image was accessible through internet login from anywhere. consulting physicians used computers connected to internet for tracing the image synchronously and giving advice. results: teleconsultations were undertaken in . % of all endoscopic procedures. teleconsultations concerned difficulties in endoscopic image interpretation in cases and decisions regarding further treatment in cases. the consulting physician solved all problems concerning proper endoscopic image interpretation. in cases the elective procedure was rejected. the elective treatment was continued in remaining cases. patients had a complication of polypectomy that was endoscopically treated. conclusions: the opinion of independent consulting physician in difficult clinical cases regarding endoscopic procedures helps to understand the endoscopic image in real time and implicates a decrease in complications after endoscopic procedures. michelle ganyo, md, robert lawson, md; naval medical center san diego introduction: a presacral phlegmon is a contained collection of infected fluid and inflammation within the bony pelvis, posterior to the rectum and anterior to the sacrum, that usually arises as a complication of surgery, malignancy, inflammatory bowel disease, ischemic colitis or perforated viscous. symptoms include low-back pain, pelvic pain and fevers. antibiotics and supportive therapy are the mainstay of treatment. however, if abscess develops, drainage is required usually by trans-gluteal percutaneous and/or surgical methods, both of which are associated with significant morbidity and mortality. endoscopic ultrasound (eus) -guided drainage of perirectal and presacral abscesses is a well described minimally-invasive approach that permits clear definition of anatomy, real-time access to the abscess and creation of an internalized fistula through placement of one or more transluminal stents. however, to date there is no published report describing endoscopic treatment of the more complicated, clinically challenging presacral phlegmon. here we present a case of a symptomatic presacral phlegmon recalcitrant to medical management that was successfully treated with an endoscopically placed retrievable, transmural, lumen-apposing metal stent. case report: this is a case-report of a -year-old, post-partum female who presented with fevers and recurrent lower back pain radiating to her rectum and vagina. her spontaneous vaginal delivery was notable for a second-degree laceration that was primarily repaired at the time of delivery months prior to presentation. her past medical history was otherwise unremarkable. radiographic imaging revealed several perirectal and presacral abscesses that were considered too small for percutaneous drainage. iv antibiotics were started and the largest abscess was targeted for eusguided aspiration. unfortunately, her pain became constant and progressed in severity. a follow-up mri a week later revealed a -cm presacral phlegmon. results: colonoscopy revealed a luminal bulge in the rectum but was otherwise normal. to permit drainage and multiple sessions of endoscopic necrosectomy, a mm lumen-apposing metal stent (lams) was placed transrectally under eus-guidance into the presacral phlegmon. endoscopic debridement with forceps and copious irrigation was performed. over the following weeks the patient reported purulent rectal drainage and resolution of her fevers and pain. repeat endoscopy revealed a normal rectum and no sign of the stent. a follow up mri showed a -cm area of heterogenous tissue in the presacral area. conclusions: although not previously described for management of a presacral phlegmon, lams appears to be a safe and effective, minimally-invasive treatment option. introduction: flexible endoscopy has evolved to include multiple endoluminal procedures such as anti-reflux procedures, pyloromyotomy, and mucosal and submucosal tumor resections. however, these remain technically demanding procedures as they are hindered by the state of flexible technology which has difficult imaging, limited energy devices, no staplers, and cumbersome suturing abilities. an alternative approach is transgastric laparoscopy, which for almost decades has been shown to be a good procedure for pancreatic pseudocyst drainage and full-thickness and mucosal resection of various lesions. we propose to expand the indications of transgastric laparoscopy by using novel endoscopically placed transgastric laparoscopy ports (taggs, kansa, usa) to replicate endoscopic procedures such as endoluminal antireflux surgery. methods and procedures: under general anesthesia female pigs (mean weight . kg) had endoscopic placement of mm-intragastric ports (taggs, kansas, usa) using a technique similar to percutaneous endoscopic gastrostomy. a mm laparoscope was used for visualization. endoflip, (crospon, inc., galway, ireland) was used to measure cross sectional area (csa) and compliance of the gastroesophageal junction (gej) before and after intervention. laparoendoscopic-assited suture plication of the gej was performed using - sutures (polysorb®). once the taggs ports were removed, the gastrotomies were closed by using endoscopic clip. at the end of the protocol, animals were euthanized. results: five laparoendoscopic-assited sewing plication were performed. the mean operative time was , min (endoscopic evaluation: . min, tagss insertion: min, endoflip evaluation+ gej plication: , min, gastric wall closure: min). in all cases, this technique was effective in achieving adequate gej plication. median gej diameter (d) and median cross-sectional area (csa) pre-plication were . mm ( . - . mm) and . mm ( - mm ). after the procedure, these values were decreased to . mm ( . - . mm) and . mm ( - mm ) respectively (p= , ). median distensibility (d) and median compliance (c) pre-plication were . mm /mmhg ( . - . mm /mmhg) and . mm /mmhg ( , - , mm /mmhg). after the procedure, these values were decreased to , mm /mmhg ( . - . mm /mmhg) and . mm /mmhg ( . - . mm /mmhg) respectively (p= , ). no intraoperative events were observed. conclusion: a hybrid laparoendoscopic approach is a feasible alternative for performing intragastric procedures with the assistance of conventional laparoscopic instruments; especially in cases where the location of the intervention limits the access of standard endoscopy or where endoscopic technology is inadequate. further evaluation is planned in survival models and clinical trials. introduction: due to previous manipulation or submucosal invasion, colonic lesions referred for endoscopic mucosal resection (emr) frequently have flat areas of visible tissue that cannot be snared. current methods for treating residual tissue may lead to incomplete eradication or not allow complete tissue sampling for histologic evaluation. our aim is to describe dissection-enabled scaffold assisted resection (descar): a new technique combining circumferential esd with emr for removal of superficial non-lifting or residual "islands" with suspected submucosal involvement/fibrosis. methods: from to , lesions referred for emr were retrospectively reviewed. cases were identified where lifting and/or snaring of the lesion was incomplete and the descar technique was undertaken. cases were reviewed for location, prior manipulation, rates of successful hybrid resection and adverse events. results: lesions underwent descar due to non-lifting or residual "islands" of tissue. patients were % m, % f, and average age (sd ± . yrs). lesions were located in the cecum (n= ), right colon (n= ), left colon (n= ) and rectum (n= ). average size was mm (sd ± . mm). previous manipulation occurred in / cases ( % biopsy, % resection attempt, % tattoo). the technical success rate for resection of non-lifting lesions was %. there was one delayed bleeding episode but no other adverse events. approximately % of patients have been followed up endoscopically to date with no evidence of residual adenoma. conclusions: descar is a feasible and safe alternative to argon plasma coagulation and avulsion for the endoscopic management of non-lifting or residual colonic lesions, providing en-bloc resection of tissue for histologic review. further studies are needed to demonstrate long-term eradication and for comparison with other methods. results: patients underwent fully covered stent placement procedures. indications for stent placement were leak in patients ( sleeve; bypass) and stricture in patients ( bypass, sleeve). five patients had stent migration. three required surgical removal, one patient endoscopic repositioning and one passed the stent per rectum. all eight patients with enteric leak successfully underwent stent placement in conjunction with diagnostic laparoscopy and drainage. all but one of these patients developed an enteric leak perioperative to index procedure. the average duration of stent treatment in these patients was days ( - days). of the patients treated for a stricture, patients ( sleeve, bypass) failed treatment and required subsequent definitive operative revision. average length of time of stent treatment in these patients was days (range, - days) and five had severe intolerance. conclusions: endoscopic stent placement of leak may require multiple procedures and carries the risk of migration; however, this therapy seems to be an effective treatment. failure rates are higher with strictures and are not as tolerated by patients. background: colonoscopy is the most commonly performed endoscopic examination worldwide and is considered the gold standard for colorectal cancer screening. the quality of examination and endoscopic treatment is affected by a number of factors that are verified by recognized parameters such as cecal intubation rate and time (cir, cit), withdrawal time, adenoma detection rate (adr) and polyp detection rate (pdr). advanced endoscopic imaging improves accurate recognition of the nature and variety of pathologic lesions, while the endoscope tips, third eye retroscope and wide-angle endoscopy allow detection of lesions located on the proximal side of the intestinal folds. the aim of the study was to assess the suitability of wide-angle colonoscopy for the detection of colorectal lesions and to analyze the functionality of a special endoscope series regarding cir, cit and withdrawal time. introduction: leak is an uncommon but serious complication of gastrointestinal surgery. when identified post-operatively, percutaneous drains are used to manage abscesses and prevent further peritoneal contamination. if drain position is suboptimal, however, the consequences of persistent leak may necessitate a formal surgical intervention in a hostile abdomen. in select situations, we have utilized natural orifice transluminal endoscopic surgery (notes) methods to enter the abdominal cavity and place/reposition drains under direct endoscopic visualization a part of our comprehensive endoscopic management algorithm for leaks. methods and procedures: a prospectively collected database was queried for patients who had undergone transluminal endoscopic drain repositioning (tedr) as part of multimodal endolumenal therapy for leak (including interventions like defect closure, enteral feeding access, or endolumenal stent placement). inadequate drainage was identified pre-procedurally by undrained fluid collections in conjunction with clinical signs of sepsis. translumenal access was obtained via the leak site and carbon dioxide insufflation was used in all cases. the peritoneal cavity was surveilled and cleared of gross debris by irrigation and suction. intraabdominal drains were located endoscopically and fluoroscopically, grasped with an endoscopic snare or grasper and repositioned adjacent to the leak site to ensure better drainage. results: four patients ( female), average age (range - ), average body mass index (range - ) were managed with tedr as a component of endoscopic treatment of full-thickness gastrointestinal leak. two patients developed leak following revisional bariatric surgery. one patient had an acutely dislodged gastrostomy tube with intraperitoneal leak after multiple laparotomies recently closed with a granulating vicryl mesh. one patient developed a leak at an esophagojejunostomy following total gastrectomy. three patients had adequate drainage after the initial tedr, while one patient required tedr on two occasions. all patients had improved drainage demonstrated by resolution of clinical signs of sepsis and resolution of fluid collections. drains were removed as clinically indicated. conclusion: intraabdominal drains are an essential element in the management of full-thickness gastrointestinal leaks, but are not always able to be adequately positioned percutaneously. transluminal endoscopic drain repositioning via a gastrointestinal defect is a viable option to avoid surgical intervention in an otherwise hostile field and is a novel practical notes application. background: epiphrenic diverticula (ed) arise from increased intraluminal pressures, often secondary to achalasia or another underlying esophageal motility disorder which causes "pulsion" physiology. ed are traditionally thought to contribute to patients' symptoms of regurgitation and dysphagia, and are frequently resected at time of heller myotomy and fundoplication done for treatment of the primary motility disorder. ed excision carries significant risks (staple line leak, pulmonary complications, mortality), and little is known regarding patients with ed and esophageal motility disorder who undergo surgical myotomy without ed resection. the goal of this study was to compare outcomes of patients with ed and esophageal motility disorder who did and did not undergo diverticulectomy at time of myotomy and fundoplication. methods: retrospective analysis of prospectively collected database from to was performed. patients with diagnosis of ed undergoing surgical treatment of symptomatic esophageal motility disorder were included. all patients underwent laparoscopic heller myotomy with toupet fundoplication by a single surgeon at a tertiary referral hospital. patients were stratified according to whether ed was excised or not excised at time of primary surgery. patient-reported symptoms were obtained from pre/post-operative clinic evaluations and mailed surveys during the follow-up period. independent samples t-test and fisher's exact test were used to compare continuous and categorical variables respectively. results: ed was identified in patients prior to surgery. primary diagnoses included achalasia (n = ), nutcracker esophagus (n= ), and diffuse esophageal spasm (n= ). ed was excised in five patients ( . %) and not excised in ten patients ( . %), with no significant difference in frequency of preoperative dysphagia ( % vs. %, p= . ) or regurgitation ( % vs. %, p= . ) between groups respectively. reasons for non-resection included ed was too proximal (n= ), patient/surgeon preference (n= ), and small ed size (n= ). the resection group did not experience any leaks and there were no mortalities in either cohort during the follow-up period. at mean clinic follow-up of days, there was no difference in frequency of residual dysphagia in patients who did or did not undergo ed resection ( % vs. %, p= . ) and neither cohort reported residual regurgitation symptoms. conclusions: this study suggests that leaving ed in place during surgical treatment of an esophageal motility disorder may achieve similar rates of postoperative symptom control. while ed excision in this study did not cause significant excess morbidity, ed resection introduces risk of leak and requires more extensive surgery that may not provide significant benefit to patients. introduction: median arcuate ligament syndrome (mals) has been described in the literature as presenting with a constellation of symptoms including nausea, vomiting, weight loss, and post-prandial epigastric pain. while many of these symptoms are consistent with foregut pathology, a cohort of patients with mals presenting with delayed gastric emptying has not been described in the literature. in this study we report on the possible association of mals with delayed gastric emptying. methods: cases of mal release were collected between and . eight patients were identified who presented with mals and underwent subsequent mal release. all patients underwent laparoscopic or robotic surgery. patients were compiled into a retrospective database and their demographic, symptomatic, imaging, and outcomes data were analyzed. background: laparoscopic fundoplication (lf) is often performed to treat paraesophageal hernia and/or gerd. care is taken to select the right patients for the operation. some patients may not improve, and others experience dysphagia or bloating after surgery. factors associated with patient satisfaction after fundoplication would be helpful during the patient selection process. methods: a retrospective review of a prospectively collected database was performed. queried patients underwent lf from to . non-elective operations and fundoplications after heller myotomy were excluded. of this cohort, patients were included only if they responded to a two-year postoperative quality of life survey. surveys were distributed preoperatively, at three weeks, at one year, and at two years. the surveys include the reflux severity index, gerd-hrql, and dysphagia score. the gerd-hrql asks about patient satisfaction with their current state ( = dissatisfied, = somewhat satisfied, = very satisfied). the cohort was divided according to their answer to this question at two years. demographics and preoperative factors were compared between the groups with kruskal-wallis and fisher's exact tests. univariable and multivariable ordinal logistic regression was performed to identify preoperative symptoms associated with satisfaction at two years. scores on the surveys over time were were also analyzed. results: a total of patients were included in the analysis (dissatisfied = , somewhat satisfied = , very satisfied = ). the only significant demographic or preoperative difference was a high number of paraesophageal hernias in the 'very satisfied' cohort (p = . ). on univariable regression, younger age and paraesophageal hernia predicted satisfaction. several variables negatively predicted satisfaction with an or \ . multivariable regression, controlled for age and hernia type, identified throat clearing, post-nasal drip, and globus sensation as preoperative symptoms less likely to result in patient satisfaction (p = . , . , and . , respectively). subgroup analysis of patients with paraesophageal hernias revealed that patients with bloating preoperatively are less likely to be satisfied at two years. survey scores over time showed all groups improving over three weeks, but while satisfied patients continued to improved, dissatisfied patients symptomatically worsened over time. conclusion: this study confirms previous reports stating atypical symptoms of gerd are less likely to improve after lf. it also shows individuals with paraesophageal hernia tend to do quite well, unless they report bloating preoperatively. patient-centered analysis such as this can be useful when discussing postoperative expectations with patients, and may reveal opportunities to individualize operative approach. objective: the study was performed to assess whether sutured crural closure or mesh reinforcement for hiatal closure yields better results with regards to symptom resolution and recurrence post-operatively. material and methods: a prospective randomized controlled trial was carried out at grant medical college and sir j. j. group of hospitals, mumbai, india. patients were randomized to receive either sutured repair or mesh reinforcement of hiatal closure. outcomes of interest were symptom resolution, quality of life scores and recurrence in the postoperative period. results: patients were recruited for the trial ( -sutured repair, -mesh reinforcement). the two groups were comparable in terms of demographic profiles, symptom severity and findings at esophagogastroscopy and manometry in the pre-operative period as well as size of the hiatal defect measured intra-operatively. post-operatively the mesh repair group had significantly better symptom resolution in terms of early satiety, chest pain and regurgitation (p\ . ) while with respect to heartburn, dysphagia and post-prandial pain there was no significant difference between the improvements demonstrated. improvement in quality of life scores after either procedure was not significantly different. recurrence was higher in the suture repair group ( vs , p. ). recurrence lead to poorer symptom severity scores as well as quality of life scores and one patient underwent re-operation. the change in the symptom severity score from baseline after the procedure at months in the subgroup population. conclusion: mesh reinforcement results in a reduced rate of recurrence and offers excellent symptom control in the short-term without a rise in complications when compared to sutured repair for the closure of hiatal defects in laparoscopic hiatal hernia repairs. material and methods: in a period from to , patients underwent laparoscopic resection ( -gastric resection, -duodenal resection), using different techniques. all patients were investigated with upper gi endoscopy, eus and abdominal contrast-ct, which allows us to get the complete evaluation of tumor, including size, location, type of growth and the gi layer. based on the findings the decision on the type of resection was made. the majority of resections were wedge or partial resections, performed using endoscopic steplers or using ultrasound scissors followed by double-suturing of gatro/duodenotomy. in the cases of tumor location on the posterior gastric wall we mobilized the the greater curvature to get a direct approach to the tumor with extraluminal growth. in the cases with intraluminal growth we used transgastric approach with small , cm incision on the anterior gastric wall for endoscopic stepler. technically the most complex procedures were in the cases of tumor location close to anatomically narrow places and muscle sphincters (gastroesophageal junction, pylorus, duodenal bulb, duodenal flexure), with high risk of stenosis and dysfunction of anatomical sphincters. in such cases we used «lifting-technique» in which we dissect serous and muscle layers circumferentially around the tumor making partial enucleation of lesion followed by total resection preserving almost all normal tissue with minimal suturing and deformity at the site of surgery. ( : ), mean age was . years (sd ± . ), patients ( %) had mis. the type of reconstruction was predominantly with a "pull-up" technique (n = , . %) followed by the kirschner-akiyama procedure (n = , . %), stapled gastroplasty was performed in patients. all the anastomosis were performed at the level of the neck and only one of the patients had a stapled anastomosis, mean operative time was min (sd ± min) including resection of the specimen. primary neoplasms were predominantly hypopharynx (n = , . %), distal esophagus (n = , %), cervical esophagus (n = , . %) and thoracic esophagus (n = , . %). histologic types were mainly squamous cell carcinoma (n = , . %) and adenocarcinoma (n = , . %). mean of hospitalization days was . (sd ± . ). no complications were observed in patients and major complications (dindo-clavien ≥iiib) were found in patients. anastomotic leak was present in patients ( . %) and perioperative mortality ( days) was . %. progressive shift to laparoscopic surgery was evidenced through the years ( - : . %, - : . % and - : . %; p = . ) and reduction in major complications (p = , ) was observed. anastomotic leaks (p = , ) and perioperative mortality (p = . ) did not show significant differences in the present study. conclusions: results in our center show that major complications decrease with time after application of minimally-invasive surgery and no differences in anastomotic leaks and mortality were seen. current data has lead us to abandon open total esophagectomy as a first-choice procedure. introduction: minimal invasive three-fields esophagectomy for minimal invasion is the surgical standard for oncological procedures and benign diseases. cervical dissection has a risk of to % in some series, of, lesion or paralysis of the rnl, but the standard in mckeon approach is %. a high level of suspicion is needed because this type of lesion has an impact on postoperative evolution and the hospital stay. main: to describe three cases of rnl post esophagectomy paralysis in three planes by least invasion. methods: in a period of years, january to june , esophagectomies for bening disease were performed. three patients ( males female) with diagnosis of terminal achalasia and stenosis secondary to caustic ingestion consulted at the minimal invasion service fundcacion valle del lili. they were schedualed for minimal invasive three fields esophagectomy, one patient without complications and early discharge ( postoperative day) but occasional dysphagia, the other two required early reintubation after de surgery with ards, patient requiered tracheostomy, the second patient could be extubated after days but with occasional dysphagia. all three had mild hoarseness after surgery. the patient who required tracheostomy was decannulated at days without complication. results: the three patients underwent endoscopy without complication in the cervical anastomosis stenosis or disorder in the emptying of the gastric tube, swallowing study without alteration and laryngoscopy with paralysis of the left vocal cord. these patients went to speech therapy with total paralysis recovery at months corroborated with laryngoscopy, without dysphagia or hoarseness. conclusion: rnl innervates the larynx and upper esophageal sphincter, therefore lesion or paresis causes symptoms such as hoarseness, dysphagia, difficulty swallowing, aspiration, difficulty in coughing, pneumonia and ards. injury has a predecessor factor in pulmonary complications and prolongation of the hospital stay. % of these patients may require some surgical procedure to restore the function of rnl. noninvasive monitoring of the laryngeal nerve decreases the risk of injury. philip case report: multiple esophageal diverticula associated with achalasia introduction: achalasia is well defined disorder of increased lower esophageal sphincter tone ( ). epiphrenic esophageal diverticulum are a rare disorder believed to result from increased intraesophageal pressure often in conjunction with a motility disorder causing functional outflow obstruction. they are a pulsion-type pseudo-diverticulum with mucosal bulging most frequently from the right posterior esophageal wall ( ) . we present a very rare case of achalasia associated with multiple esophageal diverticula successfully treated with laparoscopic heller myotomy with dor fundoplication. case presentation: a year old woman presented with years of dysphagia, chest discomfort, regurgitation, and weight loss. esophagoscopy showed a patulous esophagus with multiple esophageal diverticula (figure ). barium esophogram demonstrated esophageal diverticula in the distal esophagus and delayed clearance of esophageal contrast (figure ). high resolution monometry revealed a hypertensive mean les, an aperistaltic body on of wet swallows, and panesophageal pressurization in of wet swallows -consistent with type ii achalasia by chicago classification ( ). we performed a laparoscopic heller myotomy with dor fundoplication. the myotomy was extended cm above the gasgtroesophageal junction and cm onto the gastric cardia. an anterior diaphragmatic defect with a moderate type hiatal hernia was repaired with two sutures, ensuring to not impinge the esophagus (figure ). at weeks post operatively the patient reports excellent results. her dysphagia and chest discomfort have entirely resolved. her eckhardt score improved from seven preoperatively to one post operatively. discussion: type ii achalasia is successfully treated in the majority of cases with laparoscopic heller myotomy and partial fundoplication ( ). however, esophageal diverticula typically require both myotomy as well as diverticulectomy for successful treatment ( ) . there is little experience with the surgical management of multiple esophageal diverticula. we propose a two stage surgical approach for these patients. we reason that the risk of esophageal leak or stenosis in the case of multiple esophageal diverticulectomies out weighs the proposed benefit. indeed epidemiologic studies indicate that the majority of esophageal diverticula are asymptomatic ( ) . in the event the patient remains symptomatic after myotomy a second stage operation with diverticulectomies would be possible. this single experience suggests that diverticulectomy may not be necessary in the case of multiple diverticula associated with achalasia. instead, treatment may be directed at relieving the functional obstruction responsible for the symptoms by performing laparoscopic heller myotomy with dor fundoplication. takahiro kinoshita, md, facs, masanori tokunaga, md, akio kaito, md, masahiro watanabe, md, shizuki sugita, md; national cancer center hospital east, japan objective: the optimal surgical approach for siwert type ii cancer is still controversial due to the anatomical complexity of the region. potential advantages of laparoscopic transhiatal approach have not been fully investigated. methods and procedures: we retrospectively analyzed consecutive patients with siewert type ii cancer who underwent laparoscopic transhiatal resection. indication of surgery is patients with siewert type ii cancer with less than cm esophageal invasion. regarding the extent of resection, basically proximal gastrectomy with the lower esophageal resection was selected, aiming at preservation of gastric reservoir function. in terms of reconstruction after proximal gastrectomy, double-tract method was performed. intraoperative peroral endoscopy was routinely employed for determination of the appropriate resection level of the stomach. esophagojejunostomy was employed by overlap method using a mm linear stapler. in order to obtain a wider operative field in the lower mediastinum, the diaphragmatic crus was dissected to widen the esophageal hiatus. results: in patients ( males and females), median operation time was minutes, and estimated blood loss was g. the rate of surgical morbidity was %, and that of anastomotic leakage was %. there was no mortality. the mean length of proximal margin was mm, and no positive margin was recorded. the -and -year overall survival rate was . % and %, respectively. conclusions: laparoscopic transhiatal resection for siewert type ii cancer is technically challenging, but appears feasible and safe when performed by an experienced surgical team. a largescale prospective study is necessary for final conclusion. introduction: mesh use for reinforcement of primary crural closure is controversial. synthetic mesh use poses a risk of erosion but there is no evidence that non-synthetic mesh is useful to minimize the risk of hernia recurrence. we evaluated a fully bioresorbable mesh made from poly- -hydroxybutyrate (p hb) for crural reinforcement after para-esophageal hernia (peh) repair. the aim of this study was to evaluate the safety and efficacy of p hb mesh at the hiatus in patients undergoing peh repair. this was a review of prospectively collected data on consecutive patients that had repair of a peh with reinforcement of the crural closure with p hb mesh. to be considered a peh at least % of the stomach was herniated into the chest. a collis gastroplasty or crural relaxing incision was added for short esophagus or crural tension when necessary. routine follow-up consisted of esophagogastroduodenoscopy (egd) at months for patients that had a collis gastroplasty and a barium upper gi study (ugi), high resolution manometry (hrm) and ph test in all patients at months. a hernia of any size identified during objective follow-up testing was considered a recurrence. overall, there was a significant difference in mean measured tension between the three subjective suture ratings by the surgeons. however, there was substantial variability and overlap amongst the surgeon's ratings (figure) . the tension necessary to approximate the crura during peh repair can be objectively measured and as expected increases progressively with anterior movement up the hiatus. while there was some correlation between a surgeon's subjective assessment of the tension necessary to bring the crura together and actual measured tension, there was wide variability and imprecision from one stitch to another. objective tension measurement may provide a more reliable assessment of when excessive force is being used to re-approximate the crura and potentially improve peh recurrence rates. ahmed introduction: paraesophageal hernia repairs are increasing in prevalence, and unfortunately carry a high recurrence rate. consequently, reoperation is expected to increase in frequency. published data on the outcomes of recurrent paraesophageal hernia (rpeh) repair is very limited. because of the technical difficulties of revisional surgery, we hypothesize that laparoscopic revisional paraesophageal hernia repairs are associated with high perioperative morbidity and poor patient outcomes. methods: all rpeh repairs performed by the foregut surgical service at our institution from to were reviewed. patients were included if their index operation was a true pehr (initial type hiatal hernia repairs were excluded, as well as multiply recurrent hernias). demographics, medical and surgical history, and operative notes from the index surgery were reviewed. details from standardized pre-operative symptom assessment, objective testing and operative details for the revisional surgery were collected. patients were routinely offered month post-operative upper gastrointestinal contrast evaluation. postoperative outcomes included a standardized symptom assessment and results of objective testing at any time after surgery. results: twenty six patients were identified who underwent repair of rpeh. demographic, operative and perioperative data was available for all patients (table ) . twenty four patients underwent followup symptom evaluation (two were lost to follow-up after the initial hospitalization). sixteen patients underwent follow-up objective testing by radiographic evaluation with contrast, endoscopy or both. these subgroups were used to calculate symptomatic and objective outcomes (table ) . conclusion: reoperative laparoscopic surgery for recurrent paraesophageal hernias is technically challenging as evidenced by long operative times. despite this, perioperative outcomes at a high volume center are good, with low morbidity and no mortality. importantly, symptomatic outcomes for this difficult problem are excellent. introduction: hypotension of the lower esophageal sphincter (hles) and the presence of hiatal hernia (hh) have both been associated with gastroesophageal reflux disease (gerd). the exact likelihood with which a hles or a hiatal hernia predict gerd continues to be defined. we hypothesize a synergistic interaction in those with hles and hh in predicting gerd as defined by a positive ph study. methods and procedures: between and , consecutive patients presenting to a surgical practice with symptoms most concerning for gerd, without prior antireflux surgery were evaluated by high resolution manometry (hrm), esophagogastroduodenoscopy (egd), videoesophagography (veg) and an ambulatory ph study. hles was defined as residual les pressure of\ mmhg, hh was defined as having been noted and measured by the radiologist, these were further categorized into any hh, - cm, [ - cm background: while clinical outcomes have been reported for antireflux surgery, there is limited data on postoperative outpatient encounters and their associated costs. the aim of this study is to evaluate the utilization of healthcare and its associated costs during the -day postoperative period following antireflux surgery. methods: we analyzed data from the truven health marketscan® research databases. patients ≥ years with an icd- procedure code or cpt code for antireflux surgery and a primary diagnosis of gerd during - were selected. only patients with continuous enrollment six months prior to the date of surgery and -days after surgery were analyzed. patients with a diagnosis of esophageal cancer or achalasia during the six-month period prior to antireflux surgery, a length of stay [ days following index procedure, a capitated plan, or patients who underwent emergency surgery were excluded. outpatient endoscopy was defined using icd- and cpt codes, and related readmission was defined by clinical classification software. introduction: the development of postsurgical gastroparesis following nissen fundoplication is poorly understood. in this study, we analyze the development of gastroparesis requiring intervention and other subsequent procedures following fundoplication and paraesophageal hernia (peh) repair procedures in the state of new york. methods: using a comprehensive state-wide administrative database (sparcs), we examined all in-patient and outpatient records for adult patients who underwent fundoplication or peh repair as a primary procedure for the treatment of gerd between the years of - . patients with an initial gastroparesis diagnosis were excluded from the analysis. through the use of a unique identifier, each patient was followed until for the subsequent diagnosis of gastroparesis or reoperation. surgical procedures for the treatment of gastroparesis included pyloroplasty, pyloromyotomy, or gastroenterostomy procedures. multivariable logistic regression models were used to identify independent predictors for having subsequent reoperation. results: a total of , patients were analyzed. this included , fundoplication patients ( . %) and , ( . %) with peh repair. in the fundoplication group, ( . %) patients had a follow-up diagnosis of gastroparesis or secondary procedure. ( . %) of the patients who underwent a primary peh repair procedure had a follow-up procedure or gastroparesis diagnosis (table ) . mean time to follow-up procedure or diagnosis was . years for the fundoplication group and . years for the peh repair group. the majority of the follow-up procedures in the fundoplication group were revisional procedures (fundoplication or peh repair) (n = , . %), while ( . %) patients were newly diagnosed with gastroparesis and/or underwent a secondary procedure for its treatment. conclusion: fundoplication and peh repair procedures have a relatively low post-operative incidence of gastroparesis following initial procedure for treatment of gerd. secondary fundoplication or peh repair was more commonly performed compared to any of the surgical procedures for gastroparesis for both procedures. further analysis of association with subsequent procedures is needed. during this procedure, gastro-esophageal reflux was evaluated and assigned to severe, moderate and slight category. if the reflux was observed slightly up to cervical esophagus, the case was assigned to moderate category. if the reflux was observed intensely up to cervical esophagus, the position was returned to head high position for the safety and the case was assigned to severe category. the anti-reflux surgery was considered in the moderate and severe categories. results: we have performed laparoscopic nissen procedure in cases. the mean operation time was min. the outcome was assessed by reflux test performed on - postoperative day, and the results showed the reflux was disappeared in every cases. median follow-up period of this study was months ( - months). in cases ( . %) ppi was restarted before months after the anti-reflux surgery. in cases ( . %) ppi was restarted after the anti-reflux surgery during the whole follow-up period of this study. the bmi of the patients had no relationship to the needed restart of ppi. to evaluate the degree of esophagitis objectively before and after the anti-reflux surgery we designed "the esophagitis score". in this scoring method, a number from - was assigned according to the degree of esophagitis along with the la classification. the results of the study have shown that the reflux esophagitis was improved obviously after the anti-reflux surgery even in the ppi restarted group (p. ). discussion: the number of gerd patients who needed anti-reflux surgery seems to be so high. to extract the patients who needed it remarkably is important. the anti-reflux surgery is most effective for the patients who really have the obvious reflux. reflux test is feasible because of its convenience and visual effects for the patients. the results of the laparoscopic nissen fundoplication were good and satisfied by the patients mostly. surg endosc ( ) :s -s introduction: fundoplication at the time of giant paraesophageal hernia repair is controversial. the proposed advantages are better reflux control and lower recurrence. disadvantages include fundoplication specific complications, might be unnecessary and may not decrease recurrence. we retrospectively reviewed giant paraesophageal hernia repairs (peh) with two point gastropexy in the fundus and body, and no antireflux procedure. data collected is postoperative gerd symptoms, postoperative proton pump inhibitors (ppis) therapy and recurrence. methods: a retrospective review of patients who underwent repair of giant peh from to december of . giant was defined as a hernia with % or more of the stomach above the diaphragm. follow up consisted of upper gi (ugi) study one year postoperatively and reflux symptom questionnaire. patients were followed every months in the surgery clinic and a ppi wean was initiated at the second postoperative visit. the primary outcome we evaluated was discontinuation of ppis. in addition, we utilized a standardized reflux scale and recurrence rates collected. chi-squared was used for statistical analysis. background: gastroesophageal reflux disease (gerd) is a highly prevalent disorder with a multitude of treatment options ranging from lifestyle modifications and medical management to surgical options. despite the numerous treatments available, there is still debate over which approach is most appropriate and effective for patients. this study aims to examine the effect of robotic hiatal hernia repair (rhhr) with the novel addition of esophagopexy in patients with gerd. methods: a single institution, single surgeon, prospectively maintained database was used to identify patients who underwent rhhr with a partial fundoplication and concomitant esophagopexy for gerd from november to july . patient characteristics, operative details and postoperative outcomes were analyzed. primary endpoint was resolution of subjective gerd symptoms and discontinuation of proton pump inhibitor (ppi). recurrence of hiatal hernia was a secondary endpoint. results: eleven patients were identified meeting the inclusion criteria (rhhr + esophagopexy) with a mean follow-up of . weeks ± . weeks. in regards to the rhhr, % underwent a partial fundoplication and the additional % underwent a re-do wrap. this patient cohort was . % female with a mean age of . ± . years. preoperative esophagogastroduodenoscopy (egd) was performed in % of patients with the study showing a hiatal hernia in . %, gastritis in . % and esophagitis in . % of patients. manometry was performed in . % of the patients showing % of these patients with esophageal dysmotility. esophagograms and ph studies were performed preoperatively in . % and . % of patients respectively. preoperatively, % of patients had a documented diagnosis of gerd and were taking a ppi and/or h blocker. after rhhr with esophagopexy, . % of patients had resolution of their gerd symptoms while . % (n = ) remained symptomatic. however, one of two patients reported a subjective decrease in symptom severity following the procedure. despite resolution of symptoms, . % remained on ppis. another % switched to h blockers and one patient discontinued all antisecretory therapy. none of the patients experienced recurrence of their hiatal hernia. conclusion: based on our data, rhhr with esophagopexy results in resolution gerd symptoms in over % of symptomatic patient. in patients with hiatal hernias and gerd, rhhr with esophagopexy does lead to resolution of symptoms, however, the majority of patients remained on ppis. long-term follow up is needed to investigate whether these patients are able to discontinue ppis and remain symptom free. chaya shwaartz, nadav zilka, mustapha siddiq, yuri goldes, md; sheba medical center, israel background: d gastrectomy for gastric carcinoma is a well-established procedure in patients undergoing surgery for gastric cancer and is the standard of care in our institution. reduced pain, early ambulation, and better cosmetics are some of the benefits of minimally invasive surgery for early gastric cancer. we aimed to describe our experience in laparoscopic d gastrectomies undertaken by a single surgeon in our institution. methods: this is a single-center retrospective review of prospectively collected d gastrectomies performed by a single surgeon. between november and february , laparoscopic subtotal/total gastrectomies were performed at sheba medical center, a tertiary center for forgut cancer. clinicopathological characteristics of the patients, surgical performance, postoperative outcomes and pathological data were collected. results: forty-five patients underwent laparoscopic gastrectomy. of these, had subtotal gastrectomy and had total gastrectomy. the median age in our series ( - ). most of the patients in our series had early gastric cancer (t - ) ( %). the mean average of dissected lymph nodes was ± . the mean operative time was ± . the postoperative complications, classified using the clavien-dindo classification. severe complications ([ cd iiia) rate was %. conclusions: laparoscopic d gastrectomy for invasive gastric cancer is safe and feasible when carried out in high-volume centers by an experienced surgeon as part of a multidisciplinary team with careful case selection and appropriate high-quality postoperative support. minimally invasive management of diaphragmatic hernias after esophagectomy: a case report introduction: esophagectomy is a common treatment for both benign and malignant pathologies of the foregut. hiatial paraconduit hernias are rare complications following esophagectomy. in this study, we review our experience with these rare diaphragmatic hernias. methods: a retrospective analysis of all patients presenting with hiatial hernia after esophageal resection at the university of oklahoma health science center between and was performed. data was abstracted from the medical record for evaluation and included demographics, symptoms, repair techniques and outcomes. no patients were excluded. results: a total of ten patients were identified to have paraconduit hernias. during this time interval, there were a total of esophageal resections performed. all patients had esophagectomy for malignant disease. seven of the patients have undergone surgery. two patients are asymptomatic and are being followed at their request, and one patient is pending elective correction. of the seven patients who underwent surgery, the median age was , with males and two females. six of the seven patients underwent minimally invasive ivor lewis esophagectomy and one had an open mckeown procedure. the median time from esophagectomy to hernia repair was months, with range from month to months. the most common presenting complaint was abdominal pain and nausea. one patient was noted to have a paraconduit hernia on postoperative day and taken to surgery for repair during the hospitalization. there was one death in a patient who presented with necrosis of the small bowel. the remaining patients all had laparoscopic approach. one patient required a hand port to reduce incarcerated colon and one patient was noted to have a cecal perforation during port closure requiring repair. all patients had herniated colon, with small intestine or pancreas herniation noted in three. repair was performed by reducing the viscera, a left phrenic relaxing incision, closure of the hiatus around the conduit and then closure of the diaphragmatic defect with mesh. at median follow up of months, there are no recurrences. conclusion: hiatal paraconduit hernias are becoming a frequent finding among survivors of esophageal cancer surgery. our study demonstrates that there is a propensity for patients who undergo minimally invasive esophagectomy to develop these hernias. the vast majority of patients can undergo laparoscopic repair. our recommendation is to perform a diaphragmatic relaxing incision and liberal use of mesh. early results appear to be favorable regarding recurrence. aim: there have been several reports illustrating the safety and efficacy of various surgical techniques in performing laparoscopic esophagojejunostomy (ej). this study aims to compare two established methods of ej anastomosis -circular stapling with purse-string suture ("lap-jack") and linear stapling technique -in laparoscopic total gastrectomy. methods: patients diagnosed with gastric cancer underwent intracorporeal ej anastomosis in laparoscopic total gastrectomy from january, to october, . cases used the circular stapler with purse-string "lap-jack" method, and patients used the linear stapling method for ej anastomosis. were matched using propensity scores, and retrospective data for patient characteristics, surgical outcome, and post-operative complications was reviewed. the two groups showed no significant difference in age, bmi, or other clinicopathological characteristics, and there was no conversion to an open procedure. after propensity score matching analysis, the linear group had significantly shorter operating time ( . ± . vs . ± . , p≤ . ) and more sufficient proximal margin ( . ± . vs . ± . , p = . ). no significant difference was found in estimated blood loss, retrieved lymph node, hospital stay, and time for first flatus. there was no postoperative mortality. early postoperative complication of the circular and linear group occurred in ( . %) and ( . %, p = . ) patients respectively. ej leakage occurred in ( . %) cases from each groups, with ( %) case from both group needing radiologic or surgical intervention. no other significant difference in early complication was found. late complication was observed in ( . %) cases (circular = linear = , p = . ) with ej anastomosis stricture in the linear group, but there was no statistical significance. conclusion: both circular stapling and linear stapling techniques are feasible and safe in performing intracorporeal ej anastomosis during laparoscopic total gastrectomy. linear-stapling technique had more sufficient proximal margin and shorter operating time. there was no significant difference in anastomosis related complication between the two groups. masahiro watanabe, masanori tokunaga, akio kaito, shizuki sugita, takahiro kinoshita; national cancer center hospital east, gastric surgery division background: although the current standard treatment for advanced gastric cancer (agc) is open gastrectomy, laparoscopic gastrectomy (lg) is increasingly performed, especially in the east. however, it is a technically demanding procedure, and the feasibility remains unclear. the aim of the present study was to clarify the feasibility of lg for agc. patients and methods: the present study included patients who underwent lg for agc between and . the indication of lg has gradually expanded in our institute, and is currently any stage gastric cancer except for gastric cancer obviously invading adjacent organs or gastric stump carcinoma. we retrospectively reviewed short-and long-term surgical outcomes of the patients. results: male/female ratio was : , and median age (range) was ( - ) years. distal gastrectomy was most frequently performed ( %), followed by total gastrectomy ( %). median operation time and intraoperative blood loss was ( - ) minutes and ( - ) g, respectively. clavien-dindo grade iii or more complication rate was . %. with a median followup period of months, the -year recurrence free survival rates of pstage ii and iii patients were % and %, respectively. conclusion: the outcomes of lg for agc are satisfactory, provided that an experienced team performs the surgery. introduction: the present study aims to evaluate the predictive value of indocyanine green (icg) for the detection and prevention of anastomotic leak following esophagectomy. anastomotic leak is a highly morbid and potentially fatal complication of esophagectomy. ensuring adequate perfusion of the gastric conduit can minimize the risk of postoperative leak. intraoperative evaluation with fluorescence angiography using icg offers a dynamic assessment of gastric conduit perfusion, and can guide anastomotic site selection. methods: a search of electronic databases medline, embase, scopus, web of science and the cochrane library using the search terms "indocyanine/fluorescence" and esophagectomy was completed to include all english articles published between and august . articles were selected by two independent reviewers based on the following major inclusion criteria: ( ) esophagectomy with gastric conduit reconstruction; ( ) use of fluorescence angiography with indocyanine green to assess perfusion; ( ) age ≥ years; ( ) sufficient outcome data for the calculation of leak rates and ( ) sample size ≥ . the quality of included studies was assessed using the quality assessment of diagnostic accuracy studies- . results: our literature search yielded potential studies, of which studies were included for meta-analysis after screening and exclusions. there were eleven prospective and three retrospective studies. the pooled anastomotic leak rate when icg was used was found to be %. pooled sensitivity and specificity for leak detection were . ( . - . ) and . ( . - . ), respectively. when studies involving intraoperative modifications were removed, pooled sensitivity and specificity were only marginally changed to . ( . - . ) and . ( . - . ), respectively. the diagnostic odds ratio was found to be . ( . - . ) across all studies and . ( . - . ) when intraoperative interventions were excluded. only three trials included a control group, giving a sample size of . in studies with a comparator group, icg was associated with an % reduction in the risk of anastomotic leak [or: . ( . - . )]. conclusions: in non-randomized trials, the use of icg as an intraoperative tool for visualizing vascular perfusion and conduit site selection, is promising. however, poor data quality and heterogeneity in reported variables limits cross-study comparisons and generalizability of findings. randomized, multi-center trials are needed to account for independent risk factors for leak rates and to better elucidate the impact of icg in predicting and preventing anastomotic leaks. objective: robotic assistance for bariatric surgery represents a novel application of a rapidly emerging technology. its safety and efficacy remains primarily characterized by smaller, singleinstitution studies. in this investigation, the influence of robotic assistance on short-term perioperative outcomes is contrasted with the more established primary multi-port laparoscopic approach for patients undergoing roux-en-y gastric bypass (rygb), using data from a national bariatric database. methods: a retrospective analysis of , robotic-assist and , laparoscopic rygb patients from the metabolic and bariatric surgery accreditation and quality improvement program national database were reviewed for differences in patient characteristics and short-term outcomes. on bivariate analysis, variables associated with primary outcomes of -day reoperation, readmission and reintervention were imputed into multivariate analyses to determine independent significance. results: robotic-assist bypass patients were older (p\. ), had a higher prevalence of comorbidities and had concomitant operations more frequently performed during surgery (p\. ). on bivariate analysis, robotic-assist patients had a higher rate of readmission than laparoscopic patients ( . % vs. . %; p=. ), but no differences in -day reoperation ( conclusion: robotic-assistance does not confer an increased rate of morbidity and mortality after rygb, and represents a feasible surgical modality for the surgeon willing to adopt the technology and accept its limitations. alicia m bonanno, md, brandon tieu, md, farah husain, md; oregon health and science university introduction: marginal ulcer is a common complication following roux-en-y gastric bypass with incidence rates between and %. most marginal ulcers resolve with medical management and lifestyle changes, but in the rare case of a non-healing marginal ulcer there are few treatment options. revision of the gastrojejunal (gj) anastomosis carries significant morbidity and mortality with complication rates ranging from to %. thoracoscopic truncal vagotomy (ttv) may be a safer alternative with decreased operative times. the purpose of this study is to evaluate the safety and effectiveness of ttv in comparison to gj revision for treatment of recalcitrant marginal ulcers. methods and procedures: a retrospective chart review of patients who required surgical intervention for non-healing marginal ulcers was performed from st september to st september . all underwent medical therapy along with lifestyle changes prior to intervention and had preoperative egd that demonstrated a recalcitrant marginal ulcer. revision of the gj anastomosis or ttv was performed. data collected included operative time, ulcer recurrence, morbidity rate, and mortality rate. statistical analysis was performed using t-test and fischer's exact test. results: a total of fifteen patients were identified who underwent either gj revision (n= ) or ttv (n= ). there were no -day mortalities in either group. mean operative time was significantly lower in the ttv group in comparison to gj revision ( . ± vs. . ± minutes respectively, p= . ). recurrence of the ulcer was not significant between groups and occurred following gj revisions and ttv. overall complication rate was not significantly different with % in the gj revision group and % in the ttv group. complications included anastomotic leak ( gj), anastomotic stricture ( gj), aspiration ( ttv), dysphagia ( gj and ttv), and dumping syndrome ( gj). conclusions: our results demonstrate that thoracoscopic vagotomy may be a better alternative with decreased operative times and similar effectiveness. however, further prospective observational studies with a larger patient population would be beneficial to evaluate complication rates and ulcer recurrence rates between groups. we present a case of a -year-old female with a history of thyroid cancer who initially presented to an outside hospital complaining of reflux, abdominal pain, early satiety, and -pound unintentional weight loss. endoscopy demonstrated a cm pre-pyloric mass; with initial biopsies of the mass demonstrating only gastric mucosa. endoscopic ultrasound and fna of the lesion also failed to elucidate its pathology. due to the pyloric location of the mass and inability to rule out invasive malignancy, we recommended a robotic-assisted transgastric submucosal resection with possible distal gastrectomy. intraoperatively we found a -degree circumferential pre-pyloric exophytic sessile tumor. frozen sections suggested a benign papillary tumor therefore we proceeded with submucosal resection. the resulting mucosal defect and gastrotomy were closed primarily with absorbable suture. final pathology showed the tumor to be a tubulovillous adenoma with high grade dysplasia arising against a background of intestinal metaplasia. the resection margins were negative for dysplasia. the postoperative course was complicated by a minor leak which did not require operative intervention and subsequent gastric outlet narrowing which required endoscopic dilation and feeding tube placement. however, the patient has recovered well and has advanced to diet as tolerated. gastric adenoma has a prevalence of . - . % in the western hemisphere. the risk of carcinomatous transformation in gastric adenomas is related to size, degree of dysplasia, and villosity. gastric adenomas are considered precancerous lesions. pre-operative pathologic diagnosis of dysplasia is often elusive as biopsies will often miss or under-grade the lesion. guidelines advocate for complete resection with either endoscopic submucosal dissection or surgical resection depending on surgeon preference and local expertise. endoscopic resection has been shown to be safe and efficacious in the removal of adenomas with good long-term outcomes. in this case the pathology of the lesion was unclear after multiple unsuccessful biopsies and required a surgical diagnosis to rule out invasive malignancy. management of gastric adenomas, while rare, may require a multidisciplinary approach between surgical endoscopy, minimally invasive surgery, and surgical oncology to achieve local control in an oncologically sound manner. we show that transgastric submucosal resection can be achieved in a minimally invasive fashion using robotic assistance. objective: parahiatal hernia is a rare type of diaphragmatic hernia with incidence of . - . %. para-hiatal hernias arises lateral to the left crural musculature adjacent to but separate from the oesophageal diaphragmatic hiatus. in view of its rare occurance and little clinical suspicion, it is almost never diagnosed clinically. the current case report is intended to depict the clinical profile of an intraoperatively diagnosed para-hiatal hernia and feasibility of laparoscopic repair of parahiatal hernias. method: laparoscopic fundoplication is frequently performed at grant medical college and sir j. j. group of hospitals, india. during one such case intraoperatively para-hiatal hernia was diagnosed. discussion: primary or true parahiatal hernias occur as a result of a congenital weakness and secondary defects follow hiatal surgery. the primary treatment of para-hiatal hernia is mesh-plasty. this is coupled with fundoplication in cases of large hernia and those symptomatic for gastroesophageal reflux disease. laparoscopic repair of these uncommon hernias is safe, effective and provides all of the benefits of minimally invasive surgery. conclusion: due to its rare occurrence, knowledge about this condition among laparoscopic surgeons is important to avoid diagnostic dilemma. knowledge about its management aids intraoperatvely to avoid performing incomplete procedure. introduction: extended indications of endoscopic resection for early gastric cancer (egc) have been widely accepted. according to current japanese guidelines, additional gastrectomy with lymph node dissection (lnd) is recommended for patients proven to have potential risks of lymph node metastasis (lnm) on histopathological findings. on the other hand, the frequency of lnm in these patients is exteremely low. the aim of this study was to elucidate the accurate risk of lnm based on the number of risk factors (rf) for possible lnm, and to compare the stratified risk of lnm with predicted risk from additional radical resection. methods and procedures: we enrolled egc patients who did not meet absolute or extended indications of endoscopic resection, and investigated the risk stratification of lnm according to the total number of lnm rfs described below; ( ) sm , ( ) lymphatic vessels invasion, ( ) undifferentiated adenocarinoma and [ mm in diameter, and ( ) [ mm in diameter and ulcer formation. we compared the stratification risk to the surgical risk that was calculated based on the japanese national clinical database (ncd) risk calculator in patients with additional gastrectomy after esd. results: the total number of lnm rfs and frequency of lnm were significantly correlated ( / rf; . %, rfs; . %, rfs, . %, rfs, . %; p. , fischer exact test). the estimated frequency of lnm was found to be lower than the predicted value of in-hospital mortality rate based on ncd in . % of / rf-patients who underwent additional gastrectomy with lnd after esd. the present study suggested that some patients must be over-indicated for additional gastrectomy with lnd, and no additional surgical treatment or less invasive surgery, such as local lnd (sentinel node navigation surgery or lymphatic basin resection), might be indicated for some patients with low number ( / rf) of lnm risk factors after esd. aims: laparoscopic proximal gastrectomy has been applied for early gastric cancer in upper third. we previously reported outcomes of laparoscopic total gastrectomy in managing this condition. in this study, we applied this modified technique for upper third early gastric cancer with double tract reconstruction. it is expected that our technique could be useful for treating these cases. methods: from april of to june of , consecutive patients with upper third early gastric cancer were assigned to undergo surgical treatment with proximal gastrectory at our hospital. we had cases of total gastrectory for upper third early gastric cancer in the same study period. background: laparoscopic total gastrectomy for remnant gastric cancer is much more difficult than common laparoscopic total gastrectomy due to severe adhesions to adjacent organs, displacement of anatomical structure. purpose: the aim was to analyze cases of laparoscopic total gastrectomy for remnant gastric cancer at the department of surgery of juntendo university urayasu hospital between november and april . method: we analyzed outcome and feasibility of laparoscopic total gastrectomy surgery for remnant gastric cancer. and we compared with laparoscopic total remnant gastrectomy ( cases) versus laparoscopic total gastrectomy ( cases) in our hospital. results: in the previous laparoscopic surgeries. we performed laparoscopic distal gastrectomy in cases, laparoscopic proximal gastrectomy in pcases, and open distal gastrectomy in cases. all cases were performed laparoscopic total gastrectomy with r-y reconstruction. case of them had been converted to open surgery due to severe adhesions. the mean operative time was min and the mean blood loss was ml. there were no intraoperative complications, and there were postoperative complications as a pancreatic fistula and a bowel obstruction. however, there were no intra-operative complications more than grade according to the clavien-dindo classification. the mean postoperative hospital stay was . days. all cases were without recurrence. thus, there were no significant differences in operative time, bleeding volumes, intra and postoperative complications and hospital stay compared with laparoscopic total gastrectomy. conclusions: laparoscopic total remnant gastrectomy can be performed with similar short-term outcomes to laparoscopic total gastrectomy, and may be feasible and safe procedure, and can become an option of therapeutic strategy. although this study was not powered to show lower recurrence rates with synthetic absorbable as compared to biologic, the . % recurrence rate is consistent with other series utilizing this mesh. it is interesting to note the difference in time to recurrence. these results suggest that while synthetic absorbable mesh may result in lower recurrence rates, recurrence seems to occur earlier. the results also suggest that deconditioning (lower bmi), and difficult cases and/or recovery may predispose to recurrence. these findings can help inform lf mesh selection and predict which patients are at higher risk of recurrence. introduction: little discussion of gastroparesis (gp) following laparoscopic paraesophageal hernia repair (lphr) has been reported in the literature. we wished to examine the incidence in our institution, and identify potential risk factors for development of gastroparesis following lphr. methods and procedures: a single institution retrospective chart review was preformed using cpt codes corresponding to paraesophageal hernia repair and fundoplication to identify patients undergoing laparoscopic paraesophageal hernia repair over a five year period ( / / - / / ) by three surgeons. emergency procedures and reoperations were excluded. in total, patients undergoing non-emergent first time lphrs were identified. size of the hiatal defect was identified when able, via either measurement between the diaphragmatic crura on ct or by medical record documentation. data obtained included sex, age, hernia type, mesh usage, and existence of specific comorbidities associated with gastroparesis. presence of gastroparesis was identified either by documentation of diagnosis via clinical judgment, or by results of gastric emptying nuclear medicine studies, with timing being no longer than months from date of surgery. independent students t-test and fisher exact test were used to determine statistical differences between the groups. results: patients undergoing non-emergent first time lphrs were identified. of these, we were able to obtain the size of the hiatal defect in patients. patients overall were diagnosed with gastroparesis, with an overall incidence of . %. when comparing all patients who developed gastroparesis to those who did not, only females comprised the group which did develop gastroparesis ( males/ females with gp, males/ females without gp, p= . ). age was also found to be greater in the group which developed gastroparesis. for patients in which the size of the hernia defect was identified, the average age was years older in the group diagnosed with gastroparesis ( step under laparoscopic view, left part of the lesser omentum was cut with preserving the hepatic branch of vagus nerve. the right crus of the diaphragma has been dissected free from the soft tissue around the stomach and abdominal esophagus. in this step the fascia of the right crus should be preserved and the soft tissue should not been damaged to avoid bleeding. after cutting the peritoneum just inside the right crus, the soft tissue was dissected bluntly to left side. then the inside margin of the left crus of the diaphragma was recognized from the right side. in this part of the procedure, laparoscope uses trocar (a), the assistant uses trocar (b) to pull the stomach to left lower side and the operator's right hand uses trocar (c). step the branches of left gastroepiploic vessels and the short gastric vessels were divided with ultrasonic coagulation and dissection device. the left crus of the diaphragma was exposed and the window at the posterior side of the abdominal esophagus was widely opened. in this part of the procedure, laparoscope uses trocar (a) at the beginning of dividing left gastroepiploic vessels, trocar (b) when dividing short gastric vessels. step the right and left crus are sutured with interrupted stitches to reduce the hiatus. from the right side, the fundus of the stomach is grasped through the widely opened window behind the abdominal esophagus. then the fundus of the stomach is pulled to obtain a degree ''stomach-wrap'' around the abdominal esophagus (fundoplication). using - non-absorbable braided suture, stitches are placed between both gastric flaps. purpose: laparoscopic gastrecomy has been widely adopted as the treatment of choice by many countries and institutions. internal hernia is a well-known complication after rouxen-y gastric bypass in the field of bariatric surgery. however, there were only a few reports of internal hernia after gastrectomy in gastric cancer patients. the purpose of this study was to analyze the incidence and clinical features of internal hernia after gastric cancer surgery in a high-volume center. method: , gastric cancer patients who underwent curative gastrectomy at seoul national university bundang hospital between january and december were retrospectively reviewed in this study. internal hernia was classified into two types, mesenteric hernia and petersen's hernia. result: patients who underwent distal gastrectomy (dg) with reconstruction by billroth ii, rouxen-y gastrojejunostomy and uncut rouxen-y gastrojejunostomy, total gastrectomy (tg) with esophagojejunostomy, and proximal gastrectomy with double tract reconstruction (pg dtr) with esophagojejunostomy and gastrojejunostomy had potential space for internal hernia. among these patients, ( . %) were determined as internal hernia by computed tomography and patients ( . %) underwent surgical treatment of internal herniation. two patients were conservatively managed. all patients suffered from abdominal pain and / ( %) patients showed nausea and vomiting. the median interval between the initial gastrectomy and surgery for internal hernia was days. mesenteric hernia was observed in cases and petersen's hernia in cases. since we started closing the mesenteric and petersen's defects from may of , there were only cases ( %) observed afterwards but there were cases ( %) before closure of the defects. conclusion: internal hernia after gastrectomy is likely underreported. although we analyzed patients with internal hernia, there might be more patients with mild symptoms who were managed conservatively by their own. a high degree of suspiciousness for internal hernia should be maintained in patients presenting symptoms like nausea, vomiting and abdominal pain after gastrectomy with potential space for internal hernia. with our experience, closure of the mesenteric and petersen's defect is helpful in reducing internal hernia. however, due to low incidence, a multicenter retrospective study is necessary. introduction: the increased incidence of anemia in patients with a hiatal hernia (hh) has been clearly demonstrated, as has resolution of anemia after hh repair in these patients. despite this, the implications of preoperative anemia on postoperative outcomes have not been well described. in this study, we aimed to identify the incidence of preoperative anemia in patients undergoing hh repair at our institution and sought to determine whether preoperative anemia had an impact on postoperative outcomes. methods and procedures: using our irb-approved institutional hh database, we retrospectively identified patients undergoing hh repair between january and april at our institution. we identified all patients with anemia, defined as serum hemoglobin levels less than mg/dl in men and mg/dl in women, measured within two weeks prior to surgery, and compared this cohort to those that had normal hemoglobin values preoperatively. specific perioperative outcomes analyzed included: estimated blood loss (ebl), operative time, need for blood transfusion, failure to extubate postoperatively, intensive care unit (icu) admission, postoperative complications, length of stay (los), and -day readmission. results: we identified patients undergoing hh repair, of which had preoperative bloodwork available for review. the average age was years and the majority of patients were female ( %, n= ). most were treated electively ( %, n= ) and with a minimally invasive approach ( %, n= ). patients ( . %) had preoperative anemia. compared to patients without anemia, patients with anemia had increased rates of failed extubation postoperatively ( . % vs. . %, p= . ), increased icu admissions ( . % vs. . %, p= . ), increased need for perioperative blood transfusions ( . % vs %, p= . ), and increased rates of postoperative complications ( . % vs. . %, p. ). although mean los ( . days vs. . days, p . ), mean operating time ( mins vs. mins, p= . ), and ebl ( ml vs ml, p= . ) were greater in the anemic group, they did not reach statistical significance, and there was no significant difference in -day readmission rate ( . % vs . %, p= . ). conclusions: anemia diagnosed on preoperative bloodwork appears to be associated with increased failure to extubate postoperatively, need for icu admissions, need for perioperative blood transfusion, and increased overall complication rate after hh repair. however, we found no significant difference in los or -day readmissions between anemic and non-anemic patients. since the majority of patients in this analysis underwent elective repairs, these results would support the preoperative treatment of anemia in patients undergoing hh repair. few studies have compared the procedures' long-term effectiveness with none looking beyond years. this study sought to characterize the efficacy of laparoscopic toupet versus nissen fundoplication for types iii and iv hiatal hernia using a telephone survey. methods and procedures: with irb approval, a review of all laparoscopic hiatal hernia repairs with mesh reinforcement performed over seven years at a single center by one surgeon was conducted. patient demographics and perioperative characteristics were recorded. hiatal hernia was classified per published sages guidelines as type iii or iv using operative reports and preoperative imaging. patients with type i or ii or recurrent hiatal hernia and patients receiving concomitant procedures were excluded. the gerd-health related quality of life survey was administered by telephone no earlier than months postoperatively. patients responded to items concerning symptom severity using a -point scale ( =no symptoms to =symptoms are incapacitating to do daily activities). symptoms surveyed included heartburn ( items), difficulty swallowing ( item) and regurgitation ( items introduction: as the thoracic esophageal carcinoma has a high metastatic rate of upper mediastinal lymph nodes, especially along the recurrent laryngeal nerve (rln), it is crucial to perform complete lymph node dissection along the rln without complications. although intraoperative neural monitoring (ionm) during thyroid and parathyroid surgery has gained widespread acceptance as the useful tool of visual nerve identification, the utilization of ionm during esophageal surgery has not become common. here, we describe our procedures focusing on a lymphadenectomy along the rln utilizing the ionm. methods and procedures: we first dissect ventral and dorsal side of the esophagus preserving the membranous structure (meso-esophagus), which contains tracheoesophageal artery, rln and lymph nodes. we next identify the location of the rln which runs in the meso-esophagus using ionm before visual contact. after that, we perform lymphadenectomy around the rln preserving the nerve. this technique was evaluated in consecutive cases (neural monitoring group; nm) of esophagectomy in prone positioning, and compared with our historical cases (conventional method group; cm background: laparoscopic hiatal hernia repair, particularly large type and type hernias, is associated with high recurrence rates. various use of overlay mesh reinforcement have been described in an attempt to improve outcomes. unfortunately, overlay use of biologic mesh continues to result in high recurrence rates, and more effective repairs employing permanent mesh raise serious erosion concerns and are therefore rarely used. we theorize that employing an interlay technique with permanent mesh (positioned between both crura) will help enhance crural closure and improve rates of hiatal hernia recurrences with minimal risk of erosion. methods: we reviewed all patients who underwent a laparoscopic hiatal hernia repair from april to august by a single surgeon from a prospectively maintained database at a tertiary care referral center (n= ). patients who underwent surgery for achalasia with concurrent hiatal repair were excluded. during this time frame, a new interlay technique of polypropylene mesh was employed upon suture closure of the crura. outcomes of repair were retrospectively reviewed. recurrence of hernia was identified by positive work up of patient's symptoms (new onset dysphagia, gerd, pain). results: a total of consecutive laparoscopic hiatal hernia repair were reported in a period of months. interlay polypropylene mesh was utilized in all repairs. patients were majority females ( . %), had a median age of and had a mean bmi of . . eleven ( . %) patients were redo repairs. majority of patients received a nissen fundoplication (n= , . %) followed by a toupet fundoplication (n= , . %). median length of stay after surgery was day. median follow up was days (range: - days). there were zero reported recurrences. conclusion: laparoscopic hiatal hernia repair with interlay polypropylene mesh appears in the short term to be a safe and durable technique to reduce the incidence of hiatal hernia recurrences. further studies are needed to assess more long term outcomes of this novel technique. zia kanani , melissa helm , max schumm , jon c gould, md ; introduction: laparoscopic fundoplication remains the current gold standard surgical intervention for medically refractory gastroesophageal reflux disease. studies suggest that on average - % of patients undergo reoperative surgery due to recurrent, persistent, or new symptoms. the primary objective of this study was to characterize the long-term symptomatic outcomes of primary and reoperative fundoplications in a clinical series of patients who have undergone one or more fundoplications. methods: patients who underwent laparoscopic primary or reoperative fundoplication between and by a single surgeon were retrospectively identified using a prospectively maintained database. patients undergoing takedown of a failed fundoplication and conversion to roux-en y gastric bypass (for morbid obesity, severe gastroparesis, or or more prior failed attempts) were excluded from the current analysis. all procedures were performed laparoscopically. patients were asked to complete the validated gerd-health related quality of life (gerd-hrql) survey prior to surgery and postoperatively at standard intervals to assess long-term symptomatic outcomes and quality of life. gerd-hrql composite scores range from (highest disease-related quality of life) to (lowest diseaserelated quality of life, most severe symptoms conclusions: patients who need to undergo reoperative fundoplication have more severe gerd-related symptoms at years post-op compared to patients undergoing primary fundoplication. however, good outcomes and morbidity rates of laparoscopic reoperation that approximate that of a primary fundoplication are possible in the hands of an experienced surgeon. adenocarcinoma of duodenum: surgical or endoscopic treatment? introduction: it is well known that the adenocarcinoma of the duodenum (adc) is a quite rare lesion infact represents % of cancer of the small bowel and % of these are localized in the periampullary area: % affect the sub-papillary tract and only % the supra-papillary segment of the duodenum. the adc may arise from duodenal polyps (familial polyposis, or gardner's syndrom or be associated with coeliac disease). until now the treatment was the pancreatoduodenectomy (for anatomo-surgical reasons and for the possibility of regional lymphonode resection). infact in my series of of such procedures, where performed for duodenal cancer. in this last years patients with adc of supra-papillary segment of the duodenum underwent endoscopic submucosal dissection (esd). the purpose of this study were to check the feasibility of the esd in treating such cases. in our experience this kind of endoscopic operation was feasible with high complication rate; perforation in cases ( . %); and bleeding occurred in case ( . %). all the complications were successfully treated endoscopically and the long-term outcomes was favorable. consitering the high rate of complications, the difficult and long procedure, the compliance of patients (c ), the general anesthesia, a very very skilled endoscopist is needed. conclusions: the esd represent a new endoscopic approach enstablished in clinical practice: end is performed following the intraluminal path ( rd space) wich, unlike the others, remain virtual and has to be created by dissecting and expanding the tissues layer between the mucosa and the muscolaris propria allowing the endoscope to gain access. the benefit of esd for treating the adc of the supra-papillary segment of the duodenum, according to our experience, must be validate in the future; a pre-operative pet-tac scan examination must be performed in order to demostred the lesion of the duodenum and if there is any limphatic involvement and no infiltration of the head of the pancreas. yoontaek lee, md, sa-hong min, md, young suk park, md, sang-hoon ahn, md, do joong park, md, phd; seoul national university bundang hospital purpose: this study summarizes the single institution experience of laparoscopic gastrectomy in advanced gastric cancer and evaluates the postoperative morbidities and long-term oncologic outcomes. methods: a total of , laparoscopic gastrectomy for advanced gastric cancer were performed at seoul national university bundang hospital between may and may . the characteristics of patients, surgical techniques, postoperative morbidities, and long-term oncologic outcomes were retrospectively reviewed using electronic medical records. results: patients required conversion to open surgery. the reasons of conversion to open surgery were advanced stage (n= ), intraoperative bleeding (n= ), adhesion due to previous abdominal operation (n= ), small abdominal cavity (n= ), associated disease (n= ), and intraoperative pleural injury (n= ). the mean hospital stay was . days for distal gastrectomy, . days for total gastrectomy, . days for proximal gastrectomy, and . days for pylorus preserving gastrectomy. the mean number of collected lymph nodes was . for distal gastrectomy, . for total gastrectomy, . for proximal gastrectomy, and . for pylorus preserving gastrectomy. the rates of postoperative complications of grade ii or more were . %. there was one case of postoperative mortality due to delayed bleeding after discharge. old age was the only independent predictor of surgical morbidities. background: intrathoracic gastric volvulus is a life-threatening condition of paraesophageal hernia. the therapeutic is a challenge because in acute volvulus it may lead to gastric strangulation and necrosis. most patients are elderly and with a significant associated medical illness which has higher morbidity and mortality of major surgery. we present a laparoscopic surgery is safe in paraesophageal hernia with acute intrathoracic gastric volvulus in a high-risk patient. case presentation: an -year-old woman with underlying of diabetes mellitus and hypertension was transferred from an outlying hospital with anemia, dysphagia, urinary tract infection and aspiration pneumonia. she had severe recurrent emesis after admission. ct scan of the chest and abdomen revealed a large esophageal hiatal hernia, and most of the stomach was in the inferior mediastinum with organoaxial gastric volvulus. endoscopy revealed flat pigmented spot gastric ulcer which compatible with cameron lesion and twisting of gastric folds without evidence of ischemia. the endoscopic reduction was unsuccessful. a laparoscopic surgery was performed and the herniated stomach was successfully reduced. the hernial sac was excised. the crura were approximated and reinforced with composite mesh. nissen fundoplication was performed along with gastropexy of the greater curve of the stomach to the abdominal wall. there was no perioperative complication. she tolerated enteral diet on a postoperative day . she had an uneventful recovery and discharged in weeks after treatment of her associated medical illnesses. she had no relapse of previous symptoms at her six-month follow-up assessment. discussion: endoscopic reduction of acute gastric volvulus may be the first option in a patient with severe comorbidities. however, if there is evidence of ischemia or failure of endoscopic reduction, surgical treatment should be considered. laparoscopic reduction and gastropexy may be a lessinvasive and viable alternative to the more aggressive surgical procedure but definitive surgery with repair hiatal hernia can be done in a selected patient. conclusion: minimally invasive treatments of acute gastric volvulus with paraesophageal hernia, either endoscopic or laparoscopic offer the option for reducing morbidity and mortality in elderly with significant comorbidities. the definitive laparoscopic surgery can be accomplished successfully and safely when it is performed with meticulous attention to the surgical technique and perioperative care. reid fletcher, md, mph, emily ramirez, rn, alfonso torquati, md, philip omotosho, md; rush university medical center introduction: the objective of this study was to evaluate the impact of an enhanced recovery after surgery (eras) program on post-operative length of stay following laparoscopic sleeve gastrectomy. eras programs have been demonstrated to improve outcomes and decrease length of stay in multiple surgical disciplines however relatively little has been published regarding the impact of eras programs in bariatric surgery. methods: an eras program for all patients undergoing bariatric surgery was implemented in february at a single institution. we retrospectively reviewed all patients undergoing laparoscopic sleeve gastrectomy between february and august . as a pre-eras historical control, we also reviewed all patients undergoing laparoscopic sleeve gastrectomy between january and december . baseline patient characteristics, additional concomitant operative procedures as well as -day readmission and complication rates were reviewed. logistic regression analysis was used in univariate and multivariate models to identify factors that predicted early post-operative discharge. data analysis was completed using stata se software (statacorp lp; college station, tx). results: eighty-five patients underwent laparoscopic sleeve gastrectomy after implementation of the eras program while patients were included in the pre-eras control group. there were no statistically significant differences in the baseline characteristics between the two groups and there were no differences in the rate of concomitant procedures performed. there was a statistically significant decrease in post-operative length of stay following implementation of the eras program from . it has been reported that laparoscopic redo surgery is effective for recurrent gerd and/or hiatal hernia after surgery. however, there has been very few reports from japan. we report an initial experience of laparoscopic surgery for japanese patients with recurrent gerd and/or hiatal hernia. among patients who had undergone laparoscopic fundoplication in our hospital from to , patients with recurrent gerd/hiatal hernia underwent redo surgery. preoperative work-up included upper gi series, endoscopy, ct, h ph-impedance and manometry. the patients consisted of women and men with a mean age of . years. the interval from the initial surgery was . months ( days- months). the types of initial fundoplication were nissen: , toupet: , anterior: . the types of recurrence were sliding hernia: and paraesophageal hernia: . one patient with recurrent sliding hernia had poor gastric motility. laparoscopic redo surgery was performed on patients. redo surgery included crural repair with mesh reinforcement: , refundoplication: (nissen-nissen: , nissen-toupet: , toupet-toupet: , toupet-lateral: ) and reduction of the incarcerated paraesophageal hernia: . additional procedure included mesh reinforcement: and pyloroplasty: . open partial gastrectomy was performed for one patient with incarcerated and strangulated hernia. operation time was min. patients was converted to open surgery. oral intake was started on the st pod and postoperative stay was . days. two patients recurred after redo surgery, one of whom underwent re-redo surgery. during the surgery, ivc was injured but rescued by open surgery. eleven patients had good outcome and patients required ppi after redo surgery. our morphological fundoplication score significantly improved after redo surgery. symptom score and acid exposure time were also significantly improved after redo surgery. laparoscopic redo surgery for recurrent gerd and/or hiatal hernia after surgery is safe and effective, although attention should be paid during surgery to avoid injury of the adjacent organs. surg endosc ( ) introduction: cameron ulcers (cu) are linear erosions or ulcerations in the gastric mucosa at the level of the diaphragmatic hiatus in patients with a hiatal hernia (hh) and are frequently associated with anemia. perioperative outcomes of patients with cu undergoing hh repair are not well described. we sought to identify the incidence of cu in patients undergoing hh repair at our institution and determine whether the presence of cu impacted postoperative outcomes. methods and procedures: using our irb-approved institutional hh database, we retrospectively identified patients undergoing repair between january and april . we identified all patients with cu found on preoperative esophagogastroduodenoscopy (egd). we compared patients with and without cu to determine if they differed in terms of preoperative anemia (defined as hemoglobin levels less than mg/dl in men and mg/dl in women). lastly, we compared outcomes between the cu group and the non-cu group, focusing on need for perioperative blood transfusion, failure to extubate postoperatively, intensive care unit (icu) admission, postoperative complications, length of stay (los), and -day readmission. conclusions: the presence of cu on preoperative egd is associated with increased rate of preoperative anemia, increased los, and increased icu admission after hh repair. although the cause of anemia in patients with hh is commonly attributed to cu, only % of cu patients were anemic, indicating that differences in outcomes may not only be attributed to a higher incidence of anemia in cu patients. the implications of cu in patients undergoing hh repair need to be further elucidated. laparoscopic heller myotomy as treatment for achalasia objective: aim of this stud was to review our experience with laparoscopic heller dor myotomy. disphagia constitutes the main symptom. diagnosis is performed by means of esophageal manometry. materials and method: over a period of years, patients were treated with heller myotomy plus dor fundoplication laparoscopically. all patients had lost weight, and there was a prevalence of females with an average age of . twenty five patients had chagas disease. they were all assessed with serial x-rays, endoscopy, esophageal manometry, and their symptoms were assessed with a - score, being the most severe. results: there was no conversion or mortality. in patients the mucosa was perforated during myotomy. the mucosa was sutured without altering the result of the treatment. average hospital stay was hours. one patient had to be reoperate because of esophageal perforation with peritonitis. sixty patients were followed up with manometric control and ph-probe testing, and only % of those had pathologic reflux. conclusions: laparoscopic treatment of achalasia is possible and reproducible, while reducing the morbility of laparotomy with relieve of patients symptoms. introduction: stent treatment in the gastrointestinal tract is emerging as a standard therapy for overcoming strictures and sealing perforations. we have started to treat patients with perforated duodenal ulcers using a partially covered stent and external drainage achieving good clinical results. stent migration is a serious complication that may require surgery. pyloric physiology during stent-treatment has not been studied and mechanisms for migration are unknown. the aims of this study were to investigate the pyloric response to distention mimicking stent-treatment, using the endoflip, investigating changes in motility patterns due to distention at baseline, after a pro-kinetic drug and after food ingestion. methods: a non-survival study in five pigs was carried out, followed by a pilot study in one human volunteer. a gastroscopy was performed in anaesthetized pigs and the endoflip was placed through the scope straddling the pylorus. baseline distensibility readings were performed at stepwise balloon distention to ml, ml, ml and ml, measuring pyloric cross sectional area and pyloric pressure. measurements were repeated after administration of a pro-kinetic drug (neostigmin) and after instillation of a liquid meal. in the human study readings were performed in conscious sedation at baseline and after stimulation with metoclopramide. results: during baseline readings the pylorus was shown to open more with increasing distention, together with higher amplitude motility waves. reaching maximum distention-volume ( ml), pyloric pressure increased significantly (p= . ) and motility waves disappeared. after prokinetic stimulation pyloric pressure decreased and motility waves increased in frequency and amplitude at , and ml distentions. after food stimulation pyloric pressure stayed low and motility waves showed increase in amplitude at distentions of , and ml. during both tests the pylorus showed higher pressure and lack of motility waves at maximum probe distention of ml. similar results were found in the human study. the pylorus seems to acts as a sphincter at low distention but when further dilated starts acting as a peristaltic pump. when fully distended, pyloric motility waves almost disappeared and the pressure remained high, leaving the pylorus open and inactive. stent placement in the pylorus results in pyloric distention, possibly changing motility. this study indicates that a duodenal stent placed over the pylorus should have a high radial force in the pyloric part in order to dilate the pylorus and diminish the contraction waves, this might reduce stent migration. introduction: cutting-edge technology in the field of minimal invasive surgery allows the application of singleincision laparoscopic surgery on gastric cancer. however, single-incision distal gastrectomy (sidg) is still technically difficult due to limited range of motion and unstable field of view-even in the hands of an experienced scopist. solo surgery using a passive scope holder may be the key in allowing sidg to be safer and efficient. we report our initial experience of consecutive cases of solo sidg. methods: prospectively collected database of patients clinically diagnosed as early gastric cancer who underwent solo sidg from october until july were analyzed. all the operations were held by a single surgeon and a scrub nurse. a passive laparoscopic scope holder was controlled by the surgeon to fix the field of view. results: the mean operation time (sd) was . (± . ) min, and the average estimated blood loss was . ± . ml. average body mass index was . ± . kg/m . the median hospital stay (range) was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) days, and the mean number of retrieved lymph nodes was . ± . . there was no conversion to multiport or open surgery. early postoperative complication occurred on % with three delayed gastric emptying, two postoperative pneumonia, one pancreatitis, and one wound complication. conclusion: solo sidg using a passive scope holder allows sidg to become more feasible by providing a stable field of view. there were no peri-operative deaths in either group. in the elective group, age was not an independent risk factor for complications (or . , % ci . - . ). conclusions: the incidence of major complications and mortality in this series were much lower than those previously reported for elective lpehr, while morbidity after emergency repair remains high. the paradigm of watchful waiting for elderly and/or minimally symptomatic patients with giant peh should be revisited. the impact of vagal nerve integrity testing in the surgical kamthorn yolsuriyanwong, md, eric marcotte, md, mukund venu, bipan chand, md; loyola university chicago, stritch school of medicine background: thoracic and gastric operations can cause vagal nerve injury, either accidentally or intended. the most common procedure, which can lead to such an injury, includes fundoplication, lung or heart transplantation and esophageal or gastric surgery. patients may present with minimal symptoms or some degree of gastroparesis. gastroparetic symptoms of include nausea, vomiting, early satiety, bloating and abdominal pain. if these symptoms occur and persist, the clinician should have a high suspicion of a possible vagal injury. investigative studies include endoscopy, esophageal motility, contrast imaging and often nuclear medicine gastric emptying studies (ges). however, ges in the post-surgical patient have limited sensitivity and specificity. if a vagal nerve injury is encountered, subsequent secondary operations must be planned accordingly. methods: from january to august , patients who had a previous surgical history of a foregut operation, with the potential risk of a vagal nerve injury, had vagal nerve integrity (vni) test results reviewed. vni test was measured indirectly by the response of plasma pancreatic polypeptide to sham feeding. the data collected and analyzed included age, gender, previous surgical procedures, clinical presentation, results of vni testing and the secondary procedure planned or performed. vni testing was compared to other testing modalities to determine if outcomes would have changed. results: eight patients ( females) were included. the age ranged from to years. two patients had prior lung transplantation and six patients had prior hiatal hernia repair with fundoplication. seven patients presented with reflux and delayed gastric emptying symptoms. one lung transplantation patient had no symptoms but his lung biopsy pathology showed chronic micro-aspiration with rejection. the vni testing results were compatible with vagal nerve injury in patients. according to these abnormal results, the plans for nissen fundoplication in patients were modified by an additional pyloroplasty and the plans for redo-nissen fundoplication in patients were changed to redo-nissen fundoplication plus pyloroplasty in patient and partial gastrectomy with roux-en-y reconstruction in patients. the operative plans in patients with a normal vni test were not altered. all patients that had secondary surgery had improvement in symptoms and or improvement in objective tests (ie signs of rejection). conclusion: the addition of vni testing in patients with previous potential risks of vagal nerve injury may help the surgeon select the appropriate secondary procedure. . we present a single-center experience with a "myotomy first" approach for all patients, regardless of diverticular size. the hypothesis is that cardiomyotomy alone will provide satisfactory symptom abatement in some patients. and mis cardiomyotomy causes minimal scarring, so a staged mis diverticulectomy is feasible at a later date if diverticular retention/stasis continues. in order to discuss this treatment algorithm we present our experience with cardiomyotomy alone for patients with epiphrenic diverticula. methods: the electronic medical record was queried for patients with esophageal diverticula who were managed with cardiomyotomy and dor fundoplication alone. pre and post-operative reflux/dysphagia questionnaires were gathered; imaging studies, operative data, complications and follow up were reviewed. results: from march of until the present, patients with esophageal diverticula were treated using the "myotomy first" approach. intraoperative esophagoscopy was done to internally visualize the elimination of the inciting spastic esophageal muscle. preoperatively, all patients complained of regurgitation, followed by dysphagia in ( %) and weight loss ( %). postoperatively, dysphagia and weight loss resolved in all subjects. regurgitation symptoms resolved in ( %) patients. the average size of the diverticula was . cm , the range was - cm . post operative esophagream's showed persistent diverticual, however most had decreased in size. there were no perioperative complications, average length of stay was . days and there were no icu admissions or returns to the or. the average length of follow up for these patients was days where all patients reported being satisfied with their results and none of them have yet desired to pursue diverticulectomy. discussion: a "myotomy first" approach resulted in excellent short term symptomatic control. none of the have retained or re-experienced symptoms of diverticular retention worthy of surgical intervention. in the age of laparoscopic surgery, an esophageal epiphrenic diverteculectomy should be staged. this step wise approach seeks to assure surgical necessity for a morbid endeavor. surg endosc ( ) :s -s the background: the two-stage oesophagectomy (ivor-lewis procedure) remains the mainstay of curative surgery for oesophageal cancers in the uk. gastro-oesophageal anastomotic leak is a potentially devastating complication of this procedure affecting perioperative morbidity and mortality. although the leak rates have improved over the years, it still remains widely variable. intraoperative reinforcement of gastro-oesophageal anastomosis with an 'omental wrap' has been proposed as a measure to reduce anastomotic leak rates. there is some data to suggest that this additional technique reduces anastomotic leak. we reviewed our single institution data to assess if the omental wrap indeed had a 'cocoon' effect in maturing the anastomosis and reducing leak rates. methods: data for all cancer oesophagectomies (ilog) performed in our institute since april - was retrospectively analysed from a prospectively maintained database. the patients were categorised into two groups. masafumi ohira; department of gastroenterological surgery, hokkaido university graduate school of medicine background: in laparoscopic surgery, both surgical technique and adequate support and traction by an assistant are highly important. this study assessed the impact of the first assistant on shortterm outcomes of laparoscopic distal gastrectomy (ldg) and laparoscope-assisted distal gastrectomy (ladg). methods: patients who underwent ldg or ladg for gastric cancer at our hospital, between november and august , were included. ldg and ladg cases of billroth i reconstruction, performed by a single surgeon accredited in endoscopic procedures, were analyzed. the cases were categorized into the following groups according to the first assistant's postgraduate years (pgy) of experience: group a, - years; group b, - years; group c, - years; and group d, [ years. short-term outcomes were compared between the groups. results: we examined cases. operative time was significantly longer in group a than in group b (p= . ). no significant differences in operative time were found between groups b, c, and d. the cases were recategorized into groups as follows: group a, the young assistant group (group y, n= ), and groups b, c, and d, the senior assistant group (group s, n= ). significant differences in operative time and method of anastomosis (circular stapler or delta anastomosis) were observed between the groups (p= . and p= . , respectively), but no significant differences in complication rates were found (p= . ). the unadjusted analysis revealed that the group, method of anastomosis, and body mass index (bmi) were significant factors associated with longer operative time. multivariate linear regression analysis with stepwise model selection using akaike's information criterion (aic) revealed that bmi and group were significant factors associated with longer operative time (p= . and p= . , respectively). multivariate analysis using these variables and the method of anastomosis confirmed the significance of bmi and group for longer operative time, but no significance was found in the method of anastomosis (p= . , p= . , and p= . , respectively). conclusions: our study showed that operative time tended to be longer when the first assistant had experience of less than pgy, but the morbidity did not increase. as with the operator, the first assistant needs adequate training to ensure a smooth operation. steven g leeds, md, marc ward, md, brittany buckmaster, pa, estrellita ontiveros, ms; baylor university medical center at dallas background: gastric contents can reach beyond the esophagus into the larynx and pharynx causing an increasingly prevalent disease called laryngopharyngeal reflux (lpr). magnetic sphincter augmentation (msa) has been used as an alternative treatment for gerd with good success, but there is no data to support its use in lpr. methods: forty-five patients with msa implants for symptomatic relief with both gerd and lpr symptoms were examined. all patients experienced at least one typical gerd symptom as well as at least one extra-esophageal symptom. this was assessed using the gerd-hrql which is questions graded - on each question, and reflux symptom index (rsi) which is questions graded - on each question. patients filled out questionnaires preoperatively, one month postoperatively (early follow up), and at months to year postoperatively (late follow up). the responses on the gerd-hrql were clustered into questions inquiring about heartburn ( ), dysphagia ( ), and regurgitation ( ) like all surgical fields there is a push towards standardization of the post operative course while maintaining safe practices. other surgical fields have streamlined recovery processes in an effort to standardize care and minimize costs. laparoscopic hiatal hernia repair is a complex procedure, but with experience and a team approach, this operation can become a streamline process. methods: a retrospective review was done for over laparoscopic hiatal hernia repairs at a single institution. aspects of post operative care such hospital floor, nursing ratio utilized, pain medication, diet advancement, use of foley catheters and length of hospital stay were tracked. statistical analysis was done to compare utilization of resources as years went on along with complications and readmissions. results: a total of hiatal hernias were performed between and . improvements were noted in nearly every field over time, including faster foley removal, decreased length of hospital stay, decreased use of patient controlled analgesics (pcas) and faster advancement of diet. furthermore these patients are now treated on a surgical floor rather than the intensive care unit or step down with a higher nurse to patient ratio, decreasing hospital cost. there were no changes in complications, reoperations or readmissions over the course of the study. conclusions: cost, length of stay and so called "advanced recovery pathways" are all the rage in the surgical literature. anytime a procedure and its post operative course can become less of a "major undertaking" and more routine, the more streamline it becomes. this comes from making a standard protocol that deescalates treatment based on what is actually needed. nearly every aspect of post operative care was simplified; length of stay and cost to the hospital was decreased while no additional complications or readmissions were accrued. the foundation of a formalized advanced recovery pathway will be implemented from these factors which were studied. background: the obesity epidemic continues to worsen. bariatric surgery remains the most effective way to achieve weight loss and resolution of comorbidities. laparoscopic sleeve gastrectomy has become the most common bariatric operation due to excellent efficacy and low morbidity and mortality. the most common complication of sleeve gastrectomy is gastroesophageal reflux disease (gerd), which can adversely impact the quality of life and lead to additional esophageal complications. recently, esophageal magnetic sphincter augmentation (linx®) has become an acceptable alternative to fundoplication for certain patients with gerd. the use of linx® in patients who previously underwent laparoscopic sleeve gastrectomy was described in a case series in . the known complications of these devices include dysphagia, need for endoscopic dilation, and device erosion. the complication profile of linx® in the setting of sleeve gastrectomy has not been reported heretofore. methods: we present a case of a patient with prior sleeve gastrectomy who received a linx® device one year after her bariatric operation due to severe gerd refractory to medical management. initial evaluation demonstrated a hypotensive lower esophageal sphincter and hiatal hernia, but no evidence of stricture or twisting. soon after linx® implantation, the patient developed progressive dysphagia and worsened reflux. repeat evaluation showed esophagitis, a moderate stricture with angulation at the incisura, and a large amount of retained food. discussion: the patient was recommended conversion to roux-en-y gastric bypass, but was deemed to be a poor candidate due to heavy smoking. thus, laparoscopic removal of the linx® device was performed with hiatal hernia repair and gastric stricturoplasty. post-operative fluoroscopic evaluation revealed improvement in the stricture, but persistent gastroesophageal reflux. the patient experienced a significant improvement in her symptoms of dysphagia, nausea, and vomiting. however, once smoking cessation is achieved, she may still need a conversion to roux-en-y gastric bypass in order to address persistent gerd. conclusion: conversion to roux-en-y gastric bypass remains the standard approach to treatment of gerd post sleeve gastrectomy. new approaches to this problem, including placement of linx®, are promising but have not been evaluated for long-term safety and efficacy in the setting of prior bariatric surgery. careful diagnostic evaluation prior to placement of magnetic sphincter augmentation device should be routinely undertaken. postoperatively, close long-term follow up is imperative, particularly in patients with prior sleeve gastrectomy. presence of linx® in a patient with prior bariatric surgery may lead to worsening symptoms if complications of initial operation are present. kazuto tsuboi, md , nobuo omura, md , fumiaki yano, md , masato hoshino, md , se-ryung yamamoto , shunsuke akimoto, md , takahiro masuda , hideyuki kashiwagi, md , norio mitsumori, md , katsuhiko yanaga, md ; fuji city general hospital, shizuoka, japan, nishisaitama-chuo national hospital, saitama, japan, the jikei university school of medicine, tokyo, japan background: esophageal achalasia is one of the primary esophageal motility disorders, and the patients suffer from dysphagia, vomiting and chest pain. timed barium esophagogram (tbe) is a convenient method to assess esophageal clearance, which we usually performed before and after surgery. meanwhile, laparoscopic heller-dor operation (lhd) has been considered worldwide as a gold standard for the surgical management of esophageal achalasia. the aim of this study is to examine the effect of preoperative clearance rate at the lower part of the esophagus on surgical outcomes in patients with esophageal achalasia. patients and method: between august and april , patients who underwent lhd at our institution were extracted from the database. out of patients, patients met our inclusion criteria; such as the patients who underwent lhd as an initial operation with complete evaluation with preoperative esophageal clearance by tbe. these patients were divided into three groups by the degree of esophageal clearance (group a: clearance rate \ %, group b: %? clearance rate \ %, and group c: %? clearance rate). patients' background, pre-and post-operative symptom scores, and surgical results were compared. before and after surgery, the standardized questionnaire was used to assess the degree of frequency and severity of symptoms (dysphagia, vomiting, chest pain and heartburn). moreover, satisfaction with operation was evaluated using the standardized questionnaire. statistical analysis was performed by using krasukal-wallis test or chi-square test, and p-value less than . was defined as statistically different. results: their mean age was . years and of them were male ( . %). one hundred and sixty-eight patients ( . %) were in group a, ( . %) in group b, and ( . %) in group c. the maximum width of the esophagus in group c was smaller than that in other groups (p= . ). as to the pre-operative symptom score, the frequency score of dysphagia was significantly lower in group c (p= . ), whereas the severity score of chest pain was significantly higher in group c (p= . ). surgical outcomes including the incidence of mucosal injury were not different among the groups. moreover, the patient satisfaction with lhd was excellent regardless of preoperative esophageal clearance. conclusion: preoperative clearance rate at the lower part of the esophagus in patients with esophageal achalasia did not affect the surgical outcomes of lhd, but the characteristics of preoperative symptoms in patients with poor esophageal clearance was low dysphagia and high chest pain. surg endosc ( ) ( . cm . cm) was made by dissecting between submucosal and muscular layers at the anterior remnant gastric wall. after creation of the double flap, the posterior esophageal wall ( cm from the edge) and the anterior gastric wall (superior edge of the mucosal window) were sutured for fixation, and . cm from the inferior edge of the mucosal window was opened, and the wall of the esophageal edge and the opening of the remnant gastric mucosa were sutured continuously. the anastomosis was fully covered by the seromuscular flaps with suturing. in latg, roux-en-y reconstruction was performed through a small incision using a circular stapler. introduction: the purpose of this study was to clarify the long-term and short-term outcomes of consecutive patients who underwent thoracoscopic esophagectomy in the prone position using a preceding anterior approach for the resection of esophageal cancer at a single institution. this method was established to make an esophagectomy easier to perform and to achieve better outcomes in terms of safety and curativity. methods and procedures: we retrospectively reviewed a database of patients with thoracic esophageal cancer who had undergone a thoracoscopic esophagectomy (te, patients) or an esophagectomy through thoracotomy (oe, patients) between january and august . to compare the long-term outcomes of te and oe, we used a propensity score matching analysis and a kaplan-meier survival analysis. to analyze the short-term outcomes of te, patients were chronologically divided into three groups: a first period group ( patients), a second period group ( patients), and a third period group ( patients). as for thoracoscopic procedure, the esophagus was mobilized from the anterior structure during the first step and from the posterior structure during the second step. the lymph nodes around the esophagus were also dissected anteriorly and posteriorly. the intraoperative factors, the number of dissected lymph nodes, and the incidence of adverse events were compared among the three period groups using a one-way anova or chi-square test. results: one hundred and twenty-three patients from each group, for a total of patients, were completely selected and paired. background: it is also difficult to anastomose using circular stapler in the narrow neck field. to overcome the problem we modified circular stapling for anastomosis. gastric juice reflux is frequently observed at the esophagogastric anastomosis. we develop and report trapezoidal tunnel method to reduce the incidence reflux. ( ) patients one hundred thirteen cases ( in left lateral and in prone position), with esophageal carcinomas underwent vats-e, respectively. esophago-gastric anastomosis is performed for cases by modified circular stapling and cases by trapezoidal tunnel method. ( ) methods at first the patients are fixed at semi-prone position and esophagectomy is performed in prone position that can be set by rotating and ports are used at the intercostal space (ics). esophagectomy and the l.n. dissection are performed with pneumothorax by maintaining co insufflation. esophago-gastric anastomosis is performed as following, i) trapezoidal tunnel method sero-muscular layer of anterior wall in the near top of gastric conduit is peeled from submucosal layer after parallel horizontal incision of sero-muscular layer, and then trapezoidal tunnel of sero-muscular layer is created. the edge of the proximal esophagus is drawn into the tunnel and esophago-gastric submucosa anastomosis is performed. to wrap anastomosis distal side of parallel line is closed. ii) modified circular stapling at first the circular stapler is introduced into the gastric conduit and joined to an anvil, and close a little. and then a joined anvil is placed into the proximal esophagus and secured by means of a pursestring suture. the gastric conduit opening is closed by a linear stapler. purpose: mesh utilization and its impact on postoperative hernia recurrence following paraesophageal hernia repair remains a polarizing topic. this analysis evaluates the recent trends in laparoscopic paraesophageal hernia repairs and analyzes the impact of operative time on postoperative morbidity. methods: the - acs-nsqip database was queried for primary cpt code for laparoscopic paraesophageal hernia repair with and without mesh ( / ). only elective cases performed by a general surgeon were included. operative time was grouped into quartiles ( - , - , - , - min) and statistical analysis was performed using anova univariate with post-hoc testing and multivariate regression modeling controlling for age, diabetes, renal disease and weight loss. this analysis was powered to detect a greater than % difference in outcomes based on mesh utilization. the outcomes of interest were composite morbidity scores and readmission rates within days of surgery. results: the database identified a cohort of , laparoscopic paraesophageal hernia repairs performed between and . average patient age was years and average patient body mass index was . mesh was utilized in % of cases per year and did not change over the study period (p= . ) however mesh utilization was %, %, %, and % within operative time quartiles - respectively (p. ). postoperative morbidity and readmission rates for each operative time quartile were . %, . %, . %, and . % (p. ) and . %, %, . %, and . % (p= . ), respectively. post-hoc testing indicated statistically significant differences in postoperative morbidity and readmission rates between quartiles and / . multivariate regression analysis documented operative time as a risk factor for postoperative morbidities and readmission, even after controlling for covariates. mesh utilization was only significant for a reduction in the rate of venous thromboembolic complications (or . , p= . ) but did not impact other morbidities or readmission rates. conclusion: this analysis suggests that patients with higher operative times have increased postoperative morbidity and readmission while mesh utilization does not impact postoperative outcomes, after accounting for the longer operative time of a paraesophageal hernia repair with mesh. introduction: gastroparesis is a chronic gastric motility disorder defined by delayed gastric emptying and symptoms such as nausea, vomiting, bloating and abdominal pain. surgical options for refractory gastroparesis include pyloroplasty, gastric stimulator insertion, and gastrectomy. the palliation from a pyloroplasty and gastric stimulator may be synergistic, however concerns remain regarding the possibility of stimulator infection when performing both procedures simultaneously. we present our initial experience of combined laparoscopic pyloroplasty and insertion of gastric stimulator. methods: gastroparesis patients diagnosed by solid gastric scintigraphy or endoscopic evidence of retained food after prolonged npo status who underwent combined laparoscopic heineke-mikulicz pyloroplasty and gastric stimulator insertion between july and july were reviewed. patient demographics, pre-and post-operative symptom scores and outcomes were collected. results were analyzed using statistical tests as appropriate. p value . were considered significant. results: seven patients underwent the simultaneous pyloroplasty and gastric stimulator insertion. six patients ( %) were idiopathic and one patient ( %) was diabetic. one patient was male and six patients were female. charleen yeo, enming yong, danson yeo, kaushal sanghvi, aaryan koura, jaideepraj rao, myint oo aung; tan tock seng hospital introduction: gastric cancer is one of the most common cancers in the asian population, with recent literature supporting the laparoscopic approach in early disease. however, the minimally invasive approach in advanced disease is still controversial. the outcomes of laparoscopic gastrectomy in the elderly have also not been extensively studied. we aim to evaluate our institution's short term outcomes of laparoscopic versus open gastrectomy for gastric cancer-with particular focus on advanced disease and elderly patients. methodology: we prospectively collected the data of all patients who underwent gastrectomies for stomach cancer from to . all patients underwent a partial or total gastrectomy with d lymphadenectomy. the decision for open or laparoscopic approach was decided between surgeon and patient. we excluded patients who underwent palliative resection. all patients were followed up for at least one year post-operatively. introduction: it was an eye-opener when the lancet brought the attention about global surgery. it is estimated that the deaths due to lack of access to surgery is far greater than deaths due to malaria, tuberculosis and hiv/aids put together. there is greater need to stress the importance in developing countries. there is a responsibility at the medical schools to enlighten students about this necessity and arouse interest in concept of global surgery. the students or surgical residents in the future are a great resource to solve this major problem. the first step would be to educate surgical residents. we need to assess the existing awareness about global surgery problem among surgical residents. we can plan a program to train the next generation surgeons. methods and procedure: all the surgical residents in our institution (victoria hospital, bangalore, india) were enrolled for this study. a total of residents were enrolled. a multiple-choice questionnaire regarding global surgery was designed. the received questionnaire was analyzed to assess the depth of knowledge about global surgery. there were multiple choice questions (mcq) and an option was provided at the end for feedback and suggestion to improve the global surgery in our country. each question carried one mark. score more than was considered the cutoff for pass and those students were termed 'informed'. results: ( . %) students cleared the cut off score of and were termed 'informed'. among this group ( %) residents scored marks. ( . %) students did not cross the cut off and were termed 'non-informed'. among these ( . %) students scored marks and did not know anything on the topic. students provided relevant suggestions and opinions to improve global surgery issue. conclusion: there is a great lacuna in knowledge about global surgery among surgical residents. we need to plan a program integrating global surgery in the syllabus of surgical training. the awareness among residents would arouse interest and participation in the future. introduction: minimally invasive surgical techniques (mists) could have tremendous applications and benefits in resource poor environment. these include but are not limited to short hospital stay, reduced cost of care, and reduced morbidity, especially related to post operative infections. there is growing interest in mists in most low and middle income countries (lmic) but its adoption has remained limited largely due to high cost of initial set-up, lack of technological backup and limited access to training among others. one of the most limiting factors is the maintenance of the vision system. an affordable laparoscopic set-up as an example will therefore go a long way in improving access to mists. methods and procedures: a common zero-degrees mm scope is attached on the camera of a low price smartphone (samsung galaxy j , samsung®, seoul, south korea). two elastic bands are used to fix the scope right in front of the main camera on the smartphone. the device is covered with sterile transparent drapes (tegaderm®, m corporate, st. paul, mn, usa). a light source is connected with a fiber optic cable for endoscopic use. the image can be seen in real time on a common tv screen through an hdmi connection to the smartphone, with a sterile drape. holding the vision system through the scope would guarantee to keep the camera in place without issues. to operate in full screen the vision was digitally zoomed at . , without losing quality (that is more related to the intensity of the light). as a collateral project we built a low cost simulator training box with the same camera to train the surgeon, obtaining a high fidelity and affordable simulation setting. results: we were able to perform the tasks of the fundamentals of laparoscopic surgery curriculum using our vision system with proficiency. in a pig model, we performed a tubal ligation to simulate an appendectomy and we were able to perform basic laparoscopic suturing. no major issue were encountered and small adjustment only were required to have an acceptable, stable and clear view. conclusion: there is growing interest in minimally invasive surgeries among surgeons in lmic, but its adoption has remained limited due to reasons such as high cost of initial set-up, lack of technological backup and limited access to training among others. an affordable laparoscopic camera system will therefore go a long way in improving access to mis in such settings. open. there were no deaths or bile duct injuries in our series. two patients undergoing laparoscopic approach were converted to open ( . %). complications, los, and gender were similar between the two groups. the laparoscopic group were significantly younger and had a significantly longer operative duration (table) . long term outcomes were not available for analysis. laparoscopic and open cholecystectomy appear safe in the setting of short term surgical missions. neither group suffered major complications. both had similar immediate outcomes. los for both groups was surprisingly similar and shorter than larger series which may possibly due to patient selection. given similar immediate outcomes and large burden of disease, the open approach should be considered. however, this cost may be extracted in terms of greater pain or longer recovery time for patients, which may outweigh the benefits. further data is needed to study pain, long term outcomes, and return to work. introduction: minimally invasive surgery relies on optimal camera control for the successful execution of operations. one disadvantage of laparoscopic surgery is that camera control is dependent on a surgical assistant's interpretation of visual cues and ability to predict the next field of focus in addition to verbal commands from the operating physician to provide the optimal view. robot-assisted minimally invasive surgery provides the operating surgeon the advantage of dictating their field of view. this study aims to utilize a video processing algorithm to determine the incidence of improperly centered field of view in laparoscopic vs. robot-assisted surgery. methods: in this study, recordings of minimally invasive resection of rectal cancer ( laparoscopic and robot-assisted surgery) were evaluated. recordings were input into matlab® video processing to generate single frames at each second interval. a single reviewer would indicate the pixel which best determined where the camera should be centered based on positioning of instruments, current action (dissection/hemostasis/traction) depicted in the frame, and previous review of recordings. pixel locations were recorded for subsequent analysis. centered views were determined as those with the identified centered position pixel lying within the center quadrant when frames were split into a uniform grid. in addition, distance of each point to the absolute center of the frame was calculated based on the pixel's x and y positions. results: individual operation data was analyzed for percent of centered pixel locations and pixel distance from the center pixel of the frame. robot-assisted surgery demonstrated higher percentage of centered views over laparoscopic surgery ( . ± . vs. . ± . ; p. ). robot-assisted surgery also demonstrated shorter distances to frame center than laparoscopic surgery ( . ± . vs. . ± . ; p. ). conclusion: robot-assisted surgery aims to resolve conflicts of cooperation that occur between surgeon and assistant in laparoscopic surgery by enabling manual visual control of the operative field by the operating surgeon. this study demonstrates that by eliminating such conflicts, optimal surgical view is more frequently obtained. surg endosc ( ) background/objective: valveless laparoscopic insufflator systems are marketed for ability to prevent loss of abdominal collapse and desufflation during laparoscopy. however, community surgeons raised concern for possible entrainment of room air, including oxygen ( ), with these systems. this study seeks to quantify o and non-medical air entrainment by a laparoscopic valveless cannula system to understand the risk of intraoperative air embolism. a communityuniversity collaborative was created to design a model and test this hypothesis. methods: an artificial abdomen was developed and calibrated to equivalent compliance and intraoperative volume of an average adult abdomen. it was connected to a flow meter, oxygen concentration sensor, and commercially available laparoscopic valveless cannula system. background: further advance of near-infrared (nir) imaging capability into greater clinical usefulness will be helped by the development of new targetable agents. to avoid issues related to dose timing and contamination, compounds that become fluorescent only at the site being targeted would be a significant advance. here we build on earlier laboratory work to show step-wise advance of the agent towards clinical trialling. methods: a novel agent (nir-aza) was tested in ex vivo colorectal specimens using two commercially available systems to determine characteristics in biological tissue. it was then trialled in a large animal cohort (n= ) to determine its performance for both intestinal perfusion assessment and lymph node mapping (both stomach and colon) using again a commercially available optical imaging system and including a direct comparison with indocyanine green. results: the novel agent was easily detectable in biological tissue in the near infrared wavelength relevant to commercial instrumentation both as a local depot tattoo and as a lymphatic tracing agent. porcine model trialling again showing excellent detection and tracking characteristics both in the circulation and in gastrointestinal tissue with clear tracking to relevant lymph nodes within minutes evident with the latter. while these studies were non-survival, there was no evidence of local tissue or systemic system toxicity in any case. direct qualitative and quantificative comparison between in situ nir-aza and icg at both intestinal and lymph basin regions showed similar levels of fluorescence. conclusion: the trial compound underwent successful testing indicating proof of earlier projected potential. this is encouraging for further work to advance to first in human testing. introduction: enhanced imaging systems have been developed to alter laparoscopic camera output to facilitate visualization during laparoscopic surgery using several novel imaging modes: clara mode reduces overexposure and reflections while brightening darker areas of the image; chroma mode intensifies color contrast to more clearly delineate blood vessels; and a combined chroma-clara mode. the ies also allows the surgeon to change imaging modes throughout the procedure as needed to facilitate different portions of the operation. we hypothesized that this technology would enhance visualization of critical structures during laparoscopic cholecystectomy (lc) compared to standard laparoscopic imaging. methods: videos and still images from an ies (karl storz endoscopy) were assessed in patients undergoing lc using the four imaging modalities. three time points were assessed: ) after adhesions were taken down but before any other dissection; ) after partial dissection of the hepatocystic triangle; and ) after establishment of the critical view of safety (cvs). seven surgeons blinded to the imaging modalities ranked each modality from (best) to (worst) for each of time points ( dissection points for cases). structures identified on achievement of the cvs were also analyzed. all statistics were performed using spss. rank data was analyzed with the friedman and wilcoxon signed rank tests. results: the median ranks of the chroma and chroma-clara imaging modalities (median [iqr] [ ] [ ] [ ] vs ( - ), p= . ) were not significantly different from each other, but both ranked significantly higher than the clara and standard modalities (median rank [iqr] [ ] [ ] and [ ] [ ] , respectively, p. ). individual surgeon preferences varied; four surgeons preferred chroma-clara, two preferred chroma, one preferred clara, and none preferred the standard mode. in addition, the cystic artery and cystic duct were visible in all cases after achieving the cvs, but the common bile duct was visible in only % of cases. conclusion: enhanced imaging system technology provides modalities that were significantly preferred over standard laparoscopic imaging on retrospective review of still and video images during lc. enhanced imaging modalities should be evaluated further to assess their impact on outcomes of lc and other laparoscopic procedures. introduction: cholangiocarcinoma is often diagnosed at an unresectable stage. endoscopic stent placement is generally performed to release the tumor-induced biliary obstruction. however, stents misplacement and migration, tumor tissue ingrowth and cholangitis are relatively frequent complications. energy-based techniques (radiofrequency ablation and photodynamic therapy) have been proposed as alternatives or in addition to the stent placement, showing controversial results. the use of laser sources in the ablation of the biliary wall has not been investigated so far. this study aims at the evaluation of the optimal power and exposure time to achieve a controlled circumferential intraluminal laser ablation of the common bile duct (cbd). methods: through a laparotomy access, the cbd of pigs was exposed and a small choledocotomy was made. a confocal endomicroscopy (ce) scanning (cellvizio) was performed through the choledocotomy, after injection of ml of sodium fluorescein. the . mm diameter circumferentiallyemitting diode laser probe ( nm wavelength) was introduced in the cbd. laser ablation was performed at w during s (n= ) or s (n= ). the power setting was predetermined on preliminary ex-vivo tests on porcine liver specimen. local temperature control was monitored through a fiber bragg grating, embedded in the laser probe. ce scanning was then repeated. the extent of the ablation was measured on hematoxilin-eosin and nadh stained slides. results: the diameter of the probe was too small to enable a single-shot circumferential ablation. there were no full-thickness perforations. after s from turning laser on, the temperature at the application site reached a plateau with minimal oscillations, and remained at mean values of . ± . °c during both and min. histology revealed that the mucosa ablation, at the contact areas, induced a consistent cellular necrosis (nadh-). ce scanning provided real-time images with a specific aspect of the post-ablation mucosa, including an alteration of the normal glandular structure and a general lack of enhanced imaging. the local application of a circumferential laser source induced a precise and safe mucosa ablation with a long-standing increase in temperature in the cbd, in this experimental trial. however, there is a need of an adapted probe, better fitting the diameter of the cbd to enable a single-shot circumferential treatment. goutaro katsuno, md, phd , yasuhiko nakata, md, phd , nobuyuki kubota, md, phd , teruo kaiga, md, phd , takao mamiya, md , masahiro yan, md , naoaki shimamoto, md , shuichi sakamoto, md, phd ; department of gastrointestinal and minimally invasive surgery, mitsuwadai general hospital, introduction: recently major developments in video imaging have been achieved for performing complete mesocolic excisions (cme) or total mesorectum excisions (tme). indocyanine green (icg) fluorescence imaging is already contributing greatly to making intraoperative decisions for keeping an intact visceral fascial layer, making suitable mesentery division lines and identifying anastomotic perfusions. the aim of this study is to present our experience with laparoscopic procedures for colo-rectal cancers using icg fluorescence imaging (lap icg-fi). patients and methods: we usually use the near-infrared (nir) laparoscopy (stryker corporation, michigan, usa) for lap icg-fi. [indocyanine green fluorescent imaging] visualization of lymph flow: icg ( . mg/ . ml) was injected into the submucosal layer around the tumor at points with a -gauge localized injection before the lymph node dissection. visualization of blood flow: after complete colorectal mobilization, the mesocolon was completely divided at the planned proximal or distal transection line. indocyanine green was injected intravenously and the transection location(s) and/or distal rectal stump, if applicable, were re-assessed in fluorescent imaging mode. results: we experienced lap icg-fi cases with colo-rectal cancer patients. tumor was located at the rectum in of them, at the sigmoid colon in , at the transverse colon in , at the descending colon in , at the ascending colon in , and at the cecum in . tnm stage was -i in patients, ii in , iii in , and iv in . the median (range) age of the patients was ( - ) years with a median (range) bmi of . ( - . ) kg/m . the lymph flow was visualized in patients ( %) intraoperatively. however, a high-quality intraoperative icg lymphangiogram was achieved in patients ( %). in high-quality lymphangiogram, the lymphatic ducts and lymph nodes were clearly visualized in real time, and this proved useful in keeping an intact visceral fascial layer as well as in making a suitable mesentery division line even in the bmi[ patients. a high-quality intraoperative icg angiogram was achieved in all patients. anastomotic perfusion was satisfactory in all cases. in patients ( . %), the use of nir+icg resulted in revision of the proximal colonic transection point before formation of the anastomosis. there were no postoperative anastomotic leakages. no injection-related adverse effects were reported. conclusion: lap icg-fi is a simple, safe and useful tool to help us complete lap cme or tme and check real-time anastomotic tissue perfusion. introduction: recently, the spread of laparoscopic surgery as a standard treatment and the development of information & communication technology have yielded abundant video data of laparoscopic procedures. these data have been accumulated and we can access them anytime, anywhere. however, the direction of how to use the abundant video data are still unclear. conventionally, surgical procedures have been performed based on surgeon's subjective decisions and skills, so called "tacit knowledge". for the purpose of objective analysis of laparoscopic procedures in video data, automatic recognition of surgical tools and understanding of surgical workflow must be the first critical step. we used convolutional neural network (cnn) which is the current trend in machine learning and computer vision tasks. methods: using video database of laparoscopic sigmoid colectomy in our institute, we performed annotation of tools and phases in every frame of the operating videos. for the tool detection, we annotated bounding boxes for both left and right tools in the videos. furthermore, phase annotation was performed by watching the videos in consultation with laparoscopic surgeons. the laparoscopic sigmoid colectomy operation passes through phases; -placement of ports and preparation, -dissection of retrorectal space, -medial approach to ima, -isolation and division of ima, -medial-to-lateral retromesenteric dissection, -lateral mobilization of left colon, -rectosigmoid mobilization, -division of mesorectum, -rectosigmoid resection and anastomosis, -finishing. we used cnn architecture to perform surgical tool detection and workflow recognition. results: we totally labeled tools used in the procedures of laparoscopic sigmoid colectomy and successfully developed tool detection system by cnn. as for surgical workflow, average times of phase - were . , . , . , . , . , . , . , . , . , . min, respectively. workflow recognition system using cnn was also successfully developed, while we needed to extract pure operating scenes in advance for efficient recognition outcomes. we've developed tool detection and phase recognition systems using cnn. we need more datasets to improve the detecting ability for future clinical uses. introduction: surgical environments require special aseptic conditions for direct interaction with the preoperative images and surgical equipment, which hampers the use of traditional input devices. we presented the feasibility of using a natural user interface (nui) for gesture control combined with voice control to directly interact in a more intuitive and sterile manner with the preoperative images and the integrated operating room (or) functionalities during laparoscopic surgery. in this study, efficiency and face validity of using this nui for medical image navigation and remote control during the performance of a set of basic tasks in the or will be assessed. methods and procedures: twenty experienced laparoscopic surgeons participated in this study. they performed basic tasks in the or focused on the interaction with a medical image viewer (osirix; pixmeo) and with the functionalities of the integrated or (or ; karl storz). these tasks were carried out by means of traditional manual interaction, using a computer keyboard and mouse and a touching screen, and using a gesture control sensor (myo armband) in combination with voice commands. this nui is controlled by the tedcube system (tedcas medical systems). time required to complete the tasks using each interaction method was recorded. at the end of the tasks, participants completed a questionnaire for face validation and usability assessment. results: the use of the nui required significantly less time than conventional manual control to show preoperative studies and information for surgical support. however, the interaction with the medical image viewer was significantly faster using the traditional input devices. participants evaluated the nui as an intuitive, simple and versatile tool that improves sterility during surgical activity. seventy-five percent of the participants would choose the gesture control system as a method of interaction with the patient's preoperative information during surgery. conclusions: the presented gesture control system allows surgeons to directly interact with preoperative imaging studies and the functionalities of an integrated or during surgery maintaining the aseptic conditions. for the traditional manual interaction, it is necessary to take into account the possible reaction time and displacement time of the technician to execute the surgeon's requests. a more personalized medical image viewer is required and with higher integration with the capabilities of the presented gesture control system. emma k gibson, bs, jacqueline j blank, md, timothy j ridolfi, introduction: following a generous left hemicolectomy an anastomosis between the transverse colon and rectum may be required. extensive mobilization and retroileal routing is sometimes necessary to create a tension-free anastomosis. retroileal routing is a technique in which a window is created in the ilieocolic mesentery. the colon is routed through this window, beneath the ileum, prior to entering the pelvis. retroileal routing is uncommon and there is no data on this technique when performed in using a hand-assisted laparoscopic technique. the aim of this study was to review our experience with hand-assisted laparoscopic left sided colon resections including retroileal routing of the proximal colon to the rectum. methods and procedures: we performed a retrospective review of a single surgeon's experience with hand-assisted laparoscopic left sided resections over a seven-year period from - . indication for operation, basic demographics, bmi, procedure time, short-and long-term morbidity, and mortality were recorded. results: a total of patients underwent a hand-assisted laparoscopic left sided resection with a colorectal or coloanal anastomosis. of these, underwent hand-assisted laparoscopic procedures with retroileal routing of the proximal colon. in each case, operations included a midline hand port incision and two mm ports in the lower abdomen. the indications for operation were diverticular disease and neoplasm in nine and four patients respectively. procedures took an average of . ( - ) minutes to complete. postoperative morbidity included intubation for co retention in one patient and a rll effusion in another patient. there were no anastomotic leaks and there were no -day or -day mortalities. conclusion: retroileal routing of the colon following left hemicolectomy occurs infrequently. a hand-assisted laparoscopic approach appears to be a safe and efficient in these technically challenging cases. objective: approximation of the diaphragmatic crus pillars is a key step in hiatal hernia repair. the dogma of successful hernia repair requires tension free approximation of tissue. there are no techniques described to measure tension across the crus closure. aim of this study is to describe a novel technique for measuring the tension exerted on crural sutures and report initial findings. methods: data was collected at institutions by the same surgeon. after hiatus dissection was complete the crus defect was measured both anterio-posterior and transverse dimension. the crus closure sutures were placed posterior and then lateral to the esophagus. the initial suture is started posteriorly with a figure of eight fashion (# ). with each subsequent stitch placed anteriorly (# and # ) or laterally (l , l ) till adequate hiatus closure is achieved. we measured tension on each suture placed as follows. conclusions: the autolap system provides improved image stability, staff interactions, and enhanced ergonomic comfort for the surgical team. it also offers cost-savings from decreased staffing requirements for hospitals that routinely use staff camera holders. the system set up of - min was less variable after cases, representing the learning curve. in addition, our approach identified problems with the system that require improvement by the manufacturer. notably, we identified significant ergonomic problems for human camera holders, which has been previously described and can be addressed by this device. background: gastric leaks continue to be a troubling predicament for physicians and patients alike. they are especially concerning after bariatric surgery. electrolyte abnormalities and dehydration continuously pose a life threatening problem in these patients. methods: this is an irb approved retrospective review of our experience with a biologic tissue mesh plug closure of gastric leaks. our interventional radiology colleagues percutaneously accessed the perigastric collection with a wire and a straight catheter was guided through the gastric wall defect and advanced over the wire until it was intraluminal. the surgeon then placed an endoscope down to the level of the gastric defect. the wire was then retrieved by the endoscope achieving percutaneo-oral wire access. the biologic tissue matrix was then measured and cut to a square and inverted into a cone like structure with a flat straight piece on the open end. the cone patch was then secured to the wire with braided polyglactin suture loop. the wire was then withdrawn back through the gastric defect pulling the plug and patch into position and placement was confirmed by endoscopy. results: we attempted closure of a gastric leak arising after bariatric surgery in six patients. five underwent successful deployment while one had premature disconnection of the plug from the wire and could not be deployed. the five who had successful deployment had immediate success and within days resumed enteral intake of liquids and resolution of the leak. two of the six patients additionally underwent covered stent placement to stent a stenotic area at the incisura angularis from the esophagus to the antrum. this stent was typically removed - weeks later. there were no complications related to the procedure or the plug. only one patient has undergone repeat endoscopy to evaluate the status of the plug. in that patient an ulcer at the plug site was visualized one month after the procedure. three months later endoscopy showed the clean ulcer had shrunk to half of the original ulcer size. conclusion: this novel minimally invasive technique utilizing ir and endoscopic placement of a biologic mesh plug into gastric leaks after bariatric surgery has been highly successful in treating chronic and subacute gastric leaks. we recommend that these endoscopic techniques be used to close gastric defects prior to operative intervention. introduction: laparoscopic surgery has spread worldwide and become a standard procedure among many abdominal surgical fields. the incidence of postoperative adhesion, which is a typical postoperative complication, is considered low compared with that after laparotomy, but once complications develop, such as adhesion-induced intestinal obstruction and chronic abdominal pain, the low-invasiveness of laparoscopic surgery may decrease markedly. while we have previously used a sheet-type absorbable barrier to prevent adhesion, it requires a technique in many cases when it is applied in the abdominal cavity. in this study, we used a spray-type absorbable barrier, which is considered simple to apply, as an adhesion-preventing absorbable barrier following laparoscopic surgery. subjects and methods: a spray-type absorbable barrier for prevention of adhesion (ad spray type l®) was applied to the dissected surface, port region, and beneath the small incised wound in patients who underwent laparoscopic surgery of the large intestine after february . the nozzle is long ( mm in length) and the angle of the tip is adjustable to some extent, so that the spray could be applied easily to the target region, even in areas in which it would be difficult to secure a work space, by rotating the shaft and finely adjusting the angle of the tip. in order for the barrier to remain in the target region, this preparation must remain viscous after application. discussion: approaches for the insertion and affixing of a conventional sheet-type absorbable barrier for the prevention of adhesion has been reported previously by various researchers. the adhesion-preventing absorbable barrier used in this study was a spray type with a long nozzle, which may have been useful because it made the laparoscopic application easy. however, its application requires some experience and time for preparation compared with the use of the sheet type, which could be disadvantageous. further accumulation of cases, including evaluation of prevention of adhesion after use of the adhesion-preventing absorbable barrier may be necessary. christopher g yheulon, md, priya rajdev, md, s. scott davis, md; introduction: evidence has demonstrated that biosynthetic glue for laparoscopic inguinal hernia repair results in decreased pain. however, the two glue sub-types (biologic-fibrin based; synthetic -cyanoacrylate based) have never been compared. this study aims to assess the outcomes of those subtypes. method and procedures: a systematic review of the medline database was undertaken. randomized trials assessing the outcomes of laparoscopic inguinal hernia repair with penetrating and glue fixation methods were considered for inclusion and data analysis. thirteen trials involving patients were identified with eight trials utilizing fibrin and five trials utilizing cyanoacrylate. results: there were no differences in recurrence or wound infection between the glue subtypes when compared individually to penetrating fixation alone or indirectly to each other. there was a significant reduction in urinary retention with fibrin glue when compared to penetrating fixation (or . , % c.i. . - . ). no studies utilizing cyanoacrylate analyzed urinary retention as an outcome. there were non-significant trends in reduction of hematoma and seroma for both glue subtypes when compared to penetrating fixation (or . , % confidence interval . - . ). conclusions: glue fixation in laparoscopic inguinal hernia repair reduces the incidence of urinary retention and may reduce the rate of hematoma or seroma formation. as there are no differences in outcomes when comparing fibrin or cyanoacrylate glue, surgeons should choose the glue that is available at the lowest cost at their respective institution. however, improvement of the optical system is necessary to further utilize this advantage. we are developing an optical lens system covering the range from macroscopic to microscopic. methods: we developed a handheld prototype created by combining the objective lens system of an optical microscope and a telescope lens. a feasibility study using a porcine model was conducted. macroscopic observation was done at a distance followed by microscopic observation in contact with tissue. first, we observed the operative field macroscopically. we then observed the serosa of the small intestine microscopically, and effects of blood flow occlusion were studied. results: ( fig. and fig. ) the same visual field as ordinary laparoscopy was achieved during macroscopic observation, while using microscopic observation it was possible to observe the complex peristaltic movements of the intestine. the minute blood vessels of the visceral peritoneum and larger, deeper blood vessels were also observed. when the mesenteric vessels were occluded, changes in peristaltic movement were seen directly. congestion in blood vessels in the deep layers of the serosa was observed. improvement in peristalsis and congestion were confirmed by restoring blood flow. this system enables direct visual observations not possible with conventional optics. this system can be utilized in both laparoscopic and open surgery. the microscopic visual information obtained by this system may help with intra-operative decision making and serve to facilitate safe and precise surgery. introduction: accurate, real-time visualization is critical for efficient, effective and safe surgery. although optical imaging using near-infrared (nir) fluorescence has been used for visualization of anatomic structures and physiologic functions in open and minimally invasive surgeries, its efficacy and adoption remain suboptimal due to the lack of specificity and sensitivity. herein, we report a novel class of compounds, which are exclusively metabolized in liver or kidney, rapidly excreted into to biliary or urinary systems, and emitted two different nir fluorescence spectrums. methods: novel, water-soluble heptamethine cyanines; compound x (biliary) and compound y (urinary), unreactive towards gluthathione and the cellular proteome were synthesized, and visualized using real-time, dual-color nir imaging device. sprague-dawley rats (n= ) and yorkshire pigs (n= ) were used to demonstrate and validate its usefulness, distributed into a control group (icg; rat n= , irdye cw rat n= ), a biliary group (compound x; rat n= , pig n= ), a urinary group (compound y; rat n= , pig n= ), and dual-labeling group (compound x&y; rat n= , pig n= ). each rat and pig received one or two of the compounds at optimized dose of . -mg/kg intravenously, fluorescence signals and bio-distributions were monitored and recorded over time. the target to background ratio (tbr) was calculated in each target systems and compared to assess sensitivity and specificity. results: compound x was rapidly cleared from liver within min after intravenous injection while the fluorescence signals in biliary system lasted up to h both in rats and pigs. compound y showed significant renal excretion up to h and the urinary signals remained up to h. they were both highly specific to target organs with tbr values of . (biliary), . (urinary) and . (cf. icg) at peak signals. these new compounds have approximately - times higher quantum yields than icg and . - . times higher specificity to kidney and liver than irdye cw. one-way anova showed significant differences between control, biliary, and urinary group (p. .) dual-labeling results also showed a complete separation of these two metabolic systems (p= . ) and a real-time display of these two systems were clearly visualized with pseudo-colored labeling inside the animal body. conclusion: we report a new generation of organ-specific, real-time fluorescent markers for intraoperative visualization, navigation and potential geo-fencing. these new compounds have significantly higher quantum yields and higher specificity to visualize kidney and/or liver than any currently available reagents. background: porcine models have been widely accepted for gastrointestinal surgery studies, due to their similarities to human anatomy, histology and physiology. devices such as laparoscopic staplers have been widely used in bariatrics and are currently the cornerstone of bariatric. there are currently few published articles regarding surgical stapler testing in porcine models by means of a survival design. the purpose of this study is to present a new model for stapler testing in porcines. we present the following study in which we asses a novel stapler's feasibility and safety, and its compatibility to currently used stapler reloads. this novel stapler, the aeon™ endoscopic linear stapler (lexington medical inc., billerica, ma. pending fda approval), has been previously tested in-vitro and in-vivo by the lexington medical engineering department in matters of mechanical function, staple line bursting pressure, staple formation and hemostasis. duffy et al. used this instrument for small bowel anastomoses in a two-week survival study in porcine models. methods and procedures: four porcine animal model was used under iacuc protocol for a -day survival study held at the fiu (doral, fl, u.s.a) research facility. all animals underwent sleeve gastrectomy using the novel stapler handle, combined with the endo gia™ (medtronic, mansfield, ma) mm-staple reloads in two of the animals and aeon™ mm-staple reloads in the remaining two. no reinforcements or oversewing of the staple line was done. these procedures were performed by two bariatric surgeons. animals were monitored perioperatively by the facility staff as per protocol. the animals were euthanized at day . post-mortem assessments were done blindly. gross evaluation and comparison of the gastric tube and their staple lines was done, as well as patency, strictures, and staple line integrity. results: stapler function was equivalent with both reload brands, no technical issues were encountered. - firings were used per animal. no intraoperative complications related to stapler function ensued. no postoperative complications were encountered. all animals survived the full length of the study- days. all sleeves were patent, no strictures or bowel obstruction were present. conclusions: in an animal survival study, a follow-up period of weeks appears to be a good benchmark for stapler testing. the use of the novel stapler for gastric resections appears feasible and safe. further studies such as microscopic examination of the staple lines, might help confirm equivalence, safety and feasibility of these products for the sleeve gastrectomy procedure. jason m samuels, md , peter einersen, md , krzysztof j wikiel, md , heather carmichael , douglas m overby , john t moore , carlton c barnett , thomas n robinson, md , teresa s jones , edward l jones, md ; university of colorado denver, denver va medical center introduction: the purpose of our study was to evaluate the impact of smoke evacuation devices on operating room fires caused by surgical skin preps. surgical fires are rare but preventable events that cause devastating injuries. alcohol-based surgical skin prep serves as the fuel for a fire ignited by electrosurgical instruments. we hypothesized that increasing air exchanges near the tip of the active electrode will reduce the concentration of alcohol thus reducing the incidence of surgical fires. methods: a standardized, ex vivo model was created with a cm section of clipped, porcine skin. surgical skin preparations tested: % isopropyl alcohol with % chlorhexidine gluconate (chg-ipa) and % isopropyl alcohol with . % iodine povacrylex (iodine-ipa). based upon previous studies, a high-risk situation was replicated with immediate energy activation in the presence of pooled alcohol-based prep. the site was draped to simulate a small surgical procedure with approximately square cm exposed. (figure ) a standard and smoke evacuating electrosurgical pencil was activated for s on w coagulation mode in the presence of % oxygen. a standard wall suction was also tested with the tip held cm from the tip of the electrosurgical pencil. a chi-square test was used to compare differences between groups. results: surgical fires were created in % ( / ) of the tests with the chg-ipa and % ( / ; p= . ) of the tests with iodine-ipa. continuous wall suction did not change the incidence of fire. the smoke evacuation electrosurgical pencil significantly decreased the incidence of fire when compared to the standard pencil and continuous wall suction for both preparations (table ) . with chg-ipa, the smoke evacuation electrosurgical pencil decreased the frequency of fire by % (figure , p. ). similarly, when using iodine-ipa, the electrosurgical pencil with integrated smoke evacuation demonstrated a % decrease in fires (figure , p. ). conclusion: alcohol-based skin preps fuel surgical fires. the use of a smoke evacuator electrosurgical pencil reduces the occurrence of surgical fires. elimination of alcohol-based preps and the use of smoke evacuation devices decrease the risk of operating room fires. brian bassiri-tehrani, md, netanel alper, md, jeffrey s aronoff, md, yaniv larish, md; lenox hill hospital ureteral stents have historically been used in pelvic surgery when anatomical or clinical considerations warrant urological expertise to aid in identifying the ureters. in the colorectal and gynecologic surgery literature, prophylactic ureteral stents appear to increase the ability to detect ureteral injuries while not being shown to prevent such injuries. with the increasingly widespread use of laparoscopy and the robotic platform in complex colorectal and pelvic surgery, the utility of stents remains unclear. one of the limiting factors regarding the use of ureteral stents in minimally invasive surgery is the lack of tactile feedback; the inability of the surgeon to directly palpate the stents. one proposed method to overcome this deficiency has been the use of lighted ureteral stents. increased operating time, increased cost, and need for specialized equipment are potential drawbacks of lighted stents. an alternative to using lighted stents in minimally-invasive surgery is to directly inject indocyanine green (icg) into the ureters after cystoscopy-guided placement of ureteral stents. intraoperative visualization of the ureters is acheived by using either the pinpoint endoscopic fluorescence imaging system in laparoscopy, or firefly integrated with the robotic platform. it is hoped that the risk of inadvertent ureteral injuries during colorectal and pelvic operations will be minimized using this technique, due to improved visualization of the ureters throughout the procedure. in this case presentation, we describe a novel use of icg in a patient undergoing a laparoscopic surgery for resection of a . . . cm pelvic mass abutting the bladder, sigmoid colon and left ureter. preoperatively, there was concern that the mass would be intimately adherent to, or even invading, the bilateral ureters based on ct scan findings. after ureteral injection of icg, visualization of both ureters was easily achieved at the time of operation, and the procedure proceeded with careful and safe dissection of the mass with visualization of the ureters at all times. though there is a paucity of studies evaluating the use of icg in the laparoscopic modality, this technique was safe, easy to employ, inexpensive and very useful to visualize the ureters intraoperatively. indeed, larger studies with appropriate sample sizes would help to further validate this novel use of icg. university of colorado -denver, va eastern colorado healthcare system introduction: operating room fires are "never events" that expose the patient to the risk of devastating complications. our group has previously demonstrated that alcohol-based surgical skin preparations fuel operating room fires. manufacturer guidelines recommend a three-minute delay after application of alcohol-based preps to decrease the risk of prep pooling and surgical fires. the purpose of this study was to evaluate the efficacy of the three-minute dry time in reducing the incidence of surgical fires. methods and procedures: a standardized, ex vivo model was used with a cm section of clipped, porcine skin. alcohol-based surgical skin preparations tested were % isopropyl alcohol (ipa) with % chlorhexidine gluconate (chg) and % ipa with . % iodine povacrylex (iodine-ipa). nonalcohol-based solutions included % chlorhexidine gluconate and % povidone-iodine "paint." an electrosurgical ''bovie'' pencil was activated for seconds on watts coagulation mode in % oxygen, both immediately and minutes after skin preparation application, with and without solution pooling. results: no fires occurred with immediate testing of nonalcohol-based preparations ( / ). alcohol-based preps created flames on immediate testing in % ( / ) of cases when pooling was present. without pooling, flames occurred in % ( / ) of cases on immediate testing. after a -minute delay, there was no difference in the incidence of fire when pooling was present ( / vs. / , p [ ) . similarly, there was no difference when pooling was not present ( / vs. / , p= ). (table ) conclusions: alcohol-based surgical skin preparations fuel surgical fires. waiting minutes for drying of the surgical skin prep did not change the incidence of surgical fire (regardless of whether there was pooling of the prep solution). the use of nonalcohol-based skin preps eliminated the risk of fire. introduction: laparoscopic port sites are associated with a significant incidence of long-term hernia formation. in addition, closure with closed loop suture may lead to increased post operative pain thereby limiting patient mobility. the development of novel trocar closure systems could offer a pathway towards quality improvement and warrants investigation. we performed a randomized controlled trial comparing a novel anchor based system (neoclose®) versus standard suture closure. methods: a prospective randomized controlled trial of patients undergoing port site closure following robotic assisted laparoscopic sleeve gastrectomy or gastric bypass was completed ( with neoclose® device and with standard laparoscopic suture closure). each patient had both the camera port and stapling port closed ( port sites in each group). primary outcome measures included the incidence of hernia ( week ultrasound), time for port site closure, and depth of needle penetration. secondary outcome measures were analog pain scoring at post op day , week and week . results: physical exam as well as ultrasound evaluation showed no hernias in either group at weeks. when compared to suture closure, the neoclose® device was associated with shorter closure times ( . ± . versus . ± . s, p. ) and needle depth penetration ( . ± . versus . ± . cm, p\ . ). the neoclose® device was associated with decreased pain at week after the operation (analog pain score . ± . versus . ± . , p. ). no difference in pain scoring was observed on post operative day or at week . conclusions: trocar site closure with the neoclose® device is associated with decreased closure times and needle depth penetration. no difference in the incidence of hernias was identified very early after operation. the neoclose® device led to decreased pain week after trocar closure which is potentially secondary to decreased tension when compared to closure with closed loop suture. long term hernia data ( year) is pending with patients scheduled for follow up physical exams and ultrasounds. federico gheza, md, mario a masrur, md, simone crivellaro, md; uic introduction: robotic instruments provides a better ergonomics during suturing compared to standard laparoscopy. minimally invasive procedures with limited need of few suture may benefit from an economically affordable device able to overcome some limitations of laparoscopic suturing. flexdex surgical recently obtained the fda approval for human use of its articulated laparoscopic needle driver. the official training provided by the company (available at https://flexdex.com/register-for-training) is a h basic dry lab. the training curriculum as well as the accreditation process is not well structured. no literature is available today on this matter. our goal was building a dedicated training, to allow a safe and predictable early use in humans. methods and procedures: the training module design and implementation was done in our minimally invasive laboratory. in the preliminary phase we define with a small group of residents and research specialists a short list of mandatory concepts to detail showing the instrument. a simple suturing task was then performed by the same group with the new device, laparoscopically and with the robot, available in our lab for training only. a more complex task, based on a dedicated self-designed high-fidelity model of urethral anastomosis was then proposed, exploring different options (one flexdex only vs two flexdex, surgeon vs assistant holding the camera). lastly, we applied the new device in animals to evaluate the usefulness of including simple tasks or entire procedures in the training curriculum. results: we were able to define a multilevel, adaptable training module including a basic information session, a dry lab with inanimate low-and highfidelity models and a pig lab. subjects with different level of expertise (medical student, resident, fellow, expert and very expert surgeon) were involved to have an extensive feedback. however, our main focus was to design a training module for laparoscopic and robotic surgeons, to safely introduce the flexdex in their practice. the only outcome for this preliminary work was collected through a "post exposure" survey. the expert surgeon that did the entire training was able to give feedback after his first application of the device in humans as well. conclusions: flexdex is a promising device, available in the united states in approved facilities only. a minimally invasive lab with high laparoscopic and robotic training experience is the ideal setting to build a curriculum. a first adaptable, multilevel, original, high-fidelity training is proposed to be validated with further studies and could be implementable for accreditation purposes. surg endosc ( ) :s -s augmenting spatial awareness in laparoscopic surgery by immersive holographic mixed reality navigation using hololens objectives: endoscopic minimally invasive surgery provides a limited field of view, thus requiring a high degree of spatial awareness and orientation. because of a d field of endoscopic view, a surgeon's spatial awareness is diminished. this study aims to evaluate the efficacy of our novel surgical navigation system of immersive holographic mixed reality (mr) using a head-mounted smart glass display hololens to enhance spatial awareness of the operating field in laparoscopic surgery. the authors describe a method of registering and overlaying the preoperative mdct imaging localization of tumors, vessels, and organs onto the real world in the operating theatre through holographic smartglasses in augmented reality (ar). methods: in this study we included laparoscopic gi, hpb, urology, and gynecologic surgeries using this system. we developed a ct-based patient-specific holographic mr surgical navigating application using hololens, that is a pair of see-through monitors built-in head-mounted display. by reconstructing the patient-specific d surface polygons of tumors, vessels, and organs out of the patient's mdct, mr anatomy was displayed on the see-through grasses three-dimensionally during actual surgery. the hololens features an inertial measurement unit which includes an accelerometer, gyroscope, and a magnetometer for environment understanding sensors, an energy-efficient depth camera, a photographic video camera, and an ambient light sensor. results: the accurate surgical anatomy of size, position, and depth of the tumors, surrounding organs, and vessels during surgeries could be measured using build-in dual infrared light sensors. the exact location between surgical devices and patient's anatomy could be traced on the pair of mr smart-glasses by satellite tracking. the gesture controlled manipulation by surgeons' hands with surgical groves was useful for intraoperative anatomical references of tumors and vascular position under sterilized environment. it allowed the user to manipulate the spatial attributes of the virtual and real anatomies. this system reduced the length of the operation and discussion time. this could support complex procedures with the help of pre-and intra-operative imaging with better visualization of the surgical anatomy and spatial awareness with visualization of surgical instruments in relation to anatomical landmarks. conclusions: the immersive holographic mr system provides a real-time d interactive perspective of the inside of the patient, accurately guiding the surgeon. this helps spatial awareness of the surgeons in the operating field and has illustrative benefits in surgical planning, simulation, education, and navigation. enhancing scene visualization is a feasible strategy for augmenting spatial awareness in laparoscopic surgery. francisco miguel sánchez margallo, phd , juan a. sánchez-margallo, phd , andreas skiadopoulos, phd , konstantinos gianikellis, phd ; minimally invasive surgery centre, cáceres, spain, university of nebraska at omaha, university of extremadura, spain introduction: new handheld devices have been developed in order to address the technical limitations and ergonomic issues present in laparoscopic surgery. the aim of this study is to analyze the surgeon's performance and ergonomics using the radius r drive instruments (tubingen scientific medical, germany) during the execution of laparoscopic cutting and suturing tasks. methods and procedures: three experienced laparoscopic surgeons performed both an intracorporeal suturing task and a cutting task on a box trainer. both tasks were repeated three times. a maryland dissector and a pair of scissors were used for the cutting task. for the suturing task, a maryland dissector and needle holder were used. conventional laparoscopic instruments and their equivalent r drive instruments were used. the order in the use of the type of instruments was randomized. execution time and surgeon's ergonomics were assessed. for the latter, surface electromyography (trapezius, deltoid and paravertebral muscles) and the nasa-tlx index were analyzed. for the cutting task, the percentage of the area of deviation from the cutting pattern (% of error) was assessed. the suturing performance was assessed by means of a task-specific validated checklist. results: surgeons required more time to perform both laparoscopic tasks using the r drive instruments. the use of both instruments had a similar percentage of deviation from the exterior part of the cutting pattern. however, the deviation from the inner part was significantly higher using the r drive instruments (conv: . ± . % vs r drive: . ± . %; p\. ). needle driving was scored lower using the r drive instruments, but quality of knot tying was similar to conventional instruments. the use of r drive increased the muscle activity of the trapezius muscles bilaterally for both laparoscopic tasks. this muscle activity also increased for the left deltoid muscle during the cutting task. surgeons stated that the use of r drive instruments leads to a higher mental and physical workload when compared to traditional laparoscopic instruments. conclusions: despite the novel and ergonomic design of the r drive laparoscopic instruments, the results of this study suggest that an improvement in surgical performance and physical workload is required prior their use in an actual surgical setting. further studies should be done to analyze the use of these instruments during other laparoscopic tasks and procedures. we believe that surgeons need a longer and comprehensive training period with these laparoscopic instruments to reach their full potential in laparoscopic practice. background/objectives: d printing has been shown to be a useful tool for preoperative planning in various surgical disciplines. however, there are only several single case reports in the field of liver surgery. this is because of problematic visualization of anatomy, difficulties in methodology and-most importantly-high costs limiting implementation of d printing. the goal of this study is to evaluate the utility of personalized d-printed liver models as routinely used tools in planning and guidance of laparoscopic liver resections. materials and methods: contrast-enhanced computed tomography images of consecutive patients who underwent laparoscopic liver resections in a single centre were acquired and processed. proper segmentation algorithms were used to obtain virtual models of anatomical structures, including vessels, tumor, gallbladder and liver parenchyma in stl (stereolithography) format. after processing files, models in parts were subsequently printed with desktop ultimaker + (ultimaker, netherlands) d printer, using polylactic acid filaments as printing material. all parts were matched together to create a mold, which was later casted with transparent silicone. models were delivered to surgical teams prior to the surgery as well as used in patients' education. results: up to now, six full-sized, transparent, personalized liver models were created before laparoscopic liver resections and used as a tool for preoperative planning and intraoperative guidance. usefulness of these models has been evaluated qualitatively with surgeons. operative data was obtained for each patient and it will be used for quantitative analysis in further study phases. costs of one model varied between $ and $ and whole process of development took approximately days in every case. conclusions: d-printed models allow precise planning in complex cases of minimally invasive liver surgery by providing high-quality visualization of patient-specific anatomy. implementation of this technology might potentially lead to clinical benefits, such as reduction of operative time or improvement of short-term outcomes. having said that, more data is needed to decisively prove these hypotheses. introduction: modern laparoscopic graspers may risk inadvertent injury to tissues, and have been shown to produce crush and puncture injuries. in addition, the force transmitted to the tissues by grasper handles can be highly variable, dependent on the orientation and amount of tissue engaged by the grasper. we have developed a novel vacuum-based laparoscopic grasper designed to reduce tissue injury from grasping. the aim of this study is to compare the incidence and severity of tissue trauma caused by vacuum-based graspers versus standard compressive graspers while manipulating tissue. we performed an in vivo surgical porcine study to assess gross and histologic tissue injury after grasping trials. grasping trials were divided equally between two adult porcine models; samples of small bowel were grasped with a standard atraumatic laparoscopic grasper (aesculap double-action atraumatic wave grasper) and were grasped with our novel vacuum grasper with varying vacuum head designs ( for head a, each for heads b and c). following grasping, the porcine model was allowed to dwell for hours prior to harvest. gross injury was graded as follows: ) no injury, ) ecchymosis only, ) serosal injury, ) seromuscular injury, and ) perforation. histologic injury was graded as follows: ) serositis, ) partial-thickness injury to the muscularis propria (mp), ) full-thickness mp injury, and ) full-thickness mp and mucosal injury. mann-whitney u test was performed to compare both gross and histologic injury scores between the groups. results: on gross assessment, no samples were noted to have injury more severe than ecchymoses following grasping. the vacuum grasper was found to cause more ecchymosis (median= ) than the compressive laparoscopic grasper (med.= , u= , p. ). on histologic assessment, the compressive grasper caused significantly more severe injury (med.= ) compared to the vacuum grasper (med.= , u= , p= . ). subgroup analysis showed that heads a (med.= , u= . , p= . ) and b (med.= , u= , p= . ) caused significantly less injury compared to the compressive grasper. head c (med.= , u= . , p= . ) also showed less injury but did not reach statistical significance. conclusion: this study demonstrates that our novel laparoscopic vacuum grasper produces less tissue trauma than standard compressive graspers. vacuum-based grasping is a viable alterative for reducing inadvertent tissue injury in laparoscopy. minimally invasive surgery centre, cáceres, spain, university of nebraska at omaha, university of extremadura, spain introduction: the aim of this study is to analyze the surgeon's performance, workload and ergonomics using an ergonomically designed handheld robotic needle holder during laparoscopic urethrovesical anastomosis in an animal model, and comparing it with the use of a conventional laparoscopic needle holder. methods and procedures: six experienced surgeons performed an urethrovesical anastomosis in a porcine model using a handheld robotic needle holder and a conventional laparoscopic axialhandled needle holder (karl storz gmbh). the robotic instrument (dex®, dextérité surgical) has an ergonomic handle and a flexible tip with unlimited rotation, providing seven degrees of freedom. the use of the surgical instrument was randomized. for each procedure, an expert surgeon evaluated the surgical performance in a blinded fashion using the global operative assessment of laparoscopic skills rating scale. besides, the quality of the intracorporeal suture was assess by a validated suturing-specific checklist. the surgeon's posture was recorded and analyzed using the xsens mvn biomech system based on inertial measurement units. the surgeon's workload was evaluated by means of the nasa task load index, a subjective, multidimensional assessment tool. the patency of each anastomosis was assessed using methylene blue. results: all urethrovesical anastomoses were completed without complications. only one anastomosis with the robotic device failed the patency test. surgeons showed similar surgical skills with both instruments, although they presented greater autonomy with the conventional instruments (p =. ). for the suturing performance, the use of the robotic device led to an increase in the number of movements during the needle driving and lower tendency to follow its curvature during the withdrawal maneuver (p=. ). the level of workload increased with the robotic device. however, the surgeon's satisfaction with the surgical outcome did not differ using both instruments. the use of the robotic instrument led to similar posture of the shoulder and wrist and better posture of the right elbow (p=. ) when compared to the conventional instrument. conclusions: the use of the robotized needle holder obtained similar results for the surgical performance and surgical outcome of the urethrovesical anastomosis when compared to the conventional instrument. we consider that aspects such as the surgeon's autonomy, dexterity in driving the needle and workload could be improved with a comprehensive training with the new device. inertial sensors can be an alternative for actual and crowded surgical environments. surgeons acquired a better body posture using the novel robotic needle holder. surg endosc ( ) :s -s introduction: temporal and spatial tissue temperature profile in electrosurgical devises, such as ultrasonic scissors and bipolar vessel sealing system, was experimentally measured, and the incidence of postoperative complications after thoracoscopic esophagectomy was assessed according to the electrosurgical devises used. methods and procedures: experiment of thermal spread: sonicision (sonic) was used for ultrasonic scissors and ligasure (ls) was used for bipolar vessel sealing system. each device was activated in order to cut porcine muscle at room temperature. temperatures of both the device blade and porcine tissues beside the device were measured using a temperature probe. each experiment was performed at least three times. room temperature was degrees. clinical analysis: the patients who underwent thoracoscopic esophagectomy with -field lymph node dissection in the prone position were selected in the study. incidence of postoperative complications after thoracoscopic esophagectomy was compared according to electrosurgical devises. bronchoscopy was used for diagnosis of recurrent laryngeal nerve paralysis (rlnp). sonic and ls was employed in and patients, respectively. material: we compared consecutive cases using d laparoscopic surgery versus cases of d conventional laparoscopic surgery from january to june . all surgical procedures were performed by experienced laparoscopic surgeons using d (einsteinvision system) and hd conventional laparoscopic optic. d-laparoscopic surgery offers the depth perception of the surgical field that is lost with the conventional ( d) laparoscopic surgery, and in many series is reported to be better in terms of surgical performance. outcome measures was operation time, surgical performance, blood looses, complications and surgeon satisfaction with the procedure. results: cholecystectomy was the most frequent surgery performed with cases ( %); hernia surgery cases ( %); fundoplication cases ( %), appendectomy cases ( %), left colon excison with colo-rectal anastomosis cases( %), and other cases ( %) wich included ovarian cyst excision, liver biopsy, prostatectomy and pediatric surgery. we compared each d procedure with a standard laparoscopy case performed by the same surgeon during the time of the study. d vs d surgical procedures outcome measures are shown in table . we found better results in operation time, surgical performance and less blood looses in favor of three-dimensional laparoscopy (. ). conclusion: d laparoscopy reduces operation time related to better performance during the procedure. depth perception facilitates dissection, intracorporeal knotting, mesh placement and colo-rectal anastomosis. surgeons reported better surgical performance and comfort during d laparoscopy; there were any reported side effects such as headache or dizziness. background: social media (some) uniquely allows international collaboration, with immediacy and ease of access and communication. in areas where surgical management is contentious, this could be a valuable tool to frame the current state, propose best practices, and possibly guide management in a rapid, cost-effective, global scale. our goal was to determine the ability to use twitter-a some platform-as an alternative surgical research tool. methods: twitter was used to host an online poll on a pre-selected controversial topic with no current consensus guidelines-pathological complete response in rectal cancer. an influential colorectal surgeon published the survey "t n rectal cancer undergoes a complete response" on two separate occasions. both polls were open for duration of three days. two methodologies were tested to increase exposure and direct towards relevant participants: first, tagging several worldwide experts, then using the well-established hashtag #colorectalsurgery and publishing during an international surgical conference. the main outcome measure was the feasibility, validity, reproducibility, and methods to further participation of a twitter survey. results: the tweet polls were posted three weeks apart. there was no cost and the time required for the process was three minutes, demonstrating the feasibility. providing three closed options to select from facilitated validity. the poll's anonymity limited knowledge of the participant's qualifications, but public comments and "retweets" came from surgeons with experience ranging from trainee to department chair. a robust volume of respondents was observed. the st post received votes, "likes", "retweets", and comments from a diverse international group ( countries). all tagged members participated in the forum. the nd received votes, "likes", "retweets", and comments. the results were reproducible, with the majority favoring option on both occasions ( % and %, respectively; p= . ). treatment recommendations, their rationale, and open questions were identified in the thread. conclusions: some can be used as a research tool, with valid, reproducible, and representative survey results. while exposure was comparable across the two methods, tagging specific members guided experts to provide more opinions than using conference and specialty hashtags. this could expand awareness, education, and possibly affect management in a transparent, cost-effective method. the anonymous nature of respondents limited the ability to make conclusions, but interest and opinion leaders for further study can be easily identified. this demonstrates the potential for some to facilitate international collaborative research. background: despite the technological advancement of a minimally invasive approach to pylorus -preserving pancreaticoduodenectomy (pppd), the morbidity is still high. among the many complications, postoperative pancreatic fistula (popf) is reported in high incidence rate, which varies from researcher to researcher, and a fistula risk score (frs) has been developed to predict the popf. the aim of this study is validate the fistula risk score in minimally invasive approach of pppd and find the other meaningful parameter for prediction of popf. method and materials: from january to august , laparoscopy attempted right-sided pancreas resection was performed on patients including robotic reconstruction in the division of hepatobiliary and pancreas at yonsei university health system. among them, patients were excluded due to total pancreatectomy (n= ), open conversion (n= ), pancreaticogastrostomy and hybrid manual anastomosis (n= ), non-measurable drain and missing datas (n= conclusions: fistula risk score is significant prediction factor of popf including biochemical leaks. in addition to the previously known frs variables, our data showed that bmi is an important predictor of popf with clinical relavancy in a minimally invasive approach of pppd. laparoscopic hemi-hepatectomy for liver tumor satoru imura, hiroki teraoku, yuji saito, shuichi iwahashi, tetsuya ikemoto, yuji morine, mitsuo shimada; tokushima university introduction: with progress of surgical technique and devices, laparoscopic liver resection became a realizable option for patients with liver tumor. major liver resection such as anatomical left or right hemi-hepatectomy has also been introduced in many centers. herein, we evaluate surgical results of laparoscopic hemi-hepatectomy for liver tumor. patients and methods: until march , consecutive patients who underwent laparoscopic or laparoscope-assisted hemi-hepatectomy (left: , right: ) were reviewed and the surgical data such as operation time, blood loss, postoperative complications were analyzed retrospectively. results: of the patients underwent left hemi-hepatectomy, cases were primary liver cancer, cases were metastatic tumor, and cases were benign tumor. pure laparoscopic surgery was performed in cases. the mean blood loss was ( - ) ml, mean operating time was ( - ) minutes and mean postoperative hospital stay was ( - ) days. the rate of postoperative complications was . % (wound infection; n= ). all right hemi-hepatectomy was performed by laparoscope-assisted method. of the patients underwent right hemi-hepatectomy, cases were primary liver cancer, cases were metastatic tumor, and cases were benign tumor. the mean blood loss was ( - ) ml, mean operating time was ( - ) minutes and mean postoperative hospital stay was ( - ) days. the rate of postoperative complications was . % (biliary stenosis; n= ). the patients with hepatocellular carcinoma were followed up for a median of ( - ) months. recurrence occurred in cases and none of them had died at the time of follow-up. conclusion: laparoscopic hemi-hepatectomy is a safe and effective procedure for the treatment of benign and malignant liver tumors. ibrahim a salama, professor; department of hepatobiliary surgery, national liver institute, menoufia university abstract background: iatrogenic biliary injuries are considered as the most serious complications during cholecystectomy. better outcome of such injuries have been shown in cases managed in a specialized center. objective: evaluatation of biliary injuries management in major referral hepatobiliary center. patients and methods: four hundred seventy two consecutive patients with post-cholecystectomy biliary injuries were managed with multidisciplinary team (hepatobiliary surgeon, gastroenterologist and radiologist) at major hepatobiliary center in egypt over years period using endoscopy in patients, percutaneous techniques in patients and surgery in patients. results: endoscopy was very successful initial treatment of patients ( %) with mild/moderate biliary leakage ( %) and biliary stricture ( %) with increased success by addition of percutaneous (rendezvous technique) in patients ( . %). however, surgery was needed in ( %) for major duct transection, ligation, major leakage and massive stricture. surgery was urgently in patients and electively in patients. hepaticojejunostomy was done in most of cases with transanastomatic stents. one mortality after surgery due to biliary sepsis and postoperative stricture was in cases ( . %) treated with percutaneous dilation and stenting. conclusion: management of biliary injuries was much better with multidisciplinary care team with initial minimal invasive technique to major surgery in major complex injury encouraging for early referral to highly specialized hepatobiliary center. introduction: simple liver cyst is the solitary non parasitic cystic lesion of the liver. teatment of symptomatic liver cyst varies from simple aspiration to hepatic resection. each treatment has its own merits and associatied complications. laparoscopic unroofing (fenestration) offers the best balance between efficacy and safety. polycystic liver disese (pcld) treatment by this method are less clear because of high failure rate. liver resection though more effective carries higher risks. treatment of hydatid disease are controversial. materials and method: simple cyst may be asymptomatic and picked up as incidental findings on ultrasound examination for other abdominal complaints. few cyst have symptoms of mass effect or with complication effect due to haemorrage, rupture, infection. on examination liver is palpable. compression over bile duct give rise to jaundice. the commonest symptoms are pain, early satiety, nausea and vomiting. simple cyst are more common in female after years of age. the cyst located antriorly inferiorly and laterally are the ideal case. investigation like ultrasonography is important. it will helps us to detect the cyst nature, will help to differentiate bet ween simple cyst from poly cystic liver disease, from neoplastic liver. in endemic area of hydatid liver disease serological test is mandatory. ct scan is important regarding details information about to localise the cyst, to identify the liver tissue arroud the cyst, relationship of cyst with the nearby vital structures, number of cyst, calcification and carcinomatous changes in its wall. aspiration of cystfluid, biological and cytological examination to rule out the presence of infection, biliary communication and malignancy. recently, ca - estimation is helpful for the differentiating the simple cyst from the cystadenoma or carcinoma. for jaundice patient ercp is impotant to locate the intraductal polyp causing the biliary obstruction or cyst causes the compression of the biliary tree. for bleeding in cyst mri is helpful. carcinoma at epithelial lining may occur. result: laparoscopic de-roofing (fenestration) less radical procedure ensures adequate drainage of cyst content into the peritoneal cavity. the cyst wall can be removed using harmonic scalpel so smoked produced and fogging of lens can be minimized. the interior surface inspected with care to exclude neoplastic growth and biliary communication. whole operative procedure, duration of postoperative recovery, hospital stay is much shorter in this procedure. large cevron incision can be avoided. no recurrence in two years follow up period. liver resection and total cystectomy theoretically minimizes the recurrence risk but invoke the a real risk of postoperative complications and death. conclusion: careful case selection and meticulous surgical skills are the two major determinants of the outcome. in the llr group, the first port was placed with an alexis® wound retractor (applied medical, usa) and free access® (top corporation, japan) at the abdominal defect made by previous sc. an additional or trocars were placed as needed. results: all patients in the llr group were treated using the laparoscopic approach. there were no other significant differences in patient background and characteristics. operative duration was similar for these groups. blood loss, complication rate, and hospital stay in the llr group were significantly decreased compared with the olr group. conclusion: in concurrent liver resection and sc, the open approach may require multiple large incisions, but the laparoscopic approach can complete procedures with a stoma wound and a few port wounds. additionally, use of a platform on the wound for sc enhances safety and efficacy for dissection of intraabdominal adhesions and a clear operative view. primary hepatic lymphoma: the importance of liver biopsy diego t enjuto , carlos ortiz , laura casanova , jose luis castro , pablo sánchez , jaime vázquez , norberto herrera , benjamín tallon , carmen jimenez ; hospital severo ochoa, hospital san rafael, hospital henares primary hepatic lymphoma (phl) is a very uncommon lymphoproliferative malignancy. it accounts for only . % of all extranodal non-hodgkin lymphoma and . % of all cases of non-hodgkin disease. the diagnosis is made when there is only liver involvement or when there is minimal non-liver disease. bone marrow, spleen, or hematologic affection should be excluded to confirm the diagnosis. we present our experience with two phl's that were correctly diagnosed thanks to laparoscopic liver biopsy. -year-old male admitted because of a -month history of right upper quadrant pain and nonmeasured weight loss. liver function tests and cholestasic enzymes showed normal values. serologic tests showed negative results for both hbv (hepatitis b virus) and hcv (hepatitis c virus). ct (computed tomography) scan showed three intrahepatic lesions in segments v, vi, and vii. ct-guided fine needle did not reach the diagnosis so a laparoscopic hepatic biopsy was performed. the final diagnosis was burkitt-like lymphoma. chemotherapy with r-chop (rutiximab, cyclophosphamide, adriamycin, vincristine, and prednisone) modality was started and completed after cycles. it is currently years since the patient was diagnosed and there are no clinical or radiological signs of recurrence. -year-old male who complained of diarrhoea and abdominal pain. chronic hb infection with no viral charge was detected. ultrasound showed heterogeneity of the whole left hepatic lobe and an mri was performed. a ten by segen centimeters lesion occupying the left hepatic lobe enhanced in arterial phase was seen suggesting adenoma. laparoscopic hepatic biopsy was completed to reach a definitive diagnosis. non-hodgkin lymphoma follicular type has just been confirmed with the histology and immuno-histochemistry. chemotherapy with r-chop should be started in the following weeks. phl's diagnosis is hard to achieve. fine needle biopsies are frequently negative because of the large area of necrosis. surgical biopsies are sometimes indispensable to get enough tissue to reach the diagnosis. phls are sometimes misdiagnosed as hepatocellular carcinoma because of its relation to hcv meaning a major hepatic resection. that is the reason why we consider that all diagnostic measures should be undertaken to rule out a different type of tumor. surgical resection is normally not needed in phls; as they are chemosensitive lesions. surgical options usually add unnecessary morbidity and mortality to these patients. chemotherapy standard treatment for phl consists on r-chop combination. pancreatic neoplasm enucleation -when is it safe? case report and review of the literature elaine jayne buckley , k molik , j mellinger ; siu-som, hshs pediatric surgery introduction: solid pseudopapillary tumors are rare neoplasms accounting for - % of pancreatic malignancies with a low risk of recurrence and metastasis. pancreatic malignancies are less common in pediatric populations, though small case series have identified that pseudopapillary tumors comprise between and % of pediatric pancreatic neoplasms. as these tumors have a low risk of metastasis, the mainstay of treatment has remained surgical excision. several surgical approaches have been described from extensive resections such as pancreaticoduodenectomy to local enucleation. we present a case of enucleation of a large pseudopapillary tumor from the pancreatic head complicated by pancreatic fistula. a literature review was performed given the rarity of this tumor to review surgical approaches, to compare complications and long-term outcomes, and to identify specific strategies to decrease the risk of pancreatic fistula. case description: a year-old female presented with months of abdominal pain. computed tomography identified a right upper quadrant mass felt to be consistent with a lipoma. follow up ct at months suggested the mass was more likely a gastrointestinal stromal tumor (gist), and surgical resection was recommended. enucleation of the mass was chosen in view of a wellcircumscribed appearance, clear operative tissue planes, and concern for long-term morbidity of a more extensive resection given the patient's young age. pathology demonstrated an . cm pseudopapillary tumor with negative margins. her post-operative course was complicated by a grade b pancreatic fistula, managed with nutritional support, external drain maintenance, and endoscopic stenting. the patient achieved healing of the pancreatic fistula after four months. results: our literature review demonstrates no difference in recurrence, mortality or morbidity between types of surgery. pancreatic fistula contributed to the majority of postoperative morbidity in all cases. recommendations for enucleation include small ( - cm) tumors with between and mm margin from the main pancreatic duct. techniques identified to minimized post-operative pancreatic fistula include preoperative imaging of the duct anatomy, preoperative pancreatic stent placement, and intraoperative ultrasound to identify the pancreatic duct. some literature supports preservation of pancreatic parenchyma, particularly in younger patients, to reduce endocrine and exocrine dysfunction given the low rates of recurrence and metastasis with this rare neoplasm. conclusion: our case demonstrates complications of enucleation of a large pseudopapillary tumor with successful multidisciplinary post-operative management. with the risk reduction strategies identified, we suggest that enucleation may be considered for pseudopapillary tumors in younger patients to preserve pancreatic parenchyma and long-term pancreatic function. introduction: recent advancements in minimally invasive techniques led to increased effort and interest in laparoscopic pancreatic surgery. laparoscopic distal pancreatectomy is a widely accepted procedure for left-sided pancreatic lesions. in other cases, the adoption of laparoscopic pancreaticoduodenectomy has been hindered by the technical complexity of laparoscopic reconstruction. hybrid laparoscopy-assisted pancreaticoduodenectomy (hlapd) in which pancreaticoduodenal resection is performed laparoscopically, while reconstruction is completed via a small upper midline minilaparotomy, is combines the efficacy of open approach, and the benefits of laparoscopic approach. the purpose of this study is to report our experience of hlapd and to define the learning curves. methods: patients with benign and malignant periampullary lesion underwent hlapd by a single surgeon between july and may were retrospectively reviewed. the clinicopathologic variables were prospectively collected and analyzed. the learning curve for hlapd was assessed using cumulative sum (cusum) and risk-adjusted cusum (ra-cusum) methods. results: the most common histopathology was pancreatic ductal adenocarcinoma (n= , . %), followed by intraductal papillary mucinous neoplasms (n= , . %), ampulla of vater cancer (n= , . %), and common bile duct cancer (n= , . %). the median operation time was min (range, - min) and the median estimated blood loss was ml. based on the cusum and the ra-cusum analyses, the learning curve for hlapd was grouped into four phases: phase i was the initial learning period (cases - ), phase ii was the technical stabilizing period (cases - ), phase iii was the second learning period (cases - ) and phase iv represented the second stabilizing period (cases - ). there was a statistical difference in terms of surgical indication between phase ii and iii (p= . ). conclusions: hlapd is a technically feasible and safe procedure in selected patients. this procedure has benefits of both open and minimally invasive procedure, and could be a stepping-stone for transition from open to purely minimally invasive pancreaticoduodenectomy. in silico investigation of the background: wilson's disease is a rare autosomal recessive genetic disorder of copper metabolism, which is characterized by hepatic and neurological disease. the gene atp b (on chromosome ) leads to wilson's disease is highly expressed in the liver, kidney, and placenta and encodes a transmembrane protein atpase (atp b), which functions as a copper-dependent p-type atpase. methods: here, the rare codons of atp b gene and their location in the structure of atp b protein was studied with rare codon calculator (racc) (http://nihserver.mbi.ucla.edu/racc/), atgme (http://atgme.org/), latcom (http://structure.biol.ucy.ac.cy/latcom.html) and sherlocc program (http://bcb.med.usherbrooke.ca/sherlocc.php). racc server identified arg, leu, ile, and pro codons as rare codons. results: results showed that cyp a gene have single rare codons of arg. additionally, racc detected two rare codons of leu, single rare codons of ile and rare codon of pro. atp b gene analysis in minmax and sliding_window algorithm resulted in identification of and rare codon clusters, which shows the difference features of these algorithms in detection of rcc. analyzing the d model of atp b protein show that arg residue constitute hydrogen bonds with glu and glu that with mutation of this residue to ser this hydrogen bonds were disrupted and may interfere in the proper folding of this protein. moreover, the side chain of arg don't forms any bond with others residues that with mutation to thr form new hydrogen bond with the side chain of arg . these addition and deletion of hydrogen bonds effects on the folding mechanism of atp b protein and interfere with the proper function of the atp b position. his forms the hydrogen bonds with the his and it seems that this hydrogen bond close together two region of this protein and it seems that has a critical role in the final folding of atp b protein. conclusions: computational study of diseases such as wilson's disease and involved genes (atp b) help us in understanding of disease's physiopathology and finding new approaches for detection and treatment. pancreatic stump leak and fistula formation are significant causes of morbidity in patients undergoing distal pancreatectomy (dp), with incidence of % to as high as % in a large systematic review. we present a case of a year old female, four months status post distal pancreatectomy and splenectomy for pseudopapillary neoplasm of pancreatic tail. patient presented to our institution with day history of left upper quadrant pain and general malaise. differential diagnosis on admission was abdominal wall abscess vs incarcerated incisional hernia. physical exam was positive for severe tenderness to palpation over a * cm cm non reducible mass in left upper quadrant with surrounding skin erythema. patient underwent a diagnostic laparoscopy and intraoperative findings revealed extensive adhesions to the anterior abdominal wall and a loop of small bowel was found adhered to the previous incision site in left upper quadrant. upon further dissection we entered a large cm cavity with saponified caseous material. the saponified material and thick tan fluid were evacuated into an endocatch bag and two large bore jackson pratt drains were left within the cavity. further examination showed that the small intestine was normal with no signs of obstruction or ischemia. fluid studies and cultures were sent and showed yeast like organisms and negative for acid fast bacillus. we report an unusual presentation of a distal pancreatectomy stump leak in the formation of an intra-abdominal saponified fluid collection four months after the primary procedure. given the high incidence of pancreatic stump leak and fistula formation after distal pancreatectomy, much effort has been made to identify factors associated with higher incidence of leaks and their usual and unusual presentations, which will be reviewed in this report. initial concerns regarding healthy donor's safety and graft integrity, need for acquiring surgical expertise in both laparoscopic liver surgery and living donor transplantation (ldlt) have delayed the development of laparoscopic donor hepatectomy in adult-to-adult ldlt. however, decreased blood loss, less postoperative pain, shorter length of stay in hospital, and excellent cosmetic outcome have well been validated as the advantage of laparoscopic hepatectomy. hence, the safety and feasibility for laparoscopic donor should be further investigated. we present initial experiences and safety for totally laparoscopic living donor right hepatectomy. in cases who received elective living donor right hepatectomy for adult-to-adult ldlt, totally laparoscopic approach was applied from may up to august . the anatomical variation of portal vein was not considered as an exclusion criteria, but all donors were with type i portal vein variation. the bile duct anomaly was preoperatively evaluated with magnetic resonance cholangiopancreatography (mrcp) and was never excluded for totally laparoscopic approach. d conventional rigid º rigid laparoscopic system was used in cases and the remaining cases used d flexible laparoscopic system. in about %, hepatic duct anomalies (type , a, b) were identified. the operation time was from hours to hours. and the time for the graft removal was within minutes. the hepatic duct transection was performed under operative cholangiography via a cystic duct and the patency of left hepatic duct was also confirmed by operative cholangiography. however, during postoperative period, bile leakage was identified in only case and resolved after the biliary stent insertion by ercp. during operation, there was no transfusion and the inflow control like pringle maneuver was not used at all. v or v were reconstructed in cases and large right inferior hepatic vein was prepared for anastomosis in cases. all grafts were removed through the suprapubic transverse incision. most donors were discharged at days after hepatectomy. during the short-term follow-up period in the donors except this case, complications were not identified. conclusively, totally laparoscopic right donor hepatectomy in elective adult-to-adult ldlt can be initially attempted after enough experiences of laparoscopic hepatectomy and ldlt. however, the true benefits of totally laparoscopic living donor right hepatectomy should be fully assessed through various experiences from multi-institutes. background: the role of neoadjuvent chemotherapy on the treatment of pancreatic cancer remains widely controversial. studies have evaluated its effect on resectability and survival; however, few have studied the consequence of neoadjuvent therapy on surgical outcomes and complications. methods and procedures: a retrospective analysis was performed utilizing the targeted pancreas module of the national surgical quality improvement project (nsqip) for patients undergoing pancreaticoduodenectomy. neoadjuvent therapy was defined by chemotherapy and/or radiation in the -days before surgery. patient demographics, operative characteristics, and -day outcomes were compared amongst patients undergoing neoadjuvent chemotherapy, radiation, chemoradiation, and no neoadjuvent therapy. both univariable and multivariable analysis were completed. results: pancreaticoduodenectomy was completed in , patients. , patients had no neoadjuvent therapy; underwent both chemotherapy and radiation; underwent chemotherapy alone, and underwent radiation alone. there were no differences in demographics or comorbidities. no difference in -day mortality was found; however pancreatic fistula formation was affected by neoadjuvent therapy. neoadjuvant radiation increased fistula formation (or: . , % ci: . - . ) while neoadjuvent chemotherapy (or: . , % ci: . - . ) was protective. conclusion: neoadjuvent therapy significantly impacts surgical outcomes following pancreaticoduodenectomy. given that pancreatic fistula formation can delay post-operative chemotherapy, it may be reasonable to refrain from neoadjuvent radiation therapy for patients with resectable and borderlineresectable disease. the influence of thickest background: the use of stapling devices for distal pancreatectomy remains controversial, due to concerns about the development of postoperative pancreatic ?stula (popf). pancreas thickness might be associated with popf, but suitable thickness of stapler remains also inconclusive in view of reducing popf. methods: we routinely use thickest endo gia™ reloads with tri-staple™ (covidien, north haven, ct) for pancreas closure during laparoscopic left side pancreatectomy (lp) since . we compared short term surgical results of the consecutive ten patients underwent lp using new stapler (ns) and patients with lp using other type of stapler (os) focusing on popf. results: no patients developed clinically relavent (cr)-popf in ns group and two patients ( . %) with os group experienced cr-popf. however, there was no difference of cr-popf between two groups. pancreas thickness on stapling point were not different between two groups ( . mm vs . mm, p= . ). in ns group, patients ( . %) developed a popf, whereas in os group, patients ( . %) developed a popf. there was also no difference of popf between groups. conclusion: the gia™ reloads with the thickest tri-staple™ allows effective prevention of cr-popf after distal pancreatectomy. however, there was no advantage over thinner stapler for lp. introduction: single-incision laparoscopic hepatectomy (silh) has been showed feasible and safe in experienced hands for selected patients with benign or malignant liver diseases. there were only small series reported and most of the procedures were minor liver resections. we herein present our experience of silh during a period of months. methods and procedures: consecutive patients underwent silh which were performed by two experienced laparoscopic surgeons with straight instruments. patient characteristics and surgical outcomes were analyzed by reviewing the medical charts. results: the patient age was . ± . ( - ) years with male predominance ( patients, . %). six patients ( . %) had liver cirrhosis proved by pathologic examinations. nine procedures ( . %) were indicated for malignancy. four major hepatectomies (over two segments) and nine minor ones were performed including seven anatomical resections. the abdominal incisions were para-or trans-umbilical except one which was along the old operative scar at lower midline, while most of them (n= , . %) was within cm in length. inflow control was carried out by either individual hilar dissection or extraglissonian approach instead of pringle maneuver. the operations were all accomplished successfully without additional ports or open conversion. the operative time was . ± . ( - ) min and the estimated blood loss was . ± . ( - ) ml. five ( . %) patients encountered complications and four of them were classified as clavien-dindo grade i. the postoperative length of hospital stay was . ± . ( - ) days. there was no mortality. conclusion: silh can be performed safely and efficaciously for selected patients with benign and malignant liver diseases including cirrhosis. not only minor but also major liver resections are feasible. this innovative procedure provides low postoperative pain and fast recovery. before adopting this demanding technique, surgeons should be familiar with both single-incision laparoscopic surgery and laparoscopic hepatectomy. better outcomes after the learning curve could be anticipated. background: laparoscopic distal panreatectomy (ldp) has been replacing the open procedure for benign or malignant diseases of the pancreas. however, it is often difficult to apply ldp for pancreatic ductal adenocarcinoma (pdac) because its aggressive invasion to adjacent organs or major vessels. objectives: the objective of this study was to report our experiences for laparoscopic extended pancreatectomy with en-bloc resection of adjacent organs or major vessels for left-sided pdac. methods: we reviewed data for all consecutive patients undergoing ldp for left-sided pdac at asan medical center (seoul, south korea) between april and december . the patients who underwent laparoscopic extended panreatectomy with en-bloc resection of adjacent organs or major vessels were included in analyses. results: of total patients, underwent laparoscopic extended pancreatectomy. there were male and female patients with a median age of . years. resected adjacent organs or vessels were as following: stomach in , duodenum in , colon in , kidney in , superior mesenteric vein in , and celiac axis in . median operative duration was minutes, and median length of hospital stay was days. pathological reports revealed the following: a median tumor size of . cm, the tumor differentiation (well differentiated in , moderately differentiated in , and poorly differentiated in ), t stages (t in , t in , and t in ) , and n stages (n in and n in ). r resection was achieved in patients, and most r resection were tangential retroperitoneal margins. postoperatively, clinically relevant postoperative pancreatic fistula was occured in patients, and there was no -day mortality. median overall survival was . months and year survival rate was . %. conclusions: although laparoscopic surgery has limitations in treating extensive diseases, some selected patients can be applicable for laparoscopic extended pancreatectomy with acceptable complication and survival rates. who underwent hepatic resection was included. these patients were divided into llr or olr. demographics, tumor characteristics, recurrence rates and over-all survival were compared between the groups. results: patients were included and grouped into llr (n= ) and olr (n= ). the average tumor number was ± for both groups, while the mean tumor size was . cm and . cm for the llr and olr group, respectively. when compared with olr, llr had lower post-operative complication rates ( . % vs . %, p= . ) and shorter hospital stay ( vs days, p= . ), although the difference was not statistically significant. overall, recurrence-free and disease-free survival was comparable between llr and olr. introduction: single port surgery has been described since with cholecystectomy, colectomy, gastrectomy, and others. nevertheless, few cases are still reported in field of hbp surgery. herein, we report single port pancreatic surgery developed from our previous experience. we had started single port surgery in , since then we have done more than cases of single port surgery using surgical glove port including cholecystectomy, appendectomy, and colectomy. because we consider this experience should develop to pancreatic surgery, cases of single port staging laparoscopy for potentially resectable and borderline resectable pancreatic cancer and cases of single port plus one port distal pancreatectomy (spop-dp) have been done in our institution. single port staging laparoscopy for pancreatic cancer. resectability was proved in ( %) out of patients while patents had unresectale factor such as small liver and peritoneal metastases that was not able to detect pre-operatively. the length of hospital days were . ± . days and the days to chemotherapy were . ± . days. single port plus one port distal pancreatectomy (spop-dp) spop-dp starts with . cm skin incision on umbilicus. subsequently, a wound retractor is installed at umbilical wound. then, a non-powdered surgical glove ( . inches) is put on the wound retractor through which three -mm slim trocars and one -mm trocar are inserted via each finger tips. a semi-flexible laparoscopic camera is inserted via the middle finger port. -mm port is used when laparoscopic us, mechanical stapler, endo intestinal clip or retrieval bag were needed. an additional -mm port is inserted at left subcostal lesion mainly used for surgeon's right hand instrument. gastric posterior wall is fixed to abdominal wall by suture instead of manual retraction. pre-compression before transection of the pancreas was done using endo intestinal clip before firing. discussion: as we have seen in these two decades, surgery has dramatically been changed by laparoscopic surgery or robotic surgery. nevertheless, because of technical difficulty and relatively high post-operative complication rate, introduction of reduced port surgery to hbp surgery has just started. spop-dp using endo intestinal clip, glove port and gastric wall hanging method is feasible. but its advantage is not clear so far, multicenter rct is highly desired to clear the benefit of reduced port surgery for pancreas. introduction: scoring systems (ss) are an essential pillar of care in acute pancreatitis (ap) management. we compared six ss (acute physiology and chronic health examination (apache-ii), bedside index for severity in ap (bisap), glasgow score, harmless ap score (haps), ranson's score and sequential organ failure assessment (sofa) score) for their utility in predicting severity, intensive care unit (icu) admission and mortality. methods: ap patients treated between july and september were studied retrospectively. demographic profile, clinical presentation and discharge outcomes were recorded. predictive accuracy of six ss was assessed using areas under receiver-operative curve (auc) with pairwise comparisons. results: patients were treated for ap. twenty-two ( . %) patients were excluded for insufficient data. / ( . %) were male and mean age was . ( - ) years. most common aetiology was gallstones ( . %). mean length of stay was . ( - ) days. ( . %) patients had severe ap, ( . %) required icu admission and ( . %) died. table below shows positive predictive value (ppv), negative predictive value (npv) and auc of six ss in predicting outcomes. pairwise comparisons revealed ranson's (p. ) and sofa (p. ) scores were superior than other ss in predicting all three outcomes. auc of sofa was greater than ranson's score in predicting severity (p. ), but similar in predicting icu admission (p= . ) and mortality (p = . ). conclusion: sofa score is superior to classical ss in predicting severity, icu admission, and mortality in ap. introduction: necrotizing pancreatitis is often a devastating sequelae of acute pancreatitis. historically several approaches have been described with variable outcome. open necrosectomy is associated with higher morbidity ( %) and mortality ( %). endoscopic necrosectomy often is tolerated well but associated with stent migration and multiple procedures. video-assisted retroperitoneal debridement is tolerated well but associated with severe bleeding if adjacent blood vessels are injured during the procedure leading to severe complications. methods: in our series, we perform a step up approach by involvement of a multidisciplinary group consisting of general surgeons, gastroenterologists, infectious disease physicians, critical care internalist, interventional radiologist and nutritional services to formulate a management plan. the necrotized pancreas is initially drained with an ir guided drain, fluid cultures sent for microbiology and treatment with appropriate antibiotics if deemed necessary. the drain is gradually upsized to a fr sized drain to form a well-defined tract for surgical debridement; a preoperative ct scan of the abdomen with iv contrast to access the location and proximity of the vasculature around the necrotized pancreas. a collaboration with the interventional radiologist to discuss possible ir embolization of splenic artery prior to surgical debridement. the patient would then undergo video assisted retroperitoneal pancreatic necrosectomy and a sump drain left in-situ at the pancreatic fossa. post-operative management in the surgical icu would be lead by the critical care internalist. results: three patients were managed by this multidisciplinary approach with excellent outcomes. one patient underwent preoperative ir embolization followed by surgical debridement; second patient underwent embolization immediately following debridement; one patient did not require any embolization but had ir on standby if needed to intervene. post-operatively all three patients recovered well. they all were tolerating good oral intake and were discharged to rehabilitation facilities. conclusion: our preliminary experience demonstrates that an early multidisciplinary plan by various subspecialties can result in a pragmatic and successful approach to this potentially catastrophic condition. introduction: liver resection with preservation of as much liver parenchyma as possible is called parenchymal sparing hepatectomy (psh). psh has been shown to improve overall survival by increasing the re-resection rate in patients with colorectal liver metastases (crlm) and recurrence. the caudal-cranial perspective in laparoscopy makes the cranial segments ( , a, , and ) more difficult to access. the objective of this systematic review is to analyze feasibility, safety, morbidity, and oncologic outcomes of laparoscopic psh. methods: a systematic review of the literature was performed. medline/pubmed, scopus, and cochrane databases were searched. a search strategy was published with the prospero registry. a systematic review was conducted on all cases reported, they were categorized by area of resection and quantitative meta-analysis of operative time, blood loss, length of hospital stay, complications, and r resection was performed. results: of the studies screened for relevance, studies were selected. because interventions or endpoints were noncontributory or reporting incomplete, were excluded. only publications remained, reporting data from patients who underwent laparoscopic psh. the highest oxford evidence level was b and selective reporting bias was common due to single center and noncontrolled reports. among them, ( . %) resections were in the cranial segments ( . %), a ( . %), ( %), and ( . %), which previously would have required laparoscopic hemi-hepatectomies or sectorectomies. the most common tumor type was crlm ( %) and the second most common tumor type was hepatocellular carcinoma ( %). feasibility of laparoscopic psh was %, conversion rate was %, and complications were seen in % of cases. no perioperative mortality was reported. no standardized reporting format for complications was used across studies. meta-analysis revealed a weighted average operating time of minutes, estimated blood loss of cc, and length of stay of days. r resections were achieved in % of cases. conclusion: laparoscopic psh of difficult to reach liver tumors are feasible with acceptable conversion and complication rate, but relatively long operating times and relatively high blood loss. in future studies, data on long term survival and specific tumor type recurrence should be reported and bias reduced. yangseok koh , eun-kyu park , hee-joon kim , young-hoe hur , chol-kyoon cho ; chonnam national university hwasun hospital, chonnam national university hospital purpose: laparoscopic surgery has become the mainstream surgical operation due to its stability and feasibility. even for liver surgery, the laparoscopic approach has become an integral procedure. according to the recent international consensus meeting on laparoscopic liver surgery, laparoscopic left lateral sectionectomy ( conclusion: this study showed that laparoscopic lls is safe and feasible, because it involves less blood loss and a shorter hospital stay. for left lateral lesions, laparoscopic lls might be the first option to be considered. keywords: laparoscopy, left lateral sectionectomy. outcome analysis of pure laparoscopic hepatectomy for hcc and cirrhosis by icg immunofluorescence in.-a propensity score analysis introduction: in laparoscopic hepatectomy, the surgeon cannot use their hand to palpate the liver lesion and estimate margin of resection. the use of icg immunofluorescence technique can show up the liver tumour and has the potential to facilitate a throughout assessment during the operation. method: between and , there were patients undergone pure laparoscopic liver resection for hcc in our hospital. patients had undergone surgery by the conventional laparoscopic approach. patients had laparoscopic hepatectomy with additional icg immunofluorescence augmented technique. the surgical outcome was compared with propensity score analysis in a ratio of : . result: patients had icg immunofluorescence assisted laparoscopic hepatectomy (group ). patients using conventional laparoscopic liver resection with propensity-matched were selected for comparison (group ). the median operation time was minutes vs minutes p= . , the median blood loss was ml vs ml (p= . ). additional tumours were identified by icg technique. patients had suspicious lesion picked up by icg technique but proven to be benign pathology on frozen section examination. the sensitivity of tumour detection by group was %. % r resection was achieved in group and group respectively. hospital stay was days vs days (p= . ), post-operative complication was ( %) vs ( . %) (p= . ) none of the patient developed icg related complication. conclusion: in the current study, the new technique showed equally good short-term outcome when compared with conventional laparoscopic hepatectomy. icg immunofluorescence augmented reality is a promising technique that might facilitate easier identification tumour during laparoscopic hepatectomy. surg endosc ( ) :s -s taking the training wheels off: transitioning from robotic assisted to total laparoscopic whipple introduction: there is a substantial learning curve to performing minimally invasive pancreatoduodenectomy (mis-pd) for surgeons who are trained in open pd. the learning curve to transition from robotic assisted pd (rapd) to total laparoscopic pd (tlpd) is not well established. methods: mis-pds performed between january and june performed by sc as a surgeon or co-surgeon were included for analysis. mis-pds were performed using a robotic assisted technique prior to august , and tlpds were performed subsequently. rapds performed prior to were excluded to limit the comparison to rapds after the initial learning curve. demographics, clinical and pathologic outcomes, operative and post-operative outcomes were compared. results: a total of rapds and tlpds were scheduled during the study period. there was no statistically significant difference in age, body mass index, or prior abdominal surgery. median time from initial clinic consultation to surgery was days for the rapd group versus days in the tlpd group (p= . ). conversion to laparotomy was required in of patients ( there were no operative complications or mortality. the mean hospital stay was ± . hours. there was no postoperative jaundice, bile leak, intra-abdominal collections or mortality. conclusion: when surgery is indicated for difficult acute calculous cholecystitis, laparoscopic subtotal cholecystectomy with control of the cystic duct is safe with excellent outcomes. however, if the critical view of safety can't be achieved due to obscured anatomy at calot's triangle, conversion to open surgery or cholecystostomy must be performed to prevent bile duct injury. scott revell, md , joshua parreco , rishi rattan, md , alvaro castillo, md ; u. miami -jfk gme consortium, university of miami, miller school of medicine introduction: over the last two decades the increasing incidence of benign liver tumors has led to the expanded need for clinicians to make therapeutic decisions regarding the utilization of open, minimally invasive and ablative techniques. the purpose of this study was to compare outcomes of the management of benign liver disease based on operative approach and pathology. methods: patients aged years or older who underwent liver surgery for benign liver tumors from to were identified in the nationwide readmissions database. patients were compared based on liver pathology, resection versus ablation, and an open versus laparoscopic/robotic approach. the outcomes of interest were in-hospital mortality, prolonged length of stay (los) [ days, and readmission within -days. univariable analysis was performed for these outcomes and multivariable logistic regression was performed using the variables with a p-value . on univariable analysis. results were weighted for national estimates. results: there were , patients undergoing surgery for benign hepatic tumors in the us during the study period. the most common pathology was benign neoplasm ( . %) followed by hemangioma ( . %), and congenital cystic disease ( . %). resection alone was performed in . %, ablation alone in . %, and resection with ablation in . %. a laparoscopic/robotic approach was used in . % of cases. the overall mortality rate was . %, a prolonged los was found in . %, and readmission within days occurred in . %. an increased risk for mortality was found with hemangioma (or . , p= . ) and congenital cystic disease (or . , p= . ). resection with ablation was associated with an increased risk of prolonged los (or . , p. ), while a laparoscopic/robotic approach was a protective factor for prolonged los (or . , p. ). patients treated with ablation alone were at decreased risk for readmission (or . , p. ). omar m ghanem, md , desmond huynh, md , tomasz rogula, md ; mosaic life care, cedars sinai, introduction: laparoscopic sleeve gastrectomy is the most commonly weight loss procedures performed worldwide. as such, there is great diversity in the techniques utilized. this study aims to identify and categorize the differences in techniques and assess the need for guidelines in this field. case description: surgeons were surveyed on the techniques they employ on biweekly basis using the international bariatric club facebook group. the survey included sleeve staple line reinforcement, preoperative work up, intraoperative hiatal dissection, bougie size, distance from pylorus to distal staple line, and intraoperative leak testing. surveys were conducted between may and july . each survey was active for weeks after which data was collected. participants were required to select a single answer per question. discussion: when surveyed on staple line reinforcement (n= ), surgeons used no reinforcement, over-sewed, buttressed, clipped as necessary, over-sewed as necessary. for preoperative work up (n= ), utilized routine endoscopy, routinely obtained upper gi series, routinely obtained both endoscopy and upper gi, and employed endoscopy or upper gi series only in patients who were symptomatic. for hiatal dissection (n= ), surgeons dissected the hiatus routinely, dissected only when obvious hernias intraoperatively, dissected only if the hernia was detected on preoperative work up, and dissected in the setting of gerd symptoms. for sleeve caliber sizing (n= ), bougie \ f was used by surgeon, bougie size f, f, f were utilized by , bougie size f and f were utilized by , bougie[ f were used by , and gastroscopes ( f) were used by . with regards to distance from pylorus to where the sleeve staple line was initiated (n= ), participants started \ cm away from pylorus, between and cm, and started [ cm from pylorus. finally, for preferred intraoperative leak test during sleeve (n= ), methylene blue was used by surgeons, air leak test by , used both, and opted for none. conclusion: this study characterizes the wide varieties in the techniques used during sleeve gastrectomy. a great number of variations exist in every parameter surveyed; however, there is little evidence comparing the effectiveness and safety of these variations. in this setting, further randomized controlled trials are necessary and should be used to construct guidelines to best optimize outcomes in this extremely common and necessary operation. yen-yi juo, md, mph, yas sanaiha, md, yijun chen, md, erik dutson, md; ucla introduction: bariatric surgeries are commonly performed in accredited centers of excellence, but no consensus exists regarding the optimal readmission destination when complications occurred. our study aims to examine the impact of care fragmentation on post-operative outcome and evaluate its causes and consequences among patients undergoing -day readmission after bariatric surgery. methods: the metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip) database was used to identify patients who experienced -day unplanned readmission following bariatric surgery. non-index readmission was defined as any readmission occurring at a hospital other than the one where initial surgery was performed. primary outcome was -day mortality after surgery. logistic regressions were used to identify risk factors for nonindex readmission and to adjust for confounders in the association between non-index readmission and -day mortality. results: a total of , patients were identified as experiencing -day unplanned readmission following bariatric surgery, among whom ( . %) were non-index readmissions. occurrence of postoperative complication during initial hospitalization was the most significant risk factor for non-index readmission (or . , % ci . - . , p= . ) in our multivariate logistic regression. the three most common reasons for readmission were similar within the two comparison groups, including nausea/vomit, abdominal pain and anastomotic leakage. similar proportion of patients underwent reoperation among the two comparison groups ( . vs . %, p= . ). even after adjusting for occurrence of complications, being readmitted to a non-index facility was still associated with a . -fold odds of -day mortality ( % ci . - . , p\. ). conclusion: non-index readmission significantly increases the risk of -day mortality following bariatric surgery. patients were more likely to visit a non-index facility if complications occurred during their initial hospitalization. further patient education is required to re-inforce the importance of continuity-of-care during management of bariatric complications and guide patient's decision making in choosing readmission destinations. introduction: sleeve gastrectomy has become the most performed bariatric surgery. removing part of the stomach causes weight loss by restricting food intake and regulating the production of incretins, particularly ghrelin. however, prognostic factors to weight loss after sleeve gastrectomy have been difficult to find. the goal of this research was to study the correlation between the volume of resected stomach and weight loss. methods and procedures: volume of resected stomach of patients undergoing sleeve gastrectomy was measured. a standard laparoscopic technique was used. calibration was performed tightly around a fr bougie, and stapling started - cm from the pylorus. the standardized technique for measurement involved insufflation with a g catheter with saline solution to a pressure of cm h o immediately after removal of the specimen. resected stomach's volume, gender, age, bmi, height and % of total weight loss (%twl) at months and year were prospectively recorded. correlation between variables was analyzed with pearson's test and linear regression models. conclusion: removed stomach was larger on men than women and its size slightly correlated to height. however, volume of resected stomach did not seem to have an incidence on short termweight loss. gastric size should not be considered as a prognostic factor for weight loss in patients undergoing sleeve gastrectomy. revisional bariatric surgery after initial laparoscopic sleeve gastrectomy: what to choose salman alsabah, eliana al haddad, ahmad almulla, khaled alenezi, shehab akrouf, waleed buhamid, mohannad alhaddad, saud al-subaie; amiri hospital introduction: bariatric surgery has been shown to produce the most predictable weight loss results, with laparoscopic sleeve gastrectomy (lsg) being the most performed procedure as of . however, inadequate weight-loss may present the need for a revisional procedure. the aim of this study is to compare the efficacy of laparoscopic re-sleeve gastrectomy (lrsg), laparoscopic roux-en-y gastric bypass (lrygb) and gastric mini-bypass surgery (mgbp) in attaining successful weight loss following initial lsg. methods: a retrospective analysis was performed on all patients who underwent lsg at amiri and royale hayat hospital, kuwait from to . a list was obtained of those who underwent revisional bariatric surgery after initial lsg, and their demographics were analyzed. introduction: the aim of this study is to identify potential risk factors or early indicators, specifically related to perioperative blood pressure, and its association with perioperative hemorrhage in the bariatric population. laparoscopic bariatric surgery in the united states has been steadily increasing over the past several years. between and , the annual number of cases has increased by %. although rare, hemorrhagic complications (hc) occur at a rate of - % and can lead to significant morbidity and mortality. by identifying factors which may place a patient at higher chance of hc, surgeons can potentially mitigate those risks. these modifications could reduce morbidity and limit the requirement of transfusions or reoperations. methods and procedures: a retrospective case-control series was performed to include all patients who underwent either laparoscopic sleeve gastrectomy (sg) or laparoscopic roux-en-y gastric bypass (gb) in at a single bariatric center of excellence. a total of patients were identified with perioperative hc. each patient was matched : for procedure, body mass index, and medical comorbidities. peak systolic, diastolic, and mean arterial pressures were compared between groups at time of admission, intraoperative, and during remainder of initial hospital stay. welch's t-tests were used for comparison between groups. results: a total of procedures were performed with de novo sg, and de novo gb. revisional bariatric cases were excluded from the study. hc occurred in ( . %) total patients, gs and gb. four patients required operative treatment for hc, were treated laparoscopically and required laparotomy. the mean diastolic pressures at time of arrival on day of surgery was higher in patients who develop hc (p= . ) and mean peak diastolic pressure intraoperatively was lower in patients who develop hc (p= . ). there was no statistical difference in peak systolic or mean arterial pressures throughout the hospital stay. conclusions: bariatric surgical patients with elevated preoperative diastolic blood pressures are at an increased risk of postoperative hc. additionally, decreased peak diastolic blood pressures may be an early indication of an hc in bariatric patients. introduction: bariatric surgery in the adult population is recognized as one of the most effective treatments for obesity and its comorbidities. nonetheless, the safety, efficacy, and substantial outcomes of bariatric surgery in young adults are still not well documented. the aim of our study is to evaluate the safety and efficacy of laparoscopic sleeve gastrectomy (lsg) in young adults (\ years old) versus older adults (≥ years old). methods: we retrospectively reviewed all patients who underwent bariatric surgery at our institution from to. propensity score matching was used in order to balance covariates, matching for common demographics and comorbidities between the younger patient population (\ years old) and the control group ([ years old). all tests were two-tailed and performed at a significant level of . . statistical software r, version . . ( - - ) was used for all analyses. results: of patients, . % (n= ) met our inclusion criteria after matching. we found . % (n= ) patients under years old and . % (n= ) patients greater or equal to years old (control group). we observed that our younger population distribution was predominantly caucasian and female, . % (n= ) and . % (n= ) respectively. the mean age was . ± . years with a preoperative body mass index (bmi) of . ± . kg/m in the younger group compared to . ± . years and a bmi of . ± . kg/m in the control group. diagnosis of diabetes and hypertension were present in . % (n= ) and . % (n= ) of our younger group, respectively. no statistical significance was found when assessing the percentage of bmi loss (% ebmil) at and months follow-up as shown in table . when comparing the % ebmil at months follow-up, the younger group had . % more ebmil than the control group (p= . ). when assessing post-operative complications we observed no statistical significance. conclusions: bariatric surgery is equally effective and safe in young adult population demonstrating a significant better %ebmil at months following bariatric surgery. following prospective studies are needed to elucidate the resolution and behavior of comorbidities in a younger bariatric population. minimally invasive conversion of sleeve gastrectomy to rouxen-y gastric bypass for intractable gastroesophageal reflux disease: short term outcome background: surgical management recommendations for intractable gastroesophageal reflux disease (gerd) after sleeve gastrectomy (sg) remain controversial. this case series demonstrates our experience with treatment of post-operative intractable gerd using minimally invasive conversion of sg to roux-en-y gastric bypass (rygb). patients and methods: this is a retrospective review of a prospective data registry (mbsaqip) from jan through sept . eleven patients, female and male, were evaluated. of the surgeries, were laparoscopic, assisted with xi da vinci robot, and assisted with si da vinci robot. all patients presented with intractable reflux on high dose ppi. three had a history of aspiration pneumonia. ± . %, respectively. one was omitted due to pending results. conclusion: several solutions exist for operative management of intractable gerd after sg including redo-sleeve gastrectomy, combined gastrectomy with fundoplication, conversion to gastric bypass or anti-reflux procedures such as linx. reports remain small in series and require further study to evaluate the consistency of results. we found minimally invasive conversion of sg to rygb is a highly effective and safe option for treatment of intractable gerd. setthasiri pantanakul, chotirot angkurawaranon, ratchamon pinyoteppratarn, poochong timrattana; rajavithi hospital background: obesity is an important health problem affecting more than million people worldwide. esophageal dysmotility is a gastrointestinal pathology associated with obesity; however, its prevalence and characteristics remain unclear. esophageal dysmotilities have a high prevalence among obese patients regardless of gastrointestinal symptoms. objective: to identify the prevalence of esophageal motility disorder in asymptomatic obese patient. materials and methods: prospective study was performed between june and march . a total of of morbid obese patients who visited the bariatric and metabolic clinic at rajavithi hospital (bangkok, thailand) underwent preoperative evaluation with high resolution esophageal manometric test with manoscantm eso (smith medical). tracings were retrospective analysis and reviewed according to chicago classification criteria for esophageal motility disorders. results: among asymptomatic obese participants, twenty five of them were female. the mean age was . ( - ) years old. most of the participants were classified as class three obesity or over. the mean bmi was . kg/m . no hiatal hernia was found and the anatomy of esophagus was normal in all patients. the mean irp was . mmhg. twenty-one patients ( . %) demonstrated high irp over normal limit ([ mmhg) . four patients demonstrated premature contraction (dl\ . second). hypercontractile esophagus was identified in patients and ineffective motility disorder was found in patients. two patients were diagnosed as distal esophageal spasm (des). two patients were compatible with type achalasia and patients ( . %) have esophageal outflow obstruction. none of the patient demonstrate incomplete bolus clearance even high irp or abnormal motility. conclusion: this study reveals a high prevalence of esophageal dysmotility in asymptomatic thai obese patients. the most common abnormality were esophageal outflow obstruction and ineffective motility. the chicago classification of esophageal motility disorder may not suitable among obese population. sitembile lee, ms , chike okolocha , aliu sanni, mdfacs ; philadelphia college of osteopathic medicine ga campus, eastside bariatric and general surgery introduction: roux-en-y gastric bypass (rygb) is the most popular bariatric procedure performed worldwide, accounting for % of all bariatric procedures. however, in patients with a body mass index (bmi) ≥ kg/m (super-super obese) the rygb procedure can be technically challenging. this has led to the adoption of a single-stage treatment such as one anastomosis (mini) gastric bypass (oagb/mgb) in the super-super obese patients. proponents of the oagb/mgb claim the clinical outcomes are comparable to the rygb. the aim of this study is to compare the outcomes of the two procedures by examining the literature. methods: a systematic review was conducted through pubmed to identify relevant studies from to with comparative data on rygb versus oagb/mgb on super-super obese populations. the primary outcome was the percentage excess weight loss (%ewl). other outcomes include operative times, complication rates and length of hospital stay. results were expressed as standard difference in means with standard error. statistical analysis was done using randomeffects meta-analysis to compare the mean value of the two groups (comprehensive meta analysis version . . software; biostat inc., englewood, nj introduction: obesity is becoming more prevalent in patients with inflammatory bowel disease (ibd). the obese body habitus increases the complexity of surgeries that are often needed to treat ibd. some surgeons may delay definitive surgical treatment because of obesity. little data exists on bariatric surgery in the obese patient with ibd. methods: we retrospectively identified patients who had known diagnosis of ibd who underwent bariatric surgery from to . demographics and post-operative outcomes were assessed. results: patients were identified: with ulcerative colitis (uc) and with crohn's disease (cd). of the uc patients, none of the patients had surgery for uc and only one was on a biologic. of the uc patients, had adjustable gastric band (agb), had gastric bypass and had sleeve gastrectomy. one patient with agb had it replaced for slip and subsequently removed for dysphagia. uc preoperative bmi average was . . postoperative bmi was . with excess weight loss (ewl) of %. average follow up was months. of the cd patients, patients had ileocolic resections and one had total proctocolectomy with end ileostomy. one was on remicade and one on mp. of the cd patients, had agb, had gastric bypass and had sleeve gastrectomy. one agb patient had conversion to gastric bypass because of dysphagia and poor weight loss. a second abg patient had band removal because of dysphagia. cd patients' preoperative bmi average was . . postoperative bmi was . with average ewl of %. average follow up was months. overall, agb patients had % ewl, sleeves % and gastric bypass %. two uc patients had post-operative flares, one immediately post op and one month post-operative. four of the band patients had dysphagia, with one replacement, two removals and one conversion to bypass. there were no leaks, intraabdominal infections, fistulas or wound infections. conclusions: uc patients appear to have higher excess weight loss compared to crohn's patients; ewl % compared to % but was not statistically significant. agb had poor results in both uc and cd patients. sleeve gastrectomy and gastric bypass results in effective weight loss for obese patients with ibd. gastric bypass in ibd patient is controversial, but may be appropriate in the right clinical setting. introduction: previous studies suggest that modest preoperative weight loss is associated with improved weight loss following bariatric surgery. however, there remains a need to investigate factors which may successfully predict preoperative weight loss among bariatric patients. methods and procedures: this analysis included patients who underwent laparoscopic roux-en-y gastric bypass (rygb), sleeve gastrectomy, or gastric banding at an academic medical center in california. data were measured at patients' consult and preoperative clinical visits. preoperative weight loss outcomes were categorized as follows: no weight loss, lost weight, or gained weight. associations between categorical sociodemographic and surgical characteristics and preoperative weight loss outcomes were assessed using the chi-square test of association. associations between continuous measures and preoperative weight loss outcomes were assessed using anova. a sub-group analysis was completed among participants who lost weight prior to bariatric surgery. wilcoxon-rank-sum and kruskal-wallis tests were used to evaluate associations between patient characteristics and the number of pounds lost. results: patients (n= , ) were predominately ages - ( %), female ( %), white ( %), and privately insured ( %). patient race was significantly associated with weight loss outcomes (p = . ): whereas % of white patients lost weight prior to surgery, only % of black patients lost preoperative weight. among privately insured patients, % lost weight. in contrast, % of patients insured by medi-cal/medicaid lost weight (p= . ). on average, lower baseline excess body weight was associated with no weight loss. patients who lost preoperative weight (n= , ) were included in the sub-group analysis. male sex (p\. ), black race (p. ), undergoing laparoscopic rygb (p= . ), no previous abdominal surgeries (p= . ), upper tertile baseline weight (p. ), waist circumference (p\. ), percent body fat (p\. ), bmi (p. ), excess body weight (p. ), and systolic blood pressure (p= . ) were associated with more pounds lost. conclusions: this study demonstrates various associations between sociodemographic and clinical patient characteristics and preoperative weight loss. given previous literature indicating the positive relationship between preoperative and postoperative weight loss following bariatric surgery, the results of this study suggest an opportunity to improve preoperative weight loss among specific groups. yen-yi juo, md, mph , usah khrucharoen, md , yijun chen, md , yas sanaiha, md , peyman benharash, md , erik dutson, md ; background: besides rate and extent of weight loss, little is known regarding factors predicting interval cholecystectomy following bariatric surgery, which are important factors in a surgeon's consideration during decision-making regarding whether to perform prophylactic cholecystectomy. in addition, no previous studies have quantified the incremental costs associated with ic. we aim to identify risk factors predicting interval cholecystectomy (ic) following bariatric surgery and quantify its costs. methods: a retrospective cohort study was performed using the national readmission database - . cox proportional hazard analyses were used to identify risk factors for ic. linear regression models were constructed to examine associations between cholecystectomy timing and cumulative hospitalization costs. background: patient-reported outcomes after bariatric surgery are important in understanding the longitudinal effects of surgery. the impact of hospital practices and surgical outcomes on followup rates remains unexplored. objective: to assess the effect of hospital-level practices and -day complication rates on -year follow-up rates of a standardized patient-reported outcomes survey. methods: bariatric surgery program coordinators in a statewide quality improvement collaborative were surveyed in june about their practices for obtaining patient-reported outcomes data one year after surgery. hospitals were ranked based on their follow-up rates between and (accounting for overall performance and improvement). univariate analysis was used to identify hospital practices associated with higher follow-up rates. multivariable regression was used to identify independent associations between -day outcomes and follow-up rates after adjusting for patient factors. results: overall, follow-up rates improved from ( . %± . ) to ( . %± . ) though there was wide variability between hospitals ( . % vs . % in ) . coordinator survey response rate was %. sixty-one percent of all surveyed coordinators perceived that surgeons prioritize high follow-up rates. when asked how long were their patients followed for, % of coordinators noted their programs provided lifelong follow-up. patient reminders about the -year survey were used by % of programs, mostly during clinic visits ( %). most programs ( %) had implemented strategies to improve follow-up rates, such as handing out the survey ( %) during clinic visits. follow-up providers included surgeons ( %), nurse practitioners ( %), and/or registered dietitians ( %). patient disinterest ( %), loss to follow-up ( %), survey length ( %), and lack of staff/ resources ( %) were the factors most commonly perceived as barriers to high follow-up rates. when compared to programs in the bottom quartile of follow-up rates, those in the top quartile were more likely to hand out the survey to patients during clinic visits ( % vs . %; p= . ) and had lower rates of risk-adjusted severe complications ( . % vs . %; p= . ), readmissions ( . % vs . %; p= . ), and reoperations ( . % vs . %; p= . ). conclusions: hospitals vary considerably in their -year follow-up rates when seeking patientreported outcomes data after bariatric surgery. there were also significant differences in programspecific practices for obtaining these data. hospitals with higher -year follow-up rates were more likely to physically hand surveys to patients during a clinic visit and had lower -day severe complication, readmission, and reoperation rates. improved -year patient-reported outcomes follow-up after bariatric surgery may be a proxy for higher quality perioperative care. david merkle , kazim mohommed , danielle r rioux , dilendra weerasinghe, md, facs ; nova southeastern university, herbert wertheim college of medicine, bariatric surgery is gaining popularity not only for its weight loss benefits, but also for its metabolic effects. we present a -year-old female patient with symptoms of neuroglycopenia, occurring -years post roux-en-y gastric bypass surgery. during one of her syncopal episodes, her blood sugar was noted to be mg/dl. continuous glucose monitoring demonstrated post prandial hypoglycemia, averaging episodes per day, with a maximum of episodes in one day. upon further evaluation, the lab results of the hba c, chromogranin a, somatostatin, and urinary sulfonylurea levels were all normal, with the c-peptide level within the upper limit of normal. ct scan of the abdomen and pelvis did not show any obvious masses in the pancreas, and since the chromogranin a level was normal, it lead to the empiric diagnosis of nesidioblastosis by exclusion. we placed the patient initially on medical management which included a carbohydrate restricted diet of g per meal, eating - small meals per day, and taking mg of acarbose three times per day. overall, symptoms have improved, and she has - episodes per month, compared with about episodes per day. we will also present the data with regards to other invasive treatment options, which are available when medical treatment options have failed, such as gastric bypass reversal versus distal gastrectomy. vertical banded gastroplasties (vbgs) were a common bariatric procedure in the s but have largely fallen out of favor due to unsatisfactory weight loss and a relatively high incidence of longterm complications such as dysphagia and severe gastroesophageal reflux disease (gerd). one of the ways to address these undesirable effects is to convert to a roux-en-y gastric bypass (rygb). the aim of this study was to assess the safety and efficacy of vbg-to-rygb conversion. outcomes of vbg revisions performed at an academic center between and were reviewed. of the vbg revisions, gastrogastrostomies were created in two patients, two underwent a planned -stage conversion, and vbgs were converted to rygbs. patients were operated on an average of years after their initial vbg. presenting symptoms were weight regain (n= , . %), dysphagia (n= , . %), or severe gerd (n= , . %). fourteen patients ( %) had a gastric staple line dehiscence. of the vbg to rygb conversions, were laparoscopic, were converted to open, were open, and were robotic-assisted. average operative time and length of hospital stay were . minutes and . days, respectively. within the first months post-operatively, twelve ( %) patients required readmission directly related to surgery, while eight ( %) visited the emergency department. eight patients ( %) required at least one unplanned operation due to complication(s) during the entire follow-up: small bowel obstruction (n= , at -week, -months, and -months), necrosis/leak of remnant stomach requiring remnant gastrectomy (n= ), tracheostomy for prolonged respiratory failure (n= ), bleeding (n= ), anastomotic leak (n= ), and hemothorax requiring vats (n= ). four patients ( %) had a contained perforation that was medically managed and five ( %) developed a gastrojejunal anastomosis stricture requiring endoscopic intervention. one patient ( . %) developed pulmonary embolism. there was no mortality directly related to surgery. complete resolution or improvement of gerd/dysphagia was appreciated in all patients in the short term follow-up. patients who presented with weight regain had a mean bmi loss of . ± . points in the median follow-up time of . months up to a year after conversion to rygb. in summary, reoperative bariatric surgeries after vbgs are complex, requiring longer operative times and length of stay. our study found % risk of severe complications requiring reoperations, compared to the previously cited % in short and long-term complications. conversion of vbg to rygb provides excellent relief of severe gerd and dysphagia and is a viable option for significant weight reduction. introduction: bariatric surgery is a safe and effective treatment for severe obesity and its comorbidities. however, concomitant splenectomy is sometimes required due to uncontrolled bleeding during the surgery. limited literature exists regarding the effects of concurrent splenectomy on outcomes of bariatric surgery. this study aimed to determine these outcomes. methods: adult patients with obesity who underwent primary, elective laparoscopic roux-en-y gastric bypass (lrygb) or laparoscopic sleeve gastrectomy (lsg) with concomitant splenectomy were identified from the metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip, ) and national surgical quality improvement program (nsqip, (nsqip, - datasets. using propensity scores (based on baseline variables), patients who underwent primary bariatric surgery were matched : to a control group (primary lrygb/lsg without concomitant splenectomy) and thirty-day postoperative outcomes were compared. continuous variables and categorical variables were categorized as medians with interquartile range (iqr) and counts with percentages, respectively. background: several previous studies have suggested a correlation between weight loss and age after bariatric surgery. objective: the aim of our study is to further address age as a preoperative factor to determine the amount of weight loss after bariatric surgery. materials and methods: we performed a retrospective analysis of outcomes of a prospectively maintained database of , obese patients who underwent either sleeve gastrectomy (sg) or roux-en-y gastric bypass surgery (rygb) at our hospital between and . we analyzed the -month, -month, and -year postoperative percent total body weight loss (%tbwl) of obese patients who underwent bariatric surgery based on their preoperative age. results: the average age of patients included in the study was years old with a range of - years. an inverse relationship between preoperative age and postoperative weight loss was observed. younger patients achieved a higher % tbwl than older patients at the -month, -month, and -year postoperative follow-up. the average %tbwl for all patients at the -month, -month, and -year postoperative follow-up periods were . %, . %, and . %, respectively. at the -year follow-up, for every decade increase in age (above the average age of ), patients lost % less tbwl. conclusion: in our study, younger patients tend to lose a greater amount of %tbwl than older patients after bariatric surgery. results: patients participated in the survey. the median age was yo (iqr: - ) and . % were females. the following responses were encountered when asked about the importance of surgery-related factors: the study population indicated the following responses regarding expectations from magnetic surgery compared to conventional laparoscopy: there was no significant evidence of different responses by demographic groups. additionally, . % of the population indicated that a surgeon performing magnetic surgery should be more skillful than a surgeon performing conventional laparoscopy. conclusion: this study represents the first report of bariatric patient's perception regarding surgery-related factors. notably, nearly % of the cohort indicated that cosmesis after surgery is an important factor, whereas the responses regarding the rest of the factors were indicated as expected. the bariatric population included in this study had a positive perception of magnetic surgery. furthermore, the population perceived that this technique is associated with better outcomes, better cosmetic results, and higher surgeon dexterity. introduction: although much is known regarding medical outcomes of metabolic surgery, less is known regarding quality of life outcomes. we hypothesized that the collection of patient-reported outcomes (pros) could help us understand quality of life in this patient population. we chose to primarily use patient reported outcomes measurement information system (promis) instruments because of their broad applicability, low cost, and ability to use computer-adapted technology to survey. methods: we implemented the routine collection of pros as part of clinical care in december, . patients were offered tablets in clinic, and were asked to complete the surveys at most of their visits. we used computer-adapted technology to decrease the length of time needed to survey. we collected the following promis instruments: depression, pain interference, physical function, and satisfaction with social roles. we also collected the gerd-hrql, a general health question, and a current health visual analog scale (vas). we retrospectively reviewed our results from december through september . results: our response rate was % over the last year of collection. in total, assessments were completed by patients. the mean scores in our total patient population were as follows: vas , gerd-hrql , general health , depression , pain , physical function , and social roles . for promis instruments, the mean for the national population is , with as the standard deviation. for the depression and pain scores a higher score is worse, while a higher score indicates better quality of life for social roles and physical function. conclusions: routine collection of patient reported outcomes can be implemented in a metabolic surgery clinic. health-related quality of life appears to be decreased in this patient population compared to the general public. further work is ongoing to learn about postoperative trends, as well as differential effects of metabolic procedures. the effect of peri-operative antibiotic drug class on the resolution rate of hypertension after roux-en-y gastric bypass and sleeve gastrectomy. results: in total, rygb and sg were included in our analysis. no significant differences were found between cefazolin and clindamycin regarding hypertension resolution rates after sg. there was a significant difference in the resolution of hypertension after rygb with the use of prophylactic clindamycin or cefazolin. as shown in figure , patients who underwent rygb and received clindamycin had a significantly higher rate of hypertension resolution compared to cefazolin. this effect started at weeks post-operatively ( . % vs . % respectively, p= . ) and persisted up to the -year ( . % vs . % respectively, p= . ). we found no significant differences in patient age, sex, number of pre-operative hypertensive medications, pre-operative bmi, or %bmi change after year to account for the significant effect of antibiotic choice on hypertension resolution. conclusion: this study represents the first clinical report to suggest an impact of the type of antibiotic administered at the time of rygb on co-morbidity resolution, specifically hypertension. future studies will be needed to confirm that the mechanism of action for this novel finding is due to the differing modifications of the gastrointestinal microflora population based on the specific peri-operative antibiotic administered. introduction: laparoscopic adjustable gastric band with plication (lagbp) is a novel bariatric procedure which combines the adjustability of the laparoscopic adjustable gastric band (lagb) with the restrictive nature of the vertical sleeve gastrectomy (vsg). the addition of plication of the stomach to lagb should provide better appetite control, more effective weight loss, and greater weight loss potential. objective: the purpose of the study was to analyze the outcomes of lagbp at months. setting: this is a retrospective analysis from one surgeon at a single private institution. methods: data from all patients who underwent a primary laparoscopic lagbp procedure from december to june were retrospectively analyzed. data collected from each patient included age, gender, weight, body mass index (bmi), and excess weight loss (ewl). results: sixty-six patients underwent lagbp. the mean age and bmi was . ± . years and . ± . kg/m, respectively. all patients were beyond the -month postoperative mark. no patient was lost to follow-up. the patients lost an average of % and . % excess weight loss (ewl) at months ( . % follow-up) and months ( . % follow-up), respectively. also, the patients lost a mean bmi of . kg/m and . kg/m at months and months, respectively. the total number of fills during the study period was , and the mean fill volume was . ± cc. dysphagia was the most common long-term complication. the mortality rate was %. conclusions: lagbp is a relatively safe and effective bariatric procedure. in light of recent studies demonstrating poor outcomes following lagb, lagbp may prove to be the future for patients desiring a bariatric procedure without resection of the stomach. the median interval between (lrygb) and reoperation is months in group a and months in group b. the median percentage of excess weight loss (%ewl) is % vs %, respectively (p= . ). patients % ( in group a) were admitted in an emergency with an acute abdomen pain. ct scan was performed in patients % and has shown signs of occlusion in all cases. the most common symptoms were abdominal pain and vomiting. the surgery was performed by laparoscopy in patients % and by laparotomy or conversion in patients %. in all cases internal hernia was reduced and closed all defects. in only one patient in (group a) small bowel at jja was resected. there was no mortality and one patient had pneumonia with acute respiratory distress which was treated medically. conclusions: the closure of mesenteric defects at (lrygb) by tight non-absorbable continued sutures is recommended because it is associated with a significant reduction in the incidence of internal hernia. introduction: laparoscopic roux-en-y gastric bypass (rygb) is a common and effective form of bariatric weight loss surgery. however, a subset of patients will fail to achieve the expected total body weight loss (tbwl) greater than % after months or experience significant weight regain despite dietary, psychiatric, and behavioral counseling. although alternative procedural interventions exist for operative revision after suboptimal rygb weight loss, laparoscopic adjustable gastric banding (lagb) provides an option with short operative time, low morbidity, and effective results. we have previously demonstrated that short-term ( -month), and mid-term ( -month) weight loss is achievable with lagb for failed rygb. the objective of this study is to report the long term year outcomes of lagb after rygb failure. methods and procedures: a retrospective review of prospectively collected data before and after rygb when available, and before and after revision with lagb was performed. background: saline filled intragastic balloons have become a common outpatient procedure for the treatment of obesity. acute dilation, ischemia and necrosis of the stomach has been described in the medical literature. gastric necrosis from acute gastric dilation is a rare but life-threatening condition, which requires timely diagnosis and management. we present a case of partial gastric ischemia with necrosis hours following placement of a saline filled intragastric balloon. postoperative complaints of bloating, nausea and vomiting are common complaints following placement of saline filled intragastric balloons and can lead to a delay in diagnosis. early diagnosis and management is essential in avoiding this life threatening complication. case report: a year old woman, bmi , comorbid conditions of diabetes mellitus underwent uncomplicated placement of a saline filled intragastric balloon for treatment of obesity. hours after placement the patient complained of cramping and bloating. hours following placement the patient developed vomiting and presented to an emergency room for evaluation. she was found to have blood glucose exceeding and a severely dilated stomach with pneumotosis on ct evaluation. ng tube decompression and icu management of the severe hyperglycemia was initiated. removal of the intragastric balloon was delayed - hours until an appropriate endoscopic retrieval kit could be obtained. endoscopic retrieval was performed without incident and near complete necrosis of the gastric mucosa was noted. the antrum was the only area spared. hours after retrieval a laparoscopic evaluation of the stomach revealed full thickness necross of the entire fundus and greater curve. indocyanine green (icg) fluorescent dye was used to assess vascular integrity of the remaining stomach and to define lines of resection. resection of the greater curvature was performed using icg florescent dye to ensure that the angle of hiss was viable and well perfused. the patient had a full recovery and subtotal gastrectomy was avoided. conclusions: spontaneous gastric distension exacerbated by gastric outlet obstruction following placement of a saline filled intragastric balloon can occur. unrecognized this condition can lead to ischemia, necrosis and perforation of the stomach. appropriate evaluation of patients following placement of intragastric balloons is essential. recognition of this condition can be delayed due to the complaints of cramping, bloating and vomiting which are typical following placement of saline filled intragastric balloons. untreated, gastric ischemia and necrosis can lead to early perforation which is associated with a high mortality rate. introduction: morbid obesity has become a growing health risk in the united states with up to % of americans suffering with obesity. bariatric surgery remains the best treatment for morbid obesity. the recent use of laparoscopic sleeve gastrectomy (lsg) as a single stage procedure has met with great success because of its quick learning curve and minimal postoperative complication rates. however, there are concerns if the lsg is an effective procedure for long-term weight loss. although criticized at first, the mini-gastric bypass (mgb) surgery has become a great option for morbidly obese patients because of the ability to lose weight with minimal post-op complications. the aim of this review is to assess the outcomes of lsg as it compares to mgb for the management of morbid obesity. introduction: we hypothesize that a jejunoileal anastomosis and partial diversion using magnamosis, a novel magnetic compression device, is technically feasible and will improve insulin resistance and metabolic syndrome similarly to patients who underwent bariatric surgery. metabolic surgery has demonstrated improvements in various parameters including insulin resistance, triglyceride levels, and cholesterol. it may be technically feasible to perform a less-invasive operation through partial diversion, and thereby stimulate an increase in incretins from the l-cells of the ileum to glean these benefits. methods and procedures: we performed a laparotomy and jejunoileal partial diversion using magnamosis in five rhesus macaques with induced insulin resistance through dietary modifications. after surgery, weight was monitored and a metabolic laboratory evaluation was performed weekly. timed tests were performed at baseline and again at and weeks postoperatively for triglyceride levels, glp- , insulin, glucose, and bile acids. the primates were followed for weeks prior to euthanasia. results are represented as mean±sem and all p-values were calculated using a two-sample students' t-test. introduction: many studies concerning individuals seeking bariatric surgery indicate a higher prevalence of psychiatric disorder in this population, both before and after surgery, however results are not conclusive. the aim of this study was to investigate changes in psychiatric health after gastric bypass surgery. methods: patients within the catchment area of the department of psychiatry of the south alvsborg hospital, operated with gastric bypass surgery during - were identified through the scandinavian quality registry (soreg). patients files were examined and psychiatric diagnoses and alcohol/drug abuse were recorded preoperatively and with a follow up time of years. results: a total of operated patients were identified. of these patients had been in contact with the psychiatric department before or after surgery. patients had attempted suicide preoperatively, but no attempts were made postoperatively, all women. patients attempted suicide postoperatively without a previous history of suicidal attempts, men woman. four patients with a preoperative history of alcohol abuse were identified, all women. these individuals did not seem to abuse alcohol/drugs postoperatively. postoperatively patients with an alcohol/drug abuse were identified, men, women. none of them had a former history of abuse. of the patient performing suicidal attempts postoperatively, men woman, had a postoperatively emerging alcohol/drug abuse. conclusion: preoperatively known alcohol/drug abuse or suicidal attempts do not seem to predispose for postoperative abusive problems or suicidal behavior. preoperative identification of individuals prone to alcohol/drug abuse or suicidal attempts seems difficult. introduction: in the past, our group has popularized models for gastric bypass, sleeve and gastric imbrication. there are currently no models to predict weight loss following single anastomosis duodenal switch. surgeons who offer this procedure are left to guess based on their limited experience how their patients will do following surgery we have developed a simple office based algorithm to predict weight loss following this procedure. method: patients met the criteria for this study. these patients underwent surgery at a single institution from june to december . non-linear regression analysis was performed to interpolate weight loss at one year. a multilinear regression was run to determine the significant variables. a model was then constructed to predict weight loss after single anastomosis duodenal switch. results: bmi, htn, gender, and the interaction between htn and dm were found to affect weight loss. the model achieved a r value of . and the average error of prediction in the model was . %ewl. conclusion: today too many surgical practices offer procedures tailored to surgeon instead of the needs of the patient. using our models predicting postoperative weight loss can be a straightforward process using easily gathered data. all surgeons should be doing this currently in their own practice to allow patient to choose targeted healthcare interventions based on patient's personal goals. surg endosc ( ) introduction: there is a long-standing practice of testing anastamosis both in upper and lower gi surgery. post-operative leaks in bariatric surgery are an uncommon but serious compilation increasing morbidity and risk of mortality. the present study looks at the practice of performing an intra-operative leak test during roux-en-y gastric bypass (rygb) and sleeve gastrectomy (sg). methods and procedures: the study was divided in two independent phases of six months and months. data was collected from all patients undergoing sg, rygb or revision rygb within those two periods. to confirm the integrity of the staple line all patients underwent a methylene blue and air test intra-operatively. this was followed by a gastrograffin swallow the morning post procedure. results: total number of patients in the study was . there were four positive intraoperative tests. one patient was a primary rygb and three patients were undergoing revision rygb. all were reinforced and subsequent recovery and gastrograffin swallow showed no leak. one revision rygb had an undetected small bowel injury distal to jejuno-jejunostomy that was not identified on intraoperative or next day imaging. we used multivariate statistical analysis to study our population sample and classified the impact of each factor or their combination with the use of principal component analysis. we used systematic clustering to identify subpopulations that have significant differences in statistical distribution. result: the main determinant of total operative time was the surgeon and the level of his assistant. prior surgeries, bmi and smoking history had a statistically significant impact on the laparoscopic time (p value. ). removing the impact of various surgeons, we detected four clusters of patients based on more than patient characteristics. we noticed total or time had two different clusters: one with a standard-deviation of - min while the other had over min. conclusion: this study may have practical implications on improving scheduling. the different comorbidities of these bariatric patients helped to stratify patients into these main cluster groups. better predictability on length of operative procedure can lead to more efficient use of or time and staff, thus ultimately leading to savings for the hospital. in addition, we used automated noninvasive tracking methods to identify phases of bariatric procedures that will allow more accurate estimated or time to efficiently schedule cases. the smart or, which is equipped with multiple noninvasive sensors, allows for error free tracking and monitoring without human interference. objectives: successful outcomes after bariatric surgery (bs) require a comprehensive educational program (cep) focused on post-surgical dietary and lifestyle changes. at our institution, patients must comply with a -week life-after-surgery program prior to surgery. since many patients are not able to participate in-person, an online cep was created to improve accessibility. to evaluate comprehension, a -question test is administered at the last preoperative visit to participants of both classes. the primary objective of this study is to evaluate the effectiveness of online versus inperson cep in terms of comprehension and post-operative weight loss. methods: patients who underwent bs from august -may were retrospectively reviewed at a single institution. all patients who underwent the in-person or online cep, completed the -question test, and had post-operative follow-up for at least months were included. baseline demographic, operative, and weight data were obtained using the electronic medical record. background: body weight loss after bariatric surgery is affected by several factors. diabetes status or preoperative body mass index (bmi) would affect the body weight loss after surgery. age and sexuality may also be the predictor. furthermore, the malabsorptive procedure is considered more effective for body weight loss than the restrictive procedure alone. we investigated the contribution of preoperative background data and procedures to the body weight loss after surgery. methods: this was a multicenter, retrospective study to validate the efficacy of bariatric surgery for morbidly obese patients in japan. patients underwent sleeve gastrectomy (lsg) or lsg with duodenal-jejunal bypass (lsg/djb) in each institution from january to december , and whose bmi was kg/m or more at the first visit were included in this study. we investigated the percent excess body weight loss (%ewl) at months after surgery. univariate and multivariate analyses were done to evaluate the predictive factors of body weight loss. we defined that %ewl more than % as well response (wr background: despite its known safety and efficacy, bariatric surgery is an underutilized treatment for morbid obesity in the united states. objective: our goal was to identify factors associated with failing to proceed with surgery despite being considered an eligible candidate by a bariatric surgery program. methods: this is a retrospective study that includes all patients (n= ) who attended a bariatric surgery informational session (bis) at a single center academic institution in . eligible candidates were identified after clinical evaluation and multidisciplinary candidacy review (mcr). we compared patients who underwent surgery to those who did not (i.e. dropped out) by evaluating patient-specific, insurance-specific, and bariatric surgery program-specific variables. univariate analysis and multivariable regression were performed to identify risk factors associated with failing to undergo surgery among eligible candidates. introduction: the elderly are a special subset of the population due to their limited physiological reserve with aging. revisional bariatric surgery is becoming more common with increase in primary bariatric procedures. data on safety, weight loss, and metabolic effects of revisional bariatric surgery in elderly is limited. the aim of this study was to assess the safety and efficacy of revisional bariatric surgery in the elderly. methods: clinical data of all elderly patients ( years and above) who underwent elective revisional bariatric surgery at an academic institute between and were reviewed. demographic data, perioperative variables, and postoperative outcomes were studied. results: a total of patients were identified with a female predominance ( : ). mean age was ± . years. mean bmi at the time of revisional surgery was . ± . kg/m . the primary indication for revisional surgery included management of postoperative adverse events (n= , . %) and weight recidivism (n= , . %). in patients with postoperative complications, the most common indications for revisional surgery were dysphagia (n= , . %), marginal ulcer (n= , . %), gastric outlet obstruction (n= , . %), and fistula formation (n= , . %). the most common type of revisions included conversion of vertical banded gastroplasty to roux-en-y gastric bypass (rygb, n= ), revision of rygb (n= ), conversion of adjustable gastric banding to sleeve gastrectomy (sg, n= ), and sg to rygb (n= ). two out of seven ( . %) patients with -day postoperative readmissions had serious complications that required reoperation. one of them underwent small bowel resection for ischemia and the other had thoracotomy for hemothorax evacuation developing secondary to a gastropleural fistula. while there was no mortality over the first days postoperatively, two patients died months after surgery due to infectious complications. in the median follow-up time of (interquartile range, - ) months, mean weight and bmi changes of − . kg and − . kg/m were observed. twenty-three ( . %) patients had diabetes at time of revisional surgery. a mean reduction of . mg/dl in fasting blood glucose and . % in glycated hemoglobin were noted between baseline and last follow-up. conclusion: revisional bariatric surgery in elderly is associated with high complication rates. our data indicate that revisional bariatric surgery can potentially alleviate symptoms and resolve complications of primary bariatric surgery. elderly patients should have their risk stratified and weighed against the benefits of surgery. anne-marie carpenter, bs, alexander l ayzengart, md, mph; university of florida introduction: bariatric surgery is the most effective treatment for morbid obesity. of all available procedures, laparoscopic sleeve gastrectomy (lsg) is now the most popular worldwide. common complications of lsg include gastroesophageal reflux, stricture, and staple-line leak. although rare, portomesenteric venous thrombosis (pmvt) and liver retractor-induced injuries are increasingly reported. we present a case of isolated left portal vein thrombus after routine lsg that was likely caused by prolonged compression of left liver lobe by the nathanson retractor. case presentation: a -year-old female with a bmi of and biliary colic due to cholelithiasis underwent lsg with hiatal hernia repair and cholecystectomy. she tolerated the procedure without complication and was discharged home on the following day. on postoperative day , she presented to the emergency department with fever and epigastric pain. contrast ct revealed an isolated filling defect within the proximal left portal vein; abdominal doppler demonstrated an acute thrombus occluding the left portal vein with normal flow in the main and right portal veins. the patient was treated with a -month course of therapeutic anticoagulation with lovenox. a complete hematologic workup did not uncover any hypercoagulable conditions. the patient recovered well and remained asymptomatic at her follow-up visit weeks after operation. discussion: pmvt is a rare surgical complication with multifactorial etiology. in bariatric surgery, evidence suggests lsg elicits more frequent pmvt compared with roux-en-y gastric bypass. a systematic review cited the incidence rate of pmvt as . - % after lsg. the mechanisms are thought to be due to pneumoperitoneum, procoagulant obese state, manipulation of portomesenteric venous system during division of the gastrocolic ligament, and postoperative dehydration. liver retraction is paramount during laparoscopic bariatric surgery to provide adequate visualization of the upper stomach and diaphragmatic hiatus. most methods of liver retraction produce significant pressure on the liver parenchyma by compressing it against the diaphragm. three types of liver injury have been documented in literature: minor congestion, traumatic parenchymal rupture, and delayed liver necrosis. uniquely, we propose an additional type of injury-left portal vein thrombosis due to compression of left liver lobe with the nathanson retractor. conclusion: the case described herein represents the first documented report of isolated left portal vein thrombosis after lsg. this is a unique presentation of retraction-related liver injury causing pmvt by mechanical compression of liver parenchyma. as surgical procedures increase in duration, intermittent release of liver retraction should be performed at regular intervals. introduction: up to % of patients experience internal hernia (ih) after laparoscopic roux-en-y gastric bypass (rygb). studies have shown that antecolic roux limb orientation, and closure of the mesenteric defect reduce, but do not eliminate, the incidence of ih. we hypothesize that despite operative differences, ih occur more frequently in patients who experience significant weight loss. this study aims to determine whether those patients who present with ih following rygb experience greater than % excess body weight loss (ebwl). methods: a retrospective chart of all patients who underwent ih repair following rygb at our institution between sept and sept was performed. all applicable cpt codes to encompass ih repair were reviewed (n= ). patients with ih repair after rygb were identified. results: of the patients, were female. the mean pre-rygb weight was lbs (sd± . ), bmi . kg/m (sd± . ). all procedures but one were performed in an antecolic configuration; the other retrocolic-antegastric. fifteen cases were laparoscopic and two were open; nine had the jejunal mesenteric defect closed, eight did not. the average weight loss from the time of rygbp to ih presentation was . lbs (sd± . ) and %ebwl from rygb to the nadir weight was % (sd± ). when evaluated by t-test, there was no statistical difference in bmi at the time of program initiation, rygb, or ih presentation, as well as number of pounds lost, %ebwl, or time to ih presentation, when comparing patients for whom the mesenteric defect was closed or not. average time from rygb to ih presentation was . years (range - days) . conclusion: in our limited cohort of patients who have presented with internal hernia after rygb, there was an average of % ebwl. this is greater than the average expected %ebwl at our institution and others, suggesting that ih may occur in patients with greater weight loss at a higher frequency. mesenteric defect closure did not appear to have any influence in this limited cohort, suggesting that weight loss is a stronger factor in ih development. we plan a more extensive evaluation in a larger cohort of patients to determine if greater %ebwl is a predictor of ih formation in patients undergoing rygb. introduction: introduction of enhanced recovery after surgery (eras) pathways has led to early recovery and shorter hospital stay after laparoscopic roux-en-y gastric bypass (lrygb) and laparoscopic sleeve gastrectomy (lsg). this study aims to assess feasibility and outcomes of postoperative day (pod) discharge after lrygb and lsg from a national database. methods: patients who underwent elective primary lrygb and lsg and were discharged on pod and were extracted from metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip) dataset. a : propensity score matching was performed between cases with pod vs pod discharge, and the -day outcomes of the propensity-matched cohorts were compared. high risk patients were excluded from the analysis. purpose: the aim of this study was to evaluate a large volume, multi surgeon bariatric surgery center producing the largest sample size to date proving efficacy (% weight loss) and safety of sleeve gastrectomy following band removal in one or two step procedures. methods: all patients undergoing conversion of lagb to lrygb ( ) and lsg ( ) regardless of one step vs two step conversion from january to january were included. a retrospective analysis of our prospectively maintained database was performed to compare outcomes in patients undergoing conversion to lrybg vs lsg after lagb to identify the outcomes. introduction: the purpose of the study was to describe the use of intraoperative indocyanine green (icg) fluorescence angiography to identify the blood supply patterns of the stomach and gastroesophageal junction (gej). we hypothesized that identifying these vascular patterns may help modifying the surgical technique to prevent ischemia-related postoperative leaks. methods: patients underwent laparoscopic sg and were examined intraoperatively with icg fluorescence angiography at an academic center from january to september . prior to the construction of the sg, ml of icg was injected intravenously and pinpoint® technology was used to identify the blood supply of the stomach. afterwards, the sg was created with attention to preserving the identified blood supply to the gej and gastric tube. finally, ml of icg were injected and pinpoint® technology was used again to ensure that all the pertinent blood vessels were preserved. results: patients successfully underwent the procedure with no complications. the following blood supply patterns to the gej were found: the incidence of overall accessory blood supply to the right-side dominant pattern was more common than expected. in about half of the cases where an accessory vessel was found in the gastrohepatic ligament, the blood flow was toward the stomach (and not the liver). furthermore, the incidence of accessory blood supply from the left side was found in % of the cases. % of patients had both the left side accessory and accessory gastric artery pattern. in these particular patients, if a concurrent hiatal hernia repair is performed, these accessory blood supplies are at risk of being injured if care is not taken to preserve them, rendering the gej relatively ischemic. conclusion: icg fluorescence angiography allows determining the major blood supply to the proximal stomach prior to any dissection during sleeve gastrectomy so that an effort can be made to avoid unnecessary injury to these vessels. background: morbid obesity, a common medical concern with significant health risks, has a prevalence of . % among u.s. adults. bariatric surgery provides effective weight loss for morbidly obese patients with improvement in their comorbid conditions. traditionally, routine intraoperative drain placement (idp) and postoperative esophagram (ugis) were thought to identify early postoperative complications. recently, these interventions have been scrutinized for their effectiveness. we hypothesized that idp and postoperative ugis do not alter outcomes in bariatric surgery and only increase hospital length of stay (los). methods: two cohorts, each consisting of patients from either or were analyzed from our institution. in the cohort, all patients had idp and an ugis on postoperative day , prior to starting a clear liquid diet. in the cohort, no patients had idp or ugis, but instead were started on a clear liquid diet on postoperative day , in the absence of vomiting. all patients in each cohort underwent either a laparoscopic sleeve gastrectomy or a roux-en-y gastric bypass. a retrospective study was performed to analyze whether there was a significant difference in postoperative complications, length of stay, and operating room time between these two cohorts. those who underwent t dm remission were less likely to be vdd at all time points. the rates of vdd appear to be slightly higher in rygb at each time points. the rates of macrocytic anemia, microcytic anemia and hypoalbuminemia were low and varied depending on surgical procedure, with no relevant increase following surgery (see figure ). conclusions: vitamin d deficiency is prevalent among diabetic patients with obesity presenting for bariatric surgery. the postoperative management was successful in addressing vdd following surgery; those who experienced t dm remission after surgery were less likely to be vdd. further prospective studies are needed to explore this relationship. surg endosc ( ) :s -s introduction: it is well known that morbid obesity is strongly associated with high blood pressure. cardiovascular risk reduction is a well studied and described result of bariatric surgery. the objective of this study is to quantify hypertension resolution in patients who underwent bariatric surgery at our institution. methods: we retrospectively reviewed all the patients who underwent either laparoscopic sleeve gastrectomy (lsg) or laparoscopic roux en y gastric bypass (lrygb) at our institution between and . we selected those patients who were on antihypertensive medical treatment and had a -month follow-up. hypertension resolution was defined as the interruption of any blood pressure medications within the follow-up period. we compared the patients who had resolution of hypertension (group ) with patients who did not (group ), based on demographics, comorbidities, and outcomes. chi-square and student t-test were used for categorical and continuous variables respectively. results: out of patients, ( . %) patients met the inclusion criteria, out of which, ( . %) had a complete resolution of hypertension within months. the patient population included in group was predominantly female n= patients ( . %), diabetic (n= , %), with a mean bmi of . ± . kg/m , a mean age of . ± . years, and a preoperative systolic blood pressure mean of ± . mmhg. the most common procedure performed was lsg with n= ( %). comparison between group and group based on age, gender, bmi, and diabetes showed no statistically significant difference. estimated bmi loss % at months, type of procedure and % ebmil showed no statistically significant difference between the groups. conclusions: rapid weight loss is associated with a drastic reduction of blood pressure. besides weight loss, we did not identify a clear correlation between risk factors when we compared patients who had resolution of hypertension with patients without resolution. further prospective studies should be done for better understand these findings. the mount sinai hospital, university of chicago introduction: for many patients, hiv has transformed from a life-threatening illness into a manageable chronic disease. reflecting trends in the general population, obesity is increasingly prevalent among hiv-positive patients. surgical intervention has shown the greatest effectiveness in treating obesity. it is unknown, however, whether physician attitudes reflect the changing trends in obesity care for hiv-positive patients. methods and procedures: medical students from the first, second, and fourth years of training were invited to participate in an irb-approved survey, handed out during didactic sessions, which was designed to assess their knowledge and attitudes regarding bariatric surgery in hiv-positive patients. self-reported demographic information of respondents was also collected. the outcome of interest was the proportion of correct responses. univariate and multivariate regression analyses were performed. results: surveys were completed by medical students. demographic covariates included the following: age, sex, race, bmi, and year of training. age, sex, race, and bmi were not statistically significant in the multivariate model. however, in both univariate and multivariate models, each additional year of training was associated with a significant increase in the proportion of correct responses (multivariate model beta coefficient= . , p. ). conclusions: obese and hiv-positive patients suffer from well-documented stigma in health care. these findings suggest that medical training corrects common misperceptions of obese and hivpositive patients, and may lead to a better understanding of the appropriateness of bariatric surgery for hiv patients. whether these attitudes are predictive of referral practices remains to be seen. introduction: obesity is a common problem worldwide with numerous associated comorbidities and is associated with an increased risk of developing some cancers. despite bariatric surgery being associated with a risk reduction for cancer development, some will develop cancer after surgery and little is known about complications which might arise during multimodality cancer treatment. here we report the case of a year-old female who developed an unusual giant marginal ulcer (mu) post laparoscopic roux-en-y-gastric bypass (lrygb) while receiving systemic chemotherapy for an early stage breast cancer. case report: in summary, a year-old female with a preoperative bmi of kg/m had an uncomplicated lrygb one year prior to her presentation. she was a non-smoker, was abstinent of alcohol and did not use nsaids, steroids or other ulcerogenic medications. eight months post procedure with a bmi of . kg/m she was diagnosed and treated with bcs plus slnb for a pt n m er/pr +ve her −ve breast cancer. one week following her third cycle of docetaxel and cyclophosphamide, she presented with two days of melena, small volume hematemesis and abdominal discomfort. the patient was resuscitated with prbc, started on a ppi infusion and had free air ruled out on a cxr. upper endoscopy was complete showing a giant mu at the gastrojejunal anastomosis, biopsies ruled out malignancy and h. pylori. subsequent ct abdomen/pelvis identified contrast extravasation from the anastomosis confirming a free perforation. broad spectrum antibiotics were started and a diagnostic laparoscopy complete. a graham patch repair utilizing omentum and abdominal washout were complete with placement of surgical drains. the patient was supported with parenteral nutrition while npo. diet was advanced after an upper gi series on post operative day showed no ongoing leak. the patient was discharged on post operative day , recovered and although further chemotherapy was discontinued she completed whole breast radiotherapy. conclusion: leaks and hemorrhage are early postoperative complications that are not seen intraoperatively in our experience. furthermore, endoscopy significantly increases mean operative time. routine use should be left to the discretion of the surgeon but should not be considered an essential step of the sleeve gastrectomy. the objective of the study: surgical site infection (ssi) following bariatric surgery contributes to patient morbidity and additional use of health care resources. we investigated whether a ssi quality control initiative in the form of a refined preoperativeantimicrobial protocol affected the rate of ssi following laparoscopic roux-en-y gastric bypass (lrygb). we reviewed all lrygb procedures performed between june and december at a single bariatric surgery centre of excellence. two preoperative antimicrobial protocols were compared. patients undergoing surgery prior to february received g of cefazolin whereas patients undergoing surgery after february , received a new antimicrobial protocol consisting of g cefazolin, mg metronidazole and ml oralchlorhexidine rinse. the primary outcome was day ssi including superficial ssi, deep incisional ssi and organ/space infection as defined by the centre for disease control. clinic charts and provincial electronic medical records were reviewed for emergency department visits, microbiology investigations and physician dictations diagnosing ssi. outcomes were assessed using a students t-test. results: two hundred seventy six patients underwent lrygb of which received the refined antimicrobial protocol and received cefazolin. the refined antimicrobial protocol significantly decreased the rate of deep incisional ssi compared to cefazolin (n= , . % vs n= , . %; p\ . ). the refined antimicrobial protocol resulted in an insignificant overall reduction in the rate of superficial ssi (n= , . % vs n= , . %; p[ . ) and organ/space infection (n= , . % vs n= , . %; p[ . ) respectively. conclusions: a preoperative antimicrobial protocol using cefazolin, metronidazole and chlorhexidine oral rinse appears to reduce the rate of ssi following lrygb. this protocol may be most effective to prevent deep incisional ssi. additional patient cases or alternative study design including a randomized control trial is required to better understand the efficacy of this protocol. background: for many years, the roux-en-y gastric bypass (rygb) was considered a good balance of complications and weight loss. according to a several short-term studies single anastomosis duodenal switch or stomach intestinal pylorus sparing surgery (sips) offers similar to weight loss to rygb with fewer complications and better diabetes resolution. however, no one has substantiated complication and nutritional differences between these two procedures over the midterm. this paper seeks to substantiate previous studies and compare complication and nutritional outcomes between rygb and sips. methods: a retrospective analysis of patients who either had sips or rygb from to . complications were gathered for each patient. nutritional outcomes were measured for each group at , , and years. regression analysis was applied to interpolate each patient's weight at , , , , , , and months. these were then compared with t tests, fisher exact tests, and chi squared tests. results: rygb and sips have statistically similar weight loss at , , , , and months. they statistically differ at and months. at months, there is a trend for weight loss difference. there were only statistical differences in nutritional outcomes between the two procedures with calcium at and years and vitamin d at year. there were statistically significantly more long term major complications, minor complications, reoperations, ulcers, small bowel obstructions, nausea, and vomiting with the rygb than sips. conclusion: with comparable weight loss and nutritional outcomes, sips has fewer short and long-term complications than rygb and better type diabetes resolution rates. introduction: the purpose of this study is to determine the risk factors that contributed to increased postoperative complications, as noted in prior studies within the publicly funded insurance population undergoing bariatric surgery. methods and procedures: data was collected via a retrospective review of the medical records of patients who underwent laparoscopic roux en y gastric bypass or laparoscopic sleeve gastrectomy from to at a single institution. for each patient, data was collected in the following categories: baseline demographics, insurance status, medical comorbidities, immediate complications, re-admissions and associated complications, and follow up out to years. results: a total of patient charts were reviewed, patients were categorized as private insurance and patients were categorized as public insurance. there was no statistically significant difference in mean patient age (private . years vs public years), sex (male:female %: % for both groups), or bmi ( vs ). there was a statistical significance in relationship status in the categories of single ( % vs %), married ( % vs %) or living with a partner ( % vs %), as well as employment status ( % vs %). when comparing comorbid conditions preoperatively there was no difference except for diabetes which was less common in the private insurance group % vs %. readmission rates for complications were significantly different as well at % vs % with public insurance patients having increased complication rates and readmissions. there was no difference in follow up percentages at each time point for the two groups. interestingly postoperative bmi was significantly different in the two groups until year out ( vs ) when the difference disappears. conclusions: our current data set confirms prior research that documented higher complication rates in public insurance patient populations without differences in long term results in regards to weight loss. it also shows that the public insurance group is possibly at higher risk for complications and readmissions postoperatively due to the lack of social support at home given that a much higher percentage of them are single or divorced, and lack employment. it is likely that this lack of support at home prompts more frequent readmissions and associated complications. introduction: gastric bypass has been an acceptable treatment for the morbidly obese patient, with proven efficacy on weight loss and remission of co morbidities, especially diabetes (t dm). laparoscopic sleeve gastrectomy (lsg) is gaining momentum as an alternative procedure for the morbidly obese patient. the aim of this study is to assess the resolution of t dm by examining hba c, bmi, fat %, and % excess weight loss in t dm patients in our lsg patients. methods: we performed a retrospective chart review of t dm patients before and after lsg, analyzing hga c, bmi, % weight loss, fat %, and diabetic medications. data was analyzed by using spss version . paired t-test was applied to see the significance of bmi, weight, fat % and hba c before and after the procedure. introduction: gastroesophageal reflux disease (gerd) is a known risk following laparoscopic sleeve gastrectomy (lsg), with up to % of patients affected by the disease postoperatively. of these patients, an unknown number progress to medically refractory gerd. due to their postsurgical anatomy, these patients have limited options for intervention. while endoluminal therapies are available, surgical revision to roux-en-y gastric bypass (lrygb) has become an accepted revisional treatment. despite this therapeutic option, many payors deny coverage for this treatment. in this study, we report outcomes of revision of lsg to lrygb and difficulties in obtaining insurance approval for the operation. methods: we conducted a retrospective review of all patients who underwent a revisional bariatric operation at a single institution between january and august . we analyzed all patients who underwent conversion of lsg to lrygb. we collected data on -day mortality and morbidities, pre-and postoperative antacid use, and the insurance approval process. results: within the study period, we identified patients undergoing revisional bariatric surgery. seventeen patients had undergone conversion of lsg to lrygb. all of these patients underwent revision due gerd refractory to maximal medical therapy. the average body mass index was kg/m , and our average operative time was minutes. one patient required laparoscopic cholecystectomy within days due to acute cholecystitis, and another patient required reoperation for control of staple line bleeding. there were otherwise no -day morbidities or readmissions. fifty nine percent stopped all antacid medication by six months, and % stopped by months. of the % percent of patient still on proton pump inhibitor therapy, none of those patients complained of reflux symptoms. of non-medicare patients, % were initially denied insurance coverage for revision. only one plan accounted for all initial approvals. twenty five percent of denied patients eventually paid out of pocket, and the remaining % ultimately secured coverage after an appeal process. with no significant differences in mortality or hospital stay. significantly shorter operative times were observed in the adolescent group ( . ± vs . ± , p. ). in univariate analysis blood transfusions and vte rates were significantly lower in the adolescent group but there was no difference after risk-adjusted logistic regression analysis. analysis of readmission data showed lower rates in adolescents compared to young adults ( . % vs . % p= . ). however, adolescents are more frequently readmitted secondary to gallstone disease ( . % vs . %, p. ). the most common reason for readmissions in both groups was nausea and vomiting with fluid/electrolyte depletion, followed by abdominal pain. conclusion: adolescent bariatric surgery is feasible and safe, with outcomes similar to that of young adults. lsg is currently the most common bariatric procedure performed in adolescents which is reasonable given the relative lack of co-morbid conditions within this group. nausea and vomiting are the most common reason of readmission in both groups, but gallstone disease is significantly higher in adolescents, suggesting that this population should be carefully screened for gallbladder disease preoperatively. further studies regarding long-term results are needed to elucidate long-term outcomes, such as the durability of comorbidity resolutions in adolescent patients. introduction: revision bariatric surgery is always considered to be associated with higher complication rates. there is currently controversy in the literature regarding one stage and two stage revisions. methods: the present study is ongoing longitudinal prospective analysis of data of revision surgery in a single unit. the revision surgery was offered after initial failed or complicated gastric band, sleeve gastrectomy and roux-en-y gastric bypass (rygb). results: there were forty-two individuals who had revision bariatric surgery. the age of the cohort of patients ranged from twenty-six to seventy-five years. thirty-three were females and nine males. all patients who were hypertensive or diabetic at the time of their initial bariatric operation had a relapse of their co-morbidity prior to their revision surgery. the two stage revisions patients had their band removed at another facility, had a compilation from the band itself or did not wish for revision surgery initially. of the two failed bypasses one had a large pouch and very short limbs. the other had a gastro-gastric fistula and ultra short limbs. there were no deaths in this study. one patient who underwent one stage revision of a gastric band to bypass had an iatrogenic small bowel injury that required a second operation. amelioration of diabetes and hypertension was seen in all who had relapsed. weight loss was good in all patients except for the those undergoing revision from short limbed to long limbed bypass. conclusion: there is enough evidence that revision surgery is feasible, and can ameliorate metabolic co-morbidities after failed band and sleeve. two staged surgery is not necessarily safer compared to one stage revision. in the present study an inadvertent iatrogenic injury occurred in one stage revision group but is not true reflection of increased complications. the association between preoperative endoscopic esophagitis and post operative gerd in sleeve gastrectomy patients samer elkassem, md; medicine hat regional hospital introduction: gerd is a common complication after sleeve gastrectomy (sg). the purpose of this study is assess the relationship between pre-operative findings of endoscopic esophagitis and postoperative gerd in sg patients. the hypothesis of this study is that patients with pre-op esophagitis are more likely to have gerd post-op than patients with no esophagitis pre-op. methods: a retrospective review of sg patients who had pre-operative endoscopy and followed prospectively for at least one year was preformed. patients were divided into two groups based on pre-op endoscopic findings: those with no findings of esophagitis (ne), and those with endoscopic esophagitis, including barretts (ee). patients were followed for at least one year, and assessed for usage of a proton pump inhibitor (ppi) usage. the two groups were compared using both student t-test and chi square test. results: a total of patients did not have any findings of esophagitis on pre-op endoscopy (ne group), and patients had findings of endoscopic esophigitis (ee). there was no difference in preoperative demographics and post-op weight loss at one year (table i) . follow-up ranged from one to years post-op. the dependency on ppi usage and de novo reflux are shown in table ii . introduction: patients with "super-super obesity", defined as a bmi≥ , are at higher risk of weight-related health problems and might benefit more than others from metabolic and bariatric surgery. however, these benefits need to be weighed against the potential for increased operative and perioperative risks. accurate data regarding these patients is critical to guide procedure choice and informed, shared decision-making. the metabolic and bariatric surgery accreditation and quality improvement program (mbsa-qip) is a national accreditation and quality improvement program, which captures clinically-rich specialty-specific data for the majority of all bariatric operations in the united states. this is the first analysis of the mbsaqip participant use file (puf) focusing on this at-risk subpopulation. introduction: sleeve gastrectomy represents one of the most common surgical procedure used in bariatric surgery. the most feared complication following laparoscopic sleeve gastrectomy is the leak that occurs at the staple line. one method to reduce the risk of leak is the use of reinforcement material at the suture line. in this study, the efficacy of sutures and fibrin glue in the prevention of staple leak has been compared retrospectively. materials and methods: a total of patients undergoing lsg between october and august at the medical faculty of firat university were retrospectively assessed using the hospital database system records. results: there were males ( %) and ( %) females, with a mean age of years (range: - y), and body mass index of kg/m . while no reinforcement material was used in patients ( %) at the suture line, reinforcement sutures or fibrin glue were used in ( %) and ( %) patients, respectively. postoperative leak occurred in patients ( . %), and ( . %) of these had no use of reinforcment material for leak prevention, while additional sutures or fibrin glue had been used in patients, one in each group ( . %). one patient died due to leak and the consequent development of sepsis ( . %). discussion: lsg is increasingly more frequently used in bariatric surgery practice. however, an increase also occurs in the rate of complications. a discrepancy exists in the published literature regarding the benefit of reinforcment the suture line on the risk of leak risk. in our patient series, patients without the use of additional material in the staple line had a significantly increased risk of leak. conclusion: despite some controversy, strong evidence exists on the effectiveness of fibrin glue in the prevention of leaks in patients undergoing laparoscopic sleeve gastrectomy. background: laparoscopic bariatric surgery has been performed safely since . in a persistent search for fewer and smaller scars, single port and acuscopic surgery or even notes have been implemented. the goal of this study is to analyze the safety and feasibility of using a low cost incisionless liver retraction compared to a standard laparoscopic retractor for sleeve gastrectomy. methods and procedures: candidates for sleeve gastrectomy that fulfilled nih criteria for bariatric surgery were selected. those younger than and/or with prior upper-left quadrant surgery were excluded. all patients signed written consent. patients were randomized : to either a standard port technique with a fan-type liver retractor through a mm port (group a); or a port technique with the liver retracted by a polypropylene suture passed through the right crura and retrieved at the epigastrium with the use of a fascia closure needle (group b). all surgeries were performed by the same surgeon. surgery length from insertion of first port to withdrawal of the last was the primary endpoint. anthropometric data, % of pre-surgical total weight loss (%ptwl), visualization of the surgical field, complications inherent to liver retraction and postoperative morbidity were recorded. background: comprehensive web and hospital based preparative patient education allow the morbidly obese patients to understand weight loss surgery, its benefits, the necessity of follow up and the risk of weight regain. while the inhouse seminars provide a face-to-face interaction with the bariatric program staff, the online seminars are easily accessible and more cost effective. the primary objective of this study is to compare demographics and weight loss surgery outcomes between patients who participated in the online vs in-house preparative seminars. methods: after obtaining institutional review board approval, a retrospective chart review was performed involving patients who underwent bariatric surgery between january and december at a tertiary care center. the patients were divided into two groups based on their choice of educational seminar, online or in-house, prior to their initial consult with a surgeon. data was collected on age, type of insurance, length of stay (los), longest follow up and change in bmi to assess weight loss. results: one hundred and eighteen patients were included in this study. eighty patients attended in-house seminar while completed online seminar. the various types of surgery (laparoscopic gastric bypass, sleeve gastrectomy, and band) were similarly represented between the two groups. there was no difference in the type of insurance policy between the groups. patients who elected to take the in-house seminar were on average years older than those who chose the online course, which was statistically significant (p. ). there were no differences in los, longest follow up after surgery, and weight loss at months between the groups. conclusions: based on mbsaqip registry data, patients age or over did not have higher odds of a -day readmission compared to younger patients after lsg or lrygb. rates of -day readmission, reoperation, and death were similar, but rates of complications (e.g. pneumonias, unplanned intubations) were higher in the older group. bariatric surgery in the elderly should therefore be performed only after careful and patient-centered selection processes. introduction: revisional bariatric surgery has become more common in recent years. it is to address short and long-term complications of primary bariatric surgery as well as the issue of weight regain. the aim of this study was to retrospectively analyze the indications for reoperation and short-term outcomes in our institution. methods and procedures: between and , patients who underwent bariatric surgery in our center were included in a prospectively collected database. demographic data, primary and revisional bariatric procedures, reasons for revisions and outcomes were recorded and reviewed retrospectively. results: a total of patients underwent bariatric surgery at our institution and % of these (n= ) were revisional bariatric surgery. we identified groups of patients according to their primary procedures: adjustable gastric band (agb), roux-en-y gastric bypass (rygbp), vertical band gastroplasty (vbg), and sleeve gastrectomy (sg). of the patients, ( %) had abg as primary procedure. of those, % had their band removed due to food intolerance and severe dysphagia and % had a conversion to either rygbp or sleeve gastrectomy (sg) due to weight recidivism. in the rygpbp group (n= ), % of the patients presented with late complications. of these, % had an acute presentation (small bowel obstruction, internal hernia, or perforated marginal ulcer) requiring emergency surgery. only % patients needed gastric bypass takedown due to severe hypoglycemia. weight recidivism was noted in % of the patients that necessitated either revising the anastomosis, trimming of the gastric pouch or gastrogastric fistula takedown. in the vbg group (n= ), % of the patients experienced weight recidivism that required conversion to rygb and % of the patients required the vbg to be taken down due to obstructive symptoms. in the sg group (n= ), % of the patients experienced early complications needing a second procedure. weight recidivism was found as the most common reason for conversion ( %) to rygbp. twenty nine percent of the patients in this group underwent conversion to a rygbp due to severe de novo gerd. introduction: our aim was to systematically review the literature to compare weight loss outcomes and safety of secondary surgery after sleeve gastrectomy (sg), particularly between roux-en-y gastric bypass (rygb) and biliopancreatic diversion with duodenal switch (bpd-ds). sg was originally developed as the first part of a two-stage procedure for bpd-ds. however, it is now the most common standalone bariatric surgery performed in the united states. the majority of sg are done as the sole bariatric operation but in %, a second operation is necessary, due to insufficient weight loss, weight regain or reflux. the most common second-stage operations are rygb at % and bpd-ds at %. there are a few small case series comparing rygb to bpd-ds as a secondary surgery after sg. these studies suggest that after failed sg, bpd-ds results in greater weight loss but higher early complication rates than rygb. we had one mortality, related in part to supra-therapeutic anticoagulation perioperatively. one patient underwent successful heart transplantation and additional patients were reactivated on the transplant list. conclusion: laparoscopic sleeve gastrectomy is effective in advanced heart failure patients for meaningful weight loss, reactivation to the transplant wait list, and ultimately cardiac transplantation. however, this complex population carries a high perioperative risk and close multidisciplinary collaboration is required. more data is needed to best optimize perioperative management of these patients. the introduction: bariatric surgery is a highly effective treatment for severe obesity. while its effect on improvement of the metabolic syndrome is well described, its effect on intrinsic bone fragility and fracture propagation is unclear. therefore, the aims of this systematic review of the literature were to examine ( ) the incidence of fracture following bariatric surgery, ( ) the association of fracture with the specific bariatric surgical procedure ( ) conclusion: it appears that the overall risk of sustaining a fracture of any type after undergoing bariatric surgery is approximately percent after an average follow up of . years. the greatest risk of fractures is associated with the bpd, with the rygb being the most favorable. fractures following bariatric surgeries tend to follow osteoporotic and fragility patterns. post-operative supplementation of vitamin d, calcium and weight bearing exercises need to be optimized, and long term follow-up studies will be needed to confirm that these interventions will indeed reduce fracture risk following bariatric surgery. background: the effect of sleeve gastrectomy on gastroesophageal reflux (gerd) remains controversial. it is currently common practice to perform a hiatal hernia repair (hhr) at the time of the sleeve gastrectomy, however, there are few data on the outcomes of gerd symptoms in these patients. the aim of this study was to evaluate the effect of performing an esophagopexy hiatal hernia repair on gerd symptoms in morbidly obese patients undergoing robotic sleeve gastrectomy (rsg). methods: a single institution, single surgeon, prospectively maintained database was used to identify patients who underwent rsg and concomitant esophagopexy for hiatal hernia repair from november to july . patient characteristics, operative details and postoperative outcomes were analyzed. primary endpoint was subjective gerd symptoms and recurrence of hiatal hernia. results: thirty-seven patients were identified meeting the inclusion criteria (rsg+hhr+esophagopexy) with a mean follow-up of . over the past years there have been several bariatric surgeries cancelled secondarily to abnormal pre-operative test results within eastern health. these surgeries are often cancelled the day before their scheduled surgery, which does not provide sufficient time to book other patients. the end result is that the or gets underutilized and the bariatric surgery waitlist grows. prior to any major surgery patients are often subjected to a routine screening process, which includes a history and physical along with diagnostic screening tests and screening blood work. a preliminary analysis was done of the first patients through the bariatric surgery program at eastern health assessing the coagulation study results and outcomes. analysis showed that out of the first patients % were found to have a history of bleeding, % were using anticoagulants preoperatively, another % were noted to have a family history of bleeding. in the preoperative blood work that was done, % were found to have an elevated ptt/ inr for which hematology ended up being consulted in % of the patients. overall this did not change the preoperative management of these patients and they went on to have their surgery. intraoperatively patient was noted to have excessive bleeding and this was found not be associated with any preoperative elevation in their coagulation studies or family history of bleeding disorders. post operatively there was bleeding in patient which required transfusion, however this too was found not to be associated with any preoperative elevation in their coagulation studies or family history of bleeding disorders. overall this initial analysis showed no difference in operative management or delay in surgery secondarily to abnormal preoperative assessment findings. further analysis of a larger population of the bariatric surgery program patients is needed in order to determine whether any changes should be made to the preoperative assessment protocol. introduction: patients undergoing bariatric surgery frequently present with various obesity-related psychiatric comorbidities, including depression. furthermore, previous literature has demonstrated a positive association between depression and cardiovascular disease, and obesity serves as an independent risk factor for cardiovascular disease. however, the relationship between preoperative depression and cardio-metabolic risk factors following bariatric surgery remains unknown. methods and procedures: this retrospective analysis utilized data obtained from patients (n= , ) who underwent bariatric surgery at a single academic medical center in california. patients underwent either laparoscopic roux-en-y gastric bypass or sleeve gastrectomy. using medical record data, patients were preoperatively categorized as follows: not depressed, history of depression but not currently on anti-depressive medication, and history of depression and presently taking anti-depressive medication. patient demographic characteristics were obtained preoperatively. clinical and biochemical risk factors for cardiovascular disease were evaluated preoperatively and and months following bariatric surgery. anova, kruskal-wallis, and chisquare tests were applied where appropriate. results: in this sample, % of patients were not depressed, % had a history of depression but were not taking anti-depressive medication preoperatively, and % had a history of depression and were taking anti-depressive medication preoperatively. at baseline, depressive history was positively associated with female sex (p\. ), older age (p\. ), white race (p\. ), medicare insurance (p\. ), previous abdominal surgery (p\. ), length of stay (p\. ), requiring an inferior vena cava filter (p=. ), total cholesterol (p\. ), and triglycerides (p =. ). on average, patients with a history of depression taking anti-depressive medication weighed less than patients with a history of depression not on medication and patients without depression preoperatively (p=. ) and (p=. ) and (p=. ) months after surgery. after six months of follow-up, preoperative depressive history was positively associated with total cholesterol (p=. ), triglycerides (p\. ), hba c (p=. ), and fasting serum concentrations of insulin (p=. ). after months of follow-up, preoperative depressive history was positively associated with higher levels of total cholesterol (p=. ), ldl cholesterol (p=. ), and triglycerides (p= . ). conclusion: a history of depression prior to surgery was associated with higher levels of total cholesterol and triglycerides at baseline and and months postoperatively. after months, preoperative depressive history was also associated with higher levels of ldl cholesterol. this study suggests that, on average, bariatric patients with comorbid depression have worse lipid profiles prior to-and up to one year after-bariatric surgery relative to counterparts without depression. yen-yi juo, md, mph, yas sanaiha, md, erik dutson, md, yijun chen, md; ucla introduction: anastomotic leak is one of the most morbid complications of roux-en-y gastric bypass (rygb), yet its risk factors are ill-defined due to the rarity of the complication. we aim to identify both patient-and operative-level risk factors for anastomotic leak after rygb using a national clinical database. methods: a retrospective cohort study was performed using the metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip) database. all adult patients who underwent laparoscopic or open rygb were included. multivariate logistic regression models were used to identify patient-and operative-level variables associated with development of anastomotic leakage. clinically relevant anastomotic leakage is defined as those that required readmission, intervention, or reoperation. introduction: hyperammonemia secondary to ornithine transcarbamylase (otc) deficiency is a rare and potentially lethal disorder. the prevalence of otc deficiency is reported to be : , to : , in the general population. otc deficiency has been reported in patients presenting with neurological symptoms after roux-en-y gastric bypass (rygb), and less than cases have been reported in the literature. the aims of this study are to examine the apparent incidence of this uncommon disorder in patients after bariatric surgery and to examine potential predictors of mortality. methods and procedures: this is a single center, retrospective study in a large, urban teaching hospital of postbariatric surgery patients who developed hyperammonemia from january to august . elevated plasma ammonia with an elevated urinary orotic acid level is accepted as consistent with a diagnosis of otc deficiency. all patients in our program are instructed on a post-operative diet containing grams/day of protein. descriptive and correlative statistics are calculated for all variables. results: between january and august , bariatric surgical procedures were performed at this single medical center. seven women with neurological symptoms had plasma ammonia levels above the upper limit of normal range. their average bmi is kg/m . two patients underwent vertical sleeve gastrectomy (vsg), underwent vsg with duodenal switch, and underwent rygb. all patients were hospitalized. the mean peak plasma ammonia level is umol/l (range: - ). the mean urinary orotic acid level is . mmol/mol creatinine (range: . - . ). there were patients with no orotic acid level checked, secondary to demise. no patient had clinical features or findings of progressive hepatic failure. there are four mortalities ( . %). serum folate and peak lactic acid levels are predictors of mortality with p-values of . and . respectively. the apparent incidence of otc deficiency is : in post-operative patients. conclusions: in our post-operative population, hyperammonemia results in a high mortality. its apparent incidence, secondary to otc deficiency, amongst bariatric surgery patients is higher than that reported in the general population. since otc deficiency is identified after multiple bariatric surgical procedures, further investigation will be important to examine potential mechanisms for its development which may include a genetic predisposition (possibly triggered by nutritional deficiencies), upper gut bacterial overgrowth (supported by elevated serum folate levels), or preexisting, subclinical hepatic dysfunction. introduction: the use of closed suction drains is associated with poor outcomes in many anastomotic operations and routine use is not recommended. in this context, intraoperative drain placement for primary bariatric surgery remains controversial. recent studies demonstrate that drains confer no benefit to patients; however, data are limited to descriptive single center experiences with low sample size. in order to characterize this practice gap, and implement evidence based recommendations, we sought to evaluate the use of closed suction drain and outcomes following primary bariatric cases using the mbsaqip registry. methods: we used data from the metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip) public use file for patients who underwent a non-revisional laparoscopic roux-en-y gastric bypass (rygb), laparoscopic sleeve gastrectomy (lsg), or laparoscopic adjustable gastric banding (lagb). we excluded patients with asa status greater than or conversion to an open procedure. we analyzed demographics, preoperative comorbidities, procedure type for patients who did and did not undergo drain placement. adjusted rates of postoperative complications and mortality were then compared based on receipt of postoperative drain placement. results: of the , included patients who underwent laparoscopic bariatric surgery, , ( . %) underwent intraoperative drain placement. drains were more often placed in patients who underwent lrygb, were older, had higher preoperative bmi, had higher preoperative asa status, and had more comorbid conditions. after patient level risk adjustment, there was no difference in rates of leaks requiring intervention ( . % versus . %, p= . ) or mortality ( . % versus . %, p= . ) for patients with and without drains. in patients who underwent drain placement, there were higher rates of transfusion ( . % versus . %, p. ), reoperations for bleeding ( . % versus . %, p= . ), all reoperations ( . % versus . %, p. ), and surgical site infections (ssi) ( . % versus . %, p. ). conclusion: our analysis demonstrates that nearly one quarter of all laparoscopic bariatric surgery patients undergo drain placement. we found that drain placement is more common in preoperatively higher risk patients and following higher complexity procedures as suggested by associated increased rates of transfusion and reoperations for bleeding. we found no benefit to drain placement in terms of interventions for clinically significant leaks or mortality. finally, patients who underwent drain placement were more likely to develop ssi suggesting routine placement is not without risk. although further prospective studies are warranted, our analysis demonstrates that drains have the potential for harm with minimal protective benefit for patients after primary bariatric surgery. sleeve gastrectomy ( % n= ) and laparoscopic roux-en-y gastric bypass ( % n= ) were the two types of surgeries done in our population. the risk of developing atrial fibrillation was calculated preoperatively and found a -fold higher risk in females and -fold greater risk in males when compared with the ideal risk for each category. at months follow-up the preoperative risk was . ± . % with an absolute risk reduction of . % corresponding to a relative risk reduction of . % with males having a more significant change at months follow-up. these findings and the electrocardiographic changes at months follow-up are better described in background: the sleeve gastrectomy (lsg) is the most popular procedure worldwide to treat obesity. among those that are obese, gerd has a prevalence of . percent. many surgeons do not perform lsg in these patients because only . percent of symptomatic patients showed resolution of gerd-like symptoms after concomitant sleeve gastrectomy with hiatal hernia repair. many surgeons perform the gastric bypass on gerd patients with hiatal hernias because they believe its superior for the resolution of gerd. when they do this they overlook the many long term complication associated with gastric bypass. also, many patients do not want the gastric bypass under any circumstances. surgeons need to be open to finding better way to reduce the high recurrent rates of gerd after lsg. materials and methods: this is a single institution, multi-surgeon, retrospective study involving morbidly obese patients in a prospectively kept data base from january of through july of . these patients all had gerd with preoperatively identified hiatal hernias on egd. all patients were dependent on anti-reflux medications. there were ( . %) males and ( . %) females. bmi ranged from to . hiatal hernias measured from cm to cm. all lsg patients received a primary crural closure, with or without gore bio a mesh placement, at least weeks prior to the sleeve gastrectomy. post-operatively, patients were interviewed for gerd symptomatology and anti-reflux medication dependency. results: of the patients, ( . %) patients had resolution of gerd-like symptoms and off all anti-reflux medications after the staged hiatal hernia repair and sleeve gastrectomy. patients ( . %) had improvement of gerd but still dependent on anti-reflux medication. patients ( . %) had no resolution or improvement of gerd. there was one post-operative complication of laryngospasm with pulmonary edema status post extubation. there were no mortalities in the series. conclusions: in this study, staged hiatal hernia repair, at least weeks prior to sleeve gastrectomy, doubled the published rate of gerd resolution from % to %. % showed improvement in symptoms at one year. this rate is comparable to gerd resolution after gastric bypass. this may be an alternative approach to hiatal hernias in the morbidly obese patient with gastroesophageal reflux disease who do not want a gastric bypass. background: bariatric surgery is a common procedure in general surgery. gastric bypass has been performed laparoscopically for over two decades and multiple techniques are described. the circular stapled anastomosis, one of the earliest methods for gastrojejunostomy, is performed in two ways: a transoral method to introduce the anvil and a transabdominal approach developed later. the former technique requires passing the anvil of the circular stapler through the mouth, down the esophagus, and into the gastric pouch. in the latter method, a gastrotomy is made, the anvil is introduced, and the gastrotomy is stapled off, creating the gastric pouch. this study aims to objectively compare the two methods of circular stapled gastrojejunostomy in terms of surgical site infection (ssi) rate. methods: a retrospective chart review of patients undergoing laparoscopic roux-en-y gastric bypass with one of two surgeons at a bariatric center of excellence in an academic hospital from january introduction: laparoscopic sleeve gastrectomy (lsg) has become the most commonly performed procedure in the treatment of morbid obesity, but there is significant variability in its performance. from national database analysis, more restrictive sleeve construction, based on smaller bougie size, has not correlated with greater weight loss. we hypothesize that bougie size is not reflective of actual restriction, or that sleeve restriction does not correlate with weight loss. we performed qualitative and volumetric analysis of immediate post-sleeve contrast studies to determine the association of sleeve restriction with post-operative weight loss and complications. methods: between and , patients underwent immediate post-sleeve contrast studies. based on standardized vertebral body height assessment by preoperative chest radiograph, sleeve diameter at intervals (including the narrowest point) was measured in mm, and the volume above the narrowest point of the sleeve was calculated. sleeve shape was assumed as dual-tiered or simple truncated cone based on morphology. sleeve restriction, morphology and volumetric analysis were associated with clinical outcomes including complications, post-op symptoms, and weight loss at months. background: variability in surgical technique resulting in narrowing at the incisura angularis, twisting along the staple line, and retention of the gastric fundus has been implicated in increased gastroesophageal reflux disease (gerd) following laparoscopic sleeve gastrectomy (lsg). standardizing creation of the sleeve based on anatomic landmarks may help produce more consistent sleeve anatomy and improve outcomes. methods: a retrospective review of all patients undergoing lsg from january to november at a single institution specializing in bariatric surgery was performed (n= ). patients underwent either traditional lsg with use of a f suction bougie to guide creation of the sleeve (n = ) or anatomy-based sleeve gastrectomy (abs, n= ). abs was performed using a gastric clamp to maintain predetermined distances from key landmarks ( cm from gastroesophageal junction, cm from incisura angularis, cm from pylorus) during stapling. patient demographics, perioperative characteristics, and post-operative outcomes were compared using chi-square and student's t-tests as required. helicobacter pylori (hp) is prevalent in up to % of the population worldwide with increased rates observed in the bariatric population. bariatric surgery has seen a rapid expansion over the last years with the growing rates of severe obesity. higher hp rates are thought to be associated with increased rates of postoperative complications including increased marginal ulceration and leak rates. accordingly, some bariatric centers have adopted routine pre-operative screening and hp eradication programs. yet, while hp correlation with gastritis and malignancy has now been well defined, its impact on patients undergoing bariatric surgery remains unclear. background: the risk of developing a hiatal hernia in the obese population is . fold compared to patients with a bmi \ . most hiatal hernias after bariatric surgery are asymptomatic and when symptoms are present they may be difficult to differentiate from overeating or maladaptive eating habits. the aim of this study was to define the risk and symptoms associated with a hiatal hernia in the post-bariatric surgery cohort. methods: a retrospective review of prospectively collected data for patients who underwent laparoscopic hiatal hernia repair who previously had primary roux-en-y gastric bypass (rygb) or sleeve gastrectomy (sg). data collection spanned a five-year interval ( / - / ). preoperative and follow up data were collected from medical records and questionnaires in the clinic or by telephone. variables obtained include age, gender, psychiatric history, pre-index procedure bmi, pre-hiatal hernia repair bmi, post-hernia repair bmi, pre and post operative symptoms, and associated morbidity. all hiatal hernia repairs were done laparoscopically, with posterior cruroplasty after circumferential hiatal dissection. results: we identified patients with a symptomatic hiatal hernia who had previously (range: - years) underwent bariatric surgery. fourteen rygb patients presented at a mean of . years compared to sg patients who presented at a mean of . years after index procedure. diagnosis was by a combination of ugi ( %), ct scan ( %) and egd ( %). mean follow up was . months (range: - months). laparoscopic hiatal hernia repair was successfully performed in all patients with % mortality. dysphagia and regurgitative symptoms markedly improved in [ % of patients however, nausea, vomiting and abdominal pain were not changed in - % of patients ( figure) . conclusion: hiatal hernia following bariatric surgery is a rare but important cause of bloating manifested as nausea and vomiting, abdominal pain, regurgitation or reflux, and food intolerance or dysphagia (barf)-and should be further evaluated with imaging or endoscopy when present. laparoscopic repair of hiatal hernia is warranted and results in resolution of symptoms in the majority of symptomatic patients. mid-term outcomes of sleeve introduction: obese patients suffer from multiple organ comorbidities which contribute to a shortened lifespan. one of the effects of obesity is thought to be pseudotumor cerebri, which is secondary to increase in intracranial pressure (icp) in the absence of an obstruction. over the past two years, we have measured icp after insufflating with a laparoscopy device. we found that icp increases dramatically and it correlates with the amount of insufflation in the abdomen. over the years, there have been studies in obese patients and intra-abdominal pressure. these studies have shown that some obese patients have an intra-abdominal pressure of - mmhg. increasing intraabdominal pressure is thought to escalate intracranial pressure (icp). the objective of this pilot study was to observed change in icp after the raising intra-abdominal pressure. method: in this retrospective chart review preliminary study, pressure in each of the patients either normal pressure hydrocephalous or high pressure hydrocephalous receiving a ventricle shunt were measured by manometer. once the shunt was placed into the ventricle, we attached a manometer to measure the opening pressure. after we accessed the abdominal cavity using the standardoptiview technique, we created a pneumoperitoneum. after achieving an intraabominal pressure of mmhg, were measured the icp using the manometer. spss software version was used for data analysis. paired t-test was applied on icp before and after the procedure. introduction: postoperative bleeding represents an infrequent, yet serious complication after bariatric surgery. differences in the rate of postoperative bleeding reported for the two most common weight loss procedures-laparoscopic roux-en-y gastric bypass (lrygb) and laparoscopic sleeve gastrectomy (lsg)-are ostensibly confounded by patient and surgeon specific preoperative, intraoperative and postoperative factors, in particular, by the utilization of staple line reinforcement or oversewing. with this understanding, we aim to use a large national database to definitively characterize differences in bleeding rates between lsg and lrygb. conclusions: after appropriate risk-matching, lsg patients have a reduced likelihood of a postoperative bleeding event compared to those undergoing lrygb. this difference is likely more pronounced with intraoperative securing of the staple line via oversew, buttress or an alternative method. these findings from a large national database represent an important consideration for surgeons and patients alike when evaluating the appropriate bariatric operation. background: bariatric surgery has shown to be the most effective treatment, with documented improvement in obesity-related comorbidities. the type of health insurance coverage plays an important role in the access to bariatric surgery, but might also affect postoperative outcomes. the objective of this study is to determine whether there is a difference in outcomes based on the type of insurance months after bariatric surgery. methods: we retrospectively reviewed all the patients that underwent bariatric surgery at our institution from to . we divided the patients into two groups, based on the type of insurance, private (group one), and public (group two). we compared demographics and months outcomes between the groups, using t-test for continuous variables and chi-square for categorical variables. we also compared months estimated bmi loss between different private insurances using anova. introduction: bariatric surgeons are now performing primary and revisional procedures on the extremes of age. there is controversy surrounding the safety and effectiveness of bariatric surgery among older age groups compared to younger age groups. to address this knowledge gap, we designed a study assessing short-term bariatric surgery outcomes among various age groupings across a large national database. methods and procedures: de-identified patient data across from the mbsaqip registry was used. age groupings were organized into young, middle-aged, and older adults (in years) as follows: \ , - , and [ , respectively. the following -day outcomes were evaluated between all possible pairwise age groupings: mortality, surgical site infection (ssi), and readmission; logistic regression was used to compare outcomes between age groupings controlling for primary vs. revisional index operation, patient factors, and procedure factors. a p value of . was deemed statistically significant. results: a total of , patients were identified (age range: to [ ); % (n= , ) underwent primary bariatric operations while % (n= , ) underwent revisional cases. older adults had significantly worse outcomes than middle-aged and younger adults, respectively, for over comparisons across all outcomes; in contrast, younger adults had significantly worse outcomes than middle-aged adults for only comparisons across ssi and readmission. for primary bariatric cases, older adults had significantly higher mortality rates than middle-aged and younger adults, respectively, in the following categories: asa , laparoscopic sleeve gastrectomy (lsg), or laparoscopic roux-en-y gastric bypass (lrygb). for revisional cases, older adults had significantly higher mortality rates than middle-aged and younger adults, respectively, in the setting of female gender, caucasian race, or asa . regarding ssi, older adults undergoing primary lrygb had significantly higher organ space infections compared to younger adults. in addition, older adults who had revisional lrygb had significantly higher deep surgical site infections compared to middle-aged adults. following primary bariatric cases, older adults had significantly higher readmission rates compared to younger adults in the presence of male gender, caucasian race, asa , copd, or after lsg. following revisional cases, older adults had significantly higher readmission rates than middle-aged and younger adults, respectively, in the setting of pre-operative chronic steroid use. conclusions: overall, older adults had worse short-term outcomes compared to their younger counterparts following primary and revisional cases. further research is required to investigate these findings with the goal of targeting interventions to improve outcomes among bariatric surgical patients. background: the obesity epidemic in the united states has been accompanied by surge in bariatric surgery. nearly , bariatric procedures were performed in the us in , % of which involved roux-en-y gastric bypass (rnygb). while rnygb has proven an effective tool in combating obesity, it also alters a patient's anatomy in a way that makes traditional ercp a difficult, if not impossible option for interrogating the common bile duct. one way to approach the post-rnygb patient with obstructive jaundice is to access the peritoneal cavity via a laparoscopic/ robotic approach followed by direct cannulation of the gastric remnant with a laparoscopic port, allowing passage of an endoscope. the aim of this study was to evaluate our single center experience with minimally-invasive transgastric ercp (tg-ercp) from to . methods: we compiled a list of all patients who underwent laparoscopically or robotically assisted tg-ercp at our institution from - . we then examined patient demographics, procedural details, postoperative outcomes, and success rate, with success defined as cannulation of the ampulla, clearance of obstruction if present (stones/sludge/stenotic ampulla), and completion imaging of the biliary and pancreatic ducts. results: patients were included in the study. cases were performed robotically ( %), and laparoscopically ( %). ercp was successful in cases ( %). all unsuccessful attempts were aborted when the endoscopist was unable to pass the scope through a tight pylorus. median time of operation was minutes ( minutes if concomitant cholecystectomy was performed, minutes if not). median length of stay after operation was days (range - days). median estimated blood loss (ebl) was ml. post ercp pancreatitis occurred in patients ( . %), and was mild and self limited in all cases. patients had postoperative bleeding requiring transfusion. both of these had concomitant cholecystectomy. discussion: in patients with biliary obstruction and anatomy not suitable for traditional ercp, tg-ercp is a viable option. it can be performed with in a minimally invasive fashion (either laparoscopically or robotically) with a high success rate and low morbidity. as the population of patients who have undergone rnygb continues to grow, so does the likelihood of encountering one with obstructive jaundice. tg-ercp, therefore, should be thought of as an essential tool in the armamentarium of the general surgeon. introduction: primary palmar hyperhidrosis (ph) is a pathological condition of over perspiration caused by body produces an excessive amount of sweat. this disorder affects to decrease quality of life of patients. thoracoscopic sympathectomy is minimally invasive and an effective procedure to treat hyperhidrosis. different of level of sympathectomy has been debate for the best outcomes. many researchers studied about short term outcomes but no empirical research evidences long term outcomes of thoracoscopic sympathectomy in thailand. this study purposed to evaluate and compare the long term clinical outcomes between patients who underwent t and t thoracoscopic sympathectomy for ph with particular attention to patient satisfaction and quality of life. methods and procedures: sixty patients with ph underwent thoracoscopic sympathectomy. patients were divided into two groups by the level of thoracoscopic sympathectomy as t group and t group. they were investigated the improvement of sweating, compensatory sweating, satisfaction and quality of life. the long-term investigation was designed to examine clinical outcomes at before surgery, six months after surgery, year after surgery, years after surgery, and last follow up days were compared within group and between of t and t group. they were subjected to telephone interview using multiple questionnaires to investigate surgery outcomes, degree of satisfaction, and quality of life improvement. results: sixty patients responded to the telephone interview. patients demographic data and also recurrence rate of ph between t and t group was not significant different (p= . ). both groups improved severity of sweating without any statistical significant. but the t thoracoscopic sympathectomy led to significantly lower incidence of compensatory hyperhidrosis when compared with t group at back and trunk sites. the t group had higher overall satisfaction than t group with was not significantly different. long term result are followed after years. conclusions: there was no difference in decreasing severity of sweating between t and t level of thoracoscopic sympathectomy. both group equally archived patient satisfaction. but the t level of thoracoscopic significantly had lower severity of ch and better quality of life in long term period. introduction: acute pancreatitis due trauma is commonest cause of pseudocyst in pediatric age. due to limited literature available and under diagnosis by pediatricians, the true incidence of pseudocyst in - age group is not known. material and methods: retrospective analysis of pediatric age ( - years) patients who underwent laparoscopic cystogastrostomy at distric teaching hospital was done. patients data, presentation, investigations, opetation done and post operative course was studied. result: total of patients ( males & females) had mean age of . years, mean weight of kg. etiollogies included blunt abdominal trauma ( ), idiopathic ( ), gallstones ( ) . average cyst diameter was . cm. laparoscopic cystogastrostomy by transgastric approach was successfully possible in cases with no conversion. cystogastrostomy was performed using sutures in patients and ultrasonic energy device in patients. gastrotomy was closed with sutures in all cases. mean operative time was minutes. post operative imaging at months revealed no persistence or recurrence of cyst. conclusion: minimally invasive laparoscopic approach for chronic pancreatic pseudocyst in pediatric age group is safe and effective strategy and should be adopted as primary modality of treatment. introduction: videoscopic neck surgery is developing despite the fact that only potential spaces exist in the neck. gagner first described the endoscopic subtotal parathyroidectomy with constant co gas insufflations for hyperparathyroidism in . the cervical approach utilizes small incisions in the neck thus making it cosmetically unacceptable and cannot be used for lesions greater than cm. the axillary approach makes it difficult to visualize the opposite lobe. the anterior chest wall approach utilizes port access at various positions on the anterior chest wall depending on the surgeon. this technique also allows bilateral neck exploration. hence we have been able to perform total thyroidectomies with central compartment clearance for papillary carcinoma and near-total thyroidectomies for large multinodular goiters, materials and methods: three incisions subplatysmal plane pneumoinsufflation with carbon dioxide (co ) ports creating a subplatysmal palne dissection begins at the inferior pole posterior dissection clipping superior thyroid vessels specimen freed up thyroid lobectomy was performed in the twenty cases. the average blood loss was ml mean operative time was min there were no complications and no cases were converted to open. there were no cases of recurrent laryngeal nerve injury or postoperative tetany. no subcutaneous emphysema, ecchymosis or hypercarbia was observed in any patient. all patients were discharged on the second postoperative day except the first on the fifth day. in conclusion this approach seems to be safe in case of unilateral lobectomy but early to say it is superior to conventional thyroidectomy especially in total thyroidectomy. introduction: laparoscopic sleeve gastrectomy (lsg) is one of the most commonly performed weight loss surgeries. prolonged hospital admissions are associated with both increased morbidity and mortality and increased strain on the health care system; studies are now investigating the safety and feasibility of outpatient lsg. this study examined a single surgeon's postoperative admission trends for patients who underwent lsg. the patients were divided into two cohorts based on the date of surgery, and we hypothesize institutional experience has a significant impact on postoperative stay and hospital readmission rate. methods: this is a retrospective study on lsgs performed by a single surgeon in a tertiary center from - . inclusion criteria: patients [ years old, bmi [ with comorbidities or bmi [ , and patient approval by the bariatric surgical program in victoria, british columbia. patients with prior weight-loss surgery were excluded. patients were discharged home on a care plan involving: nurse and surgeon telephone follow-ups within one week post-surgery. patients were divided into two cohorts: cohort a (procedures between - inclusive) and cohort b (procedures between - inclusive). results: patients were included in this study: females ( . %) and males ( . %). the mean preoperative age was . ± . years, and the mean preoperative bmi was . ± . kg/m . the average postoperative discharge day for the population was day . ± . and the average or time was . ± . minutes. one patient in cohort b was re-admitted pod with a diagnosis of postoperative edema managed conservatively and is included in the analysis as pod . a second patient in cohort b returned to hospital (pod ) for abdominal pain and was managed conservatively as outpatient. conclusion: there was a significant difference in the average postoperative discharge day between patients in cohort a and cohort b who underwent lsg with patients in cohort b requiring a shorter average admission time. this study suggests that with increasing institutional experience and a postoperative discharge plan, patients undergoing lsg may be discharged on postoperative day one safely. surg endosc ( ) introduction: minimally invasive techniques have revolutionized the art of the surgical practice. the laparoscopic approach to cholecystectomy has become the gold standard and is the most common laparoscopic general surgery procedure worldwide. in an effort to further enhance the advantages of laparoscopic surgery, even less invasive methods have been attempted, including smaller and fewer incisions. the objective of this study was describing our results of years of needlescopic cholecystectomy. methods: since march all patients that underwent to needlescopic cholecystectomy micro-laparoscopic procedure with instruments of mm were included in this study in a prospective database and the information was analyzed. results: between march and september , needlescopic cholecystectomies have been done at texas endosurgery institute in san antonio, texas by a single surgeon. % of the patients were female. the average age was . (range of - years old). average operating time was . minutes (range of - minutes). the minute operation required laparoscopic cbd exploration, accounting for the extended time. average estimated blood loss (ebl) was cc (range of - cc). % of cases required conversion to standard mm cholecystectomy and was completed without incidents. all patients were followed up at weeks, weeks, and months after the procedure. only patient presented with a hernia at the umbilical site. otherwise no wound, bile duct, bile leak, bleeding or thermal injury complications were identified. conclusions: micro-laparoscopic procedures with mm instruments in this specific procedure of needlescopic cholecystectomy is safe and feasible, and is a cosmetic alternative to the standard laparoscopic cholecystectomy. there's still less report about thyroid cancer cases in toetva. this study reviews all cases of thyroid cancer which surgery were performed. there were cases of toetva in thyroid cancer and cases of opened thyroidectomy. objective: to review and report in terms of surgical outcome, complication, post-surgical treatment and recurrence in all cases of thyroid cancer surgery, especially in toetva technique. material and methods: from march -july in police general hospital, a total of patients underwent toetva with cases of toetva in thyroid cancer and cases of opened thyroid surgery in thyroid cancer. all patients were recorded in multiple parameters. results: this study have total of thyroid cancer cases which cases ( %) were male and cases ( %) were female, with an average age of . most clinical presentation was thyroid mass or nodule which was at cases ( . %), case ( . %) was non-toxic goiter and case ( . %) was grave disease. the clinical presentation mean time was . years ( weeks- years). there were cases ( . %) with a mass at right lobe, cases ( . %) with a mass at left lobe, and cases ( . %) with mass at both lobes. the size of thyroid mass was . ± . centimeters ( - centimeters). there were cases ( . %) had euthyroid, case ( . %) had subclinical hyperthyroid, cases ( . %) had subclinical hypothyroid, and cases ( . %) had hyperthyroid. for type of surgery, there were cases ( . %) of toetva surgery and cases ( . %) of opened total thyroidectomy. most patients at cases ( . %) didn't have any post-operative complication. and there were hypothyroid cases ( . %), transient hypocalcemia with no symptom cases ( . %), and transient hoarseness cases ( . %). after toetva surgery performed, cases ( . %) were redo completion thyroidectomy, cases ( . %) were transaxillary completion thyroidectomy, cases ( . %) were redo toetva, and case ( . %) deny for reoperation. and cases ( %) didn't have any complication after redo surgery, cases ( . %) were hypothyroid, cases ( . %) were hypocalcemia and hypoparathyroid, and case ( . %) was transient hoarseness. after did thyroidectomy, ultrasound neck shown that cases had no residual or recurrence thyroid mass, cases had residual thyroid tissue. all cases received radioactive iodine ablation. radionuclide total body scan showed no evidence of distant functioning metastasis. conclusion: three-year short-term followed up toetva in thyroid cancer has shown less complication and no recurrence cancer. objective of the study: sentinel node navigation surgery (snns) in gastric cancer has been investigated for almost two decades in an effort to reduce operative morbidity. indocyanine green (icg) with enhanced infrared visualization is one technique with increasing evidence for clinical use. we are the first to systematically review and perform metaanalysis to assess the diagnostic utility of icg and infrared electronic endoscopy (iree) or near infrared fluorescent imaging (nifi) for snns exclusively in gastric cancer. methods and procedures: a search of electronic databases medline, embase, scopus, web of science and the cochrane library using search terms "gastric/stomach" and "tumor/carcinoma/cancer/neoplasm/adenocarcinoma/malignancy" and "indocyanine green" was completed in may . all human, english language randomized control trials, non-randomized studies, and case series were evaluated. articles were selected by two independent reviewers based on the following major inclusion criteria: ( ) diagnostic accuracy study design; ( ) indocyanine green was injected at tumor site; ( ) iree or nifi was used for intraoperative visualization. the primary outcomes of interest were identification rate, sensitivity and specificity. titles or abstracts were screened after removing duplicates. the quality of all included studies was assessed using the quality assessment of diagnostic accuracy studies- . results: ten full text studies were selected for meta-analysis. a total of patients were identified with the majority of patients possessing t tumors ( . %). pooled identification rate, diagnostic odds ratio, sensitivity and specificity was . ( . - . ), . ( . - ), . ( . - . ) and . ( . - . ) respectively. the summary receiver operator characteristic for icg+iree/nifi demonstrated a test accuracy of . %. subgroup analysis found improved test performance for studies with low risk quadas- scores, studies published after and submucosal icg injection. iree had improved diagnostic odds ratio, sensitivity and identification rate compared to nifi. heterogeneity among studies ranged from low (i \ %) to high (i [ %). conclusions: the idea of snns in gastric cancer is intriguing because of the potential to limit operative morbidity. we found encouraging results regarding the accuracy, diagnostic odds ratio and specificity of the test. the sensitivity was not optimal but may be improved by a carefully planned and strict protocol to augment the technique. given the limited number and heterogeneity of studies, our results must be viewed with caution. objective: to evaluate the feasibility, cost effectiveness and safety of single incision laparoscopic surgery using routine laparoscopy instruments. method: cases of acute appendicitis and cases of symptomatic gallstone disease were included in study. cases were enrolled in study and prospective observational study was performed. ruptured appendicitis/abscess formation were excluded from study. similarly empyema gallbladder/gallbladder perforation were also excluded. results: total cases included; cases of appendicitis and cases of symptomatic cholelithiasis. mean age of appendectomy group was . ± . years and mean age of cholecystectomy group was . ± . years. in our study, mean operative time for sil appendectomy was . ± . min. post-operative fever was noted in cases ( . %). mean post-operative pain as per vas score taken after hours, on pod was . . average post op stay in hospital was . days, port site infection occurred in one case ( . %). patient satisfaction score obtained on the scale of - on one month follow up was . , while scar cosmesis score was . . in our study, cases underwent sil cholecystectomy, of which were male ( . %) and were females ( . %), and mean age of patients was . yrs. mean operative time in our study was . min, mean post-operative pain taken on pod as per vas score was . , mean post-operative hospital stay was . days, port site infections occurred in cases. post-op fever was noted in cases, post-operative patient satisfaction score obtained at month follow up was . and scar score of . on the scale of - . no case required drain placement and conversion. conclusion: sils can be performed using conventional laparoscopic instruments especially in a government setup where per capita economic burden to patient will be less. though it has more operative time, it has comparably less post-operative hospital stay, causes less pain, and has significantly more patient satisfaction regarding post-operative scar and cosmesis. since sils has more patient acceptance and satisfaction, it can be offered to all patients undergoing laparoscopic surgery. it is very useful in government setup where lower economic class of patients will also benefit, irrespective of unavailability of special instruments and financial constraints, as it can be performed using routine laparoscopic instruments. in the year we started to practice the pericardic window by laparoscopy to diagnostic of head injury hidden in precausal trauma, although lucketally for our society, this type of injury has decreased considerably, we have achieved an important number of patients and in the last year we have performed the procedure for another type of pathologies and also diversified the approach route according to the case. objective: sharing accumulated experience in years in the pericardic window practice by laparoscopy or thoracoscopy. material and methods description of cases results: during this period, we have accomplished cases of laparoscopic pericardal window with two unique ports for the diagnosis of head injury in trauma precordial, additionally there were practiced windows through traumatic trauma of which have been derived in treatment of cardiac injury on this way, without performing open approach. in another scenario, we have performed pericardial spill treatments for different causes by minimally invasive via. no complication or mortality associated with the procedure has been presented. conclusions: the pericardic window performed by a minimally invasive surgery is an effective, replicable strategy for the management of diagnosis and the medical and traumatic treatment of this pathology. patient selection is key and work in multidisciplinary groups guarantees good results. introduction: for the transabdominal preperitoneal repair (tapp) for groin hernia, single port surgery (sps) has been reported to reduce the abdominal wall damages. to reduce the length of the umbilical scar and to keep the view of triangulation, we use one needle forceps plus sps. patients and methods: from may to july , consecutive tapp patients were retrospectively investigated. there were male and female. we use two mm ports ( for the scope and for the operator's right hand forceps) through an umbilical multi-channel port and additional mm needle instrument is pierced above the pubic bone. a mm flexible scope allowed us to keep the triangular formation easily. we studied the safety and usefulness of this method from the viewpoints of operation time and the complications. results: median operation time of single side hernia ( cases) was min ( - ) and the bilateral case ( cases) was min ( - ). five cases needed one or two additional mm ports, and one case with severe preperitoneal adhesion due to the previous prostate cancer surgery was converted to open method because of the venous bleeding. other complications were spermatic cord injury and postoperative seroma that required the percutaneous puncture. umbilical scars and the pierced needle instrument scars became gradually invisible within or months. there were no incisional hernia nor wound infections in our series. these data was comparable to the conventional laparoscopic hernia repairs. conclusions: operation scars of this method had better cosmesis than the conventional tapp or sps tapp, and there were no differences between our sps-tapp with one needle foerceps and conventional method in operation time and the complication rate. our method was demonstrated as a less invasive approach for laparoscopic groin hernia repair. clinical application: fj clip is a stainless steel that can be used to hold organs in the abdominal cavity. it is available in two sizes: mm and mm. the device is short, it has a strong grasp, and it causes no or only negligible organ damage. we have used fj clip in the performance of local gastric excision (n= ), colectomy (n= ), and cholecystectomy (n= ) with no resulting difficulty. f loop plus is a g stainless steel loop-like device into which we can insert φ . mm nt alloy thread, which we draw out extracorporeally via simple puncture. laparoscopic total and proximal gastrectomy. we made a small incision at the umbilicus and inserted a -mm camera port and -mm metal cannula. we placed two (left and right) epigastric ports. retraction of the left hepatic lobe was easy with use of the -mm fj clip and a -mm penrose drain. for # lymph node dissection, we used the fj clip to grasp the upper part of the stomach, inserted the f loop plus from the upper right abdomen. for # dissection, we grasped the pyloric vestibule and pulled it leftward. for dissection of the upper edge of the pancreas, we grasped the left gastric arteriovenous pedicle and pulled it toward the abdomen. the fj clip's grasp and traction exerted on the stomach wall were strong and effective, and there was little organ damage. reconstruction (roux-y) or double tract were performed within the abdominal cavity by hand-sewn purse string suture of the esophageal stump, insertion of an anvil, and use of an automated anastomosis device. we have experienced total and proximally cases to date, but there have been no complications, and both intraoperative bleeding and operation time were within normal limits. conclusion: we believe the fj clip and f loop plus will replace conventional forceps for various tasks in reduced port gastrectomy. introduction: pulmonary anatomical resection is considered as standard treatment for early staged lung cancer. uniportal video-assisted thoracoscopic surgery (uvats) has recently showed favorable surgical outcomes, but remains technically demanding, especially in a complex procedure such as anatomic segmentectomy. needlescopic instruments facilitates complex laparoscopic surgeries with nearly painless and scarless postoperative outcomes, however, its utilization of thoracoscopic surgery were mostly for minor procedures such as bullectomy and sympathectomy. we presented our initial experience of lung cancer surgery performed by uniportal vats and additional needlescopic instruments, and we also compare the operative results with conventional uniportal vats. methods: from december to august , consecutive patients with lung cancer undergoing anatomical lung resections including lobectomies and segmentectomies were reviewed retrospectively. of these patients, patients received conventional uniportal vats (uvats), and patients received needlescopic-assisted uniportal vats (na-uvats). we compared the peri-and post-operative outcomes in these groups. results: there was no significant difference in demographic, anesthetic, or operative characteristics in two groups except for age. the mean operation time was statistically less in the na-uvats group ( . ± . min vs . ± . min, p= . ). the intraoperative blood loss was significantly less in the na-uvats group ( . ± . ml vs . ± . ml, p= . ). there were two major pulmonary arterial bleeding events and one conversion to thoracotomy in the uvats group. the hospital stay, duration of chest tube drainage and post-operative pain scale were comparative in the two groups. conclusion: under the assistance of additional needlescopic instruments, uniportal vats can be performed more efficiently and safely without compromising its benefit in less postoperative pain and early recovery. purpose: we applicated the v-loc into abdominal wall closure in single incision laparoscopic appendectomy (sila) from . the aim of our study is to present our experience of abdominal wall wound closure technique using barbed suture in sila and comparision of perioperative outcomes with conventional method of layer by layer abdominal wall closure after sila. methods: from august to june , sila was performed on patients with acute appendicitis at the department of surgery, hallym sacred heart hospital. under approval of institutional review board, data concerning demographic characteristics, operative outcomes, postoperative complications were compared between both v-loc closure group and conventional layer by layer closure procedures. in v-loc closure group, after removing the appendix, divided linear alba was closed using unidirectional absorbable barbed suture v-loc - with continous running fashon. begins at the end of incision, and coming back with reinforced running. subcutaneous closure was also done using same thread, and the subcuticular suture along incision line was performed with remaning portion of v-loc. results: the demographic data of patients's characteristics were similar between the two groups. the use of barbed suture significantly reduced the suturing time for abdominal wall closure (p= . ) compared with conventional suture. the postoperative incision length was significantly shorter in v-loc group than conventional group (p= . ). the rate of surgical site infection were similar in both group. no incisional hernia were noted in both group with median follow up periods of . months. the total costs of the procedure were comparable in both group under korean drg system. the use of barbed suture in abdominal wall closure in single port laparoscopic appendectomy is safe, and feasible method, reduces the suturing time, thereby decreasing the total operation time, and incision length with cosmetic effect. angela m kao, md, michael r arnold, md, julia e marx, paul d colavita, md, b todd heniford; carolinas medical center introduction: morgagni hernia is an anteromedial congenital diaphragmatic hernia seen in approximately in live births and rarely identified in adulthood. patients may be asymptomatic, have intermittent symptoms, or present acutely with incarceration/obstruction. given this, surgical repair is recommended, but a standardized technique has not yet been described. methods: a prospectively collected hernia-specific database was queried for all adult morgagni hernias performed at a tertiary hernia center. demographics and peri-operative data were compared. ( ) repair. the most common ( . %) method of repair included suturing mesh to the diaphragmatic portion of the defect and securing the anterior-inferior edge to anterior abdominal wall with transfascial sutures and/or tacks. four patients ( . %) underwent primary repair. average defect and mesh size was . cm and . cm , respectively. three patients ( %) underwent a concomitant paraesophageal hernia repair. mean ebl and length of stay was ml (range - ml) and . days (range - days). postoperative morbidity included transient postoperative hypoxemia ( patients) and pleural effusion ( ) . there was no mortality, mesh complications or recurrences with a mean follow-up of months. conclusions: morgagni hernias patients were more often older, obese, and women. these hernias remained unrepaired in % of patients despite their having had previous abdominal surgery. a laparoscopic or robotic approach offers an effective hernia repair with minimal complications, short hospital stay, and excellent long-term results for both elective and acute operations. mesh repair, sutured to the diaphragm and sutured/tacked to the abdominal wall, appears to be a very successful means to repair larger defects. introduction: hydatidosis is a zoonotic disease caused by echinococcus granulosus. it is endemic in the mediterranean, south america and middle east. it is a systemic disease wherein lungs are the second most common organ involved, after liver. radio-imaging plays an important role in diagnosing and determining the extent of the disease. surgical enucleation of cyst has been the classical treatment for this disease. bilateral lung involvement has been traditionally treated by median sternotomy or a bilateral thoracotomy. video assisted thoracoscopic surgery (vats) is an effective surgical approach in such settings. materials and methods: at our center, we have operated cases of pulmonary hydatidosis thoracoscopically over the past years. in all cases, area around the cyst was cordoned off with . % cetrimide soaked gauze pieces. a pericystotomy is performed with ultrasonic shears & the germinal membrane is delivered en masse into an endo-bag. an air leak test after saline instillation into the cavity, is a standard part of the procedure. for those cases with cysto-bronchiolar communications, the defect was sealed by either suturing or glue application. traditionally, bilateral cases & cysts larger than cm in size were tackled by an open approach. but, in our experience, cyst size, bilaterality & presence of complications are not contraindications for vats. all cases are administered perioperative albendazole ( mg twice a day, administered for three cycles of days each, with a gap of days in between) which helps in preventing recurrence and also takes care of any inadvertent intra-operative spillage. introduction: minimally invasive surgery (mis) is the standard approach for most of the surgical procedures performed by general surgeons. traditionally the majority of operations for trauma are performed open due to the complexity of the cases, however, trauma surgeons are expanding their armamentarium to include mis in a variety of acute procedures. we report our experience with the application of laparoscopy in a variety of trauma cases. methods: a retrospective review of trauma cases performed between / - / . during that time laparoscopic cases were performed after traumatic injury. patient demographics, injury severity (iss), injury mechanisms, the types of procedures and outcomes will be described. means and standard deviations were calculated and t test were performed. a p value of . was statistically significant. results: demographics-a total of trauma cases were performed laparoscopically during the study period. the majority were male, n= and the age was sd . obesity was documented in %, hypertension or cad was in %, and substance abuse was in %. blunt trauma was in % and penetrating %. the iss was sd . surgical procedures-the majority, %, of the procedures were completed laparoscopically. non-therapeutic laparoscopy was performed in %. repair of diaphragmatic or traumatic abdominal wall hernias were %. hematoma evacuation and control of bleeding was %. control of solid organ bleeding and repair was performed in %. intestinal repair occurred in %. for the cases that required open conversion iss was sd vs. laparoscopic cases iss was sd , p= . . outcomes: the overall length of stay was days sd . there was n= late death in a poly-trauma patient that required open conversion for complex solid organ and intestinal injuries. there was n= case of a community acquired pneumonia, and n= case of a recurrent pneumothorax. conclusions: a descriptive series of trauma operations approached with mis techniques is described. this cohort had high injury severity and a predominance of comorbid conditions. laparoscopy was successfully applied in the majority of cases for a variety of therapeutic procedures and mortality and morbidity was low. mis is safe and is gaining momentum for application in traumatic injury. objectives: laparoscopic distal gastrectomy for early gastric cancer is a standard treatment in japan described in guidelines. the surgical procedure has been shifting from laparoscopic assisted to complete laparoscopic surgery. in this study, we evaluated the outcomes and safety of the laparoscopic assisted distal gastrectomy. methods: for the marking of the oral side transecting line, the clipping at oral side of cancer lesion was performed by gastro-endoscopy before surgery. the lymph node dissection (d +/d ) is performed laparoscopically. as the dissection of the pancreatic superior region, the assistant hold the left gastric artery and keep the good view by retracting the pancreas. the common hepatic artery and proximal side of splenic artery are exposed. both sides of the left gastric artery and vein are exposed. left gastric vein and left gastric artery are cut after clipping and sealing. lymph node dissention of hepato-duodenal ligament is done and right gastric artery is cut after clipping and sealing. minor curvature of upper gastric wall is exposed (no , dissection). billroth i reconstruction by the circular stapler (cdh) is performed. through the upper median incision with cm, operator pulls out the stomach and transects the oral side of stomach with linear stapler after palpating the clips. duodenum is transected after purse string suture. gastroduodenal anastomosis is performed by cdh. results: two hundred cases were analyzed. the operation time, blood loss and the conversion to open surgery rate were minutes, ml, and . %, respectively. as postoperative complications, anastomotic failure, pancreatic fistula and postoperative bleeding were %, . % and %, respectively. the reoperation rate was %. one surgical death due to cerebral infarction was experienced. there were no patients with ppm (pathological proximal margin) positive and too much pm distance. frequency of abdominal wall incisional hernia and ileus were % and %, respectively. conclusion: although there is the disadvantage that small laparotomy can be made in the upper abdomen, laparoscopic assisted distal gastrectomy with billroth i reconstruction in our procedure is enough good from the viewpoint of the precision of proximal margin, and the incidence of serious complications. introduction: minilaparoscopy (mini) is a modality of minimally invasive surgery that attempts to produce less surgical trauma to the abdominal wall by reducing the diameter of surgical instruments to mm. searching for better outcomes in inguinal hernia repair, surgeons have looked for new and less invasive alternatives such as single-incision surgery, single-port surgery and mini. minilaparoscopic transabdominal preperitoneal hernia repair (mini-tapp) demonstrates some of the known advantages of mini general surgery procedures such as enhanced visualization, improved dexterity and great cosmetic outcome. it is safe and reproducible since it does not differ from standard laparoscopy. introduction: the celiac plexus is a structure located in the retroperitoneum, at the level of the lumbar vertebra, which is located in the prevertebral region and has sympathetic fibers. patients with advanced gastrointestinal cancer and associated pain, one of the management strategies is pain control. neurolysis of the celiac pleural by laparoscopy was first reported in humans in in patients with advanced pancreatic adenocarcinoma with excellent results. experience will be shown in the simplification of the technique for the procedure. method: neurolysis of the celiac pleura was performed in patients with advanced gastrointestinal cancer, stomach %, pancreas % liver % other %, no complications associated with the procedure, pain improvement was achieved in % of patients after process. the standardization of the technique by laparoscopy and its simplification, has made this procedure that is replicable and safe. description of the technique: patient in french position, technique of trocars, umbilical trocar mm and trocars of mm paraumbilical, staging laparoscopy is performed and sampling if necessary, is identified in the region of the lowercurvature of the stomach, the celiac trunk and the emergence of the left gastric artery are identified and cc of % alcohol diluted to the medium in the lateral fatty bearing are instilled through a pericranial under direct vision, verifying the non-arterial instillation of the alcohol. there were no complications related to the procedure. results: we report the experience of one group who underwent celiac pleura neurolysis in patients with advanced gastrointestinal cancer, gastric cancer %, pancreatic cancer %, liver cancer % and another %. the most frequent pathology report was adenocarcinoma, % of the patients were managed at hours with sustained effects, up to months of follow-up. with a significant decrease in pain medication. only patient required new laparoscopic neurolysis because of difficult-to-manage pain. the operative time of this procedure was minutes. the standardization of the technique, the use of low cost inputs, makes this type of procedure easily replicable with goodresults in pain management in cancerpatients. conclusions: mis is offered as one of the fundamental tools for the management of palliative procedures in gastrointestinal cancer. neurolysis of the celiac pleura with standardization of the technique, use of low cost elements, and the surgeon's skills make this procedure an option of management and control of pain in patients with advanced gastrointestinal cancer, is easily replicable, economical and insurance. background: the non-absorbable polymer clip offers a solution to the disadvantage of traditional metallic clip. due to its metallic property, it is not only expensive but also causes artifacts on imaging studies and often migrates into cbd. this study compares the traditional standard metallic clip with hem-o-lock used in laparoscopic cholecystectomy (lc) in regard of the safety and efficacy?. material and methods: this study includes patients who underwent lc implementing metallic clip (mc) and patients implementing hem-o-lock clips (h )?. both clips were applied to cystic duct and artery, then the gallbladder was dissected from the liver bed by diathermy. the intraoperative and postoperative parameters were collected including duration of the operation and complications?. results: the median operative time was not statistically different between the mc and the hc group ( . vs . minutes, respectively; p= . ) with no significantly less incidence of bile spillage ( vs. , p= . ) . no statistically significant difference was found in the incidence of postoperative complications between both groups ( vs. , p= . ). no postoperative bile leakage was encountered in both groups. conclusion: hem-o-lock clip provides a complete hemobiliary stasis and a secure cystic duct and artery control. its cost effectiveness is also attractive while provides efficacy equivalent to that of the standard metallic clip. introduction: most of the blunt thoracoabdominal injury patients always have multiple organ injuries. plan of definite treatment depends on the preoperative diagnosis. in isolating diaphragmatic traumatic injury without others organ injury laparoscopic approach is helpful, decrease a length of hospital stay as well as decrease a wound complication. authors describe the laparoscopic treatment of the patient who had rupture of a diaphragm from blunt trauma in an emergency setting. methods and procedures: a years old man presented with motor vehicle accident and mechanism of injury was blunt thoracoabdominal injury. he complains about chest tightness and tachycardia. complete evaluation and ct scan ware performed. stomach was herniated to the left chest and diaphragmatic ruptured was found neither others great vessels nor solid organs injury. the laparoscopic approach was desired and left diaphragm was repair by non-absorbable sutured without intraoperative complication. results: the patient has been discharged days post-operative with full recovery. chest x-ray was taken before discharge, in out-patient department weeks as well as months after discharge which shown no diaphragmatic herniation. conclusion(s): laparoscopic approach in isolated traumatic ruptured diaphragm patients is safe and should be considered. short-term outcome of laparoscopy-assisted distal gastrectomy with roux-en-y reconstruction through mini-laparotomy for gastric cancer since , we have introduced laparoscopy-assisted distal gastrectomy (ladg) with b-i reconstruction through mini-laparotomy. regarding to reconstruction, roux-en y reconstruction are also one of the choice in ladg, however, the technical feasibility has not been well documented so far. the purpose of this study was to compare the short-term outcome of ladg with roux-en-y reconstruction through mini-laparotomy compared to that of ladg with b-i anastomosis. between and , patients who underwent ladg for gastric cancer in oita university were enrolled in this retrospective study. since , the roux-en-y reconstruction has been performed as a standard method in our department. these patients were divided two groups based on anatstomosis; roux-en-y (r-y) group (n= ) and billroth i (b-i) group (n= ). baseline characteristics, operative results (including complications) and pathological results were evaluated. there were a considerably greater number of patients with advanced clinical stage and having ≥t invasion in the r-y group. estimated blood loss was lower in r-y than in b-i (p. ) and operative time was longer in r-y than in b-i (p. ). there were no significant differences in all grade intra-operative complications (p= . ). in addition, there were no significant differences in all grade post-operative complications between the two groups except internal hernia. hospital mortality was % in each group. ladg with r-y reconstruction through mini-laparotomy was technically feasible as well as ladg with b-i anastomosis. utilization of laparoscopy associated with blunt abdominal trauma: the nationwide inpatient sample - kenneth w bueltmann , marek rudnicki ; advocate illinois masonic medical center, chicago, il, university of illinois introduction: the incidence of trauma and its heavy burden upon the healthcare system remain strong. paradigm shifts in the management of these cases has, however, improved the mortality in such cases. it can be expected that improvements in management, when combined with the benefits of laparoscopy, will demonstrate positive impacts upon treatment outcomes. methods: the nationwide inpatient sample was referenced for inpatient stays for the years to . abdominal trauma cases were selected and identified as hollow (ho) or solid organ (so) type, and as blunt or penetrating. the trauma subset was then scanned for the presence of discrete laparoscopic procedures, laparotomy, and converted cases, and flagged accordingly. conclusion: utilization of laparoscopy in treatment of intraabdominal solid and hollow organs injury increases over time. although current analysis based on available hcup nis data include any procedures done during post-traumatic hospitalization, its results can lead to conclusion that minimally invasive technique is being utilized in increased fashion. introduction: single incision laparoscopic (sil) surgery is a laparoscopic procedure which leaves a single small incision in navel, and has been reported to be less invasive than and as safe and efficient as the conventional multiport laparoscopic (mpl) surgery. the long-term rate of incisional hernia after sils colectomy is unknown, and the risk factors of incisional hernia formation is not fully elucidated. methods and procedures: this is a retrospective from a prospectively collected database. the investigation took place in a high-volume multidisciplinary tertiary private hospital in japan. introduction: laparoscopic approach in the acute surgical care setting continues to be underutilized. we aim to report the successful diagnostic and therapeutic use of laparoscopy in the management of a nontoxic patient presenting with acute abdomen and to highlight the benefits of a minimally invasive approach without added morbidity. case report: presented is a -year-old male with history of cad s/p cabgx two years prior and no abdominal surgical history who presented to the ed with sudden onset severe, diffuse, abdominal pain of six-hour duration with n/v. there was no trauma to the abdomen. he had mildmoderate hypertension, but was otherwise hemodynamically stable. on examination, the patient was in severe distress and writhing in pain. fast exam was unable to be performed secondary to pain. cta of the abdomen revealed mesenteric abnormalities with associated small bowel edema in the rlq suspicious for small bowel ischemia. he was taken to the or for diagnostic laparoscopy. he was found to have an omental adhesive band to the abdominal wall with herniation of the small bowel through the small opening. approximately cm of ischemic, nonviable small bowel was resected and anastomosed intracorporeally. he tolerated the procedure well and was discharged home on post-operative day . discussion: primary omental related internal herniation of small bowel is exceedingly rare. there have been only few cases reported in the literature ( , , , ) . two were diagnosed on exploratory laparotomy, one on diagnostic laparoscopy and one at autopsy. the one who underwent diagnostic laparoscopy did not require bowel resection. in presenting this case, we hope to illustrate the role of laparoscopy in the management of acute abdominal pain due to bowel compromise. introduction: morgagni hernias are a rare finding in the adult population, and represent - % of all congenital diaphragmatic hernias. multiple approaches to these rare hernias have been described in the literature. here we present a novel technique of laparoscopic trans-abdominal repair using a combination of the endo-close device (medtronic, minneapolis, mn) and the ti-knot (lsi solutions, victor, ny.) methods: in a patient with a large left anterior diaphragmatic defect we performed trans-abdominal suturing utilizing the endo-close to perform primary closure of the defect, using the ti-knot to secure the pledged sutures along the anterior fascia. due to the size of the defect ( cm) this primary repair was buttressed with polyester mesh. in a second patient with a smaller ( cm) classic right-sided anterior diaphragmatic defect we similarly performed laparoscopic trans-abdominal suturing using the endo-close to traverse both the anterior and posterior fascia and the ti-knot to secure the sutures in order to perform a primary repair of the hernia. both patients presented had an uneventful postoperative course and no indication of recurrence at months. conclusions: morgagni hernias present unique technical challenges. in our experience the combined use of trans-abdominal suture with laparoscopic knot replacement device allowed for completion of both cases laparoscopically with minimal tension on the repairs. feasibility of concomitant laparoscopic splenectomy and cholecystectomy in situs inversus totalis: first case report worldwide ibrahim a salama, md, phd; department of hepatobiliary surgery, national liver institute, menoufia university introduction: situs inversus totalis is a rare anomaly characterized by transposition of organs to the opposite site of the body. combined laparoscopic splenectomy and cholecystectomy in those patients is technically more demanding and needs reorientation of visual-motor skills. presentation of case: herein, we report a year old girl presented with yellowish discoloration and left hypochondrium and epigastric pain diagnosed as hereditary spherocytosis (hs). the patient had not been diagnosed as situs inversus totalis before. the patient exhibit a left sided "murphy's sign" and spleen palpable in right hypochondruim. diagnosis of situs inversus totalis was confirmed with ultrasound, computerized tomography (ct) and magnetic resonant image (mri) with enlarged right sided spleen and presence of multiplegall bladder stones with no intra or extrabiliary duct dilatation. the patient underwent combined laparoscopic splenectomy and cholecystectomy as treatment of hereditary spherocytosis (hs). discussion: feasibility and technical difficulty in diagnosis and treatment of such case pose challenge problem due to the contra lateral disposition of the viscera. difficulty is the laparoscopic technique encountered in skelatonizing the structures in calot's triangle, which consume extra time than normally located gall bladder with right sided standing surgeon and the position changed to left sided standing surgeon during splenectomy. in review up to date medical literature this is the first case reported worldwide. conclusion: provided that the technique is performed by an experienced surgical team, concomitant laparoscopic splenectomy and cholecystectomy in situs inversus totalis is a safe and feasible procedure and may be considered for coexisting spleen and gallbladder disease as in hereditary spherocytosis (hs) as changes in anatomical disposition of organ not only influence the localization of symptoms and signs arising from a diseased organ but also imposes special demands on the diagnosis and surgical skills of the surgeon. objective: to identify the preference among medical students of the following surgical approaches: open surgery, conventional laparoscopy, minilaparoscopy (mini), single incision laparoscopic surgery (sils), natural orifice transluminal endoscopic surgery (notes), and robotic surgery. methods: an online google questionnaire was filled by medical students of different years in medical school. before answering the questionnaire, they watched an online video showing the different techniques, its advantages and disadvantages. the questionnaire consisted of questions about the hypothetical situation where the participants were going to be submitted to an elective cholecystectomy and they could decide which technique they would prefer. all statistical analysis was performed using the r software program, version . . . the chi-squared test was performed for categorical variables where appropriate. a p value . was statistically significant. results: one hundred and eleven medical students answered the survey. ( . %) were female and men. most of the students were between to years old ( . %). they were in the first four years of medical school. when asked if they would consider notes or single incision even if they know that they are new procedures and with not completely established security standards, . % ( ) answered that they wouldn´t consider with no difference between gender (p= . ). when asked if only conventional laparoscopy, robotics or mini were offered, which one they would choose: % of women and . % men chose mini first (p= . ). about the factors that they would consider the most important when choosing the surgical technique, they answered safety first ( . %), followed by the surgeon´s experience with the procedure ( . %), with no statistically significant result between genders (p= . ). when asked if they would consider an open technique even with the other techniques available and compared according to their year in medical school, students closer to finishing medical school would not consider it, with a statistically significant result (p= . ). regarding the most important factors they would consider and compared by year in medical school, safety and experience of the surgeon performed best, with a statistically significant result (p. ). conclusion: among the available surgical approaches, minilaparoscopy tends to be the preference among women medical students who considered safety the most important aspect. the closer they get to the end to medical school, the less they consider the open technique. background: extension of the single incision for the purpose of specimen removal in singleincision plus one additional port laparoscopic surgery (sils+ ) can undermine the merits of sils + , either by increasing wound-related morbidity or by destroying cosmesis. methods: we retrospectively analyzed the clinical outcomes of patients underwent elective sils + anterior resection, either with transanal specimen extraction (tase, n= ) or transumbilical specimen extraction (tuse, n= ), for colorectal cancer from january to june . this study included patients with a tumor diameter less than cm, measured by preoperative computer tomography. results: both groups were similar in patient's basic information and oncologic condition. most surgical data and postoperative clinical variables were comparable between tase and tuse group, except for increasing operative time in tase ( . + . vs. ± . min, p= . ) and reducing wound complications in tase ( % vs . %, p= . ). dosage requirement of narcotic analgesics was not inferior in tase group compare to tuse group. no significant differences were observed in conversion rate, perioperative and overall morbidity between the two groups. conclusion: although sils+ with tase prolonged operative time compare to with tuse, implement of tase is expected to provide benefit of reduced wound-related morbidity in patients with a tumor diameter less than cm. medhat ibrahim, md; al-azhar university, naser city, cairo, egypt purpose: morgagni hernia (mh) is a rare condition. mh is less than % of surgically treated diaphragmatic hernias in infants. there is no specific symptom for the maorgagni hernia. open surgical repair was the golden stander before the introduction of the laparoscopic surgery in the children and infant. there are many different laparoscopic techniques for mh repair have been reported. i report laparoscopic repair of mh in five infants using primary sutures closure with inrta-corporeal knot tying and ethicon secure strap device. this study is an evaluation of the safety and efficacy of this new laparoscopic technique of mh repair in infants with it is short-term outcomes follow up. patients and methods: five infants with mhs underwent laparoscopic repair by hernia sac excision then two primary sutures, non-absorbable proline through the full thickness of the anterior abdominal wall and the posterior rim of the defect, intra corporeal sutures knot tying, ethicon secure strap device which was used to complete the colures of the defect. there was no insertion of chest tube or drain. results: five infants with mh were operated upon. there were males and female. all cases were left side mh, male-female ratio was : . intraoperative and postoperative analgesia requirement was minimal (paracetamole mg/kg/rectal suppository/ hours for the first hours). ceftriaxone mg/kg single dose at the anesthesia induction. all operations were completed laparoscopic. all infants started and tolerated oral regular feeding with in hours from surgery. none of the patients developed intraoperative or postoperative complications. the maximum follow-up was months (mean, months). all patients are in good health without recurrence or port site compilation. conclusion: this easy save technique of mh repair is reducing the operative time and postoperative hospital stay. it is minims the need of postoperative analgesia, anti biotic. the early oral feeding is also a good benefit. the introduction: transumbilical single port laparoscopic appendectomy (tspla) is the most popularized single port surgery in the world. it provides more cosmetic benefits than conventional laparoscopic surgery. however, single port appendectomy requires longer operation time and advanced surgical skills. we aimed to investigate the learning curve for tspla. material and methods: data were collected from patients who underwent tspla by single surgeon between march and february . the learning curve were analyzed using a cumulative sum control chart (cusum) for operation time and complication. results: a total of patients were included in this study. mean operation time is . ± . minutes. there was no open or multi-port conversion. based on cusum for operation time, learning curve were cases. conclusions: tspla is a safe and effective alternative procedure. the learning curve could be overcome safely without major complications. our results suggest that the cases are sufficient to achieve surgical skills for tspla. introduction: anastomotic leakage (al) is a life threatening complication after minimally invasive ivor lewis esophagectomy (tmie ile) and has diverse treatment strategies such as conservative treatment, endoscopic treatment and surgery. however, there is no consensus on which treatment strategy is best. the aim of this study was to analyse various therapeutic strategies for al and their outcomes. methods and procedures: this retrospective multicentre study was performed in three highvolume hospitals. all patients that developed al after tmie ile in the period of january -july were included. the different endoscopic (stenting, clipping and suction-drainage) and surgical treatments and their success-rate were described; success was defined as clinical improvement after primary treatment. primary endpoint was the time until oral feeding was resumed. secondary endpoints were hospital stay and the total amount of surgical, endoscopic and radiologic interventions. results: in total patients that developed al were identified; four patients received antibiotics only. in the remaining patient, endoscopic treatment was performed as primary treatment in %; % received primary surgical treatment. basic variables were similar in these groups. median postoperative day of diagnosis of al was day in the endoscopic-group and day in the surgical-group (p= . ). admission to the icu as a result of the leakage was necessary in % in the endoscopic-group versus % in the surgical-group (p. ). however, median icu-stay was significantly shorter in the endoscopic-group ( days versus days, p= . ). success-rate of the primary treatment was similar; % and % respectively (p= . ). primary and secondary endpoints were comparable for both the endoscopic-and surgical-group; median time until oral feeding was resumed was days and days respectively (p= . ), median total hospital stay days and days respectively (p= . ) and the median number of interventions was in both groups (p= . ). conclusion: endoscopic treatment appears to be a safe and efficient therapy for al after tmie ile. a patient-tailored approach based on the condition of the patient and the morphology of the leak can be adapted to avoid surgery in a selection of patients. this may prevent surgical reoperations and reduce icu admissions. background: lymph node (ln) dissection around recurrent laryngeal nerve (rln) is one of the most important and difficult procedure in esophageal cancer surgery because of high rate of ln metastasis and risk of rln palsy. especially around left rln, the surgical area is far and narrow by thoracic approach which tends to results in insufficient ln dissection. therefore, we tried to remove this ln by imaging lymphatic chain to dissect sufficient ln. surgical procedure: we perform thoracoscopic esophagectomy by semi-prone position using - mmhg thoracic air pressure. after dissection of right rln ln, middle and lower esophagus, encircle the esophagus at the level of bifurcation of bronchus and pull toward right side by tape to dissect the dorsal and left side of upper esophagus. dissect the tissue including left rln ln from trachea by pulling esophagus up to dorsal side and try to move this tissue toward dorsal side of left rln so that this rln ln tissue can recognize as the "lymphatic chain". to increase the mobility of esophagus, cut the esophagus at the level of aortic arch and pull further up this upper esophagus to dorsal side. cut the esophageal branch of rln and separate this lymphatic chain from rln. at the end of thoracic procedure, this lymphatic chain is attached to upper esophagus. after the upper esophagus has pulled out from cervical site, lymphatic chain can easily recognize at the esophageal wall. result: we performed this lymphatic chain procedure in cases. to evaluate this procedure, cases of conventional method by same prone positioned esophagectomy was used for control. there was no statistical difference between these two groups in amount of blood loss (lymphatic chain: conventional= ml: ml, p= . ), rate of rln palsy ( . %: . %, p= . ). although the thoracic operation time was extended in some degree ( min: min, p= . ), number of dissected ln was increased ( . : . , p= . ) and recurrence along left rln has been relatively fewer by this method ( . %: . % p= . ). conclusion: ln dissection around left rln would be easy and sufficient by imaging lymphatic chain. further improvement is needed to secure this procedure and further evaluation should be done to support this data. introduction: to evaluate the role of robotic assisted surgery as part of an appropriate patient work-up and treatment of ipmn and its consistency in terms of perioperative and long term results. few reports described singular minimally invasive procedures for ipmn. this study aims to describe a comprehensive, oncologically adequate treatment of ipmn in a minimally invasive unit with an extremely high robotic penetrance. methods and procedures: we retrospectively analyze our database of resected ipmn between and . this case series includes consecutive, unselected patients: all candidates with a preoperative diagnosis of ipmn were approached robotically. results: among robot assisted pancreatic resections, we identified patients with ipmn. one was excluded for having less than months follow-up, so patients were included and analyzed. they underwent duodenopancreatectomy in cases, distal pancreatectomy in cases and central pancratectomy in . all but one indications followed the most updated available guidelines (sendai from to and fukoka from to ; american gastroenterology association guidelines were used for comparison only). one patient was operated even if the guidelines were suggesting to follow up, because of a strong familiar cancer history. the final pathology for this patient was high grade dysplasia. in another patient we were inside fukoka's recommendations, but outside aga guidelines and the final pathology was adenoma in chronic pancreatitis. postoperative morbidity was . ( low grade complications, one grade a pancreatic fistula, now considered a biochemical leakage only) and mortality was zero. one conversions to open surgery occurred only: a dp in jehowah's witness with a bulky mass behind the portal vein. the mean follow up was months (range: - ), with only one loss to follow up after months for a high grade dysplasia. conclusion: in hepatobiliary pancreatic minimally invasive centers the treatment of ipmn can be grant following the same principles of major cancer centers, with comparable results. large unbiased studies are needed to evaluate if a minimally invasive approach could modify the ratio between operated and surveilled patients. reducing the use of catheters, tubes and imaging after hiatal hernia surgery significantly reduces length of hospital stay sophia s oswald, candice l wilshire, md, brian e louie, md, ralph w aye, md, alexander s farivar, md; swedish medical center introduction: historically, standard post-operative management of patients undergoing laparoscopic hiatal hernia surgery has been placement of a foley catheter and nasogastric tube (ngt) at the time of surgery with removal early on postoperative day (pod) one, at which time an upper-gastrointestinal series study (ugi) would be performed. we initiated a quality improvement project, seeking to assess if we could safely forego placement of foley and ngt along with the ugi, unless clinically indicated. our aim was to determine if this decreased overall length of stay (los), and how often and which demographic of patients needed placement of foley or ngt postoperatively. methods and procedures: we reviewed patients who had undergone laparoscopic hiatal hernia surgery between and under a single thoracic surgeon. patients were excluded for poor esophageal motility (peristalsis \ %), previous esophageal surgery, and presence of a paraesophageal hernia (peh) with over % of the stomach contained in the chest. eligible patients were further stratified into two groups: fast track and non-fast track. fast track was defined as patients who left the operating room (or) with no foley or ngt, and did not receive a routine ugi on pod one. non-fast track was defined as patients who left the or with a foley and ngt and received a routine ugi on pod one. los was measured in hours from the start of surgery to the time of discharge. results: of the patients included, were categorized as fast track and as non-fast track. the two groups were similar in terms of age, gender, bmi and asa; however, the fast track group had fewer paraesophageal hernias and shorter surgery times [table] . the hospital los, however, was significantly shorter in the fast track group, even though there were more postoperative urinary catheters utilized. no patients in fast track group needed an ngt placed or ugi ordered during initial stay. conclusion: in more straightforward laparoscopic hiatal hernia surgery, surgeons can safely forego ngt and foley placement, as well as ugi evaluation the following morning. these initiatives may translate to a quicker discharge from the ward, and may allow safe transition to performing these cases in hour ambulatory outpatient setting. further evaluation of additional interventions and patient education to decrease los are underway. the conclusion: laparoscopic surgery seems to be a safe and feasible option, with long-term benefit for primary tumor resection with metastatic colorectal cancer, but optimal treatment has yet to be defined. the canadian association of gastroenterology (cag) has implemented the colonoscopy skills improvement (csi) program across canada with a goal of improving colonoscopy quality. the programs' efficacy has not yet been formally assessed. this retrospective cohort study was performed on fourteen endoscopists practicing in a tertiary referral center who have undergone csi training between october and december . procedural data were collected before and after csi training. data were extracted from the electronic medical record (emr) and entered into spss version . for analysis. student's t-test was used to compare groups for continuous data; chi-squared tests were used for categorical data. data were collected for a total of procedures; were done before csi training and procedures since csi training. our sample size provided % power to detect a mean difference in adr improvement of %. the most common indication for colonoscopy was family history of colorectal cancer in ( . %) patients. while age ( . yrs v. . yrs, p. ) and gender ( . % male v. . % male, p= . ) were similar, they were statistically different between groups. groups were comparable in terms of indication, and completion rate ( . % v. . %). adr improved significantly after completing the course ( . % v. %, p. ). an improvement was also noted in both polyp detection ( . % v. . %, p. ) and polyp removal ( . % v. . %, p\ . ). we have seen a significant increase in adr at out institution since implementing the csi program. gastric stomach cancer is a rapid major cause of cancer-related death globally, have higher incidence in men and it is noticeable by its heterogeneity. a lot of studies have expressed out the molecular basis of this cancer, include pathogenesis, invasion and metastasis. the invention of new technologies has help to bring out several novel biomarkers that have diagnostic and prognostic value. therefore, this review centers on biomarkers for the early diagnosis, treatment and prognosis of gastric cancer, elaborate the clinical important of serum tumor markers in a patient with this cancer as well as checking the growths, prognosis together with epigenetic changes and genetic polymorphisms. a deep and rigorous search was carried out in pub med/medline using specific words; "gastric cancer", with "tumor marker". our search yielded important reports about related topic from books and articles that were published before the end of september . conclusively, scientists are utilizing time and resource to salvage this nemesis which is of global burden. classical and novel biomarkers are important for treatment as well as pre-post diagnosis of gc. major causes for this disease are cigarette smoking, infection by helicobacter pylori, atrophic gastritis, male sex, and high salt intake. the treatment of which early diagnoses is of important to the management, after pathological diagnoses by stage prognosis and metastatic setting, although the outcome proved not so good includes chemotherapy, and oral medication are oxaliplatin, capecitabine, cisplatin and -fluorouracil ( -fu). introduction: emergent appendectomy is the standard of care in usa based on tradition rooted in theory that delaying surgery allows for progression of disease and poorer outcomes. antibiotic treatment alone has been shown feasible in the treatment of uncomplicated appendicitis. in clinical practice surgical treatment can be delayed due to a multitude of medical and logistical reasons. this study evaluates the relation between timing of surgery to outcomes. methods and procedures: consecutive adult patients undergoing appendectomy in a teaching community hospital were risk stratified using the acs risk calculator. time from imaging to incision defined early and delayed groups. statistical analysis was used to determine association between risk level, timing of surgery and outcomes. results: % of patients in this study were considered high risk. average time to incision was . hours. shorter time to incision was associated with a statistically significant lower length of stay (p. ). for every hours in surgery delay, one day was added to the length of stay. no statistical difference was found between time to incision and other outcome variables of clinical complications, conversion to open appendectomy or frequency of complicated appendicitis. length of stay was longer than predicted by acs risk calculator in both high and low risk groups. a multidisciplinary, obesity-focused approach improves diagnosis of obesity-related illnesses: a new paradigm for the care of patients with obesity roderick olivas, aaron brown, md, racquel s bueno, md, cedric s lorenzo, md; university of hawaii -department of surgery introduction: patients suffering from the burden of obesity are at significant risk for medical problems that lead to premature death and disability. we hypothesize that a multidisciplinary bariatric team will be better equipped to recognize and diagnose these conditions. this study hopes to quantify that a patient focused approach leads to increased recognition of obesity-associated comorbidities, thus improving quality of care and surgical outcomes. methods and procedure: a retrospective medical chart review of patients who underwent bariatric surgery from / / to / / was performed comparing patient problem lists obtained from their primary care providers upon entry into the bariatric program, and the final problem list generated after evaluation by the program's multidisciplinary team. the total number and specific comorbidities identified before and after multidisciplinary team evaluation was analyzed with a paired t-test and manova, respectively. comparison of the number of comorbidities identified against specific patient demographics was conducted using paired t-test. results: a total of patient charts were selected and met inclusion criteria. the sample consisted of % women and % men; the mean age was . ; the mean bmi was . ; % were morbidly obese (bmi ) and % were obese . the total number of comorbidities identified after evaluation by a multidisciplinary team was significantly greater (p=. ), with the average number of comorbidities diagnosed before and after being . and . , respectively. a significant increase (p. ) in the identification of comorbidities before and after evaluation were noted for all demographics, and no disparities regarding gender, age, marital status, employment status, bmi, or ethnicity where identified. conclusion: patients with obesity unknowingly suffer from many obesity-associated comorbidities simply because their health care providers have failed to recognize the existence of these conditions. surprisingly, this include diseases that are highly associated with obesity, such as osa and t dm, for which obese patients should be screened. although the root of this dereliction is yet to be determined, insufficient obesity-focused education and inherent weight bias among providers must be considered. assessment by a multidisciplinary bariatric team resulted in the identification and treatment of an increased number of comorbidities in this patient population. increased recognition of obesity-related comorbidities improves quality of care, which can translate into improved surgical outcomes. introduction: it is known that surgical residents suffer from sleep deprivation. no recent study evaluated the type and number of calls received at night. lately, burn out, depression and suicide have been the subject of interest in studies and media because of the higher rate among the residents compared to general population. the objective of our study is to evaluate junior resident's level of fatigue and the quantity and quality of calls received during on-call nights in general surgery at chus. methods and procedure: transversal study conducted on junior residents that were on-call in general surgery at the chus between april and august , . the participants detailed all the calls received between pm and am on an database created on the application handbase and completed a daily calendar of their on-call night noting all the tasks they did every half hour (surgery/consultation/sleep). the level of fatigue was evaluated at the end of the night at am with a visual analog of sleep scale on a score over points. results: the level of fatigue / (tired) or / (exhausted) was reached in closed to % of the oncall nights. the median number of calls by night was and the median duration of sleep was only . hours. the median lenght of uninterrupted sleep was . hours by night. among the total nights and calls analyzed, % were ''not pertinent'' and % were ''reportable in the morning''. more than % of the nights had at least one call ''not pertinent'' or ''reportable in the morning'' that have interrupted the junior resident's sleep. the level of fatigue was significantly correlated to the number of calls received during the night (spearman's rho=+ . , p. ) and to the number of uninterrupted hours of sleep (spearman's rho=− . , p. ). conclusion: the level of fatigue is very high among the junior residents in general surgery. many of the calls received during the night are not pertinent or could have been delayed to the morning. our results lead us to the conclusion that interventions and recommendations should be made to raise nurses and resident's awareness about the situation to reduce the unnecessary calls and the level of fatigue of the residents. we hope that on-call resident sleep will be better preserved and that will result in fewer health issues for them (burn out, depression, suicide). without interruptions: does twitter level the playing field? heather j logghe, md , laurel milam, ma , natalie tully, bs , arghavan salles, md, phd ; thomas jefferson university, washington university, introduction: frequent interruption of women in conversation has long been noted anecdotally, and studies confirm that women are interrupted more often than men. such interruptions can diminish perceptions of authority and compromise women's self-confidence. on twitter, users cannot be interrupted in the same way they can be in live conversation. thus the platform may provide a means for women to overcome this obstacle. to determine the degree to which women surgeon leaders utilize twitter compared to their male colleagues, we examined the twitter accounts and activity of the leaders of three national surgical societies. methods and procedures: lists of surgeons holding leadership positions in three surgical societies; the american college of surgeons, the academic association of surgery, and the society of american gastrointestinal and endoscopic surgeons, were obtained and duplicate names were deleted. table details the organizations and leadership positions included. the twitter accounts of these leaders were then identified and confirmed by reviewing the accounts for surgical content. account duration was calculated from the join date. the number of tweets, accounts following, followers, and likes were recorded for each account. outliers were defined as two standard deviations from the mean. results: one hundred sixty-eight men and women surgeon leaders were identified. forty-nine percent of the men and % of the women were found to have twitter accounts. mean account durations for men and women were similar, . years and . years, respectively. outliers for total tweets ( men, women), accounts following ( men), followers ( men), and likes ( men) were excluded from analyses. almost all positive outliers were men. there were no negative outliers. overall, excluding the outliers, there were no significant differences between men and women in any metric. conclusion: among leaders in the surgical organizations analyzed, a higher percentage of women than men have twitter accounts. those with the greatest number of tweets, accounts following, followers, and likes, however, are overwhelmingly male. thus, although women in this sample were more likely than the men to have twitter accounts, men were more likely to gain influence through their accounts. increasing women's influence in this public forum may position them as much-needed role models for the current and next generations. surgical societies may help reduce the disparity in women's representation in surgical fields through education of their members on how to use social media. introduction: the aim of this study was to report the perioperative morbidity and short-term outcomes of a case series of robotic-assisted laparoscopic transabdominal preperitoneal (tapp) inguinal hernia repairs. methods and procedures: a retrospective review (january through december ) of patients who underwent either unilateral or bilateral robotic-assisted laparoscopic tapp inguinal herniorrhaphy by two attending surgeons was performed. patient demographics, perioperative morbidity, operative time, and follow-up data were analyzed. results: patient demographics are summarized in table . mean operative times for unilateral and bilateral inguinal herniorrhaphy were . ± . and . ± . minutes, respectively. mean robot console times for unilateral and bilateral inguinal herniorrhaphy were . ± . and . ± . minutes, respectively. postoperative complications included urinary retention ( . %), conversion to open repair ( %), and delayed reoperation ( . %). no major bleeding, surgical site infection (ssi), or mortality was observed. at first follow-up visit ( ± days), symptoms/signs included groin/scrotal swelling ( %), seroma ( %), groin pain ( %), burning ( %), numbness ( %), and persistent urinary retention ( %). % of patients required a second follow-up visit. two patients underwent reoperation for suspected recurrence but instead a cord lipoma was found without a hernia defect. conclusions: robotic-assisted tapp inguinal herniorrhaphy can be performed with operative times and short-term outcomes similar to those published for open technique. the robotic-assisted tapp inguinal herniorrhaphy is a safe and an efficient minimally invasive surgical option with lower ssi risk and better cosmetic results. gunnar nelson, nathan lau, phd; virginia polytechnic institute & state university introduction: the fundamentals of robotic surgery (frs) and fundamentals skills of robotic surgery (fsrs) are universal curriculums covering a range of topics to assure a high level of surgical skills for optimal patient outcomes. this assurance of skills should include management and response to adverse events. thus, we reviewed frs and fsrs to identify any gaps in educational contents pertaining to how surgical teams are trained to handle adverse events in robotic surgery. methods and procedures: we conducted a literature search through google scholar, journal of robotic surgery, and plos one on frs and fsrs from to . we reviewed articles on preparing medical professionals in handling adverse events during robotic surgeries. besides the two curriculums, we also surveyed the literature on the characteristics of the adverse events and responses of the medical team. this literature survey provided a basis for recommending additional education contents to frs and fsrs. results: in our review, the frs contains modules consisting of an introduction to robotic surgery, with cognitive, psychomotor, and team training/communication skills. meanwhile, the fsrs contains different tasks, half of which on human-machine interaction and another half on operative interaction. both curriculums appear to lack contents on managing adverse events in robotic surgery. according to fda data, , adverse events were reported per , surgeries, of which (i) % relates to broken pieces of surgical instruments falling into patients, (ii) . % pertains to burning holes in tissue from electric arching, and (iii) . % relates to unexpected operations of the instrument such as power outage and issues with electrosurgical units. thus, medical professionals should be trained to manage common adverse events in robotic surgery. for frs, augmenting the five current scenarios in the communication section with common adverse events (i.e., broken pieces falling into patients) would minimize complications under abnormal circumstances. for fsrs, the most logical method would be augmenting the operative interaction tasks with adverse events to train medical professionals. conclusion: we discovered universal curriculums on robotic surgery lack education contents for training medical professionals to manage adverse events and out of the , procedures, ( . %) pertained to device malfunction. to protect the patient's health, universal curriculums must incorporate contents preparing medical professionals in responding to adverse events, particularly device malfunctions, during robotic surgeries. introduction: this retrospective study was performed to evaluate the safety and feasibility of the new senhance robotic system (transenterix) for laparoscopic cholecystectomies. we report the first single-institutional experience utilizing this new robotic platform. methods: approximately robotic cholecystectomies were performed using the senhance robotic system. the senhance surgical system is a new robotic platform that consists of a cockpit, manipulator arm and a connection node ( figure ). this new system provides robotic surgery with numerous advantages including eye-tracking camera control system, haptic feedback, reusable endoscopic instruments, and a high configuration versatility due to total independency of the manipulator arms. patients were between and years of age, eligible for a laparoscopic procedure with general anesthesia, had no life-threatening disease with a life-expectancy of less than month and a bmi\ . a retrospective review of a variety of prospectively collected pre-, peri-and postoperative data including but not limited to patient demographics, intraoperative as well as postoperative complications was performed. cholecystectomies were performed by expert level laparoscopic surgeons. results: the standard laparoscopic technique and setup was easily applicable to the senhance robotic system for this particular surgery. operative time and perioperative complications were comparable to reports of standard laparoscopic cholecystectomies. there was no significant learning curve detected in our case series. conclusion: we report the first experience with laparoscopic cholecystectomies using the new senhance robotic system. there were no major perioperative complications and operative time was comparable to standard laparoscopic cholecystectomies well reported in the literature. this case series suggests that the senhance robotic system can be safely and easily used for laparoscopic cholecystectomies by experienced laparoscopic surgeons. background: the ergonomic benefits or robotic surgery for the health of the surgeon are widely touted as benefits of this technique, though concern remains over a perception of increased risk of injury to patients, particularly in the novice robotic surgeon. injury to the bedside surgeon and assistants due to robotic movement can also occur, though not previously reported. we describe a finger fracture to the bedside surgeon due to entrapment between robotic arms and discuss potential risks to the surgeon in robotic procedures. procedure: a distal pancreatectomy and splenectomy was performed utilizing the davinci si system (intuitive surgical, inc., sunnyvale, ca). during the operation, hemorrhage was encountered which required an instrument exchange that was delayed by self-testing failures. after the instrument was validated and advanced into the field by the bedside surgeon, the operator abruptly took control of the device to reposition. the external portion of the active arm was then rapidly and forcefully propelled laterally toward a stationary retracting arm. the bedside surgeon's hand was still engaged on the instrument being inserted and became trapped between the two arms, leading to a right middle finger crush injury. results: the bedside surgeon sustained a fracture to the distal phalanx at the insertion of the flexor tendon with significant hyperextension of the joint. there was temporary paresthesia of the fingertip. while flexor tendon function was preserved and surgery was not required, the surgeon was required to maintain continuous splinting and was unable to return to full duty for a total of weeks. the surgeon has mild residual hyperextension. conclusions: while complications to the patient have previously been attributed to the robotic platform, this case demonstrates that there are other inherent hazards to members of the operative team. as is natural with all indirect visual surgical techniques, the operator becomes intensely focused on the internal view and instruments in the field. this spatial separation is accentuated on the robotic platform as the isolated console provides a complete visual field immersion, no tactile feedback, and a disconnect between the rapid, sizeable outward arm motions need to produce small internal movements. given the need for maximum dexterity internally, the device doesn't have external proximity sensors to prevent arm-arm or arm-operator collisions. while many bedside operators report anecdotes of collisions with the device, this case reveals the forces involved at the human-machine interface can lead to more significant injuries. robtic approach to non-midline abdominal wall hernias: a single institution experience from a high volume center emily benzer, do, j. stephen scott, md, facs; university of missouri introduction: the objective of our study was to evaluate our experience with robotically repaired non-midline abdominal wall hernias at a high-volume robotic surgery program. we also will discuss the technical advantages of the use of robotic technology in repair of these unusual hernias which have typically had higher recurrence rates then midline hernias. laparoscopic approach for lateral ventral abdominal wall hernia (spigelian) and lumbar hernia has been described, however the success of robotic assisted repair for these hernias has yet to be determined. methods: a retrospective case analysis of all robotic abdominal hernia cases between june and june at an academic institution with a single high volume robotic surgeon was performed. the operative details of robotic repair of non-midline abdominal hernias, patient demographics, length of stay and smoking status were recorded and analyzed. the technical advantages of the use of robotic technology for example circumferential fixation of the mesh, ease of intracorporeal suturing, and the use of wristed instruments to gain better angles for posterior fascial release were evaluated. results: a total of cases were identified. the average age of the patients was . years (range - years) and patients were predominantly female ( %). spigelian hernias represented % (n = ) and lumbar hernias % (n= ). all patients had primary closure of their defect and patients ( %) had a posterior myofascial release performed. mesh types placed included polypropylene uncoated (n= ), polypropylene coated (n= ), and biologic (n= ). with uncoated polypropylene mesh placed had peritoneum closed over the mesh. the average length of stat was . days (range - days). there were no recurrences identified over a mean follow up period of . months (range . - . months). conclusion: robotic assisted repair of non-midline abdominal wall hernias is a viable option in the elective setting with no recurrences noted in this case series. the technical advantages of using robotic technology were identified and discussed in detail. these advantages theoretically improve outcomes in these patients however further analysis on long-term outcome and costs will have to be determined in future studies. the inguinal hernia repair has seen several critical improvements in recent times due to the implementation of new techniques, including laparoscopic repair, as well as robotic repair. with over , inguinal hernia repairs performed annually, it is important to identify the safest and most patient-friendly method. for surgeons, robotic assisted laparoscopic surgery is gaining in popularity for its dexterity and d visualization. but despite the growing interest in robotic hernia repairs, there is a scarcity of literature to support its superiority over open inguinal hernia repair. this study hypothesizes that patients who undergo robot assisted laparoscopic inguinal hernia repair will have decreased immediate post-operative pain, shorter recovery room stays, decreased narcotic requirement, and overall decreased pain at follow up compared to open inguinal hernia repair. in this study, we performed a retrospective analysis of patients who underwent either an open or robotic assisted laparoscopic inguinal hernia repair at stamford hospital, from july -july . the following characteristics were analyzed for both subsets of patients: gender, bmi, type of repair, operative time, recovery room time, immediate post-operative pain, and post-operative pain at follow up. our study demonstrated longer average operative time for patients undergoing robotic hernia repair compared to open repair, which was statistically significant (p value=. ). patients who underwent robotic inguinal hernia repair spent less time in the recovery room compared to patient who underwent open repair. in addition, patients in the robotic hernia group required less narcotics in the recovery room compared to patients who underwent open repair (p value = . ). there was no statistically significant difference between lengths of hospital stay between the two groups. this study highlights several possible advantages of robotic inguinal hernia repair, including lower post-operative pain scores, less narcotic usage required in the post-operative period, as well as shorter recovery room time. the results from this study should increase interest in investigating the superiority of robotic inguinal hernia repair. future plans for study involve comparing robotic to laparoscopic repair. in addition, we plan to continue to follow the study patients to look at additional qualitative metrics, including time to return to work and time to return to daily activities. introduction: buccal mucosal grafts (bmg) are traditionally used in urethral reconstruction. there may be insufficient bmg for applications requiring large amounts of graft, such as urethral stricture after gender affirming phalloplasty. rectal mucosa is an alternative with less post-operative pain, no impairment in eating and speaking, and larger graft dimension. laparoscopic transanal minimally invasive surgery (tamis) has been described by our group. due to the technical challenges of harvesting a sizable graft within a confined space, we adopted a new approach using the intuitive da vinci xi® system. we demonstrate the feasibility and safety of a novel technique of robotic tamis (r-tamis) in the harvest of rectal mucosa for the purpose of onlay graft urethroplasty. methods and procedures: irb approval was obtained. three female-to-male transgender adults (age range: - years) presenting with post-phalloplasty urethral strictures underwent robotic rectal mucosal harvest. the procedure was first rehearsed on an inanimate model using bovine colon. the surgery was performed under general anesthesia with the patient in lithotomy position. the gelpoint path transanal access platform was used. the rectal mucosa was harvested by the robotic instruments after submucosal hydrodissection. specimen size harvested correlated with clinical surface area needed for urethral reconstruction. following specimen retrieval, flexible sigmoidoscopy was used to ensure hemostasis. the rectal mucosa graft was placed as an onlay for urethroplasty. results: there were no intraoperative or postoperative complications. average graft size was cm (range: - cm). every case had excellent graft take for reconstruction. all patients recovered without morbidity or mortality. they reported minimal postoperative pain and all regained bowel function on the first postoperative day. all reported significantly less postoperative pain and greater quality of life in comparison to prior bmg harvests. the procedure has been refined to increase efficiency and decrease operative time by maintaining adequate insufflation, retraction of the mucosal graft, and maintaining graft integrity. conclusions: to our knowledge, this is the first use of r-tamis for harvest of rectal mucosal graft. our preliminary series indicates the robotic approach is feasible and safe. it constitutes a promising minimally-invasive technique to employ in urethral reconstruction. demonstrated feasibility and avoidance of the challenging recovery associated with bmg harvest warrants further application and long-term evaluation of this procedure. prospective studies evaluating graft success, donor site morbidity and long-term outcomes are needed. introduction: the proportion of robotic minimally invasive procedures that are being performed annually is growing rapidly, specifically in the field of general surgery. a robotic approach to minimally invasive procedures potentially confers a number of benefits ranging from a magnified viewing field to greater attenuation and translation of hand movements leading to improved stability and maneuverability. it is paramount that a robust curriculum is designed for training surgical residents in robotic techniques. the aim of this project is to assess the current state of robotic surgery training at the ohio state university, with specific regard to whether it is currently temporally effective in addition to establishing a baseline against which the robotic surgery curriculum can be compared. methods and procedures: data were obtained for cases performed at the ohio state university hospital east, between january and september of . case time, date, type, and attending surgeon were recorded and tracked for review. of the cases, were cholecystectomies, were unilateral inguinal hernia repairs, and were bilateral inguinal hernia repairs-for a total of procedures included in the analysis. chief residents were trained in two-month blocks, beginning in january of . mean console operative times for the first and second months were compared for cholecystectomies as well as unilateral and bilateral inguinal hernia repairs. results: mean console time decreased for cholecystectomies (− . %; n= ), bilateral (− . %; n= ) and unilateral (− . %; n= ) inguinal hernia repairs from month one to month two. there was a large amount of variance across training blocks, but there was a systematic improvement in operative time across the training period. average operation length was shortest for cholecystectomies (m= . min), followed by unilateral inguinal hernia repairs (m= . min), and finally bilateral inguinal hernia repairs (m= . min). discussion: this preliminary data suggests that residents are able to decrease their robotic operation time over the course of the two-month rotation. although sample sizes were relatively small for each block, the consistency of the trend supports this conclusion. further data collection will allow for more precise estimates in the future, and stronger conclusions to be drawn. these results show that rapid improvement is possible and provide motivation to establish robotic surgery curricula for general surgery residents nationally. robotic pancreas-sparing treatment of pancreatic neuroendocrine tumors: three case reports and review of the literature alessandra marano, giorgio giraudo, stefano giaccardi, desiree cianflocca, diego sasia, felice borghi; santa croce e carle hospital introduction: pancreas-sparing resections would be the ideal procedure in case of small pancreatic neuroendocrine tumors (p-nets) reducing the risk of exocrine and endocrine insufficiency. compared to standard resection, this type of surgery is safe and feasible without increasing the risk of postoperative complications except the overall rate of clinical pancreatic fistula (pf), which did not result in higher mortality or overall morbidity. robotic surgery for pnets enucleation has been rarely described but initials experiences have shown that this approach is associated with favorable outcomes. the aim of this study is to describe three cases of dv®si™ pancreatic enucleation for p-nets located in the uncinate process, in the body and in the posterior aspect of the tail of the pancreas, respectively. a brief review of the literature regarding the application of robotics for pnets enucleation is also included. methods and procedures: this study includes patients undergoing dv®si™ enucleation for pnets with a maximum diameter no more than cm and a distance between tumour and main pancreatic duct (mpd) greater than mm. at surgery, exposure of the pancreas was achieved by separation and traction of the gastrocolic and gastropancreatic ligaments. the pancreas was explored: an intraoperative ultrasound was used ensuring negative margins and leaving the mpd intact. thus, a cross-stitch through the tumour was made routinely in order to pull the tumour. enucleoresection was carried out with monopolar scissors and bipolar forceps. the tumour was placed into a specimen bag and removed from the trocar port. a drain was always left. results: median total operative time was min. no conversion neither intraoperative complications occurred. median length of stay was . days. two patients presented a pf grade a (classification isgpf) while a pf grade b occurred in case of pancreatic tail net enucleation. final pathology revealed two insulinomas and one non-functioning net of the pancreatic body. at a median follow-up of months no pancreatic insufficiency, reoperation or tumour reoccurrence was observed in all cases. the robotic approach for the treatment of p-nets is safe and feasible and, in selected cases, it may extend the indications of minimally invasive pancreatic-sparing surgery. in particular, the robotic approach provides a more precise dissection and may ensure negative margins and the mpd intact. these preliminary results are consistent with literature data about over robotic pancreatic enucleations for p-nets that shows favourable surgical outcomes, especially if compared with those of open surgery. introduction: rectal cancer continues to be a surgical challenge. new technologies must be incorporated into practice and, at the same time, oncologic surgery and overall outcomes must be improved. the use of da vinci robotic surgery systems has spread rapidly in the field of rectal cancer treatment showing several technical advantages and favorable outcome compared to laparoscopy. since the introduction of the robotic platform in our institution in , we have adopted a single-docking robotic technique for rectal resection. the aim of this study is to present our standardized technique and to analyse the clinical outcomes of the first robotic rectal procedures. methods and procedures: prospectively collected data reviewed from consecutive patients who underwent single docking totally robotic (da vinci® si™) dissection for rectal cancer resection between june and august under eras program. robotic rectal surgery was performed without changing the position of the robotic cart but only the robotic arms are repositioned between two phases: ) vascular ligation, and sigmoid colon to splenic flexure mobilization; and ) pelvic tme. results: there were men ( %) and the median age was years (range- - ). thirty-five patients had neo-adjuvant chemoradiotherapy whilst patients had bmi [ . procedures performed included anterior resection (n= ) and abdominoperineal resection (n= ). protective ileostomy was performed in patients. the median operating time was min (range- - ). there was one conversion and two intra-operative complications (one bladder lesion and one ureteral lesion, respectively). median length of stay was . days (range, , and readmission rate was %. thirty-day mortality was zero. anastomotic leak rate was %, and all patients except by one were managed conservatively. the mean lymph node harvested was (sd± . ). radial margin was negative in all patients. at median follow-up of months, there were no local recurrences. the single docking robotic technique is a safe and feasible approach for rectal surgery: in our study it has demonstrated favourable clinical outcomes and the adoption of a standardized stepwise approach was useful especially during the initial learning phase. to the best of our knowledge, this is the largest series from italy to report this standardized approach and the short-term clinical and oncological outcomes. in the complex laparoscopic surgical procedure, there is a problem such as that the laparoscope and the surgical instruments interfere with each other because multiple instruments is concentrated in one place. this problem is significantly appear in the laparoendoscopic single site surgery. therefore we suggested multi degrees of freedom (dof) manipulator with mantle tube for assisting laparoendoscopic surgery, which manipulator has two flexion and one telescopic mechanisms actuated by wire. it is possible to insert any thin surgical instruments such an endoscope the mantle tube of the multi dof manipulator, which the manipulator can let those surgical instruments access the operative field from different axis with other instruments. the use of this manipulator has two advantages, one of which is avoidance of fighting between instruments and laparoscope. the other is that become possible to ensure a satisfactory field of vision in the operative field. in this report, we assumed that this multi-dof manipulator is used as laparoendscope. in order to evaluate the performance of this manipulator, the operation time of the test in the abdominal cavity simulator (fasotec inc.) was measured. the test is a contact test to multiple-targets, which is a test that bring a forceps contact multiple-targets in the abdominal cavity simulator according to the defined pattern. as a general comparison and evaluation target for this measurement result, it is compared with the case using the same access method as the conventional rigid endoscope. in this test, the number of contacts between forceps and laparoendoscope were recorded by using electrical device. subjects (n= ) are adult men who trained the peg transfer in the above simulator. it was compared of total operating times of the test and the field of vision obtaining each device. from these results, using the suggested manipulator device rather than using rigid laparoscope a satisfactory field of vision is obtained, and it is possible to short the operating time approximately seconds, and to small the number of contacts significantly. therefore it was shown that the effectiveness using the suggested manipulator device. for this reason, use of this device is expected to facilitate the complex surgical operation. additionally, it is performed para ablative operation of swine liver tissue in the abdominal cavity simulator, as previous step of clinical test. the operative field in this test was surveyed, the refinements of this manipulator for improvement performance were described in this report. yoshiyuki usui, md, phd, ichiro akiyama, md, phd, hironori kunisue, md, phd, hideaki mori, md, phd, tetsuya ota, md, phd; okayama medical center background and methods: we have performed approximately cases of gasless endoscopic thyroid surgery since for years. this surgery was performed through a small subclavian incision and using a wire traction and inserting an endoscope. we have modified and improved our surgical techniques by inventing various surgical instruments. here we introduce four newly invented surgical instruments, chronologically. results: we made u-retractor ( ), u-trocar ( ), u-kelly forceps ( ), and u-suction tube retractor ( ). all surgical instruments were modified from conventional surgical instruments. the u-retractor was a piercing retractor, each end of which had a sharp tip and a retractor. this retractor was inserted from the -cm working port outside the body and retracted the muscles effectively. the u-trocar was reversely set from inside to outside to make the working space wider. the u-kelly forceps which had a special ratchet were made to dissect loose connective tissue around the thyroid gland avoiding injury of the recurrent laryngeal nerve. the u-suction tube retractor facilitated a wider working port and eliminated the mist created by the ultrasonically activated scalpel effectively. recent data showed no difference of operative time, hoarseness, blood loss and hospital stay between conventional thyroid lobectomy and gasless endoscopic lobectomy. conclusion: gasless endoscopic thyroid surgery has been improved in the last years. this procedure made the excision of not only benign thyroid tumors but also small thyroid carcinomas. this operation is still cost effective, because almost all surgical instruments are reusable and is a satisfactory experience to both the patients and surgeons. objective: to put forward the importance of complete (r ) resection for the treatment of retroperitoneal tumors increasing overall survey. methods: in this study; patients having the diagnosis of retroperitoneal tumors with different histopathological subtypes whom were hospitalized in emergency surgery department of istanbul medical faculty between the years of and were evaluated retrospectively. the database of the department was analyzed. operational backgrounds, histopathological results, radiological evaluations, and assesments about relapses, and overall survey were obtained from the medical archieve. results: the average follow-up time was , years. all of the patients included into the study were undergone operations. the average time of hospital stay was calculated as days. of the patients were found to have positive surgical margins in their histopathological evaluations. overall mortality rate of the study was % ( / ). we have observed a direct correlation between complete (r ) resection and disease-free survival. patients having relapses had worse prognosis in terms of overall survey ( % mortality rate). after having done the statistical evaluation, surgery was found to be the main determining factor for the assesment of overall survey. conclusion: reference to an experienced and multidisciplinary surgical center after an early diagnosis has upmost importance for the treatment of retroperitoneal tumors. surgical approach constitutes the main element in the management. overall survey is directly correlated with complete (r ) resection. novel fluorescent dyes for real-time, intraoperative, organ-specific visualization of biliary and urinary systems using dual-color near-infrared imaging ; children's national health system, nih/nci p multidisciplinary approach for management of necrotizing pancreatitis: a case series prabhu senthil-kumar university of alberta, centre for the advancement of minimally invasive surgery introduction: the objective of this study was to systematically review the bariatric surgery literature to understand how weight loss is reported. the incidence of obesity has increased globally. according to the world health organization more than million were obese in . in the last decade, bariatric surgery has been increasingly utilized as an effective treatment option for severely obese patients. currently, bariatric surgeries are among the most commonly performed operations. the primary outcome of such procedures is weight loss which has been shown to vary according to the type of surgery. however, there are different methods used to report weight loss which makes it difficult to directly compare outcomes between studies. a previous review by dixon et al. in revealed a wide heterogeneity in weight loss reporting. however, there have been no recent reviews on the reporting of weight loss in bariatric surgery. methods: a search of the medline electronic database was performed for studies published in using search terms gastric bypass/sleeve gastrectomy, weight, human, and english. articles were selected by two independent reviewers based on the following inclusion criteria: ( ) adult participant ≥ years predictive factors for excess body weight loss after bariatric surgery in japanese obese patients takeshi naitoh hypertension resolution after rapid weight loss: a single institution experience cristian milla matute reoperative bariatric surgery: analysis of indications and outcomes: a single center experience iman ghaderi objective: to observe the effects of duodenal-jejunal transit on glucose tolerance and diabetes remission in gastric bypass rat model. method: in order to verify the effect of duodenal-jejunal transit on glucose tolerance and diabetes remission in gastric bypass, twenty-two type- diabetes sprague-dawley rat model established through high fat diet and low dose streptozotocin (stz) administered intraperitoneally were assigned to one of three groups: gastric bypass with duodenal-jejunal transit (gb-djt n= ), gastric bypass without duodenal-jejunal transit (rygb n= ) and sham (n= ). body weight, food intake, blood glucose, as well as meal-stimulated insulin, and incretin hormones responses were assess to ascertain the effect of surgery in all groups. oral glucose tolerance test (ogtt) and insulin tolerance test (itt) were conducted three and seven weeks after surgery. results: comparing our gb-djt to the rygb group, we saw no differences in the mean decline in bodyweight, food intake, and blood glucose -weeks after surgery. gb-djt group exhibited immediate and sustained glucose control throughout the study outcomes with sham operation did not differ from preoperative level. conclusion: preserving duodenal-jejunal transit does not impede glucose tolerance and diabetes remission after gastric bypass in type- diabetes sprague-dawley rat model is bariatric surgery effective for comorbidity resolution in super obese patients? methods: a retrospective analysis of outcomes of a prospectively maintained database was done on obese patients with a diagnosis of at least one or more of the following comorbidities-t dm, htn, osa, or hld-at the time of initial visit who had undergone either a sleeve gastrectomy (sg) or a roux-en-y gastric bypass (rygb) at our hospital between and . the patients were stratified based on their preoperative body mass index (bmi) class: bmi methods: we retrospectively reviewed all patients that underwent laparoscopic sleeve gastrectomy (lsg) at our institution from - . common demographics and comorbidities were collected as well as creatinine, preoperatively and up to hours after surgery. the renal function was calculated using the ckd-epi formula, derived and validated by levey et al. acute kidney injury was defined as an increase in serum creatinine by ≥ . mg/dl within hours after surgery. all tests were two-tailed and performed at a significant level of . . statistical software r, version . . ( - - ) was used for all analyses. results: of the patients reviewed conclusion: the impact of laparoscopic sleeve gastrectomy in renal function is evident within the first hours after surgery. patients undergoing lsg, especially patients with baseline chronic kidney disease stage ≥ are at increased risk of developing acute kidney injury in the perioperative setting the body mass index (bmi), fasting plasma glucose (fpg), glycosylated hemoglobin (hba c), serum triglyceride, serum cholesterol and blood pressure of all patients were measured before and at months after surgery. the results were collected and analyzed. results: patients suffered from metabolic disease undertook lsg surgery successfully (a mean age of years), were male and were female. all of patients suffered from obesity and the mean bmi of them was . ± . kg/m before surgery. among them, patients had type diabetes mellitus (t dm), patients had hypertriglyceridemia (htg), patients had hypercholesterolemia (hc) and patients had hypertension. the mean bmi of patients at months after surgery was . ± . kg/m and decreased significantly (p. ). the mean excess weight loss (ewl%) of patients was . %± . %( %* %) at months after surgery. the average levels fpg, hba c of t dm patients at months after surgery were . ± . mmol/l, . %± . % methods: we retrospectively reviewed all patients who underwent bariatric surgery from to . we assessed kidney function using the chronic kidney disease epidemiology collaboration (ckd-epi) and cardiovascular risk using framingham risk score (frs) equation pre-operatively and at and months of follow-up. our population was divided into two groups: patients with ckd stage ≥ (gfr\ ml/min) and patients with normal gfr. significance. results: of the , patients reviewed, . % (n= ) met the criteria for ckd-epi glomerular filtration rate (gfr) and framingham risk score (frs) calculations. after matching, patients ( . %) were left to analyze, % (n= ) of which had a laparoscopic sleeve gastrectomy. eighty-six patients ( %) had an impaired kidney function (ckd≥ ) (group ) and patients ( %) had a normal gfr (group ). common demographics and comorbidities after matching are described in table . the mean creatinine in group was . ± . mg/dl versus . ± . mg/ dl in group (p
). glomerular filtration rate was . ± . ml/min in group and ± . ml/min in group . furthermore, when the frs was calculated at months follow-up, patients with impaired kidney function had an absolute risk reduction of . % corresponding to a relative risk reduction (rrr) of group . the percentage of estimated bmi loss was found to be similar in both groups ( . ± . and . ± . respectively p= . ). conclusions: bariatric surgery, especially lsg, has a positive impact on kidney function particularly in patients with chronic kidney disease stage or greater. despite these patients having a higher preoperative cardiovascular risk, they showed similar risk reduction when compared to patients with normal kidney function at months of follow-up the impact of socioeconomic factors and indigenous status jerry t dang only ( . %) patients underwent urgent conversion for management of complications after sg. three patients had intraoperative complications necessitating blood transfusion. fourteen ( . %) patients required readmission within days postoperatively. six patients ( . %) required surgical interventions including for gastrointestinal leak, for hemodynamic instability, for a cecal perforation, and for a small bowel obstruction. there were no mortalities within the first year of revisional surgery. in patients with bmi[ kg/m at the time of revisional surgery, at the median postoperative follow-up of (interquartile range, - ) months, a median (interquartile range, - ) kg/m reduction in bmi was observed. overall, ( . %) patients had persistent type diabetes at time of revisional surgery. improvement of diabetes was observed in patients ( . %) after conversion of sg to rygb. among patients with gerd symptoms, subjective symptomatic relief was reported at the last follow-up. conclusion: weight recidivism is the most common indication for revision of sg objective: to evaluate laparoscopic mini-gastric bypass in the treatment of morbid obesity. method: three hundred patients with a mean bmi of . . kg/m underwent a laparoscopic mini-gastric bypass between to . a laparoscopic approach with five trocar incisions was used to create a long narrow gastric tube; this was then anastomosed ante-colically to a loop of jejunum cm. distal to the ligament of treitz peri-operative and short-term follow-up results up to does age or preoperative bmi influence weight loss after bariatric surgery? one-way anova or the kruskal-wallis test was used to compare continuous data across all groups. subsequent analysis of categorical data was achieved by chi-square or fisher's exact test. statistical significance was accepted as p. . results: a total of patients ( % male) were analyzed. average age and preoperative bmi were . ( . ) years and . ( . ) kg/m , respectively. preoperative comorbidities included: diabetes ( . %), hypertension ( . %), hyperlipidemia ( . %), previous myocardial infarction ( . %), obstructive sleep apnea ( . %), chronic obstructive pulmonary disease ( . %), gastroesophageal reflux ( . %), tobacco use ( . %). the asa classes of patients undergoing sg were ii ( . %), iii ( . %), and iv ( . %). the follow up rate at , and months was . %, . %, and . %, respectively. the -day mortality and readmission rate were % and . %, respectively. the %ewl was not different among age groups at , or months for the total, male, or female cohorts. among preoperative bmi groups, %ewl was not different in any cohort at or months, but was different at months for the total cohort (p. ) and female cohort (p\ . ), and trended toward significance in the male cohort (p= . ). the highest %ewl was found to be in patients with preoperative bmi of - . there was no difference in -day mortality or readmissions among groups a crp≥ mg/dl had a sensitivity for a complication of % and a specificity of %. primary bariatric surgery patients with a post-operative complication had higher crp levels compared to those who did not ( . ± . mg/dl vs . ± . mg/dl; p= . ). there was no difference in crp levels for patients with a -day reoperation or readmission. there were no mortalities. conclusions: bariatric surgery patients with elevated post-operative crp levels are at increased risk for -day complications. the low sensitivity of a crp≥ mg/dl suggests that a normal crp methods and procedures: the patients, who formed the previously published cohort, were contacted and their charts were reviewed. follow-up visits, symptom severity scores, and any subsequent medical or surgical interventions were collected. symptoms were assessed using the symptom severity score (sss) and the gastroparesis cardinal symptom index (gcsi) questionnaires. success was defined as a sss of or less. results: out of original patients, patients ( males, females) were available for follow-up ( patients declined participation, were lost to follow-up, patient was deceased, and was excluded after undergoing esophagectomy for unrelated indication) mbbs ; grant government medical college and sir jj government hospitals methods and procedures: twenty-six nh patients with dm were prospectively randomized to undergo either lrygb or lsg. patients were followed for -years with primary end points consisting of total weight loss (twl), percent excess body weight loss (%ebw) and impact on dm as measured by fasting blood glucose (fbs) and hba c. in addition, baseline, week, and , , , , and months post-operative levels of glucagon-like peptide (glp- ), peptide yy (pyy), leptin, and ghrelin were collected. results: a total of / patients completed follow-up. the %ebw at year for lrygb and lsg were % and %, respectively. resolution of dm occurred in / patients, the remaining three subjects were in the lgs arm. pre-operative fbs in lrgyb and lsg groups, were and , respectively. pre-operative hba c in the lrygb and lsg groups, were . and . , respectively. fbs at year for lrygb and lsg were and , while hba c for lrygb and lsg were . and . , respectively. a consistent post-operative decrease in fbs was only seen in lrygb. lrygb ghrelin percentages increased at , , and months, while levels decreased in lsg. leptin percentages decreased in both groups. the ppy levels remained relatively unchanged in both groups. lrygb glp- levels increased at week, , , and months. lsg glp- trends were similar except at months where glp- levels decreased. conclusion: lrygb and lsg resulted in equivalent post-surgical weight loss and resolution of dm in the nh population video assisted thoracoscopic thymectomy (vats) has emerged as a minimally invasive alternative to the standard transsternal approach. we present herewith the surgical and neurological outcomes after vats their operative time, blood loss, conversion rate and post operative parameters like intensive care unit (icu) stay, inter-costal drainage (icd) indwelling time, hospital stay were recorded. neurological outcomes were assessed based on myasthenia gravis foundation of america (mgfa) post intervention status classification. statistical analysis was done using stata software. results: ninety patients underwent thoracoscopic thymectomy during the study period. vats was done through right approach in ( . %), left approach in ( %) bilateral approach in patients ( %) and subxiphoid approach in ( . %). there was conversion to open approach in ( . %) patients due to dense adhesions at westchina hospital of sichuan university were included. all of the operations were performed by a single skilled surgeon. we divided our patients into two groups based on whether isao was used. of them, patients received isao for lps and patients received lps without isao. surgical skills and safety were evaluated. results: there were no significant differences in preoperative patients characteristics of the two groups. significantly less intraoperative blood loss( . ± . ml vs . ± . ml; t=− . , p= . ) were observed in group of isao conclusions: isao is technically feasible, safe surgical skills for patients reveived lps, and its represents an effective method to decreased intraoperative blood loss. p modular laser-based endoluminal ablation of early cancers: in-vivo dose-effect evaluation and predictive numerical modelling giuseppe endoscopic submucosal dissection enables en-bloc removal of early gastrointestinal neoplasms. however, it is technically demanding and time-consuming. laser-based ablation (la) techniques, are limited by the lack of depth penetration control and thermal damage (td) prediction. our aim was to evaluate a predictive numerical modelling (pnm) of the td to preoperatively select the optimal power and exposure time enabling a controlled ablation down to the submucosa (sm). additionally, the ability of confocal endomicroscopy (ce) to provide information on the td was assessed at the histology, there was an increased damage depth per higher j applications. the r value at . j was . ± . , and was significantly lower when compared to energies from j (r= . ± . ; p. ) up to j ( . ± . ; p\ . ). safe m and sm ablations were achieved applying lower p settings ( . and w), at different t values, leading to an mp impairment only in and % of the cases, respectively. ce provided relevant images of the td, consisting in architecture's distortion and disappearance of the gland's contours. the predicted damage depth we also analyzed early gastric cancer patients who received lpg-ip with cm jejunal interposition. anastomosis procedure was overlap method for eshophagojejunostomy and gastrojejunostomy, feea for jejuno-jejunostomy. results: the comparison between otg/opg-ip shows no significant difference in perioperative complications and qol scores, significant smaller body weight loss in opg-ip group. lpg-ip group also shows good result in short term outcomes. consideration: as comparison in open surgery implies superiority in jejunal interposition, we have introduced lpg-ip. esophagogastrostomy after proximal gastrectomy is simple but has a risk for sever gerd symptoms, no optimal procedure for reconstruction after proximal gastrectomy has yet been established. although laparoscopic jejunal interposition is relatively complicated in procedure, we can safely perform in combination with common anastomosis techniques. conclusion: body weight loss in otg-ip group is smaller compared to otg group consecutive patients with early gastric cancer underwent solo spdg (n= ) and mldg (n= ) performed by same surgical team. solo spdg can be defined as practice in which a surgeon operates alone using camera holder. mldg usually requires two or three surgical assistants. the inclusion criteria in this study were (i) pathologic proven stage i-ii gastric cancer (ii) no other malignancy (iii) more than d lymph node dissection (iv) r surgery. one-to-two propensity score matching was performed to compensate for the differences between two groups. results: after the propensity score matching, solo spdg (n= ) and mldg (n= ) patients were selected. mean operation time ( ± . vs ± . mins, p= . ) and estimated blood loss (ebl) ( . ± . vs . ± . ml, p= . ) were significantly lower in the solo spdg group than in the mldg group. the hospital stay and the use of pain control were similar between the two groups. although the initiation of semi fluid diet was similar, the time to first flatus was earlier in the solo spdg adhesional omental hernia: a case report an unexpected cause of small intestinal obstruction in crohn's disease strangulation inguinal hernia due to an omental band adhesion within the hernia sac: a case report omental adhesion, intestinal herniation, and unexpected death in the elderly small bowel obstruction secondary to greater omental encircling band-unusual case report the median operative time was min. the median postoperative hospital stay was . d. histological examination of the tumors revealed carcinomas, adenomas, and carcinoid. complications occurred in ( %) patients, viz., ssi (two patients), pancreatic fistula (two patients), bleeding (two patients), passing failure (one patient), and cholangitis (one patient). however, no severe postoperative complications (clavien-dindo classification grade or higher) were reported in these cases. conclusion: our cases showed that duodenal tumor resection using lecs enables curability through a minimally this study aimed to compare the outcomes of tltg with those of latg by using a meta-analysis. methods: we searched pubmed, embase, and cochrane library in may, to locate prospective or retrospective studies on surgical outcomes of tltg versus latg. the outcome measures were postoperative complications such as anastomosis leakage and anastomosis stenosis, operation time, blood loss, time to flatus, time to first oral intake, and postoperative hospital stay endoscopic thyroid lobectomy: our early experience at tertiary care hospitals of lahore univariate analysis was performed followed by logistic regression to identify independent predictors for the primary outcome. results: forty-six out of ( %) patients referred for gp required jt insertion to treat malnutrition. etiology of gp included: % idiopathic, % diabetic, % post-surgical. thirty-six patients ( %) reported severe daily symptoms. twenty-five patients ( %) had successful return to oral intake while ( %) required prolonged feeding access, reinsertion of a jt or tpn initiation. on multivariate analysis patients who had a pyloroplasty (p= . , or . ) and those who were married (p= . , or . ) were found to be independent predictors of successful discontinuation of tube feedings. on subgroup analysis -hour gastric emptying time normalized after pyloroplasty (p= . ) in patients which had a successful re-initiation of oral intake while persistent gastric emptying refractory to pyloroplasty was associated with failure. the group of patients who underwent pyloroplasty did not differ in terms of demographics, marital status (p= . ) and preoperative gastric emptying (p= . ) from those who did not. gp etiology (p= . ) psychiatric conditions (p= . ) and substance abuse laparoscopic transabdominal repair of morgagni hernia rebekah macfie average procedure length was . minutes. average hospital length of stay was . days, with all patients tolerating a regular diet prior to discharge. our -day readmission rate was / ( . %). / ( . %) patients required repeat egd evaluation for either recurrence of symptoms or impacted food bolus. at week follow-up, / patients ( %) complained of dysphagia and / patients ( %) had eliminated ppi from their daily medication regimen. at month follow-up, / patients ( %) complained of dysphagia and / patients ( %) had eliminated ppis. at year follow-up, / patients ( %) complained of dysphagia and / patients ( %) had eliminated ppis. conclusion: as a recently introduced surgical option, no long-term data exists detailing the linx procedures ultimate success rates and complication profile mini-laparoscopic vs traditional laparoscopic cholecystectomy: preliminary report deniz atasoy since the introduction of minilaparoscopic cholecystectomy (mlc) in , it gained little interest that could be attributed to decreased durability of the reduced size instruments, poorer optical resolution and smaller jaws of the instrument tips. our aim was to compare the outcomes of mlc with traditional laparoscopic cholecystectomy (tlc) one developed choledocholithiasis on postoperative day one and after ercp the course was uneventful. the other patient developed choledocholithiasis and acute pancreatitis on the sixth postoperative day and was treated conservatively. the stone in the ampulla had fallen by itself without a need for ercp single-incision plus one additional port laparoscopic surgery for colorectal cancer with transanal specimen extraction: a comparative study two patients had a previous attempt of hernia repair, one with mesh. one patient did not have any immunosuppression due to hiv infection, whereas the other were on cyclosporine, tacrolimus and/or mycophenolate mofetil. there were two laparoscopic and two open cases, mean operative time was . minutes ( - ), mean blood loss was ml ( - ). mesh used were biological porcine dermis in one case, polypropylene with absorbable hydrogel barrier in three cases. mean mesh length and width were cm ( - ) and . cm ( - ) respectively. one patient underwent a component separation, though none of the patients had the fascial defect closed. there were no intra-operative complications. three patients were readmitted for hyperkalemia, abdominal pain, and seroma respectively. neither recurrences nor reoperations were reported. mean follow-up was . days ( - ) conclusion: post liver transplant incisional hernia repair is feasible either laparoscopic or in an open fashion. because of the size and location of the defect, fascial closure is unlikely achievable. the use of standard techniques and materials give a similar result of the non-transplant population. p technique of esophagojejunostomy using orvil after laparoscopy assisted total gastrectomy for gastric cancer shinichi sakuramoto there was a significant difference in mortality between the two time-periods, / patients died during - and / died during - (p= . ). those who died were significantly older ( years ( - )) than the survivors ( y ( - )) (p= . ). five of the patients who died in the previous group died without any intervention. / of those who had an acute open necrosectomy died. surgical necrosectomy correlated significantly with mortality (p= . ). the only patient who died in the recent group died without any intervention. none of the patients receiving minimal invasive drainage in this group died until now only cases in adults and fewer than cases in children have been reported in world literature, with surgical management being the only option. an innovative, minimally invasive laparoscopic excision of the abdominal sac was performed and the scrotal component was managed by jaboulay's procedure. this is probably the first case report in world literature describing laparoscopic management of hydrocele-en-bissac. case report: a year old male presented with complaints of bilateral hydrocele and swelling in right lower abdomen since one year. computed tomography of the abdomen revealed an encysted hypodense lesion with enhancing walls along the right side of pelvis, anterior to the psoas muscle and extending through the internal ring into the right inguinal region upto the scrotal sac; measuring . cm . cm suggestive of an encysted hydrocele of cord associated with hydrocele of both scrotal sacs excessive gastric resection may result in postoperative deformity of the stomach, with consequent gastric stasis in food uptake. to minimize the resection of stomach tissue, especially for lesions close to the esophagogastric junction or pyloric ring, we have developed laparoscopic wedge resection (lwr) with the serosal and muscular layers incision technique (samit) for gastric gastrointestinal stromal tumors. this samit is simple and does not require special devices. purpose: the purpose of this study was to clarify whether lwr with samit for gastric gists is technically feasible in term of short-term outcome methods: all patients who went through lsg in our department between / to / have been evaluated for bleeding complications, after implementation of anti-bleeding policy: blood pressure was controlled to mmhg during stomach resection and staple line was reinforced throughout it's length with a running - absorbable v-lock suture. drains were used selectively. results: out of patients who went through the procedure ( . %) suffered hemorrhagic complications: patients had? hb[ gr%. patients received - red blood pc's. no patients were re-operated for bleeding. patients were readmitted for infected hematoma and had ct guided drainage. one patient ( . %) suffered from leak. conclusion: implementation of anti-bleeding policy in lsg is very effective. there is no need to use expensive buttress material to achieve these results. drains can be used selectively. the impact of this policy on leak rate needs to be fifty procedures immediately prior to, immediately after, and eight months after completion of training were included for each endoscopist. data were extracted from the electronic medical record and entered into spss for analysis. student's t-test was used to compare groups for continuous data, and chi-squared tests were used for categorical data. data were collected for procedures. patient groups pre, post, and eight months after csi training were comparable in terms of age ( . yrs, . yrs, and . yrs), sex ( it's in the bag; can stoma output predict acute kidney injury in new ostomates? robert fearn colostomy output stabilised rapidly, whilst ileostomy output increased progressively throughout the first postoperative days as can be seen in chart . twelve patients ( %) developed aki during index admission. length of stay was significantly greater in the aki group at ( % ci - ) days vs ( - ) days. highest daily stoma output was non significantly higher in the aki group ml ( % ci - , ml) vs , ( - , ml) as was mean daily stoma output at ml ( - , ml) vs ml ( - ml) (chart ). seventeen patients ( %) were readmitted for any reason, ( %) specifically for aki. in total patients ( %) developed aki within three months of their stoma surgery only of whom had developed aki during their index admission. all patients who developed aki following their index admission were ileostomy patients. conclusion: acute kidney injury in new stoma patients is associated with prolonged hospital stay and readmissions with associated morbidity and healthcare costs consecutive laparoscopic bariatric operations were performed, including primary roux-en-y gastric bypasses (lrygb), primary adjustable gastric bands (lagb), primary sleeve gastrectomies (lsg) and secondary bariatric surgeries and revisions. all bariatric procedures were approached laparoscopically ( procedures were stapled and were nonstapled). the mean patient age was years ( - ), females represented % and mean bmi was . kg/m ( - ). there were no perioperative mortalities, no conversions to open surgery and no intraoperative blood transfusions. there we two major intraoperative complications (hypopharyngeal perforation- , malignant hyperthermia- ). mean hospital stay was . days ( - days). eleven patients ( . %, in gastric bypass group and one in lsg group) required -day reoperations for postoperative complications (staple line gastrointestinal bleeding- , anastomotic leak- , strangulated port site hernia- , unexplained severe abdominal pain- , intestinal obstruction- , and intraabdominal abscess- ). there were no long term ( -year) mortalities in patients that required reoperation. there was one transfer to another institution. the dynamics of further improving safety was such that there was no complication on the recent consecutive stapled procedures and the mean hospital stay was . days ( - days). detailed subgroup analyses will be provided. conclusions: with well-controlled and structured pre-, intra-, and post-operative care, laparoscopic bariatric surgery can be performed with minimal reoperations and zero mortality in a teaching institution does concomitant placement of a feeding jejunostomy tube during esophagectomy affect quality outcomes? md, facs; icahn school of medicine at mount sinai background: placement of a feeding jejunostomy tube (fj) is often performed during esophagectomy. few studies, however, have sought to determine whether concomitant placement affects postoperative outcomes of esophagectomy of these, ldg was performed patients and odg was performed . we compared elderly patients (aged years or more) with younger patients in each operative procedure. (ldg: elderly , younger ; odg: elderly , younger ) preoperative comorbidity and surgical results were analyzed. multivariate analysis was performed to detect predictive factors for postoperative complications. results: in both ldg and odg groups, the operative time and amount of blood loss did not differ, while comorbidity was more common in elderly patients than in the nonelderly, and there were fewer retrieved lymph nodes in elderly patients. the incidence of all postoperative complications did not differ between both groups in each procedure, and there were no significant differences in the time to first flatus or postoperative hospital stay. however, in terms of specific postoperative complications, respiratory complications were more frequently observed in eldery group with odg significantly (p= . ), while not with ldg group. in multivariable analysis, age was not independent predictor of postoperative complications. conclusion: odg for eldery patients requires attention particularly in postoperative respiratory complications. ldg is a safe and less invasive treatment for gastric cancer in elderly patients who have greater comorbidity. p examining the role of preoperative ineffective esophageal motility in laparoscopic fundoplication outcomes tyler hall there were no significant differences in complications or recurrence rates. preoperative quality of life measures did not vary between the cohorts nor did postoperative scores at three weeks or six months. patients with % ineffective clearance exhibited worse gerd-hrql scores one and two years postoperatively conclusion: preoperative ineffective esophageal motility was shown to result in comparable short-term quality of life following ars. however, gerd-hrql scores at one and two yearsshowed worse outcomes in patients with preoperative iem robotic surgery as part of oncologically adequate ipmn treatment: indications, short and long term results federico gheza eligible patients who had minimally invasive surgery were stratified in multiport laparoscopic and robotic cohorts, and included if they had poi/sbo after surgery. comparative analysis assessed the demographic, perioperative, and postoperative outcomes. the main outcome measures were the incidence rate, associated variables, and time to ileus/ sbo across the mis platforms. results: during the study period total patients were reviewed- laparoscopic and robotic. postoperatively, ( . %) laparoscopic and ( . %) robotic patients suffered from poi/sbo laparoscopic sbo occur significantly later after the index procedure than robotic sbo ( conclusions: the rate of poi/ sbo is considerable and comparable across laparoscopic and robotic approaches. however, there are distinct differences in the severity, time to occurrence, and impact on quality measures, such as los and readmissions between laparoscopic and robotics. this information could be an important factor in which approach the surgeon choses laparoscopic surgical procedure was standard with using laparoscopic linear stapler. responses to surgery were evaluated a month after the operation based upon the american society of hematology evidence-based practice guidelines for itp. results: there was no open conversion in this study. the mean operation time and blood loss were min and g, respectively. there was no case using blood transfusion during and after operation. with regard to complications, one patient ( %) had a postoperative pancreatic fistula that did not require percutaneous drainage. positive responses, including the complete and partial remissions, were achieved in % ( / ). the mean follow-up duration was months, and the -, -, and -year relapse-free survival rates were % for all three time points. conclusions: the present study demonstrated that ls for itp can provide good long-term outcomes two cases of conversion from sp-c to open surgery were excluded. all procedures were followed postoperatively for a minimum of months, and wound complications such as bleeding, fat lysis, infection, or hernia were recorded. patients were classified as having a wound complication or not. results: pure transumbilical sp-c was completed . %, additional trocars were used in . %, and the rate of conversion to open surgery was . %. after a median follow-up of . (range, - ) months few cases performed with hand assist, notes, or single-incision. utilization of robotics was highest for bpd/ds ( of , cases, . %). the greatest number of robotic-assisted cases were sleeve gastrectomy ( , of , , . %) and gastric bypass ( , of , cases, . %). relatively few operations were converted to a different approach (see table). operative time was longer when using robotic approaches for both sleeve ( . vs . minutes, p. ) and bypass ( . vs . , p\ . ). postoperative los was no shorter when using robotic-assistance (see table). unadjusted -day outcomes revealed slightly higher rates of readmission for both operations when using robotic-assistance (see table), and slightly higher rates of complications after robotic sleeve gastrectomy p comparision of perioperative and survival outcomes of laparoscopic versus open gastrectomy after preoperative chemotherapy: a propensity score-matched analysis adjustment for potential selection bias in the surgical approach was made with propensity score-matched (psm) analysis. perioperative and survival outcomes were compared between the lag and og groups. results: in total, patients were identified from the database. after psm analysis, patients who underwent og were one-to-one matched to patients who underwent lag in the setting of nact. these two groups had similar outcomes in terms of intra-and postoperative complications and -year overall survival. however, the lag group had a longer operation time (p= . ) and lower estimated blood loss (p= . ). moreover, compared with patients in the og group, those in the lag group had fewer days until first ambulation conclusion: the present study indicates that lag performed by well-qualified surgeons for treatment of locally advanced gastric cancer after preoperative chemotherapy is as acceptable as og in terms of oncological outcomes. p outcomes of laparoscopic antireflux surgery for gastroesophageal reflux disease: effectiveness and economic benefits kyung won seo, phd; kosin university college of medicine purpose: laparoscopic antireflux surgery (ars) is an alternative treatment option for gastroesophageal reflux disease (gerd) in the world. however, the effectiveness and economic feasibility of ars versus medical treatment is unknown. this study was performed to evaluate the effectiveness and economic benefits of ars. methods: nine patients with gerd were treated using laparoscopic ars between and . surgical results and total cost for surgery were reviewed. results: seven men and women were enrolled. preoperatively, typical symptoms were present in patients, while atypical symptoms were present in patients. one patient underwent partial fundoplication due to absent peristalsis and the other underwent nissen fundoplication. postoperatively, typical symptoms were controlled in of patients, while atypical symptoms were controlled in of patients. overall, at months after surgery, reported partial resolution of gerd symptoms, with achieving complete control. the average cost of ars for nine patients was usd. conclusion: laparoscopic ars is effective for controlling typical and atypical gerd symptoms. the cost of ars may be more economical over the long term compared to medical treatment since laparoscopic surgery is reported to affect respiration and circulation, we should take indication of lag for elderly patients into consideration carefully. indication of lag for elderly patients, however, is still controversial. the aim of this study is to assess the safety and validity of lag for elderly patients. method: medical records were retrospectively reviewed for patients who underwent lag for gastric cancer between and . in this study, patients over years of age were defined as elderly patients. patients were divided into two groups according to age; group a (age ≥ , n= ), group b (age \ , n= ). preoperative characteristics and postoperative outcomes were analyzed. two-tailed student's test and/or pearson's chi-square test were used for statistical analysis. results: there were no significant differences in male/female ratio and body mass index between two groups. number of patients whose asa physical status was ≥ , and/or performance status was ≥ did not differ total gastrectomy ( . vs . %, p= . ), proximal gastrectomy ( vs . %, p= . ). intra-operative blood loss, operating time, and number of harvested lymph nodes did not differ between the two groups. as for postoperative complications such as intra-abdominal abscess ( . vs . %, p= . ), anastomotic leakage ( vs . %, p= . ), significant difference was not observed between the two groups. in addition, respiratory and cardiovascular complication was not observed in elderly patients. incidence of clavien-dindo classification ≥grade ( . vs . %, p= . ), and postoperative hospital stay ( . vs . days, p= . ) did not differ. conclusion: short-term outcomes of lag in elderly patients were not different from those in young patients the essential role of the transcystic duct tube (c-tube) during laparoscopic common bile duct exploration (lcbde) towakai hospital introduction: laparoscopic common bile duct exploration (lcbde) is a standard surgical procedure for the treatment of common bile duct stones (cbds). however, there are some problems associated with cbd drainage after operations even if performing with the primary closure. therefore, we developed a new drainage tube, c-tube, which contributes to shorter drainage periods and reduces perioperative complications. method: c-tube is a type of bile drainage tube which is fixed to the cystic duct with an elastic band. closing the duct with an elastic band as soon as c-tube is removed prevents bile leakage from the stump of the cystic duct. the essential roles of this tube include: . assisting suturing during operations, . use during intra-and post-operative cholangiograpy, . assisting post-operative endoscopic sphincterotomy when necessary we included patients from -years prior to our intervention and compared this with patients who had follow-up after implementation. we excluded patients having revisions, gastric banding, and patients whose primary surgeon had left during the data collection period. we analyzed demographics and follow-up rates at , , , , and months. chi-square test was used to evaluate for significance, and results were corrected for multiple comparison. results: patients met inclusion criteria in the pre-intervention group, and in the postintervention group. of those, were analyzed for the year follow-up visit. the pre-intervention group had males, females, and an average age of . approximately / of the surgeries performed were sg, / were rygb. the post-intervention group had males, females, average age of . approximately half of the post-intervention cases were sg while the rest were rygb. conclusion: bariatric surgery is a useful tool in aiding weight loss and improving comorbidities. it is essential that patients receive long-term follow-up and monitoring to achieve these goals. our program now uses a system of phone call reminders for scheduled visits, as well as calls and letters for annual visits surgeon's evaluation of an intraoperative microbreaks web-app workload questions were modified nasa task load index (physical demand, mental demand, and complexity) and procedural difficulty on - ( =maximum impact) scales. primary outcomes were the impact of microbreaks on surgeons' physical performance, mental focus, pain/discomfort and fatigue with checkboxes for improved, no change and diminished. secondary outcomes were microbreaks impact on distraction level and workflow disruption using a - ( =maximum impact) scale. descriptive statistics were calculated for median and interquartile ranges (iqr) of these responses. results: seven surgeons ( male, female), with a median (iqr) surgical experience of ( . , ) years, completed ten surgical days with a median (iqr) operative duration of ( , ) minutes/surgical day. the median number of microbreaks/surgical day was . the median (iqr) for mental demand, physical demand, surgical complexity and difficulty are shown in table . following each surgical day, surgeons reported / improved physical performance situs inversus totalis (sit) is inherited in an autosomalrecessive fashion with complete abnormal transposition of thoracic & abdominal viscera. its incidence varies from in to live births. for those undergoing surgery, laparoscopic approach is preferred as it avoids inappropriate incisions. however, due to mirroring of the viscera, the surgeon faces constant visio-spatial disorientation during laparoscopy. p ''how to be a surgeon and not dying trying'' control of basic physiological parameters in perioperative phase second main variable: blood pressure (bp) with manual measurement sleeve. preoperative bp and immediate postoperative bp were measured, we were not able to measure intraoperative bp due to the lack of consent of the surgeons involved for the use of other devices different from the heart rate band. secondary variables: years from graduation, years of practice, age, body mass index (bmi), number of medical co-morbidities, number of jobs, sleeping hours the night before. we took measurements to surgeons during a laparoscopic cholecystectomy. results: the mean preoperative heart rate was . bpm. the mean minimum intraoperative heart rate was bpm. the mean maximum intraoperative heart rate was . bpm ( % with tachycardia at the surgery). the mean immediate postoperative heart rate was . cpm. the mean heart rate minutes after the postoperative phase was . cpm. at the immediate preoperative phase % of surgeons had elevated bp level (usual normotensives) articles were randomly selected and the gender of the first and last authors determined. results: of the bariatric surgery publications reviewed, only % of first authors and . % of last authors were female surgeons. even though the proportion of female authors has increased over time, this is not proportional to the increase in the number of female surgeons or surgery residents (figure ). discussion: female surgeons are under-represented in bariatric surgery research. the number of female surgeons and residents has a continuous up trend over the last few decades our survey also included the validated quick-dash (disabilities of the arm, shoulder, and hand) questionnaire for upper-limb symptoms and the ability to perform certain physical activities. the quickdash is scored into two components: disability/symptom score, and the optional work module, which represent the impact of disability on daily activities and work responsibilities, respectively. both scores range from - , with a higher score indicating greater disability. surgeons were grouped according surgical focus (open, lap, or ra), and comparisons were made between groups. surveys with more than % of responses missing were excluded. statistical analysis were done using spss . , with α= . . results: completed surveys were evaluated (open: n= , lap: n= , ra: n= ). the survey response rate was %. . % of respondents were general surgeons, and mean age was ± . years. surgeons reported an average of ± . cases performed per month ra: . %, p= . ). likewise, there were no differences in the mean disability similarly, there was a positive correlation between mean work scores and reported pain in the upper-limb for lap and ra, both p. . conclusions: this nationwide survey revealed a similar prevalence of pain in the upper-limb among surgeons performing open, laparoscopic and robotic-assisted procedures. likewise, similar disability scores were reported between the three surgical groups. older surgeons performing laparoscopic and robotic-assisted approaches reported a higher impact of upper-limb problems interfering with their daily activities, unlike open surgeons. among all surgeons who reported pain in the upper-limb, laparoscopic and robotic surgeons were more likely to report that this pain interferes with their work activities an analysis of subjective and objective fatigue between laparoscopic and robotic surgical skills practice p d laparoscopic versus robotic gastrectomy for gastric cancer: comparisons of short-term surgical outcomes lin chen, xin guo patients who underwent d-lag (n= ) or rag (n= ) for gastric cancer were enrolled. the clinicopathological factors and short-term surgical outcomes were compared with retrospectively analysis. results: the clinicopathological factors between the two groups were well matched. postoperative recovery factors including the days of first flatus, days of eating liquid diet and hospital stay were similar. the rate of postoperative complications between the two groups were with no statistical differences in the subgroups of patients with total gastrectomy, d-lag had less blood loss and shorter operative time than rag (p= . and p. ), while for distal gastrectomy, blood loss and operative time showed no statistical differences. conclusions: this study suggests that d-lag is a novel and acceptable surgical technology in terms of surgical and oncological outcomes. d-lag is a promising approach for gastric cancer therapy methods: patients underwent robotic surgery between the beginning of to first half of in turkey were included. data were obtained from a prospectively maintained database. patient, surgeon and hospital identifiers were encrypted. parameters were operation type, operation year, robotic system used (s, si, xi), hospital volume and surgeon volume. high volume robotic colorectal hospital and surgeon was defined as the caseload within the forth interquartile ( th- th) based on the median value. results: there were colorectal procedures. surgeons performed robotic colorectal surgery at hospitals. ( . %) and ( . %) procedures were performed with the s-si and xi platforms respectively. hospitals have both of the si and xi platforms. hospitals are the si, hospitals are the xi hospital currently. the number of robotic colorectal operations increased gradually by years (figure ). the median numbers of colorectal procedures were (range - ) and (range - ) per hospital and per surgeon respectively among those hvrcs, the numbers of si and xi users were and respectively. the surgeons who performed more than procedures continued to use robot in their practice except one surgeon who stopped at . only left colectomies and no right colonic resection were performed before introduction of the xi platform first robotic cases and implementation of a robotics curriculum in a general surgery residency domenech asbun armonk ny) and utilized student's t test and chi-square. we also performed a linear regression analysis to determine the effect of or time, robotic surgery, and diagnosis on operating room costs and postoperative length of stay. results: laparoscopic and robotic cholecystectomies were performed. demographic parameters (age, gender, medical comorbidities, preoperative albumin and bmi, surgical history and smoking) were comparable. primary diagnosis was significantly different (chi-square . ), driven by more acute cholecystitis in the laparoscopic group. / robotic cases and / ( . %, p = . ) laparoscopic cases were converted to open ( for adhesions, for failure to progress, and for visualization of anatomy after adjusting for or time and diagnosis, robotic surgery was associated with a $ increase in costs robotic surgery is independently associated with increased or cost, but individual hospital systems must decide if this additional cost outweighs increased robot utilization and training benefits for physicians and staff robotic abdominal wall hernia repairs: technical considerations and lessons learned inguinal hernia repairs (ihrs) comprised the majority ( . %) of cases ( . % male, mean age . , mean bmi . ). there were unilateral ihrs with an average operative time of . ± . min and an average ebl of . ml. there were bilateral ihrs with an average operative time of . ± . min and average ebl of . ml. thirteen ihrs were combined with umbilical hernias and two with incisional hernias. average operative time for combined procedures was . min and average ebl was . ml. fifty-five incisional hernias were repaired robotically ( . % male, mean age . , mean bmi . ), four of which were retrorectus and two of those required transversus abdominis release. median hernia size was cm ( - cm). mean operative time was . ± . min and average ebl was . ml. twenty-three ventral/umbilical hernias were repaired robotically ( . % male, mean age . , mean bmi . , median size . cm ( - cm), mean operative time . ± . min, average ebl . ml). one spigelian hernia (operative time min, ebl ml) and one parastomal hernia (operative time min, ebl ml) were repaired robotically. there were no major complications and only groin seroma requiring percutaneous aspiration. nine patients required conclusion: this study demonstrates improved outcomes of robotic inguinal hernia repair compared to an open or laparoscopic approach. robotic hernia repair showed overall lower -day complication and readmission rates, and shortened los. while open approach had the highest rate of opiate use we retrospectively investigated consecutive overweight gc patients (bmi≥ ) underwent distal gastrectomy with d lymphadenectomy ( for rag and for lag) performed by two surgeons. the clinicopathological and surgical features were compared between groups. the cutoff point for initial phase (phase i) and stable phase (phase ii) were determined by cumulative sum (cusum) curve of operation time. results: generally, the surgical outcomes including postoperative complication rate, duration of postoperative hospital stay and lymph nodes harvest in the overweight patients have comparable results between rag and lag groups. the cutoff determining phase i and ii according to the cusum figure for rag group was and cases for surgeon a and b, respectively. and comparison analysis showed that the operation time of phase ii rag was significantly shorter robotic-assisted transabdominal preperitoneal inguinal herniorrhaphy: a single-center experience including perioperative morbidity and short-term outcomes patient factors, treatment factors, and outcome measures were collected in an attempt to gain insight and to generate ideas to potentially improve outcomes. results: there were no operative complications. six patients ( %) had failed gastric pacemaker placement prior to intervention. nine patients ( %) reported improvement in their symptoms and overall quality of life. four patients ( %) reported no improvement in symptoms and required additional intervention for symptom control and supportive care (one underwent roux-en-y gastric bypass, three underwent laparoscopic jejunostomy feeding tube placement to maintain nutrition). conclusion: robotic-assisted pyloroplasty is a safe option that improves symptoms and quality of life in % of our patients patients were matched into cohorts by procedure type. outcomes were analyzed using unpaired t-test and fisher's exact test. results: cost data was available for patients undergoing ras or la procedures. significant increases in equipment, labor, and overhead costs resulted with ras vs. la. variable-labor and variable-overhead costs were significantly higher in la procedures. higher supply costs and longer procedure time was seen with ras in all cohorts however, total -day costs were not significantly different in any group. conclusion: ras led to significant increases in fixed clinical, operative and pathologic factors were reviewed and analyzed. results: seventy patients underwent robotic surgery for rectal cancer during the study period. the locations of tumor were upper rectum, lower rectum. the procedure were as follow, high anterior resection in , low anterior resection in , isr in , apr in patients. eight patients underwent bilateral lymph nodes dissection (llnd). the procedures were performed successfully in all cases. mean age was . years, and % of the patients were men, and the mean body mass index was . (range, . - . ) kg/m . median operative duration was ( - ) minutes. median blood loss was ( - ) ml. median postoperative stay was ( - ) days. mean harvest lymph node number was . ( - ). surgical margins were negative in all cases. there was one conversion due to bleeding during the llnd and anastomotic leakage occurred in two patients. morbidity was %. there was no mortality postoperatively in this series. conclusion: in early series of the selected patients, this technique appears to be fesible and safe when performed by surgeons skilled in laparoscopic colorectal surgery the inactive electrode was placed touching small bowel to simulate accidental thermal injury. the bowel tissue at the site of temperature change was immediately resected and examined histologically for tissue injury. student t-tests were used for all comparisons with a p-value less than . considered statistically significant. results: comparison of the laparoscopic and robotic techniques are displayed in table . energy transfer was quantified using energy leak (per ma), which in these tests averaged . degree celsius change ( % ci . - . ) at the inactive electrode. surface temperature heated to a maximum of . degrees celsius, more in the robotic system than laparoscopy but still clinically negligible. pathology results from in vivo testing showed only thermal injury to the serosa without deeper mural injury. conclusions: stray energy transfer occurs in both laparoscopic and robotic surgery in amounts that are measurable but without clinical relevance. the average change in tissue temperature is less than degrees celsius laparoscopically and less than degrees robotically. while the robotic surgery appears to transfer more stray energy, no significant bowel injuries were caused in either group. p robot assistance can improve the performance of laparoscopic extensive concomitant adhesiolysis: results from a large observational study federico gheza outcomes compared were operative time, conversion rate, overall complications, gastrointestinal (gi) related complications (wound infection, abdominal abscess, anastomotic leak, ileus and small bowel obstruction), hospital length of stay, and -day re-admission rate. two sample t-test was used and p. was considered statistically significant. results: fifty-five robotic colectomies were matched with laparoscopic counterparts based on type of operation: right colectomy (n= ), sigmoidectomy (n= ), low anterior resection (n= ), proctocolectomy (n= ), transverse colectomy (n= ), abdominoperineal resection (n= ), and total abdominal colectomy (n= ) we assessed if technical obstacles of laparoscopic suturing were decreased and if laparoscopic skills overall were improved. surgical outcomes were compared relative to our historic values; we assessed procedure time and operating room efficiency, including set up and turn-over times. results: overall, the d/flexdex system permitted a greater improvement in working speed, superior optical visualization, and better suture handling compared to standard laparoscopy. all surgeries were completed without any complications. historically, we considered laparoscopic suturing to be complicated and inefficient. we relied on tacking devices for mesh fixation, suturing was previously completed with large cumbersome straight laparascopic devices. however, with flexdex and endoeye flex d, tacking devices have been eliminated and suturing technique improved. the mean total procedure times remained comparable for inguinal and hiatal hernia surgeries, and slightly longer for ventral hernias. operating room efficiency, including mean set up and turn-over times also remained unchanged. the acquisition cost for both the olympus endoeye flex d laparoscopic imaging system we performed a cost analysis which showed an average total cost of $ , for laparoscopic sleeve gastrectomy and an average of $ , for robotic assisted. the total reimbursements were $ , for laparoscopic sleeve gastrectomy and $ , for robot assisted. this translated to an average contribution margin of $ , for laparoscopic vs $ , for robot assisted. we analyzed these differences for bypasses as well. laparoscopic bypasses averaged minutes laparoscopically vs robotically. we found an average cost of laparoscopic $ , vs robot assisted $ , , with a contribution margin of $ , laparoscopic vs $ , robot assisted. conclusions: in our study we noted increased operative times with robot assisted operations, especially bypasses which could be explained by increased use of the robotic system for difficult cases such as revisional bypasses. the impact of cost is especially important in this financial climate, and judicious use of resources becomes important when determining surgical approach average or time for rih was minutes compared to lih which was minutes. average intraoperative cost for rih was $ , compared to lih which was $ . of note, one lih was converted to open, whereas none of the rih required conversion. average los was . hours for rih compared to . hours for lih. postoperative pain at one week follow up was the same between both groups. two postoperative surgical site occurrences (sso) occurred in the lih group ( groin seromas), whereas no ssos occurred in the rih group. eleven ventral hernia repairs were examined, were robotic (rvh) and were laparoscopic (lvh). average or time for rvh was minutes compared to minutes for lvh. average intraoperative cost for rvh was $ , compared to lvh which was $ , . no procedure from either group required conversion to open. average los was . hours for rvh, and . hours for lvh. again, postoperative pain was the same at one week follow up for both groups. there were no postoperative complications noted in either cohort. conclusion: operative time and procedural costs for rvh and rih repairs were shown to be longer and more expensive when compared to their laparoscopic counterparts. however, with increased operative experience using the robotic platform, surgical time did show a decreasing trend does robotic system have advantages over laparoscopic system for distal pancreatectomy? results: a total of consecutive patients underwent minimally invasive distal pancreatectomy (ldp n= ; ra-ldp n= ). most common pathologic finding was pancreatic ductal adenocarcinomas ( cases). there was no in-hospital mortality or cases of conversion to open surgery in this study. spleen-preserving approach was performed more often in the ra-ldp ( %) than in the ldp ( . %) groups (p= . ) both groups showed no significant differences in the total number of lymph nodes, number of positive lymph nodes, tumor differentiation, tumor stage, and resection margins. conclusions: ra-ldp is a safe and feasible approach that has an advantage of performing spleenpreserving distal pancreatectomy, with perioperative and short-term oncologic outcomes comparable to those of ldp. p robot-assisted alpps technique mike fruscione right portal vein embolization was not feasible secondary to the proximity and size of the right hemi-liver tumor burden relative to the right portal vein. the pre-operative planned procedure was a right trisectionectomy and microwave ablation of the segment lesion. results: using the da vinci xi surgical system (intuitive surgical, inc.) the right portal vein was dissected, doubly-ligated, and divided. the liver parenchyma was split from the inferior edge to the dome mm medial to the falciform ligament and down to the middle hepatic vein which was preserved to maintain adequate venous outflow. the patient was discharged home on post-operative day two. on post-operative day six, ct volumetrics demonstrated a flr of %. on post-operative day seven, a second stage alpps procedure was performed where the right hepatic artery, middle and right hepatic veins and right hepatic duct were ligated and divided. segments a/b, , , and were removed. the patient was discharged home on post-operative day five they were asked to answer demographic questions and rate their comfort level ( =not comfortable, =very comfortable) with aspects of robotic surgery. paired t-tests and wilcoxon tests were used to assess whether there were changes in comfort level before and after labs, and chi-square goodness of fit tests were used to assess whether dry lab (using inanimate objects), wet lab (using a porcine model), or simulator modules were thought to be most helpful in obtaining specific robotic skills. results: the survey response rate was % (n= ). ninety-one percent of residents felt that robotic surgery is not intuitive. prior to simulation, % of residents felt inadequately prepared to safely operate on the robotic console. following simulation, % felt better prepared and more confident to participate in robotic surgery for the first patients whom we treated (the first-stage group), we invited a visiting expert from a high-volume center to perform the procedure jointly with our hospital's surgeons by using a dual console. for the subsequent patients (the second-stage group), the procedure was performed by our hospital staff alone. in this report, we describe our experience of introduction of robotassisted colectomy and discuss issues for the future. patients and methods: the operative procedure was sigmoid colectomy, low anterior resection, and intersphincteric resection. the median number of lymph nodes dissected was . . the mean operating time was minutes for the first-stage group and minutes for the second-stage group. the median console time was minutes for the first-stage group and minutes for the second-stage group, with no significant differences between the two groups. the mean operating time other than console time was minutes for the first-stage group and minutes for the second-stage group, significantly longer in the latter group. the mean amount of hemorrhage was . g in the first-stage group and g in the second-stage group. no significant differences were found between the two groups in the mean length of postoperative hospital stay. none of the patients in either group developed a complication of clavien-dindo grade iii or higher. conclusions: the use of dual console system was particularly useful for the introduction of robotassisted surgery in our hospital. for the patients whom we treated, we found almost no difference in console time between the first-and second-stage groups. the high-quality instruction received via the dual console was considered to have had a beneficial effect on the operators' learning curve. however, the operations that were set up other than console time, such as roll-in and docking, took significantly longer in the second-stage group when the proctor was not present select specimens from each trial were immediately resected and evaluated for histologic thermal injury. experiments were repeated times based to detect an expected difference of five degrees. student t-tests were used for all comparisons with significance set at . . results: stray energy transfer was higher in the single incision setup compared to the traditional setup (figure ). stray energy in the assistant grasper caused . ± . °c of temperature change in the standard configuration, and . ± . °c in the single incision configuration (p= . ). doubling energy output to w amplified the same finding robotic single-site cholecystectomy of cases: surgical outcomes and comparing with laparoscopic single-site procedure jae hoon lee incisional hernia occurred one case in each group. rssc is safe and feasible procedures. with accumulating of experience, rssc had more short operative time than sslc. comparing to sslc, rssc is relatively suitable to acute gallbladder disease and high bmi and requires a minimal learning curve to transition from traditional multiport to single-port robotic cholecystectomy. p initial experience using da vinci xi robot in colorectal surgery anna r spivak, do, john marks, md; lankenau medical center introduction: the xi robot has been developed to facilitate multiquadrant abdominal surgery. this report presents initial experience to evaluate feasibility and safety of xi robot in colorectal surgery. methods: all cases performed on xi robot were prospectively entered into a robotic database that was queried for colorectal cases performed from intraoperative complications were encountered in cases ( . %), requiring conversion to laparoscopy. none were converted to open. mean length of largest incision . cm. median ebl ml. there was no mortality. there were ( . %) immediate postoperative morbidities: postoperative abscess, bowel perforation, two postoperative bleeds, two hernias, two hematomas, smv thrombosis, small bowel obstruction. perioperative blood transfusions were required in . % of cases. there was one anastomotic leak. median time from surgery to low residue diet and discharge was days. conclusion: initial experience shows robotic colorectal resection with da vinci xi learning curve for robotic sleeve gastrectomy and roux-en-y gastric bypass: achieving equivalence to laparoscopy residents and fellows participated in an analogous fashion in both arms of the study, and patients undergoing re-operative bariatric surgery were excluded. results: a total of patients undergoing rsg (n= ) or rrygb (n= ) were included. for the overall robotic cohort, median age was (range - ), % were american society of anesthesiologists (asa) score , % were asa score , and mean body mass index (bmi) was ± with no differences between procedures. there were no conversions to open. there was one patient with portal vein thrombosis after rsg which occurred in the th rsg and one patient who underwent re-operation in the immediate post-operative period for hemorrhage at the gastro-jejunal anastomosis in the rrygb group; this occurred in the th rrygb. there were no leaks, strictures, or mortalities in either group. mean length of stay was days± for rsg with no difference based on number of procedures performed. in the rrygb group, los decreased after the first five procedures from days± to days±(p= . ). for both procedures, operative time decreased by number of procedures performed (figure). equivalence to lsg in operative time ( minutes± ) was reached after eight robotic procedures were included. the da-vinci xi® was used for the operations. age, gender, body mass index (bmi), asa score, indication for surgery, urgency of procedure, type of procedure, docking number, operation time, estimated blood loss, complications, short (≤ days) and long term ([ days) complications were evaluated. results: patients ( females) were included. median age was . median bmi was , median asa score was . total and completion rrp-ipaa were performed for and patients respectively. the indications were as follows: medical refractory uc (n= ), cancer/dysplasia (n= ), fulminant colitis (n= ), toxic megacolon (n= ), medical treatment resulting in growth retardation (n= ), medical treatment refractory bleeding (n= ). patient with toxic megacolon had an emergent operation. the median docking number was and for completion and total rrp-ipaa respectively. median operative time was minutes. median blood loss was ml. all patients had a stapled ileal j pouch anal anastomosis. all patients had a diverting loop ileostomy at the time of ipaa creation. no intraoperative complications were observed. no conversion to open surgery was needed. the median time to flatus was day. the median time to oral intake was day. patient had a laparotomy on postoperative day due to intra-abdominal bleeding. patient had a bleeding from ileostomy which was treated endoscopically. superficial surgical site infection was observed in patients. patient had a pouchitis managed with oral antibiotics. patient had an ileus responded to conservative treatment. patient had a per-anal bleeding stopped spontaneously. patient had a urinary tract infection responded to antibiotics. patients had pouchitis, patient had a perianal fistula requiring a loop ileostomy and a parastomal hernia was developed in another patient in long term follow up ) were significantly different between the two groups. , pairs undergoing primary and pairs undergoing revisional procedures were successfully matched. robotic gastric bypass was associated with a significantly longer operation length than laparoscopic gastric bypass for both primary (median difference minutes, p. ) and revisional (median difference minutes, p. ) procedures overall, there were no significant differences in anastomotic/staple line leak, -day readmission, reoperation, re-intervention, total event, and mortality rates between matched cohorts. conclusion: when controlling for patient characteristics, those undergoing primary and revisional lrygb and rrygb had no difference in early morbidity. despite the prolonged operative duration, the robotic approach was not associated with any clinical benefit or increased complications for primary or revisional gastric bypass surgery preoperative risk factors were collected. we focused on perioperative outcomes and in hospital complication rate. results: thirty-three patients underwent robot assisted giant hiatal hernia repair at our institution. patients ( %) were years and older and patients ( %) had a bmi higher then. there were no significant differences in patient characteristics between the groups. no patient underwent conversion to open or standard laparoscopy. no mortality was observed and no transfusions were needed. four patients ( %) had a complication, two of them were older than years old. three of the four patient ( %) that had a complication were obese. there were no statistical differences in mortality % and . % of them were with s-si and xi platforms respectively. the median numbers of procedures were (range - ) and (range - ) cases per hospital and per general surgeon respectively. the high volume surgeons (higher than th percentile) performed ( %) of the cases. the xi platform has been the main tool for colorectal surgery only (figure ). conclusions: while xi platform significantly increased caseload in general surgery by facilitating performance of colorectal surgery, its preference in other general surgical fields is not superior to si laparoscopic inguinal hernia repair (tapp) -first experience with the new senhance robotic system robin schmitz ; intuitive surgical inc, loma linda university medical center introduction: crohn's disease is an incurable inflammatory disorder that can affect the entire gastrointestinal tract. while medical management is considered first-line treatment, approximately % of patients with crohn's disease require surgery within years of their initial diagnosis. traditionally, surgery has been performed via an open approach with poor adoption of minimally invasive technique. the aim of this study is to demonstrate the feasibility of robotic-assisted approach as a minimally invasive option for surgical management of crohn's disease and compare the perioperative outcomes with traditional laparotomy. methods: patients who underwent elective resection of the intestine for crohn's disease by roboticassisted or laparotomy approach from to q were identified using icd- codes from premier healthcare database. all the procedures were performed by either general surgeons or colorectal surgeons. since hospital characteristics were comparable between the two cohorts before propensity-score matching, : matching was performed using patient characteristics such as age, gender, race, charlson index score and year of the surgery to create comparable cohorts. sample selection and creation of analytic variables were performed using instant health data (ihd) platform (bhe methods: we conducted a retrospective analysis of , mis inguinal hernia repairs ( , robotic, , laparoscopic) from through with data collected in the premier hospital database. patient, surgeon, and hospital demographics of robotic and laparoscopic inguinal hernia repairs were compared. the adjusted odds ratio of receiving a robotic procedure was calculated for each of the demographic factors using a multivariable logistic regression model. statistical significance was defined as p. . sas software version . was used for statistical analysis. results: the odds of a procedure being robotic increased from inguinal hernia repair is one of the most common general surgery procedures with over , performed annually in the united states. when compared to traditional open inguinal hernia repair (oihr), laparoscopic inguinal hernia repair (lihr) has been associated with faster postoperative recovery rates and lower postoperative pain. with advances in the robotic platform, robotic inguinal hernia repair (rihr) is an available technique that is currently being explored. this study examines lihr and rihr as described in literature to see if one is superior to the other. study design: search terms: ''inguinal hernia repair surgical complications including hematomas ( . %), seromas ( . %), and trocar site infection ( . %) resolved with antibiotics, with a . % postoperative complication rate. conclusion: rihr repair is a safe alternative to lihr, with fewer postoperative complications and a faster recovery time. however, operative time as well as or room time is significantly longer, which may increase overall cost laparoscopic or robotic approach were chosen on a schedule availability basis. data was collected prospectively and it involved anthropometric data, presence of type diabetes mellitus (t dm), % of preoperative total weight loss (%ptwl), surgical time, postoperative length of stay, -day complications, and need for readmission or reoperation. comparison between groups was carried on with t-test for continuous data and with chi-square test for dichotomous variables. a p lower than . was considered significant. results: overall sagb were performed, laparoscopic and robotic. a long and thin gastric pouch was created calibrated by a fr bougie and a . cm antecolic antegastric gastrojejunal (gj) anastomosis was groups (laparoscopic vs robotic) were comparable regarding age ( vs . years, p= . ), bmi ( . vs kg/m , p= . ), %ptwl ( . vs . %, p= . ) and % with t dm ( vs there were fewer men in the laparoscopic group ( . vs % there were ( . %) major complications in the laparoscopic group: bleedings from the gj anastomosis, one of which required reoperation, severe dumping syndrome, gerd requiring revision and gj stricture that underwent relaparoscopy. the only complication ( %) in the robotic group was an acute pancreatitis. readmission rate was % in both groups and reoperation rate was % for laparoscopic and % for robotic surgeries. conclusions: totally robotic sagb with manual gastro jejunal anastomosis was safe and feasible in this early experience compared to laparoscopic approach multi degrees of freedom manipulator with mantle tube for assisting endoscopic and laparoscopic surgical operations masataka nakabayashi, phd , yuta hoshito, masters student p step by step anatomic mapping during laparoscopic transabdominal adrenalectomy lateral flank approach ranbir singh steps analyzed were: right adrenalectomy: step ) mobilize liver; ) medial dissection; ) adrenal vein isolation; ) inferior dissection; ) adrenal off kidney; ) detachment. left adrenalectomy: step ) division splenorenal ligament; ) develop plane pancreas/kidney; ) mobilization medial/lateral borders adrenal; ) adrenal vein isolatoin; ) dissection adrenal off kidney; ) detachment. structures were identified as yes/no and results expressed as percentage total n of cases seen at each step. results: structures identified at each step are shown (table) incisions were made at the oral vestibule under the inferior lip. a -mm trocar was inserted through the center of the oral vestibule with two -mm trocars above incisors. the subplatysmal space was created down to the sternal notch, and carbon dioxide was insufflated at pressure mmhg to maintain the working space. parathyroidectomy was performed using laparoscopic instruments. intraoperative parathromone levels were measured minutes after excision of gland. primary end-points were the success rate in achieving the cure from hyperparathyroid state and hypocalcemia rate. secondary end-points were operating time, scar length, pain intensity assessed by the visual-analogue scale, analgesia request rate, analgesic consumption, quality of life within postoperative days (sf- ), cosmetic satisfaction, duration of postoperative hospitalization, and cost-effectiveness analysis. result: one patient experienced a transient recurrent laryngeal nerve palsy which was spontaneously resolved within month. no permanent recurrent laryngeal nerve injury was found no mental nerve injury or infection was found. conclusion: with highly sensitive localising sestamibi and ct scans, focussed exploration is the current standard of treatment. among all minimally invasive surgeries, toepva is a feasible, safe, and almost pain-free surgical option when combined with intraoperative parathormone monitoring for patients with hyperparathyroidism indocyanine green is a water soluble nontoxic compound exhibiting near infrared renal function and long-term survival. indocyanine fluorescence helps in assessing vascular flow, tissue perfusion and aberrant anatomy and thereby leads to lower conversion rates in partial nephrectomy. we aim to present our experience in patients who underwent partial nephrectomy over years. materials and methods: of the partial nephrectomies performed at our institution, were done by laparoscopic approach alone and rest by patients who underwent llr for whole hepatoma in our facility, underwent llr for a solitary hepatoma and were divided into "before standardization" (bs; n= ) and "after standardization" (as) groups (n= ). patient background, characteristics, and perioperative outcomes were compared between these groups. procedure: we chose the devices according to phases of liver transection. a soft-coagulation monopolar device was used for marking surface. an ultrasonically activated device was used for transection of the liver surface within a -cm depth. crash and sealing with biclamp were indicated for deep-phase transection. the cavitron ultrasonic surgical aspirator was used if the lesion was close to the major glisson's sheath or the major hepatic vein. results: no significant differences in the patients' background were found between the two groups. the operative durations were min ( - min) and min ( - min) in the as and bs groups, respectively, with a significant difference (p. ). the blood loss volumes were cc ( - cc) and cc ( - cc), respectively (p= . ). the lengths of hospital stay after llr were days (range, - days) and days ( - days), respectively, with a significant difference iwao kitazono, phd , kentaro gejima , hizuru kumemura , akira hiwatashi , yuichiro nasu , fumisato sasaki , akio ido , yutaka imoto ; cardiovascular and gastroenterological surgery, kagoshima university graduate school of medical and dental science, digestive and lifestyle disease, kagoshima university graduate school of medical and dental science introduction: in locally-treatable gastrointestinal tumors, laparoscopic endoscopic cooperative surgery (lecs) is a minimally-invasive technique that can avoid excessive resection of the gastrointestinal tract. objective: to share our therapeutic guidelines and surgical technique of lecs for gastroduodenal tumors. subjects: nineteen patients who underwent lecs for gastroduodenal tumors ( patients with gastric tumor and patients with duodenal tumor).[results] ) gastric tumors ( gist, glomus): . site of lesion was u ( patients), m ( ), or l ( ), . operative procedure was acquired in a stepwise manner from classical lecs ( patients) to inverted lecs ( ) to non-exposed endoscopic wall-inversion surgery: news ( ). . operative outcome revealed no postoperative complications. ) duodenal tumors ( adenoma, m cancer, ectopic pancreas): . site of lesion was bulbus duodeni ( patient), superior part ( ), or descending part ( ); . operative procedure was esd followed by laparoscopic continuous suture in a single seromuscular layer for patients with preoperatively confirmed or suspected cancer, or full-thickness resection followed by albert-lembert suture along the short axis for patients unable to undergo esd. in all cases, c-tube was placed to prevent bleeding and perforation at the site of resection due to exposure to bile; . operative outcome included successful endoscopic hemostasis upon bleeding from exposed vessel on postoperative day in patient and anastomotic leak in patient. the event of anastomotic leak resolved after days of bile drainage through c-tube and conservative therapy. compared with patients who underwent esd alone, those who underwent lecs had significantly larger diameters of resected specimens and tumors (p. ) but no significant difference in the incidence of postoperative bleeding and delayed perforation. conclusion: for gastroduodenal tumors, lecs is a minimally-invasive and safe therapeutic option as it combines advantages of both laparoscopy and endoscopy. in particular, c-tube placement for bile drainage was effective in reducing exposure of the suture site to bile as well as supporting drainage after anastomotic leak. introduction: in japan, transurethral balloon catheters (tuc) are currently inserted in most surgical patients to maintain a urine outflow route and to measure the urine output both intraoperatively and postoperatively. however, tuc insertion not only causes postoperative pain but can also lead to urinary tract infections. temporary suprapubic catheters (spc) are used in the field of obstetrics and gynecology as a method of postoperative management to avoid performing transurethral procedures. in the field of surgery, especially in laparoscopic surgery, spc also considered how it would be a useful way to reduce patient suffering. here we report our prospective study on whether an spc can be safely inserted as a substitute for tuc during laparoscopic-assisted colectomy. subjects and methods: the subjects in this study were patients who underwent laparoscopic surgery for primary colorectal cancer from to , and who would normally have had their urinary balloon catheter removed early after surgery. during surgery, an angiomed cystostomy set was installed for patients who gave their consent to participate in this study as an alternative to a urinary balloon catheter. we prospectively collected patient information including sex and age, in addition to other perioperative data, such as, time required for cystostomy, complications accompanying cystostomy, sense of discomfort or pain associated with the vesical fistula after surgery, the time of the removal of the vesical fistula, the frequency of releasing the vesical fistula, postoperative complications. results: our subjects included cases who gave their informed consent to have an spc inserted. an spc was inserted into the remaining case. the mean surgical duration was min, and the spc insertion was performed at a mean of min after the start of surgery. insertion required a mean duration of . s. the bladder of one case ( . %) was perforated, and hematuria was observed at the time of insertion in two cases ( . %), but surgery completed without any incident. six out of cases ( . %) demonstrated neither urinary urgency nor independent urination on the day the catheter was clamped. however, the clamp was released two to four times, and draining of an average of ml urine, urinary urgency, and independent urination were confirmed - days later. conclusion: spc is a procedure that avoids crossing the urethra and its associated disadvantages. here we were able to demonstrate that the procedure can be safely used in laparoscopic surgery patients.our objective is to devise methods for proper port placement to overcome the ergonomic challenges. procedure: patients with sit were operated laparoscopically in our hospital in the period of may to november , males suffering from cholelithiasis without cholecystitis and female with acute appendicitis. after thorough review of literature and proper planning, the patients were posted for surgery. for laparoscopic appendectomy, a thorough initial diagnostic survey is performed on introducing a scope through the umbilical port and confirming the exact location of the appendix. the two working ports are introduced accordingly, which is usually a mirror image of the standard port sites. the appendix was visualised in the left iliac fossa and after meticulous dissection, the appendix and mesoappendix were divided using an endostapler. the operative time was minutes and there were no intraoperative or postoperative complications.the port placement for laparoscopic cholecystectomy in such a case is trickier as the anatomical variation and the contralateral disposition of the biliary tree demand an accurate dissection and exposure of the biliary structures to avoid iatrogenic injuries. it is important to conform to the principles of triangulation during port placement. the mirror image of -port placement is convenient for left-handed surgeons. whereas, to make the procedure comfortable for right-handed surgeons, the working ports need to be shifted caudally with the surgeon standing between the patient's legs. the mean operative time was minutes and there were no minor or major intraoperative or postoperative complications.conclusion: ergonomic comfort is vital to a smooth procedure. while mirroring ports suffices for appendectomy, all other procedures require forethought for port placement. it should be noted that ambidexterity is a desirable skill in the operating room for a laparoscopic surgeon.priscila r armijo, md, chun-kai huang, phd, gurteshwar rana, md, dmitry oleynikov, md, ka-chun siu, phd; university of nebraska medical center introduction: the aim of this study was to determine how objectively-measured and self-reported fatigue of the upper-limb differ between laparoscopic and robotic surgical training environments. methods: surgeons at the sages conference learning center, and at our institution were enrolled. two surgical skills practical environments were utilized: ) a laparoscopic training-box environment (fls) and ) the mimic® dv-trainer (mimic). two standardized surgical tasks were chosen for both environments: peg transfer, and needle passing. each task was performed twice. objective fatigue was evaluated by muscle activation and fatigue, and comparisons were made between fls and mimic, for each surgical task. muscle activation of the upper trapezius, anterior deltoid, flexor carpi radialis, and extensor digitorum were recorded during practice using surface electromyography (emg; trignotm, delsys, inc., boston, ma). the maximal voluntary contraction (mvc) was obtained to normalize muscle effort as %mvc. the median frequency (mdf) was calculated to assess muscle fatigue. subjective fatigue was self-reported by completing the validated -scale score piper fatigue scale- (pfh- ) before and after practice. statistical analysis was done using spss v . , with α= . . results: this abstract represented the performance of trainees (fls: n= , mimic: n= ) as part of larger cohort of the study. for peg transfer, emg analysis revealed that mimic had a significant increase in mean muscle activation for the upper trapezius and anterior deltoid, both p\ . . conversely, practice with fls led to significantly more muscle fatigue than mimic for the same muscle groups (upper trapezius: p= . , anterior deltoid: p= . ), represented by a significantly lower mdf. similarly, for needle passing, mimic had a significant increase in mean muscle activation for the upper trapezius (p= . ) and anterior deltoid (p= . ), but practice with fls significantly induced more muscle fatigue effort for anterior deltoid (p= . ). survey analysis revealed a significant decrease in self-reported fatigue after performing fls tasks (before: . ± . , after: . ± . , p= . ), but no difference after mimic tasks (before: . ± . , after: . ± . , p= . ). conclusions: although different muscle groups are preferentially required in the performance of fls and mimic, our analysis for both surgical tasks showed practice with mimic required more activation of shoulder muscles, whereas practice with fls could lead more muscle fatigue for the same muscle groups. interestingly, surgeons reported improved or no change in perceived fatigue after the tasks, despite of having an increase in muscular activation and effort. subjective selfreport fatigue might not truly reflect the level of fatigue when trainees practice surgical tasks using fls or mimic. objective: to investigate the prevalence of musculoskeletal (msk) injuries in bariatric surgeons around the world. background: as the popularity of bariatric surgery increases, efforts into improving its patient safety and decreasing its invasiveness have also been on the rise. however, with this shift towards minimal invasiveness, surgeon ergonomic constraints have been imposed, with a recent report showing a - % prevalence of physical complaints in surgeons performing laparoscopic surgeries. methods: a web-based survey was designed and sent out to bariatric surgeons around the world. participants were queried about professional background, primary practice setting, and various issues related to bariatric surgeries and msk injuries. results: there were responses returned from surgeons from countries around the world. . % of the surgeons have had more than years of experience in laparoscopic surgery, . % in open and . % in robotic surgery. % of participants reported that they have experienced some level of discomfort/pain attributed to surgical reasons, causing the case load to decrease in . % of the surgeons. it was seen that the back was the most affected area in those performing open surgery, while shoulders and back were equally as affected in those performing laparoscopic, and the neck for those performing robotic, with . % of the surgeons reporting that this pain has affected their task accuracy/surgical performance. a higher percentage of females than males reported pain in the neck, back and shoulder area when performing laparoscopic procedures. supine positioning of patients evoked more discomfort in the wrists, while the french position caused more discomfort in the back region. only . % sought medical treatment for their msk problem, of which . % had to undergo surgery for their issue, and . % of those felt that the treatment resolved their problem. conclusion: msk injuries and pain are a common occurrence among the population of bariatric surgeons, and has the ability to hinder performance at work. therefore, it is of importance to investigate ways in which to improve ergonomics for these surgeons as to improve quality of life.introduction: the use of robotic technology is rapidly increasing among general surgeons but is not being routinely taught in general surgery residency. we aimed to evaluate our first robotic cases during which time we developed a robotic surgery curriculum incorporating residents. methods: the first robotic cases performed at our institution from - by two surgeons were analyzed. a residency curriculum was developed and instituted after the first months. it consisted of online modules offered by intuitive surgical resulting in certification, simulator training, hands on workshops for cannula placement, docking, instrument exchange, camera clutching and other introductory tasks. patient demographics, type of procedure, resident involvement, total operative and console times, comorbid conditions and complications were evaluated. unpaired t tests were performed for statistical analysis. results: females and males comprised this series with an average age of years ± . the majority of patients, % had comorbidities, with a predominance of hypertension, % and diabetes, %. the bariatric patients had an average bmi of ± . a variety of procedures were performed including hernias, foregut and bariatric. residents participated in % of cases. there were no differences in total operative and console times in cases with residents except bariatric procedures. there were complications in this series; postoperative ileus, gallbladder fossa hematoma and an enterotomy. there was one early conversion to open in a complex foregut case and no deaths in this series.conclusions: we report our initial experience of robotics in a variety of general surgery and complex foregut cases. the implementation of a robotic surgery program and residency curriculum was safe with similar outcomes related to operative times and complications. as mis expands with the application of robotics in general surgery, residency curriculums will need to be revised. further data is needed to determine residency learning curves between robotics and laparoscopy.background: robotic surgery has made a large impact in the fields of urology and gynecology. its use is significantly increasing in the fields of general and bariatric surgery. evidence remains unclear as to the clinical impact on outcomes, and significant questions remain as to the impact of cost. our goal was to evaluate the economic impact of robotic surgeries in general and bariatric surgery at our institution. methods: this study is a retrospective analysis of minimally invasive general and bariatric procedures done at a single institution from january through june . we performed a cost and reimbursement analysis of robotic versus conventional laparoscopic surgery. the cost evaluation included operative time, operating room costs, length of stay and overall hospital expenses. in addition, we looked at reimbursement and the contribution margin per cpt code. results: our study included a total of patients who underwent laparoscopic and robot assisted general and bariatric surgeries. the average time duration for laparoscopic surgeries was minutes vs minutes for robot assisted. we performed a cost analysis which showed an average total cost of $ , for laparoscopic and an average of $ , for robot assisted. the total reimbursements were $ , for laparoscopic and $ , for robot assisted. this translated to an average contribution margin of $ , for laparoscopic vs $ , for robot assisted. for general surgery we found an average cost of laparoscopic $ , vs robot assisted $ , , with a contribution margin of $ , laparoscopic vs $ , robot assisted. for bariatric surgeries we found an average contribution margin of $ , for laparoscopic vs $ , for robot assisted. conclusions: robotic surgery has been associated with higher costs and longer operative times. in this economic climate of increased cost awareness with institutions under increasing financial pressures, judicious use of resources becomes important when determining surgical approach. although cost of robot assisted surgery may decrease with time, other quality factors may be important in patient selection. although there is no clear evidence that institutions lose money with robot assisted surgery, in our experience the contribution margin is lower with robot assisted surgery as compared to conventional laparoscopy.introduction: this retrospective study was performed to evaluate the safety and feasibility of the new senhance robotic system (transenterix) for inguinal hernia repairs using the transabdominal preperitoneal approach. our series is the first experience in the field of general surgery utilizing this new robotic platform. methods: from march to september , inguinal hernia repairs in patients were performed using the senhance robotic system. the senhance surgical system is a new robotic platform that consists of a cockpit, manipulator arm and a connection node (figure ). this new system provides robotic surgery with numerous advantages including eye-tracking camera control system, haptic feedback, reusable endoscopic instruments, and a high configuration versatility due to total independency of the manipulator arms. patients were between and years of age, eligible for a laparoscopic procedure with general anesthesia, had no life-threatening disease with a life-expectancy of less than month and a bmi \ . a retrospective chart review was performed for a variety of pre-, peri-and postoperative data including but not limited to patient demographics, hernia characteristics, intraoperative and postoperative complications. results: male and female patients were included in the study. median age was . years (range - years), and median bmi was . (range . - . kg/m ). median docking time was minutes (range - minutes), and median operative time was minutes (range - minutes). two cases were converted to standard laparoscopic surgery due to robot malfunction and intraoperative bleeding respectively. one patient developed a postop seroma that did not require any further intervention. conclusion: we report the first series of laparoscopic inguinal hernia repairs using the new senhance robotic system. compared to previously published conventional laparoscopic or robotic tapp hernia repairs these data suggest similar outcomes in operative time and perioperative complications. additionally there was no significant learning curve detected due to its intuitive applicability. therefor the senhance robotic system can be safely and easily used for tapp hernia repairs by experienced laparoscopic surgeons. this is a video presentation of years old female, who presented with suprapubic pain and mass to gynecology office. she has a history of robotic hysterectomy and sbladder sling operation years ago. this was complicated with peritonitis and long icu stay, due to what she was called ''bowel injury'' but treated only conservatively with antibiotics and subsequent abscess drainages at that time. she has occasional appearing nodule and pain at the left suprapubic region. ct ordered by gynecology read as abdominal wall hernia with long sigmoid diverticuli in hernia. also there was small amount of subcutaneous air at the tip of herniated diverticuli. after antibiotic treatment and improvement, colonoscopy shows, actually the diverticuli is the limb of the sling going through the simoid and anchored in subcutaneous fat on abdominal wall ahich represents clocutaneous fistula as gets infected. clip was placed on sling and repeat imaging comfirmed that the localion of this sling fits to location of so called ''hernia'' the sling limb was resected robotically and colon was repaired with side stapling of clolonic wall. the abdominal wall defect is repaired with long term absorbable suture. as far as we have found, the presentation and treatment of this complication is unique and could not find a similar case to guide us for the plan. background: robot-assisted surgery using da vinci surgical system (dvss) is thought to have many advantages over conventional laparoscopic surgery. it was reported that the use of the surgical robot might reduce surgery-related complications, then a multi-institutional historically controlled prospective cohort study on the feasibility, safety, effectiveness and economical efficiency of robotic gastrectomy (rg) for resectable gastric cancer was conducted in japan. this study evaluated the safety of rg using dvss xi. methods: this single-center, prospective phase ii study included patients with resectable gastric cancer (umin ). the primary endpoint was the incidence of post-operative complications greater than grade iii according to clavien-dindo classification during one month after surgery. the secondary endpoints included all adverse events and completion rate of robotic surgery. results: from oct to jan , patients were enrolled for this study. the incidence of post-operative complication greater than grade iii was %. the overall incidence of adverse events was . % (grade i; . %, grade ii; . %). no patient required conversion to laparoscopic or open surgery; thus, the rg completion rate was %. conclusion: this study suggested the introduction of rg using dvss xi for gastric cancer seems to be safe and feasible. priscila r armijo, md , dmitry oleynikov, md , sages robotic task force* ; university of nebraska medical center, sages robotic task force introduction: while robotic companies continue to aggressively market and promote the use of robots in general surgery, little is known about how this technology is employed by general surgeons, and what is expected of this technology from both novice and experts in the field. the aim of this study is to evaluate the needs of general surgeons who are new to robotic surgery and the needs of established robotic surgeons. methods: the sages robotic task force survey, a one-page survey, was designed and sent electronically to all sages members. questions regarding fellowship training, area of expertise, robotic simulation and in clinical case use, services offered in the current hospital, mentorship, likelihood of switching to a different approach, and expectations for the robot were included in the survey. two groups were created based on previous use of davinci® system in a clinical scenario, or not. statistical analysis was conducted using ibm spss v. . . , using fischer's exact and pearson's chi-squared tests where appropriate. results: sages members answered the survey. surprisingly, respondents ( %) had used the davinci® in a clinical setting. among these, ( %) had additional fellowship training, compared to ( %) in the non-clinical use group, p= . . of all surgeons with additional fellowship training, the great majority ( %) had specialization in advanced gi, mis and bariatric surgery, followed by colorectal ( %). most surgeons are performing less than cases per month using the robotic system, and with the majority of cases performed using the platform being hernia repairs ( %), followed by foregut-related procedures ( %). interestingly, from all the surgeons who replied the survey, only . % are planning to switch from open procedures to its robot counterpart, whereas . % are planning to adopt robotic-assisted procedures rather than laparoscopy. conclusions: the majority of sages members who responded to the survey have used the davinci® in a clinical setting in the past. surgeons who stated they perform mainly laparoscopic procedures were likely to continue to adopt robotic techniques, whereas those who perform open hernia repair for example were not very likely to switch to robotic approach. while the use of the robot may be enabling surgeons who used to perform mostly open procedures in the urology or gynecology fields, laparoscopic skills predict robotic utilization in general surgery. hernia and foregut appear to be the most common procedures that are being utilized.aim: while conventional multiport laparoscopic splenectomy has become gold standard for some hematological or splenic diseases, reduced-port laparoscopic splenectomy (rpls) including singleincision laparoscopic splenectomy (sils) is regarded as highly challenging. herein, we describe the technical refinements for safe rpls especially for patient with splenomegaly. methods: in all cases, access was achieved via a . -cm mini-laparotomy at the umbilicus into which a sils tm port or e-z access ® with three -mm trocars was placed. a -mm flexible scope, an articulating grasper, and straight instruments were used. our rpls is characterized by the followings: a) early ligation of the splenic artery to shrink the spleen, b) application of our original "tug exposure technique," which provides good exposure of the splenic hilum by retracting (tugging) the spleen with a cloth tape, and c) safe introduction of stapler under the guidance with a flat drain into the splenic hilum. results: rpls patients ( men and women, ± years old) comprised hematological disorder (n= ), splenic disease (n= ), and liver cirrhosis (n= ). in patients ( %), rpls was successfully completed: sils in and sils plus one additional port only in patients. conversion to open surgery was necessary in patients including liver cirrhosis with remarkable collateral varicose veins around the spleen. operation time and blood loss were ± min and ± g, respectively. weight of the extracted spleen was heavier than normal and ± g (maximum g). no intra-or postoperative complication occurred. the postoperative scar was nearly invisible. conclusions: rpls might safely be performed even for splenomegaly (up to , g). however, care should be taken for cirrhotic patient with collateral veins. rpls can be the procedure of choice even in the patients with splenomegaly and who are concerned about postoperative cosmesis. the aim of this feasibility study was to evaluate laparoscopic sn biopsy for laparoscopic snns in early gastric cancer patients. subjects and methods: this study includes patients with ct n m (primary tumor \ cm) gastric cancer who underwent laparoscopic sn biopsy in conjunction with radioisotope and dye methods between jan. and jul. . first, we looked for green-dyed sns after injection of indocyanine green (icg) without near-infrared light system, and then tried to detect the radioactivity of sns using a hand-held gamma probe inserted through a small incision at the umbilical port. after the areas where sns were distributed were resected, a gastrectomy with prophylactic lymphadenectomy was performed according to the gastric cancer treatment guidelines of the japanese gastric cancer association. we looked for undetected sns in the resected specimen at the back table. results: among cases, there were ( %) in which sns were not detected in the resected specimen. there were cases in whom sns were detected in the resected specimen. in both cases, the primary tumors were located in the middle and greater curvature of the stomach. in case , laparoscopic sn biopsy identified the left ( sb) and right ( d) greater-curvature lymph node (lns) as sns, however, lesser-curvature ( ) and infrapyloric ( ) lns remained as sns in the resected specimen. in case , the left ( sb) and right ( d) greater-curvature lns were identified as sns intraoperatively, while the lesser-curvature ( ) ln remained as an sn in the resected specimen. the sns overlooked with laparoscopic sn biopsy method were detected by radioisotope only. no cases had ln metastasis, and the -year relapse-free survival rate of these patients was %. conclusions: our feasibility study of laparoscopic sentinel node biopsy for early gastric cancer showed that we should search for sns of the lesser curvature carefully even if the primary lesion is located at the greater curvature. key: cord- -vgjz ts authors: nan title: th international congress of the european association for endoscopic surgery (eaes) sevilla, spain, – june date: - - journal: surg endosc doi: . /s - - -x sha: doc_id: cord_uid: vgjz ts nan laparoscopic cholecystectomy is one of the most commonly performed operations worldwide. bile duct injury (bdi) is a rare but very serious complication of the procedure, with a significant impact on quality of life and overall survival. the high frequency of bdi with laparoscopic cholecystectomy was first considered to be a consequence of the initial learning curve of the surgeon, but it later became clear that the primary cause of bdi is misinterpretation of biliary anatomy. intraoperative cholangiography (ioc) has been advised by many authors as the technique reduces the risk of bdi. however, the procedure has inherent limitations and is therefore reserved for select cases. fluorescent cholangiography using indocyanine green(icg) is a novel approach, which offers real-time intraoperative imaging of the biliary anatomy. a comparative study was contacted by administering icg intravenously or intrabiliary during the operation. forty patients scheduled to undergo an elective lap. cholecystectomy were randomly divided in two groups: in group a icg was administered in a dose . mg in ml solution intravenously hour before surgery. in group b icg was injected intrabiliary in a . mg/ml solution mixed with the patient's bile. also, we observed and analysed the following parameters, liver function, b.m.i, asa score and possible complications, before and after operation. results: group a. intravenous icg was administered in patients. there was no any reaction and the extrahepatic biliary anatomy was identified well. there was no bdi or any complication related to the procedure. group b. icg was injected intrabiliary in patients during the laparoscopic procedure. in all but one patient the extrahepatic biliary tree was delineated very well. in one patient part of icg solution was injected into the gallbladder wall and this resulted in a partially confusing image. there was no bdi and no postoperative complication conclusions: fluorescence cholangiography can be used during laparoscopic cholecystectomy to obtain fluorescence images of the bile ducts following intrabiliary injection during the operation orintravenous injection h before the procedure. the later technique is more easy to perform and does not require catheterization of the biliary tree. endoscopia digestiva chirurgica, policlinico universitario ''a. gemelli'', rome, italy; ihu, strasbourg, france; camma group, icube, university of strasbourg, cnrs, ihu strasbourg, strasbourg, france; ircad, strasbourg, france; digestive and endocrine surgery, nouvel hopital civil, university of strasbourg, strasbourg, france; digestive and endocrine surgery, ihu-strasbourg, strasbourg, france aim: surgical societies are united in promoting the critical view of safety(cvs) during laparoscopic cholecystectomy(lc). nonetheless, reports have shown a discrepancy between the operative reports and the correct application of cvs, which may explain the stability of bile duct injury rates. therefore, surgeons and computer scientists at our institution are developing a machine-learning algorithm to automatize cvs assessment. however, the lack of a consistent cvs video assessment framework limits the ability to generate data to train the artificial intelligence. here we describe and test a method for cvs evaluation in videos. method: between march and july , consecutive videos of lc performed at nouvel hospital civil(strasbourg, france) were recorded. two independent reviewers assessed the achievement of cvs in the s video sequences preceding clipping of cystic duct and artery. in addition to the 'doublet view' method, a 'binary' video evaluation method was tested: each of the criteria composing the cvs( structures entering the gallbladder, clearance of the hepatocystic triangle and lower part of the cystic plate) was classified as achieved or not. if the criteria were met, then the cvs was considered achieved. inter-rater agreement for cvs and for each of the criteria was evaluated. results: twenty-two videos( fundus first and partial lc, and broken videos) were excluded from the cvs analysis. cvs elements were assessable in all but one s videos sequences( . %). after mediation, cvs was achieved in / ( . %) of lc. the cystic plate was identified in only . % of videos. inter-rater agreement using the doublet view vs. the binary method was as follows: . %(? = . ) vs. . %(? = . ) for cvs achievement, . %(? = . ) vs. . %(? = . ) for the structures, . %(? = . ) vs. . %(? = . ) for the hepatocystic triangle and . %(? = . ) vs. . %(? = . ) for the cystic plate ( fig. ) . conclusions: reliable cvs assessment is crucial to generate consistent data for machine-learning algorithms aiming at decreasing bile duct injury after cholecystectomy. our binary cvs video assessment method showed higher inter-rater reliability than the doublet view, originally described for assessment of photos. further studies are on going to validate the cvs assessment in videos and support our initial results. surg endosc ( ) the vital role of surgeries in healthcare requires a constant attention for improvement. surgical process modeling is an innovative and rather recently introduced approach for tackling the issues in nowadays complex surgeries, involving complex logistics, much technology, and large teams. surgical process modeling allows for evaluating the introduction of new technologies and tools prior to the actual development and is beneficial in optimization of the treatment planning and treatment performance in operating room. in this study, we first discuss the concepts associated with surgical process modeling, aiming to clarify them and to promote their use in future studies. next, we apply these concepts to analyze the procedure of challenging interventions, minimally invasive liver treatment (milt) methods, with the ultimate goal of improving and optimizing the treatment procedure. the procedure model of current treatment activities and planning of various milt methods and the associated techniques, are analyzed and combined into a generic procedure model of milt, which provides a firm foundation for qualitative and quantitative analysis of different milt procedures. the generic procedure model is validated by data from erasmus medical center (rotterdam, the netherlands) and oslo university hospital (oslo, norway) . the proposed procedure model is designed to be a basis for improvement of the procedure and to determine how and where the new technologies can be best, effectively and efficiently, employed in the clinical practices prior to and/or during actual development of the new technologies for milt. as a conclusion, the current work illuminates the importance of surgical process modeling for improving different aspects of treatment procedures and provides an overview of various modeling strategies that can be used to establish surgical process models. the generic procedure model of various milt methods, including laparoscopic liver resection, laparoscopic liver ablation and percutaneous ablation, is introduced and validated which is a basis for introduction of the optimized procedure model of milt objective: to determine the most appropriate time to start total laparoscopic living donor right hepatectomy (tldrh) based on the experience with laparoscopic liver resection (llr). summary background data accumulation of experience in llr is essential before starting tldrh to ensure donor safety. methods: we retrospectively reviewed data of and consecutive patients who underwent llr and donor hepatectomy, respectively, between and . operative outcomes of laparoscopic major hepatectomy (lmh) were compared between two periods based on tldrh introduction (phase i - vs phase ii . learning curve of llr was evaluated using the cumulative sum (cusum) method to determine the optimal time of tldrh introduction. conclusion: accumulating an experience of at least lmh cases is needed in low-volume lt centers before starting tldrh to ensure donor safety. introduction: the number of surgical adverse events is still too high. an important number of these adverse events occur within the operating room (or) and are in fact preventable. in order to reduce adverse events in the or, we simply need to know what went well and what can be done better. the aim of this study was to analyze and debrief a predefined selection of surgical procedures, with the use of an operating room 'black box', to identify commonly observed safety threats and resilience support events. methods: in the period - , predefined gastro-intestinal laparoscopic cases were recorded by the or black box'. the postoperative surgical team assessment record (star) questionnaire was used. the recordings were analyzed by specifically trained raters, using the systems engineering initiative for patient safety (seips) model of work system and patient safety to identify relevant safety threat and resilience support events. qualitative data analysis was used to identify the most commonly discussed events during the team debriefings. results: in only . % (n = ) of times or team members, when asked direct following surgery, indicated that they had noticed aberrations (n = ) during the case. a mean number of . (sd . ) relevant positive and negative events (e.i. aberrations) per surgical procedure were identified using the black box performance report. on average, . (sd . ) of events identified by the black box were rated as safety threats. most events discussed during the team debriefings were related to communication. conclusion: these results once again highlighting the importance of clear and closed-loop communication in the operating room. theatre staff underestimated the number of aberrations occurring in the or, when asked to retrieve from memory. postoperative structured team debriefing may be important for resolving incorrect assumptions between operating team members to avoid future unnecessary miscommunication. background: the eaes has recently published an intraoperative adverse event classification to assist the direct measurement and routine reporting of minimal access surgery interventions. we aimed to explore the clinically validity and reliability of the classification. methods: a prospective evaluation utilising case videos and clinical data from a completed multi-centre laparoscopic total mesorectal excision surgery randomised controlled trial was performed (isrctn ). enacted adverse events identified with the observational clinical human reliability analysis technique were graded with the eaes classification by two blinded, independent assessors. test-retest reliability was explored using grades previously applied during the development of the classification with intraclass correlation co-efficients calculated. clinical validity was assessed using -day morbidity events, the clavien-dindo classification and the highest eaes grade per case. results: laparoscopic cases ( h of surgery) contained error events which were all successfully categorised. excellent inter-rater and test-retest reliability was seen (icc . , % ci . - . , p \ . and icc . , % ci . - . , p \ . respectively. % of patients experienced post-operative morbidity (median event, range [ ] [ ] [ ] [ ] [ ] [ ] . labelling analysed cases by their highest eaes classification grade gave % grade , % grade and % grade procedures. % of grade cases developed a morbidity event, but this significantly increased in grade and operations ( % and %, p = . ). the number of complications and highest recorded clavien-dindo grade increased with each additional grade ( . ± . vs. . ± . vs. . ± . , p = . and median vs. vs. , p = . respectively). anastomotic leak and re-operation were correctly captured by the allocated eaes grade ( . % vs. . % vs. %, p \ . and % vs. % vs. %, p \ . respectively). there was a significant rise in length of stay observed with increasing eaes grade (median vs. vs. days, p \ . ). conclusion: in the context of major laparoscopic surgery, the eaes intraoperative adverse classification is seen to be a clinically valid and reliable assessment method. psychological medicine, nuhs, singapore, singapore aims: neurobiological feedback in surgical training could translate to better educational outcomes such as measures of learning curve. the variation in brain activation of medical students when performing laparoscopic tasks before and after a training workshop is not properly studied before and we planned to do this using functional near infrared spectroscopy (fnirs) which is a non-invasive optical brain imaging tool that measures cortical oxygenation change which is used as a marker of pre-frontal cortex activity (pfca). methods: this randomised controlled trial examined the pfc activity differences in two groups of novice medical students during the acquisition of basic laparoscopic tasks. 'trained-group' had standerdised oneto-one training on the tasks, while the 'untrained-group' had no prior trainining and was just shown a video of the tasks. the pfca was measured pre and post intervention using a portable fnirs device. primary outcome was the difference in the pfca pre and post intervention. secondary outcomes were the differences in pfca between the tasks and between the sexes. results: trained and untrained medical students with an equal sex distribution and a comparable age distribution were invovlved in the study. all students were right handed. trained group had a significantly attenuated pfca in the 'precision-cutting' (p = . ) and 'suture-insertion' (p = . ) tasks compared to the untrained group. subgroup analysis based on sex revealed significant attenuation in pfca in trained females compared to untrained females across of the laparoscopic tasks: 'pegstransfer' (p = . ), 'precision-cutting' (p = . ), 'suture-insertion' (p = . ). no significant pfca attenuation was found in male students who underwent training compared to untrained males. conclusion: a standardised laparoscopic training workshop promoted greater pfca attenuation in female medical students compared to males. this suggests that female and male students respond differently to the same instructional approach. these results may have implications for surgical training and education such as a greater focus on one to one surgical training for female students and use of pfca attenuation as a form of neurobiological feedback as a measure of learning curve in surgical training. robot assisted versus laparoscopic advanced suturing learning curve e. leijte , i. de blaauw , c. rosman , s.m.b.i. botden surgery, radboudumc, nijmegen, the netherlands; pediatric surgery, radboudumc, nijmegen, the netherlands aims: compared to conventional laparoscopy, robot assisted surgery is expected to have most potential in difficult areas and demanding technical skills as minimally invasive suturing. this study was performed to identify the differences in the learning curves of laparoscopic versus robot assisted advanced suturing method: novice participants, with the knowledge of basic surgical procedures, were recruited and performed three suturing tasks on the eosim laparoscopic augmented reality simulator or the robotix robot assisted virtual reality simulator. each participant performed an intracorporeal suturing, tilted plane needle transfer and anastomosis needle transfer task. to complete the learning curve, all tasks were repeated for maximal twenty repetitions or until a plateau was reached three consecutive times. clinical relevant and comparable parameters regarding time (seconds), movements and safety were recorded. intracorporeal suturing was used to visualize and compare the learning curves between the groups. results: forty-six participants completed the learning curve, of which laparoscopically and robot assisted. when comparing the suture time, the plateau was reached much faster in the robot assisted group ( - repetitions) than the laparoscopic group ( ) ( ) ( ) repetitions) as shown in figure . there was a significant difference in 'time per suture', during the whole learning curve with median values of versus (first knot), versus (fifth) and and (eighteenth), all with a p \ . . however, the parameter 'adequate surgical knot' was reached earlier in the laparoscopic group than in the robot assisted group. first: % versus %, fifth: % versus %, and eighteenth: % versus %. when assessing the 'needle out of view' parameter, the robot assisted group scored a median of . and . s during the first, respectively eighteenth knot, and the laparoscopic participants had their instruments out of view for and s during the first respectively eighteenth knot. conclusion: the learning curve of minimally invasive suturing can be reduced with the use of robot assisted surgery, with a specific reduction in operation time. the rate of adequate knots seemed to remain lower in robot assisted surgery, although this could be due to the virtual reality aspect of the simulator. introduction: endoscopic sleeve gastroplasty (esg) is a novel promising bariatric endoscopy treatment. gastric volume reduction and delayed gastric emptying are the mechanisms driving weight loss. however, little is known about the factors influencing the effectiveness of weight loss overtime. the present study aims at evaluating the correlation between endoscopic suture appearance and excess weight loss (ewl%) at and months follow up. patients and methods: all patients who underwent follow-up endoscopy at and months after esg were included. esgs were classified in groups according to endoscopic appearance of the gastric sutures: optimal (group ) when all stitches were in place and tights; suboptimal (group ) when one or more stiches were displaced; loose (group ) when all the sutures were completely disrupted. bmi at enrollment and ewl% at and months were recorded and compared to the endoscopic appearance. results: a total of patients were included in the analysis. at months, ( . %) patients had an optimal esg, ( . %) had a suboptimal sleeve and ( . %) had complete sutures failure. bmi at enrollment and ewl% were respectively . ± . and . ± . % for group , . ± . and . ± . % for group and . ± . and . % ± . % for group . twenty five patients had months egds: ( %) presented an intact esg and were classified in group , ( %) in group and ( %) in group . twelve months ewl% was respectively . ± . %, . ± . and ± . %. initial bmi significantly correlated with suture status at both (rho - . ; p \ . ) and months (rho - . ; p = . ) follow-up. furthermore, the sutures' appearance itself correlated with ewl% at both time points (rho ? . ; p = . and rho . ; p = . respectively). conclusion: our preliminary results show that the aspect of the endoscopic suture has a significantly impact on ewl% at and months after esg. furthermore, bmi at enrollment seems to predict endoscopic suture duration overtime. larger studies and longer follow-up are needed to further validate our preliminary findings. background and aim: endoscopic sleeve gastroplasty(esg) is a relatively novel endoscopic procedure that reduces the gastric lumen with proven less complications and less months weight loss compared to laparoscopic sleeve gastroplasty (lsg) . at present there are no studies investigating the role of multidisciplinary approach in esg. the aims of the present study were to evaluate the role of multidisciplinary assessment(ma) prior esg, weight loss outcomes, quality of live improvements and adverse events. material and methods: from may to may all patients that underwent esg were retrospectively evaluated from a prospective database. until september before esg only psychiatric evaluation was requested, while after this date we adopted the guidelines of the italian society for obesity surgery and all patients were evaluated on a multidisciplinary fashion prior esg. the multidisciplinary team was composed by:gastroenterologist, surgeon, psychiatrist, endocrinologist and dietitian. patients were divided in two groups:group were patients with esg before ma and group were patients with esg after ma. we compared this two groups in terms of weight loss outcomes, quality of live improvements and adverse events. quality of live was measured with the bariatric analysis and reporting outcome system(baros).all procedures were done with the apollo overstitch suturing system(apollo endosurgery) and a double channel gastroscope olympus tgif- (olympus japan).all procedures were done in general anesthesia and with insufflation of co . all patients had ambulatory visit t , and months after esg and weight loss outcomes were measured in terms of excess weight loss (%ewl),the total body weight loss (%tbwl) and baros scale were assessed. statistical analysis was done with chi-square test and \ . value was considered significant. results: patients were identified ( female; mean age . , range - ) . mean bmi at inclusion was . (range . - . ). mean %ewl and %tbwl at months was . and . respectively (table ) .non procedure related complications were observed. comparing the two groups there was significant(p \ . ) difference in terms of %ewl and %tbwl (table ) ,with better results in group . there was also a significant improvement in the baros scale in the patients in group . conclusions: ma before esg has a fundamental role in terms of better procedure outcomes for both weight loss and quality of live in obese patients. gastroenterology, hadassah medical center, jerusalem, israel, israel aims: the over-the-scope clip (ovesco) is a novel endoscopic tool that enables non-surgical management of gastrointestinal defects. the aim of this study was to report our experience with ovesco for patients with staple line leaks following laparoscopic sleeve gastrectomy (lsg). methods: a prospectively maintained irb-approved institutional database was queried for all patients treated with ovesco for staple line leaks following lsg from to . primary outcome was complete resolution of leak following ovesco as defined by return to complete oral nutrition and no evidence of leak on imaging. secondary outcome was the number of additional endoscopic or surgical procedures needed following ovesco. results: twenty-five patients ( males, females) were treated with ovesco for staple line leaks following lsg. the median age was years (range - ), and mean body mass index was kg/m . nine patients ( %) were referred from an outside hospital. the median time from index operation to leak diagnosis and from leak diagnosis to ovesco was days (range - ), and days (range - ), respectively. all patients had upper staple-line leaks near the gastroesophageal junction. initial treatment included antibiotics- patients; computed tomography guided drainage and antibiotics- patients; and laparoscopic drainage- patients. ovesco led to final resolution of leak in patients ( %) within days of clip deployment (range - ). leaks which persisted following ovesco were eventually resolved with a combination of ovesco and stent- patients ( %), total gastrectomy and esophago-jejunostomy- patients ( %), and endoscopic suturing- patient ( %). one mortality was noted in a patient who suffered multiorgan failure. the number of additional endoscopic sessions ranged from to (median ). no procedure related complications were noted. all patients were treated with total parenteral nutrition and the total length of stay was days (range - ). conclusions: despite its low success rate, ovesco should be part of the bariatric surgeon's non-surgical armamentarium in treating staple line leaks following lsg. r. bademci , r. vilallonga , p. alberti , r. renato , c. yuhamy , s.s. cordero , l. posadas general surgery, istanbul medipol üniversitesi, istanbul, turkey; bariatric surgery, vall d'hebron, barcelona, spain background: in cases of morbid obesity, treatment is generally applied as either a surgical or endoscopic approach. the number of primary obesity surgery endolumenal (pose) procedures is increasing but the reliability and effectiveness is unclear as yet. the aim of this study was to present a series of cases that required revision surgery due to pose failure and to reveal possible alternative surgeries. materials and methods: a retrospective comparison was made of the data of obese patients with pose failure and conversion to surgical procedures between and in respect of operation, medical illness and bmi results. results: the patients comprised % females, % males with a mean age of . ± . years and mean follow-up period of . ± . months. on average, patients lost . ± . kg, with an average excess weight loss of . %. conclusion: no firm conclusions can be drawn from such a small group. although sg seems to be a safe procedure and should be considered as the first technique to be applied following pose failure, it is possible to perform gastric bypass on patients with this endoscopic precursor. introduction: the population of post bariatric surgery patients is rapidly increasing worldwide. due to the altered anatomy post roux-en-y gastric bypass (rygb), conventional endoscopic management for choledocholithiasis is challenging. these patients are now commonly managed by means of a laparoscopic assisted ercp. although effective, this requires significant resource utilization and potential morbidity related to the need for surgical intervention. we present our preliminary experience with a purely percutaneous management of choledocholithiasis in bariatric patients post-rygb. methods: a retrospective single center review identified five patients with choledocholithiasis after bariatric rygb who underwent percutaneous cbd access and treatment by interventional radiology. four patients underwent percutaneous transhepatic cbd access while one patient underwent percutaneous trans-cholecystic cbd access. in three of the five patients conscious sedation alone was sufficient to perform the procedure. results: all patients had radiologically confirmed choledocholithiasis and were clinically symptomatic prior to intervention. the biliary tree was successfully accessed percutaneously and cleared in all five patients. in the four patients where a percutaneous transhepatic access was utilized, three patients required only fluoroscopic balloon sphincterplasty and sweep of the cbd to clear the ductal stones, while the fourth required percutaneous cholangioscopy assisted lithotripsy for clearance. in the fifth patient with non-dilated intrahepatic bile ducts a trans-cholecystistic approach into the cbd was utilized with percutaneous cholangioscopic assistance to clear the ductal stones. all procedures were completed successfully with no post procedure complications. conclusion: percutaneous clearance of cbd stones in bariatric patients presents a minimally invasive alternative to current surgical practice. the use of conscious sedation and the purely percutaneous approach may potentially reduce morbidity and resource utilization for this increasingly common clinical scenario. laparoscopic narbona-arnau procedure to control the gerd after lsg- years results of a prospective study i.c. hutopila, c. copaescu background: after the laparoscopic sleeve gastrectomy (lsg) alone or associated with calibration of the esophageal hiatus, for some patients the reflux symptoms worsen postoperatively due to development of a hiatal hernia (hh) or due to the recurrence of the hh previously repaired. for these situations, when the conservative treatment fails, are proposed some surgical solutions, one of them cardiopexy with teres ligament-narbona arnau. objective: is to establish a standardized laparoscopic technique for cardiopexy using the teres ligament (narbona arnau technique) and to analyze the procedure's outcomes. methods: the study was performed in a bariatric and metabolic center of excellence-ponderas academic hospital. all the patients undergoing narbona arnau procedure to control gerd after lsg since were included and prospectively analyzed. the selection criteria included lsg patients, presenting hh and symptomatic gerd. preoperative investigations were upper gastrointestinal endoscopy, radiological contrast study, ph-metry, computed tomography with oral contrast. results: patients were included into the study. gerd and hh were preoperatively documented in all the cases. one patient was excluded after years of follow up after being converted to a laparoscopic roux-en-y gastric bypass, for intense relux symptoms. no incidents during surgery. for cases laparoscopic narbona arnau technique was performed concurrent with re-sleeve gastrectomy and gastric curvature plication. without postoperative complications. postoperative follow-up at months, , and years, the percentage of patients without gerd symptoms and free of treatment with ppis was , %, , %, , %, respectively . %. at years postoperatively the upper gi endoscopy showed remission/ improvement of the degree of esophagitis for patients. for the same period of follow-up, the ph-metry highlighted a normal value of demeester score for . % o patients (all the patients had preoperatively high de meester scores). no objective signs of hiatal hernia recurrence at imagistic investigations and upper gastrointestinal endoscopy were encountered. conclusions: complete preoperative evaluation is mandatory for choosing the optimal intervention. laparoscopic narbona arnau technique after lsg is proved to be a good option for the treatment of symptomic gerd, but further studies with high-volume patients are necessary. introduction: the aim of this study was to investigate the influence of baseline glycated hemoglobin level (hba c) level in bariatric patients on postoperative outcomes. we found scarce of clinical data regarding influence of baseline hba c on bariatric surgeries postoperative morbidity and readmission what was inspiration to conduct this multicenter retrospective study. methods and procedures: retrospective cohort study analyzed patients who underwent laparoscopic: sleeve gastrectomy (sg), roux-en-y gastric bypass (rygb) or mini-gastric bypass (mgb) for morbid obesity in seven referral bariatric centers. patients were divided into groups depending on preoperative hba c : hba c \ . %; . - . % and c . %. primary endpoints: influence of hba c level on perioperative ( -days) and postoperative ( -months) morbidity rates, operation time, length of hospital stay (los) and readmission rate. results: study group included , % females and % males. median age was ( - ) years. median hba c was . ( . - . ). hba c \ . % was present in % patients, hba c . - . % in %, and hba c c . % in %. percentage of male patients increased in groups from % in hba c \ . % to % in hba c c . % significantly. same tendency through groups we observed in case of bmi and age. uncontrolled diabetes (hba c c . %) was present in . % patients, while . % patients were not on antidiabetic medications despite having hba c c . %. median operative time in patients was significantly longer than in hba c \ . % and hba c . - . %. -days morbidity rate was . % and did not differ groups significantly, as -months morbidity rate (excl. -days) of . % . los did not differ groups significantly. patients having hba c in range of . - . % and with hba c c . % did not have significantly increased odds for perioperative morbidity, -months postoperative morbidity as compared with those with hba c \ . %. patients with hba c c . % had increased or for prolonged los as compared to those with hba c \ . % (or . ; % ci . - . ). hba c did not influence or for readmissions. patients with baseline hba c c % had significantly increased chances for hospital readmission (or . , % ci . - . ). conclusion: baseline level of hba c did not influence chance for perioperative morbidity, -months postoperative morbidity and prolonged los. patients with hba c c % have increased chance for hospital readmissions. surg endosc ( ) :s -s introduction: surgical resection is crucial for curative treatment of rectal cancer. through improvements in treatment and minimally invasive techniques, -year survival improved to over % of patients. the most recently introduced surgical technique is robotic-assisted surgery (ras). ras and conventional laparoscopy (cl) seem equally effective in terms oncological control. however, ras possibly provides further advantages e.g. d vision or the endowrist function, which have the potential to maximize the precision of surgery and thus has benefits for functional outcomes such as sexual function as well continence. therefore, the aim of this systematic review and meta-analysis was to compare functional outcomes of cl and ras for rectal cancer. materials and methods: this review was done according to the prisma and amstar guidelines andregistered with prosper-o(crd ). the search was planned with the pico criteria and conducted on medline (via pubmed), web of science and central. two independent reviewers first screened titles and abstracts and then eligible full-texts. inclusion criteria were original studies, comparative studies for cl vs. ras for rectal cancer as well as reporting of functional outcomes. quality assessment was done with the newcastle-ottawa-scale for non-randomized studies and the cochrane tool to assess risk of bias for randomized trials. results: the search retrieved hits, of which studies with patients met inclusion criteria. preliminary results yielded a lower rate of urinary retention for ras (odds ratio (or)[ %-confidence interval (ci)] . [ . , . ] ) while there were no differences for ileus (or[ci]: . [ . , . ] ). erectile function (iief) showed no differences after (mean difference (md)[ci] . [- . , . ] , (md[ci] . [- . , . ] ) and months (md[ci] . [- . , . ] ). in terms of urinary problems (ipss) there were no differences postoperative (md[ci] - . [- . , . ]) and month postoperative md[ci] - . [- . , . ] ), but advantages for the cl group after months md[ci] - . [- . , - . ] ). discussion: ras and cl seem to provide similar functional outcomes after rectal cancer surgery. however, the results need to be interpreted carefully as none of the studies had any functional outcome defined as primary endpoint. future studies should evaluate both surgical approaches in terms of functional outcomes and should be appropriately powered. methods: from april to november , laparoscopic right colectomy with intracorporeal anastomosis were performed in our surgical department. all patients in both groups were perioperatively managed using an eras pathway. seventy-two patients had the enterotomy closed with a single layer running suture of filbloc tm (assut europe). these patients were matched with patients who underwent intracorporeal right colectomy with enterotomy closed with a 'hybrid' double layer technique (first layer interrupted stitches in maxon tm - (covidien), second layer using a running suture in pds tm .intraoperative variables, anastomotic leak rate, morbidity and mortality rates were analyzed. results: the two groups were homogeneous with respect to demographics, body mass index (bmi), american surgical association score (asa) as well as for tumor stage. in the barbed group, median operating time was . min vs . min in the hybrid group (p = . ). anastomotic leak occurred in ( . %) patients in the hybrid vs ( . %) patients in the barbed group (p = . ) all patients required a reoperation. intraoperative findings at shows in ( . %) cases in the hybrid group a leak at the enterotomy closure, while an intact staler access was observed in both patients in the barbed group. no difference was observed with respect to non-infectious complications between the two groups (p = . ). patients in the hybrid group experienced a longer hospital stay when compared to the barbed group (p = . ). a re-admission occurred in the hybrid due an intraabdominal collection, while no re-admission was observed in the barbed group. no patient died in the postoperative period. conclusion: our results shows that the use of knotless barbed suture for enterotomy closure after laparoscopic intracorporeal right colectomy is safe, reproducible and associated with shorter operative time. aims: the accurate measurement and staging of rectal cancer, in particular the distal margin of low rectal tumours, is of paramount importance to optimise oncological surgical resection whilst preserving function. it is well recognised that the lower the tumour, the greater the technical challenges, operative time and the possibility of a temporary or permanent stoma. accurate localisation of the tumour is also essential to assist the multi-disciplinary team when considering neo-adjuvant chemoradiotherapy (crtx). the objective was to compare tumour height as reported on magnetic resonance imaging (mri) with endoscopic measurement. methods: a retrospective analysis of rectal tumour heights on pre-operative endoscopy and mri in patients undergoing radical colorectal surgery with curative intent over years from january . rectal tumours were identified as within cm of the anal verge (av). all mri measurements were reported by one of two specialist gastrointestinal radiologists. measurements were taken from the lowermost point of the tumour to the av. endoscopic measurements were as recorded by endoscopists including rectal surgeons, general surgeons, gastroenterologists and a clinical nurse specialist endoscopist. results: records of eighty one patients with histologically confirmed rectal adenocarcinoma were reviewed. median age was years ( to ). twenty three patients had or more endoscopies. on mri the median tumour height from the av was . cm ( . - cm) . on endoscopy the median tumour height was cm ( - cm) . on comparing endoscopy with mri, the median difference was cm ( - cm) . for over a third of patients ( %) tumours were lower on mri than endoscopy, median difference . cm ( . - cm) . only rectal surgeons documented tumour height in relation to the rectal folds. the majority of the repeat endoscopies were performed by surgeons to locate tumours more accurately pre-surgery. on no occasion was it documented whether the tumour had been measured during insertion or withdrawal of the endoscope. conclusions: precise localisation of rectal tumours is imperative to plan complex surgery and give informed counsel to patients. this study demonstrates the urgent need for a standardised protocol for all endoscopists to use while recording the distal extent of rectal tumours. objectives: the aim of the present rct was to compare the incidence of genitourinary (gu) dysfunction after elective laparoscopic low anterior rectal resection and total mesorectal excision (lar ? tme) with high or low ligation (ll) of the inferior mesenteric artery (ima). secondary aims included the incidence of anastomotic leakage and oncological outcomes. background: the criterion standard surgical approach for rectal cancer is lar ? tme. the level of artery ligation remains an issue related to functional outcome, anastomotic leak rate, and oncological adequacy. retrospective studies failed to provide strong evidence in favor of one particular vascular approach and the specific impact on gu function is poorly understood. methods: between june and december , patients who underwent elective laparoscopic lar ? tme in italian nonacademic hospitals were randomized to high ligation (hl) or ll of ima after meeting the inclusion criteria. gu function was evaluated using a standardized survey and uroflowmetric examination. the trial was registered under the clinicaltrials.gov identifier nct . results: a total of patients were randomized to hl (n ) or ll (n ). gu function was impaired in both groups after surgery. ll group reported better continence and less obstructive urinary symptoms and improved quality of life at months postoperative. sexual function was better in the ll group compared to hl group at months. urinated volume, maximum urinary flow, and flow time were significantly (p \ . ) in favour of the ll group at and months from surgery. ultrasound measured post void residual volume and average urinary flow were significantly (p \ . ) better in the ll group at months postoperatively. time of flow worsened in both groups at months compared to baseline. there was no difference in anastomotic leak rate ( . % hl vs . % ll). there were no differences in terms of blood loss, surgica l times, postoperative complications, and initial oncological outcomes between groups. conclusions: ll of the ima in lar ? tme results in better gu function preservation without affecting initial oncological outcomes. hl does not seem to increase the anastomotic leak rate. introduction: robotic single-site cholecystectomy (rssc) has been known to have some advantages such as reducing stress of the surgeon compared to single incision laparoscopic cholecystectomy (silc). however, there are few studies comparing the perioperative outcomes of these two operative methods. patient and methods: between march and february , rssc and silc were performed for benign gallbladder disease in our center. propensity score matching was performed to control variables including sex, age, body mass indes (bmi), diagnosis, american society of anesthesiologist (asa) score and cohorts were selected among the silc group through : matching. the perioperative data of these patients were analyzed retrospectively. the diagnosis was classified into acute cholecystitis, chronic cholecystitis, and gallbladder polyp. results: patient demographics between the two groups were evenly matched. total operation time including docking time was slightly longer in rssc group ( . min vs. . min, p \ . ), but real working time except the docking or set-up was shorter in rssc group ( . min vs. . min, p \ . ). conversion to additional robotic arm or additional port was frequent in silc group ( vs. cases, p = . ). intraoperative bile spillage rate ( . % vs. . %, p = . ) and postoperative hospital stay ( . days vs. . days, p = . ) were comparable in both group. conclusion: both surgical procedures performed safely. but the rssc demonstrated the better performance of the operation with shorter working time and the advantage of overcoming unexpected difficulties during the surgery with low conversion rate compared to silc. even though laparoscopic cholecystectomy(lc) is the gold standard procedure for cholelithiasis, patients are still suffering from various causes of pain. one of main causes is high pressure by pneumoperitoneum which makes peritoneal stretching and diaphragmatic irritation. however, there are few well-designed studies for evaluating pneumoperitoneum. therefore, we conducted a study to compare the postoperative pain after lc at serial different pressure methods. a prospective randomised double blind study was done in patients with benign gallbladder disease. they were divided into groups. each patients underwent lc with different pneumoperitoneum method; group a: far-low ( - mmhg), goup b: low ( - mmhg) and group c: standard pressure ( - mmhg). three groups were compared for pain intensity, duration, analgesic requirement and complications. post-operative pain score was significantly least in far-low pressure group as compared to low or standard pressure group during late periods ( , h). but, there were no pain score difference between far-low and low groups during early period ( , , , h) even though scores of standard group were significant higher than those of low group. number of patients requiring rescue analgesic doses and intraoperative complications were not significantly different among groups. this study demonstrates reducing the pressure of pneumoperitoneum results in reduction in intensity of post-operative pain. this study also shows that low pressure technique is safe with comparable rate of intraoperative complications. however, in immediate postoperative period, there is limitaton of pain relief after low pressure surgery. therefore, there may need new alternatives for pain. background and aim: anatomical hepatectomy with the glissonian approach is widely accepted as an important technique to ensure surgical safety and curability of the carcinoma. however, the histomorphological structure of the hepatic connective tissue is not sufficiently understood by surgeons. this study aimed to clarify the hepatic connective tissue structure using modern tissue imaging and analytical techniques. materials and methods: in total stained thin slices were loaded onto the computer and were reconstructed as dimages and analyzed. results: when the liver capsule enters the liver at the hepatic hilum, it becomes a sheath which envelops the portal pedicle. the hepatocytes in a row that constitute the periportal limiting plate at the edge of the hepatic lobule are firmly supported by the framework of the reticular fiber. the hepatic lobule and the portal area are in contact via the periportal space of mall. the framework of the limiting plate plays a role of a capsule of hepatic lobule (proper hepatic capsule) on the side in contact with the portal area. the binding site between the hepatic capsule and proper hepatic capsule (ppbs) is loose binding and is a layer that is easy to apply to surgical procedures. in order to enter between the liver capsule which became the sheath of the portal pedicle and the proper hepatic capsule at the hepatic hilum, the liver capsule must be dissected to reach the surface of the proper hepatic capsule. then, on the one hand, the portal pedicle is firmly gripped and pulled, on the other hand, the hepatic parenchyma covered by the proper hepatic capsule is pushed to expand between the portal pedicle and the liver parenchyma. at this time, the portal area (glisson's sheath) branched from the sheath of the portal pedicle into the gap of the hepatic lobule breaks like a string. with this dissecting plane, dissecting layer can reach to the next branch of the portal pedicle without entering into the portal pedicle or liver parenchyma. conclusion: understanding the connective tissue constituting the liver and conducting surgery turns the laparoscopic systematic hepatectomy into a standardized procedure. background: postoperative pancreatic fistula (popf) is the primary contributor to morbidity after distal pancreatectomy (dp). to date, no techniques used for the transection and closure of the pancreatic stump showed a clear superiority over the others. this study aimed to compare the rate of popf after pancreatic transection conducted with the reinforced stapler (rs) and ultrasonic dissector (ud) following dp. method: consecutive patients underwent dp from to were retrospectively reviewed. we included dps where pancreatic transection was performed by rs or ud and excluded dps extended to the pancreas head. to overcome the absence of randomization, we conducted a propensity matching analysis according to risk factors for popf. results: overall, patients met the inclusion criteria. the rs was employed in patients and ud in cases. after the one-to-one propensity matching, patients were selected from each group. the matched rs and ud cohort have no differences in baselines characteristics except for the mini-invasive approach, that was more common in the ud group ( % vs. %, p = . ). overall, patients ( %) developed a popf, a grade b ( %) and ( %) a grade c. in the rs group the rate of popf was % (n = ) and the ud group % (n = ) with a p \ . . conclusion: the results of this study suggest that the use of rs for pancreatic transection, reduces the risk of postoperative pancreatic fistula. a randomized trial is needed to confirm these preliminary data. aim: this study compares clinical and cost outcomes of robot-assisted single port and open longitudinal pancreaticojejunostomy (rlpj and olpj) for chronic pancreatitis. single incision mis needs more manual skills than conventional multiport operation. the advantage of better operation course is d vision and dedicate instrument. this paper aims to evaluate the feasibility and safety of the robot-assisted single incision with single port platform for chronic pancreatitis. materials and methods: clinical and cost data were retrospectively compared between open and ralpj. we collected patients since july, to september, . the patient was supinely placed in reverse trendelenburg position. the assistant surgeon was located between patient's legs. under general anesthesia a trans-umbilical . cm skin incision was made. a single incision advanced access platform with lagis port, glove portÒ (nelis, s. korea) and gelpoint combined with the da vinci si and xi surgical system (intuitive surgical, sunnyvale, ca, usa) pure or plus one was performed. the three arms, no. , no. , and da vinci scope, were in dwelled through the glove portÒ. pneumoperitoneum of mmhg was established through the port. a rigid -degree up scope was used during operation. results: twenty-one patients underwent lpj: open and ralpj. no robot-assisted cases converted to open were noted. patients undergoing ralpj had less intraoperative blood loss, a shorter surgical length of stay, less postoperative pain and lower medication costs. operation supply cost was higher in the ralpj group. no obvious difference in hospitalization cost was found. conclusions: versus the open approach, ralpj performed for chronic pancreatitis shortens hospitalization, less postoperative pain and reduces medication costs; hospitalization costs are equivalent. a higher operative cost for ralpj is mitigated by a shorter hospitalization and less pain control. robotassisted puestow procedure using single port platform is feasible and safe method. the total procedures by da vinci robotic system are safe and easily performed in highly selected patients. general surgery, hospital universitario infanta sofia, madrid, spain; general surgery, hospìtal quirón la luz, madrid, spain aims: the concomitant presence of abdominal wall midline hernias and diastasis recti is frequent. diastasis recti might be a risk factor not only for having but for recurrence of midline hernias. most open surgical procedures not consider the treatment of both pathologies, nor laparoscopic most spread out approaches. the author presents a novel endoscopic, extraperitoneal and retromuscular hernioplasty technique and its preliminary results. methods: a serie of patients is presented. a ct abdominal wall study is performed preoperatively. they all presented abdominal wall midline hernias in presence of a [ . cm concomitant diastasis recti. there were females and males. a totally endoscopic, extraperitoneal and retromuscular repair was performed, that included a midline anatomic restoration, tension-free hernia gap closure, omphaloplasty and skin treatment, if needed in every case. the tension-free massive-meshed hernioplasty included a bilateral totally endoscopic posterior components separation when needed. no drainages were used. all procedures included a bladder catheterization. results: all patient were dispatched within a period under h. no reoperations were needed in postoperative period. postoperative pain was measured by an eva scale. % of the patients have no pain medication after - h dispatching from hospital. % of the patients have a skin suffusion or hematoma. a male patient presented a temporary abdominal asymmetry due to a unilateral posterior component added to his technique. the mean following-up is to months ( - months) . no recurrence was observed. conclusions: preliminary results demonstrate this new approach to be a safe, feasible and a reproductible procedure. the 'terra' novel technique could provide of a new minimally invasive approach to abdominal wall midline hernias repair in the presence of a diastasis recti. only time and new results can predict the spreading out of this 'third way'. results: this study comprised males and females. mean age was years (range - years) and mean body mass index was . gh and mh were found intraoperatively in and cases respectively. mean operative time for all hernias (gh/mh) was min (range - min); min for gh (range - min); and min for mh (range - min). in . % of cases, hernia operative measurement was larger than preoperative size, especially in cases of incisional hernias ( . %). in . % of cases, laparoscopy found additional abdominal wall defects previously undetected by physical examination and by us-and/or ct-scan. a composite mesh and a non-composite mesh (up to cm in size) were used in . % and . % of cases respectively. the ethicon securestrap?? absorbable fixation device straps for sm fixation were employed in . % of cases. mean length of hospital stay was . days. mean follow-up time was months (range - months). in our study, there was one early (\ days) postoperative seroma ( . %), plus one late, small ( cm) symptomless recurrence, but neither needed reoperation. conclusion: the sutureless sm technique facilitates intra-abdominal introduction, as well as the handling and fixation of large/very large meshes. this new approach is safe and fast, even in cases of gh/mh repair. aims: any ventral hernia (vh) combined with rectus muscle separation (rms) must be repaired along with repairing the rms, otherwise there is a high risk for hernia recurrence. open rms repair is vast and traumatic surgery and laparoscopy is not effective. at a new era of repairing abdominal wall hernia by assisted endoscopy started with wolfgang reinhold's milos procedure. these procedures are somewhat complexed and real reconstruction of the linea alba (la) was limited, which done better by ferdinand koeckerling's elar technique. we perfected the elar technique to be fully endoscopic with wide mesh fusing to the muscles immediately by fibrin glue: extended endoscopic hernia & linea alba reconstruction glue (eehlarglue), achieving a low traumatic mis for vh and rms with excellent surgical and cosmetic results. methods: our eehlarglue is a totally endoscopic based technique used since . penetrating with optiview trocar and co pressure to the anterior rectus sheet (ars) level is followed by an extensive endoscopic dissection of the sub-cutaneous fat tissue from the ars. three trocars are inserted at the supra-pubic line enabling the dissection up to the xiphoid and costal margins laterally. any hernia sac is dissected, and the content reduced back to the abdominal cavity. relaxing incisions of the ars are performed longitudinally in the lateral aspect. the la is reconstructed by running two layers of non-absorbable sutures from xiphoid to pubis. a light mesh x cm is applied over the repair and the mesh is fused immediately to the muscles by fibrin glue. results: patients underwent the eehlarglue with follow up of months. all had significant rms of - - cm combined with primary or recurrent vh. recovery was smooth with - days of simple analgesics and return to regular activity within - days. no one had recurrent vh, but two males had limited rms and two early cases seroma formation. conclusions: our eehlarglue enables endoscopic vh repair and la reconstruction with extrastrength received by immediate mesh fusion to muscles with fibrin glue. thus, achieving low traumatic mis, easy recovering and very effective results-a perfect solution for patients with vh combined with rms. results: twelve blinded prospective rcts were used. when compared to tep repair, tapp repair has comparable seroma formation rates (chi = . ; (p = . ); ci - . , . ; i = %) and post-op pain at h (chi = . ; (p = . ); ci - . , . ; i = %). however, tep repair is associated with a significantly shorter operative time (chi = . ; (p = . );ci . , . ; i = %), post-op pain at hour (chi = . ; (p = . ); . , . ; i = %) and shorter hospital stay (chi = . ; (p = . ); ci . , . ; i = %). conclusion: tep is significantly better than tapp repair with regards to operative time, post-op pain at h and hospital stay. there is no significant difference with regards to post-op pain at h and seroma formation. background: primary hyperhidrosis (ph) is a neurological condition characterized by excessive sweating most often of the face, palms or axillae . palmar hyperhidrosis is treated through sympathetic chain clipping or transection .we aiming to compare the efficacy and results obtained with both techniques. patients and methods: sixty four patients underwent of sympathetic procedures from march to february . the patients were categorized into two groups: right sided transection sympathectomy and left sided clipping . patients were evaluated to compare the rates of success, satisfaction, compensatory sweating and recurrence either with transection or clipping of the t andt ganglion .mean follow up was ? _ months. results: sixty four patients males and females undergoing electro-coagulation sympathectomy on the right side and clipping on the left side. with mean age was years (range to years). all patients had balanced demographic data . no statistical difference between the two groups according rate of success. compensatory sweating was observed in patients ( . %) overall with cases of severe unsatisfied compensatory sweating. recurrence was reported in one case with transection and cases in clipping. satisfaction was occurred in cases in transection group and cases in clipping group .pnumothorax was occurred in cases in transection group compared to one case in clipping. no gustatory sweating and over dryness were reported in both groups. conclusion: both thoracoscopic sympathetic transection and clipping of t t ganglion are safe and effective procedure in palmar hyperhidosis treatment. with no differences regarding recurrence rate,satisfaction and incidence compensatory sweating. keywords: thoracoscopic sympathectomy,palmar hyperhidrosis, clipping, compensatory hyperhidrosis. introduction: primary ventral hernias and ventral incisional hernias pose a challenge for surgeons throughout the ages. even though minimally invasive surgery and hernia repair have evolved rapidly, there is no standardized method that adequately decreases postoperative complications. hybrid hernia repair is a surgical repair, which has not been adopted widely. it combines both a laparoscopic and open component allowing sac excision, primary defect repair as well as laparoscopic mesh insertion. aims: to evaluate the short-term and long-term outcomes of hernia recurrence for patients undergoing hybrid ventral repair (hvr) for the treatment of primary and incisional ventral hernias. methods: between october- and june- , hybrid vhr was performed in -patients at st mary's hospital, imperial college london. the medical records of these patients were reviewed retrospectively for demographics, comorbidities, prior surgeries, body mass index (bmi), hernial defects, hybrid technique used; mesh selection, operative time, complications and recurrences over a -year follow-up. results: twenty-four patients who underwent hybrid vhr were included with surgery performed by two surgeons. the mean age is -years with a mean bmi of . kg/m . % had incisional hernias and % had primary hernias. the number of hernia defects ranged from to , with the average mesh size used was x cm. extensive adhesionolysis was performed in % of patients. -day postoperative complications; patients developed post-operative seroma, paralytic ileus in , pain control in and urinary retention in patient. there were no conversions to open procedures. the mean length of hospital stay was -days. none of the patients developed chronic pain and only one recurrence over the -year follow-up period. conclusions: the hybrid technique for vhr is safe and feasible, and has important benefits over an open or purely laparoscopic approach, including a low rate of seroma formation, chronic pain and fiveyear hernia recurrence. future investigation may include randomized controlled trials, to fully evaluate the benefits of hybrid vhr, with careful assessment of patient-centred end-points including quality of life and postoperative pain. surgery, medical faculty-university of tetove, tetove, macedonia; general medicine, medical faculty-university of tetove, tetove, macedonia; anestesiology, medical faculty-university of tetove, tetove, macedonia; surgery, clinical hospital-tetove, tetove, macedonia laparoscopic cholecystectomy is widely used operative technique and it's characterized with less postoperative hospitalization and side effects. duration of the hospitalization after laparoscopic surgery depends on several factors of which pain and physical weakness are the most important. dexamethasone is well known; not only for its anti inflammatory effects but at the same time for analgesic and antiemetic effects, although the mechanism of this effects are not clarified yet. objectives: the aim of our study is the evaluation of analgesic effect of dexamethasone on reducing postoperative pain after laparoscopic surgery. patients and methods: in this study, patients aged - years old undergoing laparoscopic surgery, were classified into two groups, patients in each group. the first group were treated with a intravenous injection of mg dexamethasone preoperatively and another dose the next day after operation. the second group received a intravenous injection of normal saline. we evaluated the dose of consumed analgesics and antiemetic's drug during the first h in both groups. results: according to our experience results the total dose of tramadol in a postoperative period in dexamethasone receiving group was smaller than in normal saline group. measure of postoperative pain was assessed using the paper-based vas scale. our result shows that the intensity of post operative pain in a period during first h, after surgery in a group of patients treated with dexamethasone was lower compared with the group of patients treated with normal saline. nausea and vomiting during the first h was significantly lower in the dexamethasone group than in the normal saline group. surgery, hospital quiron sagrado corazon, sevilla, spain; surgery, hospital virgen macarena, sevilla, spain; surgery, hospital virgen del rocio sevilla, sevilla, spain aims: closing the defect (cd) during laparoscopic ventral hernia repair (lvhr) could be related to a reduction of seroma formation or bulging (hernia mesh) compared to conventional lvhr. but tension of the midline may contribute for some authors to a higher incidence of pain, recurrence in medium size defects and suggest to perform a component separation (cs) for restoring the midline in medium-large defects.we have developed a new technique for restoring the midline in medium ventral hernias (lira technique) and weanalyzed our results in terms of pain and recurrence compared to our conventional cd series (ccd). methods: we conducted a prospective controlled study of lvhr with ccd from january to december and a prospective controlled study performing lira technique from january to january . we analyzed and compared both techniques in medium size defects ( - cms) in terms of postoperative pain ( , days, , months and year) using a visual analogue scale (vas), bulging (return to prior distance among rectus muscles with the mesh in the sac in ct that didn't need surgical treatment)and recurrence (by physical examination and tomography). results: ccd was performed in patients (mean age was . ± . years old and mean bmi was . ± . kg/m ) and lira technique in patients (mean age was . ± . years old and mean bmi was . ± . kg/m ). the mean average follow-up in both series was year. mean average vas in ccd was . ± . ( day), . ± . ( days) . ± . ( month) . ± . ( months ) and at year. in lira series vas was . ± ( h) . ± . ( days), . ± . ( month), ( months) and ( year) . there are cases of bulging in ccd series and recurrence. bulging and recurrence were absent in lira series. conclusions: lira technique might be a safe procedure in medium size defects for restoring the midline in lvhr, and could be related to a lower pain rate compared to ccd with no recurrence or bulging. surg endosc ( ) :s -s background: the desire of pediatric surgeon to reduce incision related morbidity and pain while achieving good cosmetic results has recently led to the introduction of single incision pediatric endo-surgery [sipes] and needlescopic surgery. intracorporeal suturing and knot tying during sipes remains challenging. the aim of this study is to introduce a novel and simple technique for intracorporeal suturing of the pediatric inguinal hernia after needlescopic disconnection of hernia sac using just needles rather than laparoscopic instruments. it is an imitation of the principles of sewing machine. methods: the first author discussed the idea of the technique with the co-authors and a demonstration was done on a silicon pad before application of the technique on children with congenital inguinal hernia [cih] for peritoneum closure after needlescopic disconnection of the hernia sac. the main outcome measurements were; feasibility of the technique, knot quality, suture placement accuracy, performance and suturing time and recurrence rate. results: the sutures were snugly applied to the ridges of silicon pad with good approximation and the knot was firmly tightened in all experiments. after applying and mastering the technique on a silicon pad, we shifted to use it on children with hernia defect. all operations were completed by the needlescopic technique without the need for insertion of any laparoscopic instruments. the time required for suturing of the peritoneum around internal inguinal ring [iir] and knot tying, decreased considerably from min s in the first operation to less than min after the fifth operation and stabilized at approximately minute s. no major intraoperative complication and no recurrence. the primary end-point was to compare clinical outcome as well as cost effectiveness study between both groups. results: a total of patients were enrolled ( of them underwent tapp and olr). drop out occurred in cases ( of tapp and of olr group). patient characteristics were statistically similar between the groups. tapp procedure had less early post-operative pain (p = . ), a shorter length of stay (p = . ) and less postoperative complications (p = . ) when compared with the olr approach. a slightly higher recurrence rate in the tapp group was found. additionally, there is a trend towards a higher postoperative quality of life and less chronic pain in the tapp group. conclusions: tapp procedure for bilateral inguinal hernia effectively reduces early postoperative pain, hospital stay and postoperative complications. cannizzaro hospital, catania, italy aim: the purpose of this study was to evaluate the long-term results in terms of safety and efficacy of a new technique to repair incisional ventral hernias with a self-gripping mesh, after a mean follow-up period of months. methods: a retrospective, single-centre study was performed from june to june . all patients undergoing elective incisional ventral hernia repair were included. hernias were diagnosed based on clinical examination at the outpatient clinic. in case of doubtful diagnosis, ct-scan was used to confirm the diagnosis. the component separation technique and, when needed, tar were performed. the self-gripping mesh was placed in sublay position (overlap cm) with the self-gripping surface face down. in all cases drainage tubes were placed in retromuscular and supraaponeurotic position. the following characteristics were collected: age, sex, body mass index (bmi), smoking, comorbidities, number of previous surgical operations, defect size (ehs classification), mesh size, postoperative complications, duration of follow-up. all patients were interviewed by telephone every six months. when patients complained recurrence or other symptoms, visits were organized and when there was the doubt of recurrence a ct-scan was performed. results: a total of patients were included in this study, males, mean age was years. % of patients had bmi [ , smokers and diabetics were respectively % and %. the mean defect size was cm . component separation technique was associated with tar in patients. in cases the size of mesh was cm, while in patients the size of mesh was cm and in cases this was cm. in the other patients the mesh sizes were tailored to defect dimensions. subcutaneous seromas occurred in patients, they were treated conservatively in cases and with percutaneous punction in cases. long-term follow-up demonstrated recurrences in one case, while in another one ct-scan revealed a bulging. no cases of mesh infection, pain or sensation of mesh. conclusions: this study with a mean follow-up period of months demonstrated that the use of self-gripping mesh in sublay position is safe and effective to treat incisional ventral hernias. aim: morgagni hernias present technical challenges. the laparoscopic approach was described at first in , however, as they are uncommon in adult life and, little data exist on the optimal method of surgical management. this study purpose was to analyse a method for laparoscopic repair of morgagni giant hernias using laparoscopic primary closure. methods: this case series describes a method of laparoscopic morgagni hernia repair using primary closure. in all patients a laparoscopic transabdominal approach was used. the content of the hernia was reduced into the abdomen and the diaphragmatic defect was closed with a running laparoscopic suture using a self-fixating suture. clips were placed at the edges of the suture to secure the pledged sutures to both the anterior and posterior fascia. demographic data as age, gender and bmi were collected. operative data (operative time, rate of conversion, blood loss) and post-operative data (short and long term complications, length of hospital stay, need of readmission and reoperation) were recorded. results: retrospectively collected data about patients were analysed. there were ( . %) male and ( . %) females. the median bmi was . ± . kg/m . median operative time was ± min. there were no intraoperative complications nor conversion to open surgery. patients began a fluid diet on the first post-operative day and were discharged after a median hospital stay of ± . days. in a median follow up of months we did not observe any recurrences. conclusions: transabdominal laparoscopic approach with primary closure of the diaphragmatic defect is a viable approach for repair of morgagni hernia. in our experience, the use of laparoscopic transabdominal suture fixed to the fascia allowed the closure of the defect laparoscopically with minimal tension on the repairs. can we predict the success of the laparoscopic approach in the adhesive small bowel obstruction? c. tellez marques, e. sebastian valverde, e. membrilla fernandez, l. grande posa, i. poves prim general surgery, parc de salut mar-hospital del mar, barcelona, spain aims: the laparoscopic approach in the acute adhesive small bowel obstruction and internal hernias (asbo) has shown superior to laparotomy in terms of morbidity and hospital stay. especially, in patients who present simple adhesions or internal hernias. according to this, the aim of the study is to determine those preoperative factors associated with simple adhesions and internal hernias, and consequently, improve the success of the laparoscopic approach in asbo methods: a retrospective study of patients who underwent urgent surgery for asbo was conducted from january to may . we compare preoperative variables between single adhesions and internal hernias vs complex adhesions. a p value \ . was considered statistically significant. results: we analysed patients who underwent surgery for asbo, ( %) by laparoscopy and ( %) by laparotomy. conversion rate in laparoscopy was . %. . % of patients presented a single adhesion or internal hernia; and . % were considered complex adhesions. sex and age did not correlate with the type of adhesions. previous surgery (p \ . ), number of previous surgeries (p \ . ), asa (p \ . ) and previous abdominal wall mesh (p = . ) were significantly associated with complex adhesions. laparoscopy as the only surgical history was significantly associated with simple adhesions (p = . ). only appendectomy (p = . ) or supramesocolic (p = . ) previous surgeries tended to present single adhesions but it did not reach statistical significance. the need for intestinal resection was not related to the type of adhesions (p = . ). there was a significant correlation between the findings in the ct (computed tomography) and the type of adhesion found (p = . ). signs of ischaemia on ct were related to the need for intestinal resection (p \ . ). in the multivariate analysis, the number of previous surgeries, asa and ct scan findings were identified as independent factors related to the type of adhesion. conclusions: according to our study, a lower number of previous surgeries, asa i-ii and internal hernia in the ct scan are associated with single adhesions and internal hernias. patient selection is a key factor for the success of laparoscopic approach in asbo. aims: there aims of this study were: (i) to compare england with the united states in the utilisation of minimal access surgery (mas) and in-hospital mortality from four common abdominal surgical emergencies (appendicitis, incarcerated or strangulated abdominal hernia, small or large bowel perforation and peptic ulcer perforation). (ii) within england to evaluate the influence of mas upon in-hospital and long-term mortality. methods: between and , the rate of mas and in-hospital mortality for four abdominal surgical emergencies were compared between the united states and england. univariate and multivariate analyses were performed to adjust for underlying differences in baseline patient demographics. results: , admissions in england for four abdominal surgical emergencies were compared to an estimated , , admissions in the united states. after adjustment for patient demographics, mas was used less commonly england for three conditions; appendicitis (odds ratio (or) . , % ci . - . ), abdominal hernia (or . , ) and small or large bowel perforation (or . , ). in-hospital mortality in multivariate analysis, was increased in england compared to the united states for three conditions; abdominal hernia (or . , % ci . - . ), small or large bowel perforation (or . , ) and peptic ulcer perforation (or . , % ci . - . ). in england, after adjustment for patient demographics, open surgery was associated with increased in-hospital mortality for three conditions; abdominal hernia (or . , % ci . - . ), small or large bowel perforation (or . , % ci . - . ) and peptic ulcer perforation (or . , . similarly open surgery was associated with increased long-term mortality for three conditions; abdominal hernia (hr . , % ci . - . ) , small or large bowel perforation (hr . , % ci . - . ) and peptic ulcer perforation (hr . , % ci . - . ). conclusions: minimal access surgery was used less commonly and inhospital mortality was increased in england compared to the united states for common abdominal surgical conditions. given the benefits of mas shown in this large study, strategies to enhance adoption of mas in emergency conditions in england need to be optimised and include appropriate patient selection and improved surgeon mas training and experience. surg endosc ( ) :s -s background: in the treatment of inguinal hernias, there is little hard evidence concerning the economic reimbursement in the diagnosis-related-group (drg) era. factors that affect whether a hospital may earn or lose financially depending on open or laparoscopic approach is still underexplored. the aim of this study is to provide a reliable analysis of in-hospital costs and reimbursements in inguinal hernia surgery. methods: this retrospective study analysed the -year experience in inguinal hernia repair in patients undergoing open lichtenstein (ol), laparoscopic totally extraperitoneal unilateral (utep) or bilateral (btep) hernia repair. demographics, results, costs and drg-based reimbursements were recorded and analysed. results: during the study period, patients underwent ol, patients utep and patients btep. the average total cost amounted to eur in ol, eur in utep and eur in btep groups (p \ . *). the hospital reimbursement amounted to eur, eur and eur in the ol, utep and btep groups respectively (p \ . *). finally, the mean hospital earnings were eur, eur and - eur for each patient in ol, utep and btep respectively (p \ . *). conclusions: in-hospital costs were higher in utep and btep as compared to ol. the drg-based reimbursement provided adequate compensation for patients with unilateral inguinal hernia, whereas hospital earnings were profitable in ol group only, and led an overall financial loss in the btep group. surgeons should be conscious that clinical advantages of the laparoscopic approach are not adequately compensated for, from an economic point of view. aims: umbilical hernias are common anatomical defects in swine which become a suitable model for surgical training and research in the field of surgical meshes. the aim of this study was to develop a surgical protocol for a successful laparoscopic implantation of stem cell-coated surgical meshes. methods: large white pigs, weighing - kg and with congenital abdominal hernia were anesthetized for the surgical procedures. non absorbable polypropylene surgical meshes were coated with fibrin glue (fg) (control group) or with fg admixed with porcine bone marrowderived mesenchymal stem cells (fg/bm-mscs). approximation of hernia's borders was performed by intracorporeal suture. the meshes were carefully rolled inside the trocar for laparoscopic implantation. the surgical implantation was performed by laparoscopy using helicoidal staples. laparoscopic inspections and biopsies of the tissue surrounding the mesh were performed at , and days post-implantation. at day , the animals were euthanized and macroscopically evaluated. ultrasonography was used at day , , and to evaluate the size of the hernia. the biopsies were then processed for the histological analysis. results: ultrasonography demonstrated that the mean size of umbilical hernias before mesh implantation was . ± . cm. a decrease in hernia mean size was observed at day and . the laparoscopic procedures allowed a successful mesh implantation in all animals. in most of cases, the implantation site did not show excessive inflammation or tissue adhesions. but one animal showed hernia maintenance. one animal had peritoneal and implant-site infection. foreign body reaction was noted in the histological analysis, although no significant difference was found between the control, and bm-msc group. conclusions: the anatomical similarities between humans and pigs in umbilical hernias make this animal model useful to: i) improve minimally invasive surgical procedures for hernia treatment; ii) evaluate new surgical meshes, and iii) introducing stem cell therapy to hernia surgical repair. the laparoscopic approach is efficient and safe for the implantation of stem cellcoated meshes. gene and protein expression analysis are required to evaluate the molecular changes between the conventional and the stem cell surgical approach. aims: fluorescence angiography with indocyanine green (icg) is used as a marker in the assessment of tissue perfusion, being more frequently used in colorectal procedures. this technology has shown to be a good technique to reduce complications related to vascular supply to the anastomosis. in esophagogastric procedures blood supply to the gastric pouch, jejunum and esophagus could be evaluated by icg fluorescence imaging. it could be also used in bariatric surgery to evaluated the anastomoses, during gastric bypass, and blood supply to the gastroesophageal junction and the angle of his during sleeve gastrectomy. methods: we have collected data during gastric resection due to adenocarcinoma and bariatric procedures that were performed by the same surgeon, using icg fluorescence to evaluate blood supply. the icg was infused before performing the anastomosis in order to evaluate the need to change the transaction line (tl). we analyzed those cases in which the tl was changed and the number of leaks in those cases that we changed this line. results: all the cases were performed by laparoscopic approach: subtotal gastrectomy (sg), total gastrectomy (tg), gastric sleeve (gs) and gastric bypass. there were no changes regarding the tl before performing the anastomosis in any of the four types of procedures (sg, tg, gs, gb). in the analyzed data there is anastomotic leak in one sg procedure ( . %). conclusions: icg fluorescence angiography could be helpful in assessing blood supply during gastrointestinal anastomosis, although we have not find an influence in the results during bariatric and gastric procedures. however, we do not have the sufficient evidence to determine the value of this technology in this entities, being needed more volume and data to improve the significance of the results. aims: hyperspectral imaging (hsi) combines a spectrometer with a camera to analyze the tissues' optical properties in a broad wavelength range, without the need for a contrast agent. it provides extensive real-time information about tissue physiology, including oxygen saturation (sto ). fluorescence-based enhanced reality (fler) is a software solution providing a dynamic, quantitative analysis of the signal evolution of a systemically administered fluorophore, during fluorescence angiography (fa) . the aim of this study was to compare the performance of hsi and fler to assess bowel perfusion, in a porcine, non-survival model of bowel ischemia. methods: in pigs, an ischemic small bowel segment was created and imaged after hour of ischemia. the imaging modalities were applied sequentially to the same area.hsi was performed first, to acquire the sto spectra, by means of the tivita tm system (diaspective vision, pepelow, germany), which provides a spectral range of - nm and a nm resolution. subsequently, fa was performed using a nir-capable laparoscopic camera (d-light p, karl storz, germany), after intravenous injection of . mg/kg of indocyanine green (icg; infracyanine, serb, paris, france). the fluorescence flow was recorded during s, then the slope of the fluorescence flow was analyzed using a proprietary software to obtain a virtual perfusion cartography. the virtual cartography was overlaid onto real-time images to obtain the enhanced reality effect. ten adjacent regions of interest (rois) were selected from hsi datasets and were superimposed to fler-generated cartographies using a custom plug-in software function, allowing for a quantitative comparison of both imaging modalities. hsi was repeated after icg injection. results: the r correlation coefficient between hsi-sto and the fler slope was . . at control hsi after icg injection, the correlation coefficient dropped significantly (r . ). the interference of icg on hsi imaging was clearly identified in the spectral curves. conclusion: sto given by hsi provided results comparable to those obtained with fler in our bowel ischemia model, without the need to inject a contrast agent. icg interferes with hsi datasets, disrupting sto values. surgical treatment is one of the most effective options for treatment of giant hiatal hernia. laparoscopic approach became is a 'gold standard' over the time demonstrating all advantages of minimally invasive techniques over the open procedures. however the utility of robotic operations still remains controversial. aim of the study: evaluate the initial experience of robotic fundoplication in compare to laparoscopic procedures. materials and methods: since the january till the december of thirty operations were operated on. mean age was . ( - ), among them ( %) were female and ( %) were males. mean bmi was . ( . - . ) . laparoscopic procedures were performed in patients ( st group), robotic procedures with davinci system were performed in patients of the second group. nissen fundoplication modified was performed in patients, toupet fundoplication was used for patients. results: the median operative time in laparoscopic group was min, in robotic group- , min. there were no statistical differences between two groups (p = . ). blood loss was minimal in both groups. mean postoperative hospital stay was . days ( - days) in the st group and , days ( - days) in the second. there were no statistical differences between two groups (p = . ). postoperative course was uneventful in all patients of both groups. surgical stress response is associated with systemic inflammatory syndrome, sepsis, multiorgan dysfunction syndrome. robotic assisted surgery has been introduced to overcome the limitations of conventional laparoscopy. this technique has potential advantages over laparoscopy, such as increased dexterity, three-dimensional view, and a magnified view of the operative field. these advantages could result in limited intra-abdominal trauma and hence in attenuated surgical stress response over conventional laparoscopy. aims: this study aimed to synthesize data on the effect of robot assisted surgery on surgical stress response. methods: electronic databases were searched with the search terms 'surgical stress', 'stress response', 'oxidative stress', 'robotic assisted surgery', 'c-reactive protein', 'interleukin ', 'interleukin ','cortisol',;'oxidative stress markers', 'antioxidants', 'antioxidant status', 'mda', 'glutathione', 'cortisol', 'acute phase response' up to and including march . results: one hundred forty studies were identified and their title and abstract were reviewed. one randomized controlled trial, six non randomized comparative studies, one experimental study and one case report met inclusion criteria. data were discordant. one prospective trial concluded that cortisol and il- were lower in laparoscopic assisted distal gastrectomy compared with robot assisted distal gastrectomy in another study comparing robotic assisted laparoscopic radical prostatectomy with open radical prostatectomy based on plasma measurements of il- , il- a and c-reactive protein, it was demonstrated that robotic assisted laparoscopic radical prostatectomy induces lower tissue trauma than open radical prostatectomy. in another study, it was reported reduced expression of genes associated with surgical stress response in patients treated with robotically assisted radical prostatectomy compared with patients treated with open prostatectomy. the case report concerned a case of polymyalgia rheumatic after robotic assisted laparoscopic prostatectomy. the experimental trial demonstrated that cortisol and substance p were significantly higher with open thoracic approach versus robot assisted thoracoscopic oesophageal surgery. conclusion: further research is needed to elucidate the effect of robotic surgery on surgical stress, based on a well standardized protocol for the measurement of surgical stress response. purpose: tissue compression is essential to prepare the tissue for proper staple formation. this study evaluates the risk factors of compression injury on the circular stapling line in vitro. methods: to reproduce the artificial bowel wall, a collagen plate was prepared by mixing collagen extracted from porcine with glycerin. artificial collagen plates with mm and mm in the thickness were made for dry and healthy condition and immersed plates in the tap water for min to make wet and edematous condition. circular stapler (cdh a, ethicon, usa) was applied in the collagen plates (dry and wet condition) and optimal compressions. compression line was evaluated for compression injury score. risk factors for excessive compressions and unacceptable injury were analyzed. results: in the dry condition, optimal compression didn't cause unacceptable injury. in the wet condition, excessive compressions were occurred in . % with optimal approximation. unacceptable injury was significantly different in proper and excessive compression cases as . % and . %, respectively. on the univariate analysis, thickness ( mm), wet condition, proximal side, maximal compression, and excessive compression were associated with unacceptable injury. on the multivariate analysis using logistic regression model, excessive compression was significant independent factor to cause tissue injury (p \ . ) and this significance was also proved in the optimal compression group (p = . ). background: minimal invasive appendectomy gained much popularity due to its better cosmoses, early recovery and less wound site infections. single incision laparoscopic appendectomy (sila) has many disadvantages such as, long operative time, bad ergonomics, surgical site infections, high conversion rate and port site hernia. needlescopic appendectomy (na) using mediflexÒ facial closure needle expected to be more superior over sila. here in we compare our results of needlescopic appendectomy with single-incision one. material and methods: one hundred and twenty patients with acute non complicated appendicitis were randomly assigned to na and sila children for each group during the period between january to october . the main outcome measurements included, demographics, operative time, intraoperative complication, conversion rate, post-operative hospital stay, surgical site infection, port site hernia and cosmetic results. results: a total of children underwent appendectomy. there were children who underwent na and children who underwent sila. there were no difference in age ( . vs . years, p = . ), weight ( . vs . kg, p = . ) and hospital stay ( . vs . days, p = . ) between the two groups. there were no intraoperative complication during the two surgical approaches. operative time for na group is significantly shorter than single incision group ( . vs . min, p = . ). no single case of conversion in na group and cases needed conversion in sila group. seven cases of sila showed surgical site infection. cases of sila group presented with port site hernia. the na group was superior as regard ergonomics. the two groups showed equal excellent cosmetic results. conclusion: needles scopic appendectomy and sila are comparable as regard cosmetic results and hospital stay. na proved to be safe, applicable, repetitive and superior over sila as regard better ergonomics, less operative time, absence of surgical site infection and port site hernia. aims: to objectively analyze the surgical performance and surgeon's ergonomics in the use of a novel flexible laparoscopic instrument during intracorporeal suture, and compare it with the use of a conventional laparoscopic needle holder. methods: three experienced laparoscopic surgeons performed five laparoscopic sutures on an organic tissue using the novel flexible instrument (flexdexÒ) and five sutures using a conventional needle holder with axial handle. the new device is based on a mechanical design with no electrical components, which transfers the surgeon's hand, wrist, and arm movements to the instrument tip in an intuitive manner. the use of the instruments was organized in a random fashion. prior to the study, participants conducted a -minute training session with the new flexible instrument. execution time and quality of the suture were assessed for each repetition. besides, flexion and radioulnar deviation of the wrist were recorded using an electrogoniometer (biopac systems, inc.) attached to the surgeon's hand and forearm. the intensity of the forearm's muscle activation was also analyzed by means of a myo armband (thalmic labs). results: surgeons required more time to perform the intracorporeal suture using the novel laparoscopic instrument ( . ± . s vs. . ± . s; p \ . ), but the quality of the suture was similar with both instruments. the wrist flexion ( . ± . °vs . ± . °; p \ . ) and wrist ulnar deviation ( . ± . °vs . ± . °; p \ . ) were significantly lower when using the flexible instrument. during the suturing tasks, the use of flexdexÒ instrument led to a higher muscular activation of the flexor ( . ± . vs . ± . rms; p \ . ) and extensor ( . ± . vs . ± . rms; p \ . ) muscle groups of the forearm. conclusions: the presented novel instrument allows surgeons to perform robotic-like laparoscopic suturing. we believe that with a longer training period surgeons could potentially reduce surgical times with this device. the preliminary results of this study suggest that the use of this new instrument provides a quality of the suture similar to that obtained with a conventional laparoscopic needle holder and an ergonomically more adequate wrist posture. aims: the intraoperative real-time evaluation of tissue perfusion is one key element for successful visceral surgery. traditionally, tissue evaluation is performed visually by surgeons. newer devices for objective quantification have in majority been based on the application of the fluorescent dye indocyanin green (icg). a novel method derived from geographic research is hyperspectral imaging (hsi). the aim of this study was the evaluation of hsi as a promising method for the evaluation of tissue perfusion and its implementation in the evaluation of the gastric conduit during esophagectomy in a porcine model. methods: the hsi camera records a dimensional data cube from a dimensional surgical situs obtaining wavelengths between and nm. the absorption at different wavelengths is tissue-specific and influenced by the amount of oxygenated haemoglobin and other pigments. a software calculates different indices in real-time including oxygen saturation. a porcine model (n = ) is used for esophagectomy with gastric conduit formation. ischemia is induced artificially by magnets simulating staplers. different shapes of the gastric conduit and anastomosis formation are evaluated for perfusion metrics in order to obtain recommendations for the optimal formation of esophagogastrostomy. conclusion: hsi is a promising method for intraoperative evaluation of tissue perfusion that does not require application or injection of any agents. the preliminary results in this study showed that the gastric conduit receives its main blood supply from the gastroepiploic arteries and not via the mucosa. further results from the current evaluations enable formation of an optimized gastric tube and esophagogastrostomy in esophagectomy. surg endosc ( ) pediatric surgery, al azhar university, giza, egypt; pediatric surgery, beni suef university, beni suef, egypt background: varicocele is one of the most common causes of infertility. many surgical interventions are used for varicocele ligation including open and conventional laparoscopic multiport or single incision techniques. the aim of the study is to present a new needlescopic lymphatic sparing varicocele ligation using mediflexÒ facial closure needle and gauge vascular access cannula. material and methods: twenty-two male children with bilateral varicocele of grade ii-iii. all children were counseled by clinical examination, doppler ultrasonography, abdominal ultrasonography, and routine laboratory investigations. testicular lymphatics were delineated by subcutaneous injection of / cm methylene blue in anterior wall of the scrotum min prior to surgery. the testicular vessels (both vein and artery) were ligated one cm above the deep inguinal ring using two mediflex needles with preservation of lympatics. the main outcome measurements included; operative time, hospitalization, testicular atrophy, hydrocele formation, recurrence of varicocele and intra or postoperative complication. results: a total of twenty-two male children with grade ii-iii varicocele subjected to needlescopic lymphatic sparing technique. twenty one were bilateral. background and aims: even if the clinical outcomes of robotic rectal resections are under investigation, the related robotic costs have not yet been well addressed, and the differences between the robotic rectal resection costs and the laparoscopic approach are still not well known. we have therefore performed a prospective comparative study of robotic rectal resections (rrr) and laparoscopic rectal resections (lrr) performed at our centre with the aim to evaluate the cost-effective outcomes of robotic versus laparoscopic surgery. study design: this is an observational, comparative prospective non-randomized study which includes patients that underwent laparoscopic and robotic rectal resection reaching a minimum of months of follow up from february to march , at the sanchinarro university hospital, madrid. an independent company performed the financial analysis and fixed costs were excluded. outcome parameters included surgical and post-operative costs, quality adjusted life years (qaly), and incremental cost per qaly gained or the incremental cost effectiveness ratio (icer). the primary end-point was to compare clinical outcome as well as cost effectiveness study between both groups. results: a total of rrr and lrr were included. the mean operative time was significantly lower in the lrr approach ( versus min; p = . ). the main pre-operative data, overall morbidity, hospital stay and oncological outcomes were similar in both groups, except for the readmission rate (rrr: . %, lrr: . %;p = . ).the mean operative costs were higher for rrr ( . versus . €; p = . ); however, the mean overall costs were similar ( . € for rrr and . € for the llr; p = . ). mean qalys at year for rrr group ( . ) was higher than that associated with lrr ( . ) (p = . ). at a willingness-to-pay threshold of , € and , €, there was a . % and . % probability that rrr group was cost-effective relative to lrr approach. conclusion: this study provides data of cost-effectiveness differences between rrr and lrr approach showing a benefit for the rrr aim: the efforts were aimed to the introduction of novel surgical technologies to overcome the intrinsic anatomical and technical constraints of rectal surgery. this was the case of the introduction into the clinical practice of laparoscopy and later on of robotic surgery for rectal surgery. however, whether robotic surgery is actually superior to laparoscopy is still debated. the aim of this study was to compare d laparoscopy and robotic surgery for rectal cancer on technical and oncological outcomes. methods: this was a single-center, prospective, randomized controlled trial. all patients more than years of age undergoing elective surgery for rectal cancer situated from to cm from the anal verge were included. patients undergoing abdominal perineal amputation and/or with t and/or m tumours were excluded. patients were randomized before surgery into two arms: arms a ( d laparoscopy) and arm b (robotic), and gave their consensus to the study. demographic data, data regarding the tumour, operative and post-operative data were collected. patients with a follow up shorter than months were excluded as well. results: twenty patients were enrolled in arm a and in arm b in the period time of one year. patients' population of the arms was homogeneous as concerns demographic characteristics and stage of the disease. robot-assisted rectal resection results in comparable operative time ( . vs min; p = . ). the conversion rate was significantly lower for arm b ( vs p = . ). postoperative morbidity was comparable between groups. hospital stay was comparable but time required to resolve post-operative ileus was shorter in arm b ( . vs . days, p = . ). overall survival and disease-free survival were comparable between arms ( . % vs . %, p = . , and . % vs . %, p = . , respectively) conclusions: d laparoscopy and robotic surgery are two viable options for rectal surgery. robotic surgery can add some in terms of post-operative outcomes and ergonomics. aim: currently, robotic surgery for rectal cancer is a surgical operation that is being performed worldwide. we also introduced robotic surgery in . however, after robotic surgery, we observed a rise in creatinine kinase (ck), which is unlikely to happen in other surgeries. we studied the postoperative complications of rectal cancer patients who underwent either robotic surgery or laparoscopy during the same period of time. methods: from january to november , patients underwent surgery using robotassisted rectal resection (da vinci si cases and xi cases) and patients underwent laparoscopic rectal resection. in this study, abdominoperineal resection, intersphincteric resection, and lateral lymph node dissection were excluded. result: the operation time for the robotic surgery group was significantly longer than that for the laparoscopic group ( min vs. min; p \ . ). the ck value of the robotic surgery group on pod was significantly higher than that of the laparoscopic group ( iu/l vs. iu/l; p \ . ). in addition, one case of compartment syndrome was observed in the laparoscopic group. there were no significant differences in age, body mass index, intraoperative bleeding, tumor invasion depth, urination disorder, or postoperative hospital stay. in robotic surgery, it is considered that the increase in ck value is caused by the extended operation time, contact of the patient's cart with the left thigh of the patient, and the extra force applied to the abdominal wall caused by the displacement of the remote center. conclusion: in robotic surgery, it is suggested that the measurement of postoperative ck value is important. therefore, an attempt to shorten the operation time and paying attention to the surgical field are necessary to improve the outcomes. aims: anastomotic leak remains as one of the most important and life threatening post-operative complications in colorectal surgery. this complication has important consequences, both acute and long term, longer hospital stay, re-intervention, and increased morbidity and mortality. among all different circumstances that have been related to this entity, blood supply is an important factor that might have influence. fluorescence with indocyanine-green (icg) is used as a marker in the assessment of tissue perfusion in colorectal surgery which might reduce the numbers of leaks. methods: a multicenter analysis of the experience of centers in spain is collected in order to assess the value of icg in colorectal anastomosis. colorectal procedures were performed using icg to evaluate vascular supply in the anastomosis. icg was infused before performing the anastomosis analyzing the number of cases in which the transection line (tl) was changed. we also analyzed the number of leaks in those cases that we changed this line. results: out of the cases performed, cases were performed by open surgery, by laparoscopy, by single-port and with transanal total mesorectal excision(tatme). the following procedures were performed: right colonic resection(rc), splenic flexure partial resection(sf), left colonic resection(lc), subtotal colectomy(sc), total colectomy(tc), hartman reversal surgery(hr), low anterior resection with partial mesorectal-escision(lar) and ultra low anterior resection with total mesorectal-escision(ular). leak rate(lr) was . % ( . %rc, . %lc, . %sc, . %lar, . %ular). overall lr was . % in colonic surgery and . % in rectal surgery. the tl was changed due to icg in . % of the cases ( . %rc, . %sf, . %lc, % tc, . % lar, . % ular), being . % in colonic resection and . % in rectal resection. the relation between leaks and the cases in which the tl was changed, were % ( . %rc, %lc, . %ular). conclusion: icg fluorescence may play a role in anastomotic tissue perfusion assessment. the lr after colorectal surgery might decrease using icg to detect the proper tl before to perform the anastomosis. however, we do not have the sufficient evidence to determine that the changing transaction line can lead to avoid leaks. surg endosc ( ) aims: to analyse the value of postoperative day crp as an early predictor of safe discharge in robotic rectal cancer surgery. methods: a retrospective analysis was performed, including patients who had undergone robotic total mesorectal excision (tme) in a single centre over a -year period (may -september . patients who had a permanent stoma (abdominperineal resections or hartmann's procedure) were exluded from the study, leaving patients for further analysis. as the los is currently used as a performance tool in assessing outcomes in colorectal surgery (with a cut-off established at days), we compared the crp values in these groups. results: fourty one percent of patients were discharged home within days. they had an earlier peak of crp on postoperative day (pod) (median . , ) . the group of patients that were discharge home after days ( %) had a crp peak on pod (median , ). on pod , the group of patients that went home within days had a lower crp ( - -vs. - -) compared to the group of patients that were discharge after days, p = . ). conclusions: a crp peak on pod in robotic tme can predict an early and safe discharge (los within days). background: purposelateral pelvic lymph node dissection (lpnd) is suggested to treat suspected lymph node metastasis in pelvic side-wall in patients with rectal cancer who underwent preoperative chemoradiotherapy (crt). however, technical difficulties make it possible that lateral pelvic lymph nodes (lpns) are not dissected completely and, thus, remained in the narrow pelvis. near-infrared fluorescence imaging (fi)-guided surgery is expected to help visualization and complete excision of nonvisible lymph nodes during cancer surgery. this study aimed to evaluate the efficacy of fi using indocyamine green (icg) to identify lpns during robotic lpnd. methods: rectal cancer patients who were suspected lpn metastasis and had received preoperative crt were prospectively enrolled. icg in a dose of . mg was injected around tumor preoperatively. all procedures were performed with a totally robotic approach. after completing lpnd, fi was checked again for identifying remained lpns and resecting them completely. results: the lpns were successfully detected in ( . %) of the patients. however, after accounting for eight cases, having finished adjusting icg injection, the lpns were successfully detected in ( . %) of patients. the fi-guided lpnd group (n = ) showed similar mean operative time for unilateral pelvic dissection and complication rate, compared to patients who underwent conventional robotic lpnd (n = ). however, the mean number of unilateral harvested lpns was . in the fi-guided lpnd group, which was greater than the mean of . in the conventional group. lpn metastasis was identified in % of the fi-guided lpnd group, which was higher than that of the conventional group, . %. conclusion: fi-guided lpnd identifies lymph nodes in pelvic side-wall with great reliability. this contributes to increased number of lpns yield compared to conventional robotic lpnd. this technique should be considered to dissect them completely by preventing subsequent missing of nonvisible lpns. aims: to compare the medium-term oncological outcomes of laparoscopic total mesorectal excision (l-tme) vs. robotic total mesorectal excision (r-tme) for rectal cancer. methods: a retrospective analysis was performed including patients who underwent l-tme or r-tme resection between - . patients with disease stage iv at diagnosis or r resection were excluded. patients were initially included, and cases of r-tme were matched based on age, gender, stage and time of follow-up with an equal number of patientswho underwent l-tme. we compared -year disease free survival (dfs) and overall survival (os). in adittion, a multivariate analysis was performed in order to idenfity independent prognostic factors for -year dfs and os. results: pathological outcomes were similar between groups. however, major complications were lower in the robotic group ( . % vs. . %, p = . ), highlighting the anastomotic leakage rate, which was . % in the r-tme vs. . % in the l-tme group (p = . ). overall, the -year dfs rate was % in the laparoscopic group and % in the robotic group (p = . ). the -year os rate was % in the l-tme groups and % in the r-tme group (p = . ). for disease stage iii, -year dfs was significantly higher in the r-tme group. os was also significantly superior in the robotic group for every stage, reaching % in the stage iii. in the multivariate analysis, r-tme was a significant positive prognostic factor for distant metastasis (or . %ci . , . , p = . ) and os (or . %ci . , . , p = . ). conclusions: r-tme for rectal cancer can achieve better oncological outcomes compared to l-tme, especially in stage iii rectal cancers. the robotic approach has demonstrated to be a significant positive prognostic factor for local recurrence and overall survival, due to the better postoperative outcomes. however, a longer follow-up period is needed to confirm the oncologic findings. university hospital for visceral surgery, university of oldenburg, oldenburg, germany; bremen spatial cognition center, university of bremen, bremen, germany aims: in clinical settings, realistic assessment of one's own abilities can enhance performance and promote patient safety, especially in surgical residents, who inevitably have to acquire skills during real surgery. this study thus implemented the global assessment of laparoscopic skills (goals) questionnaire with the aim to explore divergences between resident self-evaluation and specialist's evaluation on laparoscopic performance, as a first step to implement the goals questionnaire as a tool for constructive and objective feedback. methods: between july and october , seven residents from the university hospital for visceral surgery at the pius-hospital oldenburg participated in this study. at the end of every laparoscopic operation where the resident acted as the primary surgeon, the resident and the supervising surgeon independently evaluated the resident's operative performance using the goals questionnaire. the five dimensions evaluated were depth perception, bimanual dexterity, efficiency, tissue handling and autonomy. a cumulative goals-score (with being the highest possible score) was calculated for n = laparoscopic operations. resident's year of training, the level of case difficulty and the type of laparoscopic procedure performed was also analysed. results: residents overestimated their laparoscopic abilities in . % of the operations (goals-scores: residents: median = , mean = . ; specialists: median = , mean = . ; p \ . ). residents in the first three years of surgical training were more likely to overestimate their performance (residents: median = . , mean = . ; specialists: median = , mean = . ; p \ . ) than those with more than three years of surgical experience (residents: median = , mean = . ; specialists: median = , mean = . ; p = . ). goals score differences did not depend on case difficulty and laparoscopic procedure. conclusions: surgical residents tend to overestimate their intraoperative laparoscopic performance when compared to specialist evaluation. overestimation was found to depend on one's own laparoscopic experience and seem to disappear with gained expertise. these results signify the importance of individually adapted training and the greater need for objective feedback for surgical residents. this approach could in return increase the skill acquisition rate of the resident and in return contribute towards enhancing patient safety. introduction: the delivery of safe surgical care is dependent of various, complex and interrelated factors. substantial data exist regarding the impact of training in human factor skills on surgical outcomes. however, except for the standardized time-out process, the best way to go about improving these skills remains unclear. the aim of this study was to gain more insights in the theatre staff's perception of human factors and their importance on surgical outcome in the operating theatre. methods: the surgical team assessment record (star) questionnaire was used to study the role of human factors, such as communication, situational awareness and organization, contributing to surgical team performance. the self-assessment questionnaire was filled out by the theatre staff, directly after the surgical procedure. conditional logistic regression was used to identify the impact of the role in the operating theatre on the yes versus no answers. results: some questionnaires were completed. the theatre staff rated their team's performance with a median of (iqr . , -point likert scale). the surgical fellows (n = ) rated their personal factors significantly lower compared to the rest of the operating team (median versus , p-value \ . ). the staff surgeon (n = ) indicated significantly more often that there were many distractions ( . %, yes n = ) and noticed aberrations ( . %, yes n = ) during the surgical procedure (pvalue \ . ) when compared to the rest of the operating team. most aberrations reported by the surgeons were related to technical performance. conclusions: human factors play an important role in the surgical environment. situational awareness may be less developed in members of operating teams, compared to the surgeon of that team. further work is needed to elucidate the impact of human factor skills on team performance. a team-based approach to safety interventions is recommended. future studies should determine what type of aberrations and distractions are most relevant and valuable to embark on with team training. dept. of digestive surgery, school of medicine, tokushima university, tokushima, japan; dept. of digestive surgery, tokushima university, tokushima, japan background: the qualitative evaluation for laparoscopic training of medical students was performed using rubric evaluation, and weak points in conjunction with the lack of anatomical knowledge were derived. to conquer these weak points, virtual reality (vr) ? augmented reality (ar) training for understanding of regional anatomy was investigated. materials and methods: one hundred and six students in th grade of tokushima univ. participated basic laparoscopic task training (gummy band ligation, beads transfer, delivery of beads, gauze excision) with training box and sham laparoscopic cholecystectomy with virtual simulator. rubric evaluation, as qualitative evaluation, which includes the evaluation standards for each maneuver were performed before and after basic task training and sham operation. the group which self-evaluation was higher in a rubric evaluation was investigated. the d image of vessels and bile duct obtained from mdct of real patient was projected in reality space with microsoft hololens. training of ar image using hololens was performed for understanding of regional anatomy. after training of regional anatomy with hololens, sham laparoscopic cholecystectomy was performed again, and quality of procedure was evaluated by rubric. anatomical questions were. results: rubric evaluation in basic task training showed no difference between self-evaluation and evaluation by tutor before and after practice. in sham laparoscopic cholecystectomy, several students showed higher score than tutor, especially in part of extension of operation field by elevation of the gall bladder, exposure of triangle of calot, and exposure of cystic duct. after ar training, all students showed high score in questions related regional anatomy during operation. especially, rubric evaluation of students who showed high self-evaluation in sham operation showed same score with tutor. conclusions: as rubric evaluation showed weak points of detailed parts of maneuver, and vr ? ar was useful for understanding details of regional anatomy for laparoscopic training. background: the eaes has recently published an intraoperative adverse event classification to aid reporting of minimally access surgery events. this includes capture of non-consequential errors. we aimed to investigate the clinical impact of these apparent 'near miss' events. methods: case videos and clinical data from a completed multi-centre laparoscopic total mesorectal excision randomised controlled trial was utilised (isrctn ). the eaes classification was applied by two blinded assessors to all enacted adverse events identified on video analysis using the observational clinical human reliability analysis technique. the total number of grade (non-consequential) errors were compared with the number and nature of day morbidity events (graded with the clavien-dindo system) and length of stay. results: cases ( h of surgery) contained error events of which ( . %) were classified as eaes grade (median per case, interquartile range - , range - ). there were significantly more inconsequential errors recorded in patients that developed any early morbidity event than those who had an uneventful post-operative recovery (median . (iqr - ) vs. ( - ), p = . ). a stepwise increase in the sum of eaes grade errors is seen for each additional day morbidity event reported ( . vs. vs. vs. , p = . ) and the highest clavien-dindo grade experienced ( vs. vs. vs. . p = . ). positive correlation is observed between the sum of eaes grade a errors and length of post-operative stay (r s = . , p = . ). conclusion: in the context of major laparoscopic surgery, near misses are commonplace and correlate with surgical outcomes. this may represent a novel surrogate assessment method for intraoperative performance. aims: diagnostic laparoscopy (dl) is an under-utilised procedure that can replace non-therapeutic exploratory laparotomies in many contexts. to date, no validated education programme for dl exists. this study seeks to evaluate the feasibility, acceptability and face, content, construct validity of the laplat curriculum (laparoscopic learning for abdominal trauma; a simulationbased curriculum for trauma dl). this is in addition to the development of a novel d-printed bench-top model for abdominal inspection. methods: this prospective and observational pilot study involved novice medical students and junior doctors. surgeons from the uk and international (n = ) were involved in a two stage delphi-process to determine the components of the training course which were used to formulate a final curriculum. in the absence of an adequate model for abdominal inspection, a novel dprinted abdominal inspection model was designed and produced. after an introductory familiarisation session as well as pre-course cognitive lectures, the novices performed tasks on a virtual reality and bench-top simulator with repetitions of each in a half-day session. outcome measures for construct validity were total time to complete task, accuracy, percentage of horizon maintained and economy of movement. face and content validity as well as acceptability was evaluated by a qualitative and quantitative survey. results: face, content and construct validity as well as acceptability was established. face validity was demonstrated in all components of the course (including pre-course cognitive content and technical tasks) in addition to content validity. all also met an acceptability threshold of / on a -point likert scale. one-way anova tests demonstrated construct validity in all tasks (p \ . ) with learning curves in reducing time observed. using a performance improvement metric, one-way anova tests showed similar rates of improvement per participant between most tasks (p [ . ). the course was rated overall mean . / (± . ). conclusion: this pilot study has demonstrated the feasibility, acceptability and face, content and construct validity of the laplat curriculum as well of the novel d-printed abdominal inspection model. randomised controlled trials are needed to establish higher-quality evidence, as part of a wider curriculum with transfer needed to the clinical environment. surgery, regional institute of gastroenterology and hepatology, cluj-napoca, romania; anesthesiology-surgical propedeutics, university of agricultural sciences and veterinary medicine, cluj-napoca, romania; radiology, regional institute of gastroenterology and hepatology, cluj-napoca, romania; urology, training and research center, prof. dr. sergiu duca, cluj-napoca, romania; general surgery, training and research center, prof. dr. sergiu duca, cluj-napoca, romania aims: to evaluate the benefits of systematical use of ex vivoliver model and ct imaging in the planning process for swine laparoscopic liver resections done by residents during training programs. methods: twenty four general surgery residents were equally divided into two groups: first one which performed laparoscopic liver resections without planning stage and the second one which systematically used anatomical data from a swine liver model and interactive ct scans d reconstructions. the planning stage included an interactive tutorial for establishing the strategy for the next resection followed by performing open liver dissection and the same resection on an ex vivoswine model. a total of twelve models were used during this step. afterwards, laparoscopic procedures were performed on sixteen anesthetized domestic pigs, two swine for every team, composed of three residents. both groups were part of a dedicated and continuous training program and used the same 'step by step' protocol for resections. results: the average time for imagistic planning was . min and for open dissection and resection was . min. all teams successfully completed the interventions and followed the standardized protocol without trainers' interventions and with no conversions. the second group obtained better results regarding the time needed for completion and blood loss. also, when the planning stage was applied the resection was more accurate and less functional parenchyma was removed. the 'warming up' by adding the imagistic and anatomical data to the core protocol offer more clarity before laparoscopic liver resections. this also makes an upgrade for our 'step by step' protocol and provides sufficient data to admit this planning stage as mandatory for laparoscopic liver resection on swine during a training program. introduction: submucosal tunnel endoscopic resections (ster) had been increasingly performed for treatment of gastric subepithelial tumors. one of the limitations for ster is the risk of incomplete tumor resection due to close dissection and bridging of tumor capsule. endoscopic full thickness resection (eftr) allowed complete resection of the tumor with margins to prevent recurrence. this study aimed to review the techniques and outcomes of eftr for treatment of gastric subepithelial tumors. method: patients who received endoscopic resection for gastric subepithelial tumors were recruited. the gastric subepithelial tumors were considered eligible for endoscopic resection with size \ mm. all patients received preoperative assessments including eus and ct scan to define the extend of tumors and the proportion of extra and intralumenal components. all the procedures were performed under general anesthesia with co insufflation. eftr started after injection with mucosal incision up to % of tumor circumference, followed by submucosal dissection to identify tumor margin. further dissection was performed using esd devices. after adequate exposure of lateral margins, incision into muscularis propria was performed to achieve full thickness resection. luminal defects were closed by either clips, clip-loop crown method or overstitch suturing. results: from to , patients received eftr for gastric subepithelial tumors. the mean age was . years, and were male. the gist were located at greater curvature ( ), cardia ( ) , lesser curve ( ) and antrum ( ) . the mean size was . mm ( - mm) . most of the eftr were performed in operation theatre while two were done at endoscopy. the mean hospital stay was . days, and mean operative time was min ( - mins). there was no conversion to laparoscopy. closure of luminal defect were performed mostly with clips ( ), followed by overstitch ( ) and clip and loop crown closure ( ) . most patients resumed full diet on day , and all the pathologies confirmed gist tumors with clear resection margins. conclusion: endoscopic full thickness resection is technically feasible and safe procedure for treatment of gastric gist. future research should focus on refining the techniques of eftr and closure of the defect. next generation endoscopic intervention (project engine), osaka university, suita, japan; gastroenterological surgery, osaka university, suita, japan; research & development, -d matrix, ltd., chiyoda-ku, tokyo, japan; research & development, fuso pharmaceutical industries, ltd., cyuou-ku, osaka, japan background: hemostatic peptides have received increased attention. self-assembling peptides (tdms) comprise synthetic amphipathic peptides that immediately react to changes in ph and/or inorganic salts to transform into a gelatinous state. since tdms do not carry a risk of infection, their clinical application as new hemostatic agent is expected to increase. the first generation of these peptides (tdm- ) is currently used as a hemostatic agent in europe. however, tdm- exhibits slow gel-formation and low retention capabilities on tissue surfaces. the second generation (tdm- ) was therefore developed to encourage faster gel-formation and better tissuesealing capabilities, and we subsequently verified its usefulness and increased performance relative to tdm- in preclinical open surgery. aim: the aim of this study was to verify the efficacy of tdm- in terms of its hemostatic effect in endoscopic surgery. materials and methods: evaluation of the hemostatic effect in endoscopic surgery (animal study) was performed using eight female ( kg) pigs in spine position. following systemic heparinization, we established a bleeding model by utilizing flexible endoscopic grasping forceps on the anterior wall of the stomach and duodenum. in the hemostasis method, an endoscope with a distal hood was brought into contact with the bleeding point, and ml tdm- was applied to the wound. after tdm- gelation, the endoscope was removed, and the acute hemostatic effect (after min) was confirmed. histologic evaluation was subsequently performed on resected specimens. results: in the endoscopic bleeding model, of the cases ( . %) showed complete hemostatic effects on the anterior wall of the stomach, whereas on the anterior wall of the duodenum, of cases ( %) showed complete hemostatic effects. moreover, none of the gels were displaced from the anterior walls of the stomach and duodenum, and histologic evaluation confirmed no infiltration of inflammatory cells. the new self-assembling peptide (tdm- ) displayed improved hemostatic effects relative to the previous generation (tdm- ) in endoscopic surgery. tdm- had potential usefulness for upper gastrointestinal bleeding. our future work will assess its usefulness for laparoscopic surgery. objective: indocyanine green (icg) is a dye used in medicine since the mid- s for different applications in ophthalmology, cardiology and hepatobiliary surgery; thanks to its selective hepatic uptake and biliary excretion, it can be used to evaluate hepatic function in patients scheduled for hepatic resection surgery. the aim of this study is to evaluate the efficacy and the feasibility of icg guided surgery in the intra-operative localization of liver tumors, comparing the pre-operative radiological aspect, the intra-operative visualization and the post-operative histopathological features of the tumors. materials and methods: icg was intravenously injected for a routine liver function test (limonÒ) in patients who underwent hepatic resection surgery for primitive and secondary liver tumors in the period between november and september . for each patient was performed an intraoperative visualization of the stain both in vivo and ex vivo, using a nearinfrared imaging system. all the images were recorded. results: a correct differentiation between liver parenchyma and tumor area was obtained in . % of cases. five patients were not evaluable due to widespread uptake or complete absence of uptake; it was probably the first cases enrolled in the study for which we were not able to set doses and timing of administration of icg. in patients in which the method had been feasible, we observed a prevalence of nodular pattern in patients with hepatocellular carcinoma ( %) and a predominance of rim pattern in both cholangiocarcinoma ( %) and metastasis ( %). furthermore, in patients with hccs well-intermediate differentiated (g -g ) was found predominantly a nodular pattern ( . %), whereas in poorly differentiated ones was prevalent a rim appearance ( %). regarding radiological correlations, the only one patient who presented an atypical radiological feature in pre-operative evaluation, showed a lesion with no icg captation in intra-operative visualization. conclusions: icg fluorescence imaging is a safe, minimally invasive and quite inexpensive method, that can be easily administered for routine evaluation of pre-operative liver function. it can be a useful support tool in the intra-operative detection of liver tumors, especially in laparoscopic surgery where it is not possible to directly touch the tissue. surgery, bundang cha medical center, seongnam-si, korea; surgery, severance hospital, seoul, korea; surgery, nhimc ilsan hospital, ilsan, korea; surgery, seoul national university bundang hospital, seongnam, korea; surgery, asan medical center, seoul, korea backgrounds & aims: robotic surgical system had been widely accepted in various surgical field with the expectations of overcoming the limitation of laparoscopic surgery. however, robotic liver resection had not generalized, so far. thus, this study aimed to evaluate the feasibility and safety of robotic major liver resection by prospective multicenter study. methods: from july to december , five surgeons who were novice in robotic liver resection but experienced a lot in open and laparoscopic liver resection in five tertiary hospitals performed cases of robotic major anatomical liver resection. perioperative patient's clinical data and surgical data were prospectively collected. results: cases of left hemihepatectomy, case of extended left hemihepatectomy, cases of right hemihepatectomy, cases of right anterior sectionectomy, cases of right posterior sectionectomy, and one cases of central bisectionectomy were performed. the most common indications were hepatocellular carcinoma for cases following intrahepatic cholangiocellular carcinomas for cases, liver metastases for cases, sarcoma for case, intraductal papillary neoplasms for cases, mucinous cystic neoplasm for case, hemangioma for case, and intrahepatic duct stones for cases. surgical resection margins for all tumor cases were negative. total average operation time was . ± . min and estimated intraoperative blood loss was . ± . ml (minimal to ml). in terms of severe surgical complication, there were cases of postoperative fluid collection treated with drainage and one case of bile leakage treated with percutaneous trans-hepatic biliary drainage. only one case out of cases was converted to the conventional open left hemihepatectomy because of bleeding. conclusions: in this study, robotic anatomic major liver resection might be safely performed even by robotic beginners but advanced open and laparoscopic liver surgeons. surgical technique: with the patient at °on right lateral decubitus, access is gained through the path of the percutaneous drainage catheter after opening of the aponeuroses of the oblique and transverse muscles of the abdomen. a mm laparoscopic trocar is inserted and a cavity is created with pneumoretroperitoneum at mmhg. it is accessed with an optic of °and mm, and the work space is extended with aspiration and hydrodissection. with mm grippers, the necrotic material is removed, washed and drained. a two light silicone probe is left, one light for drainage and another one for washing. results: the mean age was . background: minimally invasive surgery has achieved worldwide acceptance in various fields, however, pancreatic surgery remains one of the most challenging abdominal procedures. in fact, the indication for robotic surgery in pancreatic disease has been controversial. the present study aimed to assess the safety and feasibility of robotic pancreatic resection. methods: we retrospectively reviewed our experience of robotic pancreatic resection done in sanchinarro university hospital. clinicopathologic characteristics, and perioperative and postoperative outcomes were recorded and analyzed. aim: this work aims to study the contact pressure between the moving capsule and a synthetic small intestine in order to provide design guidance for prototyping the self-propelled capsule robot for small-bowel endoscopy. method: since small-bowel peristalsis consists of peristaltic contraction and wave distension, the contacts between the capsule and the small intestine are multimodal. we consider three contact cases for the capsule robot. case : the capsule moves on a flat small intestinal surface; case : the capsule moves in a collapsed intestine with a flat surface support; and case : the capsule moves in a surrounded small intestine. by considering these three contact cases, experimental testing and finite element analysis (fea) were conducted by measuring the contact pressure between the small intestine and the capsule. introduction: traditional laparoscopic instruments have limited degrees of freedom and are not ergonomic. this results in severe limitations in performing complex, and even simple tasks in surgery, limiting many surgeons from performing a variety of minimally invasive procedures. handx tm is a hand-held, electromechanical smart instrument with robot-like features. the instrument is composed of a sophisticated user interface that enables unrestricted hand movement, and a novel, motor driven articulating tool that is controlled by the interface. the instrument is . mm in diameter, lightweight, and can be easily moved between laparoscopic trocars and perform complex motions in the surgical field. after the regulatory process was completed we have tested the device clinically through a structured, approved, clinical trial. materials and methods: after irb approval patients were recruited to the trial. we have included a variety of procedures that require suturing and complex tissue manipulation. two experiences surgeons performed all procedures. after completing each procedure the surgeons completed a detailed standard usability (sus) questionnaire. results: procedures were completed successfully without complications or device malfunction. there were female and male patients with an average bmi of . procedures performed were right hemicolectomis with intra-corporeal anastomosis, paraesophageal hernia repairs and fundoplication, diagnostic laparoscopies, tapp procedures, ventral hernias with fascial suturing, and laparoscopic cholecystectomies. the average performance score was . / . the results suggest that the handx device is safe and easy to use and may offer a simple solution for enhancing minimal invasive surgery capabilities and possibly reduce conversion rates while maintaining current standard surgery flow.the handx could potentially extend the surgeon's abilities to access hard to reach anatomy and perform complex maneuvers and present a cost-effective alternative to large console-based robotic systems. objective: endoscopic submucosal dissection (esd) has become widely accepted treatment for rectum neuroendocrine neoplasm. the aim of this study is to evaluate the safety and efficacy of esd with dental floss-assisted suspension traction for rectal neuroendocrine neoplasm. methods: we retrospectively reviewed the medical records of the patients, who underwent esd for rectum neuroendocrine neoplasm at endoscopy center of zhongshan hospital, fudan university. the data of operation time, r resection and adverse events were collected analyzed.in dfs-esd group: after the mucosa was partly incised along the marker dots, the next step was to construct traction device, similar to others in esd, with dental floss and hemoclip. the dental floss was tied to any arm of the metallic clip. the hemoclip was attached onto the incised mucosa, another hemoclip was attached onto normal mucosa opposite to the lesion in the same way. the submucosa was clearly exposed with the traction of dental floss and the resection could proceed. results: patients were enrolled in the study. there were patients treated by esd with dental floss-assisted suspension traction and patients treated by conventional esd. the average tumor size was ( . ± . )cm in both group. the operation time was . ± . min in conventional esd group and ( . ± . ) min in dfs-esd group (t = . , p = . ). according to pathological grading about rectal neuroendocrine neoplasm, there were grade (g ) and grade (g ) in conventional esd group while grade (g ) and grade (g ) in dfs-esd group (? = . , p = . ). among cases in this study, all the basal resection margins were negative, the en blot resection rate was % and the curative resection rate was %. however, pathological results showed tumor tissue close to the burning margin in cases of conventional esd group and in cases of dfs-esd group (? = . , p = . ). conclusions: esd with dental floss-assisted suspension traction for rectum neuroendocrine neoplasm can assist exposing tumor borders, provide good vision during the procedure and offer clearer anatomic structure, so as to simplify operation, reduce operation time and ensure the negative basal margin. it is especially suitable to be promoted in primary hospitals. surg endosc ( ) aims: force feedback and assessment provides detailed insight into tissue manipulation skills. the aim of this study is to evaluate learning curves for basic laparoscopic skills based of force and motion learning curve patterns. morevover, we aimed to detect the favourable time span for this curriculum for each individual trainee. methods: in this prospective cohort study, first year surgical residents participated in a three week at home training course. a mobile box training was equipped with forcesense system for objective force, motion and time based assessment. the system provides seventeen unique metrics. the training goal was set by the mean score of proficient laparoscopic surgeons. each repetition was captured and made available for analyses. continuous force feedback was provided during training. curve fitting was used to estimate the learning curve plateau and the number of repetitions needed to approach the plateau phase and to reach proficiency level. finally, a comparisson between novices and experts was executed. results: a total of attempts, executed by residents were captured and analyzed. significant improvement of motion analysis parameters (e.g. path length and time) was observed for all training tasks, except for the fifth tasks. tissue manipulation skills (i.e. maximum and mean applied force) significantly improved by training tasks , and . learning curve analysis revealed various shapes and lengths of the individual learning curves. a large range in learning curve plateaus was found between trainees and between tasks. each trainee managed to accomplish the preset goals within three weeks. conclusion: force-and motion based assessment provides insight into both tissue manipulation and instrument handling skills. when combined in learning curve analysis, these parameters effectively show progression towards proficiency for each individual trainee over time. we emphasize the variation in learning curves between trainees. therefore, we recommend individually tailored courses provided with objective force-and motion-based learning curve tracking. aims: the posterior retroperitoneoscopic adrenal access represents a challenge in orientation and working space creation.the aim of this experimental acute study was to evaluate the impact of computer-assisted quantitative fluorescence imaging on adrenal gland identification and perfusion assessment in the posterior retroperitoneoscopic approach. methods: six pigs underwent synchronous (n = ) or sequential (n = ) bilateral posterior retroperitoneoscopic adrenalectomy (pra, n = ). fluorescence imaging was obtained via intravenous administration of ml of indocyanine green (icg) using two near-infrared camera systems. fluorescence-based visualization of adrenal glands before vascular division (n = ), after main vascular pedicle ligation (negative control, n = ) or after adrenal division (n = ) was followed by completion adrenalectomy. one of the animals had undergone icg injection h previously, during another study. the dynamic evolution of fluorescence signal intensity over time was recorded and analyzed using a proprietary software. the computed color-coded perfusion cartography was superimposed onto real-time images obtained by corresponding left (l) and right (r) camera systems. the slope of fluorescence signal intensity evolution over time in the regions of interest (roi) served to assess adrenal perfusion by means of quantitative fluorescence signal analysis. results: in the retroperitoneum, the adrenal glands were promptly highlighted after primary intravenous icg administration or showed an increase in fluorescence signal intensity upon reinjection (both glands in a recovery pig and one gland in the sequential approach). after left adrenal main vascular pedicle ligation, the gland displayed low perfusion (blue; rois a -a in figure ), while a weak fluorescence signal after completion adrenalectomy suggests perfusion via collateral vessels. with intact vascular supply, the caudal segment of the right adrenal (a ) gland showed a significantly higher perfusion rate (red) than the ischemic cranial segment (a ). quantitative analysis of logarithmic fluorescence intensity showed a statistically significant difference between perfused and ischemic zones (p = . ) allowing to assess gland vascularity. kidneys (k) and adrenal glands showed distinct perfusion curves ( figure ). conclusions: prior to dissection, fluorescence imaging allows to easily discriminate the adrenal gland from surrounding retroperitoneal structures. during adrenal gland surgery, icg injection complemented by a computer-assisted quantitative analysis helps to distinguish between wellperfused and low-perfused segments. giant adrenal tumors:technical considerations and surgical outcome a. giordano, g. alemanno, c. bergamini, p. prosperi, v. iacopini, a. dibella, a. valeri sod chirurgia d'urgenza, aou careggi, firenze, italy objectives: giant adrenal tumors are tumors with size more than cm. these are rare cancer associated with malignancy in % of cases. the size of these tumors is an important topic in literature because of their higher probability of malignancy and possible technical limitations of laparoscopic approach. we report our center's experience on laparoscopic adrenalectomy. materials and methods: in the last ten years we performed about adrenalectomies for benign and malignant adrenal tumors. of these were giant tumors. the medium size was . cm ( - cm). tumors were on the left adrenal gland and on the right. there were women and men, the average age was ( - years). of these cancers were laparoscopically removed and with open approach. cases of open conversion. results: betweenn the tumors laparoscopically removed we recorded cases of carcinoma, endothelial cysts, adenomas ( with aldosterone and with cortisol hypersecretion), myelolipomas, pheochromocytomas and metastases from lung carcinoma. the surgical outcomes in these patients were optimal in terms of good pain control and hospital stay (median days). the average time of the intervention was min with very low blood less ( ml). no postoperative complications were recorded. the removal of the adrenal gland necessitated or trocars. in the dissection and resection phases we always used radiofrequency scalpel. the follow up after and months didn't show local recurrences. conclusions: laparoscopic adrenalectomy offers significant advantages over the open approach. the size of these tumors is still at the center of debate for the choice of the technique. the tumor size is only a predictive parameter of possible malignancy. the laparoscopic approach is a safe and feasible method in terms of surgical and oncological outcomes also for the giant adrenal tumors, only if performed by expert surgeons and in high-volume centers. vascular or adjacent organs infiltration is a contraindication to the laparoscopic approach. aims: adrenal gland size greater than cm is considered a contraindication to laparoscopic adrenalectomy (la). aim of the present case-control study is to compare the surgical outcomes in patients undergoing la for adrenal gland measuring = cm versus = . cm in diameter. methods: from january to august , las were performed in the two authors' centers which follow an identical treatment protocol. eighty-one patients with an adrenal gland size = cm (intervention group) were included in the study. based on body mass index (bmi) class [ kg/m ) , lesion side (right or left), surgical technique (anterior transperitoneal for right and left-sided lesions, anterior transperitoneal submesocolic for left-sided lesions) and lesion type (conn-cushing, pheocromocytoma, primary adrenal cancer or metastases, other type of lesion), patients with an adrenal gland lesion measuring = . cm in diameter were included (control group) and paired to the intervention group. results: comparing the intervention and control groups, statistically significant differences were observed in mean lesion size ( conclusions: the only significant difference between the two groups was the operative time which was longer in the intervention group. conversion and complication rates were also higher in the intervention group but the difference was not statistically significant. based on the present data, adrenal gland size measuring more than cm in diameter is not a contraindication to a laparoscopic approach. ; and orthopaedics and urologists for the remaining . %.the costs from these claims, differed from to % of the total damage burden per year. the review of medical charts of claims related to laparoscopic gynaecologic surgery showed that % of claims were filed for visceral and/or vascular injuries ( % bowel injuries, % ureter). % of the injuries were entry-related. a delay in diagnosing injuries was the primary reason for financial compensation. conclusion: evaluating and learning from complications and claims will improve medical health care. in contrast to overall trends and developments considering medical claims, claims concerning laparoscopic surgery decreased, possible due to a rising learning curve. considering laparoscopic surgery, extra caution is required at moment of entry and the early recognising complications and at pre-operative counselling from patients. the aim of the study was to determine indications and contraindications for laparoscopic splenectomy in abdominal trauma patients and to analyze results of the operations. patients and methods: the study involved patients with spleen injury grade iii who were admitted in our institute in the years of - . the patients were divided on two groups. laparoscopic splenectomy was performed in patients (group i) and 'traditional' splenectomy was carried out in patients (group ii). there was no difference in the demographic data and trauma severity between the two groups.non-invasive investigations, such as laboratory investigations, serial abdominal ultrasound examinations (us), x-ray in multiple views and computed tomography (ct) had been performed before the decision about necessity of an operation was made. results: patients after laparoscopic operations had better recovering conditions compare to patients with the same injury after 'traditional' splenectomy. neither surgery related complications no mortalities were registered in both groups. laparoscopic splenectomy was more timeconsuming operation than 'traditional' splenectomy. we suggest that as experience of laparoscopic splenectomy is gained the operation time will be reduced. conclusion: laparoscopic splenectomy is a safe feasible operation in patients with spleen injury. the operation is indicated in patients with spleen laceration more than cm of parenchymal depth with moderate continuing bleeding or expanding hematoma and contraindicated in patients with hemodynamic instability and high bleeding rate (more than ml/h on serial us examinations). the isolated hydatid disease of the spleen is a quite rare condition, liver and lungs being the most common locations. the treatment requires usually splenectomy, open or laparoscopic. there are few reports in the literature describing a spleen-preserving type of surgery. we present a case of a female patient, y.o., with a large cystic lesion of the spleen, cm in diameter. lab tests and ct scan confirmed that is a hydatid cyst. after albendazole treatment and vaccination the patient was referred to us for surgical treatment. the procedure was performed under general anesthesia and laparoscopic approach was performed with the intention to preserve the spleen. after the cyst was identified and adhesiolysis was done, the area was isolated from the rest of the abdominal cavity with sponges with a betadine solution in order to prevent contamination. a needle aspiration of the cyst allowed the evacuation of ml of purulent content, an indicator of a dead cyst. betadine solution was injected into the lesion. laparoscopic excision of the cyst was performed using advanced electrocoagulation devices and the spleen removal was not deemed necessary. two drainage tubes were placed in the remnant cavity. an abdominal ultrasound was performed in the third postoperative day and no collections were identified. the postoperative outcome was uneventful; the patient was discharged in the th postoperative day. the conclusion is that in selected cases, with the cyst located in the anterior part of the spleen, with proper equipment and experienced laparoscopic teams, the cyst can be successfully treated without splenectomy. deep neuromuscular block was induced with rocuronium . mg/kg. in group , forty patients were enrolled for reversal of profound neuromuscular block during thyroid surgery (sugammadex mg/kg, after identification of vagus nerve). in group , thirty-five patients were enrolled profound neuromuscular block during thyroid surgery(without reversal of nmbd). tof-watch acceleromyograph was recorded in response to adductor pollicis muscle for ulnar nerve stimulation in patients with both groups; recovery was defined as a train-of-four (tof) ratio = . .to prevent laryngeal nerve injury during the surgical procedures, all patients were neurophysiologically detected using ionm. results: the total duration of surgery was higher in group than group ( . ± . , . ± . ;p \ . ). the mean time to recovery of the tof ratio to . was higher in group than group ( . ± . , . ± . ; p \ . ). the mean duration of vagus reverse (v : , milisecond) was higher in group than in group ( . ± . , . ± . ; p \ . ). no significant difference was found between left and right v -v and r -r values in group following nerve monitoring, whereas in group , a significant difference was found between left v -v , left r -r and right v -v values ( introduction: oeosphagogastric oncology trials have often lacked robust methods of monitoring and surgical quality assurance (sqa), leading to difficulty in interpretation of trial results. this study aims to assess expert opinion regarding challenges to sqa in oncology trials and potential mitigating strategies. method: a purposive international cohort of expert stakeholders with experience in oncology trials were recruited including: surgeons; oncologists; trial methodologists, and; trial managers. semi-structured interviews were thematically analysed using grounded theory. spss was utilised to assess differences between trial stakeholders' opinions. results: emergent themes were identified and consensus themes emerged on qualitative analysis of stakeholder responses. key consensus challenges to implementation of sqa in oncology trials included: insufficient resources; limitations of surgical volume in centre selection; differing oncological beliefs and resistance to change adoption; overly prescriptive protocols and standardisation contributing to difficulty in surgeon recruitment; and cultural factors leading to difficulties in providing and receiving feedback. seminal consensus mitigating strategies to overcome challenges to sqa in oncology trials included: trial centre selection according to case volume (n = , %); requirement for specific centre attributes for inclusion in trials including specialist centre designation and participation in national audit (n = , %); consideration for surgeons learning curve in surgeon selection (n = , %); flexible standardisation of trial operating (n = , %); operation manual utilisation to aid standardisation of surgical interventions (n = , %); case monitoring using video (n = , %) or photographs (n = , %); direct intraoperative observation by an expert (n = , %), and; histopathological assessment of resected specimens (n = , %). other methods of monitoring surgical quality advocated included: recording post-operative outcomes; lymph node yield; case report forms; and real time data monitoring (n = , %). oncologists were significantly more likely to state the importance of standardisation of surgery in oncology trials (p \ . ), and trial methodologists significantly more likely to advocate consideration of surgeons' learning curve in surgeon selection (p \ . ). conclusion: surveying international expert stakeholder opinion revealed a wide variety of perceived challenges across all domains of surgical quality assurance. proposed mitigating solutions require consensus opinion to formulate a framework to aid design of sqa measures within future oncology trials. research group did not register a single case of ega leakage while patients in control group (? \ , ). had the leakage which was stopped by means of 'endovac' system. there were cases of esophagus postoperative strictures which developed months after the surgery in the research group which was less than in the control group which saw cases of strictures of ega (? \ , ). months after surgery, the number of post-operative strictures increased in both groups, but was lower in the research group and amounted to cases in the research group and cases in the control group (? \ , ). there were cases of esophagus postoperative strictures which developed months after the surgery in the research group which was less than in the control group which saw cases of strictures of ega (? \ , ). neither of the groups had any cases of post-operative mortality. purpose: to investigate the prognostic effects and risk factors of the omission and delay of postoperative chemotherapy of ii/iii gastric cancer (gc), with the goal of providing a reference for interventions of related departments. methods: the clinicopathological data of patients undergoing radical gastrectomy for ii/iii gc were collected and retrospectively analyzed. we defined the chemotherapy delayed until more than days after radical gastrectomy and the complete omission of chemotherapy as unacceptable chemotherapy initiation (uac group), while the chemotherapy conducted within days of radical gastrectomy was defined as acceptable chemotherapy initiation (ac group). the survival between the two groups was compared, and the trends and risk factors of uac were analyzed. results: the total number of patients who underwent totally laparoscopic distal gastrectomy with uncut roux-en-y and delta shaped billroth-i anastomosis was and , respectively. the mean reconstruction time was longer in uncut roux-en-y than in delta shaped billroth-i, ( . ± . vs. . ± . min, p \ . ). the uncut roux-en-y was used more cartridge than delta shaped billroth-i anastomosis ( . ± . vs. . ± . , p \ . ). however there was no significant differences in operation time, estimate blood loss, number of retrieved lymph node and postoperative course between reconstruction methods. postoperative complications more than clavien-dindo grade iiia occurred in cases ( . %) of postoperative early complications and cases ( . %) of late complications. the endoscopic findings showed excellent short and long-term outcomes in terms of very low incidence of bile reflux and reflux-induced remnant gastritis in uncut roux-en-y compared with delta shaped billroth-i anastomosis. conclusions: uncut roux-en-y gastrojejunostomy was a useful reconstruction method with totally laparoscopic distal gastrectomy for cancer, especially for diverting enteral contents from the remnant stomach and preventing remnant gastritis. therefore, it is recommended for young patients with early stage disease who have a long time to live after distal gastrectomy for cancer. operative technique: the seromuscular layer above the tumor is dissected, while the mucosa is kept unbroken. when seromuscular layer is dissected all around the tumor, the full layer is lifted, and the mucosa is stretched. the mucosa is then transected with a stapling device to execute fullthickness resection of the specimen. finally, the seromuscular defect is repaired by hand-sewn suture. results: since december , clean-net has been performed in patients with gastric smts. all tumors were resected en-blocwithout rupture. the average operation time ranged from to min with an average of . min. the postoperative course was uneventful. microscopically the surgical margin was tumor-negative (r resection) in all cases. the margin width was small with an average of . mm ± . . conclusions: clean-net is a useful option in the laparoscopic surgical treatment of gastric smt, when excessive sacrifice of the healthy gastric wall surrounding the endophytic tumor should be avoided. background: the type of fundoplication-complete or partial is still controversial for the surgical treatment of gerd. laparoscopic toupet ( wrap) fundoplication has less post op dysphagia and gas bloating compared to nissen fundoplication ( wrap) and is advised to be the procedure of choice when esophageal manometry findings are abnormal, however it is considered by some less effective and more difficult to perform. the aim of this research was to determine in the functionality and efficacy of the different types of fundoplication. methods: explanted pigs stomachs weighing - kg were studied. two different studies of the les were performed: distensibility and failure point (occurrence of reflux according to volume added to the stomach). for both studies we first disrupted the lower esophageal sphincter using a rigiflex tm dilating balloon. we then performed three different fundoplications-nissen, toupet, dor and measured the distensibility of the egj after each fundoplication. the failure point was determined following each fundoplication type. results: we used pig stomachs for the distensibility study and pig stomachs for the failure point study. there was no statistically significant difference between the nissen and toupet fundoplications when distensibility was measured, however the egj was more distensible following dor fundoplication (p = . for nissen, . for toupet). when the failure point was measured, nissen fundoplication was significantly more effective than toupet, and toupet was significantly more effective than dor (p = . ,p = . respectively) conclusions: we studied the differences between the mechanical effects on the egj following three different fundoplications, encompassing , , and of the esophagus. we demonstrated that there is a significant difference between dor fundoplication and nissen/toupet when distensibility was measured. there was no difference in the distensibility of the egj following a or wrap. there was, however a significant difference of effectiveness between all three fundoplications. these findings suggest that the and fundoplications have similar functionality while the wrap mechanically prevents possible reflux and support proponents of toupet fundoplication rather than nissen due to the similar functional results while decreasing the post op dysphagia and gas bloating complications. surg endosc ( ) aim: to describe patients undergoing surgical treatment of incident gastro-oesophageal reflux disease and the use of anti-reflux treatment in a danish population-based cohort. methods: all adult danes - undergoing upper endoscopy and receiving a diagnosis of gerd within days were identified. patients with previously diagnosed gerd, peptic ulcer-disease, barrett's oesophagus or cancer of the gastrointestinal tract were excluded. in this study, only patients undergoing anti-reflux surgery within two years of gerd-diagnosis were subsequently included. age, sex, charlson comorbidity index (cci), anti-reflux surgery (primary and re-operative) and endoscopic dilatation were identified using the danish national patient registry. mortality was identified using the national civil registry. pharmacological treatment of gerd (proton pump inhibitors, h \ su \/su-blockers and other prescription anti-reflux drugs) as well as use of nonsteroid anti-inflammatory drugs (nsaid) and anti-thrombotic treatment were identified using thethe danish national prescription registry. all data was linked on an individual level using the unique identification number that all danish citizen are assigned to at birth or first immigration. results: a total of first-time fundoplications were performed, hereof . % performed laparoscopically (n = ) and . % performed using open technique (n = ). at one-year followup, . % (n = ) had undergone endoscopic dilatation and . % (n = ) had undergone reoperation. the -day mortality was \ . %. patients had a median age of years ( - years) and were predominately male ( . %-n = ). a total of . % had cci (n = ). diagnoses were gerd with esophagitis ( . %-n = ), gerd without esophagitis ( . %, n = ) and gerd without specification ( . %, n = ). before initial endoscopy, , % (n = ) used at least one type of anti-reflux drug, dropping to . % (n = ) in the year after anti-reflux surgery. however, even when censoring patients with barrett's esophagus or peptic ulcer disease after initial endoscopy and patients undergoing concomitant treatment with nsaids or antithrombotic drugs, . % still used at least one type of anti-reflux drug after surgery. conclusion: in this population-based study, anti-reflux surgery was safe and lowered the use of pharmacological treatment. however, even when adjusting for competing reasons for use of antireflux drugs, . % used at least one type of anti-reflux drug one year after surgery. the new approach to perform nissen fundiplication m. paranyak, v. grubnyk surgery, odessa national medical university, odessa, ukraine nearly % of patients who undergo laparoscopic anti-reflux surgery at long-term follow-up need for surgical reintervention mostly because of hiatal hernia (hh) recurrence, wrap migration or disruption. purpose: the aim of our prospective study was to evaluate and compare several technics of wrap fixation and determine whether modified nissen fundoplication(mnf) reduce failure rate in the long term follow up. materials and methods: this was a prospective, randomized, controlled trial. from november to october one hundred and thirty-eight gerd patients who underwent anti-reflux surgery were divided into two groups. excluded criteria for our study ware diagnosed hiatal hernia (hh) type iii. in the i group which include patients we performed the following manipulations: nf was supplemented with suturing wrap to the diaphragmatic crura ( patients) on each side using two non-absorbable stitches. such technique permit us to create more symmetrical wrap. in case of weak conditions of crura or short esophagus ( patients) fundoplication wrap was sutured to the body of stomach using two non-absorbable stitches on each side. control group ( patients) underwent classic nissen fundoplication (nf) without wrap fixation. all patients were assessed before and after surgery using validated symptoms and quality of life (gerd-hrql) questionnaires, -h impedance-ph monitoring and barium-swallow. results: baseline characteristics were similar between groups. there were no conversion to open procedure or mortality. mean hospitalization was . days ± . days. at , months (range -- ) of followup, the overall rate of complications after mnf was , % ( hh reccurence) and nf , % ( hh reccurence, slipped wrap). patient in mnf group show significant improvement in gerd-hrql score, from . ± . (preoperatively) to . ± . (postoperatively) (p? \ ? . ). complete ppi independence was achieved in %. in the ii group of patients mean gerd-hrql score decline from . ± . (preoperatively) to . ± . (postoperatively), postoperative ppi treatment was necessary in %. conclusions: according to our study mnf minimized risk of slipped wrap and intrathoracic migration of the wrap and can make positive impact on reducing the failure rate of laparoscopic anti-reflux surgery. aims: comparative evidence across laparoscopic antireflux procedures does not exist. aim of this project was to identify direct comparative evidence between laparoscopic antireflux procedures and synthesize evidence using network meta-analytical methods. methods: the databases of medline, amed, central, opengrey were interrogated. pairwise meta-analyses for each pair of interventions using a random-effects model and network metaanalysis in stata was performed using the mvmetacommand and self-programmed stata routines. differences between direct and indirect evidence were explored by comparing direct and indirect estimates though computing the inconsistency factor within each closed loop of evidence. the ranking probabilities for all treatments of being at each possible rank for each intervention were computed using the mvmetacommand in stata. a hierarchy of the competing interventions was obtained using rankograms. quality of evidence was assessed using grade-nma and the cinema application. results: forty-three publications reporting on randomized trials and some patients were identified. the network of treatments formed a closed loop between °, °and anterior °; and star network between °and other treatments; and between anterior °and other treatments. laparoscopic °, °, anterior °and anterior °were equally effective in the control of heartburn and this was supported by low quality of evidence according to grade-nma. the odds for dysphagia were lower for anterior °(high quality evidence), anterior °( moderate quality evidence), °(moderate quality evidence) and proton-pump inhibitors (moderate quality evidence) compared to °. the odds for gas-bloat were lower for °and anterior °compared to °(low quality evidence). the odds for regurgitation, morbidity and reoperation were similar across treatments, albeit these were associated with very low quality evidence. anterior °had a % probability of being the best treatment in terms of dysphagia. conclusion: under consideration of treatment effect estimates, evidence quality as assessed with grade-nma and other parameters, anterior °, anterior °and °should be preferred over °. further research needs to focus on the comparison between °and °/ °. aims: we have recently demonstrated that the tension of crural closure can be reliably measured intraoperatively (alsgbi conference december ). the aims of this study were to further characterise tension at the diaphragmatic hiatus from our prospective pilot study of patients. methods: a prospective analysis was performed of patients undergoing laparoscopic hiatal hernia repair between april and december . patients underwent crural tension measurement intra-operatively. patients had a pre-operative ct scan of the abdomen within one-year of surgery. hiatal surface area (hsa) was measured intraoperatively and a sauter-fh universal digital force gauge was used to measure the tension of crural closure during cruroplasty. outcome measures included the mean tension of the crural closure and the presence of muscle splitting during the cruroplasty. results: for all patients, the mean crural tension measurement was . n and the mean hsa was mm . pre-operative ct was positively correlated with post-dissection intra-operative hsa (r = . , p = . ), however, strength of association was weak (r = . ) and ct consistently overestimated the size of hiatal defect intra-operatively (mean of differences mm , p = . ). crural tension was positively correlated with age (r = . , p = . ), hiatal height (r = . , p \ . ), hiatal width (r = . , p \ . ) and hsa (r = . , p \ . ). crural tension was correlated to the hiatal width to height ratio to assess the shape of defect and there was positive correlation (r = . , p = . ). tension was calculated for the posterior and anterior halves of the suture cruroplasty. anterior tension was significantly higher when compared to posterior tension ( . n vs . n, p \ . ) . patients had evidence of muscle splitting during the cruroplasty. the group with muscle splitting were significantly older ( vs , p = . ), had larger hsa ( mm vs mm , p \ . ) and higher crural tension ( . n vs . n, p \ . ). the lowest observed mean crural closure tension causing muscle splitting was . n. conclusion: there is now a possibility to optimise this operation with objective measures years after it was first described. initial findings suggest that crural closure up to * n could be the permissible tension threshold for suture cruroplasty and higher tension may benefit from the use of mesh reinforcement. background: endoscopic submucosal dissection (esd) and endoscopic full thickness resection (eftr) are advanced endoscopic techniques which can be time consuming using traditional endoscopic instruments. a new endosurgery platform, designed by fortimedix surgical, was developed featuring flexible articulating instruments to use in combination with a standard flexible endoscope. the platform is intended to perform endoscopic cutting, dissecting, and hemostasis. aim: evaluate feasibility of the platform in the upper gi-tract. project description: the platform was tested in a dry esophageal model as well as a second series with a porcine esophagus and stomach. the system has an external docking station affixed to the operative table to stabilize both flexible instruments for the right and left hand of the surgeon. at the tip of the endoscope, a cap containing instrument lumens is attached to allow advancing and removing the flexible instruments. the endoscope with the cap and instrument lumens attached is advanced via an overtube with outer diameter . mm. in the first series, flexibility and range of motion of the endeffectors was assessed. additionally, the ability to advance the instruments to the intraluminal target area from the docking station and along the scope was evaluated. in the second series, the functional capabilities of the system and instruments were evaluated in a porcine model. preliminary results: : in the dry model, the platform was adequately deployed to the target then range of motion was tested as well as cutting and grasping gastric wall with instrumetn triangulation achieved. the grasping forceps provided enough force to pull the mucosal wall and expose the dissection plane. in the pig model, the distal esophagus and stomach could successfully be accessed and platform deployed. esd was performed using newly designed flexible articulating scissors, dissection-hook, and graspers with good triangulation and sufficient grasping force with traction/counter-traction. the new fortimedix surgical endo-surgery platform applied to a standard flexible endoscope is feasible to perform esd. future studies are planned to determine learning curve and compare it to traditional endoscopic instruments. background: in laparoscopic surgery, we usually observe the organs in the same direction to avoid a mirror-image situation. therefore, we are unable to recognize how far the dissection has proceeded on the other side of the target organs or lesions, especially when the plane of dissection is under the mesentery or organs. this becomes a problem not to understand how far the dissection has progressed and how much more dissection is needed. aim: to solve this problem, we developed a laparoscopic device with tip illumination. project description: the device is configured by the long and narrow part made of polycarbonate resin and a battery-powered light-emitting diode to illuminate the tip by shining light through the polycarbonate resin. during the surgery, the tip of the device is inserted into the deepest part of the dissection area, and the transmitted light indicates how far the dissection has progressed. the tip of the device has a prism structure and light is emitted in a direction perpendicular to its axis. tip position can thus be more clearly identified even with insertion in the same direction as the laparoscopic view. to verify the utility of this instrument, laparoscopic surgeries were performed in a porcine model and cadavers. preliminary results: we performed some laparoscopic surgery such as the medial-to-lateral approach to the white line of the left side of the descending colon for sigmoidectomy, dissection of the posterior surface of the pancreas to the upper edge of the pancreatic body or splenic artery for distal pancreatectomy, and the separation of the anterior surface of the inferior vena cava from the liver to the area between the right and middle hepatic vein for right hepatectomy. we quickly and easily identified the deepest part of the dissection area even if identification had been difficult using other techniques such as placing gauze in the deepest position, inserting forceps into the dissection area or simply depending on the experience of the operator. background: recent advancements within surgery have seen artificial intelligence transform traditional approaches. robotic assistive devices have demonstrated particular success, as safe and cost effective, and are widely supported via industry and local government as a step closer to the future standard of practice. an example of seamless and touchless robotic assistive technology is based on touchless and interactive eye tracker glasses worn by the surgical team thereby enabling the team to perform wider surgical tasks, more efficiently and reduced human error. we introduce a perceptually-enabled, smart operating room (smart-or) based on a novel real-time framework for theatre-wide d gaze localisation in a mobile fashion. this framework enables dynamic gaze based user interaction with a robotic scrub nurse to facilitate meaningful practical integration of human and technology intra-opertively. aims: we tested participant acceptability of a novel robotic scrub nurse during simulated surgery. project description: surgeons performed segmental resection of pig colon and handsewn end-to-end anastomosis while wearing eye-tracking glasses to select surgical instruments on a screen. the robotic scrub nurse(rn) picked up and transferred the instrument to the surgeon. the study compared human nurse(hn) vs rn. gaze-screen interaction was based on a d gaze framework we developed with synergy of conventional wearable eye-tracking, motion capture system and fixed in space rgb-d cameras for real-time d reconstruction of the environment. nasa-tlx and van der laan's technology acceptance questionnaires were collected and analysed using anova. preliminary results: overall, teams of surgeons(st) and scrub nurses(sn) participated. nasa tlx feedback for st and sn revealed no significant difference between in mental, physical or temporal demand. importantly, st and sn reported no significant difference in task overall performance. st reported more significant frustration with rn vs hn. van der laan's scores showed positive usefulness and satisfaction scores in using the rn platform. overall, all outcomes were more positive by sn vs rn. conclusions: this is the first platform of its kind. overall, quantitative and qualitative feedback was positive. the source of frustration has been understood and we believe it can be improved by appropriately modifying robot behaviour. importantly,there was no difference on perception of performance. background: endoscopic tumor resections in the gi tract may be facilitated by more advanced instruments for dissecting and suturing. we have focused on developing an endoscopic suturing technique using a standard flexible pediatric endoscope with new, flexible instruments allowing for complex end-effector movements. aim: perform flexible endoscopic suturing using a standard flexible scope in the gi tract project description: a standard flexible pediatric endoscope and a standard gastroscope were used for testing the new technique. via an overtube, the endoscope and newly designed fortimedix surgical flexible instruments (needle holder; grasper) with a diameter of mm were inserted into the esophagus. suture training was performed in an experimental setting in a box in the dry lab and porcine model . the flexible needle holder was advanced into the esophagus next to the scope, and a suture of the esophageal wall was performed, followed by extracorporeal knot-tying with knots. the test series consisted of training with both resident trainees and surgeons to evaluate the learning curve. each participant performed sutures on the box model and in the pig-esophagus. feasibility, duration of the different steps, and handling problems were documented. preliminary results: test series (box training on esophago-gastric explant) with prototype showed good feasibility. suturing was possible in out of attempts. median duration for single bite: min ( - ); knot-tying: min ( ) ( ) ( ) ( ) ( ) ( ) ( ) . test series (training in pig-model) with prototype showed improved feasibility with better flexibility of instrument shaft: median duration of double bite: min ( - ); knottying: min ( ) ( ) ( ) ( ) ( ) , overall duration intraluminal esophageal double bite suture and closing with knots: median duration: min ( - ). the new flexible endosuture instruments seem feasible to use and perform dependable intraluminal sutures. the training period and learning curve is short and the objective is to apply this system clinically for closure of perforations and fistulas. school of mechanical and aerospace engineering, nanyang technological university, singapore, singapore; general surgery, national university hospital, singapore, singapore; gastroenterology, national university hospital, singapore, singapore; surgery, chinese university of hong kong, hong kong, hong kong background: ideally, endoscopic suturing should mimic surgical closure as the latter is stronger than most endoscopic closure devices. however, endoscopic suturing is challenging due to the confined endoluminal space and lack of dexterity of current endoscopic instruments. we have developed a novel robotic suturing device to overcome these problems. aim: this animal study aims to demonstrate the feasibility of this device in closing perforations. method: the trial was conducted on an anaesthetized live pig. a double-channel colonoscope was first inserted into the rectum. following saline lift, a mm submucosal incision was created in the rectum to simulate a perforation. the robotic suturing device and grasper were inserted into the two colonoscope channels, allowing the endoscope to remain in position for tool exchanges or needle reloading. both the effectors were intuitively tele-operated by the user via a robotic master console. this robotic suturing device manipulated a curved, double-point needle (with a cm - vicryl suture) to penetrate tissues at desired orientations. the needle could be switched between both jaws of the device through a locking mechanism. this facilitated passing the needle through tissues to form stitches or through suture loops to form surgical knots. the articulated joints and five degrees of freedom allowed dexterous steering to reach targets and triangulation with other tools in a confined space. the robotic grasper facilitated handling of tissue and suture. result: a total of four running stitches were performed and secured with a surgical knot by passing the needle through suture loops. the suture was cut and the needle was removed by the robotic grasper through the channel. min and min were required to stitch and tie the knot respectively. there was no complication. conclusion: our novel endoscopic robotic device can suture perforations resulting from complex endoscopic procedures. as our suturing method is similar to laparoscopic and robotic suturing, closure using our device is expected to be as strong as a surgical through-and-through closure. when developed further, this device can be used to close full-thickness resection sites and orifices in transluminal endoscopic surgery. modelling a collaborative robot with the ieee sdc standard for combined focused ultrasound and radiation therapy j. berger, m. unger, l. landgraf, a. melzer medical faculty, university hospital leipzig, innovation center computer assisted surgery, leipzig, germany background: surgical robotics require a smooth integration into the operating room (or) . for this propose the ieee sdc(service-oriented device connectivity) standard has been developed in the or.net project. in preparation for a combined focused ultrasound and radiation therapy (fus-rt) we have shown concepts and evaluations to position ultrasound and interventional devices with collaborative kuka arms. however, the safe and intraoperative cooperation with multiple different or-devices (e.g. an irradiation unit) requires a more sophisticated exchange of the robot's information and functionality. aim: to realize a safe clinical integration, the aim of this work is to implement and evaluate a dynamic connection between the kuka robots and other devices using the vendor-independent sdc communication standard. project description: a kuka lbr iiwa r robot (kuka ag, germany) was modeled inside the sdc standard for medical device communication. the interconnection with other devices was implemented and evaluated on a mobile platform to position a clarius l wireless ultrasound transducer (clarius mobile health corp, canada). all necessary information of the robot was represented in the medical device description of the sdc standard to be shared via network. for each joint of the robot arm the position, torque, stiffness, damping, velocity and functional-states were represented, resulting in a total of parameters. the software was implemented in c ?? on a standard pc accessing the kuka controller cabinet with ros (robot operating system) via ethernet. the accessibility of each parameter, as well as activation commands for planning and movement were tested with an sdc-consumer application. preliminary results: the sdc-provider functionality of the robot was successfully implemented, allowing for dynamic changes of the robot state during interventions. all appliances (sdc standard compatible) in the robots network can react to state changes and send movement and planning commands to the robot via activations. after testing, % of the defined parameters are safely accessible. implementing the medical device communication for the kuka robot enables its integration into any networked operation room that supports the sdc standard. it is, therefore, ready to be set up and evaluated for the application of fus-rt in a clinical environment. background: assessment of perfusion of the left colon with fluorescence during anterior resections for cancer changes surgical decisions in up to % of cases. use of fluorescence has been shown to be associated with lower leak rates, and improved short-and long-term outcomes with reduced costs. given the high incidence of colorectal cancer, fluorescence-guided perfusion assessment could be of great importance in contemporary surgical practice. however, there is currently no standardisation of this technique which represents a significant limitation to widespread adoption. aim: to standardise fluorescence-guided perfusion assessment in rectal anterior resection through a computer vision algorithm. project description: videos were collected by a single surgeon in a referral centre for colorectal cancer treatment. perfusion assessment was used before proximal colon division to identify the best location for transection. a bolus of indocyanine green was injected intravenously and a near-infrared camera used to assess perfusion through fluorescence. photographs of fluorescent imaging of the colon were analysed using a non-supervised learning algorithm called 'k-means clustering'. the first step was to digitally subtract all background pixels, leaving only the area of interest of the colon. this area was then subsegmented into 'clusters' corresponding to perfused and nonperfused areas. a mathematical model was applied based on the sub-clusters centres to select the area for transection with optimal perfusion of the proximal colon. preliminary results: representative images of proximal colon under perfusion assessment were presented to expert surgeons. the optimal point for transection was selected based on their clinical judgement on previously delimited areas indicated by random letters. this was compared with the results from the automated segmentation using the algorithm ( fig. ). the area identified for section by the algorithm included the area selected by the expert surgeons in . - % of test cases. these results need to be further validated due to high risk of overfitting. next steps include the collection of multicentre data with a standardised fluorescence perfusion assessment. after robust training, the algorithm will be validated on real-time clinical data to ensure improved outcomes for patients, which is our ultimate goal. background: endoscopic submucosal dissection (esd) is a flexible endoscopic technique that allows for an en bloc removal of lesions of the gastrointestinal (gi) tract. these procedures are typically time consuming due to the difficult control of the tools, and they often require around min for removing lesions, that can reach - cm in diameter. the probability of intestinal perforation exceeds % and the hemorrhage risk ranges from . % to . %. a flexible robotic endoscope may offer a solution to overcome these limitations, by improving the degrees of freedom (dof) and operational efficiency. aim: within this clinical panorama, the aim of this project is presenting the development of a novel miniaturized robotic device to be coupled to the tip of a traditional endoscope for the surgical dissection of gi neoplasms. project description: the robotic platform consists of the miniaturized robot, the actuator housing (hereafter called external platform), the control unit and the master console (i.e.,two geomagic touch phantom) to allow the user driving and control (figure a ). during the operation, one surgeon stands close to the patient to maneuver the endoscope for exploring the gi tract and reaching the target area. another surgeon operates the miniaturized robot through the master console, carrying out the surgical procedure. the robot has been designed to be coupled to the tip of traditional flexible endoscopes of . mm in diameter. it exploits the flexibility of the endoscope for navigation through the intestine and integrates two-active robotic arms (i.e.,cautery and gripper) extending the dofs, and thus enhancing the efficiency during complex tasks such as manipulation and surgical tissue dissection. furthermore, the endoscope provides the optical system for visual feedback and one working channels for conventional instruments. preliminary results: firstly, a mock-up that faithfully reproduces the miniaturized robot has been realized using a d printer machine (projet mjp , d system, inc.) to verify the feasibility of the design solution. after verifying the potentiality of the d printed prototype, a final device, with the same features (i.e.,dof and geometry) of the d printed prototype, has been designed, fabricated and assembled ( figure b ). background: virtual and augmented reality has been widely used in many fields mainly for entertainment purposes. we think that it could be beneficial to use augmented reality in medical practice. aim: the aim of this study was to evaluate usefulness of d holographic images of patients anatomy displayed using augmented reality goggles during endovascular aortic repair (evar). project description: one of the major challenges during endovascular procedures is working on two dimentional x-ray images of three dimentional vascular anatomy. using d holograms of patients anatomy could be beneficial during the evar procedure and could make the orientation in vascular anatomy easier for surgeon. we performed two endovascular aortic repairs with the assists of microsoft hololens -smart glasses using augmented reality. we used carna life application created by polish company medapp. it was one of the first use of holograms during vascular procedures in the world (second and third stent-graft implantation using holographic imaging in the world). results: two patients with abdominal aortic aneurysms, -years old male and -years old female, were operated on. holograms of patient's anatomy made from preoperative angio ct scans by polish company medapp were displayed during the procedures using microsoft hololens. holograms could be displayed in any place and configuration using augmented reality, which means that the images did not interfere with the surgeon's field of vision. microsoft hololens use voice commends which permits the surgeon staying sterile. stent-graft implantations were successful. both patients were discharged three days after the procedure and the hospitalization was uneventful. seeing precise patient's vascular anatomy reconstructions in three dimention certainly helped us to navigate in a vascular tree. we believe that in the future this technology would enable to reduce the operation time and need for radiation. background: interaction with electronically controlled operating room (or) systems embedded in modern surgical environments is everyday practice for surgeons performing minimally invasive surgery (mis). while there is a non-sterile operating nurse available in the or, capable of interacting with these systems upon request by the surgeon, this indirect control is mostly slow, prone for error and disrupting surgical workflow. facing an unanticipated and unwanted outcome may cause distress emotions. distress emotions are undesirable when performing surgery, since they may impact available cognitive workload. furthermore, they may result in negative communication, hampering or-team empowerment and effective leadership. both factors are known to negatively influence quality and safety in the or. aim: the aim of the tedtrial is to investigate what setup best enables surgeons to interact with the endoscopic operating room setup during surgical procedures. as a result, disruptions of workflow, delays and errors may be reduced. outcome parameters will be objectified using medical data recorder (mdr) derived output and biometric analysis using hexoskinÓ. subjective evaluation of outcome parameters is done using questionnaires. project description: the tedcubeÓ system is a plug-and-play device enabling wearable sensors to act as a wireless alternative for a regular computer mouse, therefore enabling direct hands-free and sterile control of the or. the study is an observational trial with three different arms: intervention group ) direct interaction by surgeon with or environment using tedcubeÓ and myo tm armband, intervention group ) direct interaction of surgeon with or environment using tedcubeÓ and plantronicsÓ wireless microphone headset. the third arm is the control group using indirect interaction of surgeon with or environment using third-person computer interaction. main endpoint of study is the number of workflow disruptions due to the operation of laparoscopic or equipment. secondary endpoints are error rate, delay, team communication, subjectively reported frustration and satisfaction with the system and objectively measured stress as symptom of frustration and anger as distress emotions. preliminary results: primary and secondary endpoints of study are compared among groups. it is anticipated that reduction of miscommunication, error and delay may result in a reduction of distress emotions. trial start is expected q . anticipating the automated intraoperative tissue recognition: intraoperative tissue classification using hyperspectral imaging and machine background: iatrogenic injuries may occur despite a sound expertise in surgical anatomy. hyperspectral imaging (hsi) is an emerging optical method, combining the use of a camera system with a spectrometer. hsi analyzes optical properties of tissues and acquires d data sets with two spatial dimensions (x, y) and one spectral dimension (?). the data sets contain information about tissue physiology, composition, and perfusion. those spectral features coupled with machine learning algorithms might allow for automatic tissue recognition. aim: assessing the ability of an hsi-based machine learning to discriminate the hyperspectral features of different tissues during neck and abdominal surgical procedures. methods and procedures: fourteen pigs underwent laparotomy (n = ) or neck dissection (n = ). twenty data sets were acquired in vivo from abdominal organs and from neck structures by means of a customized hyperspectral camera (diaspective vision, germany). different anatomical structures were manually outlined by a surgeon using an image manipulation software (gimp). each pixel contained a hyperspectral curve and each curve was composed of bands (from to nm with a nm resolution). the curves were normalized using the standard normal variate method. a logistic regression machine learning (ml) algorithm was used to train the model to discriminate tissues, based on the hsi spectral features. the efficacy of the prediction model was tested using the k-fold (k = ) cross-validation. results: a large number of tissue-related hyperspectral curves could be extracted ( thyroid, vagal nerve, fatty tissue, cartilage, carotid artery, muscle, carotid vein, portal vein, biliary tract, gallbladder, hepatic artery, pancreas, duodenum, abdominal adipose tissue). the algorithm used min to 'learn' all data sets, and prediction was provided as an immediate output. overall, prediction accuracy was and % for neck and abdominal structures respectively. in particular, biliary ducts could be identified with a % accuracy and the vagal nerve with an % accuracy (see figure for details). background: a gaze-controlled robotic endoscope is innovative technology with myriad potential applications in the rapidly advancing field of flexible endoscopy. improvements to the current flexible device to allow examination of the gastrointestinal tract whilst minimising procedural discomfort and complications are desirable. aim: to use a gaze contingent framework to manipulate a flexible endoscope through a simulated upper gastrointestinal tract (ugit) model. description: a flexible gastroscope (karl storz pks) was attached to a ur axis robotic arm (universal robots), mounted onto a rail and placed on top of a surgical table. two cogwheel shaped dials were d printed and placed onto the up/down and left/right wheels on the head of the gastroscope ( figure ). robotization of these controls was achieved by using two motors (dynamixel rx- f) to steer the distal tip. this system allows users to operate a robotised flexible endoscope using gaze control. gaze interaction with the screen was based on a d gaze framework we developed with the synergy of conventional wearable eye-tracking, motion capture system and fixed in space rgb-d cameras for d reconstruction of the environment. users are able to control endoscope movements without handling the device. the distal tip of the gastroscope was controlled using eye gaze technology. the ur robot was used to enable shaft rotation (initiated by fixed head movements) and linear movements were triggered using a joystick handle (up for forward movement, down for endoscope withdrawal). pause and retroflexion of the endoscope are achieved by moving the joystick left and right respectively. users were asked to navigate an endoscope through an ugit model (chamberlain group) simulating a diagnostic gastroscopy using gaze control and targeting ten points scattered through the stomach. results: four expert endoscopists and one novice used gaze control to successfully navigate a gastroscope through a simulated ugit. all were able to intubate the oesophagus and accurately locate ten targets placed in the fundus, body, antrum and pylorus of the stomach. conclusion: gaze control endoscopy is a feasible concept. it allows ergonomic, user-friendly and intuitive control whilst maintaining the benefits of a flexible endoscope. background: image-guided needle biopsies and histopathological evaluation are the gold standard for the diagnosis of liver neoplasms. most often, however, these are reserved for suspicious, but not diagnostic, situations. radiomic may help to characterize tumor biology by correlating imaging features with relevant tumor-biology information. features derived from radiomic analysis may provide complementary information to support clinical decisions, especially in situations where tissue analysis cannot be performed or is inconclusive. aim: the goal of our technology is to exploit computational capabilities for image analysis in order to identify radiomic features useful for characterizing liver lesions and to identify relevant information related to patient prognosis. project description: patients derived from an internal database and patients randomly extracted from the cancer archive liver dataset were included in this study. lesions were extracted from those volumes using expert annotations ( secondary vs primary; well differentiated vs non-well differentiated). lesions were then split into training and testing sets. first order statistical features were computed and a lasso regression step was performed to reduce the number of features. both logistic regression and random forest models were built using cross-validation to predict the target classes on the test set. preliminary results: only features namely the energy and the volume of the lesion were sufficient, when combined in either model, to predict the differentiation grade on the test set with an f -score of . (± . ). we are currently working on the addition of higher order statistical features to the analysis in order to differentiate primary from metastatic tumors and identify complementary features that may assist clinical decisions in patients with inconclusive hepatic lesions. objective of the technology or deviceideally, the use of medical simulators could provide trainees with initial background information about indications for procedures, endoscopic technique, and early hands-on training experience that could shorten the initial critical learning curve. rationale for using ex vivo models is that in the beginning of the learning curve, the most important issue is having an initial exposure to the basic movements and maneuvers. our objective of is to create a stomach model from renewable polymer, which would closely simulate normal human stomach with gastric pathology for endoscopic diagnostic or interventional skill acquisition/evaluation. description of the technology and method of its use or application stomach model is based in several steps; the first one is in the in-silicodesign of the overall shape, after that we d print the positive two halves of it. the interior detail is obtained shaping the d printer parts with ceramic putty. once concluded, this elaborated part will serve as a template in order to build injection bleeding moulds. in the injection bleeding moulding a mesh is placed between layers in order to provide structural attachment points as stiches or several pathological models that will be incorporated after the casting process. we have developed for these instance polyp moulds, fistulae structures in order to attach endoscopic clamps. the two halves are closed once the pathological models are placed inside via a thermic-fusing and stitching creating a leak proof stomach model. preliminary results if available: our models were evaluated by international experts in ircar/ihu france in interventional endoscopy course and were favorable accepting for next trails in these prestigious institutions. conclusions: future directionsa new endoscopic training model of stomach was made and will be evaluated and validated for feasibility in mastering diagnostic and interventional endoscopic skills. clinical trials will be necessary to compare the ability of the simulator to perform training compared with traditional methods of training in endoscopic procedures. background: endoscopes are the eye of surgeons in minimally invasive surgery (mis). conventional endoscopes are mostly chopstick-like and are steered by the assistant. this limits the field of view and results in issues such as endoscope-instrument fencing, surgeon-assistant coordination. existing robotic endoscope holder enables solo-surgery, however endoscope remains blocking the instrument movement and impairs the operational safety. flexible endoscope such as the endoeye provides angulation at the tip and could enlarge the field of view. however, its steering the view is much more complex compared to the rigid endoscope. aim: to provide an intuitive robotic flexible endoscope with enhanced safety. project description: in this work, we present a robotic flexible endoscope for mis with enhanced safety. in the proof-of-concept system, it contains a flexible endoscope module and a robot manipulator. the endoscope contains a proximal rigid shaft and a distal flexible bending section. it is installed onto the patient side manipulator (psm) of the da vinci research kit (dvrk). visual servoing is adopted to achieve autonomous instruments tracking. during the tracking process, movements of the manipulator as well as the endoscope are minimized to save space for the operation and avoid instrument-endoscope fencing. the endoscope could also be controlled by the surgeon. a foot pedal is used to switch between the tracking-mode and control-mode. preliminary results: a prototype was developed and tested experimentally. in tracking a volume of * * mm , the spaces required by the flexible endoscope are . % (inside the trocar) and . % (outside the trocar) of that occupied by the rigid endoscope. evaluation with the fls tasks involved subjects. all of the participants completed the tasks under the tracking-mode without failure. in the ex-vivo test with porcine stomach, the endoscope successfully guided the detection, dissection and knotting autonomously. background: fluorescence imaging allows to visualize deep-seated anatomical structures, using a deeper tissue penetration of near-infrared (nir) compared to visible light. the most commonly used fluorescent substance, indocyanine green (icg), is not naturally excreted by the urinary system and requires retrograde stent placement and injection. lighted catheters have been proposed to help visualise the ureter. fluorescent dye-coated ureteral catheters could well represent a more effective and less expensive solution. icg is unsuitable for coating materials. aim: to develop a stable fluorescent coating for catheters to be used intraoperatively, working in the same nir window as icg, to facilitate its use with clinically available systems. project description: the coating was developed based on poly(methyl methacrylate) (pmma), a biocompatible polymer, and on specifically designed fluorescent dyes exhibiting icg-like optical properties. three nir dyes (substances a, b, and c) were tested in order to find the optimal one, in terms of fluorescence signal intensity, and were compared to icg in a polymer form and to an icg-based reference card (green balance tm ). the fluorescent coating was applied onto common ureteral stent materials: hydrophilic-coated ultrathaneÒ, silicone-coated latex, and pvc. the coating process involved cycles of immersion into the respective dyes blended in pmma polymer (icg, substances a, b, and c), followed by a drying phase. the various tubes were partly inserted into a porcine ureter, next to the icg-based reference card. images were taken in white light and nir modes using the d-light p camera system (karl storz), at a fixed camera-to-target distance. the fluorescence signal intensity was measured for the different regions of interest (each material/coating combination inside and outside of the ureter, reference card) using proprietary software and normalised against the reference card. preliminary results: the signal intensity was significantly higher for all new substances as compared to icg. substance a showed the strongest fluorescence signal intensity among the tested coatings in all tested conditions and materials and was identified as the ideal candidate to undergo further evaluation and in vivo testing. background: endoscopic resection(er) of early gastric cancers provides tremendous patient advantages. however, post-resection findings of deeper sub-mucosal(sm) and/or lympho-vascular invasion can necessitate a second, surgical intervention. we propose that pre-resection evaluation of the submucosal architecture under the tumour can provide critical information for staging and operative planning. we evaluate three techniques to assess the submucosal architecture underlying the gastric mucosa in a pig model. aim: to evaluate three needle-based methods of evaluating the sm before er. project description: acute pigs were used. a simulation of sub-mucosal tumours (endoscopically and eus visible bleb) by injecting the sm with cc of undyed nac. a linear eus was use for all procedures. the tumours were marked and labelled according to geography. methodology: after creating the tumours, anterior lesions were evaluated using the following g needle-based modalities: confocal microscopy(cm) using the through-the-needle cellvizio (mauna-kea) system; mini-biopsy(mb) using the micro-biopsy forceps moray (us endoscopy) and fine-needle biopsy(fnb). results: cm examinations were video recorded in all a positions. submucosal vascular visualisation was possible in all cases, excellent in / . mb was performed in lesions with a total of biopsies obtained from each lesion (total = ). fnb was performed once in the anterior lesions and twice in the posterior lesions with different needle brands. therefore, there was a total of biopsies collected. passes were performed in each biopsy (total = ). each pass constituted - insertion/withdrawal movements combined with fanning, slow pull technique, no suction and suction ( - cc air negative pressure) to collect the material. all material were sent to an animal anatomo-pathologist blinded to the acquisition method. mean time of confocal examination was min sec ( ' ' '- ' '') . mbtook a mean time of min and fnb was a mean of min for each biopsy. cm identified different patterns of vessels in relation to the probe position (superficial/reticular, middle cross-roads or deep/longitudinal). conclusion: eus-fnb, cm and mb are three potential methods to assess the sub-mucosal space underlying the gastric mucosa. cm offered the most architectural information but required more time to perform. these method's may have a role in better staging patients for appropriate er. background: the overall and disease-free survival of patients with rectal cancer is dependant on its staging, and adequate selection of the treatment strategy. mri has a proven efficacy in rectal cancer local staging and recognition of the adverse prognostic features. however, it can be difficult to utilise it as a navigation tool for surgeons, as it represents a complex three-dimensional pelvic space with a series of individual two-dimensional images. d image reconstruction has been successfully adopted in other surgical fields to overcome these limitations. aim: our primary aim is to develop a bespoke automated generation of patient-specific d pelvic models, which will improve surgical planning and navigation, patient interaction and surgical education. true-size, rotatable d models will offer a more realistic three-dimensional representation of the surgical space and its complex relationships, allowing for a more confident surgical rehearsal and potentially better utilisation of minimally invasive techniques in rectal cancer management. our secondary aim is to develop a large multipurpose database of the d models of male and female pelvis in health and in the disease. project description: our multidisciplinary team consists of colorectal surgeons, radiologists specialising in pelvic mri imaging and computer scientists. virtual d pelvic models are generated based on standard d dicom mri images routinely used for rectal cancer staging, which guarantees the high fidelity of cancer delineation. segmentation of the pelvic anatomy is performed with the use of itk-snap, an open-access, multi-platform software. machine learning technology is then employed to automate the d model generation, making it time-efficient, allowing for its clinical application. preliminary results: in the initial stage, using the manual segmentation, we have created ten models of normal male and female pelvic anatomy. a good inter-rater agreement level was found, which proves reproducibility of the approach applied. various machine learning algorithms are being explored to fully automate the process of d model generation, which will allow for their use in clinical practice and in development of the d colorectal database. the technology will be further implemented in creation of dynamic models of functional pelvic floor disorders. surgery, toho university omori medical center, tokyo, japan; surgery, neuchâtel hospital, neuch tel, switzerland background: laparoscopic gastrojejunostomies are time-consuming and require a specific training. alternatively, sutureless anastomosis can be achieved by means of endoscopically delivered magnetic rings. objective of the study: assessing the feasibility and reproducibility of an endo-laparoscopic gastrojejunostomy technique, using magnets coated with a fluorescent biocompatible polymer. methods and procedures: four pigs ( acute, survival models) and one cadaver were included in this study. the anastomotic device was composed of two magnetic rings ( x x mm; attraction force newton), each one attached to a cm long thread. the distal ring was inserted endoscopically into the first duodenum, and the extremity of the thread was clipped to the gastric mucosa. twenty-four hours later, a two-port laparoscopy ( mm, mm) was performed, using a near-infrared (nir) laparoscope (d-light-p; karl storz). the magnet's position in the jejunum was detected thanks to the transluminal fluorescence of the dye. magnetic interaction with the metallic tip of the laparoscopic grasper allowed to catch the ring and bring the bowel loop to the future anastomotic site on the gastric wall. simultaneously, the proximal magnet was delivered to the gastroesophageal junction using a flexible endoscope. the magnet was carefully advanced into the stomach allowing precise connection with the distal ring. in one cadaver the procedure was repeated. the sole variation was that, in order to reach the second jejunal loop, the distal magnet was placed using a gastroscope inserted through a transgastric port. in two acute animals, the distal magnetic ring was introduced into the jejunum via an enterotomy. the anastomotic procedure (from the distal magnet detection via fluorescence to the magnetic connection using a hybrid approach) was reiterated times. survival animals were followedup for days and underwent control endoscopies and ct-scans. results: the procedure was easy to standardize and reproducible, with a mean anastomotic procedure time of . ± . min. there were no technical problems and magnetic connection could be precisely directed in all cases, at both the anterior and posterior gastric wall. no complications occurred during the survival period and the anastomoses were patent by day . transluminal fluorescence allowed for a rapid detection of the magnet. colorectal cancer is the fourth most common cancer in high-income countries counting [ . deaths worldwide. survival rate reaches % in case of early diagnosis, falling down to % in case of advanced stage. conventional colonoscopy screening is limited by invasiveness, pain and often need of sedation. wireless capsule endoscopy enables inspection without discomfort, but passive locomotion often leads to incomplete and/or false negative results. the european endoo project (grant agreement ) aims to develop a novel system that overcomes most of the drawbacks of conventional colonoscopy, maintaining accurate and reliable diagnosis and therapy. the system is composed of an active robotic platform that magnetically drives a soft-tethered capsule; magnetic guidance is achieved through the magnetic localization of the capsule in combination with a closed-loop control that maintains an optimal and safe link between the capsule and the magnetic end-effector. a stereoscopic camera is integrated in the capsule for enhanced diagnosis though d reconstruction and automated detection of lesions/pathologies. the different modules of the endoo medical platform are illustrated in the figures. the robotic guidance systemconsists of an anthropomorphic manipulator that controls the capsule through an external permanent magnet. the robot, positioned on a dedicated trolley, is equipped with sensors for performing safe human-robot collaboration. the medical workstationincorporates: screens, buttons and pedals for visualization and command initiation, a joystick for system teleoperation and a back-end for fluidic control and data communication. the soft-tethered capsuleembeds an internal permanent magnet, magnetic sensors, an accelerometer, white and infrared illumination and an hd stereoscopic vision system with two wide-angle customized optics. a controller serves as the main control unitfor performing real-time communication and closed-loop control of the robot, localization system, capsule and physician commands. the synergistic cooperation of academic, industrial and clinical partners within the project allowed to develop and validate the system in in-vitro \/i [ , exvivoand preliminary cadaver sessions, performing comparisons with state-ofthe-art commercial colonoscopes. in conclusion, the endoo medical platform provides: reduced procedural pressures, user-friendly procedures, similar functionalities and performances of commercial devices, comparable procedural times and considerably lower costs with a new painless approach. background: this study is aimed at the comparison of the process of manual and robotic-assisted positioning of the electrode performing radiofrequency ablation with the usage of multifunctional robot-assisted surgical platform. under the control of the surgical navigation system. the main hypothesis of this experiment was that the use of a collaborative manipulator will allow to position the active part of the electrode relative to the center of the tumor more accurately and from the first attempt. we also check the stability of the electrode's velocity during insertion and consider some advantages in ergonomics using the robotic manipulator. methods: sphere-shaped tumor phantoms measuring mm in diameter were filled with contrast and inserted in cow livers. livers were used for the robotic experiment and an equal quantity for manual. the livers were encased in silicone phantoms. analysis of ct data gave the opportunity to find the entry and the target point for each tumor phantom. this data was loaded into the surgical navigation system that was used to track and record the position of the rf-electrode during the operation for further analysis. results: standard deviation of points from the programmed linear trajectory totaled in the average . mm for the robotic experiment and . mm for the manual operation with a maximum deviation of . mm and . mm respectively. standard deviation from the target point was . mm for the collaborative method and . mm for manual method. the average velocity was . mm/s for the manipulator and . mm/s for the manual method, but the standard deviation of the velocity relative to the value of the average velocity was . mm/s and . mm/s respectively.thus, in two criteria out of three, the manipulator is superior to the surgeon, and equality is established in one. surgeons also noticed advantages in ergonomics performing the procedure using the manipulator. conclusions: this experiment was produced as part of the work on the developing of the robotic multifunctional surgical complex. we can confirm the potential advantages of using robotic manipulators for minimally invasive surgery in case of collaborative practice for cancer treatment. surg endosc ( ) background and aims: laparoscopy has reduced tactile feedback compared to open surgery. in neuropsychological literature there is increasing evidence that visual and haptic information converge to form a mental representation of an object. through the combination of these inputs, this representation is believed to be more refined and robust. we investigated whether tactile exploration of a lifelike anatomical object before executing a laparoscopic action on this object in a laparoscopic box trainer improves performance of this action. description: a randomized prospective cohort study with two groups (a ? b) of ten laparoscopically naïve medical students was conducted. we compared the groups for baseline characteristics and performance, using a basic laparoscopic task (post and sleeve). to investigate the effect of haptic exploration, students performed ten repetitions of a laparoscopic needle action on a lifelike silicone caecum model (applied medical, rancho santa margerita, usa). group a did a pre-test visual exploration of the model. in group b manual exploration of the anatomical model was added to the visual exploration before executing the task. the box trainer was equipped with the forcesense tm (medishield, delft, the netherlands) system for skill assessment using objective force, motion and time parameters. results: baseline characteristics and-laparoscopic performance were comparable (p [ , ) . performances of trials on the anatomical model were captured and parameter outcomes were compared between groups. significantly less force (maximal force, maximal impulse, mean force and force volume) was exerted by the 'touch' group (p \ . ) (fig. ). this group also completed the task with less distance travelled by the instruments (p \ , ). there was no significant difference in time needed to complete the task (p = , ). conclusion: this study showed that, when performing a laparoscopic task on an anatomical model, pre-task haptic exploration of the model results in the use of significantly less force and less movement. adding haptic exploration to a laparoscopic training curriculum could therefore result in more efficient and more refined learning of laparoscopic actions. this, in turn, could lead to better, quicker and safer performance of laparoscopic operations. . esophagogastroscopy was performed before gabe and -week post-procedure assessing gastric abnormalities. weight and fasting plasma ghrelin were obtained at baseline, -, -, -and -months post-index procedure. after months, the sham group was unblinded and received gabe. both gabe and sham crossover to gabe groups were followed for months and received lifestyle therapy (behavioral-diet education). preliminary results: gabe was successful in all patients with no serious complications. significant, progressive weight loss was observed at and maintained at months. ghrelin in gabe group decreased by % ( . pg/ml) compared to baseline and months levels. weight-loss was approximately . % greater in the gabe group versus sham at months ( table ) . itt = intent-to-treat, pp = per-protocol analysis preformed using independent-sample t-test and à paired-sample t-test conclusions: gabe using eles is safe, accompanied by significant and so far maintainable weight loss. gabe using the eles demonstrated a reduction in ghrelin levels. aims: transanal total mesorectal excision (tatme) is the latest colorectal approach that continues to be in the spotlight. this study aims to describe the technique in depth by identifying and understanding technical advantages, errors and adverse events. methods: detailed video analysis using observational clinical human reliability analysis (ochra) was completed on clinical tatme cases performed by international surgeons. error frequency and error pathways leading to adverse events were described. tatme expert surgeons were interviewed and engaged in a workshop to elicit error-reducing mechanisms. results: overall technical errors and adverse events per procedure on average occurred ± . (range -- ) and ± . (range - ) times respectively. inadequate insufflation and poor camera optics were the most frequent set-up problems. instrument handling errors consisted most commonly of excessive grasper movement during the pursestring phase ( times total), inappropriate force applied ( times) with the energy device during the rectotomy, inappropriate force with the grasper ( times) and excessive movement with the energy device ( times) during tme dissection. incorrect dissection planes were created during tme dissection mostly due to insufficient retraction ( times) which didn't allow adequate exposure of the tissue planes. the most frequently occurring consequence was bleeding (mean: times per procedure). rectal perforation ( cases), vaginal wall injury ( cases), and prostatic injury ( cases) were also recorded. adverse events regularly occurred as a result of poor set-up/exposure, inappropriate retraction and/or instrument movement and incorrect plane surgery. error-reducing mechanisms and 'technical tips' describe specific steps and actions, both set-up/equipmentrelated and technique-related, that aim to prevent errors from occurring and avoid adverse consequences. ochra and individual feedback with error-reducing mechanisms developed by this study have been implemented into the national training programme for tatme. conclusion: tatme is an advanced complex procedure during which technical errors and their consequences are not infrequent. tatme requires knowledge of anatomy 'bottom-up', familiarity with its specialised equipment and technical skill working in a narrow space. appropriate structured training and mentorship are therefore recommended. surg endosc ( ) objective: insufficient vascular supply is one of the main causes of anastomotic leak in colorectal surgery. icg has been shown to provide information on tissue perfusion, identifying a well-perfused location for colonic and rectal transections and thus possibly reducing the leak rate. objective of this study is to evaluate the usefulness of intraoperative assessment of anastomotic perfusion using intraoperative indocyanine-green dye (icg) angiography in patients undergoing left-sided colon or rectal resection with colorectal anastomosis. methods: this randomized trial involved patients undergoing laparoscopic left-sided colon and rectal resection randomized : to intraoperative icg or to subjective visual evaluation of the bowel perfusion without icg (clinicaltrials.gov nct ). the primary aim was to assess whether icg angiography could lead to a reduction in anastomotic leak rate. secondary outcomes were possible changes in the surgical strategy and postoperative morbidity. results: after randomization, patients were excluded. accordingly, patients were included in the analysis; in the study group, and in the control group. icg angiography showed insufficient perfusion of the colic stump, which led to extended bowel resection, in cases ( %). an anastomotic leak developed in patients ( %) in the control group and in patients ( %) in the study group (p = n.s.). conclusion: intraoperative icg fluorescent angiography can effectively assess vascularization of the colic stump and anastomosis in patients undergoing colorectal resection. this method led to further proximal bowel resection in cases, however its role in reducing anastomotic leak rate should be studied in further research. endoscopic sleeve gastroplasty (esg) is a promising endoscopic bariatric procedure carried out with the application of transmural sutures resulting in a gastric reduction and gastric shortening. sutures are placed in u shape fashion, from the incisura to the fundus, which is preserved, using an over the endoscope suturing platform (overstitch, apollo endosurgery, austin, texas, usa). the choice of right lankmarks for suturing the gastric wall is extremely important for the efficacy and safety of the procedure. flexible endoscopy suffers from little anatomical reference points. correct spatial relation to precisely target the insertion of the helix device used for retraction and correct orientation of the full thickhness tissue bite require a good undrestanding of the anatomy of the stomach and sourrounding organs including vascular structures that could be inadvertently injured (left lobe of the liver, gallbladder, spleen, short gastric vessels, pancreas, transverse colon). surgeons by training can 'see' the anatomy beyond the gastric wall and undrestand whether they work in a safe layer or whether an underlying structure should be spared. this video illustrates all the potential risks realted with a wrong chioce of endoscopic landmarks when performing esg with respect to gastric and abdominal anatomy. introduction: central bisectionectomy, anterior sectionectomy, and posterior sectionectomy are technically demanding procedures in minimally invasive approach because of difficult expoure and extensive parenchymal transection planes. with limited robotic instruments including absence of cusa, these procedures have been rarely perfomed by robotic approach. method: consecutive robotic central bisectionectomy, anterior sectionectomy, and posterior sectionectomy were performed. patients were all males and were , , and -years-old, respectively. pathologic diagnoses were all hepatocellular carcinomas of each . , . , and . cm diameter. operative settings were identical for the three kinds of procedure. the patients were placed in supine with a reverse trendelenburg and right side elevation. umbilical -mm camera port, three -mm ports and additional -mm assistant port were used. glissonian approach and icg fluorescence image clearly demarcated the resection planes. parenchymal transection was performed using the maryland bipolar dissector and harmonic scalpel. the rubber band self-retraction method and third arm of robot system helped for stable and excellent exposure of surgical planes result: there were no conversions to laparoscopic or open surgery. the operative time was , , and min and estimated intraoperative blood loss was , , and ml. the pathologic surgical margin was . , . , and . cm. the length of stay after surgery was , , and days and there were no postoperative complications. conclusion: robotic central bisectionectomy, anterior sectionectomy, and posterior sectionectomy are still demanding procedures with long operative time. however, these procedures could be performed safely in regard to short-term perioperative outcomes. robot surgical system provided several benefits for anatomical hepatectomies including a stable and excellent operative field and clear surgical planes. suprapubic hernias (less than cm above the pubic arch in the midline) require important anatomical knowledge because of complexity of their repair and low incidence, by approximately % of all hernias. the problem to repair this type of hernias is that inferior margin of the defect is very close to pubic symphysis, consequently, mesh overlap is often inadequate. treatment of suprapubic hernias is controversial because of limited evidence in the literatura. this video shows the case of a -year-old female patient with suprapubic hernia with a defect of x cm. we performed a laparoscopic repair with a bilateral peritoneal flap of the groin region (as it is perfromed during tapp) for proper view of the pubic symphysis, cooper's ligaments, epigastric and major vessels, nerves and meticulous dissection the space of retzius. the defect was repaired by reconstructing the middle line with a running sutures. subsequently, titanium helical tacks were used to fix the mesh to the pubis and cooper and following the double-crown technique having special attention when fixing the mesh near to inguinal chanal, due to the possibility of causing chronic pain. the peritoneal flap was fixed over the mesh with abdsorbable fixation devices and seal with fibrin glue. laparoscopic repair of suprapubic hernias can be considered as the first option in treatment, because it endeavors to join the advantages of a minimally invasive approach and it is associated to low recurrence. the main advantages are that allows a proper visualization the anatomy and a proper fixation of the mesh. background and aim: thoracoscopic esophagectomy has been performed for two decades and becomes widely spread. we evaluate our cases who undergone the thoracoscopic esophagectomy and consider the future prospective of this operation.transient recurrent laryngeal nerve palsy after lymphadenectomy in this surgery is not rare and induces not only hoarseness but also aspiration or pneumoniae. new method to avoid this complication is desired. patients and methods: patients who received thoracoscopic esophagectomy in our institute from march to october were enrolled and studied retrospectively. operative indication is an all of the clinically resectable cases including with a neoadjuvant treatment or definitive chemoradiotherapy before surgery. overall survival rate of the patients with thoracoscopic approach and with thoracotomy until was analyzed. long term outcome of the patients with thoracoscopic esophagectomy was compared to the result from comprehensive registry of esophageal cancer in japan. short term results of the perioperative parameters were analyzed between left lateral decubitus position and prone position.we had introduced intraoperative nerve monitoring system for prone esophagectomy from . results: there was no significant differences of the survival rate between thoracoscopic group and thoracotomy group based on pathological stage. year survival without neoadjuvant treatment was . % (pstagei), . %(pstageiia), . %(pstageiib), . %(pstageiii), respectively. year survival rate of cstageii and iii with neoadjuvant chemotherapy was . % and year survival rate of the salvage esophagectomy after failure of definitive chemoradiotherapy was . %. every outcomes are as good as any reported results in esophagectomy. in the comparison of the lateral position with the prone position, total blood loss was significantly lower in prone position. inflammatory response after surgery was improved more rapidly in prone group, therefore, prone position is recommended as a minimally invasive procedure for thoracoscopic esophagectomy. transient recurrent laryngeal nerve palsy was observed % of patients. conclusion: thoracoscopic esophagectomy will develop further as a standard operation for esophageal cancer. nerve monitoring is useful for detecting recurrent nerve and avoiding nerve injury. background: laparoscopic total mesorectal excision (tme), in a wide female pelvis is usually technically easier than in a narrow male pelvis. however, this is not always the case, as the uterus and adnexae may obscure the views and hinder safe dissection, especially in obese patients. techniques such as graspers through additional ports or suspension with sutures through the broad ligament may potentially cause injury or need additional ports/assistants. aim: we present a novel technique using a self-retaining gynaecological uterine manipulator to improve access during deep pelvic laparoscopic surgery in female patients. technical tip: the operation is commenced in the standard manner for a laparoscopic rectal excision. once pelvic dissection is commenced, whenever it is felt that uterine retraction would be advantageous (depending on the level of the rectal tumour, size of the uterus and ovaries, obesity etc.) a self-retaining uterine manipulator (as shown in the video) is used. the tip of this disposable device is introduced into the uterus after dilatation of the uterine cervix. once the balloon at the tip has been inflated, the instrument is secure and hence there is no need for active manipulation by an assistant. the shaft can be rotated to allow anteversion/retroversion of the uterus to varying degrees as required to aid dissection. as the video depicts clearly, it acts as a self-retaining retractor for the uterus and is removed at the end of the operation. though the procedure is being demonstrated by a gynaecologist in the video, the instrument is quite easy to insert and some of our colorectal team have been trained as well. conclusion: the self-retaining uterine manipulator is an efficient tool for uterine retraction in laparoscopic rectal surgery and we have been using it routinely in tme in females for the past years, with no complications. this was previously published as a technical tip in the journal of minimal access surgerybut has never been submitted for peer review as a video. the authors present a video of two clinical cases treated by trans-axillary endoscopic approach. methods: a years-old male and a -year-old male presented with intermittent dysphagia and frequent reflux (class ii of lahey). one had a history of recurrent respiratory infections. the disease was characterized by oesophagogastroscopy (egd) and oesophagogram. trans-axillary approach with areolar port. step-by-step as follows: (i) dissection anteriorly to the pectoralis major muscle (ii) isolation of the anterior border of sternocleidomastoid muscle (iii) omohyoid muscle's isolation (iv) identification of the thyroid's upper pole (v) zd isolation (vi) myotomy of the cricopharyngeal muscle (vii) zd's resection with stapler and its withdrawn with sac. results: both cases progressed without complications. complete local recovery was verified in both cases one month after the procedure. conclusion: this technique seems feasible and reproducible, allowing zd diverticulectomy with a better cosmetic result and perhaps lower surgical site infections (ssi). in the authors' knowledge, this approach to dz has never been published. background: gastric leak occurs in - % of patients who undergo roux-en y gastric bypass (rygb) for morbid obesity. the pathophysiology may be related to gastric ischemia, fistula, or ulcer.gastric leak is a severe complication of gastric bypass (gbp) that is associated with significant morbidity and mortality. fistula may have several clinical impacts, depending on patientrelated factors, fistula characteristics, onset time, and therapy proposal. abdominal drainage, gastrostomy, and revisional surgery constitute the traditional approaches to dehiscence and fistula closure, with variable results. methods: we present a video of a clinical case of -year-old lady with body mass index of kg/m who underwent roux-en-y gastric bypass and h later presentedtaquicardia and right cuadrantum pain. the ctscan inform a apical leak at the gastric pouch level. the video shows the relevant aspects of a revisional surgery and the key points to drain the fistula and close de defect laparoscopically. results: after monts, the patient achieved succesful results, defined as a stabel clinical situation with image evidence of gastric fistula remision. conclusions: gastric bypass (gbp) is one of the most efficient bariatric interventions in morbidly obese patients. the most severe risk of this procedure seems to be the staple line leak, and the management of this complication can be very arduous. without any guidelines it is very difficult to determine the right procedure addressing the staple line leak after gbp. laparoscopic sleeve gastrectomy (lsg) has become the most commonly performed operation worldwide as a primary bariatric/metabolic procedure. however, conversion to other surgical procedures such as roux-en-y gastric bypass (rygb) or one anastomosis gastric bypass (oagb) have been described as treatment options for inadequate weight loss after lsg and unresolved co-morbidities or complications such as leak, stricture, and severe gastroesophageal reflux disease (gerd). we present two clinical cases of weight regain and severe gerd and dysphagia, which account for the main indications to reversal of lsg to either oagb or rygb. aims: we show in the video the surgical technique that we perform by laparoscopic aproach, in order to construct a roux-en-y polipropilene banded gastric bypass lrygb-b. methods: we are performing this procedures within a prospective randomized trial that is design to compare the long term results of lrygb-b versus the standard laparoscopic roux-en-y gastric bypass.the video shows our technique in a case of a years old female with a bmi of kg/m . first we create a vertical gastric pouch of about - ml, and a polypropylene mesh ( x mm) is placed - mm proximal to the anastomosis around the gastric pouch, with the help of a laparoscopic band retractor. after that a cm roux-en-y limb is constructed in an antegastric antecolic fashion, been the lenght of the biliary limb cm. a mm gastroyeyunal anastomosis is performed with a linear stapler, and the enterotomy and gastrostomy are closed with a / barbed running sutures. jejunojejunostomy anastomosis is constructed in similar fashion, but with a lenght of - mm. the petersen space and the mesenteric defect are closed with polipropilene / sutures. results: patients has been operated following this technique, and there has been no complications related to the polipropilene band. (the ramdomized prospective trial is still ongoing). conclusions:the video shows a reproductible easy way to perform a lrygb-b using a polipropilene mesh. introduction: a -year old female patient presented at our clinic two years after initial rouxen-y gastric bypass. she had had a preoperative bmi of , and had a significant weight loss which resulted in a bmi of , at two years postoperatively. she currently suffered from severe dumping with glycaemia levels dropping to mg/dl. pharmacological treatment with metformine, sandostatine and acarbose did not yield any results. on top of these problems she felt less restriction, could eat large portions and had gained kg in the last three months. objective: the usual approach for severe dumping-related hypoglycemia would be to undo the gastric bypass. this patient however was extremely anxious to regain weight, so we sought other options. we assumed that by adding more restriction and slowing down the emptying of the gastric pouch we could alleviate some-if not all-of the dumping related symptoms and prevent further weight regain. methods: in this video we present the banding of a gastric pouch for severe dumping after rouxen-y gastric bypass. results: although unconventional, the banding of the pouch yielded excellent results. the slower pouch emptying and reduced portions resulted in a near complete remission of all symptoms. as an additional benefit we found a slight weight loss of four kilograms six weeks postoperatively. conclusion: the usual treatment of severe dumping-related hypoglycemia would be an undo of the gastric bypass. in this case however the patient was extremely anxious to regain weight, being very pleased with the results her gastric bypass had yielded. in agreement with both the patient and treating endocrinologist we attempted a different approach. the slower pouch emptying and increased restriction offered another way to alleviate the dumping and deep hypoglycemia while concomitantly resulting in weight maintenance. aim: the aim of this video is to present a novel surgical technique to avoid stent migration after endoscopic placement in patients with leakage subsequent to laparoscopic sleeve gastrectomy (lsg) . methods: this video shows the case of a patient (bmi , kg/m ) who developed an upper gastric leakage days after lsg. a ct scan showed a small leakage at the eg junction complicated by intra-abdominal abscess. a ct guided percutaneous drainage of the abscess was performed. a stent placement was attempted endoscopically three times and failed for migration. we decided to place laparoscopically a non adjustable gastric ring (nagr) around the stomach, in order to avoid stent migration.first of all the stent is replaced endoscopically in order to cover the fistula tract. the patient is placed in a half sitting position and the pneumoperitoneum was obtained using a veress needle in left subcostal space. a port technique is used as in standard laparoscopic sleeve gastrectomy.the procedure starts with the mobilization of adhesions, the fistula is identified in the upper part of the tubule.the gastric tubule is isolated and the lesser omentum is opened. the blunt needle at the tip of the ring is passed retrogastrically, a tourniquet can be useful is the positioning turn out to be difficult. the nagr is then closed over the gastric tubule containing the stent. a drain is finally placed. results: the stent was removed after weeks. a gastrointestinal ct scan with oral contrast showed a complete resolution of leakage. after months the patient was in a good condition with bmi , kg/m . the stent was endoscopically removed after weeks. a gastrointestinal ct scan with oral contrast showed a complete resolution of the leakage. after months the patient was in a good condition with bmi , kg/m . conclusions: this new technique is feasible and effective, as shown in this video; however the nagr can lead to complications, so a strict follow up is needed and if any complication appears, should be considered to remove laparoscopically the ring. introduction: in this case, we will discuss the case of a year old male patient who underwent a laparoscopic cruraplasty and gastric plication resulting in a weight loss of kg. other medical history reported insulin-dependent diabetes, reflux esophagitis and sleeping apnea with cpap. two years after gastric plication the patient presented with passage problems, gastro-esophageal reflux and epigastric pain. to this end a swallow test was performed revealing a large fundus with a restricted passage of contrast. due to the persistent complaints and the abnormal findings on barium swallow a surgical re-intervention was needed. objectives: despite the current bmi of and the age of the patient, conversion from a gastric plication to a roux-en-y gastric bypass was performed. several other surgical options were considered, including an undo of the gastric plication or a dilatation with a resizing of the fundus. methods: in the video we describe the laparoscopic approach for a conversion of a gastric plication to a roux-en-y gastric bypass. results: at months follow-up the patient showed a weight loss of kg and the resolution of his earlier symptoms. the patient had a normal oral intake without any gastro-esophageal reflux or epigastric pain. conclusion: after a gastric plication, partial loosening of the sutures and stenosis are both wellknown complications. as presented in the video, it is apparent that a laparoscopic undoing of gastric plication is not as straightforward as it seems. firm adhesions between folds can compromise the procedure and inhibit a complete separation of the tissues. we believe that in these cases the best surgical approach is to convert to a roux-en-y gastric bypass. laparoscopic sleeve gastrectomy (lsg) is a relatively new surgical approach in the weight loss surgeon's armamentarium. in literature there is a consensus about the importance of mobilizing completely the gastric fundus before transection. the resg (revised sleeve gastrectomyresleeve) may be a valid option for failure of primary lsg. we focused the attention on the consequences that can have an incomplete resection of gastric fundus during an operation of sleeve gastrectomy and how they can be solved by the repetition of this procedure. a sleeve gastrectomy was performed in an obese -year-old woman (bmi = ). three days after the operation, an upper gi x-ray with gastrografin did not show any abnormalities. three months after the surgical procedure, the woman referred frequent episodes of vomiting and a significant weight loss ( kilos). an upper gi x-ray with gastrografin demonstrated the presence of multiple communicating cavities of the gastric fundus. the esophagogastroduodenoscopy (egd) showed that the gastric tube close to the esophagogastric junction was separated from a recess ( - cm in diameter) by an incomplete septum. a severe hypokalemia and consequent ecg abnormalities were treated with intravenous infusion of potassium. then, we performed a laparoscopic operation. the gastric tube was completely released along the suture line of the previous operation and, especially, the posterior surface of the upper part until the left crus of diaphragm became evident. under the guide of the bougie, the recess was removed. results: the clinical course was regular, and the patient was discharged on third post-operative day after an upper gi x-ray with gastrografin which demonstrated the absence of leakage and a normal gastric tube. after year, the patient was very satisfied with the operation. conclusions: the complete mobilization of the gastric fundus allows to see clearly which part should be resected to obtain an adequate gastric tube and facilitate a correct placement of the stapler. in our experience, in patients with a residual fundus, an upper gi x-ray with gastrografin and an egd are needed to exclude the presence of stenosis. then, a resleeve gastrectomy is an efficient and safe procedure to treat this post-lsg complication. weight regain is one of the main problems in bariatric surgery. we have many surgical option but when we evaluate patients with long follow up and bmi of superobese patient before the first surgery, the weight recidivism can arrive up to - % at years.in most cases the first surgery is a restrictive procedure, and in many cases sleeve gastrectomy.here we present a case of weight regain after laparotomic super-magenstrasse (that we consider like a sleeve gastrectomy except for remnant removal) with a big incisional hernia. after a complete multidisciplinary re-evaluation we decided to perform an oagb (one anastomosis gastric bypass) but in this case we decided to create a functional exclusion to the duodenal transit by positioning a minimizer ring. this solution is effective in food diversion and guarantee gastric and duodenal endoscopic exploration in case of need. we think that this technique can represent an option to take in account for selected cases. at the end of bariatric procedure we perform a laparoscopic repair of incisional hernia with mesh in the hope to avoid future surgery and post operative small intestine herniation. patient rejected additional bariatric procedures and in fact she has gained kg two years later (bmi . ). conclusions: lagb gastric erosion is uncommon ( . - %) . intraoperative (such as perigastric approach) and patient related factors (smoking, alcohol…) have been described as risk factors. the most frequent clinical presentation is weight loss failure; band and port issues (such as infection) are also frequent. erosion is infrequent to present as an acute event (\ %: peritonitis, abscess…) or asymptomatically (\ %). diagnosis is mostly performed under upper endoscopy. the most common therapeutic technique is removal of the band (by endoscopy or surgery), repair of the stomach, if needed, and band replacement (at least three months later). some authors have performed immediate replacement but the incidence of recurrent erosion seems to be higher. other options are lagb removal alone or conversion to different bariatric procedure. for endoscopic removal, it has been advised to wait until the band buckle is in the stomach and is sometimes very difficult. replacement of the band is not associated with weight regain. she reports years of evolution presenting moderate intensity heartburn that was exacerbated during the night as well as submit occasional rejurgutation. the intensity of the symptoms is attenuated by maintaining a diet without irritants and improving feeding times. denies hematochezia, unintentional reduction of weight, dysphagia or early satiety. the patient has suffered from obesity since childhood, after pregnancy she had progressive weight gain and difficulty in controlling blood sugar, so she is scheduled a gastric bypass roux-en-y . preoperative endoscopy was performed, evidencing submucosal tumor in the gastroesophageal junction at of the dentary arch, approximately cm in diameter. an endoscopic ultrasound was performed, demonstrating subepithelial lesion of the gastroesophageal junction, hypoechoic, with well-defined borders, pseudobilobulated, . cm x . cm, and dependent on the external muscular layer. a fine needle aspiration is performed in which spindle cells are identified, leiomyoma is likely diagnosed. it is programmed for laparoscopic resection of submucosal gastric tumor, gastric bypass and laparoscopic cholecystectomy. a tumor at the level of the gastro esophageal junction of approximately . cm is identified in the surgery, which can be resected by laparoscopy without complications. the patient is discharged after days of postoperative stay. the final histopathological result: leiomyoma of . cm with free edges. cd (-) gog (-) caldesmon (?)s (?). background: fifty percent of patients who have undergone gastric bypass, posterior reversal and sleeve gastrectomy and finally complete hiatoplasty presents symptomatic gastroesophageal reflux disease. surgical reinforcement of the lower esophageal sphincter is necessary to prevent acid reflux. here, we describe ligamentum teres cardiopexy, a surgical technique that reinforces the lower esophageal sphincter and restores its competence with a new valve, in patients with previous conversion of sleeve gastrectomy to gastric bypass and hiatal hernia repair. methods: we present the surgical techhnique performed to a patient with initial gastric bypass who underwent sleeve gasterctomy for hipoglycemias and hiatoplastia for severe gerd. persistent gerd requested to undergo ligamentum teres cardiopexy. in this procedure, the ligamentum teres is released from its umbilical connection and the hernia reduced by manual traction, freeing the last - cm of esophagus in the abdomen. the distal ligamentum teres is fixed with one stitch to the apex of the angle of his, one at the gastroesophageal junction, and one joining the gastric fundus to the esophagus. the remainder of the ligamentum teres is fixed over itself with four to six stitches, forming a necktie cardiopexy. the procedure concludes with diaphragmatic crus closure. results: after months, the patient achieved successful results, defined as resolution of gerd, no protonpump inhibitor (ppi) use, and manometry measurement over mmhg after surgery. conclusions: ligamentum teres cardiopexy combined with closure of the gastric crus is a late alternative treatment for gastroesophageal reflux disease in patients with previous sleeve gastrectomy and hiatal hernia. general surgery, ponderas academic hospital, bucharest, romania introduction: as metabolic surgery techniques evolve during the years, we have to face more and more patients with complications ands uboptimal results after the older/initial procedures. vertical banded gastroplasty(vbg) is one of those procedures that gain momentum during the initial experience in bariatric surgery, but has proven to have dissapointing results and a lot of complications, nowadays surgeons having to deal with difficult revisional operations. aim in this video: we want to present from our experience the difficulties encountered during the revisional surgery, rouxen y gastric bypass (rygbp)aftervbg, and the tips and tricks that will make this a safer and easier procedure. objective: after thorough preoperative assessment and a review of the literature multiple treatment options were considered. the procedure of choice ended up being a laparoscopic adjustable gastric banding, with the objective to achieve optimal weight loss with the lowest risk for complications. methods: in this video we present the placement of an adjustable gastric banding in a patient with a cirrhotic liver and portal hypertension and the possible pitfalls. results: postoperatively there were no complications and patient had a satisfying weight loss both months and year postoperatively. in a short review of the literature we've found that bariatric surgery is feasible in patients with portal hypertension as long as the patient is not decompensated or has bleeding varices. conclusion: cirrhosis and portal hypertension are no absolute contraindication for banding, sleeve or rny gastric bypass as long as the patient is not decompensated or has bleeding varices. the type of surgery is dependent on patient and surgeon-related factors. the aim should be to achieve optimal weight loss with the lowest possible surgical risk in this type of patients. surg endosc ( ) :s -s introduction: in this case, we will discuss on a year old female patient who had undergone a laparoscopic nissen fundoplication years ago due to gerd grade b. because of morbid obesity a n-sleeve gastrectomy was performed year ago resulting in a weight loss of kg. at presentation she had regained all the lost weight, resulting in a bmi of , . the patient history also reported insulin-dependent diabetes and obstructive sleep apnea with cpap. gastroscopy was performed showing a large residual fundus but no esophagitis. on the subsequent upper gi series a relatively wide sleeve with an intact nissen-collar was detected. objectives: a laparoscopic conversion to a roux-en-y gastric bypass was performed. other potential surgical treatment options are a sadi procedure or a sleeve gastrectomy with transit bipartition (santoro procedure). methods: in the video we describe the laparoscopic approach for a conversion of a n-sleeve to a roux-en-y gastric bypass. results: at month follow-up the patient presented with a weight loss of kg. the patient had good restriction on oral intake and did not have any reflux-related symptoms or complaints. conclusion: conversion from a n-sleeve to a roux-en-y gastric bypass is a challenging procedure. the largest pitfall during the creation of the gastric pouch is to staple a double fold of the nissen fundoplication. we believe that in these rare cases of weight regain after n-sleeve, the best surgical approach is to convert to a roux-en-y gastric bypass. four years later, in , a laparoscopic conversion to roux-en-y gastric bypass was performed because of weight regain. she now presents with satisfactory and stable weight loss over the last few years. she was recently diagnosed with a brca- mutation for which she underwent a bilateral ovarectomy and mastectomy. the patient's brother was also diagnosed with this mutation and died of pancreatic cancer at the age of . genetic counseling advised a twoyearly follow-up because of an increased risk up to % of developing pancreatic cancer. control gastroscopy showed a normal esophagus and gastric pouch. control ct scan revealed hypertrophic stomach creases in the excluded stomach. these results prompted a laparoscopy-assisted gastroscopy of the excluded stomach which uncovered hypertrophic stomach glands and intestinal metaplasia on biopsy. methods: in this video we demonstrate the laparoscopic approach for complex revisional bariatric surgery. conversion from rny gastric bypass to a sleeve gastrectomy in a patient who already underwent a vbg. the focus of the video is on a manual gastro-gastrostomy with partial gastrectomy of the fundus and part of the stomach where the old vbg-band was placed. results: after , months follow-up the patient had no complaints and a stable weight. upper gi series shows a normal passage of contrast through the sleeve gastrectomy. conclusion: endoscopic surveillance of the remnant stomach and echo-endoscopy of the pancreas is no longer possible after rny gastric bypass. in cases where the need for such a surveillance arises after a rny bypass a patient-tailored approach is necessary. in our patient a laparoscopic conversion from a rny gastric bypass to a sleeve gastrectomy was performed. this approach keeps the patient's wish for weight loss intact while enabling further surveillance through natural-orifice endoscopy. a -year-old morbidly obese japanese woman with a body mass index of kg/m suddenly complained of swallowing difficulty months after laparoscopic roux en y gastric bypass surgery with retro-colic roux limb route. an internal hernia of the defect of the transverse mesocolon was suspected by computed tomography, and emergency intervention was performed. the surgery revealed no internal hernia. however, strong inflammation and adhesion were observed between the transverse mesocolon and the retrocolicroux limb. in addition, the roux limb on the oral side of the adhesion site was dilated and bent.the adhesion between the transverse mesocolon and the flexed roux limb was dissected, linearized and re-fixedby suturing to the transverse mesocolon. however, since the difficulty of oral intake persisted re-do surgery was performed again. after resecting the roux limb involved in the severe inflammation, a 'new' roux limb was lifted to the cephalad via the ante-colic route. finally, the gastric pouch and roux limb were re-anastomosed with - absorbable sutures in an interrupted full thickness single layer manner. in the present case, we experienced difficulty with both adhesiolysis and determining the accurate target line to resect at the 'old' gastrojejunostomy. however, blocking the blood flow of the 'old' roux limb facilitated the accurate recognition of the target line. esofagogástrica, cirugía general y ap digestivo, hospital regional universitario de málaga, malaga, spain; hepatobiliopancreática, hospital regional universitario de málaga, malaga, spain introduction: marginal ulcer is one a serious complications after a bariatric gastric bypass. tobacco, non-steroidal anti-inflammatory drugs (nsaids) and helicobacter pylori (hp) infection are known risk factors. methods: we present a -year-old women operated years before of bariatric surgery with a gastrojejunal (gy) bypass technique due to intraoperative dehiscence of the staple line after attempting a vertical gastrectomy (sleeve). she has persistent vomiting and epigastralgia from months after the intervention, affecting his quality of life. upper gastrointestinal endoscopy (uge) was performed, describing an ulcer in the gy anastomosis. she started hp eradication treatment, treatment with proton pump inhibitors (ppis), tobacco and nsaids were discontinued, but she had slight improvement. after months the uge was made again, which show peptic esophagitis and marginal ulcers. the plasma gastrin level was normal. due to the persistence of symptoms despite conservative treatment, we decided reoperation by laparoscopy. we found herniated bowel in petersen space, which were reduced and the space was closed. we proceeded to truncal vagotomy. the gy anastomosis was resected ( fig. ) and performed again. finally, we perform antrectomy. the pathological anatomy showed ulceration. she was diacharged home on the th postoperative day without any complications. results: a marginal ulcer after bariatric surgery appears in the jejunal mucosa of the g-y anastomosis. the symptoms are epigastric pain, nausea and vomiting. acid, tobacco, nsaids and hp infection has an important role in their development \ sup [ . \/sup [ the first treatment is medical, discarding out the risk factors, but if it is not effective, it will be surgical, resecting the previous anastomosis. the usefulness of vagotomy is debatable, but the percentage of success increases. in our case, we perform antrectomy to avoid retained antrum syndrome. the hernia through petersen space is a cause of intestinal obstruction and abdominal pain as the case presents. although we believe that the symptoms were mainly caused by the marginal ulcer, the internal hernia was probably a symptomatic cause. conclusion: the treatment of a marginal ulcer is medical, eliminating the risk factors, but if it is not effective, the surgery is indicated. results: bowel's measurements and confection of the gastric pouch are identical in both cases. in the first case, intestinal anastomosis is performed in the inframesocolic compartment once small bowel has been divided. in the second case, such union is made next to the gastrojejunal anastomosis with the bowel uncut, making the section once no leakage has been found conclusions: laparoscopic roux-en-y gastric bypass is currently considered one of the technique of choice in the surgical treatment of morbid obesity. there are variations and alternatives for its realization. to know them can allow to individualize the technique to each type of patient. we present a clinical case of a year old female. she had a vertical banded gastroplasty procedure (in another clinic) years ago with an initial weight loss of kg in a period of months. she was seen at our clinic because she was suffering from dysphagia to solids and general diffuse abdominal pain for the last month. at physical exam we found a bmi of and nothing else called our attention. we did an upper gi endoscopy and egd transit; we concluded that a gastric bypass would offer her the best results. therefore, we converted her vertical banded gastroplasty into a gastric bypass laparoscopically. she had an uneventful postoperative period and was discharged home without complications. aims: sadis emerged as a modification of biliopancreatic diversion with duodeno-ileal switch (bpdds) in which after sleeve gastrectomy (sg), the duodenum is anastomosed to an ileal loop in a billroth-ii fashion. sadis has promising outcomes for weight loss and comorbidity resolution in morbidly obese patients avoiding the high morbidity of biliopancreatic diversion with duodenal switch. clinical case: -year-old patient, subjected to bariatric surgery two years ago, including a sleeve gastrectomy (sg). despite this operation and dietary and hygienic modifications, the patient gained weight in recent months, reaching a bmi of kg/m and an overweight of kg. an endoscopy was carried out on her, which provided evidence of a gastric remnant of moderate size with flexible tissue, normal peristalsis, and fast disposal speed. the case was discussed in a joint session, leading to the decision to apply revision surgery. the decision was taken to apply sadis, a novel technique that had never been used before in andalucia. the rate of weight re-gain after the use of classical techniques such as sleeve gastrectomy (sg) or the roux-en-y gastric bypass (rybg) is considerable high. revision surgery due to weight re-gain is necessary in many of these cases. sadis emerged as a simplified alternative to the use of bpdds as revision surgery following a gv due to weight re-gain with good short-term results, in terms of both weight control and comorbidity control. since only one anastomosis needs to be applied, chirurgical time diminishes, as well as the rate of surgery-related complications. moreover, it could be used, through laparoscopy, for patients who have undergone previous, complex abdominal surgery. conclusion: sadis showed a promising short-term weight loss outcome and comorbidity resolution rate but long-term data are missing and there is currently a high level of technical variability. on the other hand, further studies are required to measure its cost-effectiveness compared to the currently popular bariatric procedures, sg and rygb. aims: the lps sleeve gastrectomy is the most common bariatric surgery technique because it has a low surgical complexity and acceptable weight loss results. however, - % of patients present with an insufficient weight loss, weight regnances, reflux or dysphagia. in these cases, it is recommended to perform a second bariatric surgery to combine a component of malabsorption such as gastric bypass or duodenal switch. the video describes the technique of a laparoscopic biliopancreatic diversion with duodenal switch with a previous laparoscopic sleeve. the objective is to describe the safety of the technique and the subsequent success of it. methods: a -year-old female patient presented morbid obesity with a bmi of after performing a laparoscopic sleeve gastrectomy in . initially, she presented a percentage of excess weight loss of %, reaching a bmi of after two years of follow-up. after this, she suffered a reganancia of all the weight lost despite diet and exercise, presenting a bmi . a study was made with tegd where no complications of the previous surgery or symptoms of gastroesophagical reflux or dysphagia were observed. the lps duodenal switch is proposed in the obesity unit committee in , without immediate postsurgical complications. the patient presented a favorable postoperative period and was discharged three days postoperatively. results: at the present time, the patient has achieved a % excess weight loss and has a bmi of . . presents good oral tolerance with stools a day without urgency. it doesn't present protein deficioncies. vitamin deficiencies are orally supplemented. the lps duodenal switch is a technique that can be performed after a sleeve gastrectomy safely in cases of insufficient weight loss or weight reganancia. the patients presented a greater weight loss after the duodenal switch than after the gastric bypass, observing a lost of excess weight of % compared to %. the differences being statistically significant. weight regain after gastric bypass is a challenging problem. a number of revisional surgical options have been reported. this is a case of a year-old woman years after lrygb. her initial bmi was , lowest after surgery- , at presentation- . the video shows a robotassisted laparoscopic conversion of rygb to loop duodenal switch. the roux limb is transected and dissected to the gastrojejunostomy. the gastrojejunostomy is resected and the gastric pouch is recreated over a bougie. the gastric blood suply is confirmed with icg. a gastro-gastrostomy is created to restore gastric continuity and a sleeve gastrectomy is performed. the duodenum is devided and a duodeno-ileostomy is created cm from the ileocecal valve. the remaining roux limb is resected. the patient recovered uneventfully. conversion of rygb to loop duodenal switch requires creation of as little as two anastomoses, in comparison to standard ds, which requires four. it is a safe option for patients with weight regain after lrygb. methods: irb approval and informed consent have been obtained. a dissection is conducted to separate the descending mesocolon of the gerota's plan from the medial aspect to the peritoneal lining to the left parietal gutter. the peritoneal layer is incised parallel to the vessel and close to the colonic wall. the dissection is continued anteriorly up to reach the resected parietal gutter. a passage into the mesentery of the upper rectum is created for the allocation of the stapler and the dissection of the rectum. these maneuvers permit to straighten the mesentery simplifying the identification and cutting of the sigmoid arteries. a caudal-to-cranial dissection of the mesentery is performed from the sectioned rectum to the proximal descending colon by a sealed envelope device. it can be very useful to mobilize the colon in any direction: laterally, medially, or upward. the dissection is performed along the course of the vessel up to the proximal colon, with progressive sectioning of the sigmoid arterial branches. the specimen is extracted by a pfannenstiel incision. the anastomosis is performed transanally with a circular stapler according to knight-griffin technique. results: we performed a laparoscopic segmental colectomy using this approach for patients with benign sigmoid lesions: diverticulitis, flat polypoid lesions (no lift-up sign), and bowel endometriosis. the mean operative time and blood loss were . ± . min and ± ml, respectively. there were not a single conversion to open surgery and no any leakage or stricture. only cases of intraluminal bleeding and case of wound infection (treated conservatively) were observed. conclusion: we consider this approach to be safe and useful for segmental colectomy to be performed sectioning the sigmoid artery close to the colonic wall. aims: to show a clinical case with a video of a patient was operated for colon cancer in hepatic angle by a single suprapubic incision (ssilrh). methods: a -year-old male assessed for abdominal pain and weight loss. on physical examination: a painful mass was detected in the upper right quadrant. the colonoscopy revealed an ulcerated lesion in the hepatic angle and the biopsy revealed a moderately differentiated adenocarcinoma. in the abdominal ct a mass of x cm was observed (figure). the patient was operated with ssilrh technique, as shown in the attached video. results: the patient was placed in the supine position and with the legs separated. the surgeon is placed between the patient's legs. a transverse incision of the skin was made in the middle line of . cm to cm above the pubis. the underlying fascia was divided transversely, the rectus abdominal muscle was exposed, a purse-string suture placed in the fascia. an mm reusable trocar was inserted for the chamber, a mm reusable flexible trocar was placed at the o'clock position and another trocar was placed at the o'clock position. the ileocecal valve was released from the peritoneal parietal foil, as well as the mesocolon right by a lateral to medial approach to the second portion of the duodenum. the hepatic angle was also dissected from lateral to medial. for the anastomosis, the mm trocar was replaced with a mm trocar and a stapler was placed. a mm °chamber was inserted through the mm flexible trocar. the small intestine was divided as well as the proximal transverse colon with endogia. an intracorporeal ileocolic anastomosis was performed. the piece was removed through the suprapubic incision. he was discharged after days without complications. the histological studies confirmed a differentiated adenocarcinoma of x x cm. the surgical margins were free, without infiltrated lymph nodes ( / ) with stage pt n . the ssilrh technique allows a complete resection of the mesocolon and complies with the oncological principles. they can present with abdominal pain, nausea, acute abdomen, symptoms of intestinal obstruction or asymptomatic with incidental diagnosis. their diagnosis can be difficult. the objective is to demonstrate the safety and efficacy of the laparoscopic approach in this infrequent pathology. material and methods: we present a video of the surgical intervention of a -year-old patient, with functional dyspepsia, with a casual diagnosis of a pseudocystic mass of the right colon after performing a ct scan: giant diverticulum of the hepatic colon angle with fecaloid content inside it under tension the patient goes to the emergency room for acute abdominal pain, pending colonoscopy, antibiotic treatment is established, and a laparoscopic approach is decided upon after the patient's evolution. results: intervention: complete laparoscopic approach, trocars. large size tumor in the right colon, diverticular in appearance, with stony content inside, with locoregional adenopathies, oncological radical right hemicolectomy, manual intracorporeal anastomosis, correct postoperative, hospital discharge. on the th day. definitive pathological anatomy: giant diverticula on areas of intense mucosal ulceration, free edges. conclusion: the laparoscopic approach of the symptomatic diverticula of the right colon is safe and effective. introduction: minimally invasive transanal surgery (tamis) is a surgical technique whose established indications are the complete exeresis of rectal polyps that are not resectable endoscopically or early rectal neoplasms with good prognosis criteria. transanal devices with gel platform facilitate dissection in this field. however, one of the drawbacks of this approach is the oscillation of the right nerve, which hinders dissection and prolongs the surgical time. material and methods: we present the case of a patient with a central depression neoformation, located cm from the anal margin in the posterior aspect of the rectum in a male patient. the lesion occupies % of the circumference and was considered unresectable endoscopically. the endoscopic biopsies showed a tubulovillous adenoma with moderate dysplasia. results: an exeresis of full thickness of the rectal wall is performed, with subsequent suture of the defect. we show in the video the use of a glove interposed in the pneumoperitoneum gum to maintain the stability of the neumorectum and the technique of dissection and suture, as well as the stability of the neumorectum with this technique throughout the procedure. the use of a glove as a reservoir to stabilize the nemorectum is an economical and easy-to-use method that can safely replace extra devices. aims: endometriosis is a gynecologic disorder defined by the presence of endometrial glands and stroma outside the uterine cavity. deep infiltrating endometriosis (die) invades mm to the retroperitoneum of the pelvic sidewalls, the rectovaginal septum, or the muscularis of the bowel, bladder or ureters. the rectum is being the most common bowel site of involvement. for symptomatic die, medical therapy should always be the first-line treatment. therefore, a minimally invasive approach using laparoscopy is considered the gold standard option and challenging aiming at complete disease excision. also, there are several advantages of natural orifice specimen extraction when compared with abdominal incision that may directly impact the postoperative results of these young patients. methods: we report a case of a -year-old female with a -month history of chronic pelvic pain, dyschezia and rectal bleeding. these symptoms were refractory to hormonal, antispasmodic and opioid therapy. magnetic resonance imaging reported a nodule x cm invading the rectal wall cm to the dentate lane. we performed a laparoscopy and we found the nodule at the uterine posterior wall invading the rectal anterior wall. the nodule was invading into the rectum in a large area so we proceeded with segmental resection and added hysterectomy and salpinguectomy because it was the preference of the patient. the anastomosis was created intracorporeally and the specimen was removed through the vagina performing in this way a totally laparoscopic procedure with natural orifice specimen extraction. results: the total operative time was h, the postoperative stay was uneventful and the patient was discharged on day four. the pathological report showed an endometrioma cm length predominantly involving colonic muscularis propria. conclusion: laparoscopic surgery is a safe and feasible approach for the surgical management of deep infiltrating endometriosis of the rectum and the gold standard for female young patients that often need multiple surgeries. in addition natural orifice specimen extraction avoids potential complications of abdominal incisions. week-day surgery, university, sapienza, ospedale sant'andrea, rome, italy; urology, clinica mater dei, rome, italy aims: we describe a case of a patient affected by a mass in the left kidney and a diverticular stenosis of the sigma. methods: a years old woman complained abdominal pain in the left flank of the abdomen and in the left iliac fossa radiated to the hypogastrium, with fever and no passing flatus. contrast enhanced computer tomography scan (ct-scan) showed a cm mass of the superior pole in the left kidney and a colonic diverticulitis with thickness of the wall and a microperforation of the sigma. she underwent to medical therapy with resolution of the diverticulitis. after weeks a laparoscopic nefrectomy and sigmoidectomy was planned. patient was positioned on the right flank. this position was kept for both the procedures. we performed four trocar accesses along the left subcostal region and a periombelical incision for the specimen extraction. results: post-operative course was uneventful. patient was discharged in post-operative day. istopathological exam showed a renal cell carcinoma confined to kidney with no positive lymph nodes and a diverticular stenosis of the sigma. laparoscopy allowed to perform two fine procedures in a critical situation using few trocar incisions and obtaining good results. background: hartmann procedure consists in a sigmoidectomy followed by a terminal colostomy. stoma is associated with complications and suboptimal quality of life, so the restoration of colonic continuity should be at least considered in any case. open restoration has been associated with significant morbidity and mortality. many authors have described the advantages of laparoscopic hartmann reversal. we want to go a step further showing our experience using a combined laparoscopic and transanal approach in an attempt to improve the surgical technique in a patient with previous abdominal surgeries and a rectovaginal fistulae. methods: the transanal and laparoscopic team work simultaneously. by the abdominal approach a pericolostomic incision is made, the distal affected colon is resected and a purse string suture is performed around the anvil of the eea mm single-use stapler with . mm staples (autosuture, covidien). a mm umbilical trocar is located for a °camera and a gelport laparoscopic system (applied medical) with two mm trocars is introduced through the colostomy wound. hard pelvic adhesiolysis was performed and splenic flexure was also mobilized.the gelpoint path transanal access platform (applied medical) is introduced through the anal canal with three trocars in a triangle position. the proximal rectum and mesorectum are dissected until the peritoneal reflexion. the previous stapler line with the resected tissue is then exteriorized throught the anus. the distal rectum is prepared with a circumferential purse string suture. the vaginal defect was sutured transanally. the proximal colon and the anvil are extracted through the rectal stump and connected to the circular stapler, performing an end-to-end anastomosis. results: the total operative time was h. the postoperative stay was uneventful and the patient was discharged on day . conclusions: as in patients with rectal cancer, dissection of the stump in hartmann reversal procedure may be better and associated with shorter operative time. as with any new surgical procedure, it is probably too early to draw conclusions but nowadays transanal combined with laparoscopic approach seems to be a safe and feasible technique to perform a hartmann reversal, especially in challenging cases. intravenous and endoluminal contrast enhanced ct revealed the presence of a large retroperitoneal fluid and gas collection, due to diverticular perforation, extended from pelvis to iliac bifurcation, involving the left urether. no hydrosoluble contrast media leakage or massive pnuemoperitoneum were present. after an initial conservative treatment without significant improvement an emergency laparoscopic left colectomy with primary anastomosis and laparoscopic retroperitoneal collection drainage was performed. the laparoscopic approach was very challenging due to the obesity of the patient and the presence of the abscess. the patient was discharged on pod after requiring re-intervention for dehiscence of the left iliac mini-laparotomy on pod . conclusion: diverticular perforation in obese patients adds a further challenge to its laparoscopic treatment and deserves an aggressive surgical approach since its outbreak. although intracorporeal anastomosis has been demonstrated to be safe and effective after right colectomy, limited data are available about its efficacy after left colectomy for colon cancer located in splenic flexure. there are few studies comparing patients who underwent laparoscopic left colectomy with intracorporeal anastomosis or with extracorporeal anastomosis. anyway literature shows that there is no significant difference between intracorporeal anastomosis and extracorporeal anastomosis about oncological result. as for right hemicolectomy, intracorporeal anastomosis seems to show a trend towards a faster recovery after surgery due to the shorter time to flatus and lower post-operative pain expressed in the mean vas scale. laparoscopic left colectomy with intracorporeal anastomosis is associated with a lower rate of post-operative complications as for right colectomy. literature results could suggest that a complete laparoscopic approach could be considered a safe method to perform laparoscopic left colectomy with the advantage of a guaranteed faster recovery after surgery. as usual further randomized clinical trials are needed to obtain a more definitive conclusion. we show a video of a years old patient with a pure splenic flexure colon cancer who underwent to a laparoscopic left hemicolectomy with intracorporeal anastomosis. case presentation: here we describe a case of a year old asthmatic and hypertensive lady with an asa score of iii who presented to emergency after a right knee replacement with a four day history of lower abdominal pain. she was septic upon arrival to the resuscitation roomimmediately prompting the hospital's local septic management protocol. a ct scan of her abdomen showed a rectosigmoid perforation with free intra-abdominal air and fluid. the patient underwent laparoscopic hartmann's procedure within h of admission. after an uneventful postoperative recovery the patient was discharged home after a total of days of hospitalisation. she was followed up at surgical outpatients with no adverse events over the course of the subsequent months. conclusion: this case exhibits the feasibility of laparoscopic hartmann's procedure as a surgical modality for hinchey stage iv diverticulitis. the positive outcome supports the claim that for experienced surgeons laparoscopic hartmann's procedure remains a safe and viable option for elderly comorbid patients in the emergency setting. introduction: mesenteric cysts are a very infrequent pathology, they usually present an anodyne clinic, and their diagnosis is reached casually. objectives: to demonstrate the safety and efficacy of the laparoscopic approach, in cases with intra-abdominal cysts of benign etiology, using material with mini-instruments, reducing surgical aggression, maintaining its safety and efficacy.material and method: clinical case: a -year-old man with no personal history of interest. in the last two months he presented episodes of pain in the right hypochondrium, exploration without findings, us-ct scan: a cystic tumor of cm. in hepatic colon angle compatible with uncomplicated benign mesenteric cyst, tumor markers and normal colonoscopy. evidence of interest is exposed. given the evolution it is decided tto. elective surgical. result: intervention: laparoscopic approach, trocars, two of . mm, optics of mm °, benign cystic tumor, with colloid content of more than cm. of diameter in antimesenteric border of colon, which is not possible to separate, mobilization and resection is carried out by endo-gia, including a portion of the colonic wall, appendectomy, extraction in a pocket. good postoperative course, alt to nd day. definitive ap: mesenteric cyst, absence of malignancy. the laparoscopic approach is a valid and effective alternative in cases of benign intra-abdominal cystic pathology, the use of mini instruments reduces surgical aggression, favoring the recovery of the patient. male, yr, wuth doblue post-operative coloanal stenosi and women, with ultralow rectal neoplatis stenosis ( cm from anla verge). both patients were discharge ater days from prosthesis positionins without pain and complications. the first patient, with protection ileostomy, showed fecal incontinence before the operation and was performed prosthesis positioning because rectal losses of infected material and fever. fecal incontinence was showed also after procedure but he had not fever. second patient, yr, with ultralow rectal tumor, after prosthesis positioning was submitted to radiotherapy and she decided for not to be operated and she survives after months in ful well-being. conclusion: endoscopic prosthesis positioning is a consolidated procedure for treatment of bowel obstruction. this study demonstrated that this procedure is safe and this kind of prosthesis is suitable for correct positioning. results: we present a case of a years old man with faecal occult blood test positivity that was diagnosed by colonoscopy of a villous lesion at cm of anal verge. biopsies were taken showing a tubulovillous adenoma with high grade dysplasia. a rectal mri was done showing the lesion fixed to the postero-lateral left side of the lumen at cm of anal verge. no pathological lymph nodes were reported. extension study was negative. the case was presented in multidisciplinary committee agreeing in local excision. in october the procedure was done without incidents. the patient was placed in lithotomic position finding a lesion occupying of the lumen. resection was done without incidents and posterior suture with continuous barbed sutures. he presented an uneventful recovery being the patient discharged in rd postoperative day. definitive pathological findings showed a ptis with negative margins. after three months of followup the patient remains with good functional results and waiting for the first endoscopic revision. conclusions: tamis is a safe and feasible technique with low morbidity that gives us an alternative for early rectal cancer or big rectal lesions much less invasive than techniques used until now. complete mesocolon excision and d lymphadenectomy are two fundamental points in the oncological surgery of right colon cancer. most of the adenopathic recurrences of colon neoplasia in tumors located in the hepatic angle and the ascending colon are located near the head of the pancreas and the vascular axis of the superior mesenteric vein due to an alleged incomplete dissection. we present a case of right colon neoplasia where we performed a laparoscopic right hemicolectomy associated with a d lymphadenectomy. we use medial to lateral dissection of the mesocolon focused on the dissection of the superior mesenteric vein with the identification of ileocolic vascularization, right colic vessels and henle's trunk. this approach is safe and facilitates a correct resection of the mesocolon, which is approached following the embryological plans and a vascular ligature near the bifurcation. the performance of an extended lymphadenectomy allows a wider resection of the mesocolon and the excision of a greater number of lymph nodes, all of which can contribute to a greater survival. the efficacy of pc treatment is related with a properly preoperative imaging diagnosis of the disease, but the poor sensitivity for identifying small peritoneal metastasis are the major obstacle to achieve a complete resection and that leads to peritoneal recurrence. imageguided surgery using icg, could represent an advance in the detection of small peritoneal nodules. there are only a few clinical studies that have analyzed the role of icg for the staging of pc, specially from ccr, and nearly in all of them the selected approach were exploratory laparotomy. this study presents a laparoscopy case, as a non-invasive way of cs in selected patients with limited pc. a new category, tis, was created for low-grade appendiceal mucinous neoplasms (lamns) that invade or push into the muscularis propia by ajcc cancer staging th ed. management of these tumors depends on stage and histology. traditionally, laparotomy was the most recommended approach, however, if laparoscopy is safe, it could be used. the laparoscopic appendectomy should be done with 'not touch' technique and a radical approach has been recently proposed for its treatment. the laparoscopic radical appendectomy should start by exploring complete abdominal cavity. grasping of appendix should not be done. complete resection of mesoappendix is obligated. cequectomy with stapled endogia is necessary. the specimen must be extracted in an endobag. methods: we report a case of a year-old female patient with a personal history of three caesarean sections. this patient was studied due to chronic abdominal pain. a computerized axial tomography was performed, showing an appendix increased in size and a thick wall. the colonoscopy evidence a lesion that protrudes from appendiceal base which is biopsied. results: a laparoscopic way was used and large and width appendiceal was viewed ( x cm). furthermore, a rounded right anexial tumor was also found. a radical not touch laparoscopic appendectomy with stapled cequectomy was done. the intraoperative study was mucinous appendiceal tumor without serose affection. the final result was ptisnx (lamn) without resection margins affected. after h of admission, the patient is discharged without incidents. conclusion(s): minimally invasive surgery in lamns is possible if it is performed with enough experience, following specific rules and tips to manage this tumors. a correct follow-up should be carried out using tumor markers and computer tomography (ct). introduction: resection of both benign and malignant colovesical fistulae can be particularly challenging and carry with it specific surgical considerations. often there is a large inflammatory mass sat within a narrow pelvis, limiting specimen mobility and consequently access to dissection plains. additionally, with the underlying inflammatory process, the ureters may be displaced anatomically and be at risk of injury. aim: to demonstrate a streamlined and reproducible approach to the laparoscopic management of both benign and malignant colovesical fistula, with specific emphasis on the different modalities for bladder repair. method: the following method portrays an overall technique which is adapted dependant on the clinical scenario and specific intra-operative findings: approach to abdominal cavity in standard fashion.identification of right ureter.poster-medial mobilisation of the mass to facilitate delivery out of the pelvis followed by visualisation of the left ureter on the medial and lateral sides before division of the fistula.division of the fistula in benign disease or resection of the bladder dome in malignant disease.transverse laparoscopically sympathetic suprapubic skin incision.vertical incision through linea alba to deliver bulky specimen.intra/extracorporeal repair of bladder dome. results: all of the considered cases were successfully completed with a laparoscopic approach, irrespective of the malignant status of the disease in question. conclusion: both benign and malignant colovesical fistula disease can make the laparoscopic approach to resection challenging, especially when encountering a bulky mass in a narrow male pelvis. the stepwise and streamlined approach considered here can help facilitate successful and safe laparoscopic completion without the necessity to convert to open. background: primary neoplasms of the retrorectal space are very rare. they are located in anatomically difficult area to be addressed, hence a complete evaluation of the lesion is required to determine the extent of resection and the appropriate surgical approach, which include posterior, abdominal and combined abdominoperineal, depending on the characteristics of the lesion. objective: to show a combined laparoscopic abdominoperineal approach of retrorectal tumor. method: we present a video of a combined laparoscopic abdominoperineal resection of a lowlying retrorectal tumor in a -year-old female without prior abdominal surgery. conclusion: retrorectal tumors are infrequent. their anatomical location can make difficult the surgical approach. preoperative imaging can provide useful information for surgical planning. in the recent years, minimally invasive surgical approach has been proposed. laparoscopic approach is feasible and safe, but it is important to select adequately the patients. background: adult intussusception is a rare clinical event representing only - % of all bowel obstruction cases and % of all intussusceptions and the occurrence of adult intussusception due to colonic cancer is even more rare. aim: we present this case of malignant colo-colic intussusception and literature review to increase the awareness of the incidence of colocolic intussusception due to colonic cancer. case report and literature review: our patient is a years old female was admitted to our hospital due to central abdominal pain, cea level of , she was further investigated with ct scan of the abdomen and pelvis which raised the suspicion of mid transverse colon intussusception due to large polypoid lesion. she was further assessed with urgent colonoscopy which confirmed mid transverse colon tumour with biopsies confirmed adenocarcinoma. laparoscopic extended right hemicolectomy with lymph node dissection was performed. upon laparoscopic exploration it was found that the colocolic intussusception was evident as described on the ct scan and as clearly shown on the video. histologically, the transverse colon carcinoma was a moderately differentiated adenocarcinoma, with no lymph node involvement ' out lymph nodes', tnm staging of pt pn pm and r resection. intussusceptions of the colon in adult are frequently found in the ileocecal portion or sigmoidal colon but rarely in the transverse colon. only two cases of adult intussusception of the transverse colon caused by colonic cancer have been reported. overall cases on literature review reported showing colo-colic intussusception due to colonic malignancy. conclusion: colo-colic intussusception due to colorectal cancer is a rare clinical event, however it should be included in the differential diagnosis of colonic obstruction. laparoscopic surgery is safe in malignant colocolic intussusception. aims: single-incision laparoscopic colectomy (silc) aims to achieve better cosmetic outcomes, less pain, and faster recovery compared to multi-port laparoscopic colectomy, but it also has several limitations, especially the technical difficulties. we report our experience with singleincision robotic right hemicolectomy via video presentation. methods: we arranged robotic-assisted single-incision right hemicolectomy for a -year-old female patient with ascending colon tumor. the operation was performed with gloveport singleport device and a three-arm da vinci robotic surgical system through a small midline umbilical incision. colectomy was proceeded by a medial-to-lateral approach along with one or two accessory instruments for maintaining sufficient bowel traction or surgical field exposure. after vessel ligation, complete colon mobilization and right side omentum division, the robotic arms were undocked to perform anastomosis extracorporeally. results: the operation was performed successfully without drainage tube placement. the total operative time was min. the bowel movement returned on post-operative day ,and the patient tolerated normal soft diet on post-operative day . she was hospitalized for days after operation. the pathology report revealed colon adenocarcinoma (t n m , tumor size . cm), and lymph nodes were harvested. conclusions: single-incision robotic colectomy (sirc) approach seems feasible and safe in treatment of ascending colon cancer. this surgical option provides less pain and wound scar for the patient. moreover, it also achieves further benefits for the surgical procedures compared to silc. reasons being, first, it has better instruments flexibility and precision with endo-wrist, as well as less instruments clashing. second, the improved camera stability achieved through the use of the robotic arm is unattainable through manual hand-controlled methods. third, roboticassisted approach gives us an ergonomic environment, which enables the operator to control the arms while sitting by the console, and also to reassign them whenever they cross each other or block the surgical view. in spite of the advantages above, we still need to sincerely consider each patient's situation for proper management. recently, indocyanine green (icg) fluorescence has been introduced in laparoscopic colorectal surgery to provide detailed anatomical information.the aim of our study is the application of icg imaging during laparoscopic colorectal resections: to identify sentinel lymph node, for studying its prognostic value on nodal status, to facilitate vascular dissection when vascular anatomy of the tumor site is unclear and to assess anastomotic perfusion to reduce the risk of anastomotic leak. after tumor identification ml of icg solution ( . mg/kg) is subserosal peritumoral injected. a full hd image s camera, switching to nir mode, in about min displays fluorescence: the sln is identified and the sln biopsy (slnb) is performed.when tumor is in difficult site, as hepatic or splenic flexure, ml of icg solution ( , mg/kg) is intravenous injected. in about - s a real-time angiography of tumor area is obtained; on this guide, vascular dissection and pedicle ligation is performed.after anastomosis, another ml of icg solution is injected to confirm anastomotic perfusion. if there is an ischemic area, a new anastomosis is performed. from november , patients were enrolled: left colectomy, right colectomy, transverse resections, and resections of splenic flexure. in ten cases, intraoperative angiography led to the identification of vascular anatomy. in two cases the anastomotic perfusion wasn't good and the surgical strategy was changed. four postoperative complications occurred, of which one anastomotic leak, due to a mechanical problem. from november , patients were enrolled to perform the slnb: right colectomy, left colectomy, transverse resection and splenic flexure resections. the sln was identified in cases. cases were found to be n to the conventional examination and were subjected to ultrastaging. icg-enhanced fluorescence imaging is a safe, cheap and effective tool to increase visualization during surgery. it's recommended to reduce the incidence of anastomotic leak, to facilitate the assessment of vascularization in order to perform oncological resections, and to perform the slnb to study its clinical role on nodal status and for the sln ultrastaging in order to identify the micrometastases. background: surgical emptying of lateral pelvic lymph nodes (llnd) is a strategy used differently when compared the approaches to rectal cancer in the west and eastern countries. there is evidence that = mm lymph nodes in lateral compartment should be removed, even in the setting of neoadjuvant chemoradiation. minimally invasive surgery with nerve-sparing technique and sharp dissection with minimal bleeding may help overcome the significant complexity of the procedure that may have been a technical obstacle to implementation in the past. the standardization of the technique may help implementation with shorter learning curves and excellent surgical outcomes. methods: a -year-old male with distal rectal cancer underwent neoadjuvant crt for a mrt cn m mremvi ? mrcrm ? disease. there was one left obturator node of mm prior to crt. following weeks of crt completion, the patient underwent tatme for the primary disease followed by left lateral node dissection by laparoscopy. results: the present video illustrates the most relevant surgical steps to perform lateral node dissection. the procedure has been didactically divided into steps. the left ureter is identified and retracted using a vessel loop (step ). identification of the common iliac vein and dissection with subsequent identification of psoas and internal obturator muscles (step ). identification and dissection of accessory vessels. ( step ) identification of obturator nerve and obturator vessels (step ). blunt dissection of obturator nerve (step ). identification and ligation of obturatory vessels. (step ) umbilical artery is skeletonized to allow identification and clearance of fatty tissue along superior vesical arteries, internal iiliac artery/vein, inferior vesical artery and internal pudendal artery (step ). postoperative course was uneventful. conclusion: standardization of lateral-node dissection for rectal cancer has paramount importance. laparoscopic lateral-node dissection for rectal cancer provides optimal anatomical view and allows safe dissection of the nodes of interest. aims: the aim of this video is to describe our technique using fluorescence to assess the lymph flow to ensure a complete mesocolic excision and central vascular ligation in order to provide expertise to contribute to the standardization of this new tool. methods: laparoscopic right colectomy with total excision of the mesocolon was proposed in all cases. for the detection of lymph flow, we injected indocyanine green dye ( milliliter of milligrams dye dilution in milliliter of distilled water) into the subserosal to submucosal layer around the tumor at point with a -gauge injection laparoscopically after trocar insertion, and observed the lymph flow using a near-infrared system (visera elite ii, olympus) after injection. we also performed a total mesocolic excision with central vascular ligation in the region where the lymph flow was fluorescently observed. results: ( %) patients were included. no intraoperative or postoperative complications presented. no adverse effects were reported due to the infusion of indocyanine green. the lymph flow was visualized intraoperatively in a satisfactory way helping the surgeon in decision making to determine an appropriate separation line of the mesentery. the section line of the mesocolon was modified in ( %) case based on the findings obtained by fluorescence. the mean operative time was ( ) min. the morphometric laboratory data of the specimens to audit the correct complete mesocolic excision were satisfactory according to the oncological standards. conclusion: fluorescence lymphography during colorectal surgery was feasible and reproducible with a minimum of added complexity. fluorescence-guided surgery may be a helpful technique for determining an appropriate total mesocolic excision in colon neoplasms. aims: this video shows our technique for complete mesocolic excision (cme) during right colectomy for cancer. methods: in this video, a years old patient underwent a laparoscopic right colectomy with cme for a cancer of the ascending colon diagnosed with a colonoscopy performed after positivity to fecal occult blood test (fobt). after ct scan staging we obtained d printed models to clarify patient's vascular anatomy. patient was placed in supine position, trocars were inserted in left quadrants as for standard right colectomy. cme is performed by sharp dissection between the visceral fascia that covers the posterior lay of the mesocolon and the parietal fascia that covers the retroperitoneum (toldt's fascia). the ileo-colic vessels are used as landmark to identify the right anterior surface of the superior mesenteric vessels. with a caudo-cranial approach, the mesocolon is sharply dissected and the root of tributaries venous is ligated, up to the inferior margin of the pancreas. the gastro-colic trunk is dissected out with ligation of the right colic vein, while the gastroepiploic vein is preserved (harvesting the sixth group lymph node). the pancreas-duodenum fascial plane is entered and all the lymphoid tissue around the vessel surface is harvested. procedure is completed with ileo-transverse intracorporeal stapled anastomosis. results: in our experience, between april and december , laparoscopic right hemicolectomies with cme were performed. we had no major intraoperative vascular lesions. no patients needed intraoperative blood transfusion. compared to our series of standard right colectomies we did not notice any significant difference in post-operative complications. the follow-up is too short to demonstrate if the cme approach has a better oncological outcome compared to standard right colectomy. conclusions: laparoscopic cme is feasible, although it requires a higher expertise level of surgical know-how. the quality of evidence is limited and does not consistently support the superiority of cme as compared to standard right colectomy. better data are needed before cme can be recommended as the standard of care for colon cancer resections. h. bando gastroenterological surgery, ishikawa prefectural central hospital, kanazawa, japan aim: in case of right-sided transverse colon cancers, it is necessary to dissect the lymph nodes around the root o f the middle colic vessels. but in this area there are dangerous organs, for example : pancreatic head, duodenum, and gastrocolic trunk. it is the point of our technique that we resect the accessory right colic vein and middle colic vein, and then dissect pancreas head and duodenum at early step of the operation. method: we perform the operation by five trocars. the first step is to transect the great omentum, and confirm the lower edge of pancreas.there are much adhesion between mesocolon of transverse colon and stomach, great omentum. it is very important to dissect the adhesion accurately. secondly, the mesocolon is incised at lower edge of pancreas. it is possible to detect the lower edge of pancreas in obese people. the anterior surface of superior mesenteric vein is exposed. the accessory right colic and middle colic vein are resected. and then front face of surgical trunk, pancreas, and duodenum is dissected caudally as possible. the superior mesentery artery is resected below the mesocolon after flip up of transverse colon. this approach is safe and feasible, because the dangerous organs are handled by direct vision. by that, extraction of intestine is easy from small incision. afer flip up of transverse colon, the mesenteric of ileum is incised. the root of ileocecal vessels is exposed and these are resected. the peritoneum of the front of superior mesenteric artery is incised, and the lymph nodes around the surgical trunk are dissected. this dissected area is easily connected with the one done beforehand. uniquely we resect the mesocolon and major omentum from the root of dissected vessels to resected side of transverse colon. and then right-side colon is dissected medial approach. conclusion: we dissect the dangerous organs in advance. that prevent major injury of them. background: good visualisation of the operative field is a fundamental requirement for safe laparoscopic colorectal surgery. over the past years of the senior author's experience, camera systems have evolved from single to three chip, high definition (hd) and most recently, the k system. in parallel, the rest of the infrastructure such as cables, processors, monitors etc. have also undergone improvements, resulting in improved image quality. aim/methods: we present a video of a case of laparoscopic total mesorectal excision (tme), performed with strict adherence to our previously published 'stepwise approach to laparoscopic colorectal surgery' which places particular emphasis on safety aspects. tme was performed in a year old male patient with history of previous abdominal as well as robotic prostatic surgery. the procedure was filmed with all components including the camera head, cables, processing unit, screens as well as the recording/mixing decks being k. multiple external k cameras were also used. live transmission to a remote audience as part of our masterclass was achieved using appropriate bandwidth and projection on to k screens. results: feedback from the operating team as well as from the live audience was that the image quality was far superior to hd systems. the k system accorded a degree of clarity well beyond usual expectations. the depth of field also appeared to be different initially, but within a few minutes of starting the procedure and acclimatisation, the effects were appreciable. the clarity of the image which showed the fine details of the dissection planes and anatomical landmarks as well as the vibrancy of the vasculature gave a distinct three-dimensional effect to the picture. this excellent visualisation added one more layer of safety and complemented our stepwise approach for a successful procedure. conclusion: the laparoscopic k system, in our practice, proved to bea beneficial visualisation tool to enhance the accuracy of dissection. vital structures appeared to be more vivid and clearer with dissection planes being more easily apparent. in our opinion the laparoscopic k system when combined with a systematic approach enhances safety, especially in complex laparoscopic colorectal surgery. accumulating evidence suggests that laparoscopic surgery for colon cancer has feasibility and efficacy equal to or over conventional laparotomy. for cases with pasthistory of laparotomy, especially history of colon resection, however, there is almost no evidence for laparoscopic recolectomy for metachronous colon cancer. since , we have been used submucosal local injection of indocyanine green (icg) around primary colorectal cancer by using intraoperative endoscopy, and complete mescolic excision (cme) have been convincingly carried out, which was clarified by completely resected icg positive area. although evidence on the oncological efficacy of icg guided surgery has not yet been clarified, since it can be easily judged whether cme is performed clearly, it is considered that icg guided surgery for primary colon cancer is useful for education. recently, we are applying this to ensure convincing cme for patients with colorectal cancer who had a history of colic resection. the representative case is as follows. a -year-old female was diagnosed as advanced sigmoid colon cancer, and laparoscopic sigmoidectomy with high tie of the inferior mesenteric artery was performed years ago. then she was diagnosed as the metachronous descending colon cancer. the feeding artery of the new tumor should be the left colic artery, however, the left colic artery was already resected and genuine feeding artery was not identified by preoperative examination. by injecting icg into submucosa endoscopically during operation, it was clearly observed that the lymphatic flow from the tumor was directed to the inlet portion of the inferior mesenteric vein (imv). re-cme was performed by ligating the inlet of imv. intraoperative icg was also useful for clarifying the borderline for adhesion detachment of pastoperation between the mesentery and retroperitoneum (figure) . interestingly, icg flow in the mesentery direct to of the anus side was disrupted clearly at the past anastomotic site. we believe that laparoscopic surgery under icg guidance is potential useful tool that can confirm evidence to date more intuitively in real time. further studies, ideally randomized controlled trials, are required for define the oncological usefulness of icg guided surgery for re-do colectomy. the operation movie will be presented at the meeting. background: laparoscopic lateral pelvic node dissection (llpnd) is a minimally invasive alternative to open surgical therapy for advanced low rectal cancer patients. in this video, we demonstrate the technique of llpnd for rectal cancer patients with suspicion of lln metastases after neoadjuvant chemo-radiation. methods: the principle of this approach is en bloc resection with bilateral peritoneum. the peritoneum is incised lateral to the ureter following the line between external and internal iliac vessels. in the next step, llpnd dissection of the regional lymph node and high ligation of inferior mesenteric vessels were performed. a contralateral llpnd was performed in the same manner as a mirrored technique. after extracting the specimen, an end-to-end double-stapled circular anastomosis was performed. results: the procedure was done safely without any complications.the surgical duration was mins, and the blood loss was ml. the number of harvested lateral pelvic lymph nodes was . the tnm stage was ypt an m . conclusion: this approach enables extended resection during lymph node dissection, allowing autonomic nerve preservation. it is maybe a helpful approach in the treatment of locally advanced rectal cancer with a lateral lymph node metastasis. aims: the aim is to present an inspection method where the anastomosis vascularity is testing simultaneously using the indocyanine green fluorescent angiography intraluminal and intraperitoneal. methods: sixty-five year old female patient underwent standard laparoscopic-assisted low anterior rectal resection for rectal carcinoma. the proximal end of the bowel and the stump of the distal rectum were checked using near-infrared fluorescence imaging with d-light camera. after making sure of adequate perfusion of the bowel, the end-to-end stapled anastomosis was performed under the laparoscopic visualisation. the d-port proctoscope was inserted into the anus. the second icg injection was administered. the perfusion of the anastomosis in transabdominal way and viability of the mucosa in transanal way was evaluated with two d-light cameras simultaneously. the anastomosis was determined cm from the anal verge. an air-water leak and tension of the bowel tests were performed. after evaluation of anastomosis viability with fluorescence imaging, after negative air-water leak and tensions testing, the decision was made by surgeon not to perform preventive ileostomy. results: the patient had no complains for the first three days postoperatively. nevertheless, crp level was growing and was . mg/l on the second postoperative day, and . mg/l on the th postoperative day. the patient complained of the pain in the right iliac area and below symphysis on the th postoperative day. the abdominal and pelvis computed tomography scan with oral contrast was performed which denied our thoughts about the anastomotic leakage. intravenous cefuroxime and metronidazole antibiotics were prescribed. the crp level was . mg/l on the th postoperative day. the patient was discharged on the th postoperative day without preventive ileostomy. conclusion: using the original, standardized colorectal anastomosis inspection method we can determine which patient doesn't need the preventive ileostomy after low colorectal anastomosis. the important causes of anastomotic leak are local ischemia and staple line defect. the purpose of this study was to investigate the combination of methods aimed to reduce the risk of anastomotic leak after anterior resections for rectal cancer. methods: we retrospectively analyzed perioperative outcomes of the first patients, who underwent modified laparoscopic anterior resection with partial mesorectal excision for rectal cancer without preventive stomy. operative technique was modified and included routine preservation of the left colic artery (fig )(aimed to improve anastomotic blood supply), manual suture invagination of the 'dog ears' (fig ) (aimed to reduce the risk of staple line defects), transperineal pelvic drainage and pelvic peritoneum reconstruction (aimed to reduce the risk of reoperation in case of leakage). anastomotic leak rate, reoperation rate, left colic artery preservation rate, additional operative time (time required for left colic artery preservation, 'dog ears' invagination and pelvic peritoneum reconstruction), blood loss, morbidity and mortality were analyzed. results: ( . %) patient developed an asymptomatic leakage, which was managed conservatively. there was no postoperative mortality and no reoperations. median additional operative time was min for the first procedures and min for the last procedures. left colic artery preservation was successful in ( . %) patients. median blood loss was ml. conclusions: additional techniques used in our modification of laparoscopic anterior resection are safe and may lead to improved perioperative outcomes. however, they are associated with increased operative time, which may be reduced with a better learning curve. introduction: parastomal hernias are a significant cause of post abdominal ostomy morbidity with an overall life-time incidence exceeding %. the complications can range from a bulge resulting in stoma bag leakage, to life threatening bowel obstruction. the prevent-trial sought to determine if prophylactic utilisation of polypropylene mesh would decrease the incidence of parastomal hernias, with initial results demonstrating that it was safe to use in permanent end stomas. aim: to demonstrate a reproducible and streamlined technique for laparoscopic parastomal hernia repair with intraperitoneal funnel mesh, and assess the outcomes with the clavien-dindo (cd) classification tool. method: parastomal hernia repairs ( colostomy, ileostomy) were considered, with the following approach adopted for each: swab sutured in stoma orifice to prevent wound contamination.sharp dissection of the stoma using parachute technique.stoma end refreshed followed by change of gloves and instruments.lateral stay sutures placed to tighten sheath later on.pneumoperitoneum temporarily created to assess/divide adhesions.funnel mesh placed in-situ, orientated in the optimal intraabdominal position, and sutured to the peri-colic fat to prevent slip.medial suture placed to narrow the sheath further.pneumoperitoneum re-created and mesh fixed in place with double crown laparoscopic tacks.redundant portion of end stoma excised and stoma formed. results: at median follow up of months: no recurrence.no reported symptoms of pain or decreased stoma functionality.one superficial wound infection treated with drainage at bedside (cd = grade ) conclusion: laparoscopic parastomal hernia repair with intraperitoneal funnel mesh for permanent end stomas yielded good outcomes in our patient cohort. a streamlined and reproducible approach ensures that the technique can be adopted for both prophylactic, primary and recurrent repair. parastomal hernias are common and can be associated with significant morbidity. when taking this into account, in conjunction with the recommendations of the initial results of the prevent-trial, one may consider prophylactic utilisation of a mesh in patients receiving a permanent end stoma. general surgery, rambam medical center, haifa, israel year old, female patient referred to our institution with common bile duct stricture, caused by iatrogenic injury during laparoscopic cholecystectomy. during last year, patient suffered from recurrent episodes of ascending cholangitis. recently, she underwent ercp and severe stricture of middle cbd was diagnosed. plastic stent was inserted through the cbd. mrcp also showed severe stricture of cbd with dilatation of biliary tree, proximal to the stricture. due to severe and resistant (did not resolved by recurrent dilatation) structure of middle cbd, she was referred to operation. patient underwent da vinci robot-assisted excision of the cbd stricture, hepaticojejunostomy and extracorporeal jejunojejunostomy of roux-an-y limb. total operating time was min. day three after operation patient started regular diet and was discharged home on day four. final pathology has shoved part of cbd with severe inflammation. aims: extrahepatic biliary duct resection for the treatment of bismuth i and ii stage klatskin tumor is the standard surgical technique [ ] . methods: a years old patient present at emergency room (er) with right upper abdominal pain with an elevation of the inflammatory markers at the blood exams and fever. the patient was submitted to a computer tomography (ct) that shows a tumor involving the lower tract of the principal bile duct. an endoscopic retrograde cholangio pancreatography (ercp) with biopsy (intraductal papillary neoplasm of the bile duct,ipnb with high-grade dysplasia) and stent placement was performed. considering the good general conditions of the patient and an absence of vascular and nodal invasion at the preoperative imaging, a minimally invasive surgical resection of the biliary tract with cholecystectomy was performed. results: a four port laparoscopic biliary tract resection with cholecystectomy was performed with lymphadenectomy of the hepatic hilum. no vascular or liver infiltration was found. the hepatic hilum was completely skeletonized. the resection of the biliary duct was performed with adequate free margin. a biliary reconstruction with roux-en-y technique was performed and a fully laparoscopic hepatico-jejunal anastomosis was done. and abdominal retro anastomotic drain was placed. the operative time was min. the postoperative course was complicated by a low rate biliary leakage that was treated conservatively. the patient was discharged at post operative day in good general conditions. the histological examination revealed a moderately differentiated in situ cholangiocarcinoma of the principal bile duct with the involving of the cystic duct with free resection margin (pt bn r ). conclusions: laparoscopic resection of the biliary tract is a challenging procedure that allows, in expert hands, to achieve in selected cases negative pathological margin, complete linfonode retrieval and entero-biliary bypass. injury to the extrahepatic bile duct during bile duct or hepatic surgery can be reduced by better real-time visualization. recently, indocyanine green (icg) fluorescence imaging has been used in laparoscopic hepatobiliary surgery. we applied icg fluorescence imaging in patient with huge hepatic cyst which severely deviated extrahepatic bile duct. the patient had received laparoscopic cholecystectomy and huge hepatic cyst stuck firmly with peri-hepatic structures including bile duct. icg fluorescence imaging correctly identified the common hepatic duct and remnant cystic duct and allowed for more meticulous and easier dissection. therefore, icg fluorescence imaging may guide a safe and accurate dissection and excision in hepatobiliary surgery. results: total patients who underwent ercp were , and . percent ( cases) had a first failed ercp and of then were unsuccesfull in the second intent of ercp. intrahospitalary stay was more than days in the percent, in the . percent was to days, with and average of days. conclusions: before, during or after lcbde, ercp remains the gold standard for manegement of choledocolitiasis confirmed by clinics, laboratory and imagenology. lcbde is a very good option that requires experience and specific skills, and especialized equipment. in years the rate of sucess in our hospital was . % and there were no posoperatory complications such as: biliar peritonitis, pancreatitis or liver abscess. aims: easier intraoperative recognition of the biliary anatomy may be accomplished by using near-infrared (nir) fluorescence imaging after an injection of indocyanine green (icg). neither radiological support nor additional intervention such as opening the cystic or common bile duct is required, making it an easy and real-time technique to use during surgery. the aim of this video is to describe our experience in fluorescence-guided cholangiography in different clinical situations. methods: intravenous injection of icg is used to illuminate extrahepatic biliary anatomy. however, the simultaneous enhacement of liver parenchyma can disturb the visualization of clinical details. the key is in the used dose of icg, the route of administration and the time since its infusion. in the first case, a scheduled cholecystectomy is shown in which a dose ( ml of mg dye dilution in ml of distilled water) administered intravenously h before the intervention was used. the second case shows an urgent cholecystectomy in which the dose ( ml of mg dye dilution in ml of distilled water) was administered intragallbladder during surgery. all patients underwent laparoscopic cholecystectomy with traditional four-port technique. all procedures were performed using a -degree mm laparoscope with nir imaging capability (visera elite ii, olympus). results: there were no intraoperative or postoperative complications. there was no increase in operative time due to the use of icg. in the first case, a clear identification of the cystic duct and the main bile duct was obtained thanks to the biliary excretion of the icg and the intravenous clearance. in the second case, the identification of the cystic duct, the main bile duct and the cystic artery occurred due to the intravesicular absorption of icg. conclusion: fluorescence-guided cholecystectomy clarifies the dissection plane. it can be considered to increase the safety of laparoscopic cholecystectomy. being aware of the doses, times and possible routes of administration is basic to universalize the technique and give it utility in different scenarios. introduction: mirizzi syndrome type is an uncommon cause of obstructive jaundice caused by an inflammatory response to an impacted gallstone in hartmann's pouch or the cystic duct with a resultant cholecystocholedochal fistula. the obstructive biochemical changes can be caused by direct extrinsic compression from the impacted gall stone or from the fibrosis caused by advanced chronic cholecystitis, or for the established fistula. objective: we present a case of a mirizzi type syndrome with choledocholithiasis which was solved by laparoscopy approach. material and methods: a -year-old female patient with no past medical history. the history of present illness begans with the presence of icteric dye since the last days; she received symptomatic treatment with poor improvement. a liver and biliary tract ultrasound was performed with report of a mm coledochus, mm wall gallbladder. then an endoscopic retrograde clolangiopancreatography was performed with successful endoscopic sphincterotomy and removal of gallstones. but the patient jaundice persisted after the procedure. the patient underwent cholecystectomy and laparoscopic common bile duct exploration, where the findings were a mirizzi type according to the csendez classification, chronic cholecistitis and choledocholithiasis. results: in this laparoscopic approach we performed a partial cholecystectomy, bile duct exploration with removal of residual gallstones. the closure of the choledocotomy was performed with simple knots using vycril . . a subhepatic drainage was left. the patient showed adequate clinical evolution. after days the patient was discharged. conclusions: it is important to properly identify the anatomy at the time of surgery to avoid injury of the common bile duct. operative treatment of mirizzi syndrome type includes either laparoscopic or open subtotal cholecystectomy or placement of a t-tube or choledocoplasty. near-infrared fluorescent cholangiography (nirf-c) is an innovative intra-operative imaging technique that allows a real-time enhanced visualization of the extrahepatic biliary tree by fluorescence. thanks to the development of laparoscopes/endoscopes with light sources emitting infrared frequencies, it is possible to visualize anatomical structures (vessels, ureters, bile ducts, etc.) through the luminous intensity of substances (fluorescein, blue of methylene, indocyanine green) which are injected into the patient. this technology may be considered as an important teaching tool for laparoscopic surgery, especially for young surgeons in their surgical learning curve and it could lead to reduce the risk of iatrogenic bile duct injuries during laparoscopic cholecystectomy. the following video is characterized by a series of intra-operative images of biliary anatomy by fluorescence, having an important educational interest, while also detecting anatomical variations of the cystic duct. a. umezawa, minimally invasive surgery center, yotsuya medical cube, tokyo, japan aims: laparoscopic cholecystectomy(lap-c) for cholecystolithiasis has become standard. however, serious bile duct injury has been reported as a complication. repeated colic and chronic inflammation in cholecystolithiasis lead to the so-called difficult gallbladder conditions, such as dense fibrosis and scarring of the tissue. dissection of calot's triangle includes the risk of bile duct injury. critical view of safety (cvs) is the most well-known land mark for safe cholecystectomy. in the revised tokyo guidelines (tg ), important land marks and bailout procedures had been proposed. those are for the difficult gallbladder which are not able to achieve cvs. methods land marks: baseline of segment of the liver and sulcus rouvier should be confirmed. the gallbladder wall itself is also useful landmark. bailout procedure: when the dissection of calot's triangle is considered impossible, bailout procedures should be considered. subtotal cholecystectomy which leave the neck is one of option. the fundus first technique is another approach. however, because fundus first technique has a possibility of leading to serious bile duct injury, it should stop by the neck. in this video, first case shows the importance of landmarks from near miss cases of misidentified injuries. second case shows bailout procedure, subtotal cholecystectomy with fundus first technique. result: in the atrophic gallbladder (case , near miss), it is liable to misidentify the junction of common bile duct as the gallbladder neck. the neck and common hepatic duct were lifted together easily. with confirming the landmark, misidentification was corrected and bile duct injury was avoided.in the case , since the calot's triangle was obscured due to repeated cholecystitis, dissection of gallbladder was performed from the bottom to the neck, and was excised with the cervical portion remained. the remaining neck was reconstituted.in each case, intraoperative cholangiography was performed, and it was confirmed that there was no bile duct injury. without postoperative complications, those patients were discharged pod as usual lap-c. conclusion: during lap-c for difficult gallbladder, the most annoying part is bile duct injury. confirming landmarks and switching bailout procedures can be contributory to avoid bile duct injury and to achieve safe lap-c. aims: choledocholithiasis is an important cause of morbidity and is present in about % of patients submitted a cholecystectomy. his treatment should be done in the same operative time, avoiding the morbidity and hospitalization time and costs of multiple procedures.the transcystic approach is preferable to prevent morbidity associated to choledochotomy.large stones can preclude this procedure. the use of laser lithotripsy to stone fragmentation is an option to provide transcystic extraction. methods: we present a video of laparoscopic transcystic common bile duct (cbd) exploration for choledocholithiasis. results: female patient, years old with a previous hospitalization for acute cholangitis with choledocholitiasis.submitted to laparoscopic cholecystectomy with intraoperative cholangiography that showed the presence of stone in distal cbd with cm size. the use of holmium laser lithotripsy made the stone fragmentation and provided his extraction by transcystic route using a basket.the patient was discharged at th postoperative day, with no complications. conclusion: the use of laser lithotripsy for large cbd stones is safe and effective, making possible the transcystic approach and preventing the choledochotomy morbidity. surg endosc ( ) :s -s gallbladder adenocarcinoma is rare and extremely aggressive. its' incidence is higher in elder females and its progression is rapid and silent with a dismal prognosis if diagnosed at advanced stages. we present the case of a years-old female with dyspeptic complaints. the abdominal ultrasound revealed a cm solid lesion of the gallbladder suspect for malignancy. the ct confirmed the presence of a vegetant mass on the free border of the gallbladder fundus with x mm. we performed a radical cholecystectomy with lymphadenectomy and liver bed excision. the post-operative period was complicated with a urinary tract infection, with full recovery after antimicrobial treatment. the histological sample revealed an adenocarcinoma of the gallbladder (t bn m ) and the patient remains asymptomatic and tumour free months after the surgery. gallbladder cancer treatment depends of the stage and clinical presentation of the disease. complete surgical excision is the only curative treatment and should include a limited hepatectomy and portal pedicle lymphadenectomy. laparoscopic surgery might be an option in early stages, although it is challenging and requires both expertise in hepato-biliary and laparoscopic surgery. seen at the emergency room for a two month history of abdominal pain associated with jaundice. she is evaluated by the surgical team and diagnosed with acute cholecystitis and moderate risk for choledocholithiasis. the initial surgical plan was cholecystectomy with intraoperative cholangiogram. during surgery, firm adhesions are found from the gallbladder to omentum. friable tissue with edema and easy bleeding. difficulty is encountered during the dissection of calots'triangle. an intraoperative cholangiogram is done through hartmans'pouch without identifying correctly the biliary tract. therefore, an endoscopic retrograde cholangiopancreatography (ercp) is done to visualize the correct anatomy. during the ercp, a stenotic common hepatic duct is found and no stones are visualized. a biliary endoprosthesis is placed. she is discharged asymptomatic. a month later, the patient is back in the emergency room with abdominal pain. after an abdominal ct scan, we found that the endoprosthesis had migrated to the th portion of the duodenum. a second ercp is done and this time we found a big stone ( . - cm) aims: when training in the residency you watch your teacher perform laparoscopic cholecystectomy with ease, and even yourself perform several steps. but as a young surgeon, when confronted with a patient with acute cholecystitis, you're filled with emotions, and you do not know where to start the gallbladder dissection. the aim of this presentation is to show to young surgeons that you can, and must achieve, critical view of safety when performing laparoscopic cholecystectomy for acute cholecystitis. methods: we present the case of a years old female patient, bmi of . , who presented with a grade ii (moderate) acute cholecystitis. following tokyo guidelines, we initiated antibiotics and general supportive care, but without clinical improvement. the patient was proposed for laparoscopic cholecystectomy. results: at initial exploration we identified a cm long gallbladder, with a thick wall, difficult to manipulate. we opted for an anterograde cholecystectomy, in our opinion the best option in acute cholecystitis. the dissection was started with hook electrocautery and then continued with a combination of blunt dissection with the aspirator and with the hook. when reaching the pedicle, blunt dissection was used in order to appreciate the anatomy of the cystic duct and cystic artery. after correct identification of these structures they ware clipped and cut. a drainage tub was then placed, and the abdomen deflated. conclusion(s): as a young surgeon, when dealing with acute you must maintain your calm, and try to achieve critical view of safety before transecting the cystic duct and cystic artery. this can be achieved with a combination of blunt and sharp dissection, keeping your camera clean and with a good collaboration with the assisting surgeon. conclusions: here, an easy and reproducible method is described for future macroscopic analysis by the surgeon following a cholecystectomy. in addition, we depict several frequent macroscopic abnormalities in order to provide surgical colleagues with some cases of abnormal macroscopic gallbladders. the left hepatectomy is a demanding and difficult procedure, still limited to reference centers. the caudal approach and exposure of the middle hepatic vein is a reliable way to achieve a safely and reproductible left hepatectomy. with this technique, exposing the middle hepatic vein, we believe that we can perform a safe and feasible laparoscopic left hepatectomy increasing the quality of this hepatectomy. we present a -year-old woman with an intrahepatic and common bile ductlithiasiswhich was previously submitted to an ercp. with an unsolved intrahepatic lithiasis the patient was proposed to alaparoscopic left hepatectomy. the minimally invasive approach for alpps in a patient with a large hepatocellular carcinoma in a liver with severe steatosis is shown. during the first stage a partial alpps is performed. pve is performed in postoperative day one. after days from the first stage both liver volume and function (by hida scan) are re-assessed. right hepatectomy (second stage of alpps) is then conducted by laparoscopic aproach. hepato-bilio-pancreatic, centro hospitalar são joão, porto, portugal a year old woman with a previous history of anxiety and catheter ablation to treat heart arrhythmias, was studied for for multiple pancreatic cysts incidentally discovered on a routine ultrasound. an mri was performed showing multiple cystic tumors throughout the pancreas, the largest of which was mm. this led to a suspicion of multi-focal, side-branch intraductal papillary mucinous neoplasm (ipmn), with minimal dilatation of the main pancreatic duct. an echo endoscopy was subsequently performed indicating probable multifocal ipmn. a fna was carried out during this procedure, with aspiration of cystic content which was sent for cea analysis and cytology. cytology was compatible with mucinous neoplasm with mild atypia and cea u/ml. a splenic preserving total laparoscopic pancreatoduodenectomy was proposed. the procedure was uneventful and the patient was discharged on the th post-operative day. pathology revealeded a mm ipmn, with severe dysplasia and foci of microinvasive ductal adenocarcinoma of mm-pt n r . indocyanine green immunofluorescence guided laparoscopic partial hepatectomy y. tai obtaining negative tumor margin during laparoscopic hepatectomy has always been a very challenging topic for surgeons in that the surgeons are not able to palpate the tumor during laparoscopic surgery. although intraabdominal echo is available, but it demands great experiences and skills. with the guidance of icg immunofluorescence, surgeons can avoid failure of not obtaining enough negative margins nor resect too much healthy liver. icg is often used to estimate the liver function prior to hepatectomy traditionally. it binds to plasma protein and has a peak absorbance at nm and emits fluorescence with a wavelength of approximately nm. icg is preferentially retained in or around biliary malignancies due to impaired biliary excretion of hepatocytes in the affected area. we performed icg immunofluorescence guided laparoscopic partial hepatectomy on a years old male who suffers from hcc located at segment and . icg was injected days prior to the operation day. while evaluation of liver is performed, it also allowed us to use a high-end laparoscopic camera system equipped with integrated filters for detection of near-infrared fluorescence. during the surgery, we were able to clearly locate the borders of malignancies through the use of integrated filters combine with icg injection. the pathology study also confirmed that the adequate tumor free margin ([ . cm) were obtained in both tumors and the patient's condition was stable as well. icg immunofluorescence guidance enables surgeons to obtain optimum result in tumor resection through laparoscopic surgery. it also has the ability to detect bile leakage. with the use of icg immuofluorescence, surgeons will have higher chances to achieve adequate negative margins. background: parenchymal sparing hepatic resection has the advantage of preserving valuable tissue in chemotherapy-treated livers, assuring an adequate future remnant volume without compromising long-term survival. moreover, the laparoscopic approach offers the decreased postoperative morbidity of minimally invasive surgery. whenever technically feasible, this kind of procedure should be considered a suitable alternative to the classic major hepatectomy for the treatment of multiple colorectal liver metastases. methods: -year old male with a previous history of laparoscopic sigmoidectomy in november for a pt n m sigmoid adenocarcinoma. a control scanner three years later showed liver metastases in segments v, viii, ii and caudate lobe. after chemotherapy (xelox), control mri and pet scans showed a good response. he was proposed for a laparoscopic parenchymal-sparing liver resection. results: total operative time was h and min with no intraoperative complications. patient presented a right atelectasis as the only postoperative complication and was resolved with respiratory therapy. he was discharged in days. pathology report showed that lesions on segment v and viii had no viable tumor ( % fibrosis) and lesions on segment ii and caudate lobe had moderately differentiated adenocarcinoma. margins were free in all the lesions. after a month follow up, the patient has no recurrence and normal liver function tests. conclusion: minimally invasive liver resection is possible in patients with multiple bilobar liver metastases and allows to perform parenchymalsparing surgery safely. difficult localization of lesions such as the caudate lobe are not a contraindication for this type of surgery. laparoscopic approach for perihilar cholangiocarcinoma is still poorly reported in the literature due to technical challenges secondary to the combination of major hepatectomy, lymphadenectomy and biliary confluence resection. despite this, in selected cases it can be a good option to provide a short term benefit to patients. the video reports the case of a perihilar cholangiocarcinoma with involvement of left bile duct and therefore requiring left hepatectomy. komagome hospital, bunkyo-ku,tokyo, japan aims: segmentectomy is an anatomic liver resection, in which the tertiary branches of the glissonean pedicles are selectively transected. however, the branching pattern of the tertiary branches varies depending on the case, particularly in segment (s ) and segment (s ). the extrahepatic approach to the glissonean pedicle from the hepatic hilum is very difficult depending on the branching pattern. furthermore, the distance of exposing the secondary branches that are to be preserved becomes longer, and there is an increased risk of biliary leakage and delayed biliary stricture due to excessive traction in laparoscopic surgery. therefore, laparoscopic s and s segmentectomy are considered technically difficult. we standardized the intrahepatic glissonean pedicle approach for laparoscopic s and s segmentectomy. methods: we standardized the intrahepatic glissonean pedicle approach for laparoscopic s and s segmentectomy. we identify the targeted glissonean pedicle intrahepatically after the parenchymal transection along the major hepatic vein or its branch running on the intersegmental plane, referring to the preoperative simulation by d imaging. (a)s segmentectomy; after the mobilization of the right lobe, the glissonean pedicles of s (g ) can be approached from the dorsal side by transecting the parenchyma between the ivc and the right hepatic vein. after the division of the g , the parenchyma is transected along the demarcation line and the rhv from the root side to the peripheral side. (b)s segmentectomy; first, the parenchyma is transected along the middle hepatic vein (mhv) from the root side to the peripheral. g is typically detected on the right dorsal side of the mhv. after the division of the g , the liver parenchyma is transected along the demarcation line and the rhv from the root side to the peripheral side. results: we have experienced cases of laparoscopic s segmentectomy and cases of laparoscopic s segmentectomy. conclusion: our approach to the g and the g is safe and very useful. laparoscopic anatomical segmentectomy of right anterior section is technically demanding because it is difficult to dissect the deep tertiary branches of right anterior portal pedicle (rapp). we present three cases of laparoscopic anatomical segmentectomy using the extrafascial and transfissural approach: ) anatomical resection of segment , ) anatomical resection of the ventral area ) anatomical resection of segment dorsal area. the extrafascial and transfissural approach means that the liver parenchyma along the fissure lines is opened, then the surgeon can confirm the glissonean pedicles and territory directly. the extrafascial and transfissural approach in laparoscopic anatomical segmentectomy of right anterior section is feasible and effective because this technique can easily be approached to the deep tertiary branches of rapp. repeated liver resection has significant role in patients with recurrent hepatocellular carcinoma (hcc) in several situations. laparoscopic redo surgery is becoming safer along with advance in surgical technique. we have performed laparoscopic re-resection for limited intrahepatic hcc recurrence. the aim of the present study was to investigate its significance comparing with first laparoscopic liver resections. subjects: patients with limited intrahepatic hcc recurrence after open hepatectomy underwent laparoscopic liver re-resection (n = ). methods: adhesion between abdominal wall and visceral organs was carefully divided, after the first laparoscopic port was safely inserted. adhesion between diaphragm and liver surface or between previous liver cut surface and colon or duodenum was also minimally dissected. approach to the glisson's pedicles at the hepatic hilum was often difficult due to previous surgical procedure, thus pringle's maneuver was generally applied. dissection of hepatic parenchyma approaching to the target glisson's branch was often preceded under the ultrasound-guidance. liver resection was performed using lcs, biclamp, and cusa using intermittent block of the hepatic inflow. operation time, intraoperative bleeding, morbidity, mortality, and postoperative hospital stay were compared with those in patients who underwent first laparoscopic liver resection during the same period (n = ). results: operation time was significantly longer in the re-resection group, possibly due to the adhesiolysis. meanwhile, no significant difference was detected in intraoperative bleeding, morbidity, mortality and postoperative hospital stay between the first and the redo surgeries. methods: the donor was a -year-old gentleman who decided to donate part of his liver to his wife suffering from viral liver cirrhosis and hepatocellular carcinoma. his bmi was . kg/m and the preoperatively estimated donor's right liver volume was ml, representing . % of his entire liver. with the recipient's weight of kg, the graft to recipient weight ratio (grwr) was . %. the liver had classic hilar anatomy except that the right posterior intrahepatic duct seperately joined to the left main hepatic duct. after isolation and clamping of right hepatic artery and portal vein, indocyanine green of . mg was injected intravenously. results: the total operation time was min and the estimated blood loss was ml without transfusion. indocyanine green fluorescence image clearly demonstrated the anatomical demarcation between the lobes and visualized the running of the biliary tree. his postoperative course was uneventful and discharged postoperative day . conclusion: real-time indocyanine green fluorescence image may be particularly helpful to delineate anatomical surgical plane and to determine the appropriate division point of hepatic duct during laparoscopic living donor hepatectomy. surg endosc ( ) the correct management of intraoperative volemic status is essential in laparoscopic liver resection in order to control bleeding and to perform even complex procedures with a good profile of safety. central venous pressure is not really reliable in laparoscopy, due to presence of the pneumoperitoneum and patient position. monitoring of haemodynamic parameters via vigileo system is a minimally invasive method to control stroke volume variation, cardiac output, cardiac index and oxygen delivery in order to optimize the anaesthesiological management by controlling venous bleeding and avoiding tissutal ischemia. introduction: non-hydatid liver cysts represent a heterogeneous group of disorders that differ in their etiology, prevalence and clinical manifestations.within them, the simple hepatic cyst is the most frequent.the majority of simple cysts are an incidental finding during the performance of an imaging test for another unrelated cause and few of them are symptomatic or are associated with complications, and surgery is not necessary in most of them. described various therapeutic approaches so far there is no consensus about the optimal treatment of simple symptomatic, complicated or growth-showing liver cysts during its follow-up. currently the laparoscopic approach is widely used for the management of cysts hepatic, with results similar to open surgery but with the advantages of laparoscopy. objectives: to demonstrate the safety and efficacy of the laparoscopic approach in the approximation of complicated simple hepatic cysts.material and method: clinical case: a -year-old female patient with a history of: giant hiatus hernia intervention with laparoscopic nissen, fibromyalgia, previous ischemic colitis. hospital admission due to pneumonia and right pleural effusion with us: simple cyst x x mm in segment v hepatic, with dilatation of biliary radicals adjacent to the cyst, distended gallbladder with irregular walls in the hepatic side. ct: cystic lesion in segment iv-v of the liver, which has increased in size, with small microabcesses adjacencies to the lesion, thickening of the gallbladder wall, to assess cholecystitis. antibiotic treatment is established with good evolution, deciding surgery. results: intervention: complete laparoscopic approach, trocars, edematous cholecystitis, large retroyuxta vesicular cyst,with thickened walls with serous content. cholecystectomy maintaining the cyst wall, puncturing and taking samples for cytology and biochemistry of the contents, resection of the cyst wall, partial flare of its internal surface, negative intraoperative biopsy, epipoplasty, with drainage placement.correct postoperative course.pathological anatomy: simple biliary cyst with negative cytology, ck ?, ck -, calretina-. conclusion: the treatment of choice of complicated simple hepatic cysts is laparoscopic.we recommend performing an intraoperative biopsy of all resected liver cysts to confirm its nature,we propose cyst enucleation as the best surgical treatment. objective: the objective of the following case is to present a patient with symptomatic polycystic liver disease, which was solved by laparoscopy approach and the management of its complications. material and methods: the case reported is about a years old female patient with abdominal pain in upper right quadrant associated to asthenia, adynamia and hyporexia. ct scan reported heterogeneous liver with multiple ovoid images with regular edges defined which the biggest one measure x x mm with volume of cc on segment and , which comprises stomach, and the other one in segment with a volume of cc and others small sized located in segment , and b. results: in this laparoscopic approach, we performed a cyst unroofing of the two biggest cysts as well as cholecystectomy because of firm and lax adhesions. the patient evolved with fever in the th day postsurgical day and biliary leaking in a volume of cc in hrs. an ercp (endoscopic retrograde cholangiopancreatography) was asked for that was carried out by finding leak at the intrahepatic biliary duct therefore; esphinterotomy with placement of plastic endoprotesis was performed. the patient evolved without complication and was discharged at the th day. conclusions: only symptomatic polycystic liver disease needs to be treated. the choice of treatment is not yet standardized, for voluminous cysts the unroofing ideally by laparoscopy is the gold standard and the ercp is the elected treatment when the biliary leak appears as a complication. introduction: laparoscopic liver resection (llr) for tumors located in the posterosuperior segments of the liver (segments (s) or ) is a challenging procedure. especially, llr for s is difficult because the access of instruments is limited, bleeding control is not feasible, major llr is sometimes required, and obtaining sufficient resection margin is not easy. to overcome this obstacles, we performed llr in s with a lateral approach using intercostal trocars. to obtain competent resection margin, llr through right hepatic vein (rhv) first approach was performed for . cm mass located near the rhv in a year old female. case: after full mobilization of right liver including all short hepatic veins and caudate lobe, rotate the whole liver completely to the left side to approach to the root of rhv. one intercostal trocar was inserted to access the lesion. parenchymal transection started from the confluence of hepatic vein and then, followed along rhv with ligating several small branches from rhv. resection margin was demarcated after localization using laparoscopic ultrasonography. after completion of parenchymal dissection using cusa and ultrasonic shears, hemostatic agents were applied and drain was inserted. operation time and estimated blood loss were mins and ml. the patient was discharged without any complication on postoperative day . final pathological assessment confirmed clear resection margin (safety margin : . cm). conclusion: laparoscopic s segmentectomy with hepatic vein first approach technique is safe and recommended to obtain better resection margin. aims: simple liver cysts are the most common cystic lesions of the liver. most are diagnosed casually in image tests such as ultrasound or computerized tomography, most of which are asymptomatic and do not require treatment. in symptomatic patients (abdominal distension with palpable mass, abdominal pain, dyspnea, jaundice, etc.) the clinical manifestations are usually due to the growth of the cysts or the compression of neighboring structures. liver function tests are usually not altered. intracystic complications occur in less than % of cases and malignancy is exceptional. in this video, we present the case of a symptomatic patient with polycystic liver disease including a large size hepatic cyst. material and methods: -year-old woman with a personal history of arterial hypertension, saos, partial hysterectomy due to endometrial cancer, who was referred to our department complaining of supraumbilical pain and abdominal distension with palpable mases. abdominal ultrasound showed cholelithiasis and multiple simple hepatic cysts. in ct scan, multiple hepatic cysts were found, the largest one of about cm of larger diameter. echinococcus granulosus serology test was negative. there was also no evidence of cancer disease in pet scan. results: a laparoscopic approach was performed with four trocars, three of mm and a hasson trocar inserted thought a umbilical small incisional hernia. aspiration and wide unroofing of the large size cyst and smaller accessible ones was done. the patient also underwent cholecystectomy with intraoperative cholangiography and umbilical eventroplasty. the patient recovered uneventfully and is asymptomatic one year after surgery. conclusion: simple liver cysts rarely require treatment. in some cases, especially in large, complicated and symptomatic simple liver cysts, surgery is indicated. laparoscopic fenestration treatment is the best choice. aims: liver resection is the preferable initial treatment option for solitary or limited multifocal hepatocellular carcinomas. surgical indications for laparoscopic liver resection (llr) are the most important consideration, like liver function, tumor size (diameter less than cm) and location (easy technical access like in the left lateral section or on the surface of the inferior region). partial liver resection or left lateral sectionectomy are the typical procedures for such tumors and are considered the best way to begin llr. with accumulating experience and technical advancement, llr has been performed for tumors larger than cm and for others locations. some requirements to perform llr are to have experience in liver surgery and laparoscopic also, adequate technology and intraoperative ultrasound. methods: a -year-old male smoker, ex-parenteral drug users with chronic hcv liver disease child-a stage. he is diagnosed with a single lesion of cm in segment iii of the liver, biopsied twice without conclusive diagnosis and with a three-phase ct suggestive of hepatocarcinoma li-rads with data of portal hypertension (pht) and mild ascites. after the study is commented on tumor committee deciding surgical intervention. results: a laparoscopic resection of segment iii was performed with trocars. liver is explored by intraoperative laparoscopic ultrasound. vascular control was performed using the pringle technique. liver transection was done with sonostar until identification of intraparenchymal segment iii vascularization, which is sectioned with endogia ( mm) with seamguard. after the resection, we perform hemostasis control with electrocoagulation and hemostatic material. intraoperative bleeding of ml. favorable postoperative evolution, high on the th postoperative day. ap: cm trabecular hepatocarcinoma moderately differentiated pt b, r resection. conclusions: llr allows major liver resections with low morbidity and mortality and the advantages of laparoscopic surgery. an efficient learning curve can be achieved by a parallel evolution of procedures and indications (according to modified bclc staging system and treatment strategy). studies suggest that llr results in less blood loss, shorter postoperative hospital stays, lower abdominal wall trauma and lower incidences of ascites accumulation and postoperative liver failure. with respect to oncological considerations, tumor margins are adequately maintained during llr. v. drakopoulos, s. voulgaris, i. iliadis, k. botsakis, p. trakosari, v. vougas st department of surgery and transplantation unit, district general hospital of athens « evangelismos » , athens, greece introduction: laparoscopic surgery is gaining acceptance in the treatment of liver metastasis. laparoscopic treatment of liver metastasis often presents technical difficulties and requires an extensive learning curve. material-method: we present the case of a year old woman presented with a liver metastasis in section of the liver. the patient had been submitted to a laparoscopic low posterior resection in february . patient underwent laparoscopic left lateral hepatectomy, with the use of three trocars (umbilical mm, and two in the midclavicular line bilaterally.) left lateral hepatectomy was conducted with the use of a linear stapler. the postoperative period was uncomplicated and the patient remains in good condition three months after surgery. conclusion: laparoscopic approach seems to be safe for treatment of liver metastasis, offering better surgical field view and less postoperative complications. year survival rate after laparoscopic hepatectomy is compared to the open approach. general surgery, chang gung memorial hospital kaohsiung division, kaohsiung, taiwan purpose: laparoscopic hepatectomy is a quickly growing method for liver tumor because of modern technology. but for the ihd thrombosis, it is still technique dependent. the video was tried to share our experience for special case. material and method: one y/o female patient suffered from fever episode and image show s cm hcc with right anterior ihd obstruction r/o tumor thrombosis, hilum ln enlargement, double right portal vein, hilum adhesion with duodenum, no ascites . lab data : no-b, no-c child a, afp , icg clearance rate . %, plt . heart, lung function exam normal. the laparoscopic right total hepatectomy and hilum ln dissection was conducted. results: laparoscopic approached was performed. the hilum ln dissection was done with vessel and bile duct isolation. hilum ln frozen show negative malignancy. hemi-vessel control was done with resecting the vessel. right hepatectomy was done with preserving middle hepatic vein. the right anterior and posterior ihd was opened and tumor thrombosis was removed from right anterior ihd carefully. the stump of ihd was closed by suture separately. the total op time was min with cc blood loss. post op minimsl bile leakage was found in the drain at day . the patient discharged at day with drain. conclusions: laparoscopic hepatectomy may be a feasible method for hcc even with ihd tumor thrombosis. surg endosc ( ) introduction: the progressive laparoscopic learning in gastric surgery and the great development of instruments and laparoscopic material that facilitates the realization of advanced procedures, has led to an increase in the use of laparoscopy in the treatment of gastric cancer. material and methods: we present the case of a -year-old man without amc with a history of ischemic heart disease who enters our surgery department for cholangitis secondary to choledocholithiasis. ercp is requested during his admission that describes a gastric lesion from which a biopsy is taken, making it impossible to access vater papilla to perform sphincterotomy and lithiasis extraction due to the existence of duodenal diverticula. the result of pathological anatomy of the gastric lesion was compatible with adenocarcinoma. negative extension study. the clinical case is presented in a committee of multidisciplinary tumors and it is decided to perform surgical intervention of both pathologies. a subtotal gastrectomy was performed with a roux-en-y reconstruction. surgical time of min. choledochotomy was performed with lithiasis extraction, as well as intraoperative exploration of the bile duct and main conduits by means of a choledochoscope. results: income of days, with a clavien ii. the definitive pathological anatomy was an ai stage with a total of isolated nodes without evidence of neoplasia in any of them, therefore it does not require adjuvant treatment. the patient is asymptomatic, with nutritional supplementation with follow-up in ccee of surgery. conclusions: in our case, there were no serious postoperative complications when performing gastric resection and bile duct exploration with drainage of the same. from the oncological point of view, the number of lymph nodes extracted and the surgical margins are similar to those obtained in patients in whom we perform open surgery; therefore, although it is a single clinical case, laparoscopy in expert surgeons is a safe and effective technique. the puestow procedure was initially proposed to alleviate the pain in patients with chronic pancreatitis and dilated wirsung duct. its objective is to provide an efficient drainage of the pancreatic fluids and, in the meantime, to preserve the pancreatic tissue and minimize the risk of endocrine and exocrine pancreatic insufficiency. aims: to describe the particular technical aspects and the efficacy of totally laparoscopic puestow procedure in patients with cystic duodenal dystrophy. methods: a years old patient presenting diffuse epigastric pain, vomiting and weight loss was diagnosed at endoscopic ultrasound and biopsy with cystic duodenal dystrophy. a conservative treatment was decided with octreotide and opioids. however, due to the persistence of symptoms surgery was performed. results: due to the association of a dilated wirsung duct, the patient was submitted to a puestow procedure. the surgical procedure was completed in a minimally invasive manner; after dissecting the anterior surface of the pancreas an intraoperative ultrasound was performed in order to identify the wirsung duct. therefore, the pancreatic parenchyma was transected along the wirsung duct, a totally laparoscopic pancreato-jejunostomy on roux en y limb being performed. the early postoperative outcome was uneventful, the patient being discharged in the sixth postoperative day. at one month and six months follow up the need for opioid treatment significantly diminished. a kinking of the enteral anastomosis required a laparoscopic intervention one year after with a very good evolution after. conclusions: totally laparoscopic puestow procedure seems to be a safe and efficient method in order to treat symptomatic patients with cystic duodenal dystrophy in whom a dilated wirsung duct is present. aims: the approach to the intraductal papillary mucinous neoplasm (ipmn) is various, from a radiological follow-up with magnetic resonance (rm) to the surgical treatment with a pancreatic resection [ ] . the surgical approach is various and depends on the localization of the lesion and on the surgical skills [ ] . methods: a years old patient was admitted at the chi possy-saint germain-en-laye with an acute pancreatitis. at the ecoendoscopy was found a pancreatic cystic at the junction of the pancreatic body and tail with a wirusng diameter of mm. a second episode of acute pancreatitis occurred a few months later. after that episode the patient was submitted to a computer tomography (ct) that found a cystic lesion of cm with an increasing dilatation of the wirsung duct. the serum ca - was ui/ml. a laparoscopic sils distal pancreatectomy with spleen conservation was performed. results: a trans-umbilical incision was performed with the positioning of the gelpoint sils platform with the placement of trocars. a distal pancreatectomy with a spleen preservation and without a standard linfadenectomy was performed. the pancreatic stump was closed with an endo-gia mm with seamguard device. any drain was placed. the post-operative course was uneventfull. a ct scan was performed in …. post-operative day which didn't show collections. the patient was discharged in -…… post'operative day. the histological examination shows an ipmn with low grade dysplasia. no invasive carcinomatoses cells were found. the distal pancreatic sils resection with spleen conservation is a feasible and safe technique that combine all the advantages of the minimally invasive laparoscopic approach with the esthetic advantages of the sils approach. pancreato-duodenectomy is a complex surgery, requiring several anastomoses to reconstruct the digestive tract. due to its technical complexity, the laparoscopic approach is not yet the goldstandard and there remains some controversy about its oncological safety. worldwide experience is limited, and its safety and effectiveness are yet under evaluation.we present the clinical case of a years-old woman with a prior history of epilepsy. she was studied due to painless obstructive jaundice and a cm pancreatic head tumour was diagnosed on imaging, causing cbd and wirsung channels' dilatation. the tumour was considered locally resectable and she was proposed for a radical pancreato-duodenectomy.we present the main steps of the surgery including the oncological resection with lymphatic basin clearance and totally laparoscopic reconstruction.the post-operative was uneventful, and the histologic sample revealed a ductal adenocarcinoma (t ) with an r resection and / lymph nodes invaded. although technically demanding, laparoscopic pancreato-duodenectomy is safe and effective requiring teams with experience both in pancreato-biliary and laparoscopic surgery. chronic pancreatitis is characterized by a progressive pancreatic fibrosis with loss of endocrine and exocrine function. one of its main symptoms is debilitating pain. surgical drainage of a dilated pancreatic duct is an option to consider in cases of refractory pain. longitudinal pancreato-jejunostomy allows an effective decompression of the pancreatic channel and a significant improvement in the quality of life. we present the clinical case of a years-old lady with a prior history of gallstones. she was treated for an acute pancreatitis in may , followed by recurrent relapses of pain and enzymatic elevation. she required opioid use for partial pain control and a significant kg decrease on body weight due to 'fear of eating'. the endo-ultrasonography and the mri revealed a chronic pancreatitis with an mm wirsung duct with ductal stones and an atrophic body and tail. we proposed a laparoscopic longitudinal pancreato-jejunostomy. the surgery was performed with trocars, with the surgeon on the right side of the patient. we performed a trans-mesocolic cm pancreato-jejunostomy. the post-operative was uneventful, and the patient was discharged on the th post-operative day, asymptomatic. laparoscopic longitudinal pancreato-jejunostomy, although effective is a technically demanding surgery but brings the benefits of a minimally invasive approach. background: preservation of spleen in distal pancreatectomy is also useful from the maintenance of platelets and the prevention of overwhelming post splenectomy infection. we have performed laparoscopic spleen preservation distal pancreatectomy: lspdp to benign and low-grade tumors of the pancreatic body tail. the aim of this study was to report our surgical experience with the method of svp: splenic vessel preservation and wt: warshaw technique of lspdp, describe our techniques with videos. method: there are three points of our surgical technique. , precede pancreatic dissection, improve the mobility of the pancreas. , confirming the courses of splenic artery and classified them into two major types. , preserving the left gastro-epiploic vessels and short gastric vessels.the postoperative cases of lspdp which performed from april to september was retrospectively studied. result: of consecutive patients were performed lspdp at our institute, were svp and were wt. ages, gender and bmi were similar for two groups. there were no significant differences in operative time, blood loss and length of stay after surgery. comparing pathological finings, wt was associated with a slightly large tumor lesion (median mm vs. . , p = . ). among the median observation period of months, splenic infarction was observed in case in svp and cases in wt. however, they were focal splenic infarctions, they did not need surgery or drainage. there were no cases in which late onset of splenic artery occlusion or esophageal / gastric varices. conculusion: after performing lspd, the function of the spleen was good in all cases. both svp and wt were safe and feasible procedures. this is the case of a -years-old lady presenting with recurrent abdominal intractable pain she has been suffering from for the last years. msct revealed pancreatic calcifications from mm to - mm and dilatation of the main pancreatic duct in the body of the pancreas up to mm. the patient underwent laparoscopic local resection of the head of the pancreas combined with longitudinal roux-en-y pancreaticojejunostomy-a technique known as frey's procedure. it is recognized as an effective therapeutic option for the surgical treatment of patients with persistent pain caused by chronic pancreatitis.after performing the posterior wall of the pancreaticojejunal anastomosis we've faced an intraoperative complication such as volvulus of the roux limb causing serious ischemia of the limb. we were forced to remove all previous sutures in order to untwist the roux limb, thereafter the pancreaticojejunostomy was started anew.the purpose of this video is to demonstrate that frey's procedure can be performed in a minimally invasive fashion, which provides all the well-known advantages of this approach. we demonstrate that even such serious intraoperative complication as volvulus of the roux limb can be managed without conversion. our center has an experience of over laparoscopic frey's procedures, however this is the first case where we encountered with such complication and we believe this is an experience worth sharing.yet we would like to underline that this approach should be used by highly skilled minimally invasive surgeons experienced in intracorporeal suturing which is the most challenging stage in frey's procedure. v. tomulescu, i. hutopila, c. copaescu spleen preserving distal pancreatectomy (spdp) is commonly applied in patients with benign or low-grade malignant tumors in the body and tail of the pancreas. two surgical techniques for spdp have been described. the first technique was described by kimura (spleen preserving distal pancreatectomy with splenic vessel preservation-spdp-svp) and preserves the main splenic artery and vein and excises the tail of the pancreas and those small, short vascular connections to the body;the second technique was described by warshaw and involves resection of the splenic vein and artery before distal pancreatectomy, and conservation of theshort spleno-colic and gastric vessels to keep normal blood flow for the spleen (spleen-preserving distal pancreatectomy with splenic vessel resection-spdp-svr). we present the case of a years old female with / mm tumor of the pancreatic tail on ultrasonography. ct scan confirmed the tumor and endoscopic ultrasonography with fna have shown a solid pseudopapillary tumor. due to the low grade malignancy we have decided to perform a laparoscopic spleen preserving distal pancreatectomy with splenic vessels preservation (lspdp-svp). for lspdp-svp the difficulty is related with the splenic vessels dissection and manipulation. primary dissection and control of main trunk of splenic artery and vein will help to quickly control bleeding during vascular rupture in small vessels dissection. optimal stapling of any tissue requires an adequate tissue compression time to allow elongation of the tissue being compressed, smooth firing of the instrument, consistent staple line formation balanced against the risk of increased tissue tearing and excessive tensile strength. this is why, for pancreatic division, we prefer choosing a cartridge loaded with higher staplers. the pancreatic stump transection line is evaluated for bleeding and when it is needed, hemostatic clips are applied. histology report confirmed a solid pseudopapillary tumor t nomxl v r at this moment with month good follow up. in conclusion lspdp-svp is safe, reproductible and demonstrated very good outcomes when certain indications are respected. surg endosc ( ) aim: advances in minimally invasive surgery has permitted to perform complex techniques by this approach, being the laparoscopic duodenopancreatectomy (lpd) one of these. the aim of this communication is to present a surgical technique video for a complete laparoscopic pd, showing the most important steps of the resective and reconstructive phase, with the anastomosis realized completely by laparoscopy. methods: a surgical technique video is presented showing the main steps for the lpd and a complete laparoscopic reconstruction with an hepaticojejunostomy, duct-to-mucosa pancreatic-jejunostomy and a gastrojejunostomy. results: an years old woman with past medial history of arteria hypertension, dyslipidemia, type ii diabetes mellitus and a breast cancer treated in with lumpectomy and axillary lymphadenectomy plus radiotherapy, recently diagnosed of and adenocarcinoma of the head of the pancreas. the ct scan showed a neoplasia localized in the head of the pancreas without extension to other organs. a laparoscopic pd was indicated after a multidisciplinary committee evaluation. a supraumibical hasson trocar was used for the pneumoperitoneum, three mm trocars and two mm trocars were used. lpd was performed. the resective phase was done following the conventional steps of the open whipple procedure and for the reconstructive phase, a child limb was used for a termino-lateral hepatico-jejunostomy with an absorbable / monofilament; a duct-to-mucosa pancreatic-jejunostomy with an absorbable / monofilament and finally a latero-lateral mechanical gastro-jejunostomy was performed. surgical time was min. postoperative course without complications and the patient was discharged on the th postoperative day. definitive anatomopathological exam: intraductal tubulopapilar neoplasia, x x mm, with wide high grade epithelial dysplasia. free margins. ptisn ( / ). conclusion: laparoscopic pd is a feasible procedure with a high technical requirement which should be performed in specialized centres with high experience in hepatobiliary surgery and in advanced laparoscopic procedures, because of its high morbidity and mortality. conclusions: robotic assistance in whipple may overcome limitations of laparoscopy and offer a minimaly invasive approach to this procedure potentially resulting in lower blood loss and less morbidity. we need further prospective randomized trials in order to determine the exact role of robotics in pancreatic surgery. aims: distal pancreatectomy is the standard curative treatment for symptomatic benign, premalignant, and malignant disease of the pancreatic body and tail. the most obvious benefits of a laparoscopic approach to distal pancreatectomy include earlier recovery and shorter hospital stay. spleen-preserving distal pancreatectomy should be attempted in case of benign disease. laparoscopic spleen-preserving distal pancreatectomy (lspdp) is expected to be less invasive than laparoscopic distal pancreatectomy with splenectomy. however, there are few reports regarding the details of the procedure for lspdp, and its safety remains unclear. this study aimed to evaluate the feasibility and safety of lspdp. methods: retrospective analysis of surgery treatment of patients was made. lspdp was conducted in the period from to in the department of laparoscopy surgery of state institution o.shalimov national institute of surgery and transplantology. the average age was : . years, the body mass index (bmi) was . ± . results: laparoscopic distal pancreatectomys was performed in % of cases, were attempted in female and male patients. postoperative pathological examinations revealed cases of serous cystadenoma in the body and tail of the pancreas, case of serous oligocystic adenoma, case of mucinous cystadenoma, case of neuroendocrine tumor (insulinoma), and case of solidpseudopapillary neoplasm. complications related to the surgery were like acute pancreatitis with -fold increase normal plasma amylase confirmed by ct- cases, fluid collection- cases, pancreatic fistula (grade a)- cases. the operation time was . min, (range - min) blood loss of . g (range - g), mean hospital stay was . days (range - days). conversion to laparotomy was in case. mortality was . conclusion: laparoscopic spleen-preserving distal pancreatectomy is minimally invasive, safe, and feasible for the management of benign pancreatic tail tumors, with the advantages of earlier recovery and less morbidity from complications. aims: a pancreatic pseudocyst is an encapsulated, mature fluid collection occurring withing the pancreas that have a well-defined wall minimal or no necrosis secondary to pancreatic injury and mediated by the enzimatic and inflammatory disruption of pancreatic tissue. it is a common complication of acute and chronic pancreatitis. we present the case of a pancreatic pseudocyst located within the body of the pancreas due to recurrent necrotic pancreatitis. the objective of this video is to show the minimally invasive surgical approach of this entity. methods: a -year-old man without medical history was admitted to hospital in the digestive service on times for acute necrotizing pancreatitis. after study in which is evidenced cholelithiasis and pseudocyst in pancreatic body of cm maximum diameter and formation of two peripancreatic collections without signs of superinfection, cholecystectomy is indicated. magnetic control cholangiography was performed after surgery and it showed an increase in the size of the pancreatic pseudocyst, suspecting wirsung's duct disruption. therefore, endoscopic retrograde cholangiopancreatography (ercp) was performed by placing a plastic pancreatic prosthesis and performing a sphincterotomy. after hospital discharge, the patient is re-admitted due to recurrent abdominal pain without analytical alteration. tc abdominal observed an increase in the pseudocyst from to cm. this case was discussed in a multidisciplinary committee and surgical intervention was decided. results: laparoscopic approach is decided and four trocars were placed. initially, a gastrostomy was performed with liquid outlet. an aspiration of the liquid and quistogastrostomy with mm endogia was made. the patient progresses favorably, being high on the tenth postoperative day, without complications. conclusions: almost every pancreatic pseudocyst improves spontaneously and needs no specific treatment. draining is indicated when secondary symptoms to compression, complications or rapidly enlarging are found. depending on the complexity of the pseudocyst, its communication with wirsung's duct and the existence of ductal injury, it may perform a percutaneous, endoscopic or surgical drainage. the goal of pancreatic debridement is to excise all dead and devitalized pancreatic and peripancreatic tissue while preserving viable functioning pancreas, controlling resultant pancreatic fistulas, and limiting extraneous organ damage. only the surgical procedure is definitive. case: a y old male presents with intermittent low retrosternal pain and progressive dyspnea with exercise since a couple of months. cardiac investigation was negative and gastroscopy showed a grade b esophagitis. he was treated medically but with only partial response. on a thoraco-abdominal cat-scan the diagnosis of a left sided bochdaleks' hernia was made. the hernia includes the left kidney (with blood vessels and ureter), transverse colon and small intestine which are positioned in the left lower thoracic cavity with the left lung considerably compressed. method: given the clear correlation between the patients' complaints and these anatomical findings, he was referred to our service of abdominal surgery. we performed a laparoscopy with the patient in lithotomy position and the surgeon between the legs. the patient was tilted to his right side. mobilization of the spleen was necessary to gain maximal access to the hernia. we were able to reduce all the herniated content, freed the margins of the defect, reduced the hernia sac and repositioned the kidney intra-abdominally. the defect was manually closed with non-resolvable stitches and covered with a mesh which was secured with tackers. result: postoperatively the patient recovered well with adequate pain relief and pulmonary support. he could leave the hospital after days. control cat-scan on day postoperatively shows an intact lining of the diaphragm with normal positioning of the intra-abdominal organs. on follow-up weeks after surgery the patient had regained normal activities and was symptom free. conclusion: a symptomatic left sided bochdaleks' hernia in adults with an ectopic intrathoracic kidney is extremely rare. we hereby state that, during a laparoscopic repair, the kidney can also be safely reduced, which has almost never been described in literature yet, enhancing pulmonary recovery, improving access for mesh placement and thus diminishing recurrence rate. aims: large incisional hernias repair involves an actual problem for surgeons to face. anterior component separation has been an important method allowing to close the fascia defects without tension while also having underlay mesh reinforcement.therefore, we present a case of incisional hernia reparation performing endoscopic anterior component separation with advantages compared with open approach. method: we present the case of a -year-old woman, bmi kg/m , with previous laparoscopic gastric sleeve and posterior reintervention using open approach. the patient presented a cm size incisional hernia m w . a ct scan was performed, confirming a midline incisional hernia containing colon, with an herniary defect of cm. full minimal invasive abdominal wall repair was proposed. a cm size incision was made in left iliac region to reach the aponeurosis of external oblique muscle. we placed a balloon trocar and subcutaneous pneumo-dissection with mmhg pressure was performed; then, we placed a mm trocar in left lumbar space. the aponeurosis of external oblique muscle was incised and anterior component separation from inguinal to subcostal area was achieved. an extensive intermuscular dissection was performed to achieve complete midline closure. we performed the same procedure on the right side. then, with laparoscopic approach using v-loc n° suture, we completely closed the midline. eventually, we placed a x cm ptfe-c mesh fixed with a double crown of tackers and fibrin glue. results: postoperatory course was uneventful and the patient was discharged h after surgery without any remarkable event during his postoperative stay. the patient has been followed up for months without any complication or recurrence in ct scan, confirming the correct minimally invasive reconstruction of the abdominal wall. conclusions: trends in abdominal wall reconstruction and complex-hernia repairs have advanced rapidly in recent years. the goal is to perform a complete abdominal wall repair with no tension in midline incisional hernias. endoscopic anterior component separation and laparoscopic eventroplasty with closure of the defect, leads to a complete wall reconstruction without tension and avoids drawbacks due to primary close defect in those patients with herniary defects wider than cm. aims: endoscopic technique is a valid and safe approach for the treatment of abdominal wall defects. to combine the advantages of complete endoscopic extraperitoneal surgery with those of sublay mesh repair we propose totally endoscopic sublay anterior repair (tesar), a safe and feasible approach for the treatment of ventral and incisional midline hernias. methods: from may to september patients were referred to our unit for clinical and radiological diagnosis of midline ventral or incisional hernia and selected for tesar. exclusion criteria were: complicated ventral or incisional hernia (i.e. incarcerated hernia), maximum defect width [ cm, contraindications to general anesthesia. the procedure consisted of suprapubic access with trocars, complete endoscopic pre-aponeurotic dissection, isolation and reduction of the hernial sac, bilateral incision of the medial rims of recti aponeurosis and dissection of retromuscular plane to create the retromuscular space, sublay non-absorbable mesh positioning and anterior aponeurosis reconstruction. one drain was always placed in the retromuscular space and one drain in the subcutaneous space. results: all procedures were completed with endoscopic approach, with no conversion to laparoscopy or open surgery. no intraoperative complications were registered. total mean operative time was ± . min. no post-operative major complications were registered. only one subcutaneous seroma was registered ( . %), and treated conservatively. the mean postoperative stay was . ± . days. at post-discharge clinical checkups drains were checked and removed when indicated. no wound complications nor recurrence were registered to date. cosmetic and functional results were successful in all patients. conclusions: tesar is a safe and feasible technique for the extra-peritoneal sublay repair of ventral hernias with totally endoscopic approach. it provides accurate hernia repair with good outcomes in terms of resolution of symptoms and post-operative complications. r. mizuno, m. kondo backgrounds: abdominal incisional hernia is found in more than % after abdominal surgery, and risk factors such as wound infection, obesity, elderly, high abdominal pressure are pointed out. laparoscopic hernia repair using intraperitoneal onlay mesh (standard ipom) is becoming widespread in japan since the insurance release in , and our hospital is actively working on it. recently, ipom plus procedure which also carries out fascia suture in addition to laparoscopic mesh placement has been introduced. aims: we report the clinical results of laparoscopic abdominal incisional hernia repair in our hospital. methods: we performed hernia repairs using a mesh for cases from january to september . of these, cases were standard ipom and cases were ipom plus. there was no significant difference in the patient background such as gender, age, bmi, etc, and in the intraoperative findings such as hernia orifice diameters and adhesions. surgical time, postoperative hospital stay, and the rate of complications such as seroma, mesh bulging, postoperative pain, hernia recurrence were compared and examined between the two groups. results: as a result, in ipom plus group, the operation time was longer and the incidence rate of postoperative pain was higher, but the incidence of mesh bulging was significantly lower. also, in some cases since , the ' u reverse stitch method ' is used as an ingenuity of fascia suture in ipom plus. conclusions: laparoscopic abdominal incisional hernia repair has the advantage of being able to reliably confirm the hernia orifice from the intraperitoneal side?it is excellent in the identification of the fragile part of the abdominal wall and in the visibility of the restoration range. with regard to the ipom plus procedure which has been introduced in the last few years, although the operation time is extended, it has usefulness such as reduction of mesh bulging. from the viewpoint of cosmetic surgery, usage of ipom plus will increase in the future. introduction: incisional hernia is one of the most common complications after abdominal surgery. several methods have been introduced, and yet, there is no consensus on the best method of repair. we present a novel method for hernia repair which uses the retromuscular sublay mesh repair through a single incision at the pubic area to improve cosmesis. methods: medical records of patients who underwent single-port retrorectal incisional hernia repair from may to december were reviewed. patients were placed in supine position and a cm incision was made in the pubic area below the panty line. a flap is made upwards until the defect is found and bilateral rectus sheathes are dissected. a mesh is then placed between the posterior rectus sheath and the muscle. results: a total of patients with midline incisional hernia underwent single-port retro-rectal incisional hernia repair. mean age was . ± . years with an average bmi of . ± . . all the patients had midline hernia defect with an average of . ± . cm. mean operation time was . ± . min and estimate blood loss was . ± . ml. there was no postoperative complication, and ( %) patients were discharged on the day of surgery. conclusion: the single-port retrorectal incisional hernia repair is safe and effective while providing good cosmesis to selected patients with incisional hernia. aims: closing hernia defect during laparoscopic hernia repair is a vast extended technique nowadays. however, this technique is associated with mesh placemnt intraabdominally in contact by the abdominal content. nowadays there is a trend to recontruct the midline and to avoid a mesh intraabdominally in those cases suitable for it, as a new step forward of minimally invasive abdominal wall reconstruction. laparoscopic sublay approach with retromuscular placement of a mesh without mechanical fixation after reconstruction the linea alba migth be considered an option in primary hernias of the midline. methods: we present a case of a year old male with an umbilcal hernia of centimeter in diameter associated with rectus diastasis. a laparoscopic approach was performed, using one and two millimeter trocars placed on the left flank. the first step was to open the lateral side of the posterior fascia of the left rectus muscles, dissecting the retromuscular plane until we reach the linea alba getting into the preperitoneal space where the sac was diseected preserving the integrity of the peritoneum. the contralateral posterior fascia was also dissected all the way to the semilunaris line. the midline was closed, including th hernia defect, using a running double loop suture (maxon-loopÒ). a self gripping mesh (progripÒ) is placed in the retromuscular space in a sublay position ( cm long, cm wide). last, we close the fascia of the left rectus muscle using a barbed suture (v-locÒ). results: surgical time was min, being discharged of the hospital on postoperative day . pain was controlled with conventional analgesia and no postoperative complications, nor seroma was detected. conclusions: sublay approach for ventral hernia can provide a midline reconstruction, reestablishing abdominal function and avoiding the use of intraabdominal meshes and traumatic fixation, decreasing postoperative complications and pain. aims: lumbar hernia is one of the rare cases that most surgeons are not exposed to. hence the diagnosis can be easily missed. this is often related to previous surgery as lumbotomies or primary in the superior lumbar triangle. this leads to delay in the treatment causing increased morbidity. we report a case of adquired lumbar hernia in a middle-aged woman repaired by laparoscopic approach. methods: a years old woman with surgical history of a myelomeningocele surgery by posterior approach over years ago, a laparoscopic left nephrectomy years ago with a left colostomy due to a left colon injury during this procedure. a hartmann reversal by laparoscopic approach months later. patient showed a large lumbar mass over cms in the left lumbar region and a large scar near to spinal cord. it was soft in consistency, reducible and expansible on coughing and straining with defined borders. computerized tomography showed a large defect in the superior lumbar fascia over cms in the grynfeltt-lesshaft triangle with the left colon inside. results: patient was placed in a full lateral decubitus position. in order to optimize exposure, a lumbar roll was placed under the lumbar region. a capnoperitoneum ( - mmhg) was built up. one mm and two mm trocars were used and positioned in the left mid axillary line. a optic was used. adhesions were removed and toldt fascia was opened in order to expose the hernia defect bounded by quadrates lumborum, erector spinae muscles, rib and serratus. hernia content was carefully extracted from the sac using a ligasure maryland (covidien medtronic-usa). hernia defect was measured and an intraperitoneal mesh (dinamesh-ipom feg textiltechnik mbh, aachen, germany) was positioned and sutured by tackers to the margins included the bone. patient was discharged in h with a low pain rate and without complications. there is not recurrence in months follow-up. conclusion: laparoscopy might be a safe and feasible approach for repairing lumbar hernias, either primary or adquired, with a low rate of pain and complications s surg endosc ( ) after pneumoperitoneum is done, three mm trocars are placed on the left flank. the defect is delimited by drawing it over the skin of the patient with aid of an intramuscular needle and intraabdominal vision. posterior fascia is opened longitudinally at its medial edge and the retromuscular space is dissected. the arcuate line of douglas and the epigastric vessels are identified. from this point, transversus abdominis fascia is sectioned cranially cm medial to the semilunar line, preserving the neuro-vascular pedicles that reach the rectus abdominis laterally. at supraumbilical level, transversus abdominis fibers advance behind rectus abdominis, so they need to be sectioned to access to the space below the ribs. lateral dissection of this space enables a tensionfree closure at midline. once the procedure is repeated on the contralateral side using two mm and one mm trocars on the right flank, a continuous suture of the posterior fascia is performed with a barbed suture. the anterior fascia is closed with a slowly-absorbable monofilament loop-type suture. finally, a double-layer polypropylene mesh is placed at retromuscular level without any suture and fibrin glue is applied. results: the patient was discharged hous after surgery. no recurrence has been presented to the moment. conclusions: the section of the aponeurotic plane from the arcuate line of douglas enables a more accurate dissection of the retrotransversus plane without sectioning its fibers except for its cranial end, preserving the innervation and vascularization of the abdominal wall. this technical modification aims to simplify a complex laparoscopic procedure allowing its estandarization. aims: the authors present a video with their standardized laparoscopic ventral hernia intraperitoneal mesh (ipom) hernioplasty procedure but introducing a novel laparoscopic technique for tension releasing while hernia gap closure and midline anatomical restoration. methods: a years old male patient with a bmi presents a symptomatic ventral hernia recurrence after a sigma colic cancer open surgery. a ct scan study showed a cm transverse diameter midline ventral hernia. a laparoscopic ipom hernia repair procedure is performed using mm instruments and a mm camera. when checking tension while midline restoration suturing, we decide to add a tension-releasing maneuver: a totally laparoscopic transverse abdomini muscle release (taltar). this maneuver allow right rectus posterior sheath to advance some distance to the midline, in order to provide a tension-free midline closure. a double-faced ready-to visceral contact mesh is now placed and fixed. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with a rate in a eva acute pain visual scale. in a year follow-up, there has no been an anatomical or clinical recurrence. no chronic pain, anatomical recurrence, lateral asymmetry, umbilical or abdominal wall complications have been reported with this technique. conclusions: depending on the patient characteristics, anatomical hernia factors and surgeon mini invasive experience, a taltar maneuver could be a safe and feasible option for releasing tension when midline anatomical laparoscopic closure. more studies are needed in order to standardized this approach. aims: when primary ventral hernia and simultaneous diastasis recti are diagnosed, there is no consensus among the international surgical community on the surgical treatment regarding indications or surgical technique. however, if diastasis recti is symptomatic of or is associated with midline hernias, the corrective surgery of both pathologies at the same time could be the most recommended option. when we only correct the herniary defect, we risk performing a reparation on an anatomically weak tissue, so the rate of hernia recurrence may increase. we propose a minimally invasive access using totally endoscopic retromuscular hernioplasty. by developing this technique, several advantages are provided, such as no peritoneal opening without intraabdominal access, no mesh fixation needed and simultaneous solving of both pathologies. method: we present the case of a -year-old man, with bmi kg/m and no previous medical history complaining of ventral hernia with associated recti diastasis. a cm size umbilical hernia was diagnosed with a cm size supraumbilical diastasis recti associated. full endoscopy retromuscular hernioplasty was proposed. a cm size incision was made in left hypocondrium, openned the anterior rectus sheath and retracted the rectus muscle. we placed a balloon trocar and open the homolateral retromuscular space after placing two mm trocars in left lumbar space and epigastric position. we crossed-over the linea alba and achieve contralateral retromuscular space. after this step, the hernia sac was reduced and we extended the dissection cm caudal to the hernia ring. both medial posterior rectus sheaths were sutured with running barbed suture n° and a x cm size light-weight, big pore, polipropilene mesh was placed in retromuscular space and unrolled properly with enough overlap. a drain was placed and the anterior rectus sheath incision was closed. results: the patient was discharged h after surgery without remarkable events during his postoperative stay. he has been followed up for months remaining asymptomatic. conclusions: totally endoscopic retromuscular ventral hernia repair in men with umbilical hernia and diastasis recti associated, is feasible and reproducible procedure with several advantages compared to traditional laparoscopic ipom in terms of pain and mesh position. aims: parastomal hernia (ph) is one of the most frequent long-term complications of stoma formation, occurring in %- % of patients. surgical treatment for parastomal hernia is the only cure but a fairly difficult field with a recurrence rate ranging from % to % of cases. due to its advantages, the number of laparoscopic mesh repairs for parastomal hernia has gradually increased over the past decade. according to this common complication, we report a case of laparoscopic reparation of ph using the sugarbaker technique. method: we present the case of a -year-old patient with surgical antecedent of laparoscopic low anterior resection due to rectal cancer, presenting in postoperative period an anastomosis leakage with severe peritonitis was identified and a laparotomy with end colostomy was performed. the postoperative course was uneventful. during the follow-up the patient showed a centimetres size paraestomal hernia, being a m w incisional hernia confirmed with ct scan.the patient underwent full laparoscopic hernia repair, performing a sugarbaker technique, exposing parastomal hernia completely to measure the hernia ring size ( centimetres) and the midline associated defect ( centimetres). a x cm size ptfe-c was selected to allow a -cm overlap over two defects. results: using this approach, the bowel loop was pushed into the abdominal wall and appropriate place between the mesh edge and the abdominal wall is left to allow the bowel loop to pass through. postoperatory course was uneventful and the patient was discharged h after surgery without any remarkable event during his postoperative stay. he has been followed up for months without realizing any clinical signs or alterations in ct scan. compared with traditional open surgical repairs, laparoscopic repair has certain advantages including its safe operation, postoperative rapid recovery, fewer complications, and lower recurrence rate. however, it still faces challenges regarding parastomal hernia treatment, and there is a need to improve existing surgical techniques. aims: nowadays, the principal disadvantages of laparoscopic approach in hernia repair are the use of intraabdominal meshes and traumatic fixation. first, intraabdominal meshes involve the contact of the prosthesis with the intestinal loops with the consequent risk of adhesion and fistula. also, using helicoidal sutures in prosthetic fixation produces adhesions to the tackers and a non-negligible incidence of chronic pain. when it comes to lead to better results, placing the mesh in retromuscular space avoids the drawback of contact with the loops, and using self-fixation meshes may decrease the rate of acute and chronic pain. accordind to this facts, we present a case of laparoscopic ventral hernia repair with transabdominal retromuscular mesh placement without traumatic fixation. methods: we present a -year-old patient with a cm diameter hernia showed in preoperative ct scan, m w , with diastasis recti associated. the patient underwent laparoscopic surgery using transabdominal retromuscular route. one mm and two mm trocar were placed in left flank. the posterior rectus sheath on the left side is opened starting cms far from the left egde of the defect. once the retromuscular space is dissected, the hernia ring is dissected and the hernia sac reduced, we continue with the dissection in retromuscular space on the side. craniocaudal dissection is achieved cm distal to the defect margins. the hernia defect with the anterior rectus sheath and the diastasis recti were closed using v-loc running suture. self-adhesive mesh was subsequently placed. the mesh should be overlap cm from the margins of the defect, covering the defect widely, with grips facing upwards. finally, we closed the posterior rectus sheath with peritoneum on the left side with v-loc running suture. results: the postoperative course was uneventful and the patient was discharged h after the surgery. after months of follow-up no clinical or radiological recurrence was showed. conclusions: the combination of laparoscopic approach, retromuscular mesh placement and the use of self-fixation meshes, seems to be an actual useful solution, combining the advantages of each item and avoiding the use of intraabdominal meshes and helicoidal sutures. aims: laparoscopic ventral hernia repair has clear advantages over open repair, including less post-operative pain and earlier return to normal activity. however, a prolonged surgeon learning curve is necessary to perform this technique effectively. robot assistance may improve outcomes of minimally invasive ventral hernia repair with improved three-dimensional visualization and enhanced dexterity with articulating instrumentation. we report a case of robotic rives-stoppa epigastric hernia repair in order to demonstrate the feasibility of the robotic approach. methods & results: a -year-old man came to our attention for the presence of a palpable mass in the epigastric region. the abdominal ct scan showed the presence of an epigastric hernia with herniation of omental content, and the presence of diastasis recti. the patient was then submitted to a rives-stoppa robotic hernia repair under general anesthesia. the da vinci-si surgical system (intuitive surgical inc., sunnyvale, ca, usa) was brought into position over the head of the patient and docked after placement of the ports. three trocars were placed in the hypogastric region along the transtubercular line. a fourth trocar was placed in the left iliac fossa and used by the assistant. the operation started with an extended adhesiolysis and hernia reduction. then, the retromuscolar dissection began by incising the posterior sheath starting from cm above the pubic symphysis. an extended dissection of the rives space was performed to create a correct housing for the mesh. the hernia defect and the diastasis recti were closed using a - absorbable barbed suture. a phasix st tm mesh (bard inc./davol inc., warwick, ri) was positioned in the retromuscular plane, and was anchored with absorbable sutures and glue. the midline incision was closed using a - absorbable barbed suture. the operative time was minute. the postoperative period was uneventful, and the patient was discharged home on the second post-operative day. conclusions: robotic rives-stoppa ventral hernia repair is feasible, safe, and effective when a standardized approach is performed. whether robotics may improve the outcomes of minimally invasive ventral hernia repairs, including lower recurrence rates, decreased post-operative pain, or shorter surgeons' learning curve, will require careful prospective investigation. aims: the authors present a video with a left chronic bochdaleck hernia classical hernioplasty repair but performing a mini invasive thoracoscopic approach and mm instruments. methods: a years old female patient come to hospital due to chronic left dorsolumbar pain. a ct scan study showed a chronic left diaphragmatic bochdaleck hernia. a lateral right decubitus thoracoscopic repair is performed using mm instruments and a mm camera. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with no pain and a clean chest x-ray. in a year time follow-up, not an anatomical or clinical recurrence has been reported. neither chronic pain or respiratory complications happened, with in this period of time. conclusions: depending on the patient characteristics, anatomical factors and surgeon mini invasive experience, left bochdaleck hernia mini invasive thoracoscopic hernioplasty repair using mm instruments could be a safe and feasible option. more studies are needed in order to standardized this approach. surg endosc ( ) abdominal wall surgery has expanded exponentially in the last decade. many techniques have been developed, mainly in minimally invasive surgery. laparoscopic ventral and incisional hernia repair (lvihr) has become a common procedure because of its feasibility and safety but unfortunately, it is not free of complications. chronic postoperative pain and bleeding are frequent complications, prolonging hospital stay and altering quality of life of the patients. absorbable or non-absorbable tacks are the usual method of mesh fixation and sometimes combined with transfascial sutures to secure the mesh. these mechanical fixations pierce the abdominal wall causing nerve or vessel injuries. some studies showed no differences between absorbable tacks, non-absorbable tacks or transfascial sutures concerning postoperative remarkably high pain. some authors consider that a non-penetrating fixation of the mesh getting an effective mesh-abdominal wall interface will reduce significantly the postoperative pain after a laparoscopic ventral hernia repair. tissue glues are used in different medical treatments and also have been used successfully for extra peritoneal mesh fixation in laparoscopic inguinal hernia repair, open ventral hernia repair but not so in laparoscopic ventral hernia repair in spite of good results published in the literature. cyanoacrylate and its derivatives are 'synthetic glues' and classified as medical devices with stronger adherent properties than fibrin glues. experimental studies have reported good results compared with suture fixationand also tissue toxicity doesn't lead to an increased foreign body reaction. some authors have studied the use of cyanoacrylate in laparoscopic inguinal hernia repair but unfortunately, clinical trial reports in ventral and incisional hernia repair were not found in the literature because the lacking of experimental studies that guarantee the safety of intra-abdominal mesh fixation and the interaction of the glue with the intra-abdominal tissue. our group developed an experimental study demonstrating the feasibility, safety and effectiveness of the cyanoacrylate using for intraperitoneal mesh fixation and after this conclusion, started a clinical study. this video shows the methodology for laparoscopic mesh fixation with only glue in our first cases. aims: small epigastric hernias, associated or not with the rectus abdominis diastasis, and small umbilical hernias are common in middle-aged women, particularly with past history of pregnancy. the aim of this video is to illustrate a new extraperitoneal approach to these clinical situations. methods: patients between the ages of and years old, with epigastric hernia orifice up to cm, with or without associated umbilical hernia (up to cm), were chosen for this procedure. the surgery begins with a vertical umbilical incision for the umbilical hernia's correction, and dissection of the pre-aponeurotic plane. two mm trocars (mini-laparoscopy instruments) are introduced at both flanks to enlarge the pre-aponeurotic plane towards the xiphoid appendix. in this way epigastric hernial defects are isolated. the surgery proceeds with defect suturing with braded suture, midline invagination and mesh placement if necessary. results: all patients had an eventful post-operative period and were discharged home at postoperative day . the aesthetic and functional results are optimal conclusion: for selected cases with high aesthetic motivation this technique seems to be feasible and with optimal cosmetic results. this technique allows the mesh placement both in-lay and onlay, protecting it from surgical site infections often present at the classical approac bochdalek hernia is a rare entity in adults. fewer than have been reported in medical literature, the majority of which were incidentally diagnosed. as such, the optimal repair of a symptomatic hernia is unknown. we present a case of adult bochdalek hernia repair. methods: a -year-old obese male patient with a years of chronic dry cough and left lung opacity in chest x-ray. a large posterior and lateral bochdalek hernia with herniation of intestinal loops and fat to the left hemithorax was seen in chest and upper abdominal ct scan. the hernia extended to mid-thorax, caused significant atelectasis of left lung. eighteen months later, due to appearance of chest and abdominal pain following a recent motor vehicle accident, a repeat chest ct was done and a slight enlargement of the hernia was shown. results: the patient was operated laparoscopically, positioned in a semi-right lateral decubitus with double lung intubation. a large left posterior and lateral diaphragmatic hernia which contained transverse and descending colon with omental fat was seen. they were pulled in to the intraperitoneal space carefully. the defect was measured to be * cm. it was reduced to * cm by suturing with a non-absorbable v-loc suture . advancing the camera to the thoracic cavity showed the left lung to be severely atelectatic. after selective recruitment lung was well expanded. a symbotex composite cm mesh was fixed to the defect area by suturs and laparoscopic tacker. the operation and post-operative course were uneventful. chest x-ray demonstrated the bowel below the diaphragm. the patient was discharged on pod . at -month follow-up, chest x-ray was normal. objective: to demonstrate the safety and efficacy of the standardized laparoscopic approach in the treatment of large parastomal hernia. currently, this approach is recognized as the one of choice in parastomal hernia pathology, being controversial which is the best technique of choice: keyhole vs sugarbaker. material and method: clinical case: a -year-old woman with a history of laparoscopic abdominoperineal amputation due to rectal neoplasia (pt n ), a year ago, with symptomatic parastomal hernia with incarceration episodes and inflamation changes in the stomal orifice.tac: large hernia parastomal with intestinal content inside. surgical treatment is decided. result intervention: complete laparoscopic approach, right lateral partial decubitus, trocars, dissection of the hernia defect and reduction of the content, partial mobilization of the pre-stomal colon, with bleeding at the level of the vascular origin, requiring careful hemostasis to avoid ischemia of the colostomy, herniorrhaphy with stitches with extracorporeal knotting, placement of polypropylene/pvdf mesh,fixed with irreabsorbable tackers with administration of biological glue at the edges of the mesh. correct postoperative, discharge at the rd day. asymptomatic and without hernia recurrence at one year of follow-up. conclusions: the technique of sugarbaker using a laparoscopic approach is a safe and effective alternative in the treatment of parstomal hernias. objetives: laparoscopic ventral hernia repair provides advantages in term of low infection rates and postoperatory stay when is compared with open repair. trends in laparoscopic abdominal wall surgery is to complete defect closure without tension in midline. closing the defect in ventral hernias wider than - cms creates high tension in midline and postoperatory pain. it's proposed different techniques to solve this drawback. laparoscopic posterior component separation makes the defects closure easier with no tension and placing the mesh extraperitoneally. methods: years old woman with previous total hysterectomy, a m m w midline incisional hernia was clinically diagnosed and confirmed with ct scan. full laparoscopic abdominal wall repair with defect closure was proposed. trocars in left side were placed and posterior rectus sheath right side in the defect margin is freed. once the lateral edge of the rectus sheath is reached, the posterior rectus sheath is incised, dividing the posterior aponeurotic sheath of the internal oblique muscle. this allows access to the plane between the internal oblique and the transversus abdominis muscles. it's is made the same steps in the left side with trocar on the right flank. the posterior rectus sheath both side is reapproximated in the midline and cms polipropilene mesh is placed and unfolded properly. it's fixed using cyanocrilate glue. one drain is left in retromuscular position and mm trocar wounds are sutured. results: postoperatory course was uneventful. hospital stay h. the drain was removed in day after surgery. after months follow-up no complication or recurrence were identified. methods: this video will show the evidence of gangrenous jejunal segment due to superior mesenteric vein thrombosis in a patient with history of breast ca on hormonal treatment.in this video, the gangrenous segment was resected and primary anastomosis was done using endogia mm. results: a second look after h revealed to be negative for any further ischemic bowel. conclusion: therefore, laparoscopy in acute abdomen is diagnostic and for treatment. introduction: gastric pseudo-volvulation is a rare entity of paraesophageal hernia that is characterized by migration of the stomach into the posterior mediastinum. this clinical-radiological picture has severe complications so in certain cases should be operated urgently. another small group of patients are asymptomatic, although the current literature recommends their regulated surgical intervention. we present a gastric pseudo-volvulation in the mediastinum, with a laparoscopic approach, showing that by systematizing the surgery, it is possible to perform this type of intervention with relative ease and safety material and methods: we present a video of an urgent laparoscopic approach in a female patient of years with a personal history of hypertension, smoking and dyslipidemia. with a hiatus hernia diagnosed more than ten years ago. he went to the emergency department due to significant symptoms of heartburn and reflux, as well as incoerctable vomiting and difficulty feeding one week of evolution. a simple abdomen and postero-anterior chest radiograph was performed, showing a paraesophageal hiatus hernia with almost the entire stomach included in the mediastinum. a thoraco-abdominal axial tomography corroborated giant hiatus hernia with pseudovolvulation and incarceration data. urgent intervention was decided by laparoscopic approach in which hiatus hernia reduction and esophageal abdominalization were performed. closure of pillars and reinforcement with bioabsorbable mesh. gastric and gastropexy toupet of anterior face to anterior peritoneum of abdominal wall. results: the patient had a post-operative h without incident, discharged with a crushed diet. the follow-up and evolution has been acceptable without notable complications. conclusion: the laparoscopic approach, in extreme cases of paraesophageal hiatus hernia with incarceration of the stomach and pseudovolvulation of it, is a correct, safe and effective alternative in experienced groups. surg endosc ( ) case report of incarcerated hiatal hernia. years old female was admited to the hospital due to severe chest pain and vomiting for about six h. physical examination and lab test showed no abberations. chest xray revaled incarcerated stomach above the diaphragm. she was rushed to the or. laporoscopic approach was used, the stomach was removed from the chest and nissen fundoplication was performed. day after surgery patient was asymptomatic, got full oral diet. she was discharged on postoperative day two, without a need of any analgetics. gastroduodenoscopy was performed weeks after surgery and showed proper image of oesophagus, stomach and duodenum, neither signs of hiatal hernia nor inflamation were present. laparoscopic approach is good way to treat incarcerated hiatal hernias and is related with shorter lenght of stay, lesser postoperative pain and better patient comfort. and it should be procedure of choice in this kind of cases. she was operated open technique using a cm long incision in right iliac fossa and the appendix was phlegmonous. the patient began feeling bad from the second day postoperative having temperature over °c, pain and increasing crp. the general condition worsened the next day when the temperature went up till . °c, extreme generalized pain and crp: . the ct abdomen control indicates signs for generalized peritonitis and rises the suspicion for a forgotten large gauze. the patient is operated using laparoscopy technique: identifying and taking out the foreign body, doing adhesiolises, extensive lavage and in the end inserting one drain in douglas. the video is presenting what king of special graspers can be used but also tips and tricks when speaking about identifying the anatomy but also dissection in acute and inflamed environment. postoperatively the patient began to feel better and in the th day was released home. conclusion: this case illustrates that even after open surgery, laparoscopy is a viable solution with the condition that there is available experience in minimally invasive surgery. introduction: foreign bodies can enter inside the human body by different mechanisms such as ingestion, aspiration, trauma or in some cases due to medical procedures. they are potentially life-threatening events, the diagnosis could be challenging and its management depends on their location. case report: a -year-old male was referred to our hospital due to chronic abdominal pain. he had cholelithiasis, medical history of acute pericarditis and past surgical history of left adrenalectomy, left nephrectomy, distal pancreatectomy and colon resection due to an adrenal adenocarcinoma (stage t n m ).abdominal radiograph showed a foreign body in the left lower quadrant of the abdomen, as an incidental finding. this was not detected in ct scans during ten years of oncology follow-up. ct scan revealed an extraintestinal metallic curved object in the right lower quadrant. this finding was not related to any surgical intervention or trauma. diagnostic laparoscopy was performed: the foreign body seemed to be a guidewire, it was included into the omentum and almost stuck to the abdominal wall. the guidewire was reached and carefully extracted through a mm trocar without any evidence of intra-abdominal organ injury. then an elective cholecystectomy was also performed due to his medical history of symptomatic cholelithiasis.the procedure lasted min. the hospital discharge was on the third postoperative day and no complication was registered. conclusion: is extremely rare to discover a guidewire that had migrated into the peritoneal space without abdominal injuries.this case report demonstrates the technical feasibility, safety and minimal postoperative morbidity associated with minimal invasive laparoscopic removal. aims: the authors present a video with their standardized laparoscopic groin hernia transabdominal preperitoneal hernioplasty (tap) procedure but using mm instruments and mm camera approach. methods: a years old male patient with a bmi presents a symptomatic bilateral groin hernia for months. us study showed an indirect bilateral inguinal hernia. a laparoscopic tap hernia repair procedure is performed using mm instruments and a mm camera. a selfgripping mesh preperitoneal hernioplasty and peritoneal flap barbed-sutured hermetic closure was performed. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with a rate in a eva acute pain visual scale. in a year follow-up, there has no been an anatomical or clinical recurrence. no chronic pain, anatomical recurrence, umbilical or abdominal wall complications have been reported with in this period of time. conclusions: depending on the patient characteristics, anatomical factors and surgeon mini invasive experience, a laparoscopic bilateral hernia repair using mm instruments, could be a safe and feasible option. more studies are needed in order to standardized this approach. results: during tapp approach a direct hernia relapse was identified, the previous mesh was included on preperitoneal space and some non-absorbable sutures to inguinal ligament were identified. stitches and nearly total mesh removal (only the part surrounding cord elements was left in place) were performed. x heavyweight polypropylene mesh was employed fixed with gubran Ò and the flap was closed with running sutures. patient was discharged uneventfully the same day. seven months later he did not need analgesics and had no physical impairment. conclusions: post inguinal hernia repair chronic pain can be severe and disabling, and is becoming more prevalent. the origin is complex and meshes and sutures could play a role. the management is multimodal and demanding. for refractory patients, surgery may be an option. laparoscopic, open and mixed approaches have been employed. they usually combine mesh removal and substitution (often in different planes) and groin nerve therapies. nowadays, triple neurectomy seems to be the most effective treatment (more than % pain relief). generally, removal of mesh alone does not lead to lasting pain relief or has worse outcomes compared with associated neurectomy. introduction: mesh repair of inguinal hernia is sometimes followed by adverse effects such as mesh migration, chronic groin pain or recurrence. removal of the mesh is necessary in selected cases. we affront this cases by tapp intervention. methods: we present a video with two intreventions of inguinal recurrent hernia by laparoscopy (tapp). we remar the points to decide explant the mesh or not to explant. the conditions to decide the explant were the proximity to the main vessels in inguinal area (espigastric and femoral vessels) and the plication of the mesh. results: and conclusion as we show in the video, the explant of the mesh is only conditioned by the plicature of the mesh for its migration and recurrence, accompanied usually with pain. we don't remove any time the mesh or the plug if it is in the triangle of doom with firm adhesions to the main vessels. we cover the previous mesh with a new ligthweigth d mesh and closing at the end the preitoneum over the new reparation. introduction: tep technique isn't a controversial area in surgical practice for inguinal hernias anymore, but a fully accepted method. the use of general anesthesia has been the mainstay of laparoscopic hernia repair, but epidural anesthesia is not a contradiction to properly selected patients. material-method: the approach of the extraperitoneal area achieved without use of a dilation balloon, but via the indroduction of the camera and the dissection of the regional structures. trocars ports were used: a mm trocar through the umbilicus for the camera, exactly as in sils (single incision laparoscopic surgery), another one mm is placed in the midline between the umbilicus and pubis, the last mm trocar is placed in the midclavicular line ipsilateral with the hernia. the key for every operation was the tension free technique with placement and fixation of a mesh x cm. in / cases the mesh was placed with tacks on the inside of the inferior epigastric artery-vein complex. all patients were dismissed from the hospital in h, no drain was placed and no major postoperative complications took place. conclusion: tep is a demanding technique with serious learning curve. the use of a dilation balloon for insertion in the extraperitoneal area is not prerequisite. tep is an appropriate method both for first appearing and recurrent inguinal hernias. epiduralanesthesia instead of general anesthesia is no a contradiction for properly selected patients. aims: the aim of this study was to investigate the effects of preperitoneal carbon-dioxide (co ) insufflation during tapp (transabdominal preperitoneal) repair. materials and methods: male patients with inguinal hernia were include in our study. we obtain laparoscopic access at the umbilicus and introduce mm port. two mm working ports are placed lateral. diagnostic laparoscopy of the entire abdomen is necessary to rule out other pathology or contraindications for surgery. using aspiration needle we insuflate carbon-dioxide ( mmhg) preperitoneal at the level of anterior superior iliac spine while decrease abdominal gas pressure to mmhg. same procedure is made lateral to the umbilical artery. results: we found that preperitoneal carbon-dioxide (co ) insufflation during tapp facilitate the future parietalisation and even can reduce operating time in future improvements of the technique. there were no intraoperative complications related to this procedure. we did not found any potential risk of the technique when is use by trained surgeons. aims: laparoscopic inguinal hernia repairs (lihr) are performed more and more frequently because they offer some advantages; however, we cannot forget their specific complications. lihr are associated sometimes with peritoneal tears that can lead to bowel obstruction. we present two cases of bowel obstruction related to peritoneal defects post tapp procedure and review peritoneal closure, bowel obstruction and options to repair defects. a year-old male was scheduled for tapp due to bilateral relapse. two x tio mesh tm fixed with securestrapÒ, employed also for peritoneal flap closure, were employed. three days later he was readmitted with bowel obstruction with ct suggesting 'adhesions'. a year-old male had bilateral tapp in another centre. seven days later he presented with bowel obstruction. ct showed metallic tackers and suggested 'adhesions' results: first case: after four days of conservative treatment failure, a revisional laparoscopy showed ileum herniation through a peritoneal defect and firm adhesions to the mesh. bowel was labouriously separated and the peritoneal defect closed with two running sutures. he was discharged on the \ sup [ th \/sup [ postoperative day and three years later he is asymptomatic. second: after two days of conservative treatment failure, on laparoscopy, ileum was filmy adhered to polipropilene mesh through a big defect on flap closure. defect was closed with interrupted sutures. as tears persisted, an omental flap was created to cover the area. patient was discharged on the th day and continues asymptomatic three years later. conclusions: lihr bowel obstructions can be divided in adhesive disease and herniation. herniation can be early (through peritoneal defects) or late (trocar site). international guidelines recommends a thorough closure of peritoneal incision or bigger tears (grade b). the closure can be achieved with staples, tacks, running suture, or glue. these last two methods are more time-consuming but less painful. running suture seems to be the best, due to its low costs, tightness and low pain but sometimes can be technically difficult. low intra-abdominal pressures (= mmhg) facilitate suturing. when a herniation appears, careful bowel management is needed and running sutures are recommended. if tears persist, an omental flap can be useful. aims: application of a single port robotic platform to perform an entirely transanal tatme/ tata. methods: the following video demonstrates how a totally transanal proctosigmoidectomy is performed using a novel, single port (sp) robotic platform was used to carry out a totally transanal proctosigmoidectomy, single port robotic tatme/tata. a -year-old female patient with a clinical t n b rectal cancer at the cm level, status post neoadjuvant chemoradiotherapy ( cgy, xeloda) is presented. shown here is the open transanal dissection followed by docking of the sp robot, implementation of the single port instruments (fenestrated bipolar forceps, cadier, scissors, camera, clip applier) through a gelpoint path to complete a totally transanal proctosigmoidectomy including transanal tatme, ima/imv transection, splenic flexure release, and left colonic mobilization, loop ileostomy, and handsewn coloanal anastomosis. results: blood loss was cc. pathology demonstrated a moderately differentiated, rectal adenocarcinoma. the total mesorectal excision was complete (grade ), margins were negative, and all lymph nodes were negative for metastatic carcinoma. the patient was discharged on postoperative day after an uncomplicated hospital course. there was no postoperative morbidity or mortality. conclusions: application of the single port robot to transanal tatme/tata (sprtatme) is presented here. while much work remains to be done to validate the sp robot's safety, this first demonstration of a totally transanal tatme/tata establishes its feasibility and utility. this single port platform stands to greatly expand the application of natural orifice transluminal endoscopic surgery (notes). as shown, the sp robot offers more than sufficient visualization, technical control, and adequate reach to perform such an operation. we present an exciting new avenue by which to complete operations in an entirely transanal fashion, which are classically performed via a combined transanal and transabdominal approach. methods: this video shows the utilization of a new robotic platform to perform transanal endoluminal microsurgery, rtem. presented here is a year old woman with a recurrent rectal adenoma at the cm level, status post a previous tem resection in october . demonstrated is the utilization of the sp robot through a gelpoint path in order to perform a partial fullthickness and full-thickness resection. the robot is introduced through a mm in diameter cannula via a four-channel face-plate. the instruments' two-jointed mobility at the elbows and wrists as well as the novel navigation system are well demonstrated. the docking of the sp robot, utilization of the dissecting devices, and closure of the defect is shown. results: sprtem was performed with a blood loss of cc, and the patient was discharged on postoperative day . there was no postoperative morbidity, mortality, or moderate/severe pain. pathology showed tubular adenoma with low-grade dysplasia in a non-fragmented specimen with negative margins circumferentially. conclusion: initial experience using the sp robot for rtem is demonstrated here. the robot provides wonderful visualization and operative control to the surgeon. articulation of the robot's wrists and arms have the potential to facilitate technical aspects of the procedure. rtem stands as an exciting development in the field of transanal endoluminal surgery. introduction: the application of robotic approach in the esophageal surgical field is in its first phase. the microsuturing and microdissection capabilites of the robotic system can potentially overcome the traditional limitation of the laparoscopic surgery thus enhancing the indications of minimally invasive surgery. methods: we have performed a retrospective analysis of our prospectively maintained database that included patients who underwent robotic-assisted esophagectomy for malignant disease between and . results: ten out of sixteen patients had squamous cell carcinoma meanwhile six had adenocarcinoma. ten mckeown's and six ivor lewis were performed. the mean operative time was min ( - ) and the median blood loss was ml ( - ). no patients required conversion nor intraoperative transfusion. the morbidity rate was / ( . %) : a transitory laryngeal nerve paresis, a pneumotorax and pneumonia. the mean hospital stay was (range - ) days. an r resection rate of . % was achieved with a mean lymph node yield of ( - ). the -year disease free survival was . %, wheres the the -year overall survival was . %. conclusions: robotic assisted minimally invasive esophagectomy (ramie) is safe and feasible, it offers promising results while preserving a good oncology adequacy. this video shows our technique for the treatment of an esophageal diverticolum using a robotic left sided transthoracic approach, followed by a heller myotomy and dor fundoplication using a transabdominal approach. our case is a year old male, who suffered from severe dysphagia, halitosis and gastric reflux who on endoscopic and radiological investigations was found to have low grade and a cm wide esophageal diverticulum, cm from the lower esophageal sphincter. initially conservative management was attempted, however following poor compliance and the persistance of symptoms after year of therapy, surgical intervention was indicated. the operation was performed using the minimally invasive robotic system of the davinci siÒ, starting with the thorax time. the patient is positioned in left side decubitus. the camera-trocar is insert in the thorax via the fifth intercostal space the, two mm and one mm robotic trocars are added. the lung is liberated from pleural adhesions and the esophagus is then prepared exposing the diverticulum which is successfully removed with an endo-giaÒ. the esophageal muscle fibers, near the suture line is reinforced with separated vicryl stitches and the resected piece is extracted via endo-bag. a fr thoracic drainage tube is then placed and the trocar accesses repaired. the patient is the put in supine position with a °anti-trendelemburg angle. three robotic trocars (two mm and one mm) are placed and the robot docking is made from the patient left shoulder. the lesser omentum is divided to visualize and prepare the gastric-esophageal junction (gej) sparing the vagus nerve. the heller myotomy is then performed for cm over the gej and cm under it. the mucosal integrity is assured via laparoscopic and contemporary gastroscopic view. the gastric fundus is attached to the distal esophagus completing the dor fundoplication. post-operative care comprehends the removal of the thoracic drainage during the first post-operative day, the pain management and the progressive realimentation. the hospitalized period lasts day and the patient was dismissed without complications occurred. the uniportal video assisted lung lobectomies gained popularity all over the world during the last years. the technique is safely applied for peripheral pulmonary lesions, under cm, but more and more complex cases are being approached while the indications continue to evolve. our aim is to present the particular aspects of this technique in an -year-old female patient with a giant bullous lesion located in the lower lobe of the right lung. the preoperative work-up for this case is presented and commented. a multidisciplinary surgical team consisting of thoracic and pediatric surgeons was involved. a single . cm length incision in the fourth intercostal space was used for the access. due to the fact that the lesion involved almost the entire lobe and the margins were very close to the hilum, we have decided and performed a right lower lobectomy. dissection and stapling were quite difficult. all the anatomical structures had small dimensions, forcing us to perform an 'artery first approach' in a very narrow space. no complications during or after surgery were encountered. the patient was discharged after four days and she went to school on the sixth day. histopathological examination showed that the lesion was a type ccam (congenital cystic adenomatoid malformation). conclussion: the uniportal video assisted lung lobectomy was safety applied for a giant bullous lesion of the right lung. aim: dunbar syndrome, celiac trunk (ct) compression syndrome, caused by median arcuateligament is a rarely diagnosed disease because of its nonspecific symptoms, which cause adelay in the correct diagnosis. the aim of the study was to demonstrate the usefulness andadvantages of laparoscopic approach in the treatment of dunbar syndrome. methods: we performed laparoscopic release of ct in the department of general, minimallyinvasive and elderly surgery in olsztyn in . all of three patients suffered from severepain of abdominal cavity before the surgery. results: in two cases, there were a complete remission of the symptoms. in one case, there was animprovement. all patients reported relief of symptoms in the first days after the operation.there were no postoperative complications. conclusions: the laparoscopic treatment of dunbar seems to be safe and feasible procedure. thelaparoscopic surgery alone can often eliminate discomfort, while angioplasty and stentimplantation are no longer necessary. introduction: the advances in robotic surgery have permitted the application of such technology to various surgical fields, one of the last of these being hernia surgery. we present a case video of the treatment of a dual-hernia using a robotic retromuscular ventral hernia repair(rrvhr) using the davinci siÒ robotic system. the case report demonstrates the evolution of the trans-abdominal robotic umbilical prosthetic (tarup) in that it utilises a 'double docking' technique to allow the positioning of a large retromuscular mesh. methodology: our patient is a -year-old male who presented with chronic epigastric pain. the abdominal ct confirmed two abdominal wall hernias; an epigastric and supra-umbilical hernia with visceral contents and wall defect diameter of cm and . cm, respectively. using the minimally invasive robotic system of the davinci siÒ we adapted the well known retromuscular mesh technique. the operation was initially intraperitoneal with access to the retromuscular preperitoneale space using a right sided longitudinal incision.(as per standard tarup technique). we proceed with the dissection of the retro-muscular space until the left lateral edges of the rectus sheath, creating a preperitoneal space for the placement of a specifically modified ultrapro polypro-leneÒ x cm mesh. following this we repositioned the davinci siÒ in a symmetrical manner, with ports placed in the retromuscular space. the mesh is positioned and the peritneum subsequently closed with a v-lock sutureÒ. finally we opted for a negative pressure jackson-pratt drain, inserted preperitoneally. results: the patient was discharged on the nd post-operative day without complication follow up continued until months post operatively during which the patient remained asymptomatic, without signs for hernia recurrance . conclusion: the technique highlighted in our video demonstrates the utility of the robotic system in hernia repair. specifically the approach proved a success as it facilites the placement of the mesh totally extra-peritoneally with closure of the posteriore sheath without tension. the added advantages are that the port-sites are distant from the mesh thus reducing infective risk. additionally this technique allows the treatment of large peritoneal defects. surg endosc ( ) aim: to analyse the performance of a robotic fellow during a robotic total mesorectal excision (tme) at the end of the fellowship, and subsequently compare it with their mentor. methods: the fellow is exposed to robotic colorectal lists per week. during the fellowship, assessment of performance is recorded in a structured proforma covering aspects of autonomy, tissue handling and dissection. at the end of the fellowship, areview of cases performed by the fellow and the mentor was carried out in a blindly manner (video footage). results: robotic tme training was divided into modules in order of complexity and the trainee had to achieve sequential proficiency in each module, before progression. docking of davinci robotic system. inferior mesenteric artery exposure and ligation, development of medial to lateral plane and inferior mesenteric vein division. left colonic and splenic flexure mobilization. pancreas identification. rectal dissection (tme). qualitative assessments were recorded by the mentor; the fellow was 'able to perform with verbal help' most of the steps from early on. by the end of the fellowship, all steps were performed in a similar manner in terms of quality and oncological integrity when compared with the mentor. conclusions: at completion ofan advanced robotic colorectal fellowship, high quality trainees can perform every step of the tme dissection in a similar manner with the trainer, when assessed blindly, without compromising oncological integrity. aims: to find safe and simple method in robotic rectal low anterior resection with low tie arterial ligation and lymph node dissection around the root of inferior mesenteric artery. methods: we performed robotic rectal low anterior resection (rlar) by davinci si system in eight patients with rectal cancer. we applied low tie arterial ligation, just caudally to the origin of the left colic artery in all cases. during the procedure, we used tilepro function of davinci si system which enabled to display two other visual informations through external inputs under the normal -dimensional surgeon console view. preoperative d-ct vessel branching simulation video and intra-operative real time ultra sound navigation view were displayed simultaneously under normal operative camera view in the surgeon console. results: left colic artery preservation was completely done in all cases. the mean time to find and expose the left colic artery from the first incision in sigmoid mesentery was min, which was drastically shorter than conventional method. this method needed lesser mobilization of inferior mesenteric artery (ima), and may be less invasive to autonomic nerve around the root of ima which is very important for ejaculation function. conclusion: robotic rectal low anterior resection with low tie arterial ligation was performed safely and in short time, using tilepro intra-operative navigation method. preoperative d-ct vessel branching simulation video and intra-operative real time ultra sound navigation view were very useful in the procedure. we present the method in video. nerve sparing tme and pelvic neuroanatomy for colorectal surgeons p. tejedor, f. sagias, j.s. khan aim: to describe the critical points in which the pelvic nerves can be damaged during a total mesorectal excision (tme) for rectal cancer and the benefits of robotic surgery for identifying these points. methods: there are critical points regarding pelvic neuroanatomy: superior hypogastric plexus (shp): located in front of l -s . the ganglionic sympathetic fibres form the right and left sympathetic trunk, travel along the anterior surface of the aorta and coalesce in the shp at the level of the inferior mesenteric artery (ima). superior hypogastric nerves: they take an anterolateral course into the pelvis. there is an avascular 'holy plane' around the rectum between these two nerves. inferior hypogastric plexus (ihp): lies over the posterolateral pelvis, almost parallel to the internal iliac arteries. this can be identified at the lower end of the rectum. neurovascular bundles(of walsh): in front of the denonvillier's fascia, at and o'clock position. they are responsible for erectile function. results: lack of knowledge or identification of key structures at these points can lead to increased risk of nerve damage and translate into poor functional outcomes. the ima is dissected up to the origin from aorta and here the shp can be seen. care is taken to avoid any damage to these structures. the tme plane is found at the back of ima as the inner most dissectible layer between mesorectum pelvic fascia. right and left superior hypogastric nerves are identified. dissection is carried out posteriorly, laterally and anteriorly. ihp is identified at the lower third of the rectum, when the dissection is about to reach the pelvic floor. care should be taken in not to go too far lateral and damage this plexus. in the anterior dissection, plane is carried in front of the denonvilliers' fascia. the neurovascular bundles can be seen at and o'clock position and the surgeon has to be careful to stay inside that plane in order to avoid damage. conclusions: the precise dissection in robotic surgery results in minimal tissue damage and better visualization and preservation of the pelvic nerves. aims: to describe and evaluate new contributions and eventual advantages of icg fluorescence to perform an icg guided bilateral pelvic lymph node dissection in a patient who underwent low-anterior-resection for rectal carcinoma. we also present the basic steps to avoid ileostomy during rectal surgery in which icg and ghost ileostomy play an important role. methods: a -year-old male patient was referred to our hospital due to abdominal pain and significant changes in usual bowel habits.colonoscopy showed a no obstructing cm middle rectal mass, which was reported as an adenocarcinoma.ct scan and mri revealed a mm polyp in the anterior rectal wall which was located cm from the anal verge. it was involving mucosa and sub-mucosa with muscularis propia invasion. no pathological lymphadenopathies or hepatic metastatic disease were found (stage t n ).a laparoscopic ultra-low-anterior resection plus icg lateral lymphadenectomy with total mesorectal excision was performed. a complete splenic flexure mobilization was performed to achieve a safe tension-free anastomosis. transection line of the proximal rectum was checked after icg intravenous injection. icg was injected around the tumor by inserting an anoscope, just before the surgery. after the dissection of the rectum, lateral lymphadenectomy was performed assisted by icg. an end-to-side anastomosis was made. and a vascular loop was passed around the terminal ileum to create a ghost ileostomy.the procedure lasted min. reactive protein c was monitored to identify an initial leak. the patient was discharge in postoperative day and no complication was detected. results: pathological exam reported a rectal adenocarcinoma. pelvic lymphadenectomy results were: negative nodes, negative nodes and negative nodes from right lymph node dissection, left lymph node dissection and rectosigmoid resection specimen respectively. no metastatic disease was found (stage t n m ). conclusions: in our experience, icg fluorescence imaging system offers important contributions to rectal surgery furthermore than evaluating vascular supply to the anastomosis. lymphatic mapping of the lateral lymph nodes and avoiding ileostomy could be a potential important use in the future. larger studies and more specific evaluations are needed to confirm its role in colorectal surgery and to find its limitations. background: robotic surgery for colorectal cancer is an emerging technique. potential benefits as compared to conventional laparoscopic surgery have been demonstrated. innovative robotic technologies have helped surgeons overcome many technical difficulties of conventional laparoscopic surgery such as hand-eye coordination, a two-dimensional view, and a restricted range of motion. robotic-assisted surgery was established as a new approach to minimally invasive surgery, overcoming these limitations. the following video shows a total robotic sigmoidectomy step by step on the basis of ourexperience. intervention: a -year-old male patient with no previous medical historyand a colon adenocarcinoma, cm from the anal verge, no distant metastases. it was decided to perform a robotic sigmoidectomy. target anatomywas located andwe proceededto the exposure of the mesenteric vessels from medial to lateral. a cautery wasused to open the peritoneum,up to the origin of the inferior mesenteric artery, and caudally past the sacral promontory.the vessels weretransected by ligasuretm. we performedthe complete release of the colon taking care to avoid injury to retroperitoneal structures. we usedligasuretm to section the mesocolon in order to prepare the transection of the proximal colon. indocyanine green was used to check the correct vascularization. an endogia tristapletm was used to divide the colon. subsequently, we sectioned the rectumand extracted the specimen through itwith no need to make any auxiliary incisions. we introduced the anvil of the suture device to perform the anastomosis. we sectionedand close the rectum with an endogia tristapletm. finally we opened the proximal colon to introduce the anvil,making a pursestring to fix it and create a side to end anastomosis. outcome: the surgery took min. the patient started oral intake h after surgery and left the hospital on the rd postoperative day. pathological examination ruled out a colon adenocarcinoma pt n . conclusion: total robotic sigmoidectomy is safe and feasible and can be a procedure of choice to achieve a good surgical qualityand avoid assistance incisions in patients with colon cancer. surg endosc ( ) with more and more data now advocating wait and watch policy for these patients which require close radiological and endoscopic follow-up but unfortunately around % of them have regrowth of tumour which will require surgical intervention. the use of robot for cancer resections is becoming more frequent especially in narrow spaces like in an obese male pelvis. the reason being better -dimensional views, more angulation of the instruments and exclusion of tremors, which in turn leads to better dissection and preservation of hypogastric nerves. in this video, we present a robotic low anterior resection for rectal re-growth in an obese -years old male patient. he was offered neoadjuvant chemoradiotherapy after discussion in mdt. he had an complete response with chemoradiotherapy and was decided to offer him watch and wait regime. unfortunately, he developed rectal re-growth in the first year of his follow up. imaging showed t lesion with no distant metastasis and was later confirmed on histology as well. after mdt discussion he was offered robotic low anterior resection. the video starts by showing the clinicopathological features of patient including his radiological and endoscopic images. robotic port sites are shown. the edited video starts with rectal dissection after ligation of inferior mesenteric artery and vein with emphasis on narrow pelvis and preservation of hypogastric nerves, seminal vesicles and intact presacral fascia. postoperative histology was ypt no and patient was discharged home after days with no postoperative complications. background: minimally invasive surgery for colon resection has improved patient outcome, however a minilaparotomy still is necessary to extract the specimen. this report describes a new approach that combine laparoscopic parellel overlap stapling left colectomy with natural orifice specimen extraction surgery, with the aim to minimize abdominal wall trauma. method: laparoscopic left colectomy for malignant diesease was performed using a standard five-port technique. after releasing the left colon via laparoscopy, divide the proximal and distal of specimen with -echelon, and put distal sigmoid colon and proximal transverse colon together. open sigmoid colon cm apart from distal margin, and incise transverse colon at proximal margin. take transverse colon and sigmoid colon side-to-side anastomosis via -echelon. incise posterior vaginal fornix to get into the abdominal cavity and extract specimen through vaginal. outcome parameters such as complications, conversions, operative time, postoperative recovery, and postoperative pain were prospectively recorded in a database. results: surgery was performed for patients with left-colonic carcinoma. no perioperative complications or conversions occurred. the median operating time was min. the median visual analogue scale score of postoperative pain was , and of patients needed analgesia on postoperative day . the median postoperative hospital stay was days. for malignancies, tissue margins were oncologically adequate, the averge number of harvested lymph nodes were . . the -week follow-up period was uneventful. conclusion: the described technique, a combination of laparoscopic parellel overlap stapling and natural orifice surgery, has the potential to avoid incision-related morbidity of the minilaparotomy in laparoscopic left colon resections. background: open surgical skills training has been well established over centuries, however, there are some significant differences in laparoscopic surgical skills training. it is an obvious advantage that the trainee and the trainer have the same view; however, some of the hurdles include the differences in tactile feedback, hand eye co-ordination, spatial awareness, depth perception and maximizing assistance. aim: we present a video highlighting some of the key challenges faced in laparoscopic colorectal surgical training, show-casing our systematic, structured approach. our approach: we have developed a structured approach starting with junior surgical trainees and progressing through to consultant level as per the levels below: level : attend courses/ workshops level : master camera work level : contra-lateral assisting level : intermediate level trainee-start operating with trainer scrubbed. the trainer is an additional member of the scrub team and stands on the same side as the trainee (does not replace any assistant) level : advanced level trainee-gradual progression from level . trainer un-scrubbed but standing next to the monitor throughout the procedure. level : trainer in theatre but out of sight of the trainee, with little interference level : progression to trainer-once proficiency is achieved at level / , the trainee is trained to become a trainer, for the junior and intermediate level trainees. within each level the complexity of the procedure increases as the trainee progresses through the level. junior trainees (years - of surgical training) are taken through levels - , intermediate (middle years of training) level or and advanced (last - years) up to levels . this way of training allows multiple members of the team to be trained simultaneously in every case. each operating list is preceded by team briefings where the role of every member of the team is clearly identified and followed by individual and collective feedback. conclusion: this training ladder proved very successful through the years. the feedback from trainees at all stages has been consistently positive. several trainees who have progressed to independent consultant practice, in the uk and abroad, are adopting this approach in their practice. introduction: despite the potential microsuturing capabilities of the robotic surgery, most of the esofago-jejunostomy after robotic total gastrectomy are still performed extracorporeal or through mechanical staplers. this can increase the cost of the procedure, the risk related to a improper functioning of the stapler. methods: we reviewed our prospectively maintained database analyzing patients from april to september , who underwent robotic total gastrectomy with hand-sewn esophagojejunostomy for gastric cancer. results: a total of patients were included in the study. the mean estimated blood loss was ml ( - ). the overall operative time was min ( - ). length of hospital stay was days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . no conversion was necessary nor anastomotic leakage occurred. the morbidity rate was / ( . %) and included a subhepatic abscess and wound infection trough pfannenstiel incision. a r resection rate was achieved in all cases. the mean of lymph node yield was ( - ). the -year disease free survival was %, the -year overall survival . %. the robotic-assisted hand-sewn esophago-jejunostomy is a safe and no time-consuming technique. it avoids the complication related to the stapler firing and it offers cosmetic benefit to the patient in terms of extraction site. introduction: colorectal endoscopic submucosal dissection (esd) is increasingly practiced for treatment of early colorectal neoplasia. however, colorectal esd is difficult to perform due to lack of retraction as well as instability especially over hepatic flexure. dilumen eip is an external flexible sheath introduced during colonoscopy to stabilize environment for esd. this video demonstrated the use of dilumen eip for performance of colonic esd at the ascending colon method and results: this is a years old lady who received screening colonoscopy and found a mm lateral spreading tumor (lst) type iia lesion at ascending colon distal to ileocecal valve. under general anesthesia, patient received colonic esd using dilumen eip. due to significant looping, the dilumen device was introduced with the techniques of double balloon enteroscopy. after identification of the lst, the balloon in the front would be deployed to the proximal to the lesion while both balloons would be insufflated and created a stable environment. the esd procedure started after submucosal injection with normal saline in mix with indigocarmine, epinephrine and hyaluronate. mucosal incision was performed over the anal side of the lesion, and after adequate submucosal dissection, clips were applied to attach the mucosal flap to the sleeve of proximal balloon and achieved retraction. the submucosa was adequately exposed for dissection using dual knife jet. this enhanced submucosal dissection especially at one area with significant fibrosis. after the procedure, complete closure of the mucosal defect was performed by clips and assisted by the front balloon. the pathology confirmed intramucosal adenocarcinoma with clear resection margins. discussion: the dilumen eip device stabilized the environment within the colon with the double balloon and provide adequate retraction for performance of colorectal esd. surgery, kobe city medical center general hospital, kobe, japan background: robotic surgery has been widely spread all over the world, but robotic gastrectomy is not common and difficult because of complex anatomy and wide-ranging operation fields. in addition, it had been performed only under a few high-volume centers for reasons of the limitation of national health insurance in japan, which means medical expenses not covered by insurance. the situation was changed from this april, so we started robotic gastrectomy to reduce complications more rather than laparoscopic gastrectomy. we report results and aim to present the methods in detail using da vinci si surgical system. methods: we place five trocars, one is umbilical endoscopy port, and other four ports are placed at the reverse trapezoid, almost fan-shaped. using the arm number , the organ can be lifted up so that sharp lymphadenectomy is able to be done by almost a scissor as the arm number while applying the countertraction by the arm number . in order to achieve a clear and bloodless lymphnode dissection while maintaining the oncological safety, we think not only the ultrasonic coagulating scissor but also the electrocautery of the scissor is very essential in robotic surgery. less postoperative complication such as pancreatic fistula or pancreatitis might be derived from robotic surgery because we can avoid pressing the pancreas during the suprapancreatic dissection of lymph nodes. the billroth i reconstruction can be performed using da vinci endowrist stapler under stable and inflexible surgical fields without needing help of surgical assistant. results: from october to december , patients with gastric cancer were operated robotic gastrectomy, included total gastrectomy. there was no conversion to open surgery and no conversion to other procedures derived from intraoperative complications, and the overall operation time is gradually decreasing from the th case. we are now on the way of learning curve shortening operation time, but robotic gastrectomy is no less safer and adequate than laparoscopic surgery. we will show our robotic procedures including lymphadenectomy around subpyloric and suprapancreatic area, and reconstruction with several important points in our video purpose: this report describes the benefits and drawbacks in the use of a novel articulating device (artisential), which has a multi-degree wrist freedom like the davinci endowrist, in performing complete single-port d lymph node dissection (lnd) in single-incision distal gastrectomy (sidg). methods: the artisential was used in performing sidg with d lnd for patients with advanced gastric cancer. all operations were performed by a single surgeon using a threedimensional camera and a passive scope holder in place of a scopist. the artisential was used mainly in the sb and suprapancreatic lnd, an area that is relatively far from the single port. in certain cases when the pancreas needed to be pushed down, such as obese male patients, the intraabdominal organ retractor was used to lift the tissue and the artisential to push the pancreas. operative results and short-term outcome were analyzed. results: twelve patients underwent the procedure without any intraoperative events, conversion to conventional laparoscopy, or surgery-related complications including postoperative pancreatic fistula. all patients underwent single port d lnd by complete exposure of the portal and splenic vein. mean operation time was . ± . mins. and mean number of retrieved lymph nodes was . ± . . the artisential was found to be useful in grasping the tissues behind the pancreas and the major arteries throughout most of the lnd. the articulating motion also allowed the narrow single-port field of view to be clearly seen without the instrument body obstructing the camera. conclusion: the use of artisential in sidg appears feasible and reproducible, and is mandatory in performing a complete d lnd in sidg. the video shows a case of laterally spreading tumour of the rectum with preoperative benign histology, paris classification -is g (granular type), ut n eus stage, kudo type iv, nice type . the neoplasm measured x cm, and extended from to cm from the anal verge, mainly located on the posterior wall. according to our local policy the indication was a transanal full-thickness excision. this was performed with the medrobotics flexÒ robotic system, used here for the first time outside the united states.the system technology utilizes an articulated multi-linked scope that can be steered along non-linear, circuitous paths in a way that is not possible with traditional, straight scopes. the maneuverability of the scope is derived from its numerous mechanical linkages with concentric mechanisms. this enables surgeons to perform minimally-invasive procedures in places that were previously difficult, or impossible, to reach. with the flexÒ robotic system, surgeons can operate through a single access site and direct the scope to the surgical target. once positioned, the scope can become rigid, forming a stable surgical platform from which the surgeon can pass flexible surgical instruments. the system includes on-board d hd visualization. the flexÒ robotic system contains two working channels to accept a number of different surgical and interventional instruments including monopolar and bipolar electrodes, scissors and graspers for tissue manipulation.the video shows the introduction of the dedicated rectoscope, the connection of the flexible robot, and the way to operate the device performing a full-thickness excision, including suturing of the rectal defect by means of two running sutures by a v-lock / thread. while illustrating the technique the authors will comment pros and cons of the use of the device. background: hepatobiliary procedures using a minimally invasive approachare demanding, especially in major hepatectomies. the use of da vinci surgical system allows to overcome some of the kinematics limitations of the direct manual laparoscopy maintaining the potential advantages of a minimally invasive approach . we herein present a case of left hepatectomy and local lymphadenectomy for hepatocellular carcinoma, carried out with the use of the da vinci xi. methodology: a -years old man with a long-lasting hbv chronic infection and ct scan and mri finding of a -cm solid neoplasia of the left hepatic lobe and gallbladder stones, was operated with the da vinci xi platform. the patient was placed in a supine position, with °anti-trendelenburg inclination. the trocars were positioned according with the intuitive indication for the upper quadrants surgery. results: the procedure was successfully completed in min.at first, an intraoperative us scan with the use of tile-pro technology was done to determinate the tumor extension. the hepatic parenchyma transaction and the local lymphadenectomy were performed with monopolar scissors and bipolar grasps. the left hepatic vein section was performed with an endoscopic vascular stapler. there were no surgical complications or need for conversion to laparoscopy or laparotomy. the post-operative course was uneventful and the patient was discharged days after surgery. conclusion: the da vinci xi can facilitate some technically demanding procedures and ultimately widen the range of application of minimally invasive surgery such as hepatic surgery. besides the well-known advantages provided by robotic surgery on d imaging, increased range of motion and augmented surgical dexterity, one of the most interesting and innovative features of robotic technology is the digitalization of the operative view; furthermore the tile-pro multiinput display allows the surgeon a d view of the operative field along with the ultrasound exam for a precise understanding of anatomy and vascularity and of tumor location. during the last few years, robotic surgery as well, as the latest innovation of minimally invasive procedures, takes its position in this particular field with the benefits of overcoming the limitations of conventional laparoscopy. our aim is to demonstrate the advantages of robotic surgery in procedures of hepatectomies, on occasion of a robotic hepatectomy performed by our team. methods: we present video fragments of a robotic left lateral hepatectomy procedure in an elderly female patient with a symptomatic gigantic haemangioma of the left hepatic lobe. we emphasize on the technical aspects and the advantages that the surgeon gains applying the robotic techniques in such procedures. results: the procedure was completed with minimal blood loss and the patient presented an uncomplicated post-operative course, with discharge on the third postoperative day, minimal need of analgesics and full recovery. conclusions: the excellent three-dimensional and high quality visualization that the robotic system offers, combined with the flexibility and the accuracy of the robotic instruments (especially on suturing), provide to the surgeon an important aid, in order to avoid serious complications, such as intraoperative bleeding and post-operative bile leaks. the restriction of the limitations of conventional laparoscopy is far more beneficial and promising for the evolution and the future of minimal invasive liver surgery. aims: the new da vinci xi surgical cart allows multi-quadrant and complex surgical interventions in a minimally invasive fashion. we present a case of robotic appleby left pancreatectomy using this platform and its specific operating bed. methods: a -years old woman with ct scan finding of a -mm hypo-vascular neoplasm of the pancreas body underwent surgery with the use of the new da vinci xi with four arms upper quadrants trocar' disposition. results: the procedure was successfully completed in min. the pancreatic body was mobilized in order to expose the portal-mesenteric axis. the gland was transected using a robotic endo-stapler as well as the splenic vein. after evaluating the patency of collateral circles with intra-operative ultrasound, the common hepatic artery and the celiac artery were transected. then we increased the right tilted position and the neoplasia was detached from the gastric body by a tangential gastric resection using the robotic endo-stapler. finally, the operation was accomplished with the transection of the posterior attachment of the spleen and the pancreatic tail. no conversion or intra-operative complications were recorded. the post-operative course was uneventful and the patient was discharged days after surgery. the da vinci xi with its specific tools helps in performing challenging procedures such as appleby operation for locally advanced pancreatic cancer. in our experience, the robotic endo-stapler permits the operating surgeon to directly control the transaction phase whereas the specific operating bed allows to perform minimally invasive multi-quadrant surgery and to obtain a better exposition of the operating field. results: the whipple procedure was successfully completed in min. thanks to the dvtm the patient's position changed during the intervention to improve the exposure, with the instruments left inside the abdomen and without undocking the robot. the dissection of the pancreatic head from the portal vein and the section of the retroportal lamina were performed with the use of the endowrist vessel sealer device. a personal modified end-to-side pancreatojejunostomy was carried out, with / prolene and gore-tex double layer suture. no intra-operative complications occurred and no conversions to laparoscopy or laparotomy were required. the postoperative course was uneventful. conclusions: the use of the new fully wristed vessel sealer extend makes easier difficult maneuvers such as the fine dissection of the pancreatic head from the portal vein and the section of the retroportal lamina, enabling an optimized approach for vessels sealing and cutting and tissue bundles. moreover, the dvtm allows patient's movements without undocking the system or removing instruments from the abdomen, enhancing the surgical workflow. background: necrotizing pancreatitis is a devastating illness which can develop in up to % of patients who suffer from pancreatitis. it carries great morbidity with an associated mortality rate between to %. many of these patients require drainage of fluid collections to treat sequela related to pain, per-os tolerance, and source control of sepsis if infected. the step-up approach to treatment of this disease has trended towards minimally invasive techniques, considering the morbidity of open debridement. as such, many centers have implemented the use of transgastric debridement via endoscopic cystogastrostomy. this technique, while effective in draining fluid and particulate necrotic tissue, has difficulty in resection of large necrotic tissue, due to instrument and anatomic limitations. current endoscopic accessories designed for polypectomy or foreign body extraction, for example, are not optimal for performing necrosectomy. to overcome this obstacle, additional access sites can be utilized to assist debridement. we describe the first laparoscopic assisted transgastric endoscopic necrosectomy through a percutaneous gastrostomy in a year old male with infected pancreatic necrosis secondary to biliary pancreatitis. aim: to investigate the feasibility of utilizing gastrostomy access to assist in debridement during endoscopic necrosectomy. methods: the patient previously underwent an open necrosectomy and gastrostomy tube placement for acute emphysematous pancreatitis. post-operatively, there was a persistent and enlarging cm infected walled-off necrosis (won). therefore, endoscopic cystogastrostomy was performed using a lumen-apposing metal stent. results: frank pus was evacuated. initial endoscopic necrosectomy was technically challenging due to the large volume of solid necrotic tissue. repeat endoscopic debridement utilized a surgical laparoscopic grasper via the gastrostomy site to aide solid debris extraction (video). this allowed for complete necrosectomy and resolution of the won. the patient did well and was discharged subsequently. conclusion: this is another emerging minimally invasive technique in the step-up approach for debridement and drainage of won. the use of the gastrostomy as a utility port for accessory instruments not only enhanced the technical aspects of the procedure but increased its efficacy as well. further experience is needed to validate the utility and reproducibility of this technique. objective: the presentation of the minimally invasive surgical approach for pancreatic necrosectomy guided by videoretroperitoneoscopy or var (video assisted retroperitoneoscopic), established in our center, as one of the option of the step-up approach treatment for acute necrotizing pancreatitis (anp) methods: the placement of the patient on the operating table should be in decubitus, with right lateral inclination, at - °on the horizontal surface. the pancreatic cell is approached using the drainage catheter previously placed by radiological control (ultrasound or ct) as a guide, which will allow access to the cavity with safety. an incision of - cm is made around the previously placed catheter, crossing the subcutaneous cellular tissue and muscular fascias, dissolving the musculature. it continues in a blunt dissection, until a loss of resistance is appreciated which generally coincides with the outflow of necrotic or purulent material. once the retroperitoneal cell is accessed, a mm trocar is placed and a pneumoretroperitoneum is performed. the -mm trocar allows the joint use of a mm and °optic and the surgical material that allows debridement and cleaning. the aspiration and hydrodissection of the necrotic material, and the extraction of the solid component of the necrosis are proceeded. once the collection is drained and the necrotic material removed, a wash and drain system is placed, like a -way foley type probe. conclusions: in conclusion, the var is an alternative surgical technique, valid and reproducible in the treatment of anp, which offers comparable results and even superior, in some series, to those of open surgery, with satisfactory results in terms of morbidity and postoperative mortality. aim: lung subsegmentectomy is suitable for small and deep, non-palpable lung nodules. since it is difficult to intraoperatively detect the arteries, veins and bronchi of the subsegment, as well as the intersubsegmental borders, complete video-assisted thoracic surgery (vats) for lung subsegmentectomy is challenging. we use preoperative three dimensional ct to detect the arteries, veins and bronchi of the subsegment before conducting complete vats subsegmentectomy, and perform intraoperative bronchoscopy to detect the bronchi and intersubsegmental borders. i would like to describe our experience of complete vats combined subsegmentectomy for a non-palpable lung nodule. methods and results: the patient was a -year-old woman. during health screening, a small groundglass opacity was observed in her right lung on chest ct. the nodule was mm in diameter and was located in s b (horizontal subsegment of the posterior segment) near s (the anterior segment). we preoperatively diagnosed the lesion as well-differentiated adenocarcinoma, and planned combined subsegmentectomy for s b and s a (lateral subsegment of the anterior segment) of the right upper pulmonary lobe. before the operation, the locations of vessels were confirmed by three-dimensional ct angiography. video-assisted thoracoscopic surgery was performed using four ports: two cm ports in the th intercostal space in the post-axillary line and in the angulus inferior scapulae line for the operator, a cm port in the th intercostal space in the mid-axillary line for the assistant, and a cm port for the camera in the th intercostal space in the mid-axillary line. the cm port was also used for removal of the resected specimen. intraoperative bronchoscopy was used for detecting the subsegmental bronchi. she was diagnosed with primary lung cancer (adenocarcinoma in situ, nonmucinous) postoperatively. the tumor was pathologically graded as tisn m . no tumor recurrence has been noted in follow-up of twenty two months. conclusions: the combination of preoperative three-dimensional ct angiography, intraoperative bronchoscopy and complete video-assisted thoracoscopic surgery can be used for performing lung combined subsegmentectomy. aims: minimally invasive surgery is increasingly widespread for the diagnosis and treatment of abdominal pathology. laparoscopy is a diagnostic resource for those cases in which mass biopsy is not approachable through image-guided puncture, and is often therapeutic in the same act. it avoids the morbidity and mortality associated with laparotomy, favoring the early treatment of malignant processes. methods: we present a case of a year old male who was incidentally diagnosed with an oval-shaped pelvic mass in the right lateral wall of the pelvis, adjacent to the vascular bundle of the right external iliac at its origin( centimeter), without sign of infiltration of surrounding structures. no other pathological findings on the abdominal computerized tomography and magnetic resonance imaging were found. due to its localization, it was not accessible to percutaneous biopsy. the first diagnostic impression was a benign tumor of the nerve sheath (schwannoma), without being able to rule out other diagnostic possibilities. to provide a definitive diagnosis the patient was subjected to an elective laparoscopic resection of the tumor. surgical procedure was performed using a millimiter and two millimeter, umbilicus for the optical system and operative on hypogastrium and left iliac fossa respectively. acleavage plan between the tumorand rightiliac vesselswas found. the exeresis of the masswas achieved, and it was extracted using an endo-bagÒ through the umbilical port site. a drain was put in the surgical bed. results: the patient had a short, uneventful post-operative course, being discharged on postoperative day . pathological examination revealed a lymphatic node with metastasis of poorly differentiated carcinoma, with suspected urothelial lineage. cystoscopy was performed with the finding of a centimeter lesion on the right ureteral orifice with calcifications on the surface. biopsies were taken, confirming the bladder origin of the tumor. conclusions: both diagnostic and therapeutic laparoscopy is useful on pelvic masses because of the direct vision into this narrow anatomical space, especially in obese patients, providinga detailed view that makes easier to isolate and spear the anatomical structures surrounding the tumor, minimizing the risk of tumor rupture and bleeding. surg endosc ( ) aim: indocyanine green (icg)-enhanced fluorescence has been introduced initially in laparoscopic surgery to provide detailed anatomical information during laparoscopic cholecystectomy and to evaluate vascular supply to garantee correct anastomotic perfussion in order to reduce the risk of anastomotic leak. the uses of icg are increasing, specially in hepatic and oncological surgery in order to identify centinel lymph node and lymphatic mapping.we propose the use of icg imaging during complex laparoscopic colorectal resection in cases presenting ureter obstruction, to prevent iatrogenic ureteral injury. methods: we present a case of a year old female previously diagnosed of pelvic endometriosis with severe pain and symptoms related with episodes of pseudo-occlusion .a colonoscopy was performed finding sigmoid cancer in an area of endometriosis in a narrow colon with difficulties to perform a complete colonoscopy that could be related to the process of pseudo-occlusion. the biopsy was informed as an adenocarcinoma.the ct-scan showed a dilatated left ureter in an area next to the sigmoid colon.we propose a preoperative strategy with a bilateral double j stent insertion, finding a ureter obstruction caused by the endometriosis.icg was injected through the ureteral catheter, guiding us during the surgery to avoid a iatrogenic ureteral injury. results: a laparoscopic left colectomy was performed. the icg allows us to follow the ureter during the surgery, disecting the colon properly from the area attached to the ureter. the prestenotic area of the ureter was marked dilatated up to two centimeters allowing the icg to identify it from the anatomic structures of the areas and guarateeing that there was not spill of icg out of the ureter avoiding a postoperative leak of urine. conclussions: when tumors, or another entities like endometriosis, produce a ureteral occlusion, icg could be injected through a j stent, allowing us to identify and to avoid an injury.icg fluorescence imaging is a safe, cheap, and effective tool to increase visualization during surgery, offering additional information of the anatomy in colorectal surgery. in this video we are going to show three cases of the robotic treatment of splenic artery aneurism and the evolution of the technology that we relied on for the preoperative planning and intraoperative navigation. our preoperative evaluation evolved from a tridimensional virtual reconstruction with augmented reality to patient specific anatomical d printed models, initially made of rigid materials and afterwards made of malleable materials, in order to reproduce hollow anatomical structures such as vessels, feasible to simulate the planned surgical plan. the choice of a robotic approach, in selected cases, allowed to restore the continuity of the splenic artery after the exclusion or excision of the aneurism, in order to preserve the spleen. aims: tumor-induced osteomalacia (tio) is a rare paraneoplastic syndrome in which patients presents bone pain, fractures and muscle weakness caused by the fibroblast growth factor (fgf- ), a phosphate and vitamin d-regulating hormone. in tio, fgf- is secreted by mesenchymal tumors that are usually benign but small and difficult to locate. when medical treatment is unsuccessful, surgical treatment is indicated and conclusive.this video shows our technique for tio surgical treatment guided by indocyanine green (icg) fluorescent angiography. methods: the patient is an -years-old woman with tio confirmed by blood sampling of fgf- , gallium- pet/ct scan and abdominal ct scan which identified a highly vascularized mm nodule in the mesentery along the ileo-colic vascular axis.the patient was scheduled for a diagnostic laparoscopy with tio removal.the patient was placed in supine position. three trocars were introduced in the left quadrants. identified the last ileal loop, following ileo-colic vessels, a small mesenteric bulge was found. the icg fluorescent angiography confirmed the localization of the ipervascularised nodule and helped to define its edges. the nodule was removed through monopolar energy and the hemostasis was optimized through bipolar energy. the specimen was extracted through an endobag using one of the trocar access. results: the postoperative course was uneventful and the patient was discharged on postoperative day . histopathological examination showed an extrasurrenalic paraganglioma. conclusion: tio are often difficult to locate for surgical removal. icg fluorescent angiography allows to facilitate tio localization and removal. the minimally invasive technique decreases perioperative morbidity and mortality. laparoscopic removal guided by icg angiography should be considered when tio needs to be removed and is difficult to locate. aim: this video shows our technique to perform laparoscopic resection of a voluminous left paraaortic paraganglioma. methods: the patient is a -years-old man with a recent medical history offever, lumbar pain and haematuria.abdomen ct scan, performed during admission at emergency department, revealed a x cm left paraaortic retroperitoneal mass with pseudo-aneurysm. after procedure of angiographic embolization (with spermatic artery sparing), the patient was scheduled for a laparoscopic resection of paraaortic tumor. the patient was placed in the right flank position. three trocars ( mm) in the abdominal midline and one trocar in left hypocondrium were placed. at initial examination of the abdominal cavity, voluminous left paraaortic mass arising in the contest of left mesocolon was found, dislocating posteriorly kidney vessels. the parietal peritoneum was divided and the paraaortic lesion was dissected on the aortic plane from medial to lateral and from down to up, preserving the inferior mesenteric vessels; the mobilization was carried on to splenic vein. the vessels, supplying the mass and arising directly from aorta, was isolated and taped with vascular clips. on the inferior margin of the lesion a large vessel, probably connected with previously embolized pseudoaneurysm, was dissected with vascular linear stapler. the mobilization was completed through difficult dissection from aortic plane and mesocolic posterior surface. the colonic perfusion was verified with fluorescence angiography. specimen was extracted through an endobag.a drain was left in pelvis. postoperative day .histopathological examination showed a morphological and immunoistochemical pattern for benign paraganglioma. conclusion: laparoscopic resection of paraaortic paragangliomas is feasible by skilled surgeon. the minimally invasive technique decreases perioperative morbidity and mortality. careful preoperative planning and surgeon's experience with vascular dissection and visceral mobilization are mandatory for a good outcome. aims: posterior retroperitoneal endoscopic approach has been considered for many years as a very complex and unsafe surgical technique. often attributed to a difficult location and visualization of retroperitoneal structures. in addition, surgeons were forced to work in a small and easily altered space due to discontinuous flow with constant changes of the retroperitoneal vision. lately this approach is emerging thanks to technological advances, mainly better visualization laparoscopic cameras and high definition screens, as well as continuous flow insufflators of co , maintaining stable and smoke-free cavity uninterruptedly. methods: it shows a management of a potentially serious complication and the reproducibility of the technique through the retroperitoneal approach. results: to operate with high pressure of neumoretroperitoneum allows to contain the hemorrhage and to value with relative serenity and security, the best surgical option to repair said injury being laborious due to the reduced workspace. conclusions: the posterior or retroperitoneal approach is feasible, safe and fast. although the possibility of injuring the vena cava in right adrenalectomy remains one of the most serious and feared complications. as shown in the video, posterior retroperitoneal endoscopic approach allows repair of vascular injury correctly and safely. methods: four patients undergoing adrenalectomy, two of them with right adrenal pathology and two left. minimally invasive access, endoscopic approach, is exposed in all of them. results: in the first two surgeries, right gland is shown. initially, transabdominal approach, which requires mobilization and separation of the liver to access the retroperitoneal space and subsequent proceed to adrenal extirpation. later, right retroperitoneal approach is observed, with a meticulous sealing of the adrenal vein prior to complete the dissection of the gland, despite the small cavity created by co . in the second part, both left adrenal approaches are exposed. transabdominal pathway is necessary to mobilize left colon and spleen to access a narrow space above the upper edge of the pancreas to locate adrenal gland. this is very different in posterior adrenal approach. conclusions: posterior or retroperitoneal approach is feasible and safe, allowing access to adrenal glands, located in retroperitoneal space, without across peritoneal cavity and its disadvantages. colon and small intestine mobilization is not necessary, with a lower rate of intestinal lesions and postoperative ileus. in the same way, liver or spleen mobilization is avoided. aims: when performing a laparoscopic adrenalectomy, especially in the setting of pheochromocytoma, one of the most important steps is to gain control of the adrenal vein early on in the procedure before great manipulation of the adrenal gland. we present the case of a year old female with episodic headaches and tachycardia and severe uncontrolled hypertension, found to have elevated plasma and urine metanephrines with ct scan localizing a . cm right sided adrenal nodule. the patient was prepared preoperatively with phenoxybenzamine until mildly orthostatic with dry mucous membranes and was taken for laparoscopic right adrenalectomy. methods: after positioning our patient in left lateral decubitus, ports were placed inferior to the costal margin. the right lobe of the liver was mobilized and retracted cephalad and the ivc was exposed. careful and meticulous dissection was carried up the ivc, however no main adrenal vein was encountered. the adrenal gland was then dissected circumferentially and was removed in an endoscopic retrieval bag. there was no difficulty in hemostasis and the patient was deemed to be hemostatic prior to withdrawal of the ports and extubation. results: our patient had no issues with hemodynamic stability and her blood pressure was within normal ranges during and following the case. her hemoglobin was stable postoperatively with . immediately post op and . on discharge. her pre-op hemoglobin was . . conclusions: our video demonstrates a right adrenal gland that was congenitally missing a main adrenal vein. it is very possible that small venous branches were taken with dissection however we believe this report to be important to note in the literature for surgeons performing adrenalectomy. surg endosc ( ) aims: adrenal cysts are the most frequently identified adrenal cysts, although they are a rare entity. typically they are presented by abdominal pain or palpable mass, but nowadays, cystic lesions of the adrenal gland are more often discovered incidentally by radiologic studies. adrenal cysts have an extensive differential diagnoses, which makes a difficult definitive diagnosis and a difficulty in later management. the management of an adrenal cyst can be summarized in three fundamental pillars: discard the functional status of the cyst, evaluation of eventual malignancy by images, and avoid possible complications (hemorrhage, infection), especially in large cysts . methods: clinical case: a -year-old male patient, with no history, studied for nonspecific pain in the right hypochondrium, without other accompanying symptoms. an abdominal ultrasound was performed, a cystic lesion in hcd without being able to identify the origin was seen. complementary explorations of interest are shown (ct), the biochemical study discards functionality of the lesion, negative serology for hydatidosis. the minimally invasive approach is the gold standard in the surgical treatment of adrenal pathology, so a laparoscopic approach is proposed for this patient. aims: endometriosis is a high incidence disease (approximately % of women) with a large impact on women's quality of life and fertility. endometriosis nodules surgical treatment is necessary every time there is evidence of active disease. the aim of this video is to present a minimally invasive technique for the resection of an endometriosis nodule from the abdominal wall. methods: a -years-old woman, with past history of endometriosis and a c-section, presents at the office with a palpable nodule at the rectus abdominis left lateral border, close to the umbilical scar. she had complaints of exuberant catamenial pain and magnetic resonance imaging (mri) showed a mm nodule compatible with endometriosis depot. this technique uses trocars ( ? mm) placed at the pfannenstiel scar. stepby-step as follows: (i) dissection of the pre-aponeurotic plane and isolation of the lesion (ii) lesion excision and its removal with sac (iii) closure of the aponeurotic defect braded suture. results: the post-operative period was uneventful and the patient was discharged home at post-operative day one. the aesthetic result was excellent and the patient was asymptomatic one month after the procedure. conclusion: endometriosis of the abdominal wall is related to previous c-section, is a rare event (incidence . - . %) and usually located in the subcutaneous fat underlying the scar. the presence of nodules in the depth of the muscle is much uncommon and particularly in this clinical case, the nodule was located cm cephalad from the previous pfannenstiel scar. this technique seems easy and reproducible in the authors' opinio. aims: general surgeons often face gynecological pathological findings, either along with other abdominal pathology, or as primitive cases that need laparoscopic expertise. with this particular presentation, our goal is to demonstrate the essential laparoscopic skills and the basic operative strategy that a general surgeon should be familiar with, in order to manage such cases. the presentation is made on occasion of a woman with multiple uterine fibromatosis of the pelvis, who was treated by our team. methods: we present video fragments of the laparoscopic excisional procedure for multiple uterine fibromyomatosis of the pelvis, highlighting the proper strategy in order to conclude the operation effectively and uneventfully, in a minimally invasive fashion. results: patients with multiple, large or other complex forms of uterine or pelvic fibromas can effectively be treated with a minimally invasive approach, with minimal blood loss, very fast recovery and minimal postoperative pain and complications. % of pregnancies require emergency surgery for a non obstetric indication, including acute appendicitis, cholecystitis, adnexal torsion, choledoco-lytiasis, hernias, intestinal obstructions, oncologic pathology or other less frequent indications. laparoscopic approach is the preffered surgical option for the patologies presented above. aims: to present the technical particularities and to analyze the outcomes of the emergency operations in pregnant women operated in hospital. method: a retrospective study including all the pregnant women operated in our hospital between - was performed. the preoperative workup and the surgical indication was discussed by a multidisciplinary medical team. the anesthesic and the obstetrical risk and their management was evaluated and specifically planned for each patient. the intraoperative and post-operative outcomes were recorded. results: patients with gestational age between weeks and weeks who underwent emergency laparoscopic procedures were included in the study. out of the cases we have performed appedectomies, cholecystectomies, adnexal torsions. with a min mean operating time, we had no major intraoperative complications; the technical challenges are presented and discussed. the hospital stay was , days ( - days). no major complications were associated with the laparoscopic approach in these cohort. one pre-term labour in a weeks gestational patient was post-operatively encountered. conclusion: laparoscopic surgery can be the first option for pregnant woman with non obstetrical surgical emergencies; challenges in diagnostic, management and surgical techniques of the multidisciplinary team are expected. the objective of this presentation is to demonstrate step by step the technique to the oncologic surgeon and gynaecologist in training, including some tips and pitfalls. this is a laparoscopic transperitoneal approach in a woman with advanced cervical cancer (figo ib ) that will be treated with exclusive radio-chemotherapy. the purpose of the laparoscopic lumbo-aortic lymph node staging is to define the irradiation field. in this indication false negative in pet ct ranges from to % (depending of the existence of pelvic fixation or not). the limits of this lymphadenectomy are: both ureters as the lateral limit of the dissection, iliac bifurcation as the caudal limit and renal veins as the cranial one. since the tumour is cervical and not ovarian, both ovarian veins are not resected. in the pathologic report, lymph nodes were examined free of cancer spread. the patient have had a radio-chemotherapy with restriction of the irradiation field on the pelvis. lymphocele is a frequent complication that only sometimes needs treatment ranging from dietary changing to percutaneous drainage. if conversion to laparotomy for bleeding this technique loose its benefice but this is a rare complication. this technique is feasible and safe but requires advanced laparoscopic skills. objectives: although extremely rare, isolated splenic metastases are being increasingly diagnosed due to the improvement of imaging, survival times, and surveillance of oncologic patients. this video alerts to the growing diagnostic dilemma with primary lesions of the spleen, particularly in patients with history of cancer, and reviews the laparoscopic splenectomy 'step-by-step'. case-report: -year-old male patient diagnosed with rectal cancer (g adenocarcinoma at cm of the anal verge) after a colonoscopy for rectal bleeding. thoracic and abdominal ctscan and pelvicmri, showed a ct n lesion, without distant metastases, except for a mm suspicious splenic lesion. cea- . ng/ml. after neoadjuvant therapy, a complete response was verified at the th week post-crt with a stable splenic lesion, and a 'watch-and-wait' program was initiated with no evidence of disease at the rd month. pet-ctscan did not show active metabolic features, despite an increase in the splenic lesion. in mdt, elective laparoscopic splenectomy was proposed and afterwards performed uneventfully. with the patient in semi-right lateral tilt, we approached the spleen inferiorly by dividing the splenocolic ligament. then we continued upwards, dividing the gastrosplenic ligament and exposing the splenic hilum, which was then carefully dissected, clipped and divided. finally the splenorenal ligament was divided and the spleen was extracted within an endobag, through a small pfannenstiel incision. pathologic report revealed a splenic lymphangioma. the patient is currently under a 'watch and wait' protocol surveillance with no signs of regrowth or relapse disease after year and months of follow-up. conclusion: one out of five colorectal carcinomas are metastatic at their presentation. isolated metastases to sites other than liver, lung or axial skeleton, are extremely rare, but can be found in the spleen. although the rare splenic secondary involvement is usually associated with breast, lung, melanoma, and gynecologic malignancies, if we consider solitary splenic metastases, colorectal and ovarian carcinomas are important sources. also, imaging including percutaneous biopsy, is frequently insufficient to clarify the nature of splenic lesions. for all these reasons, the decision-making process about this issue can be a true challenge, and will probably end up with laparoscopic splenectomy. therefore, surgeons must be familiarized with a standardized technique. sarcoidosis is a multisystem disease of unknown etiology characterized by the formation of noncaseating granulomas. sarcoidosis should be considered in the differential diagnosis of lymphoid disease. indications for diagnostic splenectomy includes a suspicion of a neoplasic process. the less invasive laparoscopic approach is the gold standard. case report: a -year-old female was referred to a general surgery department to complete a study to rule out lymphoid neoplasia. followed by hematology for cytopenias. biopsy of bone marrow and adenopathies were negative for lymphoid process. patient presented ct with multiple solid ( - mm) lesions in spleen, in thorax showed no pathological changes. laparoscopic splenectomy was performed. access with optical trocar, in mammary line. triangle -mm trocars after pneumo under vision. section with ligasure of gastroesplenic ligament with short vessels and phrenic-splenic ligament. identification and preservation of pancreatic tail. section of splenic vessels at hilar level (branches) with ligasure. lower pole release. release of posterior part with gerota and diaphragm. incision by aid helps in bag without fragmenting. review of hemostasis, extraction of trocars under direct vision. intraoperative findings: spleen with normal external appearance, not megalic. postoperative evolution: satisfactory. first hours without incidents and with analytical control without anemization. tolerance and mobilization starts without incidents. the histopathology report shows granulomas formed by epithelioid histiocytes with the presence of multinucleated giant cells of the foreign body type, in some perisinusoidal granulomas the giant cells with the presence of asteroid bodies in their interior. the material has been revised with the extension of special studies. conclusion epithelioid granulomatosis, non-necrotizing, which suggests sarcoidosis. the procedure lasted min. the hospital discharge was on the next postoperative day and no complication was registered. conclusion: splenectomy can be performed in a classic way, but at present the less invasive laparoscopic approach is the gold standard. indications for splenectomy include splenic tumours of unknown origin, suspicion of a neoplastic process, and splenomegaly. sarcoidosis should be considered in the differential diagnosis for lymphoid disease. postoperative pathological examination confirms the diagnosis. week-day surgery, university, sapienza, ospedale sant'andrea, rome, italy aims: we describe an interesting case of a female patient affected by a suspected echinococcus granulosus large cyst of the spleen. methods: a years old woman complained abdominal pain and a sense of gravity in the upper left abdominal quadrant. computed tomography scan(ct-scan) showed a centimetre (cm) cyst of the spleen with thickness of the wall and contrast enhancement uptake referred to an echinococcus granulosus cyst. the sierological blood test assessment, antigens and antybody markes, for echinococcus granulosus infection was negative. a laparoscopic procedure was planned. the patient was positioned on the right flank, four trocars were inserted along the left subcostal region of the abdomen: one millimetre (mm) trocar for camera, one mm for the assistant, and two of mm for instruments. a periombelical minilaparotomy was performed for the specimen extraction. results: post-operative course was uneventful. patient was discharged in third post-operative day. istopathological exam showed a simple epithelial cyst of the spleen. conclusions: laparoscopy is safe and feaseable in case of large cyst of spleen in condition of unclear nature of the cyst. laparoscopy permits to explore the abdominal cavity and to assess the cyst characteristics in a lack overlap between the radiological exam and blood test examination. surg endosc ( ) we describe laparoscopic splenectomy for recurrent splenic cyst after laparoscopic marsupialization and partial resection of splenic cyst. the patient was a -year-old woman with abdominal discomfort and with a -cm palpable mass in the left upper and inferior quadrant. she undergone years ago in another country a laparoscopic operation for splenic cyst. abdominal computer tomography revealed a cystic lesion of the spleen with concomitant huge splenomegaly. serology and oncological marker were negative. we performed laparoscopic splenectomy for the recurrent splenic cyst. the operation took min. histologic examination of the resected spleen revealed a chronic hematoma. the patient had no abdominal symptoms during months of follow-up. postoperative long term follow-up and examination by ultrasound or computed tomography is required after surgical treatment for splenic cyst to exclude the possibility of recurrence after spleen-preserving surgery. hand-assisted surgery is a recognized technique that combines the advantages of laparoscopic approach with the tactile feedback of the laparotomic one. it proved beneficial especially for the treatment of megaspleens due to lymphoma localization, thanks to safer handling of splenic vessels, major bleeding control and more effective detachment of superior splenic pole from the diaphragmatic dome. here we show an hand-assisted splenectomy for megaspleen reaching the omolateral anterosuperior iliac spine due to lymphoproliferative disease, in which the hand, inserted through a right subcostal minilaparotomy, was very useful during the dissecting manoeuvres, the splenic artery recognition and ligation and the isolation of the superior pole of the spleen from the gastric fundus and diaphragm. in any case of huge spleens, the specimen bagging is very difficult to perform in a pure laparoscopic way, not to mention the inexistence of capable endobag; besides, a minilaparotomy would be necessary for the spleen extraction. hand-assisted approach allow to overcome this not underestimable technical difficulty, reducing operative time with similar aesthetic and functional results to that of laparoscopic approach. aim: the evolution of technology and its application to the minimally invasive surgery of the thyroid gland offers new surgical techniques, like the transaxillary approach. this new procedure is still being implemented in our environment and has recently begun to be incorporated into our surgical practice. the objective of this case is to explain step by step how to carry out a right transaxillary endoscopic thyroidectomy and emphasize in the most relevant tips to take into account. also, current indications and limitations of this technique will be addressed. methods: a -year-old woman is referred for evaluation of a right thyroid nodule without any associated symptomatology. the blood test shows normal thyroid profile. cervical ultrasound is performed identifying a . cm single right nodule with well-defined edges and presence of peripheral vascularization. no other nodules are identified. fine needle aspiracion (fna) of the nodule describes a bethesda iii. after evaluation, a right transaxillary endoscopic thyroidectomy was performed. results: dissection begins in the subcutaneous plane above the pectoralis major muscle until identification of the sternocleidomastoid muscle. dissection continues towards the prethyroid muscles in order to perform a lateral approach of the thyroid gland. section of the upper pole allows better exposure of the recurrent laryngeal nerve (rln) which is being monitored intermittenly. identification and preservation of the parathyroid glands is the next step. surgery is completed with the section of the inferior pole of the thyroid along with the istmus. the postoperative period was uneventful and patient was discharged at h after surgery. final pathology revealed a cm nodule without malignancy. conclusion: surgical treatment of the thyroid gland by transaxillary approach may be indicated in previously selected patients with benign pathology, offering the advantages from minimally invasive techniques (shorter recovery time, shorter incision length, etc.). further research is required to make a better assessment of the minimally invasive approaches in thyroid surgery. we present the video of a thoracoscopic esophageal leiomyomaenucleation. it has been widely demonstratedthe advantages of theminimally invasive approach in surgery. esophageal thoracoscopic surgery has been suggested as an alternative to open procedures, presenting less surgical trauma, lower risk of bleeding, less postoperative pain, lower wound infection and lower pulmonary morbidity, showing similar oncologic outcomes. although leiomyomas are the most commonof benign tumors of the esophagus, they are relatively rare, presenting an incidence of - per . autopsy series. in our case, the patient was diagnosed of leiomyoma located at the medium third of the esophagus. he referred a history of months of dysphagia for solid and liquids and retrosternal pain. the complementary studies were esophagoscopy, esophagography, ct and endoscopic ultrasonography. the patient was operated by a thoracoscopy approach using ports. it was completed the enucleation of the tumor following the closure of the muscular layer. methylene blue test confirmed no leaks. the patient was discharged on third day postoperative developing no incidences. pathology report: leiomyoma cm size, actin and desmin positive; s- , cd and cd negative. we want to demonstrate the advantages of a minimally invasive approach in this kind of pathology. aims: this video shows our technique to perform thoracoscopic enucleation of large esophageal leiomyoma. methods: the patient is a -years-old woman with a six months history of progressively worsening dysphagia. chest ct scan revealed a cm lesion of middle esophagus with extrinsic compression of mucosa and no increased fdg uptake on fdg-pet scan. barium swallow study showed a lateral deviation of thoracic esophagus due to extrinsic compression. endoscopic ultrasound confirmed the suspicion of esophageal leiomyoma. patient was scheduled for a thoracoscopic enucleation of esophageal tumor. she was placed in prone position and one-lung ventilation was employed. three trocars were placed in intercostals spaces on right hemithorax. azygos vein was identified and transected between vascular clips. esophagus was circumpherentially isolated from mediastinal structures. after myotomy, the lesion was dissected from submucosal-mucosal layer. since air leak test excluded injury of internal layer, muscular layer was closed with a continuous suture. the specimen was extracted through an endobag. a drain was left in place. results: the postoperative course was uneventful and the patient was discharged on postoperative day . final pathological examination confirmed esophageal leiomyoma. conclusion: thoracoscopic surgery in prone position allows removal of large esophageal tumor with several advantages. the minimally invasive technique decreases perioperative morbidity and mortality. introduction: spontaneous esophageal perforation is life threatening disease and requires emergent surgical treatment. recently, the efficacy of minimally-invasive surgery such as laparoscopic and thoracoscopic surgery for esophageal perforation has been reported. we report a novel technique of minimallyinvasive abdominal and left thoracic approach (malta) for spontaneous esophageal perforation. case presentation: -year-old male, who had been under hemodialysis due to iga nephropathy, complained of chest pain after vomiting several times. since the ct scan showed left hydropneumothorax and pneumomediastinum, and the gastrografin study demonstrated extravasation from left side of esophagus, we diagnosed him with the spontaneous esophageal perforation and planed emergent surgery. the patient was placed in the reverse trendelenburg position, and the legs were split, with the left side of the upper body lifted in order to perform thoracoscopy and laparoscopy simultaneously. first, we explored the thoracic cavity through a mm port in the left th intercostal space and added other ports. we identified the rupture site mm in size on the left wall of the lower esophagus and sutured the mucosa and the muscle layer with a running suture respectively. we covered the perforation section with pericardium fat and irrigated the cavity with physiological saline. then transferred to the abdominal cavity, no contamination was found in the abdominal cavity. a feeding tube was inserted into stomach through the round ligament of the liver and the operation was completed. the total operative time was min and the amount of intraoperative bleeding was ml including pleural effusion. postoperatively, the patient experienced left empyema pleurae but no other severe complications and was discharged on postoperative day . conclusion: we experienced a rare case of spontaneous esophageal perforation of a patient under hemodialysis. malta is an effective procedure for emergent esophageal operation because of great visual field of the chest and abdominal cavity without expanding contamination. introduction: digestive caustic injury is associated with high morbidity and mortality with stenosis in the long term. surgical treatment involves resection of the esophagus and reconstruction with the stomach, colon or jejunum. coloplasty provides several advantages but its vascularization is complex and involves anastomosis. classically, vascular assessment was achieved by palpation through laparotomy and color evaluation. indocyanine green (icg) allows a minimally invasive intraoperative angiography in real time. methods: a -year-old female with medical history of caustic ingestion and subsequent esophagogastric stenosis, carrier of feeding jejunostomy. . thoracoscopy (prone position): dissection of the esophagus from the hiatus to the upper thoracic inlet. . laparoscopy: patient in the supine position, placement of five trocars. total non-oncological gastrectomy, post-pyloric section of the duodenum and omentectomy were completed. mobilization of the righ, transverse and descending colon. measurement of the transverse colon with a tape (distance from the neck and the esophageal hiatus). individualization of the righ, middle (with its branches) and left colic arteries and placement of clamps at the right colic, right branch of the middle colic and left colic arteries. cc of icg were injected allowing for an assessment of the colon vascularization. section of the right branch of the middle colic artery. proximal section of the ascending and distal colon near the splenic angle, preserving the marginal arch. silk point to join the staple line of the descending colon and the pylorys. side-to-side mechanical antiperistaltic anastomosis between the distal endo of the coloplasty and the jejunum. finally an anastomosis between the ascending and descending side-to-side mechanical anastomosis using an assistance incision in the left flank was performed. . cervical dissection: extraction of the surgical especimen under laparoscopic control. vascular assessment with icg is performed before and after the side-to-side anastomosis is performed. results: there were no intraoperative complications. the patient was discharged on postoperative day . discussion: we describe the first case of total minimally invasive colonic interposition with icg assessment of the vascularization. this technique, although technically demanding, avoids the drawbacks of the open surgery and allows for a precise assessment of the vascularization of the graft. surg endosc ( ) introduction: large pedunculated fibrovascular polyps are uncommon, mostly benign, intraluminal massess, usually located in the upper esophageal tract. most frequent reported clinical manifestation is dysphagia, followed by regurgitation, chest pain and intestinal bleeding. ct scan, and mri are the key in the diagnostic work-up revealing a sausage-shaped intraluminal mass. endoscopy with ultrasonography and biopsy add important information for the diagnosis and pedicle location. surgical excision is deemed due to potentially life-threating complication related to airway obstruction. the most frequent polyp resection is performed through cervical esophagotomy or by direct esophagectomy. however, this approach is related to a high morbidity and mortality rate. in the last years, few excisions have been reported by a endoscopical approach with a lower post operative complication. material and methods: this video shows the surgical steps of a trans-oral endoscopic surgical resection of a giant ( cm) pedunculated polyp in a year old man. the procedure was performed under general anesthesia. a flexible endoscope probe was used and the distal end of the polyp was extracted through the oral cavity with a loop. the endo-gia stapler was used to cut the base of polyp and finally removed. the anatomo-pathological study confirmed the diagnostic of a fibrovascular polyp with no evidence of malignancy. results and conclusions: the patient had an uneventful recovery with no recurrency at years of follow up. this minimally invasive approach is a safe and feasible procedure to treat large esophageal fibrovascular polyps avoiding the complications related to more aggresive procedure. introduction: leiomyomas are the most common mesenchymal tumors affecting the esophagus and they usually grow in the mid to distal third of it. they tend to be asymptomatic, but sometimes they can grow to enormous size and produce dysphagia. case report: -year-old male asymptomatic patient was referred to our hospital due to an incidental finding. ct scan revealed a x mm rounded submucosal tumor on the dorsal side of the lower third of the esophagus. upper gastrointestinal endoscopy revealed a cystic lesion in the lower esophagus cm from the incisor teeth, with normal overlying mucosa. an endoscopic-ultrasound-guided fine-needle-aspiration of the mass was performed, which was reported as a likely leiomyoma.conservative treatment was performed, no growth was detected during eleven years of follow-up. but it became symptomatic, the patient complained of progressive dysphagia caused by compression so surgical resection was decided.laparoscopic enucleation of esophageal leiomyoma was performed. the tumor was reached by transhiatal dissection. a careful dissection of the mass was performed, preserving the vagal branches. an intraoperative endoscopy was performed to verify the integrity of esophageal mucosa and that the tumor was completely resected. the muscular layer was sutured after enucleation using absorbable suture material and the hiatus was closed with non-absorbable suture material. a dor fundoplication was also performed. a swallow test with a water-soluble contrast was obtained on postoperative day one. no pathological findings were found so the patient was asked to drink.histopathological exam revealed a tumor measuring mm mm mm consistent with leiomyoma.the procedure lasted min. the hospital discharge was on the third postoperative day and no complication was registered. conclusion: surgical excision is the mainstay of treatment and is recommended for symptomatic leiomyomas and those greater than cm. this case report demonstrates the technical feasibility, safety and minimal postoperative morbidity associated with minimal invasive esophageal surgery. introduction: total esophagectomy by means of minimally invasive surgery has proven to be a valid and effective alternative for performing this procedure. however, this procedure is not implemented in most centers. objective: demonstrate the technique of a total esophagectomy by endoscopic surgery for a benign esophageal stenosis. material and methods: clinical case: a -year-old female patient diagnosed with double esophageal peptic stenosis, treated on several occasions with endoscopic dilation by digestive, showing in the last endoscopy: severe esophagitis with stenosis impassable to cms. additional tests of interest are exposed. resolved: intervention: right thoracoscopy in prone position, dissection and complete mobilization of the thoracic esophagus, section of the azygos vein, pleural drainage. laparoscopic time, trocars, gastrolysis respecting the right gastroepiploic vessels, broad kocher until the cava is identified, vascular section of the vessels left gastric, full mobilization of the stomach, subxiphoid minilaparotomy, beginning of the cervical time with dissection of the cervical esophagus, section and fixation of this to a tube, externalization of the piece by abdominal route, creation of the gastric tubular with successive loads of gia, ascending posterior mediastinal plasty with manual esophago-tubular anastomosis, with placement of drainages and feeding jejunostomy. right operative with radiological control with gastrografin on the th day, discharge from hospital on the th day. asymptomatic one year after surgery, with radiological control without alterations. conclusions: the approach of esophageal peptic stenosis with minimally invasive surgery is safe and effective, adding the advantages inherent to this type of technique. (figs. , ) , showing a cystic lesion in the gastric submucosa with a well defined, medial and superior to the lesser curvature of the stomach with exophytic growth. it causes extrinsic compression of the cardia in the gastric body. within the differential diagnosis are gastric duplication cysts or gastrointestinal stromal tumor. a biopsy was taken, discarding the presence of neoplastic cells. finally, a study of digestive transit showed extrinsic compression at the cardial level, which causes difficulty in passing contrast. a laparoscopic approach was performed beginning with the dissection of the abdominal esophagus. the presence of a cystic lesion on the anterior face of the abdominal esophagus was identified (figs. , ) . we proceeded to the complete resection of cyst. the surgery was completed with °f undoplication anterior dor. the patient went home on the rd day without incident. results: duplication cysts are congenital malformations of the gastrointestinal tract contiguous with the esophagus, which can communicate with the esophageal lumen. most are diagnosed in childhood, but when it is diagnosed in adults, they used to be symptomatics. it is more frequent in men. although the pathogenic mechanism is unknown, it is caused by an anomaly during embryonic development. they are located in the thoracic esophagus, at the level of the lower and posterior mediastinum and, less frequently, in the abdominal esophagus, as in our case. they can give digestive symptoms(epigastralgia, vomiting) or respiratory symptoms. the diagnosis is made with eda, ct, and ecoendoscopy of choice, although it may be incidental. the treatment of choice in symptomatic patients is complete resection or cystic enucleation. in asymptomatic surgical is not defined, because it can cause complications, and, malignization due to degeneration is very infrequent. conclusion: the most of duplication cysts are diagnosed in childhood, although it's more frequent in adults to be symptomatic. surgical treatment can cure this disease. however, the choice between these becomes difficult in young patients, where the low incidence does not allow get series of long patients and decisions must be based on results achieved in adults. objetivos: to demonstrate the safety and efficacy of the laparoscopic approach in this infrequent pathology, pointing out the importance of having standardized the procedure to achieve better results. material and methods: case report: a -year-old man with progressive dysphagia until almost complete afagia, with clinical, endoscopic, radiological and manometric diagnosis,compatible with typical primary achalasia. chagas negative serology,we show the complementary studies of interest.dilatation is not performed, preoperative symptomatic treatment with calcium channel inhibitors. intervention: laparoscopic approach, trocars, aberrant left hepatic artery with signs of severe esophagitis,opening of the gastroesplenic-hepatic ligament, no retroesophageal window, dissection of the hiatus and inferior mediastinal, preservation and mobilization of the left hepatic artery and the anterior vagus,meticulous disection of the cardia,standardized myotomy: first proximal cms. with adequate simultaneous traction of both edges of the myotomy, then distal myotomy including - cm, including selectively the distal oblique fibers of les, tutorization with fouché and methylene blue to confirm good step and absence of leakage, dor-type funduplication, pts on each side, fixed to both pillars, hiatalmediastinal drainage. egd on the st day of normal po, dischargeat rd day, asymptomatic and with normal radiological control at year of age. conclusions: laparoscopic mh should be the first therapeutic option, in patients with primary achalasia, even in young patients. the length of myotomy, especially distal to ueg is one of the most important aspects of surgery, most authors (pellegrini) recommend that the myotomy extend - cm in the stomach, even up to cm below the ueg to achieve an effective disruption of the eei.the standardization of the procedure is fundamental to increase safety and effectiveness in these more complex cases. aims: the surgical treatment of giant hiatal hernias is a complex and demanding procedure, not only in terms of performing the operation in a minimally invasive abdominal fashion by avoiding thoracic approaches, but also concerning the management of large hiatal defects which contribute to high recurrence rates. our aim is to present our surgical technique for the reconstruction of such hiatal hernias, exploiting the benefits of the robotic approach and also to highlight the technical aspects of non-absorbable mesh placement in order to bridge effectively the hiatal defects. methods: we present video fragments from a procedures selected from a series of cases of robotic reconstruction of giant hiatal hernias performed by our team, in which a non-absorbable meshes were utilized to restore the hiatal gap. we emphasize on the clear benefits of robotic surgery in these cases and on the strategy of how to avoid high recurrence rates. results: all of our patients, who underwent reconstruction of giant hiatal hernias with this particular technique, experienced very good early post-operative results, very short hospital stay and no recurrence in a -month follow-up. conclusions: the robotic approach for the treatment of large hiatal hernias offers great advantages to both surgeons and patients, by eliminating the restrictions of conventional laparoscopic surgery, minimizing intra-operative incidents and post-operative complications. large hiatal defects are very effectively closed with the use of advanced suturing techniques and non-absorbable meshes in a tension-free bridging fashion. aims: mckeown esophagectomy is commonly used for invasive esophageal carcinoma. as the morbidity and mortality rates for esophagectomy are persistently high, minimally invasive esophagectomy in prone position is expected to reduce respiratory postoperative complications. there is still limited experience for the use of minimally invasive approaches in patients undergoing surgery after neoadjuvant chemoradiation and many concerns about the feasibility, safety, and oncological outcomes of these procedures are still present. methods: we present the case of a -year-old female with a middle third esophageal squamous cell carcinoma, who received neoadjuvant chemoradiation. she underwent laparoscopic and thoracoscopic (prone position) mckeown esophagectomy with hand-sewn esophagogastric anastomosis through a left lateral cervical incision. results: the operation was completed successfully, with no conversion to open surgery. the operative time was h with minimal blood loss and the patient was fed on day and discharged on day post-op. r resection was achieved and the number of total harvested lymph nodes was ( positive nodes, n ). conclusions: minimally invasive mckeown esophagectomy in patients with esophageal cancer and prior chemoradiation is feasible and safe procedure with acceptable oncological outcomes. results: preliminary results demonstrated that minimally invasive ivor-lewis esophagectomy procedure, provided of a better postoperative pain control and less respiratory complications. in order to standarise our procedure, the video shows how three different types of esophagogastric anastomosis are performed, depending on the patient characteristics, anatomical factors and safety and comfort for the surgeon: manual termino-terminal, mechanical termino-terminal and mechanical latero-lateral. conclusions: in our way to standardization, we are still looking for the best type of anastomosis, even though, we find out that, manually performed anastomosis are easier to performed, when the section in esophagus is lower, involving medium and inferior third. in the other hand, mechanical termino-terminal anastomosis seemed to be an ideal option for upper sections. more studies are needed in order to standardized one anastomosis, for all cases. because esophagectomy with radical lymphadenectomy is highly invasive, thoracoscopic esophagectomy (te) is attracting attention as a less invasive procedure. we first performed te with the left decubitus position in . in we developed a hybrid of the prone and left lateral decubitus positions for te, and a total of patients underwent te with a hybrid position . we introduced te with a hybrid position for the following three reasons: ( ) mobilization and lymphadenectomy around the middle and lower esophagus are easier in the prone position. thanks to artificial pneumothorax and the gravity, the middle and lower mediastinum are opened, and which give us good surgical field. ( ) lymphadenectomy along the left recurrent laryngeal nerve (rln) is more reliable and precise when performed in the left lateral decubitus position. we can dissect lymph node around the rln higher position in the upper mediastinum. ( ) unexpected events requiring conversion to thoracotomy (e.g., massive bleeding, injury of other organs, dense intrathoracic adhesion, resection of adjacent organs) are easier to deal with in the left lateral decubitus position. the patient is fixed on the operating table with the semi-prone position and we can easily change patient positions from the left lateral decubitus position to the prone position and vice versa using rotation system of the operation table. the upper mediastinal procedure including lymphadenectomy along the right and left rln is performed with the patient in the left lateral decubitus position, while the middle and lower mediastinal procedures are performed with the patient in the prone position with artificial pneumothorax ( mmhg). theabdominal procedures wereperformed by hand-assisted laparoscopic surgery (hals) and gastric tube reconstruction through aposterior mediastinal route was performed as a standard surgical procedure in our institution. the magnifying effect of thoracoscope enables us to perform more precise surgery and preserve nerve and vessels, and a hybrid position is thought to be feasible and effective methods. ivor lewis esophago-gastrectomy is a standard procedure for the treatment of distal esophageal cancer. among the years, the surgical community standardized the mininvasive abdominal phase. the thoracic phase is much more complex because usually all surgeons get in trouble in the phase of esophago-gastric anastomosis. in fact, is still very difficult and tricky to perform a mechanical circular anastomosis due to problems with the correct handling of the circular stapler through the minitoracotomy and is also difficult to place the anvyl in the proximal esophagus. the linear anastomosis (side-to-side) is a little bit easier but not so effective as the circular anastomosis in terms of leak rate. we think that robotic approach with its endowrist can allow us to overcome these limits and that a tailored double layer hand sewn esophago-gastric anastomosis could be the right choice. we treated patients with this approach and they were all uneventful in the post operative period except for a case of chylothorax we treated successfully with lipiodol injection in inguinal lymphnodes. we need more cases to analyze the technique in terms of leak rate and major complications but we think this is a promising and cost-effective procedure for robotic approach. aim: anastomotic leakage is one of the most dreaded complication after esophagectomy. indocyanine-green near-infrared angiography (nir-icga) intraoperative use has been recently introduced for visceral perfusion evaluation. in this video we present our technique for gastric conduit fashioning according to the nir-icga blood supply evaluation in a total minimallyinvasive ivor-lewis esophagectomy. methods: a years-old man affected by a siewert adenocarcinoma (ct , n , m ) underwent a preoperative neoadjuvant treatment according to cross protocol. at restaging ct-scan no more pathologic nodes were evident (ct , n , m ). the patient was submitted to a total minimally-invasive ivor-lewis esophagectomy. surgical procedure: after pneumoperitoneum induction and trocars insertion, the lesser omentum was opened and a lymphadenectomy at stations a, , , and performed. the esophagus was dissected at diaphragmatic hiatus with lymphadenectomy at station , and . the larger omentum was opened along the right gastroepiploic arcade, that was preserved, the short gastric vessels divided and gastric fundus mobilized. by evaluating the presence of an intense fluorescence at nir-icga, a tailored partial tubulization of the stomach was performed with multiple linear stapler firing. a right thoracoscopy was performed through trocars. the azygos vein ligated and divided. the mediastinal pleura was opened and the esophagus was dissected entirely with an en-bloc excision of nodes at stations , and . nodes at stations , , and were removed separately. the esophagus was sectioned above the azygos vein level and a purse-string fashioned. the cardia and the gastric tube were pulled up and a minithoracotomy performed. a new nir-icga was repeated to verify the good blood supply and tailor the site of anastomosis on a well perfused area. the stomach was opened and a circular stapler inserted. after the end-to-side esophago-gastric anastomosis fashioning, the tubulization was completed by linear stapler firing and the specimen removed. results: the post-operative course was uneventful and the pathologic examination revealed a cardial adenocarcinoma (ypt ,n , r ). conclusions: nir-icga is an interesting and easy-to use tool for surgeons. nevertheless in literature is still not clear which is the best parameter to measure the blood supply. large studied are needed. aim: uses and application of indocyanine green (icg) fluorescence in the field of surgery are growing exponentially. the safety and feasibility of its usage has been proven in several areas and various pathologies of surgery and surgeons are starting to incorporate it into their common practice. however, there are still several aspects to define regarding this technology. we present different uses of icg in the specific area of esophageal cancer. methods: we used icg fluorescence at different moments of a two-field minimally invasive esophagectomy. first of all, peritumoral injection of icg may offer a lymphatic mapping, both in the abdominal phase of the surgery and the thoracic one, improving lymph node dissection by allowing a more targeted and less morbid approach that includes all relevant nodal stations. at the moment of the gastric section, intravenous injection provides assessment of gastric conduit perfusion, therefore optimizing the construction of the graft to avoid the inclusion of poorly perfused areas that may increase the risk of leak of the anastomosis. besides that, the esophagogastric anastomosis can be tested in the thoracic phase of the operation in order to check an adequate perfusion and prevent further complications. results: we consider that icg fluorescence is a promising technology that could be easily introduced in the surgical routine of the esophageal surgeon as an instrument to assess the anastomosis perfusion. icg is also feasible in detecting lymph node drainage from the esophagus, although its technique of application needs to be defined. conclusions: icg fluorescence has opened a new world of possibilities in all the different surgical specialties. its use in the esophagectomy is safe, simple and feasible. in a near future, its application to esophageal cancer surgery could improve survival by predicting and preventing anastomotic leak and guiding in a tailored lymphadenectomy. further research is needed to demonstrate these promising applications. introduction: the oesophagectomy is currently still mandatory in the curative treatment of the malignant oesphagic pathology. this procedure is defined by important morbidity and mortality. the minimally invasive approach aims to reduce the complications without repercussion on the oncological outcomes, however it's not exempt from them being a demanding surgical technique like it is. aim: we present the video of three complications after a three-stage oesophagectomy (mckeown-like) with the thoracic stage via thoracoscopy and the minimally invasive surgical solution for both of them. methods: all three cases represent a three-stage oesophagectomy for malignant esophageal pathology. the first one was a -year-old male who suffered an intraoperative left main bronchus injury. the second case was a -year-old male with no intraoperative complications whatsoever. nonetheless, on the second postoperative day, milky drainage started to appear through the thoracic tube. the third and final case represents an intraoperative hemorrhage, which is the most common complication of this kind of surgery. results: the first case was diagnosed and treated intraoperatively with the use of an adhesive matrix. during the postoperative period the patient showed no further complications. the second case was a chylothorax, diagnosed on the second postoperative day. it was treated initially with conservative measures. due to bad evolution, he underwent surgery on the tenth postoperative day. we can see how we ligated the stump of the thoracic duct in the original surgery and then how we repaired the unexpected leak. after the second surgery, the patient was discharged on the sixth day. the last patient was also diagnosed and treated intraoperatively successfully, with no repercussion whatsoever in the postoperative time. conclusions: the minimally invasive surgery has many advantages in the upper gastrointestinal field. it is a demanding technique, so it is important to be able to treat the complications that may arise with this approach. surg endosc ( ) aims: the use of icg fluorescence is incrising in surgery, mainly as a test of vascular supply in colonic anastomoses. during the last years, other potential uses have been described, such as the identification of the sentinel lymph node and lymphatic mapping in oncological surgery. these advances could allow a better staging in order to decide the most appropriate treatment to each patient. gastric cancer is one of the fields where this could play a key role in the near future. we present a case of a patient who underwent a laparoscopic total gastrectomy with icg-guided d lymphadenectomy, where a personalized lymphatic mapping was performed. methods: a -year-old male patient underwent gastroscopy for gastric discomfort, and a gastric carcinoma was detected at the greater curvature of gastric body. endoscopic biopsy was informed as diffuse type gastric adenocarcinoma. the preoperative staging was completed with echoendoscopy and ct-scan (t bn m ). we decided to perform a laparoscopic total gastrectomy with icg-guided lymphadenectomy. the preoperative day, a gastroscopy was performed to inject . mg of icg in four submucosal areas around the tumor. results: intraoperatively, the lymphatic mapping marked by icg was checked, allowing the identification of the territory of drainage of the tumor to lymph nodes at the lesser curvature, the greater curvature and the splenic artery. a d lymphadenectomy and a total laparoscopic gastrectomy with roux-en-y reconstruction was performed. during the lymphadenectomy, we were able to observe marked lymph nodes in territory , and also observed that the paraaortic lymph node behind the celiac trunk did not become green and the lymphadenectomy at this area was not continued. the patient presented no postoperative complications, and was discharged on the seventh postoperative day. the histological results showed a diffuse type gastric adenocarcinoma pt n and isolated lymph nodes, being one of them possitive (corresponding to the adenopathy marked at the greater curvature). conclusions: lymphatic icg-mapping in gastric cancer is a potential revolutionary advance that could ensure a correct lymphadenectomy, avoiding lymph node understaging. it is necessary to continue carrying out studies that will allow developing protocols to define appropriate lymphadenectomy based on icg-mapping. introduction: petersen's hernia is one of severe postoperative complication after gastrectomy, which may result in massive resection of small intestine. it is considered an essential proscedure to close the petersen's defect for all cases after such reconstruction after gastrectomy as roux-en y method. we report a case of petersen's hernia after radical gastrectomy, which was repaired laparoscopically. patient: the patient was -year-old male, who underwent laparoscopic distal gastrectomy for gastric cancer (d lymph node dissection followed by roux-en-y reconstruction) two years ago. the closure of petersen's defect was not performed in the initial operation. he was aware of abdominal pain and visited emergency unit in our hospital. abdominal ct scan showed internal hernia of petersen's hernia. surgical procedure: in laparoscopic examination, dilation of small intestinal and mastoid ascites was observed. massive small intestine including y-limb entered into petersen's defect from left to right side. we carefully pulled through the small intestine and confirmed absence of ischemic change in the whole small intestine. then the petersen's defect was closed by continuous suturing with - non-absorbable barbed suture. results: the operation time was min and the estimated blood loss was ml. oral intake was started from the next day of the operation. there was no postoperative complication. the patient was discharged on the th postoperative day. conclusion: we could safely perform laparoscopic repair for petersen's hernia. regarding technical points in the procedure, it is important to judge the direction of the small intestine into the petersen's defect, to manage the dilated small intestine gently, and to close the petersen's defect by laparoscopic suturing. introduction: in this case we present a -year old male with a history of morbid obesity, sleep apnea and psychiatric affliction including alcohol and nicotine abuse. in he underwent a laparoscopic roux-en-y gastric bypass. the results were satisfactory, with no complications post-surgery, and a steady weight loss over time (pre: kg, post: kg). in november , he presented with complaints of dysphagia and weight loss ( kg in months). laryngoscopic examination by the otorhinolaryngologist was negative. he was referred to gastro-enterology for gastroscopy. biopsies showed a mildly differentiated adenocarcinoma of the gastro-esophageal junction with submucosal invasion. objective: after negative staging assessment, multiple treatment options were considered. the route of choice ended up being a laparoscopic radical gastrectomy with esophago-jejunostomy, with the objective to achieve optimal oncological results. methods: the procedure is demonstrated in this video. the gastric pouch as well as the remnant stomach, greater and lesser omentum were resected laparoscopically. due to the invasion of the carcinoma into the distal esophagus, a segment of the esophagus was resected as well. following anatomopathological examination on frozen section, the resection margins were reported malignancy free. results: postoperatively, there were no complications. ct scan with contrast showed no signs of leakage. anatomopathological examination confirmed the tumor to be a mildly invasive and poorly differentiated adenocarcinoma with local signet-ring cell differentiation (pt bn ). there was no need for adjuvant therapy. oral intake was sound. conclusion: adenocarcinomas of the gastric pouch are rarely seen following gastric bypass. this patient presented with complaints of dysphagia, and an adenocarcinoma was diagnosed. consequently, the patient had a total gastrectomy at our hospital. the surgery was performed laparoscopically, and was executed with success. to conclude, it is feasible to treat adenocarcinomas after gastric bypass laparoscopically via total gastrectomy and omentectomy. year old, female patient presented with upper abdominal discomfort and microcitic anemia. an ulcerative lesion was found on gastroscopy examination in body of the stomach (near the grate curvature). biopsy was done and pathology result showed poorly differentiated adenocarcinoma. chest computed tomography (ct) was without any significant findings. abdominal ct showed the lesion in stomach without enlargement of regional lymph nodes. her blood laboratory examinations were within normal limits, including serum cea. patient underwent laparoscopic total gastrectomy with modified d lymphadenectomy and roux-en-y esophagojejunostomy. total operating time (ort) was min. three days after operation, patient has developed none st elevated mi and respiratory failure. she was intubated. on day after operation she was extubated, on day patient started regular diet and was discharged home on day . final pathology result confirmed poorly differentiated adenocarcinoma of the stomach. this video shows our favourite technique for laparoscopic d subtotal gastrectomy. we usually perform the procedure with or trocars; after the coloepiploic detachment we perform the gastric transection first! this manoeuvre provides a perfect view for the lymphadenectomy. at the end of our dissection we transect the duodenum with seamguard reinforcement. before going on with the reconstructive phase, we prepare the roux en 'y' with double loop technique, usually without dividing the mesentery. then we remove the specimen through a periumbilical - cm minilaparotomy; we think it's important anyway to check margins and distance from the tumor before going on with the reconstructive phase. from the same minilaparotomy we retrieve the prepared limb for roux en y and we perform a side to side mechanical linear anastomosis outside.then we proceed with performing the anastomosis between the gastric pouch and the alimentary limb by laparoscopy. we like very much this technique for the increased exposition of tissues during lymphadenectomy. laparoscopic roux en 'y' d surg endosc ( ) a body-tc was performed in which axillary, mediastinal adenopathies and images suggestive of hepatic metastases were identified. the biopsy confirms a gastrointestinal stromal tumor. the case discussed in a multidisciplinary committee and the pet-ct study was completed, subcardial gist t n m was diagnosed. neoadyuvance was decided with imatinib for one month and surgery was performed using a laparoscopic approach. the approach was performed with trocars ( mm supraumbilical, two mm subcostal left and right, mm subxifoid and mm left flank). gastrectomy was performed with d lymphadenectomy following the oncological principles of subcardial tumors. the piece was removed in a bag by extending the mm port to mini-laparotomy. esophagogastric anastomosis was performed by hand assisted circular mechanical suture. methylene blue test was carried out. no nasogastric tube left, but drainage tutoring the esophagogastric anastomosis was left. results: the postoperative evolution was favorable. oral tolerance without incidents at fourth postoperative day. the patient was discharged without incidences on the seventh postoperative day. the pathological study of the piece was reported as subcardial gastrointestinal stromal tumor cm with respected surgical margins and lymph nodes free of malignancy, postoperative diagnosis of t n m . one month after surgery, the patient has adequate oral tolerance. she does not report gastroesophageal reflux and at months remains asymptomatic and with good evolution. conclusions: laparoscopic proximal gastrectomy is a technique that is not currently used but can be performed through a laparoscopic approach. it is a safe technique with good clinical and oncological results, especially in the early gastric cancer and gastrointestinal stromal tumors. however, long-term studies are necessary. laparoscopic gastrectomy is a perfectly safe option nowadays for the treatment of gastric cancer. every year the percentage of the laparoscopic approach is rising not only in the east but also in the west. we present a case of a year old female patient with a gastric tumor of the antrum-g adenocarcinoma with a ct n m staging. we perform a subtotal laparoscopic gastrectomy with a d lymphadenectomy and roux-en-y anastomosis. the patient begin clear liquids on the first post operative day and was discharged on the th. the final anatomopathological result of the specimen was a adenocarcinoma (g )-pt n . there were nodes resected all negative. the case was discussed in multidisciplinary team and was decided for clinical follow up with no further treatments. the patient was evaluated one month after surgery with no complaints and will continue the follow up. upper gi surgery, university of verona, verona, italy laparoscopic endoscopic cooperative surgery is an option in medium size submucosal cancers invading the muscular layer, mainly in border area were wedge resections are nor feasible.in this video we report a case of prepiloric gist treated with news technique (nonexposed endoscopic wall-inversion surgery).we think that this technique is feasible and safe and should be considered a valid option with a view to preserving the organ. aim: laparoscopic wedge resection or partial resection is a safe and feasible stomach preserving approach to gastric submucosal tumors (smt) such as gastrointestinal tumors (gist), and it has been widely performed recently. however, it should not be applied to the tumors at cardia in order to avoid stenosis or disruption of anti-reflux mechanism. we have introduced percutaneous endoscopic intragastric surgery (peigs) for smt at cardia since to preserve function of cardia. we will report the tips, techniques, and clinical result of our peigs. methods: from september to august , seven patients with smt at cardia underwent peigs in our hospital. we insert the mm port at umbilicus and investigate the abdominal cavity at first. then the incision is extended to . cm and lap-protector tm is equipped with the site to perform mini-laparotomy. using peroralendoscopy, a stomach was insufflatedand incision is made at an anterior wall of gastric body under direct vision. additional lap-protector tm is placed into the stomach so that the stomach is fixed on the abdominal wall. it enables us to keep direct access to gastric lumen and stable operative field. ez access tm is attatched on lap-protector tm , and intragastric operation is started. subsequently, two trocars are inserted using funada's gastropexy instrument. tumor is dissected by using energy devices, avoiding injury of capsule of the tumor. the defect of the gastric wall is closed by intragastric suturing. we should take particular care not to damage egj during the suturing, inserting peroral endoscopy in and out. results: the mean operation time was ( - ) min and the amount of intraoperative bleeding was . ( . - . ) ml. the maximum diameter of tumors was ( - ) mm. one case was low risk gist and otherwise were leiomyoma. the postoperative course was uneventful in all cases, without leakage or stenosis. total hospital stay was ( . - ) days. no patient had symptoms of esophagitis. conclusions: peigs for smt at gastric cardia is a feasible and safe approach, preserving function of cardia. our procedure achieves great stability and excellent visualization during the operation, which may have led to the fine surgical results. laparoscopic lymphadenectomyin gastric cancer is considered a feasible and safe procedure. data on the compliance of lymphadenectomy in the various lymph node stations is not yet well understood; moreover it is not clear if there are particular conditions relate to the patients impairing the oncological results. this video reports the use of the icg for the lymph node dissection of station number in a case of obese patient and a case of a cirrhotic patient. fist patient, m.a., was a year old man with distal cancer ct n and a bmi. second patient, t.d., was a year old man with distal cancer ct n and a alcoholic cirrhosis child b . in both cases, intraoperative endoscopy was performed to min before the onset of lymph node dissection. . mg of icg was injected into the submucosal layer in quadrants of the primary tumor. a laparoscopic subtotal gastrectomy was conduced with d lymphadenectomy. lymph note navigation were analyzed by novadaqÒ detector. using navigation system we removed the n basin. in both cases dissection were effective and pancreatic surface were easily detectable. number of lymph nodes retrieved was in the case of obese patient and in the case of cirrhotic patient. pathological tnm were pt n ( / n ?) in the first case and pt n ( / n ?) in the second. no n ? metastases were detected in n station for both cases. no pancreatic fistula was recorded. icg lymph node navigation system should be considered a valid support for the surgeon for completion of a correct lymphadenectomy in surgical challenging cases. aims: morgagni hernia is the rarest of congenital diaphragmatic hernias ( - %). its presentation is rare in adults and its finding is usually incidental. it was first described by giovanni b. morgagni in . it is located in the anterior region of the diaphragm. it is caused by a congenital defect in the fusion of the transverse septum of the diaphragm and the costal arches. the need for surgery depends on the presentation, it is recommended early repair before the development of complications. classically, the surgical approach was thoracotomy or laparotomy. currently, the tendency is to use a minimally invasive approach, which has shown good results, lower morbidity and faster recovery. the need to repair the defect with a mesh is controversial, recommended when it is not possible to close the defect without tension. the objective of this video is to demonstrate the safety and efficacy of the laparoscopic approach for the repair of this type of hernia, as well as the different types of mesh that can be used. aims: the treatment of the non-metastatic gastro intestinal stromal tumour (gist) is the surgical resection [ ] . the duodenal gastro-intestinal stromal tumour (gist) is a relatively rare clinical entity. from all the resected gist, only % are duodenal [ ] . the clinical presentation could vary from the most common acute gastro intestinal (gi) bleeding, chronic anaemia, but also acute abdomen caused by tumour rupture and intestinal obstruction. methods: a years old patient present at the emergency department of the chi poissy-st germain-en-laye (paris, france) with acute gastrointestinal bleeding. at the laboratory exams the haemoglobin was . g/ dl. the patient perform a computer tomography (ct) which shows two hyper vascularised lesion at the th duodenum; this lesions has an intra and extraluminare growing. the ct scan didn't show any other abdominal lesions. the patient were submitted to a minimally invasive surgical operation with the multiport laparoscopic technique. results: the resection of the th and th duodenum and of the first centimetre of jejunum was performed with a four trocar laparoscopic technique. a latero-lateral duodeno-jejunal mechanical anastomosis was performed. the operative time was min. the patient start with oral alimentation on the third post-operative day after a ct scan with oral contrast that was negative for anastomotic dehiscence and collections. the post-operative course was globally uneventful and the patient was discharged at fifth post-operative day without complications. the histological examination shows a low risk gist, cd positive and with a ki- inferior of % (classification tnm th edition pt aims: gastric volvulus are a rare condition that sometimes represent a diagnostic challenge for surgeons. here we present the video of a recent case of a gastric volvulus in our area that was treated with a minimal invasive approach. methods: we report the case of a -year-old woman who was admitted in the emergency room (er) with epigastric transfixing pain and impossibility to vomit that had started h prior to the admission. the physical exam showed good vital signs, and her abdomen was soft, with a tendency to tenderness with palpation in the epigastrium without guarding or rigidity. her lab tests were normal and the conventional radiology showed a double gastric bubble. we run an urgent computed tomography scan (ct scan) and a upper gastrointestinal (gi) endoscopy that showed a big type ii hiatal hernia that was complicated with a gastric volvulus. results: first, a nasogastric (ng) tube was placed for decompression of the stomach at the time when the endoscopy was made. the patient experienced a great improvement of the pain with that initial measure and remained stable. after almost a day of appropriate resuscitation, she underwent urgent surgery: we performed a hernia reduction, resection of the hernia sac, hiatal closure and a gastropexy and nissen fundoplication. the patient suffered no complications in the immediate postoperative time and was discharged after six days. conclusion: gastric volvulus are an uncommon emergency that we need to keep in mind. a simple abdomen x-ray can be very helpful, given that the double gastric bubble sign is a typical sign of this condition. it's always mandatory to perform an upper gi endoscopy in order to get to the diagnose and place a ng tube promptly. the surgery can be safely delayed in stable patients with no signs of ischemia, and a laparoscopic approach is associated with a shorter hospital stay and good long-term outcomes in this kind of patients. aims: during laparoscopic treatment of hiatal hernias the dissection can be complicated, but even more so the closure of the pillars, especially in giant hiatus hernias with a large defect. the use of prosthesis is controversial due to the lack of long-term studies and the possibility of secondary complications. the aim of this video is to demonstrate the safety of mesh hiatoplasty in hiatus hernia surgery. methods: we present the case of a -year-old woman with hypertension, hypothyroidism and right hemicolectomy for neoplasia years ago. the patient presented with malnutrition, with a weight loss of kg in the last months. a gastroscopy was performed in which a large hiatus hernia, that caused gastric volvulation, was shown. the upper gastrointestinal oral contrast study revealed esophageal tertiary waves and good passage to the gastric chamber, with an organoaxial volvulation of the stomach that was completely included in the thoracic cavity. results: through a five trocar laparoscopic approach, a large paraesophageal type iv hiatal hernia ( cm hiatal orifice) with complete herniation of the stomach and greater omentum to the mediastinum was observed. after reduction of the hernia content, complete dissection with partial resection of the sac was performed. an extended mediastinal dissection of the esophagus, with descent of the esophagogastric junction until achieving an abdominal esophagus of - cm, was carried out. after posterior and anterior phrenorrhaphy with nonabsorbable sutures, dislacement of the right pillar without diagragmatic opening was evidenced. it was decided to reinforce the hiatorraphy using a c shape composite mesh fixed with nonabsorbable sutures. the procedure was completed with a nissen-type fundoplication. postoperative course was uneventful and the patient remains without hernia recurrence months after the intervention. conclusion: prosthetic reinforcement in hiatal hernia repair can be a very useful resource in large hiatal defects in which a stress-free hiatus closure cannot be achieved. however, its use must be individualized according to the characteristics of the patient, the quality of the tissues involved and the result of simple hiatorraphy. aims: heller myotomy is an advanced laparoscopic surgical technique for the treatment of achalasia, a rare disease in which long time is needed to achieve the learning curve. surgical simulation, using animal models with an anatomy similar to humans, could help improving surgeon performance in a shorter time. the aim of our video is to show an ex-vivo and in-vivo animal model for heller myotomy training. methods: a cadaveric porcine model was developed using a tissue block including the esophagus and the stomach, without the diaphragm, mounted in a physical laparoscopic simulator. training procedures were also performed in an in vivo porcine model. experiments were carried out in the ' jesús usón' minimally invasive surgery centre in cáceres. results: the surgical technique is described step by step, first in the esophagus-stomach ex-vivo model and later in the live animal model. conclusion: surgical simulation using cadaveric an live animals offers a realistic representation, allows training in a safe environment, and can be very useful for advanced laparoscopic training in low incidence pathologies. introduction: esophagic perforation is one of the least frequent complications after laparoscopic nissen fundoplication, but it remains one of the most dreaded because of its morbidity and mortality rates and its technically difficult reparation. aims: to present how the authors dealt with an iatrogenic esophagus perforation post laparoscopic nissen fundoplication. methods: the authors report the clinical case of a -year-old woman who underwent a laparoscopic nissen fundoplication because of a symptomatic large hiatus hernia in a different hospital. the second postoperative day and after resuming oral intake, she presented respiratory and hemodynamic instability. she needed a chest tube that drained purulent content. the patient was referred to our hospital for clinical management. an urgent ct scan with oral contrast was performed without showing any leakage. results: in spite of the results, as the patient was unstable, she underwent an emergent diagnostic laparoscopy. it was found a small anterior esophagus perforation with right mediastinic collection. a running suture of the perforation was carried out and the nissen fundoplication was converted to a dör fundoplication. the operative time was min. she went to the intensive care unit for ten days. five days after the surgery, she was given methylene blue with no exteriorization through the drainages. as postoperative morbidity, she suffered from a right pneumonia and a thoracic collection that was treated by thoracic surgeons. the patient was finally discharged on the th postoperative day. she did well at home. she attended follow-ups without clinical reflux or any other particular condition. conclusions: esophagic perforation is a life-threatening complication that can be managed laparoscopically if it is detected soon after surgery and an expertise is available. surgical treatment of achalasia fails in - % of patients. the most frequent responsible cause is a previous incomplete myotomy, followed by fibrosis and aspects related with antireflux procedure. revisional surgery can represent a greater difficulty and a challenge. in this video we show revisional surgeries after heller's myotomy with dor fundoplication for achalasia. all cases presented a recurrence of the symptomatology and a revisional surgery was proposed. surgeries were characterized by the presence of a herniation of the previous fundoplication, fibrosis around the prior myotomy and/or the formation of a pseudodiverticulum. we show the steps followed and the aspects to consider during the dissection. these cases demonstrate that laparoscopic reoperation for achalasia is feasible, even after open surgery. aims: upside-down stomach (uds) is a rare type of large paraoesophageal hernia, characterized by migration of the entire or large parts of the stomach into the posterior mediastinum. uds is associated with severe complications like strangulation or volvulus development, possibly leading to acute gastric outlet obstruction and incarceration. surgical repair is the only curative treatment option and therefore recommended for these patients. standard procedure includes a hiatoplasty followed by an anti-reflux procedure. in a variety of studies, the use of mesh proved to be superior with respect to reduction of anatomical recurrences. methods: a -year old woman presented to us with reflux symptoms, dysphagia, dyspnea and tachyarrhythmias. she reported a weight loss of kg in the last months. ct scan and esophagogastroscopy showed a large paraoesophageal hernia, measuring approximately cm, with intrathoracic uds. results: we performed a laparoscopic hernia repair with dissection of the hernia sac from the posterior mediastinum, tension-free intrabdominal reposition of stomach and distal esophagus and hiatoplasty with biologic mesh (tutomesh tm ) augmentation. finally, a toupet fundoplication was performed to recreate the antireflux valve. in consequence of pronounced adhesions, a lesion of the muscularis of the distal esophagus occurred during surgery. the esophageal mucosa was unaffected. we treated the lesion laparoscopically with a simple interrupted suture (vicryl tm - ). an intraoperative patent blue v leak test did not identify any leaks. the recovery was uneventful. the patient was discharged on day after surgery and was free of symptoms in her follow-ups. conclusions: patients with large hiatal hernias and uds can be treated successfully and effectively with laparoscopic mesh repair. intraoperative complications can be handled laparoscopically in a safe manner. iatoplasty followed by nissen fundoplication represent the gold standard treatment of hiatal hernia; however it has been reported high hernia recurrence rate, especially in case of giant hiatal defects. in order to reduce recurrence rate, various techniques of hiatoplasty reinforcement have been implemented, such as prosthetic materials apposition. however, in literature have been reported various mesh complications such as esophageal and proximal stomach erosion and late esophageal perforation after ischemia, especially in case of synthetic non absorbable materials.in this video we are going to show the repair of a huge hiatal hernia by hiatoplasty and positioning of an absorbable biosynthetic 'bio a' mesh which is replaced by connective soft tissue over six months, therefore decresing complications and recurrence rate.as usual, we start with the mobilization of gastric fundus and isolation of diaphragmatic pillars by sectioning the aderences between them and herniated viscera. we proceed then with intrabdominal esophagus mobilization and higher mediastinal dissection in order to obtain an adequate esophageal lenght. after the exposition of the hiatus, we approximate the pillars with some non absorbable stitches and we reinforce the hiatus positioning a 'u' shaped bio a mesh over the esophagus, simply fixed to crura with single stitches. then we go on performing nissen fundoplication.the use of this prosthetic material appears to be cost-effective; first series in literature show very low complication rate and less recurrences of hiatal hernia than hiatoplasty without reinforcement. this video demonstrates the technique of laparoscopic identification and complete dissection of the sac of a totally intrathoracic stomach. identification of the sac is performed centrally by scoring the peritoneum overlying the arch of the diaphragmatic hiatus. the inferior edge is retracted and series of blunt dissection is undertaken. once the areolar tissue of the avascular plan is visualized; a raytec sponge is placed and advanced cephalad to expose the extra-saccular space. this raytec is kept in place to allow carbon dioxide to infiltrate and further delineate the anatomy. we then proceed with dissection at the left crus and right crus. complete reduction of the stomach can be achieved without grasping it. this can be performed by applying caudal retraction on the sac. this maneuver exposes the plane outside the sac. this space is divided into two compartments (right and left) separated by a septum which indicates the proximal extent of the sac. this plane is avascular and blunt dissection can easily free the hernia sac from the mediastinal structures and the pleura. this was followed by excision of the sac and hiatal repair which is reinforced with bioabsorbable mesh. the proximal short gastric vessels were then divided and a standard toupet fundoplication was performed. v -upper gi-reflux-achalasia introduction-objectives: the mixed giant hiatus hernias with paraesophageal component are hernias of difficult surgical correction, the laparoscopic approach of these implies a greater experience of the surgical team, given the complexity involved in its management, being the recurrence a complication that neither the use of meshes in this surgery has been able to avoid. in very high-risk patients, the gastric anterior pexy (boerema) may be an alternative to treat or alleviate the symptoms of these large hiatal hernias, although the frequency of recurrence with this technique is very high. material and methods. objective: the objective of the following case is to present a patient with type- hiatal hernia, barrett's esophagus without dysplasia and situs inversus. method: a laparoscopic partial fundoplication was performed on a -year-old male patient with a history of situs inversus totalis, who was seen in our general surgery service presenting a clinical history of retrosternal pain, heartburn and regurgitation. an endoscopy was performed, which reported hiatal hernia type and incompetence of lower esophageal sphincter, with squamocolumnar junction biopsies with report of barrett's esophagus without dysplasia. results: surgical time was programmed, for toupet type fundoplication; there were lax adhesions from omentum to wall, the lax hiatus and already known situs inversus. partial funcuplication was performed, with the usual technique adapted for structural anatomical abnormality, postoperative course without complications, oral initiation at the next day, drainage penrose type was set draining just a little serohematic liquid, diet was progressed and patient was discharged on the third postoperative day without complications. conclusion: situs inversus is the mirror image of situs solitus, which presents subdivision in situs inversus totalis, which is the most usual form, characterized by the mirror location of the intraabdominal and thoracic organs including the heart; in the case presented, the patient was referred with situs inversus and barrett's esophagus, performing laparoscopic fundoplication. the gold standard of surgical management is laparoscopic in benign esophageal pathology. gastroesophageal reflux disease (gerd) is a condition that reduces the quality of life and can causedisorders associated with acid reflux, such as bronchial asthma, barrett's esophagus and esophagealadenocarcinoma. gerd is often caused by existing of hiatal hernia. in rare instances gerd is associatedwith type iv hiatal hernias in which the part of stomach and other organs migrate into mediastinum.nowadays this condition can cause problems for some surgeons.patient was a -year-old man. he was diagnosed with hiatal hernia in . the condition had beenhaving asymptomatic course until . patient takes omeprazole mg for years. he startedexperiencing chest pains when inhaled and dyspnoea in june . co-morbidities were: arterialhypertension, chronic obstructive pulmonary disease (copd) and obesity (body mass index was . kg/m ). the posterior mediastinum contained the part of stomach, large bowel and small bowelaccording to chest roentgenography. sizes of esophageal hiatus were mm. in our clinical centerwas performed laparoscopic removal of hernia, cruroraphy, mesh repair of the esophageal hiatus andnissen fundoplication in july. during the surgery stomach, the part of small intestine, greatomentum and transverse colon were relocated into abdominal cavity. after cruroraphy, repair of theesophageal hiatus with prolene mesh was performed. the patient was in intensive care during h.total enteral feeding was initiated on second day. patients had been discharged within days aftersurgery. patient was re-examined by a surgeon in september , there were no signs of a reccurence.this case report shows an efficiency and feasibility of the laparoscopic approach to the treatment gerdassotiated with a large defect in the phrenoesophageal membrane, allowing other organs, such as colon,great omentum and small intestine to enter the hernia sac. aims: the authors present a video with their standardized laparoscopic hiatal hernia repair and anti-reflux nissen procedure but using mm instruments and mm camera approach. methods: a years old female patient with a bmi presents a symptomatic gastro esophageal reflux disease (gerd) for years. manometry and ph-metry showed a lack of esophageal motor disorders and a severe acid pattern with a . demeester score. panendoscopy study showed a cm hiatal hernia and a los angeles-grade esophagitis. she decided not to go with ppi treatment anymore. a laparoscopic hiatal hernia repair and standardized nissen procedure is performed using mm instruments and a mm camera. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with a rate in a eva acute pain visual scale. in a year follow-up, there has no been an anatomical or clinical recurrence. no chronic dysphagia, anatomical recurrence or abdominal wall complications have been reported with in this period of time. conclusions: depending on the patient characteristics, anatomical factors and surgeon mini invasive experience, hiatal hernia and anti-reflux mini invasive standardized repair using mm instruments, could be a safe and feasible option. more studies are needed in order to standardized this approach. background and aims: parastomal herniation a very frequent complication in stoma patients. in isolated parastomal hernias (type i or iii)* a laparoscopic sugarbaker repair with intraperitoneal mesh is our preferred technique. if a concomitant incisional hernia is present (type ii or iii)* we currently opt for a retromuscular mesh repair as described by pauli. we adopted a minimal invasive approach using the robotic platform. methods: we performed a robot-assisted laparoscopic pauli repair for a wide incisional henria needing component separation and a small parastomal hernia (type ii)*. a non-slit retromuscular mesh was placed after a bilateral tar (transversus abdominus release) and lateralization of the colon. results: the operation was performed robot-assisted laparoscopically with x trocars without the need to convert to an open procedure. the procedure lasted min. the patient was discharged from the hospital on post-operative day three. one month after the repair our patient presented with a parastomal skin infection for which she received surgical cleaning and wound dressing. ct scan three months postoperative shows good positioning of the mesh with a reinforced abdominal wall. conclusions: the modified sugarbaker repair in parastomal herniation is feasible following a pauli approach (retromuscular mesh positioning) completely laparoscopic, albeit robotically assisted. short-term follow up is promising. long-term postoperative follow-up in these patients is needed. methods: case presentation results: a -year-old caucasian female patient was admitted on emergency due to a progressive alteration of her physical condition and weigh loss, caused by intermitent and intense epigastric pain and vomisments. symptomes occurred several years prior to admittance, but used to be mild and rare. during the last months, the described episodes became more intense, lasted longer and occurred more frequently. percutaneous ultrasound raised the suspicion, while thoraco-abdominal ct revealed an enormous intrathoracic morgagni hernia and gastric volvulus inside the hernial sack. after a careful preoperative preparation (restoring of the nutritional and hydric inballances, amelioration of respiratory parameters), laproscopy confirmed the diagnosis; we gently reintegrated the herniated organs from the thoracic hernia into the abdominal cavity (small bowel, large omentum, transverse-doloco-colon, and thisted stomach); a laparoscopic exploration of the hernial cavity was followed by a thorough hemostasis. do to patient's frailty, we decided to leave the hernial sack in situ. surgical direct repair of the defect technique was done by using extracorporeally tied separate sutures through separate skin incisions. the postoperative outcome was completelly uneventful; patient was discharged on postoperative day . barium contrast at mounths followup showed a slight esophageal diskynesia, but normal gastroduodenal passage; due to aerocolia, the normal position of the transverse colon could be confirmed aswell; no signs of reccurrence where detected. conclusions: although very rare, a morgagni hernia should be suspected and included in the differential diagnosis of patinets with dispeptic syndrome and epigastric/thoracic symptomes. thoraco-abdominal ctscanning is the imagistic technique of choice. laparoscopic approach became the gold standard, being far mora addvantageous as compared to laparotomy or thoracoscopy. direct suture with extracorporeally separate sutures through separate skin incisions was chosen for being less time consuming; for the same resons, the hernial sack was left in situ, with no consequences so far. aims: the giant fibrovascular polyp is one of the rarest benign lesions of the oesophagus. the most common locations of origin are the upper oesophagus or crico-pharyngeal region. the lesion is more common in elderly population, particularly men. symptoms include dysphagia, odynophagia. management options include surgical resection or endoscopic removal with endoloop. we aim to demonstrate the optimal management of these rare lesions using an endoscopic approach. method: we demonstrated the management of -year old patient with a giant oesophageal polyp, excised by minimally invasive endoscopic resection. the patient was placed in supine position and tracheal intubation was performed under general anaesthetic before an endoscopic approach was taken. the oesophago-duodenoscopy visualised a cresenteric shaped lumen due to an intraluminal mass occupying two thirds of the oesophageal diameter. the procedure was a multidisciplinary approach with the upper gi surgical and gastroenterology consultants. the polyp stalk was located in the oesophagus at the level of t and an endoloop was manipulated to surround the polyp. the polyp was then separated from the stalk by cauterisation and resected from the patient. the stalk was then injected with adrenaline to prevent haemorrhage. results: the excised specimen was a cm polyp with stalk originating from the t -t level. histology confirmed diagnostic suspicions of a benign pedunculated fibrovascular polyp. the polyp was covered by non-keratinising squamous epithelium with a discoloured tip demonstrating ulceration. there was no evidence of dysplasia or neoplastic process. the video shows a case of laterally spreading tumour of the rectum with preoperative benign histology, paris classification -is g (granular type), ut n eus stage, kudo type iv, nice type . the neoplasm measured x cm, and extended from to cm from the anal verge, mainly located on the posterior wall. according to our local policy the indication was a transanal full-thickness excision. this was performed with the medrobotics flexÒ robotic system, used here for the first time outside the united states. the system technology utilizes an articulated multi-linked scope that can be steered along nonlinear, circuitous paths in a way that is not possible with traditional, straight scopes. the maneuverability of the scope is derived from its numerous mechanical linkages with concentric mechanisms. this enables surgeons to perform minimally-invasive procedures in places that were previously difficult, or impossible, to reach. with the flexÒ robotic system, surgeons can operate through a single access site and direct the scope to the surgical target. once positioned, the scope can become rigid, forming a stable surgical platform from which the surgeon can pass flexible surgical instruments. the system includes on-board d hd visualization. the flexÒ robotic system contains two working channels to accept a number of different surgical and interventional instruments including monopolar and bipolar electrodes, scissors and graspers for tissue manipulation. the video shows the introduction of the dedicated rectoscope, the connection of the flexible robot, and the way to operate the device performing a full-thickness excision, including suturing of the rectal defect by means of two running sutures by a v-lock / thread. while illustrating the technique the authors will comment pros and cons of the use of the device. surg endosc ( ) the video shows the use of a barbed suture of novel concept. this first prototype thread developed together with assut europe (roma, italy) is characterised by a bidirectional / barbed suture, of cm length overall, with two needles mm in diameter, and circumference. in order to fix the thread to the tissue, on the exact midline of the thread a small ( mm) ball of the same material of the thread is fused with heat. this button as the entire thread is made of polydioxanone, a monofilament material for long-term absorption, self-retaining. this small advancement offera a consistent advancement in endorectal surgery helping in making transverse wound closures as easy as never before. the first stitch is placed on the transverse midline of the rectal wall defect, this way approximating proximal and distal edges. the button keeps the thread under tension allowing the completion of a running suture towards one of the two lateral ends of the would. when the first end is reached the needle is cut and the other needle is grabbed in order to perform the other half of the running suture keeping the right tension on the thread, with no risk of lumen stenosis. the two lateral ends of the suture are self-blocked passing them through the last stitch. no need for clips, knotting or buttonholes to pass through. while illustrating the technique the authors will comment pros and cons of the use of the device. background: the usefulness of robotic surgery has been largely reported; however, there are not enough reports on gist's treatment. the aim of this study is to report a single center experience on gastric gist's robotic resection. gastrointestinal stromal tumor (gists) are the most common mesenchymal tumors (overall incidence - % of all gastrointestinal malignant tumor). they are most frequently located into the stomach. complete surgical resection still remains crucial for patients with gists. in cases of difficult localization of tumor (e.g. posterior wall, his angle) and bigger tumor (more than cm in diameter), there still exist apparent difficulty and limitation to conduct laparoscopic resection. robotic assistance provides wide movements of its arms and was recognized particularly useful in case of difficult tumor localization, especially for those positioned at the posterior side of the stomach wall or around the lesser curvature. methods: six consecutive patients were analyzed focusing on safety (conversion/complications rate; hospital stay), oncological (margin resection, recurrence rate) and feasibility (operative time, technical tip and tricks) profile of robotic approach. results: the mean operative time was ± min, the mean hospital stay ± days. conversion rate was nihil. no intra and post-operative (mean follow-up months) complications were registered. in all cases, the resections were classified as r . conclusions: our experience supports the usefulness of robotic system in case of gist located at anatomically difficult gastric portion, such as lesser curvature or fundus close to gej, confirming the excellent oncological outcomes ( % r ) and the encouraging safety profile ( %). regarding the operative time our data are similar or better as compared to those reported by the previous literatures and didn't differ by the most recent published data for laparoscopic gastric resection. the anatomical hand-sewn reconstruction performed by precise hand-sewn suture instead of the usage of mechanical staplers represents a real great advantages of robotic resection. in our series, no patients suffered from stenosis or leakage after operation. background: we describe the use of a different suture from those historically used in the elaboration of a widely spread surgical technique such as the nissen fundoplication, for the treatment of pathological gastroesophageal reflux or symptomatic hiatal hernias. in our unit we have implemented the use of / irreabsorbable barbed suture to close the diaphragmatic pillars and the °fundoplication with the same continues suture. aim: the objetive of the use of the irreabsorbable barbed suture in the nissen fundoplication is to shorten the surgical times, which would achieve benefits for the patient and the institution, increasing the number of ambulant patients and the number of patients to be operated the same surgical session. objective: the goal of the present study was to demonstrate that intraoperative icg fluorescent imaging is a safe technique that can be used in laparoscopy establishing the exact location of the lymphocele and reducing intraoperative risks. method: fifty milligrams of icg dissolved in ml of saline solution was injected via percutaneous drainage placed into the lymphocele to decompress transplanted kidneys weeks before a laparoscopic lymphocele marsupialization procedure. results: during the first exploratory laparoscopy, in the flank and right iliac fossa, near the renal grafts, fluorescence was identified in raised areas that were the internal side of the lymphocele lobes. the lymphocele wall was dissected and ml of serous fluid was aspirated after puncturing. a cm breach was then made in the cyst wall using the ultracision harmonic scalpel (ethicon us). afterwards, a pedicle of the omentum in the lymphocele core was interfered with and fixed by stitches. conclusions: laparoscopic surgery seems to be the preferred surgical option for the treatment of primary symptomatic lymphocele after kidney transplantation. intraoperative icg fluorescent imaging is a safe technique to establish the exact location of the lymphocele and reduces the risk of damaging urinary structures during surgery. conclusions: this method proved safe and risk-free, easily reproducible and without the need for a different toolkit than the one of a modern operating theatre. the preliminary analysis shows a strong correlation between the perfusional data so far obtained and the early outcome of the graft. thus opening the way to further analysis aimed to a future better management of post-operative immunosuppressant and support therapy. these results are quite encouraging, even if our study is at an initial stage. conclusions: results show a persistent dread across specialities regarding ai. rather than be seen as threat, ai should be embraced by clinicians as it will ease the ever-increasing daily workload faced. this will enable clinicians to focus their skills on patient centred activities, interventional procedures and development. despite current regulatory hurdles, ai implementation in medicine is unavoidable. this coming revolution presents a unique opportunity for endoscopist and radiologist to refocus their expertise in novel areas. project description: from february to july a three-armed proof of concept study was conducted at three hospitals, in three groups of patients; recurrent ventral hernia, aortic aneurysm, and healthy controls. patients were measured once at the outpatient clinic using an electronic nose based on three metal oxide sensors. measurement data were compressed to low-dimensional vectors using a tucker like algorithm, and used to train an artificial neural network (ann) to provide a classification between patients (? ) and healthy controls (- ). preliminary resultsa total of patients (hernia n = , aneurysm n = ) and controls were included in the study. based on receiver operating curve (roc) analysis, the ann could differentiate between recurrent hernia patients and controls with the following details: area under the curve (auc) . , sensitivity . , specificity . . aortic aneurysm patients and healthy controls could be differentiated with an auc of . , sensitivity of . , and specificity of . . the aeonoseÒ enose can reliably distinguish patients with weak collagen (recurrent hernia and aortic aneurysm patients) from healthy controls. validation of these results in a prospective cohort study is required before clinical application of the device. background: laparoscopic and endoscopic cooperative surgery (lecs) has been performed gastric submucosal tumor (smt) or duodenal tumor. although minimally invasive surgery using thoracoscopy has been the usual approach for esophageal smt, the treatment method combined thoracoscopy and endoscopy has not been established. in addition, submucosal endoscopic tumor resection (set) for esophageal smt was reported using the technique of submucosal tunnel. aim and project description: we planned to resect large esophageal smt located in the upper or middle thoracic esophagus by a combined endoscopic and thoracoscopic approach. because set is only recommended for tumors up to mm in size owing to the limited submucosal space available and the left thoracic approach is restricted by the aortic arch and the trachea. preliminary results (case presentation): a -year-old woman was diagnosed with a benign schwannoma of length mm originated from either the submucosal or the muscular layer of the middle thoracic esophagus by endoscopic ultrasonography, enhanced computed tomography, and ultrasound-guided fine-needle aspiration biopsy. since the tumor had increased in the years and she had a symptom of dysphagia, we planned to resect it by a combined endoscopic and thoracoscopic approach. on endoscopy in the supine position, a submucosal tunnel was created mm proximal to the cranial edge of the tumor, and its only oral end was dissected from the mucosal and muscular layers. this was followed by the resection of the entire tumor by left-sided thoracoscopic procedure in the prone position. endoscopic mucosal closure was achieved by using clips. no postoperative complications were observed. large benign esophageal tumors can be safely excised with minimally invasive surgery by using a combination of endoscopy and thoracoscopy. background: haemorrhage remains a major cause of morbidity and death in all surgical specialties. in laparoscopic surgery. relatively small amounts of blood can obscure the view of the operative field and increase the risk of injury to surrounding structures. excessive bleeding often leads to longer hospital stays, increased healthcare service utilisation, and higher healthcare costs, among other negative consequences. aim: the aim of this study was to analyse the feasibility of purastatÒ, a new synthetic haemostatic device, made of self-assembling peptides in laparoscopic colorectal surgery. project description: this was a prospective observational non-randomised study. consecutive patients undergoing laparoscopic colorectal surgery were enrolled. informed consent was obtained from all patients inclusion criteria was the need employ a secondary method of haemostasis when traditional methods such as conventional pressure or utilization of energy devices to control the bleeding were either insufficient or not recommended/ appropriate due to proximity to ureter, pelvic/sacral veins and other important structure. preliminary results: twenty patients were enrolled ( males ( %) and ( %) females). mean age was years (± , years). all patients were undergoing elective laparoscopic colorectal cancer surgery ( right hemicolectomy, sigmoid colectomy, anterior resection). we utilised mls of purastatÒ in all patients. the mean area of application was , cm (± . cm ) with the amount of purastatÒ needed per centimetre being . mls. the mean time to apply the product was secs (± s), whereas the mean time to achieve haemostasis was , secs (± . s). there were no post operative complications in this cohort of patients. mean operative time overall was min (± . min). none of the patients experienced delayed post-operative bleeding and the mean hospital stay was days (± . ). in conclusion, according to the purpose of this preliminary study, we have demonstrated that purastatÒ can be easily used in laparoscopic surgery and it is a safe, effective haemostatic agent. this is a feasibility study and additional controlled studies would be useful in the future. during last three years the laparoscopic method of surgery in the presence of common bile duct stones was carried out. after performance of intraoperative cholangiography and visualization of stones in the common bile duct laparoscopic, choledochotomy and bile duct stones extraction was undertaken in patients, using flexible choledochoscopy control. in all patients with gallbladder stones was then performed laparoscopic cholecystectomy. results: laparoendoscopic intervention on common bile duct was successfully performed in patients ( . %) and the operation was completed by common bile duct drainage by kehr. in patients due to technical difficulties conversion to open surgery was carried out. postoperative morbidity in the form of bile leakage were diagnosed in patients ( . %). in three cases they stopped spontaneously in - days after the operation. patients were operated on repeatedly and additional suturing on choledocholithiasis was carried out. postoperative mortality was . %. the death of the patient of years was caused by acute cardiovascular failure. institute for image guided surgery, ihu-strasbourg, strasbourg, france; hepato-digestif, nouvel hôpital civil, strasbourg, france; image guided surgery, nouvel hôpital civil, strasbourg, france; gastroenterology, chu-besancon, besancon, france; gastroenterology, chu-lyon, lyon, france; gastroenterology, clinic de trocadero, paris, france background: eus is difficult to learn and has a steep learning curve. therapeutic eus (teus) is even more so. simulators, ex-vivo models and phantoms are the most common current teaching modalities but are felt by many to be unsatisfactory for high-level training. aim: we designed a training curriculum for teus that uses high-fidelity animal models and present a validation study performed by teus experts. project description: different simulated pathologies were created in each of acute pigs. teus experts performed therapeutic procedures in two or more animals over two days. each intervention was evaluated simultaneously using a structured survey by an non-expert observer. data included demographics and procedure details as well as likert-scale evaluation of the quality, realism and education utility of the simulations. global evaluation of the experience was captured from the experts as written comments. all data was consurrently registered and subsequently analysed by two blinded surgical educators. methodology: three types of models were created using surgical access: -tumors (injection of types of hydrogel), -retro-gastric collections ( - cm long intestinal loops filled with oatmeal, oil-water and gel), -obstructions (bile duct and ureteral ligations days prior to experience). gastric, pancreatic and liver tumor models were used for fna and fnb practice. retrogastric fluid collections and choledochal/ureteral obstructions were used for cyst gastrostomy, hepaticogastrostomy, gallbladder drainage and kidney drainage. results: experts age: - , median intervention time min , total of interventions evaluated, overall quality of experience: ( %) ranked - (excellent), ( %) from - (good), ( %) from - (poor), / procedures were successfully completed. models were rated good to excellent quality ( - ) in ( %), poor quality in ( %). for % ( ) of the interventions the model was considered not good enough to be repeated (solid retrogastric tumor and peripheral hepatic lesion). conclusion: high-fidelity live animal models with simulated pathologies are considered to be excellent training tools by experts and may provide a better learning experience for teus. surg endosc ( ) in our short videos, we present scenarios in which this technology helped: to distinguish a significantly dilated cystic duct from the cbd, to identify an anterior cystic artery in the contest of acute inflammation, to identify a luksha duct, to exclude a leak after endoloops positioning on cystic duct. intra-operative augmented visualisation of biliary anatomy with indocyanine green cholangiography is an essential technology tool with the potential to extend the hour window of safety for emergency cholecystectomies, with significant logistics benefits. introduction: endoscopic resection of subcardial polyps has its limitations; especially when it is necessary to dry out the entire gastric wall. uniportal intragastric surgery is a good alternative for the exeresis of subcardial premalignant lesions with gastric preservation. patient and method: we present a video with two cases. technique: we perform a laparoscopy to explore the entire abdominal cavity, then we open a . cm hole in the great curvature; a cm incision is made in left hypochondrium and the uniportal device is placed inside de stomach. we inject serum in the submucosa with a endoscopic needle. when submucosa is completely separated from muscular lay; a submucosal exeresis can be made; but when there is not a complete separation after injection, then we perform a entire wall resection with a cm circular margin. if a complete wall resection is made, then we close the defect with a barbed suture. results: case : years old male with a , cm subcardial polyp, preoperative biopsy was informed as severe dysplasia. in the laparoscopy we saw an unknown lesion in the great curvature that looked like a gist. we placed the uniportal device intragastric and proceed to the submucosal serum injection. as the submucosal lay was completely separated from muscular, then we performed a submucosal exeresis. we close the gastrotomy and made the resection of the great curvature lesion with endostapler. the pathological analysis confirmed the severe dysplasia in subcardial lesion and gist in the great curvature lesion. patient was discharged with no complications after days. case : years old male with a cm subcardial polyp. preoperative ecoendoscopy suggested a muscular layer infiltration but only severe dysplasia was found in the previous biospsy. laparoscopy did not found more lesions, and uniportal intragastric device was placed. a complete wall resection was made, and the defect was closed with manual barbed suture. pathologyst confirmed severe dysplasia and unaffected margin. patient was discharged with no complications after days. conclusion: uniportal intragastric surgery is feasible and safe and may be useful for subcardial premalignant lesions when endoscopic resection is very difficult or not feasible. introduction: the role of icg in bariatric surgery is still unclear. knowing the lack of perfusion in the gastric pouch could be of great interest in revisional surgeries; but the question remains: is the current icg technology reliable enough to make intraoperative decisions in bariatric surgery? methods: we have carried out a check of tissue perfusion with icg fluorescence in several cases of primary and revisional bariatric surgery. a solution of , mg/kg was injected intravenously and icg fluorescence was performed. we looked for the correct staining of the entire gastric pouch and the intestinal loop trying to rule out areas of tissue ischemia. results: the cases in which the test was performed showed a minimal delay of - s between the intestinal loop stain and the pouch. a correct staining was observed in all but one case shown in the video. is the case of a years-old male patient who was operated years earlier in another center; at that time he underwent a sleeve gastrectomy. we evaluated the patient for persistent gastroesofhageal reflux of years of evolution with esophagitis. we offered revisional surgery to perform gastric bypass and hiatal closure. fundus was dilated so a funduplasty was performed instead of using endostappler in the vertical side of the pouch. manual anastomosis gastric bypass was performed. when the icg test was performed, a corner of the pouch does not stain green (an area of , cm) . so the decision was to resect that part and redo the anastomosis or wait and see. it was decided not to resect and the patient was discharged two days later with no complications and good outcome with a months follow up. conclusion: icg fluorescence may be useful in bariatric surgery in the future but more evidence is needed of its usefulness in making intraoperative decisions. background: lymph node status is one of the key prognostic factors in patients with colorectal cancer, and remains the most important selection criteria for adjuvant chemotherapy. it is believed that at least % of node negative patients will suffer disease recurrence within the first years after surgery. this may be due to understaging of lymph node status. sentinel lymph node mapping is widely used for staging of breast cancer and melanoma, with injection of colloid tc and isosulfan blue (ib). however, indocyanine green (icg) fluorescence guidance is a new technical approach to this issue, with promising results for detection of aberrant lymphatic drainage outside of the planned resection. the icg lymphography has the advantage of offering a good visualization of the lymphatic channels but there are problems in order to identify the lymphatic nodes. aim: the objective of the experimental study is to investigate the possibility to detect the sentinel lymph nodes after the injection of different solutions with indocyanine green in the subserosal colonic layer in the pig. project description: twelve female large white pigs were operated with laparosocpic approach and spies optic filter (karl storz, germany). indocyanine green was injected in the subserosa of the colonic wall ( ml at points). lymphatic flow was observed at - - - - and min, searching for the migration of the icg by the lymphatic channels and its introduction in the sentinel nodes. preliminary results. the identification of the sentinel nodes is very difficult with the solution of icg-sterile water. with this technique we can see the lympjatic channels but not the lymphatic nodes. the adition of % human albumin as a transporter of the icg is very helpful for the correct identifiaction of the lymphatic channels at - - min and the correct visualization of the lymphatic nodes at min after the bowel injection. addition of other transporters like dextran solutions may be helpful too but the time to the correct visualization is longer. there was significant difference among the three groups as regards postoperative les and postoperative ph metery.the incidence of persistent dysphagia was significantly higher in the group i. postoperative gerd symptoms were significantly higher in group iii ( . %. p \ . ). recurrent achalasia was significantly higher in group i ( patients . %, patients in group ii ( . %) and nil in group iii (p \ . ). in conclusion: longer myotomy on the gastric side ([ . cm) ensures complete division of the les with better outcomes in term of resolution of dysphagia but may be associated with higher postoperative gerd. therefore, a myotomy length of . to . cm on the gastric side provides a balance between relieve of dysphagia and development of postoperative gerd. c/t scan of his abdomen revealed a large groin hernia with signs of small bowel obstruction and collapsed distal bowel. emergency theatre was organised for this patient with anaesthetic assessment prior to his surgery. initial plan of local exploration with possibility of small resection was changed once he was under full anaesthetic with muscle relaxation. his abdominal girth provided an opportunity to utilise laparoscopic intervention as an initial approach. laparoscopy with degree lens revealed moderately distended loops of small bowel and a large omental mass along with a loop of small bowel incarcerated in right direct inguinal hernia site. background: robotically assisted surgery is a rapidly developing modality of minimally invasive surgery with proven advantages in the management of cancer. despite its increasing prevalence, there is still an ongoing debate regarding its future role in colorectal surgery. while the prospective randomised multi-centre studies provide research evidence for its potential efficacy, an assessment of its effectiveness and realistic outcomes in everyday clinical practice can add an important perspective to this discussion. the international robotic colorectal registry will allow to compile and pool the international robotic colorectal experience. aims: the aim of the international robotic colorectal registry is to monitor the safety and outcomes, as well as the quality of specimen of robotically assisted colorectal surgery for malignant and benign diseases of the colon and rectum. the primary endpoint is a composite oncological failure. the secondary endpoints include anastomotic leak, resection margin involvement, conversion rate, operative time, post-operative -day morbidity and mortality, long term oncological outcomes, quality of life, functional outcomes and cost-effectiveness. project description: the international robotic colorectal registry is a multicentre web-based, online secure database. the registry has been awarded an ethical approval by a relevant national committee. all surgeons performing robotic or robotically-assisted surgery are invited to participate. the data collected includes patient demographics, cancer characteristics, operative details, histology of the specimen, wound healing, post-operative therapy, readmission, quality of life and functional (bowel, urinary and sexual) outcomes. all the sensitive patient information is encrypted before its introduction into the database. preliminary results: so far, twenty robotic colorectal centres have joined the international robotic colorectal registry. the preliminary results will be published once patients have been enrolled. univariate and multivariate analyses will be performed to identify possible risk factors for poor outcome.the possibility to record open, laparoscopic or other minimally invasive colorectal procedures will facilitate comparison of the outcomes of the robotically assisted surgery and other modalities. the registry will also allow each surgeon enrolled to monitor their skill progression and outcomes over the time. results will be published in surgical literature and presented internationally. background: gastric outlet obstruction (goo) due to benign strictures is an uncommon surgical entity today. this paucity relates to the decrease in its aetiological factors in the modern era as well as to advances in both prevention and medical as well as endoscopic treatments of such condition. the most common of causes relating to peptic ulcer disease, has been subdued for decades with quality acid control medications. on the other hand advances in gastroscopic dilatations skimmed even more the frequency of these cases from arriving to surgical intervention. aim: this presentation gives an update on the standing of this pathology and its surgical management today. it will also shed a light on our early experience in this condition at the royal hospital of muscat in oman. project description: a case series of all patients with goo, who were surgically managed between and results: there were a total of patients, males and females. the cause of obstruction was peptic ulcer disease in , corrosive injury in , iatrogenic perforation in and idiopathic hypertrophic stenosis in . emergency presentation was seen in . management included jaboulay pylorpolasty in , resection in (distal gastrectomy in , total gastrectomy in ) or a bypass (gastrojejunostomy) in . in of the above, the procedure was done by laparoscopy. post operatively, temporary gastric paresis delayed recovery in , however all symptoms resolved in . there were no recurrences at minimum of years of follow up. in spite of advances in medications and gastroscopy interventions, we still seem to identify this condition within our population. although infrequent, they demand awareness from surgeons since they could be managed successfully, especially laparoscopically, with minimal morbidities and early recovery. the introduction of advanced laparoscopy to the unit's setup in recent years, made such option feasible with satisfactory and durable outcomes. background: gists of the upper gi are found mainly in the stomach ( - % of cases) and small intestine ( %). duodenal gists however, comprise a smaller subset with a frequency of to %. the optimal surgical procedure for duodenal gist is still evolving. since wide margins and extensive lymphadenectomy are not required, restrain from more radical resections in this area would be a valid option. aim: this is a video case report of a patient with a gist involving the third part of the duodenum treated by laparoscopic lateral duodenectomy and end-side roux-en-y duodenojejunostomy case report: years lady presented with recurrent mid abdominal postprandial pain with anorexia, nausea and occasional vomiting an ultrasound showed well defined hypoechoic mass of . . cm at the right para-aortic region . ct scan defined the mass as retroperitoneal, intimately related to the pancreas uncinate process and the third part of duodenum with no clear cleavage line between them. an mri endorsed the diagnosis of gist of the duodenum. she was operated upon through a laparoscopic lateral duodenectomy including the gist at the third part of the duodenum. a frozen section confirmed the clear margins . reconstruction was done by a roux-en-y duodenojejunostomy with the alimentary limb taken cm from the dj flexure. she had an uneventful post operative recovery and was discharged well. the histology confirmed a low grade gist tumour hence no further treatment was needed. at follow up six months later, she was doing well and gaining weight. conclusion: complex anatomy of the pancreatico-duodenal area makes conserving the duodenum for tumours rather than a major resection a challenging option. in our case however, with the disease in the third part being of a moderate size, a lateral duodenal wall resection including the mass was possible rather than a segmental resection. this procedure could be an ideal choice for benign, moderate sized tumours in the third and fourth part of the duodenum. background: during laparoscopic colectomy, laparoscopic lymph node dissection and extracorporeal intestinal anastomosis is commonly performed. an umbilical incision of - cm and wide-range mobilization of the intestinal tract is required for extracorporeal anastomosis. previously, we introduced intracorporeal overlap anastomosis in june as a minimally invasive treatment. here, we report its short-term outcomes. aim: we retrospectively compared the surgical outcomes of cases of extracorporeal anastomosis and cases of intracorporeal anastomosis, all of which were performed between june to may . procedures: after lymph node dissection and sufficient mobilization of the intestinal tract, the proximal and distal intestines were resected perpendicularly to the intestinal tract with a -mm linear stapler. the anastomosis was performed after the specimen was extracted from an umbilical incision. the opposite sides of the mesenteric margin cm from the staple line of the one intestinal tract, and cm from the staple line of the other intestinal tract, were marked, and then the respective intestinal tract was positioned to join the opposite mesenteric sides together. an insertion hole was made in the intestinal tract at the marked site. side-to-side anastomosis with a linear stapler was performed, and then the insertion hole was closed with a linear stapler after several temporary sutures. preliminary results: in the extracorporeal anastomosis group, the mean operation time, blood loss, and post-operative days were min, ml, and . days, respectively. furthermore, there were three intraoperative cases of bleeding ( . %), and two postoperative cases of lymphorrhea ( . %) that occurred. however, in the intracorporeal overlap anastomosis group, the mean operation time, blood loss, and post-operative days were min, ml, and . days, respectively. additionally, there were no cases of intraoperative complications, and only one postoperative case of lymphorrhea ( . %). conclusion: intracorporeal overlap anastomosis in laparoscopic colectomy is safe and feasible, and can be used as a minimally invasive treatment. nowadays d-printing it's not a new technology any more but with an exponential developing. there are beliefs that in % of everything that will be produced will be d-printed. in medical field this technology knows the same exponential developing. first used in orthopedics and maxilo-facial surgery now d-printind is used in many other fields for different reasons, like preoperative training models, surgical special instruments, in medical education, etc.. liver surgery is in continuous developing and this is the reason why we need experimental liver model for training and testing. a best liver experimental model should heave liver consistency, to be flexible, to heave the same ultrasound feedback, to be cheap and easy to be reproduced. this is why we developed a liver experimental model made of gelatin by a simple recipe, using a dprinted mold, created after a human liver ct-scan. first was made the segmentation of the liver. after segmentation we create the d virtual liver model and the negative image of the liver, which was used for creation of the pieces of the liver mold, with connections between them and a hole on the top to pour the gelatin solution. ( ) ethical concerns on learning and training with real patients and substitutives such as animals, and ( ) reconciling time devoted to learning with clinical practice, considering the european work time directives. simulation in medical education is and has been the preferred route to address both pedagogical needs. virtual simulation has proven to be a valid tool for training; however, current systems restrict usage to tasks and modules offered, without possibility of personalization. we present the minimally invasive surgery simulator scenario editor (mis-sim) an environment where users can create, edit and run virtual reality tasks designed for medical training. the environment features an editor allowing users to develop learning tasks, defining its learning objectives and task goals in an easy way. a first proof of concept has been implemented for surgical training and training activities (demostrators and short courses) have been carried out in three european sites: spain, the netherlands and hungary. during training activities, different exercises have been created and uploaded to the contents' database. trained technical skills include handeye and bimanual coordination, instrument handling and pulling. preliminary results with users have shown mis-sim training potential, although some functionalities should be made easier. personalization has been highlighted as the key added value of mis-sim with respect to the current competitions in the market: the ability for target users to use virtual reality based learning tools while remaining in complete control of the learning process. mis-sim aspires to break the barrier between vr and medical education by empowering users to create their own tasks. with mis-sim teachers/course creators and learners (healthcare professionals & future healthcare professionals) will benefit from an innovative tool to ( ) create personalised medical learning contents tailored to preferred learning styles, allowing the creation of individualize learning paths; ( ) improve the efficiency of training by focusing on the training needs of the learners and ( ) share and sell vr-based didactic contents. c. tiu , p. sánchez-gonzález , m. chmarra , d. gutiérrez , c. guzmán-garcía , l. sánchez-peralta , g. wéber , f. sánchez-margallo , b. pagador , j. dankelman aims: currently surgical training is largely based on the improvement of technology enhanced learning solutions. the progress of engineering and the diversification of training facilities outside the operating theater results in an even greater contribution of technology in the future. the main reasons to encourage these changes are increased efficiency of simulators and directly increased patient's safety. the goal assumed by the easier project is to develop multi-skill, online platforms for minimally invasive surgical (mis) procedures-based on common pedagogical principles with reference value in a multinational space. the platform will allow the connection of external assets (such as simulators) to centralize all training data from residents. this work presents the milestones of the project during its first year of life. methods: the consortium's activity started with a knowledge elicitation process organizing brainstorms and workshops including experts in mis and interventional techniques, from spain, romania and hungary. this experience led to the formulation of a questionnaire that was implemented online and sent via email to surgeons and residents from the participating countries. results: accumulated experience was used to define the pedagogical needs of the platform. the pedagogical needs form the starting point for defining the technical requirements and specifications. based on them, the design of the platform has been achieved, including its architecture and communication protocol between external assets design, facilitated by the use of state of art educational standards. discussions and conclusions: next steps include the implementation of the easier platform, as well as the definition of cases studies selected by the clinical partners in spain, romania and hungary to solve applications of the platform dedicated to cholecystectomy, lumbar puncture and arthroscopy. a pedagogical model, built by the experience of the consortium, is being used to guide instructional design of the course. finally, results will be validated in a multi-center validation study.the easier project will create a training platform with reference value for european surgery. the structure of the consortium, based on the confluence between collectives with clinical, technological and pedagogical experience, will generate a complex learning tool in surgery embodying technology-based training systems with clinical experience. background: the treatment of groin hernia is an important part of our daily surgical activity. aim: we proposed to evaluate outcomes of the laparoscopic trans abdominal pre peritoneal treatment (tapp) of the groin hernia. project description : one hundred and fifty patients who underwent a tapp for a groin hernia were included in a retrospective study between january and november . results: the gender ratio was . the average age was , years. twenty percent of patients had a history of abdominal surgery. the operative indication was a unilateral hernia in % of cases, associated with an umbilical hernia in % of cases, a recurrent groin hernia in % of cases and a bilateral inguinal hernia in % of cases. the conversion rate was . %. the hernias were classified according to the ehs classification in l type in % of cases, l in % of cases, l in % of cases, m %, l r in % of cases and f r in % of cases. a contralateral inguinal hernia was discovered in % of patients. a polypropylene mesh x cm was fixed by a stapling in % of cases and by a suture in % of the cases. the average operation time was min. the hospital stay average was , day. an antalgic treatment was prescribed in % of patients. the average time to return to normal physical activity was days. a postoperative seroma was noted in % of patients. no cases of mesh suppuration were noted. chronic pain was noted in two patients. no recurrence was noted with an average follow-up of months. conclusion: laparoscopic treatment of the groin hernia by tapp had good results concerning the postoperative pain, early recovery of physical activity and aesthetic damage. a larger setback is needed in our study to evaluate the recidivism rate. background: surgeon's training in ultrasound is viewed and understood differently in different parts of the world. if in united states the need for surgeons' training was accepted and taken over by the american college of surgeons, in europe the practice is completely different from one country to another, from one city to another, from one department to another within the same premises hospital. in some european countries, surgeons currently use ultrasound for diagnosis-especially in urgency, for follow-up, intraoperative, or as guidance for many surgical gestures. during this time, access to ultrasound of other surgical specialties-gynecology, urology, ophthalmology-is considered natural. material and method: once the decision to initiate an ultrasound course for surgeons was taken, a team of experts with technical or clinical expertise in ultrasound was organized. at the initiative of the technology commission, the courses were to be organized at the eaes congresses or others communication events endorsed by eaes. starting from the importance of each ultrasound application in surgery, it was decided to develop different modules to solve different training needs. at this time, the course offered at seville covers the capitols like abdominal ultrasound, guided punctures and trauma. a module dedicated to intraoperative ultrasound is under construction and will be available in november . the course has a skill abilities dominant character, two thirds of it being thought of as a hands-on application on stationary, classical ultrasounds with large screens and also on small size wireless actual devices. results: after his debut in frankfurt in , the course was resumed in london and in bucharest, twice. in this process, new modules and better teamwork skills have been developed. the participants' satisfaction quizzes, coming from all continents, were really encouraging. for the intraoperative ultrasound module the team approach is unique. students will have the opportunity to practice on live animals both laparoscopic and open abdominal procedures conclusions: the ultrasound for surgeons course initiated by eaeas was received with interest. the team will seek to inspect the real needs of training surgeons in this field and will complement and diversify the current platform surg endosc ( ) preliminary results: a total of procedures ( - , ± . ) were carried out in patients. the indications included acute ( leaks following esophageal resection, rupture of the strictured anastomosis following pneumatic dilatation) and chronic conditions (esophagopleurobronchial and gastropleurobronchial fistulas following the resection of esophageal diverticulum and sleeve gastrectomy). the initiation of the therapy was in , and day in case of acute conditions, and after years of the duration of the unsuccessful therapy in chronic cases. the successful closure was observed in patients, patient passed from mods and ards. in case, the initiation of evac was provided as a combined surgical and endoscopic intervention (ct proven distant intraabdominal abscesses). in chronic cases, was discontinued due to the haemophagocytic syndrome of unknown etiology, in the second one, success in reduction of the lesion and symptomatology with long term duration was observed following just applications of evac, despite minimal remanent leakage. the success is to our experience linked to early initiation of the therapy and presumes complex intensive care. the future investigation should specify the timing including preemptive use of evac and the combination of evac with other endoscopic, interventional and surgical therapeutic modalities. the aim of this feasible study is to investigate complete robotic esophagectomy with total mediastinal lymph node dissection (retm) only by the robotic arms. methods: the patient is placed hemi-prone position with one lung ventilation by blocking balloon tube under general anesthesia. the robotic trocar for st arm of da vinci xi surgical system is placed in the th intercostal space (ics) on the scapular line, the trocar of nd arm is placed in the th ics on the posterior axillary line, rd arm trocar is placed in the th ics on the middle axillary line, th robotic arm trocar is placed rd ics on the middle axillary line, and assistant trocar only for taking in and out of gauze is placed in the th ics on the middle axillary line. on the upper mediastinal lymph node dissection, robotic camera exchanges from nd to rd robotic arm to close and identify anatomical structures. esophagectomy with lymph node dissection starts from middle and lower mediastinum to upper mediastinum including along bilateral recurrent laryngeal nerves. all procedure perform under close-up view along the robotic enhanced anatomy to preserve organ functions. background: complete stenosis of the duodenal lumen secondary to a surgical suture in the treatment of a duodenal ulcer is a rare complication. the usual surgical resolution corresponds to a gastrojejunostomy associated or not, to an antrectomy with vagotomy, as a treatment for the peptic disease. the endoscopic resolution of this complication requires the use of complex maneuvers and specific therapeutic instruments. aim: to describe the endoscopic resolution of iatrogenic occlusion after raffia of perforated duodenal ulcer. description: a -year-old man was admitted to the emergency service for four days of pain and abdominal distension associated with abundant retention vomiting. performed ten days ago of a perforated duodenal ulcer, in which manual raffia was performed in two planes and drainage. abdomen and pelvis ct showed great distension and diffuse thickening of the gastric wall. the endoscopy showed abundant gastric retention content, pylorus, and bulb edema, and complete closure of the duodenal lumen, secondary to suture material; it was possible to count three suture threads. with a tipped papillotome and electrocautery, all the suture threads were sectioned, identifying a filiform opening through which a hydrophilic guide is inserted under fluoroscopy until it is sure to overcome the stenosis; we dilated the trajectory with a dilator of mm in diameter and cm in length. with a contrast medium, we observed an adequate trajectory and installed a partially covered duodenal metal stent (hanaro stent) of cm in length by mm in diameter in order to sustain the dilation. he was sent to home with inhibitors of the proton pump. after two weeks, the stent was removed, without complications. he was controlled two weeks after withdrawal, and, with pharmacotherapy, the patient was asymptomatic, making a normal life. conclusion: in this case, the result was positive. satisfactory results can prevent major surgery, which reduces the risk and possible complications. a new range of non-invasive medical tools with a remarkable improvement on the existing market. a manual laparoscopy, with the important novelty of having a bending head with a high degree of movement. this head can get multiple spatial positions to work in surgery and is very easy to use, with the same scissors thimble who controls it, so its performance and learning is very simple and intuitive. the tools can be easily reusable and they can be cleaned and sterilized by ordinary methods, very ergonomic and lightweight . the generic type models we initially have developed are focused in general surgery, but gradually we will develop new applications and different heads for specific medical conditions such as arthroscopy, laryngoscopy, otolaryngology, ophthalmology, orthopedics . its operation is simple and functional, simply moving the scissors thimbles, where they have a dual role, combining the head tilt and the action of opening and closing of this is achieved.the design of this tool allows us to work with some degrees of unparalleled freedom from the existing tools. our instruments replicate the movements of the robot with a simple handheld mechanical instrument, our philosophy is to position our instruments in between a long empty field between the surgeon and the robot. the tip of our instruments are providing the surgeons with angulations impossible to reach with the traditional instruments unless applying huge movements from the hands of the surgeon. we consider that this devices will have a very fast acceptance from the market as this robotic type movements can be managed by the surgeon through the traditional mm trocars, without the need to change to a new surgical technique, just with the traditional method and a very brief training. background: diverticula of the middle thoracic esophagus are infrequent, its etiology may be secondary to traction or pulsion mechanisms. when the etiology is a mechanism of pulsion, they are associated with esophageal motor disorders and its prevalence is estimated between . % and . % of the population . they are rarely symptomatic and the diagnostic is usually incidental. the most common symptoms are episodes of food impaction, chest pain or bronchoaspiration.diverticulectomy and esophageal myomectomy by minimally invasive approach is the treatment of choice in those with large size or associated symptoms. aim: to describe a clinical case of esophageal diverticulum solved by minimally invasive surgery approach. clinical case description: a -year-old patient with a history of epilepsy and hypothyroidism consulted for atypical chest pain and dysphagia to liquids and solids. a study with esophagogastroduodenoscopy was performed: cm from the dental arch, a large wide-mouth diverticulum was identified. we complete the study with an esophagram with barium: voluminous diverticulum in the right lateral face of the middle esophageal and a thoracic ct scan showing esophageal diverticulum located in the carina, from . x . cm to cm from the esophagogastric junction. due to the suspicion of associated motor disorder, high resolution manometry was performed showing a significant motor disorder with alteration of peristalsis and exit obstruction with incomplete relaxation of the inferior sphincter and superior hypertonic sphincter. preliminary results: the patient underwent surgery: diverticulectomy and complete esophageal myotomy by thoracoscopy minimall invasive approach. the patient evolved favorably and was discharged after days with a previus control esophagram without pathological findings. currently, it remains asymptomatic months after surgery. , rectus muscles are re-approximated from xiphoid to pubis using laparoscopic running self-locking, pds sutures to restore anatomy and physiologic function of the abdominal wall. unlike the standard access to the abdominal cavity executed with lateral access, the lap-t technique is performed through sopra-pubic aesthetic approaches, using one mm and one mm bariatric ( cm) instruments laterally, and one mm camera in the middle.the entire procedure is performed in gas-less laparoscopy, with laryngeal mask and intra-peritoneal liquid anesthesia. the repair is consolidated placing an intra-peritoneal semi-absorbable mesh. preliminary results: in all cases abdominal functioning was successfully restored; no higher pain related to the continuous laparoscopic suturing has been reported compared to bridge ipom laparoscopic repair, while allowing for a more physiologic outcome and a stronger repair. the use of miniaturized instruments allowed for minimal tissue trauma and accurate surgical gestures; the tiny trocar sites did not require skin suturing and might reduce the risk of trocar hernias. no intra operative bleeding, no seroma formation, chronic pain, nor mesh infection have been recorded. % follow up at months, % at months with no recurrences observed. the lap-t technique allowed for a sound and anatomic reconstruction, reduced trauma, faster recovery and more satisfactory aesthetic results. surg endosc ( ) background: anastomotic leakage is a serious complication, associated with significant morbidity and mortality. one possible cause of anastomotic leakage is insufficient vascular supply. markers of sufficient perfusion include pink color of the bowel wall, visible peristalsis, palpable pulsations and bleeding from the marginal arteries. these signs are subjective and may be misinterpreted even by experienced surgeons. aim: the assessment of bowel perfusion with the use of indocyanine-green fluorescence angiography might be helpful in decreasing the number of anastomotic leaks. project description: we report a case report of a middle-aged patient without significant medical history who was treated by transanal total mesorectal excision (tatme) for rectal carcinoma. the patient underwent neoadjuvant treatment with radiochemotherapy. during the surgical procedure, indocyanine-green fluorescence angiography showed adequate perfusion of the bowel. the postoperative phase was uneventful and the patient was discharged home on the th postoperative day. preliminary results: indocyanine-green fluorescence angiography is a safe, cost-effective and feasible tool for assessment of tissue perfusion during colorectal resections. background: to properly learn how to perform a laparoscopic suture, along with safe tissue handling, applying an appropriate magnitude of the force on the tissue is essential. for this reason, it is fundamental to investigate and validate if training with real-time visual force feedback improves the suturing performance of laparoscopic novice surgeons. capturing all of the forces applied in laparoscopic in surgery in an unobtrusive way has been difficult in the past. sensor has supplied a novel force-sensing film (forcefilm) that can detect all of the forces applied with laparoscopic instruments without changing the surgical workflow or operation of the instruments. aim: to evaluate the effect of visual force feedback on surgical performance, applied force and surgeon's ergonomics during training of laparoscopic suturing using the sensor technology (sensor medical laboratories ltd.). methods: twenty novice laparoscopic surgeons participate in this study. they perform a laparoscopic suture on an ex vivo stomach tissue from a pig. participants are assigned, in a random fashion, to either group that receives visual force feedback (a) or the control group (b) without visual force feedback. five training trials (t -t ) are carried out in order to assess the learning curve. in addition, an evaluation pretest (t ) and posttest (t ), without visual force feedback but recording the force applied, will be performed after the training trials. the applied force on the tissue and visual force feedback of each instrument are provided by means of the sensor technology. it accurately measures the forces exerted on the tissue from the instrument tip and wirelessly communicates the force information to the surgeon via visual force-feedback. during each trial, several parameters are evaluated such as execution time, applied force, surgical performance, and mental and physical workload. preliminary results: laparoscopic training using visual force feedback leads to an improvement of suturing skills with a reduction of the applied force and therefore providing a potentially positive effect on patient outcomes and surgeon's ergonomics. background: hiatal hernia is a frequent disorder, characterized by a protrusion of any abdominal structure other than the esophagus into the thoracic cavity through a widening of the diaphragmatic hiatus. current anatomic classification is mainly based on the location of the gastroesophageal junction and the presence of a true hernial sac, differentiating sliding from paraesophageal hernias. there is no solid evidence to support an association between gastric carcinogenesis and peh. however, chronic reflux is considered as one of the strongest risk factors of developing adenocarcinoma of the esophagus and proximal stomach. aim: herein, we report a case of an -year-old caucasian female with dysphagia, regurgitation and heartburn accompanied by an iron deficiency anemia, a remarkable total body weight loss and recurrent lower respiratory tract infections due to microaspirations. subsequent work-up with ct, upper endoscopy and barium esophagram confirmed the presence of synchronous distal gastric adenocarcinoma and a giant paraesophageal hernia with complete intrathoracic stomach. after mdt discussion and keeping in mind the patient's age and comorbidity, a d laparoscopic distal gastrectomy with a synchronous hernia reduction with posterior cruropexy was scheduled. project description: the patient was placed in a supine position, five thoracoscopic ports were introduced, and a diagnostic laparoscopy of the abdominal cavity was performed. the stomach was identified through the dilated hiatus into the left thorax. the hernia sac was dissected away from mediastinal structures, then excised to untwist the stomach. after reduction of the stomach to abdominal cavity, a total d ? gastrectomy with a roux-en-y reconstruction was performed. maintenance of optimal vision during minimally invasive surgery is crucial to maintaining operative awareness, efficiency and safety. hampered vision is commonly caused by laparoscopic lens fogging (llf) and lens condensation which has prompted the development of various antifogging fluids and warming devices. numerous tricks have been proposed to overcome this issue, such as heating the scope into a sterile thermos flask filled with hot water, or using one of the commercially available antifogging solutions. however, whether one method is superior to another remains elusive. as most surgeons know, none of these tips are totally efficient, as they don't treat the cause: the temperature difference. taking into account this need, we have developed ehs (endoscopy heater system), a thermoadjustable system by microcontroller, which is implemented in the manufacturing process of the rigid endoscope focused on laparoscopy. the technology enables the self-modulation of the temperature of the endoscope within the different conditions during the surgery, avoiding the % of laparoscopic fogging. with the adoption of ehs surgeons get a clear field of vision avoiding continues repetitions of extraction and insertion of the endoscope in the body during the intervention. in this way the risks of the patients are reduced with a more efficient and shorter duration procedure. ehs also represents an alternative that meets sustainability criteria by reducing energy costs and eliminating much of the waste currently generated by this procedure. therefore, this innovation will disrupt the laparoscopic device market by enhancing safety and effectiveness without introducing new components that could complicate surgical procedures. case report: we presented the case of a year old women with chronic coloenteric fistula. conservative treatment was unsuccessful. the orifice was then closed with two subsequent clips, and the patient recovered well. to our knowledge, this is the first successful case of coloenteric fistula treatment with ovesco discussion: ovesco system is a technique that enables the closure of gastrointestinal defects (perforation sites, leaks, fistulas) . after the system application, the patient can be treated at home as was the case with our patient. a successful closure of the leak or fistula is possible when no extraluminal abscess is present. in our case, we had a cavity (previous sinus or abscess) that drained into the small bowel, thereby forming the coloenteric fistula. this allowed us to succeed with a fistula closure, as the cavity could drain into the small bowel conclusions: looking through the reports, one notes that the success rate of the otsc system procedure for insufficiency of anastomosis or colorectal fistula was - %, but only nine successful reports of chronic colorectal fistula were found). a % success rate is reported if the clip is placed within a week of occurrence of the leak . on considering the financial side, clips could reduce costs and time of hospitalization and avoid patients having to undergo a surgical repair . the major advantage of ovesco clips seems to be their ability to grasp more tissue compared to the standard clips and their strong grip on the wound margins because of their sharpened teeth. the drawback of the clips in fistula sealing is their incomplete grasp when the tissue is fibrotic. most authors agree that ovesco is not very appropriate for fistulas larger than - mm. inguinal hernia repair is one of the most performed interventions in minimally invasive surgery. in this opportunity we report a new technique through the use of innovative devices such as the robotic clamp and magnetic deviceswith this technique and thanks to the magnetized devices and the robotic clamp we have demonstrated to reduce the surgical time between - min as well as to optimize the ergonomics of the surgeon.we explain the technique with a demostrative video and exposition of the devices that are necessary for make it.with this new technique we get a greater capacity of mabiobra for dissection of the peritoneum and later a greater facility for the suture of the same in the repair of the inguinal hernia. a motorized and computerized laparoscopic tool that can be customized to the specific surgeon and procedure a. szold aim: surgeons have different levels of skill and use instruments for different tasks, but laparoscopic instrument are commonly simple mechanical instruments that allow limited degrees of motion, and the same instruments are used regardless of the surgeon or the task. robotized articulating instruments so far have added degrees of freedom, but perform in a standard way for all users and procedures. technology: human xtensions has developed a \ u[hand-held \/u [ motorized smart laparoscopic instrument, that was recently introduced in human procedures. the device has several features that enable to customize it to the user and procedure. the degrees of freedom can be reduced from to , the scale of rotation motion has options that can control both speed and range of rotation, a feature especially useful for the variable types of suturing tasks. results: the variable features were tested in different procedures requiring suturing and grasping. the combination of all optional settings made the instrument customizable to the different skill levels of the surgeons. as such, it enabled to control the complexity of the device and take the surgeon through the learning curve until full control of all features was achieved. in addition, the combination of different controls was used for performing specific tasks requiring different levels of maneuverability. in september , the results of a bomss survey regarding the routine use of pre-operative bariatric surgery were published. they found that % of units surveyed considered routine preoperative ogd completely unnecessary. as part of newly launching bariatric services in a single isolated centre we protocoled that all bariatric patients had to undergo pre-operative ogd, including a clo test, and reviewed if the ogd findings had influenced our surgical choice of operation and any necessary treatment before surgery. all patients embarking on the bariatric programme since its launch in jan to sept were included and had an ogd. the results of these ogds and all the clo tests were reviewed. these ogds were all performed by a single consultant to minimise any potential subjective differences. of the patients, ( %) tested clo positive of which had normal findings on ogd. patients had a hiatus hernia, gastritis, oesophagitis, gastritis and oesophagitis, had other findings e.g. ulcers, polyps' or nodules. the positive clo patients underwent eradication of h pylori. studies have shown that this is a treatable and preventable cause of gastritis/ gastric cancers and potential surgical complications causing prolonged hospital stay in % of patients. knowing about the presence of a hiatus hernia prior to surgery also contributed to the surgical planning, including allowing time for the concurrent correction of the hiatus hernia in the operation. all patients with demonstrable oesophagitis ( %) had their operative choice changed to roux en y gastric bypass thus aiming to prevent post-operative reflux which would have been exacerbated had they undergone a sleeve gastrectomy instead. carrying out a pre-operative ogd had a significant impact in operative choice and additional treatment before surgery and therefore should be advised in all patients. general surgery department, ahievran university, kirsehir, turkey the majority of fatalities worldwide in people under the age of years are caused by trauma . blunt mechanisms account for . to . %of injuries [ ] [ ] [ ] [ ] ,with the abdomen being affected in . to . % of all traumatic injuries. this case contribute to the literature: a patient with sleeve gastrectomy has distorted anatomy at duodenogastric junction, if has bat,her/his small bowel perforation(sbp) will occur on more distal segment. this a unique case before unpublished. years old female who had sleeve gastrectomy years ago presented to emergency department sustained blunt abdominal trauma (bat). when she arrived pyschical exam (pe) revealed an abdominal guarding, tenderness, normal vital signs but those increased h later. wbc values also increased h later. hb was normal.fast showed in cm thickness fluid early in douglas pauch (dp), mm in supravesical, mm diameter in dp h later. abdominal ct: mm diameter fluid in interloop, dp free air at th h from the accident. a diagnostic laparoscopy(dl) was done with diagnossed acute abdomen.there were a sbr-located cm from treitz, intraperioneal fibrin deposits and fluid-repaired with a primary suture.the patient discharged on days without any event. repeat ct scans are recommended for patients with initial suspected bowel injury. we could not do this; cause ct exam could taken in rush hours only but we did repeatly pe that peritoneal iritation signs increased,resulted a dl,surgical therapy. according to the literature; dl may be a good treatment option in these patients, to reduce morbidity or mortality, time to surgery has been emphasized. long interval between presentation and surgery was found to be associated with complications. very few reports of isolated jejunal transection following blunt abdominal trauma have been published in literature. the literature mentioned; the patients with sbp are hemodinamically stable on arrival to the hospital like our case are, a rupture of the jejunum was seen just distal to the duodenal-jejunal flexure but there were a perforation cm below treitz ligament and caused me to think the patient had sleeve gastrectomy and some brid around gastric and duodenal proximal jejunal part of intestines and also caused a new descended treitz ligament. normally external forces across to spine produce a blast effect on small bowel between treitz and ileocolic ligament. introduction: sasi bypass is a novel metabolic/bariatric surgery operation based on minigastric bypass and santoro's operation.it can be offered for patients with weight regain after sleeve gastrectomy. sleeve gastrectomy (sg)is a commonly performed bariatric procedure.weight regain following sg is a significant issue.yet,the understanding of this phenomenon is still unclear.rates of regain ranged from . % at years to . % at years.sasi bypass was an option for some candidates having sg done years back and failed to achieve the required weight loss or having weight regain.in sasi bypass, resleeve gastrectomy of the dilated gastric pouch is done followed by a side to side gastro-ileal anastomosis. the aim of this study is to report the clinical results and the outcomes of sasi bypass as a therapeutic option for patients with weight regain after sg methods: we conducted a retrospective study for morbidly obese patients having history of sg done more than years back and failed to achieve and/or to maintain the required bmi. exclusion criteria:patients with recent history of laparotomy(less than months). procedure was done at sidra hospital in kuwait from november to november . using ports, resleeve gastrectomy was performed over fr bougie tube starting cm above the pylorus then gastro-ileal anastomosis (side to side)was performed cm above the pyloric ring to an ileal loop counted cm from the ileocaecal valve. data was collected from the patients including:weight loss progress,laboratory full results. discussion and results: during the study period: morbidly obese patients with a mean bmi of ± kg/m were evaluated. -%ewl(excess weight loss)reached % at one year. -diabetes was cured in the known diabetic patients (type )within months,and the one known type diabetic patient had better control and less insulin daily doses(results were guided by glycated haemoglobin results every months). follow up laboratory results were normal in % of patients (all were kept on regular vitamins and proteins supplementation)-one patient had postoperative leak(day )from the anastomotic line that was treated conservatively. conclusion: sasi bypass is a promising operation that offers a good weight loss for morbidly obese patients having weight regain after sg conclusions: our study demonstrates a good agreement between the degree of liver steatosis and monocytes fat accumulation as well as between plin levels in liver and circulating monocytes. this suggests that ectopic fat deposition is a generalized feature of insulin resistance in obesity. sg reverses monocyte fat accumulation and restores insulin signalling, which correlates well with insulin sensitivity. moreover, circulating mmp levels significantly dropped after sg suggesting that the state of generalized inflammation characterizing obesity normalizes. her stomach stapled,a foreign tube like body was seen on cut surface of the stomach.the foreign body seen in dissected stomach wall is the tube is in placed a gastric banding insuflation tube.a laparatomy was made and the tube is extracted her stomach sutured primarily,nasogastric decompression,peritoneal drainage was made.her peritoneum was drained.she has a septic condition, leave in icu for a long period.her general status being well and discharged from hospital in days.we learn after the operation not before;she had a gastric adjustable banding and extraction the gastric band, but the tube of gastric band is not removed. alkhaffaf et al present a case of fistulation of the lagb tubing into the jejunum a review of the published data to identify the salient learning points with this and similar rare complications fistulation from lagb tubing is a rare complication that tends to follow removal of an infected port. the clinical presentation is nonspecific, rendering the preoperative diagnosis difficult. the tube and band can be removed laparoscopically, with closure of the small bowel fistula site. securing the tubing to the abdominal wall fascia after intentional detachment from the port might reduce the incidence of this complication.katherine j et al report a late and rare complication of a small bowel obstruction in a -year-old woman from an lagb placed for years. although not a common complication, one that could easily see the safety record of lagb patients tarnished if this small subgroup of patients is not acted upon promptly by emergency departments' unfamiliar lagb surgery. in our case we already made esophagogastroduodenoscopy before operation, ofcourse take past medical history from the patient.the patient hide past operation (gastric banding and removing band and port but leaving insuflation tube). ). there was no difference in the two groups regarding follow-up rate. basic demographics were the same, and other long-term results were similar between the groups. regression models for both post-op complications and failure as defined by baros score did not show that gender is a risk factor. discussion and conclusions: in our study, revisional sleeve surgery were similar. we did not see any significant difference in post-op complications, success of the operation as defined by baros, or subjective feeling of the patients. we do believe that gender-specific outcomes should be taken into consideration in optimizing patient selection and preoperative patient counseling, and that in the case of a sleeve post a band gender is not a risk factor for complication or failure of the procedure. objective: the internal hernia is a rare but a potentially fatal complication of laparoscopic roux-en-y gastric bypass (lrygb). the aims of this study are: ( ) to determine the impact of mesenteric defects closure on the incidence of internal hernia after lrygb; ( ) to determine the symptoms, characteristics and management of internal hernias after lrygb. the median interval between lrygb and reoperation was months in group a and months in group b. the median percentage of excess weight loss (%ewl) was % vs %, respectively (p = . ). the median percentage of total weight loss (%twl) was % vs %, respectively (p = ns). patients, % ( in group a), were admitted to the emergency room with acute abdomen pain. a ct scan was performed in patients, %, and showed signs of occlusion in all cases. the most common symptoms were abdominal pain and vomiting. the surgery was performed using laparoscopy in patients, %, and using laparotomy or conversion in patients, %. conclusions: the closure of mesenteric defects during lrygb is recommended because it is associated with a significant reduction in the incidence of internal hernia. our study intends to analyze the long term results of sleeve gastrectomies performed by d laparoscopic approach. materials and methods: a prospective cohort study was conducted to perform gastric sleeve for morbid abesity. all surgeries were performed by the same surgeon over a period of two years. the operating surgeon is a senior most laparoscopic surgeon with vast experience in laparoscopic surgery. during two years period, cases were operated using d laparoscopy system. scientific calculation was done using spss release . windows software. results: patients, female ( %) and male ( %), with median age of . ± . ( - ) the excess weight lost (ewl) was % in the first year, % in the second, % in the third, % in the fourth, % for the fith year, % for the sixth and % for the seventh. postoperative complications were stenosis of the sleeve always located in the incisura and treated with endoscopic dilatation except one that required conversion to oagb. three leakages, all of them reoperated with drainage and introducing prosthesis by endoscopy in the same act. we have never had a postoperative bleeding of the sleeve. conclusions: d gastric sleeve laparoscopy is a safe and feasible technique for morbid obesity and related pathologies. the ewl is correct in long time. complications are rare but is necessary to have a good level of suspicious in order to a rapid solution. the worst complication is the leak of the sleeve. the oversewn of the gastric section is a good technique to avoid this complication. surg endosc ( ) aims: leak is one of the common complications of laparoscopic sleeve gastrectomy that result prolongation of hospital stay, morbidity and even mortality. methods: i report new approach for the treatment of leaks presented to me post laparoscopic sleeve gastrectomy with laparoscopic roux en y bypass to the leak site at the level of gastroesophageal area. this new approach is possible and feasible, and avoids stenting due to high failure rate, prolonged hospitalization and saves life of patients. results: all leaks healed days from surgery due to well vascularized small intestinal patch, except for leaks that healed after weeks of conservative treatment. aims: analyse the effect of one anastomosis (mini) gastric bypass (mgb/oagb) in the treatment of gastro-esophageal reflux in patients previously submitted to laparoscopic sleeve gastrectomy (sg). methods: a retrospective analysis was performed on the data of patients who underwent mgb/ oagb after a previous sg at policlinico san marco, italy, from january to june . a total of patients, female and males ( % f/ % m) underwent mgb/oagb after sg, due to the development of significant gastro-esophageal reflux disease (gerd), refractory to proton pump inhibitors (ppi), detected with the gerd questionnaire (gerd-q) and esophagogastroduodenoscopy (egds). in three patients ( %) a weight regain was also observed (mean bmi . kg/m , range . kg/m - . kg/m ). mean patients age was . ( - years old). before sg none of the patients had declared symptoms of gerd or was subjected to a therapy with ppi, preoperative egds did not show signs of esophagitis. mean bmi of the patients who developed gerd without weight regain was . kg/m ( kg/m - . kg/m ) at the time of surgery, with a medium ewl% of % ( . - . %). patients were treated unsuccessfully with ppi for at least six months before programming revisional surgery. mean gerd-q score was . results: after mgb/oagb, with a mean follow up of months ( - months), mean bmi was . kg/m and gerd-q score was . however, five patients out of ( %) developed an anastomotic ulcer or a grade c esophagitis. we did not observe any post-operative immediate complication nor any death. conclusion: mgb/oagb is a simple, effective and safe surgical procedure for patients who underwent a previous sg and who developed gerd, with satisfactory results in the short and medium post-operative time, even if there is still concern regarding the complications linked to biliary reflux. v.s. kyosev aims: laparoscopic adjustable gastric band (lagb) was one of the common techniques in bariatric surgery worldwide. the advantages included the possibility of regulation, ease of placement, acceptable weight loss and low rate of perioperative complications. a late complication of lagb is penetration of the gatric band through the gastric wall and migration into the lumen of the stomach. hereby, we present three cases of gastric band migration following lagb. methods: from to we observed cases of gastric band migration in between and years after lagb placement. the patients were hospitalized in surgical department complaining of sudden sharp epigastric pain, nausea and vomiting, with symptoms onset in the last few days. all patients underwent abdominal ultrasound examination, x-ray investigation of the abdomen with oral contrast administration, fibrogastroscopy. in cases the imaging studies revealed gastric band migration into the stomach's lumen and in case-obstruction of the jejunum by the gastric band. all patients underwent laparoscopic surgery. results: two of the patients underwent gastrotomy, extraction of the gastric band and roux-and y-gatric bypass. the patient with jejunal obstruction underwent laparoscopic enterotomy, extraction of the gastric band and cholecystectomy due to concomitant cholecystitis. two of the patients had no additional perioperative complications and were discharged at the th postoperative day. one patient developed fever, left pleural effusion and partial insufficiency of the gastrointestinal anastomosis in the early postoperative period without the need of surgical treatment. the patient was discharged on th postoperative day. all patients were prescribed a diet and monthly blood test of ion balance. conclusions: lagb was one of the most common treatment methods due to the epidemic spread of morbid obesity in western countries. detailed knowledge on possible lagb complications is essential for the treatment of these patients. the diagnosis of lagb complications is often delayed due to its relative rarity and nonspecific clinical manifestations, but in most of the cases it requires emergency surgery for management of life-threatening conditions. results: there was conversion in patients (short mesentery of the small intestine). such postoperative complication like anastomotic leak in patient ( . %) and staple line bleeding in patient ( . %), which was managed laparoscopically. compensation for type diabetes was achieved in ( %) patients, improvement was recorded in ( . %), dyslipidemia in ( . %) and ( . %) patients, arterial hypertension in ( . %) and ( %) patients respectively, what led to metabolic syndrome resolution in ( %) patients. the liquid is allowed to take after day. average postoperative hospitalization- . ± . days. %ewl for months %. conclusions: laparoscopic mini gastric bypass is an effective method of surgical correction of body weight and metabolic disorders in patients with morbid obesity and allows to receive an adequate and stable correction of arterial hypertension, lipid and carbohydrate metabolism, which are components of a metabolic syndrome. introduction: over time laparoscopic sleeve gastrectomy lsg has become the most popular bariatric operation worldwide. a critical step during lsg is ensuring sleeve-size consistency. gastrisail device (gastric positioning system) is a three in one surgical device replacing the standard bougie used in lsg for the application of suction, decompression and to serve as a sizing guide for gastric sleeve creation. the aim of this study is to evaluate the possible merits of gastrisail device in lsg over the standard laparoscopic sleeve gastrectomy. methods: a prospective study of patients randomly divided into two groups: group a composed of twenty patients who undergo lsg with the use of gastrisail and group b composed of twenty patients who undergo lsg with the standard bougie without the use of gastrisail comparing both according to operative time, consistent sleeve formation, delineation and visualization, intraoperative and post-operative complication rates, the lenght of hospital stay,gastric pouch design and percentage of excess weight loss (%ewl). results: regarding intraoperative time, the mean time was . ± . and . ± . for group a and b respectively,while no patients in group b had consistent sleeve formation, patients ( %) had consistent sleeve formation. delineation and visualization were accomplished in % of group a patients, was not accomplished at all in group b patients. the alignment of the stomach was reached in patients in group a but no patients at all in group b, the mean of hospital stay was . ± . and . ± . for group a and b respectively, the smaller tube design illustrated by gastrograffin x-ray at rd post-operative day was accomplished in patients ( %) and patients ( %) in group a and b respectively. there was no significant difference in %ewl in both groups. conclusion: the use of gastrisail device is superior to the standard lsg in consistent sleeve formation, visualization, delineation and good alignment and accomplishment of a small tube design while no significant difference in %ewl. bariatric surgery has spread all over the world. since japan has few patients with morbid obesity compared with western countries, it has been implemented only in limited facilities. however, bariatric surgery in japan is rapidly spreading recently, and many facilities are about to install bariatric surgery. effects of bariatric surgery are known to last for a long time, but some cases require reoperation which is called revision surgery due to late complications or rebound. because of thick subcutaneous and visceral fat, open surgeries are not even always a good solution to make surgery easier in morbid obese patients and all procedures must be completed laparoscopically. therefore, especially in revision surgery, the incidence of complications tends to be increased. as the number of bariatric cases to be increased in japan, cases requiring revision surgery is likely to increase. in revision surgery, it is necessary to select the procedure according to patient condition, and it is necessary to familiar well with those procedures. we will present cases that underwent revision surgery in our department and show the clinical outcome. we have done four revision surgeries after sleeve gastrectomy so far. operative indications are mid-gastric stenosis and rebounding disease. for stenosis cases, we performed roux-en y gastric bypass with distal stomach resection, and for rebounding cases, we performed re-sleeve gastrectomy with duodenal-jejunal bypass. average interval from initial operation to revision surgery is months in rebounding cases, and months in stenosis cases. duration of operation was min in average, and mean estimated blood loss was ml. no postoperative mortality was observed. in rebounding cases, excess bmi loss at year after surgery was . % in average, and both cases achieve diabetes remission at year. one cases of mid-gastric stenosis required a nutritional support with formula diet temporally. in particular after sleeve gastrectomy, revising to roux-en y gastric bypass, re-sleeve gastrectomy, and adding the duodenal-jejunal bypass will be the main techniques. along with an increase of bariatric surgery in japan, it is necessary to acquire sufficient knowledge and skills to carry out revision surgery. methods: we present the case of a year old woman who underwent lsg after lagb removal and lgcp. the patient underwent preoperative endoscopy and barium swallow, with no sign of stomach perforation or erosion. we emphasize that the patient, had undergone three operations of gastric band placement, gastric band removal and gastric plication before sleeve gastrectomy. however, a successful lsg was achieved. results: no severe postoperative complications were mentioned. conclusion: weight loss in the first year was % of the excess weight.sleeve gastrectomy after gastric band removal and gastric plication, for morbid obesity seems to be safe and efficient, especially in casesof absence of gastric erosion. surg endosc ( ) department of surgery, patients were observed with serious septic complications many years after gastric banding operation. we detected a female dominance ( female, male) in patients with a mean age of . years. the leading symptoms were: dysphagia, upper abdominal tenderness and pain, spontaneous fistula formation, fever, masked septic signs, bowel and urinary obstruction. patients underwent video-endoscopy, chest and abdominal ct (computed tomography), fistulography and cystoscopy. results: in still morbid obese patients, laparoscopic procedures were performed with a conversion rate of %: atypic gastric and cardia resection in cases, gastric suture in cases, small bowel resection and suture in - cases. in one case, fistulectomy, abscess evacuation and combined urinary bladder suture and drainage were carried out. the duration of the surgeries were over h with minimal blood loss (\ ml). the foreign bodies were completely removed in every case. intraoperative complication was not occurred. early physiotherapy were promoted, oral feeding were gradually built up from the th postoperativ day depending on the type of the operation. early postoperative complications included recurrent fistula formation (n = ) and wound infection (n = ). all the fistulas were closed after conservative treatment. average hospital stay was days, regular check-ups were held on the rd, th and th months of follow up. conclusion: gastric banding is the most common, routine and safe technique for the treatment of morbid obesity. the development of late, severe septic complications draws attention to the crucial importance of follow up. the surgical management of these patients is recommended in specialized centers in regard to difficult operative conditions and atypic treatment options. aims: single anastomosis duodeno-ileal bypass with sleeve gastrectomy (sadi-s) has been proposed as an alternative to biliopancreatic diversion with duodenal switch (bpd-ds) in order to maintain the outcome of the original procedure simplifying the technical complexity and to avoid potential complications. moreover, it potentially represents the more natural second step bariatric procedure after sleeve gastrectomy (sg). we aimed to report the initial experience with sadi-s of our high volume bariatric center. methods: retrospective analysis of patients who underwent bariatric procedure between july and november was conducted. the primary aim was the evaluation of the safety of sadi-s, defined as the rate of postoperative complications. the secondary endpoint was the bariatric efficacy of the procedure, defined as percentage excess weight loss (%ewl). results: among patients who underwent bariatric procedures at our institution ( . %) patients were scheduled for sadi-s. all patients had multiple comorbidities. initial indication for sadi-s was failed sg in patients (median pre-sg bmi . kg/m \ sup [ \/sup [ ; median months after initial operation respectively) and primary procedure in patients (median pre-operative bmi . kg/m ). the surgical procedure was accomplished with robotic-assisted approach in cases (median operative time min) and with laparoscopic trocars standard approach in the remaining cases (median operative time min). the duodeno-ileal anastomosis was fashioned using a double layer hand-sewn running sutures. no patients showed early post-operative complications, the median postoperative stay was days. at a mean follow up of months the median %ewl was . . to date no patients experienced surgical. one patient develop wernicke encephalopathy months after surgery, but he was non-compliant to multivitamin supplementation. conclusions: at least in a high volume bariatric center sadi-s, both as second step after sg and as primary surgical option, seems to be a safe and effective bariatric metabolic procedure based on solid physiopathologic principles. on the other hand, longer follow-up is necessary to support the use of this procedure as a better alternative to bpd-ds. m.r. elkeleny , a. abo khozima git and bariatric surgery, faculty of medicine, alexandria university, alexandria, egypt; git surgery department, faculty of medicine,alexandria university, alexandria, egypt four bariatric cases . female patient with intragastric balloon, minor leak from the balloon leading to ballon migration to the jejunum;hance, small bowel obstruction occured. emergency diagnostic laparoscopy was done, enterotomy and extraction of the balloon, direct repair of enterotomy and balloon extraction through mm port site. . male patient presented after days of lsg with small bowel obstruction due to entrapment of small bowel loop through one of the port sites;therefor, emergency laparoscopy was done with reduction of the herniated segment and closure of the port site. . female patient presented with stricture of the ogj after re-sleeve gastrectomy managed by balloon dilatation which recur after weeks .she was managed by expandable metallic stent for weeks with good response and the stent was removed. . -year-old male patient presented with sever peripheral neuropathy following months after sleeve gastrectomy, and the patient was getting worse; thus, he used wheel chair. he has been making good progress on vitamin b complex injections. the aim of our study was to compare histopathological findings of gastric specimens to preoperative clinical symptoms and to conclude about the need for ugi endoscopy as a routine prior to surgery. methods: the last two years, morbid obese patients were selected to undergo laparoscopic sleeve gastrectomy (lsg) in our institution. for the needs of our study, all of them had ugi endoscopy and were reviewed for upper gi symptoms. histopathological reports obtained according to our protocol, after surgery. results: gastric histology from specimens revealed: no findings in / patients ( . %), gastritis in / patients ( . %) and focuses of incomplete intestinal metaplasia without dysplasia in / patients( . %). finally, two minor leiomyomas with low cellular proliferation rate were fully excised in a patient's specimen. there was no inconsistency between preoperative symptoms and gastric histology, while leiomyomas found were no reported to ugi endoscopy due to size. conclusions: some of the patients with clinical features of food intolerance, gastroesophageal reflux disease, and peptic ulcer disease had finally findings in histopathology of their stomachs. history of helicobacter pylori infection implements a raised incidence of mucosa pathology as well. because only one case revealed carrying significant pathology (leiomyomas), we consider that is safe to proceed with surgery in an otherwise asymptomatic patient based on his previous medical records and blood tests. aims: splenic abscess following laparoscopic sleeve gastrectomy (lsg) is a rarely seen complication. the aim of our study was to present a case of splenic abscess in a morbid obese patient who underwent lsg. as the main concern in these cases is leakage from the staple line, we present our diagnostic and treatment approach. methods: a -year-old, female morbid obese patient (bmi . kg/m ), without any predisposing risk factors, underwent elective lsg in our department. following an uneventful course, she was discharged at the nd postoperative day. however, at the th postoperative day, she readmitted to our unit with high temperature of . o c, left upper quadrant tenderness and leukocytosis. contrast computed tomography (ct) revealed an abscess at the upper pole of the spleen , cm in maximum diameter, without leakage from the staple line. results: the patient was treated with broad-spectrum antibiotics and radiological percutaneous drainage of the abscess. although there was a partial clinical improvement, a week later, a new ct scan revealed the continuous presence of the abscess. despite the stable general condition of the patient a laparoscopic splenectomy was performed and a gradual recovery was followed. the presence of splenic abscess without splenic trauma or leakage from the gastric staple line, is an extremely rare complication and only a few cases have been previously reported. the cause has not yet clarified, but the proposed mechanism involves infarction of the spleen, due to vascular compromise and subsequent infection. most of the reported splenic abscesses were diagnosed during the late postoperative period. in our report we present a case of early onset, hence highlighting the need of clinical awareness for early diagnosis and treatment. introduction: obese surgical patients with obstructive sleep apnea (osa) have a higher risk of peri-and postoperative desaturations and subsequent morbidity and mortality. currently, the best perioperative management of patients without known osa remains unclear. although routine osa screening has been advocated, sleep studies are costly and time consuming. we hypothesized that bariatric patients can be safely monitored on a surgical ward by continuous postoperative pulse oximetry without preoperative screening for osa. objectives: to evaluate outcomes of continuous postoperative pulse oximetry without preoperative osa-screening, and to compare the results to outcomes of patients with osa and continuous positive airway pressure (cpap) treatment. methods: all patients who underwent bariatric surgery between and were included in this single-center retrospective cohort study. all patients were postoperatively monitored with continuous pulse oximetry on the surgical ward. patients with less than two documented saturation measurements were excluded. patient files were reviewed for osa diagnosis, cpap usage and perioperative details. primary outcomes were -day complication rates, intensive care unit admissions due to cardiopulmonary causes and postoperative desaturations of spo \ %. secondary outcomes were icu admissions following all causes, length of stay and rates of reoperation and readmission. results: in total, patients were included. patients ( %) were preoperatively diagnosed with osa, ( . %) were cpap users. complications occurred in . % of patients without osa and in . % with osa(p = . ). desaturations were documented in . % and % (p \ . ), respectively. in both groups, patient was admitted postoperatively to the icu for cardiopulmonary causes that could be related to osa (p = . ). both recovered without further complications. icu admissions, regardless of cause, occurred in . % of patients without osa and in . % with osa(p \ . ). no significant difference between groups was observed in complications based on clavien dindo classification, length of stay, reoperation-and readmissions-rates. conclusions: these findings suggest that continuous postoperative pulse oximetry without preoperative osa-screening is a safe perioperative management strategy for bariatric surgical patients. future studies are needed to assess cost-effectiveness of pulse oximetry vs. routine preoperative osa-screening in a prospective clinical setting. background: the pathology of colon is one of the most pressing and socially significant problems of modern health care, because it leads to reduction of the working population employed in manufacturing, in some cases to disability and reduced quality of life. mini invasive surgery of the colon has a great advantage: speed recovery, shorter hospital stay and better cosmetic results, the quickest return of patients to work. as a result, mini invasive endovideosurgery is firmly established in clinical practice of coloproctology. objective: the choice of optimal surgical method for treatment of colostasis, achievement of favorable outcomes of treatment. introduction: laparoscopic roux-en-y gastric bypass (rygb) is one of the most important bariatric surgical procedures performed worldwide and it can produce an important loss of weight with reversal of metabolic disorders like diabetes and dyslipidemia. even though it has good results, some complications occur after gastric bypass. a rare but serious complication of rygb is the so-called postprandial hyperinsulinemic hypoglycemia. its prevalence has been estimated less than % of cases and its pathophysiology remains unclear. methods: the aim is to present a case series of reversal surgery in patients with severe hiperinsulinemic hypoglycemia after rygbp in the hospital general universitari de la vall d'hebron. unit of endocrine-metabolic and bariatric surgery (eac-bs center of excellence for bariatric and metabolic surgery by ifso). it is a retrospective analysis of a prospective database same surgical team. we present in this study, the main features of those patients. results: between and , patients underwent a laparoscopic reversal procedure to normal anatomy and age mean was year ( years to years). mean preoperative body mass index (bmi) was . kg/m (range - . kg/m ) and were women. all patients presented hypoglycemia symptoms years after and the longest was years after the procedure. the first step of the standard approach was a laparoscopic reversal to normal anatomy with resection of the alimentary rygb limb in cases. a concomitant sleeve-like gastrectomy (sg) was added. four patients presented postoperative complications: gastrogastric anastomosis leak ( ) introduction: laparoscopic sleeve gastrectomy is the most performed bariatric procedure, but complications might interfere with patient's long-term evolution based on its compliance and tolerance, surgical attitude and unpredictable evolution. materials: we present the case of a female obese patient, with type ii diabetes mellitus and blood hypertension, with multiple, sequential bariatric minimally-invasive interventions: sleeve gastrectomy in complicated by postoperative acute gastric dilation and mediogastric stenosis, reoperated for viscerolysis and cholecystectomy, with endoscopic gastric dilations, initially converted to functional one anastomosis gastric bypass ( cm limb), with a non-adjustable gastric ring positioned instead of stapled division. the last operation was complicated months after by persistent biliary gastro-esophageal reflux, chronic abdominal pain, and gas bloat syndrome. in the patient underwent conversion to laparoscopic r-en-y gastric bypass, with gastro-enteral anastomosis resection, band removal and viscerolysis. results: conversion to r-en-y was complicated by biliary leakage post-viscerolysis, treated with laparoscopic approach in the \ sup [ th \/sup [ po day. after multiple surgical and endoscopic interventions, the patient presents short-term favorable outcomes, with no reflux or abdominal pain, with further weight loss and diabetes improvement. conclusion: bariatric surgery has unpredictable evolution in same cases, and conversion to r-en-y seems to be the best solution. lgcp is widely used in developing countries due to its lower cost and good results. material and methods: we performed in our department lgcp for morbid obesity. excess weight loss (%ewl) was % at month after surgery and % at one year. in cases revision surgery was needed for different complications and in cases for inadequate weight loss or weight regain after month follow up. in cases we performed sleeve gastrectomy (in cases after taking down the plication) and in cases we performed a re-plication in one row. results: the rate of revision surgery was % overall and % for inadequate weight loss (excess weight loss \ %) or weight regain. major complications occurred only in one patient (leak with abscess) but it was solved by laparoscopy. minor complications as vomiting and nausea appeared in patients ( %) and were solved with medication. after one year follow up %ewl in these cases was %. conclusions: revision surgery after lgcp is possible. a new plication or sg was the option in our series with good results. further studies are needed to evaluate the use of lgcp in the armamentarium of bariatric surgery. background: roux-en-y gastric bypass (rygb) is one of the most commonly performed bariatric procedures around the world.however, rygb it sometimes carries the risk of rarebut serious long-term complications such as malnutrition and liver failure. we report a case of laparoscopic reversal of rygb. methods: in march , a laparoscopic rygb was performed for a -year-old female without comorbidities and with a bmi of kg/m . all laboratory test results at the preoperative evaluation were within the normal range. abdominal ultrasound revealed moderate hepatic steatosis and oral endoscopy a hiatal hernia with grade b esophagitis. one year later, patient experienced an important weight loss of kg (from to kg) with a bmi of kg/m . however, patient presented general weakness, abdominal pain, ascitis lower extremitiy edema, anemia, progressive caloric and protein malnutrition, vitamin (a, d), mineral (copper) and folic acid deficiencies, nonalcoholic steatohepatitis (nash) and liver function was progressive worsening. results: a laparoscopic reversal of gastric bypass was performed. the operation was successfully performed via laparoscopy. operating time was min. postoperative was uneventful and patient discharge home at day . hepatic biopsy revealed nash with steatohepatitis of % (fibrosis f - / ). eight months after reversal of gastric bypass, patient has improved her clinical situation (no asthenia), maintains of weight ( kg) and has improved her nutritional status and liver function parameters. conclusion: laparoscopic reversal of rygb is technically feasible and might be performed safely after thorough preoperative evaluation in carefully selected patients with malnutrition and liver failure. conclusion: laparoscopic sleeve gastrectomy it's a safety obesity procedure before major abdominal hernia repair. it's a minimally invasively technique with an absence of anastomoses. these factors prevent fewer complications, without using the small bowel, and skin problems and allow resolution of obesity-associated co-morbidities. body weight loss after surgery may be an opportunity to repair the severe loss of domain incisional hernia. bibliography borbély, y., zerkowski, j., altmeier, j., eschenburg, a., kröll, d. and nett, p. general surgery, benhazi medical center, benghazi, libia, general surgery, royal bahrain hospital, manama, bahrain obesity is a worldwide epidemic with an increasing incidence trends and as a consequence obesity related health problems become priority to healthcare authorities in all the countries. laparoscopic gastric plication is an emergent restrictive procedure which claimed to be low cost because they do not need staplers and carries less complications as compared to laparoscopic sleeve gastrectomy. we present here a years female who was operated for morbid obesity four months back where she underwent laparoscopic gastric plication with no immediate post operative complication and her wight loss was adequate. two days before presentation to our emergency department she started to complains of sever attacks or upper abdominal pain and vomiting.clinical examination was unremarkable apart of abdominal tenderness in left upper abdomen. all blood routine were normal and all inflammatory markers were within normal range.ct abdomen showed large cystic lesion around the greater gastric curvature containing fluid and raised possibility of collection. patient was admitted to hospital, in despite of medical treatment her pain persists and necessitate immediate laparoscopic exploration. gastro-gastric hernia at the greater curvature through loosen ethibond suture that was used to plicate the stomach in the previous surgery. we released the suture to liberate the strangulated stomach which is not gangrenous. re-plication was not possible because of the extensive gastric wall edema and as preoperative discussion with the patient she refused conversion to sleeve gastrectomy no intervention was done. post surgery patient was free of symptoms and tolerating oral diet and discharged home on third post operative day with no complications. gastro-gastric herniation could progress to gastric wall gangrene which will result in high morbidity and even mortality. high index of suspicion is required to diagnose the condition . preoperative patient counseling is important to explore the surgical options if deemed necessary to convert to another bariatric procedure. k. chouillard, a. d'alessandro, l. chahine background: bariatric surgery is the best available, long-term treatment for morbid obesity. currently, laparoscopic sleeve gastrectomy (sg) is the most commonly performed bariatric procedure in france. despite its safety and efficacy, long-term complications of sg are not rare including gastro-esophageal reflux disease (gerfd), twisting, stenosis, insufficient weight loss, and weight regain. the goal of this study was to analyze the pattern and short-term results of surgical revision in patients with sg. methods: revisional bariatric surgery, regardless of its motivation, was always a multidisciplinary decision after clinical, biological, endoscopic, and radiological assessment. patients who had revisional surgery after sg were retrospectively identified and subsequently divided in subgroups according to preoperative body mass index ( we aim to present the management and the particular aspects of the surgical technique in a gastrobronchial fistula after gastric sleeve . the mean time between intervention and diagnosis is . - . months. methods: between and , laparoscopic gastric sleeve resections were performed in our bariatric center. we had one case of gastrobronchial fistula associated with an inferior lobe abscess of the left lung, diagnosed months after the gastric sleeve. the patient was subject for medical treatment for h, than a laparoscopic intervention was performed in order to drain the lung abscess and the gastric fistula and to place a feeding jejunostomy. . months after this intervention ( . months after gastric sleeve) a laparoscopic roux-en-y fistulojejunostomy was performed. the evolution was monitorized with blood tests, upper gi contrast series and ct scans. results: the surgical drainage of the lung abscess, along with the antibiotherapy, controlled the infection and allowed the lung cavity to reduce in size, and thus the drainage tubes introduced in the thorax through the diaphragmatic orifice were retracted progressively. also, the feeding jejunostomy allowed a proper nutrition for the patient with a good recovery. however, . months after the drainage intervention, the gastric fistula was not healed, and a decision to interrupt the communication with the lung cavity was made, by creating a laparoscopic fistulojejunostomy. after this, the evolution was favorable, with the healing of the lung cavity, oral feeding was permitted and the jejunostomy was suppressed. conclusions: the treatment of the gastrobronchial fistula is complex (medical, endoscopic or surgical), phased and long lasting until healing. surgery was our initial choice for treatment due to the existence of the lung abscess, which needed to be drained. key words: gastrobronchial fistula, lung abscess, laparoscopy, fistulojejunostomy s.i. filip, i. hutopila, c. copaescu introduction: leakage remains one of the most dreadful complications in metabolic surgery. the main cause of leakage is poor tissue oxygenation due to inadequate vascular perfusion. the study of intraoperative tissue perfusion in real time due to icg enhanced fluorescence could provide valuable information for the surgeon in order to prevent postoperative fistula. aim: to present our experience in using icg enhanced fluorescence in laparoscopic bariatric surgery material and method: in cases of gastric sleeve, cases of gastric bypass and in cases of revisional surgery or redo cases we used intraoperative icg mediated fluorescence to assure the optimal vascularization of the involved tissues. in our video we present intraoperative aspects before and after using icg in different cases. results: in all cases of primary gastric sleeve and gastric bypass with intraoperative use of icg we did not encounter inadequate perfusion. in one case of redo gastric bypass after failed vertical banded gastroplasty for morbid obesity despite intraoperative laparoscopic normal aspect of the gastro-jejunal anastomosis, icg mediated fluorescence allowed to identify an unexpected ischemic anastomosis and we could prevent consecutive postoperative leakage. discussion: presented cases are discussed and result with referral to literature is made. conclusion: intraoperative use of icg is a valuable tool in assessing the perfusion of the tissues and provide essential information for the surgeon in order to avoid postoperative leakage. , including patients, hemostasis with clips has been performed in all cases. however, among these cases nine patients required reoperation for early postoperative bleeding. in five cases a bleeding source from the stapled line was identified while in cases no identifiable source was found. during the second period ( to present) patients were submitted to bariatric surgery and hemostasis was performed by over sewing with a running suture. among these cases reoperation for postoperative bleeding was needed in cases ( . %), but no bleeding from the staple line being encountered ( %). the difference has statistical significance. no significant complications related to the use of this type of reinforcement were encountered. conclusions: over sewing the gastric stapled line in bariatric surgery is superior to hemostatic clip application in preventing the postoperative bleeding from the stapled line postoperative bleeding. a protocol of active search of the bleeders during the bariatric procedure should be implemented and respected in all the cases. gastroenterological surgery, saitama medical university international medical center, hidaka-shi, saitama, japan intestinal endometriosis is a rare disease which is associated with about to % of patients with endometriosis, and it is favorable to the rectum and sigmoid colon. here we report cases (shown in the table) underwent laparoscopic resection for intestinal endometriosis. there were no postoperative complications in all cases, and all patient was discharged on - \ sup [ th \/sup [ postoperative day. before the operation, of patients were diagnosing intestinal endometriosis, and it was difficult to preoperatively diagnose. among them, the symptoms at the time of menstruation were clear was one case. in case of submucosal tumor, preoperative diagnosis seems difficult. additional image examination at menstruation may be useful for diagnosis. d dissection was performed for case , , because malignant disease could not be denied as a preoperative diagnosis. of them were strongly doubted endometriosis in surgical findings. in intestinal endometriosis surgery, pelvic adhesions and fibrosis are often advanced. in the sigmoidectomy, the average operation time was min and the blood loss was ml. in the rectal resection, the average operation time was min and the blood loss was ml. in case and , pelvic adhesion was severe, residual rectum could not be straightened, and side to side anastomosis was performed. in intestinal endometriosis surgery, intestinal anastomosis method should be considered flexibly. conclusion: laparoscopic surgery for intestinal endometriosis was safe, but technically difficult because of fibrosis and adhesion. it is important to accurately diagnose from clinical symptoms and image also intraoperative findings. anastomotic method should be decided according to the case. aim: the aim of the study was to identify and highlight some of the complications one can encounter in bariatric surgery-specific-sleeve gastrectomy and discuss the therapeutic options one has at his disposal. methods: the study was retrospective. we identified a number of patients which had a sleeve gastrectomy done in our clinic for a year period. of these had important surgical complications encountered during the surgery or in postoperative care. results: the group included patients, with an average bmi of [ kg / m . average hospital stay was days, with an average of . days which increased to days when fistulas were encountered. the most frequent surgical complications were bleeding from the gastric suture ( ) and gastric fistula ( cases). other complications encountered were wound hematoma. surgery was required in of the cases of bleeding and of the fistula cases required reintervention. one case was resolved with endoscopic stenting. conclusions: laparoscopic gastrectomy is considered a safe procedure with good results for the patient. although complications are rare they pose a series of technical difficulties for the surgeon due to the weight of the patient and frequent comorbidities which come with obesity. a thorough understanding of the symptoms and good follow-up ensures the best results. aims: to achieve additional weight loss or to resolve band-related problems, a laparoscopic adjustable gastric banding (lagb) can be converted to a laparoscopic roux-en-y gastric bypass (rygb). there is limited data on the feasibility and safety of routinely performing a single-step conversion. we assessed the efficacy of this revisional approach in a large cohort of patients operated in a high-volume bariatric institution. to the best of our knowledge this series represents the largest single-center study on conversion from lagb to rygb methods: between october and december , a total of patients who underwent lagb removal with rygb were identified from a prospectively collected database. in all cases, a single-stage conversion procedure was planned. the feasibility of this approach and peri-operative outcomes of these patients were evaluated and analyzed. results: a single-step approach was successfully achieved in ( . %) of the patients. during the study period, there was a significant increase in performing the conversion from lagb to rygb single-staged. no mortality or anastomotic leakage was observed in both groups. only patients ( . %) had a -d complication: most commonly hemorrhage (n? = ? / ), with no significant difference between the groups. conclusion: converting a lagb to rygb can be performed with a very low morbidity and zero-mortality in a high-volume revisional bariatric center. with increasing experience and full standardization of the conversion, the vast majority of operations can be performed as a single-stage procedure. only a migrated band remains a formal contraindication for a one-step approach. surg endosc ( ) . six months after surgery the mean hrql score, was ( - ) in patients underwent to lsg and . ( - ) in patients underwent lgb. twelve months after surgery the mean postoperative questionnaire score was ( - ) in patients who underwent lsg. at ph-manometry the mean percentage time of acid reflux in orthostatism was . (range - . ) and in clinostatism . (range . - . ). the mean demeester score at the distal electrode was . ( . - . ). conclusions: in asymptomatic patients, complete gerd evaluation before bariatric surgery allows better selection of surgical procedure, to reduce the postoperative occurrence of severe or de novo gerd. postoperative gerd evaluation provides useful data regarding the impact of lsg on gastroesophageal reflux. a larger patient sample size is required. aims: vertical calibrated gastrectomy (usually know as gastric sleeve) as unique technique gives better results than the roux y bypass in terms of improvement of anthropometric measures, reduces comorbidities and has a lower rate of postsurgical complications, with an improvement of quality of life. material and methods: an observational, longitudinal, retrospective and comparative study with patients, aged - years,during a period of years. everyone must comply with the protocol of the unit. demographics of the population and the anthropometric data will be measured in the presurgical consultation, the month and the year after the surgery: weight, height, bmi, weight loss percentage,bmi percentage and percentage of excess weight lost. we took data on the cardiovascular risk by the framingham score. the quality of life is measured by baros scale. mayor comorbidities are hypertension, diabetes, dyslipidemia. complications will be measured in absolute frequencies. for de statistical study, we apply type t student or chi square being statistically significant p equal to or less than . . results: there was not statistically significant difference between the techniques of surgery month (p = , ), but they were evident to the year of the same (p . ). not gender or age differences were apparent. mayor complications did not appear in gastrectomy (no leaks), highlighting the number of bleeds with this surgical technique. the bypass there were two leaks. there was no statistically significant difference in cardiovascular risk (p = , ) between the two techniques. there was a more significant decrease in number of comorbidities in gastrectomy against the bypass, with a total disappearance of patients with dyslipidemia. there were no statistically significant differences in baros score, although it was higher in gastrectomy. conclusions:-the vertical gastrectomy as unique technique can be considered superior in the short term, as well as safe, according to the aec quality parameter. we think it will be necessary to continue their studies into the medium-long term. aims: analyze the impact of different bariatric surgeries technics in carbohydrate metabolism and pancreatic beta cell population of none obese adult wistar rats. methods: we used twenty healthy not obese adult wistar rats divided in five groups randomly assigned. each with n = . the control groups were divided into fasting control (f) and sham (surgical control). the surgical groups were separated into vertical gastrectomy (gs), % resection of the middle small bowel (ri ) and gastric bypass (gb). in each group was assessment: beta cell mass modifications, pancreatic islets histomorphometry, proliferation, apoptosis and neogenesis in beta-cell pancreatic population; intraperitoneal glucose test tolerance, body weight and food intake. statistical analysis as evaluated using mann whitney test. results: the malabsorptive and restrictive group have a significantly smaller increase weight than the control groups. the intraperitoneal tolerance glucose test reports incremental glucose area under curve (auc) was significantly higher in the malabsorptive group and lower in the restrictive group compare to the control groups during the second (p \ . ) and third (p \ . ) month of the study. the beta-cell mass was significantly higher in the ri group compared with control groups respectively. there was a significantly increased number of beta-cell per pancreatic insulin positive area in gs and gb. proliferation was significantly increased in ri and gb group, and significantly decreased in sg compared. there was no significantly difference during apoptosis assessment among surgical and control groups. in neogenesis differences between groups were assessed qualitatively by the presence pdx - expression, being higher in rygb. the endocrine pancreas in our model is altered by the anatomical and functional conditions arising from surgical techniques. carbohydrate metabolism conditions are affected by temporary adaptive processes due to surgical alternatives. there is a hyperplasia and hypertrophy of the beta cells in surgeries with a malabsorptive component, as well as greater neogenesis. these results could explain part of the existing relationship between the enteropancreatic axis and the existing incretins. m. buza, c. copaescu introduction: nowadays, we have high volumes of obese patients for whom surgery is the answer, but unfortunately the psychological evaluation has no standard recomandation in preoperative evaluation of bariatric patients. it is argued that surgery success, in addition to the operation itself, relies on behavioral changes and that one of the goals of the preoperative assessment is to prepare the patient for the postoperative period, aiming to optimize surgical results. aim: although no formal standard exists in the literature, there is growing recognition of the important elements to be addressed and the appropriate means for collecting the necessary data to determine psychological readiness for these procedures. methods: information regarding the components of the clinical interview and the specific measures used for psychological testing are discussed. given the limited data on predicting success after surgery, determining psychological contraindications for surgery is addressed. additionally, the multiple functions served by the psychologist during this assessment procedure are highlighted along with the value of this procedure in the patients' preparation for surgery as well as the postoperative follow-up. in our center of excelence for bariatric and metabolic surgery (coe) we introduced since a mandatory pre-and postoperative psychological evaluation for all patients addressing the metabolic program. results: psychological evaluation of patients before bariatric surgery is a critical step, not only to identify contraindications for surgery, but also-and more so-to better understand their motivation, readiness, behavioral challenges, and emotional factors that may impact their coping and adjustment through surgery and the associated lifestyle changes. postoperative follow-up is necessary. the psychological evaluation of the patient undergoing bariatric surgery is an invaluable piece of the larger pre-and post-surgical assessment, aiming better results in the short and long term after bariatric surgery. introduction: a mesenteric cyst is defined as a benign abdominal tumors that is located in the mesentery of the gastrointestinal tract, identified in * of , hospital admissions. mesenteric chylous cysts are rare pathologic entities that often present with unspecific symptoms. the preoperative diagnosis requires all the common abdominal imaging techniques. usually the correct diagnosis may be made only at the operation stage or during the histological examination. all mesenteric cyst should be resected in order to avoid their complications, complete surgical resection is recommended and curative in the majority of cases with a low risk of local recurrence. the laparoscopic approach is the gold standard in the treatment of intraabdominal mesenteric chylous cyst. laparoscopic resection provides less pain, shorter hospital stay, and early recovery for the patient. case report: we report a case of -year-old saudi woman who presented to our clinic complaining of upper abdominal pain and mass in the epigastrium for one week, no history of nausea, vomiting, or recent changes in bowel habits. her medical and family histories were clear and she had never had any abdominal interventions. abdominal palpation revealed a smooth-surfaced mass palpable in the left upper quadrant, ultrasonography and with computed tomography of the abdomen revealed an approximately mm unilocular cyst closely related to the mesentery in the left side of upper abdomen not related to the pancreas .the cyst was excised by laparoscopy complete surgical excision to avoid recurrence within healthy borders, it is contained milky white fluid. the histopathological findings were chronic inflamed mesenteric cyst. a review of the literature considering this rare entity was also performed to evaluate our treatment strategy. conclusion: mesenteric chylous cysts represent a diagnostic challenge and they should be considered when a physician encounters an intraabdominal mass. usually the correct diagnosis may be made only at the operation stage or during the histological examination. the treatment of choice is the complete surgical excision that can be safely performed by laparoscopy. surg endosc ( ) background: diverticulum of appendix is relatively rare, and appendiceal diverticulitis was reported to have a higher risk of perforation than appendicitis. in the us and europe, because of the high risk of perforation, preventive appendectomy is recommended to appendiceal diverticulosis, even if the patient has no abdominal pain. methods: we retrospectively reviewed the records of post-operative patients, who were diagnosed appendicitis or appendiceal diverticulitis on the pathological findings in our institution from january to october . all patients were performed computed tomography (ct) before operation. patients underwent laparoscopic surgery, including appendectomy, cecal resection, ileocecal resection and right hemicolectomy, while patients underwent open surgery. total of cases of appendiceal diverticulitis were analyzed in our study. result: patients had abdominal pain before surgery. patients were diagnosed appendiceal diverticulitis by preoperative ct. all patients underwent laparoscopic surgery ( appendectomy, cecal resection, and ileocecal resection). on the pathological findings, perforation of appendix was found in patients and the pseudo type of diverticula with no muscle layer was found in all patients. patients with appendicitis were treated surgically during the same period. among them, a perforation of appendix was found in cases. the perforation rate was . %. on the other hand, the perforation rate of appendiceal diverticulitis was . % in our study. conclusion: the perforation rate of appendiceal diverticulitis was higher than of appendicitis in our study. for the examination of the treatment strategy, including preventive appendectomy, the accumulation of more cases will be expected. case presentation: a -year-old man was referred to our hospital with right lower quadrant abdominal pain for days. his fever was . °c. his white blood cell count was , / ll, and c-reactive protein level was . mg/dl. ct revealed multiple diverticula of cecum and appendix. micro-abscess and free air were found around appendix. we diagnosed this case as appendiceal diverticulitis and laparoscopic appendectomy was performed. a perforation was found in resected appendix. microscopic study revealed a pseudo-diverticulum. the inflammation of appendix was stronger in serous membrane side than in mucosa side. this finding accorded with appendiceal diverticulitis. introduction: in order to reduce the abdominal trauma and the length of scar incisions (also during laparoscopic surgery) many approaches during the last decade has been proposed, such as single access laparoscopic surgery (sals). the aim of our paper was to update the data of our previous paper with a greater cohort of patients and a longer follow-up, also showing the single access laparoscopic left colectomy (salc) technique in particular with inferior mesenteric artery preservation imap (valdoni's technique). materials and methods: we made a retrospective analysis from october and october of all patients who underwent a sals approach for colorectal disease in the department of general and mininvasive surgery of san camillo hospital of trento. statistical analysis was performed using ibm spss statistics . continuous data were expressed as mean ± standard deviation (sd). categorical data were expressed as absolute number and percentage. the results are presented as -tailed values with statistical significance if p values \ . results: from october until october , salc for colorectal surgery were performed in our unit. of this , were for left colectomy. in cases we performed an imap. the salc with imap were performed only in case of benign disease. the mean operative time was . ± . . only one intraoperative complication were recorded, that was a splenic capsule tear, resolved with apposition of fibrillar haemostats. according to clavien dindo classification there were in particular grade ii complications, a bleeding solved with blood transfusion and one pancreatitis solved with medical therapy; grade iiia complications that was anastomotic bleeding solved endoscopically (the two complications raised in patients with imap) and iiib complications due to anastomotic leakage which needed reoperation. the mean length of incision was . ± . cm. logistic regression did not show any correlation between imap and any complications. conclusion: in conclusion, salc is a safe but very challenging technique which need a longer learning curve than the conventional laparoscopic one. in laparoscopic colectomy, also, imap seems to be safe and effective without correlation with post-operative complications also if performed in single access laparoscopic approach. aims: to describe an infrequent anatomical variation that can give rise to diagnostic and therapeutic difficulties. methods: patient with ivermark syndrome (situs ambiguus and polysplenia) with acute appendicitis and bibliographic review results: a -year-old male who consulted for flank and right hypochondrium pain of h of evolution, associated with nausea without vomiting, no fever noir other symptoms. to the physical examination good general condition. painful to palpation selectively on the flank and right hypochondrium, with involuntary defense and positive decompression at this level. the signs of rovsing and psoas were negatives. in the analytical performed leukocytes of , with neutrophils in % and rpc (reactive protein c) in mg/l. abdominal ct (computed tomograph): cecum and the ilio-cecal valve were visualized at the subhepatic level with tubular structure on the side and seemed to correspond to the cecal appendix which is increased in size ( mm), with findings suggestive of acute appendicitis. sigma and descending colon located in right hemiabdomen. second per duodenal portion located anterior to the superior mesenteric artery. superior mesenteric vein located to the left of the superior mesenteric artery, rotating around it, (radiological signs compatible with intestinal malrotation). no free fluid collections nor pneumoperitoneum. laparoscopic appendectomy on phlegmonous acute appendicitis without incidents. correct post-operative course, being discharged at h; the pathological anatomy was reported as acute appendicitis in phlegmonous. conclusions: ivermark syndrome is a genetic alteration with a multifactorial inheritance pattern, characterized by an alteration in the situation of the mesenteric vessels, which leads to abnormal rotation of the intestine during the embryonic period and alteration of the situation of different intra-abdominal organs, without a specific pattern that is pathognomonic, is associated with congenital heart anomalies between and %. reaching adulthood only between and % of them. a case of acute appendicitis is presented in a patient with this anomaly, which can lead to diagnostic and therapeutic difficulties due to the anatomical variations involved. abdominal tomography is the image method that provides the best performance for the diagnosis of acute pathologies in this type of patients. background: the clinical manifestations which occur in relation to decompression during scuba diving are variable. mild symptoms have often been reported in gastrointestinal tract. this is one of the severe cases with gastrointestinal barotrauma. ischemic colitis caused by air embolism very rare, therefore it is to be reported and discussed. case presentation: a -year-old man visited our emergency room with diffuse abdominal pain and bloody diarrhea days ago. the patient was a skilled diver who took seafood through diving for years. two days before presenting, the patient had severe abdominal pain just after diving for h at a depth of meters. he was immediately transferred to a local hospital for hyperbaric oxygen therapy, but there was no improvement with the symptom. abdomen ct angiography showed terminal ileal, ascending, sigmoid colonic and rectal decreased enhancement with wall thickening. sigmoidoscopy showed diffuse huge ulcerative lesions and ischemic changes on mid rectum and sigmoid colon. emergent subtotal colectomy and temporary loop ileostomy were done, and pathologic findings revealed diffuse mural infarct with serosal abscess formation in whole colon and transmural infarct in terminal ileum. conclusion: surgical approach could be one of the treatment options, though it depends on severity of the symptoms and the patients' conditions. colonic lipomas are extremely uncommon benign tumours, with an incidence ranging between . % and . %. although they are most frequently asymptomatic, when colonic lipomas are [ ?cm, they may present symptoms such as constipation, abdominal pain or rectal bleeding. most colonic lipomas typically occur in middle aged women and are located in the ascending colon and the caecum, while occurrence in other parts of the colon and rectum is rare. in this case report, we describe a lipoma that caused descendent bowel intussusception. a -year-old male presented with longstanding history of constipation. personal history of interest included active smoker, hypertension, hypercholesterolemia, psoriasis with joint affectation and reiter syndrome. he had had no previous surgery. he attended the emergency services on th july with a two-day bowel obstruction, without fever or nausea, being attended by our surgical emergency unit. he had been assessed during the previous months by gastroenterology, with a colonoscopy that showed a cm submucosal lesion that partially occluded descendent bowel, with inconclusive biopsy. an abdominal contrast-enhanced computed tomography (ct) was performed, confirming a welldefined mass located in splenic flexure of descendent bowel, conditioning a large bowel intussusception, nevertheless with no obstructive acute signs. the surgery was scheduled a few weeks later, performing a laparoscopic segmental resection with primary anastomosis including oncologic margins. the patient evolved satisfactorily in the postoperative period and was discharged six days after the surgery without any complications. likewise, he was monitored on a regular basis at our outpatient department and was free of symptoms at the -month follow-up visit. the histological analysis revealed a cm ulcerated lipoma affecting % of bowel circumference. the molecular study, using fluorescent in situ hybridation (fish) showed no mdm gene amplification. laparoscopic segmental resection of the large bowel is a safe and feasible technique for the treatment of large bowel intussusception caused by a colonic lipoma. the complete removal of the lipoma will condition the prognosis. furthermore, in the future, endoscopic surgery using colonoscopy could be employed when having a certain preoperative diagnosis of lipoma. surg endosc ( ) introduction: acute appendicitis is one of the most common abdominal surgical emergency, the diagnosis of which mostly relies on conventional methods such as physical examination and blood tests. the use of ultrasonography and ct abdomen aids in more precise diagnosis especially in patients with atypical presentation or in elderly. aim: this study aims to evaluate the ability of the neutrophil/lymphocyte ratio (nlr), platelet/lymphocyte ratio (plr) and mean platelet volume (mpv) in predicting the diagnosis of acute appendicitis. methods: retrospective analysis of prospectivly maintained data of all patients ( ) admitted with acute appendicitis to the emergency department at a tertiary hospital in the middle east between january till september . medical records and database of patients,who had appendicectomy for clinically and radiologically proven appendicitis, were reviewed. the retrieved data included patient's demographic and laboratory values of white blood cells (wbc), neutrophil (n), lymphocyte (l), and platelet (p) along with their ratios for comparison. results: spss version was used for tabulating the data. the recommended cutoff value of the nlr, plr and mpv in predicting the diagnosis of acute appendicitis was decided by using receiver operating characteristic (roc) curve analyses. at least for nlr, the confidence interval (ci) was . which is percentage of the positive values, since the confidence limit was between to %. our results showed that the laboratory parameters were fairly significant since the confidence interval was . in predicting the diagnosis in our population. conclusion: although appendicitis is a clinical diagnosis but laboratory parameters specially nlr, plr and mpv can be used as an adjunct in the diagnosis of acute appendicitis. literature is scarce concerning the validity of such parameters in our part of the world and prospective randomized controlled trials are needed to prove the efficacy of such rationale. objective: tumors of the cecal appendix represent a subset of colonic neoplasms whose early diagnosis is a real clinical challenge. correspond to . % of all gastrointestinal tumors and their prognosis depends on the type of injury, being the most frequent variety the carcinoid type. appendix involvement in endometriosis is rare, accounting for % of all endometriosis cases, and sometimes mimicking cecal tumors. methods: a -year-old woman with a history of hypothyroidism due to autoimmune thyroiditis and atrophic gastritis with gastric neuroendocrine tumors resected by endoscopy that in the digestive unit reviews, tac with double contrast was requested, showing a lobulated lesion in the cecum adjacent to the ileocecal valve, with contrast enhancement of approximately mm, suggestive of tumor. the colonoscopy evidenced a protruding appendicular osteum with inflammatory aspect that was biopsied. the pathological anatomy of the biopsy reports chronic congestive colitis with edema of the own blade and minimal acute activity, with moderate local eosinophilia.the case was presented in the multidisciplinary oncology committee and it is decided, due to the patient's background, to perform surgery on the lesion. laparoscopic right hemicolectomy was performed, with extracorporeal latero-lateral mechanical anastomosis with endogia signiaÒ mm. results: the patient evolves favorably, with good oral tolerance and depositional habit. she is sent home at the sixth postoperative day. the pathological anatomy reports tumor injury in the appendicular ostium compatible with endometriosis at the base of the cecal appendix implantation, ruling out malignant tumor pathology. conclusions: gastrointestinal tract endometriosis represents - % of cases, being most frequently located in the rectal-sigmoid region. appendix involvement in endometriosis is rare, accounting - % of all endometriosis cases and presents a preoperative diagnostic challenge, because sometimes mimicking a carcinoid cecal tumor. in our case, due to the patient's history, we assumed that the cecal lesion was a carcinoid tumor, so we performed a laparoscopic right colectomy, but if we had known that it was an endometriosis, we could have performed an appendectomy, although in both cases the laparoscopic approach gives us some benefits compared to the open approach aims: the natural history and predictive factors associated with chronic anastomotic complications have not been clearly studied. the aim of this study was to evaluated the predictive factors related to chronic anastomotic complications methods: from january to december , a total of patients who underwent anastomotic leakage were enrolled in this study. all patients underwent anterior resection with or without defunctioning stoma due to colorectal cancer. the patients received follow-up by clinical examination and abdominopelvic computed tomography (ct). they underwent a follow-up ct every months for the first year and then every months for the next years after that. complicated group (cg) underwent chronic anastomotic complications. normal group (ng) didn't underwent chronic anastomotic complications like stricture, fistula, chronic sinus, etc. results: there were no significant differences in gender, age, preoperative chemoradiotherapy and operation type between two groups. low rectum lesion and defunctioning stoma at the time of primary surgery were more frequent in cg (p = . , . ). there were no significant differences in type of anastomotic leakage, international leakage grade and ct findings at the time of diagnosis of anastomotic leakage. however, abnormal ct findings at the time of month were more frequent in cg group (p \ . ). in multivariate analysis, abnormal ct finding at the th months was only significant factor related to chronic anastomotic complications. conclusions: abnormal ct findings at the th month associated with prediction of chronic anastomotic complications. aims: acute appendicitis is the most common cause of acute abdomen requiring surgical intervention in the world. nowadays, standard treatment of acute appendicitis involves a surgical approach, eitherlaparoscopic or open.the purpose of the present study is to evaluate the safety of a discharge within less than h after performing appendectomy as a result of an uncomplicated acute appendicitis. conclusions: patients who undergo appendectomy (open or laparoscopic) for acute uncomplicated appendicitis, without surgical incidents and an adequate social/family network, can be discharged in less than h without a higher risk of post-operative complications or readmissions than patients with longer postoperative stays. it will be necessary to conduct more prospective studies with higher level of evidence that could corroborate our results. aims: median arcuate ligament syndrome (mals), also known as the celiac axis compression syndrome, is a rare condition caused by to the compression of the celiac trunk and the nerves located in this area (celiac plexus) by the median arcuate ligament. it is believed that mals is caused by the median arcuate ligament compression of the celiac plexus nerves over the celiac trunk, but another probably cause may be the lack of blood flow to the organs supplied by the celiac artery, however, this theory is controversial. the first clinical sign of mals is the apparition of postprandial abdominal pain in the upper abdomen. this typical pain forces patients to avoid eating, which can lead to loss weight (often more than pounds). other associated symptoms may include nausea, diarrhea, vomiting and delayed gastric emptying (a delay in food moving from the stomach into the small intestine). in relation to this uncommon condition, we present a clinical case of laparoscopic management of mals. methods: we present a -year-old patient with complaints of recurrent epigastric pain, postprandial vomiting and loss weight. blood tests and gastroscopy were performed to help ruling out more common causes of his symptoms, such as gastroesophageal reflux disease (gerd), gastritis or gastroparesis. as a part of the differential diagnosis, mals was suspected and a mesenteric doppler ultrasound was ordered to check blood flow through the celiac trunk and evaluate a possible compression of the celiac plexus. also, an angio-ct scan was also performed to confirm the diagnosis. once the mals was diagnosed, we decided to perform a laparoscopic approach as definitive surgical procedure. results: the patient was discharged h after surgery with no remarkable events during his postoperative stay. he has been followed up during months, remaining asymptomatic. conclusions: laparoscopic approach in mals offers a superior visualization during the surgery and involves lower morbidity in compare to open approach, which makes it an optimal treatment for this condition. aim: pilonidal sinus is a common disease with annoying and often painful symptoms. traditional surgical techniques for its treatment are characterized by either intense postoperative pain and prolonged wound-healing periods (wide resection, marsupialization) or unsatisfying aesthetic results (advancement or rhomboid flaps). 'endoscopic pilonidal sinus treatment' (epsit) is a new minimally invasive technique which utilises the meinero scope, primarily designed for the endoscopic treatment of complex perianal fistulas in a technique known as vaaft. we present our experience and outcomes in three treatment centers in northern greece. methods: between july and november we treated patients with pilonidal sinus using the epsit technique. the mean age of patients was , and % of them were male. patients were treated in the acute phase with the presence of pilonidal abscess. all operations were performed by two laparoendoscopic surgeons specifically trained in the technique. most patients were treated on a day-case basis. postoperative wound care included daily tract irrigation with ml of saline for a total of days. results: there were no immediate postoperative complications. medium postoperative pain was . on a vas scale. % of patients were discharged on the same day, patients remained in hospital for one day mainly due to social reasons. return to daily activities was immediate. in a maximum follow-up of months we observed recurrences. conclusions: epsit is a promising minimally invasive technique for the treatment of pilonidal sinus. what makes it mostly attractive is the minimal amount of postoperative pain, the excellent cosmetic result and the fast recovery with return to daily activities. introduction: isolated acute chylous peritonitis is a rare event. when presented as an acute abdomen warranting surgical intervention, it is often difficult to determine the cause pre-operatively. here, we report a case of acute chylous peritonitis due to meckel's diverticulitis presented with the clinical features suggestive of acute appendicitis. presentation of the case: a -year-old female presented with abdominal pain and clinical features consistent with acute appendicitis underwent diagnostic laparoscopy. she was found to have four-quadrant chylous peritonitis and ileus caused by an inflamed meckel's diverticulum adhered underneath a loop of small bowel and mesentery leaking chyle. after uneventful postoperative recovery, she was discharged at post-operative day two with oral antibiotics and was advised to take a low-fat diet. aims: perforated diverticulitis with purulent peritonitis (hinchey iii) has traditionally been treated with surgery including colon resection and stoma (hartmann procedure) with considerable postoperative morbidity and mortality. laparoscopic lavage has been suggested as a less invasive surgical treatment. methods: a -year-old woman with a -day history of abdominal discomfort exacerbed during the last h. ct scan showed neumoperitoneum accompanied by free fluid and a cm collection adjacent to descending colon showing diverticula suggestive of covert perforation. after h of non-response to medical treatment, associated with the impossibility of percutaneous drainage through interposition of intestinal loops, colon and lumbar vessels, urgent surgical intervention is decided. results: laparoscopic lavage of all quadrants was performed with saline, l or more, of body temperature, until clear fluid was returned. two non-suction j-pratt drains were placed. intravenous antibiotics were continued for a minimum of h, then oral antibiotics were continued for week. oral fluids were commenced on the first postoperative day and solids were subsequently introduced, depending on clinical progress. conclusion: laparoscopic management is reasonable alternative to the traditional open resection for hinchey grade ii-iii perforated diverticulitis with generalized peritonitis. this approach has a low mortality rate despite patient co-morbidity and disease severity. benefits include stoma avoidance and minimal wound infection. subsequent elective resection is probably unnecessary and readmission in the medium term is uncommon. background: constipation and fecal incontinence are common annoying complications after pull through procedures for hirschsprung disease (hsd). many causes could be the etiology of these problems. perineal descent syndrome could be the major hidden cause of these complications. the aim of this study is to evaluate the role of perineal descent syndrome in the development of post pull through constipation and fecal incontinence in addition to evaluate the role of laparoscopic rectopexy for treatment of these problems. \ b[patient and methods: \/b [ patients treated with pull through for hsd over the period of five years. out of the patients presented with constipation and fecal incontinence. patients with constipation and patients with fecal incontinence. rectal exam, anorectal manomety, defecography, contrast enema, rectal biopsy, emg, proctoscopy and endorectal ultrasound were performed to all patients. patients with stricture, missed aganglionic segment, injured internal anal sphincter, and loss of the sensory mucosa above the dentate line were excluded from the study. anterior wall rectopexy was performed for anterior wall rectocele. posterior wall rectocele was treated by retro rectal mesh rectopexy. emg is repeated weeks and months after surgery. outcome measurements included constipation, fecal incontinence and pudendal nerve latency. results: cases of post pull through constipation and fecal incontinence. patients with constipation and patients with fecal incontinence. patients with stricture, patients with missed aganglionic segment, patients with loss of anal sensory sensation and patients with injured anal sphincter were excluded from the study. defecography showed patients with anterior rectocele ( males and females) and patients with posterior rectocele ( males and females). the patients mean age . ± . years . emg showed prolonged pudendal nerve conduction in all cases. anterior wall and retro rectal rectopexy were performed laparoscopically without complications. constipation was resolved in all patients after surgery. all patients showed fully control in defecation. pudendal nerve latency decreased in all patients. conclusion: perineal descent syndrome proved to be a major hidden cause of post pull-through constipation and fecal incontinence. laparoscopic rectopexy showed a good solution of these complications. cystic lymphangioma is a rare entity. the surgical indication is determined by the size and symptomatology, and consists of the complete exeresis of the tumor. the laparoscopic approach is feasible in these cases, allowing a broad visualization of the anatomy, accessibility to the retroperitoneum in the context of a minimally invasive approach and a better recovery of the patient, without providing an increase in morbidity compared to the conventional. in this way we defend as a technique of choice laparoscopic surgery against these rare tumors for the general surgeon in the abdominal cavity, betting on a minimally invasive surgery. aims: laparoscopicposterior sutured rectopexy is one of the accepted treatment options for fullthickness rectal prolapse. recently, reduced port surgery(rps) has beenan emerging concept that, compared with conventional multiple port surgery (mps), yields reduced postoperative pain and improved cosmesis. the aim of the study is to evaluate the feasibility and safety of rps for fullthickness rectal prolapse. methods: rps was performed by single-incision plus one puncture, using internal organ retractor(ior) to secure operative field. straining one ior by - strings in - directions makes it possible to retract the internal organs three-dimensionally. this multi-directional flexible retraction could secure good operative field. from to , patients (rps: cases, mrs: cases) underwent laparoscopicposterior suture rectopexyfor total rectal prolapse. shortterm outcomes were compared between the two procedures. results: there was no significant difference between rps and mps in median operative time ( vs . min, respectively, p [ . ). the median blood loss volume was not significantly different between rps and mps groups ( . vs . ml, p \ . ). the duration of median hospital stay after surgery was not significantly different between two groups ( . vs days, respectively, p [ . ). the frequency of complications after surgery were not different between them. conclusions: reduced port lap-rectopexy can be a good therapeutic option for total rectal prolapse. a prospective, randomized, controlled trial should be conducted to confirm the superiority of this procedure over mps. the piccolo project proposes a new compact, hybrid and multimodal photonics endoscope based on optical coherence tomography (oct) and multi-photon tomography (mpt) combined with novel red-flag fluorescence technology for in vivo diagnosis and clinical decision support. for its development it includes different phases of validation. within this framework, the present study has as main objective: to characterize a model of rat colonic hyperplasia, which will be used for the development and validation of the previously mentioned endoscopic technology. secondary objectives: procure the reproducibility of the model chosen and determine the optimal time, after induction of the model. material and methods: animals (rattus norvegicus), wistar, males and females \ -yearold, randomly distributed. group (n = ): by laparotomy, a non-resorbable suture (silk / ), not stenosing, is placed through the wall of the colon. group (n = ): by endoscopy, a . mm long segment of a polymeric catheter is inserted, which is fixed to the wall of the colon by means of a suture. group (n = ): by means of endoscopy, a self-expanding and uncoated metallic stent are placed in the colon. group (n = ): a superficial laser resection of the colonic mucosa is performed by endoscopy. group (n = ): as an extension of the most optimal model. weekly, the animals were anesthetized again to perform a colonoscopy, which determined the degree of mucosal growth in descending colon and colonic biopsies were extracted weekly ( weeks). results: group . growth around the sutures after the second follow-up, diagnosed as hyperplastic polyps after a histopathological analysis. aim: the role of laparoscopy in the management of generalized appendicular peritonitis is controversial. this is due mainly to the lack of scientific data. through this study and a laborious bibliography research, we proposed to report our experience in terms of postoperative results, in the laparoscopic treatment of generalized appendicular peritonitis and to try to identify the risk factors associated with the occurrence of global morbidity and conclude on the feasibility of this technique in its treatment. methods: we conducted a retrospective study including all cases of generalized appendicular peritonitis managed laparoscopically, in the general surgery department of charles nicolle hospital between january and december . results: we identified patients. the mean age was . years. one fifth of the cases required a midline conversion ( . %). the mean operative time was . ± , min. the overall morbidity rate was % including surgical complications. there were no deaths. in uni-variate analysis, comorbidity, crp [ mg /l, operative time exceeding min and midline conversion were significantly associated with postoperative morbidity. co-morbidity, diabetes, asa score [ , delay of consultation [ days, intra-abdominal abscess and operative time exceeding min were significantly associated with medical complications. the univariate analysis also revealed that crp [ mg /l and midline conversion were predictive of surgical complications.the multivariate analysis identified the midline conversion as the only independent factor significantly associated with post operative morbidity (odds ratio = . , % confidence interval [ . - . ] ). conclusion: based on our results, it appears reasonable to continue the laparoscopic management of diffuse appendicular peritonitis. however, enhance this technique is basic in order to reduce midline conversion rate and to shorten operative time, which can lead to post operative complications. aims: currently, acute appendicitis is the most common surgical emergency. laparoscopic appendectomy is the usual procedure to treat acute appendicitis. the aim of this study is to evaluate the safety of electrocoagulation in the treatment of mesoappendix in laparoscopic appendectomy. methods: we have retrospectively studied a prospective database of operated patients of appendecectomy in emergency surgery unit. we have reviewed laparoscopic appendectomies from june st, to december st, . the mesoappendix was electrocoagulated in every laparoscopic appendectomy. the statistical analyses has been done with spss . version. results: our group consists of patients of which . % were male and . % were female. the average age was . years with a standard deviation of . % and p was . years. the most common total stay was day ( patients). the usual post-operative stay was one day ( ). we classified the diagnosis in complicated apendicitis ( patients) and no complicated apendicitis ( patients). the conversion rate was . % ( ). the main surgical complications were: surgical wound infection ( . %); intraabdominal abscess ( . %); and bleeding ( %). only one of the patients that suffered bleeding had complicated appendicitis. the medical complications were catheter sepsis ( . %); respiratory infection ( . %); cardiologicals ( , %); and paralytic ileus ( . %). the treatment of mesoappendix with electrocoagulation is safe and effective since the complications rate is very low. even so, it would be necessary to conduct more prospectives randomized studies in order to get enough evidence about the treatment of mesoappendix with monopolar electrocoagulation. introduction: the difficulty of resection of the rectum is determined by its anatomical relationships, intimately in contact with the bladder, seminal vesicles, prostate and urethra in the case of the male, vagina in the woman and nerve structures that will give defecatory, genital and urinary functionality. this structure creates a big impediment due to problems of visualization and difficult dissection, in such a way that conventional surgical techniques instigates a series of complications derived from this difficulty. we propose a new approach in rectal surgery in patients with inflammatory bowel disease. material and methods: a -year-old man with a history of ulcerative colitis developed a severe acute outbreak refractory to treatment. a total laparoscopic colectomy with a terminal ileostomy was performed in . in he was notified for reconstruction. we evidenced a rectal stump of about cm with signs of inflammatory disease at the mucosal level. a transanal proctectomy was performed with confection of 'j-pouch' and ileoanal anastomosis about cm from the anal margin by laparoscopy. the postoperative courses favorably, being discharged on the sixth day. currently in follow-up in digestive and general surgery, he is asymptomatic and he has an optimum level of quality of life valued by the sf- weeks after the intervention. conclusions: our service introduces the transanal approach to the performance of proctectomy in cases of inflammatory disease, a technique that provides clear advantages by improving visualization and the identification of anatomical structures. in this way, a safe dissection of the pelvis is achieved, adjusted to the serosa of the rectum, with preservation of the mesorectum and the hypogastric plexus, and with the consequent improvement of the genital and urinary function. the result is an equally safe surgery, which implies little increase in operative time and with better and shorter postoperative recovery.the conservation of the pelvic innervation avoids disorders of ejaculation, vaginal lubrication and bladder and rectal motility. the transanal approach for the performance of proctectomy provides benefits in terms of the preservation of the hypogastric plexus, minimizing the anatomical difficulties involved in rectal surgery and maintaining urinary and sexual function. aims: to evaluate the feasibility and outcomes of laparoscopic appendicectomies in both simple and complicated appendicitis, given the increasing trend towards a laparoscopic approach in the last four decades for the treatment of acute appendicitis. we present data from a district general hospital over a -year period. methods: we retrospectively analysed a single consultant's continually updated database of laparoscopic appendicectomies between / / and / / ( months). patient demographics, investigations, intraoperative findings and postoperative outcomes were recorded and analysed. complicated appendicitis was defined as the formation of appendiceal mass or abscess with or without perforation and peritonitis. results: cases of laparoscopic appendicectomies were identified during the specified period. the median patient age was (range - years). true positive rates for uss and ct were % and %, respectively. the rate of negative appendicectomies was %. transanal minimally invasive surgery (tamis) has been used for the treatment of rectal neoplasms such us benign polyps and early rectal cancer. when the tumour is located in the upper rectum or close to the rectosigmoid junction, this approach may be technically dificcult.we present a video of a tamis resection of a large polyp located cm from the anal verge. after properative examination and ct and mri were performed, the patient was prepared for surgery, and a trasnanal minimally invasive surgery was proposed.resection of the polyp was performed with the aim of an endogia and conventional laparoscopic materials. total resection of the polyp with free margin was possible. the postoperative pathology report confirmed a high grade displasia villo-tubular adenoma with a lesion free margin. tamis resection of tumours located above the rectosigmoid junction may be a safe and feasible technique in selected patients. aims: pelvic organ prolapse (pop) is a very relevant problem for women's quality of life and has a prevalence of about % defined by symptoms and up to % when established by physical examination. nowadays, sacrorectopexy for posterior pop and sacrocolpopexy for apical pop are considered the gold standard techniques. recently, we have seen that laparoscopic lateral suspension is a feasible procedure for apical pop, obtaining a success rate higher than % at one year. these results are similar to what we can achieve with sacrocolpopexy. methods: we herein present the case of a -year-old woman with apical and posterior pop, this was provoking an important impact on her quality of life, with obstructive defecation (needing digitations) and urinary incontinence. we proposed sacrorectopexy for her posterior pop and laparoscopic lateral suspension for her apical pop. in the video we can see how we perform a ventral mesh sacrorectopexy, following d'hoore technique; and a laparoscopic lateral suspension with preperitoneal dissection, following the technique described by the team headed by dubuisson and veit-rubin. we used laparoscopic ports ( , , . and . mm). results: patient was discharged home on the second postoperative day and has not had any sign of recurrence or extrusion after more than two years of follow-up. in addition, she has not suffered lower urinary tract symptoms, constipation or pain. conclusions: we present a case in which we have carried out a laparoscopic lateral suspension instead of a sacrocolpopexy for an apical pop, obtaining good short-term and long-term results. we consider it is very soon to assess this technique's efficacy and it has to be validated in studies with larger source of patients. nevertheless, we think this procedure might become an excellent alternative to sacrocolpopexy for apical pop. aims: laparoscopy is a minimally invasive approach with low morbidity. the aim is to show the usefulness of the laparoscopic approach for massive intra-abdominal abscesses, which it is controversial. we report three patients who underwent emergency laparoscopy for peritonitis or massive intra-abdominal abscesses not amenable to percutaneous approach that were suspected to be caused by acute diverticulitis. methods: all patients had diagnosis of acute diverticulitis (hinchey ii-iii grade) with pelvic abscesses situated between sigma and bladder or diffuse peritonitis. the patients with hinchey ii grade had failed conservative management with antibiotics. they underwent emergency laparoscopy under general anaesthesia, with three abdominal ports. intra-abdominal abscess cavities were exposed and the purulent exudate was sampled and aspirated. copious irrigation was performed under direct vision and thorough examination without other findings. the procedure was completed laparoscopically in all cases. results: all patients had favourable evolution. one of them had a properly drained faecal fistula which changed to a purulent fistula on the twentieth postoperative day. this patient underwent laparoscopic left colectomy three months later because he had have a new episode of acute diverticulitis. other two cases showed very good clinical evolution, without evidence of fistula in postoperative period and they were complete asymptomatic one month later. conclusion: in our experience laparoscopic drainage is a feasible, safe, and effective for the treatment of pelvic abscesses and diffuse peritonitis secondary to acute diverticulitis. n. pinheiro, a. ziegler introduction: solitary rectal ulcer syndrome (susr) is characterized as a rare disease whose pathophysiology remains uncertain. it was first described in by cruveilhier and his clinicopathological feature was reported in by mandigan and morson, where he is associated with defective disorders, internal rectal prolapse, and psychological changes. according to works about % of the patients are asymptomatic. when symptomatic the diagnosis can be made through physical examination, clinical history and, often, confirmed by endoscopy with biopsies. treatment depends on the severity of the symptoms and the existence of associated rectal prolapse. according to the literature, conventional surgical options include local excision, rectal mucosectomy, retopexy, and segmental colonic resection. rolato: a -year-old male complaining of anal bleeding at bowel movements years ago. he performed, several times, conservative treatment, but without improvement. he sought proctological care and underwent colonoscopy, in which he showed an ulcerated lesion on the anterior wall of the distal rectum. new investigation with videodefecogram revealed colorectal intussusception with associated mucosal prolapse, being considered the factor causing the ulcer. elected by the sacropromontofixação. evoluiu with improvement of anal bleeding, mucorrhea and anal discomfort. after a proctological examination, which was normal, a control colonoscopy performed after months of surgery revealed rectal mucosa, with residual scarring and disappearance of the submucosal nodule present in the initial examination. reassessed after months, the patient is asymptomatic. conclusion: rectal solitary ulcer whose causal factor was a colorectal prolapse (intussusception) with mucosal exteriorization through the anal canal, which was individually treated with sacropromontofixation. j.p. mali, p.j. mentula, a.k. leppäniemi, v.j. sallinen approximately - % of patients diagnosed with colonic diverticulitis have an intra-abdominal abscess as a complication. abscess diameter of - cm is generally accepted as a cut-off determining the choice of treatment between antibiotics alone and percutaneous drainage. the aim of this study was to analyze the treatment choices and outcomes of patients with diverticular abscesses. this was a retrospective cohort study which was conducted in helsinki university hospital, an academic teaching hospital functioning as secondary and tertiary referral center. patients with computer tomography-verified acute left-side colonic diverticulitis with intra-abdominal abscess were collected from a database containing all patients treated for colonic diverticulitis in our institution during - . altogether, suitable patients were included in analyses. those treated primarily with percutaneous drainage or antibiotics alone ( and patients, respectively) were further compared in regards to treatment results. the main measured outcomes were need of emergency surgery and -day mortality. abscesses under mm were mostly treated with antibiotics alone with high success rate ( out of , %). in abscesses over mm, the use of emergency surgery increased and use of antibiotics alone decreased with increasing abscess size, but the proportion of successful drainage remained at - % regardless of abscess size (figure ). there were no differences in failure rate, -day mortality, need of emergency surgery, permanent stoma, recurrence, or length of stay in patients treated with percutaneous drainage versus antibiotics alone, even when groups were adjusted for potential confounders. white blood cell count = . * /l, abscess diameter = mm, and corticosteroid medication were independent risk factors for failure of treatment with antibiotics alone. patients without these risk factor had % and patients with one risk factor had % success with antibiotics alone. percutaneous drainage as treatment for large abscess does not seem to be superior to treatment with only antibiotics. majority of patients with abscesses over mm in diameter undergo surgery as primary intervention. introduction: even today, 'chronic appendicitis' is a clinical term that is not widely accepted nor well documented amongst the medical community. its etiology is the presence of a mass (e.g. fecal mass, hyperplasia of lymphatic tissue, etc.) that continuously and partially obstructs appendix lumen. it is presented as a low intensity, intermittent, with exacerbations and remissions, abdominal pain that is located at the right iliac region. the pain lasts up to several months and it is usually underestimated by the patient. its diagnosis is based on imaging examination. appendectomy is the treatment of choice for chronic appendicitis. the operation is challenging for the surgeon who has to cope with an intensively inflamed area around the appendix without the ease of access to that area. purpose: to present our laparoscopic approach to a chronic appendicitis case and to review the literature. case report: a -year-old woman is hospitalized due to chronic appendicitis. the patient was treated conservatively with the use of intravenous antibiotics in two separate hospital admissions dated and months back respectively. eight weeks after the last exacerbation, she underwent a laparoscopic appendectomy. results: even though the procedure was planned six months after the first episode, the laparoscopy revealed a severe inflammation of the appendix, which was extended to the caecum and the surrounding preperitoneal tissues. although the difficultness of the operation it was completed successfully laparoscopically. the histological examination confirmed without any doubt the existence of 'chronic appendicitis'. the patient was discharged uneventfully the third postoperative day. conclusions: chronic appendicitis is an existing clinical entity that the surgeon may come through during his career. in the hands of experienced laparoscopic surgeon, the laparoscopic approach is feasible and safe. introduction: ventriculo-peritoneal shunting (vps) used in the treatment for hydrocephalus is associated with several complications.the exact cause of such extrusion is not known. visceral perforation is an unusual but serious complication with consequeces such as peritonitis, meningitis or encephalitis. management involves prompt removal of shunt, intravenous antibiotics, an adequate recovery gap so that cerebrospinal fluid culture is sterile and then followed by shunt replacement on opposite side. aim: multidisciplinary approach of extrusion of vps through anus by laparoscopic and external ventricular drainage. case exposure: a -year-old woman had a vps inserted year ago after excision of gangliocytoma due to lhermitte-duclos disease. she was admitted in the emergency department without symptos after trans-anal protrusion of vps catheter. the neurological and abdominal evaluation was normal. laboratory tests did not reveal disorders and abdominal ct-scan suggested perforation, itshowed the insertion of the end of the catheter in sigma, without pneumoperitoneum or intraabdominal free fluid. cranial ct-scan did no describe sings of hydrocephalus. the patient underwent an emergencysurgical intervention. first of all, antibiotic therapy was initiated and neurosurgery's team was performed an external ventricular drain and they disconnected the proximal catheter side. after that, an exploratory laparoscopy was performed. it revealed a microperforation and collection beside to an appendix' base due to the proximity with the catheter. additionally, the catheter was freed from adhesions at the point of entry into the colon and after careful dissectionwe release the vps from colon with a . cm transmural trajectory at the sigmoid level. no free fluid was seen and rest of the bowel appeared normal. the distal end was removed through the anus and the proximal end through a laparoscopic port. we performed a laparoscopic segmental cecum resection and an extracorporeal colo-colonic anastomosis was performed for a mini-pfannestiel laparotomy of assistance. there were no complications in the postoperative period, being discharge on the th day. conclusion: the multidisciplinary approach and the laparoscopic support in the diagnosis and treatment of patients with colon perforation caused bay vps catheter is a feasible and safe option in third level centers. background: acute appendicitis continues to be the most common source of complicated intraabdominal infection worldwide. the high incidence of postoperative complications and dissatisfaction with the results of treatment in cases of complicated appendicitis and peritonitis gave the reason for conducting this study. aim: to evaluate the effect of different laparoscopic trocars position in case of laparoscopic appendectomy for diffuse appendicular peritonitis for the incidence of postoperative complications methods: the results of laparoscopic treatment of patients with acute appendicitis complicated by diffuse peritonitis were analyzed. the first group consisted of ( %) patients operated by triangulation access (type trocar placement according sages guidelines for laparoscopic appendectomy (sages qla). the second group consisted of ( %) patients operated by sectorisation access (type sages qla). postoperative complications were classified by clavien-dindo classification. results: the duration of the operation for the analyzed groups was . ± . vs . ± . min. there were no deaths among this group of patients. the incidence of postoperative complications for both group was . %. postoperative complications in the triangulation and sectorisation group were % and . % respectively (p . ). clavien-dindo iiib complications were noted in . % (n- ) patients and presented with intra-abdominal abscesses (iaa). all patients with iaa were operated in sectorisation group. conclusion: sectorisation trocar placement increases the incidence of intra-abdominal complications for laparoscopic appendectomy for diffuse appendicular peritonitis. introduction: the diverticular disease of the colon is a chronic entity with a variety of abdominal symptoms that can present with recurrent episodes of acute diverticulitis (ad). the prevalence of diverticulosis is not influenced by gender and increases with age, which, according to the increase in life expectancy, explains the accumulation of cases in western countries. the classic diagnostic-therapeutic algorithm of the disease is it has been based on the hinchey classification, the use of antibiotics and the intervention of hartmann (ih) at the acute time and elective colectomy in the multirecurrent cases. the use of laparoscopy with washing and drainage is actualymore extended in cases with peritonitis. objectives: to demonstrate the safety and efficacy of the laparoscopic approach, in cases with diverticular disease complicated by severe inflammatory plastron with 'covered' perforation, with several recurrent episodes. material and method: case report: a -year-old man with ap-diverticulitis years ago with complete resolution and normal control colonoscopy. he presents in the last two months three compatible episodes of acute diverticulitis, exploration with plastron-mass in hypogastrium without defense, tac-marked thickening of a segment of cms. of medium sigma, collection not drainable in mesosigma, of cm, which loses the plane of cleavage with loops of thin neighbors with a linear tract that suggests fistulization. evidence of interest is exposed. given the evolution, it is decided surgical elective treatment. result intervention: preoperative ureteral double catheterization, laparoscopic approach, is exposed by video, rectosigmoid resection by diverticular plastron, with negative io biopsy, mechanical colorectal anastomosis. good postoperative course, discaharge at th day. defini-tive ap: perforated diverticulitis, absence of malignancy. the laparoscopic approach is a valid and effective alternative in cases of complex and severe diverticular disease. aim: tamis resection has been described for the treatment of rectal neoplasms, wether benign or early malignant tumours. since tamis appearance, many different indications have been reported.we aim to show an special indication as seen in this video of a tamis resolution of a rectal stenosis non treatable by endoscopy. method: we present a video of a female patient, previously treated for a large rectal adenoma treated by trasnanal apporach, with a postoperative sepsis which required lateral colostomy and trasanal drainage. after surgery, the patient suffered from a rectal stenosis which couldn' t be solved by endoscopy, so the patient was sent back for a surgical treatment.we decided to performed a trasnanal apporach by tamis and a long and circunferiential stenosis around cm from the anal verge was seen.we performed a rectotomy by electrocautery in the posterior rectal wall until the perirectal fat was seen and the stenosis was passed. a dilatation with a foley catether was also performed. results: postoperative course was uneventful and after months she was prepared for colostomy closure with no complications and remains asymptomatic nowadays. conclusion: tamis approach of rectal stenosis may be a safe and feasible technique in selected cases if conservative treatments fail. iatrogenic endoscopic colon perforation it is a severe, but rare complication of colonoscopy. the incidence of this complication is estimated to be . - . % for diagnostic colonoscopies and . - % for therapeutic colonoscopies. the management of these complications depends on the size of the lesion,the time elapsed between the lesions were produced and diagnostic of the lesions and associated pathology. the treatment can be consevative,endoscopic or surgical(clasic/ laparoscopic) in our sevice in last years we treated cases with iatrogenic colon perforation after diagnostic colonoscopies. all lesions were at sigma level. one case was admission in our service at days after a diagnostic colonoscopy.the pacient was operated clasic,in emergency,we found a fecaloid peritonitis,a perforation at sigma level.we made a colostomy, lavage, drainage but the pacient died after days. in cases we made the operation at maximum h after the lesion was diagnosticated by the endoscopist(directly visualisation).we didn't made radiologic investigation.the pacients were operated laparoscopic,we made suture,lavage,drainage. evolutions of the pacients were good. conclusion: iatrogenic colonic perfortion are rare,but severe complication. laparoscopic surgery can be a choice in treatment of this complication introduction: complicated diverticulitis with fistula is responsible for about % of surgical procedures in diverticular disease and is commonly found in patients with diverticulitis of the sigmoid colon. colovesical fistulas are the most frequent ( %), with highest incidence in males. only a third of these patients have a history of diverticulitis. in most cases, treatment is surgical, and colectomy is performed, whether or not in association with vesical recession. case report: year old male with pneumaturia and fecaluria for the preceding months. the colonoscopy identified a diverticulitis of the sigmoid colon and the subsequent pelvic mri suggested a colovesical fistula. the cystoscopy was not able to identify any fistulous opening, but a double j catheter was placed in the left ureter, as surgical treatment had been proposed. a subsequently abdominal pain motivated a preoperative ctscan which revealed a pneumoretroperitoneum and a fluid collection near the left ureteral tract. the multidisciplinary team on the case decided to perform a percutaneous nephrostomy, followed by an exploratory laparoscopy. the fistula tract was identified and a laparoscopic sigmoidectomy with partial cystectomy was performed, as well as a ureterorenoscopy (with double j replacement). there were no intra or postoperative complications, and the patologic repport had no signs of malignancy. video of the surgical procedure is presented. conclusion: a laparoscopic approach to complicated diverticulitis with colovesical fistula is safe and effective when performed by experienced colorectal surgeons. introduction: diverticular disease is characterized by its high prevalence, being one of the most frequent causes for hospital admission when it comes to gastrointestinal pathology. even though it is more frequent in older patients, there has been an increase in incidence amongst lower age groups. the approach to the disease has also suffered changes in the last few years, showcasing a tendency for less invasive options, deferring elective surgery to later in the course of the disease. this study examines the therapeutic approach to diverticular disease in our hospital. methods: retrospective analysis of demographic data, therapeutic options and surgical outcomes in patients admitted for diverticular disease between january and june . results: patients(n = )were included in the study: ( %)were male and ( %) were female, with an average age of years. patients( %) underwent medical treatment, with surgery reserved for the remaining patients, of which were emergencies, with the other being elective( %).about % of patients were only admitted once, %were admitted twice and % had or more episodes. for the single-admission group, the most common treatment was medical( %of cases), as was the case for the group with or more episodes(in which %of cases were subjected to medical treatment). in the patient group with episodes, %were submitted for surgery, most of which elective.as far as the surgically operated group is concerned, no statistically significant differences were found with regards to patient sex. age, however, was significantly greater for the group that underwent emergency surgery vs that submitted to elective ( . years vs . years,p \ . ).the most common procedure overall was colic recession, with hartmann's operation standing out as the most frequent for the emergency surgery group. length of hospital stay was again higher for the emergency group (vs elective; vs ,p \ . ),as well as the morbidity rate. no statistically significant differences were found with regards to mortality rate. conclusion: knowledge of the natural history of diverticular disease led to changes in the approach to treatment, with a tendency to adopt a less aggressive therapeutic. despite controversy around aspects such as selection of patients for elective surgery, among others, it is key to the approach to diverticular disease that existing recommendations are taken into account, treatment is individualized and outcomes are closely monitored. surg endosc ( ) aims: the objective of this analysis is to establish if there is differences after the procedure of laparoscopy appendicectomy comparing the use of endoloop (el) vs. endostapler (es) in complicated and non-complicated acute appendicitis. methods: we performed a retrospective analysis of a prospective database of patients from february to june . we divided the patients in two groups: depending on that the procedure of the appendicectomy was with endoloop or endostapler. the groups were created selecting patients in order to be homogeneous as to perforation appendix rate, thus a propensity score was performed for sex, age and perforation rate. an univariant analysis was carried out in regard to the differences in the use of el vs es in the apparition of abdominal complication, as well as hemorrhage, ileo, surgical wound infection, collection, reintervention or hospital readmission.qualitative variables were expressed in terms of absolute frequencies and percentages and mean values and standard deviation were used to express quantitative variables. introduction: intramural haematomas can develop anywhere within any the gastrointestinal tract . these are most frequently associated with blunt trauma above the level of the sigmoid colon and very rarely occur in the rectum . spontaneous, non-traumatic haematomas are a rare clinical condition usually secondary to haematological blood disorders or anticoagulant therapy . case summary: a -year old gentleman presented to the emergency department with a day history of worsening lower abdominal pain and bloody stool. he presented twice within the previous week with worsening, generalised abdominal pain. the patient had been taking regular aspirin and clopidogrel following insertion of coronary artery stents. on clinical examination, he was guarding with a distended, generally tender lower abdomen but all observations were stable, afebrile. an initial computer tomography of the abdomen reported pneumoperitoneum with haemorrhagic ascites; a differential diagnosis being perforated sigmoid colon with a large localised haematoma. the patient underwent an emergency laparotomy and hartmann's procedure (appendicectomy, sigmoid colostomy and rectal stump). he recovered well with no significant post-operative complications. histology reported the rectal perforation macroscopically associated with an opened haematoma and no evidence of malignancy. the appendix shows reactive appendicitis with serousal inflammation background: ulcerative colitis (uc) is one of the risk factor of developing sporadic colorectal cancer. approximately % of uc patients develop an acute attack of severe colitis, and % of these patients require colectomy. one third of the patients will not respond to steroid therapy. thus, a long-term follow-up has been recommended. case report: we reported a single case of completed years follow of colorectal cancer related ulcerative colitis on years old female patient undergoing emergency operation ( staged total colectomy and j-pouch ileo-rectal anastomosis) after year no responsed of medical treatment before, presenting with bloody diarrhea and anemia. there was no post operative complication reported. pathologic finding was early adeno carcinoma, closed follow up was done each year and for another five years later, no progression of the disease was found in this period and the patients has good quality of life after this procedures. is becoming a standard and feasible surgical method worldwide. % of patients with crohn's disease (cd) and % patients with ulcerative colitis (uc) will require an operation during their life. over the last decade, there have been many studies documenting the safety and feasibility of the laparoscopic approach for ibd in well-selected patients. methods: patients with a cd with the tight stenosis in the distal ileum and/or ileo-colon or various colon and rectum stenosis, patients with uc with ineffective medical therapy, steroid dependence or dysplasia underwent the lcs. from to , ileocolic resections, hemicolectomies, subtotal colectomies and restorative proctocolectomies with ileopouchanal anastomosis were performed either totally laparoscopically or laparoscopically assisted (n = ).the average time of the procedure was min ( - min), average blood loss ml ( - ml) and the conversion to laparotomy was in . %. average return time of the bowel function was . days ( - days) and the average hospital stay was . days ( - days). complications occurred in patients ( . %). cases of the early ileus due to adhesions, cases of the anastomotic bleeding threated conservatively, case of the instrumental perforation of the small bowel, cases of the incisional hernia in minilaparotomy and wound infections occurred. conclusion: in well-selected patients with ibd, thanks to superior short-and long-term outcomes, the laparoscopic approach should be considered a safe and effective method when performed by experienced surgeons. supported by mo . aim: over % of patients with crohn's disease (cd) will require a surgical resection within years of their diagnosis and one quarter will have another resection for disease recurrence. laparoscopy should by preferred approach in surgery in cd due to reduced morbidity, faster recovery time, shorter hospital stay and reduction in adhesions and hernial formation. methods: patients with cd with the tight stenosis in the distal ileum and/or ileo-colon or various colon stenosis were indicated for the laparoscopy. from january to november we performed ileocolic resections, hemicolectomies and subtotal colectomies either totally laparoscopically or laparoscopically assisted. the average time of the procedure was min ( - min). the average return time of the bowel function was . days ( - days) and the hospital stay was from to days. complications occurred in patients ( . %) . in cases early ileus developed due to adhesions, in case was anastomotic bleeding threated conservatively, the incisional hernia in minilaparotomy occurred in cases and wound infections occurred. conclusion: minimally invasive surgery is becoming a gold standard in cd. it is safe and feasible in well-selected patients thanks to short-and long-term outcomes. laparoscopic approach for recurrent disease is still in debate. supported by mo . aim: the aim of the study was to observe when laparoscopy is avoided when treating surgical complications of crohn disease. methods: we did a retrospective study which included all of the patients diagnosed and operated in our clinic for complications of crohn disease during a period of years. results: we identified a number of patients operated for complications of crohn disease. of these were operated by minimally invasive procedures. we observed that laparoscopy was avoided in the case of intestinal fistulas (p = , ). also when sepsis associated the surgical complication-laparoscopy was avoided (p = , ). age under years represented another factor to avoid laparoscopy (p = , ). conclusions: although laparoscopy offers numerous advantages careful selection of the patients is of utmost importance so the safety of the procedure can be ensured. retroperitoneal sarcoma represents approximately - % of all sarcomas and less than . % of all neoplasia. radiotherapy and chemotherapy still do not represent valid therapeutic alternatives; therefore radical surgery remains the only valid option. complete surgical resection is the only potential curative treatment modality for retroperitoneal sarcomas. the ability of complete resection of a retroperitoneal sarcoma with tumor grading remains the most important predictor of local recurrence and disease-specific survival. hypoglycemia is a rare but potentially lifethreatening presentation of soft tissue tumors the etiology of hypoglycemia may be difficult to diagnose, assays for insulin-like activity (ila) were found to be high in the extract of tumor tissue, while insulin was not detected in significant concentration neither in the same extract nor in his serum. the most likely mechanism of hypoglycemia appears to be production of insulin like substance and increased utilization of glucose by the tumor. laparoscopic surgery represents an alternative technique for radical resection of such tumors rather than traditional surgery. only few cases of retroperitoneal tumors resected laparoscopically were reported in the literature. we report a rare case of years old male presented to ed unconscious due to hypoglycemia.he was resuscitated and admitted for further investigations. hypoglycemic attack recurred again during the same evening of admission. initial investigations were within normal except for serum glucose mg/dl ( . mmol/l). his tsh, glucagon & cortisol levels were within normal, insulin and c-peptide levels were undetectable. only hypokalemia ( . meq/l). he tested negative for the anti-insulin antibodies. his abdominal ultrasound as well as his ct scans showed the presence of a large retroperitoneal tumor ( cm cm cm) with a heterogeneous contrast effect. a glucose supplement was required to maintain the plasma glucose level within normal limits during which complete resection of the tumor which was performed laparoscopically. diagnosis of such hypoglycemia inducing retroperitoneal fibrosarcoma represents great challenge especially when patients presents only with hypoglycemia and no other abdominal symptoms, management using minimal invasive technique to resect and remove such tumors from the retroperitoneal region shows superiority in recovery and limitation of complications when done by experienced surgeons. solitary fibrous tumor (sft) is a rare fibroblastic mesenchymal neoplasm, tipically arising from the pleura, less frequently from other anatomic sites. sft is an indolent neoplasm, but it have been described cases of greater aggressiveness in terms of local recurrences and more rarelly of distant metastases. among the various extrapleural sites, intrabdominal, retroperitoneal localization is the most common site, followed by the pelvis soft tissues and parenchymatous organs. the most common clinical finding of intraabdominal localization is a palpable mass, and the pain is the most frequently associated symptom. the diagnosis is performed by imaging, but the histological as well as immunohistochemistry characterization of the lesion is the latest goal. furthermore, histological features are used to attempt to identify the patient with a hight risk of malignant evolution of the tumor. the gold standard treatment is surgical approach, meanwhile there are no evidences about the efficacy of any adjuvant treatment. we present the case of a -year-old man affected by symptomatic tfs arising from mesosigma treated by surgical radical excision. finally, we propose a review of the literature of last decade. background: laparoscopic right hemicolectomy involves making an additional incision to remove the specimen and perform the anastomosis. recently, natural orifice specimen extraction surgery (noses) has been reported as an alternative approach without any additional incisions or extensions, may lead to better outcomes compared to conventional laparoscopic right hemicolectomy. in this video, we aimed to evaluate the safety and feasibility of noses for laparoscopic right hemicolectomy. methods: we describe the technique with transvaginal specimen extraction and d lymph node dissection in laparoscopic right hemicolectomy by this video. we performed intracorporeal anastomosis combined with a transvaginal route of specimen extraction after medial-to-lateral mobilization. transverse transvaginal posterior colpotomy was performed under aid with visualization. the specimen was pulled into the sterilized plastic bag, passed transvaginally. the vaginal incision was then closed with a running suture. results: the operation time was min and the hospital stay was days. an excellent postoperative recovery was demonstrated and has shown future potential for less incision. the pathologic tnm stage is t n m . conclusions: this video has shown that laparoscopic right hemicolectomy with the noses technique is feasible and safe for selected cases. the long-term benefits of this procedure need to be more evaluated. recently, indocyanine green (icg) fluorescence has been introduced in laparoscopic colorectal surgery to provide detailed anatomical information.the aim of our study is the application of icg imaging during laparoscopic colorectal resections: to identify the sentinel lymph node (sln) to search micrometastases that can be missed with the conventional pathological exam, and to assess anastomotic perfusion to reduce the risk of anastomotic leak. after tumor identification ml of icg solution ( . mg/kg) is subserosal peritumoral injected. a full hd image s camera, switching to nir mode, in about min displays fluorescence: the sln is identified and the sln biopsy (slnb) is performed.after the transection ml of icg solution is injected to confirm the stumps perfusion. if there is an ischemic area, a new resection is performed.after the anastomosis is performed, another bolus of icg is intravenous injected to confirm the anastomotic perfusion.when the sentinel node is negative for cancer metastases by conventional histological examination, ultrastaging is performed by serial sections. when no micrometastases are identified on these sections, immunohistochemical techniques are applied. from november , patients were enrolled: left colectomy, right colectomy, transverse resections, and splenic flexure resections. in two cases, one left colectomy and one right colectomy, the anastomotic perfusion wasn't good and the surgical strategy was changed. four postoperative complications occurred, of which one anastomotic leak, due to a mechanical problem. from november , patients were enrolled to perform the slnb: right colectomy, left colectomy, transverse resection and splenic flexure resections. the sln was identified in cases. cases were found to be n to the conventional examination and were subjected to ultrastaging. the serial sections showed micrometastases in two cases. in the other cases the immunohistochemistry was performed but the exam is still in progress. icg-enhanced fluorescence imaging is a safe, cheap and effective tool to increase visualization during surgery. it's recommended to assess the anastomotic perfusion in order to reduce the incidence of anastomotic leak, and to perform the slnb for the sln ultrastaging in order to identify micrometastases. methods: for the last years, tem was performed on patients' with early rectal cancer. there were women and nine men, age to . localization of tumors was - cm from anus. mean size of tumors was . cm. full thickness excision was performed in all patient with suturing of mucosa. during follow-up in three patients' metastasis in lymph nodes of mesorectum were detected. all of these patients were re-operated: laparoscopic colectomy with total mesorectal excision (tme) was done. for the last year in patients with early stage rectal cancer we used indocyanine green (icg) with fluorescent imaging for mapping sentinel lymph node. icg was injected in four quadrants to submucosa around the tumor. during the laparoscopy, sln was detected and removed with morphological examination. results: among nine patients in patients, sln was negative. tem was performed in these patients with good results. after - months no recurrence or metastasis were detected in these patients. in two patients with positive sn laparoscopic tme was performed with low colorectal anastomosis. anastomotic complication was occurred in one patient. conclusion: tem procedure is highly effective in selected group of patients with early rectal cancer. mapping and examination of sln can clarify indication for the tem in the patients with early rectal cancer. purpose: laparoscopic surgery for colorectal cancer provides better short-term benefits and similar long-term outcomes compared with conventional open surgery. unlike minimally invasive surgery, natural orifice specimen extraction (nose) can provide additional advantages by reducing morbidity and postoperative pain related to the surgical extraction site. this study aimed to evaluate the efficacy and safety of a nose procedure using needlescopic instruments for colon cancer surgery. methods: between november and february , patients underwent laparoscopic nose using needlescopic instruments. the first port for the camera was placed at the umbilicus. a -mm or -mm port was inserted in the right lower quadrant. a -mm or -mm port was inserted in the right upper quadrant. individual needlescopic forceps for the assistant were inserted into left upper and lower quadrant ports. thus, a total of ports were placed. the superior rectal artery and inferior mesenteric vein were ligated with clips, and colonic mobilization was performed using a medial to lateral approach. after rectal stump irrigation, the distal rectum was transected using an endoscopic linear stapler. the proximal colon and associated mesentery were transected. after the rectal stump was opened, a wound retractor was pulled through the anus and inserted in the rectal lumen. the resected specimen was transanally extracted through this route. an anvil was intracorporeally attached to the proximal colon, and the open rectal stump was reclosed using an endoscopic linear stapler; colorectal anastomosis was then performed using a double-stapling technique. results: of the patients, were male and were female, with a median age of years ( - years). median body mass index was . . the tumor site was in the sigmoid colon in patients and rectosigmoid colon in patients. median operative time was min and blood loss was ml. there was no conversion to open surgery. no postoperative complication was observed. median postoperative hospital stay was days ( - days). conclusions: nose surgery using needlescopic forceps is an easily performed type of reducedport surgery with a conventional port arrangement. this procedure is feasible for the selected patients. introduction: splenic flexure colon cancer accompanying obstruction is usually managed stent insertion as a bridge to surgery and left hemicolectomy, or subtotal colectomy. however, stent insertion can fail more often than in sigmoid colon because it requires longer colonoscopic approach in the circumstance of impossible bowel preperation. although subtotal colectomy has advantage in the aspect that it is -stage treatment, it needs open surgery in most cases, right colon has to be sacrificed without oncologic neccesity, and preoperative staging and evaluation can be insufficient. despite colostomy is reluctant procedure when considering quality of life, in splenic flexure colon cancer obstruction, we can obtain prompt stabilization of patient state, suffient time to preoperative staging and evaluation, and also we can achieve minimally invasive surgery by using colostomy site as mini-laparotomy and close colostomy before discharge. colostomy site, tumor location, and minilaparotomy site for next radical surgery have to be considered comprehensively before making colostomy incision. colostomy site has to be appropriate as mini-laparitomy site for feasibility of laparoscopic left hemicolectomy and the colostomy has to be included in the specimen with caution to prevent unneccessary lengthening of the specimen. we experienced cases which were treated succefully in this strategy and report them. result: temperary loop transverse colostomy and laparoscopic left hemicolectomy via colostomy site in splenic flexure colon cancer obstruction has advantage of quick stabilization of patient's status, suffient preoperative staging and evaluation, achieving minimally invasive surgery, and also rapid colostomy closure before discharge. our tatme procedure for locally advanced low rectal cancer following chemoradiotherapy y. nakamoto , r. okamoto , f. kimura , h. yanagi , t. nakajima , h. yoshie , n. yamanaka surgery, meiwa hospital, nishinomiya, japan, surgery, yoshie clinic, itami, japan background: short-course chemoradiotherapy using hyper-fractionation method (scrt; gy/ fraction/ days ? s- or xeloda) is performed to secure circumferential resection margin (crm) due to tumor shrinkage, reduction of cancer cells with viability, reduction of radiation hazard for resectable locally advanced lower rectal cancer (more t or n ). the patient underwent radical surgery after one month of scrt. for more locally advanced lower rectal cancer (t or n ), induction chemotherapy is performed before scrt. for patients with poor efficacy of chemotherapy, we also do normal fraction gy radiotherapy. methods: we introduced tatme from last august, and cases were performed so far. in all cases temporary stoma has been constructed, and intersphincter resection (isr) is based on partial isr avoiding total isr considering postoperative anal function. if possible, colonic j-pouch is added, and pelvic floor repair may be added for esr cases and older people. at first one team preceded with the anal operation and shifted to the abdominal procedure, now it is done with two teams with the advantage of getting good visual field from both sides when there is difficulty identifying the right dissecting layer. tatme is very useful in cases such as large tumor, obesity, and narrow pelvis. furthermore, when it is difficult to identify the dissecting layer by scarring after crt, it is more possible to control the crm/drm of cancer. results: cases of isr, case of apr, case of tpe were performed, and in of these cases lateral lymph node dissection was also performed (one side , both sides ). postoperative complications were anastomotic leakage, pelvic floor infection, perineum infection, and bowel obstruction. conclusions: tatme for locally advanced lower rectal cancer is useful even after chemotherapy and scrt. background: although many studies have demonstrated similar perioperative outcomes for single-incision laparoscopic surgery (sils) and conventional laparoscopic surgery (cls) for colon cancer, few have directly compared the costs of them. we aimed to compare costs between sils and cls for colon cancer. methods: we analyzed the clinical outcomes and overall hospital costs of patients who underwent laparoscopic surgery for colon cancer from july to september at severance hospital; were used for analysis after propensity score matching. the total hospital charge, including fees for the operation, anesthesia, preoperative diagnosis, and postoperative management was analyzed. results: the total hospital charges were similar in both groups ($ . vs. $ . , p = . ). however, the patients' total hospital bill was higher in the sils group than in the cls group ($ . vs. $ . , p \ . ) mainly due to the difference of the cost of access devices. there was no difference in the additional costs associated with readmission due to late complications between the two groups ($ . vs. $ . , p = . ). conclusions: sils for colon cancer yielded similar costs as well as perioperative and long-term outcomes compared with cls. therefore, sils can be considered a reasonable treatment option for colon cancer for selective patients. aims: technology improvements in medicine allow the development of new minimally invasive approaches. despite every single advantage of these new devices they also can cause technical problems and difficulties for the surgical team. well known from last few years-laparoscopic assisted transanal total mesorectal excision for distal rectal cancer is perfect example for a quite new procedure, based on the combination of forgotten old surgical principles and technology advances. the aim of the study is to analyze the rate of technical problems during the procedure and to measure the impact of them on the operative time. methods: we conducted prospective observational study related to technical problems during the procedure. for the period between september and november in the department of endoscopic endocrine surgery and coloproctolgy at military medical academy-sofia have been performed laparoscopic assisted transanal total mesorectal excisions. we used standard local preoperative work up and postoperative care protocols. we defined technical problem as intraoperative event different from complication leading to delay in operative time. every technical problem during the procedure was recorded and time for resolving the problem was measured in seconds. results: overall technical problems occurred in of the cases. most of them were related to the insufficient smoke evacuation during the cases. the second most common technical problem were the excessive rectal stump spasms during the procedure-this complication occurred in of the patients. mean delay of the procedure related to technical problems is min. in our series we experienced only one intraoperative complication which was specimen perforation during the dissection. three complications occurred in postoperative period-two urinary retentions and one perianastomotic abscess, without need of reoperation. conclusion: technical problems during the procedure can be source of delay in operative time. correct use of devices in operating room is the key to reduce technical issues. technical problems can increase the rate of intraoperative near miss events and complications during the transanal total mesorectal excision. surg endosc ( ) aims: anastomotic leak after rectal cancer surgery constitutes a severe complication associated with poorer oncologic outcome and quality of life. preoperative assessment of the risk for anastomotic leak is a key component of surgical planning, including the opportunity of creating a defunctioning stoma. methods: studies on rectal cancer surgery published between and were systematically reviewed according to the preferred reporting items for systematic reviews and meta-analyses of individual participant data (prisma-ipd) guidelines. with the aim to generate a score for anastomotic leak, all available per-operative covariates were used as independent factors in a logistic regression model with anastomotic leak as dependent variable. a receiver operating characteristic curve (roc) analysis was generated. we selected as threshold the value that allowed a missing rate of anastomotic leak \ %. the predictive power of the previously selected cut-off was validated in an independent set of patients. results: twenty-six centers provided individual data on patients. with a threshold value of the roc corresponding to . in the training set, the area under the roc curve (auc) was . (p \ . ). sensitivity and specificity of the model's probability [ . to identify anastomotic leak were . % and . %, respectively. accuracy of the threshold value was confirmed in the validation set with . % of sensitivity and . % specificity. conclusions: we trust that, with further refinement using prospective data, this nomogram based on preoperative risk factors may assist surgeons in decision making. the score is now available online (http://www.real-score.org). in ( . %) cases laparoscopic interventions were performed in patients with diverticular colon disease. in the group of patients with colorectal cancer localization of the tumor in the right parts was observed in ( %) patients, in the left-in ( %), in the rectum- ( %). results: in the adenocarcinoma of the sigmoid colon, performed a left laparoscopic hemicolectomy ( cases) and resection of the sigmoid colon ( ). was executed high clipping and intersection of the lower mesenteric vessels, aorto-iliac lymphatic dissection. in the standard scope, lymph node dissection was performed with removal and testing of not less than epi-, para-and mesocolical lymph nodes (max ). the average length of the laparoscopic stage is ± min. laparoscopic right hemicolectomy ( cases) was performed in accordance with the principles of cvl (central vascular ligation) and cme (complete mesocolic excision). intracorporal ileotransversoanastomosis was formed by a semimanual method with endogia universal and v-lock suture material. the average length of the laparoscopic stage was ± min, the open phase was ± . in the tumor of the lower and middle ampullary parts of the rectum ( cases) after neoadjuvant chemoradiotherapy, was executed a laparoscopic total mesorectumectomy. conclusions: the use of minimally invasive technologies in colorectal surgery provides a complete revising of the abdominal organs, adequate scope of resection and lymph nodes dissection in surgical interventions. background: it is thought that complete mesocolic excision (cme) improves the oncologic outcomes for colon cancer. but, precise mesenteric mobilization from retroperitoneum and safe ligations at the origins of central vessels are considered to be technically difficult in single port surgery(sps). to resolve this problem, we utilize retro-mesenteric medial approach for right side colon cancer. herein, we introduce this technique and assess its outcomes. operative procedure: the multi-trocar platform is placed in the umbilical site. d laparosopy is inserted from one of this channels. the surgeon manipulates instruments via the other channels. st step: right colonic mesentery is mobilized medial to lateral from the head of the pancreas and retroperitoneum along the embryonic plane. nd step: the origins of ileocolic and right colic vessels are divided and central lymph node dissection is achieved. rd step: hepatic flexure is taken down from cranial. and right lateral attachment is dissected away and cme is achieved. th step: specimen is extracted and anastomosis is performed using a functional end to end anastomosis extracorporealy. results: from april to december , consecutive patients underwent sps-cme with right side colon cancer. there were in stage i, in stage ii, in stage iii and in stage iv. the mean operative time was min. the mean estimated blood loss was ml. there was no conversion to open surgery. additional port was placed in patients ( . %). intraoperative bleeding was occurred in patient. anastomotic leakage was observed in patient ( . %), intestinal obstruction ( . %) and wound infection in ( . %). conclusion: these results suggest that retro-mesenteric medial approach in single port surgery with right side colon cancer is useful and safe technique. aims: this multicenter, randomized controlled trial (simple trial) aimed to investigate the quality of life (qol) and patient satisfaction of single port laparoscopic surgery (spls) for colon cancer, compared with multiport laparoscopic surgery (mpls). methods: patients with histologically diagnosed adenocarcinoma in cecum, ascending and sigmoid colon were eligible for this trial. eligible patients were randomly assigned to the spls or mpls group at a ratio of : . qol was measured with the eortc qlq-c third edition (korean version) preoperatively and postoperatively at month , , and . in addition, patient satisfaction was surveyed with a five-point questionnaire at postoperative month. to exclude the impact of adjuvant chemotherapy on qol, subgroup analysis for patients with or without adjuvant chemotherapy were carried out. (clincaltrials.gov identifier: nct ) results: total patients were randomly allocated into the spls group (n = ) and mpls group (n = ). in total patients, global health status and five functional scale steadily increased and nine symptom scales also gradually improved over time. but, nausea/ vomiting and appetite loss temporally deteriorated at postoperative month. pain score was significantly worse in the mpls group ( . in the spls group vs. . in the mpls, p = . ) at postoperative month and appetite loss score was significantly worse in the spls group ( . vs . , p = . ) at postoperative month. except for that domains, all the other items of qol between groups were not different until postoperative months. patient satisfaction was significantly higher regarding the operation (p = . ) and the abdominal wound (p = . ) in the spls group. in patients without adjuvant chemotherapy, some items of qol (global health status, physical functioning, role functioning, emotional functioning, fatigue and pain) were significantly better in the spls group at postoperative month. since postoperative month, all of qol domains (except pain score) were similar between groups. conclusion: although postoperative pain was temporarily better in the spls, most of qol domain were similar between the spls and the mpls group until postoperative month. in patients without adjuvant chemotherapy, spls showed better outcomes in some of functional scales and symptom scores at postoperative month. coloproctological surgery, juntendo university, tokyo, japan; gastroenterological surgery, juntendo university, tokyo, japan introduction: laparoscopic surgery causes less postoperative pain compared with pain after laparotomic surgery, and its low invasiveness should be considered for pain control. we have previously controlled postoperative pain by epidural anesthesia. in this study we compared postoperative multimodal analgesia centering on acetaminophen in patients who underwent laparoscopic colorectal cancer surgery with the conventional method. subjects: the subjects were patients who underwent laparoscopic colorectal cancer surgery between january and june . surgery was performed under epidural anesthesia in patients and multimodal analgesia in : periodic acetaminophen administration ? transverse abdominis plane (tap) block in , periodic acetaminophen administration ? local anesthesia of the wound in , and periodic acetaminophen administration ? intravenous patient-controlled analgesia (ivpca) in . the operating roomoccupying time, postoperative pain (nrs), frequency of taking analgesics as needed, and postoperative nausea were investigated for days after surgery and the duration of urethral catheter placement and postoperative intestinal movement were investigated in the epidural anesthesia and multimodal analgesia groups. results: while the time from entering the operating room to initiation of surgery was significantly shorter, the time from completion of surgery to leaving the room was significantly longer in the multimodal analgesia group. there was no difference in the operating room-occupying time. the frequency of postoperative pain was significantly lower in the multimodal analgesia group on postoperative day (pod) . the frequency of taking analgesics as needed was significantly lower in the multimodal analgesia group on pod , , and . no significant difference was noted in the duration (number of days) of urethral catheter placement or postoperative nausea between the groups. regarding postoperative intestinal movement, discharge of gas occurred significantly earlier in the epidural anesthesia group. the total number of incidents of complications in the epidural anesthesia group was . discussion: in laparoscopic colorectal cancer surgery, the effect of multimodal analgesia centering on periodic administration of acetaminophen without epidural anesthesia for postoperative analgesia was sufficient compared with the effectiveness of epidural anesthesia. this approach to analgesia may be useful because none of the potential complications of epidural anesthesia occur. surg endosc ( ) in the last years the application of new technologies like d vision or virtual reality have provided to surgeons the possibility of establish a preoperative surgical plan of each surgery and of each patient. these advances are specially useful in minimally invasive colorectal surgery due to the variability in location, anatomical relationship with other organs and vascular variants of these type of surgeries. the aim of our work is to built a digital -dimensional virtual model of the colorectal ct scan imagen of patients with colorectal cancer. the virtual models are obtained from the preoperative ct scan. the ct scans that we use to this work are general electric healthcare revolution gsiÒ and siemens somatom perspective Ò and the size of each image is mm. a medical software let us build a reconstruction of colorectal digital images where a radiologist has marked the exact image of the tumor so we obtain a d reconstruction which can provide an enhanced understanding of crucial anatomical details like the exact location of the tumor and the relationship with other organs and structures of the patient which can be selectively displayed or hidden. this information has an important applicability into clinical practice since it lets surgeons estimate the colorectal anatomy, tumor size and relationships, providing key landmarks to choose the most appropiate surgery, the best trocar location and a safer dissection specially in some cases whose location can change the kind of surgery radically. we present some cases where virtual models were crucial for the preoperative and intraoperative surgical plan, showing the potential interest of these d reconstructions in colorectal surgery. in conclusion the ct scan colorrectal image reconstruction can provide an enhanced understanding of crucial anatomical details of the colon and tumor location and relations which could contribute to choose the best surgical option and to improve safety in colorectal surgery. background: anastomotic leakage (al) after colorectal procedures are a common surgical experience and represents a significant burden both for patients and surgeons. the incidence of al has been reported to vary between . % to up to %, with rates for the colon and rectum of - % and - %, respectively. they, not only add to potential postoperative patient morbidities and to overall costs of postoperative patient care, but also are considered a quality indicator in colorectal surgery. aim: we aimed to evaluate the clinical burden associated with anastomotic leaks following colorectal surgery. methods: we conducted a retrospective analysis of colorectal patients who underwent conventional or laparoscopic colorectal surgery for colorectal cancer (crc), from january st, to december st, in a single colorectal centre (centro hospitalar de leiria). patient demographics, intraoperative and postoperative aspects were collected and analysed. all statistical analysis will be conducted using stata software (statacorp lp). results: in our cohort of pts, developed a clinical al ( . %), mostly males ( %), with an average age of ± . . male gender and conversion were independent risk factors. the group with al had a higher lohs ( . days vs, . -p \ . ). out of al have been detected after the discharge. the mean diagnostic day was the eighth, and mode estimated at day . when compared with a control group, wcc, eosinophils and crp were statistically significant different in al group, at day and . conclusion: in the present study, no statistically significant risk factors for al in crc surgery were detected, except for male gender and conversion. clinical methods and biomarkers were useful for early diagnosis. technology combined with experience and common sense may be the embodiment of the clinical method. conclusions: our regional screening program has significantly improved early diagnosis and quickened surgical treatment of crc. thanks to this, we obtained an earlier stage at diagnosis, a less invasive surgical approach, and a lower rate of complications and emergency surgery need were obtained also with an improvement in both os and dfs. introduction: surgeons are increasingly being faced with the problem of treating elderly colon cancer patients. we evaluated the outcome of silc in patients of over years with colon cancer with a propensity score matched comparison to assess its perioperative and long-term oncological outcomes. methods: this retrospective cohort study analyzed our experience with silc for colon cancer over years. eighty-seven patients of over years with colon cancer who electively underwent silc were included in this study (elderly group). eighty-seven patients were then chosen out of a collective of patients less than years old in a propensity score matched design (younger group). short-term clinical outcomes in both groups were compared and verified its long-term oncological outcome. results: american society of anesthesiologists score and post-operative complication rate were significantly higher in elderly group. however, the other short-term clinical outcomes including post-operative hospital stay were equivalent in two groups. the rates of -year cancer specific survival were . % in elderly group and . % in younger group, respectively, and the -year overall survival rates were . % and . %, respectively. no significant differences were seen between two groups. conclusions: our initial experiences suggested the oncological and clinical safety of silc in patients of over years with colon cancer. however, further studies are needed to demonstrate the advantages of this procedure compared to conventional laparoscopic colectomy. aim: some clinical trials have reported the safety and efficacy of laparoscopic colectomy for colon cancer. on the other hand, transverse colon cancer was excluded in these trials because of the difficulty of laparoscopic colectomy for transverse colon cancer. in this presentation, we report the tips for laparoscopic colectomy for transverse colon cancer. tips: in our department, transverse colon cancers has been resected by laparoscopically so far. to complete cvl and cme, lymph nodes around middle colic artery should be resected, however many important structure, duodenum, pancreas, superior mesenteric vein (smv) and so on, may be obstacles. this is most difficult point for this surgery. our surgery is as follows. mobilization of ileum and ascending mesocolon from caudal sideconfirm duodenum and pancreasexpose smv and ligation root of ileocecal artery and veindissect lymph nodes around smv and ligation of middle colic artery and accessary right colic veinconfirm pancreas from caudate side of transverse mesocolon and incise the peritoneum along the caudal side of the pancreasdissect lymph nodes sufficiently by dissection from both side of transverse mesocolonmost important point. to dissect lymph nodes safely, confirmation from both side of transverse mesocolon is necessary and dissection should be performed along important structure, smv, pancreas and so on. introduction: we have developed and previously reported single-incision plus one port laparoscopic anterior resection of the rectum (sils ? -ar) as a reduced port surgery in which we can utilize the incision for drainage as an additional access route for laparoscopic procedures including the transection the lower rectum. a consecutive experience from its introduction of sils ? -ar for rectal cancer is reviewed, and its -year oncological outcomes are evaluated retrospectively. methods: one hundred and forty-one patients ( female) with a mean age of . years adopted the sils ? procedure for rectal cancer. a lap protector (lp) was inserted through a . cm transumbilical incision; an ez-access was mounted to the lp and three -mm ports were placed. a -mm port was inserted in the right lower quadrant. results: one hundred and thirty-six patients ( . %) completed with sila ? -ar. the tumor locations in the rectosigmoid, rectum above the peritoneal reflection (ra), and rectum below (rb) were , and , respectively. the median follow-up interval was months. aims: colovesical fistulae came from inflammatory disease or cancer and do have a significant morbidity. the most common location is the sigmoid colon and the most common aetiology is diverticulitis. the treatment of choice is a surgical procedure. the aim was studying compare laparoscopic approach in patients diagnosed by benign (diverticulitis) and malignant (colon adenocarcinoma) colovesical fistulae. methods: from january to march all characteristics of surgical patients with diverticular and colon adenocarcinoma colovesical fistulae were reviewed. patient details (sex, age, symptoms, diagnosis, medical history and anaesthetic risk), surgical approach, hospital stay and complications were recorded. both groups were compared with significance level set at p \ . . results: nine laparoscopic ( %) and open approaches ( %) in diverticular colovesical fistulae were performed, with a conversion rate of %. the procedure done was sigmoidectomy. there were also performed laparoscopic ( %) and open approaches ( %) in colon adenocarcinoma colovesical fistulae. the procedures done were sigmoidectomy, pelvic exenteration, left colectomy, low anterior resection and loop colostomy. comparison between the two groups didn't show significant differences in characteristics but did show significant differences regarding the approach, with more cases performed by open approach in colon adenocarcinoma colovesical fistulae (p = . ). conversion rate didn't show significant differences. patients diagnosed for malignant colovesical fistulae had more complications, cases ( %), ( %) i-ii and ( %) iii-iv-v according to clavien dindo classification, manifesting significant differences (p = . ). laparoscopic approach didn't show significant differences regarding complications. conclusions: generally, surgical approach with colonic resection and partial or total cystectomy is the treatment of choice in colovesical fistulae, although vesical resection can be avoided if it is suspected benign aetiology. whenever laparoscopic approach is performed by experienced surgeons, is feasible in colovesical fistulae and the morbidity and mortality numbers are acceptable. laparoscopic approach allows the advantages of a minimally invasive treatment but implies clinical trials to stablish stronger evidence. aims: laparoscopic right hemicolectomy became the standard of care for treating cecum, ascending and proximal transverse colon cancer in many center. most centers use multiport laparoscopic colectomy with extracorporeal resection and anastomosis (mce). single-incision laparoscopic colectomy with intracorporeal resection and extracorporeal (sci) remains controversial. the aim of the present study is to compare these two techniques using propensity matching analysis. methods: this study analyzed patients who underwent laparoscopic right hemicolectomy including mce surgeries and sci surgeries from december to december . short-term outcomes were recorded. postoperative pain was evaluated using a visual analogue scale (vas) and postoperative analgesic use as outcome measure. results: the length of skin incision in the sci group was significantly shorter than in the mce group: median (range) ( - ) cm verses ( - ) cm (p \ . ). the vas score after surgery was significantly less in srhi than in mrhe. significantly fewer patients required analgesia after srhi after surgery. there were no significant differences in operative time, intraoperative blood loss, the number of lymph nodes removed and postoperative courses between the groups. the cost effectiveness was significantly cheaper in srhi than in mrhe. conclusions: sci for right colon cancer is safe and technically feasible. sci reduces the length of skin incision and postoperative pain compared with conventional mce. aim: this study was designed to clarify the utility of laparoscopic surgery for advanced lower rectal cancer after neoadjuvant chemoradiotherapy (ncrt). patients and methods: we investigated -year disease-free survival rate, operative outcomes and recurrence risk factor in patients with lower rectal cancer (ct - , n - ) who underwent laparoscopic surgery after ncrt from to december in kitasato university hospital. results: of patients, patients underwent low anterior resection (lar), patients underwent intersphincteric resection (isr) and abdominoperineal resection (apr). there were anastomotic leakage, and urinary disorder and sexual dysfunction. ypcr rate was . %, but patients ( . %) had recurrence ( liver, lung and lymph node and local recurrence; there is some overlapping). ypt and lymph node metastasis were detected as a recurrent risk factor. the -year relapse-free survival rate (rfs) was . % and the -year overall survival rate (os) was %. conclusion: in this examination, ypt and lymph node metastasis were risk factor for recurrence. the operative outcomes, -year rfs and the -year os are relatively good results. we will conduct further follow-up, and it is necessary to investigate a long term prognosis. laparoscopic surgery is warranted for rectal cancer after ncrt. surg endosc ( ) introduction: synchronous colorectal neoplasia presents an incidence ranges from % and %. classically its surgical treatment consisted in the realisation of a subtotal colectomy (stc), however, several authors have proposed that in certain occasions the realisation of two segmental resections with two anastomoses was not accompanied by an increased risk of anastomotic failure. the objective of this study was to compare the feasibility and safety of the laparoscopic approach of synchronous colorectal neoplasia using two different techniques: stc versus two segmental resections with two anastomoses. methods: we retrospectively reviewed the clinical data of patients over years of age who underwent colorectal surgery between and at a single center. we included patients with a synchronous colorectal neoplasia who underwent laparoscopic surgery, either stc or double resection (dr). results: a total of patients met the inclusion criteria. mainly males ( %) with an average age of years, with a scale of the american association of anesthesiologists superior to ii in % and with an average body mass index of kg / m . the mean operative time was min in the dr and min in the stc, the stc resulted in a higher conversion rate ( % vs %) and intraoperative bleeding ( % vs %), in addition to a postoperative period with more complications, only % of the patients undergoing stc didn't present any complication while % of the patients with a dr didn't present any complication. % of the stc presented anastomotic failure and only % of the dr. the mean hospital stay was days in the dr and . in the stc. in the dr, an average of cm of colon was resected with an average of . lymph nodes, while in the stc, cm of colon was resected with an average of . resected nodes. conclusions: the double resection with two anastomosis is a less aggressive surgery, with fewer complications and a shorter hospital stay, providing similar oncological results. there were no differences in morbidity, re-operations or hospital stay. regarding tumor stage there were no differences between the three groups. as for the resected nodes, we found a mean of in stc, in lc and sr with no statistical difference. there were no differences in the affected nodes among groups. in our patients we didn't find differences in the recurrences rate or in the distant metastases rate.the average follow-up was months (range: - ), with no differences in overall survival. conclusion: segmental resection of splenic flexure neoplasias is safe and feasible, with no differences in morbidity or in the oncological outcomes compared with more aggressive surgeries. introduction: the evaluation of perfusion in colorectal anastomosis is still a field of study and progress for the development of new modalities that allow reducing the ratio of dehiscence or anastomotic leakage (al) in said surgery. our objective with this work is to highlight the utility of indocyanine green (icg) in the said evaluation after colo-rectal surgery. methods: we present a series of cases of colorectal surgery (benign and malignant disease) intervened in the period between and . the population sample has been homogenized according to age criteria, risk factors and comorbidity. a retrospective database has been developed with the spss v. software for the evaluation of the results obtained. the primary outcome measure was al rate with at least month of follow-up. results: a significant reduction in the incidence of al was observed in patients who underwent colo-rectal surgery (p = . ). low al rates were shown in rectal cancer surgery (p = . ). there was no significant decrease in the al rate when colorectal procedures for benign and malignant disease were combined. conclusions: the use of the image by fluorescence with indocyanine green is a safe, reproducible and relatively simple method with which to evaluate the perfusion of the colorectal anastomosis as well as reduce the rate of anastomotic leak in the postoperative period. large well-designed randomized control trials are needed to provide evidence for its routine use in colorectal surgery. introduction: currently colonoscopy is the gold standard investigation for colonic evaluation. although caecal intubation is one of its quality indicators, it is not attained in up to % of cases. this remains a significant concern. limited data are available on the follow-up of patients with incomplete colonoscopy. aims: to assess colonoscopy completion rate, the reasons for incomplete colonoscopy, and the methods used to complete colonic evaluations after incomplete colonoscopy. methods: we performed a retrospective study of incomplete colonoscopies in our unit over a one year period ( ) these results compare favorably with published data. few statistically significant differences between groups suggest varying modalities of treatment broadly result in similar qol. this data highlights a need for well-delivered support programmes for specific issues, for example stoma care and sexual dysfunction. future studies will need to include a baseline questionnaire to truly measure the impact of surgery and measure quality in an increasingly elderly and comorbid population. splenic flexure cancer (sfc), comprising the tumours raised in the distal transverse colon and proximal descendingcolon, accountfor to %of all surgically treated colorectal cancers.in cme forsfc, dissection of both the transverse and descending mesocolon must be considered. however, the use of laparoscopic surgery as a curative treatment for sfc, has never been investigated in adequate controlled trials, because of difficulty in deciding on the appropriate operative procedure, as well as technical difficulties with laparoscopic lymph node dissection. the aim of this multicenter study is to evaluate the oncologic effectiveness of laparoscopic segmental resection with cme with for cancer located at the splenic flexure. we performed a retrospective analysis of all cases of sfc treated with a laparoscopic segmental resection with cme in five different institution. intra and post operative were evaluated. patientes were evaluated, the mean operative time was . ± . min. a total of ( . %) conversions occurred, due to splenic artery lesion, one for difficult adesyolisis and three due to locally advanced tumour. recurrence was observed in ( . %) patients. there was a significant association between disease stage and recurrence (p \ . ) with a higher proportion of stage iv patients in the recurrence group ( . % vs . %). at days follow-up no mortalitywere recorded.during a median follow-up of months (range - ), deaths occurred (all of them for disease progression). keplan mayer curves showed a compareble suvival with other colo-rectal cancer. in conclusion, laparoscopic segmental resection with cme and cvl seems to be an oncologically safe and effective procedure for treatment of sfc. it may be regarded as the standard surgical method for elective management of this disease. in the future, more tailored patient-and tumor-specific segmental resection might be achieved with the use of routine lymph node road mapping. it is very important to establish a minimum number of lymph nodes to analyse for a correct staging. it has been established as . the treatment of colorectal cancer is essentially surgical. the review of the medical literature indicates that laparoscopic colorectal surgery is a safe procedure that has not found significant differences in the survival rate from open surgery. aim: the aim of our study is to compare the outcomes of laparoscopic and open resection for colorectal cancer surgery evaluating lymph node assessment. methods: the patients were collected in our hospital during the period from / / to / / and the number of lymph nodes obtained in lymphadenectomy has been studied comparing the laparoscopic and laparotomy approaches. results: interventions were performed. were laparotomic, were laparoscopic and converted laparoscopic (fig ) . the average number of nodes found in these interventions was , . nowadays, the recommendations to obtain a proper lymphadenectomy is to find more than lymph nodes. analysing our procedures, surgeries had obtained a good lymphadenectomy. according to the approach, , % of the interventions ( ) are laparotomy, , % ( ) are laparoscopic procedures and , % ( ) are by reconverted laparotomy (figure ). the average number of lymph nodes isolated was similar. laparotomy approach found , nodes while , nodes were found in laparoscopy. converted laparoscopy found , ( figure ). conclusion: the treatment of colorectal cancer is essentially surgical. today, there are a lot of studies that support that laparoscopic surgery has a survival rate similar to laparotomy surgery. according to our study, the data collected indicates that the number of isolated lymph nodes in both approaches is very similar. to sump up, laparoscopic colorectal surgery is safe and has demonstrated oncological adequacy comparable to open approach and better short-term outcomes due to a less invasive approach. background: laparoscopic low anterior resection highlights the advantages of laparoscopic surgery (better surgical field, less bloodloss, less postoperativepain, better cosmeticresult). defunctioning ileostomy prevents anastomotic leakage in low rectal cancers, butincreases morbidity, degrades thequality of life and requires a second surgery for its closure. method: in the last months we performed laparoscopic low anterior resections for rectal cancer, whithout performing any protectiveileostomy, afterchecking the anastomosis intraoperatively( men, women. average age: years). the typical placement of trocars included one supraumbilical mm trocar, two right sided mm trocars in the midclavicular line, one mm in the left midclavicular line and one mm trocar in the suprapubic midline which is also used for specimen removal, after a cm transverse extension of the incision. we present themain stages of the procedure (dissection andmesorectal excision, division of the rectum with linear stapler using the 'chinese hat-parnex' technique, creation of an end-to-end intracorporeal anastomosisusing circular stapler under direct laparoscopic vision). results: no major postoperative complication was observed. the mean operative time was min ( - ) and free surgical margins were achieved. in one case a conversion to open surgery occured. the average length of hospital stay was days ( - ). conclusions: the laparoscopic approach facilitates access to the middle and lower rectum, total mesorectal excision and avoidance of ileostomy if possible. it is a demanding operation with extended learning curve, and requires adequate experience in laparoscopic surgery and colorectal surgical oncology. background: in colorectal cancer, local excision is an attractive treatment option, but additional resection is considered when lymph node metastasis(lnm) is expected at high rate. in lower rectal cancer, advanced surgery techniques are required, so it is often difficult to make judgments. the aim of the current study is to assess the reliability of laparoscopic surgery for submucosally invasive rectal adenocarcinoma (pt ) analyzing short-term outcomes and long-term survival. method: this cohort study analyzed patients who underwent laparoscopic rectal resection for submucosally invasive rectal adenocarcinoma (pt ). conversion rate and functional and oncologic outcomes were analyzed. data on long-term results and survival were evaluated. result: surgical procedure was low anterior resection / intersphincteric resection / abdominoperineal resection: / / , and conversion to open surgery was needed for ( . %) patients. sphincter-preserving procedures were performed in ( . %) patients. there were no perioperative mortalities and positive resection margin. the mean length of hospital stay was . days. complications beyond clavien-dindo grade iii occurred in ( . %) patients,the anastomotic leakage rate was . % ( / ). the positive lymph node metastasis rate was . % ( / ). high tumor budding (p = . ), lymphatic invasion (p \ . ), and mucinous /poor histological differentiation (p = . ) were significantly associated with lymph node metastasis on univariate analysis. on multivariate analysis, only lymphatic invasion was associated with lymph node metastasis (p \ . ).the median follow-up time was months (range, - months), recurrence free survival rates was . % ( / ). conculusion: the outcomes of this study suggest that laparoscopic surgery can be used for safe and radical resection of submucosally invasive rectal adenocarcinoma (pt )?and the absence of lymphatic invasion, budding, and mucinous /poor histological differentiation are each associated with low risk of lnm. risk stratification models integrating these factors need to be investigated further. conclusions: this study highlights the complex nature of sarcopenia, as well as its common incidence. minimally invasive surgery had a higher incidence of sarcopenia than that of open surgery when both were performed within an enhanced recovery setting. despite colorectal patients being a typically well-nourished cohort at low risk of complications, there may well be benefit from interventional strategies such as perioperative immunonutrition or pre-habilitation to reduce the incidence of this poor prognostic indicator. backgrounds: urinary dysfunction is frequently observed after rectal resection and justifies urinary drainage. the concept of enhanced recovery after surgery (eras) has been widely spread from the early s. however, the optimal duration of postoperative urinary drainage is unknown. aims: the aim of this study was to comprehend short-term outcome of early removal of urinary catheter after robotic rectal surgery (rrs). patients and methods: (patients) the data of consecutive patients who underwent rrs at two hospitals between april and november were retrospectively reviewed. the main indication of rrs was the patients who need rectal mobilization with autonomic nerve preservation regardless of benign or malignant disease. perioperative management: none of the patients received epidural anesthesia for postoperative analgesia. our basic principle was to remove urinary catheter on postoperative day (pod) . after removal of urinary catheter, trans-urethral catheterization (tuc) was performed in the following situations: ) no autonomous urination over h after removal ) the decrease in urine volume (\ ml/ hr) ) the appearance of subjective symptoms like abdominal distension. when tuc was required even once, residual urine volume was measured with ultrasonic examination device since then. results: twenty seven male and female were included. the median age of patients and bmi were years old and . kg/m , respectively. the surgical procedures included anterior resection (n = ), intersphincteric resection (n = ), abdominoperineal resection (n = ), hartmann's procedure (n = ), and total coloproctectomy (n = ). only one patient received lateral pelvic lymph node dissection. urinary catheter was removed on pod in cases ( . %), on pod in cases ( . %). although tuc was needed in three cases ( . %) immediately after removal, tuc was no longer needed within three days in all three patients. late dysuria was observed in two cases ( . %), and bladder overdistension was suspected in these two cases. conclusions: our study showed that urinary catheter could be safely removed on pod after rrs. however, careful follow-up observation to avoid bladder overdistension is essential after removal. introduction: intersphincterian low rectal resection is a valid alternative to lower rectal cancers located at about - cm from the anus. methods: we present cases from our personal experience for tumors localized - cm from the anus. of them required preoperative radiochemotherapy. in cases, abdominal surgery was performed laparoscopic, having the surgical specimen extracted transanal. lone star device was used for the perineal procedure in all cases. cases required a manually, separate wires anastomosis; the others cases benefited from mechanical anastomosis performed endoanal with - mm circular stapler. we performed complete mesorectum excision in all cases, ligation at the origin of inferior mesenteric artery, complete mobilization of left splenic flexure and lateral protective ileostomy. all pacients underwent inspection rectoscopy before transit reintegration, and cases were reintegrated over a period of - weeks, except for cases which developed a colo-anal fistula, that closed under conservative treatment over a period of - months. results: there were no postoperative anal incontinence. in one case, a relative anal stenosis occured, which required endoscopic dilation. there was case of tumor recurrence and required abdominoperineal resection. conclusion: literature data sustain a - / ratio for very low rectal resection versus rectum amputation. the limit resection under the tumor is accepted as . cm. very good functional results by considering oncological principles, is a sustainable argument for choosing this kind of procedure as an alternative of rectum amputation. in the few studies conducted on crcs, the reported rate of sln micro-metastases is up to - %. the aim of this ongoing prospective study is to assess the predictability of the ex-vivo nirf sln mapping and of the research of micrometastases in nnd crc patients to propose adjuvant chemotherapy. materials and methods: fifty-eight patients undergoing standard oncological crc laparoscopic resection have been prospectively enrolled in two centre. as previously described by the authors, the intact surgical specimen was extracted and opened longitudinally and ml of indocyanine green (icg; mg/ml) was injected submucosally at four corners around the tumor in order to identify the lymphatic pathway and the slns. each sln presenting as negative at conventional histological analysis, was further investigated with ultrastaging techniques including serial sectioning and additional immunohistochemistry, in order to detect the presence of micrometastases. results: thirty patients were n ? , and were nnd. overall, a total of lymph nodes were retrieved. a total of sln were identified (mean . per case) and of those were nnd. after ultrastaging investigations, micrometastatic cases were found in nnd patients. the patients were so upstaged to n . sln located deeper in the mesenteric and mesorectal fat could easily be identified by nirf (even after nchrt). conclusions: in our preliminary series, the ex-vivo nirf sln mapping rightly predicts the status of loco-regional nodes, as confirmed by the histological investigations. the micrometastases' identification let selected patients to undergo the adjuvant treatment with the aim to reduce the risk of recurrence. ( / ) in lateral node positive group and . % ( / ) in lateral node negative group. four of local recurrence were lateral lymph node recurrence. two patients recurred the other lateral side of previous lpl, then they were laparoscopically resected and no recurrence ( , months). two patients recurred the same side after lpl were not curable because of liver metastasis and extensive invasion to the common iliac vessels. conclusion: selective lpl for rectal cancer was safe and good local control for lateral lymph node positive patients. also curable local recurrence resection was possible for non-treated lateral lymph node recurrence. intestinal malrotation is an embriologic anomaly generally discovered in the first months of life due to bowel obstruction. adult presentation is rare and its association with colon cancer is far more rare. we report a case of a years old man affected by asymptomatic intestinal malrotation incidentally found during an abdominal computed tomography (ct) performed for retroperitoneal colonic perforation in a patient with an endoscopically diagnosed aenocarcinoma of the caecum and a large polyp of the descending colon. preoperative vascular anatomic study allowed us to plan a laparoscopic approach safely also with adequate lymphoadenectomy. the abdominal cavity was entered throught a right flank mm optical trocar on the transverse umbilical line. three additional mm trocars were placed in right iliac fossa, right and left hypocondrium respectively. exploratory laparoscopy confirmed midgut malrotation and a fresh flogistic area at the descending colon perforation site. caecum and ascending colon were on midline and attached due to adhesions to sacral promontory. ileocolic artery (ica), middle colic artery (mca) and ima were selectively ligated but not at their origins due to aberrant anatomy. laparoscopic subtotal colectomy with intracorporeal stapled ileosigmoid anastomosis were carried out (endogia mm, double layer / polyglicolic acid suturing of the breech). the anisoperistaltic nature of the anastomosis is due to the disposition of the mesenterium which did not allow an isoperistalting orientation of the two resected stumps. the specimen was extracted throught a pfannestiel incision. the postoperative course was complicated by intestinal obstruction conservatively treated with slow bowel function's restoration. the patient was discharged from the hospital in th postoperative day. unexpectedly specimen histology revealed two villous adenomas with high grade dysplasia. lymphnodes were retrieved from the specimen (ptisn ). to date our case is the only fully laparoscopic colonic resection reported in literature in malrotation as well as the first intracorporeal stapled ileo-sigmoid anastomosis for such disease. the median hospital stay was days. in-hospital mortality was nil. the overall morbidity was %. the median length of follow-up was months. conclusions: our preliminary results suggest that robotic-assisted surgery for colorectal cancer can be carried out safely and according to oncological principles. robotic surgery is advantageous for both surgeons (in that it facilitates dissection in a narrow pelvis) and patients (in that it affords a very good quality of life via the preservation of sexual and urinary function in the vast majority of patients and it has low morbidity and good midterm oncological outcomes). in rectal cancer surgery, the robotic approach is a promising alternative and is expected to overcome the low penetration rate of laparoscopy in this field. aims: postoperative inflammation have been reported as one of the independent prognostic factors in several types of malignancies.the aim of this study is to clarify the impact of laparoscopic approach on postoperative inflammatory status after surgery for colorectal cancer, and to analyze the association between postoperative inflammation and prognosis in patients with colorectal cancer. methods: a total of patients with stage l-lll colorectal cancer (crc) who underwent curative surgery were retrospectively analyzed. the maximum crp value measured between the times of surgical resection and discharge was defined as 'max crp'. the optimal cut-off value of max crp that best predicts rfs was determined to be mg/dl by the minimum p-value approach. methods: trainees working in this firm were responsible for data collection. patients who underwent emergency surgery during the calendar year of had the following details collected-the presence or absence of a complication in the -day post-operative period, the type of complication and description of complication along with the grade of the complication (see fig. .) . patients who underwent intermediate to major surgery were followed up at outpatients and were specifically asked for the occurrence of complications from the point of discharge up until the outpatient appointment. with one centralised national hospital-the people who were discharged and subsequently experienced considerable or major complications invariably represented back to hospital via the a&e department. results: a total of emergency surgeries were performed by this surgical firm in , % of these being done laparoscopically. of these cases- patients experienced post-operative complications within the first days after their procedure. this equated to a complication rate of . %. the most common complications were abdominal pain, nausea & vomiting, and wound infection. there were complications for each of these categories. post-operative bleeding occurred in cases with fistulas or leak of an anastomosis occurring in cases. death of a patient occurred in instances once as a result of post-operative bleeding from the site of anastomosis after a whipple's procedure, the nd occurred subsequent to post-operative bleeding from a peptic ulcer and in the rd case occurred in an instance of faecal peritonitis as a result of anastomotic failure after a roux-en-y bypass for a patient with pancreatic malignancy. conclusion: the davien-clindo classification proved to be simple, efficient and useful in analysing post-operative outcomes. the results indicate that despite the emergency setting & elderly cohort of patients-minimally invasive surgery proved to be a safe and viable option. conclusions: in this prospective study, we observed greater rates of detection of adenomas among endoscopists. screening colonoscopy on symptomatic and/or high risk group for crc is valuable in early detection and the prevention of crc. large sample size and long period of screening colonoscopy was needed. limitation of our study was the small sample size and no use of high detention endoscopy. results: the mean intraoperative blood loss volume was significantly less in the lap group than in the open group ( vs. ml, respectively, p \ . ). the mean operative time was not significantly different between the lap group and the open group ( vs min, respectively, p = . ). the incidence of severe postoperative complication (grade or higher in the clavien-dindo classification) was lower in the lap group ( / ( %) vs / ( %), respectively). the mean postoperative hospital stay was significantly shorter in the lap group than that in the open group ( vs. days, respectively, p = . ). conclusions: lap-tpe can be a safe and feasible procedure. background: amyloid light chain (al) amyloidosis is a rare protein deposition disorder with an incidence ranging between - cases per million people. it can present insidiously with localized or multisystem symptoms and usually occurs later in life. prognosis is poor as al typically presents at an advanced stage. intestinal pseudo-obstruction is a rarely reported complication of al amyloidosis. here we report a case of al amyloidosis which was identified during surgery for intestinal pseudo-obstruction. case presentation: a year old male presented to the emergency department with a month history of abdominal pain and distension, as well as marked swelling of his lower limbs. this had worsened in the previous weeks and he had developed intermittent diarrhoea. ct showed ileitis with marked dilation of the proximal small bowel. laparatomy revealed small bowel that was grossly distended that rapidly developed multiple petechiae and subsequent haematomas upon handling. two days later a repeat laparotomy was performed and . m of ishaemic small bowel was resected. histology showed amyloid deposition with positive congo red staining. subsequent cardiac events led to an echo being performed that showed concentric left ventricular hypertrophy attributed to amyloid deposition within the myocardium. free serum light chain ratio was sent and confirmed the diagnosis of al amyloidosis. he has recently been started on a treatment regimen consisting of cyclophosphamide and dexamethasone. discussion: systemic al amyloidosis frequently involves the gastrointestinal tract, typically presenting with chronic diarrhoea and associated malabsorption. only case presenting with pseudo-obstruction has been reported in the literature. al amyloidosis presents insidiously with non-specific symptoms depending on which organs are affected. treatment aims to prevent further deposition of protein within the organs. prognosis is determined by the organs that are affected and the extent of protein deposition within them. cardiac involvement holds the worst prognosis ultimately causing sudden cardiac death. the mainstays of management are early identification and treatment implementation to prevent protein build up and subsequent organ failure. conclusion: a diagnosis of amyloidosis should be considered in patients with intestinal pseudo-obstruction to expedite the diagnosis of al amyloidosis and improve survival. aim: in the management of locally advanced rectal cancer (larc), the achievement of a complete total mesorectal excision (tme) with clear resection margins was demonstrated to be the main predictor of overall and disease-free survival. predicting surgical difficulty in larc patients may be of particular importance to choose the best surgical approach. this study proposes a mri-based score to identify preoperatively larc patients with a high risk of having a difficult surgery. methods: this is a retrospective study based on the european mri and rectal cancer surgery (eumarcs) database, including patients with mid-low larc who were treated with neoadjuvant chemoradiation therapy and laparoscopic tme with primary anastomosis. data on pre-treatment and restaging through magnetic resonance imaging were available for all patients. surgical difficulty was defined as high or low grade taking in to account operative (e.g. duration of surgery), and postoperative factors (e.g. hospital stay). score accuracy was evaluated by estimating sensitivity, specificity and area under the receiver operating characteristics curve (aroc). results: seventeen ( . %) of larc patients were graded as high surgical difficulty. the eumarcs score was developed using the following significant predictors of surgical difficulty: bmi [ , interspinous distance \ . mm, ymrtstage = t b, and male sex. the score ranged from to . the cut-off score to best differentiate patients with a high probability of difficult surgery was = points. this cut-off value showed the best balance in sensitivity and specificity. the eumarcs score demonstrated high accuracy (aroc: . ) conclusions: the eumarcs score was found to be sensitive and specific in predicting surgical difficulty in larc patients who were candidate for laparoscopic tme. the score has the advantage of considering patient and cancer related characteristics that can be all assessed preoperatively and it can be useful in the decision making process. this score has not yet been externally validated. background: recently published two non-inferiority randomised control trials has raised questions on laparoscopic surgery for rectal cancer, showing lower quality pathological specimens to those achieved using an open technique. locally advanced rectal cancers add to the level of difficulty for laparoscopy approach. our study was aimed to assess feasibility of laparoscopic rectal surgery, comparing short term outcomes, quality of surgical specimen, morbidity and mortality, between propensity score match groups of locally advanced and early rectal cancers. methods: prospectively acquired data from consecutive patients undergoing laparoscopic surgery for rectal cancer at the minimally invasive colorectal unit in united kingdom between and . locally advanced rectal tumours were identified as t b or t with pre-operative mri scans. all the patients were operated by the same team and the procedures were performed laparoscopically. : propensity score matching was performed to create a perfect match in terms of tumour height. results: total of laparoscopic rectal resections were performed during the study period, out of which patients had locally advanced (la) disease and were propensity-score matched for tumour height with non-locally advanced (nla) patients. median operative time was higher for the la surgery group ( min vs min p = . ). however, conversion to open surgery (p = . ), readmission (p = . ), re-operation (p = . ), clinical anastomotic leak (p = . ) and -day mortality rates (p = . ) were all equivalent between the two groups. r resection was achieved in % of la group as compare to % of nla group (p = . ). conclusion: this study demonstrate that standardised approach to laparoscopy is safe and feasible in locally advanced rectal cancers. comparable post-operative short-term clinical and pathological outcomes were seen between la and nla groups. aims: the application of colorectal cancer screening programs, has showed a decrease in recurrence and mortality. for this reason, these programs are being implemented at a national level in the different spanish regions, as has happened in our community.to present the initial short-term results on the morbidity of the immediate postoperative period to days of colon cancer, mortality and hospital stay after the implementation of a screening program in our center. methods: a retrospective study was performed. patients aged between and years were included in the study, diagnosed with colon cancer. they underwent minimally invasive surgery, in most cases, with any type of colonic resection, from january to december . all patients were diagnosed, conventionally or through a screening program, the latter according to the plan implemented in our community. the sample was divided into two groups of patients according to the way of being diagnosed (group si screening = patients, group no screening = patients) and they were compared according different variables: dependent factors of the patient, factor of type colon cancer, factors of colon cancer resection and follow-up. results: both groups were comparable in all study variables. regarding the variables included in the follow-up, no statistically significant differences were found in terms of postoperative mortality-clavien-dindo v. however we found differences statistically significant in postoperative morbidity (p = . ) and in its classification according to clavien dindo i-iv (p = . ). the complications analyzed independently, such as anastomotic dehiscence (p = . ) or postoperative ileus (p = . ), have also presented significant differences, unlike surgical wound infection (p = . ). conclusion: at our center, the application of the screening program has not influenced in the initial stage of colon cancer or its surgical approach. however, we have found a lower overall morbidity rate and minor complications, justified by a lower incidence of anastomotic dehiscence and postoperative ileus. background: colorectal carcinoma is one of the most common malignancies. surgery is the only definitive method to achieve cure for this illness and can be performed via an open or a laparoscopic approach. the pros and cons of each approach have been discussed extensively, with the oncologic efficiency of the laparoscopic approach being one of the leading topics. objective: the aim of this study was to establish oncological non-inferiority of the laparoscopic approach to colorectal cancer. primary outcome measure was defined as number of harvested lymph nodes. secondary outcome measures were medium-term disease free and overall survival as well as length of hospital stay, time to oral feeding and short-and long-term complication rate. methods: this was a single center retrospective chart review. all consecutive patients who underwent colon or rectal resection due to colorectal carcinoma at hadassah medical center between the years - were included. patients who were operated on for recurrent disease or who had metastatic disease at the time of surgery were excluded. patients were divided into three groups according to the surgical approach: laparoscopic, open or converted. medium-term oncological outcomes were the same for all groups. time to oral feeding, length of hospital stay, short-and long-term complication rate were all significantly improved in the laparoscopic group. conclusions: we were unable to prove non-inferiority of the laparoscopic approach regarding the number of harvested lymph nodes. however, all surgical approaches yielded a high number of harvested lymph nodes which is most probably oncologically sufficient, as reflected by the non-existent difference in medium-term oncological follow up. this study supports previous studies showing the superiority of the laparoscopic approach regarding short term recovery and overall complications rates. aims: two non-inferiority randomised control trials have questioned the utility of laparoscopic surgery for rectal cancer by failing to prove that pathological markers of high quality surgery are equivalent to those achieved by open technique. we intend to present short and long-term postoperative outcomes from the largest single surgeon series of consecutive patients undergoing laparoscopic tme for rectal cancer. we describe the standardised laparoscopic technique developed by the principal surgeon, and the short-term outcomes from three surgeons who were trained in and subsequently adopted the same approach. methods: prospectively acquired data from consecutive patients undergoing surgery for rectal cancer by the principal surgeon (ap) at the minimally invasive colorectal unit in portsmouth between and were analysed along with data acquired between and from surgeons (tq,nf,ah) at three further international centres. end-points were overall and diseasefree survival at years, and early post-operative clinical and pathological outcomes. results: consecutive patients underwent laparoscopic tme surgery by the principal surgeon (ap). at years overall survival was . % (dukes' a = . %; b = . %; c = . %); disease-free survival was . % (dukes' a = . %; b = . %; c = . %). post-operative length of stay, lymph node harvest, mean operating time, rate of conversion, incomplete resection, major morbidity and day mortality were not significantly different between the principal surgeon and those he had trained when subsequently in independent practices. conclusion: laparoscopic tme produces excellent long-term survival outcomes for patients with rectal cancer. a standardised approach has the potential to improve outcomes by setting bench-marks for surgical quality, and providing a step-by-step method for surgical training. results: analysis of association of tumor location (sigmoid, right or left colon), operation time, blood loss, extraction site, type of surgical sutures used for wound closure with postoperative complications or specimen quality either did not show significant correlation or could not be conducted due to data nature. unexpectedly, a significant difference was demonstrated between two surgical teams in terms of hernias. majority of cases- ( . %) were performed by surgeon (s ), surgeon (s ) operated on ( . %) patients, nevertheless minilaparotomy closure was usually performed by junior members of the team. conversion rate was . % for s and . % for s (p = . ). operation time and blood loss were smaller in s group compared to s ( . ± . min vs . ± . min, p = . and . ± . ml vs . ± . ml, p = . respectively). specimen quality and early postoperative complications did not differ. postoperative hernia rate was . % for s and . % for s (p = , ). both surgeons used the same specimen extraction sites and materials for wound closure. hernias were more frequent after vertical minilaparotomy- % ( of patients), and in converted patients , % ( of patients), compared to , % ( of ) in transverse minilaparotomy group. there was no association of hernias and wound infections. conclusions: our study demonstrates, that besides consultant dependent surgical surrogates, steps which are often performed by other members of surgical team (such as wound closure) may contribute to complication rate as well. more thorough supervision of wound closure may be needed. aims: laparoscopic complete mesocolic excision (cme) right hemicolectomy is considered a demanding procedure and it is actually adopted in few centers from the west. the aim of the present study is to analyze the safety of laparoscopic cme right hemicolectomy and to compare its short-term results with standard right hemicolectomy in a single western center. methods: prospectively collected data from patients who underwent laparoscopic cme right hemicolectomy between june and november were retrospectively analyzed (cme group) and compared with data from patients submitted to standard laparoscopic right hemicolectomy between april and november (s group). results: no differences were observed between the cme and the standard right hemicolectomy groups in terms of clinical characteristics. in the cme group, . % of patients were = years old, . % of patients were asa class , . % of patients had = comorbidities, . % of patients had bmi [ and . % of patients had = previous abdominal surgeries. no differences were observed in terms of duration of surgery ( ± min vs. ± min; p = . ) and intraoperative complications ( . % vs. . %; p = . ) between cme and s groups; mean blood loss was lower in the cme group ( . ± . ml vs . ± . ml, p = . ). the percentage of overall ( . % vs. . %; p = . ) and severe (clavien-dindo = ) complications ( . % vs. . %; p = . ), redo surgery ( . % vs. . %; p = . ) and readmission ( . % vs. . %; p = . ) was comparable between cme group and s group. a significant difference was observed in the length of specimen ( ± mm vs. ± mm; p \ . ) as well as in the length of proximal ( ± mm vs. ± mm; p = . ) and distal margins ( ± mm vs. ± mm; p = . ) in favor of the cme group. the number of lymph nodes harvested was slightly higher in the cme group ( . ± . vs. . ± . ; p = . ) as it was for the percentage of cases with less than retrieved lymph nodes ( . % vs. . %; p = . ), although these differences did not reach statistical significance. conclusions: this study represents one of the few western experiences demonstrating the safety of laparoscopic cme right hemicolectomy. cme technique showed good short-term results and better quality specimens when compared with the standard procedure. aim and background: peritoneal dissemination of colorectal cancer (pc) makes the complete resection of cancer lesions impossible. in such cases, multidisciplinary therapy is essential with mainly chemotherapy. preoperative diagnosis of pc is usually uncertain by ct or mri image. for diagnosis of pc needs surgical materials with laparotomy. but the laparotomy and resection of pc with general anesthesia tends to make impossible for immediate chemotherapy. less invasive diagnosis of pc is necessary and expected.endocytoscopy (ec) makes the histological diagnosis with precise images gained by high magnification (x ). as a preliminary examination, ec diagnosis for resected specimens of pc were evaluated. methods: two cases of pc diagnosed in operation were evaluated. under general anesthesia, laparotomy was conducted. peritoneal dissemination lesions obviously diagnosed as pc were resected. immediately the lesions were stained by methylene blue solution for to s. ec observation was done according ec classification ) and ecv classification ). results: in two cases, ec observation was successfully done. images of dilated surface microvessels of a nonhomogeneous caliber or arrangement were observed in nbi ec corresponding to ec-v . histopathological diagnosis of resected specimens was metastatic colorectal carcinoma in peritoneum in both cases. conclusions: histological diagnosis for pc is gained by ec with resected specimen. as the result of this investigation, ec examination via camera port in laparoscopic operation might be possible for diagnosis for pc of colorectal cancer in vivo. aims: the aim of this presentation is to demonstrate and analyze surgical complications, arising during laparoscopic colorectal resections for cancer and to analyze the reasons of adverse events. methods: we demonstrate videos from our surgeries, where different types of complications occurred and share our classification of types of mistakes, that may lead to intraoperative complications and ways to prevent them. results: we divide mistakes in laparoscopic colorectal resections into two large groups-'false strategy' and 'dangerous techniques'. the first includes poor diagnosis, too extensive or insufficient extent of surgery and improper enthusiasm in using platforms. prevention of first type mistakes is in thorough training and peer-review of each consultant practice. second type of mistakes includes two subtypes : 'faulty habits'-use of unsafe techniques (blind port insertion, poor vascular exposure prior to clipping, not obtaining 'critical views', unsafe use of energy and stapling devices etc.) and 'failure in a certain case'-when despite correct general approach a complication occurred (misinterpretation of fascial layers or vessels). prevention of 'faulty habits' lies in supervised training in high volume colorectal departments including dedicated surgical devices training. to avoid 'failure in a certain case' standardization of surgical procedure is essential, as the most efficient way to prevent this type of mistake is 'pattern recognition'ability of a surgeon to compare the picture he sees during a procedure with a 'standard' view, he used to have during previously performed standard surgeries-this is apparentely impossible when every procedure is done differently. regular reviews of own surgeries recording and other surgeons' procedures may also fascilitate pattern formation. in case a complication occurres we use the four step course of action: preservation of the view, temporary control, decision on conversion, permanent control. conclusion: as popularity of laparoscopic colorectal resections is growing rapidly the number of intraoperative compliactions is increasing as well. we demonstrate videos of complications and our approach to classification of possible mistakes. systematic aproach to reasons, underlying certain mistakes helps to produce a strategy to reduce intraoperative complication rate. introduction: the drains placement inside the abdominal cavity has traditionally been carried out to evacuate hematic remains or postoperative collections. there is no scientific evidence of the prophylactic use of drainage in elective colorectal cancer (ccr) surgery to avoid anastomotic complications or other complications. however, it is traditionally used. when the anastomotic leak is produced, it is generally agreed that drainage system should be used for therapeutic purposes. aims: the aim of this study is to evaluate the effectiveness of the use of prophylactic drainage in elective surgery of ccr. we would check if they avoided the appearance of complications, and if they are useful when the anastomotic leak appears. methods and results: we analyzed the data collected in our hospital from / / to / / . we studied the number and type of interventions in which prophylactic drainages were placed, the appearance of anastomotic complications and if these drains were effective. interventions were performed during this period of time. % of these procedures had used prophylactic drainage ( interventions). this percentage was up to % in patients who have performed a left colon surgery as a sigmoidectomy or rectal procedure. during this period, there were cases of anastomotic leakage. in all of them had been placed drainage but only of them were effective. conclusions: we have seen that prophylactic drainage is a common practice independently of the location of the anastomosis. the last multimodal rehabilitation guidelines recommended the nonuse of drains systematically above the peritoneal reflection with a high level of scientific evidence. they cause discomfort to the patient and delay early mobilization. however, it may be useful to use drains in the first h of a pelvic floor procedure. there is not enough evidence to show sistematic drainage after colorectal anastomosis prevents complications of the anastomosis or other complications. aims: colonic cancers of the splenic flexure is uncommon and associated with poor prognosis. several studies were published aimed to identify the optimal surgical option for the best oncological outcomes. however, whether an extended colectomy or a segmental resection is required is still controversial. the aim of this study is to analyse the outcome of the two different approaches through the experience of a single centre. materials and methods: retrospective data of consecutive patients with diagnosis of colonic cancer situated at the splenic flexure of our department between and were analysed. based on type of surgical procedure, patients were enrolled in arm a (segmental resection) and arm b (extended resection). arm a patients were treated with segmental resections with a wide mobilisation of the transverse and descending colon and ligation of the left colic artery, sparing the middle colic artery and the inferior mesenteric artery. functional lateral to lateral anastomosis was performed extracorporeally. arm b patients were treated with more extended colectomies, both associated with central vascular ligation. results: out of patients included, were allocated in arm a and in arm b. patients' population of the arms was homogeneous as concerns demographic characteristics and stage of the disease. operative time was comparable ( , min vs min, p = , ). the length of the specimen was significantly shorter in arms a ( , vs , , p = , ) . the number of harvested lymphnodes did not differ between the two groups ( , vs p = , )postoperative short term complications was comparable in both arms ( vs , p = , ). no postoperative mortality was observed. overall -year survival and disease free survival rates were similar in arm a and b ( . % vs . %, p = , and , % vs , %, p = , ). hospital stay was similar in the two groups (p = , ). conclusions: despite a shorter length of surgical specimen after limited resections, postoperative complications, lymph node harvest, and survival were comparable in both.in our opinion the extracorporeal anastomosis is functional to both the achievement of a cleaner operative field and a better control of the resection margins. incidence of neuroendocrine tumours in the rectal area has increased in recent years.before the onset of minimally invasive colorectal surgery, these lesion had to be treated by a more radical technique when not suitable for endoscopic resection.selection of the cases is mandatory in order to achieve good results not only surgical, but also oncological. we present our series of neuroendocrine tumoirs treated by tamis approach, including technical aspects, deffect closure techniques and data regarding pathological findings.all cases were low grade carcinoid tumours. resection with free margin was obtained in all cases. defect closure was performed in all cases. the tumours were settled , and cm form the anal verge. postoperative course was uneventful, ann no adyuvant therapy was needed.tamis apporach for rectal neuroendocrine tumours is a safe and feasible technique. proper selection of the cases is mandatory in order to achieve good results. surg endosc ( ) aim: to assess the safety and efficacy of single layer of barbed vs double layer 'hybrid' (interrupted and running) suture for the closure of anastomotic stapler access enterotomy after laparoscopic right colectomy with intracorporeal anastomosis. methods: from april to november , laparoscopic right colectomy with intracorporeal anastomosis were performed in our surgical department. all patients in both groups were perioperatively managed using an eras pathway. seventy-two patients had the enterotomy closed with a single layer running suture of filbloc tm (assut europe). these patients were matched with patients who underwent intracorporeal right colectomy with enterotomy closed with a 'hybrid' double layer technique (first layer interrupted stitches in maxon tm - (covidien), second layer using a running suture in pds tm - (ethicon). intraoperative variables, anastomotic leak rate, morbidity and mortality rates were analyzed. results: the two groups were homogeneous with respect to demographics, body mass index (bmi), american surgical association score (asa) as well as for tumor stage. in the barbed group, median operating time was . min vs . min in the hybrid group (p = . ). anastomotic leak occurred in ( . %) patients in the hybrid vs ( . %) patients in the barbed group (p = . ). all patients required a reoperation. intraoperative findings show in ( . %) cases in the hybrid group a leak at the enterotomy closure, while an intact staler access was observed in both patients in the barbed group. no difference was observed with respect to noninfectious complications between the two groups (p = . ). patients in the hybrid group experienced a longer hospital stay when compared to the barbed group (p = . ). a re-admission occurred in the hybrid due an intraabdominal collection, while no re-admission was observed in the barbed group. no patient died in the postoperative period. aims: lymph node status is one of the key prognostic factors in patients with colorectal cancer, and remains the most important selection criteria for adjuvant chemotherapy. it is believed that at least % of node negative patients will suffer disease recurrence within the first years after surgery. this may be due to understaging lymph node status. sentinel lymph node mapping is widely used for staging of breast cancer and melanoma, with injection of colloid tc and isosulfan blue (ib). however, indocyanine green (icg) fluorescence guidance is a new technical approach to this issue, with promising results as it is not influenced by body mass index or lymphatic invasion. intraoperative fluorescence icg navigation also aims for detection of aberrant lymphatic drainage outside the planned resection. the icg lymphography has the advantage of offering a good visualization of the lymphatic channels but there are problems to identify the lymphatic nodes. our objective with this study is to rate the use of the intraoperative lymphogram in cases of elective colorectal surgery to evaluate if there were changes in the surgical attitude regarding the performance of lymphadenectomy. methods: indocyanine green was injected into the submucosal layer around the tumor at points with?a -gauge localized injection before lymph node dissection and the lymph flow was observed at , and min after injection, using a near-infrared camera system. in addition, a complete mesocolic excision with central vascular ligation guided the region where the lymph flow was observed to be fluorescent. the following table summarizes the procedures carried out as well as the lymphadenectomy performed before and after the use of icg. in brief, after the application of intraoperative icg it was observed that in % of patients additional lymph nodes were obtained after the expansion of the surgical plan, moreover % affected lymph nodes were spotted after the expansion of the surgical plan. conclusions: intraoperative real-time visualization of the lymph flow using indocyanine green fluorescence imaging during laparoscopic colon cancer surgery is feasible and a helpful technique for lymph node mapping which may lead to intraoperative changes in lymphadenectomy. tamis resection of rectal tumours has proven to be a sefe and feasible technique, specially for lesion located in the mid and low rectum.when the tumour is located in the upper rectum, and specially near the colorectal junction, tamis resection may be more difficult, not only due to technical aspects, but also due to the risk of a free perforation, specially when a full thickness resection is performed.we present our results of tamis resections of lesions located around the colorectal junction. four resections where performed with the aim of an endostpaler in order to achieve full resection without the risk of a free colonic perforation.in cases, an abdominal combined laparoscopic exploration was made, in order to help and assure proper resection of the lesion as well as avoiding intraoperative complications.distance from the anal verge ranged from to cm.postoperative course was uneventful in all cases, and a complete specimen resection was obtained in all cases.tamis resection of tumours located in the rectosigmoid junction may be a safe and feasible technique in selected patients. methods: between january to april , patients with diagnosis of right colon adenocarcinomas underwent right hemicolectomies. the data was analysed for patients demographic, histology, type of surgical approach, intraoperative details (length of surgical procedure, blood loss, blood transfusion, conversion rate) and short-term post-operative outcomes including complications. introduction: postoperative ischemic colitis is a life-threatening vascular gastrointestinal condition, that mainly occurs after cardiovascular surgery. we present a surprising case following a laparoscopic rectum resection. case report: a -year-old diabetic patient with upper rectal adenocarcinoma undergoing laparoscopic anterior rectal resection (partial mesorectum excision) and mechanical anastomosis following chemotherapy / radiotherapy. after h postsurgery he presented abdominal pain, distension and fever. on adominal computed tomography (ct) scan (contrast enema) no anastomotic leakage (al) finfings were revealed. neither digital palpation nor proctosigmoidoscopy ( th day) showed al signs. the patient clinical situation improve with conservative treatment (antibiotics, digestive rest …), c-reactive protein levels decreased and the blood cultures were negative. on the th day he was discharged presenting semiliquid stools. eight days later he needed hospital readmission: air and feculent/purulent discharge from the previos abdominal drainage orifice. ct scan: no evidence of dehiscense found although rectum and sigmoid colon distention and an image of a 'large fecaloma' were observable. on the th day of hospitalization he expulsed a large malodorous segment of tissue with necrotic asppearance (image) through the anus with surprising histologic features: 'complete-thickness necrotic colonic wall'. further rectosigmoidoscopy: complete anastomosis, signs of ischemic colitis proximate to the anastomosis and a fistulous orifice. surprisingly, the patient progressed favorably, being discharged the th day for ambulatory control with a low debit enterocutaneous fistula. histopathological diagnosis: ypt n m . follow up: the fistula discharge quantity increased maintaining diarrheal stools through anus along with persistent anemia and malnutrition. a exploratory laparotomy was schedule. fistulous tract towards a small stenotic segment of colon inmediately proximal to the colorectal anastomosis was identify and resected. finally a terminal colostomy was performed. subsequent postoperative without incidents. currently the patient is asymptomatic. comments: it seems indisputable that a colon segment, proximal to the anastomosis, was necrosed and expelled through a colorectal anastomosis. the mechanism seems inexplicable to us. it is even more disconcerting that there was no disruption of the anastomosis. objectives: fluorescence-guided surgery has emerged as a new imaging modality to improve the detection of liver and lymph node metastasis in colorectal cancer. in right-sided colon cancer, the standard lymphadenectomy should reach the ileocolic vessels and the right branch of the middle colic vessels. the purpose of this study is to perform an objective estimation of lymphatic drainage and metastatic lymphonodes in right-sided colon carcinoma through indocyanine green (icg) lymphography. methods: patients with right-sided colon adenocarcinoma were included, excluding those in stage iv, t and those who underwent urgent surgery. cc of icg peritumoral were injected using a peripheral intravenous catheter at the beginning of the intervention. the lymphatic drainage mapping of the tumor was identified. lymphadenectomy of the ileocolic vessels and right branch of the middle colic vessels was performed extending it to the left branch and origin of middle colic vessels if it was shown in the mapping. results: patients were included. the average age was . in patients the tumor was located in the ascending colon and in patients in the hepatic angle. in patients, the mapping showed lymphatic drainage to ileocolic vessels and right branch of the middle colic vessels. in patients ( %) it showed drainage to the left branch and origin of the middle colic artery, therefore extended lymphadenectomy was performed at that level. in patients, the postoperative period was uneventful. patient presented infection of the surgical wound and another patient developed a cm perianastomotic collection treated with percutaneous drainage. the anatomopathological report showed nodal metastasis in of the patients ( %) in whom lymphatic drainage was observed in the territory of the middle colic vessels with icg. these patients presented the tumor in the hepatic angle. therefore, of the patients with right-sided colon carcinoma ( %) presented nodal metastasis in the territory of the middle colic vessels. conclusions: fluorescent lymphography may improve the results of lymphadenectomy in colon cancer. in patients with tumors of the hepatic angle, lymphadenectomy extended to the left branch and origin of middle colic vessels, could be an adequate alternative. introduction: over the last decade, the common principles of surgical treatment in colon surgery are central vascular ligation (cvl) and complete mesocolic excision (cme). however, the superior mesenteric vessels anatomy, while performing the right colectomy is characterized by wide variability, which can lead to complications, especially during minimally invasive surgical intervention. objective. the purpose of this study is describing vascular variations around the superior mesenteric artery and vein-middle colic, right colic and ileocolic vessels, henle trunk in the laparoscopic right colectomy. materials and methods: the study was held in the 'dobrobut' clinic and o. o. bohomolets national medical university, department of general surgery (kyiv, ukraine) during the - period. patients were included to the study, females ( . %), males ( . %) in the average age of , ± , years. all the patients underwent the laparoscopic right colectomy (cme ? cvl) with d lymph node dissection. recorded video materials from each laparoscopic right colectomy were analyzed during the study. results: ileocolic vessels were the most stable. there were typical anatomical position in all cases. . % of cases, ileocolic vein was identified anteriorly to the ileocolic artery, while . % being posteriorly. right colic vein was absent in . % of cases. right colic vein drainage was to henle trunk and inferior mesenteric vein in . % and . % respectively. the right colic artery was present in % of patients, it's origin was superior mesenteric artery in . % and . % the middle colic artery. the middle colic vein was present and drained to superior mesenteric vein in % of cases. same as the middle colic artery with the superior mesenteric artery origin. henle trunk was present in . %, gastro-pancreato-colic trunk in . % of cases gastro-pancreatic trunk in . %, gastro-colic in . %. conclusions: knowing the options of surgical vessels anatomy, while performing the right colectomy, altogether with surgeons preparation, using the ct-scan data can reduce the risk of iatrogenic damage and complications risks. introduction: the enhanced recovery after surgery (eras) protocol was designed to accelerate convalescence, reduce morbidity and shorten the length of hospital stay (los). one of its major interventions is balanced perioperative fluid therapy. the impact of this single intervention on short-term outcomes is widely discuss. aim: the aim of this study was to assess the impact of perioperative fluid therapy on short-term outcomes. material and methods: the analysis included consecutive prospectively registered patients operated laparoscopically for colorectal cancer between november and january . patients were divided into two groups: balanced (= ml) or unbalanced ([ ml) perioperative fluid therapy. all patients were treated according to eras protocol. study outcomes were: recovery parameters, morbidity rate, los, -day readmission rate. results: group consisted of and group of patients. there were no statistically significant differences between the groups in terms of demographic and operative parameters. morbidity was lower in group ( . % vs . %, p = . ). patients in group were discharged home earlier than in group ( vs days, p \ . ). moreover, we observed differences in recovery parameters between the groups: tolerance of an oral diet on the st postoperative day ( % vs. %, p = . ) and patient mobilization on the day of surgery ( % vs. %, p = . ). -day readmission rate was lower in group ( . % vs. %, p = . ). conclusion: a balanced perioperative fluid therapy on the day of surgery may be associated with faster convalescence, lower morbidity rate, shorter los and lower -day readmission rate. methods: a retrospective analysis was performed including patients who underwent lcs or ocs for cancer treated as emergency in a single centre between and . patients who underwent palliative surgery were excluded. lcs were : propensity score-matched based on pposum and stage of disease with ocs. short-term outcomes included oncological quality, length of hospital stay (los) and postoperative mortality. for long-term outcomes, -year overall and disease free survival (os and dfs) rates were analyzed. results: during the study period, a total of emergency colorectal resections were performed. of them, % (n = ) were coloniccancers. lcs were matched to an equal number of ocs.median age was ( ) years and % were females. median follow-up was ( ) months. the majority of resections were right hemicolectomies ( %), followed by sigmoid resections ( %) and subtotal colectomies ( %). operative time ( ( ) background: total mesorectal excision (tme) offers the best reported rates for local recurrence and survival in patients with rectal cancer. our series from a single high-volume center, assessed the feasibility, safety and long-term oncologic adequacy of laparoscopic total mesorectal excision methods: we reviewed the prospective database of consecutive unselected patients undergoing laparoscopic tme for rectal cancer between and at the department of general surgery, onze-lieve-vrouwziekenhuis hospital (olv), campus aalst, belgium. the objective of the present study was to evaluate the effectiveness of laparoscopic tme, with an emphasis on perioperative variables and long-term oncological outcomes. results: pts with mid and distal rectal cancer up to cm from the anal verge had laparoscopic tme resection. patients ( . %) underwent a sphincter-preserving surgery and the remaining patients ( . %) had an abdominoperineal resection. end-to-end anastomoses: pts ( %), j-colonic pouch: pts ( % introduction: the rica clinical pathway (intensified recovery in abdominal surgery), also called surgical multimodal rehabilitation, is the application of a series of perioperative measures and strategies in those patients who are going to undergo a surgical procedure with the objective of reducing secondary stress to the surgical intervention. in this way, we achieve a better recovery of the patient and significantly reduce complications and morbidity. objective: s to analyze, through our database of patients undergoing crc, the percentage of postoperative ileuses and the following quality indicators: the postsurgical hospital stay, the anastomotic leak, and the infection of the surgical site. to check if the implantation of the rica pathway has meant an improvement in our postoperative hospital stay and with that, a lower sanitary cost. methods and results: we analyzed the data collected from those patients who underwent ccr in our hospital between / / and / / , during which time we implemented the rica clinical pathway. the average hospital stay was days. of the patients, . % presented anastomotic leak, . % infection of the surgical wound and . % paralytic ileus. we have verified how the average hospital stay increases with the appearance of anastomotic leak ( . days), infection of the surgical wound ( . days) and paralytic ileus ( . days). when we divided this -month period into two halves to see the impact of the implantation of the clinical pathway, we obtained the following results: the post-surgical hospital stay in the period from / / to / / was . . the stay from / / to / / was . . the implantation of the rica clinical pathway is providing us with important advantages in our clinical practice, with greater postoperative comfort and an improvement in our quality indicators, such as the decrease in the average hospital stay of our patients. on the other hand, after starting its implementation we have encountered the resistance to change clinical habits and the one that requires a multidisciplinary participation, so adherence to this is being progressive, and requires periodic audits to reinforce and consolidate our achievements, and identify our points of improvement. however, tem has not yet achieved widespread use. recently, transanal minimally invasive surgery (tamis) using single-port surgery devices has been reported. initially facilitated by existing single-port surgery devices, two platforms for transanal access, the gelpoint Ò path (applied medical, rancho santa margarita, ca, usa) and the sils tm port. the gelpoint Ò path is the only platform to be specifically designed for tamis y tatme. objetive: in the present study, usesa gelpoint Ò path was performed in patients with lower rectal neplasms. results: complete full-thickness excision was performed in all cases of tamis and free margins over rectal cancer. on two cases no neoplasm was visualizad. the patient characteristics, operative techniques and operative outcomes were evaluated. the mean age of the patients was . years (range - ). the mean operating time were min (range - ). patients was selsted for tatme, for tamis and two patients for evaluation and biopsy if was necesary. additional transabdominal rectal resection was not performed, and adjuvant chemoradiotherapy was performed in all cases. tamis using a gelpoint Ò path was revealed to be easy and safe to perform. although only a small number of cases were treated, and the operation was demonstrated to be sufficiently feasible. conclusion: gelpoint path is a good tool for colorectal surgery in tatme, tamis and evalluation of anastomoses or de novo lesions introduction: several improvements in rectal cancer treatment, in the last decades, resulted in a markedly increased survival. nevertheless, surgery remains the prevalent treatment and to % of operated patients experience some kind of functional abnormalities. as nowadays we acknowledge the importance to focus not only on survival rates but also on quality of life, we craved for a precise, reproducible, simple, clear and user-friendly tool for evaluating bowel function in rectal cancer patients after sphincter saving operation. therefore, we performed a thorough translation with cultural adaptation of the patient reported outcome tool, low anterior resection syndrome (lars) score, to the portuguese language (lars-pt) and population. methods: according to the current international recommendations, we designed this study encompassing three main phases: (i) cultural and linguistic validation to european portuguese; (ii) feasibility and reliability tests of the version obtained in the previous phase; and (iii) validity tests to produce a final version. the questionnaire was completed by patients from six portuguese colorectal cancer units, and completed it twice. results: the portuguese version of lars score showed high construct validity. regarding the test-retest, the global intraclass correlation showed very strong test-retest reliability. looking at all five items, only items and presented a moderate correlation. lars score was able to discriminate symptoms showing worse quality of life in patients submitted to preoperative radio and chemotherapy. conclusion: lars questionnaire has been properly translated into european portuguese, demonstrating high construct validity and reliability. this is a precise, reproducible, simple, clear and user-friendly tool for evaluating bowel function in rectal cancer patients after sphincter saving operation. therefore, his sistematic use should be implemented. oesophagectomy is the mainstay of curative treatment for oesophageal cancer and post-oesophagectomy diaphragmatic hernia (podh) represents a potentially life-threatening surgical complication characterized by an underestimated occurrence rate and unknown related risk factors. this study analyses the experience of two tertiary designated centers in order to evaluate key elements concerning development and treatment of podh. a cohort of consecutive patients affected by a clinically resectable oesophageal cancer (any t, any n and m ) underwent ivor-lewis oesophagectomy between march and april according to three different approaches: totally open incision procedure (oilo), hybrid (hilo) and totally mininvasive to esophagectomy (milo). all population was retrospectively observed in the context of a postoperative calendarised follow-up in order to record the incidence and postrepair results of podh. patients underwent ivor-lewis oesophagectomy for cancer and ( . %) developed podh within a median follow-up period of months ( - ). surgical repair was generally applied by the mean of laparoscopic cruroplasty ( %) with a conversion rate of %. postoperative morbidity did not include early recurrences but exclusively cardio-pulmonary complications ( patients) with one case of respiratory failure leading to death. the discharge was reached after a median hospital stay of days ( - ) while recurrences ( %) occurred over a median followup period of . months. a wide univariate analysis identified statistically significant associations between podh occurrence and the administration of preoperative chemoradiotherapy, the complete pathological response (cpr) and a lymph node harvest (lnh) larger than stations (p-value of . , . and . respectively). the strong influence of an extended lnh was confirmed by the multivariable analyses ( . ) along with cpr which should however be considered as longer survival-related bias. the minimally invasive surgery and the neoadjuvant chemoradiotherapy represent a considerable part of multimodal treatment for oesophageal cancer presenting a not statistically significant association with podh development while a lnh including more than nodes resulted to be an independent risk factor mirroring the extent of surgical demolition in oesophagectomy. l. barbulescu aim: to asses the safety and effectiveness of robotic total meso-rectal excision vs laparoscopic total meso-rectal excision and to analyse the primary outcomes. methods: the operative, post-operative and oncological outcomes were evaluated to assess the effectiveness of both techniques of tme. in our center were performed robotic rectal resections and laparoscopic resections from january to present. results: the rtme was associated with longer operation time, early bowel movements, lower risk of conversion and shorter hospitalization. the statistical equivalence was seen between rtme and ltme for non-oncological variables like blood loss, morbidity and reintervention risk. the oncological variables such as number of harvested nodes and positive circumferential resection margin risk were also comparable in both groups. the length of distal resection margins was similar in both groups. conclusion: rtme in patients with rectal cancer was associated with a lower rate of conversion and less incidence of urinary retention. the operative time in rtme was significantly longer than in ltme. the initial oncological and function outcomes of rtme seem to be equivalent with ltme. c. athanasiou aims: two randomized controlled trials failed to show non-inferiority of the laparoscopic total mesorectal excision (ltme) compared to open. ltme becomes particularly challenging in low rectal cancers and in narrow pelves. many surgeons report that robotic tme (rtme) may be beneficial in that setting. our aim was to systematically review the literature and compare the pathologic outcomes of open, laparoscopic and robotic tme for rectal cancer methods: medline, embase, scopus, cochrane library and web of knowledge databases were searched for randomized controlled trials (rct) reporting patholologic outcomes of open, laparoscopic or robotic tme with no language restriction. our primary outcome was quality of tme on macroscopic assessment of the specimen. secondary outcomes included positive circumferential resection margin, distance to radial margin, number of lymph nodes and positive radial margin. the included studies were quality assessed and the jadad score was reported. the grade approach was used to rate the certainty of each network estimate. results: fourteen rcts were included in our study. seven rcts compared the otme to the ltme, six compared the ltme and rtme and one study the otme to the rtme. no statistical significant difference was found in quality of tme when the the ltme was compared to the otme or = . ( . , , ) or the rtme or = . ( . , . ) . no difference was found in pcrm for the laparoscopic or = . ( . , . ) or the robotic approach or = . ( . , . ) when compared to open. distance to radial margin and number or lymph nodes didn't differ between the groups. conclusions: no significant advantage on pathologic specimen quality has been found with the robotic approach. the ltme doesn't seem to compromise the quality of the specimen. h. samura , j. arakaki , k. sugata , y. hori , y. nagamine , f. kohagura , h. motonari , s. kameyama , t. ishimine division of digestive and general surgery, urasoe general hospital, okinawa, japan; department of surgery, urasoe general hospital, okinawa, japan colorectal cancer often invade adjacent organs and it is known that prognosis improves with resection of the involved organ. we report our experience of invaded adjacent organ resection, which include seminal vesicle, uterine and bilateral appendages, posterior wall of the vagina and bladder wall. method: although the range of resection is predicted by image study preoperatively, at the time of operation, it was decided by palpation with a forceps. each operation is evaluated by operation time, blood loss, blood transfusion volume, postoperative complication, postoperative hospital stay, and short term prognosis. result: resection cases of seminal vesicle, posterior vaginal wall, uterine and bilateral appendages and bladder wall were , , and , respectively. the results are shown in the order of seminal vesicle / vaginal posterior wall / uterine / bladder. median age was , , and years old. the median operation time was , , , min, the median blood loss was , , , ml, and only one case of uterine and bilateral appendages resection required the blood transfusion. the average postoperative hospital stay was , , , days. nine cases have postoperative complication, that include delayed wound healing, anastomotic leakage and rectovesical fistula, postoperative ileus, chyle ascites and neurogenic blodder. all of those were improved with conservative treatment. the mean hospital stay in complication cases was days ( - ) and ( - ) days without complications. the median observation period was days ( - ), and there was no local recurrence. all of the case of stage iv were dead. there was no local recurrence and all patient without stage iv are alive, it seems that the resection range was sufficient. conclusion: even with adjacent organ invasion colorectal cancer, it was possible to determine the resection line by palpation with laparoscopic forceps manipulation, and possible to resect margin free of cancer. laparoscopic low rectal resection with/without diverting ileostomy p. ihnát, m. tesar, p. ostruszka, p. gunková, p. vávra background: the construction of diverting ileostomy (di) is recommended to avoid septic complications of anastomotic leakage. the aim of our study was to assess the benefits and risks of di constructed during laparoscopic low anterior resection (lar). methods: retrospective clinical cohort study was conducted in university hospital ostrava, czech republic. all patients undergoing laparoscopic lar with tme because of rectal cancer within a -year study period were assessed for study eligibility. results: a total of patients ( patients without di, patients with di) after laparoscopic lar were enrolled into the study and underwent analysis. both study subgroups were comparable in terms of demographic and clinical features. postoperative -day morbidity was significantly lower in patients without di ( . % vs. . %, p = . ). anastomotic leakage frequency was higher in patients without di ( . % vs. . %, p = . ); surgical intervention was necessary in . % of patients without di. stoma-related complications were noted in . % of patients with di; some patients had more than one complication. surgical intervention because of stoma-related complications was needed in patients ( . %). distinctive complications of di laparoscopic construction (small bowel obstruction due to di semi-rotation around its longitudinal axis) was noted in patients ( . %). mean stoma period (interval between lar and di reversal) was more than months in our study; only . % of patients were reversed without delay (= months). postoperative morbidity after di reversal was . %; re-laparotomy was needed in . % of patients. conclusions: despite benefits of di in protecting low rectal anastomosis, ileostomy construction remains fraught with many stoma-related complications and long stoma periods associated with significantly decreased quality of life. aims: single port laparoscopic is a minimally invasive surgical technique that joint the cosmetic advantages with the well recognized benefits of the standard laparoscopic approach [ ] . we describe a laparoscopic single port hartmann reversal in a patient by the use of the umbilical colostomy site for surgical access [ ] . methods: a years old patient was submitted to a laparoscopic single port hartmann procedure with an trans-umbilical colostomy for a recurrent sigmoid volvulus that was treated at the beginning by endoscopic de-rotation. after three months the patient was reevaluated for a hartmann reversal with a laparoscopic single port technique. after routine skin preparation and laparoscopic setup, the colostomy is mobilized from its mucocutaneous border, and the anvil of a circular stapler is secured to the distal lumen. by the use of a gelpoint system with trocars, the intra-abdominal adhesiolisis in performed. the splenic flexure is mobilized to achieve a sufficient mobilization of the left colon that allows the fashion of a tension free anastomosis. the rectal stump is mobilized to the mid rectum, starting from the posterior mesorectal fascia around to the anterior rectal wall. a tension-free colorectal anastomosis is secured with a standard circular mm stapling device inserted transanally. the colostomy wound is closed. the operative time was min. results: the postoperative course was uneventful, the patient was discharged at forth postoperative day, oral intake started on postoperative day three. conclusions: single port laparoscopic hartmann reversal thought the umbilical stoma site is a minimally invasive surgical option that is safe in selected patients and offer the best cosmetic results. [ the progressive evolution of surgical techniques and oncologic protocols on rectal cancer disease facilitates surgeons to challenge the skills for anus preservation in low rectal cancer surgery. the laparoscopic surgery is already one of the best ways to reach the pelvic floor and to try procedures, which were previously difficult to apply through open surgery. the anastomotic leakage has particularly high occurrence if the anastomosis is performed in the anal or distal rectum area. it is evident that although the fecal diversion does not decrease post operatory mortality, it significantly reduce the risk of anastomotic leak and the risk of a second major surgery when the leak occur. diverting stomas are low-risk procedures from a technical point of view, but they potentially expose the patients to postoperative morbidity, impacting the patients' quality of life. it is not easy to decide whether the fecal diversion is needed or not. this decision must be made on a case to case basis, trying to apply the stomas only when they are really needed. we report our initial experience by living a transmesenterial cotton loop around the pre terminal ileum which extremities are turned out usually through the lateral trocar wound in laparoscopy or by applying a dedicated mini incision in open surgery. the purpose is to perform (in case of suspected fistula), a mini invasive diverting procedure, by widening the loop wound and by pulling up the ileum in a lateral loop ileostomy. we applied this procedure to consecutive patients with low colorectal anastomosis and in two of them we performed a lateral loop ileostomy with good results. we believe this can be an alternative that needs to be standardized. purpose: sarcoidosis is a chronic, multisystem inflammatory disorder with unknown aetiology characterised by noncaseating granulomas within involved organs. gallbladder involvement in sarcoidosis is extremely rare and literature review revealed only reported cases to date. in this paper, we present a case of gallbladder associated sarcoidosis. method: a -year-old lady was known to the clinic for regular surveillance of liver steatosis and incidental gallbladder polyps. the largest polyp was mm at presentation in and has grown to mm in . in view of worsening symptoms of biliary colic and growing polyps, a laparoscopic cholecystectomy was performed. results: laparoscopic cholecystectomy was unremarkable and specimens of the gallbladder and lymph nodes were sent for histology. histological examination revealed chronic cholecystitis with polypoid cholesterolosis of the gallbladder and noncaseating granulomata within a lymph node, which strongly suggest sarcoidosis. conclusion: in conclusion, we report a case of incidental finding of gallbladder sarcoidosis over the course of treatment of biliary colic and symptomatic gallbladder polyps. therefore, the definitive treatment for patients with symptomatic gallbladder sarcoidosis is a cholecystectomy. the surgical management of cholelithiasis can be associated with significant morbidities. despite the relatively low incidence of bile duct injuries during laparoscopic cholecystectomy, the total number is large due to the high frequency of the operation. the subtotal cholecystectomy with its variants is a well known bailout strategy to the surgical community. however, there is no agreement on when and how to perform these procedures. indeed, the majority of surgeons will adopt these solutions when there is a struggle to identify the critical view of safety. this struggle results increases the risk of injuries. we hypothesize that a primary intent gall bladder lithotomy and disconnection (glad) when the dissection of the gb pedicle is anticipated difficult dissection is a safe and feasible strategic option. methods: out patients elevtively admitted to aberdeen univesity hospital with gall stone disease between march and november , consecutive patients were operated with glad procedure based on intraoperative criteria. the primary outcome was the operative time. secondary outcomes were length of hospital stay, the criteria to do this procedure will be explained, the outcomes will be listed. indocyanine green is a molecule that becomes fluorescent when excited struck with light of a specific wavelength in the infrared spectrum (nir-infrared), allowing the visualization of anatomical structures in which it has accumulated. the aim of the study is the application of icg enhanced fluorescence in laparoscopic cholecystectomy in order to identify the anatomy of the biliary tract, to reduce the risk of iatrogenic lesions and the conversion rate. the study involves laparoscopic cholecystectomy for cholecystitis and gallstones of main biliary tract. the evening before the surgery, a vial of icg ( mg) diluted in ml of saline solution was intravenous injected. during the procedure, after opening the calot triangle, switching to the nir mode on the camera, the anatomy of the biliary tract and in particular of the main biliary tract is visualized. the cystic duct and cystic artery are isolated, their section between clips is cut and the cholecystectomy is performed. from january patients were enrolled: cases of acute cholecystitis, cases of gallstones of main biliary tract, undergoing preoperative ercp. in cases of cholecystitis, the angiography allowed the visualization of the main biliary tract. in one case, an abnormal course of the cystic duct was identified. in two cases of gallstones of common bile duct, it favoured the visualization of the biliary tract anatomy. all cases were completed with laparoscopic technique. there were no intra-and post-operative complications. icg-enhanced fluorescence is a safe, effective, cheap and rapid tool that can also be applied in small hospitals with no need for training. its use does not extend the time of surgery and allows the visualization of the anatomy of the biliary tract, especially in situations where it can be altered by reducing the conversion rate and potentially the risk of iatrogenic lesions of the main biliary tract. case presentation: patient is a year old female with no significant past medical or surgical history presented to the emergency department with a day history of worsening sharp right upper quadrant pain with associated nausea, vomiting, and po intolerance. the pain started a few months prior, however it was self-limited with diet modifications. an ultrasound demonstrated a contracted gallbladder with a mm gallbladder wall. white blood cell count was within normal limits and total bilirubin was slightly elevated to . mg/dl. no palpable mass was noted on physical exam. an mr cholangiopancreatography was performed which demonstrated a dilated gallbladder measuring . x . cm, a severely thickened gallbladder with a small intramural collection and multiple gallstones. the patient proceeded with a laparoscopic cholecystectomy. intraoperatively, the omentum was densely adhered to the gallbladder and needle decompression of the gallbladder was unsuccessful due to the wall thickness. the gallbladder was subsequently removed without any complications. patient's remaining hospital course was uncomplicated. surgical pathology returned demonstrating acute on chronic cholecystitis. discussion: cholecystomegaly or 'giant gallbladder' disease is a rare pathology encountered in the surgical world. there have been few reported cases, most of which occurred in the elderly ([ years). kuznetsov et al. defined an enlarged gallbladder to have a volume of - cc and a giant gallbladder as exceeding cc (the average weight of the liver). the etiology remains unknown, however certain factors exist to allow the gallbladder to reach this size without life-threatening sequela. preoperative imaging, such as mr cholangiopancreatography, is important to differentiate biliary pathology and delineate anatomy. removal of the gallbladder is recommended to prevent the development of complications like cholangitis or bowel obstruction. the cause of cholecystomegaly still remains uncertain and warrants further research. the management and treatment remains similar to acute cholecystitis. aims: mini-laparoscopic cholecysectomy (mlc) is considered to be the best variant of minimizing surgical trauma and improving cosmesis in laparoscopic cholecystectomy. the most challenging techniqual step of mlc is clipping the cystic duct. it may be impossible or unsafe when diameter of cystic duct exceeds mm, which is common in severe chronic colecystitis or acure cholecystits. there is very limited data in the literature about the use of mlc in acute cholecystits. the aim of study was to access the first results of new technique of mlc. methods: five women with the mean age of years ( - ) underwent mlc. the st -mm troacar was inserted in the umbilicus and used for the camera and removal of the gallbladder. the nd -mm troacar was inserted in subxyphoidal area and used for the main working instruments, including medium-large polymer clip-applier (hem-o-lok type). the rd and th -mm troacars were placed in right subcostal area and used for mini-graspers (karl storz). in initial procedures we used conventional -mm clip-applier with adopted medium-large titanium clips. to improve safety, we aplied -mm hem-o-lok type clip-applier for the last patient with acute cholecystitis. in this case the diameter of cystic duct was , mm. the clipping was performed successfully. the -mm drain was placed via subcostal troacar incision. also, in this case we applied original technique of removal of the bladder using wound retraction instrument (karl storz). results: in all the cases there were no intra-or postoperative complications. the mean duration of procedures was min ( - min). the postoperative stay was days in every patient. the patients estimated their pain on postop day as 'almost absent' and cosmethic results mo postop as 'exellent'. conclusions: . new technique of mlc alowed to perform the clipping of cystic duct safely, which is essential in acute calculous cholecystitis. was conducted in department of surgery lumhs jamshoro. all the patients having age = year of age, either gender presented with history of abdominal pain, nausea and vomiting and were diagnosed as cholelithiasis included in the study and were planned either for mini-laparoscopic cholecystectomy and conventional laparoscopic cholecystectomy were explored for outcome while the patients with empyema gallbladder, gangrene, mucocele gallbladder and adhesions were excluded from the study. results: during one year study period, total five hundred patients were diagnosed as cholelithiasis with means age . ± . (sd). of five hundred, ( . %) were underwent for mini-laparoscopic cholecystectomy with ( . %) were males and ( . %) were females. the outcome were measured as postoperative pain (vas) . ± . , size of wound (umbilical mm, epigastrium mm and subcostal mm), excellent cosmetic results, mean ± sd for hospital stay (hrs) and operative time (minutes) was . ± . and . ± . , early return to work ( . %), minor oozing ( . %), port size hernia ( . %). remaining ( . %) were underwent for conventional laparoscopic cholecystectomy with ( . %) were males and ( . %) were females. the outcome were measured as postoperative pain (vas) . ± . , size of wound (umbilical mm, epigastrium mm and subcostal mm), mean ± sd for hospital stay (hrs) and operative time (minutes) was . ± . and . ± . , early return to work ( . %), port size hernia ( . %) along with zero ( %) mortality. conclusion: it has been concluded that mini-laparoscopic cholecystectomy is superior and feasible than conventional laparoscopic cholecystectomy and has decreased early postoperative incisional pain, avoided late incisional discomfort and safe procedure with nearly scarless wounds with superior cosmetic effect especially for young female patients. objective: to determine the outcome of immediate versus late laparoscopic cholecystectomy in acute cholecystitis at tertiary care hospital hyderabad / jamshoro sindh pakistan patients and methods: the descriptive case series study of one year ( - ) was conducted in department of surgery lumhs jamshoro. all the patients having age = year of age, either gender presented with history of abdominal pain, nausea and vomiting and were diagnosed as acute cholecystitis (cholillthiasis) included in the study and were planned for laparoscopic cholecystectomy and were explored for outcome as immediate (within h) and late components ([ weeks). the frequency and percentage was calculated for categorical variables and mean ± sd was calculated for numerical variables. as this was descriptive case series so there was no any statistical test of significance was applied. results: during one year study period, total one hundred patients were diagnosed as acute cholecystitis with means age . ± . (sd). of one hundred, % were females and % were males. the immediate outcome reported as tissue fragile %, pancreatitis %, slipage of ligature of cystic duct %, empyema gallbladder %, mucocele % and gangrenous gallbladder % while the late outcome reported as adhesions %, cholecystoduodenal fistula and mirizzi syndrome % and %, gallstone ileus %, perforated gallbladder % and cholidochiolithiasis % while the mean ± sd for hospital stay (days) in immediate as . ± . while in late outcome (days) during acute cholecystitis . ± . and after surgery ( weeks later) as . ± . respectively. conclusion: it has been concluded that early lc for acute cholecystitis with cholelithiasis is safe, low cost and feasible intervention and offering the additional benefit of shorter hospital stay and reduce the economical burden. surg endosc ( ) :s -s general surgery, chang gung memorial hospital kaohsiung division, kaohsiung, taiwan background: the treatment of common bile duct (cbd) stones is challenging while unclear hepatic hilum anatomy especial experience of previous laparotomy. a minimally-invasive approach choledocholithotomy is feasible, but can be difficult and converted for the unclear anatomy of the biliary tree. near-infrared (nir) cholangiography by systemic administration of indocyanine green (icg) can enhance the visualization of the biliary tree anatomy but is limited by the high intensity of background fluorescence signal coming from the liver. nir fluorescence cholecysto-cholangiography by direct biliary tree administration of the icg can enhance the biliary tree without background noise signal. we created the nir cholangiography via different route according to patient situation : systemic circulation or biliary tree injection to see the feasibility of those application. material and method: ten patients who suffered from obstructive jaundice due to cbd stone and patients received percutaneous biliary tree drainage as first treatment and patients received endoscopic biliary tree drainage. those patients received laparoscopic choledocholithotomy as definite treatment after acute infection phase. patients received biliary tree icg injection via drain tube and patients by systemic injection. visualization and fluorescence patterns around cbd was recorded. results: in our series, one patient received previous gastrectomy and patients had previous biliary tree surgery. background: laparoscopic cholecystectomy (lc) has become the gold standard for the treatment of gallstone disease. multiple studies have confirmed its safety, lc at index admission is still not widely practiced in ireland. we present our experience in performing index cholecystectomy at cuh after the start of acute care surgery program in may . aim: the aim of this study is to determine the safety of laparoscopic cholecystectomy at index admission, complications,re-admissions, and los. methods: electronic records, theatre records and imaging reports were searched to enroll all patients who underwent lc for gallstone disease at index admission from may to october . patient demographics, indication for surgery, postoperative complications, readmission and conversion rate were recorded.in addition timings of mrcp and ercp, imaging findings, and los were also noted. results: a total of patients underwent lc during the study period. median age was years ( - ). male to female ratio was : . . ( %) patients had acute cholecystitis, ( %)had acute biliary pancreatitis, ( . %)biliary colic and ( . %) had cholecystitis with signs of cbd obstruction. ( . %)patients had obstructive jaundice and one with adenomyomatosis. patients ( %) had preop mrcp while ( %) underwent pre-op ercp. all except patients undergoing ercp had preprocedure mrcp. patients had pre-op cholangiograms. in terms of complications, ( . %) patients had bile leak and one( . %) had re-operation. one patient had the post-op hematoma which was drained percutaneously, one patient had procedure abandoned because of bradycardia upon induction of anesthesia. there was no common bile duct injury, no conversion to open and no days mortality was reported. the average length of hospital stay has been days. ( to days). conclusions: laparoscopic cholecystectomy at index admission for cholecystitis, choledocholithiasis, and biliary pancreatitis, has been a safe and feasible treatment option in our hospital. a safe practice can be ensured by adherence to a care pathway and a multidisciplinary, consultant-led service. index cholecystectomy service can be provided safely across the country to prevent diseaserelated morbidity and multiple re-admissions in patients awaiting interval surgery. when to use the two-stage surgery to treat choledocholithiasis: the size aims: the treatment of choledocholithiasis has been provided by various of studies worldwide. the most common accepted minimal invasive treatment was two-stage treatment using endoscopic retrograde cholangiopancreatography before or after laparoscopic cholecystectomy(ercp ? lc), and one-stage treatment with laparoscopic exploration of the common bile duct(lcbde). in fact, despite several large studies have been published in recent years, the debate for the ideal treatment of choledocholithiasis is way from being concluded. we aim to find the proper treatment option for the patients with variable sizes of choledocholithiasis. methods: we retrospectively analyzed patients who underwent treatments for cholidocholithiasis in our institute between january , and july , . the patients who received either ercp and lc in the same admission, and the patient who received lcbde, irrespective of trans-cystic(ltcbde), or choledochotomy(lcd), were included. the data was analyzed with chi-square test and mann-whitney u test. results: the stone size of the ercp ? lc group is significantly smaller than the lcbde group. we further analyzed the ercp failure case, and the group of stone size [= . mm has a significantly higher rate of procedure failure. the failure rate is increasing with the stone size. conclusions: both the treatment of lcbde and ercp ? lc have similar safety and success rate, and the rate of residual stone was also similar in both group. however, the failure rate for ercp is significantly increased when stone size is larger than . mm in this study. aims: the xanthogranulomatous cholecystitis (xc) is a rare entity that can cause doubt in the choice of surgical treatment, because of differential diagnosis with gallbladder carcinoma (gc). methods: a -year-old patient presented acute abdominal pain in the right upper quadrant, nausea and low-grade fever with signs of peritonitis. he had elevated pcr, leukocytosis with neutrophilia. abdominal ultrasound showed an acute xanthogranulomatous cholecystitis. a laparoscopic cholecystectomy was decided but it was converted to open surgery due to the difficulty in the dissection, with fundus embedded in the hepatic bed and intraoperative finding of hilar adenopathic conglomerate .the postoperative period was torpid, with abdominal pain, jaundice, elevated bilirubin and enzymes of cholestasis. postoperative abdominal tomography showed injury in the iv segment of the liver suggestive of neoplasia. metastatic adenopathic conglomerate at the hepatic hilum caused extrinsic biliary obstruction with hepatic failure later so an internal-external drain was placed in the bile duct. the patient was died a week later. the pathological anatomy reported a stage four of gc. results: xc is a rare, non-neoplastic, inflammatory and destructive entity of the gallbladder wall, considered a variant of chronic lithiasic cholecystitis. it may be due to extravasation of bile or ulceration of the mucosa, causing an inflammatory reaction and fibrosis, with xanthomatous cells. the prevalence is to % in the resected gallbladders. it is more frequent in - years oldfemales. its clinical presentation does not have specific characteristics that differ from cholelithiasis, except for the weight loss. radiologically it is characterized by nodular thickening and increased attenuation of the vesicular wall with signs of cholecystitis, indistinguishable from a vc. the xanthogranulomatous inflammatory foci infiltrate the hepatic parenchyma, having an invasive behavior; hence, it mimics a neoplastic disease. the confusion in diagnostic and the risk of gc (up to %) makes treatment contentious. conclusions: the xc can simulate an advanced gc that sometimes makes us wonder if we should perform a radical surgical treatment; when presented in an emergency situation, our therapeutic decision can focus on solving the acute problem and be conditioned by the patient's general condition. single port transumbilical laparoscopic surgery (sptls) is a techinque that has been around for about years. although the enthusiasm for this type of surgery seems to have diminished in recent years it is expected to rise considering the recent development of sophisticated devices for its execution . we report retrospectively our year experience with procedures performed by sptls technique. in a private practice setting in mexico city. procedures include cholecystectomy ( ), appendectomy ( ), inguinal hernia tapp and tep ( ), hiatus and esophageal ( ) , sleeve gastrectomy ( ), colon ( ), gyn ( ) . different access platforms were employed. we explain our selection criteria for the application of the technique and describe the evolution of the instruments employed during the past years, from laparoscopic conventional to curved and bendable; regular scopes to extra long telescopes with different angles. or time, top bleeding, conversion rate, the need to employ an extra trocar, complications, pathology reports, scheduled or urgent kind of surgery and length of hospital stay were recorded from the beggining; patients variables such as bmi, asa status, tep risk, satisfaction with the procedure and other were recorded . we describe the evolution of our technique, and our learning curve with cholecystectomies. we compare our group of sptls transvaginal assisted laparoscopic hysterectomy (tvalh) patients vs tvalh multiport patients. we explain the feasibilty, and efficiency of the procedure in our hands compared to other series. background: in japan, the severity of acute cholecystitis(ac) is assessed by the severity classification of the tokyo guidelines (tg ). the value of c-reactive protein (crp) is not included in the severity classification criteria. the first line treatment, according to tg , for mild (grade i) to moderate (grade ) ac is laparoscopic cholecystectomy, but laparoscopic surgery may not be feasible in some cases due to adhesion or local inflammation of the gall bladder. aim: the aim of this study is to assess the effect of crp on the open conversion rate in laparoscopic cholecystectomy for acute cholecystitis.method: we conducted a retrospective study. patients who were diagnosed with ac and treated with emergent laparoscopic cholecystectomy between june and may in our institution are included. we set the cutoff value for crp at mg/dl and compared the open conversion rate. secondary endpoints are amount of bleeding, operation time, post-operative course (peak in body temperature and inflammatory markers) and the frequency of complications according to the clavien-dindo classification. results: out of patients had a crp value greater than or equal to mg/dl. the median crp values for the crp \ group and crp = group were . and . , respectively. the open conversion rate of the crp = group was significantly higher than that of the crp \ group ( / , / , p = . ). the most common reason for these conversions was local adhesion ( / ) . there were no differences in the amount of bleeding, operation time, post-operative course, and frequency of complications with clavien-dindo grade ii or higher. background: reports about clinical value of fluorescent cholangiography using indocyanine green (icg) during single-incision laparoscopic cholecystectomy (silc) were increasing. we report clinical value and pitfalls of fluorescent cholangiography during silc for the patients with the infraportal type of the right posterior bile duct. methods: our silc procedure utilized the sils-port with an additional -mm forceps through the umbilical incision. before silc, ml of icg ( . mg) was administrated by intravenous injection. for fluorescent cholangiography, icg fluorescent laparoscope system was used. results: we performed fluorescent cholangiography during silc in patients with the infraportal type of the right posterior bile duct. all procedures were completed successfully. the interval from the injection of icg to the first obtained fluorescent cholangiography before the dissection of calot's triangle ranged from to min. detectability of infraportal type of the right posterior bile duct before dissection in claot's triangle was . % (n = ) and that during dissection in calot's triangle was . % (n = ). the infraportal type of the right posterior bile duct could be identified under fluorescent cholangiography only when it joined into the common hepatic duct. conclusions: utilization of fluorescent cholangiography can lead silc to safe even for the patients with the infraportal type of the right posterior bile duct. its benefit is emphasized when the infraportal type of the right posterior bile duct joins into the common hepatic duct. aims: due to the development of laparoscopic surgery and the progress made in surgical treatment ofhydrocephalus, surgeons may come across patients with ventriculoperitoneal (vp) shunt, as candidates for laparoscopic procedures. according to this fact, we report a case of an unusual complication of laparoscopy surgery that can appear in this kind of patients. methods: we present a case of a -year-old man with medical history of normotensive hydrocephalus with vp shunt, that came to the emergency room complaining of abdominal pain and fever since two days. blood test showed an elevation of infection parameters and inflammatory markers, and the ultrasound study revealed an emphysematous cholecystitis. therefore, we decide to carry out an emergency laparoscopic cholecistectomy. the patient did not present any adverse event during the surgery or the immediate postoperative period, being discharged the third postoperative day and evaluated ambulatory one month after the surgery with no complications. two months after surgery, the patient returned to the emergency room presenting alteration in consciousness and fever. results: during the study of the pacient, an abdominal ct was performed, showing a complete section of the vp shunt in the subcutaneus space of the upper abdominal wall and intraperitoneal migration of the remaining catheter. the patient was transferred to neurosurgery to carry out an emergent replacement of the ventriculoperitoneal shunt. after surgery and intravenous antibiotic treatment, the patient evolved favourably and was discharged a few days later. conclusions: the rate of serious complications associated with a laparoscopic approach is overall low and up to % of them occur during the abdominal access for camera or port placement and may not be recognized until postoperative period. vp shunts should not be a contraindication for laparoscopic surgery. however, laparoscopy approach must be carry out with good anesthetic and monitoring facilities and taking several previous considerations, such as verifying the proper functioning of the vp shunt, identifying the path of the catheter within the abdominal wall to avoid inadvertent damage to the catheter during trocar placement and ensuring that the intraperitoneal portion of the catheter is not twisted or obstructed prior to decompression of the abdomen. surg endosc ( ) introduction: since advantages of robotic surgery is being more emphasized, robotic cholecystectomy (rc) cases are increasing. ajou group had introduced a method called which technique places the trocars transversally on the bikini line and it makes cosmesis and pain beneficial. however, rc with low incision port has several limitations. therefore, we changed port placement which may be a one of safe tehniques for rc. method: this study retrospectively reviewed data for patients who received rc with port changing method (rcpc, n = ) and rc with low incision port (rcli, n = ) from february -february and surgical variables were analyzed. results: patients in both groups had similar demographic features and indications for surgery. the rcpc group required no conversions to conventional robotic surgery and no additional operation, whereas the rcli group had one incisional hernia ( . %) and two bowel perforation ( . %) cases. length of stay ( . ± . vs. . ± . days, respectively; p = . ) did not significantly differ between the rcpc and scli groups. however, the rcpc group had shorter operative time ( . ± . vs. . ± . min; p = . ) than the rcli group, although the parameters mentioned above were not statistically significant. conclusion: robotic cholecystectomy with bikini line incision has some limitations even though it has cosmetic benefits. whereas robotic surgery with changing port method is one of safe and feasible procedures for performing robotic cholecystectomy. also nothing more to say that it gains cosmesis effect and escapes complications. mini surgery, odessa medical university, odessa, ukraine the aim of the study was to optimize the diagnostic and therapeutic tactics for yatrogenic injuries of the extrahepatic bile ducts. methods: patients were examined. typical manifestations were jaundice, cholangitis, biliary peritonitis, external biliary fistula, subhepatic abscess.cholecystectomy main cause of damage.a visual, manual and x-ray examination of the hepato-choledochus and cholangioscopy were performed. ultrasound, endoscopic retrograde cholangiopancreatography, fistuloholangiography or percutaneous transhepatic cholangiography play a leading role in diagnosing. the results: high damage to the bile duct was detected in . % of patients, low-in . %.percutaneous transhepatic drainage under ultrasound control was performed in . % of patients.emergency laparotomy, sanation of the abdominal cavity and external drainage of the bile ducts were performed with bile peritonitis. recovery operations produced . % of patients. reconstructive interventions were performed in . % of patients after - weeks after the first stage. the covery operations were successful in . % of patients. . % of the sick had complications in the form of biloma. a scar stricture formed in . % of patients after - months. patient underwent recanalization of the stricture zone with a dilatation balloon through interchangeable transhepatic drainage. balloon dilatation was performed retrogradely through the large duodenal papilla in patients. deaths in the postoperative period was not observed. conclusions: the surgical team should be strengthened by an experienced surgeon when intraoperative diagnosis of yatrogenic damage to the bile ducts.the operation should be completed by external drainage of the bile duct and the abdominal cavity in the absence of an experienced specialist.recovery operations are shown only with lateral injury of the ducts.the patient must be sent to a specialized institution for radical surgical treatment after stabilization of his general condition. aim of the study sub-hepatic bile collections, biloma and hematoma are rare complications and we present our experience in treatment this complications. material and methods: from laparoscopic cholecystectomy performed in our clinic, three patients (two women and one men) to whom it was performed laparoscopic cholecystectomy, came back two weeks later after they were released from the hospital because of epigastric discomfort, fever and nausea. results: clinical examination after rehospitalization showed tenderness in the epigastrium and right subcostal region. in all patients were measured high levels of leukocytosis and crp . an ultrasound examination of the abdomen revealed a large hypoechoic collection in the sub hepatic space, after the abdominal ct scan was performed, the density of the collection did not indicate the presence of blood in two patients. percutaneous drainage of the collection in both patients was realized under us guidance and - fr catheter was inserted in the sub hepatic region. in the first patient cc of bile-stained liquid, and in the second patient cc of biliary liquid was drained. in a third patient h after surgery signs of significant hypotension and limited tenderness at the right subcostal region occurred. a complete blood count (cbc) showed a decrease in the level of haemoglobin to . g%. ultrasound examination revealed a fluid collection in the sub hepatic space, which is also confirmed by computed tomography. laparotomy was performed and the large sub hepatic hematoma was evacuated. after that the fr abdominal drain was inserted into the sub hepatic space. the postoperative course of all three patients was not complicated. conclusion: sub hepatic biloma and hematoma are rare complications of laparoscopic cholecystectomy, while early diagnosis followed by percutaneous drainage or open laparotomy is the only way to resolve these complications. ( ), hemoperitoneum . % ( ) . the average number of days of hospitalization was . days. there was no mortality at days. conclusion: in the emergency setting the rendezvous technique has an adequate success rate of cannulation and clearence of the bile duct, an acceptable surgical time, few complications, these being more frequent in those patients with inflammation of the gallbladder and without associated mortality at days. there is a need for controlled randomized studies with a greater number of patients recruited and follow-up to determine the usefulness of this technique. intraoperative cholangiography could serve as a fundamental solution to avoid the bile duct injury during laparoscopic cholecystectomy. however, it is difficult to identify the cystic duct to which the contrast catheter should be inserted in cases with high degrees of adhesion around the calot's triangle. in these cases, it is not possible to conduct cholangiography from the cystic duct. for these types of cases, intraoperative cholecystography may serve as an option. however, since the bladder is a bag-like organ that expands when liquids are entered, directly inserting a contrast dye into the bladder would make the bladder itself expand, which makes it impossible for to maintain enough pressure in the contrast dye to flow into the cystic duct, extrahepatic bile duct, and intrahepatic bile duct. also, since it is difficult to control leakage of the contrast dye from the catheter insertion site, it is not possible to obtain enough images to sufficiently understand the anatomical characteristics of the bile duct in many cases. therefore, cholecystography is not generally recognized as a method to be used during surgery. in our facility, we insert the contrast catheter through the bladder after stretching the gallbladder neck as much as possible, hold the gallbladder neck with a removable intestinal clamp, and then apply the contrast dye to the bile duct. through this method, it is possible to insert enough contrast dye into the cystic duct, extrahepatic bile duct, and intrahepatic bile duct to understand the anatomical characteristics of the bile duct, allowing us to obtain appropriate images of the biliary tract. because this method uses equipment that is highly versatile, we believe that it is inexpensive and convenient. during this presentation, we will also conduct a case presentation of the methods of bladder contrasting that we utilize in our facility during laparoscopic cholecystectomy. introduction: retrieval of a thick walled gallbladder during a difficult laparoscopic cholecystectomy (lc) for an acute or chronic calculous cholecystitis can be exasperating. it increases operative time and often necessitates enlargement of mm port to deliver the specimen. the 'in-situ cholecystotomy', which we wish to call the 'delhi maneuver' is very helpful in improving the ergonomics of specimen retrieval, saves time and conserves cosmesis. patients & methods: one hundred and ten patients of acute or chronic calculous cholecystitis were placed randomly in groups. a disposable transparent plastic bag was used in all cases to retrieve the gallbladder specimen through the - mm port using a rampley's sponge holding forceps. retrieval was done using conventional technique in patients (group b). the delhi maneuver was used in the remaining patients (group a). it involved cutting the gall bladder inside the plastic bag in a certain fashion, delivering the gallstones in the bag, and removal of gallbladder preceding the stones. the retrieval time, number of insertions of sponge holder, any rupture of plastic bag as well as the number of cases needing port enlargement were noted. results: the average time taken by delhi maneuver (group a) was min as compared to was min by conventional method (group b). the number of insertions of sponge holder ranged from - in group a (mean ) and - in group b (mean ). four patients needed port enlargement in group a ( %) while patients needed enlargement in group b ( . %). there were incidences of bag rupture in group a ( %) and in group b ( %). the delhi maneuver improved the ease and speed of specimen extraction at laparoscopic cholecystectomy for thick walled gallbladders. it also decreased the need for port enlargement for specimen retrieval. the bile duct injuries are a very complex desease to confront, the inciian managment is to clasificate the injury and to identifie the mechamism of the injury. it's important for the optimal heal of the patient to have a multidisciplinary approach including internal medicine, surgery, endoscopy and interventional radiology specialists. the laparoscopic cholecystectomy responsible for %- % of them.this is a retrospective study on the incidence, classification and management of bile duct injuries in a private sector hospital in monterrey nl. mexico. in this study, bile duct injuries were identified in years of experience in a single center. were categorized using the strasberg classification. variables were evaluated such as type of injury, mechanism of injury, hospital stay, if the surgery was scheduled or of emergency, the moment in which the surgeon evidenced the injury, the way in which the surgeon became aware of the injury performed. the type of management that was given to this lesion was also studied and the days of intrahospital stay and the number of reinterventions or procedures performed were compared.the average age of the patients was years, patients belonged to the female sex, although there were lesions of all kinds in this work, there was a greater incidence in strasberg type a lesions, which represented % of the lesions. the most common diagnosis presented was cholecystolithiasis. in surgeries the evidence and repair of the bile duct was in the same intervention aims: bile leak is a rare but recognised complication after laparoscopic cholecystectomy. this usually occurs after a difficult procedure complicated by adhesions, unusual anatomy or if the surgeon is inexperienced or unfamiliar with the anatomy. this video aims to demonstrate the laparoscopic diagnosis and treatment of this complication particularly for surgical trainees. methods: we report a case of significant bile leak occurring soon after a straightforward laparoscopic cholecystectomy due to very short cystic duct (cd). the procedure was carried out uneventfully but the cd was clipped flush with the bile duct. the patient was discharged on the day of surgery feeling well but readmitted with abdominal pain h later. results: after readmission the patient underwent a ct demonstrating only a small amount of fluid suggestive of a small collection. she was treated conservatively but suddenly deteriorated and a repeat ct confirmed significant intraperitoneal fluid. a diagnostic laparoscopy was carried out urgently confirming a cd stump bile leak where the clips had sloughed off causing the leak. two litres of bile was aspirated with copious irrigation and a latex t-tube inserted into the cbd. patient made a full and rapid recovery. conclusions: this is a rare complication and learning opportunities for trainees are therefore infrequent. this video demonstrates a successful laparoscopic approach to management of postoperative bile leak showing t-tube insertion technique and highlighting the need for careful cd closure techniques during laparoscopic cholecystectomy when the duct is very short. about - % of bile duct stones could not be extracted using conventional endoscopic techniques (baloon, sphincterotomy). there is lower success rate in elderly patients; among the biggest challenges are intrahepatic stones, size of stone is large, etc. aims: to present the case of a recurrent intrahepatic lithiasis and its management using spyglass choledochoscopy.to expose, other cases and the main outcome and complications of other difficult cases of bile duct stones that solvedusing this choledochoscope vs. the traditional one and the beneffits. we present a case of years old male who presented with cholangitis caused by an intrahepatic stone that required multiple sessions of endoscopic retrograde cholangiopancreatography with spyglass for clearance. one year later, he presented again with cholangitis, that required another session of spyglass lithotripsy and cholecistectomy. conclusions: besides ercp, there are different approaches to treat difficult bile duct stones, as transhepatic percutaneous drainage, surgical techniques, or other endoscopic techniques (doubleballoon, enteroscopy). ercp and sphincterotomy are the first step of endoscopic treatment with more than % of success rate, and a low mortality and morbility rate; other steps include some lithotripsy techniques, or the use of biliary stent as a bridge before definite treatment. spyglass is a visualization & intervention system used when common ercp has been unsuccessful, and it is first line for better and direct image of biliary ducts, with °range of motion, with multiple advantages like the concomitant use of lithotripsy devices. aims: the number of elderly people has increased, because of the strong association between age and gallstone disease, both prevalence and incidence of this disease are increasing. this presentation aims to review our current management options of octogenerian patients with acute cholecystitis. methods: we retrospectively analyzed octogenerian patients who were admitted to the our hospital with the diagnosis of acute cholecystitis between january and october . the patients were initially allocated to four different treatment groups as follows: immediate surgery, delayed surgery, medical treatment and cholecystostomy. differences in the outcomes between the treatment groups were evaluated. results: there were males ( . %) and females ( . %) with a mean age of . years (range - years). the patients had different co-morbid diseases, especially hypertension ( , . %) cardiovascular disease ( , . %) and diabetes mellitus ( methods: a retrospective observational study where were analyzed patients older than years who underwent urgent surgery for ac who fulfilled an indication for surgery according to tokyo guidelines . the type of cholecystitis, stay and postoperative complications, the type of intervention, the conversion rate, the need for reoperation and re-admissions in patients older than years were analyzed and compared with those of patients operated on for cholecystitis younger than years. outcomes: a total of patients were registered, older than years ( %) and younger ( %). in cases, cholecystitis were complicated ( . %), cases older than years ( . %) and in cases younger than years ( . %). the approach was laparoscopic in % of the cases older than years, with a conversion rate of . %, not finding statistically significant differences with younger than years ( % laparoscopies with . % of conversions). % of patients older than years had some type of postoperative complication, not finding statistically significant differences in patient younger than years ( %); being the most frequent complication the intrabdomintal abscess ( . % of patients [ years, and . % of those \ years = '' span = '' [ being not statistically significant with % ci. any patient older than years required re-entry after discharge, compared to patients younger than years who were re-entered, not being statistically significant; and any patient older than years required reintervention, while it was necessary to reoperate patients younger than years ( %), being not statistically significant. mortality was very low, finding case in older than years ( . %) and case in younger ( . %), not obtaining statistically significant differences. the postoperative stay in patients younger than years of age has a median of days and in older than years a median of days, not finding statistically significant differences with % ci conclusions: laparoscopic cholecystectomy is safe and effective in the treatment of elderly patients with (ac), there being no differences with younger patients. introduction: significant bile leak is an uncommon but serious complication of laparoscopic cholecystectomy. our study aims to evaluate the efficacy of relaparoscopy in treating symptomatic bile leak and biloma formation. material and methods: patients presenting with postoperative bile leak after different operations on extrahepatic biliary tree from january to december were reviewed retrospectively (in total, , laparoscopic surgical interventions were performed for the period under study). the sites of bile leaks were the cystic duct stump in thirty seven patients, the bile ducts of luschka in fifty two, liver beds in cases after hepatectomy, in had small injury of cbd, and seven patients with tubular stenosis of the common bile duct. results: three main approaches of mini-invasive treatment of bile leakage was used: ( ) percutaneous puncture with or without drain under ct-scan or ultrasound guidance in patients; ( ) endoscopic management in patients (in patients ( . %) were managed with ercp alone and fifteen ( . %) were treated with a percutaneous intervention followed by ercp. endobiliary stent placement was performed after es in patients and without es in twenty seven patients ( ) relaparoscopy has been performed in patients, in cases of biliary peritonitis. conclusions: relaparoscopy was the ultimate method of treating postoperative complications of laparoscopic surgery in . % of patients. in general, this method, as well as laparoscopic intervention, is highly effective in the diagnosis and correction of postoperative complications, with minimal surgical trauma for the patient, with great therapeutic effect and subsequent rapid social rehabilitation of patients. introduction: laparoscopic operations have already become routine, even for pancreatoduodenectomy for periampular cancer. for unresectable cases, endoscopic bibliary stenting or hepaticojejunostomy are usually used. these methods are quite expensive and may be accompanied by complications. materials and methods: laparoscopic cholecystogastroanastomosis was performed in patients with unresectable periampullary cancer. there were females and men and average age was , . the indications for surgery in all patients was unresectable periampullary cancer and biliary hypertension with preserved patency of the cystic duct. the level of bilirubinemia ranged from to lmol/l (the average level was , lmol/l). we used -port technique. optical trocar was placed in the right iliac region, one mm above the navel and one mm in the right hypochondrium after punction gallblaber and aspiration of bile, we cut the apex of the gallbladder and gastric antrum up to . cm and performed cholecystogastroanastomosis with barbed-suture v-loc. results: we had not conversion to open surgery. the average operation time was min. postoperative stay was average days and on median follow-up of month. post-operatively, there were no major morbidity and nil mortality. we had cases of leakage of bile through drainage for up to - days, which spontaneously stopped. all patients showed a decrease in the level of bilirubinemia. patients were later radical operated (pancreatoduodenectomy), while they did not have such phenomena as cholangitis, pancreatitis, inflammation of the hepatoduodenal ligament elements, which we often observe after endoscopic biliary stenting. conclusions: laparoscopic cholecystogastroanastomosis is safe, effective and feasible for patients with periampular cancer and obstructive jaundice. aims: surgeons with the expertise and resources to perform laparoscopic common bile duct exploration often prefer the 'one stage approach' over endoscopic retrograde cholangio-pancreatography (ercp) for the management of common bile duct (cbd) stones. this case series aims to evaluate the effectiveness of lcbde in a single benign upper gastrointestinal (gi) unit. methods: all patients with suspected and confirmed pre-operatively cbd stones who underwent a lcbde between january and october were included. lcbde was performed on the basis of pre-operative suspicion of cbd stone confirmed by intra-operative imaging. results: patients with confirmed choledocolithiasis had lcbde during this time period. the indications for lcbde were deranged liver function tests, dilated cbd or confirmed stones on preoperative imaging. median age was (range - ), % of whom were female. % of patients had confirmed cbd stones pre-op. % of cases were performed as emergencies and conversion rate to open was . %. choledocotomy was performed in % of cases. in % of these t-tube was left in situ. transcystic approach was used in the remaining %. despite positive intraoperative imaging no stones were found on cbd exploration in cases ( %). in patients stones were unable to be cleared with lcbde. the overall morbidity was %. % of patients had gallstone related complications. overall mortality was % (due to bile leak). / patients required re-intervention with re-look laparoscopy (n = ) or ercp (n = ). patients re-presented within months with cbd stones. overall median length of stay was days. conclusions: our case series demonstrates that lcbde is an effective and safe treatment for choledocolithiasis in both the elective and emergency settings. complication rates are comparable with therapeutic ercp ( % specific complications) followed by laparascopic cholecystectomy ( % day morbidity). the variability in anatomic location of subvesical bile ducts puts them in danger during hepato-biliary operations. its prevalence varies between % and %. the origin and drainage of these ducts were limited mainly to the right lobe of the liver, but great variation could be seen. some authors think of them as small bile ducts that drain directly into the body of the gallbladder; others consider them to be networks of miniscule bile ducts between the liver capsule and the gallbladder. recent studies suggest that clinically relevant bile leaks complicate approximately . - . % of cholecystectomies. injury to a subvesical duct is one of the most common causes of cholecystectomy associated bile leak and occurs as often as major bile duct injuries and leaks from the cystic duct stump. indeed, recent studies suggest that about % of clinically relevant bile leaks are caused by inadvertent injury to a subvesical bile duct. there are four types of subvesical bile ducts, including ( ) superficial variations of segmental and sectorial bile ducts, ( ) superficial or intercommunicating accessory bile ducts, ( ) hepaticocholecystic ducts, and ( ) aberrant bile ducts.we present a case of year old patient who developed a coleperitoneum after a routine daycase colecystectomy due to the inadvertent injury of a hepatocholecystic duct. a superior comprehension of ductal anatomy is essential in preventing and managing operative injury to the subvesical ducts, although some times is unavoidable. nowadays, the diagnosis of liver cancer is primarily radiological, as recommended by the principal international societies. in doubtful cases or due to the clinician needs, diagnostic evaluations can eventually be completed with a liver biopsy. the goal is to perform the examination, or the examinations, that guarantee the most elevated sensibility and specificity levels being as little invasive as possible. nevertheless, even using the best radiological tools, the diagnosis is not certain, due both to device limitations and radiology experience. recently, various diagnostic algorithms have been proposed, relating with contrast enhancement characteristics, different radiological techniques, blood examinations and cross evaluations from different radiologists. one of the most recent algorithm purposed is liver imaging reporting and data system (li-rads), that evaluates ct and mri imaging to classify hepatic lesions in different diagnostic categories, in order to perform a better and more precise diagnosis of hcc or other liver benign or malignant lesion. through a retrospective study, we evaluated and compared preoperative imaging and post-operative histological reports. results reveal that li-rads routine use increases hcc diagnosis up to %. background: we previously developed a modified difficulty scoring system (dss-ihd) of laparoscopic liver resection (llr) for patients with intrahepatic duct (ihd) stone. we validated dss-ihd in patients who underwent llr for hepatolithiasis. methods: dss-ihd was based on the extent of liver resection ( to ), stone location ( to ),atrophy of liver parenchyma ( to ), ductal stricture \ cm from the bifurcation ( to ), and combined choledochoscopic examination for remnant ihd ( to ). results: the dss-ihd ranged from to and divided to -level groups of low group (score * ; n = ), intermediate group ( objective: improving the surgical treatment of patients with cholangiogenic abscesses of the liver through the application of minimally invasive technologies. material and method: in the presented study presented results of treatment of patients with biliary liver abscesses. surgical interventions for hepatic abscesses were performed simultaneously with the elimination of the primary pathological process of the biliary system, which caused the occurrence of cholangitis, or in the near future (up to days) after biliary drainage drainage. among patients with biliary liver abscesses, treated with minimally invasive methods, revealed abscesses of the right hepatic lobe, -abscesses of the left hepatic lobe, -abscesses and right and left hepatic lobes. single abscesses were detected in patients, and in -two or more abscesses. in terms of liver abscesses, more than cm were detected in patients, more than cm in patients. drainage of the biliary tract was carried out endoscopically transpapillary and (if the endoscopic approach was unsuccessful) with transcutaneous transhepatic approach. results: drainage under ultrasound guidance was performed on patients with solitary and patients with two or more cholangiogenic abscesses of the liver. laparoscopic interventions were performed on patients. among the patients operated on using minimally invasive technologies, occurred complications ( . %). patient died due to the development of biliary sepsis ( . %). conclusion: percutaneous drainage of liver abscesses under ultrasound control is appropriate not only for single abscesses, but also for their larger number, which has many advantages over other interventions. it was proved possibility of simultaneous drainage of liver abscess and bile duct. percutaneous drainage of the liver abscess, drainage of the biliary tract and laparoscopic surgical intervention are complementary aspects in the treatment of liver abscesses of biliary origin. after laparoscopy residual calculus can be removed endoscopically in more favorable conditions after stabilization of the patient's condition is achieved and the infection-associated disorders are eliminated. in case of localization of abscesses in the marginal segments of the liver, laparoscopic atypical resection of the liver with an abscess is most desirable. general surgery, rambam medical center, haifa, israel background: recently robotic surgery has emerged as one of the most promising surgical advances. despite its worldwide acceptance in many different surgical specialties, the use of robotic assistance in the field of hepatobiliary (hbp) surgery remains relatively unexplored. our study presents single institution's initial experience of robotic assisted surgery for treatment of benign hepatobiliary pathologies. methods: a retrospective analysis of a prospectively maintained database on clinical outcomes was performed for consecutive patients that underwent robotic assisted surgery for benign hbp disease at rambam medical center during - . results: there were robotic assisted surgical procedures performed for benign hbp pathologies during the study period. there were anatomical robotic liver resections for symptomatic hemangiomas, cases of giant liver cyst, robotic assisted surgery for type i choledochal cyst, case of benign (iatrogenic) common bile duct (cbd) stricture, cases of robotic (cbd) exploration due to large intra choledochal stones and cases of cholecystectomy for cholelithiasis. the median postoperative hospital stays for all procedures were . days (range - days). general morbidity (minor) was %. there was no mortality in our series. conclusion: robotic surgery is feasible and can be safely performed in patients with different benign hbp pathologies. further evaluation with clinical trials is required to validate it's real benefits. most liver cysts are asymptomatic and tend to have a benign clinical course. however, symptomatic or complicated liver cysts sometimes require surgical intervention. needle aspiration is safe and can be the lease invasive procedure, this procedure is however associated with a high failure rate and rapid recurrence. surgical approach is the crucial and provides definitive treatment for such cysts. thirteen cases were nominated from shonan kamakura general hospital between january and december . mean age and body mass index (bmi) were . and . , respectively. all patients have had any complaint such as upper abdominal pain, dyspnea, and fever. two cases were clinically diagnosed as the infectious cyst and serum crp was elevated before surgery. additional cholecystectomy was planned for one case of chronic cholecystitis with gallbladder stones. all cases were prompted the reduced port surgery (rps) and cases were performed rps with trans-vaginal approach (hybrid notes) and case was chosen in single port surgery. cyst unroofing was performed for all cases. mean operation time and blood loss of all cases were . min. and . ml, respectively. no surgical complication has been occurred in all cases, an infectious cyst case was however required additional drainage for infectious control after surgery. although statistic difference was not shown, fewer blood loss and shorter hospital stay was seen in non-infectious cases, compared to laparotomy cases. mean hospital stay after surgery of whole cases, non-infectious cases, infectious cases was . , . , . days, respectively . no recurrence of any symptom was shown in any cases in observation period ( - days) . laparoscopic unroofing is the definitive treatment for the complicated or symptomatic liver cyst. however, for the infectious cyst, infection control such as intensive drainage and/or administration of antibiotic before surgery may be needed to avoid additional treatment, leading to longer hospital stay. laparoscopic unroofing of liver cyst can be the first choice for symptomatic or complicated liver cyst. also, reduced port surgery can be nominated to achieve less invasiveness. introducction: laparoscopic liver resection (llr) has been increasing since it was first reported in . three international expert consensus conferences on llr surgery were held in louisville, ky, usa, in , morioka, japan in and southampton, uk, in . while most initial minimally invasive liver resections were typically done for benign lesions in anterior o left segments, llr is currently being applied for major anatomic resections, malignancy, cirrhosis and liver donor hepatectomy. clinical case report: this is a -year-old male patient with a history of hta and liver cirrhosis due to hepatitis b virus. hepatocarcinoma is diagnosed in liver segment vi with a size of cm . in the digestive study the patient presents a child a stage, meld \ , without signs of portal hypertension. complete analytical with normal afp and cea . markers. after presentation of the patient in a multidisciplinary committee and being a stadium according to the early bclc classification, laparoscopic surgery with segment vi resection was decided. discussion: laparoscopic liver resection is becoming widely accepted for the treatment of hepatocellular carcinoma. liver resection is a first-line option in very early and early-stage disease. many meta-analysis have shown that llr is better than open liver resection in terms of short-term outcomes for patients with child-pugh a cirrhosis, solitary tumors, and minor resections. in the long-term setting, the results demonstrate that a minimally invasive approach is comparable to an open approach in terms of overall. in conclusion, the current evidence conclude than llrs for hcc are safe and may be considered a standard practice in specific settings. results: there were women ( %) and men ( %). the age of patients ranged from to years. the patients underwent complex examination including abdominal ultrasound, esophagogastroduodenoscopy, and some of them underwent ct (computed tomography). all patients in the first stage were performed antegrade external drainage of biliary tracts with x-rays of the biliary tracts, and specifying the level and extent of the block.total miniinvasive interventions were hold. two patients in connection with the uncoupling of equity ducts were performed antegrade bilobar stenting with preliminary split external bile release.there were complications after carried out interventions in cases, which were associated with dislocation of holangiostomic drainage in patients ( . %); with acute cholecystitis in patient ( . %); with hydrothorax in patients ( . %); perihepatic biloma in case ( . %). patient ( . %) had a recurrence of obstructive jaundice due to germination of endobiliary stent in the late period after stenting. lethal outcome appeared in patient. conclusions: ultrasound examination allows us to determine the level of obstruction of the biliary tract, to substantiate the tactical position in the application of mini-invasive technologies. antegrade miniinvasive technologies in the treatment of tumor lesions of the proximal bile ducts allow timely and effectively stop biliary hypertension and to determine further treatment strategy. acknowledgements this study was supported by the russian science foundation under project ? - - . background: repeat hepatectomy is an effective treatment, with long-term surgical outcomes for recurrent hcc and colorectal liver metastasis(crlm). however, the efficacy of a minimally invasive surgical approach for recurrent liver tumor is not yet confirmed. the purpose of this study is to examine the efficacy of laparoscopic repeat hepatectomy(lrh) compared with open repeat hepatectomy(orh) for recurrent liver tumor. we retrospectively analyzed the clinicopathological features and short-term surgical outcomes between lrh and orh. methods: from to , patients with liver cancer underwent repeat hepatectomy. of those patients, patients underwent partial hepatectomy, patients were undergone laparoscopically, and patients underwent open hepatectomy. we compared the clinicopathological and surgical parameters in the lrh group with those in the orh group. results: there were no significant differences in patients' gender, age, viral infection status, child-pugh classification, tumor size, tumor number, and tumor location in the two groups. the operative times were similar, but blood loss was significantly lower in lrh group ( vs. ml, p \ . ). the postoperative hospital stay was significantly shorter in the lrh group ( . vs. . days, p = . ). postoperative complications(cd = a) were observed only in the orh group, with a complication rate of . %. conclusions: we demonstrate that lrh reduces blood loss and postoperative complications compared with orh. lrh might be a feasible and effective procedure for the selected patients. background: the liver is the most common site of metastatic disease with up - % of all cancers having the potentiality for sending liver metastasis during the disease. consequently, increasing value for surgical resection of hepatic deposits of different types of cancers, the need for accurate evaluation of the extent of hepatic metastasis was established for choosing the most suitable patients for surgery and in planning the extent of hepatic resection. the aim of this work is to evaluate the role of intra-operative ultrasound in the detection of hepatic deposits in intra-abdominal malignancies with special emphasis on its accuracy, sensitivity, specificity. patients and method: this study was carried out on thirty patients who were admitted to the gastrointestinal surgery unit, main alexandria university hospital with intra-abdominal malignancies for whom elective open surgical intervention was recommended in the period from st of september till the th of march . results: in the present study consisted of males ( . %) and females ( . %). their mean age at admission was . ± . years. six of the included patients ( %) were found to have hepatic lesions by using ious including the four cases ( . %) already detected by preoperative imaging. two cases ( . %) were newly discovered in the operative room by using ious. conclusion: the current study has proved that ious demonstrates superior lesion detection over the various non-invasive preoperative imaging modalities causing significant impact on change of the planned surgical strategy laparoscopic approach to the liver has become an integral part of surgery. two consecutive international consensus meeting recommends major hepatectomy has been on the expert hands. tumors located in the right posterior section are considered to be difficult for laparoscopic resection. patients and methods: since , until , cnuhh has been performing laparoscopic hepatectomies including major hepatectomies. among major ones, there are rh, lh, rps, ch, and as. we analyze data on patient demographics, tumor characteristics, operative date, and posterior outcome retrospectively. results: during - , laparoscopic rps were performed. the diagnosis were hcc in and crlm in patients. median operative time was min, and median blood loss was ml. no blood transfusion was occurred. median tumor size was mm, and median resection margin was . mm. six of the patients ( %) were cirrhotic on pathology. there was no conversion and was no postoperative mortality. median hospital stay was . days. conclusion: laparoscopic rps is known challenging procecedure. strict preoperative planning and operative procedure is mandatory. even though it should be performed by the experienced hands both on hepatic surgery and laparoscopic skill, it can be an good option for treatment of the tumor locating over right posterior section. purpose: previously we developed a new sponge (named endoractor) as an organ retraction device in laparoscopic surgery in and have reported that it is useful in various surgical procedures including rectal surgery we confirmed that it is also useful in laparoscopic radiofrequency ablation of the liver in terms of pulling and protecting organ, so we report it materials and methods: a case is an -year-old female with liver cirrhosis. she had primary hepatocellular carcinoma in s lesion with a diameter of . cm very close to the inferior vena cava and middle hepatic vein root and in s lesion with a diameter of . cm we thought she could not put up with hepatic resection because of her poor hepatic reserve capacity. and we could not expect treatment effect by embolization therapy since contrast effect was poor. so we decided to select ablation therapy in the puncture and ablation of the s tumor, since there was concern about the thermal damage of the middle hepatic vein and the cooling effect by the inferior vena cava, we would dissect the right coronary mesentery sufficiently and pull the liver apart from the inferior vena cava and the middle hepatic vein as much as possible using our endoractor also, in the puncture and ablation of the s tumor, it was feared that the stomach would be thermally damaged, so we would place endoclactor between the liver and the stomach to protect the stomach results: when ablating the s tumor, we could pull the liver securely without slipping, so we did not cause thermal damage to the middle hepatic vein. and there was no cooling effect by the inferior vena cava, so we could obtain sufficient cautery margin. in ablation of s tumor, we were able to puncture by stabilizing the lateral segment of the liver on our endoractor, and avoid thermal damage of the stomach conclusion: it seems possible to perform safe and reliable puncture and ablation by using our endoractor as well in laparoscopic radiofrequency ablation surg endosc ( ) surgical reinterventions in patients with complicated hepatic hydatid cysts usually occur as a result of diagnostic or technical failures during the initial procedure. according to recent studies, the most common complication after liver hydatid cyst surgery is local sepsis at the residual cavity and long-term biliary leak. we report the case of a -year-old male with a history of liver hydatid disease four years before the current episode, admitted in our surgical department for intense upper right quadrant pain. abdominal ultrasonography, ct and mri scans revealed three cysts in the gastrosplenic ligament, in liver segments vii-viii, and ii-iii respectively, sized between and cm. the intraoperative aspect during laparoscopy was strongly suggestive for liver hydatid disease. laparoscopic fenestration with tunneling for the hepatic cyst in segment viii, partial cystectomy in the left liver lobe and ideal cystectomy in the gastrosplenic ligament were performed. postoperatively, the patient displayed a constant biliary drainage output of - ml from the cavity remnant in the segment viii. conservative therapy for external biliary fistula and concomitant treatment with albendazole for months were initiated. evolution was slowly favorable with decreased biliary drainage to ml two months after surgery and complete symptom resolution five months after hospital discharge. aims: this study aimed to evaluate the effectiveness of fluorescence imaging with indocyanine green (icg) during laparoscopic deroofing of hepatic cysts. methods: this was a single-center, case-control study. we included patients who underwent laparoscopic deroofing between november and october . imaging with and without icg fluorescence was performed in (icg group) and (non-icg group) patients, respectively. icg was intravenously administered between min and . h before surgery. we performed a standard laparoscopic procedure. we detected a thin bile duct on the hepatic cyst on using intraoperative icg fluorescence imaging. we adjusted the resection line of the cyst wall and ligated the bile duct at the point at which it crossed the resection line. data on age, sex, cyst size, resected cyst size, operative time, estimated blood loss, post-operative hospital stay, complications, and recurrence were compared between the groups. results: the mean cyst size was ± . and ± . mm, the mean resected cyst size was ± . and ± . mm, and the mean operative time was . ± . and ± . min in the icg and non-icg groups, respectively. using icg fluorescence imaging, the bile duct was detected on the cyst wall in patients ( %). all surgeries were completed laparoscopically, and no post-operative complications occurred in either group. recurrence of the hepatic cyst occurred in one patient ( %) of the non-icg group. conclusions: fluorescence imaging with icg is used widely in hepatobiliary surgery for intraoperative identification of biliary and vascular anatomies. this method does not require complicated techniques or instruments. icg fluorescence imaging may facilitate the prevention of intra-or post-operative complications, such as biliary leakage, in laparoscopic surgery. in this study, icg fluorescence imaging was found to be effective in detecting the bile duct on the cyst wall intraoperatively, allowing for wider resection of the cyst and avoiding inadvertent injury. our study suggests that wider resection of the cyst wall might prevent recurrence of hepatic and that icg fluorescence imaging could ensure procedural safety. abdominal ct showed: large hepatic cyst ( x , x cm size), with no malignity signs, that occupies practically the whole right liver, causing subsegmentary atelectasis of the middle lobe, superior and inferior cava vein compression, and displacement of right kidney, pancreas and right atrial. due to breath involvement, a percutaneous drainage is performed achieving clinical improvement and reduction of the size of the injury. the patient was released but a cyst superinfection occurred; once this problem was solved, the drainage was removed. results: in light of the complication, surgical treatment was decided, which confirmed the large cyst located in right posterior hepatic segments with tight diaphragmatic adhesions. we carried out the cyst evacuation and a wide laparoscopic resection of the cyst walls, until the posterior area of the cava vein, combining supra and infrahepatic access. the patient was released on the sixth postoperative day and continues asymptomatic. conclusions: simple cysts can be approached in a no surgical way (punction-aspiration with/ without sclerosing products injections) or in a surgical way (cyst wall fenestrations, cystectomy or liver resections). a conservative treatment will obtain symptomatic relief but with a high risk of recurring. recurrence is the main drawback of unroofing. cystectomy is the better option but may be too complicated depending on the cyst's location. to our patient, we carried out a wide laparoscopic unroofing (even though its posterior localization) to minimize recurrence possibilities. in conclusion, laparoscopic resection of the cyst wall is a simple and effective approach in symptomatic or complicated cases. background: single-incision laparoscopic surgery or laparoendoscopic single-site surgery is emerging as an alternative to conventional multiple-incision laparoscopic surgery. it has a potential benefit of less postoperative pain and faster recovery compared with conventional multiple-incision laparoscopic surgery. single-incision laparoscopic hepatectomy (silh) has been reported in only a few small series and the majority were minor resections. case report: a y/o male patient is a case of chronic viral hepatitis b and early cirrhosis of liver. two atypical hepatocellular carcinomas (up to . cm in diameter) located at the junctions of segments & and segments & were impressed by liver magnetic resonance imaging (mri). we performed single-incision laparoscopic anatomical hepatic resection of the right posterior section via a -cm transverse incision on the right middle abdominal wall. inflow control was carried out with an extra-glissonian approach before parenchymal transection. the glissonean pedicles of segments and were divided by linear staplers respectively as well as a major branch of the right hepatic vein in segment . the operative time was min and the estimated blood loss was ml. the pathologic examination revealed two foci of hepatocyte dysplasia with a safe margin of cm. the patient was discharged eight days after the surgery uneventfully. conclusion: single-incision laparoscopic anatomical right posterior sectionectomy is feasible and safe by experienced laparoscopic surgeons. it provides a fast recovery but needs a long operative time. the mortality in the patient with liver cirrhosis is very high. the aim of this work was to decrease mortality and morbidity by using endoscopic local heamostasis and laparoscopic operations, in the patients with bleeding from cirrhosis by variceal bleeding. methods and material: we observed patients with cirrhosis complicated by variceal bleeding during years. there were patients with child phue a, ones with child phue b, ones with child phue c. all the patients were performed prolonged endoscopic heamostasis with conservative therapy. the main methods that we used were the ligation in cases, sealing in cases, sclerotherapy in cases. in cases we couldn't stop the bleeding with band ligation method and introduce the danis stents into esophagus and stopped the bleeding successfully. to prevent the re-bleeding we performed the laparoscopic dissection the abdominal part of esophagus with suturing the venous vessels, coagulations and dissection of short gastric vessels between stomach and spleen, clipping the left gastric artery and vein in the patients. in patients we performed laparoscopical suturing the variceal veins by introducing the laparoscopic trocars into the stomach. in cases with varices vien of stomach, with non-effective local endoscopic heamostasis we performed laparoscopic resection the fundal part of stomach. results: endoscopic local heamostasis were successful (in %) in cases. the relapse of bleeding were in patients. patients died. there was no mortality after laparoscopic operations. there were cases for trocar wounds infection, cases of subphrenic abscess. goals: the advance of laparoscopic surgery also includes the more complex procedures of abdominal surgery such as those affecting the liver and pancreas. there are multiple indications that laparoscopy has in hepatobiliopancreatic surgery, both in benign and malignant pathologies. material and methods: we present the video of a -year-old male patient with a history of right hemicolectomy due to disease-free intestinal lymphoma who, in the control analysis by his attending physician, detects the elevation of tumor markers. an extension study was started showing a hepatic lesion in the caudate lobe with a pathological anatomy suggestive of hepatocarcinoma and an adenopathy suspicious for malignancy adjacent to the right renal vein. the clinical case is presented in a multidisciplinary tumor committee and it is decided to perform surgery. a laparoscopic caudate lobe resection was performed, previously performing intraoperative ultrasound and a lymphadenectomy of the portal territory, vena cava and exeresis of adenopathy of the right renal vein. introduction: major vascular complications during laparoscopic surgery occur approximately in one in cases, but mortality rate can reach - %. most major vascular injuries lead to conversion to laparotomy but successful laparoscopic repair is also possible. simulation training improves laparoscopic performance and possibly reduces surgeons mental strain. materials & methods: during two editions of advanced laparoscopic training course participants had a task to control a major vessel damage (damage). before the task an educational video explaining the methods of obtaining haemostasis was shown. the algorithm of the 'damage' task was as follows: without previous preparation a cm injury of a major vessel was done with l-hook electrocautery. after the injury participants were free to control the damage the way they wanted. heart rate of the participants was measured with an ear electrode. measurements were carried out times-before the injury, immediately after, and afterwards obtaining vessel control. after participants were interviewed for their feelings after the 'damage' task. results: there were vessel injuries in animals. one animal died during the 'damage' task min after desuflation due to relapse of bleeding. there was no conversion to open procedure. temporary vessel control was obtained with different methods. all participants used vicryl . or pds ii . suture for final hemostatic purposes. heart rate of the participants before injury were - ± . bpm, immediately after the injury it rose to - ± . bpm, and after obtaining vessel control were in the range - ± . bpm. a statistically significant difference was found between the ratio of the first and second hr measurement (p = . , t = - . ), and second compared to the third (p = . , t = . ) measurement. participants judged their experience on a -point scale ( - was not helpful at all; -was extremely educative). the educational value of the task received points in cases and points in one case. conclusion: participants feel stress during major vessel bleeding even in animal model, and this stress can result in a serious intraoperative mental strain and significantly increase heart rate. participants found the 'damage' task very useful for their daily practice. the aim of study was to improve the results of treatment of patients with hepatic echinococcal cysts by using of argon plasma coagulation. methods: the analysis of treatment results of patients was put into the basis of this study. it was ( . %) men and ( . %) women in total. an average age of them was . ± . years. the main difference between groups was a way of liver parenchyma coagulation in order to make reliable hemostasis. in main group the final stage of surgical intervention on liver was argon plasma coagulation. it was performed to ( . %) patients. alternatively, monopolar coagulation was performed to ( . %) patients (comparison group). results: in main group in the . % cases pericystectomy was conducted. the resecting surgeries was performed to . % cases. in comparison group was conducted in . % cases. in early postoperative period in main group the complications were observed in . % of cases. the same parameter was . % in comparison group. it led to relaparomies. the forming of external biliary fistulas was observed in ( . %) patients in main group and in ( . %) patients in comparison group. however, all the fistulas have closed spontaneously on th- th day in both groups. hernias of abdominal wall and peritoneal adhesions that manifested by intestinal obstruction of different degree were considered as complications of late postoperative period. these values were % and . % in main group versus % and . % in comparison group, respectively. the resection of hepatic echinococcal cysts with further application of argon plasma coagulation on the cyst bed was accompanied by complications quantity decrease in patients that underwent surgery in early as well as in late postoperative period. in this case more positive dynamics of functional liver values improvements was observed. aims: indocyanine green (icg) fluorescence imaging has been reported as a reliable and safe navigation tool in laparoscopic hepatectomy. however, the factors affecting the sensitivity of tumor detection with icg fluorescence imaging is relatively unclear. the aim of the present study is to analyze the factors of successful icg fluorescence in laparoscopic hepatectomy. methods: this is a retrospective single-center study. this study population consisted of laparoscopic hepatectomies from january to november undertaken at kurashiki central hospital. we excluded patients whose tumors were located more than mm from the liver surface, those who did not receive icg fluorescence imaging, and those who were not injected with icg dye ( . mg/kg) intravenously within days of surgery. the pinpoint endoscopic fluorescence imaging system was used to detect the tumor location. we evaluated the relationship between successful fluorescence and the timing of injecting icg before operation, tumor size, icg r , liver damage and bmi. results: following exclusion, patients were eligible for analysis. among the tumors resected, icg fluorescence imaging detected tumors ( . %), including hepatocellular carcinomas and liver metastases. icg fluorescence imaging detected all tumors in the patients injected with icg to days before hepatectomies . icg fluorescence imaging detected all tumors which were more than mm in diameter. there was no relationship between indocyanine green fluorescence with icg r , liver damage and bmi. conclusions: the injection of icg to days before operation and a tumor size of more than mm can be factors in successful fluorescence in laparoscopic hepatectomy. introduction: cysts in the liver have a wide variety of aetiologies. it is important to characterize the cystic lesion before treating it. the simple cyst has a low prevalence and is more frequent in women. fenestration is a useful option for the treatment of simple cysts in selected patients. case presentation: a -year-old woman was referred to our hospital with a one-year history of intermittent, right upper quadrant pain, with no other associated symptoms. computed tomography and magnetic resonance imaging showed a large cyst ( , x , cm) in the right of the liver. the cyst presented lobulated morphology, smooth edges and well delimited. there were other smaller cysts in the left lobe. hepatic function in blood analysis was normal. biomarkers, tumor markers and hepatitis virus markers were negative. outpatient follow-up and symptomatic treatment of pain was decided. after six months of follow-up, the pain persisted, so surgical treatment was proposed. a laparoscopic fenestration was performed, widely resecting the free wall of the cyst. there was no evidence of a connection to the bile duct. there were no complications. on days she was discharged. discussion: some giant hepatics cysts become symptomatic due to mass effect. persistence of pain is an indication of surgical treatment. laparoscopic fenestration is an alternative for the management of simple hepatic cysts. aim: laparoscopic liver resection for malignant pathology such as colorectal cancer metastases has been a matter of discussion for several groups in the last years. it has been proposed as a safe and feasible treatment but subjects like short and long term outcomes and oncologic results have not been adequately assessed. methods: we performed an observacional retrospective study of patients undergoing laparoscopic liver resection for colorectal metastases in our center. from november to november a total of patients underwent laparoscopic liver resection. data for resection margin, hepatic and extrahepatic recurrence and both disease free survival and overal survival were collected. patients were discussed in a multidisciplinary group with oncologist, radioterapic oncologist and surgeons. the surgical procedures were perfomed by the same team in all the cases to minimize bias. results: a total of patients ( . %) were non resectable at the time of surgery.the mean overall survival was months with a maximum of months. we got a mean of disease free survival in our patients of . months. the hepatic recurrence was %, most of them in high risk patients, and from this group . % underwent a new liver resection. major complications took place in patients ( . %) two biliar leaks, one bowel perforation, two hepatic failure, one evisceration and three respiratory insufficiency needing urgent surgery in three of the cases. mean hospital stay was . days. a mean of days of this stay were in an intensive care unit. conclusions: laparoscopic liver resection for colorectal liver metastases could be a feasible technique when perfomed by trained surgeons. it improves the postoperatory recovery with a reduction of hospital stay and less postoperatory pain without increasing the development of major complications or mortality in the first days after surgery. we got good oncological results that have been improving with the experience acquisition of the surgical team. aged to underwent surgery for cirrhosis with massive refractory ascites child c ( - ), without obvious signs of hepatic encephalopathy. major etiological factors were: viral hepatitis c ( patients ( . %)), b ( patients ( . %)), b ? d ( patients ( . %)), toxicity ( patients ( , %) ). to prevent possible bleeding at the first stage, endoscopic filling of esophageal varices with fibrin glue was performed in patients ( . %). after testing the effectiveness of varices filling, in the following - days decompression surgery of thoracic lymphatic duct was performed under local anesthesia to improve lymphatic drainage from liver and abdominal organs. simultaneously, laparoscopic sanitation of abdominal cavity was performed, with complete evacuation of ascites fluid, rinsing and drainage. fractional post-surgery rinsing was repeated daily for - days towards removing peritoneum edema and improving its absorptive properties. results evaluation was performed , and months after surgery, based on criteria of liver reserves and ascites volume. results: post-surgery mortality from liver failure was . % ( patients) . other patients died of the same cause the following - months. annual survival rate was . %. complete ascites regression over - months after surgery was noted in patients ( . %), significant regression and stabilization in ( . %), moderate regression with need for periodic decompressive laparocentesis in cases. in all patients, functional liver reserves and life quality significantly improved. conclusions: the use of the given technique of refractory ascites correction, in patients with depleted liver cirrhosis, by laparoscopic sanitation with post-surgery fractional rinsing of abdominal cavity, with simultaneous decompression of thoracic lymphatic duct showed very high efficiency and deserves establishment as a clinical practice. t. urade, hepato-biliary-pancreatic surgery, kobe university, kobe, japan aim: anatomical liver resections guided by a demarcation line after portal staining or inflow clamping of the target territory were established as essential methods for the curative treatment of hepatocellular carcinoma (hcc) and then subsequently applied to other malignancies. however, laparoscopic anatomical liver resection (lalr) is much more difficult to reproduce these procedures and to confirm demarcation of the hepatic segment visually on the monitor. recently, laparoscopic fluorescence imaging system has been used as a tool for real-time intraoperative navigation in llr. the aim of this study is to demonstrate how to perform lalr using indocyanine green (icg) fluorescence imaging. methods: three patients underwent pure lalr using icg fluorescence imaging. the following operative procedures were performed: partial liver resection for hcc, segmentectomy for liver metastasis and right anterior sectionectomy for hcc. in all patients, preoperative d simulation images from dynamic ct were reconstructed using a d workstation to decide on cutting points of the glissonean branches. after mobilization of the liver, intraoperative ultrasonography was performed to identify the location of the tumor and glissonean pedicles corresponding to the tumor-bearing hepatic region. we dissected or transected the hepatic parenchyma to encircle the glissonean pedicles. after clamping or closure of them, . mg of icg was injected intravenously to identify the boundaries of the hepatic segments under near-infrared light. parenchymal transection was started according to the demarcation on the liver surface. the lateral aspect of the parenchymal transection was carried out based on the demarcation between non-fluorescing and fluorescing liver parenchyma as far as possible. results: in all the cases, demarcation lines on the liver surface could be visualized clearly after injection of icg. in addition, boundaries of cone units, segments and sections could be recognized to some extent because the tumor-bearing hepatic region became non-fluorescing parenchyma during parenchymal transection. these procedures were completed successfully, and the postoperative courses were almost uneventful. aim: sintrahepatic cholangiocarcinoma is the second most common primary liver cancer after hepatocellular carcinoma (hcc). although the laparoscopic approach of these tumours is not frequent due to its complexity, it is performed increasingly by hepatic surgeons.traditionally, the abdominal surgery in cirrhotic patients has been reserved to selected cases secondary to the high rate of complications. the advance on the treatment of the hcc on liver cirrhosis and the higher safety when performed by laparoscopic approach has encourage some surgeons to extend surgery to child b-c or portal hypertension patients. methods: we present a male of years old, diagnosed in of liver cirrhosis accompanied with portal hypertension. on mri in was found a solid lesion of mm located on segment ii hepatic. biopsy confirmed the diagnostic of intrahepatic cholangiocarcinoma. after a liver function evaluation (child c, meld ), an hepatic chemoembolization was performed. sequentially ct scans indicated a complete radiologic response. after years of follow up, mri showed a recurrence of mm between segment ii and iii of the liver.on multidisciplinary committee liver resection was decided due to suitable liver function and low aggressiveness of the tumour. a laparoscopic left lobe liver resection was performed. sonastarÒ and ligasure tm were used to perform the liver transection and endo gia tm for portal and hepatic veins sections. the surgery develop was complicated due to trend to bleeding that finally was achieve through cauterization. results: early after the surgery, the patient presented a haematic debt through the drain of cc accompanied of hypotension, therefore an emergent surgery was indicated. an exploratory laparoscopy was performed finding hemoperitoneum and diffuse bleeding of the liver surface that was controlled. the patient had a proper recovery and was discharged on the th day post-surgery. the analysis of the specimen showed a . cm cholangiocarcinoma with a . cm margin of resection. conclusion: there is an augmented risk of complications on liver resection of cirrhotic patients with portal hypertension. the laparoscopic approach allows to reduce potential complications, despite bleeding continuous to jeopardize this surgery, this option could be proposed on selected patients. introduction: accessory spleen itself is found in approximately % to % of the population. most ( %) are located near the splenic hilum but intrapancreatic accessory spleens (ipas) are the second most frequent location ( . %) of accessory spleens. in adults, ipas are clinically silent. they may become clinically important because of their radiographic similar appearence of cancer. intrapancreatic accessory spleen is a rare cause of pancreatic pseudotumors and is located in the pancreatic tail in approximately % to %. ipas can be difficult to differentiate radiologically from hypervascular pancreatic tumors such as pancreatic endocrine neoplasms because theycan share a similar enhancement pattern. as a result, most of the reported cases of ipas have been diagnosedonly after distal pancreatectomy was completed. material and methods: we present the case of a -year-old male patient with a history of large vessel vasculitis followed-up for rheumatology, which showed a pancreatic nodule in a control ct so he was referred to digestive for study. an echoendoscopy was performed. it showed, at the level of the tail, in the third distal, a lesion of x mm, hypoechoic, with rounded morphology and well-defined edges that can not be biopsied given the absence of adequate window for the realization of fine needle aspiration biopsy (fnab). based on these radiographic findings, the differential diagnosis included a pancreatic endocrine tumor. due to the high suspicion of malignancy and the absence of biopsy, he was referred to general surgery for scheduled surgery. a laparoscopic corporocaudal pancreatectomy was performed without incidents and the definitive histology showed an intrapancreatic accessory spleen in the pancreatic tail that excluded the presence of cancer. conclusion: intrapanceratic accesory spleen is a challenging diagnosis to make and it should be included in the differential diagnosis of pancreatic neoplasm. its early identification precludes surgical resection. however, the preoperative diagnosis of ipasmay be difficult, and distal pancreatectomy is a safe and relatively simple operation, most of the reported cases of ipas being diagnosed correctly only after surgery there are various options for treating pps. this paper describes our tailored and methodological approach to laparoscopic drainage of pancreatic pseudocysts based on an anatomical classification. methods: we adopted the laparoscopic approach in patients who had pps requiring surgical drainage. the laparoscopic method had been decided according to preoperative computed tomography (ct) and intraoperative findings. the results shown represent median (range). the aim of this work was to decrease mortality and morbidity in patients with combined trauma. methods and material: for years patients were brought to our clinic with combined trauma. everybody was performed ct and ultrasound examination. patients were performed open laparatomic operation due to massive liver rupture, spleen rupture and massive trauma of bowels, pancreas and kidney with massive bleeding. in circumstances we didn't found the trauma of the abdominal organs and the massive abdominal bleeding after ct observation. those patients were cured conservatively. in circumstances with combined trauma after ct examination we performed laparoscopic operation. in circumstances from the patients, who we started laparoscopic operation in, we conversed to laparotomy, due to massive liver rupture, and trauma spleen and hollow organs. in those circumstances we performed urgent laparotomies with suture ligation of bleeding points, suturing of liver and hollow organs and drainage of abdomen cavity. results: we performed laparoscopic operation in patients. in circumstances with trauma of liver we performed laparoscopic electro coagulation and argon-plasma coagulation. in circumstances with trauma of liver we performed electro coagulation with packing the omenture to its surface. in circumstances with trauma of spleen we performed argon plasma coagulation and used fibrin glue. after laparotomic operations mortality were in circumstances, morbidity were in patients. after laparoscopic operation mortality were in circumstances of severe combined trauma with multiple abdominal trauma and morbidity in patients. conclusion: laparoscopic operations in patients with combined trauma decrease mortality and morbility. aims: in laparoscopic distal pancreatectomy, getting away liver and stomach from the surface of the pancreas is sometimes difficult. when we separate the pancreatic body from the retroperitoneum, we must not injure the pancreas to prevent breaking a tumor. when we cut the dorsal side of the spleen from the retroperitoneum, we rarely cut into the spleen accidentally. based on our experiences, we gradually explored a set of procedural operation steps to resolve these problems. our three-step maneuver simplifies the procedure and improves the efficiency and safety of laparoscopic distal pancreatectomy. methods: as the first step, to get away the liver we sutured the round ligament of liver and crus of the diaphragm using - pds and the both ends were tugged form the outside of the body through both side of the xiphoid process. and the stomach was hung from the outside using two nylon thread like a bridge, so we could see the surface of the pancreas body with a good view. the second step was a rolling up maneuver of the pancreas. when we separate the pancreatic body and tail from the retroperitoneum, we rolled the pancreas with gauze for use in laparoscopic surgery and lifted the gauze up in only one assistant's forceps. then we could find the correct line for dissection clearly. the last step was a hanging maneuver of the spleen. when we cut the dorsal side of the spleen from the retroperitoneum, we hanged the hilum of spleen with cotton tape. with this technique we could find easily the correct line to dissect. results: the operation time was h and min and the estimated blood loss was a little. we did not injure the tumor or spleen in this operation. the patient recovered uneventfully after short hospitalization. conclusion: our three-step maneuver can be effective to perform laparoscopic distal pancreatectomy. about - % of patients with pancreatic collections will develop walled off necrosis, with an associated - % mortality. there are multiple options for intervention and drainage, usually the outcomes after endoscopic drainage are related with the nature of the collections. aims: to evaluate and present the rol of endoscopy in pseudocyst and walled off necrosis treatment, and favorable outcomes. methods and results: we present a case of a years old male, who presented biliary pancreatitis treated with cholecystectomy and transoperative cholangiogram weeks ago. he continued with persistent abdominal pain; his ct scan showed a big walled off necrosis; he was taken to surgery for an endoscopy-assisted laparoscopic cystogastrostomy with necrosectomy, he was discharged days po. conclusions: the step-up management of walled off necrosis has proven to be a better option than conventional surgical or endoscopical techniques alone; by reducing complications and mortality vs conventional necrosectomy. the use of endoscopic treatments reduce the pro-inflamatory response. drainage of walled off necrosis can be done by a transpapilar or transmural endoscopic apporach each one with its own advantages. some authors avoid the use of endoscopy in walled off necrosis because of a higher rate of complications, re-interventions and a greater lenght hospital stay. in our experience, we have achieved excellent results with this combined technique. nearest and long- patients underwent chemotherapy after electroporation procedure. day mortality was . % (n = ) in electroporation group. it was found that erreversible electroporation improved local recurrence-free survival ( and months, respectively, p = . ) and distant recurrence free survival ( and months, respectively, p = . ) . overall survival was and months, respectively (p = . ). conclusion: irreversible electroporation of locally advanced pancreatic cancer is safe. four month chemotherapy followed by surgical procedure is associated with good local response and better overall survival compared with chemotherapy alone. these data will be validated in further multicenter study. introduction: pancreatic pseudocysts are the most frequent complication of acute or chronic pancreatitis. usually asymptomatic, they can be managed conservative or, in case of complications, by several methods, endoscopic, percutaneous or by surgery. material and method: we present the case of a years old patient known with an episode of acute pancreatitis five years ago, who was hospitalised now for an upper gastrointestinal bleeding with hematemesis. the upper endoscopy showed a subcardial bulking with an erosion of the posterior gastric wall, with signs of recent bleeding, managed by clipping. patient work-up showed a cm pancreatic pseudocyst at endoscopic ultrasound. taking into consideration the history of the patient, the size and the complication of the cyst, the patient was proposed for a drainage intervention. results: a minimally invasive approach was decided. using ultrasonography guidance, a posterior gastrotomy was performed with the cystotome, establishing the comunication with the pancreatic pseudocyst. dilatation of the path with mm cre baloon, with partial evacuation of turbid liquid. the drainage consisted in pigtail fr plastic stents. the patient was discharged the following day in a good health condition.the endoscopic ultrasound control at weeks showed complete resolution of the pancreatic cyst and was followed by stent removal. the endoscopic drainage of the pancreatic pseudocyst represents the first treatment option as an alternative to the surgical intervention, being minimally invasive, with low risk and fast recovery. clinical case report: a -year-old man was admitted to the hospital with a diagnosis of severe acute pancreatitis and multi-organ failure. during the first month patient has in uci and non invasive procedures were attempted: enteral feeding by a nasoduodenal tube was started and antibiotics were administered to control sepsis. on day , percutaneous drainage was performed for large retroperitoneal abscess. on days, endoscopic transgastric necrosectomy was performed and the left collection was resolved. due to the multi-organ failure persistence and the evidence of size increase of the right retroperitoneal collection, a vard was decided.the right collection was accessed following the previously pigtail catheter. a mm trocar was placed to create retro-pneumoperitoneum with a pressure between - mmhg. a trocar of mmhg was placed, purulent content was aspirated and a debridement was performed. irrigation and aspirate was performed with normal saline and povidone-iodine solution. drainage was used to perform washes with physiological saline and urokinase.on days, the ct confirmed collection resolution. on days he was discharged. after months, the patient is in good clinical condition. discussion: drainage of the retroperitoneal abscesses via laparotomy is highly invasive and risky. vard enables radical necrosectomy and drainage less invasively. in this patient, the complete resolution of the right collection is obtained with retroperitoneal debridement without complications. we conclude that careful retroperitoneal necrosectomy is a valid alternative for the management of right collections. aims: in this study we analyze laparoscopic approach for hepatocellular carcinoma in order to clarify iwe can take advantage in some outcomes as complications, postoperative recovery or long-term survival outcomes. methods: a retrospective case consecutive study has been taken analyzing: age, sex, body max index, comorbidity, surgical extension and tumor size. the outcomes analyzed were: operation time, intraoperative blood loss, blood transfusion, postoperative morbidity and mortality, intensive care stay, hospital stay, tumor size, r resection, conversion rate, early reintervention, disease-free survival rate, overall survival rate results: in this study patients were analyzed males and females with ages between and years (mean age ) and diverse comorbidities: arterial high pressure ( / ; %), diabetes ( / ; , %) ; dislipemy ( / ; , %) , hepatophaty measured as liver cirrhosis ( / ; , %). all of them underwent laparoscopic liver surgery, in cases non-anatomical resection was performed while in the other a segmentectomy was performed. in cases the laparoscopic was strict, in and assistance incision was needed. operative time was - min (mean: min). blood loss mean was , g/dl and only intraoperative transfusion were needed. massive blood loss was reported in case. postoperative medical complications were observed: hepatic failure and renal insufficiency and in case we observed a postoperative hemorrhage that needed an urgent reintervention. the mean of intensive care stay was day and hospital stay was . days. about oncological outcomes r resection was achieve in / ( %), r in / ( %). at years / cases were free disease, dead by progression of disease and dead by other causes. aim: the purpose of this study is to analyze our initial experience with laparoscopic duodenopancreatic resection. introduction: laparoscopic procedures have advanced to represent the new gold standard in many surgical fields. laparoscopic pancreatoduodenectomy and laparoscopic distal pancreatectomy(ldp) are advocated to improved perioperative outcomes, including decreased blood loss, shorter length of stay, reduced postoperative pain and expedited time to functional recovery. however, the indication to minimally invasive approach for pancreatic surgery is often benign or low grade malignances. material and method. the steps of ldp procedures are similar to the open procedure. we perform destructive part of procedure totally laparoscopically and we prefer to do reconstructive part of procedure using hand-assisted techniques. for the period - , we have been perform pd, ( %) we have done with laparoscopic approach. ( %) of patients were operated totally laparoscopic and ( %) of patients were operated by handassisted techniques. results: a significantly higher conversion rate was encountered when lc was done - weeks after es, as compared to week after ercp. it is estimated that pancreatitis after ercp affects roughly three to percent of patients and many endoscopists quote a post-ercp pancreatitis rate of - %. however, - % is probably a more realistic answer for the majority of ercp endoscopists. wise endoscopists inform their patients that there is a spectrum of post ercp pancreatitis severity, from mild ([ % of cases) to severe ( - % of cases). in mild forms, pancreatitis after ercp may resolve itself. conclusion: endoscopic retrograde cholangiopancreatography is a procedure used to diagnose and treat disorders involving the pancreatic and bile ducts. acute pancreatitis is the most common and feared complication of endoscopic retrograde cholangiopancreatography. the assumption is that the duration of the laparoscopic method is longer, but on the other hand the patient have better wound healing and fewer possibility of developing postoperative hernia . the postoperative period is much more simple due to the significantly shorter hospitalization and the faster recovery, and according to patients the level of pain is much smaller as well. however the oncology results are the same. introduction: spiegel hernias are a rare, representing only between . % and % of all abdominal wall hernias. due to its location, below the spiegel line, its diagnosis requires a high index of suspicion. the physical examination only detects % of the spiegel hernias and, in many occasions, imaging tests are necessary for the diagnosis. goals: our objective is to describe the case of an urgent laparoscopic repair of a case of high grade bowel obstruction secondary to a spiegel hernia. material and methods: we present the case of a -year-old male patient with no medical history that comes to the emergency department of our center due to an eight hour evolution of abdominal discomfort associated with nausea without vomiting or other symptoms. the patient was afebrile and hemodynamically stable at all time. on physical examination, the abdomen is soft and depressible, painful on the left flank where a tumor compatible with spiegel's hernia is palpable. in the blood count there is no leukocytosis nor alteration of inflammatory parameters. an abdominal computed tomography (ct) scan was requested from the emergency department which demonstrated a high-grade small bowel obstruction caused by an entrapped loop of distal jejunum conditioned by a left-sided spiegel hernia. given the situation, an informed consent was obtained, and the patient was taken to the operating room for emergency laparoscopic repair. we performed a laparoscopic hernioplasty with ventralpatch mesh between oblique major and transverse and primary closure of defect in continuous suture. after this, the evolution of the patient is favorable, with good oral tolerance and re-establishment of intestinal transit, being able to be discharged h after surgery. the spiegel hernia is a rare entity that requires a high index of suspicion for its diagnosis. despite the limited evidence published in the literature on the laparoscopic repair of incarcerated spiegel hernias, the studies published so far suggest that the laparoscopic repair is a valid alternative to the classic approach when it is performed by a well-trained laparoscopic surgeon. introduction: repair of lateral abdominal wall hernias (both primary and incisional) can be challenging due to the complexity of anatomy, issues with fixation and the low incidence of such cases. a good understanding of abdominal wall and retroperitoneal anatomy, coupled with proficient laparoscopic technique is essential for successful repair via the minimally invasive approach. methods: a retrospective review of a prospectively maintained database was performed to identify patients with lateral abdominal wall hernias who underwent laparoscopic repair from january to july . results: patients with hernias were identified ( primary, incisional). mean patient age was (range - ) and mean bmi was . kg/m (range . - . ). according to ehs classification, the incisional hernia defects were located at subcostal (l , n = ), flank (l , n = ), iliac (l , n = ) and lumbar (l , n = ) regions. background: it is commonly admitted that laparoscopic surgery has the advantage of abdominal wall preservation. however, the increased use of laparoscopy has resulted in certain complications specifically associated with the laparoscopic approach, such as trocar-site incisional hernia. until today, it is not finally clarified 'patient-dependent' factors contributing to the occurrence of postoperative hernia after laparoscopic abdominal surgery. methods: between and , patients were operated due to trocar-site incisional hernia in one surgical centre. 'the patient-depending' factors which caused postoperative trocar site incisional hernia data was collected and retrospectivily analysed. results: port site incisional hernia occurred in % ( patients) after the use of trocars with mm or larger diameter. the presence of metabolic syndrome was the decisive factor in the development of postoperative incisional hernia in % ( patients). in % ( patients) the postoperative hernia occurred on the background of a long cough symptoms caused by chronic obstructive pulmonary diseases. the cause of postoperative hernia in % ( patients) of patients was the condition of lifting a one-time severity or heavy physical work. in % ( patients) of postoperative patients hernia developed due to prolonged constipation of chronic inflammatory colon diseases. conclusions: thus, when the aponeurosis of the trocars is adequately closed, the reason of the occurrence of postoperative hernias was caused by patient-dependent factors which increase intra-abdominal pressure. for this method, small midline incision cm in length - cm away from hernia orifice was carried out initially. dissection of intraperitoneal adhesion was carried out by sils with sils device. subsequently after closure of initial laparotomy unilateral anterior rectus sheath was incised from the same incision and dissection of retro-rectus space up to preperitoneal space was done under laparoscopic vision. dissecting the other side was carried out by same fashion. initial dissection of linea alba could be done by open surgery from initial incision. further dissection of linea alba, retro-rectus space, and hernia orifice was carried out by sils. defect closure of anterior and posterior rectus sheath using barbed suture was also done by sils and self-grip mesh was inserted. additional trocar to assist retro-rectus dissection, defect closure, and decompression of intraperitoneal cavity was inserted as required. aims: the laparo-endocsopic approach of inguinal hernia contiue to bring many clarifications concerning inter-parieto-peritoneal space of this region through in vivo exploration, obtained by magnification by means of specific optic intrumentation. our study aimed to revalue the in vivo fascias, to establish their embryological correspondences and to reunite the variable nomenclature existing in the classical anatomy of this region. these observations find their applicability in tapp and tep hernia procedures, as the old anatomical descriptions are no longer operative. methods: we have tried to identify the structures that delimit the anatomical regions of retzius and bogros in recording of tapp procedures performed on men, on the right side, for small indirect hernias on patintes with clear view of the structures. additional, a review of literature on this subject has been performed through a search in the detabases according to the following keywords: bogros space, retzius space, preperitoneal approach, urogenital fascia. results: retzius and bogros are the medial and lateral compartments of the inter-parietalperitoneal space, located between the transversal fascia and the parietal peritoneum. these narrow, virtual spaces are best highlighted today with the help of insufflation techniques during laparo-endoscopic procedures. a competent and careful dissection confirms a 'deep and superficial' stratification, highlighting embryonic relics derived from the uro-genital fascia: urinaryprevesical fascia and spermatic fascia. in addition, the real retzius space is located previously and the real bogros space is located behind this strcuture. the confluence area of the two spaces is a critical point of laparo-endoscopic dissection, its non-recognition may 'wander' the dissection. conclusions: literature data in this topic reflects a certain terminological confusion using general terms such as 'preperitoneal tissue' or 'arreolar tissue' to denote what we consider to be the urogenital fascia or its prologations. the data obtained were synthesized in several drawings and diagrams very useful in training surgeons to use tapp / tep techniques. aim: spigelian hernia containing epiploic appendage is really rare entity. in this paper, we present a very rare case of spigelian hernia involving epiploic appendage performed laparoscopic hernia repair. case report: a -year-old woman presented to the emergency department with sudden onset abdominal pain in the left lower quadrant. on physical examination, she had a small, palpable tender mass in the left lower abdominal quadrant. temperature and white blood cell count were normal. an inflamed epiploic appendage with an oval shape, a fatty core, and a central thin hyperdense line in the hernia sac was detected on abdominal computed tomography. its intraabdominal relationship with the normal wall of the sigmoid colon was well appreciated (figure a, b) . diagnostic laparoscopy was performed. (figure ) adhesions between the sac and epiploic appendage are released using sharp dissection. a peritoneal flap is then created (figure ). laparoscopic tapp repair was used without closing the defect (figure ) . the patient was discharged on th days uneventfully. aims: morgagni's hernia is an in infrequent, congenital, anterior or retrosternal diaphragmatic defect. the right side is the most frequently affected, up to % of cases. it represents between and % of congenital diaphragmatic hernias. in childhood, they usually attend asymptomatically or with respiratory symptoms. up to % are diagnosed in adulthood, incidentally or after gastrointestinal obstruction debut. the treatment is surgery, which can be by laparoscopic or open approach.we present a case of laparoscopic approach with intra-abdominal mesh placement of giant morgagni's hernia diagnosed in senile age. methods: -year-old woman with a history of advanced alzheimer's dementia, partially dependent in daily life activities and institutionalized who consulted for intermittent episodes of oral diet intolerance associated with vomits of one month of evolution. abdominal examination was anodine. chest radiograph revealed a right lower lung field mass with fluid collected. thoracoabdominal scan showed small bilateral pleural effusion and large, right anterolateral morgagni's hernia, which contains dilated segment of transverse colon and greater omentum . results: laparoscopic approach was performed. hernia was reduced and hernia sac was removed. the defect was repaired with a dual-component (absorbable and non absorbable) mesh anchored with intracorporeal suture. patient recovered and was discharged days after surgery. conclusion: laparoscopic approach for morgagni's hernia reapir is secure and offers the advantages of less post-opertive pain, faster recovery and short postopatory stay. introduction: recently, laparoscopic operations for ileus are increasing. we have undergone laparoscopic operation to adhesive ileus with umbilicar incision at the beginning. the umbilicar incision at the beginning makes it possible to secure the laparoscopic field by peeling the adhesion under direct view, and makes it easy to repair damage to the intestinal tract. surgical procedure: at first, the umbilicus - cm incision was made and peeled the adhesion as much as possible under direct vision. secondly, ez access was set and inserted one mm port, therefore laparoscopic operation was performed with or pieces of mm ports. when the repair or resection of small intestinal due to damage is necessary, it is pulled out through the ez access. objective: to investigate the possibility of problems of laparoscopic ileus operation to adhesion ileus by umbilicar incision at the beginning. introduction: small bowel obstruction (sbo) during pregnancy is a rare condition with an incidence of . - . % and in around % of cases it is most caused by adhesions from previous abdominal surgery. other diagnosis, such as, hernias, malignancy, volvulus or intussusception are extremely rare. when sbo occurs in pregnancy, it carries a significant risk to mother and fetus. its diagnosis of can be difficult to make as symptoms are often attributed mistakenly to the pregnancy. goals: a case report of congenital bowel obstruction during the second trimester of pregnancy handled by laparoscopy. material and methods: we report the case of a year old woman with a history of chronic lung disease, pregnant because in vitro fertilization ( ? weeks) who attended the emergency department with abdominal pain and bloating accompanied by nausea and vomiting for two days. on physical examination she showed a distended, soft, depressible and painful abdomen without peritonism. laboratory tests were normal. a nasogastric tube was placed with generous output fecaloid intestinal contents. abdominal ultrasound by expert radiologists in abdomen showed a moderate amount of free abdominal fluid with normal uterus moderate and sbo to the ileum because of intestinal adhesion. this results were confirmed with an magnetic resonance imaging (mri). results: the patient was operated by laparoscopic approach with three trocars. the main problem was discovered. we founded a congenital adhesion which conditionated the obstructive syndrome. postoperative recovery was uneventful and the patient was discharged h after surgery. conclusion: the non-obstetrical acute abdomen in pregnant patient is a reality that occurs in one of every pregnancies. its diagnosis in more difficult than in nonpregnant patients requiring or high index of suspicion. the laparoscopic approach of acute abdomen during pregnancy is a valid and safe option, even in the early hours after diagnosis of bowel obstruction when it is performed by a well-trained laparoscopic surgeon. aim: intestinal malrotation (im) without midgut volvulus in adults is a rare clinical entity, which is the result of an incomplete rotation of the small bowel during embryogenesis, due to the nonlysis of the ladd bands. these ligaments spread between the duodenum and caecum and do not allow the gastrointestinal tract to take its normal position into the peritoneal cavity. im appears in to - newborns and is usually asymptomatic. diagnosis is usually made in the first month, and presents with findings of an acute abdomen, small bowel ileus and volvulus. im in adults is a rare entity. most of the times it is asymptomatic, but it can cause chronic abdominal discomfort and constipation. we present the laparoscopic management of an adult patient with intestinal malrotation. methods: our patient, a year old female, presented to the emergency room with a -month history of abdominal pain and nausea. all blood tests were normal. an abdominal mri showed intestinal malrotation without volvulus. due to persisting symptoms, she underwent a diagnostic laparoscopy with complete lysis of the ladd bands. the only unusual finding was a slight oedema of the duodenum. results: her symptoms settled postoperatively and she was discharged on the nd postoperative day. since her discharge, she has not developed any similar abdominal pains or complaints. conclusions: symptomatic intestinal malrotation in adults is an unusual clinical entity, but it is definitely one of the differential diagnoses we need to consider in case of chronic abdominal symptoms. the management consists of the division of the ladd bands, and this procedure can be performed safely with laparoscopy. many small intestinal obstructions are due to adhesions after laparotomy, but small bowel obstructions without history of open surgery is relatively few. in diagnostic imaging such as preoperative ct examination, the cause is diagnosed to some extent, but details are sometimes unknown unless operative observation is actually made. in many institutions, laparoscopic surgery is also actively introduced into the operation to relieve bowel obstruction, and its effectiveness is beginning to be recognized. we examined the usefulness of laparoscopic surgery for patients with small bowel obstruction without history of laparotomy from experience in our hospital. aim: from december to october , we searched cases of laparoscopic surgery for a small bowel obstruction without previous laparotomy at our hospital, and clinical findings, surgical results, and postoperative course were examined. results: there were ten cases. eight men and two women. the median age was years ( - yrs.) . reasons for intestinal obstruction were adhesions cases, internal hernia cases, persimmon stones case, small intestine tumor case. four cases of adhesions were emergency surgery. there were cases of emergency surgery and waiting surgery. five laparoscopic operations were completed and five cases during laparotomy transition. the median surgical operation time was min ( - min), and the median bleeding amount was g ( - g). there was no fatal case after operation, only one complications of ileus. the median length of hospital stay was days ( - days) . conclusion: laparoscopic surgery for intestinal obstruction with no history of laparotomy was thought to be a safe and effective procedure. although the transition to laparotomy would be higher in case of emergency, but there was no case of large incisional laparotomy. conclusions: laparoscopic surgery for sbo reduces postoperative complications and contributes to shortening the postoperative hospital stay and to decreasing the rate of recurrences, although it is a retrospective study, which is a safe and a useful approach. furthermore, first episode of sbo without previous operation seems to be an appropriate indication for laparoscopic surgery. background: postoperative adhesion after abdominal surgery may cause intestinal obstruction, chronic pain, or female infertility, which constitutes the major problems after surgery. adhesion formation are reported to be reduced by laparoscopic surgery and the use of anti-adhesion barriers. seprafilm composed of sodium hyaluronate carboxymethylcellulose bioresorbable membrane has been widely used to date, especially in open surgery. the characteristics of seprafilm, which is easily stick when wet, conversely brittle when dry cause it difficult to deliver into the abdominal cavity via the small incision in laparoscopic surgery. therefore, seprafilm is not much used in laparoscopic surgery. although various methods of insertion of seprafilm have been reported, some need special devices, or some acquire skill. methods: we adopted the pre-moistening technique for the replacement of seprafilm in consecutive cases of laparoscopic gastrointestinal surgery. a sheet of seprafilm was cut into equal pieces. to soften the sheets, one of the pieces was placed on a folded wet gauze until it became naturally curled then it was reversed, and the same procedure was repeated. softened sheet is easily to deliver into the abdominal cavity via a small incision by pushing with digital finger. moistened sheet expands naturally in the abdominal cavity. one or two pieces were needed to cover the incision. this process took only a few minutes. results: in all cases, the sheets were successfully introduced into the abdomen and spread widely enough to cover the incision. there have been no adverse effects, no postoperative complications, or gastrointestinal obstruction due to adhesion in the observation period of median two years. conclusions: short term outcomes were good after applying this technique. however, to record the incidents of intestinal obstruction and chronic pain, over years observation is indispensable. long term follow-up studies are required to clarify the usefulness of the anti-adhesive barrier in gastrointestinal surgery. b. east, rd department of surgery, motol faculty hospital, prague, czech republic aim: since when the ipom acronym was used for the first time our views at intraperitoneal mesh positioning has changed several times. despite growing evidence on its possible long term consequences it is still preferred method at some centres for large number of patients. the aim of this study is to point out the pitfalls of this method but also show that ipom is a good technique but only for highly selected cohort of patients. methods: this is a review of the literature focusing on the indications and complications of ipom pointing out controversies among the published articles over last two decades. some mesh material characteristics are being discussed as they are basic for understanding this complex and highly sensitive issue. results: a wide range on indications of ipom from little umbilical to large incisional hernias is advocated by many. however, some opinion leaders promoting this technique as universal and ideal for everyone just few years ago are advising to avoid it if possible lately. a necessary overlap has also been questioned recently. despite improving anti-adhesion barriers and methods of fixation in may a surgical mesh has become classified as risk class iii by the eu parliament and council on medical devices hoping to prevent physiomesh like incidents in the future. the need for post market registries and long term follow up is obvious. conclusion: us as surgeons implant a mesh in our patients and therefore we should be aware of its possible long term effects. no mesh on the market has a long term safety evidence especially in the intraperitoneal space. ipom is a good technique but possess a significant risk of long life complications and therefore should be spared only for those unfit for other methods of repair, patients with too high mesh infection risk, obese or older patients. introduction: acute appendicitis in elderly patients is relatively uncommon and could represent an underlying neoplasm. hence patients over the age of are often referred for a follow-up colonoscopy after management of acute appendicitis. the current routine use of computed tomography (ct) scans in the evaluation of suspected acute appendicitis in elderly patients prior to surgery coupled with intra-operative findings at laparoscopy question the role of follow-up colonoscopy for these patients. aims: to determine the role and optimal timing of colonoscopy in early detection of colorectal neoplasia after treatment of acute appendicitis in elderly patients. methods: all patients aged years and above with confirmed appendicitis admitted to our hospital during the period / / to / / were included. follow-up colonoscopy, diagnosis of colorectal neoplasia and its location in this patient cohort was evaluated. results: number of people aged and above in olol who had appendectomies from the dates / / to / / = . out of them / ( %) had full colonoscopy within years of the appendectomy.of them of the colonoscopies done were maleand were females. / ( %) of these colonoscopies were completely normal. colonoscopy identified colorectal carcinoma in ascending colon ( . %). other pathologies identified included: benign polyp ( %), polyp with low grade dysplasia ( %) and others ( . %) (lymphocytic colitis, ulcerative colitis, medication related ulceration, diverticulosis, melanosis coli, haemorrhoids). conclusions: in elderly patients above years of age: there may be an increased risk of colorectal cancer after acute appendicitis. only % of this patient cohort underwent colonoscopy after appendectomy. the current recommendations suggest the need for follow-up colonoscopy in elderly patients post acute appendicitis. further studies are needed to decide whether routine colonoscopy is indicated after acute appendicitis patients over years. introduction: it is generally accepted that the main aetiology of appendicitis is obstruction due to appendicoliths in adults and lymphoid hyperplasia in children. in contrast, incidental appendicoliths have been reported to occur in up to % of the asymptomatic population. controversy still exists regarding the association of appendicolith and appendicitis. is the appendicolith a causative factor or merely an incidental finding? aims: to determine the association between the presence of appendicolith and acute appendicitis (perforated or non-perforated) vs healthy appendix. methods: we collected the data retrospectively from the electronic records of all appendicectomies performed between january and december in our institution. data collected included: age, sex, appendix histology and the presence of appendicolith. interval or incidental appendicectomies were excluded from this study. we analysed the data using spss software version . results: during the study period appendectomies were performed (males: , females: , age range: - years). cases were histologically confirmed cases of acute appendicitis and of these, were perforated. a normal appendix was identified in cases. the remaining cases were due to chronic appendicitis, sub-acute appendicitis, lymphoid hyperplasia, parasitic infestation, and neoplasm. appendicolith was found in cases, of which were found in a normal appendix and were found in an inflamed appendix. out of the cases of appendicolith with normal appendix: cases were aged between and years old, cases were aged between and years old and case was aged between and years old. out of cases of appendicolith with acute appendicitis, cases were aged between and years old, cases were aged between and years old and case was aged over . conclusions: appendicolith may merely be an incidental finding and is not the primary cause of appendicitis. no significant correlation between gangrenous/perforated appendicitis and the presence of appendicolith. contrary to popular belief appendicoliths are more common in paediatric appendicitis than in adult cases. further research is recommended. over the last years, patient satisfaction surveys have gained increased popularity. nowadays, respect for patients' needs is central to our health care system. hospitals use patient satisfaction surveys to assess quality of care. many hospitals routinely survey patient satisfaction but relatively little data has been published. our acute surgical assessment unit operates from am to pm monday to friday and in its first year saw surgical patients, of whom were discharged and were admitted to the hospital for further management. aims: to assess the levels of satisfaction of patients attending asau at our lady of lourdes hospital. methods: a random sample of patients seen in the asau was surveyed to determine their level of satisfaction and the experience they had whilst attending asau. a novel self-reported patient satisfaction questionnaire was developed and used to assess patients' opinion regarding the treatment they received, the doctor's explanation of their condition, the waiting time and the service in asau. also the questionnaire encouraged patients to suggest improvements to the service. aim: sintestinal obstruction is a very common cause of presentation to an emergency department. the most common cause in patients with prior abdominal surgery are adhesions, but the list of differential diagnosis is large. internal hernia is a very rare cause of obstruction, with a reported incidence of between . and . %. the herniation related with broad ligament defects is even more uncommon. methods: we report the case of a -years-old woman with antecedents of liver transplant, tubal ligation and appendectomy. the patient was admitted refering abdominal pain in the epigastrium of h duration, accompanied by nausea and vomiting. on physical examination, abdomen was depressible, tender in the right low quadrant, without evidence of peritoneal irritation. laboratory studies were normal except for an elevated leukocyte count with a left shift. computed tomography (ct) revealed dilated small bowel loops with a transition point in right lower quadrant. radiological diagnosis was intestinal obstruction, with fibrous adhesion as the most probably aetiology. management was conservative at the beginning, with intravenous hydration, nasogastric tube and administration of gastrografin (diatrizoate) without a good response. results: at h, an exploratory laparoscopy was perform, finding dilatation of small bowel loops and a cm defect in the right broad ligament in which a segment of ileum was herniated. ileal segment was liberated without evidence of ischemia. the hernial defect was closed by laparoscopy with simple silk stitches. the postoperative course was excellent, tolerating oral feeding next morning. the patient was discharged h after surgery. conclusions: internal hernias of the broad ligament are an extremely rare cause of intestinal obstruction, but must be added to the differential diagnosis for female patients due to the risk of intestinal strangulation and perforation. even if clinical and radiological diagnose is difficult, ct is the best tool to delineate the cause and location of the obstruction. laparoscopy allows reduction of the hernia and closure of the defect with minimal invasiveness. because of that, the laparoscopic approach of bowel obstruction should be considered as the first choice if there is the suspicion of an internal hernia, without signs of necrosis or perforation. the laparoscopic approach is a safe and effective tool in the management of postoperative complications. it is well tolerated in critically ill patients and avoids respiratory and wound related morbidity associated with laparotomy. it also reduces diagnostic delay and a considerable number of unnecessary laparotomies, with a high resolution rate and minimal morbidity. it thus represents a valid and necessary alternative in surgeon's armamentarium. in the management algorithm of our institution we always choose the laparoscopic technique as the fisrt tool in case a reoperation is necessary. , small bowel obstruction ( . % vs . %), and colorectal cancer obstruction ( . % vs . %) was found higher for acs unit group, and also progressively higher during the last years. conclusion: according to our study, laparoscopic approach in abdominal emergencies shows an upward trend, and surgeons from acs units seem to have higher rates of laparoscopy than general surgeons in emergency procedures. background: incarcerated and strangulated hernias present a major problem in emergency medicine. there is scarce data about the role of laparoscopy in the management of these patients. laparoscopic repair offers the benefits of the ability to survey the incarcerated organ and to evaluate its viability, apart from the obvious advantages of laparoscopic surgery. the use of mesh repair in these emergent operations is also a major concern, due to the un-sterile conditions in which they are performed. objective: to evaluate the safety and short-term efficacy of laparoscopic emergent repair of incarcerated hernias. methods: retrospective review of prospectively collected data of all the patients who underwent emergent laparoscopy due to an incarcerated hernia between november and october . results: during the study period, patients underwent emergent laparoscopy due to incarcerated hernias ( females, males). had incarcerated inguinal hernias, and had incarcerated umbilical hernias. mean age was . . all inguinal hernias were repaired in the tapp approach, and using an absorbable mesh. all umbilical hernias were repaired using the ipom approach. patients had bowel obstruction, had incarcerated omentum, and one patient had incarcerated urinary bladder. patients underwent resection of an ischemic organ ( bowel, urinary bladder, omentum). mean hospital los was . days. during the follow up period there were no mortalities, and no recurrences. one patient had a wound infection that resolved with antibiotics. conclusion: laparoscopic emergent repair of incarcerated hernias is a safe and feasible approach. further studies with longer follow up time need to be conducted, in order to evaluate the added benefit of the laparoscopic approach. gibraltar is a small overseas british territory with a residential population of approximately , inhabitants, that increases up to , daily due to incoming tourists and cross-frontier workers. as a geographically isolated center we have to provide a varied service including emergency surgery, and elective operating such as colectomies, gastrectomy's etc. one of the challenges faced is the limited stock of red blood cell (rbc) units within gibraltar and reliance on platelets (plt) from across the border from spain. given the immanent brexit we need to prepare for the challenges we will face in these times of political and distribution uncertainty. a prospective audit of all blood use within gibraltar was carried out over months. the number and type of units requested, the number of units given, the speciality, location and indication for requests was recorded. introduction: the use of laparoscopic surgery in abdominal emergencies, such as in trauma, has had a slow acceptance. the advantages with this approach include less postoperative pain, faster recovery, quicker return to everyday activities, and fewer complications. we have collected the cases and indications of laparoscopy in abdominal trauma in the main hospitals in the andalusian capitals and compared with the national registry material and methods: a total of patients who underwent laparoscopic surgery in the main hospitals of seville, cordoba, malaga, cadiz, huelva, jaen, granada and almeria were analyzed. they have been compared with the traumas archived nationally by the spanish association of surgeons taking into account age, sex, score of the american society of anesthesiologists, hemodynamic stability and mechanism of injury. the intra and postoperative variables were compared between groups. results: at the national level, the main cause of abdominal trauma were traffic accidents, therefore, it was the patients who had a greater number of laparoscopies ( . %), followed by stab wounds ( , %) and run over ( . %). in our series, the average age of the patients is years and % are male. only eco-fast was performed in % of the patients, being positive in . % of the cases. as they were stable patients, in % of the cases a tac was possible. in our data, % of the laparoscopies were performed for therapeutic purposes as well as being diagnostic, thus avoiding a posterior laparotomy. conclusion: slaparoscopic surgery for abdominal trauma, either blunt or penetrating, is safe and technically feasible in hemodynamically stable patients. we found that laparoscopic surgery was associated with shorter operative time, lower estimated blood loss and faster return to normal diet. based on our findings we establish the indications of laparoscopy in these patients aims: submucosal aneurysm of small intestine is extremely rare, but its rapture can be lifethreatening. due to the unstable hemodynamics and unknown site of bleeding, emergency laparotomy has been widely performed for the rupture. we will present case reports and show the strategy for minimally invasive treatment for ruptured aneurysm. methods: we experienced two cases of ruptured submucosal aneurysm resected by laparoscopic surgery. case is a -year-old male who was taken to our er with massive hematochezia. ct showed arterial bleeding in the small intestine and angiography revealed bleeding from the ilial artery. selective embolization using gelatin sponge and micro coil was performed and hemostasis was obtained. video capsule endoscopy found the hemispheric elevated lesion with protrusion at the top in the ileum. using balloon assisted enteroscopy, the site of aneurysm was marked with injecting india ink, which allows surgeons to accurately and easily identify the part of small intestine with aneurysm. subsequently, a single incisional laparoscopic assisted partial ileectomy was performed for the purpose of definitive diagnosis and preventing re-bleeding. the ileum with aneurysm was easily identified in laparoscopic exploration owing to the marking, and it was taken out from the incision to perform resection. case is a -year-old female who was transferred to our emergency department with sudden onset of massive melena. ct and angiography were perfomed, and bleeding from the rd jejunal artery were confirmed. subsequently, therapeutic embolization was performed in the same way as case . enteroscopy revealed submucosal elevation similar to case in the jejunum. we carried out endoscopic tattooing, followed by single incisional laparoscopic assisted partial jejunectomy. results: the operative time in case and case were min and min, respectively, and the amount of blood loss was both ml. the postoperative course was uneventful in both cases. case was discharged on the postoperative day , and case was on postoperative day . conclusions: our experience indicates that ruptured submucosal aneurysm of the small intestine can be effectively managed by a laparoscopic surgery with combination of therapeutic embolization and enteroscopic evaluation, which is safe and minimally invasive. background: laparoscopic bilateral inguinal hernia repair may be completed with one large selffixating mesh crossing the midline in front of the bladder. no studies have investigated in detail whether preperitoneal mesh placement induces temporary or more lasting urinary symptoms. methods: urinary and hernia related symptoms were evaluated preoperatively and postoperatively at , and months in patients using the iciq-mluts questionnaire and eurahs-qol score. results: voiding symptoms and bother scores were unchanged at or months, but there was significant improvement at months compared with preoperative findings (symptoms p \ . ; bother score p \ . ). incontinence symptoms improved at month (p \ . ) but not at or months, with a bother score significantly improved at month (p \ . ) and months (p \ . ). diurnal and nocturnal frequency did not change significantly postoperatively, but months nocturnal bother score was decreased (p \ . ). eurahs-qol scores showed significant improvement in all domains for all measurements compared to previous measurements. postoperative symptoms were improved at months, compared with preoperative pain scores (- . ), restriction of activity (- . ) and cosmetic scores (- . ) these findings were statistically significantly (p \ . ). at months, there were no patients with severe discomfort (score = ) for any of domains. no recurrences were diagnosed with % clinical follow-up at months. conclusion: placing a large preperitoneal self-fixating mesh for bilateral groin hernia repair did not cause new urinary symptoms and demonstrated significant improvement in voiding symptoms at months. incontinence and nocturnal bother score were significantly improved. introduction: tep/tapp hernia repair is an increasingly widely used surgical methods for minimally invasive treatment of inguinal hernia. tep advantages to tapp are noincision of the parietal peritoneal sheet, therefore no need for its recovery-sewing or sticking at the end of the procedure, and no need for attachment of the prosthetic mesh to the structures of the anterior abdominal wall, which results in a reduction in the financial cost of operation.various types of meshes with different characteristics are used, depending on the surgeon's preferences.the aim of this study is to highlight mesh-related postoperative complications, which can be serious and life-threatening. material and methods: a retrospective cohort study of cases of unilateral or bilateral tep and tapp hernia repair performed at the university hospital for the period - with a study of early and late postoperative complications potentially causally related to the implanted prosthetic mesh and methods of their treatment. results: for a -year period tapp ( bilateral) and tep ( bilateral) have been performed. three complications (clavien-dindo iva, ivb and v) were found, of which were early postoperative (up to pod)-one in tapp- pod small bowel adhesive ileus due to suture dehyscense of the peritoneal sheet and adhesion of a bowel loop to the surface of polypropylene mesh.one in tep- pod-a large preperitoneal hematoma with haemorrhagic shock at years old female in anticoagulant therapy-an open revision of the preperitoneal space and definitive haemostasis; followed in pod established bladder lesion from erosion from the edge of self-locking polypropylene mesh. suture and drainage performed, but the patient died of decompensation of concomitant diseases. a late complication- months after bilateral tep-erosion of soft polypropylene mesh of sigma (probable undetectable lesion of the peritoneum) with faecal peritonitis-hartmann procedure with laparostoma followed by restitution but persistent chroniosepsis with established abscess in retzii. months after-revision with abscess incision and extraction of infected meshes. discussion: use of biologic meshes is quite expensive, however synthetic non-resorbable meshes implanted in preperitoneal layout is a prerequisite for specific severe postoperative complications. inguinal hernia repair is one of the most performed procedure all over the world, with more than million procedures performed each year, it represents one of the top three most performed procedures. the lichtenstein procedure is one of the first procedures that a young trainee in general surgery learn, not only for its reproducibility and for the great numbers of procedures that could be done in each department, but also because during inguinal hernia repair the trainee learn a lot of skills which are the basis of major surgical interventions. the surgeon's performance for any procedure could be evaluated by way of established learning curves that can predict the minimum number of procedures required to reach the same intra and post-operative outcomes as an experienced surgeon performing the same technique. the aim of our multicentre study was to analyse how many cases are required to stabilize operating time (ot) and intra and post-operative complication rates over the course of the learning curve period for a lichtenstein procedure. from january to december all lichtenstein procedures from four different institutions were recorded in a prospective maintained computer database. the results of the first consecutive procedures performed by three different trainees (group a; group b; group c) were compared with the same numbers of procedures by two senior surgeons of the same institutions (group e, group f). cusum analysis was performed to evaluate the achieving of learning curve. no differences in terms of biometric and hernia type were recorded between the five groups. cusum analysis showed that the trainees achieve the learning curve between the - procedures. no intra or post-opertive complications were recorded during the training period.in conclusion after our analysis we found that at least procedures are needed for the trainees to achieve the learning curve for lichtenstein procedures. background: since its first description in the s, the total extraperitoneal (tep) technique has established itself as a popular endoscopic method for the repair of inguinal hernias. the tep repair is generally viewed as a technically-demanding procedure requiring adequate experience to minimize and handle complications. in this case report, we describe an uncommon complication of urethral injury, which was successfully repaired laparoscopically. case report: mr r is a year old gentleman with no significant past medical history who presents to the department of general surgery, tan tock seng hospital, with a two-month history of a reducible right inguinal hernia, associated with some tenderness. an ultrasonography confirmed the diagnosis of a fat-containing indirect right inguinal hernia. in view of persistent pain, mr r was counseled for a laparoscopic repair of his right inguinal hernia. as mr r was able to empty his bladder just prior to surgery, no urinary indwelling catheter (idc) was inserted. an infra-umbilical incision was made to access the posterior rectus sheath and a balloon was used to bluntly dissect the pre-peritoneal plane. on inspection of the operating field, persistent pooling of blood was noted in the retropubic space. careful inspection revealed a defect in a tubular structure just inferior to the bladder neck. an idc was inserted, which confirmed a . cm defect in the pre-prostatic urethra. decision was made for primary repair using absorbable sutures in two layers. the bladder was subsequently filled via the idc, which did not reveal any leak. we then completed the right inguinal hernia repair using a mesh. mr r made an uneventful recovery and was discharged on post-operative day with instructions to keep the idc in-situ for two weeks. the idc was removed after two weeks and a micturating cystourethrogram was performed, which showed no filling defects along the urethra and no contrast leaks. discussion: though uncommon, urethral injuries can be a complication of laparoscopic tep repair. the key to managing these complications is in the early identification of such injuries intra-operatively. with early recognition and careful assessment, such complications can be managed laparoscopically with minimal post-operative morbidity. aim: the purpose of this study is to report surgical technique and outcome of hybrid tapp procedure (a combination of tapp and ipom) for inguinal hernia patients complicated with preperitoneal space adhesion. methods: hybrid tapp procedure is applied if peritoneal dissection or closure of the peritoneum is difficult due to severe adhesion. the peritoneum should be dissected as much as possible. for the site where adequate dissection was achieved, the collagen mesh is placed outside the peritoneum. in the part where dissection was difficult it is placed inside the peritoneal cavity. in order to prevent mesh migration, the mesh should be directly fixed to the cooper's ligament with a tacker. for this purpose, the peritoneum around the cooper's ligament must be well-dissected, even if it is strongly adhered, so that the ligament can be exposed. the crucial points in the hybrid tapp procedure are fixation of the mesh and prevention of the bowel herniation into the preperitoneal space. at the site where peritoneal dissection is possible, the mesh is directly fixed on the fascia using a tacker. if it is difficult, the mesh is placed in the peritoneal cavity and fixed over the peritoneum. if there is a risk of migration along with peritoneum, transcutaneous full-thickness fixation can be performed using non-absorbable sutures. the preperitoneal space should be closed tightly as soon as possible in order to prevent the bowel herniation into the preperitoneal space. at closure of the preperitoneal space, the peritoneum is fixed on the collagen mesh using non-absorbable sutures. objective: show a tapp approach using a self-fixating mesh( x cm. progrip tm laparoscopic self-fixating mesh, medtronic) with bipolar peritoneal defect sealing, avoiding the use of tackers and performing an easy and sutureless peritoneal closure. material and methods: years old male, asa ii, medical history of beta-latacm allergy, high blood pressure, dyslipidemia and bilateral knee surgery. diagnosed of bilateral inguinal hernia at consultation due to inguinal disconfort. surgical site infection prophylaxis with iv vancomycin. balanced general anesthesia. supine decubitus position with shoulder supporting to allow a forced trendelemburg. degree optical device with trocars disposition: one mm umbilical trocar and mm trocar in both flanks, same distance and height to umbilical trocar. peritoneal opening and flap creation with monopolar energy, blunt maneuvers and pneumoperitoneum dissection. anatomical landmarks identification(cooper's ligament, epigastric and iliac vessels, hernia defect and spermatic cord elements). reduction of hernia sac content(pseudosac in this case, direct hernia) and complete peritoneal dissection to achive a correct mesh placing. mesh is folded in parts(one inferior part, two superior parts) in vertical axis outside the abdomen to facilitate the posterior intraabdominal maneuvers. introduction: into abdominal cavity with grasping forceps and correct unfolding mesh assesment: medially(pubic bone), caudal(cooper's ligament) cranial(more than cm of hernia defect/ deep inguinal ring) and lateral(anterior superior iliac spine). finally, we use a bipolar forceps to close de peritoneal defect. in order to facilitate this step, its necessary to decrease pneumoperitoneum pressure and to use the grasping forceps to bring together both peritoneal flap edges prior to bipolar energy sealing. results: min. surgical procedure. h hospital discharge, no complications. routine outpatient follow up(week, month, months and month later) with an epididymitis episode months after surgery(treated with oral ciprofloxacin). conclusions:-this procedure is an easy implementation technique once the intraabdominal mesh unfolding procedure control is reached.-the use of a self-fixating mesh avoid the use of tackers and its potential disadvantages(e.g. increasing postoperative pain).-bipolar peritoneal sealing offers a quick, easy, cheap and safe peritoneal closure, avoiding the contact of the mesh with the viscera in the same manner. results: we performed procedures within patients. the average age was years. twenty six percent of hernias were bilateral, , % were inguinoscrotal and % in the right side. the median asa score was . the conversion rate was , %. the average duration of the procedure was , min min. overall morbidity was %. there were seromas ( , %) . on -year follow-up, one recurrence ( , %) was found and chronic postoperative pain in one case . we had no mortality. in the univariate analysis, male sex, inguinoscrotal hernias, hernias classified as nyhus a were significantly associated with overall postoperative morbidity. a chronic obstructive pulmonary disease was the only variable significantly associated with the occurrence of medical complications. conclusion: given these results, the tapp technique is a good alternative in the treatment of groin hernias. however, enhancing this approach is essential to reduce the operating time and the postoperative outcomes. introduction: studies have emphasized the impact of a strong safety culture on patient outcomes. consequently, many interventions focus on improving the safety culture, of which teamwork and safety climate are important ingredients. it is known that differences in culture and safety attitudes may also impact teamwork. implementations of safety interventions, such as a ' black box', are dependent upon these differences. the aim of this study was to assess the safety culture at the operating theatre complex, along with the theatre staff's attitude towards a specific quality improvement intervention, a black box in the operating room as a tool for structured team debriefing. methods: the validated dutch version of the hospital survey on patient safety culture was administered to all healthcare professionals working in the operating room complex at one academic medical centre. this survey was supplemented with questions regarding the use of a 'black box', a medical data recorder in the operating room, to measure the staff's attitude towards this quality improvement tool and its potential contribution to patient safety. aims: the aim of the study was to compare two methods of treatment of dunbar syndrome: thelaparoscopic release of median arcuate ligament alone and the hybrid method consisting ofsurgery and percutaneous stent implantation to celiac trunk. methods: we performed laparoscopic release of ct in the department of general, mini-mallyinvasive and elderly surgery in olsztyn in - . all of patients suffered from severepain of abdominal cavity before the surgery. three patients underwent doppler percutaneousangioplasty of the ct with stent implantation one month after the laparoscopy. results: all patients reported relief of symptoms in the first days after the operation. in two cases fromboth groups, there were a complete remission of the symptoms. in one case respectively,there was an improvement. there were no postoperative complications. the results of both methods do not show the differences therefore the surgery alone seems tobe a safe and feasible procedure. it increases the comfort of the patient and brings theopportunity for normal functioning. the method of wedge resection of lungs in patients with limited forms of chemo-resistant pulmonary tuberculosis is developed. in order to evaluate the efficacy, patients underwent surgery (the main group). for comparison, the data on similar operations in patients, made according to the traditional method (with the help of a cardboard weaving machine yo- ) were selected. compared the duration of the stage of resection itself, the frequency of need for additional hemostasis of the parenchyma sutures, the degree of deformation of the pulmonary tissue in the seam area, the frequency of postoperative complications and reoperations, the duration of postoperative inpatient treatment. the developed method, in comparison with the traditional one, has the following advantages: simultaneously leak proofness and hemostasis with minimal electrothermal damage to tissues are provided and there is no need for additional hemostasis, there are no negative effects of manual stitching of parenchyma of lung with abandonment of foreign material, a significant reduction in the duration of wedge resection of the lung from . to . min, a decrease in the number of postoperative pulmonary-pleural complications is achieved by . % and caused by them reoperations-by . %, shortening the duration of postoperative inpatient period of treatment from . to . days. introduction/aims: laparoscopy is a diagnostic and therapeutic resource that is largely used in elective gastrointestinal surgery due to its well-known advantages over the classic open approach. nevertheless, there is still some discussion about its application in emergency surgery. our aim is to analize the use of the laparoscopic approach by the members of the surgical emergency unit from our medical center. methods: a descriptive research based on the data of patients who required emergency surgery, that was performed by the members of the surgical emergency unit of a spanish hospital between november and may , was conducted. these data were analyzed according the pathology that motivated the surgical procedure and the chosen form of surgical approach (open versus laparoscopic). results: out of the patients in whom emergency surgery was performed, suffered from a pathology that actually allowed the laparoscopic treatment. laparoscopy was used in . % of these patients. according to pathology, the most common were acute appendicitis and cholecystitis, in which the laparoscopic approach was used, respectively, in % and % of the cases. regarding other less frequent pathologies, such as gastroduodenal perforation, bowel obstruction, diverticulitis and pancreatitis, laparoscopy had a less significant role. according to the year, a general tendency to increase the use of the laparoscopic approach was found, most notably in the cases of acute appendicitis and cholecystitis (with rates above % in ). conclusions: despite our positive results in the terms of the implementation of the laparoscopic approach in emergency surgery, there is still room for improvement, especially in regards of the less common pathologies. furtheremore, additional studies are needed in order to identify the factors that have had an effect, in favour or detriment, in the development of emergency laparoscopy in our center. aims: laparoscopic surgery, which produces small scars, has become widespread. when performing surgery through small laparoscopic incisions, a surgeon manipulates tools inserted into the abdomen through ports. for minimally invasive accurate procedure, the port as the pivot point should be stabilized on the abdominal wall. however, these laparoscopic incisions are loaded while manipulation because it is difficult for the port to be fixed on. thus, it is necessary for the patient friendly manipulation to be fixed the port mechanically. we developed a new pivot restraint device (prd) attached to a trocar for guiding the tool. the purpose of this study is to evaluate both of reducing the operating time and the load of the port with the prd experimentally. methods: the prd uses gimbal mechanism for two rotating axes and a linear guide mechanism for the insertion axis though into the forceps. in the experiment, the left hand forceps with or without the prd and the right hand forceps without the prd were set on the training box. the box had a measuring system created with a pressure sensitive sensor for the continuous force (resolution . n, fps) applied to abdominal wall fulcrum. the experiment task was performed as following three steps. ( ) the surgeon lifted the g weight for s at the initial position using the right hand forceps. ( ) the weight was transferred from the right hand forceps to the left hand forceps, and held for s. ( ) the weight was moved to the predetermined position, held for s, and returned to the initial position. the surgeons were five endoscopic specialists and five non-specialists. the operating time and the time ratio exceeded n for the left hand forceps were measured. two grouped datasets with or without the prd were compared using two-sided t-test. results: the prd was associated with both of reducing the operating time ( . s vs. . s; p \ . ), and the load of the port ( . % vs. . %; p \ . ) at the statistical analysis. conclusion: the prd could be used for reducing the operating time and the load of the port in minimally invasive accurate procedure. background: pathophysiological changes during laparoscopic surgery and positive pressure pneumoperitoneum (pp) may include (beside cardiovascular changes) elevated intra-thoracic as well as intracranial pressures. however, the possibility of physiological and functional cerebral impairment under pp is still debated. aim: to study the effects of pp on brain activity during different modes of anesthesia and ventilation during laparoscopic cholecystectomy (lc). patients and methods: thirty patients undergoing elective lc were divided to those who were ventilated by intermittent positive pressure ventilation (ippv, pt.) and by high frequency jet ventilation (hfjv, pt.). in those under hfjv we used total intravenous anesthesia (tiva). in those under ippv we either used inhalational anesthesia or tiva. intra-ocular pressures were detected in both eyes, trans-cranial doppler was used to measure the changes in flow of the middle cerebral artery, and cerebral oxygenation (o saturation) was measured too. each parameter was detected during anesthesia before surgery, several times during surgery under pp and after co evacuation. a novel computerized signal analysis by a continuous recording through a single electrode was done to explore cerebral cognitive activity during surgery. results: all surgeries went uneventful and without complications, pp was set to mmhg, and each patient was positioned in a degree anti-trendelenburg posture. cerebral perfusion and oxygenation were not changed significantly during pp. intra-ocular pressures decreased during anesthesia and increased during pp, but to a lesser extent under tiva. however, pressures during pp did not exceed pre-surgical values. we did not observe changes in cognitive brain activity during pp, although enhanced cerebral activity was seen under hfjv. conclusions: increased intra-abdominal pressure during laparoscopic surgery was not accompanied by decreased cerebral functions, maybe due to cerebral circulatory auto-regulation. changes in cerebral cognitive functions under hfjv might be explained either by the different cerebral effects of tiva in comparison to inhalational anesthesia, or due to dissimilar hemodynamic changes during hfjv. aims: gallstone ileus (gi) is a rare complication of cholelithiasis and accounts for . - % of small bowel obstructions. intermittent and non-specific presentation often results in late diagnosis. the triad of rigler is pathognomonic (pneumobilia, small bowel obstruction and ectopic gallstones), so an image test is usually mandatory in order to assure the diagnose. our aim is to expose our experience regarding this topic to show that a minimally invasive approach is feasible in selected cases. methods: since january we treated cases of gi, of whom ( %) underwent laparoscopic surgery. in all cases a ct was made to reach diagnosis. enterolithotomy alone is our preferred procedure for the resolution of this pathology. here we present a descriptive analysis of our data in those cases where a laparoscopic treatment was attempted. epidemiological variables, surgical technique, postoperative complications, days until hospital discharge, recurrence, etc. has been collected. results: % of patients were female( ) and % male ( ). mean age was . size of gallstones varied from to mm and ct located them all in the ileum. two conversions to open surgery were made ( %), in one case because the gallstone could not be found and in the other case due to the need of an intestinal resection. in two cases ( %) la aparoscopic-assisted surgery was performed using a pfannestiel incision for the gallstone extraction and enterorrhaphy. only one case was total laparoscopic approach ( %). two cases needed an intestinal resection and anastomosis, one of them was complicated with a leak that needed reintervention. there were two cases of recurrence during the follow-up time. hospital stay varied from to days, mean of days. conclusion: the widespread use of ct facilitates early diagnosis with high sensitivity detecting rigler's triad. a totally laparoscopic procedure might be ideal for patients specially with solitary stones even though a laparoscopic-assisted approach is an easier technique for surgeons with less experience in laparoscopic surgery. although experience in minimally invasive surgical treatment of gi is still developing, it may be recommended in selected cases and experienced hands. introduction: most of surgical interventions in hospitals in the world, where laparoscope is used, it is common that the vision inside the human body is constantly interrupted by fogging in laparoscope tip. the laparoscope fogging is caused by the difference of temperatures between the optic tip and the abdominal cavity. material and method: we replaces the traditional laparoscope for the ehs (endoscope heater system) with resistance between the internal and external tube that maintains the temperature of laparoscope at ( - °celsius) without modifying the external architecture of traditional laparoscope. results: ehs does not generates any waste like other anti-fog systems, like liquids, plastics covers or electric heater. reduces intervention time, can keep same instruments or accessories for the intervention. all of the above means a saving of resources with have a positive environmental impact. conclusions: the discomfort transmitted by surgeons about the fogging in laparoscopy tip make success of the product and it will replace the current laparoscope which is fogged. aim: synchronous locally-advanced low rectal cancer and prostate adenocarcinoma represent a rare condition and a challenging situation for colorectal surgeons and urologists. the simultaneous resection of both adenocarcinomas after long-course chemoradiation therapy combines two major surgical procedures associated with a potentially increased postoperative morbidity. in the other hand, simultaneous resections minimize the risk of difficult dissections, which are expected if the two procedures are scheduled sequentially. in the past decade, robotic-assisted minimally-invasive surgical techniques have been increasingly used to treat both rectal and prostatic malignancies. especially in case of prostatic malignancy, the robotic approach is considered the treatment of choice because it is associated with significantly lower blood loss and transfusion rate, and much greater functional outcomes compared to laparoscopy. methods: we present the case of a -year-old male patient (bmi: . ) diagnosed with a histologically proven locally-advanced rectal adenocarcinoma (ct an ) located at cm from the anal verge and concurrent histologically proven prostatic adenocarcinoma [gleason score of ( ? )] located in the postero-basal right lobe. the preoperative total-body computed tomography (ct) scan showed no evidence of metastatic disease. after discussion in a multidisciplinary meeting, the patient received a long-course neoadjuvant chemoradiation therapy (ncrt). at the restaging positron emission tomography / magnetic resonance imaging (pet-mri), the rectal lesion was classified as ymrt n . preoperatively, the surgical difficulty was assessed as high, based on the calculation of the eumarcs score (equal to / ). moreover, due to the high-risk status of the prostate cancer (gleason ), it was decided not to preserve the neuro-vescular bundles during the radical prostatectomy. results: the patient was operated on after weeks from completion of ncrt by using the da vinci robot system si with a single docking approach, as previously described, in order to address both cancers. conclusions: this video shows the main surgical steps of the simultaneous robotic resection of the low rectal adenocarcinoma first, of the prostatic carcinoma then, and the mechanical colo-anal anastomosis followed by drain positioning and ileostomy. this video demonstrates the perioperative safety and feasibility of the minimally invasive robotic approach in case of extended and challenging oncologic resections. general surgery, rambam medical center, haifa, israel year old, male patient presented with melena, without abdominal pain, nausea or vomiting. patient underwent colonoscopy and tumor was found in ascending colon (near the hepatic flexure). biopsy from the tumor has showed moderately differentiated adenocarcinoma. his blood laboratory examinations were within normal limits except of hgb level- . . cea and cea - were normal. abdominal computed tomography was normal . patient underwent da vinci robot-assisted right hemicolectomy with extracorporeal anastomosis. total operating time was min. three days after operation patient started regular diet and was discharged home on day four. final pathology result confirmed diagnosis of moderately differentiated adenocarcinoma. introduction: one of the goals of colorectal surgery is to decrease the number of leaks once an anastomosis has been performed. this life-threating entity after elective surgery has been related to the clinical history of the patients, the location of the tumor and to technical reasons, specially due to tension in the anastomosis or to lack of vascularization. tension could be identified during surgery, while vascular supply is evaluated by the surgeons based on a subjective analysis of the color of the colon/ileum. fluorescence tries to make these subjective parameter more objective in order to avoid an anastomosis with lack of vascularization, decreasing the numbers of leaks related to this factor. patients and method: the study presents a quasi-experimental analysis made from january to october in two hundred and eighty-five patients who underwent elective colerectal surgery, performing either a colo-rectal, ileo-rectal or intracorporeal ileo-colic anastomosis. vascular supply was eveluated using indocianyne green (icg) in one hundred and forty-five patients, while one hundred and forty subjects were operated in a previous period without using this technology, being considered the control group. the number of time that the attitude changed and the number of leaks were collected. results: out of the cases performed, were right colectomies (rc), left colectomies (lc) and rectal excision (re). in % the transection line was changed ( , % in rc, , % in lc and , % in rr) . in comparison with the control group, the icg group had a significantly less indicence of anastomotic leak compared to the control group ( , % vs. , %, p = , ), lower rate of terminal stoma after reoperation ( , % vs. , %, p = , ), a shorter length of hospital stay ( days vs. days, p = , respectively), and a low morbidity and mortality. conclusions: the rate of leaks after colorectal surgery decrease using icg to detect the proper transsection line before to perform the anastomosis in comparison with control group. these findings might influence in the final results although it is necessary in the future to find a system that provides greater objectivity by quantifying icg. aims: anastomotic leaks continue being one of the most important complications when a colorectal surgery is performed. this complication is usually related to the level and type of resection, the patient clinical history and surgical technique, where tension and vascular supply are the most important. indocyanine green (icg) fluorescence angiography seems to be helpful in order to evaluate the vascularization at the resection margins. methods: we have collected data on colorectal procedures that were performed by the same surgeon using icg fluorescence angiography to evaluate vascular supply to the anastomosis. in order to asses in which of the different type of colorectal procedure has more value to be used, we analyzed the type of surgical procedure, the percentage change in the resection margin and the number of anastomotic leaks (al). results: all of the cases were performed by laparoscopic approach: left colonic resection (lc), right colonic resection (rc), splenic flexure partial resection (sf), low anterior resection with partial mesorectal escision (lar), ultra low anterior resection with total mesorectal escision (ular) and total colectomy (tc). there was a change of transection line (ctl) in lc ( , %), rc ( %), sf ( , %) and ( , %) in rectal anastomosis (lar, ular and tc). as far as al we found: lc ( , %), rc ( %) and , % in rectal procedures. lc, sf and rectal procedure showed more ctl and less al, while rc showed less ctl and more al. conclusion: icg fluorescence angiography as an additional tool to try to reduce the anastomtic leak rate seems to have more value in the procedures that involve the left colon and the rectum, since that is where we have observed the greatest number of ctl, this could be explained by the riolan's arcade and the variability of the vascular anatomy. however, it seems that this is a line of research should continue developing with longer and larger studies, so in that way we can have more significant results. retrorectal tumors ara rare and often found incidentally. the majority of retrorectal tumours are benign, but they have potential for malignant transformation and therefore should be resected when found. a case of a -year-old female patient with a retrorectal tumor is showed. the tumor was found incidentally on ct scan of the abdomen for evaluation of non specific right side abdominal pain. a mri was also performed and imaging was informed as a probably congenital retrorectal tumor (tailgut cyst) there was no evidence of involvement or invasion of other structures the tumor was palpable at rectal examination. a transanal minimally invasive surgery (tamis) approach was proposed. preoperative preparation was done with a full mechanical and oral antibiotic bowel preparation. preoperative parenteral antibiotics werw administred. under general anesthesia, lithotomy position. the contour of the tumor is not visible due to the small size. palpation of tumor and placement of clips to lolocate was done. placement of gel point path and rectal insufflation. a longitudinal incision was made to the posterior left side of rectal wall. the insufflation of the perirectal extraperitoneal space allowe for excellent exposure of the tumor. the tumor was disected with ligasure. then the tumor was extracted transanally.the proctotomy was closed in a single layer with reabsorbible monofilament continuous suture (pds). no complications after the procedure. the patient was discharged at days. discusion: traditionally, the retrorectal tumors have been resected using a posterior parasacrococcygeal approach, an abdominal approach or a combined abdominal and posterior approach. with the advent of minimally invasive surgery, laparoscopyc approach has been described too. however, tamis approach is feasible, with low pain, morbidity, fester recovery and excellent cosmetic (no scare) results. it can be accomplished using standard laparoscopic equipment, with transanal access. we think that perhaps it could be the gold standar approach for this tumors. aimes: robotic-assisted laparoscopic surgery (rals) is a promising advanced technology that can overcome the inherent limitations of conventional laparoscopic surgery (cls). its advantage includes free-moving multijoint forceps, a motion scaling function, high-quality three-dimensional imaging, and stable camera work by an operator. this study aimed to clarify the short-term outcomes of rals for rectal tumors. methods: this study group comprised patients who underwent rals for rectal tumors (cancer in patients and gastrointestinal stromal tumor in patient), excluding ones with distant metastasis from november through december . the clinicopathological findings and short-term outcomes in rectal tumors were analyzed. results: the median operative time was min ( - ). the median console time was min with a median blood loss was ml ( - ). conversion rate was . % ( / ). the median postoperative hospital stay was days ( - ). patients ( . %) had postoperative complications. patients ( . %) had lymph nodes metastases. the mean harvested lymph node was . . the r resection rate was % ( / ). conclusions: these results suggest that rals for rectal tumors is safe and feasible, and the perioperative outcomes are acceptable. introduction: anastomotic healing defects are a feared complication which might have a fatal impact on the patient. fundamental conditions for proper anastomotic healing include sufficient blood supply. fluorescent angiography using indocyanine green in the spectrum of near infrared light facilitates the monitoring of tissue perfusion during a surgery. aim: a presentation of the results of our non-randomized study in which we assessed prospectively obtained data from a perioperative assessment of anastomosis perfusion by fluorescent angiography using indocyanine green during robotic rectal cancer surgery. method: thirty patients with rectal cancer, who underwent a robotic resection with primary anastomosis, were consecutively included in the study between april , and june , . the study included patients facing a least invasive surgery with a guaranteed payment by a health insurance company. during the surgery, we monitored and assessed the quality of the perfusion of the resection line of the sigmoid colon and subsequent anastomosis by means of fluorescent angiography using indocyanine green in the spectrum of near infrared light. the data were obtained prospectively and subsequently analyzed. results: between april , and june , , we consecutively included rectal cancer patients in the project: men and women. monitoring of the perfusion of the resection line and anastomosis was successful in all cases and perfusion quality was satisfactory across the sample. perfusion insufficiency requiring a change in the resection line level or anastomosis adjustments was not detected with any patient. in two cases ( . %) of tme, we gave up the planned protective ileostomy owing to quality perfusion of the anastomosis. one patient ( . %) suffered from defective anastomosis healing without clinical symptomatology (type a). we found no technical complications related to fluorescent angiography or undesirable effects due to the application of indocyanine green. conclusion: even though we did not register insufficient perfusion in our sample and hence we did not have to change the resection line level or adjust the anastomosis, we may state that fluorescent angiography performed by an experienced colorectal surgeon may potentially reduce the frequency of complications linked to defective anastomosis healing.supported by mo aims: the aim of our study is to demonstrate whether robotic surgery has any influence on the reduction of complications in the aged population undergoing rectal cancer. methods: we performed a retrospective analysis of a prospective database of patients who underwent robotic surgery for rectal cancer. we divided our population in groups: under year old, between and year old and above year old. we recorded complications in each group intra and post procedure. qualitative variables were expressed in terms of absolute frequencies and percentages and mean values and standard deviation were used to express quantitative variables. the analysis of data was applying fisher's exact test or chi-squared test for qualitative variables and variance analysis or student'-t test for quantitative variables. statistically significant values of p \ . underwent multivariate logistic regression analysis. results: the present study included patients ( males).seventy seven patients were under year old, patients were between and year old and patients were above year old. the analysis showed conversion rates of . %, . %, . %, and complication rate of . %, . %, and . % in each group. univariate analysis showed no differences between the three groups. nevertheless, there were statistical differences from bmi, asa and neoadjuvant therapy. in multivariant analysis only neoadjuvant therapy was significant. conclusions: robotic approach do not decrease complications in elderly population. introduction: it has been described the advantages of total transanal mesorectal excision (tatme), with better visualization and access to the lower rectum. we use this access whith the gel point path device, to repair a rectovaginal fistula with stenosis of low rectal anastomosis in two patients, that would be difficult by conventional abdominal approach method: we show our surgical technique for repair a rectovaginal fistula with stenosis of low rectal anastomosis in two female patients operated due to rectal neoplasia. one of the patients underwent prior chemo-radiotheratpy. rectoscopy and image test was performed at the patients prior the intervention. no recurrence signs are recorded at mri.we describe the operation technique: a new anterior rectal resection was performed with a combined transanal (gel point path) and abdominal minimally invasive approach. redo anastomosis whith eea stappler was performed, vaginal repair and epiploplasty. the intervention was especially laborious due to the fibrous tissue. pathology: fistulous path without tumor infiltration in the two patients. at two months, a opaque enema show permeability and absence of leaks in the two patients. the ileostomy was closed at three months. discusion: we believe that transanal access through the gel point path can be a good option for rectovaginal fistula and stenosis of low rectal anastomosis, allowing a better visualization and acces, and making more easy a very difficult intervention. introduction: tamis or transanal minimally invasive surgery for polyp resection has increased fame for several situations in which adenomas with or without dysplasia cannot be removed with conventional colonoscopy. in this video we show the step by step technique performed with the da vinci xi system. material and methods: in this video we show the setting and the location of the patient-side cart and the arms to perform the resection of polyps in different patients and how to develope the procedure. results: after placing the patient-side cart the arms are connected to ports and the camera, double fenestrated grasper and scissors are connected to the arms through a transanal gel-port device. a line is described around the polyp with monopolar energy to determine the place of the dissection. the scissor is exchanged by a robotic harmonic wrist instrument and the complete dissection is performed. the wound is closed using a robotic needle holder and a suture. results: transanal robotic surgery could be safely performed after a standardized technique is stablished. aims: robotic rectal cancer surgery has demonstrated to obtain at least the same results than laparoscopic surgery. however, robotic surgery is associated with high rates of costs, specially when conversion to opened surgery occurs. the goal of this study is to create a predictor nomogram of conversions for robotic rectal cancer surgery. methods: we performed a retrospective analysis of a prospective database of patients who underwent robotic surgery for rectal cancer from october to november . we performed a bivariant analysis and detected the variables which were related with the conversion: body mass index (bmi) and the t. we divided the patients of the population in two groups depends on obesity (bmi of kg/m ) and on t (t - /t - ). we registered conversions in each group calculating the pretest risk. we performed likelihood index (lr ?/-) for under and above kg/m of bmi, adding in a second step the lr of t; obtaining the prediction index for four groups by using a standardize nomogram. results: the present study included patients ( males). were under bmi of kg/m and above. regarding t, were with a tumor of t - and with t - . the analysis showed a conversion rate of the statistical sample of %. univariant analysis showed significative differences in the bmi (p = . ) and t (p = . ). a nomogram was performed; as regards the bmi, the positive likelihood index in the group of bmi [ a prediction index of conversion of % (lr ? , ) and in bmi \ the prediction index of conversion is % (lr- , ). adding the t group data, for bmi [ and t - the conversion prediction rate is . % (lr- , ); for bmi [ and t - the conversion prediction is % (lr ? , ). bmi \ and t - the conversion prediction is % (lr- , ); imc \ and t - , the conversion prediction is %. conclusion: a standardize nomogram with the variable bmi and t facilitates the selection of patients for robotic surgery in rectal cancer avoiding conversion to open surgery. background: d-laparoscopy is proven to improve performance in dry laboratory settings, especially for novice surgeons due to better depth perception. however, the benefits for experienced laparoscopic surgeons are still discussed. aim: the aim of this study is to compare the results of right hemicolectomy (rc) using a conventional ( d hd) laparoscopic system with rc performed using a d laparoscopic system in terms of duration, complications and results. material and methods: from all laparoscopic right hemicolectomies performed in our clinic we selected all procedures performed by the same team of consultant surgeons using the same technique and divided them in groups. the study group comprised of all patients operated using our d einstein vision . system; all other patients which were operated using our standard wolf hd laparoscopy system comprised the control group. all patients were retrospectively analyzed in terms of patients characteristic, or time, duration of operation, intra-and postoperative complications, length of hospitalization, pain score, necessity of analgesics and number of lymph nodes retrived. risk factors for complications (bmi, smoker, diabetes, copd, bph) were also registered. results: there were patients included in the study group, while the control group comprised of patients. mean operation time in the study group was . min in the study group, while mean or time was . min. mean operation time in the control group was . min, while mean or time . min. one reintervention was noted in the control group and two in the the study group; no conversion to open surgery was noted. there were no significant differences regarding patient characteristics, pain score, wound complications, hernia rate, length of hospitalization or number of lymph nodes removed. conclusions: there were no significant differences regarding the outcome of rc using d laparoscopy; total or time was significantly higher in the study group due to the time needed to set up the d-laparoscopy unit. this is biased by the fact that the d system needs to be set up manually while the conventional hd system is integrated in the or. also, there was no significant difference in complication rate. background/purpose: robotic approach can be a treatment option for patients with pelvic recurrence after primary resection for rectal cancer. however, data regarding patient selection, complication rates, and oncologic outcomes are rarely reported. we aimed to present initial experience and to evaluate feasibility, safety, and oncologic outcomes of robotic salvage surgery for recurrent rectal cancer. methods: ten patients who underwent robotic salvage surgery for local recurrence at the anastomotic site, lateral pelvic side-wall, or lateral pelvic lymph nodes (lpns) were retrospectively evaluated from a prospectively maintained database. results: two patients underwent pelvic mass excision with en bloc resection of anastomosis and redo-anastomosis, and eight patients underwent lateral pelvic lymph node dissection (lpnd) for lpn metastasis; one of these eight patient underwent additional en bloc resection of anastomosis. all patients achieved r resection. the median operation time was min and the median estimated blood loss was ml. there were no conversions. as for intraoperative complications, one patient experienced ureter injury during lpnd because the metastatic lpn was closely abutting to the ureter. the median hospital stay was days. in six patients who underwent lpnd, the median number of harvested lymph nodes was (range - ) and the median number of metastatic lymph nodes was (range - ). with median follow-up months, one patient developed lung and pelvic recurrence at months after salvage operation and seven patients remained in disease-free state at the last follow-up. conclusion: initial experience of robotic salvage surgery for pelvic recurrence in rectal cancer indicated that it is safe and feasible. therefore, the robotic approach can be considered as a treatment option for the treatment of local recurrence in selected patients. introduction: there is uncertainty regarding the effects of simulated patient death. several reports showed increased cognitive load and poorer learning outcomes, and others increased performance without causing stress to learners. we have not found any report studying the impact of animal death in the simulation lab. methods: this was an observational cohort study to assess the emotional and cognitive load of surgeons who experienced animal death in the simulation lab. seventy-four faculty and residents from different surgical specialties training minimally invasive surgery participated in the study. one cohort consisted of surgeons whose animal died during surgery, and the other by those whose animal survived. emotions were assessed using the scale for mood assessment and cognitive load with nasa task load index. results: twenty percent of participants experienced mortality while training anti-reflux surgery ( cases) and other procedures ( cases). causes of death included intraoperative pneumothorax (n = ), hemorrhage (n = ), and cardiac dysrhythmias (n = ). participants exposed to animal death had higher levels of sadness and anxiety, and lower levels of happiness (p [ . ). cognitive load was slightly higher in the exposed cohort (p [ . ). conclusions: these findings suggest that mortality in the animal lab do not have a significant effect on cognitive workload and emotions of surgeons training complex laparoscopic procedures. introduction: the visuospatial profiles of expert laparoscopic surgeons remain unaccounted in the current literature for as the influence of visuospatial ability on laparoscopic learning has mainly been investigated in medical students or novice surgeons and using simulators as means of performance measurement. such knowledge is critical, as without understanding how clinical experience may impact visuospatial processes in surgeons, we hinder our efforts to utilize the available knowledge to support surgical education for the future. this study is aiming to explore the development and influence of visuospatial processes on intraoperative laparoscopic learning. method: the study reports the interim baseline results from the ongoing longitudinal study throughout a -year period of training on laparoscopic surgery. data from surgeons including residents undergoing training were captured and compared to specialists who are working in departments of general and visceral surgery at two large hospitals. the mean experience of the surgical residents was years. the mean laparoscopic experience among the senior surgeons is years, with each surgeon performing an average of laparoscopic procedures per week. visuospatial ability was tested using mental rotation test (mrt), guay visualization of views tests (gvvt), spatial perspective taking and spatial orientation test (ptost) and pictorial surface orientation (picsor). spearman correlation coefficient was used in this study with a p-value of significance at \ . . results: senior surgeons have an overall good visuospatial profile, in the sense that they performed close to optimum on all measurement scales. the spearman rho revealed a significant correlation between scores on gvvt and picsor (r = . , p = . ) and between ptost and picsor (r = -. , p = . ). a significant correlation between years of laparoscopic experience and ptost score was also observed (r = . , p = . ). when comparing residents and senior surgeons, no significant difference on the mrt was observed (m = . , sd = . ), nor between baseline scores of senior surgeons and resident surgeons on all tests. conclusion: the results of this study carry important clinical and theoretical implications, as the results hint towards the idea that intraoperative laparoscopic experience lends little to no influence over the development of visuospatial ability. learning models and laparoscopic technical skills, how to adapt each case to improve objectives: according to da. kolb learning is the result of how people perceive and then process what they have perceived. the aim of this study is to identify the personal characteristics of learning in of the participants in a course of laparoscopic technical skills according to the styles described by kolb. methods: between june and november , participants performed a h course distributed over five consecutive days performing laparoscopic manual intestinal anastomosis in endotrainer. they all filled in kolb's learning style test adapted to spanish. the anastomoses were performed in 'ex-vivo' swine intestines. in each anastomosis we evaluated the quality at the end and execution time. the test and quality variables were analyzed through statistical studies. results: in our study, % of the participants were women and % wew men. %were staff surgeons and % were resident. the median age among residents was years and among the staff years. the most frequent learning model in the sample studied was converging ( %). the predominant model among women was assimilating ( %), which, however, represented only % in men. in men, converging model was predominant ( %). among the staff, the most frequent model was diverging ( %). adaptation style prevailed among residents ( %), being rare among the staff ( %). the mean time of the anastomosis was min for both the adapter model and the assimilator, min, for the convergent and divergent models. the quality of the anastomosis performed by each participant was % for the adapter model, % for the assimilator model, % for the convergent model and % for the divergent model. the predominant style in our study was convergent. among women, the most frequent model was assimilator wheras in men it was the least frequent. in the residents, the most frequent model was adapter however, it was very rare in adjuncts. among residents we do not find divergent styles. the highest quality of the anastomosis was achieved by those who worked with an assimilating style. knowing previously the training style we can individualize the teaching methodology in order to improve competences. aims: assess whether laparoscopic appendicectomies (la) are a superior option to open appendicectomies (oa). specifically, comparing the time taken, complication rates and whether it is more appropriate to perform an la overnight, as opposed to oa. finally, to find out how a range of outcomes differs between different grades of surgeon. methods: an information request was sent to the clinical coding department to derive patient identification numbers for all appendicectomies over a ten-month period ( total surgeries). these numbers were then inputted into the hospital information system where the electronic operation note is present, and specific outcomes were derived and analysed. results: % of operations were oa and % were la. mean la times for consultants, sas and spr were . , and min respectively and oa , and min respectively. their respective conversion rates were %, % and %. oa had a complication rate of . %, la was . %. conclusion: oa are performed more than la. spr doctors had the slowest completion times for la but the lowest conversion rates. sas doctors had the fastest completion times for la and oa but higher conversion. la takes longer than oa but has lower complication rates; key factors when performing at night. key statement: laparoscopic appendicectomies require more surgeon-hours and have the potential to be converted to open, however the rates of complications and serious complications are significantly lower. background: paper based resources have been the standard sources for information for centuries. however, more and more people (patients and staff alike) are looking online for information. while the internet often provides excellent resources, there is often conflicting and confusing material of doubtful veracity. trainee staff and patients/carers should be able to access reliable resources whenever and wherever they are. the aim of this project was to create a high-quality resource fulfilling these needs. aim: we present a video demonstrating our integrated colorectal education website ( http://www.colorectaleducation.com/). our approach: high quality health care provision requires highly trained staff as well as wellinformed patients. information resources for these two groups are usually accessible from different repositories. our integrated website provides a common platform for all those involved in colorectal surgery, to use, learn and reflect on. users are directed to separate sections for patients and colorectal professionals. multiple disclaimers prevent patients accidentally stumbling across clinical/ operative information, whilst providing access to those who wish to do so. trainees struggle with balancing their educational needs with their service commitments. this website gives them the opportunity to view detailed operative training videos on the go. many of videos are chapter based allowing them to stop and re-start with ease. modules are also available for nurses providing them access to relevant educational material. the modular design of the website allows us to build upon it with more topics planned to be added over the next eighteen months. the resource also has detailed chapterised videos for patients due to undergo various colorectal procedures. all have been approved by a multi-professional panel including patients and are designed to provide information, offer support and to allay any anxiety. videos with the care pathway and previous patients' experiences are accessible on demand. conclusion: on demand information has now become the norm with the use of smart phones/ tablets. this website provides patients, surgical trainees and other healthcare professionals access to information and education in a clear and reliable format anywhere in the world. colorectal education, on demand and just a click away! objective: in the last decade the growing interest in robotic surgery is evident as shown by several published articles. the aim of the present study is to evaluate the main outcome of a single center experience and to describe the organizational system we have progressively established in our center in order to improve the development of robotic program in all surgical area. materials and methods: we report a case series of patients who underwent robot-assisted surgery at sanchinarro university hospital since the beginning of the program (october ) until november main patient demographic characteristics, type of surgery, peri and postoperative data and follow-up were evaluated. results: a total of robotic procedures were performed for a total of patients. the prevalence of malignant disease was %. a total of pancreatic surgery were performes; liver resections (mean operating time: min); gastrectomy (mean operating time min); esophagectomy (mean operating time: min); colorectal resections ( rectal resections, sigmoidectomy hemicolectomies right, left colectomy) (mean operating time: min); nissen procedures (mean operating time: min), esofagheous myomectomy for achalasia (operating time: min); adrenalectomy (mean operating time: min); three biliary surgery for benign desease, splenectomy. eight partial resection of the duodenum, one yeyunal resection, one mesenteric cyst resection and retroperitoneal tumor have been performed. conversion rate was %, total morbidity have been %. there has been no peri and postoperative mortality up to days after surgery. the average hospital stay and intensive care were respectively days (range - days) and . days (range - days). conclusions: the organizational model defined in our center is facilitating the constant and progressive development of the robotic program. a broad and flexible availability of the robotic system, a progressive increase of young surgeons joining this technology as well as the institutional and departmental economical effort are the points with which the robotic system may increase its development in a surgical department. aims: endoscopic surgery has been widespread in the field of general surgery. however, in japan, there is no standard program for endoscopic surgery training, and its competency has not been considered for the acquisition of board certified surgeon. the purpose of this survey was to investigate the current situation of endoscopic surgery training and autonomy of young surgeons for endoscopic surgery in japan. methods: the survey was planned to target general surgery members of the japan society for endoscopic surgery (jses) who was post graduate year or less. after approval by the ethics committee of jses, the request for the participating in survey was mailed to object members. questionnaire responses were available in print or online media. the contents of the questionnaire consisted of items, about the conditions of endoscopic surgical training, experienced case number, and the self-assessment of autonomy from to point by zwisch scale in specific procedures of endoscopic surgery. results: the total response rate was . % ( / ). sixty five answers were excluded due to inadequate response and answers were analyzed. of the questionnaire respondents, % were male and % were female. the ratio of board certified surgeon was %. although % of the teaching hospitals had simulators for basic training of endoscopic surgery and % of the respondents practiced basic skill of endoscopic surgery, only % teaching hospitals had specific training programs for endoscopic surgery. the surgeons who operated cases of laparoscopic appendectomy and inguinal hernia repair and cases of laparoscopic cholecystectomy, right hemicolectomy and sigmoidectomy, felt confident to perform each procedure independently. regarding with laparoscopic rectal resection and gastrectomy, even though the surgeons who had cases of experience, they didn't had confidence to perform those procedures independently. conclusions: this study is the first national survey to investigate the status of endoscopic surgery training in japan and the autonomy of young surgeons for endoscopic surgery. in order to develop a training system for not only basic skills but also advanced procedures of endoscopic surgery, cooperation of each teaching hospital, academic surgical society, medical specialty board is necessary. currently there is a debate about what is the most optimal work schedule for residents of general surgery, it is important to respect the free time of residents to avoid burnout, however it is also important have enough exposition to clinical cases that allow a satisfactory development in the clinical practice. this becomes even more important when we talk about the learning of surgical skills. this is where the laparoscopic simulation industry opens a large area of opportunity, for a reasonable price it is possible to practice basic laparoscopic skills without compromising patient safety. this is a pilot study that was carried out during the period from january to june , in a public hospital in monterrey, nl, mexico, the composition between the execution of the standardized exercises of the fls (fundamental laparoscopic surgery) in an endoscopic simulator was performed to residents of general surgery (from first to fifth year) hrs before being on call vs these same residents post call. a series of questions was asked to each resident in each measurement, so in this way they answered the same questions twice, then a comparison of the results of both questionnaires was made. the results of the exercises were assessed and rated by the same person using the criteria established in the fls for the scores of each exercise and for the final grade. an average age of years was obtained, measurements were taken of residents of which are male and female. on average, the residents before be on call performed the exercises with h of having slept while the post call performed the exercises with . h of having slept, the residents before be on call had on average . h without sleep while the post call had h without sleep. the average number of hours worked per week is h, measured by the time in and out of the hospital. in this study, conclusive results were obtained regarding the null relationship of sleep deprivation with the performance of laparoscopic skills in surgical residents. aim: 'precision cutting' is one of skills tasks of the fundamentals of laparoscopic surgery (fls) program, which is cutting a circle on a piece of gauze under laparoscope and assessed by completing time (maximum time limit: s). there is no definition of quality of the final product. the aim of this study is to develop an assessment tool of laparoscopic precision cutting and test its reliability. method: an assessment tool of laparoscopic precision cutting was developed with four items based on completion, degree of deformation, degree of being pulled, and overall appearance of the final product of laparoscopic precision cutting by experts' meetings. the scale of each item was points likert scale. a descriptive sheet with a legend and a text description for each scale (fig) was attached for assessors' reference. for our high school entry medical students, they gained hands-on experiences of laparoscopic skills first time by attending a -hour course at minimally invasive surgery training center, national taiwan university hospital (ntuh). we invited students to participate this study after this training. we collected participants' final products of ' precision cutting' station and assessed them by using this assessment tool. this study was proved by institutional review board, ntuh (irb no: rinb). results: students were enrolled between february to june . two non-medical assessors and a senior surgeon were invited to assess the products. the mean score and cronbach' s alpha value of each item were as followed: completion . ± . , . ; degree of deformation . ± . , . ; degree of being pulled . ± . , . ; and overall appearance . ± . , . . conclusions: in summary, we successfully developed an assessment tool for laparoscopic 'precision cutting' and showed its reliability. the tool could provide qualitative descriptions for objective feedbacks. validating this tool in a large scale is undergoing. purpose: to evaluate whether the participants who experienced this scenario could recall an interventional scenario for testing trainees' situational awareness and intra-operative decision making when they participated this training again. methods: we designed an iodm training course for junior surgical trainees and nurses by using live pigs since sep . in the first simulation, we created an interventional scenario and then provided an educational session. a researcher disconnected the ekg monitor on purpose for creating a scenario that the pig would lose vital signs when the team nearly finished a diagnostic laparoscopy. if the team did not aware the situation after . min, a researcher would remind the team (fig). we used a new developed assessment tool of iodm and an assessment tool for nontechnical skills for surgeons (notss) for self-evaluations and objective assessments. we also discussed with them about their reactions while encountering this interventional scenario. results: between sep to june , teams participated this training and experienced this interventional scenario. fourteen nd year surgical trainees have experienced it before. only one participant ( %) recalled it and made a quick decision while encountering this interventional scenario again. the results of iodm assessment and notss did not show statistical difference comparing their self-assessments in the first and second year. based on the analysis of the discussions, most of them remembered this this interventional scenario and reminded themselves to react it properly before the simulation. however, when they were the primary surgeon of diagnostic laparoscopy, they focused on performing this procedure and tutoring their junior trainee. they had no capacity in their brain to notice the change of vital signs. in addition, although they increased their situation awareness in clinical settings after the st time iodm training, they did not show this ability in the simulation. conclusions: recalling of an interventional scenario for testing situational awareness of surgical trainees was very poor ( / , %) among the nd year surgical trainees. qualitative analysis of discussions showed their brain capacities were occupied by performing new procedures and tutoring others. how to enhance trainees' situational awareness should be addressed. aims: a well-designed learning curve is essential to measure the progress of surgical abilities. learning curves are very important to test the skills of trainees. however, there are still no welldefined criteria for developing good learning curves. as a result, many authors use subjective evaluation criteria. the purpose of this review is to analyse this field of surgical education and to identify the key criteria for good learning curves. methods: learning curves were investigated in the field of laparoscopic and robotic minimally invasive surgery. surgery of appendectomy, cholecystectomy, cholectomy, inguinal hernia repair and gastrectomy were considered. the type of surgery, the year of publication, the design of the study, the surgeon's experience (resident, young or senior), the surgical technique, the number of patients involved in the study and the suggested learning curve by the different studies were taken into account. in the selection of articles, more importance was given to those based on the activity of young surgeons or residents. results: the literature analysis showed conflicting results. the different learning curves for the same surgery may be due to the different evaluation criteria considered. only a few studies investigate the learning curves of young surgeons and residents. conclusions: the data available in the literature on learning curves are contradictory. several factors need to be evaluated in order to create more accurate learning curves. we suggest the introduction of checklists with a score for each parameter to be examined, in order to develop more objective and standardized learning curves. aim: the uk training programme for transanal total mesorectal excision (tatme) has completed its first round of training. the study aim was to design a reporting platform that provided trainees with video-assisted feedback in a clear, concise and useful manner to support their training. methods: an established method of video analysis called observational clinical human reliability analysis (ochra) was used to assess the surgical performance of the trainees during their clinical tatme cases. a reporting form for the ochra results was designed identifying areas of difficulties in each procedure and providing error reduction mechanisms. this was piloted during the national training programme for tatme in the uk. results: the ochra reporting form underwent three modifications before the content and format was agreed upon. the final version is divided into three sections: a. case details, b. ochra findings, and c. suggested error-reducing mechanisms. for part b the tatme procedure was divided into four phases of the operation: . pursestring, . rectotomy, . tme dissection, and . connected phase when the abdominal and transanal teams work together synchronously. for each phase, ochra findings described the most frequently occurring technical inaccuracies/errors, number of consequential errors/adverse events and the most frequent and serious consequences encountered. suggested error-reducing mechanisms in part c were developed and established by an expert workshop and individual interviews with international surgeons experienced in tatme.trainee and mentor feedback stated that the reporting form had a clear format, easy to follow and understand. the error-reducing mechanisms were particularly useful and allowed the trainee to focus on improving specific technical aspects in their subsequent cases. conclusion: video analysis using ochra can provide a wealth of information on surgical performance, especially for trainees at the start of their learning curve. as an exploratory study, validation of the reporting platform is required; however, its potential to offer detailed, individualised feedback to enhance training is promising. laparoscopic pelvic surgery training program-using a new concept d-printed versatile pelvi-trainer r.c. elisei , f. graur , c. popa , e. mois , l. furcea , n. al hajjar general surgery, bistrita emergency county hospital, bistrita, romania; general surgery, regional institute of gastroenterology and hepathology ,,prof. o. fodor,,, cluj-napoca, romania pelvic laparoscopic surgery (rectal, urological, or gynecological laparoscopic surgery) is an advanced surgery which require advanced skills, not easy to acquire. there are a lot of training programs for advanced laparoscopic skills but many of them are not affordable for most of surgery residents in eastern europe, where the training programs are far behind from those in western europe. because of that those training programs need to be improved and optimized. in the european union we want equal and high skilled surgeons. this is why we designed a new concept of pelvi-trainer, a versatile one in order to offer the residents the possibility to achieve advanced laparoscopic skills like perfect coordination, precise movements, ability to cut and suture after a well defined route, all of them in the pelvis tight space. we d-printed this pelvitrainer which has multiple characteristics: cheap and easy to produce, easy to be used, versatile because offer the possibility to achieve the skills named above, and many others, but also to train on real ex vivo animal rectum (suine model). we also believe that with a proper training a medical student and a young surgery resident are able to achieve the same skills like experienced surgery residents or specialists. in order to demonstrate that we need a study to compare the time to perform or more exercises in this new concept pelvi-trainer by the medical students, young and experienced residents and surgery specialists. what we want to achieve with this training program project is to have more and more skilled surgeons in advanced laparoscopy and an equal laparoscopic surgery training all over the country, close to the level of training in the western europe. also we want this training program to make a standardization of the pelvic laparoscopic surgery training first in our country and then in other countries if possible. aims: the objective of this systematic review is to provide an evidence-based overview of the different components of laparoscopic training curricula, emphasizing the value of objective forcebased assessment and how this in implemented in modern laparoscopic training. methods: bibliographic databases of pubmed and embase were searched till april to identify studies reporting on evidence-based laparoscopic skills training. abstracts of retrieved studies were reviewed by two authors independently and those meeting the inclusion criteria were selected for full-text review. results: the search yielded a total of individual records. a total of articles were included. the articles were divided into nine different categories, which include 'metrics', 'benchmark criteria', 'measurement systems', 'timetable', 'training modalities', 'camera settings', 'training tasks', 'serious gaming', and 'competition'. a descriptive analysis of the data is provided. motion analysis parameters, such and path length and time are frequently validated and used for assessment. the results of validation studies on tissue manipulation parameters, such as maximum force and mean force show proved their discriminating power between different levels of proficiency. however, implementation of these metrics remain restrained. conclusions: numerous studies on laparoscopic skills training have been conducted over the years. nevertheless, no consensus is reached towards the use of objective assessment tools. although the value of validated metrics is described well, implementation of objective metrics is limited. we recommend to consider objective force-and motion metrics for feedback and assessment during laparoscopic skills training. surgery, regional institute of gastroenterology and hepatology, cluj-napoca, romania; anesthesiology, university of agricultural sciences and veterinary medicine, cluj-napoca, romania; radiology, regional institute of gastroenterology and hepatology, cluj-napoca, romania aims: the aim of the study was to create a new easy learning method of swine liver anatomy for residents in training. based on human liver surgical anatomy we put 'face to face' the similar structures and also the differences using ex vivo porcine models and ct reconstructions from live pigs. methods: having in mind the human liver anatomy, in the first stage we used data obtained from dissection of twelve porcine liver models to create an anatomical pattern, which summarized the most important surgical information. in the second stage, anatomical data obtained from ct scans of twelve living anesthetized pigs were analyzed. the ct reconstructions and volumetry data were added to the gross anatomy pattern to create a more complex learning module. results: the residents established the most frequent description of swine liver anatomy by putting together the information from ex vivo model dissection. the liver parenchyma is divided into four main anatomic lobes: left lateral, left medial, right medial and right lateral. all those lobes are connected only in the posterior part, which allows a very good separation between them by deep fissures. just as in humans, we found eight distinct segments with independent vascularization and biliary drainage. portal vein has a specific 's' shape; in most cases hepatic artery was found like a trifurcation and extrahepatic biliary tree has a very thin wall. in the right hemi-liver, the inferior vena cava passes through the liver parenchyma. most frequent, we found five hepatic veins which are running completely intraparenchymal. the imagistic data offered a very useful d reconstruction with anatomical positions of the vascular-biliary tree and liver segmentation and gave us the possibility to create practical scenarios for resections. perhaps the most important information was to discover and see the section plan and to calculate the volume of the remaining liver after resection. conclusions: the anatomical-imagistic pattern based on \ i[ex vivo \/i [ model disections combined with imagistic data offers a unique mindset before intervention. the concept 'human \ i[vs \/i [ swine' to create an easy method of learning for residents in training can be applied to swine liver anatomy. the learning of surgery is traditionally based on the behaviourist model . goals are set, standards of care fixed, with regular assessments of the level achieved. the teacher exercises control over the student, imposing rules and models, supported by 'reinforcing' actions (reward or punishment). the theory of skinner's program of education, from , is reflected in surgical learning. it foresees a gradual progression by level of difficulty, following a transmission-imitation model . these theories seem currently outdated to face the new challenges of medicine and surgery and to keep up with technological developments. bruner, one of the theorists of the constructivist model, proposed in a method of collaborative learning between those who teach and those who learn. the goal of the method was to improve strategic problem solving. the comparison between various perspectives (between teacher and student), allowed the learner to better absorb knowledge and improve critical thinking. in kapur published on the theory of 'productive failure'. this model makes the error of a single person useful for all his colleagues, privileges the practice of theoretical knowledge, contextualised learning as opposed to abstract learning, and 'guided' practice compared to a 'guided' theory. bruner and kapur's systems favour creativity, critical analysis of a problems origin, and the practical use of knowledge. they represent a hypothesis of learning, based on constructive discussion and a continuos 'give-take' feedback system. in order to put these new models into practice in the clinical context, one may hypothesise and propose the adoption of a formal discussion of clinical cases that are complicated or difficult. thereby making the theoretical lessons more collaborative, intuitive and inclusive. in the surgical field, one could adapt such a concept to surgery simulation, virtual reality and anatomical models. aim: large hiatal hernias have a surgical indication when the patients suffering disabling symptoms such as anaemia, dyspnea, chest pain, gastric reflux. several studies showed that in the case of large hernias the placement of a prosthesis was safe and could protect against recurrence. mini-invasive surgery is the preferred approach for hiatal hernia repair and anti-reflux procedure and the toupet fundoplication has been shown to be the best surgical technique for the hiatal hernias repair.the laparoscopic approach is currently the surgical gold standard but is burdened by technical difficulties especially in the case of large hiatal hernias. the robotic system is designed to overcome some technical difficulties of laparoscopy and the studies available in literature report the safety and effectiveness of the robotic approach in complex hiatal hernias repair. methods: we present the case of a grade iv hiatal hernia treated with a robotic approach in a years old woman (bmi: kg/m ). the medical history consisted of a road accident with a probable mechanism of deceleration, three years before. the patient had been suffering from dyspnea for three years. due to the recent discovery of an anaemia, the patient was subjected to an endoscopic examination with the identification of a voluminous grade iv hiatal hernia. a subsequent computed tomography (ct) scan showed also the partial herniation of the transverse colon. results: the patient underwent to surgery by using the da vinci robot system siÒ (intuitive surgical, sunnyvale, usa) with a single docking approach. the surgery consisted in the liberation of the hernial sac, the placement of a goretex prosthesis and the packaging of a toupet fundoplicatio. the surgery was performed without complication. conclusions: the robotic approach in the hiatal hernia surgery seems to be a valid alternative to laparoscopy, especially in complex cases. the surgical ability in robotic surgery is of paramount importance. general thoracic surgery, kawasaki municipal hospital, tokyo, japan aim: video-assisted thoracoscopic surgery (vats) with carbon dioxide (co ) for mediastinal surgery is known to improve the visualization of medaistinal space. we report our experiences with two cases that underwent vats thymectomy using co insufflation under the one-lung ventilation general anesthesia by double lumen tube. methods: the instruments that were used for vats thymectomy were only the -mm -degree rigid thoracoscope, maryland jaw energy device, cotton made-dissectors, and straight endoscopic grasping forceps. they were used through sealed ports designed for laparoscopic surgery. lowpressure co insufflation set at mmhg were used for compression of surround tissue of mediastinal tumor during the releasing procedure. results: the patients were an -year-old male and a -year-old female. thoracoscope with the mmhg co insufflation provides excellent visualization of the medaistinal space and operation could be done smoothly without any hemodynamic compromise. their pathological diagnoses were thymic cancer and thymoma, type b . the operative times were min and min. the postoperative courses were uneventful and the patients were discharged on day th and rd . conclusion: we have just begun to routinely use co insufflation for mediastinal tomorectomy and present our early experiences of successful vats thymectomy by utilizing co \ su \/su insufflation. aims: this retrospective study aims to evaluate the feasibility of single-incision thoracoscopic surgery (sits) for primary spontaneous pneumothorax (psp), using a novel multichannel port (x gateÒ). methods: between october and november , ten patients who underwent sits using x gateÒ. nine patients were male and was female, with mean age of . ± . years old. a . cm incision is placed in the middle axillary line on the th or th intercostal space, depending on the lesions. postoperative outcomes of these patients were compared with those of patients with psp who underwent conventional three-port video-assisted thoracic surgery (vats). results: there were no conversions from sits to vats. mean operative time of sits group was significantly shorter than that of three-port vats group ( . ± . min vs . ± . min, p = . ). mean number of staplers used in surgery was . ( ) ( ) ( ) ( ) in sits group and ( ) ( ) ( ) ( ) ( ) in vats group (p = . ). mean duration of postoperative drainage was also shorter in sits group ( . ± days vs . ± . days, p = . ). no recurrence and wound infection were observed in sits group. conclusion: sits using x gateÒ is feasible when performed for selected patients with psp. x gateÒ provides good visualization of intrapleural space and esthetic outcomes, as well as a superb maneuverability by decreasing mutual interference of surgical instruments. although conventional three-port vats for psp is well established, sits using x gateÒ can be a permissible alternative. further examinations are required to evaluate efficacy of sits using x gateÒ. aims: haemorrhage remains a leading cause of potentially preventable death in trauma. in particular non-compressible torso haemorrhage is approximated to cause - % of mortality in civilian trauma patients with otherwise survivable injuries and % in war setting. we performed a literature review to assess the potential for using endovascular stenting in traumatic venous injuries and explore the evidence of their efficacy and safety with different venous injury patterns. methods: systematic online search of pubmed performed using key words'endovascular stent', 'venous injury', trauma, penetrating, blunt, abdominal and pelvic. inclusion criteria included all studies that explored the use of endovascular stents following traumatic abdominopelvic venous injuries. english language studies were used. results were presented according to prisma guidelines. results: of the studies generated by the search,there were only four case reports in the literature documenting the use of endovascular stents in traumatic venous injuries dating back to and most recently . the four cases included three retrohepatic ivc injuries, two secondary to blunt trauma and one penetrating; whilst the final case a blunt injury at the ilio-caval bifurcation. all four cases reported successful deployment of stents via the femoral or internal jugular veins, with subsequent resolution of haemorrhage. length of time taken for stent insertion ranged from to min. three of four patients made full recoveries and discharged from hospital, with one patient subsequently dying of a brain injury independent of the successful venous stent insertion. no complications were reported at up to months follow up in remaining cases including stent leak, stenosis or migration. conclusion: endovascular venous stents have been used successfully in managing complex abdominopelvic traumatic venous injuries. in particular retrohepatic venous injuries refractory to hepatic packing and vessel embolization, which are not amenable to direct surgical repair due to anatomical location. however before endovascular stenting can be added to the arsenal of interventional radiologists for abdomino-pelvic trauma, further development of stents custom made for venous injuries as well as prospective studies examining their long term safety and outcomes is needed. tracheal papilloma is a rare neoplasm growing from the tracheal or bronchial epithelium and has no specific clinical presentations. this is a -year-old female who complained of progressive dyspnea for about months. physical examination was unremarkable and the there was no abnormal finding by the chest plain film. chest computed tomography was arranged and revealed a mass lesion located at the tracheal lumen with more than % luminal obstruction. we used fiberoptic bronchoscopy to evaluate the airway and found a mass lesion with pedicle originated from the posterior tracheal wall. cryotherapy was considered for the tumor mass removing to establish a patent airway. the pathologic report revealed tracheal papillomatosis without any malignant component. dyspnea was immediately improved and the patient chose closely observation after the bronchoscopic cryotherapy. aims: recent advances in laparoscopic surgery, both in techniques and instrumentation material, have led to the emergence of innovative technological fields, among which robotic surgery stands out.one of the handicaps of this surgery is its high cost as well as the long learning curve. in this stage a new tool arises, the flexdex semi robotic arm, which combines the precision and the range of movements of robotic surgery with the greater availability, simplicity of use and learning of conventional laparoscopic surgery.the objective of this study is to evaluate the efficacy and safety of the flexdex device in different laparoscopic procedures. methods: flexdex's is a three-axis gimbal technological device integrated in a conventional laparoscopic instrument that translates the surgeon's hand, wrist, and arm movements from outside the patient into corresponding movements of an end-effector inside the patient's body.the greater accessibility provided by the flexdex allows the surgeon to perform sutures in areas of difficult access where mobility with conventional laparoscopic instruments is not optimal. the comfort of the surgeon remains fundamental in any type of surgery, even more when we are in anatomical locations with complex access, especially for the realization of sutures. here is where surgical innovation instruments such as flexdex provides ergonomic comfort for the surgeon and improves the patient's safety, especially in high-risk situations, such as when performing anastomosis. results: we present a prospective series of laparoscopic procedures carried out by the same surgical team being the initial experience in our environment in the use of the flexdex semi robotic arm for the realization of complex anatomical sutures.this is a case series of patients to whom different surgical techniques requiring manual suture have been performed. these being tapp procedures, nissen-type fundoplicature and reinforcements of colorectal anastomosis. it is important to note that in none of the cases complications were recorded conclusions: flexdex can provide an excellent alternative to the robotic systems in complex surgical procedures, offering surgeons the precision and control they desire while maintaining the balance of cost, outcome and patient benefit. background: a new single-port device (fsis-flexible-single-incision-surgery) is presented. this new platform has three working channels, two for rigid instruments and one for the flexible endoscope. the channel for flexible instruments offers a pneumatic sealing to avoid the air's leak of the cavity (abdomen, rectum, vagina) . in this study the preclinical data are shown testing the feasibility and safety for laparo-endoscopic instruments. methods: experimental evaluation of feasibility and safety in two stages. in the first stage a working channel with pneumatic sealing was tested in simulators to use a flexible endoscope. in the second stage (animal model) the single incision device that makes possible to use laparoscopic instruments and flexible endoscopes was tested. the measured variables were: time of the procedure, co employed, adverse intraoperative events, grip's losing, losing of pneumatic sealing, feasibility and safety of the procedure for the surgeon. results: the hysterectomy and double adnexectomy was done with a median time of . min. the median of the co consumption was . litres. only in one case ( . %) the surgeon had problems with the abdominal navigation of the endoscope that was easily solved. the grip's lose wasn't a major problem. the median size of the skin incision was . cm. the median surgeon' score for the feasibility was and for the safety was . . conclusions: the surgeons considered that the use of the device was very feasible and safe. the fsis-device is a universal platform for single-incision-surgery for surgeons and gastroenterologists and for abdominal, rectal and vaginal access. aim: despite the near-infrared fluorescence (nirf) via the intravenous administration of indocyanine green (icg) improves the visualisation of the cystic duct (cd) and the extrahepatic biliary tract (ebt), the back fluorescence of the liver reduces the signal-to-noise ratio.we have modified the technique of nirf cholecystocholangiography with intragallbladder icg injection by using the arrow-karlan tm balloon cholangiography catheter instead of the purse string at the gallbladder's fundus. this procedure allows a high rate of visualisation of the ebt, with few cases of icg leakage.aim: of this study is to confirm the feasibility of this different technique and to analyse the icg spillage from the gallbladder and to identify the ebt. methods: we enrolled nine patients undergoing laparoscopic cholecystectomy for cholelithiasis. the gallbladder was perforated with the cholangiogram catheter, the balloon inflated with . ml of saline and tightened. the bile was drained and the icg bolus injected. a titanium clip was the placed on the catheter strict closely to the gallbladder in order to prevent the catheter dislocation. results: the cd and the ebt were visible before dissection in / and / patients respectively. after dissection the cd was visible in all the patients and the ebt again in / patients. there was only one icg spillage due to a tardive positioning of the clip. in a case of inflamed gallbladder this technique helped in the identification of the dissection plane. conclusions: our preliminary results of this ongoing study confirm the feasibility of this different approach as a possible alternative to the purse string and a good visualisation of ebt. introduction: robotic-assisted surgery is a promising technique for overcoming the limitations of laparoscopic surgery, especially with regards to complex and advanced surgical procedures. here, we describe the establishment and implementation of our robotic upper gastrointestinal (gi) and hepato-pancreato-biliary (hpb) surgery program within our center of excellence for minimally invasive surgery as well as the first-year results. method: robotic-assisted surgery was performed using the davinci xi surgical system tm and performed by two surgeons specialized in minimally invasive surgery (db and tk). our robotic surgery program of upper gi and hpb surgery was established in three steps: ( ) first, surgical procedures with easier degree of difficulty were performed robotically, including cholecystectomy, minor gastric resections and fundoplications. ( ) then, pancreatic distal resections, enucleations, adrenalectomies and atypical liver resections were robotically performed, as procedures with moderate degree of difficulty. ( ) finally, advanced and highly complex procedures were performed, including right hemihepatectomy, complex pancreatic head resections (including portal vein resections), total gastrectomy and esophagectomy. data collected from july till july were retrospectively analyzed with regard to conversion rate, morbidity (clavien dindo grade £ ) and mortality. results: within the first year, a total of robotic assisted upper gi and hpb resections were performed. the first step of establishing our robotic surgical program included eight procedures. here, conversion rate, morbidity and mortality were %. within the second step of establishment procedures were performed. conversion rate, morbidity and mortality were %, % and %. the last step included of advanced and highly complex procedures. these procedures resulted in a conversion rate of %, % morbidity and % mortality. conclusion: our stepwise approach enables a safe implementation of a robotic surgical program for upper gi and hpb surgery with low morbidity and no mortality even for highly complex procedures. however, highly complex procedures required a high conversion rate, which might be caused by the early stage of experience. the standard surgical procedure of choledochal cyst is a complete excision of the cyst with rouxen-y hepaticojejunostomy and laparoscopic surgery had been increasingly used. this is still a challenging way to perform anastomosis due to the small diameter of bile duct and the possibility of bile leak or stricture. robotic system can overcome the shortcomings of laparoscopy with providing three-dimensional view, magnification, and articulated instruments. from jan to dec , patients underwent robotic cyst excision and hepaticojejunostomy by single surgeon. we reviewed the clinical data and compared with laparoscopic outcomes of early (from to ) and late (from to ) group, retrospectively. patients of robotic series were all female with mean age . years and bmi . . the mean size of cyst was . . cm, and todani type ia , ic and iva , respectively. total trocars were used with robotic working arm and assist and camera. the mean operative time . ± . min, and it was similar with late laparoscopic group ( ± . min) and significantly shorter than early group ( ± . min).there were no open conversion in robotic and late laparoscopic group, however, the early laparoscopic group involved % of conversion rates. the hospital length was ± . days in robotic group, and it was similar with late group ( ± . ) and more shorter than early group ( . ± . ). in robotic series, postoperative complications occurred patients. one case included cholangitis which was resolved after conservative treatment. bile leakage was developed in patient, and treated with drain that inserted intraoperatively. last cases showed incisional hernia at postoperative months, and was corrected by laparoscopic herniorrahphy. complications (n = ) in late laparoscopic group included hepaticojejunostomy stricture and stone, bleeding of jejunal branch, portal vein thromobosis, acute pancreatitis, and adhesive ileus. there were no mortaility case in any groups.robotic surgery of choledochal cyst is a safe and feasible option with short-term results that are comparable to laparoscopic approach. general surgery, sanchinarro university hospital, madrid, spain background: the incidental detection of benign to low-grade malignant small pancreatic neoplasms increased in the last decades. the surgical management of these patients is still under debate. the aim of this paper is to evaluate the safety and feasibility of robotic enucleations. methods: we retrospectively reviewed our prospectively databases from november . demographics, pathological characteristics, perioperative outcome, and medium-term follow-up of patients who underwent robotic pancreatic enucleations were collected. results: patients were included. the mean age of the patients was years ( - ). the median body mass index was ( - ). ten lesions were located in the pancreatic head, in the pancreatic body, in the pancreatic tail. operative time was min (range - ), no intraoperative transfusion were needed and in one patient conversion to open approach was needed. in three patients grade b pancreatic fistula occurred. the mean postoperative stay was , days. conclusions: robotic enucleation is a feasible and safe approach, with low incidence of morbidity. the results of surgical treatment of patients with pulmonary tuberculosis were evaluated depending on the prevalence of the tuberculosis process and the type of surgical intervention used. according to the results of the questionnaire, people operated on pulmonary tuberculosis in the period from to years ago, the frequency of cases of tuberculosis reactivation, the complicated course of the remote postoperative period, as well as the mortality and causes of lethal outcomes were assessed. it was found that after sublobular resection and lobectomy, treatment failure was noted at . %, relapse of tuberculosis- . %, pleural empyema- . %, bronchial fistula- . %, cardiovascular insufficiency-in . % operated. the mortality rate was . % with a total clinical efficacy of . %. after combined resection and bylobectomy, treatment failure was noted at . %, relapse of tuberculosis- . %, pleural empyema- . %, bronchial fistulae- . %, cardiovascular failure- . % operated. the mortality rate was . % with a total clinical efficacy of . %. after pneumonectomy, treatment failure was noted at . %, relapse of tuberculosis- . %, pleural empyema- . %, bronchial fistulae- . %, cardiovascular failure- . % operated. the mortality rate was . % with a total clinical efficacy of . %. robotic reduced-port splenectomy using single-site platform j.h. lee background: in the era of minimal invasive surgery, single incision laparoscopic splenectomy can offer some advantages compared to conventional laparoscopic splenectomy. but it requires expertise in minimally invasive techniques due to technical difficulties. the da vinci robotic reduced-port splenectomy using single-site platform permits greater freedom of movement and higher levels of accuracy than previous laparoscopic surgery through two small incisions. methods: we performed a retrospective review of all patients who underwent robotic reduced-port splenectomy using single-site platform at our institution between january, and november, . one cm periumbilical incision was made for glove port insertion and the other incision was made at left side of abdomen for additional mm port insertion.the surgical technique is much same as open procedure. short gastric artery was ligated, firstly. splenic artery and vein were ligated individually. during the surgery, any stapling device was not used. vessel sealer was used for hemostasis and mobilization of spleen. a specimen was removed through umbilical port site within lap-bag. result: eight patients ( female and male) with median age of . years underwent robotic reducedport splenectomy using single-site platform (one case with combined robotic cholecystectomy for gall bladder stones without additional trocar). the indications were; hematological disease (n = ), splenic mass (benign n = , malignant n = ). preoperatively measured spleen size was ranged . cm to cm (mean cm). there were no intraoperative complications and open conversion. mean operative time was min. (range - min) including docking (mean min) and console time (mean min) mean blood loss was under ml. mean hospital stay was . days after surgery. one patient underwent oral anticoagulation therapy only for portal vein thromobisis without any symptoms, and thromobisis was resolved at month follow-up ct scan. there were no clavien-dindo class iii or above postoperative complication. conclusions: robotic reduced-port splenectomy using single-site platform seems to be feasible and effective. it seems to overcome certain limits of previous robotic or conventional single-site laparoscopic splenectomy and single-site only robotic splenectomy. we think mm additional port allows to use endo-wrist da vinci instruments such as vessel sealer which enhances dissection efficiency andsafety of procedures. aims: inguinal lymph node dissection carries an important risk of post-operative complications, mainly related with wound complications and long term lymphedema. the minimally invasive approach aims to reduce the morbidity of this procedure, avoiding the traditional groin incision but still allowing a full access to the lymph node basin. the authors aimed to describe their videoassisted inguinal lymph node dissection (vilnd) cases, comparing the surgical outcomes with a sample of open inguinal lymph node dissection (oilnd) cases. methods: we performed a retrospective descriptive study that compared the data from patients submitted to vilnd since (the year in which this technique was first performed in our institution) with the patients submitted to oilnd in and . gynaecologic and urologic malignancies were excluded. the statistical analysis was performed using spssv Ó, with a p value \ . indicating statistical significance. results: a total of cases of inguinal lymph node dissection were analysed, . % of which vilnd (none of them requiring conversion to the open approach). melanoma was the primary tumour in % of patients. the vilnd and oilnd groups had no statistically significant difference between them regarding age, body mass index, smoking status or the reason for lymph node dissection-clinically detected lymph node vs. positive sentinel node biopsy. the mean of isolated lymph nodes in the vilnd ( . ) and oilnd ( . ) groups was also not statistically different (p = . ). there was no difference in the rate of post-operative seroma, wound dehiscence or lymphedema. the rate of surgical site infections was higher in the oilnd group- % vs. . % during post-operative hospital admission (p = . ); . % vs. . % after discharge (p = . ). conclusions: in our population of patients we conclude that the main advantage of the videoassisted approach regarding surgical morbidity lies in the reduction of the infection rate, as the published literature also confirms. the equivalent number of lymph nodes retrieved in both groups points toward the oncological safety of the minimally invasive procedure, that we hope to study further in the future after a longer follow up period. objectives: to evaluate the clinical feasibility of tumor localization technique with radio-frequency identification (rfid) clip marker methods: we developed the proto-type rfid integrated endoscopic clip (rfid-clip) and probe to detect it on serosa surface during the laparoscopic surgery. a pig weighing kg was used as the specimen for the in-vivo test. endoscopist performed the application of the rfid-clip on porcine gastric mucosa. after then, the surgeon tried to find the location of rfid-clip using the detection probe and marked with the electrocautery. after the gastrectomy with cm margin (each to proximal and distal), we confirmed the prediction of rfid-clip location and accuracy of resection. results: rfid-clip location was detected and recorded on the exact site of clip application. detection range was very short and we confirmed there are almost no differences between actual clip location and our prediction. this result might arise from using the low-frequency rfid tag to increase the accuracy through reduction of the range. however, some rfid-clip were not detected because of the issue of clipping trouble, not rfid tag. conclusions: this is a basic study to evaluate the clinical usefulness and feasibility of the new localizing technique. we confirmed the possibilities of this system and it could be the helpful option to provide the information of exact location for the minimally invasive surgery or early gastrointestinal tumors. background: the advantages of laparoscopic posterior retroperitoneal adrenalectomy (lpra) have been described in the literature. the aim of this study was to compare the clinical outcomes of lpra and robotic posterior retroperitoneal adrenalectomy (rpra) and determine the differences that could affect the outcomes. methods: we retrospectively analyzed adrenalectomy cases at asan medical center from to . there were lpra and rpra cases, and their clinicopathological features and surgical outcomes were compared. results: in lpra, there was a positive relationship between operation time and male gender, early period of experience, adrenal tumor size, and pheochromocytoma. in rpra, adrenal tumor size and pheochromocytoma were the only factors affecting the operation time. when the adrenal tumor size was = . cm, the operation time of lpra was shorter than that of rpra (p = . ). when the tumor size was [ . cm, there was no significant difference in the operation time of lpra and rpra (p = . ). conclusions: rpra is a feasible and technically safe approach for benign adrenal diseases. the use of rpra could benefit patients with large tumors and provide comfort by overcoming the factors contributing to a longer operation time in the laparoscopic technique. methods: twenty years experience at the american university of beirut medical center for laparoscopic adrenalectomy. a total of cases were done laparoscopically with no conversion and minimal complication. the average operative time is mins.the video will show the various steps used for lap redo (lt) adrenalectomy for a cm pheochromocytoma using the lateral position and through trocars. attempt to remove the pheochromocytoma in iraque was complicated by cardiac arrest treated successfully and patient referred to the american university of beirut medical center. results: patient had smooth postoperative course following laparoscopic adrenalectomy and patient discharged days later with no complications. conclusions: even large adrenal masses can be completed laparoscopically in advanced experienced centers in laparoscopy. surg endosc ( ) aims: the adrenocortical of uncertain malignancy neoplasm is a spectrum of classification for adrenal tumors whose histopathological diagnosis is uncertain. clinical case: we present a year old patient with constitutional syndrome and severe hypercortisolism and hypokalemia reason why she was admitted to icu for episodes of ventricular fibrillation. no other medical history of interest except refractory hypertension to treatment. the tc showed a left adrenal mass of . . cm with microcalcifications, areas of necrosis and hemorrhage, no infiltrating, without disease to distance. the surgery was a laparoscopic left adrenalectomy with no evidence of infiltration and no lymph nodes. the histopathology lesion presented a dense proliferation cellular of cortical type, with incomplete fibrous, without vascular or capsular invasion, with a % ki ; positivity vimentin and cd . all epithelial markers, were negative. all this leads to the diagnosis of a neoplasm of uncertain malignancy potential adrenocortical. during the postoperative period, the patient presents a crisis of adrenal insufficiency that was treated with intravenous replenishment corticoidea and later orally with good clinical response. discussion: the adrenal carcinoma has a low incidence ( . %), incidence peak around the years, the most frequent is the mixed secretory. they are - % of the adrenal incidentalomas. it is usually presented to the diagnosis as a locally advanced tumor with metastases (to liver, lung, retroperitoneal ganglia and bone). may present clinically due to hormonal hyperproduction; or be non-functioning tumors. the adrenal carcinoma poses a great difficulty at the time of the diagnosis pathological, and includes as differential diagnosis to other abdominal tumors. the distinction between corticoadrenal adenoma and adrenal carcinoma is sometimes difficult, so it has been defined a spectrum of intermediate category called adrenocortical neoplasm of intermediate or uncertain malignancy. it is obtained with the weiss criteria, being necessary at least of them for confirm the diagnosis of adrenal carcinoma. this category has a low risk of local recurrence or metastasis, but it needs a narrow follow-up. conclusion: adrenal carcinoma of uncertain malignancy implies a new category in those tumors of difficult classification. aims: multiple endocrine neoplasia type (men ) is an autosomal dominant disorder with an estimated prevalence of per , in the general population. among patients suspected to have a pheochromocytoma, the diagnosis is rarely confirmed and only % is presented bilaterally. we present bilateral laparoscopic adrenalectomy in patients with men . method: a -year-old woman with a family history of medullary thyroid cancer and breast cancer. personal history: hypertension, medullary thyroid cancer, breast cancer, laparoscopic cholecystectomy. appendectomy. after a study by endocrinology and suspicion of bilateral pheochromocytoma, discussing the case in a multidisciplinary committee, bilateral adrenalectomy was decided by laparoscopic approach. selective alpha- -adrenergic blocking agent (doxazosin) were utilized before surgery. under general anesthesia left adrenalectomy was performed first in right lateral decubitus position. mmhg pneumoperitoneum was started with the verres needle and trocars ( mm umbilical, mm subxifoid and mm left subcostal).once dissection was completed the gland was placed in a plastic bag and extracted through one of the trocars incisions, then the position of the patient was changed to left lateral decubitus for the right adrenal approach. another right subcostal mm trocar was used. adhesiolysis of previous cholecystectomy was performed to right adrenal approach. adrenal veins were divided between metallic clips.no drainage was employed. results: the procedures were successfully performed without conversion. surgical time was min and hospital stay was days. had a clinical reversion with control of blood pressure monitored by endocrinology conclusions: currently, the laparoscopic approach is the technique of choice for the management of adrenal pathology.lateral decubitus transperitoneal approach is the procedure of choice in most cases. bilateral laparoscopic synchronous adrenalectomy is feasible and safe with good results as in our patient. traditionally the treatment of hyperparathyroidism for patients with familial hyperparathyroidism was subtotal parathyroidectomy or total parathyroidectomy and auto transplantation. in the era of minimally invasive parathyroidectomy, the removal of only abnormal glands guided by preoperative localizing studies has been suggested. aims: this systematic review aimed to investigate the role of focused minimally invasive parathyroidectomy in the treatment of patients with familial hyperparathyroidism. methods: electronic databases were searched with the search terms 'men i', 'familial hyperparathyroidism', 'men a','hyperparathyroidism-jaw tumor syndrome', 'parathyroidectomy', 'minimally invasive ', for the time period up to and including december . full publications, including clinical trials randomized or not, retrospective studies, case series, case reports that provided relevant data met inclusion criteria. results: thirty five possibly relevant studies were identified. abstracts were reviewed and fifteen articles were excluded. twenty studies, that met inclusion criteria were retrieved in full text and included in the systematic review, including three retrospective cohort studies i.e. two presenting data on meni associated hyperparathyroidism and the third study on familial hyperparathyroidism and seventeen small case series or case reports. the two retrospective studies on meni hyperparathyroidism included patients treated either with focused minimally invasive parathyroidectomy or with the conventional approach. these studies presented conflicting data with one supporting and the other negating the focused minimally invasive parathyroidectomy due to the failure of localization studies to identify enlarged parathyroid glands in a great number of patients. conclusion: undoubtedly, the idea of minimally invasive parathyroidectomy in patients with hereditary and familial hyperparathyroidism is interesting. this idea is especially challenging in the case of meni. existing data suggest that focused mimimally invasive parathyroidectomy is feasible under the condition of exact preoperative localization studies. the main advantage of this approach is the minimization of the risk of postoperative hypoparathyroidism. however, data are limited and further research is needed before valid conclusions can be drawn on the suitability of this approach. objective: resection of pheochromocytomas is a challenging procedure due to hemodynamic lability, tumor vascularity and malignant potential.given the technical challenges for resection of large pheochromocytomas, there were hesitations about using the laparoscopic approach for these tumors during the first decade of laparoscopic surgery. however, improvement in imaging modalities,better pharmacological preparation,advances in anaesthesia and laparoscopic surgery rendered laparoscopic surgery for pheochromocytomas safe and efficient. our aim was to evaluate surgical outcomes in patients with pheochromocytoma and to validate the role of laparoscopic surgery in the treatment of these tumors. design: a total of procedures for pheochromocytoma were performed between january -september . the preoperative diagnosis, operative details, complications, length of hospital stay, morbidity and follow up were retrieved from the hospital records of patients who underwent adrenalectomies for benign and malignant adrenal tumors in the same period. preoperative localization was established in all patients with computerized tomography (ct) or magnetic resonance imaging (mri), while iodine - -metaiodobenzyguanidine(mibg) scan was reserved for ambiguous cases where paraganglioma or metastatic disease was suspected. endocrinological evaluation and complete adrenal dynamic testing were performed to determine whether the tumor was functional or not. results: eighty-seven tumors were removed from patients. one patient with meniia underwent bilateral resection of pheochromocytomas in two stages. tumor size in laparoscopic procedures ranged from . cm to . cm (mean . cm). forty-three patients had benign disease, potentially malignant (based on pass), malignant with metastasis. eight were in the context of a familial syndrome. sixty -eight patients underwent laparoscopic adrenalectomy, patients had open approach from the start for recurrent pheochromocytoma or large benign tumor, patient had open approach due to inoperable malignant pheochromocytoma and patients had conversions from laparoscopic to open procedure. nine patients received sodium nitroprusside intraoperatively to treat hypertension. one patient developed pulmonary embolism, and succumbed month later. there were no recurrences for the benign tumors during the follow-up period. conclusions: laparoscopic resection of pheochromocytomas despite its increased level of difficulty compared to that of other adrenal tumors, is a safe and effective procedure. aim: the concept 'large' in transperitoneal lateral laparosopic adrenalectomy (tlla) has been evolving along time, ranging from to - cm depending on different authors. on the other hand, some authors discourage laparoscopic surgery in larger tumors due to the increased risk of malignancy in those larger than - cm, referring to malignancy in out of or cases. paragangliomas are rare tumors originated in extra-adrenal chromaffin cells, with an incidence of - cases per million inhabitants. they can appear in any location between neck and pelvis. sympathetic paragangliomas are usually functional and catecholamines producers. we present a movie of surgical intervention of a -year-old patient who, in study for refractory hypertension, presented paraganglioma producing norepinephrine, whose approach was performed laparoscopically. -year-old woman studied by nephrology for refractory hypertension. on physical examination, only obesity standed out. in blood exams, levels of normetanephrine were observed in plasma of pg/ml and aldosterone pg/ml. abdominal scintigraphy was performed in which there was no evidence of increased activity at adrenal level. abdominal ct shows retroperitoneal extra-adrenal tumor of inter-aortocava location immediately below renal vessels with dimensions of . . cm. after preparation, she was operated. laparoscopic access was performed under exhaustive monitoring. an heterogeneous, polylobulated tumor of cm, located interaortocava, intimately adhered to left renal vascular pedicle, was observed. a cattell-braash and kocher maneuver was performed, with exposure of inferior cava and aorta to iliac bifurcation. complete tumor excision was performed after clipping arterial and venous tributary branches. after the operation, the pacient presented favorable evolution being discharge on the second postoperative day with good control of blood pressure levels. laparoscopic approach of retroperitoneal paragangliomas is a safe technique, which allows minimally invasive access, with consequent improvement in postoperative results. the exact location of lesions and their relationships with surrounding structures, as well as their functional behavior, are very important when considering the best therapeutic strategy for these patients. we present the case of a -year-old obese male patient referred for adrenalectomy after being diagnosed with left adrenal incidentaloma. abdominal mri showed a . / . / . left adrenal mass with normal hormonal levels. after preoperative workup, the patient underwent standard laparoscopic adrenalectomy. the lateral to medial dissection and mobilization of the spleen and pancreatic tail was difficult due to the abundance of peritoneal and pararenal fat. the anatomy was peculiar: the bulky pancreatic tail was located well inferior to the splenic hilum and was visible throughout the intervention and the spleen was quite elongated-long axis = cm. the exposure of the adrenal gland was therefore cumbersome. the operating time was min and blood loss ml. the abdominal drainage was maintained for h. before discharge the patient underwent a control abdominal us examination that only showed a thin line of left pleural fluid. the patient was readmitted days after discharge for chest pain, fever ( . °c) and malaise with no abdominal signs. the emergency ct scan diagnosed left basal pneumonia with minimal pleural effusion and a / cm fluid collection between the spleen and diaphragm while the blood test showed leukocytosis. the patient was treated for pneumonia with an apparent clinical benefit for three days and lowered white cell count but his condition worsened during the forth day. repeat abdominal us demonstrated that the abdominal collection increased in size therefore the patient underwent emergency surgery. during laparoscopic exploration, the collection was unveiled as being pancreatic juice (more than times the normal serum levels of lipase and amylase). after thorough lavage, two drainage tubes were positioned in the left subphrenic space. the postoperative course was uneventful under antibiotic treatment for pneumonia and pancreatic antisecretory medication. the patient was discharged after days with minimal pancreatic drainage and the drainage tube was extracted after more days. the aim of the study was to develop the algorithm and the choice of the method of endoscopic treatment of a combined pathology of uterine leiomyoma and adenomyosis depending on the reproductive plans. methods: the study involved patients with a combined pathology of uterine leiomyoma and adenomyosis. indications for conservative myomectomy were: the size of the uterus is more than weeks. pregnancy; multiple leuomatous nodes and adenomyotic foci up to cm in size; hemorrhagic and pain syndromes, anemia, compression of the adjacent organs; suspected node malfunction; submucous leiomyoma deforming the uterine cavity with foci of adenomyosis; subserous, cervical isthus nodes and foci of adenomyosis; the presence of endometrial hyperplasia, tumors of uterine appendages; growth rate of uterine leiomyoma more than weeks pregnancy for the year; the growth of uterine leiomyoma on the background of drug treatment; infertility associated with leiomyoma and uterine adenomyosis.the laparoscopic myomectomy of the subserous node on the 'leg' with a size of more than cm and nodes of more than cm of intramural location is shown with an interest in preserving the organ.the hysterectomy is indicated for women after years of age who insist on hysterectomy, with a combination of uterine leiomyoma with atypical endometrial hyperplasia. results: the conservative myomectomy and removal of adenomyotic foci were performed in ( . %) patients: from hysteroscopic access- , vaginal access- , laparoscopic access- , abdominal access- in the presence of reproductive plans.the hysteroscopic myomectomy was performed in ( . %) patients, hysterectomy in ( . %) patients: from laparoscopic access- , from vaginal access- , from abdominal access- in the absence of reproductive plans. conclusions: the choice of surgical treatment of uterine leiomyoma and adenomyosis depends on the reproductive plans of the woman and the severity of the lesion.the laparoscopic method of treating a combined pathology of uterine leiomyoma and adenomyosis in the presence and absence of reproductive plans is a priority for women. surgery, policlinico ,,paolo giaccone,,, palermo, italy background: breast cancer in females represents the most frequent neoplasm in all age groups. the risk of getting breast cancer (mc) increases with age. the brca and the brca genes (tumor-suppressor genes, autosomal dominant transmission at high penetrance) alone justify from % to % of cases of hereditary breast cancer. methods: from january to june we have analyzed patients with brca mutation. all patients had in common a genetic mutation of brca or brca tumor suppressor genes. results: the frequency of germline mutation on brca ( patients: %) was identical to brca gene ( patients: %). of the analyzed patients were women ( . % of patients) brca and brca , and men ( . %) all with brca mutation. conclusions: prophylactic surgery must be seen as a way to put the patient in the condition to implement the most appropriate treatment. further studies will be necessary to support the validity of prophylactic surgery in patients with mutations in brca and brca genes. introduction: laparoscopic hysterectomy is a safe surgical technique for removing the uterus with or without including the ovaries and fallopian tubes. laparoscopic surgery of endometrial cancer is a safe method, with the mean time of recovery being two days only. material-method: the case of a yr old woman with metrorrhagia and anaemia (ht , %) due to adenocarcinoma of the endometrius is presented. the patient underwent a laparoscopic hysterectomy and oophorectomy. trocar ports were used during the procedure (a mm transumbilical port, similar to the port used in single incision laparoscopic operations, two mm ports at the level of the anterior superior iliac spines, and a mm port in the middle of the imaginary line between the pubic symphisis and the umbilicus). the uterine vessels and the uterine ligaments were ligated and dissected by using a thermal energy source. the patient's postoperavite course was uneventful. the patient continues to be in good condition, months post-surgery. conclusion: laparoscopic hysterectomy seems to be a safe method for addressing endometrial cancer, as it offers the surgeon a better surgical field, is tissue friendly and causes fewer postoperative complications. it is considered to be a less traumatic operative method, as due to zooming in the picture there is greater accuracy in handling the tissue, and blood loss is minimal. m. shahin background: hysterectomy is one of the most frequently performed surgical procedure. though there are three approaches in hysterectomy (open, vaginal and laparoscopic), still there are controversies regarding the optimal route for performing it. methods: this prospective comparative study included obese patients subjected for panhysterectomy as a treatment. the forty-two patients were allocated into two groups: group (a) subjected to laparoscopic pan-hysterectomy, group (b) subjected to open pan-hysterectomy. results: there was significant difference between the two groups regarding mean operative time, blood loss, analgesic requirements and hospital stay, while no significant difference regarding intra-operative complications. conclusions: laparoscopic hysterectomy in obese patients has emerged as a viable, safe and better alternative to open hysterectomy amongst appropriately trained surgeons. general: endometriosis in the inguinal region is rare. the usual presentation is that of a woman in the reproductive age group. it accounts for . - . % of patients affected by endometriosis. the groin swelling is usually slow growing, painful with exacerbations during menses. the incidence of inguinal endometriosis on the right side is - % as compared to the left. aim: to present our laparoscopic approach for the treatment of the diagnostic dilemma. case presentation: a -year-old woman presented with a palpable mass in the right groin. the swelling was associated with a dull aching pain. the patient was suffering from increasing pain over the swelling during menstruation. she had undergone cesarean section some years ago and the scar had healed by primary intention. mri scan revealed a nodular hypoechoic lesion at the level of the internal inguinal ring with the absence of vascular flow around the lesion. results: since inguinal endometriosis was in the differential diagnosis and it may be associated with pelvic or intraperitoneal endometriosis, a laparoscopic approach was decided. the procedure was successfully completed laparoscopically following the transabdominal preperitoneal approach. the endometriosis was found, after dissecting the internal inguinal ring, firmly adhered to the round ligament. it was excised en bloc with the round ligament. a preperitoneal polypropylene mesh was inserted to protect for future inguinal hernias due to extensive dissection at the level of the internal inguinal ring. no intraperitoneal endometriosis was appreciated. histopathology revealed endometriosis of the round ligament. the patient was uneventfully discharged the next day. on follow up the patient was asymptomatic. conclusions: round ligament endometriosis is a rare entity. it is a disease of specific interest to the physician. it can be confused with an inguinal hernia and thereby pose a diagnostic dilemma. we recommend considering endometriosis in the differential diagnosis of groin swellings in women. the transabdominal preperitoneal approach is feasible and safe in the hands of an advanced laparoscopic surgeon. introduction: sentinel node biopsy is the newest accepted method for surgical staging of early stage endometrial and cervical cancer. aim: to evaluate the role of the technique of indocyanine green (icg) identification of the sentinel lymph nodes in cases of early endometrial cancer. material and method: five patients with early endometrial and cervical cancer were introduced in a prospective study. icg was locally injected during the laparoscopic exploration. novadac pinpoint near to red technology was used. guided biopsies were performed into the marked sentinel nodes and histological results were evaluated. results: sentinel lymph nodes were easily identified by using icg and near-infrared technology. technical details are described. no associated complication was encountered. conclusion: sln mapping using icg in uterine cancers is demonstrated as an effective and safe procedure. laparascopic extraction of an intraperitoneal gossypiboma following c/s and a retroperitoneal gossypiboma following pyeloplasty n. ozlem general surgery department, ahievran university, kirsehir, turkey gossypibomas are forgatten foreign bodies,iatrogenic.their symptoms are different where they are. they extracted with laparotomy in the past but now we can some article mentioned their extraction was made with laparoscopy. case : y o female has abdominal pain after c/s for . years. a gossypiboma was extracted with laparoscopy above umblicus.a superficial surgical site infection existed,drained,subsided. case : yo m had a pyleoplasty operation years ago.a gossypiboma was extracted with retroperitonescopy,no postoperative event. basibuyuk et al reported retroperionescopic extraction of a gossypiboma from single port in first time.althoug every effort taken the incidence of foreign body detected in the body is about . - . %.they are most frequently localized in the intraabdominal cavity followed by tracheobronchial area,pleural cavity,pararenal area,vagina,spinal chord, neck, femur,breast,bladder,pancreas,and they may cause local irritation,and infection.tactile sense is absent in laparoscopy. all radiologic examinations(usg ct pet mri etc) be used to detect.we used usg ct.in the end laparoscopy make the diagnosis and remove gossypibomas in our cases with less postoperative pain and cosmosis. justo et al the computerized tomography (ct) scan is the most useful method for diagnosis; however, sometimes the preoperative diagnosis remains uncertain even after the imaging exam. in that case, laparoscopy arises as a valuable diagnostic tool, as well as a prompt treatment option. concerning gossypiboma, prevention is preferred rather than treatment. notwithstanding, there is no highly reliable prevention system. counting sponges is a method based on staff communication during the surgery with only % sensibility. routine surgical postoperative x-ray (spox) constitutes an early detection system, but the need to incorporate a radiopaque marker and to expose the whole surgical field to maximize its efficacy limits its use. more recently, electronic dispositives based on barcode detection and other technological adjuncts for counting sponges are being developed. none of these prevention systems are reliable when used alone. our education and research clinic was a state hospital before. no surgeon followed above instruction.but now we use all. multiple procedures and surgical teams, long operations and non-elective operations are the evidenced risk factors.c/s operation was learned full opened of ostium of cervix of the patient. urology, japan, nagoya, japan aims: some scoring systems have been suggested to standardize the renal tumor characteristics. among them, renal score is widely used in partial nephrectomy. whereas diameter-axis-polar (dap) score was developed to be more significantly related with postoperative renal function. our study compared dap score with renal score in robotic partial nephrectomy (rpn) outcomes. methods: records of patients who underwent rpn at nagoya daini red cross hospital between april to october were analyzed retrospectively. those include three oncocytomas. accordingly, we calculated the estimated glomerular filtration rate (egfr) just before rpn and month postoperatively in patients. we compared two nephrometry scores with warm ischemic time and change in egfr. results: in our institution, four surgeons performed rpn. according to dap score, patients were high, were middle and were low. according to renal score, were high, were middle and were low. the median warm ischemic time was min ( - ). the median egfr decreased from . ( . - . ) to . ( . - . ) ml/min/ . m . there were no significant differences in warm ischemic time and percentage change in egfr between renal score groups (p = . and . ) but significant differences between dap score groups (p \ . and p \ . ). univariate and multivariate analyses were used to identify factors influencing postoperative renal function. that confirmed that dap score was independent poor predictors of change in egfr after rpn. conclusions: dap score is simpler estimate system than renal score. our study suggested that dap score is a useful scoring system for preoperative evaluation of renal tumor for rpn. further investigation is needed to better understand preoperative dap score. aims: retroperitoneal primary tumors comprise a great variety of neoplasm with different histological typologies, with insidious clinical symptoms and little specificity in most cases. its diagnosis is established through imaging tests and anatomopathological study is needed so complete surgical resection is the treatment of choice. the aim of the video is to demonstrate the safety and efficacy of the minimally invasive approach in patients with retroperitoneal lesions. methods: a -year-old female patient who, in the course of an abdominal pain at the right iliac fossa suspected of possible acute appendicitis, is diagnosed with a right retroperitoneal tumor, compatible with primary neurogenic tumor on a ct. radiographic imaging is a key component of the evaluation of a patient with a retroperitoneal mass, a ct scan is necessary to evaluate the primary site as well as to rule out metastatic disease. after complete biochemical study, nonfunctioning tumor is determined. the study is completed with mri where the lesion is located below the right kidney, in front of the right psoas muscle and lateral to the inferior vena cava, and without contact with these structures. ??it is in intimate contact with the ovarian vein. the complementary tests and iconography of interest of the case are exposed. surgical intervention is proposed with a laparoscopic approach. results: full minimally invasive approach in left lateral decubitus position: trocars-lateral laparoscopic transabdominal approach. laparoscopic liberation of the right colon, kocher maneuver until the inferior vena cava is visualized, identification of a tumor of approximately cm in the right infrarenal region, lateral to the right ureter, which includes the gonadal vessels. resection of the tumor in block with margins previous dissection and clipping of the proximal and distal gonadal vessels with ligasureÒ. the patient presented a successful postoperative recovery, being discharged h after the intervention. definitive result of the specimen: leiomyosarcoma, grade of the fnclcc with negative margin. the laparoscopic approach is a safe and effective technique in the approximation of retroperitoneal tumors, a radical oncological criterion is always needed with correct margins of resection especially in those of uncertain etiology. we started endoscopic thyroidectomy using the lifting method in and have developed single incision endoscopic thyroidectomy (siet) via chest (c-) or axillary incision (a-) by our original retractor since . we created a new approach in . recently, we have applied this method to parathyroid surgery. in this study, we present our method and results in parathyroid surgery with regard to surgical outcome and patients' complaints. method: endoscopic parathyroidectomy of c-siet was performed in patients with hyperparathyroidism (primary , secondary ) in new approach (mean age , male female ). single parathyroid adenoma was diagnosed using ultrasonic device, preoperatively. the patient is placed in a supine position with the neck extended. mm vertical incision is made in anterior chest. flexible endoscope (olympus co. japan) is used through mm trocar detached the retractor. in new approach, the parathyroid and thyroid are exposed through the avascular space between sternal head and clavicular head of sternocleidomastoid muscle. both of the skin and sternal head are lifted up by our original retractor (takasago medical co. japan). parathyroid adenoma behind the thyroid is resected using an ultrasonic scalpel. i would like to present our c-siet procedure. results: no scars in the neck were left in all cases. benign and hemi lateral parathyroid adenoma sized from mm to mm (mean: . mm) were operated. mean operation time is min. in new approach. there was no complication. parathyroid hormone levels decreased in all patients immediately after operation. conclusion: it is a little possible to make recurrent nerve palsy in this approach. new approach is useful to operate and make the working space wider without stress to find out of parathyroid adenoma. our original retractor can be introduced easily in most hospital, because it is not so expensive. most of women satisfied cosmetic results because of hidden scars. objectives: radiofrequency ablation (rfa) is a novel and developing technique for the treatment of parathyroid hyperplasia/adenoma in the context of secondary hyperparathyroidism (hpt) to chronic kidney disease (ckd) and there is little literature on the subject. the purpose of this study is to determine its usefulness by contributing a case carried out in our hospital. methods: we selected a case of secondary htp in a patient of years old with ckd who presented a parathyroid adenoma detected clearly by ultrasound scanning. the patient was dismissed for surgery due to high surgical risk due to his comorbidities. rfa of a right inferior parathiroid adenoma was performed. intact parathyroid hormone (ipth) was measured before arf and min after de procedure, calcium and phosphorus were measured the day after. the treatment was considered effective if ipth levels decreased at least % min after rfa and calcium levels decreased the day after. results: ipth level before rfa was pg/ml. ipth level after min of rfa was pg/ ml, this meant a % reduction (normal values - pg/ml). calcium levels were from . at the baseline to . the day after (normal values . - . mg/dl) and phosphorus from . to . mg/dl (normal values . - . mg/dl). the patient presented dysphonia as a complication that improved with corticosteroid therapy. we are currently waiting for the next analytical controls at , and months after the proceidure. conclusions: rfa of parathiroid adenomas for treating secondary hpt in patients with ckd is feasible in selected patients. this treatment may reduce the morbidity that surgery supposes, it is developed in an outpatient regime avoiding hospital admission and this contributes to a reduction of health costs. however, a longer follow-up is necessary to verify the good results in our case. splenectomy is one of the treatment strategy for advanced portal hypertension due to liver cirrhosis. after splenectomy, thrombocytopenia is dramatically ameliorated, and liver function parameters have also been improved in several clinical settings. however, the mechanism underlying such a phenomenon remains unclear. the aims of the present study was to analyze histological changes of the liver after splenectomy in human, and to speculate the underlying mechanism. subjects and methods: cirrhotic patients with hepatocellular carcinoma (hcc) who had undergone laparoscopic splenectomy prior ( weeks- months) to hepatic resection were analyzed (n = ). non-tumorous liver specimens obtained at hepatectomy were histologically investigated. liver tissues from cirrhotic hcc patients who underwent only hepatectomy were used as controls (n = ). results: after splenectomy, significant leukocytosis, especially increase in monocytes, was observed in addition to thrombocytosis. in the non-cancerous liver tissues, many round-shaped cd -positive macrophages accumulated after splenectomy, while this phenomenon was merely observed in patients without splenectomy. the macrophages were cd ? (m marker) and cd -cd ? , suggesting their anti-fibrotic population. the accumulated macrophages existed around fibrous scar as well as ck ? epcam ? cells spreading out from the ductular reactions (dr). as a result, the number of ki -positive hepatocytes significantly increased after splenectomy. the amount of platelets detected in the liver did not change even after splenectomy. finally, remarked attenuation of the established liver fibrosis was detected after relatively long duration. the accumulated macrophages expressed metalloproteinase (mmp)- and fibroblast growth factor (fgf)- , suggesting these molecules may possibly participate in resolution of established fibrosis and hepatocyte proliferation. conclusion: splenectomy in cirrhotic patients with portal hypertension ameliorate liver fibrosis, and stimulate liver regeneration. the mechanism possibly include hepatic accumulation of anti-fibrotic cd -positive macrophages and stimulation of dr-derived ck ? epcam ? progenitor-like cells. in patients with advanced splenic fibrosis, splenectomy could be a feasible therapeutic modality. the paper tries to establish the role and the opportunity of using laparoscopy in regard with abdominal contusions, as well as its indications or contraindications, combined in a therapeutic algorithm. we analyzed two groups of patients with abdominal contusions divided over two -year periods, - ( patients) and - ( patients) respectively. we have separated the two periods because starting from we have established a strategy for dealing with cases of abdominal contusions where we included diagnostic and / or therapeutic laparoscopy and nonoperative management. the investigation was done by fast echography, ct scan, simple abdominal radiography, peritoneal lavage puncture, and sometimes arteriography. in the second period we determined the diagnostic and therapeutic laparoscopy indications: suspicion of hollow or parenchymal organ injury, or mesentery injury, the presence of hemoperitoneum or fluid in the peritoneal cavity in a stable patient without major hemorrhage, apparent with unique injuries, without immediate vital risk and without other associated severe trauma. we have associated in this last period the nonoperative management for patients with grade and lesions of parenchymal organs that do not have fluid in the peritoneum, or only a very discreet quantity. in the first period, all patients were treated by classic surgery, resulting in unnecessary laparotomies where no visceral lesions were revealed. in the second period, we applied non-operative management to patients out of , patients with grade and splenic injuries, and patients with grade and hepatic lesions. diagnostic laparoscopy was performed in cases, in of them without evidence of lesions, and in other cases of grade lesions no therapeutic action was required. therapeutic laparoscopy was required for one case of splenectomy and one of hepatorrhaphy. diagnostic laparoscopy is useful in abdominal contusions, if certain indications are followed and in selected patients. in our study, with the introduction of modern therapeutic strategies, unnecessary laparotomies were completely avoided, some lesions being even treated by laparoscopy. the new algorithm introduced allowed % of patients to avoid laparotomy. aims: about cases of splenic hamartoma have been described in the literature since it was first described by rokitansky in , it is a rare benign tumor. it is usually a casual finding in laparotomies or autopsies. they are usually asymptomatic, but there are few symptomatic splenic hamartomas and they can be associated with haematological alterations, being in some cases associated with spontaneous splenic rupture and acute abdomen, two thirds of them have multiple tumors. there are no specific data that allow the preoperative diagnosis of this entity, which is performed after the anatomopathological study of the surgical specimen, which must be extracted entirely, this together with the size of the spleen makes the laparoscopic approach difficult. the aim of this video is to demonstrate the surgical technique of a complete laparoscopic approach for this type of lesions, without the need for assistance laparotomies (handport). methods: clinical case: a -year-old man admitted to internal medicine due to fever and left lumbar pain. additional explorations of interest are discussed, including: thrombopenia of probable peripheral origin secondary hypersplenism (fna of bone marrow), ct: splenomegaly with splenic masses, which deform the splenic contour, compatible with atypical hemangiomas, without being able to discard other vascular splenic tumors. results: complete semi-laparoscopic approach, trocars, multilobulated splenomegaly ( x cm.), mechanical vascular section, complete bag extraction after minilaparotomy on the left flank. the patient presented a successful postoperative recovery, being discharged on the th po day. abdominal ultrasound at st week with portal vein thrombosis, which resolves after treatment with heparin. definitive result of the specimen: multiple splenic hamartoma. asymptomatic one year after surgery. the laparoscopic approach is a valid and effective alternative to splenic benign tumor lesions. the size does not contraindicate this type of approach, although the complete extraction of the spleen is recommended for its pathologic study. we recommend eco-doppler control per week, given the risk of portal thrombosis with an existing laparoscopic post-splenectomy. objectives: splenic cysts are a rare entity, currently described between - cases in literature. a female patient's case is hereby presented, giant splenic cyst treated by conservative laparoscopic surgery obtaining good results. method: years old female, without any relevant medical history, examined after abdominal pain on the left hypochondriac region, nausea, postprandial swelling and mass sensation. after exploration the presence of such mass was ratified, the rest of exploration found no relevant findings, no record of previous traumatism nor any other relevant incidence. diagnosis was made through ultrasound and computerized tomography, the existence of a big splenic cyst is confirmed, cm by cm, on the superior section of the spleen, negative results after parasitism test, normal haemogram, coagulation and biochemistry levels. patient was intervened using laparoscopic surgery, performing the deroofing technique on the cyst (two liters of orangey amber serous liquid that was sent for analysis) as well as extirpation of superior wall of the cyst, which was sent to pathological anatomy, a saline solution was used to cleanse the cavity, omentum and drainage were then set in place. results: patient evolved satisfactorily, hospital discharge and drainage withdrawal after h. regular check-ups, after and months, patient presents no symptoms nor recurrence. pathological anatomy confirmed primary splenic cyst and the extracted liquid as cystic. conclusion: splenic cysts are primary ( %) or secondary ( %). diagnosis is performed through imagery tests, cat scan the being standard test used. regarding her treatment there is no clear consensus, due to the fact that up to a few years ago, complete splenectomy was the recommended treatment, techniques with preservation of the spleen are currently being widely recommended through laparoscopy in literature. among the conservative techniques percutaneous aspiration, with or without the injection of a sclerosing agent, partial splenectomy, marsupialisation, cystectomy, decapsulation, unroofing or fenestration can be found. the main issue is recurrence rates. few cases of primary giant splenic cysts treated by laparoscopic decapsulation can be found in literature, this treatment being simple and quick to perform, resenting a recurrence rate lower than other techniques such as aspiration and marsupialization. introduction: technology's progress and its application in the minimally invasive surgery of the thyroid gland offers us new surgical approache's like the transaxillary approach. this new technic still unusual in our environment and has recently begun to be incorporated into our surgical practice. the objective of this case is to explain step by step how to carry out a right transaxillary thyroidectomy and emphasize in the most relevant tips to take into account. also we going to review the main limitations we observed so far. statement of the case: we present the case of a -year-old woman referred for evaluation of a left thyroid nodule without associated symptomatology. the blood test shows normal thyroid profile. cervical ultrasound is performed identifying a . cm single right nodule with welldefined edges and presence of peripheral vascularization . no other nodules are identified. fna of the nodule describes a bethesda iii. after evaluation we decide to perform a left transaxillary thyroidectomy. discussion: surgical treatment of the thyroid gland by transaxillary approach may be indicated in previously selected patients, offering the advantages from minimally invasive techniques (shorter recovery time, shorter incision length, etc.). surely, more evidence and experiencie is required to make a better assessment of minimally invasive approaches in thyroid surgery. surgery, taipi city hospital, yan-ming branch., taipei, taiwan; surgery, taipei city hospital, taipei, taiwan the first endoscopic thyroidectomy was performed in using a cervical approach. since then, various remote-access method, have been developed for thyroid surgery to avoid scarring of the neck. trans axillary approach(taa),bilateral axillo-breast approach(baba),and retroauricular approach(raa) are common in use. the main benefit of these procedure is that there are no visible scar that is one of the drawbacks of conventional kocher's incision. however,these methods require more dissection and longer operation time than conventional thyroidectomy transoral thyroidectomy(tovet) is a new approach and has become popular in recent years, however,most surgeons peformed a single procedure because of the limited patients and the learning periods sine ,more than cases were performed,patients received endoscopic thyroidectomy(et) procedure at our hospital. we compare the surgical procedure of bilateral axillo-breast approach(baba) with transoral vestiblar approach(tovet) in our hospital both performed by one single surgeon .the surgen has expended eaqual amounts of time with these two procedures. the patient seletion process,operation time, operation procedure and approach,learnig experience, consmetic effect,onaologic consideration and surgical outcome were discussed yhroughly. presenting a case of a thyroid metastasis from an ovarian carcinoma, we conducted a review of the literature without finding similar reported cases. case: a -year-old woman consults for progressive asthenia, weight loss and ascites. abdominal ct finds a conglomerate in the pelvis involving the ovaries and peritoneal implants, the largest up to cm. an omental epigastric lesion biopsy and paracentesis is performed resulting in adenocarcinoma and omental metastasis from ovarian neoplasm, associated with ca of . patient starts neoadjuvant therapy with carboplatin-paclitaxel. in image controls there is a favorable response. three months later, intervention was carried out; laparotomy hysterectomy ? double anexectomy ? omentectomy ? appendectomy ? pelvic and paraaortic lymphadenectomy.the anatomopathological study shows a low-differentiated endometrioid carcinoma, omentum infiltration and absence of metastatic lymphatic involvement. while getting the maintenance treatment with bevacizumab the patient presented symptoms of arthritis and hypercalcemia was detected ( . ) with pth . a gammagraphy was performed and an increased uptake area was detected in the lower pole of rtl, suggestive of a parathyroid adenoma. we initially proposed the possibility of performing radiofrequency ablation but in a previous thyroid ultrasound we visualize nodular lesions in rtl compatible with adenoma and a mass in the superior mediastinum that seems to correspond the area of greatest uptake in the gammagraphy so finally the procedure is dismissed and surgery is proposed. during the intervention we found a hard consistency nodule in the inferior pole rtl and lymphadenopathies of hard consistency in right vi area that are sent for intraoperative anatomopathological study with the result of adenocarcinoma metastasis without identifying origin. a total thyroidectomy, parathyroidectomy and central ganglion drainage is performed with the result of a parathyroid adenoma, lymphatic invasion of ovarian-grade latent carcinoma and extensive vascular permeation by carcinoma of the thyroid. the patient maintains oncological treatment with carboplatin-caelix. in the last follow-up, the pth and calcemia remains normal. conclussion: although some cases of neoplasic thyroid involvement associated with struma ovarii have been published, no cases similar to the one described are found, neither in our experience, which is why it is an exceptional case. the aim of the study was to evaluate the effectiveness of the use of embolization of the splenic artery in order to prevent portal bleeding. methods: the study included patients, who had esophageal varices bleeding, which developed as a result of decompensated cirrhosis of the liver of various etiologies of classes b and c according to child-pugh. patients were divided into groups. the main group included ( . %) patients who underwent endoscopic ligating of bleeding varix and in order to prevent recurrence of bleedingembolization of the splenic artery with gianturco coils. the comparison group consisted of ( . %) patients who received only drug therapy. to assess the effectiveness of the treatment, the patient's condition was monitored for months. results: the average age of patients in the comparison group was . ± . years. using only drug therapy, we stopped bleeding in ( . %) patients. in all cases, at the end of treatment, we received an improvement in clinical and laboratory parameters. ( . %) patients died. the duration of treatment was . ± . days. the average age of patients in main group was . ± . years. performing endoscopic ligation of bleeding varices, we stopped bleeding in ( . %) patients. in all cases, at the end of treatment, we received an improvement in clinical and laboratory parameters. ( . %) patients died. the duration of treatment was . ± . days. a statistical analysis of mortality and duration of treatment revealed a significant difference (p \ . ) between the groups in both indicators. after splenic artery embolization in all cases managed to achieve a reduction in blood flow of - %. after months among patients in the comparison group, bleeding relapse occurred in ( . %) cases. in the main group, this indicator was . % ( patients). the indicator in the main group was significantly (p \ . ) different from the same indicator in the comparison group. conclusion: performing embolization of the splenic artery in patients after endoscopic hemostasis of variceal bleeding allows to reduce the pressure in the portal system, which in turn leads to a decrease in the frequency of bleeding recurrences. thoracoscopic esophagectomy for aortoesophageal fistula y. ebihara, t. shichinohe, y. kurashima, s. murakami, surgery ii, hokkaido university, sapporo, japan background: aortoesophageal fistula (aef) is an uncommon but one of highly fatal conditions. there are surgical, endoscopic and interventional radiological treatment options, however, definitive treatment is the surgical intervention. video-assisted thoracoscopic surgery (vats) has been gradually accepted as a substitution for thoracotomy to reduce the invasiveness of the surgery as radical surgery for esophageal cancer. we aimed to evaluate a feasibility of vatsesophagectomy (vats-e) for aef in this study. introduction: achalasia is the most common motility disorder of the esophagus. heller's cardiomyotomy associated with a antireflux technique is the treatment of choice in patients with this disease; however, a small group of patients could present a recurrence of the symptoms being necesary a new surgery, what is an important challenge for most of the surgeons. we report the case of recurrence after a laparoscopic miotomy and dor fundoplication as a paradigm for the appropiate management in this kind of patients. methods: a years old female, who underwent a previous miotomy and a dor fundoplication in due to an achalasia.six years after surgery, the patient showed epigastric pain and dysphagia. the study of the patient was performed with: barium swalow, phmetry, manometry, ct-scan and mri showing a recurrence of her disease.the patient was transfered to our center where she underwent a new surgery.the key points of the new surgery includes the next steps: dissection of the previous adhesions, dissection of the dor's partial fundoplication, avoid dissection of the anterior esophageal wall at the leve lof the hiatus (the area of previous myotomy) in order to avoid perforation of the esophagus, lateral and posterior dissection of the distal esophagus, lateral myotomy at the rigth wall of the esophagus and a toupet's funduplicatury. all of thisis procedures are done under intraoperative endoscopy in order to confirm a good passage to the estmach and to identify a perforationic supervision. results: following theseis steps several patients have been operated in our center with excellent results. in all of these cases, including the patiente presented previously, the symptoms have dissapeared. conclusions: achalasia is a rare motility disorder of the esophagus, being recurrences an important challenge for surgeons. a great proper therapeutic strategy using the different diagnostic exams and the supervison by a group of experts in this kind of entity are the basis in order to obtain good results in these situations. aims: re-do fundoplication is usually performed for recurrent reflux symptoms due to wrap failure or recurrent hiatus hernia. conversely, persistent dysphagia may occur early due to tight wrap/crural repair which should be avoided by good surgical technique. a small group of patients however may suffer progressive dysphagia due to weakening motility (especially in older patients), fibrosis of the wrap or a combination of the two. this video demonstrates the successful treatment of this problem with a laparoscopic conversion from nissen to posterior toupet fundoplication. a year old man underwent an uncomplicated laparoscopic nissen fundoplication in with complete resolution of reflux symptoms. he re-presented years later, still free of reflux but suffering progressive dysphagia and troublesome regurgitation. investigations demonstrated intact wrap and no mechanical obstruction, but confirmed low-amplitude peristalsis. a trial endoscopic dilatation improved symptoms for days before recurrence, suggesting likely wrap fibrosis (which would reduce elasticity and impede passage of food bolus), justifying consideration for a conversion from nissen to toupet. results: this video demonstrates the expected adhesions between fundoplication and inferior surface of left lobe liver, mobilisation and division of the nissen fundoplication, and reconstitution of a posterior toupet fundoplication. the patient made a good recovery and was discharged the following day. three-and six-month follow-up confirmed complete resolution of symptoms with no recurrence of reflux. conclusion: laparoscopic re-do surgery for late-onset progressive dysphagia is a safe and viable option. patients must be thoroughly investigated and carefully selected for an appropriately tailored procedure. they should also be advised of the increased risks associated with re-do surgery. the anatomy can be unpredictably distorted by variable adhesions and this operation should therefore only be performed by laparoscopic surgeons experienced in both primary and re-do fundoplication. methods: i report unusual iatrogenic injury of cervical esophagus that resulted with complete resection post total thyroidectomy for papillary ca of thyroid patient presented days post surgery to our center. the video will show the steps used to treat this unusual complication by neck exploration, laparoscopic trans hiatal esophagectomy with creation of gastric tube with preservation of the right gastroepiploic artery and the neck anastomosis between the cervical esophagus and stomach. were open and minimally invasive esophagectomies. of the patients, were for squamous cell carcinoma, were adenocarcinoma and were of other histological diagnosis such as gastrointestinal stromal tumor and schwannoma. the median length of stay for patients who underwent minimally invasive esophagectomies was days ( to days) while the median length of stay for patients who underwent open esophagectomies was days ( to days). the minimally invasive group had a shorter icu stay of day. for day morbidity, the minimally invasive esophagectomy group had patients who encountered anastomotic leaks, with post operative pneumonia while the open esophagectomy group had patient with anastomotic leak, patient with post operative stricture and patient with delayed gastric emptying. there were mortalities in the minimally invasive group while there were no mortalities in the open group. conclusion: our data show that patients who underwent minimally invasive esophagectomies had a shorter duration of hospitalization with similar perioperative morbidity rates. minimally invasive esophagectomy is a viable surgical option for a select group of patients. aims: there has been an increasing tendency towards minimally invasive surgery for esophageal cancer. our aim was to evaluate the results of the thoracoscopic approach (ta) and compare them with the ones of open approach (oa) at our institution. methods: retrospective review of all patients who underwent esophagectomy due to esophageal cancer (adenocarcinoma or squamous cell) between and were included. patients with siewert iii tumors and those who didn't need a thoracic approach were excluded. results: during the study period were performed esophagectomies, through ta. in . % of these, the abdominal stage was done by laparoscopy. when comparing ta versus oa, there were no statistically significant differences in the baseline characteristics of the two groups (mean age, median body mass index, ecog performance status, asa score, smoking status, diabetes mellitus, pulmonary disease, histologic type, clinical staging and neoadjuvant chemo and radiotherapy). regarding outcomes, there were no significant differences in need of intraoperative transfusion, median intraoperative blood loss, operative time and length of stay. although not significant, in ta group there was a tendency for higher overall morbidity ( . % versus . %, p = . ); major morbidity-ctcae - ( . % versus . %, p = . ); anastomotic leak ( . % versus . %, p = . ) and re-intervention rate ( . % versus %, p = . ). on the other hand, in ta group there was a tendency (although not significant) towards lower rate of respiratory complications ( . % versus . %, p = . ), lower rate of r margins ( . % versus . %, p = . ) and higher median of lymph nodes removed ( versus , p = . ). conclusions: in our series, outcomes of ta were similar to oa, with a tendency towards lower respiratory complications, lower rate of r margins and higher number of lymph nodes removed in ta group. the impact of these findings in survival remains to be seen. the tendency towards higher morbidity may be related to the learning curve, since this were the first cases performed at our center. background: esophagectomy is a surgical procedureburdened by a high morbidity rate. the effect of minimally invasive (mi) approach on elderly patients is still not clear. aim: of this study was to analyze the impact of mi approach on post-operative course according to the patient age. methods: a consecutive series of patients underwent to elective oncological esophagectomy between and . all data were entered into a prospective database. patients submitted to -flield or trans-hiatal esophagectomywere excluded andonly ivor-lewisopen, hybrid or totally minimally invasive esophagectomywere. patients were stratified according to age in groups:group a(= years) patients, group b ([ and \ years) and group c (were = years) .clinical and pathological factors influencing surgical outcome were evaluated. complications were classified according to clavien-dindo (cd). results: as expected outcomes worsened with patients age(cd = b: . % group a, % group b and % group c. p = . ), mortality ( % group a, % group b and . % group c. p = . ) and length of stay ( days group a, days group b and days group c. p = . ).a statistically significant higher incidence of anastomosticleaks was observed among patients submitted to totally mi esophagectomy in group c vs a and b that were respectively , %, % and %. major respiratory complications were not statistically different among these three sub-group. conclusions: old age has a significant impact on outcomes afteresophagectomy. in this subset of patients a mi approachcould also increasepostoperative morbidity. elderly patients should be carefully selected before to be submitted to mi esophagectomy. introduction: esophagectomy is a major surgical procedure with morbidity and mortality related to the patient's condition, stage of the disease, complementary treatments, and surgical experience. minimally invasive esophagectomy (mie) may lead to a reduction in perioperative morbidity and mortality with very good quality of life. material and method: we present the experience of the center of excellence in esophageal surgery regarding totally mie through thoracolaparoscopic modified mckeown three-stage approach followed by esophageal reconstruction by gastric intrathoracic pull-up and cervical esophagogastric anastomosis used for the treatment of thoracic esophageal cancer. results: in the last years, mie was performed initial, in our clinic with extracorporeal preparation of the gastric conduit with reduced lung complications and hospital stay. we introduced the totally minimally invasive esophagectomy with laparoscopic-assisted feeding jejunostomy using a d high definition camera. operative times were: thoracic- min, abdominal- min and cervical- min with a total of min. the augmented d high definition image provided an excellent visual field, that allowed an accurate identification of dissection plans and extensive periesophageal and perigastric lymphadenectomy. the short-term outcomes of the totally minimally invasive esophagectomy procedure were very encouraging with early feeding on jejunostomy and the control of cervical anastomosis was usually performed in the th day postoperative and the patients were discharged in the th day postoperative without any symptomatology. at the first and third-month follow-up was not reported any major complications. the long-term oncological results are being evaluated. conclusions: the totally minimally invasive approach using advanced technology of endoscopic surgery allowed for these patients a simple postoperative evolution, no major complications, and a good recovery after an extensive surgery. the solid experience in open esophageal surgery of the upper gastrointestinal surgeons provides a fast learning curve of complex minimally invasive surgical procedures with reduced perioperative morbidity. long-term follow-up should confirm the results from the literature regarding the survival, which is expected to be for these patients at least equivalent with outcomes after open esophagectomy. introduction: esophageal fistulas, benign or malignant, represent a real challenge for the surgeons and gastroenterologists, regarding the treatment and the outcome. in these cases, endoscopic treatment is the first line approach, being less invasive and sometimes avoiding the need for surgery. this includes clips, stents, glue and even suture. material and method: we have analyzed esophageal fistulas in patients with benign or malignant pathology, diagnosed and treated in the first months of . the management of this complication included a self-expandable esophageal metallic stent. we have evaluated the diagnosis, the surgical intervention, the timing until the development of the leak, the localization and management of the fistula. results: were postoperative leaks and spontaneous esophageal fistulas. the localization was cervical in one case, thoracic in cases and abdominal in cases. for the postoperative fistulas, in patients the treatment included at least one surgical reintervention with lavage and drainage, beside the insertion of an esophageal metallic stent. in the other cases, endoscopic treatment and antibiotic therapy was enough. in cases, the stent migrated needing repositioning. days mortality was %, both patients from postoperative group. conclusions: esophageal fistulas represent a severe complication, usually in patients already immunocompromised. endoscopic management, including self expandable esophageal metallic stent, can be the main approach, by stopping the contamination and by permitting the early per oral feeding. disadvantages include the possibility of migration and the need of removal after - weeks. methods: five hundreds and one patients with esophageal cancer who underwent mie from to at our department were eligible. we considered the risk factors of complications of pneumonia, anastomotic leakage, and hoarseness after surgery, and the risk factors of difficulty of surgery. results: the risk factors of postoperative complications in univariate analysis were more than years old (odds ratio: . , p = . ), more than ii in asa-ps (odds: . , p \ . ), more than g of bleeding (odds: . , p = . ), more than min. of operation time (odds: . , p \ . ), and colon reconstruction (odds: . , p = . ). the one in multivatiate analysis was more than ii in asa-ps (odds: . , p = . ). the risk factors of much bleeding were colon reconstruction (odds: . , p \ . ), and more than of lymph node dissection (odds: . , p = . ). the risk factors of long operation time without cervical lymph node dissection were neo-adjuvant therapy (odds: . , p \ . ), more than of lymph node dissection (odds: . , p = . ), and colon reconstruction (odds: . , p \ . ). the ones with cervical lymph node dissection were more than pstage iii (odds: . , p \ . ) and more than of lymph node dissection (odds: . , p = . ). conclusions: considering those risk factors, we should perform perioperative management more carefully. method: sa -year-old man with a tobacco and alcoholic habit was suspended for years, under treatment for arterial hypertension, who consults for a logical dysphagia of months of evolution. he is diagnosed of stenosing esophageal distal third epidermoid carcinoma txn m . it is decided to place a prosthesis that is effective and subsequent neoadjuvant qt-rt, after weeks of its completion the surgery is performed. results: the surgery is performed in times, initially by laparoscopy. the esophageal hiatus and the greater curvature are dissected preserving the right gastroepiploic, and lymphadenectomy of the celiac trunk with pedicle section of the left gastric. gastric plasty is performed with a section of lesser curvature towards fundus. it is continued by thoracoscopy. a section of the azygos vein is performed, dissection of the esophageal middle and lower third and lymphadenectomy. gastric plasty is promoted, proximal esophagus section and latero-lateral intrathoracic gastro-oesophageal anastomosis. the anatomopathological study reports ypt and pn with / adenopathies, and disease-free surgical margins. he was discharged without complications on the th day and did not require re-entry. conclusions: ivor-lewis endoscopic surgery is safe and meets oncological criteria in selected patients with distal esophageal neoplasia and performed by an experienced esophagogastric unit. background: the rates of thoracoscopic esophagectomy performed in the prone and left lateral decubitus positions are similar in japan. we retrospectively reviewed short term outcomes of thoracoscopic esophagectomy for esophageal cancer performed in the left lateral decubitus position under artificial pneumothorax by co insufflation in a single institution. this study aimed to evaluate the feasibility of applying this procedure. methods: between july and december , patients with esophageal cancer underwent thoracoscopic esophagectomy in the left lateral decubitus position under artificial pneumothorax by co insufflation. the thoracic procedure is performed as follows:the lymph nodes around the right recurrent laryngeal nerve are dissected. on the cranial side, the lymph node dissection is advanced to the level of the inferior thyroid artery. then, the assistant rotates the trachea toward the ventral side, and the lymph nodes around the left recurrent laryngeal nerve are dissected. the middle and inferior mediastinal lymph nodes are dissected including supradiaphragmatic lymph nodes and the dorsal lymph nodes around the thoracic descending aorta. then, the esophagus is transected using an automatic suture device. finally, the tracheal bifurcation area lymph nodes are dissected. we retrospectively analyzed these patients. results: the completion rate of thoracoscopic esophagectomy was . %, and the procedure was converted to thoracotomy in five patients, due to hemorrhage,severe adhesion. the mean intrathoracic operative time, intrathoracic blood loss, and number of dissected mediastinal lymph nodes were . min, . ml, and . , respectively. postoperative complications included pneumonia ( . %), anastomotic leakage ( . %), and recurrent nerve paralysis ( . %). postoperative ( d) mortality was / ( . %) due to ards and nomi, respectively. conclusions: standardization of the procedure for thoracoscopic esophagectomy in the left lateral decubitus position under artificial pneumothorax by co insufflation, with a standardized clinical pathway for perioperative care led to favorable surgical outcomes. introduction: recently thoracoscopic surgery has become widespread even in chest procedure in thoracic esophageal cancer surgery. as an advantage of minimally invasive esophagectomy, it is possible to perform sophisticated procedures due to its magnified visual effects. on the other hand, short-term perioperative safety and oncological safety are still unclear. in cases where abnormal anatomy or comorbidity in the thoracic cavity is observed, it is thought that it is necessary to carry out thoracic surgery which ensures safety while keeping in mind the transition to transthoracic surgery. here, we report on esophageal resection of the thoracic esophageal cancer accompanied by a mm saccular aneurysm inside the aortic arch. patient: a -year-old man visited a nearby doctor with a chief complaint of discomfort during swallowing. upper gastrointestinal endoscopy examined middle cervical esophageal cancer and received referral to our hospital. ct revealed a mm saccular aneurysm inside the descending aorta in contact with the thoracic esophagus. preoperative diagnosis was middle thoracic esophageal cancer; -iic ct bn m stageia (uicc th). we performed thoracoscopic esophagectomy and lymph node dissection as curative surgery. the anterior surface of the aorta was exposed from the lower mediastinum and descended ascendingly, reaching the lower end of the saccular anus at the head level of the lower pulmonary vein. peeling off the esophagus dorsal side along the margin of the saccular sac and performing esophageal resection. conclusion: we reported thoracoscopic esophageal resection for thoracic esophageal cancer with chest descending aortic saccular aneurysm. thoracoscopic surgery, which can fully exploit close magnification effect, seemed to be useful for anatomically disqualified cases. introduction: anastomotic leakage from oesophagojejunal (oj) anastomosis after total gastrectomy is associated with a high morbidity and mortality rate. leakage rates reported vary between ?% and ?% but lack of consensus in management. in the past, it often required surgical intervention or radiologically abscess drainage that will keep patients fasted with external drain for a long duration. recently, variable endoscopic options-oesophageal stents, clips, fibrin glue and endoluminal vacuum therapy had been introduced with variable outcomes. here, we presented a case of oj anastomotic leak management with combination innovative endoluminal and radiologically technique to insert double pig-tailed catheter. aim: to introduce the feasibility of double pig-tailed catheter for drainage and management of oj anastomosis leak. a year old man presented with two months history of dysphagia. upper endoscopy (ogd) showed suspicious cardio-oesophageal lesion. histology biopsy confirmed with adenocarcinoma. ct-scan of thorax, abdomen and pelvic showed irregular thickening at cardiooesophageal junction with regional lymphadenopathy. no distant metastases. he underwent uneventful d total gastrectomy. on th post-operative day, patient had spike fever and newly developed atrial fibrillation. urgent ct-thorax, abdomen and pelvis with oral omnipaque. it showed lower mediastinal gas-containing fluid adjacent to oj anastomosis within the left retrocrural space suspicious for leak. ogd evaluation showed pin-hole oj leak. guidewire inserted via endoscopy into left retrocural space under radiologically guidance. double pig-tail fr cm subsequently inserted via seldinger approach over guide wire. the proximal end of pig-tail pushed into left retrocural space and distal end positioned into efferent jejunal limb with crocodile jaw through endoscope. diluted contrast injected and passed down to efferent limb with minimal leak. outcome: after double pig-tail insertion, patient started on clear feed on st day post-insertion. one week later, he was started on full feed. repeat upper endoscopy and stent removal done two weeks later. contrast injection showed small blind ended sinus tract from anastomosis toward left pleural space without obvious leak. conclusion: radio-endoscopic is a novel minimally invasive technique that allows insertion of double pig-tailed internal drainage to control oj anastomosis leak. it allows early enteral nutritional feeding and avoid external drainage. background: the number of gastric cancer (gc) survivors, especially long-term survivors, is increasing. how best to evaluate the diseasespecific survival (dss) of gc survivors over time is unclear. we aimed to assess changes in the conditional survival of patients with gc after curative intend gastrectomy and the evolution of the impact of well-known risk factors. methods: clinicopathological data from , patients who underwent curative intend resection for gc at four specialized centres (three in china and one in italy) and from the surveillance, epidemiology, and end results (seer) database were retrospectively analysed. changes in the patients' -year conditional disease-specific survival (cs ) were analysed. we used time-dependent cox regression to analyse which variables had long-term effects on dss and devised an accurate, dynamic dss predictive model based on the length of survival. results: the median follow-up time was months, and disease-specific death occurred in , cases ( . %). the dss of the patients after surgery was dynamic, and most of the disease-specific deaths occurred within the first years after surgery. based on -, -, -, -and -year survivorships, the cs of the population increased gradually from % to . %, . %, . %, . %, and . %, respectively. subgroup analysis showed that the cs of patients who had poor prognostic factors initially demonstrated the greatest increase in postoperative survival time (e.g., n b: . %- . %, ? . % vs. n : . %- . %, ? . %). time-dependent cox regression analysis showed the following predictor variables constantly affecting dss: age, the number of examined lymph nodes, t stage, n stage and site (p all \ . , years after gastrectomy). the influence of prognostic factors on dss and cs changed dramatically over time. based on data from several large global centres, we developed an effective model for predicting the dss of gc patients based on the length of survival time. this model can provide personalized long-term follow-up strategies for patients. methods: we retrospectively analyzed clinicopathological data for rgc patients who underwent radical gastrectomy from centers. the prognosis prediction performances of the ajcc th and ajcc th tnm staging systems and the trm staging system for rgc patients were evaluated. web-based prediction models based on independent prognostic factors were developed to predict the survival of the rgc patients. external validation was performed using a cohort of chinese patients. result: the mean number of retrieved lymph nodes was . , and in . % of patients, the number was = . the predictive abilities of the ajcc th and trm staging systems were no better than those of the ajcc th staging system (c-index: ajcc th vs. ajcc th vs. trm, . vs. . vs. . ; p [ . ). within each staging system, the survival of the two adjacent stages was not well discriminated (p [ . ). multivariate analysis showed that age, tumor size, t stage and n stage were independent prognostic factors for overall survival (os), disease-specific survival (dss) and disease-specific survival (dfs). based on the above variables, we developed web-based prediction models, the huang os model, the huang dss model and the huang dfs model, which were superior to the ajcc th staging system in their discriminatory ability (cindex), predictive homogeneity (likelihood ratio chi-square), predictive accuracy (aic, bic), and model stability (time-dependent roc curves). the stratified analysis showed that regardless of whether more or fewer than lymph nodes were retrieved, the predictive performances of the web-based prediction models were still better than those of the other three staging systems. a decision curve analysis showed that the huang model provided better net benefits than the other three staging systems. external validation showed predictable accuracies of . , . and . , respectively, in predicting os, dss and dfs. conclusion: the ajcc tnm staging system and the trm staging system did not enable good distinction among the rgc patients. we have developed and validated visual web-based prediction models that are superior to these staging systems. objective: to perform competing risk analysis and evaluate cancer-and noncancer-specific mortality in patients with gastric cancer after radical surgery. methods: a total of patients from our department (as training set) and a total of patients from the surveillance, epidemiology, and end results (seer) database (as validation set) were enrolled in the study. the cumulative incidence of cancer and noncancer-specific mortality was determined by univariate and multivariate competing risk analysis. results: the five-year cancer-and noncancer-specific cumulative incidence of death (cid) in the training set were . % and . %, respectively, which were significantly lower than that in the validation set ( . % and . %, respectively). multivariable analysis showed that age, tumor site, tumor size and ptnm stage were independent predictors of gastric cancer-specific mortality and overall survival, whereas age was an independent predictor of gastric noncancer-specific mortality. noncancer-specific cid surpassed cancer-specific cid for ptnm stage i patients after approximately years of surgery, but never for stage ii and iii patients. moreover, for stage i patients, the time point when noncancer-specific cid surpassed cancer-specific cid become earlier as age increasing, with only . years after surgery for patients more than years of age. conclusions: age is an independent predictor of gastric cancer-and noncancer specific mortality and overall survival for patients after radical surgery. for patients with stage i gastric cancer, noncancer-specific mortality is a significant competing event, with an increasing impact as age increases. aim: of the study was to analyse the possibility of function preserving gastrectomy based on the sentinel lymph node (sln) concept. methods: during last years in two clinics odessa national medical university we used mapping procedures in the patients with early gastric cancer. there were men and women, age to years, mean age . ± . years. blue dye was injected into quadrants of the submucosal layer surrounding the primary lesion using an endoscopic puncture needle in patients. blue lymphatic vessels and blue-stained lymph nodes can be identified by laparoscopy within min. of the blue dye injection. we used . % indocyanine green in patients, which we injected by intraoperative endoscopy. new technology indocyanine green (icg) fluorescent imaging was used for sln mapping in this patients. results: amany patients, in which we used blue dye for mapping sln, positive sln was in patients, negative-in patients. in all patients distal gastrectomy (dg) was performed with d lymphdissection. from patients with negative sln in patients metastasis in other lymph nodes were detected.among patients in whom we used icg fluorescent mapping positive sln were detected in patients. laparoscopic-assisted distal gastrectomy with d lymph node dissection was performed in these patients. in patients with negative sln partial wedge resection was performed in patients, segmental pylorus preserving gastrectomy was performed in patients. during follow-up period from to months no recurrences or metastasis were detected in these group of patients. qol in this group of patients was much better, than in patients with conventional distal gastrectomy. conclusions: icg fluorescent method is highly effective for detection of sln. in the patients with early gastric cancer function preserving gastrectomy based on sln navigation may be promising strategy to achieve better results. laparoscopic procedure taking advantage of robotic gastrectomy for gastric cancer to prevent pancreatic fistula gastrointestinal surgery and surgical oncology, ehime university, toon-city, japan backgrounds and aims: analysis of japanese national clinical database (ncd) showed that laparoscopic gastrectomy(lg) had rather increased pancreatic fistula (pf) compared with open gastrectomy. on the other hand, last year, multicenter collaborative research result of robotic gastric cancer surgery(rg)was shown that the complications including pf were significantly decreased as compared with lg. in this study, we have employed a new easy to use device in lg to minimize pf during suprapancreatic lymph nodes dissection requiring pancreatic retraction and compared with conventional lg and rg. materials and methods: internal organ retractor (aesculapÒ) to grasp the gastropancreatic fold and the suprapancreatic peritoneum to imitate davinci's forceps was guided with a thread outside the body. patients(jan. * nov. ) were divided into three groups as follows, group lg- (n = ), lg using the standard devices, group lg- (n = ), lg using organ retractor, group rg (n = ). amylase value in drain(d-amylase) and the volume in drainage, intraoperative bleeding, postoperative hospital stay, incidence of cd (] grade iii) were compared among three groups. results: data are indicated as lg- /lg- /rg(mean ± sd), respectively. on the day and third day after surgery, d-amylase were ± / ± / ± and ± / ± , ± (iu/l). d-amylase was significantly lower in lg- and rg group than in lg- the day after surgery. the operation time was significantly longer in rg, ± / ± / ± (min). bleeding volume and hospital stay did not differ among groups. pancreatic fistula (cd ] grade iii)was observed only in lg- group at (%) . discussion: pf(grade]cdiii), which may lead to mortality, occurred in lg- group. a significant elevation of d-amylase on the st postoperative day was prevented in lg- just like rg, which seemed to lead to prevent pf afterwards. the multijoint forceps is known to be an advantage of rg but it cannot be reproduced by lg using a linear forceps. however, another advantage such as vertical grasping and lifting of the gastropancreatic fold at rest could be mimicked by lg using this device, which seemed to enable a safe lymph node dissection and lead to reduce the pancreatic damage. conclusion: this inexpensive and easy to use method taking the advantage of rg seems to reduce surgeon's fatigue and tissue damage(pf). the study presents comparison of perioperative outcome between different surgical approaches for gastric adenocarcinoma (ac). methods: retrospective cohort of patients that underwent gastrectomy for (ac) at rambam hospital during - . patients data was collected based on demographic characteristics, bmi, operating room time (ort), number of lymph nodes (ln), length of hospitalization (loh), and perioperative complications. results: study population included patients after total gastrectomies, of them robotic and partial gastrectomies, of them robotic. age, gender and bmi were similar between patients who underwent any type of procedures. median length of hospitalization (loh) for robotic total gastrectomy was . days and it was significantly shorter than both laparoscopic total gastrectomy (ltg) . days (p = . ) and open total gastrectomy (otg) . days (p \ . ). similar significant differences in (loh) between the groups were observed among patients who underwent partial gastrectomy, but the comparison between robotic and laparoscopic procedures was limited due to small numbers of (lpg). median(ort) was significantly longer among robotic gastrectomies compared to open, the difference was min in total gastrectomy group and min in partial gastrectomy group (p \ . for both differences), but the difference in(ort) between laparoscopic and robotic procedures were smaller and non-significant. the number of dissected (ln) was similar between the procedures in total gasrectomies. in partial gastrectomies, the number of dissected (ln) was even higher among both laparoscopic and robotic gastrectomies compared to open (p \ . ).) conclusions: robotic total and partial gastrectomies for gastric (ac) are associated with oncologically adequate lymphadenectomy and faster patient recovery, but longer ort. objectives: during esophagojejunostomy using a circular stapler after latg, placement of the anvil head via the transabdominal approach proved difficult. the authors report on a method modified for laparoscopy-assisted, esophagojejunostomy performed by placing the pretilted anvil head(orvil) via the transoral approach. methods: between january and november , esophagojejunostomy was performed using orvil in patients after latg. the anesthesiologist introduced the anvil while observing its passage through the pharynx. during the anastomosis, we kept the jejunum fixed in position with a silicone band lig-a-loops, thereby preventing the intestine from slipping off the shaft of the stapler. results: esophagojejunostomy using the orvil was achieved successfully in all patients. no other complications, such as hypopharyngeal perforation and/or esophageal mucosal injury, occurred during passage. the postoperative complications of anastomosis were leakage in two patients and stenosis in patients, in whom mild relief was achieved using a bougie. conclusions: esophagojejunostomy using the orvil is a simple and safe technique. gastrointestinal tract surgery, fukushima medical university, fukushima-shi, japan; surgery, ohara general hospital, fukushima-shi, japan background: juvenile polyposis of the stomach is a very rare disease, and its malignant potential has been reported previously and total gastrectomy has been recommended as a standard treatment. recently, the usefulness of laparoscopic surgery for this case has been reported, however this type of surgery is thought that maintaining the surgical space is difficult because of distended and thickening stomach. case presentation: eight years ago, a -year-old woman who had no family history of gastrointestinal polyposis had been diagnosed with gastric polyposis and polyp-related anemia and received twice endoscopic submucosal dissection to early gastric cancer in another hospital. she had received an annual upper gastrointestinal endoscopy and she had taken iron supplements for anemia caused from the occasional bleeding from the polyps. however, the number of the polyps had increased over time. because she had a loss of appetite, she admitted to our hospital. enhanced computed tomography showed gastric wall thickening and multiple gastric polyps without lymphadenopathy or distant metastasis. colonoscopy showed no specific findings. she was diagnosed as the juvenile polyposis of the stomach, and she received laparoscopic total gastrectomy with roux-en y esophagojejunostomy. in operative findings, although there were the excessive distention and congestion of the stomach, standard laparoscopic surgery could be performed. the resected specimen revealed multiple variously sized polyps throughout the stomach except for lesser curvature and fundus and the histopathological examination revealed that all polyps were hyperplastic polyps without containing cancer. she was discharged on postoperative day . we successfully performed laparoscopic surgery to treat a rare case of juvenile gastric polyposis. introduction: we report a novel technique for combined use of laparo and thoracoscopy for faradvanced adenocarcinoma of esophagogastric junction (aeg). case presentation: a 's years old man presented with far-advanced aeg. an esophagogastroduodenoscopy revealed a type lesion with the entire circumference around esophagogastric junction (egj). contrast radiography revealed a severe stenosis in the egj and wall irregularity from egj to cardia. computed tomography revealed a stenosis of egj, suspected invasion into the left side diaphragm and some lymph nodes metastases at the abdomen. we diagnosed siewert type ii aeg (ct an m , cstage iiia : japanese classification of gastric carcinoma ver. ). surgical technique :the patient was placed in the reverse-trendelenburg position with the left upper body lifted and legs spread, under general anesthesia. the tumor was huge, exposed from the serous membrane and invaded the left crus. first we performed from laparoscopic proximal gastrectomy using five ports. then, three ports were added in the th, th, and th intercostal spaces with the patient in the same body position, and performed thoracoscopic lower esophagectomy under artificial pneumothorax with intrathoracic pressure of - mmhg, which allows the ventilation of both lungs. the lower esophagus was resected under the thoracoscopic view to ensure an adequate margin. following this resection, intrathoracic esophagojejunostomy was performed by using the laparo-and thoracoscopic techniques. the operative time was min, and the blood loss was g. he was discharged on the th day after the operation without any postoperative morbidity. the histopathological diagnosis was pt bn am , p , pstage iv. after adjuvant chemotherapy with capecitabine and oxaliplatin, ramcilumab monotherapy is undertaken now. ct revealed solitary lung metastasis in months after the operation. conclusion: malta for locally advanced aeg invading the surroundings could be performed safely. introduction: despite being the pioneer in laparoscopic surgery, europe did not have similar surgical experience compared to east asia due to decreased exposure to gastric cancer. several studies on minimally invasive gastrectomy for gastric cancer have been conducted in europe. however, some of them did not analyse total gastrectomy as a distinct entity combining both distal and total gastrectomies; moreover, most of them do not provide data on full five-year follow up for each patient. baltic countries stand in between east and west in terms of gastric cancer incidence: incidence rate per , is . in united kingdom, . in lithuania and . in japan. this exposure to gastric cancer provides unique opportunity to investigate the role of laparoscopic gastrectomy. therefore, a case-control study was designed to evaluate laparoscopic (ltg) versus open total gastrectomy (otg), comparing short-term surgical and long-term oncologic outcomes. surgery, jeju national university, school of medicine, jeju, korea; surgery, chosun university, school of medicine, gwangju, korea objective: although mcv (mean corpuscular volume) levels are known to be associated with the prognosis of various diseases, few study investigated mcv as prognostic factor after gastric cancer surgery. the aim of this study is to address the prognostic value of mcv in gastric cancer who underwent curative gastric cancer surgery. methods: patients (june -december ) with stage i, ii, and iii cancer were consecutively included in this study. all patients underwent curative gastric cancer surgery including subtotal gastrectomy or total gastrectomy. overall survival (os), disease-free survival (dfs) and postoperative complications rate were compared between mcv [ group and = group. results: of all patients, the mean mcv was fl (normal range, to fl). the dfs was significantly higher in the high-mcv ([ ) than low-mcv group(= ) (p \ . ) group. there was no significant difference in postoperative complications when compared with clavien-dindo scale. the survival rate of the high mcv group was higher but there was no significant difference. conclusions: mcv may be a predictive factor after gastric cancer surgery. unlike previous studies, patients with low mcv group showed lower dfs. more research is needed on the significance of mcv in variety of disease. methods: and materials. for years we observed cases with gist of stomach and duodenum. seven patients were brought to clinic with the bleeding and two patients were brought to clinic with vomiting and compensate stenosis. in all circumstances we done the ct, mrt and endoscopic examinations of stomach and duodenum with biopsy . in two circumstances we performed endoscopic operation. in one circumstance we successfully take off the gist from the duodenum endoscopically. during the operation we use the endoscopic instruments. in another circumstances,after endoscopic excision the tumor appear the bleeding which was stopped by endoscopic local heamostasis, by putting clipps on the vessels. in circumstances the tumors were in stomach. in circumstances we performed laparoscopic wedge resection the tumors by staplers. in circumstances when the tumor was very big and situated in the fundus of stomach, we performed laparoscopic resection of the fundal part of stomach by using laparoscopic staplers and 'liga sure' sealing. in circumstance we took off the tumor by putting laparoscopic trocars inside the stomach for instruments and for visualization tumor. after excision the tumor and took it of the stomach we sutured the holes in the stomach. we have no mortality after laparoscopic operation. there were no malignisation in all circumstances. we have cases morbidity. in circumstance the bleeding from the stomach that was stopped endoscopically. in circumstance there was wound infection. the aim of the study to decrease the morbidity in the patients with perforated ulcers of the stomach and duodenum. we observed patients with perforated ulcers of stomach and duodenum. women were , men were . average age about years. patients had perforation ulcer of stomach and duodenum. patients had perforations with bleeding. all patients were divided in two groups. the first groups patient operated laporocopically, in the second group patients operated traditionally. results: there were no mortality in the group that operated laparoscopically. in the group that were operated traditionally one patient died after rebleeding. the average stay in hospital in the group that were operated laporoscopically about days. in the groups with traditional operations, were about days. the morbidity in the first group were in cases. pneumonia in cases, suppuration of the troacar points were in cases. in the second group pneumonia were in cases, suppuration of the operation wound were in cases, subdiaphragmatic abscess was in cases. conclusion: laporoscopic operation in during treatment decrease the mortality, morbidity and hospital staying in the patients the perforated ulcer of stomach and duodenum . of the patients of the third group ( . %) were operated about ulcer rebleeding in the hospital, and ( . %)-about the profuse bleeding ulcer. noonr patient had recurrent bleeding. the average treatment time for patients in group was . ± . days. conclusions: the development of hemorrhagic shock in patients with peptic ulcer bleeding significantly increases the risk of rebleeding and mortality. the application of endoscopic hemostasis allows to reduce the risk of rebleeding and mortality compared with conservative antiulcer therapy. surgical treatment can achieve reliable hemostasis, but accompanied by higher mortality and longer duration of hospital treatment. tan tock seng hospital is second largest hospital in singapore. it is affiliated to two medical schools in singapore and it is a training hospital for both undergraduates and postgraduates. minimally invasive surgery for both benign and malignant diseases of upper gastrointestinal tract becomes more and more popular nowadays. in our department, all the residents have to view the step by step instructional videos of mininally invasive surgeries before they can assist in the cases or perform on their own under the supervision of consultant surgeons. the viewing of the instructional videos help them with better understanding of the procedures. the viewing of videos help them with the importance of steps, standardization of steps. with the help of instructional video, they can not only assist better in the surgery but also reduce the learning curve when they start doing the procedure themselves after the graduation from the residency programme. this is the step by step instructional video of laparoscopic repair of perforated duodena ulcer for surgeons-in-training rotated to our department. in general duplication cysts are rare developmental congenital disorders of the gi tract. three morphological criteria should be met in order to confirm the pathological diagnosis: . they should be attached to the stomach's wall and should be the continuation of it, . at least one of the muscle layers of the stomach's wall should be included and .it should have normal gastric mucosa. the treatment is either enucleation or partial gastrectomy. aim: present our minimally invasive approach to a rare prepyloric submucosal cystic lesion causing gastric outlet obstruction. case report: a -year-old female with vomiting, weight loss and in bad general condition was diagnosed after a full work-up (blood tests, endoscopies, eus, ct and mri) with a submucosal cystic tumor. this cyst first was thought to be a duplication cyst. since the patient was young, our intention was to offer the least invasive surgical technique in order to spare gastrectomy and billroth anastomosis. results: the procedure was completed laparoscopically with enucleation of the cyst through a gastrotomy on the anterior wall of the stomach. after the enucleation of the cyst the gastric mucosa was sutured back and then the gastrotomy was closed with continuous sutures. the result of the pathological report confirmed a rare case of a heterotopic pancreatic cystic lesion. the postoperative course of the patient was uneventful and was discharged with instruction for her diet the th postoperative day. the patient months post-operative has no symptoms. conclusion: in such benign conditions and especially in young patients, gastrectomies could be avoided if possible and give their place to less invasive approaches in order to reduce lifelong risks and morbidity. trangastric enucleation of the cyst although a demanding approach is safe and could be considered as a 'gentler' technique with reduced morbidity. background: pancreatoduodenectomy is considered to be very invasive for early superficial duodenal tumors (sdts), which have a lower risk of lymph node metastasis. partial duodenal resection with endoscopic submucosal dissection for sdts is an attractive technique but it is associated with a high risk of complications. the full-thickness resection of the duodenum wall including laparoscopic and endoscopic cooperative surgery has risk of spreading tumor cells and digestive juices into the abdominal cavity. we have developed novel technique for sdts to decrease the risk of exposure to abdominal cavity of tumor cells and digestive juices, called nonexposed duodenum laparoscopic and endoscopic cooperative surgery (neo-dlecs). aim: the aim of this study is to evaluate the feasibility and safety of neo-dlecs for sdts. surgical procedure: the attachment of the transverse mesocolon was freed from the head of the pancreas and retroperitoneal tissues under laparoscopy. the duodenum and the head of the pancreas were mobilized from the retroperitoneum using the kocher maneuver. a standard esd was performed for the sdt using endoscope. the serosa of the esd ulcer bed was reinforced using the laparoscopic hand-sewn suturing technique in the seromuscular layer around the resected area. after completing the procedure, the endoscope was inserted and passed over the resected area to confirm that there was no stenosis or leakage. methods: ten consecutive patients with sdt underwent neo-dlecs in our institute between march and march . the clinicopathological features of the patients and surgical outcomes were prospectively collected and retrospectively analyzed. results: pathological diagnosis was adenocarcinoma for six patients, adenoma for three patients, and neuroendocrine tumor grade for one patient. the median tumor size was ( - ) mm. the median operative time was . ( - ) min. the median blood loss was ( - ) g. there were no conversions to open surgery in this series. intraoperative perforation was found in two cases during the esd procedure. however, all perforations were closed and reinforced using hand-sewn sutures. no postoperative complications were above grade in the clavien-dindo classification system. conclusions: neo-dlecs is safe and feasible and can be an option for surgical sdt resection. aims: wilkie's syndrome is caused by the entrapment of the rd part of the duodenum between the aorta and the superior mesenteric artery (sma). surgery is indicated for chronic cases and failure of conservative management, being reported a laparoscopic duodenojejunostomy as a minimally invasive option. methods: all cases treated by laparoscopic duodenojejunostomy in our centre because of chronic wilkie's syndrome were recorded. results: females and male underwent a laparoscopic duodenojejunostomy, with a mean age of years (range - ). all patients presented abdominal pain, and weight loss was identified in most of them. a reduced aortomesenteric angle measured by ct scan was the key for the diagnosis (mean angle . degrees, range - ). conventional laparoscopic approach was performed in two patients, the other two patients underwent a sils port approach. mean time of surgery was . min (range - ) and length of stay was days (range - ). after a mean follow-up of . months (range - ), patients improved their symptoms. conclusions: surgery is the mainstay in complicated or refractory cases of sma. laparoscopic duodenojejunostomy has the advantages of the laparoscopic approach (including rapid recovery time, reduced post-operative pain and shorter hospital stay) and it is feasible, safe and effective. in mexico in , gastric cancer represented the rd cause of death; it may manifest in a variety of histologic, anatomic, and genetic patterns, which influences the surgical approach. until now gastrectomy with curative intent is the only treatment that offers potential cure in gastric cancer. in recent years, laparoscopy has emerged as an important modality in the surgical management. in multiple trials no significant difference in recurrence, long-term survival and disease-free survival was observed when compared to the standard open gastrectomy. we present the case of a year old man. with a smoking history of pack years, suspended years earlier. he presented unspecific upper gastrointestinal symptoms; an upper endoscopy was made observing a suspicious depressed lesion of cm located in the greater curvature between the body and the antrum, the biopsy resulted in a poorly diferentiated signet-ring cell carcinoma of the stomach. an endoscopic ultrasound and a thoracoabdominal ct scan showed no evidence of enlarged adenopaties or metastatic disease. initially a diagnostic laparoscopy was made, there was no evidence of carcinomatosis, nor free intraperitoneal fluid; so the greater omentum was dissected towards the splenic and hepatic flexure; a d lymph node dissection was performed, and a subtotal gastrectomy with reconstruction of roux en y was done; intraoperative endoscopy was done to identify the lesion, so adequate margins could be obtained. the patient had a good post operative evolution and was discharged home at th day tolerating oral intake. minimally invasive techniques have proved equivalency of oncologic results when compared to the conventional approach; these techniques are becoming the preferred approach in the treatment of well-selected patients with gastric cancer and have a role in definitive staging, curative resection, and lymphadenectomy. appropriate selection of patients and optimal technical approach are paramount for good outcomes. most data of laparoscopic gastrectomy come from eastern countries, where the prevalence is higher; however western experience is growing along with evolution and development in surgical instruments and new technology. wilkie syndrome is a rare cause of high intestinal obstruction, resulting from the compression of the duodenum between the abdominal aorta and the superior mesenteric artery. the main symptoms are nausea and vomiting, weight loss, early satiety, abdominal distension and epigastric pain. historically, the barium study and arteriography were the diagnostic tests used; more recently the angiotac has shown greater sensitivity. the diagnostic criteria are: dilated duodenum, duodenal compression by the superior mesenteric artery and aortomesenteric angle less than degrees. patients with an acute condition usually respond to conservative treatment (decompression, correction of hydroelectrolyte alterations, nutritional support…). however, those with chronic symptoms usually require surgery preferably with laparoscopic approaches of duodenojejunostomy or the strong's procedure. the strong procedure mobilizes the duodenum by dividing the ligament of treitz. once the duodenal-jejunal junction is mobilized, the duodenum is positioned to the right of the superior mesenteric artery and it is preferred because it provides less morbidity due of the maintaining of the integrity of the gastrointestinal tract, but it has a failure rate of %. gastrojejunostomy allows gastric decompression, but does not relieve duodenal compression, so digestive symptoms may persist, leading to the appearance of a blind loop syndrome or recurrent peptic ulcers. on the other hand, the duodenojejunostomy, which according to some series may be the procedure of choice, may obtain a success rate higher than %. we advocate to initiate the surgical approach with the strong procedure and if it fails to perform to a duodenojejunostomy. during this procedure, gastro-esophageal reflux was evaluated and assigned to severe, moderate and slight category. if the reflux was observed slightly up to cervical esophagus, the case was assigned to moderate category. if the reflux was observed intensely up to cervical esophagus, the position was returned to head high position for the safety and the case was assigned to severe category. the anti-reflux surgery was considered in the moderate and severe categories. results: we have performed laparoscopic nissen procedure in cases. the outcome was assessed by reflux test performed on - postoperative day, and the results showed the reflux was disappeared in every cases. median follow-up period of this study was months ( - months) . in cases ( . %) ppi was restarted before months after the anti-reflux surgery. in cases ( . %) ppi was restarted after the anti-reflux surgery during the whole follow-up period of this study. the bmi of the patients had no relationship to the needed restart of ppi. to evaluate the degree of esophagitis objectively before and after the anti-reflux surgery we designed 'the esophagitis score'. in this scoring method, a number from - was assigned according to the degree of esophagitis along with the la classification. the results of the study have shown that the reflux esophagitis was improved obviously after the anti-reflux surgery even in the ppi restarted group (p \ . ). discussion: to extract the gerd patients who really need anti-reflux surgery is important. reflux test is feasible because of its convenience and visual effects for the patients. the results of the laparoscopic nissen fundoplication were good. background: laparoscopic paraesophageal hernia repair with fundoplication has become more and more popular nowadays due to less morbdity and mortality with shorter length of hospital stay. discussion: tan tock seng hospital is the second largest hospital in singapore. it is affiliated to two medical schools in singapore and it is a training hospital for both undergraduates and postgraduates. in our department, all the residents have to view the step by step instructional videos of mininally invasive surgeries before they can assist in the cases or perform on their own under the supervision of consultant surgeons. the viewing of the instructional videos help them understand the procedures better. the videos can also help them recognize the important steps and standardized safe approach. with the help of instructional video, they can not only assist better in the surgery but also reduce the learning curve when they start performing the procedure themselves during their training period. this is the step by step instructional video of laparoscopic paraesophageal hernia repair with fundoplication for surgeons-in-training who are posted to our department. conclusion: the step by step instructional video on laparoscopic paraesophageal hernia repair with fundoplication can help the surgeons in training reduce their learning curve and improve their surgical skills so that they can perform the procedure safely. the human immunodeficiency virus (hiv) is a neurotropic virus. there have been reports of patients with hiv who have esophageal motility problems, sometimes associated with opportunistic infections. the absence of contractility is defined as a major motility disorder according to the chicago v . classification, which is characterized by normal esophagogastric union relaxation and % peristalsis failure. we present the case of a -year-old male patient with a history of acquired immunodeficiency on treatment with efavirenz, emtricitabine and tenofovir. he presented progressive dysphagia, gastroesophageal reflux and pyrosis of months of evolution. physical examination showed no alterations. upper endoscopy is done reporting a normal esophagus and diffuse chronic gastritis. the esophagogram reported inadequate esophageal motility with contrast stasis and a delayed emptying. the esophageal manometry reported an upper esophageal sphincter with high resting pressure. the middle and distal esophagus showed absence of peristalsis with a pan-esophageal pressurization pattern. the lower esophageal sphincter presented normal resting pressure and borderline relaxation ( %). the integrated relaxation pressure was less than mmhg. the diagnostic impression was absence of contractility (chicago classification v . ).medical management was initiated with inhibitors of the proton pump, isosorbide dinitrate and injections of botulinum toxin without success. it was decided to program the patient for a heller myotomy with toupet fundoplication. a trans-surgical endoscopy revealed a complete myotomy with no leakage or obstruction. the patient went home on the second postoperative day tolerating a solid diet.heller myotomy by laparoscopy with partial fundoplication is safe in the treatment of patients with hiv and esophageal motility disorders, reporting a mortality of . %. the effect of endoscopic treatments prior to surgery is controversy aims: epiphrenic diverticulum represents an infrequent entity and it is usually associated with esophageal motility disorders, such as achalasia, distal esophageal spasm, nutcracker esophagus or hypertensive lower esophageal sphincter. nowadays, epiphrenic diverticulectomy, esophageal myotomy and partial fundoplication is the gold standard technique; although it supposes a challenging procedure and it may provoke lots of complications. approach for diverticulectomy usually depends on the distance from the upper border of the diverticulum's neck to gastroesophageal junction, considering that thoracoscopy should be carried out when this distance is more than cm. methods: we presentthecase of a -year-old male patient, with a bodymass index of anda medical history of diabetes, smoking and alcoholism. his symptoms were mainly regurgitation and dysphagia. upper endoscopy showed esophageal dilatation and the presence of a diverticulum with its neck cm over the gastroesophageal junction. ct scan confirmed these findings and manometry showed achalasia. in the video we can see how we perform a laparoscopic diverticulectomy with esophageal myotomy and dor fundoplication. results: patient was discharged home on the second postoperative day with no complication. after more than two years of follow-up, he has not suffered regurgitation, heartburn, dysphagia or chest pain. conclusions: we present a case with an epiphrenic diverticulum secondary to achalasia in which we performed a laparoscopic diverticulectomy, esophageal myotomy and dor fundoplication. some authors suggest that the correction of the underlying motility disorder is the key in the management of these patients and they do not recommend concomitant diverticulectomy for all cases. however, we consider that the complete procedure, adding diverticulectomy, supposes the gold standard and it is feasible to perform for teams which are skilled in esophageal and gastric laparoscopic surgery, despite its high morbidity rates. purpose: a laparoscopic wedge resection for a gastric submucosal tumor closed to gastroesophageal junction or involved to gastroesophageal junction is technically challenging and more aggressive compared with tumors in other sites of the stomach. a gastroesophageal reflux disease would be more prevalent after laparoscopic wedge resection of a gastric submucosal tumor in gastroesophageal junction because of the destruction to low esophageal sphincter. we hypothesized that a prophylactic anti-reflux surgery after this surgery would be less prevalent the gastroesophageal reflux disease (gerd) and more improve the quality of life of the patients. the aim of this study is to analyze our experience with prophylactic anti-reflux surgery after laparoscopic wedge resection for a gastric submucosal tumor of gastroesophageal junction materials and methods: we retrospectively collected data from patients who diagnosed with submucosal tumor of near the gastroesophageal junction underwent laparoscopic wedge resection between january and december . the patients were divided into groups according to operation with prophylactic anti-reflux surgery (group a) and without one (group b). results: there were no difference in the frequency of the preoperative gerd symptoms between the groups, whereas postoperative gerd symptoms and postoperative use of acid suppressive medications were more frequent in the group b (p = . , p = . ). however, there were no differences in the follow-up endoscopic findings in terms of reflux esophagitis and hill's grade between the groups. in group a, postoperative mean low esophageal sphincter (les) pressure was . ± . . the les pressure was dropped until mmhg in the only one patient. however, there was no reflux symptom in this patient. conclusions: the prophylactic anti-reflux surgery after laparoscopic gastric wedge resection of gastroesophageal junction is an effective method of prevent gastroesophageal reflux symptoms. background: the most critical obstacle is a pancreatic leakage(pl). the most cause of pl might be an activation of pancreatic juice by the mixing of pancreatic juice and intestinal fluid because of the anastomosis technique, the difference of anastomosis between pancreatic duct and caliber of jejunum, and the topple of jejunal mucosa. aim: in this study, we devised the new anastomotic method of pancreato-jejunostomy, so called ' pancreatic stent sliding guide' (pssg) method using a pancreatic duct stent. we would like to demonstrate its method and results. (operative procedure) the cases of hybrid laparoscopic pancreatico-duodenectomies (pd) were done by shuriken-shaped umbilicoplasty with pssg. the pancreatic duct stent, which is fit for a diameter of pancreatic duct, is used for the direct puncture without any incineration. the aims of direct puncture are both the avoidance of the enlargement of anastomotic opening and disturbance of blood flow. the contralateral of anastomotic opening is also punctured and the stent is pulled out of the jejunum. the - pds with the needles at both ends is used for anastomotic thread. firstly, the eversion anastomosis of posterior wall is done by sliding the needle on the stent. and then the anastomosis of anterior wall is done by the same way. the stent of contralateral side is cut and the hole is closed. materials and methods: the cased of pancreato-jejunostomy by pssg method were done by february . the average of patient's age was y.o. the disease of patients were pancreatic cancer (n = ), bile duct cancer (n = ), and papilla vater cancer(n = ). the pancreatic leakage by the isgpf were grade : ,a: ,b: ,c: respectively. in the same periods, we underwent the more ten cases of open pd by pssg method. the pl were only one case of grade a and there were none of clinical pl. conclusion: our new device of pancreato-jejunostomy by pssg might be very effective for the decrease of pl from the view point of machanisms of pl even for laparoscopic pd. year old, male patient presented with upper abdominal discomfort and pain, without nausea, vomiting or weight loss. an sub mucosal lesion was found on endoscopy examination in first part of the duodenum. endoscopic ultrasound has showed . cm sub mucosal lesion in first part of duodenum (anterior wall and close to pylorus). cytology examination from the lesion has showed neuroendocrine tumor. computed tomography of abdomen and chest were normal. his blood laboratory examinations were within normal limits. patient underwent da vinci robotic partial gastrectomy with intra corporeal billroth ii gastrojejunostomy. total operating time (ort) was min. three day after operation patient started regular diet and was discharged home on day fife. final pathology report confirmed diagnosis of carcinoid tumor with ki less than %. surg endosc ( ) :s -s p -robotics & new techniques-education integrated education for colorectal disease-a digital solution for a digital age united kingdom aims: surgical plume has problem in poor visibility of the operative field, inclusion of harmful chemical substances, and biological risk. it is desirable that plume should be removed appropriately to minimize these risks. we assessed whether these problems can be solved by using commercialized evacuator semi-quantification of residual chemicals in the abdominal cavity: was performed using industrial smoke tester by aspirating the intra-abdominal plume onto filter papers and digitizing the stains. ( ) detection of dna in the exhausted gas from the evacuator: the hepa filter, which was interposed at the inlet or outlet of the evacuator, was analyzed using pcr method to detect any dna derived from porcine tissues. results: ( ) laparoscopic visualization: judgement score were . vs. . for ec and . vs. . for us (evacuator: on vs. off, both p \ . ), indicating the visualization was significantly better in the use of the evacuator on both devices general surgery, royo villanova hospital general surgery minimally invasive surgery centre, jesús usón minimally invasive surgery centre methods: i report my experience at the american university of beirut medical center for laparoscopic adrenalectomy cases, left adrenalectomy and cases for right adrenalectomy. three out of the series are large adrenal of cm, and all of these were completed laparoscopically.the video will show the steps of this procedure.a large rt. adrenal mass measuring cm, wt. gm was removed laparoscopically using trocar techniques. the lateral position facilitated the exposure and ease of dissection. the mass was removed by extending one of the trocar site with muscle splitting using endocatch mm. results: patient was discharged home days after surgery. the operative time was hour. pathology revealed carcinoma with no involvement of the capsule or vascular invasion patients (male: n = ; female: n = ) underwent minimally invasive adrenalectomy (tp: n = ; rp: n = ) at our institute. mean patient age was . years ( - years). besides comparing operative (intraoperative blood loss, previous abdominal surgeries, conversion rate, operative time, tumor size) and perioperative factors (time of hospitalization, time to oral intake, histology, postoperative complications) in each group, perioperative outcomes of a learning curve (lc)-the first procedures in both groups-was also analyzed in terms of tumor size, significantly larger lesions were removed with tp (tp: . ± . mm vs rp: . ± . mm; p = . ). the number of asa (american society of anesthesiologists) ii patients were significantly higher in the tp group while there were significantly more asa iii patients in the rp group conversions ) showed no significant difference. the analysis of lc showed a significant difference in previous abdominal surgeries min vs rp: . ± . min; p = . ] all favoring the tp approach. conclusion: both methods proved to be feasible and safe in terms of minimally invasive adrenalectomy. based on our own experience the tp approach resulted in improved operative time and conversion rates to demonstrate the safety and efficacy of the laparoscopic approach in the treatment of large splenomegaly. currently, this approach is recognized as the one of choice in benign splenic pathology, being controversial in the face of a massive splenomegaly or neoplastic pathology. material and method: clinical case: a -year-old man followed in the dept. of internal medicine for a hepatosplenomegaly of probable lymphoproliferative origin. additional explorations of interest are provided. result: intervention: complete laparoscopic approach, right lateral partial decubitus, massive splenomegaly, ? cm, splenuncle of - cm that is resected, section of short vessels, dissection of the splenic hilum, vascular section with endogias, splenectomy with full extraction in a pocket through reduced laparotomy in the left flank for anatomopathological study the aim of this video is to demonstrate the safety and efficacy of the laparoscopic approach in the treatment of large splenomegaly. currently, this approach is recognized as the one of choice in benign splenic pathology, being controversial in the case of a massive splenomegaly or neoplastic pathology it can transform into adenocarcinoma. patients and methods: between and we performed laparoscopic nissen fundoplication (lars) in cases of gerd. in cases of gerd patients be was proved by endoscopy and histological examination. the demeester score was higher ( . versus . , p \ . ), and bile re?ux was measured more frequently among the be patients on the other hand during the . years long endoscopic follow up early barrett carcinoma developed in patients, . months after the lars. both patients underwent a limited surgical resection of the distal esophagus and esophagogastric junction, regional lymphadenectomy, and reconstruction by interposition of an isoperistaltic jejunal segment. there were no complication. histological examination was shown pt n stage disease in both cases. oncological follow up was months long ( . y) and both patients are still disease free. conclusions: although lars can affect regression in a part of be patients, progression to adenenocarcinoma can also occur. endoscopic surveillance is important in the case of be to recognize early cancer, to perform limited surgical resection with low morbidity and long overall-and disease free survival gastric cancer development a nomogram for predicting the conditional probability of survival after d lymphadenectomy for gastric cancer this study aimed to devise a nomogram to predict the conditional probability of cancer-specific survival (cpcs) in gastric cancer (gc) patients after gastrectomy with d lymphadenectomy. methods: clinicopathological data for , gc patients who underwent d lymphadenectomy in a large-volume eastern institution (the training cohort) were analysed. cancer-specific survival (css) was predicted using cox regression models. a conditional survival nomogram was constructed to predict cpcs at and years post-gastrectomy. two external validations were performed using a cohort of , chinese patients and a cohort of italian patients. results: in the training cohort, the -year cpcs was . % immediately post-gastrectomy and increased to . %, . %, . % and . % at , , and years post-gastrectomy, respectively. multivariate cox regression analyses showed that age; tumour site, size and invasion depth; numbers of examined and metastatic lymph nodes; and surgical margins were independent prognostic factors of cancer-specific survival (all p \ . ) and formed the nomogram predictor variables. internal validation showed that the conditional nomogram exhibited good discrimination ability at and years post-gastrectomy (concordance index, . and . , respectively) gastric cancer does non-compliance in lymph node dissection affect oncological efficacy in gastric cancer patients undergoing radical gastrectomy? univariate and multivariate analyses revealed that non-compliance was an independent risk factor for os. logistic regression analysis demonstrated that the extent of gastrectomy, primary tumour site, history of intraperitoneal surgery, bmi and open gastrectomy were independent preoperative predictive factors for non-compliance. cox analysis demonstrated that age, pt, pn, and the extent of gastrectomy independently affected os in patients with noncomplaint lymphadenectomy. however, os was significantly better in the compliant group than in the non-compliant group regardless of the recommendation for chemotherapy. stratified analysis demonstrated that os was significantly better in chemotherapy patients than in patients without chemotherapy and stage ii patients (pt n /n m and pt n m ) in whom chemotherapy was not recommended. conclusion: non-compliance is an independent risk factor after radical gastrectomy for gc we prospectively collected and retrospectively analysed the medical records of patients with proximal gc who underwent lspsd. the data were split / , with one group used for model development and the other for validation testing. results: of the patients enrolled in this study, ( . %) required laparoscopic haemostasis treatment. a multivariate analysis determined the following preoperative adverse risk factors for the model group: gender, preoperative n stage, and terminal branches of the splenic artery (spa), and we developed a scoring system based on these findings. each of these factors contributed point to the risk score. the intraoperative laparoscopy hemostasis rates were . , . , . , and . % for the low-, intermediate-, high-, and extremely high-risk categories, respectively. there were statistically significant differences among groups (p \ . ). with the increase in risk, both blood loss volume (blv) and operative time (min) of lspsd increased significantly (p \ . ).the area under the receiver operating characteristic curve for the score of intraoperative laparoscopic haemostasis was . . the observed and predicted incidence rates for intraoperative laparoscopic haemostasis were parallel in the validation set. conclusions: this simple we compared the survival of src patients with that of tubular adenocarcinoma patients according to bmi. results: the -year survival of src was significantly worse than that of wmd (p \ . ) but superior to that of pd (p \ . ). bmi-stratified analysis showed that in the high-bmi group, the prognosis of src was similar to that of wmd (p [ . ) and better than that of pd (p \ . ). in normal-bmi patients, src had a worse prognosis than wmd (p \ . ) but a more favorable prognosis than pd (p \ . ). src among low-bmi patients displayed much poorer survival than did both wmd (p \ . ) and pd (p = . ). multivariate analysis indicated that the risk of death was lowest for src patients with a high bmi and highest for src patients with a low bmi baseline characteristics were compared in a -patient rspshl cohort and a -patient lspshl cohort. one-to-four propensity score matching was performed to determine between-group differences. result: in total, patients were matched, including patients who underwent rspshl and who underwent lspshl. no significant differences in baseline characteristics were observed between these groups after matching. significant differences in total operative time, estimated blood loss (ebl), splenic hilar blood loss (shbl), splenic hilar dissection time (shdt), and splenic trunk dissection time were detected between these groups (all p \ . ). furthermore, no significant differences were evident between rspshl and lspshl in the overall noncompliance rate of lymph node (ln) dissection ( the highest body temperature within week after operation was used to establish diagnostic thresholds for high body temperature and low body temperature, which was obtained by x-tile software. the study used cox regression to analyze the influence of high body temperature on -year dfs. results: a total of patients were included in the analysis. the diagnostic threshold for high body temperature was defined as °c; patients with a high postoperative body temperature were allocated to the high temperature group (htg), while another patients were allocated to the low temperature group (ltg) cao department of gastric surgery, fujian medical university union hospital, fuzhou, china background: laparoscopic surgery for remnant gastric cancer third step: baring of the right side of the esophagus. fourth step: exposure of left gastroepiploic vessels and lns dissection in the splenic hilar area. fifth step: baring of the left side of the esophagus. the above procedure was performed for rgc patients with stage ct - an /? disease. results: there was no conversion to open surgery. mean operation time was . ± . min, mean blood loss was . ± . ml, and mean times to first flatus p -upper gi-gastric cancer a novel prognosis prediction model after gastrectomy for remnant gastric cancer: development and validation using international multicenter databases fuzhou, china; department of gastrointestinal surgery the model calibration was accurate in predicting -year survival. dca showed that the model has a greater benefit. the results were also confirmed by bootstrap internal validation. in external validation, c-statistics and dca showed good prognostic performance in patient datasets from participating institutions. moreover, we verified reliability of the model in an analysis of patients with different eln counts p -upper gi-gastric cancer a novel abdominal negative pressure lavage-drainage system for anastomotic leakage after r resection for gastric cancer while risk of gastric cancer for ppi users was higher than non-ppi users when duration between - year, = year, = year and = year. the risk of gastric cancer when duration = year(rr = . )and duration = year(rr = . )are higher than risk of gastric cancer when duration between - year (rr = . ). according to location subgroups meta-analysis,risk of non-cardiac gastric cancer for ppi users higher than non-ppi users conclusion: based on a systematic review with meta-analysis, we found the correlation between long-term use of ppi and the risk of gastric cancer and long-term use of ppi may increase the risk of non-cardiac gastric cancer when duration = year p -upper gi-gastric cancer age-adjusted charlson comorbidity index (acci) is a significant factor for predicting survival results: there were patients included in the analysis. the high-acci and low-acci groups had significant differences in preoperative abdominal surgery history, asa grade, tumor size, tumor stage, histologic type, age and comorbidity (all p \ . ). the incidence of postoperative complications was . % in the high-acci group and was significantly higher than that in the low-acci group (p = . ). the overall survival rate (os) and cancer-specific survival (css) rate in the low-acci group were both higher than those in the high-acci group (p \ . ). univariate and multivariate analyses showed that the acci was an independent risk factor for os and css (p \ . ). furthermore, a combination of the tnm staging system and acci showed a trend toward higher prognostic value and higher auc for os and css than the tnm staging system alone (p \ . ). conclusions: the acci was an we aimed to investigate the clinicopathological features and prognosis of patients with mgc and the impact of postoperative adjuvant chemotherapy on long-term survival. methods: the clinical and pathological data of patients diagnosed with gastric adenocarcinoma and undergoing radical gastrectomy from stratified analysis showed that, in advanced gastric cancer (agc), the -year os rates of mgc without adjuvant chemotherapy and sgc without adjuvant chemotherapy were . % and . %, respectively, with a statistically significant difference (p = . ). the -year os rates of advanced mgc after adjuvant chemotherapy and of advanced sgc after adjuvant chemotherapy were . % and . %, respectively, and the difference was not statistically significant (p = . ). the -year os rate of advanced mgc after adjuvant chemotherapy was significantly higher than that of patients without adjuvant chemotherapy ( . % vs. . %, p = . ). conclusions: mgc is a poor prognostic factor after radical gastrectomy for gastric cancer background: whether the tumor-node-metastasis (tnm) staging system is suitable for patients with node-negative gc is still controversial. the modified staging system established by rpa showed good prognostic performance in a variety of cancers. the application of rpa has not been reported in the prognostic prediction of gc. methods: node-negative gc patients who underwent radical resection at fujian medical university union hospital (n = ) and sun yat-sen university cancer center (n = ) with an at least -year follow-up information were selected as the training set. rpa was used to develop a modified staging system. patients from the surveillance, epidemiology, and end results databases (n = ) were selected as the external validation set. results: the -year overall survival (os) rates of patients with th ajcc-tnm stage ia-iiia in the training set were ia %, ib %, iia %, iib % and iiia %. multivariate analysis (mva) showed that larger tumor size, older age, and deeper depth of invasion were independent risk factors for os in patients with node-negative gc (all p \ . ). patients were reclassified into rpa i, rpa ii, rpa iii, and rpa iv stage based on rpa, the -year os rates were %, %, %, and %, respectively, with significantly difference (p \ . ). two-step mva showed that the rpa staging system was an independent predictor for os (p \ . ) were retrospectively collected. patients were classified into two groups according to bmi of \ kg/m ( patients; high bmi group) and = kg/ m ( patients; low bmi group). for these patients, clinicopathological variables were analyzed using propensity score matching to mitigate the selection bias: sex, age, asa physical states, clinical stage, laparoscopy-assisted total gastrectomy (latg) or totally laparoscopic total gastrectomy (tltg), d lymph node dissection, combined resection of other organs, method of anastomosis, jejunal pouch reconstruction. the surgical results and postoperative outcomes were compared and examined between the two groups. results: a total of patients were matched for the analysis. contrary to our expectations, there were no differences in the surgical results about operative time and estimated blood loss (low bmi . ± . min, high bmi . ± . min; p = . , low bmi . ± . g, high bmi . ± . g; p = . , respectively). furthermore, there was no significant difference in postoperative outcome of complication (clavian-dindo [ iiia) and the length of postoperative hospital stays (low bmi cases, high bmi cases baiocchi general surgery, university of brescia-spedali civili, brescia, italy background and aim: recently indocyanine green (icg) was introduced in clinical practice as a fluorescent tracer. the use of icg for sentinel lymph node (ln) mapping was investigated in lots of fields such as breast methods: we conduced a single center prospective trial. we included patients with gastric cancer candidate to surgery. icg was injected intraoperative or the day before surgery, via submucosal or subserosal. total or subtotal gastrectomy was performed open, laparoscopic or video-assisted access. during gastric cancer standard lymphadenectomy we studied lymphatic flow and ln bright in vivo and ex vivo japan introduction: in japan, the number of elderly patients with gastric cancer has been increasing in correlation with the increase in average age of the population. the aim of this study is to assess the safety and efficacy of laparoscopic gastrectomy for cancer in elderly patients compared with the short-term outcome in the nonelderly. method: we reviewed patients who underwent laparoscopic gastrectomy (dital gastrectomy,proximal gastrectomy,total gastrectomy)between ).the incidence of advanced cancer(stageiior more)was higher in elderly patients there were no significant differences in the operating time,blood loss and postoperative hospital stay. there were no significant differences in the incidence of postoperative morbidity. conclusion: in elderly patients, there was a tendency of reduction surgery being selected according to individual condition, but there was no significant difference in the short-term outcome.hence,we conclude that laparoscopic gastrectomy is indicated even in elderly patients. p -upper gi-gastric cancer improved technique of vacuum therapy and carried out ltg . a patient factor (the gender, the age and bmi), an operation factor (operation time, the bleeding amount, lymph node dissection and conjurer), a coincidence related complication (clavien dindo classification, sutural insufficiency of grade more than , anastomotic stricture, anastomotic region bleeding and reflux esophagitis) and the post-operatively length of stay were considered . result: cs crowd met cases ( . %) and ls cluster ( . %) cases. years old of age medians ( - ), men and women were examples ( . %), examples ( . %) and bmi median . ( . - . ) by a patient factor, and a significant difference didn't admit by two groups for days, the post-operatively average length of stay was days by ls group by cs group. conclusion: operation time was short for a coincidence by linear stapler more than a coincidence by circular stapler in comparison of an esophagoenterostomy way in ltg on the day before the operation, we endoscopically clipped several points located cm proximal to the tumor edge to cover about half of the tumor. after lymph node dissection, we incised the stomach with an endoscopic linear stapling device,including the previously placed clips. reconstruction was performed in all patients who underwent billroth i or roux-en-y procedures. result: no complications were observed during pre-operative endoscopic clipping or intraoperatively p -upper gi-gastric cancer small intestinal tumors after laparoscopic surgery in our hospital small intestinal tumors are rarely observed, accounting for about - % (malignant cases: - %) of all gastrointestinal tumors. therefore, occasionally, their diagnoses can be difficult. however, recently, capsule and balloon endoscopes have been widely employed were examined regarding patient backgrounds, diagnostic methods, pathological findings, postoperative courses, and prognoses. results: the subjects consisted of males and females, with a mean age of years. their chief complaints were black stools the median distance from the treitz ligament or bauhin valve was cm ( - ) postoperative complications were abdominal abscess ( cases; . %) and surgical site infection (ssi), hemorrhage, and paralytic ileus ( case each; . %). pathological diagnoses were lymphoma metastatic small intestinal tumor ( cases; . %), and granuloma, lipoma, peutz-jeghers polyp, clear cell sarcoma, malignant mesothelioma, and ectopic pancreas most patients were diagnosed in bleeding, complicated by anemia and black stools. however, as most tumors were relatively close to the treitz ligament and bauhin valve, almost a half could be diagnosed with a small intestine endoscope before surgery patients were classified as popf and no-popf according to their grade b or c popf status. popf was diagnosed according to international study group of pancreatic fistula (isgpf) criteria or clinical findings. patient characteristics, intraoperative parameters, electrosurgical device type, pathological findings, and early postoperative outcomes were compared. electrosurgical devices were classified asthunderbeat (tb) or laparosonic coagulating shears (lcs) based on energy sources. results: eighteen patients developed grade b or c popf. among them, ( . %) and ( . %) were diagnosed with popf according to isgpf criteria and clinical findings ), operation time (p = . ) and electrosurgical device type (p = . ) were significant risk factors for popf following lag ) and tb device (or, . ; % ci ) were independent risk factors for popf following lag. conclusions: operation time and tb use significantly affect the risk of popf and should be considered in future clinical studies. p -upper gi-gastric cancer feasibility and nutritional benefits of double flap with no-knife stapler reconstruction after laparoscopic proximal gastrectomy for gastric cancer were analyzed. receiver operating characteristic curves were generated, and by calculating the areas under the curve(auc) and the c-index, the discriminative ability of crps during different periods were compared, including pre-crp, postoperative days , , and postoperative maximum crp (post-crp max ). a decision curve analysis was performed to evaluate the clinical utility. result: ultimately, patients were included this study and the median follow-up time was ( - ) months. for postoperative recurrence, the auc and c-index of pre-crp were . and . , respectively, significantly higher than the other crps, all p \ . . among = ''''''the = '''' post-crps = '''' post-crp = '''' sub = similar findings were observed for overall survival. conclusion: both pre-crp and post-crp max , cheap and easily obtained, are independent predictors of recurrence for gc. act significantly prolonged the rfs for stage ii/iii gc patients with high-prep p -upper gi-gastric cancer robot-assisted gastroduodenal surgery: a single center experience robot-assisted gastroduodenal surgery (ras) was introduced to overcome the technical limitations of conventional laparoscopy. it provides a d-amplified view to the surgeons and an increased ability to control the operative field by manipulating optics, as well as enhanced mobility and precision of instruments. the aim of the present study is to evaluate the main outcome of a single center experience in gastroduodenal robotic surgery. materials and methods: we report a case series of patients who underwent robot-assisted gastroduodenal surgery at sanchinarro university hospital between conclusions: robot-assisted gastroduodenal surgery is a safe and feasible technique in experienced centers with advanced robotic skills. in the literature, there are only few reports of robotic assisted gastroduodenal resection. further studies are necessary to better confirme our results. p -upper gi-gastric cancer atypical methods: retrospective review of ogd reports before and after the introduction of the new guidelines. inclusion criteria: all elective ogds. exclusion criteria: emergency ogds and elective therapeutic ogds. data recorded: patient demographics, endoscopist, indication, number of photos, anatomical site photographed, pathology identified and whether pathology photographed or not. results: ogds reviewed, before the guidelines (group ) and afterwards (group ). the most common indication was reflux ( %) in group and anaemia ( %) in group clinical utility of systematic pre treatment staging laparoscopic exploration methods: all locally advanced gastric adenocarcinoma managed in surgical oncological unit between st january and th november were prospectively enrolled in the study. in the absence of emergency surgery or preoperative contraindications, all patients with curative intent underwent either preoperative chemotherapy followed by surgical exploration in the intent of curative gastrectomy (g)or systematic pretreatment laparoscopic exploration (l) benkabbou surgical department the patient background (age, gender, bmi) and c-stage of the preoperative factor were matched using propensity score matching method, and the surgical results were compared and examined. results: thirty rg groups matched rag cases. the operation time (rag / lg) was significantly longer in the rag group as . ± . min / . ± . min (p \ . ). amount of blood loss was not significantly different each other; ml / ml (p = . ). pathologically t a case was involved in cases in rag and cases in lg. the extent of lymph node dissection (d ? / d ) was / cases in both groups conclusions: rag in our clinical experiences can be safely introduced and short-term results are comparable to those of lg. verification of superiority of robotic surgery including long-term results seems to influence the future of robotic surgery conclusions: totally laparoscopic gastrectomy is feasible method in terms of surgical outcomes. furthermore, totally laparoscopic total gastrectomy is not technically difficult in advanced gastric cancer such as early gastric cancer and safety method. key words: gastrectomy, reconstruction, laparoscopic surgery, stomach neoplasm aims: meckel's diverticulum (md) is one of the most common congenital anomalies of the small intestine caused by an obliteration defect of omphalomesenteric duct. the objective of this study was to review surgical treatment and clinical outcomes of md, and evaluate the safety and feasibility of minimal invasive surgery (mis) in md. methods: we performed a retrospective analysis of medical record for patients who underwent meckel's diverticulectomy at six hallym-university-affiliated hospitals between d), as well as the average of drainage stay. patients who underwent laparoscopic repair required significantly less parenteral analgesics than the open group.the mean postoperative stay was significantly shorter for laparoscopic group (mean, . d) than the open one.morbidity of medical and surgical complication was higher in open groups ( vs ). the most common complication in both groups was medical complication.more case of pneumonia was occurred in open groups compared to laparoscopic groups methods: a retrospective study using our prospective database was designed to analyse all the resected md in our centre. epidemiological data, clinical setting, diagnostic test and histological results were reported. results: md was resected in patients, males and females, with a mean age of . years ( . - . ). in cases, a laparoscopic approach was chosen. eighty-seven percent of the patients had a presurgical imaging test (ultrasounds, ct-scan or meckel's scan) background: perforated peptic ulcer (ppu) is a substantial health problem with significant postoperative morbidity up to % and mortality up to % worldwide. aims: this study aimed to estimate the sensitivity scoring systems for prognosis morbidity of patients operated for ppu with diffuse peritonitis. methods: a total of patients were underwent emergency repair for ppu with diffuse peritonitis in pirogov russian national research medical university's surgical clinics during - years. different scoring systems used to predict outcome in ppu patients were identified: boey score, peptic ulcer perforation (pulp) score, asa, mannheim peritonitis index (mpi), world society of emergency surgery sepsis severity score (wses score). to quantify the strength of the concatenation of prognostic score and morbidity we use odds ratio (or) with % ci ), respectively. pulp score and asa score have good prognostic value in relation to morbidity, but less than boey, mpi and wses sss. patients with pulp [ had or with % ci of p -upper gi-gastroduodenal diseases gastrostomy tube placed by laparoscopy as a new therapeutic option for continuous intestinal infusion treatment with levodopa/ carbidopa we present year outcomes of our initial consecutive patient cohort. methods: patients were identified in a prospectively maintained irb-approved database ( - ). post-operative eckardt scores and a -point validated system questionnaires were obtained via telephone interviews one patient required reoperation for failed myotomy. the mean eckardt score at years was . (± . ), with all fourteen patients having an eckardt score \ . all patients reported significant improvement in their quality of life. classic gerd symptoms (heartburn and regurgitation) were present in ( . %) patients. proton-pump inhibitors are being used by % of with patients with excellent symptom control. seven patients returned for a repeat egd (median . yrs) with patients having normal anatomy and having la grade a esophagitis ( patient on ppi). barrett's esophagus was not detected. conclusion: long-term results from our early experience with lhm are excellent and durable with only one patient requiring re-intervention in years until recently the esophagectomy was the only choice in treatment of patients with end-stage achalasia. developing of minimally invasive techniques such as a laparoscopic heller miotomy and peroral endoscopic myotomy (poem) allowed to use them as a treatment options. aim: to present an experience of treatment of patents with end-stage cardiac achalasia. materials and methods: since . till the laparoscopic heller myotomy was performed in , and esophagectomies were performed in patients with failed previously myotomy made in other clinics. gastric tube was used to replace the esophagus in patients underwent esophagectomy after skeletization of crura posteriorly to esophagus, two separated rectangular patches of parietene progrip mesh (covidien) measuring x . - cm were attached to the posterior surfaces of the crura. the patches were fixated themselves due to special hooks. than continious twodirections suture was placed through both crura along with the patches using self-gripping v-loc - suture (covidien). the same suture was used for construction of nissen fundoplication wrap . cm long. aditional anchoring stich through the wrap and esophageal wall was placed using ti-cron - suture (covidien). d laparoscopy was used while suturing using richard wolf epic system. results: all the procedures were performed successfully. there were no cases of bleeding from the suturing points either from the crura and the fundus wall. there were no crural dehiscence while suturing, even if the distance between crura was more than cm. the mean duration of suturing facilitated by d laparoscopy was min (range, - min) for crural repair, and min (range, - min) for fundoplication. there were no excessive postoperative pain in all the patients. there were no disphagia month postop in every patient. conclusions: . the new technique of posterior buttress of crural repair using small patches of parietene progrip mesh and v-loc suture showed feasibility and safety. . the use of d in such case most commonly manifested symptoms are cough, sore throat, hoarseness, dysphonia, globus and only % patients with lpr have typical gerd symptoms. also ppi therapy are less effective in patients with lpr in comparison with patients which have typical features of gerd. purpose: to compare the outcomes between surgical treatment and conservative therapy in patients with laryngopharyngeal reflux. materials and methods: for the period chesarev faculty surgery # , federal state autonomous educational institution of higher education i.m. sechen, moscow, russia p -upper gi-reflux-achalasia a case of a primary parahiatal hernia associated with a type i hiatal hernia emergency county hospital parahiatal hernia is a rare disease that occurs when an abdominal organ protrudes through an opening adjacent to an anatomically intact esophageal hiatus. the herniated organ is usually the stomach, although cases of omental and colonic herniation exist we report the case of a -year-old woman which accused epigastric pain, starting years prior, pseudo-angina, heartburn and bloating. based on imagistic findings the patient was diagnosed with a parahiatal hernia and an associated type i hiatal hernia. patient underwent surgery and a cm diameter defect in the diaphragm lateral to the left crus was discovered, through which - % of the stomach had herniated. the hiatal orifice was slightly enlarged but anatomically intact, with an associated small sliding hiatal hernia. we performed closure of the defect, hiatoplasty and a floppy-nissen fundoplication pneumatic dilation with mm balloon was performed under general anesthesia. radiological contrast control and endoscopy reevaluation revealed a perforation just above the squamo-columnar junction. a minimally invasive approach was decided. an fully covered esophageal stent was inserted. radiological control after days reveals left pleurisy and migration of the stent. the same day was performed an endoscopic repositioning of the stent with clip fixation. left pleural puncture was performed and clear fluid was extracted. the condition of the patient got worse and she was transferred on icu ( . ). we performed left pleurostomy and initial exploratory laparoscopy-no intraperitoneal lesions. due to difficult transhiatal access to the inferior mediastinum the surgery was converted to open-perisophageal mediastinal abscess was found, evacuated and drainage and jejunostomy were performed. after a week, the patient presented progressive altered condition, febrile syndrome. thoraco-abdominal ct-scan showed left pleural effusions. left pleurostomy was performed, with extraction of fetid fluid. continuous lavage was instituted. on st of august, the pleurostomy tube drained gastric content, and the clinical examination revealed signs of generalized peritonitis. laparotomy was performed with lavage, drainage and posterior decompression gastrostomy. results: postoperative evolution was favorable, with the suppression of pleural drainage in . and discharge in . with alimentation exclusive on jejunostomy. one month later, she had normal clinical and radiological examination evaluation of efficacy was performed with reflux symptom index (rsi) specific for extraesophageal symptoms, subjective satisfaction and occurrence of dysphagia and gas-bloat syndrome. a rsi score [ was considered as pathological. results: rsi significantly decreased after surgery ( and msa as compared with total fundoplication; , % of patients were satisfied with surgery: a comparison between techniques showed superiority of msa objective: to demonstrate the efficacy of hiatorraphy without the use of meshes in thegiant paraesophageal hiatus hernia, as well as the standardization of our technique, with thetechnical steps that we make successively. material and method: clinical cases: -year-old man,with symptomatic hiatal hernia with progressive intolerance and dysnea. egd: the stomach rotated in a giant hiatal hernia.gastroscopy not completed due to endoscope loop formation within giant hiatal hernia with gastric volvulation. ct: large hiatal hernia,combined volvulation (axial axial mesenteric organ), the stomach in a right subpulmonary situation. results: intervention: laparoscopic approach.hh of large paraesophageal size,double organoaxial-and-mesenteric volvular component,gastric walls very thickened and adhered to the mediastinum.reduction of all content and the sac,is adhered to the pleura, extended med-iastinal esophageal disection, up to vein pulmonary and get enough abdominal esophagus and rule out the presence of an short esophagus,posterior-anterior and left tutorized modified hiatorraphy with stitches in ''u'' with non-absorbable suture on teflon reinforcement patches.nissen fixed to both pillars, intramediastinal drainage.egd at the st day with esophageal stenosis due to inflamation of the nissen, resolved with medical treatment. dischage at th day.asymptomatic and without radiological recurrence after months of follow-up. conclusions: in giant and paraesophageal hiatus hernias, modified primary hiatorraphy together with mediastinal esophageal dissection extended can be an effective and safe alternative, and can be advised as a technical gesture prior to a collis nissen and-or placement of a hiatal-hiatoplasty mesh united states of america aim/background: prescribed opioids for pain control have been implicated as major contributors to addiction through their illicit use. efforts to reduce opioid prescriptions and measure their impact on outcomes are novel. we analyzed how patient outcomes are affected with reduced opioid prescriptions following laparoscopic foregut surgery narcr: %), length of hospital stay (narcs: . days vs. narcr: . days), -day readmission rates (narcs: % vs. narcr: %) and perioperative complication rates. additionally, no significant qol outcome differences between the groups were reported at one month postoperatively. conclusion: our study supports reducing opioid prescriptions as a strategy to counter illicit drug use and addiction patients who underwent paraesophageal hernia repair at a tertiary referral center were analyzed retrospectively. demographic data, asa classification, characteristics of peh, onset of symptom, dysphagia severity score, characteristics of fundoplication (partial vs. total; laparotomy vs. laparoscopy; emergency vs. elective) and surgical outcome (length of stay, complication and -day mortality) were recorded and reviewed. results: patients were included; % were female (mean age of . years old and mean body mass index of . ). mean onset of symptom was . weeks after peh repair, dysphagia severity scores were changed . from . . conclusion: in our series, the dysphagia severity scores reduced after surgery upper gi surgery, the catholic university of korea mary's hospital, incheon city, korea p -upper gi-gastric cancer general surgery biopsy from the mass has showed poorly differentiated signet ring cell adenocarcinoma. chest computed tomography revealed mm thoracic aortic aneurism. abdominal computed tomography showed mm infra renal aortic aneurism and no evidence of metastatic disease general surgery year old, female patient presented with upper abdominal pain, weight loss ( kg during last three month), without nausea or vomiting biopsy was done and pathology result showed intestinal type, her -negative adenocarcinoma of the stomach. chest and abdominal computed tomography (ct) were normal. endoscopic ultrasound (eus) revealed cm lesion with invasion to the muscularis propria (mp) she was treated by neo adjuvant chemotherapy ( cycles carboplatine ? fu). patient underwent laparoscopic partial gastrectomy with modified d lymphadenectomy and billroth ii gastrojejunostomy. total operating time (ort) was min. three day after operation patient started regular diet and was discharged home on day fife. final pathology result confirmed intestinal type, modified differentiated adenocarcinoma of the stomach economou st surgical department general, visceral and transplant surgery, section minimally invasive surgery, heidelberg university hospital, heidelberg, germany; department of surgery, iuliu hatieganu university, cluj-napoca, romania; general and visceral surgery, klinikum mittelbaden, baden-baden, germany surgery, toyonaka municipal hospital, toyonaka city, osaka, japan; gastroenterological surgery, osaka university, osaka, japan; next generation endoscopic intervention, osaka university, osaka, japan aims: uncomplicated healing of anastomoses in colorectal surgery is the basis for early adjuvant oncology therapy. the basis for proper healing is good blood flow. we use by robotic surgery foor control the firefly by intuitive. since january , icg has used for blood flow in laparoscopic bowel surgery for the d-light system of storz. method: use of icg wants to accurately determine the resection line for free colon operations based on good blood circulation. we use icg pulse in two batches and color detection using d light from storz to verify blood flow. the first dose is given after the skeletalisation of the intestines intraabdominally and the second after the colic anastomosis to verify its vitality. results: in the period under review we performed laparoscopic operations on the free colon, % of the operations were elective. we had . % leakage across the set, however, in the subset of elective operations, we had only a leak of . %. conclusion: in an unselected set of colorectal operations, leakage was . %, but only . for elective operations. in our group there was a clear effect of using icg in elective laparoscopic resections with an intracorporal anastomosis, the effect was not shown in others, probably due to leakage were factors other than blood flow. objective: right hemicoloctomy (rhce) is the first choice in treating the right colon cancer. complete mesocolic excision with extended lymph node dissection at the roots of superior mesenteric artery (sma) branches enables removal of all lymphatic tissue and prevents local recurrence. previously variability of sma branches was demonstrated. the aim of presented study was to compare the distribution of sma branches in two ethnically different cohorts methods: preoperativect scans with vascular d reconstruction were assessed in patients ( - years) from russia and patients ( - years) from turkey with right colon cancer operated in - . the distribution of ileocolic artery (ica), right colic artery (rca) and middle colic artery (mca) was investigated. results: ica and mca could be found on ct scans in all patients, whereas rca had significantly different distribution between patient cohorts: it was visible in ( %) of turkish patients and only in ( %) of russian patients (p = . ). conclusion: these results suggest that there might be ethnical differences in sma branches distribution. in turkish patients all named sma branches ate visible on ct scans in %, whereas in russian patients only in %. the majority of patients from russia don't have rca. ica and mca could be found in all patients regardless ethnicity. knowing the variant of sma branching before the operation can help plan extended lymph node dissection. the national training programme in laparoscopic colorectal surgery (s-micras-lapserb) in serbia was set up to introduce standardized and structured training in laparoscopic colorectal surgery. method: an assessment based structured training programme (lapserb) started in . series of hands on supervised workshops were conducted for four different hospitals using the structured training by single trainer. this study aims at retrospective analysis of prospectively collected data for patients undergoing colorectal resections. we look at short-term clinical and pathological outcome of patients within laparoscopic colorectal resections performed in national training program. results: during the period november until november , laparoscopic colorectal resection was performed in ( male and female) patients. mean age of patients was . ( - ). the most common indication was colorectal cancer ( patients, . %), ( . %) patients were operated due to the colorectal polyps not suitable for endoscopic resection and ( . %) was operated due to ibd. there were ( . %) right colonic, ( . %) left colonic /tme and ( . %) other resections. average number of lymph node harvested in patients with colorectal carcinoma was . . there were / ( . %) r resections mean duration of hospital stay was . days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . postoperative complications were encountered in / patients ( . %). overall, mortality rate was . % ( / ). conclusions: this study demonstrates successful and safe adoption of laparoscopic technique for colorectal resections. short term clinical and pathological outcomes are compared to published data and shows wider adoption at the national level. standardization of operative technique and structured training remains the key in success. introduction: femal adnexal tumor of probable wolffian origin (fatwo) was first described in . it is a tumor of mesonephiris wolffian duct origin. fatwo is rare tumor which is usualle benign. in the literature has been reported cases and only cases of recurrent disease. next rare tumor in pelvic localisation is sex cord-gonadal stromal tumor of sertoli cells. methods: we present case report of women who presented metastatic fatwo with duplicity of sertoli tumor. results: -year old women underwent years ago exstirpation of tumor in the left broad ligament. histologically there was rare benign fatwo. this year was indicated adnexectomy at gynecology department. during the operation was done bilateral adnexectomy and discovered tumor of anterior wall on upper rectum. microscopic examination showed sertoli tumor on the left ovary. afterward we completed next examinations. colonoscopy without any abnormality. on ct scan was tumor cm without contact to rectum wall, without distant metastasis. the same was described on rectal ultrasonography-normal wall of rectum, tumor probably from uterus. at diagnostic laparoscopy was tumor mass cm, with necrosis arising from anterior wall of the rectum. next small metastasis on pelvic peritoneum. we performed debulking of this big tumor and metastasectomy, there was no infiltration to muscularis propria of the rectum. patient did not have any postoperativ complications. microscopic examination of the rectal tumor and small peritoneal metastasis showed metastatic fatwo. after weeks she underwent laparoscopic second look operation. there were small metastasis on pelvis peritoneum. we removed two biggest metastasis and the rest was destroyed wit j plazma. microscopic examination showed in this metastasis sertoli tumor. conclusion: our patient has metastatic fatwo and sertoli tumor. fatwo is so rare, that in the literaure is not enough information for observation or adjuvant therapy. in one case was described imatinib mesylae (gleevec) therapy with good results. surgeons must be ready to meet new diagnosis. bochdalek hernia is a type of congenital diaphragmatic hernia. in most cases, it is diagnosed during the neonatal period. we present a case of laparoscopically treated congenital bochdalek hernia that led to jejunal strangulation in an adult . case: an fifty eight year old obese(bmi = . ) female admitted for gradually worsening right flank pain, vomiting and respiratory distress for one day. there was no trauma history to chest or abdomen. the past medical history were old cva, well controlled hypertension and dm. she was hemodynamically stable. her right flank was very tender. there was no abdominal distension. initial cbc tests showed leukocytosis ( , /ul) , but electrolytes were normal. chest pa revealed right side diaphragmatic hernia. there was poorly enhanced, herniated small bowel in the right hemithorax on chest ct scan. the patient was taken for emergency operation. on laparoscopy, the normal liver was displaced leftward because of herniated bowel. there was incarcerated jejunum and omentum which could not be reduced. so we widened a cm sized posterolateral diaphragmatic defect first, and then we could reduce the strangulated jejunum( cm in length) and omentum. there was no hernial sac. the defect was closed with -o prolene. finally, the strangulated jejunum was resected and anastomosed extracorporeally. the hospital progress of the patient was not eventful. on post-operative day , the patient was allowed soft diet. the patient was discharged on post-operative day without any complication. conclusion: congenital diaphragmatic hernia is an uncommon condition in adults, but you should keep in mind the diaphragmatic hernia as a cause of intestinal obstruction and respiratory distress in an adult. prompt surgical intervention is required to a favorable outcome. laparoscopic repair of bochdalek hernia is a good management option. aims: intestinal obstruction is one of the most frequent abdominal conditions in the emergency department (ed). up to % of patients having undergone a laparotomy will have an episode throughout their lives, of which % up to % will respond to conservative management. the laparoscopic approach is widely accepted and supported by the studies published up todate. it is recommended in patients with suspected single band, who have less than one previous laparotomy and less than h of clinical evolution. our objective is to validate in our experience that these premises are the appropriate ones in the election of candidates for a minimally invasive approach. methods: we present a series of cases admitted with symptoms compatible with adhesive intestinal obstruction in the ed of a third level hospital during months. all patients underwent abdominal ct to rule out the possible causes of obstruction. emergency surgery was indicated because of failure of a conservative medical treatment or for the findings of the complementary tests. results: a initial laparoscopic approach was performed in the patients, with a conversion rate of % of the cases (resection and anastomosis was required in patients, due to loop suffering or intestinal tumor not seen in the ct). among the patients who required laparotomy, % had more han h of clinical evolution before de surgery and % had free fluid in the tc. as surgical complications: intestinal perforations were produced secondary to the manipulation. there was recurrences of obstruction in the following months. conclusion: the laparoscopic approach is feasible in selected cases and experienced hands. acording to our results it is recommended to perform it in only in patients with less than h evolution and with a single band image in the ct without free fluid. the intestine should be explored avoiding the manipulation of the most extensive loops to prevent complications and keep in mind the possible conversion to laparotomy in case of complications. aim: the aim of the study was to evaluate whether physiologic and operative severity score for the enumeration of mortality and morbidity (possum) is useful to predict the risk of complications in patients older than years. methods: we performed a retrospective study of patients older than years old diagnosed with acute abdomen who were admitted to the department of general, minimally invasive and elderly surgery in olsztyn between may and october . results: the most common disagnosis was ileus. the mortality rate in surgery department was %.after relocation to the intensive care unit, the overall mortality rate was . %. the patients who died a short time after surgery had mortality rates greater than % and morbidity rates greater than % according to possum. conclusions: this study shows that possum seems to be a valuable scale to predict the risk of death after surgery in older patients. patients with higher mortality and morbidity scores should be very carefully selected for surgery. aims: diaphragmatic hernia in adulthood is rare. the most common causes are blunt and penetrating trauma. we present an intraoperative video of the laparoscopic repair of an adult onset, non-traumatic, diaphragmatic hernia in a patient with splenomegaly. method: a year old woman was referred to upper gastrointestinal surgery with epigastric burning and pain in the left side of her chest, radiating to the left shoulder, for one year. there was no recent or distant history of trauma. she has a past medical history of treated hepatitis c, cirrhotic liver disease, splenomegaly, thrombocytopenia and iron deficiency anaemia. gastroscopy was interpreted as a fundal diverticulum. ct abdomen/pelvis with intravenous (iv) contrast showed this to be a left diaphragmatic defect with herniated stomach causing a volvulus, which lay immediately above an enlarged spleen. a ct three years prior to this showed no diaphragmatic hernia. the patient had some symptomatic relief with a proton pump inhibitor and oral antacids, however due to her persistent symptoms surgery was undertaken. the patient had laparoscopic repair of the diaphragmatic hernia. ports were as follows: mm umbilical, mm left upper quadrant, mm right upper quadrant, mm left iliac fossa. a left posterior diaphragmatic defect was found, just above the enlarged spleen, containing the incarcerated fundus of the stomach. the hernia was reduced by gradual dissection of the sac. a cm nonabsorbable polypropylene mesh (proceed) was used to cover the defect a cm margin. this was tacked in place with protack. a single french robinson drain was left in situ. results: the procedure was uncomplicated. oral diet was introduced on post-operative day and the drain was removed and patient discharged on day . there were no post-operative complications. conclusions: the video shows an effective dissection of a left sided diaphragmatic hernia and mesh repair, overcoming multiple technical challenges secondary to splenomegaly and portal hypertension. aims: creating laparoscopic anastomosis is a challenging surgical skill with high clinical relevance. to assure efficient training and enhanced learning curves, constructive and objective feedback is essential. currently there is no appropriate instrument to assess the surgical performance while creating laparoscopic anastomosis. the aim of this study is to develop and validate the anastomosis-objective structured assessment of technical skill (a-osats) score. methods: to obtain an international expert consensus for a procedure specific checklist (psc) for laparoscopic anastomosis, a modified delphi survey with an integrated analytic hierarchy process is currently being performed. each a-osats sub step is assigned a specific weight to determine its importance to the final outcome of the anastomosis. to validate the a-osats score, a laparoscopic side-to-side small bowl anastomosis with a linear stapler and hand-sewn closure of the enterotomy was chosen and is performed by surgeons with varying degrees of laparoscopic experience on a live porcine model. all performances are recorded and rated twice using the a-osats by two blinded experts. results: the final a-osats score includes a weighted psc developed by the modified delphi survey and the already validated global rating scale of previously published osats scores. four key steps (bowel placement, creation of enterotomies, stapling, closure of enterotomy) and sub steps, as well as their definitions, were established during the delphi survey. to validate the a-osats, surgeons ( experts, intermediates, novices) have participated in the study so far. preliminary results showed significant differences between all three levels of laparoscopic experience (novices: . ± . ; intermediates: . ± . , experts: . ± . ; p \ . ) for the overall a-osats score as well as the psc itself (novices: . ± . , intermediates: . ± . , experts: . ± . ; p = . ). conclusions: the a-osats is a weighted score that objectively assesses surgical skill during the creation of laparoscopic anastomosis. preliminary results confirm construct validity of the proposed score. furthermore, by offering the possibility to differentiate single aspects during the procedure, the a-osats allows focused feedback to enhance one's performance. minor changes in weights are expected after the last round of the delphi survey. interand intrarater reliability will be assessed after final inclusion of all participants.aims: to apply augmented reality technology from three-dimensional colon models as preoperative planning method in colorectal surgery. method: from three-dimensional anatomical models of the colon we have developed holograms of augmented reality. the models were obtained from ct images (siemens somatom perspective Ò) with abdominal image cuts with mm thick. the recovery of the images was in dicom format and the processing to achieve the three-dimensional reconstruction was performed with the programs osirixÒ and horosÒ, which made a complete segmentation of the colon surface, and a modification of the image density. in this way models d were obtained of the isolated colon, and in relationship with the bone structure. the application colon d ar was designed (increased hyper experience-visualizer with slam technology) creating a hologram of augmented reality to scale : from each three dimensional model to make a projection of it on the abdomen of the patient by modifying the position in height of the reconstruction, using the bone pelvis as anatomic reference point to calibrate the placement of the hologram. results: in the preliminary phase (from october to december ) holograms of augmented reality were developed in patients with colorectal cancer (right colon, left colon, transverse colon and rectum) to complement the radiological reconstruction with the virtual model. in the application phase (from january ) the holograms developed are going to be applied as a method to improve preoperative study. conclusions: three dimensional reconstruction of the tumor in the preoperative plan of colorrectal surgery combined with hyperreality technology allows to develop models of augmented reality in order to improve colon anatomy knowledge and to plan the surgical technique.aims: laparoscopic adrenalectomy has become the standard of care for most adrenal masses. we report a case of laparoscopic adrenalectomy for left adrenal adenoma. methods: we present the case of a -year-old caucasian female patient with an asymptomatic, left-sided adenoma, that was incidentally detected during abdominal ultrasound. no headaches, palpitations, tachycardia, tremor, dizziness or vomiting were reported. pre-operative blood tests confirmed that the tumor was a non-secreting one and a ct-scan revealed a . . cm left adrenal mass. laparoscopic surgical excision was proposed. the patient was placed in semilateral right-sided decubitus position. four trocars ( epigastric- mm & subcostals- mm & mm) were used, without the use of a liver retractor. the adrenal vessels were clipped not only with the standard laparoscopic clips, but also with the hem-o-lok ligation system. results: the operation lasted for h with minimal blood loss. the patient's post-operative course was uneventful and she was finally discharged four days post-operatively. histology report ensured that it was adenoma of the adrenal cortex. aims: since the first laparoscopic adrenalectomy in (gagner), the laparoscopic lateral transabdominal approach has proved to be the one of choice. it provides an easy anatomical orientation, overall the technique is similar to other traditional laparoscopic procedures. on the other hand, the posterior retroperitoneoscopic adrenalectomy (pra), described in (waltz), has proven to be a safe technique and effective for the surgical management of several adrenal pathologies. the advantages include direct access to the adrenal gland, without the need for visceral mobilization or lysis of adhesions from previous abdominal operations and the ability to perform a bilateral adrenalectomy without repositioning the patient. currently there is controversy about which is the approach of choice, having to take into account the learning curve necessary for the retroperitoneal approach and the reduced number of patients with adrenal pathology subsidiary of surgical management. the objetive is to demonstrate the safety and efficacy of the standardized laparoscopic approach of the left adrenal gland with trocars for selected cases. methods: clinical case: -year-old man, resistant hypertension despite concurrent use of three antihypertensive agents, with biochemical and radiological diagnosis of left adrenal adenoma with primary hyperaldosteronism. demonstrative video of the technical steps in a standardized way that we propose for laparoscopic left adrenalectomy only using trocars. results: full laparoscopic surgical approach in right lateral decubitus position: trocars-lateral transabdominal approach. steps: . laparoscopic liberation of the splenic flexure of the colon for the colo-spleen-pancreato-gastric en block mobilization until identification of the left pillar, . dissection of the medial border of the gland, identification of left renal and diaphragmatic vein, as well as the adrenal vein which is dissected and clipped, . dissection of the lateral edge of the adrenal gland, . lower pole dissection of the gland completing the resection with ligasureÒ. the patient presented a successful postoperative recovery, being discharged h after the intervention. asymptomatic, the patient does not need antihypertensive drugs at year follow-up. conclusion(s): the standardization of the procedure allows reducing the number of trocars, maintaining the safety and effectiveness of the minimally invasive approach. aims: cortical-sparing adrenalectomy is a suitable treatment for hereditary and sporadic bilateral pheochromocytoma, in cases of low risk of malignancy, to reduce the possibility of adrenal insufficiency assuming the chance of local recurrence. the aim of the study is to analyze the functional results of partial adrenalectomy by retroperitoneal endoscopic approach in singleadrenal patients or patients requiring bilateral adrenalectomy. methods: prospective study between january and october including pheochromocytoma patients diagnosed with low risk of malignant mutations. all patients agreed to be included in the study. experienced endocrine surgeons who have been trained in minimally invasive endocrine surgery performed the procedure using the same surgical technique. demographic variables and clinical characteristics were collected, subsequently carrying out the descriptive analysis of the data. results: a total of eight patients were registered, five associated with men type syndrome and three in the context of vhl syndrome. retroperitoneoscopic resection was performed without laparoscopic or open conversion and no postoperative complications; the average hospital stay was . days. preservation of the functional cortex without corticosteroids was achieved in ( . %) of out cases with a follow-up of . ± months. today, these seven patients have a preserved adrenal function without hormone replacement. conclusions: cortical-sparing adrenalectomy by the retroperitoneal endoscopic approach, in expert hands, is safe and feasible for the treatment of hereditary and sporadic pheochromocytoma in a context of low malignancy, making it possible to avoid the need for corticoid replacement in most cases. biomedical sciences, university of west attica, athens, greece partial adrenalectomy has been suggested for patients benign adrenal tumors especially in the case of hereditary syndromes, like multiple endocrine neoplasia type , von hippel-lindau disease and neurofibromatosis type i. aims: this systematic review aimed to investigate the role of partial adrenalectomy in the treatment of hereditary pheochromocytoma. methods: electronic databases were searched with the search terms 'men ii', 'von hippel lindau', 'neurofibromatosis', 'laparoscopic partial adrenalectomy', 'robotic assisted partial adrenalectomy' for the time period up to and including december . full publications, including clinical trials randomized or not, retrospective studies, case series, case reports that provided relevant data met inclusion criteria results: thirty five possibly relevant studies were identified. abstracts were reviewed and fourteen articles were excluded as they were review articles or articles presenting data on open partial adrenalectomy. twenty one studies, that met inclusion criteria were retrieved in full text and included in the systematic review. eight studies presented data on partial adrenalectomy in patients with von hippel lindau including two case series with median follow up ranging from to . years and six case reports. thirteen studies presented data on partial adrenalectomy in patients with men ii, including two case series and eleven case reports. recurrence rate was estimated at about % for pheochromocytoma. overall steroid dependence rate was estimated at %. conclusion: minimally invasive partial adrenalectomy is a therapeutic option especially in patients with heritable pheochromocytoma, given that tumors are often bilateral, tumors are commonly benign and severe morbidity and mortality may be associated with life-long steroid replacement therapy such as the possibly lethal addisonian crisis . however, data are limited, follow up is not standardized and not appropriately reported and rcts are difficult to be done due to the rarity of the disease. a multinational registry on the short term and long term outcomes of partial adrenalectomy in hereditary pheochromocytoma would be a significant source of knowledge. results: patients were operated on after an average of months with complaints. in both groups, the leading symptoms were severe dysphagia and severe regurgitation. no intraoperative complication was detected. in the transoral group, one patient had to be reoperated on for bleeding, another patient developed pneumonia in the transcervical group. the average duration of the surgeries ( . vs. min, p \ , ), the time to oral feeding ( . vs. . days, p \ , ) and the mean hospital stay ( . vs. . days, p \ , ) were significantly shorter in the transoral group than the transcervical group. patients were completely symptomless postoperatively. after transcervical treatment, complaints were developed in cases (moderate dysphagia and hoarseness). after transoral surgery, recurrent symptoms were observed in patients, had to be reoperated transcervically due to severe regurgitation. conclusion: transoral stapler diverticulostomy is a fast procedure and offers short hospital stay especially in comorbid, aged patients and intermedium diverticulum size. in the long term, some of the patients may require reintervention due to persistent regurgitation. the transcervical approach has higher perioperative morbidity, which can be performed in patients with less than cm or large diverticulum size. aims: complex hiatal hernias, either implicating large hiatal defects or concerning cases of recurrence, often need apart from the primary closure of the hiatal gap, the re-enforcement of the crura with the use of meshes. our aim is to demonstrate the surgical technique for the on-lay placement of the absorbable mesh (phasix tm st mesh /bard) in challenging cases, presenting both the laparoscopic and the robotic approach. methods: we present video fragments from procedures of laparoscopic and robotic reconstruction of complex hiatal hernias, performed by our team, in which an absorbable mesh was utilized in an on-lay fashion. results: patients having undergone a minimally invasive surgical approach (laparoscopic or robotic) for the treatment of complex hiatal hernias with the use of an absorbable mesh, had an uneventful post-operative course and very short hospital stay and recovery time. the -month follow up revealed no recurrences or late complications. conclusions: treating complex cases of hiatal hernias with a minimally invasive approach can be proven quite challenging, with high recurrences and possible complications rate. a proper surgical technique, either laparoscopic or better (based in our primary experience) robotic, by experienced surgical teams and the use of meshes with the right strategy, minimizes the complications, offers all the benefits of minimally invasive surgery and reduces the recurrence rates. aims: several flexible endoscopic techniques for symptomatic zenker's diverticulum have been developed during the last decade. thulium laser has limited tissue penetration and may decrease the risk of perforation. this study reports the first use of thulium laser through flexible endoscopy for cricopharyngeal (cp) myotomy. aims were safety and efficacy of flexible endoscopic thulium laser myotomy and quality of life (qol) changes after treatment. methods: a retrospective review of a prospectively collected database of patients who underwent thulium laser septum division for symptomatic zenker's diverticulum was done. demographic data, presenting symptoms, diverticulum characteristics, and intraoperative data were analyzed. functional outcome swallowing scale (foss) and m.d. anderson dysphagia inventory (mdadi) questionnaires were administered to determine severity of dysphagia and its effect on qol, both preoperatively and during follow-up visits. all the operations were carried out under general anesthesia. a continuous laser configuration and an emissionpower of w was used in non-contact mode. once the mucosa was opened, the fibers of the cricopharyngeal muscle were divided until the buccopharyngeal fascia was visibile. results: between march and september , patients ( males) underwent flexible endoscopic cp myotomy with thulium laser. mean age was ± . , mostly males ( . %). seven patients ( . %) presented with recurrent diverticulum after previous transoral or open treatment. mean diverticulum size was . ± . cm. preoperative main symptoms were dysphagia ( . %), regurgitation ( . %), and cough ( . %). foss score was = in patients ( . %). mean mdadi global and composite score were . ± . and . ± . . complete division of the septum was achieved in all patients. mean hospital stay was . ± . days. there was only one perforation treated conservatively. no -days mortality was observed. at median follow-up of months, foss was = in ( . %) patient and mdadi global and composite score were . ± . and . ± . . all main symptoms were significantly reduced and qol significantly increased. conclusions: flexible endoscopic approach with thulium laser is a safe and effective treatment option for zenker's diverticulum either as a primary treatment or as a rescue therapy. objective: this study sought to explore prognostic factors for patients with borrmann type iv gastric cancer and to establish a predictive model for survival benefit of postoperative adjuvant chemotherapy in such patients. method: this study reviewed the clinical data of patients who underwent curative surgery at fujian medical university union hospital from to for borrmann type iv gastric cancer using a prospective database. cox regression analyses were performed to identify prognostic factors that formed the basis for a nomogram and risk groups. establishment of risk groups to identify patients with borrmann type iv gastric cancer who would benefit from adjuvant chemotherapy. results: patients who underwent r resection were included in this study.multivariate analysis showed that bmi, tumour differentiation, pt stage, pn stage, and asa score were independent prognostic factors. patients in the act-group had longer os than patients in the sagroup, although the p-value for this difference was marginally above the threshold for statistical significance ( . % vs. . %, p = . ). stratified analysis showed that there was no significant difference in os between the act-group and the sa-group for each ajcc stage (stage ii: . % vs. . %, p = . ; stage iii: . % vs. . %, p = . ).a nomogram was established based on these independent risk factors, and nomogram scores were used to divide all patients into a high-risk group (score [ ), an intermediate-risk group ( \ score = ) and a low-risk group (score = ).further stratified analysis based on ajcc stage showed that the -year survival rate was higher in the adjuvant chemotherapy group than in the surgery alone group for low-and intermediate-risk patients in each ajcc stage, while high-risk patients in stage iii did not significantly differ. objective: this study sought to explore the prognostic factors for smoking patients with gastric cancer and to establish a predictive model for the survival benefit of postoperative adjuvant chemotherapy in such patients. methods: we studied patients who were diagnosed from september to september at union hospital of fujian medical university. cox regression analyses were performed to identify prognostic factors. the kaplan-meier method was used to assess the effect of smoking history on the benefit of adjuvant chemotherapy after gastric cancer surgery. a decision tree algorithm was used to identify smoking patients who benefited from postoperative adjuvant chemotherapy. results: the median follow-up time for the whole group was . months, and the average age of all the included patients was . years.multivariate analysis showed that age (p \ . ), bmi (p \ . ), degree of tumor cell differentiation (p \ . ), and ajcc stage (p \ . ) were independent risk factors for the prognosis of smoking patients. based on these independent risk factors, a decision tree model for the benefit of adjuvant chemotherapy for smokers with gastric cancer was established, and the smoking patients were divided into the low-risk patients . %), medium-risk patients ( - year os, . %) and high-risk patients ( - year os, . %) (p \ . ). conclusion: cigarette smoking may reduce the efficacy of adjuvant chemotherapy after gastric cancer surgery. our decision tree model is simple and effective for identifying smokers who would benefit from adjuvant chemotherapy. objective: our study investigated the effect of lymph node (ln) noncompliance on the longterm prognosis of patients after laparoscopic total gastrectomy (ltg) and explored the risk factors of ln noncompliance. methods: the clinicopathological data of gastric cancer (gc) patients who underwent ltg with d lymphadenectomy from june to december were prospectively collected and retrospectively analyzed. the effects of ln noncompliance on the long-term prognosis of patients with gc after ltg were explored. results: the overall ln noncompliance rate was . %. ln noncompliance was significantly correlated with age, bmi, asa score, tumor size, macroscopic tumor type and tnm staging (p values \ . ). the survival rate of patients after ltg with ln compliance was significantly superior to that of patients with ln noncompliance (p = . ). the stratified analysis of tnm stage indicated that there was no difference between the os of stage i patients with ln compliance and those with ln noncompliance; os of stage ii/iii patients with ln compliance was significantly better than that of those with ln noncompliance. cox regression analyses showed that ln noncompliance was an independent risk factor for os. logistic regression analysis showed that high bmi ([ kg/m ) was an independent risk factor for preoperative prediction of ln noncompliance in cstage ii/iii patients. compared with patients with a low bmi (bmi \ kg/m ), those with a high bmi were more likely to show ln noncompliance during surgery, especially during the dissections of # , # a and # a ln stations. conclusion: ln noncompliance was an independent risk factor for poor prognosis in patients with advanced gastric cancer (agc) after ltg. patients with high bmi were more likely to have ln noncompliance, especially during the dissections of # , # a and # a ln stations. ln tracing was recommended for these patients to reduce the rate of ln noncompliance. aim: to study the differences in pathology, survival, and recurrence between special remnant gastric cancer (srgc) and nonspecial rgc (nrgc). method: a total of rgc patients were analyzed in hospitals in china from january to july .we compared the -year overall survival (os) disease-free survival (dfs) rates and used two-step regression explore the influence of the rgc categories on patient outcomes. results: all of the patients divided into srgc group (group s) (n = ) and nrgc group (group n) (n = ). the r resection rate and lymph node (ln) dissection number of group s were significantly higher than group n (p \ . ). the difference in -year os was not significant (p = . ), but the -year dfs of group s was worse than group n (p = . ). twostep multivariate analyses showed nrgc was an independent risk factor for poor dfs. of the patients who had undergone r resection, patients ( . %),suffered recurrence, and the recurrence rate of group s was significantly higher than group n (p = . ), moreover, the ln recurrence rate of group s was significantly higher than group n (p = . ). cox regression analysis showed that age, ca level, n stage and category of rgc were independent risk factors for rgc recurrence. conclusion: srgc has a higher r resection rate and ln dissection number than nrgc, but among patients who had undergone radical gastrectomy, srgc patients had worse dfs and a higher tendency for ln recurrence; thus, they should be treated differently in the clinic. objective: the aim of this study was to report our institution's experience with a novel abdominal negative pressure lavage-drainage system (anplds) for anastomotic leakage (al) after radical gastrectomy (rg) for gastric cancer (gc). background: al is a severe complication associated with high morbidity and mortality after rg for gc. the optimal creation of drainage in al patients after rg remains controversial. methods: the study enrolled patients who underwent r resection for gc at our institution between and . anplds was routinely used for patients with al after january . al rates and postoperative outcome were compared before and after the anplds therapy. we used multivariate analyses to evaluate clinicopathological and perioperative factors for associations with al and failure-to-rescue (ftr) after al. results: al occurred in patients ( / , %), leading to deaths. the al rate was similar before ( - , period ) and after ( - , period ) the implementation of anplds ( . % vs . %, p = . ). age and malnourished were independently associated with al. the ftr rate and abdominal bleeding rate after al occurred were respectively . % and . % for the entire period, but compared with period , it significantly decreased at period ( . % vs . %, p = . ; . % vs . %, p = . , respectively). what's more, only anplds therapy was an independent protective factor for ftr after al. conclusion: our experience demonstrates that anplds is feasible and cost-effective for the management of al after rg for gc. objective: to apply the principles of the 'metro-ticket' paradigm to develop a novel tnm staging system (ntnm) for gastric cancer (gc). background: the 'metro-ticket' prognostic tool for hepatocellular carcinoma has been proven to predict outcome, but a similar concept has not been investigated for gc. methods: the ntnm considered the distance from the origin on a cartesian plane incorporating the pn (x-axis) and pt (y-axis) stages. gc patients undergoing radical resection at fujian medical university union hospital (fmuuh) (n = ) were included. the ntnm was validated using external cohorts from the sun yat-sen university cancer center (sysucc) (n = ) and surveillance, epidemiology, and end results (seer) (n = ) databases. results: ntnm classes with the same distance from the origin have same stage; the stage increases with this distance. among all patients, . % (n = ) were restaged in the ntnm compared with the th edition of the ajcc-tnm classification; . % (n = ) were downstaged in the ntnm compared with the th edition. the ntnm provides significant survival differences between stages (all p \ . ). the survival difference between stages ib and iia was especially large for the ntnm (p \ . ) compared to the th and th editions (p = . ). the concordance index and hazard ratio increased successively with the ntnm stage. similar findings were observed in both external cohorts. conclusion: compared with the ajcc-tnm classification, the 'metro-ticket' ntnm for gc is easier to remember and provides some improvements; therefore, the ntnm may be considered for adoption in future editions of the ajcc-tnm classification. objective: to investigate the prognostic value of complete blood count (cbc)-based biomarkers for patients with resectable gastric cancer (gc). methods: patients with gc who underwent curative resection between december to december were included. estimated area under the curve (auc) and multivariate cox regression models were used to identify the best cbc-based biomarker. time-dependent receiver operating characteristics (t-roc) analysis was used to compare the prognostic impact. results: based on multivariate analysis, the lymphocyte-monocyte ratio (lmr) and hemoglobin (hb) level were the independent prognostic factors (both p \ . ). based on the lmr and hb level, we established the cbc-based inflammatory score (cbcs). higher cbcs was associated with older age, female sex, higher american society of anesthesiologists (asa) score, proximal tumor location, larger tumor size, later stage and vascular involvement (all p \ . ). univariate analyses showed that higher cbcs was also associated with poorer overall survival (os), which was consistent in each stage (all p \ . ). multivariate analysis revealed that the cbcs was a significant independent biomarker (p \ . ). furthermore, t-roc curve of the cbcs was superior to that of the prognostic nutritional index (pni), systemic immune-inflammation index (sii), modified glasgow prognostic score (mgps) and c-reactive protein/albumin ratio (crp/ alb) throughout the observation period. conclusion: preoperative lmr and hb were optimal cbc-based biomarkers for predicting os in gc patients after curative resection. based on the lmr and hb, we developed a novel and easily obtainable prognostic score called the cbcs, which may improve the prediction of clinical outcomes. purpose: the aim of this study was to evaluate the prognostic value of the eighth ajcc tnm staging classification for patients with gastric cancer who had already survived for years. patients and methods: patients who underwent radical gastrectomy at a large eastern center were considered. the prognostic value of staging systems were assessed and compared. additional external validation was performed using a dataset from the surveillance, epidemiology, and end result (seer) database. results: the -year overall survival (os) rate for patients in the training set was . %. with the prolongation of the survival time after surgery, the -year os improved significantly (p \ . ). however, there were no significant differences in survival curves among patients who have survived years after surgery. the auc and c of the eighth ajcc classification for predicting of -year os decreased gradually after surgery and appeared stable after years. for patients who survived years after surgery, we constructed a new tnm staging system (ntnm) according to the survival curves of t stage and n stage. a -step multivariate analysis showed that ntnm, age and sex were independent prognostic factors. the ntnm demonstrated superior prognostic stratification, with higher c-statistic and likelihood ratio chi-square scores and lower aic values than those of the ajcc classification. similar results were observed in the external validation set. conclusion: the ntnm predicted an additional survival more accurately than did the ajcc classification for patients who have survived years after surgery; this may guide decisions regarding surveillance. objective: to investigate the relationship between preoperative sarcopenia and systemic inflammation and evaluate the prognostic impact of these factors on patients with resectable gastric cancer (gc). methods: patients with gc who underwent radical gastrectomy between december and december were included. a multivariate cox regression analysis was performed to identify the prognostic factors. a novel prognostic score (slmr) was developed based on preoperative sarcopenia and the lymphocyte-monocyte ratio (lmr), and its prognostic value was evaluated. results: in total, patients with resectable gc were included in the study. on multivariate analysis, preoperative sarcopenia and the lmr were shown to be independent prognostic factors (both p \ . ). a low lmr was an independent predictor from sarcopenia (p \ . ). based on preoperative sarcopenia and the lmr, we established the slmr. an elevated slmr was associated with older age, higher asa scores, larger tumor size, advanced stages and vascular invasion (all p \ . ). multivariate analysis revealed that the slmr was a significant independent predictor (p \ . ). we incorporated the slmr into a prognostic model that included tumor size and tnm stage and generated a nomogram, which accurately predicted -and -year survival for gc patients. objective: to explore whether adjuvant chemotherapy is still needed in patients aged less than years with pt n - and pt / n gastric cancer. methods: multi-center cohort data of patients with gastric cancer who underwent radical gastrectomy were analyzed. kaplan-meier curves and cox regression were used to analyze the relationships between chemotherapy and prognosis. additionally, nomograms to predict the benefit of chemotherapy were established. results: in total, , patients with pt n - and pt / n gastric cancer were included. patients ( . %) were aged \ years. the -year overall survival (os) was not significantly different between the \ years of age group and = years of age group ( . % vs. . %, respectively; p = . ). lymph node (ln) metastases (hr . ; p = . ) and ln dissection number \ (hr . ; p \ . ) were independent risk factors for the os of patients aged \ years. adjuvant chemotherapy did not improve the -year os for patients aged \ years with pt n - and pt / n gastric cancer (p = . ). however, chemotherapy showed a significant benefit (p = . ) when there were ln metastases and/or ln dissection number was \ . two nomograms were constructed, and the calculated difference was the potential benefit of adjuvant chemotherapy for the patients aged \ years. conclusions: ln metastases and ln dissection number \ were independent prognostic risk factors of patients aged \ years with pt n - and pt / n gastric cancer. patients with these risk factors may benefit from the addition of adjuvant chemotherapy. objective: the choice of reconstruction after distal gastrectomy remains controversial. we have performed roux-en-y (r-y) method after laparoscopic distal gastrectomy(ldg) as a standard since , but we have performed billroth ii (b-ii) method in an increasing number of cases, depending on the patient. we retrospectively investigated the outcomes of patients with b-ii method after laparoscopic distal gastrectomy in our hospital. methods: patients who underwent b-ii and r-y reconstruction after ldg from january to december were included. the patient characteristics, surgical outcomes, and postoperative outcomes between the procedures were retrospectively analyzed. we also compared extend of gastritis on endoscopy and loss of body weight after surgery at year. results: b-ii / r-y : / . b-ii was selected in the elderly patients with poor asa-ps (p \ . ). in surgical outcomes, operative time was shorter for b-ii than r-y (p \ . ), and blood loss was also smaller (p = . ). in postoperative outcomes, there were significant differences in complications (?grade ) (b-ii vs. r-y: . vs. . %, p = . ) and length of stay (b-ii vs. r-y: median . vs. -day, p \ . ). there was significant difference in presence of gastritis between b-ii ( . %) and r-y ( . %) (p \ . ), but no significant difference in loss of body weight (p = . ). conclusion: b-ii reconstruction may be an adequate procedure for high-risk cases because of its shorter operative time and the absence of severe complications. background: numerous studies have shown that the short-term efficacy of three-dimensional ( d) laparoscopic radical gastrectomy (lg) is comparable to that of two-dimensional ( d)-lg. whether d-lg affects the recurrence pattern after surgery has not been investigated. using data from a prospective clinical trial, the present study compares the recurrence patterns between d-lg and d-lg. methods: from january to april , a total of patients were recruited for the clinical trial (nct ). the recurrence types, the first recurrence time and recurrence-free survival (rfs) were compared between the two groups. multivariate analyses of factors associated with rfs were performed to identify whether d-lg affects the recurrence patterns. results: ultimately, patients were analyzed ( in the d-lg group and in the d-lg group), and there were no differences in the clinicopathological data between the two groups. distant metastasis was the most common type of recurrence. there were no significant differences between the two groups in the recurrence types, the first recurrence time or rfs (all p [ . ). according to the th american joint committee on cancer tumor-node-metastasis (tnm) staging system, both groups were stratified into pathological (p) i, ii, and iii stages. the stratified analysis showed that there were no statistically significant differences in rfs between the d group and the d group among patients in each subgroup (all p [ . ). the multivariate analysis of rfs showed that pathological tnm (ptnm) stage and lymphovascular invasion were independent risk factors (all p \ . ). the multivariate analysis of post-recurrence survival (prs) showed that adjuvant chemotherapy was an independent protective factor (p = . ). conclusions: distant metastasis was the most common type of recurrence after lg. the postoperative recurrence patterns, rfs and prs after d-lg were similar to those after d-lg. purpose: the aim of this study is to evaluate the efficacy of delta-shaped anastomosis compared to circular stapler anastomosis in laparoscopic distal gastrectomy with billroth i reconstruction (ladg-bi). method: this is a single-center randomized controlled study. eligibility criteria included histologically proven gastric adenocarcinoma in the lower third of the stomach, clinical stage i tumor. patients were preoperatively randomized to circular stapler anastomosis or delta-shaped anastomosis. the primary endpoint is the number of analgesics use during days after surgery. we compared the surgical outcomes of the two groups. postoperative qol was evaluated using the postgastrectomy syndrome assessment scale- . this trial was registered at the umin clinical trials registry as umin . results: between december and september , patients (delta-shaped anastomosis , circular stapler anastomosis ) were enrolled. there was no difference in the number of analgesics use during day after surgery (median : delta-shaped anastomosis vs. : circular stapler anastomosis, p = . ). there was no difference in the overall proportion with in-hospital grade ii-iiib surgical complications ( %: delta-shaped anastomosis, %: circular stapler anastomosis). there was no operation-related death in either arm. regarding postoperative qol evaluated month after surgery, diarrhea subscale was significantly worse in delta-shaped anastomosis than in circular stapler anastomosis. conclusion: we did not demonstrate the advantage of delta-shaped anastomosis in terms of postoperative pain. since delta-shaped anastomosis tended to cause postoperative abdominal symptoms related to diarrhea, we should carefully apply the delta-shaped anastomosis to ladg. introduction: the use of a three-dimensional( d) camera for laparoscopic surgery has been reported in literature. however, there are only few comparative studies demonstrating its benefits, and no reports on the application of d vision to single-incision laparoscopic surgery. this study aims to compare d vision to the previous two-dimensional( d) system in solo single-incision laparoscopic distal gastrectomy(sidg). methods: medical charts of gastric cancer patients who underwent solo sidg from february to december were retrospectively reviewed. patients were grouped into either d group or d group depending on the type of camera used. all the operations were performed by a single surgeon using a flexible camera(olympus, japan), fixed onto a passive scope holder without the use of a scopist or an assistant. operative data, postoperative outcome, and early complication were analyzed. results: ninety had their operations under d vision and used the d scope. in both groups, there was no difference in age, body mass index, staging, and other demographic or histopathologic criteria. operative time was significantly faster in the d group( . ± . vs. . ± . mins., p = . ) and ebl was also less( . ± . vs. . ± . ml, p = . ). patients in the d group started small fluid diet faster( . ± . vs. . ± . postoperative days, p = . ), and were discharged faster( . ± . vs. . ± . postoperative days, p = . ). early complication was also less in the d group( . % vs. . %) but there was no statistical significance(p = . ). conclusion: the use of the d camera improves operative outcome and hospital stay in patients undergoing solo sidg. the frequency of anastomotic leakage after gastrectomyreaches - %. at the same time, mortality in this group of patients reaches %, and the use of aggressive methods of surgical treatment for the treatment of anastomotic leakage increases the mortality rate from to %. since , vacuum-assisted closure has been used to treat anastomotic leakage of various localizations. the essence of this method is based on the creating a local negative pressure, which is transmitted to the drip cavity through a special porous spongy system. the negative pressure created in the closed cavity, allows you to remove exudate, helps to reduce tissue swelling, improvesmicrocirculation, which in turn contributes to the development of granulations and wound healing with separation of the fistulous course. failures in using the method of vacuum therapy in anastomotic leakage are associated with the great difficulty of delivering a polyurethane sponge with a drainage tube to the leakage zone. in this regard, we have developed an improved method of endoscopic local vacuum therapy, in which the delivery of a polyurethane sponge was carried out with the help of a thread through a pharyngeal ring, a leakage zone and brought out through a drainage tube. this technique has been successfully used in the treatment of four patients with anastomotic leakage after operations on the upper part of the digestive tract. for complete healing of the cavity of the leakage and defect of the organ wall, it took , , and sessions of replacing the vac system, respectively (average . ± . ). there were no complications during the endoscopic local vacuum therapy. when the control endoscopic studies after months after the completion of the treatment at the site of defects of the seams of the anastomoses formed tender scar tissue without signs of narrowing of the organ. aims: enhanced recovery after surgery pathways are safe and effective for patients undergoing gastrectomy. this study aimed to identify perioperative factors influencing the adherence to the protocol, the postoperative course, and the consequent length of stay. methods: between and , patients were referred to our institution for gastric cancer. among these, patients underwent atypical gastric resection and were excluded from this analysis. were assigned to either total or distal gastrectomy and represent the study population. all patients were managed with a standardised perioperative pathway according to eras principles. according to data from the literature and based on our clinical experience, patients with optimal adherence to eras protocol may fit the criteria for discharge within ninth postoperative day, that was considered our ideal threshold for hospital discharge. data were retrospectively collected and analysed from a prospectively maintained database. statistical analyses were performed using spss version for macintosh. the v test, with a significance level of . , was used to investigate the association between the outcome and perioperative categorical variables. when parametric assumptions were met, student's two-tailed t-test was used to compare the means of continuous variables; otherwise, the mann-whitney test was performed. a significance level of . was chosen. logistic binary regression with a backward selection procedure and selection criteria of p-value \ . were exploited to determine significant predictors. results: preoperative, intraoperative and early postoperative variables were considered. among all, multivariate regression analysis revealed that incomplete preoperative immunonutrition, failure to extubate the patient at the end of surgery, intraoperative crystalloids infusions [ ml and blood transfusion [ ml, surgery duration [ min, and failure to mobilise patients within h from surgery were associated with delayed discharge. the logistic regression model was statistically significant (p \ . ) and correctly classified . % of cases. sensitivity and specificity were . % and . %, respectively. conclusions: results seem to be clinically rational and focus the attention on the importance of some perioperative clinical issues for the management of postoperative course. these variables could be considered as clinical goals to be reached in order to get an early discharge. objectives: the purpose of this study is to confirm the safety of laparoscopic gastrectomy with intraperitoneal cisplatin administration as a treatment for advanced gastric cancer with potential for peritoneal seeding. methods: from july to august , patients with advanced gastric cancer who underwent ip chemotherapy after diagnostic laparoscopy were retrospectively studied. all patients underwent laparoscopic gastrectomy with ip chemotherapy or ip chemotherapy alone after a diagnostic laparoscopy. gastrectomy was performed for palliative purposes even with seeding. results: the average age of the patients was years. eight patients ( . %) had preop chemotherapy. curative resection (r ) was performed in patients ( . %). in diagnostic laparoscopy, cytology was performed in patients ( . %) and cy was ( . %). peritoneal metastasis was detected in patients ( . %). of the total cohort, the year os rate was . % and the median survival time was months. in the case of stage iiib and below, the -year os rate was %, but it was % in stage iiic-iv group. when the r resection group and the r - resection group were compared, the -year os rates were . % and . %, respectively. hematological toxicity such as neutropenia was not seen in all patients. the mean hospital stay was . days and adjuvant chemotherapy was performed in patients ( . %). background: radical proximal gastrectomy (pg) and lymph nodes dissection are indicated for selected gastric cancers at the upper third of the stomach. with the advent of laparoscopic surgeries, more and more pg were performed by laparoscopic apporaches. in the past years, our team has accomplished and reported the oncological outcome of laparoscopic distal gastrectomies in cases of clinical stage i gastric cancer in taiwan. through the evolution of surgical trechniques and team work, we have cruised the learning curve of laparoscopic gastrectomy and reconstruction. materials and methods: in this report,we would like to present our surgical experience of laparoscopic proximal gastrectomy for gastric cancer patients. from to , pateints with gastric cancer underwent laparoscopic gastrectomies by the same surgical team at the national taiwan university. among them,six consecutive pateints (male:female = : ) with gastric adenocarcinoma of the upper stomach underwent laparoscopic pg in . the demographics, dissection, reconstruction methods and peri-operative outcome are presented. all six patients tolerated the procedure well, onepatient had mild anastomotic stenosis and improved with one session of endoscopic dilatation. one patient needed temporary proton pump inhibitor for controlloing acid reflux. four of the patients were pathological stage i, and the rest two pateint were stage iia and iiia disease. there was no tumor recurrence until now. summary: laparoscopic proximal gastrectomy is technically safe for treating upper third gastric cancers. the long term oncological outcome deserve further observation. introduction: open gastrectomy (og) has long been the preferred surgical approach worldwide for treatment of gastric cancer (gc). nowadays, several randomized, prospective trials have confirmed improvements in postoperative outcomes for laparoscopic gastrectomy (lg) compared to open procedures, with similar oncologic outcomes. however, most part of these studies comes from the eastern countries. material and methods: a prospective non randomized study was conducted with all patients operated of gc at ramón y cajal university hospital from january to december . over patients enrolled, patients underwent lg and og. textbook outcome was defined as the percentage of patients who underwent a complete tumour resection with at least lymph nodes in the resected specimen and an uneventful postoperative course, without hospital readmission. results: a textbook outcome was achieved in . % of patients operated of gc. the outcome parameter 'no severe postoperative complication' had the greatest negative impact on the textbook outcome. a statistically higher number of patients with early cancer ( % vs. . %) and subtotal gastrectomy ( . % vs. . %) were found in the laparoscopic group. no statistically differences were found between open and laparoscopic approach regarding operating time, rate of microscopic margin positivity, hospital stay, number of retrieved lymph nodes, complications, reinterventions, mortality and readmissions. no statistical differences in textbook outcome were found between both groups ( . % vs. %; p = . ). conclusions: laparoscopic gastrectomy for treatment of gastric cancer seems to be safe and feasible with similar textbook outcomes compared to open gastrectomy. introduction: laparoscopic surgery has been increasing for treatment of gastric cancer. however, standardization of this minimally invasive approach has not been reached yet because of its technical difficulties and the concern about oncological safety. the aim of the study was to analyze the outcomes of our learning curve in this complex surgical technique. material and methods: the first consecutive cases of laparoscopic gastrectomy (lg) performed at our hospital from november to february were enrolled. patients were divided into two groups based on the period they were operated. training phase (tp) was considered between and ( cases) and more-developed phase (mdp) between and ( cases). conversion, lymphadenectomy and retrieved lymph nodes (ln), hospital length of stay, mean operative time, complications, reintervention and mortality rates were compared between the two phases of learning curve. results: the number of retrieved ln was higher in the mdp ( ± , vs. , ± , ; p = , ). furthermore, we have also found less complications ( , % vs. , %; p = , ), a decreased reintervention rate ( , % vs. , %; p = , ) and overall mortality ( , % vs. %; p = , ) in the mdp. there were no significant differences in conversion rate, mean operative time, and hospital length of stay between phases. conclusion: although we consider that our learning curve is not completed yet because the average of monitored parameters have not reached a steady state, the improvement on surgical parameters and postoperative course in the last two years have showed our results are near to the best results published in the literature. aims: lymph node (ln) dissection proves to be essential for oncological gastrectomy, given that the presence of ln metastases is very high, even for early gastric cancer ( . % for t a and . % for t b). this way, d dissection for advanced gastric cancer and d ? for early gastric cancer are the gold standard procedures. some teams are using indocyanine green (icg) lymphography to improve their ln dissections, claiming that this technique facilitates the harvesting of small fluorescent ln that, otherwise, would be difficult to identify by conventional laparoscopic methods. methods: we herein present the case of a -year-old man with a t b distal gastric cancer. endoscopic ultrasound discarded the presence of metastatic ln and ct scan showed no distant metastases. icg was administrated endoscopically the day before the surgery, an amount of mg was injected along the submucosal layer around the tumour. in the video we can see how we perform a laparoscopic distal gastrectomy with d ? ln dissection and roux-en-y reconstruction. icg lymphography helped us to complete our expected ln harvesting, especially for groups (infrapyloric) and (left gastric artery). thanks to this technique, we could resect ln that we might have obviate during a usual laparoscopic procedure. results: patient was discharged home on the sixth postoperative day without complications and with adequate oral tolerance. conclusions: we present a case in which we have performed a laparoscopic distal gastrectomy with d ? dissection and roux-en-y reconstruction. we used icg lymphography to help us to improve our ln harvesting. although it is soon to assess if this technique may increase the number of retrieved ln and in which stations might be more useful, we consider this is a harmless method that may help gastric teams to complete their expected ln dissections. introduction: gastrointestinal stromal tumor (gist) represents around . % to % of gastrointestinal neoplasms, with the mesenchymal tumor being more frequent than the digestive one.the gist can be produced from the esophagus to the anus, at any point, being the stomach of ( to %) and the small intestine ( to %) more frequent sites.it is characterized by the expression of the tyrosine kinase growth factor receptor,cd ,differentiating it from other mesenchymal tumors,which do not express it.it is accepted that its origin corresponds to the interstitial cells of ramón y cajal,which act as a pacemaker for intestinal motility.they are very heterogeneous tumors, which vary in size,morphology and biological behavior,being neoplasms with uncertain malignant potential.the incidence is between the fourth and sixth decades,being the distribution by gender similar. clinical case: female patient of years,who goes to the general surgery service,as interconsultation,after a veda,by dyspepsia.it is reported stomach:ceiling mucosa without alterations,at the level of the greater curvature is seen a tumor of cm,hard to the touch with the biopsy forceps,slightly irregular covered with mucosa of normal appearance. computed tomography: stomach body:rounded image of nodular aspect which does not present heterogeneous enhancement after administration intravenous iodine contrast extending to peritoneal region, measures x x mm liver:hypodense image without heterogeneous enhancement adjacent to this,a mm rounded image that is suggested to be studied with nmr. gadolinium nmr liver hypodense image with well-defined limits without heterogeneous enhancement of cystic aspect. gastric roof,heterogeneous formation,which enhances with gadolinium mmx m-mx mm,having to discard a gist. surgical technique laparoscopic partial gastrectomy. pathological anatomy and immunohistochemistry . cm injury with net edges.uncertain malignant fusocellular nodule, cd ??? actin-dog ??? s -no mitosis or invasion of the mucosa is observed. conclusion: a case of stomach gist is presented,which,the main symptom was dyspepsia,being the clinical presentation very variable,in relation to the place in which it is located. there is fletcher criteria for the risk of malignancy,this being less than . cm,very low risk,less than cm, the patient evolved favorably,without surgical complications.aims: to present the surgical procedure of resection of the lesser gastric curvature and its pedicle with laparoscopic surgery, fulfilling oncological criteria, carried out in the general surgery service of the hospital of torrecárdenas. methods: an -year-old man with prostate cancer treated with complete hormonal block and epoc, who consults for rectal bleeding of week of evolution. it is diagnosed of gist in gastric lesser curvature, x x cm, very vascularized and infiltrates the wall producing marked imprint on the fundus. it is tributary of left gastric artery. precise blood transfusion and presents hemodynamic stability, is decided surgical resection scheduled. results: the surgery is performed by laparoscopy, with a tumor of approximately cm, which is dependent on the lesser curvature. the esophageal hiatus and the lesser curvature are dissected with section of the left gastric pedicle. atypical gastrectomy of the lesser curvature including gist, making a gastric sleeve dependent on the greater curvature. the anatomopathological study reports pt pn with lymph nodes without adenopathies, and disease-free surgical margins. he was discharged without complications on the th day and did not require re-entry. conclusions: the laparoscopy surgery for atypical gastrectomy of lesser curvature is safe and meets oncological criteria in selected patients and performed by an experienced esophagogastric unit. aims: to present the surgical procedure of total gastrectomy with d lymphadenectomy with laparoscopic surgery, fulfilling oncological criteria, carried out in the general surgery service of the hospital of torrecárdenas. methods: a -year-old male with a tobacco habit who consults due to epigastric pain and constitutional syndrome of months of evolution. it is diagnosed of gastric adenocarcinoma t n m . neoadjuvant qt is decided, after weeks of its completion, scheduled surgery is performed. results: the surgery is performed by laparoscopy, showing a stenosing tumor in at gastric antrum of approximately cm. dissection of the greater curvature with section of the right gastroepiploic at its birth and duodenal section is performed. dissection of the lesser curvature with d lymphadenectomy, section of the pedicle of the left gastric and the distal esophagus. the transit is restored with latero-lateral esophageal-jejunal anastomosis and jejunojejunostomy. the anatomopathological study reports ypt a and pn with / adenopathies, and disease-free surgical margins. he was discharged without complications on the th day and did not require reentry. conclusions: the laparoscopy surgery for total gastrectomy with complete d lymphadenectomy is safe and meets oncological criteria in selected patients and performed by an experienced esophagogastric unit. background: in gastric cancer surgery, to secure surgical margin, it is necessary to accurately judge the position of the tumor. however, with conventional marking clips, it is difficult to identify the exact location of the tumor during laparoscopic surgery. purpose: we investigate whether icg (indocyanine green) fluorescence navigation method is effective and safe for determination of cutting line in laparoscopic gastrectomy. patients and methods: subjects underwent laparoscopic gastrectomy (including robot-assisted surgery) based on the icg method for gastric cancer in the period from april to december . the day before surgery, icg diluted times ( . ml of reagent ? . ml of distilled water) was injected at cm from the tumor edge and . ml at the four submucosal layers around. then clip to the same part. gastrectomy based on standard surgery is performed, and the position of the tumor and spread of icg are confirmed by icg fluorescence navigation during operation, and a cutting line is determined. the extent of icg from the tumor is again measured with the excised specimen and compared with the pathological margin. results: among the patients who underwent intraoperative pathological examination, they were negative in all cases except one. the spread of icg was . cm on average, and considering the marking position ( cm) from the tumor edge, securing of . cm or more was possible. the operation time was . ± . min and the estimated bleeding loss was . ± . ml. conclusion: laparoscopic gastrectomy with icg method can evaluate tumor position and spread easily and in real time during operation and it was effective for determining the cutting line in laparoscopic gastrectomy. epstein-barr virus (ebv) has been known as one of causal virus of gastric cancer. ebv-related gastric cancer considered to be about % of the entire gastric cancer, and it is rare that ebvrelated gastric cancer has multiple lesions. the patient was years old female. she was diagnosed with upper gastrointestinal endoscopy with lesion in the lower major stomach body, lower anterior wall of the stomach body, rear wall in the middle part of the stomach, rear wall in the middle part of the stomach, and lesser curvature of the stomach angle, as a result of biopsy, adenocarcinoma was observed from the former four. the patient underwent a robot-assisted total gastrectomy. adding a newly found lesion, the histopathological diagnosis was pt b in the lower major stomach body, pt b in the lower anterior wall of the stomach body, pt b on rear wall in the middle part of the stomach, pt b on rear wall in the middle part of the stomach, and pt a in lesser stomach body, pn , pstageia. pathological examination results showed that the four lesions were positive for tumor cells in eber in situ hybridization and were considered to be ebv-related gastric cancers. she was discharged on the th day after the operation without any postoperative morbidities.there has been no sign of recurrence without postoperative therapy for months. results: a -year-old female with no medical history of interest or allergies to medications, who consulted for palpable mass at mesogastric level to the left of the midline associated with abdominal pain of - months of evolution, without concomitants or relationship with the intake, valsalva or physical efforts, without change in the depositional habit or toxic syndrome. the abdominal ct (computed tomography) revealed a cystic mass in jejunum mesentery, defined edges, about cm in diameter and that does not capture contrast; likewise, there is no ascites, retroperitoneal adenopathies or other intra-abdominal or pelvic masses, radiology recommends completing the study with abdominal mri (magnetic resonance imaging) that informs of possible lymphangioma at the level of the jejune mesentery. surgical exeresis was decided, which was carried out by laparoscopic approach, with emptying of the lesion and enucleation of the lesion without incidents, the postoperative evolution was favorable being discharged at h. the pathological anatomy reported fibro-adipose tissue with presence of lymphatic dilatations associated with a cystic lesion without epithelial lining, with serous fluid and abundant macrophagic reaction compatible with mesenteric lymphangioma. conclusions: the mesenteric cyst is a rare pathology with an incidence ranging from / , to / , , predominating in the fourth decade of life. it is defined as any cystic lesion in the mesentery, and is subdivided according to its origin into lymphatic, mesothelial, urogenital, dermoid, and enteric and pseudocysts. most of the time they are asymptomatic although they can (as in our case) present with abdominal pain and even produce complications such as intestinal obstruction, volvulus, intracystic hemorrhage, infection, rupture, and even malignant transformation. for the diagnosis, the palpation can be of great help, showing mass of well-defined limits and partially mobile. the imaging test of choice is abdominal ultrasound / abdominal ct, supplemented by magnetic resonance imaging. the recommended treatment is surgical exeresis, considering laparoscopy as the first option; if it is complete, it can be considered as a curative treatment. purpose: gastrostomy(og) is an alternative method for nutrition support, especial for the patients with oral-esophagus route obstruction or dysfunction. the most operation were conducted by young surgeon or residents. laparoscopic gastrostomy(lg) was a new coming procedure and the skillful suture techniques were needed. the most the residents can't be qualified for this operation. we designed the method for laparoscopic gastrostomy to provide the traning opportunity of suture skill training and guarantee the patient's safety. material and method: laparoscopic gastrostomy procedure was done with two mm trocar. the lower body of stomach was chose. four point around gastrostomy wound were chose for subcutaneous fixation. the straight needle with - prolene was inserted into peritoneal cavity from upper point, then punctured the sero-muscular layer of stomach. the needle was retrieved out from the same point by guidance of gauge needle. the same way was used for other three points. one purse string around gastrostomy was created by one hand suture method and fastened by köckerling knot tier after insertion of fr foley tube. finally, the peritonization was finished by hand tie externally and knot were keep in subcutaneous layer material-method: we present the case of a year old woman who presented with melena, hematemesis, anemia (ht . %) and being haemodynamically unstable. after the stabilization of the patient, a gastroscopic examination followed, where it revealed a tumor of the fundus (adenocarcinoma). the patient was submitted to laparoscopic total gastrectomy and oesophagojejunal anastomosis, omega type (o), and intestinal anastomosis braun, with the usage of trocars (umbilical mm as inserted in laparoscopic surgery of a single incision, and two mm in the midclavicular line bilaterally). the oesophagojejunal anastomosis was conducted with the use of a linear stapler for the posterior wall and the convergence of the anterior wall with laparoscopic sutures in two layers. patient remains in well condition, months after the operation. conclusion: laparoscopic approach seems to be safe for treatment of gastric cancer of the fundus and of the gastroesophangeal junction, as it offers better surgical field view and less postoperative complications. the restoration of the continuity of the gastrointestinal tube with anastomosis of w type is considered safe alternative to the classic roux-en-y anastomosis. git and bariatric surgery, faculty of medicine, alexandria university, alexandria, egypt; git surgery, faculty of medicine, alexandria, egypt background: superior mesenteric artery syndrome is best described as compression of the third part of duodenum by the superior mesenteric artery, resulting in obstruction. the study of this rare medical condition was carried out since decades yet remain obscure. this study aimed to analyze different clinical presentations, diagnostic modalities, treatment approaches and outcomes, as well as to emphasize the importance of long term follow up. methods: thirty-five superior mesenteric artery syndrome cases were collected retrospectively from a facebook group called 'superior mesenteric artery syndrome awareness & support'. a questionnaire was designed using google form to obtain the demographics, presenting symptoms, risk factors and co-morbidities, investigations, means of treatment and the outcomes. data was entered into microsoft office excel for statistical analysis. results: the median age at diagnosis was years. the median body mass index was . kg/ m ;. the median time interval from symptom onset to initial diagnosis was months. the major presenting symptoms were abdominal pain ( . %), nausea ( . %), and vomiting ( . %). abdominal computed tomography scan with contrast ( . %) was commonly used for confirmation of diagnosis. thirteen cases ( . %) were congenital. thirty patients ( . %) had received treatment. the overall management success was only . %. surgical management ( . %) was the most used regimen. conclusion: diagnosis of superior mesenteric artery syndrome is established after a thorough assessment of the clinical presentations and confirmation with suitable imaging modalities. the choice of treatment should be dependent on the causes and severity as different patients respond differently to therapy. recurrence is possible in all patients thus a long-term follow up is required. aims: in the last hundred years much has been written on peptic ulcer disease and the treatment options for one of its most common complications: perforation. laparoscopic repair of perforated peptic ulcer has been gaining popularity in recent years. treatment for perforated ulcer can be performed laparoscopically in % of cases, making it possible to avoid a median laparotomy which can lead to wound infection and late eventration. methods: a -year-old male presented to emergency room with a three-hour history of progressively worsening epigastric pain and nausea. physical examination revealed rebound tenderness compatible with an acute abdomen. a ct scan showed: important pneumoperitoneum unable to define the drilling point; distended stomach with plenty of fluid inside and dense content fundus / body suggestive of active arterial bleeding . results: the patient was emergently taken to the operating room for diagnostic laparoscopy . perforation shown in greater gastric curvature associated blood remnants. gastrotomy for clot removal is done without observing active bleeding. the gastrotomy was repaired using standard stitches. all exudate was aspirated and the peritoneal cavity was irrigated with warm saline solution the patient had an uncomplicated post-operative course. jp drain was removed and he was discharged one week after surgery. conclusion: the role of laparoscopic surgery in emergencies is well documented. laparoscopic approach is indicated in any case of suspected gastroduodenal perforation and seems to offer the same advantages as for the vast majority of laparoscopic procedures. laparoscopic surgery may therefore have a real place in the treatment of perforated peptic ulcer. the aim: of our study was to evaluate of effectiveness of local injection of platelet-rich plasma for treatment of peptic ulcer bleeding with hemorrhagic shock in experiment. methods: the study was performed on wistar rats according to local and international rules for working with experimental animals. the average weight of animals was ± grams. in all animals our modification of type acetic acid ulcer (susumu okabe, ) was modeled. we randomly divide all animals in groups. rats with only modeled ulcer were included in group . rats with modeled ulcer and hemorrhagic shock after - . ml blood sampling were included in group . in group we included rats with modeled ulcer and hemorrhagic shock and performed local injection of platelet-rich plasma (local periulcelar injection of . ml of autologous platelet-rich plasma). on st, th and th day measurement of the ulcers square and morphological study were performed. results: the data we have received demonstrate a tendency of decrease of ulcers' square in all groups with time flow. we also compared sizes of ulcerative defects in all groups at every point of the study. on the st day of investigation there were no differences (p [ . ) between ulcers' square in all groups. on the th day we found out more rapid decrease of size in group (p [ . ). however, this tendency had no statistical significance. on the th day difference was larger and it was statistically significant this time (p \ . ). also the better ability to stimulate the activity of fibroblasts and revascularization in the young connective tissue with improving oxygenation in the ulcers and enhancing of cell proliferation, differentiation and accelerating of maturation of connective tissue and healing of ulcers was demonstrated in group . conclusion: platelet rich plasma reduces inflammatory response and stimulates proliferation of gastric epithelial cells on th day with the restoration of secretory activity and epithelialization of ulcers in . % of experimental animals on th day, the activation of the fibroblastic reaction during the all experiment and decreasing of ulcers' square. h. fujii, depat. of surgery, japanese red cross fukui hospital, fukui, japan introduction: in conjunction with charmant, a local eyeglass frame manufacturer, we developed novel devices called the fj (free jaw) clip to grasp organs in the abdominal cavity and the f (free) loop plus to pull thread extracorporeally from within the abdominal cavity. product summary: the fj clip is used to grasp organs in the abdominal cavity, a stainless steel, removable forceps for use in laparoscopic surgery. it provides a strong grip but rarely crushes organ tissue. the clip comes in two sizes, one for use in a -mm port and the other for use in a -mm port, and in two lengths, . mm and . mm, respectively. to pull out thread tied to the fj clip, we developed the f loop plus, which is a g by -mm-long special stainless needle with f . -mm niti alloy thread which is used pull suture threads from inside the abdominal cavity to outside the body. case: we performed cases of reduced port laparoscopic and endoscopic cooperative surgery (lecs). we performed reduced port surgery (rps) by making a . -cm incision at the umbilicus, inserting trocars ( mm and mm), and inserting another trocar ( mm) at the left side of the abdomen. we expanded the left hepatic lobe with a -mm fj clip for penrose drain placement, grasped the front wall of the gastric body with a -mm fj clip, applying traction toward the legs to pull up the tissues around the tumor, and resected all layers of the tumor via oral endoscopic submucosal dissection technique. the resected area was closed with a suturing device or interrupted sutures in the abdominal cavity. a year-old female was admitted to the emergency department with complaints of abdominal cramping pain, back pain and diarrhea for one day. she also had fever, ever up to °. in these two weeks, she felt occasionally epigastric pain. her past medical history included hypertension. on physical examination, she was conscious and alert. abdominal examination revealed diffuse tenderness and knocking pain over right flank. laboratory tests indicated an degraded white cell count of /cumm with % band forms, c-reactive protein of mg/dl and abdominal liver function tests (alanine aminotransferase: u/l, alkaline phosphatase: u/l, gammaglutamyl transferase: u/l) without hyperbilirubinemia. abdominal x-ray showed paralytic ileus. our presumptive diagnosis was acute peritonitis, based on the patient's symptoms. empirical antibiotics were administered immediately, and a computed tomography (ct) imaging study was performed. the ct scan showed a stick like foreign body noted between ventral side of pylorus and smv lumen, about . cm in length and associated with perifocal infiltration and segmental smv thrombus formation. (fig. ) however, there is no obvious pneumoperitoneum and no evident ascites is associated. an emergency exploratory laparotomy was performed, revealing stomach perforation at posterior wall with a cm fish bone thourgh pancreas into smv. localized inflammation and fibrosis were identified without obvious fluid accumulation( fig. - ) . removal of fish bone and simple closure of stomach perforation were performed. blood cultures revealed bacteroides thetaiotaomicron. three weeks later, she received a follow-up ct scan which showed smv obliteration with chronic pylephlebitis. aim: here we present a case report about the endoscopic treatment for iatrogenic gastric perforation secondary to a chest tube insertion. methods: a case report of a -year-old male with history of a road traffic accident. described injuries were severe brain injury with gcs \ at pre-hospital care arrival, thoracic injury with several rib fractures on the left hemithorax and hypoventilation on the left side. prior to hospital transfer a chest drain was inserted on the left side, and the patient was intubated. results: at hospital admission, the patient was hemodynamically stable and connected to a mechanical ventilator. thoracic exam showed persistent hypoventilation on the left chest. no other abdominal or pelvic injuries were found in the physical exam. a frontal chest x-ray revealed pneumothorax and the chest tube was not viewable. a further ct scan showed the chest drain placed in the abdominal cavity, into the stomach, besides a subdural hematoma, comminuted pelvic fracture of the pubic rami and a left sacroiliac fracture. during the first h in the icu, neurological worsening was observed, and a new cranial ct revealed enlargement of the subdural hematoma, for what the patient underwent decompressive craniectomy, with improvement thereafter. following a five-day period of stabilization after surgery, the patient was evolving satisfactorily, and the removal of the intragastric chest drain was considered. endoscopy was performed to confirm the placement of the drain, and it was removed under direct vision. approximately twenty five centimeters of the catheter were visualized in the gastric lumen, and then successfully removed. the patient recovered well and was discharged from icu to medical hospital ward after fourteen days, and a week later he was discharged home. conclusion: endoscopic management for gastric perforation after a chest drain insertion may result effective and can prevent open surgery morbidity. aims: intestinal infusion treatment with levodopa/carbidopa (duodopa) is a therapeutic option concerning the advanced parkinson disease cases with no response to the conventional treatment. the drug requires carrying out a gastrostomy either by percutaneous endoscopy way, or by laparoscopy -if the first one is not possible-. later, a duodenum-yeyunum tube is placed in order to infuse the duodopa gel continuously by a portable bomb. in this report, we explain the laparoscopic gastrostomy technique. method: sin this report, we include two patients with advanced parkinson disease: the first one is a year-old female patient suffering from an important gait disorder; and the second one is a year-old male patient with uncontrolled motor fluctuations. in both cases, a percutaneous endoscopic gastrostomy was proposed, but neither was feasible because of the non-traslumination between the gastric and the abdominal wall. under general anesthesia, neumoperitoneum by veress needle was performed. three main trocars and one accessory were placed. at the level of the gastric antrum, a cm incision was conducted to insert a gastrostomy tube, to be the guide for the drug infusion catheter. next, the gastrostomy is fixed to the abdominal wall by the stamm technique, externalizing the catheter through the accessory trocar in the medial line. results: on the first post-operative day, a duodenum-yeyunum tube is placed by endoscopic control through the gastric device. both patients got well satisfactorily, and no complications were described; and they develop a total normal life within the limitations of their underlying disease. conclusions: the duodopa intestinal infusion shows a significative improvement for the advanced parkinson disease symptoms, compared with oral medication; appreciating positive results referring to life quality. when the catheter placement by endoscopy way does not seem posible, gastrostomy by laparoscopy constitutes a valuable surgical option for the treatment of this kind of patients. peptic perforated ulcus (ppu) is a common surgical emergency and laparoscopic repair has been introduced as an alternative to open repair. it has shown good results and allows closure and peritoneal lavage, just like the open repair does but with the advantages of a minimally invasive surgery. the objective is to report the outcome of laparoscopic ppu in our hospital. methods: from january to october , patients with a clinical diagnosis of ppu were assigned to undergo laparoscopic repair. this retrospective study included all husm patients who underwent laparoscopic ppu repair by emergency surgeons. minimum follow-up of months is carried out. results: of the patients in this series, % were men and % were women, between and years of age at the time of surgery, average of years. the time between the manifestation of symptoms and surgery was [ to h in % of patients. in patients there was a history of previous ulcer or non-steroidal anti-inflammatories intake and up to % were smokers. a ct scan was performed in all cases to reach the diagnosis primary closure with simple suture plus omental patch was the elected technic ( %). the approach was performed with trocars in %, trocars in % and in case. cases ( %) were gastric ulcer, duodenal cases ( %) and in one case no perforation was found. the conversion rate was %, in two cases due to technical difficulty and in the other case because the level of the perforation was not found. the median postoperative stay was days although there were cases with intrabdominal complications. there was an exitus due to a metastasic pulmonary neoplasia diagnosed in the immediate postoperative period. there were no cases of recurrence in the follow-up time. conclusion: in most centers, including ours, the rate of laparoscopic management has gradually increased along with the improvement of technical skills. improvements in the outcome of laparoscopic ppu repair are to be expected with more experience surgeon and a good selection of the cases. general surgery, jzu hospital ,,sveti vracevi,, bijeljina, bosnia-herzegovina introduction: diverticulum is an outpouching of a hollow organ. gastric diverticulum is rare form od this disease. incidence of detection varies depending on investigation method. it has been reported in . % cases of autopsies, . % cases of gastroduodenal roentgenographies with contrast, and . - . % cases of upper endoscopies.small diverticula are usually asimptomatic, but bigger diverticula can cause variable symptoms such as abdominal pain, feeling of epigastric fullness right after meal, feeling of discomfort in upper parts of abdomen, and severe 'foetor ex ore' .diagnosis is usually established in procedures such as gastroduodenal roentgenographies with contrast, upper endoscopies and abdominal ct scan. case report: a -year-old woman came to our hospital because of feeling of discomfort and mild pain in upper abdomen that lasted for last year. diagnosis is established after ct scan of abdomen and upper endoscopy procedure. initially she has been prescribed conservative therapy (proton pump inhibitors). since the symptoms persisted, laparoscopic resection of the gastric divertuculum was performed using endogia stapler. considering the feeling of discomfort and abdominal pain dissapeared, the patient was discharged from hospital on the fourth postoperative day. conclusion: asymptomatic gastric diverticula doesn't require treatment. since gastric diverticulum can have complications such as bleeding, perforation and neoplasia, patient without symptoms should be monitored. initial therapy for symptomatic diverticula is conservative therapy (proton pump inhibitors). if conserative therapy doesn't procude expected results, laparoscopic resection of the diverticulum should be considered. introduction: the acute perforation of a gastric ulcer is a serious entity that requires urgent surgical treatment in most of the occasions, it is increasingly accepted that the approach of choice is laparoscopic, depending, above all, on the time of evolution of the process. objectives: to demonstrate the safety and efficacy of the laparoscopic approach in the perforation of a pyloric peptic ulcer, even in cases of severe peritonitis, by means of a standardized procedure, insisting on the sequential thorough washing of the cavity.material and methods: we present a video of the surgical intervention of a patient with acute abdomen, with a history of nsaid ingestion, exploration and ct-analysis compatible with perforation of hollow viscus, probably of gastric origin. results: intervention: complete laparoscopic approach, trocars. severe biliopurunitic peritonitis, by pyloric perforation 'acute', liquid culture, suture of the perforation, epipoplasty, sequential thorough washing of the cavity with physiological saline and placement of drainages.correct postoperative period, discharge from the hospital on the th day after completing antibiotic treatment. endoscopy and helycobacter test are performed on an outpatient basis with normal results. conclusion: the laparoscopic approach is safe and effective in acute and complicated gastric ulcer disease, even in cases of severe peritonitis. surgical procedure: the clean-net procedure involves the selective dissection of both the serosa and muscle layer using a laparoscopic monopolar endoscopic scissor. the preserved mucosal layer provides a mechanical barrier between the gastric lumen and peritoneal cavity that aids in the prevention of peritoneal cavity contamination with gastric contents. tumors are observed with an upper gastrointestinal endoscope with the injection of indocyanine green (icg) into peri-tumoral submucosal layers at points. selective seromuscular dissection is performed using a laparoscopic electrocautery monopolar scissor. the mucosa surrounding the gist is then resected using a endoscopic mechanical stapler to prevent exposure of the gastric lumen to the peritoneal cavity and peritoneal tumor cell seeding. results: there were males and female, and the average age was years. the operation time was min, the average bleeding volume was . ml, the postoperative hospital stay was . days. the mean tumor diameter was . mm, the final histopathological diagnosis was gist, schwannoma. there were no postoperative complications of clavien-dindo classification or more. conclusion: clean-net was found to be safe and useful for the treatment of gastric smt with ulceration. year outcomes: laparoscopic heller myotomy stands the test of time aims: laparoscopic cardiomyotomy leads to excellent relief of dysphagia in % of patients and avoids thoracotomy or laparotomy. methods: we present a video illustration of the procedure that was modified at the american university of beirut medical center. so far, patients underwent laparoscopic cardiomyotomy, age range of to years, with males and females. most of them have had previous balloon dilatation. results: all cases were successfully completed laparoscopically without complications. followup of months to years revealed excellent results with complete resolution of symptoms and no need for further medications. this will result in minimal post-operative pain and very short recovery period and is associated with low complication rate. conclusion: cardiomyotomy for achalasia is ideal for laparoscopic approach. magnification allows for precise division of muscle fibers. the new technique of hydro dissection and enseal for division of esophageal muscle allows for completion of the procedure without injury of the mucosa. therefore, adequate release of the obstructing segment followed by anti reflux procedure toupet will lead to excellent results with minimal morbidity and no mortality. aims: laparoscopic repair of huge hiatus hernia methods: twenty two cases of huge hiatus hernia presented to the american university of beirut medical center. patients underwent through trocars in the upper abdomen reduction of the hernial sac from the chest. special care was taken in the dissection of the mediastinum to keep the thoracic fascia and pleura intact. the defect was sutured primarily by -ethibond sutures reinforced by onlay prolene mesh u-shaped was fixed at the rt. and lt. crus and a floppy nissen fundoplication performed . results: the video presentation includes the technical aspects and the method of reducing and repair of huge hiatus hernia.aim: nowadays, there is little experience in the world of applying robotic surgical system (rss) in treatment of patients with hiatal hernia (hh) and reflux-esophagitis (re). the aim of study was to determine the possibility and feasibility of using rss in treatment of patients with hh. materials and methods: a total of patients underwent robot-assisted hh repair without mesh, followed by fundoplication with our original method ( °full symmetric wrap). the clinical and technical analysis did not reveal any advantages over similar laparoscopic procedures, so we abandoned the use of rss for hh type i, and these patients were excluded. there were ( %) patients with hh type iii and ( %) type iv. the surgeries were performed by experienced robotic upper gastrointestinal surgeon and conducted with the davinci si surgical system (intuitive surgical, sunnyvale, ca). results: average operation time was ± ( - ) min. the respondents' mean age was . ± . years (range - ) and bmi was . ± . (range . - . ) cm/kg . average blood loss was ± ( - ) ml. average hospital stay was ± . ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) days. the average follow-up time was ± . ( - ) months. postoperative x-ray imaging and upper gi endoscopy was conducted in all ( %) patients. there was no hh recurrence diagnosed. we did not observe a relapse of hh or clinical manifestations of re in the early (less than days) and long-term (more than months) postoperative periods. conclusion: we can conclude that robot-assisted surgery is safe, appropriate and justified in patients with hh type iii and iv. all procedures performed to the patients with giant hh revealed clear technical advantages of rss over similar laparoscopic operations: an enlarged d hd image, bendable instruments with endowrist technology allowed for precise dissection of tissues (hernial sac, cicatricial adhesions) in a narrow anatomical space-posterior mediastinum without damage to pleura, pericardium and vagus nerves. we believe that use of rss in treatment of patients with reflux esophagitis and/or hh type i is unjustified, due to the lack of proven advantages over laparoscopy. introduction: the presence of major anatomical obstacles -such as massive caudate lobe-in the confined operative field of laparoscopic hiatal hernia repair (lhhr) poses significant challenge to the foregut surgeon. aim: to provide a safe alternative for lhhr using a laparoendoscopic approach. method: this patient is a year old female, with bmi of . . her past medical history includes diabetes, hypertension and hyperlipidemia. she had gerd for years. her egd showed x cm hiatal hernia and class b esophagitis. manometry showed ineffective esophageal motility. we used the classic five ports approach for lhhr. we found a massive caudate lobe which was comparable to the size of an already enlarged left lobe of the liver. the operative strategies: terminating the procedure proceeding with the standard approach and taking the risk of bleeding from the caudate lobe itself or the inferior vena cava (ivc) with possible catastrophic outcome. using the laparoendoscopic approach. the following three steps facilitated the performance of safe and effective surgery. additional liver retractor this improved exposure and minimized manipulation of the caudate lobe. extracorporeal sliding arthroscopic knots (esak) esak are similar to the knots used in endoloop. they are tied extracorporeally and require a single insertion of the knot pusher as they do not unravel. transoral incisionless fundoplication (tif) we performed tif to avoid a limited operative field and to prevent excessive tissue manipulations associated with laparoscopic fundoplication. tif also preserves the angle of his and produces partial fundoplication which has less side effects of dysphagia and gas bloat syndrome. results: the operative time was min (lhhr min and tif min). there were no complications. patient discontinued omeprazole which she used daily for years. at months follow up, her gerd related quality of life (hrql) and gerd symptom (gerss) scores were ( vs ) and ( vs ). conclusion: the laparoendoscopic repair of hiatal hernia in the presence of anatomical obstacles is safe and effective. longer follow up is needed to assess the durability of this repair. gastroesophageal reflux disease (gerd) is a condition that reduces the quality of life and can causedisorders associated with acid reflux, such as bronchial asthma, barrett's esophagus and esophagealadenocarcinoma. gerd is often caused by existing of hiatal hernia. nowadays, some surgeons haddifficulties with the laparoscopic approach to treatment of recurrent hiatal hernias.patient was a -year-old man. he requested medical assistance with dysphagia, nausea after eatingand heartburn getting worse in a horizontal position. conservative treatment was not effective.transthoracic nissen fundoplication was performed in . the main complaints of the patientpersisted during the postoperative period. the upper half of stomach and s-like curved esophagus werelocated in the mediastinum according to multislice computed tomography of the thorax in august.in our clinical center was performed laparoscopic cruroraphy, cardiomyotomy and nissen fundoplicationin november. during the surgery the normal anatomical position of stomach has been restored, s-like curve of esophagus has been removed; a gastric cuff (collis-nissen) has been created and anteriorand posterior cruroraphy has been performed. the patient was in intensive care during h. anasogastric tube feeding was continued during the first h. passage of the contrast through theesophagogastric junction was free within h after surgery. patients had been discharged within days after surgery.this case report shows that at the current stage of surgery laparoscopic approach can be useful not onlyfor treatment of primary hiatal hernias-but also for treatment of recurrent ones. aims: laparoscopic heller myotomy procedure, completed with an anti-reflux procedure is technically demanding. we report a case of laparoscopic heller myotomy followed by a dor anterior fundoplication. methods: this is the case of a -year-old caucasian woman with gradual dysphagia for solids and liquids, accompanied by severe regurgitation and chest pain. an initial diagnosis of achalasia was made in , with the use of manometry and barium swallow. endoscopic dilatations were attempted pre-operatively with no clinical improvement. decision was made to perform a laparoscopic heller myotomy, combined with a standard dor anterior fundoplication. a -ports operation took place (one intra-umbilical mm trocar-single incision laparoscopic surgery (sils) technique, two -mm subcostal trocars, and one another mm subcostal trocar for the use of liver retractor). the operation lasted h and min. results: no post-operative complications were noted. the post-operative swallow test showed improvement of the esophageal patency. the patient started a liquid diet three days later and was discharged six days post-operatively. two months later the patient presented no complications. conclusions: heller's myotomy has demonstrated good long-term results in the treatment of esophageal achalasia and the laparoscopic approach has been well established in the last two decades. it is a very demanding operation to perform and the disease is relatively rare, making the learning curve difficult to achieve. aims: achalasia is a type of motor disorder of the esophagus due to degeneration of ganglion cells in the myenteric plexus, leading to failure of relaxation of the lower esophageal sphincter, accompanied by a loss of peristalsis in the distal esophagus. the association of a long-term achalasia and a large size hiatus hernia is an infrequent entity. among the therapeutic options is medical treatment, endoscopic treatment and surgical treatment associated with an antireflux procedure. the laparoscopic approach being the more indicated due to its better results in terms of morbidity, mortality and recurrences. the aim of the video is to show the effectiveness and safety of the laparoscopic approach in this infrequent pathology, pointing out the importance of performing a standardized procedure. methods: -year-old male patient, with personal history of chronic ischemic heart disease and obesity, diagnosed with long-term achalasia with moderate dilatation of the esophagus associated with giant hiatus hernia. the complementary explorations and iconography of interest are exposed. results: intervention: complete endoscopic approach, trocars. reduction of hernial content into the abdominal cavity, dissection of the hernial sac and esophageal lipoma. extended mediastinal esophageal dissection. complete resection of both the sac and lipoma, respecting the posterior vagus. heller's myotomy of cm, including cm distal to the ueg, perforation of mm of the mucosa at the ueg level, suture and blue methylene verification of the sealing. hiatorraphy and dor-type anterior fundoplication as antireflux technique. correct postoperative, with egd control on the rd po day and discharge on the th. asymptomatic at months after surgery. conclusion(s): for achalasia laparoscopic heller myotomy with a partial fundoplication should be the treatment of choice in patients who are at low surgical risk. the length of the myotomy, especially distal to ueg is one of the most important aspects of the surgery, to achieve an effective disruption of the les. the presence of a giant hiatus hernia makes the procedure difficult, increasing the risk of complications, such as perforation. standardization is essential to increase safety and efficacy in these complex cases. purpose: there is evidence that the application of mesh-reinforced hiatal repair has resulted in a significant reduction in recurrence rates in comparison with primary suture repair, at least in short-term follow-up. however, and instead of this, the standard of care for repairing large paraesophageal hiatal hernias (lphh) remains controversial because no clear guidelines are given regarding indications, mesh type, shape and position. the aim of this study is to evaluate our short-term outcomes in management of lphh with a biosynthetic monofilament polypropylene mesh surrounded by a high-purity and adherent titanium dioxide surface coating to enhance the biocompatibility (tio mesh tm ). methods: a retrospective study was conducted on our institution between december and october . data were collected on patients with lphh greater than cm in which a laparoscopic repair was carried on by primary suture and additional reinforcement with a tio mesh tm . clinical and radiological recurrences, dysphagia and mesh-related complications were investigated. results: there were females and males with a mean age of years (range, - years). all operations were completed laparoscopically. median postoperative stay was days. after a mean follow-up of months, patients developed clinical recurrence of reflux symptoms ( . %) and radiological recurrences ( . %). there were no mesh-related complications. conclusions: the use of tio mesh tm for laparoscopic repair of lphh is suited and with a reasonably low recurrence rate in this short-term study. additional long-term studies with enormous numbers carried out for years will be necessary to affirm whether this mesh is convenient in the prevention of recurrences and mesh related complications. background: surgery for refractory gastroesophageal reflux disease (gerd) has a satisfactory outcome, however sometimes fundoplication fails and redo surgery is required. several publications have investigated the feasibility of performing reoperative fundoplications using laparoscopic techniques. the aim of this study was to describe our experience in laparoscopic redo fundoplications in the last years. material and methods: we retrospectively reviewed consecutive patients who required laparoscopic redo fundoplication from january to august .the indications were recurrent symptoms of gastroesophageal reflux disease (gerd) ( . %), recurrent symptomatic paraesophageal hernia ( . %), dysphagia ( . %) and acute volvulus ( . %). results: all redo fundoplications (basically toupet . % and nissen . %) were completed laparoscopically. the mean operative time was min (range, - . min). a mesh was placed in % of cases. intraoperative and postoperative complication rates were . % and . % respectively. the mean hospital stay was days (range, - days). one patient ( . %) from the laparoscopic group required a third operation-one for acute recurrent paraesophageal herniation of the redo wrap one month after surgery, which was repair laparoscopically again. symptomatic outcome was successful in . % without any kind of proton bomb inhibitors therapy. conclusion: laparoscopic redo fundoplication is technically feasible and clinically effective with a reasonable low rate of postoperative complications p -upper gi-reflux-achalasia objectives: in recent years, balloon dilatation (bd) for diseases requiring correction of the impaired patency of the sphincter zones of the esophagogastroduodenal region has become widespread. purpose: to assess the effectiveness of the use of the balloon dilatation in patients with impaired sphincter zones of the esophagogastroduodenal region. materials and methods: in the institute department of surgery for the period from to , bd was performed in patients. of them diagnosed with achalasia of cardia (ac): - stage, - stage, - stage, - stage. patients diagnosed with pylorospasm, patients had compensated stenosis and patients had subcompensated ulcerative pyloroduodenal stenosis. there were males, females, average age ( . ± . ). bd was performed under endoscopic and / or x-ray control by 'boston scientific' balloons with a diameter of - mm, mm and mm, a course of - sessions with an interval of - days and a cylinder exposure of - min. evaluation of bd was performed using esophagogastroscopy, balloon manometry and x-ray passage of barium. results: in the course of the study, the existing indications were refined and new indications were developed for performing an endoscopic bd in pyloroduodenal stenosis and in ac. in patients with stage - ac, a positive result was noted in . % of cases already after the first session of bd. recurrences of ac after bd for up to years were established in ( . %) patients: at stage -in . %, at stage -in . %, at stage -in . % and at stage -in . %. repeated bd courses in case of ac recurrence in ( . %) cases turned out to be ineffective. recurrence of pyloroduodenal subcompensated stenosis was diagnosed in . % of cases in the period of months after performing bd. conclusions: bd is an effective method for correcting the permeability of the sphincter zones caused by the pathology of the esophagogastroduodenal region. keywords: balloon dilatation, achalasia of cardia, pylorospasm, ulcerative pyloroduodenal stenosis, recurrences. introduction: the reoperation in antireflux surgery significantly increases morbidity and mortality up to - %, reaching rates of % in patients undergoing or more surgeries. the advantages of laparoscopic surgery used in this surgical technique have amplified its acceptance and use, resembling its results in terms of feasibility, safety and efficacy of laparoscopic surgery to open surgery.objective: :evaluate the currently literature about antireflux surgery reintervention, focusing on the main indications of re-intervention, type of approach and morbidity and mortality of laparoscopic antireflux surgery. material and methods: a literature search was conducted in two electronic databases, med-line and embase. the search was limited to the period to . terms were used in relation to the procedure or intervention and the underlying disease. we chose observational studies (cohort, cases and controls and series of cases), where the main indication for antireflux surgery would have been gastroesophageal reflux disease. results: a total of studies were selected, most of them were case series ( . %), cohort studies ( %) and case-control studies ( . %). a total of patients. the main indications were anatomical faults, of these failures, recurrent hiatus hernia and sliding occupy the highest percentage, while physiological failures, failure in esophageal and gastric motility occur more frequently. the main type of approach was laparoscopic in %, the conversion rate was . % and the open approach was reserved for complex cases with more than one re-intervention . %, for abdomen . % and chest . %, this last for cases with high esophageal lesions that can not be repaired via trans-abdominal.the main complications were injuries to hollow viscera, such as: esophagus and stomach among others. these complications are related to the complexity of the procedure. mortality has remained low up to . %, however, the cause of death was due to medical complications and not related to the procedure. conclusions: this systematic review on reoperation in reflux surgery has confirmed that morbidity after reoperation surgery is higher than after primary surgery and reoperation indications increase with the use of new technologies (manometry) and the laparoscopic approach continues on the rise, with great adaptation to its use and improvement in results. aims: eras protocol is not commonly used in acute emergency procedures. elective lc is commonly performed as one day surgery, while in an emergency setting of acute cholecystitis, the in hospital stay averages , days. the aim of this trial is the application of eras protocol in patients with acute cholecystitis, undergoing laparoscopic cholecystectomy. methods: a randomized prospective trial was conducted in first surgical department of sismanogleion g.h.a. the study included patients, who were admitted with acute cholecystitis and underwent lc into h from their admission. preoperatively, they all received crystalloid isotonic solutions and antibiotics. . % were submitted to ercp, preoperatively, due to choledocholithiasis. the postoperative care included early mobilization into h after surgery, early fluid intake (into h) and early liquid food intake (into h). they all received systematically antibiotics, analgesics and antiemetic on demand. asa score was not an exclusion criterion. results: conversion to open procedure was necessary in . % of patients, whom were excluded from the study. all the rest were discharged into h from the surgery with the guidance to receive oral antibiotics for more days. readmission was necessary for patients, one week after the operation. the first one presented with bile leak and submitted to ercp with stent placement and percutaneous drainage of the intrabdominal collection. the second one presented with choledocholithiasis and underwent ercp with balloon catheterization. conclusion: it is commonly accepted that eras protocol in elective procedure enhances the postoperative recovery while reduces the in hospital stay and cost. in emergency condition eras cannot be applicated preoperatively. however, a modified post surgery application seems to have advantages equal to those observed in elective procedures. aim: laparoscopic cholecystectomy is the gold standard for the treatment of symptomatic cholelithiasis. administration of one single dose of chemoprophylaxis before an elective laparoscopic cholecystectomy is a broadly accepted practice. however, its value is currently questioned, especially in low risk patients. method: this study was conducted in a high volume surgical department. one hundred and twelve patients submitted to elective laparoscopic cholecystectomy were included in this research. a written consent was acquired after thorough patient briefing. half the patients that underwent surgical operation received one dose of antibiotics min prior to the incision and the other half did not receive any chemoprophylaxis. results: the age ranges from to years old. commonest concomitant diseases were arterial hypertension, type ll diabetes, hypothyroidism and respiratory deficiency. approximately % of patients were smokers and % were obese (bmi [ ). the duration of the operations was between and min. intra-operative gallbladder rupture was observed in patients (rate %). all the patients were discharged the first post-operative day and their monitoring continued for more days. in the chemoprophylaxis group, no surgical site infection or other major complication was observed. from the group that did not receive any antibiotics, one patient developed surgical site infection and specifically infection of the surgical port in the epigastrium, which was treated with drainage of the abscess and oral antibiotics administration. no other complications were recorded. conclusion: our study concluded no statistically significant difference between the two patient groups, which depicts that chemoprophylaxis may not be necessary in elective cholecystectomy operations. on the contrary, antibiotics increase the cost of hospital stay and are often accompanied by multiple mild or severe side effects. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - whai nr authors: nan title: esp abstracts date: - - journal: virchows arch doi: . /s - - - sha: doc_id: cord_uid: whai nr nan ofp- - intraoperative evaluation of breast cancer sentinel lymph node metastasis with an automated molecular detection method as an alternative to standard pathological evaluation f. beca * , e. rios, p. pontes, i. amendoeira * centro hospitalar de são joão, anatomic pathology, porto, portugal objective: one-step nucleic acid amplification assay (osna) from sysmex® is an automated system to detect sentinel lymph node (sln) metastasis in breast cancer patients. this assay is based on the detection of cytokeratin mrna amplification by reverse-transcription loop-mediated isothermal amplification. traditionally intraoperative evaluation of sln is accomplished by cytology and/or frozen section, accounting for heavy workload to pathology department. the objective of this study is to compare the osna assay with our standard processing for intraoperative sln evaluation. method: sln from patients with ck + and invasive primary carcinomas where simultaneously evaluated by cytology and/or frozen section and by osna assay. results where compared with gold-standard definitive h&e evaluation (serially sectioned at μm intervals). results: the sensitivity and specificity of osna assay was . % and . %, respectively with a positive predictive value (ppv) and a negative predictive value (npv) of %. for standard intraoperative evaluation, specificity was % and sensitivity was . %. conclusion: osna assay showed similar specificity and higher sensitivity than standard pathologic examination. these findings suggest it can be used as an accurate tool for intraoperative evaluation of slns metastasis and possibly contribute to reducing the need for re-intervention for axillary lymph node dissection. objective: antracycline-based chemotherapy represents a standard of care in breast cancer patients especially who overexpress her- . topoisomerase a has been considered a molecular target for antracyclines and its co-amplification with her- genes has been proposed. in our study we investigated if topoisomerase a overexpression can be used as a molecular marker on predicting response to antracycline-based chemotherapy. method: breast cancers tissues from patients underwent to antracycline-based neoadiuvantchemotherapy and treated with adiuvant therapy were collected into tissues microarrays, and classified according to pathological stage, er, pr, her- and topo a status. the expression of topoisomerase a has been correlated to response to chemotherapy. results: our results show that topo a expression is significantly higher in neoplasms with larger size, nodal metastasis, scant er end pr receptors, and amplification of her- gene (p < , , t-test). cancers in witch high level of topo a has been found exhibit a better clinical response to treatment with anthracycline (p < , , t-test). therefore topo a expression could be considered both as predictive and prognostic marker. conclusion: the employment of topo a as a predictive marker in clinical practice may be useful for the medical therapy of non-endocrine-responsive patients candidate to undergo treatment based on anthracycline, that is no free from adverse effects as cardiomyopathy and leukemia. ofp- - stem cells in triple negative breast cancer and associated in situ lesions m. comanescu * , m. dobre, g. bussolati * institutul victor babes, dept. of pathology, bucharest, romania objective: triple negative breast cancer (tnbc) (negative for expression of estrogen and progesterone receptors (er, pr) and her /neu protein) represent a subtype of breast cancer associated with poor prognosis and highly aggresive behaviour. genetic characterization of stem cell (cd , ....) in this type of carcinoma might provide important data concerning origin and evolution. data of the literature focused specifically on tnbc but, despite the interest shown to stem cells recently, there are no data concerning the genetic characterization of stem cells in the context of cell biology of tnbc as compared with associated dcis. method: we investigated, through immunohistochemistry, the expression and distribution of several stem cell/related antigens, exploring the association of tnbc with dcis and comparing the presence of stem cells in the invasive and in the in situ component. results: the multiplicity of parameters to be detected for characterising stem cells and of the diagnostic materials that are available would prevent a straightforward approach and a sequential combination will have to be planned. conclusion: optimization of detection, identification and characterisation of tumourigenic breast cancer stem cells might permit further identification of targeted treatment. over-and undergrading of breast cancer on core biopsies in comparison to surgical specimens t. decker * , c. focke, d. gläser * d. bonhoeffer medical center, dept. of pathology, neubrandenburg, germany objective: to evaluate the relevance of histological grading (hg) based on core biopsies (cb) for clinical decision making in breast cancer (bc) we evaluated the concordance with hg from surgical specimen (ssp) and the reasons for under-or overgrading. method: cb and related ssp of bcs were prospectively graded according to the nottingham grading system (ssp: g , g and g ). cb/ssp agreement rates and positive predictive values (ppv) of cb based hg were calculated. rates of over-and underestimation of glandular differentiation (gd), nuclear pleomorphism (np) and mitotic activity (ma) were calculated. results: cb/ssp agreement rates came out with % for g , % for g , and % for g . the ppvs of cb based hg were % for g , % for g and % for g . the overgrading and undergrading rates on cb were % and %, respectively. main causes for undergrading in cb were underestimated ma ( %) and a combination of underestimated np and ma ( %). conclusion: whereas overgrading on cb is an exception, undergrading derives largely from underestimation of proliferation. agreement of hg between cb and ssp ranges from almost perfect for g , moderate for g to slight for g tumours. ofp- - comparative histopathology of mammary alterations in women and dog v. deckwirth * , p. kronqvist, m. lintunen, a. sukura * university of helsinki, dept. of veterinary pathology, finland objective: companion animals such as dogs share the same environment with people. mammary tumours are the most prevalent spontaneous neoplasia in intact bitches. more animal models are needed for human mammary tumour research. our objective was to describe equivalent non-neoplastic and neoplastic mammary gland alterations in woman and dog. method: formalin-fixed, paraffin-embedded archived human and canine mammary gland tissue samples. epidemiological data were collected and analyzed from human (n= ) and canine (n= ) patients for the years - . histological comparison was performed with he-stained tissue sections and immunohistochemistry. applied antibodies included ck / , erα, pr, ki , her , p , sma, ecadherin and calponin. results: several comparable benign and malignant mammary gland alterations were identified, consisting of lobular, tubular and ductal as well as mesenchymal of origin. most of these have not been described previously. the epidemiological results show also shared characteristics. conclusion: although there are canine specific lesions there also exist equivalent entities. our results indicate the female canine as a suitable model for translational research. ofp- - st. gallen intrinsic subtyping of breast cancer: influence of proliferation assessment methods c. m. focke * , d. gläser, k. finsterbusch, t. decker * dietrich bonhoeffer klinikum, abt. pathologie, neubrandenburg, germany objective: the st. gallen conference strongly recommends intrinsic subtyping of breast cancer (bc) for therapeutic decisions. we analyzed the influence of different proliferation assessment methods on the bc subtype distribution. method: intrinsic subtyping was performed according to the st. gallen criteria on bcs. proliferation was assessed by mitotic activity index (mai) (area: , mm², cut-off ≥ ) and by several variants of ki -labeling-index counting any nuclear staining of ( ) tumor cells within the hot spot (ki - ), ( ) tumor cells in hpf in the tumor periphery, including the hot spot (ki - periphery), and ( ) tumor cells in hpf, including hot spot, cold spot and an intermediate area (ki - spectrum) using the recommended cut-off of < %. results: of carcinomas, % were triple-negative, % her +, % luminal b her +, and % special type. the rates of bcs after different proliferation assessment methods were: mai % vs. %; ki - spectrum: % vs. %, ki - periphery: % vs. % and ki - : % vs. %. conclusion: subtyping of luminal a and luminal b her neg bcs according to st. gallen is highly influenced by the method of proliferation assessment used. its biological relevance seems to be not unequivocal. ofp- - the utility of cytological smears allied to the osna method for intraoperative analysis of sentinel lymph-node metastasis in breast cancer patients s. foreid * , m. martins, h. pereira, m. oliveira * loures, portugal objective: one-step-nucleic-acid-amplification (osna) is a recently introduced pcr method to evaluate intraoperative sentinel lymph-node metastasis in breast cancer. it assesses the whole lymph-node in a -minutes protocol. many studies have reported it to be more sensitive than frozen section and/or cytology. yet, it is more expensive and takes longer to perform. method: between and at chlc, we have performed the intraoperative analysis of sentinel lymph-node using both osna and cytological smears in a group of patients. results: lymph-node evaluations were done. cases were osna positive ( micrometastasis and macrometastasis). of those, were also positive for tumor cells on cytological smears. cases were negative by both methods and cases were detected by osna but not on cytological smears. importantly, all macrometastasis diagnosed by cytology were confirmed by osna. of notice, in this series, % of the positive cases detected by osna were also detected by cytological smears. conclusion: like previously reported, osna showed a greater sensibility than cytology to diagnose sentinel node metastasis. however, the majority of metastasis can be detected by cytological smears alone. our results suggest that a less expensive and faster cytological analysis should precede osna, and if positive, the latter method could be disregarded. tuesday, september , . - . objective: tumor markers may be concentrated intraductally in early breast cancers. method: to test this hypothesis, her -ecd and ca - of the needle washout fluid from benign lesions, dcis, and invasive carcinomas of the breast were measured using chemiluminescence. the measuring limit for her -ecd ( . ng/ml) and that for ca - ( u/ml) were used as the cutoff values, respectively. results: the proportion of patients with any biomarker elevation was . % in cases of benign lesions, . % in dcis, and . % in invasive carcinomas. thus, biomarker elevation was most frequent in dcis, followed by invasive carcinomas (p< . ). her -ecd values over ng/ml or ca - values over u/ml were s een exclusively in dcis or invasive ductal carcinomas with an extensive intraductal component, supporting the hypothesis. conclusion: our approach may be useful to identify a subset of breast cancer patients who are suitable for intraductal molecular targeted therapy. ofp- - biological significance of proliferation and her overexpression in luminal/oestrogen receptor-positive breast cancer d. jerjees * , a. green, a. benhasouna, a. alshareeda, r. abduljabbar, f. barros, c. nolan, i. ellis, e. rakha * university of nottingham, dept. of oncology, united kingdom objective: in this study, we have compared the biological significance of proliferation; assessed using ki labelling index (ki -li), with respect to her expression in oestrogen receptor positive (er+) breast cancer to assess the impact on growth fraction and biological characteristics of luminal breast cancer. method: well-characterised er + breast cancers were assessed for expression of a large panel of biomarkers (no= ). results: % of the cases showed high ki -li (> %) and % showed her over-expression and both were positively associated with younger age, higher tumour grade, lymph node stage and shorter outcome. both markers were associated with up-regulation of er-coactivators (cd , carm , pi kca&tk ), p-cadherin, p and with lower levels of er expression and down-regulation of er-related genes (pgr, ar&gata ) and brca . neither of the two markers was associated with expression of basal-associated cytokeratins. high ki -li was associated with down-regulation of luminal enriched markers including luminal cytokeratins, muc , gcdfp- , fhit, and transcription and differentiation errelated genes (foxa and tff ). in contrast, her expression was associated with up-regulation of luminal cytokeratins, gcdfp- , fhit and e-cadherin. conclusion: unlike high ki -li, her overexpression is not associated with the down-regulation of luminal enriched genes. increased growth fraction in er + tumours may be driven by different mechanisms in her +&her -disease. ofp- - loss of drosha expression is associated with poor survival in breast cancer patients s. khoshnaw * , t. abdel-fatah, c. nolan, z. hodi, e. rakha, d. macmillan, i. ellis, a. green * university of nottingham, breast cancer pathology, united kingdom objective: drosha is a protein that plays a key role in the biogenesis of micrornas which are well known to be deranged in human breast cancer. the purpose of this study was to investigate the hypothesis that drosha is significant in the development and progression of breast cancer and has clinical relevance as a potential predictive and prognostic target. method: we have examined drosha protein expression, using microarray and immunohistochemistry, in a well-characterised series of unselected invasive breast cancer patients (n= ) with long-term follow-up and documented expression characteristics for a large range of biomarkers of known relevance in breast cancer. furthermore, a smaller cohort of selected breast cancer cases (n= ) was also investigated for differential drosha protein expression in distinct stages of tumour progression, including in situ (dcis), invasive and metastatic components. results: drosha cytoplasmic and nuclear expression was lost with cancer progression, and was associated with loss of brca expression. multivariate statistical analyses showed that drosha cytoplasmic expression was an independent predictor of breast cancer specific survival, tumour recurrence and metastases. conclusion: drosha cytoplasmic expression in breast cancer is an independent predictor of patient outcome, tumour recurrence and metastasis. these observations imply that alterations in mechanisms of mirna regulation can influence tumour behaviour. objective: determination of proliferation in breast carcinomas is highly prognostic and can have treatment implications. however, there is no consensus on issues such as counting area or cut-off values. our aim was to examine and compare ki- counts in various tumor areas, the novel mitosis marker phh , and standard mitotic count. method: we examined a nested case-control series (n= ) as part of the population-based norwegian breast cancer screening program. mitotic count (mitosis per mm ) was assessed on h&e-sections. the percentage of ki- positive nuclei among tumor cells was recorded for both hot-spots (hs; highest proliferation) and cold-spots (cs; lowest proliferation). phh counts (per mm ) were evaluated by immunohistochemistry. results: the upper quartiles for ki- -hs, ki- -cs, phh and mitotic count consistently showed the strongest associations with known unfavorable tumor features (high histologic grade, er negativity, her positivity, ck / +, p-cadherin+). univariate survival analysis showed comparable prognostic strength of ki- -hs, phh count and mitotic count. in multivariate analysis, mitotic count was the strongest prognostic factor among these. conclusion: by upper quartile, ki- -hs, phh and mitotic count all showed strong associations with various tumor features. in multivariate survival analysis, mitotic count was the strongest proliferation marker. objective: ki li is a good marker of breast cancer prognosis. we aimed at comparison of performance of ki antibodies. method: breast cancer cases were retrieved. immunohistochemical staining for ki clones ( - , b , mib , polyclonal, sp ) performed in tma-s in automated immunostainer. evaluation performed on digital slides utilizing linear - frequency score. objective: in breast cancer, classification systems are used to assess pathological response to neoadjuvant chemotherapy. biomarkers are still needed to better predict the efficiency of primary systemic therapy. dysregulation of connexin (cx) channels (gap junctions) is involved in carcinogenesis and tumor progression. method: we have correlated cx expression and the pathological response in neoadjuvant treated breast cancers. fifteen of the human cx isotypes were pre-screened. cx , cx , cx and cx were found widespread and tested in tissue microarrays of breast cancers prior to and after neoadjuvant chemotherapy. pathological response was characterized according to the ewgbsp, cps-eg, miller-payne, sataloff, and nsabp classification systems. results: only the cps-eg classification had prognostic relevance by score - cases presenting better overall survival (p= . ) than those scoring - . cx levels prechemo correlated with hormone receptor status pre-and post-chemo and had inverse correlation with her levels pre-chemo. reduced cx expression (< %) post-chemo correlated with better overall survival (p= . ). moderate or higher cx expression (> %) pre-and post-chemo was also associated with better survival in the ewgbsp tr b (ppre= . ), sataloff tb (ppre= . ; ppost= , ) and miller-payne g (ppre= . ; ppost= . ) subgroups. conclusion: classifications combined with testing for cx and cx expression can improve prediction of neoadjuvant chemotherapy in breast cancer. ofp- - atypical vascular lesions after radiation therapy for breast cancer: a clinicopathologic study and outcomes of cases h. gobbi * , c. fraga-guedes, m. mastropasqua, m. kneubil, e. botteri, g. viale * ufmg, dept. of anatomic pathology, belo horizonte, brazil objective: we assessed clinicopathologic features and outcomes of cases of atypical vascular lesions (avl) in patients submitted to radiation therapy for breast carcinoma. method: data from all patients were retrieved from the files of the european institute of oncology (eio), milan. histopathological review of all cases was carried out. results: all patients were women (median age: year, range: - year). median lesion size was . cm (range: . to . cm). median latency interval from radiation to avl diagnosis was months (range: to months). . % of cases presented as an erythematous unique papule confined to superficial/middermis with proliferation of thinwalled, vascular channels, lined by a single layer of bland endothelial cells. no prominent nucleoli, mitoses or blood lakes were seen. patches of chronic inflammatory infiltrate accompanied all avl. one patient ( . %) developed local recurrence, and two patients ( . %) developed angiosarcoma in the previous biopsy site, and months later. margin involvement was associated to unfavorable outcome. conclusion: our data suggest that post-radiation avl and angiosarcoma may represent a spectrum of the same disease process. the development of local recurrence and subsequent angiosarcoma indicate that avl must be completely excised, with free surgical margins, and patients must be accompanied by clinical exams and imaging. objective: gastric dysplasia is classified as gastric/foveolar and intestinal/adenomatous according to morphological (architectural and cytologic) features. the immunophenotypic classification of dysplasia, based on the expression of mucins and cd , recognizes three major types: gastric (muc ac and/or muc ); intestinal (muc and/or cd ); hybrid (gastric and intestinal markers). cdx is a transcription factor responsible for early intestinal differentiation. aldh is recognized as a stem cell marker in several organs. method: nineteen cases of dysplasia were classified as gastric/foveolar (n= ) or intestinal/adenomatous (n= ) and graded as low-grade (n= ) and high-grade (n= ). immunophenotypic classification showed gastric (n= ; %), intestinal (n= ; %) and hybrid (n= ; %) phenotypes. results: cases classified by morphology as gastric/foveolar-type displayed gastric ( %) or intestinal ( %) immunophenotype, whereas intestinal/adenomatous-type cases displayed gastric ( %), intestinal ( %) or hybrid ( %) immunophenotype (p= . ). high-grade dysplasia was identified in cases with gastric ( %), intestinal ( %) and hybrid ( %) immunophenotype (p= . ). cdx expression was significantly lower (p= . ) in gastric ( %) than in intestinal and hybrid immunophenotypes ( %). aldh expression was displayed only in gastric ( %) and hybrid ( %) immunophenotypes (p= . ). conclusion: gastric dysplasia is heterogeneous and morphology is insufficient to elucidate cell differentiation. high-grade dysplasia, decreased cdx expression and high aldh expression are associated with gastric immunophenotype. ofp- - tumor budding in pancreatic cancer: are we missing important prognostic information? d. born * , i. zlobec, m. montani, b. gloor, a. lugli, e. karamitopoulou-diamantis * universität bern, inst. für pathologie, switzerland objective: tumor budding is defined as single tumor cells or small cell clusters at the invasive front of gastrointestinal (including colorectal, gastric and ampullary) carcinomas and is linked to adverse prognosis. to date it has not been reported in pancreatic ductal adenocarcinomas (pdacs). we assessed the presence and prognostic importance of tumor budding in pdac. method: whole-tissue sections of pdacs with full clinico-pathological and follow-up information were stained with pancytokeratin. tumor budding was assessed in high-power fields (hpfs) by two pathologists and considered high-grade (hg) when an average of > buds was counted. measurements were correlated to the patient and tumor characteristics. results: inter-observer agreement was strong (icc= , ). hg-budding was found in . % of cases and was linked to advanced pt-stage (p= . ), lymphatic invasion (p= . ) and decreased disease-free and overall survival (p< , and p= , ). there was no association with pn, pm, r-stage or vascular invasion. in multivariate analysis the prognostic effect of hg-budding was independent of lymphatic invasion, pn and r-stage [p< , , hr ( % ci)= , ( , - , )]. conclusion: tumor budding occurs frequently in pancreatic cancer, is an indicator of worse outcome and adds independent prognostic information. routine use of tumor budding would help to better stratify patients into prognostic subgroups. ofp- - epstein-barr virus and p in the development of gastric cancer v. genitsch * , m. cathomas, l. terracciano, l. tornillo, a. lugli, a. marx, g. sauter, f. carneiro, f. hofstädter, n. willi, g. cathomas * kantonsspital baselland, abt. pathologie, liestal, switzerland objective: epstein-barr virus (ebv) is associated with a subset of gastric cancers (gc) . in this study, the prevalence of ebv and its presence in the development of gc were assessed. method: ebv was evaluated in gc by tissue microarrays using in situ hybridization for eber (n= ). non-tumorous, dysplasia, intramucosal and invasive carcinomas and metastasis of ebv positive gc were analyzed (n= ). p expressen was evaluated in ebv positive and a subset of ebv negative gc. results: . % of gastric cancers were positive for eber. all invasive cancers showed diffuse eber positivity and only lymph nodes revealed a focal loss of eber. in gc with foci of atypia/low grade dysplasia, no or only focal eber positivity was observed. p was overexpressed in of ebv negative but not in ebv positive gc ( . % vs. %; p= . ). conclusion: ebv is diffusely expressed in invasive gc and only occasionally lost in metastasis. in low grade dysplasia, however, ebv is often focal or missing, indicating that ebv infection may be associated with progression from low grade dysplasia to invasive cancer and this progression is p independent. ofp- - p x r attenuates colitis associated carcinogenesis p. hofman * , t. juhel, m. ilie, o. bordonne, o. boyer, x. hebuterne, s. adriouch, v. vouret-craviari * chu de nice, lpce, france objective: it is now well established that patients with inflammatory bowel disease presented a higher risk to develop a colon cancer. our previous works characterized the link that exists between bacterial infection and inflammation and highlighted the role of the microenvironment on the onset of chronic inflammation. here, we wished to understand the role of inflammasome, a complex of proteins that is activated by microbial motifs and the purinergic receptor p x and involved in the maturation of il- β. method: we treated p x r knockout mice to induce acute colitis ( % dss) or colitis associated colon cancer (aom and dss). results: we showed that p x r ko mice are more resistant than wild type mice to a dss-induced colitis. further, we demonstrated that p x r ko mice are highly susceptible to inflammation driven colon tumorigenesis. in addition, we showed that tumors from p x r ko contained more neutrophils (cd b+/ly g + cells) and less macrophage (cd b+/ly g-) than tumors obtained from wt mice. conclusion: we will present results on the characterization of the pro tumoral tumor associated neutrophils and the molecular mechanisms that are linked to the presence of such immune cells. ofp- - iel counts and distribution in normal duodenum, non-gse ielosis and gse: do we need a cut-off? b. a. karabork * , s. yuksel, b. savas, a. ensari * ankara university of medicine, dept. of pathology, turkey objective: intraepithelial lymphocytosis (ielosis) is a characteristic feature of gluten-sensitive enteropathy (gse), though, the cut-off is still debatable. we aimed to evaluate the number and distribution of iels in normal duodenum, and in non-gse ielosis and gse. method: the study group comprised of a spectrum of normal duodenal biopsies (n= ), non-gse ielosis (n= ), type (n= ) and type (n= ) serologically diagnosed gse cases. the number of iels/ enterocytes and distribution on h&e and cd -immunostained sections, were assessed for each group. chi square test was used for statistics. results: iels increased significantly through the spectrum ( , in normal, , , in type , , in type ) on h&e and cd , respectively (p< , ). iel counts ≥ on h&e and ≥ on cd had a sensitivity of , % and %, and a spesificity of , % and , %, respectively, for gse. iel distribution was diffuse in type ( , %), type ( %) but focal in non-gse ( %) (p< , ). conclusion: the current cut-off for iel counts seems to be sensitive for ielosis, though, not specific for gse and needs to be accompanied by the diffuse distribution of iels. ofp- - geographic analysis of the protein expression of metastasis suppressor gene rkip and its clinical relevance in colorectal cancer v. koelzer * , e. karamitopoulou, h. dawson, a. lugli, i. zlobec * university of bern, inst. of pathology, switzerland objective: loss of raf- kinase inhibitor protein (rkip) expression has been implicated in disease progression in several tumor types. the aim of this study was to evaluate rkip expression in colorectal cancer stratified by histological "zones" and to determine the zone responsible for its clinical relevance. method: immunohistochemical expression of rkip was assessed in colorectal cancers using whole tissue sections. four different areas were evaluated using highpower-fields (hpfs) each: normal mucosa, tumor center, invasion front and tumor buds. the average expression for each zone was assessed for its clinical relevance. results: expression of rkip was diffuse in normal mucosa and progressively lost towards the tumor center and front ( %, % and % rkip-positivity, respectively; p< . ). only % of tumor buds were rkip-positive. rkip loss in the tumor center only corresponded to more frequent lymph node positivity (p= . ), distant metastases (p= . ), lymphatic invasion (p= . ) and more advanced tnm stage (p= . ). rkip loss was highly prognostic (hr ( % ci): . ( . - . ); p= . ) independently of tnm and therapy. conclusion: the clinical relevance of rkip is restricted to the tumor center where it acts as an independent prognostic factor. its absence in tumor buds provides further evidence to support their hypothesized metastatic potential. ofp- - outcomes of neoadjuvant chemoradiation for rectal carcinoma g. ozgun * , f. oz atalay, n. ugras, o. yerci * uludag university, dept. of pathology, bursa, turkey neoadjuvant radiation and chemoradiation is currently the treatment of choise for patients with locally or advanced carcinoma of the rectum. patients treated with neoadjuvant chemoradiatheraphy with good clinical response and tumor regression present a controversial management dilemma. to assess the effects of chemoradiation on tumor regression and disease free survival, consecutive series of patients receiving neoadjuvant chemotheraphy or chemoradiation and a control series of patients without pretreatment were studied. the overall survivals and disease free survivals were compared in terms of variables such as tumor regression grade, stromal response in tumor bed, state of lymph node metastasis, and the localization of the tumor in between patients treated with chemoradiation and the control group. neoadjuvant treatment is one of the treatment modalities in rectal carcinomas. we want to present our experience between and in our institution. key words: rectal carcinoma, neoadjuvant treatment, pathologic complete response ofp- - identification of tissue microvascular invasion biomarkers in hepatocellular carcinomas by maldi imaging mass spectrometry n. poté * , t. alexandrov, j. le faouder, s. laouirem, j. belghiti, j. m. camadro, v. paradis, p. bedossa * paris, france objective: microvascular invasion (mvi), a major predictive factor of tumoral recurrence and mortality in patients with hepatocellular carcinoma (hcc), is only detectable on pathological examination. so far, there is no reliable tool to identify mvi prior to surgical procedures. maldi imaging mass spectrometry (ims) represents a new analytical tool to provide the relative abundance and distribution of the whole proteins expressed in a tissue section. the aim of this study was to compare, using maldi ims, the tissue proteome of hcc without and with mvi in order to identify surrogate biomarkers of mvi. method: a total of hcc samples obtained from surgical specimens, for which frozen samples were available, were retrospectively collected. two groups of tumors were defined ( hcc without mvi; hcc with mvi) and were analysed by maldi ims. a statistical comparative analysis of acquired mass spectra was then performed in order to identify protein peaks differentially expressed between the two groups. results: protein peaks were differentially expressed between the two groups, all overexpressed in hcc with mvi. protein characterization of some of these peaks is in progress. conclusion: these results highlight the potential of maldi ims to uncover new biomarkers in liver carcinogenesis. the identification of mvi biomarkers would be helpful in the therapeutic strategy of patients with hcc. ofp- - a histopathological scoring system can predict the recurrence of hepatocellular carcinoma in liver transplant patients treated with transarterial chemoembolization f. rosini * , f. vasuri, d. malvi, p. baldin, w. f. grigioni, a. d´errico-grigioni * university of bologna, dept. of anatomic pathology, italy objective: aim of this study is to define the histopathological features predictive of hepatocellular carcinoma (hcc) recurrence in patients treated with orthotopic liver transplantation (olt) after tumoral transarterial chemoembolization (tace), in order to establish a predictive "score" applicable by pathologists for recipient risk stratification. method: we retrospectively enrolled patients ( total neoplastic or necrotic nodules) who received tace for hcc downstaging before olt. the following data were collected: number of neoplastic/necrotic nodules, microscopic thrombosis, the residual neoplastic tissue (rnt), and dysplastic nodules. sizes, percentage of necrosis, edmondson's grade, clear cell features were also collected for each nodule. results: median follow-up was days: ( %) hcc recurrence cases were recorded. statistically rnt (best cutoff cm , p= . ), the total nodule number (best cut-off ≥ , p= . ), and the neoplastic thrombosis (p= . ) were the only variables predictive of hcc recurrence. scoring + each parameter, recipients with (n= ) had % recurrence rate; recipients with +/ + (n= ) had %; recipients with + (n= ) had % recurrence rate (p< . ). we were able to stratify olt recipients with tace-treated hcc in risk groups based only on histopathological analysis. ofp- - discovery of a new histopronostic factor in rectal adenocarcinoma treated by radiochemotherapy followed by surgical resection a. sannier * , j. lefèvre, f. bretagnol, d. cazals-hatem, y. panis, p. bedossa, n. guedj * paris, france objective: neoadjuvant radiochemotherapy (rct) followed by surgical resection became the treatment for locally advanced rectal cancer. pathological diagnosis is important for the prediction of prognosis and adjuvant treatment. study aim was to identify histopronostic factors in a consecutive series of patients treated by rct and surgery. method: patients were included, follow-up, tumor morphologic pattern and modified rectal cancer regression grade (m-rcrg) were assessed. univariate and multivariate analysis were used to assess predictors of disease-free survival (dfs). results: -years dfs was %. in univariate analysis, ypt, budding, calcifications, circumferential margin, node involvement, invaded margin, vascular emboles and perineural involvement were pronostic factors (p< . ). in multivariate analysis, presence of calcification in tumor bed (p= . ) and small circumferential margin (p= . ) were the only two independent factor of worse dfs. mrcrg was not correlated to dfs. among the mrcrg tumor, dfs was significantly better in patient with ypt than in other ypt stages (p= . ). conclusion: presence of calcification in tumor bed is a new major histopronostic factor described for the first time in rectal cancer. our results raised the question of whether ypt stage or histological tumor regression is more important in the prediction of patient prognosis. tuesday, september , . - . objective: the most widely used system for histological grading of colorectal cancer (crc) is based on the degree of gland formation, despite significant interobserver variability and low prognostic value. herein we analyzed the prognostic significance of a grading system based on the presence of poorly differentiated clusters in stage i crc. method: poorly differentiated cancer clusters were assessed by two independent pathologists in stage i crc characterized or not by disease progression. tumors with < , to , and > clusters were classified as g , g , and g , respectively. the prognostic value on disease-free survival and the association with other clinicopathologic characteristics of the conventional and novel grading systems were analyzed. results: k statistics for inter-observer variability in the assessment of histological grade based on poorly differentiated custers was . (good). high histological grade assessed with the novel system, but not with the traditional one, represented a negative significant prognostic factor for disease-free survival and it was significantly associated with venous invasion, lymphatic invasion, budding, invasive growth and nodal micrometastases. conclusion: we suggest that a tumor grading system based on the number of poorly differentiated clusters has a stronger power to stratify stage i crc patients by prognostic outcome than conventional grading. ofp- - cytokeratin : a marker for braf mutated colorectal carcinomas? s. gurzu * , z. szentirmay, i. jung * university of targu mures, dept. of pathology, romania objective: cytokeratin +/cytokeratin − (ck) is used as the characteristic immunophenotype of colorectal carcinomas (crc) . some new studies suggested that aberrant pattern ck /ck can be identified in colorectal cancer with microsatellite instability (msi). our aim was to establish which factors may determine changing in this immunophenotype. method: in crc, randomly selected, we performed immunohistochemical stains with ck and ck and analyzed the microsatellite status and braf mutations with real time pcr. results: from the crc, were msi and mss (microsatellite stable) cases. % of mss cases diffusely expressed ck without ck expression. from the msi cases, presented braf mutations. in msi cases with braf mutations, ck was focally expressed or negative and ck was diffusely expressed. conclusion: cytokeratin positivity may be used to select braf mutated msi colorectal carcinomas. both ck and ck should be used for differential diagnosis of colorectal cancer. ofp- - intratumoral budding in preoperative biopsies predicts local and distant metastasis in colorectal cancer patients a. lugli * , m. hädrich, d. inderbitzin, m. borner, i. zlobec * medizin. universität bern, inst. für pathologie, switzerland objective: in , the term "intratumoral budding, itb" was used to describe the presence of tumor buds within the main tumor body and was correlated to worse clinical outcome in colorectal cancer patients. here, we further elucidate the potential clinical role of itb in pre-operative biopsies using pan-cytokeratin stained tissues and a quantitative scoring system. method: pre-operative biopsies from patients with colorectal cancer underwent immunohistochemistry for pancytokeratin (ae /ae ). itb were counted in the area of densest budding ( ×) and classified as high-grade when > buds/hpf were observed based on receiver operating characteristic (roc) curve analysis. results: high-grade itb occurred in . % of cases and was associated with right-sided tumor location (p= . ), more advanced pt (p = . ) and pn (p< . ) classifications, distant metastasis (p = . ), higher tumor grade (p= . ) and lymphatic invasion (p= . ). the specificity and positive predictive value for lymph node metastasis was . % and . %, respectively. disease-free survival was significantly worse in patients with high-grade itb ( -year survival= %) in comparison to patients with low-grade itb ( -year survival= %) (p= . ). conclusion: the assessment of itb in pre-operative biopsies is predictive of local and distant metastasis in corresponding resections and should be considered in daily management of colorectal cancer patients. ofp- - alpha-methylacyl-coa (amacr) in gastric cancer -correlation with clinicopathological data and disease free survival a. mroz * , m. kiedrowski, z. lewandowski * cmkp, gastroenterology and hepatology, warsaw, poland objective: diagnostic and prognostic significance of alphamethylacyl-coa (amacr) has been established in many human cancers. its correlation with clinical and pathological data in gastric cancer has not been fully elucidated and its impact on surveillance has not been studied thus far. method: we analyzed consecutive gastric cancer cases in terms of amacr immunohistochemical expression and clinical/pathological characteristics and followed patients' postoperative history. results: amacr was expressed in / gastric cancers ( . %). we did not find correlation between amacr expression and gender, age, location, histological type, ptn staging, vascular and nerve sheaths invasion. overall disease-free survival tended to be worse in amacr positive patients (p= . ), and in adenocarcinoma subgroup it was significantly shorter (p= . ). conclusion: amacr expression might represent promising adverse prognostic factor in gastric cancer, particularly in adenocarcinoma histological type. ofp- - development of a rabbit monoclonal antibody for determining hent status and predicting response to gemcitabine in pancreatic ductal adenocarcinoma m. raponi * , j. isaacson, j. ranger-moore, j. clements, b. richardson, s. ormanns, k. winter, a. allen, r. andersson, v. heinemann, a. p. dicker * clovis oncology, san francisco, ca, usa objective: human equilibrative nucleoside transporter (hent ) is the primary membrane channel through which gemcitabine (gem) enters pancreatic tumor cells, and patients whose tumors have low expression of hent may derive little benefit from gem therapy. method: an analytically validated rabbit monoclonal antibody immunohistochemistry (ihc) in vitro diagnostic (ivd) for assessing the degree of hent expression in pancreatic adenocarcinoma using an automated ihc platform has been developed. results: using primary tumor samples (n= ) from a randomized controlled clinical adjuvant trial (rtog - ), we developed and verified a hent scoring methodology and cut-off for identifying patients least likely to benefit from gem (hent -low). the distribution of hent expression was similar between these samples and an independent set of pancreatic adenocarcinoma specimens from the aio-pk study of which were confirmed metastatic biopsies. overall, using this cut-off, approximately two thirds of pancreatic tumors displayed low-hent expression across the different data sets. conclusion: a robust hent ihc ivd has been developed using a rabbit monoclonal antibody and identifies two thirds of primary and metastatic pdac cases as having low expression of hent . ofp- - hypoxia activates pancreatic stellate cells: development of an organotypic culture model of thick slices of normal human pancreas v. rebours * , m. albuquerque, p. ruszniewski, p. lévy, a. sauvanet, v. paradis, p. bedossa, a. couvelard * beaujon hospital, pancreatology unit, clichy, france objective: pancreatic stellate cells (psc) promote oncogenesis by modulating cell proliferation. aim-to evaluate the early activation of psc in case of hypoxia in normal human pancreas. method: an organotypic culture model of thick sections of human pancreas has been developed and validated. slices of pancreas ( μm) were prepared from surgical specimens and cultured in hyperoxia conditions. half of the samples underwent an initial phase of culture in normoxia ( % o ) to reproduce hypoxia. the total duration of culture was h. cell viability, hypoxia, apoptosis and activation were monitored. results: sections per specimen were cultured. analysis was performed at baseline, h, h and h. morphological analysis showed gradual appearance of ductal/acinar dedifferentiation. at h, foci of necrosis were identified. hypoxia was confirmed by the expression of hif and ca at h ( % and % of labeled cells). apoptosis was limited, acinar cells expressed caspase at and h. analysis of proliferation using ki index showed significant activation of psc at h (× /baseline) and at h (× / baseline). activation of psc was confirmed by smooth muscle actin immunochemistry. conclusion: organotypic culture of normal human pancreas is possible with optimized cell viability at h. hypoxiainduced activation of psc occurs very early. objective: ipn are still poorly characterized precursor lesions of bile duct adenocarcinoma. we present a multicenter study with focus on morphology, subtyping and prognosis and provide a detailed molecular analysis in the stepwise progression from low-grade to high-grade lesions and invasive carcinoma. method: fourty-five patients with biliary ipn were included into the cohort, twenty-two patients with conventional adenocarcinoma of the bile duct served as control cohort. subtyping was performed based on histomorphology and expression profile of mucins and cdx expression. furthermore, low grade lesions (n= ), high-grade lesions (n= ) and invasive components (n= ) of ipn as well as conventional bile duct adenocarcinomas (n= ) were investigated individually for alterations of common oncogenic pathways using immunohistochemistry, real-time polymerase chainreaction and direct sequencing. results: ipn are mainly detected at preinvasive stage ( %), pancreato-biliary and intestinal type are the most common subtypes ( and %, respectively). detailed molecular analysis showed that p overexpression, k-ras mutation, loss of heterozygosity of p locus and nuclear ßcatenin expression are early events and loss of smad and overexpression of egfr and her are later events in the tumor progression of ipn. conclusion: stepwise alterations of common oncogenic pathways are required for progression of biliary ipn from non-invasive precursors to invasive carcinoma. ofp- - influence of braf mutations and rac b/rac mrna ratio or protein expression on outcome in patients with metastatic colorectal cancer (mcrc) treated with firstline chemotherapy m. cuatrecasas * , v. alonso-espinaco, p. jares, c. horndler, a. castells, j. j. lozano, j. maurel * hospital clinic, barcelona, spain objective: metastatic colorectal cancer (mcrc) patients with braf mutation v e present poor overall survival (os). rac b, a rac spliced variant, is overexpressed in crc and impairs apoptosis by activation of nuclear-factor-kb. we evaluated if rac b was an independent prognostic factor in mcrc. method: we examined braf (v e) mutational status, rac b by immunohistochemistry, rac b/rac expression ratio by mrna rt-pcr, and mismatch repair (mrr) deficiency by microsatellite instability (msi) analysis, in mcrc patients treated in firstline therapy with folfox or capox from three spanish institutions. we assessed whether these biomarkers were independently predictive of progression free survival (pfs) or os. results: % crc had braf mutations, % msi-h, % high rac b/rac ratio, and % rac b protein overexpression. five of crc ( %) with braf mutations had high rac b/rac ratio/protein vs / ( %) without braf mutation (p= . ). all braf mutated or rac b/rac high were mss. low rac b/rac ratio and braf wt had higher response rate ( % vs %; p= . ). multivariate regression analysis identified ecog ps as a significant variable for pfs and ldh levels, ps and high rac b/rac ratio for os. conclusion: high rac b/rac expression ratio or immunoexpression constitutes a marker of poor os and a potential marker of acquired chemo-resistance in mcrc treated with oxaliplatin-based therapy. objective: annually more than . new cases of papillary thyroid cancer, that is % of malignant tumors, are registered all over the world. method: there are clinical and morphologiсal methods: immunohistochemistry (mmp- , mmp- , timp- , timp- ), light, transmission, electron, probe and scanning microscopy with the trace element analysis. it was studied cases of thyroid cancer since. results: at the papillary thyroid cancer progression matrix metalloprotease and specific tissue inhibitors of metalloprotease (timp- ) increased in the cytoplasm of tumor cells, but the concentration of timp- decreased. tumors with low degree of ultrastructural differentiation were presented by cells with electron-dense dark cytoplasm and cells with poor cytoplasm organelles and differently formed nucleus with numerous deep invaginations. high level of some matrix metalloproteaseexpression (ММР- ) and low level of timp expression (Тimp- ) were observed in current cases more ( % and % respectively), than at more differential papillary thyroid cancer ( % and %). at performance of atomic force microscopy it was shown that connection between cells had become weak at the decreasing of malignancy degree. it was revealed the significant increasing of oxygen, magnium, potassium, calcium in tumor nodes. conclusion: conducted comlex morpho-chemistry analysis display diagnostic patterns of papillary thyroid cancer. objective: cytological features of btn-ph, sometimes interpreted as papillary carcinoma (pc) on fine-needle aspiration biopsies (fnab), have not been studied extensively. method: available material from btn-ph was reviewed retrospectively and compared with pcs. results: fnab diagnoses were: non-diagnostic (n= ), benign (n= ), aus/flus (n= ), suspicious for follicular neoplasm (n= ), suspicious for malignancy (s-pc) (n= ), and malignant (p-pc) (n= ). the extent of papillary hyperplasia on histology was higher in s-pc/p-pc diagnostic categories ( %; p= . ; range: - %). on cytology, papillary structures, present in % of bpn-ph cases, were more frequent and numerous in the s-pc/p-pc group ( %; p= . ). focal nuclear atypia was seen in all s-pc/p-pc cases, including rare grooves ( . %), enlargement/crowding ( %), chromatin clearing/rare pseudoinclusions ( %), but were less than in pcs (p< . ). nuclei were smaller in btn-ph than in pcs (mean diameter/range (μm): . / - vs . / - ; p= . ). all btn-ph tested (n= ) were immunonegative for hbme- , galectin- , and keratin- . conclusion: btn-ph diagnosed as s-pc/p-pc often show papillary structures and focal nuclear features of pc but they are significantly less than in classical pcs, stressing the need to apply strict criteria for pc diagnosis. ofp- - genetic alterations in glucagon cell adenomatosis t. henopp * , m. anlauf, s. biskup, g. klöppel, b. sipos * medizin. universität tübingen, inst. für pathologie, germany objective: glucagon cell adenomatosis (gca) was recently recognized by us as a multifocal neoplastic disease of the endocrine pancreas unrelated to men . multiple micro-and a few macrotumors are found on the background of a hyperplasia of glucagon cells. the disease may cause unspecific abdominal symptoms and only rarely a glucagonoma syndrome. recently a mutation in the glucagon receptor (gcgr) gene was described in one gca patient. method: the extracted dna of five patients with gca was sequenced and the gcgr gene analyzed for mutations. results: sequencing of the gcgr gene revealed germline mutations in three out of five patients. one patient shows two different heterozygous point mutations in the hyperplastic alpha cells as well as in the non-tumorous tissue leading to two premature stop codons. one patient harbors a homozygous stop mutation. the third patient shows two homozygous missense mutations of the gcgr gene that most likely also led to a dysfunction of the gcgr. in the two other patients no germ line mutations of the gcgr gene were detected. these variants were not identified in healthy subjects. conclusion: the finding of germ line and somatic "loss of function" mutations of the gcgr gene in three of five patients with gca suggests that a change in the signalling function of the gcgr may cause glucagon cell adenomatosis via glucagon cell hyperplasia. ofp- - timp- expression and hypoxia in papillary thyroid carcinoma: relationship to brafv e mutation and clinical behavior m. ilie * , s. lassalle, p. brest, c. bonnetaud, a. bozec, n. guevara, j. haudebourg, i. birtwisle-peyrottes, j. santini, p. hofman * chu de nice, lpce, france objective: brafv e causes up-regulation of timp- , promoting cell invasion in papillary thyroid carcinoma (ptc). hif- α is regulated by hypoxia but also by brafv emediated signaling pathway in ptc. we assessed the impact on clinical behavior in ptc of timp- , hif- α and the hypoxia-inducible caix and caxii. method: the protein expression was assessed by western blot in two cell lines, tpc- /brafwt and bcpap/ brafv e. tma-immunohistochemistry analysis was used to study protein expression in ptc samples. braf status was analyzed by pyrosequencing. data were correlated with clinicopathological variables of patients. results: higher expression of all proteins was detected in bcpap exposed to hypoxia. timp- expression displayed % sensitivity and % specificity for identifying braf mutation (p< . ), and was associated with pt-stage (p= . ), pn-stage (p= . ), and multifocality (p= . ). hif- α expression was correlated to pt-stage (p= . ). caix expression was related to pn-stage (p= . ), and both caix (p= . ) and caxii (p= . ) were highly associated with vascular invasion. conclusion: timp- protein expression is reliable surrogate of braf mutated status in ptc. timp- and hypoxiaregulated proteins have potential for future use as predictors of the malignant change in ptc, and warrant further investigation as new therapeutic targets for the treatment of highly aggressive form of ptc. objective: mtc are usually aggressive tumours which account for - % of all thyroid carcinomas. most of mtc are sporadic, the remaining tumours are due to hereditary forms with germline activating mutations of the ret proto-oncogene. the aim of this study was to assess the microrna profiling expression in both sporadic and hereditary forms of mtc. method: a total of frozen mtc from patients with welldocumented clinico-pathological parameters were used for microarray analyses ( hmtc and smtc) using human microrna mircoarray kit version (agilent technologies). after identification of a set of mirna of interest, validation was done using qpcr. results: when comparing hmtc harbouring germinal ret mutation with smtc, seven mirnawere significantly deregulated (mir- , − a, − c, − a, − - *, − b, − ). the most highly discriminative mirna in tumoral samples (> fold change) identify mir- , − - p, − , − c and − . moreover, mirna could segregate mtc with good prognosis and better clinical outcome (mir- , − , − , − and − ) . conclusion: a specific microrna signature in hmtc can be identified. moreover, detection of micrornas in tissue samples might be of interest for improved clinical management and treatment of patients with mtc. ofp- - focus on the follicular variant of papillary thyroid carcinoma: combination of the immunohistochemical markers ck , hbme and tpo is associated with histopathological diagnosis, better than molecular markers objective: the follicular variant (fvptc) is the major variant of papillary thyroid carcinoma (ptc), ranging from % to % of all ptc. it remains however a problematic entity. some consider that the encapsulated fvptc (enfvptc) have an excellent prognosis like follicular adenomas (fa); others showed that enfvptcs had potential for hematogenous spread like follicular carcinoma. its place in the spectrum the multivariate cox regression the flt -itd mutation (p= . ) was an independent poor prognostic factor, whereas the goodness-of-fit of the minkowski fractal dimension an independent favorable prognosticator (p= . ). the karyotype had no influence on survival. conclusion: flt -itd and chromatin fractal characteristics were more important as prognostic factors for overall survival than karyotype and gene methylation status. financial support: fapesp and cnpq. objective: the prevalence of cyclin d (cyd ) positive bcells with mantle cell lymphoma (mcl) phenotype in the mantle zones of reactive lymph nodes ("in situ" mcl/mclis). and related minimal mcl infiltrates in reactive lymphoid tissues of healthy individuals and in mcl patients are unknown. method: all . reactive lymph nodes from unselected consecutive surgical specimens of patients without a history of lymphoma obtained over a months´period were stained for cyd . additionally, all morphologically reactive lymph nodes and extranodal lymphoid infiltrates of mcl patients from to were studied. samples predating the lymphoma diagnosis for at least months were available from / ( . %) patients. results: a mclis was not identified. however, in four mcl patients, an early manifestation of mcl with mantle zone growth pattern was detected retrospectively, antedating the lymphoma diagnosis for - months. in six mcl patients, only small groups of cyd positive cells in morphologically reactive extranodal infiltrates were detected more than months before the diagnosis of mcl (range - months). conclusion: mcl "in situ" is an extremely rare phenomenon in morphologically reactive lymph nodes. in mcl patients, however, immunohistochemically detectable infiltrates of mcl cells antedating the lymphoma diagnosis were found in a significant proportion of cases ( / = %). ofp- - e f- 's relationship with tumor growth in hodgkin's lymphoma is regulated by p status e. georgiadi * , g. dimtsas, t. p. vassilakopoulos, v. g. gorgoulis, i. a. doussis-anagnostopoulou * larisa, greece objective: e f- is a member of the e f family group of transcriptional factors that play a major role in cell cycle progression and arrest, as well as in apoptosis. in different tumors e f can demonstrate opposing roles, acting either as an oncogene or as a tumor suppressor gene. method: we have previously established e f- 's immunohistochemical expression in hodgkin lymphoma (hl) and its correlation with p expression, although no relationship with either proliferation or apoptosis was demonstrated. a further investigation in a larger series of cases of primary hl with the addition of new apoptotic techniques and the use of p expression as an indicator of p functional status was undertaken. results: following stratification of our cases based on p functionality, e f- was inversely correlated with the proliferation marker topoiia. independently of p , high levels of e f- expression showed a linear trend with apoptosis and an inverse trend with proliferation (borderline p value in both cases). conclusion: both e f- and p activity is upregulated in hl, however it seems that p status is a major regulator of the relationship between e f- expression and tumor growth. alternatively, e f- may use p -independent pathways to induce apoptosis. objective: ptld represent a spectrum of usually ebv-driven lymphoplasmacytic proliferations in the settings of immunodeficiency associated with allograft. ebv infects cells in latent or lytic forms. although viral oncogenes are expressed in latency programs, lytic ebv replication is required to develop lymphomas in mouse models. the role of intratumoral ebv replication in ptld has never been addressed before. method: a series of ptld was reviewed and ebv latency genes were explored in all cases and included eber - , lmp and ebna . moreover, two early lytic genes involved in ebv replication, bzlf /zebra and ead were also analyzed. all clinical and pathological data were collected. results: the median age was years ( - ) with a male predominance ( m: f). ebv infection was observed in cases ( %). ebv replication was observed in % of the cases. these were more likely to be polymorphic ptld with latency iii in the context of stem cell transplantation. moreover these cases had atypical clinical presentations such as brain involvement or disseminated disease with aggressive behavior (os: months vs. months). conclusion: ebv replication occurs in tumor cells of ptld-patients associated with poor outcome and may be share common morphological and phenotypical features. in routine praxis, their differential diagnosis might represent a challenge requiring a specific work-up of immunohistochemical (ihc) and fluorescent in situ hybridization (fish) analyses. method: paraffin sections from cd +/cd + blastic b-nhl were examined for cyclin-d , cd , bcl- and mum- ihc expression together with fish method using probes for ccnd rearrangement and/or igh/ccnd translocation. the final diagnosis was established according to the criteria of who classification. results: / cases were diagnosed as bmcl showing cyclin-d positivity and ccnd rearrangement, other analysed ihc markers were negative. five of these cases were polyploid. / cases were diagnosed as cd + dlbcl in spite of coexpression of cyclin-d in of them, as they showed various cd , bcl- and mum- expressions and missed ccnd gene rearrangement. conclusion: cyclin-d positivity may appear in other than mcl lymphomas due to changes at transcriptional or posttranscriptional level. in spite of overlaping phenotypes, fish analysis of ccnd represents an effective tool to increase the diagnostic accuracy to distinguish cd + dlbcl from bmcl. supported by vega grant nr. / / and projects ceprii (imts: ) and mdcc (imts: ) co-financed by eu sources. ofp- - still's disease associated lymphadenopathy: report of two cases resembling malignant lymphoma with review of possible morphological findings k. kamarádová * , m. nova, m. kalinova, v. campr * prague, czech republic objective: still's disease (or systemic onset juvenile idiopathic arthritis, sojia) is a systemic inflammatory disorder characterized by fever, rash, and arthritis. it has a peak of incidence in early childhood with rare occurence in adulthood (adult-onset still's disease, aods). several other symptoms as hepatosplenomegaly, leukocytosis and lymphadenopathy may be associated possibly mimicking malignant lymphoma. method: we present two cases of still's disease associated lymphadenopathy in a child and in an adult simulating morphologically peripheral t-cell lymphoma and classical hodgkin lymphoma respectively. results: histological, immunohistochemical and molecular examination revealed non-neoplastic lymphadenopathy with atypical paracortical t-cell hyperplasia with immunoblastic reaction in the former and burnt-out histiocytic pattern in the latter, both falling into a broad spectrum of reactive lymph node changes associated with still's disease. the lymph node lesions in still's disease can be histologically divided into four major categories: ) simple atypical paracortical hyperplasia, ) burnt-out histiocytic pattern, ) exuberant immunoblastic reaction in paracortical hyperplasia and ) follicular hyperplasia. the second and the third pattern may closely resemble malignant lymphoma. extended and careful clinical examination with relevant laboratory findings and possible complex histochemical and molecular analysis are necesarry to rule out the suspition of lymphoma. objective: intravascular large b-cell lymphoma (ivlbcl) is a rare form of diffuse large b-cell lymphoma characterized by exclusive or preferential intravascular growth. ivlbcl often goes clinically unrecognized. we report three clinically unsuspected cases of classic variant ivlbcl. method: cases diagnosed as ivlbcl were collected from the pathology files of germans trias i pujol hospital. the diagnosis was reviewed in agreement with who criteria and immunophenotypic and clinical data were gathered. results: a -year-old male and an year-old-male showed neurological symptoms leading to progressive deterioration and death. a -year-old female presented with fever of unknown origin and died days after admission. at autopsy there were no gross findings suggestive of a neoplastic process in any instance. histologic study showed intravascular large b cells with vesicular nuclei involving predominantly the central nervous system in the first two cases and the adrenal glands, liver, and spleen in the third case. conclusion: the three cases of aggressive, advanced-stage lymphoma herein described illustrate how the clinical diagnosis of ivlbcl remains elusive due to its low frequency and variegated clinical presentation and how the pathologic diagnosis of ivlbcl continues to be highly dependent on autopsy examination. objective: the world health organisation (who) currently recognises four categories of ptld, which are lymphoid/ plasmacytic proliferations occurring due to immunosuppression after organ transplantation. the aim of this study was to examine the clinicopathologic characteristics of ptld occurring after renal transplantation in the republic of ireland. method: the national renal transplant registry from to (inclusive) was cross referenced with the national cancer registry to identify renal transplant recipients who had developed ptld up to the present day. review of pathology and chart review was conducted for each case. results: of , patients ( , allografts), cases of ptld were identified, with pathology review possible in cases. there were ( %) monomorphic ptld ( diffuse large b cell {dlbcl}, anaplastic large cell, burkitts-like, myeloma], hodgkin-type ptld, ( %) polymorphic ptld and ( %) early lesion. in addition, there was mantle cell lymphoma and lymphomatoid granulomatosis. the annual incidence of ptld was estimated as . % ( % ci . - . ). median time to diagnosis was . years (range to . years). conclusion: similar to other studies, monomorphic ptld is the most common type, with dlbcl representing the majority of cases. of interest, early onset ptld was very uncommon in this study. monday, september , . - . , forum hall ofp- oral free paper session head and neck pathology ofp- - human papilloma virus associated oropharyngeal carcinoma is strongly correlated with cancer of unknown primary s. ihrler * , g. assmann, p. zengel, m. mollenhauer * medizin. universität münchen, institut für pathologie, germany objective: human papillomavirus (hpv) associated oropharyngeal carcinoma has been identified as a distinct entity within squamous cell carcinoma of the head and neck and is preponderantly restricted to palatinal tonsils and base of tongue. these two primary locations have for long -and hpv-associated carcinomas have recently -been associated with the clinical situation of cancer of unknown primary (cup). method: to investigate the relationship between hpv and cup in detail, we studied consecutive patients who initially presented as cup and were finally diagnosed with carcinomas of these two locations. results: twenty-one carcinomas ( %) proved to be positive for high-risk hpv (p and polymerase chain reaction). they were frequently very small (smallest: . cm; cases: ≤ . cm; on average . cm) and located in a deep, submucosal position. conclusion: this demonstrates an overrepresentation of hpv-associated carcinoma in patients who were initially diagnosed with cup, supporting a strong causal relationship between hpv-association and cup. the frequent manifestation as cup presumably is caused by the unusual predisposition for small size and submucosal location, combined with frequent and early lymphatic metastization. in order not to miss these small, clinically occult carcinomas, consequent interdisciplinary cooperation and meticulous histological work-up is mandatory. ofp- - methylthioadenosine phosphorylase inactivation depends on gene deletion in laryngeal squamous cell carcinoma a. nadal * , l. conde, i. vilaseca, l. alos, m. bernal-sprekelsen, a. cardesa * hospital clínic barcelona, dept. of pathology, spain objective: methylthioadenosine phosphorylase is an essential enzyme for the methionine and adenosine salvage pathway in normal cells, frequently inactivated in many different human cancers. the mtap status could be important for tumor cell sensitivity to adjuvant chemotherapy. to date there are no reports on mtap status in laryngeal carcinoma. method: a series of laryngeal squamous cell carcinomas was investigated for mtap mrna expression through reverse transcription and quantitative pcr (qpcr) as well as for mtap gene deletion and/or promoter hypermethylation through qpcr and methylation-specific pcr respectively. results: low mtap mrna expression was found in % of cases, associated with mtap gene deletion in % (p< . ) but not to mtap promoter hypermethylation, indicating that in this tumors gene deletion is the main mechanism of mtap inactivation. neither low mrna expression nor gene deletion was associated with any of the clinicopathologic parameters investigated. conclusion: given the significance of mtap status for cell sensitivity to different chemotherapeutic regimes, our results suggest that determination of mtap inactivation could be useful for adjuvant therapy selection in laryngeal squamous cell carcinomas. ofp- - ercc , p and ki- immunohistochemical expression as predictive and prognostic markers in head and neck squamous cell carcinoma treated with platin-based induction chemotherapy h. roussel * , m. housset, h. tournat, p. ravel, s. hans, c. badoual * hegp, dept. de anatomie-pathologie, paris, france objective: ecc enzyme has been associated with resistance to platinum-based chemotherapy. the purpose of this study was to evaluate the role of ercc expression with p and ki- as predictive and prognostic markers in the response to platin-based induction chemotherapy in patients with head and neck carcinoma (hnscc). method: patients treated from to by an induction chemotherapy regimen for hnscc were included retrospectively. we assessed response to treatment, progression-free survival (pfs) and overall survival (os). results: % and . % of hnscc showed low expression of f and fl ercc . no correlation was found between the two clones (p= . ). in the patients treated with cisplatin- fu chemotherapy, a low expression of f ercc was associated with a better response (p= . ). over expression of p , ( . % of cancers of the oropharynx) was correlated with a better os (p= . ) and a better pfs (p= . ). conclusion: these results suggest that ercc expression might be a useful predictive marker of hnscc in patients treated by cisplatin-based chemotherapy. the f ercc clone appears to be the best for immunohistochemistry. our study confirms the prognostic value of the over expression of p in carcinoma of the oropharynx. objective: tumor-stroma ratio or proportion of tumor (pt) has been presented as a prognostic factor in colorectal and breast adenocarcinomas, but there is no information about squamous cell carcinomas (scc) and laryngeal carcinomas in particular. method: eighty-five laryngeal carcinoma cases were included in this series. five digital images of the tumor sections were obtained (h&ex ). percentage of epithelial tumor component was determined by software allowing the pathologist's selection of tiny areas as carcinomatous and stromal region for statistical analysis as a prognostic marker. results: median follow up was months (range - ). the mean pt was ( , + , ) . there was no difference for pt when tumor grade and stage were considered. although statistically insignificant, the mean pt was the lowest ( , + , ) for subglottic carcinomas and cases with perinodal invasion ( , + , ), and highest in pn cases ( , + , ) but there was no statistical significance. the over all and disease free survival analysis did not reveal significance for pt. only pathological stage was an independent factor for over all survival (p= , ). conclusion: although there might be an association with adverse prognostic factors and low tp, the findings in this series does not support pt as a prognostic marker in laryngeal carcinomas. ofp- - tumoral microvasculature in ameloblastoma subtypes c. h. siar * , k. h. ng * university of malaya, dept of. oral pathology, kuala lumpur, malaysia objective: angiogenesis is essential for tumoral growth and progression. the ameloblastoma is a benign but locallyinvasive odontogenic neoplasm with distinct behavioural characteristics of its subsets. whether angiogenesis contributes to a more aggressive course in these variants remains unclear. the aim here was to determine and compare the tumoral microvasculature in different ameloblastoma subtypes and to speculate on their significance. method: immunohistochemical staining for vascular markers (cd , cd , cd and vegf) was performed on archival tissues of cases solid/multicystic (sma), unicystic (ua), desmoplastic (da) and recurrent ameloblastoma (ra). mean microvessel density (mvd) at the tumour advancing front and centres were obtained. results: vegf was heterogeneously expressed in the tumoral epithelium in all ameloblastoma subtypes. protein localization was membranous/cytoplasmic. mean mvd was slightly higher in sma compared to ua but the difference was not significant (p> . ). mean mvds were not significantly different between primary and recurrent ameloblastoma; and between tumoral centre and advancing front of each subtype (p> . ). conclusion: although ameloblastoma subtypes are distinctive in their clinicopathologic presentations and behaviour, their tumoral microvasculature is not different. this suggests that angiogenesis is not a major factor influencing the progression of these ameloblastoma subsets. ofp- - an immunohistochemical study of e-cadherin and snail expression in laryngeal squamous cell carcinoma a. tanoglidi * , m. tsopanomichalou, c. barbatis, c. kostopoulos, o. pantzartzi, v. gorgoulis * hellenic red cross hospital, dept. of pathology, athens, greece objective: snail and (slug) are zinc-finger transcription factors, repressing the e-cadherin in epithelial tumors. published data in head and neck squamous cell carcinoma show either inverse relationship or unrelated co-expression, but e-cadherin reduction/loss and snail overexpression seem to confer an aggressive phenotype. method: thirty cases of laryngeal squamous cell carcinomas (lsccs) (gradei: , gradeii: , gradeiii: ) and one large cell neuroendocrine carcinoma (nec) were studied using double staining immunohistochemical method for e-cadherin (mab-dab) and snail + slug (pab-ap). the colocalization of both molecules was assessed and the expression was compared to the adjacent normal or dysplastic epithelium and the histological grade. results: normal squamous and dysplastic epithelium show strong nuclear snail expression and complete e-cadherin membranous positivity. reduction/loss of snail with or without e-cadherin membranous expression is observed in / ( %) lsccs with a tendency for inverse relationship with e-cadherin at the invasive front. / ( %) lsccs strongly express nuclear snail, without absolute relationship to the reduction/loss of e-cadherin, with a tendency for snail negativity in grade ii ( %). the nec is nuclear snail negative but cytoplasmic positive. conclusion: snail and e-cadherin may be retained, reduced or lost in lsccs, without a definite inverse relationship. these immunophenotypic combinations need further investigation. ofp- - non-sebaceous lymphadenoma of salivary glands: proposed development from intraparotid lymph nodes and risk of misdiagnosis c. weiler * , a. agaimy, p. zengel, s. ihrler * munich, germany objective: non-sebaceous lymphadenoma (nsla) is a recently described, rare benign salivary tumour composed of lymphoid and epithelial components, definitionally lacking sebaceous differentiation. method: nine cases of nsla were immunohistochemically stained for ck / , ck , ck , ck , p , and ki . results: all tumours ( males, females, mean age years) were located in the parotid gland and showed intimate intermingling of lymphoid tissue with islands of epithelium with a wide spectrum of histological differentiation. the immunohistochemical profiles mirrored the epithelial differentiation; tumours with basaloid or lymphoepithelial differentiation strongly expressed ck / , ck , p , while tumours with ductal differentiation showed strong positivity for ck /ck and ck / /ck /p in luminal and basal cell layers, respectively. a hilus structure with salivary inclusions or d - (podoplanin) positive marginal sinus were identifiable in and of the cases, respectively, confirming origin within intra-/periparotid lymph nodes. six cases were initially misdiagnosed as other benign (n= ) or malignant (n= ) tumours. conclusion: our study provides strong evidence that nsla belongs to the group of salivary tumours that pathogenetically develop from embryonic salivary inclusions in intra-/periparotid lymph nodes. knowledge of the wide histological spectrum of this rare tumour is important in order to avoid misdiagnosis. ofp- - crtc /maml fusion transcript in central mucoepidermoid carcinoma of mandible: diagnostic/ histogenetic implications d. bell * * md anderson cancer center, dept. of pathology, houston, tx, usa objective: mec typically arises from major/minor salivary glands. intraosseous salivary carcinomas are extremely rare ( - % of all mecs reported). the t( ; ) and its crtc / maml fusion transcript have been identified in mec at different sites and are associated with development of a subset of these tumors. we report examples of central mec of the mandible, including a case with a history of primary retromolar mec. method: rt-pcr and dna sequencing analyses used to study microdissected components of central mec and noncentral mec. of the central mec tumors, arose from ectopic salivary rests; others appeared to be of glandular odontogenic origins. results: we identified crtc /maml in central mec arising from ectopic salivary rests. this fusion transcript was not detected in non-central mec or in another central mec arising from a glandular odontogenic etiology. conclusion: central mec can manifest the fusion transcript in a subset of central mec originating from ectopic salivary rests, and may have diagnostic/histogenetic roles in the future analysis of this entity, given the absence of the fusion transcript in mec with glandular odontogenic precursors. since the initial clinical and radiological diagnosis in three central low-grade mecs was a benign odontogenic cyst, our findings support a future role for the fusion analysis in initial diagnostic efforts. ofp- - salivary duct carcinoma: morphological and immunohistochemical study of cases m. vazmitel * , a. dubrovskij, r. smoljakova, s. rjabceva * minsk, belarus objective: an evaluation of morphological and imunohistochemical features of salivary duct carcinoma (sdc). method: cases sdc were retrieved from tumor archives collected at belarusian cancer centre from to years. immunohistochemistry, fish. results: a man to women ratio was : ; median age , years (average . tumors arose in the parotid (n= ), submandibular (n= ) glands. sdc developed from pleomorphic adenoma (n= ) or de novo (n= ), there were foci of ductal carcinoma in situ (n= ), sialodochodysplasia (n= ) and pagetoid spread (n= ) additionally to usual features of sdc. as well, tumors demonstrated acinic cell carcinoma-like (n = ) and micropapillary growth patterns (n= ), oncocytic (n= ), clear cells (n= ), apocrine (n= ) and mucinous (n= ) changes. immunohistochemically, tumors were characterized by ar-expression (n= ), her /neu overexpression (n= ), of them score + in cases. ck and s- positivity, stat negativity was detected in one case with acinic cell carcinoma-like pattern. foci of sdc in situ were highlighted by ck and calponin. amplification of c-erb -gene was found in case, etv /ntrk gene was not identified in acinic cell carcinoma-like sdc. conclusion: salivary duct carcinoma, like breast cancer, is characterized by wide variability of morphological patterns and immunohistochemical features reflecting possible molecular heterogeneity, which need further investigation. sunday, september , . - . objective: quantification of protein expression based on immunohistochemistry (ihc) is an important step for translational research and clinical routine. however, routinely used eyeballing scoring systems are time-consuming and subject to significant intra-and interobserver variability. aim of our study was to explore, whether an image analysis software proves sufficient as an alternative tool to assess protein expression. method: prostate cancer specimens were stained with one nucleus specific marker (i.e., erg), one cytoplasmic specific marker (i.e., slc a ), and one marker expressed in both compartments (i.e., tmprss ). a pathologist visually quantified all stainings, applying a four-step scoring system. for digital quantification, an image analysis software (tissue studio v. . ) obtained a continuous spectrum of staining intensity. results: for each of the three antibodies we found a strong correlation of the eyeballing protein expression score and the score of the image analysis software (correlation coefficient of . , . , and . for erg, slc a , and tmprss , respectively, p< . ). conclusion: our data suggest that tissue studio is a powerful tool for the quantification of protein expression in ihc stainings. further, since the digital analysis is precise, it might help to overcome intra-and interobserver variability and increase objectivity of ihc based protein assessment. ofp- - is quality of histological and cytological slides a palatable issue?: the taste project m. comanescu * , g. bussolati, a. sapino, g. butur, f. schmitt, f. feoli, t. tot, e. ovcin * institutul victor babes, dept. of pathology, bucharest, romania objective: the taste (technological platform and repository of high quality images and slides) project aims at developing a novel tele-pathology system by means of modern icts (information and communication technology) and is addressed to technicians and doctors. method: histological and cytological preparations are the basis for pathological diagnosis performed in daily practice throughout europe in a number of several millions per year and correctness and reproducibility of such diagnosis are heavily dependent on the technical quality. yet, quality is variable in different places and countries, related to school level, technicians' dedication, standard of apparatuses and reagents. variation in technical quality of the preparations prevents their open circulation at european level and precludes optimal diagnosis. results: the taste project tackles these problems by building-up an ict environment taste system where professionals from different countries will submit, via web, "virtual slides" of their own preparations to a panel of internationally recognized experts who will give comments and suggestions on the quality of the preparation. conclusion: the present approach (unprecedented at world level) will fuel a web-based community, aimed to a levelling and improvement of histopathological and cytopathological preparations, thus leading to an innovative training and more reproducible diagnosis, a basic requisite for disease treatment. of the whole atlases. virtual microscope positions can be stored in the memory and users can navigate quickly back and forward similarly to web pages. all these features are available through any web browser without need to install any additional software. results: in addition to the current atlases (dermatopathology, fetopathology, pathology of the newborn and bone marrow), an interdisciplinary atlas of pathology for pregraduate students of medicine is under development. it will contain full size annotated virtual slides. the atlases are free of charge, but registration is required. in order to ease the access, the atlases are connected to national academic identity federations. the members of institutions connected to identity federations can use theirs home credentials without revealing them to the atlases. conclusion: the atlases can serve not only to the students as a source of information, but they can be used by teachers as a source of teaching material. proving experiment of international virtual slide telepathology, japan-vietnam and japan-china trial i. mori * , s. aida, y. osamura * international university of health, dept. of pathology, tokyo, japan objective: the communications between asian countries are growing. in the field of medical information and technologies, the necessity of communication is also required. in this work, as communication of pathology field, we tried to prove that international remote pathology diagnosis is possible using virtual microscopy (vs) telepathology. method: we put vs scanner and server to ) our hospital, tokyo, japan, ) cho-rai hospital, ho-chi-minh city, vietnam, and ) chinese rehabilitation research center, beijing, china. pathologists go and look the vs through leased optical line circuit and make diagnosis. discussion was made through teleconference system. results: the vs image maintained adequate quality for pathology diagnosis even looked cross-border. the diagnoses matched well between hospitals. the response of vs viewer was also good this time. when we use too much band width to the teleconference system or we sent too many data in the background, the response deteriorate, and was not good enough for diagnosis. conclusion: it was confirmed that the international vs telepathology diagnosis is now reached to a stage of actual use at least when connected by leased optical line circuit. challenges and solutions in the setup of a findings database for a large scale tissue collection r. reihs * , h. müller, s. sauer, k. zatloukal * medizin. universität graz, inst. für pathologie, austria objective: many medical centres have acquired through the last years huge data collections of great relevance for biomedical research. in order to utilize this knowledge it is necessary to analyse this records in a structured way. in our use case the starting point were approximately . million pathological findings of a non-selected patient group. method: we developed software tools for the classification of medical records using a phonetic a multi-level spell correction module and an ontology term extraction and decision tree text classification system. with the help of a visual editor a comprehensive decision tree ( nodes) was set-up by a team of bioinformaticians and medical experts. results: with our tool set we achieved in the icd- classification a f-score of , % (precession , % and recall , %) for the interpretation of the classification results we developed visualization tools and applied the whole software suite at several test cases, e.g. the analysis of a colon data-set with . findings over years. conclusion: we developed an automatic classification and visualization system and applied the results in a series of research projects. the software suite is currently used in several research projects and can be easily adjusted to specific phrases used in different institutions and languages. an image repository for decision support o. eichhorn * * aperio, vista, usa objective: pathology is a visual field, and pathologists make decisions based on visual information. an online repository of easily searched, well-categorized pathology images can be of immense use to pathologists, scientists, educators, and students. method: individuals and institutions all over the world have accumulated collections with thousands of slides representing a huge variety of pathologies. some collections are digitized and available online, while others remain libraries of glass slides, with a variety of formats for case information and other metadata. working with collectors to publish their slides and aggregating them together can yield an amazing decision support resource. results: long-term archival storage is a key issue for organizations adopting epathology. a cloud-based repository can provide an inexpensive and scalable solution. epathology is increasingly used for remote consultations. cases managed by online consultation networks can be de-identified and contributed to a common repository, increasing the value of the decision support tool while providing a long-term archive. conclusion: this talk will present concepts for an image repository for decision support, and discuss technical and business considerations for making it a reality, including ways to curate image metadata, organizational principles and search tools, relationships with users and contributors, access methods, and security and privacy considerations. ofp- - perfect diagnostic accuracy using pathomorphological diagnosis spectra constructed with the help of whole slide imaging k. furuya * , t. maeda, k. kito * ehime prefectural central hospital, dept. of pathology, matsuyama, japan objective: pathomorphological diagnosis (pd) is important for estimation of clinical trials and new medical equipment such as elastography. however, interpretation of pathomorphological findings is often subjective, and pathology publications show only a limited number of photographs. because pathomorphological changes occur in succession, we attempted to construct pd spectra to improve pd accuracy. method: pd spectra are defined as the series of pathomorphological images arranged in the order of disease progression or cell cycle procession. the images from the whole slide imaging (wsi) server were arranged in order in power point files at a fixed magnification, and pd spectra were constructed: chronic hepatitis staging, nuclear grading of hepatocellular carcinoma, nuclear grading of breast cancer, and ki- labelling index (li). the numbering of all images in the pd spectra corresponded with that of the wsi images so that the whole slides could be viewed alongside. next, these tools were tried out by people. results: these tools brought on perfect accuracy with regard to staging and grading, and very high precision of ki- li for breast cancer. conclusion: pd spectra are valuable not only for pd but also as learning tools and have a worldwide applicability for measuring pd online. ofp- - color correction of immunohistochemistry stained tissue section images by histogram transfer according to control tissues s. sarioglu * , s. seyrek, m. sakar * dokuz eylul university, faculty of medicine, izmir, turkey objective: for semiquantitative and quantitative analysis, previous normalization attempts of images of tissue sections are not satisfactory. in this study we evaluated, if color correction can be achieved by histogram transfer depending upon control tissue image (cti) differences. method: images from colon and placenta sections stained by anti-cd were used as cti and/or sample tissue images (sti). thirty-four but one (standard stained slide with images scti and ssti), was stained for different durations and dilutions than the standard procedures. digital images taken by a ccd camera connected to a light microscope without normalization was stored at a computer. software was prepared in order to find the histogram difference between two ctis and transfer the difference to the sti for achieving a corrected sti (corsti). ssti (one image) and sti and corsti ( images each) were semiquantitatively scored by two observers in blind fashion and the sti and corsti scores were compared with ssti score. results: the wkappa was , for two observers. the sti semiquantitative score was same as the ssti in , % of the sti but this was , % for corsti. conclusion: it seems histogram transfer depending upon ctis may be a valuable tool for color correction of tissue section images. sunday, september , . - . objective: there is a crucial need for identification of markers to choose the best treatment schedule and monitor the effectiveness of treatment in metastatic melanoma (mm) . the detection of circulating tumor cells (ctc) correlates with prognosis in different cancer subtypes. ctc can be detected by direct and indirect methods such as cytopathological approach, rt-pcr or immunomagnetic separation using the cellsearch (cs) method. method: from june to december , blood was drawn from patients with mm and controls patients and processed into h by the cs method. the primary end-point was the overall survival (os) of mm patients. results: enumeration of at least cmc was effective in . % of patients with mm (n= ) and in % of controls patients (n= ). nine patients ( %) had ≥ cmc. none of the controls patients had more than one cmc. mean os time was . months among patients with mm and < cmc (n= ) and mean os time was . months among patients with mm and cmc ≥ (n= ) (p= . ). conclusion: our results demonstrate that detection of cmc correlates with os in melanoma. thus, cmc monitoring should be developed to ensure the best treatment follow-up for patients with mm. ofp- - significance of circulating tumor cells (ctcs) detection using the cellsearch system in patients with locally advanced head and neck squamous cell carcinoma (hnscc) a. bozec * , m. ilie, e. long, o. dassonville, g. poissonnet, j. santini, e. chamorey, f. peyrade, k. benezery, a. sudaka, e. selva, p. hofman * centre antoine lacassagne, dept. of surgery, nice, france objective: the significance of ctcs in patients with hnscc is debated currently. we evaluated the potential detection of ctcs using the cellsearch (cs) assay tm (veridex, nj, usa) in hnscc patients and to identify the clinical factors predictive of the presence of ctcs in this population. method: forty-nine patients with locally advanced hnscc were included. the presence and number of ctcs were determined using the cs system in all patients prior to the initiation of therapy, in patients months after treatment, and in healthy individuals. results: the cs system was able to detect the presence of ctcs in of patients ( %) before therapy and in of disease-free patients ( %) after therapy. no ctc was found in the control group. when considering the presence of ctcs before or after therapy, the presence of ctcs was statistically associated with patients' age (p= . ; t-test) and n-stage (p= . ; fisher's exact test). conclusion: ctcs are identified in a relatively low proportion of patients with locally advanced hnscc and correlated with initial lymph node involvement. ctc detection could be a future useful prognostic tool to adapt the treatment intensity in hnscc patients. the diagnostic utility in identifications of an aneuploid chromosomal (ch.) pattern by using urovysion™: a fluorescence in situ hybridization (fish) commercial test, to improve the efficacy of cytology in peritoneal effusions l. baron * , m. postiglione, c. trombetta, f. quarto * p.o.s. leonardo, s.o.c. di anatomia patologica, castellammare di stabia, italy objective: the challenge in diagnosis of effusion is in differentiation of reactive and neoplastic mesothelial or metastatic cells and identification of primary neoplasia. the difficulties in this differential diagnosis derived from the low sensitivity (~ %) and specificity of effusion cytology. by identification of tumor associated aneuploidy fish could enhance the cytodiagnostic yield in effusions. method: we used urovysion™ (abbott), a fish commercial kit, designed for identifications of ch. , , polisomy and p loss in urothelial tumors. it has been applied to detect chromosomal aneuploidies as a marker of malignancy in effusions with or without known source primary tumors. also was evaluated reproducibility of molecular alterations with primary tumors and any their specific aneuploidies. results: fish positivity was observed in ( %) of cases, whereas were positive at cytology ( %). overall results of cytology and fish together rised to %. conclusion: urovysion™ test could be an useful tool to distinguish malignant cells in inconclusive effusions cytology but it doesn't seem to be used to provide definitive indications about primary neoplasia. ofp- - loss of imprinting of isulin-like growth factor (igf ) in colon carcinomas leads to the cell cycle genes activation -hints to intense proliferation d. belharazem * , j. kunanz, a.-k. henne, p. kienle, p. ströbel * inst. of pathology, university of medicine, mannheim, germany objective: the insulin-like growth factor (igf ) gene is regulated by imprinting in normal tissues. the loss of imprinting (loi) results in the bi-allelic expression of igf . its increased activity has been associated with many cancers including colorectal cancer (crc). to investigate the significance of igf in crc, normal tissue and tumor biopsies from patients were analyzed and correlated with clinical outcome and the kras and pik status. method: the igf g/a gene polymorphism and imprinting status were examined by restriction fragment length polymorphism of dna derived from normal tissues and tumors then of rna from heterozygous cases. igf protein analysis as well as an rna microarray were performed. results: % of the analysed cases were heterozygous. loi was detectable in both normal tissues and tumors in % of cases. tumors with loi had significantly higher igf protein levels. gene expression analysis revealed significance of cell cycle progression and mitosis genes in loi. conclusion: igf loi is a common and early change in patients with crc and in normal tissues, provides possibly a preneoplastic change. however increased igf protein levels as well as a different cell cycle specific gene expression profile were detected in tumors with loi. future studies must be performed to find out, whether such subsets of tumors differ in their response to chemotherapy or alternative therapies. objective: novel high-throughput technologies has revealed numerous genomic abnormalities in b-cell non-hodgkin lymphomas (nhl). this study aimed to detect point mutations involving ezh and cd b in a population of b-cell nhl by applying a multiplex mutation assay to minimal residual material from needle aspirates, and to determine the evolution of their mutational status overtime using all sorts of available samples from these patients. method: dna was extracted from residual cytological material stored on fta® cards as well as from archived cytological and histological specimens. the presence of point mutations was investigated by a specifically developed assay utilizing massarray spectrometry and confirmed by direct sequencing. results: all samples from b-cell nhl cases were successfully analyzed. mutations in ezh (y ) and cd b (y ) were detected in . % and % of the samples, respectively. mutational status varied in one third of the positive cases, with either gain or loss of mutations over the course of time. conclusion: minimal material from lymph node fine needle biopsy is a reliable source for high-throughput multiplex analysis for the detection of point mutation involving ezh and cd b. whenever possible, the most recent sample should be tested to verify the current status of the disease. ofp- - microrna expression profile identifies a distinct molecular signature between myc translocation-positive and negative burkitt lymphoma cases g. de falco * , a. onnis, f. morettini, f. fuligni, c. bellan, e. rogena, p. p. piccaluga, l. leoncini * university of siena, dept. human pathology and oncology, italy objective: the molecular hallmark of burkitt lymphoma (bl) is a dysregulation of myc oncogene, due to one of the three translocations leading to ig-myc fusion. however, about % of bl cases lack an identifiable myc rearrangement, although no significant difference of myc expression among cases has been observed. additional mechanisms alternative to translocations were explored by microrna profiling. method: ten myc translocation-positive and ten myc translocation-negative formalin-fixed and paraffin-embedded bl specimens were used for this study. for microrna and gene expression profile, the samples were hybridized on the mircury™ lna array and the illumina dasl whole genome assay, respectively. results: our results identified a panel of four micrornas which are differentially expressed between the two groups. importantly, these micrornas control relevant biological processes, such as the angiogenesis, apoptosis and cell proliferation, according to gene ontology categories. furthermore, the impact of microrna dysregulation on the gene expression pattern identified genes which more likely are regulated by the selected microrna. conclusion: using this approach, we showed a clear-cut microrna and gene signature between myc translocationpositive and negative bls. the identification of specific altered micrornas may represent an alternative molecular mechanism leading to myc over-expression in the absence of genetic alteration in cancer. ofp- - droplet digital pcr: a highly sensitive assay for b-raf, k-ras and egfr mutation detection j. frampton * , c. thorne, v. spivey, j. goodall, c. lowe * horizon discovery, dept. of diagnostics, cambridge, united kingdom objective: droplet digital™ pcr (ddpcr™) enables the absolute quantitation of nucleic acids in a sample. horizon discovery's patented gene editing technology enables it to manufacture reference material that is genetically defined and validated for the allelic frequency of the mutation. these reference standards were used to test the sensitivity of bio-rad's qx ™ droplet digital™ pcr platform. method: a panel of defined genomic dna allelic standards and formalin fixed paraffin embedded (ffpe) cell line samples were used in this study. this panel covered egfr (g s, t m, l r and l q), k-ras (codon ) and b-raf (v e and v k) mutations containing allelic frequencies between . % and %. droplet digital™ pcr was performed using mutation specific taqman custom snp assays. results: there was a strong correlation between the predicted and actual allelic frequencies even down to . %. the allelic frequency called by the ddpcr™ platform was highly reproducible between experiments particularly in the - % dilution range. conclusion: this study has demonstrated that the droplet digital™ pcr qx ™ platform can detect allelic frequencies down to at least . % using genomic dna purified from cell lines and down to at least . % using genomic dna extracted from ffpe cell lines. ofp- - chromothripsis and focal copy number alterations determine poor outcome in malignant melanoma t. gaiser * , d. hirsch, r. kemmerling, j. camps, t. ried * medizin. universität mannheim, inst. für pathologie, germany objective: genetic changes during tumorigenesis are usually acquired sequentially. however, a recent study showed that in - % of all cancers a single catastrophic event, termed chromothripsis, can lead to massive genomic rearrangements confined to one or a few chromosomes. method: in order to explore whether the degree of genomic instability and chromothripsis would influence prognosis in cancer, we applied high-resolution array comparative genomic hybridization to malignant melanomas (mm) that were, despite comparable conventional clinical and pathological parameters, defined by a profoundly different clinical course. results: we observed a striking association between both number and structure of chromosomal aberrations and outcome. mm associated with good prognosis showed only few chromosomal imbalances (average . alterations per case), predominantly presented as whole chromosome or chromosome arm gains and losses. mm with poor prognosis harbored significantly more chromosomal aberrations ( . per case; p= . ). these aberrations were mostly focal events, which culminated in two cases in a pattern consistent with chromothripsis. conclusion: here, we describe for the first time the phenomenon of chromothripsis in primary mm and reveal a link between focal copy number alterations and chromothripsis with poor outcome in mm patients (p= . ), providing a genetic approach to predict outcome in histopathologically indistinguishable mm. ofp- - targeting endometrial stromal sarcoma: histone deacetylase and pi k/akt/mtor signaling p. quan * , e. lederer, i. halbwedl, h. denk, k. zatloukal, j. haybaeck * institute of pathology, medical university of graz, austria objective: endometrial stromal sarcoma (ess) is a gynecological malignancy with few therapeutic options. up-regulated histone deacetylase (hdac) was observed in ess patients. the hdac inhibitor saha reduced ess cell growth by inhibiting mtor. the pi k/akt/mtor signaling cascade, vital to cancer growth is a critical target in cancer therapy. this study aimed at investigating if hdacs interact with the pi k signaling in ess pathogenesis and thus might serve as targets for ess therapy. method: tissue microarrays were used to determine hdac expression in ess specimens. western blots revealed protein expression levels of hdacs and pi k related molecules in two independent ess and one control cell lines (ess- , mes-sa and hesc). results: elevated hdac and levels were found in ess tissues and cell lines. increased cell growth and akt/mtor cascade hyperactivation were detected in ess cells. saha reduced growth of all cells, and induced cell death in ess cells. saha reduced phosphorylated ebp in all cells, but only inhibited activation of akt and p s k in ess- cells. conclusion: hdacs are linked to pi k signaling during ess pathogenesis. saha reduced cell growth via inhibiting pi k/akt/mtor components suggesting that a combination of saha with pi k inhibitors might be effective in treating ess. ofp- - usefulness of linking biobanking field and animal model: high successful rate of human primary non-small cell lung carcinoma (nsclc) xenografts in a model system separated by distance and time m. ilie * , l. blot, v. hofman, e. long, m. nunes, c. butori, e. selva, a. merino-trigo, n. vénissac, j. mouroux, p. vrignaud, p. hofman * chu de nice, lpce, france objective: with the ongoing need to improve therapy for nsclc, there has been an increasing interest in the development of reliable preclinical models to test novel therapeutics. the aim of this study was to evaluate the rate of establishment of patient-derived nsclc xenografts in the context of a long-distance research network. method: fresh surgically resected nsclc specimens were addressed from the human biobank hospital (nice) to the animal facilities (sanofi, vitry-sur-seine). shipment was performed in aqix medium at room temperature. within h post surgery, tumour fragments (~ mm ) were subcutaneously implanted in female scid mice. the growing tumours were passaged in new mice (≤ passages). the xenografts were histologically checked to eliminate human or murine lymphoma. results: overall, nsclc samples were implanted leading to ( %) nsclc xenografts. the rate of tumour growth was higher in non-adenocarcinoma specimens ( / ; %): / ( %) for squamous cell carcinoma, / ( %) for large cell carcinoma, and / ( %) for pleomorphic carcinoma, when compared to adenocarcinoma samples ( / ; %). conclusion: we report a high successful rate of xenotransplantation established from patient-derived nsclc tissues. our biobanking model system, regardless to extended time and distance, provides a stable and reliable animal model in human lung cancer research. monday, september , . - . objective: we tried to interpret "the job" of mf in different stages of tumor growth by monitoring the dynamics of their distribution. method: on a rat model of experimental tumorigenesis using bp fibrosarcoma cells injected intraperitoneally, we performed a semiquantitative analysis of the recruitment of mf in the tumor compartment (microenvironment) as well as in peripheral blood. results: we observed a slow increase of mf count in the initial phase of tumor growth followed by a slow decrease in the late phase within the tumor compartment. simultaneously peripheral blood showed constant increase throughout the experiment resulting in typical leukocytosis accompanying developed tumors. conclusion: increased mf in the initial phase could reflect their enrollment to "prepare a suitable microenvironment for tumor seeding". their decrease began after the tumor size was macroscopic, thus it was time for them to carry out a "different job". in the mean time their amount in peripheral blood started becoming significant, this is also the time when tumor -host interactions reach a systemic level. in futher experiments we will attempt to verify our assumption: when the tumor flourish in the host organism, it uses informed, transformed and recruited macrophages for growth and building construction of the tumor, and for communication with distant organs on a regulatory level. ofp- - wwtr and cyr are early markers of barrett's esophagus malignant progression m. mesquita * , j. cardoso, a. pereira, s. braga, m. bettencourt-dias, p. chaves, j. pereira-leal * ipolfg, dept. of pathology, lisboa, portugal objective: barrett's esophagus (be) is the major risk factor for esophageal adenocarcinoma (ea). be has a low risk of progression to ea, being imperative to identify markers to stratify the risk. our aim was to look for potential early biomarkers of be malignant progression. method: three available microarray datasets were analyzed with r statistical computing software complemented with bioconductor. biomarker prioritization included: ) expression barcode . ; ) filtering using differential expression analysis. candidate genes were validated by qrt-pcr on rna from paraffin embedded samples (rna-pes) of be patients with high-grade dysplasia/adc diagnosed during surveillance and of their index endoscopy dysplasia-free samples. as controls, we used samples from be patients who have not progressed. results: under conservative criteria, we identified upregulated genes that distinguish ea-progressed from nonprogressed be samples. a second filter, followed by qrt-pcr validation, trimmed the candidates to markers (wwtr and cyr ). qrt-pcr on time-series rna-pes of ea-progressed and ea-free be samples showed these genes are up-regulated years before the development of ea as compared to patients who did not developed ea. conclusion: wwtr and cyr were identified as early risk markers for be neoplastic progression and may have a potential role in be risk stratification. combined microrna in situ hybridization and immunohistochemical detection of protein markers b. nielsen * , t. møller, k. holmstrøm * bioneer a/s, dept. of molecular histology, hørsholm, denmark objective: micrornas are small noncoding rnas that constitute a novel group of biomarkers with exciting functions in cell differentiation, proliferation and apoptosis by mediating degradation or destabilization of target mrnas. mir- is highly prevalent in malignancies. previous reports on mir- in situ hybridization (ish) in colon and breast cancer identified expression in the stromal cell population that was related to recurrence and cell proliferation (nielsen et al., clin exp metastasis , : ; rask et al., apmis , : ) . here, we present a histological assay platform that allows parallel localization of micro-rnas and protein markers. method: routinely processed paraffin embedded breast cancer samples were analyzed. microrna ish probes were detected with fluorescent tyramine reagents and primary antibodies with compatible immunofluorescence. the reagents were introduced on a semi-automated platform, histoflex, which has a precise temperature control unit and utilizes continuous liquid flow. results: by combination of mir- ish and pdcd immunofluorescence we found differential expression in many but not all mir- positive cells. using the histoflex, the double fluorescence assay was reduced from to h without loss of sensitivity and specificity. conclusion: a -fold faster assay platform was developed and our in vivo findings support the assumption that pdcd is a target of mir- . the tnf-superfamily members april and baff and their receptors (baffr, bcma, taci) are differentially expressed in tumors and normal tissues and exert specific effects related to cell fate and differentiation v. pelekanou * , v.-i. alexaki, g. notas, m. kampa, e. stathopoulos, a. tsapis, e. castanas * university of crete, pathology, school of medicine, heraklion, greece objective: classically, tumor immune-related microenvironment was synonym of inflammatory cell infiltrate. recently, the detection of synthesis and secretion of immune mediators by tumor cells highlighted tnf-superfamily (tnfsf) members as key counterparts of cell fate-related signals. here, we assayed the synthesis-expression-function of a subset of tnfsf ligands and receptors (baff, april, baffr, bcma, taci) in normal epithelial tissues and tumors and resident adult mesenchymal stem cells. method: tissues (breast, cns, kidney, skin, adipose tissue) have been assayed by means of immunohistochemistry and real-time pcr. cell models were used for signaling, transcriptomics and functional assays. results: all tumors expressed this tnf-sf subset. baffr is absent, while its specific ligand baff is ubiquitously expressed; contrairewise, april, bcma and taci are expressed in tissue-specific manner. april relays on tumor evolution/grade and is involved in tumor proliferation/differentiation/apoptosis signaling through nfκb-related, or a novel pathway, implicating jnk/foxo a/gadd , triggering transcriptional events. conclusion: autocrine/paracrine effects of april and its receptors bcma and taci in normal/tumoral parenchyme and the recruitment/attraction of immune-related cells could orchestrate tissue response, regulating cell fate, differentiation, inflammation, tissue remodeling and cancer, with potential tailored-therapy application. objective: mutation analysis of kit and pdgfra genes is currently used in gastrointestinal stromal tumors (gist) as an important step in diagnostic protocol. method: dna samples obtained from parafin-embedded biopsy material of gist patients diagnosed in - were screened for mutations in exons , , and of kit and , and of pdgfra. results were tested for associations with clinical parameters of the tumor. results: causal mutations (according to in silico analysis with polyphen- predictor) were identified in . % of patients, most frequently in kit exon ( . %). the kit exon , pdgrfa exons and , kit exon , pdgfra exon and kit exon mutations appeared with frequencies . %, . %, . %, . %, . % and . % resp. genotype-phenotype correlation analysis revealed statistically significant association between intestinal localization of tumors and presence of kit exon p. - _dup mutation and gastric localization of tumors and presence of pdgfra exon p. d v mutation. conclusion: centralized and standardized gist diagnosis offers clinically and prognostically relevant informations and is necessary for the indication of appropriate targeted therapy of gist patients. supported by projects cepr ii (imts ) and mbcc (imts ) at cu jfm co-financed by eu and by grant novartis slovakia. objective: an association between cystic fibrosis transmembrane regulator (cftr) gene mutations and infertility may occur. this study investigated the frequency of mutations in the cftr gene, of a group of consecutive patients candidate for assisted reproductive techniques with the aim of identify subjects carriers of the most severe ones. method: we screened healthy subjects ( females and males) for cftr gene mutations and ivs -polit polymorphism utilizing the cftr inno-lipa amplification kit including both general and italian regional strips. results: cftr mutations were detected in . % of the patients, a percentage similar to that reported in the general population. the most common mutation was Δf /n observed in . % of patients. no difference in the gender distribution was evidenced. in the large group of patients analyzed . % were wt, . % carrier of one mutation, and interestingly . % compound heterozygous. conclusion: our data support the relevance of an accurate determination of mutations in the cftr gene in order to inform the couple of their carrier risk and the possibility on having affected child. moreover our findings highlight the potential of genetic screening as a tool to identify possible compound heterozygous subjects without cf-like symptoms. objective: a patient's response to cancer treatment can depend on their biomarker status. therefore it is important that laboratories perform their biomarker tests well. in an international external quality assessment scheme for kras mutation analysis on ffpe slides was set-up. method: this scheme was run for years by the coordination center, a medical and technical expert and scheme organizers under the umbrella of esp. participants received samples. genotype results were analyzed and labs with good performance were listed on the esp website. written reports of were evaluated in detail. results: in , % of participants identified all samples correctly. results seemed to improve over the years from . / to . / . genotype results from laboratories that participated for years appeared to improve overtime, although this couldn't be statistically proven. in the reports some important elements weren't always present. for example, the patient's name was only present in %, correct hgvs nomenclature in % and interpretation of results in less than %. conclusion: kras-eqa probably facilitates improvement of laboratory testing. the quality of reports of kras tests needs to be improved. a guideline for reporting molecular pathological results would be a good instrument for both participants and assessors. objective: microsatellite instability (msi) due to mismatch repair (mmr) deficiency is reported in a fraction of colorectal cancers (crcs) ( - %) complicating ibds (ibd-crcs). our recent findings argued for the existence of yet unknown mechanisms underlying mmr-deficiency in ibd-crcs, different from those involved in sporadic and hereditary msi crcs. here we hypothesized that over-expression of mir- and mir- , two inflammation-related mirnas that target core mmr proteins, could constitute a mechanism underlying mmr deficiency in ibd-crcs. method: we compared the expression of both mirs in ibd-crcs and non-ibd hereditary or sporadic crcs using nonneoplastic ibd and healthy mucosa samples as controls. results: overall, mir- and mir- were significantly over-expressed in both non-neoplastic and neoplastic mucosa of ibd patients as compared to healthy controls. mir- alone was preferentially expressed in msi vs. mss ibd-crcs. in contrast, mir- was poorly deregulated in non-neoplastic and neoplastic tissues of non-ibd msi or mss crcs. conclusion: mir- , alone or synergistically with mir- , may favor the emergence of msi ibd-ccrs. since its deregulation was not limited to neoplastic tissues but extends to non-neoplastic normal mucosa as well, the hypothesis of a mir- field defect promoting mmr deficiency and yet msi-driven transformation can be proposed. ofp- - pi k, akt and pten expression in enucleated brazilian uveal melanoma cases using tissue microarray g. freeman * , d. begnami, a. damascena, j. neves, s. nonogaki, f. soares * são paulo, brazil objective: in the world literature, little has been published on the molecular pathways in primary uveal melanoma or subsequent development of metastases. the pi k pathway has been implicated in regulation of apoptosis, cell cycle regulation, transcription and translation. method: we used immunohistochemistry with tma to investigate expression of pi k, akt and phos-akt, and pt in over formalin fixed paraffin embedded blocks of uveal melanoma from the a.c. camargo hospital, são paulo, brazil from to . fish was performed for pten copy numbers. results: in this study, neither immunohistochemistry nor fish results showed a statistically significant difference between the primary tumors and the tumors which metastasized. statistical analysis of all tumors together (tma) revealed only one molecular marker (pten), which gave results close to statistical significance. conclusion: this is the first large study of brazilian patients for pi k, pten akt expression by immunohistochemistry and pten using fish. expression values for molecular markers chosen did not reach statistical significance, although pten values were close. use of fish to distinguish metastatic from non metastatic cases also came close to statistical significance. these results suggest that the expression of pten in uveal melanoma may be a good topic to investigate further. objective: chronic antibody-mediated (camr) and chronic t-cell-mediated rejection (ctcmr) represent predominant reason for late graft dysfunction. both categories share similar morphological features. we ask the question whether there are differences between camr and ctcmr on the molecular level. method: graft biopsies (> m) were performed and evaluated according to the banff classification. biopsy specimens with camr (n= ), ctcmr (n= ) and from protocol biopsies with normal histology (n= ) were stabilized in the rna-later. using the taqman low density array, the intrarenal expressions of genes relating to immune response (b-cell activation, t-cell activation, chemokines, growth factors, immune regulators and apoptosis) were analyzed. results: both categories of chronic rejection were associated with up-regulation of many genes in comparison with the control group: chemokines (ccl , ccl , cxcl , cxcl , cxcl ), growth factor tgfb , mhc class ii (hla-dma, hla-dmb, hla-dr, ubd), and in t-cell dependent mechanisms (cd , cd , lag ), including cytotoxic t-cell associated transcripts (gbp , gzmk). in hierarchical clustering, camr and ctcmr gene expression profiles were similar. conclusion: chronic rejection very probably involves prolonged cooperation of innate immunity and allospecific immune response. our study showed that ctcmr and camr do not differ on the molecular basis. ofp- - podocyte loss and glomerulosclerosis in inducible mouse model of podocin mutation-related nephrotic syndrome i. simic * , m. tabatabaeifar, g. mollet, c. antignac, s. weber, f. schaefer * universitätskinderklinik, nephrologisches labor, heidelberg, germany objective: mutations in nphs gene, encoding podocyte specific protein podocin, cause hereditary nephrotic syndrome. knock-in mice carrying the r q mutation, murine analogue of the most common human mutation r q, show developmental arrest of podocytes and renal failure at neonatal age. the aim of this study was to quantify renal histopathological changes in mice with postnatally induced r q hemizygosity. method: c bl/ mice with nphs flox/r q, cre + genotype were injected with tamoxifen for days to induce hemizygosity for r q-mutant podocin. tissue samples were collected at defined intervals after induction. renal morphology was evaluated by quantitative histology, immunohistochemistry and electron microscopy. results: animals developed proteinuria within week that progressed into renal failure and advanced uremia at week - . the number of podocytes per glomerulus started decreasing at week ( ± vs. ± , p= . ), whereas glomerular sclerosis index increased from week ( . ± . vs. . ± . , p< . ). interstitial changes included fibrosis (up to % of section area in end stage renal disease), tubular atrophy and dilatation. conclusion: our results implicate that the expression of mutated podocin in induced r q-podocin mice leads to podocyte loss that precedes interstitial damage and glomerulosclerosis. quantification of histological changes will enable better evaluation of the efficacy of different pharmacological approaches directed to improvement of podocyte viability and attenuation of glomerulosclerosis. ofp- - heavy chain deposition disease in kidney biopsies a. vizjak * , j. mraz, j. lindic, d. ferluga * university of ljubljana, faculty of medicine, slovenia objective: heavy chain deposition disease (hcdd) is a rare and not yet fully explored monoclonal immunoglobulin-related disorder. method: we studied the histopathology in kidney biopsy cases of hcdd, representing a . % prevalence among native kidney biopsies. results: hcdd was diagnosed in kidney biopsies of women (mean age . years). light microscopy showed diffuse nodular glomerulosclerosis ( / ), associated with mesangial proliferation ( / ) and capillary aneurysms ( / ). immunofluorescence showed abundant mesangial deposits and ribbon-like deposits along glomerular, capsular, tubular and vascular basement membranes, positive for heavy chain igg ( / ) and igg ( / ), with deleted gamma ch domain ( / ). complement c and c q stained positive in all cases. by electron microscopy, punctate and powdery electron-dense deposits were found on the same locations. conclusion: immunofluorescence examination of kidney biopsies, including testing for immunoglobulin heavy and light chains, is crucial for diagnosis of hcdd. our study confirmed that hcdd is peculiar among monoclonal immunoglobulin deposition diseases not only because of its rarity but also because of uniform histomorphologic pattern of nodular glomerulosclerosis with pronounced capillary aneurysms and significant proliferation due to complement activation. deletion of the heavy chain ch domain and its significance in the pathogenesis has to be emphasized. ofp- - two cases of anca associated vasculitis with geographical necroses in the kidney tissue a. bartonova * , e. honsova, r. rysava * ikem, dept. of pathology, praha, czech republic objective: anca associated vasculitides (aav) are rare systemic autoimmune diseases affecting small to mediumsized blood vessels. in kidney biopsy samples, aav usually demonstrate pauci-immune necrotizing crescentic glomerulonephritis (gn). method: among cases of aav evaluated at ikem during years, had a very unusual pattern of renal involvement. results: the first patient had been treated for recurrent otitis media with unilateral hearing loss. he also suffered from artralgias and hematuria. an ultrasound study revealed a tumor-like mass in his kidney. during exploratory surgery, the lesion appeared to be infiltrative and a nephrectomy was performed. the second patient presented a granulomatous inflammation in a scar. a month later she developed multiple kidney and spleen "abscesses" with negative hemoculture, and underwent a nephrectomy with splenectomy. in both cases, the final pathological diagnosis was necrotizing granulomatous vasculitis of small and medium-sized vessels, with large geographical necroses of kidney tissue simultaneously with necrotizing crescentic gn. tests for anca antibodies were positive. no microbial pathogens including mycobacteria were detected. objective: polyomavirus nephropathy (pvn) is a common complication after renal transplantation. virus control is achieved by a reduction of immunosuppression allowing an effective t cell-mediated antiviral immune response. the morphology of resolving pvn has not been investigated. method: protocol biopsies of patients with pv viremia treated by reduction of immunosuppression only were included and scored according to banff criteria. the extent of interstitial inflammation was estimated as % of cortex. the number of tubular cross sections with sv + cells per mm of biopsy length was counted. findings were grouped as pre-, increasing, decreasing, and post-viremia. results: during the phase of decreasing viremia, we found a significant increase in the tubulitis score, the extent of tubules with intraepithelial lymphocytes, and interstitial inflammation (p< . ). these, to a lower extent, persisted after virus clearance. the number of sv + tubules correlated with the virus load in the serum, but sv immunohistochemistry was frequently negative ( / cases), especially if viremia was below geq/ml. conclusion: resolving pvn is characterized by a self-limiting acute interstitial nephritis. our findings are important because the diagnosis of interstitial rejection depends on the same morphological criteria. therefore, acute interstitial rejection cannot be diagnosed with certainty during pv viremia. ofp- - neural cell adhesion molecule and fibroblast growth factor receptor positive interstitial cells increase in interstitial fibrosis in different renal diseases j. markovic-lipkovski * , c. müller, s. cirovic, s. tatic, d. mitrovic, g. müller * university of belgrade, faculty of medicine, serbia objective: ncam + cells with dendritic morphology are rarely present in normal renal interstitium. the aim of this study was to evaluate presence of ncam + cells in kidney biopsies of different renal diseases with and without interstitial fibrosis (if). further immunophenotyping of ncam + renal interstitial cells was performed. method: kidney biopsies, after routine diagnosis, were stained applying antibodies against ncam clone c .d or cloneeric . for double immunofluorescence, antibodies against ncam cloneep y and fgfr , alpha beta integrin, alphasma, cadherin , cadherin were used. by rpcr, different ncam isoforms ( , , ) were detected using specific primers in renal tissues with and without if. results: ncam + interstitial cells were identified in cases: % lupus nephritis, % focal segmental glomerulosclerosis, % iga nephropathy, % membranoproliferative glomerulonephritis, % membranous glomerulonephritis. ncam + cells coexpressed fgfr and alpha beta integrin, and were increased in if. ncam + cells did not coexpress alphasma, cadherin and cadherin , although they were closely colocalised. all normal renal tissues tested for rpcr showed presence of all ncam isoforms, however ncam lacked in some tissues with if. conclusion: ncam + renal interstitial cells coexpress fgfr and alpha beta integrin and are increased in renal diseases with if, mostly in diffuse proliferative lupus nephritis. in contrast to normal kidneys, in renal tissue with if ncam is present in a lesser extent. reproducibility for c d immunohistochemistry in renal allografts: results from the banff trial m. mengel * , s. chan, j. climenhaga, p. randhawa, h. regele, y. kushner, r. colvin * university of alberta, laboratory of medicine, edmonton, canada objective: detection of c d is crucial for diagnosing antibody mediated rejection, yet formal reproducibility studies are limited. method: we conducted an international multi-center trial to assess the reproducibility for c d immunohistochemistry on paraffin-sections. a tissue microarray (tma) was constructed comprising kidney allograft specimens representing negative, focal, and diffuse c d positive cases. participants stained the tma slides, evaluated their stains and entered their scores online. stained slides were returned for centralized panel scoring. weighted kappa statistics were used to determine reproducibility. results: inter-institutional reproducibility, i.e. the product from variability between observers and staining methods was low (mean kappa . ). inter-observer reproducibility was fair (kappa . ), while inter-laboratory reproducibility was moderate (kappa . ). inter-observer reproducibility could be significantly improved by omitting the banff c d grading schema and only considering +/− calls (kappa . ). scoring only c d+/− inter-laboratory reproducibility improved considerably (kappa . ). higher dilutions of the primary antibody were associated with worse reproducibility. fixation < h or fixation in ethanol had significant negative impact on inter-laboratory reproducibility. conclusion: c d results reported from paraffin section are highly variable between institutions. simplification of the grading schema would improve reproducibility between observers. technical reproducibility between laboratories is acceptable but could further be improved by standardizing protocols. objective: heparanase is a predominant mammalian enzyme that cleaves heparan sulfate, the key polysaccharide found in the basement membranes and at cell surface. heparanase is overexpressed in the diabetic kidney; however, its role and mode of action in diabetic kidney disease remains largely unclear. method: applying heparanase-null mice we found that deletion of the heparanase gene protects diabetic mice from diabetic nephropathy (dn). recombinant heparanase enzyme and a specific heparanase inhibitor (sst ) were used to explore its mode of action in several in vivo and in vitro models. results: there is essential involvement of heparanase in the pathogenesis of dn. deleting the heparanase gene protects diabetic mice from dn and administration of specific heparanase inhibitor decreases the extent of albuminuria. in vitro, heparanase enhances macrophage activation by diabetic milieu components, and thus increases the kidneydamaging properties of macrophages. conclusion: our results validate the role of heparanase in dn and reveal the mechanism of heparanase action emphasizing its function in coupling chronic inflammation, macrophage activation and diabetic kidney injury. these findings will help in developing effective strategies to disrupt the heparanasedriven sequence of events in diabetic kidney disease, and in designing novel therapeutic interventions in dn. sunday, september , . - . , terrace ofp- oral free paper session neuropathology ofp- - microvascular angiogenesis occurs in a subset of only the arteriovenous types of vascular malformations and is more abundant in men than in women l. b. meijer-jorna * , c. m. van der horst, c. m. van der loos, a. c. van der wal * medical center alkmaar, symbiant pathology center, netherlands objective: episodic volume expansion may complicate the very slow growth pattern of congenital vascular malformations. we investigated the role of microvascular angiogenesis in this process of sudden growth. method: resection specimens of symptomatic vascular malformations were screened for presence and extent of sheets or clusters immature microvessels, interpreted as microvascular angiogenesis. microvessel density (mvd), mast cell density (mcd) and ki labelling index of endothelial cells (ec) were assessed immunohistochemically and quantified. extent of angiogenesis was correlated with the type of vascular malformation and clinical characteristics. results: of cases, were arteriovenous malformations (avm), were venous (vm), and were lymphatic (lm). microvascular angiogenesis was observed in % of all vascular malformations, of which % appeared to be avm. mvd, mcd and ki labelling indexes of ec were significantly higher in immature vessel areas. moreover, in affected patients these angiogenic responses were far more extensive (in terms of area involvement) in men than in women (p< . ). conclusion: microvascular angiogenesis appears a specific feature of the arteriovenous type of vascular malformations, and is much more extensive in men than in women. we suggest that these microvascular responses may contribute to onset of symptoms due to a mass forming effect. ofp- - the impact of ventricular assist device prior to transplantation on acute cellular rejection and antibody-mediated rejection in cardiac allografts with due consideration of seasonal behaviour: a prospective study k. wassilew * , e. potapov, c. knosalla, t. krabatsch, m. hummel, r. hetzer * deutsches herzzentrum berlin, germany objective: this study evaluated the impact of bridge-to-transplant ventricular assist device support on development of acute cellular (acr) and antibody-mediated rejection (amr) with due consideration of seasonal behaviour in cardiac allografts. method: we studied consecutive right ventricular endomyocardial biopsies (emb) between / and / prospectively. paraffin-embedded sections were evaluated for acute cellular rejection, endothelial cell swelling and capillary deposition of c d, c d and iga/m/g. the effects of vad (n= ) on acr and amr, classified according to the ishlt, were studied for seasonal effects and compared to results of emb harvested from patients without vad support (n= ). results: our results did not reveal significant differences between the two groups in any given parameter. a positive correlation was found for endothelial cell swelling and capillary c d deposition; the data failed to show a correlation between c d and c d deposition or c d deposition and endothelial swelling. complement and immunoglobulin depositions seemed to be more pronounced but without statistical significance in autumn and winter. conclusion: our results demonstrate only statistically insignificantly more pronounced capillary complement deposition in autumn and winter. the use of vad did not predict development of amr or acr. the c d staining does not add to the pathological diagnosis of amr. relevant changes of the molecular profile in the recurrence of glioblastomas with respect to the correspondent primary tumors m. idoate * , j. echeveste, r. diez valle, m. montanana, j. sola, t. labiano * university of navarra, dept. of pathology, pamplona, spain objective: in the literature, there is not enough information about changes of relevant molecular parameters in the recurrent glioblastoma. method: the study included a total of grade iv astrocytomas (oms) and their correspondent recurrences in a series of patients treated by -ala guided surgery. all patients received similar treatment. a comparative histologic and molecular study which included loh of pten region, egfr amplification sish, methylation of mgmt by msp-pcr and sequencing of egfr variant iii mutation on representative tumor samples was obtained. results: the recurrences appeared adjacent to surgical cavity in seven cases. the mean time between the diagnosis of primary and the recurrence was days ( - ). the primary glioblastomas showed loh q in % of cases, hypermethylation of mgmt in %, egfr amplification in % and egfrviii in %. in five cases ( %) the molecular profile of the recurrences was different to the primaries and most of them ( %) in the group of vaccines treated glioblastomas. the molecular profile change included one to several of the studied parameters. in one case all of the molecular parameters had changed. conclusion: the molecular profile change of the recurrences of glioblastomas in respect to the primaries is a frequent event that could be due to the selection of tumor cells due to both heterogeneity and treatment effect. objective: there is not literature reference on the prognostic significance of the proliferative activity in the border of tumor. a large series of patients with glioblastomas operated by fluorescence guided surgery with uniform treatment and enough follow-up have been studied. method: a total of samples of different fluorescence quality from glioblastomas, primaries and recurrences, were studied. for each tumor, the maximum value of ki- was determined by a semiquantitative counting by two observers and by an autoanalyzer. these values were compared with relevant oncologic parameters. results: the results of ki according both counting methods were concordant. the ki max values of red (center), pink (intermediate) and blue (border) fluorescence samples obtained by quantitative analysis were % % and %. in the cox regression analysis for overall survival, the ki value was an independent prognostic factor, stronger than other relevant clinical parameters studied (p= . ). in the kaplan-meier for a ki cut-off of %, the median overall survival was . months against . months (p= . ). for the patients with recurrent glioblastomas the overall survival according to ki values was also significant (p= . ). conclusion: the evaluation of the proliferative activity by ki- in the border of tumor defined by -ala fluorescence is a relevant independent prognostic marker of primary and recurrent glioblastomas. objective: according to the levels of estrogen receptor (er) expression, primary breast carcinoma (bc) cases are divided into two groups: er-positive and er-negative (godhirsch et al. ) . in metastatic bc, and especially in brain metastases (bm) of bc, the role of er expression is not clearly defined. aim: investigate er expression levels in brain metastases of bc and evaluate its prognostic significance. method: surgically obtained tumor specimens from sixty bc female patients (median age , ranges - ) with bm bc were stained with antibodies to er (dako). ihc expression was evaluated according to the semi-quantitative method. survival was estimated by means of kaplan-meyer curves. statistical analysis was performed using staitis-tica . software. results: er expression was detected in % of patients ( / ), and % ( / ) were er-negative. median time period between primary diagnosis and bm development was significantly (p< . ) longer in er-positive group compared to er-negative group ( and months, respectively). recurrent bm developed in both groups with a similar frequency ( % and %, respectively). general survival was significantly (p= , ) higher in er-positive patients compared to that of er-negative group. conclusion: er expression is considered to be an important prognostic factor for development of bmbc. nevertheless, further studies involving more patients with bmbc are required. ofp- - dopamine and alpha synuclein interplay in neurodegeneration: a rat animal model g. stoica * , g. lungu, n. bjorklund * texas a&m university, veterinary pathobiology, college station, usa objective: a spontaneous autosomal recessive rat model for neurodegeneration was developed in our laboratory. these rats demonstrate progressive increases in alpha synuclein (α-syn) in the brain mesencephalon followed by loss of dopaminergic terminals in the basal ganglia and motor impairments. method: histology, immunohistochemistry, transmission electron microscopy were used for morphological analyses. for α-syn assessment real-time pcr and western blot were used. dopamine measurements were performed by ion mobility mass spectral (imms). results: the severity of pathology is directly related to the overexpression of α-syn and parallel decrease in dopamine (da) level in the striatum (st) of affected rats. the neurodegeneration in this model is characterized by the presence of perikarya and neurites lewis bodies (lb) and diffuse marked accumulation of perikaryal α-syn in the substantia nigra (sn), brain stem, and striatum along with neuronal loss. light and ultrastructural analyses revealed that the process of neuronal degeneration is a "dying back" type. the disease process is accompanied by gliosis and release of inflammatory cytokines. conclusion: decrease dopamine and overexpression of α-syn in the brain mesencephalon may provide a naturally occurring animal model for parkinson's disease and other synucleinopathies that reproduces significant pathological, neurochemical, and behavioral features of the human disease. sunday, september , . - . objective: the mitotic index in thin primary melanomas replaced clark level in the staging system of the ajcc. however, the recommended quantification of proliferation by hot spots on he stains is criticized. an alternative may be the immunohistochemical proliferation marker phosphohistone h (phh ) visualizing all mitotic cells. and, when combined with the melanocytic marker mart , phh insures quantification of proliferating melanocytes, only. method: primary melanomas with a median follow-up of years for patients with event-free melanoma were included. phh /mart stains were performed by an indirect sequential immunoenzymatic technique. the number of phh /mart positive cells was counted in a fixed -mm frame in the dermal area with the highest concentration of positive cells (hot spots). results: in multivariate analysis, phh in hot spots was a strong independent prognostic marker for recurrent disease (hr= . , % ci, . to . ; p=. ) and melanoma-specific death (hr= . , % ci, . to . ; p=. ), when corrected for primary tumor thickness and ulceration. conclusion: cellular proliferation is an independent prognostic marker in primary cutaneous melanoma. however, accurate quantification is crucial for correct clinical staging. for this purpose, proliferation indices of the novel phh /mart double stains seem very promising. objective: some of the more common benign vocal fold lesions are polyps, nodules and polypoid corditis (reinke's edema). the majority of vocal fold pathology develops in the mucosal layer of the vocal fold, precisely in reinkes space. basic science and clinical research over past decades has led to advances in our understanding of benign laryngeal lesions. the aim of our study was compared contact telescopy findings with histological analyzes in a patients with reinke's edema. method: the our study included consecutive patients with clinical diagnosis of reinke's edema. videoassisted contact telescopy was performed in all cases under general anesthesia and mucosa was stained by methylene blue. tissue biopsies were taken from areas exhibiting visible pathological changes and sent to routine histopathology and immunohistochemical analyses. we analyzed expression of pancytokeratin antibodies, vimentin and cd . results: in the all case of reinke's edema, contact telescopic scans showed a change of direction or a disappearance of regular distribution and morphology of blood vessels (irregular shapes, positions, and patterns, apparently running in random directions) in vocal fold mucosa. marked pathological changes were noted by contact telescopic scans, confirmed with histopathology analyses. conclusion: contact telescopy is a useful additional diagnostic tool regarding reinke's edema. ofp- - volunteering in malawi: a snapshot of surgical pathology in sub-saharan africa s. berezowska * , t. tomoka, s. kamiza, d. a. milner jr., r. langer * universität bern, institute of pathology, switzerland objective: the breadth of material found in surgical pathology services in african countries significantly differs from the common spectrum of "the west". we report our experience in the pathology departments of blantyre and lilongwe, malawi. method: during a six-week period cases were processed ( histology, cytology). results: the vast majority of cases showed significant pathological findings (n= ; . %): ( . %) were non-tumoral conditions and ( . %) benign tumors or tumor like lesions. the large group of malignancies (n= ; . %) comprised pediatric tumors (e.g. rhabdomyosarcoma, small blue round cell tumors), and adult tumors. amongst women (n= ), squamous cell carcinomas (scc) of the cervix uteri predominated (n= ; . %), followed by breast carcinomas (n= ; . %), and esophageal scc (n= ; . %). males (n= ) most often showed scc of the esophagus (n= ; . %), and scc of the urinary bladder (n= ; . %). lymphomas (n= ) and kaposi's sarcomas (n= ) were less frequent. conclusion: providing pathology service in a low resource country may be handicapped by lack of personal, inadequate material resources, or insufficient infrastructure. rotating volunteers offer a bridge for capacity building of both personnel and the local medical service; in addition, the volunteer's horizons are broaden professionally and personally. objective: antinuclear antibodies (ana) are routinely tested by indirect immunofluorescence (if) and immunoblot. in this paper we aim to retrospectively analyze results of both methods. method: on two cohort groups: a (a total of samples), and b (a total of samples) we performed if on commercially obtained hep- cell, and immunoblot on (strips euroline). results: if revealed at least different morphological patterns of ana staining, which we grouped into easily distinguished categories: speckled nuclear fluorescence, homogenous nucleoplasmic fluorescence, multiple nuclear dots, nucleoli staining, and scattered nucleoplasmic dots. we compared these groups according to frequency, type of antibody and correlation with immunoblot resuts. we summed the characteristics of each if category, and noticed that both cohorts revealed . % and %, respectively of sera, which did not show positive if at an acceptable dilution (only by : ) or were fully negative in if test, but were clearly positive in immunoblot test. this was especially alarming with anti-scl- antibodies, which could be barely recognized in if test, but were clearly present in of positive stained strips (cohort a) and/ or out of positive stained strips (cohort b). ofp- - macrophages and their subtypes in tumorigenesis and growth o. el-hassoun * , l. maruscakova, z. valaskova, m. kopani, j. jakubovsky, i. hulin * alphamedical laboratories, dept. of pathological anatomy, martin, slovakia objective: it has been established that macrophages (mf) play a basic role in the tumor microenvironment. still, the definition of the function of mf with relation to tumors is still vague, mainly when mf are observed acting occasionally as pro tumor and elsewhere as anti tumor. method: in this paper we attempt to sum up the accumulating observations of mf function with respect to the microenvironment and the tumor entity. results: through the different stages of tumorigenesis and growth, macrophages are engaged in various signaling cascades that tame them to perform a certain function in a given microenvironment. this makes mf a multi-program cell with unique adaptivity. we assume that without the macrophages tumors cannot progress. conclusion: the available mf subtype analysis is still inadequate and doesn't bear into account the dynamics of tumorigenesis and growth. our presented mf schemes provide an insight on the tumor microenvironment with respect to stage. a complex view on the signaling cascades affecting mf is needed to formulate a novel classification of mf based on their programming. this consequently can be the basis of the development of "anti-mf therapy" or on a larger scale "anti-tumor microenvironment therapy". ofp- - lodox x-ray is an invaluable asset in autopsy procedures l. liebenberg * * university of cape town, dept. of forensic medicine, south africa objective: the field of autopsy performance has faced numerous challenges internationally. minimal background information on the deceased, various religious and personal objections regarding cutting a deceased body as well as health hazards face the pathologist regularly. lodox x-ray is a huge help for the pathologist. method: lodox x-ray (low dose x-ray) using the statscan has proven invaluable in busy forensic pathology laboratories in cape town, south africa. the digitally captured radiology images render a wealth of clear, recognizable, easily documented and user friendly images for use in diagnosis and court evidence. results: the use of the statscan has revolutionized the process of forensic autopsy procedures in cape town, south africa. the radiology images obtained in an unusually user friendly and time efficient way have improved efficacy, efficiency, accuracy and pro-active occupational health safety in our forensic laboratories. conclusion: lodox x-ray is a proven invaluable special investigation in forensic, and also academic, autopsy examination procedures. sunday, september , . - . objective: castration resistant prostate cancer (cr-pca) is the most aggressive form of prostate cancer (pca) posing a significant therapeutic challenge. our aim was to perform whole exome-sequencing on cr-pca/normal paired formalin fixed paraffin embedded (ffpe) samples using the solid next generation sequencing platform (ngs). we identified two promising genes-ywhaz and ptk , that could serve as novel potential therapeutic drug targets in pca. method: genomic dna was sequenced from cr-pca/ normal paired ffpe samples. a set of amplified/deleted genes were validated using fluorescence in-situ hybridization (fish) assays using a pca progression cohort consisting of cases for localized pca, patients with primary pca and corresponding ln metastasis, and samples for crpca. results: exome-sequencing identified regions of deletions/ amplifications, including nkx . , pten, cmyc and ar genes, known to play a role in pca. we identified several amplified genes as druggable targets such as ptk and ywhaz. for ywhaz we identified . % amplification in localized pca, . % amplification in ln metastasis and . % amplification in crpca. for ptk we % amplification for localized pca and % for both ln metastasis and cr-pca. conclusion: this is the first study to use exome-sequencing approaches on ffpe cr-pca to understand the biology of disease and its plausible treatment options. a study of d-np (p ) expression in tumours of stratified epithelium d. nonaka * * the christie hospital, dept. of histopathology, manchester, united kingdom objective: p can be expressed by a minority of adenocarcinomas. Δnp (p ), isoform of p , has recently been reported as more specific in lung squamous cell carcinoma (scc) than p , and it appears to be a more reliable marker for scc. there is no comprehensive study on p expression in tumours of different histotypes. method: tumours of various hisotypes were studied, including tumours of stratified epithelium such as scc ( cases) of the skin, head/neck, lung, cervix, and oesophagus, urothelial carcinomas ( ), cutaneous basal cell carcinomas ( ), thymomas ( ) and thymic carcinomas ( ), adenocarcinomas ( ) from multiple organs, germ cell tumours ( ), brain tumours ( ), lymphomas ( ), and sarcomas ( ). p immunostains were performed on tissue microarrays and staining extent was evaluated as focal ( - %) and diffuse ( - %). results: all but one tumours of stratified epithelium diffusely expressed p . the negative case represents spindle cell scc. other tumours except for basal/squamous component of two teratomas, one breast metaplastic carcinoma, and squamous component of adenosquamous carcinomas were all negative for p . conclusion: p is specific in tumours of stratified epithelium and its sensitivity appears comparable to p . p can serve as a marker for tumours of stratified epithelium. objective: although formaldehyde is the standard fixative used in routine histopathology, it is not considered an "ideal" fixative. formaldehyde presents carcinogenic properties, a slow fixation and produces cross-links with proteins and nucleic acids. formaldehyde-free fixatives are commercially available. method: from to we used glyo-fixx (thermo scientific, us), a glyoxal-based fixative, as formaldehyde substitute, analysing surgical pathology specimens and autopsies. all samples were processed and stained routinely. moreover we performed immunohistochemistry by an automated immunostainer and special stains with standard techniques, where needed. in tumoral cases we fixed cm sample in neutral buffered formalin and compared the results with gyo-fixx fixation. results: glyo-fixx fixation needed less hours than formaldehyde and not harden the tissues. on gross examination of the adipose tissue, lymph nodes were easily found, due to a more marked whitish appearance. morphologically we have not found differences between formalin and glyo-fixx, except for the eosinophils degranulation. special stains resulted similar to those with formaldehyde fixations. almost all antibodies not required pretreatment, but needed adjusts in the standardized protocol for formaldehyde. conclusion: our experience demonstrated glyo-fixx is a good non-toxic alternative to formaldehyde in routine pathology, capable to preserve morphology and protein integrity of the tissues. liposarcoma with solitary fibrous tumor-like dedifferentiated areas: clues on differential diagnosis m. aizpurua * * huvh/ics, dept. of pathology, barcelona, spain objective: well-differentiated liposarcoma (wdls) can undergo dedifferentiation to nonlipogenic sarcomas. solitary fibrous tumour (sft) characteristics have not been highlighted as a pattern of dedifferentiated liposarcoma (dls). however, this does occur and could be the cause of diagnostic pitfalls in atypical locations or partial tumoral resections. the aim of this comparative study is to demonstrate that some dls's may show morphologic features resembling sft's, and furthermore to describe the clue features supporting differential diagnosis. method: study comprised wdls-dls and sft ( malignant). histological features were systematically reviewed, and cd /cd /s /bcl immunostains and mdm fish-analysis were evaluated in all cases. results: there are many overlapping characteristics between dls and sft (table ). significant differences between both tumor types were observed in lipoblast-like cells (lpc); mature adipocitic cells within tumor (mac); stromal bands surrounding adipose tissue with atypical cells (sac); patternless (pa); keloidal-collagen (kcm) and haemangiopericytoid-pattern (hp). diagnostic accuracy of different features is shown in table . conclusion: the dedifferentiated component in some liposarcomas may resemble sft. in the dls/sft differential diagnosis, immunohistochemistry may be confusing. presence of wdls areas is the main diagnostic clue and mdm fish amplification is successful in distinguishing dls from sft. osteoblastoma and diagnostics pitfalls w. ouahioune * * ehs douera, dept. of pathology, algiers, algeria objective: osteoblastoma is a rare benign osteoblastic tumor with a potential for local bone destruction and aggressiveness. the most common site of osteoblastoma is the vertebral column particularly the posterior elements and the sacrum. method: in this study we present nine cases of osteoblastoma and the principal differential diagnosis. results: on a period of years, nine cases of osteoblastoma had been diagnosed including seven males and two females. the age of those patients ranged from to year old. the tumor was localized in the spine in five of the nine cases and the other ones in the long bones. the radiological diagnosis of osteoblastoma was made in just two cases. the diagnosis was made by histology in eight cases. all our patients had been treated with curettage. on the nine patients, just one of them had developed two successive recurrences. conclusion: osteoblastoma is a rare benign tumor which is rarely diagnosed by radiology alone. the pathologist should always suspect an osteoblastoma in front of a vertebral localization of any tumor. objective: congenital hepatic fibrosis (chf) is a developmental disorder of the portobiliary system. clinical findings usually include enlarged liver, well-preserved hepatocellular function and portal hypertension. chf is frequently associated with hepatorenal fibrocystic disease. method: a -year-old woman was referred to our hospital for further evaluation of hepatomegaly, and portal hypertension was found. laboratory tests were all negative. mri identified hepatosplenomegaly with hypertrophic left medial segment, high uptake nodular areas and homogeneous boundary liver. needle biopsy was performed. results: the biopsy demonstrated slight portal fibrosis and fibrous septa with proliferation of numerous biliary ducts, some of them containing inspissated bile. it was also noticeable the few number of portal vein branches. there was no inflammation or hepatocyte necrosis. therefore, our diagnosis was congenital hepatic fibrosis. we correlated these results with radiology. magnetic resonance cholangiopancreatography showed peripherally dilated biliary intrahepatic branches contrasting with preserved caliber of main intrahepatic branches. these findings were also diagnostic for chf. conclusion: the low prevalence of chf makes it hard to think about this entity in patients with portal hypertension and normal laboratory findings. the histological differential diagnosis must be done mainly with cirrhosis, but also with idiopathic portal hypertension and, in small biopsies, with biliary hamartoma. results: a total of cases of digestive tract lymphoma were reported ( males, females) with a sex-ratio of . . the patient age ranged between and years with a median age of years. stomach location was the most frequent with % of cases followed by intestine ( %) and colon ( %). among the digestive tract lymphomas, % were of marginal zone type (malt) and % were large b-cell lymphoma. according to the ann arbor staging system, % of patients were diagnosed at stage i and ii. conclusion: lymphomas of the digestive tract are still frequent in the centre of tunisia. the malt type lymphoma is main reported histological type. the association with the helicobacter pylori should be considered. collecting duct carcinoma in the west of ireland: a rare experience a. shalaby * , c. e. connolly * university hospital galway, dept. of histopathology, ireland objective: collecting duct carcinoma is a rare renal neoplasm arising from the epithelium of bellini's ducts in the distal part of the nephron. method: we describe our experience of this entity in university hospital galway with a series of four cases diagnosed between and . results: three patients aged between and years presented with either lymph node or bone metastases without evident abdominal mass. in each case the biopsy from the metastatic lesion showed adenocarcinoma with an immunohistochemical profile suggestive of renal origin and a renal mass was subsequently confirmed by radiology. the fourth patient was an year old male who presented with haematuria. abdominal ct scan revealed a . cm mass in the right kidney extending into the renal vein. biopsy of the renal mass in all cases confirmed adenocarcinoma with a tubular pattern with positive cytokeratin and vimentin staining and cd negative staining. one patient was treated with nephrectomy with post-neoadjuvant chemotherapy and died months after surgery. the other three patients died few months after their diagnosis. conclusion: collecting duct carcinoma is a rare renal neoplasm that can be difficult to diagnose on core biopsies, however, it can be identified based on radiological findings, gross, microscopic, histochemical and immunohistochemical features. objective: wilms tumors (wt) is the most frequent renal tumor of children. genetic alterations have been suggested as associated factors but the exact pathogenesis of wt is not fully characterized. tissue factor (tf) is a glycoprotein which happens to be a key receptor for factor vii/viia and a primary initiator of coagulation. tf has also been associated with angiogenesis. recent evidence pointed out an important role for tf in cancer progression and metastasis. method: in the present investigation the differential expression of tf in wt was assessed by real-time pcr of rna retrieved from paraffin sections using microdissection. results: different histological components of wt were analysed. the results revealed that tf was upregulated in blastema and epithelial components as compared to nonneoplastic tissue ( . and . -fold respectively, p< . ). stroma and non-neoplastic tissue presented low levels of tf expression. tf expression in wt metastatic lesions was also significantly upregulated as compared to non-metastatic wt. microvessel density was positively correlated with tf expression (r= . ). conclusion: as described in other tumors, tf seems to play a significant role in the behavior of wt. further investigations are warranted to better understand the pathways by which tf exerts its effects on tumor progression and its potential as a target for therapy. objective: biliary atresia (ba) is the most common neonatal cholestatic disorder and the prime indication for liver transplantation (ltx) in children. histopathological markers in liver biopsies emerge promise as indicator of early ltx in patients with ba. method: ductular proliferation, ductal plate malformations and type i, iii, iv and v collagen deposition were evaluated on kasai portoenterostomy (kpe) and liver transplantation (ltx) biopsies from children with ba. formalin fixed and paraffin embedded liver biopsies were stained with hematoxylin-eosin, picrosirius-polarization and immunofluorescence methods. there were analyzed liver histoarchitecture, biliary ductus and collagen deposition in hepatic compartments. pathologic findings were graded according to a -point semi-quantitative severity-based scoring system. impact of these markers was tested on ltx time (< year and > year). results: median age of kpe was weeks (range - ) and of ltx was months (range - ). in kpe liver biopsies, ductular proliferation, ductal plate malformations and collagen deposition were increased but these parameters presented no association with clinical evolution for early ltx. furthermore, collagen v prominent deposition was found along of hepatic sinusoids and type i, iii and iv were more frequent in portal compartment. conclusion: these results suggest that histopathological parameters evaluation presented may not determine early ltx in biliary atresia. the role of hofbauer cells on villous vasculature in early fetal losses e. Özer * , y. arman karakaya * dokuz eylul university, dept. of pathology, izmir, turkey objective: the aim of this retrospective study is investigate the role of hofbauer cells in early fetal losses. method: the slides obtained from archieval blocks of missed abortion (ma, n= ) and blighted ovum (bo, n= ) cases and unwanted pregnancies materials (control group, n= ) were stained by immunuhistochemical methods using cd and cd antibody to label hofbauer cells and endotelial cells, respectively. results: the mean number of vilous hofbauer cells was found to be significantly higher in both bo and ma in contrast to the control group (p= . and p<< . , respectively). however it was not significantly different between bo and ma (p= . ). chalkey method revealed no statisticaly significant difference in the control group in comparison with ma and bo in (p= . , p= . , respectively). higher microvessel scorring were found in ma in contrast to bo and the control group (p= . and p= . , respectively). however, there was no difference between the control group and bo (p= . ). conclusion: we think that hofbauer cells may be of biological importance in early fetal losses and play a role on defective vasculature formation in ma. placental vegf and its receptors expression patterns in preeclampsia k. pavlov * , e. dubova, a. shchegolev, g. sukhikh * v.i. kulakov scientific center, dept. of pathology, moscow, russia objective: placental angiogenesis anomalies play an important role in some complications of pregnancy development, including preeclampsia (pe). vegf and its receptorsone of the key placental angiogenic factor. aim: to evaluate patterns of vegf and its receptors (vegfrs , and ) expression in placentas from pe complicated pregnancies. method: we performed complex morphological and immunohistochemical study of term placentas from mild preeclampsia (mpe) cases ( st group), term placentas from severe preeclampsia (spe) cases ( nd group) and term placentas from uncomplicated pregnancies (control group). results: we revealed significantly increased syncitial vegf expression levels in preeclamptic placentas terminal villi and these changes were much prominent in spe. we also detected insignificantly increased endothelial vegf expression levels in preeclamptic placentas terminal villi. vegfr- syncitial expression levels in spe terminal villi were significantly higher in compare to the mpe and control groups. vegfr- endothelial and syncitial expression levels were significantly lower in both pe groups terminal villi and these changes were much prominent in mpe. patterns of vegfr- expression in preeclamptic and control groups were multidirectional. conclusion: revealed patterns of vegf and its receptors expression point on altered placental angiogenesis in pe and reflect different degree of such alteration in mild and severe pe. objective: chorionic villi vascularity disturbances, caused by abnormal expression of angiogenic factors could correlate with maternal and fetal complications in diabetic pregnancies. method: aim: to evaluate the patterns of vegf receptors (vegfr , vegfr and vegfr ) expression in placentas from gestational diabetes (gd) and type diabetes (d ) pregnancies and correlation between them and some clinical parameters in newborn (placental weight (pw), newborn weight (nw), placental/newborn weight index (pnwi), st day of life blood glucose test results ( bgtr)). term placentas from d (n= ) and gd (n= ) and term placentas from normal pregnancy (control group) were studied. results: in both d and gd group we revealed significantly higher level of vegfr (p= . ), vegfr (p= . ) and vegfr (p= . ) endothelial expression in terminal villi. difference in vegfr expression among d and gd groups was also significant (p= . ) as difference in its expression between d and control group (p = . ). among clinical parameters we only revealed significant increase in bgtr in diabetic groups (p = . ) with marked difference between d and control group (p = . ). we revealed multidirectional correlation between vegf and vegfr expression and nw in d and gd groups. conclusion: revealed changes reflect placental angiogenesis disturbances influence on intrauterine fetal development. microvillus inclusion disease (mvid) is a disorder of defective intracellular trafficking and disrupted epithelial cell polarity c. thoeni * * division of cell biology, medical university innsbruck, austria objective: mvid is a congenital enteropathy characterized by loss of microvilli and formation of microvillus inclusions (mi) in enterocytes. mvid is caused by mutations in the myo b gene, coding for the myosin vb (m b) motor protein involved in intracellular transport and maintenance of epithelial cell polarity. we examined the effects of loss of m b in enterocytes of mvid patient and in a caco- cell model. method: the expression and localization of cell membrane transporters (cd , na/k atpase) and various cell organelle markers : endosomal rab gtpases [rab , , , , ] ; early (eea ) and late endosome (lamp ), and golgi (giantin)) were analyzed using multilabel immunoflourescence and confocal microscopy of a duodenal biopsy from mvid patient and in caco- cells following m b si rna knock-down (hum mut : - , ). results: depletion of m b in both enterocytes and caco- cells resulted in disruption of epithelial cell polarity with loss of apical microvilli, formation of mi; mislocalization of transporters as well as aggregation of epitopes for different rab gtp ases, early and late endosomes. conclusion: m b plays a critical role in polarized organization of enterocytes in mvid -a disorder characterized by defective intracellular trafficking and altered endosomal sorting. caco- cells provide an excellent model to study the pathogenesis of mvid. ofp- - beta-catenin expression and mutational analysis of ctnnb gene in pediatic adrenocortical tumors (act) r. alaggio * , p. dall'igna, a. martines, e. lalli, r. boldrini, v. d´onofrio, g. esposito * università di padova, dipt. di pathology, italy objective: act have an unpredictable clinical behaviour, and no liable histological or molecular parameters are available to predict outcome. the activation of wnt/β-catenin pathway, involved in tumor growth and progression in adult act has been explored only in brasilian pediatric act. the aim of this study is the investigation of the possible prognostic role of β-catenin accumulation and/or ctnnb (β-catenin gene) mutations also in italian pediatric act. method: β-catenin immunostaining and mutational analysis of ctnnb gene at exons and , when possible, were carried on a series of act ( malignant and benign, according to wieneke classification), from patients enrolled in the italian pediatric rare tumor (trep) study. results: immunostaining for β-catenin showed membrane/ cytoplasmic staining in cases, benign and malignant, and nuclear staining in malignant. no mutations of ctnnb gene were found in the tumors analyzed. conclusion: ctnnb gene mutations do not appear to be involved in pathogenesis of pediatric act. the accumulation of protein might be related to different mechanisms. its presence in the majority of malignant act suggests a possible role in tumor progression. ofp- - neuroblastoma presenting like a wilms' tumor with thrombus in inferior vena cava: a case series h. sartelet * , g. gaetan, a. ouimet, c. lapierre, p. teira * chu sainte justine, dept. of pathology, montreal, canada objective: neuroblastomas and wilms' tumors are frequent pediatric solid tumors. the first is frequently detected in the adrenal gland and the second develops in the kidneys. the extension through the vena cava and the lung metastases are frequent in wilms' tumors and are rarely in neuroblastoma. we present the cases of three children with abdominal tumors with thrombus in the inferior vena cava and pulmonary metastases were discovered yet demonstrated a stage neuroblastoma. method: the three male patients were between and months old. they presented an abdominal mass, near the superior pole of the kidney. thrombus of the vena cava was evoked on imaging studies in all cases and pulmonary metastases were always found. catecholamine metabolites were present in the first case and negative in the two others. two out of three patients had a radical nephrectomy. results: the pathological analysis always found a neuroblastoma poorly differentiated or undifferentiated without mycn amplification and confirmed the tumoral thrombus in the second case. the evolution of the two first patients was unfavorable and the third is alive. conclusion: invasion of the inferior vena cava and pulmonary metastases in children with neuroblastoma is uncommon and can modify the surgical management. ofp- - epcam -a marker for tufting enteropathy (te) and a useful tool in the differential diagnosis of congenital enteropathies c. thoeni * * division of cell biology, medical university innsbruck, austria objective: congenital enteropathies (ce) are characterized by villous atrophy and disruption of apical microvilli in duodenal enterocytes. te is characterized by focal eptithelial tufts and mutations in the epcam gene, coding for epithelial cell adhesion molecule, important for cell-cell contacts. microvillus inclusion disease (mvid), shows loss of apical microvilli and formation of intracytoplasmatic microvillus inclusions. mvid is caused by mutations in the myo b gene, important for intracellular transport and organization of epithelial cell polarity. in this study, we identified a new epcam mutation in a patient with te and used anti-epcam antibody as a marker for the diagnosis of te and distinction from mvid. method: we used immunohistochemistry (ihc) with monoclonal epcam antibody (mouse monoclonal ab for epcam, ncl esa/leica microsystems) on routinely processed duodenal biopsies from patients with te, mvid and age matched controls. immunostaining for e-cadherin served as reference. results: epcam expression was completely absent in the biopsy from te patient homozygous for a novel epcam mutation (c c>g, ser x). e-cadherin showed normal expression and distribution pattern in the enterocytes. in mvid, expression and distribution pattern was comparable to controls for both epcam and e-cadherin. conclusion: loss of epcam is specific and a sensitive marker for confirmation of te. epcam antibody is useful in the differential diagnosis of ce. objective: in pulmonary adenocarcinoma egfr mutation analysis has become an important part of the diagnostic work-up. frequently the tissue of bronchoscopies is of limited diagnostic value. the aim of the present study was to evaluate the impact of rose on brochoscopy specimens with regard to egfr mutation. method: rose was used for adequacy of the specimen and for separating adenocarcinomas and carcinomas nos from other neoplasms in the lung. if it was not possible to retrieve material for histology, cytology and cellblock were made. if adenocarcinoma or carcinoma nos was suspected in rose, further two biopsies or two cell blocks were obtained. egfr mutation analysis was performed by pcr (light cycler ; egfr rgq pcr kit; qiagen). results: malignancy was diagnosed in of cases by histology or on cytology with and without cell blocks. adenocarcinomas (n= ) and carcinomas nos (n= ), scc (n = ), sclc (n = ), and rare tumors (n= ) were diagnosed, respectively. among the group of adenocarcinomas and carcinomas nos (n = ), egfr mutation analysis was performed in ( , %) cases and showed mutations in ( , %) and "wild type" in ( , %) tumors. no material was left in ( , %) cases. conclusion: rose supports bronchoscopic procedures to retrieve adequate specimens for tumor diagnosis and subsequent egfr mutation analysis. objective: patients with fibroblast growth factor receptor (fgfr ) amplified squamous cell lung cancers (l-scc) are treated in phase i clinical trials using small molecule inhibitors (smi). scc of the lung share common molecular alterations with squamous cell head and neck cancers (hn-scc). aim of our study is to assess if hn-sccs also harbor fgfr amplifications. furthermore, we aim to inhibit cell proliferation of fgfr amplified hn-scc cell lines using a smi. method: the cohort consists of patients suffering from hn-scc, of these suffering from metastatic disease. primary tumors and metastatic tumors were assessed for fgfr copy number status using fluorescence in-situ hybridization (fish). we tested cell lines for fgfr amplification status and inhibited these with smis. objective: the pulp of pequi has high levels of antioxidants properties. method: eighteen male balb/c mice divided: animals received by gavage , μl/mg/day of pequi oil (control + cbcoil= ) during days. after days, of these mices received two doses of , g/kg intraperitoneal of urethane (urethane + cbc oil= ). the other animals were only submitted to two doses of , g/kg intraperitoneal of urethane (urethane group= ). after days, groups were sacrificed. antioxidant activity was evaluated in the lung tissues by tbars (thiobarbituric acid-reactive substances), cat (catalase) and sod (superoxido dismutase) test. dna damage was estimated by comet test. results: the lung parenchyma from urethane groups without oil and with oil showed neoplasic formations induced by the chemical carcinogenesis in contrast with control + cbc oil group. the results of tbars test showed a significant decrease of lipid peroxidation in the urethane + cbc oil, than urethane group. the cat and sod test didn't show a significant difference. comet assay showed a significant decrease of dna damage in urethane + cbc oil when compared with urethane group. conclusion: the antioxidant components in the pequi oil diminish the oxidative stress status and dna damage in chemical carcinogenesis, suggesting that this type of strategies may have a greater impact in lung cancer treatment. financial support: fapesp. ofp- - an algorithm for gene mutation analysis in lung cancer m. comanescu * , c. iosif, m. dobre, l. buburuzan, g. bussolati, f. vasilescu, c. ardeleanu * institutul victor babes, dept. of pathology, bucharest, romania objective: somatic alterations of k-ras, egfr and alk which are increasingly requested in order to predict response to personalized therapies in lung cancer are mutually exclusive and are represented in over % of lung adenocarcinomas. the approach to be followed for planning analyses is not standardized in the literature. method: we have studied by molecular methods (pcr-rflp for k-ras, direct sequencing for egfr and fish for alk) formalin fixed, paraffin embedded cases of lung adenocarcinomas of different subtypes. results: based on rational, biological and economic considerations we started with k-ras analysis by pcr-rflp, which showed mutations in cases ( with mutation on codon and one case with mutation on codon ), the residual cases were all analyzed for egfr mutations. fish for alk translocation followed in egfr-wild cases. conclusion: the analysis for k-ras mutations allows to select the significant percentage (approximately % of adenocarcinomas) when egfr mutation analysis by direct sequencing can be postponed. k-ras and egfr wild cases will then undergo fish analysis for alk translocation. acknowledgements study conducted with the support of the following project: project persother -smis-csnr: / . ; with the support of sectoral operational programme "increase of economic competi-tiveness" priority axis : research, technological development and innovation for competitiveness. ofp- - gene mutation analysis in adenosquamous carcinomas of the lung m. comanescu * , g. bussolati, c. ardeleanu, l. daniele, g. gaina, c. luca, a. sapino * institutul victor babes, dept. of pathology, bucharest, romania objective: adenosquamous carcinomas of the lung constitute a rare and aggressive variant of lung cancers. the pressing interest in evaluating the mutational status in lung carcinomas for predicting responsiveness to targeted therapies is presently focused on adenocarcinomas (of different sub-types), which makes preliminary histological typing a crucial step in order to select cases to be genetically analyzed. hence the interest in deciding if adenosquamous carcinomas should be included among adenocarcinomas or, viceversa, if they should be interpreted as a variant of squamous cancers and excluded from the process of gene analysis. method: we have thus collected, from our two institutions a large number ( cases) of cancers showing the histological definition of adenosquamous carcinomas according to the who criteria and performed gene analysis for k-ras (codons , ) and egfr (codons , and ) mutations. the detection of rearrangements of the alk gene by fish was also performed. the results indicate that k-ras and, specifically, egfr mutations are detectable in a fraction of these tumors. in conclusion, adenosquamous carcinomas should not be denied the chance of genetic analysis eventually leading to a targeted therapy. acknowledgement project persother -smis-csnr: / . romania. ofp- - pathology of the lung progenitor cells and their niches in idiopathic interstitial pneumonias and lung sarcoidosis s. demoura * , e. kogan, v. tuong, o. kichigina * moscow, russia objective: the aim of the study was to investigate the morphological and the molecular-biological changes of the lung progenitor cells and their niches in idiopathic interstitial pneumonias (ipf) and lung sarcoidosis (ls). method: we performed an immunohistochemical study on open lung biopsies from patients (usual ipf - , desqvamative interstitial pneumonia- , nonspecific ipf - , cryptogenic ipf - , ls - , control - patients). immunochemistry was done on step paraffin sections with monoclonal and polyclonal antibodies: apo-cas, pcna, , , pdgf, fgf, igf ii, cd , mmp , , , , timp , cd , sma, ema. results: deep injury of clara cells together with pneumocytes ii and their nishes were found in usual ipf, sk with prominent ipf and desqvamative interstitial pneumonia. myofibroblast proliferation, neoangiogenesis, adenomatosis and fibrosis with high production of tnfα, tgf-β, pdgf, fgf, igf ii, cd , mmp , , , , timp accompanied proliferation of these epithelial cells. while in other variants of ipf pathological changes were localized in the interstitium, vessels and basal bronchiolar epithelial cells. conclusion: localization of injury and inflammation in progenitor cells niches results in pathologic reparation, sclerosis and precancer lesions. ofp- - a small immunohistochemical panel allows for accurate diagnosis of primary and metastatic lung cancer in biopsy specimens d. felizardo * , r. henrique, a. l. cunha * instituto de oncologia porto, dept. of pathology, coimbra, portugal objective: precise subclassification of lung cancer, mostly performed in biopsy or fine needle-aspiration specimens, is required for appropriate therapy. moreover, distinction between primary and metastatic carcinoma is critical. thus, the role of immunohistochemistry (ihc) has been emphasized, although an optimal ihc diagnostic algorithm has not been firmly established. herein, we evaluated the performance of the ihc panel used at our institution in subclassification of lung cancer and identification of metastatic carcinoma. method: cases of non-neuroendocrine lung carcinoma, diagnosed from march to april were selected. ihc was performed for ck , ck , ttf- and p . resection specimens were compared with the respective biopsy. results: of cases analysed, ( . %) were diagnosed as lung adenocarcinoma, ( . %) as epidermoid carcinoma, ( %) as other forms of nsclc, and ( . %) as metastasis. importantly, % of primary lung adenocarcinomas were initially suspected to be metastasis. in cases submitted to surgical resection, ( . %) were correctly diagnosed in the biopsy, revealing a substantial agreement (κ-value= , ). conclusion: our ihc panel allows for reliable subclassification of lung carcinomas in most cases and is decisive for appropriate diagnosis in patients suspected of lung metastasis, which is critical issue in a cancer institute. ofp- - rationale for treatment of metastatic squamous cell carcinoma of the lung using fgfr inhibitors f. göke * , a. franzen, a. schroeck, v. scheble, r. kirsten, r. menon, d. goltz, d. boehm, w. vogel, s. perner * universitätsklinik bonn, inst. für pathologie, germany objective: we previously identified amplified fibroblast growth factor (fgfr ) as a therapeutic target for small molecule inhibitor (smi) therapy in squamous cell lung cancer (l-scc), resulting in currently running clinical trials treating patients with stage iii disease. as most patients present with metastatic stage of disease, we attempt to demonstrate fgfr amplification in lymph node metastases of amplified primary tumors. our study aims to give a rational to include these patients in a targeted smi therapy. method: we assessed formalin-fixed paraffin-embedded (ffpe) primary l-scc samples. samples were primary tumours with corresponding ffpe lymph node metastasis. the biotin-labelled fgfr target probe ( p . to p . ) was used to determine the fgfr amplification status performing fluorescence in situ hybridization (fish). results: of assessable metastatic l-scc, samples displayed fgfr amplification ( %). all of these primary tumors also harbored fgfr amplification in their lymph node metastasis. non-amplified tumors never displayed fgfr amplification in corresponding metastases. conclusion: we found fgfr amplification not only in primary l-scc, but also in corresponding lymph node metastasis, suggesting that this genetic aberration is a clonal event in tumor genesis. our study provides data indicating new therapeutic possibilities for patients suffering not only primary, but also metastatic fgfr amplified scc lung cancer disease. kras mutation was a g to t transversion in % of the smoker nsclc population and a g to a transition in % of the non smoker nsclc population. mutations were noted at codon ( %), codon ( %) and codon ( %). a kras mutation was a negative prognostic factor with a hazard ratio for death of . ( % confidence interval, . - . ). a mucinous histological subtype was observed in more than % of kras mutated tumours. conclusion: kras oncogene substitution must be accurately determined in primary lung adenocarcinoma for correlation with tumour behaviour and clinico-pathological parameters. objective: there is an urgent need for diagnosis of nsclc at its early-stages and for improving the survival rate of patients. micrornas, small non-coding rnas, are frequently deregulated in nsclc. this study aimed to explore plasma micrornas for diagnostic value, and evaluated the correlation between expression profiles of plasma micro-rnas and disease-free survival (dfs) in nsclc patients. method: we selected eighteen most frequently expressed micrornas in nsclc. total plasma rna including micrornas was isolated and reverse-transcribed into cdnas. the level of micrornas was determined by quantitative real-time rt-pcr in resectable nsclc patients and matched cancer-free individuals. the correlation between the expression of micrornas in plasma and patient dfs were examined by log-rank and cox analysis. results: expression levels of mir- ,− ,− ,− a, − ,− ,− ,− ,− , and let- f in the plasma of nsclc including stage-i patients were significantly higher compared with controls (p< . ). the combination of these micrornas yielded % sensitivity and % specificity (auc= . ) in discriminating nsclc patients from controls. the levels of mir- , − , − a, − , − and mir- a were significantly associated with dfs (p< . ). conclusion: our results suggest that high expression of plasma mirnas signature would provide potential noninvasive blood-based biomarker for the prognosis of nsclc. monday, september , . - . objective: intratumoral disorganized neo-vasculature induces oxygen fluctuations which contribute to tumour growth and metastatic potential. although the activation by hypoxia of the carbonic anhydrases caix and caxii is well known, responses of these proteins under reoxygenation remain to be elucidated. method: in this study we evaluated the effects of hypoxiareoxygenation on caix and caxii expression and cell proliferation in a and h lung adenocarcinoma cell lines. we further investigated by immunohistochemistry on tissue microarray the value of the combined expression of these proteins to predict outcome in nsclc patients. results: caix expression was maintained at high level after reoxygenation in contrast of the rapid caxii downregulation, whereas the cell proliferation rate was significantly increased. survival analyses showed that high caix/ low caxii was associated with high cumulative incidence of relapse and with poor overall survival of nsclc patients (p< . ). conclusion: our results provide insight into understanding dynamic responses of caix and caxii expression under tumour cells reoxygenation and demonstrate a critical role for reoxygenation on caix and caxii levels that may select for aggressive lung cancer phenotype. these findings suggest that caix and caxii play selective roles in tumour progression and emphasize their significant prognostic and potential therapeutic value. ofp- - alk-gene rearrangement: a comparative analysis on circulating tumour cells (ctcs) and tumour tissue from lung adenocarcinoma patients m. ilie * , e. long, c. butori, v. hofman, c. coelle, v. mauro, k. zahaf, c.-h. marquette, j. mouroux, p. paterlini-bréchot, p. hofman * chu de nice, lpce, france objective: until now the alk status in ctcs isolated from lung cancer patients has not been characterised. we assessed the alk status in ctcs detected in lung cancer patients and correlated the results to the alk status defined in the corresponding tumour tissue. method: lung adenocarcinoma patients showing ctcs isolated using the isolation by size of epithelial tumour cell method were screened for their alk status both in tumour samples and in ctcs. alk break-apart fluorescence in situ hybridisation (fish) and immunochemistry using an anti-alk antibody were done on ctcs and compared with results obtained on corresponding tissue specimens. results: patients showed alk-gene rearrangement and strong alk protein expression in ctcs and corresponding tumour samples. negative results were found for patients in ctcs and corresponding tumour samples. conclusion: we demonstrate that the alk status can be determined in ctcs from lung cancer patients both by immunocytochemistry and fish analysis. a strong correlation was found for the alk status obtained from corresponding tissue specimens. these results favour non-invasive, alk-gene status pre-screening on a routine basis on ctcs isolated form lung cancer patients and open new avenues for real-time monitoring for adapted targeted therapy. ofp- - detection of egfr mutations and eml -alk rearrangements in lung adenocarcinomas using archived cytological slides e. imyanitov * , n. mitiushkina, a. iyevleva, a. poltoratskiy, a. ivantsov, a. togo, i. polyakov, s. orlov, d. matsko, v. novik * n.n. petrov institute of oncology, st. petersburg, russia objective: while the molecular analysis of egfr and alk in archival lung cancer tissues is relatively well established, the acceptability of genetic investigation of cytological material in clinical routine remains a subject of debate. method: islets of malignant cells were visually located on the archived cytological slides, lysed in situ by the drop of sds-containing buffer, and subjected to the standard dna and rna extraction. examination of paraffin-embedded tissue blocks from the same patients was done in parallel. results: cytological/histological lung adenocarcionoma sample pairs underwent the analysis for egfr mutation. cytological and morphological samples failed to produce dna. concordance for the wild-type and mutation status was observed in / and / informative pairs, respectively; pair was non-interpretable; and pairs had mutation only in the cytological or histological material, respectively. rna extraction followed by rt-pcr analysis for the eml -alk translocation was done for the pair; failures were observed for cytological and histological samples. / informative pairs were concordant for the norm, contained identical translocations, and were noninterpretable. pair demonstrated alk rearrangement in the tissue block but not in the histological slide. conclusion: archived cytological slides appear to be well suitable both for egfr and alk analysis. objective: non-small cell lung cancer (nsclc) represents a heterogeneous group of cancers consisting mainly of squamous cell carcinoma (scc) and adenocarcinoma (ad). increased sirtuin (sirt- ) expression leads to deacetylation of p that could be important in the pathogenesis of lung cancer. differences of the molecular mechanisms in nsclc subtypes may follow subtly different pathways to tumorigenesis. the aim of our study was compare sirt- expression in lung adenocarcinoma, squamous cell carcinoma and control group. method: patients with stage lung cancer were enrolled in the study. patients had adenocarcinoma, but patients had squamous cell carcinoma. lung tissue for control group were selected from autopsy cases. immunohistochemical and western blot methods were used to evaluate sirt- expression in lung tissue. results: obtained results showed that patients with lung cancer had increased sirt- expression compared to control group ( ± vs. ± cells/mm , p< . ). in addition, patients with squamous cell carcinoma had increased sirt- positive cells compared to patients with adenocarcinoma ( ± vs. ± cells/mm , p= . ). conclusion: lung cancer is characterized by an increased sirt- expression which ir more prominent in squamous cell carcinoma. objective: transplantation is the only treatment for several end-stage lung diseases but limited by chronic allograft dysfunction particularly obliterative bronchiolitis (ob) and its correlate bronchiolitis obliterans syndrome. the development of preclinical models is crucial to better identify immunological/non-immunological mechanisms leading to ob. [group b and c] post-transplant, animals were sacrificed. in animals from group c, cyclosporine was administered at sub-optimal dose. lung rejection was graded according to the working formulation of ishlt and the presence of circulating donor-specific (dsa) antibodies determined by flow cytometry. results: in group a, acute rejection (ar) or ob occurred in % and % of animals, respectively. ar occurred in - % of animals in group b and c respectively. ob was detected in % and in % of group b and c respectively. high levels of dsa igg were observed in cases with ar. conclusion: a novel model of pulmonary ob was developed in the rat. to obtain a reproducible onset of ob, shortterm and sub therapeutic cyclosporin administration appears indispensable, at least in our species combination. ofp- - epithelial dysplasia and lung cancer in end-stage idiopathic pulmonary fibrosis: padova experience n. nannini * , f. lunardi, e. balestro, e. rossi, m. loy, m. saetta, f. rea, f. calabrese * university of padova, italy objective: idiopathic pulmonary fibrosis (ipf) is associated with increased risk of lung cancer. the prevalence of high grade dysplasia/lung cancer was studied only in a small number of ipf patients. the aim of our study was to investigate the prevalence of precancerous/cancerous changes and their relationship with both metaplastic changes and clinical data. method: native lungs from ipf patients were studied. the degree of honeycomb changes and squamous, cuboidal and bronchial cell metaplasia were graded (score: - ). the presence or absence of precancerous/cancerous changes were also evaluated. results: three patients showed neoplastic transformation ( %) and nine high grade dysplasia ( %) ("cancer" group). the "cancer" group had similar smoking history, sex, age and duration of disease than the "no cancer" group. all lungs showed metaplasia, the score of squamous (p= . ), cuboidal (p= . ) and bronchial cell (p= . ) metaplasia was significantly higher in the "cancer" than in the "no cancer", while the honeycomb score was similar in the two groups. conclusion: advanced ipf patients have a high prevalence of high grade dysplasia/lung cancer, complex epithelial metaplasia, particularly squamous type, is more frequent in "cancer" group, independently from all clinical parameters, including smoking history. ofp- - primary pulmonary adenocarcinoma with enteric differentiation, morphologically indistinguishable from metastatic colorectal adenocarcinoma: case report with a history of metastatic colon adenocarcinoma s. percinel * , p. celepli, h. nalbant, y. yuyucu karabulut * ankara, turkey objective: according to a computerized medline search in the english literature, the present case is thought to be the first primary pulmonary adenocarcinoma with enteric differentiation, completely resembling metastatic colorectal adenocarcinoma morphologically, with a history of metastatic colon adenocarcinoma. results: a year old man with a history of colon adenocarcinoma which was detected in underwent radiologic evaluation. a chest computed tomography scan revealed a mm solitary nodule in the left lower lobe which enlarged in year. the patient underwent left lower lobe wedge resection. macroscopically, an irregular whitish nodule of . cm in greatest diameter was detected. microscopically, the nodule was entirely composed of glandular and papillary structures, some of which had a cribriform pattern, lined by tumor cells that were cuboidal to tall columnar with nuclear pseudostratification, eosinophilic cytoplasm, brush-border, luminal necrosis and nuclear debris. tumor cells were diffusely positive for cytokeratin (ck) and negative for thyroid transcription factor- , surfactant protein-a, cdx- and muc . only a very few tumor cells stained for ck . conclusion: enteric morphology with consistent expression of ck and a scattered positivity for ck helped in the distinction from metastatic colon adenocarcinoma and favored the diagnosis of primary pulmonary adenocarcinoma with enteric differentiation. expression of cxcr and cxcl in pulmonary carcinoids s. seiwerth * , l. brcic, a. sepac, a. zanko * zagreb school of medicine, pathology, croatia objective: cxcr and its chemochine ligand cxcl seem to play an important role in the process of tumor metastasis and, possibly, homing of metastatic tumor cells. the chemochine seems to be responsible for creating microenviromental predispositions for survival of metastatic cells, in a similar way it is done for developing immuno-competent cells. in contrast, expression of cxcl in primary tumor cells, is thought to be associated with lower metastatic potential. cxcr is expressed in a wide range of tumors, and is thought to be crucial for the metastatic process and tissue-specific spread firstly of breast and prostate cancer. the role of cxcr signaling has been poorly evaluated in carcinoids in general and not at all in pulmonary carcinoids. method: immunohistochemically we investigated the expression of cxcr and cxcl in pulmonary carcinoids. results: together tumors ( typical and atypical) where investigated. in there was a metastatic process (in typical and atypical). ligand cxcl expression was negative in all of the metastatic tumors and in two without known metastasis in contrast to only two non-metastatic tumors showing negative reaction. cxcr positivity was found in both metastatic and non-metastatic carcinoids. the other investigated parameters where present in both metastatic and non-metastatic tumors. objective: to evaluate col v and decorin expression in pulmonary tissue and to characterize biochemical profile of colv from lung fibroblasts culture from ssc patients. method: we evaluated col v and decorin expression and tridimensional reconstruction ( d) of patients with ssc without pulmonary hypertension that underwent surgical lung biopsy and as control was obtained lung fragments from normal individuals who died from trauma. col v amount in lung sections was evaluated with immunofluorescence. to biochemical characterization of col v from lung fibroblasts culture was used quantitative immunoblot. results: it was found that the structure of col v fibers was distorted and strongly thickened in lung tissue from ssc patients compared with thin fibers pattern in the healthy controls. decorin was distributed around col v fibrils in the bronchovascular interstitium and vascular walls. histomorphometric analysis of ssc lung demonstrated increased expression of both col v and decorin when compared to the control (p < . ). the semiquantitative imunoblot detected an increased high molecular weight colv fraction in patients when compared to the control. conclusion: the over expression and unusual organization of colv fibers with biochemical changes associated to increased decorin indicates that matrix signalization pathway is involved in colv fibrillogenesis process in ssc pulmonary fibrosis. objective: probe-based confocal laser endomicroscopy (pcle) is a new method used during bronchoscopy by means of special miniprobe alveoflex and based on the visualization of intraalveolar structures which possess autofluorescence. aim: to compare the visual signs of a healthy and pathologically changed lung tissue received at pcle in patients with infiltrative and local lung nodules with the diagnosis, delivered by light microscopy. method: an autopsy and surgical material was fixed in % neutral formalin solution and was analyzed by studying with a new method for visualization of structures. histological specimens were studied at that spaces, where the pcle was applied. we compared our results by using qualitative method. results: normal lung tissue structures include alveolar septum with the high light emission and light-negative spacesthe alveolar spaces. in case of pneumonia alveolar septum were saved, but the light density of alveolar spaces was higher in compare with the normal tissue. in case of alveolar proteinosis we observed unique globules, which had the highest light emission. we found out several authentic signs, which are representative for each kind of pathological feature. moreover, we revealed some other characteristics, which help us to distinguish some types of lung cancer. conclusion: pcle can be used as an additional method of noninvasive diagnostics in vivo. increased copy nember of alk gene is not associated with increased immunoreactivity of alk protein in lung adenocarcinomas t. balharek * , a. farkasova, z. kviatkovska, k. scheerova, p. tilandyova, z. hutka, l. plank * comenius university, dept. of pathology, martin, slovakia objective: anaplastic lymphoma kinase (alk) gene rearrangements represent an important predictive marker and promissing therapeutic target in small subset of non-small cell lung carcinomas (nsclc). immunohistochemical (ihc) screening of alk abnormalities in nsclc was reported to have variable reliability. we analyzed association between alk immunoreactivity and increased number of alk gene copies often seen in nsclc. method: we examined clinically selected egfr negative lung adenocarcinomas. alk protein expression was detected by ihc using antibody clone alk (dako). alk gene status was assessed by fluorescent in situ hybridization using lsi alk dual color rearrangement probe (abbott) and spec alk/eml tricheck probe (zytovision). results: increased alk gene copy number was identified in cases, of them showed cytoplasmic alk positivity. in of these cases we detected rearrangement of alk locus, once represented by alk-eml fusion combined with more complex cytogenetic abnormalities. remaining third alk + case was negative for alk rearrangement. conclusion: ihc seems to be useful method for initial screening of alk rearrangements in nsclc. there is no clear association between alk protein expression and number of alk gene copies. prognostic relevance of alk copy gains or amplification in lung adenocarcinomas remains to be determined, together with role of alk-inhibitors in those cases. expressions of egfr, ercc- , ß tubulin iii, rrm- in advanced non-small cell lung carcinoma n. bassullu * , e. namal, i. turkmen, r. yasar, p. y. korkmaz, z. akcali, g. demir, g. b. dogusoy * istanbul bilim university, dept. of pathology, turkey objective: egfr, ercc , rrm and βtubuliniii predicts sensitivity to therapeutic agents and provide prognostic information in nonsmall cell lung cancer (nsclc) which is the most frequent worldwide cause of cancer death. method: we investigated the expressions of egfr, ercc , βtubuliniii, rrm immunohistochemically in advanced nsclc cases and their correlations with other pathologic features. results: the age distribution was - with an average of , . male/female ratio was / . ercc protein was detected in nuclei of carcinoma cells in patients ( , %) (h score > ). Βtubuliniii expression was detected in cytoplasm of cancer cells in patients ( , %) (score ≥ ). ercc and βtubuliniii expression were not associated with pathological factors. rrm was negative (score< ) in ( %) cases and negativity was significantly correlated with male gender (p< , ). egfr was negative (score< ) in ( , %) cases and negativity was nearly correlated with absent necrosis (p< , ). no significant correlations were found between egfr, ercc , βtubuli-niii, rrm and the pathological parameters. conclusion: in our study, as a first step, egfr, ercc , βtubuliniii, rrm showed no significant correlation with pathologic features. we look forward to obtain further results in the next study consisting of correlations with follow ups. proliferative markers in idiopathic pulmonary fibrosis: clinical, radiological and functional significance e. parra * , m. cornati, v. capelozzi * fmusp, dept. of pathology, são paulo, brazil objective: natural course of idiopathic pulmonary fibrosis (ipf) could be predicted by proliferative markers of the fibrotic process, such as myofibroblasts and interleukins (il)- and il . our primary aim was to determine whether these proliferative markers influence the course of ipf course measured by a radiological/functional score. method: twenty-eight patients with biopsy-proven ipf disease, who underwent pulmonary evaluation by high-resolution computed tomography (hrct) fibrosis score and pulmonary function tests were studied. five normal lung tissues (nlt) were included biomarkers in lung tissues were detected by immunohistochemistry and quantified by histomorphometry for myofibroblasts alpha-smooth muscle actin (α-sma), anti-interleukin (il)- and il- . results: myofibrobalst amount, il- and il- expression were higher in ipf than in nlt (p< . ). myofibroblast expression of α-sma was positively correlated to il- and il- expression. lung tissue from patients with high hrct fibrosis scores expressed significantly greater α-sma+, il- and il- when compared with patients with low hrct fibrosis scores (p< . ). negative correlations were found between myofibroblasts α-sma + and vc and dlco. conclusion: proliferative markers, detected by immunohistochemistry, in lung tissue allowed recognizing a dichotomous distribution of hrct fibrosis course and influenced pulmonary function tests, suggesting that they may be promising markers of prognosis in these patients. objective: epithelioid mesothelioma is the most common histologic type of the diffuse malignant pleural mesothelioma, also having the best prognosis. although a great variety of histological patterns within epithelioid type has been described, a clear impact of histological subtyping on clinical outcome is unknown (kadota et al., .) . here we compared median survival of six histological subtypes of epithelioid mesothelioma. we examined hematoxylin and eosin-stained slides of patients diagnosed with epithelioid mesothelioma. according to previously described predominant histological features we grouped them into six subtypes: trabecular, solid, microglandular, tubulopapillary, micropapillary and pleomorphic. results: the median survival of all patients with epithelioid mesothelioma was . months. the best median survival was in trabecular and micropapillary subtypes (each months, n= and n= , respectively), followed by tubulopapillary ( months, n= ), microglandular ( months, n= ) and solid ( months, n= ) subtypes. the worst median survival was in pleomorphic subtype ( months, n= ). conclusion: epithelioid type of diffuse malignant pleural mesothelioma shows a great diversity of histological patterns that likely have an impact on the clinical outcome and patient's survival. further investigations of genetic variations among different subtypes may provide valuable information for better understanding of pathogenesis of these tumors. objective: genetic instability resulting in both aneuploidy and polyploidy are discussed to be involved in prostate cancer (pca) development and progression. aim of this study was to comprehensively characterize the ploidy status and proliferation levels in pca with regard to disease progression. method: using fish, we assessed localized pca, pca with corresponding lymph node metastases, and hormone-refractory distant metastases for losses and gains of all chromosomes. the proliferation rate was assessed using phh and ki immunohistochemistry. results: we observed significant increases in aneuploidy with advancing tumor stage (p< . ). chromosomes x, , y, , , and were most frequently numerically altered. increased levels of proliferation were significantly associated with the extent of aneuploidy and tumor stage (p< . ). combining aneusomy of chromosomes , , , and x with phh immunoreactivity resulted in a prediction model for lymph node metastases with a sensitivity of . % and a specificity of . %. conclusion: we present evidence that genomic instability leading to aneuploidy is an important factor in pca progression. furthermore, we demonstrate that increased ki- and phh expression are potential indicators of metastatic disease. lastly, we suggest a new approach to preoperatively determine lymph node metastasized disease in pca. ofp- - erg protein expression and genomic rearrangement status in primary and metastatic prostate cancer: a comparative study of two monoclonal antibodies m. braun * , d. goltz, z. shaikhibrahim, w. vogel, d. boehm, a. dobi, n. wernert, g. kristiansen, s. perner * universitätsklinik bonn, inst. für pathologie, germany objective: overexpression of the erg protein is highly prevalent in prostate cancer (pca) and most commonly results from gene fusions involving the erg gene. recently, an n-terminal epitope targeted mouse and a cterminal epitope targeted rabbit monoclonal anti-erg antibody have been introduced for the detection of the erg protein. here, we are the first to compare the mouse erg-mab to the rabbit erg-mab for their concordance on the same pca cohort. furthermore, we assessed if the erg protein expression is conserved in lymph node and distant metastases. method: we evaluated tissue microarrays of specimens containing localized pca, lymph node, distant metastases, and normal prostatic tissues. we correlated the erg protein expression with the erg rearrangement status using an erg break-apart fluorescence in-situ hybridization (fish) assay and ihc of both erg antibodies. results: erg protein expression and erg rearrangement status were highly concordant regardless of whether the mouse or rabbit erg-mab was used ( . % versus . %, respectively). conclusion: this is the first study to comprehensively compare the two erg-mabs. by demonstrating a broad applicability of ihc to study erg protein expression using either antibody, this study adds an important step towards a facilitated routine clinical application. ofp- - improved method of detecting the erg gene rearrangement in prostate cancer using combined dual-color chromogenic and silver in-situ hybridization m. braun * , j. stomper, d. boehm, w. vogel, n. wernert, g. kristiansen, s. perner * universitätsklinik bonn, inst. für pathologie, germany objective: the recently detected erg rearrangement revealed as a recurrent and prevalent prostate cancer (pca) specific event. to detect this alteration, fish is the method of choice. however, fish harbors disadvantages for widespread adoption in clinical practice. subsequently, the chromogenic in-situ hybridization (cish) and the enzymatic metallography silver in-situ hybridization (sish) emerged as promising bright-field alternatives. we aimed to develop a combined cish and sish (cs-ish) gene break-apart assay on the example of the erg gene. method: we assessed and compared pca and benign specimens for their erg rearrangement status applying a dual-colour fish and cs-ish erg break-apart assay on consecutive sections. results: we observed a highly significant concordance ( , %) between fish-based and cs-ish-based results (pearson's correlation coefficient . , p< . ). conclusion: we demonstrate that the erg rearrangement status can reliably be assesed by cs-ish. further, we confirm that the cs-ish technique combines the accuracy and precision of fish with the ease of bright field microscopy. we developed a tool which allows a broad spectrum of applicants to study the biological role and clinical utilization of erg rearrangements in pca. moreoever, our study is the first proof-of-principle for bright-field cs-ish gene fusion or break-apart assays. ofp- - rearrangement of the ets genes etv- , etv- , etv- and elk- is a clonal event during prostate cancer progression m. braun * , z. shaikhibrahim, w. vogel, d. boehm, r. menon, n. wernert, g. kristiansen, s. perner * universitätsklinik bonn, inst. für pathologie, germany objective: ets gene rearrangements are frequently found in prostate cancer (pca). recently, we observed that erg rearrangement in primary pca transfers into lymph node metastases, suggesting it to be a clonal expansion event during pca progression. here, we investigated whether this also applies to the less frequent ets genes, etv- , etv- , etv- and elk- . method: using break-apart fish assays, we evaluated the status of above mentioned ets gene rearrangements on a cohort comprising of primary pca, corresponding lymph node and distant metastases. results: etv- , etv- , etv- and elk- rearrangements were found in %, %, % and % of the primary pca, respectively, and in %, %, %, % of the corresponding lymph node metastases, respectively. rearrangements of etv- and etv- were not found in any of the distant metastases cases, whereas etv- and elk- rearrangements were found in % and % of the distant metastases, respectively. conclusion: our results suggest that rearrangement of the less frequent ets genes is a clonal event during prostate cancer progression. our findings provide insights into potential clonal expansion events during pca progression and may have significant implications in understanding the molecular basis of the metastatic cascade of pca. ofp- - comparative analysis of two prostate biopsy systems: a study of cases f. e. costa * , r. dias, j. r. vizcaíno * centro hospitalar do porto, braga, portugal objective: it's expectable that the higher the number of prostate biopsies the greater the probability of prostate cancer detection, presumably improving the diagnostic accuracy and treatment decision. method: under this assumption, since may/ , santo antónio's hospital implemented a prostate mapping system, increasing the number of biopsies performed from - to - and processing each core independently. we intend to establish a comparison between the traditional prostate biopsy system (ts) and the prostate mapping system (ms). for this study, all biopsy slides and corresponding surgical specimens from patients subjected to ms and patients subjected to ts were reviewed. gleason score, percentage of tumor present in prostatic tissue (expressed as small ≤ %, medium - % and large ≥ %) and presence of tumor in prostate apex and base were analyzed. results: comparing ms with ts, the correlation coefficient was , % and , % for gleason score and , % and , % for the percentage of tumor present in prostatic tissue, respectively. mapped biopsies were able to detect the tumor in the apex and base in % and % of cases. conclusion: these results suggest that prostate ms improves the diagnostic accuracy of prostate cancer and has a good ability to predict the presence of tumor in prostate apex and base. ofp- - frequency and prognostic significance of tmprss -erg gene fusion in lymph node positive prostate cancers a. fleischmann * , i. zlobec, t. visakorpi, g. thalmann * universität bern, inst. für pathologie, switzerland objective: tmprss -erg incidence and prognostic significance in lymph node positive prostate cancer are virtually unknown. method: a tissue-microarray was constructed from hormone-naïve nodal positive, surgically treated prostate cancers containing samples from all gleason patterns (gp) present in every primary tumor (pt) and corresponding lymph node metastases (met). tmprss -erg status was determined by fluorescence in-situ hybridization and correlated with various histomorphological tumor features (gleason score, stage, cancer volume, nodal tumor burden) and biochemical recurrence-free, cancer-specific and overall survival. results: tmprss -erg fusion was present in . % (homogeneous . %, heterogeneous . %) of the pt and in . % (homogeneous . %, heterogeneous . %) of the met. percentage of tmprss -erg in gp / / of pt and met were: %/ %/ % and %/ %/ %. concordance in tmprss -erg status between pt and met was poor (kappa . ) showing . % and . % of cancers with gene fusion solely in the pt and met, respectively. tmprss -erg fusion was not correlated with histopathological tumor features and predicted late biochemical recurrence independently (p= . ) when present in pt. conclusion: tmprss -erg fusion in pt is more frequent and its distribution more heterogeneous compared to met. the gene fusion in primary tumors independently predicts late biochemical recurrence. ofp- - wnt and shh pathways activation in penile carcinoma a. silva * , t. almeida, f. soares, m. buim * hospital ac camargo, investigative pathology, são paulo, brazil objective: penile carcinoma (pc) is rare in developed countries, accounting for less than % of all neoplasms in men, and biological characteristics of this tumor are poorly known. wnt and shh pathways are important for cell proliferation, differentiation and survival, and therefore, play a role in carcinogenesis of various organs. the goal of this study is to investigate the expression profile of wnt and shh pathway proteins in pc, characterizing the expression of target proteins from wnt (wnt- , wnt- , wnt- gsk β, β-catenin, d cyclin, mmp , c-myc, cd ) and shh (shh, smo, gli, egfr) pathways. method: for that, samples of pc were obtained from the files of anatomic pathology department from a.c. camargo hospital (brazil) and submitted to immunohistochemistry. results: we observed that wnt- and wnt- were expressed in and cases, respectively, out of the evaluated samples. strong shh expression was detected in / cases, whereas weak and negative expression was seen only in one case each. smo and gli- proteins were expressed in almost all cases ( / ), and also d cyclin, b-catenin and egfr were frequently expressed. conclusion: these preliminary results suggest that wnt and shh pathways may be active and participating in the progression of penile cancer. ofp- - expression of the multidrug resistance protein correlates with longer psa relapse free survival and androgen receptor and forkhead box a protein expression in prostate cancer m. montani * , g. kristiansen * medizin. universität bern, inst. für pathologie, switzerland objective: multidrug resistance protein (mrp ) a transmembranary transport protein has shown to be expressed in prostate cancer cell lines and cancer cell specimens and turned out to be among the highest upregulated genes in an arraybased transcription analysis. its expression is regulated in an androgen dependent manner, possibly modulated by forkhead box a (foxa), an androgen receptor (ar) co-activator. therefore, we investigated its expression in a large cohort of neoplastic and non-neoplastic prostate tissues (n = ) and evaluated its prediction of psa relapse free survival (rfs). method: tma (n= ) stained for mrp , foxa, ar, er, psa, ki- : radical prostatectomies (rpe) (n= ), castrate resistant prostate cancer (crpc) (n= ), metastases (n= ) and non-neoplastic (n= ). psa-rfs of patients; mean months. results: mrp expression decreases with tumor progression into castrate resistant disease, correlates with psa, ar and foxa expression and inversely correlates with gleason score. moreover, a strong mrp expression is significantly associated with a longer psa rfs in rpe patients. normal tissues from the transitional zone show a weak mrp expression compared to the peripheral and central zones. conclusion: mrp expression seems to predict psa relapse free survival in prostate cancer patients. since its expression is androgen dependent it decreases with tumor progression into crpc. ofp- - synchronous angiomyolipoma and renal tumors in patients without tuberous sclerosis: clinico-pathological study of cases f. j. queipo * , r. a. carías, Án. f. panizo, m. l. gómez-dorronsoro, f. j. pardo * clínica universidad de navarra, pathology, pamplona, spain objective: simultaneous existence of aml and renal neoplasia is frequent in tuberous sclerosis (ts) patients, but uncommon in non-ts cases. method: a total of cases of coexistent renal neoplasia and aml in non-ts patients were identified. clinico-pathological features were studied. results: patients: m/ f (mean age , year; range: - ) . mean aml size: , cm (range: , - , ). the main size of the renal neoplasms was , cm (range , - ). aml morphology: leiomyomatous, classic-triphasic, lipomatous, and epithelioid. three cases had multifocal aml. cases had ipsilateral tumors associated with aml: ccrcc ( sarcomatoid), chrcc, ro, tfe rcc, hybrid (ro-chrcc) renal-cell tumor, urothelial carcinoma, and case had tumors: mtsc-rcc and concomitant prcc. cases had contralateral tumours associated with aml: chrcc and ro. one patient had bilateral tumors associated with aml: an ipsilateral ro and a contralateral ccrcc. the median follow-up was , mths (range , - , ) : all patients were alive without disease. conclusion: the coexistence of renal tumors with aml is a rare event, usually incidental. if aml is found incidentally together with other renal tumors, it is important to exclude ts retrospectively. ofp- - primary renal synovial sarcoma: a clinicopathologic, immunohistochemical and molecular genetic study of cases j. schoolmeester * , j. cheville, a. folpe * mayo clinic, dept. of anatomic pathology, rochester, mn, usa objective: primary renal synovial sarcoma (r-ss) is a rare malignant neoplasm. approximately cases have been described, chiefly as case reports or as part of relatively limited small studies. we studied the clinicopathological, immunohistochemical (ihc) and molecular genetic features of well-characterized r-ss. method: all available slides for institutional and consultation cases of r-ss were reviewed. an ihc panel (tle , bcl , cytokeratins, s , cd , ini ) was performed. rt-pcr for ss -ssx / or fish for ss rearrangement was performed. follow-up was obtained. results: the patients ( m, f) ranged from to years (mean ). all tumors were of monophasic fibrous type. ihc results: all cases were strongly positive for tle and bcl ; focally positive or negative for cytokeratins; negative for s and cd ; and showed retained ini expression. molecular genetic results were: ss -ssx ( cases), ss -ssx ( cases), syt rearrangement ( case). follow-up ( cases): dead of disease, alive without disease. conclusion: our data show a striking overrepresentation of the ss -ssx fusion subtype among r-ss, in contrast to other ss, in which the ss -ssx fusion subtype accounts for two-thirds of cases. the reasons for this difference are unclear. the prognosis for r-ss appears similar to that of ss of more common locations. objective: new data related to the diagnosis and therapy of lung cancer have also affected the position of the pathologist in this area. principal changes in its classification have appeared in relation to adenocarcinoma and partly also in the large cell type of lung cancer. method: presented data represent a five-year retrospective study of a series of cases of lung cancers analyzed in our institute. besides routine he staining the following immunohistochemical reactions were used: antibody against ttf- , surfactant a, chromogranin, synaptophysin, ck , ae /ae , p , βe , cd and napsin a. results: using the who, classification of lung cancer, an unusual phenomenon of the return of squamous cell cancer (scc) prevalence over adenocarcinoma has been documented, which in the earlier years has equated the scc. the difference in obtaining the tissue samples, and changes in the geographical origin of certain patients of eastern slovakia may partly explain these differences. though the new travis classification (j thorac oncol, ) was not used, there prevailed mixed forms also in our series. conclusion: it is important to respect new information regarding the classification of lung cancer, especially in the category of adenocarcinoma. the constructive collaboration of pathologists with clinicians is recommended. objective: extranodal marginal zone lymphoma malt lymphoma and carcinoid are neoplasms occurring most frequently in the gastrointestinal tract and respiratory system. although each of them occurs relatively frequently and separately, the simultaneous appearance of these two neoplasms is exceptionally rare. method: we report an exceptional synchronous association of malt lymphoma and typical carcinoid in the lung. results: a -year-old male with sjögren's syndrome presented with cough and chest pain with no improvement after antibiotherapy. bronchial fibroscopy was normal. computed tomography showed a diffuse interstitial pneumopathy with a persistent left lower lobe nodule suspected of malignancy. a surgical resection of the nodule was achieved. histopathologically, it measuring . cm in diameter and was composed of association of malt lymphoma and typical carcinoid tumor which were focally admixed. immunohistochemical stains were strongly reactive to endocrine marker in carcinomatous component. tumor cells were diffusely cd positive in lymphomatous component. assessment of extent of lymphoma revealed a gastric location. the patient received chemoptherapy. he's still alive since months. conclusion: the best of our knowledge, this is the first report of such a collision lung tumor at the same anatomical site. the aim of this study is to describe the pathogenesis and clincicopathological feartures of such exceptional association. objective: primary mucoepidermoid carcinoma is rare comprise less than % of all lung tumors. it is characterized by the presence of squamoid cells, mucin-secreting cells and intermediate type. on the basis of morphological and cytological features, it is divided into low and highgrade types. method: we report a retrospective study of cases diagnosed during a -year period. diagnosis was made by histological examination of specimen obtained from lobectomy in cases and pneumonectomy in cases. no patient had history of salivary mucoepidermoid carcinoma. results: there were men and women ranging in age from to years. computed tomography showed a well circumscribed tumor arising in bronchus in all cases. a bronchial fibroscopy showed a main, lobar or segmental mass. bronchial biopsy revealed a non-small-cell carcinoma in only one case. histologically, tumors were classified into low grade mucoepidermoid carcinoma and high grade mucoepidermoid carcinoma. patients with low grade carcinoma remain alive after surgery alone. patients with high grade carcinoma received chemotherapy, four of them developed distance metastasis. conclusion: mucoepidermoid carcinoma is a rare primary malignancy of the tracheobronchial tree which is difficult to diagnose by limited biopsy. the prognosis is variable and depends upon the histological grade. objective: primary pulmonary malt lymphoma is considered to originate from malt of the bronchus secondary to autoimmune or inflammatory processes. although, it comprises more than two-thirds of all primary non-hodgkin's lymphoma of the lungs, it is a rare entity and accounts for less than % of all lymphomas. method: we report a retrospective study of cases of malt pulmonary lymphomas diagnosed during an -year period. diagnosis was made on surgical pulmonary biopsy in cases, bronchial biopsy in other cases and on lobectomy in one case. results: there were women and men ranging in age from to years. two patients had a history of sjoren's syndrome and one had a history of previous mammary carcinoma. computed tomography revealed bilateral nodules in patients, lung mass in patients and an area of consolidation in one patient. fiberoptic bronchoscopy showed bronchial stenosis in only cases. morphologically, the neoplasms had features typical of malt lymphoma. one patient was treated with surgery alone and received chemotherapy. five patients remained alive while one patient presented with recurrence. conclusion: pulmonary malt lymphomas are characterized by an important dissociation between clinical expression and radiological pattern. therefore, histological documentation is mandatory to ensure diagnosis. objective: malignant mesothelioma (mm) is an aggressive tumour with a poor prognosis. carbonic anhydrases and their inhibitors offer an opportunity for developing novel anticancer drugs, as well as diagnostic and prognostic tools. carbonic anhydrase ix (caix) is a membranous metalloenzyme involved in cell adhesion and ph homeostasis. it is a direct target of hypoxia-inducible factor and serves as a marker of hypoxia. this study was designed to assess systematically caix expression in mm of pleura and peritoneum, and their benign counterparts. method: mms of pleura ( epithelioid, biphasic, sarcomatoid) and peritoneum ( epithelioid), and normal or reactive pleural samples were analyzed. caix expression was determined using immunohistochemistry. membranous immunoreactivity was evaluated semiquantitatively. specimens were divided into five subgroups according to the staining pattern and intensity. results: , % ( / ) of mms expressed caix. all epithelioid mesotheliomas showed at least a weak focal ( , %, / ), but predominantly a strong diffuse ( , %, / ) staining with caix antibody, without any perinecrotic pattern. sarcomatoid mesotheliomas were negative. normal mesothelial cells were diffusely positive. conclusion: these data suggest that mechanisms of caix overexpression in mm are different than due to hypoxia and appear promising in prospective use of specific therapeutic caix targeting in advanced mesothelioma. objective: eml -alk crizotinib therapy needs validation at lower cost and rapid answer in pathology routine. method: histological/who and ck , ttf , ck . , cd /chromogranin and vimentin panel classifications with alk (clone a , novocastra laboratories ltd, newcastle, united kingdom) were applied to paraffin sections of bronchial-pulmonary carcinomas: adenocarcinomas, epidermoid carcinomas, pleomorphic carcinomas (mixed type adenocarcinomas with large/giant/fusiform cells), neuroendocrine carcinomas (nec) ( combined large cell nec with adenocarcinoma and with epidermoid carcinomas; sclc chromogranin positive combined with adenocarcinoma) and adenosquamous carcinoma. results: the applied antibodies specified bronchial pulmonary carcinomas subtypes clearly. in over years old nonsmoking females mixed type adenocarcinomas alk expression was over %: acinar, solid, micropapillary and microacinar patterns; one glandular mucinous pattern (mucinous ba pattern) and one ba pattern, all expressing ttf- . conclusion: in this study, / adenocarcinomas of older women had alk protein expression, only one with a mucinous pattern. as protein positivity cases comprise a lower number, fish described by s. lantuejoul seems to be the most appropriate method. it is now necessary to decide whether kras and egfr mutations have to be determined together and/or select ttf- positive adenocarcinomas (from terminal respiratory unit) raised by this approach. objective: the year survival of bronchial-pulmonary carcinomas remains poor, between % and %/men and % to %/women. treatment orientation is influenced by clinical staging and morphological classification in biopsies of % of the cases. method: this study comprised surgical specimens where we compared immunohistochemistry in-between adenocarcinomas ( ), epidermoid carcinomas ( ) and the heterogeneous groups of large cell neuroendocrine carcinoma ( ), small cell lung cancer ( ), large cell carcinoma ( ), adenosquamous carcinoma ( ) and pleomorphic carcinomas ( ) with max f-fluordesoxiglucose (fdg), a clinical parameter based in pet to preview diagnosis and prognosis. results: we found significant differences (p= . ) between ttf- positive and negative adenocarcinomas where the f-fdg capture was lower in ttf- positive cases, indicating lower metabolic activity. ttf- negative adenocarcinomas have similar and higher metabolic activity as epidermoid carcinomas. the other histological types have fdg capture similar in between the two defined groups. conclusion: immunohistochemical and f-fdg analysis correlate with clinical differences between adenocarcinomas and epidermoid carcinomas, where ttf- negative adenocarcinomas are biologically similar to epidermoid carcinomas, requiring a different medical approach as well as molecular pathology particular interpretation. these results strain the classification of bronchial ttf- negative adenocarcinomas because they are different from the terminal respiratory unit ttf- positive adenocarcinomas. objective: according to researchers pneumonias are characterized by increase number of alveolar macrophages population after increased migration of monocytes from bone marrow and blood into the alveolar space. method: we examined observations of pneumonia with bacteriological, histological and blood monocytes examination in all cases. bacteriological study was performed at the institute of antimicrobial chemotherapy. histological examination was performed in the smolensk regional institute of pathology. results: in the foci of pneumonias we discovered mainly mixed gram positive and gram negative microorganisms. ten observations were characterized by an increase in the absolute number of blood monocytes. fifty observations did not reveal excess levels of blood monocytes. in all cases with an increase in the number of blood monocytes, gram negative bacteria were isolated. in cases with normal number of blood monocytes some of the microorganisms were gram positive. conclusion: gram negative bacteria result in activation of the monocytes and their entry from the bone marrow into the blood more than gram positive bacteria. objective: bronchial carcinoids and schwannomas are uncommon, slowly growing, low-grade neoplasms. carcinoid tumors are thought to arise from neuroendocrine/kulchitsky's cells of bronchial epithelium. schwannomas are thought to originate from schwann cells. clinically they may be asymptomatic or present with non resolving recurrent pneumonia, hemoptysis, respiratory distress. conclusion: we present two cases of respiratory distress in two teenage patients. pulmonary hamartomas mimicking metastatic carcinomas h. erdem * , l. yilmaz aydin, a. k. uzunlat, m. oktay, a. n. annakaya, u. yilderim, f. basar * duzce university, dept. of pathology, turkey pulmonary hamartomas are usually found incidentally and they mimick metastic tumours. we report a case of a year-old man who admitted to chest disease service for cough. he had laryngectomy operation formerly and diagnosed as squamous cell carcinoma. chest-x-ray and computed tomography revealed pulmonary nodules. it was removed but there was not metastatic carcinomas. this case was presented because it can be difficult to make diagnosis when the patient has malignancy. objective: experimental models of pulmonary fibrosis (pf) has been proposed and its later phase tend to the resolution in different degrees depending on the drug/ strain/inflammatory-pattern. thus the maintenance of these mechanisms may participate in the pf-progression. our objective was to determine the immune-fibroticpattern in models of pulmonary fibrosis in the late stage ( d). method: distinct animal models were used, including balb/c, c bl/ and il ra-ko-c bl/ mice by bleomycin and paraquat. we analyzed the amounts of total peribronchiolar-interstitial collagen (tpc-tip) by picrosirius and col -col by immunofluorescence through the morphometric evaluation. these data were validated by rt-pcr. results: the tpc did not differ between the treated groups. tic was higher in the c bl/ strain, independent of the absence of il- ra. the col -immunoexpression was higher in control and blm-treated il ra-ko-c bl/ than in wild-mice and lower in blm-balb/c mice. the col immunoexpression was higher in blm-balb/c. likewise, the col gene expression was higher in the il ra-ko mice and lower in the blm-balb/c. conclusion: the perpetuation of fibrosis in pf-susceptiblemice can be modulated by il- -dependent col -hiperexpression and by col -subexpression. this suggests that col is an important component responsible for the development of pf. the prognostic role of filamin a protein expression in patients with non-small-cell lung cancer m. gachechiladze * , j. skarda * palacky university, pathology, olomouc, czech republic objective: an actin-binding protein filamin a (flna) serves as a scaffold in various signaling pathways. recently, it has been reported that flna interacts with brca protein and is required for efficient regulation of early stages of dna repair processes. as dna repair proteins are important prognostic markers for non-small-cell lung cancer (nsclc) patients, we aimed to investigate the role of flna protein expression in nsclc, as well as its correlation with brca protein expression. method: we performed a preliminary study of fnla and brca protein expression in nsclc patients. formalin-fixed paraffin-embedded tissue sections were stained by immunohistochemistry using antibodies against flna c-terminus (ep y, lsbio), brca n-termunus (ms ) and against phosphorilated forms of brca at ser and ser (abcam). staining intensity was estimated semi-quantitatively and correlated with all available clinico-pathological factors. results: flna expression was significantly higher in cancer, compared to normal lung tissue. positive correlation has been revealed between flna and brca phospho-ser expression. also, year overall survival rate was higher in patients with strong flna expression. conclusion: according to our preliminary study results the prognostic role of flna protein expression deserves to be a subject for further studies in patients with nsclc. ps- - pulmonary nodular lymphoid hyperplasia ("pulmonary pseudolymphoma") -the significance of increased numbers of igg positive plasma cells d. guinee * , a. gerbino, s. murakami, m. koss * virginia mason medical center, pathology, seattle, usa objective: pseudolymphomas of the skin, breast, and lacrimal glands show an increase in igg positive plasma cells. we hypothesized that a similar increase in igg positive plasma cells occurs in pulmonary nodular lymphoid hyperplasia (pnlh). method: immunohistochemical stains for igg and igg were performed in cases of pnlh, cases of balt lymphoma, cases of intraparenchymal lymph nodes (ipl) and case of follicular bronchiolitis (fb). the mean number of igg -and igg-positive plasma cells and the igg /igg ratio was obtained from a manual count of three separate high power fields (hpfs) of areas of highest cellularity. results: the average number of igg positive plasma cells and the igg /igg ratio was increased in pnlh (case : /hpf, ratio: . , case : /hpf, ratio: . ). in comparison, average igg positive plasma cells per hpf were much lower in balt lymphoma (range= - . /hpf, ratio= - . ), ipl (range igg / hpf= - , ratio= - . ) and fb ( /hpf, ratio= . ). conclusion: the increase in igg positive plasma cells and igg /igg ratio in pnlh aids in diagnosis and supports our current understanding of pnlh as a distinct form of reactive lymphoid proliferation. the relationship between pnlh and igg -related sclerosing disease requires further study. ps- - calretinin, ck / , ttf- , cea, ber-ep , and cd : a useful combination of immunohistochemical markers for differentiating pleural mesothelioma from pulmonary adenocarcinoma n. gursan * , m. calik, e. demirci, s. sipal, b. gundogdu, c. gundogdu * ataturk university, medical faculty, erzurum, turkey objective: the distinction between pleural epithelial mesothelioma and peripheral lung adenocarcinoma involving the pleura is still an important diagnostic problem for surgical pathologists. the aim of our study was to identify the most specific and sensitive markers for the positive identification of mesothelioma. method: paraffin-embedded blocks from surgical material of pleural epithelial mesotheliomas and pulmonary adenocarcinomas were retrieved from the files in our department. the primary antibodies used in each case were the following: antibody anticalretinin, ema; cea, berep , ttf- and cd . results: of the mesotheliomas, % stained for calretinin, . % for ck / , . % stained for ema and ae /ae . of the lung adenocarcinomas, . % cases showed reactivity for calretinin, . % for ck / , . % for cd , all for ttf- , % for cea, . % for ber-ep , . % for ema, and all for ae /ae . conclusion: calretinin were the highly specific positive mesothelial markers, whereas ck / showed high sensitivity but low specificity. among negative markers, we advocate the use of ttf- , cea and cd which were the most specific in differentiating mesotheliomas from adenocarcinomas. cyclooxygenase- expression in nsclc i. kern * , e. sodja, m. rot * university clinic golnik, slovenia objective: overexpression of cox- correlates with aggressive disease of nsclc. aim of our study was to determine the cox- expression levels by two methods. method: analysis was done on consecutive surgical specimens of nsclc fixed in paxgene tissue system. relative quantification of cox- mrna expression was performed by quantitative rt-pcr using intron-spanning primer-probe set. cox- protein expression was assessed by immunohistochemistry (ihc). scoring was performed using an intensity-extent system, both parameters on the scale - and multiplied to give ihc index. results: there were cases of adenocarcinoma, cases of squamous cell carcinoma and one typical carcinoid. cox- mrna expression was detectable in all specimens. the median cox- mrna expression value, normalized against he internal reference gene gadph, was , (range , - , ). we observed cytoplasmic and membranous immunohistochemical reaction patterns. average ihc index was , . there was positive correlation between mrna and protein cox- expression (r = , ). adenocarcinoma cases had average relative mrna expression value , and ihc index , , while squamous cell carcinoma had average relative mrna expression value , and ihc index , . conclusion: cox- is expressed in nsclc with various ihc reaction patterns. adenocarcinoma and squamous cell carcinoma have different cox- expression levels. objective: the akt/mammalian target of rapamycin (mtor) pathway is up-regulated in many human cancers, and agents targeting the mtor pathway are in various stages of clinical development and application. method: expression of pakt and mtor was studied by immunohistochemical analysis of surgically resected nonsmall cell lung cancer (nsclc) specimens on a tissue microarray (tma). results: the results were correlated with clinicopathological features. expression of mtor showed a strong correlation with the expression of pakt (p< . ) and was significantly associated with female gender, tumor size ≤ cm, adenocarcinoma (adc), non-smoker status, and lower pathological stage. expression of pakt was correlated with older age (≥ ), adc, non-smoker status, and lower t stage. univariate survival analysis revealed that the mtor and pakt positive group had a significantly longer cancer-specific survival than the mtor and pakt negative group (p= . and p= . , respectively). coexpression of pakt and mtor correlated with better prognosis than either single or double negative pakt and mtor groups (p= . ). however, multivariate analysis proved that mtor and pakt expression are not independent prognostic factors for cancer-specific survival. conclusion: expression of pakt and mtor expression is more significantly associated with adc than squamous cell carcinoma (scc) and expression of these proteins is associated with better prognosis. ps- - pak immunohistochemistry marker in non-small cell lung cancer (nsclc) a. kucukosmanoglu * , o. d. İlara colakkadıoglu, k. bakir * university of gaziantep, pathology, turkey objective: pak is a member of p -activated kinase family. many human tumor express and activate this family. because of their role in cell transformation, they become therapeutic targets. they have important roles in cell survival, cell proliferation and cell migration. they affect cell growth and tumor invasion. method: cases with nsclc reported in gaziantep university pathology department between and , reviewed retrospectively. immunohistochemically nuclear and cytoplasmic pak stainings were considered positive and scored according to degree of staining. results: our cases included squamous cell carcinoma, adenocarcinoma, adeno squamous carcinoma and large cell carcinoma. there was no significantly associated between tumor types and pak staining. of patients showed recurrence and of cases showed nuclear staining but there were no statistically significance (p: , ). cases died from the disease and in of these cases nuclear stainig was very intresting, inspite of statistical results. conclusion: in this study pak staining patern and score were compared with tumor type, size, recurrence, mortality and lymph node involvement in nsclc. and we found no statistically significance. of case with nsclc stained with pak . these results suggest that pak was expressed in lung like prostat, plasenta and breast. pleural fibroelastosis is a similar spectrum of histopathology in chronic fibrosing lung disorders l. marcal * , e. r. parra, l. antonangelo, v. capelozzi, f. vargas, e. c. nascimento, v. teodoro, k. c. silva * lia junqueira marçal, são paulo, brazil objective: parenchymal fibroelastosis in chronic fibrosing lung disorders has been much investigated, but little attention has been directed at the visceral pleura (vp) participation in these situations. our aim was to verify whether elastic deposition accompanies collagen deposition in the repairing process of chronic lung injury. in this work we studied the distribution of these fibrous components of vp in bullous disease type i and ii, smoking-related interstitial fibrosis and usual interstitial pneumonia (uip). method: we employed histochemical methods on conventional histological slides. we measured, by image analysis, the content of fibres of the collagenous and elastic systems of the visceral pleura in histological slides sampled from surgical lung biopsies and bullectomy, using the picrosirius-polarization method and weigert's resorcin-fuchsin stain, respectively. results: four groups were studied: i, patients with spontaneous pneumothorax due to type i bullous disease; ii, patients with spontaneous pneumothorax due to type ii bullous disease; iii, smoking-related interstitial fibrosis; and iv, patients with idiopathic pulmonary fibrosis. the first two groups were used as controls. patient diagnosed with idiopathic pulmonary fibrosis ( ), in which experimental transplantation was performed of type ii pneumocytes, ingress to the hospital for further study groundmass of the right lung. in the postmortem study a gr. mass was identified, the mass was formed by sarcomatous cells habit, undifferentiated and pleomorphic, with the presence of multiple implants and adhesions in rib cage, diaphragm, pericardial fat. ihc profile was performed: vimentin (+), ema focally (+), wt , ck , ttf , ckae /ae , s , cd , cd and cd (−) and low proliferation index, the diagnosis undifferentiated malignant tumor. with inconclusive ihc profile, so blocks were sent to clinic barcelona hospital, for study. the estimated prevalence of idiopathic pulmonary fibrosis is cases per , population in men, with a mortality of % at years of diagnosis. cancer rate of in transplanted patients is within the range between % and %. could the appearance of this tumor be related either to transplanted pneumocytes or it is inmunosupression the main risk factor? current treatments for idiopathic pulmonary fibrosis are not effective, which requires the study and development of new therapeutic options, using fibroblast proliferation inhibitors, as well as prospective studies, in the long term, the evolution of these patients and the development of secondary entities to it. objective: mediastinal hemangiomas are rare tumors accounting for , % of all mediastinal tumors. these tumors are challenging because of the lack of clinical and radiologic specific signs. their diagnosis is mainly based on microscopic study. method: we report a -year-experience of a single institution. we describe cases of mediastinal hemangioma. results: our study contained men and women with a mean age of . years. symptoms consisted mainly in chest pain. neurologic signs were observed in patients. surgical treatment was performed in all patients dealing with a total resection in patients. the most used surgical approach was a posterolateral thoracotomy. video-assisted thoracic excision was tried in one patient and was converted into a median sternotomy. microscopic examination concluded to a mediastinal hemangioma in all cases. all the patients presented no complications after a follow up periods varying from months to years. conclusion: mediastinal hemangiomas are rare tumors whose diagnosis is based on microscopic findings. their surgical management may be challenging because of their connection to the adjacent structures. video-assisted thoracic excision is being more frequently used by experienced surgeons. these tumors are benign with a good behavior. objective: actinomycosis is an infectious disease caused by actinomyce israeli in % of the cases. it is mainly observed in alcoholic patients and affects mainly cervicofacial, abdominal and thoracic regions. pulomnary actinomycosis accounts for % of the localizations and is caused by the inhalation of septic particles causing granulomatous lesions, the extension to the adjacent organs and a cutaneous fistulae. it represents a real pitfall and mimics malignant lesions, tuberculosis or other infectious diseases. method: we report a retrospective study about cases of pulmonary actinomycosis diagnosed over a -year-period. results: we describe the cases of men aged between and years who presented with respiratory signs. radiologic investigations showed in all cases parenchymal masses with irregular margins mimicking malignant processes. positivie diagnosis was based on microscopic examination. the treatment was based on surgical excision associated to a medical treatment without complications after a follow up period ranging from months to months. conclusion: pulmonary actinomycosis shares usually the same clinical and radiologic features as malignant lesions. positive diagnosis is based on microscopic findings. it inducesusually surroundng organs and may affect, in some cases, the vital prognosis. over expression of hyaluronan syntehase- activity has impact in the remodeling process and survival evolution in patients with idiopathic pulmonary fibrosis e. parra * , v. capelozzi * fmusp, dept. of pathology, são paulo, brazil objective: the idiopathic pulmonary fibrosis (ipf) is a terminal illness characterized by unremitting extracellular matrix (ecm) deposition in the lung. in this regard, the myofibroblasts and the ecm components such as collagen and hyaluronan (ha) have an important role in the fibrosis. we analyzed the expression of has (ha synthase ), has , has and hyaluronic acid receptor (cd ) by epithelial and myofibroblasts cells in patients with ipf and we correlated with a survival. method: has- , has , has and cd epithelial and myofibroblast expression were evaluated in surgical lung biopsies from patients with ipf in minimal and severe fibrosis by the point-counting technique. impact of these markers was tested on pulmonary functional tests and follow-up until death from ipf. results: has and cd expression were significantly increased and directly associated with severe fibrosis. myofibroblast has activity was indirectly associated to dlo/va (r= − . ; p= . ). kaplan maier curves determined a higher risk of death for patient with high ha (> . %) expression than in low expression (log rank p= . , figure) . conclusion: the increased has- activity in epithelial and myofibroblast cells have impact in the remodeling process and the survival evolution, suggesting that strategies aimed at preventing the effect of this ecm component may have a greater impact in patient's outcome. financial support: fapesp, cnpq. ps- - viral antigens are more frequently observed in acute interstitial pneumonia than in other types of idiopathic interstitial pneumonias e. parra * , g. c. dos santos, f. weisshaupt stegun, c. dos santos cirqueira, m. i. seixas duarte, v. l. capelozzi * fmusp, dept. of pathology, são paulo, brazil objective: the etiology of idiopathic interstitial pneumonias (iips) remains unclear. many researches assumed that viruses can represent an important factor of aggression to the lung provoking fibrosis. in this regards, we researched the presence of virus infections in patients with iips. method: biopsy samples from patients with idiopathic interstitial pneumonia (ipf); patients with nonspecific interstitial pneumonia (nsip); patients with acute interstitial pneumonia (aip) and patients with idiopathic cetrilobular fibrosis (iclf) were used to investigate by microarray and immunohistochemistry the presence of measles virus (mv), hepatitis-c virus (hcv), adenovirus (adv), respiratory syncytial virus (rsv), epstein-barr virus (ebv), herpes i and ii viruses (hvi and hvii). results: we detected the epithelial alveolar infection by mv and cmv in . % and . % in aip, respectively. endothelial cmv infection was observed in % of iclf group. when we compared with the age of these patients, patients with ≤ of years old had more infection by mv and cmv than the group with ≥ years old. the other viruses were not detected in the different groups. conclusion: the viral infections observed in aip and iclf groups reinforce the possible viral participation in these pulmonary diseases. financial support: fapesp. abnormal up regulation of cyclooxygenase- is observed in idiopathic pulmonary fibrosis e. parra * , f. lin, w. teodoro, a. p. velosa, v. l. capelozzi * fmusp, dept. of pathology, são paulo, brazil objective: several factors modulate fibroblast proliferation and collagen deposition in pulmonary fibrosis diseases, including cyclooxygenase (cox). the main of this study was observed the relationship between cox and cox in surgical lung biopsies from patients with idiopathic pulmonary fibrosis (ipf). method: twenty four patients ( ± . year) were characterized with ipf. immunohistochemistry and histomorphometry were used to evaluate the amount of cox and cox expression in lung specimens. the expression of these markers was tested with their pulmonary function tests. results: significantly a higher amount of cox- was observed in ipf patients principally in normal and vascular areas (p= . ) when compared with control group (figure) , contrasting with similar amounts of cox- in both groups. an important negative correlation was observed between total lung cox- expression and dco/va (r= . , p= . ) in ipf patients. conclusion: higher vascular expressions of cox probably mediated the inflammatory reaction in patients with ipf and have an important impact with pulmonary function tests, suggesting that a participation in the pathway of ipf. financial support: fapesp. error barr shows the correlations between cox- expression in normal and vascular areas of ipf compared with control group ps- - multiplex ligation probe-dependent amplification (mlpa) as an ancillary method for the diagnosis of malignant pleural effusion e. parra * , d. rosolen, l. kulikowski, r. dutra, v. l. capelozzi, f. vargas, m. acencio, l. antonangelo * fmusp, dept. of pathology, são paulo, brazil objective: a definitive diagnosis provided by the finding of malignant cells in pleural fluid (pf) can be established in around % of patients with pleural malignancy. however, underdiagnosis risk in cytological suspicious cases is high, which makes the cytological diagnosis quite limited. this is an important clinical problem, especially if we consider that some patients, in bad clinical conditions, can not be submitted to a guided thoracoscopic biopsy. method: using multiplex ligation probe-dependent amplification (p -mrc-holland) we have studied sequence variations of egfr gene and amplifications/deletions of chromosomal regions frequently associated to tumors (atg b, pahs, pros, nsd , and cdgif genes). results: forty-three malignant pf samples from patients with different cancers were evaluated, even in those cases with scarce pellet cells. four benign pleural effusions were used as control. gene sequence changes were observed in ( . %) cases, while others copy number abnormalities were found in ( . %). conclusion: the findings suggest that mlpa could be considered an alternative tool to detect molecular genetic changes in malignant pleural effusions, since this technique is relatively low expensive and not time consuming. our next challenge is to find the best combination of probes capable to recognize malignant cells of any origin in fresh samples of pf. multiple primary pulmonary myopericytoma: case presentation n. petre * , f. pop, d. leonte, s. bedereag * medcenter bucharest, dept. of pathology, romania objective: myopericytoma is described as a perivascular myoid tumor which usually develops as a solitary mass in the subcutaneous tissues of the extremities, in adults. method: the chest radiography of a -year-old woman showed multiple, bilateral lung nodules. after the surgical excision of one of them, the histopathologic examination revealed a well circumscribed nodule, composed of fusiform or oval cells, with eosinophilic cytoplasm and ovoid nuclei, without atypia, in a concentric pattern, intimately associated with thin-walled vessels, within a myxoid stroma. results: the spindle cells were diffusely positive for vimentin, smooth muscle actin (sma) and occasionally for desmin. cd and cd were positive only in endothelial cells. ki was positive in less than % of neoplastic cells. conclusion: the histological and immuno-histochemical findings led to the diagnosis of multiple primary pulmonary myopericytoma. ps- - five cases of lung pneumocytoma: clinico-pathological, immunohistochemical and ultrastructural study i. rodriguez * , a. panizo, b. larrinaga, i. amat * c. hospitalario de navarra, dept. of pathology, pamplona, spain objective: lung pneumocytoma (lp), also called sclerosing hemangioma, is an uncommon tumor of uncertain histogenesis. method: we analyzed the clinical, morphological, immunohistochemical and ultrastructural features of lp. results: all patients were women, with median age of year (range: - ). mean size was mm (range - mm). all tumors were located within the right lung (upperlobe: ; middle-lobe: ; lower-lobe: ). all cases showed solid, papillary, sclerotic, and hemorrhagic patterns. the tumors were composed of cells types: pale polygonal and surface cells. ema, ttf , and pr were observed in both type cells. the surface cells showed also positivity for ck , napsin-a, and surfactant-a, whereas the polygonal cells for vimentin. surface cells had short microvilli and lamellar bodies in their cytoplasm. polygonal cells contained abundant microfilaments and rough endoplasmic reticulum. all patients were alive and well without recurrence at the last follow up. conclusion: lp is likely to be an epithelial tumor with a differentiation toward type ii pneumocytes, which exhibits various architectural patterns. the ihc profile provides useful clues for the diagnosis of this lung neoplasm when typical features are absent. all our cases had an excellent prognosis with no evidence of recurrence following surgery. rare tracheobroncheal lesions diagnosed by small bronchoscopic biopsies a. sasmaz * , s. yuksel, h. nalbant, p. celepli, e. kadan, s. percinel, s. dizbay sak * ankara, turkey objective: six different, rarely seen tracheobronchial lesions, taken by bronchoscopy as small fragments were discussed with an emphasis on morphological findings and differential diagnosis. results: six patients were men and two patients were women with an age range of to years. most of the lesions were symptomatic and discovered as small masses by bronchoscopy. the tracheobronchial lesions varied from polypoid, nodular, well-demarcated, fragile masses to nodular thickenings. microscopically, a polyp composed of a fibrovascular stroma with scattered mononuclear inflammatory cells was compatible with fibroepithelial polyp. mature adipocyte proliferation in two cases, s- and cd positive polygonal or ovoid cells with abundant eosinophilic, granular cytoplasm having small, hyperchromatic nuclei, smooth muscle actin and desmin positive spindle cells in interlacing fascicles within a myxoid and collagenous stroma with mononuclear inflammatory cells, cd and cd positive thin-walled vascular channels, filled with red blood cells and s- positive spindle cells having palisading of wavy nuclei in two cases, located in the submucosa were main findings of lipoma, granular cell tumor, inflammatory myofibroblastic tumor, cavernous hemangioma and schwannoma, respectively. conclusion: tracheobronchial biopsies, even with small fragments of lesions can allow to render rare specific histopathological diagnoses, some of which need to be confirmed by immunohistochemistry. objective: small cell lung carcinomas (sclc) are aggressive neoplasms that give very early distant metastases. matrix metalloproteinases (mmps) are a family of endopeptidases which degrade extracellular matrix and modulate cell adhesion, playing a crucial role in cancer cell invasion and metastasis. the aim of the present study is to investigate the immunohistochemical expression of mmp- , mmp- (type iv collagenases) and mmp- (interstitial collagenase) in patients with sclc. method: formalin-fixed, paraffin-embedded lung biopsy specimens from patients with sclc (m:f= : , median age= . ) were immunostained for mmp- , mmp- and mmp- (anti-mmp- polyclonal antibody, anti-mmp- kda collagenase iv antibody, anti-mmp- kda collagenase iv antibody, spring bioscience). results: immunohistochemical expression of mmps was detected in the cytoplasm of neoplastic cells. immunoreactivity was designated positive when > % of neoplastic cells were stained. overall, mmps were expressed in the majority of sclc. specifically, mmp- was positive in % ( / ), mmp- in . % ( / ) and mmp- in . % ( / ) of sclc. non-neoplastic lung parenchyma did not stain for mmps. conclusion: the majority of sclc express immunohistochemically mmp- , mmp- and mmp- . given the role of mmps in cancer progression and metastasis, their expression in sclc may contribute to their aggressive course. objective: number of pulmonary adenocarcinoma (adc) patients incresease. treatment of non-small cell pulmonary carcinoma depends of its histological type in era of target molecular therapy particulary of adc: diagnosis of adc is established on small-sized pulmonary biopsies obtained on bronchoscopy and by transthoracic needle pulmonary biopsy. the aim of this study was to evaluate optimal panel of monoclonal antibodies in diagnosis of pulmonary adc on small-sized biopsy pulmonary samples. method: analysis of small-sized biopsy pulmonary samples. diagnosis of adc was established on hematoxillyn-eosin stained (h&e) samples and confirmed immunohistochemicaly by thyreoid transcription factor- (ttf- ), napsin-a, surfactant b and cytokeratin (ck ). descriptive statistical method (%) was used. results: ttf- specificity was %( / ), napsin-a- %( / ), surfactantb - %( / ) and ck - %( / ) in adcs. two monoclonal antibodies were positive in % ( / ) adcs, one of them was necessary ttf- or napsin-a. three monoclonal antibodies were positive in %( / ) and in %( / ) adcs, respectively. there is not statistical significans in number of monoclonal antibodies for diagnosis of adc. conclusion: no one monoclonalantibody is specific for one histological type of carcinoma and its origin. ttf- , napsin-a, surfactantb and ck belong in optimal panel for diagnosis of pulmonary adenocarcinoma. ps- - expression of cd , e-cadherin and bcl- in lung cancer i. strumfa * , a. abolins, j. gekis, k. pavlovs, a. vanags, j. grusina-ujumaza, g. volanska, j. gardovskis * riga stradins university, dept. of pathology, latvia objective: lung cancer represents major problem in oncology as the incidence and mortality is high (parkin et al., ) . molecular studies could reveal additional targets for intervention or prognostic evaluation. however, the published data are controversial (renouf et al., ; leung et al., ; maraz et al., ; ko et al., ) . method: consecutive lung cancer cases ( ) were retrieved by retrospective archive search. the diagnostics has been performed systematically in accordance with who classification (travis et al., ) . the expression of cd , e-cadherin and bcl- was analysed by immunohistochemistry and evaluated semiquantitatively. results: the intensity of bcl- expression was highest in small cell cancer, reaching the mean value . . no or very low bcl- expression was found in adenocarcinoma, carcinoid and squamous cell cancer. the membranous expression of e-cadherin showed significantly higher mean intensity in adenocarcinoma ( . ) and carcinoid ( . ) than in small cell cancer ( . ) and squamous cell cancer ( . ). cytoplasmic e-cadherin expression was observed in small cell cancer. cd expression was widespread and frequent in non-small cell lung cancer. conclusion: the main histological types of lung cancer show different immunophenotype. bcl- expression is found in small cell cancer. the expression of e-cadherin is characteristic in adenocarcinoma and carcinoid. cd is expressed in non-small cell lung cancer. localized nodular pulmonary and thoracic cage amyloidosis i. strumfa * , a. abolins, j. gekis, k. pavlovs, a. vanags, b. strumfs, g. volanska, j. gardovskis * riga stradins university, dept. of pathology, latvia objective: localised amyloidosis represents a rare differential diagnosis of neoplasms. however, in some cases amyloid deposits are manifestation of tumour itself. to increase awareness about these uncommon but important differential diagnostic issues, we show here three well-characterised cases of thoracic cage or pulmonary localised nodular amyloidosis. method: the cases were identified by systematic retrospective archive search, - . congo red stain, polarisation microscopy and immunohistochemical analysis was applied. results: there were males and female among the patients diagnosed with thoracic cage or pulmonary amyloidosis. the patients were - years old and had no history of previous malignancy. clinically, either lung cancer or sternal osteochondroma was suspected. radiologically, - nodules measuring . - . cm were found in lungs ( cases, one of these being bifocal) or sternum ( ). surgical treatment was applied in all cases. amyloid deposits were identified by congo red stain and polarisation microscopy. al type of amyloid was confirmed. in case of bone lesion, the plasma cells were monoclonal justifying the diagnosis of multiple myeloma. conclusion: localised amyloidosis can involve lung or sternum as tumour-like mass. the lung lesions can be single or multiple. association with haematologic neoplasm is likely if bone is affected. clonality analysis of plasma cells surrounding amyloid deposits is mandatory. pleural localization of castleman disease: a rare entity e. tastekin * , f. oz puyan, e. isler, u. usta * trakya university, dept. of pathology, edirne, turkey objective: castleman disease (cd) comprises a heterogeneous group of disease with various clinical presentations and prognosis. by definition; cd is a lymphoproliferative disorder of the mediastinal lymph nodes and extranodal tissues with follicular and interfollicular abnormalities. pleural localization is an unusual presentation of cd. method: a -year-old man applied to the hospital because of cough. chest x-ray and ct images showed an irregular, × × mm sized intrapleural-extrapulmoner mass in the paramediastinal region. surgical specimen was consisting of a × × . cm sized, irregular-round shaped, gray colored calcified mass. microscopically, a lymphoid proliferation with distorted follicles and increased dendritic cells (with cd ) was detected. lymphocyte depleted germinal centers resembling "onion skin" appearance with penetrated sclerosing hyalinized blood vessels were also seen. interfollicular region was composed of proliferating blood vessels lined by plump endothelial cells and few lymphoplasmositer infiltration. there was heterogeneous positivity with cd and cd . expanded mantle zones showed diffuse bcl positivity. ki proliferation index was low. the mass was diagnosed as "castleman disease, hyaline vascular type". typical appearance of the follicular and interfollicular abnormalities allowed definitive differentiation from mesothelioma and solitary fibrous tumor of the pleura. conclusion: pleural cd is a rare lesion but should be considered especially on young patients in the differential diagnosis of pleural masses. objective: to evaluate foxp expression in bronchus-associated lymphoid tissue (balt) and correlate with the inflammatory process and collagen content in the lung tissue in an experimental model of scleroderma (ssc) after type v collagen (col v)-induced nasal tolerance. method: female new zealand rabbits (n= ) were immunized with mg/ml of col v in freund's adjuvant (im). after days, six immunized animals were nasally tolerated with col v ( νg/day), during days (im-tol). animals (n = ) only tolerated served as control (ct). foxp expression in balt and inflammatory cells in pulmonary interstitium were evaluated by point counting method. types i, iii and v collagen gene expression were evaluated by real-time pcr. results: im-tol when compared to im presented decreased lymphocytes, macrophages and monocytes and types i (p= , ) and v (p= , ) collagen mrna expression in pulmonary tissue. t lymphocytes foxp were expressed in % of im-tol and , % of ct (p= , ). additionally, balt was higher expressed in im-tol in relation to ct. conclusion: col v-induced nasal tolerance in ssc model induces foxp regulatory t cells in balt which can trigger an immune regulatory mechanism resulting in decreased inflammation and collagen expression. it suggests that col v tolerance could be a promising therapeutic for human scleroderma treatment. igg -related lung disease t. tichy * , h. hornychova, j. jankova, j. skarda, b. krajsova * university hospital olomouc, inst. of pathology, czech republic objective: igg -related lung disease is one of the manifestations of igg -related systemic disease. obliterative vasculitis is considered an organ specific feature of igg -related disease in the lung. we report a case of a -year-old man with multifocal subpleural consolidations in both lungs. histologic examination revealed igg -related lung disease with obliterative lymphoplasmacytic vasculitis. method: histological sections from paraffin blocks were used for hematoxylin-eosin and special stains (elastica-masson trichrome, pas, geimsa, grocott), for immunohistochemistry (antibodies against kappa and lambda light chains, cd , cd , igg, igm, iga, igg , lmp ) as well as for eber in situ hybridisation and pcr. results: the lung tissue was irregulary fibrotic with dense lymfoplasmacytic infiltration concentrated in the walls of pulmonary arteries. some arteries were obliterated by intimal fibrosis. infiltrating lymphocytes were small and without atypias. the majority of lymphocytes were cd positive, nearly all plasmocytes showed igg positivity. igg /igg ratio was %. eber in situ hybridisation was negative and monoclonal reaarangement of t cell receptor or immunoglobulin heavy chain genes were not detected. conclusion: igg -related lung disease is rare. obliterative vasculitis and high percentage of igg positive plasmocytes are main histological features of this entity. objective: primary lung carcinoma with heterotopic osteocartilaginous formation is exceedingly rare. to date only cases have been reported in the literature and the pathogenesis is not fully investigated. we report a case of pulmonary adenocarcinoma with osteocartilaginous formation with immunohistochemical staining of bone morphogenetic proteins (bmps). method: a -year-old man was admitted to our hospital for a lung tumor in his left upper lobe. an open lung biopsy followed a lobectomy was done. the resected tissue was fixed with % formalin and embedded in paraffin. sections, cut from paraffin blocks, were subjected to hematoxylin and eosin (he) staining and masson-goldner staining, in addition to immunohistochemical staining. results: the tumor was poorly differentiated adenocarcinoma with heterotopic ossification. some of the tumor cells were positive for bmp- , , and . some of mesenchymal cells in tumor interstitium were positive for osteoblast (ob)-cadherin or bmp receptor-ia. conclusion: we report a case of primary lung adenocarcinoma with heterotopic osteocartilaginous formation and reviewed previously published reports. to our best knowledge, this was the first case that not only bmp was expressed in tumor cells but also ob-cadherin or bmp receptor in some mesenchymal cells of tumor interstitium. expression of transforming growth factor ß and e-cadherin in lung adenocarcinoma j. yoo * , s. y. park * st. vincent's hospital, pathology, suwon, republic of korea objective: there is evidence supporting the concept of tumor progression from pulmonary adenocarcinoma in situ (formerly bronchioloalveolar carcinoma, bac) to adenocarcinoma with varying degrees of invasion. the aim of this study was to investigate the role of tgfß in tumor invasiveness in lung adenocarcinoma, and to determine the potential relationships between its expression and immunophenotypes of cell adhesion molecules. method: tumor samples (n= ) from adenocarcinoma in situ (n= ), minimally invasive adenocarcinoma (formerly bac with ≤ mm invasion, n= ), and lepidic predominant invasive adenocarcinoma (formerly mixed adenocarcinoma showing non-mucinous bac features with > mm invasion, n= ) were examined for the expression of tgfß , e-cadherin, n-cadherin, and h-cadherin proteins using immunohistochemistry. results: twenty-five tumors ( %) were positive for tgfß . the frequency of immunoreactivity in patients with adenocarcinoma in situ, minimally invasive adenocarcinoma, and lepidic predominant invasive adenocarcinoma was % ( / ), % ( / ), and % ( / ), respectively (p= . ). loss of e-cadherin expression was more frequently observed in invasive adenocarcinomas than in adenocarcinomas in situ (p= . ). tgfß expression showed a statistically significant correlation with h-cadherin expression (p= . ), but not with e-cadherin expression (p= . ). conclusion: these results suggest that tgfß and e-cadherin may play an important role in invasive progression of lung adenocarcinoma through regulating epithelial-to-mesenchymal transition. ps- - lin a expression as prognostic indicator in lung adenocarcinomas v. zolota * , v. tzelepi, g. psiouri, i. lilis, n. panagopoulos, c. sirinian, c. scopa * medical school of patras, dept. of pathology, greece objective: lin a is implicated in stem cell pluripotency by blocking let- mirnas and oct- is a transcription factor highly expressed in embryonic stem cells. the present study investigates the prognostic significance of both markers in lung adenocarcinomas. method: we evaluated, by immunohistochemistry, lin a and oct- a expression in formalin-fixed, paraffin-embedded tissues from lung adenocarcinomas. immunoreactivity was scored as +, < % positive tumor cells (ptc); +, - % ptc; +, - % ptc; and +, > % ptc and was compared with clinicopathologic features and overall survival. results: regarding lin a expression % of tumors were recorded as +, % as +, % as + and % as +. the stratification for oct- expression was % ( +), % ( +), % ( +) and % ( +). lin a and oct- expression was not significantly associated with age, gender and tumor grade or stage. lin a + expression predicted a shorter overall survival (p< . ) on univariate analysis. oct- was not associated with patients' prognosis. cox multivariate analysis showed that age, tnm and lin a + expression were independent prognostic factors of survival. conclusion: lin Α expression appears to be an independent predictor of poor outcome in lung adenocarcinomas. further studies are warranted in order to investigate its role in stratifying patients at increased risk for poor outcome. comparison of the mutation status of egfr and kras on pulmonary adenocarcinoma and corresponding brain metastasis objective: egfr and kras mutation statuses have been known associated with the sensitivity of tyrosine kinase inhibitor treatment in pulmonary adenocarcinoma. however, the mutation analyses were usually performed on the primary tumors only, due to the availability of the tumor tissue. method: to compare the kras and egfr mutation statuses between pulmonary adenocarcinomas and corresponding metastases, we performed direct sequencing, followed by scorpion arms method on wild-type cases for egfr analysis, and allele-specific real-time pcr for kras analysis, on the paired samples of pulmonary adenocarcinoma. results: thirty-one ( . %) pulmonary adenocarcinomas and ( . %) brain metastases out of the paired specimens have egfr mutations, and ( . %) paired specimens were concordant for the egfr mutation status between the primary and metastasis. in addition, ( . %) pulmonary adenocarcinomas and ( . %) brain metastases out of the paired specimens have kras mutations, and only ( . %) paired specimens were concordant between the primary and the metastasis. conclusion: the status of egfr mutation is relatively consistent between primary and metastasis comparing to that of kras mutation in pulmonary adenocarcinomas. however, discordance for the mutation statuses does happen. accordingly, repeat analysis is recommended if tissue from metastatsis or recurrence is available. objective: we tried to measure and analyze characteristics of neuroendocrine tumors in lung by image analysis and help to diagnose them. method: it was analysed that sixteen cases of typical carcinoid tumors, five cases of atypical carcinoid tumors, fifteen of small cell carcinomas, fifty one cases of large cell neuroendocrine carcinomas. we analyzed the nuclear area, perimeter, major axis and minor axis using i-solution image analyzer software package. results: the mean nuclear area was . μm in the typical carcinoid tumors, . μm in the atypical carcinoid tumors, . μm in the small cell carcinomas, and . μm in the large cell neuroendocrine carcinomas. after the statistical results, every method was effective to distinguish large cell neuroendocrine carcinoma from other tumors and the circumferences of nucleus was the most effective to distinguish among them. conclusion: pulmonary neuroendocrine tumors were the nuclear morphologic differences of each tumors. therefore, diagnosis that considers morphologic differences of pulmonary neuroendocrine tumors contributes to increase reproducibility and accuracy. sunday, september , . - . an abundant growth of lactobacilli may result in lysis of vaginal epithelial cells, named as cytolytic vaginosis. this cytolytic process may cause the symptoms as seen in candidiasis. we observed pap smears to evaluate if cytolysis has a relationship with infertility. method: in the pathology department of mardin maternity hospital, - period, we examined pap smear cases suffering from the similar syptoms as candidiasis. of the smears, were diagnosed as "cytolytic vaginosis". no growth was observed in cultures. results: the number of the cases suffering from the infertility was (% . ) among cases. in the cytolytic vaginosis group this ratio was % . (n= ). the ratio of the infertil cases of cytolytic vaginosis group over general population was significantly higher (p< . ). conclusion: our results are in favour of supporting the hypothesis of the relation between cytolytic vaginosis and infertility. lactobacilli are thought to have inhibitive role in fertility by changing the vaginal ph and adhering to the epithelial cells so inhibiting the sperm penetration. penicillium and aspergillus spp. on pap smears from the surprising origin i. i. akgun * , b. a. borsa * mardin maternity hospital, dept. of pathology, turkey objective: fungal organisms are commonly seen on smears. some unusual species as aspergillus and penicillium are so rare and generally seen via contamination. in this study, we investigated the origin of the many extraordinary fungal organisms seen on pap smears in series in only a few months. method: in pathology department of mardin maternity hospital, we observed both smear and vaginal discharge materials, came from the same hospital but different clinicians, for months. results: smears came from clinician a and smears from clinician b. curious fungal organisms with huge branching hyphas and macrochonidias were seen in smears among smears ( aspergillus and penicillium species were recognized morphologically). no growth was observed in cultures. interestingly, among all smears came from clinician b, there was no abnormal funguses. the smear samples with unusual fungal components all came from the same gynecologist. this made us strongly consider the probability of contamination. conclusion: penicillium and aspergillus spp. are extremely rare in vaginal smears. in our study, these funguses are thought to be airborne passed from the thick layer of the mold spreading on the ceiling of the office. objective: nearly all cervical cancers are related to human papillomavirus (hpv) infection. hybrid capture -hc (qiagen, hilden, germany) is the method to determine the presence of high risk hpv (hr hpv) in cytology samples with the best clinical sensitivity. method: in our study we compared the results of the hpv test in thinprep® cervical samples using first hc and secondly cervista (hologic, madison). samples were divided in two groups for the statistical analysis. the first group were hsil cases confirmed by biopsy (n = ) and the second group cytology samples reported as negative, asc-us or lsil (without biopsy or biopsy, not hsil) (n= ). results: overall, concordance between the two techniques was % ( / ). there were cases hc + cervista−, and cases hc −cervista+. the average hc viral load in discordant cases was . . in our series the hc sensitivity for ≥ cin was % ( / ) and the specificity was . % ( / ). cervista results were . % ( / ) and . % ( / ), respectively. in the hsil cases the coincidence was . % ( / ). conclusion: cervista results showed good clinical sensitivity and high concordance with hc , with a slightly lower specificity. in conclusion, according to our data the determination of hpv with cervista is comparable with hc . mammary analogue secretory carcinoma of salivary glands: cases including cytology, histology, ihc, em, rt-pcr and fish p. farrajota * , e. tani, c. carvalho, j. wejde, g. elmberger * centro hospitalar do porto, dipt. do anatomia patologica, portugal objective: mammary analogue secretory carcinoma (masc) of the salivary glands was described in by skálová et al. in a series of cases. we present new molecularly confirmed cases with focus on detailed cytomorphological features and correlation with special studies. method: a year old man and a year old female presented with history of slow growing parotid and submucosal buccal tumors, respectively. two fine needle aspiration biopsies (fnab) were performed in each case. the surgical resection specimens were routinely processed and special studies applied on paraffin tissue section. the fnab smears were retrospectively studied. results: all smears showed similar features of abundant proteinaceous background, moderate cellularity including macrophages and irregular and variable sized groups of cells with slightly enlarged round hypercromatic nuclei and abundant bluish vacuolated cytoplasm frequently containing small reddish granulations. the first fnab reports confirmed salivary origin and the second ones alert for possible neoplastic nature, advising surgical resection. electron microscopy performed in the parotid case caracterized the granules as mucigen. subsquent pathologic evaluation and molecular confirmation was carried out. conclusion: masc is a low grade malignant tumor capable of simulating a benign salivary gland process so it is important to know its cytological characteristics. adenosquamous lung carcinoma: the importance of cyto-histologic correlation d. felizardo * , a. l. cunha, r. henrique, c. lobo * instituto de oncologia porto, dept. of pathology, coimbra, portugal objective: adenosquamous lung carcinomas (asc) are uncommon and aggressive tumors. identification of a squamous component within a nsclc is critical as it excludes anti-vegf therapy. likewise, recognition of an adenocarcinoma component is also relevant because it prompts the search for egfr mutations. since % of lung cancers are diagnosed in small biopsies/cytological specimens, cytohistological correlation is crucial for correct diagnosis. thus, we aimed at determining the relevance of combining cytologic and histologic findings in initial diagnostic workup of asc. method: we searched for cases of asc diagnosed at our institution from march to april . cytologic and histopathologic findings were evaluated and their accuracy for diagnosing asc was assessed. results: of cases of lung cancer in biopsy, ( %) corresponded to probable asc and in cases cytologic examination was also performed. in cases, cytology was not conclusive, but in cases it demonstrated cytomorphological features of probable asc. we emphasize one case in which only the squamous component was valued by histology, but cytology provided clues that prompted immunohistochemical analysis which led to the diagnosis of probable asc. conclusion: cyto-histological correlation augments the diagnostic accuracy in asc of the lung, emphasizing the complementarity of both procedures. the impact of the bethesda system for reporting thyroid cytopathology (tbsrtc): a retrospective study of aspirates with emphasis on the prior "indeterminate" objective: the material of this study is consisted of selected fna biopsies performed in the last mounths period and followed by the endocrinology department. method: the bethesda system of reporting thyroid cytopathology was used. slides were stained with papanicolaou preparations. results: totally cases were examined, of which were female and were male. age of the patients varied between and . cases were diagnosed as nondiagnostic or nonsatisfactory. cases diagnosed as consistent with a benign follicular nodule. only cases were diagnosed as a consistent with lymphocytic thyroiditis in corelation with the clinical features. only case diagnosed as a granulomatous thyroiditis. diagnosis as "atypia of undetermined significance or follıcular lesion of undetermined significance "was made only in cases whereas "follıcular neoplasm or suspicious for a follıcular neoplasm" was diagnosed in case. "suspicious for malignancy" diagnosed in cases. cases were diagnosed as malignant. conclusion: although diagnostic terminology and morphologic criteria for cytologic diagnosis of thyroid lesions are well established in the bethesda system, difficulty in categorizing the cases and underestimation or overdiagnosis are quıite frequent issues. to solve this problem, retrospective studies which include surgical and cytopathologic reports, to examine slides by at least two different pathologists in different times will be helpfull. metastatic pulmonary adenoid cystic carcinoma: report of a case diagnosed by fine-needle aspiration cytology p. karabagli * , s. ozbek * selcuklu medical faculty, dept. of pathology, konya, turkey objective: a rare case of adenoid cystic carcinoma of ceruminal gland with pulmonary metastasis presented with characteristic cytological and histological findings. method: the biopsy specimen of the patient, a year-old man with painfull, growing mass in his external auditory canal was interpreted as an adenoid cystic carcinoma. he was treated surgically and received post-operative radiotherapy. after years, a distant metastasis relapse was observed. computed tomography (ct) of the chest depicted multiple spiculated masses suggestive of metastases throughout both lung fields, the diameter of the largest measuring cm. ct guided percutaneous transthoracic fine needle aspiration biopsy was performed from the largest lesion. results: cytological examination of the aspirates revealed large spherical hyaline globules representing basement membrane material surrounded by neoplastic cells. the cells were cohesive, closely packed, and had uniform round to oval hyperchromatic nuclei with scanty cytoplasm. these features were suggestive of an adenoid cystic carcinoma. conclusion: patients with adenoid cystic carcinoma could be frequently encountered with disease recurrence confined to the lung. fine-needle aspiration cytology provided a conclusive diagnosis of adenoid cystic carcinoma. objective: often some of a fine needle aspiration (fna) sample is left behind in the hub of the needle when the specimen is ejected on the slides ( figure a) . method: we have designed a device that collects this material. it consists of a plastic adaptor that has a core of polyvinyl alcohol foam protruding from its lumen at one end ( figure b) . the other end of the adaptor then fits on to the syringe (figure c) . as the fna is being performed the sample material flows up the lumen of the needle's shaft and emerges into the hub of the needle where it is absorbed into the tip of the foam core ( figure d ). smears are made by ejected the specimen from the needle onto a slide by air pressure from an attached syringe. the air passes through the foam forcing some of the sample in the foam back down the needle but some remains behind. the device is then removed and placed in a formalin specimen pot. once fixed the core is pulled from the adaptor ( figure e ), processed and sectioned as for routine histology specimens ( objective: the presence of dcs has been studied histologically in ptc, but their presence and potential diagnostic value in thyroid fnas has not been evaluated. method: we assessed the presence of dcs in cytological samples of histologically confirmed ptcs (n= ) and benign thyroid nodules (btn) (n = ) using cd a. corresponding ptcs (n= ) and btn (n= ) from surgical excisions were stained with both cd a and langerin. results: cd a + dcs were identified in / ptcs on cytology ( %). they were either isolated in the background or more typically closely associated with tumor cell clusters. the ptc cases with the least dcs corresponded to the follicular variant on histology. in contrast, most btn ( %) lacked cd a + dcs. when dcs were present, they were primarily isolated in the background although / cases ( %) contained rare dcs among tumor cells. tumor-infiltrating dcs and background dcs were both higher in ptcs than in btn, but only the former was statistically significant (p< . and p= . respectively). similar findings were found on histology where all ptcs contained cd a + and langerin + dcs while only / btn ( %) contained rare dcs. conclusion: dcs are present in fnas of ptc, typically among tumor cell clusters, while they were absent or rare in fnas of btn. thus dcs may be useful as an additional diagnostic marker for ptc. significance of p immunostaining in postmenopausal women with atypical squamous cells a. repse fokter * , k. gornik kramberger, s. hutter Čelik, s. sramek zatler * celje general hospital, dept. of pathology and cytology, slovenia objective: the evaluation of postmenopausal pap smears can often be challenging. degeneration associated with atrophic vaginitis, hyperchromatic crowded groups, parabasal cells with organophilic cytoplasm or variations in nuclear size may be falsely interpreted as squamous atypia or even more severe lesion. method: the study included postmenopausal papanicolaou patients ( smears) with the initial cytological diagnosis of asc-us or asc-h. the smears were decolorized and immunostaining for p (ink a) was applied. in patients ( smears) tissue biopsy and histological examination was performed and four asc-us cases had cytological follow up only. results: among cases with histological examination positive p reaction was found in patients ( smears). the histological diagnoses were: cin ( ), cin - ( ), cin ( case), cin ( patients/ smears), ais ( case) and invasive squamous carcinoma ( patients). two p positive patients with three smears and initial negative histology had cin after years. among p negative patients there were two with cin and two with normal histology. four asc-us patients without histological examination had normal cytological follow up. conclusion: p is a useful marker in detecting of clinically significant cervical lesions in postmenopausal women. endoscopic-ultrasound guided fine needle aspiration cytology of intraductal papillary-mucinous neoplasms of the pancreas: report of two cases and review of the literature e. tejerina gonzález * , a. lópez garcía, c. gonzález lois, f. fernández garcía, a. herreros de tejada, e. sanz * hu puerta de hierro-majadahonda, dept. de anatomía patológica, spain objective: intraductal papillary-mucinous neoplasms (ipmn) of the pancreas are mucin-producing tumors recently recognized as a distinct entity among other mucinous pancreatic tumors. endoscopic-ultrasound guided fine needle aspiration (eus-fna) is a sensitive technique which can be very useful in the accurate diagnosis of these neoplasms. method: we present a case of a -year-old female with recurrent episodes of acute pancreatitis and a -year-old male with abdominal pain. tc showed a mm-cystic tumor at the uncinated process of the pancreatic head and cystic dilatation of the main pancreatic duct, respectively. in both cases eus-fna was performed. results: smears showed medium-to-large, cohesive groups and complex papillary clusters set in a clean, mucoid background, composed of cuboidal or columnar epithelial cells with abundant clear or mucinous citoplasms. a honeycomb arrangement was occasionally seen. although nuclei were predominantly medium-size and uniform, some groups showed some nuclear enlargement and pleomorphism with visible nucleoli. a diagnosis of "mucinous neoplasm with papillary pattern" was rendered in both cases, followed by partial pancreatectomy. the histologic examination was consistent with ipmn. conclusion: the cytologic features of intraductal mucinous papillary neoplasms of the pancreas, evaluated by eus-fna, seems to be quite characteristic of these tumors and allow to suggest a correct preoperative diagnosis. objective: the human igh locus at chromosome q is frequently involved in different translocations of non hodgkin lymphoma (nhl), and the detection of any breakage involving the igh locus should identify a b-cell nhl. the split signal igh fish-cish dna probe (ifcd) is a mixture of two fluorochrome-labeled dnas that binds the telomeric and the centromeric segments, on the igh breakpoint respectively. we tested the capability of the ifcd to detect igh translocations and diagnose b-cell nhl on cytological samples. method: fifty cytological specimens from lymphoproliferative processes were tested using the ifcd and the results compared to light chain assessment by flow cytometry (fc), igh status by pcr, and to clinical-histological data. results: ifcd analysis detected positive, negative and inadequate cases; there were true positive ( %), true negative ( %), false negative ( %) and false positive cases ( %). comparing the sensitivity of the ifcd with fc and pcr, the highest sensitivity was obtained by fc followed by ifcd and pcr. conclusion: the ifcd is effective in detecting any translocation involving the igh locus; it can be employed on different samples from different b-cell nhl whereas it is not useful to classify the specific entities. sunday, september , . - . objective: morphometric analysis of the glands and inflammatory cells proportions in colon mucosa may be crucial for distinction of subtle changes. the most important step is a recognition and proper segmentation of the separate glands, even branched, atrophic or distorted. method: new sequential algorithm has been proposed, based on mathematical morphology transformations, such as extended regional maxima, hit-or-miss, opening and closing operations. for lumen opened glands snake algorithm with closing has been applied to surrounding elements with sequential elimination of the closed contours under decreasing area criterion. proposed method has been used to evaluate cases of diversion colitis to compare specimens sampled from functional part of colon and defunctioned distal one. results: the appropriate results were obtained for specimens with different section planes with efficiency %. in comparison of the b to a was observed decreased participation of epithelium in the glands area measured in deep region (an average % instead %). in addition, the area of the gland was slightly reduced from % to % of the mucosa. conclusion: the proposed method can be useful to automatic morphometry analysis of the gland shape in colon mucosa. the recognition of the opened glands improves previously designed algorithms. objective: quantitative analysis of the immunohistochemical histological samples plays an important role in the diagnosis and prognosis of many cancers. the commercial and non-commercial software's to computerized image analysis of microscopic images based in several approaches. method: the advance segmentation scheme usually consists of preprocessing, nuclei extraction and classifier blocks. when the preprocessing step includes filtering and conversion to the most discriminative representation and the classifier can be realized by various tools, especially support vector machine as a state-of-art, nuclei extraction is the most crucial part of any algorithms. we compare single thresholding methods such as otsu, kurita, minimum error, entropy thresholding, with the sequential thresholding approaches combined with area criteria, local threshold value or extended regional maxima. all of them are accompanied by watershed method and filtration. results: for ki- stained meningioma specimens in the fields of view without overlapping nuclei the most advance methods are adequate. when increasing the cell number, only sequential thresholding with area criteria and extended regional maxima give acceptable results. the mean relative error is . ± . and . ± . % respectively. conclusion: the results confirm the efficiency of using sequential methods for segmentation of cell nuclei. for best results, use sequential thresholding with area criteria. ps- - nhs improvements: enhanced it project for histopathology f. mayall * * musgrove park hospital, dept. of cellular pathology, taunton, united kingdom objective: many histopathology laboratories are using antiquated software that hobbles their performance. we developed open source filemaker pro based histopathology reporting software with innovations based on lean principles to enhance work flow. these include: -colour coded visual workflow control -"one-click" extrawork requests with order tracking -user defined template reporting -reporting of complex cases using benchmark profomas -exportable data-sets using open database connectivity -easy local customisation and enhancement by the user. results: the software was used by staff to report more than histopathology specimens at two histopathology laboratories. many of these cases were complex cancer resection cases requiring key data capture. the histopathology staff using the software were surveyed on their experience of using the software. the key steps in lean it development: -start with a small idea -develop software with multiple plan, do, study, act (pdsa) cycles -ask users for more ideas for enhancements -recruit others into the project by allowing them to use the software and experience its benefits -standardise new reporting process by achieving agreement between users -design a project that requires minimal financial investment this open source software is available for download from www.freedp.org. sunday, september , . - . objective: myxopapillary ependymomas (mpe) often occur at the filum terminale but occasionally occur outside the central nervous system (cns). common site for these extraneural tumors is sacrococcygeal area. method: a cocygeal mpe with radiologic, gross and microscopic features will be presented. a year old female admitted neurosurgery department with painful coccygeal mass. magnetic resonance imaging revealed a coccygeal, well-demarcated, non-enhancing mass of × cm in diameter. the mass did not have any connection with the spinal cord. it was completely excised and grossly the cut surface of the encapsulated mass was soft. microscopically the tumor was comprimised of myxoid stroma with pseudopapillary structures around hyalinized vessels. the tumoral cells showed intracytoplasmic dot-like positivity with ema. there were no mitosis, and the ki labeling index was low. conclusion: extraspinal mpes are rare and the most common location is sacrococygeal area. these masses are often preoperatively misdiagnosed as pilonidal sinus. unlike the cns mpes, extraspinal mpes have a potential to metastasize even if they do not show any anaplastic morphological feature. long term follow-up is recommended as metastases can occur up to years after initial presentation. objective: cerebellar liponeurocytoma (cl) is a rare neoplasm with neuronal, astrocytic and lipomatous differentiation arising in adults. the entity is considered as a grade ii neoplasm in the last who classification of central nervous system tumours. we show two typical cases of this entity stressing the spectrum of histological changes and its differential diagnosis. method: patient is a -year-old woman with a history of headaches and instability. ct scans and mri showed a cm in diameter relatively ill-defined, poorly enhanced, round tumour mass in the left hemisphere. patient is a -yearold woman with a history of headaches and instability. mri showed a . cm in diameter well-defined triangular lesion in the right hemisphere. results: complete surgical resection was achieved in both cases. both tumours showed highly cellular neoplasms composed by round to polygonal cells with scant cytoplasm and round nuclei with fine chromatin pattern. mitoses were not seen. well differentiated lipomatous cells grouped in irregular nests were intermingled with the predominant neuronal component. conclusion: cerebellar liponeurocytomas are located in the cerebellar hemispheres and pursue a non aggressive clinical course, with local recurrences detected mainly after incomplete resections. the typical adipous tissue is the result of a lipidization process and not a true metaplasia. objective: giant cell tumor of bone is an uncommon neoplasm that accounts for about % of all bone tumors. it usually involves long bones and is very rarely encountered in the skull where it mostly involves the sphenoid and temporal bones. it is a benign neoplasm but can be locally aggressive. method: we present a case of a year old woman with history of multiple sclerosis whose routine ct scan revealed a mass with corrosive features and calcification situated at the petrous portion of her left temporal bone. a biopsy was sent to our laboratory. results: histopathologic examination revealed a neoplasm composed of sheets of oval mononuclear cells, with uniformly distributed nuclear chromatin, evenly intermixed with numerous giant cells that contain a variable number of nuclei with features similar to those of mononuclear cells. cytoplasm of either cell type was eosinophilic and cell borders were indistinct. mitoses were scarce and regular. foamy histiocytes, hemosiderin and microscopic nodules of cartilage formation were also noted. conclusion: although giant cell tumor is a benign neoplasm its' localization in this case posses a threat that should be managed surgically and by adjuvant radiotherapy if complete excision is unobtainable. leptin and leptin receptor expression in pituitary adenomas g. ayranci * , t. avsar, a. seker, t. kilic, s. bozkurt * marmara university, dept. of pathology, istanbul, turkey objective: leptin is a regulatory hormone which is mainly synthesized by adipocytes and regulates body fat mass. leptin also has regulatory function on anterior pituitary. in this study, we have investigated the expression levels of leptin (ob) and its receptor (ob-r) in various types of pituitary adenomas and normal anterior pituitaries. method: pituitary adenoma cases between and were selected. immunohistochemistry for leptin (ob) was performed for each cases of null cell, gh, acth, prolactin and fsh/lh secreting adenomas. western blot was performed for leptin receptor (ob-r) in cases consisting of all five subtypes. results: immunohistochemical staining showed greater immunoreactivity for leptin in normal pituitaries compared to adenomas. except for acth-secreting pituitary adenomas, all other four subtypes showed widespread and intense staining for leptin (p< . ). null and fsh/lh-secreting subtypes were mainly showed cytoplasmic and nuclear staining. western blot analysis showed leptin receptor expression in all types of adenomas except null cell. objective: primary malignant brain tumors often present local invasive growth. the study aimed to estimate the expression of kai protein, mmp- , cd v and their correlation in gliomas of different grade of malignancy. method: expression of mmp- , cd v and kai was evaluated on formalin fixed paraffin-embedded tissue blocks divided into: pilocytic astrocytoma (n= ), fibrillary astrocytoma (n= ), anaplastic astrocytomas (n= ), anaplastic oligodendrogliomas (n= ), glioblastoma multiforme (n= ) and normal brain tissue (n= ) using immunohistrochemistry. results: mmp- and cd v was observed frequently in gliomas with high grade of malignancy. kai immunoreactivity was mainly observed in specimens with low degree of malignancy. mmp- and cd v expression was significantly increased when the degree of malignancy of the gliomas increased (p< . ). whilst kai expression increased when the degree of gliomas malignancy decreased (p = . ). positive correlations between mmp- and cd v (p= . ) and inverse between kai and mmp- expression (p= . ) were found in gliomas. these results indicate that association between mmp- and cd v expression may increase dissemination of tumor cells. whereas, high expression of kai protein might suppress the function of mmp- in gliomas. pediatric glioblastoma: a case report a. barin * , s. ekici, c. altunkaya, s. yilmaz, g. s. yalcin, g. hatipoglu, m. caydere, h. ustun * ankara education hospital, dept. of pathology, turkey objective: glioblastoma is the most common malignant tumor of central nervous system in adults which characterized by malignant pleomorphic astrocytic cells with marked nuclear atypia, mitotic activity, necrosis and/or microvascular proliferation. glioblastoma may manifest at any age but usually affects adults especially older than years. in this case, we present a pediatric glioblastoma case because it has seen rarely under years and it is so important owing to its prognosis. method: an -year-old boy has had headache for days. according to radiological imagings × mm in diameter infiltrating mass has seen in temporal lobe contains × mm in diameter cystic component and contrast enhancement was noted. clinically grade astrocytoma has thought primarily and he has been operated. results: in microscopic examination nuclear atypia, mitotic activity and microvascular proliferation has seen without necrosis. objective: gliosarcomas are biphasic neoplasms composed of a glioblastoma admixed to a sarcomatous component with different lines of differentiation. histogenesis of these tumors is still discussed. our objective is to specify pathological characteristics of this neoplasm its related to or not to glioblastoma. method: cases of gliosarcomas diagnosed in our department of pathology. clinical, radiological, therapeutic and follow-up data were reviewed. histological features and immunohistochemical results were also established. results: female patients with a median age of years (ranging - years). intracranial hyperpressure and paralysis are main symptoms. the brain imaging showed frontal, fronto-parietal and intra ventricular expansive process, respectively. microscopically, a biphasic pattern, with anaplastic astrocytic cells alternated with malignant mesenchymal areas, demonstrating a fibrosarcoma pattern, in two cases and rhabdoïd pattern in one case. immunohistochemical stain confirmed the diagnosis of gliosarcoma in each case. patients were treated by surgical excision; one patient was lost on follow-up. one patient died by postoperative complications. conclusion: clinical, radiological and follow-up features of gliosarcomas share great similarities with glioblastomas. histopathological, histochemical and immunohistochemical studies are helpful in accurate diagnosis. cytogenetic and molecular data support a monoclonal origin for these tumors. objective: case of biphasic synovial sarcoma of nerve in a year-old female located between flexor muscles and radial diaphysis and adherent to median nerve and ulnar artery is presented. method: tissue was fixed in formalin, embedded in paraffin and stained with haematoxylin and eosin. periodic acid-schiff stain without diastase pre-digestion was obtained. ventana antibodies were employed: ttf- , er, ema, s , ck , ck and mnf . ss -ssx fusion gene transcript was detected with conventional rt-pcr and qrt-pcr. results: macroscopically nodule had smooth circumscribed borders, grey-yellowish colour and measured , × × , cm. histologically it was delimited by a dense sclero-hyaline capsule and constituted of numerous glands lined by one layer of cuboidal-columnar cells showing eosinophilic cytoplasm and round to ovoid nuclei with a single small nucleolus. they contained dense eosinophilic strongly pas and ema positive material. glands were immersed within tightly packed spindle cells. mitoses were scanty. necrosis was absent. ema strongly stained most of the glandular as well as scanty spindle cell elements. chromosomal reciprocal translocation t (x; ) with positive signal for ss -ssx transcript was seen. conclusion: synovial sarcoma of nerve is a rare condition that has to be distinguished from histologically similar lesions. rabdoid meningioma: presentation of a rare case i. dimitriadis * , a. heva, i. matzarakis, a. sekouli, p. sakellariou, l. sakkas * hospital of thessaloniki, dept. of pathology, greece objective: in , keppes and later perry suggested the term rhabdoid meningioma which was adopted into the who classification (grade iii) in . we present a rare case of a rabdoid meningioma and study the clinicopathological characteristics of the neoplasm. results: Α -year-old woman was submitted in surgical excision of an endocranial tumour (d: cm) in our hospital. the histological examination revealed entirely rabdoid morphology. the neoplastic cells expressed the following immunohistochemical phenotype: s +, vimentin+, ema+, gfap−, pr+, hmb −, a /ae +(focally), ck / +(focally), sma−, ki- (< )%. conclusion: rabdoid phenotype represents an indicator of malignant transformation regardless of the tumor's histogenesis. expression of ki- antigen correlates with recurrence even in meningiomas with minimal atypical features, which should prompt closer surveillance after excision. rabdoid meningiomas behave aggressively and have bad prognosis contrary to the classic meningioma. prognosis is influenced by it's complete excision and the expansion within the intracranial cavity. it has to be distinguished from glioma, melanoma and metastatic carcinoma. the patient after year is in generally good health, and is followed up by oncologists. atypical extraventricular neurocytoma (case report) s. ersöz * , u. yazar, a. reis, k. kuzeyli * karadeniz technical university, pathology, trabzon, turkey objective: central neurocytomas are rare intraventricular tumors with neuronal differentiation, typically located in the ventricles while tumors located outside the ventricles designated as ''extraventricular neurocytomas'' (evn). neurocytomas are classified as atypical if they exhibit a mib- labeling index > % or atypical features, like focal necrosis, vascular proliferation, and increased mitotic activity. we here report a case of atypical evn of the left frontal lobe. method: a -year-old woman patient admitted to hospital for headache. ct scan showed a large partially calcified cystic mass. mr imaging showed circumscribed, solid mural nodule within the cystic mass. the patient underwent a left frontal craniotomy. specimens were fixed in % formalin and stained with hematoxylin and eosin. for immunohistochemistry, sections were incubated with monoclonal antibodies to synaptophysin, neurofilament protein, map- , nse and gfap. the proliferative index was assessed with mib- . results: histopathological examination revealed a neuronal neoplasm composed of uniform cells in solid sheets. small necrosis areas and mitotic figures were evident. immunohistochemical examination revealed positive results for synaptophysin, neurofilament protein, map- , and nse. gfap staining was negative. mib- index was calculated as %. conclusion: in general, central and extraventricular neurocytomas have a good prognosis. atypical extraventricular neurocytomas are quite rare like this case with malignant histopathological properties. meningioma and schwannoma associated with neurofibromatosis type : a case report of rare genetic disorder o. e. gürer * , r. tuncer * akdeniz university, dept. of pathology, antalya, turkey objective: neurofibromatosis is a group of genetic disorders referred as phakomatoses. there are two main forms called nf and nf which have different incidence, molecular characteristics, and clinical exhibitio. both have higher risk for the development of certain rare malignant tumors that occur in the brain, nerves or spinal cord. method: here, we present a rare case in which multiple meningiomas and vestibular schwannomas diagnosed in the same operation. results: a year old woman presented with hearing loss clinically. she underwent surgical treatment for tumoral lesions in two different localization in the same session. one of them resected from pontoserebellar location was diagnosed as a schwannoma and the other one located on dura was diagnosed as a meningioma respectively. conclusion: nf is far less common than nf and strict diagnostic criteria have been described each of them. since difficulty to detect genetic abnormalities and no single criteria is pathognomonic, clinical characteristics much more valuable for differentiation. knowledge of associated clinical features will help to correct diagnosis and to predict much more devastating clinical course for nf . diagnostic confusion: central neurocytoma n. gursan * , e. demirci, m. calik, f. daloglu, c. gundogdu * ataturk university, medical faculty, erzurum, turkey objective: central neurocytoma is a rare intraventricular brain tumor that affects young adults and presents with increased intracranial pressure secondary to obstructive hydrocephalus. they are usually located in the supratentorial periventricular region. because of some clinical and radiological findings cns neurocytomas were confused with other intraventricular lesions. method: a -year-old male was admitted with chronic headache with months duration on july , , when there were no neurological deficits or physical abnormalities. computed tomographic (ct) scans showed × mm a mixed density mass with amorphous calcification in the right lateral ventricle, which was irregularly enhanced by contrast medium. light microscopy showed sheets of monotonously neoplastic cells with uniform round-to-oval nuclei and inconspicuous nucleoli. the cytoplasm was clear or eosinophilic with indistinct border. capillary networks were well developed and divided the tumor cells into groups. no nuclear pleomorphism or mitosis was seen. we present a case of intraventricular neurocytomas confirmed by immunohistochemical studies. conclusion: this rare tumor causing diagnostic confusion, discussed by the literature. a case of oligodendroglioma with neuronal differentiation t. hirose * , s. nobusawa, y. nakazato, a. sasaki * tokushima pref. center hospital, dept. of diagnostic pathology, japan objective: we report a case of oligodendroglioma showing marked neuronal differentiation. method: a -year-old female visited an emergency room because of an attack of convulsion. imaging analyses disclosed a calcified, . × cm tumor in the right frontal lobe. results: the resected tumor was composed of a mixture of oligodendroglioma-like (olg) and gangliocytoma-like (gc) areas. in the former, fried-egg appearance and more cellular nodules were recognized. in the latter, there were numerous small neuron-like cells with basophilic cytoplasm. olg areas showed immunoreactivity for olig , gfap and mutant idh , while gc areas were positive for synaptophysin and neun. ki- labeling index was about % in cellular nodules of olg areas. at least one allelic loss of p/ q was detected in . / . % cells in olg areas and . / . % in gc areas with fish analyses. furthermore, an identical mutation of idh (g a, r h) was demonstrated in both olg and gc areas. conclusion: genetic abnormalities including p/ q loss and idh mutation indicate that this case is an oligodendroglioma associated with prominent ganglion cell differentiation. oligodendrogliomas with neuronal differentiation like our case may suggest a close relationship between oligodendroglial progenitor cells and neuronal cells. gemistocytic astrocytoma with granulomatous inflammation o. ipci * , e. atik dogan, t. ozgur, h. gokce, n. yilmaz, a. yilmaz * mustafa kemal university, dept. of pathology, hatay, turkey objective: the coexistence of granulomatous inflammation and astrocytoma is extremely rare. hemostatic agents are used to control intraoperative bleeding in many surgical subspecialties, including neurosurgery. there are occasional reports of granulomatous reaction to biomaterials. method: we aimed to present a year old female patient, who was operated because of the tumor at left frontoparietal region. tumor was histopathologically reported as oligoastocytoma. however, no granulomatous inflammation was detected beside the tumor. after months, patient was reoperated because of tumor recurrence, abscess and inflammatory necrotic tissue beside the tumor. histopathologically, gemistocytic astrocytoma with granulomatous inflammation and abscess formation was reported. no micro-organism had grown in any kind of cultures. at our neurosurgery department surgicel is used after craniotomy as hemostatic agent. it's raw material is cellulose and absorbed in about months. results: in the literature, germinoma accompanied by granulomatous inflammation have been reported. however, astrocytic tumors don't express such an association. the patient doesn't use any drugs forming granuloma reported in the literature. conclusion: we encountered that surgicel causing granulomatous inflammation in very few cases in the literature. the findings might be secondary to surgicel that used in the first operation. objective: astrocytic tumors show various molecular and genetic alterations in its development and progression. glioblastoma multiforme (gm) develops as either de novo or by progression from diffuse astrocytomas (da) or anaplastic astrocytomas (aa) by genetic alterations. method: loss of heterozygosity (loh) and microsatellite instability (msi) was investigated in cases of astrocytic tumors (das [n= ], aas [n= ] and gms [n= ]) with microsatellite markers and microsatellite markers harboring p and pten, respectively. the patients' age ranged from to years (mean, years). results: loh was statistically significant in aas and gms, compared to that of das (p= . ). loh for p and loh-h group for q were statistically significant in aas and gms (p= . and p= . ). msi rate in das, aas and gms was detected in . %, . %, . %, but it showed no significant correlation with prognosis factors. msi and msi-h rates were correlated with younger (〈 years) group (p= . , p= . , respectively). conclusion: alterations on pten and p may contribute to the development and progression of astrocytic tumors. especially, loh-h for q and p is considered as clinical application of discriminating the aas and gms from das. msi might be involved in the tumorigenesis of relatively young patients. objective: charcot-marie-tooth disease (cmt) iii or dejerine-sottas syndrome is a severe hereditary demyelinating motor and sensory neuropathy presenting in infancy or early childhood with delayed motor milestone and extremely slow nerve conduction velocities. method: among unrelated korean cmt patients, mutational screen for well known cmt genes reveals three patients with pmp de novo mutation showing dss. the histopathological findings of distal sural nerves are analyzed in all patients twice; cases and with years interval and case with years. results: on semi-thin transverse sections, all the remaining axons (thinly myelinated or demyelinated) are surrounded by classic or basal lamina onion bulbs (obs). the first biopsy of case ( years-old) shows obs ( , /mm ), which consists of , ( . %) mfs with obs and , ( . %) demyelinated axons with obs. the second biopsy shows obs ( , /mm ), consisting of ( . %) mfs with obs, ( . %) demyelinated axons with obs, and ( . %) obs with no discernible axons. cases and show similar histopathologic findings with main differences in numbers of obs. conclusion: although the differences noted in two biopsies are probably due to age of the patients, correlations with clinical symptoms will be helpul for management of the patients. objective: two mutations of the seipin gene (n s and s l) have been reported and known to cause neurodegenerative disorders. we performed whole exome sequencing in two charcot-marie-tooth families and identified three patients with two causative heterozygous mutations (n s and s w) and s w is a novel mutation. method: the histopathological findings of distal sural nerves are analyzed in two patients (cases and ) with s w novel mutation (fc ) and a patient (case ) with n s mutation (fc ). results: transverse semi-thin sections reveal increased numbers of myelinated fibers (mfs) ( , /mm ; , / mm ; , /mm ) with increased small mfs forming regenerative axon clusters and loss of large mfs. histogram shows unimodal distribution pattern. cases and show the similar ranges and average of diameter of mfs ( . . μm, . μm). case shows more numbers of small mfs ( . ~ . μm, . μm). ultra-structural examination reveals mfs with myelin abnormalities, pseudo-onion bulb formation including single axon or axon clusters, and thick mfs. conclusion: the findings are consistent with axonal neuropathy with features of demyelination. additional cases will be necessary to know the significance of differences between these cases. immunohistochemical subtyping of primary and secondary glioblastomas k.-s. lee * * seoul national university hospital, republic of korea objective: glioblastomas (gbls) may develop de novo (primary gbl; p-gbl) or through progression from lowgrade or anaplastic astrocytomas (secondary gbl; s-gbl). for subtyping of gbls, we tried immunohistochemical anaysis. method: we collected cases of gbls in snubh and snuh. the mean age at the time of the primary surgery was . years (range - ). immunohistochemical studies were performed for egfr, p and idh- . results: according to clinical history, gbls consisted of primary and four secondary gbls. however, egfr(+)/ p (−) immunohistochemical features of p-gbl consisted . % and egfr(−)/p (+) immunohistochemical features of s-gbl consisted . % among cases. egfr(+)/p (+) was noted in . % and egfr(−)/p (−) in . %. immunohistochemical expression of idh- was noted in cases ( . %) out of cases. expression of idh- and egfr(−)/p (+) showed positive correlations with young age. characteristic features of s-gbl were noted in . % of clinically p-gbl. conclusion: we analyzed immunohistochemical subtypes of korean glioblastomas. combination of egfr and p was unsatisfactory but combination of egfr, p and idh- can be a good tool for immunohistochemical subtyping of glioblastomas. (also known as erbb- ) is a -kd transmembrane glycoprotein with tyrosine kinase activity. her is expressed in some human malignancies and can be a potential target for therapeutic intervention with selective inhibitors. there are only a few studies on the relationship between meningioma and her expression, and the results are different as well. the aim of this study was to determine this relationship. method: seventy-two paraffin blocks of meningioma were selected randomly and immunohistochemical staining was then performed for each specimen. results: thirty-one of the meningiomas were her -positive. her expression was observed in ( %) of the grade ii/iii, and ( . %) of the grade i meningiomas. conclusion: consequently, her expression was detected in % of meningiomas. no significant difference was seen between grade i and ii/iii meningiomas, primary and recurrent tumors, and males and females from the point of view of her expression. objective: in our previous papers we studied accumulation of iron and proteoglycans in the brain. the aim of this analysis is to build on our previous results alongside other authors to investigate the reason for the presence of excessively accumulated iron observed in parkinson disease (pd). method: postmortem samples taken from the globus pallidus were prepared for light microscopy for iron and proteoglycans detection. patients had neither iron metabolism disorders nor had clinical signs of neurodegeneration. the samples were analysed using electron paramagnetic resonance (epr) to investigate the bounds between iron and proteoglycans. results: epr measurements confirm the presence of complex bounds between fe(iii) and proteoglycans. conclusion: previous data showed a possible dependance between accumulated iron, proteoglycans, ferrireductive alpha-synuclein and lewy bodies. we propose a mechanism of toxic iron accumulation in the brain in pd. this cyclic process of amplification is presented by onward steps. it is completed by our own observations into one cycle. the initiator of this vicious circle is probably an impirement of the equilibrium in the presence of useful amount of iron. understanding these relations could bring new insights to the neurodegenerative disease. role of pdgfr overexpression in schwannoma, and its diagnostic and therapeutic implications t. neuman * , y. fellig * hadassah medical center, dept. of pathology, jerusalem, israel objective: platelet-derived-growth-factor-receptors (pdgfr) are transmembrane tyrosine kinase receptors that function as relay points for signaling pathways. they play a key role in numerous processes that affect cell proliferation, tumor genesis, cancer invasion, metastasis, and modulation of apoptosis. recently, pdgfr has been demonstrated to be overexpressed in schwannoma in vivo, and in a small series of acoustic neuromas. however, its phenotypic expression in different variants of schwannoma remains largely unclear. method: immunohistochemical staining was used to detect pdgfr expression in archived formalin-fixed, paraffin-embedded schwannoma tissue samples (n= objective: pancreatic ductal carcinoma (pdc) has one of the worst prognoses. up to % of pancreatic cancers are diagnosed at the locally advanced or metastatic stage. however, brain metastases from pancreatic carcinoma are extremely rare since less than cases have been reported in the literature since . method: we report a case of a brain metastasis from pancreatic adenocarcinoma occurred in a years old women. results: -year-old woman developed severe neurological symptoms and progressively decreased consciousness. ct revealed cystic tumor in cerebral peduncle and medulla oblongata. stereotactic biopsy revealed metastasis of carcinoma. the patient died from of the twelfth day after stb because of recurrent hemorrhage. autopsy revealed well-differentiated papillary adenocarcinoma in the head of pancreas and with multiple metastases in liver and two metastases in medulla oblongata and cerebral peduncle. ihc staining for cytokeratins , and , mucin and ac types was strongly positive. ki labeling index was %. conclusion: we reported an autopsy case of brain metastases from pancreatic cancer, in which the patient initially developed symptoms of a neurologic disorder without exhibiting any symptoms of pancreatic disease. the use of bone morphogenetic proteins (bmp ) with nunostrucnure grey implant in reparation of bone tissue defect t. pavlova * , l. pavlova, a. nesterov, i. goncharov, d. kolesnicov * belgorod state university, dept. of pathology, russia objective: the matters of materials development of scull bones defect plastic are obviously necessary this time. the experience in use of nanocostructive titans, composites, bone morphogenetic proteins was accumulated. method: implant, made of nanoconstructed titan grey, covered by layer (gelatin, dextran), layers ( -gelatin, dextran, -hydroxyapatite, collagen, dextran) and by layers with use of bone morphogenetic proteins bmp- . the experiment was conducted on rat-males. in order to study the regeneration processes it were used light, fluorescence, probe and scanning microscopy with the trace element analysis. results: the most active changes were registered in the experimental group with use of composite from titan grey with layers of covering and bmp- . the covering compound increase the rate of regeneration processes due to creation calcium and phosphorus ion depot and perform support function for again-formed tissue. increase in the concentration of calcium, phosphorus, sodium, magnesium in trabeculae of bones in comparison with matrix bone at term of weeks testify about active regenerative processes; the mature smooth surface with structured relief of bone trabeculae and diploic veins were detected. conclusion: results can be used in neurosurgery, traumatology-orthopedics, dentistry, plastic and cosmetic surgery. alpha-synuclein (as) pathology of the peripheral autonomic nervous system (pans) in parkinson disease (pd) and other lewy body diseases (lbd) t. ribalta * , e. tolosa, j. navarro otano, e. gelpi * hospital clinic of barcelona, dept. of anatomic pathology, spain objective: pd and other lbd have been associated with as aggregates in the central nervous system (cns). however, autonomic dysfunctions may appear at any time in the course of the disease. our objective was to investigate the distribution of as in the pans in synucleinopathies. method: we examined pans structures obtained at post mortem from subjects ( female, age range - , mean ) at our brain bank with a diagnosis of pd ( ); lewy body dementia ( ); alzheimer's disease ( ); mixed dementia or other ( ). a complete neuropathological study of pans was performed, including dorsal spinal ganglia, vagus nerve, paraspinal sympathetic ganglia, mesentery, adrenals, digestive and genitourinary systems, heart, and skin. routine and immune stains for as and tyrosine-hydroxylase were applied. cases were semiquantitatively assessed. results: % of cases showed as pathology in the cns. of them, as aggregates were additionally present in the pans in %. a gradient in involvement was observed, being the paraspinal chain (particularly the stellate ganglion) the most constantly involved structure, followed by digestive system, adrenals, and gu system. conclusion: these findings indicate that as aggregates are extensively found in pans in synucleinopathies. the highest expression is found in the paraspinal ganglion chain and decreases in other pans regions. our results provide valuable information about potential development of new diagnostic and therapeutic strategies. objective: chordoid meningioma is an uncommon histopathological variant of meningioma. method: we report one case of chordoid meningioma occurring in adult patient. paraffin embedded tissue was stained with hematoxylin-eosin. we used immunohistochemical markers such as cea, ema, vimentin and tle . results: a -year-old woman was hospitalized with clinic of an intracranial hemorrhage. axial computed tomography (ct) of the brain revealed a . * . * . cm lesion with perilesional edema in the left temporoparietal region. foci of intratumoral hemorrhage were also seen. compression of the left lateral ventricle and midline shift to right side was seen. the patient underwent a left temporoparietal craniotomy for resection of the tumor and died days after surgery. histologically, sections revealed sheets, trabeculae and lobules of tumour cells scattered in a pale basophilic myxoid matrix. some of these cells exhibited characteristic cytoplasmic vacuolization. a typical focal meningiomatous pattern was also observed. the tumor cells were diffusely positive for epithelial membrane antigen, vimentin and a strong nuclear immunoreactivity for tle . these cells showed negative immunostaining for cea. conclusion: chordoid meningioma should be distinguished from chordoma, chondrosarcoma, metastatic mucinous carcinoma and other variants of meningioma. morphology of non-specified encephalopathy in cases of polymerase chain reaction proved presence of human herpesvirus- s. roga * , i. strumfa, s. kuleznova, s. chapenko, s. rasa, m. murovska * riga stradins university, teaching department, latvia objective: the morphology of non-specified encephalopathy is a complex medical problem. human herpesvirus- (hhv- ) infection can be discussed as a predisposing factor. the aim of the present study was to investigate the presence of beta hhv- in non-specified adult encephalopathy cases. method: the blood, meninges and brain tissue were obtained in adult (aged - years) autopsies including cases with encephalopathy and cases in the control group. tissues were submitted for routine histology including haematoxylin-eosin stain. the presence of hhv- genome (dna) was analysed by nested polymerase chain reaction (npcr), hhv- variants by restriction endonuclease analysis. results: the gross and microscopic structure did not reveal any specific changes. in the encephalopathy group, hhv- dna sequence was found in meningeal tissues ( / cases; p= . ), in brain tissues ( / cases; p = . ), and both in brain and meningeal tissues ( / cases; p= . ). in the control group, the viral dna was identified in meningeal tissues ( / cases), in brain tissues ( / cases), both in brain and meningeal tissues ( / cases). hhv- b variant was detected in all cases. conclusion: on the basis of the present study it can be concluded that hhv- is a pathogenic factor that can predispose to encephalopathy. pathological variants of brain metastases of breast carcinoma and their prognostic and predictive role in different age groups d. objective: it is extremely rare that the intramedullary spinal cord metastases (iscm) are the first manifestation of cancer. method: in database of neurosurgery biopsies from to two cases of iscm were found without preoperative signs of primary neoplasm. surgically removed tissue was stained with hematoxylin-eosin and immunohistochemically using following markers: ae / ae , ck , ck , mammaglobin, ttf- , gfap, s- and vimentin. results: one patient was male aged and another female aged . they presented with neck pain and rapidly progressive upper and lower limb weakness. imaging analysis showed contrast-enhancing mass at the level c -c in male and c -c in female, reported as an ependymoma. grossly, tumor tissue was of soft consistency and grey white color with yellowish foci of necrosis. histopathology revealed moderately differentiated adenocarcinoma with distinct border to surrounded glial tissue. in both cases, tumor cells were immunopositive for ae /ae , ck and ttf- indicating primary lung origin. postoperatively primary cancer was found in upper lobe of the left lung in both cases, without evidence of lymphadenopathy and other distant metastatic lesions in female. conclusion: diagnosis of iscm in both cases without preoperative signs of neoplasm in other organs was surprising. immunohistochemical analysis was essential for determination of cancer origin. objective: tetraspanin cd is a positive effector of cancer invasion and metastasis in many tumor types. method: we investigated the protein expression of cd in cases of who grade i to iv gliomas. additionally, we performed o -methylguanin-dna methyltransferase (mgmt) methylation analysis using real-time methylation-specific pcr in glioblastomas, and the prognostic significance of these biomarkers in glioblastomas was evaluated. results: cd was overexpressed in a significant proportion ( . %) of glioblastomas, while it was not deetected in most of grade i to iii glial tumors except for rare overexpression in anaplastic astrocytoma ( / , %) and oligoastrocytomas ( / , %). cd overexpression was closely associated with mgmt methylation (p= . ), and it was a prognostic factor for predicting worse overall survival (os; p= . ) and progressionfree survival (pfs; p= . ). combination of cd overexpression and mgmt methylation better stratified the patients' os (p= . ) and pfs (p= . ). in multivariate analysis, cd overexpression was an independent prognostic factor for predicting os over mgmt methylation (p= . ). conclusion: cd seems to have a critical role for high grade progression in astroglial tumors. cd is a good tissue marker for predicting worse prognosis in glioblastomas. results: a gross total resection of the tumor was achieved. histological examination revealed a paucicellular tumor with lobulated architecture and abundant myxoid stroma, containing stellate or spindle cells lacking mitotic activity. alcian blue and mucicarmin histochemical stains were diffusely and strongly positive. immunohistochemical analysis showed diffuse reactivity for vimentin and scattered cells positive for cd . stains for ema, gfap, s protein, cytokeratin and smoothmuscle actin were negative. conclusion: primary intracranial myxoma should be distinguished from other myxoid intracranial tumors such as myxomatous meningioma, epithelioid hemangioendothelioma or sarcoma through appropriate pathological and inmunohistochemical analysis. a metastasic cardiac myxoma should also be ruled out through cardiac evaluation including an echocardiography. the distinction between this entity and the reported neurothekeoma of the meninges needs to be reevaluated. holocord pylocitic astrocytoma associated to syringomyelia j. trillo-tinoco * , t. garibay-huarte, e. gómez-apo, m. a. rodríguez-florido, l. chávez-macias, j. e. olvera-rabiela * hospital general de mexico, dept. of surgical pathology, mexico city, mexico objective: spinal intramedullary tumors sometimes extend both superiorly and inferiorly along almost the entire cord, and those diagnosed as pilocytic astrocytoma are rare. the syringomyelia is an ependimary or periependimary cavitation of the spinal cord and is considered a suffering of degenerative and irreversible type, % of the cases is associated with intramedullars tumors. method: a three-year-old girl with history of headache, progressive weakness, months before, she was hospitalized for pneumonia, had impairment of neurologic functions and died of cardiac arrest. the autopsy was performed. results: the neuropathologic study revealed a intramedullary holocord tumor with secondary syringobulbia, cervical and lumbar syringomyelia. histopathological examination of all specimen resulted in diagnosis of a pilocytic astrocytoma. although no signs of atypia were present, an elevated proliferative activity of endothelial vessels was noted. conclusion: gross total resection of holocord and longitudinally extensive intramedullary spinal cord tumors can be achieved with preservation of long-term neurological function and also solved the syrinx. objective: epithelial cellular adhesion molecule (ep-cam) has been studied in many tissues and neoplasm, including thyroid and thyroid tumors. this is a preliminary study to assess the immunohistochemical expression of ep-cam in thyroid tumors using ber-ep . we examined the expression of ep-cam using the monoclonal antibody ber-ep in cases of thyroid tumors, including adenomas, papillary carcinomas, follicular carcinomas, medullary carcinomas, poorly differentiated carcinomas, and anaplastic carcinomas. we assessed the positivity as a predominantly membranous staining of the cells, and was scored according to the estimated percentage tumor cells in the total tissue section (negative: - %; positive: ≥ %). results: ber-ep expression was detected in normal thyroid tissue (perilessional), in all the adenomas, follicular carcinomas, and medullary carcinomas. papillary carcinomas showed in % lacking areas of expression, coinciding histopathologically one case with poorly differentiated component. all of the poorly differentiated and the anaplastic carcinomas were negative. conclusion: the expression of ep-cam, using ber-ep , was related to normal thyroid tissue and well differentiated neoplasms. our study suggest that lost of expression is associated to dedifferentiation. this results match up with the literature. in addition, clinical data and follow up are required to correlate focal areas of lost of expression of ber-ep with dedifferentiated areas of the tumor. the pathological effects of estradiol valerate on testis tissue: size and weight in male rat f. bidhendi * , r. ahmadi * imamkhomeini hospital, dept. of radiology, tehran, iran objective: studies show that estrogens can influence reproductive system differentiation. the main aim of this study was to determine pathological effects of estradiol valerate on testis histology and morphology in male rats. method: adult male albino wistar rats were divided to control and estradiol valerate ( μg/kg/day) receiving groups. estradiol valerate was applied subcutaneously. after weeks, testes were excised and studied morphologically and histologically. data were statistically analyzed and compared between groups using anova. results: our findings revealed that estradiol valerate injection resulted in reducing of testes weight and size (p< . ). semeniferous tubules were apparently deformed in estradiol valerate receiving animals and cellular density was also reduced. number of spermatocytes, spermatids and sperms was decreased in estradiol valerate receving rats compared with control animals (p< . ). conclusion: estradiol valerate has considerable pathological effects on testes morphology and histology in male rats. key words: estradiol, testes. are tumor-dimension, galectin- and cyclind useful to characterize oncocytic adenomas and carcinomas?: mutations in about % of cases. the first clinical manifestation of this tumour is often represented by lymphatic metastases. method: twelve cases of ptc and respective lymph node metastases were retrieved from our archive from to present. formalin fixed paraffin embedded tissue sections were stained with routine hematoxilin-eosin and representative tissue areas for both tumour and metastases were microdissected and dna was extracted. after pcr amplification the mutational status of braf and ras was determined by dna sequencing. results: seven cases of twelve ( %) showed a braf-v e mutation. interestingly, only in three these cases ( %) there was concordance in the mutational status between primary tumour and metastases. moreover, all metastase of wild type carcinomas were also wyld type. conclusion: even with an overall good prognosis, ptc is characterized by high incidence of lymph node metastases. as braf mutational status correlates well with prognosis and tumour progression, a correct molecular assessment is of paramount importance. our data may indicate that, because the possibility of discrepancy in the mutational status between primary tumour and metastases, molecular analysis should be performed on the primary tumour. objective: diabetes may cause chronic and non-healing diabetic foot ulcers (dfu) decreasing the welfare of patients. some neuropeptides, substance p and neurotensin (nt), may act as inflammatory modulators and improve wound healing. natural biopolymers, chitosan derivatives, are receiving great attention as powerful wound dressing materials for wound healing applications due to their favorable properties. the work aim was to use -methyl pyrrolidinone chitosan (mpc) as a platform for the delivery of nt. method: diabetes was induced by intraperitoneal injection of mg/kg streptozotocin. mice were anesthetized and two mm excision wounds were created dorsally. mpc alone, nt alone, mpc loaded with nt or pbs were placed daily on wounds. histological analysis of skin, at days , and , was done through h&e and masson´s trichrome stainnings. results: in diabetic mice, the healing process was slower, showing an engulfment of acute inflammatory cells that triggered macrophage activity when compared with controls. at day , mpc treatment leaded to a faster healing with retraction of the wound site, nt induced a slower noncontracting healing and combined application delayed inflammatory repair with persistent neutrophils. conclusion: at day , all treatments induced a total healing however, mpc + nt reduced neutrophils infiltration compared with mpc alone. acth producing nasal paraganglioma: case report j. cassis * , a. galzerano, m. chorão * hospital egas moniz, dept. of anatomy pathology, lisboa, portugal objective: female patient with chronic sinusitis and hypertension presents with lipothymia, dizziness, polyuria and polydipsia. laboratory studies showed hyperglicemia, leucocytosis, hypokalemia, hypercortisoluria and elevated serum acth levels. head mri revealed tumor in the right nasal cavity with implantation at the cribiform plate of the ethmoid bone. method: microscopically the tumor had lobular architecture with big, round cells and with eosinophilic granular cytoplasm. these cells were nse+, synaptophysin+, cd +, cd + and acth+. surrounding the tumor lobules were several spindle cells s- + (sustentacular cells). results: the lobular growth pattern, the presence of sustentacular cells (s- positive) , and the acth producing granules favours an acth producing nasal paraganglioma diagnosis. conclusion: head and neck paragangliomas account for , % of the tumors in this region, and are mainly located in the carotid bodies. paragangliomas arising in the nasal cavity are rare, only cases reported so far. they affect the middle or upper turbinates, and also the etmoid, maxillary and sphenoidal sinuses. we report the third case in literature of a acth producing paraganglioma in the nasal cavity. sporadic aggressive silent somatotroph pituitary adenoma in the young: report of two cases l. chinezu * , a. vasiljevic, s. achard peyregne, g. raverot, e. jouanneau, j. trouillas * emergency county hospital, pathology, tg. mures, romania objective: silent pituitary adenomas are infrequent tumors in adults and exceptional in young. we describe two cases of -year-old girls with pituitary adenoma without genetic history, revealed by visual field defects, with panhypopituitarism in one patient. in both cases, mri showed giant pituitary adenoma, with invasion of the cavernous and sphenoidal sinuses. hormone assays revealed normal gh, prl and igf plasma values. surgery was incomplete in both cases. results: both tumors presented a diffuse pattern. the cells exhibited large nuclei with prominent nucleoli. by immunocytochemistry, focal and low percentages of gh-and prl-immunoreactive cells were observed respectively in both tumors (gh= - % and prl= - %). cga was positive. cytokeratin and others antibodies against pituitary hormones were negative. both tumors had high proliferative indexes: ki- > % ( - %) and elevated mitotic number ( - mitoses). detection of p was also positive ( . - . %). the cases were diagnosed as atypical adenomas, according to the who classification. conclusion: the pathological diagnosis of these aggressive gh-prl tumors has to be taken into account by the clinician to choose the optimal therapeutic strategies. despite of the important side effects of the radiotherapy, this aggressive treatment might be proposed earlier, even for the young patients, to avoid the tumoral progression. intrauterine growth retardation with high fat diet in rats markedly disturbs islet morphology characterized by peri-islet inflammation, fibrosis and haemosiderosis j. dahlstrom * , v. delghingaro-augusto, l. madad, a. chandra, e. bean, c. simeonovic, c. j. nolan * act pathology & anums, dept. of anatomical pathology, garran, australia objective: pre-and post-natal factors such as intrauterine growth retardation (iugr) and high fat (hf) diet contribute to type diabetes (t d). our aim was to determine if iugr and hf diets interact in t d pathogenesis. method: a surgical model of iugr (bilateral uterine artery ligation) in sprague-dawley rats with sham (sh) controls was used pups were fed either hf or chow (ch) diets from weaning. serial measures of body weight and glucose tolerance were made. at weeks, rat pancreases were harvested for histological assessment. results: iugr vs sh pups weights were % lower. hf diet caused excess weight gain, dyslipidaemia, hyperinsulinaemia and mild glucose intolerance not further aggravated by iugr. markedly abnormal islet morphology was evident in / sh-ch, / sh-hf, / iugr-ch and / iugr-hf rats (chi-sq, p= . ). abnormal islets were characterised by larger size, irregular shape, peri-islet inflammation with cd positive cells and marked haemosiderosis. overall beta-cell mass was not altered by iugr, with a trend for it to be mildly increased in both hf-fed groups. conclusion: hf and iugr independently and together contribute to islet injury. the marked islet haemosiderosis associated with iugr and hf diets warrants further investigation as iron is toxic to β-cells. objective: adreno-hepatic fusion (ahf) is defined as adhesion of the liver and right adrenal gland with or without a fibrous capsule dividing both organs. we report a surgical case of ahf in which a virilising malignant adrenocortical tumour protruded into the liver mimicking a hepatic mass. method: a -years-old woman presented with mid-right abdominal pain and marked hirsutism. computed tomography (ct) revealed an apparently intrahepatic solid tumour suggestive of a giant hepatic adenoma. the right adrenal gland was not visible on ct and the right kidney showed marked downwards displacement. a radical right adrenohepatectomy was performed, with a satisfactory outcome. results: grossly, a giant, partially encapsulated mass was seen to protrude from the right adrenal gland into the liver. microscopically, the tumour was constituted by atypical clear cells with nuclear polymorphism, necrosis and moderate mitotic activity. tumour cells expressed vimentin and melan-a protein. conclusion: to our knowledge, this is the first description of a malignant adrenal tumour in the setting of ahf. as illustrated by this case, awareness of ahf as an entity and attention to its distinctive gross and histologic features are essential to avoid confusion between adrenal and hepatic lesions, especially when imaging studies have provided misleading findings. objective: cushing's syndrome (cs) is a rare disease, resulting in the majority of cases from acth hypersecretion. % is of pituitary origin, % of adrenal origin, and only % from ectopic acth production. method: we reported a rare cause of ectopic acth dependent cs, caused by a pheochromocytoma. results: a -year-old male was hospitalized for severe high blood pressure, depression and weight loss. biological investigation revealed serious hypokaliema, severe acth dependent hypercortisolism with elevated urinary free cortisol secretion, loss of diurnal variation, and excess plasma acth level. ct-scan revealed a nodular lesion in the right adrenal gland with hyperplasia in the left one. the nodular lesion was assumed to be a pheochromocytoma based on the elevated serum and urinary catecholamine and metabolites and local uptake in right adrenal gland in i-mibg scan. right adrenalectomy was performed. macroscopic examination revealed a cm, well-circumscribed, tan-brown tumor, associated with diffuse adrenocortical hyperplasia. histological examination confirmed the diagnosis of pheochromocytoma, without signs of aggressiveness. the tumor cells were immunopositive for chromogranin a, synaptophysin and acth. after surgery, catecholamine secretion returned quickly to normal level. biological and clinical cs regression was noted. conclusion: despite the rare association of cs with pheochromocytoma, preoperative diagnosis is required to an appropriate therapy. a rare case: extralobar pulmonary sequestration mimicking neuroblastoma g. emiroglu * , n. Özsan, s. tiryaki, a. Çelik, y. ertan * ege university, dept. of pathology, izmir, turkey objective: extralobar intraabdominal pulmonary sequestrations are rare congenital malformations which are characterized by disorganized and nonfunctioning pulmonary parenchyma. these lesions have no communication with the bronchial tree and pulmonary arteries. they receive their blood supply from the systemic circulation. method: we describe a days old male infant admitted to the hospital with a left subdiaphragmatic, × × mm, hiperechogenic and, solid mass that was identified during ultrasonography on the th week of gestation. results: abdominal computerized tomography demonstrated a × × mm, solid, vascularized, encapsulated, mostly cystic suprarenal mass with no calcification reported to be highly suspicious for neuroblastoma. the mass was completely excised. gross pathologic examination revealed a well circumscribed spongy lesion that mimicked a lung tissue. on microscopic examination, a disorganized lung tissue that was composed of alveoli, alveolar ductus and bronchioles was seen. normal adrenal tissue was not observed. later, we learnt that the lesion's arterial blood supply was from the abdominal aort. based on these findings, our diagnosis was intraabdominal extralobar pulmonary sequestration. conclusion: intraabdominal extralobar pulmonary sequestrations should be kept in mind in cases of the adrenal masses as the surgical resection is the adequate treatment method for these lesions. axillary lymph node metastasis of papillary thyroid carcinoma showing anaplastic transformation with cutaneous metastasis s. erkilic * , a. ozkur, u. elboga, f. celenk, m. kanlikama * gaziantep university, medical faculty, turkey objective: papillary thyroid carcinoma (ptc) rarely metastasizes to axillary lymph nodes. although anaplastic transformation (at) may occur in the cervical lymph node metastasis from ptc, it is rarely observed in the metastatic axillary lymph nodes. furthermore, cutaneous metastasis from anaplastic thyroid carcinoma (atc) is extremely rare. method: a -year-old male patient who had operated for multinodular goiter years ago presented with neck swelling on the right side. residual thyroid tissue was detected and the lesion was removed surgically in combination with a right neck dissection. histopathologic examination was consistent with ptc and metastatic lymph nodes. the patient received radioactive iodine therapy. afterwards, the patient presented with a right axillary mass years after the first operation and underwent surgical excision. results: histopathologic examination showed ptc with anaplastic transformation. histopathologically confirmed multiple skin metastases from atc emerged in thoracic and abdominal regions months after the last operation. patient died months after the diagnosis of anaplastic carcinoma. conclusion: although most ptcs show an indolent course and have a favorable prognosis, distant metastasis and anaplastic transformation of the metastatic lymph nodes may occur even more than many years after the primary treatment. local "bystander"effect of gene therapy on human tumor cells of medullary thyroid carcinoma in vivo l. feketeova * , m. poturnajová, l. kucerová, p. babál * comenius university, inst. of pathology, bratislava, slovakia objective: gene therapy acts on change of prodrug into cytotoxic drug inside tumor cells transfected by foreign enzyme. it uses also "bystander" effect on surrounding tumor cells without enzyme. we used yeast cytosine deaminase (ycd) in combination with -fluorocytosine ( fc) converted into -fluorouracil ( fu) and its metabolites. medullary thyroid carcinoma (mtc) is a malignant tumor often with therapy resistant distant metastases. patients with sporadic form have metastases at the time of diagnosis, mostly in bones, lungs and liver. the aim of this study was to evaluate the efficiency of ycd/ fc in mtc treatment using xenotransplants derived from model tt cell line in nude mice. method: tumors were immunohistochemically stained with polyclonal antibody anti-egfp (enhanced green fluorescence protein) and monoclonal antibodies anti-pcna and anti-ki . positivity was semiquantitatively evaluated. results: diffuse positivity for pcna was seen in untreated and treated tumors, respectively. positivity for ki- was diffuse in untreated and only sporadic in treated tumors. conclusion: diffuse pcna positivity in treated tumors suggests that the tumor cells stopped in s phase. scattered positivity of ki- after treatment document suppressed proliferation mediated by ycd/ fc gene therapy, which implies therapeutic application in patients with metastatic mtc. (itms , vega / / , vega / / ). ps- - e-cadherin/ß-catenin immunoexpression in thyroid carcinomas k. ivanova * , e. onal, j. ananiev, t. vlaikova, m. gulubova * trakia university, general and clinical pathology, stara zagora, bulgaria objective: the aberrant activation of wnt signaling pathway may be a common denominator for the development of thyroid tumorigenesis. it was announced that the loss of ecadherin rather than β-catenin mutation represents a crucial event in determining the degree of differentiation of thyroid carcinomas. the aim of the study was to evaluate the expression of e-cadherin and β-catenin in the thyroid cancer tissue and to correlate these data with some histological and clinical parameters of the tumors. method: we investigated patients, having thyroid tumorspapillary, follicular, anaplastic and oncocytic carcinomas immunohistochemically with antibodies against e-cadherin and β-catenin. survival analyses were done. results: e-cadherin expression was focally retained in the tumor cell membranes and in the tumor cell cytoplasm of the papillary, follicular and oncocytic thyroid cancers, weather in anaplastic cancers it was almost lost (p= . , and p= . , respectively, fisher's exact test). the expression of β-catenin in tumor cytoplasm and membrane in papilary cancers was higher as compared to that in the other tumors (p= . , and p= . , respectively). conclusion: not surprisingly, the presence of aberrant expression of e-cadherin and/or β-catenin in thyroid cancer has been associated with better patients' prognosis and more well differentiated tumor histology. correlation between estrogen receptor and progesterone receptor with some prognostic factors in papillary thyroid carcinoma m. jalali nadoushan * , r. amirtouri, a. davati * shahed university, dept. of pathology, tehran, iran objective: the more prevalence of papillary thyroid cancer in women shows the probability of the role of sex hormones in the cancer. the aim of this study was determination of relation between sex hormones receptors and some prognostic factors. method: we studied patients with pathology report of papillary thyroid carcinoma after thyroidectomy between and . the specimens were stained immunohistochemically for er and pr. the other informations such as sex, age, tumor size and lymph nodes involvement obtained from the patients documents. results: the average age of patients was . + , . . % t of samples were er positive while this was . % for pr. the percentage of lymph nodes involvement was . %. the size of tumors was . + . cm. there was a direct relationship between female sex and positivity of er (p≤ . ). but there was no significant relationship between er and pr with age, tumor size and lymph nodes involvement. conclusion: it seems to be that er is more prevalent in females but for showing of its role in prognosis, further studies are recommended. objective: type diabetes mellitus (t dm) treatment aims to control metabolic effects, preserve pancreatic function and reduce complications, such as nephropathy. this study intends to evaluate the effects of sitagliptin, a dipeptidyl-peptidase- inhibitor, in pancreatic and renal lesions in zucker diabetic fatty (zdf (fa/fa)) rats, an animal model of t dm. method: male obese diabetic zdf (fa/fa) rats, -weeksold, were treated with vehicle or sitagliptin ( mg/kgbw/ day) for weeks, and compared with lean control zdf rats (n= each). biochemical parameters as well as pancreatic and renal lipid peroxidation and histopathology profile were assessed. specimens we stained with haematoxylin-eosin and periodic acid of schiff, and examined by light microscopy. lesions were evaluated by a semiquantitative rating. endocrine/exocrine pancreas and renal glomerular, tubulointerstitial and vascular lesions were assessed and scored ( / - ). results are mean ± sem; anova and duncan's post-hoc analysis (p≤ . was considered as significant). results: sitagliptin improved metabolic parameters, reduced lipid peroxidation (p< . ) in both organs and significantly prevented major diabetic pancreatic and renal lesions in obese diabetic zdf rats. conclusion: sitagliptin seems to comply with the three main objectives of t dm therapeutic management. underlying molecular mechanisms deserve further elucidation, but could be related with metabolic improvement and reduction of oxidative stress. objective: thyroid tumors of uncertain malignant potential (tt-ump) include follicular and well-differentiated tumors of ump (ft-ump/wdt-ump), as it refers to the presence of questionable capsular/vascular invasion or incompletely developed papillary thyroid carcinoma (ptc)-type nuclear changes. however, a diagnosis of tt-ump is difficult in most cases. we aimed to investigate whether immunohistochemistry (hbme- , cytokeratin- , galectin- , cd and p ) provides additional information concerning such lesions. method: we performed an immunohistochemical analysis on wdts-ump and fts-ump. results: in the wdt-ump group, hbme- was positive in / ( . %) cases. cd , a marker whose expression is reduced in thyroid carcinoma, showed a "malignant" profile (no expression) in / ( . %) cases. / ( . %) cases were positive for both antibodies. one case showed the coexpression of hbme- , cd , galectin- and cytokeratin- . only one ft-ump case was positive for hbme- . the follow-up data revealed no distant metastases or persistent disease. conclusion: tt-ump demonstrated very heterogeneous immunohistochemical profiles. wdts-ump revealed a certain tendency toward a ptc profile, suggesting a possible pathogenetic link between these two entities. however, immunohistochemistry is to be regarded more as a supporting factor, while morphological criteria should always prime in the diagnostic decision. results: there was a positive correlation between tpcfv and hmwck, ck , hbme , galectin , fibronectin (p< . ), but there was no correlation with tpcfv and ret/ ptc (p> . ). hbme- and ck stained strong and diffuse positive in tpcfvs but weak and focal in fas. our study suggests that morphologic features combined with immunohistochemical panel of hmwck, ck , hbme- , galectin- and fibronectin can help to distinguish benign and malign thyroid neoplasms and tpcfv from follicular adenomas. ret/ptc expression has been nonspecific but its detection can be a useful tool combined with immunohistochemistry for diagnosing tpcfv. morphological features of the system mother-placentafetus during pregnancy on diabetes mellitus t. pavlova * , v. petrukhin, e. malutina, a. nesterov, l. pavlova, i. goncharov, d. kolesnicov * belgorod state university, dept. of pathology, russia objective: combination of diabetes mellitus and pregnancy has a special concern. method: it were studied pregnants with diabetes mellitus type and pregnant with gestational diabetes mellitus (gdm). the methods were used: light, transmission, electron, probe and scanning microscopy with microelement analysis. results: the pregnancy at diabetes mellitus type and gdm proceeds with complications: treat of pregnancy termination ( , % and , %), hydramnion ( , and , %), preterm birth ( , and , %). it was displayed decreasing of oxygen in mother's erythrocytes. at the diabetes mellitus in uterus of women in birth there are violations of spiral arteries as well as circulatory disorders (stasis, sludge, thrombosis and diapedetic bleedings), that is significantly expressed at gestosis. tendency to cell form changing is observed in erythrocytes at diabetes mellitus, especially at diabetes mellitus type . the frequency of placental insufficiency in pregnant with diabetes mellitus type were %, at gdm- %. the gestosis accession were resulted to birth's increasing of children with prenatal injury of central nervous system at diabetes mellitus type in mother and diabetic fetopathy at gdm. the clinical and morphological parallels of the system mother-placenta-fetus were presiced ad diabetes mellitus type and gdm. sclerosing mucoepidermoid carcinoma of the thyroid in a year-old female a. polónia * , l. santos, c. eloy, r. celestino, p. soares, m. jácome, c. lobo * ipo-porto, pathology, rio tinto, portugal objective: sclerosing mucoepidermoid carcinomas (smecs) are low-grade malignant tumours with both squamous and mucinous differentiation representing less than % of thyroid malignancies. smecs are usually associated to hashimoto thyroiditis and classically disclose eosinophilia. results: a year-old female with hashimoto thyroiditis presented a . cm nodule in the left lobe of the thyroid. the total thyroidectomy specimen disclosed a firm, well circumscribed, non-encapsulated tumour. microscopically, the tumour was composed by anastomosing clusters of squamoid cells in a sclerotic background without eosinophils. intra-cytoplasmatic pas/diastase positive material was focally found. the tumour cells expressed ae .ae , ttf- and p whereas no expression was observed for thyroglobulin, calcitonin and cd . the ki- labeling index was %. in the molecular analysis no mutations were detected in braf or (k-, n-, h-) ras genes nor ret/ptc or pax /ppargamma rearrangements. the remaining thyroid showed hashimoto-type thyroiditis. a cervical lymph node metastasis was identified. the patient was treated with radioactive iodine and is alive without signs of disease after months of follow-up. conclusion: smecs may not disclose the classical eosinophilia and represent rare low-grade tumours that can give rise to metastases. the most frequent molecular alterations found in thyroid tumours were not detected in this case. ps- - alk and bmp- overexpression as a cause of ossifying papillary thyroid carcinoma objective: ossification is an often encountered finding in papillary thyroid carcinoma (ptc). we hypothesized that osteogenic signaling may be related to osteogenesis of ptc. bone morphogenic protein (bmp)- is the most osteogenic subtypes among bmps. and as a cellular receptor, activin receptor-like receptor (alk) has been emphasized in bmp- induced osteogenic signaling. we investigated the expression of alk and bmp- and their correlation with ossification in ptc. method: alk and bmp- expression were investigated by immunohistochemistry in tumors and adjacent normal follicles of ptcs with bone formation and ptcs without bone formation. alk and bmp- expression were further verified by quantitative real time polymerase chain reaction in each group of cases of ptcs with or without bone formation. results: alk and bmp- immunoreactivity were increased in ptc with bone formation when compared to those without bone formation (p< . and p= . ). both mean values of alk and bmp- mrna expression were elevated in ptcs with bone formation compared with those without bone formation (p= . and p< . ). conclusion: alk and bmp- overexpression may be underlying the molecular alteration that accounts for osteogenesis in ptc. objective: thyroid is an extremely rare site of metastases. renal clear cell carcinoma (rccc) is one of the most frequent primary malignancies causing thyroid metastases as a single nodule ( %) or multiple nodules ( %). we report the case of a -years-old female patient affected by multinodular goiter, who was diagnosed with rccc, treated with nephrectomy. method: due to compressive symptoms, years after nephrectomy, the patient underwent thyroidectomy and the thyroid was sent for histology. results: histology showed multinodular goiter. in the largest hyperplastic adenomatous nodule (left lobe, diameter mm ), multiple solide yellow-orange areas were detected grossly. these areas were microscopically composed of sheets of large cells with clear cytoplasm and small hyperchromic nuclei, with a focally infiltrative growth pattern. at immunohistochemistry clear cells were negative for ttf , hbme , galectin- and positive for cd . conclusion: this is a rare case of multiple rccc metastases within an adenomatous nodule in goiter. clinical diagnosis of rccc metastases to thyroid is extremely difficult, even more if metastases grow in multinodular goiter. it should be always suspected in patients with a clinical history of rccc. objective: follicular carcinoma, oncocytic variant, is a rare type of thyroid carcinoma. we examined a case of oncocytic carcinoma misdiagnosed as medullary carcinoma on fineneedle aspiration (fna) because it was associated with amyloid material. method: a -year-old female rheumatoid arthritis (ra) patient had a . cm mass in the right lobe of her thyroid, which showed no enhancement effect on ct scan and was diagnosed as a "cyst". fna was performed, and she subsequently died of unrelated causes. an autopsy was performed. results: cytology: large and small round-shaped tumor cells were present with round nuclei with granular chromatin. double-or triple-nucleated cells and amyloid material were observed. histology: oncocytic round cells proliferated diffusely in the fibrous capsule. extra-capsular invasion and vascular infiltration by the tumor cells were recognized. electron microscopy: the cytoplasm of tumor cells was full of mitochondria. therefore, we diagnosed this as follicular carcinoma, oncocytic variant. amyloid deposition was also observed in several other organs. conclusion: amyloid deposition was caused by amyloidosis secondary to ra. narrowing of the tumor feeder artery due to amyloidosis may have prevented early enhancement on the ct image. objective: whipple's disease is an infectious disease caused by tropheryma whipplei, an ubiquitous gram-positive actinobacteria. the incidence of the disease is less than per milion. we report five new consecutive cases, four males and one female, diagnosed in our clinic from august to january . method: diagnosis was reached with the help of gastroduodenal endoscopy and histopathological examination of the duodenal biopsies, by lymph node biopsy and by electron microscopy. results: the main symptoms were arthralgia, weight loss and diarrhea. the endoscopic aspect of the small bowel mucosa varied from congestion, granularity of the mucosa to whitish plaques. all patients showed pas positive, diastase resistant, ziehl-neelsen negative macrophages in the lamina propria of the duodenal mucosa and, in one patient, suspected for lymphoma, in an abdominal lymph node. the diagnosis was confirmed by electron microscopy in all cases. classic whipple's disease was the diagnosis in all five cases, but one patient showed involvement of the endocardium and two patients showed lymphadenopathies. conclusion: clinical evolution was favorable under long-term antibiotics (cephtriaxone/trimethoprim-sulfamethoxazole) and follow-up biopsies in three patients showed a normal endoscopical mucosa and a reduced but persistent number of pas positive macrophages in the duodenal mucosa. results: eleven cases of nen of gastrointestinal tract were identified. they were men and women with a mean age of . years. the most common localization was the appendix ( cases). the other cases were localized in the small intestine, the ileo caecal valve, the fundus and the bulb. the mean tumor's size was . mm. mitoses were absent in the majority of cases ( , %). all the tumors were well differentiated classified as grade in cases and grade in cases. the tumor was aggressive in cases. the small intestine's net was multifocal infiltrating the subserosa with lymph node metastasis classified pt n . the ileo caecal valve's net was also aggressive with lymph node and hepatic metastasis classified pt n m . all patients underwent surgical treatment. conclusion: gastrointestinal nens are complex tumors whose incidence is rising and whose treatment requires precise classification and risk stratification. multiple duodenal stromal tumors associated with neurofibromatosis- g. benkhedda * , y. lamouti * chu frantz fanon, dept. of pathology, blida, algeria objective: gastrointestinal stromal tumors (gist), most commonly occur sporadically, but there seens to be some increased tendency for these tumors to develop in patients with neurofi-bromatosis (nf ). there is no histological difference between the nf assocaciated cases and the sporadic cases. however, tumors associated with nf frequently show multiplicity. method: a case of multiple duodenal gastrointestinal tumor arising in a year old mal with nf is reported. the abdominal exploration revealed multiple solid nodules in the duodenum, and the pancretecticoduodenectomy was performed. results: macroscopy: the resected segment of the duodenal showed seven suberosal solid masses. the largest mass measured , cm× cm× cm, and is coupled with the bors lower pancreas but remains limited by a capsule. the cut surface was smooth and white in appearance. microscopy: the tumors were composed of interlacing fascicles of the uniform spindle cells with elongated cytoplasm. the tumor cells lacked pleomorphism, and mitotic figures were absent. immunohistochechemistry: the tumors cells were diffusely positive for cd , cd , and negative for desmin, aml and ps . conclusion: gist are rarely noted in association with neurofibromatosis- . duodenal gist are most frequently diagnosed in the workup of symptoms not specific to these masses. duodenal resection is rarely indicated except in the case of duodenal gist and early-stage adenocarcinoma or if the tumor appeared involve the pancreatic parenchyma on preoperative imagings. periampullary adenomyoma: a true trap diagnosis g. benkhedda * , y. lamouti * chu frantz fanon, dept. of pathology, blida, algeria objective: adenomyoma is a term generally applied to nodular lesions showing proliferation of both epithelial and smooth muscle components. it is usually presented as biliary obstruction. most cases are misdiagnosed as adenoma or carcinoma by preoperative endoscopic or radiologic. therefore, it is frequently treated with extensive surgery. method: we report a case of a year-old man with an adenomyoma located in the ampulla of vater diagnosed by endoscopic piecemeal resection. results: on histologic examination, the lesion consisted of hyperplastic glandular lobules, mainly located in the muscle layers of the vaterian system. the lobular formations consist of small glands surrounded by myofifroblastic, fibroblastic proliferation, sparse capillaries and inflammatory cells. ihc: ki : rare cells with a positive nuclear staining were presenting in the epithelial and mesenchymal components aml: the myofibroblastic of most spindle cells was confirmed by a strong cytoplasmic expression. conclusion: real incidence of adenomyoma of the vaterian system is difficult to appreciate as different names (adenomyoma, adenomyomatosis, myoepithelial hamartome) are used to designate the same histological lesion. adenomyoma was diagnosed only in adult patients -mean age: y). the histogenesis is still a subject of controversy. the most widely accepted hypothesis is that the lesion may represent a form of incomplete heterotopic pancreas. adenomyoma is considered as benign and slow growing, but its potential neoplastic nature cannot be excluded. however, the histogenesis of these tumors is subject to further study. carcinosarcoma of the digestive organs has been reported to exhibit aggressive behavior. carcinosarcomas in digestive organs have been reportedly associated with a poor prognosis. , however, some cases that have been treated with a curative operation showed long-term survival. analysis of her expression level in gastric carcinomas m. bialas * , a. sinczak-kuta, a. lazar, k. urbanczyk, k. galazka, j. szpor, s. demczuk, d. adamek * jagiellonian university, dept. of pathomorphology, krakow, poland objective: the expression of her an oncoprotein belonging to tyrosine kinase family belongs to recently evaluated prognostic and predictive factors in gastric cancer. overexpression of her is observed in about - % gastric cancers. according to many authors, overexpression and/or amplification of her correlates with poor prognosis. the aim of our study was to analyze her expression in gastric cancer in material routinely examined in pathomorphology department, jumc. method: we have analyzed cases of gastric cancers. the material came from females and males, age: - . immunohistochemical reactions were performed automatically on benchmark bmk classic (ventana) using path-way her- /neu ( b ) antibody. scoring system for her expression was ranged from to + where: and + were regarded as a negative, + as equivocal and + as positive. results: most cases of gastric cancers were her -negative ( / ): including score ( patients) and + ( patients). three cases were equivocal and two cases showed + expression level. the lower level of her expression in the analyzed material in comparison with literature could be related to a relatively small group of cases but one cannot exclude that there exist some other factors that stand behind this and surely the investigations should be continued. ps- - mapkap kinase overexpression influences prognosis in gastrointestinal stroma tumors, associates with copy number variations on chromosome , and expression of p map kinase and etv p. birner * * medizinische universität wien, institut für klinische pathologie, austria objective: etv has been proposed to be activated by kitmutations in gastrointestinal stromal tumors (gists). aim of the study was to evaluate the role of etv and associated proteins in gist. method: expressions of etv , mapkap kinase (map-kapk ), phosphorylated p map kinase (pp ), phosphorylated msk , phosphorylated rsk , cop and kit were determined immunohistochemically in gists. sequence analysis of kit, pdgfra and mapkapk and fish of etv and chromosome was performed. results: prominent etv expression was seen in % of gists, but no correlation with clinical outcome was found. correlation of etv expression and kit mutation was seen in % of cases. mapkapk overexpression (n = / . %) correlated with pp expression (p= . ) and alterations of chromosome (p= . ). in one cases with high makapk expression, a mapkapk gene mutation was found. all relapsing gists with very low/low risk showed high mapkapk and kit expression. map-kapk overexpression was an independent prognostic factor for disease free survival (p= . ). conclusion: etv is not universally overexpressed in gist and seems to be induced also by other pathways than kit-mutation. mapkapk -overexpression is associated with shorter survival in gist. patients with low risk gists overexpressing mapkapk might profit from adjuvant tyrosine kinase inhibitor therapy. gastrointestinal stromal tumors: comparison of two risk stratification systems in a multicenter study of turkish cases g. bulbul dogusoy * * gayrettepe florence nightingale, dept. of pathology, istanbul, turkey objective: a nationwide database was performed for gastrointestinal stromal tumors (gists) in a large series of primary gists surgically treated at centers all around turkey. the aim of this multicenter study was to analyze and compare the performance of the national institute of health (nih) and armed forces institute of pathology (afip) risk criteria to determine the ideal risk stratification system. method: statistical analysis of a nationwide database is consisted of age, gender, location, risk groups, histopathologic features and the results of cd , cd , desmin, sm actin, s- protein, and ki immunohistochemistry. results: in cases registered in database, male to female ratio was . and mean age was . years. most common location was stomach ( . %) followed by small intestine, omentum-peritoneum, large intestine, and esophagus ( . %, . %, . %, . % respectively). comparison of the two risk-stratification systems demonstrated that proposed modified afip seems to be better when compared with nih system. many histopathologic and immunohistochemical findings showed significant correlation with risk groups of afip, even with 'not sufficient data' group (p< . ). the results of this multicenter study demonstrates that although follow up results are not provided, afip risk criteria seems to be more useful in prognostication for gists among the two systems. objective: her /neu overexpression or amplification is an important biomarker for identifying patients with intestinal-type gastric cancer who respond to therapy with trastuzumab. moreover, intestinal type gastric cancer shares many phenotypic and molecular genetic changes with colorectal cancer. in particular a progression from chronic gastritis to intestinal metaplasia, dysplasia, and finally malignant transformation is probably the sequence of gastric carcinogenesis. somatic mutation of k-ras gene is common in colorectal cancer, being found in more than one-third of cases, but it seems to have, mostly in intestinal-type gastric cancer, a low incidence ( - %). the purpose of this study is to assess her gene amplification and k-ras mutational status in a series of intestinal-type gastric carcinoma patients. method: twenty paraffin embedded gastric cancer specimens were tested for her amplification by chromogenic in situ hybridization (cish) and k-ras mutational status (codon e ) by pcr-rflp. results: six ( %) cases were her amplified. only one case ( %) was found to have k-ras mutation (codon ), but it was her not amplified. conclusion: the frequency of k-ras mutation and her amplification are in agreement with other studies on this topic. in our study, the two seem mutually exclusive events. objective: gastric carcinoma is related with cancer genetic susceptibility that can be investigated as single nucleotide polymorphisms (snps) and as cytokine genes are known to predispose to malignant disease, several polymorphisms of interleukin- (il- ) gene have been reported to in some may be associated with tumour progression including inhibition of malignant epithelial cells apoptosis and stimulation of angiogenesis. method: the aim of this study was to clarify the association between il- polymorphisms and the risk of gastric cancer and chronic gastritis development or maintenance. pcr-ssp genotyping for il- - c>g polymorphism was performed in biopsies of gastric carcinoma and in biopsies of chronic gastritis. results: there was association between il- - c allele (p = , ) and - cc, low producer, genotype (p = , ) and gastric carcinoma, whereas il- g allele (p= , ) and il- gg (p< , ), high producer, genotype was associated with gastritis. conclusion: we conclude that il- - , low producer genotypes, may have an important role in gastric carcinogenesis and the polymorphism study of this molecule could be a good marker for gastric carcinoma susceptibility when high grade dysplasia is seen in biopsies. objective: in helicobacter pylori gastritis, constant antigenic stimulation triggers a sustained b-cell proliferation. errors made during this continuous dna replication are corrected by the dna mismatch repair mechanism. failure of this mechanism has been described in hnpcc and results in a replication error phenotype. inherent to their instability during replication, microsatellites are the best markers of this replication error phenotype. we aimed to evaluate the role of defects in the dna mismatch repair mechanism and microsatellite instability (msi) in gastric mucosa-associated lymphoid tissue (malt) lymphoma. method: we examined microsatellite loci (bat , bat , d s , d s , d s , tgfb, bat , d s , d s and d s ) for instability in patients with malt lymphomas. in addition, these tumors were also immunostained for mlh , msh and msh , as well as screened for the presence of t( ; )(q ;q ) by real time polymerase chain reaction (rt-pcr). results: we found msi in / ( %) lymphomas, with one tumor displaying high levels of instability. msi occurred in both t( ; )(q ;q )-positive and -negative tumors. conclusion: our data suggest that a mmr-defect may be involved in the development of gastric malt lymphomas, and mutations in the msh mmr gene or hypermethylation of the msh promoter might be associated with msidriven gastric lymphomas. mitosis-specific marker phospho-histone h in the diagnosis of gastrointestinal stromal tumors a. dolzhikov * , a. tverskoi, k. hizhnyakov * regional pathology hospital, dept. of oncomorphology, belgorod, russia objective: the assessment of proliferative activity is one of the major parameters in the proper grading of gastrointestinal stromal tumors (gists). in the low level of mitotic activity it is difficult to calculate dividing cells correctly. phospho-histone h (phh ) is a recently introduced immunomarker for mitotic cells. method: immunohistochemical study of cases of gists of different malignant potential, statistical analysis. results: the count of phh -immunoreactive cells demonstrates the strong correlation (gamma , ; p< , ) with malignant potential of gists detected by standard parameters. it clearly separates true mitotic cells from apoptotic and piknotic nuclei. in all cases phh count was slightly higher than the mitotic index in h&e stained slides. we have found that pph demonstrated the two types of staining: nucleosomal dot-like type and mitotic homogenous type. the first type, probably, reflects the fraction of cells just before the prophase, which cannot be identified in h&e slides. conclusion: immunostaining of pph is a useful additional marker in detection of proliferative activity in gists, helping to identify true dividing cells correctly. ps- - her assessment in gastric carcinoma using ihc and fish p. drev * , b. gazic, j. contreras * institute of oncology, dept. of pathology, ljubljana, slovenia objective: her in gastric cancer (gc) should be assessed following guidelines -recommended testing algorithm employs ihc as a screening tool and is followed by ish to clarify equivocal cases. reported incidences of her + gc vary substantially and discordant results are frequent. in a series of gc her was assessed by ihc and fish to determine incidence of her + gc, concordance of the methods and reevaluate the recommended algorithm. method: her was assessed by both ihc (pathway b ™) and fish (pathvysion™) in gc samples. reactions were evaluated according to guidelines. her was considered positive in case of strong protein expression ( +) and/or gene amplification (ratio ≥ . ). frequencies of ihc and fish scores, incidence of her + gc and concordance (chi-square) of applied methods were analysed. results: ihc score distribution: neg ( ) objective: gastric glomus tumors are rare neoplasms originated from modified smooth muscle cells of the glomus bodies. such lesions present a diagnostic challenge in biopsy material. herein, we report a case of gastric glomus tumor in a -year-old woman. method: a -year-old woman presented with refractory epigastralgy. a gastrointestinal stromal tumor of the gastric antrum was suspected. the diagnosis was made after ultrasound-guided endoscopic biopsy, followed by an endoscopic submucosal resection. results: the biopsy showed tight convolutes of capillarysized vessels surrounded by collars of small, rounded cells set in a hyalinized, myxoid stroma. immunohistochemically, these cells were positive for smooth muscle actin and type iv collagen (pericellular pattern), synaptophysin (focal positivity) and negative for cd , cd , desmin, s and cytokeratins. the diagnosis of gastric glomus tumor was rendered. the resection specimen revealed a submucosal well-circumscribed × , × , cm nodule with identical histological features. the patient was disease-free months postoperatively. conclusion: visceral glomus tumors are rare neoplasms. when they arise in the gastrointestinal tract, the stomach (antrum) is the most frequent location. the differential diagnosis includes net, epithelioid gist and hemangiopericytomas. they usually behave in a benign fashion, although malignant cases have been reported. surgical excision is the standard treatment. immunohistochemical evaluation of replication protein-a in gastric cancer: clinicopathological associations h. gakiopoulou * , e. fourtziala, g. levidou, a. stofas, n. alexakis, p. korkolopoulou, e. patsouris * university of athens, st dept. of pathology, greece objective: replication protein a (rpa ) is required for stabilization of single-stranded dna at early and later stages of dna replication being thus critical for eukaryotic dna replication. in this study, we investigated for the first time the immunohistochemical expression of rpa protein in a series of gastric carcinomas in relation with clinicopathological parameters (age, sex, lauren's histologic classification, histologic grade, lymphovascular invasion, tumor size, depth of invasion (t), lymph node metastasis (n) and stage). method: a standard immunohistochemical method and a semi-quantitative evaluation for the detection of rpa labeling index (li) were applied. results: nuclear rpa immunoreactivity was seen in all carcinomas with a mean value of . %. statistical significant correlations emerged between: . rpa li and low tcategory (p= . ) . rpa li and absence of lymph node metastasis (p= , ). rpa li was higher in cases without lymphovascular invasion; however this association did not reach statistical significance. conclusion: the widespread expression of rpa in gastric carcinomas suggests that this protein is implicated in gastric cancer growth. the observed significant associations between rpa li and low t as well as n tumors could imply that rpa might offer a growth advantage in the early stages of gastric cancer progression. pigmented histiocytic "pseudotumoral" reaction due to endoscopic tattooing of the duodenum with india ink r. hadhri * , c. objective: india ink has been used for endoscopic tattooing to facilitate localization of a luminal abnormality at the time of surgery or repeat endoscopic examination. recognition of this phenomenon is important to prevent misinterpretation of this _nding as other cause of black tissue deposits. some of them can be easily excluded by microscopic examination; the others, however, necessitate special stains or, as illustrated by our observation, an adequate clinical information! method: a -year-old man was referred for enteroscopy for microcytic anemia. the procedure revealed a cm flat umbilicated lesion of the duodenum. endoscopic tattooing was performed to guide surgical excision. results: histologic examination of the surgical specimen revealed a tubulovillous adenoma with low-grade dysplasia. the submucosa showed numerous aggregates of large cells containing a heavy black pigmentation. special histologic stains were inconsistent with melanin or iron. this submucosal cellular infiltration was linked to the preoperative use of india ink when the notion of tattooing was "kindly" provided by the surgeon! conclusion: despite a striking and misleading appearance, the microscopic finding of such phenomenon does not represent a pathologic state. nevertheless, communication between enterologists, surgeons and pathologists are mandatory to assure prompt recognition and avoid unnecessary investigations. objective: gastritis cystica polyposa is a unique lesion found on occasion at the stoma of a gastrojejunal anastomosis. however, gcp is rarely found in an unoperated stomach. method: a -year-old woman with abdominal discomfort and vomiting. physical examination, routine hematological examination and biochemical tests were within normal limits, exept mild anemia. results: upper gi endoscopic examination revealed a pedanculated polyp, in the greater curvature. endoscopic ultrasonography revealed a polypoid heteroechoic mass with cystic area and × mm diameter in posterior wall of gastric body. polypectomy was performed without any complications. histological examination of the protruding lesion revealed some misplaced cystic glands were entrapped in dense disorganized bundles of smooth muscle of muscularis mucosa surrounded by a rim of lamina propria. these findings were consistent with gastritis cystic polyposa. objective: human papillomavirus (hpv) infection is a known risk factor for the development of squamous cell carcinoma (scc) of the cervix, the oropharynx and the anogenital region. the aim of our investigation was to assess the prevalence of hpv dna in patients with esophagus cancer in the western population. method: formalin-fixed paraffin-embedded blocks from consecutive patients who underwent esophageal endoscopic mucosal resection or esophagectomy for scc were tested for the presence of hpv dna by polymerase chain reaction using consensus primers gp +/gp +. viral genotyping was determined by type-specific primers. results: among the total of cases, no hpv dna was detected in cases ( . %). cases were tested hpv positive ( . %) , of which was hpv +, was hpv + and were non-high-risk hpv type. the non-high-risk hpvs were detected in patients with previously treated scc of the mouth (n= ) and the oropharynx (n= ). patients with high-risk hpvpositive tumors had no history of oropharyngeal cancer. conclusion: our study revealed the presence of oncogenic hpv genotypes in a subset of esophageal cancer. the low rates of viral infection detected suggest that hpv unlikely represents a significant etiologic factor in esophageal carcinogenesis. further studies are needed to confirm these data in larger populations. objective: gastric cancer is the second leading cause of cancer mortality in the world. amplification of her- /neu oncogene has become an important biomarker for identifying patients who will respond to her- targeting therapy. the rate of her positivity in gastric cancer is variable, ranging from % to %. objective: gastrointestinal stromal tumors (gists) are primary mesenchymal tumors that arise in the gi tract. only - % of gists occur in the duodenum. here, we report a rare case of duodenal gist with extramural growth that mimicked a pancreatic tumor. method: a years old male patient was admitted to our hospital for duodenal ulcer and gastrointestinal bleeding which was diagnosed in an another clinic by endoscopy. abdominal mr of the patient showed a × , cm necrotic mass in the uncinate process of the pancreas which was excised by whipple operation. results: macroscopically, the resected specimen consisted of solid mass that was connected to the patient's duodenal wall but not to the parenchyma of the pancreas. microscopic examination of the tumor showed spindle shaped and epithelioid cells with mild nuclear pleiomorphism. immunohistochemistry revealed that the cells are strongly positive with cd , with focal expression of cd and sma. conclusion: gists are low-grade malignant mesenchymal tumors of the gi tract and are believed to originate from the neoplastic transformation of the cajal cells, which are located between the longitudinal and circular layers of the muscularis propria. they most frequently arise from the second part of the duodenum where they push or infiltrate into the pancreas. in our case report, the patient's tumor exhibited extramural growth and mimicked a pancreatic tumor. mantle cell lymphoma (mcl) is relatively a rare subgroup of non-hodgkin lymphoma. we have experienced an uncommon case of mcl. a -year-old man was admitted to emergency service for upper gastrointestinal bleeding as melena. gastric corpus wall thickness found increased and splenomegaly determined at whole abdominal ultrasonography. endoscopic examination revealed subsequently, cm diameter polypoid lesion at corpus anterior wall and cm dimensioned elevated and vascularised lesion at corpus posterior wall. endoscopic biopsy had reported as active gastritis and mucosal reactive hyperplastic changes with ulcerative background. after than total gastrectomy and splenectomy specimens examined diffuse infiltration of monotonous medium-sized, atypical lymphoid cells with hyperchromatic nuclei. these cells were positive for cd , cd , cd , bcl- and cd a, but negative for cd , cd and bcl- . atypical lymphoid cells were present even in splenic hilus and lesser curvature lymph nodes. we reported this case as primary gastric mantle cell lyphoma according to morphologic and immunohistochemical staining features. we report a -year-old man who had been searched for bicytopenia (anemia and thrombocytopenia). bone marrow examination revealed extensive bone marrow metastasis of signet ring cell carcinoma. pas, d-pas and mucin histochemical markers performed to bone marrow sample which were all positive. immunohistochemical markers were positive with muc- , muc- ac, muc- (focal staining), ck , ck , ema, cea and negative with vimentin, cd , chromogranin. according to morphologic and immunohistochemical results we thought primarily gastrointestinal system malignancy, particularly stomach carcinoma. upper gastrointestinal endoscopy performed subsequently, hemorrhagic ulcerative lesion had seen and taken biopsies. endoscopic biopsy results showed metastatic signet ring cells origin from stomach. we report this case because bone marrow metastasis can be found commonly in some malignant tumors but diagnosing a nonhematologic malignancy from bone marrow is an unusual event. the pathological effects of salvia officinalis extract on serum level of alkaline phosphatase in male rats e. abdollahi * , r. ahmadi * tehran university, iran objective: studies show that there is association between salvia officinalis extract administration and liver or heart functions. the main aim of this study was to determine the effects of salvia officinalis extract on serum level of alkaline phosphatase in male rats. method: male wistar rats were randomly divided into control, normal saline receiving and salvia officinalis extract ( , or mg/kg/day) receiving animals of in each group. after a period of weeks, blood samples were collected using cardiac puncture method. following serum collection, serum alkaline phosphatase levels were measured by spectrophotometry method. data were statistically analyzed and compared between groups using anova. results: the results indicated that serum alkaline phosphatase levels were significantly increased in salvia officinalis extract ( , or mg/kg/day) receiving animals compared with control rats. conclusion: our findings show that salvia officinalis extract is enhancer of serum alkaline phosphatase according to which, impairing effect of the extract on certain tissues is conceivable. the pathological effects of waterpipe smoking on serum levels of cea, alkaline phosphatase or creatine kinase in male and female rats r. ahmadi * , m. mafi * islamic azad university, dept. of physiology, hamedan, iran objective: various studies show that smoking can influence serum levels of tumor markers such as carcino embryonic antigen (cea) and kinase or phosphatase enzymes. the main aim of this study was to determine the pathological effects of waterpipe smoking on serum levels of cea, alkaline phosphatase (alp) or creatine kinase (ck) in male and female rats. method: male and female wistar rats were randomly divided into control and waterpipe smoking groups of in each. after a period of weeks, blood samples were collected using cardiac puncture method. following serum collection, serum cea, alkaline phosphatase or creatine kinase levels were measured. intestinal tissue was also examined histologically. data were statistically analyzed and compared between groups using anova. results: the results indicated that serum cea, alp or ck levels were significantly increased in male and female waterpipe smoking animals (p< . , p< . or p< . , respectively). there was not gender effect on serum cea, alp or ck levels. there were also histological changes in intestinal tissue including increased tissue plasma cells infiltration and inflammation. conclusion: our findings show that waterpipe smoking is an enhancer factor of serum cea, alkaline phosphatase or creatine kinase levels, according to which, damaging effects of waterpipe smoking on various tissues, particularly intestine should be considered seriously. the effects of acute or chronic immobilization stress on serum level of creatine kinase and alkaline phosphatase in male rats m. alinavaz * , r. ahmadi, m. mafi * islamic azad university, nutrition, tehran, iran objective: studies show that immobilization stress has a variety of effects on serum levels of liver enzymes. the main aim of this study was to determine the effects of immobilization stress on serum level of creatine kinase and alkaline phosphatase in male rats. method: male wistar rats weighing ± g were randomly divided into control, acutely or chronically immobilized animals of in each group. animals were immobilized for h/day or h/day for a period of weeks or week in chronically or acutely immobilized groups, respectively. blood samples were collected using cardiac puncture method. following serum collection, creatine kinase or alkaline phosphatase level was measured by spectrophotometery method. data were statistically analyzed and compared between groups using anova. results: the results indicated that serum creatine kinase level was significantly increased in rats enduring acute or chronic immobilization compared with control animals (p< . ), however, there was not significant difference between serum alkaline phosphatase levels in immobilized animals compared with control rats. conclusion: immobilization stress may leave pathological effects in liver or other organs leading to enhanced serum creatine kinase level. immunoexpression of lactoferrin in bone metastases and corresponding primary carcinomas v. barresi * , a. ieni, g. giuffrè, g. branca, g. tuccari * university of messina, dept. of human pathology, italy objective: with reference to primary bone tumors, we previously found lactoferrin (lf) immunoreactivity in chondroblastomas, chondromyxoid fibromas, giant cell tumours and osteoid osteomas, while no immunoexpression for this protein was detected in chondrosarcomas and osteosarcomas. herein we aimed to analyze lf distribution in bone metastases from cancers of different sites. method: lf immunohistochemical expression was investigated in formalin-fixed and paraffin-embedded specimens of human bone metastatic lesions as well as in the primitive corresponding carcinomas. the primitive sites of carcinomas were: breast ( ), prostate ( ), kidney ( ), lung ( ), colon-rectum ( ), uterus ( ). a lf intensity-distribution (id) score was calculated for each case by multiplying the values of the area staining positivity and the intensity staining. results: lf immunostaining, with variable id scores, was encountered in / ( %) metastatic bone lesions. immunoreactivity for lf was found in primary carcinomas with a percentage of neoplastic cells ranging between tand %, although this positivity decreased in breast carcinomas ( . %) and was totally absent in lung cancers. the immunohistochemical concordant evidence of lf in bone metastases and corresponding primary carcinomas strongly supports the hypothesis of an autoctone production of this protein by the neoplastic elements themselves in order to get a greater availability of iron for their increased turnover. results: it was observed a negative correlation between il /il and il /foxp concentration in the sf as measured by fc, before the treatment. in the sm, il and mmp were strongly expressed in macrophages, fibroblasts, endothelial cells and the extracellular matrix of control cases, but reduced significantly in the treated cases (p< , ). il and foxp positive cells remained costantly in a reduced number in the ms. conclusion: as the proinflammatory cytokines decreased after the therapy, we can conclude that these are more efficiently influenced than some immune cells participating in the autoimmune process. histological evaluation of the spleen after acute bleeding followed by blood replacement with two different physiologic solutions m. cabral * , a. l. ortiz, c. venâncio, j. objective: chondromyxoid fibroma is a rare benign lesion accounting for less than . % of all primary neoplasms of bone. a study was conducted to assemble a national study group for rare entities and share the experience of different centers. method: the data was collected from the pathology archives of referral hospitals in turkey. results: among the cases enrolled in the study, . % (n= ) of them were male. median age was ± yearsold (range: - ). the most frequent localizations were tibia ( %, n= ), femur (n= , . %) and pelvic bones (n= , . %), followed by feet bones ( . %), fibula ( . %), humerus ( . %), hand bones ( . %), cranium ( . %), costa ( . %), radius ( . %), scapula ( . %), and vertebrae ( . %). although the cases with appendicular skeleton involvement was younger than the others (median age: . ± . vs ± . respectively) no significant correlation was found between age, gender and localization. conclusion: cases displayed a wide age-range with a slight male predominance. the most frequent localizations were tibia, femur and pelvic bones. rare localizations such as temporal frontal bones, vertebrae and scapula were also observed. the study, which may serve as a preliminary work for future studies was presented to share our experience on this rare entity. histological changes and granulocytes redistribution in adjuvant arthritis l. feketeova * , p. jancová, p. objective: surgical treatment options of malignant tumors of the knee includes reconstruction with incorporation megaprosthesis. inguinal lymphadenopathy due to the lymphatic uptake of metal debris has been described, and may be clinically confused with tumor metastasis. method: we report the case of a -year-old woman with inguinal lymphadenopathy caused by metallic debris from a knee mega prosthesis for malignant fibrous histiocytoma. results: the histopathological changes seen in lymph node were metal debris containing sinusoidal macrophages in a background with numerous epithelioid granulomata in the remaining lymph node. conclusion: the identification of regional lympadenopathy in patients with past history of malignancy usually indicates metastatic disease. post-prosthesis lymph node histiocytosis resembling metastatic disease is described, and that is why we need resect and examine lymph nodes with the use of polarized light microscopy to identify birefringent particles of prosthetic debris for an accurate histologic diagnosis. objective: chordoma is a slow-growing malignant bone tumor that exhibits notochordal differentiation. nearly % of cases occur in the sacrococcygeal region and in the base of the skull. the remaining cases develop in the mobile spine, predominantly in the cervical and lumbar vertebrae. we report a rare case of paravertebral mediastinal chordoma without bone destruction. method: case report. results: a -year-old japanese woman was admitted to hospital after a tumor was incidentally detected on a plain chest x-ray image. the tumor was located in the paravertebral region of the mediastinum and did not show any destruction of the thoracic vertebra radiologically. the tumor was clinically diagnosed as a benign neurogenic tumor and the tumor was easily removed surgically. microscopically, the tumor mainly consisted of tumor cells with extensively vacuolated cytoplasm, arranged in cord-and nest-like fashion in a myxoid matrix background. immunohistochemically, the tumor cells showed diffuse positivity for pancytokeratin (ae /ae ), vimentin. the tumor cell nuclei were positive for brachyury, which is a key transcription factor of notochordal development. conclusion: these results confirmed the tumor to be an extraosseous chordoma in the paravertebral mediastinal region, which is rather an extremely rare location for usual chordoma. ps- - testicular papillary mesothelioma: a case with borderline features s. mavropoulou * , z. tatsiou, p. nasos * general hospital, laboratory of pathology, xanthi, greece objective: well-differentiated papillary mesothelioma occurs rarely in the paratesticular region, with only a few published case reports. method: we describe a case of a -year-old man who initially presented with discomfort in the left testis and underwent resection of a hydrocele in the left testis. a hydrocelectomy was performed, during which a pedunculated mass, , cm in greatest dimension, was found attached to the testis. results: microscopically, the mass was composed of multiple branching papillary structures with fibrovascular cores covered by a single layer of low cuboidal to cuboidal cells with predominantly bland nuclear and cytologic features and rare microscopical necrosis. immunohistochemical staining for calretinin and cytokeratines / was positive and proliferative marker ki was < %. accordingly the diagnosis of a well-differentiated papillary mesothelioma was made. the patient has not received additional therapy and is disease free months after diagnosis. conclusion: in conclusion, we report a rare case of a welldifferentiated papillary mesothelioma of the tunica vaginalis of the testis. the combination of benign and semimalignant characteristics can make the diagnosis of such a lesion problematic and pathologic distinction from malignant mesothelioma is crucial, although it may be difficult because of the variability of associated histologic features. objective: lipoma of the bone is a rare benign adipocytic tumor that arises intraosseous and rarely on the cortex or on the surface of the bone (parosteal lipoma). parosteal lipoma affects the long bones diaphysis of adults over years old. method: we present the case of a years old female with clinical diagnosis of femoral exostosis. conventional radiographs shows the presence of a cm length area of lucency on the femoral metaphyseal surface with a periostal reaction at the base of the lesion. results: gross examination reveals a cm osseous tumor, whitish on the surface and yellow on the section. on microscopic examination there are mature adipocytes with small foci of bone scattered throughout the adipocytes and hyaline cartilage at the perifery of the lesion. the gross and microscopic examinations correlated with conventional radiographs led us to diagnosis of parosteal lipoma. conclusion: this case is interesting being a rare bone tumor, the young age of the patient and the location on the surface of the bone. application of scanning acoustic microscope to evaluate lymph node lesions k. miura * * hamamatsu university, school of medicine, japan objective: a scanning acoustic microscope (sam) is a device that uses ultrasound (frequency, > khz) to image an object or tissue. because it is known that the harder the tissue, the more the speed of ultrasound, sam can provide data on the elasticity of cells and tissues. method: we compared lymph node lesions between acoustic and light microscopic images to evaluate the usefulness of sam. results: sam system discriminated lymph node components and demonstrated distinct acoustic images of the lymph nodes such as cancer metastasis, lymphomas, granulomatous diseases, and deposition diseases such as amyloidosis. areas with desmoplastic reactions associated with cancer invasion or post-inflammatory fibrosis showed the greater speed of sound than normal lymph nodes. these results corresponded well to those obtained using the conventional microscope. conclusion: sam provides the following benefits: ( ) images are acquired in only few minutes without requirement for staining, ( ) imaging pattern is similar to that of light microscope, and ( ) speed of sound from each lesion is digital and statistical analysis is possible among diseases. although resolution of sam is little lower than that of light microscope, the sam can be an ancillary tool for histological diagnosis and clinical research. objective: parkinson's disease (pd) is considered one of the major neurological disorders of the population, and there is increasing data provides enough evidences confirming the involvement of free radicals and other reactive oxygen species (ros) in a number of physiological and pathological processes. the aim of the present study was to evaluate, the effect of therapy in patients with parkinson disease on biomarkers of oxidative stress, such as products of lipid peroxidation by two different methods: electron paramagnetic resonance and visible spectrophotometry. method: the study was performed in blood samples of patients with pd -with therapy, patients with pd -without therapy and healthy volunteers as controls. the products of lipid peroxidation were measured as malondialdehid (mda), spectrophotometrically by thiobarbituric acid (tba) method. the levels of lipid radicals were determinated ex vivo at room temperature on an x-band emx-micro spectrometer, bruker, germany. results: by the present study we reported higher levels of oxidative stress in pd patients without therapy compared to those with therapy. these results were comfirmed by the epr method. conclusion: the increase of oxidative stress, in pd patients' might be an additional reason for many secondary complications. objective: primary aneurysmal bone cyst (abc) is a histologically complex and mainly cystic lesion that accounts for % of all primary bone tumors. information regarding its clinical presentation and management in hands and feet remains sparse. method: the medical records abc in hands or feet in hospital la paz pathology department from to were retrospectively reviewed and compared with existing data. we also propose pathological criteria for differential diagnosis between abc and giant cell reparative granuloma (gcrg). results: ten abc in hands or feet were identified, out of ( . %), five tumors were in metacarpals, in metatarsals and in phalanx. radiographs showed expansile lytic lesions in metadiaphyseal region, sometimes with aggressive appearance. histologically, abc showed a mixture of blood-filled spaces with connective tissue septae containing osteoclast giant cells and foci of osteoid. scattered areas of so called "blue bone" were present in cases ( %). there were two solid variants. five patients underwent resection and curettage, three of which relapsed ( %). conclusion: abc should be considered in the radiologic differential diagnosis of hands and feet tumours because these lesions can even mimic malignancies. although clinicopathologically some authors consider gcrg is related to the abc solid variant, we believe they are different entities. carb- is the superior anti-cd monoclonal antibody in optimized protocol settings r. røge * , s. nielsen, m. vyberg * inst. of pathology, aalborg, denmark objective: immunohistochemical detection of cd is important in diagnosis of hodgkin lymphoma and may be relevant in classification of renal tumours. in four tests with - participating laboratories conducted by the nor-diqc external quality assessment scheme only - % of cd stains were sufficient, mainly because of too diluted primary antibody concentration, insufficient hier and less successful antibody clone. the purpose of this study was to evaluate three anti-cd antibodies based on vendor and inhouse optimized protocols. method: multitissue blocks with various malignant lymphomas, renal tumours and normal tissues (n= ) were stained with three concentrated (conc) antibodies (carb- , mma and by ) according to predetermined in-house optimized protocols on two staining platforms. ready-to-use (rtu) solutions of carb- and mma were also examined. extension and intensity of stains was scored using the h-score method. results: carb- conc with an in-house optimized protocol gave the highest h-scores in classical hodgkin lymphoma, renal tumours and normal kidneys. carb- rtu and mmaconc gave slightly lower scores, while mmartu and by conc gave the lowest scores and a large proportion of false negative reactions. all in-house optimized protocols gave better staining results than vendor protocols. conclusion: the importance of antibody selection and protocol optimization in immunohistochemical laboratories is emphasized. objective: studies show that there is association between stress and pathophysiological changes in reproductive system. the main aim of this study was to determine the pathological effects of immobilization stress on testes tissue and serum testosterone level in male rats. method: wistar rats were randomly divided into control, acutely or chronically immobilized groups of in each. animals were immobilized for h/day or h/day for a period of weeks or week in chronically or acutely immobilized groups, respectively. after weeks, blood samples were collected using cardiac puncture method. following serum collection, testosterone level was measured by radioimmunoassay method. the effect of immobilization stress on testes histology was also examined. data were statistically analyzed and compared between groups using anova. results: serum level of testosterone was decreased in acutely or chronically immobilized rats compared with control animals (p< . ). in histological study, semeniferous tubules were significantly deformed and cellular concentration was reduced in immobilized rats compared with control animals (p< . ). the number of spermatocytes, spermatids or sperms was also decreased in immobilized rats (p< . ) and this pathologic change was more prominent in acutely than chronically immobilized rats. conclusion: this report underlines the importance of studying the archive material in order to thoroughly comprehend a single museum object. this handling of matters will help to turn anatomical collections into a unique teaching tool for modern medical practice and a noteworthy documentation of scientific, artistic and historical value. objective: extramedullary plasmocytoma is an uncommon plasma cell tumor localized preferentially in the upper aerodigestive tract, with no evidence of underlying multiple myeloma. it accounts for less than % of head and neck tumours. method: we report a case of a -year-old male patient with reccurent left-sided epistaxis and nasal obstruction. ct-scan showed a left maxillary mass eroding the lateral wall of the nasal cavity. the tumor was surgically removed. results: histologic examination showed a diffuse infiltrate of neoplastic cells in the submucosa, arranged in a scant vascularized stroma. the neoplastic cells were large to medium-sized with amphophilic cytoplasm, irregular nuclei and proeminent, eosinophilic nucleoli. mitotic figures were frequent. imunohistochemical stains were performed in order to make a differential diagnosis: the tumor cells were positive for cd and cd and they expressed cytoplasmic immunoglobulin with kappa light chain restriction. most of the tumor cells were also cd and ema positive. conclusion: even though extramedullary plasmacytoma of the sinonasal tract is rare, it should be included in the differential diagnosis. the cooperation among the otorhinolaryngolists, pathologists and hematologists are required to manage the patients effectively in order to provide optimal treatment. objective: sinonasal malign melanoma is a rare antity, mostly arising from nasal cavity and accounting for < % of all sinonasal tract neoplasms. method: we report two patients complaining of nasal obstruction and epistaxis. endoscopy revealed partially haemorrhagic mass obliterating nasal passage. in one patient diagnosis was done as a plasmacytoma by pathologist in an another hospital. multiple biopsies were taken. microscopically, the tumour was consisted of medium sized cells, which had high nuclear to cytoplasmic ratio with pleomorphic nuclei containing eosinophilic nucleoli and intranuclear cytoplasmic inclusions. the cytoplasms contained variable amount of melanin pigment. mitosesincluding atypical forms-were easily detectable. there were mild inflammatory infiltrate against the tumour and rare tumour necrosis. one was showed pseudopapillary growth pattern and lymphovascular invasion. results: immunhistochemically, tumour cells showed immunreactivity for s- , vimentin, hmb- and melan-a(mart- ). tumour cells were negative for cd- , cd- , cd- , cd- , sma, pan-ck and desmin. submandibulary lymph node metastasis was observed in one patient. this patient has undergone chemotherapy of temozolomide and cures of regional radiotherapy. for the other patient, there were no metastasis detected by pet-ct. conclusion: sinonasal malign melanoma is a rare malignacy, but this entity could be kept in mind to avoid missdiagnosis with other tumors sharing similar morphology. objective: significant changes in the voice occur after years. vocal quality is dependent on the vocal fold (vf) tissue biomechanics that derive from the extracellular matrix composition and organization. we studied muscularlamina propria interface of human vf in the aging. method: two authors evaluated density of vessels and the thickness of the deep and muscle layer by he and ihc (collageniv) with semiquantitative score. these data were validated by point-counting morphometric method. results: with the aging of the vocal folds we identified increased of vessels density in the muscle and deep layers, increasing the matrix density and thickness of the deep layer, and "dissection" of muscle fibers by dense connective tissue. the progressive structural changes in musclelamina propria interface play a crucial role in the remodeling and vocal quality. the increased density of vessels and matrix in aging may contribute to the preservation of vocal function by the physiological repairing. ps- - "lymphoepithelioma-like" thymic carcinoma in parotid gland m. bugdayci * , g. atay, b. sozeri, g. guler tezel * hacettepe university, pathology, ankara, turkey objective: we report a -year-old woman presented with a slow growing painless mass beneath the right ear. a fineneedle aspiration revealed whartin tumour. the patient underwent parotidectomy and histopathological examination showed thymic carcinoma with areas of thymoma. method: histopathological examination was done according to conventional protocols. immunohistochemical stainings were performed using lsab methods. panck, cd , cd , ck / , ck , vimentin, ki- , cd , bcl- , cd a, s , sma, tdt antibodies were used. results: histopathologically, a well circumscribed, nodular, hybride neoplasm finely seperated from parotid gland had two components, uniform spindle cells in basaloid form lobulated by fibrous septae and large epitheloid cells with vesicular nuclei and meganucleoli, suggesting thymoma and thymic carcinoma, respectively. prominent lymphocytic reaction with germinal centers, high mitotic activity, small necrotic foci and microabcesses were also seen. immunohistochemisty revealed panck, vimentin, cd and cd a positivity. the diagnosis of "lymphoepithelioma-like" thymic carcinoma was made. conclusion: thymic carcinoma is a rare tumor most commonly located in the anterior mediastinum. thymic carcinoma which originates from ectopic rests of thymic tissue caused by defective migration of the embryonic thymus, is extremely rare. in the presented case a thymic carcinoma was found in parotid gland which is an unusual site. objective: recent in vitro and in vivo studies have shown that several malignant tumors express an alternatively spliced variant of the receptor of growth hormone -releasing hormone (sv ), which operates by a ligand-dependent and independent manner. method: nine ( ) adenocarcinomas, pleomorphic adenomas and warthin tumors were studied by immunohistochemistry for sv expression and visualized by diaminobenzidine staining. results: sv expression was cytoplasmic and was detected in / malignant ( %) and / ( %) warthin tumors. however, only / ( %) pleomorphic adenomas expressed sv (p< . , x -test). immunoreactivity ranged from mild to intense in all positive specimens, with the exception of warthin tumors at which only intense immunoreactivity was recorded. conclusion: our study, for the first time reports the presence of anti-sv immunoreactivity in tumors of the salivary glands. furthermore, the high association of sv expression with the malignant as opposed to the benign neoplasms implies a role of sv in the progression of the disease. a surprising finding of our study was the high positivity for sv exhibited by the warthin tumors that implies biological similarities between these histopathological entities of the salivary glands. these results imply that the use of antagonistic analogs of ghrh merit further investigation. objective: sialadenoma papilliferum (sp) is a rare, benign neoplasm of salivary gland origin which manifests as an exophytic papillary excrescence of the mucosa. indeed, sp is both an exophytic proliferation of papillary stratified squamous epithelium above the mucosal surface and an endophytic salivary ductal proliferation beneath the mucosa. it arises predominantly in minor salivary glands and usually affects patients in the age range of - years, with reports in young patients being exceedingly rare. method: we report a case of a previously healthy yearold man diagnosed with a nodular mass in the upper lip buccal mucosa. the tumour was excised and submitted for microscopic examination. results: histologic examination revealed a biphasic proliferation of papillary stratified squamous and salivary ductal epithelia, both underneath the mucosal surface. conclusion: in this unique case, as the classical sp, the tumour has a biphasic proliferation of squamous and ductal epithelia. however, unlike the classical sp, both epithelia grow under the mucosal surface. as a result, it didn't manifest as an exophytic proliferation, but as a nodule. we excluded squamous papilloma, inverted ductal papilloma, intraductal papilloma and mucoepidermoid carcinoma, the principal entities in the differential diagnosis of sp, and concluded it to be a sp with inverted pattern. interest of histopronostic classification in three grades in the therapeutic management of primary epithelial parotid carcinoma v. results: microscopic examination showed a biphasic tumor with a prominent myxoid stroma and tumor cells with clear cytoplasm involving pleural tissue. immunohistochemistry showed tumor cells positive for keratin, s protein, smooth-muscle actin, p protein, vimentin. histological and immunohistochemical features confirmed the diagnosis of epithelial-myoepithelial carcinoma. the patient underwent a right parotidectomy years earlier for removal of . cm mass diagnosed as emc. conclusion: epithelial-myoepithelial carcinoma of salivary gland is a rare low-grade malignant neoplasm with a potential for local recurrence and metastases. rare metastases for lungs, kidney and brain were reported. we described an additional case of pleural metastasis. objective: salivary defense system recognized as an important protective factor of the child oral environment. research data reveal that bacterial infection, dental caries and periodontal diseases have a higher incidence in cases of salivary glands dysfunction. the aim of present study was to find out the morphological peculiarities of the fetal parotid gland in cases of restrictions of its intrauterine growth (iugr) at late gestation. method: parotid gland of twenty human fetuses with iugr from the late spontaneous abortions material were compared with fifteen fetal glands in cases of induced abortions due to psychological reasons (control group). tissue samples were stained with hematoxylin and eosin. stereological examination was done to find out volume fractions of parotid gland structural components. results: results have shown the reduction of the area of acini, large collected ducts, striated and intercalated ducts in iugr group. the volume fractions of vessels were also lower than on controls. the foci of immature secretory lobes in cases of growth restriction occupied wider zones within loose, poorly cellular, fibrous stroma. conclusion: our study demonstrates the delay of the parotid gland structural maturation in pregnancies complicated with iugr. impaired growth and secretory gland's dysfunctions may cause pathological changes in oral ecosystem of a child. objective: dermal fillers are injectable products commonly used in aesthetic medicine. this type of treatment, which often replaces traditional surgical procedures provides satisfactory cosmetic results. it is known, however, that there is a risk of undesired effects at the site of injection of the product or even at a distance. literature reports numerous cases of orofacial injuries caused by dermal fillers. the aim of this study is to evaluate two of the most widely used facial dermal fillers for aesthetic purposes in an effort to identify adverse reactions they may produce. method: two of the most commonly used materials for aesthetic purposes by dermatologists and plastic surgeons, polymethylmetacrylate and hialuronic acid were injected in rat tongues: % polymethylmethacrylate (n = ) and mg/ml hyaluronic acid (n = ), compared to an inert solution for control (n= ). after , and days, local clinical and histological alterations were analyzed. results: the following factors were verified: intensity of the inflammatory response (h&e), amount of newly formed blood vessels (ihc) and macrophages and collagen fibers density (picrosirius). results showed that both filling materials triggered local inflammatory response to a greater or lesser degree. objective: the aim of this study was to compare clodronate and zoledronic acid regarding their influence on the repair of surgical wounds in maxillae (soft tissue wound and tooth extraction) and their relation to osteonecrosis. method: thirty-four wistar rats were allocated into three groups according to the treat-ment received: (i) animals treated with zoledronic acid, (ii) animals treated with clodronate and (iii) animals that were given saline solution. all animals were subjected to tooth extractions and surgically induced soft tissue injury. histological analysis of the wound sites was per-formed by means of hematoxylineosin (h&e) staining and immunohistochemical staining for receptor activator of nuclear factor-kb ligand (rankl), osteoprotegerin (opg), von willebrand factor, and caspase- . results: the zoledronic acid group showed higher incidence of non-vital bone than did the clodronate group at the tooth extraction site. at the soft tissue wound site, there were no significant differences in non-vital bone between the test groups. rankl, opg, von willebrand factor, and caspase- did not show significant differences between the groups for both sites of surgical procedures. conclusion: both of the bisphosphonates zoledronic acid and clodronate are capable of inducing maxillary osteonecrosis. immunohistochemical analysis suggests that the involvement of soft tissues as the initiator of osteonecrosis development is less probable than has been pointed out. objective: the aim of this study was to evaluate the expression of cd a, cd , cd , cd , caspase- , tryptase and basement membrane thickness in oral lesions of lichen planus (lp) and lupus erythematosus (le). method: oral lesions of lp (n= ) and le (n= ) were biopsied. after confirmation of diagnosis, the specimens were submitted to pas and immunohistochemistry. results: cd a expression was significantly greater in epithelium and connective tissue in lp. epithelial cd and cd did not differ between the diseases, but were greater in connective tissue in lp. cd expression was greater in both epithelium and connective tissue in lp. caspase- did not differ between the groups, whereas tryptase was greater in le epithelium. basement membrane thickness did not differ between lp and le. conclusion: cd a, cd , cd , cd , caspase- and tryptase expression and the basement membrane thickness are not definitive criteria for differential diagnosis of oral lichen planus and lupus erythematosus. objective: odynophagia may be caused by inflammatory/ infectious conditions and tumors arising within the oropharynx. we present an unusual case of a follicular dendritic cell sarcoma (fdcs) of the tonsil manifesting with odynophagia. method: an year-old man presented with odynophagia refractory to medical therapy; a . cm left tonsillar mass was noted on inspection. bilateral tonsillectomy was performed. a panel of antibodies was applied on paraffin tissue section. results: grossly, the left tonsil featured a . cm well circumscribed grey lesion. histologic examination showed a proliferation of spindle to ovoid cells arranged in fascicles with swirling qualities admixed with scattered lymphocytes. frequent mitoses and apoptotic bodies were seen but no "tumoral" necrosis was recognized. tumor nests were separated by thin stromal septi containing a prominent glioblastoma-like microvascular proliferation with focal formation of glomeruloid tufts. the tumor cells were immunoreactive for cd , cd and vimentin. objective: salivary gland tumors reveal a broad morphology and immunohistochemistry may be helpful. method: we examined mucoepidermoid (mec), adenoid cystic (adc), asinic cell (acc) and salivary duct carcinomas (sdc); myoepitheliomas (me), basal cell (bca), pleomorphic adenomas (pa) and warthin tumors (wt) for sma, calponin, s , cd , gfap, p , cea, gcdfp , glut , be , ck , ck , cd and galectin , using tissue microarray. results: tumors expressing myoepithelial markers were adc (sma, calponin), bca (sma, calponin, p ), pa (sma, calponin, s , cd , gfap, p ), me (s , cd , gfap), mec (p ) and wt (p ); pa being the only tumor expressing all of the markers in the panel. gfap, s , ck , p , ck / and galec-tin expressions were higher in benign and ck was higher in malignant tumors (p < . ). some of our findings helping in differential diagnosis are as follows: in differentiation of pa from adc: gfap, cd , gcdfp positivity; higher expression of s ; lower expression of ck and cd favors pa. in differentiation of pa from bca: gfap and cd positivity favors pa. in differentiation of mec from sdc: cd , ck , p and ck / positivity favors mec. conclusion: we conclude that salivary gland tumors may be well characterized by using distinct markers in each differential diagnosis. objective: lobulary capillary hemangioma (lch) is a benign, rapidly growing vascular lesion of the skin and mucous membrans. it may rarely present as a mass of considerable size and thus entirely fill the nasal cavity. it occasionally appears in the nasal region as a pedunculated or broad base mass. trauma and hormonal influences are the most common presumed etiologic factors. lch usually involves the gingiva, lips, tongue, and buccal mucosa. the most common symptoms are epistaxis and nasal obstruction. method: in this report; a case of years old boy operated surgically of lcd of the nasal septum, which occurred post delivery was described. results: on examination, a red pedunculated bloody swelling , * cm was noted arising from the posterior part of the nasal septum to nasopharinx. histopathologically lch has characteristics consistent with lobular prolifeation of capillaries in a fibromyxoid stroma. conclusion: we emphasise that rarely seen lch must be kept in mind in the differantial diagnosis of a rapidly growing mass, who is cured surgically. follow up months later showed no recurrance. although the head and neck is not an uncommon region, the nasal cavity is extremely rare sites for lch in children. objective: claudins are integral transmembrane proteins of tight junctions, critical for maintenance of cell adhesion and polarity. altered claudin expression has been detected in carcinomas and correlates with tumour progression. actinic cheilits is a pathologic condition that affects the lower lip, caused by chronic exposure to solar radiation; it corresponds to the early phase of squamous cell carcinoma. we investigated claudin patterns in phases of actinic cheilitis, as alterations in adhesion molecules are considered important during the progression of this process. method: immunohistochemistry against claudins − , − , − , − and − was performed in cases of actinic cheilitis/squamous cell carcinoma; results were analysed qualitatively. results: actinic cheilitis (low grade intraepithelial squamous cell carcinoma), and the invasive front of superficially invasive squamous cell carcinoma and in situ squamous cell carcinoma were negative for claudins − , − and − . claudin − was present on all epithelial layers in most cases evaluated. claudin − was present in all epithelial layers in actinic cheilitis, but negative in cases of superficially invasive and deeply invasive squamous cell carcinoma. conclusion: altered expression of claudins is present in ac from its incipient phase throughout the progression of the disease to invasive squamous cell carcinoma. objective: metastases to the jaw bones are uncommon and are most likely to arise from primary lung, breast, prostate or kidney tumours. jaw bone metastases from a primary oesophageal carcinoma are especially rare, with only seven reports published in the literature. method: here, we describe the case of a year-old male patient where years elapsed between the diagnosis and successful treatment of a poorly differentiated, stage pt n primary oesophageal adenocarcinoma and re-presentation with jaw pain due to a metastatic mandibular deposit. results: the morphological appearance of the metastasis and immunohistochemical positivity with ck , ck and cdx strongly supported an adenocarcinoma of upper gastrointestinal tract origin. conclusion: this case is of particular interest as there is an unusually long time between the detection of the primary oesophageal adenocarcinoma and diagnosis of metastatic disease. the longest period of time we have found for this reported in the literature is months, although it is known that some oral metastases may appear more than years following the primary tumour diagnosis. melanotic oncocytic metaplasia of the nasopharynx -a report of three cases and review of the literature j. s. lee * , j. y. na * chonnam national university, hwasun hospital, pathology, jeollanam-do, republic of korea objective: melanotic oncocytic metaplasia of the nasopharynx is a rare condition which is characterized by the presence of usually a small, brown to black colored pigmented lesion around the eustachian tube opening. although it is a benign lesion, it may be clinically misdiagnosed as malignant melanoma. microscopically, melanotic oncocytic metaplasia is a combination of oncocytic metaplasia of the epithelium of the gland and melanin pigmentation in its cytoplasm. method: in our present study, we report three cases of melanotic oncocytic metaplasia of the nasopharynx. results: all the three cases occurred in men and were presented as multiple black pigmented lesions around the torus tubarius. microscopically, mucous glands with diffuse oncocytic metaplasia and numerous black pigments were observed. no cellular atypia was observed. immunohistochemically, the scattering of s- proteinpositive, and hmb- -negative dendritic melanocytes was evident. conclusion: this is the first report of cases of oncocytic metaplasia of the nasopharynx in korea. objective: angiokeratoma is a rare cutaneous lesion. mucosal involvement is occasionally found as part of a more generalized cutaneous disease. isolated angiokeratoma in the oral mucosa is extremely rare with only a few cases reported thus far. method: we report the case of a -year-old female presented with a painless lesion on the left bucal mucosa, of months duration. on clinical examination, a solitary purple lesion of approximately mm in diameter was found. results: the lesion was excised and the histological examination revealed a tumour involving lamina propria, composed of large dilated vascular spaces, lined by normal appearing endothelium and filled with blood or with fibrin thrombi. the overlying epithelium showed variable degree of acanthosis and hyperkeratosis. accordingly the diagnosis of angiokeratoma was made. on clinico-laboratory examination no angiokeratomas were found anywhere on the skin as well as no other malformation or metabolic disorder. the patient received no further treatment and years later remains disease free. conclusion: in conclusion we report a case of a solitary angiokeratoma of the oral mucosa. although rare, it should be included in the differential diagnosis when evaluating any lesion in this location and further investigations should be performed to rule out a metabolic or systemic disease. results: histopathologically in macroscopy, the cystic lesion was determined as a small part of tissue in white -grey color, in sizes of , × , × , cm. in microscopic evaluation, the epithelial tumor cells were formed mostly tubular, partially in solid form and broadly making pseudopapillary pattern in stromal tissue. in the luminal structures formed by tumor cells, there has been mucin as dark basophilic stained with pas/alcian blue (ph: , ). the tumor cells are strong and have common positivity with ck , and have negativities with ck (b-sa method). in the focal area that the tumor cells got the solid pattern, synaptophysin and chromogranin to evaluate the neuroendocrine differentiation wasn't determined. conclusion: the lesion in the medial part of the tympanic membrane of the patient was reported as colesteatom and the lesion in the middle ear was reported as middle ear adenoma. histopathologic evaluation for helicobacter pylori as a possible etiopathogenic factor in chronic tonsillitis e. Özgün * , d. altinel, a. albayrak, a. tan, s. sayhan, n. bozlak * nevehir devlet hastanesi, dept. of pathology, turkey objective: helicobacter pylori is the major gastric pathogen which has an important role in the etiopathogenesis of chronic gastritis. we investigated the presence of helicobacter pylori as an extragastric reservoir in the tonsillectomy specimens to display if it is an etiologic factor in the development of chronic tonsillitis. method: in the current study, cases with chronic tonsilitis were examined in bilateral tonsillectomy specimens. objective: salivary duct carcinoma (sdc) is a rare, aggressive malignancy with poor prognosis. its histomorphology is distinctly reminiscent of the ductal carcinoma of the breast. method: a -year-old man was admitted with a mass of the right preauricular area. the mass had been enlarging steadily for the past months. computed tomography (ct) of the neck revealed × × cm contrast enhancing solid mass with irregular borders at the right parotid region. thorax ct, abdominal and thyroid ultrasonography were unremarkable. as the aspiration cytology was malignant right parotidectomy and right cervical lymphadenectomy was performed. results: histopathological examination showed a mixture of ducts, nests and cords of cells often embedded in a desmoplastic stroma with comedonecrosis of some ductal structures. the tumor cells were polygonal with vesicular nuclei, prominent nucleoli and eosinophilic, oncocytic cytoplasm. the tumor margins were lobulated and irregular with perineural invasion. mucin stains were negative. we noted high proliferative index and cerbb overexpression. the tumor was classified as a salivary duct carcinoma, oncocytic variant of parotid gland. the resection margins were negative and the lymph nodes were reactive. conclusion: as high proliferative index and cerbb overexpression are predictive factors, close clinical follow up is recommended for the risk of local recurrences and metastasis. immunohistochemical expression of bcl- and ki- in oral lichen planus and leukoplakia with different degrees of dysplasia f. pigatti * , l. a. de assis taveira, c. t. soares * university of são paulo, oral pathology, brazil objective: the oral lichen planus (olp) is a chronic inflammatory disease of unknown cause, and its malignant potential is a very controversial issue. therefore, the aim was to evaluate the immunohistochemical expression of apoptosis-related proteins and cell proliferation in olp and epithelial dysplasia in order to investigate changes related to carcinogenesis and emphasize the importance of long-term follow-up of patients with olp. method: for this purpose, we selected samples of olp, samples of leukoplakia with epithelial dysplasia, and samples of normal oral mucosa as controls. the evaluation of the expression of bcl- and ki- was conducted in accordance with the immunoperoxidase technique. results: there was also a high expression of blc- protein in inflammatory cells in olp lesions and leukoplakia with epithelial dysplasia. the expression of ki- marker was higher in all analyzed tissue levels in the lesions of olp and leukoplakia with epithelial dysplasia when compared with the control group. objective: pleomorphic adenoma (pa) is the most common benign salivary gland neoplasm of the major and minor salivary glands. pleomorphic adenoma was shown sometimes to undergo malignant transformation in its natural course. carcinoma ex pleomorphic adenoma (cpa) is a rare salivary gland malignancy that may develop from either a long-standing primary or a recurrent pa. the genetic mechanisms involved in the progression of adenoma to a carcinoma is still unclear. to identify the predictors of disease, more knowledge about their genetic profiles is necessary. this study aimed to characterize alteration in the dna copy number of pa and cpa. objective: leukoplakia is an oral premalignant white lesion. although its etiology may vary, smoking has been implicated as a possible risk factor. the p protein has been shown to inhibit kinases, and it is known that its expression is decreased during carcinogenesis. the reduced expression of p has been correlated with poor prognosis in carcinoma. in this study a role for the smoking habit in the expression of this protein was investigated. method: forty cases clinically diagnosed as oral leukoplakias and that presented a mild to intense degree of epithelial dysplasia and could not be diagnosed as any other disease were selected. twenty cases were from current smokers (more than cigarettes/day for at least year) and neversmokers. thirty-six cases of leukoplakia without dysplasia were used as controls. histological sections of each lesion were subjected to the estreptoavidin biotin immunohistochemical method for detection of p . results: a semi quantitative analysis was performed and the results showed that the expression of p was independent of the smoking status of the patient (p= , ), using kruskal-wallis and mann-whitney tests. although not statistically significant, due to the small number of cases, the results indicate that the counting of p in leukoplakia correlates inversely with degree of epithelial dysplasia. low-level laser therapy may influence on the akt/mtor signaling pathway in oral cancer cells f. sperandio * , f. giudice, l. correa, d. pinto jr., s. de sousa * university of sao paulo, dept. of oral pathology, brazil objective: distinct cells respond differently to low-level laser therapy (lllt), while the exact molecular mechanisms involved in cell proliferation or growth inhibition, after light stimulation remain poorly understood. although lllt has shown promising results in accelerating wound healing and preventing or treating oral mucositis, there is no evidencebased consensus of what this energy could cause in cancer cells. this should be highly pondered when an oral cancer patient is treated with lllt due to oral mucositis, for example. method: two tongue squamous cell carcinoma cell lines (scc and scc ) were utilized to find the effect of low-level laser irradiation on the akt/mtor signaling pathway. laser irradiation ( and nm) consisted on mw of power and energy densities of , and j/cm . after a single irradiation the most significant energy densities found with mts assay were employed to analyze akt/mtor signaling pathway related proteins through immunofluorescence and western blotting. results: beyond modifying the growing rates of cancer cells, low-level laser irradiation was able to induce different variations in the studied pathway, however a direct correlation among the proteins was not found. conclusion: lllt may act on akt/mtor/cyclin d signaling pathway, which has a widely recognized role in head and neck cancer progression. mucins as predictors of recurrent pleomorphic adenoma of salivary glands: an immunohistochemistry analysis of over cases t. teshima * , r. ianez, c. coutinho-camillo, s. lourenço * são paulo, brazil objective: pleomorphic adenoma (pa) is the most common tumor of salivary glands and, despite its benign behavior, the recurrence after primary surgery is significant. in attempt to find a marker capable to predict the recurrence of this lesion, this study aims to analyze the expression of mucins muc , , , ac and in cases of pa, considering that mucins have been related to tumour growth of some organs. method: this study was performed in cases in pa, which of them presented recurrence after the initial surgery. all the primary tumors were histologically processed and submitted to the immunohistochemistry reaction. the antibodies for muc , , , ac and were used and then analyzed with conventional optical microscope. results: muc was the only mucin significantly expressed in most of all cases ( %), being present within ductal lumen and cytoplasm. the other mucins showed a focal positivity in few cases, where muc , ac and were cytoplasmic, while muc was expressed in ductal lumen and blood vessels. objective: drug resistance remains a major problem in the treatment of nsclc cancer patients for both, conventional chemotherapeutic and novel biological agents. intrinsic or acquired resistance is caused by a variety of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. recent data showed that drug resistance mechanisms might also be regulated by micrornas (mirnas). method: we tested lung cancer samples. the total rna was isolated from individual specimens and then rt-pcr and pre-amplification were performed. we detected levels of mir- , mir- a, mir- b, mir- , mir- , mir- *, mir- , mir- - p, mir- p, mir- x, mir- - p, mir- p, mir- , mir- and mir- using taq-man® microrna assays by lightcycler® real-time pcr system. we assigned obtained results using statistical methods. results: our results suggest that mir- - p and mir- are involved in apoptosis. the higher levels of mir- - p correlate with bcl- positivity and pro-apoptotic protein bax seems to be regulated by mir- . the level of lrp protein responsible for drug resistance in lung cancer patients seems to be regulated by mir- . conclusion: within our cohort of nsclc patients we did not find any correlations between the expression profiles of the abovementioned mirnas and survival. the modified methacarn fixation as an excellent preservation of histology, protein immunoreactivity and rna integrity in paraffin embedded tissue specimens p. babal * , r. milcheva, p. janega, p. celec * comenius university, dept. of pathology, bratislava, slovakia objective: fixation techniques preserving morphological fidelity, immunoreactivity and integrity of nucleic acids may have a high impact on both basic and applied research and diagnostic pathology. we investigated the effect of formalin, absolute ethanol and methanol; ethanol supplemented with acetic acid and modified methacarn fixative on the tissue morphology and immunoreactivity of different types of tissues and the preservation of rna fragments of different lengths. results: the modified methacarn fixation provided a histomorphological quality comparable to the formalin-fixed tissue specimens. the immunoreactivity was superior in the buffered than in the untreated formalin and the preservation of protein antigenicity in normal and pathologically changed tissues tested with several antibodies was excellent with the use of alcohols with acetic acid. the acidic ethanol and the modified methacarn fixative showed the best preservation of the integrity of rna in satisfactory quantity and quality of fragments up to bp, which was reliable for relative evaluation of gene expression. objective: cflip prevents the apoptosis by caspase inhibition, its overexpression correlates with the progression of different tumors. thymomas and thymic carcinomas are thymic epithelial tumors, in which the regulation of apoptosis is still unknown. we investigated the role of cflip in regulating the viability of thymus carcinoma cell line c using an rna interference. the cell line hacat was used as a control. method: c and hacat cell lines were transfected with an established "short hairpin" pires/puro cflip-shrna expression vector. cflip suppression and its effect on pro and antiapoptotic molecules were analyzed using q-pcr and western blot. apoptosis was quantified using the flow cytometry. results: the c-shcflip, but not the shcflip-hacat, showed sensitivity to trail (tumor necrosis factor-related apoptosis-inducing ligand), accompanied by an overexpression of both the pro-apoptotic protein noxa (p< - ) and the anti-apoptotic proteins birc , birc and xiap compared to the non-transfected cells (p< - ). conclusion: cflip shrna seems to induce apoptosis in the thymus carcinoma cell line. simultaneously antiapoptotic proteins (aips) birc , birc and xiap were up regulated in order to protect the cell from apoptosis. could these aips provide the way to escape the cell death? aips selective inhibitions could represent a promising therapeutic approach for malignant thymic carcinomas. objective: nucleolin is a multifunctional dna-, rna-and protein-binding protein, involved in fundamental aspects of transcription, cell proliferation and growth. it is located in the nuclei/nucleolus, cytoplasm and on the cell surface. the present study aimed at optimizing the histological identification of nucleolin. method: mamary invasive ductal carcinoma μm paraffin sections were pretreatead with pronase at room temperature ( - min) to epitope exposure. biotinilated peptide against nucleolin was applied ( / . mm dilutions), incubated at room temperature for min/overnight at °c; after phosphate-buffered saline, peptide binding was identified by peroxidase-conjugated streptavidin and , -diaminobenzidine tetrahydrochloride applied according to manufacturer's instructions before haematoxylin counterstaining. results: pretreatment for min at . mm of biotinilated peptide showed only nuclear expression, as mm/ min pretreatment with overnight peptide incubation; positive cytoplasmic expression was obtained also after min incubation/room temperature. at . mm concentration for min epitope retrieval, cytoplasmic and nuclear positivity raised over % expression, independently of peptide exposure (overnight/room temperature). conclusion: for optimizing peptides/antibodies, we have to be aware that nuclear/cytoplasmic expressions depend on digestion and peptide concentration. histological morphology is also important and discordant results may be erroneously obtained. ps- - kras testing in clinical laboratory: optimizing targeted therapy l. cheng * , l. miravalle * indiana university, dept. of pathology, indianapolis, usa objective: activating mutations in the kras gene are found in more than % of colorectal tumors, where they are associated with a poor response to anti-epidermal growth factor receptor therapies. mutation testing techniques have therefore become an urgent concern. several methods for kras mutation detection have been described in the literature. most of these are laboratory developed tests and only a few commercial assays are currently available. method: we studied the performance characteristics of a kras mutation detection assay on the abi- xl genetic analyzer using a new commercial mutation detection kit. samples were analyzed in parallel by different reference laboratories using alternative methodologies. various sample types were used including formalin-fixed paraffin-embedded tissue, fine-needle aspirates, and cyst fluid specimens. results: a high level of agreement ( % correlation for formalin-fixed paraffin-embedded tissue and fine-needle aspirate samples and % correlation for cyst fluid specimens) was obtained despite the use of different methodologies. conclusion: shift termination assay is a simple, robust, and sensitive method for the identification of kras mutations in wide variety of specimen types. objective: micrornas (mirs) are small rnas that modulate protein expression via post-transcriptional regulation of mrna. they are related to malignancy in several tumors. deregulation of mirs expression has been described in high grade astrocytomas of adult patients, however there is scarce information in pediatric patients. in this work we quantified the expression profiles of mirs in pediatrics astrocytomas. method: total rna was extracted from astrocytomas and normal brain (nb) tissues, formalin fixed paraffin embedded. the expression levels of mir- , mir- - and mir- were quantified by using mirna-specific taqman mirna assays. results: the mir- - was more abundant in nb against to mir- and - (p< . ). mir- and mir- - were significantly down-regulated in all grades compared to nb (p< . ) but in grade iv was more decreased: -and -fold, respectively. in patients with recurrent tumor, the expression of mir- was lower (p< . ). live patients expressed high levels of mir- (p< . ) and mir- (p < . ) against deceased. conclusion: micrornas are differentially expressed between astrocytomas and nb. the low expression of mir- and - could be a potential marker in recurrent pediatric astrocytomas, and both are associated to less survival. embedded tissue is useful to describe and evaluate expression of molecular markers of malignancy. objective: isolation of genomic dna from formalin fixed paraffin embedded (ffpe) tissues is a critical step for molecular diagnostic assays. horizon diagnostics has generated ffpe cell line reference standards containing defined mutant allelic frequencies, enabling the quality control of both assay sensitivity and dna extraction. method: a panel of x-man™ (gene-x, mutant and normal) cell lines were developed using our patented gene editing technology (genesis™) including; b-raf v e, v k; egfr Δe -a , t m, l r, l q; k-ras g a, g c, g d, g r, g s, g v, g d; pi kα e k, e k, h r. ffpe blocks containing specific allelic frequencies including: %, %, %, % or % were generated and sections cut. dna was extracted using five different extraction methods and analysed using droplet digital™ pcr. results: mutant alleles could be detected using droplet digital™ pcr at each defined allelic frequency and the reproducibility of each test was very high. the allelic frequency was consistent throughout each block. the total dna yield from each section was consistent using the same extraction method but varied between methods. conclusion: this study has demonstrated horizon diagnostic's ffpe reference standards have a highly accurate defined mutation specific allelic frequency together with a consistent dna quantity. transcript variants and isoforms of the phosphatase subunit ppp ca and its regulatory binding partners in haematological malignancies g. grech * , c. saliba, b. shawn, r. avellino, p. j. m. valk, r. delwel * university of malta, dept. of pathology, msida, malta objective: the importance of feedback mechanisms involved in suppression of growth factor-induced signals is gaining importance both to understand molecular mechanisms of disease and also as potential therapeutic targets. our previous studies show that erythroid differentiation can be blocked by constitutive expression of the pp a inhibiting subunit, alpha . the aim of this study was to identify variants and transcript isoforms of ppp ca and the inhibiting subunits alpha and set, using ( ) cell lines derived from haematopoietic disease, and ( ) objective: epithelial cells grow by proliferation and adhesion, which will form an organ/tissue. stat is involved in cell proliferation and various cellular events. in the present study, we studied the role of stat in cell adhesion in hepatocytes. method: stat knockout cells (s ko cells) and liverspecific stat ko (l-s ko) mice were generated. cellular adhesion was analyzed by light/electron microscopy. expression of adhesion molecules was examined by immunohistochemistry and western blot analysis. protein expression and cellular adhesion were also examined in the pre-/post-hepatectomy liver tissue in control and l-s ko mice. results: mrna and protein of e-cadherin were not expressed in s ko cells. il- up-regulated e-cadherin in control cells but not in s ko cells, and induction of constitutively active mutant of stat restored e-cadherin level in s ko cells. interestingly, membrane-bound beta-catenin expression was not affected, but released to cytosol in s ko cells. s -ko cells showed almost normal proliferation but did not form cell cluster due to lack of e-cadherin. electron-micrograph confirmed lack of intercellular adhesion structures (desmosome) in s ko cells. also in l-s ko mice, hepatocytes lack desmosome structure and cell adhesion. objective: alk positive anaplastic large cell lymphoma (alcl) is characterized by anaplastic lymphoma kinase (alk) expression, most commonly associated with the t ( ; )(p ;q ), fusing the alk and nucleophosmin (npm) genes. however, in a significant proportion of cases the alk gene has a number of other than npm translocation partners. the aim of the study was to detect the npm/ alk fusion gene and the other possible fusion genes. method: npm/alk was detected by rt-pcr, npm/ alk negative lymphomas were analyzed by alk specific rapid amplification of ´cdna ends ( ´race). we prepared q-rt-pcr assay for quantification of Á lk mrna. molecular findings were correlated with i-fish. results: we analysed alk positive alcl. chromosomal breakpoints affecting the alk locus were detected by i-fish in all patients. the npm/alk was detected in / patients. ´race identified atic/alk in , cltc/alk in patients. other fusion genes (tpm /alk, tpm /alk, alo /alk) were found in one patient. in all specimens, overexpression of ´alk mrna suitable for the minimal residual disease (mrd) detection was found. objective: a year-old girl, presented with an osteolytic epiphyseal lesion of the distal ulna. based on histopathological examinations, the lesion was classified as giant cell tumor of bone (gct). four years later, the girl developed a second lesion at the same site. the histopathological examination revealed aneurysmal bone cyst (abc) with solid spindle celled and giant celled areas which raised a question of a recurrent gct or a primary abc. the aim of this study was to analyze the clonality of these two processes. determination of maternal and paternal x chromosome activation status is useful in the diagnostic analysis of nonrandom x inactivation patterns. method: the human androgen receptor (humara) gene polymorphism assay was used to identify the clonality of these two processes. results: the patient was identified as heterozygous for the humara allele. the gct and abc samples exhibited a monoclonal pattern, but one of them was with maternal and the other with paternal x chromosome origin. conclusion: the findings of this case report demonstrate the clonal behavior of both lesions with different clonal patterns. the above investigation proved to be helpful in distinguishing between recurrent gct and de novo abc in the field of previous surgical intervention. . in all specimens gen her /neu was quantifed agains reference gen by qrt-pcr method (lyghtcycler® ii, roche). results: the results of cytogenetic analysis have been showed in % that didn´t corespond with the results of qrt-pcr quantification. on the contrary the results of ihc was correlated with the results of qrt-pcr in %. methods sish vs fish has in % different of results. in regard to the quality of ish signals the sish is more preferable for determination her /neu. conclusion: a pilot study conducted on a group of patients showed that among the methods used in the process of determining her /neu amplification are significant differences, even in evaluation and interpretation of test results. regardless of the histological type of tumors (diffuse and intestinal type) as problematic for the evaluation of samples proved to be endoscopic route. the use of molecular methods in diagnosis of malignant melanoma of biopsy s. libor * , p. dundr, s. lísová, c. povýšil * general faculty hospital, inst. of pathology, prague, czech republic objective: distinction between benign and malignant melanocytic lesions commonly represents a big challenge for the pathologist. in this case it appears as a useful auxiliary method of fluorescence in situ hybridization (fish). the aim of this study was by using fluorescently labeled probes detection of numerical changes occurring in malignant melanoma and thus supply or confirm the diagnosis. method: the retrospective study included samples tissue with an established histological diagnosis ( x malignant melanoma, benign melanocytic lesions x). for detection was used probe vysis lsi rreb /lsi myb/lsi ccnd / cep (abbott molecular, usa), olympus ax fluorescent microscope (immersion lens x). in the area of the tumor were calculated signals of individual probes and evaluated according to the manufacturer's instructions. results: evaluable results were obtained in / cases ( %) of the melanomas were and benign melanocytic lesions. in all four cases of malignant melanoma have been burdened with genetic changes, mainly in the ccnd (in %) gene amplification and preb (in %) and myb (in %). in contrast, for all benign melanocytic been demonstrated normal findings. conclusion: dna abnormalities detected by fish occurr in the vast majority of malignant melanomas but are not seen in benign nevi. this fact make the fish test an important extra step in the differential diagnosis melanocytic lesions with ambiguous or borderline histological findings. objective: egfr is a receptor on the cell membrane with tyrosine-kinase activity and which is a regulator of proliferation, apoptosis, angiogenesis, tumor invasion. he is found to be overexpressed in some lung cancer histological subtypes. men is a tumor suppressor gene, with a role in cellular growth and differentiation, dna reparation, and apoptosis. method: surgically resected specimen from patients (men n= ; women n= , age ± ) with lung cancer were studied: carcinoid tumors (ct)- , small cell lung carcinomas (sclc)- , large cell neuroendocrine carcinomas (lcnec)- , adenocarcinomas (ac)- , and squamous cell carcinomas (scc)- . the histological subtype, ptnm stage, and gene expression of men and egfr in tumor and normal lung tissue were evaluated. results: overexpression of egfr was observed in %(scc- %, ac- %, sclc- %, lcnec- %, ct- %). decreased expression was observed in %(lcnec- %, sclc- %, ct- %, ac- %, scc- %) (p< . ). overexpresssion of men was observed in % of all tumors (sclc- %, ct- %, scc- %, lcnec- %, ac- %) (p> . ). significant correlation between overexpression of men and early stage scc was observed (p= . ). conclusion: egfr is a target for therapy with monoclonal antibodies, so the tumors that overexpress egfr can be considered for treatment with these drugs. men can eventually be a marker for good prognosis and a potential target for therapy. objective: homeobox genes encode transcription factors controlling cellular proliferation and differentiation. altered expression of prox homeobox gene is related to many cancers, including breast, esophagus, lymphatic and oral. method: after overexpression of prox gene in scc cell line, total rna was extracted from three overexpressing-prox clones (oc) and one control-transfection cell clone (cc). microarray analyses were performed using the whole human genome k according to the manufacturer's instructions. genes with a fold change of greater or lower than . in oc versus cc, were considered as increased or decreased, respectivelly. gene ontology (go) was used to assign biological process related to significantly differentially expressed genes. results: down-regulated genes mmp , timp and notch were further validated by qrt-pcr. comparative gene analysis of oc and cc revealed up-regulated and down-regulates genes. pathways induced upon prox overexpression in go terms included vascular development control, cellular adhesion, regulation of proliferation, among others. mmp , timp and notch expression by qrt-pcr showed reduced expression leves in oc. these genes are commonly overexpressed in oral squamous cell carcinoma and have been related with metastatic tumors and worse prognosis. objective: barrett´s esophagus is the unique known precursor for esophageal adenocarcinoma (eac), in a gradual progression to dysplasia. our objective is to determine the correlation between the hypermethylation in cpg islands of the promoter region of p tumoral suppressor gen in epithelial dna and the histopathological pattern, as possible biomarker for risk of progression. method: . a study was performed about the evolution of precursor lesions, in a group of patients diagnosed following the vienna classification. . p hypermethylation is analyzed in paraffin embedding samples, through laser microdissection, dna extraction, pcr amplification, pyrosequencing and quantification. results: . of cases with to biopsies remained as nd, id or lgd, and cases progressed to hgd and/or eac. . within the control group the methylation grade is . % and in the diagnostic groups is . % (nd), % (id), . % (lgd), . % (hgd) and . % (eac). conclusion: . % remained stable, but those reaching hgd all progressed to ace (only in % of the latter, hgd is recognized with h&e). . methylation´s grade is higher in all diagnostic groups comparing to the control group, progressively increased as the dysplasia grade found with h&e, so it may be a good biomarker for neoplastic progression. objective: in high risk hpvs the expression of oncoprotein e is responsible for the degradation of p , while e inactivates prb and causes the progression to s phase of cell cycle, both sustaining the conversion to and the maintenance of malignancy. the aim of this study is to compare the performance in the detection of the e and e mrna expression of hr-hpv using the nuclisens easy q test (biomerieux) or hpv oncotect (incelldx) a test based on flow cytometry-fish method. method: we enrolled patients positive for hr-hpv dna and/or pap smears. all subjects underwent a colposcopy histological evaluation and were tested both for the nuclisens easy q test and hpv oncotect. results: out of subjects resulted positive for rna expression. the patients were divided into two cohorts based on the histological diagnosis: low grade lesions (cin ) and high grade lesion (cin +). patients with cin were with nuclisens easy q test and with hpv onco tect, those with cin + were with either tests. conclusion: these preliminary results suggest that hpv oncotect have a better specificity than nuclisens easy q test, while more samples need to assess the difference in sensitivity. detection of pik ca/akt mutations in human meningiomas a. saetta * , g. tomara, i. chatziandreou, p. tsioli, e. el-habr, i. sakalidou, g. vretakos, e. boviatsis, p. korkolopoulou, e. patsouris * university of athens, st dept. of pathology, greece objective: the pi k/akt pathway is a major signaling pathway frequently activated in human cancer due to pik ca and akt gene mutations thus representing a potential therapeutic target and prognostic biomarker. in this study, we examined the mutational status of pik ca and akt in meningiomas. method: meningiomas were screened for activating mutations in "hot spot" exons , of pik ca using real time pcr and high resolution melting analysis. pik ca wild-type samples were analyzed for mutations in exon of akt . the mutations were verified by sequencing and/or pyrosequencing results: mutations were detected in out of samples ( %) in exon of pik ca and were identified as p.e k and p.s f. regarding exon , out of samples ( , %) showed the following mutations: p.r q, p.t t, p.h v, p.m i, p.h r ( cases), p.h t. finally, in exon of akt , mutant cases were detected ( %) all identified as p.e k. in % of the patients the activation of the pi k/akt pathway is due to mutations in the two examined genes. conclusion: aberrant activation of the pi k/akt pathway due to pik ca and akt mutations is commonly observed in human meningiomas and these genes could be considered as potential targets in new therapies for cancer. study of pi k/akt/mtor pathway in urothelial bladder carcinoma a. saetta * , n. prekete, e. trigka, g. levidou, m. karlou, p. pavlopoulos, p. korkolopoulou, e. patsouris * university of athens, st dept. of pathology, greece objective: deregulation of the pi kinase-akt/mtor pathway is a frequent event in tumorigenesis. we examined the possible significance of the components of this pathway in bladder urothelial carcinoma (uc). method: cases with bladder uc were screened for mutations in exons , of pik ca gene and exon of akt by pcr-sscp, hrma, sequencing and/or pyrosequencing. the expression of p-mtor, p- e-bp , p-p s k, p-akt, fgfr and p-erk was evaluated by immunohistochemistry. results: , % of the cases were mutant in pik ca gene and % in akt . cases with wild type akt displayed higher fgfr receptor expression (p= . ). p- e-bp expression was more frequent in low grade (p= . ) and in pta-t tumors (p = . ). furthermore, superficial tumors presented higher levels of p-p s k expression (p = . ) and lower levels of p-akt expression (p = . ). in multivariate survival analysis, p- e-bp immunoexpression emerged as an independent prognostic factor of adverse survival (hr = . , p= . ), along with tumor grade and t-category. conclusion: activation of pi k/akt/mtor pathway in bladder uc is not exclusively related to the presence of akt and pik ca mutations. expression of p- e-bp could serve as an independent prognosticator. comparison of dna extraction methods of formalin fixed, paraffin-embedded archival tissues b. senguven * , e. baris, t. oygur * gazi university dental faculty, dept. of oral pathology, emek, turkey objective: formalin fixed, paraffin-embedded (ffpe) archival tissues are valuable resources for many molecular studies. the goal of this study is identify the optimal method for dna extraction from ffpe tissues. method: human gingival tissues which has obtained from patients with gingival hyperplasia were used. serial sections of μm thickness obtained using a standard microtome. half of the sections were deparaffinized on glass, the other half collected directly to a , ml tube. in order to identify the optimal method for dna extraction, we compared phenol-chloroform protocol and dna extraction mini kit. the duration of proteinase k digestion were also compared. spectrophotometric evaluation of the yield and purity of dna was conducted. to deteminate the amplifiablity of extracted dna, three different bp fragment of the beta-globin gene were amplified using pcr. results: the phenol-chloroform method had the lowest yield and purity. deparaffinized specimens on glass, digested for h and isolated using mini kit had the highest yield. amplification of the bp fragment of beta-globin gene was successful in all samples. conclusion: according to our results deparaffinization on glass, increasing the time of proteinase k digestion and using commercial kits for isolation seems the best method to obtain amplifiable dna from archival specimens. microrna signatures associate with fallopian tubal implantation in humans r. shao * * gothenburg university, physiology and endocrinology, sweden objective: micrornas are small non-coding rna molecules that regulate a large number of cellular pathways and deregulation or altered expression of mirnas is associated with many disease states. the function of the fallopian tubes appears to involve orchestrated spatiotemporal alterations in transcriptome profiles, the regulation of tubal gene expression and function by mirnas may thus be of primary importance in tubal ectopic pregnancy. method: both implantation sites and non-implantation sites of fallopian tubes from women with ep and decidual biopsies from women undergoing therapeutic surgical termination of pregnancy were collected and analyzed by mirna array. the unique mirna profiling results were validated by taqman qrt-pcr, and bioinformatics' analysis was employed to further predict the mirna targets. results: a total of mirnas were differentially expressed in implantation sites compared with those in non-implantation sites of fallopian tubes after comparison of decidual mirnas, among which were up-regulated while are down-regulated. the mir- was significantly increased, whereas let- i, mir- , and mir- were significantly decreased. differentially expressed mirnas were predicted to be related with several signaling pathways in normal intrauterine implantation. conclusion: our findings establish an mirna signature associated with tubal implantation and provide the experimental basis for further understanding the molecular and cellular mechanisms of initiation and development of tubal ep in humans. lung is an organ that can sense any perturbation of the air, due to its constant interaction (long live) of the air. our hypothesis was that lung could be an organ sensor of climatic change observed today. method: this preliminary study analyzed the expression of isoforms of the genes of hsp . the cases were chosen from archive of autopsies from to , with different diagnosis, and were matched in sex and age with a same number of cases chosen from to . the expression was studied by rt-pcr from lung tissue embedded in paraffin, samples of each decade. results: the expression levels of hsp were normalized to β-actine. we found expression in six cases of - : five for hsp aa - , and for hsp ab , one case expressed both. their matched cases in - never expressed any isoform. the cause of dead in all cases doesn't fit with any pattern. conclusion: with these results we propose than the lung could be a sensor of global warming, and the expression of these genes could be molecular markers of this climatic change. apoptosis associated genes and their role in predicting responses to neoadjuvant breast cancer therapy d. tvrdík * , h. skálová, p. dundr, l. stanek, c. povýšil, l. petruželka * general university hospital, institute of pathology, prague, czech republic objective: neoadjuvant chemotherapy is used in the treatment of breast carcinoma because it substantially reduces the size of the primary tumor and lymph node metastases. this present study is aimed at the investigation of biomarkers that can predict a pathologic response to the therapy. method: the transcriptional profile of key apoptosis genes was evaluated in both pre-therapeutically obtained tumor tissue by core needle biopsy and in specimens removed by final surgery, using a pathway-specific real-time pcr assay. results: on the basis of a hierarchical cluster analysis of significantly changed genes, we divided patients into good and bad prognosis groups, which correlate well with progression-free survival. in the good prognosis group, we found a statistically significant downregulation of the expression of mcl and igf r genes after neoadjuvant treatment. we also found a statistically significant overexpression of bcl l , bcl af , casp , casp , casp , cideb, fadd, hrk, tnfrsf , tnfsf and cd genes. in contrast, we found upregulation of igf r after the treatment in the group with poor prognosis. conclusion: as we have shown, gene expression profiling after neoadjuvant chemotherapy is a valuable research tool for investigating molecular markers, which may better reflect tumor biology and treatment response than standard prognostic and predictive factors. objective: non-small cell lung carcinoma (nsclc) is one of the most serious cancers. identification of genetic changes (mutations, amplifications or rearrangements) within egfr, kras and alk oncogenes, associated with nsclc, allows choosing the patients, benefit from biological therapy with tyrosin kinase inhibitors. method: dna is isolated from formalin fixed paraffin embedded specimens or cytology specimens. mutation detection is performed by real-time pcr, fragment analysis, primerextension analysis and mutant-enriched pcr. wt-egfr patients (e.g. with no mutation detected) are tested for alk gene rearrangement and egfr gene polysomy or amplification using the fluorescence in situ hybridization (fish) method. results: since / till / , dna samples were analyzed. out of these, patients ( , %) were found to be positive for activating mutations within egfr gene. since / till / , patients were analyzed for alk gene rearrangement, egfr gene polysomy or amplification. alk gene rearrangement has been proven in ( , %) cases, alk gene amplification in ( , %) cases and egfr gene polysomy or amplification in ( , %) cases. conclusion: determination of genetics changes in tumor can provide powerful tool for setting up strategy and therapeutic protocols in nsclc patients. objective: a year old gentleman, ex-smoker with a history of asbestos exposure presented to the emergency department with a day attack of severe abdominal pain and bilious vomiting. history of abdominal pain, lethargy, and weight loss over the last months and years of recurrent pleural effusion. pleural aspirates and a videoassisted thoracic biopsy were negative. method: x-ray confirmed small bowel obstruction and an emergency laparotomy performed. the terminal ileum was intussuscepted into the caecum causing proximal obstruction. a right hemi-colectomy was performed. results: on gross examination there was intussusception of the terminal ileum secondary to a polypoid mass at the ileo-caecal valve. histology showed an oncocytic tumour with neuroendocrine features arising from the serosa of the small bowel. the tumour cells were positive for ck , calretinin, and vimentin but negative for ck , chromogranina, cd and ttf- . the features were in keeping with a malignant peritoneal mesothelioma infiltrating the small bowel wall. conclusion: peritoneal malignant mesothelioma is an uncommon tumour which rarely causes mechanical small bowel obstruction. to our knowledge this is the first case of localised primary peritoneal mesothelioma presenting with intussusceptions. giant mesenteric cystic lymphangioma in adult; rare tumor, unusual location and uncommon age of occurrence: a tunisian case report s. attafi * , w. ajouli, n. bouchiba, a. chouchene, m. h. balti, k. bellil * fsi hospital, dept. of pathology, marsa, tunisia objective: cystic lymphangioma is a rare benign neoplasm arising from the lymphatic system. it occurs as a result of congenital malformations of the lymphatics, leading to the obstruction of local lymph flow and the development of lymphangiectasia. lymphangioma is common in pediatric patients, but it is extremely rare in adults, with only about cases reported in literature. most lymphangiomas are found in the head and neck; intraabdominal and specially mesenteric locations are very unusual. the aim of this work was to study its clinical, histological and therapeutic features and their diagnostic difficulties. method: we report the case of a years-old woman, who presented a painful syndrome of the right iliac fossae (rif). the physical examination found a mass of the rif. abdominal ultrasonography and magnetic resonance image showed a mesenteric cystic formation. at laparotomy, a large cystic tumor of the caecal mesentery, measuring cm, was found. histopathology showed a fibrous and thin walled cyst, lined by flat low lying epithelium with surrounding tissue of scattered lymphoid cells. results: the diagnosis of cystic lymphangioma was retained. conclusion: mesenteric lymphangiomas are very rare, but they can cause acute abdomen that requires an emergent surgery. therefore, they should be included in the differential diagnosis of cystic intra-abdominal lesions raises several possibilities, including both malignant and benign soft tissue tumours. objective: the spleen metastases from colon cancer are rare conditions and usually associated with extensive disease. there are only eight reports in english-language literature of isolated splenic metastases from colorectal carcinoma, which generally metastasize to regional lymph nodes, liver and abdominal peritoneum. method: we report a case of multiple splenic metastases in a -year-old woman. results: in november , the patient had undergone right hemicolectomy, lymphadenectomy and chemotherapy for stage iii tubular adenocarcinoma of the ascendant colon with positive pericolic lymph nodes. in december , she was admitted in our hospital for diffuse abdominal pain and intestinal transit disorders. computed tomography of the abdomen revealed multiple metastases of different sizes in liver and spleen. during the surgery, which was performed with the goal of curing metastatic disease, there were found two nodular hepatic secondary tumors, and numerous spleen metastases, with diameters from . cm to . cm. the histopathological evaluation of splenectomy specimen revealed a metastatic tumor deposit, histologically similar to the primary tubular adenocarcinomas of colon. conclusion: this is the ninth documented case of splenic metastasis from colon cancer. previously reported cases of this type were isolated tumor, this one being the first reported with multiple splenic metastases. the role of fatty acid synthase in inflammatory bowel disease (ibs) e. bas bozkurtlar * , n. ozkan, a. e. kedrah, c. celikel * marmara university, dept. of pathology, istanbul, turkey objective: it was demostrated that fas expression increases not only in mucosa involved by active colitis but also in normal mucosa of ulcerative colitis (uc) patients. our aims were to evaluate the role of fas expression in differential diagnosis of ibd, to search for any possible change during the progression of the disease in patients with clinical and endoscopic follow-up. method: among colonoscopic biopsy samples of uc cases, samples were classified as remission, as resolution, and as active period; samples of crohn disease (cd) cases were classified as active period. fifteen cases with normal endoscopic and morphologic findings comprised the control group. results: between uc and cd active periods and control group, a significant difference was found (p= , ). there was a statistical correlation between fas expression of basal crypt of normal mucosa in uc active period and the disease duration (p= , ). conclusion: the increasing in fas expression in patients with ibd can not be explained only by the inflammation. fas expression can not aid us in the differential diagnosis of uc and cd, but it seems that it is related to the extent of inflammation in uc. the relation between the disease duration and an increase in fas expression active uc, might have a significant role in carcinogenesis in uc. micropneumatosis -an (un) usual finding in gastrointestinal specimen? k. blaue * , m. plauth, j. knolle * städtisches klinikum dessau, pathology, dessau-roßlau, germany objective: a rather rare disease, micropneumatosis describes the findings of cyst-like cavities devoid of lining epithelium in the gastrointestinal wall. micropneumatosis is often secondary to intestinal bacterial infection or mechanical factors. this study evaluates the incidence of micropneumatosis in our institute. method: we analyzed all cases of micropneumatosis obtained in our institute between and . available data were retrieved from patients' records. results: out of . specimens obtained between and , cases ( . %) presented with micropneumatosis ( female, male; mean age years, age range to years). most common site was the colon ( cases), followed by stomach ( ), small intestine ( ) and greater omentum ( ). conclusion: micropneumatosis represents a rare but harmless diagnosis in gastrointestinal specimen. due to the histological picture, which may be confused with dilated lymphatic vessels, submucosal lipomas, and, to the untrained eye, even with signet ring cell carcinoma, incidence may even be higher. in unclear cases, where there seems to be a endothelial layer, immunohistochemistry may be necessary to confirm diagnosis of micropneumatosis. this study shows that micropneumatosis should be considered in differential diagnosis in every age and sex. predicting lymph node metastasis in pt colorectal cancer -a meta-analysis providing rationale for therapy decisions s. bosch * , i. nagtegaal * radboud university nijmegen, pathology, netherlands objective: we conducted a meta-analysis of published reports on the predictive value of risk factors for lymph node metastases (lnm) in pt colorectal cancer in order to provide a rationale for choosing follow-up or radical surgery after local excision. method: local excision is an attractive treatment option for colorectal cancer, but is only safe in the absence of lnm. several pathological factors have been associated with lnm, however it remains unclear how to integrate these in clinical decision making. results: a pubmed search revealed studies totaling patients. strong predictors of lnm were lymphatic invasion (rr . [ % ci . - . ]), budding (rr . [ % ci . - . ]), and high grade histology (rr . [ % ci . - . ]). deep submucosal invasion was also strongly associated with lnm, however in a risk stratification model this factor was of limited added value. conclusion: the absence of lymphatic invasion, budding and high grade histology may justify withholding radical surgery. the independent role of submucosal invasion depth is probably limited. models for risk stratification based on these factors need to be validated. objective: dendritic s + cells in intestines have been described in more studies. their connection to s + fibres of nervous system is lesser of an object of interest. method: we have processed the samples of intestinal mucosa of people with crohn's disease and colitis ulcerosa by the means of form-paraffine technique and anti s antibodies. results: in lamina propria and in submucosa at crohn's disease were s + the fibres of various width and orientation. outside of them the diffusely scattered round s + mononuclear cells could be seen. contrary to colitis ulcerosa the granulation tissue at crohn's disease contains in the area around abscesses a thick accumulation of s + cells and this is similar also in the formed lymphoid tissue. conclusion: the positive fibres can be seen in submucosa at the edge as well as in lpm between the bases of the glands. in between the plexus/ring and longitudinal muscle there are massive bands of connective tissue which contains bundles s + of ganglion cells and nerve fibres. the fibres are present in ring as well as in longitudinal muscle. in this area s + fibres can also be seen in vessel walls. numerous bundles of granulatory tissue do not contain positively reacting substance. in the basal part lamina propria there are visible s + cells as well as long, smooth, positively reacting fibres. ps- - ncf -deficient mice with impaired oxidative bursthave a more agressive progression of dextran sulfate sodium (dss) -induced colitis l. carvalho * , t. rodrigues-sousa, a. alarcão, a. f. ladeirinha, m. souto-carneiro * amadora, portugal objective: intestinal inflammatory disease (iid) as a primary immunodeficiency depends on mutations in the nadph oxidase complex, responsible for the production ofreactive oxygen species (ros). one of the most common clinical patterns in iid is chronic colitis. ncf -mutation in mice leads to deficiency in ros, rendering them susceptible to autoimmunity. here we studies how ros-deficiency in ncf -mutant mice influenced the immune response to dss colitis, its recovery and answer to a second induction. method: colitis was induced in wild type (wt) and ncf mutant (ncf ) b .q mice by administration of . % dss in the drinking water for week. after week recovery, dss was administered for another week. mice were sacrificed at days , , and , the colon was removed and folded into a swiss roll. sections of the colon were stained with he, and monoclonal antibodies against b cells (b ), cd + t cells; and macrophages (mac /cd b) were applied. results: colitis was more severe in ncf than wt mice, with epithelial dysplasia, hyperplasia of peyer's patches and poor epithelium recovery (hyaline scars). at all time-points the amount and location of colonic b cells, t cells and cd b + cells was distinct between groups. these results suggest that ros are crucial for leukocyte recruitment and tissue-repair in dss-induced colitis. histopathological pattern of polypoid lesions of colon in albanian population g. cekodhima * , a. cekodhima, a. beqiri, g. andrea * university hospital maria teresa, dept. of pathology, tirana, albania objective: our aim was to study histopathologic pattern of the polypoid lesions in albania. method: we studied lesions, polypectomies and colorectal surgery in patients in tirana. results: there were observed polypoid lesions of large intestine in males and females. ( . %) were adenomatous polyps, ( . %) were hyperplastic, ( %) were inflammatory polyps with ulcerative colitis, ( . %) were inflammatory ( . %) were juvenile polyps, ( . ) non hodgkin's lymphoma and ( . ) was fibrolipoma. tubular adenomatous polyps were the commonest polyps. they were more common ( . %) in male population as compared to female ( . %). high grade dysplasia was present in ( . %) and malignant change in ( %). the size of the polyps range from . to cm. conclusion: adenomatous polyps were the most frequently found polyps in our study; the approximately % were advanced lesions. the role of cancer stem cells in biology and prognosis of colon cancers a. f. Çiçek * , Ö. Öngürü, m. gamsizkan, a. günal * gülhane military medical academy, dept. of pathology, ankara, turkey objective: this study was designed as a retrospective clinicopathological observation based on immunohistochemical and statistical findings to show the relationship between the disease progression and the intensity of the stem cell population within the tumor for colorectal cancers. method: for this purpose, we investigated colorectal carcinoma cases, retrospectively. paraffin embedded blocks was obtained from pathology archive and demographical patients' data from the patient's files of gastroenterological surgery department. immunohistochemically, we used cd and musashi- antibody to determine the stem cells within the tumor. we noted the age of the patients, histopathological diagnose, tumor location, grade, tnm status, clinical stage, disease-free-survival and the outcome. then we statistically compared all prognostic data with the immunohistochemical findings results: all of the cases were immunoreactive for both antibodies, furthermore we found a significant statistical correlation (p= . ) between cd expression value and patient outcome. when the value of cd expression was high, clinical outcome was poor. in addition, there was a relation between high musashi- expression and poor outcome. conclusion: based on these findings we reported that overexpression of both cd and musashi- antibodies may be a poor prognostic factor in colorectal cancers. immunohistochemical evaluation of vegf expression in colorectal carcinomas d. crisan * , m. florea, d. fodor * university of medicine and pharmacy, pathology, cluj-napoca, romania objective: vascular endothelial growth factor (vegf) is an important angiogenic glycoprotein secreted by the tumor cells and host cells which proved to be a powerful prognostic factor in various human cancers. the aim of our study was the evaluation of vegf expression in colorectal carcinomas using immunohistochemistry, in order to identify the relationship between the presence of the protein in the tumor cells and a series of morphological parameters. method: we analyzed consecutive surgically removed colorectal carcinomas. immunohistochemistry was done on formalin-fixed and paraffin embeded tissue sections, using the anti-human vegf-a monoclonal antibody (clone vg , dako). the extent and intensity of staining were graded and used to calculate the immunoreactive score for each case. statistical analysis was performed with spss (statistical package for the social sciences), using nonparametric tests. the level of statistical significance chosen was p < . . results: vegf was expressed in all tumors, with a heterogeneous distribution. the only parameters that correlated with high vegf positivity were the extent of the tumor necrosis and the presence of lymph node metastases (p = , ). conclusion: high levels of the vegf expression may be an indicator of poor prognosis in colorectal carcinomas. collision tumour of the appendix: mucinous cystadenoma and carcinoid -report of a case c. dastamani * , a. paraskeva, e. carvounis, t. theodosopoulos, a. kondi-pafiti * aretaieion hospital, dept. of pathology, athens, greece objective: appendiceal carcinoids are usually located at the tip of the appendix and occur as incidental findings. epithelial neoplasms of the appendix are uncommon and consist of mucinous adenomas, carcinomas and neoplasms with features of both carcinoid and adenocarcinomas. method: a case of dual mucinous cystadenoma and carcinoid of the appendix is reported. results: the patient, a -year old-asymptomatic female presented with a cystic right iliac fossa mass. the excised specimen was appendix cystically dilated measuring up to , cm in diameter. the luminal content consisted of viscid mucin. the base of the appendix was thickened (up to , cm) and firm. microscopically the cystic part of the specimen had features of mucinous cystadenoma with adenomatous epithelium extending as well over the thickened appendiceal wall. at that area the wall was involved by a well differentiated neuroendocrine neoplasm who gr i (carcinoid). the neoplastic cells infiltrated the entire thickness of the appendiceal wall, the mesoappendix and reached the serosal surface. the two neoplasms where separate and there was no transitional zone between them. conclusion: dual carcinoid/epithelial neoplasia is a rare occurrence in the appendix. the prognosis appears to be no worse than for either of the two components alone. gastrointestinal stromal tumor (gist) of the anal canal: a case report l. de carvalho * , p. a. teixeira, v. g. siqueira, m. pereira, t. or, e. pereira * centro universitário lusíada, dept. of pathology, santos, brazil objective: gastrointestinal stromal tumors (gist) are mesenchymal tumors derived from interstitial cells of cajal. they are found more frequently in the stomach, small intestine while colon and rectum represent unusual sites. gist's of the anal canal are extremely rare. method: -years-old woman who presented bleeding, pain and constipation for several months. the rectal examination revealed a well defined mass located within cm of the anal verge. the magnetic resonance imaging confirmed a well-circumscribed mural mass without adenopathy. the local excision of the tumor was performed for pathological study. results: gross examination showed a . × . cm fibrous mass. histological examination revealed a spindle cell tumor with moderate atypia and mitotic count of mitosis/ hpf. neoplastic cells showed marked positivity for c-kit and cd and negativity for muscular markers. a diagnosis of gist with intermediate risk of aggressive behavior was made. conclusion: gists of the anal canal are extremely rare with only few cases reported in the literature. we described an additional case of gist of the anal canal with histological and immunohistochemical study. intestinal graft versus host disease with giant cells b. doganavsargil * , a. vink, e. j. petersen, g. j. arnold offerhaus * ege university, school of medicine, izmir, turkey objective: graft-versus-host disease (gvhd) is a common complication of allogeneic bone marrow transplantation in which immune cells in the transplanted marrow attack the immunocompromised host. it can occur in either an acute or in a chronic form and may affect various organs. intestinal gvhd is particularly important because of its frequency, severity and its effects on the general condition of the patient. method: here we report a -year-old man who developed gvhd, months after receiving allogeneic non-myeloablative peripheral blood stem cell transplantation for refractory chronic lymphocytic leukemia. colonic biopsies revealed an edematous and congested lamina propria, dilated crypts lined by flattened epithelium, increased apoptotic figures and vanishing crypts. notably there were some giant cells in between crypts. however subsequent sections and special stains didn't show any granuloma formation or presence of an infectious agent. results: to our knowledge presence of giant cells in gvhd hasn't received proper attention before. although we don't know the actual importance or clinical consequences of the entity yet, we report this case to increase awareness of this feature within the context of gvhd. we have seen more cases with giant cell gvhd afterwards; comparison with other cases of gvhd without giant cells may help clarifying their significance. differential mutation patterns of kras and braf in adenomatous and serrated neoplastic sequences o. erdogan * , b. savas, a. ensari * ankara university, faculty of medicine, turkey objective: colorectal carcinomas develop through adenomatous or serrated neoplastic sequence. different molecular mechanisms underly these morphologic sequences which may be translated into different therapies. we aimed to assess adenomatous and serrated neoplastic sequences in terms of kras and braf mutations. method: the study group comprised of adenomatous polyps (aps, n= , . %), conventional adenocarcinomas (conca, n = , . %), serrated polyps (sps, n = , . %) and mucinous adenocarcinoma (mucca, n= , . %). kras mutation was assessed for exon and exon using pyrosequencing while braf v e mutation was analysed using allele specific pcr. chi square test was used for statistics. results: kras mutations were observed in . % of carcinomas and . % of polyps. kras mutation rate was significantly higher (p< . ) in conca ( %) than in mucca ( %), and in aps ( %) than in sps ( . %). braf mutation was found in one mucca ( . %) while all sps were mutated. tsas had the highest kras mutation rate ( . %) in comparison to ssa/ps ( . %) (p< . ) whereas ssa/ps and hps had significantly higher rates of braf mutation ( . %, . %, respectively) than tsas ( . %) (p< . ). conclusion: kras seems to take part in adenomatous sequence while braf, seem to play a significant role in serrated neoplastic progression of the colorectum. objective: quiescent ulcerative colitis (q-uc) is morphologically characterized by typical architectural and cellular mucosal changes that define the process as chronic. it is not clear which of these changes are more ubiquitous in q-uc. our aim was to evaluate which classical histological findings of q-uc are more prevalent in rectal samples. method: rectal biopsies were collected from patients with clinical and endoscopical q-uc. the biopsies were evaluated for mixed inflammation in the lamina propria, crypt architectural abnormalities, basal plasmacytosis, fibrosis, and paneth cell metaplasia as well as for features of active disease (cryptitis, lamina propria neutrophils and erosion). results: forty-five patients ( % female; median age years) were included. thirteen ( , %) biopsies showed focal activity and four cases ( , %) had criteria for active disease. epithelial distortion, chronic inflammatory infiltrate and basal plasmacytosis emerged as features present in , and % of biopsies. overall, biopsies were histologically normal (no chronic features, no active disease). conclusion: rectal mucosa from patients with clinical and endoscopical q-uc can show microscopic active disease, which illustrates that endoscopy alone may be insufficient to identify quiescent disease. in q-uc, the most frequent changes (present in over % of the biopsies) were epithelial distortion and chronic inflammatory infiltrate. vacuum-based preservation of colorectal cancer specimens: a comparison with formalin fixation j.-f. fléjou * , s. el-naderi, p. cervera * hôpital saint-antoine, dept. de anatomie pathologique, paris, france objective: in pathology, an alternative to immediate fixation in neutral buffered formalin (nbf) is vacuum sealing and cooling (vsc) ("tissue safe" system). there have been few evaluations of vcs. method: we assessed msi, kras, braf in colorectal cancers, conserved with vcs before fixation ( cases with surgery in a hospital distant from our centre), or immediately fixed in nbf ( cases with surgery during the same period in our hospital). dna was extracted from paraffin embedded tissue. msi was assessed by mlh and msh immunohistochemistry and pcr; kras and braf were screened by multiwell-plate based real-time pcr (lightcy-cler® , roche), confirmed by sanger sequencing. results: there was no difference regarding morphological analysis. immunohistochemistry was interpretable in all cases, with negative cases in the vcs group ( mlh ), negative cases in the nbf group ( mlh , msh ), and a % correlation between immunohistochemistry and pcr. dna extraction was possible in all cases. kras and braf mutations were detected in and cases in the vcs group and and cases in the nbf group, respectively. conclusion: we show that analysis of msi, kras and braf is feasible in surgical specimens after vcs. this procedure can be an alternative to formalin fixation. clinical significance of cd -positive m macrophage in colorectal cancer s. fushimi * , m. matsumoto, s. takahashi, t. ogino, j. itakura, t. ito, a. matsukawa * okayama university, dept. of pathology, japan objective: tumor-associated macrophages are divided into two phenotypes, termed m /m macrophages. m macrophages promote tumoricidal responses whereas m macrophages assist tumor progression and metastasis. in this study, we have analyzed a clinical significance of m macrophages in excised sections from patients with colorectal cancer. method: patients who had surgical resection for colorectal cancer between and were identified from a prospective database. tissue sections with adenocarcinoma in pt category were employed in this study. the sections were stained with anti-cd , a marker of m macrophages. the relation between the staining pattern and tumor budding or patient prognosis was examined. results: fifty-two patients ( male, female) with a median age of years (range - ) were studied. the tumors were classified into four subtypes according to the staining patterns of cd -positive cells, i.e. sparse type (n= ), invading-tip type (n= ), peri-nest type (n= ), and dense diffuse type (n= ). only a dense diffuse type was related to the high degree of tumor budding. the prognosis with a dense diffuse type demonstrated a poor prognosis as compared to the other types. conclusion: even the case with comparable tumor depth, cd -positive macrophages with dense diffuse distribution was related to the poor prognosis. objective: the treatment of locally advanced rectal cancer has shown a significant development over the last decade. the total mesorectal excision and the use of pre-operative radio (chemo)-therapy are leading the patients to a gradual improvement of local control of the disease. the aim of this study is to evaluate the pathological tumour response to pre-operative treatment through correlation between the clinical stage (ctnm) and the pathological stage (yptnm). method: since , patients with locally advanced rectal cancer, assessed by high resolution magnetic resonance imaging (hr-mri) and tnm staging system, have been evaluated by the colorrectal multidisciplinary team. pre-operative radio (chemo)-therapy was mandatory before undertaking standard surgery. surgical specimens were evaluated according to the vikingo project's protocol implemented by the spanish association of surgeons. results: patients completed the pre-operatory treatment. after evaluation of clinical and surgical study specimens, we observed variable degree of downstaging in ( , %) patients, of whom showed complete response. the ( , %) remainig patients were assessed as stable disease. no local progression of tumours was observed. conclusion: in this study, approximately % of patients, who were staged by hr-mri and underwent preoperative radio (chemo)-therapy for locally advanced rectal cancer, showed some degree of downstaging. we remark the fact that nearly % of patients reached complete response. the density of macrophages in colorectal cancer is inversely correlated to tgf-beta expression and patients' survival m. gulubova * , i. manolova, j. ananiev * trakia university, general and clinical pathology, stara zagora, bulgaria objective: the role of macrophages in colorectal cancer tumorogenesis is complex because they can both prevent and promote tumor development. we investigated cd infiltration in tumor tissue and its correlations with proteins ot tgf-beta signaling pathway. method: a nonselected panel of primary tumors of colorectal origin was investigated immunohistochemically with antibodies against cd , tgf-beta , smad , smad , tgfrii and levels of tgf-beta were measured by elisa. results: lower cd infiltration in tumor nests was associated with expression of tgf-beta (chi = . , р = . ) and smad (chi = . , р= . ) in tumor cells and with tgfrii expression (chi = . , р= . ) in tumor cells membranes. there was not correlation between cd cell numbers in tumor tissue and tgf-beta serum levels. we have found higher frequency of liver metastases in patients with lower infiltration with cd in invasive margin (chi = . , р= . ). the survival time was shorter for patients with low cd infiltration in tumor nests and invasive margin, compared with the survival time for patients, with higher cd infiltration in both tumor compartments (p< . ). conclusion: the increased levels of tgf-beta in the tumor have an immunosuppressive effect on cd infiltration. inflammatory myofibroblastic tumor of the colon: the th reported case s. gurzu * , t. bara, i. jung * university of targu mures, dept. of pathology, romania objective: inflammatory myofibroblastic tumor (imt) is a very rare tumor of the colon in which the diagnosis is especially based on immunohistochemistry. in the colon, we report the th case of imt. method: case presentation results: we report the case of a -year old male who presented with symptoms suggesting colon cancer. a mm protruded tumor was endoscopically described in the ascending colon. right hemicolectomy was performed. macroscopically the tumor was well defined, covered by normal mucosa with a central depressed area, suggesting a gist (gastrointestinal stromal tumor). microscopically spindle cells with fascicular arrangement admixed with mononuclear leukocytes were observed. the tumor cells expressed vimentin, sma, desmin and alk and were cd and cd negative. intense angiogenesis and cd expression in the endothelial cells were also observed. conclusion: in the imt of the colon, clinical endoscopic and gross feature can imitate a carcinoma or a gist. the ihc pattern of imt can offer informations about its histogenesis. based on the immunophenotype, we hypothesize that this tumor seems to occur from the the pluripotent stromal cells, cd positive which can be either differentiated in the interstitial cells of cajal (telocytes), which are also cd positive or, during differentiation, can loss cd positivity. objective: determining the fraction of tumor cells in colon carcinoma samples analyzed for kras mutation is important for choosing the proper testing modality. however, when asked to determine tumor cell fraction in tissue samples, different pathologists give considerably different estimations, possibly leading to erroneous interpretation of kras mutation analysis results and poor treatment choices. method: we developed a free, easy to use computer program that estimates tumor cell fraction on colon carcinoma slides that are immune-stained with anti-cytokeratin antibody. sixty samples were evaluated by the program and results were compared to actual measurement of tumor fraction. results: the tumor cell fraction estimated by the computer program showed highly significant correlation with the actual measurements (r= . , p< . ). additionally, we found that a short calibration step prior to beginning the computer estimation increased the accuracy of the results. in four cases ( %) there was some discrepancy between the computer estimation and the actual measurements, however, this was attributed to lower quality immunohistochemical staining. conclusion: in conclusion, we believe that this program can be used for standardizing the evaluation of tumor cell fraction in colon carcinoma, and that its use might aid in making better diagnosis and treatment choices for these patients. magnetic resonance imaging assisted tumour block selection in colorectal cancer c. hunter * , g. brown, l. temple, m. abulafi, a. arnaout * croydon university hospital, dept. of colorectal surgey, united kingdom objective: inadequate sampling may result in understaging in colorectal cancer. fat clearance techniques and whole mount sections are time consuming and costly. we hypothesise that high resolution magnetic resonance imaging (mri) of the colorectal cancer specimen may aid histological sampling and result in upstaging. method: patients undergoing resection are prospectively recruited, and randomised to "conventional histology" or "mri assisted histology". in addition to routinely selected tumour blocks, additional blocks and any additional lymph nodes are selected with the aid of specimen mri or visual inspection and palpation according to group. t stage, depth of extramural invasion, distance to resection margin, lymph node (ln) number and involved ln are compared between the two groups. results: to date, of patients have been recruited. we present the methods and preliminary results. there is a trend towards greater t stage and ln yield. no difference in n stage, extra-mural vascular invasion or involved non-peritonealised resection margin has yet been observed. conclusion: early results suggest that specimen mri may upstage colorectal cancer specimens by aiding tumour block selection. we will complete recruitment of colorectal cancer patients over the next months to determine whether observed differences are significant. diverticular disease of transverse colon is rare and perforation may occur in crohns disease c. hunter * , a. arnaout * croydon university hospital, dept. of colorectal surgey, united kingdom objective: diverticular disease commonly affects the sigmoid colon in the west and the right colon in asia. perforation is uncommon affecting / per year. perforated diverticular disease of the transverse colon is therefore very rare. we present a case of crohns disease complicating transverse diverticular disease that lead to perforation and unfortunate death. method: radiological and histopathological features are presented with a review of the literature results: to the best of our knowledge, this is the first reported case of crohns disease complicating diverticular disease of the transverse colon. we suggest that distal stricturing leading to increased intraluminal pressure or increased weakening of the colonic wall due to coincident crohns fissure and diverticulum are possible pathogenic mechanisms. conclusion: crohns disease complicating transverse diverticular disease is uncommon, but may be one pathogenesis leading to the rare event of transverse colon diverticular perforation. results: there were episodes in patients. they start between day and ( ± days). duration ranged from to days ( ± ). the three moderate and the three mild ar reversed with treatment. of the severe ar, caused the failure of the graft. five episodes de ar began as moderate or severe directly. severity in the course of each episode was fluctuating. sixteen indeterminate rejections were seen in grafts (did not reach definitive criteria). they lasted - days (mean: ± days). conclusion: -acute rejection is the most common cause of morbidity in the intestinal transplant ( % of the grafts). -it can develop at any time and relapse. -the duration and severity of the episodes are fluctuating. -in our series, % reversed, but severe rejection often lead to graft loss ( %). histopathological study of intestinal grafts lost in a series of adult transplants. hospital octubre. madrid. method: we studied explants and autopsy. the autopsy was not authorized in one death. results: seven grafts were lost in patients ( multivisceral). two types of failure were seen. a) local: severe acute rejections (ar) (days , , ) and lymphoproliferative syndrome (lps) in day ; b) systemic: sepsis (days , , ). ar showed types of morphology: ulcerative, pseudomembranous and with disappearance of the villi preserving the crypts. lps was a high-grade b lymphoma and affected the graft and the lymph mesenteric nodes with venous thrombosis that cause ischemic necrosis. the sepsis were due to acinetobacter, to adenovirus in the graft and pulmonary aspergillosis and to pseudomona aeruginosa with necrohemorrhagic pancreatitis. conclusion: -the incidence of igl was %. -the cause of igl was local (ar and lps) in % and sepsis in %. -severe ar was the most frequent cause of failure. -histological examination properly determine and document the causes of igl. cap polyposis: report of two cases r. ivanova * , d. kyoseva, g. trifonov, r. nikolov * hospital of endoclinology, laboratory of pathology, sofia, bulgaria objective: cap polyposis is a rare colorectal disease characterized by mucoid, bloody diarrhea associated with multiple inflammatory polyps covered by a cap of fibrinopurulent mucous. the disorder was first described in and up to date a small number of cases has been reported in the literature. method: we report two cases with multiple polyps of rectum and sigma, which were histologically diagnosed as cap polyposis. results: the cases were two males on age of years and years. in both cases there was a history of mucoid and bloody diarrhea. on the basis of colonoscopy findings, in the young patient there was a broad differential diagnosis including m. crohn, inflammatory pseudopolyps and cronkhite-canada syndrome. in the second case the clinical diagnosis was polyps of sigma. endoscopic biopsies and polypectomy were done. in both cases, the histology showed typical histological features of cap polyposis -polypoid lesions containing elongated, tortuous and often distended crypts covered by a cap of inflammatory granulation tissue. conclusion: the recognition of this rare disease is of practical significance because its clinical symptoms have some similarity with inflammatory bowel disease or irritable bowel syndrome. histopathological findings in consecutive appendectomies: a retrospective analysis j. jeruc * , z. dolenc stražar, v. jovic, a. cerar, n. zidar * university of ljubljana, faculty of medicine, slovenia objective: appendectomy is one of the most common surgical procedures. histology usually confirms the clinical suspicion of acute inflammation, but sometimes other clinically relevant diagnoses are made. the aim of our study was to audit the appendectomies at our institution and summarise atypical pathological findings with emphasis on benign and malignant tumours. method: we reviewed the histopathology results of consecutive appendectomies received in a -year period. results: excluding three cases of metastatic process, appendiceal tumour was found in cases ( . %), which is a higher rate than reported in the literature. there were four endocrine tumours, all g , three cases of classical adenomas, cases of low-grade appendiceal mucinous neoplasm (one associated with pseudomyxoma peritonei) and three cases of carcinoma (one mucinous adenocarcinomas, one intestinal type, one undifferentiated carcinoma). we also diagnosed one case of multicystic mesothelioma. endometriosis was diagnosed in seven cases. in cases appendicitis was associated with diverticula, in cases changes were suggestive for crohn's disease and in one case for cystic fibrosis. negative appendectomy rate was . % that falls within the range reported in the literature. conclusion: histopathological examination of appendectomy specimens may reveal many different conditions not previously suspected; therefore, it should be performed in all cases. results: cdx , muc , muc , muc ac, and muc expression was observed in . % ( cases), . % ( cases), . % ( cases), . % ( cases), and . % ( cases), respectively. while siac patients with cdx expression showed less nodal metastasis (p= . ), those with muc expression tended to have siacs with nodular or infiltrative growth (p = . ), poor differentiation (p = . ), and more lymphatic invasion (p= . ). muc ac expression was associated with siacs with well differentiation (p= . ) and frequent pancreatic invasion (p= . ). patients with cdx +/muc -had more polypoid (p= . ) and well differentiated (p= . ) tumors and a significantly better survival (median, . months, p< . ) than those with other immunophenotypes (cdx −/muc +, cdx +/ muc +, and cdx −/muc −). patients with cdx expression (median, . months) had a significantly better survival than those without cdx expression ( . months) by univariate and multivariate analyses (p< . ). conclusion: cdx expression is an independent good predictor of survival in surgically resected siac patients. small neuroendocrine tumor of appendix with metastasis in the ileocecal lymphnode: a case report g. kalan * , s. tušar, n. zidar * general hospital jesenice, dept. of pathology, slovenia objective: neuroendocrine tumors (net) of appendix are believed to behave less aggressively than nets at other sites. separate staging criteria have therefore been proposed, and tumor size > cm appears to be the dominant criterion for aggressive behavior in appendiceal nets. however, it is controversial whether radical surgery is indicated in tumors measuring > cm. we report a case of a -year-old girl with acute appendicitis in whom appendectomy and right-sided hemicolectomy were performed. method: surgical specimens were sampled and processed according to standard histological and immunohistochemical (ihk) procedures. results: in the base of appendix, there was a tumor measuring . cm in diameter, focally invading mesoappendix, with free resection margins. microscopically, tumor exhibited characteristic features of net g , with positive ihk for chromogranine and synaptophysin, and ki index < %. ihk for podoplanin revealed focal lymphovascular invasion. in the right-sided hemicolectomy specimen, there was no residual tumor, metastasis was found in one ilecoecal lymphnode. conclusion: our case shows that appendiceal net smaller than cm can metastasize to regional lymphnodes. ihk against podoplanin might be helpful in searching for lymphovascular invasion, helping to separate it from the retraction clefts, thus providing additional information concerning risk factors for a more aggressive behavior. lipomatosis of ileocaecal valve causing small bowel obstruction mimicking crohn´s disease m. kalman * , p. szépe, j. marcinek, t. balhárek, l. plank * university hospital martin, dept. of pathology, slovakia objective: lipomatosis of ileocaecal valve is a rare cause of small bowel obstruction. method: we describe a -year-old male treated for crohnś disease and clinical and imaging presentation of chronic small bowel stenosis and obstruction. results: a right hemicolectomy was performed, and hypertrophy of bauhin´s valve causing intestinal obstruction was found. microscopical examination of the specimen revealed morphological changes consistent with lipomatosis of the ileocaecal valve. the ileum and colon were macroscopicaly unremarkable and histomorphological features of crohn´s disease were absent. conclusion: many patients with lipomatosis of the ileocaecal valve are asymptomatic, or the lipomatosis gives causes insignificant symptoms only or rarely obstruction may occur. then, as in the presented case, the surgical resection with ileo-colic anastomosis is the only effective treatment. overexpression of cxcr in tumor buds is a strong predictor of lymphatic invasion and lymph node metastasis in colorectal cancer e. karamitopoulou-diamantis * , d. kassahn, i. zlobec, v. koelzer, h. dawson, a. lugli * universität bern, inst. für pathologie, switzerland objective: cxc chemokines enhance tumor cell survival and proliferation. especially cxcr promotes tumor development by stimulating angiogenesis and favoring metastasis. in contrast, cxcr is angiostatic and may exert an anti-tumor effect. since tumor budding is linked to vascular/lymphatic invasion and lymph node (ln) and/or distant metastasis in colorectal cancer (crc), here we explored the expression of cxcr + in relation to tumor budding. method: a multiple-punch tissue microarray of crcs with full clinicopathological information including therapy underwent immunohistochemistry for cxcr and cxcr . expression was evaluated in tumor-center, -front and -buds and correlated to clinical data. results: cxcr -expression was homogeneous throughout tumor-center, -front and -buds (average %) and was unrelated to clinicopathological features or survival. cxcr -expression was high in tumor-center and -front but reduced (p< . ) in buds ( %, %, and %, respectively). maintenace of cxcr -expression within buds was predictive of ln-positivity (p< . ) and lymphatic invasion (p< . ) but not of venous invasion or distant metastasis. cxcr -positivity in buds was associated to poor outcome (p= . ) in ln-negative crcs (p= . ). conclusion: maintenance of cxcr -expression in buds has a profound effect on tumor aggressiveness and prognosis in ln-negative crc-patients. this would help in establishing a tumor budding "profile" especially linked to lnmetastasis in crc. objective: cox- expression was investigated in colorectal cancer and colorectal precancerous lesions. its expression in adenomas is associated with increasing size and neoplastic potential. little is known about cox- expression in subtypes of serrated colonic polyps, which constitute substantial number of colon cancer precursors. the aim of the study was to assess cox- expression in serrated polyps of the colon and to investigate its potential discriminative role. method: consecutive serrated polyps were analyzed. they included: traditional serrated adenomas (tsa), sessile serrated polyps (ssp) and hyperplastic polyps (hp). cox- expression was assessed semi-quantitatively ( - ). kruskall-wallis test was used for the comparison of cox- expression between serrated categories (α= . ). objective: in colorectal cancer (crc), tumor buds represent a more aggressive tumor cell type at the invasive front with apparently low proliferation. the aim of this study was to determine the proliferation potential of tumor buds by comparing ki staining across different tumor areas and adjacent normal tissue. method: whole tissue sections from patients with crc underwent immunohistochemistry for ki . high-powerfields were evaluated for each of the following regions: normal mucosa, tumor center, tumor front and tumor buds. ki -positivity was correlated to patient outcome. results: average ki -positivity was . % in normal mucosa and significantly higher in the tumor center ( . %; p < . ) and invasion front ( . %; p < . ). strikingly, only . % of all tumor buds showed ki -positivity (p< . ). although ki -positivity in the tumor center or front was unassociated with clinicopathological features or patient survival, the greater the number of ki -positive tumor buds, the worse the prognosis. this effect was also found after adjusting from tnm stage (hr ( % ci): . ( . - . ); p= . )). conclusion: a marked absence of ki staining is found in most tumor buds, suggesting a substantially decreased proliferation rate. however, the association of ki -positivity with worse prognosis in % of cases points towards a heterogeneous population of tumor buds. kayexalate and intestinal necrosis: an underrecognised entity p. luís * , a. alves, c. ferreira * hospital de santa maria, dept. de anatomia patológica, lisboa, portugal objective: there are only few cases of intestinal necrosis secondary to administration of kayexalate described in the literature, most of them from postoperative patients, having the largest study only patients in years. we report our experience in the last years with a brief review of the pathophysiology and histologic aspects of the lesions induced by the kayexalate. method: between and march seven patients were diagnosed with ischemic necrosis related to kayexalate administration, five in colon biopsy and two in resection specimens. they're comorbidities and clinical evolution was extracted from the medical records. results: the patients' age ranged between and years. in one case we had the clinical information of chronic renal insufficiency. in three of them, there was no previous history of renal impairment. one patient developed symptoms after one single administration of kayexalate. the clinical presentation ranged from abdominal pain, gastrointestinal bleeding and three of them had a fatal outcome. the administration of kayexalate can have associated risks with different clinical impact in any patient. we must be aware of the related complications, as the histological identification of the kayexalate crystals indicates the etiology of the ischemic lesions to the clinicians and alerts them to the dangers of this therapeutic. objective: egfr plays an important role in colorectal cancer (crc) progression and represents a natural target for molecular anticancer drugs. in this study we evaluated consecutive crc aimed to analyze k-ras mutational status in relationship to: different clinical-pathological parameters, egfr immunohistochemical (ihc) expression, response to cetuximab treatment. method: k-ras mutations and egfr expression were determined by direct cycle sequencing and ihc respectively in surgical specimens and biopsies. ihc findings were evaluated using four different score systems. results: the distribution of k-ras mutations did not significantly vary between surgical or bioptic specimens or with respect to different anatomic tumor localization. in contrast, k-ras is more frequently mutated in egfr negative/low score tumors than in positive ones (p< . ). focusing on the surgical treated crc patients, we found a higher percentage of k-ras mutations in t crc (p= . ) and in younger patients (p= . ). finally, we observed that % of primary crc, concomitantly evaluated with their paired metastases, changed k-ras mutational status during progression. conclusion: our data showed that tumor size, patient age and egfr ihc expression significantly influenced k-ras mutations. interestingly, we observed that cetuximab treated patients, had a better clinical outcome when egfr presented a high ihc score. multiple immunohistochemical investigation of signaling pathways in colorectal cancer t. micsik * , l. kopper, t. krenács, z. nagy, o. horváth * semmelweis university budapest, st department of pathology, hungary objective: despite the recent advances in therapeutic armamnet colorectal cancer (crc) remains one of the leading cancer deaths worlwide. crc has a heterogenous molecular background with known prognostic and predictive markers, but the whole picture is rather complex with many unexplored connections. method: we performed multiple immunohistochemical stainings on tissue microarrays (tma) made of crc cases to study the correlations between egfr-ras, cell cycling, apoptosis, cell adhesion and tumor invasion pathways. we evaluated digitized slides using matrix scores considering both the frequency and positivity of cells and statistical analysis followed. results: we found strong linear correlation between positivity and (p- , p- ) phosphorylation of the egfr receptor and p positivity detected with different clones. furthermore, phospho-nf-kb p positivity was strongly linked to survivin-expression. conclusion: activation of egfr-related pathways including nf-kb is a key factor in crc-growth. the strong link between cancer promoting egfr signaling and the mutational lack of the p driven apoptosis suggests a deadly cooperation, which can be further aggravated by forced tumor survival through survivin. correlation of these markers potentially predict poor disease outcome but can be specifically targeted by the upcoming new molecules of tailored therapy. objective: kras mutational status is important in colorectal carcinoma (crc). it is unclear, however, which is the most informative tissue source for study in crc cases showing more than one tumor mass. to explore this issue we determined kras status in a series of primary (pt) and metastatic crcs. method: a total of tumors belonging to patients ( males and females) with ages ranging between and years (average, years) were studied. kras mutation hotspots in codon and were analyzed by polymerase chain reaction and sequentiation. wt-kras cases were confirmed with the kras stripassay tm. objective: lynch syndrome (ls), the most frequent form of hereditary colorectal cancer (crc), is caused by germline mutations in the mismatch repair system genes. a recently identified mechanism involving the epcam gene is responsible for . % of msh -negative ls cases. we herein explore epcam protein expression in ls-associated, msh -negative crcs to evaluate its potential value in the algorithmic approach to ls population screening. method: we studied a total of msh -negative crcs from different patients in whom we were able to perform a complete germline analysis. expression of both msh ( : dilution, clone g - , pharmingen) and epcam ( : dilution, clone ber-ep , dako) was evaluated by immunohistochemistry (ihc). results: nine patients showed a deleterious germline mutation that involved the msh gene in three instances and the epcam gene exon in six instances. all patients harbouring the epcam mutation belonged to the same family. of the crcs, epcam expression loss was seen in only five, all of them were from patients with a germline epcam deletion. conclusion: due to the high specificity of epcam protein loss for identifying ls patients with an epcam deletion, we recommend adding epcam ihc to the ls diagnostic algorithm in msh -negative crc cases. objective: colorectal carcinoma with signet ring cell component (crc-src) is a rare and distinct subtype with little molecular information. we investigated the frequency of braf mutation in crc-srcs and its relation with clinicopathologic parameters. method: we categorized tumors into groups - %, - %, - % and > % according to signet ring cell component. genomic dna was isolated from parafin blocks and analyzed for braf v e mutation by polymerase chain reactionrestriction fragment length polymorphism (pcr-rflp). results: eleven of cases showed braf mutation ( . %). the results were also confirmed with sequence. no statistically significant differences were found in clinicopatologic parameters between braf wild-type and mutant crc-src. on the other hand, when we adjusted age, gender, percentage of signet ring compenent and stage, we found a statistically significant increased risk in braf mutant group compared to braf wild-type in cox regression analysis (hr= . , % ci= . - . , p= , ) conclusion: braf mutation is frequent in crc-srcs. this finding may support its diverse molecular pathogenesis and could have important therapeutic implications for those patients. to clarify the relation of braf mutation with clinicopathologic parameters and prognosis in crc-src, studies with multi-instutional larger series are needed. coexisting lipomas and adenocarcinoma of the colon: case report g. orgun sonmez * , u. bayol, o. akman, s. cumurcu, m. olmez * tepecik research and training hospital, izmir, turkey objective: lipomas of the large bowel are rare, they are invariably submucosal and therefore may intussuscept. infrequently lipomas may also present in the form of multiple polypoid masses in the colon. we present a -years old female with submucosal lipomas and a coexisting adenocarcinoma of transverse colon. method: -years old female patient was admitted to hospital because of rectal bleeding for months. colonoscopic examination revealed mucosaly intact polypoid tumoral masses and an ulcerated tumoral mass in transverse colon. biopsy of the ulcerated mass was diagnosed as adenocarcinoma. extended right hemicolectomy was performed. results: grossly, ulcerated tumor was , × × , cm and there were polypoid tumors in cecal area of the right colon which were . - . - . cm in their greatest dimensions. polypoid tumors were yellow, lipomatous on cut surfaces. microscopically ulcerated tumor was composed of adenocarcinoma morphology and the other polypoid tumors were composed of submucosal mature adipocytes with a thin capsule around them. the diagnosis was colonic adenocarcinoma (pt n m ) and submucosal lipomas of colon. the patient had no additional therapy and she is healthy with no evidence of recurrence. conclusion: this case is presented to remind that there may be multiple lipomas of colon with coexisting malignant epithelial tumors. objective: primary liposarcoma of the small intestine is exceedingly rare. to our knowledge, only cases have been reported so far. we report a case of the primary myxoid liposarcoma of the ileum. method: the patient was -year-old woman. ct and mri revealed a -cm-intrapelvic mass, which was proved to be a tumor of the distal part of the ileum, and, therefore, partial ileectomy was performed. results: the tumor was . × . × . cm in size, relatively well circumscribed and located within the intestinal wall without invasion to the neighboring tissues. microscopically, the tumor had mixture of cellular and myxoid areas, showing proliferation of short spindle and stellate shaped cells in a delicate capillary vasculature. univacuolated tumor cells and a small number of lipoblast-like cells were also seen. immunohistochemically, these proliferating cells were positive for s- protein. according to these pathological findings, we diagnosed the tumor as myxoid liposarcoma. fish methods showed the tumor had chromosomal translocation t( ; )(q ;p ). conclusion: the differential diagnosis of this includes other spindle cell tumors such as gist and myogenic tumors. but we could arrive at the proper pathological diansosis following the above morphological features and the specific chromosomal translocation. we will also discuss the clinico-pathological features of the cases including ours. prognostic significance of selected immunohistochemical markers in colorectal cancer e. paltseva * , o. samofalova, y. gorbacheva, p. tsarkov * first moscow state medical university, pathology, russia objective: the aim of our study was to identify a combination of markers whose expression is predictive of invasion and metastasis of colorectal carcinoma. method: the expression of e-cadherin, β-catenin, tenascin c, kai- , epidermal growth factor receptor (egfr) and vascular endothelial growth factor (vegf) was studied immunohistochemically and correlated clinicopathologically in primary colorectal carcinoma cases ( with metastatic lymph nodes). patients ( %) developed haematogenous metastases at median , months postoperatively ( - ). results: expression of following markers significantly correlated with depth of tumour invasion: cytoplasmic accumulation of e-cadherin (p= , ), nuclear staining for β-catenin (p= , ), increased tenascin c expression (p= , ), and loss of egfr and vegf expression (p= , and , , respectively). decreased tenascin c and kai- expression associated with the presence of lymph node metastases (p= , and , , respectively). weak cytoplasmic e-cadherin expression correlated with distant metastases (p= , ). conclusion: our results suggest that abnormal expression of proteins involved in cell adhesion and migration (e-cadherin, βcatenin, tenascin c, egfr) and angiogenesis (vegf) may be related to the invasion of colorectal carcinoma. detecting the expression of e-cadherin, tenascin c and kai- probably possesses clinical significance in evaluating lymph node and distant metastasis and predicting the prognosis of colorectal cancer. objective: in colorectal adenocarcinomas (crcs) microsatellite instability (msi) can be documented by immunohistochemical detection of mismatch repair proteins (mmr). the aim of the present study is to identify the percentage of msi-positive crcs, in the epirus region of greece and compare it to the international data. method: a total of cases of sporadic crcs, adenomas and hyperplastic polyps, formalin-fixed paraffinembedded, were examined immunohistochemically using standard methods (en vision system), with the antibodies mlh (clone es ), msh (clone pu ) and pms (clone m r g) (novocastra-menarini). immunostained sections were evaluated semi-quantitatively, taking in account the percentage of neoplastic positive cells and estimating nuclear staining intensity, compared to the internal positive controls (normal epithelium, lymphocytes). results: all hyperplastic polyps and adenomas expressed the examined mmr. two out of crcs were negative for msh and pms , while one of them was also negative for mlh . interestingly, one of these two crcs had an adjacent adenoma, which also showed negative staining for msh and pms . conclusion: immunohistochemical staining for mmr is possible in archival material. loss of mmr varies, depending on the protein examined. in crcs from the epirus region the estimated loss of at least two mmr is %. objective: matrix metalloproteinases (mmps) are a family of zinc-dependent neutral endopeptidases collectively capable of degrading essentially all extracellular matrix (ecm) components. genetic alterations in mmp- and mmp- have been implicated to play an important role in colorectal carcinogenesis (crc). the aim of this study was to investigate the hypothetic correlations between the mmp- and mrna expression and the clinicopathologic variables of the crc. method: the expression of mmp- and - mrna was assayed by rt-pcr for tunisian sporadic colorectal adenocarcinomas. results: mmp- was highly expressed in the cases. mmp- was not detected in cases and moderately or highly expressed in cases. absence of mmp- mrna expression was significantly related to an advanced tumoral invasion stage (pt ) (p= . ). conclusion: our study suggests that the overexpression of mmp- could be the result of an imbalance in the system of transcriptional regulation of mmp- , which would involve the loss of expression or activity of its inhibitor: timp- . a variable expression of transcripts of mmp- could be explained by the hypothesis of hypermethylation of the promoter or degradation of its transcript after protein translation. relationship between matrix metalloproteinases and promoter polymorphisms and colorectal cancer risk objective: the carcinogenic schema of colorectal cancer (crc) follows a multistep process governed by mutational events that affect multiple micro-environmental factors, including matrix metalloproteinases (mmps). this present work aims to contribute to a better understanding of mmp- and - promoter polymorphisms involvements in the genesis and progression of sporadic crc in tunisian population. method: the mmp- and mmp- promoter genotypes were determined by pcr-rflp in tunisian patients with sporadic colorectal cancer and healthy control subjects. results: no significant associations were found regarding mmp- (- c/t) polymorphism and crc susceptibility. there was a connection between the mmp- - promoter polymorphism and gender (p = . ). the mmp- (− c/t) was significantly associated with crc (or= . [ . to . ]). no implication of mmp- - promoter polymorphism in the clinicopathological parameters of crc was evident. conclusion: our findings suggest that the mmp- promoter polymorphism was not a predictive factor for the crc occurrence while the mmp- - ct genotype was a risk factor for crc susceptibility. hodgkin's lymphoma in distal rectum in a patient with crohn´disease treated by biologic therapy -a case report r. sampaio * , f. costa, e. diane, j. palla garcia, j. r. vizcaino * anatomia patológica, valpaços, portugal objective: rare cases of hodgkin`s lymphoma have been reported in the setting of inflammatory bowel disease. method: we report a case of hodgkin`s lymphoma in a years old male patient with corticosteroid-dependent crohn´disease (cd) who had been medicated with azathioprine for many years. in the last years he began periodical therapy with infliximab, till the time when he complained of worsening of symptoms and the appearance of asthenia, tenesmus and weight loss. infliximab was withdrawn and replaced by adalimumab. a colonoscopy with several biopsies revealed the presence of a hodgkin lymphoma. results: the histological examination revealed granulation tissues where we identified atypical cells (reed-sternberg cells) with the following immunohistological staining: cd +, cd +, lmp + lca-, cd -, cd -, alk-. these results in conjunction with the morphological aspects and the clinical history of cd favored the diagnosis of hodgkin lymphoma in a patient with preceding cd. the patient was subjected to radical colectomy. postoperative pathological examination showed cd with involvement of the distal rectum by hodgkin's lymphoma. conclusion: the present report may serve as a reminder to clinicians of the possibility of occult lymphoma in patients with colonic cd treated with biological therapy. objective: dna mismatch repair (mmr) status in sporadic colonic cancers (ccs) had provided valuable prognostic and predictive information in the appropriate clinical setting; however there are no definitive guidelines for microsatellite instability (msi) testing. we evaluated the expression of mmr proteins and correlated them with outcome in a cohort of patients with sporadic colon cancer. method: we reviewed cases of sporadic stage ii colonic cancer from abril th to december th . clinical data including tumor grade, prognostic factors, therapy and outcome were searched for. tissue microarrays using a manual beecher system were constructed and stained with antibodies for mlh , msh , pms and msh . results: % ( ) of ccs had loss of expression of one or more mmr proteins, including defect in protein . % ( ), proteins , % ( ) and tree proteins , %( ). the mean overall survival was months with no significant differences in patients between microsatellite stable and unstable tumors. conclusion: we detected a fairly low prevalence of the loss of immunohistochemical expression of mmr proteins. this result might have influence our mean overall survival between microsatellite stable and unstable tumors. the pathological effects of orchidectomy on intestine tissue and serum level of carcino embryogenic antigen s. sattari * , r. ahmadi * azad university of iran, science dept., hamedan, iran objective: carcino embryogenic antigen (cea) is a wellknown tumor marker influenced by carcinogenic changes in intestine tissue. the purpose of this study was to determine the effects of orchidectomy on serum level of cea and histological changes of intestine in male rats. method: male wistar rats were randomly divided to control, sham, uni-orchidetomised and biorchidectomised groups of in each. weeks after operation, blood samples were collected using cardiac puncture method. serum cea level was measured using elisa method. the effect of orchidectomy on intestine tissue was also histologically studied. the results were statistically analyzed using anova. results: serum level of cea was significantly increased in orchidectomised rats compared with control animals (p< . ). the increase in serum cea level was more in biorchidectomised than uniorchidectomised rats (p< . ). there was also an enhancement in tissue lymphocytes, plasma cells and inflammation in intestine of orchidectomised animals. the results showed that orchidectomy results in pathological changes in intestine tissue leading to increased serum cea level. the plasma cells density in pararectal lymph nodes of patients with rectal cancer after neoadjuvant therapy m. sezak * , n. ozsan, b. pehlivanoglu, s. ozkok, t. yoldas, b. daganavsargil * ege university, dept. of pathology, izmir, turkey objective: neoadjuvant chemoradiotherapy (nt) is standard procedure for locally advanced rectal cancer. in this study, we compared the morphology of lymph nodes and the amount of plasma cells in the interfollicular region of lymph nodes in rectal tumour surgical excision material, between two groups who had received and not received nt prior to surgery. method: fifty cases with nt and forty cases without nt were included. the number and diameter of lymph nodes were recorded. the morphology of the lymph nodes were evaluated, and the percentages of interfollicular plasma cells were demonstrated immunohistochemically (cd ). tumor regression grade was assessed using mandard scoring system. results: average number and diameter of lymph nodes were greater in patients without nt (p< , ). atrophy of germinal centers was noted as % in nt group, versus %. the amount of plasma cells in the interfollicular region was found to be higher than fifty-one percent in % of nt group versus % (p< , ), as well. complete response rate (mandard's: ) was %. no significant association was found between the amount of plasma cells and tumor regression. conclusion: nt leads to a decrease in the volume of the lymph nodes and atrophy of germinal centers, conversely, causes stimulation of the proliferation of interfollicular plasma cells. adenomyoma of the ileum: pathohistological features with reference to pathogenesis b. snezana * , b. andrejic, n. solajic * clinical center of vojvodina, center for pathology, novi sad, serbia objective: adenomyoma is a rare benign non-neoplastic tumor-like lesion. it originates from abnormal embryonic buds but its pathogenesis has not been fully elucidated. method: a -year old man with severe pain, intussusception and ileus underwent a resection of a part of the intestine containing a tumor mass acting as a lead point. tissue was analysed using h&e, histochemical and immunohistochemical stains. results: lesion located in the mucosa, submucosa and muscularis propria consisted of glandular structures varying in size and morphology, the larger ones containing papillary projections and the smaller ones being similar to brunner's and peribiliary glands, both of them surrounded by smooth muscle bundles. the lesion was not accompanied by ectopic pancreas, and it indicated a diagnosis of adenomyoma of the ileum. glandular elements were ck + and ck − and cdx- −, opposite to intestinal mucosa. conclusion: adenomyoma is a mass lesion rarely found distal to the duodenum, which bares close clinical and morfological resemblance to a tumor and some non-neoplastic conditions. cytokeratin expression favors the heterotopic pancreas theory of pathogenesis, but abnormal interaction between the endoderm-and mesoderm-derived tissues can not be excluded. smoothelin in biopsies of colorectal carcinomas as marker of muscularis propria invasion p. stoemmer * , p. torres-galea * gemeinschaftspraxis pathologie, forschungslabor, augsburg, germany objective: staging of colorectal carcinomas is of eminent importance for prognostication and treatment; preoperative distinction between high grade dysplasia, invasion of submucosa (with permigration of the muscularis mucosae mm) and infiltration of muscularis propria (mp) is difficult in biopsies, due to similarities of smooth muscle cells in mm and mp. smoothelin is a robust marker of the mp in normal colon, not expressed in mm.we analysed it value for distinguishing mp and mm near carcinomas. objective: tuberculosis can affect any part of the gastrointestinal tract. anal localization, in particular, has a very low incidence ( , %). method: a year-old male presented, complaining of perianal pain, constipation, weight loss of kilos the last months and night sweating. physical examination was normal, except for an ulcerated lesion in the anal region. the patient was submitted to computerized tomography (ct) and incisional biopsy of the lesion. results: the ct of the chest revealed multiple, scattered, calcified nodules in the upper zones of both lungs and histological examination showed confluent, non-necrotizing, epithelioid granulomas with the presence of langhans' multinucleated giant cells. acid-fast bacilli were detected both within the anal lesion and in the sputum. mantoux test was positive. the patient had a history of pulmonary tuberculosis years ago. a diagnosis of anal tuberculosis was made and the patient was put on a four drug anti-tuberculous regimen. in months the symptoms improved and the perianal lesion healed. conclusion: a tuberculous origin must be considered when the cause of anal and perianal lesions is unclear. therefore histological and bacteriological confirmation is essential in order to avoid undue delay in diagnosis and treatment. the relation of braf v e mutation and microsatellite instability in colorectal carcinomas i. turkmen * , n. bassüllü, b. toptas, t. Öztürk, r. yasar, p. korkmaz, n. saygili, o. Öztürk, g. demir, g. dogusoy * istanbul bilim university, dept. of pathology, istanbul, turkey objective: colorectal carcinogenesis is associated with various morphological and molecular pathways. microsatellite instability (msi) pathway has familial and sporadic forms. among crc's % of the sporadic cases are associated with msi and these have hypermethylation of the mlh promoter associated with braf v e gene mutation. familial forms do not show braf v e mutation. method: we investigated the microsatellite instability (msi) by immunohistochemical study of mlh , pms , msh and msh together with the braf mutation presence and their correlation with standard histopathological parameters in sporadic crc cases. results: the age distribution was - with an average of , . immunohistochemical expression loss of mlh , pms , msh and msh was seen in , %, , %, , % and , % of the total cases respectively. msi was seen in , % of the cases. the braf v e gene mutation was seen in cases and among these cases ( , %) showed msi, while msi frequency was , in non-mutated group. and this was statistically significant (p < . ). msi was found significantly correlated with grade and lymph node number (p< . ). conclusion: our study confirmed that sporadic cases of crc with msi are associated with the braf v e gene mutation. msi is correlated with histologic grade and dissected lymph node number. colon biopsy diagnostics may reliably be performed using virtual microscopy objective: virtual microscopy using whole slide images (wsis) is a feasible alternative to optical microscopy, offering major advantages for pathology practice. the present study aims to prove reliability of this promising technique for colon biopsy diagnostics. method: colon biopsies (n= ) were assessed (into main diagnostic groups) using both glass slides and wsis by pathologists and residents. two of the pathologists having ample experience using wsis, scored the biopsies in a primary diagnostic setting. for each case the criterion standard diagnosis was defined based on glass slide diagnoses. accuracy was defined as the percentage of concordance with the criterion standard. kappa statistics were calculated as a measure of observer agreement. results: the overall concordance rates were . % for wsis and . % for conventional microscopy. the intraobserver (wsis versus glass slides) agreement was good to excellent, with kappa values ranging from . to . (mean . ) and was higher than the interobserver agreement for glass slides (mean . ). concordance with the criterion standard varied less between wsis and glass slides in the diagnoses of pathologists with virtual experience. conclusion: this study showed good diagnostic accuracy and reproducibility for wsis, indicating this technology may be used for colon biopsy diagnostics. intestinal occlusion due to colonic lipoma: a case report f. vukmirovic * * clinical center of montenegro, dept. of pathology, podgorica, montenegro objective: lipomas of the digestive tract are rare benign tumor and most often found incidentally during a colonoscopy, computed tomography scan, surgery or autopsy. lipomas of the colon were first reported by bauer in and are most often located in the ascending colon. the incidence of this lesion is estimated between . and . % and represents . % of the colonic benign lesions. we report a case of patient with symptoms of ileus due to colonic lipoma. method: a -year-old man patient was urgently admitted with symptoms of ileus. right hemicolectomy was performed, and clinical impression was that it was a malignant tumor. results: examination of the cecum we found polypoid whitish yellow tumor, size × , cm. on histology the tumor was composed of mature adipose tissue without cellular atypia. the tumor was located into submucosa. larger parts of the surface mucosa was eroded. the postoperative course was uneventful and intestinal passage was quickly established. conclusion: although colonic lipomas seldom cause severe symptoms in patients and are easily removed by endoscope while they are small, severe symptoms like abdominal fullness, intestinal obstruction, intestinal bleeding and intussusceptions may appear as a lipoma grows larger. in some cases lipoma may clinically mimic colonic carcinoma. prognostic impact of lymph node ratio outperforms positive lymph nodes and lymph nodes harvested: a time-dependent analysis in mismatch repair-proficient and -deficient colorectal cancers i. zlobec * , e. karamitopoulou, l. terracciano, d. inderbitzin, a. lugli * university of berne, inst. of pathology, bern, switzerland objective: we compare the prognostic strength of the lymph node ratio (lnr), positive lymph nodes (+lns) and collected lymph nodes (lncoll) using a time-dependent analysis in colorectal cancer patients stratified by mismatch repair (mmr) status. method: stage iii-iv patients were included. multivariable cox regression analysis and time-dependent receiver operating characteristic (troc) curve analysis were performed. the area under the curve (auc) over time was compared for the three features. results were validated on a second cohort of stage iii-iv patients. results: the auc for the lnr was . and outperformed + lns and lncoll by - % in both mmr-proficient and -deficient cancers. lnr and + lns were both significant (p < . ) in multivariable analysis but the effect was considerably stronger for the lnr [lnr: hr= . ( % ci: . - . ); +lns= . ( % ci: . - . )]. similar results were obtained for patients with > lncoll. an optimal cutoff score for lnr = . was validated on the second cohort (p< . ). conclusion: the lnr outperforms the + lns and lncoll even in patients with > lncoll. its clinical value is not confounded by mmr status. a cut-of score of . may best stratify patients into prognostic subgroups and could be a basis for the future prospective analysis of the lnr. prognostic value of tumor-stroma ratio in rectal adenocarcinomas s. zoidze * , r. scheer, j. klaase, m. elferink, a. baidoshvili * laboratorium pathologie oost, enschede, netherlands objective: recently, tumor-stroma ratio (tsr) has been identified as a strong predictor for survival in colorectal cancer. despite an identical biology clinical implications are quite different for colon and rectal cancer regarding to anatomical differences. method: tsr was estimated on h&e stained histological sections of patients who underwent resection for rectal adenocarcinoma between and . none of these patients had received neoadjuvant chemo-or radiotherapy. the tsr was determined, by two independent investigators, in different layers of the rectal wall at the point of highest tumor infiltration and at the border of the tumor. tsrs were categorized into three categories: tsr-low, tsr-moderate and tsr-high. results: patients with stage i and ii disease (t - n ) and tsr-high showed significantly better year survival rates for overall survival compared to tsr-low and tsr-moderate (p= . ) and a trend to a better disease specific survival (p= . ) and disease free survival ( . ). in a multivariate cox regression analysis the tsr remained an independent prognostic factor for overall survival, when adjusted for age, pt-status and grading. conclusion: tsr as a prognostic tumor characteristic can be used to identify patients with a good and a poor outcome in lymph node metastasis negative cases. objective: goblet cell carcinoid (gcc) of appendix vermiformis is a rare neoplasm that share histological features of both adenocarcinoma and carcinoid tumor. while its malignant potential remains unclear, gcc's particularly show transmural dissemination and are more aggressive than conventional carcinoids. patients usually present with acute appendicitis. they usually lack the formation of a well-defined tumor mass; thus, it is somewhat difficult to accurately assess their size. method: case presentation: fifty years old male patient applied to the emergency service with abdominal pain and laparatomy was performed with suspicion of acute appendicitis. grossly, conjestion and exudation on the distal edge of appendectomy material were seen. a × cm lesion which was spreading into peripheral adipose tissue was seen on the cut surface. microscopically, tumor was composed of goblet cell groups resembling "signet ring cells". tumor was infiltrated into muscularis propria and mesoappendix. immunohistochemically, chromogranin, synaptophysin, cytokeratin , mcea and p were stained positive. ki (mib ) proliferation index was %. conclusion: adenocarcinoid of the appendix is a rare tumor, which is very difficult to diagnose preoperatively and even macroscopically, making histological examination essential. immunohistochemical staining is required for definitive and differential diagnosis. here, we present this rare case with literature reviewed. normal colon tissue and colon carcinoma show no difference in heparanase promoter methylation d. hershkovitz * , y. peerless, e. simon, e. sabo, o. ben-izhak * rambam health care campus, dept. of pathology, haifa, israel objective: heparanase, the sole heparan sulfate degrading enzyme, has a role in cellular invasion. accordingly, a large number of studies have demonstrated an association between heparanse expression and tumor stage and patients' prognosis. in colon carcinoma, heparanase shows increased expression in tumor compared to normal tissue and its expression correlates with the presence of metastasis. one of the regulatory mechanisms of heparanase expression is methylation on its promoter. in the present study we evaluated the role of heparanse promoter methylation in colon carcinoma. method: analysis of heparanse promoter methylation was done on samples of colon carcinoma as well as sample of normal colonic mucosa. dna was extracted from ffpe tissue and subjected to bisulfite conversion. the relative fraction of methylated and unmethylated dna was evaluated with real-time pcr. results: the fraction of methylated dna was %± . in the colon carcinoma group, and . %± . in the normal colon group (p= . ). only one case in the normal group and one case in the tumor group showed more than % methylation in the heparanase promoter. conclusion: we did not find any difference in heparanase promoter methylation between colon carcinoma and normal colonic mucosa, suggesting that heparanase overexpression in colon carcinoma is mediated by other mechanisms. objective: among b-cell non-hodgkin's lymphomas, neural cell adhesion molecule/cd expression is exceptional. the aim of this study is to report unusual cd (neural cell adhesion molecule, ncam) expression on diffuse large b cell lymphoma (dlbcl). method: the panel of antibodies included cd , cd , cd , bcl- , bcl- , mum- and cd . results: a total of cases of dlbcl were identified and one case was also detected positive for cd . the subject was female, years old. the patient had presented generalized lymphadenopathy. she had not documented involvement of extranodal sites. the case is alive with disease year after diagnosis and chemotherapy treatment. objective: a case of mediastinal t-cell lymphoblastic lymphoma with indolent clinical course in a -yearold man was studied by immunohistology and molecular biology. method: formalin-fixed sample. histology with ematox-ilin&eosin and giemsa; immunohistochemistry by apaap method. t-cell receptor gamma rearrangement by biomed protocols. results: after one fainting episode the patient was found to have pericarditis caused by a xx-cm-diameter mediastinal mass. the first biopsies were unsuccessful and the third diagnostic one was done months later. in this time-period the patient was well without symptoms and the mass stable. after two mediastinal biopsies with unspecific features (castleman disease?), the diagnostic one showed fibrosis, abundant small reactive lymphocytes (cd +/cd +) and areas with diffuse small-medium size lymphocytes positive for cd , tdt, cd , cd , lmo and high ki . t-cell receptor gamma genes were monoclonally rearranged. conclusion: indolent t-lbl is exceedingly rare: for this reason in our case lmo was a key marker for the diagnosis which was delayed for the presence of fibrosis and reactive b cells. objective: granulocytic sarcoma (gs), also termed myeloid sarcoma or chloroma, is a rare malignant solid tumor resulting from the extramedullary proliferation of myeloblasts or immature myeloid cells. gs most frequently occurs in patients with acute myeloid leukemia, myeloproliferative neoplasms or myelodysplasia. gs rarely presents in the absence of systemic myeloid disease. gs most commonly occurs in the soft tissues of the head and neck, bone, skin and less often in the central nervous system and spinal cord. results: a year old woman presented with month history of weakness of both legs. mri of the thoracal spine showed cm diameter epidural mass compressing the dural sac in the spinal canal at the t - levels. decompressive laminectomy and tumor removal were performed resulting in neurological improvement. histologically neoplastic cells have round nuclei with finely dispersed chromatin and scant cytoplasm proliferating in a diffuse pattern. immunohistochemically, tumor cells were positive for mpo, cd , cd , cd . diagnosis was gs without bone marrow involvement. conclusion: we report the case of gs due to unusual localization and nonleukemic presentation. results: there were males and female with a median age of years ( - years). physical examination shows splenomegaly in all cases and hepatomegaly in cases, hematological parameters shows lymphocytosis with peripheral hairy cells in all cases. flow cytometric immunophenotyping was done in cases, and confirmed the diagnosis of hcl with cd c, cd +, cd +, cd +, and cd -, cd -, and cd -. bone marrow biopsy was done in all cases, showing massive infiltration with villous cytoplasmic projection cells that were cd positive. the treatment consists of blood transfusion in all cases, associated with splenectomy in cases, only patient was treated with ( cda: cladribine) with a complete remission after a follow up of months. conclusion: hcl has consistent cytologic histologic and immunologic features that make classification reliable and reproducible; it remains problematic because of the variety of the disorders and the differential diagnosis of splenic lymphoma of the marginal zone. objective: primary cns lymphomas (pcnsl) are uncommon tumors in immunocompetent patients, they represent up to % of non hodgkin lymphomas (nhl) and - % of all brain tumors. the majority of pcnsl are diffuse large b cell lymphomas, - % are t cell lymphomas, in rare instances low grade b cell lymphomas. we determine clinical characteristics, histological findings and treatment outcome of pcnsl. method: five cases of pcnsl occurring between and were retrospectively reviewed. results: they were males, female, median age years, range ( - ). most common symptom was neurological deficits ( cases). mri of the brain revealed an expansive tumor affecting the parietal bone in cases, the temporal bone in cases and the frontal bone in case. immunohistological finding showed large b-cell nhl in cases, anaplastic lymphoma in cases, and large-cell immunoblastic lymphoma in case. treatment consists on exclusive tumoral resection in cases, surgery and chemotherapy ( case), complicated by death in all these cases. surgery followed by high dose chemotherapy and radiotherapy ( case) with complete remission after a follow-up of years. conclusion: chemotherapy followed by involved field irradiation appears to be an adapted therapy. objective: primary cutaneous non-hodgkin's lymphoma (pcnhl) is defined as lymphoma limited to skin without extra-cutaneous involvement at presentation. we describe here epidemiological and histo-pathological aspects of pcnhl. method: retrospective review of clinical data of pcnhl patients diagnosed in hematology department, frahet hached hospital, sousse ( . results: fourty three patients with pcnhl, median age: years ( - ), sex-ratio was . . the site of cutaneous involvement was upper lumb in cases, lower lumb in cases and disseminated in cases. pcnhl was small t cell lymphoma in cases, large b cell lymphoma in cases, anaplastic, cd + cd + hematodermic neoplasm, and follicular type in case respectively. according to tnm classification cases were t bnxmx, cases were t n m . after first line therapy, patients in cr, pr was obtained in case and failure in cases, patients relapsed within to months (median months), all of them were disseminated cases. conclusion: our study indicate that the extend of cutaneous involvement at the time of diagnosis is a significant prognostic factor in pcnhl. objective: follicular lymphomas (fls) account for one third of non hodgkin lymphomas (nhls) in adults. the disease is characterized by a response to initial treatment, followed by relapses, sometimes associated with histologic transformation into high grade nhl. analyze epidemiological characteristics of patients with fl. method: retrospective study of of adult fls diagnosed in hematology department ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . results: median age was years ( - ), sex-ratio: . the most frequent clinical symptom at diagnosis was lymphadenopathy in cases. involvement of the spleen in cases. b symptoms were present in cases. performance status was more than in cases. according to the international prognostic index (flipi), patients have low index, have intermediate index, and have high index. grade fl was seen in the majority of cases. treatment modalities varied over time and according to the disponibility of drugs especially to anti cd monoclonal antibody. cr was obtained in cases, pr in cases; patients relapsed within - months. histologic transformation was seen in cases, we have noted deaths related to the disease in cases. conclusion: treatment results in fl might not only be improved by more effective induction regimens but also by maintenance treatment. objective: cd and fli- are widely used for their diagnostic utility in ewing sarcoma/peripheral neuroectodermal tumor (es/pnet). cd expression has been documented in a variety of tumors, including lymphoid malignancies of t cell origin. also fli- expression is common in different tumor types. however, few studies have investigated the cd and fli- immunoreactivity in diffuse large b-cell lymphoma (dlbcl). we aimed to determine the frequencies of cd and fli- immunohistochemical expressions in dlbcl which may lead to a misdiagnosis. method: cd and fli- expressions are retrospectively investigated by immunoperoxidase staining in prechemoterapy primary tumors of dlbcl cases. results: out of cases, cd and fli- expressions were observed in ( . %) and ( . %) tumors, respectively. concomittant expressions of cd and fli- proteins were found in / ( . %) cases. the present study revealed that cd and fli- are frequently expressed in dlbcl, thus dlbcl should be considered in the differential diagnosis of cd + and fli- + neoplasms. primary cutaneous marginal zone lymphoma with biclonality e. beretouli * , t. koletsa, a. bouzakis, s. mavropoulou, g. karkavelas, i. kostopoulos * ahepa hospital, dept. of pathology, thessaloniki, greece objective: primary cutaneous marginal zone lymphoma (pcmzl) represents a b-cell lymphoma which presents with papules, plaques or nodes. the presence of a monotypical light chain b cell population supports the diagnosis of pcmzl. we describe a case of pcmzl which showed different light chain restriction in two heterochronous lesions. method: Α year-old male patient presented in with a cutaneous nodule in the anterior surface of the tibia, which was totally excised. the patient did not show other lesions since , when a new cutaneous nodule located in the right ankle, was appeared. immunohistochemical analysis was performed in both biopsies. results: histological and immunohistochemical findings of both biopsies were consistent with marginal zone lymphoma of the skin with lambda light chain restriction in the biopsy of the first lesion and kappa light chain restriction in the biopsy of the second lesion. blood laboratory studies, chest and abdominal computerized tomography scans revealed no evidence of systemic involvement by lymphoma. conclusion: the finding of both kappa and lamda light chain restricted b cell populations in pcmzl is unusual. the monoclonal light chain switching in pcmzl is rare and its pathogenesis is discussed. objective: microvessel density (mvd) has prognostic significance in some malignancies. little information exists about angiogenesis in mantle cell lymphoma (mcl), although antiangiogenic drugs are used experimentally. prognostic factors of mcl are needed as outcome and therapy are heterogeneous and often unsatisfactory. the aim is to assess mvd in mcl and to evaluate its prognostic significance. method: mcl specimens (ffpe) were examined by immunohistochemistry with anti-cd antibody. mvd was quantified using systematic uniform random sampling and unbiased counting frames. clinical data were analyzed in kaplan-meier survival curves and log-rank test. results: median survival (ms) of all patients was months, median progression-free survival (mpfs) months. mean mvd was , microvessels/mm , median , /mm . dividing cases into quartiles: < , /mm , < , /mm , < , /mm and ≥ , /mm ( , , , cases), ms and mpfs did not differ statistically significantly between these groups (p= , ; similar for therapy groups). visible differences are only between the fourth quartile and the rest: objective: plasmablastic lymphoma (pbl) is a rare aggressive subtype of diffuse large-b-cell lymphoma which occurs primarily in the oral cavity of hiv-positive patients. it is extremely rare in immunocompetent patients. method: a total of cases of pb were retrospectively analyzed. all cases had tissue available for immunohistochemistry and in situ hybridization. results: the age range of the patients was - years (median ) and all were men. three patients were hiv negative. hiv + patients: tumors involved the oral cavity (n = ), and small intestine (n= ). hiv-patients: tumors were located in bone marrow (n= ) and lymph nodes (n= ). all tumors were composed of monomorphic plasmablastic cells. tumors were negative for cd and cd , while they showed diffuse positivity for cd , cd , mum and ema. by ish, monoclonal light chain restriction was detected. ebv (eber) was positive in all cases. the median overall survival was , months regardless of the intensity of chemotherapy. only one patient is alive month after diagnosis. conclusion: pbl may appear in non immunocompromised patients and other locations than oral cavity. any patient diagnosed with pbl should be tested for hiv. patients who had hiv-negative pbl have lower rates of oral involvement. spinal granulocytic sarcoma preceding clinical manifestation of acute myeloid leukemia v. cemerikic-martinovic * , n. drndarevic, n. andjelkovic, p. djordjevic, d. jovanovic, r. nikolic, t. martinovic * beo-lab, dept. of pathology, belgrade, serbia objective: granulocytic sarcoma is a rare malignant neoplasm of primitive myeloid cell origin, most commonly found in association with acute myeloid leukemia. granulocytic sarcomas generally occur in the soft tissues, bone, and skin. method: we report a case of a -year old man with spinal granulocytic sarcoma manifesting as rapidly progressive paraplegia preceding clinical manifestation of acute myeloid leukemia. results: magnetic resonance imaging revealed a tumor in lumbal spine (l -l ). baseline laboratory data were normal. he underwent emergent laminectomy and the tumor was totaly resected. histological examination of lesion demonstrated diffuse proliferation of large atypical cells with round nuclei with delicate chromatin and one or more prominent nucleoli. immunohistochemical studies revealed positive staining for lca, cd , cd , cd , lysozyme and cd while myeloperoxidase, lymphoid, neuroendocrine markers, s- and cd were negative. the final histological diagnosis was granulocitic (monoblastic) sarcoma. no chromosomal aberrations were detected by fish. a month later, acute myelomonocytic leukemia was diagnosed upon a peripheral blood and bone marrow examination showing an increased number of abnormal monoblasts. chemotherapy, with multiple regimens for leukemia in combination, did not affect the tumor and the patient died. conclusion: granulocytic sarcoma should be considered in the differential diagnosis of spinal tumors. objective: survivin, a member of the inhibitor of apoptosis family might play an important role in the pathogenesis of diffuse large b cell lymphoma (dlbcl). we investigate clinical and prognostic significance of survivin expression in nodal dlbcl. method: biopsy specimens obtained from patients with newly diagnosed nodal dlbcl treated with immunochemotherapy (r-chop) were immunostained for survivin. results: survivin immunoexpression (> % positive tumor cells) has been found in ( . %) patients. significant difference in immunoexpression was noticed between gcb and non-gcb subtype of dlbcl (p= . ). there was no significant association with ipi, "bulky"disease, ki- immunoexpression or other clinico-pathological parameters. univariate analysis showed that survivin expression was unfavorable factor for therapy response and shorter survival in patients with dlbcl (p= . and p= . , respectively). patients with survivin overexpression experienced relapse more often that the patients without expression of this apoptotic protein ( . % vs. . %), but this difference did not reach statistical significance (p= . ). the results of this study showed that disregulation of survivin expression had the important role in determination of course of disease in pateints with nodal dlbcl treated with r-chop. therefore, survivin represent potential therapeutic target in dlbcl. langerhans cell histiocytosis of the jaws: a report of ten cases with an analysis of the mechanism of eosinophilic infiltration y. objective: langerhans cell histiocytosis (lch) frequently presents eosinophilic infiltration. here we report ten cases of lch that involved the jaws and our investigation of the mechanism underlying eosinophilic infiltration in lch. method: we evaluated the ccl- /eotaxin- expression of lch cells via immunohistochemical staining. toluidine blue staining was used to inspect the densities of mast cells, with ten periapical granuloma specimens serving as a control group. results: every patient was classified as having single-system lch, even though acute lymphoblastic leukemia occurred in one patient during lch treatment. the ratio of mandible to maxilla lch was : . the jaw lesions were the earliest manifestation in seven patients with multifocal lch. toluidine blue staining revealed that the number of mast cells in lch lesions was not significantly higher than in periapical granulomas. however, upon immunohistochemical examination, most of the patients showed diffuse positivity for eotaxin- in lch cells, while few eosinophils/ t cells were immunoreactive. conclusion: we surmise that the eotaxin- expression of lch cells may be relevant to eosinophilic infiltration in lch. further studies of the eotaxin- functions including its influence on the immature state of lch cells may be needed to understand the pathogenesis of lch as well as the role of tissue eosinophilia in lch. method: we report a case of sant in years-old man presented with abdominal discomfort and pain in a left lumbal region. abdominal computed tomography scan revealed a massive haematoma extended from spleen to the enlarged left kidney and left retroperitoneal space. the patient also had a spontaneous rupture of right kidney years ago. results: macroscopically, the cut surface of spleen showed multiple well circumscribed red brown nodules. microscopically, sant consists of multiple well-circumscribed angiomatoid nodules showing plump endothelial cells and extravasated erythrocytes. nodular formations are surrounded by a variable lymphoplasmacytic infiltrate, spindle cells and collagenous stroma. immunohistochemical staining displayed endothelial phenotype that resembled splenic capillaries (cd +/cd +/cd −). expression of cd was also present. conclusion: the differential diagnosis of sant includes splenic hamartoma, inflammatory myofibroblastic tumor, littoral cell angioma and hemangioendothelioma. it has been postulated that sant represents a peculiar hamartomatous transformation of splenic red pulp in response to an exaggerated nonneoplastic stromal proliferation. sant has a benign clinical course and splenectomy has been supposed as curative. collision tumor of menningioma and non hodgin malignant lymphoma of cerebellum h. erdem * , a. k. uzunlar, u. yildirim, a. sav, m. dosoglu * duzce university, dept. of pathology, turkey primary central nervous system lymphoma (pcnsl) constitutes a rare group of extranodal non-hodgkin's lymphomas (nhls), primarily of b cell origin, whose incidence has markedly increased in the last three decades. immunodeficiency is the main risk factor, but the large majority of patients are immunocompetent. this report presents the case of a -year-old woman with a collision tumor of primary malignant lymphoma and meningioma in the cerebellum. collision tumor of primary malignant lymphoma and meningioma have not been described in the literature. the morphological aspect is interesting with regard to the problem of collision tumors. prognostic significance of immunohistochemical expression of the angiogenic molecules vegf-a, vegfr- and vegfr- in patients with classical hodgkin lymphoma e. georgiadi * , g. dimtsas, p. karakitsos, t. vassilakopoulos, i. thymara, p. korkolopoulou, e. patsouris, c. kittas, i. doussis-anagnostopoulou * larisa, greece objective: classical hodgkin lymphoma (chl) is characterized by the presence of a small percentage of malignant hodgkin and reed-sternberg (hrs) cells amongst a reactive background. the role of angiogenesis in chl is still unclear. the aim of the study was to evaluate the expression of vegf-a, vegfr- and vegfr- and their correlation with clinicopathological parameters and prognosis. method: the immunohistochemical expression of vegf-a (vg , dako), vegfr- (rb- , neomarkers) and vegfr- (sc- , santa cruz) was studied in a large cohort of patients with chl and the results were correlated with clinical characteristics and patient outcome. results: the neoplastic hrs cells expressed vegf-a, vegfr- and vegfr- in . %, . % and . % of the cases respectively and their expression levels were intercorrelated. expression of vegfr- and vegfr- was significantly and positively correlated with early disease stage, absence of b-symptoms, wbc < . , alb ≥ , ips ≤ . vegfr- was additionally positively correlated with male gender and esr < . all three molecules were statistically correlated with ramifications of blood vessels but not with microvessel density. conclusion: based on our results, we could speculate that, in contrast to solid tumours, the process of angiogenesis is probably an early event in neoplastic progression in the context of hodgkin's lymphoma. ps- - p upregulation is associated with proliferation and is not capable of inducing apoptosis in hodgkin's lymphoma e. georgiadi * , g. dimtsas, t. p. vassilakopoulos, v. g. gorgoulis, i. a. doussis-anagnostopoulou * larisa, greece objective: p is a tumor suppressor protein described as the "guardian of the genome". in a normal cell, p is inactivated by its negative regulator, mdm . once activated, p induces cell cycle arrest and apoptosis by activating various cell cycle related genes, like waf / cip encoding for p . although mutations that deactivate p are the most common genetic alteration found in cancer, they are rare in the context of hodgkin's lymphoma (hl). method: p immunohistochemical expression was investigated in a subset of cases of primary hl. the study of the p -downstream protein p was used as an indirect way of analyzing its functional status. p expression was correlated with proliferation, apoptosis, clinicopathological data and prognosis of the patients. results: p showed a median value of . %, suggesting an upregulation of the p gene in hl, and a significant correlation with p (p= . ) and the proliferation marker mib expression (p= . ). conclusion: although p overexpression is a frequent finding in hl and there is indication of its functionality, this upregulation does not lead to apoptosis. it can be speculated that this is possibly due to mdm -p interactions. ps- - simultaneous gastric adenocarcinoma and b cell lymphoma of the stomach f. gerin * , k. turkoz, b. kantarcioglu, c. ataizi celikel * marmara university, dept. of pathology, istanbul, turkey objective: the simultaneous association of gastric carcinoma with gastric lymphoma is a rare event. method: we describe a years old man, who had been diagnosed as marginal zone lymphoma in a cervical lymph node biopsy years previously.no endoscopic examination was evaluated then. there was no pathological appearance in the gastrointestinal system by ct scanning and patient was considered nodal marginal zone lymphoma. patient had taken cycles of chemotherapy which consisted of rituximab, cyclophosphamide, vincristine, and prednisolone. , years after completion of chemotherapy, a gastric wall thickening compatible with gastric lymphoma or linitis plastica was detected on ct and gastroscopic examination was indicated. endoscopic examination revealed an erosive, fragile, hemorrhagic, malignant-looking lesion with a diameter of . cm in the cardia. besides, rugae of fundus and corpus were severely rough and hyperemic suggesting a diffuse malignant infiltration. multiple biopsies were taken from cardia, fundus and corpus of the stomach. results: cardiac samples showed pancytokeratin positive signet ring cells which contain neutral and acidic mucine in microscopic examination. samples of corpus and fundus did not show similar carcinoma cells, instead, there was diffuse infiltration of atipical lymphocytic infiltration consistent with marginal cell lymphoma both morphologically and immunophenotypically. conclusion: co-occurrence of carcinoma and marginal zone lymphome is a rare event and dramatically exacerbates prognosis of a patient with such an indolent lymphoma. assessment of demographic data, staging and experesion of cd , cd , cd , bcl- in reed-sternberg cells of hodgkin lymphoma cases in their first admission in urmia imam komini hospital b. ilkhanizadeh * , p. mazlomi * imam komini hospital, dept. of pathology, urmia, iran objective: immunohistochemistry plays an important role in diagnosis of hodgkin lymphoma. beyond diagnostic importance of immunohistochemistry, so far many studies have investigated the correlation between cellular markers and the prognosis of patients with hodgkin lymphoma. amongst other markers, cd is one of the most studied marker with a lot of controversy around it. method: retrospectively eligible patients entered the study. clinical and laboratory data and immunohistochemistry findings analyzed for possible correlation between disease stage and other parameters. results: in our study . %, . % of cases respectively expressed cellular markers cd and cd while only . % of cases expressed cd . furthermore statistical analysis revealed that cd was inversely correlated with disease staging. (p= . ). in contrast we didn't find any relation between cd or cd positivity with disease staging. (p= . , p= . ). however in our study expression of cd was not related to stage or other parameters of poor prognosis which proposes that in our patients this marker possibly was not related to disease prognosis. conclusion: therefore we suggest that cellular marker cd is not beneficial beyond ips factors and its usage should be confined to diagnostic purposes. primary naturall killer cell lymphoblastic leukemia/ lymphoma of central nervous system g. karagkounis * , t. argyrakos, e. ronne, e. dimitriadis, d. papadopoulos, g. stranjzalis, d. rontogianni * athens, greece objective: a -year old woman with a -year history of a multifocal lesion occupying the cerebellum who periodically received cortisol and etoposide as it was considered tumor-forming multiple sclerosis. method: two years after initial clinical diagnosis a stereotactic biopsy was performed. immunohistochemistry and in situ hybridization were performed on the tissue sample. results: the perivascular (virchow-robin) space of microvessels was occupied by middle sized lymphocytes with blastic chromatin and scant cytoplasm. the neoplastic cells were negative for: . b-cell markers: cd , cd , cd , cd a, pax- , . myelogenous differentiation antigens: mpo, cd , cd , . plasmacytoid dendritic differentiation antigens: cd , tcl , . blastic cells markers: cd , cd . t-cell markers: cd , cd with restricted expression of cd and cd in a few cells, . cytotoxic enzymes tia- , granzyme-b and perforin. all cells expressed intensely cd and tdt and some of them cd . in situ hybridization for ebv virus (ebers) was negative. pcr analysis: t-cell receptor and immunoglobulin genes were in germ line. bone marrow was negative. objective: lymphomas constitute , cases on inhabitants annually, its frequency grows. method: this work presents epidemiological, morphological analysis of nodal/exstranodal lymphomas. analysis of data from izhevsk cancer center during - was carried out. results: lymphoma appeared in . % among first diagnosed malignant tumors. the panel of monoclonal antibodies was used for tumor immunophenotype identification. nodal lymphomas presented cases: -hodgkin lymphoma (hl), non-hodgkin lymphoma (nhl). exstranodal lymphomas presented cases, various variants of nhl were more frequent. small lymphocyte lymphoma - , diffuse large b-cell lymphoma (dlbcl) - , follicle center lymphoma - , mantle cell lymphoma - were frequent among nodal lymphomas. t-cellular nhl presented cases ( -peripheral t-cell lymphoma, -angioimmunoblastic tcell lymphoma). among other organs of lymphatic system lymphomas appeared in tonsils ( ), thymus ( ) and spleen ( ). exstranodal nhl were registered in stomach ( ), rarely in other organs -testis ( ), central nervous system ( ), nose ( ), breast ( ). as rule dlbcl was more frequent ( ). conclusion: results coincide with data on lymphoma's frequency and morphological characteristic in other regions. in situ mantle cell lymphoma in nasopharynx t. koletsa * , f. tsiompanou, c. poulios, g. karkavelas, i. kostopoulos * auth, dept. of pathology, thessaloniki, greece objective: the nasopharynx normally contains abundant lymphoid tissue and it can be the site of both lymphoid hyperplasia and lymphomas. we present the first case of the recently described in situ mantle cell lymphoma (mcl) in nasopharynx. a year-old woman presented with nasal obstruction in . a mass located in nasopharynx was found. biopsy was sent for histological examination. a new biopsy was taken years later. histological, immunohistochemical and fish studies were performed. results: histological examination of both biopsies revealed lymphoid hyperplasia, characterized by cd and cyclind positive cells of mantle zones, findings consistent with in situ mcl. the diagnosis of in situ mcl confirmed by fish analysis for t( ; ). the patient examined thoroughly and has remained free of an overt lymphoma to the present day. conclusion: pathologists and otorhinolaryngologists should be aware that in situ mcl may be observed in nasopharynx. it may be appropriate to perform cyclind immunostain, even in cases with clinical and histological findings compatible with lymphoid hyperpasia. the patients should be examined thoroughly, since in situ mcl may accompanied by an overt lymphoma in other sites or it may be a precursor of an overt mcl. multisystem langerhans' cell histiocytosis coexisting with metastasizing adenocarcinoma of the lung a. lovrenski * , m. panjkovic, z. eri * institute for lung diseases, dept. for pathology, sremska kamenica, serbia objective: langerhans' cell histiocytosis (lch) is an uncommon disease of unknown etiology characterized by uncontrolled proliferation and infiltration of various organs by langerhans' cells. to our knowledge, this is the first case reporting an association of multisystem lch with metastasizing adenocarcinoma of the lung. method: we present the case of a -year-old man, heavy smoker, with dispnea, cough, hemoptyses, headache and ataxia, who died shortly after admission to our hospital. on the autopsy, we found tumor in the posterior segment of the right upper pulmonary lobe as well as a right-sided occipito-parietal lesion which penetrated into the right ventricle resulting in internal and external hematocephalus. histologically and immunohistohemically, the diagnosis of primary lung adenocarcinoma with brain metastasis was made (tumor cells showed positivity for ck and ttf- which confirmed the diagnosis). light microscopic examination of the other organs showed lch involving the pituitary gland, hypothalamus, spleen, mediastinal lymph nodes, and lungs. immunohistochemical studies revealed cd , s- and cd a immunoreactivity within the langerhans' cells. conclusion: multisystem form of lch with extensive organ involvement was an incidental finding, while the metastatic lung adenocarcinoma to the brain, that led to hematocephalus, was the cause of death. langerhans cell histiocytosis associated with extramedullary hematopoiesis in a -year-old male patient l. lungoci * , l. elena, b. flavia, m. rodica * city hospital, pathology, timisoara, romania objective: langerhans cell histiocytosis is a rare disease histologically characterized by the proliferation of langerhans cells. method: a -year-old male came to our attention presenting with erythematous-squamous lesions all over the body, axillary and inguinal adenopathies and hepatosplenomegaly. we have taken a cutaneous and an axillary lymph node biopsy. results: microscopic examination of the cutaneous fragment revealed the presence of a dermal infiltrate of large cells with an eosinophilic cytoplasm and vesicular, nucleolated nuclei with an irregular nuclear margin, raising suspicion of a cutaneous lymphoma. immunohistochemical stains for cd , cd , cd a, cd , cd , cd , cd , and bcl- were negative, and the previous diagnosis was not confirmed. the microscopic examination of the axillary lymph nodes showed foci of extramedullary hematopoiesis, raising suspicion of a lymphoproliferative process, but the immunohistochemical stains and the paraclinical data didn't confirm it. based on the intense positive reaction for cd , s and ki- showed by the tumoral cells from the prelevated tissues, correlated with the clinical and paraclinical findings, a diagnosis of langerhans cell histiocytosis was established. conclusion: the particularity of our case consists in the presence of extramedullary hematopoiesis in the lymph nodes of a patient diagnosed with langerhans cell histiocytosis. unusual primary cutaneous presentation of b-cell chronic lymphocytic leukaemia s. mavropoulou * , z. tatsiou, g. xanthopoulidis, a. goutzouvelidis * general hospital, laboratory of pathology, xanthi, greece objective: b-cell small lymphocytic lymphoma/chronic lymphocytic leukaemia (b-cll) is a low-grade lymphoproliferative disorder and cutaneous lesions are rarely the presenting findings. method: we report the case of a -year-old woman who presented in winter with erythematous plaque on her nose, of months duration. laboratory data showed elevated white blood cell count ( . /mm ) with small mature lymphocytes predominance ( %). results: histopathologic examination of the skin lesion reveals infiltration of the reticular dermis and subcutis, consisting mostly of small lymphoid cells, without epidermotropism. immunohistochemical examination revealed positivity for cd , cd a and cd in the neoplastic cells. according to the laboratory and histopathologic findings the diagnosis was consistent with b-cll with a primary cutaneous presentation. a staging bone marrow aspiration and biopsy showed % infiltration by a clonal b-cell proliferation with typical cll immunophenotyping. ct-imaging was normal, so stage i according to binnet classification was confirmed. the patient received cycles of chemotherapy and remained in remission for year, when the skin lesion recurred in winter, at the same location. conclusion: in conclusion we report an extremely rare case of subclinical b-cll with cutaneous presentation. it is important to maintain a high index of suspicion for a lymphoproliferative process in skin lesions with atypical lymphocytic infiltration. myeloid sarcoma mimicking nasal polyp s. mungan * , u. cobanoglu, s. ersoz, g. guvendi, m. yilmaz * karadeniz technical university, pathology, trabzon, turkey objective: a myeloid sarcoma is a tumour mass consisting of myeloid blasts with or without maturation occuring at an anatomical site other than the bone marrow. every site of the body can be involved, skin, lymph node, gastrointestinal tract, bone, soft tissue and testis. % cases presents at multiple anatomical sites. method: the current case is year old woman presenting with nasal polyp and underwent polypectomy. the polyp was , cm in diameter and had a bloody appearance. by microscopic examination there was a diffuse infiltration of neoplastic cells under the cilliated epithelium. the mitotic index was high in neoplastic lymhoid cells. immunohistochemical study was helpful for the differential diagnosis. objective: the aim of this study was to describe the epidemiology and anatomo-clinical features of the ear, nose and throat (ent) lymphomas in the centre of tunisia. method: all ent lymphoma cases diagnosed in the pathology department, farhet hached hospital, sousse during a -year period were analyzed. results: a total of cases of ent lymphoma were reported ( males, females) with a sex-ratio of . . the age at diagnosis varied from to years with a median age of . years. the majority of ent lymphoma cases ( %) were of b cell type including large b-cell lymphoma ( %) and small-cell b lymphoma ( %). nasopharynx and tonsils were the most involved areas with % and % of cases, respectively. according to the ann arbor staging system, these lymphomas are diagnosed in the stage ii ( %) followed by stage i ( %). conclusion: the most frequent lymphoma developed in ent region was b cell lymphoma, especially located in nasopharynx and tonsils. results: a total of cases were reported ( males, females) with a sex-ratio of . . the median age was years (range: - years). lymph node lymphomas were the most frequent accounting for . %, followed by ent, mediastinum, and haematopoietic system ( . % each one); cutaneous and digestive location were less frequent ( . % each one). among the patients, ( . %) had had hodgkin's lymphoma and ( . %) had had non-hodgkin's lymphoma (nhl). diffuse large b-cell lymphoma was the most diagnosed ( %) followed by burkitt lymphoma ( . %) and t-cell lymphoma ( . %). according to the ann arbor staging system, . % of patients were diagnosed in an advanced stage. method: we present a case of composite ailt and diffuse large b-cell lymphoma (dlbcl) a -year-old woman with multiples lymph nodes. biopsy of inguinal node was performed. three months later the patient had enlarged cervical node which was removed. results: firstly, the inguinal lymph node was diffusely infiltrated by small-medium size lymphocytes with scanty cytoplasm. the nuclei were irregular. several large cells were observed in a background of prominent arborizing high endothelial venules. the small cells were cd positive. the large cells were cd a, cd positive. theses cells showed nuclear positivity for eber by ish. secondly, the cervical lymph node showed large areas of necrosis admixed with pleomorphic and medium size t lymphocytes. there were a population of neoplastic ebv + pleomorphic b lymphocytes; some of them with rs-like morphology. we performed b and t-cell clonality studies. we detected tcr-gamma t-cell and igh b-cell gene clonal rearrangement. conclusion: occasionally igh gene rearrangement has been detected but only a few cases have demonstrated, morphologically, composite ailt and ebv-associated diffuse large b-cell lymphoma (dlbcl). typhlitis as initial manifestation of granulocytic sarcoma of the appendix: a case report p. rohani doost * , b. ilkhanizadeh, f. noroozinia, a. objective: myeloid sarcoma is a localized mass of myeloblasts or immature myeloid cells involving any extramedullary site. reports in the literature documenting leukemic infiltration of appendix are uncommon and extremely rare when symptoms mimicking typhlitis. method: a -year-old man with a history of relapsed acute myelogenous leukemia (fab classification, m ) admitted for receiving induction chemotherapy. after days of chemotherapy, he presented with fever, nausea, vomiting, abdominal pain and severe neutropenia ( . × /l as absolute neutrophil count). this manifestation was attributed to typhlitis and he received antibiotic therapy, but he came back days later with acute abdomen prompting exploratory laparotomy which revealed inflamed appendix. results: histologic examination showed diffuse appendiceal wall infiltration of mononuclear cells with medium-to-large vesicular nuclei, conspicuous nucleoli and pale eosinophilic cytoplasm. immunostains showed the diagnosis of granulocytic sarcoma with myeloperoxidase, leukocyte common antigen, cd , cd and cd positivity and epithelial membrane antigen, keratin, cd , cd , cd , cd a and cd negativity. conclusion: our case declares that physicians and surgeons should be aware of granulocytic sarcoma in the differential diagnosis of mild tenderness on palpation of the abdomen as a complication of acute myelogenous leukemia. objective: angioimmunoblastic t-cell lymphoma (aitl), one of the most frequent entities among peripheral t-cell lymphoma, is characterized by lymphadenopathy, b-symptoms, and an aggressive behaviour. method: we reported cases of aitl during a period of years ( ) ( ) ( ) ( ) . results: the age of patients varied between and yearold. they hadn't any pathologic history. two patients presented with a multiple cervical lymphadenopathy, and weight loss. the other patient had fever and sweat. physical examination found multiple lymph nodes with hepatosplenomegaly in patients. abdominal ultrasound and ct-scan showed diffuse lymphadenopathy with features raising the possibility of lymphoma. the diagnosis of aitl was confirmed by a microscopic and an immunhistochemical study of lymph node or liver biopsy. patients received a cure of chemotherapy conclusion: angioimmunoblastic t-cell lymphoma (aitl) is an aggressive non-hodgkin t-cell lymphoma. aitl is frequently associated to immunological and hematologic diseases such as autoimmune hemolytic anemia, vasculitis, rheumatoid arthritis, and autoimmune thyroid disease. its natural history has been the subject of controversy, considered for many years to be a nonmalignant disorder or a dysimmune disease, until the clonal nature of aitl was proven by molecular studies. although patients are usually treated with corticosteroids, chemotherapy, and/or plasmapheresis, outcomes were dismal. objective: low grade lymphomas (indolent) are commonly located on extranodal sites. nodal location are relatively of low incidence. our aim is to describe the epidemiological status of nbcl and to assess their pathological features in the center of tunisia. method: we report cases of indolent nbcl diagnosed in the department of pathology, during a period of years ( - ) . clinical, pathological and immunohistochemical (large panel of antibodies), treatment and outcome data are collected. results: the mean age was year old ( and years). the sex ratio was . . nbcl was . % of all n h lymphomas and . % of indolent nbcl of any sites. nodal enlargement and hepatomegaly were the main symptoms. small lymphocytic lymphomas were the most frequent variant ( %), followed by follicular lymphomas ( %). mantle cell lymphoma and marginal zone b cell lymphoma were rare ( % and %, respectively). they were exclusively of b cell type. % of cases were in stage iii-iv. % of patients received chemotherapy. % of recurrence and % of death were found. conclusion: nbcl in the center of tunisia are relatively uncommon. small lymphocytic lymphoma is the most frequent in our population. they are still of relatively worse prognosis. objective: bone marrow metastases can be found in some malignant tumors, but diagnosing a nonhematologic malignancy from marrow is an unusual event. herein we present a case of metastatic signet ring cell carcinoma with an unknown primary site. method: a year-old woman admitted to the hospital with weakness and bone pain. positron emission tomography/computed tomography (pet/ct) revealed disseminated bone lesions showing increased metabolic activity. bone marrow biopsy and aspiration were performed. results: bone marrow biopsy revealed extensive necrosis and nearly total replacement of normal bone marrow elements by atypical tumoral cells. tumoral cells were arranged individually or in small clusters, having hyperchromatic, eccentric located nuclei and abundant cytoplasm with signet ring cell morphology. even after thourough investigation of all the systems, a primary site of malignancy could not be detected. she received nine cycles of chemotherapy. craial mri revealed multiple metastatic lesions. months after the initial diagnosis, she died of disseminated metastatic lesions. conclusion: even after thorough investigations, a primary site of malignancy could not be detected. in the literature, all the presented cases had very short survival varying from days to a few months. our patient lived for months with multidiciplinary approach. objective: approximately % of follicular lymphomas (fls) are bcl negative and - % of fls lack a translocation t( ; ). previously we have found that negative bcl staining in cases that do have a t( ; ) is caused by mutations in the epitope for the bcl- antibody, but this is never the case in bcl- negative, t ( ; ) negative cases. now that translocation detection is more commonly performed we noticed that many of the cases that were referred to us as translocation negative fl are actually often nodal marginal zone lymphomas (nmzls) with extensive follicular colonization. method: t( ; ) negative cases that on h&e sections look like fl were analyzed by morphology and immunohistochemistry, including antibodies against cd , bcl- , ki- , and cd . results: careful observation of the bcl- staining pattern revealed that follicles that appeared positive at low power contained negative cells at high magnification. the amount of negative cells varied from follicle to follicle and was associated with the extent of ki- positivity. conclusion: based on morphology and immunohistochemistry we reclassified many cases of bcl- positive, t( ; ) negative fl into nmzl. although there is presently no positive marker for nmzl, we believe that such cases are responsible for most t( ; ) negative fl. objective: recent studies do not provide a definite answer on the origin and pathogenesis of lichen planus (lp). histologic findings are the same, regardless of the area involved. electron microscopy is useful to confirm the diagnosis in atypical cases and monitoring lp development. this study aimed an ultrastructural characterization of keratinocyte degeneration in pemphigoides, hypertrophic, and follicular lp types correlating this with levels of gelatinolytic activity caused by metalloproteinase- (mmp- ). method: skin biopsies were processed conventionally and examined in a jeol transmission electron microscope. mmp- expression was detected immunohistochemically. results: lp pemphigoides demonstrated intact basal keratinocyte cell membrane, and the lamina densa lining blister cavity correlating with moderate basal mmp- expression of weak intensity. hypertrophic type demonstrated good preservation of keratinocytes and their junctions along with strong basal and suprabasal mmp- expression of moderate intensity. both types demonstrated diffuse and strong mmp- immunostaining in dermal lymphohistiocytic infiltrate. a hypertrophic type revealed an increase of expression within dermal sweat glands comparing with lp pemphigoides. a diffuse and strong epidermal and follicular mmp- staining was noticed in follicular variant of lp. conclusion: ultrastructurally keratinocyte involvement displays deviations in various types of lp, and these changes correlate with levels of gelatinolytic activity caused by mmp- . objective: clevudine is a recently introduced anti-viral agent that shows efficacy against hepatitis b virus related chronic liver diseases. however, it has been reported that certain patients who have received long-term administration of clevudine exhibit myopathy involving mitochondrial abnormalities. to make a differential diagnosis congenital mitochondrial myopathy and drug induced mitochondrial myopathy, we investigate ultrastructural findings of drug induced myopathy. method: we studied histopathological features of myopathy focused on the various ultrastructural mitochondrial abnormalities found in patients with long-term clevudine therapy. results: in every case, ragged red fibers and multinucleated fibers with eosinophilic granules were observed. additionally, type fiber atrophy was found in cases. mainly concentrated in subsarcolemmal or inter-fibrillar areas, the abnormal mitochondria were enlarged and swollen, showing a variety of morphological types. most of the abnormal mitochondria indicated structural abnormalities in cristae, including the apparent decrease in the number, concentric lamellar pattern, and structure that is branched or lattice-like. conclusion: from the overall analysis, clevudine-induced myopathy is characterized by ragged fibers showing proliferation of abnormal mitochondria with various forms of inclusion bodies and abnormal cristae. another particular feature of interest is the presence of multinucleated fibers, which, in most cases, are filled with abnormal mitochondria. ultrastructural features of human mature oocytes subjected to cryopreservation s. a. nottola * * university la sapienza, dipt. die anatomia histol., rome, italy objective: oocyte cryopreservation protocols have not been fully optimized yet. our aims were to evaluate and compare the ultrastructure of human mature oocytes frozenthawed (f/t) after slow freezing and vitrification. method: the oocytes, fixed at sampling (fresh controls) and after freeze/thawing, were processed for light and transmission electron microscopy (lm and tem) observations. results: by lm, both fresh and f/t oocytes were rounded cells surrounded by an intact zona pellucida (zp) and containing uniformly distributed organelles. by tem, numerous vacuoles were found in f/t oocytes after slow freezing. on the contrary, vacuoles were only occasionally detected in f/t oocytes after vitrification, and in fresh controls as well. amount and density of cortical granules (cgs) appeared abnormally reduced in f/t oocytes, irrespective of the protocol applied. conclusion: in conclusion, a) cryopreservation currently ensures a good overall preservation of the oocyte; b) however, vacuolization appears as a recurrent form of cell damage during slow freezing, whereas the quasi absence of vacuoles seems the most relevant marker of quality in vitrified oocytes; c) premature cg exocytosis -and the consequent hardening of the zp -seems a non-specific, ubiquitous phenomenon occurring during freeze/thawing, suggesting the appropriateness of the use of icsi as the preferred insemination method after cryopreservation. tuesday, september , . - . results: there were women and men with a median age of (range - years). symptoms were dominated by abdominal pain, jaundice, vomiting, and alteration of the general condition. radiologic investigation showed multiloculated cystic mass of the pancreas, associated to splenic thrombosis and bone metastasis in one case. in gross, we received multilocular cysts of to cm in wall, their wall was fibrous, and thick, the internal surface showed many papillary projections and mural nodules. there was no communication with the duct system. on histologic examination, cysts had a fibrous wall lined by mucinous atypical cells showing frank anaplasia, wall's cyst was invaded by anaplastic glands; the stroma was of ovarian type in all cases. conclusion: pmc are uncommon neoplasia, their diagnosis is based on the histopathologic exam, their diagnosis is better than duct carcinoma and depends on the extent of tumour invasion. heterotopic pancreas of the gallbladder associated with acute cholecystitis o. akman * , e. e. pala, u. bayol, m. emiroglu * tepecik training research center, dept. of pathology, izmir, turkey objective: pancreatic heterotopia is a rare entity, which is commonly found in the stomach, duodenum, jejunum and meckel's diverticulum. heterotopic pancreas is extremely rare in the gallbladder. despite being a congenital condition, it takes years to become symptomatic. it can be associated with cholecystitis or cholecystolithiasis. method: a -year-old female patient was admitted to emergency service with abdominal pain. on physical examination, right upper abdomen was tender without defense or rebound. abdominal ultrasonography revealed small sized gallstones and pericholecystic fluid. magnetic resonance cholangiography showed dilated intrahepatic and extrahepatic bile ducts. laparoscopic cholecystectomy was performed. results: cholecystectomy specimen was × × mm. on gross examination we noted a few milimetric yellow colored stones and mm yellow solid intramural nodule. the mucosa was covered with fibrinopurulent exudate. microscopic examination revealed acute cholecystitis and aberrant pancreatic tissue consisting of acini and ductules. the phlegmonous inflammation of the gallbladder infiltrated the aberrant pancreatic tissue. the patient recovered completely after cholecystectomy. conclusion: we found this case worth reporting because, pancreatic heterotopia of the gallbladder is a rare, clinically silent entity unless complicated with gallstones and acute cholecystitis. expressions of c-erb-b , egfr, p , pten, mtor, pi k in hepatocellular carcinomas and adjacent liver tissues method: fifty hcc cases were stained immunohistochemically with these markers. correlations between the markers and pathologic characteristics were analyzed. results: the cases had an average age of , . male/female ratio was / . hbv is more common in advanced stages and right lobe location (p< . ). tumor size is significantly larger in patients older than years (p< , ). no membranous c erb b staining was seen while cytoplasmic positivity was present in % and significant correlation was found with multiplicity (p< . ) and p positivity (p< , ). egfr membranous positivity was present in % and significantly correlated with stage (p< . ). p was negative in % and pten is reduced or absent in %. all markers were similarly expressed in adjacent noncancerous tissue. no significant correlations were found between pten, mtor, pi k and the pathological parameters. conclusion: in our study, as a first step, none of the markers among c-erb-b , egfr, p , pten, mtor, pi k was found significant in correlation with pathologic features. we look forward to obtain further results in the next study consisting of correlations with follow ups. the value of echo guided liver biopsy in the positive diagnosis of a rare primary liver tumorpathological approach g. becheanu * , v. herlea, v. serban-barbu, m. dumbrava, s. enache, g. smira, c. angelescu, a. pop, m. diculescu * bucharest, romania objective: hepatic angiomyolipomas are rare mesenchymal hepatic tumour. the most important problem about their diagnosis on biopsy specimens is to exclude a hepatocellular carcinoma. method: we present two cases of hepatic angiomyolipoma diagnosed in fundeni clinical institute, bucharest. both females patients, and years old, were imagistically diagnosed with a cm hepatic left lobe tumor in the first patient, respectively a . cm hepatic right lobe tumor in the second one. both tumors were echo guided biopsied. the biopsy specimens measured about mm, respectively mm. the tumors were surgical removed. the biopsy and resection specimens were analyzed in light microscopy, including immunohistochemistry. results: initial diagnosis on he stain was cell variant hepatocellular carcinoma, in the mm biopsy, respectively suspicious for angiomyolipoma in the second case. further immunohistochemical studies showed positivity for vimentin, hmb , melan a, s and actin, and negativity for och e , ck , ck / , cea, cd , ki and factor viii in both biopsy specimens, consistent with hepatic angiomyolipoma diagnosis, confirmed on surgical specimens too. conclusion: diagnosing a hepatic angiomyolipoma is not easy to do especially on a biopsy. the size of biopsy specimens plays a very important role in a correct diagnosis. results: a -year-old woman presented with a -day history of nausea, fever and diffuse abdominal pain. ct scan revealed a well circumscribed small lesion of the pancreatic body. on arterial phase imaging, the mass was of low density relative to the pancreas without marked contrast enhancement. based on these findings, the patient underwent surgical resection of this mass and a distal spleno-pancreatectomy was performed. microscopic examination showed a well circumscribed but non encapsulated soft tissue proliferation consisted of blood vessels lined by a single layer of uniform flattened cells, with dilated lumen filled with red blood cells and inflammatory cells. immunohistochemical staining confirmed the vascular nature of the lesion. the diagnosis of pancreatic hemangioma was made. conclusion: adult pancreatic hemangioma is an extremely rare tumor. the review of all published cases showed that this tumor often do not demonstrate the contrast-enhanced ct features typical of an hemangioma, so a poor arterial phase enhancement cannot rule out pancreatic hemangioma and the histological examination is very important in these cases. objective: hepatocholangiocarcinoma (hcc-cc) is an uncommon form of primary liver cancer with features of hepatocellular and biliary epithelial differentiation. it accounts for . - . % of all primary liver carcinomas. many of the demographic and clinical features of this tumor remain unclear. it was previously suggested that coexisting chronic liver diseases were rarely seen in patients with hcc-cc. method: we report a case of hcc-cc diagnosed in patient with cirrhosis. results: a year-old man presented with ascites, jaundice, vomiting and symptoms of portal hypertension. on abdominal ct scan the right hepatic segment was heterogeneous, hypervascular in the late arterial phase and low-attenuated on portal venous phase. based on a these clinicoradiological informations, diagnosis of infiltrating hepatocellular carcinoma was made and hepatic biopsy was performed. histopathologic examination showed, in addition to features of liver cirrhosis, the presence of malignant proliferation composed of tubular structures as well as microtrabecular and compact foci. on immunohistochemical stainings the tumor cells expressed cytokeratins and . cells lining the tubular structures reacted with cytokeratin allowing the diagnosis of hepatocholangiocarcinoma. conclusion: hepatocholangiocarcinoma is a rare primary liver cancer. preoperative noninvasive diagnosis with conventional radiography is often difficult especially when occurring in patients with liver cirrhosis or other hepatic chronic disease. the diagnosis is frequently made only after histological and immunohistochemical examination. morphological changes in different zones of the gallbladder in cholelithiasis m. bokhodir * , t. vervekina * tashkent medical academy, dept. of patological anatomy, uzbekistan objective: gallstone disease remains a serious problem of modern surgery. an important aspect of the problem is the question of the urgency of surgical intervention, depending on the condition of the gallbladder. method: it was carried out the analysis of case histories and excised gallbladder with different forms of cholelithiasis. . % were women in the age of - years. acute catarrh cholecystitis was found out in % cases, acute phlegmonousin % cases, acute gangrenous -in % cases, chronicin % and chronic relapsingin % cases. results: all forms of cholecystitis characterized by a maximum thickness of the body wall, minimal at the bottom of the gallbladder. in all forms adipose tissue inflammation in the connective tissue layer was increased and it was found out the atrophy of the mucosa. in the inflammatory infiltrate in acute forms of inflammation were found neutrophilic and basophilic leukocytes: with simple acute cholecystitis -in the bottom, and with acute phlegmonous and gangrenous cholecystitis -in the area of the body of the gallbladder. in the cases with chronic cholecystitis were dominated mononuclear cells in all areas, and in the cases with chronic relapsing -neutrophilic and basophilic leukocytes in the bottom and the body, and mononuclear cells -in the neck of the gallbladder. ps- - histological features of chronic hepatitis c in hemodialysis patients i. delladetsima * , a. kokkori, v. sypsa, m. psichogiou, s. sakellariou, j. boletis * athens university, st dept. of pathology, greece objective: chronic hepatitis c (chc) in hemodialysis patients has not been thoroughly investigated, despite its high frequency. the present comparative study aims to highlight the histological features of chc and its association with putative pathogenetic parameters in this specific group of patients. method: sixty-one biopsies of hemodialysis patients and biopsies from the general population with chc were comparatively evaluated for the severity and specific histological features of chc. results were examined in relation to age, time of dialysis, viral load and genotype. results: patients on hemodialysis were older than patients of general population (p= . ), showing a similar genotype distribution (p= . ) and lower viral loads (p= . ). chc on hemodialysis was significantly milder according to stage (p= . ), activity and its parameters (p< . ). significantly reduced was also the frequency of lymphoid aggregates (p< . ), bile duct damage (p< . ) and steatosis. (p= . ). severity of hepatits was not associated with time on hemodialysis. in multivariate analysis the differences were independent of age, which was associated with more severe disease. steatosis was associated with hemodialysis duration and age. conclusion: chc in hemodialysis patients is significantly milder than in general population. limited necroinflammatory activity and absence of immune mediated lesions are indications of defective immune response although involvement of low viral load cannot be overlooked. ps- - inflammatory (myofibroblastic) pseudotumor of the liver: case report e. demiralay * , s. altaner, h. Özdemir * baskent university, dept. of pathology, istanbul, turkey objective: inflammatory pseudotumor of the liver (ipt) is a rare, benign tumor-like lesion of which etiology is not fully known. it is confused with primary and metastatic malignant tumors as it seems as an occupying mass on radiologic examinations. method: thirtyeight-year-old male patient who had fatigue and dyspepsia was detected to have elevated liver enzymes and hepatomegaly. on computed tomography, two masses of which middle is mildly hypometabolic, measuring × mm in right liver lobe and × mm in left liver lobe were detected. biochemical tests were normal except elevated ast and alt levels. ultrasound-guided needle biopsy was done for two masses. results: on microscopic examination, a lesion composed of abundant fusiform fibroblastic cells and vascular structures among inflammatory cells composed of lymphocytes, plasmocytes, histiocytes and eosinophillic leucocytes and including sharp margins between regenerated liver tissue was observed. on immunohistochemical examination, sma was stained focal positive in fibroblastic cells. staining with desmin, s- , panck, cd , ki- and cd was not seen. conclusion: ipt does not have a specific radiologic finding and definite diagnosis is made with pathologic examination. this case is presented as it is rarely seen, radiologically resembles to malignant tumors and for review of pathological differential diagnosis. enhancer of zeste homologue (ezh ) expression in malignant and benign hepatic tumors k. dezso * , v. szabó, e. bugyik, k. schlachter, s. paku, z. schaff, p. nagy * semmelweis university, st dept. of pathology, budapest, hungary objective: the immunohistochemical demonstration of enhancer of zeste homologue (ezh ) proved to be a useful marker in several tumor types, including hepatocellular carcinomas.in order to recognize the diagnostic value of this protein in hepatic tumors we have investigated the presence of ezh in the most common liver neoplasms. method: the presence of ezh has been studied by standard immunohistochemistry in several formalin-fixed paraffin-embedded tumor samples (hepatocellular adenoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, metastatic liver tumors and primitive childhood tumors). results: forty of hepatocellular carcinomas, of cholangiocarcinomas, of hepatoblastomas and of metastatic liver tumors stained positively, but all the investigated hepatocellular adenomas (n = ) and proliferating biliary structures were negative. the other primitive childhood tumors that were examined all expressed ezh . conclusion: based on these results ezh is a sensitive marker of malignancy in hepatic tumors regardless of their histogenesis. in routine surgical pathology ezh could be helpful to diagnose hepatocellular carcinomas and it might be the first marker to distinguish transformed and reactive cholangiocytes. ps- - nuclear ploidy as a marker of intraductal papillarymucinous pancreatic neoplasms malignancy e. dubova * , o. mishnev, a. shchegolev * v.i. kulakov scientific center, dept. of pathology, moscow, russia objective: intraductal papillary-mucynous pancreatic neoplasm (ipmn) represents , to . % of all tumors of exocrine pancreas. our aim was to study nuclear ploidy of malignant and benign ipmns cells. method: we studied pancreatic surgical specimens from females and males. we performed histological, morphometrical and ploidy analysis of all specimens. results: ipmn cells nuclear perimeter and size was higher than in normal pancreatic duct cells. this parameters were higher in malignant noninvasive ipmn than in benign tumor. nuclear size and perimeter of invasive malignant ipmn were in intermediate position between benign and malignant noninvasive tumor. mean nuclear ploidy (mnp) of normal ductal pancreatic cells was . c. mnp of benign ipmns was . c, borderline tumors - . c. mnp of noninvasive malignant ipmns was . c, of invasive ipmns - . c. we also revealed that aneuploidy coefficient (ac) of benign ipmns was (there weren't aneuploid nuclei in its cells). ac of borderline ipmns was . . maximal level of ac was in malignant noninvasive ipmns whereas ac of invasive ipmns was lesser. conclusion: nuclear morphometric parameters and mean nuclear ploidy level as well as aneuploidy coefficient could be used as additional criteria for determining malignant potential of ipmns. ps- - an intraductal papillary mucinous neoplasm of the pancreas with concomitant neuroendocrine tumor: a case report j. garcia * , f. e. costa, r. sampaio, r. dias, a. duarte, j. ramon vizcaíno * lisbon, portugal objective: intraductal papillary mucinous neoplasms (ipmns) are a recently classified pancreatic neoplasm with an increasing incidence. ipmn of the pancreas is a lesion consisting of mucin-producing cells with neoplasic potential, characterized by papillary intraductal growth and varying degrees of cytologic atypia. neuroendocrine tumors of pancreas (pnet) are rare pancreatic neoplasms comprising - % of all pancreatic tumors. pnets express neuroendocrine markers, however the true cell or cells of origin are not fully understood. pnets are classified as functional or nonfunctional based on the presence or absence of a specific clinical syndrome associated with hormone oversecretion. intraductal papillary mucinous neoplasm of the pancreas with concomitant neuroendocrine tumor are a highly rare occurrence. method: a literature review of ipmns and pnet was made, as well as a review of the clinical data of a patient with ipmn and concomitant pnet. results: a -years-old caucasian female, underwent a laparoscopic distal pancreatectomy with splenectomy for ipmn. macroscopic examination revealed discrete dilatation of pancreatic ducts and nodule in the pancreas tail. hystologic examination revealed neoplasic lesion of intraductal papillary-mucinous in the body and tail of pancreas with moderate dysplasia (ipmn borderline) and neuroendocrine tumor grade (who ). conclusion: this case reports a highly rare occurrence. glypican and agrin expression in hepatocellular carcinoma and cholangiocellular carcinoma: a immunochemistry study n. gursan * , h. balta, b. gundogdu * ataturk university, medical faculty, erzurum, turkey objective: glypican (gpc ), which plays a role cellular growth, differentiation and migration is one of members of glypican family. although gpc expression in the liver, glypican upregulation of the vast majority of tumors are available. agrin is found the surface on the cells and the extracellular matrix, the micro-vessels method: in this study, formalin-fixed, paraffin-embedded surgical specimens from patients with hepotocellular carcinoma and patients with cholangiocellular carcinoma diagnosed between and years in ataturk university, faculty of medicine, department of pathology were studied with immunohistochemistry agrin and gpc . results: we detected agrin in around bile ducts and blood vessels within the portal areas in the normal liver. İn the malignant hepatocellular carcinoma (hcc) is seen in a dramatic increase in the quantity of agrin. gpc immunpositivity showed in % of hcc and % of cholengiocellular carcinoma. agrin immunpositivity showed in % hcc and % of cholangiocellular carcinoma. conclusion: gpc and agrin, that is useful in early diagnosis of hcc cases; cholangiocellular carcinoma, or not enough was understood to be very limited. value of glypican , hep par and alpha fetoprotein in diagnosis of hepatocellular carcinoma n. gursan * , h. balta, b. gundogdu * ataturk university, medical faculty, erzurum, turkey objective: glypican showed high expression of the embryonic liver and intestine, is silenced in normal adult tissues. it is an oncofetal protein. İn general, oncofetal proteins, while having a critical role in tumor progression or immunotherapy is a potent tumor marker used as a target. alpha fetoprotein, clone c localized, normally produced by gestational age, fetal liver and yolc salc, glycoprotein structure emerging marker for patients with hcc. hep par antibody was developed in using fixed liver as immunogen. method: İn this study formalin-fixed, paraffin-embedded surgical specimens from patients with hepatocellular carcinoma diagnosed between and years in ataturk university, faculty of medicine department of pathology were studied with immunohistochemistry gpc , heppar , and afp. gpc , heppar and afp expression was divided into categories negative (negative or weak under % tumor cells cytoplasmic staining) and positive (and % over of tumour cells) moderate or strong cytoplasmic with membranous accentuation. results: immunpositivity at gpc , hep par and afp were %, %, % respectively. objective: fibroblastic growth factor receptor (fgfr) family is known to be related to the development and progression of various types of cancers. the aim of this study is to determine the clinical implication of fgfr expressions in patients with hepatocellular carcinoma (hcc). method: immunohistochemical analysis was done in cases of hcc using tissue microarray. diffuse cytoplasmic staining for fgfr , , and in more than % of tumor cells were designated as positive. the results were analyzed in terms of various clinicopathologic parameters. results: on univariate analysis, the overall survival rates of patients with fgfr expression ( cases, . %) were significantly lower than those with no expression (hr . , % ci . - . , p< . ). however, the overall survival rates of patients with fgfr expression ( cases, . %) were significantly higher than those with no expression (hr . , % ci . - . , p< . ). there was no statistical significance between patients' overall survival rates and expressions of fgfr ( cases, . %) or fgfr ( cases, . %). on multivariate analysis, only fgfr expression is independently associated with reduced os (fgfr : hr . , % ci . - . , p< . ). conclusion: the fgfr expression can be used as an independent prognostic factor in patients with surgically resected hcc. objective: hurp (hepatoma upregulated protein) is a putative oncogene, overexpressed in many human cancers, including hepatocellular carcinoma. it has also been shown recently that high hurp protein levels correlate with resistance to chemotherapeutic agents. in the present study we investigate the expression of hurp and its correlation with pancreatic adenocarcinoma, prognosis and patient survival. pancreatic adenocarcinoma is one of the most aggressive types of cancer and represents the fourth most common cause of death, either in the male or in the female population of the united states of america. method: hurp immunoreactivity was assessed by immunohistochemistry in a series of primary pancreatic adenocarcinomas. in parallel, hurp expression was examined in normal pancreatic tissues. statistical analysis related hurp expression levels with clinicopathological characteristics and survival. results: results showed a positive correlation between hurp overexpression and grade as well as lymphovascular invasion. all non malignant biopsies were negative. furthermore, positive expression of hurp appeared to be an important independent prognostic factor too, related with poor survival rates. conclusion: our results showed that hurp overexpression is associated with poor prognosis in pancreatic adenocarcinoma and indicated a diagnostic potential of this protein. its role in the carcinogenetic process awaits further elucidation. hepatocellular carcinoma in patient with hepatic porphyria: a case report j. marcinek * , p. szépe, t. balhárek, m. kalman, l. plank * university hospital martin, dept. of pathology, slovakia objective: hepatocellular carcinoma (hcc) is frequently associated with liver cirrhosis or chronic hepatitis of various etiology, less often with other chronic hepatopathies, including hepatic porphyria. we present a case report of hcc diagnosed in patient with clinically unrecognized porphyria cutanea tarda (pct). method: a -year old male was admitted to hospital complaining of a pressure below low costal margin, anamnesis of a trauma year ago and clinical suspicion of a posttraumatic intrahepatic haematoma. by a surgery, a partialy necrotic and hemorrhagic tumor mass cm in diameter was removed. results: the mass corresponded histologicaly to moderately differentiated clear cell hcc, resembling renal clear-cell carcinoma. the surrounding parenchyme showed steatohepatitis with fibrosis and distinct intracellular porphyrin crystals. conclusion: development of hcc is a well documented complication of pct, a disorder of porphyrin biosynthesis. pct can be inherited (autosomal dominant trait), the acquired form (sporadic pct) is more common. known etiological factors of pct include toxins, alcohol abuse, estrogens or chronic viral hepatitis, or association with other hepatic diseases like iron overload (including hereditary haemochromatosis) or chronic hepatitis c, increasing the risk of malignancy. however, in the presented case none of these associations was recognized and its etiology remains obscure. objective: signet ring cell carcinoma is an extremely rare type of gallbladder carcinoma composed overwhelmingly ( %) of signet ring cells. only a few cases of this histologic type have been published and detailed knowledge of this disease is not available. method: we report a case of signet-ring cell carcinoma of the gallbladder in a -year-old woman who was admitted with epigastric pain. under the preoperative diagnosis of cholangiocarcinoma, which was based on findings of ct scan and ultrasonography. cholecystectomy was performed. results: microscopic examination revealed a signet ring cells tumor that arose from the sub epithelial layer of mucosa and involved all the layers of gallbladder. nuclear atypia and mitoses were present. periodic acid schiff (pas) stain highlighted the intracellular mucin in the tumor cells. lympho-vascular emboli were detected in the subserosal layer. the cystic duct surgical margins were invaded. conclusion: gallbladder adenocarcinomas are seen frequently, but signet-ring cell carcinoma is a rare entity. owing to the location of the gallbladder, dissemination of the tumour to the adjacent tissues is usually presented at the time of the diagnosis. it is necessary to exclude a gastric or colonic signet ring cell carcinoma secondarily involving the gallbladder. objective: gastrointestinal stromal tumors (gists) are mesenchyme neoplasms of the gastrointestinal tract which express cd . gists can occur in the entire length of gastrointestinal tract, but very seldom can also arise in omentum, mesentery and retroperitoneal space, kidney, urinary bladder etc. there they are located adjacent to stomach or intestine, but not originated from the latter. these tumors are designated as "extra-gastrointestinal stromal tumors" (egists). there are only description of the primary egists reported in the literature. method: we report a case of a pancreatic egist in a year-old female patient. results: ct scan showed a mass in the head of pancreas. grossly encapsulated tumor, cm in diameter, was found in the head of the pancreas (figure ) . histologically, tumor demonstrated spindle-cell pattern consisted of distinct fascicles and bands. the number of mitoses was - / high power fields in "hot-spot" areas. ihc revealed strong positivity for cd , cd in neoplastic cells and negative for sma, desmin, s- . ki labeling index was %. conclusion: we presented a rare case of pancreatic gist. the tumor has very good prognosis. hepatoblastoma is classified as epithelial ( %) or mixed epithelial/mesenchymal ( %). epithelial hepatoblastoma is further divided to pure fetal ( %), embryonal ( %), macrotrabecular ( %) and small cell undifferentiated ( %). aim: to increase the awareness regarding possibility of hepatoblastoma in adult age. method: we review four cases of hepatoblastoma epithelial type diagnosed in pathology department of fundeni clinical institute between and . immunohistochemistry was done in all cases: cea, vimentin, ck / , ck , ck , ki , ttf , cd , cd , cd , muc a, alpha-fetoprotein. results: two of the patients were males and two females with age ranging between and years. the tumors varying in size from to cm. on microscopic examination the tumor was composed mainly of epithelial elements. the pathological diagnosis was epithelial hepatoblastoma epithelial type. conclusion: hepatoblastoma is a rare tumor in adult age and epithelial type is the rarest from all type of hepatoblastoma in adult age. the pathological diagnostic is quite difficult even using immunohistochemistry because none of the markers are not specific for hepatoblastoma. submassive hepatic necrosis with regenerative nodules: a series of cases y. rodríguez-gil * , j. salazar, j. delgado sánchez, g. lópez alonso, c. ibarrola de andrés, f. colina ruizdelgado * hospital universitar de octubre, dept. of surgical pathology, madrid, spain objective: confluent hepatic necrosis is the morphological correlate of fulminant liver failure. a group of these livers develop a combination of areas of necrosis and regenerative macronodules (rmn). method: sequential biochemical and clinical data and tissues from two groups of patients were compared. group : cases of submassive necrosis associated with rmn; group : cases with total confluent necrosis without nodular regeneration. results: group : mean age was , years (ranged months- years); group mean age was , years (ranged months- years) (p> , ). a longer course ( , ± , days vs , ± , , p< . ) and higher maximum serum bilirubin levels ( , mg/dl ± , vs , mg/dl± , , p< . ) were observed in group . serum ast, alt and gamma-gt were lower in group (p> . ). morphologically livers from group showed well defined regenerative green-yellow nodules with large regenerative hepatocytes in acinar pattern without surrounding fibrosis. the remaining parenchyma showed confluent necrosis, haemorrhage, mixed inflammatory infiltrate and ductular proliferation. conclusion: a special anatomoclinical form of subacute liver failure was characterized by regenerative macronodules in a background of extensive confluent liver necrosis. illness duration was longer and had higher maximum bilirubin levels than those without rmn. immunohistochemical study of muc gene family in pancreatic cancer g. setdikova * , o. paklina * moscow, russia objective: investigation of the muc gene in pancreatic ductal adenocarcinomas (pda). method: in the present study, we examined the expression of muc , and ac types by immunohistochemical analyses in pda from patients. overall survival curves were drawn by the kaplan-meier method. for all analyses, p< . was considered to be statistically significant. results: in our cases most of the pda was presented as muc +/muc ac + phenotype - % cases ( / ). the group with intestinal phenotype mucin, which characterized by positive expression of muc and cdx , was only % ( / ). the group which characterized by positive expression only muc ac (gastric phenotype), was % ( / ). the survival rate of patient better in group with muc expression. cumulative survival at months after surgery was . and the median postoperative follow-up period was months. the most aggressive behavior was pda with expression only muc ("true pancreatobilliary type"). cumulative survival at months after surgery was . and the median postoperative follow-up period was months. conclusion: the mucin profile as a prognostic factor is important not only for intraductal pancreatic mucinous neoplasm, but and for ductal pancreatic adenocarcinoma. ps- - hepatocellular carcinoma microvessels density depends on tumor differentiation: radiologic-pathologic correlations a. shchegolev * , e. dubova, u. tumanova * v.i. kulakov scientific center, dept. of pathology, moscow, russia objective: prognosis and recurrence level in hepatocellular carcinoma (hcc) depends on microvessels density. method: we performed ct and pathological correlations in cases of hc ( cases of highly (h-hcc), cases of moderate (m-hcc) and cases of low differentiated hcc (l-hcc)). results: vessels number in w-hcc in compare to m-hcc was higher by . % (p< . ). total vessel area was higher in m-hcc and l-hcc than in w-hcc by . % and . % accordingly. we observed positive correlation between ct density of h-hcc and number and total vessel area. correlation between h-hcc ct density and mean vessel area was negative. we also revealed modest negative correlation between m-hcc density and the number vessels in all phases of ct. negative correlation between total and mean vessel area and m-hcc density was revealed in native and arterial ct phases in compare with venous and delayed phases where positive correlation was revealed. in all phases of ct we revealed strong negative correlation between l-hcc density and the number of vessels whereas correlation between l-hcc density and mean vessel area was strongly negative. conclusion: we revealed decreasing in angiogenic activity in hccs neoplastic progression and growth. ct signs correlate with hcc histological differentiation. ps- - primary sporadic liver schwannoma: an extremely rare diagnosis r. silva * , c. eloy, j. m. lopes * centro hospitalar de são joâo, serviço de anatomia patológica, porto, portugal objective: primary liver schwannoma (pls) is extremely rare. results: case report: a -year-old female with previous history of cholecystectomy, appendectomy, arterial hypertension and diabetes mellitus was admitted due to an intrahepatic nodular lesion identified in routine ultrasound examination. she was submitted to partial hepatectomy with postoperative uneventful evolution. macroscopically, the specimen disclosed a . cm nodular, well circumscribed, yellowish tumour. histologically, the tumour displayed an expansive growth pattern, comprising short bundles of spindle cells with mild atypia, without mitotic figures or necrosis. immunohistochemically, the tumor cells expressed diffusely vimentin, s- and gfap, in the absence of ae /ae , cam . , cd , and hmb ; ki- index was %. staging procedures did not disclose evidence of any other tumor. there was no personal or family evidence of neurofibromatosis. these features are consistent with sporadic pls. conclusion: schwannoma is a rare benign tumour in the gastrointestinal tract with few cases reported in the liver. the clinical presentation is usually an upper abdominal pain but they can be asymptomatic. secondary cystic degeneration and hemorrhage are common in large tumours. differential diagnosis by imaging evaluation includes several benign and malignant, namely metastatic, tumours. therefore, the pathological examination is crucial for the diagnosis of primary liver schwannoma. metastases of hepatocellular carcinoma to the costa and soft tissue: a very rare entity e. tastekin * , t. d. yalta, o. yalcin, t. ciftci * trakya university, dept. of pathology, edirne, turkey objective: hepatocellular carcinoma (hcc) is the most common primary tumor of the liver with a potential of metastasis primarily to the lung, abdominal lymph nodes and bone. however, metastases to the costa, soft tissue are rare. method: an -year-old man was admitted to hospital with weakness, anorexia. chest x-ray and ct-scan demonstrated a × cm sized, intrathoracic mass on the lateral-side of the left .costa and also infiltrating the axillary soft tissues.a secondary mass with the identical radiological features was also detected within the c -vertebra. whole-body ct-scan was planned.a × cm sized nodular, hipodense mass was detected in the upper segment of the liver. interestingly, none of these masses demonstrated a significant pathological fdg-uptake in the pet.a trucut biopsy was taken from the axillar. microscopically, large tumoral nests were observed in connective tissues. polygonal shaped tumoral cells were found to have granular cytoplasm, pleomorphic nuclei. immunohistochemistry results: positive cytoplasmic reaction with heppar, ttf- ; canalicular staining with polyclonal-cea, cd ; weak cytoplasmic staining with pankeratin, b . ; negative reaction with vimentin, afp, ck , ck . results: these histomorphological, immunohistochemical findings strongly supported to the diagnosis of hcc metastasis. conclusion: hcc that is, in general, morphologically similar to mesenchymal and epithelial tumors, should be considered among the differential diagnosis of unknown primary tumors. morphological justfication of using pulsed electric discharge in surgical treatment of echinococcosis m. tussupbekova * , d. kaliyeva, e. turgunov * medical university karaganda, dept. of pathological anatomy, kazakhstan objective: echinococcosis is characterized by lesions of internal organs, severe complications, often lead to disability and death. method: new method of processing of the residual cavity with impulsive electric discharge after echinococcectomy was experimentally developed and introduced into clinical practice. the effectiveness of the method is confirmed by morphological examination of operational bioptic of the liver tissue. results: patients underwent surgical treatment with the new method (patent of the republic of kazakhstan ¹ ). parasitic cysts with an inner germinal layer and protoskoleks, outdoor layer -chitinous shell with a productive reaction with lymphocytes infiltration, single eosinophils, separate groups of hydatid bubbles with their invasion in the liver tissue were detected in the histological examination of material taken intraoperatively prior processing of residual cavity with electropulse discharge. fibrinoid necrosis, destruction of germinal elements, single lymphoid infiltration, hyperemia of blood vessels, absence o inflammatory reaction were marked in the histological material after processing by impulsive electric discharge. the results showed that echinococcectomy with processing of the residual cavity with impulsive electric discharge is an alternative for pericystectomy and liver resection. objective: congenital peribronchial myofibroblastic tumor is a rare, solid mesenchymal tumor of the fetus and neonate, usually associated with nonimmune hydrops fetalis. we report the case with clinical, radiological and pathologic findings and review the other cases in the english language literature. method: we present a case of cpmt, whom a right lung mass was detected in intrauterine life. days after delivery by cesarean section, right lobectomy was performed. results: the tumor was limited to lung, and composed of spindle cells, proliferating around bronchial unit. central necrosis and - / hpf was present. patient is well months after surgery. conclusion: congenital peribronchial myofibroblastic tumor is a rare solid pediatric tumor of lung which was named differently in the past, such as congenital fibrosarcoma, congenital leiomyosarcoma, congenital mesenchymal malformation of the lung, neonatal pulmonary hamartoma. albeit it resembles sarcoma with high cellularity, mitosis and necrosis features microscopically, no additional therapy to tumor resection is needed. so it is important to keep in mind this scary looking, but innocent tumor both in prenatal and postnatal evaluation. morphologic alteration of metastatic neuroblastoma in bone marrow after chemotherapy g.-e. bae * * samsung medical center, dept. of pathology, seoul, republic of korea objective: the aims of our study are to evaluate the histologic features of metastatic neuroblastoma in bone marrow (bm) in comparison with those of primary neuroblastoma, and to compare the histologic characteristics of metastatic neuroblastoma in bm before vs. after chemotherapy. method: total biopsies from children diagnosed as stage neuroblastoma with bm metastasis were examined histologically; primary neoplasm biopsies, bm biopsies before chemotherapy, primary neoplasm excision biopsies after chemotherapy, and bm biopsies after chemotherapy. results: ) primary neoplasms were classified as neuroblastoma poorly differentiated (n= ), neuroblastoma differentiating (n= ), ganglioneuroblastoma intermixed (n= ) and neuroblastoma type unclassifiable (n= ). ) metastatic foci in bm before chemotherapy were composed of undifferentiated and/or differentiating neuroblasts but not ganglion cells, in neuropil but not schwannian stroma. ) metastatic foci of bm after chemotherapy showed differentiation such as ganglion cells and schwannian stroma, which was more prominent after more cycles of chemotherapy. ) metastatic neuroblastomas in bm after chemotherapy were as mature as or less mature than those in primary neuroblastomas after chemotherapy. conclusion: metastatic neuroblastomas in bm initially consist of more immature components than primary neuroblastomas, whereas they become differentiated as primary neuroblastomas after multi-cycle chemotherapy. morphometric evaluation and clinical correlations in malignant small round cell tumors c. bansal * , a. gupta, a. kumar, a. srivastava * csmmu, dept. of pathology, lucknow, india objective: nuclear size increases in malignant tumors and reflects dna content, ploidy and proliferation index. present study investigated; could nuclear morphometery differentiate histomorphologically similar paediatric malignant small round cell tumors in h & e stained sections for using in a poor resource country? method: morphometric analysis was done in confirmed but difficult to differentiate round cell tumors histomorphologically and were analyzed by cell images from different areas in each section, using leica q win images software. results: nuclear measurements were obtained for retinoblastoma ( ), hodgkin lymphoma ( ), wilms tumor ( ), medulloblastoma ( ), ewing's sarcoma ( ), alveolar rhabdomyosarcoma ( ), malignant hemangiopericytoma ( ), non-hodgkin lymphoma ( ) and neuroblastoma ( ). amongst the retinoblastomas, maximum mean nuclear area percent ( . ) was seen in cases with nerve involvement and metastasis, followed by cases with only nerve involvement ( . ) and smallest area ( . ) was in non-nerve involving, non metastatic cases. wilms tumor cases with metastasis had higher nuclear area ( . ) than non metastatic ( . ). non-hodgkin lymphoma nuclear area ( . ) was more than hodgkin's ( . ). amongst all tumors, minimum value ( . ) was seen in malignant hemangiopericytoma. conclusion: morphometric evaluation in paediatric malignant round cell tumors have generated useful data, and needs further multicentric confirmation for implementation. objective: survivin, a bifunctional protein that regulates cell division and suppresses apoptosis, may play an important role in tumorigenesis. the aim of this study was to determine survivin expression patterns in wilms tumor (wt) and to analyze it in relation to stage, prognostic category and histological type. method: immunohistochemical expression of survivin was analysed in cases of primary wt and in normal kidney specimens uninvolved by the tumor. results: fifty one out of cases of wt ( . %) showed decreased cytoplasmic survivin expression compared to the expression in normal kidney tissue. decreased cytoplasmic expression (in all components of wt) of survivin was found significantly more often in low stage compared to high stage wts ( . % vs. . %; p= . ). tumors of intermediate risk group showed more often decreased cytoplasmic expression of survivin in comparison to high risk group, but the difference was not significant. decreased survivin expression was found more frequent in wts with diffuse anaplasia and in epithelial wts compared to other histological types, but without statistically significant difference. conclusion: decreased survivin cytoplasmic expression may be associated with the favorable prognosis wt. objective: hepatic tumors accounted % of congenital neoplasms. mesenchymal hamartoma of the liver is a rare benign tumor of children. we report a case of hepatic mesenchymal hamartoma in a premature male neonate. method: a premature neonate born at gestational weeks from year-old mother, g p with non-consanguineous marriage; prenatal ultrasonography showed a fetal macrosomia, a highly vascularized abdominal mass occupying two-third of the abdomen and displacing the bowel loops associated with poly-hydramnios. the newborn examination showed abdominal firm lump. ultrasound found and anterior highly vascularized mass displacing the left lobe of liver. the infant died few hours later. a complete autopsy was performed. results: external examination showed a male neonate anatomically of - week having an increased periombilical diameter and a macrosomia, ascitis, pulmonary hypoplasia, cardiomegaly, pleuropericardial effusion, liver tumor developed in left lobe and dilation of renal vessels. histologically the tumor showed a mixture of normal liver tissues with blood or lymphatic vessels, bile ducts within an abundant edematous and myxoid stroma. conclusion: in neonate and fetus, prenatal diagnosis is possible by ultrasonography. large tumors can affect the viability of the newborn. adequate excision is curative in most of cases. ps- - ncam polysialylation as potential initiator of differentiation and proliferation of renal progenitors in human fetal tissue s. cirovic * , j. tadic, n. radunovic, c. müller, g. müller, j. markovic-lipkovski * inst. for pathology, nephropathology, belgrade, serbia objective: objective: neural cell adhesion molecule (ncam) is widely expressed on mesenchymal and early tubular epithelial cells during kidney development although with still undefined function. ncam can be polysialylated and as psa-ncam has been shown to be involved in proliferation and migration of neuronal cells during brain development. the aim of this study was to evaluate the presence of psa-ncam in nephron precursors in relation to the expression of renal progenitor and proliferation markers. method: human fetal and neonatal kidneys were analyzed using double-immunofluorescence (dif) staining and western blot (wb) analysis. specific antibodies against ncam, psa-ncam, epcam, cd and ki- were applied. results: on wb only fetal tissue samples have bands with ncam at to kda which suggest that ncam molecule is polysialylated; dif analysis of fetal tissue show psa-ncam + ki- + cells in all structures known as nephron precursors. while in neonatal tissue psa-ncam and ki- were positive only on rare single cells in intersticium. conclusion: psa-ncam expression appears to characterize a very early stage of induced nephron progenitors differentiating from ncam + epcam-mesenchymal cells. according to psa-ncam localization and coexpression with ki- during development and its practically absence in neonatal tissue, suggest that psa-ncam present potential initiator of proliferation and differentiation of renal progenitor. objective: cystic hygroma (ch) typically develops in utero, late in the first trimester to early in the second trimester of gestation. many of the fetuses with ch present additional malformations commonly associated with chromosomal anomalies. method: a review of fetal autopsies performed over a year period revealed cases of ch ( , %). the results of cytogenetic analysis and the prenatal ultrasound findings were retrieved and compared to the autopsy findings. results: fetal death was due to therapeutic abortion in / cases, intrauterine death in / cases, and spontaneous abortion in / cases. cytogenetic analysis was available in cases. the results showed an abnormal karyotype in cases ( cases of turner syndrome and cases of trisomy ). the mean size of ch was , cm. other findings suggestive of the cause of fetal death were diagnosed in / cases ( , %). the most common autopsy findings were hydrops and central nervous system anomalies. the autopsy findings were in agreement with the prenatal ultrasound findings in / cases, while in cases ( , %) additional findings were detected during autopsy. conclusion: our study is confirms the strong correlation between ch and chromosomal anomalies of the fetus. immunohistochemical expression of e-cadherin in primary and metastatic nephroblastoma cases i. franckevicha * , r. kleina * university children´s hospital, dept. of pathology, riga, latvia objective: in nephroblastoma cases association between decreased e-cadherin expression and higher stage of tumor (safford sd, ) and lower expression in metastatic tumors (alami j, ) was described. some authors considered that e cadherin is not likely to play tumor suppressor role in nephroblastoma ( shulz s, ) . purpose of this study was to compare the expression of e-cadherin in metastatic and primary tumor cases in latvia. method: cases of primary tumors, metastases and case of relapse were analyzed immunohistochemically using visualization system envision. the number of e-cadherin positive structures per field was assessed (magnification× ). comparison between primary tumors and metastatic/ relapse tumors groups was made using mann-whitney test. results: in primary tumor group the number of positive structures in the field ranged from to , average . , sd . . in the metastatic tumor group, the number of positive structures varied from to , average . , sd . . comparison of expression observed in both primary and metastatic tumors groups showed the decreased e-cadherin expression in metastatic tumor group (z= − . , p= . ). conclusion: expression of e-cadherin is lower in metastatic tumor group that may suggest about negative correlation between it and higher tumor grade. ps- - testicular fibrous hamartoma: a case report a. kilitci * , f. yilmaz, s. yanik, h. ozturk * abant izzet baysal university, dept. of pathology, bolu, turkey objective: fibrous hamartoma (fh) of childhood is uncommon benign tumor. they are generally seen in the head and neck region, gastrointestinal system and lung. as in the case we report, they may also occur in other unusual sites such as groin and testis. the clinical presentation is almost always a mass or swelling, however our case was admitted to our hospital because of a left testicular atrophy. we report a fh in the testis, which has a rarely location and clinical presentation. method: a -year-old male presented to department of pediatric surgery because of left testicular atrophy. left orchiectomy was performed. results: in macroscopic examination, testis dimension was . × . × . cm. its cut surface was smooth and dirtywhite. in microscopic examination, the tumor showed disorganized matur tissue that composed of fibrocollagen stroma, vessels, muscular and adipose tissue. by this findings, it was diagnosed as testicular fibrous hamartoma (tfh). conclusion: in conclusion, tfh should be always kept in mind with testicular atrophy not only testicular mass or swelling. and knowledge of this particular type lesion is important to distinguish the fh of childhood from other situations in testis such as testicular torsion, incarcerated hernia, malign neoplasm, etc. in order to allow a correct diagnosis and avoid inadequate treatment. placental pathologic features in diabetes and hypertension p. luís * , a. costa-silva, a. alves * hospital de santa maria, dept. de anatomia patológica, lisboa, portugal objective: the aim of this study was to evaluate the most frequent placental findings in diabetes and hypertension and their main differences. method: retrospective study of selected placentas from to weeks gestation, in a universe of placentas examined over the last years ( % associated with hypertension, , % with diabetes and , % with both diabetes and hypertension (dwh). results: in diabetes the most frequently found abnormalities were immature villi ( %) and infarction ( %). in dwh the most frequent lesions were the immature villi ( %) and inflammatory lesions (either acute or chronic) ( %). in the hypertension group the most frequently found lesions were infarction ( %) and accelerated maturation of the villi ( %). in % of placentas in diabetes, % in dwh and only % in hypertension, no lesions were found. the incidence of fetal death was % in diabetes, % in dwh and , % in hypertension. when evaluating placental weight, small placentas were more frequent in the hypertension group ( %), and large placentas were more frequent in diabetes ( %). conclusion: our findings may contribute to evaluate the consequences of diabetes and hypertension in fetal outcome. as inflammatory conditions are usually not directly attributed to diabetes or hypertension, placental examination may help in diagnosis of associated pathology like infection. placental villi morphometry in preeclampsia k. pavlov * , e. dubova, r. shmakov, a. shchegolev * v.i. kulakov scientific center, dept. of pathology, moscow, russia objective: terminal villi structure abnormalities could play an important role in preeclampsia (pe) and its complications development. our aim was to perform comparative morphometric study of the placentas from mild and severe pe pregnancies. method: complex morphological and morphometric study of term placentas from mild pe (mpe) cases ( st group), term placentas from severe pe (spe) cases ( nd group) and term placentas from uncomplicated pregnancies (control group) was performed. results: we revealed significant decreasing in terminal villi size in both preeclamptic groups in compare to control. this decreasing was much prominent in spe group. we also observed significant terminal villi perimeter decreasing in both pe groups without any difference between mpe and spe groups. morphometry of cd stained specimens revealed significant decreasing in mean capillary number in both pe groups. single capillary area and perimeter were significantly lower in mpe and spe groups and these changes were much prominent in spe group. total villous capillary area and perimeter were significantly lower in both pe groups with minimal values in spe group. degree of villous capillarisation was significantly lower in both pe groups. conclusion: revealed features reflect villous structure changes in preplacental hypoxia, caused by mild and severe preeclampsia. sudden intrauterine death: the usefulness of autopsy f. portelli * , e. orlando, e. di stefano, e. maresi * university of palermo, section of pathology, italy objective: we investigated in autopsies of stillbirths the usefulness of autopsy even in the absence of risk factors and/or apparent anatomical/clinical causes which could explain the death. method: from january to december (institute of pathology, paolo giaccone-palermo) autopsies on dead uterine foetuses were performed. cases were abortions (< weeks), were stillbirths (≥ weeks). in all cases the autopsy included a macroscopic and microscopic examination both of the foetus and the placenta. the final diagnosis of death was based on both the morphological and clinical data. of the stillbirths, cases were "risk factor pregnancies", non. results: the stillbirth mortality was classified as follows: -sudden intrauterine unexplained death (siud,"sine materia" and absence of risk factor autopsies), cases; -explained intrauterine death ("cum materia" with or without risk factor autopsies), cases; -borderline intrauterine death ("sine materia" and presence of risk factor autopsies), cases. conclusion: in the absence of autopsy in / cases it was possible to establish the cause of death based on the presence of risk factors. in the absence of risk factors the autopsy showed a certain anatomical cause in / , only in / it was "sine materia". the role of "traditional" and "tomography" autopsy in foetal congenital heart disease (chd) f. portelli * , f. p. busardò, l. gutsul, l. averna, e. orlando, e. maresi * university of palermo, section of pathology, italy objective: the role of systematic autopsy in foetal chd is to identify the morphology of cardiac and possible associated extracardiac malformations (ecm) and the correlation between morphology, suspected clinical etiology and genetics. we evaluated the role of autopsy in foetal chds with/without prenatal diagnosis. method: from january to december (institute of pathology, paolo giaccone -palermo), foetal autopsies were performed. the autopsy protocol used was "tomography" for abortions, the "traditional" technique was adopted for stillbirths. results: in cases chds were identified. in cases chd diagnosis was made only through autopsy, in after a certain ( ) or suspect ( ) clinical/genetic diagnosis. the etiology of chd associated to ecm ( ) was: chromosomic type ( ), syndromic/sequence type ( ), association type ( ). the etiology of chd without ecm ( ) was never syndromic. conclusion: "traditional" and "tomography" autopsy plays a key role in the diagnosis and counselling of chd, either when it represents the only diagnostic tool ( , %) or when it is preceded by a clinical/genetic study. in the latter cases, its value depends on the detection of ecm, useful to "consolidate" a suspect clinical diagnosis ( , %) and to "complete" a malformative picture etiologically known thanks to a clinical/genetic analysis ( , %). objective: deregulation of cell cycle control is a hallmark of cancer. we have examined protein expression and gene amplification of cyclin a in wilms tumor (wt) and to analyze it in relation to tumor stage, prognostic group and histological type. method: real-time quantitative pcr was used to detect gene amplification of cyclin a in tumor tissue from patients with wt, while immunohistochemistry was applied to detect protein expression of the same cyclin. results: cyclin a gene amplification was found in out of the ( . %) cases of wt. cyclin a protein overexpression was detected in all four cases, but was also found in . % of cases without detectable gene amplification. so, there was no significant correlation between cyclin a gene amplification and protein overexpression. all cases with amplification of cyclin a were of favorable histological type, intermediate risk group and three out of four cases were low stage wts. on the other hand, overexpression of cyclin a was found significantly more often in high stage wts compared to low stage wts (p= . ). conclusion: cyclin a gene amplification might be associated with the favorable prognosis of wt (low stage, intermediate risk group and non-anaplastic tumors). the gold standard to diagnose is lung biopsy or necropsy. up to % of cases have mutations or deletions in the gene foxf (cr q . ) who plays a crucial role in the development of the lung vasculature. method: we report a case of a term newborn female affected of acd/mpv who was diagnosed by a lung biopsy. she died at days of life. autopsy and molecular diagnosis were also performed results: in the biopsy and autopsy specimens a decline in the number of capillaries in the alveolar septa and detachment of the epithelial lining was observed. secondary proximal plexiform arteriopathy, muscularization of arterioles and venous-venular dilatation and proliferation were evident. a pathogenic mutation in the gene foxf (frame shift mutation in the first exon) confirmed our diagnosis. conclusion: acd is defined by a decrease in capillaries with alveolar septal thickening and hypertrophy of the middle muscular layer of arterioles. mpv suggests an imbalance of angiogenesis. the foxf mutation helps to prenatal diagnoses of high risk families and to give the diagnosis to patients who fail to perform biopsy or autopsy. objective: prognosis of rhabdomyosarcoma (rms) has improved significantly over the last years. overall survival (os) is> % in the majority of patients; even though, children with metastatic tumors have a dismal prognosis with an os < %. method: we analyzed in cases, the possible correlation of notch activation with histology, presence of metastasis and outcome. immunohistochemistry (ihc) was performed for the notch downstream effectors hes and hey . results: hes was strong or moderate in % of the cases. hey was positive in %. out of alveolar rms (arms) were positive for hes and was positive for hey . both stains were negative in the fusion-negative arms. in embryonal rms (erms), eight out the were hey positive and of the were hes positive. the patients whit metastases had staining for hes and hey . patients who died showed hes (+). of the patients in remission showed positivity for hes and for hey . conclusion: hes expression was found in the majority of rms while hey was more erms specific. is no correlation between pathway activation, metastasis or outcome. the blockage of the pathway with specific inhibitors could offer a new therapeutic option for this patients. a renal epithelioid angiomyolipoma in a young woman with tuberous sclerosis complex, cortical tubers by neuroimaging, facial angiofribromas and lung lymphangioleiomyomatosis j. trillo-tinoco * * hospital general de mexico, dept. of surgical pathology, mexico city, mexico objective: tuberous sclerosis (ts) is a genetic disorder affecting cellular differentiation and proliferation, which results in hamartoma formation in many organs like skin, brain, lung, kidney and heart. method: a seventeen-year-old female with history of facial angiofibromas a years before, right renal tumor diagnosed a year before as theratoid-rhabdoid tumor, with chemoradiation as adjuvant treatment, cortical tubers by neuroimaging a few months before, and diagnosis of lung lymphangioleiomyomatosis recently. after last diagnostics, we reviewed again the renal tumor, new orientation of tissue, with new histological sections were performed. (fig) results: at histological examination, the kidney showed an infiltrating tumor, very cellular, consisting mainly of polygonal cells with eosinophilic cytoplasm, other multinucleated similar to the ganglion cells, also small hamartomatous areas with smooth muscle, fat and blood vessels proliferation. the tumor cells expressed hmb , mart- , sma, vim and cd , ckae / were negative. (fig) conclusion: the majority of renal angiomyolipomas is sporadic and to % occurs as part of ts, and their partnership is more close with epithelioid variant, recently, a rare entity with aggressive behavior, difficult histological characterization and poor prognosis. pulmonary mast cells in sudden infant death syndrome (sids) c. zaharia * , c. loddo, p. schmidt, r. m. bohle * university of saarland, inst. of pathology, homburg, germany objective: several theories of the underlying mechanisms of sids have been proposed, one of them is focusing on shock including anaphylaxis. increased concentrations of mast cell tryptase in post mortem blood have been observed without increased mast cell numbers in lung tissue. the aim was to evaluate the age-related distribution of pulmonary mast cells in infants dying of sudden infant death syndrome and controls. method: infants (up to year of age) who died of sids and controls who died of other non-pulmonary causes were examined. peribronchial mast cells exhibiting tryptase immunoreactivity were evaluated and quantified in high power fields in lung sections. results: the number of mast cells in peribronchial regions amounted to , (± , )/mm in children aged month up to months. mast cells in sids cases were , (± , )/mm . the difference was not significant (p= , , student'st test). conclusion: it is unlikely that increased pulmonary mast cells are indicators of sids. the role of mast cells in sids remains controversial. trophoblast apoptosis in placentas from pregnancies complicated by preeclampsia s. zekic tomas * , i. kuzmic prusac, d. roje, i. tadin * clinical hospital centre split, pathology, croatia objective: to assess trophoblast apoptosis separately in cytotrophoblast, syncytiotrophoblast, total villous trophoblast and syncytial knots, as well as to investigate the expression of apoptotic factors fas ligand (fasl), bcl- and proliferation marker ki- in trophoblast of placentas from preeclamptic patients. method: the study included placental samples from preeclamptic and normal pregnancies. for the detection of apoptosis and proliferation antibody m and antibody against ki- antigen were used. expression of fas ligand and bcl- was assessed using semi quantitative hscore method. syncytial knots were expressed as the number of syncytial knots per individual villus and as the total number of syncytial knots in each placental sample. results: apoptosis in all stages of trophoblast differentiation, number of syncytial knots per individual villus and the total number of syncytial knots were significantly higher in preeclamptic placentas than in control group placentas. fas ligand expression was significantly less, and bcl- expression significantly greater in the villus trophoblast among the study subjects compared with controls. there was no difference in the trophoblast proliferation between groups. conclusion: our findings might suggests that increased apoptosis and syncytial knots formation combined with reduced fas ligand expression could be involved in pathophysiological mechanisms of preeclampsia. contribution of fetal autopsy for diagnosis of meckel-gruber syndrome m. jo * , i. guerra, g. perez de nanclares, p. morales, j. j. aguirre, z. s. quintero, c. gomez, n. t. villagra * hospital txagorritxu, pathology, vitoria, spain objective: meckel-gruber syndrome (mgs) is a lethal rare autosomal recessive malformation. the six implicated genes encode proteins involved in primary cilia function. groups of families in finland, india and north of africa have been identified. method: a fetus, xy, karyotype, therapeutically aborted at weeks with alobar holoprosencephaly in ultrasound. fetal autopsy was performed. genetic counseling was proposed to the family revealing moroccan origin and consanguinity, the parents were first cousins. results: a male fetus showing cyclopia, proboscis and a single opening with two rudimentary eyes was the external morphology. histological examination confirmed holoprosencephaly and also showed bilateral corticomedullary renal multicystic and periportal hepatic fibrosis with bile duct dilatation. the main diagnosis was mgs although other ciliophaties and non-ciliopathies conditions were considered, such as bardet-bield, joubert, smith-lemli-opitz syndrome and trisomy . however, the pathological characterization, parent's consanguinity and their north african origin makes mgs the likely diagnosis.a genetic study on paraffin-embedded material was requested, the poor quality of dna stopped definitive genetic diagnosis. conclusion: the pathologist may encounter atypical cases that require morphologic diagnosis to determine the type of underlying mutation and provide genetic counseling to parents.a meticulous autopsy is necessary to establish the diagnosis of mgs. fresh material would have to be frozen in order to make current diagnostic techniques of molecular pathology. mesenteric cysts in the pediatric age group r. jankovic * , j. sopta, m. stojanovic, b. lekic, b. jovanovic, z. stojsic * university of belgrade, faculty of medicine, serbia objective: mesenteric cysts are extremely rare lesions arising with an incidence of / , admissions in children. clinically, mesenteric cysts are generally comprehended as a unique diagnostic entity, although they exhibit histological diversity. the objective of this study was to determine the incidence and the histology of the mesenteric cysts in the pediatric age. method: all cases of mesenteric cysts operated at the university children's hospital belgrade over the -year period of to were reviewed using pathology reports from the files of the institute of pathology. histological slides were re-examined and immunohistochemistry was applied, when necessary. results: a total of cases of mesenteric cysts were identified. cysts of lymphatic origin (cystic lymphangiomas) were recorded in patients ( %), cysts of enteric origin -in patients ( %): duplication cysts and one isolated enteric cyst of the mesenterium. only one example of the cyst of mesothelial origin, i.e. benign cystic mesothelioma was diagnosed ( %). the most frequent site was mesenterium and mesocolon ( %), followed by omentum ( %) and the retroperitoneum ( %). conclusion: cysts of enteric origin are easily recognized. it is important to differentiate between cystic lymphangioma and cystic mesothelioma due to their different natural history. objective: myxoid liposarcoma belongs to the group of soft tissue sarcomas with lipomatous differentiation. breast is a rarely affected (only . % of breast sarcomas), and often misdiagnosed. method: a -years old woman presented with a painful timorous lump of the left breast. mammography showed oval, lobulated lesion between medial quadrants, while ultrasonography revealed hipoechogenic, inhomogeneous nodule. patient underwent core biopsy which was histologicaly inconclusive and followed by quadrantectomy with excision of both medial quadrants. results: a lump ( cm) was visible on the skin surface. serial sectioning revealed solid gray-white tumor with cystic and prominent necrotic areas. the tumor was located in the deep mammary tissue an it infiltrated the overlying dermis without involving the epidermis. it consisted of atypical stelate and spindle cells with infrequent mitoses and low ki- proliferative index. on the periphery mature adipocytes and lipoblasts were present. stroma was abundant, myxoid with plexiform capillary pattern. the myxoid substance stained slightly alcian-blue positive and tumor cells showed cytoplasmic immunopositivity for s . phylodes tumor was excluded because of absence of epithelial component. conclusion: myxoid liposarcoma has a distinct morphology, rarely confused with other soft tissue tumors, although on cytological smears or biopsy samples it may be unrecognized. objective: synovial sarcoma poses a difficult diagnostic challenge since it can be confused with other benign or malignant entities. method: four biphasic and one monophasic synovial sarcomas were studied. we used the break apart/split signal kit (vysis) to detect the t(x; ) translocation and we performed immunohistochemistry for tle- , ini- , d - , cd , cd , bcl- , ema, ck , cd , s- , desmin and claudin- . results: immunohistochemically we observed: tle- +( / ), ini- ( / ) with reduced nuclear and ( / ) with no expression, d - +( / ), cd +( / ), cd +( / ), bcl- +( / ), ema focal expression ( / ), ck −( / ), cd −( / ), s- −( / ), desmin−( / ), claudin- + ( / ). fish revealed the split signal between the centromeric and telomeric end of the syt gene. conclusion: t(x; ) translocation remains the diagnostic hallmark of synovial sarcoma. ini- which is typically negative in atypical teratoid/rhabdoid tumor, epithelioid sarcoma and myoepithelial carcinoma seems to be reduced or even negative in synovial sarcomas. the focal expression of d - must be considered when the differential diagnosis includes mesothelioma. finally tle- is a very sensitive marker for synovial sarcoma. ps- - "mixed aneurysmal bone cyst" and "simple bone cyst", represent a different group of cystic lesion e. ayhan cinar * , b. doganavsargil, m. sezak, f. oztop * ege university, pathology, izmir, turkey objective: there are some mixed cysts (mc) with overlapping histological features of aneurysmal bone cyst (abc) and simple bone cysts (sbc). method: we reviewed pure abc, sbc and "mixed cysts (mc)", and compared them by nonparametric tests. results: mcs, mainly showed two paterns as "type : sbc with secondary abc foci (n= )"and "type : cysts with fully developed sbc and abc areas (n= )". the median age for abc, sbc, and mc were ± . (range: - ), ± . (range: - ) and ± . years old (range: - ) respectively. mcs were more frequent in males than abc and sbc (p= . ) and the most frequently involved bones were humerus-femur-pelvic bones in descending order (p= , ). "type mc" showed more frequent cementum-like amorph material (p< . ), ectatic venules (p= . ), calcifications (p= . ), less cholesterol deposition, necrosis and no different fracture and osteoid matrix when compared with type mcs and sbcs. conclusion: cysts with overlapping features of abc and sbc are not uncommon. though they usually represent "sbcs with secondary abc component", they have dissimilarites with ordinary sbcs which needs to be further clarified with larger series. objective: percent of tumor necrosis after neoadjuvant chemotherapy, determined by detailed specimen mapping, has a high prognostic value. however both the mapping and grading systems has some practical problems. method: cases were reviewed for potential pitfalls in handling and reporting processes and correlated with radiological findings by nonparametric-tests. results: most of the tumors were osteosarcoma ( . %) and ewing sarcoma ( . %), located in femur ( %), tibia ( , %) and humerus ( . %). cortex, soft tissue and joint invasion was observed in %, . % and . % of the cases, respectively. a median of . tumor blocks ( - ± , ) were submitted for histology. tumors were totally necrotic in cases ( %) (huvos grade-hg-iv), hg iii in . %, hg ii in . % and hg i in . % of the cases. overall good (hg iii-iv) and poor responders (hg i-ii) correlated well with radiological findings (p< . ). discrepant cases showed extensive oedema and congestion enhancing contrast medium, patchy necrosis unabling accurate histologic evalation, or failure to prove tibiofibuler joint involvement because of sagittal sectioning (p< . ). conclusion: evaluating chemotherapy responce is a laborious work. radiologic orientation prior to grossing and applying morphometric technics may enhance more accurate evaluation. atypical ewing sarcoma/primitive neuroectodermal tumor with unusual melanocytic differentiation -a case report p. buzrla * , j. dvorackova, i. urbanovska, h. bielnikova * inst. of pathology, ostrava-marianske hory, czech republic objective: atypical ewing sarcoma/primitive neuroectodermal tumor with the melanocytic differentiation is a very rare, malignant tumor which occurs in infants and adolescents. it is localized mainly in soft tissues. its biological behavior is aggressive, but its response to the chemotherapy is prognostically good. method: a -month old male is presented with the × cm tumor of the soft tissue, localized in the right front over the cranial margin of the orbit. the tumor was surgically exstirpated and then sent to the histological investigation with the additional immunohistochemistry and the fish. results: microscopically, the tumor is hypercelullar and consisted of oval to polygonal cells with the vesicular nuclei, the prominent nuclei and the amphophilic cytoplasm. in other parts of the tumor, there are smaller neoplastic cells with hyperchromatic nuclei. some neoplastic cells contain the melanin pigment in the cytoplasm, which was positive in fontana-masson method. the immunohistochemical stains for vimentin, ae / , ck , cd , hmb- , fli- were positive. the fish investigation demonstrated the translocations (t ; , q ;q ) in % of neoplastic cells and (t ; , q ; ) of % ones. conclusion: in literature, the ewing sarcoma/pnet showing the myogennic differentiation is usual, but the melanocytic differentiation is rare and can be confirmed not only immunohistochemically, but also by fish investigation. objective: poly(adp-ribose) polymerase- (parp- ) is a nuclear enzyme involved in the repair of dna single-strand breaks. parp- inhibitors may be efficient in therapy of malignancies. this report evaluates the expression of parp- in primary testicular germ cell tumors and correlates expression patterns with histological subtypes and patient/ tumor characteristics. method: group of patients with testicular germ cell tumors were investigated for parp- expression by immunohistochemistry, scored by the multiplicative quickscore (qs) method and compared to parp- expression in normal testis. results: we observed higher expression of parp in testicular tumors compared to normal testis (mean qs= . vs. . , p< . ). the parp- overexpression (qs> ) was most often detected in intratubular germ cell neoplasia itgcn ( % of specimen with parp- overexpression), compared to . % of normal testicular tissue specimen. there was no association between parp- expression and clinical variables. conclusion: parp- expression is higher in tumor tissue than in normal testis. parp- could represent a novel treatment target in tgcts and the assessment of parp- expression in tumor samples may lead to the consideration of tgcts patients for parp inhibitor therapy. supported by / -nou- , vega / / and itms: cofinanced by european regional development fund. objective: gardner fibroma (gaf), a rare lesion, typically occurs in infants, children and young adults, with predilection for the trunk (particularly paraspinal region) and no gender predominance. gaf is an ill-defined plaque-like mass, with rubbery consistence, usually asymptomatic, ranging from . to cm. although benign, - % of the cases may be associated with apc mutations, familial adenomatous polyposis (fap) and/or gardner syndrome (gs). we report cases of gaf, which allowed the identification of families with gs. results: case : -month-old boy, with nodules in the left paraspinal region, the largest with . cm. case : month-old boy, presenting a . cm ill-defined deep right scapular mass. both lesions were infiltrative, paucicelular densely collagenized proliferations of bland spindle cells. a gaf diagnosis was made and clinical/genetic investigation advised. apc gene sequentiation revealed, in both cases, a frameshift germline mutation in exon . additionally, case presented a del( )(q q ). heredogram showed typical manifestations of gs: case : mandibular osteomas, epidermoid cysts, "soft-tissue tumours", colo-rectal carcinoma. case : hepatoblastoma, colo-rectal carcinoma. conclusion: given the high association with gs and the fact that it can be its first manifestation, gaf is considered a sentinel lesion for this syndrome. therefore, an accurate diagnosis is of the utmost importance. prognostic significance of bcl- , c-myc and survivin in synovial sarcoma d. demir * , b. yaman, y. anacak, b. keçeci, g. kandiloğlu, t. akalın * ege university, pathology, izmir, turkey objective: in this study, we evaluated synovial sarcoma cases, who had been referred to our tertiary tumor center during the last years. we applied bcl- , c-myc and survivin as immunohistochemical markers and evaluated the relation with conventional prognostic findings and prognosis for those patients who have follow-up. method: in this study; ten-year tumor free survival rate was % reflecting the agressive behaviour of synovial sarcoma. tumor grade was the most valuable prognostic input. progression free survival (pfs) was months for gradeii cases ( cases) and months for gradeiii cases ( cases) (p= , ). results: immunohistochemically, there was weak relation between bcl- staining intensity with prognosis. overall survival was months for weak or negative cases ( cases), months for focally intense cases ( cases) and months for diffuse and intense cases ( cases) (p= , ). there was also weak relation with c-myc staining pattern with prognosis. overall survival was months for c-myc negative cases ( cases), months for cytoplasmic positive cases ( cases) and months for nuclear positive cases ( cases) (p= , ). there was no relation between survivin and prognosis. conclusion: in conclusion; tumor grade is the most valuable prognostic parameter in synovial sarcomas. immunohistochemically c-myc and bcl- staining have weak relation with synovial sarcoma prognosis. immunohistichemical profile of primary and recurrent desmoids e. dubova * , t. sidorenko, a. shchegolev, a. adamyan * v.i. kulakov scientific center, dept. of pathology, moscow, russia objective: desmoids is a locally recurrent and invasive but not metastatic tumor. our aim was to study immunohistochemical profile of primary and recurrent desmoids. method: complex morphological study of abdominal desmoids. all the tumors were divided into groups: primary, first recurrent and second and more recurrent cases. we used antibodies against β-cathenin, cox- , apc, survivin and ki- for immunohistochemical study. results: nuclear and cytoplasmic β-cathenin expression levels were significantly higher in recurrent than in primary desmoids. apc cytoplasmic expression level was also significantly higher in recurrent tumors. we revealed only cytoplasmic cox- expression in desmoids, and its level was significantly higher in recurrent tumors with the activity increasing accordingly to the number of recurrences. survivin expressed both in nuclei and cytoplasm of the tumor cells and its expression levels were significantly lower in recurrent desmoids. conclusion: revealed immunohistochemical properties of primary and recurrent desmoids reflect tumor transformation and progression and could be used as additional prognostic markers in this disease. objective: many bone and soft tissue sarcomas (bsts) are aggressive tumors with fatal prognosis. the importance of angiogenesis for the growth and progression of solid bsts is now well recognized. a variety of chemokines have been described that either promote (angiogenic) or inhibit (angiostatic) angiogenesis. the aim of this study is to characterize the expression profile of some chemokines in a series of xenotransplanted human bsts. method: one ewing sarcoma (es), grade chondrosarcoma (chs), osteosarcoma (os), synovial sarcoma (ss), fibrosarcoma and gastrointestinal stromal tumor (gist) were xenotransplanted into the backs of nude mice (athymic balb-c nude mice). when the tumor size reached the cm, animals were sacrificed and tumors analyzed for the expression of cxcl / / , cxcl and cxcl , using two-color staining fluorescence on each slide under a confocal microscope (olympus fv ). results: we observed that angiostatic chemokines (cxcl and cxcl ) presented higher expression than angiogenic (cxcl / / ) chemokines in es, grade chs, os and gist whereas fibrosarcoma and ss were more positive for angiogenic chemokines. conclusion: the expression profile of angiostatic and angiogenic chemokines depends on the type of bsts and could be related to their different biological behaviour. other elements such as angiogenic or pro-inflammatory markers should also be considered. objective: the wide range of differential diagnostic possibilities of rhabdomyosarcomas (rms) shows the need for more specific and sensitive markers. low survival rates of high risk rms patients require new prospective therapeutic targets. method: archival material ffpe samples of rms ( embryonal -erms, alveolar -arms) and non-rms soft tissue tumors were evaluated by immunohistochemistry for myogenin, myod , egfr, vegf, cox- , p-akt and p-mtor expression. the presence of pax / -fkhr forming translocations, myogenin, myod , gamma subunit of fetal acetylcholine receptor (achr) and k-ras mutational state were determined by rt-pcr. results: / erms and / arms showed expression of myogenin, myod and gamma achr. non-rms tumors were negative. translocations were only found in arms. egfr expression was characteristic for erms, without the presence of activating k-ras mutation. strong expression of vegf was detected in all samples. p-akt and p-mtor showed overlapping expressions in , % rms. in most of the samples weak cox- positivity was demonstrated. conclusion: myogenin, myod and the fetal achr are specific and sensitive diagnostic markers of rms. rms subtypes are identifiable by pax / -fkhr detection and probably egfr expression. the results indicate vegf, egfr, cox- and akt-mtor pathway directed therapy to be considered in rms. supported by itms . immunohistochemical analysis of potential targets in desmoid tumor therapy a. janegova * , z. hlavata, p. babal, p. janega * comenius university, dept. of pathology, bratislava, slovakia objective: desmoid tumors (dts) are clonal fibroblastic/ myofibroblastic proliferations. although histologically benign, desmoids are locally invasive and often have an unpredictable clinical course. dts are infrequent lesions, but they have a high incidence in patients with familial adenomatous polyposis (fap). the treatment of dts needs to be individualized. method: to explore the molecular bases of potential pharmacologic targets in dts we evaluated the immunohistochemical expression of steroid hormone receptors (era, erb, pr) and cox- protein in sporadic (n= ) and fapassociated desmoid tumors (n= ) together with gi adenomas of fap patients (n= ). results: nuclear erb expression was found in / dts. all adenoma samples showed nuclear erb positivity, which was weaker than in the surrounding normal epithelial cell. era and pr expression were lacking in all samples. cox- was found in / dts. adenomatous polyps showed intense cox- expression compared to surrounding normal mucosa. conclusion: high incidence of erb positivity in dts supports the usage of hormonal therapy in these lesions. open question is the effect of anti-estrogen therapy in dt patients with adenomatous polyps, as in adenomas estrogen seems to have preventive potential. cox- expression suggests the benefit of anti-inflammatory treatment in dts adenomatous polyps. supported by itms: . retroperitoneal sarcomas: clinicopathological features in a series of cases e. kairi-vasilatou * , a. tsagkas, a. melloy, a. paraskeva, a. kondi-pafiti * aretaieio nosokomeio university, dept. of histopathology, athens, greece objective: retroperitoneum is the less common site of origin accounting for approximately % of soft tissue sarcomas. method: between january and december , our hospital's records of patients with retroperitoneal sarcomas were retrospectively studied. results: the patient median age was years and there was no sex predominance. median tumor size was . cm (ranging from to cm) with % of them being larger than cm. the most common histological type was liposarcoma ( / - , %), followed by leiomyosarcoma ( / - , %) and undifferentiated soft tissue sarcoma ( / - , %). the remaining tumors ( , %) included chondrosarcomas, well-differentiated fibrosarcomas, pecoma, hemangiopericytoma, ewing sarcoma and malignant peripheral nerve sheath tumor. of the sarcomas were high grade ( %) and ( %) low grade. -year recurrence rate was , %. the -and -year overall survival rates were , % and , % respectively. seven patients received adjuvant chemotherapy. conclusion: the most commonly encountered histologic subtypes are liposarcoma and leiomyosarcoma, which are consistent with the results of the present study. complete tumor resection at first operation is the only treatment factor that consistently predicts improved survival. abdominal desmoid tumor y. lorenzo mahia * , m. san martin alonso, b. iglesias rodriguez * hospital maixoeiro, anatomia patologica, vigo, spain objective: abdominal desmoid tumours are rare benign neoplasms. they are commonly found in the mesentery, while they are rarely found in the intestinal wall. most cases are sporadic, although there is a link with colonic polyposis, trauma, and oestrogen. they predominate in - year-old women. this present case is notable due to its location in the jejunal wall, possible relation with previous surgery, and the age and sex of the patient. method: we present a -year-old male patient previously operated for umbilical hernia years before. he came to the consultation on noticing an abdominal mass. since the initial suspicion was of jejunal wall gist tumour, surgery was performed. results: the histopathological and immunohistochemical findings, support the diagnosis of desmoid tumour. conclusion: desmoid tumours consist of fibroblastic monoclonal proliferation developed from aponeurotic muscle structures. some authors consider them non-neoplastic processes given their limited aggressiveness while others classify them within distinct low-grade sarcomas. their origin is not well established, although there are known factors involved such as mutations in the apc gene or beta-catenin and trisomy and . the originating cell, the myofibroblast, is involved in post-traumatic cellular regeneration. this explains why we find these tumours associated with previous surgery. treatment of advanced dermatofibrosarcoma protuberans with imatinib mesylate with or without surgical resection w. michej * * cancer centre institute of warsaw, dept. of pathology, poland objective: dermatofibrosarcoma protuberans (dfsp) is a rare soft tissue sarcoma of the skin characterized by the presence of specific col a -pdgfb fusion protein, which appears as a consequence of the t( ; ) (q ;q ) translocation. method: the aim of the study was to perform an analysis of patients with advanced dfsp treated with imatinib, with or without surgery. we examined patients ( male, female; median age years) with locally advanced/initially inoperable and/or metastatic dfsp treated with imatinib - mg daily between / and / . all diagnoses were ascertained cytogenetically (fluorescent in situ hybridization). median follow-up time was months (range: - ). results: metastases were present in cases (two lungs, two soft tissue, two lymph nodes). fibrosarcomatous transformation was confirmed in patients. after treatment with imatinib overall responses were: partial responses, stable diseases ( %) and progressive diseases ( %). seven patients ( %) after resection had residual disease confirmed by pathologic examination and remained free of disease. conclusion: we proved that anti-tumour effect of imatinib in dfsp with presence t( ; ) had in most cases good responses. imatinib therapy may in some cases leads to tumour resection because of lesser size. objective: at an estimated incidence of cases per million per year, osteosarcoma is the most common malignant primary bone tumor. method: we conducted a retrospective study to identify the osteosarcoma cases diagnosed at major tertiary care hospitals in turkey. results: our study group was made up of cases: men, women, aged - years (mean . year). all patients had been diagnosed with skeletal osteosarcoma between and at one of the tertiary care referral center. tumor was most frequently located in femur ( . %) followed by tibia. apart from the long bones, pelvic and gnatic bones were the next in location, . % and . % respectively. there were ( . %) secondary osteosarcomas, related to previous irradiation and various underlying diseases. for the histological types conventional osteblastic intramedullary tumors were most prevalent, making . .% of the cases. chondroblastic and telengiectatic osteosarcoma are the next common histologic types. surface tumors were detected in ( . %) cases. rare histological types like small cell, epitheloid, chondroblastoma like and fibrous dysplasia like were also reported. conclusion: this study is conducted as a preliminary work to form the basis of a pathologic database for the osteosarcoma cases diagnosed in our country. objective: kaposi's sarcoma is a rare disease likely associated with human herpes virus infection, and occurs predominantly in jewish, mediterranean and middle eastern men. since there is a paucity of reports on the pattern of its occurrence in tunisia, we here analysed the epidemiological pattern and anatomoclinical features. method: we retrospectively studied consecutive cases of kaposi's sarcoma diagnosed in the pathology department, farhet hached hospital, sousse during a -year period. results: kaposi's sarcoma represented % of soft tissue sarcomas. there were ( . %) females and ( . %) males (male-to-female ratio: . : ). median age at diagnosis was (range: - years). the age distribution showed that elderly (≥ years) were the most affected patients with a frequency of . %, followed by patients aged - years ( . %), and patients under years ( . %). the most common location was the lower limbs, particularly the distal lower extremity ( . %), followed by contiguous location ( %), and soft tissue, nos ( %). conclusion: kaposi's sarcomas were more frequently diagnosed in elderly. the distal lower extremities were more involved. objective: rhabdomyosarcoma is the most common soft tissue sarcoma in the first two decades of life. in this study, we analysed the epidemiological pattern and antomoclinical features of rhabdomyosarcoma in central tunisia. method: we retrospectively studied all cases of rhabdomyosarcoma diagnosed in the pathology department, farhet hached hospital, sousse during a -year period. results: there were ( . %) females and ( . %) males (male-to-female ratio: . : ). median age at diagnosis was (range: - years). rhabdomyosarcoma was more frequently diagnosed in childhood ( %) than in adults ( %). in children, the tumour size was higher than cm in % of cases, the embryonal subtype was the most frequent ( %) and the two most common sites of disease were the head and neck ( %) and genito-urinary tract ( . %). in adults, the tumour size was higher than cm in % of cases, the pleomorphic subtype was the most diagnosed ( %), and limbs were the most involved sites ( %). conclusion: rhabdomyosarcomas is more frequently diagnosed in children than in adult. head and neck locations were the most involved and embryologic type was the most diagnosed. in adults, rhabdomyosarcomas were more frequently localized in limbs and diagnosed as pleomorphic type. microarray-based dna methylation study in ewing sarcoma of bone h.-r. park * , y.-k. park * hallym university, sacred heart hospital, anyang, republic of korea objective: alterations in the dna methylation pattern are a hallmark of malignancy and also of ewing sarcomas. however, most epigenetic studies in ewing sarcoma have focused on the analysis of few candidate genes and comprehensive studies are required. method: here, we report for the first time a microarraybased dna methylation study of cpg sites of cancerrelated genes in ewing sarcomas. we used illumina's goldengate methylation cancer panel i microarray. results: using appropriate controls (n= ), we identified a total of cpg sites hypermethylated in ewing sarcoma. most of hypermethylated genes are related with cell adhesion, cell regulation, development, and signal transduction. we compared the methylation mean of each tumor according to the survival data. the methylation mean was significantly higher in the alive patient group ( . ± . ) compared to the dead patient group ( . ± . ) (p= . ). however, the methylation mean was not significantly correlated with age, sex, or tumor location. we selected the most popular hypermethylated genes, gdf , osm, apc, and hoxa , but, their methylation levels were not significantly correlated with the survival data. conclusion: we have characterized the dna methylation profile of ewing sarcomas and detected cpg sites that were significantly hypermethylated in ewing sarcomas. these might therefore play an important role in the development of ewing sarcomas. pleomorphic and dedifferentiated leiomyosarcoma associated with lynch syndrome: a case report h. quiceno * , f. j. queipo, r. carías, j. j. sola, f. j. pardo * clinic university of navarra, anatomical pathology lab, pamplona, spain objective: the lynch syndrome or hereditary non-polyposis colorectal cancer (hnpcc) is a hereditary syndrome that predisposes to different types of colonic and extracolonic cancer, mainly in endometrium, stomach, ovary, and hepatobiliary and urinary tract. occasional sarcomas have been reported in hnpcc patients. method: we describe a pleomorphic and dedifferentiated leiomyosarcoma of the gluteus in a patient with lynch syndrome. we study her clinical, pathology, immunochemistry and molecular alterations. results: we present a year old woman with previous colorectal, endometrium and breast cancers in a lynch syndrome with mutations in exons and of mlh , which appeared with a right gluteus mass. grossly, it was encapsulated, whitish-yellowish with necrotic areas and measured , cm. microscopically, the tumor was composed by a diffuse and polymorph spindle cells with fascicular pattern, focally myxoid and zones of necrosis and large hyalinization. the cells had large nuclei, sometimes giant multinucleated, with a brisk atypical mitosis activity. there was ihq variability: positivity to desmine, msa, calponin and negative to caldesmon and sm. there was loss of msh and msh repair proteins. conclusion: we must suspect a lynch syndrome relational sarcomas if we found one in a patient with hnpcc in order to test to mismatch repair proteins. immunohistochemical review of synovial sarcomas, including expression of tle : a "new" marker, with molecular confirmation in cases b. rekhi * , r. basak, s. desai, n. jambhekar * tata memorial hospital, dept. of pathology, mumbai, india objective: synovial sarcoma displays a wide clinicopathological spectrum and a specific translocation t(x; )(syt-ssx)(p . ; q . ). cost constraints limit molecular confirmation in every case. lately, tle has been recognized as a useful ihc marker. herein, we present ihc review of synovial sarcomas, including tle expression. method: forty-two synovial sarcomas included monophasic spindle-cell type ( ) ( . %), biphasic ( . %), calcifying ( ) ( . %) and poorly-differentiated type ( ) ( . %). tumors were confirmed with molecular analysis. tle immunostaining was graded from , +, +, +, with + or + grades interpreted as positive staining. results: on ihc, various synovial sarcomas were positive for ema ( / )( . %), ck ( / ) ( %), ck/mnf ( / )( . %), bcl ( / )( . %), mic ( / )( . %) and tle ( / )( . %), while negative for cd ( / ). among other tumors, tle was positive in schwannomas ( / ) ( %), neurofibromas ( / )( %), mpnsts ( / )( %) and pnets ( / )( %). sensitivity and specificity of tle for synovial sarcoma was . % and %. % of pnst ( % of schwanomas, % of mpnst and % of neurofibromas). in contrast, her positivity was observed in % of other mesenchymal tumors ( % uterine and % non-uterine leiomiomas, % uterine and % nonuterine leiomiosarcomas, % ups). conclusion: her overexpression is frequently found in pnst, including % of mpnst. due to the crucial role of her receptor in cell signalling as a main activator of the pi k pathway, these results support the rationale of developing new therapeutic approaches in mpnst. objective: nonneural granular cell tumour (nngct) is a rare neoplasm described in by leboit et al. till now, no more than cases have been presented in the literature, nearly all were limited to the skin. we report the first nngct of the breast and present its morphological and immunohistochemical characteristics. method: a year-old female underwent lumpectomy because of tumour of the breast. after routine histological examination, the immunohistochemical studies were performed using commercially available antibodies against cytokeratins (ae /ae ), cd , cd a, s , cd , smooth muscle actin, desmin, miogenin, inhibin, and ki antigen (mib ). results: microscopically, the tumour was composed of ovoid and polygonal cells with abundant granular, eosinophilic cytoplasm. the cells presented mild or moderate polymorphism and trace mitotic activity. they were arranged in diffuse sheets with no evidence of nesting or fasciculation. the histological texture of the tumour resembled that of classical granular cell tumour of abrikosoff. however, the tumour cells were strongly immunoreactive for cd and inhibin, whereas the reactions against s protein as well as other antigens studied gave negative results. the value of ki index did not exceed - %. conclusion: the differential diagnosis of granular cell myoblastoma (abrikossoff tumour) should include the nonneural granular cell tumour. low grade fibromyxoid sarcoma: a study of cases k. shelekhova * , a. konstantinova * petrov´s research institute for oncology, st. petersburg, russia objective: low grade fibromyxoid sarcoma (lgfms) is a specific type of fibrosarcoma with deceptively banal appearance and malignant behavior. method: a year retrospective study revealed lgfms cases. morphological and immunohistochemical analysis was performed. follow-up information was obtained for cases. results: all tumors occurred in adults (mean year). they developed in neck ( ), extremities ( ) and trunk ( ). beside classical morphology of lgfms following features were observed: necrosis ( ), rosettes ( ), areas of increased cellularity ( ), foci of epithelioid cells ( ), marked nuclear pleomorphism ( ), prominent myxoid change ( ), invasive border ( ), bone formation ( ), focal retiform pattern ( ) and sclerosing epithelioid fibrosarcoma-like areas ( ). immunoprofile was the follow: vimentin ( %), ema ( %), cd ( % focally), s ( ), sma( ), desmin ( ), ae /ae ( ). two patients were without evidence of disease, one developed lung metastasis after year. two patients developed secondary tumours in and years after initial diagnosis. there were differences in the location and histologic features compared with primary ones. secondary tumors were less cellular, without necrosis or epithelioid cells. behavior of secondary tumors did not fit to the classical concept of tumor progression that implies increase of malignancy. conclusion: lgfms is a distinctive but unusual type of soft tissue sarcoma demonstrating a wide histologic spectrum and behavioral range. immunohistochemical and mutational study of synovial sarcomas k. specht * , m. bettstetter, g. keller, h. rechl, r. von eisenhart-rothe, h. höfler, m. straub * technische universität münchen, inst. für pathologie, germany objective: synovial sarcomas are mesenchymal tumors of unknown histogenesis. their molecular signature is a specific t(x; )(p . ;q . ) translocation. no effective targeted therapies are currently available. the aim of this study was to evaluate the expression and mutational status of potential molecular therapeutic targets. method: molecularly confirmed cases of synovial sarcomas were included in this study. immunohistochemical stainings of the egf-r family (egf-r, her /neu, her ), and signaling molecules implicated in the mtor pathway (akt, mtor, pten), as well as e-cadherin and snail was performed. in addition, cases were screend for mutations in the egfr, pik c, b-raf, k-ras, and n-ras genes. results: egf-receptor family members as well as e-cadherin and snail are important for defining the tumor phenotype by determining epithelial-mesenchymal transition of synovial sarcomas. activation oft the mtor pathway is seen in a significant number of cases. mutations of the genes studied are an overall rare event in synovial sarcomas and other types of sarcomas studied. conclusion: egf-r expression is found in many synovial sarcomas, however, mutations of egfr or downstream molecules appear to be rare. activation of mtor pathway is frequently seen in synovial sarcomas. the benefit of targeted therapy against these genes in synovial sarcomas remains to be determined. collagen v induces differentiation of rabbit adipose tissue-derived stem cells in chondrocyte-like phenotype w. teodoro * , i. brindo da cruz, a. p. velosa, s. carrasco, c. goldenstein-schainberg, r. fuller, e. parra, v. capelozzi * faculdade de medicina da usp, disciplina de reumatologia, são paulo, brazil objective: stimulated mesenchymal stem cells (mscs) have capacity of differentiation in many cell types. it is being used in degenerative diseases treatment protocols. we evaluated the collagen v (col v) and collagen xi (col xi) influence in the differentiation of rabbits adipose tissue-derived mscs in a chondrocyte-like cell phenotype. method: mscs isolated of new zealand rabbits adiposetissue were maintained in culture by weeks. colv, colxi and colv/xi ( μg/ml) were added to culture during h. the cells aggregates were stained with toluidine blue, alcian blue and picrosirius. chondrocyte-like phenotype was confirmed by immunofluorescence to cd , vimentin and collagens i, ii and iii. results: mscs stimulated with colv expressed proteoglicans and collagen, when compared with colxi and colv/ xi and control. in the presence of colv, mscs was capable to increase collagen ii expression confirming its chondrocyte-like cell phenotype. in contrast, mscs cultured with colxi and colv/xi express collagen i and iii. conclusion: the data suggest that colv may facilitate the differentiation of rabbit adipose tissue-derived stem cells into a chondrocyte-like phenotype. further studies are urged in order to evaluate the influence of colv in the ability of chondrocytes to remodel osteoarthritic joint surface at ultra structural and molecular levels. objective: xrcc is essential for dna base excision repair, single strand break repair and nucleotide excision repair. method: we evaluated xrcc immunohistochemically in early stage breast (n= ), ovarian (n= ), gastric (n= ), colorectal (n= ) and pancreaticobiliary cancers (n= ). pre-clinically, we evaluated a panel of xrcc deficient and proficient chinese hamster ovary and human cancer cell lines. double strand break repair (dsb) inhibitors targeting atm (ku ), dna-pkcs (nu ) and atr (nu ) were evaluated for synthetic lethality and cisplatin alone or in combination with dsb inhibitors for chemopotentiation. results: in breast cancer, xrcc loss ( %) was associated with a -fold increase in risk of death and metastasis (p< . ). in ovarian cancer, xrcc positive tumours ( %) were more resistant to platinum chemotherapy (p= . ). xrcc positivity conferred a fold increase of risk of death (p= . ) and independently associated with poor survival (p= . ). pre-clinically, ku , nu and nu were synthetically lethal in xrcc deficient compared to proficient cells as evidenced by dsb accumulation, g /m cell cycle arrest and apoptosis. xrcc deficient cells were hypersensitive to cisplatin which was enhanced by dsb repair inhibitors compared to in proficient cells. conclusion: conclusions: xrcc deficiency in human tumours may be suitable for synthetic lethality application and exploited for cisplatin chemotherapy potentiation. analysis of bcl oncoprotein expressing breast cancer by molecular subtype a. abolins * , i. strumfa, z. simtniece, a. vanags, g. trofimovics, j. gardovskis * riga stradinš university, inst. of oncology, latvia objective: breast cancer is the most common malignancy in western women. despite the progress in morphological investigation, active research is devoted to potentially important targets for prognosis and intervention. bcl oncoprotein represents such factor. method: consecutive breast cancer cases were examined by routine protocol approach. the bcl oncoprotein expression was detected immunohistochemically. expression was considered positive if it was in more than % of tumour cells. the molecular subtype ( objective: breast cancer is the most common malignant tumour of latvian women (www.csb.lv). correct diagnosis, including identification of tumour histogenesis, is the prerequisite for appropriate treatment. method: consecutive breast tumour cases were selected by systematic retrospective archive search and were examined by breast cancer panel. if expression of oestrogen and progesterone receptors and her protein was negative, mammaglobin, cytokeratin ae /ae , vimentin, cd , cdx , cytokeratin , ttf- and melanosome protein hmb- were detected. results: five patients ( . %, % ci= . - . %) with secondary breast tumours were identified among cases. breast was affected by metastatic small cell lung cancer ( ), malignant neuroendocrine tumour of small intestine ( ) as well as by epithelioid melanoma metastasis in breast tissues ( ) or intramammary lymph node ( ). analysing medical records, multiple synchronous and/or metachronous metastasis, involving brain, kidneys and ovaries, were found in all patients with haematogenous tumour metastases in the breast. in contrast, the lymphogenic tumour spread was isolated. conclusion: . lymphogenic or haematogenous metastasis rarely ( . % of malignant breast tumours by morphology) can develop in breast tissues. it should be taking into account when planning the differential diagnostic approach, especially immunohistochemistry. . haematogenous metastases in breast are associated with a wide synchronous or metachronous extramammary tumour spread. the prognostic significance of tumor-associated stroma in invasive breast carcinoma objective: fibroblasts in the stromal component of a tumor may influence tumor progression in various organs. the prognostic significance of tumor-infiltrating lymphocytes is also frequently reported. however, the prognostic significance of the stromal component in breast cancers, particularly those of high grade, has not been established. method: in this study, we analyzed surgically resected specimens from patients with breast carcinoma, including high grade tumors, for tumor-stroma ratio, dominant stroma type (collagen (c), fibroblast (f) or lymphocyte (l) dominant type), and central fibrosis on hematoxylin-eosin stained histological sections. we correlated these features with clinical prognosis. results: among the specimens examined, ( . %) were of c type, ( . %) of f type, and ( . %) of l type. central fibrosis was found in tumors ( %). the dominant stroma type was a significant prognostic factor on univariate and multivariate analyses, together with t classification, nodal status and bloom-richardson grade. tumorstroma ratio and central fibrosis did not predict survival on multivariate analysis. even in high-grade tumors, relapse-free intervals differed significantly according to dominant stroma type. conclusion: conventional hematoxylin-eosin stained tumor slides may contain more prognostic information than previously thought; in particular, the dominant stroma type in invasive breast cancer may potentially be used to predict outcome. ps- - pathology in breast implants substitution l. alfaro * , j. serra, j. m. ibañez * valencia, spain objective: health alert concerning breast implants brand pip led to a review of patients harboring these prosthesis and in many cases replacement by new ones. anaplastic lymphoma described in these patients has been an additional problem to be faced by plastic-surgeons and pathologists. method: ninety two cases of women with breast implants were studied. most of them had pip implants (although no information of prosthesis type was available in all cases). eighty of them presented with ruptures of different size. after implant replacement, histopathologic analysis of fibrous capsules and liquid from seromas in periprosthetic cavities was carry out. following fda recommendations, immunohistochemical studies to rule out lymphoma was performed in cases. results: fibrous capsules showed synovial metaplasia in all cases. morphology was practically identical that in real articular synovial cells, and occasionally intracavitary nodules were seen detached from the surface in a process similar to synovial chondromatosis. no cases of lymphoma were seen and no expression of cd or alk markers occurred. a case of pericapsular ductal invasive carcinoma was discovered. conclusion: synovial metaplasia seems to be very common in capsules around breast implants. development of conventional breast carcinoma is probably much more frequent than lymphomas independently of the possible implant influence. post-radiation angiosarcoma of the breast: report of a case a. apostolaki * , m. sofopoulos, s. tsitsiou, e. pigadioti, n. mylona, n. arnogiannaki * agios savvas hospital, dept. of anatomical pathology, athens, greece objective: post-irradiation angiosarcoma generally occurs after breast conservation and radiation therapy. it affects the dermis of the breast within the radiation field. the incidence of post-radiational angiosarcoma is about . %. method: an -year-old woman who had undergone conservative surgery and radiotherapy for breast cancer years ago presented with multiple red-purplish papules on the skin of her right breast. excisional biopsy followed by simple mastectomy was performed. results: excisional biopsy revealed a neoplasm composed of highly pleomorphic cells with prominent nucleoli forming solid areas and neoplastic slit-like vascular channels. many mitotic figures and some individual apoptotic cells were also present. the tumor cells were positive for endotelial cell markers (factor viii, cd , cd ) and negative for ck . the gross inspection of the mastectomy specimen showed multiple reddish-purple papule-like lesions on the skin, spreaded in an area of × cm. sectioning showed numerous homogenous slightly hemorrhagic white nodules measuring from to mm located in the dermis. histopatology and immunohistochemistry findings were consistent to those of the excisional biopsy. infiltration of the subdermis was noted. conclusion: diagnosis was high grade post-irradiation angiosarcoma. simple mastectomy is the treatment of choice. adjuvant chemotherapy should be considered in high grade neoplasms like this one. prognostic factors in invasive lobular carcinoma of the breast g. askan * , g. ayranci, n. Özkan, h. kaya, u. ugurlu * marmara university, dept. of pathology, istanbul, turkey objective: determination of molecular features in breast carcinomas, such as hormone receptor expression, can guide clinicians to the optimal choice of therapy. in this study, the relationship between the histologic grade, pathologic stage and the prognosis of invasive lobular carcinoma of the breast, and the tissue expression levels of er, pr, her- /neu, p , bcl- , ki- and e-cadherin was investigated. method: cases of invasive lobular carcinoma of the breast, from to , were included in this study. a single best representative paraffin block was selected for each case and h&e staining and immunohistochemistry procedures were performed. ki-kare test was used for statistical method. results: of cases had classical lobular carcinoma, cases had tubulolobular, pleomorphic, signet ring cell, or apocrine features. the median age of patients was years. er and pr were positive in cases. all cases were negative for e-cadherin. ki- was greater than % in cases with pleomorphic lobular carcinoma. p positivity increased with grade. in cases p was negative and bcl- was positive. bcl- and p were positive in and cases respectively. conclusion: in contrast to the literature, there was no correlation between bcl- status and other molecular markers, including p . rapid immunohistochemistry in intraoperative sentinel axillary lymph node evaluation p. baldin * , m. c. cucchi, y. ishikawa, v. eusebi, m. p. foschini * university of bologna, dept. of anatomic pathology, italy objective: sentinel lymph node (sn) examination is the current procedure to establish the status of axillary lymph nodes in breast cancer. to avoid a two step delayed surgical procedure, a reliable and quick method of sn evaluation is advocated. rapid immunohistochemical technique (uich) for keratin has been only recently proposed. aim of this study is to apply keratin uich in frozen sections (fss) of sns. method: a consecutive series of (series a) sn cases was studied at fs level followed by two sections stained with keratin uihc. all procedure requires min. for comparison consecutive cases (series b) of sn were studied with fs only. all residual tissue from both series was paraffin embedded (ps). results: series a: sns showed tumour involvement in cases ( . %). in only cases ( . %), ps sections evidenced additional neoplastic cells (micometastases) not seen with keratin uihc, that led to a delayed axillary dissection. series b: ps sections revealed metastatic deposits (micrometastases) not seen in fs in . % of cases. conclusion: fs coupled with keratin uhic of all the entire lymph node accurately evidences carcinoma cells in sns. high concordance of her in situ hybridization methods with abbott fish j. boers * , l. krol, c. netjes, h. meeuwissen, c. prinsen, c. van krimpen, e. van der logt, j. bart, e. schuuring * isala klinieken, dept. of pathlogy, zwolle, netherlands objective: we conducted a comprehensive concordance study of ish methods with abbott fish in a large series of breast carcinomas. method: tissue micro arrays (tma) were constructed by taking tissue-cores from praffin blocks of primary breast carcinomas. up to cases were analyzable in ish assays. scoring was performed by two independent observers without knowledge of the other ish data according the asco-guidelines for her -testing. cases were considered positive when the ratio was ≥ . . discordant cases were reviewed and scores were reassigned on consensus of opinion. concordance and cohen's kappa score were calculated in relation to fish, abbott. results: in cases analyzable with abbott her fish, . % were her -positive. concordances (kappa-scores) of the other assays were: dako fish . % ( . ), dako duocish . % ( . %), zytovision fish . % ( . ), zytovision duocish . % ( . ), single probe sish ventana . % ( . ), dual probe sish ventana . % ( . ). conclusion: conclusion: concordance of her ish assays with abbott fish were shown to be . % or higher. in this study, dako assays had a lower kappa score with abbott fish than ventana or zytovision assays. which is the best method to measure multiple breast cancer? m. boros * , c. marian, o. pop, s. stolnicu * umph targu mures, pathology, romania the size of the breast tumor is relevant in a patient s management also affecting the prognosis. for unifocal lesions, tumor staging depends on the maximum diameter of the tumor, whereas in multiple lesions, this issue is not standardized. the aim of this paper is to study which is the best method in the assessment of the tumor size in multiple invazive carcinomas (multifocal and multicentric) in correlation with the lymph node metastases developement. two different assessments of the tumor size (diameter of the largest focus=ld, and combined, aggregate diameter of all the foci=ad) were used in primary invasive breast lesions ( multiple, unifocal) and compared with the nodal status (chi-test). the use of combined tumor focus upstaged ( . %) patients with multiple tumors ( upstaged from pt to pt and from pt to pt ). there was no difference in nodal positivity based on pt status between ld and ad. we observed a statistically significant difference in the mean diameter of the largest focus between the unifocal and multifocal group ( , vs , mm) (p= , ). aggregate diameter in multiple breast cancer is not correlated with an increase of axillary metasases and should not be used for staging. central nervous system metastases in women with invasive breast carcinoma, not otherwise specified, are associated with estrogen receptor status e. cambruzzi * , a. g. reginatto, c. g. zettler, k. l. pêgas, v. grings, j. m. venites, c. a. coelho * ulbra and ufrgs, dept. of pathology, porto alegre, brazil objective: central nervous system metastases (cnsm) from breast cancer (bc) are relatively common and can present as the first site of disease progression. lymph node status and tumor size are regarded as important prognostic indicators for disease-free and overall survival in bc. the aim of this study was to investigate prognostic/predictive pathological data in bc that could define a high-risk group to develop cnsm. method: the authors evaluated female patients with invasive breast carcinoma, not otherwise specified, previously submitted to setorectomy/mastectomy, in order to determine lymph node status, tumor size, histologic grade, estrogen receptor status (er) and immunoexpression of her /neu. of these cases, patients developed cnsm. results: the patients who developed cnsm were younger (median age . ± . years/p= . ) and more likely to have t n disease than patients with no cnsm. the presence of encephalic disease was associated with er (p= . ). lymph node status (p = . ), tumor size (p = . ), histologic grade (p= . ), and her /neu expression (p= . ) were not significant risk factors. conclusion: although the literature data discriminate that her /neu overexpression in bc is related with cnsm, the authors suggests that these lesions can be related to er too. efforts to determine other risk factor for development of cnsm may be warranted. androgen receptors and sex hormone serum levels in breast carcinoma: study of the ordet cohort l. cimetti * , s. sieri, a. m. chiaravalli, n. sahnane, f. sessa, c. riva, c. capella * university of insubria and ospedale di circolo, varese, italy objective: androgens and androgen receptors (ar) are involved in breast cancer (bc) pathogenesis. high testosterone serum levels increase the risk of developing mainly er + bc, especially after menopause, although androgen role in tumor progression is not clearly elucidated. method: correlations between serum sex hormones and clinico-pathological features of bc arisen among , women previously recruited for ordet study were investigated. prediagnostic estradiol, testosterone (free/total) and shbg serum levels were available. immunohistochemistry for ar was evaluated along with er, pr, her- and mib- . results: ar + was found in . % of bc. higher estradiol (p= . ), free testosterone (p= . ) and shbg (p= . ) were seen in premenopausal patients. in dead patients higher free testosterone was observed (p= . ). no correlation was found for hormone levels vs stage, histotype and grade. higher shbg was seen in pr-rich tumours (p= . ). her- +/er-cases showed a trend for a higher total testosterone (p= . ) and shbg (p= . ). testosterone, estradiol and shbg were similar in ar + and ar-tumors. among triple negative, ar + tumours showed higher free testosterone (p = . ). bc with few ar + cells showed higher total testosterone (p= . ) and a worse outcome (p= . ). conclusion: our results confirm the role of ar in bc and suggest the androgen involvement in tumour progression. mitotic count in metastatic breast carcinoma to lymph nodes: preoperative study a. córdoba * , l. gomez, f. vicente, i. amat, c. llanos, d. guerrero * hospital navarra, ap, pamplona, spain objective: axillary lymph node metastasis (alnm) is one of the most important prognostic factors in breast cancer. the reasons why tumours are capable to result in axillary metastasis remain unclear. the evaluation risk of alnm would improve the treatment planning. we study the metastatic breast carcinoma to the lymph node to obtain information about the metastatic risk. method: we study patients with metastatic breast carcinoma to the lymph node diagnosed preoperatively by needle biopsy. we study the mitotic average (× hpf), metastasis size, positive lymph nodes, total lymph node studied, lymph node ratio (nº of positive node/total nodes nº), primary tumour size and grade. results: all the cases with high mitotic count were associated with macrometastasis. we didn't find any relation between the mitotic rate or the metastatic size with the number of lymph node affected, and the lymph node ratio. breast carcinomas g , t , < mitosis/hpf lymph nodes showed - % of positive lymph nodes. conclusion: we tried to obtain information from the metastatic breast carcinoma to the lymph node to predict the axillary status. in our cases we couldn't predict the lymph node involvement based in the tumour size, grade, metastatic size, and metastatic mitotic rate. the preoperative study of a breast tumour and their lymph node metastasis don't allow predicting the lymph node status in our series. ps- - rassf a hypermethylation is associated to the presence of tumoral cells detected by one-step nucleic acid amplification (osna) a. córdoba * , f. vicente, n. perez janices, j. perez vizcaino, e. gochi, n. torrea, d. guerrero * hospital navarra, ap, pamplona, spain objective: one-step nucleic acid amplification (osna) is used in routine clinical use for sentinel lymph node biopsy (slnb). it consists of the molecular quantitation of a tumoral marker (cytokeratin-ck mrna). gene hypermethylation is one of the most common mechanisms of inactivation of suppressor genes. rassf a gene, is a region frequently hypermethylated in breast cancer. method: patients with breast cancer were included in the study, and a total of lymph nodes were analysed. results: %, . % and . % of the tumours were of low, intermediate and high-grade, respectively. in . % of the cases there are not tumoral cells, in contrast to . %, . %, . %, . % that showed low number of cells/itc (< copies/sample), micro-(< copies/sample) and macro-mtx, respectively. there is a very clear association between rassf a hypermethylation and the presence of tumoral cells (p= . ), being more frequent in macro-mtx compared to the rest of groups (p= . ). rassf a hypermethylation is also correlated to unfavorable histologic grade in the tumour (p= . ) and lymph node involvement at the diagnosis ( . ). conclusion: the analysis of rassf a hypermethylation could provide additional information to osna to detect tumoral cells in lymph nodes. the spare tissue material derived from osna could be good material to consider new molecular studies. results: of these, patients ( , %) had palpable tumor at presentation, ( , %) with palpable axillary adenopathy. in cases ( , %) the tumor diameter was over cm at the time of diagnosis. in cases ( , %) the histological type was infiltrative ductal carcinoma, with cases having high grade. most of the tumors expressed er and pr and were her- negative (none was triple negative). however, in cases ( , %) the ki- index was more than %. conclusion: young women with breast cancer have been shown to have a poorer prognosis because of the high grade and hormonal status. our study shows some similar aspects, but the hormonal status is totally different from the dates in the literature, revealing a possible better response to hormonal treatment and a better survival. immunohistochemical predictive markers for trastuzumab resistence a. cuesta diaz de rada * , e. honrado franco, m. baltasar moreira, f. m. izquierdo garcia * hospital de león, anatomía patológica, spain objective: her positivity defines a clinically challenging subgroup of patients with breast cancer with variable prognosis and response to therapy. the main aim of this study is to identify immunohistochemical markers to predict trastuzumab resistence. method: tumours from patients with invasive ductal carcinoma, who were previously treated with trastuzumab, were included in a tissue microarray and stained for er, ki , p , cyclin d , p and for her and sish (silver in situ hybridization). results: after years of follow up, % of patients with p negative tumours were disease free (p . ) and % were alive (p , ) and the patients cyclin d positive, % were alive (p . ) and % were disease free (p . ). among her +, % did not amplify (polysomy ). moreover, % of treated patients were her − + and did not amplify by sish. conclusion: the best immunohistochemical markers to predict a good response to trastuzumab treatment were p negative and/or cyclin d positive. all her positive + tumours should be confirmed by hybridization, since polysomy is found in %, and these tumours should not be treated with trastuzumab. variations in the immunostaining techniques could induce trastuzumab treatment to patients with her non amplified tumours. ps- - cd expression in breast carcinoma p. czapiewski * , j. szade, a. zaczek, m. welnicka-jaskiewicz, w. biernat * medical university of gdansk, dept. of pathology, poland objective: cd is a membraneous protein that is expressed widely among various soft tissue tumors. there is growing evidence that its expression in some carcinomas correlates with epithelial to mesenchymal transition (emt) and is a poor prognostic factor. its importance in the breast carcinoma remains unsettled. method: the analysis was performed on breast cancer samples from patients. tumors were graded histologically according to the nottingham system. cd expression was scored by grading system used for her- . only cases showing grade ( +) were regarded as positive. additionally, expression of estrogen (er) and progesterone receptor (pr), her- , e-cadherin, vimentin, twist, ki- , c-myc, cyclin d and topoisomerase alfa was performed. expression of the cd was correlated with all these markers and with clinical outcome. results: expression of cd was observed in patients and correlated significantly with negative pr status (p= , ), higher histological grade (p= , ), positive twist (p= , ) and topoisomerase alfa expression (p= , ). there was also a trend toward higher frequency of cyclin d positivity (p= . ). no impact on prognosis for cd expression was found. conclusion: expression of cd correlates with high histological grade, negative progesterone receptor status and expression of certain emt and proliferation markers, but it does not influence prognosis in breast carcinoma. primary plasmacytoma of the breast: a case report l. de carvalho * , g. g. monteiro, l. casagrande, t. ricci, t. lebrão, v. tarricone, p. dinamarco * centro universitário lusíada, dept. of pathology, santos, brazil objective: extramedullary plasmacytomas of the breast are extremely rare, especially those that are not associated with multiple myeloma and they can mimic mammary carcinoma. method: case report: -year-old woman presented a palpable mass in the left breast previously diagnosed as a lobular carcinoma by core biopsy performed in other service. in our institution a conservative surgery with lymph node dissection was proposed. results: frozen section was required and the gross examination showed a . × . × . cm firm-elastic nodule with surgical margins free of tumor and the microscopic examination showed tumor cells with abundant cytoplasm with hyaline appearance. the paraffin sections showed a proliferation of plasma cells with moderate atypia. the immunohistochemical study confirmed the diagnosis of plasmacytoma. there was no axillary lymph node involvement. there were no bone lesions in the additional investigation. conclusion: primary plasmacytomas of the breast are extremely rare and have to be included in the differential diagnosis with breast carcinomas especially in material of core biopsy. sentinel lymph node in breast cancer: form morphology to molecular examination l. di tommaso * , b. fernandes, b. fiamengo, c. navligu, p. spaggiari, s. manara, c. a. garcia etienne, g. masci, a. testori, c. tinterri, m. roncalli * irccs istituto clinico humanitas, dept. of pathology, rozzano, italy objective: sentinel lymph node (sln) examination is a standard in breast cancer treatment. it can be performed on formalin-fixed paraffin-embedded material (ffpe) or on frozen sections (fs). ffpe or fs suffer two drawbacks: ) partial examination; ) operator's dependence. to avoid these limitations, a molecular technique (osna) targeted to quantify a tumoral fingerprint (ck ), has been introduced. our aim is to compare the performance of ffpe, fs and osna. ps- - her assessment in invasive breast cancer using ihc and fish: results from consecutive cases p. drev * , b. gazic, j. contreras * inst. of oncology, dept. of pathology, ljubljana, slovenia objective: her in invasive breast cancer (ibc) should be assessed according to recommended algorithms employing ihc as screening tool and ish only in equivocal cases. therefore there is little data on possible missdiagnosis in discordant cases. method: her was assessed by both ihc and fish in consecutive ibc. distribution of ihc and fish scores, incidence of her + ibc, concordance and level of amplification in discordant cases were analysed. results: ihc distribution: neg( ) . %, neg( +) . %, equivocal( +) . %, pos( +) . %. fish distribution: nonamplified . %, amplified . %, equivocal . % of which in . % ratio was ≥ . . . % ibc were her + and . % were double-equivocal. . % of equivocal( +) were amplified. discordance was infrequent ( . %) (p< . ): among neg( ) and neg( +) , % and , % were amplified, while among pos( +) . % were nonamplified. ihc negative tumors, representing . % of all her +, were amplified, however in . % ratio was ≤ . . conclusion: % ibc are her +. application of two standardised methods results in excellent concordance and enables detection of all her + ibc, while recommended algorithms lead to missdiagnosis in % of her + ibc, but in these amplification is low-level. case report: unusual breast cystic lesion in a -yearold female p. farrajota * , p. cusati, d. esteves, c. dias, c. carvalho, g. falconieri * centro hospitalar do porto, dipt. do anatomia patologica, portugal objective: papillary carcinoma of the female breast may exhibit a broad phenotype. we present an unusual case of a pseudoencapsulated invasive papillary carcinoma featuring transitional cell features. method: a specimen of internal right female breast biquadrantectomy was routinely processed and a panel of antibodies applied on paraffin tissue section. results: grossly, the specimen showed a . cm cystic lesion with fibrino-hematic material and multiple pinkish-grey papillary structures, the largest measuring cm. the histological examination revealed a malignant epithelial papillary proliferation reminiscent of transitional cell carcinoma, with high nuclear grade, frequent mitotic figures, multiple areas of invasion and rare necrotic foci. tumor cells were immunoreactive for p , βe and ki- ( %) and negative for other stains including her /neu, estrogen and progesterone receptors. our differential diagnosis included a metastasis from a primary urothelial carcinoma, an intraductal papillary lesion, a metaplastic carcinoma and a papillary adnexal neoplasm, all reasonably excluded after careful clinico-pathologic evaluation. conclusion: metastatic involvement of the breast is uncommon but it should be considered if tumor phenotype is inconsistent within usual or "special type" breast carcinoma. yet, some rare primary misleading lesions are difficult to recognize. ps- - volume measurement of female sprague-dawley mammary tumors induced by n-methyl-n-nitrosourea: comparing ultrasonography and caliper a. faustino-rocha * , c. teixeira-guedes, j. pinho-oliveira, r. soares-maia, r. arantes-rodrigues, b. colaço, r. ferreira, p. oliveira, m. ginja * utad, dept. of veterinary sciences, vila real, portugal objective: n-methyl-n-nitrosourea (mnu) is a chemical carcinogen frequently used to induce mammary tumors in female rats, which experimental evaluation requires the monitoring of tumor's volume. for this purpose several methods are described, namely: caliper and ultrasonography measurement. the aim of this work was to compare data obtained by caliper and ultrasonography. method: twelve female sprague-dawley rats with . ± . g body weight were intraperitoneally injected with mnu ( mg/kg) at days of age. thirty-six weeks after mnu administration forty-one tumors volume was determined by caliper (vito®) and ultrasonography (logiqp ®, general electric healthcare) measurement. the tumor volume (v) was calculated according to the following formula v=π.〖s_ ^ 〗^.s_ ⁄ , being s_( ) and s_ the tumor diameters (s_( )<s_( )). results: the means tumor caliper and ultrasonography measures were . ± . cm and . ± . cm , respectively. the icc for scoring reproducibility was . ( % confidence interval, . to . ) and paired-sample t-test was significant (p< . ). conclusion: usually mammary tumors in rat are measured by caliper. however, our results showed that there are volume differences between both methods studied. ultrasonographic method is more accurate and more precise than caliper. therefore, we recommend the use of ultrasonography to rat tumor evaluation. objective: there is a significant percentage of patients with breast cancer and her receptor overexpression that do not respond to trastuzumab-directed therapy. the aim of this paper is to study the alterations of one of the her signalling pathways related to trastuzumab response in breast cancer. method: the study comprised cases of breast carcinoma in different stages of evolution with her overexpression (herceptest +) or gene amplification (fish). all patients were treated with trastuzumab. the assessment of pten and pakt expression was based on staining intensity and percentage of stained cells. we also determined the molecular alterations in pik ca. results: our series showed a statistically significant association between a positive expression of pten and the absence of pik ca mutations with better overall survival from treatment onset with trastuzumab (p= . and p= . , respectively). the association with the expression of pakt was statistically significant if survival is evaluated from the pathological diagnosis of the tumor (p= . ), pakt positive tumors had worse survival. objective: expression of mammaglobin a (mg-a) in breast cancer has been reported to be of prognostic value and to be associated to hormone receptor-positivity. mg-a is also useful in the diagnosis of metastatic breast cancer. the present study evaluates the differential mg-a expression in two subsets of primary breast cancer (metastasizing vs. non-metastasizing) and in their correspondent metastases. method: immunohistochemical analysis of mg-a (clone - a ) was performed in non-metastasizing primary breast tumors and paired primary and metastatic breast cancers. a percentage of % of stained tumor cells was considered positive. the expression of mg-a in the different groups, together with other clinicopathologic factors (grade, size, hormone receptors, her , etc.) was analyzed by t-test. results: expression of mg-a was significantly higher in estrogen receptor-positive (er+) tumors (p< . ). a trend was noted for er + metastasizing primary breast cancers to show a higher expression of mg-a, compared to those nonmetastasizing (p= . ). no significant differences were noted in the expression of mg-a between metastasizing primary tumors and their correspondent metastases, with a good correlation (p= . ). conclusion: these results suggest that mg-a might be of prognostic value in determining a subset of er + breast cancers prone to progression. larger series are needed to confirm these observations. concordance of st. gallen intrinsic subtypes in core biopsies and related surgical specimens of breast cancer c. m. focke * , t. decker * dietrich bonhoeffer klinikum, abt. pathologie, neubrandenburg, germany objective: to evaluate the relevance of intrinsic breast cancer subtyping (ibcs) on core biopsies (cb). results: samples were tested in . score was found in . % cases, + in . %, + in . %, + in . %. the amplification was revealed in . % of + cases, . % tumors were +/fish+. cases were tested in : score − . %, + . %, + . %, + . %. the amplification was seen in . % + cases and . % tumors became +/fish+. there was no significant distinction using % and % cells score system for + tumors, whereas for groups + and + it was. conclusion: asco/cap scoring criteria developed in have no effect on her -positive tumors detection but increases number of cases with equivocal result, what require retesting with ish. microvessel density in triple-negative breast cancer: characteristics and prognostic implications h. gobbi * , m. de brot, r. rocha, f. soares * ufmg, dept. of anatomic pathology, belo horizonte, brazil objective: we assessed topography and characteristics of blood vessels in triple-negative breast cancer (tnc) and examined their association(s) with clinicopathological features, basal-like phenotype and influence on patient prognosis. method: blood vessels were assessed in tissue sections from tncs stained for cd . slides were scanned using scanscope® xt and analysed with aperio image-scopetm, microvascular density v algorithm. microvessel density (mvd) was assessed in three areas: intratumoural (it), peripheral tumoural (pp) and peritumoural (pt), and correlated with clinicopathological features and prognosis. results: the majority of blood vessels were located largely at the peripheral tumoural and peritumoural areas. tumours with higher total mvd and pt-mvd were significantly associated with the presence of recurrence (p = . ; . ), distant metastasis (p = . ; . ), hematogenous metastasis (p= . ; . ); and shorter overall survival (os; p= . ; . ) and disease free interval (dfi; p= . ; . ). high pt-mvd correlated with a higher stage at diagnosis (iii and iv; p= . ), lymph node (ln) metastasis (p= . ), basallike phenotype (p = . ), high histologic grade (p = . ), presence of distant metastasis (p = . ), and shorter os (p = . ). conclusion: blood vessels are mainly located at the peritumoural and peripheral areas in triple negative breast cancers and seem to have a major role in tumour progression by representing means for disease dissemination. expression of cd and cd in primary ductal invasive carcinoma of breast and metastatic lymph nodes m. gudadze * , k. kankava, t. javakhishvili, m. sarishvili, g. burkadze * central university clinic, dept. of pathological anatomy, tbilisi, georgian, republic of georgia objective: cancer stem cells are believed to have certain role in progression, metastatic potential and outcome of ductal invasive carcinoma of breast. method: the archived materials of - years from the department of pathological anatomy, n. kipshidze central university clinic were used for the research. cases of ductal invasive carcinoma were chosen for the study. the cd , cd immunohistochemistry (biocare medical) was used for staining. evaluation was performed using spss software. results: the results showed: . % of grade ii cases and . % of grade iii cases were lymph node positive. cd positivity in grade ii tumors was seen in . % of primary site and . % of metastasis; in grade iii tumors- % of primary site and . % of metastasis. cd positive cases in grade ii- . % for primary tumor and % in metastasis, for grade iii- . % for primary tumor and . % for metastasis. the results show that cd positivity is significantly less frequently seen in metastasis than in primary tumor; cd positivity is more frequently seen in metastasis for grade ii cases. this indicates, that cells with stem cell phenotype decrease in metastasis reflecting heterogeneity between primary and metastatic sites, which should be taken into consideration for assessing progression and treatment. ps- - solid neuroendocrine breast carcinoma: three case reports b. ince alkan * , s. yuksel, e. kadan, s. percinel, s. dizbay sak * ankara university, turkey objective: it seems that neuroendocrine breast carcinoma (nebc) is underrecognized in routine practice. world health organization classification of tumors of the breast defined nebc as a subtype of invasive mammary carcinoma in which > % of the tumor cells express neuroendocrine markers. three cases compatible with solid nebc are discussed with an emphasis on identifying features useful in recognition of this tumor type. results: the patients were , and years old women. all patients presented with a palpable mass, two in the right and one in the left breast. microscopically, infiltrating, solid cohesive nesting pattern with delicate sinusoidal vasculature or peliosis and papillary pattern were observed. the tumor cells were round or polygonal with abundant granular, eosinophilic cytoplasm. the nuclei had hyperchromatic/vesicular or "salt and pepper" chromatin. two cases had ductal carcinoma in situ component. all cases were positive for synaptophysin and chromogranin a in > % of tumor cells. while a positive status for estrogen receptor was detected in all cases, progesterone receptor was observed in two cases. none of the tumors displayed her overexpression. conclusion: morphological clues suggestive of nebc must be recognized for immunohistochemical confirmation for appropriate classification of this clinically distinct subtype of invasive breast carcinoma. objective: automation of her fish may improve her gene testing. the aim of our study was to evaluate an automated her fish assay for assessing the her genomic status. method: core biopsies of invasive breast carcinomas were analysed in parallel using the manual pathvysion™ her- dna probe kit and the automated leica her fish system for the bond™ instrument. to assess intermethod agreement, concordance analysis was performed for various numerical and categorical parameters. results: carcinomas with all her immunohistochemical scores were included ( +: ; +: ; +: ; +: ). using either her /cep ratio > . or ≥ . as criterion for her amplification, high levels of concordance were observed between automated and manual fish (concordance rate %, k coefficient . ). high levels of inter-method agreement were also found for her copy number, cep copy number, her /cep ratio, the percentage of carcinoma cells with her /cep ratio > . , and the presence of her genetic heterogeneity, her clusters and cep polyploidy. conclusion: her testing using automated fish is feasible on breast carcinoma core biopsies. automated her fish using the leica her fish system for bond is an alternative to manual her fish in evaluating the her status of primary invasive breast carcinomas. myofibroblastoma of the breast: presentation of three cases e. kairi-vasilatou * , c. dastamani, a. tsagkas, a. paraskeva, a. kondi-pafiti * aretaieio nosokomeio university, dept. of histopathology, athens, greece objective: myofibroblastoma is a rare, usually solitary, benign spindle cell tumour composed of myofibroblasts. it affects both genders equally. method: three cases diagnosed in our laboratory are presented. results: two patients were postmenopausal females ( and years old) and one was male ( years old). the tumour was , cm in greatest diameter in the first case, cm in the second and cm in the third. microscopically, all neoplasms were circumscribed and consisted of uniform, bland spindle cells separated by broad bands of hyalinized collagen. the neoplasmatic cells had abundant eosinophilic cytoplasm, oval nuclei and were arranged in fascicles. cellular atypia was found only in one case and mitoses were scarce. immunohistochemically, all neoplasms were strongly positive for vimentin, cd and bcl- , while the expression of desmin and sma varied. s- protein was negative. the neoplasms of the female patients were strongly positive for er and negative for pgr. the stains for estrogen and androgen receptors were not carried out in the case of the male patient. conclusion: differential diagnosis can be complex. fibromatosis of the breast, nodular fasciitis, myoepithelioma, myofibrosarcoma, solitary fibrous tumor, inflammatory myofibroblastic tumour should be excluded based on the histological and immunohistochemical findings. method: using light-and transmission electron microscopy fragments of mammary gland tumors, received intraoperative, from patients - years with infiltrative ductal breast cancer were studied. results: most of the mast cells were localized in the areas of infiltrative growth, at a background of lymphohistiocyte infiltration and in peripheral part of the tumor. intratumourous mast cells were totally degranulated. the mast cells with intact structure at a background of cells and tissue destruction were also in mammary gland. in destructive mast cells the nucleus with electron-dense circle and chain of granules were observed. on transmission electron microscopy their deformations and the decrease of density were revealed. the mast cells were of elongated of rounded shape with large nucleus and invaginations of karyolemma. the nucleus occupies almost completely the cell's area. it has a ring of large but pale granules. small protuberances were observed on the surface of the cell membrane. their number decreased in direct proportion to the size of must cells and their remoteness from microvessels. such protuberances are necessary for mast cells in moving. destructively changed cells with the signs of swelling, vacuolization and clasmatosis were frequent in breast cancer. objective: determination of eligibility of breast cancer patients for treatment with anti-angiogenic drugs has been always considered as a challenge for oncologists. each mutation in the genes of proliferation phase enhances the angiogenesis of tumor. we aimed to determine the effect of concurrent mutations of her- and tp on angiogenesis. method: women affected by invasive ductal carcinoma (idc) sporadic breast cancer were included. immunohistochemical study was performed with her- , tp , ki- and annexinv markers. angiogenesis index was semiquantitatively calculated by mvd-cd technique. statistical associations between parameters were evaluated. results: prevalence of her- positive and tp positive cases were . % and . %, respectively. . % of patients showed concurrent mutation of these genes. concurrent mutation led to significant increases in both angiogenesis and proliferation and a significant decrease in apoptosis. there was no statistically significant association between concurrent mutation and tumor grade. conclusion: this study demonstrates that most of the tissue prognostic factors are poor in concurrent mutation of both genes. also, our study illustrates that the concurrent mutation correlates with a higher angiogenesis and thus an increased risk of recurrence. we can conclude that in priority setting for administration of anti-angiogenic agents, patients with concurrent mutations are more eligible. adenoid cystic carcinoma in male breast: a case report e. kimiloglu sahan * , u. karinoglu, a. akyildiz igdem, n. erdogan * taksim´s hospital, dept. of pathology, istanbul, turkey objective: adenoid cystic carcinoma of the breast is a rare variant of breast cancer that accounts for . % of all breast carcinomas and occurs commonly in women between the ages of to . in the literature, only a few examples have been reported in men. it is well-differentiated tumour with favorable prognosis and generally presents as a painful breast mass. method: here, we are presenting a years old male patient with a × mm diameter solid mass on left breast retroareolar region. on fine needle aspiration biopsy, there were 'atipical proliferating ductal epitelial groups'. then, hookwire localization and excision have been performed. microscopically, there were tubulary and cribriform islands composed of basaloid type cells with eosinophilic cytoplasm and myxoid material in the cribriform spaces at the center of the islands. we used immunohistochemical markers such as p , s , ck , cd , ck and smooth muscle actin for differential diagnosis. results: the diagnosis was 'adenoid cystic carcinoma'. the margins of the tumor were positive, so radical mastectomy was performed for treatment. conclusion: because adenoid cystic carcinoma of the breast in male is a very rare example, we present our case here. comparison between the bond oracle her immunohistochemical system, the polyclonal her dako antibody and chromogenic in situ hybridization in breast carcinoma h. kourea * , v. tzelepii, i. nikolatou, p. ravazoula, v. zolota * university of patras, dept. of pathology, greece objective: the sensitivity and specificity of immunohistochemical (ihc) methods for her testing are very important given the therapeutic implications. this study compares the concordance between oracle and the her dako polyclonal antibody (her ), in breast carcinomas (bcs) that were equivocal ( + or not evaluable/ne), by her staining, considering as gold standard the chromogenic in situ hybridization (cish). method: bcs (n= ), problematic by her staining ( − / + or ne), and studied by cish in our institution, and additional consecutive bcs were stained with her and oracle, and scored separately by three pathologists. consensus scoring for each ihc method and cish results were recorded. descriptive statistics and measurement of the cohen's kappa coefficient were performed. results: the overall agreement between the tests in a × analysis shows a concordance in % of cases (κ= . ). among the cases studied by cish, equivocal were and cases, for her and oracle, respectively. seventeen her equivocal cases were negative with oracle and cish. in problematic cases, using cish as gold standard, the sensitivity, specificity, positive and negative predictive values for her and oracle were , . , . , and , . , . , , respectively. conclusion: in problematic cases, oracle testing shows higher specificity and positive predictive value. stromal p and ki expressions of the mammary phyllodes tumors: are they the clues in determination of tumor grade? u. kucuk * , u. bayol, e. e. pala, s. cumurcu * tepecik training hospital, dept. of pathology, izmir, turkey objective: conventionally growth pattern, stromal overgrowth, stromal cellularity, stromal mitotic activity are the main parameters in grading of phyllodes tumors. recent studies revealed that p and ki expressions are both correlated with grade of phyllodes tumors of the breast. method: we searched for p and ki expression rates of benign and malignant phyllodes tumors in our archival data and correlated them with conventional parameters such as stromal cellularity and mitotic activity rates. benign, malignant phyllodes tumors were reevaluated as regards stromal cellularity (low/moderate and high), mitotic activity (low and high), p expression (low, moderate, high), ki expression (low and high) rates. statistical correlation amongst the whole parameters were searched with chi-square test. results: stromal cellularity, mitotic rate, p and ki expression rates were all closely correlated (p = . - . ) for benign and malignant histologic subgroups. ki expression was significantly correlated with histologic subgroups, stromal cellularity and mitotic rate (p= . - . ). similarly p expression was correlated with histologic subgroups, stromal cellularity and mitotic rate (p= . - . ). conclusion: both ki and p expression rates are statistically significantly correlated with grade of mammary phyllodes tumors, so they can be used in determination of tumor grade, especially for differential diagnosis of benign and malignant ones. quantitative measure proliferative markers by image analysis of invasive ductal carcinoma a. kudaybergenova * , s. kalantarli * rscrct, dept. of immunohistochemistry, st. petersburg, russia objective: we analyzed a total number of tumor cells) in invasive ductal breast carcinoma, proliferative activity (% ki -positive cells) and mitotic index (% phh positive cells) to establish absolute quantity tumor cells per sq.mm of histological slide and relations this measure with proliferation and mitosis. method: the study included patients diagnosed with breast carcinoma from baku oncology hospital during the - . after whole slide scanning by mirax scanner ( dhistech, budapest) of he, ki and phh stained slides we juxtaposed all three slides in one screen to the found area with maximal ki level in tumor and the corresponding area in other slides. morphometric analysis was performed using the pannoramic viewer software ( dhistech, budapest). for each case we analyzed a total number of tumor cells in mm , number of ki and phh positive cells. results: mean tumor cells in mm of histology slide was +\− cells, median - cells, +\− ( %) were positive for ki and +\− ( %) were in mitosis. there was moderate correlation between cell density and ki r= , (p= , ) and phh r= , (p= , ). conclusion: by analysis of breast cancer, was established a total tumor cell per mm and main proliferative characteristics for invasive ductal carcinoma. objective: adenomyoepithelioma of the breast is a very rare benign tumor with biphasic proliferations of epithelial and myoepithelial elements. it is morphologically and immunohistochemically identical to epithelial-myoepithelial cell carcinoma of the salivary gland. the histologic criteria of malignant ame is not well-established because of the rarity of ame. we report a case of malignant adenomyoepithelioma in a -year-old woman. method: on ultrasonography, a well-marginated and lobulated solid mass was found at liq of the right breast.. an ultrasono-guided core biopsy was performed. the diagnosis of core biopsy was myoepithelial lesion. she subsequently underwent a wide local excision of the lesion. results: microscopically the tumor mass was composed of biphasic patterns which showed formation of tubules lined by an inner layer of ductal epithelial cells surrounded by proliferation of myoepithelial cells that also formed solid nests. but this tumor had foci of infiltrating margins and proliferation of spindle cell components. also noted are numerous mitotic figures and increased mylepithelial ki- positivity ( - % of tumor cells). conclusion: breast lesions which have predominently myoepithelial cells can be divided into myoepithelial hyperplasia, adenomyoepithelioma and malignant adenomyoepithelioma. tavassoli divied ame into tubular, papillary, and solid subtypes. the criteria of malignant ame is not wellestablished but some criterias can apply to make a diagnosis of malignant ame. objective: neoadjuvant chemotherapy (nact) is available for patients with breast carcinoma. however, resistance to chemotherapy is still a main cause of mortality. method: differentially expressed genes were identified from previously published studies that examined chemoresistant and chemoresponsive cell lines or patients with breast carcinoma. the expression of selected gene products was assessed in tissue microarray slides comprising post-nact resection specimens from breast carcinoma patients using immunohistochemistry, and analyzed according to the molecular subtype and residual cancer burden (rcb) grade. results: most cases were positive for abcb ( . %) and myc ( . %), but negative for top a ( %). rcb-ii cases expressed much higher levels of muc (p= . ) and clu (p= . ) than rcb-iii cases. positive expression for calr (p= . ) and lgals (p= . ) was observed more often in triple negative types than in luminal types, and cytoplasmic cdkn b expression was observed more often in luminal types (p= . ). conclusion: positive expression of abcb and myc and negative expression of top a in the residual carcinoma after nact implies general resistance to nact. expression of calr, lglas and cytoplasmic cdkn b may be associated with resistance depending on the subtype. expression of muc and clu can be used to predict the rcb grade or response to nact. primary and metastatic melanoma of the breast-review of cases i. liepniece-karele * , l. osipova, m. sperga, s. isajevs, a. grjunbergs, j. eglitis * riga ecuh, dept. of pathology, latvia objective: primary breast melanoma is a very rare tumour accounting for < % of all malignant melanomas. the malignant melanoma can be with different manifestation in the breast (primary breast tissue or primary breast skin melanoma as well as metastatic melanoma). method: in this study cases of breast melanoma were identified from our records of the past years ( . % of all breast cancer cases). a histological and an immunohistochemical (ihc) study was performed using antibodies against ckae / , hmb- and melan-a on both the biopsy and operation material. results: obtained results showed that in cases pigmented, epitheloid and spindle cell melanoma, but in one case epitheloid cell amelanotic melanoma was found. by ihc the melanoma cells expressed hmb in three cases, melan-a and s in all cases. no expression of ckae / was observed. conclusion: careful histological and immunohistochemical examination of malignant tumour is essential for adequate diagnosis, follow-up and treatment of breast melanoma. stem cell expansion in ductal carcinoma in situ of breast c. lopes * , a. paula, o. marques, a. rosa, a. rema, f. carvalho * icbas, dept. of pathology, porto, portugal objective: the aim is to study the ability of a cell marker panel -aldh , cd and ki to identify breast stem cells in no malignant and ductal carcinoma in situ. method: double-color triple-immunohistochemistry -to aldh , cd and ki -was done in paraffin embedded tissue specimens from patients arrayed in tissue microarray blocks. statistical was done using chisquare probability test: differences were considered significant when p< . . results: significantly higher immunoreactivity was seen in dcis than in benign lesions of breast (p< . ) with used markers. in a total of specimens, cd +/aldh +/ ki -cells were identied in cases. the distribution was as follows: dcis ( / ); fibroadenoma ( / ), atypical hyperplasia ( / ); other benign lesions ( objective: data from in vitro and clinical studies suggest that casr expression can be associated with the development of bone metastases. most probably casr stimulates production and secretion of pthrp via egfr pathway. the aim of our study was to assess expression of casr in the primary breast cancer and correlate it with the risk of bone metastases. method: we have analysed patients with the breast cancer. bone metastases were diagnosed in cases, patients died without skeletal involvement (control group). casr expression was assessed in primary tumors using tissue microarray (tma) and immunohistochemical technique (polyclonal antibody pierce bio. pa - ). to evaluate cytoplasmatic casr expression we have used - point scale in which was defined as uniformly strong or medium staining in more than % of tumor cells. results: strong or medium staining in more than % was identified in most studied cases, however it was more predominant in patients diagnosed with bone metastases than in the control group ( , % vs , %), p= , . conclusion: expression of casr is common in primary tumors of patients with disseminated breast cancer irrespectively of the metastatic site. however, the patients with bone involvement have higher rate of expression, that is borderline significant statistically. as a result, a group of patients with very high risk of bone dissemination might be separated. bcl expression is associated with centromere alterations in luminal b breast cancer a. matsionis * , i. pavlenko, a. petrov * rostov refional institute, dept. of experimental pathology, rostov regional institute objective: bcl is an important established prognostic parameter in human breast cancer (bc) and chromosome centromere (cep ) copy number is proposed to be the same too. we evaluated bcl expression in different bc's molecular subtypes in relation to increased cep level (cep > per nucleus). method: immunohistochemistry for bcl assessment and fluorescence in situ hybridization for detection of cep alterations were used. statistical analysis was performed with fisher's exact test. results: a total of cases of female invasive bc's were analyzed ( ) ( ) . the tumour subtypes were as follows: luminal a (er/pr+, her /neu-, ki < %) - ; luminal b (er/pr+, her /neu-, ki > % or er/pr+, her /neu+) - ; her /neu+(er/pr-, her /neu+) - ; triple-negative - . in our study group, bcl -cases were preferentially er-in agreement with previous reports. however, , % tumours of luminal b subtype were bcl -too and all of them had increased cep copy number (p= , , rφ= , ). we therefore hypothesized that not only er had influenced bcl in luminal b bc but cep alterations also. conclusion: centromere alterations are associated to bcl downregulation in luminal b bc's. the mechanisms responsible for that remain to be established but the underlying cause could be promoter methylation or transcriptional repression. outcome of excision of radial scar diagnosed on core biopsy: a single centre analysis f. menezes * , m. caldas, n. coimbra, c. leal * ipo porto, dept. de anatomia patologica, portugal objective: radial scar (rs) is a sclerosing lesion of the breast which may be associated with a spectrum of epithelial proliferative lesions and carcinoma. it is frequently subject to biopsy when presenting as a mammographic abnormality. the need to excise rs diagnosed on core biopsy (cb) remains controversial. the aim of our study is to determine the frequency of upgrade in diagnosis after excision of rs. method: a retrospective study of rs diagnosed on cb in our department between / / and / / was performed. results: cases were retrieved, all women, with a median age of years. of these, pairs of cb/resection specimens were obtained. cb diagnosis was rs without atypia in cases, rs with atypia in cases and rs with in situ carcinoma in cases. after excision, / ( , %) cases diagnosed as rs without atypia were upgraded: to atypia, to in situ carcinoma and to invasive carcinoma. of those cases diagnosed on cb as rs with atypia, / ( , %) were upgraded: to in situ carcinoma and to invasive carcinoma. conclusion: our results support the excision of all lesions diagnosed on biopsy as rs. *fm and mc are joint first authors. outcome of excision of papillary lesions diagnosed on core biopsy: a single centre analysis f. menezes * , m. caldas, n. coimbra, c. leal * ipo porto, dept. de anatomia patologica, portugal objective: papillary lesions of the breast (plb) comprehend a spectrum of entities with different morphologies and malignancy risk, which present a diagnosis challenge on core biopsy (cb). although diagnostic accuracy has improved with immunohistochemistry, the need to excise benign plb remains controversial. the aim of our study is to determine cb diagnosis accuracy in plb, and subsequent need to excise all lesions. method: a retrospective study of plb diagnosed by cb in our department between / / and / / was performed. results: cases were identified, with a median age of years. cb diagnosis was benign in cases, some kind of atypia found in , and malignant lesions ('in situ' and encysted/invasive types of carcinoma) in . surgical excision was performed on cases, including all cases with carcinoma. following excision, / and / benign cb results were respectively upgraded to atypical and malignant; / with atypia were upgraded to malignant. overall, % ( / ) of benign and atypical plb cb diagnosis therefore missed malignancy. conclusion: our results show that, despite good correlation between cb and excision diagnosis, some cases of carcinoma are missed on cb, so the excision of all plb remains advisable. *fm and mc are joint first authors. myoepithelial carcinoma of the breast: a case report i. michalopoulou manoloutsiou * , b. christoforidou, p. xirou, v. bostani, e. goupou, i. themeli, f. patakiouta * theagenion hospital thessaloniki, dept. of pathology, greece objective: myoepithelial carcinoma of the breast is an extremely rare tumor, composed purely of myoepithelial cells, predominantly spindle, with identifiable mitotic activity. method: we report a case of a year-old female patient with a palpable, well demarcated lump in her left breast, measuring , cm in its maximal diameter. surgical excision and axillary lymph node dissection were performed. results: histologically, the tumor displayed an infiltrating growth pattern and consisted of spindle cells, that appear to emanate from myoepithelial cells of ductules entrapped in the center of the lesion. mitotic activity did not exceed mitotic figures/ hpf. immunohistochemically, the tumor cells revealed positivity for p , cytokeratin / , cytokeratin βΕ , smooth muscle actin, cd and s protein, whereas they were negative for desmin, cytokeratin , cd , hmb , estrogen, progesterone receptors and her- oncoprotein. approximatelly % of tumor cells showed nuclear positivity for mib- /ki- . all axillary lymph nodes were free of metastases. conclusion: myoepithelial carcinoma of the breast is a potentially highly aggressive neoplasm and its differential diagnosis is fairly broad, including metaplastic spindle cell carcinoma and a variety of myofibroblastic lesions. objective: we investigated the differential expression of several biological markers between primary invasive breast carcinomas and their paired lymph node metastasis analyzing separately epithelial and stromal components. method: representative samples of idc and paired compromised lymph nodes were arrayed in a tma and selected markers: hormonal receptors, her- , proliferation (p , pakt, pmtor, tgfβ ) , motility (cd and cxcr) and basal markers (ck , ck , c-kit) were evaluated by ic. results: in the primary tumor, p , mib- , tgfβ , cd and cxcr were more expressed in epithelial cells (p< . ), while pakt, pmtor, c-myc and c-kit showed a similar frequency in both components. hormone receptors, her- /her- and cytokeratins were not expressed in stromal cells. the proliferative biomarkers were concordant in the epithelial component. cd frequency was similar but cytoplasmic cxcr as opposite to nuclear was predominantly expressed in lymph nodes (p= . ). stromal cells from lymph node showed a reduced frequency of cd (p= . ) and c-myc (p= . ) when compared to the stromal component of primary tumors. conclusion: epithelial cxcr expression may facilitate lymph node metastasis whereas the low frequency of cmyc and cd in the lymph node stromal component indicated decreased proliferation enhanced motility of stromal cells in this site. expression of hypoxia-inducible factor- a and associations with vascular endothelial growth factor expression, high microvessel density and features of aggressive tumors in african breast cancer h. nalwoga * , j. b. arnes, h. wabinga, l. a. akslen * university of bergen, the gade institute, norway objective: breast cancer in africans is reported to have poor clinical outcome. whereas hypoxia-inducible factor- α (hif- α) expression has been linked to treatment failure and poor prognosis in breast cancer, there is a lack of reports about hif- α expression in africans. the aim of this study was to evaluate hif- α expression in relation to vascular endothelial growth factor (vegf) expression, angiogenesis, and other tumor characteristics in an african population. method: in total, we analyzed breast cancers by immunohistochemical staining. we determined microvessel density (mvd), proliferating microvessel density (pmvd), and vascular proliferation index (vpi) in the most vascularized areas as well as expression of hif- α and other biomarkers using tissue microarrays. results: expression of hif- α (in / tumors; %) was associated with vegf expression (p< . ), mvd (p = . ), high tumor grade (p= . ), high ki- proliferative rate (p< . ), and p expression (p= . ). conclusion: there is a high expression of hif- α in this series of breast cancer which is strongly associated with vegf expression and increased mvd. more studies are required to assess the therapeutic implications of hif- α expression in this population. the patient underwent lumpectomy. the gross specimen had a tan grey firm nodule of × × mm. histological examination revealed a proliferation made of signet ring cell and glandular structures, with islands of goblet cells. immunohistochemistry revealed strong positive staining with ck and ck , and a sparse positive staining with synaptophysin and chromogranin.er, pr and her were negative. the patient underwent appendicectomy with a final diagnosis of breast localization of a primary occult appendiceal gcc. conclusion: the differential diagnosis between primary carcinoid tumor of the breast and signet ring cell carcinoma metastatic to the breast is often controversial in surgical pathology. diagnoses need to be made correlating clinical and histological examination in difficult cases in which there is not a diagnosis of carcinoid tumor elsewhere. their histological appearance may mimic ductal adenocarcinoma of the breast. the distinction is important due to differences in management and prognosis. determination of her gene amplification by chromogenic in situ hybridization (cish) in breast carcinoma m. neagu * , c. ardeleanu, g. butur, a. florin * national institute of pathology, bucharest, romania objective: detection of her neu amplification is an integral part of breast carcinoma diagnostics to decide therapy. method: we study cases of breast carcinoma embended paraffin tissues, both cish and fish were performed on each case using (spot-light her ) for cish and both her- and chromosome probes for fish (vysis). sixty tumor cells were evaluated in each case. the scoring system and interpretation of cish -invitrogen. results: concordance between cish and fish was found in , % cases, considering fish as gold standard, sensitivity of cish was . % and specificity %. cish is more practical alternative due to lower cost, no requirement of fluorescence microscope, use of existing bright-field microscopy and techniques it s similar to ihc, archivable and quantitative results, it s easy to observe both the tissue morphology and the gene amplification evaluation of the cases analyzed, showed similar results for both methods. two cases were discordant. in these cases, low-level amplification was suggested by cish but nonamplification by fish. conclusion: our results, suggest that cish is a useful technique to determine her- /neu oncogene status, in breast carcinoma for paraffin embendded tissues, is a highly accurate, reproducible and practical technique, with a high sensitivity. usual ductal hyperplasia with central necrosis, microcalcifications and multiple foci of pseudoinvasion arising in a radial scar: a potential diagnostic pitfall a. nechifor-boila * , s. stolnicu * university of medicine and pharmacy, tirgu-mures, romania objective: the diagnosis of radial scar (rs) is often difficult, especially when associated with proliferative changes, pseudoinvasion and necrosis. method: a -year-old woman was referred to the surgery department for a palpable mass in her left breast, regarded as a possible fibroadenoma on ultrasonography. results: lumpectomy was performed and the macroscopy revealed a tan-white mm diameter lesion, with irregular margins and firm consistency. on light microscopy, a sclerosing lesion, with a stellate arrangement of ducts surrounding a central fibro-collagenous zone was identified. haphazardly arranged, distorted ducts associated with massive intraductal proliferation, highly suspicious of infiltrative were also present in the center of the lesion. the proliferating ducts showed slit-like, irregular, secondary lumens, several containing central necrosis and a heterogenous cell population. the presence of central necrosis and pseudoinvasion were, however, worrisome. immunohistochemistry for p demonstrated the presence of myoepithelial cells surrounding all the areas with pseudoinfiltrating features, while ck / displayed a heterogenous, mosaic-like positivity, characteristic for usual ductal hyperplasia (udh). conclusion: because pseudoinvasion in benign rs can easily be misinterpreted as invasive carcinoma, immunohistochemistry is mandatory to establish the presence of myoepithelial cells. although rare, central necrosis may occur in udh and should not be used as a single diagnostic criterion of malignancy. phenotypical and morphological heterogenity of breast cancer: our experience j. nieslanik * , j. dvorácková, m. uvírová, Š. laciok, r. ondruššek, d. Žiak * cgb laboratory a.s., dept. of pathology, ostrava, czech republic objective: breast cancer is the most frequent malignant tumor in women with a rising incidence. about new cases are diagnosed in czeck republic every year, up to % die on it´s account. in cgb laboratory are investigated of malignant breast tumors every year. we evaluate the morphology, grade of differentiation and phenotype properties-hormone receptors expression, overexpression and amplification of her- /neu gene. % of carcinomas posses significantly morphologically and phenotypically heterogenic tumor population in one and the same tumor leasion. results: the most often sign of phenotype heterogenity found is hormone receptors expression or overexpression of her- /neu gene. often we see two morphologically different tumor populations coexisting. the most interesting cases from our practice are on the poster. conclusion: as for now, studies about the origin of heterogenity inside the same leasions, did not find a single theory to resolve it. a possible answer gives the theory of tumor stem cells and the model of clonal evolution. our observations demonstrate and may explain the practical reason of different phenotype properties found in core cut biopsy where one population may be held, against the heterogenic properties of the tumor shown when resected as a whole. high grade infiltrating carcinoma with squamous features -a case report d. r. novac * , c. ardeleanu, s. taban, f. cadariu, a. dema * municipal clinical hospital, anatomical pathology, timisoara, romania objective: apocrine carcinoma is a rare and histologically distinct type of invasive breast carcinoma. method: the patient, a years-old woman was admitted to the hospital for a tumor mass in the upper internal quadrant of the left breast. tissue fragments from the quadrantectomy specimen were routinely processed by paraffin embedding. for the immunohistochemical (ihc) study we used the following antibodies: ck , androgen receptors (ar), estrogen receptors (er), progesterone receptors (pr), high molecular weight cytokeratin (hmwck), p , gross cystic disease fluid protein- (gcdfp- ), en vision system, visualization with diaminobenzidine. results: the gross examination revealed a nodular, well delineated mass of , / , / cm, firm, gray-beige with areas of necrosis on cut section. microscopically, the tumor had a predominantly solid, partially cystic and less tubular growth pattern. the tumor cells had abundant eosinophilic granular cytoplasm and pleomorphic high-grade nuclei. a small tumor contingent presented squamoid features. ihc stains showed the following profile: ck +, ar+, gcdfp- +, er-, pr-, p /hmwck + (focal/zonal). a diagnosis of high-grade infiltrating apocrine carcinoma with squamous differentiation was established. conclusion: there are only a few cases of apocrine carcinoma on record and our case is even more special because of his peculiar aspect, the squamous differentiation. the effects of treadmill exercise in the number and weight of mammary tumors chemically induced in female sprague-dawley rats: preliminary results j. oliveira * , c. teixeira-guedes, a. faustino-rocha, r. soares-maia, r. arantes-rodrigues, m. j. pires, m. ginja, p. oliveira, r. ferreira * utad, dept. of veterinary sciences, vila real, portugal objective: we hypothesized that moderate exercise in treadmill may affect the mammary tumor development. n-methyl-n-nitrosourea (mnu) is a commonly used carcinogen to induce mammary carcinomas. the aim of this study was to evaluate the influence of treadmill exercise in the development (number and weight) of female rat mammary tumors. method: in this experimental protocol were used female sprague-dawley rats. mnu was intraperitoneally administered at days of age in a dose of mg/kg. animals were randomly divided in two groups: sedentary (n= ) and exercised (n= ). the exercise program was started after carcinogen administration. animals were exercised in a treadmill control le ® after an initial period of familiarization. thirty-six weeks after mnu administration animals were sacrificed and tumors were counted and weighted. results: sedentary and exercised group presented and tumors, respectively. pearson chi-square value was not significant (p> . ). the mean tumors weight of sedentary group ( . ± . g) was lower than exercised group ( . ± . g). the difference between groups was not significant (p> . ). conclusion: we observed that exercised group showed minor number of tumors, however the lesions presented higher volume. future morphological and biochemical analysis of tumors will allow a better understanding of the relation between mammary cancer and physical exercise. objective: primary non-hodgkin's lymphomas is an uncommon disease representing approximately . % of all reported malignant mammary neoplasms. clinically they are mainly observed as solitary lesions but may also be seen as multiple foci. herein, we present a very rare case with the diagnosis of multifocal malignant lymphoma of the breast with a detailed clinicopathologic evaluation. method: a -year-old-female patient with a right palpable breast lump admitted to the hospital. mammography and ultrasonography findings showed different foci of hypoechoic solid mass forming lesion in the right breast. excisional biopsy has been performed. results: in the histopathological evaluation, both lesions demonstrated diffuse infiltration of mammarian tissue with foci of necrosis; mitotically active tumor cells with large nucleus and prominent nucleoli. the immunohistochemical analysis revealed diffuse and strong lca, cd , cd , focal cd positivity while pancytokeratin, ema, cd , sma were negative. the case was diagnosed as diffuse large b-cell lymphoma. conclusion: breast is an uncommon site for primary malignant lymphomas. we report a very rare case with a diagnosis of multifocal primary non-hodgkin's lymphoma of the breast. ps- - cytological, histopathological and clinical correlation at differential diagnosis of granulomatous mastitismalignancy t. ozgur * , e. atik, s. toprak, h. gokce, n. sengul, m. temiz * mustafa kemal university, medical faculty, antakya, turkey objective: idiopathic granulomatous mastitis (igm) is a rare disease that is difficult to diagnose by only radiological methods and clinical findings. method: first case; a years old woman with a well circumscribed mass of cm diameter at right breast applied to our hospital general surgery outpatient clinic. in the examination of fine needle aspiration biopsy (fnab), palisaded epitheloid histiocytes and scattered atypical cells have been seen on a bloody and inflammatory background. we suggested excisional biopsy. second case; a years old woman applied to our hospital general surgery outpatient clinic with a sore mass in her left breast. the mass has been excised for malignancy suspect. foci of abscesses, active chronical inflammation, areas of hemorrhage and numerous granulomas containing epitheloid histiocytes are observed microscopically. results: in microscobic evaluation of the excisional biopsy of the first case prediagnosed as granulomatous mastitis invasive ductal carcinoma has been our diagnosis. in the second case there have been numerous granulomas containing epitheloid histiocytes, microscopically. our diagnosis was granulomatous mastitis in this patient prediagnosed as malignancy. conclusion: especially fnab findings are easy to confuse with malignancy. we wanted to emphasize to clinician and pathologist to be more alert on distinction of granulomatous mastitis and malignancy. objective: triple-negative-breast-cancer (tnbc) that accounts for - % of all breast carcinomas is defined by the lack of estrogen receptor, progesterone receptor, her expression with agressive clinical behavior. tnbc is categorized into basal like subtype which is characterized by the expression of basal cell markers and normal breast subtypes. method: we studied on immunohistochemically tnbc patients to determine egfr, cytokeratine / (ck / ), p , ki , gcdfp expression patterns by immunohistochemistry, her /chromosome gene status by fish. results: most of the tumors ( , %) were invasive ductal carcinomas. p , ki , gcdfp mean positivity rates were , %; , %; , % respectively. gcdfp positivity was noted in cases of which were ck / (−). the cut-off value for ck / positivity was %. egfr immunoreactivity was grouped into , + as negative; +, + as positive categories. ck / was positive in , %, egfr was positive in , % of the patients. the relation between ck / and egfr expression was statistically significant (p < . ). her fish was negative in all cases. conclusion: as a result gcdfp alone is not a useful marker to detect the metastasis of basaloid type breast cancers. ck / and egfr coexpression can be used to diagnose basaloid tumors with % cut-off value. objective: lobular neoplasia (lns) of the breast include atypical lobular hyperplasia (alh) and lobular carcinoma in situ (lcis). because lns do not present typical clinical or radiologic findings, the diagnosis is incidental. many reports suggest that ln is not only a risk factor of invasive lobular carcinoma but also is as a precursor. it is important to know about the incidence of lns and associated disease in korea. method: a total cases of breast biopsy or excision from five major hospitals in daegu were reviewed independently by pathologists of the daegu breast pathology study group. the incidence of alh and lcis, associated disease, the presence of microcalcification and necrosis were reviewed. all suspected cases of lns were confirmed on e-cadherin immunohistochemical staining. results: only cases out of cases ( . %) revealed lns. associated disease were cases of invasive ductal carcinoma, cases of in situ ductal carcinoma, cases of invasive lobular carcinoma, cases o f other benign disease, and cases of florid ductal hyperplasia or columnar cell change. conclusion: the incidence of lns was similar to western women. incidental lns were frequently associated with invasive ductal carcinoma and further excision is suggested if it was present on the needle biopsy specimen. silver-enhanced in situ hybridization (sish) detection assay for her gene status determination in breast carcinoma: a four year experience in our laboratory f. patakiouta * , g. c. balis, v. theodorou, e. triantafillidou, c. fotiou, i. boukovinas, c. andreadis, g. sibilidis * "theagenion" cancer hospital, dept. of pathology, thessaloniki, greece objective: assessment of her status in breast cancer is important in the clinical management of patients and can be identified by a number of methods. in this study we present the results of the sish technique used in our laboratory in the last years. method: we analyzed cases of invasive breast carcinoma, including core biopsies, which were previously characterized immunohistochemically (cb ) as equivocal on a protein level (her +). all cases were evaluated by bright-field sish using the automated ventana benchmark xt machine. evaluation was independently performed by two pathologists (fp, gb) based upon the algorithms of the manufacturers and asco/cap guidelines. results: out of cases the her gene was amplified in cases ( . %), while in cases ( . %) there was no gene amplification. fourteen of the cases ( . %) were characterized as equivocal. the concordance between the two pathologists was high ( . %). conclusion: the sish method is new, fully automated and very rapid. furthermore the bright-field sish signal does not decay and can be visualized by any standard bright-field microscope even after long periods of time, making it particularly suitable for routine application in surgical pathology. objective: breast fibromatosis (bf) represents . % of breast tumours that simulate carcinoma. the aim of this study is to characterize the cases of bf diagnosed in our institution. method: bf cases diagnosed from to were evaluated with clinicopathological and immunohistochemical parameters. we also studied mutations in exon of the gene for β-catenin in cases. ultrastructural study (us) was performed in case. results: there were women between and years old. radiologically they showed deeply located hypoechoic irregular lesions. size ranged between and cm. histologically they were characterized by an ill-defined spindle cell proliferation without epithelial elements or atypia. immunohistochemical study was negative for ae /ae , ck / , p , cd and bcl . actin was diffusely positive in / , and s and desmin focally positive in / . β-catenin showed cytoplasmic( / ) and nuclear( / ) expression. the us showed double smooth muscle and myofibroblastic differentiation. three patients showed mutations in exon of the β-catenin gene ( with substitution at codon (t a) and deletion of base pairs (a _e del)). conclusion: focal desmin expression and us suggest smooth muscle differentiation in some bf. all cases showed cytoplasmatic positivity for β-catenin but nuclear expression was seen in only % of cases. % of the valuable cases showed genetic alterations of β-catenin. objective: breast cancer is a heterogenous disease consisting of distinct entities characterized by different gene expression patterns. gene expression profiling of breast cancer revealed major subtypes, including hormone receptor (hr) positive luminal a and b, her- positive and basallike breast cancer. most of the basal-like carcinomas are triple negative (hr and her- negative). as most triple negative breast cancers express egfr, mutations in the signal transducing cascade of ras/raf/map kinase might play a role in tumor progression. the aim of this study was to determine the incidence of kras and braf v mutations in triple negative breast carcinomas. method: total genomic dna was obtained from formalin-fixed, paraffin embedded, triple negative breast tumors. kras was analyzed by pyrosequencing of codon , , . braf (exon , v ) mutations were analyzed with autogenomics infiniti braf assay on the autogenomics infiniti analyzer. results: we found kras mutation in only one case ( . %), apocrine carcinoma which was gly asp (ggt> gat) mutation. the braf v mutations were not detected in all analyzed tumors. conclusion: we concluded that kras mutation was rare in triple negative breast carcinomas and high frequency of egfr overexpression in this subtype might be related to other pathways in egfr signaling. the pre-lymphatic system and the lymphatic network of the breast in menstruating and in menopausal women: a comparative study s. popovska * , i. ivanov, t. betova, t. dineva, z. ivanova * medical university hospital, dept. of clinical pathology, pleven, bulgaria objective: the pre-lymphatic system was initially described in the s by casley-smith and florey. it consists of cd positive fibroblasts lined stromal spaces communicating with the lymphatic capillaries. the aim of the following investigation was to compare the lympho-vascular density and pre-lymphatic network density of breast tissues in the context of the menstrual status of the patients. method: formalin-fixed, paraffin embedded tissue specimens from patients with primary breast cancer were studied. tumor-free tissue materials were selected. tissue processing and immunohistochemical staining with d - and cd antibody was performed in accordance to standard laboratory protocols. quantitative evaluation of the lymphatic and pre-lymphatic system was performed. the kruskal -wallies test was used to compare differences between the median values of the studied variables. statistical significance of the differences was considered if p< . . results: the pre-lymphatic network was found to be significantly more dense in menstruating, compared to menopausal women k-w= . ; p< . n= . lymphatic vascular density was higher in menstruating compared to menopausal women k-w= . ; p= . ; n= . conclusion: involution changes in the pre-lymphatic and lymphatic system of the breast were found to appear in postmenopausal women. this may be the explanation for the slow local progression of breast cancer observed often in elder patients. comperative study of herceptest and fluorescence in situ hybridization results in breast carcinoma: indeterminate ( +) group must not be a wide range uncertainty category y. saglican * , u. ince * acibadem maslak hospital, dept. of pathology, istanbul, turkey objective: herceptest is an exclusively used immunohistochemistry method for testing her /neu overexpression in breast carcinomas. the purpose of this study is to select the proper patients candidate to in situ hybridization, with an indeterminate her score. method: a retrospective analysis of cases with invasive breast carcinoma were evaluated for her status using immunohistochemistry and fluorescence in situ hybridization. İmmunohistochemistry of cases that had been previously evaluated by nine different pathologists in our department, were reviewed by two experienced pathologists. interpretation of the immunohistochemistry results was made without knowing the first results. results: compared to the first evaluation; cases with score , + and +correlated with the review of immunhistochemistry and none of score and + tumors were positive with in situ hybridization. gene amplification was detected in cases; of them with +score. three of + tumors and of + cases were negative with in situ hybridization. discordance was detected in cases in + score; of them were reclassified as negative and were technically unsatisfactory. conclusion: using a different cut-off value for indeterminate her results and training of pathologists to minimaze the interpretational differences, will maximize the accuracy, while not submitting to unnecessary molecular tests. objective: the excision repair cross-complementation (ercc ) enzyme plays an essential role in the nucleotide excision repair pathway and is associated with resistance to platinum-based chemotherapy in different types of cancer. the aim of the present study was to evaluate the clinicopathological significance of ercc expression in breast cancer patients. method: we used immunohistochemical to analyze ercc expression in a tissue microarray from breast carcinomas. ercc expression analysis was available for cases and was correlated with clinicopathologic factors and outcome data. results: ercc -positive was observed in ( . %) cases and was correlated with smaller tumor size (p= . ) and with positivity for estrogen receptor (p= . ). ercc expression did not correlate with overall and disease-free survival rates. the majority ( . %) of special histological types of invasive breast carcinomas was positive for ercc compared to invasive ductal carcinomas (ercc -positive in . % of the cases). similarly, triple negative breast cancers (tnbc) were more frequently negative for ercc ( . % of the cases) compared to the non-tnbc ( . %). conclusion: ercc expression correlated significantly with favorable prognostic factors, such as smaller tumor size and er-positivity, suggesting a possible role for ercc as a predictive and/or prognostic marker in breast cancer. a study of cd positive basal/myoepithelial cells in a consecutive series of in situ lobular neoplasia of the breast s. shousha * , g. forbes * charing cross hospital, dept. of histopathology, london, united kingdom objective: we have previously reported a few sporadic cases of in situ lobular neoplasia (iln) associated with marked proliferation of cd positive basal/myoepithelial cells. method: twenty consecutive cases of pure iln and dcis were studied. new sections were cut and stained for e-cadherin and cd . results: one out of the cases showed foci of invasive lobular carcinoma in the new cut sections. all iln cases showed increased proliferation of cd positive basal cells, varying from a focal mild increase to a marked proliferation surrounding or mingling with the lobular cells. cd positive cell proliferation was least observed in a case of iln with central necrosis and in foci of iln seen adjacent to the invasive carcinoma. the invasive tumour cells were cd negative. all cases of dcis had a thin layer of cd positive myoepithelial cells, that was incomplete or absent in some foci. conclusion: our findings confirm the presence of a unique relationship between excess proliferation of cd positive basal cells and iln; an association which is not seen in dcis or invasive carcinoma. this is particularly interesting as recent evidence indicates that cd is involved in the regulation of mammary stem and 'sphere forming' cells. objective: primary neuroendocrine carcinomas (nec) of the breast are defined by the who classification as a group of neoplasms that express neuroendocrine markers in more than % of the cells. these tumors can exhibit different morphologic appearances including solid, nested or alveolar pattern, papillary or mucinous differentiation or conventional ductal structures. the aim of this study is to identify histomorphological features useful in the recognition of this tumors. method: invasive breast carcinomas were reviewed, none of them previously typified as nec. were ductal carcinomas ( grade i, grade ii and grade iii) and lobullilar carcinomas. in all cases immunohistochemical staining for the neuroendocrine marker synaptophysin was performed. results: eight of the cases showed intense immunoreactivity for synaptophysin. all of them were predominantly composed of solid, confluent nests and cords of cells with medium-to high grade nuclear atypia. two cases showed intense desmoplastic or sclerotic stromal response. a colloid component was identified in another two cases. conclusion: due to its morphologic variability, mammary nec is frequently underrecognized. a predominantly solid pattern of confluent nests and trabeculae associated with a prominent sclerotic stromal response or colloid differentiation should prompt to perform additional immunohistochemical staining in order to exclude neuroendocrine differentiation. objective: association between centrosome abnormalities and response to chemotherapy has not been fully elucidated in breast carcinomas (bc). here we analysed the association between aurora kinase a (aurka) and gamma-tubulin (gt) and response to neoadjuvant chemotherapy (pst) in patients treated with bc. method: the immunohistochemical expression of aurka and gt was analysed in core biopsies of bcs taken before administration of pst. aurka expression was analysed using a modified allred-like scoring system (intensity and percentage of staining combined). cells containing one or two centrosomes as determined by gt were considered negative, whereas cells containing more than two centrosomes were regarded as positive. centrosome amplification was graded in each specimen as follows: negative ( - % of cells); weak ( - %); moderate ( - %); and strong ( %<). pathological response rates were assessed using chevallier's classification. results: centrosome amplification was significantly higher in patients achieving complete response when compared with the cases where partial remission or no response was defined (p= . ), whereas we found no significant correlation between the expression of aurka protein and response rate to neoadjuvant chemotherapy (p= . ). conclusion: bcs showing centrosomal amplification as determined by gt present higher response rates to neoadjuvant chemotherapy, but need further validation. breast granulocytic sarcoma with aleukemic presentation: a case report o. tzaida * , p. giagkazogloy, p. repousis, m. kotsopoulou, i. kasselaki, c. valavanis, i. iacovidou * anticancer hospital metaxa, peiraias, greece objective: granulocytic sarcoma (gs), a rare solid tumor of immature myeloid cells in exrtamedullary sites, usually occurs during the natural course of acute myelogenic leukemia (aml). rarely is presented without overt hematological disease. the breast is an uncommon site of localization. method: we report a case of breast gs with aleukemic presentation, condition that is extremely rare and requires a high index of suspicion for diagnosis. a year old woman, presented with a solitary, non-tender, palpable left breast mass. a lumpectomy was done. results: histopathology revealed breast parenchyma diffuse infiltration, in a targetoid pattern, by immature small cells intermingled with eosinophils. lobular carcinoma and lymphoma were considered in differential diagnosis. the myeloid origin of the neoplasm was established by immunohistochemical analysis that revealed lca, mpo, cd , hladr, cd (kp ) positivity. there was no evidence of leukemia in the peripheral blood and bone marrow. the patient was treated as aml with systemic chemotherapy and months after the diagnosis she is without evidence of disease. conclusion: breast gs is a challenging situation both for pathologists and hematologists. available evidence strongly favors the application of systemic chemotherapy despite the appeared localized nature of the disease. method: paraffin blocks of tumor tissue of breast cancer cases collected at our cancer centre for years. the immunohistochemical study was performed by standard techniques using following antibodies (dako): er, pr, her- , egfr, ck / , ck , ki . results: all cases were classified into the subtypes: luminal a (n = ; . %); luminal b (n = ; . %); her- -positive (n= ; . %); basal (n= ; . %); negative (n= ; . %). conclusion: luminal breast cancer phenotype predominates in our collection, predictive/prognostic value of molecular genetic subtypes is planned to be investigated. we recognized statistically significant differences (p< , ) in five-year survival rates between tumors of luminal (a, b) and basal subtypes. proliferating trichilemmal cyst with atypical cytological features r. g. wright * , c. gallivan, t. molden-hauer, r. liang * gold coast hospital, pathology queensland, australia objective: proliferating trichilemmal cysts are relatively common occurring within the scalp of elderly women. proliferating trichilemmal cysts of the breast, however, are rare. these cysts are benign with very few demonstrating malignant transformation. method: we report a case of proliferating trichilemmal cyst of the breast with atypical cytology which prompted excisional biopsy. cytological assessment was useful in this case as it indicated excisional biopsy rather than a more extensive excision. results: smears produced from the fna showed sheets of atypical epithelial cells with foamy histiocytes, multinucleate cells and fibrotic material with evidence suggestive of fat necrosis. a cell block was acellular. a specific diagnosis was not made. an excision specimen was advised and a proliferating trichilemmal cyst was demonstrated. objective: a large clinical value has determination of molecular subtype of breast cancer (bc). to clarify morphological and molecular biological similarities and differences between bc depending on age, we compared features between tumors of younger and older women. method: patients with bc are included in research: from till years, among them patients were till years. er, pr, her- /neu, p , p , ki , ck / , p , bag , mcl , ps , vegfr, her- were analyzed in all cases by immunohistochemistry. results: the younger patients with bc had higher expression of p , p , p , bag , mcl , ki- , vegfr, her- (p< , ) than older patients. there was a basal-like molecular subtype of bc -fold more frequent compared with older patients ( , % versus , %; p< , ). threeyears overall survival in patients till years was by , % and a five-year overall survival was by , % lower than in patients over years (p< , ). objective: breast her ish relies on correct enumeration of her /cep signal. however, as with immunohistochemistry, there are technical pitfalls which may render ish difficult/unreliable to interpret. the uk neqas has established a 'technical' ish module to provide feedback to laboratories using both chromogen and fluorescence based tehcniques. method: unstained slides consisting of breast cancer cell lines were distributed. laboratories were asked to demonstrate her gene amplification using their routine assay, and then return the slides for assessment. chromogen based methods were assessed around a multi-header microscope with assessors scoring each slides, whilst the fluorescent ish method was assessed by a single individual. assessors scored the 'readability' of each slide, without counting the probes, and provided feedback where the hybridisation technique could hinder interpretation. results: cish based methods showed acceptable staining in % (n= ) of cases. staining problems ranged from loss/poor her /cep signal, non-specific staining and morphological damage. the fish pilot assessment showed an acceptable rate of % (n= ), with the low pass rate being attributed to poor preservation/quenching of fluorescence signal. conclusion: greater emphasis needs to be placed on the 'readability' of an ish slide, prior to carrying out the process of enumeration. tuesday, september , . - . objective: intravenous leiomyomatosis is a very rare growth pattern variant of leiomyoma in which nodular masses of tumor grow within venous channels. occasionally the tumor can extend to vena cava and the right heart. method: we present a case of a -year-old woman, who was admitted in the emergency room with rapidly evolving exertional dyspnea. cardiac ultrasonography revealed a "big mass in the right chambers". she was submitted to a right atriotomy with resection of part of the tumor, which was sent for intraoperative consultation. results: grossly, the tumor was polypoid, firm, with a smooth surface. the frozen section showed a lesion composed of tortuous vessels and in some areas a fibrillar eosinophil extracellular matrix and others with spindle cells. no significant atypic or pleomorphic cells, mitosis or necrosis were observed. the diagnosis was deferred for definitive paraffin sections. in the definitive h&e and with immunohistochemical stains, the case was diagnosed as an intravenous leiomyomatosis, and the diagnosis was confirmed in the hysterectomy specimen. conclusion: intravenous leiomyomatosis with cardiac involvement is an extremely rare condition. clinical information is essential for the correct diagnosis in frozen section. characterization of a specific mechanism for late loss of cardiac allograft: the antibody mediated rejection (amr) as a major factor of cardiac allograft vasculopathy (cav) p. bruneval * , c. toquet, a. loupy, p. pouvier, a. cazes, j.-p. duong van huyen * france objective: the rational of this study was to investigate explanted failing grafts in the light of new concepts in rejection mechanisms, namely amr. method: this retrospective multicentric study collected explanted cardiac grafts failing ≥ year posttransplantation. the vasculature of the grafts was assessed from epicardial coronary arteries to myocardial microcirculation. immunohistochemistry was performed for c d complement fraction deposition and cd -positive macrophages in the explanted grafts and previous endomyocardial biopsies. donor specific antibodies (dsa) were retrospectively assessed using luminex sa technique. results: a pure classical coronary atherosclerosis pattern was observed in / ( %). a pure pattern of cav was present in / ( %) and a mixed pattern associating cav and atherosclerosis features in / ( %). interestingly the cav pure and mixed patterns were associated with vascular inflammation within the arteries and/or the microcirculation with c d deposits and macrophages ( / ) in the explanted grafts versus nil ( / ) in pure atherosclerosis pattern (p< . ). furthermore they were associated with previous amr episodes on endomyocardial biopsies ( / ) and by positive dsa ( / ) versus nil in pure atherosclerosis pattern (p< . and p= . , respectively). conclusion: in failing cardiac grafts cav lesions are associated with makers of amr. cav should be the consequence in coronary arteries of an ongoing amr process. results: in our cases increase in fibrous and adipose tissue concordant with age, indicating an age related nature, were detected. fibrous and fatty tissue infiltration appeared at the age of . fatty infiltration started at the age between and at sinoatrial node. in cases calcification and in cases inflammation was observed. amyloid accumulation was not present. in cases myocardial infarction not involving ccs was seen. in case fibroelastoma was detected. conclusion: in turkish population age related fibrosis and fatty infiltration in ccs appeared at the age of and increased with age. fatty infiltration in sinoatrial node started at a younger age than that of reported in the literature. in cases whom the cause of death could not be determined we could not detect lethal pathologic features. however we think that examination of the ccs will improve the quality of autopsy diagnosis. periadventitial adipose tissue in human coronary atherosclerosis: a neurotrophin study p. ghenev * , p. panayotov, g. chaldakov, l. aloe * medical university varna, dept. of pathology, bulgaria objective: recent evidence demonstrates that epicardial adipose tissue, including coronary periadventitial adipose tissue (tunica adiposa), are paracrine sources of bioactive mediators (adipokines, no, h s) which may be involved in coronary atherogenesis. because of the increasing interest for extra-neuronal effects (inflammation, wound healing, lipid and glucose metabolism) of the neurotrophins nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf), including in human coronary atherosclerosis in autopsy samples, the aim of the present study is to evaluate the expression of ngf and bdnf and their receptors (trka and trkb, respectively) in cardiosurgery biopsy samples of pericoronary adipose tissue. method: immunohistochemistry of ngf, bdnf and their trk receptors. results: ngf, bdnf, trk a and trkb expression was lower in periadventitial adipose tissue of atherosclerotic coronary arteries as compared to non-affected coronaries. conclusion: we provide the first evidence for a possible role of neurotrophins in the molecular remodeling of tunica adiposa in human coronary atherosclerosis. this study is dedicated to rd anniversary of rita levi-montalcini, the nobel laureate for the discovery of ngf. objective: in normal myocardium, apoptotic myocytes are usually absent, or if affected, with most of positive cells per million. method: the apoptosis was measured by the tunel method in patients with dilated cardiomyopathy (dcm) and myocarditis (mc), and expresed by apoptotic index (ai) in patients within both groups, and to correlated with ejection fraction (ef) and with different morphological stages of these entities. statistical analisys was performed, and p values ≤ . were considered statistically significant. results: ai in mc cases was , + , , and in dcm cases the value was significantly higher , + , (p= . ). in the group mc patients, we didn't found statistically significant correlation between ai and different values of ef (p= . ). the analysis of the value of ai between patients with different morphological stages of mc didn't show statistically significant correlation (p= . ), as well as in dcm (p= . ). conclusion: apoptosis play a significant role in dcm and mc but its significance in the progression to heart failure has still to be established. objective: cardiac lesions following blunt chest trauma are multiple and of various degrees of severity. "cardiac contusion" is a difficult diagnosis, especially antemortem and if signs of thoracic trauma are not evident on external habitus and on the underlying internal plans of cardiac area. is the contusion a cause of death per se? method: the authors present two cases of road accident male victims, aged and year-old, submited to complete postmortem examination with ancillary diagnostic methods. results: no traumatic lesions or underlying pathology was found at the autopsy. epicardial and myocardial haemorrhagic foci were histologicaly documented. toxicology was negative. conclusion: cardiac contusions are caused by one of the following mechanisms: ) heart compression between bone structures, ) sudden acceleration-deceleration movements and ) sudden thoracic/abdominal pressure increase. they may be asymptomatic or lead to rhythm disturbances responsible for unexpected death ( %) . they seem to be underdiagnosed, thus suspecting the entity "contusio cordis" is crucial, not only antemortem for correct surveillance and treatment, but also postmortem to adequately establish causality nexus in cases of post-thoracic trauma deaths. the influence of bone marrow-derived multipotent stromal cells on myocardial scar healing in experimental myocardial infarction l. kakturskiy * , t. fatkhudinov * institute of human morphology, central laboratory of pathology, moscow, russia objective: previous studies have shown the benefits of intracoronary infusion of bone marrow-derived multipotent stromal cells (bm-mscs) in heart disease. our aim was to study engraftment, differentiation of bm-mscs and its role in myocardial reparation. method: acute myocardial ischemia was produced by transient occlusion (total of min) of the proximal left anterior descending coronary artery and followed by reperfusion. bm-mscs were isolated, expanded by standard and labeled with pkh (sigma). cells were cd and cd positive. days after occlusion during cross-clamping of aorta the cells were administered into the cavity of the left ventricle at the concentration × in saline solution. in day, , and weeks after transplantation the labeled cells were detected in the cryosections and heart morphometry was performed. results: bm-mscs were detected only in the scar tissue and had a fibroblast-like phenotype. they neither differentiated into cardiomyocytes nor into the cells of blood vessels. in weeks after transplantation the scar thickness was higher in the group with cell transplantation (p< . ). conclusion: engrafted bm-mscs promote myocardial fibrosis only in the scar, but not in the perifocal myocardium, provided strengthening of the scar, remodelling of outflow tract, and improvements the myocardial function. objective: from january to december (institute of pathology and legal medicine, paolo giaccone -palermo) autopsies were performed on males and females who died of a massive pte. method: systematic autopsy. results: saddle pulmonary thrombosis in cases, in isolated in both the main pulmonary arteries. in all cases the transverse heart diameter was , - cm superior to the longitudinal one, the interventricular septum became rectilinear with a right ventricular dilation. the coronary artery anatomy was: critical stenosis through fibroatheromatous plaque of the dominant right coronary artery (rca, ), non dominant left coronary artery (lca, ); non critical stenosis of rca ( ), lca ( ); absence of stenosis ( ). histological ventricular examination showed hypoxic-ischaemic and/or reperfusion and overload lesions. in all cases, the lungs showed plurifocal occlusive thrombosis of intramural pulmonary arterial (ipa) vessels associated with adjacent infarctual necrosis ( ); acute partially haemorrhagic basal oedema ( ) and desquamative macrophagic alveolitis ( ). conclusion: in all cases of massive pte the thrombosis of ipa vessels associated with an adjacent infarction ( %) is constant. this suggests that death is nearly always preceded or triggered by one or more episodes (clinically silent or with non specific symptoms) of thromboembolism of ipa vessels. cardiac findings in routine fetal autopsies: more than it meets the eye? e. rios * , c. bartosch, c. ramalho, j. monterroso, o. brandão * centro hospitalar s joão, pathology, porto, portugal objective: congenital heart disease (chd) is the most common malformation in newborns. our aim was to evaluate the spectrum of chd in consecutively performed fetal autopsies and to correlate prenatal and postmortem diagnoses. method: a retrospective study of fetal autopsies was performed in a tertiary referral hospital. chd was classified in seven categories: left ventricular outflow tract obstruction-lvoto, right ventricular outflow tract obstruction-rvoto, septal defects, connection anomalies, conotruncal anomalies, complex anomalies and others. cardiac defects were also classified as isolated or associated with others anomalies. results: chd was identified in ( . %) fetuses. most common categories were septal defects and complex anomalies. associated anomalies were found in fetuses ( . %). in this group, septal defects were the most common chd, frequency being significantly higher than in the group of isolated cardiac anomalies (p= . ). comparison of prenatal and postmortem diagnoses ( cases) showed complete or partial agreement in and cases ( % and %, respectively) and complete disagreement in cases ( %). in the latter group, prenatal diagnosis had not been done by a pediatric cardiologist. conclusion: the high prevalence of chd in lost pregnancies highlights the importance of systematic fetal autopsy performed by a specialized pediatric pathologist. objective: the appearance of pathological calcification, which leads to significant changes of vascular wall, which underlie the development of atherosclerotic complications, has a great importance in the morphology of atherosclerosis development. purpose of the work is the study of pathological biomineralization in heart valves affected by atherosclerosis. method: the study was conducted on sectional material of mitral and aortic valves obtained during autopsy. the tissue of heart valves was studied by methods of histology, electron microscopy, x-ray diffraction, infrared spectroscopy. results: macroscopically mitral and aortic valves are thickened, opaque, dull whitish, sometimes with deformity and ulceration. histology observed the formation of atherosclerotic plaques, fibrous changes, and inflammatory infiltration. according to x-ray phase analysis biominerals in heart valves are represented by apatite crystalline phase. sizes of crystals have distinct age dependence. the results of infrared spectroscopy revealed absorption bands of carbonate apatite replacement; in all instances they correspond to the type of substitution b (co -replaces po -). conclusion: the study of pathological mineral formations on human heart valves by the range of morphological and physicochemical methods show that they are aggregates of interacting organic and mineral components, their ratio changes with "maturation": the organic component decreases and the mineral component, represented by carbonate containing hydroxyapatite doped with chlorine grows while improving, so the process is dynamic. relationship between myocardial injury, oxidative stress mechanism and sepsis/septic shock in infants submitted to surgery for congenital heart defects m. silva de oliveira * , e. medeiros floriano, p. henrique manso, r. nilsson sgabieri, j. guilherme klamt, e. zangiacomi martinez, w. vilella de andrade vicente, s. gusmão ramos * university of são paulo, dept. of pathology, ribeirão preto, brazil objective: a progressive ventricular dysfunction caused by ischemic myocardial injuries remains one of the leading causes of death during the postoperative course in congenital heart disease (chd). the aim of this study was to investigate the role of oxidative stress in these myocardial injuries. method: myocardial injuries and oxidative stress mechanisms were assessed by histopathology and immunohistochemistry and quantified by morphometrical analyses. results: myocardial injury was observed in pediatric patients submitted to surgery for chd with cardiopulmonary bypass, followed by lethal exit. oxidative stress mechanisms were directly related to these particular types of myocardial injuries. importantly, -hydroxynonenal ( -hne), a marker of lipid peroxidation, is strongly expressed, especially in irreversible myocardial lesions. although morphologically similar, myocardial injuries observed in patients who evolved with sepsis in the peri-operatory period exhibited a completely different set of oxidative stress mechanisms. increased concentrations of nitrotyrosine protein adducts were observed in these patients, suggesting that peroxynitrite-mediated protein nitration may be the predominant oxidative stress mechanism found in these situations. conclusion: the underlying mechanisms of these lesions seem to be related to the development of ischemia or ischemia/reperfusion followed by oxidative stress mechanisms that vary depending on whether sepsis was present. while the exact mechanism is not fully understood, it has been suggested that endogenous catecholamine release could have a role in this process. objective: a significant decrease of the mean cardiomyocyte dna content and increased numbers of diploid cardiomyocytes after ventricular unloading was demonstrated, suggesting a numerical increase of cardiomyocytes. the heart harbours several stem cells populations including c-kit (cd ) + cells and side population cells (spc), that might proliferate after unloading and generate diploid cardiomyocytes. it was tested, whether there is an increase of abcg + spc and cd + cells after unloading. method: in paired myocardial samples (before and after lvad), the number of cells with immunoexpression of abcg- , c-kit/cd and mef- was assessed by immunohistochemistry and morphometrically determined. results: a significant increase of spc and cells with coexpression of c-kit and mef- after unloading was observed (p= . ). a significant positive correlation between both spc and cells with coexpression of c-kit and mef- expression was observed (p= . and . ). no correlation was found between the number of spc and the mean cardiomyocyte dna content. conclusion: spc are significantly increased in the myocardium after ventricular unloading, suggesting a role of stem cell proliferation during "reverse cardiac remodelling". these cells might proliferate and commit to different cell lineages such as cardiomyocytes or endothelium, and thus ameliorate cardiac function. role of elastin gene polymorphism in ascending aorta aneurysm development in patients with essential hypertension v. zakharova * , v. dosenko, m. kostiv, e. rudenko * cardiovascular surgery institute, pathology, kiyv, ukraine objective: there is supposition about genetic propensity in the ascending aorta aneurysm (aaa) development. aim: to investigate role of elastin gene polymorphism (rs ) in aaa development in essential hypertension (eh). method: dna samples from persons with aaa and eh ( group), with eh only ( group) and healthy subjects ( group) were examined. polymorphism rs was studied with real-time pcr. histological examination of aaa walls was performed in group. results: distribution on genotypes a/a, a/g, g/g was the following: group - . %, . %, . %; group - %, %, %; group - . %, . %, . %. fragmentation of elastic membranes and media collagen component strengthening were revealed in persons with a/a, a/g, g/ g genotypes in . %, . %, . % respectively. persons with g/g genotype have more frequently ( . %) than others (a/a - . %, ag - . %) signs of inflammation, which promote aaa development parallel to eh. conclusion: we obtained evidence that elastin gene polymorphism (rs ) plays important role in eh and aaa development. in persons with allele a in rs risk of eh and aaa development is higher than in g/g genotype. mostly allele a in rs is accompanied with elastic membranes fragmentation and their substitution by collagen that decreases arterial wall elasticity. tuesday, september , . - . objective: our aim is to inform on the frequency, clinical aspects, and histopathological features of the tumor metastatic to the orbit. in adults, the most common tumor metastatic to the orbit are carcinomas, breast carcinoma the most frequent. less common are the metastases from gastrointestinal tract, kidneys, and prostate. cutaneous malignant melanoma is the most common nonepithelial tumor metastatic to the orbit. method: orbital metastatic tumors may occur at any age, but most often between and years of age. most reported statistics reveal a slight predominance of women, accounted for by the fact that breast carcinoma is causing metastases most frequently. a frequent finding in the cases of metastatic carcinoma is enophthalmos that is associated with scirrhous carcinomas. we present two cases of orbital metastatic tumors in which the primary tumors were a basal cell carcinoma and a cutaneous malignant melanoma. results: as to the metastasis from a basal cell carcinoma, the primary tumor was located in the external angle of the eyeball and metastasized the external / of the orbit. in the case of malignant melanoma, the primary tumor was located in the upper lid and invaded the orbital floor and fat. in both patients eyeball enucleation was performed. conclusion: we chose to present these cases given the differential diagnostic challenge and rarity of their location. uveal melanoma a. dumitriu * , s. dumitriu * umf iasi, dept. of neurology, romania objective: uveal melanoma which arises from melanocytes residing in the stroma is the most common primary intraocular cancer in adults. we report a case of uveal melanoma presenting as complicated cataract in a years-old male. results: the right eye was blind for months. the patient underwent extended enucleation and histopathology was consistent with uveal melanoma. conclusion: we present this case in order to provide the medical community a basic reference that would help to make further progress in this rare disease, which remains difficult to treat. results: there were more women than men ( % versus %). metastases developed in % (total group mean follow up of . year). mixed tumors were most common overall ( . %): for non metastasizing tumors there were . % mixed, . % fusiform and . % epithelioid types. for metastatic tumors there were . % mixed, . % epithelioid and . % fusiform types. the difference between cell type and metastatic development was significant in all but epithelioid compared with mixed tumors. epithelioid type had a relative risk of mortality of . and mixed had . compared to fusiform cell type. conclusion: our patients were younger, with larger tumors and different histological proportions from those of the collaborative ocular melanoma study. five year survival times were similar to those for europe. uveal melanoma between preserving vision and prolonging survival. pathological contributions to a better clinical management m. mera * , l. blaga * umf cluj-napoca, anatomic pathology, romania objective: uveal melanoma has a high metastatic potential which is invariably fatal. there is an ongoing debate on the impact of ocular treatment on metastatic disease and survival. improved prognostication is needed to identify cases that could avoid unnecessary loss of vision while avoiding unethical care. our study is aimed at contributing to the developing of reliable tools to assess the metastatic risk. method: an accurate prediction should include both clinical features and histologic parameters as biopsy techniques continuously advance. cases of uveal melanoma, either reviewed or prospectively managed in our pathology department, were included in a log-logistic multivariate survival model that we have considered would best accomodate our data. the performance of the model was assessed by bootstrap re-sampling. results: extravascular matrix patterns, optic nerve invasion, tumorinfiltrating lymphocytes, tumor cytomorphology, nucleolar size and the mitotic count together with initial visual acuity have been found to be the most important predictors, ahead of the traditional tnm stage. the results are provisional but encouraging. conclusion: there is still need for multidisciplinary approach and multicenter collaboration in an effort to elucidate the uncertainty related to the best management of one of the most traumatising types of cancer. orbital hemangiopericytoma: a case report e. omoscharka * , k. lankachandra * university of missouri, dept. of pathology, kansas city, usa objective: hemangiopericytomas are rare vascular tumors arising from zimmerman's pericytes, which ubiquitously surround blood capillaries and post-capillary venules. they rarely occur in the orbit, accounting for only % of all orbital neoplasms. in most cases the primary presentation is progressive proptosis. method: a -year-old female was first seen for the evaluation of a right orbital mass which has been causing proptosis. associated symptoms included peripheral vision loss in the right eye, persistent headache with occasional balance problems. a palpable, . × . cm well circumscribed mass with a smooth surface, resting on the inferior orbital wall was completely removed through right anterior orbitotomy. h&e stained histologic sections were studied. in addition, tissue sections were subjected to immunohistochemical stains for cd , cd , bcl , sma, desmin, ema and s- . results: the mass was composed of spindle cells with very low mitotic activity. staghorn vascular channels were evident, and in several areas the tumor cells invaded the pseudocapsule. the tumor cells were positive for cd , cd and bcl- supporting the histopathologic diagnosis of benign hemangiopericytoma. conclusion: orbital hemangiopericytoma is a slow growing tumor with a potentially malignant behavior, and a high local recurrence rate if incompletely excised. complete, intact removal prevents tumor recurrence and provides a good outcome. expression of sod family in rat lacrimal gland after intermittent light exposure c. l. zamfir * , f. e. zugun, e. cojocaru, r. o. temneanu, r. folescu, m. spataru * university of medicine iasi, dept. of histology, romania objective: any disturbance of chronobiological rhytms represents a potential stressor, acting like a strong oxidant trigger. sod family (cuzn-sod, mn-sod and ec-sod), is one of the most significant enzymatic complexes involved in antioxidant defense. monitoring local sod distribution in lacrimal gland after intermittent light exposure can be able to provide insights into the distinct links of antioxidant adaptative mechanisms.so long as lacrimal gland morphology and functions are dependent to a normal light exposure, mobilisation of its own antioxidant resources, especially sod, is an usefull starting point in antioxidant ophtalmic therapy. method: immunohistochemistry was performed in microscopic analysis of glandular fragments prelevated from male wistar rats, randomized in control/intermittent light exposed groups for days. there have been used specific antibodies for each enzymatic member of sod family. blood samples were prelevated to control sod levels. the study respected all imposed ethical criteria. results: lacrimal glandular tissue exhibits a strong cuzn-sod immunoreactivity, a moderate one for mn-sod and only a small ec-sod reactivity, while the level of sanguin sod is high, as a marker of oxidative aggression. conclusion: oxidative stress may be engagged in a deep alteration of lacrimal gland. mobilisation of sod, depending on their specific glandular location and tissular receptivity, should be regarded as a promising alternative for antioxidant therapy. tuesday, september , . - . objective: liposarcoma is the most commonly diagnosed soft tissue sarcoma in adults and occurs predominantly in the lower limbs and retroperitoneum. primary mediastinal liposarcomas are rare accounting for less than % of all mediastinal tumors. method: we report a retrospective study of cases of mediastinal liposarcoma during an -year period. diagnosis was made on mediastinal biopsy in cases and on resected specimen in the other cases. results: there were men and a women ranging in age from to years, explored for respiratory symptoms. radiological findings showed an anterior inhomogeneous mediastinal mass in all cases with extension to left hemithorax in one case. a surgical resection was achieved in only cases but was incomplete. histopathologically, tumors were classified into well differentiated lipoma-like liposarcoma, one myxoid liposarcoma and one mixed liposarcoma (sclerosing liposarcoma with myxoid liposarcoma). all patients received adjuvant radio or chemotherapy. one of them presented lung metastasis during the follow up and another died from respiratory failure. the two other are still alive. conclusion: mediastinal liposarcomas include a heterogeneous group of bulky tumours, the progression of which depends on the histological type. histopathologic examination is always necessary as much for diagnosis as prognosis. mediastinal epithelioid hemangioendothelioma with a highgrade clinical course a. konstantinova * , k. shelekhova, v. f. klimashevsky * st. petersburg state university, russia objective: epithelioid hemangioendothelioma (eh) is a rare neoplasm usually presenting in soft tissues. about twenty cases of eh have been reported in the mediastinum. most of these mediastinal eh exhibited an indolent course. results: a -year-old woman presented with focally encapsulated mass, . × cm in size, in the anterior mediastinum. the tumour of bone density was adhered to the lung tissue and occluded left subclavian vein. microscopically, the tumor consisted of foci of so-called blister cells typical for eh, anastomosing cords of small epithelioid cells embedded in myxohyaline matrix with haphazardly distributed metaplastic bone and hemorrhagic cellular stroma. spindling of the tumor cells was prominent. the tumor cells demonstrated moderate atypia, but mitotic figures were not found. immunohistochemically, cells of primary tumor were positive for fli , vimentin, cd and cd . focal staining with ema was observed. multiple metastases were subsequently found in the liver and lungs. metastatic deposits had the same morphology as a primary tumor. we present the case of rare mediastinum epithelioid hemangioendothelioma with peculiar histological features such as prominent spindling of neoplastic cells and abundant metaplastic bone formation in aggregate with aggressive clinical behavior that exhibited a profound metastatic poteintial. objective: thymic carcinomas comprise rare malignant epithelial neoplasms, which exhibit a disproportionately large variety of growth patterns. we present three cases of primary thymic carcinomas, including two squamous cell carcinomas and one papillary adenocarcinoma. method: all three patients were males, aged , and years, and presented with mediastinal masses measuring cm, . cm, . cm in maximal diameter respectively. results: microscopically the first two tumors were poorly differentiated squamous cell carcinomas, immunohistochemically positive for cd and cd , whereas the third tumor was a papillary adenocarcinoma, positive for cd and negative for cd and ttf- . immunohistochemical features of the tumors were supportive of thymic derivation. conclusion: primary thymic carcinomas are rare malignant tumors with clear-cut atypia, largely lack of organotypic features and a very diverse differentiation. squamous cell carcinoma is the most frequent subtype. they are often a diagnosis of exclusion, since metastases to thymus and anterior mediastinum, mainly from the lung, are far more common. immunoreactivity to cd , cd and cd may support the thymic origin of neoplastic squamous cells. treatment options include surgical excision, radiation and/ or chemotherapy depending on tumor stage and patient's condition. the prognosis is generally poor with squamous cell carcinomas having a slightly more favorable outcome. wednesday, september , . - . objective: surgical blood loss can lead to ineffective tissue perfusion of vital organs. physiologic solution chosen for blood volume replacement may be determinant for preserving renal integrity. our aim is to study kidney histopathological changes in a hemorrhagic model, followed by intravascular volume replacement with ringer's lactate or hydroxyethylstarch (hes) / . solutions. method: thirty one pigs under general anaesthesia with propofol and remifentanil underwent haemorrhage at a volume of ml kg- , over min. after a waiting period of min, intravascular volume was replaced using hes / . (g ) and ringer's lactate (g ). one hour after, pigs were euthanized with iv potassium chloride and sixty two renal samples were taken for histopathological examination, using pas staining. renal damage was assessed for glomerular, tubulointerstitial and vascular lesions. results: mean arterial pressure reached mmhg after bleeding, and recovered for values above mmhg in both groups after volume replacement. histopathological lesions observed in g were more frequent than those in g . conclusion: hes / . may reduce the incidence of histopathological lesions secondary to renal hypoperfusion after severe bleeding when compared with ringer lactate. results suggest that the reestablishment of intravascular volume with hes / . may preserve renal integrity secondary to blood loss. expression of aeg- , p and its clinicopathological significances in the malignant lesions of renal cell carcinomas h. erdem * , m. oktay, u. yildirim, a. k. uzunlar, m. a. kayikci * duzce university, dept. of pathology, turkey objective: astrocyte elevated gene- (aeg- also known as metadherin) is associated with various aspects of tumor malignancy. the aim of this study was to investigate p relationship between aeg- and prognostic parameters. method: this study was made paraffin blocks (tumoral samples), which were histopathologically diagnosed at department of pathology from to . subtypes of the cases were ( %) clear cell renal cell carcinomas (rcc) and ( %), non-clear rcc respectively. by immunohistochemical analysis, we investigated aeg- , p expression in carcinomas of kidney and we determined its relationship with clinicopathological parameters. results: there were significant relationship between increased aeg staining score and tumor capsule (p= . ), lymphovascular invasion (p= . ) and significant relationship between the increased diameter with the increase of p (p= . ). there were significant correlation between increased diameter of tumor and degree of increase the fuhrman (p= . ). conclusion: high aeg- , p expression correlates with prognostic parameters in the rcc. in addition, aeg- , p expression in rcc may be associated with tumor progression. relationship of cd , cox- and p in renal cell carcinomas with survival and other prognostic parameters: a tissue microarray study h. erdem * , u. yildirim, a. k. uzunlar, m. a. kayikci * duzce university, dept. of pathology, turkey objective: renal cell carcinomas (rcc) is the seventh most common human malignancy. rcc is now recognized to be a complex neoplasm consisting of several different tumor subtypes, each with distinct genetic and clinical features. the aim of this study was to investigate the expressions of cyclooxygenase- (cox- ), p and cd in rcc that has different clinicopathologic characteristics. method: this study was conducted on a total of paraffin embedded kidney samples (tumoral samples), which were histopathologically diagnosed at department of pathology from to . ihc stains for cox- , p and cd were performed on tissue microarray using standard procedures. results: there were significant correlations between cox- and subtype (p= , ), cox- and diameter (p= , ). significant relationships were found between p and age (p= , ), p and diameter (p= . ). besides, there were significant correlations between cd and furhman grade (p= , ). conclusion: cd , cox- , p expression correlates with prognostic parameters in the rcc patients. in addition, cox- , p , cd expression in rcc may be associated with tumor progression. objective: ebv related malignancies (skin cancers, lymphomas, kaposi sarcomas) complicates organ transplantation ebv-associated smooth-muscle tumors are rare. method: among kidney grafted patients, three developed ebv-smt. case . female y, with ebv-smt (generalized) case . female y, breast phylodes sarcoma, reviewed diagnosis: ebv-smt. case . male year, severe abdominal pain, gut tumor-like nodules were excised suspection of tbc. ebv-smt was microscopic diagnosis. results: progressive weight-loss all patients, chronic cough in patient , chest x ray multiple pulmonary lesions, on ct-scan paravertebral lesions. transthoracic biopsy: spindle cells proliferation with mixed cellularity eosinophilic cells intermingled fascicles resembling smooth muscle, mononuclear inflamatory cells and capillarie evoqued myofibroblastic tumor. no mitosis, mild nuclear atypia. negative immunohistochemestry: cd , cd , cd , ema, cytokeratin, s , cd . alpha-smooth muscle actin diffusely positive, evb-lmp negative. in situ hybridization : ebv + nuclei. ebv-smt was established. conclusion: smt in immunocompromised are ebv associated. primary target of ebv are b lymphocytes, may infect smooth-muscle through receptor for ebv cd mechanisms of ebv related-tumor genesis is integration of ebv-dna within alk locus in tumor cells. alkgene rearrangement and expression; associated with inflammatory myofibroblastic tumors, anaplastic large-cell lymphomas etc. clonally of multifocal ebv-smt using southern blot showed, multiple tumors constitute independent primary lesions. method: the degree of glomerulosclerosis was scored into - , mesangial proliferation was scored into (absent) and (present), and the degree of crescents was scored into - . glomerular injury score (gis) was obtained by adding the above scores and grouped into categories (group , score - ; group , - ; group , - ). results: serum creatinine level was significantly increased ( . ± . , . ± . , . ± . mg/dl, p= . ), estimated glomerular filtration rate (egfr) was significantly decreased ( . ± . , . ± . , . ± . ml/min, p= . ) and proteinuria was significantly increased as gis increased ( . ± . , . ± . , . ± . g/ h, p= . ). interstitial fibrosis of more than % of cortical area increased as gis increased ( . , . , and . %, p< . ). when a multivarate analysis was done, gis group was the most important predictive factor of egfr (p= . ) and proteinuria (p= . ). objective: the aim of the study is to evaluate the histopathological finding and the c d staining patterns of the patients who were biopsied due to graft dysfunction (gd) after initial well-function within month of deceased donor kidney transplantation (ddkt). method: histological analysis and c d immunostaining were performed on needle core biopsies. results: thirty-four patients (mean age: ± years, male: %) were included. histological analysis revealed acute rejection (ar) (n: ), acute tubular injury (ati) (n: ), allograft infection (n: ), borderline changes (n: ), normal morphology (n: ), and donor-related changes (n: ). c d staining was detected in % ( of ) biopsies; staining patterns were diffuse (n: ), focal (n: ) and minimal (n: ). diffuse (n: ) and focal (n: ) c d positivity accompanied of cases of ar. in remaining patients having non-ar histological picture, diffuse or focal c d positivity were detected in and cases, respectively. peritubular capillaritis was detected in . % ( / ) of biopsies, of which four had c d diffuse, one had c d focal positive. conclusion: beyond cold ischemia time-induced ati, immunological causes including antibody mediated process may play an important role in early impairment of graft function after ddkt. immunohistochemical analysis of the renal interstitial fibroblasts s. kostadinova-kunovska * , r. jovanovic, m. bogdanovska, v. janevska, l. grchevska, g. petrushevska * faculty of medicine, inst. of pathology, skopje, macedonia objective: renal fibrogenesis is a process common to all progressive kidney diseases. the main executive cell of this process is the fibroblast, by secreting and remodeling the extracellular matrix. the number of fibroblasts is minor in healthy kidney interstitium, but it increases during the process of fibrosis. their morphology and immunophenotype vary due to different intrinsic and extrinsic factors, thus making their identification and visualization, as well as determination of their origin, very difficult. method: we performed morphological and immunohistochemical analyses on kidney biopsies with primary glomerulopathy and interstitial fibrosis, using the following antibodies: vimentin, α-sma, s a , cadherin and cd . results: interstitial fibrosis with focal, rather than diffuse distribution, was present in all analyzed cases. the total interstitial fibroblast population was positive for vimentin, majority of the cells were positive for s a , and a smaller proportion of cells were positive for α-sma, cadherin and cd . furthermore, different cells in the fibroblastic population showed positivity for different markers. conclusion: the above stated observations contribute to the theory that different subpopulations of fibroblasts, with different origin, take part in the renal fibrogenesis. soluble epoxide hydrolase inhibition reduce blood pressure and organ damage independently of nitric oxide (no) in mice with goldblatt two kidney, one clip model ( k c) p. kujal * , l. kopkan, l. cervenka, z. vernerova * charles university, third faculty of medicine, prague, czech republic objective: investigate the role of no in the blood pressure (bp)-lowering effects of soluble epoxide hydrolase (seh) inhibition in k c model. method: the endothelial no synthase gene knockout mice and their wild-type controls were used. renal concentrations of epoxyeicosatrienoic acids (eets) and dihydroxyeicosatrienoic acids (dhets) were measured in nonclipped kidney. renal no synthase activity was determined by measuring the rate of formation of l-[ c]citruline. results: treatment with the seh inhibitor caused the same bp decrease that was associated with increase in daily sodium excretion in both types of mice. the ratio of eets/dhetes in the nonclipped kidney was increased and did not alter renal nos activity. seh inhibition reduced significantly glomerular and tubulointestitial injury. conclusion: bp-lowering effects of chronic seh inhibition in k c mice are associated with normalization of the reduced availability of biologically active eets in the nonclipped kidney and their direct natriuretic actions. total inflammation in -month surveillance renal transplant biopsies is associated with decreased renal function and de novo class donor specific antibody z. laszik * , s. chandran, f. vincenti * ucsf, dept. of pathology, san francisco, usa objective: the goal was to correlate inflammation and acute rejection (ar) with renal function and antibody status in -month renal transplant surveillance biopsies. method: relative risks of inflammation and ar in month biopsies was calculated by multivariable poisson regression. results: ar was seen in . % (n= ), borderline change in . % (n= ), and c d positivity in . % (n= ) of all biopsies. total cortical inflammation (ti), present in . % (n= ) cases, was associated with ml/min/ . m lower egfr at months ( % ci= − . , − . ). de novo hla class was identified in . % (n= ) and class dsa in . % (n= ), with most having mfi values < . increased risk of ar was seen with higher levels of de novo class (rr . , % ci= . , . ) or class dsa (rr . , % ci= . , . ). class (but not class ) dsa was associated with a higher risk of ti (rr . for ti-score ≥ , % ci= . , . ) at months. conclusion: both ti and ar in -month surveillance kidney transplant biopsies are strongly associated with de novo hla class dsa. total inflammation at months correlates with decreased kidney function. objective: tenofovir disoproxil fumarate has been used in the treatment of hiv patients producing occasional renal dysfunction and fanconi syndrome. method: a -year-old patient was referred with severe acute renal failure and important metabolic acidosis and glycosuria with normoglycemia in urine analysis, altogether with urinary alkaline ph and proteinuria with a protein/ creatinine ratio of . he was hiv and b positive diagnosed years earlier. his medication regimen consisted of lopinavir, ritonavir, tenofovir and lamivudine. the patient had a history of nausea, vomiting and some diarrhea with low fluid intake for a week. besides slight dehydration, the physical examination was unremarkable. renal ultrasound showed normal kidneys. with adequate fluid restoration, the renal function improved but other metabolic alterations like nephrogenic diabetes remained. results: renal biopsy revealed toxic acute tubular necrosis predominantly involving proximal tubules with prominent eosinophilic inclusions within proximal tubular cell cytoplasm, which represented giant, abnormal mitochondria, consistent with tenofovir toxicity. some histochemichal studies (cox and sdh) were performed. conclusion: although prospective clinical trials have demonstrated a low incidence of renal toxicity with tenofovir, there are several such reports in the literature. current recommendations suggest close monitoring of renal function after initiation of therapy with tenofovir, specially at the beginning and when used in combination with certain antiretroviral agents. objective: nutcracker syndrome (ns) is caused by compression of the left renal vein between the aorta and superior mesenteric artery. the main presenting symptom of this rare entity is haematuria, with various degrees of proteinuria. the diagnosis of ns syndrome is primarily by imaging and can sometimes be challenging. method: a -year-old male was referred to nephrology with a months history of intermittent gross haematuria. all blood test including blood cell count, biochemistry, immunoglobulin electrophoresis, antinuclear antibody were normal. urinary analysis revealed numerous red blood cells and a protein/creatinine ratio of . . a previous cystoscopic examination was normal. renal ultrasound revealed a diffuse increased echogenicity. results: renal biopsy showed a normal histology with no immunofluorescence deposits. ct scan revealed dilatation of the distal left renal vein with narrowing between the superior mesenteric artery and the aorta. conclusion: ns is a rare entity causing haematuria. there are few reported cases with histology, although it usually shows no abnormalities. the proposed mechanism for the haematuria is an abnormal communication between the submucosal venous plexus and the calyceal system presumably induced by renal venous hypertension. banff initiative for quality assurance in transplantation (bifquit): reproducibility for bkv immunohistochemistry in renal allografts m. mengel * , s. chan, j. climenhaga, h. regele, b. colvin, p. randhawa * university of alberta, laboratory of medicine, edmonton, canada objective: detection of bk virus associated large t-antigen is crucial for the diagnosis of polyomavirus nephropathy. method: in an international multi-centre trial we assessed the inter-observer and inter-laboratory variability for bk immunohistochemistry. a tissue microarray was constructed comprising specimens representative of the whole analytical spectrum from negative over mild to strong sv positive cases. participants at centers stained the tma slides using local protocols. participants evaluated their slides following a provisional banff grading schema. details regarding local staining protocols and evaluation scores were collected online. stained slides were returned for centralized panel re-evaluation. weighted kappa statistics were used to determine the variability. results: the bk inter-observer reproducibility was substantial (mean kappa . ), but inter-laboratory reproducibility was below chance (kappa − . ). separating components of bk evaluation schema into stain intensity and stain percentage showed no significant improvements in reproducibility. however, collapsing the proposed bk scoring schema into a simple positive/negative call improved bk inter-laboratory variability to . . conclusion: these results indicate a significant variability between laboratories for detecting the sv large-t antigen by immunohistochemistry in paraffin sections. any proposed grading schema for bk nephropathy, which is dependent on percentage and intensity of nuclear staining, will essentially not be reproducible between laboratories. objective: cryopyrin-associated periodic syndrome (caps) is a rare hereditary inflammatory disorder with three differents phenotypes: familial cold autoinflamatory syndrome, muckle-wells syndrome and neonatal-onset multisysten inflamtory disease caps results from a mutation of the nlrp gene ( q ) coding for cryopyrin, which forms intracellular protein complexes (inflammasomes). results: case report: year old women with chronic renal disease stage (creatinin , mg/dl and proteinuria mg/ h) who developed urticarial episodies since months old related to cold exposure. after years old patient describe associated to these episodios bilateral arthritis (knes, ankles and elbows), conjunctival inflammation, shivers, and asthenia, more frequently in winter and precipitated by cold exposure, air conditioning, stress and menstruatión. aditionaly the patient developed in last years bilateral hypoacusis a kidney biopsy showed a amorphous, acellular and acidophilic material congo red positive deposits at interstition, arteries and arterioles and less frequent at glomeruli. this material displays apple green birefringence by polarizad light microscopy. diagnosis: secondary renal amyloidosis conclusion: the clinico-pathologial findings of this case are compatible with muckle-wells syndrome. secondary amyloidosis is a severe complication which occurs in % of muckle-wells cases. amyloidosis is cause by increase of c-protein and a amyloid during the episodies before described and deposition of the a amyloid in differents tissues. glomerulocystic disease associated with thrombotic microangiopathy in two kidney allografts j. m. mosquera reboredo * , e. vazquez martul * complexo hospitalario universitario, dept. of pathology, la coruña, spain objective: glomerulocystic kidney disease (gckd) is a rare condition usually congenital and reported in infants and young children. only few cases of adquired gckd had been reported often following hemolytic uremic syndrome (hus). method: histological study of two kidney graft explants. results: we present two cases of kidney transplant who developed hus in allografts. both cases showed at the histological examination typical vascular and glomerular changes of thrombotic microangyopathy (tma). a cystic transformation with increase of urinary space and retraction of glomerular tuft was frequently observed. chronic transplant vasculopathy was found in the two cases. humoral active rejection was also demonstrate in one case with moderate peritubular capillaritis and glomerulitis and diffuse c d deposition at peritubular capillaries. conclusion: in a few cases gckd appear to develop after another kidney disease includes single case reports of gckd associated with mesangial glomerulonephritis, wegener's granulomatosis, progressive systemic sclerosis, after hus (include some case in adult patient). our two cases represent a initial stage of adquired gckd. the ethiopathogenetic relationship is not clear but some authors propous that cystic dilatation of the bowman's capsule associated to tma/ hus may be secondary to ischemic mechanism. case report: cytomegalovirus gastritis in renal transplanted man f. noroozinia * , k. makhdoomi, a. esmaeili, a. saffarifard * emam khomeini hospital, dept. of pathology, urmia, iran objective: cmv is an important pathogen in immunocompromised hosts, including patients with aids, neonates and transplant recipients. this infection develops in - % of transplant patients. upper gi symptoms in solid organ recipient are common ( %) and clinical signs are more serious in % of cases. method: we report a year old man with end stage renal disease underwent kidney transplantation from a cmv negative donor on june . after . months he admitted with fever, generalized body pain, oral aphtous ulcers and epigastric pain accompanied by malaise. endoscopic examination revealed multiple antral erosions with surrounding erythema. clinicopathological investigations revealed cmv viremia with a ig-m antibody titer and cmv gastritis confirmed by histopathological examination. results: the patient was started on intravenous (iv) ganciclovir mg/kg per day every h initially for weeks, afterwards the fever decreased; cell blood counts throwback to normal ranges and general condition of the patients improved. conclusion: cmv infection develops in - % of the transplant patients. the colon and stomach are the most common sites of gastrointestinal infection. though the rate of gi affliction by cmv is high, localization to the gastric antrum is not common. immunohistochemistry study of c-kit expression in renal cell carcinoma f. noroozinia * , f. abbasi, z. yekta, f. meisami, a. saffarifard * emam khomeini hospital, dept. of pathology, urmia, iran objective: renal cell carcinomas include about - % of adults neoplasms and - % of all renal tumors. in many cases, it is possible to distinguish. rcc subtypes on the basis of hematoxylin-eosin staining alone. however, overlapping morphologic features pose some difficulties in making a proper diagnosis. to render an accurate diagnosis, additional methods like immunohistochemichal staining against c-kit have been recommended. method: we reviewed cases of rcc diagnosed during years. formalin fixed, paraffin embedded specimens was available in cases. the expression of c-kit was evaluated using immunohitochemistry. results: six cases of clear cell type ( . %), of papillary type ( . %), and of chromophobe type ( %) were positive for c-kit that considering chi-square test there is significant relevation between rcc's subtypes and c-kit expression (p: . ). from cases with renal vein invasion, showed positive expression of c-kit ( . %) and in cases with no venous invasion, c-kit expression was detected in ( . ), so no significant relation was found between renal vein invasion and c-kit expression. conclusion: the expression of c-kit in rcc may have diagnostic significance. objective: igg -rsd shows abundant igg -positive plasma cells, diffuse fibrosis, and increased serum igg levels. although igg -rsd typically results in autoimmune pancreatitis (aip), any organ may be involved. thus, igg -rsd tin often goes unrecognized in the absence of aip. this is an igg -rsd tin case with mediastinal lymph node and pulmonary involvement. method: a -year-old male presented with rapidly progressive renal failure, hypergammaglobulinemia, hypocomplementemia, and enlarged mediastinal lymph nodes and bilateral pulmonary nodules on ct. renal and mediastinal lymph node biopsies were performed and igg ihc was done on both after standard techniques. results: the renal cortex and medulla showed diffuse interstitial fibrosis with tubular atrophy and abundant plasma cells ( igg -positive cells/hpf), numerous lymphocytes and some eosinophils. if showed igg, c , kappa, and lambda granular deposits in tubular basal membranes and bowmann capsules. lymph node follicular and paracortical hyperplasia with abundance of mostly igg positive plasma cells was seen. an igg -rsd diagnosis was rendered and high serum igg levels were demonstrated. steroid therapy resulted in lymph node and lung nodule reduction. conclusion: tin with abundant plasma cells and diffuse interstitial fibrosis, especially if accompanied by hypergammaglobulinemia, hypocomplementemia, or extrarenal involvement, should suggest igg -rsd and prompt serum igg level determination and renal igg ihc. objective: a -year-old woman was admitted for chronic renal failure. clinical examination was normal. biological explorations showed creatinin clearance around ml/min, tubular proteinuria with bence jones κ protein. serum immunoelectrophoresis identified abnormal monoclonal immunoglobulin g and κ-light chains (lc). bone marrow histology was normal. method: kidney biopsy revealed diffuse intracytoplasmic vacuoles in the proximal tubules resembling osmotic nephrosis. distal tubules, glomeruli, vessels and interstitial compartment were normal. immunofluorescence (including anti-κ and -λ staining) was negative. electronic microscopy (em) revealed intracytoplasmic immunoglobulinic crystals containing κ lc inside the vacuoles, leading to the diagnosis of light chain proximal tubulopathy (lcpt). results: lcpt is a rare complication of dysglobulinemia. it may be associated with crystals within the cytoplasm of proximal, less frequently distal, tubular cells, consisting more frequently in κ lc. rarely, diffuse tubular vacuolization is present, often indistinguishable from osmotic nephrosis. in our case, there was no proximal tubular dysfunction and immunofluorescence was negative. the first evocated diagnosis by light microscopy was "osmotic nephrosis". however, we failed to identify any causal factor. finally, the diagnosis was performed by immunoem. conclusion: in conclusion, before a picture of osmotic nephrosis without obvious cause, em and immunoem may be helpful for the diagnosis of lcpt, often revealing a dysglobulinemia. opportunistic infections in renal transplantation -a case series r. sampaio * , r. dias, p. farrajota, a. coelho, t. almeida, a. duarte, j. r. vizcaino * anatomia patológica, valpaços, portugal objective: oporto's hospital centre is one of the portuguese hospitals with more renal transplantation activity (performed since ). although increasingly rare, opportunistic infections (oi) in transplanted patients remain a major diagnostic challenge and are associated with high mortality rate. method: in order to evaluate the incidence of oi in renal transplant patients and to identify the location of infection, the respective techniques of diagnosis used and the survival time after infection, we conducted a retrospective study using the nephrology department's database on renal transplants. we consulted the registries from to . results: we investigated cases and found cases of oi caused by herpes virus (n= ), cytomegalovirus (n= ), polyomavirus (n= ), aspergillosis (n= ), alternaria (n= ), mucormycosis (n= ), candidiasis (n= ), tuberculosis (n= ), cryptococcus (n= ) and pneumocystis (n= ). the lung and urogenital system were the most affected systems. kidneys were affected in cases respectively by mucormycosis, tuberculosis and cryptococcus. of all the cases of oi, were diagnosed by the department of anatomic pathology ( by biopsy; by cytology; by biopsy and cytology). conclusion: in many cases the diagnosis could only be performed through histologic/cytologic examination. prompt diagnosis and treatment are necessary to avoid life threatening complications and may greatly improve prognosis. papillary renal cell carcinoma with osseous metaplasia and bone marrow elements: a case report l. l. santos * , a. polónia, r. henrique, c. lobo * ipo porto, dept. of pathology, viana do castelo, portugal objective: renal cell carcinomas might display foci of calcification and even ossification. this rare event has been reported mostly in the clear cell variant. herein, we present the case of a year-old man, previously diagnosed with colon cancer, incidentally found to have a calcified mass in the kidney, which was interpreted as non-characteristic for renal cell origin. results: radical nephrectomy was performed. a . × . × . cm tumor was found, with a heterogeneous brown cut surface, containing areas of necrotic tissue and extensive calcifications. histopathologic examination disclosed papillary structures covered by small cells with scant cytoplasm, dispersed among mature bone tissue enclosing marrow elements. epithelial tumor cells were imunorreactive for cytokeratins, cd and pax , and negative with hmb- . hale`s colloidal iron was negative. trisomy of p, p and p was detected by fish. a diagnosis of prcc type , fuhrman grade , with extensive osseous metaplasia was rendered. conclusion: ossification of renal cell tumors is rare, occurring mostly in clear cell type and the underlying mechanism is unclear. these tumors show atypical radiological features and might be confused with non-renal cell tumors. to the best of our knowledge, this is the first report of prcc with osseous metaplasia and bone marrow elements. the effect of doxycycline on glomerulosclerosis in / renal ablation s. sarioglu * , d. sonmez, a. celik, f. saglam, o. yilmaz, e. koraltan, z. cavdar, g. oktay * dokuz eylul university, faculty of medicine, izmir, turkey objective: the effect of matrix metalloproteinase (mmp) inhibitors in segmental sclerosis is unknown. the aim of this study is to investigate the effect of a mmp inhibitor, doxycycline, on glomerulosclerosis (gs) in renal ablation nephropathy. method: fourteen of the female wistar albinos were / nephrectomised. doxycyline was given to half of each group ( mg/kg/day total days). after sacrification, the gs, mmp- , mmp- , timp- expressions were analyzed histopathologically. pro and active mmp- and - were analyzed by gelatin zymography. timp- and timp- were measured with elisa assay. results: doxycycline administration to the / nephrectomy group improved gs, but did not inhibit glomerular mmp- or cortical pro-and active-mmp- and pro-mmp but increased timp- and timp- expression in all groups in cortical tissue. mmp- expression and gs were increased in all groups receiving doxycycline. conclusion: we have demonstrated improved gs in renal ablation model by doxycycline administration but also doxycycline has an unexpected adverse effect. the effect of doxycycline on the expression of mmp- and - cannot explain the improvement in gs, but increased cortical tipm- and - may be an important contributing factor for inhibition of mmps. digitally reinforced hematoxylin-eosine polarization in diagnosis of renal amyloidosis s. sen * , b. sarsik * ege university, faculty of medicine, izmir, turkey objective: systemic amyloidosis is a rare disorder, characterized by extracellular accumulation of congo-red (cr) positive fibrillar amyloid protein deposits. the kidney is the most commonly affected organ by systemic amyloidosis. cr staining which increases the positive birefringence of the weakly birefringent unstained amyloid. in this study we investigated potential power of digitally reinforced birefringence of routine hematoxylin-eosine (he) slides on the renal biopsies. method: we reviewed he stained slides for polarization. sixty five amyloidosis cases were diagnosed by renal biopsy from to at our laboratory. all biopsies were evaluated by light and immunofluorescence microscopy. slides were reevaluated blindly using a microscope (olympus bx ) attached polarization filter and connected to a digital camera (olympus dp , sal). depositions which show green birefringence on he with digitalized microscopy were considered as positive and results were confirmed using cr. results: of the cr confirmed amyloid positive biopsies, showed green birefringence with he. of the cr confirmed amyloid negative biopsies, two were considered as false positive. the sensitivity, specificity, positive and negative predictive values were estimated as %, %, % and %, respectively. conclusion: we concluded digitally polarized he sections can be used as a fast and first step diagnostic method for renal amyloidosis. sirolimus ameliorates cyclosporin-induced nephrotoxicity in a rat modelfocus on renal lesions, oxidative stress, inflammation, proliferation and angiogenesis j. sereno * , a. m. romão, m. teixeira, b. parada, c. mega, h. vala, e. t. lemos, f. teixeir, f. reis * ibili, medicine faculty, laboratory of pharmacology and experimental therapeutics, coimbra, portugal objective: sirolimus (srl) have been pointed as a feasible option for minimize the use of cyclosporin a (csa), especially because of putatively less nephrotoxicity. this study aimed to characterize the histological lesions and the molecular pathways implicated in csa-induced nephropathy and prevention when converted to srl. method: the following groups (n= ) were tested during weeks: vehicle, csa, srl and conversion (csa weeks + srl weeks). bp and hr were monitored. blood was collected and kidney gene expression of markers of inflammation, proliferation, angiogenesis and oxidative stress were assessed. histology: h&e, pas and gordon & sweets staining. statistics: anova and post´hoc tests (p< . ). results: after week of csa treatment, there was important kidney lesions, including glomerular, tubulointerstitial and vascular: mesangial expansion, atrophy, bowman capsule enlargement, hyaline cylinders formation, tubular calcification and vascular congestion, as well as arteriolar vacuolization and arteriolosclerosis. conversion to srl, ht and tachycardia were reduced, accompanied by amelioration of kidney dysfunction and lesions (glomerulosclerosis and tubulointerstitial fibrosis), together with reduction of oxidative stress, proliferation and angiogenesis. conclusion: in conclusion, srl ameliorates csa-induced nephrotoxicity in a rat model, which might be due to protection against oxidative stress, proliferation and angiogenesis, but these mechanisms deserve better exploitation. acknowledgements: fct(sfrh/bd/ / ). non-lupus "full-house" nephropathy in serbian population in last six years j. vjestica * , s. cirovic, s. tatic, r. naumovic, s. simic-ogrizovic, j. markovic-lipkovski * inst. for pathology, medical faculty, belgrade, serbia diffuse glomerular and sometimes focal mesangial staining of immune complex depositas (iga, igg, igm, c , and c q), also known as "full-house" pattern commonly indicates lupus nephritis. however, some non-lupus nephropathy also can be present with a "full-house" immunofluorescence pattern, mimicking lupus nephritis. the aim of this study was to define the clinicopathological spectrum of originally non-lupus "fullhouse" nephropathy. biopsies from january till december were analyzed in order to identify all renal biopsies cases showing "full-house" pattern. the study included "full-house" renal biopsy diagnosiss. from all analyzed cases ( %) had lupus nephritis expressing "full-house" pattern and ( %) cases had non-lupus "full-house" nephropathy. in the absence of clinical and/or serological evidence of systemic lupus erythematosus (sle), at the time of renal biopsy, in cases diagnose was membranous glomerulonephritis (gn), than mesangioproliferative gn, membranoproliferative gn, iga nephropathy, rapidly-progressive gn and membranous/membranoproliferative gn cases. nonlupus "full-house" nephropathy is present in broad spectrum of different types of gn, predominantly in cases of membranous gn. the possibility of "full-house" nephropathy preceding the emergence of overt sle remained to be clarified. objective: although the renoprotective effects of prostacyclin have been demonstrated in many studies, the protection mechanisms of prostacyclin in chronic kidney disease, especially at the terminal stage, are still remained unclear. in the present study, we performed pathological and pathphysiological analyses of prostacyclin renoprotective effects using a stable prostacyclin analogue, beraprost sodium, in the disease kidney of anti-gbm glomerulonephritis (gn) rats. method: beraprost was administrated from weeks after induction of gn. the condition of renal microvascular network and localization of apoptotic cells were examined using renal vascular corrosion casts, immunostainings and tunel-staining. the intracellular apoptotic signaling pathway was analyzed by western blot and qpcr. results: in the kidney of beraprost-treated rat, significantly high density of renal microvascular network was maintained, and apoptosis of vascular endothelial cells was suppressed even at the terminal stage of anti-gbm gn. pathophysiological analyses revealed that transcriptional and post-translational modifications of bcl and xiap, which were anti-apoptotic proteins in mitochondria dependent apoptotic pathway, were occurred in the kidney of beraprost-treated rat. conclusion: these results suggested that prostacyclin protects renal vascular network by inhibiting mitochondria dependent endothelial apoptosis, and it play an important role for preservation of renal function in the chronic kidney disease. wednesday, september , . - . objective: although villous adenomas commonly occur in the gastrointestinal tract villous adenomas of the urinary tract, including the bladder, are infrequently encountered. method: we report a case of urinary bladder villous adenoma in a -year-old man. the patient was undergone cystoprostatectomy because of diagnosis infiltrative urothelial carcinoma in the transurethral resection of bladder. results: histopathological examination of cystoprostatectomy specimen there was no urothelial carcinoma. a , cm polypoid mass was seen at the dome of the urinary bladder. histopathology confirmed that this tumor was a villous adenoma with a polypoid growth of the glandular epithelium consisting of small tubular glands, dilated cystic glands or papillary fronds lined by a columnar epithelium. the glandular epithelial cells displayed mild nuclear atypia and nuclear pseudostratification with some mucus cells admixed. the patient was diagnosed with a rare case of villous adenoma of the bladder. over the past months of follow up, the patient is alive and no metastasis. conclusion: patients with isolated villous adenomas in the urinary bladder have an excellent prognosis and surgical resection is curative. however, it is uncertain whether an untreated lesion might eventually develop into an adenocarcinoma. therefore, close follow up is recommended because of the possibility that this condition might be premalignant. caprin overexpression in urothelial carcinomas of bladder b. akkaya * , z. cetin, s. berker-karauzum, m. baykara * akdeniz university, school of medicine, antalya, turkey objective: caprin encoded by cytoplasmic activation/ protein- gene located in p chromosome region. it has been reported that caprin is associated with cell proliferation in various types of cell lineages. method: we researched whether caprin might be overexpressed or not in urothelial carcinoma of bladder and its overexpression could be correlated with clinicopathologic parameter (age, sex, invasion). fifty urothelial carcinoma of bladder ( infiltrative; non-infiltrative) were stained by immunohistochemically in tissue microarrays. results: the expression of caprin was observed in infiltrative urotelial carcinoma cases (% ) and non-invasive urothelial carcinoma cases (% ). age range was - . eight patients were female, patients were male. conclusion: in the literature caprin overexpression was reported in different types of tumors including esophageal, stomach, prostate, lung, liver. caprin overexpression might be correlated with the cellular proliferation potential. to determine of importance of caprin - overexpression new studies are necessary. objective: pseudohyperplastic squamous cell carcinoma of the penis (pscc) is a low grade tumor with specific clinical and pathological features. this very uncommon tumor occurs in association with lichen sclerosus, the main location is foreskin, and the high degree of differentiation may difficult its discrimination with pseudoepitheliomatous hyperplasia. method: a -year-old male presented a penis lesion involving glans and foreskin, clinically suspicious of malignancy. size was , × , cm and a conservative resection was done. pathological study was performed, and hpv detection with a commercially available kit: pcr amplification and reverse hybridization with probes to hpv types. results: the lesion showed a non-verrucous well differentiated squamous cell tumor. upper layers lacked any atypia but infiltrative growth was evident in basal layers, with atypical cells and mitosis. lichen slerosus changes were evident bordering the tumor. hpv was negative for all types studied. no further treatment was employed and after a months follow-up no recurrence has been observed. conclusion: pscc should be taken in mind when handling penis tumors. a correct differentiation from benign lesions and a knowledge of its low grade to avoid overtreatment will benefit patients. lichen sclerosus and not hpv seems to play a precancerous role. evaluation of sunitinib malate and meloxicam as single agents or in combination in bladder cancer cell lines r. arantes * , r. pinto-leite, c. lopes, l. santos, a. colaço, p. oliveira * utad, dept. of veterinary sciences, vila real, portugal objective: currently accepted for the treatment of advanced renal cancer, sunitinib malate is a small molecule inhibitor of the vegfr family, with ability to regulate tumor growth, progression, angiogenesis and metastasis. several reports have suggested that encouraging effects can be achieved by combining cox- inhibitors with anticancer agents. the goal of this work was to evaluate the effects of sunitinib malate and meloxicam isolated and combined on three human bladder cancer cell lines. method: t , and ht cells were treated with several concentrations of sunitinib malate and meloxicam, as single agents or in combined schedule. their influence on cell proliferation was determined by mtt method after h of treatment. control samples were processed in the same way as treated samples but in drug-free medium. absorbance values of each well were read at nm using an elisa plate reader. results: a reduction in cell proliferation rate was observed when all cell lines were treated either with sunitinib malate or meloxicam isolated. simultaneous exposure to both agents enhances the inhibition of cell proliferation. statistical significances were obtained when treatment groups were compared with control group. conclusion: these results suggest a potential clinical application of sunitinib malate in combination with meloxicam on bladder cancer. bcl- expression in prostate carcinomas b. balinisteanu * , a. dema, s. taban, c. lazureanu, d. herman, s. ursoniu, a. loghin, a. vaduva * municipal hospital, pathology, timisoara, romania objective: the disturbance of apoptosis represents an important event in the genesis of tumors with different localization. the study of anti-apoptotic protein bcl- expression from the perspectives of the prognostic and predictive value in prostate cancer has led to inconsistent results, even contradictory. method: expression of bcl- was analyzed in groups of prostatic carcinoma: localized, locally advanced and with distant metastases. for histological grading of carcinomas we used gleason score. classification of the tumors into prognostic subgroups was made according to nccn guidelines. for the immunohistochemical study we used anti-bcl- antibody (clone ), envision system, visualization with diaminobenzidine. the results of immunohistochemical reaction were assessed by evaluating the extent and intensity of immunostaining. statistical analysis was performed using stata . . results: of the analyzed cases of prostate carcinomas showed bcl- over-expression: , % localized carcinomas, , % locally advanced carcinomas and , % carcinomas with distant metastases (p< . ). although most of the bcl- positive tumors were poorly and moderately differentiated, the correlation between bcl- over-expression and tumor grade did not show statistical significance (p= , ). conclusion: bcl- overexpression in advanced prostate carcinomas suggests involvement of this marker in the progression of tumors in this location. urothelial carcinoma of the bladder: a clinicopathologic study of cases g. benkhedda * , s. khalifa, y. lamouti * chu frantz fanon, dept. of pathology, blida, algeria objective: urothélial carcinoma (uc) accounts for nearly % of urinary bladder tumors.a variety of histological variant of uc have been recently recognosed. some variants have prognostic and therapeutic implications. the aim to this study is to assess the pathological features from our series and to compare our results that of the literature. method: we retrospectively studied patients who were diagnosed histopthologically with urothelial carcinoma using the who classification system. results: the mean age of patients at diagnosis was (range, - years). , % were male ( h/ f). all tumors were classified as urothelial carcinomas: , % urothelial neoplasm with squamous differentiation, , % with glandular differentiation, , % urothelial tumors nested and % sarcomatoid. in this study most tumors were grade ( cases) and stage pt . conclusion: adult urothelium has the capacity to undergo several pathways of phenotypic cellular and structural differentiation as a result of the embryological origin of the bladder from the multipotent tissues of the cloacal endoderm and the mesodermal wolffian ducts. the clinical course of bladder cancer varies depending on the histological type of neoplasm, grade and stage of the tumor. hight-grade muscle-invasive urothelial cancers and tumors schowing variant microscopic morphology have in general hight mortality and poor prognosis. comparison of insignificant cancer detection rates in prostatectomies performed following and -core biopsy schemes u. berber * , a. haholu, i. yilmaz, z. kucukodaci, d. demirel * gata heh, dept. of pathology, istanbul, turkey objective: widespread use of extended biopsy protocols have increased the prostate cancer detection rates. besides this improvement, whether detection of clinically insignificant cancer detection rates are increased by extended biopsy protocols is not well documented. in the study, we aimed to compare the rates of insignificant cancers found in prostatectomy specimens performed following and -core biopsy protocols. method: retrospectively, we investigated the low volume/ low grade (lv/lg) prostate cancers in prostatectomy specimens. tumors volumes were calculated digitally as multiplying total tumor areas by mm for average block thickness, and corrected for tissue shrinkage by multiplying a factor of . . results: of the prostatectomies, were performed following sextant technique, and were performed after -core protocol. review of the h&e stained sections revealed insignificant cancers. number of lv/lg tumors found in and -core groups were ( . %) and ( . %), respectively. conclusion: when compared to sextant technique, detection of lv/lg tumors were significantly raised in prostatectomies performed following -core protocol, and this increase points out the need for new approaches in patient management to avoid overtreatment after extended biopsy protocols. ps- - impact of total core length for cancer detection in a lateral zone targetted -core prostate biopsy scheme u. berber * , a. haholu, z. kucukodaci, i. yilmaz, d. demirel * gata heh, dept. of pathology, istanbul, turkey conclusion: total core length is significantly associated with cancer detection rates and may be used as a reliable adjunctive tool in deciding repeat biopsies for patients with negative biopsy result. objective: primitive neuroectodermal tumors (pnets) are highly malignant tumors of neuroectodermal origin. we report a case of renal pnet in a -year-old male with a -month history of intermittent hematuria.. he underwent a right radical nephrectomy. macroscopically, the inferior pole was replaced by a multinodular, grey, glistening tumour measuring . / / cm, with foci of necrosis and hemorrhage. method: serial histological sections have been assessed using hematoxylin-eosin and van gieson stain and the indirect immunohistochemical analysis for antibodies: mnf , vim, cd , nse, mic /cd . results: histological examination revealed a uniform population of undifferentiated, small-to medium-sized tumor cells, arranged in alveolar-insular patterns, with round to oval nuclei, small nucleoli, numerous signs of mitosis and scattered apoptosis, geographic zones of necrosis. dispersed cells showed cytoplasmic vimentin positivity favouring the diagnosis of pnet. few tumoral cells appeared positive for mnf . expression of cd was positive in a large number of tumoral cells and an area of the tumour exhibit a milder reaction of positive nse. mic was positive with moderate staining in almost all tumoral cells. conclusion: diagnosis is based on histology and immunohistochemistry but pathological evaluation can be challenging because of the differential diagnosis with other small round cell tumors. nonamyloid fibrillary glomerulonephritis: presentation of two cases e. beretouli * , g. dimas, g. karayannopoulou, t. koletsa, d. grekas, g. karkavelas * ahepa hospital, dept. of pathology, thessaloniki, greece objective: fibrillary glomerulonephritis (fgn) is a rare disease, characterized by fibrillar deposits in the mesangium and the glomerular capillary loops. these deposits do not have an amyloidlike cross-β structure and are readily distinguishable from amyloid by the larger thickness of fibrils and lack of congo red staining. method: we report two cases of a -and -year-old women, who presented with severe nephrotic syndrome, rapidly progressive chronic kidney disease and lymphoproliferative disorders. glomerular crescents were present in about % of both renal biopsy specimens ( of and of glomeruli, respectively). immunohistochemical analysis, immunofluorescence and an electron microscopy (em) studies were performed. results: renal biopsy showed a deposition of an amyloidlike extraneous substance in the mesangium, as well as within the glomerular basement membranes. congo red staining was negative. the em examination revealed fgn. conclusion: fgn must be included in the differential diagnosis of rapidly progressive chronic renal disease. em confirm the diagnosis of fgn, which suggests a poor outcome. objective: urachal carcinomas represent less than % of bladder-related cancers. most are adenocarcinomas, but urothelial, squamous and small cell carcinomas may occur. there are pathological criteria for assessing an urachal origin. a specific staging system is lacking for these tumors. method: a mass of the bladder dome was found in two and year-old men. a year-old woman presented an urachal cyst. ct-scan revealed no other tumors and partial cystecomy was performed. pathological examination showed partially cystic adenocarcinomas, one enteric type g , and two mucinous type g . two tumors extended into the bladder mucosa and one was limited to the urachal cyst. surgical margins were negative. immunostainings were positive for ck , ck , cdx , cytoplasmic beta-catenin, and negative for p . results: with a follow-up of , months and years, all patients are free of disease. conclusion: no tnm classification exists for urachal carcinomas. specific staging systems have been proposed by sheldon, and more recently by the mayo clinic. two cases are sheldon iiia/mayo clinic ii, and one sheldon ii/mayo clinic i. since stage, grade and surgical margins are the main prognostic factors, a clear and relevant staging system is needed for these rare carcinomas. objective: renal cell carcinoma (rcc) with two different histologies must be included in the unclassified group (who classification). whether these cases should be included in this histological subgroup or be considered hybrid rcc is a matter of debate. we report one of such cases in which papillary and chromophobe phenotypes meet. method: a cm in diameter asymptomatic left renal mass was discovered incidentally in the radiological follow-up of a breast carcinoma diagnosed years before in a yearold woman. follow-up showed right adrenal gland metastasis. patient died months later. results: grossly, tumor well circumscribed, tan-yellow, with haemorrhagic and necrotic areas. the neoplasm showed two different histologies clearly defined: one showed solid nests of polyhedral cells, with eosinofilic cytoplasm, central and hyperchromatic nuclei, occasional mitosis recapitulating chromophobe rcc. the other component presented a well defined tubulopapillary growth pattern typical of papillary rcc. focally sarcomatoid transformation, with tumor necrosis and chronic inflammation. by immunohistochemistry, chromophobe and papillary areas retained their specific phenotypes. conclusion: hybrid renal carcinomas do exist, but they are most probably hidden in the unclassified group of renal tumors. however, the exact histological context for which a renal neoplasm deserves the name "hybrid" remains to be defined. objective: renal cell carcinoma with thyroid-like follicular pattern is a rare histological subtype of renal carcinomas that has been very recently described. method: a . cm in diameter asymptomatic left renal tumor was discovered during the study of a macroscopic hematuria in a years old man. the lesion was organconfined. results: grossly, the tumor was a well circumscribed, solid, brown and homogenous intraparenchymatous nodule. proliferating cells were arranged in a microfollicular pattern with colloid-like material resembling thyroid adenoma. cells displayed low grade nuclear features and had eosinophilic cytoplasm. some areas showed a solid pattern of growth resembling an oncocytoid neoplasm. by inmunohistochemistry, the tumor was negative for thyroglobulin and ttf , and positive for ema, ck , ae /ae , and ecadherin. conclusion: renal cell carcinoma with microfollicular thyroid-like features has been very recently identified in the literature. there is no agreement on the exact nature of this neoplasm so far, and the who classification of renal tumors still does not consider this phenotype as a distinct histological subtype. anyway, the tumor must be distinguished from metastatic thryroid carcinoma, another quite unusual condition. evidence for clonal fibroblast proliferation and autoimmune process in idiopathic retroperitoneal fibrosis l. cheng * , j. clevenger, a. lopez-beltran * indiana university, dept. of pathology, indianapolis, usa objective: we sought to determine if idiopathic retroperitoneal fibrosis is clonal process and if it is an autoimmune, or igg -driven, process. method: thirty cases of idiopathic retroperitoneal fibrosis, in whom known causes of retroperitoneal fibrosis were excluded and those for which paraffin blocks were available, were included in this study. we performed clonality analysis in female patients. genomic dna samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser capture microdissection. results: eight of information cases ( %) showed nonrandom x-chromosome inactivation, or a clonal process. of the patients for which igg analysis was performed, ( %) were positive for igg -positive plasma cells and all were negative for alk. of the patients, for which both clonality analysis and igg analysis were performed, were clonal and igg negative ( %), were clonal and igg positive ( %), were nonclonal and igg positive ( %), and were nonclonal and igg negative ( %). conclusion: our data indicate that a significant proportion ( %) of idiopathic retroperitoneal fibrosis cases in females is associated with a clonal expansion of fibroblasts. in addition, a subset of idiopathic retroperitoneal fibrosis cases could be classified in the igg -related sclerosing disease spectrum. although a conclusive association with malignancy, urologic disorder, or systemic disease has not been established, often the lesion carries a challenging clinical differential diagnosis that includes malignancy. we examined clinical and histopathologic characteristics in patients. medical records were assessed for presentation, clinical diagnosis, associated urothelial carcinoma, radiation treatment, tobacco use, immunologic/urologic disorder, and treatment strategy/outcome. results: the mean age was (range - years). presenting symptoms were: pain ( %), hematuria ( %), and dysuria ( %), in contrast to asymptomatic ( %). clinical diagnosis favored malignancy in % of cases. concurrent or subsequent urothelial carcinoma was present for five patients ( %), though none developed urethral carcinoma. histologic features included mixed hyperplastic urothelial and squamous lining, overlying a variably fibrotic, edematous, inflamed, and vascular stroma. invaginations of urothelium extending into the stroma were common ( %), showing rounded nests with cystic or glandular luminal spaces, similar to urethritis cystica/glandularis, without intestinal metaplasia. two lesions included an organizing thrombus, one with intravascular papillary endothelial hyperplasia. twenty patients were treated with topical medications without resolution. three lesions recurred ( %) after excision. conclusion: urethral caruncle is an uncommon lesion that may clinically mimic benign and malignant conditions, making tissue diagnosis critically important. human papillomavirus (hpv) is not involved in urothelial tumorigenesis l. cheng * , r. alexander, a. lopez-beltran * indiana university, dept. of pathology, indianapolis, usa objective: the purposes of this study were to investigate the possible role of human papillomavirus in the development of squamous cell carcinoma of the urinary bladder and to determine if p expression could serve as a surrogate marker for human papillomavirus in this malignancy. method: forty-two cases of squamous cell carcinoma of the urinary bladder and cases of urothelial carcinoma with squamous differentiation were investigated. hpv infection was analyzed by both in situ hybridization at the dna level and immunohistochemistry at the protein level. p protein expression was analyzed by immunohistochemistry. results: human papillomavirus dna and protein were not detected in cases of squamous cell carcinoma ( %, / ) or cases of urothelial carcinoma with squamous differentiation ( %, / ). p expression was detected in ten cases ( %, / ) of squamous cell carcinoma and nine cases ( %, / ) method: a review of clinical data from an adpkd patient with tcc admitted in our hospital and a review of current literature regarding adpkd and tcc were made. results: the patient is a year-old woman with chronic renal failure due to adpkd, on hemodialysis for years. she was admitted with left lumbar pain, hematuria and fever. radiological exams revealed various complex cysts with dense material that did not enhance with contrast, compatible with hematic cysts in the context of adpkd. the whole clinical findings suggested cystic infection complicated with sepsis, so nefrectomy was performed. macroscopic examination of the ressected kidney revealed a white granulous nodular formation with . cm diameter in the renal pelvis. histological examination confirmed a highgrade papillary tcc with parenchymal infiltration (pt ) and extense scamous differentiation. conclusion: the present case illustrates that tcc can occur in adpkd, despite its rarity. it can be difficult to successfully diagnose tcc on adpkd based on clinical-radiological findings. objective: carcinoid tumor is a very uncommon neoplasm in the kidney. we report the histopathologic and immunohistochemical (ihq) study of two new cases. method: case : year-old female with a cm asymptom-atic¨cystic¨mass discovered in a routine exam. the patient underwent tumorectomy. case : year-old female with a cm renal mass who underwent left nephrectomy. two months later a needle biopsy confirmed carcinoid tumor metastasis in the liver. both patients currently free of disease. results: both tumors showed similar histologic features. neoplastic cells were grouped in nests, ribbons, and pseudoglands with rosette-like appearance and showed eosinophilic granular cytoplasm and chromatic nuclei. low mitotic index. ihc: diffuse/intense cytoplasmic staining for cd , cd , ae /ae , chromogranin and synaptophysin. conclusion: primary renal carcinoid tumor is rare in the clinical practice. the histological findings correspond to a well differentiated neoplasm and are similar to carcinoid tumors in other locations, which makes the diagnosis feasible even without previous personal experience. ihc confirms the diagnosis. it usually behaves as a low grade neoplasm, but recurrences and metastases do occur. only single cases and short series have been published so far. there is no accumulated experience to establish long term prognosis. prostatic adenocarcinoma occurring simultaneously with large cell neuroendocrine carcinoma of the urinary bladder: an extraordinary collision tumor p. czapiewski * , m. sieczkowski, m. matuszewski, k. krajka, w. biernat * medical university of gdansk, dept. of pathology, poland objective: radical cystoprostatectomy is a standard surgical procedure for male patients with muscle-invasive urinary bladder (ub) carcinoma. vast majority of these tumors are urothelial carcinomas, while large cell neuroendocrine carcinoma (lcnec) is a very uncommon tumor with less than reported cases. invasive prostate carcinoma is incidentally detected in up to % of cystoprostatectomy specimens. it is usually well differentiated and shows low propensity for dissemination and local recurrence. method: clinical and pathological description of an extremely rare collision tumor composed of lcnec of the urinary bladder and a high grade acinar prostate carcinoma. results: a -year-old male patient was admitted to the urology department due to severe gross haematuria. cystoscopy revealed large tumor of the posteriorinferior wall of the ub with involvement of the left uretheral orifice. in the radical cystoprostatectomy specimen lcnec of the ub, involving bilaterally prostate and spermatic vesicle (pt a), was diagnosed. additionally both lobes of the prostate were involved by the acinar prostatic carcinoma (pt c, gleason score + = ). conclusion: high-grade prostate carcinoma may rarely coexist with ub tumors of uncommon histology. objective: despite of numerous researches in the field, data regarding immunohistochemical (ihc) expression of androgen receptor (ar) in prostate carcinomas, in terms of prognosis and therapy, are quite controversial. method: the ihc expression of ar was analyzed on groups, each of primary diagnosed prostate carcinomas: localized, locally advanced and distant metastasized. the anti-ar antibody (clone ar ) was used. for each tumor was determined the percentage of ar-positive nuclei, assigning a staining score ( to ). a value of % was considered to be discriminat o r y b e t w e e n t u m o r s w i t h h i g h a n d l o w a r expression. results: all the analyzed carcinomas showed ar. . % of tumors had high ar expression and . % showed low ar expression. / localized tumors (one tumor vanished during processing), / locally advanced and / distant metastasized tumors showed high ar expression (p= . ). / well-differentiated, of moderately differentiated and / poorly differentiated tumors showed high expression of ar (p= . ). conclusion: although not reaching statistical significance, the ar expression tended to correlate with the stage of disease and with the degree of differentiation in terms of an increased expression of ar in advanced, moderately and poorly differentiated tumors. objective: prostate lesions with atypical stromal component are rare and poorly characterized in terms of evolution and prognosis. method: we retrospectively analyzed cases of unusual prostate tumors diagnosed on prostate needle biopsy. the biopsies were performed due to an elevated serum psa and/ or phenomena of bladder outlet obstruction. for the immunohistochemical (ihc) diagnosis there were used the following antibodies: ck ae /ae , psa, psma, vimentin, sma, er, desmin, pgr, cd . results: two of the three patients, one and the other years-old, with serum psa level of ng/ml and . ng/ml respectively, were diagnosed with sarcomatoid carcinoma, the former presenting with liver metastases at diagnosis. in evolution, the death of the first patient occurred months after the diagnosis and the second patient refused surgery. the third patient, years-old, with a nodular prostatic mass of . cm, was clinically suspected to have an unusual malignant prostatic tumor considering that, despite his ng/ml of serum psa, he presented multiple bone metastases. the histopathological diagnosis was stromal tumor of uncertain malignant potential. conclusion: the prostate lesions with atypical mesenchymal component are rarities in urologic pathology. an accurate diagnosis relies on meticulous pathological examination techniques and supplemented, for rare cases, with other clinical and laboratory data. immunohistochemistery study in a case of nephrogenic bladder adenoma m. r. farzaneh * , a. safaei, a. amin shareefi * shiraz, iran objective: nephrogenic adenoma is a rare benign lesion of bladder that may be confused with malignant lesions. there is strong relation with urinary tract irritation and intravesicl instrumentations. nephrogenic adenoma was initially thought to originate from urothelial metaplasia however no solid proof is available. method: we present this years old lady with urinary problem. cystocopic examination show a sessile mass and biopsy show circumscribed proliferation of tubules, cysts, and papillae lined by cells with low cuboidal to columnar epithelial cell. nephrogenic adenoma can be a significant diagnostic pitfall as certain histological features, such as the presence of enlarged nuclei with prominent nucleoli results: immunohistochemistery study showed strong reactivity to ck , p s, cd , and ema but negative for ck , psa, and p . conclusion: we recommend that any lesion in cystoscopic examination should be followed and immunohistochmistery examination some times is mandatory to differentiate them from malignant tumors. regulators of apoptosis and the cell cycle are overexpressed in bladder cancer metastases and may predict survival a. fleischmann * , r. seiler, a. perren, t. george * universität bern, institut für pathologie, switzerland objective: expression of biomarkers and their prognostic relevance may differ between primary tumours (pt) and its metastases (met). there is little information about this phenomenon concerning apoptosis and cellcycle associated biomarkers in urothelial bladder cancer (ucb). method: nodal positive patients (n= ) with ucb underwent cystectomy and lymphadenectomy. immunohistochemical expression of bcl- , bcl- , mdm- , p and cyclind was quantified in tissue microarrays constructed from pt and corresponding nodal met. results: frequency of mdm- positivity increased from pt ( %) to met ( %, p < . ). this trend was not significant for bcl- (pt: %; met: %, p= . ) and bcl- (pt: %; met: %, p= . ). median percentage of p and cyclind immunostained cells increased significantly (p< . ) from pt ( %/ %) to met ( %/ %). mdm- and cyclind expression were positively correlated in pt (p< . ) and met (p< . ). p and mdm- positivity were inversely correlated in pt (p< . ). only high cyclind expression in the metastases predicted early death significantly and independently (p= . ). conclusion: biomarkers of apoptosis and cell-cycle associated are up-regulated in the metastases indicating differences in survival and proliferation of cancer cell compared to primary tumours. both tumor components may harbor different prognostic information and are not necessarily surrogates for each other. evaluation of histopathologic and histomorphometric changes of testicular tissue and gonadotropins levels following consumption of methylphenidate in male mice z. ghahri saremi * , s. fazelipour, z. tootian, m. shafii * tehran, iran objective: one of the most common psychiatric disorders in children is adhd (attention deficit hyperactivity disorder), which is treated extensively by methylphenidate. this study investigates the assessment of the effects of methylfenidate on histopathologic and histomorphometric changes of testes and serum level of gonadotropin in adulthood which produces gametes and has importance for future generations. method: in this study adult male mice (balb/c) were used. after determining the body weight, the animals were divided randomly into two experimental groups of and one control group. the experimental groups received ritalin via gavage as follow: the group received mg/kg/day and the group received mg/kg/ day for a period of days. after evaluation of body weight, general anesthesia was used for taking blood samples from the heart in order to measure gonadothropins levels in serum. then for the purpose of body weighing and measuring of diameter of germinal epithelium the testes were removed and the possibility of any pathologic changes was considered. results: the results showed that methylphenidate with different doses could decrease germinal epithelium and also body weight significantly. besides some significant changes in serum gonadotropins, without any pathological changes were observed. conclusion: our findings demonstrated that methylphenidate administration in adulthood due to influence of enhanced computed tomography and magnetic resonance imaging was characterized as × × cm sized welldemarcated left renal middle-superior polar mass with central necrosis and cystic changes. the contralateral kidney was normal. he underwent left radical nephrectomy by thoracoabdominal approach. gross examination of the specimen revealed yellowish white × × cm sized mass, with multifocal necrozis and cystic changes. histopathology of these resection specimens; scattered spindle cells and foam cells, fine vascular network and necrosis. immunohistochemistry revealed cd -positive xanthoma cells. the tumor also stained positive for vimentin, cd , but negative for cytokeratin, ema, desmin, sma and myod . a diagnosis of mfh was made. conclusion: mfh is a primitive mesenchymal tumor with some histiosytic and fibroblastic differentiation primary renal mfh is an extremely rare lesion. because this malign mesenchymal tumor is indistinguishable clinically and radiologically from renal cell carcinoma diagnosis and histopathology of this rare lesion are discussed. hyaline ring granulomas in the urinary bladder: a case report n. hammer * , n. gatt, j. de gaetano * swatar, malta objective: hyaline ring granulomas, or pulse granulomas, are rare inflammatory responses to vegetable matter, characterised by aggregates of hyaline rings and other inflammatory components around vegetable matter. the vast majority occur in the oral cavity and only a few specific extra-oral cases have been reported. we present a case of pulse granulomas in the wall of the urinary bladder, occurring in a year-old gentleman with a concurrent history of diverticulitis and colo-vesical fistula. pulse granulomas were recently described in the bladder in association with interstitial cystitis, in mesocolonic fat and mesocolonic lymph nodes in association with inflammatory bowel disease and in enterocutaneous and recto-salpingeal fistulas in association with diverticulitis. to our knowledge this is the first case reporting the direct association of the occurrence of bladder pulse granulomas in relation to diverticulitis. method: the tissue sections were fixed with % buffered formalin and stained with h&e. results: histopathological examination revealed several hyaline ring granulomas within the outer muscle layer and serosa of the urinary bladder, together with hyaline ring granulomata within the outer wall of the sigmoid colon associated with diverticula. conclusion: it is important to differentiate pulse granulomas from parasites, hyaline ring vasculopathy, granulomatous inflammatory disorders and even malignancy. objective: micropapillary carcinoma (mpc) is associated with poor prognosis, and may lead to immediate cystectomy irrespective of stage. we study mpc, with "classical" histologic features, to "stage-matched urothelial carcinoma (uca), not otherwise specified (nos)", and other uca with divergent differentiation to compare outcome. method: cases from patients with mpc, uca with squamous (sq) or glandular (gl) differentiation, small cell carcinoma (smca), and nested variant (neca) variant of uca were identified. histologic and clinical data including stage and outcome were collected. results: all groups show male predominance and similar age range. mpc showed the highest rate of nodal metastases (table ) . patients with mpc tumor volume of > % showed the highest rate of nodal metastases of all other subgroups. the majority of patients with mpc died, a large percentage from unknown causes. compared to mpc, sq showed similar presentation at higher stages and similar survival, but a lower propensity for nodal metastases. in our cohort, gl presented at lower stages, showed slightly better survival, and a lower rate of nodal metastases, when compared to mpc. our cohort included fewer cases of smca and neca. however, both patients with neca died of disease. conclusion: mpc, particularly when it represents > % of tumor volume, shows higher rate of nodal metastases than remaining subgroups, and is associated with a larger percentage of patient deaths. objective: müllerian-like stroma with er/pr expression is typical of renal mixed epithelial and stromal tumor and has been described in kidneys with obstructive pathology. wt overexpression is not reported in this setting. method: immunohistochemical study for er, pr and wt in autosomal dominant polycystic kidney disease (adpkd) (n= ), acquired cystic kidney disease (ackd) (n= ), xanthogranulomatous pyelonephritis (xp) (n= ), and renal lithiasis (rl) (n= ). controls: fetal (< w, n= ; > w, n= ) and adult autopsy kidneys (n= ). results: stromal er was found in % of adpkd, ackd, xp and rl; pr expression in % of adpkd, ackd and rl and % of xp, and nuclear wt in % of xp and rl. er were negative in % of controls. we found pr expression in % of fetal controls of second trimester. wt was negative in the stroma of adult controls ( %). % of fetal controls showed wt expression in the peripheral cortex. conclusion: er and pr expression is frequent in kidneys with chronic obstructive and inflammatory pathology. wt is expressed occasionally. more studies are needed to determine whether these findings are a consequence of renal obstruction or they are involved in its pathogenesis, as well as their potential therapeutic implications. telomerase expression in urothelial carcinomas of the urinary bladder: does it make sense for carcinogenesis or prognosis? d. kankaya * , s. kiremitci, k. zengin, c. tuygun, a. sertcelik * ankara university medical school, dept. of pathology, turkey objective: human telomerase reverse transcriptase (htert) has been reported as poor prognostic marker in several cancers. in the present study, we examined htert expression in urothelial carcinoma (uc) to investigate whether it has a role on the carcinogenesis or prognosis. method: immunohistochemistry was performed to detect htert protein expression in tisse microarray blocks consisting of cores of papillary uc (n = ) and infiltrating uc (n = ) cases. nucleolar staining was considered and staining scores (intensity × distribution) were determined. tumors were grouped as low or high in terms of histological grade, and as early (ta, t ), or late stage (t -t ) in terms of pathological stage. results: htert expression was significantly higher in the papillary uc group (p= . ). tumors in late stage were more likely to show low staining scores of htert (p= . ). no correlation was found with tumor grade and recurrence rate. no effect on survival.was detected. conclusion: these findings indicated an association of htert protein with early stage ucs as its expression significantly decreased with muscularis propria invasion. incidental multifocal renal cell carcinoma in graft nephrectomy with cellular rejection findings: concurrence of papillary renal cell carcinoma and renal carcinoma associated with xp . translocation d. kankaya * , s. kiremitci, a. ensari, a. sertcelik * ankara university medical school, dept. of pathology, turkey objective: malignancy represents the second main cause of death in renal transplant patients and increase markedly by years after transplantation. method: a year old male who has been on hemodialysis for months for chronic renal failure, underwent living kidney transplantation from his brother. he received immunosupressive therapy of siclosporin and prednisolon. twelve years later, due to the impairment of renal functions hemodialysis was started again and ultrasonography revealed chronic renal parenchymal disease. graft nephrectomy was performed with the pre-diagnosis of chronic rejection. results: macroscopic examination revealed two well-circumscribed tumor nodules with mm and mm in largest diameter. microscopically, both tumor nodules showed tubulopapillary organization. larger tumor consisted of clear cells with tfe positivity, whereas the small one showed eosinophilic cells with diffuse cytokeratin and amacr positivity, without any tfe expression. concurrence of papillary renal cell carcinoma and renal cell carcinoma associated with xp . translocation was reported. non-tumoral renal paranchyme revealed interstitial inflammation, tubulitis and transmural arteritis with fibrinoid necrosis compatible with type iii cellular rejection. conclusion: as far as we know this is the first case in the english literature demonstrating concurrence of papillary renal cell carcinoma and renal cell carcinoma associated with xp . translocation in a graft nephrectomy. the effect of postoperative intravesical bcg and mitomycin c therapy on recurrence in superficial bladder cancer d. kankaya * , k. zengin, a. sertcelik, c. tuygun, n. sertcelik * ankara university medical school, dept. of pathology, turkey objective: to evaluate the efficacy of two mostly used intravesical agent, bcg and mitomycin c, in case of tumor recurrence. method: between and , we performed tur-bt to patients whom pathology results were superficial bladder cancer. patients were treated with intravesical bcg once for week weeks then monthly up to one year beginning from . day postoperatively. patients were treated with intravesical mitomycin c, beginning from first h postoperatively and once a week for weeks. patients didn't get any further treatment, and excluded from study. results: for bcg group, expected disease free interval was . months, and . months for mitomycin c group. when two groups compared for expected disease free interval, statistically significant difference observed (p= . ). for bcg and mitomycin c group, recurrence was seen in and patients, respectively. for patients treated with bcg, and years disease free survival rate was % . and % . , respectively. in mitomycin c group and years disease free survival rate was % . and % . . conclusion: intravesical adjuvant bcg maintenance therapy is more effective for tumor reccurence than intravesical mitomycin c therapy. papillary cystadenomas of the epididymis: case presentation p. katafygiotis * , l. abou-asabeh, a. nomikos, s. chranioti * hospital of voula, dept. of pathology, athens, greece objective: papillary cystadenomas of the epididymis are rare, first described by sherrick in . they are considered hamartomatous lesions rather than neoplastic and may be seen as intrascrotal swellings in a wide age range. we report here the case of a -year-old male who presented with a swelling of the right testis over the past year. his past medical history was otherwise unremarkable. method: ultrasonography revealed the presence of an epididymal cyst. grossly the cyst had a cm diameter with a cyst wall of . cm thick attached to a part of epididymis measuring × cm filled with clear fluid. results: histologically the cyst showed pseudopapillary structures lined by monotonous clear cells. there was mild nuclear pleomorphism, no mitotic activity and no necrosis. immunostaining for ae /ae , ema, were positive, as well as focally for ck / whereas calretinin and cea was negative. similar pattern of staining was seen in the epididymal parenchyma. conclusion: papillary cystadenomas are benign neoplasms and there have been no reports of recurrence or metastasis in the recent literature. bilateral disease is often associated with von hippel-lindau syndrome. our patient received no further treatment and remains asymptomatic and well months after the intervention. effect of neoadjuvant sorafenib treatment on histology of clear cell renal cell carcinoma and occurrence of circulating tumor fragments g. kats-ugurlu * * umc st. radboud, dept. of pathologie, nijmegen, netherlands objective: clear cell renal cell carcinoma (ccrcc) generally presents with a micronodular phenotype (mp) due to high expression levels of vascular endothelial growth factor (vegf-a). earlier we have shown that mp is associated with shedding of multicellular tumor fragments (mtf) into the circulation and pulmonary metastasis. we hypothesized that vegf inhibition will destroy mp resulting in less mtf. method: ccrcc patients were treated for weeks by daily administration of sorafenib ( mg bid). three days after therapy, nephrectomy was performed and kidneys were perfused via the arteria renalis. venous perfundate was filtered and processed to agarcytoblocks for mtf. treatment effects were studied using immunohistochemistry. results: all tumors were ccrcc as demonstrated by high vegf-a expression. none of the tumors showed mp after sorafenib treatment. tumors showed large areas of necrosis and fibrinoid necrosis of the blood vessels, concomitant with profound perivascular inflammation. / ccrcc patients ( %) had mft vs % in a control group (p= . fisher's exact test). individual tumor cells in the mtfs showed increased mitotic activity. conclusion: sorafenib destroys mp in ccrcc, attacks tumor vasculature, causes extensive necrosis and inflammation. post-treatment mtf are increased in venous perfundate. care should therefore be taken with neo-adjuvant sorafenib treatment of ccrcc. objective: claudins are major transmembrane proteins of tight junctions. as the disruption of their function have important impact on tumorogenesis, invasion and metastasis. claudins became a focus of interest for targeting therapies. although their expression profiles have been studied in many organs, researches on claudin expression in bladder are in limited number. the aim of this study is to present the differential expression of claudins in invasive urothelial cell carcinoma (iucc), noninvasive papillary urothelial carcinoma (npuc), carcinoma in-situ (cis), papillary urothelial neoplasm of low malignant potentiall (punlmp) and control group (cg). results: cases ( iucc -further divided into: muscle invasive ucc, ucc with lamina propria invasion-, npuc, punlmp, cis and cg) were evaluated with claudin- , , . interestingly, high claudin- and - score in cg ( %, %), decreased significantly in all non-invasive lesions (mean %, %). claudin- expression appeared to decrease in iucc vs. others (% vs mean % ). conclusion: higher expression of claudin- in low-grade and non-invasive lesions may be used as a diagnostic tool. in terms of claudin- and - , their decreased expression in non-invasive lesions when compared to cg and their trend to show more increased expression in iucc needs to be studied further in larger studies. can we rely on alternative sampling method of radical prostatectomy specimens? t. d. kokenek-unal * , a. s. oguz-erdogan, m. alper * yb research and training hospital, dept. of pathology, ankara, turkey objective: prostatic adenocarcinoma is the most common cancer and second leading cause of cancer death in men. the incidence of prostate cancer has been increasing because of efficiency of modern cancer scanning programs and residual awareness of the patients. for that reason, there is a considerable increase in the number of prostatectomy specimens in the university and research hospitals.it is costly and time-consuming procedure and causes in turn increased workload. the aim of this study is to compare the results of total and alternative sampling methods and to delineate the differences if there are any. method: totally embedded radical prostatectomy specimens were randomly selected and evaluated for key pathologic parameters. these cases then were reevaluated with limited sampling. the limited sampling method was built to include similarly embedded apical margins, bladder neck margins and seminal vesicles. in addition to that new slices were selected by skipping every other slice as differently from totally sampling protocol. results: the % reduction was achieved in number of blocks. the concordance rates between two sampling methods were % , % . , % . , and % respectively for gleason scores, perineural invasion, extraprostatic extension and pathologic stages. conclusion: although the limited sampling protocol provides statistically significant results, because of critical role of pathological assessment in treatment of prostatic adenocarcinoma, they can be found unsatisfactory for many pathologists. objective: initially described in embryonic neuroepithelium, nestin is an intermediate filament involved in cell differentiation and transiently related to vimentin, keratin and glial fibrillary acidic protein (gfap). in tumoral and vascular proliferation, nestin is recognized in stem/progenitor cells. the octamer-binding transcription factor (oct- ) is a biomarker of this lineage. since there are few data concerning the presence of nestin in leydig cells and testicular tumors, our aim was to investigate a series of leydig cell hyperplasia and tumors. method: cases ( hyperplasia, adenomas, malignant leydig cell tumors and lymph node metastases) and controls were immunostained with anti-nestin, vimentin, keratin, gfap and oct- antibodies. results: twenty-six ( %) cases (tumors and hyperplasia) were nestin positive with mostly weak, finely granular cytoplasmic staining. four ( %) were negative, ( %) not interpretable. vimentin was expressed in twenty-seven ( %) cases. seventeen ( %) were focally keratin positive. only three ( %) showed oct- positivity. all were gafp negative. conclusion: in leydig cell hyperplasia, adenomas and malignant tumors nestin and vimentin are expressed frequently, sometimes with keratin. this would be a sign of epithelialmesenchymal transition. further investigations are needed to understand the relationship with the malignant potential of these tumors. morphological changes of testis in atherosclerosis n. krupnov * , a. astrakhantsev * bureau of forensic medical exp., ryazan, russia objective: up to now in the field of morphology of reproductive system of men the problems of testis involution and morphogenesis at atherosclerosis haven't been thoroughly investigated. we have investigated histologically testis of died patients aged - with general atherosclerosis and testis of men aged - with minimal manifestations of atherosclerosis died in an accident. results: the absolute volume rete testis of patients being ill with atherosclerosis decreases by - %. absolute volume of convoluted seminiferous tubules and interstitial tissue decrease by - %. in testis there are zones of focal sclerosis of seminiferous tubules, whose area goes as far as . % of shear section area. it was revealed that on the periphery of sclerosis zones there is a reduction of transaction area of seminiferous tubules by - % with sertolli cells quantity reduction by - %. it has been stated the downward changes in index of spermatogenesis in convoluted seminiferous tubules by - . %, accounted for the quantity reduction of all kinds of cells. conclusion: morphological changes of testis at general atherosclerosis characterize atherosclerotic testiculopatie, caused by chronic ischemia of testis. a leydig-cell tumor in a cryptorchid testis: report of a case e. lambropoulou * , a. datsis, p. morfaki, g. charalambopoulou * general hospital of messologhi, dept. of pathology, greece objective: leydig cell tumor is a rare form of testicular neoplasm, representing only - % of all testicular tumors. according to accepted medical knowledge, this type of tumor isn't linked to cryptorchidism, unlike germ cell tumors of the testicles. herein, a case of leydig cell tumor in a cryptorchid testis is described. method: a -year-old, unmarried man was admitted for surgical repair of inguinal hernia. his past medical history included untreated bilateral cryptorchidism first noted in childhood, as well as poliomyelitis that presented at age two. the past surgical history and review of systems were noncontributory. a ct scan of the abdomen and pelvis revealed two × cm testicles at the level of the inguinal canal. he underwent bilateral inguinal orchiectomy combined with surgical repair of his inguinal hernia. results: the right testis contained a solid, well-circumscribed, round, tan-colored tumor . cm in diameter. the histological appearance was that of a leydig cell tumor exhibiting no mitotic figure. there was no clinical or radiological evidence of metastatic spread. conclusion: although cryptorchidism is considered to be a risk factor for developing germ cell tumors, there have been a few reported cases of leydig cell tumors with a history of cryptorchidism. our case adds to the evidence that there may be a link between the two conditions. objective: cd , e-cadherin, β-catenin are "cell adhesion molecules" and appear to influence development, inflammation, cancer invasion and metastasis. we studied the expression of these cams in prostatic adenocarcinoma (pca), high grade prostatic intraepithelial neoplasia (hgpin) and nodular adenomatous hyperplasia (nh). method: specimens of radical prostatectomies were assessed. these cams were determined by immunohistochemistry. all sections included pca, hgpin, and nh. the expression of these markers was evaluated with three scores. the correlation of immunopositivity with gleason score and tnm stage was investigated. results: cd was strongly expressed in . %, . % and . % of nh, hgpin and pca, respectively. e-cadherin immunostaining was higly detected in . %, . % and . % of nh, hgpin and pca areas while β-catenin immunostaining was exclusively membranous in . % of nh and nuclear/cytoplasmic in . % and . % of hgpin and pca areas. all markers were unrelated to gleason score (p= . ). cd and e-cadherin immunopositivities were inversely associated with tnm stage (p= . and p= . respectively); such an association was not observed in β-catenin (p= . ). conclusion: cd and e-cadherin decreased expression is probably associated with invasive potential of prostate cancer. β-catenin staining pattern in neoplastic lesions differs from that in non-neoplastic prostate lesions. results: the loss of ecad expression was significantly higher at the tif when comparing with tscp and nnm. the loss of ecad was correlated with histological grade, infiltrative pattern, lymph node metastasis, perineural and vascular invasion. the vimentin expression showed association with histological grade, infiltrative pattern, t stage, lymph node metastasis, perineural and vascular invasion. conclusion: the loss of ecad and the gain of vimentin expressions occur more frequently at the tumor invasion front and are associated with classic factors of poor prognosis and low survival rates. prognostic value of immunohistochemical markers in bladder cancer i. m´sakni * , a. chaabane, f. bougrine, b. laabidi, a. bouziani * rades meliane, tunisia objective: the tumor stage and grade of bladder tumors are the major elements to define the prognosis. however, it is sometimes difficult to identify an infiltration of chorion or detrusor muscle. moreover, the evaluation of tumor grade is subjective and not reproducible. method: our objective is to study the prognostic value of the expression of proliferating cell nuclear antigen (pcna), ki antigen, the tumor suppressor gene p , the protooncogene c-erb b , the receptor for epidermal growth factor (egf-r), the apoptosis suppressor gene bcl , carcinoembryonic antigen (cea) and epithelial membrane antigen (ema). results: the study showed that the pcna expression was significantly associated with the early recurrence (p= . ) and the tumor stage (p= . ). the mib expression was correlated to the early recurrence (p= . ), and tumor progression in stage and/or grade (p= . ). the c-erbb expression showed significant association with the tumor grade (p= . ). the prognostic value of other markers has not been proven. conclusion: these findings may be useful providing better classification of bladder tumors thus the better management of patients. the c-erbb expression contributes to refine the tumor grading. pcna and mib can predict the early tumor recurrence; they could be relevant for the determination of endoscopic controls rhythms of patients. objective: primary urinary bladder neuroendocrine carcinoma (pubnec) is a rare tumor characterised by an aggressive behaviour and poor prognosis. method: we report five cases of pubnec diagnosed in the departement pathology of farhat hached hospital between and . results: our population is composed by four mens and a woman. all patient are heavy smokers. the most common presenting symptom is hematuria and dysuria in all cases. one patient presented a complete urinary retention. a cystoscopic examination with transurethral resection was performed in all cases. the pathological examination with use of immunohistochemical markers of neuroendocrine differentiation were consistent with a large cell neuroendocrine carcinoma in four cases and a small cell neuroendocrine carcinoma in a case. a cystoprostatectomy was made in two cases followed by chemotherapy. conclusion: the clinical presentation of pubnec is similar to other bladder cancers and is characterized by advanced stage at diagnosis and rapidly progressive disease. the diagnosis of poses several problems: a vesical metastasis has to be excluded and such lesions have to be differentiated from transitional cell carcinoma, lymphoma, paraganglioma and peripheral nerve neuroblastoma. there is no gold standard for the management of patients affected due to low disease frequency ps- - igg -associated inflammatory pseudo-tumor (ipt) of the ureter: a case report a. marando * , g. d´ambrosio, f. catanzaro, f. sessa * university of insubria, dept. of surgical, varese, italy objective: igg -associated inflammatory pseudo-tumor (ipt) is a novel clinico-pathologic entity characterized by intensive infiltration of igg -positive plasma cells, associated with systemic igg -related sclerosing disease. many reports described igg -related ipt in various locations such as pancreas, salivary glands, liver, breast, lung and recently also ureter. method: it is described a case of ureteral ipt with pathologic and immunohistochemical features of igg -related ipt, fibrohistiocytic type. results: the study case is a -year-old female with severe stenosis of the left ureter and hydronephrosis, who underwent to nephroureterectomy and endoscopic resection of multiple lesions in the bladder. the histological examination showed transmural fibrosing inflammatory lesion of the affected ureteral wall, with abundant plasma cells intermixed with many histiocytes, lymphocytes, fibroblasts and scattered eosinophils. the majority of infiltrating plasma cells were positive for igg . bladder lesions showed similar histological features. the diagnosis was igg -related ipt, fibrohistiocytic type. conclusion: igg -related ipt of ureter is extremely rare, with only few cases reported in literature. recognition of this entity is clinically relevant because this type of ipt can be treated with steroid therapy and may be associated with sclerosing autoimmune disease in other organs. a rare case of malignant fibrous histiocytoma of the urinary bladder s. mavropoulou * , z. tatsiou, i. amplianitis, p. nasos * general hospital, laboratory of pathology, xanthi, greece objective: malignant fibrous histiocytoma (mfh) is an extremely rare malignant mesenchymal neoplasm of the urinary bladder with only a few well-documented cases reported in the english literature. method: we report the case of an -year-old man who was brought to our hospital due to sudden massive haematuria. catheterization failed to obtain haemostasis so, a suprapubic incision and direct exploration of the bladder was performed. a large solid tumor was found with a shaggy haemorrhagic surface and biopsies were taken. results: histological examination revealed an infiltrating malignant neoplasm composed of variably pleomorphic ovoid neoplastic cells with eosinophilic cytoplasm, bizarre tumor giant cells and prominent stromal osteoclastic giant cell reaction. immunohistochemical examination was negative for cytokeratin, desmin, smooth muscle actin, psap, nse, s protein and hmb whereas it was positive for cd in a large number of tumor cells. accordingly, the diagnosis of undifferentiated pleomorphic sarcoma with histological features compatible with giant cell mfh of the urinary bladder was made. the patient died days after the diagnosis. conclusion: in conclusion, mfh of the bladder should be kept in mind when facing with an undifferentiated malignant tumor. despite the poor prognosis, early diagnosis and aggressive salvage therapy might offer the chance of long-term survival in selected cases. partial nephrectomy experience at a single tertiary-care oncology centre: a clinicopathologic study of cases s. menon * , g. bakshi, h. tongaonkar, v. noronha, a. joshi, k. prabhash, s. desai * tata memorial hospital, dept. of pathology, mumbai, india objective: partial nephrectomy (pn) is replacing radical surgery as a gold standard in the treatment of small renal masses. intra-operative frozen consultation for margin status is aimed at achieving a disease free state in order to reduce chances of recurrence. method: a retrospective clinicopathologic analysis of consecutive pns performed at our institute from to was undertaken. results: sixty cases of pn were analysed. male to female ratio was . : . median age was years. six cases were benign: oncocytomas ( ), angiomyolipoma ( ); while cases were malignant: renal cell carcinoma (rcc) -conventional ( ), papillary ( ), chromophobe ( ), mucinous tubular spindle cell carcinoma ( ) and one case of primitive neuroectodermal tumour. in patients, the renal tumour was a second malignancy. median tumour size was . cm with cases of stage pt . intra-operative margin was positive in cases. mean margin for all cases was . mm. median follow-up was of months. none of the tumours recurred or metastasized during follow-up. conclusion: conventional rcc is the commonest histology in pn cases. frozen section analysis has a definite role in achieving margin free status. pn is not associated with increased risk of local recurrence in small renal tumours. clear cell tubulopapillary renal cell carcinoma: a clinicopathologic study of two cases g. muñiz * , a. corominas, n. cerda, a. perez, v. caamaño, m. gonzalez, l. etxegarai, j. i. lópez * hospital universitario cruces, dept. de anatomía patológica, barakaldo, spain objective: cleal cell tubulopapillary renal cell carcinoma has been recently identified as a low grade renal cell tumor with distinct histological features. method: patient : year old female with a history of fibromyalgia and persistent loin pain. a right renal mass, . cm in diameter, was discovered in the rheumatologist's follow up. right nephrectomy was performed and the patient is free of disease months later. patient ; year-old female with a history of diabetes mellitus type , hypertension and renal failure grade with a . cm in diameter renal tumor in the routine studies. she underwent tumorectomy. results: grossly, both were cystic tumors with gelatinous fluid and white-yellowish solid areas. tumors showed a tubular and papillary architecture. proliferating cells displayed clear cell cytoplasm and hyperchromatic nuclei placed in the luminal side. ihc showed positive staining with e-cadherin, ema and ck . conversely, cd , cd and amacr were negative. conclusion: clear cell tubulopapillary renal cell carcinoma should be considered a distinct subtype of renal cell carcinoma according to its unique morphologic and inmunohistochemical features. the few cases reported so far behave in an indolent course. survivin expression in renal epithelial tumors: its usage in the differential diagnosis of eosinophilic renal epithelial tumors a. ozcan * , n. yigit, o. onguru, b. a. firat, s. ozaydin * gulhane military medical academy, dept. of pathology, ankara, turkey objective: the differential diagnosis of renal tumors can be problematic due to overlapping morphologic features. the purpose of this study was to assess the potential contribution of survivin expression in the differential diagnosis and determination of therapy modalities of these tumors. method: this study consisted of chromophobe (chrcc), clear cell (ccrcc) and papillary (prcc) renal cell carcinomas, and oncocytomas. sections were stained against survivin antibody. results: prccs and ccrccs showed diffuse and strong survivin expression. survivin expression was strikingly prominent in type prccs and cystic ccrccs. in ccrccs, survivin expression was more pronounced in low grade areas than high grade and sarcomatoid areas. in chrcc, survivin expression was more limited and weaker than that of oncocytomas and other malignant renal tumors. in non-neoplastic renal tissue, survivin expression was more pronounced in podocytes and atrophic tubules than other nephron parts. conclusion: our findings suggest that survivin may contribute to the differential diagnosis of renal tumors because of the partially unique staining patterns. it was purposed that knockdown of survivin reduced growth, induce apoptosis and enhance in vitro radiosensitivity of rcc cells. taken together, to be known different survivin expression patterns in renal tumors may help to determine new therapeutic strategies for rccs. objective: there are several tumor-like lesions and miscellaneous neoplasms of rete testis. we present a case with adenomatous hyperplasia of rete testis (ahrt). method: the patient was years old with undescended testis and referred to our hospital. there was no clinical or endocrin abnormalities. cryptoorchidism was unilateral and the other testis was normal. right orciectomy was performed and sent to pathology labarotory for examination. results: there was no tumoral lesion in gross examination but in microscobic examination there was gland like tubular structures. some of these were back to back position with little intervening stroma and mild to moderate atypia. ema and pancytokeratin immunohistochemistry findings with morphology confirmed the diagnosis of ahrt in this case. conclusion: ahrt is a rare proliferatif lesion and can be confused with malignancy. it is incidentally realised in microscobic investigation. it may present as a very small lesion detected in microscobic examination or solid-cystic mass lesion which is macroscobically evident. clinic history, localization, histologic features and immunohistochemistry are criterias for differentiating these lesions.we present this case for both surgeons and pathologists with its importance to be confused with malignancy. objective: prostate cancer is the second leading cause of death in men. the localized disease often responds to conventional therapies like androgen ablation via castration and/or administration of chemical inhibitors but advanced disease resistant to any curative therapies is still challenge for investigators. there are increasing efforts to enhance the possibility of finding positive and sensitive immune markers for diagnosing and treating prostate cancer. method: we applied immunohistochemical markers; amacr and inos. formalin-fixed parafin embedded tissues of prostate needle biopsy specimens diagnosed as prostate adenocarcinoma between and years were enrolled in the study. results: amacr expression has been found in ( . %) and inos expression in ( . %) of prostate adenocarcinomas.no significant relationship of amacr and inos has been obtained (p> . ). there was no significant correlation of histopathologic grade of the tumors with amacr and inos expression (p> . ). conclusion: the expression of amacr and inos might be important diagnostic immune markers for prostate adenocarcinomas especially in needle biopsies when the quantity and quality of tissue are limited. a tissue microarray study of napsin-a expression in renal tumors a. panizo * , f. j. queipo, j. j. sola, j. pardo * hospital de navarra, anatomia patologica, pamplona, spain objective: napsin-a is an aspartic protease present lung, renal, and thyroid cells. there are few studies evaluating napsin-a in renal neoplasms. therefore, we studied ihc expression of napsin-a in a wide spectrum of renal tumors. method: ihc for napsin-a (rabbit polyclonal antibody) was performed in a series of cases of primary and metastatic renal tumors on tma. cytoplasmic immunoreactivity was scored: intensity ( - +) and extent (% of tumor cells: - ). the scores were added: positive case: combined ihc score> ; negative if combined score of or less. objective: the diagnosis of prostatic carcinoma can be challenging on needle core biopsies. the aim of this study was to assess the utility of alpha-methylacyl-coa racemase (amacr/p ) antibody cocktail for prostate cancer diagnosis. a prospective analysis of consecutive radical prostatectomy specimens and prostate needle biopsy semples was performed to select histological sections showing foci of minimal prostatic carcinoma, high grade prostatic intraepithelial neoplasia (hgpin) and benign mimickers of prostatic carcinoma (atrophy, adenosis). method: serial histological sections were stained with hematoxylin and eosin, van gieson and immunomarkers: amacr and p using a prediluted antibody cocktail. results: the cocktail was very useful in highlighting prostatic carcinoma associated with hgpin (flat or cribriform) and distorted foci of minimal carcinoma. amacr was positive with moderate and strong staining in almost all cases for which the immunohistochemical result converted the atypical diagnosis to a final cancer diagnosis. the cases whose diagnosis was changed from "atypical" to cancer were all highly suspicious for cancer based on he histology and negative basal cell markers. conclusion: this cocktail would be of diagnosis utility when limited tissue is available for histopathological evaluation of small diagnostically difficult foci (prostate needle biopsy and surgical specimens). overexpression of cytokeratin , ki- and topoisomerase-ii-a can significantly stratify the recurrence risk in patients with bladder cancer after transurethral resection s. petrov * , k. malkhasyan, r. khasanov * kazan cancer center, dept. of pathology, russia objective: the current predictive models based on main clinical tumor features are not accurate for the biggest bladder cancer patient group, who underwent the transurethral resection (tur). method: overall patients with primary urocarcinoma after tur were included in this retrospective study. the follow up plan in all cases included cystoscopy and biopsy every months in the first years. the recurrence criteria were cystoscopical and pathological confirmation of the tumor growth. in all cases using tma technique (tma master, dhistech) there was done the ihc expression study of p , p , ck , e-cadherine, b-catenin, cd v , ki- ( % cut-off), topo-ii-a ( % cut-off) and her , as well c-erb-b amplification study (cish). results: there were no association found between the her expression and c-erb-b amplification in urocarcinoma patients. in multivariate regression analysis only ck , ki- and topo-ii-a showed the significant prognostic power in recurrence prediction. these markers were used to develop the powerful predictive index for bladder cancer patients after tur. conclusion: although the her overexpression is relatively common event in bladder cancer, c-erb-b gene amplification isn't main mechanism of its realization. the ck , ki- and topo-ii-a are promising prognostic markers for recurrence and should be validated in further prospective study. solitary fibrous tumor of the urinary bladder associated with a high-grade urothelial invasive carcinoma. a case report a. pitino * , s. squillaci, c. spairani, m. ferrari, m. f. cosimi, w. fusco, c. rossi, f. montefiore, g. l. bigatti, v. la paglia * san giacomo hospital, division of anatomic pathology, novi ligure, italy objective: solitary fibrous tumor (sft) is an unusual spindle cell neoplasm, which can exceptionally occur in the urinary bladder. method: we present a case of urinary bladder sft in a year-old man who complained of pelvic pain. cystoscopy revealed a large protruding, fleshy mass at the anterior wall of the bladder. a biopsy was first misclassified as inflammatory myofibroblastic tumor (imt). subsequent complete transurethral resection was performed. results: macroscopically, a × × cm greyish lobulated firm polypoid tumor was seen. microscopically, it consisted of uniform spindle cells with elongated tapered ends nuclei forming a patternless growth in a collagenous background. mitotic figures were rare. immunohistochemically, the neoplastic cells were positive for cd and bcl- , and negative for α-sma, desmin, ck ae / ae and alk- . the bladder urothelium showed foci of high-grade transitional carcinoma with lamina propria invasion. conclusion: initially, sfts were thought to be of mesothelial origin. then, these tumors have also been observed in extrapleural and extraserosal sites, which suggests a mesenchymal cell origin. the differential diagnosis should always include other spindle cells lesions such as sarcomatoid carcinoma, leiomyosarcoma and imt. to date, this is the first reported case of association of urinary bladder sft and high-grade urothelial invasive carcinoma. renal oncocytomas with unusual features: clinicopathological study of cases f. j. queipo * , h. d. quiceno, f. j. pardo, Án. f. panizo, m. l. gómez-dorronsoro, g. aisa, f. j. monzón, e. mejía, c. del agua, j. alfaro * clínica universidad de navarra, pathology, pamplona, spain objective: oncocytoma (ro) is a benign renal neoplasm, with a wide morphologic spectrum and excellent prognosis. recently, it has been described worrisome morphological features. method: ro treated in our centres were reviewed, and we focused on identifying the worrisome and the atypical features. results: we identified ro with at least one of the worrisome feature. patients: m/ f (mean age , year; range: - ). mean tumor size: , cm (range: , - , cm); right kidney: , and left: cases. invasion into the perinephric or renal sinus fat was the most frequent worrisome feature: tumors ( , %) and focal chromophobe carcinoma-like areas in cases ( , %). lymphovascular invasion, entrapped renal tubules-glomeruli, necrosis and mitosis were found in ( , %) respectively. all but two ros had at least worrisome features. followup was available for all cases (median months; range - ): all patients were alive without recurrence or metastasis. conclusion: ro is a tumor which often can show worrisome and atypical morphology. it is necessary to know and recognize the worrisome features to prevent diagnostic errors, if otherwise typical oncocytoma morphology is present. despite these atypical morphological data, the prognosis is excellent. ki and p expression in urothelial carcinomas and clinicopathologic correlation a. ribeiro * * chlc, epe, serviço de anatomia patológica, lisboa, portugal objective: the most important predictive parameter for the biological behaviour of urothelial carcinoma is histological grade, except for depth of invasion. the aim of this study was to investigate the expression of p oncoprotein and ki antigen in a series of transitional cell bladder carcinoma with papillary morphology (ptapapillary carcinoma and pt ) with histological grade and recurrence. method: this study included cases diagnosed with urothelial carcinoma with papillary morphology (pta, pt ). immunohistochemical expression of ki and p were examined in each case, and were graded accordingly to the percentage of cells stained, in low, moderate and high-expression groups. results: as described previously in other publications the expression of p and ki has a relationship with histological grade. we also noted that of the recurrences, were associated with moderate to high expression of either p or ki , or both. conclusion: we concluded that p and ki expression combined with histological grade and pathological stage may be helpful in assessing more accurately the biological behaviour of urothelial carcinoma. and the overexpression of p and ki are related with an unfavourable prognosis. objective: prostatic stromal hyperplasia with atypia is a rare lesion with fewer than cases reported worldwide. it can be mistaken for sarcoma because of the presence of atypical, bizarre cells. its malignant potential is uncertain. the follow up study of every case is important. method: a -year-old man with previously diagnosed atypical prostatic adenostromal hyperplasia with atypia of stromal cells was hospitalized with urinary obstruction after transurethral resection of prostate made years ago. repeat turp was performed and tissue specimens were investigated and compared with the initial biopsy. results: the second biopsy showed the benign hyperplastic prostatic glands with atypical, bizarre, frequently multinucleated giant stromal cells between them. they displayed intense immunoreactivity for actin, vimentin and androgen receptors. the microscopical picture was identical to that seen in the first specimens. the nuclear abnormalities looked like in an atypical symplastic leiomyoma of myometrium. no evidence of sarcomatous or carcinomatous transformation, mitotic index evaluation was noted. conclusion: this case maintains the viewpoint that the prostatic adenostromal hyperplasia with stromal cell atypia is a benign lesion. but it can recur and requires the repeat turp or radical surgery. cancer risk in patients with precancerous lesions of the prostate y. rogov * , v. zakharava, t. liatkouskaya, e. cherstvoy * belarusian medical academy, dept. of pathology, minsk, belarus objective: prostatic intraepitelial neoplasia (pin) and atypical small acinar proliferation (asap) has a high predictive value as markers for prostate cancer (pca). method: pca-risk in patients with precancerous lesions has been assessed on biopsy material in patients having morphological suspicious to pca. suspicious foci were estimated with use of cocktail amacr + hwc + p . results: according to our results revealing of precancerous lesions in biopsy specimens has been associated with pca identification in re-biopsies (f= , ). within the first- years the overall incidence of pca in re-biopsies made % in the group of precancerous lesions and %in the asap group. life-time-without-pca median made up -years with no reliable difference between pin and asap groups (ww= , ; p= , ). thus, the cumulative share of patients without pca in the asap group formed %- %- %- %- % at the end of the first-second-thirdfourth-fifth year of supervision respectively. conclusion: within the first years pca risk makes % in the general group of precancerous lesions of the prostate and % in asap group with no difference between asap and pin groups. Сumulative share of patients without pca in rebiopsy specimens decreased from % to % during these years. benign prostatic hyperplasia and prostate carcinogenesis after the chernobyl accident in ukraine a. romanenko * , a. chekalova, a. yurakh, p. harkonen, s. vozianov * institute of urology, kiev, ukraine objective: the prevalence as well as immunohistochemical (ihc) study of latent, incidentally found prostate cancer (lpc) as well as precancer lesions, in patients, who underwent surgery for bph were studied. method: bph samples were obtained by prostatectomy from ukrainian patients consisting of patients from areas without radio-contamination (control group ) and of patients living in cs contaminated areas of ukraine (group ). ki- , p , p kip- , p and bcl- proteins were ihc investigated in bph from all patients. results: the incidences of lpc (gleason score ), chronic prostatitis, pia and pin were . , . , , and . % in group ; . , . , . and . % in group , respectively. greatly elevated levels of p , ki- , bcl- associated with decreased levels of p kip- and p in areas of pia and less lpc and pin in group to compare with group patients were obtained with statistically significant differences. conclusion: our study suggests that chronic long-term lowdose radiation exposure might result in the increase of chronic inflammation and it is now found to be associated with increased incidences of pia and pin in bph accompanied by p , p kip- and bcl- alteration which in turn could lead to prostate carcinogenesis. eosinophilic globules in rete testis mimicking yolk sac tumor in a testicle with seminoma e. ronne * , t. argyrakos, d. rontogianni * evangelismos hospital, dept. of pathology, athens, greece objective: the recognition of a non-seminomatous component in an otherwise typical testicular seminoma changes the choice of adjuvant chemotherapy. method: a -year old male underwent orchiectomy for a testicular mass. serum tumor markers levels (β-hcg/afp/ ldh) were normal. results: macroscopical examination revealed a . cm white-yellow tumor. the tumor was composed of the characteristic for seminoma homogenous, clear cells arranged in nests and islands separated by septa and infiltrated by lymphocytes. immunohistochemically the tumor cells were positive for plap, oct / , cd and d - and negative for cd and ck . . there was also a regular pattern of tubular structures infiltrated by the seminoma cells while retaining a low columnar type epithelium. the presence of sphaerical eosinophilic globules (pas+/dpas+/afp−) within the tubular lumina was strongly reminiscent of the hyaline globules of yolk sac tumor. the tubular structures were positive for ck . and negative for afp, glypican- and oct / . conclusion: the presence of tubular structures with sphaerical eosinophilic globules creates a suspicion for a yolk sac tumor component. the absence of afp and glypican- expression, the regular pattern of the tubular structures and their continuity with rete testis excluded this suspicion. hyaline eosinophilic globules in rete testis should not be confused with the globules produced by yolk sac tumors. beta-catenin expression and ctnnb mutations in a series of wilms tumours r. santi * , p. pinzani, f. salvianti, g. baroni, m. pepi, g. nesi * university of florence, pathological anatomy section, italy objective: ctnnb mutations have been found in - % of wilms tumour (wt) cases. nuclear beta-catenin protein has been detected by immunohistochemistry in a higher proportion of wts, thus suggesting alternative genetic pathways leading to beta-catenin activation in these neoplasms. method: sixteen renal wts and secondary wt localizations were retrospectively investigated. the series included paediatric patients, females and males, with a mean age of . years and a year-old female patient. immunohistochemical analysis of beta-catenin was performed and findings were reported for each neoplastic component (i.e. epithelium, stroma and blastema). tumour dna was extracted for direct sequencing analysis. results: the majority of wts showed moderate to strong membranous staining for beta-catenin in the epithelial ( . %) and the blastemal ( %) components. nuclear betacatenin expression was observed in combination with cytoplasmic staining in the mesenchyma and/or the blastema of two primary renal tumours. in these cases the deletion of codon p.s del and the missense substitution p.t a were detected. conclusion: preliminary results of this ongoing study highlight beta-catenin cytoplasmic and membranous expression in the tumour cells of primary and metastatic wts, with few cases demonstrating nuclear expression. in our series, betacatenin nuclear expression was invariably associated to ctnnb mutation. a preliminary study on o -methylguanine-dna methyltransferase and type transglutaminase expression profile of renal cell carcinomas b. sarsik * , b. pehlivanoglu, d. tunali, a. simsir, e. gokmen, s. sen * ege university, faculty of medicine, izmir, turkey objective: o -methylguanine-dna methyltransferase (mgmt) repairs o -methylguanine in dna, therefore provides a tumor supressor effect. type transglutaminase (tgase- ) is a multifunctional enzyme involved in many biological processes. few data are available in the literature on their expression in kidney cancers. in this preliminary study, we evaluated immunohistochemical expression of mgmt and tgase- in renal carcinoma cases. method: forty cases of renal carcinoma including ten clear cell, ten chromophobe cell, ten papillary and ten urothelial carcinoma were randomly selected. thirty-one patients were male and average age was , . staining intensity and percentage of staining tumor cells were scored. total score was categorized as weak, moderate and strong. results: strong mgmt positivity was demonstrated in cases ( %). eight cases ( %) showed strong tgase- expression. fifty percent of clear cell carcinomas strongly expressed mgmt and tgase- . no correlation was found between mgmt and/or tgase- expression, tumor size and grade. eighty percent of urothelial carcinomas strongly expressed mgmt. conclusion: the prognostic value of mgmt and tgase- as well as their potential role in treatment response have been investigated recently. mgmt and tgase- may be prognostic factors in renal carcinomas. further investigation is required to verify our findings. three dimensional topographic analysis of cases of radical prostatectomy a. n. seo * , k. s. lee, g. choe * seoul national univ. bundang, pathology, seongnam-si, objective: prostate cancer is typically mutifocal, and there has been no report about the exact number of prostatic carcinomas in each case. we have performed topographic analysis using three dimensional mapping technique. method: we established data base including cases of radical prostatectomy consisting of individual adenocarcinomas, and performed comprehensive pathologic analysis. objective: tmprss -erg gene fusion is the most common genetic alternation in prostate cancer. it is associated with the expression of oncogene erg protein. recently, the immunohistochemical staining method that using anti-erg antibody was verified strong correlation with erg protein which is the product of genetic alteration. aim of this study is to declare that the relationship between erg expression and clinicopathological factor. method: otal cases of radical prostatectomy specimen were assessed. all cases were constructed tissue microarray and immunohistochemical staining was performed. results: erg-positive rate was . % ( / ) and significantly higher expression of erg expression was observed in the subgroup that has lower gleason score. (p< . ) analysis with the histologic pattern of prostate adenocarcinoma, tumors with discrete glandular unit (gleason pattern ) is shown higher frequency of erg expression (p= . ). conclusion: erg-positive case was smaller than that of western population (about %) and other factors including age, tumor volume, initial psa level, pathological stage and margin status were not significantly related with erg expression. in conclusion, positive rate of erg immunohistochemical staining is meaningful higher in the tumors with wellformed gland that is represented by lower gleason score. solitary fibrous tumour of the kidney mimicking renal cystic neoplasm t. tichy * , j. skarda * university hospital olomouc, institute of pathology, czech republic objective: solitary fibrous tumour (sft) can develop at any anatomic site, but in the kidney is described rarely. in general, sft forms an unencapsulated solid mass. we report a case of a -year old woman with benign sft of the left kidney. the tumour showed extensive pseudocystic change and mimicked renal cystic neoplasm. method: a nephrectomy specimen showed cystic tumour beneath renal capsule and in peripelvic adipose tisssue. histological sections were used for hematoxylin-eosin and for immunohistochemistry (antibodies against vimentin, smooth muscle actin, desmin, s- protein, cd , cd , bcl- , cd , ck , ae -ae , ema). results: microscopically the tumour showed uniform spindle cell proliferation with expansive tumour margins, without necrosis or hemorrhagies. mitotic activity was per high power fields. cystic spaces without epithelial lining contained eosinophilic proteinaceous fluid. imunohistochemically tumour showed difuse positivity for cd , cd , bcl- , vimentin and stained negatively for s- protein, cytokeratins, ema, smooth muscle actin, desmin and cd . conclusion: sft of the kidney is infrequent. some renal sft can undergo pseudocystic transformation and mimic renal cystic neoplasm both clinically and macroscopically. objective: renal angiomyolipomas are mesenchymal tumors that comprised of adipose tissue, smooth muscle like cells and abnormal thick walled blood vessels admixed in various proportions. epithelial renal angiomyolipomas are rare variants and may exhibit atypia. in the literature epithelioid renal angiomyolipomas with atypia are reported to have malignant potential. method: left radical nephrectomy was performed due to renal mass with flank pain and hematuria. results: in gross examination; . × . × cm gray white mass with occasionally necrotic areas at the middle portion of left kidney renal sinus and perinephritic fatty tissue, macroscopically. microscopic eveluation reveals; tumoral lesion comprised of adipose tissue, smooth muscle and blood vessels, showing areas of epitheloid morphology and moderate to severe degree of nuclear atypia. in addition to histomorphology positivity hmb- immunohistocemistry elaborated to the diagnosis of "epitheloid renal angiomyolipoma with atypia". conclusion: this case has been presented due to its infrequent occurrence and malignancy potential. correlation of minute focus of prostate adenocarcinoma on random multifocal needle biopsy with radical prostatectomy specimen a. urbanskiy * * russian research centre for ra, pathology, st. petersburg, russia objective: this work attempts to determine the importance of small foci of prostatic cancer in random multifocal needle biopsy specimens. method: patients with a microscopic focus confined to a single core specimen (which defined as tumor less than mm with a gleason score of or less) were identified from a retrospective review of needle biopsies of the prostate. twelve of these subsequently under went radical retropubic prostatectomy at our centre. clinically significant tumors were defined as those with volume greater than . cc. results: average tumor volume was , ± , cc (range , - , cc). in , % ( ) of the cases was less , cc (range , - , cc, mean volume - , ± , cc). there was ( , %) of patient with extraprostatic extension. conclusion: our data have shown high frequency of revealing insignificant tumours (mean ± %; Р= , ). however, about % of patients had clinically significant tumors warranting definitive therapy. the smallest focus of cancer on needle biopsy is not a guarantee of a clinically insignificant tumor. ps- - e-cadherin in mice: expression in normal urothelium, pre-neoplastic and neoplastic urothelial lesions c. vasconcelos nóbrega * , c. costa, r. arantes-rodrigues, a. henriques, h. vala, a. colaço, l. santos, c. lopes, p. oliveira * escola sup. agrária viseu, dzerv, portugal objective: e-cadherin is an adhesion molecule that promotes the integrity and stability of the urothelium. a decrease in its expression is associated with more aggressive tumour phenotypes, with the ability to invade and metastasize. our aim was to describe the expression of e-cadherin in normal urothelium and in urothelium with pre-neoplastic and neoplastic lesions of icr male mice. method: urothelial lesions were chemically induced by nbutyl-n-( -hydroxybutyl) nitrosamine in icr mice, and evaluated by immunohistochemistry in order to determine the staining pattern of e-cadherin. results: in normal urothelium, . % of e-cadherin expression was at the cellular membrane level. in simple hyperplasia, the same pattern was observed in . % of lesions. nodular hyperplasia exhibited a mixed pattern ( % membrane and % cytoplasmic pattern). in . % of dysplasia, a cytoplasmic pattern was seen. on invasive carcinoma the majority of invasive urothelial cells exhibited a pattern of membrane and cytoplasmic staining. on squamous metaplasia it was observed a membrane pattern on basal and intermediate layers, and a loss of immunoreactivity in the most superficial ones. conclusion: e-cadherin is a valuable tool for investigating the cellular adhesion status of the urothelium in mice. neoplastic lesions exhibited an abnormal, heterogeneous staining pattern. new potential prognostic and predictive factors in conventional clear cell renal carcinoma p. latalova * , p. flodr * fmd pu and fh olomouc, dept. of clinical and molecular pathology, czech republic objective: biological behavior of conventional clear cell renal carcinoma (ccrc) is associated with tumour stage and grade. cancer progression with metastasis is part of a process epithelial-mesenchymal transition (emt) and is joined with invasiveness due to adhesion and cytoskeleton change. the central role of the emt mechanism is attributed to snail factor. the intermediate filament expression is changed through the progression of many types of neoplasms. these changes are organ or tissue specific and depend also on the degree of malignant transformation (genome instability acceleration). the expression changes of cytokeratin (ck ) are signs of aggressive biological behaviour in some tumour types -e.g. colorectal and breast carcinoma (downregulation of expression) or ccrc (upregulation of expression). the expression of snail and ck molecules and mrna ck levels correlate to stage and grade of ccrc (according messai et al. ) . objective: study was based on the expression of immunohistochemical markers (ck , ck , vimentin, snail, cd ) in cases with evaluation of the primary tumour and its metastasis. results: the difference and similarity of intensity and percentage of positive neoplastic population between the primary and the secondary carcinoma will be presented. conclusion: snail and ck seem to be potential tumour progression factors and may be included in so called personalised medicine. objective: sarcomatoid carcinoma (sc) of the prostate is a rare variant of prostatic cancer representing less than % of prostate tumors. tumors are most commonly composed of an admixture of both malignant glandular and spindle cell elements. the sarcomatoid component can vary from % to %. method: we report the case of a -years-old patient with an invasive tumor of the prostate for which he had a radical prostatectomy. histopathological examination showed that the tumor was responding to an undifferentiated proliferation made of beaches and clusters of pleomorphic cells usually spindle-shaped, with eosinophilic cytoplasm, and elongated or oval nuclei, which are very irregular, sometimes monstrous, multinucleated, basophils with numerous mitotic figures. in some places, there were areas of glandular differentiation and foci of comedocarcinoma. immunohistochemical study showed immunoreactivity of spindle and pleomorphic cells with cytokeratin and vimentin. results: the diagnosis of sarcomatoid carcinoma of the prostate-grade gleason + was confirmed. conclusion: sc of the prostate is an exceedingly rare tumor. retrospective analyses render prostate sc as one of the most aggressive prostate malignancies. the prognosis is dismal regardless of other histologic or clinical findings. extra-uterine extension (figo stage ii-iv) were significant parameters of poor prognosis. no multivariate analyses were performed due series limitation. conclusion: detailed pathology evaluation, including histological subtype, and figo stage are essential in the adequate management decision/prognosis evaluation of u-lms. objective: over % of women with endometrial cancer show signs of uterine bleeding, so most cases are diagnosed at an initial stage. this study covers two patients who presented the first signs of this disease in an exceptional manner. method: case- a -year-old woman was afflicted with a six-month toxic syndrome. an omentum tumor was identified in the x-ray. eleven days after the excision, she demonstrated oliguria, dysuria, and scant uterine bleeding. the pelvic ultrasound revealed a uterine mass. case- a -year-old woman demonstrated disabling right inguinal pain. after an x-ray examination of her pelvis, a metastasis was suspected. a biopsy confirmed it. the immunochemistry pointed to a gynecologic, pancreatic or intestinal tumor. the computer tomography scan revealed a uterine mass. results: the first case only suggested an estromal gastrointestinal tumor or a mesothelioma, misdiagnosing and missing the endometrial carcinoma. the second case was uncommon as bone metastasis is found with solid tumors but seldom occurs with endometrial cancer and even less so as a first sign. conclusion: we must consider endometrial cancer at signs of uterine bleeding, however, further investigation is required in order to consider the % who do not show the common signs. extrapelvic endometriosis presenting as a retroperitoneal tumor a. alves * , p. luís, a. ribeiro, m. ferreira * hospital santa maria, servico de anatomia patologica, lisboa, portugal objective: endometriosis is a benign disease characterized by the presence of functional endometrial tissue in ectopic locations. retroperitoneal, liver or kidney involvement are extremely rare. method: we present a case of a -year-old woman, which was referred to surgical consultation because of a retroperitoneal mass that was found on a routine abdominal ultrasonography (us). a subsequent ct scan showed a cm tumor involving the liver, right kidney and adrenal gland. the gynecological us revealed ovaries with normal size and two probable uterine leiomyomas. she was submitted to a tumorectomy with segmental hepatectomy and nephrectomy. results: grossly, the tumor was × × cm, invaded the kidney, the liver and a segment of diaphragm. it was white, fasciculate and with hemorrhagic focus. histologically the tumor was composed of a biphasic proliferation of endometrial glands and endometrial stroma, both without atypia. the case was diagnosed as deep infiltrating extrapelvic endometriosis. with a years follow-up, the patient is doing well and without evidence of disease. conclusion: despite being an extremely rare presentation, endometriosis can involve retroperitoneal organs and simulate a malignant neoplasm, even without a previous history of pelvic involvement. objective: malignant melanoma of the female genital tract is a multifocal disease resulting from a disorder of melanocytes cotyledonoid dissecting leiomyoma is a benign smooth muscle tumor which can mimic a malignant lesion due to its alarming aspect. there are some variant forms of leiomyomas with an unusual infiltrative growth pattern. due to its worrying appearance of the gross specimen, it is often mistaken for malignant lesion and it is important to be aware of this entity not to over treat this benign smooth-muscle neoplasm. we report a case of a years old woman who underwent hysterectomy because of the suspect of a tumor growing from the right lateral uterine wall. on gross study the tumor measured mm in diameter and was nodular, brown in color and irregular on cut section. histological examination showed a nodular tumor composed of smooth muscle cells with a storiform pattern which dissects the uterine wall. the tumor was highly vascularized, included a myxoid component and showed some areas with invasion of vascular components. cellular atypia, mitosis and necrosis were absent. the patient has had a good clinical course, without relapses until the date, what supports the fact that these tumors have a benign clinical behavior, even though they may have a malignant appearance. uterine leiomyoma with lymphoid infiltration a. n. deger * , c. kocak * s.b. dpu. kec. ea hastanesi, patoloji bolumu, kutahya, turkey objective: although leimyomas of the uterus are common, lymphoid infiltration of leimyomas is a rare occurrence. we presented a case of a -year-old woman whose leiomyoma was diffusely infiltrated by lymphocytes. lymphoid infiltration was analyzed with immunohistochemical methods and infiltration was found to be polyclonal type. method: a -year-old woman with abnormal uterine bleeding was prediagnosed uterine leiomyoma and she was applied total abdominal hysterectomy and bilateral salphingo-oopherectomy. the analysis of the sample sent to pathology lab showed two leiomyomas. hematoxylin eosin -stained slides were prepared after sampling. ımmunohistochemical stains were applied. results: microscopic examination of leiomyoma in greater diameter revealed a lesion with well-circumscribed borders composed of interlacing fascicles of bland monomorphic spindle cells diffusely infiltrated by lymphoid cells. the infiltrate not extended into the surrounding myometrium. immunohistochemical analysis showed positive staining with cd , cd , cd . it was seen that inflammatory infiltration was polyclonal and involved t cells, b cells and histiocytes. leiomyoma with lymphoid infiltration was diagnosed. the postoperative course was uneventful within a month follow up period. conclusion: leiomyoma with lymphoid infiltration first described in by ferry et all. from that date on, the literature involves few cases. because it is rare and differential diagnose with malignant lymphoma is crucial, we presented the case and reviewed the literature. objective: vulvar intraepithelial neoplasia is divided into two groups: usual type and differentiated type. the differentiated vulvar intraepithelial neoplasia, which is frequently seen with invasive squamous cell carcinoma, can be confused with some benign lesions. the aim of this study is to analyse p , p , and ki- expression characteristics of different histological types of vulvar intraepithelial neoplasia, invasive squamous cell carcinoma, and benign lesions of the vulva. method: in this study, immunohistochemical analysis of vulvectomy cases with p , p , and ki- was performed. results: of patients who underwent vulvectomy, nine had invasive squamous cell carcinoma and nine had vulvar intraepithelial neoplasia. four additional vulvar intraepithelial neoplasia lesions were found accompanying the invasive squamous cell carcinomas. nine benign lesions were found accompanying the invasive squamous cell carcinomas and vulvar intraepithelial neoplasia. mean ki- proliferation index was . % in the usual type of vulvar intraepithelial neoplasia cases and . % in the differentiated vulvar intraepithelial neoplasia cases. no p expression was present in benign lesions. conclusion: ki- pi does not recognize the usual type or differentiated type of vulvar intraepithelial neoplasia. p positivity can be of value in distinguishing differentiated type vulvar intraepithelial neoplasia from benign lesions. an audit of surgical pathology reports of endometrial carcinoma: experience from a referral centre in india k. deodhar * , b. rekhi, s. menon, b. ganesh * tata memorial hospital, dept. of pathology, mumbai, india objective: the aim was to see, compliance to minimum data information in carcinoma endometrium reports, in a team of pathologists; and also to analyze these parameters e.g. tumor size, type, grade, depth of myometrial invasion, lymph node yield, ptnm stage etc. method: during the period of - , from the files of pathology department of our hospital, reports of operated carcinoma endometrium cases were retrieved and analyzed for various, above mentioned, parameters. results: the median age was . years and median tumor size was cm. endometrioid adenocarcinoma was the commonest type ( . %); followed by mmmt ( . %) and serous carcinoma ( . %). grade was the commonest tumor grade ( . %). less than half of myometrial invasion was seen in % of the cases, =/> half myometrial invasion was seen in . % of cases. (information-not available in cases). parametrial involvement was seen in . % cases. the ptnm stage was not mentioned in . % reports. the median lymph node yield was . conclusion: the compliance to adhere to minimum data information in carcinoma endometrium reports is generally good. lymph node yield is reasonable. parametrial involvement and mentioning of ptnm staging is to be done more meticulously. use of proformas/checklists is recommended. extraovarian granulosa cell tumor: a case report i. efstratiou * , s. pervana, e. pazarli, d. alataki * papageorgiou hospital, dept. of pathology, thessaloniki, greece objective: extraovarian granulosa cell tumor is a very rare neoplasm. we report a case of a tumor located in mesocolon. method: a -years-old woman presented with abdominal pain and fever. abdomen ct showed a mesocolic mass adherent to the left kidney and the spleen. she underwent resection of a cm long segment of the left colon with a circumscribed mesocolic tumor measuring × cm. the tumor was infiltrating the subserosa of the large intestine. intraoperatively normal ovaries have been identified. results: histologically the tumor is composed by monomorphous cells in solid nests developing cavities which contain serous or hemmorhagic fluid. the tumor cells have scanty cytoplasm and often grooved nuclei. mitoses are very rare. the tumor cells were immunoreactive for vimentin, inhibin and progesterone receptors. the postoperative course was uncomplicated and the patient is free of disease months later. conclusion: light microscopic and immunohistochemical features of the tumor are similar to those of ovarian granulosa cell tumors (gct). since the tumor was resected in toto and the mitotic activity of the cells is very low, we expect a favorable prognosis. extraovarian gct are very rare and only cases have been published during the last years. probably these tumors arise from residual tissue of the genital ridge (so-called secondary mullerian system). detection of amplification of terc and tert genes in cytology samples from cervical neoplasias by fluorescent in situ hybridization a. farkasova * , e. kudela, t. balharek, p. zubor, j. danko, l. plank * jfm cu, dept. of pathology, martin, slovakia objective: increased telomerase activity represents an early event in cervical carcinogenesis allowing cell immortality by recovering chromosomal telomeres. thus, detection of terc and tert gene amplification might represent a diagnostic and valuable prognostic biomarker of cervical neoplasias. method: cervical smears from patients classified according to bethesda as nilm (n= ), asc-us (n= ), l-sil (n= ), h-sil (n= ) and scc (n= ) were analysed for terc ( q ) and tert ( p ) gene amplification by fluorescent in situ hybridization (fish) in cells per slide using a four-color fish probe (fhact™). results: the numbers of terc and tert copies, average ratio of gene copies and average number of cells with ≥ terc gene copies were highest in scc, followed by h-sil, l-sil, asc-us and nilm cases. correlation between terc/tert amplification intensity and oncocytological findings was statistically significant (p< . ). conclusion: fish analysis of terc and tert genes could be effective tool for the diagnosis of cervical neoplastic lesions. using together with cytology and hpv dna testing it can achieve higher sensitivity and specificity to discriminate h-sil and invasive carcinomas from l-sil lesions. objective: ovarian stromal hyperplasia (osh) is characterised by non-neoplastic overgrowth of the ovarian cortical. mild hyperplasia of the cortical and medullary stroma is found in the ovaries of about one-third of perimenopausal and postmenopausal women. it is nearly always diffusely bilateral. we report a case of osh associated with endometrial carcinoma (ec). method: a -years-old caucasian woman, obese, smoker, with fatty liver, gilbert´s syndrome and cholelithiasis. she had endometrial curettage biopsy for dysfunctional uterine bleeding for month, which was diagnosed of complex atypical endometrial hiperplasia. a total abdominal hysterectomy whith bilateral salpingo-oophorectomy were performed. results: the diagnosis was well differentiated endometrioid ec and bilateral osh with focal stromal hypertecosis. conclusion: osh of moderate to severe degree may be found in women with disorders associated with androgenic and estrogenic manifestations including ec, obesity, hypertension, and glucose intolerance, but these findings are less frequent and less obtrusive than in stromal hyperthecosis. obese woman are at risk for developing endometrioid ec as a result of the increased capacity in adipose tissue to convert androstenedione to oestrone, and testosterone to oestradiol. a relationship in the origin of hormone-dependent endometrial pathology may exist between osh, blood androgen levels and ec. detection of micrometastases in para-aortic lymph nodes in patients with carcinoma of the uterine cervix after negative frozen section analysis l.-c. horn * , c. kellner, r. scherling, m. höckel, j. einenkel * medizin. universität leipzig, institut für pathologie, germany objective: previous studies considered the presence of micrometastases (mm) in pelvic lymph nodes as clinically relevant prognostic indicator. method: frozen section analysis was performed in all cases. after fs-examination nodes were examined by one h&e-stained slide. all nodes without metastatic disease after frozen section and permanent section examination were subject of the present study. patients and pan were enrolled and immunohistochemical staining using two cytokeratin-cocktail antibodies (ae /ae and kl-) was performed. results: in one case, one single node showed micrometastasis, representing an incidence of . % of the studied cases and . % of the examined lymph nodes. in three cases benign endosalpingiosis was seen. the patient with mm is alive without evidence of disease months after surgery. itc were not observed. conclusion: the frequency of mm in pan is very low. there are only limited data regarding their prognostic impact within the literature. after careful examination of all removed pan using h&e-staining (and step sectioning), immunohistochmeical ultrastaging cannot be recommend for routine use. serous tubal in situ carcinoma (stic) in tubal and primary peritoneal carcinomas l.-c. horn * , k. leonhardt, s. kafkova, j. einenkel * medizin. universität leipzig, institut für pathologie, germany objective: serous tubal in situ carcinoma (stic) has been defined as one important precursor of pelvic serous cancer. morphologically, stic is defined by are cytologic atypia, high proliferative index and strong staining for p . method: the present study evaluates the presence of stic and p -signature in consecutive cases of prophylactic salpingo-oophorectomy in women with brca- -mutaion (bso), macroscopically inconspicuous tubes of patients with primary tubal cancer (tc) and cases of primary peritoneal cancer (ppc) using immunohistochemistry against ki- and p (clone do- ). results: the frequency of p -signature and stic was % and % in cases of prophylactic surgery, % and % in tc and % and % in ppc. conclusion: stic and p -signature as precursor lesions of pelvic serous cancer is seen in macroscopically inconspicuous fallopian tubes in unilateral tc in patients with elective bso and patients affected by ppc. we propose that the sectioning and extensively examining the fimbria protocol be applied to all cases with ppc, tc and in women with prophylactic bso. objective: primary adenocarcinoma of the vulva is rare, and enteric differentiation is excepcional. only a few cases of neoplasms of pure intestinal-type in the low genital tract have been reported. they are considered to arise from cloacal remants or from intestinal heterotopia. method: we present the case of a year-old woman, with a histerectomy performed years before. on clinical examination a polipoid lesion in the vulvar vestibule was noticed. biopsy revealed a tubular adenoma of intestinal type with a focus of adenocarcinoma. the first histologic interpretation was a metastatic intestinal tumor. clinical examination, recto-colonoscopy and magnetic resonance imaging of the abdomen excluded this possibility. results: the immunohistochemical study showed reactivity with citokeratin but didn't with citokeratin . all this led to conclude that it was a primary intestinaltype adenocarcinoma of the vulva arisen from an adenoma. conclusion: on our knowledge there are less than reported cases of intestinal adenoma in the genital tract. they have been described also in the vagina and cervix but only a few developed an adenocarcinoma. it is important to be aware of this tumor type and to distinguish it from metastatic colorectal adenocarcinoma in order to plan appropriate treatment. ligneous cervicovaginitis and endometritis: case report m. koyuncuoglu * , e. dogan * dokuz eylul university, dept. of pathology, izmir, turkey objective: ligneous (wood-like) disease is a rare chronic pseudomembraneous inflammation of the mucous membranes which may also affect genital tract. the underlying pathogenesis is still unclear and an effective method of treatment has not yet emerged. method: here we present a years old female patient presented to our clinic with years of unexplained infertility. her diagnostic infertility work-up revealed no abnormality. however, at gynecologic examination there was a thick and hard granulation tissue at the cervix extending to the posterior vaginal wall. transvaginal ultrasound was unremarkable except for increased endometrial thickness. results: cervicovaginal and endometrial biopsy with diagnostic hysteroscopy was performed which demonstrated ligneous inflammation of both cervix and vagina. endometrial biopsy was reported as chronic non-specific endometritis with dense fibrin deposition. conclusion:. the disease may also affect other organs including oral cavity and eyes. this unusual condition is difficult to treat and lack of awareness makes the diagnosis also problematic. objective: differential diagnosis lm vs stump may be controversial, especially regarding necrosis evaluation; few studies address the relation between different treatments and their morphological effects. we aim to characterize a series lms and putative stumps harboring necrosis. method: consecutive putative stumps ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and a series of lms with necrosis diagnosed at chsj & ipop-porto. clinical files, particularly previous hormonal treatment (ht), gross specimen and histology features. results: putative stumps and lms with necrosis. lms mean age at diagnosis: years; mean size: . cm ( . - ); % with other lms; treatment: myomectomy ( %) and hysterectomy ( %); % peri-partum, . % post-uterine artery embolization; ( %) previous ht: progestin ( %), oral contraceptive-oc ( %), hormonal-iud ( . %), and medical assisted reproduction ( . %). stumps mean age at diagnosis: years; mean size: cm ( . - ); % with other lms; low mitotic index-mi (mean . ; median / hpf); slight cell atypia; variable ischemic/ tumor-type necrosis. treatment: myomectomy ( %), hysterectomy ( %) and radiotherapy ( %); ( %) previous ht: progestin ( %), oc ( %), hormonal-iud ( %); no recurrence in remaining cases (n= ): median follow-up: . months. conclusion: lms with necrosis and stump may display overlapping features. diagnosis of stump should consider previous ht, detailed morphology (necrosis type/atypia/mi), to prevent overdiagnosis of stump. positivity. plap and chromogranin were negative. ki- nuclear positivity was found in less than % of sertoli cells and in about % of leydig cells in hyperplastic areas. karyotype was xy. conclusion: although, surgical pathologists encounter tfs rare in a clinical practice, they should be aware of this condition, especially in lack of relevant clinical data, when it could be interpreted as a tumor. objective: frozen section (fs) diagnosis of ovarian mucinous tumors can be difficult due to the size of these tumors, heterogeneity and potential risk of metastasis from gastrointestinal (gi) neoplasms. method: we reported ovarian mucinous tumors submitted for (fs) evaluation between january and avril was conducted. results: fs and final pathology results were collected. the average tumor size was , cm ( - cm). the fs and final pathology diagnosis were concordant in , % ( / ) of the cases. of the ( , %) discordant cases, one ( , %) was downgraded and cases ( , %) were upgraded. of the tumors interpreted as borderline mucinous tumors (bmt) on fs, ( , %) were malignant at final diagnosis ( ovarian, gi), ( %) remain as bmt and ( , %) was benign. of the benign tumors on fs, cases ( , %) were upgraded to bmt at final diagnosis. tumors with a malignant diagnosis on fs ( cases) were % concordant with final diagnosis. conclusion: our study showed a , % rate of discordance between fs and final diagnosis. given that gi origin is a possible finding, intraoperative assessment of the appendix should be performed in all mucinous ovarian tumors. uterine müllerian adenosarcoma: a clinicopathologic study of cases a. nasfi * , l. charfi, k. mrad, s. sassi, r. sellami-dhouib, r. doghri, m. driss, i. abbes, s. nechi, w. jomaa, k. ben romdhane * salah azaiez institute, dept. of pathology, tunis, tunisia objective: müllerian adenosarcoma (ma) is a distinctive type of uterine tumor, traditionally regarded as a low-grade variant of mixed müllerian tumors. method: thirty one cases of adenosarcoma were examined during a period of years (may -february . results: thirty tumors were of the uterine corpus and one of the cervix. the mean patient age was , years (range: to year). the main clinical manifestations were vaginal bleeding and pelvic pain. physical examination showed cervical/vaginal mass or pelvic mass. treatment was known in cases: patients underwent hysterectomy with bilateral salpingo-oophorectomy in cases and lymphadenectomy in cases. tumor size ranged from , to cm (mean: . cm). microscopically, sarcomatous overgrowth was found in cases ( %), heterologous elements in ( %). eleven cases ( %) had myometrial invasion involving the internal half of the myometrial thickness in cases, and more than % in cases ( %). the cervical tumor presented as an endocervical polyp without invasion of the cervical wall. conclusion: uterine ma are low-grade neoplasms capable of local recurrence and much less commonly distant metastasis. surgical excision is the main treatment strategy with a good prognosis in the early stage disease. the most important adverse prognostic factors are deep myometrial invasion and sarcomatous overgrowth. spectrum of epithelium changes in adenomyosis n. nizyaeva * , e. kogan, t. demura * scientific center of obstetric, dept. of pathology, moscow, russia objective: adenomyosis (am) is a very common gynecological disorder. despite high incidence of the disease epithelial changes precise developmental events leading to the condition remain controversial. the aim of the study was to investigate and compare epithelium morphologicaly and markers expression of proliferation, apoptosis, invasion and neoangigogenesis in am foci. method: this study was done on biopsy samples of uterus taken from women with adenomyosis. immunohistochemical staining of tissues was performed with antibodies to apocas, ki- , mmp- , timp- , claudins , (cl , ), e-cadherin, cox- , egfr, vegf. results: four variants of epithelium changes were found in am foci such as proliferative type, hyperplastic type with and without atypia, and atrophic type. times during years and investigated using routine light microscopy and immunohistochemistry (ihc) to control the treatment response. results: in the course of progestin treatment the complex endometrial hyperplasia with intraepithelial neoplasia became histologically less complicated and than normal. ihc profiles and ki- expression had the same dynamics. in the youngest patient the unusual gland cell atypia with focal calcification was observed one time and hysterectomy was considered. conclusion: the study confirms that the repeated curettages with conservative treatment and histological studying can be successfully used in some cases of complex endometrial hyperplasia with intraepithelial neoplasia. value of ki , p and ck markers in differentiating cervical intraepithelial neoplasia and benign lesions a. safaei * , m. pourjabali, f. sari aslani, m. momtahan * shiraz university of medical sciences, iran objective: the cervical cancer is one of the most common cancers among women worldwide. diagnosis of cin affected by high rates of discordance among pathologists. therefore, we need to other adjunct methods for accurate diagnosis of cin versus benign lesions in equivocal cases. the aim of this study was evaluation of ki- (mib- ), ck and p ink a (p ) markers by immunohistochemical method in differentiating cin from benign cervical lesions. method: seventy-seven cervical biopsies that originally diagnosed as non-cin (n = ) and cin (n = ), were reviewed by three pathologists and re-classified as non-cin (n = ) and cin (n = ), based on agreement between at least two of three, to obtain a consensus diagnosis. consensus diagnosis was defined as the "gold standard". then immunostaining for ki , p and ck was performed on all cases and their results were compared with original and consensus diagnosis. results: the overall agreement between original and consensus diagnosis was . % (kappa = . , p-value < . ). the sensitivity and specificity of ki immunostaining were . % and . % and for p were . and . %, respectively. the sensitivity and specificity of ck negative staining for cin detection were . % and . % respectively. conclusion: we recommended using ki and p markers as complementary tests for differentiation between dysplastic and non-dysplastic lesions. ps- - wt expression in ovarian borderline and malignant surface epithelial tumors m. tahamtan * , f. sari aslani, a. safaee * shiraz, iran objective: wilms tumor gene product, a tumor suppressor gene, now is considered to have oncogenic functions. there seems to be differences in wt expression among surface epithelial ovarian tumor subtypes. method: immunohistochemistry for wt was done on serous & mucinous cystadenocarcinomas, borderline serous & borderline mucinous tumors, endometrioid ovarian carcinomas, clear cell carcinomas, malignant brenner tumor, metastatic adenocarcinomas and endometrial adenocarcinomas. a tumor was considered negative if< % of tumor cells were stained. positive reactions were graded : +, - %; +, - %; +, - %; +, - %. results: of serous cystadenocarcinomaes, ( . %) were positive, showed reactivity of< % of the tumor cells and one were negative. all borderline serous tumors were positive. all mucinous tumors, endometrioid carcinomas, clear cell carcinomas, metastatic adenocarcinomas and primary endometrial carcinomas were negative. the single malignant brenner tumor were positive for wt . conclusion: wt is a good marker to distinguish primary ovarian serous carcinomas from other surface epithelial tumors (especially endometrioid subtype) and metastatic carcinomas (especially endometrial serous carcinoma), other than malignant mesothelioma. degree of expression is not an indicator to separate high grade borderline serous tumors from low grade ones. objective: fatwo is a rare neoplasm originating from the mesonephric duct remnants that occurs predominantly in the broad ligament but also in the ovary. it is considered a lowmalignant potential tumor. however local recurrences and metastases have been reported. method: a year-old female, on routine gynecological physical examination, followed by pelvic ultrasonography and computer tomography was diagnosed with a mass at the right ovary. a salpingo-oopherectomy was performed. on gross examination a well-circumscribed, solid and focally cystic tumor measuring , × × , cm arose in the ovarian hilus. results: microscopically the tumor consisted of mediumsized, ovoid to polygonal cells arranged in a solid, tubular and sieve-like pattern. some cystic spaces were lined by low-cuboidal cells and contained amorphous, eosinophilic material. cellular atypia and mitoses were rare. the tumor cells were positive for vimentin, inhibin-a (focally), cd , cytokeratins / , and (focally) and negative for cytokeratin , epithelial membrane antigen, carcinoembryonic antigen and a-fetoprotein. conclusion: histopathological and imunohistochemical findings consistent with an ovarian fatwo. differential diagnosis includes endometrioid carcinoma, clear-cell carcinoma, sertoli-leydig cell tumors (retiform variant) and rete ovarii adenoma. surgical excision is the optimal treatment. radiation therapy, chemotherapy or even targeting molecular therapy is questionable. müllerian adenosarcoma is a rare mixed tumor of low malignant potential. usually presented as a large endometrial polyp in postmenopausal women. they are associated with tamoxifen or radiation therapy. microscopy shows a mixture of benign glandular epithelium and low-grade endometrial sarcoma is typically concentrated around the glands. the differential diagnosis is made with the adenofibroma, but now is doubted the existence of this tumor and is considered more of a distinct adenosarcoma. the treatment is total hysterectomy. sisters of and years with a history of dysmenorrhea. hysteroscopy was performed to the eldest one, in which multiple polyps are observed and partially resected. the pathological diagnosis was mixed mullerian tumor, adenofibroma/adenosarcoma. total abdominal hysterectomy was performed. histological examination showed a mixed tumor with glandular component and a low-grade sarcomatous, scarce mitosis, mild atypia and no necrosis. the diagnosis was: mullerian adenosarcoma. a year later, a hysteroscopy and endometrial biopsy was performed to the youngest sister, that showed endometrial polyps with stromal predominance, and no significant atypia. with the diagnosis of mullerian adenosarcoma, and taking into account family history, total hysterectomy was performed. adenosarcoma is a rare tumor whose family presentation is not described in the literature. gliomatosis peritonei is associated with frequent relapse but not affects overall survival in patients with ovarian immature teratoma n.-r. yoon * * samsung medical center, dept. of pathology, seoul, republic of korea objective: gliomatosis peritonei (gp) associated with ovarian teratoma has known to have no adverse prognostic effect. we investigated the clinicopathological features of ovarian teratoma associated with gp, and compared immature teratomas (its) with gp to its without gp. method: we investigated patients with ovarian teratoma with gp and patients with it without gp, who were diagnosed at samsung medical center (seoul) from january to august . results: six patients with it with gp ( . % of patients) had recurrence. when it with gp (n= ) was compared to it without gp (n= ), patient of it with gp showed larger tumor size (median, cm vs. median cm) (p< . ), more frequent relapse ( %, / vs. . %, / ) (p= . ), and frequently elevated pre-operative ca level ( %, / vs. %, / ) (p= . ). survival curves showed significantly shorter relapse-free survival in patients of it with gp (p= . ). two-year relapse-free survival rates were . % and . % in it with gp and it without gp, respectively. however, all patients except one case of it with gp alive. conclusion: ovarian it with gp was characterized by larger tumor size and frequent elevation of preoperative ca level, and gp was associated with frequent relapse in patients with ovarian it. corelation between histopathologic diagnosis with p and ki immunostaining, cytologic features in cervical neoplasia c. altunkaya * , s. yilmaz, a. barin, s. ekici, g. s. yalcin, m. caydere, h. ustun * s.b ankara e. a.h, pathology, turkey objective: frequency of cervical carcinoma decreases nowadays since cytologic screening methods have been using extensively. the incidance of cervical intraepithelial neoplasia increases. cytologic diagnosis og these lesions are substantially important. so we aim to identify the role of p and ki immunostainings for predictive factors of cervical neoplasia. method: the study was conducted january to january . ninety-three cases (mean age , ; range - years) diagnosed as squamous intraepithelial lesions were included, objective: there are several prognostic factors in melanoma. it was suggested that her- expression may influence the tumor behavior. the aim of the study was to investigate the relationship of her- expression with various prognostic factors. method: her- expression was evaluated in melanomas, without metastases and with lymph node and with distant metastases. membranous, cytoplasmic and nuclear her- expression was separately analysed. the staining intensity and percentage of positive tumor cells were evaluated. results: the cytoplasmic staininig was seen in all cases, with average intensity of and percentage of positive cells ranging from % to % (mean . %). the percentage of cytoplasmic her- positive cells was inversely correlated with clark and breslow stage (r=− . and r=− . ). cytoplasmic her- reaction was significantly stronger in cases with lymph node metastases ( . vs. . p< . ). in cases a dot-like cytoplasmic reaction was seen. membranous positivity was seen in cases ( %). conclusion: her- may play a role in melanoma progression. it may be involved in lymphatic dissemination. further studies of prognostic significance of her-expression in melanoma are needed. hypopigmented mycosis fungoides with unusual vitiligolike presentation in child h. erdem * , n. buyukbabani, h. turan * duzce university, dept. of pathology, turkey objective: mycosis fungoides (mf) is the cutaneous t cell lymphoma. classical, clinical and histopathological findings of mf are detected in most of the patients. however, some of the patients have defined atypical mf. hypopigmented mf (hmf) is one of the atypical forms. hmf could be misdiagnosed with clinical and histopathological examination. hmf is considered mistakenly vitiligo. therefore, it should be considered differential diagnosis. mf is usually seen in the middle aged and elderly. the occurrence of mycosis fungoides in children is very rare. method: an -year-old girl child attended dermatology clinic with complaints of pruritus and hipopigmented patches. lesions was performed punch biopsy and reported hmf. conclusion: herein, this case presented because it was considered vitiligo as clinically and diagnosed hmfas histopathologically. reducing block sampling in wide local excisions for melanoma c. fives * , c.c.b.b. heffron * cork university hospital, dept. of histopathology, ireland objective: it is established practice that wide local excision (wle) is undertaken for the further management of cutaneous melanoma, however, definitive guidelines for macroscopic sampling have not been established. our aim was to determine whether our sampling of wle specimens was adequate, inadequate or excessive and to establish guidelines for these specimens. method: cases which underwent initial biopsy and subsequent wle in were identified. we recorded the specimen size, macroscopic appearance, number of blocks sampled and margins of the original biopsy. results: there was wide variation in the number of blocks sampled (range - ). residual melanoma was identified in of the cases ( . %) which had clear margins on the original biopsy. of these, had evidence of a pigmented lesion on macroscopic examination. the remaining cases had margins of mm or less on the original excision. no subsequent surgery was performed on these cases. conclusion: our study has shown that in wle specimens with no evidence of a macroscopic lesion and in which margins of the original biopsy were clear by greater than mm, little is to be gained from extensive sampling. reduced sampling would result in saving laboratory resources with a predicted % block reduction in our laboratory alone. functioning oxyphil adenoma of parathyroid gland: a case report m. genadieva -yordanova * , s. hristova, a. vlahova, g. todorov, b. miserliovska, s. yordanov * alexandrovska hospital, dept. of pathology, sofia, bulgaria objective: the most frequent cause of primary hyperparathyroidism are adenomas of the parathyroid gland, the majority of which are composed of chief cells. oxyphil adenomas are uncommon and account for % of functioning parathyroid adenomas. up until year they have been considered non-functional. method: laboratory findings revealed calcium levels of . prior to operation. the serum parathyroid hormone level was . pg/ml. a ct scan of the neck showed normal size thyroid lobes with heterodense structure with an oval hypodense lesion in the lower right pole of the thyroid. results: at surgery a mahagony-brown mass measuring mm in diameter was removed and reported as oxyphil adenoma on frozen section. histopathological examination revealed encapsulated adenoma composed of oxyphil cells with abundant, granular pink cytoplasm and a rim of normal parathyroid tissue. mitotic figures were absent. postoperatively, a decrease of the serum calcium down to . mmol/l was declared. the followup period was unremarkable. conclusion: there appear to be a growing evidence that a big part of oxyphil adenomas of parathyroid gland can produce parathyroid hormone and contribute to the cases of primary hyperthyroidism. effectiveness of uvb phototherapy on mycosis fungoides using histologic guitart criteria in iranian patients a. ghanadan * , a.-h. ehsani, h. seirafi, m. khiabani * tehran university, razi hospital, iran objective: mycosis fungoides (mf) is the most common primary cutaneous lymphoma. mycosis fungoides often develops slowly over many years, presenting with a generalized erythroderma, skin patches or plaques. the diagnosis of mf requires the integration of clinical and histopathologic findings. narrowband uvb (nbuvb) is widely used to treat mf. to evaluate the effectiveness of therapy the histopathologic findings before and after nbuvb reviewed using guitart criteria. method: we enrolled patients ( women, men; age range, - years; mean age, . years) with clinically and histologically proven mf. the patients received nbuvb phototherapy three times a week. a biopsy was performed months after onset of the treatment and guitart criteria used to scoring mf before and after therapy. results: phototherapy was reduced the primary intraepidermal atypical lymphocytes (p value= . ), dermal atypical lymphocytes (p value= . ), epidermotropism (p value= . ), density of infiltration (p value= . ) and lymphocytic infiltrate without inflammatory features (p value = . ) in all patient. but the relationship between the reticular fibroplasia of papillary dermis and phototherapy was not proved (p value= . ). overall score of patients was significantly lower after phototherapy (p value= . ). conclusion: our data suggest that nbuvb therapy is reduces histiologic score of mf and is effective for the treatment of iranian patients. nodular colloid degeneration of the skin: report of three cases a. ghanadan * , k. kamyab-hesari, m. daneshpajouh, k. balighi, m. khosravi * tehran university, razi hospital, iran objective: nodular colloid degeneration (ncd) is a rare dermatological disorder and also a rare type of colloid milium. the degeneration may be related to sun exposure. method: three cases of ncd were enrolled from the archive of dermatopathology department of razi hospital during - . results: in this report, three cases, all presented with multiple plaques and nodules in the nose and the face, are depicted. histologically, these nodular masses were homogeneous, with eosinophilic clefted materials expanding the papillary dermis and extending into the deep dermis. histochemical review showed the reactivity of the colloid materials via the pas, crystal violet and methyl violet staining. all the three cases were finally diagnosed as nodular colloid degeneration. conclusion: ncd is a rare disease but it should be considered in any cases with a history of long exposure to the light. we suggest the long term exposure to the sun as an etiologic factor thus, sun protection would be the most preventive and available treatment. glypican- protein expression in melanoma: a immunocytochemistry study n. gursan * , h. balta, b. gundogdu * ataturk university, medical faculty, erzurum, turkey objective: glypican- (gpc ) is a cell surface heparan sulfate proteoglycan. serum gpc was shown to be expressed in % of melanomas (ms) but gpc expression in melanoma tissues had not been investigated.in this study, immunohistochemical analysis of gpc protein expression was investigated in histologic sections from melanoma tissues. method: melanoma patients, twenty patients with insitu melanoma, twenty patients stage and stage i melanoma, twenty patients stage ii and stage iii melanoma. all cases were stained with anti-gpc antibody. gpc expression was divided into categories: negative (negative or weak cytoplasmic staining) and positive (moderate or strong cytoplasmic with membranous accentuation). results: gpc immunopositivity. showed in , % of melanomas. gpc expression at stage , i, ii, iii were . %, . %, . %, % respectively. method: we report the case of a year old man presenting with an erythematous plaque of the right hemithorax. results: macroscopically the lesion had irregular borders and was mildly infiltrative with a violet hue. microscopic examination revealed tumor cells within lymphatic spaces, without infiltration of the adjacent stroma. the histologic and immunophenotypic characteristics of these cells were compatible with a carcinoma of lung origin. ct scan revealed a lung mass as well as multiple liver and chest wall metastases. conclusion: malignancies originating from the breasts, lungs and large bowl are the most common to involve the lymphatic net of the skin. melanoma on the other hand is more frequently presenting with lymphangitic invasion, whereas inflammatory carcinomas may also affect lymphatic vessels and may be confused with erysipelas. the revelation of the primary origin is not always easy and differential diagnosis should also include gynecological malignancies, kidney and urinary bladder carcinomas. in our patient the diagnosis was established on the grounds of histology and immunohistochemistry, and was supported by the clinical and radiological findings. objective: inverted follicular keratosis (ifk) is almost always a solitary lesion occuring mainly in adult life. men are affected twice as often as women. pathologically, they can be confused with a variety of lesions, both benign and malignant. squamous carcinoma is the most serious differential diagnosis. method: we decribe a retrospective study about cases of ifk diagnosed over a -year-period ( - ) . results: we collected lesions, all treated by surgical excision. haemalum eosin sections were studied in all cases. all lesions were single. five, of them, were situated on the face and two on the scalp. all the patients were adults with a mean age of years, average between and years. they presented mostly as asymptomatic papules and all were small lesions. the different sections of the lesion showed skin well delineated endophytic epithelial proliferations with inverted papillomatous and acanthotic components containing several circumscribed squamous eddies. they was no atypia, mitotic activity necrosis or stromal invasion. conclusion: ifk pose very real diagnostic problems unless one is aware of this entity. in fact, they may mimic malignant lesions especially squamous cell carcinoma, both clinically and pathologically. primary cutaneous follicular lymphoma with prominent spindle cell areas. so called spindle cell follicular lymphoma. case report g. ivády -szabó * , t. strausz, e. tóth * national institute of oncology, dept. of surgical oncology, budapest, hungary objective: spindle cell type variant of cutaneous follicular lymphoma is a rare histologic variant of primary cutaneous follicular lymphomas. it is caracterised by the presence of spindle and bizarr cells. we present a case of a years old man who had a nodule on his scalp and two smaller nodules on his frontal region. we examined an excisional biopsy specimen of the forehead. method: he, stains all and immunohistochemical stains were used. results: histology revealed dense lymphoid infiltrate showing nodular pattern in the dermis and in the subcutis. the lymphoid infiltrate predominantly composed of large centrocytes. there were areas where the neoplastic cells show spindle cell morphology with bizarre nuclei and in these areas the stroma were myxoid, mucinous. with immunohistocemical stains the neoplastic cells were positive for cd , bcl- , and negative for cd , cd , vimentin, cd , s , actin, desmin, ae /ae . cd showed residual network of follicular dendritic cells in the background. conclusion: spindle cell lymphoma is a very rare variant of cutaneous follicle centre cell lymphoma and the presence of the spindled bizarre cells can cause differential diagnostic problems. the main differential diagnostic entities are primary or metastatic spindle cell sarcoma and spindle cell melanoma. careful morphological and immunohistochemical analysis are required to the correct diagnosis. the histopathologic and immunohistochemical features of mycosis fungoides b. a. karabork * , a. okcu heper, s. yuksel, i. kuzu * ankara university of medicine, dept. of pathology, turkey objective: the histopathologic diagnosis of early mycosis fungoides (mf) is often difficult. the lesions can mimic a variety of inflamatory dermatitis. we aimed to establish and draw attention to the most frequent histopathologic and immunohistochemical features of mf. method: we reviewed skin biopsies of clinicopathologically diagnosed mf cases at medical university of ankara. we looked for a) epidermotropism, atypical lymphocytes, morphologic features in the epidermis, dermoepidermal junction and dermis b) immunohistochemical staining ratios of cd , cd , cd , cd , cd and expression loss of cd , cd . results: atypical lymphocytes) ( %), dermal fibrosis ( %), epidermotropism ( % single cells, % linear arrangement, % pautrier's microabscesses, % 'haloed' lymphocytes), epidermal acantosis ( %), basal vacuolar degeneration (focally %, marked %) and a perivascular lymphocytic infiltrate were the most common and important features. dermal edema ( %) extravasated erythrocytes ( %), spongiosis ( %), eosinophils ( %) and necrotic keratinocytes ( %) were the less seen and non-specific ones. immunohistochemical results correlated with a ratio of % cd , % cd positive mf. expression loss of cd was seen in %, cd in % of cases. conclusion: diagnosis of early mf requires the correlation of morphologic, immunohistochemical and clinical features. also due to the disease's heterogenity, biopsies from different locations and rebiopsies will enhance the diagnosis. connexins of cutaneous melanocytic tumours g. kiszner * , z. buday, i. teleki, e. varga, i. b. nemeth, i. korom, t. krenacs * semmelweis university, st dept. of pathology, budapest, hungary objective: connexins (cx) form transmembrane channels that can transport ions and small regulatory molecules between adjacent cells. they also function as hemichannels and through protein interactions and are involved in the control of cell replication and maintenance of multicellular homeostasis. method: we have tested the expression of connexins in melanocytic tumours using immunohistochemistry in tissue microarrays of common and dysplastic nevi, and primary and metastatic malignant melanomas. results: cx was not found in melanocytic tumours despite expressed in the basal epidermis. cx . reaction showed punctate cell membrane staining in % of naevi in their superficial regions including atypical nests, and displayed cytoplasmic staining in % of melanomas. low levels of cx were revealed in the cytoplasm of > % of naevi and melanomas but only % and % showed membranous positivity, respectively. cx perinuclear/cytoplasmic immunostaining was observed in % of naevi and % of melanomas and as cell membrane reaction in % and %, respectively. punctate cx reaction was detected in vertical tumour nests in % of naevi, while only % of melanomas proved positive and showed cytoplasmic cx delocalization. conclusion: therefore, most tested connexins were significantly down-regulated in malignant vs. benign melanocytic tumours that possibly contribute to their malignant phenotype. quantitative follow up study of cd a, cd and cd positive cells in multiple basal cell carcinoma cases after combined treatment r. kleina * , i. truksane, j. kisis, i. franckevica * riga stradins university, dept. of pathology, latvia objective: in last years appear more cases with multiple basal cell carcinomas (bcc). skin immune system reaction is especially important in nonsurgically treated cases. aim of study is to evaluate the dynamics of cd a, cd , cd marked cells in bcc and in skin adjacent to tumour ( and mm) before and after the treatment with cryotherapy and imiquimodum. method: from bcc cases with multiple tumours investigated immunohistochemically, in dynamics we have characterized patients. antibodies for cd a, cd , cd + cells in derma and epidermis were used. they were evaluated in fields of vision ( ×) before and after treatment. % imiquimod cream and double freezing was used. control group: three normal skin samples. statistical analyses of results were done. results: after treatment were erythema, then crust and exfoliation. microscopically expressed fibrosis instead bcc was found. amount of immune cells before and after treatment were: cd ± , / ± , (also in epidermis), langerhans cells ± , / ± , , cd ± , / ± , . conclusion: all immune cells of skin react to the combined treatment of bcc in radius of mm. there is significant statistical difference between the numbers of langerhans cells in normal epidermis and in bcc cases treated with cryomethod and imiquimodum. regulatory t-cells in invasive and in situ squamous cell carcinoma of the skin and actinic keratosis h. kourea * , a. stravodimou, v. tzelepi, h. papaioannou, h. papadaki, c. scopa * university of patras, dept. of pathology, greece objective: regulatory Τ-cells (Τregs) participate in tumor tolerance and facilitate tumor growth and foxp transcription factor is necessary and sufficient for their development and function. we investigated the presence of t-regs in invasive (in) and in situ (is) squamous cell carcinoma (scc) of the skin and actinic keratosis (ak). method: tregs were identified using immunohistochemistry for foxp and recorded using image analysis in cases of inscc (and their adjacent is, ak or benign tissue (bn), when present), in cases of is and cases of ak (and their adjacent bn tissue). statistical analysis was performed using the paired t-test. p-values < . were considered statistically significant. results: in inscc cases, Τregs in the tumor were more numerous than the adjacent Βn (n= ) (mean vs. , p < , ). additionally, adjacent is had more tregs than adjacent ak (n= ) ( vs. , p= , ). significant differences between inscc, and is or ak were not observed. in is and ak cases, Τregs were more numerous than the adjacent bn ( vs. , p< , and vs. , p< , ), respectively. conclusion: treg infiltration of the skin increases early from the precancerous ak, indicating early involvement of tregs in the development of scc. objective: mastocytosis is a rare disorder and its true incidence is unknown. skin is most commonly involved, followed by the bones and the gastrointestinal tract. method: five men presented with many brown lesions in the trunk and one woman with multiple redish brown nonpruritic macules in the extremities. we used immunohistochemistry for c-kit and tryptase for the definite diagnosis. the expression of cd and cd were also evaluated. results: histology showed five cases of urticaria pigmentosa with severe diffuse infiltration of the papillary dermis by mast cells and one case of telangiectasia macularis eruptiva perstans (tmep) with scattered mast cells around dilated capillaries and venules of the papillary dermis. the mast cells were positive to c-kit and tryptase and only in one case there was a coexpression of cd and cd > % of the mast cells. in that case involvement of bone marrow was observed and the diagnosis of indolent systemic mastocytosis was established according to who criteria. conclusion: urticaria pigmentosa is the most common form seen in adults while tmep is an uncommon form that occurs exclusively in adults. the skin involvement in indolent systemic mastocytosis is a part of the spectrum of the disease. ps- - subvisual nuclear characteristics are different between keratoacanthoma and keratoacanthoma-like squamous cell carcinoma k. metze * , m. tabai, r. adam, i. watanabe, a. de moraes, m. cintra * university of campinas, pathology, brazil objective: the differential diagnosis between keratoacanthorna and keratoacantoma-like squamous cell carcinoma may be extremely diffcult. our objective was to study whether computerized image analysis could be helpful. method: in patients biopsies of keratoacantoma-like lesions were taken at admission. one month later surgical excision was performed in growing lesions, whereas regressing lesions were left untreated. a final diagnosis was established combining clinical and histological evaluation, digitalized images from ki- immunostained and hematoxylin counterstained sections of first biopsies were obtained. tumor nuclei were marked interactively. an inhouse computer program analyzed the geometric relations between the nuclei. nuclear gray values and their histogram entropy were calculated. results: keratoacnthomas and keratoacantomalike squamous cell carcinomas entered this investigation. basic variables of the geometric analysis did not differ between the two entities regardless whether all nuclei or only the ki positive ones had been examined. chromatin gray values were significantly lower and their histogram entropy higher in the keratoacantoma-like squamous cell carcinomas. conclusion: basic geometric variables do not seem to be different beteen both lesions, but a shift in the gray value histogram to lower values with increased entropy in carcinomas indicated important differences of the chromatin structure, supported by fapesp and cnpq. non-infectious erythematous papular and squamous lesions of the skin in our institute, with clinicopathologic correlation t. ozgur * , a. c. dogramaci, e. atik, s. hakverdi, m. yaldiz, z. a. tas * mustafa kemal university, medical faculty, antakya, turkey objective: non-infectious erythematous, papular and squamous lesions of the skin are basic lesions that pathologists differentiate in routine laboratory examinations. our aim has been to analyse these lesions by pathologic and clinical findings in our institute with determining clinicopathologic correlation. method: in our study cases prediagnosed as erythematous, papular and squamous lesion by dermatologists and evaluated in pathology laboratory between and have been reviewed. results: the lesions comprised % . of the total load of surgical pathology and . % of total number of skin biopsies. the highest percentage was in the - year age group ( . %) with a female predominance of . %. the limbs were most frequently involved ( . %). psoriatic lesions were the commonest ( . %), classic generalized plaque variant psoriasis ( %) being the most frequent. correlation with the histopathologic diagnosis was positive in . % cases and negative in . % cases. conclusion: the contribution of histopathology to the final diagnosis was significant. it confirmed the diagnosis in . % cases and gave the diagnosis in . % cases. ps- - vulvar lichen sclerosus: a misnomer for an entity with decreased fibrillar components and increased amorphous components in extracellular matrix remodeling e. parra * , c. a. pires de godoy, v. l. correia feitosa, w. teodoro, a. p. velosa, v. l. capelozzi * fmusp, dept. of pathology, são paulo, brazil objective: the hyalinization of subepidermal skin is one of the histopathological characteristics of the vulvar lichen sclerosus (vls). it was found that patients with vls present autoantibodies against the extracellular matrix protein (ecm- ) and the deficiency of this protein is responsible for the development of a different disease, the lipoid proteinosis. this disease shows a similar histology with vls and has better characterized morphology. method: we analyzed vls patients biopsies and the control group was composed by vulva samples from authopsy. the biopsies and control samples were analysed by immunofluorescence for collagen i, iii and v and th total collagen fibers by picrosirius staining. the elastic fibers were stained with verhoeff and the proteoglycans and glycosaminoglycans with periodic acid-schiff and alcian blue. collagen quantification was performed through image analysis. results: it was observed a significant reduction in all studied collagens as well as in the elastic fibers. on the other hand, the proteoglycans were increased in the vls biopsies. conclusion: this study did not found an increase in collagen sclerosis that would justify the term used for the ls. it was observed a predominance of the edema area probably caused by the increase of glycoproteins as in lipoid proteinosis. combined high-grade basosquamous carcinoma and malignant melanoma of the scalp (malignant basomelanocytic tumor) metastasizing to the breast. a case report a. pitino * , s. squillaci, c. spairani, f. ottelli zoletti, m. f. cosimi, m. ferrari, a. tropiano, p. c. rassu, f. tuo, p. maiocchi * san giacomo hospital, division of anatomic pathology, novi ligure, italy objective: background. basal cell carcinoma (bcc) is a very low grade, usually not metastasizing skin malignancy, which needs to be radically excised. methods. an year-old woman was treated for a × , cm multinodular mass in the scalp. one month later an ultrasonography of the left breast showed a , cm mass which was excised with clean margins. axillary lymph nodes were free of metastasis. results: results. the dermal tumor presented well demarcated basaloid epithelial nests with squamous differentiation focally connected with the epidermis. they showed peripheral palisading and high nuclear grade with pleomorphism, brisk mitotic activity and multiple prominent nucleoli. atypical cells, arranged as strips, nests or isolated elements, were observed at the tumor edge, separated by a grey-zone from the overlying malpighian epithelium. immunostains were positive for cytokeratins, melanocytic (s- /hmb- / mart- ) and neuroendocrine (cd /nse/cromogranin) markers. the , cm breast tumor corresponded to a high grade metastatic bcc with reactivity for epithelial markers, s- and cd . objective: pyoderma gangrenosum (pg) was described at first by brocq and named by brunsting et al. in . superficial granulomatous pyoderma (sgp) was described as a variant of pg in . we present three cases of this rare variant. method: there were two women and one man with age ranging from to years. all of them had a long history of a slowly enlarging, eritematous and ulcerative plaque in different areas including leg and breast skin. a biopsy was performed in all cases. results: the superficial dermis showed neutrophilic inflammation with an admixture of granulomatous inflammation and sinus formation. there wasn't vasculitis and fat tissue was not affected. conclusion: sgp is a rare variant of pg. diagnosis of this entity generally is made on the basis of skin biopsy results with all features mentioned previously and a typical clinical appearance. most of times, these lesions are diagnosed as granulomatous dermatitis, thinking in infectious pathology. this is the most important differential diagnosis, because sgp is responsive to corticosteroids. the pathogenesis of pg is unknown, though is now believed to altered neutrophil chemotaxis and some authors suggest that sgp may be a delayed-type hypersensitivity to an unknown antigen. pathergy is the inciting factor in several patients. high concordance in braf status between native-braf malignant melanoma and matched lymph node metastases a. santos briz * , e. godoy, l. arango, p. antúnez, m. yuste, c. roman, e. fernández, m. d. ludena * hospital universitario salamanca, dept. de anatomía patológica, spain objective: the discovery of selective v-raf murine sarcoma viral oncogene homolog b (braf) v mutation as an oncogenic mutation in cutaneous malignant melanoma (mm) has changed the treatment paradigm for melanoma. selective braf inhibitors have demonstrated response rates far higher than standard chemotherapeutic options. braf mutation analysis is usually performed on primary tumour tissue; however, no conclusive data are available on the concordance of test results between primary tumours and corresponding metastases. we assessed the concordance of braf mutation status in a study of primary brafnative tumours and their corresponding lymph node metastases. method: patients with histologically confirmed nonmutated braf mm who underwent surgical resection of the primary tumour and positive selective lymph node biopsy or lymphadenectomy were included. mutation status was determined by means of a realtime pcr assay (cobas braf v mutation test, roche molecular systems). results: there was a % concordance of the braf results in primary tumor and corresponding metastases in analyzed pairs. conclusion: our findings show total concordance in braf status between primary braf native tumors and their paired metastases, and suggest that acquisition of braf v mutation in metastases from primary native tumors is a rare event. dermatoscopic and histologic score correlation in atypical spitz/reed nevi m. saravia * , z. pellicer, j. m. martin, c. monteagudo * hospital clinic university valencia, dept. of pathology, spain objective: different dermatoscopic scores of pigmented skin lesions have provided a valuable tool in daily routine practice to differentiate malignant from benign lesions. however, these semiquatitative methods have a high rate of false positives in spitz/reed nevi. we have compared semiquantitative histopathologic and dermatoscopic findings in a series of spitz/reed nevi. method: we collected cases ( female and men) of atypical spitz and/or reed nevi. all cases were microscopically confirmed. a histologic index (hi) was constructed scoring the following findings: symmetry, sharp demarcation, architectural dysplasia, melanocytic atipia and nest size, and compared with validated dermatoscopic scores (abcd score, -point checklist and menzies score). distributions of these scores for the different dermatoscopic patterns were also analyzed. results: median age at presentation was years (range to ). the hi varied from to points. we found a strong positive correlation between the hi and menzies score (r= , , p= , ). in addition, hi values differed significantly when the multicomponent dermatoscopic pattern group was compared with the other patterns (u = , z =− , , p = . ). conclusion: menzies is the only dermatoscopic score which strongly correlates with histologic findings in spitz/reed nevi and may be useful in the diagnosis of atypical lesions. correlation of nonmolecular and molecular subtyping of inherited epidermolysis bullosa k. veselý * , h. bucková, l. fajkusová, m. veselá, b. jerábková * st. anne's hospital, dept. of pathology, brno, czech republic objective: inherited epidermolysis bullosa (eb) is a heterogeneous group of hereditary mechanobullous diseases with skin blister formation of variable severity and numerous extracutaneous manifestations. eb comprises three main groups and many subtypes and their recognition is important for prognosis and genetic counselling. method: we examined skin biopsy samples of patients with eb using transmission electron microscopy, immunofluorescence antingen mapping (am) and their peripheral blood samples by mutational analysis. results: in the total number of cases, concordant results of molecular and nonmolecular methods were found in cases ( %). in patients ( %) mutational analysis did not confirm the results of ultrastructural and am analysis. conclusion: strong correlation in the results obtained by different methods was observed. using combination of nonmolecular and molecular diagnostics methods high diagnostic accuracy can be achieved. granulomatous slack skin (gss): a case report with evidence of t cell clonality i. yilmaz * , h. baloglu, u. berber, z. kucukodaci, m. arcila * gata heh, dept. of pathology, istanbul, turkey objective: gss is a very rare variant of mf. we describe an additional case of this rare disorder in a yo female that has been approved by tcrb&g tcell clonality assay. method: a yo female with a single bulky skin lesion in the inguinal fold which had first appeared y before and enlarged up to cm, referred to plastic surgery for esthetics. h&e section of the deep skin biopsy of lesion showed a granulomatous infiltrate in the dermis. nuclear atypia and epidermotropism is not significant. but there is numerous multinucleated histiocytic giant cells. elastophagocytosis are present. tcrb&gt cell clonality assay showed a clonal rearrangement in tcr gamma gene. she was referred to dermatology for treatment and now she is getting uva phototherapy. results: the diagnosis of gss is difficult in early stages of the disease. clonal rearrangement of tcr genes can be a useful diagnostic tool in early stages of the disease. conclusion: - % of patients with gss carry a risk for the development of a second malignancy. gss may be associated with lymphoproliferative disorders. therefore, follow up of the patient is important. this case report supports that gss is an indolent variant of mf due to clinical, histological and t-cell gene rearrangement results. unusual occurrence of multicentric reticulohistiocytosis with systemic involvement in childhood s. zurac * , c. dobrea, a. diaconeasa, a. colita, c. solovan, c. arion, f. staniceanu * colentina university hospital, dept. of pathology, bucharest, romania objective: multicentric reticulohistiocytosis (mr) with systemic involvement is an extremely rare disease afflicting mainly adults. method: we report a case of sr arising in a year month old boy with concomitant celiac disease. results: since - months, he developed several brown to dark-red skin nodules (up to . cm) located mainly on face and arms; current episode include hemorrhagic syndrome, severe pancytopenia and massive hepato-splenomegaly. bone marrow aspirate ruled out acute leukemia and tezaurismoses; bone marrow biopsy and skin biopsy showed important infiltration with numerous large cells with abundant fine granular/ground glass eosinophilic cytoplasms in a reactive inflammatory stroma (lymphocytes, few plasma cells and few eosinophils); bone marrow cells were mononucleated, cutaneous cells were both mono&multinucleated; tumor cells immunophenotype was consistent with histiocytes (cd +, s- protein + (faint), cd a-, langerin-) both in bone marrow and skin biopsies; no hemophagocytosis was seen. mr with systemic involvement of skin, bone marrow, liver and spleen was diagnosed. cyclophosphamide therapy was instituted with initial diminishing of splenomegaly; latter on, despite adding cyclosporine and etoposide, no further impact on pancytopenia and hepatosplenomegaly was recorded. conclusion: complete hematologic examination is mandatory for patients diagnosed with cutaneous reticulohistiocitosis, irrespective of their age, in order to identify mr and institute proper treatment. primary cutaneous follicle centre lymphoma: a case report and review of literature k. diamantopoulou * , c. zorzos, c. eftychiadis, i. famellos, g. piagkos, g. karamanis, i. babalis, s. binder, h. mahera * general hospital kat, pathology, athens, greece objective: primary cutaneous follicle centre lymphoma (pcfcl) is an indolent, primary cutaneous b-lymphoma with an excellent prognosis but a high incidence of reccurence. apart from standard surgery and local radiotherapy, targeted strategies with anti-cd (rituximab) has introduced new treatment modalities. method: woman, aged , with solitary skin lesion on her face. after surgical excision, histo-and immunohistochemistry with the abc system on paraffin embedded tissue was performed. results: microscopically, a non-epidermotropic perivascular and periadnexal neoplastic infiltrate with a follicular growth pattern was observed. the lesion consisted of predominantly medium-sized centrocytes and several centroblasts enmeshed in a network of cd (+) dendritic cells. immunohistochemically, the neoplastic lymphoid cells were cd , cd a, bcl and bcl positive. cd , mum and ig were all negative. the patient received one cycle of rituximab and months later is free of disease. conclusion: primary cutaneous b-cell lymphomas (cbcl) account for % of cutaneous lymphomas. pcfcl is one of the three major categories of cbcl, recently classified by who, (the other two: pcmzl and pcdlbcl, leg-type). bcl- , except for systemic follicular lymphoma, it can also occur in pcfcl, as in our case. pcfcl, usually locally treated with surgery and radiotherapy, could possibly offer a candidate for anti-cd targeted therapy, especially in multifocal lesions. objective: thirteen and ten years old male patients admitted with months interval, having diffuse hemorrhagic papules on extremities and trunk. the first has not responded to methorexate previously. the second had varicella infection months before and, his rushes spreaded out recently. method: multiple punch biopsies of the first patient revealed epidermal necrosis, basal vacuolisation and subepidermal diffuse infiltrate composed of cd and mostly cd positive t cells showing a striking angiocentric distribution. there were cd positive large cells with vesicular nuclei both in epidermis and dermis. some of the vessels exhibited lymphocytic vasculitis with fibrinoid necrosis. punch biopsy of the second patient revealed subcorneal pustule, dyskeratosis and basal vacuolisation, papillary dermal edema, erythrocyte extravasation and severe lymphocyte-rich cellular reaction infiltrating the interface. lymphocytes were cd and mostly cd positive. in between, there were large cells with vesicular nuclei exhibiting cd positivity. results: diagnoses were pleva based on the vascular and interface changes in accordance with clinical findings. both patients responded to oral antibiotics. conclusion: pleva is among the reactive conditions that can simulate cd positive lymphoproliferative diseases. although cd positive lymphomatoid papulosis is defined in pediatric patients, no response was seen to oral antibiotics. are hmb- and mib results reliable for safe diagnosis of nevi? z. yusifli * , o. kurtulan, Ç. aydin, m. bugdayci, Ö. gököz * hacettepe university, pathology, ankara, turkey objective: melanocytic lesions having subtle features suspicious for malignancy create considerable difficulty in decision-making.process. lack of deep hmb- positivity and low dermal mib- index can be reliable tools in association with morphology. method: twenty-seven acquired melanocytic lesions in which hmb- and mib- have been performed are reviewed to determine the importance of these markers in routine diagnostic practice. asymmetry, focal loss of maturation, presence of mitoses, epidermal consumption and lymphocytic inflammation were the morphological features that have created difficulty especially when seen alone in a bland appearing nevus. results: all cases with epidermal consumption and/or mitoses and nearly half of cases with inflammation, asymmetry and focal loss of maturation was associated with.focal deep dermal hmb- positivity. thirteen cases had an mib- index of ≤ %. two of cases with mib- index of > % had also hmb- positivity and/or inflammation. the case with a mib- index of % was an acral nevus bearing of the evaluated morphological features. conclusion: focal deep dermal hmb- positivity goes parallel with epidermal consumption and dermal mitoses, mostly used prevailing criteria of malignancy in melanocytic lesions but it's expression is not adequate for diagnosis of melanoma. low mib index can be a reliable marker of benignity. objective: leptospirosis in humans usually courses with hypokalemia and hypomagnesemia, and the putative mechanism may be related to nitric oxide production. methylene blue is a known inhibitor of inducible nitric oxide synthase, and have beneficial effects on clinical and experimental sepsis. method: serum creatinine and ionic changes were evaluated at various time points ( , , and days) in hamsters. we also determined the effect of methylene-blue treatment when used as adjuvant therapy combined with a late start of standard antibiotic (ampicillin) treatment. results: rather than k and mg depletion, hyperkalemia and hypermagnesemia were observed during acute infection. these findings are probably associated with an accelerated progression to renal failure, since this model is not feasible to mirror the supportive therapy (including fluid expansion) that retards progression to the oliguric/hyperkalemic state. infected hamsters at day presented diffuse tubular cell swelling with mild or no nephritis. at days and they showed variable degrees of acute tubular changes, regeneration of tubular epithelia and nephritis. survival and renal pathology did not differ among different treatment groups. conclusion: adjuvant methylene blue had no effect on the survival, renal pathology or mg and k serum levels during acute-phase leptospirosis in hamsters. occupation of the striated muscle fibre by trichinella spiralis is associated with increased intracellular sialylation p. babal * , r. milcheva, d. ivanov, s. petkova * comenius university, dept. of pathology, bratislava, slovakia objective: the knowledge about glycoproteome in skeletal muscle is limited and most of the information come from studies on aberrant glycosylation in inherited muscle diseases. this work describes the intracellular changes in sialylation of skeletal muscle fiber during the process of its transformation into a nurse cell after occupation by the nematode trichinella spiralis. results: the study was performed at defined time points post infection. lectin histochemistry with tml, mal and sna detected increased production of sialylated glycoproteins within the affected fibers, and it was evaluated by acidic ninhydrin reaction in muscle homogenates. increase of total sialyltransferase activity was estimated by measurement of incorporated cmp-n-[ c]-acetylneuraminic acid, and immunohistochemistry showed higher expression of α- , -sialyltransferases ii and iv within the affected fibers. sna lectin affino-blots showed at least four protein bands with approximate molecular weight between and kda, which were more reactive to sna compared to their counterparts from the control samples. conclusion: it is evident that skeletal muscle injury induced by trichinella activates biosynthesis of glycoproteins bearing sialic acids, which are not present in the normal muscle fiber. however, the protein identity, function and the biological significance of these sialoglycoproteins remain to be elucidated. bg po objective: protozoal and helminthic infestations of the central nervous system (cns) are rare and their incidence is less than %. they are more common among children, the elderly and immunocompromised individuals and tend to follow a fatal course. method: we report cases of cerebral amebic abscess occurring in immunocompetent men. results: they were men aged of and years old. headache; altered mental status and fever were common presenting symptoms. in the cases the diagnosis of cerebral abscess was suspected on cerebral ct scan. histopathologic analysis revealed extensive areas of necrosis and hemorrhage with granulomatous lymphoplasmacytic inflammatory infiltrate. other conditions such as malignancy and tuberculosis. it occurs normally in the mouth and tonsils, however, it can sometimes occur in the chest, abdomen, pelvis or other areas of the body. method: we describe a retrospective study about cases of actinomycosis diagnosed over a -year-period ( - ) . results: the cases were divided in cervicofacial actinomycosis, genito-urinary tract actinomycosis, cutaneous actinomycosis, abdominal actinomycosis and cases occur in central nervous system. all patients were symptomatic. the most frequent symptom consisted in fever. the diagnosis was based on histologic study in all cases. tissue histologic studies show suppurative and granulomatous inflammatory changes, connective proliferation, in which bacteria form typical granular colonies composed of radiating gram positive filaments. conclusion: diagnosis of actinomycosis is usually made retrospectively by means of histologic examination of surgically obtained specimens, but rarely preoperatively. it is generally treated with long term antibiotics. expression of angiogenesis factors in placentas with intrauterine transmission of hiv a. kolobov * , e. musatova, v. karev, v. zinserling, d. niauri * st. petersburg, russia objective: placenta plays an important role in the prevention of the mother-to-child transmission of hiv during pregnancy. the purpose of this investigation was to study expression cd , bfgf and tgfβ in human placentas in cases of intrauterine hiv infection. method: group a - placentas from women and children with hiv infection. in control -group b, were placentas from women without hiv infection. on paraffin slices expression of cd , bfgf, tgfβ and p was evaluated. we counted the area of positive cells related to the square of the slice. results: in all placentas with hiv infection p has been found in placental macrophages and villi's vessels. in group a expression of cd in endothelial cells was , ± , % of the slice's square (in control - , ± , %) (p> , ). expression of bfgf in villi's stroma was , ± , % (in control - , ± , %) (p> , ). expression of tgfβ in villi's vessels was , ± , % (in control - , ± , %) (p< , ). conclusion: thus, placentas in cases with intrauterine transmission of hiv are characterized by increased expression of tgfβ the significance of this fact is still to be evaluated. objective: unilocular cystic echinococcosis which echinococcus granulosis causes is seen frequently in the animal husbandry practised underdeveloped countries. method: the unilocular cystic echinococcosis cases which were seen in the eastern part of turkey, in years between . . and . . , except faculty of medicine of atatürk university are presented. results: organ distribution: female: liver (sd: . ), lung (sd . ), spleen (sd . ), abdomen (sd . ), kidney (sd . ); male: liver (sd . ), lung (sd . ), spleen (sd . ), brain (sd . ), soft tissue (sd . ) . conclusion: the frequently seen organs in both gender are liver and lung. the rarely seen organs were spleen , brain , abdomen , kidney , vertebrae , soft tissue , pleura , bone , breast . the others were very rare: eye , gallbladder , thyroid , neck , ovary , testis . relation of the length of appendix and parasitosis: preliminary report a. kurt * , s. a. Özmen, i. erdem * bölge egitim ve arastirma hastanesi, dept. of pathology, erzurum, turkey objective: is there an effect of the length of appendix vermiformis on the settlement of the bowel parasites? we have decided to research. method: it was studied retrospectively for not to be under the influence. randomly chosen from the archives, parasitic appendix ( women + men) and appendix vermiformis ( women and men) lengths were compared. the last appendix materials of the appendixes which include parasites ( e.vermicularis, a.lumbricoides, t.saginata) and operated any causes were examined. placeholder conditions such as fibrous obliteration and tumour were excluded. the premeasured length of the appendix in the series ( + = ) were listed with the gender. results: appendix length in the control series: the length of appendix in men: . the length of appendix in women: . the length of appendix in both gender: . cm. the length of parasitic appendix: the length of appendix in men: . the length of appendix in women: . the length of appendix in both gender: . . objective: on the basis of recently detected receptors for erythropoietin on the surface of mscs we hypothesized that introduction of epo together with mscs may enhance their effect and improve the results of sepsis treatment. method: wistar male rats were randomized into groups with animals in each: group -the healthy controls, - groups were intraperitoneally introduced bacterial lps mg/kg. group - got × allogeneic mscs, group - . μg of recombinant epo-beta, group -mscs and epo in the same doses. the morphological study of liver, spleen, thymus, lung and kidney was performed. histological specimens were evaluated by qualitative, semiquantitative and quantitative methods. results: in lung tissue the thickness of alveolar septums progressivly reduced from group to .in kidney and liver tissue the dystrophy of hepatocytes and nefrocytes and a blood vessel's dilatation in studied groups also reduced.in lien tissue we observed hyperplasia of white pulp in groups and . structural features of thymus were connected with patterns of t-lymphocyte proliferation and differentiation. conclusion: combined treatment with epo and mscs can reduce acute lung injury and kidney damage, cause hyperplasia of lymphoid tissue and enhance the immune response more than separate treatment in an experimental model of sepsis in rats. mycobacterial spindle cell pseudotumor of the liver a. alves * , a. costa, a. fernandes, g. do carmo, i. tavora * hospital santa maria, servico de anatomia patologica, lisboa, portugal objective: spindle cell pseudotumors are rare manifestations of mycobacterial infections. we describe a case in the liver in an immunocompromised patient with tuberculosis. method: a -year-old man, with active substance dependency, hiv- infection known for years and acquired immunodeficiency syndrome for years, was receiving tuberculostatic medication for disseminated tuberculosis and investigated for persistent fever. a liver ultrasonography confirmed a moderate hepatomegaly and showed multiple dispersed nodules in both lobes, with to cm of greater diameter. results: a needle biopsy from one of the nodules showed a proliferation of spindle cells without atypia, grouped in intersecting bundles, with immunoreactivity to vimentin, cd and lysozyme and negative for cd , ps , sma and desmin; a ziehl-neelsen stain revealed several acid fast bacilli in the spindle cells. they were pas negative. conclusion: pseudotumoral lesions in hepatic tuberculosis usually correspond to granulomatous inflammation. a spindle cell proliferation, like that seen in other organs are more frequently associated with non tuberculous mycobacteria, is very rare. we found only another case in the literature of liver spindle cell pseudotumor associated with tuberculosis. this lesion must be differentiated from mesenchymal neoplasms. sixteen months follow-up in a treated case of whipple disease (wd) c. popp * , m. petre, g. micu, a. bastian, r. chirculescu, r. mateescu, f. staniceanu * colentina university hospital, dept. of pathology, bucharest, romania objective: wd is a rare, multi-systemic infection caused by a ubiquitous environmental bacteria -tropheryma whippleithat affects the duodenum and small intestine but also the brain, endocardium, skin, lungs and joints. method: we are presenting a years old man with a years history of diarrhea, malabsorption and weight lost (about kg). endoscopical examination revealed an unspecific, yet unusual pattern of lesions: small, yellow, multiple, slightly elevated patches involving duodenum and jejunum. histological examination revealed numerous foamy macrophages in lamina propria with pas (+), ziehl-neelsen (−) material in cytoplasm. results: treatment with trimethoprim/sulfamethoxazole was initiated with a spectacular response: after days of therapy the patient had normal stool and in months he already gained back kg. after year of treatment the biopsy confirmed a partial remission with fewer macrophages persisting in lamina propria of duodenum. after months of treatment (march ), very few macrophages with pas (+) material are still identified on duodenal biopsy. conclusion: wd is a potentially fatal disease with unspecific clinical and endoscopical signs that can be diagnosed on duodenal biopsies. histological follow-up is a useful tool of managing the treatment and histopathological confirmation of the absence of foamy macrophages is mandatory in discontinuing therapy. small cell urothelial carcinoma (scuc) comtherapy. method: we report two cases of scuc. both of them refer to year-old males, smokers. they presented with gross hematuria. transurethral resection bladder tumor (turbt) specimens showed scuc, followed by cystoprostatectomy and pelvic lymphadenectomy, confirming the initial diagnosis. immunohistochemistry was used on paraffin embedded tissue. results: microscopically, the tumor cells show a diffuse architectural pattern with occasional nesting, are small, uniform, with scant cytoplasm, nuclear crowding, nuclear molding, inconspicuous nucleoli and finely stippled chromatin. numerous mitoses, azzopardi phenomenon and foci of necrosis are present, as well as areas of urothelial carcinoma. tumor cells are strongly (+) with ker ae /ae , cam . (dot-like perinuclear pattern), cd (the most consistent neuroendocrine marker), p , ki- ( %), focally (+) with synaptophysin and (−) with chromogranin, ker βe , ker , ker , ttf- , lca. conclusion: pathologic and immunohistochemical data identify scuc. differential diagnosis includes metastasis of a small cell carcinoma from another site rare variant: primary renal malignant fibrous histiocytoma n primary leiomyosarcoma of the kidney: three cases b renal pelvic and ureteral dilatation was seen in the first case because of the mass effect. microscopically the alternating spindle tumor cells were detected which were positive with actin, desmin, caldesmon and negative with s , pan-ck, cd and cd congress hall foyer rd floor ps- poster session gynaecological pathology although rare, u-lms is the most frequent malignant gynaecological mesenchymal tumor, usually disclosing unfavorable prognosis. u-lms diagnosis may be controversial. in this study we evaluated clinical-pathological features/prognostic factors of u-lms. method: all consecutive u-lms diagnosed ( - ) at chsj and ipo-porto and with available material were reevaluated. the clinical files were reviewed, including demographic, clinical, imaging, surgical, staging, and follow-up parameters. the pathology features, including immunohistochemistry evaluation, were re-evaluated and the tumors staged according to most recent consensus criteria. results: the mean age at diagnosis of the cases was years ( - ); % were multiparous and % postmenopausal women. the majority ( %) had symptoms at diagnosis, mostly abnormal bleeding. histological classification displayed: % spindle, % epithelioid, % myxoid, . % pleomorphic, and . % mixed u-lms. treatment included surgery ( %), radiotherapy ( %), and chemotherapy ( %). overall median survival was months portugal objective: u-lms is the most frequent malignant gynaecological mesenchymal tumor, disclosing dm results: our series: ( . %) with metastasis; literature: with metastasis. global mean age at diagnosis: years ( - ); initial treatment: hysterectomy ( %), radiotherapy ( %), and chemotherapy ( %) pancreas, vulva/vagina, salivary gland, oral cavity, heart, mediastinum, adrenal, and breast; % with metastatic related symptoms. overall median time to first metastasis: months (ci % . - . ). pancreas, heart and mediastinum were the earlier metastatic sites microglandular adenocarcinoma (mga) of the endometrium, a rare subtype of endometrial mucinous adenocarcinoma, has histologic similarities to the microglandular hyperplasia (mgh) of the cervix. it should not be misdiagnosed as cervical mgh especially in a small endometrial biopsy specimen. method: eight cases of mga of the endometrium and cases of control mgh of the cervix were immunostained for ki- , cd , p , p , p , vimentin, estrogen receptor (er), progesterone receptor (pr) and pten. hpv dna analysis was performed using paraffin-embedded mga tissue by polymerase chain reaction amplification endometrial carcinoma (ec) displays variable outcomes. method: immunohistochemistry for p , ki , egfr, her- (%positive), pten, estrogen and progesterone receptors (er/pr), perk, pjnk (h-score results: ade: er/pr mean h-score from / to / , from g to g , for pten, and for perk and pjnk, two adeg > % p and one % ki . ads: % > % p and % > % ki , / for er/pr, for pten, and for perk and pjnk. adcc: none er/pr; , and for perk -regulated) compared to ulm and myometrium, several known to be involved in cell-cycle and/or apoptosis pathways (p , rb, e f, cyclin-d /e) and/or metastasis. conclusion: albeit some overlap, mirna expression profile of ulms is different from ulm but relatively similar to non-ulms. ulms and non-ulms share deregulation of several mirnas that may be linked to specific cell-cycle/ apoptosis pathways %) included rhabdomyoblastic ( ), cartilaginous ( ), adipocytic ( ) & sex-cord-type ( ). four cases had leiomyomas; alivewith-disease (mean, . months) and free-of-disease (mean, months). conclusion: adenosarcomas are uncommon endometrial endometrioid carcinoma is the most common malignant tumor of the uterine corpus usually developing in postmenopausal women. more seldom it occurs in young reproductive age and sometimes develops from an endometrial intraepithelial neoplasia (ein). the ein diagnosis and treatment in such patients have some difficulties. method: we have studied curettage tissue specimens from three women with ages , and , with abnormal uterine bleeding and obesity. the severe complex endometrial hyperplasia with low grade intraepithelial neoplasia was revealed in all cases polypoid glands with sarcatomatous stroma (phylloides-like pattern). b. periglandular condensation. c. sarcomatous overgrowth. d. decidualization. e. cartilagenous dedifferentiation. f. rhabdomyoblastic dedifferentiation (inset). g. vimentin positivity. h. desmin in rhabdomyoblasts. i. er positive glands. which were examined histopathologically, and in terms of cytologic features, p and ki immunoexpression. results: cervical smear samples from patients were diagnosed as agus, ascus, asc-h, hgsil, lgsil cases three types are generally acknowledged: circumscriptum (or capillary), cavernous, and cystic. -years-old female. method: case report: a -years-old woman presented to the department of obstetrics and gynecology at our hospital. for uterin myoma, she underwent total abdominal histerectomy and bilateral salpingo-oophorectomy congress hall foyer rd floor ps- poster session dermatopathology atypical spitz tumor or spitzoid melanoma: which morphological criteria ensure the diagnosis? method: thirty-one spitzoid lesions were reviewed by a scoring system based on seven selected criteria reported previously appreciating characteristics of spitzoid lesions. as a prevailing criteria, presence of deep mitoses had a score of ; ulceration, asymmetry, epidermal consumption, solid growth pattern, loss of maturation, pigment heterogeneity were scored as for each. final diagnosis was sm for scores - , ast for - , sn for . results: the most frequently seen criteria, loss of maturation was solid growth pattern and pigment heterogeneity were nearly equally seen in sm and ast. first diagnoses of sn switched to ast, to sm, and of ast to sm. conclusion: presence of deep mitoses is the single criteria present only in sm. loss of maturation, epidermal consumption and ulceration can be used to differentiate ast from sm basal cell carcinoma (bcc) of the skin is the most common form of human cancer. sometimes the histological appearances of benign adnexial neoplasm of hair follicle apparatus tumors (trichoepithelioma, trichoblastoma) are diffucult to distinguish from bcc. the aim of this study is to investigate the usefulness of cd and p in distinguishing bcc and benign adnexial neoplasm of hair follicle apparatus tumor. method: this study included bcc and benign adnexial neoplasm of hair follicle apparatus tumor. we used immunohistochemical stains for p and cd in all cases. results: in bcc cases the expression of cd was noted in tumor cells in cases objective: a widely accepted adequacy criteria for histopathologic evaluation of prostate core biopsy protocols has not been defined yet. selection of patients for repeat biopsies after a negative initial biopsy result is also a matter of debate. we aimed to evaluate the impact of total core length for detecting prostate cancer in a -core transrectal ultrasound guided biopsy scheme. method: retrospectively, we investigated the core lengths of patients underwent initial prostate biopsy by a lateral zone targetted -core protocol between and . results: cancer was identified in ( . %) patients. mean total core length for positive and negative patients were . cm and . cm, respectively. cancer detection rates in groups for total core length < cm, - cm, and > cm were . % ( / ), . % ( / ), and . % ( / ), respectively. when a threshold of cm was selected for total core length, overall cancer detection rates for < cm and ≥ cm groups were . % ( / ) and . % ( / ), respectively. conclusion: although molecular confirmation remains its diagnostic gold standard, tle , ema, bcl- , mic , cks and cd constitute an optimal for sy-novial sarcoma. awareness of tle expression in other tumors and its correct interpretation are necessary. objective: malignant peripheral nerve sheath tumors (mpnst) comprise - % of sarcomas. prognosis is poor and the search for new treatments is ongoing. her is a crucial receptor for neuregulin signalling in schwann cells but to date, has not been studied in sarcomas. our aim was to determine the prevalence of her receptor expression in soft tissue tumors, including mpnst. method: her expression was evaluated by immunohistochemistry in a total of benign and malignant mesenchymal tumors, including neurofibromas, schwanomas, mpnst and other lesion (uterine and non-uterine leiomiomas and leiomiosarcomas, synovial sarcomas, undifferentiated pleomorphic sarcomas (ups)). results: her overexpression was found in % of cases. significantly, her positivity was present in ps- - breast cancer in young czech women: an institutional review f. sobande * , a. ryška * dept. of pathology, hradec kralove, czech republic objective: breast cancer in young women poses unique diagnostic and management challenges however, very few large studies investigating clinico-pathologic and immunophenotypic characteristics within this group exist and varied, arbitrary age-limits are used to define 'young'. method: an institutional archive search was performed for breast cancer diagnosed in women < in [ ] [ ] . age at diagnosis, tumor histological type and grade, estrogen and progesterone receptor (er and pr), her /neu, ki- and p status as well as clinical course during the follow-up period were recorded. patients were sub-classified into agegroups of -year intervals for statistical analysis. results: ninety of carcinomas were diagnosed in women < . they frequently displayed aggressive morphology, high proliferative activity, increased expression of p and high incidence of lymph node metastasis. we found a statistically significant decrease in her /neu over-expression with increasing age using as the cut-off (< : %, ≥ : %; p= . , chi-square test). no significant age-related differences in er or pr expression were observed within the group of women < . conclusion: we show that should be used as the cutoff for defining young women; in whom breast cancer is most likely to over-express her /neu. the mechanisms behind this finding and its significance are yet to be elucidated. objective: osna is a molecular assay for lymph node in breast carcinoma (bc) based on m-rna detection of ck . the aim of this study is to evaluate the rate of negative o heterogeneous immunohistochemical expression of ck in bc. method: ck was assessed in consecutive samples. cases were categorized as positive, negative and heterogeneous. other parameters recorded where histological type and grade (nottingham) and molecular subtype (st. gallen ). results: cases ( . %) were ck +. ten ( . %) were ck -and ( , %) were heterogeneous. there were intraductal carcinomas, heterogeneous and ck -. among invasive carcinomas, there were ck -: ductal, papillary and metaplastic. three cases corresponded to g , g , g and t mi not available. five cases were luminal a(la), luminal b/her -(lb-), her + and triple negative (tn). among heterogeneous cases, were ductal, metaplastic, and atypical medullary carcinoma. eleven were g , and g . four were lb-and cases were tn. conclusion: all lobular carcinomas expressed ck ck was negative in . % and heterogeneous in . % of bc. they represent a heterogeneous group but half of these cases are either g or tn. % of heterogeneous cases were tn and % lb-with no la cases in this subgroup. case report of an extremely rare tumor of the breast: mucoepidermoid carcinoma f. staniceanu * , c. socoliuc, a. birceanu, e. gramada, l. nichita, r. andrei, a. croitoru * colentina university hospital, dept. of pathology, bucharest, romaniaobjective: staniceanu and socoliuc are first authors in equal proportions mucoepidermoid breast carcinoma, classified in category of purely epithelial metaplastic carcinomas, is rarely encountered with only a few female cases described in literature. it is composed by mucous, intermediate and epidermoid cells, similar to its salivary gland counterpart. method: we present the case of a years old man with a , / , cm breast tumor located under the nipple, poorly circumscribed, with increased consistency and central cystic area containing serous fluid. tissue samples were preserved using % formalin. all sides were stained using hematoxylin-eosin. immunohistochemistry was performed using labeled streptavidin-biotin method. results: microscopic examination reveals islands and sheets of polygonal cell with clear/pale cytoplasm, "signet cells" figures, areas of squamous metaplasia and centrally located hyperchromatic nuclei, some with visible nucleoli and rare mitoses. tumoral parenchyma contains ducts and cysts of varying sizes, with pale eosinophilic luminal accumulation pas, d-pas and alcian blue positive (mucin). immunohistochemical tests were performed. differential diagnosis was made among several benign and malignant lesions, the final diagnostic being mucoepidermoid carcinoma, low grade. conclusion: mucoepidermoid carcinoma of the breast is an extremely rare entity; however its specific morphologic and prognostic features (similar to its salivary gland counterpart) allow proper diagnostic. endomyocardial biopsy value in differential diagnosis of hypertrophic cardiomyopathy j. markovic * , i. ranisavljevic katuca, s. glumac, r. jankovic, j. d. vasiljevic * clinical center of serbia, dept. of pathology, belgrade, serbiaobjective: endomyocardial biopsy (emb) may be used to diagnose different types of cardiomyopathies, to monitor transplant rejection, evaluation of myocarditis, heart failure of unknown origin, arrhythmias, drug toxicity and heart neoplasmas. the aim of this case is to show how noninvasive cardiac investigations are sometimes not sufficiently conclusive for distinguishing between hypertrophic obstructive cardiomyopathy (hocm) and the other cardiac diseases.method: thirty-one representative left ventricle biopsies were obtained following suspected hocm. samples underwent routine standard and special staining procedures. five histologic parameters were used for assesment (disarray and hypertrophy of myofibers, myocardial short runs, perinuclear halo and bizarre nuclei and fibrosis) and graded from to , presenting in summary histological hocm index (hhi-ranging from to ). results: the histological findings in emb of thirty-one patients, in ( , %) patients hocm diagnosis was confirmed, in ( , %) patients hhi was under or equal to % ( ) so hocm diagnosis couldn't be confirmed, amyloidosis in one ( , %) patient, myocarditis in ( , %) patients, dilatative cmp in ( , %) patients and unspecific cardiac changes in ( , %) patients. conclusion: a definitive diagnosis can be obtained by means of a multidisciplinary approach including emb findings. morphometric study of structural changes in the heart of people who abused alcohol for more than years l. mitrofanova * , e. naumova * almazov federal heart, blood and cardiomorphology, st. objective: the information about structural changes in the heart of people abused alcohol (paa) is very contradictory. method: we studied hearts of paa (> g per day), hearts of patients with ischemic heart disease (ihd) and hearts of healthy persons. at the morphometric study of heart we measured the mean cardiomyocyte diameter (mcd), the relative areas of fatty infiltration (fi), fibrosis, cardiomyocytes. immunohistochemistry was performed using antibodies against cd -antigens to count the number of antigen-positive cells per mm². the average heart weight, the mcd of the paa were not significantly different compared to the ones of the control group. the average relative area of fi of rv for the group of paa was ± % and was significantly different compared to the one of both the control group and the group with ihd. the relative area of rv fibrosis in the group of paa was . ± . % and was not significantly different compared to the ones of the other two groups. the mean number of cd + lymphocytes/mm² of % of paa was more than . conclusion: the paa did not have myocardial hypertrophy. % of them had chronic myocarditis. marked fi in paa was significantly higher than the other two groups. objective: the aim of the study was to evaluate the expression of angiotensin-converting enzyme (ace- ) in left ventricular myocardium in the presence of chronic myocardial ischemia. method: heart specimens with persistent ischemia (n= , pre-infarction ischemic heart disease (ihd) group) and postinfarction scar (n= , post-infarction ihd group) from dissected males and heart explants (n= , end-stage ischemic heart failure group) were studied. heart specimens (n= ) selected at autopsy from individuals who died from accidents were used as controls. the slides of myocardium were incubated with polyclonal antibody against the ace- ( : , ab , abcam). results: ace- expression in cardiomyocytes of the preinfaction ihd and post-infarction ihd groups did not differ from the controls and was increased only in the end-stage ischemic heart failure group (p< . ). ace- expression in vascular endothelial cells, smoth muscle cells and macropages of the pre-infarction ihd group did not differ from the controls; but the number of ace- expressing cells in the post-infarction ihd and in the end-stage ischemic heart failure groups was greater, as compared with the pre-infarcion ihd group and the controls (p< . ). conclusion: expression of ace- is increasing in progression of ischemia-induced myocardial dysfunction. thrombotic occlusion of the peripheral pulmonary arteries: anatomical "trigger" of the massive pulmonary thromboembolism (pte) f. portelli * , f. p. busardò, l. gutsul, p. fleres, p. pugnetti, e. orlando, e. maresi * university of palermo, section of pathology, italy objective: tubulocystic carcinoma of the kidney (tcrcc) is a rare variant of renal tumor showing unique gross and microscopic features. several recent studies favour its inclusion within the papillary renal cell carcinoma (prcc) group. method: patient was a year-old man with a left asymptomatic renal mass incidentally discovered by sonography. patient was a year-old man with aortic estenosis and coronary disease. a right renal mass was discovered in the work up for aortic valve replacement and coronary bypass. results: both tumors were well circumscribed masses with "sponge-like" appearance. on light microscopy, the tumors were composed of ectatic tubules and cysts of variable size, lined by a single layer of tumor cells with eoshinophilic cytoplasm, apocrine appearance, rounded nucleus sometimes overlapping, and prominent nucleoli. ihc showed positive staining with ck , e-cadherin, cd , and amacr. conclusion: tcrcc has been recently recognized as a distinct subtype of prcc by some authors. in fact, some few cases reported have histological transition between tubulocystic and papillary phenotypes on routine histological sections. additionally, tcrcc shows trisomies of chromosomes and . the exact location of this tumor within the who classification is a matter of debate. objective: our purpose is to evaluate her protein expression in urothelial carcinoma of the bladder. her /neu is a tyrosine kinase receptor (family of human epidermal receptors), showing overexpression in a large variety of tumor cells. method: bladder biopsy and cystectomy specimens were included in this study. the age of the patients ranged from to (mean ). among them ( . %) were male and ( . %) female. all cases were reevaluated histopathologically according to who grading system. her protein expression was detected by immunohistochemistry (cb ) using the same criteria as for breast cancer. results: all bladder neoplasms were low-or high-grade urothelial carcinomas. her protein overexpression was observed in ( . %) cases ( +: cases and +: cases), while ( . %) cases were negative ( : cases and + : cases). among her positive cases ( %) were high-grade carcinomas, while ( %) was low-grade carcinoma. among negative cases ( %) were lowgrade carcinomas, while ( %) were high-grade carcinomas. conclusion: these findings indicate that her protein overexpression is predominately correlated with high tumor grade in urothelial carcinoma of the bladder and patients with these characteristics could be potential candidates for targeted therapy. mucinous adenocarcinoma of the renal pelvis: a case report and review of the literature m. otani * , t. tsukamoto, h. serizawa * tokyo medical university, division of diagnostic pathology, japanobjective: primary mucinous adenocarcinoma of the renal pelvis is extremely rare and about cases have been reported to date. we present a case of mucinous adenocarcinoma followed by renal cysts. method: the patient is a -year-old male. a renal cyst in the right upper region was found incidentally. he had no symptom at that time. after years, he came to the hospital because of hematuria and back pain. the examination revealed that the renal cyst was enlarged accompanied with solid lesion. the high serum level of cea indicated malignancy and tumorectomy was performed. results: macroscopically, a renal cyst ( × mm) in the right upper portion was adjacent to a solid mass ( × mm). the tumor showed papillary growth ( × mm) in renal pelvis. microscopically, the tumor was consisted of adenocarcinoma with tubular or papillary structure, spreading from cyst wall to pelvis. the tumor cells contained mucin and some showed signet-ring celllike feature. conclusion: clinically, high serum level of cea is a marker of mucinous adenocarcinoma originated from renal pelvis. in our case, the tumor has already from pelvis to cyst wall. we thought it originated form renal pelvis, however, the renal cyst is also a candidate for its origin. results: an cm tumour was revealed in douglas cavity, histologically, deciduoid morphology was predominant with abundant atypical mitoses and massive neutrophyls infiltration. immunohistochemical positivity: calretinin, cytokera-tin - , cam . , cytokeratinae ae , e-cadherin, vimentin, hmbe , ca and negativity: cd , cd a, melan-a, myod , wt- , cd , hmb , actin, thrombomodulin, desmin, alpha-fetoprotein, moc , cerb- , estrogen and progesterone. normal dna-repair proteins expression. conclusion: mdm is highly aggressive neoplasm, differential diagnosis includes: squamous carcinoma, gist, deciduosis pseudotumoral, anaplasic large cell lymphoma, trophoblastic tumour, oxyphilic variant of ovarian clear cell carcinoma. this case represent a rare variant of mesothelioma, in which, was possible a complete resection with a probably better pronostic unlike most of these cases. objective: we evaluated morphological features of male gonad in an adult patient with tfs with an accent on its immunohistochemical properties. method: our years old patient had a primary amenorrhea. external genitals were female, without pubic and axillary hair. us confirmed cystic formation of the left gonad and lack of uterus. tumor markers were normal but testosterone was high. cystic formation of the left gonad was surgically removed, initially specified as a left ovary, as well as the other gonad. pathohistological examination and karyotyping was done. results: immunohistochemical analyzes of the gonad sample reveal that sertoli cells showed positivity for inhibin α, vimentin, cd , ckae /ae and calretinin and leydig cells showed positivity for inhibin α, vimentin, cd , ckae /ae and calretinin, and weak positivity for s- , synaptophysin i nse. immunoreactivity for cd in hyperplastic areas of leydig cells showed the most intensive conclusion: we conclude that cd and p expression may be a useful adjunct marker in distinguishing bcc and benign adnexial neoplasm of hair follicle apparatus tumor. objective: pigmented purpuric dermatosis (ppd) comprises a group of vascular disorders of unknown etiology. histologically, it is characterized by lymphocytic capillaritis in the papillary dermis. method: a retrospective and monocentric study concerning the cases of ppd diagnosed in the department of anatomopathology la rabta over years (from to ). these cases were studied clinically and histopathologically. hepatitis c and b profile was carried out to evaluate a possible relation. results: cases of ppd have been diagnosed; men and women, aged from to years. only one patient tested positive for hepatitis c with an elevated viral load. she had a profuse eruption involving the trunk and the limbs of years duration, contrary to the other patients in whom only the lower limbs were affected. she has been treated by alpha interferon and ribavirin with a disappearance of the eruption within months. in the five other patients, the lesions resolved spontaneously within a period of to months. conclusion: hepatitis c may play a role in the pathogenesis of ppd especially when the lesions are profuse and longstanding. in our patient, the disappearance of lesions months after the onset of interferon alpha therapy with a significative decrease of viral load is an argument supporting our hypothesis. objective: fibrolipomatous hamartoma of nerve is a benign lesion, very rare in medical practice, occurring in children or young adults. case report: a year old woman, g. n., presented with a slowly growing plantar mass in the right foot that had been present for years. neither the patient nor members of her family had a history of neurofibromatosis or other soft tissue tumors. she had no pain at rest, but she had shooting pain while running or walking. physical examination revealed a mobile, tender mass located in the plantar soft tissue. method: after surgical excision, the lesion was well circumscribed, / / mm, white colour with yellow areas. tissues specimens were fixed in % buffered formalin, routinely processed and embedded in paraffin, sectioned at μm, then stained routinely by hematoxylin -eosin, than examined by light microscopy. results: the lession is characterized by proliferation of mature fatty and fibrous tissue within the epineurium of the nerve accompanied by prominent concentric perineurial fibrosis. the histopathological diagnosis was fibrolipomatous hamartoma of nerve. conclusion: the tumour usually affects nerves of the upper extremities, most commonly the median nerve. involvement of other nerves is rare. the lesion is benign and the recommended treatment is surgical excision. objective: leishmaniasis refers to the spectrum of diseases caused by leishmania species, which are protozoa of order kinetoplastida. clinically leishmaniasis is divided into visceral (kala-azar), cutaneous, mucosal, and mucocutaneous syndromes. the final diagnosis of the disease, lesions of the parasites, as well as shown smear'lerde factor confirmed by histopathological and clinical evaluation. method: in our study, patients with the diagnosis of leishmania cutaneous between and in department of pathology, ataturk university faculty of medicine were presented. results: four ( . %) cases were females and ( . %) were males. age ranging of cases was found from to years and median age . ± . years. the lesions were found on the face in patients. facial skin biopsy specimens were taken in all cases. giemsa-stained touch preparation of the skin biopsy revealed amastigotes inside macrophages, consistent with leishmaniasis. lesions of patients who have long been different from the rest of them were those who migrated to the cukurova region. conclusion: leishmaniasis is public health problem in southeastern anatolia region of turkey, especially in sanliurfa and Çukurova region. autoimmune blistering skin diseases in east anatolia n. gursan * , o. ceylan, h. bilen, n. akdeniz, m. atasoy * ataturk university, medical faculty, erzurum, turkeyobjective: autoimmune blistering skin diseases can be divided into pemphigus diseases and subepidermal bullous diseases. in pemphigus, the autoantibodies are directed against intercellular contact structures; in the pemphigoid diseases, the autoantibodies are directed against adhesion molecules of the basal membrane zone. method: a retrospective study of cases of autoimmune blistering skin diseases obtained over a period of years from st october to st december in the department of pathology, medical faculty of ataturk university was designed. results: in the present study, the mean age was years and male: female ratio was , . the most commonly affected sites were the buccal mucosa and the palate. % of cases, the oral cavity was the primary site of involvement. in histopathological examination we showed intraepithelial vesicle formation, subcorneal separation with acantholysis, and the presence of tzanck cells in biopsy specimens. conclusion: the incidence of autoimmune blistering skin diseases in our department increased in the past years. pemphigus vulgaris is the most common of these diseases. % of autoimmune blistering skin diseases was pemphigus diseases. objective: acute febrile neutrophilic dermatosis (sweet syndrome) is the most common neutrophilic dermatosis. a dense dermal neutrophilic infiltrate is the usual histological findings. the absence of histological image of vasculitis has been considered a characteristic of the disease and a distinctive feature of the other neutrophilic dermatoses. however, recent reports suggest that vasculitis should not exclude the diagnosis. method: this retrospective study examines cases collected over a period of years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . we studied its clinical and histological characteristics. skin biopsy specimens were reviewed to determine the prevalence of vasculitis. results: sweet syndrome is significantly more common in women than in men (sex-ratio= / ). the patient's mean age was years. disease developed most often in autumn. atypical presentation was noted in patients. skin biopsy revealed a sweet syndrome pattern in all cases. the prevalence of vasculitis (vessel wall damage with fibrinoid necrosis) was % ( of the patients). conclusion: our series of patients is particular by the frequency of atypical clinical presentation and the presence of vasculitis in histologic examination, a feature that shouldn't exclude the diagnosis. it was suggested that vasculitis in sweet syndrome represents an epiphenomenon rather than a primary immune-mediated process. dermal lymphangitic carcinomatosis as a primary manifestation of lung carcinoma: report of an unusual case c. hadjileontis * , e. angelidou, a. kafanas * serres general hospital, dept. of pathology, greeceobjective: although dermal lymphangitic metastases are reported to range between and , % of all visceral carcinomas, their presence as a primary manifestation of the tumor is rather unusual. different structures (isolated or forming small clusters) with the morphological characteristics of amoeba trophozoites were identified in the areas of necrosis with and without an inflammatory reaction. the diagnosis of cerebral amebic abscess was then made. the patients received antibiotic treatment. conclusion: cerebral amebic abscess is rare but has poor prognosis. it should be considered as a diagnosis for any patient with subacute and/or chronic meningoencephalitis without evident bacterial etiology. more effective antibiotic drugs can be chosen for post-surgical treatment; furthermore, better survival may be achieved by diagnosing the disease in early stages. morphometric characteries of women placenta which can be different forms of syphilis m. bokhodir * , g. raimnazarova, z. parpiev * tashkent medical academy, dept. of patological anatomy, uzbekistanobjective: many literary information give us about getting down coefficient of fertitity -placenta at women which suffer from pregnancy with syphilis. method: verified data analysis about morphometric placentae and it's comparison with newly-born child at women which suffer from different forms of syphilis. results: research placenta with syphilis show us the area of placenta surface is decreased, it was formed primary syphilis − ,+− , sm, secondary − , ± . sm and with latent form of syphilis , ± . (at control . ± , sm p> o.l), at that time as a mass, placentary cell was been bigger and it formed (at primary- . ± . gr and at secondary- , ± , gr and at latent form of syphilis . ± . gr, then in control ( , ± , ) but all this different were been statistically unreliable (p> , ). comporision mass of placenta and mass newly born-child show us corfficient of fertitity-placenta is make up ( , ± , ) and actually it is not different at control fact group. conclusion: the change coefficient of fertitity -placenta was typifying for until penicillin era, that time. when actual condition, treatment syphilis with antibiotic to bring on development phathomorphism placentae with out different coefficient of fertitity -placenta. influenza b virus-associated pneumonia: report of one fatal case associated with sickle-cell trait l. de carvalho * , l. avila, h. camargo, c. torriso, r. penny, a. sementilli * centro universitário lusíada, dept. of pathology, santos, brazil objective: influenza virus pneumonia complicated with acute respiratory distress syndrome is rare and has a high mortality rate. patients with sickle cell trait are asymptomatic, however, complications with increased risk of thromboembolism can occur. method: case report: a -year-old woman with no preexisting medical conditions presented nonproductive cough and fever for days. for the next days, developed acute respiratory failure. she was treated for septic shock and acute respiratory distress syndrome without success with death. expectoration and blood cultures were negative and the real-time polymerase (rt-pcr) of the thracheal swab was positive for influenza b virus. results: the autopsy showed a diffuse necrotizing alveolitis and intra-alveolar hemorrhage with pulmonary infarction. there was no histological evidence of bacterial pneumonia. an evident blood cell sickling was observed associated with sickle cell trait. rt-pcr was also positive for influenza b virus in pulmonary tissue and the immunohistochemistry was negative for influenza a virus. conclusion: severe hypoxemia caused by the acute respiratory distress syndrome was a possible cause of the blood cell sickling and thrombotic events. objective: lupus vulgaris (also known as "tuberculosis luposa") are painful cutaneous tuberculosis skin lesions with nodular appearance, most often on the face around ears nose, eyelids and lips, cheeks. ıt is still a major health problem in developing countries. cutaneous tuberculosis is a rare form of extrapulmonary tuberculosis. lupus vulgaris (lv) is the most common form of cutaneous tuberculosis. lv should make differential diagnosis other granulomatous disease. all three cases of old female patient. conclusion: herein, three cases were presented which they treatment with correlation clinical and histopathological examination. objective: actinomycosis is a rare, chronic, and slowly progressive granulomatous disease caused by filamentous gram positive anaerobic bacteria from the actinomycetaceae family. it is often misdiagnosed because it can mimic conclusion: the mean length of appendix in both series was about . cm. there were no important difference of the length of either parasitic appendixes or all cause appendectomy materials. in this study, it is concluded that there isn't an effect of the length of appendix on the settlement of bowel parasites. objective: cystic hydatid disease (chd) is an infection produced by larvae of the parasite platyhelminth echinococcus granulosus. living in a rural area is an important risk factor for this disease. it is still an endemic disease in some regions of the world, particularly in many mediterranean countries. the organs most commonly affected are the liver and the lungs. method: we present a new case of multiple thoracic hydatidosis. results: we present the case of a -year-old caucasian man who presented chest pain and dyspnea. chest-x-ray showed a well limited cardiac opacity of the upper lobe and of the right lung. chest-ct-scan showed a mediastinal cyst evoking a hydatid cyst in the left ventricle associated to a right pleural effusion. the patient underwent surgery that consisted in the drainage of multiple pericardial cysts, cystectomy of a left ventricle cyst and right pleural cystectomy. microscopic examination showed a pleura-pulmonary and cardio-pericardial hydatidosis. the patient was treated with albendazole and did not present complications or recurrences after year of follow up. conclusion: multiple thoracic hydatidosis is rare. cardiac location is the most challenging because it is difficult to manage and is life threatening. treatment modalities are mainly based on surgery. preventive measures are necessary to avoid disease recurrence. objective: lymphadenopathy is frequently present in hiv positive patients, especially in those with severe immune deficiency and consequently elevated risc of opportunistic infections and neoplasms. considering the large spectrum of possible differential diagnosis a thorough lymph node analyses should be performed. method: we examinated peripheral lymph nodes from patients from latero-cervical, axilar, inguinal and supraclavicular regions; nodes ( , %) had diameter less then cm. results: histopathological findings indicated mycobacterial infection in patients ( , %), reactive hyperplasia in patients ( , %), lymphoma in patients, metastasis from kaposi sarcoma in and supurative inflamation in . clinically suspected lymphoma was microscopically infirmed in all cases. cases of tuberculosis, of lymphoma and of kaposi sarcoma metastasis were identified, all of them without prior clinical suspicion we identified tuberculosis, lymphoma and metastasis in , % of nodes smaller then cm. conclusion: in % of cases lymph node biopsy identified tuberculosis or malignancy, and established a reliable diagnosis in various clinical suspicions. we consequently emphasize the importance of lymph node examination in the evaluation of lymphadenopathy in hiv positive patients, even is not prominent. stăniceanu and nichita are first authors in equal contribution. the importance of autopsy in hiv infected patients objective: nowadays possibilities of medical investigations offer miriads of accumulating data regarding hiv infected patients status. correlation and/or validation of these informations with pathological findings is mandatory to establish in order to ensure appropiate management of hiv infection. method: we performed a comparative study between clinical/paraclinical and autopsy findings in some of the most frequent afected organs in hiv infection; we studied in hiv positive patients autopsied between and ; all patients had no or incipient (up to year) antiretroviral treatment. results: patients presented lung pathology, clinico-pathological correlations revealing discrepancies, total concordances and partial concordances. most of these inconsistencies referred to opportunistic infections, clinically suspected and pathological refuted in cases and clinically unsuspected and pathological identified in cases. clinically suspected neurological alterations were microscopically infirmed in , % of cases, while cases presented unsuspected lesions. opportunistic infections were key: cord- -fio cjj authors: nan title: peripheral nerve society meeting july – , sitges, barcelona, spain date: - - journal: j peripher nerv syst doi: . /jns. sha: doc_id: cord_uid: fio cjj nan the peripheral nerve society was founded in from two groups of academic investigators, peripheral nerve study group and peripheral neuropathy association of america, interested in the basic biology and function of the peripheral nervous system and its application to the clinic. their invite only biennial meetings involved - attendees in cloistered settings organized by shoestring and local initiative. from this, we have grown remarkably. we now have an annual meeting of over people including meetings within the meeting for the special interest groups in inflammatory, diabetic and hereditary neuropathy. with this substantial growth and the success of jpns, the journal of the peripheral nervous system the society continues to flourish. has proven to be a year full of exciting changes for the society. pns has transitioned from a biannual, to an annual meeting. next year, the meeting will be taking place at the renaissance baltimore harborplace hotel from - july in baltimore, maryland. the development of a new website has been completed, please visit www.pnsociety.com to see the new face of the society. finally, pns has adopted new executive staff. with their guidance and the leadership of an active and diverse board of prominent professionals in the field the peripheral nerve society continues to grow and anticipates more exciting changes in the year to come. the peripheral nerve society provides annual meetings, teaching courses, guidelines, and other resources to aid in the education of members. becoming a member of pns means collaborating with prominent global professionals in the field to develop and provide the best treatments for people with peripheral nerve diseases and setting standards of care within the field. please participate in our future by joining the pns, volunteering for a project aligned with your interests and sending your ideas for the future to the executive office, or board member. peles e . department of molecular cell biology, rehovot, israel. two schwann cell-dependent mechanisms control the presence of na + channels at the nodes of ranvier: i. clustering of the nodal complex by glia-derived proteins and ii. restriction of nodal proteins within the nodal gap by the paranodal junctions. these mechanisms depend on specific cell adhesion molecules that mediate the contact between myelinating glia and their underlying axons at the forming nodes and the paranodal junction. during myelination, na + channels initially clustered at heminodes that border each myelin segment. this process requires gliomedin, nrcam and neurofascin (nf ), three cell adhesion molecules (cams) that mediate the interaction between schwann cell microvilli and the axon. na + channels clustering activity of gliomedin is tightly regulated by two distinct and functionally opposing proteolytic events. while the clustering activity of gliomedin is enhanced by its shedding from the surface of schwann cells by a furin protease, its activity is negatively regulated by bone morphogenetic protein /tolloid-like (bmp /tld), and tolloid-like (tll ) metalloproteinase. cleavage by these enzymes restricts the activity of gliomedin to the nodal area and prevents the formation of ectopic clusters along axons that are devoid of myelin segments, as well as below the myelin internodes. hence, proteolytic processing of gliomedin facilitates, yet limits, the clustering of na + channels to specific sites along the axon in a timely manner. furthermore, axon-glial contact mediated by gliomedin and nf at the nodes, not only plays a role in na + channel clustering during development, but also contributes to the long-term maintenance of na + channels at nodes of ranvier. in addition to clustering by gliomedin, the distribution of na + channels is restricted between two growing myelin segments by the flanking paranodal junction. at this site, axon-glia contact is mediated by a distinct set of cell adhesion molecules (i.e., caspr, nf and contactin) that also delineate the underlying axonal and glial cytoskeleton. this paranodal junction-dependent restriction of na + channels to the nodes is mediated by the spectrin-based paranodal axonal cytoskeleton. illa i . neuromuscular unit, neurology department, hospital santa creu i sant pau, universitat autònoma de barcelona, barcelona, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune disorder of the peripheral nerves with clinical and immunological heterogeneity. currently, the diagnosis of cidp is based on clinical and electrophysiological criteria and does not take into consideration the presence of immune biomarkers. several autoantibodies against proteins of the node of ranvier in patients with cidp have now been described. these antibodies define specific cidp subtypes sometimes referred to as nodopathies and can have diagnostic and prognostic implications. anti-contactin (cntn ) antibodies. we have described the presence of antibodies to cntn in a small subset of patients with cidp. these patients shared a phenotype and have poor response to ivig. the anti-cntn antibodies are predominantly igg . pathological studies from skin and sural nerve biopsies of patients show morphological changes in the paranodes. experimental data supporting the pathogenicity of anti-cntn igg antibodies include: a) demonstration in vitro that the antibodies disrupt the binding of the cntn -caspr complex to neurofascin- (nf ); b) intraneural injections of antibodies progressively and specifically disrupt the paranodal axo-glial junction; and c) chronic infusion of antibodies induced clinical and electrophysiological worsening in animals with experimental autoimmune neuritis (ean). anti-nf antibodies. antibodies to neurofascins were first reported in patients with guillain-barré (gbs) and cidp and subsequently, antibodies specific to the nf isoform were found in a small group (< %) of patients with cidp. studies by us and confirmed by others have demonstrated that patients with cidp and anti-nf antibodies have a distinct phenotype that often includes a low-frequency tremor and poor responses to ivig. the autoantibodies are predominantly of the igg subtype. the passive transfer of monoclonal anti-neurofascin antibodies (which recognize all neurofascin isoforms) to mice with ean strongly exacerbated the severity of the pathology, but no studies have yet demonstrated that patient-derived anti-nf igg antibodies are pathogenic. a pathogenic role of the antibodies is however supported by sural nerve biopsies from patients with cidp and anti-nf antibodies that showed paranodal demyelination in the absence of inflammation, the loss of septate-like junctions and, the interposition of cellular processes between the paranodal loops and the axolemma. these alterations are reminiscent of those found in nfasc-null mice suggesting that anti-nf antibodies may specifically disrupt the nf -cntn -caspr complex at the paranodes. antibodies to other nodal proteins. recently neurofascin- and neurofascin- were reported as the main targets of autoantibodies in five patients with igg reactivity against the nodes of ranvier; the antibodies were predominantly igg . these patients presented with clinical features distinct from those in patients with anti-nf igg antibodies. four of these patients had subacute onset of sensory ataxia without tremor. the presence of anti-caspr antibodies has been reported in two patients with inflammatory neuropathies, one classified as cidp, the other as gbs. both patients had intense neuropathic pain. the skin biopsy from both patients showed paranodal disruption. some patients whose sera show nodal or paranodal reactivity in teased nerve fiber preparations have antibodies against other nodal proteins, such as gliomedin or neuronal cell adhesion molecule (nrcam) . the skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. indeed some mechanoreceptors can detect even nanometer-scale movements. movement is transformed into electrical signals via the gating of mechanically-activated ion channels at sensory endings in the skin. the sensitivity of piezo mechanically-gated ion channels are controlled by stomatin-like protein- (stoml ), which is required for normal mechanoreceptor function. under pathophysiological conditions following nerve injury or diabetic neuropathy the slightest touch can produce pain. it is at present unclear whether peripheral changes in sensory mechanotransduction may underlie hypersensitivity associated with neuropathic pain. here we have examined the role of the stoml modulation of piezo channels in mechanoreceptors and nociceptors to under pathophysiological conditions. we recently developed small molecules that act as inhibitors of stoml function. peripheral application of stoml inhibitors can alleviate hypersensitivity in models of neuropathic pain. our data strongly suggest that tactile evoked pain in models of peripheral neuropathy may be at least partly driven by sensitization of sensory mechanotransduction driven by stoml . coleman m . john van geest centre for brain repair, cambridge, uk. axons are lost early in many neurodegenerative disorders of peripheral and central nervous system. the degeneration of transected axons (wallerian degeneration) can be slowed tenfold by overexpression of a variety of nad-synthesizing enzymes, such as isoforms of nmnat or the related mutant fusion protein, wld s . wallerian degeneration is also delayed by deletion of tlr adapter protein sarm , a protein recently reported to promote nad degradation. it is important to understand fully the mechanism of wallerian degeneration because related mechanisms contribute to axon loss in a number of disease models, including models of peripheral neuropathies, parkinson's disease, multiple sclerosis and glaucoma. new data also suggest a role in hereditary spastic paraplegia. while depletion of nad is an attractive hypothesis for the mechanism of wallerian degeneration, especially as nad can be increased by dietary methods, it cannot explain a number of key observations. fk , an inhibitor of nampt, blocks the nad salvage pathway and strongly depletes nad, including within axons. however, instead of killing axons as the nad hypothesis would predict, it does precisely the opposite: it phenocopies the protective effect of wld s . moreover, ectopic expression of the bacterial enzyme nmn deamidase, a protein absent in mammals, protects injured axons both in transgenic mice and in primary neuronal cultures, but it has no effect on nad levels either under basal conditions or in degenerating nerves. these observations fit better with a proposed toxic role for the nad synthesis intermediate nmn, a model that can also explain the protective effect of wld s . a full understanding of the pathway should identify a number of points where intervention could be a treatment for multiple axonopathies. as with any medical discipline, expansoin of knowledge about the fundamental science behing a disorder of the human nervous system comes part and parcel with a change in our understanding of the epidemiology of any given disorder or groups of disorders. recent advances in our fundamental understanding of inflammatory neuropathies of the peripheral nervous system have been accompanied by drastic changes in our understanding of the neuroepidemiology of these disorders -the specific populationss affected by peripheral neuropathies, as well as the varying importance / contributions of select peripheral neuropathies to the overall burden of peripheral nervous system (pns) disease, and how this shift in epidemiological understanding influences the clinical approach to diagnosis and management of patients with pns disease. the past few decades have witnessed a paradigm shift in many aspects of pns disease diagnosis and treatment; from the association of human immunodeficiency virus (hiv)-associated neuropathies; to the increassing recognition of hereditary / familial peripheral neuropathies; to the increased recognition of specific neuropathies such as multifocal motor neuropathy with conduction block. in addition, timely events such as the recent, and increasingly irrefutalbe evidence for a link between zika virus and a guillain-barré syndrome, and the rather unexpected resurgence of peripheral neuropathies due to previously 'exotic' etiologies such as lepromatous neuropathy require prompt clinical attention. this plenary session aims to describe the evolving neuroepidemiology of peripheral nervous system disorders, and how these changes may influnece the clinical approach to the diagnosis, prognostication, and treatment of otherwise 'unusual' periphal nerve diseases. oxaliplatin chemotherapy for colorectal cancer is seriously limited by neurotoxic side effects which are not fully understood. oxaliplatin-induced peripheral neurotoxicity (oipn) comprises an acute syndrome and a chronic sensory neuropathy. the acute symptoms, notably cold hyperalgesia, have been attributed to transient ion channel dysfunction, and the worse they are the more severe the chronic neuropathy that ensues. we designed a combined in vitro and in vivo project, using neurophysiology to better understand the pathogenesis of oipn. in the in vitro study, differentiated f cells (rat drg neurons x mouse neuroblastoma n tg- cell line) were incubated for and hours in . m oxaliplatin, and their electrophysiological properties studied by patch-clamp. the treated f cells showed relatively depolarized resting membrane potentials, significantly decreased firing frequencies, and increased sodium current densities. moreover, a decrease in erg (ether-à-go-go-related gene) potassium current was also evident. in the in vivo study, we applied nerve excitability testing (net) to a wistar rat model of oipn. to investigate the acute syndrome, we compared behavioural and neurophysiological data of animal cohorts (controls and oipn rats, n= each) before and after oxaliplatin administration ( mg/kg, iv). twenty-four hours after the injection we observed differences between the groups in behaviour (cold plate test, p= . ) and in superxcitability of motor axons (p= . ). to investigate the chronic neuropathy, we compare a control group (n= ) with a treated group (n= , oxaliplatin mg/kg twice weekly x weeks, iv). both groups are studied with behavioural, neurophysiological (sensory and motor nerve conduction studies, net), and pathological (caudal and sciatic nerve, skin biopsy, drgs) methods. data are collected at baseline, end of treatment and weeks after treatment; to obtain a full net profile of all significant changes. in this highly translational approach to oipn, the in vivo net changes in the acute and chronic rat models can be matched on the one hand to findings from in vitro experiments, and on the other to clinical data, since net is also easily applied in humans. of these results. in denmark there is a unique situation to conduct epidemiological studies facilitated by the danish civil registration and the danish national hospital registry (dnhr). this enables us to identify all gbs patients in denmark in a given period. from the same period as the igos cohort was included we have identified all gbs patients admitted to or seen in outpatient's clinics of hospitals in denmark (september st to december st ). records from the population based danish cohort will be reviewed for demographic and clinical data and compared to the patients included in igos from denmark, as well as with the igos europe/america cohort. during this period patients from denmark have been included in igos. the danish group is comparable to the europe/america group not counting the danish patients (n= ) of the igos cohort in regard to sex and age at entry, gbs disability score at nadir, and percentage of patients needing mechanical ventilation. in the danish igos group % are males, the median age is ( - ) years, the mean(sd) gbs disability score at nadir . ( . ), and % of the danish group needed mechanical ventilation. in the europe/america group % are males, the median age is ( - ), mean(sd) gbs disability score at nadir . ( . ) and % of the group needed mechanical ventilation. at the meeting we will compare and present data from the danish population based cohort as well as epidemiological data. chronic inflammatory demyelinating polyneuropathy (cidp) is a common autoimmune disease of the peripheral nervous system (pns) that causes sensorimotor impairment. mice with a dominant autoimmune regulator gene (aire) g w mutation on the non-obese diabetic (nod) background (nod.aire gw/+ mice) develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. in sapp, demyelination is caused primarily by th t cells; however, the contributions of nerve-resident cells such as schwann cells are poorly understood. we identified a population of non-hematopoietic, integrin alpha + (itga +) cells in the pns that increases in frequency and number during sapp. these itga + cells coexpress numerous schwann cell markers including sox , p , s b, myelin protein zero, and peripheral myelin protein , suggesting that itga + cells are schwann cell-like. additionally, during sapp, these itga + cells upregulate the extracellular matrix protein periostin (postn), which has recently been shown to promote macrophage recruitment and activation in inflammatory disease and cancer. our data indicate that macrophages are pathogenic during sapp. therefore, we hypothesized that itg a+ cells promote macrophage recruitment during sapp via postn production. to test this hypothesis, we performed in vitro chemotaxis assays. conditioned media from nod.aire gw/+ nerve promoted significantly more macrophage chemotaxis than conditioned media from postn −/− nerve. furthermore, postn recombinant protein was sufficient to induce macrophage chemotaxis in vitro. our findings show that itg a+ schwann cell-like cells mediate macrophage chemotaxis by upregulating postn during sapp and suggests postn as a novel target for the treatment of cidp. "wear-off" frequency will be analyzed by assessing the proportion of subjects with any given degree of gs and rods intracycle fluctuation and the proportion of cycles in which gs and r-ods fluctuation occurs. to determine the extent of "wear-off" the degree of difference between maximum and minimum gs, r-ods, tugs, onls, and vas scores will be analyzed. currently subjects from different sites have been enrolled ( sites eligible for enrollment). this interim study report will provide preliminary representative data, demonstrating ivig "wear-off" effects on gs and other outcome measures. by better understanding the frequency and extent of ivig treatment-related fluctuations we expect that these results will help facilitate development of cidp treatment optimization strategies. we also expect that this information will be important in forming hypotheses to be tested in future studies (for example, comparing different dosage intervals, optimal ivig taper guidelines, or assessing the long-term outcome of short-term cycle to cycle clinical fluctuations). neuropathic pain is a frequent feature of peripheral neuropathy causing a significant impact on patients' quality of life and health care costs. resolving the genetic architecture of painful neuropathy will lead to better disease management strategies, risk stratification, and counselling. therefore, we aim to develop a reliable technique to rapidly and accurately re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. whole exome sequencing of thousands of samples remains expensive for clinical use. several targeted enrichment approaches are currently available to selectively enrich for genomic regions of interest. in this study, we compared the sensitivity, specificity, targeting efficiency, reproducibility of performance and cost effectiveness of truseq ® custom amplicon-next generation sequencing (tsca-ngs) and molecular inversion probes-next generation sequencing (mips-ngs) methods. for both methods, we constructed a targeted enrichment kit to capture the coding and exon-flanking intron sequences of nine sodium channel genes (scn a, scn a-scn a, and scn b- b) expressed in nociceptive neurons. probes were designed for the two methods using their respective informatics pipelines. in total, patients with diabetic and idiopathic neuropathy were tested by both methods. among the patients, patients were tested previously by sanger sequencing for scn a-scn a. approximately kb was captured and sequenced. % of the targeted regions showed an average coverage of ≥ x in tsca-ngs, and % in mips-ngs. we managed to identify potential pathogenic mutations and polymorphism variants by mips-ngs and tsca-ngs. moreover, we observed a perfect agreement ( %) between sanger sequencing data and those obtained using mips-ngs and tsca-ngs. both ngs approaches showed user-friendly software to design probes and exhibited a similar on-target efficiency. although the overall coverage per region varied across different dna samples, it was sufficient to detect any variant in these regions. mips-ngs has more versatile assay design, demonstrated a high degree of flexibility with probes re-placement and > x cheaper than tsca-ngs. mips-ngs is a reliable, flexible, and inexpensive method to detect genetic variations in thousands of patients. in our centers, this technology is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients. alonso-jiménez a , , belvis-nieto r , diaz-manera j , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; centro para la investigación biomédica en red para enfermedades raras, ciberer, madrid, spain; neurology department, hospital universitario dexeus, barcelona, spain. most acute demyelinating polyneuropathies have an immune-mediated pathogenesis and are included within the guillain-barré syndrome spectrum. occasionally, other mechanisms such as metabolic, infectious or toxic may lead to gbs-like presentations. thermatrim ® and pura alegria ® are different brands of the same illegal slimming product that is sold through online vendors and in which the exact composition is unknown. here we present a patient with an acute demyelinating polyneuropathy secondary to the intake of the slimming product "pura alegría". a year-old woman with no remarkable medical history reported days history of distal numbness in her feet that progressed in one week to her knees and her left hand. she had had an upper respiratory tract infection ten days prior to these symptoms. the neurological examination showed absent distal vibratory and arthrokinetic sensations and arreflexia in lower limbs, decreased vibratory sensation in her hands and a ataxic gate. the lumbar puncture showed . g/l of proteins with no cells. the emg fulfilled diagnostic criteria for acquired demyelination. intravenous immunoglobulin therapy was started but the symptoms kept worsening and corticosteroids were started. the patient mentioned then the slimming product. a brain mri showed diffuse leukoencephalopathy that was asymptomatic. steroids and the pura alegria slimming products were withdrawn and the patient recovered completely after one year of follow-up. "pura alegría", "thermatrim" and "thermatrim plus" are slimming products that were forbidden in spain after several cases of acute leukoencephalopathy and acute polyneuropathy. exact composition is unknown although the spanish drug agency detected the pesticide malonoben, a tyrosin kinase inhibitor, among the components. since nine cases of pura alegria/thermatrim neurological toxicity, including leukoencephalopathy and polyneuropathy have been described. all cases had good outcomes after treatment withdrawal, although recovery is slow and may be incomplete. our case highlights the need to carefully consider drug toxicity, including dietary supplements, in the differential diagnosis of gbs specially when evolution does not follow typical patterns. alvarez s , klein d , martini r , moldovan m , , krarup c , . center for neuroscience, university of copenhagen, denmark; department of neurology, developmental neurobiology, university hospital würzburg, germany; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. charcot-marie-tooth neuropathy type a (cmt a) resulting from peripheral myelin protein kda (pmp ) overexpression is the most common hereditary motor and sensory neuropathy in humans. the transgenic pmp (pmp tg) mouse line c carrying copies of the human pmp gene, has a slowly progressing neuropathy phenotypically like cmt a with thin and abnormally thick myelin profiles and supernumerary schwann cells. in addition, pmp tg nerves showed activated macrophages leading to axon-myelin compartment disruption and maldistribution of k+ channels (kohl b et al, am j pathol. ; : ) . the aim of the present study was to investigate the motor axon excitability in pmp tg versus wt littermates. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle and "threshold-tracking" the plantar compound muscle action potential (cmap). at age months, when the post-developmental maturation was nearly complete in the wt, the pmp tg cmap showed an increase in latency by %. the cmap amplitude was decreased by %, although the mean motor unit size (mscan method) appeared unchanged indicating a lack of collateral sprouting. furthermore, pmp tg showed abnormalities in both passive cable properties and voltage dependent parameters. at age month, the cmap latency of pmp tg was increased by % as compared to wt. in contrast to this marked conduction slowing along the tibial nerve from to months of age, the progression of excitability changes localized at ankle appeared modest. nevertheless, when pooling data from to months, the increase in pmp tg latency was correlated (spearman p< . ) with an increase in accommodation half-time during depolarizing electrotonus (+ % of threshold) from to ms and a reduction of the late subexcitable period of the recovery cycle from to % of threshold, both changes consistent with a redistribution of k+ currents consistent with the maldistribution of k+ channels. our data suggest that in the pmp tg cmt a model, a functional, thus potentially reversible abnormality in k+ channel distribution, accumulates along the nerve and aggravates the conduction impairment due to impaired myelin formation and maintenance. alvarez s , krarup c , , moldovan m , . center for neuroscience, university of copenhagen, denmark; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. mice heterozygously deficient of myelin protein p gene (p +/−) show a mild progressive dysmyelinating neuropathy, with conduction slowing and impaired excitability, phenotypically similar with charcot-marie-tooth disease type b (cmt b). we found that in p +/− the accumulating myelin abnormalities were paralleled by progressive changes in voltage-dependent motor axon function resulting in neurotoxic membrane depolarization (rosberg mr, et. al. neurobiol dis. : ) . the aim of this study was to investigate the relationship between demyelination and motor axon function in p +/−. demyelination of the right sciatic nerve by topic lysophosphatidylcholine (lpc) application was carried out in p +/− and wild-type (wt) mice, in year (mature) and years (aged) groups. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle (distal to lpc demyelination) and "threshold-tracking" the plantar cmap responses. live imaging studies by cellvizio (mauna kea technologies, paris, france) confocal laser endomicroscopy were carried out in transgenic mice expressing the fluorescent reporter yfp in peripheral nerve axons under the thy promoter. in mature wt the sciatic morphological and electrophysiological demyelinating features following lpc could be readily observed at hours but disappeared by weeks. no morphological changes could be observed at the tibial level. consistently, no conduction or excitability changes could be observed at the right tibial neve level as compared to the left tibial nerve in wt. in contrast, in p +/− the motor axon function was impaired at the tibial nerve level at weeks after sciatic lpc demyelination. in mature p +/−, although the cmap amplitude appeared preserved, the distal motor latency was prolonged whereas the excitability measures showed reduced deviations during threshold electrotonus and increased refractoriness at the expense of superexcitability of the recovery cycle, both consistent with membrane depolarization. furthermore, in aged p +/− the delayed tibial conduction was associated with a drop in cmap amplitude and a prolongation of the strength-duration time constant. taken together these data suggest that focal demyelination aggravates membrane dysfunction along the entire motor axon in p +/− providing a novel experimental model to explore the link between demyelination and axonal membrane dysfunction in cmt b. amass l , li h , gundapaneni b , schwartz j , keohane d . pfizer inc., new york, ny, usa; inventiv health inc., burlington, ma, usa. a number of factors can influence disease progression in transthyretin familial amyloid polyneuropathy (ttr-fap), a rare, fatal, hereditary amyloidosis. this analysis evaluated the specific role of baseline neurologic severity on neurologic disease progression in ttr-fap. a predictive model was created based on longitudinal data from val met patients who participated in the tafamidis (a selective ttr stabilizer) clinical development program. data from the intent-to-treat population of the double-blind, placebo-controlled registration study (tafamidis group, n= ; placebo group n= ) and its two consecutive open-label extension studies in which all patients received tafamidis were used. the second extension study is ongoing, but a formal, prospectively-planned interim analysis was conducted with the cut-off date of december , . this analysis focused on the first months of treatment for the overall study cohort analyzed. the neuropathy impairment score-lower limbs (nis-ll) was used to assess neurologic functioning at baseline and at subsequent study visits. a linear mixed-effects model for repeated-measures (mmrm) analysis, with baseline nis-ll, treatment, and their interactions with time as fixed effects, was used, and the slope and intercept for each patient were included as random effects. patients were primarily caucasian with early-stage neurologic disease (baseline nis-ll mean [standard deviation]: tafamidis, . [ . ]; placebo, . [ . ] ). across both groups, disease progression increased with increasing levels of baseline severity (nis-ll) (p< . ). however, the predicted magnitude of change from baseline to month for tafamidis was consistently less than that for placebo across a range of observed baseline nis-ll values, suggesting a disease-modifying effect of tafamidis. similar findings were observed for the nis-ll muscle weakness subscale. this mmrm analysis in patients with val met ttr-fap demonstrates that disease progression strongly depends on baseline neurologic impairment and highlights the disease-modifying effect of tafamidis across a range of baseline levels of neurologic severity. clinicaltrials.gov identifiers: nct , nct , nct . amino h , misawa s , sekiguchi y , shibuya k , watanabe k , suichi t , kuwabara s . department of neurology, chiba university, chiba, japan. guillain-barré syndrome (gbs) is a potential life threatening neurological disorder and respiratory insufficiency is one of the critical complications. eramus gbs respiratory insufficiency score (egris) is a method for predicting the chance of respiratory insufficiency in gbs. however, clinical characteristics and courses can vary for subtypes of gbs, whose occurrences differ for each region: acute inflammatory demyelinating polyneuropathy (aidp) is very common in european countries, whereas acute motor axonal neuropathy (aman) is frequently seen in asian countries. the aim of this study is to investigate the usefulness of egris in japan, where aman is more common than in the netherlands. clinical and electrophysiological profiles of consecutive gbs cases, who visited our hospital within days after symptoms onset between and , were reviewed. of the gbs patients, % were classified as aidp and % as aman according to the electrodiagnosis criteria by ho and colleagues. higher egris scores correspond to higher risk of respiratory insufficiency in total of the gbs patients, as well as in aidp patients. however, in patients with aman, egris scores did not always match the chances of respiratory insufficiency: up to % of the patients with low risk of egris showed respiratory failure, whereas only % of the patients with high risk of egris needs intubation/mechanical ventilation. in aman, associations with mechanical ventilation were seen for rapid progression (shorter duration between onset and hospital admission), more decreased vital capacity, and more frequent autonomic involvement. egris is useful also for japanese gbs patients. however, for aman patients, it should be used with discretion. another score to predict respiratory insufficiency might be required in asian countries. anandan c , litchy wj , laughlin rs , leep hunderfund an , naddaf e . mayo clinic, rochester, usa. in patients with suspected ulnar neuropathy, nerve conduction studies (ncs) are commonly requested to help with diagnosis and localization. however, routine ncs are often normal or not localizing. ulnar ncs recording from the first dorsal interosseous muscle (ncs-fdi) is thought to increase the diagnostic yield of electrodiagnostic testing, although not commonly considered. we developed a quality improvement strategy to routinely perform ulnar ncs recording from the abductor digiti minimi muscle (ncs-adm) as well as ulnar ncs-fdi in all patients referred for suspected ulnar neuropathy. we utilized the dmaic (define, measure, analyze, improve, control) model of process improvement to define our problem and create a map of the current process for ulnar neuropathy diagnosis in our electromyography laboratory. we determined baseline performance via review of distal sensorimotor polyneuropathy (dpn) is the most common complication of diabetes and risk factors beyond hyperglycemia have proven important particularly in type diabetes (t dm). only few prospective studies from early-stage t dm exist. we aimed to study the development of dpn during the first years after a screening-based diagnosis of t dm. from the addition-denmark study participants were eligible for this study. dpn was assessed by the michigan neuropathy screening instrument questionnaire (mnsi) at four time-points during follow-up. dpn was defined by a mnsi score ≥ . participants ( %) were positive in mnsi at baseline and thus excluded from this study. by kaplan-meier plot we evaluated the cumulative incidence of dpn and in cox proportional hazard models we calculated hazard ratios (hr) for the intervention groups in the addition trial and for various covariates proposed to influence the development of dpn. models were adjusted in steps for intervention group, age, sex, baseline mnsi, lipid-lowering and anti-hypertensive treatment. this study cohort consists of participants ( % men) with a median age of . years (p ;p : . ; . ) and median baseline hba c of . (p ;p : . ; . ). a cumulative incidence of % was seen during years of diabetes. there was no statistically significant difference in hr between the intervention groups or by sex but a significantly higher hr of . ( %ci: . ; . ) was seen for age (per year). the highest hr was found for a history of cardiovascular disease (myocardial infarction or stroke) up to ten years prior to the diabetes diagnosis with a hr of . ( . ; . ). weight, waist circumference, body-mass index and methylglyoxal (log transformed) showed modest but statistically significant associations with incident dpn with standardized hrs of . ( %ci: . ; . ), . ( %ci: . ; . ), . ( %ci: . ; . ) and . ( %ci: . ; . ) respectively. this study demonstrates a fairly low cumulative incidence of dpn in people with screen-detected t dm and provides evidence that macrovascular disease, obesity and oxidative stress are important risk factors for dpn even at the earliest stages of t dm. andermann syndrome, also known as agenesis of the corpus callosum and peripheral neuropathy (accpn), is an autosomal recessive disorder with a broad spectrum of mild to severe neuromuscular and psychiatric consequences. the gene variants causing disease were first identified in french-canadian families. in the present study, we intended to phenotype and genotype a series of non-french-canadian familial cases presenting with charcot marie tooth disorder associated with agenesis/dysgenesis of the corpus callosum. for this purpose, seven families, of consanguineous marriage, were studied. patients were clinically and para-clinically investigated using mri and electrophysiology (mncvs). for some, a sural nerve biopsy was taken. microsatellite markers around the accpn locus were used in two large families; followed by sanger sequencing of all the exons and intron-exons boundaries of the gene, in one patient from each of the families. the age at onset of the disease was at birth in the patients from the largest consanguineous family ( affected individuals). the biopsy from one patient showed a severe demyelinating neuropathy with many hypomyelinated fibres and mostly secondary axonal changes. these finding were compatible with electrophsiological data where the mncvs are of demyelinating range. dysgenesis of the corpus callosum in one patient and agenesis in another sib were revealed by mri. we identified one homozygous truncating mutation in this family. interestingly, no causative variant was found in a patient from another family and showing homozygous haplotype. two different heterozygous variants were identified at one hit in two patients from two non-consanguineous families. genetic investigations will be continued to identify the possible second hit. in the remaining families, no variant was found. the negative family cases will be subjected to ngs. at this stage, it is tempting to speculate on the genetic heterogeneity of accpn in our series. baba m , suzuki c , ogawa c , tomiyama m . department of neurology; diabetes center, aomori prefectural central hospital, aomori, japan. in we introduced a staging system of severity of diabetic polyneuropathy (dpn) by nerve conduction study (ncs) of the lower limb: sensory ncs of the sural nerve and motor ncs of the tibial nerve. the system consists of five stages; ncs- (normal): no abnormalities, ncs- (mildly abnormal): presence of delay of mcv, scv, minimal f-wave latency, or positive a-wave, ncs- (moderately abnormal): decrease in sural snap less than uv, ncs- (severely abnormal): decrease in plantar muscle-cmap to - mv, ncs- (ultimately abnormal): plantar muscle-cmap lost or less than mv with trace of sural-snap. to examine validity of the system, we conducted -year prospective observation on development of diabetic foot (df) by the ncs staging system. in addition, occurrence of ischemic heart disease (ihd) and stroke (is), and death of neuro-vascular events were also counted. n - , we carried out ncs in diabetics, and categorized them by the ncs staging system: % was ncs- , % was ncs- , another % was ncs- , % was ncs , and % was ncs- . we then followed them and prospectively counted the occurrence of df, ihd and is in patients (mean age ys). the occurrence of df during the following years was; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- : %. occurrence of any of df, ihd and/or is was as follows; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- %. there was no death from nsc- , − , and − groups (n= ), while two from ncs- group (n= ) were found dead in a bed or on a driver's seat, and other two from ncs- group (n= ) died of sudden cardiac arrest or infection after foot amputation. in summary, the present ncs grading system seems to work satisfactory not only for diagnosis of severity of the current dpn, but also for prognostic prediction of the dpn-related foot and vascular events. bacon c , feely sme , shy me . university of iowa hospital, iowa city, ia, usa. for patients with charcot-marie-tooth disease, also known as hereditary motor and sensory neuropathy, the rasch modified cmtnsv is a validated measurement of symptoms and impairment. the score is comprised of nine parameters of a clinical examination, including nerve conduction studies (ncs) . each parameter has an individual score ranging from zero to four, with the composite score having a maximum of prior to rasch modification. for patients who do not complete a ncs, the cmt exam score (cmtesv ) is used, with a maximum score of before rasch modification. one parameter of the cmtesv is the vibration sense. using a rydel tuning fork, a care-giver measures vibration sense in a patient's feet, ankles, and knees and a score is determined by the severity of reduced vibration sense. in cmt a patients, % received a score of out of , noted by a reduced vibration sense at the knee. in order to capture a more sensitive vibration measurement, we tested ways of taking the total raw score of the rydel tuning fork at each point of vibration sense, the toe, ankle, and knee bilaterally. scores range from zero to , with reflecting full vibration sense. in this modified measurement, vibration sense scores varied in wider distribution. for cmt a patients captured in this studied, a modified vibration sense score results in a potentially more sensitive total cmtesv score. bae dw , an jy . st. vincent's hospital, the catholic university of korea, suwon, korea. diabetic lumbosacral radiculoplexus neuropathies (dlrpn) are usually subacute painful, monophasic, asymmetrical lower limb neuropathies with incomplete recovery due to ischaemic injury and microvasculitis. the diagnosis relies mostly on clinical suspicion and characteristic electromyographic findings. however, in acute phase, neuroimaging has more important diagnostic significance than electrophysiological studies. here we describe mri and ultrasound findings in a -year-old woman with dlrpn who was diagnosed with diabetes three years ago, but has not received any other treatment. she had a -day history of acute-onset severe pain with weakness of muscles innervated by left femoral and obturator nerve and decreased sensation in left l -l dermatome. nerve conduction studies showed reduced amplitude in left femoral nerve and electromyography showed only increased insertional activity in left iliopsoas muscle and no volitional activity in muscles innervated by left femoral and obturator nerve. ultrasound revealed increased cross-sectional area (csa) of left femoral and lateral femoral cutaneous nerve. mri showed enhancement in left l , nerve roots and proximal femoral nerve and increased signal intensity in left iliacus and iliopsoas. we diagnosed her with dlrpn and started corticosteroid. nerve ultrasound has not been previously reported in a patient with cabin air in commercial airliners originates from aircraft engines or auxiliary power units. this bleed air may occasionally be contaminated with hydraulic fluids and engine oil that contains a number of potentially hazardous chemicals including tricresyl phosphate (tcp). over the last years reports are emerging about aircrew members that experience symptoms such as tingling or burning of extremities in addition to headache, and vertigo. this "aerotoxic syndrome" is controversially discussed in the literature and has been attributed to exposure to organophosphate contaminated cabin air. since tcp has been associated with peripheral neuropathy we aimed to determine the frequency of peripheral neuropathy among frequent flyers. civilian air crew members and frequent flying passengers (m:f = : , median age years, median exposition time in aircrafts of . hours) were examined at the university hospital of cologne or at the frankfurt airport (iata code fra) by a detailed questionnaire of past medical conditions, a standardized neurological examination and nerve conduction studies of sural, tibial, and ulnar nerves. we identified subjects with clinical and electrophysiological evidence for large fiber peripheral neuropathy. incidence of peripheral neuropathy was not correlated to exposition time in aircrafts. in addition subjects showed signs of ulnar neuropathy, subjects reported abnormal vibration sensation, subjects suffered from gait imbalance and individuals reported tingling of extremities. our study shows a . % prevalence of large fiber peripheral neuropathy among frequent flyers. comparison of these data with prevalence rate in an age-matched control group will reveal a possible association of chronic exposure to cabin air and risk for peripheral neuropathy. the high incidence of the symptom tingling in our cohort warrants further studies to determine the risk for small fiber neuropathy in this condition. intravenous immunoglobulin (ivig) are human igg derived from plasma pools of healthy donors. although there are studies in literature evaluating their effectiveness in different pathological animal models, there are no data about their possible role on bortezomib (btz)-induced peripheral neuropathies. female wistar rats were treated following a preventive schedule (btz and ivig co-treatment for and weeks) and a therapeutic schedule ( weeks of btz treatment followed by a -week ivig-btz co-treatment). caudal nerve conduction velocity (ncv), plantar and dynamic tests were performed at different time points. animals were sacrificed after ws (acute phase) or ws (chronic phase) and tissue samples (dorsal root ganglias -drg-, sciatic nerve, caudal nerve, skin) were collected for morphological, morphometrical and immunohistological analysis.in the preventive schedule, ivig was not able to rescue caudal ncv reduction caused by btz neither after nor after weeks of co-treatment. same results were observed in the therapeutic schedule. on the other hand, the evaluation of mechanical allodynia and cold hyperalgesia showed that ivig injection protected from btz effect in both treatment schedules. morphometric analysis evidenced that, even if not statistically significant only the preventive schedule has a tendency to protect the caudal nerve from btz damage. this result is consistent with the morphological evaluation of the nerve. also, intra-epidermal nerve fibers density was preserved in the preventive schedule but not in the therapeutic one. finally, sciatic nerve and drg macrophage infiltration levels tended to be reduced in the therapeutic schedule and were brought back to ctrl (rats not treated or injected with ivig alone) levels in the preventive one. in conclusion, we were able to demonstrate for the first time that ivig treatment especially used as preventive treatment option may reduce btz-induced neuropathic painful pointing out the possible role of inflammation in the pathogenesis of this invalidating pathology. this work was supported by kedrion spa. we studied the prevalence, the molecular cause and clinical presentation of hereditary motor neuropathies in a large cohort of patients from the north of england. detailed neurological and electrophysiological assessments and next generation panel testing or whole exome sequencing were performed in patients with clinical symptoms of distal hereditary motor neuropathy (dhmn, patients), axonal motor neuropathy (motor cmt , patients) or complex neurological disease predominantly affecting the motor nerves (hmn plus, patients) . the prevalence of dhmn is . affected individuals per . inhabitants ( % confidence interval: . - . ) in the north of england. causative mutations were identified in out of index patients ( . %). the diagnostic rate in the dhmn subgroup was . %, which is higher than previously reported ( %). we detected a defect of neuromuscular transmission in cases and identified potentially causative mutations in patients with demyelinating multifocal motor neuropathy. many of the genes were shared between dhmn and motor cmt , indicating identical disease mechanisms therefore we suggest changing the classification and include dhmn also as a subcategory of cmt. abnormal neuromuscular transmission in some genetic forms provides a treatable target to develop therapies. barohn rj , gajewski b , pasnoor m , brown l , herbelin l , kimminau k , jawdat o , parks c , shlemon p , dimachkie mm and the pain-controls study team . the university of kansas medical center, kansas city, ks, usa. cryptogenic sensory polyneuropathy (cspn) is a common slowly progressive neuropathy that affects adults and presents with significant neuropathic pain for which multiple medications have been tried including antiepileptics, antidepressants, topicals and narcotics. a web based survey among neuromuscular experts suggested pregabalin as being more effective than other medications, however there are presently no comparative studies to assess the most effective medication. the objective of this study was to determine which of the pharmaceutical therapies (pregabalin, duloxetine, nortriptyline or mexiletine) is most effective for neuropathic pain and best tolerated in cspn. to achieve this objective we performed a prospective randomized open labelled comparative effectiveness adaptive design study of cspn patients through the patient centered outcomes research institute (pcori). cspn patients who fulfilled the inclusion and exclusion criteria were enrolled into this study. patients underwent a baseline neurological evaluation and randomly assigned to one of the neuropathic medications for months. the primary outcome is the change in likert-like pain scale. the secondary outcomes included nih pain interference scale, nih fatigue interference scale, nih sleep disturbance scale, sf- and adverse events. the outcome measures are performed at baseline, month , and . statistical analysis using bayesian adaptive design developed by berry consultant software will be performed to determine winner and losers (winner = greater than point improvement in pain). total number of patients to be enrolled is . recruitment has been challenging and a number of recruitment techniques have been used. to date, there have been patients screened, patients randomized from us sites. anticipated completion of enrollment by june and end of final patient assessment by september . interim analysis performed after first patients completed their months as part of bayesian adaptive design analysis and occurs every weeks. the distribution of randomization of patients to the medications at last adaptive design randomization was . % to medication , % to medication , . % to medication and . % to the th medication. this study may give physicians and patients evidence for future management of cspn patients. transthyretin amyloidosis (attr), which encompasses a group of disorders with significant clinical variability, is caused by transthyretin (ttr) derived amyloid deposition. the clinical aspects of autonomic nervous system involvement in attr are only partially known. the ongoing, multinational, longitudinal, observational transthyretin amyloidosis outcomes survey (thaos) provides the opportunity to expand our understanding of dysautonomia in attr. data from all symptomatic subjects enrolled in the thaos registry with a diagnosis of attr (cut-off date: january , ) were assessed for the presence and temporal course of autonomic symptoms, genotype and phenotype associations, and clinical burden according to the frequency and severity of symptoms. of symptomatic subjects enrolled in thaos, ( . %) had autonomic symptoms at enrollment including: gastrointestinal ( subjects, . %), urinary ( , . %), erectile dysfunction ( , . %), orthostatic hypotension ( , . %), xerophthalmia ( , . %) and dyshydrosis ( , . %). subjects with autonomic manifestations, compared with those without, were younger (mean age [standard deviation, sd] of . [ . ] vs . [ . ] years), with a longer duration of attr symptoms ( . [ . ] vs . [ . ] years). autonomic dysfunction was less common with wild-type attr ( of subjects, . %) than in mutation groups: val met ( / , . %); non-val met/non-cardiac ( / , . %); and "cardiac mutations" (val ile, leu met, thr ala, or ile leu mutations; / , . %). similarly, time (mean [sd] , years) from first attr symptoms to onset of autonomic symptoms, was longest for wild-type attr ( . [ . ] ) followed by "cardiac mutations" ( . [ . ]), non-val met/non-cardiac ( . [ . ]), and val met ( . [ . ] ). autonomic symptoms were present at disease onset in over a third of subjects ( , . %) . autonomic dysfunction was less frequent in subjects with cardiac phenotype ( of subjects, . %), than with mixed ( / , . %) or neurologic ( / , . %) phenotypes. the burden of autonomic symptoms (mean [sd] ) varied by genotype, val met ( . [ . ] , non-val met/non-cardiac ( . [ . ], "cardiac mutations" ( . [ . ]), wild-type attr ( . [ . ]), and by phenotype, mixed ( . [ . ]), neurologic ( . [ . ]), cardiac ( . [ . ]). dysautonomia is common, and a significant burden, in subjects with hereditary forms of attr. its prevalence is higher in val met than in other genotypes, and in the neurologic or mixed phenotypes. the objective of this study is to assess the usefulness of motor unit number index (munix) technique in charcot-marie-tooth type a (cmt a) disease and to test correlation between munix and clinical impairment. munix technique was performed in abductor pollicis brevis (apb), abductor digiti minimi (adm) and tibialis anterior (ta) muscles in the non-dominant side. a munix sum score was calculated by adding munix of these muscles. muscle strength was measured using the mrc (medical research council) scale. disability was evaluated with several functional scales including cmt neuropathy score version (cmtnsv ) and overall neuropathy limitation scale (onls). cmt a patients with known pmp gene duplication were enrolled. the munix of the adm, apb and ta muscles were correlated with the mrc of the corresponding muscle (p< . ). munix sum score was correlated with clinical scales: cmtnsv (r=− . , p< . ), onls (r=− . , p< . ). in conclusion, munix correlates with muscle strength and clinical measurements of disability in cmt a patients. the munix technique evaluates motor axonal loss and correlates with disability. the munix sum score may be a useful outcome measure of disease progression in cmt a. the objective of this study was to assess the usefulness of mri in charcot-marie-tooth type a (cmt a) disease and to test correlation between muscle fat fraction and clinical impairment. mri was performed in the non-dominant lower limb of cmt a patients and healthy controls. fat fraction of tibialis anterior muscle, cross section area and volume of sciatic nerve were determined. muscle strength of dorsiflexion was measured using a dynamometer. disability was evaluated with cmt neuropathy score version (cmtnsv ). cmt a patients with known pmp gene duplication were enrolled. fat fraction of tibialis anterior muscle was significantly increased in patients compared to healthy controls. it was correlated with muscle strength (r=− . , p< . ) and cmtnsv score (r=− . , p< . ). cross section area and volume of sciatic nerve were significantly increased in patients compared to healthy controls. in conclusion mri fat fraction correlates with muscle strength and clinical measurement of disability in cmt a patients. it may thus be a useful outcome measure of disease progression in cmt a. basta i , peric s , cobeljic m , bjelica b , bozovic i , kacar a , nikolic a , rakocevic stojanovic v , stevic z , lavrnic d . neurology clinic, clinical center of serbia, school of medicine, university of belgrade, belgrade, serbia. there is a complete lack of data about epidemiological and clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) in serbia and surrounding countries. furthermore, there is a striking scarcity of information about quality of life (qol) in cidp patients and all qol studies have been conducted in countries with high standards of health care. in august we have designed the iness (inflammatory neuropathy study of serbia) in order to comprise as many patients with cidp from serbia, republic of srpska (bosnia and herzegovina) and montenegro covering population of more than nine million people. our first aim is to analyze overall impact of cidp on physical, mental and social areas of life measured with generic, symptom specific and disease specific questionnaires -sf- , inqol and cap-pri, respectively. furthermore, we aim to analyze influence of the disease on patients' working status and presence of depressive mood measured by beck's inventory. following features of patients are included: sociodemographic data, clinical aspects of the disease, level of disability, severity of sensory symptoms, presence of comorbidities, electrophysiological characteristics, as well as fatigue, autonomic symptoms and neuropathic pain. we intend to define the most significant predictors of decreased qol in order to focus on patients with the highest risk and to improve care of cidp. we also want to see if cidp patients in complete remission as per clinical findings still have reduced quality of life. we have recruited patient so far and we expect to include around subjects overall. we will present the first data of the study at the pns meeting . beaudonnet g , prud'hon s , cauquil c , labeyrie c , not a , bouilleret v , adams d . neurophysiology chu bicêtre, le kremlin bicêtre, france; neurology chu bicêtre, le kremlin bicêtre, france. familial amyloid neuropathy (fap) is a life-threatening disease of autosomal dominant inheritance due to transthyretin (ttr) gene mutation, a liver-produced protein. current treatments slow down its natural course and are indicated from the very first objective symptoms. we aimed to evaluate two neurophysiological markers: sympathetic skin response (ssr) and heart rate variability (hrv) in the early detection of sympathetic damages due to fap. ssr and hrv were assessed in ttr gene mutated patients with neither clinic nor electroneuromyographic abnormalities and controls matched on gender and age. cases were recruited consecutively from current care in the french reference center for rare diseases of bicetre university hospital. ssr was recorded on the two palms and on the sole of the left foot with to stimulations between and ma. hrv was registered during three conditions of seconds each: normal breathing, deep breathing ( cycles of seconds of inspiration and seconds of expiration) and valsalva manoeuver during seconds. valsalva ratio, defined by the ratio between the longest and shortest rr intervals, was significantly higher in the control groups after bonferroni correction (means of . and . , respectively, p< . ). there was no significant difference between the two groups for any ssr parameter, although means of amplitudes were systematically higher in controls than among cases. our results confirm that autonomic nervous fibers are damaged early in both clinical and electroneuromyographic asymptomatic patients mutated on the ttr gene. valsalva ratio seemed to be the most discriminative marker. long-term follow-up with test repetition and confrontation with cardiologic assessment will help to precise how these tests could be used in current care. they might help to identify high risk patients to propose them an appropriate early treatment and could be used to follow treatment efficacy. familial amyloidotic polyneuropathy (fap) was originally characterized by andrade as an axonal neuropathy which subsequently was found to be associated with a number of mutations in the plasma protein transthyretin (previously named prealbumin). it is now recognized that cardiomyopathy may be a significant factor in a majority of patients with the hereditary form of transthyretin amyloidosis (fap) and many of the transthyretin (ttr) mutations are associated with cardiomyopathy with no or minimal signs of peripheral neuropathy. attr-wt also called senile cardiac amyloidosis and senile systemic amyloidosis is recognized as late-onset, usually in the th or th decade of life, and the fact that the majority of patients are males. transthyretin neuropathy proven by nerve biopsy has been rarely reported in this population. here we report our experience with patients having attr-wt characterized by cardiomyopathy but also with varying degrees of peripheral neuropathy. clinically, the neuropathy appears as typical axonal or mixed axonal/demyelinating neuropathy as is seen in fap. pathologically, two types of ttr deposition have been found, ( ) intraneural ttr amyloid deposits as seen in fap are present in some patients and ( ) other patients have extensive vascular deposition of amyloid in both perineural arteries and veins without deposits within nerve trunks. in conclusion, peripheral neuropathy may definitely be a part of the attr-wt clinical presentation and with the increase in numbers of attr-wt cardiomyopathy patients being identified, it is important to ascertain whether any evidence of peripheral neuropathy is due to the amyloidosis and not to compounding syndromes such as diabetes mellitus type ii. besora s , santos c , izquierdo c , martinez-villacampa m , simó m , bruna j , , velasco r , . neuro-oncology unit, hospital universitari de bellvitge-institut català d́oncologia, ĺhospitalet, barcelona, spain; medical oncology department, institut català d́oncologia, ĺhospitalet, barcelona, spain; department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. oxaliplatin (oxa) is the first-line chemotherapy agent in the treatment of colorectal cancer (crc). oxa-induced peripheral neuropathy is the most frequent long-term side-effect. retreatment with oxa is frequently considered in patients as salvage treatment. patients receiving oxa-based chemotherapy regimen at least twice at our institution between and were reviewed. the aim of this study was to investigate whether retreatment with oxa increases the risk of developing or worsening previous oxa-induced peripheral neuropathy. the severity of neuropathy was measured by national cancer institute-common toxicity criteria (nci), total neuropathy score (tns) © and nerve conduction studies. one hundred twenty-five crc patients were included. median age was years. after first-line oxa-based chemotherapy, . % of patients developed neuropathy according nci, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. severity of neuropathy was grade ( . %), grade ( %), and grade ( . %). median time to retreatment with oxa was months. frequencies of neuropathy before retreatment were as follows: . % grade , . % grade , and . % grade . after retreatment, severities of neuropathy were . % grade and . % grade . no patient developed grade . . % of patients did not develop neuropathy. peripheral neuropathy was the reason for stopping prematurely treatment after first-line and retreatment in . % and . % of patients, respectively. worsening of previous nci score was observed in . % of patients. the great majority of patients ( . %) remained within the same nci score than before retreatment after median [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients that did not develop neuropathy after first treatment (n= ), only and patients developed grade and , respectively, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients who initially developed grade and neuropathy, no differences in tnsc © scores just before and after finishing retreatment with oxa were identified ( [ - ] vs [ - ], p= . ). retreatment with oxa in crc patients is a feasible option even in patients who developed moderate or severe neuropathy previously. lack of worsening of previous neuropathy is observed in the great majority of patients. neurological monitoring of patients candidates to retreatment with oxa should be considered. bessaguet f , sturtz f , magy l , , desmouliere a , bourthoumieu s , demiot c . ea -myelin maintenance & peripheral neuropathy, faculties of medicine and pharmacy, university of limoges, limoges, france; department of neurology, reference center for rare peripheral neuropathies, university hospital of limoges, limoges, france. prolonged pressure and the resulting local ischemia are widely accepted as the primary etiology of skin pressure ulcers (pus) but precise mechanisms of their formation remain unclear. in this study, we wanted to study the potential role of sensory small nerve fibers in regulation of inflammation during pus formation. to achieve this goal, we developed a mouse model of a purely sensory neuropathy and this was induced by resiniferatoxin (rtx). in this model, seven days after a single injection of rtx ( g/kg; ip), mice present a thermal and mechanical hypoalgesia associated with large substance p (sp) and calcitonin gene-related peptide (cgrp) depletion without neurodegeneration. this model mimic quite well what is observed in early stages of sensory nerve fiber defect. studies have shown that sp and cgrp are involved in cutaneous inflammation regulation. in fact, these neuropeptides are released by sensory fibers and are pro-inflammatory mainly through recruitment of immune cells and vasodilation. thus, we studied gene expression of pro and anti-inflammatory cytokines by a rna array approach during pus formation in control and rtx mice. seven days after a single injection of rtx, epidermis, dermis and subcutaneous tissue layer were pinched with magnetic plates during hours. pressure induced a stage pus. gene expression was evaluated in each compressed area h after pressure. results showed mainly a down-regulation of gene expression in pus of rtx mice compared to control mice. a decrease of cgrp/sp in skin sensory small fiber increased pus formation associated with an increase of interleukins (il)- , il- , il- and il- expression and a decrease of il- expression in rtx mice. supplementary experiments with rt-qpcr for each cytokine will be necessary to confirm these preliminary results. these observations suggest a cgrp/sp role in regulation of cytokines expression during pus formation. the new inflammatory profile exhibited in this study might help in the design of new treatments improving the quality of life of neuropathic patients prone to developing bedsores. bhattacharyya bj , jayaraj nd , belmadani a , ren d , rathwell ca , hackelberg s , miller rj , menichella dm . department of neurology, northwestern university, chicago, il, usa; department of pharmacology, northwestern university, chicago, il, usa. painful diabetic neuropathy (pdn) is one of the most common and intractable symptoms of diabetes, affecting % of diabetic patients. the hallmarks of pdn are neuropathic pain and small fiber degeneration, manifested by the loss of dorsal root ganglion (drg) nociceptor axons. neuropathic pain is associated with nociceptor hyper-excitability in the absence of physiologically appropriate stimuli. in states of neuropathic pain, drg nociceptors become increasingly responsive to a variety of excitatory influences, including inflammatory cytokines. in particular, we have shown that stromal cell derived factor- (sdf- ) and its receptor cxcr are necessary for the generation of neuropathic pain in mouse models of pdn. however, the molecular mechanisms leading to the hyper-excitability of drg nociceptors in pdn are unknown, as are the mechanisms leading to small fiber degeneration. this fundamental gap in our knowledge represents a critical barrier to progress in developing novel therapeutic approaches for pdn. the objective of this study is to identify the molecular cascade linking cxcr /sdf- chemokine signaling to drg nociceptor hyper-excitability, neuropathic pain, and small fiber degeneration in pdn. drg nociceptors can be identified by a series of molecular markers, including expression of the sodium channel na v . . indeed, > % of na v . -expressing drg neurons are nociceptors. feeding mice a high fat diet (hfd) for several weeks induces glucose intolerance, obesity, and mechanical allodynia, a particular pain hypersensitivity associated with pdn. using the hfd model combined with dreadd receptor technology, we have shown that reducing excitability of na v . -expressing neurons prevents and reverse neuropathic pain, neuronal calcium overload, mitochondrial dysfunction, and small fiber degeneration. furthermore, we have shown that cxcr receptors are necessary for neuropathic pain and small fiber degeneration in pdn. taken together these data demonstrate that na v . nociceptor hyperexcitability in pdn is driven through the activation of cxcr receptors. inhibition of hyperexcitability can prevent and reverse the development of pdn. furthermore, these observations will advance our understanding as to how changes in excitability, calcium influx, and mitochondrial dysfunction in nociceptors contribute to neuropathic pain and small fiber degeneration in pdn, which is a critical barrier to progression for effective and disease modifying treatment for pdn. bianco m , terenghi f , gallia f , nozza a , scarale a , fayoumi mz , giannotta c , morenghi e , nobile-orazio e . poems (polyneuropathy, organomegaly, endocrinopathy, m protein and skin changes) syndrome is an unusual multisystem disease with neurological disability due to a severe disabling polyneuropathy, with high mortality by multiorgan failure. peripheral blood stem cell transplantation (pbsct) is considered the treatment of choice for poems while lenalidomide is the most promising therapy for patients not eligible for pbsct. the aim of the present study was to compare the long-term effects on clinical, biological and neurophysiologic parameters in patients with poems treated with lenalidomide or pbsct. the clinical, biological and neurophysiologic data were reviewed in poems patients treated with pbsct (n: ) or lenalidomide (n: ). the mrc sumscore on muscles, onls scale, vegf serum levels and nerve conduction studies were assessed before (t ) and after (t ) and years (t ) of treatment and the differences were compared using anova. combining the two groups of patients, there was a significant improvement after treatment in the mean mrc sumscore (t = ± ; t = ± ; t = ± ; p = . ), in the mean onls score (t = . + . ; t = . + . ; t = . + ; p = . ), in the ulnar mean distal motor latency (t = . ± . msec; t = . ± . msec; t = . ± . msec; p = . ), distal compound muscle action potentials amplitude (t = . ± . mv; t = . ± . mv; t = . ± . mv; p = . ), motor conduction velocity (t = . ± . m/sec; t = ± m/sec; t = . ± . m/sec; p= . ) and serum vegf levels (t vs t : p = . ; t vs t : p = . ). the difference was also significant when we separately analyzed patients treated with lenalidomide and pbsct and there was no difference between the two groups in any of the analyzed parameters. treatment with pbsct and lenalidomide significantly and similarly improved clinical, biological and neurophysiologic parameters in patients with poems syndrome up to two years. since pbsct may not be suitable for all patients, lenalidomide may represent an effective and a valuable alternative in these patients or in those relapsing after pbsct inducing a prolonged clinical, biological and neurophysiologic improvement. bis d , tao f , abreu l , sleiman p , hakonarson h , zuchner s and inherited neuropathy consortium. dr. j.t. macdonald department for human genetics, hussman institute for human genomics, university of miami, miami, fl, usa; center for applied genomics, the children's hospital of philadelphia, philadelphia, pa, usa. inherited peripheral neuropathies are clinically and genetically heterogeneous diseases that can cause distal muscular atrophy and sensory loss. alleles in over one hundred different genes have been shown to cause peripheral neuropathies; yet, greater than % of axonal neuropathy patients do not receive a genetic diagnosis. large scale exome studies are now beginning to be sufficiently powered to perform mutational burden analysis. this approach compares damaging allele frequencies of cmt cases with a control group to identify additional causes for neuropathies. this approach will also identify genes that require an oligogenic inheritance to cause a phenotype. in a deviation from the classic linkage-based and heuristic variant filtering approaches to gene identification, we are performing burden analyses in a large cohort of cmt families. in known neuropathy genes, we saw that neuropathy cases carried on average . rare, non-synonymous variants, while unrelated non-neuropathy controls harbored . variants (p= . e- , mann-whiney u-test). enrichment of rare, non-synonymous variants in cmt disease genes within inherited peripheral neuropathy cases suggests the presence of multiple weaker alleles in individual patients. we also performed an unbiased exome-wide gene-based burden analysis and ranked genes after multiple testing correction. several new candidate genes were identified that need further follow up conformational studies. a number of known cmt and related genes were observed in the list of top candidates. we are currently analyzing additional aspects of this sample and are actively seeking more cmt exomes to enlarge our study. in summary, statistical methods traditionally reserved for more 'common' phenotypes' increasingly are becoming available for rare disease genetics. c bl/ mice were treated with bortezomib alone or in combination with monastrol. neuropathic changes were assessed by nerve conduction studies and histological analysis. analysis of axonal morphology was performed with light and electron microscopy. anti-neoplastic properties of monastrol alone and in combination with bortezomib were assessed in different blood cancer cell lines. prolonged treatment with bortezomib induced a sensory neuropathy in mice. significant changes in axonal morphology correlated with reduced function of peripheral nerves. the administration of monastrol substantially ameliorated morphological features of axonal alterations and sensory neuropathy. cytotoxicity studies in blood cancer cell lines showed no interference of monastrol with the cytostatic effects of bortezomib. our data indicate that monastrol may alleviate bortezomib induced neuropathy. the favorable cytotoxic profile of monastrol makes it an interesting candidate as neuroprotective agent to prevent bortezomib-induced neuropathy. boczonadi v , meyer k , gonczarowska-jorge h , , bartsakoulia m , roos a , , bansagi b , zahedi rp , talim b , bruni f , kaspar b , , lochmüller h , boycott km , müller js , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; rinch, columbus, oh, usa; leibniz-institute für analytische wissenschaften-isas-e.v., dortmund, germany; capes foundation, brazil; department of pediatrics, hacettepe university children's hospital, ankara, turkey; dbbb,university of bari aldo moro, bari, italy; department of neuroscience, the ohio state university, columbus, oh, usa; department of genetics, cheo, university of ottawa, ottawa, canada. while autosomal dominant mutations in gars, encoding the glycyl-trna synthetase, have been identified in patients with charcot-marie-tooth peripheral neuropathy (cmt d) and distal spinal muscular atrophy type v (dsma-v), autosomal recessive mutations cause mitochondrial disease affecting skeletal muscle and heart. gars is a bi-functional enzyme and it is responsible for normal protein translation both in mitochondria and the cytoplasm. in this study we have focused on the mitochondrial function of the gars by investigating a mouse model (gars c r) , human fibroblasts and induced neuronal progenitor cell lines (inpcs). mild mitochondrial abnormalities were detected in skeletal muscle of the gars c r mice while no other tissues were affected. control and patient fibroblasts harboring gars mutation were directly converted into inpcs. we identified tissue specific impairment of mitochondrial function in neuronal cells carrying not only recessive but also dominant gars mutations, suggesting neuron-specific effects of mitochondrial alterations.comparative proteomic analysis of inpcs showed significant changes in mitochondrial proteins. furthermore, the reduction of the vesicle-associated membrane protein-associated protein b (vapb) and its downstream pathways in gars-deficient inpcs suggests that altered mitochondria-associated endoplasmic reticulum (er) membranes (mam) may also contribute to the motor neuropathy. boczonadi v , king ms , bansagi b , roos a , , eyassu f , borchers c , lane m , ramesh v , lochmüller h , pyle a , griffin h , smith ac , chinnery pf , , alan j robinson aj , edmund rs kunji ers , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; mitochondrial biology unit, medical research council, cambridge; leibniz institute of analytic sciences (isas), dortmund, germany; uvic-genome bc proteomics centre, vancouver, canada; paediatric neurology, royal victoria infirmary, newcastle upon tyne foundation hospitals nhs trust, newcastle upon tyne, uk. members of the mitochondrial carrier family (slc ) transport nucleotides, keto acids, amino acids, fatty acids, co-factors and inorganic ions across the mitochondrial inner membrane. several inherited diseases with very variable clinical presentations are associated with dysfunctional mitochondrial carriers. we report a patient with childhood-onset spinal muscular atrophy and mitochondrial myopathy caused by a homozygous mutation in slc a , encoding the mitochondrial oxodicarboxylate carrier (odc). the mutation renders the carrier dysfunctional and, consequently, -oxoadipate cannot be imported into the mitochondrial matrix. computer modelling of the metabolic defect caused by the mutation predicted that the impaired transport leads to accumulation of -oxoadipate, pipecolic acid and the known neurotoxin quinolinic acid, which were precisely confirmed by targeted metabolomics in serum and urine. exposure of -oxoadipate and quinolinic acid reduced the level of mitochondrial complexes in sh-sy y cells in-vitro suggesting a possible pathomechanism. here we demonstrate that -oxoadipate and quinolinic acid are toxic for spinal motor neurons and their increased levels may contribute to neuropathy. and families have been reported. interestingly, most mutations target the same amino acid residue (k e, k m, k n, k t) in the highly conserved -crystallin domain of the hspb protein. the spectrum of diseases caused by mutations in the hspb gene was recently expanded to distal myopathy. hspb is ubiquitously expressed, but is highly expressed in motor neurons and muscles. the hspb is a chaperone that participates in clearing misfolded poly-q containing proteins such as mutant huntingtin and ataxin- involved in respectively huntington's disease and spino-cerebellar ataxia. hspb directly interacts with the co-chaperone bag and their role in chaperone-assisted selective autophagy is well described. to delineate the molecular deficits and functional consequences of hspb mutations we generated a knock-in (ki) mouse model for the k n missense mutation mimicking the neuropathy phenotype. we observed that homozygous mutant mice (hspb k n/k n ) develop a progressive axonopathy, with decreased compound motor action potential amplitudes, and loss of large and medium myelinated axons. this results in locomotor deficits with an impaired performance at the rotarod and grip strength tests. at the ultrastructural level, the hspb -ki model displays severe signs of axon degeneration and a clear myofibrillar myopathy, as observed in some patients with hspb mutations. interestingly, hspb positive aggregates were found in the sciatic nerve and gastrocnemius muscle of our mutant mice. additionally, our model allowed us to generate hspb knock-out (ko) mice using the same targeting vector. strikingly, the homozygous hspb -ko animals do not show any sign of axonopathy and display a much milder myopathy than the hspb -ki animals. these data suggest that part of the pathomechanisms is due to toxic gain-of-function of the mutant protein. boutahar n , reynaud e , nasser y , camdessanché jp , antoine jc . university hospital, saint-etienne, france. dysimmune sensory neuronopathies (snn) depend on neuron cell death induced by an inflammatory reaction in dorsal root ganglia. we have recently identified the intracellular tyrosine kinase (trk) domain of the fibroblast growth factor receptor- (fgfr ) as the target of antibodies in a subset of patients with non paraneoplastic snn. fgfr is one of the four fgfrs and has been involved in sensory neurons maintenance during development and cell death induction after axotomy. fgfrs ligand fixation results in the activation of several intracellular pathways through adaptator protein interactions with the trk domain. in particular ras activation may lead to cell proliferation or apoptosis through erk / or p map kinase signaling. the p mapk pathway is also involved in neuronal cell death induced by nmda and ampa receptor activation. as fgfr is a cell surface protein, human antibodies may interfere with the receptor functioning as a growing number of evidence has showed with other cell surface antibodies in neurological diseases. to test this hypothesis we developed an in vitro model using fvbn mice cortical neurons culture exposed to a rabbit polyclonal antibody reacting with the trk domain of fgfr . comparatively to normal rabbit iggs, the fgfr antibody induced neuron cell death in a dose dependent manner. neuron cells were exposed to fgfr antibody concentration leading to - % cell death in absence or presence of the p mapk inhibitor sb and the expression of fgfr , glur subunit of ampa receptors and nr subunit of nmda receptor was measured by quantitative rt-qpcr. the fgfr antibody induced an upregulation of fgfr while the glur and nr subunits were modulated. these changes were prevented in presence of sb . these preliminary results indicate that anti-fgfr iggs may interfere with the functioning of the intracellular domain of the protein and the expression of nmda and ampa receptors through the p map kinase pathway. this model may be used to test the effect of human anti-fgfr iggs in vitro. braathen gj , tveten k , holla Øl , busk Øl , hilmarsen ht , svendsen m , høyer h . section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. next-generation sequencing (ngs) has during the last years entered the clinical diagnostics. ngs has proven to be very efficient in the diagnostics of disorders where multiple genes can be involved. our ngs-based targeted gene panel consists of hereditary neuropathy genes, i.e. mostly charcot-marie-tooth genes. this study is a retrospective study of clinic samples received between may and february . we describe the identified novel likely pathogenic sequence variants, according to international guidelines. in this period we identified novel, not previously described, likely pathogenic sequence variants in the following genes: aars, fgd , gan, hint , litaf, lrsam , mme, mpz, nefl, pmp , sbf , sh tc and yars. there is now a large range of genes causing hereditary peripheral neuropathies and many likely pathogenic sequence variants. likely pathogenic sequence variants are not only identified in old well established neuropathy genes but also in the newer genes like mme. modelling brown-vialetto-van laere syndrome in c. elegans mutations in slc a and slc a , which encode the riboflavin transporters rfvt and rfvt . patients with rfvt deficiency exhibit proximal and distal limb weakness, sensory ataxia, diaphragmatic paralysis, optic atrophy, sensorineural deafness and bulbar palsy. riboflavin is critical for the biosynthesis of flavin mononucleotide and flavin adenine dinucleotide, essential cofactors for carbohydrate, amino-acid and lipid metabolism. mutations in slc a reduce or abolish rfvt expression resulting in impaired riboflavin uptake into sensory and motor neurons. high-dose riboflavin treatment can improve or stabilise a patient's condition, however the optimum dose and long term effects of riboflavin treatment, and disease pathomechanisms remains poorly understood. to further understand the pathophysiological consequences of slc a mutations, we propose developing an animal model for bvvl. caenorhabditis elegans (c. elegans) are small round transparent nematodes extensively used for studying the genetics and molecular biology of neurodegenerative diseases. there are two c. elegans riboflavin transporter genes, rft- and rft- . based on protein sequence homologies and expression profiles for both genes, rft- is the ortholog of rfvt . the expression of rft- is regulated by riboflavin availability and knock-down of the rft- gene by sirna perturbs c. elegans development. our aim is to develop a knock-in c. elegans model of bvvl. human rfvt and c. elegans rft- protein sequences were aligned with clustal omega to identify conserved amino acid residues associated with bvvl mutations. the amino acid involving the l p rfvt mutation is conserved in rft- (residue l ). to create our model, we will introduce the l p rft- mutation into the rft- locus in c. elegans genomic dna using crispr/cas- technology. this bvvl c. elegans model will allow us to explore the pathogenic consequences of rfvt deficiency underlying motor nerve degeneration and to evaluate drug therapy regimes by determining the optimal riboflavin dose and treatment initiation, and trialing other compounds that may improve benefits seen with riboflavin supplementation. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune-mediated inflammatory disease of the peripheral nervous system. cidp can present chronic progressive or relapsing-remitting courses and can predominantly affect motor but also sensory nerve fibers causing weakness of proximal and distal muscles. it represents the most common chronic autoimmune neuropathy and is pathologically characterized by focal inflammatory-mediated demyelination followed by axonal degeneration. recently, we have developed a new animal model for cidp, the chronic-ean, induced in lewis rats by active immunization with s-palmitoylated p ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) peptide. this model fulfills electrophysiological criteria of demyelination with axonal degeneration, a result confirmed by immunohistopathology. the late phase of the chronic disease is characterized by an accumulation of il- + cells and macrophages in sciatic nerves and as well as high serum il- levels. it is a reliable and reproducible animal model for cidp that can now be used for translational drug studies for chronic human autoimmune-mediated inflammatory diseases of the peripheral nervous system, particularly cidp, for which, there is a real need for new immunotherapies. the aim of this study was to test the therapeutic efficacy of ivig and a recombinant fc fragment (fcrec) in this new cidp animal model. treatments with ivig and fcrec proved effective in preventing further progression of cidp in rats. the therapeutic treatments not only decreased the maximal clinical scores of the cidp rats compared to albumin treatment but also abolished the disease chronicity. interestingly, a better efficacy of fcrec treatment compared to ivig was demonstrated at histological level, with the myelinated fibers well preserved and the greatly reduced accumulation of macrophages and il- + cells in sciatic nerves. ivig and fcrec therapeutic activities in this model can also be followed by measurement of antibodies in the serum and could therefore be used as biological markers. the current study provides for the first time direct evidence that ivig is effective in the treatment of cidp rats and suggests that a novel fcrec compound is more effective than ivig. it will contribute to the development of more effective and safer drugs for the treatment of autoimmune peripheral neuropathies like cidp. each time, one measurement with supramaximal stimulation according to the conventional ncs and another measurement with % enhanced stimulation intensity were conducted. we analyzed the results with special consideration of minimal and maximal fiber velocities and of the velocity with maximal fiber proportion. as a result, we detected statistically significant differences between cidp patients and controls in all these parameters. compared with controls, cidp patients had slower minimal and maximal fiber velocities and also the spectrum of motor conduction velocities was definitively shifted to slower velocities. slower minimal conduction velocities could be detected in some cidp patients by using the enhanced stimulation intensity. interestingly, in some cidp patients the conventional ulnar ncs were normal, but the collision technique showed fibers with a conduction velocity of less than m/s, indicating demyelination. to our knowledge, this is the first study using hopf's collision technique systematically in cidp patients. significant differences between the variation of ulnar motor nerve conduction velocity of cidp patients and controls could be detected. this method also showed signs of demyelination in some cidp patients with normal ulnar ncv. by enhancing stimulation intensity above the threshold of supramaximal stimulation in conventional ncs, the collision technique may be even more valuable. the clinical course of guillain-barré syndrome (gbs) is highly variable and some patients may develop treatment-related fluctuations (trfs) as an indication of ongoing disease activity and temporary treatment effect. other patients present as gbs, but subsequently develop repetitive relapses as an indication of acute-onset chronic inflammatory demyelinating polyneuropathy (a-cidp). this distinction is important because treatment may differ. we determined the frequency and clinical presentation of patients with gbs-trf and a-cidp in the first patients included in the international gbs outcome study (igos) with a follow-up of at least months. thirty-eight patients ( %) were excluded because of alternative diagnoses. of the remaining patients, ( %) had at least one trf, ( %) had a-cidp. preliminary analysis showed no significant differences between the groups (total-gbs, gbs-trf and a-cidp) for sex, age, sensory symptoms, cerebrospinal fluid results and mechanical ventilation. a-cidp patients had a median age of years (iqr - ), % was male, and all patients were treated in the acute phase of the disease (intravenous immunoglobulins (ivig) ( ), plasma-exchange (pe) ( ) or methylprednisolone (mp) ( )). gbs-trf patients has a median age of years (iqr - ), % was male and all patients received treatment (ivig ( ) and pe ( ). gbs-trf patients showed more antecedent events ( % versus %, p= . ), a higher gbs disability score (≥ ) at nadir ( % versus %, p= . ) and less frequently developed ataxia ( % versus %, p= . ) than patients with a-cidp. onset to nadir was longer in a-cidp than in gbs-trf ( days (iqr - ) versus days (iqr - ), p= . ) and the total gbs group ( days (iqr - ), p= . ). the time until the first clinical deterioration tends to be longer in the a-cidp patients (median days (iqr - ) versus median days in the gbs-trf group, not significant). the diagnosis a-cidp was made after a median of days . in conclusion, this first analysis identified distinctive characteristics of gbs-trf and a-cidp in support of a different pathogenesis that may help with early identification of these disorders in clinical practice. additional results will be presented at the conference. chronic inflammatory demyelinating polyneuropathy (cidp) is a disorder with a highly diverse clinical presentation, electrophysiological phenotype, response to treatment and outcome. this heterogeneity may indicate the presence of distinct subtypes of cidp, which may have a different pathogenesis and require more personalized treatment. the international cidp outcome study (icos) is a prospective, observational, international multi-center study that aims to describe this variation in clinical and electrophysiological subtypes and to define the clinical and biological determinants of these subtypes, disease activity, treatment response and outcome. in addition icos aims to provide an infrastructure for conducting new (therapeutic) studies in cidp, similar to the international gbs outcome study (igos). all patients fulfilling the efns/pns ( ) diagnostic criteria for cidp can be included in icos, independent of age, duration and severity of disease or treatment. we collect information on neurological deficits, diagnostic characteristics, various validated clinical outcome measures, previous and current treatment and we collect biomaterials (dna, cerebrospinal fluid, nerve biopsies and repeated serum samples). icos was started as a pilot study in dutch university centers. by february , patients were included in icos, patients recently diagnosed with cidp and previously diagnosed patients. included were ( %) males and ( %) females with a median age of years (iqr - ). the current cohort consists of classic (sensory-motor) cidp, madsam and pure motor variants. of the patients diagnosed in the past, patients ( %) were treated (intravenous immunoglobulins (ivig) ( ), prednisolone ( ), subcutaneous immunoglobulins (scig) ( ), dexamethasone ( ), plasma-exchange ( ) and ivig with methylprednisolone (mp) ( )). the recently diagnosed patients all received treatment (ivig ( ), dexamethasone ( ), plasma-exchange ( ) and patients were treated in a pilot study with ivig with methylprednisolone (mp)). the protocol has been evaluated and adjusted and will be shared with other researchers via the inflammatory neuropathy consortium and igos consortium. our aim is to include at least cidp patients worldwide with a minimum follow-up period of years. dominant mutations in glycyl trna synthetase (gars) cause inherited axonal neuropathy (charcot-marie-tooth type d). mutations in the mouse gars gene cause a similar phenotype, and represent valid disease models. we routinely use two mouse strains, one with a severe neuropathy, and one with a milder, later onset neuropathy. using these mouse models, we are exploring the mechanisms through which gars mutations cause peripheral axon degeneration. efforts include ribosome tagging to isolate ribosome-associated mrnas specifically from motor neurons, and non-canonical amino acid tagging to visualize and isolate newly synthesized proteins. taking advantage of the anatomy of motor neurons, we are able to analyze cell bodies separately from peripheral axons in both of these approaches. in addition, we are using these mouse models for preclinical studies testing gene therapy approaches to treat cmt d. consistent with our previous genetic studies in mice, knockdown of the mutant transcript, while preserving sufficient levels of the wild type, is a very successful approach when administered before the onset of neuropathy. we are now testing this approach in mice after the onset of symptoms, and in mice carrying a mutant gars allele associated with human disease. these studies are potentially translational, and also address mechanistic questions such as the timing and cell autonomy of the pathophysiology. burnor e , yang l , hao z , patterson k , quinn c , scherer ss , lancaster e . the university of pennsylvania, philadelphia, pa, usa; the second xiangya hospital, central south university, hunan, china. autoantibodies to two isoforms of neurofascin (nf and nf ) have been reported in patients with guillain-barre syndrome (gbs) or chronic inflammatory demyelinating neuropathy (cidp). it is not clear which of these responses reliably distinguish autoimmune neuropathies from other severe neuropathies, which responses are transient versus persistent, or which responses may target multiple isoforms of neurofascin. in addition, approximately % of neuropathy patients have no known cause, and it is unknown whether a subset of these patients may have autoantibodies to neurofascins. we have studied cohorts of patients with autoimmune neuropathy (n= ), genetic neuropathy (n= ), and idiopathic neuropathy (n= ) for igg and igm responses to neurofascins. neurofascin antibodies were found in ( %) of patients with autoimmune neuropathy, and ( . %) idiopathic neuropathy patients, but only % ( of ) in patients with genetic neuropathy. follow-up serum samples were available for positive cases. persistent responses were associated with chronic neuropathy while transient responses were seen in gbs or with remission of cidp. most patients had responses specific to either nf of nf . however, a particularly severe, treatment-resistant form of cidp, approaching a locked-in state, was seen in a patient with a unique response to all three isoforms of neurofascin (nf , nf , nf ). treatment of this patient with rituximab resulted in clinical improvement and resolution of the neurofascin antibody response. in conclusion, autoantibodies to neurofascins distinguish autoimmune neuropathies from severe genetic neuropathies, but the clinical phenotype may depend on the persistence and isoform specificity of the immune response. antibodies to the common domains shared by nf and nf may portend a severe but treatable neuropathy. a subset of idiopathic neuropathy patients may have an autoimmune mechanism. burns j , , sman ad , cornett kmd , wojciechowski e , , walker t , menezes mp , , mandarakas mr , rose kj , , bray p , , sampaio h , farrar m , , refshauge km , raymond j and the fast study group. university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia; university of new south wales, sydney, australia. exercise has undisputed benefits for human health and potentially as a treatment for neuromuscular disease. but there is also a risk of harm due to overwork weakness. we report the results of a -month randomised, double-blind, sham-controlled trial evaluating progressive resistance exercise of foot dorsiflexor muscles in pediatric cmt. sixty patients ( cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx ) aged - years were randomly assigned to undergo -weeks ( sessions) of moderate-intensity progressive resistance exercise or sham exercise. the primary endpoint was change in isometric dorsiflexion strength between groups assessed by hand-held dynamometry (expressed as a z-score based on age-and sex-matched normative reference values, positive values indicate an improvement in strength). the primary safety endpoint was change in muscle and fat volume of the muscles responsible for dorsiflexion by mri between groups. secondary outcomes were function (balance, long jump, -min walk test), walking ability ( d gait analysis), self-reported ankle instability, parent-reported quality of life (physical summary, psychosocial summary and global impression of change scores), and adverse events. fifty-five ( %) children completed the trial. adherence was comparable between exercise ( %) and sham ( %) groups. while patients experienced muscle soreness during training in the exercise group, adverse events did not differ between groups. the mean z-score for dorsiflexion strength increased in the exercise group by % at -months (from − . ± . to − . ± . ) and decreased in the sham group by %, mirroring the natural history of cmt (from − . ± . to − . ± . ). between-group ancova-adjusted difference at -months was . ( %ci, . to . ; p= . ). the mean scaled scores for mri muscle volume and fat volume were comparable between groups at months (p> . ). the global impression of change scores favoured the exercise group at -months (p= . ) and -months (p= . ). there was no other measurable effect of exercise. pre-specified subgroup analyses according to age ( - years vs. - years) showed a larger treatment effect with exercise in adolescents. targeted progressive resistance exercise was effective at halting progression of dorsiflexion weakness without detrimental effect on muscle morphology or other signs of overwork weakness in children with cmt. byung-nam y , yoon-ho h , suk-won a , seok-jin c , jung-joon s . inha university hospital, incheon, south korea; seoul metropolitan government boramae medical center, seoul national university, seoul, south korea; joong ang university hospital, seoul, south korea; seoul national university hospital, seoul, south korea. a years old female patient visited the hospital with weakness in both upper limbs from months under the suspicion of motor neuron disease with the finding of extensive denervation changes on electromyography. whole spine mri showed ventral nerve rootlet enhancement in the c and t bundles. the emg had confirmed active denervation changes in the muscles innervated by bilateral c ∼c roots. the cerebrospinal fluid culture test showed a protein (csf) elevation to mg / dl. she got a steroid pulse therapy. she had the symptoms of dry mouth and dry eyes during the recent years. a salivary gland scan test was performed for the possibility of sjögren's syndrome, and as a result, absorption of the contrast agent in both parotid and submandibular glands was decreased. the shammer test showed mm on the right side and mm on the left side. she was diagnosed of sjögren's syndrome. after weeks, overall strength improvement was observed, and bilateral shoulder abduction was improved to mrc grade or higher. the follow-up spine mri also showed that the initially seen ventral nerve root enhancement was disappeared. the case had visited the hospital with major symptoms of weakness and atrophy of the muscles, showing similar pattern to motor neuron disease, and was diagnosed as inflammatory polyradiculopathy and confirmed as primary ss during differential diagnosis. this case suggests that primary ss may induce inflammatory polyradiculopathy, which shows motor symptoms as major symptoms rather than sensory symptoms, and that a fast and accurate diagnosis is needed in terms that it can be treated with steroids and appropriate immune suppressive agents. traumatic injuries to peripheral nerves are frequent. however, effective pharmacological treatments are lacking. curcumin, a polyphenol found in rhizomes of curcuma longa, has been shown to develop antioxidant, anti-inflammatory, and neuroprotective properties. however, due to its poor hydrosolubility and its extensive metabolism, the use of large curcumin doses is required for therapeutic purpose. the aim of the present study was to investigate the effects of a local infusion of a low curcumin dose on nerve regeneration and functional recovery after sciatic nerve injury in rats. the experiments were conducted in g sd male rats submitted to unilateral sciatic nerve crush at d . curcumin was solubilized in polyethylene glycol and continuously administered using osmotic pumps ( . mg/day until d ) with a catheter delivering the drug near the lesion site. functional analyses using von frey, beam walking, static sciatic index (ssi) and grip strength tests were carried out at d (reference test) and every week after injury (d , d , d , d and d ). in addition, an evoked electromyogram was performed at d and d . after euthanasia (d ), nerve and muscle samples were collected and analyzed by light and electron microscopy. functionally, a significant improvement of the mechanical sensitivity (+ %) was observed at d in the curcumin-treated group (n= ) vs. vehicle group (n= ). in curcumin-treated group, skillful walking and finger spacing of the ipsilateral paw (ssi) were fully restored respectively at d and d contrary to vehicle group. furthermore, curcumin treatment improved the grip strength recovery (+ % at d ). the electrophysiological results indicated a full recovery of motor nerve conduction velocities (mncv) after days of curcumin treatment, while mncv remained altered in vehicle group ( % of the mncv at d ). morphometric analysis of nerve sections using g-ratio showed an improvement in the thickness of the myelin sheath in curcumin treated animals (+ % vs. vehicle group). histological investigation of gastrocnemius muscle indicated decreased neurogenic lesions in curcumin group. proteomic analysis is currently under investigation to understand the mechanisms involved in curcumin effects. our data could lead to the development of new therapeutic strategies in peripheral nerve regeneration using low doses of curcumin. previous studies suggest that the metabolic syndrome (mets) is associated with distal symmetrical polyneuropathy (dsp), and that diabetes, pre-diabetes, and obesity are the main metabolic drivers. the aim of this study is to investigate the association of mets components with retinal and cognitive function in a bariatric surgery cohort prior to surgery. patients were recruited from the adult bariatric surgery clinic at the university of michigan and lean controls from a research website (no mets components based on ncep/atpiii definition). participants underwent extensive metabolic phenotyping including a glucose tolerance test and fasting lipid profile. dsp was defined using the toronto consensus definition of probable clinical neuropathy. retinal function was measured with frequency doubling technology perimetry (average mean deviation), and cognitive function with the nih toolbox (composite score). univariate linear regression models were used to evaluate the association between mets components and retinal and cognitive function. to date, we have recruited bariatric surgery participants and lean controls. in the bariatric population, the mean (sd) age was ( . ) with % female compared with a mean age of . ( . ) with % female in the lean group. in the bariatric group, . % had diabetes, . % pre-diabetes, and . % normoglycemia. the dsp prevalence was % in lean controls, . % in normoglycemic, . % in pre-diabetic, and . % in diabetic bariatric participants (p< . for trend). retinal function was . ( . ), − . ( . ), − . ( . ), and − . ( . ) (p= . for trend), and cognitive function was . ( . ), . ( . ), . ( . ), . ( . ) (p= . for trend) in these same groups for lean controls, normoglycemics, pre-diabetics and diabetics, respectfully. pre-diabetes (− . , %ci: − . ,- . ) was the only mets component associated with retinal function, and waist circumference was the only one associated with cognitive function (− . , %ci − . ,- . ). dsp and retinal function, but not cognitive function decline with worsening glycemic status. similar to previous data for dsp, pre-diabetes and obesity are associated with retinal and cognitive function respectively. interestingly, while clinical dsp is common in this population, clinical retinopathy and dementia are not, indicating that dsp may be the first metabolic complication in the morbidly obese. and anti-cntn antibody-positive cases were confirmed by cba and were of igg subclass in half of them. by cba we identified additional / ( %) anti-caspr seropositive patients, whose isotype is currently being tested. sera of anti-nfasc and anti-cntn igg seropositive patients and patients with anti-caspr antibodies stained paranodes by indirect immunofluorescence on mouse teased nerve fibers. of note, seronegative patients for known antibodies showed reactivity against node and/or paranodes. compared to other seronegative cipd patients, seropositive patients had more frequently subacute onset of the neuropathy and a younger age at onset, particularly for nfasc or caspr antibodies. weakness was more severe and was often associated with proprioceptive loss, sensory ataxia and tremor. neuropathic pain was not a feature of caspr -seropositive patients. frequent findings were increased distal motor latencies and temporal dispersions on nerve conduction study and a higher protein level in csf. finally seropositive patients tended to have a higher disability and showed worst response to ivig. rituximab was effective in one patient with anti-nfasc antibodies and two patients with anti-cntn antibodies showed good and persistent recovery after cyclophosphamide. prevalence of antibodies was % in italian cidp patients and their presence was associated with distinctive clinical features. their determination, followed by characterization of igg subclass in positive cases, has clear clinical impact, by helping to guide therapeutic choices. the reactivity against nodal and paranodal components in sera from patients without known antibodies suggests that other targets could play a role in the autoimmune response in cidp and they still need to be identified. mutations in amynoacyl trna synthetases (arss), enzymes that catalyse the covalent attachment of amino acids to their cognate trna, are responsible for autosomal dominant cmt , intermediate cmt (cmt-i) and dhmn. we report the case of a male of italian ancestry who first presented with bilateral ankle clonus at three months, followed by toe walking and ankle instability. the ankle clonus subsided during adolescence. in the third decade he developed progressive walking difficulties followed by distal sensory loss. neurological examination at the age of revealed a steppage gait, distal lower limb weakness, decreased pinprick to the ankles, and reduced vibration sensation at the knees. reflexes were brisk in the upper limbs, reduced at the knees and absent at the ankles. muscle tone was increased in the lower limbs and plantar responses were extensor. nerve conduction studies revealed an axonal neuropathy. brain and spinal cord mri were normal. sanger sequencing of pmp , gjb , mpz , gdap and mfn were negative. sureselect focused exome sequencing (agilent technologies, santa clara ca, usa) demonstrated a c. g>a, p.arg his mutation in aars. the p.arg his mutation in aars has previously been reported in families with intermediate or axonal motor-sensory neuropathy, and in one case was associated with sensory-neuronal deafness. cns involvement has not previously been described with this mutation. mutations in aars have been associated with a range of phenotypes including cmti, cmt and dhmn with variable age on onset ranging from to years (mean years). of note, the aars p.gly arg mutation has been reported in a family with cmt and pyramidal signs. this study provides further evidence that pyramidal tract involvement can be an early feature of cmt n due to mutations in aars, further expanding the spectrum of arss-associated phenotypes. small fibres in the skin are vulnerable to damage in metabolic or toxic conditions such as diabetes mellitus or chemotherapy resulting in small fibre neuropathy and associated neuropathic pain (np). whether injury to the most distal portion of sensory small fibres due to a primary dermatological disorder can cause np is still unclear. recessive dystrophic epidermolysis bullosa, (rdeb) is a rare condition in which mutations of proteins of the dermo-epidermal junction lead to cycles of blistering followed by regeneration of the skin. damage is exclusive to the skin and mucous membranes, with no known direct compromise of the nervous system. it is increasingly recognised that most rdeb patients experience daily pain, the aetiology of which is unclear but may include inflammation (in the wounds), musculoskeletal (due to atrophy and retraction scars limiting movement) or np. in this study we investigated the incidence of np and examined the presence of nerve dysfunction in rdeb patients. around three quarters of patients presented with pain of neuropathic characteristics which had a length dependent distribution. quantitative sensory testing of the foot revealed striking impairments in thermal detection thresholds combined with an increased mechanical pain sensitivity and wind up ratio (temporal summation of noxious mechanical stimuli). nerve conduction studies showed normal large fibre sensory and motor nerve conduction however skin biopsy showed a significant decrease in intraepidermal nerve fibre density. autonomic nervous system testing revealed no abnormalities in heart rate and blood pressure variability however the sympathetic skin response of the foot was impaired and sweat gland innervation was reduced. we conclude that chronic cutaneous injury can lead to injury and dysfunction of the most distal part of small sensory fibres in a length dependant distribution resulting in disabling np. these neuropathic pain (np) following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons which begins to normalise over time. we investigated the composition and distribution of shaker type potassium channels (kv channels) within the nodal complex of myelinated axons following injury. at the neuroma that forms after damage, expression of kv . and . (normally localised to the juxtaparanode) was markedly decreased. in contrast kv . and . , which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in the rat and in humans. within the dorsal root (a site remote from injury) we also noted a redistribution of kv channels towards the paranode. blockade of kv channels with dtx after injury reinstated hyperexcitability of a-fibre axons and enhanced mechanosensitivity. changes in the molecular composition and distribution of axonal kv channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury. members of the francophone anti-mag cohort group are listed in "appendix". appendix polyneuropathy with anti-mag igm antibodies is classically progressive, predominantly sensory, and distal with ataxia and sometimes postural-intention tremor. we assessed clinical, biological, electrophysiological, and histopathological features in patients with igm gammopathy and anti-mag antibody titres higher than btu. we focused on characteristics of patients according to the anti-mag antibody titres at diagnosis. we retrospectively and prospectively analysed standardized report forms of patients from fourteen french-speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset and last follow-up was . years ( . - . ). about . % of patients presented with a "variant" clinical phenotype independently of anti-mag titres (< or ≥ btu). this included acute or chronic sensorimotor polyradiculoneuropathies, paucisymptomatic sensory polyneuropathy and multifocal neuropathy. at the most severe disease stage, . % of patients were significantly disabled. anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in respectively , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. sixteen percent of patients did not meet the electrodiagnostic criteria of definite demyelinating neuropathy, independently of anti-mag titres (< or ≥ btu). nerve biopsy, performed in nineteen patients, provided support to the diagnosis of anti-mag neuropathy in some particular issues (low titres of anti-mag, unusual clinical or electrophysiological phenotype). we assessed the degree of probability (probable, possible or uncertain) that patient neuropathies are directly related to anti-mag antibodies, according to anti-mag titre, electrophysiological data and nerve biopsy characteristics if available. it appears uncertain in patients with low anti-mag titres ( . % of the whole population). the clinical phenotype didn't appear to be different according to anti-mag antibody titre. many of the patients with low anti-mag titres presented "genuine" anti-mag neuropathy as demonstrated by edx studies, clinical presentation and sometimes nerve biopsies. for a small proportion of these patients, a direct relation between neuropathy and anti-mag antibodies is uncertain due to atypical clinical presentation, axonal neuropathy pattern or nerve biopsies, and positivity of antigangliosides antibodies. we assessed therapeutic management, response to immunotherapies and adverse events in a cohort of patients presenting with igm gammopathy and anti-mag antibody titres higher than btu. we retrospectively and prospectively analysed standardized report forms of patients from french speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset to diagnosis and last follow-up were respectively . ( - ) and . years ( . - . ). anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. seventy eight percent (n = ) of patients received immunotherapies. transient response to ivig or plasma exchanges at six month was observed in less than and % of patients respectively. chemoimmunotherapies and rituximab were more frequently administered in the group of patients with malignant hemopathies (n = ) compared to mgus (n = ) (mean lines of therapy = . , range - , sd . versus . , range - , sd . , p = . ). more than % of patients (n = ) received rituximab monotherapy. clinical worsening, mostly transient and reversible, was observed in / patients after rituximab. clinical response to rituximab at months and/or during - months follow-up period was observed in . % of patients, and correlated with anti-mag titre ≥ btu ( / responder versus / non-responder patients, p = . ). at - months follow-up, responder patients presented shorter symptom duration compared to non-responders, though not significant after logistic regression ( . years, range . - . , sd . in responder patients versus . years, range . - . , sd . , in non responder patients, p = . ). in some cases, electrodiagnostic studies were recorded during rituximab treatment follow-up and showed in responder patients a clear improvement of motor conduction velocities. these data may support another clinical trial to study benefice of rituximab in anti-mag neuropathies early in the disease. it raises issues about the value of incorporating electrodiagnostic parameters as end-points. schwann cells (scs) are essential for axon integrity and myelination in the uninjured pns. after pns injury, scs function as "first responders" , undergoing phenotypic re-programming and orchestrating many processes that lead to functional nerve repair. receptors in scs that contribute to sc repair programs remain incompletely understood. we identified a member of the ionotropic glutamate receptor (igr) family, n-methyl-d-aspartate receptor (nmda-r), in scs that is upregulated after nerve injury and acts as a co-receptor with ldl-receptor related protein (lrp ). lrp is a well-known regulator of the sc response to pns injury. herein, we used pcr to profile igr expression in cultured rat scs. obligate receptor subunits required for assembly of nmda-rs, ampa-rs, and kainate receptors were identified. treatment of rat scs with - microm glutamate or . - . microm nmda robustly activated akt and erk / . the response was transient and bimodal; glutamate concentrations greater than microm failed to activate cell-signaling. phosphoprotein profiling demonstrated other cell-signaling and transcription factors regulated by glutamate in rat and human scs, including p s -kinase, glycogen synthase kinase- , ribosomal s kinase, c-jun, and creb. activation of cell-signaling by glutamate in scs was blocked by eliminating calcium from the medium, by the selective nmda-r antagonist, mk , and by genetic silencing expression of the obligate nmda-r nr subunit. phosphoinositide -kinase/pi k functioned as an essential upstream activator of both akt and erk / in glutamate-treated scs. by activating pi k and erk / , glutamate promoted sc migration. glutamate ( microm) or nmda ( microm) injected into crush-injured sciatic nerves robustly activated p-erk / in both myelinating and non-myelinating scs in vivo. these results identify igrs as potentially important cell-signaling receptors in scs that may promote axon-glial interactions. understanding the function of sc nmda-r is important given current efforts to develop nmda-r-targeting drugs for patients with pain, depression, and alzheimer's disease. while frequently overlooked in a therapeutic context, scs are extremely important in the pathogenesis of chronic neuropathic pain. if these drugs modulate the activity of sc nmda-r and sc physiology, the response to pns injury may be altered and the possibility that neuropathic pain develops increased. candayan a , atkinson d , durmus tekce h , parman y , jordanova a , battaloglu e . department of molecular biology and genetics, bogazici university, istanbul, turkey; center for molecular neurology, antwerp university, antwerp, belgium; istanbul medical school, istanbul university, istanbul, turkey. charcot-marie-tooth (cmt) disease is a group of inherited peripheral neuropathies affecting one in individuals worldwide. the disease presents both clinical and genetic heterogeneity. so far, mutations in genes and loci are associated with cmt with autosomal dominant, autosomal recessive, x-linked and mitochondrial inheritance. despite the advances in genetic testing, approximately % of all cmt patients worldwide remain without a molecular diagnosis. we have investigated unrelated cmt patients of turkish descent, in all of which pmp duplication has been excluded previously. we used multiplex amplification of specific targets for resequencing (mastr) assay to sequence exonic regions of common cmt genes. recurrent mutations were identified in cases in mfn , gjb , mpz and hint genes. we have also identified novel variants in cases in mfn , pmp , gars, aars, ighmbp and gdap genes, all of which are very rare or not present in the variation databases and are predicted to be pathogenic by in silico tools. familial segregation analyses are ongoing for novel variations. mfn and gjb genes were the most commonly mutated causative genes in this cohort. cases without molecular diagnosis after the mastr testing are candidates for further analyses such as whole exome sequencing or whole genome sequencing. outcomes of the current study and our previous experience with turkish cmt patients suggest a high genetic heterogeneity. our insight is that different genetic strategies or larger panels are essential to determine the causes underlying cmt especially in regions where rare recessive types of the disease can be observed due to high frequency of consanguineous marriages. capoccitti g , giannini f , ginanneschi f , casali s , insana l , rossi a . university of siena, siena, italy. several efforts have been made to elaborate new electrophysiological criteria for early diagnosis of guillain-barré syndrome (gbs) and to differentiate demyelinating (aidp) from axonal (aman/amsan) forms. the aims were to verify the diagnostic power of total cmap (tcmap) duration, firstly applied in gbs field. this parameter was compared with commonly used neurophysiological measures, including negative phase cmap duration (ncmap), and was added to modified rajabally criteria. we reviewed the clinical and electrophysiological data of patients with gbs (level or of brighton clinical criteria). each patient underwent at least two neurophysiological studies, the first within weeks, the second between and weeks from symptom onset. at least four motor and three sensory nerve conduction studies were recorded for each test. regarding early diagnosis, the binary logistic regression model with multiple variables, including ncmap duration, showed that the features of predictive model presenting greater significance (p < . ) were tcmap duration, sural sparing and a-waves. among these, tcmap duration showed greater significance (p = . ). the tcmap was diffusely prolonged in aidp compared to aman/amsan, already in first examination and confirmed in the second one. roc analysis for tcmap duration in aidp vs. aman/amsan showed: cut-off . ms, auc . , ppv . %. we propose the tcmap duration as a new useful electrophysiological measure for early diagnosis of "generic" gbs and for early differentiation between aidp and aman/amsan. moreover, the prolongation of tcmap, the presence of a-waves and sural sparing represent a strongly diagnostic predictive triad of aidp. aifm (apoptosis-inducing factor, mitochondrionassociated- ) has captured great attention from biomedical researchers due its critical role in the regulation of cell apoptosis. this flavoprotein is typically located in the mitochondrial intermembrane space where it is associated with respiratory chain complex-i. upon a cell-death insult, aifm is cleaved into a kd protein that is released into the cytosol. the kd peptide may enter the nucleus to trigger chromosome condensation and fragmentation, initiating a caspase-independent pathway of apoptosis. however, this nuclear translocation may be blocked by cytosolic heat-shock protein- (hsp ) that binds with the fad domain (aa - ) of aifm . mutations in aifm gene have resulted in several clinical phenotypes, including a family with cmtx (glu val). clinical deficits in these patients usually involve multiple organs. in this study however, we identified a family with a novel missense mutation (phe leu) in aifm that developed a late-onset sensory motor axonal polyneuropathy by nerve conduction criteria. the proband and affected siblings exhibited distal muscle weakness and atrophy with normal cognitive and cranial nerve functions. there was no obvious phenotype from other organs. interestingly, this phe leu mutation affects a highly conserved amino acid at the center of the fad domain. we hypothesize that this mutation impairs the binding between aifm and hsp , leading to an enhancement of cell-death signaling. this family therefore provides a unique opportunity to explore how altered apoptotic signaling affects peripheral nerve system. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr chronic inflammatory demyelinating polyneuropathy (cidp) can be the key symptom leading to diagnosis of associated lymphoma. patients with diagnosis of cidp according to efns/pns criteria associated with b cell lymphoproliferative disorders (bld), in one center, between november and november were included. demographical, clinical, nerve conduction, immunological, histological data and response to treatment were recorded retrospectively. eight patients ( men), median age yo were included. onset of polyradiculoneuropathy was either chronic (n= ) or acute (n= ). neurological condition led to diagnosis of bld in all but one case, because of onset (n= ) or worsening of neuropathy (n= ). clinical presentation was that of cidp in patients or pseudo-canomad in and plexopathy in one. lymphoma type was: lymphoplasmacytic (n= ), diffuse large b cell (n= ), small lymphocytic (n= ), marginal zone (n= ), unclassified small b cell (n= ). only patients presented with lymphadenopathies. bld was diagnosed in all patients on myelogram or bone marrow biopsy, performed because of cytopenia (n= ), atypical (n= ) or severe (n= ) neuropathy. monoclonal gammapathy was identified in / patients (igm n= , igg n= ). neuromuscular biopsy was performed in patients and disclosed endoneural infiltration in . anti-neuronal or antigangliosides antibodies were positive in patients. none of the patients presented a systemic autoimmune disease, hemolytic anemia associated with bld (n= ). immunomodulating treatment was administered in all patients (ivig n= , plasma exchange n= , steroids n= ) and immunosuppressants (n= ). immunochemotherapy for lymphoma was initiated because of lymphoma type or severity in cases, in cases because of the associated neuropathy. median follow-up was of months after treatment initiation. four out of patients treated within months of neurological onset improved as well as one out of patients whose preexisting neurological condition had worsened. two patients presented neurological relapse during progression of lymphoma. two patients died. unusual presentation of cidp -i.e., rapid progression or treatment failure -should lead to further testing for associated lymphoma. because general symptoms and lymphadenopathy often lack, diagnosis requires analysis of bone marrow with lymphocyte phenotyping. early treatment with immunochemotherapy was associated with better prognosis in our series. cardiac scintigraphy is a useful tool for the diagnosis, prognosis and pre-symptomatic early detection of familial amyloidosis-associated neuropathies three main types of familiar amyloidosis: transthyretin (ttr), apolipoprotein a and gelsolin. cardiac involvement is a leading cause of morbidity and mortality; one new described mutation strongly related with isolated cardiac amyloidosis is the ttr val ile. the discovery of tests that allow early diagnosis of cardiac involvement in amyloidosis and to infer about the etiology of the disease is of major importance. in a cohort of patients with different types of familiar ttr amyloidosis, we aimed to assess the role of mtc- , -diphosphono- , -propanodicarboxylic acid ( mtc-dpd) in detecting myocardial amyloid infiltration. we enrolled four patients diagnosed with late familiar amyloidosis, which mutations were documented at deoxyribonucleic acid analysis: three patients with ttr val met mutation and one patient with ttr val ile mutation. three patients were asymptomatic for cardiac involvement and one patient (val ile mutation) had a previous diagnosis of heart failure. myocardial uptake of mtc-dpd scintigraphy was semiquantitatively and visually assessed at five minutes and three hours.the uptake of mtc-dpd highly demonstrated amyloid in cardiac area in two out the three cases of ttr val met and in ttr val ile. ttr val ile case presented the highest uptake due to the exclusive deposition of amyloid in cardiac area resulting in severe heart failure. in hereditary transthyretin-related amyloidosis, including the mutations ttr val met and val ile, mtc-dpd cardiac scintigraphy can identify infiltration even in asymptomatic individuals, allowing an early diagnosis of cardiac compromise in this group of diseases. we can consider that this non-invasive test would be a tool with potential importance in the diagnosis, prognosis and pre-symptomatic early detection of cardiac amyloidosis, giving emphasis on its applicability in familial forms of amyloidosis. cervellini i , galino j , zhu n , birchmeier c , bennett dl . ndcn university of oxford, oxford, uk; max-delbrück-center for molecular medicine, berlin, germany. erk/mapk pathway has a critical role in pns development since its involvement in many physiological processes. sustained erk / mapk activation in schwann cells enhances myelin growth during development and overcomes signals ending myelination leading to a continuous myelin production. however, strong activation of erk has also been shown to cause schwann cells dedifferentiation and demyelination in vivo. our aim was to investigate whether a mild activation of this signalling pathway in adult schwann cells (scs), by expression of gain of function mek dd allele, could have a beneficial role in remyelination and regeneration after injury. erk/mapk activation in adult scs in plpcreer t ;mek dd mutant mice, did not affect myelination during development. following sciatic nerve injury, wallerian degeneration was enhanced in mutants pushing towards a dedifferentiation stage of scs as previously described. however, mapk activation was detrimental during regeneration with a delay in functional recovery and a negative impact in both myelinated and non-myelinated fibres compared to controls. one month after injury the total number of axons in mutant sciatic nerves was half of the controls. although no differences in g-ratio have been found in the two groups, mutants presented a higher number of myelinated axons showing myelin disruption with start of myelin decompaction, lack of cajal bands, abundant sc processes surrounding axons and a shorter sc elongation, as seen by decreased internodal distance. in addition, we found a negative effect of mapk activation also in small diameter axons with the presence of abnormal remak bundle structures, reduced number of c-fibres/remak bundle and a significant decrease in intra epidermal nerve fibres density in the skin. we concluded that mild mapk activation has a different role in development and remyelination where negatively affects axon survival, myelin stability, remak bundle formation and small fibres regeneration. cervellini i , galino j , zhu n , bao lu , bennett dl . ndcn, university of oxford, oxford, uk; harvard medical school, boston, ma, usa. neprilysin (nep) is an endopeptidase which has been of interest due to its potential role in neurodegeneration and pain as a consequence of its ability to degrade amyloid and substance-p respectively. nep expression is not limited to cns and it has been reported to be expressed in schwann cells, nodes and schmidt-lanterman incisures. our interest in this gene was related to recent findings that have associated homozygous and heterozygous nep mutations with charcot-marie-tooth type- . in old mice lacking nep subtle morphological changes have been reported. our aim was to determine whether nep expression was modulated by nerve injury and to investigate its role in axon regeneration and re-myelination. we find that nep gene expression was decreased after nerve crush and furthermore was dependent on the growth factor (and pro-myelin signal) neuregulin- . in control mice nep expression was transiently reduced and returned to baseline at day after injury, in neuregulin- knock-out (ko) mice, in which re-myelination was impaired, the expression was still decreased at day . in assessing behavioural measures of locomotor and sensory function one month after sciatic nerve crush, nep ko mice showed a functional regeneration comparable to wt, as seen by sciatic functional index measurement, beam and toe spreading tests. the only significant difference we observed between wt and ko was in the sensorial test, showing ko mice recovering faster in the pinch test by days after crush. the results for all the tests at baseline did not differ between the two groups. detailed histological analysis of nerve repair was undertaken using electron microscopy. there was no difference between wt and ko in total axon number, g-ratio, axon diameters and other myelin features one month post crush. in summary, although nep expression is regulated by nerve injury in a neuregulin- dependent fashion this endopeptidase is dispensable for axon regeneration and re-myelination after nerve injury in the rodent. diabetes neuropathy is a common complication of diabetes, and neuropathic pain has a detrimental impact on quality of life. this study investigated sensory nerve excitability properties to elucidate the axonal changes of diabetic neuropathy. a total of diabetes patients ( type ii, and type i) were enrolled in this study. clinical assessment, nerve conduction studies, and nerve excitability testing data were analyzed to determine axonal dysfunction in diabetic neuropathy. among those patients, seventeen subjects had complained of spontaneous painful sensation over feet or hands (painful cohort), and seventy-eight patients had no sensory symptoms or decreased the sensation over foot (non-painful cohort). sensory nerve excitability of the painful cohort showed reduced late subexcitability (p= . ), increased superexcitability (p= . ) in compared to the non-painful cohort. there is no difference in disease duration, blood glucose levels (hba c) between these two cohorts. these findings suggested the possible pathogenesis of painful sensory axons might be hyperpolarized or slow potassium channels dysfunction. these insights our further understanding of painful diabetic neuropathy, and may provide a basis for neuroprotective or therapeutic approaches for painful polyneuropathy. the main purpose of this study was to assess the clinical feasibility of diffusion tensor imaging (dti) for the diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp). between march and december , we prospectively enrolled patients with definite cidp according to the efns criteria and two control groups: healthy volunteers matched on age and sex and patients with cmt- a. using a -t magnetic resonance imaging scanner, we obtained dti scans of brachial plexus of these groups and prepared fractional anisotropy (fa) maps, and compared these values between groups. adc values and cervical nerve roots diameters on stir sequences were evaluated too. two neuroradiologists, blinded to clinical informations, reviewed mri studies independently. in all patients with cidp, we also performed clinical evaluation and electroneuromyography. significantly decreased fa values (p< . ) and increased adc values were observed in cidp patients compared with healthy subjects. there is no significant difference between cidp and cmt group. inter-observer concordance was excellent for fa values ( c= . ; p< . ) and moderate for adc values ( c= . ; p< . ) and cervical nerve root diameters ( c= . ; p< . ). there is a significant correlation between fa and disease duration (r = − . , p < . ), inclusion mrc score (r = . , p < . ) and between fa mesured on c c and incat score at inclusion (r = − . , p < . ). no significant correlation is observed between fa and electrophysiological indices. compared with healthy subjects, cervical nerve root diameters were significantly increased (p< . ) in patients with cmt and cidp. contrary to fa values, moderate level of concordance was found between inter-observers measurements of diameters (cclin = . ). our preliminary data prove the clinical feasibility and reproductibility of dti for the evaluation of plexus and cervical nerve roots in patients with cidp. cheng yj , teng a , goh ejh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. the sural-sparing pattern of the sensory nerve action potentials (snap) of guillain-barré syndrome (gbs) has been attributed to greater immunological injury of the blood-nerve barrier at its most vulnerable regions. we asked if entrapment sites, such as median nerve at the wrist, are more predisposed than the distal nerve endings to such injury. we compared the median snap with radial snap measured antidromically at digit in gbs patients whose nerve conduction study showed the sural-sparing pattern. the terminal nerves at digit are of similar length, but those of median nerve are prone to compression, often subclinically, at the carpal tunnel while those of radial nerve are not. we defined the sural-sparing pattern as a greater decrease in median and or ulnar snap than that of the sural, compared to age and height-matched normal controls. a total of gbs and miller fisher patients from our institution's database were studied. patients had the sural-sparing pattern, of whom had pre-existing carpal tunnel syndrome. of the remaining patients with sural-sparing, had abnormal median snap at digit , while had both abnormal median and radial snaps at digit . none had isolated abnormality of the radial digit snap. among the cases that had abnormal median and radial snaps at digit , the mean percentage decrease when compared to age and height matched norms was greater in median nerve compared to radial nerve ( % and % respectively). of the patients without sural-sparing pattern, had normal snaps; patient had inexcitable sensory nerves while the other had a length-dependent decrease in snap. in the latter patient, unlike those with sural-sparing, there was no differential decrease of median snap over radial snap at digit . our findings suggest that the disruption of blood nerve barrier at entrapment sites rather than the distal nerve endings may underlie the pathophysiology of the sural-sparing pattern seen in gbs. chiba a , uchibori a , gyohda a . kyorin university, tokyo, japan. serum igg anti-gq b antibody is the most specific biomarker for fisher syndrome and its related disorders (fs-rd), but approximately - % of the patients are seronegative for it in conventional assays (gq b-seronegative). some molecules need ca + cation to interact with their ligands, and antibodies with such a property (ca + -dependent antibodies) are reported. we have found that such a ca + -dependency is also present in igg anti-gq b antibody, and majority of gq b-seronegative patients with fs-rd have this type of antibodies. in patients with final clinical diagnoses as fs-rd (fisher syndrome, guillain-barré syndrome with ophthalmoplegia, bickerstaff brainstem encephalitis, and acute ophthalmoplegia), were seropositive for igg antibodies against gq b-related antigens (isolated gq b in , and gq b-conatinig complexes in two) in conventional elisa using phosphate-buffered saline. in the remaining patients, eight ( %) turned positive for igg antibody against gq b-related antigens (isolated gq b in seven and gq b-conatinig complexes in one) in elisa using ca + -added tris-buffered saline. the reaction strengths increased depending on ca + concentration, and reached to nearly maximum level in the physiological concentration. all the patients with the ca + -dependent antibodies were also positive for igg antibody against gt a-related antigens, suggesting that the terminal disialo residue common to both the gangliosides would be important as an epitope also for the ca + -dependent antibodies. in the patients with ca + -non-dependent antibodies, only two showed increased titers of igg anti-gq b antibody by adding ca + , and showed significantly decreased titers. this difference in the effect of ca + -addition between ca + -dependent and ca + -non-dependent antibodies suggests that ca + would not be just an enhancer of the antigen-antibody reaction. there are four single bonds between the two pyranose rings in the terminal disialo, and those rotatable bonds make it possible for the disialo structure to take various conformations. a molecular model shows that the distance between two minus-charged carboxy groups in the disialo could vary from nearly zero to approximately , pm and that the disialo would take specific conformations, if divalent ca + cation, which size is approximately pm in diameter, interacts with these two minus-charged groups. the ca + -dependent antibodies might recognize such particular conformations of gq b. charcot-marie-tooth disease (cmt) is a genetically and clinically heterogeneous disorder with variable inheritance modes. it is characterized by loss of muscle tissue and touch sensation, predominantly in the feet and legs but also in the hands and arms in the advanced stages of disease. as several molecules have been reported to have therapeutic effects on cmt, depending on the underlying genetic causes, exact genetic diagnostics have become important for executing personalized therapy. aminoacyl-trna synthetase (arss) genes encode enzymes responsible for charging trna with corresponding amino acids. arss are ubiquitously expressed, essential enzymes responsible for performing the first step of protein synthesis. specifically, arss attach amino acids to their cognate trna molecules in the cytoplasm and mitochondria. recent studies have demonstrated that mutations in genes encoding arss can result in neurodegeneration, raising many questions about the role of these enzymes in neuronal function. mutations in six cytoplasmic ars genes have been reported as the cause of cmt. this study was performed the whole exome sequencing to identify genetic defects in korean cmt patients from unrelated families. variants were sorted with cmt gene list that includes almost genes were related cmt neuropathy, and additionally sorted wes data as ars genes. capillary sequencing for family members and more than controls revealed five novel mutations, c. g>a (p.d n), c. c>t (p.s f), and c. c>a (p.p h) in gars; c. c>a (p.p t) in mars; _ ga>at (p.d i) in yars gene in each family. the mutation sites were well conserved between different species and each mutation were located in the well-conserved catalytic domain or between two catalytic domains or anticodon-binding domain. in silico analysis predicted all mutations may affect protein function. clinical features were similar to those reported in other countries, but differed in terms of age at onset and degree of disability. we believe that those novel ars mutations are the underlying causes of the each family. a -year-old man presented with a -year-history of weakness in biltearal upper limbs. he was complaining of intermittent fasciculation of upper and lower limbs with gradually worsening of paresthesia for years. dysphagia and dysarthria were also presented years ago. there was no patient affected muscle weakness and bulbar symptoms in his family members. in neurological examination, the patient had weakness in biltereal upper and lower limbs (mrc grade ) and prominent distal sensory loss were combined in length dependant pattern. deep tendon reflexes were absent on bilataral biceps and knee joints. in nerve conduction study, there was consistent with demyelinating sensorimotor polyneuropathy. molecular diagnostic analyses those spinobulbar muscular atrophy (sbma) and mutation related to peripheral myelin protein (pmp ) gene were performed and confirmed expansion of expansion of a polymorphic cag in androgen-receptor (ar) gene and deletion of pmp gene. smba, also known as kennedy disease, is an adult-onset, x-linked recessive trinucleotide, polyglutamine (poly-g) disorder caused by expansion of a polymorphic cag tandem-repeat in exon of ar gene on chromosome xq - . charcot-marie-tooth disease (cmt) is the most common hereditary neuropathies and cmt cases with motor conduction velocities(mcvs) of upper limb below m/s are defined as demyelinating (cmt ) and those with mcvs above m/s are defined as axonal (cmt ). most families with cmt linked to duplication of pmp gene on the short arm of chromosome ( p . ), called cmt a. the reciprocal deletion of pmp gene is a responsible genetic defect in % of hereditary neuropathy with liability to pressure palsy (hnpp). these "classical" phenotypes of cmt a and hnpp have been considered which are determined by different mutation mechanism of the same gene. however, an overlap of cmt a and hnpp due to pmp gene deletion was reported that suggestion the phenotype of hereditary neuropathies may differ variably. herein, we report a patient who simultaneously presented clinical and electrophysiologic features of smba and cmt a with genetical confirmation of cag expasion and deletion of pmp gene. charcot-marie-tooth type a disease (cmt a) is an inherited peripheral neuropathy stemming from overexpression of pmp protein in schwann cells due to the duplication of the pmp gene. this leads to abnormal schwann cell differentiation and dysmyelination, eventually leading to axonal loss and muscle wasting. no approved treatment is currently available for cmt a. we conducted a systems biology level analysis of the signaling network putatively underlying the processes driving cmt a pathology. based upon this, we identified and tested three repurposed drugs -baclofen, naltrexone and sorbitol -alone and in combination to determine their ability to rescue aberrant myelination in cultures derived from cmt a transgenic rats overexpressing pmp gene. to this end, we studied a validated in vitro co-culture model of sensory neurons and schwann cells adapted to -well culture plates. this model allows measurement of the appearance of myelin proteins as an index of the physiological process of in vivo myelination. total myelin length was quantified with an automatic image analyzer following pmp immunostaining. we first determined the full dose-response curves of single drugs, emphasizing their promyelinating activities. we then tested binary combinations of very low and inactive doses of each drug and compared these to the activity of the combination of the three, namely pxt . whereas combination of any two drugs was not significantly active at the doses tested, combination of all three produced a synergistic improvement in myelination. these findings clearly demonstrate the necessity of using pxt over its single components and highlight the value of pleiotropic combinational repurposing of drugs at low doses as a novel approach for rapid drug development in cmt a and other disorders. autonomic dysfunction is frequently observed in guillain-barré syndrome (gbs) and affects approximately % of the patients. it has been shown that the sweating function can be impaired in gbs. the aim of the present investigation is to summarize the current knowledge on sweating disturbances in gbs patients. we have used appropriate terms to systematically search for references published until and indexed in the following databases: medline, embase, lilacs and cochrane. the inclusion criteria were a diagnosis of gbs and a description of the methods used to test the sudomotor function. the search was limited to the english language. relevant information about study design, methods of assessing the deficit of sweating, patient's characteristics and main results were collected. we selected original references for the final analyses. the majority of the studies were cross-sectional in nature and there were two longitudinal studies. the severity of sweating impairment varied according to the applied method, ranging from normal to almost sympathetic nervous system failure. in seven research papers, the sympathetic skin response was used to evaluate the sudomotor function in patients, and approximately % demonstrated abnormal results. however, researchers used different stimulation protocols and parameters to interpret their results. regarding whole-body sweating test, four research papers applied the thermoregulatory sweating test in patients and they showed areas of anhidrosis on the lower limbs in all of them. eight patients presented sweating impairment on the upper limbs and abdominal wall. results on the sudomotor axon reflex test suggested a length-dependent pattern of sweating loss according to one case report. in another study, eight gbs patients were tested only on the distal leg and foot dorsum and the authors proposed an association between post-ganglionic sudomotor function and antiganglioside antibodies. the present literature review showed that the studies of sweating disturbances in gbs patients included only small cohorts. future studies with larger patients sample sizes are necessary to investigate the patterns of sweating loss in gbs and their changes along the follow up. funding: grants # / - and # / - , são paulo research foundation (fapesp). sensory neuronopathy (sn) represents a rare subgroup of peripheral neuropathies characterized by degeneration of primary sensory neurons at the dorsal root ganglia on the spinal cord. depending on the neuronal population affected, its clinical presentation may manifest as gait ataxia, proprioceptive sensory loss and positive and negative sensory symptoms. although a few reports have mentioned areas of anhidrosis in sn, we were not been able to find previous case series studies on the sweating function in sensory neuronopathies. the aim of the present investigation was to study the whole-body distribution of sweating on both anterior and posterior surfaces in patients diagnosed with sn. quantitative sensory testing for cold and warm sensation threshold (method of levels) was performed on the dorsum of the hands and feet in a randomized order. we tested the thermoregulatory sweating using a sweat chamber ( to ∘ c air temperature and % relative humidity). the oral and skin temperature was monitored and the test time did not exceed min. in order to study the sudomotor axon reflex we employed the q-sweat device on standardized body sites. the test was performed on both sides, simultaneously. we included seven patients (three male; mean age . years) with a mean disease duration of . months (range - ) and a confirmed diagnose of sn. patients presented an asymmetrical loss of cold and warm threshold on hands or feet compared with healthy control (p< , ). regarding the tst results, we found a striking variation of sweating disturbances, ranging from small areas of anhidrosis on the trunk to complete failure of the sympathetic nervous system. two patients underwent the axon reflex test and there was an asymmetrical and mostly distal pattern of sweating loss in one of them and a distal-symmetrical on the second one. our findings indicate a great variability of sweating losses in sn, not overlapped to the sensory loss areas. currently, we are testing more patients in order to confirm our results. funding: grants # / - , # / - and # / - , são paulo research foundation (fapesp). diet, exercise, and inflammation are established modulators of peripheral nervous system function, including pain. prior work examining exercise consistently demonstrates a benefit on heightened pain from a number of acute and chronic pain models. in the present work, we investigated several parameters of peripheral nerve function relevant to pain in rats bred for high (high capacity runners, hcr) or low running capacity (low capacity runners, lcr). the longtime selective breeding of these rat substrains has created divergent intrinsic aerobic capacities and predisposition of metabolic conditions between lcr and hcr rats. examination of the role of sex in the development of chronic pain has established key differences in males and females. to understand gender specific differences, this study focused on female rats to understand the role of metabolic status and peripheral nerve function in females. our analysis identified numerous parameters of peripheral nerve function relevant to pain and neuropathy that are different among lcr and hcr female rats. lcr female rats display reduced hind paw mechanical sensitivity, increased hind paw intraepidermal nerve fiber density and trka-positive epidermal axons, increased numbers of langerhans and mast cells in the hind paw dermis, and increased overall fat mass relative to body weight compared to female hcr rats. examination of sensory and motor nerve conduction velocities, thermal sensitivity, and mrna expression of selected genes relevant to peripheral sensation found no differences between hcr and lcr females. together these results suggest that a genetic component of aerobic capacity and metabolic status can influence sensory sensitivity and specific aspects of inflammation and immune responses in peripheral tissues, which may lead modify the animal's responses to tissue damage and painful stimuli. the lcr and hcr rat model will provide a useful model in the future to assess the involvement of metabolic status in the development of pain. ivig is often considered treatment of first choice in chronic inflammatory demyelinating polyradiculoneuropathy (cidp) because of its rapid onset of action and its relatively safe long-term adverse event profile. clinical trials published so far focused on a loading dose of . g/kg ivig and/or a standard maintenance dosage of . g/kg ivig once every weeks, but have not investigated different dosing options. this study is a prospective, double-blind, randomized, parallel group, multi-center phase iii efficacy study and will be conducted in centres in canada, eu, russia, ukraine and australia. adult patients with definite or probable cidp according to the efns/pns criteria will be enrolled and randomized : : to receive either . g/kg or . g/kg or . g/kg ivig (panzyga ® ) for seven maintenance infusions at -week intervals during the dose-evaluation phase. the starting loading dose will be . g/kg ivig (panzyga ® ) for all patients. primary objective: efficacy measured as percentage of responders (decrease in adjusted incat score by at least point) in the . g/kg ivig (panzyga ® ) arm (given every weeks) at week as this should corroborate the existing and published evidence on efficacy of ivig in cidp. secondary outcome: percentage of responders at week in the . g/kg and . g/kg ivig (panzyga ® ) arms relative to baseline and compared to the . g/kg arm. the procid study aims to confirm published clinical results obtained with the . g/kg standard dose and will in addition evaluate one higher and one lower maintenance dose, with the aim to offer cidp patients a more adequately dosed and effective treatment policy. * este medicamento no se encuentra comercializado en españa. understanding the rate of disease progression in patients with charcot-marie-tooth disease (cmt), both within and between subtypes is important for clinical prognosis and is crucial for clinical trial design. due to the progressive nature of cmt, intervening at the earliest stages of the disease is a priority. measuring progression of a disease with both motor and sensory deficits requires a multi-item composite scale. the cmt pediatric scale (cmtpeds) is a well-tolerated psychometrically robust -item scale measuring fine and gross motor function, strength, sensation and balance for children and adolescents aged - years with cmt. the aim of this study was to determine the rate of disease progression of children and adolescents within and between genetic subtypes of cmt. ( female) participants aged - years enrolled in the inherited neuropathies consortium were included in this study. demographic, anthropometric and diagnostic information were collected at baseline and -year follow-up. disease progression was measured with the cmtpeds. on average cmtpeds scores progressed at a rate of . points over -years ( % change from baseline, p< . ). there was no difference in rate of disease progression between males and females. of the most common genetic subtypes, participants with cmt a/pmp duplication progressed by . points ( % change from baseline, p< . ), nine participants with cmt b/mpz mutation progressed by . points ( % change), six participants with cmt a/mfn mutation progressed by . points ( % change) and seven participants with cmt c/sh tc mutation progressed by . points ( % change). participants with cmt a progressed faster than those with cmt a (p= . ). children with cmt a progressed consistently during childhood and adolescence while children with cmt b and cmt a progressed faster during childhood than adolescence. overall, children with cmt progress at a significant rate over -years according to the cmtpeds. understanding the rate at which affected children deteriorate is essential for adequately powering clinical trials of disease-modifying interventions. queen square, london, uk; department of neurology, university of iowa carver college of medicine, iowa city, ia, usa; department of neurogenetics, the national hospital for neurology and neurosurgery, ucl institute of neurology, london, uk; department of molecular neuroscience, ucl institute of neurology, london, uk and national hospital for neurology and neurosurgery, queen square, london, uk. in recent years, targeted ngs panels have changed the diagnostic work-up in patients with inherited neuropathies. however, there is limited data on the impact of targeted ngs panels on the diagnosis of cmt patients in everyday practice. the aim of this study was to investigate the impact of targeted ngs panels on the diagnosis of cmt across two tertiary referral centres in the united kingdom (london) and united states (iowa). in london, patients with a diagnosis of cmt (previous pmp duplication and common cmt genes excluded in appropriate cases) underwent targeted ngs panel sequencing covering genes associated with cmt and additional genes associated with hsp or als. a variable number of genes, ranging from to , were analysed depending on the clinical phenotype of the patients. a definite molecular diagnosis was achieved in cases ( %) including pathogenic and likely pathogenic mutations in sh tc ( cases), gjb ( cases, including cases with mutations in the promoter and 'utr regions), gdap ( cases), fgd ( cases), aars ( cases), ighmbp ( cases), mpz ( cases), nefl ( cases). vus were further identified in patients. the diagnostic rate was higher in demyelinating cmt cases ( / , %), compared to cases with axonal cmt ( / %), dhmn ( / , %) and hsn ( / , %). in iowa, patients were investigated by ngs panels covering to genes associated with cmt. a molecular diagnosis was reached in / ( %), and in particular / ( %) demyelinating and / ( %) axonal cmt cases. the most frequent genes identified were gjb ( cases), mfn ( cases), sh tc ( cases) and ighmbp ( cases). vus were identified in patients, including cases with novel variants in aars, warranting additional testing such as segregation of the variant in the family or functional validation studies. in clinical practise, targeted ngs panels represent an effective approach for the diagnosis of cmt. the lower diagnostic rate in london is likely to be due to prior sanger sequencing and exclusion of mutations in common cmt genes in this patient population. more than genes are known to cause cmt and an even larger number are known to cause peripheral neuropathy as part of a more complex neurological disorder. despite the use of custom panels, a significant proportion of patients with inherited neuropathy have no molecular diagnosis. the aim of this study was to investigate the diagnostic yield of a disease-associated gene exome (sureselect focused exome, agilent technologies, santa clara ca, usa) in the diagnosis of cmt and in cases with complex neurological syndromes associated with neuropathy. thirty-one patients with molecularly undiagnosed inherited neuropathy were analysed with sureselect focused exome sequencing. six patients had a more complex phenotype including learning difficulties, cerebral white matter changes, ataxia and pyramidal tract involvement. a genetic diagnosis was achieved in / ( %) of cases by detecting a mutation in cmt-associated genes mpz ( cases), aars, nefl, bscl , bicd and trpv . of note, six cases had mutations in genes which are not covered by currently available diagnostic targeted ngs panels, including kif a, polg, mme ( cases), dnajb , and a novel candidate gene. the average coverage was higher compared to the usual coverage of whole-exome sequencing; % of the targets were covered at x or more, and % of the targets were covered at x or more. this study provides evidence that the sureselect focused exome is a useful tool for the diagnosis of cmt and complex neurological disorders and provides further insight into the phenotypic spectrum of genes associated with inherited neuropathy. changes in the cis-regulatory sequences of a gene's untranslated regions (utr) are increasingly recognised as a significant cause of inherited disease in humans. for example, variants in the non-coding region of gjb account for % of all mutations in our cohort of cmtx patients. one of the biggest challenges in analysing the large number of non-coding variants in a gene is identifying those that are disease-causing and those which are polymorphisms. the aim of this study was to implement a reliable method for the in-vitro functional validation of non-coding variants in the promoter and 'utr regions of gjb . in our cohort of cmtx patients we have previously identified seven mutations (c.- _ inst, c.- g>a, c.- t>g, c.- t>c, c. t>c, c.- g>c, c.- c>t) in the promoter region and one novel mutation in the 'utr (c. c>t), which were considered likely to be pathogenic based on the clinical phenotype, segregation in affected family members and absence in control databases. we have now generated a luciferase-based reporter system and optimised it in a hela cell line. mutations in the promoter region were generated by site-directed mutagenesis using a commercially available gjb promoter clone (genecopoeia). our preliminary results show a reduction of luciferase activity for the c.- t>c and c.- _ inst mutations compared to the wild-type promoter. this difference was increased when transcription factors sox or egr were co-transfected with the t>c and c.- _ inst mutations respectively. validation of other variants is currently ongoing. if successful, our study will provide a useful tool for the validation of mutations in non-coding regions of gjb . moreover, it will constitute a proof-of-principle approach to the functional validation of non-coding variants in other cmt genes known to cause disease by a loss of function. recessive and dominant mutations in leucin-rich repeat and sterile alpha-motif-containing (lrsam ) have been associated with cmt p. lrsam is a ubiquitin e ligase containing a ring domain in its c-terminal, which is crucial for correct protein folding and ubiquitination activity. to date, the majority of dominant mutations reported have resulted in a frame shift disrupting a major portion of the ring-domain, although point mutations in this domain have also been described. the aim of this study was to report the prevalence, clinical features and genetic findings of patients with cmt p in our centre. we performed targeted next-generation sequencing in genetically undiagnosed cmt patients and identified cases with heterozygous mutations in lrsam ( . %) from unrelated families. the mutations identified included frameshift insertions and deletions, a non-frameshift deletion and non-sense and missense point mutations. all of the mutations were novel and were located in or flanking the ring domain. the average age of disease onset was in the rd decade but an earlier onset was reported in two cases. four had a positive family history in keeping with autosomal dominant inheritance. symptoms at presentation were heterogeneous and encompassed distal numbness, unsteadiness, distal weakness of upper or lower limbs and foot deformities. positive sensory symptoms, including tingling and shooting pains, and cramps were also frequently reported. neurological examination showed mild to moderate distal atrophy and weakness, with early ankle plantar flexion involvement in three patients. loss of vibration and reduced joint position sense were often prominent in the lower limbs and appeared to be disproportionate to the degree of weakness and impairment of pinprick sensation. ankle jerks were absent but knee and upper limb reflexes could be normal or brisk. after an average disease duration of years, all but one patient was able to walk independently. nerve conduction studies showed a sensory and motor axonal neuropathy with normal conduction velocities. our study highlights that mutations in lrsam are a relevant cause of cmt and are associated with prominent large fibre sensory loss. in recent years, the implementation of ngs panels for the molecular diagnosis of cmt has increased the number of patients with a genetic diagnosis. nevertheless the interpretation of a particular variant as disease causing can be challenging especially when multiple variants are identified in a single patient. we report two illustrative cases of such challenges. the first index case was born of non-consanguineous healthy parents. he presented with falls in early childhood. over the years he developed foot deformities and progressive length dependent weakness. he had multiple orthopaedic operations to his feet. the past history was also notable for kyphoscoliosis, sensorineural deafness from the age of and bilateral cataracts. nerve conduction studies at the age of revealed a demyelinating neuropathy consistent with the clinical phenotype of cmt . his younger brother had a similar, although more severe phenotype. a litaf (c. c>t,p.pro ser) mutation was identified by sanger sequencing and was present in both affected brothers but also in the unaffected sister. ngs for cmt -associated genes was therefore performed and identified two compound heterozygous pathogenic mutations detected in sh tc (c. c>t, p.arg *; delg, p.arg serfs* ), which segregated with the disease in the family. the second case describes a brother and sister with early onset demyelinating cmt associated with scoliosis and cranial nerve involvement. the male proband underwent ngs and a single previously reported pathogenic intronic splice-site mutation in sh tc (c. - a>c) was found. relative read-depth analysis of ngs was performed to look for possible copy number variants in sh tc , thus identifying a deletion of exon , which was confirmed by long pcr. cruz-velásquez gj , miramar-gallart md , alarcia-alejos r , roche-bueno jc , rodríguez-valle a , capablo-liesa jl . neurology service, university hospital miguel servet, zaragoza, spain; genetics unit, clinical biochemistry service, university hospital miguel servet, zaragoza, spain. charcot-marie-tooth disease (cmtd) defines a clinical and genetically heterogeneous group of inherited peripheral neuropathies characterized by chronic motor and sensory impairment. the type cmtd- also known as cowchock syndrome, is the product of the mutation in the apoptosis inducing factor mitochondria associated gene (aifm ). it is a slowly progressive, recessive, x-linked disease characterized by axonal neuropathy, deafness, and cognitive impairment. our purpose is to describe new cases, brothers, children of non-consanguineous parents, with a characteristic phenotype and a new mutation in the aifm gene. both siblings present from childhood, progressive weakness in lower limbs with diffuse amyotrophies. needing tenotomy before the year due to equinovarus foot. likewise they develops sensory deafness and one of them requires unilateral support at and the other wheelchair at , this one need a pacemaker for an atrioventricular block at . with brain functions and normal language, sensorial deafness, proximal and distal weakness in the four limbs with intense amyotrophies, predominantly distal. tactile and painful sensitivity decreased in glove and sock pattern. an extensive metabolic and biochemical study was normal. the electroneurography demonstrates an axonal neuropathy without response in most of the nerves explored. the electromyography shown a myogenic pattern with distal predominance. brain mri was normal in both cases. through a genetic study by exoma targeting genes associated with cmt and inherited related neuropathies was identified the homicigosis mutation in the aifm gene (p.glu lys), located in the chromosomal region xq . (cmtx ). cowchock syndrome is a rare entity, with few cases described in the literature. the in silico analysis indicates in of the predictors used (provean, sift, polyphen , lrt, mutationtaster, mutationassessor and condel) , that it is a deleterious variant. we audited all patients with peripheral neuropathy caused by a paraprotein, who received treatment and attended king's college hospital peripheral nerve service between - . patients were identified retrospectively from our database of patients attending the peripheral nerve outpatient clinic. clinical information was obtained retrospectively from hospital electronic patient records. we excluded patients with poems syndrome or in whom the neuropathy was not felt to be caused by the paraprotein. we identified patients with a diagnosis of paraproteinaemic neuropathy. we excluded four who did not fulfill the diagnostic criteria and eleven who had received no treatment or were under diagnostic study. we included patients in the final audit. ( %) had igm paraprotein. the haematological diagnosis was monoclonal gammopathy of undetermined significance (mgus) in %, waldenstroḿs macroglobulinaemia %, and lymphoma or plasmacytoma %. after treatment, overall ( %) patients improved neurologically, ( %) stabilised, and ( %) worsened. in the patients who received more than one type of treatment, we analysed outcomes according to the most powerful treatment received. patients received rituximab alone of which ( %) improved, ( %) stabilised and ( %) worsened. nine patients received rituximab combined with cyclophosphamide or bendamustine, of which ( %) improved, ( %) stabilized and ( %) worsened. eight patients received intravenous immunoglobulin, of which ( %) improved, ( %) stabilized and ( %) worsened. four patients received other chemotherapy, of which improved, stabilised, worsened. two patients received corticosteroids and both worsened. there was improvement in / ( %) with mag antibodies and / ( %) without. there was improvement in / ( %) with mgus and / ( %) with haematological malignancy. there was improvement in / ( %) with kappa light chains and / ( %) with lambda. factors associated with better outcome (by univariate analysis) were negative mag antibodies, kappa light chain, and haematological malignancy. there was no significant difference between treatments in the proportions who improved. cumberbatch m , cox a . addenbrooke's hospital, cambridge, uk. treatment of patients with autoimmune neuropathies such as chronic inflammatory demyelinating polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) has centred on the use of intravenous immunoglobulin (ivig). however, ivig therapy is associated with systemic side-effects, treatment wear-off effects and regular hospital attendance. subcutaneous immunoglobulin (scig) is an efficacious alternative that can be flexibly dosed, self-administered at home and avoids the 'peaks' and 'troughs' observed with ivig. these factors may combine to improve patient satisfaction and alleviate hospital capacity issues. here, we report on clinical and patient experience of switching from hospital-based ivig to home-based manual push scig for the treatment of cidp and mmn. this was a clinical case series of patients ( cidp, mmn; mean age . years) who were clinically stable on ivig and wished to switch to manual push scig. starting scig dose was equivalent to the final ivig dose for each patient (mean . g/kg week). clinical efficacy (medical research council sum score, -m walk, modified inflammatory neuropathy cause and treatment score, overall neuropathy limitations scale, romberg test) and patient-reported outcomes ( -item short form health survey , life quality index [lqi] ) were assessed at baseline and at regular intervals until the final visit ( - months after switching). at baseline, patients cited 'convenience' as their primary reason for switching to scig. eight patients completed the full assessment period and successfully undertook administrations at home (via hospital-at-home service in cases). dose adjustments, based on clinical need, were required in patients. treatment efficacy and patient quality of life, measured by sf- , were maintained after switching to scig; overall patient satisfaction, measured by lqi, increased from % to %. in the lqi, 'convenience', 'travel time/cost' and 'interference-work' were significantly improved (p< . ) after switching to scig therapy. adverse events included mild erythema and localised swelling, as expected for a ml subcutaneous injection. these findings suggest that manual push scig therapy is a viable alternative to ivig for patients with cidp and mmn, as it maintains disease stability, is more convenient for patients and may help ease hospital capacity concerns. cumberbatch m , soares regua d , cox a . addenbrooke's hospital, cambridge, uk. intravenous immunoglobulin (ivig) is used to treat a number of chronic autoimmune neurological diseases. in most centres, infusions are given at slow rates as there is a perception that this reduces the risk of adverse events (aes). this results in longer in-patient admissions, or frequent day case attendances, impacting on both the patients' quality of life and hospital capacity. however, there is little evidence to suggest that slow infusion rates are required. we used the manufacturer recommendations to optimise infusion rates and reduce the time patients spend in hospital. we report a retrospective audit which describes the impact of different ivig infusion rates on patients' clinical condition. the audit comprised three -month assessment periods: january-june (cohort ; infusion rates of . ± . ml/kg/hr, n = ); january-june (cohort ; . ± . ml/kg/hr, n = ) and july-december (cohort ; . ± . ml/kg/hr, n = ). clinical data were reviewed to determine: patient demographics, duration of infusion; time spent in hospital; and incidence of aes. the three cohorts were well matched in terms of patient demographics ( patients were treated in all treatment periods). cohorts and had significantly shorter treatment episodes than cohort ( . and . vs. . hours, p< . ), spent less time on the unit over the month period ( . and . vs. . hours, p< . ) and had fewer admissions/patient ( . and . vs. . , p< . ). the overall incidence of confirmed aes (mainly headaches) was similar across the cohorts (cohort : %; cohort : %; cohort : %). these findings indicate that increases in ivig infusion rate are well tolerated and significantly reduce treatment time, which benefits patients and offers potential cost savings and reduced pressures on hospital capacity for healthcare providers. cunningham me , yao d , meehan gr , barrie ja , willison hj . university of glasgow, glasgow, uk. one mechanism of injury in the acute motor axonal neuropathy (aman) form of guillain-barré syndrome (gbs) is the attack of peripheral nerve axons by anti-ganglioside antibodies (agabs). rodent models have demonstrated that that binding of these antibodies activates the complement cascade, resulting in the insertion of the terminal component, membrane attack complex (mac) into the axonal membrane. complement activation also results in the release of anaphylatoxins, which are known to recruit phagocytic immune cells to the site of injury. our current in vivo mouse model of agab and complement-mediated injury are acute and severe, resulting in respiratory distress over several hours of such magnitude to warrant termination of experimental procedures. to observe and potentially target immune cell infiltration following agab and complement-mediated injury, a subacute model extending over days is required. here, we demonstrate the development of such a model. to compare differences in immune cell infiltration subacutely under control and injury conditions, mice with endogenous expression of egfp in monocytes and macrophages underwent a modified agab and complement-mediated injury, resulting in a less severe phenotype than previously published models. six days following injury, immune cells in the diaphragm were compared by immunofluorescence and flow cytometry. flow cytometry found overall presence of neutrophils was significantly increased in the diaphragm. macrophages were also increased in injured mice, although did not achieve statistical significance at this timepoint. these results were reflected in immunofluorescent staining of the diaphragm where egfp+ macrophages were quantified surrounding the neuromuscular junction (the primary injury target in this model). the development of an extended mouse model of agab and complement-mediated injury is important, since acute models do not take into consideration either the late-term effects of complement-mediated activation at the nerve membrane, or the recovery phase. future studies will look at the effect of inhibiting complement activation on the presence of immune cells in distal motor nerves. family- is a large australian family with an autosomal dominant form of dhmn (dhmn : omim % ) -a group of length-dependent neurodegenerative disorders affecting the lower motor neurons leading to chronic disability. we recently reported a novel . mb chromosomal insertion within the dhmn locus which we hypothesise is likely to cause disease by dysregulating the expression of one or more nearby genes. studying gene dysregulation in peripheral nerve disease is challenging as the relevant tissues (spinal cord and peripheral nerve) are not easily accessible in patients. therefore, alternative strategies are needed to elucidate the disease mechanisms and pathways involved in peripheral nerve degeneration. to address this problem, we have devised a two-tiered strategy to assess dysregulation of candidate genes using patient lymphoblast and fibroblast cell lines. these cell lines can be easily established, are minimally invasive to obtain, and will harbour the natural mutation and genetic background of patients. our strategy firstly uses lymphoblast gene expression profiles as an initial screening tool to prioritise candidate genes for assessing altered expression. differentially expressed genes will then be modelled in c.elegans where behavioural and nerve morphology can be assessed. using rt-pcr, we have screened dhmn candidate genes in patient and control lymphoblast cell lines. eighteen candidate genes were expressed in lymphoblasts. twelve of the eighteen genes were prioritized for further analysis based on expression in both lymphoblast and neural tissues. quantitative analysis using qrt-pcr taqman assays revealed that ube c, was differentially expressed between patients and controls. it is important that patterns of differential expression can be recapitulated in neural cell-specific models. as part of our second strategy, we have generated patient and control induced pluripotent stem cell derived motor neurons (ipsc-mns) from reprogrammed fibroblasts. using this model, we will perform rna-seq and qpcr experiments to examine disease-relevant alterations in gene expression in neural tissue. we predict that utilization of these two strategies will shed light on the pathogenic mechanisms underlying the dhmn insertion and provide useful insights of pathways leading to peripheral nerve degeneration. the outcome of guillain-barré syndrome remains unchanged since plasma exchange and intravenous immunoglobulin were introduced over years ago. pathogenesis studies on gbs have identified the terminal component of complement cascade, the membrane attack complex, as a key disease mediator and thus a therapeutic target. the inhibition of complement in guillain-barré syndrome (ica-gbs) trial looked at the first use of c pathway inhibition with eculizumab in humans with gbs in a randomised, double-blind, placebo-controlled trial. its primary outcome was to look at safety and tolerability of administration concomitantly with ivig and in the context of severe (gbs disability score or greater) disease. participants were recruited for a month period, with regular follow up. subjects were screened, with ( %) being randomised. the two main causes for failure to proceed were participant concerns around eculizumab side effect profile, specifically the meningitis risk, and also intercurrent infection precluding treatment. five received eculizumab for four weeks, alongside standard intravenous immunoglobulin treatment, with receiving placebo. the safety outcomes, monitored via adverse events capture at each trial visit, showed eculizumab to be well tolerated and safe when administered in conjunction with ivig. the most common adverse events were mild derangement in transaminases or infection. there were no infusion reactions. primary and secondary efficacy outcomes were captured via gbs disability scores, mrc sum scores, rasch overall disability scores and overall neuropathy limitation scores. for the primary efficacy outcome at weeks after recruitment, of placebo and of eculizumab-treated subjects had improved by or more grades on the gbs disability score. all patients had improvements in other measured parameters. this trial highlights the challenges in recruiting acutely unwell patients, due to time constraints and intercurrent infection. although the small sample size precludes a statistically meaningful analysis, these pilot data indicate further studies on complement inhibition in gbs are warranted. charcot-marie-tooth disease (cmt) affects about one in . people. currently more than genes have been identified, with the most different phenotypes. the majority of cases in western countries are autosomal dominant and classified as demyelinating and axonal according to electroneuromyography (enmg). the clinical condition is characterized by weakness and predominant sensory changes in the feet and hands. sometimes there are different phenotypes. recently, variants in heterozygotes in the hars gene (histidil-trna synthetase) have been described associated with cmt called type w.to report a case of cmt-sensitive phenotype with a probable new mutation in hars gene (p.leu arg).a male patient, adopted son, caucasian, drug addict, for three years suffered pain in lower limbs, of great intensity, refractory to drug treatment. the examination showed retrognathism, abolition of patellar and achilleas reflexes, painful and thermal anaesthesia and apalesthesia in the feet.the enmg showed reduced sensory action potentials in sural and superficial fibular nerves. laboratory investigations for painful polyneuropathy of thick and fine fibres was normal. sural nerve biopsy revealed axonal predominance neuropathy. exome sequencing revealed a mutation in the hars gene with a pathogenic variant in heterozygosity, with replacement of the amino acid leucine at position by arginine. our patient, although we did not know the antecedents, presented painful polyneuropathy, whose genetic research, although not unequivocal, indicated a variant called cmt w. few cases of this variant were described, with several mutations. our case revealed mutation hitherto unknown (p.leu arg). we conclude by the importance of a thorough genetic evaluation, in cases of sensory polyneuropathy of unknown cause. small fiber neuropathy (sfn) is a condition that affects the small a -and c-fibers, leading to severe neuropathic pain and autonomic dysfunction. several sodium channel gene mutations have been found in patients with sfn, with scn a-gene mutations being the most frequent. because current available sodium channel blockers are not selective for na v . , these treatments often result in numerous side effects. lacosamide is an anticonvulsant that targets specific sodium channels with a slow-inactivation state, while sparing those with normal activity. several mutations of the scn a-gene with an impaired slow-inactivation of na v . have been found in patients with sfn. therefore, a positive effect of lacosamide on pain reduction in these patients is expected. the primary objective of this study was to determine the effect of lacosamide versus placebo on pain in subjects with scn a-associated sfn. secondary objectives were to determine the effect of lacosamide on autonomic symptoms, sleep interference, and quality of life, and to examine the safety and tolerability. the lacosamide-efficacy-'n'-safety in sfn (lenss) study was a randomized, placebo-controlled, double-blind, crossover-design study. subjects were randomized to start with lacosamide and end with placebo or vice versa. during both of the two phases of the study, the subjects were treated for a period of eight weeks of mg bid, preceded by a titration period, and ended by a tapering period. patients filled in a pain diary twice daily and scored a set of validated questionnaires on autonomic symptoms, sleep interference, and quality of life at multiple study visits. in total patients with scn a-associated sfn were included between november and february . the subjects had a median age of years, ranging from to years. sixty percent of the patients were female. the final results of the study, including the primary and secondary outcomes, will be presented. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces must be in intimate contact with nerve fibres to have a proper function, but it has been shown that this is compromised due to the foreign body reaction (fbr). this fbr is the first response of the nonspecific immune system against an implanted device and is characterized by a first inflammatory phase followed by a second antiinflammatory and fibrotic phase. this process results in the formation of a tissue capsule around the interface causing function loss due to the physical separation between the active sites of the electrode and nerve axons. taking this into account, here we have tested several anti-inflammatory drugs such as dexamethasone, ibuprophen and maraviroc to reduce macrophage activation as well as clodronate liposomes to reduce monocyte/macrophage infiltration. moreover, sildenafil have been administered as an antifibrotic drug to reduce collagen deposition in a fbr model with longitudinal parylene c-based intraneural devices implanted in rat sciatic nerve. briefly, animals were systemically treated with dexamethasone, ibuprophen, sildenafil, maraviroc or clodronate liposomes for two weeks, and nerve damage, inflammatory reaction and matrix thickness around the implant were assessed. treatment with dexamethasone, ibuprophen or clodronate liposomes significantly reduced the inflammatory response in the nerve in comparison to saline group while sildenafil or maraviroc had no effect on iba positive cells infiltration in the nerve. however, only dexamethasone was able to significantly reduce the matrix deposition around the implant after two weeks of treatment. these results support the idea that inflammation triggers the foreign body response in peripheral nerves and a potent anti-inflammatory treatment with dexamethasone could have a beneficial effect on lengthening intraneural interfaces lifespan. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces functionality decreases over time, among other factors, due to the foreign body response (fbr), which encapsulates the implanted devices and physically separates the active sites from the nervous tissue. here we have studied the fbr to parylene c or polyimide thin devices implanted in rat sciatic nerves, assessing thickness of the tissue capsule, signs of inflammation and nerve damage. we have characterized the responsible cells of this response and several molecular mediators over months of implant to find differences between the fbr to both materials. after weeks of implant, the inflammatory response due to the surgery was already decreased, whereas in the implanted nerves it reached its highest levels to then decrease at chronic time points. besides, the amount of foreign body giant cells (fbgc), as a result of macrophage fusion, found in the tissue capsule around the implant also increases progressively to reach a maximum after weeks. on the other hand, molecular analysis of the environment revealed a peak of inflammatory cytokines during the first day of implant to return to standard levels thereafter. however, an increase on ccls molecules was found at later time-points for both materials. with regard to the capsule thickness, all the devices were surrounded by a tissue deposition which appeared soon after the implantation. however, in the case of polyimide devices, the tissue capsule showed a peak weeks after the implant and signs of remodeling thereafter, while the parylene c devices showed a second increase from to weeks in comparison to polyimide devices. immunohistochemical and electron microscopy analysis revealed two different cell types implicated in the fbr in nerve to both materials: macrophages, in close contact with the interface, and fibroblasts which appear after weeks surrounding the tissue capsule. although further analyses are needed to elucidate the differences in the fbr to parylene c and polyimide polymers, these results can help to determine therapeutic targets in order to reduce this response and to improve the intraneural interfaces lifespan. delmont e , antoine jc , paul s , boucraut j , attarian s . referral centre for als and neuromuscular diseases, marseille, france; referral centre for neuromuscular diseases, saint etienne, france; immunology laboratory, saint etienne, france; immunology laboratory, marseille, france. peripheral neuropathies with antibodies against myelin associated glycoprotein (mag) are chronic sensory neuropathies characterized by the presence of an igm monoclonal gammopathy and high levels of anti-mag antibodies. these antibodies recognize a specific epitope called human natural killer (hnk ) shared by nk lymphocytes and several components of the peripheral nerve (mag, p , pmp , sgpg, phosphocan). recently an elisa test has been developed to detect antibodies against hnk epitope. our objectives were to determine the sensitivity and the specificity of anti-hnk antibodies in the diagnosis of anti-mag neuropathy and to know if these antibodies were correlated with the severity of the disease. anti-hnk antibodies were assessed in anti-mag neuropathies and in negative controls: chronic inflammatory demyelinating polyradiculoneuropathies (cidp), miller fisher syndromes, sensory neuronopathies, length-dependant axonal sensory polyneuropathies, healthy controls. in anti-mag neuropathies, were recorded age, disease duration, incat sensory sum score (iss), overall neuropathy limitation scale (onls), rasch-built overall disability scale (rods), mrc sum score, anti-mag antibodies titer, peak dosage of the igm monoclonal gammopathy. anti-hnk antibodies were measured with gangliocombi™ mag elisa test and anti-mag antibodies with anti-mag autoantibodies elisa test both from buhlmann company. anti-hnk antibodies were positive in / anti-mag neuropathies, and in / controls (sensitivity %, specificity %). in anti-mag neuropathies, anti-hnk titer was correlated with sensory deficiency evaluated with the iss score (r= . , p= . ) and with disability evaluated with the rods (r= − . , p= . ) and onls scales (r= . , p= . ). anti-hnk titers were not related to age, disease duration, mrc sum score, anti-mag antibodies titer, peak dosage of the paraproteinemia. anti-mag antibodies titers were associated with none of the characteristics of the patients with anti-mag neuropathy. anti-hnk antibodies have good sensitivity and specificity in the diagnosis of anti-mag neuropathy. compared to anti-mag antibodies, their value is that their titers are related to the disease severity. these results need to be confirmed in a larger prospective cohort. chronic inflammatory demyelinating polyradiculoneuropathy (cidp)is a heterogeneous and treatable immune-mediated disorder that critically lacks biomarkers to support diagnosis. recent evidences indicate that paranodal proteins (contactin- , contactin-associated protein- , and neurofascin- ) are the targets of autoantibodies in a subset of patients with cidp showing distinct clinical presentations. particularly, these biomarkers appear to have clinical relevance and help to orientate therapeutic choice. here, we examined five patients presenting an igg reactivity against the nodes of ranvier and the axon initial segment. using a proteomic approach, cell-based assays and elisa, we identified neurofascin- (nfasc ) and neurofascin- (nfasc ) as the main targets of autoantibodies at the nodes of ranvier. four patients displayed predominantly antibodies of the igg subclass, whereas one patient presented igg antibodies that activated the complement pathway in vitro. these antibodies recognized different epitopes than the previously described anti-neurofascin- igg suggesting different pathogenic functions. accordingly, patients with anti-nfasc / igg showed a distinctive clinical presentation. most patients had a severe phenotype associated with conduction block or decreased distal motor amplitude. tremors or neuropathic pain were not observed. four patients presented with a subacute-onset and sensory ataxia. of interest, the neuropathy occurred concomitantly with nephrotic syndromes in two patients and with an igg -related retroperitoneal fibrosis in one patient. this suggested that autoantibodies could be responsible for the occurrence of both disorders. intravenous immunoglobulin and corticosteroids were effective in three patients, and one patient improved following cyclophosphamide and rituximab treatment. clinical remission was found to correlate with the depletion of anti-nfasc / antibodies and the loss of igg reactivity toward the nodes of ranvier. in addition, recovery of conduction block and of distal motor amplitude were observed following remission and suggested a nodo-paranodopathy. our data demonstrate that nodal antigens are the target of autoantibodies in a subgroup of patients with cidp. this emphasizes that the pathogenic mechanisms involved in chronic immune-mediated demyelinating neuropathies are broad and may include dysfunctions of the nodes of ranvier. mutations in the neurofilament heavy (nefh) gene have been recently identified as a rare cause of autosomal dominant, axonal charcot-marie-tooth disease (cmt ). the clinical spectrum of this condition remains to be delineated. we report two french families with an axonal, predominantly motor, dominantly inherited form of cmt caused by two previously unreported mutations in the nefh gene. twelve patients belonging to two different families were included in the study. they displayed an axonal motor and sensory neuropathy, with no mutations in known axonal cmt genes. a remarkable feature in all patients was the early involvement of proximal muscles of the lower limbs, occurring approximately to years after the onset of motor deficit. proximal weakness affected predominantly the iliopsoas muscle, whereas quadriceps and hamstring muscles were relatively preserved. muscle weakness and muscle wasting progressed rapidly, with most of the patients requiring walking assistance after years of disease evolution. three patients in family had brisk reflexes. nerve-conduction velocity studies displayed evidence of a motor and sensory axonal neuropathy predominantly affecting the lower limbs. original deletions of nucleotides near the end of the coding sequence of nefh were identified: in family , c. _ del (p.lys argfs* ), and in family c. _ del (p.lys glyfs* ) causing a frameshift. interestingly, this frameshift leads to the loss of the terminating codon and to the translation of additional amino acids encoding a cryptic amyloidogenic element, suggesting that this type of mutations could induce protein aggregation. consistently, we showed that overexpression of the mutated forms of nefh in a human neuroblastoma cells induced the formation of protein aggregates. we also observed that it triggered caspase activation and apoptosis. using electroporation of chick embryo spinal cord, we confirmed in vivo that mutated nefh formed aggregates and triggered apoptosis of spinal cord neurons. altogether, this suggests that these mutations in nefh cause protein aggregation and neurotoxicity in neurons expressing nefh. progressive loss of such neurons would explain the early motor involvement and the pyramidal signs observed in some patients. our results provide a physiological explanation to the presence of cmt and als clinical features in affected patients. del valle j , , delgado-martínez i , righi m , santos d , , cutrone a , bossi s , d'amico s , micera s , , navarro x , . neuroprosthetic devices that are aimed to restore sensorimotor limb function of amputee patients require highly selective electrodes designed to establish a tight relationship with the nerve, allowing the bidirectional transduction of signals between nerve fibres and the interface and enabling close-loop control from the user. differently from extra-or intraneural interfaces, regenerative nerve electrodes are designed to enable electrical interface with regrowing axonal bundles of injured nerves, aiming to achieve high selectivity for recording and stimulation. however, most of the developed designs pose an obstacle to the regrowth mechanisms due to low transparency and cause an impairment of the nerve regeneration. in this work, we present a novel double-aisle planar regenerative electrode, a new type of highly transparent, non-obstructive regenerative electrode, which allows the selective stimulation and recording of separated nerve fascicles. the design consists of a thin and flexible double-sided electrode longitudinally inserted across a conduit thus creating two separated aisles in which regenerating fascicles can independently regrow after nerve transection. electrodes implanted in acutely transected nerves of rats showed the capability of selectively stimulating and recording different fascicles inserted in the aisles. moreover, chronic implantation of the electrode in a nerve gap of mm after sciatic nerve section allowed for fascicle regeneration and reinnervation of distal muscles as confirmed by the high number of myelinated axons inside each aisle, good biocompatibility, and adequate nerve conduction. in addition, three and six months after implantation, independent stimulation and recording of each separately regenerated fascicle were possible. our results demonstrate the potential contribution of the doubled-aisle regenerative electrode to selectively interface different fascicles of an injured nerve with no deleterious effects on nerve regeneration. therefore, this multi-aisle regenerative electrode may be suitable for neuroprosthetic applications, such as prostheses for the restoration of hand function after amputation or severe nerve injuries. demichelis c , garnero m , franciotta d , cortese a , callegari i , mancardi gl , schenone a , leonardi a , benedetti l . department of neuroscience, rehabilitation, ophthalmology, genetics, maternal and child health, university of genoa and irccs aou san martino-ist, genoa, italy; laboratory of neuroimmunology, irccs, "c. mondino" national neurological institute, university of pavia, pavia, italy; u.o. neurology, asl imperiese, imperia, italy. querol et al. showed that neurofascin (nf ) antibodies identify a chronic inflammatory demyelinating polyradiculoneuropathy (cidp) phenotype characterized by severe polyradiculoneuropathy, poor response to intravenous immunoglobulins (ivig), and disabling tremor. neurological improvement after therapy with rituximab has been previously reported in three patients with cidp with igg anti-nf antibodies. herein we describe the acute-onset of a case of cidp positive for nf igg antibodies resistant to conventional therapies and responsive to rituximab. the patient is a year-old woman who presented acute onset ataxia and gait disturbances; her symptoms progressed over two weeks and distal weakness, numbness and paresthesias appeared too. the nerve conduction study was suggestive for a motor-sensory polyradiculoneuropathy mainly demyelinating. the cerebrospinal fluid analysis showed elevated protein level and normal cellular count. the patient was initially diagnosed with guillain-barré syndrome (gbs) and treated with plasma exchange without improvement. an ivig cycle was started with a partial relief but at the time of admission to the rehabilitation center the patient still had a marked weakness in all four limbs. after six months she presented a further clinical deterioration and she was restricted to wheelchair. there was no response to additional treatment with ivig, while pulse corticosteroid treatment determined a significant clinical improvement. during the next months, despite the maintenance of steroid therapy, the patient presented a progressive deterioration and she was again restricted to wheelchair. postural and intention tremor appeared at upper limbs and became progressively more disabling. anti-nf ab dosage resulted positive. rituximab was administered at a dosage of mg/m /weekly for weeks. after three months the tremor improved, allowing her to eat independently and the patient was able to walk with bilateral support. antibodies anti-nf were negative. after six months she walked without support and she was able to stitch crochet. as previously reported, in this case a cidp positive for igg nf developed severe polyradiculoneuropathy with predominant distal weakness, ataxia, disabling tremor and resistance to conventional therapies. interestingly the onset was gbs-like. the correct identification of these cidp subtypes has diagnostic, prognostic and therapeutic implications. rituximab con be useful in these patients. demir Ö , yazıcı t . department of neurosurgery, university of gaziosmanpaşa school of medicine, tokat, turkey; department of neurosurgery, kent hospital, giresun, turkey. it is still challenging problem to maintain motor and sensory functions of peripheral nerve after nerve transection. after the nerve injury, calcium concentration in the damaged area increases. then the calcium ions act like cytotoxic agents in the damaged area. nifedipine is calcium channel blocker. we aimed to investigate the effects of nifedipine on nerve regeneration by modulating calcium in the damaged area. twenty-four swiss albino male rats were divided into two groups. left sciatic nerve transection surgery was performed to the all rats in both groups. then the all transected nerves were sutured primarily with epineural interfascicular method. in the experimental group, the anastomosis sites were wrapped with a piece of gel foam soaked into diluted nifedipine solution. in the control group, the anastomosis sites were wrapped with a piece of gel foam soaked into saline solution. we evaluated the effect of nifedipine by using functional, electro-physiological and histopathological studies after the surgeries. in the postoperative second week, walking test was performed and sciatic function index was calculated. in the postoperative third week electroneuronography (enog) was performed. there are significant differences between two groups. nifedipine improved nerve recovery functionally (p< . ) and electro-physiologically (p< . ). in the postoperative fourth week, we performed histopathological examination. in the experimental group with nifedipine there were more organized axons that reached the aim. we conclude from these results that nifedipine is an effective nerve protective agent when used locally at the anastomosis site after the transection of the nerve. the lack of effective, disease-modifying therapies for cmt highlights the need for novel preclinical models suitable for drug discovery. studies in rodent models of cmt tend to be time-consuming, and findings so far have translated poorly into clinical trials. primary and induced pluripotent stem cell (ipsc)-derived neuronal cultures are an established model of neurological diseases. however, due to the random distribution of neuronal bodies and neurites that happen when plating these cells, this system is not ideal to investigate axonal, length-dependent processes like peripheral neuropathies and particularly cmt. to optimize this well-established model system, we developed a robust human platform to study axonal morphology and physiology based on motor neuron neurospheres. we differentiated motor neurons from human induced pluripotent stem cells, purified them by magnetic sorting and cultured them in suspension until they formed neurospheres. floating neurospheres can be maintained in agitation for months as a reliable source of motor neurons. after neurospheres are platted, axons rapidly grow out of them in a radial fashion, resembling dorsal root ganglia cultures. this configuration allows for a better visualization of axons in imaging studies and for continued axonal growth over at least a -day period. axons grew at an average rate of micrometers/day and reached up to cm in length. neurospheres can be fixed and stained allowing for morphological analysis and investigation of protein distribution in axons. this system is also ideal for time-lapse imaging to study axonal transport of organelles and neurofilament kinetics. lastly, our motor neuron neurosphere system lends itself well for high content screening platform. neurospheres can be plated in -well plates where multiple compounds can be tested and the axons easily imaged by a high content screening microscope. in summary, we developed a new platform to investigate motor axons in vitro, which are particularly useful to study length-dependent processes such as inherited peripheral neuropathies and may facilitate the identification of new therapeutic compounds using high content screening systems. mutations in the neurofilament light chain (nfl) gene cause autosomal dominant axonal charcot-marie-tooth neuropathy (cmt e). nfl is a major component of the neuronal cytoskeleton, and is believed to function in conjunction with nfm and nfh to provide structural support for the axon and regulate axon diameter. despite the significant advances in understanding its biological basis, there is still no effective, disease-modifying therapy for cmt e, in part due to the paucity of preclinical models suitable for drug discovery. the development of novel preclinical platforms that can faithfully mimic mechanisms of axonal degeneration in vitro would be an essential and valuable resource to better understand the biology of cmt e and identify potential targets for therapy development. to address this, we generated control and cmt e patient-derived motor neurons and cultured them in suspension until they formed neurospheres. immunostaining of cmt e neurospheres with nfl and tubb antibodies revealed numerous areas of nfl accumulation in n s cmt e axons, resembling the accumulations of mutant nfl protein seen in the processes of catecholaminergic neuronal cell line cad overexpressing several nfl mutants. further analysis demonstrated that areas of nfl accumulation were also immunopositive for nfh, pnfh and nfm and that at least nfl and nfm co-localized in the same areas of deposits. taken together, these results demonstrate that abnormal axonal neurofilament distribution is a feature of cmt e ipsc-derived motor neurons and involve all three neurofilament subunits. we also developed an image analysis routine to allow for automatized quantification of neurofilament distribution. preliminary quantification of nfl signal intensity revealed that axons from patients have a weaker nfl signal compared to control axons, but present several signal peaks above the range observed in controls, which related to the areas of nfl accumulation. these results suggest that nfl accumulates in certain regions of cmt e axons but is reduced in the areas with no accumulation. these findings can be readily adapted into a high content screening platform and will be used to identify compounds able to reverse this axonal phenotype. in summary, we identified a strong axonal phenotype in human cmt e motor neurons with potential as a screening platform for drug discovery. nodal and paranodal proteins have been identified as antigens in peripheral inflammatory neuropathies, however the frequency and clinical relevance of antibody responses against these targets remain poorly investigated in gbs. patients with acute onset inflammatory neuropathies were identified by exploration of the local databases of the departments of neurology and the institute of neurology of the medical universities in innsbruck and vienna. patient data, electrophysiological classification and presence of anti-gangliosid antibodies were retrospectively retrieved by review of patient records. only patients with typical clinical presentation and electrophysiological results consistent with one of the subtypes of gbs were included in the study. among forty-nine patients, thirty-five were classified as aidp, six aman, three amsan, three mfs, and two pharyngo-cervico-brachial gbs. of the included patients had anti-ganglioside-antibodies. ten patients with the initial suspicion of aidp had a disease duration of more than months and were reclassified as cidp. all patient and twenty sera of control patients with non inflammatory polyneuropathy were screened by an optimized tissue based assay using rat brains for immune responses against surface antigens, and by cell-based assays with transfected hek cells for antibodies against contactin (cntn ), contactin (cntn ), contactin-associated-protein (caspr ) and neurofascin- (nf ). in the tissue based assay some of the patients showed a light neuropil staining. none of gbs patient's sera had antibody reaction to cntn , cntn , caspr or nf in cell-based assays. among the cidp patients, two patients demonstrated reactivity against cntn with similar clinical presentation as previously described. none of the control patients had any antibody reaction to the performed tests. our results suggest that antibody responses to cntn , cntn , caspr or nf are absent in austrian gbs patients, although more patients will be screened to substantiate these preliminary results. furthermore, it remains to be established whether antibodies against cntn may predict a chronic course in acute onset inflammatory neuropathies. a few variants of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described with a frequency of - %. their relation and possible evolution into typical-cidp remain unclear, as is their treatment response possibly because of differences in diagnostic criteria. we used the data from a web-based database on italian patients with cidp to determine the frequency and characteristic of these variants, the possible evolution into typical-cidp, and their treatment response. all the patients were assessed at study entry and the disease course before inclusion was analyzed. by february- , we included patients ( men, women), aged - years (median years) with a mean disease duration of . years (range . - years) and complete data available from . based on the clinical data and our revised diagnostic criteria, patients ( %) were classified to have atypical cidp at onset and for the following two years including with dads ( %), with motor cidp ( %), with sensory cidp and cisp ( . %), with lewis-sumner syndrome ( . %), and with recurrent cranial neuropathy. at study entry, patients ( %) had progressed into typical cidp after - years (median years) while ( %, % of total) still had atypical cidp after . - years (median years) with a similar proportion of progression ( - %) within each group. the diagnosis of atypical cidp at entry fulfilled efns/pns criteria in ( %). csf studies were diagnostic in / ( %) patients, nerve biopsy in / ( %), and nerve imaging in / ( %) tested patients. similarly to typical cidp, % of treated patients with atypical cidp improved after treatment with a proportion of response varying from % to % in the different forms. most patients with sensory or motor cidp had however an unsatisfactory response to steroids. this study shows that he proportion of patients with atypical cidp varies during the course of the disease with almost % of the patients evolving into typical cidp within years from onset. in addition, response to treatment is frequent in atypical cidp even if not all the forms respond to the same therapies. only few studies investigated the frequency of antecedent events and comorbidities in patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and little is known on the role of possible predisposing factors, dietary, and lifestyle habits, on the onset and progression of the disease. we used the data from a web-based database on italian patients with cidp to determine the frequency of antecedent events and comorbidities and the possible role of predisposing factors including lifestyle and dietary habits and exposure to toxic agents, using a structured questionnaire. partners of patients served as controls. impairment was evaluated using the mrc sumscore and disability with incat and r-ods scales. logistic regression was used to calculate odds ratio (or) with % confidence interval (ci) for the risk of cidp. sex and disease-duration were included as covariates. by february- , patients were enrolled, with complete data on patients for antecedent events and comorbidities and patients and controls for lifestyle habits. ninety-two patients ( %) reported an antecedent event, mostly infection or vaccination ( %). one or more comorbidity were present in % of the patients including hypertension ( . %), thyroid disorders ( %) and diabetes ( . %) and in % influenced the choice of initial therapy. exposure to toxic environmental agents (odds ratio [or] = . ; % ci, . - . ), cigarette smoke (or = . ; % ci, . - . ), and dietary supplements (or = . ; % ci, . - . ) were associated with a higher risk of cidp while rice consumption was associated with a reduced risk (or = . ; % ci, . - . ). concerning disease severity, more severely affected patients more frequently consumed raw-meat (or = . ; % ci, . - . ) and white meat (or = . ; % ci, . - . ), while rice (or = . ; % ci, . - . ) and soft drink consumption (or = . ; % ci, . - . ) and physical activity were associated with lower disability (or = . ; % ci, . - . ). this study confirms that comorbidities are frequent in patients with cidp and often influence the choice of initial therapy. in addition preliminary data show that toxic exposure and some lifestyle and dietary habits may influence the onset and progression of cidp. doppler k , schuster y , weishaupt a , sommer c . department of neurology, university hospital würzburg, würzburg, germany. autoantibodies against the paranodal protein contactin- have recently been described in patients with cidp. in most patients, autoantibodies of the igg subclass are predominant and are supposed to be pathogenic. the role of igg anti-contactin- is so far unclear. in the present study, igg of three different patients, one with igg anti-contactin- , one with a low titer of igg anti-contactin- and one with a high titer of igg anti-contactin- , and of controls were injected into the sciatic nerves of lewis rats. nerve conduction studies of the injected nerve and motor and sensory testing were performed before and after injection. conduction blocks and motor deficits were detectable in the two patients with high titers of igg and igg , not in the patient with low titers. the percentage of conduction blocks was . % in rats injected with igg of the igg patient and % in those injected with igg . motor deficits were detectable in both patients with conduction blocks but were most apparent in the patient injected with igg of the igg patient. no differences in sensory testing were observed. conduction blocks and motor deficits improved after five days and were normal after seven to eight days. our data give the first evidence of pathogenicity of igg anti-contactin- autoantibodies, not only igg . igg of the igg patient induced a more severe clinical and electrophysiological phenotype compared to the igg patient. remarkably, this reflected the clinical phenotype of the patients, as the igg patients showed an acute-onset of sensorimotor symptoms at the time of blood withdrawal whereas the igg patient presented with a more chronic course of disease. doppler k , frank f , koschker a-c , reiners k , sommer c . department of neurology, university hospital würzburg, würzburg, germany; endocrinology and diabetes unit, department of medicine i, university hospital würzburg, würzburg, germany. axoglial dysjunction and paranodal demyelination have been discussed as potential mechanisms of nerve fiber damage in diabetic neuropathy. studies on human tissue are limited, as nerve biopsies are invasive and only rarely performed in patients with confirmed diabetic neuropathy. skin biopsy has recently been suggested as a good tool to analyze paranodal and nodal changes of myelinated fibers. in the present study, we analyzed the paranodal and nodal region in myelinated fibers of skin biopsies of patients with diabetic neuropathy, patients with diabetes mellitus without neuropathy, and normal controls. immunofluorescence of skin sections with antibodies against caspr, neurofascin, sodium channels and myelin basic protein was performed to assess paranodal/nodal architecture, segmental demyelination and myelinated nerve fibers. staining with antibodies against protein gene product . was used to quantify unmyelinated nerve fibers. we found an increase of elongated ranvier nodes and a dispersion of neurofascin at the distal leg in patients with diabetes mellitus with and without neuropathy and at the finger in patients with diabetic neuropathy. an increased dispersion of caspr was only found in biopsies of the finger in patients with diabetic neuropathy. our data show that skin biopsy is an appropriate tool to analyze nodes of ranvier in patients with diabetes mellitus. structural nodal changes are detectable in diabetic neuropathy, and even in diabetic patients without neuropathy. dourado me , fernandes u , vital al , ramos e , urbano jc , sena a , queiroz jw , jeronimo smb . federal university of rio grande do norte, natal, brazil. the erasmus gbs outcome score (egos) is a validated prognostic model that uses acute phase and easy-to-obtain clinical characteristics to determine outcome at months in patients with gbs. this study aims to assess the validity of egos in rio grande do norte, brazil, and to compare with another european study. data collected prospectively from a cohort of patients with gbs of rio grande do norte, brazil, between june and august , was assessed. ninety patients were excluded for missing data or diagnoses of miller fisher syndrome and atypical forms of gbs. to calculate the egos, the gbs disability score was assessed in the second week of disease and at months. to compare this study with the european one in independent group proportions, we used the student's t-test, being considered statistically significant p< , . the patients included were divided in four groups based on egos. thus, patients had egos between and ; had egos between . and . ; had egos and had egos between . and . in the first, second, third and fourth group, ( %), ( . %), ( . %) and ( . %) of the patients were unable to walk independently after six months of the disease, respectively. overall, of the patients analyzed, ( . %) had poor outcomes in this study. in the european paper, based on the same group division, of ( . %), of ( %), of ( %) and of ( %) were unable to walk independently. comparing both studies, the patients of this study were younger, more seriously ill in the first weeks and with more sensitive deficits. there were no difference relative to sex, cranial nerves deficits and presence of anti-gangliosides antibodies. using the student's t-test for ability to walk after months according to egos stratification, we achieve in the first group p= . ; in the second p= . ; in the third p= . ; and in the fourth p< . .the egos did not have a good capacity to predict the ability to walk after months of gbs in rio grande do norte, brazil. historically, guillain-barré syndrome (gbs) epidemics are rarely seen. between and , we treated and followed cases of gbs in the state of rio grande do norte with a yearly incidence of . / , . no seasonality was observed. the mean age of the patients was years (range, - ), with % of the cases younger than years. demyelinating variant was the most frequent subtype of gbs. in march , the first report of autochthonous transmission of zika virus (zikv) was determined in natal, brazil. later that month, we documented an increase in incidence of gbs in natal, brazil. the incidence in was of . / . . of the cases of gbs diagnosed in , were diagnosed from march through may, which coincided with the outbreak of zikv in natal, brazil. eighteen patients ( % of the cases) had a history of rash and fever prior to onset of gbs symptoms, with the median age of years (range, - ) . the electroneuromyography studies of these patients indicated that ( . %) had acute inflammatory demyelinating polyneuropathy, ( . %) had acute motor axonal neuropathy, and ( . %) was inconclusive. the mean time from onset of zikv infection symptoms to onset of the gbs were days (range, - ). the mean time of nadir was days (ranged, - ) . cranial neuropathies were present in patients ( . %). nine patients were bedridden ( %) and ( . %) required mechanical ventilation. the mean protein content of the central spinal fluid was . g/l, with the white blood cell count below /mm in all patients. they were all treated with intravenous igev. they all improved quickly. anti-gm was negative in all patients. rt-pcr was negative for dengue, chikungunya and zika. serum mac-elisa igm for zika and dengue was made in patients and it had % of positivity. prnt for zika and dengue had % positivity. in summary, we report a geographically and temporally defined cluster of gbs associated with an outbreak of acute rash in the state of rio grande do norte, brazil. as the prevalence of diabetes mellitus continues to increase worldwide, diabetic complications represent a growing burden to patients and society. distal symmetrical polyneuropathy (dsp) is a common complication that affects up to % of diabetic patients. dsp reduces patient quality of life due to chronic pain, ulcerations, and may lead to lower extremity amputations. despite its high prevalence, the mechanisms underlying diabetic dsp are poorly understood and several mechanisms are believed to play a role. we hypothesize that diabetic dsp arises from microvascular complications characteristic to diabetes. specifically, capillary dysfunction -disturbances in capillary flow patterns -is a likely candidate to explain development of dsp, as it can limit oxygen and nutrient delivery to nervous tissue, causing nerve dysfunction and damage, and thus development of dsp. we will study this hypothesis utilizing the state-of-the-art blood flow imaging techniques to visualize and quantify endoneureal blood flow and then link these findings with measures of dsp (e.g. nerve conduction velocity; intra-epidermal nerve fibre density) in animal models. we will include several animal models of diabetic dsp caused by either type or type diabetes. two photon microscopy and optical coherence tomography allow visualisation and quantification of capillary transit times and blood flow within peripheral nerves at high resolution. we hypothesise that changes in blood flow patterns and subsequent impairment of nutrient and oxygen delivery to nervous tissue precede the onset of diabetic dsp. if our experiments support this prediction, we will attempt to develop interventions that improve capillary blood flow to prevent or delay the development of dsp. duman o , saracoglu m , haspolat s , bozkurt o . department of child neurology, akdeniz university hospital, antalya, turkey. axonal polyneuropathies are very heterogeneous group of diseases which are very rarely seen during infantile age. some of them may be accompanied by developmental retardation, severe muscle weakness and progressive course. we aimed to present two cases of axonal sensorymotor neuropathy with infantile onset and atypical course. our -year-old boy patient was admitted to our clinic for progressive gait loss since one month. he was the first offspring of consanguineous parents with normal prenatal and natal history. electromyography revealed axonal sensorymotor polyneuropathy. metabolic and cerebrospinal fluid (csf) examinations for etiology were all normal. brain and spinal magnetic resonance imaging (mri) were normal. he had partial benefit from oral steroid treatment. in the course of disease along with four neuropathy attacks he had significant benefit from serial intravenous immunoglobulin treatments in two years clinical course. a month-old girl who is the first offspring of nonconsanguineous parents was admitted to the clinic for acute tetraparesis. axial sensorymotor polyneuropathy was detected in the electromyography. metabolic and cerebrospinal fluid (csf) examinations were normal. she had three more acute polyneuropathy attacks during ivig cessation period. both patients revealed with serial immunoglobulin treatments but unresponsive to riboflavine treatment. we aimed to discuss our rarely seen and the pathogenesis is not completely understood cases' course. serial ivig treatment may be helpfull for such patients' treatment. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominantly inherited disorder caused by mutations of the transthyretin (ttr) gene. the mutant amyloidogenic transthyretin protein causes the systemic accumulation of amyloid fibrils that result in organ dysfunction. ttr-associated fap is a progressive and fatal disease, if left untreated, and should be considered in the differential diagnosis of any person presenting with a progressive polyneuropathy, particularly with accompanying autonomic involvement. the clinical, electrophysiological, histopathological, and genetic characteristics of patients from turkey ( female, male) from eleven families with polyneuropathy and mutations in ttr were evaluated. two patients had no family history of ttr-fap and were considered as sporadic cases, and the remainders were familial cases displaying an autosomal dominant inheritance pattern. sequence analysis of the ttr gene revealed five mutations (p.val met, p.glu gln, p.gly glu, p.glu gly and p.gly glu). most common mutation was p.val met (in unrelated families). mean age at disease onset was . ± . years (range - years). the most commonly reported initial complaint was paresthesia in the feet (asymmetric in three patients). four patients ( male) with the p.glu gln mutation presented with carpal tunnel syndrome. two patients with the p.gly glu mutation showed episodes of dysarthria and hemiparesis, consistent with this genotype. seven patients died during the follow-up period as a result of systemic involvement. this study suggests that our cohort of ttr-fap patients from turkey exhibits clinical and genetic heterogeneity. ebenezer gj , liu y , judge dp , cunningham k , truelove s , carter nd , sebastian b , byrnes k , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; division of cardiology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. effect of amyloid deposition on cutaneous nerves was assessed in subjects with pathogenic ttr variants and control subjects. three groups of subjects each including ttr-fap patients, age/gender-matched healthy subjects and disease controls as well as ttr mutation carriers without neuropathy (ttr-nopn) and with al-amyloid underwent neurological examination and mm skin biopsies. micron sections were stained with anti-pgp . , anti-ttr and congo red. amyloid burden with imagej, intraepidermal (ienfd) sweat gland (sgnfd) and pilomotor densities (pmnfd) measured and correlations between amyloid burden, fiber subtype, neuropathy impairment score-ll (nis-ll) and nis sensory subscore were evaluated. ienfd, sgnfd, and pmnfd were all significantly reduced in ttr-fap patients vs. healthy controls while mutation carriers had intermediate reductions. lower nerve fiber densities were associated with nis-ll (p< . ). congo red staining revealed brilliant red amyloid deposits with apple-green birefringence within dermal collagen, sweat glands, and arrector pili muscles. amyloid infiltration was observed in the endoneurium and perineurium of small fiber sensory and autonomic nerves that innervate sweat glands and arrector pili muscles. cutaneous amyloid deposition was detected in % of ttr-fap and not in healthy or disease controls subjects. both al and / ttr-nopn subjects were congo red positive. amyloid burden was inversely correlated with ienf (p< . , r=− . ) sgnf (p< . , r=− . ), pmnf (p= . , r=− . ) distal leg densities, and correlated with nis-ll (p= . , r= . ) and nis sensory subscore (p= . , r= . ). wild-type ttr staining was less prominent in pathogenic ttr carriers. the diagnostic sensitivity and specificity to detect amyloid in skin were % and % in ttr-fap. the repeat measurement of the amyloid burden from the same section with imagej was r = . , p< . and different sections from the same biopsy was r = . and p< . . we conclude that endoneurial amyloid contributes to sensory and autonomic nerve injury. amyloid burden correlated strongly with sensory/autonomic axon densities and nis-ll. skin punches offer a convenient alternative to establishing a tissue diagnosis. amyloid burden is an attractive biomarker marker for ttr-fap and treatment effect. the study was supported through a grant from pfizer. ebenezer gj , truelove s , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. we investigated differences of unmyelinated sensory nerve fibers in the distal limb among healthy-weight subjects (bmi < kg/m ) and overweight/obese subjects (bmi ≥ kg/m ), aged - years. subjects underwent neurological examination and mm skin punches from distal leg (dl), thigh (dt), and proximal thigh (pt) sites, from which micron sections were stained with anti-pgp . antibody; intraepidermal nerve fiber density (ienfd; fibers/mm) and epidermal thickness were assessed. a second dl biopsy was processed for electron microscopic examination and both thick and thin sections were examined for ultrastructural changes. multivariable linear regression models were used to assess the effect of age, gender, height and weight. after controlling for height, age, and obesity, females were found to have lower distal leg ienfd (− . ; p= . ). increasing age and height were significantly associated with decreasing dl ienfd, with decreases of − . fibers/mm per years (p<. ) and − . fibers/mm per cm (p< . ), respectively. even after controlling for height, being overweight/obese was associated with reduced dl ienfd, with . fibers/mm lower dl enfd than healthy-weight individuals (p=. ). these findings remained consistent across distal thigh and proximal thigh enfd, though not all associations remained significant. the epidermis was thicker in obese subjects across the lower limb, most pronounced at the distal leg (μm, mean± sd, healthy-dl: ± . , dt: . ± . , pt: . ± . , obese-dl: . ± . , dt: ± . , pt: . ± . ). under em very few intact dermal nerve bundles were identified at the proximal thigh sites. the atrophic and degenerating axons were seen with perineurial infiltration by dense collagen in obsess/overweight subjects but not age/gender matched controls. obesity further accelerates attenuation of epidermal nerve fibers across the lower limb even after controlling for other associated factors. echaniz-laguna a , geuens t , petiot p , péréon y , adriaenssens e , haidar m , capponi s , maisonobe t , fournier e , dubourg o , degos b , salachas f , lenglet t , eymard b , delmont e , pouget j , juntas morales r , goizet c , latour p , timmerman v , stojkovic t . strasbourg university hospital, strasbourg, france; peripheral neuropathy group, vib department of molecular genetics and institute born bunge, university of antwerp, antwerpen, belgium; lyon university hospital, lyon, france; nantes university hospital, nantes, france; hôpital de la pitié-salpétrière, paris, france; nice university hospital, nice, france; marseille university hospital, marseille, france; montpellier university hospital, montpellier, france; bordeaux university hospital, bordeaux, france. in this study, we describe the phenotypic spectrum of distal hereditary motor neuropathy caused by mutations in the small heat shock proteins hspb and hspb and investigate the functional consequences of newly discovered variants. among unrelated patients with distal motor neuropathy, we identified mutations in hspb ( index patients/ ; . %) and hspb ( index patients/ ; . %) genes. patients have slowly progressive distal ( %) and proximal ( %) weakness in lower limbs, mild lower limbs sensory involvement ( %), foot deformities ( %), progressive distal upper limb weakness ( %), mildly raised serum creatine kinase levels ( %) and central nervous system involvement ( %). we found a broad range of disease onset with some patients presenting with foot drop at the age of years, and others presenting symptoms only after years. disease progression was slow in all patients, and even with a disease duration of more than years patients were still able to walk. none of our patients were wheelchair dependent. muscle pathology, nerve pathology and electrophysiology showed in all cases a slowly progressive, mostly symmetrical and predominantly distal motor axonal neuropathy. mild sensory involvement was observed upon nerve conduction studies, mostly the lower limbs, in % of cases. we identified hspb and hspb mutations, including respectively and not previously reported. transmission was either dominant ( %), recessive ( %) or de novo ( %). three missense mutations in hspb (pro ser, gly asp, gln arg) cause hyperphosphorylation of neurofilaments, while the c-terminal mutant ser leu triggers protein aggregation. two frameshift mutations (leu fs, ala fs) create a premature stop codon leading to proteasomal degradation. two mutations in hspb (lys met/asn) exhibited increased binding to bag . we demonstrate that hspb and hspb mutations are a major cause of inherited motor axonal neuropathy. mutations lead to diverse functional outcomes further demonstrating the pleotropic character of small heat shock proteins. eftimov f , querol l , rajabally ya and on behalf of all participants of the st enmc workshop. academic medical center, amsterdam, the netherlands; hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; aston university, birmingham, uk. although chronic inflammatory demyelinating polyneuropathy (cidp) is a treatable neuropathy further research is urgently needed to define the diagnostic clinical and electrophysiological boundaries of cidp and its subtypes, and to define the role of biomarkers in supporting the diagnosis, monitoring disease activity and predicting response to treatment and outcome. in recent years, several national registries and biobanks have been developed to enable systematic data collection in cidp. an international registry with large number of patients is needed to allow answering many important questions and develop validated prognostic models to predict outcome in individual patients with cidp. at the inflammatory neuropathy consortium (inc) meeting in , the inc members agreed that a european neuromuscular center (enmc) workshop would be the ideal setting to reach a consensus on the infrastructure of database and biobanks. the st enmc workshop will take place on may - , with participants representing different countries. primary objective of the workshop is to reach a consensus on inclusion and exclusion criteria, core sets and recommended sets of clinical data, diagnostic data and follow-up points and a manual of operations for collection of biomaterials. a secondary objective is to construct an infrastructure to allow sharing data between different databases and biomaterials. conclusions of the consensus meeting and outline of further perspectives will be presented at the peripheral nerve society meeting in . eichinger kj , burns j , cornett k , bacon c , shepherd m , mountain j , sowden j , shy r , shy me , herrmann dn . clinical outcome assessments that measure functional ability are important endpoints for clinical trials. dr. burns has led the development/validation of a functional outcome assessment (cmtpeds) for individuals with charcot marie tooth disease (cmt) ages - years in the inc rdcrn. the cmtpeds is reliable and sensitive to change. however a validated functional outcome measure (fom) for adults with cmt is needed. our data in - year-olds indicated that the cmtpeds could be modified for adult use. however, some items of the cmtpeds (e.g. balance beam and jumping) have floor effects in adults with cmt. we have developed an adult cmt-fom modeled on the cmtpeds, and refined based on literature review, patient interviews, a large-scale cmt patient survey and expert opinion. the cmt-fom is a performance-based scale comprising items that are combined to form a composite score to quantify functional ability of adults with cmt. the cmt-fom shares items with the cmtpeds. four items were added to measure functional abilities relevant to adults (sit to stand, meter walk/run, stair climb, and timed up and go test). the cmt-fom scoring mirrors the cmtpeds. to generate a score ranging from - , raw item scores are converted to z scores, based on age-and sex-matched normative reference values form the norms project and categorized to a - likert along a continuum of impairment levels. we have conducted a pilot study of the cmt-fom in adults with cmt a ( male, female, age . ± . yrs) of differing severity (cmt exam score (cmtes) range - ). the cmt-fom is feasible, individuals were able to complete all items, and takes minutes to perform. the mean cmt-fom score was . ± . (range - ). concurrent validity of the cmt-fom is supported by an association with the cmtes (r = . ). the overall score did not demonstrate floor or ceiling effects. in summary the adult cmt-fom is well-tolerated and captures upper and lower limb strength, dexterity, balance, speed, ambulation and endurance. the cmt-fom requires validation in a large longitudinal cohort, prior to application in clinical trials. estilow t , glanzman am , burns j , cornett kmd , menezes mp , shy r , moroni i , foscan m , pagliano e , pareyson d , laurà m , bhandari t , muntoni f , reilly mm , finkel rs , sowden j , eichinger k , herrmann dn , shy me , yum sw , ramchandren s and on behalf of the inherited neuropathies consortium. cmt is associated with progressive impairment of the hands. reducing this impairment by treating children who are in the early stages of the disease is crucial. studies assessing measures of cmt hand function in children and their associations with patient-reported outcomes are lacking. we analyzed the upper extremity items from the cmt pediatric scale (cmtpeds) and pediatric cmt quality of life scale (pcmt-qol) in children ages - years enrolled in the inherited neuropathies consortium, to explore the relationships between measures of hand function (impairment, activity, and activities of daily living), and patient-reported outcomes. weak grasp ( %), hand pain ( %), and tremor ( %) were prevalent impairments. performance on activity level tasks, the nine hole peg test ( hpt) and functional dexterity test (fdt), were impaired in % and % of the cases, respectively. patients reported difficulty "sometimes" to "always" in opening a jar/lid ( %), zipping/buttoning ( %), writing ( %), carrying a plate without spilling food ( %) and putting on shoes ( %). patients reporting tremor showed significant differences on the hpt (p=. ). grip strength was shown to have a moderately significant correlation with performance on the fdt (r=. ; p<. ). stepwise multiple linear regression showed that grip strength (beta=−. ; p<. ), hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to open a jar (adjusted r =. ; p<. . grip strength (beta=−. ; p<. ), and fdt (beta . ; p<. ) were predictive of ability to carry plate without spillage (adjusted r =. ; p<. ). grip strength (beta=−. ; p<. ), hpt (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to put on shoes (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to zip/button (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and hpt (beta . ; p<. ) were predictive of ability to use a pen/pencil (adjusted r =. ; p<. ). children with cmt present with frequent limitations in adl performance impacting qol. the upper limb measures of the cmtpeds are associated with hand performance and interventions to improve grip strength and reduce pain should be investigated further with respect to their impact on improving function, and ultimately qol. evans me , morrow jm , wastling s , sinclair cdj , fischmann a , shah s , emira ak , hanna mg , yousry ta , thornton js , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; neuroradiological academic unit, ucl institute of neurology, london, uk; university of basel hospital, basel, switzerland. responsive outcome measures are needed in charcot-marie-tooth disease (cmt) to allow adequately powered clinical trials to test novel therapeutics. we have shown high responsiveness of mri quantified intramuscular fat accumulation in calf muscles of cmt a patients over months. the aim of the present study was to assess the responsiveness and longitudinal validity of quantitative mri over a year follow-up period. we undertook two further sets of quantitative mri, myometric and clinical assessments in the original mrc centre cmt a quantitative mri cohort. mri sequences included fat quantification using the point dixon fat-water separation method, t quantification and magnetisation transfer imaging. of the patients with genetically confirmed cmt a were assessed at baseline ( male, mean age . ± . years), underwent repeat assessments a median on months (data already published), underwent repeat assessments at a median of months, and underwent a final assessment at a median of months. the primary outcome measure currently being analysed is mean calf muscle fat fraction at a single axial slice a fixed distance distal to the knee joint. results of this analysis and correlation with clinical measures will be presented at the peripheral nerve society meeting. fainmesser y , dori a , , drory ve , . department of neurology and neuromuscular service, tel-aviv medical center, tel-aviv, israel; department of neurology, sheba medical center, ramat gan, israel; sackler faculty of medicine, tel aviv university, israel. the use of anabolic drugs by those who wish to increase lean body mass is widespread and not well supervised medically. a years old man was referred for evaluation of a slowly progressive sensory and motor disturbance in the distribution of the left ulnar nerve. his symptoms began months after repeated self-injections of anabolic steroids and vitamin e into the biceps and triceps brachii muscles bilaterally. his examination showed increased muscle mass of the injected muscles with a hard-rubbery consistency, atrophy and weakness of left interossei, mild weakness of the bilateral biceps brachii and sensory loss in an ulnar nerve distribution. nerve conduction studies showed a left ulnar neuropathy with reduced motor and sensory response amplitudes and denervation in the left first dorsal interosseus, as well as mild myopathic changes with significantly reduced insertional activity in both biceps muscles. mri of the soft tissues of the arms showed massive fibrosis and infiltration of fat in the arm muscles compressing the ulnar and median nerves on the left. neurolysis of the left ulnar nerve was performed, and the ulnar nerve was found enclosed in a fibrotic mass throughout the entire length of the upper arm. the brachial artery and the median nerve were similarly enclosed in fibrotic tissue and were released. a biopsy of the affected muscles showed muscle necrosis and fibrosis. the patient was treated with physiotherapy and losartan with only mild improvement in the consistency of the muscles, but without clinical improvement in ulnar nerve function, and worsening of nerve conduction. this case illustrates severe peripheral nerve damage due to entrapment in massive muscle fibrosis following improper intramuscular injection of anabolic steroids for cosmetic purposes. fabrizi gm , testi s , høyer h , braathen gj , squintani g , bertolasi l , ferrarini m , taioli f , cabrini i , pancheri e , cavallaro t , tonin p . department of neuroscience, biomedicine and movement, university of verona and department of neuroscience, aoui verona, italy; section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. inherited diseases of nerve and muscle may overlap phenotypically or coexists as facets of the same disorder. two families whose probands were initially diagnosed with a lower motor-neuron (lmn) syndrome and a hereditary distal motor neuropathy (dhmn) turned out to represent a vcp (valosin-containing protein)-related syndrome and a gne (udp-n-acetylglucosamine -epimerase/n-acetylmannosamine kinase)-related "distal myopathy" . both conditions shared the presence of rimmed vacuoles (rv) in muscle biopsies. in family , the year-old male proband had a lmn syndrome manifesting at age years (wasting/weakness, cramps, fasciculations of thigh muscles, later involving the distal upper limbs). his year-old brother had lower limb weakness and diffuse pain in bones/joints since age . the father was diagnosed with a "muscular dystrophy" thirteen years before dying a years; by age years he had developed a behavioural frontotemporal dementia (ftd). electrodiagnosis (edx) disclosed myopathic changes with ongoing denervation together with a mild sensory-motor axonal polyneuropathy in the proband, and a chronic sensory-motor axonal polyneuropathy in the brother. mri showed fatty replacement of weakened muscles in both siblings and diffuse changes of bones consistent with paget disease (pd) in the elder sibling. in family , a year-old african woman was affected by weakness and wasting starting at the distal lower-limb muscles at age years and soon progressed proximally; parents were not consanguineous and two sisters out of six siblings, deceased in their thirties for post-partum complications, had a similar disease. edx mainly disclosed a neurogenic process with ongoing denervation. muscle biopsies from the three family- patients and from the family- proband showed a myopathy with rv. next-generation sequencing demonstrated a heterozygous c. c>t change of vcp leading to a known pathogenic p.arg cys substitution in all family- patients, and two compound heterozygous c. g>a and c. g>a changes of gne in the family- proband leading to known p.ala thr and p.ala thr substitutions. vcp is known to cause autosomal dominant amyotrophic lateral sclerosis- , charcot-marie-tooth disease type y or inclusion body myopathy (ibm) with pd and ftd (ibmpfd); gne causes the autosomal recessive nonaka myopathy (alias ibm ). both cases emphasize the clinical and neurophysiological heterogeneity of those disorders. fargeot g , vandendries c , labeyrie c , viala k , theaudin m , adams d . neurologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neuroradiologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neurologie, groupe hospitalier universitaire pitié salpêtrière, paris, france. the diagnosis of cidp is often challenging, especially when electrophysiological signs of demyelination are lacking. plexus mri has documented nerve abnormalities in small series of typical cidp but its contribution in patients with no electrophysiological signs of demyelination remains to be proved. we report the results of plexus mri in a serie of patients suspect of having cidp without efns pns definite electrophysiological criteria. we did a retrospective study of patients consulting in kremlin bicetre and pitié salpétrière hospital. lumbar or brachial plexus mri (or both) were performed and we assessed nerve trophicity, t -stir signal intensity and gadolinium enhancement as well as the topography of abnormalities. a consensus diagnosis was made by a group of experts (based on clinical data and other supportive criteria) and allowed us to classified patients in "cidp" or "other diagnosis". the practical contribution of plexus mri to the diagnostic algorithm has been studied. diagnosis of cidp was made in patients. mri was abnormal in % of cidp patients and showed nerve roots hypersignal/hypertrophy/enhancement in respectively . / . / . % of patients. the pattern of abnormalities was often asymetrical ( . %), diffuse ( . %) or multifocal ( . %). after unblinding, the mri confirmed the diagnosis of experts in . % of patients and changed the diagnosis in % of patients. plexus mri has shown to be useful in our serie to confirm the diagnosis of experts or to modify it in patients ( %) . the "classical" pattern described in definite cidp (diffuse nerve root hypertrophy and hypersignal) was documented in % of our cidp patients whereas less typical pattern (focal or multifocal abnormalities, hypersignal without hypertrophy) was found in %. plexus mri seems usefull when facing patients suspected of having cidp when electrophysiological criteria are not met: both symetrical diffuse or asymetrical multifocal patterns can be found and should be always correlated to the clinical examination and other supportive criteria. the specificity of such abnormalities remains to be studied. feely sme , rebelo a , abreu l , tao f , bacon c , zuchner s , shy me . university of iowa, iowa city, ia, usa; dr. john t. macdonald department of human genetics and hussman institute for human genetics, miller school of medicine, university of miami, miami, fl, usa. mutations in bcle-associated athanogene (bag ) have been shown to cause a distal myofibrillar myopathy and cardiomyopathy that can severely affect children or only affect adults depending upon the particular mutation. children with severe cardiomyopathy and myopathy have also developed axonal peripheral neuropathy, consistent with the known localization of bag in neurons as well as in muscle. we have identified two large autosomal dominant families with adult onset charcot-marie-tooth disease (cmt ) with the identical novel missense mutation pro ser, a codon previously shown to cause severe or mild myopathy depending on the amino acid substitution. these families expand the phenotypes caused by mutations in bag to include cmt and provide an additional example of adult onset cmt that may previously have been diagnosed as chronic idiopathic axonal neuropathy (ciap). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. myelin protein zero (mpz), expressed only by myelinating schwann cells, has sequence alterations that have previously been reported to cause demyelinating, intermediate, and axonal forms of charcot marie tooth (cmt) disease. we describe a rare duplication in mpz which is causing an early onset, demyelinating form of cmt in our patient. patient was a product of a normal pregnancy and delivery. early milestones were delayed. she began walking at months of age. she was not able to keep up with her peers and was the slowest runner. she started wearing smos at years of age and afos by years of age. she was diagnosed with scoliosis at years of age and started wearing a brace at years. her examination at years of age showed that her fdi, apb, and adm were all / bilaterally. weakness in her lower extremities included / foot eversion and / great toe dorsi flexion. she had tight heel cords bilaterally. pinprick sensation was reduced throughout in her upper and lower extremities and absent at her toes bilaterally. vibration sensation with a rydel tuning fork was absent at her toes and ankles and reduced at her knees and fingers. nerve conduction studies were performed and revealed no responses in all sensory nerves tested with the exception of the radial nerve which had normal latency and mildly slowed conduction velocity ( m/s). prolonged latencies, demyelinating range slowing (between - m/s), and low cmap amplitudes in almost all segments of the median and ulnar motor nerves were also observed. these findings were consistent with a hereditary sensorimotor demyelinating polyneuropathy. she was diffusely areflexic and her total cmt pediatric score (cmtpeds) was / which is in the severe range. her cmt neuropathy score (cmtns) was / in the moderate range. the duplication of mpz has previously been identified as a rare cause of cmt b. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. mutations in egr cause a severe, demyelinating form of cmt, cmt d. we describe a novel mutation in egr which led to extreme variability in severity in a family. proband was a year old girl who was the product of a normal pregnancy and delivery. early milestones were on time. problems with walking started at years of age. at . she was unable to use stairs, run, or jump. at she was wearing bilateral afos. by she was using a wheelchair and started breathing assist at night. she lost arm lift but could still hold a pen. by she could not ambulate independently and breathing assist was required day/night. she lost the ability to write and developed a head drop. by years she could not sit up. on exam her head and neck muscles were / . upper limbs, deltoids, biceps, wrist ext/flex, finger ext, fdi, and adm were / bilaterally. triceps were / ; finger flexors and apb were / bilaterally. lower limbs were / and she had contractures on the right. sensory examination was normal. she was diffusely areflexic. ncvs were absent. both the cmtns and cmt pediatric score were severe at / and / . exome sequencing revealed a r l variant in egr which was likely pathogenic. the probands' mother also had this mutation. she had no reported symptoms at the age of with strength at / throughout with the exception of left foot eversion which was +/ and great toe dorsi flexion which was -/ bilaterally. sensory exam showed a decrease of vibration and pinprick sensation at left toe. she had a normal gait, tandem gait and could toe walk, but not heel walk. she was diffusely areflexic. ncvs showed absent sensory responses. motor ncvs showed her latencies were prolonged and velocities reduced ( - m/s). cmtns was mild ( / ). additional family members who had the r l mutation were evaluated including maternal grandmother who was moderately impaired ( / ) and maternal aunt who was severe ( / ). no other mutations that could cause another known neuropathy or myopathy were identified and mitochondrial sequencing was normal. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). fisgun a , luan x , hoke a . johns hopkins university, baltimore, md, usa. molecular mechanisms that underlie slow distal axonal degeneration seen in chemotherapy induced peripheral neuropathy (cipn) are unclear. however, several identified molecular targets suggest shared mechanisms with wallerian degeneration. since spontaneous mutation in wlds mice and genetic deletion of sarm gene lead to slow wallerian degeneration, we asked if wlds or sarm knockout (ko) mice are resistant to distal axonal degeneration induced by several chemotherapy agents. we chose chemotherapeutic drugs from different classes of agents, paclitaxel (taxane), cisplatinum (platin-based drugs) and bertozomib (proteasome inhibitor), to model cipn in mice. primary outcome measure was evaluation of epidermal nerve fibers in the hind paw plantar footpads. secondary outcome measures included thermal sensation and nerve conduction studies. sarm ko mice were almost % protected against development of sensory neuropathy but the protection in wlds mice was partial. this study confirms the pivotal role sarm plays in mediating axonal degeneration and identifies inhibition of sarm activity as a potential therapeutic target for prevention of cipn. florio f , scapin c , ferri c , feltri ml , wrabetz l , d'antonio m . myelin biology unit, san raffaele scientific institute, milan, italy; hjkri-university of buffalo, ny, usa. myelin protein zero is the most abundant structural protein in myelin of the pns. in humans, more than mutations in p are associated with hereditary neuropathies. deletion of serine causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in mice (wrabetz et al., ) . p s del protein is misfolded and is retained in the er where it gives rise to a dose-dependent unfolded protein response (upr) (pennuto et al., ) . the upr results in the activation of transcriptional and translation control programs that reduce protein synthesis and increase the folding and degradative capacity of the cell. usually, when this first response is not sufficient the cells may activate apoptosis resulting in cell death. however, in p s del schwann cells there is no cell death suggesting that these cells may respond differently to chronic stress. transcriptomic analysis performed on p s del nerves showed increased expression of transcription factors normally present only in the early phases of differentiation such as c-jun, sox and id (d' antonio et al. ) . in order to understand the role of the expression of these factors in the peripheral nerve myelination we used ex vivo and in vivo approaches and we showed that sox and id act as negative regulators of myelination. these results suggest that the expression of sox and id may contribute to the hypomyelination observed in p s del mice. as such, we reasoned that their ablation could ameliorate the phenotype. surprisingly, the ablation of these factors in the p s del mouse severely worsens the neuropathy bursting schwann cell differentiation and increasing the expression of both p wild type and mutant allele with concomitant exacerbation of the upr. this suggests that the overexpression of early differentiation factors in schwann cell under chronic er-stress is an adaptive mechanism that limits differentiation and reduces the expression of toxic proteins. intravenous immunoglobulin (ivig) is the treatment of choice for the guillain-barré syndrome (gbs). the working mechanism of ivig in gbs is undefined, but most likely all potential effects are dose dependent. the pharmacokinetics (pk) and pharmacodynamics (pd) of ivig in gbs are highly variable between patients and a rapid consumption or clearance of ivig is associated with poor recovery. in the current study we developed a model to predict the pk of a standard dosage of ivig ( . g/kg for consecutive days) in individual patients with gbs. non-linear mixed-effects modelling (nonmem) was used to construct a model based on a cohort of gbs patients, with a total of sequential serum igg levels. the final model accurately describes the day to day increment in igg levels during the -day course and the initial rapid fall and graduate decline to steady-state levels thereafter. we explored several potential covariates that improved the predictive capabilities and decreased the between-subject variation in the model. the model including these covariates were evaluated successfully (bootstrap analysis) and through numerous simulation studies each based on (simulated) gbs patients. in conclusion, a first accurate and robust nonmem model for the pk/pd of standard ivig treatment in gbs was developed. the model can be used to predict the pk in individual patients applying a few simple baseline characteristics. in addition, the effect of different treatment regimens of ivig in gbs on a population pk/pd level can be simulated. this modeling technique is a new tool to optimize the pk in individual patients and the study design for new trials with ivig in gbs. forese mg , pellegatta m , rivellini c , podini p , quattrini a , previtali sc , taveggia c . axonal neuregulin (nrg ) type iii is an essential instructive signal for peripheral nervous system (pns) myelination, as its expression determines whether axons are myelinated as well as the thickness of the myelin sheath. we recently demonstrated that gamma-secretase cleavage of nrg type iii generates an axonal intracellular fragment, which translocates in the nucleus to upregulate the expression of the prostaglandin d synthase (l-pgds) gene in neurons. l-pgds catalyzes the conversion of prostaglandin h into prostaglandin d (pgd ). we also showed that specific inhibition of l-pgds activity impairs in vitro myelination. accordingly, myelin in l-pgds null mice is noticeably thinner, thus indicating that l-pgds is a new modulator of developmental pns myelination. previous studies have shown that prostaglandins are involved in the process of wallerian degeneration (wd) and axonal regeneration after injury. thus, to determine whether l-pgds and pgd could be important in pns regeneration and remyelination, we performed sciatic nerve crush injury in months old l-pgds null and wild type control mice and we analysed nerves by morphologic, biochemical, histological and molecular approaches at different time points (t) after crush. we focused on three phases: degeneration (t -t ), axon regeneration (t -t ) and remyelination (t ). our results indicate that in l-pgds null mice the amount of myelin proteins synthesized after crush as well as the number of remyelinated fibers do not change, suggesting that l-pgds might be dispensable for remyelination. however, we observed an increased number of macrophages in null nerves during regeneration (t ), possibly as a consequence of an increase in the blood-nerve barrier (bnb) permeability, indicating potential alteration in the regeneration process in l-pgds null mice. these results suggest that l-pgds could have a different role in developmental pns myelination and after injury. whether other prostaglandins and synthases might compensate for l-pgds activity is currently under investigation. fornasari be , , raimondo s , , ronchi g , , crosio a , budau ca , el soury m , muratori l , , tos p , battiston b , geuna s , , gambarotta g . department of clinical and biological sciences, university of turin, italy; neuroscience institute cavalieri ottolenghi, turin, italy; microsurgery unit, health and science city, cto, turin, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. to repair nerve gaps following severe peripheral nerve injuries, chitosan tubes were proved to give good results, comparable with those obtained with nerve autografts, the gold standard technique. to further improve peripheral nerve regeneration using chitosan tubes, a conduit enrichment strategy was developed using longitudinal skeletal muscle fibres, which have been previously shown to be good fillers in the "muscle in vein" experimental paradigm, where they played a trophic and a structural role. to this aim, rat median nerve gaps were repaired using two different conduits: mm chitosan tubes filled with a longitudinal piece of pectoralis major muscle ("muscle in tube") and hollow chitosan tubes. samples were harvested at early time points ( , , , days) for biomolecular and morphological analysis, and later ( months) for stereological analysis. autologous nerve grafts were used as gold standard positive control in the early time points. biomolecular analysis carried out on in vitro degenerating muscle and on "muscle in tube" at early time points show that the muscle produces high levels of soluble isoforms of neuregulin , a key factor for schwann cell survival and activity, usually released by schwann cells after nerve injury. functional assay and stereological analysis carried out on the distal part of regenerated nerve months after nerve repair, show no significant differences in the regeneration outcome between hollow chitosan tube and "muscle in tube" groups. therefore, we conclude that for short gaps (≤ mm), both hollow chitosan tube and "muscle in tube" are good techniques to repair nerve defects and we suggest that the "muscle in tube", which spontaneously releases neureg-ulin , might be a promising strategy to promote regeneration when the gap is longer or the repair is delayed in time. foucquier j , bertrand v , jouve e , truillet r , mandel j , laffaire j , blin o , magy l , lehert p , , hajj r , guedj m , cohen d , attarian s . pharnext, issy-les-moulineaux, france; aix marseille université, aphm, marseille, france; hôpital dupuytren, limoges, france; university of melbourne, melbourne, vic , australia; faculty of economics, louvain, belgium. charcot-marie-tooth disease type a (cmt a) is a rare disease belonging to a group of inherited, progressive, chronic motor and sensory peripheral neuropathies. the charcot-marie-tooth neuropathy score (cmtns) (shy et al., ) and the overall neuropathy limitations scale (onls) (graham & hughes, ) are considered as the main clinical scales for evaluating progression of disability associated with cmt. as cmt a is a slowly progressive neurodegenerative disease, the choice of endpoints and their ability to monitor small changes over time remain a major concern for clinical drug development. with this in mind, we studied a cohort of french cmt a patients with a follow-up ranging from months to . years resulting from the merge of two multicentre clinical trials (micallef et al., ; attarian et al., ) and a non-interventional study (unpublished) . the sensitivity to change of both cmtns and onls were assessed using a mixed effect model estimating annual progression with time in years, cmtns or onls baseline value as covariates, study centre as a fixed factor and patients as a random effect to account for the repeated measures. disease progression was estimated to be + . points per year on the cmtns (p = . ) and + . points per year on the onls (p = . x − ), both corresponding to a deterioration of impairment and disability. while both endpoints have similar and favourable properties, our set of observations led us to conclude that the onls could be more promising to monitor disease progression in cmt a. frasquet m , , lupo v , mas f , , vílchez r , chumillas mj , , espinós c , sevilla t , , . hospital universitari i politècnic la fe, valencia, spain; instituto de investigación sanitaria la fe, valencia, spain; centro de investigación príncipe felipe, valencia, spain; eresa, valencia, spain; centro de investigación biomédica en enfermedades raras (ciberer), valencia, spain; departamento medicina, universitat de valencia, valencia, spain. mutations in the bicd gene are a cause of dominant spinal muscular atrophy, lower extremity predominant (smaled). we report six patients belonging to three spanish families who carry three different novel mutations in the bicd gene. we describe clinical, electrophysiological and magnetic resonance imaging (mri) data. we provide results of muscle biopsy of one patient and skin biopsy for the study of epidermal nerve fiber density (enfd) of other two patients. genetic diagnosed was reached using a gene panel for genetic testing of cmt and dhmn. three novel mutations in the bicd gene that segregated with the disease were detected: p.val gly; p.tyr his and p.s l. the most frequent clinical phenotype consisted of mild weakness in proximal muscles of lower limbs combined with foot deformities. one patient had prominent sensory symptoms and abnormalities on sensory examination. other two patients had minor sensory abnormalities on examination. in one patient sensory and motor nerve action potentials were reduced, in the rest of patients electrophysiological studies showed normal motor and sensory nerve responses, with chronic denervation predominantly in muscles of lower limbs. mri studies at the level of tight and calf were performed in all patients. the most affected muscles were rectus femoris, vastus lateralis and medial gastrocnemius. mri studies at the level of feet were obtained from five patients and showed that there was not fatty infiltration in intrinsic foot muscles. mri at the level of pelvis muscles performed in four patients showed marked fatty infiltration of gluteus medius muscle in two of them. muscle biopsy performed in one patient showed myopathic features. skin biopsy was performed in two patients of the same family. in the older patient, who had minor sensory abnormalities on examination, there was a marked reduction of enfd that followed a length-dependent pattern. in conclusion, we report three new pathogenic mutations in the bicd gene. in our study we include mri findings at the level of pelvis and feet, which allow us to better define the pattern of muscle involvement related with this gene. our results also raise the subject of a possible sensory involvement in the disease. hereditary neuropathy with liability to pressure palsies (hnpp) is an autosomal dominant disorder that usually results from deletions in the pmp- gene. the neuropathy is unique in that it manifests with recurrent mono-neuropathies at common sites of compression. while spontaneous recovery from episodes of nerve injury usually occurs, it is often incomplete, and over time patients may develop a length dependent polyneuropathy. given the relapsing/remitting nature of hnpp symptoms, standard clinical scores, such as the cmt neuropathy score, are not effective at capturing the severity or progression of the disease. a specific tool is therefore needed for measuring clinical severity of hnpp in preparation for emerging clinical trials. in the current study, we evaluate a new pilot measure, the hnpp severity score (hnpps). the score is composed of patient reported questions addressing current and prior sensory and motor symptoms, and the impact of symptoms on quality of life, followed by items based on a motor examination. total scores vary from - , with higher scores indicating increased disease severity. in this study, the hnpps was administered to patients with genetically confirmed hnpp at the ucl institute of neurology. subjects included males and females with a mean age of years (+/− , range - ). the mean hnpps was . points (+/− . , range - ) and the data did not demonstrate major skew. the cronbach alpha for the hnpps was . , and items based on the physical examination showed the least variability. a modest correlation was observed between the hnpps and the cmt examination scores (pearson correlation . , ci . - . ). we conclude that the hnpps may be a useful measure of clinical severity in hnpp, and should be refined in larger patient cohorts. fridman v , sillau s and on behalf of the inherited neuropathies consortium (inc) . university of colorado hospital, aurora, co, usa; university of iowa hospitals and clinics, iowa city, ia, usa. the most common of the hereditary neuropathies (hn) is cmt a, an autosomal dominant demyelinating neuropathy that results from duplications in the pmp- gene. recent advances in defining the pathomechanisms of the disease have led to an increasing number of potential therapies; however, the absence of reliable natural history data and the paucity of sensitive clinical outcome measures have been barriers to effective clinical trials. the charcot marie tooth neuropathy score (cmtns) was developed to quantify impairment and measure progression in hn. it was observed that while the score discriminates well between mildly and severely impaired patients, it tends to cluster together patients in the middle range of severity. to improve the score's sensitivity, rasch analysis-based weighted category responses were developed. we report a longitudinal study of weighted cmtns and cmt examination scores (cmtes) over a three-year time frame in patients with cmt a. baseline, one year, two year and three year wcmtnsv /wcmtesv scores were available for / , / , / and / patients respectively. mean wcmtns (sd)/wcmtes (sd) scores were as follows: . ( . )/ . ( . ) at baseline, . ( . )/ . ( . ) at one year, . ( . )/ . ( . ) at two years and . ( . )/ . ( . ) at three years. a mixed regression model showed significant change in wcmtns and wcmtes at years (mean change from baseline at years was . points (p=. ) for wcmtns and . points (p=. ) for wcmtes. significant change as compared to baseline was also seen at years (mean change from baseline . points (p= . ) for wcmtns and . points (p= . ) for wcmtes. we conclude that weighted cmtnsv scores show change over the first three years of the inherited neuropathies consortium natural history study and are a helpful measure of progression in cmt a. fridman v , novak p , david w , macklin ea , mckenna-yasek d , walsh k, oaklander al , brown r , hornemann t , eichler f . massachusetts general hospital, boston, ma, usa; university of massachusetts medical school, worcester, usa; university hospital zurich, zurich, switzerland. hsan is an autosomal dominant, severe sensory motor polyneuropathy caused by mutations to serine palmitoyl-coa transferase. mutations shift the substrate preference from serine to alanine leading to formation of neurotoxic -deoxysphingolipids ( -deoxysl). treatment with high-dose l-serine has been shown to reduce -deoxysl accumulation and improve neuropathy in transgenic hsan mice. we report a two-year, delayed-start, placebo-controlled clinical trial evaluating the safety and efficacy of oral l-serine in hsan . eighteen hsan subjects were equally randomized to l-serine ( mg/kg/d) or placebo for year. at -weeks, the placebo group crossed-over and all participants took open-label l-serine for one additional year. sixteen subjects completed their -week visit, and no serious adverse events related to l-serine were reported. participants randomized to l-serine experienced a decline in charcot marie tooth neuropathy scores (cmtns) over year relative to placebo (− . units, % ci − . to − . , p = . ). both groups improved in the second year of the study, with a diminished difference in cmtns at weeks (− . units, % ci − . to . , p= . ). skin biopsies from the distal leg site were largely devoid of intra-epidermal nerve fibers (ienf), but at year, a greater increase in ienf density was seen in participants on l-serine versus those on placebo (median change of vs. fibers/μm , p= . ). -deoxysl levels declined among participants on l-serine versus those on placebo after one year of treatment ( % decrease in deoxysphinganine vs. % increase on placebo, p < . ), and placebo participants experienced similar declines in -deoxysl levels after crossing over to l-serine. we conclude that l-serine is a safe and potentially efficacious treatment for hsan . fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the relationship between guillain-barré syndrome (gbs) and malignancy is uncertain. under the diagnostic criteria of paraneoplastic neurological syndrome (pns) by euronetwork, , neuropathy with no definite onconeural antibodies identified due to gbs has been classified as "non-classical" paraneoplastic disorder. we retrospectively analyzed data of consecutive patients admitted with gbs from birmingham, u.k. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) to calculate the relative cancer risk using different definitions and determined characteristics of malignancy-associated gbs. patients were classified according to definitions: ) all cases of malignancies excluding preceding diagnoses > year before gbs and with no evidence of malignant disease activity at the time of gbs diagnosis, and ) malignancies diagnosed > years post-gbs onset as per pns criteria. total number of gbs patients with malignancy was ( . %). a total of patients ( . %) fulfilled requirements for inclusion as malignancy-associated gbs as per existing criteria for pns. associated malignancy consisted of angioimmunoblastic t-cell lymphoma ( ), poorly differentiated squamous cell carcinoma of nasal septum ( ), gastric adenocarcinoma ( ), hepatocellular carcinoma due to hepatitis b ( ), rectal carcinoma with liver metastasis ( ) and myelodysplastic syndrome ( ). malignancy was globally commoner in our gbs cohort compared to the general population (odds ratio: . ; ci: . - . ; p = . ). however, this was unconfirmed if paraneoplastic criteria were applied. gbs patients with cancer were significantly more likely to be older (p = . ), hyponatremic (p = . ) and demonstrate more electrophysiological axonal loss (p < . ). cerebrospinal fluid (csf) protein levels were lower in the malignancy group (p = . ) and neurological improvement was less likely (p = . ). in-patient mortality was significantly higher in patients with malignancy (p < . ). none of the patients in the malignancy group had positive anti-ganglioside antibodies or anti-neuronal antibodies (anti-hu, yo, ri, crmp ). we conclude global cancer risk is higher in gbs than in the general population, although definition-dependent. application of the strict published criteria for paraneoplastic syndrome reduced the number of cases and suggested absence of a link. malignancy requires consideration in elderly, hyponatremic subjects with normal csf protein, severe electrophysiological axonal loss who fail to improve post-treatment. fu liong h , aude-marie g , anne-catherine an , emilien d , julien c , shahram a , yusuf r . anti-myelin associated glycoprotein antibody (anti-mag) neuropathy is a slowly progressive neuropathy resulting in disability through distal sensory more than motor deficits and tremor. there are currently no evidence-based treatments for anti-mag neuropathy and the effect of the disease on quality of life (qol) in this patient population is unknown. our objective was to assess determinants of qol in patients with anti-mag neuropathy. the sf- questionnaire was assessed in patients, from marseille, angers (france) and birmingham (united kingdom). routine clinical evaluations included mrc sum score, incat sensory score, inflammatory rasch built overall disability score (i-rods), ataxia score, jamar grip dynamometry, timed meter-walk, neuropathic pain symptom inventory (npsi) score, and fatigue severity score (fss). there were males and females. mean age was . years (sd: . years). mean disease duration was . years (s.d.: . years). there were no significant differences between the french and u.k. cohorts in terms of gender distribution, age, disease duration, anti-mag antibody titre. all physical assessments, including mrc sum score (p= . ), jamar grip dynamometry (p= . ), onls (p= . ), irods (p= . ), incat sensory score (p= . ), -meter timed walk (p= . ), ataxia score (p= . ), tremor score (p= . ), were comparable. prevalence and/or severity of pain (p= . ), fatigue (p= . ), restless legs syndrome (p= ) and cramps (p= . ) were also similar. physical component summary (pcs) and mental component summary (mcs) of the sf questionnaire were significantly lower than in reported normal subjects of both countries (p< . ). all sf- qol domains correlated with i-rods, except mcs for which significance was however approached (p= . ). pcs correlated with mrc sum score, ataxia score, timed m-walk, tremor, jamar grip dynamometry, npsi pain score, fss and level of social support. mcs correlated exclusively with fss and level of social support. in multivariate regression, pcs was associated independently with i-rods (p< . ) and npsi pain score (p= . ), whereas mcs was associated independently with fss (p= . ). qol is accurately predicted in anti-mag neuropathy by the i-rods and fss, lending support to their use in clinical and research settings. effective measures to improve qol should include tremor and neuropathic pain treatment, fatigue management and improved social support. fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the electrophysiological diagnosis of guillain-barré syndrome (gbs) is dependent on a number of abnormalities affecting different parameters in peripheral nerves on nerve conduction studies (ncs). diagnostic sensitivity varies with different electrodiagnostic criteria, practices as well as extensiveness of nerve study. however, the most efficient method of performing electrophysiology for guillain-barré syndrome (gbs) is unknown. we retrospectively analyzed electrophysiological data of consecutive gbs patients from birmingham, uk ( - ), studied ≤ weeks post-onset. we first identify abnormal nerves from various regions which produced gbs electrodiagnosis using the recently described criteria by rajabally et al. we subsequently used pre-established hypothetical nerve conduction study protocols to determine the potential optimal method of achieving electrodiagnosis. we found the sensitivity of electrophysiology for each gbs subtype was dependent on the upper and lower limb nerves tested. in acute inflammatory demyelinating polyneuropathy (aidp), abnormalities were predominant in the arms, whereas leg abnormalities predominated in axonal gbs. in aidp, the most common abnormal parameters were distal motor latency ( . %) and conduction block ( . %), and the most frequently affected nerve was the median ( . %). prolonged f-waves were present in % and f-waves were absent in %. mcv was the least frequently abnormal ( . %) with demyelinating range slowing, significantly lower than cb or dml prolongation (p< . in both cases). in axonal gbs, reduced motor amplitudes ( . %) and conduction block ( . %) were the most common parameters, and the most frequently abnormal nerve was the tibial ( . %). f-waves were absent in . %. . % of all motor nerves were unexcitable, significantly more common in lower limb nerves compared to upper limb nerves (p= . ). on comparison of different hypothetical ncs protocols ( unilateral protocols with nerves, with nerves as well as the protocols with -nerves and with exclusive bilateral upper limb and lower limb testing), unilateral -nerve (median, ulnar, common peroneal and tibial) testing produced the highest diagnostic sensitivity for both aidp ( . %) and axonal ( . %) gbs. electrodiagnostic sensitivity in gbs is thus dependent on nerves tested and parameters considered. each subtype preferentially involves specific nerves and parameters. these findings may help per-procedure interpretation, improve electrodiagnostic sensitivity, and reduce patient discomfort. funakoshi k , nagashima t , kokubun n , hirata k , yuki n . department of neurology, dokkyo medical university, tochigi, japan; department of neurology, mishima hospital, nagaoka, japan. recently, igg anti-ganglioside complex (gscs) antibodies have been reported in patients with guillain-barré and miller fisher syndrome. some researchers (nobile-orazio et al., ) reported igm anti-gscs antibodies in multifocal motor neuropathy (mmn) and other chronic immune-mediated neuropathies. in ten of eleven mmn patients with anti-gm antibody, there was a decreased reactivity to gm /gd a compared to single antigen gm . similarly, in one of two chronic inflammatory degenerative polyradiculoneuropathy (cidp) patient with anti-gm antibody, there was a decreased reactivity to gm /gm , gm /gd a, gm /gd b, gm /gt b compared to gm . these relationships were defined negative interaction. in one cidp with anti-gd b antibody, anti-gm /gt b and -gm /gt b reactivity increased although gm and gm were negative (positive interaction). in japan, on the other hand, this correlation remains unclear. sera were investigated from one mmn patient ( y/o male) with anti-gm , and anti-gd a antibody, one cidp patient ( y/o female) with anti-gm , and one subacute sensory motor polyradiculoneuropathy ( y/o male) with anti-gd b, and anti-gq b. igm antibodies to gscs gm /gd a were tested with a mixture of gm and gd a (each pmol/well) as antigen. anti-gm /gd a antibodies were judged to have positive interactions when the optical density was . greater than the sum of the antibodies against individual gm and gd a, and negative interactions when the optical density was % or less compared to the antibodies against individual gm or gd a. antibodies to at least one combination of two of the six gangliosides (gm , gm , gd a, gd b, gt b, and gq b) were similarly tested and judged for positive or negative interactions. in one mmn, anti-gm /gd a had negative interactions. in one cidp, anti-gm /gd a, and anti-gm /gq b had negative interactions. in one subacute sensory motor polyradiculoneuropathy, various anti-gscs antibodies including gd b or gq b had negative interactions. in the cidp patient mentioned above, anti-gm /gt b or anti-gm /gt b had no positive interactions. the relationship between this attenuated reactivity presumably driven by adjacent gangliosides and the mechanism of chronic immune-mediated neuropathies needs to be clarified. galino j , cervellini i , zhu n , stöberl n , fricker fr , lee g , hütte m , lalli g , bennett dl . the nuffield department of clinical neurosciences, john radcliffe hospital, university of oxford, oxford, uk; wolfson card, king's college london, guy's campus, london, uk. schwann cells (scs) are the myelinating cells in peripheral nerve system and are important for normal peripheral nerve development and repair. rala and ralb are small gtpases that have been implicated in neural tube closure, neurite branching and have been described as upstream effectors of proteins involved in cell migration and membrane dynamics. due to their potential role in sc function here we investigated, by genetic ablation in transgenic animals of one or both gtpases, their importance in sc function in adulthood and in nerve repair. we showed that ral gtpases are dispensable for sc function in the naïve state. however ral signalling (provided by rala or ralb) is required for effective remyelination of axons following nerve injury. moreover, absent ral signalling produced defects in axon reinnervation of distal organs and a delay in motor function recovery after nerve injury. we also studied the ral dependent cellular mechanisms that may be responsible for impaired sc remyelination and noted abnormal sc lamellipodia formation that prevent normal axial and radial axon remyelination. this work demonstrates for the first time a novel mechanism for ralgtpases that controls sc lamellipodia formation and their importance in normal sc function during peripheral nerve repair. garcía-lareu b , , , ariza l , cobianchi s , , chillón m , , , , navarro x , , , bosch a , , . tumor necrosis factor (tnf) alpha has been implicated in the pathogenesis of diabetic peripheral neuropathy (dpn), among other inflammatory demyelinating diseases and neuropathic pain. tnf alpha is a pro-inflammatory cytokine that can act at several stages in the demyelination process. it is produced by schwann cells (scs) in the peripheral nervous system (pns) after nerve injury and released into the local environment to attract and activate macrophages at the site of injury, contributing to wallerian degeneration. in vivo studies demonstrated a local inflammation in the sciatic nerve of rats after injection of tnf alpha, followed by demyelination and axonal degeneration. furthermore, the application of tnf alpha resulted in acute mechanical hiperalgesia, a main characteristic of neuropathic pain and therefore tnf alpha is postulated as a biomarker for painful changes after nerve injury. with the aim to characterize tnf alpha effects in chronic neuropathic pain and in diabetic neuropathy, a transgenic mouse model overexpressing tnf alpha cdna under the peripheral myelin protein p promoter was generated. here we characterized the overexpression of tnf alpha in myelinated scs at different stages of myelination (postnatal days , and ) showing that high levels of tnf alpha in sciatic nerve leads to the downregulation of the major pns myelin proteins (p , mbp, pmp , mag) compared to wild type mice, correlating with the loss of structured myelin and an increase in p ntr, a marker for immature and non-myelinated scs in the sciatic nerve. iba staining showed high levels of macrophage infiltration in both sciatic nerve and spinal cord tissues, compared with wild type animals. stress conditions were induced by sciatic nerve crush surgery after which recovery and subsequent remyelination were delayed in the transgenic mice, as evaluated by the sciatic functional index (sfi) and electrophysiological tests. on the other hand, mechanical and thermal nociception seemed to be unaltered, with or without lesion. this model could be helpful in the characterization of the role tnf alpha in pain development, injury and dpn as well as in developing efficient therapeutic strategies to modulate such pathological conditions. garcía-sobrino t , , blanco-arias patricia , , vidal-lijó mp , quintáns bea , , sobrido mj , , pardo j , . department of neurology, hospital clínico, santiago de compostela, spain; neurogenetics research group, instituto de investigaciones sanitarias (idis), santiago de compostela, spain; genomic medicine group (u ), centre for biomedical network research on rare diseases (ciberer), spain; department of neurophysiology, hospital clínico, santiago de compostela, spain. charcot-marie-tooth (cmt) disease is a genetically heterogeneous group of hereditary motor and sensory neuropathies. more than genes were involved in the disease pathogenesis. the objective of this study was to assess the genetic distribution of cmt disease in galicia (northwestern spain). patients were diagnosed as cmt if they had a consistent neurological and/or neurophysiological examination or if they had sensory motor neuropathy with a positive family history. a total of cmt adult patients ( % females) were evaluated with a median age of [ - ] years. the molecular diagnosis was achieved in patients ( %) with a higher success in cmt ( %) than cmt ( %). globally, pmp duplication was the most frequent finding ( %), followed by mutations in mpz ( %), mfn ( %), gjb ( %) and gdap ( %). in cmt , with expception of the pmp duplication, pathogenic variants in egr , nefl and mpz gene were most common. pathogenic variants in mfn and gdap accounted for % of cmt patients. several patients referred to our institution as cmt were diagnosed as hereditary motor neuropathy (hmn) and pathogenic variants in the bscl gene were the most frequent. pathogenic variants not previously related to cmt were identified in mpz, mfn , gjb , egr , nefl and sh tc genes. sporadic or autosomal recessive (ar) cmt accounted for the % of all diagnoses. the genetic epidemiology of cmt in galicia follows a similar pattern to other populations, although some remarkable features are axonal gdap and demyalinating egr pathogenic variants as well a seemingly elevated proportion of ar cases. mutations in mpz gene are associated to a wide range of phenotypes. the r c, in particular, is associated to a severe and early onset disease. some cmt patients respond to immunosuppressive or immunomodulatory treatment, some of them harbouring mpz mutations. we present the case of a -year-old brazilian female with a severe cmt b (r c) that presented an acute episode of worsening after a febrile exanthematic disease. she complained of tingling, stopped walking without support, and could not raise her arms. her emg showed an asymmetrical reduction of motor cv, reduced amplitudes, severe temporal dispersion and possibly conduction block. csf protein was mg%, with normal cell count. diagnosed as gbs, she received ivig. curiously, she markedly improved, regaining the functional status of youth, recovering functions that she had long lost. on emg, motor amplitudes improved significantly, but cv remained the same. reviewing carefully her past history, we identified some motor fluctuations along her adult life. at age , while pregnant, she developed positive sensory symptoms, became unable to get up from the chair and could not walk without assistance. after delivery, she improved, but did not return to baseline. one year later, she presented a new transitory functional worsening after an influenza illness. her parents were healthy but two out her children have a severe cmt. her first daughter never walked and died at the age of six. six months after treatment with ivig, she faced a new relapse. we pulsed with corticosteroids, with great response. she remains stable to date, four months after treatment. the episodic fluctuations and the evident response to treatment clearly suggest an associated immunomediated process. the fast improvement of the amplitude with maintenance of cv suggests that another mechanism in addition to demyelination and axonal degeneration is involved. we propose the existence of an associated channelopathy. funded by cnpq, faepa, pronas (ministry of healthy) gbs is an acute, immune-mediated neuropathy that comprises three major subtypes: aidp, aman, and amsan. on clinical grounds, this distinction is based on nerve conduction studies (ncs): aidp is a demyelinating neuropathy, aman is an axonal motor neuropathy, and amsan, an axonal sensory and motor neuropathy. recently, it has been demonstrated that axonal gbs, in addition to axonal degeneration, may present conduction block (cb) that can progress to axonal regeneration or revert, characterising a reversible conduction failure (rcf), a finding usually associated to good prognosis. patients presenting axonal cb are frequently diagnosed as having aidp, a mistake that can be avoided with two sets of ncs, one as early as possible and the second after three weeks of disease. following these recommendations, we retrospectively classified a brazilian group with gbs followed in our institution that had been submitted to two ncs sets. from september to january , patients fulfilled clinical criteria for gbs at clinical hospital of ribeirão preto and had more than one ncs accomplished. at least four motor nerves (median, ulnar, peroneal, and posterior tibial) and five sensory nerves (medial, ulnar, radial, sural, and peroneal superficial) were evaluated in each examination, according to routine procedures. first ncs revealed patients with aidp ( %), cases of axonal gbs ( aman, amsan, %), and patients with equivocal results ( %). at follow-up study, patients ( %) had their classification changed. the main shifts were from aidp to axonal group and from equivocal results to aidp. aidp increased to % (n = ), axonal gbs increased to % (n = , aman, amsan), and there were no more equivocal patients. although the majority of studies have shown that aidp is more frequent in western countries while axonal gbs predominates in eastern countries, we found a different pattern of distribution in our population, with predominance of the axonal subtypes. the considerable increase of axonal gbs at follow-up studies reinforces that serial emg are mandatory for accurate diagnosis of gbs subtypes. prospective studies are now being carried out in order to confirm these results. in amyloid light chain (al) amyloidosis, misfolded light chain (lc) accumulates and causes progressive peripheral neuropathy (pn) and progressive failure of critical organs such as the heart and kidneys. progressive ascending sensorimotor neuropathy is often a related clinical finding. the deposition of toxic lc amyloid in peripheral nerves is associated with paresthesias, pain, muscle weaknesss, orthostatic hypotension, and diarrhea or constipation in approximately % of patients with al amyloidosis. there are no approved treatments for al amyloidosis. current therapeutic approaches for al amyloidosis are intended to limit lc production but do not directly target misfolded lc deposited as amyloid in organs. we report phase / trial results of neod , an investigational monoclonal antibody that targets misfolded lc and may neutralize circulating lc aggregates and clear insoluble deposits. trial inclusion criteria were one or more plasma cell-directed treatment completed before enrollment, partial or greater hematologic response to any previous therapy, and persistent organ dysfunction. neod was administered intravenously every days during a dose-escalation phase ( patients; . , , , , , , or mg/kg in a + study design) and an expansion phase ( patients; mg/kg). we assessed safety, tolerability, pharmacokinetics, immunogenicity, organ responses based on consensus criteria, and pn responses using the neuropathy impairment score-lower limb (nis-ll). neod treatment was not associated with dose-limiting toxicities or serious adverse events, drug-related discontinuations, or antidrug antibody development in any patient (n = ). of patients with measurable pn at baseline (n = ), % achieved pn response based on the nis-ll score after months of treatment, which resulted in a median % nis-ll score reduction (mean baseline nis-ll, . ). in a best response analysis, % of cardiac-evaluable patients (n = ) and % of renal-evaluable patients (n = ) met respective criteria for organ response. improvements in neuropathy have not been previously shown in al amyloidosis. these results demonstrated that monthly neod infusions were safe, well tolerated, and associated with responses across three different organ systems. gervais cbl , ross ma , goodman bp , khoury ja , muzyka i , smith be . mayo clinic in arizona, scottsdale, az, usa. this work describes clinical, examination and electrophysiologic findings in a cohort of patients with length dependent sensorimotor peripheral neuropathy (ldsmpn) with ventral abdominal sensory loss. ldsmpn affects the longest nerve fibers, namely those innervating structures in the feet and hands. what is less well appreciated is that length dependent involvement of sensory nerve fibers in ldsmpn from the thoracic segments gives rise to ventral abdominal sensory loss. consecutive patients seen for ldsmpn (n= ) were evaluated for the presence or absence of ventral abdominal sensory loss. demographic variables, symptoms and quantitative neurologic findings (neuropathy impairment score [nis]) were examined using descriptive statistics. final diagnoses were noted. ventral abdominal sensory loss to pinprick (which was asymptomatic in all patients tested) was documented in / ldsmpn patients ( . %), mean age was . years (range - ), m:f gender ratio was . ( : ), mean nis was . (range - ), ncs/emg abnormalities were found in / patients (the remaining showing objective evidence of small fiber sensory involvement). ldsmpn patients without ventral abdominal sensory loss (n= ) had a mean age of . (range - ), m:f of . ( : ), and mean nis of . (range - ). no patient ( / ) had dorsal torso sensory loss between the shoulder and buttock levels on either side. diagnoses of the ldsmpn patients with vs. without ventral abdominal sensory loss included charcot marie tooth or other hereditary neuropathy (n= vs. ), abnormal carbohydrate metabolism (n= vs. ), idiopathic (n= vs. ), hypothyroidism (n= vs. ), inflammatory (n= vs. ) and other (n= vs. ). ventral abdominal sensory loss appears to be common in patients diagnosed with ldsmpn of a variety of causes including inherited neuropathies; in addition to those innervating distal limb territories, distal sensory fibers from the thoracic region represent another category of length dependent involvement in ldsmpn; ) the clinical examination of ldsmpn should include the ventral abdomen. gibbons c , garcia j , casasola m , freeman r . beth israel deaconess medical center, harvard medical school, boston, usa. objective: to determine the relationship between measures of autonomic function, electrochemical sweat conductance (esc) and intra-epidermal (ienfd) and sudomotor nerve fiber density (sgnfd). background: structural and functional measures of small fiber neuropathy have been studied in patients with diabetes, but little information comparing these techniques exists. design/methods: we studied patients with diabetes (ages ± yrs, gender f) and healthy control subjects (age ± yrs, gender f). subjects underwent examination scores (nis-ll), quantitative sensory testing, autonomic testing (heart rate variability, valsalva maneuver, tilt test), esc, and millimeter punch skin biopsies at the distal leg and distal thigh for ienfd & sgnfd. results: there were strong correlations between exam scores (nis-ll) and biopsy ienfd (r=− . , p< . distal leg; r=− . , p< . distal thigh), and sgnfd (r=− . , p< . distal leg; r=− . , p< . distal thigh). moderate correlations were noted between exam scores and qst (r values . - . , p< . ), ienfd and qst (r= . - . ). modest correlations were noted between esc and parasympathetic function (r= . - . , p< . ). modest correlations were noted between esc and ienfd (r= . , p< . , but only at the distal leg) and esc and nis-ll (− . , p< . ). no correlations between exam scores and autonomic function were noted. no correlations were detected between esc and sgnfd (r=− . - . ) or esc and sympathetic adrenergic function (r= . ). conclusions: differences between tests are expected based on our understanding of the pathophysiology and natural history of diabetic neuropathy. exam scores, qst results and biopsy results do correlate, and are consistent with prior studies. in contrast, there was little relationship between tests of autonomic function and exam scores, similar to prior studies and our understanding of autonomic function and differences in p. esc had little correlation with either exam scores or biopsy ienfd or sgnfd. there was a modest correlation between esc and parasympathetic function. the exact relationship between esc and diabetic peripheral neuropathy is not clear, and further research studies are needed to determine the role this technique has in clinical practice. objective: to determine the optimal tissue thickness of skin biopsy sections for studies of cutaneous nerve fibers. background: although analysis of intra-epidermal nerve fiber density (ienfd) is routinely reported using μm thick tissue sections, many recent studies of peripheral alpha-synuclein deposition use or μm frozen sections, or μm paraffin embedded tissue sections. design/methods: we compared the results of biopsies from patients with parkinson's disease (pd), using tissue sections each of , and μm thickness. tissues were stained with pgp . and stained for phosphorylated alpha-synuclein (p-syn). the total number of dermal structures (hair follicles, sweat glands, pilomotor muscles) were quantified, nerve densities analyzed, and the frequency of p-syn positive results. we also studied μm paraffin embedded tissue samples from patients with pd. results: in the biopsies of patients with pd there were no differences in the number of sweat glands, hair follicles or pilomotor muscles in , or μm sections. there were significantly fewer blood vessels noted in and μm sections compared to μm sections (p< . ). ienfd and sgnfd declined with tissue thickness (p. . , all) and there was increased variability in results in thinner tissue sections. there was a highly significant reduction in p-syn positive sections in thinner tissue sections (p< . , all tissues compared to μm sections). paraffin embedded tissue sections had significantly lower nerve densities and positive p-syn results (p< . ) compared to all frozen tissue sections of , and μm thickness. thinner tissue sections carried a greater risk of false positive result or indeterminate results due to difficulty interpreting overlap with pgp . . conclusions: tissue sample thickness plays a critical role in interpretation of skin biopsy results. thinner tissue sections, or paraffin embedded tissue sections, do not provide equivalent data and significantly underestimate nerve densities and positive alpha-synuclein results with increase false positive results despite similar numbers of dermal tissue structures. ace- is an investigational protein therapeutic that acts as a localized ligand trap for myostatin and other negative regulators of muscle growth. local injection of ace- into the gastrocnemius muscle of wild-type, mdx, and sod mice produced dose-dependent increases in muscle mass and force without systemic effects. in a phase single-center, double-blind, placebo-controlled dose escalation study in post-menopausal women, unilateral injections of ace- into the rectus femoris (rf) or tibialis anterior (ta) muscle were generally safe and well tolerated. mean percent changes from baseline in muscle volume of the injected muscle were + . % in the rf and + . % in the ta at the highest dose administered with minimal changes observed in the contralateral side and placebo-treated subjects. frequent related aes (≥ %) included injection site pain, pain in extremity, injection site discomfort, and muscle twitching, with similar incidence in ace- and placebo-treated groups. all aes were grade - and reversible. together, these preclinical and clinical results support further studies of ace- in myogenic and/or neurogenic diseases with focal loss of muscle strength and function, including cmt. a phase study is ongoing in facioscapulohumeral muscular dystrophy (fshd). study a - is an ongoing multicenter, two-part, phase study to evaluate the safety, tolerability, pharmacodynamics, efficacy, and pharmacokinetics of ace- in patients with cmt and cmtx. part is open-label and will enroll up to dose-escalating cohorts ( patients per cohort); part is randomized, double-blind, and placebo-controlled, and will enroll an additional patients. ace- will be administered bilaterally to the ta muscle once every three weeks for up to five doses. a safety review team will meet periodically throughout the study to review safety data and make dosing recommendations, including the recommended dose level for part . eligible patients must have genetically confirmed cmt or cmtx with mild-moderate weakness in ankle dorsiflexion. safety and tolerability will serve as the primary outcome for part , muscle volume evaluated by mri for part . additional outcome measures of interest include strength by quantitative muscle testing, function by motor tests, and quality of life by the cmt-health index questionnaire. our study was aimed to evaluate the functional and morphological consequences of cellular and humoral responses in chronic inflammatory demyelinating neuropathy (cidp), using extensive standardized high-resolution sonography (hrus) and nerve conduction study (ncs) protocols in incident treatment-naive patients. we enrolled consecutive, newly diagnosed, treatment naive patients with cidp. in addition to all relevant clinical examinations, all patients underwent a standardized ncs and extensive hrus protocol, of median, ulnar, tibial, fibular and sural nerves. we assessed standard nerve and fascicle size, and echogenicity. we found focal sonographic enlargements in multiple nerves and nerve segments with and without ncs abnormalities. the degree of nerve hypertrophy was not associated with presence of ncs features of demyelination, i.e. / ( %) of median nerve segments showed enlargement without strong decrease in motor conduction velocity and / ( %) hypertrophic median nerve segments revealed no conduction block. a lower distal cmap of median nerve was related with lower mrc sums-scores (p < . ). we found no correlation between age, disease duration or mrc sum-score and nerve size. cellular and humoral responses in cidp may lead to nerve enlargement along the length of nerves, that can be detected by hrus, whereas ncs allows identification of its' specific focal disruption in nerve function. the most common complication of diabetes is peripheral neuropathy, which has prevalence as high as % and is characterized by damage to neurons, schwann cells and blood vessels within the nerve. the concept of schwannopathy as an integral factor in the pathogenesis of diabetic neuropathy is re-emerging, and it is now known that schwann cells cultured in hyperglycemic environments underproduce neurotrophins and exhibit loss of axonal associations, further indicating a non-optimal glial cell activation and function. furthermore, the increased expression of p ntr in myelin sheaths around fibers that are susceptible to axonal degeneration in diabetic neuropathy suggest an important role for this molecule in disease progression. with this project, it is our main goal to evaluate how disruption of p ntr signaling in the schwann cells affects the pathophysiology of diabetic neuropathy. by using a fluorescent live/death cell viability assay, our preliminary data indicate that wild-type schwann cell cultures present increased cell death rate h after stimulation with high levels of glucose. the p ntr has a highly recognized role in the activation of death signals and when absent, we observed that schwann cells are significantly more resistant to apoptosis in hyperglycemic conditions. the role of p ntr receptor signaling in neuron-schwann cell communication and myelination under in vitro diabetic conditions was investigated with primary schwann cell-sensory neuron co-cultures. after eight days of ascorbic acid stimulation, both under euglycemic and hyperglycemic conditions, myelination was assessed by confocal microscopy using specific markers for neurons and myelin. results highlight a compromised ability of wild-type schwann cells to myelinate axons when exposed to a hyperglycemic environment, which was even intensified in co-cultures with schwann cells lacking the p ntr . to complement the in vitro studies, we are modeling type diabetes in a p ntr schwann cell conditional ko mouse model and plan to investigate nerve mrna expression profile to disclose genetic regulation depending on this receptor signaling and its modulatory role in endoneurial hypoxia and neuroinflammation. the results from this project will provide an integrated vision of how impaired schwann cell activity guides neuropathy progression. gondim faa , barreira aa , cruz mw , cunha fmb , de freitas m , frança mc jr , marques w jr , nascimento ojm , oliveira asb , pereira rc , pupe c , rotta ft , schestatsky p . panelists on behalf of the scientific department of peripheral neuropathy, brazilian academy of neurology, brazil. neuropathy is one of the most common neurological manifestations of several diseases and sfn has been progressively receiving more attention in the medical literature. the aim of this study is to generate a set of recommendations to define and diagnose sfn in brazil. a group of neurologists, members of the scientific department of peripheral neuropathy from the brazilian academy of neurology reviewed a preliminary draft prepared by the first author that was distributed by email. the panelists got together on . . at the city of fortaleza, brazil, to discuss and finish the text for the first submission of the manuscript. sfn can be defined as a subtype of neuropathy characterized by selective involvement of unmyelinated or thinly myelinated sensory (sometimes also autonomic) fibers. it is usually characterized by sensory (pain/dysesthesias/pruritus) or combined sensory and autonomic complaints, associated with an almost entirely normal neurological examination (except for sensory changes). electromyography is normal. a growing list of medical conditions has been linked to sfn, although there is no evidence-based literature to support the use of any specific set of screening tests to diagnose the etiology of sfn (the panelists will suggest a basic screening panel). sfn may also serve as a fallacious but useful terminology to uncover discrepancies in the normal values from different neurophysiology laboratories. in brazil, skin biopsy is not usually performed and initial forms of leprosy may have predominant small fiber involvement. there are several tests to demonstrate involvement of small sensory and autonomic fibers. skin wrinkling test, sympathetic skin responses & heart rate variability (conducted on emg machines) and thermoregulatory sweat test may be low-cost screening alternatives. after the final meeting on . . , we finished the first draft for submission to arquivos de neuropsiquiatria (together with a translation to portuguese as supplementary material), the official journal of the brazilian academy of neurology to serve as a source for the definition and diagnosis of sfn in brazil. the final draft will be submitted after presentation at the pns meeting in barcelona on . . the lower cranial nerves (lcn) include the paired glossopharyngeal, the vagal, the accessory and the hypoglossal nerves. these are involved in the execution of swallowing, speech, phonation, tasting, as well as sensory and autonomic functions. lcn can be affected in central and peripheral nervous system diseases. this study investigates the use of ultrasound (us) to detect lesions of the peripheral course of lcn. in addition mri can be used in unilateral lcn local lesions to demonstrate indirect signs of nerve lesions such as muscle atrophy. the patients were examined in supine position with the neck in maximum extension with either a regular or a portable us system (logiq e and logiq e ge healthcare, milwaukee, wisconsin) and high frequency transducers ( to mhz). according to the detected pathology longitudinal or transversal images were recorded. a series of exemplary observations are demonstrated. in one case a paraganglioma in the glossopharyngeal nerve at the carotid sinus was found. in another patient an us of the thyroid gland revealed a nerve tumor, which was later identified to be a schwannoma. in another case multiple neurofibroma were identified in the vagal nerve during a routine neurofibromatosis screening. us has become also important in investigations of lesions of the accessory nerve, following damage by surgery. nerve continuity and scar formation can be distinguished. lesions of the hypoglossal nerve can be caused by tumor infiltration. in one patient an infiltration of the nerve by a squamous cell carcinoma, and in another case by a low grade sarcoma were detected. in addition mri demonstrated an atrophy of the tongue. identification and assessment of the lcn from the point of exit of the skull to their endpoint can be pursued by us techniques, which have been described for the individual nerves. us is easily applicable and -in addition to the "static" image of the mri -allows assessing nerve positions during different movements, as well as detecting muscle movements as fasciculations. mri can also be used to detect not only more proximal lesions, but also changes of the skeletal muscle by denervation. grümme l , hattenhauer st , wolffram k , kleinschnitz c , mausberg ak , stettner m . university hospital essen, essen, germany; heinrich-heine-university, düsseldorf, germany. in chronic inflammatory demyelinating polyneuropathy (cidp), t cells are suspected to play a crucial role in myelin destruction. cd and cd t cells contribute to the inflammatory process, while the exact mechanism of myelin damage is still under debate. to elucidate the molecular interaction of t cells and myelin sheets, we compared neuritogenic and control cells in a live imaging in vitro model. the myelinated fibres of rat dorsal root ganglia (drg) served as model for the peripheral nervous system. drgs of embryonic (e ) lewis rats were cultured; myelination was initiated after one week in vitro and continued for two additional weeks. lewis rats immunized with p and neuritogenic t cells of the lymph nodes were obtained ten days after immunization. control t cells were prepared from healthy rats. for vital tracking, neuritogenic t cells were stained with orange cell tracker, while the control cells were labelled with cfse. myelin detection in vital cultures was assessed by incorporation of c fatty acids conjugated with a fluorophore into the myelin layer. experiments were performed in a conditioned microscope chamber, the two t cell populations were added simultaneously to the drg culture and migration was tracked using live cell imaging. we observed differing migration patterns for neuritogenic and control t cells. the velocity as well as the directionality was altered. after initial contact, the non-neuriotgenic t cells in close proximity to myelin subsequently decreased over time, while the numbers of neuritogenic t cells close to myelin remained stable. long term incubation with neuritogenic t cells affected the myelin integrity in regard to the intermodal length as well as the myelin ratio. further experiments will elucidate the specific effects of neuritogenic t cells to decipher the role of t cells during inflammation in the pns, which could be useful in developing targeted therapies. gundapaneni b , sultan mb , keohane dj , schwartz j . inventiv health inc., burlington, ma, usa; pfizer inc., new york, ny, usa. transthyretin familial amyloid polyneuropathy (ttr-fap) is a rare, life-threatening disorder caused by protein destabilizing ttr gene mutations, broadly classified as val met (the most common mutation worldwide) and non-val met genotypes. tafamidis, a highly-specific ttr-stabilizer, is the only medicine approved to delay neurologic progression in ttr-fap. the objective of this post-hoc analysis was to compare the effects of tafamidis on neuropathy progression in patients with val met and non-val met genotypes. val met patients were participants in a randomized, double-blind, placebo-controlled clinical trial of tafamidis, while non-val met patients were participants in an open-label tafamidis study. patients were grouped into three cohorts: val met tafamidis (n= ); val met placebo (n= ); and non-val met tafamidis (n= ). baseline disease severity and change in disease severity from baseline to month was assessed using the neuropathy impairment score-lower limbs (nis-ll). the effect of tafamidis in the val met and non-val met cohorts versus the val met placebo cohort was determined using a mixed-effects model for repeated measures (mmrm). at baseline, patients in the non-val met cohort were older, had longer symptom duration, and more advanced neurologic impairment than the val met cohorts. at month , the baseline-adjusted mean ± standard error change in nis-ll was comparable between the val met tafamidis and non-val met tafamidis cohorts ( . ± . and . ± . , respectively). these changes were smaller than that observed in the val met placebo cohort ( . ± . ; p= . vs val met and p= . vs non-val met) indicating less disease progression. based on predicted values from the mmrm analysis, the size of the change in nis-ll across the full range of baseline nis-ll scores was remarkably similar in the val met tafamidis and non-val met tafamidis cohorts and was consistently smaller than that observed in the val met placebo cohort. moreover, in all three cohorts, as baseline nis-ll increased, the predicted level of disease progression also increased. in conclusion, while controlling for baseline disease severity, tafamidis delayed disease progression to a comparable extent in val met and non-val met patients. the similar trajectories of disease progression across val met and non-val met patients suggest that these two genotype groups may be more similar than previously considered. clin-icaltrials.gov identifiers: nct ; nct . gutmann l , shy m . university of iowa, iowa city, ia, usa. post tetanic potentiation (ptp) is a physiological phenonmenon seen with disorders of the neuromuscular junction (nmj). it is caused by the influx of ca++ into the terminal axon during the tetanus resulting in an increased number of acetylcholine (ach) vescicles released by each axonal action potential. in myasthenic syndromes it results in improved nmj function by increasing the probability of achieving a large enough end plate potential to generate a muscle action potential. ptp has different appearances depending on whether the nmj defect is pre-synaptic or post-synaptic. in pre-synaptic defects (the most common being lambert-eaton syndrome) there is an increased amplitude of the muscle action potential after a tetanus that persists up to minutes. prolonged ptp has now been reported in disorders, one on a genetically determined neuropathy/myasthenic basis and the other on an acquired toxic origin. prolonged ptp up to minutes was reported in families with a motor neuropathy (having leg weakness and foot deformites) and a congenital myasthenic syndrome caused by a heterozygous mutation of the synaptotamin ii (syt ) gene (c t>g p.pro [p.asp ala] and c g>a [p.pro leu]). electrophysiological testing showed features of a presynaptic defect with prolonged ptp persisting up to minutes. the same phenomenon has been noted in the acquired pre-synaptic defect caused by botululinum toxin. the ptp continued up to minutes. syt is the synaptic vescicle calcium sensor in the terminal axon, allowing for fusion of ach containing vescicles with the presynaptic membrane and the synchronous release of ach. the fusion requires a complex assembly process involving snare proteins. both the syt gene mutation and botulinum toxin affect normal syt function, the mutation by altering amino acids in the calcium-binding domain and the toxin by binding to syt as well as gangliosides gd a and gt b on the neural membrane. the mechanism for the ptp prolongation remains unknown. prolonged ptp appears to be a unique physiological abnormality resulting from altered syt . this phenomenon has been decribed to occur in syt mutations causing congenital motor neuropathy/myasthenic syndrome and botulism. the abnormality may represent a physiological marker for a presynaptic nmj defect involving altered syt . hachisuka a , , senger j , curran m , chan km , . division of physical medicine and rehabilitation, university of alberta, edmonton, canada; department of rehabilitation and medicine, university of occupational and environmental health, kitakyushu, japan; division of plastic surgery, university of alberta, edmonton, canada. background: motor unit number estimation (mune) techniques are valuable tools in neuromuscular disease. among them, the multiple point stimulation (mps) is one of the most common used. contamination by distant single motor unit potentials (smups) generated by neighboring muscles is a potential confounding factor. this is particularly problematic in ulnar neuropathy, one of the most common neuropathies in humans. reason being that the ulnar nerve innervates the majority of hand muscles. the goals of this study are to test the hypotheses that ) distant smups all have an initial positive deflection and ) elimination of smups generated by distal muscles will significantly lower the mune results in the hypothenar muscles. methods: to address the first hypothesis, we tested subjects by stimulating their median nerve while recording smups simultaneously over the hypothenar and thenar muscles. for the second hypothesis, we carried out mps mune of the hypothenar muscles using multi-channel recordings placed over ulnar innervated intrinsic muscles across the hand. when a smup with an initial positive deflection was detected at the hypothenar electrodes, its original was systemically tracked through all the recording channels. results: in the first series of experiments, in accordance with the dipole theory, all smups recorded at the hypothenar recording electrodes had an initial positive polarity. in the second series of experiments, of the studies carried out in subjects, distant smups generated by muscles other than those in the hypothenar eminence represented ± . % (mean±sd) of the overall sample. mune calculated using only smups generated by the hypothenar muscles was ± , compared to ± if all smups were included (p < . ). the extent of increase in mune was highly correlated with the proportion of distant smups found in each study (r = . , p < . ). conclusion: in contrary to some studies suggesting that smups from distant muscles could have an initial negative deflection, we found all smups from distant muscles had a positive deflection. exclusion of those smups from the sample had a significant impact on the mune results. hagiwara w , konno s , kihara inoue m , fujioka t . toho university, tokyo, japan. matrix metalloproteinase (mmp) plays crucial roles in developing immune-mediated neuritis as guillain-barré syndrome (gbs) and its animal model experimental autoimmune neuritis (ean). to investigate the intraneural expression of mmps during ean and the effect of a phosphodiesterase- inhibitor cilostazol (clz) on it, ean rats were treated with either mg/kg/day of clz or vehicle from one day post immunization (dpi). to induce ean female lewis rats were immunized with synthetic peptide from bovine p protein. cauda equina (ce) were removed in several time points, total rna was extracted and reverse-transcribed to obtain cdna that was subjected to real-time pcr analysis for expression of mmp- , mmp- and tissue inhibitor of matrix metalloproteinase- (timp- ) messages. mmp- and mmp- messages peaked at dpi, that is presymptomatic phase of ean. all rats developed motor paralysis at dpi, mmp- and mmp- messages subsided at this moment. however, timp- message reciprocally increased at dpi, persisted through dpi. treatment of clz suppressed motor paralysis of ean significantly. mmp- message peaked at dpi and mmp- message peaked at dpi. on the other hand, both messages at dpi were suppressed compared to untreated ean rats. timp- message in clz treated rats peaked at dpi coincided with motor paralysis peak. both mmp- and mmp- messages might result in subsequent upregulation of timp- that finally downregulates mmps activity and inflammatory process. clz treatment suppressed and delayed expression of mmps and facilitate timp- expression, resulting suppression of ean. the precise mechanism of expression of mmps and timp- remain unclear, however, that mmp- and mmp- messages peaked at dpi suggests involvement of pro-inflammatory cytokines such as tumor necrosis factor-alpha or interleukin- . clz might suppress these cytokines resulted in mmps down regulation. mmp- less affected by clz and thus stimulated timp- expression at dpi. clz might rational treatment for immune-mediated neuropathy via mmps modulation although further investigation especially in-vivo study is needed. haidar m , de winter v , asselbergh b , bouhy d , timmerman v . peripheral neuropathy research group, vib, university of antwerp, antwerp, belgium. the small heat shock protein hspb (hsp ) gene is ubiquitously expressed and encodes for a chaperone protein with essential cellular functions. our lab was the first to identify missense mutations in hspb responsible for axonal charcot-marie-tooth neuropathy (cmt f). since then we became interested in understanding the physiological functions of hspb and its association with cmt neuropathies. we demonstrated the involvement of hspb in microtubule stability. because of the link between autophagosome formation and its intracellular transport, and microtubules stability, we believed that the macro-autophagy process could be regulated by hspb . macro-autophagy is a cellular housekeeping process during which autophagosomes target, envelop and degrade aberrant protein aggregates and damaged organelles. there is strong evidence for an essential role for autophagy in the maintenance of neuronal homeostasis; hence its impairment can lead to a neuropathic condition. our data indicate that macro-autophagy is disrupted by hspb cmt-causing mutations. combining novel microscopy and interactomics techniques we unravelled the way different cmt-causing mutations in hspb impair the autophagic pathway. our data present the impairment of autophagy as a possible pathomechanism for cmt-causing hspb mutations. hajjar h , gautier b , berthelot j , gonzalez e , gess b , young p , tricaud n . institute of neurosciences of montpelllier, inserm, university of montpellier, montpellier, france; universitätsklinikum münster, klinik für schlafmedizin und neuromuskuläre erkrankungen, münster, germany. cmt a, the most common of charcot-marie-tooth diseases, results from the duplication of peripheral myelin protein (pmp ) gene. this gene encodes for a small protein of kda, pmp , mainly produced by schwann cells and the excess of pmp leads to demyelination. there is no cure for this disease but one approach for a treatment is gene therapy. a transgenic rat model exists for cmt a, which possesses copies of the mouse pmp gene. our goal is to provide a proof of principle for gene therapy in peripheral nerves using this rat model of cmt a. our strategy is to reduce the overexpression of mouse pmp protein in rats schwann cells using short hairpin rnas (shrnas). shrnas are small non-coding rnas that specifically bind to targeted mrnas resulting in their degradation. we tested for the efficiency of several shrnas targeting mouse pmp in vitro to find two shrnas that reduce pmp levels. the shrnas have been cloned in an adeno-associated serotype (aav ) viral vector together with green fluorescent protein in order to detect infected cells. aav was selected for its high transduction rate of myelinating schwann cells, for its good diffusion and low immunogenicity. we plan bilateral injections in the sciatic nerve of control and diseased rats. the efficiency of this gene therapy will be checked by assessing muscle strength (grip test), way of walking (catwalk), mobility (rotarod) and nerve conduction velocity of treated cmt a rats versus non-treated. the process of myelination and myelin maintenance in schwann cells will be analyzed by biochemistry and electron microscopy. biochemical tests include western blot for pmp protein expression in sciatic nerve, immunohistochemistry for pmp protein expression in myelinating schwann cells and pcr for mrna pmp expression. if the therapy is successful in rats, it could possibly be later on used in clinical trials. ultrasound is a widely-used tool in diagnosing carpal tunnel syndrome (cts). several different methods for sonographical evaluation of median nerve damage exist, such as calculating the ratio of cross sectional areas (csa) of median nerves in various sites. this enables detection of actual nerve swelling proximal to the carpal tunnel as an expression of median nerve damage inside the carpal tunnel (as seen in cts). in comparison to other diagnostic methods no data exist about the prevalence of cts like changes in an unselected population. a series of non-selected fresh cadavers were examined. arms of fresh, non-embalmed whole body cadavers were examined. the medical record did not allow to obtain conclusive information on the peripheral nerves. using a regular ultrasound system with a mhz transducer, median nerves were identified and tracked along their course in the forearms. csa measurements of the median nerves were performed at two sites in each arm: .) halfway between the elbow joint and wrist, .) directly proximal to the carpal tunnel. csa ratio was calculated with the following formula: csa ratio = csa wrist (cm ) csa forearm (cm ) csa ratio < . was found in ( . %) arms, csa ratio ≥ . in ( . %) arms and csa ratio ≥ in ( %) arms. csa ratio ≥ . was detected in . % of women and % of men. the overall mean (± sd) age was . ± . years. men ( . ± . ) were significantly younger than women ( . ± . ; p = . ). a weak but significant correlation between age and csa ratio was found in women (spearman -− . ; p= . ), but not in men (p= . ). the mean bmi for csa ratio ≥ . was . ± sd . . based on a csa ratio ≥ . as a criterion for cts, the present ultrasound results are consistent with the average cts prevalence reported in previous studies, which were obtained with electrophysiological methods. this study on a large unselected series of cadavers confirms the comparability of both methods. instrumental activities of daily living (badl and iadl), falls and gait patterns in the large, prospective, population based rotterdam study. in total, participants of this study (mean age years, % women) underwent a polyneuropathy screening involving a symptom questionnaire, neurological examination and nerve conduction studies. screening yielded four groups: no, possible, probable and definite polyneuropathy. participants were interviewed about badl (stanford health assessment questionnaire), iadl (instrumental activities of daily living scale) and frequency of falling in the previous year. in a random subset of participants, gait was assessed with an electronic walkway (gaitrite). associations of polyneuropathy with badl and iadl were analyzed continuously with linear regression, and dichotomously with logistic regression. history of falling was evaluated with logistic regression and gait changes were evaluated with linear regression. we found that participants with definite polyneuropathy had more difficulty in performing badl and iadl than participants without polyneuropathy. polyneuropathy related to worse scores of all badl (especially walking) and three iadl components (housekeeping, traveling, and shopping). participants with definite polyneuropathy were two times more likely to fall, and these falls more often resulted in injury. participants with polyneuropathy had worse gait parameters on the walkway, including lower walking speed and cadence, and more errors in tandem walking. in summary, chronic polyneuropathy is strongly associated with significant impairment in daily life. recognition of polyneuropathy and related disability is very important in order to inform, support and possibly treat patients, and to prevent future falls and dependence in daily functioning. patients with polyneuropathy often suffer from tingling sensations, numbness, weakness and pain. these symptoms are used in several screening questionnaires, most of which were developed for high-risk patient groups, such as individuals with diabetes mellitus. in most tools equal weights are applied to all symptoms, while some might be more informative than others. we evaluated the diagnostic value and frequency of occurrence of individual symptoms of chronic polyneuropathy and constructed and validated a simple screening questionnaire that can reliably help to diagnose polyneuropathy in low-risk patient groups. in a multi-step procedure, we initially compiled a twelve-item questionnaire concerning symptoms of polyneuropathy. the questionnaire was completed by polyneuropathy patients and controls (headache, transient ischemic attack, multiple sclerosis). we calculated the sensitivity, specificity and likelihood ratios of each individual symptom. next, stepwise multivariable logistic regression was used to create a compact model, able to discriminate cases from controls using only the most informative symptoms. a simple scoring system was subsequently developed based on the regression coefficients of this reduced model. external validation was subsequently conducted in a population of cases with chronic idiopathic axonal polyneuropathy and controls without polyneuropathy. performance was assessed with discrimination (area under the curve, auc), and calibration. numbness and tingling feet were most frequently reported by polyneuropathy patients and had the highest sensitivity. feeling as if walking on cotton wool and allodynia of the feet had the highest specificity. multivariable logistic regression yielded a model that contained these four symptoms, complemented with balance problems and tingling hands. based on this regression analysis, the erasmus polyneuropathy symptom score (e-pss) was created, a score ranging from to . this polyneuropathy symptom score had a good performance (auc . ) in de derivation set and proved to be valid in the external population (auc . ). in this study, we created a simple, validated polyneuropathy symptom score (e-pss) that takes both the individual value of only six different symptoms and its frequency into account. this tool can be helpful as screening instrument in clinical practice and for future studies on polyneuropathy. multiple sclerosis (ms) is traditionally viewed as a central nervous system disease. to date, there is no unequivocal evidence implicating involvement of the peripheral nervous system (pns). this study aims to prove whether the pns is additionally affected and if so, to detect, localize and quantify these peripheral nerve lesions in patients with multiple sclerosis (ms) by applying high-resolution mr-neurography (mrn) with large anatomical coverage in combination with standard electrophysiological and neurological tests. we prospectively enrolled patients with confirmed ms (> years), two patients with clinically isolated syndrome (cis), and age-/sex-matched healthy volunteers. any other potential causes for a concomitant polyneuropathy were excluded. all ms patients underwent detailed neurological and electrophysiological testing. tesla mrn with large anatomical coverage from lumbar plexus and spinal nerves down to ankle level was performed in all participants by using fat-saturated, t -weighted turbo-spin-echo (tse) sequences (tr/te / ms) and a dual echo tse sequence for t -relaxometry (tr ms; te /te / ms). a d t -weighted, fat-saturated space sequence (tr ms; effective te ms) was used for imaging of the lumbar plexus. manual segmentation of spinal/sciatic/tibial/peroneal nerves was performed on a total of , axial slices. besides evaluation of nerve t w-signal, detailed quantification of nerve lesions by analyzing morphometric (nerve caliber) and microstructural markers (proton-spin-density and t -relaxation-time) was conducted. mean lesion load at thigh level was higher in ms ( . ± . ) vs. controls ( . ± . ;p< . ). nerve proton-spin-density was also higher in ms (tibial/peroneal: . ± . / . ± . ) vs. controls (tibial/peroneal: . ± . / . ± . ;p< . ). in contrast, t -relaxation time was significantly higher in controls (tibial/peroneal: . ± . / . ± . ) vs. ms (tibial/peroneal: . ± . / . ± . ;p< . ). proximal tibial and fibular nerve caliber was also significantly higher in ms (tibial: p< . ; fibular: p= . ). for the first time, pns lesions in ms patients could be visualized and quantified in vivo by high-resolution mrn. lesions are indicated by an increase of proton-spin-density and a decrease of t -relaxation-time. nerve caliber as a morphometric criterion also significantly increased. this proof-of-concept study may offer new insights into the pathomechanism of ms and might have future implications on therapeutic approaches. immunoglobulin g (igg) fc-gammars confer diverse effector functions by linking the cellular and humoral arms of the immune system that has been involved in the pathogenesis of guillain-barré syndrome (gbs). in the post-polio era, the polymorphisms of fc-gammar and their relevant knowledge have become one of the main targets for new therapeutic strategies for the treatment of gbs patients. differences in severity and frequency of gbs subtypes found between south-asian and western populations can be attributed to their genetic susceptibility. therefore, we aimed to determine fc-gammar polymorphic alleles (fc-gammariia: h /r ; fc-gammariiia: v /f ; fc-gammariiib: na /na ) and their possible link with gbs on the currently available large gbs cohort in bangladesh. fc-gamma r polymorphisms of gbs patients and healthy controls were genotyped using sequence-specific pcr. for validation, we carried out the sequencing of some samples for fc-gammariia and fc-gammariiia alleles. no significant differences were found regarding the distribution of fc-gammar genotypes and allele frequencies in gbs patients and controls. fc-gammar-h/h- genotype was significantly predominant in patients with severe disease compared to patients with mild disease (p= . , or, . ; % ci, . - . ). no other significant associations were found in gbs patients for candidate alleles and disease severity. fc-gammariiia-f/f- was found to be significantly predominant in anti-gm antibody positive gbs patients compared to anti-gm antibody negative patients (p= . , or, . , % ci, . - . ). fc-gammariiia-v alleles were significantly higher in patients with poor prognosis when compared to patients with good outcome (p= . , or, . , % ci, . - . ). no significant association of fc-gammariiib genotypes and alleles were found with gbs patients, disease severity and disease outcome. extensive subgroup analysis revealed no significant association in genotype and allele frequencies between aman and aidp subtype. in conclusion, igg fc-gammar polymorphisms do not constitute significant risk markers for susceptibility to gbs, however homozygous fc-gammariia-h might be involved in the severe form of gbs. in addition, fc-gammariiia-f/f- might play an important role in the molecular mimicry against nerve gangliosides in gbs. further studies that enroll a large number of patients (e,g. igos) are required to confirm the present findings from different geographical areas. hayes jm , o'brien pd , backus c , feldman el . department of neurology, university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common complication observed in patients with impaired glucose tolerance and type diabetes. male mice fed a high fat diet (hfd) develop metabolic impairments and pn serving as an appropriate animal model to study pn development and progression. it is well documented that female mice fed a hfd display a degree of protection against hfd-induced metabolic changes with mice retaining relatively normal insulin sensitivity. this protection is attributed to differences in fat accumulation and to the anti-diabetic effects of estrogen. based on these sex-dimorphisms we hypothesized that hfd-fed female mice would also exhibit resistance to developing pn. in the present study male and female c bl /j mice were fed either a standard diet ( % kcal fat; sd) or a high fat diet ( % kcal fat; hfd) from wk. at wk, wk and wk, neuropathy phenotyping was performed on all groups complemented with longitudinal metabolic assessments including insulin tolerance testing (itt). neuropathy phenotyping consisted of hindpaw latency to heat stimulus, motor and sensory nerve conduction velocities (ncvs), and terminal intraepidermal nerve fiber (ienf) counts. assessment of insulin resistance through itt demonstrated that during early hfd feeding, female hfd-fed mice exhibited relatively normal insulin responsiveness, while male hfd mice exhibited insulin resistance. despite this finding, wk female hfd mice displayed a similar pattern of pn to that of their male counterparts, with similar fold-changes in hindpaw latency and sensory and motor ncvs. therefore, although female hfd-fed mice exhibit resistance to hfd-induced metabolic changes, they display a pn comparable to male hfd-fed mice suggesting that systemic insulin resistance does not mediate pn. further studies are underway investigating the role of insulin signaling in the peripheral nerves of female hfd-fed mice. anti-mag (myelin-associated glycoprotein) neuropathy is a disabling autoimmune peripheral neuropathy caused by monoclonal immunoglobulin m (igm) autoantibodies that recognize the carbohydrate epitope hnk- (human natural killer- ). this glycoepitope is highly expressed on adhesion molecules, such as mag, present in myelinated nerve fibers. since the pathogenicity and demyelinating properties of anti-mag autoantibodies are well established, current treatments aim at a reduction of autoantibody levels. however, the therapies applied so far are primarily immunosuppressive and lack selectivity and efficacy. we therefore hypothesized that a significant improvement of the disease condition could be achieved by selectively neutralizing the pathogenic anti-mag antibodies with carbohydrate-based ligands mimicking the natural hnk- glycoepitope. in an inhibition assay, a mimetic (mimhnk- ) of the natural hnk- epitope inhibited mag-binding by pathogenic igm antibodies from patient sera, however only with micromolar affinity. therefore, considering the multivalent nature of the mag-igm interaction, polylysine polymers of different sizes were substituted with the mimetic. with the most promising polylysine glycopolymer pl (mimhnk- ) the inhibitory effect on patient sera was improved by a factor of up to , per epitope, consequently leading to a low nanomolar inhibitory potency. since clinical studies indicate a correlation between the reduction of anti-mag igm levels and clinical improvement, an immunological surrogate mouse model for anti-mag neuropathy, producing high levels of anti-mag igm, was developed. the observed efficient removal of these antibodies with the glycopolymer pl (mimhnk- ) represents a first step towards an antigen-specific therapy for anti-mag neuropathy. hinder lm , mendelson f , backus c , feldman el . university of michigan, ann arbor, mi, usa. in the united states % of children and young adults are obese and at risk of developing prediabetes. prediabetic patients largely develop the same macro-and microvascular complications as patients with type diabetes, including peripheral neuropathy (pn). moreover, recent clinical data suggest normoglycemic obese patients develop pn. central obesity, characterized by excess fat storage in visceral white adipose tissue, leads to systemic metabolic dysfunction largely due to an imbalance between pro-inflammatory/anti-inflammatory adipokine production. subcutaneous adipose tissue is considered 'benign', but adopts a visceral-like phenotype in response to metabolic stress, with reduced thermogenicity, reduced brown adipose identity, and increased pro-inflammatory gene expression. the popliteal adipose tissue (pat) depot, corresponding to subcutaneous adipose, is adjacent to the peripheral nerve affected in pn, contains the lymph node for lymphatic drainage of the hind limb, and expands following local, sterile hind paw inflammation. the aim of the current study was to characterize pat changes in the high fat diet (hfd) mouse model of obesity and pn, and consider its contribution to peripheral nerve dysfunction. we previously reported c bl /j mice fed % hfd from - wk develop obesity, 'prediabetes' and pn; and switching mice back to a standard diet from - wk improves metabolic and pn phenotypes. at and wk pat was bilaterally dissected and the lymph node removed. the left pat was processed for histomorphometry, and the right pat for rt-qpcr. at wk hfd was associated with a significant shift in adipocyte size-frequency distribution, with a greater number of larger adipocytes. switching the hfd mice back to standard chow from - wk restored the size-frequency distribution towards age-matched controls. rt-qpcr was performed to assess changes in thermogenicity (ucp ), brown adipose identify (cidea) and sterile inflammation (saa ). at and wk hfd pat had reduced thermogenicity and brown adipose identify, and increased sterile inflammation. this switch towards a visceral-like phenotype was reversed in the hfd mice switched back to standard chow. in summary, hfd-induced changes in pat histomorphometry and adipose identity closely associate with pn phenotype. these preliminary data suggest a potential role for pat-nerve signaling in pn. hinder lm , backus c , hayes jm , feldman el . university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common and debilitating complication of obesity and diabetes that triggers pain and loss of sensation. substantial nerve damage occurs in many patients prior to noticeable symptoms and no treatments are currently available; therefore, there is a critical need to identify treatment strategies that impact the underlying disease pathogenesis. our in vivo fluxomics data in the bks-db/db mouse model of type diabetes (t dm) and pn suggest that 'metabolic reprogramming' occurs in the t dm nerve to downregulate mitochondrial oxidative phosphorylation of substrates derived from glycolysis and fatty acid beta-oxidation. therefore, we hypothesize that distinct systemic metabolic alterations occur in obesity and diabetes which induce tissue-specific metabolic reprogramming within the peripheral nerve, altering fuel utilization and ultimately leading to tissue dysfunction. we contend that identifying conserved bioenergetic profiles across mouse models of pn will provide insight into key pn mechanisms. the current study utilized two mouse models of pn: the % high fat diet (hfd) mouse model of obesity and prediabetes at wk of age ( wk hfd), and the leptin receptor-deficient bks-db/db model of t dm at wk of age. mitochondrial function was determined in primary dorsal root ganglia (drg) neurons and sural nerve tissue from both models using the seahorse xf analyzer. resting mitochondrial oxidative metabolism was upregulated in drg neurons from mice with pn, with increased resting atp production and maintained mitochondrial coupling. in contrast, resting atp generation was decreased in sural nerve from mice with pn, with decreased coupling efficiency. relative spare respiratory capacity was attenuated in both drg neurons and sural nerve from mice with pn, indicating that mitochondria were less able to increase respiration in response to an energetic challenge. moreover, mitochondrial copy number was unchanged in drg neurons, but decreased in sural nerve tissue of mice with pn compared with respective controls. these data suggest a change in absolute number and function of sural nerve mitochondria, and a conserved cross-model proximal-distal bioenergetic profile in pn. we are currently exploring the relationship between these changes and pn pathogenesis. hoang ttn , umapathi t . hospital, ho chi minh city, vietnam; national neuroscience institute, singapore. ho chi minh city (hcmc) is the biggest metropolitan city in southern vietnam. its population is more than ten million. adult patients with neurological disorders are seen at six city public hospitals, including , hospital. there has been no systematic study of guillain barré syndrome (gbs) at hcmc or in vietnam in general. we are in the process of starting a prospective gbs database that we hope to expand to the other public hospitals at hcmc. here we describe our experience from . we saw gbs patients at , hospital. most of the cases were admitted in the rainy season, from late april to november, when mosquito-borne flavivirus infections are more common. patients were seen in the first week of illness and reported antecedent fever. two patients had diarrhea. diagnoses were made largely on clinical features, cerebrospinal fluid analysis and nerve conduction study. clinical findings include limb weakness, numbness and vii cranial nerve palsy. extraocular eye movements were affected in one patient. none had respiratory involvement severe enough to require artificial ventilation or intensive care. there were no pure miller-fisher syndrome cases; we suspect this might be related to the mild deficits that did not prompt hospitalization. nerve conduction studies showed typical features such as loss of f waves and abnormal blink reflex. the electrophysiology of patients' was dominated by demyelinating changes, one case was largely axonal and the remaining patient had normal electrodiagnostic study. repeat nerve conduction studies were not feasible because of limited resources. neurologists use corticosteroids as the main treatment. intravenous immunoglobulin and plasma exchange are costly and not reimbursed by medical insurance. we are currently preparing to systematically study gbs in southern vietnam, specifically with regards to possible role of antecedent flavivirus infections. we are also exploring the possibility of using low volume plasma exchange as a feasible cost-effective therapeutic modality. about % of patients with guillain-barré syndrome (gbs) treated with intravenous immunoglobulin (ivig) or plasma exchange deteriorate after initial improvement or stabilization -a phenomenon that is termed treatment-related fluctuation (trf). it is important to distinguish acute onset cidp (a-cidp) from gbs-trf during early course of the disease, because their therapeutic strategies and prognoses are different. herein, we describe a patient with gbs-trf, but with an extended progression phase that exceeds weeks. a -year-old woman was admitted due to acute onset progressive leg weakness and diplopia that had developed weeks prior (onset, d ). on neurological examination, right facial palsy was also observed. lower extremity weakness was moderate in proximal and distal muscles (mrc grade iv) with absent knee and ankle jerks. sensory examination revealed no abnormality in all modalities. she denied any recent diarrhea or upper respiratory infection, and vaccination. albumino-cytologic dissociation was noted in csf analysis; white blood cell count of / l and protein level of . mg/dl. nerve conduction study revealed demyelinating sensorimotor polyneuropathy with prolonged distal latency and conduction blocks. anti-ganglioside antibodies (gm igm/g, gd b igm/g, and gq b igm/g) were all negative. following ivig treatment, she was discharged with considerable improvement (d ). days later, she was re-admitted due to deterioration of leg weakness and hand clumsiness (d ). after another ivig treatment, she was discharged with clinical improvement (d ). days later, however, she was admitted again (d ) due to another considerable deterioration with four extremity weakness being worst at this time (mrc grade ii-iv in upper extremity, grade ii in lower extremity). a-cidp was considered given the progression phase exceeding weeks, but, we decided to give another treatment with ivig instead of a switch to corticosteroids because of uncertainty regarding distinction between a-cidp and gbs-trf. she was significantly improved following ivig treatment, and finally discharged (d ). thereafter, there has been no further deterioration during long-term follow-up of year. conclusively, this is a rare case of gbs with extended progression phase and trf. we propose that this could be referred to as subacute inflammatory demyelinating polyradiculoneuropathy (sidp) with trf. hsieh s , chao c . national taiwan university hospital, taipei, taiwan. transthyretin (ttr)-related familial amyloid polyneuropathy (fap) constitutes a major etiology of adult-onset hereditary neuropathies worldwide, in particular, a mutant ttr of ala ser (ttr-a s) in taiwan, the most common cause of acquired genetic neuropathy with adult onset (> years of age) of taiwanese patients. fap is a pan-modality neuropathy involving motor, sensory, and autonomic components of the peripheral nervous system with early involvement of small fibers as a major symptom. the early symptoms of fap are sometimes minimal and difficult to ascertain, mainly related to the fact that conventional electrophysiological examinations were not sensitive enough to detect small fiber neuropathy. skin biopsy with quantification of intraepidermal nerve fibers (ienf) has become one of the standard approaches to diagnose small fiber sensory neuropathy based on pathological documentation of nociceptive nerve degeneration. to explore the issue of early biomarkers in fap, we performed skin biopsy and compared ienf density with parameters of nerve conduction studies (ncs) and quantitative sensory testing (qst) on subjects ( men, aged . ± . years) with genetic confirmation of ttr-a s: patients and carriers. the ienf densities were significantly reduced compared to the age-and gender-matched controls in carriers ( . ± . vs. . ± . fibers/mm, p = . ) and patients ( . ± . vs. . ± . fibers/mm, p = . ). the latter was consistent with our previous report (neurology, : - , ) . the abnormal rate of ienf density was significantly higher than that of ncs and qst, respectively. in conclusion, there was significant skin nerve degeneration in carriers with ttr-a s. compared with qst and ncs, ienf density assessment had the highest abnormal rate and highest sensitivity to detect neuropathic changes in the early stage of fap. charcot-marie-tooth type p (cmt p) has been associated with frame-shift mutations in the ring domain of lrsam (an e ligase). this study describes families with a novel missense mutation of lrsam gene and explores pathogenic mechanisms of cmt p. this american family with dominantly inherited axonal polyneuropathy reveals a phenotype similar to those in previously reported non-us families. the affected members in our family co-segregated with a novel missense mutation cys arg that alters a highly conserved cysteine in the ring domain. this mutation leads to axonal degeneration in the in vitro neuronal cell-line. moreover, using protein mass spectrometry, we identified a group of rna binding proteins (including fus, a protein critically involved in motor neuron degeneration) that interacted with lrsam . the interactions were disrupted by the cys arg mutation, which resulted in reduction of intranuclear rna-binding proteins. a knockin mouse of cys arg has been created for further explorations of cmt p mechanisms and therapeutic development. together, our findings suggest that the mutant lrsam may aberrantly affect the formation of transcription machinery. given a similar mechanism has been reported in motor neuron degeneration of amyotrophic lateral sclerosis, abnormalities of rna/rna-binding protein complex may play a role in the neuronal degeneration of cmt p. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr ). this double-blind, multicenter, parallel-group trial randomized ( : ) adult participants (n= ) with chronic inflammatory demyelinating polyradiculoneuropathy being treated with intravenous immunoglobulin (ivig) or corticosteroids to . mg fingolimod (n= ) or placebo (n= ) once-daily. previous treatment was discontinued (ivig) or tapered (corticosteroids). in the total trial population, there was no significant difference between the groups in the primary outcome, time-to-first confirmed worsening (≥ -point increase on the adjusted inflammatory neuropathy cause and treatment [incat] scale vs. baseline), or time-to-first any worsening (confirmed on incat assessment or unconfirmed). no significant difference between the two treatment groups was shown on secondary outcomes; change from baseline in grip strength and rasch-built overall disability scale (r-ods) at six months or trial end. analyses of pre-specified subgroups were performed for primary and secondary outcomes. this trial evaluated the efficacy and safety of fingolimod in chronic inflammatory demyelinating polyradiculoneuropathy (cidp). corticosteroids, intravenous immunoglobulin (ivig) and plasma exchange are recognized treatment options but no other immunomodulators demonstrated efficacy in a controlled trial. fingolimod has been shown to be efficacious and is approved for the treatment of relapsing multiple sclerosis. results from experimental autoimmune neuritis in rats suggested that it might show an effect in cidp. in this double-blind, multicenter, parallel-group trial, cidp participants receiving ivig or corticosteroids were randomized to once-daily fingolimod . mg or placebo ( : ). participants were stratified by inflammatory neuropathy cause and treatment disability (incat) scores and prior treatment. previous ivig treatment was discontinued after one final course before randomization. previous corticosteroid treatment was tapered over weeks. the primary outcome was time-to-first confirmed worsening (≥ -point increase on the adjusted incat score versus baseline). secondary outcomes included change in grip strength and rasch-built overall disability scale (r-ods) score from baseline to month and at trial end. the trial was stopped for futility by an independent data monitoring committee after a pre-planned interim analysis based on pre-specified criteria. in all, participants received fingolimod (ivig: , corticosteroids: ; age: . ± . years [mean±standard deviation]; male: . %); received placebo (ivig: , corticosteroids: ; age: . ± . years; male: . %). the percentage ( % confidence interval) of participants free from confirmed worsening at the trial end was not significantly different between fingolimod ( . % [ . %- . %]) and placebo ( . % ( . %- . %); p= . ). in the first days, approximately % participants experienced worsening. at trial end, approximately % participants had no worsening. there was no significant difference after six months or at the trial end (whichever occurred earlier) in the secondary endpoints. adverse events were reported in / and / participants in the fingolimod and placebo group, respectively. there were no deaths. nine participants in the fingolimod group and in the placebo group had serious adverse events. adverse events leading to trial drug discontinuation occurred in ( %) participants on fingolimod and none on placebo. no new safety signals emerged in this trial. acknowledgment: the authors consulted for or were employed by the study sponsor novartis pharma ag, basel, switzerland. myelin sheath enwraps non-nociceptive mechanoselective abeta-afferents transmitting touch/vibration sense. a prominent reduction in the mechanical stimulus required to evoke a withdrawal response in rodents, a phenomena interpreted as mechanical allodynia, arises due to peripheral nerve/myelin damage. evidence has emerged that nerve injury-induced mechanical allodynia depends on the adaptive immune/t cell activity in female but not male rodents. having previously demonstrated both the release of the cryptic - peptide regions of myelin basic protein (mbp - ) following sciatic nerve chronic constriction injury (cci) and the direct, robust and t cell-dependent ability of the pure mbp - peptides to induce mechanical allodynia after injection into the intact sciatic nerve, we hypothesized that mbp - contributes to sexual dimorphism in mechanical allodynia. the pure mbp - wild-type (wt), its histidine (his) mutant or scramble peptides were administered into an intact sciatic nerve fascicle in male and female rats or mice, followed by von frey testing. intra-sciatic mbp - -wt peptide induced robust and lasting allodynia in females. in contrast, males responded with a brief and mild decline in mechanical sensitivity for one day post-injection of both wildtype and control peptides. the algesic ability of mbp - -wt was diminished in the his mutant. we here present the molecular changes in the sciatic nerve, drg and the spinal cord after the intra-sciatic mbp - injection in male and female animals. in addition, using the biotin-labeled mbp - peptide and the hrp-labeled goat anti-rat igg/igm antibodies, we developed an elisa to quantitatively assess seropositivity for the specific anti-mbp - peptide igm/igg autoantibodies in female and male rats post-cci. human serum from female patients with multiple sclerosis was used for control. our work corroborates the findings of sexual dimorphism of mechanical hyperpathia and suggests its potentially autoimmune nature in females. iijima m , nishi r , ikeda s , kawagashira y , koike h , sobue g , katsuno m . nagoya university, nagoya, japan. non-obesity diabetic (nod) b - knockout (ko) mice are characterized by chronic and progressive neuritis and expected as models of immune-mediated neuropathies, especially cidp. hindlimb-predominant weakness due to inflammatory demyelination followed by axonal degeneration begins from around twenty week-age in all female mice until thirty week-age. to clarify the efficacy of immunoglobulins as immune-regulating therapeutics and the similarity of pathogenesis of human cidp, we injected intraperitoneally human-derived immunoglobulins (ipig, mg/mg bw/week) and saline as a control to totally forty female mice. clinical and pathological estimations in sciatic nerves were performed in time series. as a result, the ipig-treated group was protected from weight loss which could be related to axon loss followed by muscle atrophy as well as inflammatory demyelination between twenty-five week-age and thirty week-age compared to the control. in addition, the pathological findings in sciatic nerves showed that ipig apparently suppressed inflammatory infiltrates. about the subsets of inflammatory infiltrates, while macrophages (cd +) and lymphocytes (cd +) highly existed and suggested to play a main role in the neuritis until thirty week-age, only macrophages naturally disappeared after thirty week-age without any therapeutic induction. immunoglobulins effectively suppressed only macrophages although that did not suppress cd + lymphocytes. in conclusion, nod b - ko mice respond to immunoglobulins in a similar manner to human cidp and this efficacy is due to the suppression of macrophage-dominant pathogenesis. therefore, macrophage-derived pathogenesis is for the main target of immunoglobulin therapy and we should focus on the lymphocyte-derived pathogenesis which might plays an important role in non-responders to immunoglobulins. ikeda s , nishi r , kawagashira y , iijima m , koike h , katsuno m , sobue g , . department of neurology, nagoya university graduate school of medicine, nagoya, japan; research division of dementia and neurodegenerative disease, nagoya university graduate school of medicine, nagoya, japan. chronic inflammatory demyelinating polyneuropathy (cidp) is an acquired immune-mediated polyradiculoneuropathy that is characterized by heterogeneous clinical manifestations. typical cidp is defined as neuropathy manifesting in a progressive manner, stepwise manner, or with recurrent symmetrical proximal and distal weakness and sensory impairment in all four limbs. although they occur at a lower proportion than the so-called typical cidp, atypical forms, such as multifocal acquired demyelinating sensory and motor (madsam), distal acquired demyelinating symmetric (dads), pure sensory, pure motor, and focal, are considered to cidp subtypes. thus far, pathological features characterizing each clinical subtype have not been fully elucidated. we analyzed clinical and pathological correlations in consecutive cidp patients who underwent sural nerve biopsy and fulfilled the definite or probable efns/pns criteria. there were male and female patients. the age at biopsy was . ± . (mean ± sd) years, and the duration from the onset of neuropathy to biopsy was ± months. fifty-five percent (n = ) of the patients were classified as having typical cidp. regarding atypical cidp, madsam (n = , %), dads (n = , %), and pure sensory (n = , %) subtypes were the major subtypes, while pure motor (n= , %) and focal (n= , %) subtypes were rare. no significant difference was found among these subtypes in terms of sex, age at biopsy, and disease duration. sural nerve biopsy specimens revealed that the densities of large myelinated fibers significantly decreased in the madsam subtype than in the other subtypes (p = . ). in addition, the variation in nerve fibers among fascicles was more conspicuous in the madsam subtype than in typical cidp (p= . ). patients with the dads subtype tended to show the formation of onion-bulbs. in conclusion, pathological findings of sural nerve biopsy specimens were different among the cidp subtypes. further studies are needed to clarify mechanisms leading to different pathological features. small volume plasma exchange (svpe) can be an affordable and potentially effective alternative form of plasma exchange. svpe is the repeated removal of small volumes of supernatant plasma over several days via sedimentation of patient whole blood. the aim of this study is to assess the clinical feasibility and safety of svpe in patients with gbs in low-income countries. twenty adult patients with gbs diagnosed as per the criteria for gbs of the national institute of neurological and communicative disorders and stroke (ninds) were enrolled for svpe at a centre in bangladesh. serious adverse events (sae) were defined as the number of patients developing severe sepsis associated with the central venous catheter (cvc) or deep venous thrombosis in the limb where the cvc is placed for svpe. the svpe procedure was considered safe if less than of svpe-treated gbs patients have a sae, and feasible if eight litres of plasma could be removed in at least of svpe-treated gbs patients. among the cases who received svpe, ( %) patients were male and the age range between to yrs. all the patients were quadriplegic and bedbound at enrolment for svpe with a median mrc score of (iqr, - ). cranial nerve involvement, autonomic dysfunction and requirement for assisted ventilation were observed in ( %), ( %) and ( %) patients respectively. electro physiologically ( %) patients were motor axonal and ( %) patients were sensory-motor demyelinating type. during the svpe none of our patients experienced sae and one patient experienced central line associated blood stream infection. common adverse effects were transient intravenous fluid responsive hypotension during the svpe sessions in ( %), cv catheter insertion site hemorrhage in ( %) and hypersensitivity reaction to fresh frozen plasma in ( %) patients. there was no hypo-albuminemia, anemia or electrolyte imbalance observed in most patients ( %) treated with svpe. improvement in one or more grade of the gbs disability score at four weeks after the onset of svpe was observed in ( %) patients. in conclusion svpe can be a safe, feasible and cost effective alternative to standard pe in the developing countries. guillain-barré syndrome (gbs) is a descriptive disease entity defined by a set of clinical, electrophysiological and laboratory criteria. various clinical phenotypes exist that may be triggered by different antecedent infectious events. although the disease appears to affect primarily the elderly in developed countries, but, scenario is different in developing countries. bangladesh has made an impressive progress towards the eradication of poliomyelitis, and no new cases have been reported since . gbs, an acute polyradiculoneuropathy, is the most frequent cause of acute flaccid paralyis. the crude incidence rate of gbs in < years of age reported here appears to be . × to × higher than that reported in the literature. we conducted a hospital based observational study including patients fulfilling the national institute of neurological disorders and stroke (ninds) criteria for gbs patients between and in dhaka medical college hospital, dhaka, bangladesh. detailed clinical, electrophysiological, serologic and microbiological data were obtained. gbs affected predominantly in young adults males (m/f= : ) living in rural areas. antecedent events were recorded in > % of patients; frequent events being gastroenteritis (> %) and upper respiratory tract infection ( %). more than % of the patients were bed-bound (gbs disability score ) at entry and about % patients required mechanical ventilator. about % patients did not receive specific treatment either intravenous immunoglobulin (ivig) or plasmapheresis due to high expensive treatment cost. % patients had died during hospitalization. % of patients had an axonal variant of gbs and evidence for a recent c. jejuni infection ( %). c. jejuni infection was significantly associated with serum antibodies to the gangliosides gm and gd a, axonal neuropathy, and greater disability. in conclusion, the majority of the patients do not receive standard treatment with ivig in view of its high price. therefore, we developed low-cost treatment strategies and conducted a safety and feasibility trials for small volume plasma exchange (svpe) on gbs patients in bangladesh. in future, it is essential to conduct a phase ii clinical trial to assess the efficacy of svpe for low-in-come countries. prognosis of guillain-barré syndrome (gbs) has not improved in last two decades. current therapies (intravenous immunoglobulin, ivig and plasma exchange, pe) had been proved to be effective on two third of patients in developed world. unpredictable and poorly understood clinical course of gbs hamper treatment development. in bangladesh, most patients affected by gbs cannot afford specific treatments with ivig or pe instead most of them receive only supportive care. therefore, we aimed to compare the outcome of ivig treated patients with supportive care patients in improvement of gbs disability score and mrc sum score by using world's largest gbs cohort in bangladesh. we conducted a prospective observational study enrolling gbs patients between and from dhaka medical college hospital and national institute of neuroscience and hospital, dhaka, bangladesh. only gbs patients ( %) received standard ivig treatment. in current analysis, ivig treated patients and age, sex and severity matched controls (supportive care only) were considered. outcome of both groups were compared using fisher's exact or chi square test and survival analysis were performed by kaplan meier method using log rank test. among patients (cases and controls), male/female ( / ), median age years, % patients were bed-bound, one-fourth patients required mechanical ventilation and % were axonal. we did not found any significant differences of treatment outcome in both cases and control groups in gbs disability score (week : p= . , months: p= . ) and mrc sum score (week : p= . , months: p= . ). survival analysis revealed, the differences of time required for independent locomotion, improvement of one gbs disability score and improvement of mrc score were not statistically significant between treatments (ivig) and supportive care patients. in conclusion, our analysis showed that standard dose of ivig use has no considerable advantage to improve specific outcome measures among gbs patients in bangladesh. as the phenotype of gbs in bangladesh is different from developed world; therefore, an efficacy trial for ivig is needed for developing countries like bangladesh or new targeted therapeutic strategies can append beneficial effects for gbs patients. isose s , , watanabe k , omori s , sekiguchi y , beppu m , shibuya k , amino h , suichi t , misawa s , kuwabara s . graduate school of medicine, chiba university, chiba, japan; national hospital organization chiba east national hospital, chiba, japan. diabetic neuropathy is a frequent cause of neuropathic pain, suggesting the small-fiber involvement. additionally, persistent peripheral pain-related inputs could cause neuronal hyperexcitability and complex interactions of the nociceptive pathways, i.e., central sensitization. to investigate the pathophysiology of neuropathic pain in diabetic neuropathy, we studied pain-related evoked potentials (preps) after selective intraepidermal electrical stimulation (ies) to a-deltaand c-fibers in diabetes patients with neuropathic pain (n= ) and without neuropathic pain (n= ). we also conducted a longitudinal study to assess changes in preps and pain profiles in patients with neuropathic pain months after the start of treatment with duloxetine. this study is registered with the umin clinical trials registry, umin . ies was applied in the hand and foot, and preps were recorded from the cz electrode referenced to the linked earlobes. we evaluated prep latencies, amplitudes, and amplitude ratios of preps after c/a-delta -fiber stimulation. in the conventional nerve conduction studies, patients with neuropathic pain significantly showed conduction slowing and decreased snap amplitudes in the median and sural nerves compared with those in patients without neuropathic pain. in pain-related sep studies, there were no significant differences in prep amplitudes and latencies after a-delta -or cfiber stimulation between the patients with neuropathic pain and without it. prep amplitude ratios after c/a-delta -fiber stimulation tended to increase in patients with neuropathic pain compared to patients without pain. after the treatment with duloxetine, c/a-delta -prep amplitude ratios were significantly decreased after both hand and foot stimulation, and as for numerical rating scale (nrs) scores as the intensity of pain. patients with less pain relief showed the tendency of higher c/a-delta prep amplitude ratios before treatment compared to patients with better pain relief. the correlation between reduction of c/a-delta prep amplitude ratios and nrs reduction did not reach statistical significance. this pain-related sep study demonstrated that abnormal cortical response in patients with neuropathic pain could improve after the treatment with duloxetine, this might reflect the cortical hyperexcitability as a central sensitization. this study is funded by shionogi & co., ltd. the sponsors played no role in the design and management of the study, collection and analysis of data, interpretation of the results, or the writing of the writing of the report. peripheral nerve injury is commonly associated with traumatic injury which is often amenable to surgery. despite improved methods in surgical repair, functional recovery remains a challenging clinical problem that often leads to significant morbidity in patients. alternative therapies that could augment surgical repair may be beneficial in functional outcomes. neuroinflammation is a complex pathway with different cellular components and cytokines that are activated following peripheral nerve injury. macrophages are responsible for the breakdown of debris following injury as well as promotion of regenerative signals. macrophage polarization is the process by which macrophages take on phenotypically distinct functions based on the local environment and signaling cues. exercise has been shown to drive macrophage polarization from a pro-inflammatory m phenotype towards an anti-inflammatory m phenotype in numerous tissues, but remains uninvestigated in the peripheral nervous system. the purpose of our study was to identify how exercise affects macrophage polarization, motor and sensory function, and neuroregeneration following sciatic nerve crush. c bl/ mice underwent sciatic nerve crush injury and were then given access to running wheels (exercised) or not given access to running wheels (sedentary) for weeks. exercised mice ran an average of . km per night. injured exercised mice were protected from the development of thermal hyperalgesia when compared to injured sedentary mice. exercised mice had fewer paw slips on beam walk testing compared to sedentary mice. no differences were measured in mechanical sensitivity or motor coordination and balance assessed by rotarod. while motor nerve conduction velocities were significantly reduced for injured mice compared to uninjured controls, motor nerve conduction velocities from injured exercised animals were significantly higher than injured sedentary animals suggesting improved nerve recovery with exercise. injured sciatic nerves from exercised mice demonstrated increased m macrophages compared to sciatic nerves from injured sedentary mice. the behavioral changes and altered macrophage polarization correlated with increased epidermal nerve fiber density, improved myelination, and increased in vitro neurite outgrowth from injured exercised animals. therefore, exercise alters macrophage polarization towards an anti-inflammatory phenotype which improves repair and recovery of the injured peripheral nerve. jack mm , shah k , everist b , reyna j , hylton p . university of kansas medical center, kansas city, ks, usa. diffusion tensor imaging (dti) has long been used to evaluate the location and integrity of white matter tracts in the brain. dti uses quantitative data and directionality of water diffusion to determine axonal connectivity of the nervous system. the technology has only recently been utilized in limited settings in the peripheral nervous system due to challenging technical factors and lack of widespread availability. magnetic resonance (mr) neurography or peripheral neurography is a technique which uses diffusion to differentiate between intraneural and perineural tissues. it allows for fascicle patterns to be visualized particularly in the setting of peripheral nerve sheath tumors. peripheral nerve sheath tumors of various pathologies cause surrounding nerves to be involved or displaced in a range of directions. this technique helps determine the anatomic location of these nerve fibers in relation to the mass, which is particularly helpful at distinguishing neuromas from schwannomas. this data is invaluable to the surgeon to ensure a safe and low morbidity operation. while this technology has benefit particularly with surgical planning, it has been underutilized due to the challenges of requiring complex software to produce fiber tracks and the inability to translate these images into the operating room. here, we utilized brainlab software that is commonly available and utilized in surgical suites to produce images of the radial nerve fiber tracts with an associated peripheral nerve sheath tumor prior to surgical resection. while the software is commonly used in the central nervous system, it has not been reported to have been used in the peripheral nervous system. this software offers a high usability and produces anatomically correct and reliable fiber tracts. this technique overcomes the reliance on highly specialized software and extensive training required for use that most other tractography software has. utilizing peripheral neurography in this case allowed for complete surgical resection without postoperative deficits. this data offers clinicians an option to investigate peripheral nerve fibers in various pathologic states, to plan appropriate operative trajectories to peripheral nerve pathology, and to improve surgical outcomes for patients with peripheral nerve sheath tumors. jacobsen b , parry g , allen j , walk d , muley s , ortega e . barrow neurological institute, phoenix, az, usa; university of minnesota, minneapolis, mn, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is an immune mediated neuropathy that is responsive to immunomodulatory agents such as glucocorticoids, intravenous immunoglobulin (ivig) and plasma exchange (pe). the specific immunopathogenic mechanisms of cidp remain unclear but there is increasing interest in nodal proteins as a site of the immune attack. even though the majority of patients respond to one of the aforementioned immunomodulatory agents there are some who are unresponsive or incompletely responsive to these first line agents and other more aggressive treatments may be necessary. cyclophosphamide and stem cell transplantation may be effective but are associated with considerable morbidity. anecdotal reports suggest that rituximab may be beneficial for some patients that fail first-line therapy, especially if they have antibodies to nodal proteins. we present four patients with intractable cidp who responded to rituximab. one of the three patients had diabetes. disease duration prior to starting rituxan was short ( months) in two patients and longer and months in the other two. all patients had failed treatment with glucocorticoids and ivig, and in two, plasma exchange and ivig-pe were also ineffective. two of the four patients were quadriparetic and non-ambulatory. two gm doses two weeks apart of intravenous rituximab were instituted in all patients. all patient tolerated the treatments well without adverse effects. all patients responded within four weeks and continued to improve at six months. other immunomodulatory agents were successfully tapered but not totally discontinued. it remains unclear whether antibodies to nodal proteins were present in these patients. in conclusion, although rituximab efficacy remains uncertain on the basis of randomized controlled clinical trials, it may be beneficial in selected patients otherwise intractable to first-line treatments. further studies are necessary to better understand which patients may benefit most from rituximab and where in the treatment algorithm rituximab should be applied. neuropathic pain is a chronic condition seen in patients suffering a direct injury to the peripheral or central nervous system or an indirect injury due to, e.g., diabetes or multiple sclerosis. current treatment options fall short of preventing or completely relieving patients of their pain. for years, research has focused on understanding the role of neurons in neuropathic pain pathogenesis while overlooking the role of supportive cells in general and satellite glial cells (sgcs) in the dorsal root ganglion in particular. these cells not only buffer the neuronal microenvironment they are also involved in controlling the electrical activity flowing through the neurons and in neuropathic pain pathogenesis. the aim of this project is to understand the role of sgcs in neuropathic pain development and thereby aid the identification of new drug targets. to purify the sgcs from adult mice we optimized a fluorescently activated cell sorting (facs) protocol. the success of our purification method was confirmed using qrt-pcr and visual inspection of the sorted cells. finally, we are running rna sequencing on sgcs after peripheral nerve injury to compare their transcriptome to that of uninjured cells at different time points. the results from our study are likely to deepen our understanding of how sgcs contribute to the development and maintenance of neuropathic pain. guillain-barrésyndrome (gbs) is an immunemediated disorder in the peripheral nervous system (pns) triggered by molecular mimicry against nerve gangliosides. one of the cell surface receptors (fas)-ligand (fasl) interaction transmits apoptotic signal to eliminate the auto-reactive b and t-cells, which generates cross-reactive antibody against nerve cells. host genetic polymorphism of fas and fasl may alter their expression and induce aberrant apoptotic response to develop gbs. therefore, we determined the single nucleotide polymorphisms (snps) of both fas receptor (− g/a and - a/g) and fasl ligand (− c/t) in gbs patients (n= ) as well as healthy controls (n= ) using the lightcycler technique. serum level of soluble form of fas and fasl was measured using commercially available sandwich elisa kit. comparison of genotype, allele and haplotype frequencies was done with the gbs subgroups based on the clinical and serological data. ag heterozygote (p= . , or= . , % ci= . - . ) and polymorphic g-allele (p= . , or= . , % ci= . - . ) of fas receptor - a/g promoter snps were significantly associated with anti-ganglioside (gm ) antibody positive gbs patients. in addition, − g-allele (p= . , or= . , % ci= . - . ) and - g/- g haplotype (p= . , or= . , % ci= . - . ) were predominantly associated with the axonal variant of gbs patients. serum soluble form of sfas (median levels pg/ml vs. pg/ml, p= . ) and sfasl (median levels pg/ml vs. pg/ml, p= . ) were found to be elevated in anti-gm antibody positive gbs patients compared to anti-gm negative patients. no significant association was found in genotypic distribution between gbs patients and healthy controls. in conclusion, fas/fasl promoter snps are not a susceptible factor for gbs but could be one of the influencing factors to develop cross-reactive anti-ganglioside antibodies in gbs patients in bangladesh. furthermore, functional studies with a larger sample size (using cohort like international gbs outcome studies-igos) are required to explain the immune pathogenic role of these snps for gbs patients. jang sy , yoon ba , shin yk , yun sh , jo yr , park ji , shin kj , kim jk , park ht . dong-a university, busan, south korea; inje university, busan, south korea. myelination is essential for the proper function of the nervous system. schwann cells, which form the peripheral myelin sheath, have the unique ability to dedifferentiate and to destroy the myelin sheath under various demyelination conditions. during schwann cell dedifferentiation-associated demyelination in wallerian degeneration after axonal injury, schwann cells exhibit myelin and junctional instability, down-regulation of myelin gene expression and autophagic myelin decomposition. however, in inflammatory demyelinating neuropathy, it is still unclear how schwann cells react and contribute to segmental demyelination before myelin scavengers, macrophages, are activated for myelin clearance. here, we show that schwann cell dedifferentiation-associated demyelination is a mechanism involved in the initial demyelination observed in a mouse model of inflammatory demyelinating neuropathy using ultrastructural, biochemical and microarray analyses. myelin uncompaction and myelin membrane instability generated by dedifferentiated schwann cells lead to autophagolysosome-dependent cytoplasmic amputation between the axon-containing myelin sheath and the schwann cell body, resulting in the formation of the "myelin corpse", thereby allowing macrophages to phagocytose the myelin corpse in the end stage of segmental demyelination. we found myelin corpse formation in inflammatory demyelination to be a process similar to the myelin rejection during wallerian degeneration, which appeared to be dependent on schwann cell autophagolysosome activation since schwann cell-specific atg knockout mice exhibited delayed myelin rejection following nerve injury. finally, lysosome inhibition in schwann cells not only prevented segmental demyelination but also delayed the progression of clinical stages by suppressing the myelin corpse formation in inflammatory demyelinating neuropathy. thus, our findings indicate that demyelination by schwann cells and macrophages might be part of a process that includes sequential divisions of labor with respect to myelin rejection and digestion, respectively. in conjunction with previous studies showing schwann cell dedifferentiation and autophagy in toxic and hereditary neuropathies, the concept of "schwann cell dedifferentiation-associated demyelination" provides insight into the development of possible therapeutic strategies to prevent schwann cell demyelination in peripheral demyelinating neuropathies. methods: electrophysiological data, the charcot marie tooth exam score (cmtes), and bmi from patients with known cmt a were obtained and analyzed. results: when controlled for age, bmi does not affect studies of ulnar motor nerve conduction in ctm a patients, but rather specific components of the cmt exam scores (cmtes, loss of pinprick sensation and motor strength in the lower extremities). discussion: bmi and clinical components of the cmtes are correlated, but it is uncertain which is the primary effect -i.e., whether the reductions in pinprick sensation and motor strength in the lower extremities lead to a higher bmi, or higher bmi results in these signs. introduction: charcot-marie-tooth disease type c (cmt c) is a rare, dominantly inherited neuropathy caused by mutations in the lipopolysaccharide-induced tumor necrosis factor (litaf) or small integral membrane protein of the lysosome/late endosome (simple) gene. methods: we present a case series comprised of patients in whom cmt c is caused by a gly ser substitution in the encoded protein. we focus on clinical presentation, electrodiagnostic analyses, and our findings in the context of previously described cases. results: the gly ser mutation causing cmt c is a mild form of cmt, as patients walked on time, had less weakness than those with charcot-marie-tooth disease type a (cmt a), had a charcot marie tooth neuropathy score (cmtns) indicative of mild disease, and had faster ulnar and median motor nerve conduction velocities compared to those with cmt a. discussion: the g s mutation in litaf seems to be clinically indistinguishable from a mild presentation of cmt a. jha mk , russell k , lee y , rothstein jd , morrison bm . johns hopkins university, baltimore, md, usa. peripheral nerves are highly dependent on metabolic energy to maintain both basic cellular functions such as axon transport, na+/k+ ion gradients, and myelination, as well as to support regeneration following injury. though glucose certainly provides some metabolic support, our recent studies have shown that monocarboxylates, such as lactate, pyruvate, and ketone bodies, also contribute to recovery from peripheral nerve injury. monocarboxylate transporters, particularly mct , are the predominate transporters for monocarboxylates in the peripheral nerve. in a recent publication, we found that mct heterozygous null mice, which express % less mct in all cells, have slowed nerve regeneration and reduced myelination following sciatic nerve crush. this study was limited by the global reduction of mct , which is widely expressed in schwann cells (sc), dorsal root ganglia (drg) neurons, endothelial cells, macrophages, and perineurial cells within the regenerating peripheral nerve. to better understand the mechanism by which mct contributes to normal nerve function and nerve regeneration, we produced and validated conditional mct null (mct loxp) mice that allow selective deletion of mct from scs, drg neurons, endothelial cells, or macrophages through mating to cell-specific cre lines. we are currently quantifying peripheral nerve development, aging, and regeneration in each of these mouse lines. following sc-, but not drg-, specific mct knockdown, sensory peripheral nerves develop demyelination by months of age, manifest by reduced myelin thickness, increased g-ratio, and reduced conduction velocity. studies are ongoing in cultured scs to determine the mechanism for demyelination. neither sc nor drg knockdown of mct impairs nerve regeneration following sciatic nerve crush. these results suggest that sc-specific mct is critical for maintaining myelin in sensory, but not motor, peripheral nerves as they age. they also suggest that mct expression in peripheral nerve cell types, other than sc and drg, is important for nerve regeneration. ongoing studies are determining the contribution of mct in other peripheral nerve cell types, particularly endothelial cells and macrophages, to normal development and regeneration following injury. our results will clarify the role of lactate and its transporter, mct , in peripheral nerve function, potentially suggesting novel targets for demyelinating neuropathies or nerve injuries. small fiber neuropathy (sfn) is a condition in which the smallest nerve fibers are affected, characterized by neuropathic pain and autonomic dysfunction. according to international criteria, sfn diagnosis is based on clinical symptoms in combination with abnormal temperature threshold testing (ttt) and/or reduced intraepidermal nerve fiber density (ienfd) in skin biopsy. skin biopsy is moderately sensitive, invasive and the process is time consuming and expensive. ttt is a widely available diagnostic tool, but lacks specificity. previous studies introduced stimulated skin wrinkling (ssw) as an objective, non-invasive diagnostic tool to detect sympathetic nerve dysfunction in sfn by means of a categorical assessment. however, our unpublished data has shown that inter-observer reliability of categorically assessed ssw is quite low. in this current study we will use a new digital method for ssw quantification: the digit wrinkle scan© (dws©). the primary study objective is to define normative values for dws© expressed as total wrinkle length per fingertip surface (mm/mm ). subsequently we investigate the applicability of dws© in patients with definite sfn, based on abnormal iefnd and/or ttt, determining the dws© sensitivity and specificity, as well as its validity. for this cross-sectional study, we will include healthy participants and patients diagnosed with sfn. eligibility is based on meeting the inclusion and exclusion criteria and providing written informed consent. skin wrinkling is induced by emla (eutectic mixture of local anesthetics) cream© application and captured by taking pictures. the primary outcome measure is total length of wrinkles per mm as shown on the photographs, which will be calculated by a new software program. patients are stratified according to age and gender. based on the results of healthy participants, normative values will be defined. inter-and intra-observer reliability will be determined. in the sfn group, additional correlation analysis will be conducted to determine the correlation between dws© and different outcome measures (sfn-symptom inventory questionnaire, visual analogue pain scale, neuropathic pain scale, sfn-rasch-built overall disability scale, ienfd and ttt). we expect to provide digitally quantified ssw (dssw©) normative values that can be used in clinical practice in the diagnostic workup for sfn. cmt , characterized by axonal degeneration, is an inherited motor and sensory neuropathy accounting for about % of total cmt patients. the cmt subtype shows on its own, a vast genetic heterogeneity with more than mutations in known genes rendering the identification of relevant drug targets and therapies very challenging. so far, only hdac inhibitors have shown promising results in mouse models for hspb mediated axonal cmt (cmt f), albeit such a single gene approach may have a limited relevance at clinical levels owing to the limited number of patients per genotype. in this study, we investigated common causative molecular players of cmt associated axonal degeneration. for this, an itraq based proteome analysis was performed on five patient's derived lymphoblasts bearing different cmt causal mutations alongwith respective age and gender matched unaffected controls. software-assisted interpretations of the obtained data led us to identify two proteins which were significantly downregulated in cmt patients compared to controls. these two proteins were then validated using western blotting and qpcr on patient derived lymphoblasts and fibroblasts. our results prompted us to unveil whether these two proteins can be used as potential biomarkers for identifying cmt patients. therefore, through a europe-wide collaboration, we constructed a cohort of cmt patients and healthy controls. these two proteins exhibited significant downregulation in this cohort suggesting a potential new role of these proteins as cmt biomarkers. remarkably, we were also able to validate the significant decrease in ineurons (neurons differentiated from patient derived ipscs) strengthening the importance of our finding and also suggesting the relevance of these proteins in the pathogensis of axonal cmt. this will be the first study involving multiple cmt causal genes at once, thereby holding the potential to offer new drug targets and potential biomarkers with wider application both clinically and pharmaceutically. mesenchymal stem cells (mscs) represents a valuable source of stem cell therapy, can differentiate into various cell types. we investigated of the neuromuscular potential of human tonsil-derived mscs (t-mscs) for neuromuscular regeneration in trembler-j mice that is considered to be a model for charcot-marie-tooth disease type a (cmt a diseases), which is involving hereditary motor and sensory peripheral neuropathies. the t-mscs differentiated toward skeletal myocytes, as evidenced by increased expression of skeletal muscle-related markers (including troponin i type , and myogenin) and the formation of myotubes in vitro. in situ transplantation of t-msc-derived myocytes (t-myocytes) into gastrocnemius in trembler-j mice, a mouse model of cmt a, enhanced motor function, as identified with recovery by a compound muscle action potential (cmap) amplitude. and the regenerated shape of the sciatic nerve and skeletal muscle by immunochemistry, without the formation of teratomas. furthermore, the expression levels of nerve growth factor (ngf) and glial cell line-derived neurotrophic factor (gdnf) were significantly increased in t-myocyte compared with t-mscs in vitro. these results indicate that the transplantation of t-myocyte can be a therapeutic option of cell therapy for the neuromuscular regeneration in hereditary peripheral neuropathy, comprising cmt a disease. kagiava a , karaiskos c , richter j , tryfonos c , lapathitis g , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory, the cyprus institute of neurology and genetics, nicosia, cyprus; department of molecular virology, the cyprus institute of neurology and genetics, nicosia, cyprus; neurology clinics, cyprus school of molecular medicine, the cyprus institute of neurology and genetics, nicosia, cyprus. x-linked charcot-marie-tooth disease (cmt x) is a common form of inherited demyelinating peripheral neuropathy resulting from mutations affecting the gap junction protein connexin (cx ). using a cx knockout (ko) mouse model of the disease, we have shown that targeted expression of virally delivered cx results in morphological and functional improvement. since patients with cmt x express mutant forms of cx in schwann cells, that could potentially interact with virally delivered wild type (wt) cx through dominant-negative effects, we also treated mutant mice expressing the t i, r w and n d mutations associated with cmt x on a cx ko background. all three mutants were localized in the perinuclear compartment of myelinating schwann cells consistent with retention in the er (t i) or golgi (r w, n d) with loss of physiological expression in non-compact myelin areas. following intrathecal delivery of the human gjb gene we could detect the virally delivered wt cx correctly localized in the non-compact myelin areas only in t i/cx ko mutant mice, but not in the other two mutants, suggesting dominant effects of the r w and n d mutant but not of the t i mutant. gjb treated t i/cx ko mice showed improved motor performance, along with lower ratios of abnormally myelinated fibers and reduced numbers of inflammatory cells in all tissues examined compared to mock-treated animals. in contrast, gjb treated r w and n d mutant mice showed only slight but not statistically significant improvement. this study provides additional proof of principle for a clinically translatable gene therapy to treat cmt x even in the presence of endogenously expressed cx mutants, since at least one er-retained cx mutant did not interfere with the expression of virally delivered cx allowing a therapeutic benefit similar to cx ko mice. however, golgi-retained mutants may interfere with virally delivered wt cx and other approaches besides gene addition may be needed for effective treatment. funding: muscular dystrophy association (grant mda to kak). kaida k , kadoya m , koike h , iijima m , takazaki h , ogata h , moriguchi k , shimizu j , nagata e , takizawa s , chiba a , yamasaki r , kira j-i , sobue g , ikewaki k . national defense medical college, tokorozawa, japan; nagoya university graduate school of medicine, nagoya, japan; kyushu university, fukuoka, japan; university of tokyo, tokyo, japan; tokai university school of medicine, isehara, japan; kyorin university, tokyo, japan. antibodies to a glial protein, neurofascin (nf) have recently been identified in approximately % of patients with chronic inflammatory demyelinating polyneuropathy (cidp), which are igg -predominant. igg anti-nf -associated cidp may be a distinct subtype from typical cidp in terms of clinical features and response to immunotherapy. however, a diagnostic criterion of anti-nf -associated cidp has not established yet. to develop optimal criteria and design the best treatment plan for the anti-nf -associated cidp, procedures for determining anti-nf antibodies should be simplified, prevalent, and reproducible, as well as being accurate. cell-based assay (cba) has hitherto been utilized for determining antibodies to nf in sera from patients, results of which have been confirmed by immunohistochemistry (ihc) using teased nerve fibers from rodents. these methods are the most reliable techniques, while not necessarily easy-to-use and easy to maintain in most laboratories. in the present study, we aimed to validate the diagnostic utility of a conventional enzyme-linked immunosorbent assay (elisa) for determination of anti-nf antibodies and the igg subclass. sera from patients with efns/pns criteria-met cidp were examined with elisa using human recombinant nf . to verify elisa results, ihc on rat sciatic nerves, western blot (wb) and cba using nf -transfected and naive hek cells were conducted. the human nf -based elisa clearly distinguished between anti-nf antibody-positive and -negative sera. fifteen cidp patients ( %) were igg anti-nf antibody-positive, which were confirmed by wb, ihc and cba studies. none of disease controls or healthy subjects had positive results. twenty-five sera randomly selected from anti-nf -negative cidp sera were also negative on cba. the anti-nf activities on elisa were significantly positively-correlated with those on cba (p < . ). analyses of clinical and laboratory findings showed that anti-nf -associated cidp was characterized by younger onset, distal dominant phenotype, tremor, sensory ataxia, higher protein levels in cerebrospinal fluid, and poor response to ivig, which were consistent with those in previous studies. this elisa combined with determination of the igg subclass is a simple and reliable method for initial screening for anti-nf antibodies. the genetic abnormality responsible for x-linked charcot-marie-toothy neuropathy subtype cmtx was recently identified by whole genome sequencing to be a kb insertion into chromosome xq . . the clinical profile of cmtx in childhood is not well described. we reviewed the clinical characteristics, neurophysiological profile and cmt pediatric scale (cmtpeds) assessments of children with genetically confirmed cmtx . cmtx was characterized by early onset, and early and progressive hand weakness. most affected children were symptomatic within the first two years of life. the most common presentation was with equinovarus foot deformity in the first year of life. cmtpeds analysis in these children revealed that cmtx progressed more rapidly ( . ± . points/ year, n= ) than cmt a and cmtx . grip strength in the second decade of life in most affected males was two standard deviations below age-and sex-matched normative reference values. the most severely affected individual was wheelchair bound at years of age and two individuals had no movement in the small muscles of the hand in the second decade of life. there was only a single symptomatic female identified and she had mild signs. nerve conduction studies showed a demyelinating sensorimotor neuropathy with motor conduction velocity in eight children while one child had a length-dependent sensorimotor axonal neuropathy. understanding the unique phenotype of cmtx is essential for directing genetic testing, as the cmtx insertion will not be detected on the snp microarrays, multi-gene panels or whole-exome sequencing currently used for the diagnosis of cmt. the early onset of disease coupled with rapid progression means that many children with cmtx will have severe disability within the first two decades of life and hence early diagnosis is needed for early commencement of rehabilitation. kapoor m , catania s , sarri-gonzales s , lunn mp , manji h , reilly mm , carr as . centre for neuromuscular diseases, national hospital for neurology and neurosurgery, london, uk; department of neurophysiology, national hospital for neurology and neurosurgery, london, uk. the conventional dosing of ivig in cidp and mmn is based on treatment trials that used bolus and maintenance dosing of ivig between - . g/kg. there are rare published articles reporting the efficacy of higher maintenance ivig doses. we present three cases of inflammatory neuropathies, who are currently stabilized on ivig doses of mg- mg/kg of ivig per month, refractory to standard dose ivig and other immunosuppressants. the first case is a year-old-lady with cidp who presented with episodes of ascending sensory disturbance, weakness, and diplopia. she had activity related fluctuations and pre-dose deterioration on g/kg/month ivig. she then had an acute deterioration with mrc sum score dropping from to even with additional plasma exchange. her bilateral foot drop (mrc grade - ) and fluctuations persisted with an increase of ivig to . g/kg/month. she is now clinically stable (ankle dorsiflexion mrc grade - , mrc sum score ) on mycophenolate and g ivig weekly ( . g/kg/month). case is a -year-old male fitness instructor with mmn and sjogren's syndrome. he presented with recurrent proximal and distal weakness that responded to g/kg of ivig and deteriorated with iv methylprednisolone. he had peri-dose fluctuations, intermitted proximal weakness, and persistent foot drop (ankle dorsiflexion mrc grade - ) at . mg/kg/month, worsening to mrc grade - on . g/kg/month and fluctuating between mrc grade - on . g/kg/month. an increase of ivig to g weekly ( . g/kg/month), has resulted in mmn rods scores of / , improved distal power and return to full capacity at work. case is a -year-old man with predominantly upper limb cidp. he received g/kg ivig without any benefit, had no response to doses of plasma exchange, doses of cyclophosphamide or dose of rituximab between and . since , he has received g/kg/month ivig with improvement of mrc sum score from to . these cases highlight that some patients require a much higher than conventionally prescribed dose of ivig, and that these doses are tolerated over years without serious adverse events. idiopathic rapid eye movement sleep behaviour disorder (irbd) has been identified as a precursor of alpha-synucleinopathies, such as parkinson's disease, dementia with lewy bodies, multiple system atrophy. several studies linked changes in cutaneous innervation with central nervous system pathology in neurodegenerative disorders. recently small fiber neuropathy and alpha-synuclein deposition in the skin found to be a potential biomarker in parkinson's disease. we evaluated the epidermal innervation of irbd patients and age and sex-matched controls from skin punch biopsies from the distal leg using pgp . immunohistochemistry. furthermore, a battery of clinical examinations were performed on patients and controls alike, including structured interviews, clinical motor and non-motor questionnaires and rating scales (e.g. unified parkinson's disease rating scale [updrs], non-motor symptoms questionnaire [nms-quest] and beck depression inventory, epworth sleepiness scale, evaluation of cognitive and olfactory functioning as well as blood samples. irbd patients, compared to controls, showed a significant reduction in intraepidermal nerve fiber density (p = . ), whereas the axon swelling ratio, did not differ between groups. patients with irbd reported non-motor symptoms more frequently than controls (updrs i, nms-quest). olfaction and daytime sleepiness differed between both groups, whereas there were no differences regarding cognition. these in vivo findings demonstrate small fiber neuropathy in irbd patients that are associated with non-motor symptoms indicating that peripheral abnormalities may occur early in irbd. they warrant larger scale longitudinal studies in order to investigate their prognostic value. katz j , lewis r , spatafora d . california pacific med center, san francisco, usa; cedars-sinai medical center, los angeles, usa; neuropathy action foundation (naf), santa ana, usa. multifocal motor neuropathy (mmn) is a rare condition that affects . in every , individuals worldwide and is associated with motor dysfunction and moderate to severe disability. the neuropathy action foundation conducted a global survey to determine the impact of mmn on patient quality of life (qol) and gaps in patient/provider educational needs. the first global mmn qol survey was an item internet questionnaire available between january and july , . the survey focused on three primary areas: timely and accurate diagnosis, the efficacy of treatment, and the impact of the disease on patients qol. the survey was completed by patients from countries. the majority of respondents said they were diagnosed between the ages of and years ( . %), more than % reported that it took more than one year to be diagnosed and more than % reported that it took - years or longer to be accurately diagnosed. with respect to treatment options : . % reported receiving intravenous immune globulin and . % reported receiving subcutaneous immune globulin therapy. other therapies being used to treat mmn were gabapentin ( . %), and pregabalin ( %). almost half ( . %) said that mmn often impacts their overall schedule. half of the participants reported that mmn often or always interferes with their employment; % had difficulty typing on a computer or using a telephone, . % had trouble concentrating, and . % said they had to work really hard to pay attention or else they would make a mistake. this is the first assessment of mmn from a patient's perspective. the survey highlighted critical issues relating to the diagnosis, management, and impact on the qol of individuals with mmn. the data also identified gaps and insights in provider education relating to proper diagnoses and management of the condition from a patient's perspective. katz j , levine t , dimachke m , barohn r . forbes norris center, san francisco, ca, usa; phoenix neurological institute, phoenix, az, usa; kansas university medical center, kansas city, ks, usa. in the united states, cidp cases are submitted to insurance companies to determine whether ivig therapy is appropriate. this is done using specified diagnostic criteria, which reduce diagnosis to a boolean analysis, where a disease can only be present or absent. this leaves no room for uncertainty, even when it truly exists. boolean criteria are useful for clinical trials, but fall short where real decisions are made under uncertainty and based on perceived cost/benefit analysis. this project attempts to elucidate root causes of uncertainty and to find solutions to this dilemma. we asked cidp experts to select a single diagnosis in cases where ivig was approved using the submitted case records. while there was agreement on many cases, in the five most "uncertain" cases no more than reviewers agreed on a single condition, who chose up to four separate entities. among these, at least three reviewers diagnosed an immune neuropathy in all five. the root cause of the disagreement, to a large degree was unclear documentation (aunts) which consisted of pasted, missing, and disorganized data. reviewers missed useful information, admitting it was too difficult to fully parse records. to resolve uncertainty, reviewers admitted to discounting certain reported datum to help fit the entity they suspected, such as reported therapeutic responses or certain electrodiagnostic/exam findings. other disagreement, however, reflected the complexity of neuropathy diagnosis, such as knowing if improvement was due to natural history or treatment, unawareness of rare presentations, or analyzing a true uncle (complex case). reviewers used bayesian (select most likely diagnoses from a list) and fuzzy logic (compare best fits to base cases). when the "best" diagnosis did not fit the base case, they had to re-interpret the data. improving review procedures requires eliminating aunts by collecting all key information and simplifying how records are presented. it also needs more advanced data methods to analyze common and rare borderline presentations (uncles like mama v pma, cidp v cspn, etc..), developing diagnostic algorithms that address real uncertainty, educating prescribers and patients on process, and creating systems that measure outcomes longitudinally after induction or tapering of therapy. keisuke y , miyuki m , motoi k , susumu k . department of neurology, faculty of medicine, kinki university, osaka, japan. anti-ganglioside antibodies are closely associated with clinical phenotype and specific symptoms in acute immune-mediated neuropathies. igg anti-gq b antibodies are specifically associated with miller fisher syndrome (mfs), bickerstaff brainstem encephalitis (bbe) and guillain-barré syndrome (gbs) with opthalmoplegia (gbs-op). in addition, ganglioside complexes (gscs) containing gq b also can be targets in such diseases, and might be associated with the clinical features. however, factors regulating clinical phenotype in those gq b-associated antibodies-positive diseases have not yet been known. for investigating the differences of antibody reactivities among those diseases, we examined, using combinatorial glycoarray, igg antibodies to ten individual glycolipids [gm , gm , gm , gd a, gd b, gq b, galactocerebroside (gal-c), lactosylceramide (laccer), ga , sulfatide] and glycolipid complexes consisting of two of the glycolipids listed above in sera from patients with gbs-op who were positive for anti-gq b antibody by elisa (gbs-op-gq b), patients with mfs with the clinical triad (opthalmoplegia, ataxia, and areflexia), and patients with bbe. by combinatorial glycoarray, overall sensitivity of antibodies to gq b and gscs containing gq b was . % ( / ) in gbs-op-gq b, . % ( / ) in mfs, and . % ( / ) in bbe, respectively. there were no significant differences in antibody reactivities between mfs and bbe. it is notable that antibodies to gscs containing gd b were more frequently found in gbs-op-gq b patients than in mfs or bbe patients (e.g., gd b/sulfatide: p= . and p< . , respectively). presence of the antibody reactivities to gscs containing gd b may possibly be related with clinical features of gbs-op-gq b, including frequent need of artificial ventilation. kennedy r , , , carroll k , , paterson k , ryan mm , , , mcginley jl , . royal children's hospital, parkville, australia; university of melbourne, parkville, australia; murdoch childrens research institute, parkville, australia. problems with walking and footwear fit are often reported by children and adolescents with charcot-marie-tooth disease (cmt). a cross-sectional, case controlled study of gait was conducted in children with cmt and typically developing (td) children. gait was assessed barefoot and in two types of the participants' own typical footwear; optimal (e.g. athletic shoes) and suboptimal (e.g. slip-on footwear). the aims were to determine differences in spatio-temporal (s-t) gait variables between children with cmt and td children; and to investigate the effect of footwear choices. twenty-nine independently ambulant children aged - years with confirmed genetic or clinical diagnoses of cmt, and age and gender matched td children participated (mean age . years; males). exclusion criteria included developmental disorders, other neuromuscular conditions or musculo-skeletal diseases that could affect gait, and lower limb injury or surgery in the preceding months. assessment included s-t gait patterns, footwear characteristics, metre run, and cmtpeds. gait was assessed at self-selected speed with an electronic walkway (gaitrite™), with trials for each condition. the primary gait variable assessed was speed; other variables included step length, step time, cadence, base of support width and step-to-step gait variability. across all footwear conditions children with cmt walked more slowly ( regeneration of cutaneous unmyelinated axons is known to be slowed in dm-patients and after -months, the density of intraepidermal nerve fibers (ienf) does not return to baseline levels after chemical or mechanical axotomy. however, the long-term outcome of regeneration in dm or control subjects is not known. additionally, it is not clear if the regeneration of sensory distal axons is length-dependent. here we measured the rate of axonal regeneration -months after chemical denervation using a capsaicin model in dm patients (n= / dm /dm ) without neuropathy, and controls. dm skin punches were performed at distal thigh at baseline, -hours post-capsaicin, and at , , and days. blood glucose and hgba c were measured at baseline, , and days. healthy controls had skin punches at both distal leg and proximal thigh at baseline, after capsaicin chemical axotomy, and days , , and . regeneration rate was significantly higher at the thigh in healthy controls ( . fibers/mm/day ( % ci: . - . fibers/mm/day) compared to dm (p= . ), but no difference between dm ( . fibers/mm/day % ci: . - . fibers/mm/day) or dm ( . fibers/mm/day % ci: . - . fibers/mm/day) (p= . ). comparing regeneration rate at different time intervals, showed that regeneration was significantly slowed between day and dm patients, while it continued with the same rate in controls. blood glucose or hga c had no effect on regeneration rate. ienfd returned to baseline in controls by -months ( % of baseline) while it is did not in dm subjects, %/ % (dm /dm ) of ienfd baseline, (p= . dm vs. controls). there was no difference in regeneration rate ienfd %-baseline by -months at distal leg and proximal thigh in controls (p= . ). these results suggest that the rate and outcome of regeneration is independent of the length of the axon. additionally diabetic patient have incomplete nerve regeneration after months regardless of diabetes type or the level of glycemic control. regeneration of axons slowed down over time in patients with dm and reached a plateau after days. kiessling p , lledo-garcia r , watanabe s , langdon g , tran d , bari m , christodoulou l , price g , smith b , byrnes w , brock m , jolles s . ucb pharma, monheim, germany; ucb pharma, slough, uk; ucb pharma, braine-l' alleud, belgium; ptx solutions ltd, london, uk; ucb pharma, raleigh, durham, usa; university hospital of wales, cardiff, uk. ucb is a humanised high-affinity monoclonal igg antibody developed to bind human neonatal fc receptor (fcrn), selectively inhibiting igg salvage and recycling. conditions such as myasthenia gravis (mg) are characterised by pathogenic igg autoantibodies; inhibition of fcrn may provide a suitable therapeutic approach. this phase i, double-blind, dose-escalating, first-in-human study (nct ) evaluated the safety and pharmacology of ucb . forty-nine healthy adults (mean age years, range - ) were randomised and received a single dose of intravenous (iv) or subcutaneous (sc) placebo (n= and n= , respectively), or a single dose of iv or sc ucb ( , or mg/kg; n= per dose, per administration). subjects were followed up until day . one placebo iv subject did not complete the study. twenty-seven of subjects ( %) receiving ucb , and / ( %) receiving placebo, reported ≥ treatment-emergent adverse event (teae) of mild/moderate intensity. severe teaes occurred in four subjects, all in the ucb mg/kg iv group (headache [n= ], back pain [n= ]). no serious aes occurred. incidence of infections was similar with ucb and placebo. the most frequently reported infection was nasopharyngitis. treatment-related teaes were reported by % of subjects receiving ucb and % receiving placebo: the most common in the ucb -treated groups were headache ( / ; %) and vomiting ( / ; %); these occurred more frequently with the iv than sc route. non-linear increases in ucb plasma concentration-time profile with increasing dose were observed with ucb . serum igg was reduced in a dose-depended manner with ucb iv and sc: decreases from baseline to day with ucb iv were . %, . % and . % for , and mg/kg doses, respectively, and . %, . % and . %, with ucb sc doses, respectively. these data indicated that the fcrn inhibitor ucb effectively reduced serum igg, with sc administration generally better tolerated than iv. further to these observations, the efficacy, safety and tolerability of ucb sc for chronic-intermittent treatment of moderate-to-severe mg are being evaluated in an ongoing phase ii, multi-centre, randomised, double-blind, placebo-controlled study (eudract - - middle east respiratory syndrome (mers) has a high mortality rate and pandemic potential. however, very little information has become available on this syndrome since it first erupted in . this study aimed to evaluate the frequency of neurological complications and their clinical presentations in mers. we reviewed the medical records of all patients who were diagnosed with laboratory-confirmed mers coronavirus (cov) infections and subsequently admitted to a single reference center for mers treatment during the outbreak in korea. in total, patients ( . %) reported neurological symptoms during or after mers-cov infection. the potential diagnoses in these cases included bickerstaff's encephalitis overlapping with guillain-barré syndrome, critical illness polyneuropathy or other toxic or infectious neuropathies. neurological complications did not appear concomitantly with respiratory symptoms, but were instead delayed by - weeks. neuromuscular complications were not rare in mers-cov-infected patients, and they may have previously been underdiagnosed. understanding neurological manifestations is important in an infectious disease like mers, because evaluation is frequently limited during treatment, but it can interfere with prognosis and sometimes require modification of treatment. kim hj , hyun jk , kim tu . dankook university, cheonan, south korea. the diagnosis of carpal tunnel syndrome (cts) is based on clinical symptoms, physical examinations and supported by nerve conduction study (ncs). ultrasonographic examinations can be performed to assess peripheral nerves with less discomfort and the surrounding anatomic structures. while the usefulness of ultrasonography (usg) in the cts has been reported, no study to date has compared the diagnostic utility of various usg findings for cts. we investigated the correlation of various usg findings to the clinical symptoms/signs and ncs findings in patients with cts. twenty-eight hands ( patients) with cts based on electrodiagnostic criteria and clinical symptoms such as tingling sensation or pain in the first to third fingers, burning sensation, paresthesia and weakness of hand grip power. all subjects were examined with usg. cross-sectional area (csa) and flattening ratio (fr) of the median nerve was calculated at level of radio-ulnar joint, pisiform and hamate. swelling ratio of the median nerve and palmar displacement of the flexor retinaculum was also calculated. clinical assessment was conducted using the boston carpal tunnel questionnaire (bctq) scale and historical-objective (hi-ob) scale. the analysis of correlation between usg findings and clinical symptom scales/ncs findings was performed using correlation analysis. the csa of the median nerve at level of radio-ulnar joint was significant correlated with bctq scale, hi-ob scale, distal motor latency, and conduction velocity (cv). the csa of the median nerve at level of pisiform was significantly correlated with hi-ob scale, distal motor latency, and cv. the fr of the median nerve at level of radio-ulnar joint was significantly correlated with bctq scale, hi-ob scale, distal motor latency, and cv. the swelling ratio of the median nerve was also significantly correlated with distal motor latency and cv. in patients with cts, csa of the median nerve at level of radio-ulnar joint was most closely related to ncs findings and clinical symptoms. so, csa of the median nerve at radio-ulnar joint might be a complementary tool for the diagnosis of cts. kitaoji t , tsuji y , ashida s , yamada t , ishii r , tanaka a , mizuno t . department of neurology, graduate school of medical science, kyoto prefectural university of medicine, kyoto, japan. the case was a -year-old woman. first, she noticed paresthesia in the right plantar eight months before admission and in the left plantar four months before admission. three months before admission, she developed muscle weakness in her feet. the muscle weakness and paresthesia extended to the lower legs in a few months. twenty days before admission, she experience difficulty in walking. on admission, the muscle weakness was observed in the legs, especially in the right tibial anterior muscle (ta). there was severe sensory disturbance and loss of deep tendon reflex in the legs. she had trouble walking due to the weakness and sensory aphasia. in nerve conduction study (ncs), conduction block was observed between the ankle and popliteal in both tibial nerves. the blood level of angiotensin converting enzyme (ace) was elevated. cerebrospinal fluid analysis was normal. there was no enhancement in the lumbar nerve roots shown on mri. gallium- scintigraphy showed hot spots on bilateral hilar lymph nodes and mediastinal nodes and biopsy of mediastinal nodes showed non-caseating epithelioid granuloma. therefore, we diagnosed her with sarcoid peripheral neuropathy by sarcoidosis. by using the nerve ultrasound, partial spindle-shaped nerve enlargement was observed at the part of conduction block in the left tibial nerve. we started the treatment with methyl prednisolone ( mg, days) and oral prednisolone therapy ( mg/kg/day). after treatment, the paresthesia and muscle weakness in the legs had gradually improved. the partial enlargement in the left tibial nerve also improved on the -hospital day. in ncs, the conduction block improved, however, the compound muscle nerve potential of tibial nerve decreased because of axonal damage. this partial spindle-shaped nerve enlargement by using ultrasound has never been reported in sarcoid peripheral neuropathy before. the nerve ultrasound may be useful for evaluation of therapeutic effect of sarcoid peripheral neuropathy. peripheral nerve injuries are still debating problems in the world because of poor recovery. there is absolutely a need for new therapeutic agents to improve outcome by altering nerve regeneration. there are some studies in the literature about some therapeutic agents that used in the cases of peripheral nerve injuries. despite these studies, an agent with clinical use has not been presented yet. in this experimental study, we aimed to analyze the effects of curcumin (cur) in the cases of peripheral nerve injuries. forty rats were randomly and equally divided into four groups. the first group was control group. rats in this group were not operated. right sciatic nerve injuries were performed to the other groups. the second group was operation group with no therapeutic agent. the third group was operation and local cur applied group. the fourth group was operation and systemic cur applied group. electrophysiological evaluations were performed with electroneurography (enog) before and after the surgeries. systemic use of cur although caused improvement in the enog values but could not make a positive contribution to the nerve regeneration statistically. additionally local use of cur made negative effect to the nerve regeneration statistically. according to our statistical results we could not recommend cur as a nerve protective agent. klein cj , wu y , jentoft me , mer g , spinner rj , dyck pjb , dyck pj , mauermann ml . mayo clinic, rochester, usa. intraneural perineurioma is a hypertrophic peripheral nerve tumor having immunoreactivity to epithelial membrane-antigen, negative for s- . the origin of perineurial cells is debated to be similar to meningeal cells. ip does not metastasize, but motor deficits accumulate over time from tumor growth in nerve and plexus. after schwannomas and neurofibromas, perineuriomas are the most common nerve tumor of young adults. a chromosome q deletion has been reported in one patient. we identified ip cases from our previously published clinical cohort with available flash frozen ip tissue for dna isolation. wes with cnv analysis and cgh microarray analysis (agilent x k superprintg ) were performed on extracted dna; had available germline dna (lymphocytes and buccal tissue). we compared the exome data against online and in-house control data (∼ , ) examining variants less than . frequencies, predicted damaging or nonsynonymous. wes identified three novel, heterozygous, damaging mutations in tumor necrosis factor receptor-associated factor (traf ) in of ( %) cases; p.l p (n= ), p.h r (n= ) and p.s r (n= ). mutations were within the wd domain, p.l p, p.h r within exon and p.s r within exon , and mapped to a limited region of traf with protein structure modeling. two of cases ( . %) showed macroduplications/deletions on multiple chromosomes, including chromosome , confirmed with cgh microarray analysis and cnv results from exome data analysis. four of ( %) had no discovered mutation. age of onset or severity did not correlate with type of mutations. this study provides strong evidence that traf is a specific tumor driver of ip. mutations in traf are also linked to benign intracranial meningiomas suggesting a shared pathogenesis and close origins of perineurial and meningeal cells. study supported by: mayo foundation and the mayo center of individualized medicine. klein i , , bobylev i , , lehmann hc , . university hospital cologne, cologne, germany; center for molecular medicine cologne (cmmc), cologne, germany. peripheral neuropathy is a common side effect of paclitaxel. clinical evidence suggests that the delivery mechanism of paclitaxel formulations influence time course and severity of paclitaxel induced peripheral neuropathy. in a preclinical model we studied access, distribution and toxicity of two paclitaxel formulations (nanoparticle albumin-bound paclitaxel (nab) and solvent-based paclitaxel) in the peripheral nervous system (pns). c bl/ mice were treated with mg/kg or mg/kg of nab-paclitaxel or solvent based paclitaxel. kinetics of paclitaxel in neurons was assessed by a newly established immunostaining technique. neurotoxicity was evaluated by functional assays and nerve morphology. paclitaxel accumulated mostly in dorsal root ganglia, whereas distal nerve segments showed only low uptake of paclitaxel. treatment of mice with the two paclitaxel formulations resulted in paclitaxel uptake mostly in nf + larger fiber neurons. in ib +, and cgrp+ small fiber neurons, paclitaxel was less frequently detected. nab-paclitaxel was incorporated more rapidly compared to solvent-based paclitaxel but neurons also showed a faster clearance of nab-paclitaxel compared to solvent based paclitaxel. functional assays and nerve conduction studies indicated that nab-paclitaxel was less neurotoxic compared to solvent-based paclitaxel. this is the first study that characterizes in detail the access of nab-paclitaxel and solvent based paclitaxel into the pns. our findings have important implications to understand the pathomechanisms of paclitaxel induced neurotoxicity and to develop neuroprotective strategies by preventing access of paclitaxel to the pns. the aim of our study was to investigate the etiology of neuropathy in patients with rheumatoid arthritis (ra). subjects were neuropathy patients with ra admitted to our department. laboratory investigations, nerve conduction studies (ncs) and sural nerve biopsy were performed. mean patient age was . years (range, - years), and mean disease duration was . years (range, - years). clinical diagnosis for neuropathy was rheumatoid vasculitis (rv) in patients, rv with acute motor axonal neuropathy (rv-aman) in and chronic inflammatory demyelinating polyneuropathy with ra (ra-cidp) in . rheumatoid factor ( / ) and rheumatoid arthritis particle agglutination ( / ) was high and c tended to be lower in the rv group (rv and rv-aman). anti-ganglioside antibodies were examined in patients, with positive results in . an rv-aman case was diagnosed with motor-dominant clinical presentation and the presence of anti-galnac-gd a immunoglobulin (ig)g antibody. no other cases with rv were examined for anti-ganglioside antibodies. positive results for anti-gm and gm igg antibody were seen in one ra-cidp patient. we evaluated sural/median (s/m) ratio) for sensory nerve action potential (snap). s/m ratio was low in rv cases ( / ) and high ( / ) in immune-mediated cases, suggesting a so-called normal sural abnormal median pattern in immune-mediated neuropathies. the rv-aman case showed a moderate value in s/m ratio. nerve biopsy revealed thinly myelinated nerve fibers in ra-cidp cases compatible with demyelination, while the rv group showed the typical pathology for necrotizing vasculitis. rv cases were treated with prednisolone (psl), intravenous methylprednisolone, intravenous cyclophosphamide and increased psl dose. ra-cidp and rv-aman were treated with intravenous ig. in conclusion, neuropathy in ra can be divided into vasculitic and immune-mediated groups. ncs, and the s/m ratio of snap with some laboratory parameters in particular, may be of use in differential diagnosis and deciding treatment strategies. koh s , wong shj , loh kw , chng ysk , pawa c , ei ma , lee bjh , subramaniam t , umapathi t . national neuroscience institute, singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university singapore, singapore; tan tock seng hospital, singapore; khoo teck puat hospital, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful autonomic neuropathy that develops with abrupt improvement in glycaemic control. typically, type or dm patients on insulin or oral hypoglycaemic agents (ohga), present with painful neuropathy and autonomic dysfunction within weeks of rapid improvement in glucose control. current emphasis to achieve good glycaemic control rapidly may inadvertently increase incidence of tind, hence the impetus to understand risk of over-zealous glycaemic control. we therefore set out to study the occurrence of tind in a dm cohort of a tertiary hospital. we screened all patients who had two hba c measurements between and . during this period, approximately patients were seen per year. we found patient-encounters that showed hba c decrease of ≥ % over months or ≥ % over months. we then used a structured checklist of tind symptoms to shortlist cases. these case-encounters were scrutinised and classified as; 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or uncertain temporal relationship to decrease in hba c; unlikely tind: alternative explanation exists for symptoms. only one case was deemed 'probable tind'-a middle-aged man with newly diagnosed type dm who presented to emergency department with palpitations and worsening 'frozen feet' sensation that disturbed sleep. his hba c decreased by . % in weeks. his symptoms improved within a month with neuropathic pain medications and resolved months later. he also developed maculopathy and proliferative retinopathy. ten months later, he developed significant proteinuria. four other cases were classified as 'possible tind' while the remaining were unlikely tind. our study is limited by retrospective design and reliance on hospital records. nevertheless, our findings suggest that tind is uncommon in a general cohort of dm patients. on the other hand, the number of patients with painful neuropathy and acute dysautonomia symptoms contemporaneous with rapid decline in hba c raises the intriguing possibility that forme fruste of tind exists and one should interrogate the rate of hba c decline in dm patients with these symptoms. koike h , kadoya m , kaida k , ikeda s , kawagashira y , iijima m , kato d , ogata h , yamasaki r , matsukawa n , kira ji , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; national defense medical college, tokorozawa, japan; nagoya city university graduate school of medical sciences, nagoya, japan; kyushu university, fukuoka, japan. we investigated the morphological features of chronic inflammatory demyelinating polyneuropathy (cidp) with autoantibodies directed against paranodal junctional molecules, particularly focusing on the fine structures of the paranodes. sural nerve biopsy specimens obtained from cidp patients with anti-neurofascin antibodies and patient with anti-contactin antibodies were assessed. these antibodies were examined using sera obtained from patients with cidp who fulfilled the criteria of the european federation of neurological societies/peripheral nerve society. thirteen cidp patients without these antibodies were also examined to compare pathological findings. characteristic light and electron microscopy findings in transverse sections from patients with anti-neurofascin and anti-contactin antibodies indicated a slight reduction in myelinated fiber density, with scattered myelin ovoids, and the absence of macrophage-mediated demyelination or onion bulbs. teased-fiber preparations revealed that segmental demyelination tended to be found in patients with relatively high frequencies of axonal degeneration and was tandemly found at consecutive nodes of ranvier in a single fiber. assessment of longitudinal sections by electron microscopy revealed that detachment of terminal myelin loops from the axolemma was frequently found at the paranode in both anti-neurofascin and anti-contactin antibody-positive cidp patients compared with antibody-negative cidp patients. patients with anti-neurofascin antibodies showed a positive correlation between the frequencies of axo-glial detachment at the paranode and axonal degeneration, as assessed by teased-fiber preparations (p < . ). in conclusion, paranodal dissection without classical macrophage-mediated demyelination is the characteristic feature of patients with cidp with autoantibodies to paranodal axo-glial junctional molecules. koike h , ikeda s , takahashi m , kawagashira y , iijima m , misumi y , ando y , ikeda si , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; kumamoto university, kumamoto, japan; shinshu university hospital, matsumoto, japan. peripheral neuropathy is the cardinal feature of familial amyloid polyneuropathy (fap), but its mechanism has not been fully elucidated. we used electron microscopy to examine schwann cells and endoneurial microvessels. sural nerve biopsy specimens from fap patients with transthyretin val met mutation were assessed. patients were consisted of early onset cases from endemic foci and late onset cases from non-endemic areas. loss of nerve fibers with or without neighboring amyloid deposition was a common feature. the amount of amyloid deposition was greater relative to the extent of nerve fiber loss in early onset cases than in late onset cases. the atrophy of schwann cells, particularly nonmyelinating cells, that were apposed to amyloid fibrils was more conspicuous in early onset cases than in late onset cases. the numbers of endothelial cell nuclei, endothelial cell profiles, and occluded microvessels were significantly increased in the fap patients compared with patients with nutritional/alcoholic neuropathies (p < . , . , and . , respectively). findings suggestive of the disruption of blood-nerve barriers, such as the loss of tight junctions and the fenestration of endothelial cells, were also more frequently found in the fap patients (p < . ), irrespective of the presence or absence of amyloid deposition. in conclusion, these findings suggest that direct insult of amyloid fibrils causes schwann cell damage resulting in the predominant loss of small-fiber axons characteristic of early onset cases. in addition, vasculopathy may also participate in the pathogenesis of neuropathy, particularly in late onset cases. kolb n , smith ag , singleton jr , beck s , howard d , dittus k , karafiath s , mooney k . university of vermont, burlington, vt, usa; university of utah health, slc, ut, usa. chemotherapy induced peripheral neuropathy (cipn) is a major cause of morbidity due to numbness, pain, and gait instability. this prospective study compares the current standard care for cipn symptom management to a new care delivery model which utilizes an automated symptom tracking program paired with a nurse practitioner led intervention triggered by moderate to severe symptoms. all participants beginning taxane or platin based chemotherapy called a telephone based automated symptom tracking program daily (symptom care at home -sch) to report chemotherapy related numbness and tingling. sch tracked the presence and severity of neuropathic symptoms and their interference with activities of daily living (adls) on a - scale. participants were randomized to two groups. the usual care (uc) group was advised to call their oncology provider for recommendations on symptom management. in the nurse practitioner (np) group, when symptom severity was ≥ participants received automated self care strategies and a call from a nurse practitioner to provide treatment recommendations based on consensus guidelines. patients participated in the study. mean duration of follow up was . ± . days with . ± . calls. the np group had fewer days with any neuropathic symptom ( . % ± . vs. . % ± . , p= . ), with moderate to severe neuropathic symptoms ( . % ± . vs. . % ± . , p< . ) or days of distress from neuropathic symptoms ( . % ± . vs. . % ± . , p= . ). on days with moderate to severe symptoms participants also reported burning ( . ± . %), weakness ( . ± . %), balance problems, ( . ± . %), and tripping ( . ± . %). there was no significant difference between groups in the interference in adls (np . ± . vs. uc . ± . , p= . ). overall the automated telephone system effectively identified neuropathy symptoms and their severity. compared to usual care in which patients must independently reach out to their care team for symptom management, sch is effective in decreasing symptom prevalence, severity and distress. kouton l , kremer l , tard c , morales r , kuntzer t , attarian s , boucraut j , delmont e . referral centre for als and neuromuscular diseases, marseille, france; neurology department, strasbourg, france; neurology department, lille, france; neurology department, montpellier, france; neurology department, lausanne, switzerland; immunology laboratory, marseille, france. antibodies against proteins of the node of ranvier have been recently described in severe chronic inflammatory demyelinating polyradiculoneuropathies (cidp). they target paranodal proteins, namely contactin (cntn ) and neurofascin (nf ). cell-based assay and elisa are available in research, but no gold standard technic is admitted for the detection in routine of these antibodies. our objective was to evaluate if flow cytometry analysis is an efficient technic to detect antibodies against ctn and nf in a large cohort of cidp patients. flow cytometry analysis were performed on a bd facs-diva. human embryonic kidney (hek) cells were transfected either with nf or cntn . sera were diluted / . antibodies anti-cntn or nf were revealed using fitc conjugated anti human igg antibodies. delta mfi (mean fluorescence intensity) was calculated as mfi of transfected cells less mfi of non-transfected cells. measures were normalized using positive controls and negative controls from healthy blood donors. sera of cidp patients from different french neuromuscular referral centres were analysed with flow cytometry. respective delta mfi were (standard deviation ) and (standard deviation ) for antibodies against nf and cntn in cidp antibodies negative patients. antibodies against nf were found in patients (respective mfi , , ) and against cntn in two other patients (respective mfi and ). isotype of these antibodies was igg in patients and igg and igg in the remaining patient. all the patient had severe cidp. four patients had poor response to intravenous immunoglobulins (ivig) and have been treated with immunosuppressive drugs. as usually reported, the patient with anti-nf antibodies had postural tremor. flow cytometry seems effective to detect antibodies against nf and cntn . compared to other assays, benefits of flow cytometry are: to analyse a large number of sera in the same time and to give objective numerical results expressed in mfi that can be compared to the results of other samples. further studies are needed to confirm that flow cytometry can be the best test to assess antibodies against cntn and nf in routine. the mechanism by which intravenous immunoglobulins (ivig) improves peripheral nerve function in multifocal motor neuropathy (mmn) is unknown. the rapid clinical improvement following ivig could be related to blocking complement deposition on gm epitopes, change in ion-channel properties of affected motor axons, or both. the present study investigated median nerve motor excitability parameters at ∘ c just before ivig administration as well as at the peak of clinical improvement in patients with mmn. the investigated nerves were characterized either by conduction block (n= ), demyelinative slowing without block (n= ), or motor axon loss (n= ). the results of motor excitability testing in mmn showed no difference between pre-and post ivig recordings. clinical assessment of apb muscle showed increase in mrc score in patients and decrease in patient after ivig administration. in patients mrc score of the apb remained the same. those findings indicate that clinical changes following ivig administration are not related to excitability parameters of affected motor axons in mmn. where impulse conduction is blocked or markedly slowed in motor axons but is normal in sensory axons. sensory symptoms or signs are usually absent but have occasionally been reported in skin areas innervated by nerves with prominent motor axon loss. although the mechanism of selective motor involvement in mmn is unresolved, it may be related to differences in antigenic properties between motor and sensory axons or differences in biophysical properties. the objective of the present study was to compare ion-channel activity in both motor and sensory axons of nerves affected by mmn. affected nerves had to have motor conduction block, demyelinative slowing on motor ncs, or motor axon loss, whereas sensory ncs had to be normal. we performed excitability tests of motor and sensory axons in affected median nerves of mmn patients and healthy controls at ∘ c. conditioning and test stimuli were delivered at the median nerve at the wrist; cmaps were recorded from the thenar muscle and snaps from the rd digit. results of motor excitability testing in mmn showed fanning-out of threshold electrotonus, decreased i/v slope, and increased superexcitability, all compatible with persistent hyperpolarization of resting membrane potential in motor axons. sensory excitability testing in mmn showed decreased subexcitability but was otherwise normal. this may indicate minimal involvement of sensory axons in mmn. krarup c , , moldovan m , , alvarez s , ciano c , pisciotta c , pareyson d . university of copenhagen, copenhagen, denmark; rigshospitalet, copenhagen, denmark; fondazione irccs istituto neurologico c. besta, (incb), milan, italy. mutations in the gene coding for myelin protein zero (mpz, p ) are associated with different forms of charcot-marie-tooth (cmt) disease. we describe a family harboring a frameshift mutation (c. dela / p.asp thrfster ) in the p gene, predicted to result in a nonfunctional p truncated very early in the extracellular domain. this offered the rare opportunity to assess the consequences p deficiency in absence of the potential gain-of-function effects of the mutations itself. conventional conduction studies and multiple measures of nerve excitability by "threshold tracking" were carried out in heterozygote parents (aged and ) and their two homozygote sons (aged and ). in the homozygous patients, all distal limb cmaps and snaps were absent. for neurophysiological assessment, the spinal accessory nerve was stimulated at the neck and cmap was recorded over the upper trapezius muscle. eight normal subjects, mean age , were used as control. the two sibs showed a severe phenotype with early onset, severe scoliosis, complete loss of distal movements and relevant proximal weakness, cmt examination score (cmtes) - / ; both heterozygous parents had very mild adult-onset neuropathy with cmtes < / . control subjects had a trapezius cmap with a latency of . ms and an amplitude of . mv. heterozygotes had a mild cmt type b phenotype, with a cmap latency of . ms and an amplitude of . mv whereas the homozygotes had a severe neuropathy with a cmap latency of . ms and an amplitude of . mv. consistently, the homozygotes had a more severe impairment in excitability with a rheobase of . ma as compared to . ma in the heterozygote and . ma in controls. deviations in excitability measures were similar to our previous reports in p +/− and p −/− mice. mathematical modeling, indicated both altered passive cable properties due to dysmyelination and depolarizing features with increased na+ currents. our data suggest that p deficiency is associated with impaired axonal na+ channel function, arguing for the translational value of na+ channel blocker treatments as found in p null mouse models. krishnarajah s , divino v , mallick r , dekoven m . csl behring, king of prussia, pa, usa; quintilesims, fairfax, va, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is a rare neurological disorder of the peripheral nervous system. the objectives of this retrospective real-world study were to compare demographic and clinical characteristics among cidp cases and matched controls and to assess cidp treatment utilization. adults newly diagnosed with cidp between / / and / / were identified in the quintilesims pharmetrics plus health plan claims database (first diagnosis date termed the index date). eligibility requirements were: confirmation of cidp (second cidp diagnosis or initiation of cidp therapy) within year of initial diagnosis, continuous health plan enrollment in the months prior to diagnosis (the pre-index) and the years following diagnosis (the follow-up), and no cidp diagnosis or use of cidp therapy in the pre-index. a total of , cidp cases met the study eligibility criteria. cases were direct-matched to controls based on age, gender, region, health plan, and payer type at index, and pre-index charlson comorbidity index score. the final sample consisted of cases matched to controls (both: mean [sd] age . [ . ]; . % male; . % commercially-insured). alternative pre-index diagnoses among cases included inherited neuropathies ( . %) and chronic acquired polyneuropathies ( . %). in the pre-index, neuropathic pain ( . % vs. . %), back pain ( . % vs. . %), and use of opioids ( . % vs. . %) and anti-convulsants ( . % vs. . %) were significantly higher among cases compared to controls (p< . for all). median total pre-index healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . ). over the follow-up, median total healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . [mean $ , and $ , ] ). cidp-related therapy costs accounted for . % of total healthcare costs for cases. the majority of cases ( . %) initiated cidp therapy over the follow-up, in a mean of . ( . ) days from initial diagnosis. half ( . %) of cases initiated treatment with corticosteroids only, while . % initiated ivig only. over the follow-up, . % of cases used any corticosteroid, while . % used any ivig. our findings suggest a substantial clinical and economic burden of cidp compared to matched controls. corticosteroids and ivig were most commonly used to treat cidp. kronlage m , baeumer p, pitarokoili k , schwarz d , schwehr v , godel t , heiland s , gold r , bendszus m , yoon ms . department of neuroradiology, heidelberg university hospital, germany; department of neurology, st. josef hospital, ruhr university of bochum, germany. objective: to evaluate large coverage magnetic resonance neurography (mrn) in chronic inflammatory demyelinating polyneuropathy (cidp). methods: in this prospective study patients with cidp and healthy controls were examined by a standardized mrn protocol at tesla. lumbosacral plexus was imaged by a t -weighted d-sequence ( mm isotropic voxel size); peripheral nerves of the upper and lower extremity by axial t -weighted turbo-spin-echo sequences ( . x . mm in-plane resolution). lesions were characterized by nerve cross sectional area (csa) and t -weighted signal (nt ). additionally, t -relaxometry of the sciatic nerve was performed using a multi-spin-echo sequence. all patients received a complementary electrophysiological exam. results: patients with cidp exhibited increased nerve csa and nt compared to controls (p < . ) in a proximally predominating pattern. roc analysis revealed best diagnostic accuracy for csa of the lumbosacral plexus (auc = . ) and nt of the sciatic nerve (auc = . ). csa correlated with multiple electrophysiological parameters of demyelinating neuropathy (f-wave latency, nerve conduction velocity) of sciatic and median nerve, while nt only correlated with f-wave latency of sciatic and not median nerve. t -relaxometry indicated that mr-signal increase in cidp was due to increase in proton-spin-density (p < . ), and not increase in t -relaxation time. conclusion: both nt and csa might aid in diagnosis of cidp, but csa correlates more robustly with electrophysiological parameters. since best diagnostic accuracy was shown for proximal nerve locations, mrn may be a useful complementary tool in select cidp cases. kühnemund j , wetzel c , bégay v , moshourab r , lewin gr . mdc & bih, berlin, germany; mdc, berlin, germany; charité, berlin, germany. damage of peripheral sensory nerves due to diabetes, herpes zoster infection, chemotherapy or trauma can cause chronic neuropathic pain. common symptoms include increased pain sensation (hyperalgesia), touch-induced pain (allodynia), paresthesia and spontaneous pain. we currently have poor understanding about the underlying molecular mechanisms at the peripheral level and treatment of patients suffering from neuropathic pain is inadequate. recent meta-analysis studies show that common first-line medications only yield nnts (numbers needed to treat) between . to . . it is still unclear to what extent allodynia can be attributed to changes in the physiological properties of intact sensory afferents. in this study, we aimed to elucidate whether changes occur in intact sensory afferents that innervate the plantar skin of the hind-paw following induction of neuropathic symptoms. this question was of particular interest considering that blocking mechanotransduction in the skin can alleviate mechanical hypersensitivity in neuropathic pain models (wetzel et al nature neuroscience ( ): - ). we used the chronic constriction injury (cci) model in mice which is behaviourally characterized by robust mechanical hypersensitivity. we made electrophysiological recordings from primary afferent neurons which had intact axons passing through the constriction to innervate the plantar skin using an ex-vivo skin-nerve preparation. we were able to record from myelinated afferent fibers and unmyelinated c-fibers with receptive fields in the control uninjured plantar skin as well as plantar skin of cci mice. we will present evidence that changes in the mechanosensitivity of sensory fibers innervating the glabrous skin may contribute to the symptoms of neuropathic pain. neuropathology, national hospital for neurology and neurosurgery, london, uk; irccs foundation "carlo besta" neurological institute, milan, italy; neurogenetics unit, national hospital for neurology and neurosurgery, london, uk; nuffield department of clinical neurosciences, oxford, uk; department of clinical neurophysiology, norfolk and norwich university hospital, uk. hsn secondary to sptlc / is a rare slowly progressive neuropathy resulting in marked sensory loss, especially nociception and significant motor deficit. despite most of the patients having the same c w mutation in sptlc , there is marked heterogeneity in the phenotype. l-serine oral supplementation has been suggested as potential therapeutic candidate however the lack of outcome measures is a major limiting factor in the initiation of a clinical trial. we undertook a natural history study to identify outcome measures that are responsive enough to be used in a clinical trial. the assessments used were cmt neuropathy score (cmtns version and cmtns version rasch modified), mri of calves and thighs, computerised myometry, quantitative sensory testing (qst), comprehensive neurophysiological assessment, proximal thigh skin biopsy for intra-epidermal nerve fibre density (ienfd), plasma dsl levels and patient based questionnaires (neuropathic pain symptom inventory and sf- v ). standardised response mean, srm (mean change/standard deviation of change) was used to compare responsiveness between tests. patients were recruited: with sptlc (c w) and with sptlc mutations. when analysed as a whole cohort, proximal calf mri fat fractions showed the most significant change over months. ienfd, plasma dsl levels, npsi and sf- v showed minimal change or the change was not in the clinically expected direction. for subsets of the remaining assessments which showed the highest responsiveness, the cohort was sub-divided into mild-moderate (cmtns ≤ ) and severe (cmtns> ) subgroups. in the mild-moderate subgroup, the greatest improvement in responsiveness was seen in computerised myometry (ankle plantarflexion: srm=− . and ankle eversion: srm=− . ). in the severe subgroup, qst (vibration detection thresholds on hands: srm=− . and face: srm=− . and pressure pain threshold on the face: srm= . ) and proximal calf mri fat fractions (srm range= . - . ) showed the greatest improvement. focusing on subgroups classified according to disease severity improved the responsiveness of some tests into the highly responsive range with mri still being the best outcome measure. this will reduce the number of participants required to power a clinical trial and might be a possible solution for designing a clinical trial for a rare, slowly progressive disease with a heterogeneous phenotype. kugathasan u , clark aj , suriyanarayanan s , laurá m , wilson e , , kalmar b , greensmith l , , hornemann t , reilly mm * , bennett dlh * . hsn secondary to sptlc / mutation is a slowly progressive sensory motor neuropathy leading to profound sensory loss with variable but often severe motor deficit. the genes sptlc and encode for the essential enzyme serine palmitoyltransferase (spt) which catalyses the rate limiting step in the sphingolipid de-novo biosynthesis. mutations in these two genes alter the substrate specificity of spt leading to the synthesis and accumulation of atypical metabolites called -deoxysphingolipids ( -deoxysl). plasma levels of -deoxysl are raised in hsn patients. deoxysphingolipids have been shown to be toxic in avian and by our group, in mammalian primary drg and motor neuron cultures. firstly, this study looked at the effects of -deoxysl on the survival and neurite integrity of human ipsc derived sensory neurons following exposure to different concentrations of deoxysphingolipids. later, we determined if there was autonomous -deoxysl production in hsn patient ipsc derived sensory neurons. sensory neurons were differentiated from human ipscs using a combination of small molecular inhibitors. deoxysphingolipids were found to be neurotoxic in this model after hours of treatment. in control lines, there is a significant reduction in neuronal survival following treatment with both -deoxysphinganine ( -deoxysa) and -deoxymethylsphinganine ( -doxmethsa). a clear dose-dependent increase in the expression of the axonal injury marker, atf , is seen with both -deoxysphngoid bases. in both instances, -deoxysa is more neurotoxic than -doxmethsa, which is similar to the findings in avian and mammalian primary neuronal cultures. autonomous -deoxysl production is seen in ipsc derived neurons obtained from three different hsn patients with the levels being significantly greater than that seen in multiple control lines. this is the first study to demonstrate that human ipsc derived sensory neurons can be used as an in-vitro model for hsn , providing a great opportunity both to probe the pathomechanisms mediating deoxysphingolipid toxicity and to test potential therapeutic agents. recent studies have demonstrated an association between autoantibodies directed against antineurofascin- (anti-nfasc ) and a subpopulation of cidp patients characterized by sensory ataxia, tremor and poor response to ivig treatment. here we report on the clinical features of three patients who developed acute changes in their phenotype during the course of their neuropathy, a potential clue to recognize a neuropathy with predominantly humoral dysimmunity. case reports: our index patient had developed sensory changes over weeks, followed by irregular locomotion, and muscle weakness with general areflexia. a first run of ivig improved the patient in a week, but he relapsed within days. a second ivig course with prednisone again normalized deficits within days. ivig runs and rituximab were still necessary to treat a nd , and then a rd relapse before obtaining complete improvement. the course of the neuropathy was months. two other patients were encountered with more chronic courses but in whom periods of worsening or improvement suggested a relapsing-remitting neuropathy, either spontaneously or following immunomodulating treatments. in all three, extensive work-ups were negative, anti-nfasc igg were positive, and detailed repeat nerve conduction studies demonstrated fluctuating conduction blocks. discussion: our report underscores that in chronic cidp patients a relapsing-remitting course could be encountered as a key feature of anti-nfasc neuropathy. this not yet described characteristic course could be of value when deciding using rituximab instead of immunomodulating treatments. kusunoki s , morikawa m , kuwahara m , ueno r , samukawa m , hamada y . faculty of medicine, kindai university, osaka-sayama, japan. anti-glycolipid antibodies are often detected in sera from patients with autoimmune neuropathies, such as guillain-barré syndrome (gbs), chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and multifocal motor neuropathy (mmn). not only individual glycolipid antigens but also mixtures of two different glycolipids (glycolipid complexes) are sometimes recognized by serum antibodies. to investigate antibody activities against large number of glycolipid complexes in serum samples from patients with gbs, mmn, and cidp, we examined igm and igg antibodies against glycolipids [gm , gm , gd a, gd b, gq b, galnac-gd a, lm , galactocerebroside (gal-c), asialo-gm (ga ), and sulfatide] and glycolipid complexes consisting of two different glycolipids listed above, by using combinatorial glycoarray. serum was obtained from patients with gbs, patients with cidp and patients with mmn, all in the acute or relapsing phase. serum was also obtained from healthy controls and patients with other neurological diseases. we investigated the relationships between the clinical features and presence of those antibodies. high titers of igg antibodies were detected almost exclusively in gbs patients. in contrast, igm antibodies were frequently present in mmn and gbs. among the anti-glycolipid complex antibodies in gbs, anti-gm /sulfatide, anti-ga /sulfatide, anti-gm /gd a, and anti-gq b/sulfatide igg antibodies were common ( , , , and patients, respectively). igg antibodies against antigens containing gm were significantly correlated with pure motor gbs (p < . ) and those against antigens containing gq b were significantly correlated with gbs with ophthalmoplegia (p < . ). in seven of the patients with anti-gq b/sulfatide complex antibodies, the antibodies were specific to the gq b/sulfatide complex rather than the individual gq b and suldfatide antigens. moreover, four patients did not have antibodies other than those to the anti-gq b/sulfatide complex. in patients with mmn, igm antibodies to antigens containing gm or galnac-gd a were present in % and . %, respectively. glycoarray is efficient for detecting antibodies against numerous glycolipid complexes in immune-mediated neuropathies. we need further investigations on other immune-mediated diseases using larger number of antigens. kuwabara s , misawa s , sekiguchi y , susumu kusunoki and the jet-gbs study group . department of neurology, chiba university, chiba, japan; department of neurology, kindai university, osaka, japan; japanese eculizumab trial for guillain-barré syndrome, chiba, japan. guillain-barré syndrome is a monophasic immune-mediated neuropathy, but a substantial number of patients with severe disease have poor recovery, even if treated with immunoglobulin. recent studies suggest that complement activation plays a pivotal role in gbs-associated axonal degeneration, and eculizumab is a monoclonal antibody that specifically binds to complement component and inhibits complement activation. jet-gbs is an investigator-led, phase , randomized, placebo-controlled trial conducted in hospitals, this trial aims to investigate the safety and efficacy of eculizumab for treatment of severe gbs. patients were randomly assigned ( : ) to treatment with immunoglobulin plus either eculizumab ( mg/day; n= ) or placebo (n= ) once weekly for weeks. the primary outcome measures are safety and efficacy (the proportion of subjects who regain their ability to walk independently at week ). the secondary outcome measures included the proportion of subjects who were able to walk independently at week , and other measures such as mrc sum scores, nerve conduction parameters. enrollment for the trial began in august , and follow-up of the last patient was completed in october . analyses will be made in april , and the results will be presented at this meeting. this trial is registered with clinicaltrials.gov identifier: nct , and funded by the japanese agency for medical research and development, and alexion pharmaceuticals inc. neurofascin , a paranodal protein in peripheral nerve, is a target antigen for autoantibodies in a subset of chronic inflammatory demyelinating polyneuropathy (cidp). anti-neurofascin antibody-positive cidp is characterized by onset at younger age, tremor, and refractoriness to ivig. we have treated four patients with anti-neurofascin antibody-positive cidp at kindai university hospital. anti-neurofascin antibody was detected by both elisa and cell-based assay in those patients. igg subclass was predominantly igg in all four cases. the median age of the patients at admission was . years [range: - years]. among the four patients, three had tremor, and two had severe cerebellar ataxia. cerebrospinal fluid protein levels were remarkably increased [median: . mg/dl, range: - mg/dl]. although ivig treatment was administered in all four patients, the responses were poor or partial. in contrast, plasma exchange (pe) was performed in all four patients and the clinical symptoms dramatically improved in two of them. corticosteroids were also effective in those two patients. sural nerve biopsy was performed in all four patients. although sensory nerve action potentials of the sural nerves from those patients were not evoked, the transverse semithin sections of sural nerves from three patients revealed only slight or mild loss of myelinated fibers. partial paranodal demyelination was observed in teased-nerve fibers from three patients. in addition, abnormal paranodal lesions such as loss of the transvers bands were observed by electron microscope in all four patients. those electron microscopic findings were not observed in control patients with anti-neurofascin antibody-negative cidp. anti-neurofascin igg antibody-positive cidp shows distinctive clinical and pathological features. labeyrie c , besson f , vandendries c , cauquil c , beaudonnet g , not a , durand e , adams d . neurologie adulte, chu bicêtre, le kremlin bicêtre, france; médecine nucléaire, chu bicêtre, le kremlin bicêtre, france; clinique bizet, paris, france; unité de neurophysiologie clinique, chu bicêtre, le kremlin bicêtre, france. study of the proximal portion of the peripheral nervous system (pns) is difficult because less accessible to electrophysiological exploration and biopsy. the indications of mri are increasing in proximal neuropathies to analyse morphology of the roots, plexus and proximal nerves: integrity of the nerve bundle, inflammation or infiltration of these structures. pet-computed tomography (pet-ct) can detect infiltration of the proximal segments of the pns, with hypermetabolism of roots, plexuses and large nerve trunks. pet-ct is also useful in detection of solid neoplasm which can infiltrates pns and primary nerve sheath tumors. however, spatial resolution of pet -ct scan is limited to explore the roots and plexuses for moderate hypermetabolisms. we believe that a mild hypermetabolism could be highlighted in inflammatory neuropathies or in mild tumor infiltrations. we performed a fusion of whole body pet (performed because of suspected neoplasia) and plexus mri (pet-mri) images in ten patients with a peripheral neuropathy for which we suspected proximal involvement. the pet-ct and mri were first read separately, then with merge of the images by a nuclear physician and a neuroradiologist. five out of ten patients presented with hypermetabolism of pns (hmpn+) on pet-mri: root (n= ), and/or dorsal root ganglia (n= ). median sul of lesions was . . among the hmpn+ patients: hypermetabolism was already apparent on pet-ct in cases, the merge invalidated abnormalities seen on pet-ct in patients and mri detected additional lesions, not visible on pet-ct, in one patient. all hmpn+ patients had hypersignal and or hypertrophy of pns on mri either diffuse (n= ), or multifocal (n= ). among hmpn-(no hypermetabolism) out of ( %) had abnormal mri ( multifocal and diffuse). gadolinium enhancement was found in all patients receiving gadolinium in the hmpn+ (n= ) and only in / patients in the hmpngroup. final diagnoses of hmpn+ patients were neurolymphoma in one ( %), and idiopathic cidp in the others ( %) with histological proof on nervous root biopsy in one of them. final diagnosis in hmpn-patients was cidp in out of and sequelae of neuropathy in relation to lymphoma without relapse for the last one. cidp was more disabling (onls≥ ) in hmpn+ than in hmpn-group % vs. %. pet-mri could be helpful to detect a proximal inflammation of the pns, and to plan further testing. further studies are needed to evaluate the prognostic value of hmpn+. lancaster e , li j , liem r , scherer ss . perelman school of medicine, university of pennsylvania, philadelphia, pa, usa; columbia university school of medicine, new york, ny, usa. heterozygous nefl n s/+ mutant mice are the first animal model of a charcot-marie-tooth disease e (cmt e). people with this mutation have a severe, early onset axonal neuropathy. axons in the mutant mice have reduced number of neurofilaments and decreased diameters. they also show early onset of tremor and abnormal hindlimb clasping behavior. we measured the compound action potentials (caps) from tails every weeks from the same cohort of nefl n s/+ mutant mice and their wt littermates from to weeks. even at the age of weeks, the amplitude of mutant caps was only ∼ % of wt caps ( ± . microv, n= vs ± . microv, n= ), and the caps stayed substantially smaller than those in wt mice for weeks ( ± . microv, n= vs ± . microv, n= ). the conduction velocity and the duration of caps in mutants were slower and wider compared to those of wt. separate cohorts of mice (n= mutants and n= wt) were sacrificed at different time points for analysis by light microscopy. in caudal nerve of mutant mice, the number of axons was significantly reduced compared to that of wt at weeks and, by weeks, it was reduced more than %. this model system may be useful for preclinical studies of treatments for cmt e since the animals show progressive neuropathy over weeks, which can be objectively measured electrophysiologically and anatomically. despite more than cmt genes identified today, at least % of cmt patients do not carry a mutation in any of these genes. progress in gene identification in recent years suggests that there are still many more cmt disease genes to be discovered; however, it has become rare to gather support from multiple large families that allow for conclusive linkage analysis. we have studied multiple extended dominant cmt families with linkage support for a gene at chromosome p . . originally a czech family yielded a two point lod score of . at this locus and a family from southern italy showed a lod score of . . whole exome sequencing of multiple family members identified missense mutations in the gene atpase na+/k+ transporting subunit alpha (atp a ). atp a has not been associated with human diseases thus far. in expression studies on teased fiber preparations we already confirmed predominant expression in the nodes of ranvier of peripheral nerves. through collaborative efforts in the inherited neuropathy consortium and beyond we identified five additional multigenerational families via exome or sanger sequencing resulting in a total of seven unique segregating missense changes: leu arg, ile thr, ala thr, asp phe, pro ala, pro thr and asp ala. five of these mutations fall into a remarkably narrow motif associated with the sodium binding structure of atp a , flanking the flexible hinge motif. functional studies in different model systems (mammalian cell lines, xenopus oocytes, patient fibroblast lines) are underway to determine whether a loss or a dominant gain-of-function represents the disease mechanism. taken together, we show strong support for a major new dominant cmt gene, atp a . this finding represents a new pathway and an attractive new target for therapy development in axonal cmt. laurá m , ramdharry g , , singh d , kozyra d , skorupinska m , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; school of rehabilitation sciences, st george's university of london/kingston university, uk; royal national orthopaedic hospital, stanmore, uk. charcot-marie-tooth (cmt) disease is the most common inherited peripheral neuropathy. foot deformities are frequent complications and orthopaedic surgery is often required. however there are no evidence based guidelines on the type or timing of the surgery. only few studies have described the long-term results of surgical procedures and evidence regarding optimal surgical management of these patients is lacking. we prospectively studied surgical management of cmt patients attending our centre. we collected data and assessed cmt patients before and after surgery. data included: history of ankle instability, pain, skin condition, details of physiotherapy and orthotic management, assessment of lower limb strength, charcot-marie-tooth examination score (cmtes), foot posture index, ankle dorsiflexion range of movement and specific questionnaires (foot index and manchester-oxford foot questionnaire, modified fatigue severity scale and modified falls efficacy scale), details of surgical procedures. patients were assessed yearly after surgery. so far patients ( males and females, age range - ) have been evaluated prior to surgery. all patients but one had genetically confirmed cmt ( cmt a, cmtx, cmt a). patients have been assessed after year, patients after years, patients after years and patient after years from surgery. a wide range of surgical procedures were performed by one dedicated orthopaedic surgeon. preliminary results showed reduction of number of falls in / ( %) patients and improvement of callosities in / ( %) patients at year follow up. there was also significant improvement of alignment of the operated foot (p= . ) and pain (p= . ). there were no significant changes in measures of strength and ankle range of motion. further analysis on a larger number of patients will be important to determine the long-term outcome of surgery. data acquired from this study will help develop orthopaedic intervention guidelines and identify areas for further research. lavigne-moreira c , oliveira mf , marques vd , onofre ptbn , dos santos acj , nascimento ojm , barreira a , marques w jr . division of neuromuscular diseases and neurogenetics, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; department of neurology, fluminense federal university, rio de janeiro, brazil. fap associated to ttr mutations is defined as a length-dependent axonal sensory and motor polyneuropathy that at early stages affects mainly the small nerve fibers, associated to autonomic and thermo-algesic sensations. the expected emg pattern was that of an axonal sensory and motor polyneuropathy, but in fact several unexpected patterns may be found. in this study we present the results found in a brazilian population with ttr mutation. patients were divided in three groups: ttr-met of early onset, ttr-met of late onset and ttr non-met . in the first group (ttr-met of early onset), ( . %) examinations were normal, ( . %) were axonal, were demyelinating fulfilling pidc criteria, one suggested a predominately motor polyneuropathy and the final one presented a lower motor neuron disease pattern. in the second group (ttr-met of late onset), ( . %) had an axonal pattern, ( . %) had an intermediate cv, ( . %) had a demyelinating pattern, had a lumbossacral pattern and the final one had no definite pattern. among the non-ttr met , two patients had the ttr -asp tyr, one presented an axonal pattern, while the second presented initially an axonal pattern, that changed to a demyelinating pattern in the second examination. patients with ile val mutation presented an axonal neuropathy associated to cts. most patients with demyelinating or intermediate pattern were treated with corticosteroids or ivig, with no satisfactory results. this small series of patients shows clearly that fap-ttr is associated to several emg patterns in addition to the expected sensory and motor axonal polyneuropathy. this variability is present in the same family and in the same patient in different occasions. clinicians should be alert to these possibilities to do not delay diagnosis and treatment. identifying the mechanisms involved in this variability could improve our knowledge of this intriguing disease. lavin tm . greater manchester neurosciences centre, salford royal hospital, manchester, uk. subcutaneous immunoglobulin (scig) has evidence from small trials for its use following intravenous igg (ivig) loading in chronic inflammatory demyelinating polyneuropathy (cidp). potential advantages of scig include stable igg levels and reduced complication rates. scig de novo can be considered as igg levels gradually rise with subcutaneous delivery and maybe beneficial for patients for whom complications are a concern. we present cases of cidp who were initiated on subcutaneous immunoglobulin g ( g/kg/month, % scig, hizentra, csl behring) following corticosteroids; without intravenous igg loading. patient is year old with a history of ihd with a progressive lower limb sensory changes and sensory ataxia over years. neurophysiology confirmed sensory demyelinating changes. he did not respond to corticosteroids. concerns were raised about the potential risk orf adverse cardiac events with iv immunoglobulin. scig therapy was introduced at g weekly. initial therapy has been well tolerated and objective measures (rods-cidp score, jamar grip strength, hole peg) have remained stable through initiation, and at months. patient is yr old male with a past history of branch retinal vein thrombosis. he developed a sensorimotor polyneuropathy over years with neurophysiology fitting efns criteria for cidp. given concerns regarding previous thrombosis, a trial of scig was given at g/week, with resolution of sensory ataxia and improvement in objective markers (jamar grip strength, rods-cidp score, hole peg) at months. unfortunately an adverse event occurred of an urticarial skin reaction. patient is a yr old male who presented with motor predominant cidp. there was no response to corticosteroids but improvement following plasma exchange. due to a past history of transient ischaemic attack, scig was started. there has been a positive response after months with improvement in walking distances and stable objective markers (rods-cidp score, grip and pinch strength). our cohort remained neurologically stable during initiation of scig, with adverse reaction. our experience would support further trials in this area regarding the efficacy of scig compared to ivig in both the short and long term. diabetic peripheral neuropathy is the most common and debilitating complication of diabetes and it is associated to neuropathic pain and non-traumatic amputations. despite the economic burden and human costs, nowadays there is not a specific treatment to cure diabetic peripheral neuropathy. hyperglycaemia and dyslipidemia-mediated oxidative and endoplasmic reticulum stress has been linked to diabetic peripheral neuropathy, among other altered pathways. in order to study in more detail the molecular mechanisms that lead to the development of diabetic peripheral neuropathy we set up in vitro models of the disease using nsc- (motoneurons) and med . (sensory neurons) cell lines or primary cultures of dorsal root ganglia and motoneurons exposed to saturated fatty acids. it has been reported that palmitic acid is able to induce endoplasmic reticulum stress and oxidative stress in multiple cell types including schwann cells and myenteric neurons, resulting in a good model of dyslipidemia-mediated stress. here we found that palmitate induces upregulation of the molecular chaperone bip/grp and the ccaat-enhancer-binding protein homologous protein (chop) mrnas and a dose-dependent downregulation of the apoptosis marker bcl- in primary cultures. moreover, palmitic acid induces loss of neuron dendrites and cell death at high doses and upregulation of heme oxygenase (ho- ) and chop at lower doses in the nsc- cell line. we are currently characterizing multiple molecules implicated in oxidative stress, endoplasmic reticulum stress and inflammation pathways in these cell types to find new therapeutic targets to treat type diabetic sensorimotor polyneuropathy, that will be subsequently validated in the db/db mouse model by pharmacological or gene therapy strategies. lee bjh , ohnmar o , wong j , koh sj , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful peripheral neuropathy and autonomic dysfunction that occurs within weeks of tight glycaemic control. therapy with either insulin or oral hypoglycaemic agents (ohga) may result in tind. the quantum and rate of decline in hba c predicts development and severity of tind. both type i and ii dm patients are prone to this iatrogenic complication. besides the expected morbidity associated with painful somatic and autonomic neuropathy, tind patients also develop life-threatening eye complications such as maculopathy. with greater awareness, we picked up at least typical cases of tind in recent few months. however, there was a fourth possible tind case, whom we feel deserves special attention. the circumstances surrounding this case are common and may go under-recognised in acute hospitals. a year-old man with -year history of poorly controlled type ii dm was admitted with a partial left middle cerebral artery stroke. one month before admission, his hba c was . %. his hospitalization was prolonged because of his considerable disability that required rehabilitation. he stayed weeks. his blood sugar was difficult to control with hyperglycaemic episodes requiring ohgas and insulin. the highest capillary blood glucose recorded was mmol/l. he also had episodes of hypoglycaemia. three weeks into hospitalization, he developed severe orthostatic hypotension. he came to our attention two months later when he was sent for autonomic screening tests for severe postural hypotension, in spite of therapy with fludrocortisone and midodrine. his hba c was . %. he had marked orthostatic hypotension suggestive of sympathetic dysfunction. we were not able to discern if he had a painful neuropathy because of his aphasia. there was no recent ophthalmology review. we suggested a diagnosis of possible tind. he was discharged with lower doses of ohga. however, day after discharge, he attended the emergency department for a syncopal episode. this case raises intriguing questions on the safety of glucose control paradigms commonly employed in patients with acute stroke and myocardial infarction as well as the possible role of major fluctuations in blood glucose in the development of tind. lee hs , kim sm . presbyterian medical center, jeonju, korea; yonsei university college of medicine, seoul, korea. the occurrence of peripheral neuropathy by a tumor within or compressing a nerve in neurofibromatosis (nf) type and is relatively well known. however, nf presenting with demyelinating polyneuropathy unrelated to tumor masses is rarely reported. we report a rare presentation of an nf patient with demyelinating polyneuropathy of subacute onset. a -year-old woman was referred to our hospital with paresthesia on both limbs months before. she was healthy and had no family history. the symptoms worsened over the next months, tremor and weakness in both limbs occurred. although she treated as a chronic inflammatory demyelinating polyneuropathy (cidp) with intravenous steroid and immunosuppressant in another hospital, symptoms were getting worse. bifacial numbness and left eyeball pain occurred weeks ago and she visited our hospital. on neurologic examination, bilateral facial sensory hypoesthesia and symmetrical both limbs weakness (mrc grade iv+) were observed. decreased touch and pinprick sensation on both limbs were observed like stocking-glove distribution. the reflexes were sluggish. on nerve conduction study, sensorimotor demyelinating polyneuropathy with conduction blocks and temporal dispersions was observed. bilateral r , r responses were prolongation on facial blink test. mri of cervical spine and brain revealed contrast enhancing tumor-like enlargement of multiple nerve roots and cranial nerves. nerve biopsy was performed on right supraorbital nerve, neurofibromatous changes were observed. the symptom deterioration was stopped without treatment. gene test dose not revealed nf type and . on review of the literature, polyneuropathy in nf patient can result from tumor masses within the proximal nerve roots, or along the peripheral nerve, or in the extramedullary lesion affecting neighboring nerve roots. thus, axonal type polyneuropathy and focal amyotrophy have been reported in nf. demyelinating polyneuropathy has not been reported before in our knowledge. although the etiology of demyelinating polyneuropathy in nf requires further clarification, some authors claim that unknown local toxic or metabolic influences of the endoneurial pathological cells on adjacent nerve fibers. in this patient, atypical symptoms were observed in cidp such as cranial nerve involvement and stocking-glove sensory distribution. in this case, a nerve biopsy can be helpful the accurate diagnosis. lee jy , yoo jh , kang dk , bae js . department of neurology hallym university, seoul, korea. acute disseminated encephalomyelitis (adem) is an uncommon post-infectious inflammatory demyelinating disorder of central nerve system, while guillain-barre syndrome (gbs) is a prototype of acute post-infectious peripheral neuropathy. previous reports regarding the coexistence of these relatively rare diseases suggest that certain immunogenicity within central and peripheral nerves may share a common autoimmune process during the disease course. a previous healthy years old man was admitted because of fever, headache, nausea and myalgia in department of infectious disease. two weeks before admission, he suffered from watery diarrhea for days and spontaneously recovered. at initial presentation, he had high fever( ∘ c), headache and myalgia and intermittent horizontal diplopia. a few days after admission, he began to complain of drowsy mentality, bilateral extremities weakness, especially lower limbs, dysarthria, bilateral facial paralysis, urinary retention, dyspnea. on neurologic examination, he had mildly drowsy mentality, symmetric muscle weakness scoring of on bilateral hip, knee flexion and finger extension, but he had no sensory symptoms. he showed gazed evoked nystagmus with no extraocular muscle palsies. deep tendon reflexes were not present. pulmonary function test revealed a severe restrictive pattern. csf studies disclosed a dissociative increase of protein contents ( mg/dl) without pleocytosis. anti ganlioside antibody assay identified an anti-gt a igg positivity in his serum. nerve conduction study (ncs) showed prolonged motor terminal latencies and slow motor conduction velocity on multiple nerves. in contrast, sensory ncs revealed no abnormal findings. imaging studies unexpectedly revealed apparently symmetrical lesions across bilateral brainstem and basal ganglia suggesting a diagnosis of adem. after both ivig and high dose steroid treatment, he remarkably recovered from disturbed mental state and motor weakness. about month after the symptom onset, he could walk with assistant aid and discharge to other hospital for rehabilitation. our case suggest that certain component of autoimmunity simultaneously result in both cns and pns inflammation. specific immunological mechanism is remained to be elucidated. although we could not conclude whether cellular component or humoral component is dominant for our case, the presence of anti gt a antibody suggest a role of humoral mechanisms. the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. leitao amf , , araújo df , marques h , pamplona l , , souza mh , , nbraga ll , , gondim faa , . the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. the classic guillain barré syndrome (gbs) is characterized by motor weakness, hyporreflexia, but limited sensory deficits. sensory variants involving either small or large fibers or both are unusual and represent a diagnostic challenge. we described patients presenting with the sensory variant of gbs and retrospectively analyzed the clinical and electrophysiological findings of patients fulfilling the criteria for sensory gbs according to oh et al. criteria. six patients were identified (mean age years: range - years). four had a previous infection. they all consulted due to distal painful paresthesias and allodynia. on examination the patients presented normal strength and normal cranial nerves through the course of the disease with reduced knee and ankle reflexes in patients. distal hyperesthesia to pinprick was identified in and one of them additionally had hyperhidrosis and constipation. two additional patients presented hypoesthesia to pinprick and temperature. one patient had distal proprioceptive sensory loss with sensory ataxia. csf albumin cytological dissociation was present in patients. nerve conduction studies (ncs) identified a sensory motor demyelinating neuropathy in patients. among the with normal ncs, had abnormal cold and warm threshold in their qst evaluation. all patients received symptomatic treatment for the neuropathic pain and only two ivig therapies. longstanding pain, fatigue or both were persistent findings in patients after a mean follow up of months. in conclusion the sensory variant of gbs is both an infrequent presentation and a diagnostic challenge. longstanding pain and fatigue are common persisting findings. the epidemic of zika virus (zikv) throughout the americas and asia, and the subsequent rise in reported cases of guillain-barré syndrome (gbs) caused worldwide concern. as of january , countries have reported evidence of mosquito-borne zikv transmission and in of these countries, a sudden increase of gbs has been reported. moreover, case studies and a case-control study further indicate that zikv may trigger gbs. however, accurate diagnosis of both zikv and gbs in many of these studies is disputed, and a comprehensive description of the clinical phenotype of gbs related to zikv is lacking. the international gbs outcome study (igos) is a prospective observational study on the factors determining the onset, clinical course and outcome of gbs. at present, a research consortium of centers from countries has included patients in igos. our aim is to investigate zikv-related gbs in igos as is already occurring in colombia. in igos-zika, data on clinical features and ancillary investigations will be collected in zikv endemic areas according to the igos protocol with some modifications. first, igos-zika has a case-controlled study design to define the association between gbs and zikv and other arboviruses. second, urine samples will be collected and additional questions on preceding events will be asked, focusing on arbovirus infections. third, a more limited follow-up is required. our aim is to recruit additional centers via the inflammatory neuropathy consortium (inc) and centers in all arbovirus endemic regions that are willing to participate. the focus of igos-zika will be on the accuracy of the diagnosis of both gbs and zikv and on defining the associated clinical phenotype, course and outcome. igos-zika provides the opportunity to combine data and biobanks from various geographical regions using a standardized protocol and to compare these data with data and biosamples already collected in igos. studying these cases will help to optimize diagnostics and care for gbs patients in arbovirus endemic countries and provides a unique opportunity to further understand the pathogenesis of gbs. moreover, this study design and network can be used to adequately respond to other future viral epidemics related to gbs. lerat j , cintas p , dzugan h , , magdelaine c , , sturtz f , , lia as , . service de biochimie et génétique moléculaire -chu de limoges, limoges, france; service de neurologie et d'explorations fonctionnelles -chu de toulouse, toulouse, france; ea -université de limoges, limoges, france. pharc syndrome is an autosomal recessive neurodegenerative pathology leading to demyelinating polyneuropathy, hearing loss, cerebellar ataxia, retinis pigmentosa and early-onset cataract. these various symptoms can occur at different ages, so that pharc syndrome can be a differential diagnosis of charcot-marie-tooth disease (cmt) associated with deafness. only abhd mutations have been reported in patients. we described the th mutation and compared our results to the literature data. we analysed by next generation sequencing (ngs) strategy using a targeted cmt and associated neuropathies -gene panel the dna of a -year old male who has suffered from demyelinating sensory and motor polyneuropathy and ataxia since the age of . bilateral sensorineural deafness was diagnosed at the age of five. bilateral congenital cataracts were operated on at the age of . a new large complex homozygous mutation, with one deletion of seven base pairs and one insertion of base pairs, was detected. by analyzing our patient data and those of the literature, we evaluated that, in pharc syndrome, sensorineural deafness always occurs as the first feature in late teens. the ophthalmological symptoms are cataracts that occur at a mean age of yo and then retinis pigmentosa at a mean age of . demyelinating sensory-motor polyneuropathy is the most variable characteristics, which occurs in the thirties. we report the first large complex homozygous mutation in pharc syndrome, which is certainly under-diagnosed. therefore, it seems interesting to include abhd in the panels of the five symptoms, especially deafness ones. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a chronic disabling disease that often improves with immune therapy. to date, the most reliable diagnostic criteria for cidp are the efns/pns revised criteria, with a reported sensitivity of % and specificity of %. we implemented a web-based database to collect data from patients with cidp followed by italian centers with expertise on cidp to determine the frequency and characteristic of cidp and it variants, the diagnostic criteria used for their diagnosis, the possible evolution into typical cidp, the association with specific anti-nerve antibodies, and their response to therapy. all the patients were evaluated at the time of inclusion and will be followed for two years to monitor their outcome and response to therapy. by february we included patients with cidp and variants ( men, women), aged - years (median ) with a mean disease duration of . years (range . - years). based on clinical symptoms, cidp was defined as typical in % and atypical in %. the diagnosis of typical cidp fulfilled efns/pns criteria in % of the patients while nerve conduction studies were not diagnostic in % (grouped as clinical cidp) or not available in %. we analyzed the frequency of supportive criteria for the diagnosis of cidp in patients with clinical cidp and found that increased csf proteins, demyelination or cell infiltration on nerve biopsy and imaging abnormalities consisting with cidp on us or nmr were present in %, % and % of the patients, respectively. a relapsing course was present in % of patients with clinical cidp, increasing the reliability of the diagnosis for cidp. in addition an improvement after one or more therapies was reported by % of the patients, with a positive response to ivig in %, steroids in % and plasma exchange in %, similarly to what observed in patients fulfilling efns/pns criteria. in % of the patients with clinical cidp two or more supplementary criteria for cidp were present. this study on a large population of patients is providing useful information that may help to revise the current diagnostic criteria for cidp. li j , cannell m , suragani r , pearsall r , kumar r . acceleron pharma inc, cambridge, usa. charcot-marie-tooth (cmt) is the most common hereditary peripheral neuropathy and is characterized by demyelination and/or axonal damage of peripheral nerves and muscle weakness. foot drop, steppage gait, and foot deformities are a typically seen in cmt patients. consequently, falls are commonly reported in these patients. improvement of dorsiflexor muscle function to prevent falls may improve quality of life and activities of daily living in patients with cmt. ace- , a locally-acting ligand trap that binds growth and differentiation factors (gdfs) and activins, has previously been shown to increase muscle mass and force in both duchene muscle dystrophy (dmd) and amyotrophic lateral sclerosis (als) mouse models. in the current study, we evaluated the therapeutic effects of ace- to improve muscle strength in the trembler (tr-j) mouse model of cmt a. these mice harbor a mutation in the peripheral myelin protein (pmp ) known to cause cmt a. seven-month old (b .d -pmp tr-j /j) mice were administered ace- ( g, twice weekly) intramuscularly to one of the unilaterally tibialis anterior (ta) muscle for weeks. the contractility of the ta muscle was evaluated during isometric contraction. all data were compared to the uninjected contralateral control hind-limb. after weeks of ace- treatment, ta muscle mass was increased by % (p< . ) and its physiological cross-sectional area was increased by % (p< . ). the increase in muscle mass correlated with an increase in strength, with maximum tetanic force and twitch force improved by % (p< . ) and % (p< . ), respectively. in addition, temporal properties during isometric contraction, such as maximum rate of contraction and relaxation, were accelerated by % and % respectively (p< . ) in the ace- -treated ta muscle compared to its contralateral hind-limb. pathological and biochemical assessment of ace- -treated mice showed enlarged myocyte area (+ %, p< . ) and reduced atrogin- mrna expression (− %, p< . ). together, these results demonstrated that ace- attenuates the degree of muscle atrophy and also improves muscle function in a mouse model of cmt a. the current study provides proof of concept for the use of ace- as a therapy for cmt to improve dorsiflexor muscle function and alleviate foot drop. we have generated a rat model of charcot-marie-tooth disease a (cmt a) harboring the p.arg trp mfn mutation, whose human counterpart results in a severe, early-onset axonal neuropathy. the mutation was made using zinc finger nuclease-mediated genome editing in fertilized rat eggs. a large cohort of mutant and wt littermates were characterized behaviorally and found to develop multiple motor deficits that worsened over time. nerve conductions of the tail (caudal nerve) was performed on a separate cohort of mutant (n= ) and wt littermates (n= ) every weeks from to weeks. mutant rats showed a progressively decline in the amplitude of the compound action potential after weeks, whereas the amplitude progressively increased in their wt littermates. separate cohorts of rats were sacrificed at , , and weeks and analyzed by light microscopy. in mutant rats, there was a reduced density of myelinated axons and active axonal degeneration in distal but not proximal nerves, and in the fasciculus gracilis of the cervical spinal cord at and weeks. these findings were not present in the -week-old cohort of mutant rats, or in wt rats at or weeks. a genetically authentic animal model of cmt a that develops a progressive, length-dependent axonal neuropathy will be a valuable tool for examining the pathogenesis and treatment of cmt a. lindborg ja , niemi, jp , defrancesco a , zigmond re . case western reserve university, cleveland, usa. traditionally the role of immune cells in nerve degeneration and regeneration has focused on the infiltration of inflammatory monocytes into the distal nerve after nerve injury and the phagocytosis by the resulting macrophages of myelin and axonal debris, thereby clearing a path for regenerating axons. therefore, it was surprising when we discovered that in ccr knockout (ko) animals, in which the entry of these inflammatory monocytes does not occur, that wallerian degeneration precedes normally. we now report that the reason for this is that neutrophils and schwann cells compensate for the decrease in macrophage accumulation. furthermore, nearly complete depletion of circulating neutrophils by systemic injection of an antibody to ly g leads to an inhibition of myelin clearance both in ccr ko and in wild type animals. on the other hand, we have demonstrated a second site of macrophage accumulation in wild type animals, namely around axotomized sensory neurons in dorsal root ganglia (drgs). blockade of that accumulation, for example as occurs in ccr ko animals, leads to a dramatic impairment of nerve regeneration. to examine the relationship between macrophages and regeneration further the monocyte chemokine ccl was overexpressed in drg neurons in intact animals by viral infection using an aav containing the ccl coding sequence. the resulting overexpression of ccl led to the accumulation of macrophages in drgs even though no injury had taken place and subsequently to an increase in the intrinsic growth capacity of the sensory neurons. examination of changes in gene expression in the drgs in these animals revealed increased expression of the cytokine leukemia inhibitory factor and an increase in its downstream signaling pathway that involves the phosphorylation and nuclear translocation of stat . strikingly, pharmacological blockade of stat activation inhibited the increase in the neurons' growth capacity produced by the virus. these results reveal unexpected interactions between immune cells and neurons facilitating nerve degeneration and regeneration and could lead to therapies to improve regeneration after injury or in disease. lin j , , qiao k , , huang j , , zhao cb , , lu jh , . institute of neurology, fudan university, shanghai, china; department of neurology, huashan hospital, fudan university, shanghai, china. we used terminal latency index (tli) as a tool in differentiation between poems syndrome and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). comparison of median and ulnar nerve conduction studies including motor conduction velocity (mcv), distal motor latency (dml) and terminal latency index (tli) were studied in poems patients, matched cidp patients and normal controls. in this cohort, the average age at evaluation was . ± . years old in poems group and that of cidp patients was . ± . years old. except the ulnar terminal latency index in cidp group, poems and cidp patients demonstrated prolonged distal latencies, low conduction velocities and increased terminal latency indexes compared with the normal group. reduced conduction velocities and higher terminal latency indexes in poems group than in cidp group was found. increased tli was demonstrated in . %(median nerve) and . %(ulnar nerve) poems and that in cidp patients was . %(median nerve) and . %(ulnar nerve). decreased tli was found in . %(median) and . %(ulnar) cidp patients and none in poems. temporal dispersion (td) and conduction block (cb) were more often seen in cidp patients with increased tli than that in poems. compared with cidp and poems showed greater slowing of the intermediate nerve segments and relatively more uniform demyelination. about % cidp demonstrated more distal conduction slowing and more td and cb especially in those with increased tli. terminal latency index combined with td and cb may be helpful in differentiating poems from cidp. lin y , sung j , chang t , jowy t . department of neurology, taipei municipal wanfang hospital, taipei, taiwan. the purpose of our study is to exam whether electrophysiology changes could be detected in prediabetes patients and to discover the possible mechanism of nerve injury in prediabetes stage. we analysis and compare the nerve excitability test data between prediabetic patients and age-matched normal control subjects. prediabetes is defined by american diabetes association (ada) as one of the three following: hba c . % to . %, fasting glucose mg/dl to mg/dl, and hour oral glucose tolerance test to mg/dl. patients with radiculopathy, myelopathy, entrapment neuropathy such as carpel tunnel syndrome, and polyneuropathy were excluded. the strength-duration time constant (sdtc) and superexcitability showed significant difference (p< . ) between two groups. we also find increased threshold electrotonus in depolarization (ted) and reduced relative refractory period (rrp) and refractoriness in . msec. these early changes in prediabetic patient are similar in nerve excitability feature of diabetic patients. however, the above changes are not found in motor axonal excitability test. our data supports that nerve excitability test may be a useful, non-invasive, and less time dependent tool to detect peripheral nerve injury in prediabetic stage. the sensory axons are more vulnerable than motor axons. superexcitability is the most sensitive parameter in prediabetes. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominant disorder caused by mutations of ttr gene and is associated with variable penetrance. ttr-fap is rare, except for endemic areas. this is a retrospective study of ttr-fap patients diagnosed at our center between - . we identified four families with different ttr mutations. in one family with v a mutation, nine family members over four generations diagnosed with ttr-fap was followed since . in the other families the index cases with different mutations were identified between - . affected family members with v a mutation developed severe progressive polyneuropathy with cachexia, with onset of the disease between ages and . three patients presented with marked visual symptoms (one patient underwent vitrectomy). nine patients died to years after disease onset. two patients (sisters) underwent liver transplantation -one died after years of disease at age , second is years old and wheelchair-bound as her symptoms continue to progress. the ttr mutations diagnosed in the index cases of three other families are: d v, f l and v m. they all presented with similar clinical picture of late-onset ttr-fap with predominant progressive axonal sensory, motor and autonomic polyneuropathy. all three index cases were men, the onset of symptoms was between - years with numbness and paresthesia in the feet followed by weakness and autonomic dysfunction. all had excessive weight loss resulting in cachexia and were diagnosed with cardiomyopathy. no patient suffered from visual symptoms. all three patients progressed to stage ii of ttr-fap -walking with assistance. time to diagnosis was . - years. due to advanced stages of their disease these patients were not suitable for therapy with tafamidis or the liver transplantation. the ttr d v (p.d v) was confirmed as a de novo mutation, which is uncommon in ttr-fap. in the remaining families carriers of ttr mutations were identified and are followed up. pedigree analysis of the family with f l mutation revealed affected members who with high probability died from ttr-fap. our study suggests that patients with ttr-fap in poland exhibits clinical and genetic heterogeneity. liu x , fan d . department of neurology, peking university third hospital, beijing, china. objective: to identify the gene mutation of chinese charcot-marie-tooth pedigrees and investigate the correlation among the clinical manifestation, electrophysiology and mechanism of different genotype. methods: we included pedigrees with cmt enrolled in our hospital from january, to december . we recorded clinical features, cmtns and electrophysiological data at diagnosis. the patients underwent mutation analysis of pmp , cx , mpz, mfn , hspb , hspb using mlpa, dhplc and sanger gene sequencing. results: we found pmp duplication pedigrees ( . %), cx pedigrees ( . %), mfn pedigrees ( . %), mpz pedigrees ( . %) conclusions: in chinese han population, the proportion of pmp duplication is relatively low, the majority of clinical manifestation is classical cmt. axonal cmt can show isolated lower extremity injury, with central nervous system involvement. hmn may be an underestimated clinical types, the identification should be done with caution in differential diagnosis. liu y , liu b , sebastian b , wozniak km , wu y , slusher b , polydefkis m . johns hopkins school of medicine, baltimore, usa. chemotherapy-induced peripheral neuropathy (cipn) is a common dose-limiting toxicity in the treatment of many cancers. most cipn studies preferentially focus on sensory fiber loss and dysfunction. here, we compared the structural and functional recovery of autonomic fibers in sweat glands (sweat gland nerve fiber density, sgnfd) and sensory fibers (intra-epidermal fiber density, ienfd) in mouse footpads after exposure to a maximum tolerated dose (mtd) of several common chemotherapy agents. additionally, we assessed footpad sweat production as a functional correlate to sgnfd reductions. female balb-c mice ( -animals/group) were treated with a mtd of four anti-tubulin drugs: paclitaxel (pca, mg/kg), ixabepilone (ixa, mg/kg), eribuline (erib, . mg/kg), vinoelbine (vino, mg/kg), or corresponding placebo given intravenously, mwf for two weeks. recovery was assessed at -hours, , , , , and weeks following the last dose. footpads were processed to visualize epidermal nerve fibers using pgp . and autonomic nerve fibers with tyrosine hydroxylate and pgp . . ixabepilone-treated mice experienced significant reductions in sgnfd at hrs, while ienfd nadir occurred at a later time point, -weeks. the recovery to baseline levels occurred more quickly for ienfd ( -weeks) than sgnfd ( -weeks). in contrast, vinorelbine and eribuline treated mice experienced a maximum deficit in sgnfd and ienfd at hrs and sgnfd recovery was slower ( -weeks) compared to ienfd ( -weeks). pca-treated animals showed more severe ienfd and sgnfd deficits compared to the other agents with both ienfd and sgnfd not recovering completely until -months. reductions in th-sgnfd were comparable or more pronounced to decreases in pgp . -sgnfd for all agents and timepoints. pca-treated animals demonstrated reductions in footpad sweat droplet number thereby providing a functional correlate. together, these data indicate that in mouse models of cipn, autonomic nerve fibers are affected more severely than sensory nerve fibers, and also recover more slowly than intraepidermal nerve fibers. autonomic dysfunction may be an important and under-appreciated consequence of chemotherapy exposure. the pi -kinase vps (pik c ) synthesizes phosphatidylinositol -phosphate (pi p), a lipid critical for both endosomal membrane traffic and macroautophagy. human genetics have implicated pi p dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating charcot-marie-tooth (cmt) peripheral neuropathy. here, we investigated the role of vps , and pi p, in mouse schwann cells by selectively deleting vps in this cell type. vps -schwann cell knockout (vps scko ) mice show severe hypomyelination in peripheral nerves. vps −/− schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. upon reaching the promyelinating stage, vps −/− schwann cells are significantly impaired in the elaboration of myelin. nerves from vps scko mice contain elevated levels of the lc and p proteins, indicating impaired autophagy. however, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in vps scko mice is caused by impaired autophagy. endosomal membrane traffic is also disturbed in vps −/− schwann cells. we investigated the activation of the erbb / receptor tyrosine kinases in vps scko nerves, as these proteins, which play essential roles in schwann cell myelination, are known to traffic though endosomes. in vps scko nerves, erbb was hyperphosphorylated on a tyrosine known to be phosphorylated in response to nrg exposure. the overall level of erbb was also decreased during myelination. our findings suggest that the loss of vps alters the trafficking of erbb / through endosomes. abnormal erbb / signaling may contribute to the hypomyelination observed in vps scko mice. lombardi r , devaux j , cortese a , dacci p , benedetti l , demichelis c , lauria g . irccs foundation "carlo besta" neurological institute, milan, italy; aix-marseille université, marseille, france; irccs c. mondino national neurological institute, pavia, italy; university of genoa and irccs aou san martino-ist, genoa, italy. the recent identification of igg anti-neurofascin (nfascin) antibodies in a group of patients has widened the spectrum of presentation for chronic inflammatory demyelinating polyradiculoneuropathy (cidp). these patients can be distinguished by disabling tremor, poor response to intravenous immunoglobulin and distal and sensory disturbances. cell-adhesion molecule nfasc and cntn are expressed on the paranodal junction (pnj) of nodes of ranvier, and play key roles on sodium channel clustering and glia-axon interactions. quantification of unmyelinated intraepidermal nerve fibers (ienf) is a useful parameter employed in small nerve fiber pathology diagnosis. in addition the immunohistochemistry evaluation of dermal nerve fibers allows to examine morphological changes of myelin sheath and ranvier nodes structure. we performed immunofluorescent colocalization studies using antibodies to visualize axons (protein-gene-product . , neurofilament, tubulin), sheath of myelin (myelin-basic-protein) and specifically nodal/paranodal/juxtaparanodal structures (pannfascin, nfascin , nfascin , caspr, cntn , potassium and sodium channels) in skin tissues from seronegative and seropositive cidp patients. we analyzed axon and myelin sheath damage, abnormal nodal-paranodal-juxtaparanodal architecture and morphometric parameter as internodal length of ranvier nodes. our results on skin biopsies from three igg nfasc -positive cidp patients revealed complete loss of nfasc staining at the paranodes, asymmetrical paranodes and widening of the nodes of dermal myelinated nerve fibers. one igg cntn -positive cidp patient showed abnormal nodal/paranodal immunostaining with different features as compared with igg nfasc -positive patients suggesting specific changes. however, such alterations were not found in four seronegative cidp patients. our data support the hypothesis that examining specific axonal and myelin markers could provide diagnostic and prognostic clues on nodo-paranodopathies. the goal of this study is attempt a possible correlation between the presence of serum autoantibodies and structural changes in nodal/paranodal regions of dermal nerve fibers in cidp patients. knowledge of autoantibodies expression in the peripheral myelinated nerves of cidp patients could serve for stratifying patients and potentially guiding personalized treatments. lunati a , lerat j , dzugan h , , rego m , magdelaine c , , bieth e , calvas p , cintas p , gilbert-dussardier b , goizet c , journel h , magy l , toutain a , urtizberea j , sturtz f , , lia as , . charcot-marie-tooth disease is one of the most frequent inherited peripheral neuropathies ( / ). so far, mutations in more than genes have been identified causing either the demyelinating form (type ) or the axonal form (type ). duplication of pmp gene is the most frequent cause of autosomal dominant demyelinating form. autosomal recessive demyelinating form is often due to sh tc gene mutations. patients suffer then from early severe neuropathy starting in the first decade. scoliosis and deafness are often observed. we analysed patients suffering from peripheral neuropathy, by multiplex-ligation-dependant-probe-amplification (mlpa), followed by targeted next-generation-sequencing (ngs) using a -gene custom panel designed for the diagnosis of charcot-marie-tooth and associated neuropathies. mutations of interest were verified by sanger sequencing. diagnosis was positive for patients. as expected, the most frequent mutation was the pmp duplication detected in patients. deletion of pmp was observed in patients and pathogenic point mutations were detected in patients. sh tc gene appeared to be the most frequently mutated with nine patients diagnosed. associated with known mutations, four new mutations have been identified: two nonsense mutations and two missense mutations. all these patients presented deafness and/or scoliosis. sh tc appears to be an important gene involved in charcot-marie-tooth disease, often associated with deafness and /or scoliosis. it is important to pay attention to these associated symptoms in charcot-marie-tooth patients in order to guide their diagnosis and to improve their medical care. lupo v , , frasquet m , , sánchez-monteagudo a , , barreiro m , alberti ma , casasnovas c , quintáns b , , , camacho a , domínguez c , sedano mj , pelayo al , pardo j , sobrino t , sobrido mj , , , sevilla t , , espinós c , . mme (membrane metalloendopeptidase) mutations, inherited in an autosomal recessive fashion, have been recently identified in japanese probands (higuchi et al. ) . thus, mme has been included in the list of cmt genes as a new autosomal recessive axonal form, cmt t (mim ), and moreover, it is considered a strong candidate for the genetic diagnosis of unsolved late-onset cmt cases. in fact, few months later auer-grumbach et al. ( ) reported european probands with autosomal dominant late-onset cmt and mutations in mme. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy using an updated version of our custom gene panel, which includes mme. in this study, we report probands with cmt , intermediate cmt or dhmn/cmt and homozygous or compound heterozygous mutations in mme, and proband with cmt and a heterozygous mutation in mme. we have identified different type of mutations: novel splice donor variant, frameshift, nonsense and missense mutations. the two nonsense changes, p.trp * and p.arg *, and the splice donor variant c. + g>a, were detected in homozygous or compound heterozygous state in patients, while the frameshift mutation, p.pro leufs* , was detected in homozygous or heterozygous in the remaining patients. strikingly, the two nonsense and the frameshift mutations had been previously reported as causative for autosomal-dominant cmt t (auer-grumbach et al. ) . out of three missense mutations, one is novel (p.his tyr), and two are reported in control databases (p.asn lys and p.arg trp). this study shows that the autosomal recessive cmt t is common in spanish population, and moreover, it suggests that screening of mme using gene panel testing could help to improve diagnosis of unclarified inherited peripheral neuropathies cases. funds: isciii (pi / , pi / ); fundació per amor a l' art. inherited peripheral neuropathies (ipns) encompass a group of disorders highly heterogeneous, clinically and genetically. charcot-marie-tooth (cmt) disease is closely related to distal hereditary motor neuropathy (dhmn) or distal spinal muscular atrophy (dsma), and some patients show additional signs associated with amyotrophic lateral sclerosis (als). targeted gene panel and exome sequencing are considered to be powerful and cost-effective tools for diagnosis of these disorders. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy: families were investigated by exome sequencing and, cases were tested using different updated versions of a gene panel . each version comprises , or ipn genes, respectively and it shows a high coverage performance: percentage of analyzable target base with > coverage was , %. both exome and gene panel capture libraries were based on sureselect capture technologies (agilent technologies), and sequencing was performed in miseq or hiseq illumina equipment. we have identified novel genes and novel mutations in known genes, broadening the phenotypical spectrum associated with ipns. exome sequencing has allowed us to identify causative gene in % of familiar or sporadic cases: morc , aars, bscl , kif a, gars, egr , fig , dnajb , drp , ighmbp , dao, sod , fig . gene panel testing has been mostly performed in sporadic cases, and it has allowed us to identify either disease-causing or candidate mutations in % of cases: kif a and bicd were the most common genes mutated. update gene panels neuro and neuro have revealed novel mutations in genes recently associated to cmt and cmt disease: mme and pmp , respectively. in sum, both strategies have helped us to achieve a more accurate clinical and genetic reclassification of these disorders, an impossible challenge using conventional sequencing methods. our study expands the clinical phenotype previously associated to known ipn causing-gene, and emphasizes that gene panels should be considered as a first diagnosis method for unclarified ipn patients. funds: isciii (pi / , pi / , pi / ); fundació per amor a l' art. magy l , mathis s , goizet s , tazir m , vallat j-m . department and laboratory of neurology, national reference center for rare peripheral neuropathies, chu limoges, france; department of neurology, chu bordeaux, france; department of medical genetics, chu bordeaux, france; department of neurology, chu algiers, algeria. charcot-marie-tooth (cmt) disease is a hereditary neuropathy with a relatively homogeneous phenotype but is genetically heterogeneous. moreover, nerve conduction studies distinguish different forms, adding another level of complexity. the current classification of cmt being difficult to understand for physicians, scientists and patients, we presented and published a proposal for updating this classification, based on inheritance, nerve conduction findings and gene/mutation involved. inputs from colleagues prompted us to conduct a survey in order to try to reach some consensus about our proposals. we conducted an internet survey between october and december . the link to complete the survey was sent several times by email with an introduction to more than people. participants were contacted through the emailing list from the last cmt meeting in venice (september ) and additional physicians and scientists who are involved in cmt care and research were contacted as well. one hundred seven people from various countries (mainly france, italy and the usa) answered the survey. most ( %) of the participants were between and years of age, % being physicians and % being scientists. the vast majority ( %) considered the proposal constituted an improvement over the historical classification whereas % wanted to keep the old one. about the order of information, % of participants thought the mode of inheritance should come first, whereas % felt the phenotype should be placed at the beginning. ninety-one percent of people thought cmt should be kept as a generic name for hereditary sensory and motor neuropathy. for pure sensory neuropathy, % favoured hsn over hsan although % thought the opposite and % of participants felt dhmn should be kept for distal motor neuropathy. about nerve conduction findings, % of participants thought the intermediate phenotype has to be kept and % favoured our proposal to replace " " by "de" (for demyelinating) and " " by "ax" (for axonal). finally, % of responders thought that genetic information should be included in the classification of cmt. overall, our proposal of a new classification received a very good appreciation from physicians and scientists implicated in the care of patients with hereditary neuropathy. mallik r , hubsch a , gaida a , barnes d . csl behring, kop, usa; csl behring, bern, switzerland; csl behring, ottawa, canada. the risk of hemolytic events (hes) with intravenous immunoglobulin (ivig) therapy appears to be linked to the isoagglutinin (anti-a and anti-b) level of the specific ivig product. using published anti-a and anti-b titers for seven ivig products and corresponding he rates reported to the eudravigilance database, we developed a mathematical model to predict the risk of he to patients receiving ivig products of given anti-a and anti-b levels. modeling was performed separately for the risk to patients with blood groups a, b, ab and o and the overall population risk was estimated assuming a blood group distribution of % a, % b, % ab and % o. applying the prediction model, we calculated the he risk for an ivig product produced via a chromatographic process (privigen ® , csl behring) a) without any isoagglutinin reduction measures ( ) ( ) ( ) ( ) ( ) ( ) ( ) , b) with an anti-a donor screening program eliminating approximately % of donors with high anti-a titers ( ) ( ) ( ) , c) incorporating an anti-a/anti-b specific immunoaffinity chromatography (iac, igisolo tm ) step in the manufacturing process (since ) and d) with both measures (b and c) combined; as well as for an ivig product produced with a cohn-like cold ethanol fractionation process (carimune ® nf/sandoglobulin ® , csl behring). isoagglutinin titers in ivig products, measured by european pharmacopoeia direct assay, were provided by dr c bellac, swissmedic, bern, switzerland. the predicted risk was highest with the chromatographically purified ivig without isoagglutinin reduction ( . cases expected per kg ivig used). anti-a donor screening reduced the predicted risk to . cases/ kg. a greater risk reduction was predicted with the iac isoagglutinin reduction step ( . cases/ kg). the combination of both methods produced little benefit ( . cases/ kg) versus iac alone. the predicted hemolytic risk with ivig produced by cohn-like ethanol fractionation was low ( . cases/ kg). an observational cohort study to confirm these hemolytic risk reductions is in progress. at present, the observed hemolytic risk for anti-a donor screening appears consistent with the prediction calculated by the model; results for iac isoagglutinin reduction are expected in . the cmt infant scale (cmtinfs) is an outcome measure of functional ability for young infants and children aged < years. cmtinfs aligns with the cmt pediatric scale and cmt neuropathy score to measure disease severity across the lifespan. to measure gross motor and fine motor function, cmtinfs comprises of two subscales: gross motor (e.g. head control, crawling, walking, jumping and hopping) and fine motor function items (e.g. grasping, reaching, tearing paper and buttoning). overall and subscale-specific function is expressed as a z-score based on normative reference values (positive z-scores indicate poorer function). a total of controls aged - months (mean age , sd m) have been assessed across australia (n= ), thailand (n= ) and usa (n= ). total cmtinfs z-scores did not differ significantly between sites (australia vs thailand) (p= . ) or gender (p= . ). data collection is ongoing and infants aged < years are eligible for inclusion. to date, infants ( % male) aged - months (mean age , sd m) with a range of cmt subtypes ( cmt a, cmt d, cmt c, cmt x and unidentified gene) have been assessed with cmtinfs. mean total z-score for infants with cmt ( . , sd . , range: − . - . ) was significantly higher than controls ( . , sd . , range: − . - . , t=− . , p= . ). differences between affected infants and controls were larger in infants older than months. infants with cmt a (cmtinfs z-score . , sd . ) and cmt c (z-score . ) were less affected than cmtx (z-score . ) and cmt d (z-score . ). the gross motor function subscale differed significantly between cmt cases and controls ( . , sd . vs . , sd . ; p=. ) and a significant difference was also observed for the fine motor function subscale ( . , sd . vs . , sd . ; p= . ). reliability, factor and rasch analysis of the cmt-infs is underway to assess validity. initial results support the sensitivity of cmtinfs in distinguishing between infants with and without cmt. preliminary analyses also suggest the scale is sensitive to genetic subtype. with increased power, cmtinfs promises to become a useful outcome measure of disease severity and function in infants with cmt. manso c , querol l , mekaouche m , illa i , devaux j . aix-marseille université, marseille, france; universitat autónoma de barcelona, barcelona, spain. contactin- , contactin-associated-protein- (caspr ), and neurofascin- (nfasc ) are essential for the formation of paranodal axoglial junctions. igg autoantibodies to contactin- , caspr , and nfasc are associated with subsets of patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp) presenting with common clinical features. anti-contactin- igg autoantibodies have been shown to be pathogenic and to affect the paranodal axoglial junctions in vivo and in vitro. by contrast, the pathogenic effect of anti-nfasc igg have not been demonstrated. here, we purified anti-nfasc igg from cidp patients' plasma and investigated their effects after passive transfer. to determine whether these antibodies can pass the paranodal barrier, we performed intraneural injections of anti-nfasc igg autoantibody. by contrast to anti-contactin- igg , anti-nfasc did not penetrate the paranodal regions after intraneural injections, but bound to the surface of the schwann cell. to perform chronic exposure, lewis rats were implanted with intrathecal catheter and anti-nfasc igg were administrated in a daily manner during three weeks. igg to nfasc , but not control igg , induced progressive clinical deteriorations characterized by gait ataxia and hindlimb paraparesis. these deteriorations were associated with nerve activity loss in motor spinal nerves and with a selective loss of the paranodal specialization characterized by the disappearance of the caspr /contactin- /nfasc complex at paranodes. the passive transfer of anti-nfasc igg thus seem to induce similar pathogenic effects as the anti-contactin- igg . however, the pathogenic mechanisms leading to paranode disappearance appear different. our findings indicate that igg directed against nfasc are pathogenic and further show that these antibodies are reliable biomarkers of a specific subset of cidp patients. better fit the needs of the consortium and improve patient experience. these enhancements include creating mobile friendly webpages, updating enrollment form content, access to a customized dashboard, and the ability of registrants to explore their data in comparison to other registrants. we will review these enhancements in depth, and demonstrate their impact on the growth and development of the rdcrn inc contact registry. martinez c , hubsch a , watson dj , shebl a , wallenhorst c , simon tl . institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl bering ag, bern, switzerland; csl behring llc, king of prussia, usa; csl bering gmbh, marburg, germany. hemolytic anemia (ha) is a complication of intravenous immunoglobulin (ivig) treatment, particularly in patients receiving high dose ivig for immune modulation, such as guillain-barré syndrome or chronic inflammatory demyelinating polyneuropathy. the primary mechanism for the increased risk is believed to be passive acquisition of anti-blood group a and b antibodies (isoagglutinins) from the ivig product. to reduce the quantity of isoagglutinins, an anti-a donor screening was implemented for the ivig privigen ® from - and donors with high titers were excluded from contribution to pooled plasma. anti-a donor screening was replaced since with an immunoaffinity chromatography step, which decreases isoagglutinins to a greater extent, but no data are available to test its clinical effectiveness. to test the effectiveness of the donor screening, two cohorts of patients treated with privigen ® before and after start of donor screening were identified from a hospital-based administrative database of us hospitals with in-and outpatient discharge diagnoses, procedures, drug utilization and laboratory tests between / and / (period ) and between / and / (period ). privigen ® dose per kg body weight was estimated from the daily quantity administered and age-and sex-specific us population body weight estimates. ha within days of privigen ® use was assessed from manual records review and the incidence rate of ha in the two periods compared. incidence rate ratios (irr) of ha were adjusted for sex, age, treatment setting, indication and dose per kg body weight using period as reference. the incidence rate of ha was . / , person-days ( % confidence interval: . - . ) in period ( has in , person-days) and . ( . - . ) in period ( has in , person-days). the adjusted irr was . ( . - . ). significantly less ha risk was found with high dose (≥ . g/kg body weight) privigen ® , irr . ( . - . , p= . ). we conclude that anti-a donor screening and exclusion of donors with high anti-a titers from plasma pools is associated with a decreased risk of ha with ivig. matsumoto a . department of neurology, keijinkai jozanki hospital, sapporo, japan. subacute myelo-optico neuropathy (smon) is the intoxication of clinoquinol with main clinical symptoms of paresthesia and spasticity of legs. these symptoms have been considered to be elicited by the disturbance of spinal cord and peripheral nerve as the intoxication of clinoquinol. however, as to the patients with smon who have been still living after the onset of disease, the examination of nerve conduction velocities are in normal ranges and the clinical symptoms of peripheral neuropathy are not observed now. in order to investigate whether the peripheral neuropathy were observed in the early stage of smon, we investigated the longtidunal changes of electrophysiological results in patients who could examine the nerve conduction studies from early stage of smon until the present time. as to the disturbance of pyramidal tract functions (myelopathy) in smon patients, the central motor conduction times were calculated by transmagnetic stimulation of motor cortex, cervical roots and lumbar roots. the peripheral nerve conduction velocities of sensory nerve were examined with the sural nerves. as the results, in patients with smon who could examine the electrophysiological examination from the early stages of smon until to years later, the central motor conduction times of leg muscles from motor cortex to lumbar roots were prolonged in the smon patients compared to the normal cases. these results suggest the presence of disturbances of conduction velocities of spinal cord. conduction velocities of sensory nerve velicities (sncvs) showed the delayed sncvs of sural nerves( - m/sec) at the first examination from the onset of - years. however from to years later, sncvs of these cases were covered to - m/sec. from these electrophysilogical examinations, it was suggested that the presenting main symptoms of smon were the disturbance of myelopathy, and the disturbance of peripheral nerve function had been recovered after onset of smon being elapsed a long time, natural killer (nk) cells are part of our innate immune system with regulatory and effector functions. they comprise the first line of defence in the recognition and destruction of virus-infected and pathologically altered cells. different studies suggest that the treatment with intravenous immunoglobulins (ivig) has an immunomodulatory effect on nk cells. ivig is a first-line treatment for various autoimmune diseases in particular in chronic inflammatory demyelinating polyneuropathy (cidp). the lack of a predictive marker for ivig responsiveness in cidp avoids the early preservation of non-responding patients. to better understand the effect of ivig in patients with cidp, we tested whether ivig treatment altered the nk cell status. additionally, we analysed if the alteration in the populations may serve as a surrogate marker in predicting the outcome of ivig treatment. using semi-quantitative pcr and flow cytometry in the peripheral blood of patients with cidp, we analysed the effects of ivig on the nk cell population before treatment initiation and h after first dose and correlated the changes with the reponsiveness to ivig. ivig administrations induced a reduction in the expression of several typical nk cell genes. interestingly, this ivig-induced reduction of nk cells was reversible four weeks after the ivig treatment. flow cytometry data revealed that ivig reduced the cytotoxic cd dim nk cell population, while regulatory cd bright nk cells remained almost unaffected or were even increased. interestingly, we found that the observed effects on nk cells almost exclusively occurred in cidp patients who responded to ivig therapy. correlation between the changes in the nk cell population and treatment efficiency suggests a crucial role for nk cells in the immunomodulatory mechanism of ivig. further studies are warranted to investigate whether the differences in the nk cell status of patients with cidp represent a reliable surrogate marker in predicting the outcome of ivig therapy. mccray b , sullivan j , woolums b , aisenberg w , lloyd t , sumner c . johns hopkins university, baltimore, usa. mutations in transient receptor potential vanilloid (trpv ), a calcium-permeable non-selective ion channel, cause charcot-marie-tooth disease type c (cmt c). trpv is unique in that it represents the only membrane-expressed ion channel in cmt and thus a potential therapeutic target. previous work has suggested that trpv mutations lead to gain of channel function and toxicity in cultured cells. neuropathy-causing mutations of trpv largely cluster in the cytosolic ankyrin repeat domain (ard) that is known to mediate protein-protein interactions, suggesting that pathogenesis may be related to disruption of such interactions. in order to identify trpv -interacting proteins, we performed two unbiased proteomics screens and identified multiple cytoskeletal-modifying proteins including syndapin- , a neuronal protein known to promote axonal outgrowth by influencing the actin cytoskeleton. in cultured cells, we have shown that trpv and syndapin- co-localize to highly dynamic actin-rich cellular processes and together stimulate robust neurite extension, but this facilitation of neuritogenesis is impaired by disease-causing mutations in trpv . we have also shown that over-expression of syndapin reduces trpv -mediated calcium influx in cultured cells and rescues toxicity of mutant trpv . in addition, syndapin over-expression suppresses mutant trpv phenotypes in a drosophila model of trpv -related neuropathy. further, we have demonstrated that treatment of drosophila with a specific trpv channel antagonist ameliorates trpv mutant toxicity. together, our data highlight the importance of trpv interaction with cytoskeletal proteins such as syndapin- in the pathogenesis of cmt c. specifically, our results suggest that mutations in trpv disrupt the normal role of trpv in regulation of cytoskeletal dynamics and that interactions with the cytoskeleton reciprocally modulate trpv channel function and influence toxicity of mutant trpv . mcgonigal r , yao d , barrie ja , crawford c , willison hj . university of glasgow, glasgow, uk. guillain-barré syndrome (gbs) is in part mediated by anti-gm ganglioside antibodies induced by preceding infections. anti-gm antibodies target plasma membrane gm that is extensively distributed in both glial and axonal membranes, particularly at the node of ranvier. antibodies deposited at this site in models of gbs are associated with complement deposition, conduction block, structural disruption of ion channels and macrophage infiltration. the wide distribution of the gm ganglioside target leads to unwanted complexity in ascribing pathological outcomes to injury of cell-specific membranes, in particular unravelling the consequence of paranodal schwann cell membrane injury on axonal function, and vice versa. to overcome this impasse, we have generated transgenic mice through glycosyltransferase manipulation that express gm exclusively in neurons or glia, thus allowing us to very specifically target and injure axonal or glial membranes with a single anti-gm ganglioside antibody. through this route we can create mouse models of both the axonal and demyelinating forms of gbs, induced by a single anti-gm antibody, thus creating otherwise highly comparable conditions. here, we show anti-gm antibody binding is restricted to the nodal axolemma in galnact −/− -tg(neuronal) mice and conversely to paranodal loops in galnact −/− -tg(glial) mice. when anti-gm antibody and a source of complement is added to a nerve-muscle ex vivo injury paradigm, there is a loss of axonal integrity (i.e. loss of neurofilament immunolabeling) when the neuronal membrane is targeted in galnact −/− -tg(neuronal). conversely, axonal integrity is maintained when the paranodal membranes are decorated by antibody and complement products ex vivo in galnact −/− -tg(glial) mice. in a passive immunisation model in vivo, galnact −/− -tg(neuronal) mice acutely develop weakness, respiratory dysfunction, associated complement deposition, and degenerative pathology in distal axons. in contrast, galnact −/− -tg(glial) mice have significantly fewer abnormalities under the same acute conditions. these data indicate the high vulnerability of axonal membranes to acute injury and underline the importance of developing specific axonal protection strategies. in summary, targeting the nodal axolemmal or glial membranes allows us to study associated nodal pathology, and determine the downstream consequences on function and axon fate, currently a major area in gbs clinical research. memon a , madani s , schultz l , grover k , arcila-londono x , sripathi n , ahmad bk . neuromuscular division, department of neurology, henry ford hospital, detroit, michigan, usa. objective: to differentiate sensory electrophysiology, tli and treatment response in patients with paraproteinemic cidp. background: low tli has been reported as a useful electrophysiological marker for mag-cidp. to our knowledge comparison of sensory electrophysiology and tli of paraproteinemic cidp subgroups have not been previously reported. methods: retrospective review(january -december ) of patients with cidp fulfilling electrophysiological criteria(aan ad hoc subcommittee and albers and colleagues).cidp patients with diabetes(n= ) were excluded. patients were divided into idiopathic (n= ) and paraproteinemic cidp(n= ). paraproteinemic cidp sub-groups: mag( ), non-mag( ) and igg( ) were compared to idiopathic cidp( ). these groups were compared for demographics, history of cancer, csf protein, sensory conductions, tli measurements and response to treatment using chi-square tests for binary and categorical variables and t-tests for continuous measures. results: there was a higher proportion of females in idiopathic-cidp compared to non-mag-cidp ( % vs %). idiopathic group having a higher proportion of patients on monotherapy( % vs %) and combination therapy( % vs %) compared to non-mag. higher mean csf protein compared to mag-cidp(p= . ) was seen in the idiopathic. the difference between idiopathic and igg-cidp was significant for overall rx response(p= . ) and rx response in patients with follow-up(p= . ). for both variables, patients in the idiopathic group had a higher proportion of patients on combination therapy and lower proportion of no treatment offered compared to patients in the igg-cidp. % of non-mag-cidp patients had a history of cancer vs % of mag-cidp. none of the other differences were significant. there were no group differences in sensory electrophysiology and tli. conclusions: sensory electrophysiology and tli may have no value in differentiating paraproteinemic cidp. csf protein is higher in idiopathic cidp compared to mag-cidp. idiopathic-cidp has a higher proportion of females compared to non-mag-cidp and a higher proportion of patients on combination therapy compared to igg-cidp. cancer screening should be considered in patients with non-mag-cidp. memon a , madani s , ahmad bk , schultz l , grover , arcila-londono x , sripathi n . department of neurology, henry ford hospital, detroit, michigan, usa. introduction: sensory electrophysiology and terminal latency index (tli) differences have been described in various cidp sub-groups. objective: evaluate electrophysiology, tli and treatment response in idiopathic and diabetic cidp. methods: retrospective review of patients with cidp who underwent electrodiagnostic evaluation (january -december . patients fulfilled electrophysiological criteria described by ad hoc subcommittee of american academy of neurology (aan) and albers et al. we excluded patients( ) with acute inflammatory demyelinating neuropathy, hereditary sensorimotor neuropathy, vasculitis and polyneuropathy with paraproteinemia. patients were divided into idiopathic( ) and diabetic( ) groups. these groups were compared for age, sex, history of cancer, csf protein, response to treatment, sensory response abnormalities and tli measurements using chi-square tests for binary and categorical variables and t-tests for continuous measures. all testing was at the alpha= . level. results: group differences for age, sex, history of cancer, csf protein and treatment response were not significant. comparing tli values in measurable responses, the difference between the two groups for tibial tli was significant (p= . ), with idiopathic group having a lower mean as compared to the diabetic. tli values differences for median, ulnar and peroneal nerves were not significant. the difference in abnormal rates of sensory responses was significant for the sural nerve with the idiopathic group having a lower rate compared to the diabetic group ( % vs %, p< . ). no differences were noted for the ulnar, median and radial nerves. tibial tli and sural sensory responses have some value in differentiating the two groups. larger prospective studies are needed to confirm our findings. ivig is an important treatment option for cidp. although recommended by treatment guidelines, little is known about the consequences of temporary ivig withdrawal to assess ongoing immunoglobulin need. path is a randomized, double-blind trial of the subcutaneous immunoglobulin (scig) igpro (hizentra ® , csl behring) in cidp. before scig randomization, subjects underwent periods of ivig withdrawal ( weeks or until pre-determined indications of clinical deterioration) and ivig restabilization (igpro ; privigen ® , csl behring). subjects not showing deterioration during withdrawal period were withdrawn from study. to proceed to scig randomization, subjects had to achieve "cidp stability" (no relevant change in incat score at last two restabilization visits and at least the same total score as at screening). subjects entered the ivig withdrawal period. ( %) of these qualified for igpro restabilization; subjects ( %) were not ivig dependent, and ( %) were withdrawn for other reasons. one subject withdrew consent before igpro dosing. at the end of the igpro restabilization period, cidp stability was achieved in % of subjects ( did not reach stability, were withdrawn for other reasons). post-study follow-up information was available for / subjects who did not reach stability: ( %) had improved to baseline clinical status and had not, meaning at least % of the subjects improved to their pre-study status. during the restabilization period, / subjects ( %) improved in at least one of the predefined outcome measures. on average, subjects improved by . points in incat total score, . points in i-rods centile score, kpa in mean grip strength (dominant hand), and . points in mrc sum score. improvement occurred with a median of days after the first igpro dose in one or more efficacy outcome measures and in % of cases after the third igpro maintenance infusion. headache and nasopharyngitis were the most frequently reported adverse events (aes) during restabilization. aes deemed causally related were mostly mild or moderate. no unexpected aes or laboratory or vital sign findings associated with igpro occurred during the study. in summary, igpro reversed neuromuscular disability and improved activity/participation after previous clinical deterioration during an ivig withdrawal period. pmp duplication is the most frequent cause of charcot-marie-tooth disease (cmt). since it discovery, more than genes have been identified to be potentially responsible for cmt disease. however only single nucleotide variations (snvs) or small indels have been described. this could be due to the new sequencing strategy (ngs), especially ngs by amplicon sequencing, for whose few convenient tools are available and easily usable to detect cnvs responsible for inherited disease. to overcome this problem, we designed "cov'cop", a user-friendly tool able to detect cnvs among amplicons sequencing data. using the run's coverage file provided by the sequencer, "cov'cop" simultaneously analyzes all the patients of the run using a two-stages algorithm containing correction and normalization levels and provides an easily understandable output, showing with various colors, potentially deleted and duplicated amplicons. we validated our method on several datasets, including those of our targeted ngs panel screening genes known to be involved in cmt and close pathologies. cov'cop detected easily pmp duplication and deletion in our patients, confirmed by mlpa. in addition, cov'cop permitted the detection of new cnvs different from the pmp duplication, in cmt patients. we confirmed these cnvs by quantitative pcr and cgh array. we present here one of these cnvs: the duplication of aars gene detected in cmt patients and we discuss the pathogenicity of this new cnv. additional cnvs responsible for cmt disease are probably still to be discovered and we believe that cov'cop will help molecular geneticists to rapidly identify them. poems (polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscraisia and vascular endotherial (vegf) overproduction. although number of therapeutic interventions for plasma cell dysorders have been applied to poems syndrome, there have been no randomized clinical trials. this phase / double blind, randomized, placebo comtrolled trial was performed to investigate the safety and efficacy of thalidomide for patients with poems syndrome who are not eligible for stem-cell transplantation. the primary endpoint was the reduction rate of serum vegf concentrations at weeks in intention to treat analysis. additional outcomes of long-term extension study included progression-free survival. twenty-five poems patients were randomly assigned to either thalidomide plus dexamethasone or placebo plus dexamethasone from nov , , to july , . one patient in the placebo group was excluded from analyses because of a protocol violation. the adjusted mean serum vegf reduction rate at weeks was . (sd, . ) in the thalidomide group compared with − . ( . ) in the placebo group (p= . ). the kaplan-meier rate of progression-free survival at months was . in the thalidomide group, as compared with . in the placebo group (hr, . ; % ci, . to . ). in the randomized study period, mild sinus bradycardia was more frequent in the thalidomide group than in the placebo group ( % vs %; p= . ). thalidomide suppresses serum vegf concentrations and lengthened pregression-free survival in poems patients who are ineligible for stem cell transplantation. although thalidomide treatment has a risk of bradycardia, the benefits would exceed the risk. this study is registered with the umin clinical trials registry, umin . montes-chinea ni , coutts m , vidal c , courel s , rebelo a , abreu l , zuchner s , saporta, ma , . department of neurology, university of miami, miami, usa; department of human genetics, university of miami, miami, usa. autosomal dominant mutations in synaptotagmin- (syt ), a synaptic vesicle protein that functions as a calcium sensor for neurotransmission, have been previously linked to presynaptic neuromuscular junction (nmj) dysfunction and motor neuropathy in two families. both pathogenic mutations (asp ala and pro leu) were located in the c b domain of syt , which is essential for neurotransmitter release at the nmjs. we report a family with a new missense mutation in the c b domain of syt and a similar phenotype characterized by a slowly progressive, predominantly motor neuropathy and evidence of presynaptic nmj dysfunction on nerve conduction studies. the index case is a year-old woman with gradually progressive weakness of her extremities. she had normal developmental milestones, but was found to have bilateral high arched feet and hammertoes and occasional falls around the age of . she gradually developed progressive leg weakness, worsening bilateral hand cramping, weak handgrip, and only mild paresthesias on distal extremities. family history is remarkable for similar symptoms reported by her maternal grandfather, two maternal uncles, her mother and a younger sister. her neurological exam revealed inability to walk on heels or toes, significant distal lower extremity weakness and absent ankle deep tendon reflexes. cranial nerve examination and coordination were normal and there were only non-specific sensory changes in the lower extremities. emg/ncs revealed normal sensory responses throughout; however, motor nerve evaluation demonstrated globally reduced amplitudes with a > % increment after brief isometric contraction. further electrophysiological evaluation with slow ( hz) repetitive nerve stimulation of the right ulnar motor nerve revealed a % decremental response in amplitude and a > % increase in amplitude immediately after a one-minute period of sustained muscle contraction, which rapidly extinguished after one minute. voltage-gated calcium channel (vgcc) antibodies and a chest ct were normal. targeted sanger sequencing revealed an ile lys mutation in syt , which is located in the c b domain and is predicted to impair protein function. syt -related neuropathy is a rare disease, but should be suspected in patients presenting with a combination of pre-synaptic nmj dysfunction (resembling lambert-eaton myasthenic syndrome) and a predominantly motor neuropathy, especially in the context of a positive family history. charcot-marie-tooth (cmt) disease affects roughly in , individuals and is described as an inherited peripheral neuropathy primarily affecting distal muscles. limited studies detail patient-reported impact of muscle weakness on functional activities. this anonymous survey was developed with input from clinical experts and patient interviews and aimed to better understand the prevalence and impact of various cmt clinical manifestations on patients' lives. the survey was administered online to the hereditary neuropathy foundation's (hnf) patient contact database and is ongoing through june . here we present preliminary data on patient characteristics and disease impact for cmt patients collected february - , . respondents were mostly female ( %) and mostly from the us ( %). median age (range) at symptom onset was years ( - years), at diagnosis was years ( - years), and at present was years ( - years). the sample was representative of all cmt types (cmt , , , ,and x). the most common physical and clinical manifestations of cmt were problems with balance ( %), ankle weakness/foot drop ( %), loss of feeling or abnormal sensation in the lower leg/foot ( %), and hand muscle weakness ( %). maintaining balance, walking long distances, and climbing up and down stairs were key challenges associated with ankle weakness/foot drop. foot drop was considered by % to be the primary factor contributing to falls, which averaged . falls to ground per month. of those with foot drop, % had bilateral weakness. a majority of respondents ( %) used some form of assistive device for mobility, including ankle-foot orthotics/below-the-knee leg braces ( %), canes/walking sticks ( %), and custom foot orthotics/inserts ( %). the most common drug therapy included pain and anti-inflammatory medications ( %). foot surgery was the most common surgical procedure received ( %) and toe surgery was the most common surgery considered ( %). key symptoms that affected quality-of-life "very much" included problems with balance ( %), ankle weakness (foot drop) ( %), and fatigue ( %). these data suggest a high prevalence of lower leg muscle weakness; therefore, therapies aimed at improving ankle weakness and the resulting foot drop and imbalance may be beneficial to patients' daily functioning and quality of life. morano m , , gambarotta g , ronchi g , , cillino m , fornasari be , , fregnan f , , tos p , cordova a , moschella f , geuna s , , raimondo s , . department of clinical and biological sciences, university of turin, orbassano (to), italy; neuroscience institute of the "cavalieri ottolenghi" foundation (nico), university of turin, orbassano (to), italy; plastic and reconstructive surgery. department of surgical, oncological and oral sciences, university of palermo, palermo, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. nerve fiber regeneration and complete functional recovery after peripheral nerve injury do not always occur and can be influenced by many factors including patient age, gender, lesion site, injury severity, size of the gap between damaged nerve stumps and time interval that elapses before performing surgical repair. the poor outcome occurring after a long delay can be due to loss of the neuron ability to regenerate, loss of the schwann cell ability to support regeneration and, of course, progressive muscle atrophy. the aim of this study was to investigate the nerve regeneration after delayed repair and to study the degenerative processes of the denervated distal nerve stump and denervated muscle. in particular, the analyses were focused on the role of nrg/erbb system, that is expressed both in nerve and in muscle tissue, during degenerating and regenerating processes. functional recovery analysis performed after nerve repair showed that only the group repaired immediately and not the groups repaired with a delay of or months, recovered partially. nevertheless, morphological analyses demonstrated that, despite the delay, the nerve fibers are still able to regenerate, even if they are fewer and smaller than the immediate repaired group. moreover, the analysis of the nrg /erbb system showed a significant decrease of soluble nrg in both degenerating and delayed-repaired nerves. the poor outcome after delayed nerve regeneration might be explained by schwann cell impairment and the consequent ineffective support for nerve regeneration. as regards denervated muscle analysis, results showed that erbb receptors expression is related to the innervated state of the muscle, with an upregulation of erbb clearly associated with denervation state. interestingly, nrg isoforms are differently regulated depending on the type of nerve injury. future experiments will be needed to address the in vivo efficacy of different isoforms of nrg both in injured nerve and denervated muscle. we planned a multicenter, prospective, randomized, single blind, controlled study to evaluate the efficacy and safety of an innovative rehabilitation protocol based on the use of treadmill training in a cmt a population. the protocol required that subjects were blindly randomized into two treatment groups, spe (three months of respiratory, proprioceptive and stretching exercises) or trespe (the same treatment plus aerobic training at the treadmill). subjects were evaluated at baseline (t ), after three month of treatment (t ) and further three months of follow up free of therapy (t ). the full assessment included: -mwt (primary outcome measure), -mwt, walk- , short physical performance battery (sppb); lower limbs dynamometric strength evaluation; berg balance scale (bbs); cmt neuropathy score; medical outcomes study short form (sf ). a total of subjects (mean age of . ± . years) were recruited. at t we found a significant improvement in both groups in the -mwt (p< . ), -mwt (p< . ), bbs (p< . ) and sppb (p< . ), while at t only the -mwt was still significantly improved (p< . ) in the spe group. no significant differences between groups were observed for any of the outcome measures. performances on walk did not significantly change during follow-up (p= . ). concerning the sf , we did not observe consistent changes during follow-up or consistent differences comparing the two treatments. in conclusion, this multicenter, prospective, randomized, single blind, controlled study shows that the combination of respiratory, proprioceptive and stretching exercises has a positive impact on the performance of cmt patients, especially regarding walking tests. the aerobic exercise at the treadmill, is well tolerated but apparently does not add any further improvement to the conventional treatment. we speculate that the relatively low clinical severity of the patients, due to the selection criteria, may have prevented a positive effect of treadmill exercise. müller m , dohrn m , romanzetti s , reetz k , gess b . university rwth aachen, department of neurology, aachen, germany. muscle mri is increasingly used in neuromuscular patients to detect changes in muscle volume, muscle fat infiltration and edema. muscle mri are mostly analyzed by qualitative means as quantitative analysis is time-consuming and not well established. here, we developed a novel method for semi-automated segmentation of muscle mri data sets. based on axial t -weighted dixon mri stacks, muscle volumes were quantified by an adapted water-shed algorithm. muscle volumes of thighs and calves were determined separately and the ratio of thigh/calf was calculated. myopathy, neuropathy patients and healthy controls were included in the study. muscle volumes determined by semi-automated segmentation were very similar to manually segmented data sets, differences being < %. this was the case for patients as well as healthy controls. the time-saving effect of automated segmentation was very strong ( vs min. per patient). muscle volumes of the thigh and also of the calf of myopathy patients showed a highly significant difference (p< . ) compared to healthy subjects. in neuropathy patients there was a just significant difference (p< . ) of muscle volumes compared to healthy patients that did not sustain in bonferroni's multiple comparison test. the ratio of thigh/calf muscle volume was significantly different comparing patients with myopathy and neuropathy (p< . ). subgroup analyses of different groups of myopathy patients showed highly significant differences (p< . ) in myositis, limb-girdle-muscular dystrophy and metabolic myopathy, compared to healthy patients, but no significant differences in-between these groups. taken together, the data shows that automated segmentation of muscle mri allows for exact and fast quantification of muscle volumes in neuromuscular patients. higher patient numbers are necessary to test differences between specific disease groups. further studies should also address the possible use as a marker of disease progress for clinical studies or therapy monitoring. murakami t , nishimura h , nagai t , hemmi s , kutoku y , sunada y . department of neurology, kawasaki medical school, kurashiki, japan; department of pathology, kawasaki medical school, kurashiki, japan. familial amyloidotic polyneuropathy (fap) is an autosomal dominant hereditary systemic amyloidosis caused by mutation of transthyretin (ttr) gene, and usually shows sensory dominant polyneuropathy and autonomic neuropathy at the initial stage. the pathogenesis of neuropathy is not well understood, and explained by several mechanisms, including such as mechanical compression, vessel occlusion, ttr toxicity and schwann cell dysfunction. we describe a sporadic patient with late-onset fap due to ttr e k. she noticed dysesthesia first in the foot at age . the symptoms were slowly progressive, and abnormal sensations were extended up to the both upper arms and the both knees at age . distal muscle weakness and atrophy was also observed in the extremities. she noticed difficulties in walking and frequent diarrhea. echocardiogram revealed diffuse left ventricular hypertrophy, suggesting cardiac amyloidosis. amyloid deposits were not detected in the endoneurim or perineurium of the sural nerve years after the onset of the disease, but a marked loss of myelinaed and unmyelinated nerve fibers was observed in it. ttr-derived amyloid deposits were confirmed in the peroneous brevis muscle, salivary gland and heart tissue. dna analysis revealed the heterozygote mutation, p.e k (e k)/c. g>a, of ttr gene, and she was diagnosis as fap. these findings suggest that the proximal parts of peripheral nervous system might be strongly involved by ttr aggregates or amyloid fibrils. blood-nerve barrier in the distal part of peripheral nerves could be preserved until later in the patient. several biopsy sites other than nerve may be helpful and necessary for diagnosis of ttr amyloidosis in mild or late-onset fap as our case. amyloidgenic element (cae) encoded by the ' utr. this study also identified a de novo c. _ dup frameshift mutation predicting p.lys glnfs* in nefh from a cmt family with atypical clinical symptom of proximal dominant weakness. this mutation is located near the previously reported frameshift mutations, suggesting a mutational hot spot. these relatively frequent deletion/duplication events with this resign might be caused by the putative hairpin structure. patient's lower limb mri revealed a marked hyperintense signal changes in the hip muscles than those in the thigh or lower leg muscles. this study also observed an anticipation pattern of earlier onset ( yrs old for mother to yrs old for daughter) and more severe symptoms in later generation. therefore, this study suggests that the stop loss and translational elongations by the ' utr of the nefh mutations may be relatively a frequent genetic cause of axonal peripheral neuropathy with the specific characteristics of proximal dominant weakness and an anticipation pattern. neil j , haghi ashtiani b , choumet v , musset l , léger jm . department of immunology, pitié-salpêtrière hospital (ap-hp), paris, france; department of neurology, national referral center for rare neuromuscular diseases, pitié-salpêtrière hospital (ap-hp), paris, france; institut pasteur, emerging diseases epidemiology unit, paris, france. the myelin-associated glycoprotein (mag) is a transmembrane glycoprotein localized in periaxonal shwann cells and oligodendroglial membranes of myelin sheaths. mag contains a carbohydrate epitope (hnk- ) that is a target antigen in autoimmune peripheral neuropathy associated with monoclonal igm gammopathy. in these neuropathies, numerous studies report the absence of correlation between the titers of anti-mag antibodies and the disease course. anti-mag titers and igm level at diagnosis are not always associated with disease severity and there is not good correlation between pre-and post-treatment anti-mag titers in patients who respond clinically to immunomodulators. mag belongs to siglec- a family and the linkage of sialic acid to the underlying sugars is an important determinant of siglec binding. mag shows high affinity for alpha- , -linked sialic acid ( , -sa).moreover, human monoclonal igm possesses heavy chain glycosylation sites at asn , , , and with sialylated olidgosaccharides and high-mannose type oligosaccharides. igm may bind to mag via these glycan epitopes as an alternative and additional route of antigen binding other than through the fab v regions. this mag-glycans igm interaction may be clinically neutral but could lead to an overvaluation of the biological results. in this study, we analyzed sera from patients with igm reactivity against mag: of them had an anti-mag neuropathy with various degrees of severity, and the last one had igm monoclonal gammopathy, strong serum anti-mag reactivity but no neurological disease. igm were extracted and purified from these sera by affinity chromatography. for each batch, an aliquot was digested by jack bean alpha-mannosidase and anti-mag reactivity was performed by elisa and indirect immunofluorescence (iif), before and after demannosylation. these extracts, tested in an iso quantitative way with regard to the original serum, showed a decrease of activity (elisa) and intensity (iif) after demannosylation. furthermore, elisa anti-mag was carried out in sera from patients with igm monoclonal gammopathy without neurological impairment: of them ( . %) showed a significant biological response. taking into account the fact that anti-mag antibodies are pathogenic (in animals models), these results support the hypothesis of neutral intermolecular interactions between igm and mag. ng cjb , ng jph , tay lb, t , umapathi . yong loo lin school of medicine, national university of singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. patients with postural orthostatic tachycardia syndrome (pots) have marked tachycardia with no associated decrease in pbp. we asked if the reflex tachycardia, which corresponds to postural dizziness in these patients, could be a result of a decrease in casp. two male patients, and years of age, with clinical features typical of pots went through a complete battery of autonomic screening tests. the sympathetic and parasympathetic responses were normal other than a heart rate increase of and beats per minute, respectively, on standing. there was no significant decrease or increase in pbp on standing for and minutes. casp was measured non-invasively by a device bpro r that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure waveform from which a numerical casp value is derived. the casp measurements for both patients did not decrease on standing for and minutes. our preliminary observation suggests that the basis of tachycardia in pots patients may not be a decrease in central blood pressure. we are proceeding to systematically study more pots patients to corroborate the above observation. we are also trying to compare the difference between pbp and casp in pots and age, gender-matched normal controls. the major limitation of the study is the model-based mathematical derivation, rather than direct measurement, of casp. we are proceeding to systematically stud y more pots patients to corroborate the above findings. ng jph , ng cjb , t umapathi . lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university of singapore, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. orthostatic hypotension (oh) is a prominent component of autonomic dysfunction (ad), the consequent hypoperfusion of vital organs responsible for considerable morbidity and mortality. these structures are exposed to casp rather than pbp. we sought to understand the relationship of casp to peripheral blood pressure (pbp) in patients with autonomic dysfunction exposed to orthostatic stress. we reviewed autonomic function tests of patients tested at our laboratory over a -year period. the patients were divided into cohorts: ( ) no ad and no oh ( ) no ad, with oh ( ) mixed ad ( ) parasympathetic dysfunction ( ) sympathetic dysfunction. casp was measured non-invasively by a device, bpro r , that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure-wave form from which a numerical casp value is derived. the difference and ratio of casp to pbp was recorded at rest and after minutes of standing. out of patients had complete data and a definitive final diagnosis. cohorts - had , , , , patients respectively. mean casp-pbp difference in cohorts - were − . , − . , − . , − . , − . respectively at minutes, and − . , − . , − . , − . , − . respectively at minutes. mean casp/pbp ratio in cohorts - were . , . , . , . , . , . respectively at minutes, and . , . , . , . , . respectively at minutes. there was no significant difference in the response of casp and pbp to orthostatic stress across abnormal cohorts to and in comparison with the "normal" cohort (p= . to . ). there was also no relationship to symptoms, namely postural dizziness. in conclusion, autonomic dysfunction does not seem to affect the casp-pbp relationship as measured by non-invasive means. the absence of true normal controls, the exclusion of significant number of patients because of incomplete data and the model-based mathematical derivation rather than direct measurement of casp are limitations that we aim to address in follow-on studies. niu jw , guan hz , cui ly , guan yz , liu ms . the department of neurology, peking union medical college hospital, chinese academy of medical sciences, beijing, china. we aimed to explore the correlation between afterdischarges in motor nerve conduction studies and clinical motor hyperexcitability in patients with voltage-gated potassium channels (vgkc) antibodies. six patients with positive serum antibodies to contactin-associated protein-like (caspr ) or/and leucine-rich glioma-inactivated protein (lgi ) were recruited, including with autoimmune encephalitis, and with cramp-fasciculation syndrome. electromyography (emg), nerve conduction studies (ncs) and f waves were performed, and afterdischarges were assessed. one patient was followed up. five patients had clinical evidence of peripheral motor nerve hyperexcitability (myokymia or cramp), and four of them had abnormal spontaneous firing in concentric needle electromyography. prolonged afterdischarges following normal m waves were present in all six patients, including the two patients who had no emg evidence of peripheral nerve hyperexcitability (pnh). in the patient who was followed up, afterdischarges disappeared after treatment with intravenous immunoglobulin (ivig). afterdischarges in motor nerve conduction study might be more sensitive than needle electromyography for detecting peripheral motor nerve hyperexcitability in patients with vgkc antibodies, and could disappear gradually in accordance with clinical improvement and reduction of antibodies. in our research, multiple sites measurement of cross sectional areas (csa) by ultrasound was performed to differentiate charcot-marie-tooth type a (cmt a) and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). twenty-eight patients with cidp, patients with cmt a, and healthy controls (hc) were recruited prospectively. consecutive ultrasonography scanning was performed from wrist to axilla on median and ulnar nerves. csas were measured at predetermined sites of each nerve. cmt a had significantly larger csas at all sites of median and ulnar nerves (all p< . ). in cmt a, csas increased gradually and homogeneously from distal to proximal along the nerve, except potential entrapment sites. cidp displayed three different morphological patterns, including mild enlargement in patients, prominent segmental enlargement in , and slight enlargement in one, among which different treatment responses were observed. all patients with mild nerve enlargement treated with intravenous immunoglobulin (ivig) were responsive ( / ), while less than half of those with prominent segmental enlargement ( / ) were responsive (p< . ). the patterns of csa enlargement were different in cmt a and cidp patients. consecutive scan along the nerve and multiple sites measurement by ultrasound could supply more detailed morphological feature of the nerve and help to differentiate cidp from cmt a. guillain-barre syndrome (gbs) mainly consists of acute inflammatory demyelinating polyradiculoneuropathy (aidp) and acute motor axonal neuropathy (aman). in aman, conduction block (cb) could be reversible or followed by axonal degeneration. we aimed to identify the correlation between existence of cb and the functional outcome for patients with gbs. gbs patients were prospectively recruited for serial electrophysiological tests and disability evaluation. all patients received treatment of intravenous immunoglobulin (ivig), and their disabilities were evaluated on the hughes functional grading scale before and month after treatment. patients were classified into aidp, aman, equivocal or normal according to electrodiagnostic criteria described by rajabally et al. aman patients who had follow-up nerve conduction studies were further classified into three groups. group was typical aman without conduction block, group had reversible conduction block, group had conduction block and subsequential axon degeneration. electrophysiological study results showed aidp, aman, equivocal and normal. probable or definite conduction block was observed in aidp patients and aman patients. aman with cb had higher reduction of hughes grade at one month ( . ± . vs . ± . ,p= . ), and lower percentage of patients with slow recovery (unable to walk independently at six months) ( % vs %, p= . ) compared with aman without cb. there were no significant differences between aidp with cb and without cb, in the reduction of hughes grade at one month. among the aman patients who were followed up, were typical aman without cb (type ), had reversible cb (type ), had cb and subsequential axon degeneration (type ). hughes grades at nadir were similar, while patients with reversible cb (type ) had the largest hughes grade reduction at one month (type - . vs type - . vs type - . ). none of the patients with axon degeneration (type ) showed rapid recovery, while % of those with reversible cb (type ) had rapid recovery (improvement by two or more hughes grades within four weeks after onset). electrodiagnosis of aman with conduction block, especially reversible conduction block, might be a marker of good recovery. we report a follow-up study of nerve ultrasound in a patient with primary neurolymphomatosis. a -year-old female presented with -months history of asymmetric limb pain, paresthesia, and weakness. electrodiagnostic studies and spinal cord mri showed an axonal neuropathy involving cervical and lumbosacral root, brachial plexus and left median nerve. detection of malignant b lymphocytes by cytology and flow cytometry of cerebral spinal fluid confirmed the diagnosis of b-cell non-hodgkin lymphoma. nerve ultrasound showed dramatic enlargement of upper, middle and lower trunks of left brachial plexus (cross sectional area-csas mm , mm , mm respectively), middle trunk of right brachial plexus (csa mm ), and proximal part of left median nerve (csa - mm ). five months later, after five chemotherapy of rituximab and high-dose methotrexate, and intrathecal injection of cytosine arabinoside and dexamethasone, the patient had clinical improvement. nerve ultrasound also showed alleviation of nerve enlargement. the csas of upper, middle and lower trunks of left brachial plexus were mm , mm , mm respectively; the csa of middle trunk of right brachial plexus was mm ; the csa of proximal part of left median nerve was - mm . peripheral nerve ultrasound could help locate the distribution of nerve involvement, and reveals disease progression. nolano m , provitera v , stancanelli a , caporaso g , saltalamacchia am , borreca i , lullo f , califano f , lanzillo b , iodice r , manganelli f , barone p , santoro l . irccs "salvatore maugeri" foundation, institute of telese terme (bn), italy; "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; center for neurodegenerative diseases (cemand), department of medicine and surgery, neuroscience section, university of salerno, italy. a peripheral nerve involvement has been demonstrated in pd with the evidence of a small fiber pathology as possible intrinsic feature of the disease and a higher occurrence of large fiber neuropathy in patients longtime treated with l-dopa. however the role that disease itself and ldopa have on small and large fiber pathology in pd is still debated. we studied morphology and function of cutaneous innervation, in idiopathic pd patients ( male, aged . ± . ), including naïve and l-dopa treated subjects without electrophysiological signs of neuropathy, with the aim to assess and characterize small and large fiber involvement and the effect of l-dopa on it. all patients underwent a screening to rule out potentially neurotoxic conditions such as glucose intolerance, dysendocrinopathies, vitamin e, b and folic acid deficiency, hepatic or renal failure, hiv or connective tissue disorders. skin biopsies were obtained from thigh, leg and fingertip from the more affected side and bilaterally from thigh and leg in patients. samples were processed with indirect immunofluorescence technique using primary antibodies to mark different sensory and autonomic fiber populations. density of intrapapillary myelinated endings (ime), meissner's corpuscles (mc) and epidermal nerve fibers (enfs) was obtained as well as a semi-quantitative assessment of sudomotor, pilomotor and vasomotor innervation. further evaluation included sympathetic skin response, quantitative sensory testing and dynamic sweat test. morphological and functional findings were compared with data extracted from our age and sex stratified normative dataset. ienf, ime, mc densities were lower (p< . ) compared to controls in both naïve and l-dopa treated patients without differences between them except for mc density that was lower in l-dopa treated subjects ( . ± . vs . ± . /mm ). a loss of autonomic nerves was also found in both groups compared to controls. significant abnormalities (p< . ) of thermal sensory thresholds, tactile thresholds, mechanical pain perception and reduced sweating output were present and similar in both groups. our work confirms in pd an intrinsic peripheral nerve pathology involving both small and large fibers. small fiber pathology isn't affected by l-dopa treatment while sensory large fibers involvement, already present in naïve patients worsens with ldopa treatment. noto y , garg n , li t , timmins hc , park sb , shibuya k , kiernan mc . brain and mind centre, the university of sydney, sydney, australia. the diagnosis of amyotrophic lateral sclerosis (als), a progressive, fatal neurodegenerative disorder defined by combined upper and lower motor neuron involvement, remains clinically based. the purpose of this study was to determine the ultrasound appearance of peripheral nerves in als patients, and to investigate whether parameters such as distal/proximal ratios of nerve cross-sectional areas (csas) may effectively differentiate disease mimics from als. nerve ultrasound of the median, ulnar, and tibial nerves was performed in als patients compared to mimic patients ( patients with peripheral nerve hyperexcitability syndromes (pnhs) and patients with multifocal motor neuropathy (mmn)). comparison of nerve and the distal/proximal ratios was undertaken by ultrasound and compared across clinical and neurophysiological parameters.compared to normal controls, csa of the median nerve at the upper arm was decreased in als (p < . ). in comparison to als mimic disorders, csa at the proximal site of the median, ulnar and tibial nerve and the forearm/upper arm ratio of the median and ulnar nerves had diagnostic values. in addition to csa of the median, ulnar, and tibial nerves, the median and ulnar nerve forearm/upper ratios may provide a useful marker in for the diagnosis of als. approximately in , individuals are diagnosed with charcot marietooth (cmt) disease, making it the most common hereditary peripheral neuropathy. there is no documented cure for cmt, however, many of those affected, report difficulty with mobility, imbalance, and weakness of the feet and hands. in the general population, patients reporting difficulty walking, falls and/or fear of falling, and poor strength are often referred to physical (pt) and occupational therapists (ot) to skillfully address the impairments and help restore function and quality of life (qol). for patients diagnosed with cmt this is unfortunately not the norm, often leaving patients without any skilled guidance on managing their functional impairments. patients ( males) with a mean age of . years ( - ), went through a progressive and skilled pt intervention over the course of months. the program included: therapeutic activities and exercise, neuromuscular reeducation, and manual techniques to address the documented deficits. patients were progressed through the program based on the borg scale. each patient was assessed prior to and post the commencement of the program with the berg balance scale, minute walk test, timed up and go, sit to stand in seconds, foot self selected and fast gait speed, the lower extremity functional scale, activities balance confidence form, upper extremity functional index, oswestry, and the sf- qol measure. the patients were seen by the same skilled pt - a week for weeks. all but patients improved in all measures taken, indicating an improvement in function and overall quality of life. participants reported a total of falls in the months prior to the initiation of the study and only fall was reported during the month pt intervention. this study makes a strong case for the utilization of skilled pt to address deficits in patients with cmt. additionally, the utilization of objective, valid and reliable outcome measures in this population may help healthcare practitioners establish baseline function and response to change. a large randomized control trial is recommended to study the effects of a specific pt intervention on outcome measures in patients with cmt. ogata h , fujita a , yamasaki r , matsushita t , kira ji . department of neurology, neurological institute, graduate school of medical sciences, kyushu university, fukuoka, japan. clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) patients with autoantibodies against neurofascin (nf) , one of the paranodal proteins, have been elucidated while the relation between anti-nf antibody levels and long-term clinical course in these patients still remains elusive. we retrospectively collected clinical, electrophysiological and immunological data of three anti-nf antibody-positive cidp patients. they were all males and their ages at onset were , , and years old. their clinical severity was evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale. anti-nf antibody levels were measured by flow cytometry using hek cell lines stably expressing human nf . after immunotherapies of various combinations, including intravenous immunoglobulin, plasmapheresis, corticosteroids and other immunosuppressants, were introduced, their clinical parameters were gradually improved. decreased or absent dtrs were normalized and grip strength was increased by more than kg. hughes functional scale scores were decreased by at least one point compared with those at nadir. ncs findings of all three patients also showed obvious amelioration. for example, their f wave latencies in the right ulnar nerve were improved from to ms, from to ms, and from to ms, respectively. anti-nf antibody levels after treatment were decreased in two patients whose pre-and posttreatment sera were available. when dose of oral prednisolone was being tapered, they experienced re-exacerbation of clinical parameters, especially dtrs and grip strength. their ncs findings and serum anti-nf antibody levels were also deteriorated. exacerbation of these laboratory data in one patient preceded his clinical fluctuation, which suggests that ncs and serum anti-nf antibody levels could be used as early disease activity markers. in this case series, not only clinical but also laboratory data support a notion that anti-nf antibody-positive cidp patients were reactive to combined immunotherapies including corticosteroids. even though various treatments were administered to them, efficacy of oral corticosteroids seemed to be dose-dependent. optimal disease activity markers and immunotherapies for long-term maintenance of remission in anti-nf antibody-positive cidp should be identified. evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale, after starting immunotherapies, ohnmar o , kamyw , ng lfp , t umapathi . university of medicine , yangon, myanmar; singapore immunology network, a*star, singapore; national neuroscience institute, singapore. singapore's zika virus (zikv) outbreak started in late august . over a period of months, we studied patients enrolled into our institution's prospective guillain-barré syndrome (gbs) database for relationship to zikv infection. we also studied gbs controls that were seen before the established outbreak and non-gbs controls. the index cases tested negative for zikv pcr in blood and urine. we proceeded to test zikv igg, igm, dengue virus (denv) igg and igm, and neutralization assays against zikv and denv. one patient with anti-gq b igg positive miller fisher syndrome had detectable zikv igm and zikv igg. the serum showed low titre denv igm and denv igg. follow-up serum at about months showed increase in zikv igg. we believe this patient has zikv-gbs. one patient with acute motor sensory axonal neuropathy and another with acute inflammatory demyelinating polyneuropathy had high zikv igm but low denv igm and igg. another patient with mfs showed high levels of zikv and denv igm but low igg. the latter two patients had gbs before the zikv outbreak in singapore. we suspect these patients could have zikv-gbs, but are awaiting convalescent sera for confirmation. two patients seen during the outbreak had detectable levels of zikv igg but serial testing showed a decline after a period of - months. the initial and follow-up sera showed raised denv igm and igg levels in one and raised igg levels in the other. in addition, both had stronger neutralizing capacity against denv than zikv suggesting that the initially detectable zikv igg levels was due to cross reactivity with previous denv infection. four patients, gbs control and non-gbs controls also showed serological response consistent with previous exposure to denv. one normal control showed nil exposure to both viruses. in summary, using various overlapping serological methods we diagnosed definite and suspect zikv gbs cases. our findings highlight ) insensitivity of blood and urine pcr to diagnose zikv-gbs ) the problems of interpreting zikv serology from cross-reaction with denv ) serial serology increases diagnostic accuracy. okar sv , ergunay k , bekircan-kurt ce , , erdem-ozdamar s , , tan e , . department of neurology, hacettepe university, ankara, turkey; virology unit, department of medical microbiology, hacettepe university, ankara, turkey; neuromuscular disease research laboratory hacettepe university, ankara turkey. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated polyradiculoneuropathy. it is believed that acute inflammatory demyelinating poliradiculoneuropathy is caused by t-cell mediated autoimmune response targeting peripheral nerve myelin. molecular mimicry plays a role in the pathogenesis of some gbs cases. this mechanism has been well demonstrated in the acute motor axonal neuropathy (aman) variant, in which autoantibodies to camphylobacter jejuni share epitopes with peripheral nerve gangliosides. this molecular mimicry mechanism can be attributed to some cases with atypical triggers such as zika virus or west nile virus infections. moreover there are accumulating clinical data for vector borne viral infections triggering gbs. we evaluated vector-borne viral infections in our gbs and aman patients. eight patients with gbs, two patients with aman and as a control group, seven patients with normal pressure hydrocephalus were included. gbs and aman was diagnosed with clinical, electroneuromyograpic and cerebrospinal fluid (csf) findings. cerebrospinal fluid serum and urine samples were examined for vector borne viral infections via generic flavivirus and phlebovirus pcr. we also documented our patients prognostic scores such as modified erasmus gbs outcome score (megos) and modified erasmus gbs respiratory insufficiency score (megris). the mean age of the patients was , ( - years) , six of them were female. all csf, serum and urine samples of our patients and control patients were negative for flavivirus and phlebovirus families. the preliminary results of our study in this our small cohort did not show any correlation between the vector-borne viral infections and gbs. further studies with broad number of patients are needed for more suggestive results. neuromyotonia (nmt) consists of spontaneous motor unit activity that reflects increased peripheral nerve excitability, leading to involuntary, persistent muscle activity, visible as muscle twitching at rest, with generalized, easily provoked cramps. since the first electrophysiological (emg) description of nmt by denny-brown and foley ( ) , there has been discussion about the origin of the abnormal electrical activity recorded in needle emg studies. we studied two patients. patient 's nmt is aggravated by cold, and he has an associated non-progressive mild polyneuropathy with demyelinating features. he is now -year-old and has been followed in our centre for years. he is negative for anti-vgkc antibodies. firstly, he was treated with carbamazepine and phenylhydantoin with poor response, but he has shown major improvement on intravenous immunoglobulin (ivig) during the last years. patient is a year-old man with nmt, followed for year, with high titters of anti-vgkc antibodies ( pmol/l; normal< ). he improved on ivig during the last months. screening for neoplasia was unremarkable in both patients (negative anti-neuronal antibodies, in particular anti-hu, anti-yo and anti-ri antibodies; normal computerized tomography scan of the chest and abdomen). in addition to routine studies, we tested synchronicity to spontaneous discharges in different motor units in simultaneous recordings made with two needle electrodes in the first dorsal interosseus muscle. time-locked fasciculations in these double recordings would represent abnormal ectopic activity initiated in a nerve trunk with ephaptic stimulation of a nearby axon. in patient , this research protocol was applied once, years after regular ivig treatment. patient was investigated before and year after ivig. both patients improved after ivig, mirrored by a striking decrease in the amount of spontaneous activity on emg. moreover, our technique did not detect synchronous spontaneous activity (time-locked fasciculations) on the second assessment, although this was predominant before treatment in patient . in nmt, abnormal discharges originate both in distal axonal branches and in more proximal segments. it appears that ivig is more effective in blocking antibody activity in proximal axonal segments, perhaps related to factors such as blood-nerve barrier, temperature or differing ion channel distributions. celiac disease (cd) is a chronic, multisystem and immune-mediated disorder characterized by small-bowel sensitivity to dietary gluten in genetically predisposed individuals. neurological manifestations may occur in about % of patients, including peripheral nerve involvement. recent growing evidence strongly suggests that peripheral neuropathy in cd may be autoimmune and associated with anti-ganglioside antibodies. a -year-old woman presented with slowly progressive weakness of her right hand and fingers extensors. medical and family history were unremarkable. on examination, muscle strength (medical research council-mrc) was scored for right wrist and finger extensors, for right abductor pollicis longus and for right ankle dorsiflexion. right triceps brachii and brachioradialis reflexes were weak, but normal elsewhere. the remainder of the neurological examination was normal. neuroaxis magnetic resonance was unremarkable, specifically with no gadolinium-enhancing lesions. motor and sensory nerve conduction studies were normal. no conduction block or abnormal temporal dispersion was found. needle electromyography showed severe neurogenic changes with abnormal spontaneous activity in right radial-innervated muscles, and chronic neurogenic changes in homolateral tibialis anterior, peroneus longus and extensor digitorum brevis. ganglioside antibody testing was positive to igg anti-gm antibody, but negative to anti-gm and other anti-ganglioside antibodies. additional blood tests were unremarkable, in particular cryoglobulin testing was negative. intravenous immunoglobulin improved weakness, as right extensors of the wrist and fingers scored (mrc). a monthly treatment was initiated, which after months was changed to every weeks to preserve function. a diagnosis of mama was established. later, cd was diagnosed in her daughter due to chronic diarrhoea. our patient underwent anti-tissue transglutaminase antibodies determination and small-bowel biopsy after years disease' duration, and a diagnosis of cd was made. gluten-free diet was started, but her neurological picture did not change after six months. in our case the presence of igg anti-gm antibody may support a causal link between mama and biopsy-confirmed cd, and the lack of response to gluten-free diet may be explained by chronic axonal injury induced by memory t-cells. this case broadens our knowledge about neurological manifestations in cd, raising a probable association with purely axonal multifocal motor neuropathies and anti-ganglioside antibodies. Õunpuu s , , pogemiller k , acsadi g , pierz k , , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa. orthopaedic surgical intervention of the foot and ankle is performed in persons with charcot-marie-tooth (cmt) for improving foot pain, ankle instability, orthosis fitting/comfort issues and shoe wear. the impact of these surgeries on ankle function during gait is not known. therefore, the goal of this study was to measure gait changes in ankle function following surgical intervention by means of computerized motion analysis. fourteen patients with cmt ( ± years pre and ± years post-operative) with bilateral orthopaedic surgery were included. all patients had a plantar fascia release plus some combination of other soft-tissue (posterior tibialis and tendo-achilles lengthenings; extensor hallucis longus, peroneus longus and anterior tibialis transfers) and bony procedures (metatarsal and cuboid osteotomies). all patients completed two gait analyses (pre and on average . years post-surgery) during barefoot walking using a standardized d motion analysis protocol. the changes in ankle kinematics and kinetics and temporal-spatial parameters were analyzed in reference to a control group of patients with cmt without intervening surgeries with . years between gait analyses as well as normal reference data. the surgical group showed a significant increase in height between the pre and post-operative analyses and no changes in walking velocity (pre: ± , post: ± , normal: ± cm/sec). similarly, the ankle kinematics and kinetics showed no changes as a result of surgery. however, there was a trend for increased peak ankle dorsiflexion (pre: ± , post: ± , normal: ± degrees). as in the surgical group, the control group showed an increase in height but no simultaneous increase in walking velocity (test : ± , test : ± cm/sec). the control group also showed no changes in ankle kinematics and kinetics suggesting that the impact of the disease is, for the most part, not noticeable over . years. the results show that surgical intervention that primarily addresses foot alignment does not negatively impact ankle kinematics and kinetics during gait. the comparison with the control group supports this finding. however, there are some other findings such as the increase in peak dorsiflexion during stance in some patients that will need to be examined further in larger cohorts. Õunpuu s , , pogemiller k , pierz k , , , acsadi g , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa. ankle-foot-orthoses (afos) are commonly prescribed for patients with charcot-marie-tooth (cmt) to improve gait and reduce ankle instability and falling. there are no studies that examine bracing outcomes using objective evaluations. the purpose of this study was to examine the impact of various afo designs on gait parameters in children and adolescents with cmt. we predicted that the afos would improve excessive and delayed peak dorsiflexion in stance and equinus in swing. fifteen patients (mean age ± years) were analyzed barefoot and with their prescribed afos by means of a standardized motion analysis protocol. a full clinical examination was also completed including strength and passive range of motion measures. the afos included solid, hinged and posterior leaf spring (tapered, less supportive) designs. sagittal plane ankle kinematics and kinetics and temporal-spatial parameters were analyzed in comparison to normal controls. walking velocity improved from ± to ± cm/sec demonstrating the functional benefits of afos. ankle plantar flexion angle at initial contact improved from − ± to − ± degrees demonstrating improvement in drop foot in swing and at initial contact reducing the risk of tripping. however, the degree of peak ankle dorsiflexion in stance remained the same and excessive at ± degrees barefoot and ± degrees with afos suggesting that in many patients the afo design was not sufficiently supportive. peak ankle plantar flexor moment showed improvement from . ± . to . ± . nm/kg highlighting the improved base of support provided by the afos that compensate for ankle plantar flexor weakness. however, peak power generation was reduced from . ± . to . ± . w/kg indicating that some of the available ankle strength was impeded with the afos. the results of this study suggest that orthoses can provide improved gait outcomes which will improve overall function. however, there are individual differences in patient impairment (strength, range of motion and bony deformity) and associated gait presentation that need to be accounted for in afo design. afos do not always function as intended due to the complex interaction between patient impairment, orthosis stiffness and orthosis design. motion analysis can assist in identifying the specific afo needs for an individual with cmt. painous c , lópez-pérez ma , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, barcelona, spain; hospital san pedro, logroño, la rioja, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune neuropathy with a heterogeneous clinical spectrum. specific autoantibodies define different clinical phenotypes. cidp with nf antibodies constitutes a specific cidp subset in which a high incidence of postural and intention tremor has been described and that responds poorly to immunoglobulins. we report a patient with an anti-nf positive cidp that presented head, voice and limb tremor that improved with immunotherapy. a -year-old man with unremarkable medical history, presented at the age of with progressive distal weakness and paraesthesia. numbness, gait ataxia and action tremor involving voice, head and limbs appeared sequentially. the emg showed features of acquired demyelination fulfilling cidp diagnostic criteria. a first course of intravenous immunoglobulins was ineffective. the weakness, ataxia and tremor worsened significantly, needing two walking aids first and becoming wheelchair bound later on. he received six plasmapheresis cycles that were also ineffective and oral corticosteroids ( mg/kg) were started with mild improvement. after corticosteroid tapering the patient developed a severe relapse and was referred to our centre. five plasma exchange cycles followed by rituximab ( mg/m , doses) were added to the corticosteroids. three months later the weakness, ataxia and tremor, including the head and voice tremor, started to improve significantly. six months later the patient presented a significant improvement and was able to walk unaided. the anti-nf antibody titres fell from / pre-rituximab to be undetectable. limb tremor is known to occur in patients with inflammatory neuropathies and, specifically in anti-nf + cidp patients but, to our knowledge, this is the first report of a cidp patient presenting with head and voice tremor ever reported. the improvement of tremor with immunotherapy strongly suggest that anti-nf autoantibodies are involved in its pathogenesis, expanding the phenotype of anti-nf specific clinical features. palaima p , peeters k , l. pelayo-negro a , garcía a , gallardo e , garcía-barredo r , de vriendt e , infante j , berciano j , jordanova a . vib and university of antwerp, center for molecular neurology, molecular neurogenomics group, antwerp, belgium; university hospital "marqués de valdecilla", santander, spain. charcot-marie-tooth disease type g is an autosomal dominant and slowly progressing inherited neuropathy which was first described over years ago. it has been attributed to a single spanish family consisting of individuals with affected members spanning four generation. initially, the genetic defect was linked to a . mb region in q -q . . however, extensive sequence and structural variant analyses using whole genome sequences (wgs) of three affected individuals did not reveal any known or novel genetic causes within the region. over the last decade, serial clinical re-evaluation of the entire pedigree was performed leading to changes to the affection status of seven individuals redefining the disease-linked region to chr q . -q . (z max = . at theta = ). additional family members were then submitted for whole exome sequencing. this has led to the identification of a novel missense variant in the e ubiquitin-protein ligase lrsam (p.cys tyr) that was previously not covered by wgs. the variants co-segregated with disease and were absent from controls. other mutations that are known to disrupt the ring domain of lrsam have been previously reported to cause both autosomal dominant and recessive cmt type p (cmt p). unlike prior reports, we demonstrated that the mutation does not influence overall protein levels of lrsam , nor of its ubiquitylation target tsg in patient derived lymphoblasts. transcriptomics analysis identified significant upregulation of another e ubiquitin-protein ligase (nedd l) and of a key regulator of axonal degeneration (tnfrsf ). notably, magnetic resonance imaging of lower-limb musculature systematically showed fatty atrophy in both clinical and subclinical mutation carriers emphasizing its use for the identification of mildly affected members. our findings demonstrate that the isolated genetic entity cmt g is caused by a missense mutation in lrsam and should be reclassified as cmt p. moreover, we reveal novel molecular players associated with lrsam dysfunction, and highlight pathways and therapeutic targets shared with amyotrophic lateral sclerosis and alzheimer disease. damage to peripheral nerves is a prerequisite for neuropathic pain. however, it remains unclear why some neuropathy patients have pain, but others with identical nerve conduction, qst and ienfd do not. we propose to examine the distribution of trpv and its co-localization with cgrp and sp in epidermal nerve fibers in patients with and without neuropathic pain. we aimed to define the expression pattern of trpv in normal healthy subjects first, and study the co-localization of trpv with cgrp and sp in normal controls and patients with painful neuropathy. skin biopsies from neuropathy patients and normal subjects were utilized. anti-trpv antibody generated in our university with support from ninds-funded dept. of neuroscience monoclonal core (ns ) was used. combined immunohistochemistry were performed to identify co-expression of trpv with cgrp, sp and pgp . . the distribution of trpv in controls revealed a proximal to distal gradient similar to that observed for ienf labeled by pgp . . trpv staining was more intense in nerve terminals in the epidermis. combined immunostaining revealed that % of pgp . labeled fibers in the epidermis were trpv +, while % of cgrp+ fibers trpv +. patients with pain had a higher density of trpv + fibers compared to that of patients with numbness. a greater proportion of cgrp+ fibers ( %) in the painful patients were trpv +. expression of trpv in controls exhibits a distal to proximal gradient. trpv expression and co-localization with cgrp were altered in neuropathic pain patients, suggesting that this receptor plays an important role in pathological states. activation of the nop-receptor (nop-r) by its endogenous ligand nociceptin/orphanin fq or non-peptide agonists modulates nociception and analgesia in neuropathic pain models. the wide distribution of nop-r and its endogenous ligand represents an attractive treatment target. since pain is the most prominent feature in pc, we defined expression of the nop-r in plantar skin biopsies and assessed whether alterations exist in pc-affected vs pc-unaffected and anatomically matched control skin. skin biopsies from k a pc and control subjects were immunohistochemically stained for nop-r. combined immunostaining for nop-r with pgp . , neurofilament h (nfh) and cgrp was used to define nop receptor expression in the epidermis and upper dermis. robust nop-r was detected in epidermal keratinocytes and a subset of pgp . + fibers in both epidermis and dermis. staining was inhibited through pre-incubation with a nop-r blocking peptide and western blot analysis using homogenized human skin tissue demonstrated a band at ∼ kd consistent with nop-r molecular weight. nop-r expression occurred in dermal nfh+ a beta-fibers in all groups though no cgrp+ fibers co-expressed nop-r. pc-affected skin had slightly lower nop-r expression than in pc-unaffected skin and a similar pattern in anatomically matched locations from healthy control subjects was observed. nop-r is expressed in human plantar skin epidermal keratinocytes as well as a subset of epidermal and dermal nerve fibers. these fibers are pgp . +, cgrp-and many co-express nfh. nop receptor is a viable pharmaceutical analgesic target in pc patients irrespective of its slight down-regulation as compared to pc-unaffected skin. this work was supported by grünenthal gmbh. pareyson d , stojkovic t , leonard-louis s , reilly mm , laurà m , parman y , battaloglu e , tazir m , bellatache m , bonello-palot n , sacconi s , guimarães-costa r , attarian s , latour p , megarbane a , schenone a , ursino g , sabatelli m , luigetti m , santoro l , manganelli f , quattrone a , valentino p , murakami t , scherer ss , dankwa l , shy me , bacon cj , herrmann dn , pisciotta c , previtali s , bolino a . milan, italy; paris, france; london, united kingdom; istanbul, turkey; algiers, algeria; marseille, france; nice, france; lyon, france; genoa, italy; rome, italy; naples, italy; catanzaro, italy; kurashiki, japan; philadelphia, usa; iowa city, usa; rochester, usa. cmt b and b are characterized by recessive inheritance, early onset, severe course, slowed nerve conduction, myelin outfoldings, association with loss-of-function mutations in myotubularin-related protein- and − (mtmr , mtmr /sbf ), respectively, involved in the metabolism of ptdins p and ptsins( , )p phosphoinositides, key regulators of membrane trafficking. in a multicentre retrospective study to better characterise cmt b, we collected a minimal dataset of information including cmtes/cmtns on cmt b patients, cmt b ( unrelated families) and cmt b ( families). cmt b patients were younger and with earlier onset than cmt b : last visit was performed at a mean age of years (sd . ; range - ) for cmt b and years ( . ; - ) for cmt b ; disease onset occurred at a mean age of . years ( . ; - ) in cmt b as compared to . years ( . ; - ) in cmt b ; delay in motor milestones occurred in / cmt b and / cmt b subjects. eleven cmt b patients became chair-bound, whereas all cmt b subjects but one are still ambulant, although with afos for patients and requiring unilateral support in two cases. both disease types are characterised by vocal cord involvement ( / cmt b and / cmt b ); respiratory involvement was seen almost exclusively in cmt b patients (n= , four requiring non-invasive ventilation and one tracheostomy, as compared to one cmt b patient on niv at age ); two cmt b subjects and an affected cmt b relative not included in the present study died of respiratory complications. glaucoma (n= ) and buphthalmos (n= ) occurred only in cmt b . cmtes/ cmtns scores were higher in cmt b patients in spite of their younger age, indicating more severe disease: cmt b = cmtes mean . (n= ; sd . ; range - / ), cmtns mean . (n= ; sd . ; range - / ,; cmt b = cmtes mean (n= ; sd . ; range - / ), cmtns mean (n= ; sd . ; range - / ). our data confirm that cmt b is more severe than cmt b . interestingly, mtmr interacts with mtmr . mtmr is a catalytically active phosphatase, whereas mtmr is a catalytically inactive protein, known to increase mtmr enzymatic activity. thus, in cmt b nerves a residual enzymatic activity of mtmr may result in a less severe clinical phenotype as compared to cmt b . the charcot-marie-tooth disease (cmt) national registry is fully operative (https:www.registronmd.it) with collection of clinical/genetic information (minimal dataset) and reporting of clinical scales; cmt patients have registered thus far and have chosen one of the reference centers; information collected during the ad hoc visit have been entered in the registry for of them. registered patients have the chance to participate to a study that requires filling online self-reported questionnaires related to five important issues: disease course and complications during pregnancy; use, efficacy and tolerability of orthotics and assistive devices; outcome of surgery for skeletal deformities; safety of anesthesia; occurrence of sleep disorders (including evaluation of fatigue, anxiety and depression). by february , patients and relatives/friends (as healthy controls) have filled the questionnaires. we are performing a first explorative analysis of results, but data collection on all questionnaires will be prolonged until novemebr , , to obtain a larger sample. pregnancy: / cmt women had at least one pregnancy; complications ranging from mild to severe occurred in / pregnancies vs / in controls. cmt worsened in pregnancies ( patients) with no recovery in instances. prenatal diagnosis was performed in / pregnancies. satisfaction related to surgical procedures for foot deformities, assessed with vas (score - ), was . ± . (n = ). repeat surgery was required in / instances. sleep: the epworth sleepiness scale questionnaire revealed abnormalities of sleep in / cmt patients ( %) and in / controls ( %). the vast majority of cmt subjects ( / ; mean . ± . ), but also of controls ( / ; . ± . ) were not good sleepers according to the pittsburgh sleep quality index (psqi) (range - , good sleep). fatigue: scores of modified-fatigue impact scale (range - , no fatigue) were higher for cmt (mean ± . ) than controls (mean . ± . ). hospital scale for anxiety and depression: / cmt subjects had mild-to-severe anxiety and / mild-to-severe depression as compared to / and / controls, respectively. data analysis on orthotics and anesthesia is ongoing. in conclusion, the first data analyses confirm that there are problems related to all the five domains explored, that will need to be specifically addressed in patients' care. supported by telethon-uildm grant gup . charcot-marie-tooth disease (cmt) is the most frequent motor and sensory peripheral neuropathy and is known to have the uniform demyelination. the traditional definition of conduction block is the reduction of negative peak of compound muscle action potential (cmap) of proximal segment relative to adjacent distal segment more than %. in this study, we tried to find the frequency of conduction block in cmt a patients and to investigate the differences of the clinical manifestation. we enrolled unrelated cmt a patients ( males and females) with pmp duplication and undertook nerve conduction studies from to . stimulation sites were wrist, elbow and axilla for median nerve study. eleven patients ( . % of all enrolled patients) had ncs features suggestive of conduction block. compared to cmt a patients without conduction block, functional disability scale was significantly higher in cmt a patients with conduction block (p < . ). however, onset age and disease duration were not different between cmt a patients with and without conduction block. in addition, conduction block was more frequently observed in distal segments than proximal segments. we suggest that the frequency of conduction block in cmt a patients is not low, and there is some heterogeneity of demyelination in cmt a patients. also, in cmt a patients, the conduction block might have relationship with clinical disability. park jg , park ms . yeungnam university college of medicine, daegu, korea. the subacute combined degeneration (scd) is diagnosed by a characteristic clinical manifestation, spinal mri, and decreased serum vitamin b levels. we report a case of scd with elevated homocysteine and methlymalonic acid level in the situation of spurious elevation of vitamin b concentration. an years old man presented with progressive gait disturbances and sensory disturbances. about a year ago, he felt both hands and feet paresthesia and gait disturbances. four months before the visit, nerve conduction tests were performed elsewhere due to symptoms. polyneuropathy was found. the laboratory test showed megaloblastic anemia. however, serum vitamin b concentration was increased to pg / ml (normal: - pg / ml). his symptoms progressed gradually. at presented, neurological examination showed spasticity of the lower limb without weakness, and vibration and position sensation were decreased in both lower limbs, and showed ataxic gait. spinal cord mri showed a lesion with a long t high signal intensity from c to t in the posterior column of the spinal cord. further laboratory test were added, then while folate was normal level but homocysteine at . umol/l (normal: - umol/l), and methylmalonic acid at . um / l (normal: - . um / l) were increased. finally, scd caused by vitamin b deficiency was diagnosed. in addition, gastric endoscopy was performed to find the cause of vitamin b deficiency and chronic atrophic gastritis was found. after cobalamine treatment for months, hemoglobin level was improved and his symptoms were all improving with a little gait disturbances. false-elevation of vitamin b level could lead to delayed diagnosis and cause irreversible changes in the nervous systems. one of the most commonly used methods to measure vitamin b concentrations is competitive-binding assay, which may result in normal levels even with vitamin b deficiency due to the effect of anti-intrinsic factor antibodies. when vitamin b deficiency is suspected, even if the vitamin b concentration is normal, additional tests of homocysteine and methylmalonic acid should be considered. parman y , durmus h , deymeer f , oflazer-serdaroglu p , battaloglu e . istanbul university, istanbul faculty of medicine, department of neurology, istanbul, turkey; bogazici university, istanbul, turkey. hereditary motor and sensory neuropathies, also called cmt neuropathies, are the most common group among the hereditary neuropathies. cmt subtypes are grouped by axonal, demyelinating or intermediate phenotype, or by autosomal-dominant (ad), autosomal-recessive (ar) or x-chromosomal inheritance. cmt- is considered axonal and characterized by distal muscle wasting, mild sensory loss and normal or near-normal nerve conduction velocities. mutations in more than genes are known to be able to cause autosomal dominant (ad) or recessive (ar) cmt- to date. the genetic heterogeneity in charcot-marie-tooth (cmt) is a challenge for genetic diagnostics. clinical clues and frequencies of mutations in cmt genes from large cohorts may help to develop strategies for efficient genetic testing. here, we present the clinical, electrophysiological and genetic features of patients from turkey, diagnosed as genetically confirmed cmt- in the department of neurology, istanbul faculty of medicine our clinic between and . genetic testing was performed for gdap by dna sequencing and samples from patients were exome sequenced (wes). twenty-one male and ten female patients from unrelated families were investigated. segregation was ar in eight, ad in six families and two were isolated cases. intraand interfamilial variability in the age of onset with a range of - (mean . ± . years) and disease progression rate were striking. slowly progressive weakness and atrophy of distal muscles in the feet and/or hands were the most common presenting symptoms. mfn was found in four unrelated ad kinship and in unrelated ar kinships and was most common gene mutated among whole cohort. gdap was the most commonly mutated gene among ar families ( / ). wes revealed further mutations in aars, nefl, kif b and hspb , however, the causative mutation could not be identified in known cmt genes in about % of patients. our data indicates the marked intra-and inter-familiar phenotype variability in cmt- as previously described in literature. many more genes causing arcmt- remain to be discovered. pasnoor m , veerapaneni k , murphy r , statland jm , kimple d , hamasaki a , glenn md , herbelin l , barohn rj , jawdat o , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. electrodiagnostic (edx) studies involving electromyography/nerve conduction study (emg/ncs) are useful for diagnosis of neuromuscular disorders. although these are safe procedures, emg and ncs often evoke anticipated pain and anxiety. there are no published research studies to support the notion that providing detailed information to patients prior to these procedures would reduce procedural pain and ameliorate anxiety levels afterwards. the objective of this study was to perform a prospective survey to assess anticipated pain and anxiety in patients presenting for edx studies, design a detailed patient education form, and assess the change in perceived pain and anxiety pre and post procedure in the standard of care (soc) education versus the printed detailed education instrument group. after completing a brief pain/anxiety questionnaire, patients were randomly assigned to either soc verbal education group or to read the detailed education form. another brief questionnaire was completed post procedure. seventy-eight patients were enrolled at the time of abstract submission, in intervention and in soc groups. mean age of patients enrolled was ± years. pain was anticipated by % patients as a visual analog scale (vas) score mean of . ± . with no significant difference between both groups (p= . ). post-procedural pain was reported in % of patients with mean pain score of . ± . and no significant difference between the two groups (p= . ). anxiety pre-procedure was reported by % patients with a mean likert-like score of . ± . score in all cases (p= . between both groups). post-procedural anxiety frequency was reduced to % with mean level of . ± . (p= . between both groups). most patients reported pain with both emg and ncs ( %). we found that edx testing evokes moderate pain in most and in some cases moderate anxiety level. the detailed education intervention did not attenuate the frequency or severity of pain. post-procedural anxiety was expectedly reduced in severity and frequency in both groups with a suggestion that anxiety severity is lesser in the detailed education group but numbers were small. further studies with more detailed questionnaire and perhaps web-based education in larger population may be useful to reduce pain/anxiety in patients presenting for edx studies. pasnoor m , roach c , barohn rj , statland j , jawdat o , dick a , glenn m , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. diagnostic criteria for chronic inflammatory demyelinating polyneuropathy (cidp) have been designed for use in clinical trials. there is limited data on the comparative diagnostic utility of these criteria in the clinic setting and it is unknown if some of these criteria are better for predicting treatment response. the objective of this study is to compare the sensitivity of various diagnostic criteria and review treatment response. after obtaining local irb approval, we performed a retrospective chart review of cidp patients seen at the university of kansas medical center between and . we abstracted the clinical, electrodiagnostic, and treatment information. we determined the frequency of patients fulfilling each of the following criteria: efns , aan, saperstein and incat. we defined treatment response based on treating physician's impression of change, patient-reported functional improvement or one point grade change in the mrc grade. fifty-three cidp patients charts were reviewed, were excluded due to missing data. mean age was . , and ( %) were female. elevated csf protein was seen in / ( %). twenty-eight ( %) fulfilled efns definite criteria, ( . %) efns probable, ( . %) aan, ( . %) incat, and ( . %) the saperstein criteria. treatment response in patients who fulfilled efns definite/probable criteria included / to ivig, / to iv or oral corticosteroids, / to mycophenolate mofetil; among patients who fulfilled aan criteria, these were respectively / , / and / ; among those meeting incat criteria / , / and / ; and for saperstein criteria it was / , / and / . half to two-thirds of patients responded to plex based on different criteria. while all cidp criteria can similarly predict ivig treatment response, the efns criteria are most sensitive for the clinical diagnosis of cidp. pellegatta m , canevazzi p , forese mg , podini p , quattrini a , taveggia c . division of neuroscience and inspe, axo-glia interaction unit, san raffaele scientific institute, milan, italy; division of neuroscience and inspe, experimental neuropathology unit, san raffaele scientific institute, milan, italy. axonal nrg type iii is an essential instructive signal for pns myelination, since its expression determines whether axons are myelinated and also the thickness of the myelin sheath. previous studies have shown that secretases' cleavage controls nrg type iii activity at post-transcriptional level. specifically, the beta-secretase bace- cleavage of nrg type iii promotes myelination, whereas the alpha-secretase tace inhibits pns myelination by inactivating nrg type iii. after a damage, the axon located distally to the site of injury degenerates and loses the myelin sheath, following a process termed as wallerian degeneration. unlike the cns, axons in the pns can regenerate and regrow. several studies have shown that regeneration is favored by the trans-differentiation of schwann cells, which start to create a permissive environment for axonal regrowth. in addition, it has been shown that nrg regulates the early stages of the dedifferentiation process and is essential for creating a permissive environment to regeneration. similarly, the beta secretase bace- promotes pns regeneration and remyelination. in the present study, we analyzed the role of the alpha-secretase tace during wallerian degeneration. our data indicate that tace is upregulated in sciatic nerves after injury, distally to the site of damage. since tace is expressed in schwann cells and axons, we analyzed the role of both glial and neuronal tace during degeneration and regeneration processes. thus, we performed crush injury in sciatic nerves of two different transgenic lines carrying a conditional deletion of tace either in schwann cells (p -cre//tace flox/flox ) or in neurons (chat-cre//tace flox/flox ). the analyses of the different phases of the wallerian degeneration in these animals suggest a role of glial tace during the early stage of the process. in fact, we observed an increased number of myelinated axons only in p -cre//tace flox/flox mice, while we did not detect substantial differences in mutant chatcre//tace flox/flox mice. these results suggest that even during regeneration tace has an inhibitory role, as deletion of tace in schwann cell likely induces the activation of a neuro-protective program that we are currently investigating. péréon y , nizon m , küry s , besnard t , quinquis d , boisseau p , magot a , mussini jp , mayrargue e , barbarot s , bézieau s , isidor b . reference centre for neuromuscular disorders; dept. of genetics; dept. of paediatric surgery; dept. of dermatology, university hospital, nantes, france. the hereditary sensory and autonomic neuropathies (hsan) constitute a clinically and genetically heterogeneous group. hsan are associated with sensory dysfunction, altered pain and temperature perception with varying degrees of autonomic dysfunction, and abnormal small fibers neurodevelopment. more than ten genes have been described so far in the hsan group. original classification encompassed four entities, but additional subgroups continue to be described. hereditary sensory neuropathies (hsan) type ii are characterized by autosomal recessive inheritance, onset at birth and self-mutilating behavior. until now, one homozygous frameshift variant, c.[ _ del], p.(lys phefs* ), was reported in arl ip (adprribosylation-like factor -interacting protein ) in a consanguineous family. patients presented with spastic paraplegia, diffuse sensory and motor polyneuropathy and acromutilation. the disorder was classified as autosomal recessive spastic paraplegia spg . here, we described a new patient with congenital insensitivity to pain, sensory neuropathy, self-mutilation, and spastic paraplegia. whole exome sequencing showed a homozygous frameshift variant c. [ _ del], p.(lys phefs* ) in arl ip . the protein harbours reticulon-like short hairpin transmembrane domains and has a role in endoplasmic reticulum shaping. the variant causes an additional c-terminus hydrophobic domain which could alter its function. arl ip interacts with atlastin- responsible for spg a and hsan type d. this report highlights the role of arl ip in pathophysiology of insensitivity to pain and spastic paraplegia. péréon y , nguyen a-l , leclair-visonneau l , fayet g , nguyen j-m , magot a . laboratoire d'explorations fonctionnelles, hôtel-dieu, nantes, france; département de statistiques médicales, hôtel-dieu, nantes, france. carpal tunnel syndrome (cts) is responsible for sensory and/or motor symptoms that may be increased by wrist flexion or extension (phalen sign). previous studies have shown that short duration (e.g. min or less) flexion or extension was not responsible for significant changes of median nerve conduction parameters. the objective is the study was to determine how prolonged extension (reverse phalen's test) affects median and ulnar nerve conduction parameters in normal subjects. after providing informed consent, normal subjects ( females, age rank - years) underwent motor and sensory testing of both median and ulnar nerves from the dominant side. edx testing was performed using standard techniques with wrist in neutral position, then during passive wrist extension ( ∘ ) for minutes, with sequential recording of both motor action potential (cmap) and sensory action potential (snap) latencies and amplitudes every minutes after extension onset. ulnar nerve conduction parameters remained unchanged in all the subjects. regarding median nerve, groups of subjects could be individualized: subjects with no changes; subjects with only significant decrease of snap amplitude (− %); subjects with both snap amplitude and velocity decrease; subjects with both cmap and snap significant changes. when present, changes did not appear before min, with snap decrease being the earliest observed abnormality. prolonged wrist extension was responsible for median nerve edx parameter modifications in / normal subjects. sensory fibers were firstly affected, corresponding to the chronology clinically encountered in cts. based upon these results, the same protocol will be used in patients presenting with mild clinical cts, in order to try and identify an additional edx technique being useful in the diagnosis of cts. one could also wonder whether the subjects with the most important changes would be more prone to present cts in the future. perez-siles g , , drew a , , ellis m , kidambi m , takata r i , speck-martins c e , hagerman k a , siskind c e , day j w , ginzberg m , nicholson g , , , kennerson m l , , . northcott neuroscience laboratory, anzac research institute, sydney, australia; sydney medical school, university of sydney, sydney, australia; molecular medicine laboratory, concord repatriation general hospital, sydney, australia; sarah network rehabilitation hospitals, brasilia, df, brazil; department of neurology, stanford health care, stanford, ca, usa; pediatric neuromuscular unit, wolfson medical center, holon, israel. mutations in the atp a gene cause of x-linked hereditary distal motor neuropathy (dhmnx). to date, missense mutations (t i and p s) in this copper transporting atpase are the only confirmed mutations causing dhmnx in two independent families. next generation sequencing (ngs), including whole exome and whole genome sequencing, is increasing the rapid detection of variants in genes known to cause disease, however the absence and size of additional families, make it challenging to determine the pathogenic or benign status of variants identified. we have recently identified new variants in atp a in patients with progressive peripheral neuropathy, suggesting further genetic heterogeneity of dhmnx. two of these new variants, pe v and pm v, are located at highly conserved amino acids within domains of atp a (a-domain and p-domain, respectively) that are critical for the catalytic cycle of the copper transporter. we have also identified a third variant (c. - a>g) located bases upstream exon that is predicted to abolish a conserved branch-site, a consensus intronic sequence necessary for the processing of immature rna during splicing. our recent investigations using both patient fibroblasts and an atp a conditional knock in mouse model for dhmnx expressing atp a t i , the orthologue of the human atp a t i mutation, showed reduced atp a protein levels and defective retrograde trafficking of atp a, as pathological hallmarks of dhmnx atp a causative mutations. to elucidate if the newly identified pe v, pm v and c. - a>g atp a variants are pathogenic, we have systematically assessed patient fibroblasts harbouring these mutations for altered parameters previously found in the pathogenic t i atp a mutation. we predict that functional characterisation of atp a using patient fibroblasts harbouring newly identified variants will provide the evidence to ascertain whether these variants are disease causing. establishing a systematic functional readout for atp a variants will improve accuracy of genetic counselling and patient management of dhmnx in those cases where genetic evidence is limited. this study represents a complementary and necessary approach to the use of ngs, for validating the pathogenic status of variants identified and for expanding the genetic heterogeneity of dhmnx. month of birth (mob) have been defined as a risk factor for more than diseases. for instance, susceptibility for multiple sclerosis (ms) seems to be associated with being born in may, while lower risk of ms is observed in those born in november. there are no studies that assessed potential association between mob and a risk for guillain-barré syndrome (gbs). we sought to determine if the risk of gbs is associated with the mob. study included gbs patients diagnosed in serbia, montenegro and republic of srpska in the period from january , until december , . mob of patients and of general population were compared. patients with gbs had tendency to be born in october ( % increase compared to general population, p= . ) and were less likely to be born in june ( % decrease, p= . ). when consider specific seasons, gbs patients were more likely to be born in winter months ( % increase, p< . ). no associations were found between month/season of birth and disease severity. results of this pilot study showed that gbs patients are more likely to be born in cold months, and less likely to be born in june. this might be explained by higher exposure to different pathogens during pregnancy in cold months. in accordance with this, it is well known that early exposure to infective agents reduces risk of allergic and autoimmune diseases. further studies are needed to test our findings in different cohorts and in regions with different climate. these results may shed new light on the disease pathogenesis. majority of patients with guillain-barré syndrome (gbs) tend to have a successful recovery, but in number of them significant long-term consequences may negatively affect their quality of life. aim of this research was to analyze the outcome of the disease one year after the acute episode of gbs. among patients diagnosed with gbs in seven tertiary centers in serbia, montenegro and republic of srpska during , subjects were retested after one year ( % males, mean age ± years). functional disability of patients was estimated based on the gbs disability scale (gds). severe form of the disease (gds score - ) was registered in % of patients at admission, % at nadir, and % on discharge. after one year follow-up period, % of patients had no symptoms of gbs, % had mild symptoms, % was able to walk but not to run, % needed unilateral support during walking, while % was wheelchair bound or bedridden. lethal outcome was registered in three patients during acute phase of gbs and in four more during one-year follow-up. paresthesias were present in % of gbs patients, musculoskeletal pain in %, and fatigue in % one year after acute phase of the disease. factors associated with the worse functional outcome (gds grade above ) after one year were: age above , preceding respiratory infection, and worse gds on discharge. one year after the onset of the disease, significant number of our gbs patients had neurological impairments including sensory symptoms, pain, fatigue and muscle weakness which may significantly affect patients' everyday functioning. phetteplace je , saade d , bacon c , shy me . university of iowa hospitals and clinics, iowa city, ia, usa. charcot-marie-tooth (cmt) disease, also known as hereditary motor and sensory neuropathy, is a heritable peripheral neuropathy caused by genetic mutations in many different genes. cmt type a is the most common form of cmt. individuals affected with cmt a commonly have distal muscle atrophy, foot deformities, abnormal gait, and reduced nerve conduction velocities caused by demyelination of the peripheral nerves. cmt a is known to be caused by a duplication of the pmp gene at p . . the proposed disease mechanism of cmt a is believed to be increased gene dosage of the pmp gene, although the exact function of pmp is not known. there have been previous reports in the literature of individuals who carry a homozygous duplication of pmp , therefore having four copies of the gene. however, many of these reports have focused on adult patients who symptoms may not vary significantly from individuals who harbor only a duplication of pmp . a month old male presented to the university of iowa hospital due to a family history of cmt a and concern for delay in independent ambulation. he was known to have both a maternal and paternal family history of cmt a, although the families were believed to be unrelated. on exam he was unable to walk independently, but could take a few steps when his hands were held. per report he began crawling and scooting at months of age. he was not found to have any structural abnormalities or atrophy in his upper or lower extremities. electrodiagnostic testing revealed a demyelinating neuropathy. only one nerve was tested, as the patient was unable to tolerate additional shocks. the peroneal nerve was found to have a nerve conduction velocity of m/s. based on the clinical presentation, electrodiagnostic examination and family history deletion and duplication testing for pmp was performed. this testing revealed a homozygous duplication of pmp , which has been previously reported to cause cmt a. this case supports the gene dosage disease mechanism, as our patient appears to be more severely affected than a typical infant or toddler affected with cmt a. piscosquito g , saveri p , provitera v , stancanelli a , ciano c , magri s , taroni mpz mutations are associated with the demyelinating early-onset charcot-marie-tooth (cmt) type b and with the axonal later onset cmt i/cmt j. paresthesias/dysesthesias and pain have been already reported in some cmt types and also in cmt i/j. we report patients ( from families, sporadic cases,) carrying the mpz c. dela mutation (p.asp thrfster ). this is predicted to be a loss-of-function mutation, leading to p haploinsufficiency. most of the subjects have genetic ancestry from puglia region (southern italy). two patients carried the mutation in homozygosity, showing a severe phenotype: onset at birth, severe scoliosis, proximal weakness, distal limb plegia, no autonomous walking, cmt examination score (cmtes) - / . all the other heterozygous patients had mild adult-onset (mean age , range - ) neuropathy, walked without aids or ankle-foot orthoses, had cmtes < / . five showed mild foot deformities, / had foot and / hand weakness (mrc never less than / ), / sensory loss in lower limbs (ll). paresthesias/dysesthesias (mostly tingling and burning in type) localized in hands and ll were reported by / subjects, neuropathic pain by / . in the homozygous patients, all cmaps and saps were absent. in all other subjects, motor conduction velocities (cv) were > m/s (range - m/s) and sensory cv > m/s (range - m/s). cmaps as well as saps were normal or moderately reduced in amplitude, indicating only slight axonal loss. we confirm that heterozygosity is associated with very mild cmt, whereas homozygosity causes a very severe neuropathy. our data suggest that paresthesias and neuropathic pain are very common and should be considered as part of the phenotype. although the clinical picture suggests a small fiber neuropathy, no such evidence is provided in the current literature. in the attempt to further investigate this field, we found in a -year-old male patient of our series that all thresholds were increased at quantitative sensory testing, and several abnormalities of small nerve fibers were present in skin biopsy. apparently, p aneuplody causes neuropathy and small fiber involvement through a mechanism different from other mpz missense mutations. poitras t , chandrasekhar a , singh v , martinez j , zochodne dw . neuroscience mental health institute, and division of neurology, department of medicine, university of alberta, edmonton, alberta, canada. a microarray mrna analysis of dorsal root ganglia examined in long-term diabetic mice yielded an unexpected upregulation of a unique set of proteins known as major urinary proteins (mups & ). originally, this family of barrel shaped proteins was described as including mediators of pheromone secretion in the urine of rodents, apparently unconnected to other functions. we identified its unanticipated expression in sensory neurons of adult mice and rats and explored its potential impact on the properties of these neurons. analysis of rat and mouse sensory neurons showed widespread and pan-neuronal cytosolic mup expression, not only in the cell body, but also in distal sensory projections located in the dermis of the footpad. to assess the role that mup might offer in the growth behaviour of adult neurons, we examined sirna-induced knockdown of mup on pre-injured dissociated drg primary cultures. mup knockdown in both species showed a significant increase in neurite outgrowth following mup sirna treatment when compared to a scrambled sequence control. mice treated with streptozotocin (stz) model features of human type diabetic polyneuropathy, including decreases in multi-fiber motor and sensory measurements, and changes in thermal sensation in the extremities. to explore the potential role of mup upregulation specifically in diabetes, we examined the impact of non-viral intranasal injection of mup sirna on indices of neuropathy in chronic type diabetic mice. following treatment, mice treated with mup sirna showed improvement in both motor and sensory nerve conduction velocities compared to scrambled controls. taken together, this data suggests that mup plays a growth-suppressive role in both diabetic and non-diabetic neurons and may also contribute to the electrophysiological and behavioral abnormalities associated with diabetic polyneuropathy. the repurposing of mups in sensory neurons identifies an interesting role for a supposed pheromone protein. [supported by cihr, cda and adi] enhancement of their outgrowth properties (singh et al, ) . here we asked whether similar activation, using low dose capsaicin, might similarly ramp up the regenerative activity of sensory neurons. using rat adult primary sensory neurons, we identified a dose dependent relationship between capsaicin exposure and their outgrowth with enhancement at lower doses and expected neurotoxicity at higher doses. doses that enhanced outgrowth were nonetheless associated with rises in intraneuronal calcium. we next tested whether a similar impact of trpv activation in vivo might exist. we used a high sciatic crush injury mouse model to assess the impact of capsaicin at low ( um) or high ( mm) doses applied directly at the site of axon outgrowth after injury. by days following the single application of capsaicin, low doses of capsaicin showed more rapid recovery of thermal, but not mechanical sensation, whereas high dose capsaicin showed no significant difference compared to vehicle treated mice. in parallel with the neurotoxicity observed in vitro, the high dose capsaicin resulted in a decrease in sensory nerve conduction velocity, while the low dose showed no significant difference when compared to the vehicle treated animals. there was no impact on motor axon recovery. taken together, subtoxic trpv activation with intraneuronal calcium mobilization, as applied to regenerating sensory axons, enhances their outgrowth after injury, and improves behavioural recovery similar to that following extracellular electrical stimulation. [supported by cihr] hereditary transthyretin (hattr) amyloidosis is a rapidly progressive, life-threatening disease caused by ttr gene variants, resulting in ttr misfolding and amyloid deposition in multiple organs, including peripheral nerves. hattr amyloid polyneuropathy has prominent small fiber involvement characterized by neuropathic pain and dysautonomia. measurement of intraepidermal and sweat gland nerve fiber density (ienfd, sgnfd) in skin biopsies has been useful in identifying small fiber abnormalities in hattr. we report the effect of patisiran, an investigational rnai therapeutic for the treatment of hattr, over a -month period on ienfd, sgnfd and dermal amyloid content from the phase open-label extension (ole) study. skin biopsies (distal leg, distal thigh) were obtained every months for up to years within the phase ole study (patisiran . mg/kg iv, q w, nct ) and analyzed in a blinded manner by a central laboratory. all analyses are exploratory; nominal p-values are reported without adjustment for multiple testing. twenty-seven patients enrolled; preliminary data indicated patisiran was generally well tolerated; resulted in sustained mean serum ttr lowering of ∼ % for > months and a mean . -point decrease in mnis+ (n= ). twenty-four patients underwent serial skin punch biopsies. at baseline, mean ienfd was . and . fibers/mm, while mean sgnfd was . and . m/mm for distal thigh and distal leg, respectively. amyloid was detected in ∼ % of skin biopsies, mean baseline dermal amyloid content . % and . % for distal thigh and distal leg, respectively, which inversely correlated with ienfd and sgnfd. overall, ienfd was stable throughout the -year treatment. sgnfd increased over time relative to baseline, reaching statistical significance at , , and months for distal thigh (p< . , all time points) and at months for distal leg (p= . ). dermal amyloid content decreased over time and reached statistical significance at , and months for distal thigh (p< . , all time points) and at , , , and months for distal leg (p< . , all time points). in summary, improvements in sgnfd and dermal amyloid content observed over a -year period suggest that these parameters have the potential to serve as biomarkers of response to patisiran treatment. polydefkis m , neuhaus s , doherty l , ebenezer gj . department of neurology, johns hopkins university, baltimore, md, usa. misdiagnosis and delayed identification of an etiology for sensory neuropathies hampers adequate management and delays initiation of therapy. we present several cases that illustrate variable presentations of amyloid neuropathy (an) and an complicated by amyloid arthropathy. a -year-old former college athlete developed painless difficulty walking. he was unable to keep up with family members during routine outings and subsequently was unable to navigate uneven ground during a hike, something he had previously excelled at. balance decreased, making bicycling difficult. neurological evaluation -months after symptom onset led to a diagnosis of peripheral neuropathy. a reversible neuropathy panel was normal though a c was elevated ( . %). over several months, his strength/sensation deteriorated despite diet improvement, losing lbs and a c improvement ( . %). upon referral, emg revealed a sensorimotor neuropathy with markedly reduced/absent sural, peroneal and tibial motor amplitudes. there was mild orthostasis. nis was . skin biopsies revealed severe ienfd, sgnfd reductions and positive congo red staining, confirmed with birefringence. a sural nerve biopsy confirmed amyloid deposits. genetic testing revealed the ttr-fap variant leu his. there was no history or suspicion for a family history of amyloid. a -year-old male with a history of controlled diabetes x years developed abrupt onset of rls months after a lymphoma diagnosis. treatment with rituximab led to radiographic resolution of the lymphoma though the patient developed progressive peripheral neuropathy confirmed by ncv/emg. new-onset early satiety, mild orthostasis and ed emerged. skin biopsies demonstrated a lichenoid pattern of amyloid deposition consistent with aa-amyloid. a -year old woman with leu his ttr-fap since developed progressive leg weakness, reduced ability to walk and low back pain. emg demonstrated progression of her sensorimotor neuropathy and superimposed multilevel radiculopathy. spine mri demonstrated severe lumbar stenosis requiring surgical decompression. postoperatively, she regained strength, walking more easily. congo red staining of vertebral bone demonstrated widespread positive staining with birefringence consistent with amyloid arthropathy. these cases illustrate to wide sprectrum of amyloid neuropathy presentation, an example of amyloid arthropathy-induced spinal stenosis and the utility of skin biopsy to diagnose and manage amyloid neuropathy. the differentiation between hereditary neuropathy and chronic inflammatory demyelinating polyneuropathy (cidp) in children is especially significant because of completely different treatment possibilities and prognosis in these conditions. the aim of the study was to compare electrophysiological abnormalities in a group of children and young adults with demyelinating neuropathy of chronic or subacute onset. retrospective analysis of clinical and nerve conduction study (ncs) data included children and young adults with charcot-marie-tooth neuropathy x type (cmtx ), children with charcot-marie-tooth neuropathy type a (cmt a) and children with cidp. in our study / cmtx had both axonal and demyelinating changes in ncs study. the aan and efns electrophysiological cidp criteria were fulfilled in / cmtx , / cmt a and / cidp patients. additionally patients with cmtx are classified with efns criteria as "probable/possible cidp". a distal compound muscle action potential (dcmap) was > ms in all cmt a and / cidp patients but none with cmtx . abnormal median/normal sural snap (amns) parameter was observed in // cidp and / ctmx patients but not any cmt a patient while abnormal sural/normal median snap (asnm) parameter was found in / patients with cidp. a difference between conduction velocities (cv) of two corresponding nerves > m/s was seen in / cmtx and / cidp patients but no one with ctm a. one patient with cmtx had especially conspicuous difference between nerve conduction in lower limbs, with axonal neuropathy and demyelinating features, and upper limbs with no changes. one -years-old patient with genetically confirmed cmtx had no abnormalities in ncs. electrophysiological data of cmtx , cmt a and cidp indicate diffuse demyelination, however in cmtx axonal changes coexist, seen in / patients, and asymmetrical abnormalities between corresponding upper and lower limb were observed in / patients. our study has showed that duration of dcmap is useful not only in diagnosing an inflammatory neuropathy but also in differentiating cmtx from cidp. however presence of the not homogenous abnormalities in ncs in patients with cmtx may mimic inflammatory neuropathy. prada v , mori l , francini l , accogli s , ursino g , gemelli c , schizzi s , grandis m , bellone e , mandich p , schenone a . department of neurosciences, rehabilitation, ophthalmology, genetics and maternal and child health, university of genoa, genoa, italy. the overwork weakness (ow) problem in cmt disease has been debated for long time. it has been reported that the non-dominant hand (ndh) of patients with cmt disease is stronger than the dominant hand (dh) as a result of ow and some authors verified this hypothesis using mrc on different muscles (van pomeren, ). more recently, piscosquito et al. ( ) found that the ow phenomenon does not exist using a dynamometer and the hole peg test, a dexterity test. we recruited cmt patients and healthy controls. we evaluated the strength of the -pinch and of the hand-grip with a dynamometer, the opposition ability with the thumb opposition test (tot) and applied an instrumental testing of hand function using the sensor engineered glove test (segt), which previously demonstrated its sensitiveness to measure severity of hands dysfunction in cmt patients. tot was significantly higher in the ndh compared to dh (ndh= , ± , ; dh= , ± , , p< . ) in cmt patients, instead there was no difference in dh and ndh in healthy controls. in the evaluation of -pinch and of the hand-grip, ndh performed slightly but not significantly better than the dh ( -pinch: dh= , ± , n; ndh= , ± , n; ± , n; ndh= , ± , n, p:ns) . in normal controls we confirmed the % rule (noguchi & demura, ). finally, segt results were similar between the ndh and dh in cmt patients but in normal controls there was a better performance in the dh. in conclusion, this study supports the existence of the overwork weakness in cmt, evident in every measurements. dexterity and overall ability of the hands impaired in the dh, probably because of compensating movements, compared with the healthy controls in the weaker hand. finally, our results support the importance of avoiding supramaximal exercises and educating the cmt patients to prevent incorrect movements, which may overload the hand muscles. prasad k , ohnmar o , teng a , cheng yj , umapathi t . national neuroscience institute, singapore; yangon general hospital, yangon, myanmar; yong loo lin school of medicine, national university of singapore, singapore. antecedent infections that have been linked to guillain-barré syndrome (gbs) include campylobacter jejuni, haemophilus influenza, epstein-barr virus, cytomegalovirus, mycoplasma pneumoniae and influenza a virus. in south-east asia, the burden of mosquito-borne flavivirus infections is considerable. even in a small country like singapore, with a population of only . million and in spite of excellent sanitation and public health measures, dengue virus (denv) infections can exceed , a year. both symptomatic and asymptomatic denv infections are associated with gbs. zika virus (zikv) infections were detected in singapore in late august . doctors working in south-east asia anecdotally report that gbs cases increase after rainy season when mosquitos breed. we are currently planning studies to estimate the hitherto unknown burden of gbs associated with denv, zikv and other mosquito-borne viruses in this region. as a preliminary step, we reviewed the gbs cases seen at our institution in for evidence of denv and zikv. three out of the tested cases were positive for denv serology. one of these was also positive for denv pcr. all these patients had symptomatic dengue. two had acute inflammatory demyelinating polyneuropathy (aidp) and acute motor axonal neuropathy (aman) subtype of gbs. none the patients tested were positive for zikv pcr in serum and urine. one patient, with miller fisher syndrome (mfs), had serological evidence of recent zikv. he did not have clinical zikv symptoms. in mfs cases, the initial detectable zikv igg was later proven to be due to cross reactivity with previous denv infection. in cases ( amsan, aidp, mfs) the initial zikv igm was raised. we await analysis of convalescent sera to decide if they indeed had zikv or the results were related to previous and co-infection with denv. our preliminary findings indicate that flavivirus infections may account for at least some of the gbs cases in singapore. the lack of symptoms in some cases and the interactions between zikv and denv antibodies make accurate diagnoses challenging. prior r , , benoy v , , vanden berghe p , van den bosch l , . ku leuven-university of leuven, department of neurosciences, experimental neurology and leuven research institute for neuroscience and disease (lind), leuven, belgium; vib-center for brain & disease research, laboratory of neurobiology, leuven, belgium; laboratory for enteric neuroscience, translational research center for gastrointestinal disorders, k.u. leuven, leuven, belgium. charcot-marie-tooth disease (cmt) is the most common inherited neurodegenerative disorder of the peripheral nervous system. it is divided into two main subtypes, a demyelinating subtype (cmt type ) and an axonal form (cmt type ). cmt is a length-dependent disease, in that it effects the longest neurons in the body, thus the muscles in the peripheral regions are affected first and foremost. moreover, the majority of cmt patients share a classical phenotype with shared pathological hallmarks, such as distal muscular atrophy, reduction in nerve conductions, etc. but also molecular pathological hallmarks, like the breakdown in the transport of organelles and vesicles in neurons in a process called axonal transport. currently, there is no cure or effective treatment available to cmt patients. histone deacetylase (hdac ) has been shown to be a key regulator in axonal transport. moreover, inhibiting hdac has been demonstrated to stabilize microtubules, which act as molecular tracks for motor proteins and facilitates axonal transport of cargos. our labs current focus is on cmt type a, the main cause of cmt, which is caused by a duplication of a segment of chromosome p . containing the gene encoding peripheral myelin protein (pmp ). pmp is mainly expressed by schwann cells, the cells that myelinate neurons in the peripheral nervous system. in the work presented, the commercial mouse schwann cell-line, sw cells, was transfected to overexpress pmp using a lentivirus vector system to mimic that of cmt a patient schwann cells. the overexpression of pmp was confirmed using immunofluorescence and western blot techniques. these transfected sw schwann cells were then co-cultured with primary mouse dorsal root ganglion neurons (drgs) isolated from adult mice. these co-cultures where then analysed after weeks in vitro for axonal transport. the investigated groups were: a co-culture of drgs + sw cells, a co-culture of drgs + sw cells transfected with pmp , and a co-culture of drgs + a sw cell-line transfected with green fluorescent protein using a lentivirus vector system, and a monoculture of drgs. furthermore, this work demonstrates that the overexpression of pmp in schwann cells can impair axonal transport in co-cultured drgs in comparison to the other co-culture groups. moreover, these axonal transport defects were able to be rescued by the treatment of a hdac inhibitor, tubstatin a. to conclude, selective hdac inhibitors have been shown to be a beneficial treatment for a number of cmt subtypes in preclinical studies in our lab and offer as a viable treatment for a currently, untreatable debilitating disease. provitera v , caporaso g , stancanelli a , piscosquito g , di caprio g , saltalamacchia am , santoro l , nolano m . "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; department of neurosciences, reproductive and odontostomatological sciences, university 'federico ii' of naples, naples, italy. the observation of rate and patterns of cutaneous nerves regrowth after mechanical or pharmacological distal axonotmesis, has proven to be an excellent tool to study human nerve regeneration in normal and pathological conditions. most of the observations, however, are referred to small fibers, possibly due to the availability of excellent models of reversible distal cutaneous small fiber axonopathy. no such model is available to study the regeneration of myelinated fibers in human. we studied regrowth of cutaneous large fibers in fingertip of patients with carpal tunnel syndrome after surgical decompression. we recruited patients (m/f / ; age . ± . years) with carpal tunnel syndrome candidate to surgery. patients underwent clinical and electrophysiological evaluation, quantitative evaluation of discriminative threshold at rd fingertip. in the same site, patients also underwent mm punch skin biopsy. skin sections were stained by immunohistochemical techniques and cutaneous innervation was analyzed by confocal microscopy. meissner corpuscles and their myelinated afferences were assessed following previously published procedures. twelve months after surgery, patients repeated functional evaluation and underwent a second skin biopsy two mm apart from the first one. mean density of mcs/mm was . ± . at time and . ± . at follow-up, mean density of myelinated endings was . ± . at time and . ± . at follow-up. however, not in all patients regeneration occurred. based on the variation of mc density between time and follow-up we were able to identify patients in which active regeneration had occurred. in this subgroup mean density of mcs/mm was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . in the same group, mean density of myelinated endings was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . we describe morphological patterns associated to nerve regrowth in the biopsies of patients in which nerve regeneration occurs. we propose an in vivo model to study regeneration of large myelinated fiber endings in human skin. in addition to the count of nerve endings, the identification of patterns associated to nerve regeneration can increase the diagnostic yield of skin biopsy. the most common type of charcot-marie-tooth disease is caused by a duplication of pmp , leading to dysmyelination, axonal loss and progressive muscle weakness (cmt a). currently, no approved treatment for cmt a patients is available. among others, preclinical therapeutic approaches aim to correct the pmp overexpression in order to ameliorate axonal loss and muscle weakness. we previously reported that the novel polytherapeutic drug pxt , a low-dose combination of baclofen, naltrexone and sorbitol, improved dysmyelination and axonal loss after long-term application in adult pmp transgenic rats, a known animal model of cmt a. interestingly, after short-term application in young cmt a rats we observed a long-lasting prevention of muscle weakness as well but without obvious effects on dysmyelination and axonal loss. improved distal motor latencies in the electrophysiological recordings and a shift in the axonal diameter distribution raised the hypothesis that therapy may improve the neuromuscular endplate pathology as another therapeutic target for cmt a. here, we report a preclinical trial in adult cmt a rats treated from postnatal week for consecutive weeks as the most effective regimen in order to facilitate a deeper understanding of the mode-of-action of pxt . long-term therapy effects were confirmed by an ameliorated behavioral phenotype, improved distal motor latencies and also nerve conduction velocity in the electrophysiological recordings. histological analysis revealed an increased number of neuromuscular endplates, which were lowered to wildtype level after pxt treatment. further investigations include detailed analysis of peripheral nerve myelin thickness, internodal and nodal length, terminal axonal sprouting at the neuromuscular endplate and muscle fiber subtyping in treated cmt a rats. charcot-marie-tooth disease (cmt) is a rare hereditary neuropathy for which novel treatments are being developed and urgently needed. only few clinical trials and natural history studies have been performed. evaluation of intervention efficacy is hampered by slow progression and lack of sensitive outcome measures. the interindividual disease variability is high and prognostic disease measures are not established for cmt patients. previously, we have identified skin-derived disease and progression biomarkers in a large european and us-based cmt a cohort. within the german charcot-marie-tooth disease network (cmt-net, www.cmt-net.de) we aim to establish easier accessible biomarkers in blood of cmt a patients. blood analysis is less invasive and can be repeatedly performed during clinical trials and routine patient care. we have started clinically assessing approximately young, adolescent and adult cmt a patients at clinical centres (münster, münchen, göttingen) including a large set of clinical outcome measures (cmtnsv , onls, mwt, mwt, walk , hpt, sf- , fss, psqi, ess, bdi-ii). we will sample blood and skin tissue once a year at , and months for an observational period of years. tissue samples will be analyzed on the transcriptional, translational and epigenetic level. we envision that the diagnostic and progression biomarkers may be used to measure therapeutic effects within clinical trials and later in clinical routine monitoring. this is the first trial testing "circulating" biomarkers from blood in a prospective observational trial in cmt a patients. igm peripheral neuropathy is a slowly progressive and heterogeneous disease with symptoms ranging from mild foot numbness and minor imbalance to severe neuropathic pain and sensory and motor dysfunction. international consensus regarding assessment and treatment of patients with igm peripheral neuropathy is lacking. this might be caused by the repeated use of suboptimal outcome measures, the small trial sizes with low numbers of treated patients, the indolent disease course needing a longer follow-up period to capture relevant changes, or the possibility that administered treatments were not aggressive enough. the imagine study is an international, multi-centre, prospective, observational cohort study, which will result in a unique collection of a large number of prospectively collected and highly standardized clinical data, and a biobank from a large population of well-defined patients with igm peripheral neuropathy. the main objective is to describe in detail the variation in clinical subtypes, clinical disease course, past and current practice of treatment, antibody titres, and clinical picture at the various levels of assessing outcome. patients being at least years, and fulfilling the international criteria for igm peripheral neuropathy are eligible. exclusion is primarily based on concomitant diseases influencing peripheral nerve function. several study parameters measuring weakness, sensation, activity and participation, ataxia, pain, and quality of life are of interest. in february an enmc kick of meeting is organized, bringing together an igm peripheral neuropathy study group. the meeting will focus on the development of a core set of outcome measures to be used in future studies in igm peripheral neuropathy. the imagine study started in the netherlands in september , and to date patients were included. the imagine study starts in the united kingdom, italy, and france in the beginning of . centres recruiting at least patients with igm peripheral neuropathy are eligible to participate in the study. during the pns global recommendations from the enmc meeting, as well as the first results of the imagine study, will be presented. puget s , paolantonacci p , burlot l lfb biomédicaments, les ulis, france; lfb biotechnologies, les ulis, france. in dysimmune neuropathies patients (mmn, cidp, gbs), ivig have become the gold standard due to their efficacy and tolerability. acute renal impairment, a rare but serious adverse event, can be induced by all ivig. its incidence is not determined precisely for each ivig. it mostly occurs in patients at risk, with pre-existing conditions (prior renal insufficiency, diabetes, mellitus, arterial hypertension, elderly > years old, dehydratation, hypervolemia, sepsis, paraproteinemia or concomitant use of other nephrotoxic drugs) and treated by immunomodulatory doses. as the number of these patients increases over the years, the choice of ivig in association with the precautionary measures will be decisive to reduce or avoid the occurrence of this adverse event. from s to s, the nephrotoxicity of ivig has been explained by tubular toxicity related to osmotic nephrosis. sucrose, a stabilizer of some ivig, has been implicated as one of the causes of this. even if several new ivig without sucrose are used, renal impairment cases have been described and studies have highlighted that sucrose is not the only ivig stabilizer associated with tubular damage. however, in a recent study, renal impairment cases ( / ) have occurred in cidp elderly patients treated by free-sucrose ivig. the occurrence of renal failure seemed to be low (only / ) for patients treated with sucrose-ivig in association with precautionary measures (hydratation, adaptation of dosage according body mass index, rate infusion). otherwise, since s, cases of renal impairment secondary to hemolysis have been increased with some ivig but would be rare for full ethanolic fractionated products as they have been identified to generate very low occurrence of hemolysis. the occurrence of renal impairment related to ivig seems to be multifactorial, need to find out all causes (mechanisms of different stabilizers, hemolysis, and concentration of ivig). registries would be helpful for physicians to define the incidence, relative risk of ivig-related renal impairment and the right ivig in association with precautionary measures in these patients at risk. the published ultrasound (us) studies on chronic inflammatory demyelinating polyradiculoneuropathy (cidp) report diffusely increased cross-sectional area (csa) of nerves. these data have not been correlated with histological patterns. to date, no further information about us ultrastructural nerve modification has been provided because of limited frequency range ( - mhz) of the us probe. the aim of this preliminary study is to evaluate the correlation between histological findings from sural nerve biopsies and us patterns found with uhfus ( mhz) from sural nerve at the ankle. four patients with cidp underwent uhfus nerve evaluation of clinically affected sural nerve before undergoing a sural nerve biopsy. us findings included: cross-sectional area of the nerve, connective tissue depth and changes in echogenicity of fascicles. those data were then compared to the histological findings obtained in transversal and longitudinal sections. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened and in two of the four patients hyperechogenicity of fascicles was observed. also, thanks to the ultra-high-resolution of the probe, a direct correlation of the histological and us images was possible, so as a direct measurement of internal microstructure of the nerve. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of sural nerve can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue, as confirmed by the histological study. in particular, we have observed a hyperechogenicity of fascicles in most severe patients; in those cases, histology confirmed an increase of endoneurial depth. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed and will be presented during the meeting. puma a , cambieri c , panicucci e , desnuelle c , raffaelli c , sacconi s . high-resolution ultrasound (hfus, mhz) of peripheral nerves is a valuable non-invasive, painless complement to neurophysiology, especially in the workup of cidp. nevertheless, the current spatial resolution of echographic images doesn't allow a detailed study of the nerves. ultra-high frequency ultrasound (uhfus, mhz) is a new tool with a - times higher spatial resolution than traditional hfus. the aim of this preliminary study is to evaluate sensory and motor nerves structural characteristics found with uhfus in patients with definite cidp. seven patients with cidp underwent uhfus nerve evaluation of median, ulnar, peroneal and sural nerves, bilaterally. us findings included: cross-sectional area of the nerve, connective tissue depth, nerve vascularization and changes in echogenicity of fascicles. patients also underwent electroneurography (eng) and plexus mri. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened. two of the seven patients presented an epineural hypervascularization, observed at the doppler evaluation; none of them was treated by iv immunoglobulines. echographic changes were present even in the absence of mri abnormalities (root hypertrophy). eng characteristics correlated with the us patterns. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of nerves can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue. in particular, uhfus allows a more detailed study of nerves, demonstrating that the structural abnormalities hit connective tissue, while -conversely to previous studies -fascicles anatomy seems to be spared. we did not show nerve vascularization except in non-treated patients. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed. purcell l , , wojciechowski e , , gibbons p , , jamil k , , menezes, m , , burns j , . sydney children's hospitals network (randwick and westmead), new south wales, australia; university of sydney, new south wales, australia; universiti kebangsaan, kuala lumpur, malaysia. - % of children with cmt are reported to have hip dysplasia. we aimed to investigate the relationship between radiological hip dysplasia indicators and walking pattern in children with cmt. thirty children ( female; . ± . yrs; ± . cm; ± . kg cmt a, cmtx , cmtx , cmt f, cmt a) underwent d gait analysis ( dga), and had an anterior-posterior pelvis radiograph within months of assessment. radiographs were reviewed by two orthopaedic surgeons, and the reliability of measures of dominant limb hip health via radiograph was assessed. of the measures, measures had an intraclass correlation coefficient > . between raters, and the more experienced surgeon's measures were used for further analysis. the measures of acetabular index (ai), centre edge angle (cea), neck shaft angle (nsa), medial joint space, head width, lateral uncoverage, migration percentage and triradiate were used to investigate correlations with kinematic and kinetic dga variables of the pelvis and dominant limb hip, knee and ankle in planes, temporal spatial parameters and gait profile score. dga data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model. gait data were compared to typically developing children ( female; . ± . yrs, ± . cm, ± . kg). five of affected children had a migration percentage > ∘ , representing % of our sample. maximum hip abductor moment in terminal stance was significantly lower than normative reference values, and was moderately correlated with of the radiographic measures (ai r=− . , p= . ; nsa r=− . , p= . and medial joint space r=− . , p= . ). walking speed (normalised to leg length) was correlated with medial joint space (r= . , p= . ) head width (r=− . , p= . ) and triradiate (r=− . , p= . the ganglionopathies are a unique group of disorders with a varied etiology which includes autoimmune disorders, paraneoplastic syndromes and toxin induced causes. it has been observed that despite extensive investigations the cause of a sensory neuronopathy is often idiopathic in approximately - % cases. electrodiagnostic criteria to diagnose a possible ganglionopathy requires at least one sensory action potential absent or three sensory action potentials < % of the lower limit of normal in the upper limbs plus less than two nerves with abnormal motor nerve conduction study in the lower limbs. ulnar sensory-motor amplitude ratio values (usmar) lower than . is useful in differentiating ganglionopathy from axonal length-dependent neuropathy. we performed a retrospective analysis of patients who were either admitted or evaluated in the outpatient department in department of neurology, nizam's institute of medical sciences, with a possible ganglionopathy as per the diagnostic criteria proposed by camdessanchéet al. consecutive patients who sought treatment during a year period with a clinical pattern of sensory neuronopathy were analysed retrospectively. we reviewed the electrodiagnostic studies of patients and calculated the ulnar sensory-motor amplitude ratio values in comparison with patients with a clinical and electrophysiological diagnosis of diabetic axonal sensorimotor neuropathy. the clinical profile of a ganglionopathy from the indian subcontinent comprised of a prominent sensory ataxia as the initial presenting clinical manifestation with a predilection towards involvement of large-fiber sensory modalities. electrophysiologically a pattern of absent snaps was characteristically encountered with a significant value of usmar less than . , enabling differentiation from axonal length-dependent neuropathy. rahman im , jahan it , nahar s , khalid mm , jahan i , hayat s , , , islam z . guillain-barré syndrome (gbs) is a post-infectious autoimmune polyradiculoneuropathy where immune response is triggered by molecular mimicry between glycolipid antigens expressed by an infective agent such as campylobacter jejuni (c. jejuni) and human peripheral nerve gangliosides. cd molecules are mhc-like structures specialized in capturing and presenting a variety of glycolipids to antigen-specific t lymphocytes. the objective of this study was to investigate the possible role of coding region polymorphisms of cd genes in the pathogenesis of gbs in bangladeshi population. single nucleotide polymorphisms (snps) of exon of cd a (* /* ) and cd e (* /* ) in well defined gbs patients and healthy controls were studied to delineate their effect in developing gbs. we genotyped exon of both cd a (cys/tryp) and cd e (gln/arg) genes through pcr-rflp. to validate these findings, direct sequencing of pcr product was performed for at least samples for each position. we found no differences in genotypes and allele frequencies of both genes in gbs patients compared to controls. however, compared to control individuals with cd a* /cd e* haplotype were . times more likely to develop gbs, whereas individuals with cd a* /cd e* haplotype had a reduced relative risk by . times. a positive association of cd a* /cd e* haplotype was observed only in axonal form of gbs ( . % vs. . %, p = < . ). haplotype cd a* /cd e* was prevalent among anti-gm antibody positive gbs patient compared to anti-gm antibody negative patients ( . % vs. . %) though it was not statistically significant. snps in cd a and cd e were not associated with antecedent c. jejuni infection, disease severity and disease outcome at months of follow up. in conclusion, cd polymorphisms are not a susceptibility or disease causing factor in gbs. conversely, increasing knowledge of this field may offer new dimension for the research to elucidate better answer for disease pathogenesis and also contribute to conduct high power meta-analysis. extensive genetic testing was performed on a unique patient, who received national media attention due to her severe cmt phenotype. the index patient was never able to walk independently, had difficulty breathing and swallowing and demonstrated aspiration by a barium swallow study. nerve conduction velocity testing revealed velocities of m/s in her upper extremity. she required continuous positive airway pressure (cpap) for an upper respiratory infection and developed bilateral vocal cord paralysis. we used the combined brief assessment of motor function (bamf) scale to evaluate her disability. her fine motor scale was a / , her upper extremity gross motor scale was a / and her lower extremity gross motor scale was / . unfortunately, she died at age in her sleep, presumably from respiratory arrest. the proband's father was asymptomatic, however, his neurological exam showed pes cavus bilaterally and mild atrophy of the first dorsal interossei muscles in his hands. his nerve conduction velocities were slowed to m/s in the upper extremities and his cmt neuropathy score (v ) was / . we performed whole exome and subsequently whole genome sequencing in the trio. comprehensive structural variant analysis for copy number variations, large deletions, and recombinations was completed by a combination of software tools. known cmt genes were excluded as the underlying cause and the only viable candidate gene remaining was sh tc . we showed expression of sh tc in peripheral nerve and schwann cells. sh tc is a paralogue to sh tc , which causes the recessively inherited dysmyelinating form cmt c. sanger sequencing confirmed the variants in our family, p.v m (c. g>a, chr : ) and p. a p (c. g>c, chr : ) , and showed the segregation of the heterozygous variations. we hypothesize that the heterozygous paternal allele had a minimal effect and let to a very mild or subclinical form of peripheral neuropathy. of interest, the recessive sh tc gene has also been shown to cause mild aberrant forms of peripheral nerve degeneration in carriers of heterozygous disease alleles. in summary, this paper will present genetic and molecular evidence from an extensive n= study proposing a novel cmt gene. charcot-marie-tooth (cmt) is an important cause of morbidity worldwide. it is a heterogeneous disease manifesting as progressive weakness, wasting and loss of feeling in a length-dependent pattern. there are an increasing number of cmt-related genes in the literature. more recently, recessive mutations in the hint gene have been reported as causative of predominant motor axonal neuropathy associated with neuromyotonic discharges on emg. one of the characteristics of autosomal recessive cmt is it varying frequency in different populations and ethnic groups. we sought to evaluate the frequency of mutations in the hint gene in a brazilian cohort with axonal hereditary neuropathy. all patients included in this study born within south east area of brazil. the group consists of consecutive patients with axonal neuropathy screened for recessive or sporadic axonal neuropathy in the neurogenetics laboratory of clinical hospital of ribeirão preto. direct sequencing of the coding region of hint gene was done. among patients screened, were suspected of having recessive axonal neuropathy without neuromyothonic discharges, and two have axonal neuropathy associated with neuromyothonic discharges on emg. we did not find any disease causing mutations among our patients. some previously reported studies reported a high frequency of mutations in the hint gene among recessive axonal neuropathies in some european countries. our results demonstrated that frequencies of mutation underlying genetic hereditary neuropathies are different between different ethnic groups and have implications for the organization of services management of cmt, for genetic counseling, and for gene flow in different world populations. funded by cnpq, fapesp, faepa, pronas (ministry of health). rodriguez-menendez v , ballarini e , malacrida a , , ceresa c , semperboni s , , meregalli c , cavaletti g , nicolini g . school of medicine and surgery, experimental neurology unit, university of milano-bicocca, monza, italy; phd program in neuroscience, university of milano-bicocca, monza, italy. bortezomib (btz) is a proteasome inhibitor widely used for multiple myeloma treatment. btz induced peripheral neuropathy (bipn) is the most frequent adverse effect. bipn in humans, is a dose dependent painful sensory neuropathy characterized by nerve axonopathy and a tendency to recover in the follow-up period after btz withdrawal. bipn affects principally dorsal root ganglia (drg) and different rodent models have shown alterations in sensory neurons, small unmyelinated axons, large myelinated axons, axonal mitochondria and schwann cells. in this work, we evaluated the effects of btz in vitro on drg neurons isolated from adult mice. our interest focused on dystonin, a protein able to interact with all the three components of cytoskeleton (microtubules, microfilaments and intermediate filaments) and able to bind map b (a mt-associated protein) through which can influence golgi apparatus morphology. there are different neuronal isoforms of dystonin and in particular dystonin-a is able to modulate tubulin acetylation and stability through interaction with map b. it is noteworthy to underline that map b expression in the mature nervous system is restricted to sensory neurons. western blot analysis demonstrated that a treatment with nm btz induces a statistically significant decrease in tubulin acetylation. this result does not go along with our previous study where we observed a tubulin polymerization increase after btz treatment. therefore, we have decided to focus our interest on soma organelle organization that is well known to be dependent from cytoskeleton structure. immunofluorescence images showed an altered distribution of acetylated tubulin in soma cytoplasm after hour treatment. moreover, confocal analysis of cis golgi (gm ) demonstrated that in btz treated neurons the normal golgi structure is lost showing a spot-like non perinuclear labeling distribution. additionally, dystonin distribution seemed comparable to that of cis golgi apparatus suggesting that btz could induce a change in dystonin localization which in turn affects golgi organization, probably through map b.these data suggest that the cytoskeleton alterations, induced by btz, could probably cause wrong maturation and trafficking alteration of golgi vesicles consequently impairing the correct sensorial function. romão tt , aleixo bfl , wedemann d , herculano fgn , prado h , cal h , pupe c , bittar c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. acute motor and sensory axonal neuropathy (amsan) is a clinical variant of guillain-barré syndrome (gbs) which has rarely been reported in association with human immunodeficiency virus (hiv) and neurosyphilis. we describe a case of a -year-old woman who started with muscle weakness in the left leg followed by weakness in the right leg and in upper limbs in one month. motor symptoms were followed by urinary and fecal incontinence. no preceding symptoms, as diarrhea or fever were referred. neurological examination showed global areflexia with impairment of vibratory and tactile modalities, as well as loss of proprioception. pinprick sensation was moderately compromised. no impairment of higher mental functions was observed. other causes of neuropathies were ruled out. cerebrospinal fluid (csf) examination showed cell/mm , increased protein level, normal glucose level and negative vdrl test. blood analysis showed hiv and positive serology tests, vdrl : and positive t. pallidum hemagglutination assay. igm cytomegalovirus antibody was negative, cd was in normal range and hiv load was undetectable. cranial and spinal cord magnetic resonance imaging revealed extensive involvement of the posterior spinal cord tracts which supports the diagnosis of neurosyphilis. nerve conduction studies showed a significant reduction in compound motor action potentials amplitudes, particularly at lower limbs, and absent sural nerve responses. electromyography revealed positive waves and fibrillation at lower limbs. treatment with methylprednisolone, human intravenous immunoglobulin and ceftriaxone has been settled, resulting in improvement of upper limbs paresis and sphincters dysfunction. antiretroviral therapy was initiated. this amsan case is associated with central nervous system involvement, encompassing an encephalomyeloradiculopathyrelated to hiv/t. pallidum co-infection. c.jejuni is the main etiological agent associated with amsan, but there are some reports of hiv/cmv co-infected patients who presented this gbs variant. facing a patient with amsan, mainly when cns involvement is present, it is of crucial importance to undergo investigation of an ongoing infectious process, such as hiv and syphilis, considering the impact of early treatment on prognosis. roodbol j , , yiu e , doets a , , de wit mcy , monges ms , van nes s , catsman-berrevoets ce , van den berg b , , jacobs bc , and on behalf of the igos-kids consortium. only a few prospective cohort studies so far have investigated the clinical presentation and course of guillain-barré syndrome (gbs) in children using age-adjusted outcome scores. in we started a study based on the igos protocol but adjusted for children with gbs (igos-kids). (pediatric)neurologists from argentina, australia and the netherlands joined forces to determine the clinical and biological determinants of disease progression and recovery in gbs in children from different geographical areas. for each age category specific pain scales are used including the comfort score for children < years old, pain faces revised age - years old and numerical rating scale in children ≥ years old and outcome measures. a new strength score was developed: gbs kids score. this score will be used and validated in addition to the mrc-sum score. the onls, r-ods and fss are also be used in igos-kids. age-dependent quality of life questionnaires were added to the igos-kids protocol, namely the pedsql and the pedsql multidimensional fatigue scale. these scales are available for children ≥ years old. an activity score validated for neuromuscular disorders for children ≥ years old was also added. currently there are children included in igos-kids, eight children from australia and five from the netherlands. additional clinicians and researchers interested in gbs in children are most welcome to participate in igos-kids. prognostic models have been developed to predict the highly variable clinical course of guillain-barré syndrome (gbs) in adults. the clinical course of gbs is equally variable in children, but the current prognostic models have not been validated in children. in this study, we aimed to identify in children with gbs the characteristics at hospital admission that predict the clinical severity and outcome. the study was conducted in two patient cohorts from europe: one (largely) german cohort of children and one dutch cohort of children. clinical information was obtained regarding preceding infection, first symptoms, neurological deficits at admission and nadir, results of additional investigations (cerebrospinal fluid and nerve conduction studies). clinical severity, course and outcome was defined by the gbs disability score, especially the ability to walk unaided, at a follow-up of month, months, months and months after onset of symptoms. univariate and multivariate regression analyses were performed initially on the two separate cohorts. combined the cohorts consisted of children (age median years, range - years) including boys ( %). the median duration between onset of symptoms and hospital admission was days (range ). pain at admission was remarkably frequent and present in ( %) children. in the combined cohorts, children ( . %) developed respiratory failure and one child died. multivariate regression analysis showed that in both cohorts strong predictors of respiratory failure available at hospital admission were cranial nerve involvement, a higher gbs disability score and a shorter period in days between onset of symptoms and admission. this information was used to develop and validate a prognostic model for children with gbs that will be presented at the conference. in conclusion, based on the analysis of two independent cohorts of patients the predictors of respiratory failure and clinical recovery were identified and validated. similar factors were identified for adult patients, although the prognosis in children in general is better than in adults. this information will be used to develop a simple prognostic model for current clinical practice to predict the chance of respiratory failure and outcome in children with gbs. the negative trials of vitamin c in cmt a have highlighted the lack of sensitive outcome measures in charcot-marie-tooth disease (cmt). neurofilaments are abundant neuronal cytoskeletal proteins and their concentration in blood is likely to reflect axonal breakdown. we therefore examined plasma neurofilament light chain (nfl) concentration as a potential biomarker in cmt. blood samples were collected from patients with cmt and age matched healthy controls over a -year period. disease severity was measured using the weighted cmt examination and neuropathy scores. plasma nfl concentration was determined using an in house developed simoa assay based on the nfl antibodies from the nf-l light elisa kit (umandiagnostics). plasma nfl concentrations were significantly higher in cmt patients (median: . pg/ml; iqr: . - . ) than in controls (median: . pg/ml; iqr . - . , p< . ) and correlated with disease severity as estimated by the weighted cmt examination (n= , r= . , p< . ) and neuropathy scores (n= , r= . , p= . ) . concentrations were also significantly higher when subdividing cmt patients by genetic subtype; cmt a (n= , p< . ), sptlc (n= , p< . ) and gjb (n= , p= . ) or into demyelinating, cmt (n= , p< . ) or axonal, cmt (n= , p< . ) forms compared to healthy controls. there was no significant difference in the plasma nfl concentration after year in patients with cmt (n= , mean difference − . pg/ml, % confidence interval − . - . ) or healthy volunteers (n= , mean difference − . pg/ml, % confidence interval . - . ) which is unsurprising as cmt is a slowly progressive disease in which the rates of axonal degeneration are likely to be constant and elevated. in summary, we have shown that plasma nfl levels are significantly raised in patients with cmt and that they correlate with disease severity. this is of relevance not only for the field of cmt but for peripheral neuropathies in general, and suggests that plasma nfl holds promise as a biomarker of peripheral neuropathy in both routine practice and clinical trials. neurolymphomatosis (infiltration of the peripheral nervous system by lymphoma cells) is a rare and usually devastating condition belonging to the spectrum of lymphoma-associated neuropathies. cerebrospinal fluid examination with cytologic examination, flow cytometry and clonality testing by pcr may show malignant cells especially when nerve root involvement is prominent. however, nerve biopsy remains a useful tool to confirm the presence of malignant cells invading the nerve. neuropathological features are important and pcr-based immunoglobulin or t-cell receptor clonality testing on nerve fragments may add notable value for the diagnosis of neurolymphomatosis, although this has not been systematically investigated. we retrospectively studied clinicopathological data and clonality results of nerve biopsy samples in patients with nl from centres, performed between and . among patients with nl, we found % of b-cell lymphoma and one t-cell lymphoma. nl was the first manifestation in , % of patients. the main clinical pattern was symmetrical progressive sensorimotor polyneuropathy in % of patients and pain was a prominent feature in %. clonality testing showed a monoclonal rearrangement in ( %) cases, oligoclonal rearrangement and no amplification in ( %). the main histological pattern was perivascular infiltration of predominant b-cells in the epineurium, without signs of vasculitis. the extent of axon loss was highly variable between patients. various chemotherapeutic regimens were used and the median overall survival was months. only one case showed a monoclonal pattern among control nerve samples from patients with other types of neuropathy including vasculitis and cidp. overall, we found that clonality testing on nerve biopsy specimens may provide decisive information on the presence of neurolymphomatosis, with a sensitivity of . %, a specificity of . %, a positive predictive value of . % and a negative predictive value of . %. we confirm the utility of nerve biopsy for the diagnosis of nl and show the great yield of pcr-based clonality testing to assess the malignant feature of peripheral nervous system lymphoid infiltrates. despite an accurate diagnosis, neurolymphomatosis still remains a devastating disease with an overall poor prognosis. in the last years, cumulative data have shown that patients with amyotrophic lateral sclerosis (als) and mouse models of the disease present loss of small epidermal and dermal nerve fibers and sensory dysfunctions, in addition to the classical motor symptoms. our objective is to characterize this small fiber neuropathy and to clarify if axonal loss involves all sort of fibers equally, or if there is some specificity. for this purpose, we performed an immunohistochemical characterization of total intraepidermal nerve endings (protein gene product; pgp . ), peptidergic (calcitonin gene-related peptide; cgrp) and nonpeptidergical nerve epidermal endings (isolectin b ; ib ) of the sod g a mouse at different stages: presymptomatic stage ( weeks), disease onset ( weeks) and in symptomatic stage ( weeks). the sympathetic sweat gland innervation was immunolabeled for vasoactive intestinal peptide (vip) from very early stage ( weeks) to disease onset ( weeks). the results showed a marked reduction of the intraepidermal nerve fibers already in the presymptomatic stage compared to the wildtype littermates (p< . ). this axonal loss affected more markedly the nonpeptidergic axons from the disease onset stage ( % axonal loss, p< . ), whereas no significant differences were found in the cgrp positive fibers ( . % axonal loss). a reduction of the sympathetic innervation of the sweat glands was also found from the disease onset stage ( % axonal loss, p< . ). in summary, we have found that nonpeptidergic and sympathetic innervation of the skin are predominantly affected in the sod g a mouse model. these findings suggest that this specificity could be used as an accessible biomarker for the disease. hereditary and inflammatory neuropathies are peripheral nerve disorders of different pathophysiology whose identification is crucial for therapeutic approach. diagnosis of cmt is easy when there is a family history, disease course is slowly progressive, neurophysiological findings are homogeneously abnormal. cases of cmt patients with inflammatory-like phenotypes leading to a misdiagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described. the presence of "red flag signs", such as slowly progressive decline, mild sensory symptoms, minimal electrophysiological progression and no response to therapy should prompt re-evaluation of the diagnosis of cidp. the introduction of nerve neuroimaging has contributed to the diagnostic work-up. we describe cmt patients ( men, women, mean age . ± . yrs, mean neuropathy duration . ± . yrs) with genetically confirmed cmt, who were initially diagnosed with cidp. neurophysiology showed demyelinating features in patients, the remaining patients had axonal features. csf analysis showed albumin-cytological dissociation in patients, oligoclonal igg bands were present in patient. nerve ultrasound in patients with demyelinating neuropathy showed diffuse increased cross-sectional area. mr-neurography in one patient confirmed diffuse nerve hypertrophy and increased signal intensity, supporting the hypothesis of a overlap syndrome. all patients were treated with immunomodulatory therapies. among patients with demyelinating features, patients underwent ivig for months, without benefit; the remaining were treated with steroids, showing temporary improvement. the patients with axonal neuropathy complaining of a progressive history, underwent ex adjuvantibus plasma exchange and ivig, without benefit. given the lack of benefit from therapies, screening for hereditary neuropathies was performed. among patients with demyelinating neuropathy, had cmt a, cmt b, cmt d. among the patients with axonal neuropathy, one was diagnosed with cmt k. in patients an overlap syndrome was present. several clinical and laboratory features (csf protein elevation), might contribute to the misdiagnosis of cidp in cmt patients. refractoriness to immune-modulatory treatment should rise the suspicion of a hereditary neuropathy. an overlap cmt/cidp syndrome may be considered, in front of acute/subacute deterioration and/or proximal muscles involvement. two patient with an overlap syndrome showed benefit after either steroids or ivig. ruiz m , tripodi sm , campagnolo m , zara g , salvalaggio a , ruggero s , toffanin e , anglani m , briani c . neurology, department of neuroscience, university of padua, padua, italy; neuroradiology, padua hospital, padua, italy. facial diplegia is a rare regional subtype of guillain-barrè syndrome characterized by rapidly progressive bilateral facial palsy in absence of other cranial neuropathies, ataxia or limb weakness. the diagnosis is based on history, clinical examination, laboratory data. the outcome of this variant appears to be better than that of classical gbs. although about % of gbs patients clinically exhibit facial nerve involvement, there are only few reports that demonstrate mri gadolinium facial nerves enhancement. a -year-old previously healthy man sought neurological advice for the acute onset of right perioral paresthesias and hyposthenia at the extensor hallucis longus bilaterally. in the previous month, during a trip to china and japan, he had suffered from gastroenteritis. at neurological examination he presented with bilateral extensor hallucis longus hyposthenia. ankle jerk reflexes were absent. one week later, he developed bilateral facial palsy. neurophysiology disclosed absence of activity in the facial nerves innervated muscles bilaterally and signs of mild partial denervation at right l metamer. csf analysis showed increased protein levels ( mg%) and . leucocytes/ l. serological tests for infectious agents were negative and serum levels of angiotensin converting enzyme were normal. antibodies to monosialoganglioside gm of igg isotype were positive ( / ). t brain mri showed, after gadolinium administration, marked bilateral enhancement of the facial nerves in their extra-and intra-canalicular segments. t lumbosacral mri scan ruled out the presence of disc diseases as well as signal modifications within conus medullaris and cauda equine nerve roots. the patient was treated with iv immunoglobulins ( . g/kg/die for days) with benefit on facial weakness. at follow-up examination months later, the patient presented a further improvement of the facial diplegia and neurophysiology disclosed a partial recovery of activity in the facial nerves, with persistent axonopatic damage. bilateral extensor hallucis longus hyposthenia persisted. in conclusion, we report on a regional subtype of guillain-barrè syndrome with the curious association of facial diplegia and bilateral extensor hallucis longus hyposthenia, and igg anti-ganglioside antibodies, that are not commonly described in facial diplegia. moreover, we provide t brain mri evidence of facial nerve involvement. rumora ae , tabbey ma , lograsso g , dolkowski j , haidar j , lentz si , feldman el . department of neurology, university of michigan, ann arbor, usa; department of internal medicine, division on metabolism, endocrinology and diabetes, university of michigan, ann arbor, usa. diabetic neuropathy (dn) is the most common complication of diabetes affecting up to % of diabetic patients. the pathogenesis of dn in type diabetes is directly related to the metabolic syndrome associated with the western diet composed of elevated levels of long chain saturated fatty acids (sfas) and low levels of medium chain sfas. long chain sfas are associated with insulin resistance and dyslipidemia while medium chain sfas have been associated with decreased lipid accumulation and improved mitochondrial function. since dn is primarily a disorder of the sensory dorsal root ganglion (drg) neurons, we sought to evaluate the impact of sfa hydrocarbon chain length on mitochondrial trafficking mechanisms that are critical for distributing atp throughout drg axon to provide energy for synaptic transmission. we hypothesize that sfas with longer hydrocarbon chains will impair mitochondrial trafficking whereas medium length sfas will not impact mitochondrial movement along the drg axon. in this study, we examined the impact of sfa hydrocarbon chain length on mitochondrial trafficking, directionality and velocity in primary mouse drg neurons. drg neurons were treated with increasing concentrations of long chain sfas, stearate and palmitate, or medium chain sfa, laurate, ranging from . to micromolar for twenty-four hours. drg neurons treated with long-chain sfas, palmitate and stearate, showed a significant decrease in the percentage of motile mitochondria whereas medium chain sfa, laurate, does not alter mitochondrial motility. we next assessed whether motile mitochondria in drg neurons treated with palmitate or stearate exhibited altered directionality or velocity of mitochondrial trafficking. palmitate and stearate treatments resulted in a trending decrease in the number of mitochondria trafficking in both anterograde and retrograde directions. furthermore, . to micromolar palmitate and stearate induced a significant decrease in mitochondrial velocity. laurate treatment, on the other hand, retained directionality and velocity of mitochondrial trafficking. these results suggest that hydrocarbon chain length of saturated fatty acids plays an important role in regulating mitochondrial trafficking mechanisms in dorsal root ganglion neurons. impaired mitochondrial trafficking in drg neurons exposed to elevated levels of long-chain sfas may play a critical role in the progression of dn. dyslipidemia is a critical factor that contributes to the development of diabetic neuropathy (dn). the progressive nerve damage associated with dn correlates with the dyslipidemic state characterized by elevated circulating levels of harmful saturated fatty acids (fas) and low levels of beneficial unsaturated fas. in dn, primary sensory dorsal root ganglion (drg) neurons exhibit energy dyshomeostasis and mitochondrial dysfunction, however, little is known about the differential impact of saturated and unsaturated fas on mitochondrial mechanisms in drg neurons. mitochondrial trafficking is an essential mechanism for transporting mitochondria throughout drg axons to provide cellular atp for neuronal function and neurotransmission. since mitochondrial trafficking is regulated by metabolic flux, we sought to determine whether saturated fa, palmitate, and unsaturated fa, oleate, have differential effects on mitochondrial trafficking in drg neurons. we evaluated mitochondrial trafficking patterns and the mitochondrial membrane potential (mmp) in primary drg neurons treated with physiologically relevant concentrations of palmitate, oleate, and combinations of both fas. primary drg neurons treated with . to micromolar palmitate induced a significant and dose-dependent reduction in the percentage of motile mitochondria. these palmitate treatments also induced a dose-dependent reduction in mitochondrial velocity but did not impact the directionality of mitochondrial movement. alternatively, . to micromolar oleate treatments did not impair the percent of motile mitochondria, the direction of mitochondrial movement, or mitochondrial velocity. since palmitate and oleate have differential effects on mitochondrial motility, we next assessed whether oleate could counter the inhibitory effect of palmitate on mitochondrial trafficking. surprisingly, oleate/palmitate mixtures at ratios of : or : prevented palmitate-induced impairment of mitochondrial trafficking. we assessed the impact of palmitate and oleate on mmp by staining palmitate and oleate-treated drg neurons with tetramethylrhodamine methyl ester. drg neurons treated with micromolar palmitate exhibited an increase in the percentage of depolarized mitochondria while mitochondria in oleate-treated drg neurons retained mmp. interestingly, drg neurons treated with : oleate/palmitate mixtures also maintained polarized mitochondria. these results suggest that saturated and unsaturated fas have a distinct impact on mitochondrial trafficking mechanisms in drg neurons and that equimolar ratios of oleate/palmitate can prevent impairment of mitochondrial trafficking. of g alpha- in m r overexpressed sensory neurons significantly reversed the m r-induced reduction in relative levels of total neurite outgrowth (mean±sd: . ± . (control) vs . ± . (g alpha- knockdown), p< . , n= neurons). further, treatment of m r overexpressing neurons with mt or pirenzepine sequestered g alpha- / proteins at the m r and significantly reversed the impaired cytoskeleton-mediated reduction in total neurite outgrowth (mean±sd: . ± . (control), ± (+ nm mt ), ± . (+ micromolar pirenzepine); n= , and neurons, respectively; for treated groups p value= . vs control by one-way anova). mt and pirenzepine also significantly restored mitochondrial trafficking and abundance of mitochondria in the distal neurites. our findings suggest a novel mechanism in which modulation of m r influences tubulin polymerization, mitochondrial distribution in nerve terminals and controls axonal outgrowth and regeneration. funded by cihr and nih. muscarinic receptors are a group of five g-protein coupled receptors (gpcrs) that are targeted by drugs for the treatment of several human pathophysiological conditions. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) antagonists of the muscarinic acetylcholine type receptor (m r) induced elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt et al., ) . one of the major cellular effectors activated by gpcrs is extracellular signal-regulated kinase (erk). the erk signaling cascade regulates a variety of cellular processes including growth and proliferation. both g protein and beta-arrestin mediated signaling pathways can lead to erk activation by phosphorylation through different kinases. activated erk in turn can phosphorylate about cellular substrates, thereby mediating diverse functions. in this study, we have analyzed beta-arrestin recruitment, as part of the receptor internalization process induced by agonist/antagonist binding. in addition, we studied phosphorylation of erk by mt and pirenzepine using isoelectric focusing with phospho-erk specific antibodies and a variety of cell-based assays including beta-arrestin and g protein (gnas/gnasl/gnaq/gna /gna /gna ) knockout cell lines. our study revealed that beta-arrestin is recruited to the m r upon mt and pirenzepine treatment. treatment with nm mt and micromolar pirenzepine significantly increased the dual phosphorylation of tyr and tyr residues of erk / in primary rat sensory neurons (p< . ) in comparison to muscarinic agonist carbachol ( micromolar) and general antagonist atropine ( micromolar). we have identified multiple distinct phosphorylation events on the m r by isoelectric focusing that are specific to mt and pirenzepine induction. further, we have shown that mt and pirenzepine-mediated erk phosphorylation is dependent on both g protein and beta-arrestin recruitment to m r. finally, we reveal that increased erk phosphorylation by mt and pirenzepine significantly (p= . and . , respectively) increased phosphorylation of camp responsive element binding protein (creb) at ser . these results show for the first time that antagonists of the m r can activate the erk signaling pathway and possibly drive phenotypic change in adult sensory neurons. funded by cihr # mop- . charcot-marie-tooth disease (cmt) is representative of inherited neuropathies affecting an estimated in people. the immense advances in gene discovery gained from next-generation sequencing (ngs) projects have revealed the extent of cmt's genetic heterogeneity, with over loci already identified. this knowledge is rapidly translated into clinical comprehensive gene testing panels, often containing over genes. such a large genomic space will invariantly yield variants of uncertain clinical significance (vus) in nearly any person tested. this rise of the number of vus creates major challenges for genetic counseling. in addition, less individual mutations in already known genes are being published as the academic merit is decreasing, and most such testing now happens in clinical laboratories. we propose to capture more of this data in the cmt field to gain a more complete collection of alleles in cmt genes, ideally in conjunction with detailed phenotypic data. this represents a rational approach to eventually reduce the number of vus. thus, we have created a unique, community-driven variant database for cmt researchers and clinicians. the inherited neuropathy variant browser provides simple, user-friendly access to currently reported cmt variation, including patient-level genotypic and phenotypic information. we have also designed an interactive rating system of genetic variation to assist the community with interpretation of vus. for the initial release, we have collected genetic variation, along with genotypic and phenotypic data when available, from published literature, clinical lab reports, and our in-house database. we highly encourage new submissions of not only observed pathogenic variation, but also variation of unknown significance. the goal is to provide a platform for the cmt community to store, share, and discuss genetic data in order to resolve variation of uncertain significance as a joint-effort. with active participation, we aim to provide the community with a more complete mutational spectrum in cmt genes to assist allelic interpretation and patient diagnosis. neurofilaments are strictly neuron-specific intermediate filaments crucial for maintaining axonal architecture. pathogenic mutations in nefl, which encodes the light chain of neurofilament, cause dominantly and recessively inherited charcot-marie-tooth neuropathy (cmt). the nefl-associated neuropathy can be either axonal (type e) or demyelinating (type f). most pathogenic nefl variants are dominantly inherited missense mutations, which are thought to cause disease by inducing nefl aggregation, leading to the disruption of axonal transport. however, investigation of disease mechanisms caused by the mutations has been complicated by the neuronal specificity of nefl. we identified a homozygous nefl variant c. c>t predicting a nonsense change p.arg * in a patient with early-onset cmt. to elucidate the disease mechanism, we used pluripotent stem cells, reprogrammed from patient's skin fibroblasts, to differentiate patient-specific spinal motor neurons. the motor neurons revealed a near complete loss of nefl mrna, and absence of nefl protein. our results establish that nefl is not essential for the development of human nervous system but its absence causes progressive axonal neuropathy. we currently profile the transcriptomic alterations of the motor neurons lacking nefl using single cell rna sequencing to identify compensatory pathways. friedreich's ataxia (frda) is the most common autosomic recessive ataxia, due to a trinucleotide expansion within the frataxin gene. within the wide phenotype, frda patients present also with an axonal neuropathy whose pathological mechanisms are not completely known. eight patients ( women, mean age . yrs, range - ) with genetically confirmed frda underwent neurophysiological and nerve ultrasound evaluation at four limbs bilaterally. echogenicity and cross-sectional area (csa) of median, ulnar, radial, peroneal, tibial, and sural nerves were recorded. mr neurography and diffusion tensor imaging (dti) analysis were performed in one patient; fractional anisotropy (fa), radial (rd) and axial (ad) diffusivity of median, radial and ulnar nerve were calculated at proximal, intermediate and distal sites. all patients presented with sensory axonal neuropathy. seven patients ( %) presented with increased csa of median and ulnar nerves at arm and axilla. mean median nerve csa at mid-upper arm was . mm (normal values < mm ), mean ulnar nerve csa at mid-upper arm was . mm (normal values < mm ). mean median nerve csa at axilla was . mm (normal values < mm ), mean ulnar nerve csa at axilla was . mm (normal values < mm ). mr neurography (performed in one patient) confirmed diffuse swelling and signal hyperintensity of median and ulnar nerves at the arm and dti analysis showed abnormal values of fa, ad and rd along the whole course of evaluated nerves thus suggesting a wide alteration of nerves structure. frda patients presented with an axonal neuropathy characterized, at ultrasound, by a nerve enlargement strictly limited to mid-upper limbs in all patients, findings that cannot be solely explained by a dying-back axonopathy, as suggested by several authors. neither a dorsal root ganglia neural loss could explain by itself our findings, because a diffuse csa reduction would have been expected. on the whole, these findings represent a peculiar feature in frda, but its pathophysiologic meaning remains unclear. inherited peripheral neuropathies are an important health concern for which there is currently no disease-modifying therapy. dogs are affected by a variety of peripheral neuropathies that are breed-specific, indicating a strong genetic component. the use of in-bred populations, such as pure-bred dogs, is advantageous for genetic dissection of disease. acquired peripheral neuropathy (apn) is an inherited late-onset generalized polyneuropathy with high prevalence in labrador retrievers. the most prominent features of apn, laryngeal paralysis and pelvic limb weakness, are associated with the longest peripheral motor nerves in the dog. the pathologic features of apn are similar to human peripheral neuropathy. our aim is to understand the genetic and pathologic features of apn for development of this condition as a naturally occurring large animal model for human disease. we performed a genome-wide association study (gwas) and short-read high-depth whole genome sequencing (wgs) to investigate the genetic underpinning of apn in the labrador. our gwas data indicates that apn is an autosomal dominant disease. the initial analysis from the wgs study resulted in a potential causal variant with an autosomal dominant pattern; this variant is associated with an axonal gene. the neuropathologic progression and histologic features of apn are poorly defined. using genetic markers from our gwas study, we are able to confidently identify labradors with pre-clinical apn, from which nerve biopsies can be obtained. preliminary analysis of biopsies from labradors suggest apn is an axonopathy. further histologic data from preclinical and symptomatic dogs is being obtained to further define the pathogenesis in this model. mutations in morc lead to an axonal form of neuropathy (cmt z). to date, nine families have been published with mutations in the morc gene, showing that this gene is frequently involved in cmt. while the recent genetic data clearly established the causative role of morc in cmt z, its phenotypic consequences in patients and role in neuronal biology remains to be clarified. therefore, we aim to look for altered genetic and biochemical pathways with a transcriptomic approach in order to investigate the role of morc in hereditary peripheral neuropathy. we have performed transcriptomic analysis using a human gene expression microarray (v x k, agilent technologies), in three hek- t cell lines: control, morc knock-down (kd) and the overexpression of the most common morc mutation, the p.r w (np_ ) (kr). differential gene expression assessment was carried out using limma moderated t-statistics. standard analysis techniques perform one test for each gene. thus, for each gene, a t-test statistic is reported together with its corresponding p-value. in this analysis we have used the conventional multiple testing p-value correction procedures proposed by benjamini hochberg to derive adjusted p-values. the preliminary results reveals that kd shows up-regulated genes involved in transmission of nerve impulse and cilium metabolism, suggesting that morc might act at this level in the peripheral nervous system. otherwise, kr shows a major alteration of main axonal metabolic pathways, including the overexpression of genes related to the generation of neuronal action potential, transport through the axon and its targeting in synapse formation. kr also shows a marked alteration of gene expression related to organization, assembly and cilium movement and with the axonemal dynein complex assembly. this study provides an important step towards understanding the pathomechanism underlying to morc p.r w and its role in cmt z. aifm encodes a mitochondria associated apoptosis inducing factor. mutations in aifm lead to a wide spectrum of neurodegenerative disorders: cowchock syndrome; a combined oxidative phosphorylation deficiency (coxpd ) with severe encephalomyopathy; x-linked deafness with peripheral sensory neuropathy; spondyloepimetaphyseal dysplasia (semd) with mental retardation; and an infantile motor neuron disease. previous studies showed severe defects in mitochondrial metabolism, related to redox function, mitochondrial fragmentation, and respiratory deficiencies. in addition, some mutations impair the protein expression of aifm and cause an increase in caspase-independent apoptosis. by targeted next-generation sequencing, we detected the aifm c. c>t (p.phe ser), in a year-boy. this mutation was confirmed in his year-old affected brother. electromyography and nerve conduction velocities studies revealed an axonal polyneuropathy with exclusive involvement of motor fibers, with an early childhood-onset. both children currently show normal cognitive and cranial nerves functions. the in silico structural modeling of human aifm showed that the mutation of a phenylalanine to serine at position disrupts the hydrophobic interaction between phe and pro , and consequently, it destabilizes an alpha-loop domain. cartoon of protein superposition between two different human aifm structures suggest that a lack of constraints in this region could affect the interaction between - helix and the - -hairpin regions, a very important stage for the functional activity of the aifm protein. patient-derived fibroblasts were used to investigate the pathological effect of the p.phe ser mutation: fibroblasts from patients show a similar mrna but different protein expression of aifm compared to healthy control fibroblasts. however, they have an aberrant morphology, from fibroblastic to polygonal shape, and they are larger than control fibroblast; mitochondria from mutant fibroblasts are markedly fragmented compared to controls; the viability of the patient's fibroblasts is lower, but it does not correlate with an increase in apoptosis. instead, it seems to be caused by an increase in the expression of genes activating the senescent program, like p and p . our study confirms that variable effect of different mutations on the protein function may contribute to the clinical variability observed in aifm patients. funds: isciii (pi / ); fundació per amor a l' art. sango k , takaku s , niimi n , yako h . diabetic neuropathy project, tokyo metropolitan institute of medical science, tokyo, japan. coculture models of neurons and schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained from the primary culture with embryonic or neonatal animals. because it is recognized that some biological properties of both neurons and schwann cells change with maturation and aging, culture systems of adult animal cells appear to mimic peripheral nerve degeneration and regeneration better than those of immature animal cells. we have established spontaneously immortalized schwann cell lines from long-term cultures of adult fischer rat peripheral nerves. one of these cell lines, designated ifrs , has been shown to retain distinct schwann cell phenotypes, such as spindle-shaped morphology with expression of glial cell markers, synthesis and secretion of neurotrophic factors and cytokines, and fundamental ability to myelinate neurites in cocultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed pc cells. our current investigation focuses on the establishment of the coculture system of ifrs cells and nsc- motor neuron-like cells. nsc- cells were seeded at a low density ( x /ml) and maintained for a week in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (bdnf, ng/ml). after overnight exposure to mitomycin c (mmc, micro g/ml), nsc- cells were cocultured with ifrs cells ( x /ml) and maintained in serum-containing medium supplemented with bdnf ( ng/ml), ciliary neurotrophic factor (cntf, ng/ml) and coenzyme q ( micro m). under this culture condition, overgrowth of nsc- cells was prevented and gradual movement of ifrs cells toward the neurites emerging from nsc- cell bodies was observed. double-immunofluorescence staining carried out at day of the coculture showed myelin protein zero-immunoreactive ifrs cells surrounding the beta iii tubulin-immunoreactive neurites. this coculture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g. amyotrophic lateral sclerosis, charcot-marie-tooth diseases and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them. santos pp , torezani gs , maciero l , pagliarini lfd , romão tt , abunahman ms , ferreira is , bittar c , pupe c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. zika virus (zikv) is a flavivirus related to dengue, yellow fever and west nile viruses, and has been recently associated to the occurrence of neurological complications in children and adults. previous studies have linked zikv to the development of guillain-barré syndrome (gbs), myelitis, meningoencephalitis and ophthalmological manifestations in adults. guillain-barré syndrome (gbs) encompasses a spectrum of post-infectious neuropathies characterized by different distributions of weakness and sensory impairment. serum anti-ganglioside antibodies are often found and are related to different clinical patterns. recently we have encountered patients in rio de janeiro, brazil, with distal edema in lower limbs, acute weakness, pain and sensory disturbances during the acute stage of an acute febrile exanthematous illness. the symptoms persist for up to days, with complete resolution afterwards, without specific therapy. blood concentrations of muscle enzymes show normal values, and electromyography and nerve conduction studies (emg/ncs) are usually unremarkable. of note, all cases have had positive rt-pcr for zika virus, indicating an illness that occurred during the viremic phase of this arbovirus infection, and complete recovery within the expected timeframe for the resolution of the systemic viremia. there is a subset of patients who developed acute weakness very early after the initial viral symptoms, with clinical, laboratorial and electrophysiological findings that substantially differ from gbs. we describe three cases with similar features suggestive of an acute infective polyneuritis (aipn). one might hypothesize that zikv might lead to a direct neurotropic injury, significant enough to cause weakness and sensory complaints, but not severe enough to cause permanent damage, resolving in conjunction with decreasing blood viremia. we consider that these patients differ from classical gbs because their illness begins during the acute febrile stage of an infective illness and the clinical course is more rapid leading to complete resolution in a few days. savransky a , mozzoni j , massaro sanchez mp , reisin r , monges ms . department of neurology, hospital de pediatria j.p. garrahan, buenos aires, argentine; department of physical therapy, hospital de pediatria j. p garrahan, buenos aires, argentine; department of neurology, britain hospital, buenos aires, argentine. our objective is to describe a series of children with guillain barré syndrome (gbs) included in the igos protocol. as part of the igos multicenter protocol, pediatric patients meeting gbs diagnostic criteria, who consulted within the first weeks of symptom onset and had parental consent to participate in the study were included. patients were also offered to participate in the extended -year protocol. all patients were evaluated according to the igos protocol. patients were recruited between october and june . twenty-four patients, eight girls, participated. ages ranged from months to years (mean . years). all patients agreed to participate in the extended protocol, which was completed by of them. eight patients completed the -year follow-up and are still under evaluation. five patients were lost to follow-up.twenty-two had the classic variant of gbs and two miller fisher. radicular pain in the back or lower limbs was reported by %. twenty-three patients underwent lumbar puncture and albumin-cytological dissociation was found in . in all cases, csf was stored for proteomic studies. all patients underwent emg showing aidp in , and amsan and aman in each. one patient with aidp developed to cidp. in patients full-spine mri was performed and cauda equina enhancement was found in every case. three patients required ucip, two with invasive and one non-invasive ventilation. all patients were treated with gammaglobulin, with a second dose at weeks in cases with a poor response. all patients who followed the protocol were evaluated with the gbs disability score. median score was at baseline and between and at the -year assessment. we describe a series of children with gbs as a part of an international protocol including patients of different ages. pain was a frequent and early symptom and could be determined despite the young age of our patients. most patients fully recovered. we were invited to participate in igos kids to better assess this age group. saysavath k , somchit v , t. umapathi . mittaphab hospital, vientiane, lao pdr; setthathilath hospital vientiane; national neuroscience institute, singapore, singapore. laos people democratic republic is a country of . million people in south east asia. largely agricultural, its capital and metropolis is vientiane. adult neurological services are concentrated at mittaphab hospital, vientiane serviced by three neurologists. on the average about six cases of guillain-barré syndrome (gbs) are seen per year. it is believed that most patients do not seek medical attention. cases appear to cluster during the rainy season. most patients present late, often at the second week of illness when recovery is unapparent after seeking treatment from traditional medicine doctors and at district hospitals. a substantial number of patients seek treatment at hospitals across the border, in neighboring provinces of thailand. common antecedent symptoms are viral prodrome and diarrhea. miller fisher syndrome appears to be rare, possibly because of the mild deficits that do not prompt patients to seek medical attention. diagnosis is made largely from clinical features and from spinal fluid analysis. nerve conduction studies are not available. patients are often treated with steroids by internists. intravenous immunoglobulin and plasma exchange are not available. common complications include pneumonia, autonomic dysfunction (fluctuating blood pressure), pressure sores and depression. patients who develop respiratory failure are nursed at a twelve-bedded intensive care units. plans are afoot to set up a prospective gbs database, systematically study antecedent infections, including of flaviviruses, and develop low-volume plasma exchange as a feasible therapeutic modality. a gene therapy approach for treating cmt c neuropathy schiza n , markoullis k , richter j , tryfonos c , kagiava a , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory; department of molecular virology; neurology clinics, cyprus institute of neurology and genetics and cyprus school of molecular medicine, nicosia, cyprus. charcot-marie-tooth type c (cmt c) is the most frequent form among recessively inherited demyelinating neuropathies and results from mutations in the sh tc /kiaa gene. sh tc mutations cause loss of function of the sh tc protein suggesting that gene replacement therapy may be useful for treating cmt c. sh tc −/− mice develop all major aspects of the human pathology including early onset progressive peripheral neuropathy with hypo-and demyelination along with decreased motor and sensory nerve conduction velocities, offering a relevant model for testing treatments for cmt c. in order to develop a gene replacement strategy for cmt c, we generated a novel lentiviral vector, lv-mpz-sh tc .myc, to drive expression of the human sh tc cdna under the control of the myelin protein zero (mpz/ p ) promoter specifically in myelinating schwann cells. a c-terminus myc tag was included to facilitate expression analysis. a control vector (mock) was also produced in which the sh tc cdna was replaced by the egfp reporter gene. we first confirmed expression of hsh tc in hela cells transfected with the pcdna -cmv-sh tc .myc vector. immunofluorescence analysis confirmed a strong expression of sh tc specifically at the plasma membrane with additional localization in a dotted pattern intracellularly. for in vivo gene delivery we used both intraneural and intrathecal injections of the lv-mpz-sh tc .myc vector in -week to -month old sh tc −/− mice. expression of virally delivered hsh tc was assessed weeks after injection. immunofluorescence analysis showed hsh tc immunoreactivity in perinuclear schwann cell cytoplasm in sciatic nerve teased fibers of sh tc −/− mice following both intraneural and intrathecal delivery, while lumbar intrathecal gene delivery resulted additionally in expression of hsh tc in the lumbar roots. real time pcr analysis confirmed hsh tc mrna expression in both lumbar roots and sciatic nerves. thus, we have developed a novel lentiviral vector for schwann cell targeted gene delivery to treat cmt c and for testing possible therapeutic effects in the mouse model of the disease. hereditary neuropathies are a group of disorders which are characterised by the systemic impairment of peripheral nerves. more than neuropathies are associated with causative gene defects [ ]. charcot marie tooth (cmt a) neuropathy is the most frequent hereditary neuropathy, triggered by a mutation in the peripheral myelin protein gene (pmp ). cmt a leads to a primary loss of myelin sheath and afterwards to a degeneration of axons [ ] . symptoms appear with the degeneration of axons, whereas demyelination is thought to be largely asymptomatic. for that reason, we investigate in the mechanisms of axonal degeneration. for our analysis, we used purified axoplasma without detectable myelin proteins of the sciatic nerves of weeks old pmp -c mice. in this early stage of the axonal degeneration sarm was significantly increased. the nad + concentration in the axoplasma was dramatically reduced. this correlates to the previous finding that sarm promotes axonal degeneration by cleavage of nad + . additional studies showed an increase of the nad + -dependent axonal protective factor sirt in pmp -c sciatic nerves. nmnat- , known as the active component of the wallerian degeneration slow gene, was unaffected in axoplasma of pmp -c sciatic nerves. summarised, these results indicate that the pathway of sarm , nad + and sirt may play a critical role in axonal degeneration in neuropathy. we report two unrelated czech patients with cmt b , both sporadic cases in the family. patient is a year old man with congenital glaucoma after ophthalmological surgeries. his early motor development was normal. at the age of years parents noted gait problems, first neurological examination was at the age of years, when emg showed diffuse motor and sensory neuropathy with severely decreased ncv ( - m/s). he developed foot deformities (pes cavovarus) and underwent corrective orthopedic surgeries at the age of years. after several dna tests for demyelinating cmt the sfb gene was sanger sequenced and a novel missense mutation p.ile asn was found in homozygous state in the patient and in heterozygous state in both parents. the patient was later tested also by ngs of a panel of all genes to be causal for hereditary neuropathies and no other potentially causal variants were detected. patient is years old man, with normal early motor development. at the age of years parents noted gait problems with distal leg weakness which progressed into distal leg plegia at the age of years. hand weakness was noticed since the age of years. he has severe atrophies of distal muscles of all extremities, is self ambulant. at the age of , emg showed unrecordable responses from nerves of lower limbs and ncv was measurable only on ulnar nerve and was m/s. at the age of years increased intraocular pressure was diagnosed and he uses anti glaucoma eye drops. after many single gene tests for demyelinating cmt, we used ngs of a panel of genes known to be causal for hereditary neuropathies and two novel heterozygous, probably pathogenic variants affecting invariant splice sites were detected: c. - a>g and c. - _ del, both confirmed by sanger sequencing. the first variant is also in the father, but the second is probably de-novo (not detected in parents, despite correct parentity). sekiguchi y , kikuchi si , konno si , sekiguchi m . department of orthopaedic surgery, fukushima medical university school of medicine, fukushima, japan. carpal tunnel syndrome (cts) is the most common entrapment neuropathy. ultrasonography can be used to detect anatomic changes in cts. more recently, it has been shown that doppler ultrasonography can detect increased intraneural blood flow in cts. the purpose of this study was to determine the most suitable finding of pre-and postoperation in cts by ultrasonography. a total of wrists of patients with nerve conduction study (ncs) proven cts were evaluated with ultrasonography. we measured the median nerve's cross-sectional area (csa) and intraneural blood flow of median nerve by ultrasonography. the correlation between these ultrasonographic measurements, ncs severity and duration of clinical cts symptoms was analyzed. the csa (mean, mm ) was no significantly reduction after successful carpal tunnel release. morphologic median nerve changes may persist for a longer period regardless of successful surgery and clinical improvement. however, intraneural blood flow is increasing after successful carpal tunnel release. we conclude that doppler ultrasonography results strongly correlate with post operated cts improvement. hence doppler ultrasonography is a useful method for functional improvement of pre-and post cts operation. senger jl , chan km , olson jl , webber ca . university of alberta, edmonton, canada. the beneficial effects of a preinjury crush conditioning lesion (cl) on peripheral nerve regeneration is well-documented in animal models. no human studies have been attempted to date, given the ethical dilemma of deliberately injuring an intact nerve, and the difficulty in predicting the timing of a nerve injury. recent studies demonstrate that hour of electrical stimulation (es) produces effects similar to cl in neuronal cultures. this, coupled with a surgical environment favoring nerve transfers, in which an intact nerve is deliberately cut to reinnervated a denervated muscle, means that es may be clinically translatable to enhance regeneration. this study hypothesizes that es prior to nerve injury will enhance nerve regeneration. twelve sprague-dawley rats were divided into four groups based on conditioning-type to the mid-common peroneal (cp) nerve: es ( ), crush ( ), sham-es ( ), and naïve ( ). one week following conditioning, they underwent a cut/coaptation of the cp nerve at the sciatic trifurcation. post-cut day , nerves and dorsal root ganglia (drgs) were collected. axonal counts of nerves stained with nf- revealed similar regeneration between es and crush ( . vs. . mm, p= . ) that was superior to sham-stimulation ( . mm) or no-conditioning ( . mm, p< . ). a greater number of axons at the distal tip were present in animals that received either type of conditioning compared to the unconditioned cohorts. drgs were stained with neuronal injury marker growth associated factor- (gap- ), and satellite cell glial cells with glial fibrillary acidic protein (gfap). significant increase in gap- expression at three days was observed in es and crush cohorts compared to sham or naïve (p< . ) cohorts. the satellite glial cells of es and crush conditioning showed a significant increase in gfap expression ( . % and . % respectively) compared to sham ( . %) and naïve ( . %) drgs. by demonstrating similar improvements in axon regeneration, this proof of principle project suggests that es conditioning may produce regenerative outcomes comparable to the classical crush injury model. in turn, this suggests that es may be a promising method for delivering conditioning lesions in clinical trials for conditioning nerves prior to surgical intervention. we report a unique case of newly developed waldenstrom's macroglobulinemia (wm) in a patient with chronic inflammatory demyelinating polyneuropathy (cidp) with antibodies against myelin-associated glycoprotein (mag) and sulfatide who was undergoing treatment with intravenous immunoglobulines (iv-ig). subsequent rituximab infusions did not have a positive impact. patients with wm can develop demyelinating and axonal polyneuropathies and few patients have anti-mag and/or anti-sulfatide antibodies. anti-mag antibodies ( % of wm) are associated with sensorimotor axon loss and demyelination and anti-sulfatide ( % of wm) with sensory axonal loss. rarely, both antibodies can be present, with a more severe clinical phenotype. cidp associated with anti-mag and anti-sulfatide antibodies can represent independent entities, not associated with wm. there are no reports to date of patients with cidp associated with anti-mag and anti-sulfatide antibodies that developed wm during immunomodulatory therapy with iv-ig. in addition, subsequent rituximab infusions after the iv-ig were stopped have not been proven beneficial, as has been previously reported for anti-mag cidp patients. seventy-six year old right-handed gentleman presented with persistent numbness in his left foot, three months following artificial disc placement in his lumbar spine. gradually he developed sensory ataxia. no radicular signs were present on exam or impingement on serial spine mri's. ncs/emg studies were consistent with a cidp variant with severely prolonged distal motor latencies. serum anti-mag and anti-sulfatide antibodies were elevated. chronic therapy with iv-ig was able to partially stabilize the symptoms; however, six years later he newly developed wm. subsequent infusions with rituximab, after iv-ig was stopped, did not improve the clinical picture or the ncs/emg findings. wm can newly develop in an autoimmune setting, such as cidp associated with anti-mag and anti-sulfatide antibodies. in this particular case, there was an ongoing immunomodulatory therapy for our cidp patient, as he had monthly iv-ig infusions. this may reflect a possible induction of pathological b cell clone proliferation during the iv-ig treatment. subsequent rituximab infusions, after the iv-ig was stopped, did not improve the symptoms or the demyelination features on ncs/emg. he continues to be symptomatic despite efforts. charcot-marie-tooth disease (cmt) is an inherited neuropathy without known cure (prevalance: : ). duplication of the gene encoding the peripheral myelin protein of kda (pmp ) underlies the most common subtype cmt a. severely affected cmt patients suffer from sensory and motor symptoms with wheelchair-boundness. the clinical phenotype is highly variable and is determined by the amount of axonal loss, but the molecular mechanisms of the disturbed neuron-glia interaction are poorly understood. risk factors have not been investigated. therefore, cmt-net, a german network funded by the german ministry of education and research (bmbf, bonn, germany) includes interdisciplinary expertise from molecular biology, neurology, neuropathology and human genetics in order to identify genetic and non-genetic risk factors of disease severity of cmt by: (i) examining the mechanisms of the disturbed axon-glia-interaction and neuronal vulnerability, (ii) identification of genetic modifiers and (iii) novel therapeutic targets, (iv) validating outcome measures in children and adults, (v) establishing a biobank and (vi) exploring the disease burden via an internationally harmonised patient registry. cmt-net includes three service structures cmt-net will focus on cmt a, but also includes rarer subforms. we will provide the scientific basis for the development of translational approaches to therapy in patients. our approach bridges cutting edge molecular screening techniques, transgenic animal models of altered axon-glia interactions (fly, chick, mouse, rat), state-of-the-art genomic technologies and human patient trials in order to understand and treat the disease aggravation in cmt. sezer g , tekol y , sezer z , . erciyes university, betül ziya eren genome and stem cell centre, kayseri, turkey; erciyes university, school of medicine, pharmacology department, kayseri, turkey; erciyes university, good clinical practice and research centre, kayseri, turkey. analgesic effects of antidepressant drugs are well known for a long time, however, their systemic side effects limit their usage as an analgesics. venlafaxine is an antidepressant drug that has different structure. our purpose was to investigate whether systemic analgesic effect has been proved drug, venlafaxine, has local peripheral antinociceptive action. we applied vanlafaxine ( μl , , , μg) to male, sprague-dawley rats' paws by intraplantar injection and also by intraperitoneal route ( , , , mg/kg) in formalin test, a model for acute and tonic pain. we also pretreated another groups of rats with mg/kg naloxone (opioid receptor antagonist), mg/kg cpt (adenosine a receptor antagonist) or saline (ip.) before μg/paw venlafaxine injection. to check the effect is local or not, we determined the blood levels of venlafaxine in at different time points after both the local and systemic applications by gc-ms method. datas were expressed as number of flinches and total time for biting/licking of the injected paw over phase ( - min) or phase ( - min) and analyzed using the student's t-test. venlafaxine induced antinociception at , and μg concentrations by the local peripheral application and at , mg/kg doses by the systemic application in formalin test and the effects were comparable. pretreatment with naloxone diminished the effect of venlafaxine in the both phases, however, it was not statistically significant. pretreatment with cpt decreased venlafaxine induced antinociception only in phase . neither naloxone nor cpt changed formalin induced nociceptive behaviors alone. this is the first that determines the peripheral antinociceptive actions of venlafaxine in rat formalin test. with roles of opioid and adenosine a receptors in this action. our results suggest that venlafaxine has local peripheral antinociceptive effect and such an activity may led to trials for to use this drug as a cream-gel formulation for analgesia in clinics in the future. topical application might permit the attainment of higher and more efficacious concentrations in the region of the sensory nerve terminal, with limited systemic side effects. shah a , hoffman em , klein cj , staff np . mayo clinic, rochester, usa. cipn is a common dose-limiting complication for patients with cancer. the long-term disease burden of cipn is compounded by increasing cancer survivorship, yet there are minimal data on long term outcomes following onset of cipn, especially in population-based studies. we utilized the rochester epidemiology project to examine incidence and disease burden of cipn among individuals of olmsted county, minnesota with neurotoxic chemotherapy exposure between and . clinical records were queried for the presence of neuropathic signs, symptoms and icd- diagnostic codes as well as for patient provided information on impairment with activities of daily living and use of pain medications. a total of individuals with incident exposure to neurotoxic chemotherapy agents between and were identified. based on aan criteria for identifying peripheral neuropathy, ( . %) of these individuals were determined to have cipn, while ( . %) controls did not. the median time from incident exposure to reported symptom onset was days (iqr . - ). patients with cipn received a neuropathy icd- diagnosis in merely cases ( . %). median survival following incident chemotherapy exposure among all cases and controls was . years with a significantly longer mean survival in cases with cipn as compared to that of controls ( . years vs. . years, p< . ). in addition to acute effects in cipn, individuals surviving greater than years following exposure to neurotoxic chemotherapy continue to self-report increased symptoms of numbness (or . , % ci . - . ) and pain (or . , % ci . . . ) of the extremities. through utilization of patient provided information, our study was able to collect data on long-term impairment associated with previous history of exposure to neurotoxic chemotherapy. our results are consistent with previous reports of the high incidence of cipn in the first two years following incident exposure. additionally, our results provide evidence of high incidence of cipn independent of individual chemotherapeutic agent used. additionally, our results indicate icd- -cm diagnostic code attribution may dramatically underestimate the magnitude of cipn. increased survival following exposure to neurotoxic chemotherapy and its long-term disease burden necessitates further study of among survivors. the utility of quantitative muscle ultrasound as a marker of disease severity in charcot-marie-tooth (cmt) disease subtypes was investigated. muscle ultrasound was prospectively performed on individual muscles from cmt patients ( cmt a, cmtx , cmt a) and compared to muscles from age and gender-matched controls. muscle ultrasound recorded echogenicity and thickness in representative muscles including first dorsal interosseus (fdi) and tibialis anterior (ta charcot-marie-tooth (cmt) disease is the most frequent inherited peripheral neuropathy, and there is currently no available cure. the most common subtype of cmt, cmt a, is completely associated with duplication of the pmp gene, which encodes peripheral myelin protein of schwann cells. previous studies of cmt a mainly relied on rodent models, and it is not yet clear how pmp overexpression leads to the phenotype in patients. based on induced pluripotent stem cell (ipsc) technology, we herein developed a brand new in vitro cell model of cmt a, called cmt a-hipscs, in the hopes of simulating the developmental progress of the disease and gaining new insights into its pathogenesis. here, we efficiently derived ncscs from cmt a-ipscs and assessed the potential of the isolated cmt a-neural crest stem cells (ncscs) to differentiate into peripheral neurons and schwann cells using defined media. we found that, unlike normal control ncscs, cmt a-ncscs rarely generated schwann cells. instead, cmt a-ncscs produced numerous endoneurial fibroblasts in the schwann cell differentiation system. we further established a pmp -overexpressing ipsc model, and obtained similar results when pmp -ncscs were subjected to schwann cell differentiation. these results suggest that the development of schwann cells in cmt a patients is interrupted by the duplication of pmp . with the exception of the demyelination-remyelination process, developmental disabilities of schwann cells should be considered as an underlying cause of cmt a. shimoi t , yamada t . international university of health and welfare, tochigi, japan, cmt japan, tokyo, japan. charcot-marie-tooth (cmt) disease is the most common hereditary motor and sensory neuropathy. our preliminary report suggests that a certain cmt patient has the recruitment disorder of motor units during muscle fatigue and this disorder may be a factor of "super fatigability" in motor neuropathy patients. if the "super fatigability" occurs, we would expect patients with this characteristic to become slower in recovery from muscle fatigue than patients without this characteristic. in order to verify this hypothesis, we measured characteristic of recovery from muscle fatigue in charcot-marie-tooth patients with electromyographic study. twenty three participants were asked to maintain their % of maximal voluntary isometric contraction (mvc) of elbow flexor until exhaustion as the fatigue exercise. in addition, the participants asked to perform s of their % of mvc at , , , , , , , , , s after the fatigue exercise as recovery tasks. the surface emg (semg) signals of biceps brachii muscle were determined during the exercise and tasks. muscle force, median power frequency (mdpf) and the root mean square of semg amplitude (rms) were used as objective parameters of muscle fatigue. borg scale was used as a subjective parameter of muscle fatigue. six of twenty three participants showed significant decrease of rms during the fatigue exercise. in consideration of this result, we compared alteration of mdpf in recovery task between six participants with decrease of rms (abnormal group) and seventeen participants with increase of rms (normal group). as the result, the abnormal group had at least s as the recovery time from muscle fatigue in contrast with s of normal group. the recovery time from muscle fatigue in subjective parameter was shorter than the time in objective parameters in each group. our data support the "super fatigability" hypothesis. and that hypothesis may induce "hidden muscle fatigue". comparison between complex regional pain syndrome type and based on electrophysiologic, imaging and clinical findings shin jy , moon jy , sung jj . seoul national university hospital, seoul, republic of korea. complex regional pain syndrome (crps) is a constant regional neuropathic pain characterized by various kinds of motor, sensory, and autonomic changes. conventionally, the crps is divided into type i and ii according to the absence and presence of nerve injury. but the pathogenesis of crps is not fully understood yet. and there is still no systematic comparative study between crps type i and ii. we compared between crps type i and ii using multimodal approaches including electrophysiologic, imaging, and clinical findings. the patients ( type i and type ii) diagnosed with crps using the international association for the study of pain (iasp) diagnostic criteria were included. type i and ii were divided by electromyography and nerve conduction study. we obtained clinical information such as continuing pain, allodynia, hyperalgesia, edema, temperature, skin color, sweating, trophic change from patients. all patients were evaluated by bone scan, thermography, quantitative sudomotor axon reflex test (qsart), quantitative somatosensory test (qst). the ratio of qsart and temperature threshold abnormality in type ii was higher compared to type i ( . % vs . %, . % vs . % respectively, p = . and . ), among clinical symptoms, sweating change significantly high in type ii compared to type i ( . % vs . %, p = . ). other electrophysiologic and imaging, clinical findings were not significantly different in both type. in this study, we identified that crps type i and ii are distinguished not only by the nerve injury but also by the sudomotor function, and qsart can serve as a good technique to differentiate between crps type i and ii. it is estimated that there are two distinct pathogenesis in crps. our results may be helpful to diagnose crps correctly and understand the pathogenesis of crps. sjogren syndrome (ss) is an autoimmune inflammatory disorder of exocrine glands resulting in xerophthalmia and xerostomia. ross syndrome is a rare entity characterized by tonic pupil, hyporeflexia, and segmental anhidrosis. we present a -year-old hispanic woman with debilitating sensory and autonomic neuropathies, and persistently elevated anti-ss-a and anti-ss-b antibodies, without the classic sicca complex. she initially developed diarrhea and an ear infection, then felt toe and later leg numbness, which eventually spread to her cheeks and tongue, over few months. four years later, her thumbs and index fingers started tingling. also, she developed orthostatic lightheadedness, tachycardia, segmental hypohidrosis of the right abdomen, and hyperhidrosis of the remaining trunk, intermittent erythema, chronic diarrhea, and a -pound weight loss. her exam demonstrated orthostatic hypotension, bilateral tonic pupils and light-near dissociation, sectoral palsy of the right iris sphincter, stocking-distribution diminished sensation to all modalities, pseudoathetosis, areflexia, dysmetria, intention tremor, romberg, and sensory gait ataxia. mri of neuraxis demonstrated t -weighted hyperintensity in the dorsal spinal cord from c to the lower thoracic level, with mild atrophy. electrodiagnostic testing was consistent with moderate-to-severe, length-dependent, asymmetric, sensory polyganglionopathies. csf showed oligoclonal bands. serology showed elevated antinuclear antibody ( : , reference < : ), ss-a ( , reference < u), ss-b ( , reference < u), and rheumatoid factor ( , reference < iu/ml) titers. the remaining workup was negative for infection (syphilis, hiv, htlv, hepatitis, lyme disease), paraneoplastic syndrome (anti-hu and ganglionic nicotinic acetylcholine receptor antibodies), pyridoxine intoxication, malignancy (chest/abdomen/pelvis ct, breast ultrasound, axillary lymph node flow cytometry, colon and esophagus biopsy), celiac disease, vitamin deficiency, autoimmune disease (anti-aquaporin and anti-gq b antibodies), and adrenoleukodystrophy. the patient received ivig and steroid with some gait improvement and currently takes mycophenolate. midodrine and fludrocortisone resolved her dizziness. this case highlights an important, treatable sensory ganglionopathy and systemic autonomic neuropathy due to sjogren syndrome, and illustrates the overlapping clinical triad of ross syndrome, which may guide future management. siles am , , assylbekova d , , diaz-manera j , , rojas-garcia r , , cortes e , , gallardo e , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, univeristat autònoma de barcelona, barcelona, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. inflammatory neuropathies are a heterogeneous group of peripheral nerve diseases that respond to immune-therapies. chronic inflammatory polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) are two chronic inflammatory neuropathies responding to intravenous immunoglobulins (ivig). b lymphocytes are involved in their pathogenesis. while widely used in clinical practice, ivig's mechanism(s) remain not completely understood. ivig are reported to lead to b-cell anergy and to increase regulatory t-cell function and frequency. regulatory b cells (bregs) are a rare subset of b lymphocytes that suppress immunopathology acting upon several target cells in the immune system. impaired breg yields have been described in a plethora of autoimmune conditions. the presence of regulatory b cells in inflammatory neuropathies and the effect of ivig therapy on their frequencies has not been studied. the aim of this study is to describe the frequencies of bregs in cidp and mmn and the effect of ivig on their frequencies. patients fulfilling diagnostic criteria for cidp or mmn and matching controls were included. pbmcs were obtained by gradient centrifugation before ivig infusion and one week after treatment. b-cells were isolated with negative selection magnetic beads, cultured and activated with the tlr agonist odn and anti-human igg+iga+igm. il- secretion capacity was assessed by flow-cytometry. twenty-eight patients were included of whom where cidp and mmn. of all patients included, received ivig and were suitable for pre and post ivig breg frequency comparisons. breg frequencies did not differ in patients (before ivig treatment) and controls (p= . , mann whitney test, two-tailed). however, the frequencies of bregs significantly increased one-week after treatment with ivig (p= . , wilcoxon matched pairs test, two-tailed). when stratifying by disease subtype, breg frequencies increased in cidp patients after ivig (p= . , wilcoxon matched pairs test, two-tailed) and in mmn (p= . , wilcoxon matched pairs test, two-tailed) although results did not reach statistical significance in mmn. this is the first study that studies the breg frequencies in cidp and the first study that addresses the effect of ivig on breg frequencies. our study provides the proof of principle that bregs could become a biomarker for response to ivig but this would need a larger and prospective study. siles am , , martínez-hernández e , diaz-manera j , , rojas-garcia r , , gallardo e , , illa i , , graus f , querol l , . paraneoplastic neuropathies (pn) are rare, immune-mediated disorders of the peripheral nerve with important prognostic implications. ectopical expression of neural antigens in the tumor leads to the development of onconeural antibodies. several autoantibodies associate to pn, including anti-hu, anti-caspr or anti-cv antibodies but a significant proportion of pn lack identifiable antigens. adhesion molecules that are autoantigens in other neuropathies, like contactin- , are present in several types of tumors. our study proposes a systematic screening of autoantibodies against neural cell-adhesion molecules and neural structures to detect novel antigenic reactivies in pn. thirty-five patients followed in our centre and at the neuroimmunology-multiple sclerosis unit at hospital clínic de barcelona, with pn fulfilling diagnostic criteria of possible (n= ; . %) and definite (n= ; . %) paraneoplastic disease were included. serum samples were obtained and tested by immunocytochemistry against contactin- (cntnt ), neurofascin (nf ) and the cntn /caspr complex. primary cultures of dorsal-root ganglia (drg) and rat schwann cells were incubated with patients' sera to detect antibodies targeting neural structures. ten individuals ( . %) presented with a tumor and a neuropathy involving both sensory and motor symptoms. the remaining patients ( . %) presented with a tumor and a classical sensory neuronopathy without anti-hu or any other onconeuronal antibody. among the latter, ( . %) patients were diagnosed with small-cell lung carcinoma. the rest of the individuals ( . %) associated diverse malignancies. we did not detect any sera reacting against cntn , nf or the cntn /caspr complex. in igg antibody screening experiments, patients ( . %) reacted against drg neurons, of them ( . %) reacting strongly, and patients ( . %) reacted mildly against rat schwann cells. in igm experiments, patients ( . %) reacted slightly against drg neurons and patients ( . %) against rat schwann cells, of them ( . %) featuring strong staining. experiments screening antibodies against motor neurons and immunoprecipitation assays are ongoing. overall, % of patients reacted strongly against either neurons or schwann cells. our study did not detect antibodies against the neural adhesion molecules cntn , nf and the cntn /caspr complex in patients with pn. however, a significant proportion of pn patients harbour antibodies targeting neural structures, which suggests that novel neoplasm-associated antigens remain to be discovered. simmons m , tao f , abreu l , zuchner s , li j . department of neurology, vanderbilt university school of medicine, nashville, tennessee, usa; hussman institute for human genomics, university of miami, miami, florida, usa. objective: despite of a shared genetic mutation of the trisomy of chromosome p (c p ), patients with charcot-marie-tooth type- a (cmt a) present with a high variability of their disease severities. the underlying cause for the variability is still unclear. in this study, we tested a hypothesis whether a second genetic mutation known to damage the nervous system is also present in cmt a patients with early onset and severe phenotypes. methods: from a cohort of patients with cmt a mutation (chromosome p duplication), we identified patients with an early onset (< or = years of age) of the disease. four of the eleven also had dna testing for a panel of known cmt-related genes and sequencing of mitochondrial dna in addition to the dna testing for c p duplication. results: besides the c p duplication, we identified three additional mutations in the four patients with early onset. the mutations were a missense mutation of arg his in mpz gene, an a g mutation in mitochondrial trna for glycin and a homozygous mutation of c p duplication. three of the four had symptomatic onset at birth. one showed symptoms at years of age. conduction velocities were severely reduced in all four patients (from to m/s). interpretation: traditional approaches to identify genetic modifiers, including snp association, assume that those modifiers are clustered in a small region of human genome and shared by the studied patients. however, our study suggests that genetic modifiers in cmt a may be highly diverse and scattered throughout the genome, which could make the conventional approach via the genetic variants association difficult. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr sjogren's syndrome(ss) is a systemic auto-immune disease that apart from exocrine glands may affect any organ. involvement of peripheral nervous system results in wide spectrum of neuropathic manifestations. the aim of our study was to evaluate the clinico-electrophysiological patterns and pathological characteristics of neuropathy in sjogren's syndrome (ss) patients presented to neuromuscular clinic in a tertiary hospital from south india. this is a retrospective study from the departments of neurology, rheumatology and pathology from nizam's institute of medical sciences. twenty one patients with diagnosis of ss and peripheral neuropathy between to were analysed. clinical records, conventional nerve conduction studies, lip and nerve biopsy reports were collected.in patients with ss associated neuropathy,male to female ratio was : . in ( . %) neuropathy was the initial manifestation,while in ( %)exocrinopathy preceded neuropathy. the patterns of neuropathy included mononeuropathy multiplex(mnm) in patients ( %),ganglionopathy in ( %),length dependant, trigeminal, autonomic neuropathy and cidp in ( %)and cranial neuropathy in ( %).eighteen( %) were seropositive..schirmer's test was positive in ( . %).nerve biopsy showed vasculitis in patients, demyelinating and axonopathy in patients each.we conclude that neuropathy is frequently the initial presentation of ss.mnm is the common pattern followed by ganglionopathy. pattern of neuropathy helps in arriving at the diagnosis of ss. confirmation of ss is not by mere serology. schirmer's test and lip biopsy are equally essential for the diagnosis especially in seronegative patients when clinical index of suspicion is high. siskind ce , tesi rocha c . stanford health care, palo alto, ca, usa; stanford university, stanford, ca, usa. here, we report the first case of a human found to have a homozygous, presumed disease causing variant in the arl ip gene, causing charcot marie tooth disease (cmt) with central nervous system findings. the proband was born at term with apgars of and at and minutes. he was found to have iugr and was hospitalized for days for weight gain. he developed respiratory distress during the admission and was intubated. due to inability to extubate, he was transferred to stanford children's hospital, where he remained for three months. noted during admission was hypotonia, areflexia, minimal voluntary movment, sinus tachycardia, and dysautonomia. brain mri found polymicrogyria and cerebral underdevelopment with generally normal-appearing brainstem, with moderate ventriculomegaly. ncs found length dependent polyneuropathy with axonal degeneration. follow up muscle and nerve biopsy found immature muscle and amyelinating neuropathy the patient had normal plasma amino acid, acylcarnitine, lactate, pyruvate, urine organic acid testing, opththalmology exam, newborn screen, and normal array cgh. genetics ordered whole exome sequencing through baylor genetics laboratory (houston, tx, usa), which found a homozygous nonsense variant in arl ip : c. c>t, p.r x. a second child had been identified by baylor with two variants in this gene. that child had hypotonia, respiratory distress and seizures, and a muscle biopsy consistent with sma. the parents of that child chose to withdraw care at months of age. our patient's parents continued with aggressive therapies, including tracheostomy and g-tube for feedings. he had several subsequent hospitalizations for respiratory distress, possible seizure activity, and buldging anterior fontenelle, but now, at two years of age, has made developmental progress and is living at home with his family. he is able to smile, reaching for toys and swatting objects. he has little voluntary movement, and no longer responds to light touch stimuli. overall, this is the first picture of a child affected with a severe amyelinating form of cmt that causes weakness, hypotonia, and possible seizures, with the main concerning feature being the severe respiratory distress that may be life threatening, but can be managed with extreme care. charcot marie tooth (cmt) disease is the most common inherited peripheral neuropathy. patients frequently ask whether pregnancy will affect their cmt, whether cmt will affect their pregnancy, the optimal delivery and whether they or their child will have a higher risk of complications during pregnancy or delivery. so far few studies address these questions. currently no guidelines exist for the management of pregnancy, delivery and postnatal care in cmt patients. the aim of the study is assess the impact of pregnancy on cmt and assess how cmt affects pregnancy, delivery and care of the new born baby. we designed a retrospective questionnaire with expert help from an obstetrician with a special interest in pregnancy in patients with medical conditions. the questionnaire is divided into four parts (prior, during, after pregnancy and delivery) and includes questions on impairment, falls, pain, fatigue and respiratory complications during those periods; type of delivery, possible complications, details of anaesthesia and difficulties looking after the baby in the first months postpartum. so far women ( pregnancies) with cmt and related disorders have answered the questionnaire. % of patients had cmt a, the remaining had various subtypes of cmt and related disorders. patients reported deterioration of cmt symptoms during pregnancy in % of pregnancies with resolution of symptoms after pregnancy in % of pregnancies. of symptoms questioned walking ( %), balance ( %), and hand function ( %) deteriorated the most. there was an increased use of orthoses and walking aids during pregnancy. the majority of women ( %) had natural delivery, % were assisted and % had caesarian sections which was similar to the uk population ( %). no complications with anaesthesia were reported. the survey is currently ongoing. we plan to survey consecutive patients. data acquired from this survey will provide valuable information on current practice and will inform future guidelines and standard of care in charcot marie tooth disease. multifocal motor neuropathy (mmn) is a slowly progressive disorder in adults, characterized by asymmetrical limb weakness, mainly affecting the arms. despite beneficial effect of immunoglobulins, weakness gradually progresses. a major determinant of muscle weakness is the degeneration of affected motor axons. treatments aiming to reduce loss of motor axons require objective tools to quantify such an effect. therefore, we applied the compound muscle action potential (cmap) scan, which is an electrophysiological method that, with increasing transcutaneous stimulus-currents, successively activates all motor units (mus) in a muscle. it captures the contribution of enlarged mus due to reinnervation by the presence of relative large discontinuities in the scan. the aim of the present study was to identify pathophysiological changes of mu-loss and reinnervation in mmn patients by means of the cmap-scan. recordings were obtained from mmn patients. cmap-scan recordings were performed in the median nerve at the wrist where motor responses were recorded from the thenar muscle. we determined the number of largest cmap-scan discontinuities by means of a novel marker, d , where a low number is indicative of mu-loss and enlarged mus. furthermore, we applied the recently developed method of professor hugh bostock for obtaining a mu-number estimate from the cmap-scan. the median peak cmap amplitude was . mv (range . - . mv) and median d was (range - ). in three mmn patients with a normal maximum cmap amplitude (> mv) a reduced d (< ) was found indicative of mu-loss and enlarged mus. furthermore, d and the estimate of mu number were significantly related (r = . , p < . , n = ). the findings suggest that the cmap-scan is a sensitive tool in detecting the underlying pathological changes of reinnervation and mu-loss in mmn, more so than standard maximum cmap amplitude. it is quick and easy to perform and has the potential to be useful for follow-up studies. smith ag , thurgood b , revere c , hauer p , aperghis a , singleton jr . university of utah, salt lake city, utah, usa. corneal confocal microscopy (ccm) directly and quantitatively assesses corneal innervation including nerve fiber length (nfl) and density (nfd). ccm has shown promise as a diagnostic test. we have previously demonstrated that ccm has a diagnostic performance for diabetic neuropathy (dpn) similar to skin biopsy with assessment of intraepidermal nerve fiber density (ienfd) and nerve conduction studies (ncs). the responsiveness of these surrogate measures to dpn progression and their relation to clinically meaningful outcomes has not been well explored. diabetic patients undergoing annual retinopathy examination were recruited. each underwent ccm, ienfd, ncs including sural sensory and peroneal motor responses, the utah early neuropathy score (uens), the norfolk quality of life -diabetic neuropathy (nqol-dn, a validated neuropathy specific qol scale), and a minute walk test ( mwt). with dpn based on symptoms ( %) or signs underwent repeat testing at months and at months. at baseline, nqol-dn correlated with sural sensory amplitude (ssa) (− . , p< . ), peroneal motor conduction velocity (pcv) (− . , p< . ) and ienfd (− . , p< . ). no ccm metric was related to qol. mwt distance correlated with ssa ( . , p< . ), nfl ( . , p< . ) and nfd ( . , p< . ). over months, there was a significant worsening in dpn signs assessed by the uens (increase . +/− . , p< . ). ssa declined . uv (p< . ) and ienfd . fibers/mm (p< . ). there was no change in any ccm metric, pcv or nqol-dn. these findings suggest measures of distal axonal integrity are most sensitive to neuropathy progression, with ienfd having the greatest responsiveness. in contrast, ccm was not responsive to dpn progression. both ncs and ienfd (but not ccm) were significantly correlated with neuropathy-specific qol, whereas ncs and ccm measures correlated with physical functioning. the responsiveness of ienfd and ssa, and their relationship to qol support their selection as endpoints in dpn clinical trials. chronic inflammatory demyelination polyradiculoneuropathy (cidp) affects in , people, and is marked by chronic autoimmune infiltration of peripheral nerves and destruction of the myelin sheath. with current therapies, only % of cidp patients achieve complete remission. to produce more effective, mechanism-based therapies, we study mice with a partial loss of function g w substitution in the autoimmune regulator (aire) gene on the non-obese diabetic (nod) background (nod.aire gw/+ ) that develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. autoimmunity can result from defective immunosuppression. the potent, immunosuppressive cytokine interleukin (il- ) is increased in the peripheral blood mononuclear cells (pbmcs) of active phase cidp patients relative to remission phase patients. further, pbmcs from cidp patients produce il- in response to the myelin protein p . despite these findings, whether il- is important for cidp pathogenesis is not known. thus, we sought to determine the role of il- in sapp. il- was highly upregulated in sciatic nerves of nod.aire gw/+ mice with sapp, suggesting it may play an immunosuppressive role in pathogenesis. however, genetic ablation of il- in nod.aire gw/+ mice lead to a paradoxical delay in disease development. age-matched il- -deficient nod.aire gw/+ mice exhibited no sciatic nerve infiltrate and no reduction in nerve conduction during electrodiagnostic studies. interestingly, the delay in sapp was specific, since the incidences of five other autoimmune manifestations in il- -deficient nod.aire gw/+ mice were unchanged relative to il- -sufficient nod.aire gw/+ controls. importantly, il- -deficient nod.aire gw/+ mice did not have colitis, which is consistent with previous studies of il- deficiency on the nod background. il- is known to perform effector functions in autoimmunity by promoting b cell secretion of immunoglobulins. however, genetic ablation of b cells did not affect neuropathy development in nod.aire gw/+ mice, suggesting b cells are dispensable for pathogenesis and unlikely to mediate the protective effect of il- deficiency. il- -deficient nod.aire gw/+ cd + t cells, which are sufficient to transfer sapp, exhibited increased activation, increased interferon gamma secretion, and preserved nerve-specific t cell activation. these data suggest t cell activation and priming are unperturbed and not the mechanism of protection. in summary, our data showed that il- was paradoxically an effector cytokine in sapp. long exercise test (let) has been used especially in myotonic syndromes and muscle channelopathies. marked decrement in compound muscle action potential (cmap) amplitude after prolonged exercise was previously reported in patients with paramyotonia congenita, hyperkalemic or hypokalemic periodic paralysis. we describe a patient with secondary hypokalemic paralysis who showed abnormal let results. a -year-old man presented with ascending flaccid paralysis which evolved in a hyperacute fashion. the patient became quadriplegic after two hours. initial laboratory evaluation revealed severe hypokalemia, with normal thyroid function. we performed electrodiagnostic studies including long exercise test as proposed by mcmanis et al. nerve conduction study was normal, but marked decrement in cmap amplitude (up to % decrease after minutes) was noted after prolonged exercise. despite oral and intravenous potassium replacement, serum potassium level was not corrected as expected. the unusual clinical course prompted for evaluation of secondary etiologies. abdomen computed tomography scan revealed a . x . cm-sized mass in the left adrenal gland. aldosterone to renin ratio was elevated, suggestive of primary hyperaldosteronism. genetic study for cacna s mutation turned negative. after receiving laparoscopic adrenalectomy, the patient experienced no further attacks, and also was able to stop his antihypertensive medication. let may show abnormal results in condition with reduced membrane excitability, even without true channelopathy. according to international criteria, the diagnosis small fiber neuropathy (sfn) is based on clinical symptoms in combination with a reduced intraepidermal nerve fiber density (ienfd) in skin biopsy and/or abnormal temperature threshold testing (ttt). the sensitivity of skin biopsy is moderate to good, although ienfd is normal in about % of patients with sfn complaints. furthermore, ttt is a widely available diagnostic tool, but lacks specificity. corneal confocal microscopy (ccm) has been described and is used in clinical practice as an objective, non-invasive diagnostic tool to detect small nerve fiber damage in patients with diabetes mellitus. this study examines the applicability of ccm in patients with sfn, and the value of ccm as an additional diagnostic tool in sfn. we will include healthy participants to compare the results with the recently published ccm normative values, and patients referred to the sfn center maastricht with the clinical picture of sfn. corneal nerve fiber density (cnfd), branch density (cnbd), fiber length (cnfl), and the tortuosity coefficient (cnft) will be determined in all participants. the results will be compared with the ienfd and ttt. preliminary results will be presented. southanalinh k , university of health sciences, vientiane capital, lao p.d.r. located in south east asia, lao pdr is a landlocked country with a population of about . million inhabitants. the health indicators are among the lowest in south east asia. the total health caregivers in consisted of , persons corresponding to a ratio of . health workers per inhabitants. the main network for health care service provision remains the public system. its health care facilities consist of four central teaching and referral hospitals; five regional hospitals, including one teaching hospital; provincial hospitals; district hospitals, and about health centers. only one in seven sick people receives modern health care treatment. most people rely on self-medication and/or reliance on self-healing. neurological care is a very new field. knowledge of common neurological disorders among both the lao population and medical staff is only beginning to be spread. there are three neurologists in the country. six neurology residents are currently being trained in a three-year program supported by the association pour la promotion des neuro-sciences au laos (association for the promotion of neuro-sciences in laos) and the asean neurological association. indeed, resources are scarce. in the peripheral nerve diseases domain for example, we have only one electromyography machine that was only temporary used when emg experts from france and from singapore came to teach residents. a significant mismatch between the provision of specialized neurologic services and the needs for them exists, especially in rural areas. also, health insurance is not available for the majority. as a consequence, patients have to bear the costs themselves, which constitutes a limit to the access of available healthcare facilities. neurologic training centers, laboratory facilities and equipments are limited. optimizing available human resources, integrating primary, secondary, and tertiary healthcare tiers and making medical treatment more affordable are need to improve neurologic care in the developing world. in certain low-income countries with limited human and financial resources, it may be difficult for governments to apply some of these recommendations on their own. in these circumstances, it is suggested that countries work with international agencies, nongovernmental organizations or other partners to put their plans into practice. spina e , topa a , iodice r , tozza s , dubbioso r , ruggiero l , santoro l , manganelli f . department of neuroscience, odontostomatological and reproductive sciences, university of naples "federico ii", naples, italy. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a disabling disease and about % of patients may become persistently disabled over time. our aim was identify clinical prognostic factors of long-term disability in a large series of cidp patients. we collected data from cidp patients with definite diagnosis according efns/pns criteria and positive response to first-line therapies (immunoglobulin or corticosteroids) including sex, age of onset, phenotype, disease duration, course of disease (monophasic/relapsing-remitting or chronic progressive) and disability at the time of diagnosis assessed using the modified rankin scale (baseline mrs). all patients had clinical assessment of disability through mrs within the last months (last mrs). ordinal logistic regression model was applied to evaluate the relationship among the clinical parameters and last mrs, considered as ordinal outcome ( - ). anova test for repeated measures was applied to test the overall effects of different course on disability accumulation while t-test was performed to evaluate inter-group differences for parametric variables. we found a significant relationship between last mrs and the course of disease [p< . , z= . , or: . ]. disability accumulation was greater in patients with chronic progressive course than those with monophasic/relapsing-remitting course of disease [p= . ]. moreover, patients with progressive course were older [p= . ]. our data suggest that chronic progressive course of disease may be a major negative prognostic factor for long-term disability in cidp patients. to note that a chronic progressive course of disease is also associated with an older age from the beginning and a more pronounced worsening over the course of disease. sprenger a , lichtenstein t , henning t , lehmann hc . department of neurology, university hospital of cologne, cologne, germany; institute of diagnostic and interventional radiology, university hospital of cologne, cologne, germany; department of neurology, university hospital of cologne, cologne, germany. an unresolved problem in the treatment of inflammatory neuropathies is the lack of valid and reliable diagnostic biomarkers to evaluate axonal damage. we investigated if "diffusion tensor imaging" (dti) and mri t w multi echo dixon imaging are eligible methods to determine proximal nerve injury in chronic inflammatory demyelinating polyneuropathy (cidp). in this prospective observational cohort study the sciatic nerve of cidp patients and age matched healthy controls was investigated. all subjects underwent multimodal mri imaging to determine fractional anisotropy (fa) and muscle fat fraction of the biceps femoris and quadriceps femoris muscle. patients were evaluated by mri, clinical examination and nerve conduction studies at baseline and after six months. the mean fractional anisotropy (fa) value was significantly lower in the sciatic nerve from cidp patients compared to controls. fat fraction of the biceps femoris and quadriceps femoris muscle were significantly higher in cidp patients compared to controls. mri outcome parameters remained unchanged after six months. our study demonstrates the utility of mri imaging to differentiate between "healthy" and functional constricted proximal nerve segments. we postulate that dti and dixon mri might be eligible methods to assess proximal nerve damage in cidp. the presence of peripheral myelin protein (pmp ) has been known for decades, but its functional role was uncovered only recently. recent characterization of pmp -deficient mice revealed a role of pmp in the lipid homeostasis of myelinating schwann cells. in this study, we analyzed the functional impact of pmp on myelination. to decipher the role of pmp , experimental demyelination was performed in myelinating dorsal root ganglia cultures, and in vivo re-myelination was assessed after experimental peripheral nerve damage. we used the myelinating dorsal root ganglia (drg) model in pmp -deficient schwann cell cultures, combined with an established de-and remyelinating protocol in order to analyze myelination in vitro. we also performed experimental nerve crush in pmp -deficient mice. morphometric parameters were defined for the in-vitro experiments and functional parameters such as nerve conduction velocity and the clinical score were additionally measured for the in vivo experiments. structural analyses of the drg cultures revealed fibers expressing myelin basic protein (mbp) and pmp , as well as fibers positive for mbp alone. in contrast to our previous in vivo data, we were also able to detect myelin segments that stained positive for pmp , but were negative for mbp. pmp -deficient drg-cultures demonstrated slightly greater nodal lengths than the control cultures. this trend was significantly augmented after in vitro de-and remyelination, which also resulted in decreased internodal lengths only now, while conserving an intact myelin structure. concomitantly, in vivo nerve crush gives rise to a more severe phenotype in pmp -deficient mice than in wild-type controls. consistent with this, nerve conduction studies showed a delay in remyelination, and analysis of semi-thin sections demonstrated an altered fiber structure in the peripheral nerve biopsies. together, these data suggest that in addition to its role in glial cell lipid homeostasis, pmp also plays a role in remyelination of the injured peripheral nervous system. anti-mag neuropathy remains a difficult diagnosis to treat given its limited therapeutic options. of all interventions, rituximab has emerged as the most effective, although its effect has been with mixed results, especially in patients with advanced axonal loss. lenalidomide is another promising immune modulating therapy, whose effect has been well demonstrated in neuropathy associated with poems (polyneuropathy, endocrinopathy, organomegaly, m-spike protein, and skin changes) syndrome, a condition that has several striking parallels to anti-mag neuropathy. the use of lenalidomide has not been previously described in anti-mag neuropathy. herein, we describe a case of lenalidomide-responsive anti-mag neuropathy in a patient with advanced axonal loss. suichi t , misawa s , sato y , beppu m , sekiguchi y , shibuya k , watanabe k , amino h , kuwabara s . department of neurology, graduate school of medicine, chiba university, chiba, japan; clinical research center, chiba university hospital, chiba, japan. polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes (poems) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscrasia and vegf overproduction. several diagnostic criteria for the disorder have been published, but sensitivity/specificity analyses, and their validation have never been performed. the aim of this study is to establish valid diagnostic criteria for poems syndrome. consecutive poems patients, seen at chiba university hospital since , were screened. of these, we have set a gold standard group of poems syndrome, based on treatment response and exclusion criteria during -year follow-up, and patients was diagnosed as having definite poems syndrome. we also collected patients with cidp (demyelinating neuropathy control) and with multiple myeloma, primary amyloidosis, or mgus (m-protein control). criteria for poems syndrome was defined as having two of the three major criteria (polyneuropathy, m-protein, and elevated serum vegf level) and at least two of the four minor criteria (extravascular volume overload, skin changes, sclerotic bone lesions, and thrombocytosis) which were determined by logistic regression analyses. according to the criteria the sensitivity was %, and the specificity was %. our results indicate that the proposed criteria have an excellent diagnostic accuracy, and are useful in clinical practice, presumably leading to early diagnosis and treatment. intraepidermal electrical stimulation (ies) is a new technique to that assesses the function of a-delta fibers in the epidermis. using this technique, we previously reported that the epidermal pain threshold was two-hold in asymptomatic diabetic patients than in normal subjects (muscle nerve : - , ). subsequently, we reported that the elevated pain threshold negatively correlated with intraepidermal nerve fiver density (jpns : s , ). empirically, it is known that lowering the skin temperature makes it less likely to feel pain. therefore, it is necessary to investigate whether the results of ies are affected by skin temperature. the aim of this study was to investigate the influence of a low skin temperature on pain threshold. we recruited subjects with a mean age of . years. for nociceptive stimulation, we used an ies method with a concentric micro-needle electrode that was developed specifically for the selective stimulation of cutaneous a-delta fibers. we placed the ies electrode onto the extensor digitorum brevis and began stimulation with intensity strong enough for the subject to feel a pricking sensation, then reduced the current in steps of . ma until no sensation was felt. we defined pain threshold as the minimum electrical intensity at which a subject felt a pricking sensation. firstly, we measured pain threshold at skin temperature above degrees celsius. then, we put an ice pack on the extensor digitorum brevis for min to lower the skin temperature, and measured pain threshold at skin temperatures below degrees. mean pain threshold values above degrees and below degrees of skin temperature were . and . ma (p< . ), respectively. our data indicated an elevated pain threshold in epidermis with a low skin temperature. one of most common methods for nociceptive stimulation is painful co laser stimulation. some co laser stimulation studies reported pain threshold increased with a low skin temperature. our result is similar to that of co laser stimulation. pain threshold using ies is very easy and non-invasive technique. it may be useful for the evaluation of small fiber neuropathy. svačina mkr , röth p , bobylev i , sprenger a , zhang g , sheikh ka , lehmann hc . department of neurology, university hospital cologne, cologne, germany; department of neurology, university of texas, houston, usa. intravenous immunoglobulins (ivig) are an effective treatment in guillain-barré-syndrome (gbs). in most patients, the optimal ivig dose and regime is unknown. in serum and ivig preparations, immunoglobulin (ig) g form igg dimers, which are assumed to consist of idiotypic/anti-idiotypic antibody pairs. however, data about kinetics of igg dimer formation in gbs are lacking. to study igg dimer formation, c bl/ mice were injected with ivig and anti-gd b antibody or pbs. blood sera were collected h, h and week post injection. a third cohort received an anti-gd a/gt b antibody and blood was collected h post injection. igg was extracted and subtyped into polymeric, dimeric and monomeric fractions using the Äkta fplc system. dialysed dimeric and monomeric igg fractions were examined for the presence of anti-ganglioside antibodies by anti-ganglioside antibody elisa. further, blood samples from gbs patients were collected before (pre-ivig) and after treatment with ivig (post ivig). serum samples were examined for igg dimers and monomers using the Äkta fplc system. in the mouse model, a maximum peak of igg dimer formation was observed h post injection. in gbs patients' samples, igg serum levels and igg dimer content was significantly higher after treatment with ivig. we demonstrate here the feasibility to assess igg dimer formation in an animal model and in gbs patients' samples after treatment with ivig. h after ivig treatment appears to be the optimal time point to assess igg dimer formation. further studies are warranted to determine the utility of igg dimer formation as surrogate marker for treatment response in gbs. svaren j , moran jj , wu x , gutmann l , shy m . university of wisconsin-madison, madison, wisconsin, usa; university of iowa, iowa city, iowa, usa. development of outcome measures for clinical trials in cmt a is a major challenge given the slowly progressive nature of the disease. outcome measures can be used to measure a) target engagement for a given therapy, as well as b) disease process and c) disease burden. several candidate therapies have been shown to reduce pmp levels in cmt a rodent models and thereby ameliorate the symptoms of pmp overexpression. measuring pmp mrna reduction in human trials has so far been limited to analysis of skin biopsies by qrt-pcr, which did not demonstrate clear elevations of pmp mrna during, nor a reduction following, ascorbic acid trials. the analysis of skin biopsies is hampered by variable amounts of schwann cells (sc) in skin biopsies, as well as the variable amount of pmp in sc as previously established by immuno-em in cmt a skin biopsies (katona et al., ) . therefore, it is important to develop optimal normalization criteria to address the variability inherent in skin biopsy analysis. ideally this will employ normalization to sc-specific genes that are not altered by cmt a status. to optimize normalization, we have performed rna-seq analysis of skin biopsies from patient and control skin biopsy samples. analysis of these data after normalization to read depth indicated that pmp levels were . fold higher in cmt a patient samples compared to control skin biopsies. however, there was significant variability in pmp levels particularly in cmt a samples, which may be due to variable amounts of schwann cells in cmt a skin. using a combination of sc-specific genes for normalization, we were able to reduce the apparent variability and optimize the differential levels between cmt a and control skin biopsy samples. we also identified other sc-specific genes that were apparently induced in cmt a skin biopsies relative to control. these studies provide a new framework for gene expression analysis in skin biopsies, enabling more precise evaluation of pmp levels in clinical trials for cmt a as a measure of target engagement. in addition, the normalization framework may also be applicable to other types of cmt. chronic inflammatory demyelinating polyneuropathy (cidp) is the most common chronic autoimmune neuropathy, with an estimated prevalence of between and per , people. it can cause temporary disability in the affected individuals and may eventually lead to permanent disability or death. cidp is commonly treated with intravenous immunoglobulin (ivig) therapy or corticosteroids. octagam ® % is licensed for cidp in france, while octagam ® % is licensed for cidp in germany and belgium. this analysis presents data from three open, multicenter, non-interventional, single-arm, non-controlled studies of a post-authorisation safety surveillance (pass) program for the subset of patients receiving octagam ® % or % for neurological indications, focusing on patients with cidp. briefly, data from in-and out-patients in austria, france, germany, and uk treated with octagam ® for neurological disorders were collected by physicians and analyzed to assess safety and tolerability of the treatment. of patients included in the three studies, patients ( . %) received octagam ® for neurological indications, of which patients ( . %; mean age . years [range - ]) had cidp. the mean dose of octagam ® per course was . g/kg bw for patients with cidp; for the other neurologic indications, the dose ranged from . (for multiple sclerosis) to . g/kg bw (for guillain-barré syndrome). premedication was not needed in . % of these patients. the development of clinical appearance since last observation (mean: every . months) was assessed for of the cidp patients by their treating physicians. the majority of observations ( . %) assessed the patients as stable and . % showed even an improved clinical appearance. only . % of the observation periods resulted in deteriorations. adverse drug reactions were rare: of the infusions received by patients with neurological disorders, . % of infusions were associated with an adr ( . % of infusions in cidp patients). overall, treatment with octagam ® was effective and well-tolerated in patients with cidp. these results are consistent with data for the overall patient population (including patients with primary and secondary immunodeficiencies, dermatological and other diseases). szepanowski f , szepanowski lp , kleinschnitz c , kieseier bc , stettner m . department of neurology, medical faculty, university duisburg-essen, essen, germany; department of neurology, medical faculty, heinrich-heine-university, düsseldorf, germany. lysophosphatidic acid (lpa) is a pleiotropic signaling lipid that acts as ligand for at least six specific g protein coupled receptors. schwann cells (sc) are known to mainly express the lpa receptor subtype. an emerging body of in vivo evidence has linked lpa with injury induced peripheral nerve demyelination as well as neuropathic pain. however, the molecular mechanism underlying its demyelinating effect has remained largely unclear. myelinated dorsal root ganglia (drg) cultures were treated either with lpa, lpa + am (lpa antagonist) or vehicle. we assessed myelin basic protein, tumor necrosis factor alpha (tnf-alpha) as well as the sc differentiation marker sox by immunocytochemistry. additionally, myelin was investigated by sudan black staining. to better understand the relevance of lpa signaling for demyelination in vivo, we performed sciatic nerve crush in c bl/ mice treated with am at mg/kg in order to study schwann cell expression of tnf-alpha, sox and sox , a marker for sc dedifferentiation, by immunohistochemistry. in drg cultures, lpa caused a significant reduction of myelin as demonstrated by both sudan black staining and immunocytochemical analysis of myelin basic protein. demyelination was paralleled by an upregulation of tnf-alpha as well as downregulation of sox . lpa mediated effects were found to be blocked by addition of the lpa receptor antagonist am . in c bl/ mice, am treatment prior to crush injury increased sox expression in scs in the distal nerve stump while reducing the number of cells expressing sox . these data indicate that lpa may be a critical factor to shift scs towards an injury-associated phenotype and contribute to the onset of wallerian degeneration. szepanowski lp , szepanowski f , kleinschnitz c , stettner m . department of neurology, university hospital essen, essen, germany. glyphosate-based formulations comprise the world's most commonly used herbicides. in non-resistant plants, glyphosate exerts toxic effects most likely via inhibition of aromatic amino acid synthesis by interfering with the shikimate pathway. while glyphosate is the active ingredient, herbicidal formulations contain several adjuvants, including polyethoxlated alkylamines (poeas). although glyphosate has long been considered safe for use in humans and animals, several studies have implicated glyphosate and/or the commonly used adjuvants in cytotoxicity, carcinogenicity and endocrine disruption. furthermore, glyphosate-based herbicide has been reported to mediate neurotoxicity in immature rat hippocampus involving glutamate excitotoxicity. however, it remains unclear whether glyphosate alone or in combination with its adjuvants may have detrimental effects on myelin integrity in the peripheral nervous system. myelinated dorsal root ganglia (drg) cultures were treated over the course of ten days with either pure glyphosate or a glyphosate-based herbicide at concentrations of . %, . % and . %. the concentration of the glyphosate-based herbicide was matched with regard to glyphosate content ( %). controls were treated with equal amounts of vehicle adjusted for the ph. subsequently, cultures were stained with sudan black and myelin content was assessed by determining the number of internodes per neurons. while glyphosate, regardless of its concentration, did not show any effect on myelin content, the glyphosate-based herbicide caused significant demyelination in a concentration-dependent manner. notably, at . %, drg cultures were completely devoid of myelin and appeared severely necrotic. these data raise the possibility that not glyphosate itself, but rather the adjuvants in glyphosate-based herbicide formulations may cause demyelination. the open question whether demyelination is a direct effect of the adjuvants or a consequence of increased cellular glyphosate uptake due to permeabilization warrants further investigation. tan cy , tan mp , yeoh ky , goh kj , shahrizaila n . department of medicine, university of malaya, kuala lumpur, malaysia. in guillain-barré syndrome (gbs), autonomic dysfunction is common and accounts for significant morbidity and mortality. there have been many studies investigating the electrodiagnosis of gbs but few have studied autonomic dysfunction in gbs. the current study comprehensively investigates quantitative autonomic function in patients with gbs and its variant. ten gbs patients were prospectively recruited and the results were compared to age-and gender-matched healthy controls. a series of autonomic function tests including computational (power spectrum analysis of heart rate variability (hrv) and baroreflex sensitivity (brs) at rest) and challenge tests (deep breathing, eyeball compression, active standing, valsalva manoeuvre, isometric exercise and ice-water hand immersion) were performed. parasympathetic function was represented by high frequency (hf) hrv, heart rate responses to deep breathing, eyeball compression, valsalva manoeuvre and active standing. sympathetic function was represented by low frequency (lf) hrv, blood pressure responses to active standing, sustained handgrip and ice-water hand immersion. in the frequency domain analysis of hrv, low frequency (lf: . ± . vs . ± . ; p= . ), high frequency (hf: . ± . vs . ± . ; p= . ) and total power spectral densities (psd: . ± . vs . ± . ; p= . ) were significantly reduced in patients compared to controls. the mean up slope ( . ± . vs . ± . ; p= . ), down slope ( . ± . vs . ± . ; p= . ) and total brs slope ( . ± . vs . ± . ; p= . ) were significantly lower in the gbs group. the diastolic rise in blood pressure upon ice-water hand immersion was significantly lower in gbs group compared to controls ( . ± . vs . ± . ; p= . ). our findings suggest that computation dependent tests (hrv and brs) were sensitive at detecting autonomic dysfunction and baroreceptor reflex insensitivity in gbs patients. in contrast, ice-water hand immersion was the only reliable challenge test making it useful as a bedside measure of autonomic function in gbs patients. sjögren's syndrome (ss) is an autoimmune disease that affects both east and west. nevertheless, we still have limited knowledge of how autoantibodies in ss affects the peripheral nervous system. in this study, we investigated the peripheral neuropathy in ss and sicca complex using the nerve excitability test, to elucidate how peripheral nerves are affected. we have enrolled a total of patients with ss or sicca complex. of these, two patients were excluded due to co-morbid carpal tunnel syndrome. each patient received clinical evaluation, examination for ssa/ssb antibodies titer, the nerve excitability test, conventional thermal quantitative sensory test, and conventional nerve conduction study. compared to normal control subjects, motor nerve excitability test of ss patients with positive ssa or ssb antibodies (n = ) were found to have increased rheobase (p< . ), increased relative refractory period (rrp) (p< . ), increased refractoriness at . ms (p< . ), increased accommodation toward depolarizing current in threshold electrotonus (te) (p< . ), and decreased superexcitability (p< . ). the sensory axonal study in seropositive ss also revealed increased rrp (p< . ), increased refractoriness at . ms (p< . ), and increased accommodation toward hyperpolarizing current in threshold electrotonus (te) (p< . ). meanwhile, in seronegative ss and sicca complex (n = ), we found no significant axonal properties changes. the present study revealed that peripheral nerves are affected differently in seropositive ss and in seronegative ss/sicca complex. in seropositive ss, motor axons tended to be depolarized, and both sensory and motor axons have increased refractoriness. the findings suggested that ssa and ssb antibodies might play a role in the inactivation of transient sodium channels. the effects of the antibodies on transient sodium channels might be the basis of peripheral neuropathies and even cardiac arrhythmias and heart block in ss. charcot-marie-tooth disease type a (cmt a), caused by the pmp duplication on chromosome p . , is the most common subtype of inherited peripheral neuropathies and affects in , individuals worldwide. while sharing the same genetic cause, cmt a patients often present great variability in their phenotypic presentation and disease severity. the cause of the phenotypic variability is largely unclear. in this study, we performed genome-wide association study (gwas) to identify novel genetic modifiers of various phenotypes in cmt a. dna samples from cmt a patients were genotyped on illumina omniexpress platform. after standard quality control, the dataset includes k markers in individuals ( individuals from european ancestry, and individuals from asian ancestry). we focused our analyses on the european population. logistic regression in plink was used to analyze the association between the clinical outcomes and patients' genotypes in an additive model. for cmt neuropathy score (cmtns), the analysis was performed using linear regression in plink, adjusting for patients' age. the analyses yielded several suggestive association signals. an association peak on chromosome was identified in difficulty with eating utensils (lead snp rs , chr : , p= . e- , odds ratio= . ). the peak is located within a non-coding gene linc . hearing loss showed an association peak on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ), located in an intergenic region near the megf gene. in foot plantar flexion, an association signal was identified in the dscam gene on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ). cmtns showed an association signal on chromosome (lead snp rs , chr : , p= . e- , beta= . ), located within an intergenic region close to dffb, c orf , and linc . while these suggestive signals require further validation, our study provides novel insights into the genetic architecture of cmt a. novel genetic modifiers may serve as potential targets for therapeutic interventions in the future. teng a , ohnmar , kalpana p , chai yh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. we present an intriguing diagnostic puzzle, that was eventually cracked serendipitously. a -year-old man was seen for bilateral ptosis. evaluation for myasthenia gravis was negative. nevertheless, a diagnosis of ocular myasthenia gravis was made and he was put on pyridostigmine. he did not respond. the ptosis progressively worsened. he sought a second opinion. at this evaluation, he was noted to have complex ophthalmoplegia without diplopia, bilateral facial weakness and mild bulbar weakness. he had no sensory complaints. the limb examination was remarkable for slightly reduced reflexes, normal strength, and other than increased vibration threshold at the toes, intact sensory examination. repeat serological and electrodiagnostic work-up for myasthenia gravis was negative. a myopathic disorder such as chronic progressive external ophthalmoplegia was considered. serum creatine kinase and lactate were normal. he underwent biceps muscle biopsy which showed increased cox-negative and sdh positive fibers, supporting the then clinical impression of a mitochondrial cytopathy. at this point, he underwent blepharoplasty to improve his vision. routine histological examination of the levator palpebrae muscle showed amyloid deposits. this prompted a review of the earlier biceps biopsy, which revealed amyloid deposits that were not appreciated before. at this point, the significance of the patient and his son's history of lattice corneal dystrophy became apparent. he also reported that his late mother had similar facial appearance as his. the patient's nerve conduction study showed length-dependent sensory axonal polyneuropathy, right carpal tunnel syndrome and bilateral facial neuropathy. he had no definite symptoms of autonomic neuropathy. cardiac evaluation was unremarkable. the final diagnosis of familial gelsolin amyloid polyneuropathy was made. genetic confirmation for the patient and his family is being planned. we highlight the key clinical features of gelsolin neuropathy. the symmetric cranial neuropathy can resemble a muscle or neuromuscular junction disorder and the relative sparing of the cardiac muscle, somatic and autonomic nerves contrasts with transthyretin-related amyloid polyneuropathy. neuropathy is one of the most common long-term complications of diabetes. furthermore, % to % of diabetic neuropathy patients will develop neuropathic pain. the pathophysiology of neuropathic pain in diabetic peripheral neuropathy is complex and not fully understood. a potential mechanism is a change in voltage gated sodium channels, such as nav . . loss of function mutations in this channel cause insensitivity to pain, whereas gain of function mutations have been linked with different pain syndromes including small fiber neuropathy. in a cohort of patients with diabetic peripheral neuropathy we investigated whether mutations in nav . were associated with diabetic neuropathic pain. twelve nav . variants were identified in nine participants all within a cohort of participants with painful diabetic peripheral neuropathy. five of these variants were previously associated with pain disorders: v l, m l; w r, r h, l v. among the other variants two of them met the criteria of potential pathogenicity based on predictive algorithms and were further studied. functional analysis by whole cell patch clamp showed that one of these variants (m t) drastically impairs the inactivation of the channel by shifting the steady-state fast-inactivation towards more depolarizing potentials. there were no phenotypic difference between those participants with pathogenic variants and those participants without pathogenic variants. no rare nav . variants were found in participants with painless diabetic peripheral neuropathy. these observations suggest that mutations in nav . may contribute to painful diabetic peripheral neuropathy. tholance y , rosier c , f bouhour , psimaras d , kuntzer t , taieb g , créange a , delmont e , camdessanché jp , antoine jc . university hospital, saint-etienne, france; university hospital, lyon, france; university hospital, paris, france; university hospital, lausanne, switzerland; university hospital, montpellier, france; university hospital, creteil, france; university hospital, marseille, france. dysimmune sensory neuronopathies (snn) encompass paraneoplastic snn and snn associated with systemic autoimmune diseases such as sjögren syndrome, lupus or inflammatory bowel or rheumatic diseases but also a number of apparently idiopathic cases. biomarker antibodies are well-known in paraneoplastic snn but are lacking in non paraneoplastic cases. from a mono-center retrospective study we identified in anti-fgfr antibody as a potential biomarker of dysimmunity in patients with idiopathic or systemic autoimmune disease associated sensory neuropathy. the identified patients were more frequently women and had a non lenthg dependent neuropathy suggestive of snn. anti-fgfr antibody was the only immunological marker in / of cases at initial work-up although / of patients eventually developed with time systemic autoimmune disease. to confirm the incidence and the clinical pattern of patients with anti-fgfr antibodies we launched a prospective multicenter french study including patients with a sensory neuropathy suspected to be a snn of no paraneoplastic, genetic or metabolic origin. we present here the results on the first included patients compared to healthy blood donors. anti-fgcr antibodies were searched by elisa using the trk intracellular domain of the protein (invitrogen©). we found patients positive for anti-fgfr antibody ( . %). these patients were women and men aged . years as a mean ( - ). the neuropathy was acute and subacute in one patient respectively and progressive in the others. six patients fulfilled the diagnosis criteria of snn and the last one had a sensory neuropathy in the lower limb with abnormal sensory action potentials in the four limbs suggesting snn without reaching the requested criteria. one patient developed uveitis which is a new symptom with anti-fgfr ab. an unclassified dysimmune context was present at the initial work up in patients and one patient developed sjögren syndrome with follow-up. as a whole the clinical pattern of these patients is consistent with that of the initially published series. the lower prevalence of positive sera may be due to more stringent criteria used for elisa but needs to be confirmed on the complete prospective series. peripheral neuropathy research registry (pnrr) neurological assessment was scored using the total neuropathy score clinical version (tnsc), comprising pinprick and vibration sensibility, deep tendon reflexes, strength and patient symptom report. compound sensory action potential (csap) amplitudes were recorded antidromically at the lateral malleolus, stimulating the sural nerve at the mid-calf. of the total sample, % reported lower limb neuropathy, with % of patients reporting 'quite a bit' or 'very much' severity of tingling and numbness in their feet. the average sural csap amplitude was . ± . v and % of patients had sural amplitudes below the lower limit of normal for age. the total tnsc score correlated with the pro fact-gog ntx score (r = −. , p<. ) and sural amplitude (r = −. , p <. ). vibration sensibility correlated with the overall fact-gog ntx score (r =−. , p <. ), and sural amplitude (r = −. , p <. ). sural amplitude correlated with patient reported severity of numbness and tingling in the lower limbs (r =−. , p <. ) but not with the overall fact-gog ntx score. patient reports of neuropathic symptoms in the lower limb correlate with both objective neurophysiological and clinical measures of neuropathy severity. identifying links between objective neurophysiological markers and patient reported outcomes are critical to assess the impact of clinical interventions. tomaselli pj , gouvea sp , nyshyama kfs , nicolau n jr , lourenço cm , marques w jr , . division of neuromuscular diseases, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; neurogenetics, department of neurosciences and behaviour sciences, university of são paulo, ribeirão preto, brazil. mutations in the gab junction beta -protein gene (gjb ) are the second most frequent cause of charcot-marie-tooth disease (cmt), accounting for approximately % of cmt cases worldwide. the gjb codes for connexin protein (cx ). in the peripheral nervous system, the cx is expressed in the schwann cells and allows intercellular traffic of ions and small molecules between opposed cells. we analysed retrospectively detailed clinical and neurophysiological data of five families carrying novel gjb mutation submitted for testing at our neurogenetics laboratory. mutations were identified by bidirectional sanger sequence analysis of gjb coding region. we identified a total of subjects from five different kindreds with novel mutations (p.a v, p.l w, p.l q, p.f s, p.r l). these five novel mutations segregate with phenotype, are located in highly conserved amino acids among gjb and other gab junction protein sequences and among different species, are not present in any public database (exac, dbsnp and genome database), and were not found in normal brazilian controls. in silico analysis, predict these variants to be pathogenic, there was no male-to-male transmission; males were more severely affected than females. four out seven female have subclinical neuropathy and were only identified after clinical and electrophysiological evaluation. the conduction velocities were in the intermediated range in the males patients and higher in the females included in this study. we describe five new pathogenic mutations causing cmtx in a brazilian population and expand the number of causative mutations in the gjb gene. funded pure neural leprosy (pnl) is a slowly progressive, predominantly sensory patchy neuropathy presenting with positive and/or negative sensory symptoms, which are usually followed over time by distal asymmetrical weakness. despite rare, monomelic involvement in leprosy has already been reported. we sought to describe the clinical and electrophysiological patterns of an unusual leprosy neuropathy presentation. clinical data were retrospectively collected from nine patients who had monomelic involvement and were referred for further investigation to the emg lab. seven out nine patients were male. four patients had a brachial plexus like presentation and five have a lumbosacral plexus like presentation. the initial complaint was hypoesthesia in four patients, tingling in two patients and hypoesthesia with tingling in two patients. severe pain was observed in just one patient. all individuals from the group of patients with lumbosacral plexus-like presentation and three with brachial plexus-like presentation had no sensory nerve action potentials (snaps) for all nerves tested in the affected limb with or without motor involvement. one patient with brachial plexus-like presentation had focal slowing of conduction velocity with temporal dispersion of both median and ulnar nerves in the affected limb. one patient with plexus-like presentation had snaps with low amplitude of all nerves in the affect limb. the diagnosis of leprosy was confirmed by nerve biopsy findings, anti-pgl antibody, and positive response to specific treatment. nerve biopsy was performed in four patients, and the bacillus was found in two. the anti-pgl antibody was positive in four patients. plexus mri was performed in two patients and was normal. we found the distribution of motor and sensory symptoms were restricted to on limb in this group of patients. as a typically patchy disorder pnl may affect any nerve, although the reason why damage are restrict to only one limb has to be elucidated. the description of these cases increases the clinical spectrum of leprosy neuropathy. this possibility should be considered in the differential diagnosis of patients with plexopathy from endemic areas after excluding other causes. funded ataxia with oculomotor apraxia type (aoa ) is a very complex disorder characterized by an early-onset progressive cerebellar ataxia with cerebellar atrophy and peripheral neuropathy and it is caused by recessive mutations in the aprataxin gene (aptx ). when the neuropathy is present, it has been described in % of cases as primarily axonal. we describe a case of aoa due compound heterozygous mutations in aptx associated with demyelinating neuropathy. the patient was born from healthy and non-consanguineous parents and presented in the first decade with progressive cerebellar ataxia, multidirectional ophtalmoparesia, oculomotor apraxia, choreiform movements of limbs and peripheral neuropathy. he had normal cognition and stopped walking at age of . blood tests were unremarkable with normal levels of leucocytes, serum proteins, immunoglobulin, cholesterol, vitamin e, and alfa-feto protein. brain mri showed severe cerebellar atrophy. the motor conduction velocity in the upper limbs was slow with preserved amplitudes. the distal latencies and the minimal f wave latencies were prolonged. there was no evidence for superimposed acquired demyelinating neuropathy. direct sequencing of the aptx gene revealed two variants, c. - a>g, p? and c. g>a; p.w x. the first variant is novel and affects a highly conserved acceptor splice site of exon . the other variant is the most common portuguese variant, the nonsense mutation w x is located in exon . parental dna was tested and confirmed the variants were in different alleles. the presence of a demyelinating neuropathy in aoa suggests that phenotypic variability in this condition may be larger than previously considered. at the same time, it increases the differential diagnosis of inherited conditions with cerebellar ataxia and demyelinating neuropathy. finally, this finding opens the functional effects of the aptx gene. funded by: cnpq, fapesp, faepa, pronas (ministry of health). topa a , spina e , iodice r , tozza s , ruggiero l , dubbioso r , esposito m , santoro l , manganelli f . university of naples "federico ii", naples, italy. we report our -year experience of subcutaneous immunoglobulin (scig) in a cohort of patients with chronic inflammatory demyelinating polyneuropathy (cidp) from a tertiary care neuromuscular center. we analyzed data from cidp patients ( males and females, mean age: ± . years; mean age at onset: . ± . years; disease duration: . ± . years) treated with scig and with a follow-up period of months. all patients were previously responders to intravenous immunoglobulin (ivig). eight patients had a typical cidp and five patients had an atypical variant of cidp. five patients switched to weekly maintenance scig therapy (continuous regimen) because of short-lasting response to ivig therapy. eight patients with a longer lasting response to ivig received scig with a pulsed regimen similar to that used for ivig (from to cycles per year); seven of them because of difficulty in hospitalization and one for allergic reaction to ivig. changes in clinical status were assessed over the period of follow-up by using clinical evaluation of muscle strength, modified rankin scale, overall neuropathy scale and inflammatory neuropathy cause and treatmentsum score. in patients we evaluated also six minute walking test, hole-peg-test and meter walking test. all the five patients treated with a continuous regimen of scig remained clinically stable throughout the follow-up period. among the patients receiving pulsed scig treatment, out of ( %) responded to scig similarly to ivig, while three patients ( . %) worsened and needed to be treated again with ivig and the other one ( . %) stopped any therapy. subcutaneously administered immunoglobulin were well tolerated and no patients complained of adverse events. in conclusion, our findings confirm that continuous scig therapy is efficacious in maintaining clinical stability in patients with short-lasting response to ivig. moreover, our data suggest that pulsed therapy with scig may represent an alternative therapeutic option for the treatment of a subset of cidp patients. touvier t , ferri c , mastrangelo r , glimcher l , , wrabetz l , , , d'antonio m . myelin biology unit, division of genetics and cell biology, san raffaele scientific institute, dibit, milan, italy; department of cancer immunology and virology, dana-farber cancer institute, boston, usa; department of medicine, harvard medical school, boston, usa; hunter james kelly research institute, university at buffalo, buffalo, usa; department of biochemistry, university at buffalo, buffalo, usa; neurology, jacobs school of medicine and biomedical sciences, university at buffalo, buffalo, usa. mpz glycoprotein is an abundant product of terminal differentiation in myelinating schwann cells. the mutant mpzs del causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in transgenic mice. mpzs del protein is retained in the endoplasmic reticulum (er) of schwann cells and induces an unfolded protein response (upr) characterized by activation of perk, atf and ire /xbp pathways. we have previously reported that activation of chop and gadd , two downstream mediators of perk, is pathogenetic in mpzs del mice (pennuto, ; d' antonio, ) but the role of the other upr branches remains to be investigated. in this study, we investigated the role of the er stress sensor enzyme ire and of xbp -a transcription factor specifically activated by ire -in mpzs del pathogenesis. we generated a new mouse model with schwann cells-specific ablation of xbp and in parallel we exploited mpzs del dorsal root ganglia (drg) explant cultures in which xbp signaling is modulated by gain/loss of function approaches. we observed that absence of xbp dramatically worsens hypomyelination and electrophysiological/locomotor parameters in young and adult mpzs del neuropathic animals. interestingly we observed that perk, atf and ire -mediated ridd signalings are upregulated in neuropathic animals lacking xbp . this suggests that activation of xbp targets have an essential role in limiting mpzs del toxicity, which cannot be compensated by other stress responses. moreover, we demonstrated in mpzs del drg cultures that inhibition of xbp splicing by u c (cross, ) decreases myelination whereas activation of xbp splicing by quercetin (wiseman, ) slightly ameliorates myelination. altogether, these data demonstrate that xbp pathway has a critical adaptive role in mpzs del neuropathy and suggest that activation of this pathway may be beneficial for cmt b and perhaps for a broad range of neuropathies characterized by upr activation. tozza s , bruzzese d , iodice r , esposito m , dubbioso r , ruggiero l , topa a , spina e , santoro l , manganelli f . department of neuroscience, reproductive sciences and odontostomatology, university of naples "federico ii", naples, italy; department of public health, university federico ii of naples, naples, italy. in cmt a patients, the clinical impairment progressively increases over time and correlates with the axonal loss. evidence has suggested that the decline of physical performance in cmt a patients may reflect a process of normal ageing. the aim of our study was to describe, by a case-control cross-sectional design, the progression of physical impairment with ageing in cmt a patients. we enrolled cmt a patients ( m; range - years) and sex-and age-matched healthy controls. to assess physical performance, all patients and controls underwent -meter walk test ( mwt), -minute walk test ( mwt) and -hole peg test ( hpt) of their dominant (d) and non-dominant (nd) sides. moreover, to assess clinical disability, impairment and quality of life in the cmt a group we used the charcot-marie-tooth neuropathy score (cmtns), the mrc sum score and the short form- (sf- ) questionnaire. the linear regression model was used to evaluate the changes over time of clinical measures in patients and controls. the chow test was used to determine whether the ageing had a different impact on clinical measures for the two groups. physical performance worsened with ageing in both patients and controls, but with a greater slope for cmt a patients [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. the rate of deterioration of physical performance was not different between patients and controls until the th year of age. after the th year of age the rate of deterioration became greater in cmt a group [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. moreover, in cmt a patients also cmtns, mrc sum score and sf- worsened with ageing and with a greater rate of deterioration after the th year of age. our study demonstrates that clinical decline in cmt a patients goes parallel to the normal ageing process until the th year of age, whereupon the clinical deterioration accelerates. tsouni p , devic p , moura b , planque e , bédat-millet al , devaux j , steck aj , delmont e , hottinger af , kuntzer t . dcn, chuv, lausanne, switzerland; centre de référence maladies neuromusculaires, hospices civils de lyon, lyon, france; cabinet médical, epinal, france; département de neurologie, chu de rouen, rouen, france; cnrs, crn m-umr , université aix-marseille, marseille, france; centre de référence maladies neuromusculaires et sla, hôpital la timone, marseille, france. new therapeutic options in immuno-oncology have allowed significant progress in the management of melanomas. treatment usually consists of a combination of two monoclonal antibodies targeting cytotoxic lymphocyte-associated protein and programmed cell death- . as a result, the immunologic barrier protecting tumor cells is overcome allowing an antitumor response. we report cancer patients with immune checkpoint inhibitor-induced neuropathies as a complication of this immunomodulating oncologic treatment. case reports: our index patient developed severe myalgia days after introduction of ipilimumab-nivolumab followed by painful paresthesias of the face and extremities day after the nd cycle of treatment. generalized areflexic quadriparesis (mrc ) with gowers'sign and distal loss of vibratory sensation were found. a -day course of ivig plus corticosteroids (cs) had no effect. three monthly ivig courses were necessary to improve the deficits at months. a survey of sfnp members revealed other patients with similar acute courses but with phenotypes varying from sensorimotor deficits with areflexia and myalgia to purely sensory ataxic forms following immunomodulating treatment. work-up including anti-nodal antibodies were negative in patients. from the other , had abnormal csf and had necrotizing myopathy. detailed repeat ncs demonstrated signs of nerve hyperexcitability and of demyelination or conduction blocks. evolution was slowly favorable following ivig and cs. discussion: our report underscores that atypical acute generalized demyelinating neuropathies are induced by these novel treatments. they may be associated with severe myalgia or other systemic toxic effects. discontinuation of the oncologic treatment depends on severity of symptoms. outcome was slowly favorable following ivig or cs, albeit slower than in the case of primary inflammatory neuropathies, probably given the long half-life of the monoclonal antibodies. gap junctions (gjs) are membrane channels found in most tissues connecting adjacent cells or different cell compartments as in schwann cells. they are involved in electrical connectivity and metabolic homeostasis allowing the passage of small molecules such as ions, second messengers, nucleotides and peptides. an important functional role of peripheral nerve connexins is suggested by their involvement in x-linked inherited neuropathy as well as in acquired neuropathy caused by oxaliplatin. although gjs play a role in electrical connectivity their specific role in the formation of the sciatic nerve compound action potential (cap) remains unclear. the aim of this study was to investigate the role of peripheral nerve connexins in the electrical responses of the mouse sciatic nerve under normal and stress conditions. for this purpose we used sciatic nerves of three different mouse models, the cx knockout (ko), cx ko and the cx /cx double knockout (dko) mice. using our ex vivo model for extracellular recordings we exposed sciatic nerves from different genotypes to three different gj blockers: octanol, -beta-glycyrrhetinic acid (gra) and octanoic acid (oa) and recorded the cap. amplitude and duration of the cap were used as an indication for the effects of the different blockers on the cap formation. all gj blockers caused a gradual decrease of the cap without any changes in the duration of the cap in all genotypes, suggesting progressive disturbance of axonal membrane excitability in the absence of one or two gj proteins. comparison of the three genotypes showed that cx may play a dominant role in the maintenance of the cap formation since nerves from cx ko mice proved to be more sensitive to the gj blockers compared to the cx ko nerves showing a faster decline of the cap amplitude. moreover the effect of gj blockers was similar in cx ko and dko nerves. finally, the effect of gj blockers on the dko nerves implies the presence of another gj protein. in conclusion, our results confirm the direct functional involvement of cx gj channels and cx hemichannels in the cap formation and indicate the existence of at least one more connexin in peripheral nerve. ca( +)-dependent anti-ganglioside antibody in seronegative guillain-barrÉ syndrome uchibori a , gyohda a , chiba a . kyorin university, tokyo, japan. we have reported ca + -dependent igg anti-ganglioside gq b antibodies in gq b-seronegative patients with fisher syndrome and its related disorders (fs-rd). in patients with fs-rd who were gq b-seronegative in conventional assays using phosphate-buffered saline without ca + , % turned seropositive for gq b-related antigens in assays using ca + -added tris-buffered saline. objective: we investigated whether ca + -dependent anti-ganglioside antibodies was present also in ganglioside-seronegative patients with other clinical disease types of guillain-barré syndrome (gbs) other than fs-rd. methods: the subjects were the following: patients with final clinical diagnosis as gbs (acute motor axonal neuropathy [aman], n = , and acute inflammatory demyelinating polyradiculoneuropathy [aidp], n = ), and patients with final clinical diagnosis as sensory ataxic neuropathy (san), n = . all subjects were ganglioside-seronegative in the conventional assays. we assayed serum igg antibodies against various gangliosides (including asialo-gm ) in elisa using tris-buffered saline as a basal buffer in both ca + -added and -non-added conditions. increase of the optical density (od) more than . in ca + -added condition compared with ca + -non-added one was taken significant, i.e. positive for ca + -dependent antibody. results: ca + -dependent antibody was negative in all aman and san patients. in aidp, the antibody titers (ods) against gainac-gd a were significantly increased in patients, and those against asialo-gm were increased in other patients in ca + -added condition. however, the titiers of those ca + -dependent antibodies were all at low level. conclusions: in clinical disease types of gbs other than fs-rd, ca + -dependent igg antibodies against ganglioside were detected in a few patients with aidp, but the positive rate and the antibody titers were low compared with case of gq b-seronegative fs-rd. ca + -dependent antibodies against ganglioside are considered to be more specific for gq b. the aim of the study was to investigate the relationship between median sensory conduction of median nerve and ulnar nerve in patients diagnosed with carpal tunnel syndrome. two hundred and eighty-six hands with carpal tunnel syndrome and hands in control group were investigated. patients were staged clinically and electrophysiologically. diagnosis of carpal tunnel syndrome was made according to the presence of paresthesia, pain in the innervation area of the median nerve, weakness and atrophy in the median nerve innervated muscles, positive phalen and tinel tests. median motor and sensorial nerve conduction study, including first, second, third finger and palm, and ulnar motor and sensorial nerve conduction of fifth finger studies were performed to all patients and control group. the ratio of distal latency and velocity of nerve conduction of first, second, third and palmar branches to fifth finger was calculated. distal latency of first, second, third finger and palm of patients with cts are longer and velocity is more slowly than controls. in addition to these findings, the velocity of fifth finger is also slower and distal latency of this one is longer than healthy subjects. the most sensitive method of classifying the carpal tunnel syndrome as normal, mild and moderate is the ratio of distal latency and velocity of second finger (p< . ). carpal tunnel syndrome is the most common encountered neuropathy. in nerve conduction studies can be used the ratio of distal latency and velocity of second finger as determine the degree of carpal tunnel syndrome. the most surprising finding of this report is the nerve conduction studies of fifth finger. the subclinical susceptibility of fifth finger can be explained by overusing of wrist. ursino g , gemelli c , grandis m , reni l , bellone e , geroldi a , gotta f , mandich p , ferrara m , schenone a dinogmi university of genoa, genoa, italy; irccs-aou san martino hospital genoa, genoa, italy. charcot-marie-tooth (cmt) neuropathy represents a clinically and genetically heterogeneous group of hereditary peripheral neuropathies characterized by chronic motor and sensory impairment. to date mutations in up to genes may cause cmt. the aim of this study is to describe our large population of cmt patients, and, within this, highlight specific phenotypes. the cmt clinic in genova, started in . during the years, patients underwent complete neurological, rehabilitative, neurophysiological examinations and genetic testing. the patients were routinely tested for common genes (as pmp , gjb , mpz, mfn ), while in specific cases we followed the candidate gene approach testing single genes based on the genotype-phenotype correlation. however, the ngs techniques were used when routine genetic testing was negative and a clear genotype-phenotype correlation could not be identified. cases are present to date in our database of cmt patients. in ( . %) patients, in spite of a clinical diagnosis of cmt, a genetic diagnosis is still lacking; ( . %) patients had alternative diagnosis (i.e hereditary spastic paraparesis etc.). instead, in patients ( . %) a defined genetic diagnosis was reached, of them being females ( . %) and males ( . %). among these, except for the more common cmt a, hnpp and cmt x phenotypes, we frequently observed patients affected by cmt b and cmt f. according to most literature data, we observed ( . %) patients with cmt b and patients ( . %) affected by cmt f. at the first visit, the cmt b phenotype was clearly length-dependent: . % patients showed impairment of the lower limbs and saving of the upper limbs; in terms of severity of the neuropathy, the mean cmtns was . and the mean age was . years. similarly, % of patients affected by cmt f, present with the same phenotype; the mean cmtns at the first visit was . and the mean age was . years. in conclusion, based on the experience of the genova cmt clinic, we describe a large population of cmt patients and a specific phenotype in cmt b and f patients, characterized by involvement of the lower limbs and selective sparing of the upper ones, which may help in addressing the diagnostic algorithm. non-freezing cold injury (nfci) develops following sustained exposure to cold temperatures, resulting in tissue cooling but not freezing. this can result in persistent sensory disturbance of the hands and feet including numbness, paraesthesia and chronic pain. both vascular and neurological aetiologies of this pain have been suggested but remain unproven. we prospectively approached patients referred for clinical assessment of chronic pain following non-freezing cold injury between february and november . of patients approached consented to undergo detailed neurological evaluations including: questionnaires to detail pain location and characteristics, structured neurological examination, quantitative sensory testing, nerve conduction studies and skin biopsy for intra-epidermal fibre assessment. of the study participants all had experienced nfci whilst serving in the united kingdom armed services and the majority were of african descent ( . %) and male ( . %). many patients reported multiple exposures to cold. the median time between initial injury and referral was . years. pain was principally localised to the hands and the feet, neuropathic in nature and in all study participants associated with cold hypersensitivity. clinical examination and quantitative sensory testing were consistent with a sensory neuropathy. in all cases large fibre nerve conduction studies were normal. the intra-epidermal nerve fibre density, however, was markedly reduced; ⋅ % of subjects having a count at or below the ⋅ centile of published normative controls. using the neuropathic pain special interest group (neupsig) of the international association for the study of pain (iasp) grading for neuropathic pain % had probable and ⋅ % definite neuropathic pain. chronic non-freezing cold injury is a disabling neuropathic pain disorder due to a sensory neuropathy. why some individuals develop an acute painful sensory neuropathy on sustained cold exposure is not yet known but individuals of african descent appear vulnerable. screening tools, such as the dn questionnaire, and treatment algorithms for neuropathic pain should now be used in the management of these patients. funded by the wellcome trust and the uk ministry of defence guillain-barré syndrome (gbs) is highly heterogeneous regarding clinical presentation, course, electrophysiological subtype and outcome. in part this variety is associated with differences between geographical regions, although this had not been investigated in a single comparative study. one aim of igos is to define the influence of the geographical origin on the heterogeneity of gbs. in igos all gbs patients within weeks of onset can participate, independent of age, variant, severity and treatment. in february , patients were included from countries. the first inclusions in igos were used in this analysis. seventy-five patients ( %) were excluded because of alternative diagnoses (n= , including a-cidp), protocol violations (n= ) or missing data (n= ). of the remaining patients, % were males and % female with a median age of years (iqr - ). at entry % presented with tetraparesis and % with paraparesis. during follow-up % needed mechanical ventilation and % died. of all gbs patients % received treatment ( % ivig, % pe). the remaining % received supportive care only (mild gbs or low social-economic status). antecedent events were reported in % of patients, including upper respiratory tract infection ( %) and gastro-enteritis ( %) as the most frequent events. the pure motor form was the predominant subtype in patients from bangladesh ( %). in europe/americas and asia (without bangladesh) the predominant subtype was the sensorimotor form ( % in europe/americas and % in asia). in asia (without bangladesh) there was a relatively larger proportion of patients with mfs/mfs-overlap syndrome ( %) than in other regions ( - %, p< . ). the proportion of patients able to walk unaided at months after follow-up was % in europe/americas, % in bangladesh and % in asia (without bangladesh). kaplan-meier analysis comparing electrophysiological subtypes of gbs (as reported by neurologist) showed that patients with inexcitable nerves or axonal neuropathy needed more time to regain the ability to walk unaided than the patients with demyelinating or equivocal result (log-rank test, p < . ). these findings demonstrate the extensive geographical differences in gbs. future igos studies will investigate the role of genetic and environmental factors that additionally could explain these differences. van den bergh pyk , attarian s , grapperon am , nicolas g , cassereau j , rajabally ya , delmont e , woodard jl , piéret f and the university of louvain gbs electrodiagnosis study group * . corticosteroids are considered as one of the first line treatments for chronic inflammatory demyelinating polyneuropathy (cidp). different types of corticosteroids are used and there are no comparative studies assessing the improvement rates, remission rates, tolerability and the side effect profiles of these treatment regimens. in addition, there are currently no reliable predictors of favorable treatment response to steroids, which would greatly ease the choice of first line treatment. in this retrospective study we will compare efficacy, tolerability and safety of three different corticosteroid regimens used as first line treatment in three large cidp centers in netherlands, serbia and italy. treatment naïve cidp patients who received either pulsed dexamethasone, pulsed methylprednisolone or daily prednisone will be included in the study. data will be extracted from patient charts. the primary outcome is the percentage of treatment responders at months after start of first treatment, in which treatment response is defined as subjects who improved after treatment and are either without treatment after six months or are still being treated with the first chosen therapy. secondary endpoints include the remission rates and in case of a relapse, the mean duration of remission to relapse; the discontinuation rate within months of treatment due to inefficacy, adverse events or intolerance; and the frequency of adverse events and serious adverse events (sae) during treatment or within month after stopping treatment. furthermore, we will explore the value of previously reported potential predictors of treatment response. results will be presented at the peripheral nerve society meeting . van lieverloo g , , musters a , adrichem m , esveldt r , doorenspleet m , klarenbeek p , van schaik i , de vries n , eftimov f . academic medical center, department of neurology, amsterdam, the netherlands; academic medical center/university of amsterdam, department of clinical rheumatology and immunology, amsterdam, the netherlands. following reports that pathogenic antibodies are present in a minority of patients with chronic demyelinating inflammatory neuropathy (cidp), we studied whether oligoclonal expansions of b-cell clones are present in patients with cidp. recently, we developed a new method for b-cell receptor (bcr) repertoire landscaping based on high throughput sequencing (hts) of rna extracted from blood. bcr repertoire was analyzed in patients with cidp: patients with active disease and starting treatment (group ), patients with stable disease using intravenous immunoglobulin (ivig) treatment in which treatment withdrawal was attempted (group ), and patients in remission (i.e. no treatment in the last months, group ). clinical parameters and sampling was performed at baseline (group , and ), at months after start of treatment (group ), at months or earlier in case of relapse in group and at baseline only in group . most cidp patients had highly expanded bcr clones, regardless of disease activity and response to treatment. however, in group , the most expanded b-cell clones at baseline showed no overlap with the expanded bcr clones after improvement. based on these preliminary data expanded bcr clones are observed in the peripheral blood of most cidp patients, regardless of disease activity (active, stable disease or remission/cure). functional characterization of these expanded clones remains to be performed. van rijs w , fokkink wjr , tio-gillen ap , brem md , jacobs bc , huizinga r . erasmus mc, university medical centre, rotterdam, the netherlands. myeloid cells, including monocytes, macrophages and dendritic cells, are critically involved in the induction of adaptive immune responses, clearance of pathogens and in the initiation of tissue repair. in the guillain-barré syndrome (gbs), macrophages are present in the peripheral nerve, where they phagocytose (damaged) myelin and axons. dendritic cells (dc) are increased in the cerebrospinal fluid of patients with gbs. however, the composition and phenotype of monocytes and dc subsets in the peripheral blood is unclear and it is unknown if these cells can be used as biomarker to monitor disease activity or response to treatment. here we investigated the frequency and phenotype of six myeloid subsets in the peripheral blood mononuclear cells (pbmc) using advanced -color flow cytometry. pbmc were isolated from patients with gbs, before and after immunomodulatory treatment, and age and gender-matched, healthy controls. the frequency of total monocytes, determined as percentage of cd + cells, was increased in gbs patients compared to controls (p< . ). the monocyte population was skewed towards more intermediate (cd +cd +; p< . ) and less non-classical (cd -cd +; p< . ) monocytes. classical (cd +cd -) and intermediate monocytes as well as cd c+ dc expressed significantly higher levels of cd compared to healthy controls. in contrast, the expression of cd and siglec- was significantly higher in the non-classical monocytes of gbs patients compared to controls. no differences were observed in the expression of cd , cd , cd and siglec- . immunomodulatory treatment strongly reduced the frequency of non-classical monocytes and all dc populations in cd + pbmc. the expression of cd , cd c and hla-dr was reduced in classical monocytes after treatment. in addition, siglec- expression was reduced in several monocyte and dc populations after treatment. in summary, our data identify significant changes in the monocyte compartment in gbs. the decrease in non-classical monocytes may suggest that these cells have migrated to peripheral tissues, promoting the differentiation of classical monocytes into intermediate monocytes. further analysis should reveal whether these changes are related to preceding infections, disease severity and response to treatment. in mme. the two mutations were absent from control databases (e.g. exac), affected highly conserved aminoacids and were predicted to have deleterious effects by in silico analysis. unfortunately, both parents were deceased and we were therefore unable to prove that the two mutations reside on separate alleles. mme encodes the metalloprotease neprilysin whose role in peripheral nervous system is still unclear. higuchi and colleagues have described japanese cmt families with late-onset sensory-motor axonal neuropathy and recessive loss-of-function mutations in mme. we report two novel missense mutations in mme in a case of late-onset cmt . we hypothesize an autosomal-recessive mode of inheritance as most likely given the clinical phenotype and the absence of a family history. anti-contactin- (cntn ) antibodies were recently identified in a subgroup of patients with chronic inflammatory demyelinating polyneuropathy (cidp) showing acute/subacute onset of severe sensory-motor neuropathy and poor response to intravenous immunoglobulin (ivig) and corticosteroids. these antibodies belong to the igg isotype and interact with cntn -neurofascin (nf ) complex at paranodes leading to loss of nodal integrity. a -year-old man presented with acute onset of distal weakness in the lower limbs, four limbs paraesthesias and sensory ataxia. at clinical examination ankle swelling was also observed. nerve conduction study showed a demyelinating polyneuropathy and cerebrospinal-fluid examination revealed cyto-albuminologic dissociation. sural nerve biopsy disclosed diffuse loss of myelinated fibres. at routine blood test serum albumin was reduced and proteinuria was gr/ hours, thus leading to the diagnosis of nephrotic syndrome. kidney biopsy showed changes consistent with membranous glomerulonephritis, together with sub-epithelial deposits of immune complexes and complement deposition. treatment with ivig and corticosteroids did not improve neurological status, while membranous glomerulonephritis showed moderate response to ivig. a six-month course of cyclophosphamide was started leading to normalization of renal function and muscle strength and partial improvement of sensory ataxia. the patient did not require any further treatment and after years his condition remains stable. cntn antibodies were tested on a recently collected patient's serum and resulted positive on both elisa and cell-based assay. the patient here reported showed the typical clinical features of anti cntn -associated cidp including older age, acute onset, severe motor impairment and sensory ataxia. the contemporary occurrence of membranous glomerulonephritis was reported in only one other case. contactin- is expressed at low levels in the kidney and a direct damage of anti-cntn antibodies could be hypothesized. alternatively, renal damage might have been secondary to unspecific immune complexes deposition. a good response to anti-cd rituximab was recently reported in patients with cidp associated with anti-cntn and anti-nf antibodies. notwithstanding this single-case observation, our report suggests that also cyclophosphamide may be considered an effective therapy in anti-cntn antibodies-associated cidp and membranous glomerulonephritis, leading to persistent clinical remission. velasco r , , besora s , santos c , sala r , izquierdo c , simó m , gil-gil m , , jiménez l , pardo b , calvo m , palmero r , clapés v , bruna j duloxetine is the only agent demonstrated effective in treating pain related with chemotherapy-induced peripheral neuropathy (cipn). patients with symptomatic cipn treated with duloxetine were retrospectively collected in a single-institution. aim of the study was to evaluate the drug's efficacy and rate of compliance. only patients with cipn with distressing positive symptoms (pain, numbness and/or paresthesia), and non-progressive disease were included. cipn was graded employing the total neuropathy score (tns © ) and national cancer institute-common toxicity criteria. response to duloxetine was assessed with patient global impression of change (pigc) scale ( : no benefit; : excellent response). consecutive first one-hundred cipn patients treated with duloxetine were analyzed. median age was ( - ). , , and received platinum, taxane, bortezomib and vincristine-based regimen, respectively. median tnsc © was ( - ). severity of neuropathy was grade ( %), grade ( %), and grade ( %). sixteen patients were on treatment with other analgesic agents. median time from finishing chemotherapy to duloxetine initiation was months [ - ]. median pigc score was [ - ]. among responders, . % and . % scored low ( - ) and high ( - ) benefit, respectively. fifty-seven ( %) patients discontinued early duloxetine due to intolerable side effects (n= ) the goal of this study was to determine whether predicted fork stalling and template switching (fostes) during mitosis deletes exon in peripheral myelin protein kd (pmp ) and causes a gain-of-function mutation associated with peripheral neuropathy in a family with charcot marie tooth disease type e. two siblings previously reported to have genomic re-arrangements predicted to involve exon of pmp were evaluated clinically and by electrophysiology. skin biopsies from the proband were studied by rt-pcr to determine the effects of the exon re-arrangements on exon mrna expression in myelinating schwann cells. transient transfection studies with wild type and mutant pmp were performed in cos and rt cells to determine the fate of the resultant mutant protein. both affected siblings had a length-dependent demyelinating neuropathy with severely slow nerve conduction velocities (< m/sec). rt-pcr studies of schwann cell rna from one of the siblings demonstrated a complete in frame deletion of pmp exon (pmp delta ). transfection studies demonstrated that pmp delta protein is retained within the endoplasmic reticulum and not transported to the plasma membrane. our results confirm that that fostes mediated genomic rearrangement produced a deletion of exon of pmp , resulting in expression of both pmp mrna and protein lacking this sequence. in addition, we provide direct experimental evidence for endoplasmic reticulum retention of the mutant protein suggesting a gain-of-function mutational mechanism consistent with the observed cmt e in this family. pmp delta is another example of a mutated myelin protein that is misfolded and thus likely to contribute to the pathogenesis of the neuropathy. in poems and cidp, distal limb nerve conduction studies are limited in identifying demyelination and detecting treatment effects in severely affected patients. blink reflex r latency may help to not only identify demyelination but also provide a meaningful treatment outcome measure especially in severely affected patients. poems and cidp patients having undergone routine nerve conductions and blink reflex testing were identified. correlation between r latency, limb nerve conduction studies and neuropathy impairment scores (nis) was calculated with treatment. blink reflexes were performed in patients ( poems, cidp; nis range: - points). overall, r latency prolongation occurred in . % of patients ( . % poems, . % cidp). patients with r prolongation (> ms) had more severely affected nerve conductions in both poems (ulnar cmap . mv vs . mv, p= . ) and cidp (ulnar cmap . mv vs . mv, p< . ). r latency correlated with nis severity in poems better than cidp (r = . vs . , p=< . vs. . ). follow-up nis and r latency evaluations after treatment were available in patients ( poems, cidp). the r latency changes were concordant with the nis changes in % of poems and % of cidp patients. in severely affected patients [ulnar cmap amplitude ≤ . mv ( . %: / )] except for one, all had prolonged r (> ms), allowing for treatment follow up and initial diagnosis. blink reflex r latencies are valuable in defining demyelination in severely affected poems and cidp patients, but also provide a sensitive, early treatment outcome measure among those same severely affected patients. watson dj , martinez c , wallenhorst c , hubsch a , shebl a , simon tl . csl behring llc, king of prussia, usa; institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl behring ag, bern, switzerland; csl behring gmbh, marburg, germany. chronic inflammatory demyelinating polyneuropathy (cidp) rarely occurs in children. clinical trials in pediatric patients have not been performed and there are little data on therapy with intravenous immunoglobulin (ivig). we performed an observational trial to investigate the risk of adverse events of special interest (aesi) with ivig, i.e. hemolytic anemia, aseptic meningitis, acute renal failure, thromboembolic events and anaphylactic reactions. the study cohort was derived from the us premier perspective database and consisted of patients < years with a diagnosis of cidp (icd- cm diagnosis code . ) treated with the ivig privigen ® (csl behring, bern switzerland) between jan and dec . we identified pediatric cidp patients: preschool children (age - years at first treatment for cidp), children ( - years) and adolescents ( - years); females and males; white, one black and two allocated to "other race". six patients had a history of other ivig use for guillain-barré syndrome, one patient for myasthenia gravis and one for immunodeficiency before the diagnosis of cidp. the mean privigen ® dose calculated from the cumulative quantity of privigen ® per treatment episode and the corresponding age-and gender specific median of the us population body weight estimate was . g/kg body weight. the number of recorded treatment episodes per patient ranged from to . using an at-risk period of days for hemolytic anemia, and days for other aesi after each privigen ® administration, no aesi were observed in the patients with cidp with a total of person-days at-risk for ha and person-days at-risk for other aesi. this observational study shows that ivig (privigen ® ) is used for treatment of cidp in pediatric patients with or without concomitant conditions and revealed no particular safety issues in this patient group. hsn- is a peripheral neuropathy most frequently caused by missense mutations in the sptlc or sptlc genes, which code for two subunits of the enzyme serine palmitoyltransferase (spt). spt catalyzes the first and rate limiting step of de novo sphingolipid synthesis. it has been shown that mutations in spt cause a change in enzyme substrate specificity which results in the production of two atypical sphinganines, deoxysphinganine (dsp) and deoxymethylsphinganine (dmsp), rather than the normal enzyme product, sphinganine (sp). levels of deoxysphingolipids are elevated in the blood of hsn- patients and this has been shown to cause the peripheral nerve damage characteristic of the disease, which affects both sensory as well as motor axons. however, the underlying pathomechanism of how deoxysphingolipids damage neurons remains elusive. here, dsp and dmsp-mediated neurotoxicity was examined in primary mouse motor and sensory neurons, by assessing cell survival and neurite outgrowth following exposure to different concentrations of sp, dsp or dmsp. the abnormal enzyme products were found to have a rapid and dose-dependent neurotoxic effect in primary neurons. we also explored the potential mechanisms that underlie deoxysphingolipid neurotoxicity, by characterizing mitochondrial function and changes in calcium handling. we found that mitochondrial dysfunction and calcium handling deficits may be key mediators of abnormal sphingolipid neurotoxicity, in both motor and sensory cell models. specifically, we revealed mitochondrial abnormalities, signs of endoplasmic reticulum stress and dysfunction of store-operated calcium channels. we propose that early deficits in mitochondria and calcium handling may underlie deoxysphingolipid neurotoxicity and thus present potential therapeutic targets for hsn- . months. one patient was excluded because the decrease in hba c was not contemporaneous with weight loss. the mean and median decrease in bmi per month was . and . respectively. the mean and median interval between surgery and decrease in hba c was . and . days respectively. records of these patients were scrutinized and classified as: 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or temporal relationship to decrease in hba c is uncertain; 'unlikely tind': when an alternative explanation exists for symptoms. only one patient was classified as 'possible tind': a -year-old woman who developed neuropathic pain in both lower limbs month after a rapid hba c decline of . % and about months after bariatric surgery. she had no documented autonomic symptoms. our study is limited by small cohort size, retrospective design and reliance on hospital records. nonetheless, it demonstrates that besides nutritional neuropathies, tind should also be considered in dm patients who develop peripheral neuropathy after bariatric surgery. in another study, we found tind is uncommon in a general cohort of diabetic patients. the occurrence of 'possible tind' in only dm patients corroborates earlier data that weight loss may act in tandem to increase the risk of tind. woolums bm , tabuchi m , sung h , sullivan jm , mamah c , yang m , blum id , wu mn , sumner cj , lloyd te . johns hopkins university, baltimore, usa. dominant missense mutations in the gene encoding the cation channel transient receptor vanilloid, family member (trpv ) cause inherited neuropathies including charcot-marie-tooth disease c (cmt c). in vitro, mutations in trpv that cause cmt c cause a gain of trpv channel function and increased intracellular calcium which subsequently leads to cellular toxicity. however, the mechanisms by which cmt c mutations in trpv lead to neuronal dysfunction in vivo remain poorly understood. we generated transgenic drosophila that express either wild-type or a cmt c causing trpv mutant (trpv r c ) to assess the effect of trpv r c on neuron function in vivo. selective expression of trpv r c in drosophila ccap neurons (n ccap ) results in a failure of drosophila wing expansion that is blocked by genetically inactivating the channel pore, demonstrating the requirement of channel function in mediating this phenotype. perforated patch clamp analysis of n ccap reveals that trpv r c causes a calcium-dependent increase in n ccap neuronal excitability. this hyperexcitability is restored to control levels by application of a trpv selective antagonist. high level expression of trpv r c causes synaptic and dendritic degeneration, both of which are rescued genetically by inactivating the channel pore or pharmacologically by feeding larvae a trpv selective antagonist. we conducted a genetic screen in n ccap and found that camkii knockdown potently suppresses the trpv r c mediated wing expansion phenotype and selectively rescues degeneration of synapses but not dendrites. we also find that trpv r c , but not controls, disrupts mitochondrial transport in axons by increasing the number of stationary mitochondria. our data demonstrate that trpv r c elevates neuronal intracellular calcium which disrupts mitochondrial transport and mediates neurodegeneration through compartment-specific calcium-mediated signaling pathways, and supports the further investigation of trpv antagonists as potential therapeutics for the treatment of cmt c. mood disorders, including anxiety and depression, are commonly observed among chronic pain patients with prevalence estimates ranging from to %. comorbidity between mood and chronic pain disorders has been linked to altered limbic regulation of the hypothalamic-pituitary-adrenal (hpa) axis. stress activates the hpa axis and can initiate and/or exacerbate symptoms related to both chronic pain and mood disorders. previous studies from our laboratory have investigated the influence of early life stress on mechanical pain hypersensitivity, visceral hypersensitivity and behavioral evidence of mood disorder later in life. here, we are testing the hypothesis that chronic stress exposure in adulthood can increase somatic and visceral sensitivity and anhedonic behaviors in a mouse strain with a genetic predisposition to anxiety. adult, female a/j mice were exposed to repeated foot shock stress for continuous days and tested for alterations in mechanical sensitivity, sucrose preference, visceromotor response (vmr) during urinary bladder distension, and serum corticosterone levels. mice that underwent shock stress had a significantly decreased mechanical withdrawal threshold in the hind paw compared to their baseline and sham group measurements. sucrose preference was measured prior to shock exposure and throughout the shock paradigm as an indicator of anhedonic behavior. mice that underwent shock stress displayed a trend toward decreased sucrose preference, indicating anhedonia, in comparison to mice in the sham group that did not display anhedonia. mice that underwent shock stress displayed significant increases in vmr during bladder distension compared to sham mice. finally, serum corticosterone levels were significantly higher in the mice that underwent shock stress compared to sham mice, indicating a stress-induced increase in hpa axis output. together these data suggest that chronic stress exposure can induce mechanical allodynia, visceral hypersensitivity, and depression-like behaviors in an anxiety-prone mouse strain. future studies will incorporate gene expression in the hypothalamus, amygdala, and hippocampus, as well as investigation of possible downstream peripheral neuroimmune modulation and neuronal morphology changes. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated neuropathy. as prognostic markers of gbs, modified erasmus gbs outcome score (megos), erasmus gbs respiratory insufficiency score (egris), and Δigg have been reported. however, the proportions of subtypes of gbs are known to be different between the western countries and japan, it remains to be elucidated whether those markers can also be applied to gbs in japan or not. we here investigated retrospectively gbs patients and determined the megos and the egris of those cases. among them, Δigg could be obtained in cases. we evaluated the prognosis using gbs outcome score (functional grade: fg) at months; we called good prognosis when fg at months was less than and poor prognosis when that was or more. as a result, in cases with higher score than at megos on admission cases ( %) had poor prognosis and in cases with higher score than at megos at day of admission cases had poor prognosis. in cases with higher score than on egris cases ( %) needed the mechanical ventilator. patients with good prognosis had higher Δigg(average: mg/dl) than patients with poor prognosis(average: mg/dl). we calculated the cut-off value of Δigg in patients, which was mg/dl. patients with higher Δigg than mg/dl could significantly walk independently at six months (p< . ). patients ( %) had poor prognosis in patients with lower Δigg than mg/dl. ( %) of patients were treated with the single cycle of intravenous immunoglobulin (ivig). other patients ( %) were treated with the combined therapies, such as intra venous methylprednisolone and/or plasmapheresis or the second cycle of ivig in addition to ivig. although there was no difference in prognosis between patients with the single cycle of intravenous immunoglobulin (ivig) and patients with the combined therapies, in the patients who had fg> and megos > on admission, the combined therapies made better prognosis than the single course of ivig (p< . ). we found that megos, egris and Δigg were also available in japan. the efficacy of the combined therapies in severe gbs patients should be investigated in the future large scale prospective studies. yiu em , , , burns j , , , menezes mp , , ryan mm , , and for the paediatric cmt best practice guidelines consortium. royal children's hospital melbourne, melbourne, victoria, australia; murdoch childrens research institute, melbourne, victoria, australia; university of melbourne, melbourne, victoria, australia; university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia. charcot-marie-tooth disease (cmt) often presents during childhood. common symptoms include weakness, limb pain and cramps, foot deformity, and balance impairment. guidelines for the optimal management of common problems experienced by children with cmt do not currently exist. development of these guidelines will provide an evidence base for the management and monitoring of children with cmt. a series of systematic literature reviews utilising the grade (grades of recommendation, assessment, development, and evaluation) approach were conducted to answer pre-specified key clinical questions related to the management of paediatric cmt. these included treatment recommendations for symptoms such as weakness, pain, balance impairment, joint deformity, and impaired upper limb function, and anticipatory monitoring for associated complications such as hip dysplasia. this yielded minimal to no evidence for the pre-specified clinical questions, and evidence-based management recommendations could not be made. consensus-based statements will therefore be formulated via a three-round delphi process, to be conducted in . the delphi panel will consist of local and international medical and allied health professionals who have experience in the management of children with cmt. efficacy of pxt in the treatment of adult patients cmt a (n= ) was shown in a multicenter, randomized, double-blind, placebo-controlled phase ii study (attarian et al. ) . pxt taken x/day, orally, for consecutive months was well tolerated and safe. significant improvement of disability was observed for the highest tested dose, thought indicative for an early, meaningful change in disease course (meta-analysis by mandel et al., ) . this formed the rationale to initiate a multicenter, randomized, double-blind, placebo-controlled pivotal phase iii study (clin-icaltrials.gov: nct ) of pxt in mildly to moderately affected cmt a patients in december . the primary objective is to assess the efficacy of doses of pxt compared to placebo on disability as measured by the mean change from baseline overall neurology limitations scale (onls) score at month and . furthermore, efficacy on the proportion of responders (i.e. improvement of onls), impairment (cmtns-v ), functional tests ( -mwt, qmt, -hpt), electrophysiological parameters (cmap, snap and ncv) and quality of life (eq- d, vas) are secondary endpoints. pursuant this study, patients will be eligible for a -month extension study, in which pxt assigned patients will continue with the previously assigned dose, whereas placebo patients will be randomized to one of the two pxt doses. the study is conducted in investigational sites in countries (eu, canada and us). in december patient randomization was completed (n= ). the screen failure rate was %, as expected ( patients were screened). the independent dsmb recommended to continue the study as planned following a safety analysis on the first patients treated for > months. preliminary baseline characteristics are based on patients (data not cleaned). the study population had a mean age of . ± . years (range - ; male . %) of which . % had a confirmed genetic diagnosis of cmt a. the mean cmtns-v was . ± . and the mean motor nerve conduction of the ulnar nerve was . ± . m/s. ten patients withdrew from the study, due to adverse events unrelated to study treatment. the last patient completing the study is expected in march . zhang g , ghosh p , lin j , ghauri a , sheikh ka . department of neurology, university of texas health science center at houston, houston, tx, usa. assessment of epidermal nerve fiber density and its structure integrity is critical for the diagnosis and evaluating the effectiveness of potential therapies in small fiber neuropathies. currently, skin biopsies, at multiple sites, are most commonly used to assess these diseases. these studies are expensive and time consuming due to cumbersome processing and quantification techniques and serial biopsies over time are often not feasible due to costs and patient acceptance. moreover, vast majority of normative data for skin biopsies in humans are available only for few distal sites and a significant proportion of patients with small fiber neuropathies have focal or regional symptoms not involving the commonly biopsied sites in the leg. live imaging could overcome these limitations and provide a noninvasive real time assessment of epidermal nerve fibers all over the body. we previously found that anti-ganglioside antibody (aga) is an effective neuronal delivery vector for transport of various cargos, such as fluorescent dyes, to peripheral nerves. in the current proof of concept study, we examined whether non-invasive multiphoton microscopy can be used to probe/image the epidermal nerve fibers in living animals after systemic and/or local delivery of fluorescently conjugated aga. we found that the individual nerve endings in skin and cornea are distinctly labeled, and visualized under two-photon microscope. the epidermal nerve fiber labelling by fluorescent-tagged aga was further validated using transgenic mice selectively expressing yellow fluorescent protein in their nervous system. in-vivo multiphoton imaging provide a tool with potential for dynamic longitudinal evaluation of small fiber neuropathies, including nerve degeneration and regeneration, without tissue removal. thus, the use of multiphoton microscopy in conjunction with fluorescently labeled aga as neuronal vector can have many research and clinical applications, such as labeling and live visualization of epidermal nerve fibers to assess small sensory nerve fibers in health and disease. zhou y , tavori h , lee s , al salihi m , fazio s , notterpek l . mcknight brain institute, university of florida, gainesville, florida, usa; knight cardiovascular institute, oregon health and science university, portland, oregon, usa. the majority of hereditary neuropathies are due to abnormalities in peripheral myelin protein (pmp ), whose genetic variants include increased expression (gene duplication), haploinsufficiency (gene deletion), or point mutations. phenotypic heterogeneity in clinical presentation is common among hereditary neuropathy patients even within the same family, the cause of which has not been determined. to investigate the role of pmp in the pathogenesis of the neuropathies we have generated and characterized pmp null (pmp −/− ) mice (amici et al., ) whose peripheral nerves show alterations in lipid metabolism (lee et al., ) . to examine the molecular changes underlying these abnormalities we determined the expression of cholesterol synthesis (srebp pathway), and cholesterol uptake, transport and efflux genes (lxr pathway). in affected nerves, we found the cholesterol synthesis pathway inhibited, while the lxr pathway, and particularly apoe and abca , upregulated at the mrna and protein levels. since pmp is expressed at low levels in the liver, the central organ for the regulation of cholesterol in the body, we studied liver tissue form pmp −/− mice. liver from pmp −/− mice showed significant hepatomegaly, clear features of microvesicular steatosis, as well as marked increase in lxr pathway genes and proteins (abca and apoe), as compared to wt. ultrastructural studies identified lipid droplets and significantly enlarged mitochondria in the liver of male pmp −/− mice, which is not due to mitochondria fusion, as the levels of mfn and remained similar to wt. as disturbed hepatic cholesterol homeostasis induces the activation of kupffer and stellate cells, we determined the extent of inflammation in nerve and liver tissues from pmp −/− mice with leukocyte (cd b) and macrophage markers (cd ). in nerve sections, we detected an increase in the number of cd b+ cells, which was confirmed by western blots. in the liver of pmp −/− mice we found a significant increase in cd -reactive kupffer cells and elevated levels of tnf-alpha. the severe dysregulation of cholesterol metabolism in nerve and liver, including neuroand hepatic inflammation in the absence of pmp − suggest that dysregulated cholesterol metabolism and inflammation may act as a disease modifier in pmp -dependent neuropathies. our aim was to ascertain frequency of and risk factors for tee, arterial (ate) and venous thromboembolic events (vte) in neuromuscular patients receiving regular ivig. we performed a retrospective case-note review of inflammatory neuropathy patients receiving regular ivig treatment. we collected the following data over a month study period this analysis suggests tee incidence is higher in ivig treated patients than comparable population-based rates. examination of tee occurrence in age and vascular risk factor matched ivig-treated and ivig-naïve individuals is required to appreciate the excess risk associated with ivig treatment. référence des maladies neuromusculaires et la sla, hôpital de la timone ha) and whether temporal dispersion (td) parameters are helpful. fifty-eight patients diagnosed according to asbury and cornblath ( ) were prospectively included between january and september . edx and were performed - (mean= ) and - days (mean= ) after disease onset, respectively. there were no differences in classification consistency between ho and ha at edx (p= . ) and edx (p= . ), but more patients were classified as aman when comparing ra with ho and ha at edx (p< . ). at edx , ra classified more patients as equivocal with ho (p< . ) and as aman with ha (p< . ) ) or edx (ho, fe p= ; ra, fe p= . ; ra with td, fe p= . ). gm , gd a, gd b and gq b igg antibodies were tested (willison at edx , only ho showed maybe more antibodies with aman compared to aidp (fe p= . , phi=− . ) and with aman compared to equivocal cases since correlation with factors associated with axonal gbs, in casu rcf and antibodies, is far from exclusive, the usefulness of edx subtype classification using specific criteria sets, remains doubtful. the frequency of rcf indicates that nodal/paranodal alterations may represent the main pathophysiology in more gbs patients than currently thought ruiz m , lessi f , cacciavillani m , riva m , salvalaggio a , campagnolo m , briani c . neurology, department of neuroscience, university of padua, padua, italy; hematology and clinical immunology unit, department of medicine, university of padua, padua, italy; cemes, data medica group, padua, italy.arsenic trioxide (ato) is highly effective in treatment of acute promyelocytic leukemia (apl). it is licensed in italy for treatment of relapsed-refractory apl and for first line chemotherapy in low risk patients. ato most frequent side effects are well described, but less is known on ato induced. we describe apl patients who were treated with all-trans retinoic acid (atra)/ato as first line therapy. the characteristics of ato induced neuropathy was prospectically analyzed by neurological evaluation using both the total neuropathy score, clinical version (tnsc) (a validated scale for chemotherapy induced peripheral neuropathy) and neurophysiological assessment. patients have been evaluated at baseline, at the end of the induction phase, at the end of ato/atra treatment and year after discontinuation of treatment. baseline neurophysiology was performed at the end of induction phase. both patients were men, respectively and -yr-old. none of the patients had previous history of neuropathy. baseline tnsc was (no clinical signs of neuropathy) in both patients. neurophysiological evaluation performed after the end of induction cycle did not reveal signs of peripheral neuropathy in both patients. patient received mg of ato during induction, total , mg. patient received mg of ato during induction, total , . both patients developed leg numbness during consolidation cycles and patient also hand numbness. tnsc at the end of therapy was in patient and in patient . neurophysiology at the end of therapy detected signs of sensitive axonal neuropathy in both patients. they received full doses of ato consolidation ( . mg/kg/day for days/week, on alternate months for total months and tretinoin weeks on weeks off). during the first year of follow-up both tnsc and neurophysiology year after the end of consolidation cycle were consistent with full recovery. our patients developed sensory axonal neuropathy during ato therapy, that clinically manifested during consolidation cycles and improved up to complete recovery during follow-up. published case reports show that outcomes may not be as good as in our patients. a multicenter prospective study evaluating the characteristics of ato-induced neuropathy in apl is ongoing. ruiz m , campagnolo m , salvalaggio a , cacciavillani m , taioli f , fabrizi gm , briani c . department of neuroscience, neurology unit, university of padua, padua, italy; data medica group, emg unit, cemes, padua, italy;mutations in the mitochondrial copper-binding protein sco , cytochrome c oxidase assembly protein, have been reported in several cases of fatal infantile cardioencephalomyopathy with cox deficiency. we identified compound heterozygous variants in sco in two unrelated patients with isolated length dependent axonal sensorimotor polyneuropathy of variable clinical severity (axonal autosomal recessive charcot-marie-tooth disease type , cmt ) by whole exome sequencing. although peripheral neuropathy has been described as a secondary feature in a few cases of fatal infantile cardioencephalomyopathy, the disease onset, clinical phenotype and survival in our patients differ significantly from the previously described cases. our patients developed predominantly motor neuropathy; moreover, they are still alive and they have not developed cardiomyopathy, which is the main phenotype and cause of death at early infancy in reported patients. both of our patients harbor mutations adjacent or near the conserved copper-binding motif (cxxxc), including the common reported pathogenic variant e k and the novel change d g. in addition, each patient carries a second mutation located in the same loop region of the protein, p t and r q. western blots from fibroblasts from these cmt patients showed reduced levels of cox , a subunit of complex iv, indicating cox deficiency. our findings demonstrate that cmt can be the predominant phenotype associated with sco mutations, pointing to a broader phenotypic heterogeneity. the mechanism linking mitochondrial respiratory chain dysfunction to isolated axonal loss of variable severity remains to be elucidated. the muscarinic acetylcholine (ach) type receptor (m r) is a metabotropic g protein-coupled receptor expressed by adult sensory neurons. cholinergic signaling through muscarinic receptors can modulate axonal plasticity in invertebrates and lower vertebrates. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) m r antagonists elevate neurite outgrowth and protect from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt, et al., ) . furthermore, we demonstrated that excessive cholinergic signaling due to m r overexpression caused a significant reduction in neurite outgrowth (calcutt, et al., ) . the mechanism of m r-antagonist driven neurite outgrowth remains poorly understood, however, we have proposed that ach constrains axonal outgrowth via m r activation. cholinergic signaling is mediated via recruitment of trimeric g proteins, of which g alpha- and g alpha- regulate cytoskeleton dynamics by control of tubulin polymerization. activated gtp-bound g-alpha proteins destabilize microtubules by increasing the intrinsic gtpase activity of tubulin. we have therefore tested the hypothesis that cholinergic signaling regulates neurite outgrowth via modulation of g protein mobilization and the dynamics of the tubulin cytoskeleton. we found that over-expression of m r in adult sensory neurons induced dissolution of the tubulin cytoskeleton in distal neurites. g alpha- expression in adult sensory neurons was significantly higher (p< . , -fold) than g alpha- . subsequent knockdown the peripheral neuropathy research registry (pnrr) is a multicenter collaborative research project sponsored by the foundation for peripheral neuropathy to advance the science in distal symmetrical polyneuropathies (dsp). the registry was designed to prospectively characterize clinical phenotype and natural history of patients with dsp and obtain biofluids to identify new causes and genetic modifiers of dsp with careful genotype/phenotype correlations, and to develop biomarkers. the enrollment in the registry is still ongoing but an interim analysis was carried at the end of december . eligible study participants were years or older with a diagnosis of idiopathic, diabetic, chemotherapy-or hiv-induced peripheral neuropathy. they were examined by a physician at one of the six consortium members (johns hopkins university school of medicine, icahn school of medicine at mount sinai medical center, beth israel deaconess medical center, northwestern university medical center, university of utah medical center and kansas university medical center). the collected data set included ( ) a detailed questionnaire, discussing their symptoms, medical history and family history, ( ) a standardized neurological examination form, ( ) electrodiagnostic evaluation and ( ) diagnostic laboratory testing. blood samples (whole blood, plasma and serum) were collected for future biomarker and genotyping evaluations. at the end of , complete data sets and blood samples were collected from patients. % had diabetic pn, % had hiv-associated pn, % had chemotherapy induced pn and % were diagnosed with idiopathic pn. detailed analysis of clinical presentation, examination findings and diagnostic investigations will be discussed at the presentation. standardized phenotyping with linked bio-specimen banking will help establish the minimum data set required for neuropathy diagnosis and support genotype-phenotype correlations with next generation sequencing technologies and development of novel biomarkers. pnrr data will improve our understanding of disease mechanisms paving the way for new therapeutic discoveries in painful and non-painful neuropathies. chemotherapy-induced peripheral neuropathy (cipn) is a major side effect of treatment, typically presenting as a sensory neuropathy. symptoms include pain, paraesthesia, and numbness in the extremities, resulting in functional impairment. increasingly, patient reported outcomes (pros) are utilized to accurately examine the impact of cipn symptoms on patient function. however, the links between objective neurological assessment and pro measures remain ill defined. this study aimed to identify links between neurophysiological measures and pros in patients treated with neurotoxic chemotherapy. assessments were conducted in patients (f= , mean age . ± . years) who had completed neurotoxic chemotherapy on average . ± months previously (platinum-based n= , taxane-based n= or taxane/platinum combination therapy n= ). patients reported the presence and severity of neuropathic symptoms via the fact-gog ntx , a validated patient questionnaire. clinical the most important neurological side effect of a large number of anti-cancer drugs is a painful peripheral neuropathy. mainly chemotherapeutics that interfere with microtubules, including the plant derived vinca-alkaloids such as vincristine, are well known to cause chemotherapy-induced peripheral neuropathies (cipn). to date, few treatments are available and focus on symptom alleviation and pain reduction rather than on preventing the neuropathy all together. for the first time, we highlight the potential of specific histone deacetylase (hdac ) inhibitors as a preventive therapy for cipn, using novel rodent models for vincristine-induced peripheral neuropathies (vipn), characterized by a sensory axonopathy. one reason so few therapies are available, is because the exact pathophysiological mechanisms are poorly understood. mounting evidence proposes axonal transport, a pathway frequently disturbed in neurological disorders, as a major player in the pathophysiology of vipn. proper axonal transport requires dynamic microtubules which are highly modulated by post-translational modifications. since vincristine interferes with the polymerization of microtubules, we reason disturbances in microtubule dynamics, and by extension axonal transport, could contribute to vipn. we illustrate that increasing acetylation of -tubulin after hdac -inhibition, can restore vincristine-induced defects in axonal transport in cultured dorsal root ganglion neurons. also in vivo, -tubulin acetylation was restored in the saphenous nerve and dorsal root ganglia, two sensory tissues that are affected by vincristine. ultimately, this correlates to a reduced severity of the neurological symptoms, both on the electrophysiological and on the behavioral level. moreover, we discovered that hdac -inhibition was not only protective against neurotoxicity, but also reduced tumor progression in a mouse model for acute lymphoblastic leukemia. taken together, our results show that hdac -inhibition is an ideal strategy to prevent vipn with beneficial effects both on the neurotoxicity as well as on tumor growth.approximately two-thirds of cidp subjects need long-term corticosteroids or intravenous immunoglobulins (ivig), with ivig being slightly preferred based on safety profiles. subcutaneous ig (scig) is an alternative option for ig delivery but has not previously been investigated in a large-scale clinical trial in cidp.we performed a randomized, double-blind trial in cidp investigating . and . g/kg weekly doses of scig igpro (hizentra ® , csl behring) versus placebo in subjects for maintenance treatment. ivig-dependent adults with definite or probable cidp according to efns/pns criteria were eligible. the primary outcome was the percentage of subjects with a cidp relapse ( -point deterioration on adjusted incat disability score) or who were withdrawn for any other reason during the -week scig-treatment period. multiple secondary endpoints were assessed. superiority of at least one igpro dose over placebo was tested one-sided using the cochran-armitage trend test for the primary outcome and the jonckheere-terpstra tests for secondary outcomes.the primary outcome occurred in % of high-dose scig, % of low-dose scig, and % of placebo subjects (p < . ); cidp relapse occurred in % of high-dose scig, % of low-dose scig and % of placebo subjects (p < . ), respectively. both scig doses were superior to placebo (low-dose vs placebo p = . ; high dose vs placebo p < . ). median incat score, mrc sum score, and grip strength remained stable in scig subjects. high-dose scig prevented the r-ods decline seen with low-dose scig and placebo (p < . ). all placebo subjects deteriorated on measures of strength and disability.causally related adverse events occurred in ( %) subjects ( % placebo, % low dose, and % high dose).scig igpro was efficacious and safe as maintenance treatment. mutations in metalloendopeptidase (mme) gene have been associated with autosomal-recessive late-onset charcot-marie-tooth type- (cmt ). to date, all patients have had at least one truncating mutation, either in homozygosity or in trans with a missense mutation. more recently, loss-of-function and missense heterozygous mutations were also identified in autosomal-dominant cmt. we report the case of a previously healthy caucasian woman, born to healthy unrelated parents, who presented at the age of thirty-nine with numbness and cold sensation in the lower limbs. subsequently she developed progressive gait disturbance and impaired hand dexterity. her homozygous twin presented at the same age with similar symptoms. the family history was otherwise uneventful, in particular neither neuropathy nor dementia were described. neurological examination at the age of fifty-three revealed a steppage gait, distal upper and lower limb atrophy and weakness, distal sensory loss and bilateral pes cavus. deep tendon reflexes were normal in the upper limbs and absent in the lower limbs. nerve conduction studies revealed an axonal sensory and motor neuropathy. a sural nerve biopsy revealed a reduction in myelinated nerve fibers and active axonal degeneration. targeted sanger sequencing of mpz , gjb , gdap , nefl, fkrp, bscl , hspb and mfn were negative. sureselect focused exome sequencing was therefore performed and identified two missense heterozygous mutations [c. g>a,p.c y;c. t>c,p.y h] or lack of efficacy (n= ). most frequently reported adverse events were cognitive ( %), gastrointestinal ( %) and genitourinary ( %). discontinuation due to perception of lack of efficacy was more frequently reported by men ( % vs % p= . ). women presented higher punctuations on pigc scale compared with men ( . ± . vs . ± . , p= . ). pigc scores were significantly higher in patients receiving taxane ( . ± . ) than platinum ( . ± . ) agents (p= . ). no significant differences according severity of neuropathy neither type of chemotherapy were observed in drop-out and retention rates. patients with long-lasting cipn (> months) reported lower pigc scores ( . ± . vs . ± . , p= . ) and higher frequency of suspension due to adverse events ( % vs %, p= . ) and less rate of continuation of duloxetine ( % vs %, p= . ). more than one-third of patients with disturbing cipn discontinued duloxetine prematurely due to intolerable side-effects. low tolerability, male gender and long-lasting cipn may limit duloxetine usefulness in the treatment of symptomatic cipn. verboon c , jacobs bc , and the igos consortium. department of neurology, erasmus medical centre, rotterdam, the netherlands; department of immunology, erasmus medical centre, rotterdam, the netherlands.the efficacy of intravenous immunoglobulin (ivig) in guillain-barré syndrome (gbs) has only been demonstrated in severely affected patients who are unable to walk independently. although there is no proof that ivig is effective in milder forms of gbs, some neurologists are treating these patients with ivig considering that even milder forms of gbs may result in poor recovery, residual deficits, fatigue or pain.we determined the effectiveness of a single course of ivig ( g/kg in - days) in relatively mild forms of gbs in the ongoing observational international gbs outcome study (igos). the gbs disability score, mrc sum score and patient reported outcome measures (prom) were compared at and weeks. ordinal logistic regression analysis was used to determine the effect of ivig on the gbs disability score, adjusted for previously identified prognostic factors.data were analyzed from the first patients enrolled in igos by december , including patients with mild gbs at entry, of which patients ( %) were treated with supportive care, while patients ( %) received ivig (start ivig after onset of symptoms in days, median , iqr - ). at baseline, patients in the ivig treated group compared to the untreated group less frequently had pure motor gbs ( % versus %, p< . ) and axonal damage or unresponsive nerves ( % versus %, p= . ), but a worse gbs disability scores at nadir (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ). at weeks, the median mrc sum scores and prom were not significantly different between treated and untreated patients. however, more patients in the ivig group showed complete recovery of muscle strength at weeks than patients in the control group ( % versus %) (p= . ) and more frequently showed full neurological recovery on the gbs disability scale ( % versus %, p= . ). additional results will be presented at the conference.based on the results of this interim analysis in observational data, we conclude that patients with a relatively mild form of gbs may benefit from a single course of ivig. despite treatment with intravenous immunoglobulin (ivig), many patients with guillain-barré syndrome (gbs) recover insufficiently. we primarily aimed to determine whether a second ivig course ( g/kg in - days) in patients with a poor prognosis improves outcome on the gbs disability scale after weeks. we included patients from the prospective, observational international gbs outcome study (igos) treated with ivig and who had a poor prognosis on the modified erasmus gbs outcome score (megos). of patients enrolled in igos, patients were eligible; patients ( %) were treated with one ivig course (control group); patients ( %) received an 'early' second ivig course ( - weeks after start first course) and patients ( %) a 'late' second ivig course (within - weeks). one week after study entry, patients receiving an 'early' or 'late' second ivig course had significantly worse gbs disability scores and mrc sum scores than controls, implying the need for adjustment of baseline characteristics. the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' group, and . ( % ci . - . ) for the 'late' group, suggesting worse outcomes with a second course of ivig compared to controls. at months, patients ( %) in the 'early' second ivig group, patients ( %) in the control group and only ( %) in the 'late' second ivig group were able to walk unaided (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' second ivig group and only . ( % ci . - . ) for the 'late' second ivig group. in gbs patients with a poor prognosis, we did not find a beneficial effect of a second course of ivig after weeks follow-up. our results suggest that an 'early' administered second ivig course might improve outcome at weeks. given the limitations of this observational study, a randomized controlled trial with a larger number of gbs patients with a poor prognosis being treated early in the disease course is needed to confirm or refute these results. vidal c , bhatt n , agudelo c , mahapatra a , saporta ma . department of neurology, university of miami miller school of medicine, miami, usa.multifocal motor neuropathy (mmn) is an inflammatory demyelinating chronic neuropathy characterized by progressive asymmetric weakness in the distribution of two or more peripheral nerves, without objective sensory loss or upper motor neuron signs. the cardinal neurophysiological finding is conduction block outside the usual sites of nerve compression. supportive clinical criteria include high titers of anti-gm antibodies, good response to ivig, increased cerebrospinal fluid (csf) protein (< g/dl) and magnetic resonance imaging (mri) with diffuse swelling of the brachial plexus. we report a case of a year old woman with a two month history of progressive muscular weakness which began in the right upper extremity, described as difficulty in gripping objects, writing and typing on the computer, followed by progressive lower extremity weakness with difficulty rising from a chair and left foot drop. she also complained of pain in her right trapezius and scapular area. she denied any tingling or numbness. on neurological examination there was right scapular winging and asymmetric weakness predominantly involving the right upper and left lower extremities. reflexes were absent throughout. lumbar puncture revealed albuminocytological dissociation (wbc and protein . g/dl). ncs revealed slow conduction velocities for both ulnar and median nerves and a median nerve conduction block at the forearm segment. sensory nerve action potentials were normal for all tested nerves. needle emg revealed acute denervation of right periscapular muscles. brachial plexus mri showed symmetric bilateral thickening from trunks to peripheral nerves. anti-gm igg/igm were negative. two days after lumbar puncture and the beginning of ivig, patient presented binocular diplopia on extreme left lateral gaze. brain mri with and without contrast showed no intracranial pathologies. after five days of ivig, patient was discharged with significant clinical improvement and recovery of the right scapular winging. diplopia improved a week after discharge. we report an atypical multifocal motor neuropathy case. although this patient fulfills all clinical criteria for mmn, we report some features usually not found in mmn such as scapular winging and a mild and transient left vi nerve palsy. mmn should be included in the differential diagnosis of scapular winging. vlckova e , , raputova j , , srotova i , , sommer c , Üçeyler n , birklein f , rebhorn c , rittner hl , kovalova e , , nekvapilova e , , belobradkova j , olsovsky j , weber p , dusek l , jarkovsky j , bednarik j , . despite many studies addressing biomarkers for pain in diabetic polyneuropathy (dpn), little is known about why it affects only a certain proportion of dpn patients. the somatosensory system plays a key role in the pathophysiology of neuropathic pain (neup) and subgroups with different sensory profiles might respond differently to pain treatment. we aimed to characterize sensory phenotypes of patients with painful and painless diabetic neuropathy and to assess demographic, clinical, metabolic, electrophysiological and psychological parameters related to the presence of neup in a large cohort of well-defined dpn subjects.this observational cross-sectional multi-centre cohort study (performed as part of the ncrnapain eu consortium) of subjects with dpn (non-painful, ndpn, n= ; painful, pdpn n= ) associated with diabetes mellitus of type and (median age years, range - years; women) comprised detailed history taking, neurological examination, laboratory tests, quantitative sensory testing, nerve conduction studies, neuropathy severity scores, and neuropsychological questionnaires. all parameters were analysed with regard to the presence and severity of neup. the presence and severity of neup were positively correlated with severity of neuropathy and thermal hyposensitivity (p< . ). a minority of pdpn patients ( . %) had a sensory profile indicating thermal hypersensitivity; this was associated with less severe neuropathy and better response to pain therapy. the presence of neup was also associated with female gender (p< . ) and with higher cognitive appraisal of pain as assessed by the pain catastrophizing scale (p< . ), while parameters related to diabetes (duration, hba c, microangiopathy) showed no influence on neup presence and severity. this study confirms the necessity of comprehensive dpn phenotyping and underlines the importance of the severity of neuropathy that should be taken into account in the stratification of patients with pdpn for analgesic treatment and drug trials.people with charcot marie tooth disease (cmt) experience slowly progressive muscle weakness, sensory loss and musculoskeletal changes over time. this leads to disability and risk of comorbidities due to inactivity. exercise is important to maintain general health but may also help to improve symptoms of cmt. we conducted a randomised controlled crossover trial of aerobic exercise to ascertain the effect of training on fitness levels, muscle strength, function and general well-being. in addition, we monitored the safety of training and feasibility of participation in this type of exercise in local community gyms. motivation, confidence and barriers to exercise were explored using qualitative interviews. the recruitment target was people. in total people with cmt a were approached to participate and were unable to commit to the trial or did not meet the study criteria on initial screening. thirty-one people underwent more detailed screening but three failed to meet the study criteria, five people withdrew before starting and three withdrew part way through the study. the data for the people who started the study were analysed using a random effects model. there was a % participation level in the training and it was well tolerated with no increases in pain or blood serum creatine kinase. an increase in vo peak (ml/min/kg) was observed in the cmt group with (pre training: n= , . ± . , % ci . to . ; post training: n= , . ± . , % ci . to . ; pre control: n= , . ± . % ci . to . ; post control: n= , . ± . , % ci . to . ) . there was wide between subject variation leading to a small overall effect size with cohen d of . ( % ci:- . to . ). a tentative regression model showed no effect of group or time point. there were no major changes in other measures of impairment, function or patient reported outcome measures. this pilot study showed that a community based model of training had a small effect on cardiopulmonary fitness, and was well tolerated with good participation. ankle-foot orthoses (afo) are commonly prescribed for children with charcot-marie-tooth disease (cmt) to manage foot drop, however the type and severity of functional impairment results in gait deviations that might require alternate orthotic designs. the aim of this study was to identify d gait patterns of children with cmt based on severity of functional weakness (based on heel walk, toe walk and foot drop items during gait using the cmt pediatric scale) to inform a design pipeline for d printed orthoses. d gait data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model in children with cmt ( male; ± . yrs, ± . cm, ± . kg), of various cmt types: cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx and compared to typically developing children ( male; . ± . yrs, ± . cm, ± . kg). data were subdivided into three groups denoting increasing severity of dorsiflexion and plantarflexion weakness: no difficulty heel or toe walking (cmt nd ), difficulty heel walking (cmt dh ), difficulty toe and heel walking (cmt dth ). the cmt nd group showed a near-normal gait pattern. the only significant differences noted at the ankle were reduced peak dorsiflexion in stance (p< . ), indicating that an orthotic intervention may not be required. in addition to reduced peak dorsiflexion, the cmt dh group demonstrated significantly reduced dorsiflexion in swing (foot-drop) and a reduced dorsiflexor moment in loading response (p< . ). this suggest, the cmt dh group would require a flexible afo to allow activation of the plantarflexors during push-off, prevent foot-drop and restore a heel rocker in loading response. in contrast, the cmt dth group presented with significantly delayed and increased peak dorsiflexion in stance and reduced plantarflexion and power at push-off (p< . ). they also had significantly increased mean knee extensor moment (p< . ) revealing early signs of 'crouch gait'. therefore, the cmt dth group would require a rigid afo to limit the amount of dorsiflexion and assist movement of the ground reaction force anterior to the knee during stance. three distinct gait patterns at the ankle were identified in children with cmt, indicating patient-specific orthotic design pathways to target specific functional impairment. wojciechowski e , , chang a , cheng t , , little d , , menezes mp , , hogan s , burns j , . university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia.children with cmt are often prescribed ankle-foot orthoses (afo) to manage lower limb impairment such as foot drop and foot deformities. they are handmade by plaster cast followed by thermoplastic moulding. this traditional approach provides limited design options, can be costly, with long outpatient wait times. d printing, also known as additive manufacturing, has the potential to transform the way orthoses are prescribed, designed and manufactured. the aim of this review was to evaluate the evidence of d printing afos compared to traditional manufacturing methods, for children with cmt. there are currently no studies evaluating the application of d printing afos for children with cmt. however, a small, but emerging evidence base exists for d printed afo's in adults from studies including healthy participants and populations with rheumatoid arthritis, post-polio syndrome, foot drop and ankle weakness secondary to injury. samples sizes ranged from - participants for studies related to in-shoe orthoses and from - for studies related to afos. the methods of d printing included sterolithography, selective laser sintering and fused deposition modelling using materials such as nylon , nylon , polylactide, polycarbonate and abs. d printed afos were comparable to traditional manufactured orthoses in terms of patient-perceived comfort, temporal-spatial parameters, plantar pressure measurements and d gait analysis. however, the effects of long-term usage and durability of d printed afos has not been investigated. d printing orthoses have potential advantages including increased design possibilities, improved productivity, higher compliance, and reduced labour needs. disadvantages include redesigning clinical pathways, limited evidence base for clinical effectiveness, limited biocompatible materials, occupational safety considerations and a high level of expertise required for software operation and fabrication of devices. further research is required to determine the feasibility of d printed afos for children with cmt, and the most appropriate and effective printing pathway, materials to improve health outcomes of affected patients. wong shj , koh sj , lee bjh , chng ysk , pawa c , subramaniam t , cheng ksa , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore; yong loo lin school of medicine, national university singapore, singapore; khoo teck puat hospital, singapore.treatment-induced neuropathy of diabetes mellitus (dm) (tind) is a complication of rapid glycaemic control. individuals present with neuropathic pain and autonomic dysfunction within weeks of improvement in glucose control. the use of both insulin and oral hypoglycaemic agents has been associated with tind. its severity is determined by the rate and quantum of hba c decline. other predisposing factors include anorexia and weight loss. we studied the incidence of tind in dm patients who have undergone massive weight loss and hba c decline after bariatric surgery. we screened electronic records of patients ( dm, non-dm) who underwent bariatric surgery between and . dm patients fulfilled the tind hba c criteria of a decrease of ≥ % over months or ≥ % over key: cord- -bie veti authors: nan title: ecc- abstracts date: - - journal: int j antimicrob agents doi: . /s - ( ) -x sha: doc_id: cord_uid: bie veti nan f spain introduction: the prevalence of erythromycin resistance (er-r) in group a streptococci (gas) has increased in spain since early s with current rates exceeding % in some regions. this study determined the emm -types associated to erythromycin resistance in spain. material and methods: isolates belonged to the sauce* surveillance collection. rapid sequence analysis of specific pcr products was used to deduce emm -types corresponding to the majority of the known gas m serotypes. pcr primers used: gasm ( ?-tattgcgct-tagaaaattaa- ?) and gasm ( ?-gcaagttctt-cagcttgttt- ?). sequencing was done with the big dye terminator mix and autosequenator (applied biosystems). dna sequences were subjected to homology searches against the bacterial dna database. results: overall, gas isolates ( er-r) were analysed. three m-types (m , st and m ) accounted for . % of the er-r isolates, whereas they just represented a . % of the ery-s. for er-r isolates the strongest association was seen with m (or / ; % ci . Á/ . ), and m was second after m only in the last temporal period of the study ( Á/ ) . no homogeneous distribution of er-r m-types by centres was seen. conclusions: few m-types (leading by m ) are responsible for the er-r in spain. but for m , the remaining er-r m types (st , m and m ) did not show a temporally nor geographically homogeneous distribution. *sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en espana' (susceptibility to the antimicrobials commonly used in the community in spain ) and is the spanish word for the willow tree. significant increase in the prevalence of erythromycin-resistant, clindamycin and miocamycin-susceptible (m-phenotype) streptococcus pyogenes in spain ( ( Á/ purpose: a variety of methods is used for a molecular typing of enterococcus spp. and related gram-positive bacteria. these include dna-based methods such as macrorestriction analysis using pulsedfield gel electrophoresis (pfge), ribotyping, and amplification-based methods such as rapid amplification of polymorphic dna (rapd) and amplified fragment length polymorphism (aflp). we used a homogeneous strain collection of transconjugants resulting from filter-matings with different antibiotic-resistant e. faecium and a recipient isolate from our lab. the influence of transferred antibiotic-resistance determinants on the outcome of different typing methods was investigated. results: fragment patterns resulting from pfge indicated minor differences between the transconjugants and the recipient. in respect to different primers used for rapd, none or only a single fragment shift was detected in the resulting fragment patterns. aflp clusters all transconjugants into a group of major relatedness, but the result was strongly dependent on the mathematical method used for cluster analysis. fragment patterns of digested plasmids showed the possession of different or only widely related plasmids in the transconjugants. conclusions: the results of this study clearly show that under certain situations typing methods commonly used for enterococci and related gram-positive bacteria come to their limits. the sasss network aims to set up a national surveillance study to obtain standardized information on antimicrobial susceptibility to various bacterial pathogens. currently, hospitals are participating in the project from different geographical regions in saudi arabia. during the st year ( ), the sasss focused on setting up this network. overall, high frequencies of resistance to antibiotics to different bacterial pathogens in saudi arabia were seen. geographical variations of resistance were noticed, which could be related to different prescribing practices. approximately, and % of escherichia coli and k. pneumoniae , respectively, were extended spectrum â-lactamases (ebls) producers. resistance of enterobacteriacae group to carbapenem and pipracillin/tazobactam is low. resistance of pseudomonas aeurginosa to various anti-pseudomonal antibiotics including carbapenem is high and alarming. methicillin resistant staphylococcus aureus (mrsa) comprised % of s. aureus isolates. no vancomycin intermediate s. aureus (visa) was detected. high level resistance to gentamicin in enterococcus were seen in % of the isolates and only % of enterococcus facieum were glycopeptide resistant. resistance of streptococcus pneumoniae to penicillin ranged between % to almost %. surveillance of antibiotic resistance on a national level is necessarily to give guidance to practicing physicians on the best agents to use. the world-wide problem of betalactam resistance (r) in streptococcus pneumoniae (sp) has been complicated by increasing r to macrolides and some older fluoroquinolones (fq) (ciprofloxacin cip). aim of our study was to evaluate rate of acquisition of resistance to different fq: cip, sparfloxacin (spx) and levofloxacin (lev) of sp strains with different levels of susceptibility to penicillin (p). fifteen strains were serially and daily passaged in subinhibitory concentrations of these four antibiotics by a gradient plate method until acquisition of resistance. clinical strains isolated from children in day-care centers were used. five strains were susceptible (s) to penicillin (p) (one reference strain, four clinical isolates: p micsb/ . mg/l): five were intermediate (i) to p (one reference strain: p mic . mg/l, four clinical strains p mics: . Á/ mg/l), five were resistant (r) to p (p mics . mg/l). mean of number of passages (n ) necessary to reach i or r level with each fq as selecting agent are in the following table: spx and lev induced resistance but more slowly than cip. our results show that rate of acquisition of resistance to fq is strongly related to alteration of susceptibility to p, probably by modification of cell wall. these results are concordant with clinical results. clinical relevance of phase variation in pneumococcal opacity: nasopharyngeal (np) colonization in children from day care centers (dcc) soa . carsenti h, mancini g, bensoussan m, dunais b, pradier ch, dellamonica p. archet hospital, infectious disease, nice, france streptococcus pneumoniae (sp) adherence to nasopharyngeal (np) epithelium is a prerequisite for induction of otitis transparent sp (t) have been shown to colonize the np of infant rats better than opaque (o) sp. opaque sp has proven more virulent than the t form during systemic infection in a mouse model. aim of this study was to evaluate phase variation in the nasopharynx of children. sp strains were isolated during a winter epidemiology study of np samples in children from family dcc. mics determinations were performed by e -test for penicillin (p), amoxicilline (amx) and ceftriaxone (cro). serotypes were performed using the quellung reaction. upon oil immersion microscopic examination short chains of six to eight cocci were noted as , '/, '/'/, '/'/'/ for absence, , , !/ chains by field, respectively. phase variation was detected on catalase trypticase soja plates, amx and cro mics and bactericidal activity was determined for pairs of o and t variants with different serotypes and susceptibility to penicillin. seventy strains of sp were screened for phase variation. nine out of with chain length , '/ had o variants while out of strains with chain length '/'/ or '/'/'/ showed o variants. proportion of o variants was predominant when chain length increased. serotype f was prevalent. bactericidal activity of o variants showed a four-to eightfold increase of mbc. o variants may be present in np of children while t are predominant form for colonization. these virulent variants with lower level of autolysis showed less susceptibility to killing by antibiotics. they may persist in np and explain the absence of eradication by active molecules. antimicrobial resistance among clinical strains of s. pneumoniae isolated from patients with community-acquired respiratory tract infections (carti) in russia soa . kozlov rs a , bogdanovitch tm a , sivaya ov a , agapova ed b , ahmetova li b , furletova b , gudkova lv b , gugutsidge b , ilyina vn b , marusina b , multich ig b , ortenberg ea b , schetinin ev b , shturmina purpose: to determine the antimicrobial resistance of pneumococci causing carti in different russian cities. methods: a total of non-duplicate strains isolated in russian cities in were studied. antimicrobials tested included penicillin (pen), amoxicillin (amo), erythromycin (ery), azithromycin (azi), clarithromycin (cla), midecamycin (mid), spiramycin (spi), clindamycin (cli), levofloxacin (lev), vancomycin (van), rifampicin (rif), tetracycline (tet) and co-trimoxazole (sxt). susceptibility testing was performed by broth microdilution with interpretation of the results according to nccls guidelines ( ) a map of bacterial resistance in a hungarian region soa . farkas a a , juhasz a b , orosi p a , miszti c c , balogh m d . a kenezy teaching hospital, hygienie, debrecen, hungary , b kenezy teaching hospital, laboratory, debrecen, hungary , c university of debrecen, microbiology lab, debrecen, hungary , d regional hospital berettyóújfalu, laboratory, debrecen, hungary background: regional trends of microbiological resistance pattern constitute basic data and qualifying criteria for effective infection control. purpose: the aim of our study was to establish an internationally compatible regional database in a hungarian county hajdú -bihar. methods: our model is the national nosocomial infections society publications' format from the u.s. published in . it contains data regarding various icu types, ambulatory patients and hospitalised patients. the same format is used for antibiotic utilisation data and device related infections' rates as well. we collected cleaned data of years Á/ from all the microbiological laboratories of our county. results: ciprofloxacin p e. coli . . . Á/above susceptibilities not significantly different from u.s. data were as follows: mr cns, streptococcus pneumoniae /penicillin and rd generation cephalosporin, pseudomonas aeruginosa /piperacillin and enterobacter spp. and escherichia coli /ceftriaxon. conclusions: this database proved to be a very useful tool for choosing primary wards of active surveillance including places for infectious disease physician's visit (icu, rehabilitation unit). additional analysis is needed at an individual institution's level for other heavily used (or useable) antibiotics and bacteria as well (aminoglycosides, beta-lactam Á/beta lactamase inhibitor combinations, nd generation cephalosporins, corynebacteria . to compare epidemiological, clinical, and immunological features of add before and after haart introduction, between the patients (p) diagnosed in Á/ , and the p detected since , in a case-control study. though the mean number of newly diagnosed aids p had a sharp drop in the haart era, from p/year in Á/ to p/year since (p b/ . ), the distribution of add and underlying immunodeficiency showed limited changes. when excluding a greater frequency of tuberculosis (tb) (p b/ . ) and wasting syndrome (p b/ . ), all other add did not show a different frequency before and after . a tendency towards a higher mean cd count at aids disease was noticed: vs cells/ml (p b/ . ), with a significant difference for candida esophagitis , toxoplasmosis, kaposi sarcoma and tb (p b/ . Á/b/ . ). the limited variation of clinical and immunological presentation are attributable to the poor impact of haart before aids recognition: . % of p detected since did not receive haart or had insufficient compliance to antiretrovirals, so that . % of p were aids presenters. during the haart era, an increase of mean age and sexual transmission was found (p b/ . ). notwithstanding the effects of haart on the natural history of hiv disease, the consequences on add distribution and related immunodeficiency were negligible, since most p could not benefit from haart before aids onset. a high clinical suspicion for add should be maintained when facing p with missed or undertreated hiv disease. radata */communication internet platform management of resistance analysis guided haart switch for implementation in clinical practice of hiv-infected individuals soa . paech v, lorenzen t, stoehr a, plettenberg a. ifi, interdisciplinary infectiology and immunology, hamburg, germany purpose: hiv-resistance analyses are indicated to prepare switch of haart in hiv-infected individuals with failure to ongoing haart regimen. specialists at several responsible sites often feel lack of complementary informations if interpretation of resistance analyses is done independent from each other. clinical benefits from resistance analysis assays are sigfnificantly higher for those physicians, who can access external advice from hiv-experts for possible treatment options. the database concept 'radata' (www.radata.de) was developed in germany to generate expert advice for implementation in haart switch. results: fifteen hiv-treatment centres, seven laboratories and high ranked authorities in hiv-medicine contribute to radata database since it is started in january in germany. hiv-infected subjects are eligible to participate at the project after presentation of failure to haart (viral load !/ c/ml). expert advice is generated after all data are evaluated and based on recommendations of Á/ external hiv-experts. observation after therapy switch is scheduled for a period of months. conclusions: radata is a novel database concept with features for evaluation of data and availability of complementary information to participating sites. the project is designed to provide its proficiency to patients and centres from germany and foreign countries. further information will be provided after the number enclosed subjects have enlarged. in vitro effects of hiv infection on abc transporter expression and antiretroviral drug efficacy soa . therefore, we evaluate in primary cultures of human monocytederived macrophages (mdm) and lymphocytes, effects: ( ) of retroviral infection and haart on the expression and activity of p-gp and mrp; and ( ) of specific inhibitors of these host proteins on antiretroviral activities of nrti, non-nrti and ip. results: on the one hand, we evidenced a transitory increase of p-gp mrna expression in lymphocytes and mdm in response to in vitro hiv infection. this was correlated to an increased p-gp cell surface expression and activity, and an increased tnf-alpha production and mrna. in contrast, no significant modulation of mrp was observed. on the other hand, psc and probenecid potentiated in vitro the anti-hiv activity of azt and indinavir. these effects were accentuated when psc and probenecid were combined. conclusion: these results showed that: ( ) hiv infection by increasing abc transporter expression could favorise the efflux of antiretroviral drugs and decrease their pharmacological effects; and ( ) specific inhibitors of these transporters could reverse these deleterious effects. effects of interferon alpha plus ribavirine therapy on frequencies of hcv, hiv and cmv specific cd -t-cell responses in peripheral blood of hiv/hcv coinfected patients after months of treatment soa . methods: two groups of patients with chronic hcv infection were studied: hiv coinfected progressors with antiretroviral therapy and hiv-negative controls. twelve hcv/hiv and hcv patients have already reached months of ifn-alpha'/ribavirine therapy. virusspecific cd -t-cells in peripheral blood were analyzed by ifngamma-elispot-assays using hiv-p , one cmv and three hcv (core, ns , ns ) antigens. results: ( ) at baseline, hcv-specific cd -th -cells frequencies were significantly lower than hiv-and cmv-specific ones; ( ) frequencies of cd -th -cells against hcv as well as against cmv were similar in the two groups; ( ) in hcv'//hiv'/, hcv specific cd -t-cell frequencies did not change between baseline and th month of anti-hcv treatment, decreased in three and increased in only one case. hiv-and cmv-specific frequencies were decreased in seven patients. similar results were observed in hiv-negative group. conclusion: ( ) hcv-specific immune responses might be more prone to tissue compartmentalization than hiv-specific ones; ( ) immune defects induced by hiv infection might not be responsible for the low level of hcv-specific responses observed in hiv-progressors; ( ) ifn-alpha'/ribavirine therapy influence on hcv-and hivspecific cd -t-cell frequencies after months of treatment will be discussed. frequencies of hiv- -p specific th cells (elispot) are correlated with plasma hiv- viral load in a cohort of lt-np and slow progressors soa . martinez v a , alatrakchi n a , costagliola d b , bonduelle o a , agut h c , autran b a , alt study group a . a hopital pitié-salpêtrière, laboratoire d'immunologie cellulaire, paris, france , b faculté de medecine saint-antoine, inserm sc , paris, france , c hopital pitié-salpêtrière, laboratoire de virologie, paris, france background: hiv- -specific t helper- cell responses have been associated with long-term-non-progression (lt-np) in hiv infection but the correlation between frequencies of hiv- -p -specific th cells and viral load has not yet been studied. we prospectively quantified these frequencies by using an ifn-gamma elispot assay in a cohort of lt-np. methods: a cohort of lt-np and slow progressors (infection !/ years and cd counts !/ /mm ) was analysable. hiv- -p specific t cells were analyzed using: proliferation, ifn-gamma eli-spot assays and ifn-gamma production in cell supernatants. results: wide ranges were observed in the frequencies of hiv- -p -specific cd th cells as assessed by elispot ( - sfc/ pbmc) with a median of sfc/ pbmc. these frequencies were negatively correlated with viral load (r /(/ . , p / . ) but not with cd counts and associated with a low level of t cell activation assessed by cd on cd cells (r /(/ . , p / . ). similar results were obtained with t cell proliferation and ifn-gamma production. conclusion: interestingly, the numbers of hiv- -p -specific th cells correlate with plasma viral load, independently of cd counts indicating that: ( ) the defect in hiv- -specific cd th cells does not reflect the global cd depletion; and ( ) these responses are strongly correlated to the control of virus replication. impact of drug Á/drug interactions on therapeutical management of active tuberculosis in hiv infected patients soa . martinez v a , truffot c b , caumes e c , katlama c c , jarlier v b , bricaire f c , jouan m d . a department of infectious and tropical diseases, pitié salpêtrière hospital, institut pasteur, unité de génétique mycobactérienne, paris, france , b department of bacteriology, pitié salpêtrière hospital, paris, france , c department of infectious and tropical diseases, pitié salpêtrière hospital, paris, france , d institut pasteur, unité de génétique mycobactérienne, paris, france since and the use of haart, management of hiv patients with active tuberculosis raised the question of drug Á/drug interactions and therapeutical management of both infections. retrospective cohort study: follow-up of hiv patients with active tuberculosis diagnosed between and . studied data included evolution of tuberculosis and hiv, cd cell counts, plasma hiv viral loads, antituberculosis and antiretroviral regimens. ninety-four percent of patients were treated by quadruple combination antituberculosis drug with rifabutin for five patients. fourteen patients were treated by double antiretroviral therapy of nucleoside reverse transcriptase inhibitors (nrtis) and by triple or more drugs (nrtis and/or nonnucleoside reverse transcriptase inhibitors nnrtis and/or protease inhibitors pis). the median follow-up was months. there was no difference on cd cell counts and viral loads in the two groups and between the patients treated by nnrtis and pis at the diagnosis of tuberculosis, at the time of antituberculosis drug discontinuation and concerning cure rates of tuberculosis. on the other hand, the plasma hiv viral load was significantly better controlled in patients with nnrtis than with pis (p b/ . ). in hiv patients with active tuberculosis receiving haart, antiretroviral combination including nnrtis allows a better control of viral replication than regimen including pis without impact of the use of rifampin or rifabutin. adherence is essential to the effectiveness of antiretroviral therapy. a pharmacy visit would improve the patient advisement. a survey was carried out over a period of months in the u.m.i.t. a self-report was distributed to patients. ninety were evaluated. for %, information's given by the clinician were sufficient and for % the treatment advice cards were useful. however, % of them would like to attend a pharmacy visit. the topics they would prefer to be tackled were drug interactions ( %), side effects ( %) and effect of forgetting ( %). the treatment was well accepted and tolerated for, respectively and % of the patients. the viral load and the cd count were well known by, respectively and %. however, inaccurate pattern of treatment was frequent ( !/ %) and bad adherence was observed: treatment forgotten occasionally ( %), regularly ( %) or inadequate attitude when the treatment was forgotten ( %). number of pills, dose frequency, length of the treatment would be risk factors of nonadherence. for the majority of patients, a pharmacy visit is necessary and beneficial. the first result shows a better understanding of the treatment, an improvement of the adherence and an enhancement of plasma concentration of antiretroviral drugs. in vivo activity of glycopeptides against s. aureus infection in a rabbit endocarditis model: is mic predictive for in vivo efficacy? soa . asseray n, caillon j, lemabecque v, jacqueline c, batard e, potel g, bugnon d. laboratoire antibiologie, faculte de medecine, nantes, france we have studied the in vivo efficacy of vancomycin (v) and teicoplanin (t) against five staphylococcus aureus (sa) strains: two methicillin-susceptible (mssa and ), two methicillin-resistant (mrsa and ) and one glycopeptide-intermediate (gisa ) strain, in a rabbit endocarditis model. mics of v and t (v/t) were . / . , / . , / , . / . , and / , for mssa , mssa , mrsa , mrsa , and gisa , respectively. the animals were randomly infected with one of these strains, then treated for days by v or t. a continuous infusion of v, simulating a mg/kg/ h human dose was used. t was infused as a continuous infusion allowing simulating a mg/kg human dose, following an initial bolus. these regimens achieved clinically relevant serum steady-state concentrations of glycopeptides ( !/ mg/ l). results were as follows: expressed in log cfu/g of vegetations (mean /sd, followed in parenthesis by the number of rabbits used). purpose: to determine the prevalence of the decreased glycopeptides susceptibility among clinical isolates of staphylococcus aureus collected from patients hospitalized in strasbourg university hospital between / / and / / . the susceptibility to glycopeptides of s. aureus isolates collected from hospital environment during approximately the same period was also investigated. methods: the susceptibility to glycopeptides was studied among the mrsa isolates, using: Á/ detection of the decreased susceptibility to glycopeptides using agar plates containing mg/l teicoplanin, Á/ detection of hetero-visa strains using agar plates containing mg/ l vancomycin, Á/ determination of the mics of vancomycin and teicoplanin using the agar dilution and the e -test strips methods. results: thirty-nine percent of s. aureus clinical isolates ( out of strains) are mrsa. no visa or hetero-visa strain was detected. six percent mrsa isolates are teicoplanin intermediate s. aureus strains. in the environment, % s. aureus isolates are methicillinresistant (five out of ). the five strains are all susceptible to glycopeptides. conclusion: the results regarding vancomycin are reassuring. however, the high rate of mrsa and the presence of teicoplanin intermediate s. aureus isolates prove that prevention and control measures need to be improved. comparative investigation of polymerase chain reaction and a conventional methods for detection of methicilin resistant staphylococcus amont clinical isolates soa . kantardjiev tv a , vacheva-dobrevski rs b , panajotov sv a , bachvarova am a , velinov ti a , levterova vs a . a national center of infectious and parasitic diseases, microbiology, sofia, bulgaria , b military medical academy, clinical microbiology, sofia, bulgaria purpose: identification on methicillin resistant staphylococci has a great clinical implication and significant impact of antibiotic therapy. the aim of this study is to compare the disc-diffusion test (ddt), oxacillin agar screen test (oast) and pcr for detection of mec a gene. fifty selective clinical isolates ( staphylococcus aureus and nine s. epidermidis ) determined as methicillin resistant by ddt were enrolled in the study. ddt was performed with oxacillin disk ( mkg/ml) on mueller-hinton agar (mha) without nacl (nccls, ) . oast was performed on mha with % nacl, oxacillin mkg/ ml, t c. these strains was genotypically characterized for the mec a gene presence by pcr method using the mec a - ?-aaa atc cat ggt aaa ggt tgg c- ? and the mec a Á/ ?-agt tct gca gta ccg gat ttg c- ? primers (gibco, brl) . results: in the group of mrs isolates, detected by pcr, positive results were as follow: s. aureus and six s. epidermidis . for six s. aureus isolates ddt and oast were positive; pcr-negative. for two s. aureus and two s. epidermidis isolates pcr was positive; phenotypic methods-negative. conclusions: accurate and rapid detection of mrs is a constant challenge for laboratories. the pcr assay (first time in our country) appears to be more reliable than routine susceptibility testing for the rapid diagnosis of mrsa infections at hospitals, particularly due to the heterogeneous resistance of many strains. . / . ( ) . / . ( ) . / . ( ) . / . ( ) . / . ( ) v . / . * ( ) . / . ( ) . / . * ( ) . / . ( ) . / . ( ) t . / . * ( ) . / . ( ) . / . * ( ) . / . ( ) . / . ( ) distribution and antibiotic susceptibilities of bacteria isolated from suspected urinary tract infections of inpatients in hungary soa . rozgonyi f, csukás z, kamotsay k, szabó d, ostorházi e, berek z, maródi c. institute of medical microbiology, semmelweis university, budapest, hungary between l january and december , a total of , urine samples were cultured % as native urine (nu) and % as uricult-plus (up) (orion diagnostica, finland) . cultivations were negative in % of nu and % of up specimens, while contamination was revealed in % of nu and % of up. in the clinical bacteriologically evaluable positive nu and up cultures, the distribution of the gram-negatives was very similar with the predominance of escherichia coli ( and %) followed by enterobacter spp. the distribution of gram-positives differed significantly according to the types of specimens. nu resulted in % group-d and % group-b streptococcus while up did and . %, respectively. third generation cephalosporins and fluoroquinolons were equally very effective against e. coli strains, while ampicillin inhibited growth of % only. carbapenems, cefepime and the fluoroquinolons were the most active against enterobacter strains. interestingly, trimethoprim'/ sulfarmetoxazole combination could inhibit more than % of enterobacteriaceae strains. piperacillin'/tazobactam ( %), imipenem ( %), and ciprofloxacin ( %) could be the drog of choice against pseudomonas aeruginosa . enterococcus strains were highly sensitive to glycopeptides ( %), nitrofurantoin ( %), imipenem ( %) and amoxicillin'/clavulanic acid ( %). antimicrobial susceptibility levels of escherichia coli isolates cultured from urine at a tertiary care teaching hospital. temporal trend and comparison between community-acquired and nosocomial urinary tract infection soa . nanetti a, manfredi r, valentini r, calza l, chiodo f. uni versity of bologna, infectious diseases, bologna, italy in order to assess the local temporal trend of antibiotic sensitivity of the most common urinary tract bacterial pathogen, all urine-cultured escherichia coli isolates were reviewed as to susceptibility profile, and specimen source (community-versus hospital-acquired infection). when evaluating sensitivity levels of community-acquired pathogens ( Á/ ), a significant resistance rise was limited to cotrimoxazole (p b/ . ) and nalidixic acid (p b/ . ), while a tendency towards increased resistance regarded norfloxacin (p / . ) (fig. ) . when community-acquired e. coli isolates were compared with nosocomial strains (tested in the years Á/ ), a greater susceptibility of community-acquired e. coli isolates was limited to cotrimoxazole versus all other compounds in the year (p b/ . ), while it was extended to amoxicillin, cephalotin, nitrofurantoin and piperacillin in the year (p b/ . ) (fig. ) . on the whole, e. coli showed an elevated sensitivity rate ( !/ % of tested strains) to nitrofurantoin, gentamicin, amikacin, and nd-and rd-generation cephalosporins, while only amoxicillin and piperacillin had a mean resistance rate !/ %, regardless of the community or nosocomial origin. a permanent surveillance of sensitivity levels of the most common pathogens responsible for infectious diseases enables to identify local antimicrobial activity and its temporal variations, and plays a key role in starting empiric therapy, pending bacterial identification and in vitro assays. conclusion: in uti, the antimicrobial agents such as st cg combined with aminoglycosides are recommended as initial treatment as well as the rd cephalosporin generation at monotherapy. in addition, the fluoroquinolones and aminoglycosides are effective in uti. prevalence of resistance mutations to antirretrovirals and relation to virological failure s . garcia f a , suarez s a , alvarez m a , martinez nm a , valera b b , pasquau j b , hernandez quero j a , maroto mc a . a hospital san cecilio, microbiology, granada, spain , b hospital virgen nieves, microbiology, granada, spain purpose: to investigate the prevalence of resistance mutations in the reverse transcriptase (rt) and protease (p) genes of hiv and to relate it with the type of virological failure (vf), we have studied patients ( % naïve or pregnant women, % were first vf, % were second vf, % more than two vf. resistance mutations were investigated using trugene hiv- genotyping kit (visible genetics). results: global prevalence of resistance mutations for rt inhibitors (rti) has been !/ % for m l, d n, k n, m v, l w, t yf, and for l i, m i, l p, a vt, l m for p inhibitors (pi). the prevalence of resistance mutations for the naïve patients studied was very low (a g, v i for rti and l i, m i, m i */all n / */and l p n / for pi). for patients on first vf only k n, m v, t yf (rti) were !/ %, as well as l i, d n, l p (pi); when patients on second vf were studied, then m l, e d, k n, m v, g a, l w, t yf, k qe (rti) and m i, l p, a t (pi) were !/ % prevalence; finally, when patients with more than two vf were studied, the following resistance mutations were !/ %: m l, d n, k r, k n, v i, y c, m v, g a, l w, t yf (rti), and l i, m i, m il, l p, a t, l m (pi). conclusions: the prevalence of primary resistance in the population studied is very low; the prevalence of mutations in the reverse transcriptase and protease genes increase in parallel to the type of virological failure. genotypic resistance in hiv- rna from patient plasma compared with rapid virus isolation and phenotypic resistance in patient pbmcs s . stuermer m, groeschel b, cinatl j, doerr hw. institute for medical virology, university clinic frankfurt, frankfurt, germany objective: to compare hiv- virus isolation in the presence of antiretroviral drugs with plasma hiv- genotyping. materials and methods: hiv- genotyping was performed using the viroseqtm vers. from applied biosystems. interpretation of genotype was done according to international standards. cd -cells were purified from patient plasma and cultivated in microtiter plates coated with anti-cd and anti-cd antibodies in the presence of different concentrations of antiretroviral drugs. virus production was measured using a p antigen assay. phenotypic activity was expressed as % reduction of p concentrations. results: seventeen samples were analyzed. for samples results were obtained from both methods, two samples could not be analysed by phenotyping and four samples not by genotyping. only / samples showed total and / samples partial concordance, / samples showed discordance between the two assays. in discordant samples the genotype gave a definite interpretation. conclusion: hiv- virus isolation and phenotyping from pbmcs may overcome the problem of currently used resistance assays, which analyse only the reverse transcriptase and the protease gene of hiv- . possible mutations in other regions may influence viral fitness and therefore contribute to the growth of the virus population present. the lack of concordance between the two assays is related to the different blood compartments used. the clinical value of resistance tests using pbmcs is under investigation. interleukin- co-operates with a new type i ifn, ifn-tau to inhibit early steps of hiv-i biological cycle s . rogez c a , clayette p a , martin m a , dereuddre-bosquet n a , martal j b , dormont d c . a cea, drm, fontenay-aux-roses, france , b inra, physiologie animale, jouy-en-josas, france , c cea, crssa, ephe, drm, fontenay-aux-roses, france background: type i interferons (ifn) exhibit efficient antiviral activities notably against hiv, but severe side effects restrict their clinical uses. ifn-tau is an ovine or bovine non-cytotoxic type i ifn which displays higher inhibitory effects towards hiv replication than ifn-alpha, particularly in human monocyte-derived macrophages (mdm). the antiretroviral activity of ifn-tau seems to involve antiviral and immunomodulatory mechanisms: il- synthesis is increased in dose-dependent manner in mdm treated with ifn-tau and a specific inhibition of il- biological activity decreases its antiretroviral efficiency. results: after a -h infection, a significant decrease of intracellular hiv rna amount was found in mdm treated with ifn-tau. in parallel, no additive inhibition was observed with ifn-tau during the elongation of proviral dna. these results suggest either an inhibition of hiv nucleocapsid uptake or an immediate hiv rna degradation, and the expression of ?, ?-oas, mxa protein and pkr was then measured. ifn-tau induced the expression of these three host cell factors. the role of il- on these different steps was evaluated and we showed that il- co-operates with ifn-tau during the very early step of hiv biological cycle. conclusion: altogether, these results evidence that ifn-tau uses the same antiretroviral pathway as others type i ifn in mdm, and that il- takes part to its inhibition of early steps of hiv biological cycle. actinomycin-d as a modulator of resistances due to cell-wall active agents like bacitracin (bc) and lysozyme (lz) s . chakrabarty an a , dastidar sg b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india it was observed that development of lzr in the lzr mutants took placed at three different levels and was accompanied with unselected, distinctive and elevated levels of bcr. similarly, bcr in bcr-mutants were also detected at three different levels. although the levels of bcr ( / mg/ml) in the bcr mutants could be raised only by persistent efforts, an increase in the levels of lzr (as cross-resistance) in the same mutants could be easily achieved. a correlation of actinomycin-d resistance with lzr and bcr of the mutant bacteria and the effects of lipase treatment on the same showed a Á/ -fold rise in actinomycin-d resistance of the lzr and bcr mutants of gram-positive bacteria compared with their correspondence wild-types. these findings suggest that lzr and bcr are controlled by several genes accounting for reduced cell-wall and cell-membrane permeability and indirectly, by phenotypic alteration of the lipid content of the cell-wall. thus, the alteration of cell-walls and membranes and a phenotypic extra lipid layer can work in conjunction with the efflux pump mechanisms finally determining the levels of drug-resistance. experimental development of drug resistance to non-antibiotics: a role of alteration of membrane fluidity and efflux systems s . dastidar sg a , mazumdar k a , asok kumar k a , chakrabarty an b . a jadavpur university, pharmaceutical technology, calcutta, india , b department of medical microbiology, calcutta university, calcutta, india drug resistance among clinical strains was studied by selecting mutants resistant to promazine (pr) and methdilazine (md). the results showed that successive step-up mutants of pr and md developed cross-resistance to several unrelated drugs, which in subsequent steps had broader resistance spectra with higher levels of resistance. experiments on the membrane fluidity or permeability of bacterial cells using diphenyl hexatrine (dph), a fluorescent probe on md-mutants showed that three was marked reduction in the membrane fluidity and permeability. when several analogues of the basic phenothiazine structure, e.g. -chlor-methyl-n -methyl-pyrrolidine (cmp), methyl- -methyl- -pyrrolidone- -carboxylate (mmpc), hydroxymethyl-n -methyl-pyrrolidine (hmp) and md with final substitution were tested for antibacterial function on different strains, highest activity was observed with respect to md. with anaerobic bacteria the resistance(s) dependent on efflux pumps showed higher levels of resistance even to md. we have found the non-antibiotic agents triflupromazine, trimeprazine and diclofenac sodium have high degree of activity against vibrios, staphylococci and pseudomonads. the explanation of such a phenomenon in terms of possible efflux pumps will be discussed. csf, plasma and urine pcr in lyme neuroborreliosis s . pícha d a , moravcová l a , lásiková Š a , marešová v a , Ž ïárský e b . a charles university, nd medical school, st clinic for infectious diseases, prague, czech republic , b department of cellular and molecular biology, charles university, rd medical school, st clinic for infectious diseases, prague, czech republic the main reason for high diagnostic value of pcr in neuroborreliosis (nb) is the direct way of spirochete detection. two sets of primers in nested pcr were used: one for plasmide gene encoding ospc protein and second for chromosomal gene s rdna. so far patients with clinically manifested involvement in nb were enrolled into the prospective designed study (being continued). the main including criterion was positive prove of intrathecal specific antibody secretion (in patients) and pcr positivity in csf (in ). all patients were repeatedly examined by neurologist and samples of csf, plasma and urine were taken: ( ) before treatment; ( ) after treatment; ( ) after months. before treatment were patients pcr positive in csf, six in plasma, and in urine. five were parallel positive in csf and plasma and four in all three body fluids. urine after treatment was positive in seven ( %) cases and completely negative after months. the pcr has had relative high sensitivity ( %), but does not rich the sensitivity of antibody index ( %). supported by grant mzcr ; . consumption of imipenem correlates with b-lactam resistance in pseudomonas aeruginosa s . lepper pm a , hö gel j b , trautmann m a , grusa e c . a department of medical microbiology and hygiene, university of ulm, ulm, germany , b department of biostatistics, university of ulm, ulm, germany , c hospital memmingen, central pharmacy, memmingen, germany purpose: in the present study we investigated the monthly consume of three anti-pseudomonas-active antibiotics, namely imipenem, piperacillin/tazobactam (pt) and ceftazidime during a period of years ( Á/ ) . the use of these antibiotics was correlated to the rate of resistance in pseudomonas aeruginosa . results: inspection of the time series for use of imipenem, ceftazidime, and pt, and the corresponding time series for resistance (each available from july to july ) indicates a remarkable coincidence between use of imipenem and resistance against the three antibiotics mentioned. pearsons's coefficient of correlation for the use of imipenem and the resistance against imipenem was . (p b/ . ), between imipenem use and pt resistance was . (p b/ . ), and between imipenem use and ceftazidim resistance . (p b/ . ). we found positive regression coefficients quantifying an association with imipenem use in the same month (p b/ . ) and with the use during the preceding month (p b/ . ). the same was true when checking dependence of ceftadizime resistance (p b/ . ) and pt resistance (p b/ . ) on imipenem use observed during the same month. neither the use of ceftadizime nor of pt could be identified as factors creating resistance to one of the three antibiotics under consideration within a reasonable period of time. conclusion: there might be a strong pressure towards resistance created by carbapenems. this could limit the use of carbapenems for initial empiric therapy. treat hard and fast: short course antibiotic treatment and its relation with patient compliance and effectiveness s . perez-gorricho bpg a , ripoll m b , pechere jc c . a niño jesus hospital, infectious diseases, madrid, spain , b insalud, outpatient consult, madrid, spain , c university of geneve, microbiology, geneve, switzerland 'treat hard and fast ': short course antibiotic treatment and its relation with patient compliance and effectiveness. finding the important implications for the way in which physicians manage patients with mild Á/moderate respiratory tract infections, and the relation of this management with the perception of antibiotic effectiveness, and the compliance with the antibiotic regimen has been the main purpose of the research. in a pan-european market research study of more than patients, designed to determine behaviour to the antibiotic management of mild-moderate respiratory tract infections, patient expectations of antibiotic therapy were identified, particularly those aspects that relate to efficacy and compliance. the study identifies three key drivers of patients perceived antibiotic efficacy: length of antibiotic course, time to onset of symptom relief and time to complete resolution of symptoms. the results demonstrate that once daily treatment for short periods is perceived by patients to be significantly more effective than longer antibiotic courses and thus better meets patient expectations of therapy. in this study, a macrolide, azithromycin, was selected as the drug therapy of shortest course, being the antibiotic with the shortest dosage schedule for common outpatient infections. the perception of efficacy with short course therapy also correlates with overall satisfaction with management by the physician and with patient compliance with antibiotic therapy. purpose of the study: group b streptococci (gbs) remain a major cause of neonatal infections. consensus guidelines have recommended an intrapartum antibioprophylaxis by amoxicillin, which has reduced the incidence of early-onset neonatal gbs infections. however, an increased incidence of beta-lactam-resistant gram-negative neonatal sepsis has been reported. the aim of our study was to analyse the consequences of this antibioprophylaxis on the intestinal microbial colonization of newborns. a study of the fecal flora was carried out on stools samples from days-old newborns divided into groups: group a intrapartum treated mothers (n / ); and group b untreated mothers (n / ). both groups were matched with regards to known factors affecting intestinal microbial colonization: gestational age, type of delivery and feeding. results: colonization by enterobacteria and enterococci was not significantly different and occurrence of amoxicillin-resistant enterobacteria was similar ( / and / in groups a and b, respectively). however, the colonization by clostridia was modified: the number of newborns colonized was significantly less important in group a than in group b (group a: / and group b: / p b/ . ). conclusion: in our study, intrapartum antibioprophylaxis did not affect intestinal colonization by aerobes but reduced significantly colonization by clostridia, potentially anaerobic pathogens. impact of an antibiotic policy restricting the use of b-lactams and macrolides on the incidence of clostridium difficile associated diarrhoea in general medical, renal and elderly patients s . boswell tc a , pacey s b , broomfield s c , westmoreland d c , yates c c . a nottingham city hospital, microbiology, nottingham, uk , b nottingham city hospital, pharmacy, nottingham, uk , c nottingham city hospital, infection control, nottingham, uk the purpose of the study: to investigate the short-term impact of a new antibiotic policy for the treatment of urinary and respiratory infections on the incidence of clostridium difficile associated diarrhoea (cdad) in hospitalised medical, elderly care and renal patients. the results obtained: a policy restricting the use of b-lactams (except parenteral penicillin), and promoting alternative antibiotics including levofloxacin for pneumonia, and doxycycline for non-pneumonic respiratory infections, was launched in july . as a result there was a significant and sustained reduction in use of aminopenicillins, cefuroxime and macrolides, with a corresponding increase in doxycycline and levofloxacin. the incidence of cdad was determined during the st months of the new policy and compared to the last months of the old policy. the incidence of cdad fell from . to . per patients, and from . to . per in-patient days (p b/ . ). in contrast, there was no change in the incidence of cdad in other specialties (surgery, oncology etc.) that had not introduced the new policy. there was no change in the incidence of nosocomial bacteraemia with quinolone-resistant coliforms or mrsa, despite the increased use of levofloxacin. conclusions: hospital-wide reduction of b-lactam and macrolide use in medical patients can result in a significant and immediate reduction in cdad. longer follow-up will determine if this effect is sustained. use of imipenem/cilastatin i.v. (tienam i.v.) for the treatment of low respiratory tract infections in intensive care units s . izzo l a , orsetti r b , boschetto a a , binda b a , della casa u a , caramanico l a , la mazza a a . a department of surgery, universitá degli studi di roma 'la sapienza','p. valdoni', rome, italy , b s. camillo-forlanini, intensive care unit, rome, italy ventilator associated pneumonia (vap) is considered the most frequent infection in the intensive care unit (icu), occurring in Á/ % of patients intubated for longer than h besides nosocomial pneumonia is a common complication in the critically ill surgical or trauma patient. inadequate treatment can lead to the complications of acute respiratory distress syndrome (ards), empyema, and lung abscess. the most important aetiological agents both in vap and in pneumonia which arise as complication in surgical or trauma patients are bacteria, whit a marked predominance of staphylococcus aureus and pseudomonas aeruginosa . the authors present their experience ( cases) on the employment of imipenem/cilastatin i.v. (tienam i. v.) as initial empirical monotherapy at the dose of mg)/ /day or g)/ / day for the treatment of the serious lower respiratory tract infection in an icu. tienam is a well tolerated broad spectrum antibacterial agent that is effective against the majority of gram-positive and gram negative aerobic and anaerobic bacteria including most pseudomonas species. except one patient deceased for causes related to his very poor general conditions and three cases in which has been necessary the addition of an aminoglycoside, in all the other patients the imipenem/ cilastatin (tienam) monotherapy has shown satisfactory clinical and bacteriological responses. clinical auditing of the impact of recommendations on antibiotic treatment s . kinoo j a , david-ouaknine f a , hacquard b a , echard y a , decazes jm b . a centre hospitalier lagny marne la vallée, lagny sur marne, france , b hospital saint louis, paris, france the aim of this study was to assess the impact of curative antibiotic recommendations on suitable prescriptions at lagny-marne la vallée hospital (general hospital, beds). two prospective exhaustive audits were made (all complete hospitalizations, excluding psychiatry, february Á/may and ) of the detailed curative antibiotic prescriptions, before and after distribution of internal recommendations. the same methodology, designed by a multidisciplinary team, was used for both periods. the same antibiotics were available at the pharmacy. the prescriptions were assessed by an infectious diseases specialist and a pharmacist using pre-established criteria: literature recommendations ( audit), internal recommendations ( audit). six hundred and fifty-six prescriptions for patients were collected and analysed in , for patients in . exhaustivity of the recovered prescriptions was over %. patient characteristics, infection sites and microbiological findings were similar for both groups. suitable prescriptions were significantly increased ( Á/ %, p b/ . ). unsuitable prescriptions (economic reasons, too short or too long course, incorrect administration, or underdosage) were significantly reduced. prescriptions for incorrect indications were unchanged and necessary combined treatment not being prescribed, increased. local recommendations improved prescriptions, but efforts have to be done in order to go on the improvement of the practice behaviour. cost-effectiveness analysis of antibiotic therapy in hospitalized patients with copd exacerbations (ae-copd) s . beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy antibiotic costs represent a high burden of total drug costs for hospital administrations. a scientific approach considering also the economic aspects of each therapeutic decision may gain optimal treatment objectives at pondered costs. in our study we retrospectively evaluated the clinical effectiveness and costs of antibiotic therapy in patients with ae-copd. from to , our retrospective study results support previous pharmaco-economic considerations according which in choosing an antibiotic regimen for ae-copd we must take into consideration the expected clinical and microbiological results without forgetting to consider the economic burden of our decisions. significant increase in fungaemia due to non-albicans candida species s . shah pm. klinikum der j.w. goethe universitaet, schwerpunkt infektiologie, frankfurt am main, germany until , predominant candida species in blood cultures was candida albicans . it accounted for . % of all candida species cultured from blood. since then we have observed a gradual increase in number of non-albicans candida species. from , onwards nonalbicans candida species out-number c. albicans . this observation is especially important as non-albicans candida species are generally non-susceptible to azole derivatives and empirical use of azoles in suspected candidaemia should not be recommended. amphotericin b is uniformally active against almost all candida species. echinocandin may be an alternative. see figure below. a search for newer antifungal chemotherapeutics s . chakrabarty an a , dastidar sg b , saha b b , basu l b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india fungal infections due to the mucor-rhizopus (m-z) group present formidable problems due to lack of appropriate and effective drugs against them, as seen in increasing number of clinical situations; death due to mycoromycosis is nearly inevitable. we analysed the biological 'weak-spots' of the mucor-rhizopus group and attempted to devise suitable drugs using their weak-spots. we have noted that like many free-living fungi, the m-z fungi are facultatively chemoautotrophic (can grow on simple sources of carbon and nitrogen and a solution of mineral salts), like the human pathogenic chemoautotrophic nocardioform bacteria. we devised a minimal medium based on that of davis and mingioli, supplemented with simple chemical compounds as sole sources of carbon and nitrogen. the key chemical here was diphenylamine with trypan blue (dpa Á/tb) and other similar sources of c and n. we found that while media free of these chemicals (controls) allowed good growth of different strains of m-z fungi, a mixture of dpa Á/tb completely prevented their growth over a wide concentration range. experiments with immunocompromised mice showed that these drugs at the concentrations used are well tolerated; mice experimentally infected with several clinical isolates of m-z fungi and receiving these chemicals showed that these fungi could not grow in vivo. in vitro activity of newer fluoroquinolones against multi-drug resistant salmonella typhimurium s . nolones resistance is being also reported. we have studied the in vitro activity of b-lactams and fluoroquinolones against multi-drug resistant s. typhimurium from human sources. material and methods: fifty multi-drug resistant s. typhimurium were tested against cefazolin, cefuroxime, cefotaxime, cefepime, ofloxacin, levofloxacin, and moxifloxacin, by the agar dilution method according nccls guidelines. results and conclusions: all the strains were resistant to four or more of the following antibiotics: ampicillin, tetracyclines, chloramphenicol, streptomycin, sulphonamides and nalidixic acid. a high proportion of strains were intermediate or resistant to amoxicillin/clavulanate. we found no resistance to cephalosporins. nevertheless, % were intermediate to first and/or second gen. cephalosporins. cefotaxime and cefepime were the most active cephalosporins (mic : . mg/l). though increasing fluoroquinolones resistance has been described among this kind of strains, no resistance to fluoroquinolones was found here. levofloxacin was the most active fluoroquinolone (mic : . mg/l), followed by ofloxacin (mic : . mg/l) and moxifloxacin (mic : . mg/l). high rates of resistance to antibiotics by salmonellae from diarrhoeic children in zliten-libya s . ghenghesh ks a , ben ali m b , abuhelfaia a b , dufani ma a . a faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya , b faculty of arts and sciences, el-ghomes university, biology, el-ghomes, libyan arab jamahiriya salmonellae are major bacterial cause of diarrhoea in libya particularly in children. included in the present study salmonella species isolated from children with diarrhoea in zliten city-libya. the children aged between a few days to years. the organisms were tested for their susceptibility to antibacterial agents using the disc diffusion method. of the isolates examined, ( %) were resistant to ampicillin, ( . %) to amoxicillin Á/clavulanic acid combination, ( %) to cefoxitin, ( . %) to chloramphenicol, ( . %) to doxycycline, ( . %) to gentamicin, ( . %) to nalidixic acid, ( . %) to trimethoprim Á/sulphamethoxazole and none ( . %) were resistant to norfloxacin. a strong relationship was observed between the availability of antibiotics in the pharmacies of the city and resistance of the isolated salmonellae to these drugs. the misuse of the antibiotics by the community may be an important factor (among others) in the emergence of these high rates of resistance by the salmonellae examined. effect of ceftriaxone along with probiotics administration on intestinal ecosystem and betalactamase activity s . bertazzoni minelli e a , benini a a , zoppi g b . a department of medicine and public health-pharmacology section, university of verona, verona, italy , b department of paediatrics, university of verona, verona, italy oral bacteriotherapy during antibiotic treatment is a much debated topic. aim: to study whether different probiotics can prevent imbalance of the intestinal ecosystem (dysbiosis) in children during therapy with ceftriaxone (cx). methods: fifty-one children (mean age . years) with febrile respiratory tract infections were treated with cx mg/kg/day iv, alone (therapy ) and along with the following preparations: saccharomyces boulardii ( ); enterococcus spp. ( ); lactulose ( ); l. casei gg ( ); l. rhamnosus , l. bifidus and l. acidophilus ( ); b. bifidum and l. acidophilus ( ); and a mixture of various lactobacilli and bifidobacteria at high concentrations ( ). faecal samples, collected before and after treatment, were analysed for microflora composition, cx concentration, and beta-lactamase (bl) activity. results: cx causes intestinal dysbiosis. no c. difficile was found. faecal bl increased after therapy in all treated groups. cx alone increased bl activity in % ( / ) of children (no activity before treatment); a higher incidence ( Á/ %) was found in groups and . after therapies , , , , and , bl activity was found in or more children. cx was detected in % of faecal samples. conclusions: the probiotics administration seems to protect against dysbiosis caused by cx and to contain the increase in faecal bl activity. the effects differ according to the probiotic administered and are peculiar to certain bacterial species. these preliminary data need further studies. comparative study of initial and acquired drug resistance in pulmonary tuberculosis in iran s . mansoori d, arami s, mirabolhasani z. nritld, infectious disease, tehran, islamic republic of iran purpose: resistant to anti-tuberculosis agents particularly multiple drug resistant (mdr) is a major obstacle in treatment tuberculosis in the world. between september and march for smear and culture positive pulmonary tuberculosis patients (old / , new / ) pretreatment susceptibility tests of isolated bacilli to inh, rif, emb and stm were performed by standard proportional method and the results were attributed to three groups: (i) newly diagnosed without any history of treatment; (ii) patients with history of treatment for one course; (iii) patients with history of treatment for two or more courses supposed to be mdr cases. the results were collected for each drug individually and different combinations of two, three and four medications. results: resistance to one, two, three and four drugs was significantly increased in group iii comparing to groups ii and i, also in group ii compared to group i. we observed a high rate of primary resistance to inh and stm in groups i and ii and a high rate of mdr (inh and rif resistance) in groups ii and iii. conclusion: the duration of bacilli exposure to antituberculosis agents in the past is a major factor in developing resistance. in contrast to who's guideline, due to high rate of primary resistance especially to stm in our area, we do not recommend addition of stm for treatment of patients whose initial four-drug regimens have been failed (group ii). donors, to understand host interactions with this bacteria, to develop new methods of diagnosis and define new vaccine candidates. nineteen tb patients and seven healthy donors were enrolled in french hospitals. cellular immune responses were evaluated by lymphoproliferation and ex-vivo quantification of specific th cells by elispot-ifn-gamma assays. four recombinant proteins of m. tuberculosis were tested: esat- , b, erp and tb b . and compared with tuberculin. we confirmed that b (but not esat- in our study) gives higher responses in tb patients compared to donors according to the results in proliferation assay (p / . ). in addition, frequencies of th cd specific cells for esat- and tuberculin were statistical different between the two groups (p / . and . , respectively). . % for b and . % for esat- of the patients tested were responders in elispot-assay versus . % for both in proliferation assay. for the new antigens erp and tb b . , no difference was observed between the two groups. in conclusion, b and esat- are recognised by a large number of our patients. they seem to be promising antigens for the development of new methods of diagnosis or for the development of new vaccines. erp and tb b . are not preferentially recognised by tb patients. other exported antigens will be tested. the incidence of tuberculosis (tb) is increasing worldwide. in recent some years, geographical differences in the incidence of tb in former yugoslavia have been observed. an important rise in tb cases was registered in the bordering region of bosnia. it is likely that poorer living conditions, influenced by war and emotional stress, may promote such rising incidence of tb. renal tuberculosis was diagnosed in patients (female , male ) from district brcko in bosnia, during the period of years, Á/ . at the same time none patient had active pulmonary tb lesions, fibrous lesions were noticed in patients, but we did not diagnose any signs of previous pulmonary tb in seven patients. seven patients developed relapse of renal tb after Á/ years of previous treatment. guided by clinical parameters, precisely done renal echosonography enabled early suspicion and searching for renal tb, by radiological and other methods. bacteriological diagnosis was performed by detection mycobacterium tuberculosis on loewenstein Á/jensen medium in patients. pcr as simple, fast and highly sensitive method enabled diagnosis in incipient stadium of disease, so antituberculous therapy could be instituted some months earlier. prompt diagnosis of renal tuberculosis (using pcr, besides standard methods) is necessary, otherwise delayed diagnosis may be dangerous. a study on resistance to first generation anti-tuberculosis drugs in mycobacterium kansasii s . mirsaeidi sm, farnia p, mohammadi f, mansoori sd, jabbari r, taghizadeh r, masjedi mr, velayati aa. national research inistitute of tuberculosis and lung diseases, infectious diseases and immunology (nritld ), tehran, islamic republic of iran purpose: this research has been performed to determine antibiotic resistance of atypical mycobacteria especially mycobacterium kansasi . results: twenty-three pigmented colonies which indicated atypical agents from nritld's mycobacterium culturebank were selected, they then underwent type identification and antibiogram for inh, rif, etb, stm. nine samples were m. kansasi and were other non-mtb, was m. gordonei , m. xenopi , mac, m. bovis , m. tererra , m. asiatioum , m. marinum and . mean age in m. kansasi cases was '/ . year and in non-kansasi cases '/ . year and in whole society ntm was . '/ . . frequency of resistance in kansasi group were to inh ( %), to rif ( %) to etb ( %), to stm ( %) and prevalence of mdr was ( %) and in non-kansasi group frequency of resistance were ( %) to inh, to rif ( %), to etm ( %) and to stm was ( %), to mdr was ( %). conclusion: a significant difference was seen between the age groups of patients who are affected with m. kansasi and non-kansasi (p b/ . ), also in frequency of resistance to first generation anti-tb drugs. m. kansasi is detected as the most common atypical mycobacterium agent in pulmonary infections and attention to antibiogram is recommended before treatment. buruli ulcer caused by mycobacterium ulcerans , is the third most common mycobacterial after tuberculosis and leprosy in west africa. nowadays, the only effective treatment is surgery. it consists in a large excision of the lesions, often followed by a skin transplant. in this study, the effectiveness of rifampin, amikacin and their combination were estimated in the treatment of mice, which were infected experimentally by m. ulcerans . after weeks of treatment with rifampin, amikacin or their combination, no more viable bacilli were found in infected tissues. the animals were kept for other months. among the mice treated with rifampin alone, two mice out of relapsed. the minimal inhibitory concentration of these isolated strains went from . to mg/ml. the dna sequence, obtained from a -pb of the rpob gene from these strains, showed a missense mutations, which affect a ser- replaced by a phenylalanine. this modification on the gene leads to an important inefficacy of treatment when rifampin was used alone. this study showed that rifampin and amikacin have a bactericidal action on m. ulcerans and that a combination of these antibiotics is necessary to avoid the selection of resistant mutants. histopathologic and electron microscopy studies of a severe isolated hiv enteropathy detected in an aids presenter. favorable response to haart introduction or . manfredi r, calza l, chiodo f. university of bologna, infectious diseases, bologna, italy advanced hiv infection was detected in a heterosexual female with a -year history of chronic diarrhoea and severe wasting, as expressed by a body weight of kg, a cd '/ count of cells/ml, and a plasma viraemia of . million copies/ml. a malabsorption syndrome was confirmed by d-xylose test, but repeated pathogen search tested negative at stool examination and light microscopy, scanning electron microscopy (em), and transmission em study of enteric mucosa. em assays detected an ultrastructural modification of duodenal mucosa never reported to date: an extensive thinning of enterocytic microvilli, disappearance of glycocalix, and large vacuolization of the enterocyte cytoplasm. two weeks after starting an indinavir-based haart, diarrhoea disappeared and our patient significantly gained body weight: kg after months, kg after , and kg after year, paralleling a cd '/ increase to cells/ml, and undetectable hiv viraemia. the subsequent -year follow-up confirmed absence of gut disturbances, a stable body weight, a cd '/ count of Á/ cells/ml, and hiv viraemia persistently b/ copies/ml. repeated endoscopy and related histopathologic and em assays documented a notable improvement of mucosal damage, with complete cure reached after years of haart. a direct intestinal localization of hiv may be responsible for severe diarrhoea, malabsorption, and wasting, though the morphological features of hiv enteropathy are still unclear. haart acts favourably also against isolated hiv-related enteropathy. kaposi's sarcoma in a non-hiv immunocompetent adult: relapsing due to the development of a squamus cell carcinoma or . sioula e a , magira ee a , georgopoulou c a , rontogiani d b , gounaris t a . a evagelismos, internal medicine and infectious diseases, athens, greece , b evagelismos, pathology, athens, greece a -year-old heterosexual hiv negative girl was diagnosed with cutaneous kaposi's sarcoma. the disease was started years earlier with the appearance of lesions on the left feet and on the right knee. absolute number of cd and cd were and cells/dl, respectively with a decreased lymphocyte proliferation. human herpesvirus type had been detected in biopsy specimens and she placed on recombinant interferon alpha- b. follow up few months later the lesions decreased in size. two years after the onset of the disease the patient readmitted because of a mass on the left paratracheal region along with mediastinitis. her body temperature was increased. the patient underwent thoracic ct scan, which demonstrated mediastinal well defined soft tissue infiltration associated with mediastinitis and a well-defined mass in the left paratracheal region. the mass biopsy revealed squamous cell carcinoma. several violaceus lesions were observed on the arms, hands and face. severe bilateral lymphedema of the legs with a reddish papules nodules and tumors from . to cm in diameter on the soles, toes and calves were present. this case illustrates the significant relapsing of the cutaneous kaposi's sarcoma soon after the appearance of and the carcinoma and the mediastinitis. a -year-old male patient with insulin-dependent diabetes underwent cardiac surgery for aortocoronary bypass years ago. two weeks after surgery he developed mediastinitis and sternal osteomyelitis caused by methicillin-resistant staphylococcus aureus (mrsa). twice, revisions and plastic surgery for sternal osteomyelitis were performed. the patient received initially treatment with vancomycin. then the patient received intravenous outpatient treatment with teicoplanin for weeks followed by by treatment with fusidic acid and then trimethoprim/sulfametrol. the fistula was closed. four months later he presented again with substernal pain and purulent discharge. the culture revealed the growth of staphylococci which were first mistaken for coagulase-negative staphylococci. after closer investigation these staphylococci were identified as small variant mrsa. computer tomography (ct) revealed multiple mediastinal abscesses. the patient was treated with intravenous linezolid mg bid for days and then switched to oral linezolid mg bid. the oral therapy was pursued for weeks under close surveillance. the patient improved substantially, the purulent discharge disappeared. the mediastinal abscesses were not detected any longer by ct at the end of treatment. the treatment with linezolid was well tolerated. platelets decreased initially but rose to normal values without treatment modification. nosocomial pneumonia due to stenotrophomonas maltophilia in a profound granulocytopenic patient hospitalized for community-acquired staphylococcus aureus severe sepsis or . radulescu a a , sasca n b , lupse m c , tatulescu d c . a university of medicine and pharmac, epidemiology, cluj-napoca, romania , b the teaching hospital of infectious diseases, laboratory, cluj-napoca, romania , c university of medicine and pharmacy, infectious diseases, cluj-napoca, romania objective: to present the diverse opportunistic infections in immunocompromised patients and treatment difficulties. findings: a -year-old women was admitted to the teaching hospital of infectious diseases cluj with a -day history of fever, myalgia, lumbar pain, hemorrhage syndrome. severe sepsis was diagnosed and the conditions that evolved in it were paronychia in a patient with chronic leukemia having prolonged and profound granulocytopenia due to aggressive treatment with ifn. the condition at admission was critical due to trombocytopenia ( b/ platelets/ml) and hemorrhage syndrome. the evolution was favorable under antimicrobial treatment (imipenem), blood and platelet transfusion, intravenous immunoglobulins, granulocyte colony-stimulating factor, antifungal prophylaxis and supportive care. blood and pus cultures revealed mssa. in the th day of hospitalization she developed bronchopneumonia and respiratory failure. the sputum culture was positive for stenotrophomonas maltophilia susceptible to ceftazidime, fluoroquinolones. treatment was unsatisfactory until introducing ticarcilline Á/clavulanate and ciprofloxacine. she had uneventful recovery despite remaining granulocyto-trombocytopenic. conclusions: treatment of infections with emerging agents in immunocompromised patients is difficult, guidance by results of susceptibility testing being misleading with a poor correlation between the tests and treatment outcome. early disseminated listeriosis in a liver transplant recipient (ltr): a rare case due to an in vitro multiresistant strain or . manfredi r, de ruvo n, vivarelli m, bellusci r, montalti r, la barba g, abtueli aden a, cucchetti a, attard l, calza l, cavallari a. university of bologna, infectious diseases, bologna, italy a ltr receiving cyclosporin, azathioprine and steroids, developed an extraordinary episode of sepsis and pleural effusion due to a multiresistant listeria monocytogenes (lm) isolate. a lm strain serov. showing the same, extensive resistance pattern (all penicillins and stand nd-generation cephalosporins), was isolated from multiple blood cultures and pleural fluid weeks after surgery, while stool exam was negative. our p spent her life in countryside and bred some animals, but denied consumption of uncontrolled food. iv cotrimoxazole administration achieved a complete clinical and microbiological cure in days. underlying immunodeficiency may prompt unusual/severe lm infection, but because of its usual community-acquired origin, lm disease remains infrequent in hospitalized p. only seven anecdotal reports of lm infection were described in ltr, Á/ , all but occurring months Á/years after surgery. an early respiratory and systemic infection caused by a community-acquired lm strain which proved resistant to first-choice antibiotics, but had a favorable response to cotrimoxazole (used only once in a ltr with lm sepsis), characterized our episode. an epidemiological survey retrieved the possible source of this usually community-acquired infection. lm should be regarded as an emerging opportunistic pathogen in ltr, and specific risk factors should be seeked. when immunodeficiency is of concern, the unpredictable sensitivity of lm should prompt in vitro assays to adjust antimicrobial therapy problems for discussion hbv Á/hcv and liver carcinogenesis: where does the viral influence end? or . pappas ga. university hospital, internal medicine, ioannina, greece hepatocellular carcinoma (hcc) is a major clinical problem worldwide, usually evolving over a long-standing liver pathology, in the latter stages in the form of cirrhosis. hbv and hcv chronic infection is a common etiology of cirrhosis, and hence, hcc. a number of studies have attempted to clarify the role of these viruses into the progression towards hcc. does their end in the stage of cirrhosis? is progression towards hcc independent of the etiology of cirrhosis (since alcoholic cirrhosis also proceeds to hcc)? do the trials with interferon alfa for patients with hbv or hcv cirrhosis exhibit a favorable result due to the antiviral properties of interferon, or is interferon exhibiting anti-oncogenic potential?, and is hcc cytokine and hormone sensitive (view ongoing trials with somatostatine analogues versus hcc)? (hence, if we treat alcoholic cirrhosis patients with interferon could we have a favorable response?). which patients with hbv or hcv cirrhosis are eligible for interferon treatment?: interferon treatment is a potential hazard for those with thrombocytopenia. how ethical is it to conduct a randomised trial where one leg of cirrhotic patients is left without antiviral therapy? and on the basis of which classification system should the two legs of such a trial be separated? moreover, do viral proteins with oncogenic potential exist (the controversy over the recently discovered hbv protein is still, unresolved)? a major topic awaiting for a major debate. to assess the role of hiv-associated campylobacteriosis (c) according to haart availability, patients with positive culture were identified since . compared with the Â/ hiv-infected p followed in the last decade, no epidemiological differences were shown, save a greater sexual exposure to hiv (p b/ . ). the introduction of haart caused a drop of frequency of c (from . to . episodes per p-year; p b/ . ), and modified clinical features, with disappearance of dissemination and mortality, reported in and patients before (p b/ . ). hiv-related immunodeficiency and disease stage were significantly related to c features before and after haart availability: p b/ . for cd and neutrophil count, p b/ . for aids diagnosis. most cases ( ) were community-acquired, but alimentary or environmental risk factors were never found. ten patients received cotrimoxazole prophylaxis (nine before ; p b/ . ), while no relationship occurred with steroid or antibiotic use, caused cases out of . a % sensitivity was found to quinolones, followed by cephalosporins ( . %), gentamicin ( . %), macrolides ( . %), and cotrimoxazole ( . %). a Á/ -day antimicrobial therapy cured p , but relapses caused by similar strains occurred in patients within Á/ weeks, all in the pre-haart era (p b/ . ). c still occurs in the haart era, probably due to its varied mode of transmission. the frequency of c is greater in hiv-infected patients, but less frequent visceralization, recurrences, and mortality characterized the haart era. objective: to determine the incidence and risk factors for nosocomial viral respiratory infections (nrvi) and involvement of human coronaviruses (hcov) in a neonatal and pediatric intensive care unit. methods: prospective observational study. nasal samples were obtained by cytological brush at admission and weekly thereafter for all hospitalized infants. nasal samples were taken monthly from staff. virological studies were performed, using immunofluorescence for respiratory syncitial virus (rsv), influenza viruses, paramyxoviruses, and adenoviruses; both immunofluorescence and rt-pcr were used for hcov detection. results: during , hcov related nrvi were detected in nn and six in children. three hcov-related outbreaks were observed (february, august and december), associated with a high prevalence of infection in staff. during august outbreak, hcovinfected nrvi were detected over hospitalized infants. seventy-five of hospitalized preterm nn with gestational age under weeks and . % of staff members were infected. risk factors for nrvi in nn were birth weight, gestational age, ventilation, oxygenation and hospitalization length. ninety-two percent of infected preterm nn were symptomatic, mainly with bradycardia and respiratory worsening. conclusions: these data provide additional evidence for a significant role of hcov in nrvi occurring in hospitalized preterm nn. strain typing and screening of dna targets to assess echinococcus sp transmission in new and old geographic endemic foci or . bart jm a , piarroux r a , dia l b , benchikh-elfegoun mc c , vuitton da a , bardonnet k a . a serf, parasitology, besancon, france , b national centre of veterinary study, parasitology, nouakchott, mauritania , c university of mentouri, parasitology, constantine, algeria purpose: cystic echinococcosis is due to echinococcus granulosus. parasite cycles depending on the main intermediate host species involved in different foci have been described promoting mixed infection in the same definitive host. strain typing is a tool to identify the main intermediate host involved via the dogs in the human infection route and to focus the control measures. many dna targets have been used to compare samples and to access the parasite cycle in different countries. but no study has compared the value of each of these targets. eight targets have been tested in mauritania where echinococcosis is an emergent disease, and in algeria where strain typing has never been done. thirty-five cyst samples from human, ovine, camel and bovine have been tested with six nuclear and two mitochondrial targets. results: the two mitochondrial targets and four out the six nuclear targets have allowed to discriminate the different foci. two strains have been found infectious to human : the 'sheep' strain in algeria and the 'camel' strain in mauritania. conclusion: although overlapping geographically sometimes, this raises the question of the respective genetic evolution of the different strains and of their involving in human infection. alveolar echinococcosis in france: an update or . bardonnet k, bresson-hadni s, bartholomot b, gérard a, watelet j, beytout j, saurin jc, piarroux r, vuitton da, who centre collaborating for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the highest prevalence rate for alveolar echinococcosis (ae) in europe has been found in france. in , a french observatory of human ae was done in order to get data that could be used to evaluate presentation, evolution and management of ae. material-methods: french cases were collected for the period Á/ . registration of every case was performed with the subject's agreement. a questionnaire was filled in by referring to the patients' medical files or to practitioners or to patients themselves. completeness of the collection of cases was ensured by multiplying the sources of information. results: two hundred and sixty nine french patients were registered. sex ratio averaged . mean age at diagnostic was . years. . % of diagnosis was performed in 'echinococcosis free' french areas. symptoms, but not always specific liver symptoms, were present at diagnosis in . % of cases. the liver was the main location of lesions in . % of cases. a wide spectrum of management of the patients was observed, accounting for regional differences. conclusion: this french observatory of human ea will facilitate a better management of the disease at the national level. it shows new epidemiological trends, and especially an extension of the endemic area. can coins and paper currency transmit bacillus anthracis ? or . ghenghesh ks. faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya anthrax is an often fatal bacterial infection caused by bacillus anthracis . recent events that began in september in us has gained the organism worldwide attention and heightened awareness of and concern about anthrax. many cases of anthrax with a number of deaths have been reported as a result of contact with envelopes, sent through postal mail, containing b. anthracis endospores. a number of studies have shown that currency is colonized with bacterial organisms, that include enteropathogens (e.g. shigella sp.), other enteric flora (e.g. escherichia coli ) and potential pathogens (e.g. staphylcoccus sp. , pseudomonas sp. and bacillus sp.). furthermore, methicillin-resistant s. aureus (mrsa) isolates that produced enterotoxin (seb) and toxic shock syndrome toxin- also been reported. all of these studies do agree on that currency may be considered as a method of spreading potentially pathogenic and pathogenic bacteria in the community. therefore, currency could also be a vehicle for spreading other highly pathogenic organisms that include b. anthracis . in addition, the introduction of the 'euro' could also allow such bacteria greater freedom to travel across the euro zone. the threat of using currency, particularly paper notes, in spreading lethal organisms should be investigated and proper measures to prevent the use of such a method by terrorists should be implemented. salvage of temporary femoral catheters for haemodialysis using antibiotics in ambulatory patients or . gerasimovska v, oncevski a, dejanov p. department of nephrology, clinical centre, skopje, the former yugoslav republic, macedonia the stay of femoral catheters (fc) for haemodialysis is typically short-term for several days. we used fc as a temporary vascular access (va) for a longer period of time in outpatients going on regular ambulatory haemodialysis, who had a problem with their permanent access. we analysed patients who were discharged from hosptal with fc. duration time of fc was between and days (average . days) with cummulative total of days. the incidence of bacteriaemia was . episodes/ catheter days. in six patients we had signs of infection, so according to our protocol we took blood cultures from peripheral vein, and from catheter (at same time) and started with antibiotic therapy (ab) systemically and locally (ab was 'locked' in catheter) with different duration of time. dominant microorganism was staphylococcus coagulasa negative, and much less staphylococcus aureus , and enterococcus.ab that were frequently used were: cefotaxim, vancomycin and ciprofloxacin. at one of six patients we removed catheter at once without trying to save the catheter. catheter tip was sent for microbiological analysis too. criteria for catheter-related bacteriemia (crb) was found in only one patient, and for possible crb in five patients after we removed the catheters. in the absence of clinical signs of infection, ab treatment was not provided for positive tip culture alone or for positive blood culture of the catheter with negative blood culture from peripheral vein. advances in meningitis education or . holt de a , tait mi b , cavanna al b , worgan-brown s a , hart b a . a the meningitis trust, stroud, uk , b the computer-aided learning unit, school of health science, university of wales, swansea, uk background: meningitis remains an important cause of death worldwide despite improvements in diagnosis, treatment and prevention. clinical and lay awareness of the disease relies on education, however educational delivery has changed and the introduction of material suitable for computer and internet application is now necessary. we have developed educational material on cdrom and on the internet applicable both at tertiary university and secondary school level. application: a computer-aided learning program on cdrom, covering all aspects of meningitis has been produced. it is suitable for undergraduate teaching of healthcare professionals from student nurse and doctors to pharmacists. in order to reach school children in a form acceptable to both pupils and teachers, we have developed a curriculum-linked website. these applications are simple to use and can be incorporated into existing courses of study, so that issues raised can be discussed with tutors and group peers. comment: the introduction of new methods of teaching and learning mean that compatible educational material must be produced. we believe that these applications, focusing on meningitis, are the first of their kind and that they offer tutors the opportunity to progress their teaching of the disease both in methodology and content. brivudin compared to famciclovir for improved therapy of herpes zoster: effects on acute disease and postherpetic neuralgia or . potentially treatment-related adverse events occurred in . % of the brivudin recipients and in . % of the famciclovir recipients (p / . ). conclusions: in zoster patients ]/ years, brivudin )/ mg and famciclovir )/ mg showed equivalent effects on prevalence and duration of phn. brivudin is as effective as famciclovir in stopping viral replication in acute herpes zoster. brivudin offers the advantage of a once daily dosage regimen while being as well tolerated as famciclovir. activity of complexes of pt(ii) and pd(ii) with pyridine- -carbaldehyde thiosemicarbazone (hfotsc) (acta virol., , , ) with selectivity index (si) . times higher than that of acyclovir (acv). in order to evaluate virus specific response and structure Á/activity relationships we continue our investigations with three pt(ii) and three pd(ii) complexes. the activity was evaluated against sensitive to acv hsv (strain bja) and resistant strains r- (hsv ) and pu (hsv ) and compared to that obtained against strain victoria (hsv ) infection. si was indicative for activity. the virus specific response was demonstrated by the fact that viruses sensitive to acv were also sensitive to pt(hfotsc) ]cl , while acv resistant viruses were sensitive to [ptcl(fotsc) ]. the structure Á/activity relationship was proved by the fact that the less active against hsv infection was [pd(fotsc)]. influenza diagnosis, treatment, and the impact of new antivirals on current treatment behaviours during influenza outbreaks or . schaetz l a , sessa a b , a hoffman-la roche f. basel, switzerland , b italian college of general practitioners, italy introduction: annual influenza epidemics severely affect individuals, families, health care systems and society. the availability of new and specific antivirals provides an opportunity for better management of influenza. methods: during the / and / influenza seasons, physicians ( Â/ /country) and public ( Â/ /country) in the usa and europe were interviewed to determine perceptions of influenza and behaviours for its treatment. results: patients recognise influenza illness as severe and identify it by symptoms of fever, muscle aches/pains and cough. physicians use these symptoms to diagnose influenza clinically ( % fever, % muscle aches/pains, % cough); their main treatment objective being to reduce complications. antibiotics for influenza treatment are broadly recommended/prescribed by about % of european physicians, whereas currently available antivirals are only recommended by %. the recommendation of antivirals by us physicians increased from % (season / ) to % ( / ) and markedly decreased antibiotic use (from to %). experience from the two influenza seasons shows that influenza antivirals are only used while the virus is circulating and that the volume of use is proportional to the size of the outbreaks. conclusions: experiences in the usa show that with prompt outbreak information antivirals can be used appropriately in times of influenza activity. influenza treatment with oseltamivir: costs and benefits for the individual as well as for society or . objective: to evaluate the effects of treatment of influenza with antivirals (oseltamivir) on health outcomes and costs to patients and society. methods: based on clinical trial data and data from the literature a simulation model has been developed. the underlying clinical pathway covers morbidity and mortality due to influenza and its specified complications. health outcome data and costs were attached to events in the model. the model compares various scenarios, which are defined by treatment schemes within defined populations and other parameters. application of the model is shown using uk unit cost data simulating an otherwise healthy adult population comparing oseltamivir with usual care. results: early treatment results in reduced morbidity, which translates into faster recovery and return to normal activities ( . days). lower morbidity and mortality make this a cost-effective intervention from a societal perspective. the analysis covers more than different scenarios and the incremental cost effectiveness ratios will be discussed. conclusion: antiviral treatment appears to be effective in terms of health outcome and cost for otherwise healthy adults from the perspectives of both the individual patient and society. however, this effect is very sensitive to time when treatment is started and the accuracy of the diagnosis of influenza. oseltamivir is well tolerated by all patient groups or . thakar b a , dutkowski r b , froelich e c , gilbride j a , ward p a . a roche global development, welwyn, uk , b f hoffman-la roche, nutley, usa , c f hoffman-la roche, basel, switzerland introduction: oral oseltamivir, the ethyl ester pro-drug of a potent inhibitor of influenza virus neuraminidase, is licensed for the treatment and prophylaxis of influenza in the usa. patients and methods: safety data [adverse events, laboratory safety evaluations] derived from clinical trials involving !/ subjects (including Â/ children and Â/ high-risk adults) and healthy volunteers in a large study investigating ecg parameters. spontaneous event reports from medwatch or yellow-card reports following use by Â/ individuals worldwide. an observational case-control study of !/ subjects with influenza-like illness treated with oseltamivir. results: oseltamivir was well tolerated in clinical trials; drug-related side-effects were limited to transient gi effects occurring in / : exposed individuals. these resolved spontaneously and caused drop out in b/ % of treated subjects. no effects on ecg parameters were noted at doses ]/sixfold above the licensed regimen. oseltamivir had no adverse effects on pulmonary function. no additional effects were identified among high-risk adults or children, or following prolonged dosing for prophylaxis. occasional reports of liver dysfunction have been documented post-marketing but causal association has not been established. conclusions: oral oseltamivir is an effective and safe antiviral suitable for influenza management in all patient groups. the decision to stop the vaccination against smallpox and the loss of specific immunity of a high proportion of the population made apocalyptic the perspective of a natural or provoked re-emergence of smallpox. therefore, it is important to improve the current capacities to prevent or to treat the orthopoxvirus infections. uracil dna glycosylase (udg) is one viral enzyme indispensable to the replication of poxviruses. udg of the copenhagen strain of vaccinia virus (vv) was characterized with the aim of defining specific inhibitors susceptible to be used as a new class of active antiviral substances on the viruses of the orthopoxvirus genus. the activity of this enzyme was analysed in real time, in an original method, on a pcr quantitative instrument by digestion of amplified dna revealed by fluorescent intercaled molecules. this technique was used to screen and select several active antiviral substances on udg. moreover, the antiviral activity was estimated by the cytopathic effect of the vv on infected vero cells. the cytotoxicity was determined by inhibition of trypan blue exclusion. the specificity of action of each tested compound was estimated by the selective index ( % cytotoxic dose/ % effective dose). two antiviral compounds were selected for their inhibitory effect on udg activity and on vv replication in vero cell culture : ('/)- -iodo- ?-deoxyuridine and -chlorouracil. these compounds are candidates for the chemotherapy of poxvirus infections. objective: to study the efficacy and tolerance of russian antiviral drugs produced from dna in a limited resources context. results: the drug derinat was produced from salmons' milt. mm of dna was Á/ kda, hyperchrome effect !/ %, protein content b/ . %. the conjugation of the dna with fe '/ resulted in a new drug named ferrovir which influences dna and rna synthesis during early stages of hiv- replication by blocking the virus's action on cells' metabolism and reduces cytomegalovirus titre in fibroblast cells for . Á/ . ig tcid . a protective effect of ferrovir against fatal herpes encephalitis mice was found. the drugs are not toxic. ic !/ mg/ml. ec of ferrovir against hiv- was mg/ml. in limited clinical trials patients received mg of drugs twice daily ( Á/ days). administration was well tolerated and no side effects were observed. derinat in . % cases of herpesvirus infection ( patients) improved the healing and shortened duration of illness. hiv-infected patients ( ) treated with ferrovir showed sustained, elevated cd '/ counts and a significant reduction in hiv- viral load (median . ig). the apparent remission was found in patients with concomitant hiv and herpes virus infection. conclusions: antivirals show good antiviral potency against rnaand dna-viruses; are well tolerated by patients and are useful in case of mixed infections; low price makes them accessible to populations with low financial resources. ortho total hcv core antigen assay can aid early prediction of response in patients treated with interferon/ribavirin or . lunel f a , veillon p b , payan c b . a ahu angers, laboratoire de bacterio-virologie, angers, france , b chu angers, laboratoire de bacterio-virologie, angers, france aim: evaluate the predictive value of total hcv core antigen assay and viral kinetics in patients with chronic hcv. methods: one hundred and twenty two patients infected by genotype , , or pretreatment viral load (bdna . , chiron) !/ meq/ml, with no previous treatment, received mu interferon (ifn) during months (m). ribavirin was given with ifn after months therapy, for months in patients with detectable rna. viral load was expressed as log (ui/ml) and hcv ag as log (pg/ml)/ ). results: pretreatment ag values were correlated with viral load (r / . ). we observed a rapid decrease of ag ( . log pg/ml) and viral load ( . log ui/ml) after m in sustained responders (sr). in patients who relapsed (rr) after ifn alone, the fall was less important ( . log pg/ml, . log ui/ml) during m . in sr and rr to combination therapy, the decrease of ag and viral load at m was, respectively, (ag: . and . log pg/ml; rna: . and . log ui/ml). we did not observed significant variation of ag and viral load in nonresponders. the negative predictive value of hcv rna and ag after m of treatment were %, and positive predictive values were and %. after month of ifn alone, the hcv ag decrease was highly predictive of sr, correlated with rna negativation and early reduction of hcv rna ( !/ log). conclusion: early measurements of total hcv core antigen are useful to predict long-term response to treatment. lamivudine in the treatment of acute hepatitis b or . vincenti a, meini m, luchi s, de gennaro m, ricciardi l, moneta s, scasso a. infectious diseases department, infectious diseases, lucca, italy acute hepatitis b is a self-limiting infection, but in some cases its course may be particularly severe. we report a case of a -years-old woman affected by acute hepatitis b treated with lamivudine. on admission in the hospital the alanino-aminotransferase was u/l, the aspartate-aminotransferase u/l, bilirubin , mg/dl, hbsag, hbcigm and hbeag were positive, hbv dna was . copies/ml. during the following days, the levels of ast and alt gradually rose; on the th day prothrombine time was %, bilirubin mg/dl and the patient developed signs of encephalopathy. four plasmapheresis were practiced without benefit, so the patient was treated with lamivudine, mg/day. after days of therapy, lamivudine was discontinued because of the appearance of diffuse maculopapular rash. at this time the results of liver function tests were normal; after four months hbsag and hbv dna were no longer detectable. in our patient lamivudine prevented an acute hepatic failure. our experience suggests a promising role of lamivudine in the treatment of acute hepatitis b, but how long such therapy have to be practiced and in which patients? prospective, controlled, clinical studies using lamivudine in patients with acute-hepatitis b are necessary. the cost-effectiveness of amantadine versus symptomatic care in the treatment of influenza or . morris s a , carman wf b , barber j c . a city university, london, uk , b west of scotland specialist virology centre, glasgow, uk , c alliance pharmaceuticals, chippenham, uk aim: to assess the cost-effectiveness of amantadine versus best symptomatic care in the treatment of influenza in the uk. methods: we constructed an economic model populated with parameters from the published literature. the model structure is the same as that used in the economic evaluation of zanamivir published by the national institute for clinical excellence in the uk. we conducted a cost-utility analysis (incremental cost per qaly gained) of amantadine versus best symptomatic care. the analyses are conducted for all adults (average-risk group) and the at-risk population (high-risk group), based on the prevalence of influenza over an average season and when the virus is circulating. the perspective is that of the nhs. results: in the average-risk group the incremental cost per qaly gained of amantadine relative to best symptomatic care is uk£ , during an average influenza season and uk£ , when the virus is circulating. for high-risk individuals the figures are uk£ , and uk£ , , respectively. the results are sensitive to the hospitalisation rate. conclusions : if the threshold for cost-effectiveness is £ , per qaly gained amantadine represents value for money in the treatment of influenza in a variety of scenarios, including the baseline for both average-risk and high-risk groups when the virus is circulating. background: surveillance studies all over the world have revealed an extraordinary increase in the prevalence of penicillin resistant streptococcus pneumoniae . the newer quinolones are believed to have broad activity against s. pneumoniae . methods: a total of penicillin resistant clinical strains isolated from patients at hacettepe children's hospital, ankara, turkey between and were tested for their in vitro susceptibility to various antibiotics that are commonly used in the treatment of respiratory tract infections. the minimum inhibitory concentrations (mics) of the penicillin, amoxicillin/clavulanic acid, doxycycline, azithromycin, clarithromycin, ceftriaxone, ciprofloxacin, levofloxacin, moxifloxacin and gemifloxacin were determined using the nccls recommended procedure for e -test. results: the range of mics, mic and mic values for all agents tested against the strains are shown in the table. gemifloxacin and moxifloxacin had the highest in-vitro activity among the quinolones tested. all strains tested were susceptible to b/ . mg/ml gemifloxacin, b/ mg/ml moxifloxacin and mg/ml levofloxacin. conclusions: there is some degree of resistance to all the drugs except the newer quinolones which were active against all isolates studied. purpose: stenotrophomonas maltophilia prevalence is growing, mainly in some hospital areas. s. maltophilia is frequently multidrug resistant. fluoroquinolone (fq) resistance varies from one to another study, but in whole resistance is moderate to high. gyra and parc qrdr partial codes have been recently described. we have studied correlations between fq-resistance and mutations in these sequences in s. maltophilia clinical strains. material and methods: gyra and parc qrdr regions from six fqresistant and two fq-susceptible s. maltophilia clinical strains were amplified and sequenced. mics of ciprofloxacin (cfx), gatifloxacin (gfx) and clinafloxacin (cnfx) were determined by the agar dilution method, according guidelines defined by nccls for p. aeruginosa . results and conclusions: mics ranges of cfx, gfx and cnfx for resistant strains were Á/ , Á/ and . Á/ mg/l. susceptible strains had mics of cfx, gfx and cnfx of , . and . Á/ . mg/l, respectively. most susceptible and resistant strains had no significant mutations in the fragments sequenced. only one resistant strain (mic of cfx mg/l) and one susceptible strain (mic of cfx mg/l) had a significant gyra mutation, the same in both strains (ile Á/val). thus, fq resistance in s. maltophilia shall derive from changes in other areas in the topoisomerases or probably from other mechanisms of resistance, such as efflux pumps. purpose: corynebacterium urealyticum is the cause of encrusted cystitis and other inespecific utis and systemic infections. it is frequently multi-drug resistant, with a high rate of resistance to fluoroquinolones (fq). the mechanisms of resistance to fqs have not been described in c. urealyticum . we describe the c. urealyticum parc gene qrdr region and its relationship with quinolone resistance. materials and methods: the activity of ciprofloxacin (cfx), levofloxacin (lfx), gatifloxacin (gfx), clinafloxacin (cnfx) and moxifloxacin (mfx) against c. urealyticum clinical strains was determined following nccls guidelines for enterococci. we amplified and sequenced their parc qrdr by standard methods. results and conclusions: five strains ( . %) were cfx-susceptible (mic . Á/ . mg/l), had mics Á/ mg/l and ( . %) were highlevel cfx-resistant (mic Á/ mg/l). cnfx was -fold more active than cfx. mfx and gfx had mics of and mg/l. all the strains, including the type strain, showed a c to t change at the position referred to wild type s. aureus parc gene, leading to a ser- -phe change, described as the main parc change in fq-resistant s. aureus . this finding suggest that this mutant sequence, as compared with parc sequences from other grampositives, might be the wild-type for this species, and might explain in part its high resistance rate, and its apparent lightness to develop high-level resistance. purpose: during routine surveillance, we identified ciprofloxacinresistant (mic!/ mg/l) pneumococcal isolates and compared clinical details and resistance patterns. results: they were isolated from sputa ( ) and blood cultures ( ) from adults, most with heart or lung disease. hospital admissions were common; half had been inpatients in the previous months. nineteen patients received quinolones in the preceding months, in part reflecting the local policy (introduced in ) of penicillin and ofloxacin for first line treatment of pneumonia. thirteen patients had radiological signs of pneumonia and were pyrexial with raised inflammatory markers. agar dilution mics for quinolones, including norfloxacin with and without reserpine, penicillin and erythromycin were performed. an increase in norfloxacin mics was noted over the period ( Á/ mg/l) to ( mg/l). fluoroquinolone efflux was suggested in three isolates. resistance to moxifloxacin (mic Á/ mg/l) was noted from onwards. all isolates were serotype v and resistant to penicillin (mic!/ . mg/l). thirty-one were resistant to erythromycin (mic!/ mg/l). conclusion: the policy of using quinolones may have contributed to the development of quinolone resistance and this cluster of isolates. the increasing levels of quinolone resistance observed raise concerns about the future use of newer quinolones for the treatment of respiratory infections. s. maltophilia has emerged in the last years as an important nosocomial pathogen, inherently resistant to most of the antimicrobial agents. new quinolones has been proposed as a treatment of choice because their enhanced activity, but several parameters (t a , atmosphere, method) can affect the results of mics. methods: we have performed mics using two different methods (agar dilution and microdilution) and different conditions: and c of temperature; atmosphere of o and co , and incubation times of , and h. a total of strains were assayed with nine quinolones following standard nccls. comparisons were made between results with Á/ and Á/ h using the x -test (a / . ). results: no differences were found between Á/ and Á/ h results with agar dilution, except with atbs in the case of mics at c co . on the contrary, almost all the atbs showed significant differences in the results with and h using microdilution method, at any condition of ta or atmosphere. comparison of mics (p values, significance level / %) with incubation times of and h at different procedure conditions. the incubation time is a parameter that seems to affect significantly the results of mics of quinolones when microdilution method is used, whereas only few differences can be encountered with the agar dilution method. results: breakpoints were used as proposed by nccls . during the study period the pneumococci resistance was noted as follows: % to pc, % to em and % to sxt. the rank order of activity of the five fqs against multi-drug resistant pneumococci was: cip (mic : mg/l), ofx (mic : mg/l), lvx (mic : mg/l), grx (mic : . mg/l), tvx (mic : . mg/l). conclusions: in romania, fluoroquinolones represent alternative treatment to beta-lactams and macrolides for first-line empirical treatment for respiratory tract infections caused by pneumococci but, continued vigilance for emerging resistance to fqs is further indicated. introduction and material/methods: susceptibility testing (semiautomated broth microdilution method, sensititre, trek diagnostics, usa, following nccls recommendations) was performed with six different quinolones to streptococcus pneumoniae isolates with a ciprofloxacin-cip */mic ]/ mg/l collected in two consecutive sauce$ surveillances in spain ( Á/ / Á/ ). nccls resistance (r) breakpoints were used ( ]/ for ofloxacin-ofl and levofloxacin-lev; ]/ for sparfloxacin-spa; ]/ for gatifloxacin-gat */and moxifloxacin-mox), but for gemifloxacin-gem */where ]/ was used. results were as follows. conclusions: for cip-r isolates gem and mox were the most active agents. gem was the only agent not influenced by cip mic increase regarding prevalence of r , with % resistance for strains with cip mic ]/ mg/l. $sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en españ a' (susceptibility to the antimicrobials commonly used in the community in spain) and is the spanish word for the willow tree. in vitro activity of gatifloxacin and seven other antibiotics against respiratory and urinary tract pathogens from the community. first results of the basic */study ps grimm h, on behalf of a european multicenter study group, institute for med. microbiology, weingarten, germany a total of centers in austria, belgium, france, germany, italy, portugal, spain and switzerland are involved in the basic study (bacterial annual susceptibility information collection). the mics of gatifloxacin (gati), ciprofloxacin (cipro), clarithromycin (clari), benzylpenicillin g (pen), amoxicillin (amox), amoxicillin/clavulanic acid (augm), cefuroxime (cur) and cefixime (cix) were determined using the microdilution method. each center is requested to investigate strains each of the following species: s. pneumoniae (spn), s. pyogenes (spy), s. aureus (sau), e. faecalis (efa), m. catarrhalis (mca), h. influenzae (hin), e. coli (eco), k. pneumoniae (kpn), p. mirabilis (pmi) and p. aeruginosa (pae). so far approximately strains are enrolled. some important mic %/percentage resistance were as follows: from the oral antibiotics tested gatifloxacin has the highest activity and broadest spectrum against all relevant respiratory and urinary tract pathogens. gatifloxacin is a promising alternative for therapy of respiratory tract bacterial infections. in vitro activity of gatifloxacin against bordetella pertussis in comparison with erythromycin, ciprofloxacin and levofloxacin ps bourgeois n, pangon b, ghnassia jc, doucet-populaire f, de versailles ch. microbiologie, le chesnay, france purpose of the study: bordetella pertussis infections are far more common in adults and adolescents than is generally estimated. however, they are often not recognised. infected or colonised adults can act as a reservoir of infection, passing it to children. fluoroquinolones are currently recommended for the treatment of respiratory tract infection in adult patients, which is usually empirical. gatifloxacin is a novel -methoxyquinolone, with a potent activity against both gram-negative and -positive bacteria. the in vitro activity of gatifloxacin was compared with those of erythromycin, the drug of choice for both treatment and prophylaxis of pertussis, ciprofloxacin and levofloxacin, against clinical isolates strains of b. pertussis including erythromycin resistant strains. results: we used the agar dilution method on mueller Á/hinton medium supplemented with % horse blood to determine the mic of each antibiotic. gatifloxacin (mic , . mg/l) was as active as ciprofloxacin and levofloxacin (mic , . mg/l) against both sensitive erythromycin (mic , . mg/l) and resistant erythromycin (mic , !/ mg/l) strains. conclusion: gatifloxacin may be an effective drug in the treatment or prophylaxis of adults with suspected or confirmed pertussis. ex vivo serum activity (killing rates) after gemifloxacin mg versus trovafloxacin mg single doses against ciprofloxacin-susceptible andresistant streptococcus pneumoniae ps calvo a a , giménez mj b , alou l a , gó mez-lus ml a , aguilar l b , prieto j a . a microbiology department, universidad complutense, madrid, spain , b glaxosmithkline, medical department, tres cantos, madrid, spain serum bactericidal activity was measured ex vivo after single dose administration of gemifloxacin (gem) mg and trovafloxacin (tro) mg to healthy volunteers in a randomized, cross-over phase i trial. blood samples were collected h (cmax) after dosing and serum killing rates were determined against a serotype penicillin (pen) Á/ciprofloxacin (cip) susceptible strain (s ) (mics of . , , . and . mg/l for pen, cip, gem and tro) and a serotype pen Á/cip resistant strain (s ) (mics of , , . and . mg/l for pen, cip, gem and tro). tubes with . ml of serum sample and . ml broth ( % todd Á/hewitt'/ % hbss) were incubated over h at c. final inocula was cfu/ml. mean colony counts for samples and controls (k ) are shown in the figure: gem exhibited higher colony counting decrease of the initial inocula, versus tro, for both strains. after h incubation, the initial inocula decrease obtained with tro and the cip susceptible strain was similar to that obtained with gem and the resistant strain, showing a lower influence of cip mic increase in the ex vivo bactericidal activity of gem versus tro. urine bactericidal activity after administration of gemifloxacin and trovafloxacin single doses in a phase i study ps garcía-calvo g a , parra a a , giménez mj b , ponte c a , aguilar l b , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain urine bactericidal activity after o.d. administration of gemifloxacin (gem) mg and trovafloxacin (tro) mg, was assessed in six adult males in a cross-over phase i trial. urine killing rates (ukr) against escherichia coli atcc (mic mg/l of . and . for gem and tro) and s. saprophyticus atcc (mic mg/l . and . for gem and tro) were performed with samples collected at , Á/ , Á/ and Á/ h. a . ml of iso-sensitest broth and . ml of bacterial logarithmic growth were added to ml sample, giving a final inoculum of cfu/ml. colony counting was performed after , , and h incubation. percentages of initial inocula reduction (iir) were calculated. mean urine concentrations measured by bioassay were (mg/l): . , . and . for gem, and . , . and . for tro. against e. coli , an iir of . % was obtained after h incubation with all samples except with tro at Á/ h. against s. saprophyticus an iir of % was obtained after h incubation with all samples except with tro at Á/ h, where bacterial regrowth was found. the maintenance over h of gem urine antibacterial activity suggests its efficacy in the treatment of uncomplicated cystitis. influence of the decreased susceptibility to ciprofloxacin on gemifloxacin versus levofloxacin efficacy in experimental pneumococcal pneumonia in guinea pigs ps garcia-olmos m a , parra a a , gimenez mj b , garcia-calvo g a , ponte c a , aguilar l b , soriano f a . a fundacion jimenez diaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain the efficacy of ciprofloxacin (cip), levofloxacin (lev) and gemifloxacin (gem) in the treatment of pneumococcal pneumonia was assessed in a guinea pig model using three strains (s) with mics (mg/l) of , and . (s ), , and . (s ) and , and (s ) for cip, lev and gem, respectively. intraperitoneal treatments started h after s. pneumoniae intratracheal inoculation, and continued t.i.d up to four doses. ten animals were included in each group. doses (mg/kg) used were , and for cip, lev and gem, respectively, in order to mimic auc - h and cmax obtained in humans after standard doses. animals that survived h after inoculation were sacrificed and colony counts were performed in lungs ( the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of nosocomial pneumonia. our aim was to study the pharmacokinetic Á/pharmacodynamic appropriateness of lfx mg iv bid in the treatment of six inpatients with ventilator-associated pneumonia (vap) ( / years; m Á/ f; / kg). blood and urine samples were collected in steadystate conditions at appropriate intervals. lfx concentrations were analysed by hplc. the aetiological agent was identified in all the cases and its in vitro sensitivity to lfx was always assessed. the results obtained mean values ( /sd) of the major pharmacokinetic parameters were: cmax, . / . mg/ml; vdss, . / . l/kg; t / b, . / . h; cl, . / . ml/min/kg; auc -t, . / . mg/ml h. cumulative urinary excretion was . / . %, confirming that lfx clearance is mainly renal. clinical cure and microbiological eradication were obtained in all the patients after a Á/ day therapy. a suprainfection due to acinetobacter anitratus insensitive to lfx occured in case. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/ ; auc/mic !/ ) in all the cases. the conclusion reached the findings suggest that lfx mg bid iv may be considered effective in the treatment of vap caused by sensitive bacteria. comparative pharmacokinetics of levofloxacin in patients with lower respiratory tract infections (lrti) being treated with sequential therapy ps pea f a , brollo l a , lugatti e b , di qual e a , dolcet f b , talmassons g b , furlanut m a . a institute of clinical pharmacology and toxicology, dpmsc, university of udine, udine, italy , b division of pneumology, sm misericordia hospital, udine, italy the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of lrti. our aim was to study the pharmacokinetic Á/pharmacodynamic appropriateness of a standard switch lfx iv/os regimen ( mg iv od for Á/ days followed by mg os od for Á/ days) in the treatment of seven inpatients with lrti ( / years; m Á/ f; / kg). blood samples were collected in steady-state conditions at appropriate intervals. lfx plasma concentrations were analysed by hplc. the aetiological agent was identified in / cases and its in vitro sensitivity to lfx was assessed. the results obtained absolute oral bioavailability was . / . , with a cmax of . / . vs . / . mg/ml after iv and oral administration, respectively. no significant difference in the main pharmacokinetic parameters was observed between the two routes. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/ ; auc/mic !/ ) in the two assessable cases. all the patients were clinically cured after a Á/ day therapy. the conclusion reached the ad interim findings show that lfx mg od may guarantee per os an exposure similar to that achievable after iv administration, suggesting that sequential therapy may be considered effective in the treatment of lrti. levofloxacine in the exacerbations of copd due to pseudomonas ae ps micheletto c, tognella s, pomari c, dal negro r, ospedale orlandi, div.pneumologia, bussolengo, italy development of antibiotic resistance in bacteria is a problem of great concern. gram-negative bacteria, including multidrug-resistant (mdr) pseudomonas aeruginosa (ps), are responsible for a significant proportion of episodes of copd exacerbations, particularly in elderly ( ). aim was to check the susceptibility to common antimicrobial treatments against ps strains isolated from bronchial secretions in patients with severe exacerbations of copd. methods: microbial investigations were conducted on specimens: spontaneous purulent sputum ( . %), and tracheobronchial aspirates ( . %, collected with a protected specimen brush). results: fifty-seven ps pathogen strains ( cfu) were identified ( . %) and tested over a -month period: ps. aeruginosa . %; ps. putida . %; ps. fluorescens . %, and burkholderia cepacia . %. the assessed susceptibility to most common antibiotics was: levofloxacine ( %), ciprofloxacine ( %), ipenem cil. ( %), ceftazidime ( %); amikacin ( %), and piperacillin'/tazobactam ( %). a much lower susceptibility was found for ticarcillin Á/clavulanic acid ( %), gentamicin ( %), and netilmicin ( %). ( ) at present, levofloxacine proves the most effective antimicrobic option for treating copd exacerbations due to ps infection; ( ) a much more efficient policy of antibiotic prescribing should be promoted in order to prevent the selection of resistant strains in these cases. these results confirm the excellent 'in vitro' activity of levofloxacin against nosocomial gram-negative pathogens, including the esbl producing strains ( % of escherichia coli , e. cloacae and k. pneumoniae were inhibited at . mg/l). levofloxacin was more rapid than ciprofloxacin to determine a bactericidal effect particularly against s. maltophilia . moreover, considering the favourable pk/pd profile, levofloxacin can represent a valid therapeutic option for the treatment of severe gram-negative nosocomial infections. moretti f, quiros-roldan e, casari s, chiodera a, viale p, carosi g. university of brescia, institute of infectious and tropical diseases, brescia, italy a -year-old man, ivdu, hiv positive was attended in aur hospital for fever and toracic pain. a x-chest radiography revealed a round lesion of cm near the lingula with central hyper-diaphan area. lymphocytes cd '/ count was cells/mm and viral load . cp/ ml. hospital stay rhodococcus equi was found in cultures of peripheral blood, faecal and sputum specimens. antibiotic treatment with oral rifampin ( mg/qd) and with intravenous imipenem ( mg tid) was started. due to the persisting fever, immodificated radiography and negativity for p. carinii , mycobacteria and bacteria in bal coltures, imipenem was substituted by parenteral vancomycin ( mg bid). after days, because of persisting fever and increase of the diameter of the lung lesion ( cm) vancomycin was sustituted by oral levofloxacyn ( mg bid), continuing rifampin. after a days course of levofloxacyn therapy the fever remitted. the patient was discharged with levofloxacyn ( mg bid) and rifampin and, after months of follow-up, a radiological control pointed-out a remarkable resolution in the lung lesion. we may suppose that levofloxacyn can be effective for the treatment of r. equi infection, even if more studies (particularly controlled studies) are necessary. liberti a a , izzo b a , loiacono l b , calabria g a , patarino t a , izzo e a . a ii department, naples, d. cotugno hospital, italy , b iii department, naples, d. cotugno hospital, italy the increasing prevalence of salmonella typhi strains with reduced susceptibility of chloramphenicol had prompted the search for other antibiotics with the same efficacy.quinolones are a class of antibiotic with an activity in vitro and in vivo against enteropathogenes. we inwestigated the use of levoxacin in two regimens of treatment of typhoid and paratyphoid infection. patients and methods: thirty-two adult patients were incluted in this study from september to april ; patients had positive culture for s. typhi and six had positive cultures for s. paratyphi . all isolated were fully susceptible to levoxacin. we compared treatment with levoxacin for days, mgr b.i.d. (group , patients), with treatment for days, mgr b.i.d. (group , patients). clinical cure was defined as defervescenze of fever by day of treatment, with an absence of complications and no clinical relapse during the followup. results and conclusion: the clinical cure rate was % ( patients) for group and % ( patients) for group ; the difference in these rates was not statistically significant. the blood cultures of all patients were sterile by day of treatment and remained so until the th month of follow-up, no subjects had clinical or microbiological relapse and all stool cultures remained negative, also. the two regimens of treatment was good tollerated and no adverse event was registered; it was concluded that levoxacin treatment for days in enteric fever is not necessary the mulidrug-resistence of s. typhi led to the use of quinolones as the first-line drug in the treatment of enteric fever. pefloxacin in the treatment of patient with acute infectious diarrhoea ps troselj-vukiae tvb a , strahinja v b , poljak i a , stojanoviae d c , nikoliae n d . a department of infectious disease, university hospital center, rijeka, croatia , b glaxosmithkline, marketing, rijeka, croatia , c dept. rijeka, institute of public health, rijeka, croatia , d dept. rijeka, maritime academy, rijeka, croatia the purpose of the study was to investigate clinical and bacteriological efficiency in and days pefloxacin treatment and to compare it with symptomatic therapy. the results obtained in patients treated with pefloxacin the therapy was clinically effective already in the third day while in the control group this happend in the th day. bacteriological eradication was noted in patients ( %) of the first and patients ( %) of second group in the th days of the treatment. they all had negative cultures and weeks after pefloxacin protocols were completed. only patients ( %) in control group had negative stool cultures in the th day of the treatment and all of them weeks after it ended. there was no statistically significant difference in clinical (p / . ) and bacteriological (p / . ) efficiency between and days pefloxacin treatment protocols. both protocols significantly differed in clinical (p b/ . ) and bacteriological (p / . ) eradication from the control group. the conclusion reached is that the efficiency of pefloxacin (quinolones) in the treatment of acute infectiuos diarrhoea and justifies their use in the more severe forms of the disease. dalhoff a a , ullmann u b , schubert s b . a bayer ag, wuppertal, germany , b university of kiel, institute of med. microbiology, kiel, germany background: the antibacterial activity of moxifloxacin (mxf) was compared to levofloxacin (lev), amoxicillin (amx), clarithromycin (cla) and erythromycin (ery) in an in vitro model. method: pharmacokinetics in bronchial mucosa (bm) and serum (s) following single oral doses of mg mxf or cla and mg lev, amx or ery were simulated using a one compartment model. bacteria tested staphylococcus aureus (nos. , ), streptococcus pneumoniae (nos. , ). aliquots were taken ( Á/ h) and plated on to brain heart infusion agar for enumeration. results: s. pneumoniae was eliminated by all agents studied. significant differences were apparent with s. pneumoniae and the s. aureus strains: objective: to compare the safety and efficacy of once-daily moxifloxacin with once-daily ceftriaxone in the treatment of cap in hiv-infected patients (pts). methods and results: in a retrospective survey, oral moxifloxacin ( mg daily)/ Á/ days) was compared to standard regimen of i.v. ceftriaxone ( g daily)/ Á/ days) for treatment of cap in hiv'/ pts. adults pts with clinical signs and symptoms of cap and consistent chest x-ray findings were included. pts had a median age of years (range Á/ and % were male). demographic characteristics were similar in both treatment groups; pts received mxifloxacin and pts ceftriaxone. clinical success rates were % for moxifloxacin and % for ceftriaxone. at a post-study evaluation approximately weeks later, moxifloxacin-treated pts and ceftriaxone-treated pts had relapsed. the adverse events reported were comparable for both treatment groups. there were four-related adverse events ( gi, headache) for moxifloxacin-treated and ( gi, skin) for ceftriaxonetreated pts. conclusion: the results of this study show that moxifloxacin as oral therapy is as effective and well tolerated as i.v. ceftriaxone in the treatment of hiv'/ pts with cap. therapy with moxifloxacin was not associated with any significant clinical or laboratory abnormalities. these data suggest that once-daily oral administration of moxifloxacin is potentially convenient and cost-effective alternative therapy for cap in pts with hiv infection. moxifloxacin in the treatment of acute maxillary sinusitis after first-line therapy failure or acute sinusitis with high risk of complications ps gehanno p a , berche p b , perrin a b , arvis p c . a ent department, bichat claude bernard hospital, paris, france , b microbiology department, necker enfants malades hospital, paris, france , c bayer pharma, medical affairs department, paris, france the efficacy and safety of moxifloxacin (mxf) mg once daily for days was evaluated in the treatment of acute maxillary sinusitis after first-line therapy failure, or acute sinusitis with high risk of complications. in this prospective, multicenter study, a total of patients with acute bacterial sinusitis confirmed by sinus x-ray were valid for efficacy analysis: one hundred and seventy five patients ( . %) had an acute maxillary sinusitis which failed to respond to a previous antibiotic therapy given for a mean duration of . days, and ( . %) had an acute sinusitis with high risk of complications (frontal: , pan-sinusitis: and sphenoid: ). ninety two patients ( . %) were microbiologically valid. clinical cure and continued clinical cure rates at Á/ and Á/ days post-therapy were . and . %, respectively. clinical cure rates at Á/ days post-therapy were . and . % in sinusitis after first-line therapy failure and in sinusitis with high risk of complications, respectively. bacteriological eradication rate during therapy (day Á/ ) was . %. at Á/ days post-therapy. bacteriological success rates were . % in patients who failed to respond to a previous antibiotic and . % of patients who had sinusitis with high risk of complications. . % of patients experienced drug-related adverse events, abdominal pain ( . %), nausea ( . %) being the most frequently reported events. mxf was rapidly effective and a well-tolerated treatment for this kind of infection. neisseria gonorrhoeae with decreased susceptibility to penicillin and ciprofloxacin: novel mutation patterns in the gyr a and par c genes of ciprofloxacin resistant isolates and plasmid profile of penicillin resistant isolates of n. gonorrhoeae in india (delhi) ps chaudhry u, saluja d. dr. b. r. ambedkar center for biomedical research, university of delhi, delhi, india commercial sex workers (csws) serve as the most important reservoir of gonorrhoea. periodic monitoring of the antimicrobial susceptibility profile of neisseria gonorrhoeae in a high-risk population provides essential clues regarding the rapidly changing pattern of antimicrobial susceptibilities. in india, such a surveillance of the in vitro antimicrobial susceptibility of n. gonorrhoeae was established in . significant increasing trend of penicillin and ciprofloxacin resistance with high mic of Á/ and Á/ mg/ml, respectively were found over the years ( Á/ ) . the molecular basis of ciprofloxacin resistance, i.e. mutations in the gyr a and the par c genes of isolates, were analyzed. four isolates (with an mic of mg/ml for ciprofloxacin) harbored triple mutations (ser- to phe, asp- to asn and val- to leu) in the gyr a gene. the third mutation of val- to leu, lies downstream of the quinolone resistance determining region of the gyr a and has not been described before in gonococcus. in addition, these isolates had a phe- to tyr substitution in the par c, a hitherto unknown mutation. the alterations in the par c gene were seen in these isolates only in the presence of changes in the gyr a gene and comprised amino acid changes at codons , , , , and . the presence of b-lactamase plasmid among the penicillinresistant isolates was determined by their plasmid profiles and further confirmation was carried out by a pcr based protocol. our findings suggest that emergence of penicillin and ciprofloxacin resistance in n. gonorrhoeae isolates from a major std center in india, indicates the need for the increased awareness and prudent use of antimicrobials. in vitro activity of newer antibiotics against methicillin-resistant staphylococcus aureus ps gutierrez zufiaurre mn, sanchez hernandez j, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: mrsa are frequently co-resistant to a number of structurally unrelated antibiotics. more than % mrsa are resistant to gentamycin, ciprofloxacin, macrolides and clindamycin. newer antibiotics active against multi-drug resistant grampositives have been developed. we have tested the in vitro activity of newer antibiotics against genetically-characterized, high level ciprofloxacin resistant mrsa. material and methods: thirty-six ciprofloxacin-resistant, gyra/grla mutant mrsa clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), gatifloxacin (gfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method, according nccls guidelines. results and conclusions: all the strains were resistant to cfx, , % were lfx-susceptible and . % were gfx-susceptible. nevertheless, mics of lfx and gfx for all susceptible strains was in the highest extreme of the susceptibility range. mfx was the most active quinolone. almost all the strains were high-level er-resistant with constitutive mlsb phenotype. tl did not improve significantly its behaviour, although it was -fold as active as er against the only er-susceptible strain. va, lin and syn had excellent activity against all the strains. they showed a very homogeneous behaviour against all the strains that were included in a range of Á/ mg/l of lin and va and . Á/ mg/l. sanchez hernandez j, gutierrez zufiaurre mn, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: corynebacterium urealyticum is the etiologic agent of encrusted cystitis and inespecific utis, and can be also involved in systemic infections. c. urealyticum is frequently multi-drug resistant, so only glycopeptide antibiotics and tetracyclines have high susceptibility rates, while fluoroquinolones resistance rates vary significantly. we have tested the in vitro activity of linezolid, telithromycin, synercid and newer fluoroquinolones against multi-drug resistant c. urealyticum clinical strains. material and methods: sixty-four c. urealyticum clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method according guidelines defined by the nccls for enterococci. results and conclusions: results confirm the high resistance rate to older fluoroquinolones and macrolides, with !/ % cfx resistance and % er-resistance. lfx was more active (mic mg/ml). mfx was the most active fluoroquinolones (mic mg/ml). tl improve its behaviour in respect to er (range . Á/ mg/ml). va, lin and syn had excellent antimicrobial activity. no resistant strains were found. mic were , . and mg/ml, respectively. mics were similar for all the strains independently of their resistance to other antibiotics plasma concentrations, urinary excretion and bactericidal activity of linezolid ( mg) versus ciprofloxacin ( mg) in healthy volunteers after a single oral dose ps wagenlehner fme a , wydra s a , onda h a , kinzig-schippers m b , sö rgel f b , naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, (ibmp), nürnberg-heroldsberg, germany purpose of the study: in a randomized cross-over study volunteers received a single oral dose of mg linezolid versus mg ciprofloxacin to assess plasma concentrations (up to h), urinary excretion (by hplc), and urinary bactericidal titers (ubt) up to h. the mean maximum plasma concentration of linezolid was . mg/ ml and of ciprofloxacin . mg/ml. the cumulative renal excretion (mean) of parent drug was %/ % for linezolid/ciprofloxacin. ubts were determined for a reference strain and five gram-positive clinical uropathogens with the following mics (mg/ml) for linezolid/ciprofloxacin: s. aureus atcc ( / . ), s. aureus (mssa) ( / ), s. aureus (mrsa) ( / ), s. saprophyticus (msse) ( / . ), e. faecalis ( / ), e. faecium ( / ). results: median ubts measured within the first h for linezolid were : for enterococcal strains and : to : for the four staphylococal strains. median ubts for ciprofloxacin were : for the two enterococcal strains, : to : for the two ciprofloxacin susceptible, : . for the two resistant staphylococcal strains. areas under the ubt Á/time-curve showed statistically significant differences only for the two ciprofloxacin resistant staphylococcal strains in favour of linezolid. conclusion: linezolid exhibited the same bactericidal activity against ciprofloxacin resistant as susceptible strains. linezolid should be tested for treatment of complicated uti due to gram-positive uropathogens in a clinical trial. whitehouse t a , cepeda ja a , tobin purpose: we performed pharmacokinetics within a double-blind, randomised trial comparing linezolid and teicoplanin in intensive care patients with known or suspected gram-positive infection. they received either mg linezolid intravenously -hourly, or mg teicoplanin -hourly for the first three doses and once daily thereafter (or every rd day if renally impaired). blood samples were collected to create serum pharmacokinetic profiles. linezolid was quantitated by hplc and teicoplanin by fluorescence polarization immunoassay. results: twenty two patients were studied in the linezolid group (m Á/f : , mean age years [range Á/ years]). median treatment duration was . days (range Á/ ). eighteen patients were treated with teicoplanin (m Á/f : , mean age years [range Á/ ]) for median days (range Á/ ). steady state peak concentrations (mean / sd) for linezolid and teicoplanin were . / . and . / . mg/l, respectively. trough concentrations at day were . / . mg/l for linezolid and . / . mg/l for teicoplanin. recommended breakpoints of staphylococcus aureus are mg/l for linezolid and mg/l for teicoplanin. accumulation occurred in one -year-old linezolidtreated patient with impaired renal function. conclusion: current recommended dosing regimens for linezolid and teicoplanin are generally appropriate in the critically ill, though a more detailed analysis is required. stamos g, lebessi e, ioannidou s, paleologou n, kallergi k, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of the study was to investigate the susceptibility of methicillin resistant staphylococcus aureus (mrsa) isolates from a -bed paediatric hospital to quinupristin/dalfopristin (q/d, streptogramin combination) and linezolid (lzd, oxazolidinone). material: we performed a retrospective analysis of mrsa strains, isolated from patients hospitalized in miscellaneous medical departments [neonatal unit ( ) , surgical wards ( ), orthopaedic wards ( ), oncology unit ( ), other wards ( ) and outpatient clinic ( )], during a -year period ( Á/ ) . the sources of isolation were pus ( ), throat ( ), nasal ( ), bronchial ( ), skin ( ), stool ( ) ear ( ) and other ( ) specimens. all isolates were sensitive to glycopeptides, while . % were resistant to gentamicin and . % to erythromycin. methods: the sensitivity testing was performed by a disk diffusion method (bbl sensitivity disks, becton dickinson), according to nccls guidelines. the breakpoint zone diameters for lzd and q/ d were ]/ and ]/ mm for susceptibility and / and / mm for resistance, respectively. results: all isolates were proved to be susceptible to both antibiotics. the mean inhibition zones were . mm for lzd and mm for q/d. conclusions: lzd and q/d are very promising antimicrobial agents, showing excellent activity against mrsa clinical isolates. the prudent therapeutic use is strongly recommended to avoid the emergence of resistance. in vitro activity of streptogramins and oxazolidinones against streptococcus pneumoniae clinical isolates ps stamos g, lebessi e, paleologou n, psatha m, sanida p, zaphiropoulou a, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of this study was to evaluate the in vitro activity of linezolid (lzd), a member of oxazolidinones and the streptogramin combination quinupristin/dalfopristin (q/d) against clinical isolates of streptococcus pneumoniae from a tertiary care paediatric hospital. material: a total of pneumococcal isolates exhibiting reduced susceptibility to common antibiotics were included in the study. the strains were isolated from middle ear fluid ( ), eye ( ), nasal ( ) blood ( ) and other ( ) cultures during the last years. the percentages of the isolates that were resistant to penicillin, erythromycin, cotrimoxazole and clindamycin were . , . , . and . %, respectively. methods: the susceptibility testing was performed by a standard disk diffusion method (bbl sensitivity disks, becton dickinson). in case of marginal results or intermediate sensitivity to quinupristin/ dalfopristin, the mic was determined using the e -test method (ab biodisk). results: all isolates were found to be sensitive to lzd. q/d was very active as well, except for two isolates that exhibited intermediate susceptibility, showing cross-resistance to macrolides and clindamycin, as well. conclusions: the new antimicrobial agents show excellent activity against resistant to common antimicrobials pneumococcal isolates, but the clinical use is not suggested, unless no other therapeutic solutions are available. linezolid is a new oxazolidinone with excellent activity against gram-positive organisms including glycopeptide-resistant strains of staphylococci and enterococci. in icu linezolid has been used for the treatment of severe gram-positive infections in a control trial. the susceptibility pattern of all gram-positive isolates from icu patients has been studied. methods: a total of specimens from icu patients were processed, were from patients enrolled in the antibiotic trial. all methicillin-resistant staphylococcus aureus (mrsa), coagulase-negative staphylococci (cons), enterococcus sp and methicillin-sensitive staphylococcus aureus (mssa) were tested. the break point for linezolid was mg/l and for teicoplanin mg/l. isolates were tested for susceptibility by e -test. results: linezolid ( isolates) mic / (mg/l) were as follows: mrsa (n / ) . / , cons (n / ) . / . , enterococcus sp (n / ) . / . , mssa (n / ) . / . teicoplanin ( isolates) mic / (mg/l) mrsa (n / ) . / . , cons (n / ) / . , enterococcus sp (n / ) . / . , mssa (n / ) . / . . all grampositive isolates were inhibited by concentrations of linezolid below the breakpoint including eight strains of staphylococci resistant to teicoplanin. conclusions: linezolid was highly active against grampositive isolates. resistance to teicoplanin was similar to other reported series. there was no emergence of resistance to linezolid. mrsa colonization is often a limiting factor for discharge from icu. clearance of mrsa is seldom achieved with conventional glycopeptide treatment. the oxazolidinone, linezolid, has excellent soft tissue and respiratory tract penetration and might be expected to eradicate carriage in some patients. we recently performed a doubleblind randomized trial in icu patients with known/suspected gram positive infection. received intravenous linezolid, mg b.d., and patients received teicoplanin mg o.d. mrsa clearance was assessed at end of treatment (eot), at -and days follow-up. results: in the linezolid and teicoplanin groups, and were known to be colonized with mrsa at study entry, respectively, while and were not. detection of clearance of mrsa colonization at eot was % for linezolid vs % for teicoplanin group (x b p b/ . ), at days it was % vs % (x b p b/ . ), and at days % vs % (ns). conclusion: short-term mrsa clearance can be achieved in significantly more patients treated with linezolid however this was not maintained at days, either because of incomplete initial eradication or recolonization. further analysis will follow when molecular typing of the isolates is completed. penetration of linezolid into bone, fat and muscle during hip arthroplasty ps lovering am, bannister gc, zhang j, macgowan ap, southmead hospital, bcare, bristol, uk there are limited data describing the concentrations and penetration of linezolid (lzd) into tissues, such as bone, that can be used to guide therapy for non-vascular infections. here we report the concentrations and penetration of lzd for bone, fat and muscle in comparison with cefamandole (cmd). twelve patients received mg lzd as a min infusion and mg cmd as a bolus injection immediately before hip arthroplasty. bone, fat, muscle and blood samples were collected at timed intervals after the infusion and assayed by a validated hplc method. for bone, peak levels of both agents occurred min after administration with mean levels of lzd . mg/kg versus cmd . mg/kg, decreasing to lzd . mg/kg versus cmd . mg/kg at min. correction for blood concentrations gave penetration of lzd % versus cmd % at min and lzd % versus cmd % at min. for fat and muscle, peak levels occurred min after infusion. mean levels were lzd . mg/kg versus cmd . mg/kg (fat) and lzd . mg/kg versus cmd . mg/kg (muscle). correction for blood concentrations gave penetration of lzd % versus cmd % (fat) and lzd % versus cmd % (muscle). we conclude that linezolid exhibits rapid penetration into bone and associated soft tissues achieving levels in excess of the mic for sensitive organisms; with a similar distribution and penetration profile to agents currently used for treatment of infections in these tissues. bacqué e a , barrière jc a , berthaud n b , desmazeau p b , dutruc-rosset g b , dutka-malen s b , ronan b b . a aventis pharma, chemistry, paris, france , b aventis pharma, disease group, paris, france xrp is a new oral streptogramin composed of semi-synthetic synergistic components in a / (w/w) association: rpr ( d-( -morpholino)methyl- d, g-dehydro pristinamycin i e ) from pi a and rpr [( r )- -deoxy- -fluoro pristinamycin ii b ] from pii b by original synthetic routes. the association has antibacterial activity against staphylococci including methicillin */mls b -resistant strains (mic range: . */ mg/ml), streptococci (mic : . mg/ml), pneumococci including multidrug resistant strains (mic : . mg/ ml), enterococci including vancomycin-resistant strains (mic : mg/ ml), m. catarrhalis and neisseria spp. (mic : . mg/ml), h. influenzae (mic : mg/ml), legionella spp. (mic : . mg/ml) and anaerobes (mic range: . Á/ mg/ml). xrp is generally bactericidal at the concentration of Á/ )/the mic against s. aureus , s. pneumoniae , h. influenzae and demonstrates consequent pae ( . Á/ !/ . h at Á/ )/mic, following an exposure of . Á/ h). mutants of s. aureus to xrp were isolated at low frequencies ( . )/ ( Á/ . )/ ( ) at )/ and )/mic while no mutant could be isolated at )/mic. these results suggest that xrp ( / w/w) is a promising compound for the treatment of community-acquired infections. ex vivo evaluation of rpr /rpr (xrp ), a new oral streptogramin ps berthaud n, diallo n. aventis pharma sa, infectious disease group, paris, france the intra cellular activity of xrp , was assessed in j murine macrophages containing ingested staphylococcus aureus (three strains) or l. pneumophila (one strain). at the concentration of )/ the mic, growth of s. aureus was strongly inhibited after a -h period of incubation (d log cfu/ml vs t controls range: (/ . Á/(/ . , according to the strain tested). at the concentration of )/ the mic, growth of intracellular l. pneumophila was inhibited after a Á/ -h period of incubation (d log cfu/ml vs t controls about (/ . and (/ . at and h, respectively). rpr and rpr alone had also inhibiting effect on bacterial growth (d log cfu/ml vs t controls after h of incubation about (/ . and (/ . , respectively). the bactericidal activity of xrp was also assessed against slowly growing s. aureus , under experimental conditions mimicking those observed in patients with an infected indwelling device (first step of infection: adherence to inert support; declared infection: biofilm model). under the experimental conditions, xrp demonstrated a rapid and potent bactericidal effect against s. aureus adherent to an inert support or included in biofilm. this effect was observed at each of the three concentrations tested ( , , and , and )/ mic, respectively). in vivo evaluation of xrp (rpr /rpr ), a new oral streptogramin ps berthaud n, huet y, aventis pharma sa, infectious disease group, paris, france the oral efficacy of xrp , was assessed in staphylococcus and pneumococcal murine infections. mice were challenged ip ( )/ , and )/ times ld ). abscesses were established by intramuscular injection of about bacteria into the right thigh of mice. pneumonia was established by intranasal injection of about bacteria. mice were treated twice a day for (staphylococcus aureus septicaemia and abscesses), (s. pneunomoniae septicaemia) or days (s. pneumoniae pneumonia). six to days post infection, for septicaemia and abscesses the results were expressed as ed , whereas for pneumonia they were expressed as the dose yielding an average survival time (ast) significantly longer than that of the untreated infected controls. xrp was efficacious in the treatment of experimental infections in mice caused by mls b -sensitive and -constitutively resistant s. aureus (ed range: Á/ and Á/ mg/kg/administration in septicaemia and abscess models, respectively). it was also efficacious in the treatment of infections caused by s. pneumoniae whatever the serotype and the resistance profile of the strains tested (ed range: Á/ mg/ kg/administration in septicaemia, ast: mg/kg/administration). these results suggest that xrp might be effective for the treatment of staphylococcal and pneumococcal community-acquired infections. xrp (rpr /rpr ), a new oral streptogramin: bactericidal activity and pharmacokinetics in a model of streptococcus pneumoniae mouse pneumonia ps berthaud n, huet y, diallo n. aventis pharma sa, infectious disease group, paris, france the bactericidal activity against streptococcus pneumoniae of xrp , a new oral streptogramin composed of two semi-synthetic synergistic components in a / (w/w) association (rpr , pristinamycin i derivative and rpr , pristinamycin ii derivative), was assessed in lungs of mice with pneumonia. mice were inoculated intranasally with about cfu of strain - (mls bresistant). eighteen hours later (t ), animals received xrp ( mg/kg p.o). the administration was repeated , , , and h afterwards. to study the influence of varying the ratio of pi to pii component administered on activity and pk parameters, ratios of rpr to rpr ranging from / to / were also administered under the same conditions. after an oral unitary administration at mg/kg, xrp , as well as ratios of rpr to rpr ranging from / to / , demonstrated strong and quick bactericidal activity in lungs. lung levels of rpr and rpr were generally equal or two times higher than blood levels. resulting rpr /rpr ratios in blood and lung, although not in accordance with the initial ratio administered, were synergistic for Á/ h in blood and for Á/ h in lungs explaining the activity observed. conclusion: based on in vitro data, telithromycin is a good candidate for the treatment of rti. in vitro evaluation of abt- , telithromycin and azithromycin against streptococcus pneumoniae , moraxella catarrhalis , haemophilis influenzae and methicillin-resistant staphylococcus aureus ps steele-moore l, berg d, barnes i, couch k, klein j, holloway w. christiana care, infectious disease, wilmington, us macrolide resistant streptococcus pneumoniae (sp) is a worldwide concern predominantly because these isolates tend to be multiply drug resistant. new agents with increased activity against these pathogens are clinically important. the ketolide class of antimicrobial agents demonstrate excellent in vitro activity against sp, even those that are macrolide resistant. the in vitro activities of the ketolides abt- (a) and telithromycin (t) were compared to azithromycin (az) against clinical isolates of sp, moraxella catarrhalis (m.cat), haemophilis influenzae (h.flu) and mrsa. organisms tested: strains of sp (including az resistant), h.flu, mrsa and m.cat. microdilution mic tests were performed following nccls recommendations using freshly prepared plates containing haemophilis test medium for h.flu, cation adjusted mueller-hinton broth (camhb) with laked horse blood for sp and camhb for m.cat and mrsa. the new ketolides, a and t had superior activity against sp including the az resistant strains (mic s: a / . mcg/ml, t / . , az / ). all compounds had excellent activity against m.cat while none demonstrated activity against mrsa. h.flu activity was comparable among a, t and az. these new ketolides are not currently approved by the fda; however t has been approved in europe. ló pez h, vidal gi, salomó n jm, scaglione m, zitto tr. centro de infectología, infectious diseases, buenos aires, argentina objective: we evaluated the impact of the initial treatment failure rate, hospitalisation and costs in outpatient treatment of adult cap in argentina comparing amoxicillin, clarithromycin and telithromycin. method: a probabilistic model was implemented in outpatient treatment of cap. we estimated an initial treatment with amoxicillin, clarithromycin or telithromycin. we assumed expected clinical cure at . , . and . %, respectively. for those patients with failure treatment we evaluated a second-line of antibiotics (amoxicillin followed by clarithromycin and clarithromycin followed by new fluorquinolone) or hospitalisation. patients with telithromycin and failure treatment must be hospitalised without a second line of outpatient treatment. costs of cap included drug's costs by days, medical visits, chest radiographies, analysis and hospitalisation. results: we estimated treatments in patients and first-line drug failure in , and patients with amoxicillin, clarithromycin and telithromycin, respectively. costs in outpatient treatment were: hospitalisation $ , and $ , . ; second-line drug $ and $ ; second-line hospitalised $ . and $ with amoxicillin and clarithromycin, respectively and hospitalisation with telithromycin $ . conclusions: telithromycin showed lower clinical failure, hospitalisation and costs in cap. some studies suggest shortening cap telithromycin treatment to days helping adherence to treatment and decreasing costs even more. the new macrolides */a good alternative of tetracycline in the treatment of mediterranean spotted fever ps popivanova nip a , petrov aip a , boykinova obb a , kazakova zkk a , baltadjiev agb b . a medical university, infectious disease, plovdiv, bulgaria , b department of anatomy, medical university, plovdiv, bulgaria mediterranean spotted fever (msf) caused by rickettsia conorii appears an endemic disease for some regions in bulgaria. frequently the disease has a severe course with multiple organ lesions. the early and adequate treatment is of extreme importance for the outcome of the disease. searching an alternative antibiotic treatment of this disease we considered macrolides for their good cell and tissue penetration and dose-dependent bacteriostatic and bactericide effect. we treated msf patients with doxycycline )/ mg/day, patients with clarithromycin )/ mg/day, as well as patients with midecamycin )/ mg/day and midecamycin acetate mg/kg/day. as a surrogate marker for treatment evaluation the effect on the febrile syndrome was used. our findings showed that by the th day of treatment the fever normalized in . , . and . % of the patients treated with doxycycline, clarithromycin and midecamycin, respectively. for the same period the patient fever decreased below c in , and . %, respectively. the intoxication symptoms were influenced within the same period equally in all treated patients. conclusions: we suggest that the new macrolides appear a good alternative of tetracycline on patients with msf. erythromycin resistance of gram /'// cocci in bulgaria. benefits of the new macrolides in the treatment of respiratory infections ps popivanova ni a , yovtchev ip b , dobreva md c , argirova ta c . a medical university, infectious disease, plovdiv, bulgaria , b medical university, ear-nose-throat disease, plovdiv, bulgaria , c medical university, microbiology, plovdiv, bulgaria in the recent years we tested erythromycin sensitivity of species of gram /'// cocci isolated from throat and nose secretions, ear and eye effusions, sputa, cerebrospinal fluid and blood cultures, vaginal and urethral secretions, urine and fecal samples from patients with inflammatory diseases of the listed organs and systems. staphylococcus aureus , staphylococcus coagulase /(//, streptococcus â-haemolyticus , enterococcus were isolated. of these microorganisms s. aureus was the most abundant. our resistograms revealed sensitivity of the gram /'// cocci in . and . % for and , respectively, and resistance of . and . %, respectively. in the tests with midecamycin and midecamycin acetat the same microorganisms showed sensitivity of . % and resistance of . %. the clinical findings showed excellent effect of the new macrolides including clarithromycin and azalides */azithromycin. we conclude the resistance of gram /'// cocci and especially of s. aureus to erythromycin increases very quickly and has reached dramatical extent. by now, the new , , and -membered ring macrolides and azalides show high antibacterial activity and good clinical effect. pappas ga, liberopoulos e, tsiara s, elisaf m, tsianos e. university hospital, internal medicine, ioannina, greece quinupristin/dalfopristin (q/d) is a novel injectable streptogramin antibiotic which initiation in therapeutics was hailed as an important step towards treatment of vancomycin-resistant enterococcus faecium (vref) species. initial reports concluded in an excellent response of vref to q/d. reports of q/d-resistant strains of e. faecium have emerged, both in usa and europe. we report two cases of e. faecium bacteremia in which the responsible isolate was not sensitive to q/d. the first patient was a woman with acute leukemia and septicemia. e. faecium was cultured from blood samples: the species was resistant to almost all antibiotics, exhibiting sensitivity only to tetracycline (!), while its sensitivity to q/d was indermediate. the second patient was a man with endocarditis, in whom blood cultures isolated e. faecium sensitive to a number of antibiotics, including ciprofloxacin and vancomycin; still the sensitivity to q/d was indermediate. q/d has not been officially introduced to the antibiotic arsenal of greek medicine. moreover, the drug has never been used in our hospital, not even experimentally. other e. faecium species isolated in our hospital have been sensitive to q/d. how much hope can be put on a drug that seems to be partly useless before its initiation? increasing reports of v/ q-resistant strains of e. faecium from all over europe raises fears that the v/d story might well end before it begins. varadinova t a , diakov t a , karagiozova d a , genova p a , pardon p b , baudry c b , quideau s b . a sofia university, virology, sofia, bulgaria , b et institut du pin, centre de recherche en chimie moleculaire, universite bordeaux , bordeaux, france vegetable tannins posses a wide range of biological activities. the aim of the present study was to evaluate the cytotoxicity of five purified vegetable tannins against mdbk cells. the maximal nontoxic concentrations (mnc) and the concentrations required to inhibit cell growth by % (cc ) were evaluated after , and h periods of action. mnc values after h indicated that compounds stimulated cell surveillance when applied in concentrations lower than . mm. cc values indicated: (i) a decrease in cytotoxicity after h as cc were up to times lower than those observed after the h period, and (ii) a re-increase in cytotoxicity when the period of action was prolonged up to h as cc were times lower than those observed after the h period. these data thus appear to reveal the capability of the investigated natural polyphenolic products to stimulate cell surveillance in a time-dependent manner. antibacterial effect survey of enoxolone on periodontopathogenic and capnophilic bacteria isolated from specimens of patients with periodontitis ps salari mh a , kadkhoda z b , sohrabi n a . a tehran university of medical sciences, pathobiology, tehran, islamic republic of iran , b tehran university of medical sciences, dentistry, tehran, islamic republic of iran objectives: most of the microorganisms associated with periodontitis are capnophilic and anaerobic bacteria. purpose of this study was to detection of antibacterial effect of enoxolone on periodontopathogenic and capnophilic bacteria. methods: in this study periodontopathogenic and capnophilic bacteria were isolated from specimens of patients with periodontitis by culture method. an anti-bacterial activity of enoxolone against these microorganisms was investigated by minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) methods. results: based on our findings the mic, mbc and lethal does (ld ) of enoxolone for actinobacillus actinomycetemcomitans , eikenella corrodens and capnocytophaga were ' , , ', ' , , ', and ' , , ' mg/ml, respectively. conclusion: our results show enoxolone has antibacterial effect on a. actinomycetemcomitans , e. corrodens and capnocytophaga spp. sakalova stn. medical university, microbiology, grodno, belarus we have synthesized diamides of dicarboxylic acids, with components such as -nitrothiazole benzolsulphamides and triazol. all the above compounds exhibited bacteriostatic activity towards some microorganisms. for further studies of bacteriostatic activity of amides and diamides of dicarboxylic acids, as well as for determination of 'structure Á/activity' relationship, we have synthesized a range of monoamides. antibacterial activity of the synthesized compounds was studied in vitro by agar dilution methods. for this purpose, approximately various gram-positive and -negative microorganisms including clinical strains of staphylococcus aureus , bacillus subtilis , serratia marcescens , escherichia coli , proteus morganii , micrococcus lisodeicticus , staphylococcus epidermidis , shigella sonnei , salmonella typhimurium , yersinia enterocolitica . minimum inhibitory concentration was expressed in mg/ml. nitazole was used as a comparison substance. analysis that new derivatives of -nitrotiasole have high antibacterial activity relative towards certain microorganisms included strains obtained from infection department's patients. results will be shown. microbial susceptibility to the essential oil of ziziphora clinopodiodes lam. ps purpose of the study: antimicrobial activities of essential oils vary from oil to oil and from one micro organism to another. the antimicrobial and chemical properties of essential oil from ziziphora clinopodiodes lam. has not been studied and hence the present study was planned to evaluate those properties against a series of micro organisms viz, escherichia coli , staphylococcus aureus , pseudomonas aeroginosa , klebsiella pneumonia , bacillus subtilis , bacillus licheniformis , streptococcus faecalis , candida albicans and saccharomyces cerevisiae . results: z. clinopodiodes lam. essential oil was found to have remarkable antimicrobial property against all the microorganisms but p. aeroginosa . the oil exhibited its best antimicrobial activity within a maximum of min. seventeen components were identified by gas chromatography and mass spectrometry (gc and gc/ms) analysis of the oil, out of which pulegone ( . %), neomenthol ( . %), cyclohexene, -methyl- -( -methenyl)trans ( . %), , -cycloheptadien- -one, , , -trimethyl ( . %), piperitone ( . %), and limonene'/ , -cineole ( . %) constitute major parts of the oil. conclusion: monoterpenes seem to have antimicrobial role. it seems necessary to explore antimicrobial properties of new harmless antimicrobial agents from natural sources as substitutes for common chemical drugs. methanol extract of carpobrotus edulis enhances killing of methicillin resistant staphylococcus aureus phagocytosed by thp- human monocyte derived macrophages and promotes the release of modulators of cellular immunity ps although alkaloids from the family mesembryanthemaceae have anti-cancer activity, species of this family have received little attention. because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, we have studied a crude methanol extract of carpabrotus edulis , a common plant found along the portuguese coast, for properties normally associated with plasma membrane active compounds. the results of this preliminary study show that the extract is non-toxic at concentrations that prime thp- human monocytederived macrophages to kill ingested methicillin resistant staphylococcus aureus and promote the release of lymphokines associated with cellular immune functions. the extract also induces the proliferation of thp- cells within day of exposure and days earlier than that induced by phytohemagglutinin. similar results were obtained with monocyte-derived macrophages isolated from human peripheral blood. the active component or components of the plant extract may be exploited as intracellular active anti-bacterials as well as modulators of cellular immunity. enhancing of erythromycin production by saccharopolyspora erythraea nur with common and uncommon oils ps hamedi j a , malekzadeh f a,b saghafi-nia ae b . a department of biology, faculty of science, university of tehran, tehran, islamic republic of iran , b shafe-e-sari co., antibiotic production co., teheran, iran enhancing effect of various oils on the erythromycin production by saccharopolyspora erythraea nur were evaluated in a complex medium consisting soybean flour and dextrin as the main substrates. the biomass, erythromycin, dextrin and oil concentrations, and ph value were measured on a daily basis. also, the kinds and frequencies of fatty acids of the oils used were determined. saturated fatty acids in the shark oil were higher than that of vegetable oils used. erythromycin concentration in the melon (cucumis melo var. inderus cultivar mashhad ) seed oil containing medium was . times higher than that of the control medium (without oil) and . times higher than that of rapeseed oil containing medium. erythromycin concentration in the other oil containing media, including rapeseed, soybean, shark (carcharhinus dussumieri ), and safflower oils was . , . , . , . time higher than that of control medium, respectively. the melon seed oil had the least enhancing effect on the biomass production, and thus decreasing the cost of the biomass separation. can varicella be eliminated by universal childhood vaccination ? */ epidemiological and economic data from germany ps wutzler p a , banz k b , neiss a c , goertz a d , bisanz h d . a institute for antiviral chemotherapy, university of jena, jena, germany , b outcomes international, basle, switzerland , c institute of medical statistics and epidemiology, technical university, munich, germany , d glaxosmithkline, pharma, munich, germany purpose: universal varicella vaccination in childhood is expected to reduce substantially the number of uncomplicated cases of chickenpox and to decrease the number of complicated cases requiring hospitalization. to generate fundamental data for decisions of the health authorities epidemiological and health-economic data were collected in two large studies. using an age-structured decision analytic model the benefits, costs and cost-effectiveness of a varicella immunization program over a period of years were assessed. results: it could be shown, that the vast majority of varicella cases occur in children aged less than years. in . % of the cases a severe course was assessed. overall incidence of complications was estimated to be . %. a routine varicella vaccination program targeting healthy children could prevent . % of varicella cases and over major complications per year provided the coverage rate would be %. under these conditions the elimination of varicella is predicted to be achievable within Á/ years. a combined measles, mumps, rubella and varicella vaccine is expected to provide the required coverage. conclusions: routine childhood varicella vaccination appears to be a highly efficient strategy to significantly reduce the sizeable burden of varicella and leads to significant savings from both societal and payer's perspective. bulgakova va, balabolkin ii, sentsova tb. scientific center of child health, russian academy of medical sciences, scientific research institute of pediatrics, moscow, russian federation objective: to estimate efficacy of vaccine influvac at children with allergic diseases. methods: twenty children aged Á/ years with allergic diseases received vaccine influvac (solvay pharma). for the control group of children, with allergic pathology did not receive this vaccine because of an intolerance of chicken protein ( children). results: all vaccinated children for the observable season of months did not get influenza. general and aboriginal reactions to a vaccine did not occur. in control group for the observable season two children were ill with influenza and four children with acute respiratory virus infection ( %). among vaccinated children there was an increase in titre to a protective level ( : and above) to all to three strains of influenza days after injection. vaccine influvac can be recommended for an immunisation against influenza of children with an allergic pathology because of efficacy and absence of side effects. ló pez h, zitto tr, vidal gi, cánepa mc, salomó n jm, scaglione m. centro de infectología, infectious diseases, buenos aires, argentina objectives: our study examines the possible economic impact of the influenza on health working adults in argentina, and the intervention cost saving with immunization. methods: this is a theoretical study based on a mathematical model. population data was published in s national statistics. the global incidence of influenza infection was estimated at %. we have estimated the direct cost on influenza infection (outpatient visits, drugs and hospitalization) and indirect cost (work absenteeism and productivity loss) and projected net saving for the Á/ year-old vaccinated group. the vaccine effectiveness was estimated at and %. the price of vaccine was $ each. results conclusion: influenza vaccination is effective in diminishing cases of flu and reducing working-day loss. its a safe and cost-effective vaccine. ló pez h, zitto tr, cánepa mc. centro de infectología, infectious diseases, buenos aires, argentina flu infection is a major cause of illness and one of the most common cause of work absenteeism, increasing institution costs, healthcare provider visits, use of drugs, and decreasing work productivity. vaccine against flu has an effectiveness between and %, in health adults. objective: evaluate the impact of flu-like respiratory tract infections in a health institution staff during year, comparing vaccinated with not-vaccinated groups. methods: we evaluated all causes of absenteeism along year ( ), based on the written note made by the professional who has evaluated ill people, selecting flu-like respiratory infection causes. we evaluated age, working days loss related to illness, and cost on vaccinated and not-vaccinated groups. results: one hundred and sixty eight of the total staff ( people) were vaccinated, of them had flu-like infection, resulting in working days lost. for not-vaccinated group, people had flu and lost days. lost cost for vaccinated group was of $ , and for notvaccinated group, $ . conclusion: we observed a decrease in working days loss and money waste related to flu-like infections on the vaccinated group. because of safety and effectiveness of vaccine against flu, the implementation of vaccination will be cost-effective for all institution staff. we studied functioning of the interferon system in children with atopic bronchial asthma (ba) at the age of Á/ years. the control group included healthy children. we investigated the interferon status (method of grigoryan s.) and serum concentrations of ifngamma (ifng) (elisa). there was a decrease in the ifn-producing ability of leukocytes to the synthesis of ifna at % and ifng at % of children with ba. serum level of ifn of children with ba during all period of illness is compared to the children without predisposition to atopy ( . / . and . / . pg/ml accordingly) was significantly decreased. production of ifna increased after using viferon (recombinant a b ifn and antioxidants). decreased ability of gammainterferonogenesis in the most children was not affected by the action of immunomodulators. there was shown interferon system's dysfunction in the development of atopy and increasing predisposition to respiratory infections and to persistent of atypical infections in children with ba. harxhi a, pilaca a, pano k. university hospital center of tirana, infectious diseases, tirana, albania congo-crimean haemorrhagic fever is viral disease with a high rate of mortality that is caused by a nairovirus, bunyaviride species. this is a zoonotic disease, which affects sporadically humans and is geographically distributed even in eastern europe and balkan. during the months of may and june , in northeast of albania were reported eight cases of haemorrhagic fever. serologic tests performed in the laboratory of reference in thesaloniki, greece confirmed the diagnosis of congo-crimean haemorrhagic fever. in the mean time, who reported the outbreak in southwest kosovo of cases suspected for haemorrhagic fever from which were confirmed laboratory as congo-crimean haemorrhagic fever. we are describing here the clinical history of one of eight cases with cchf in albania. from the epidemiological point of view this case was considered peculiar, as it was the only one hospitaly acquired, and due to the gravity of the haemorrhagic syndrome was admitted at the intensive care unit at infectious diseases service, university hospital center of tirana. results: in the study were included patients ]/ years of age with documented hbsag-carrier ]/ months (average age */ . years, male */ %, female */ %). hbv dna in serum was tested by qualitative and quantitative pcr (commercial test-system ampli-sens hbv). hbeag, hbeab, hbsag were detected by elisa (hoffmann la roche). hbv dna by qualitative pcr was detected in % patients, by qualitative pcr was detected in % patients in the concentration ]/ copies/ml, in . % ]/ copies/ml, in . % ]/ copies/ml, in . % ]/ copies/ml ( fig. ). hbv dna level distribution among the hbsag carriers. elevated ( . the upper limit of normal) alt level was determined in . % of the hbv dna negative and . % of the hbv dna positive patients. hbeag was detected in . % of the hbv dna positive patients and had not been determined in the hbv dna negative patient. eleven percent of patient had the combination of the biochemical, serological and virology criteria, which are typical for active chronic hepatitis b (hbsag-carrier !/ months, hbv dna ]/ )/ copies/ml, elevated alt). conclusion: in smolensk % of the hbsag-carriers have viral replication confirmed by qualitative pcr. eleven percent of them have active chronic hepatitis b. kandemir o a , polat a b , kaya a a . a medicine of faculty, mersin university, clinical microbiology and infectious disease, mersin, turkey , b medicine of faculty, mersin university, pathology, mersin, turkey the exact potential of nitric oxide in the pathogenesis of chronic viral hepatitis is not known. the elevated nitric oxide production is assumed to be responsible for the pathological changes in many inflammatory conditions, mainly via peroxynitrite, a potential oxidant that is produced by the reduction of superoxyde anion with nitric oxide. the intensity and the distribution of the immunohistochemical staining of intrahepatic inducible nitric oxide synthase were studied in the biopsy specimens obtained from patients with viral hepatitis and patients with elevated transaminase levels from other etiology. hepatic inducible nitric oxide synthase staining was significantly more intense in the viral hepatitis group (p / . ). inducible nitric oxide synthase staining levels correlated well with the severity of the viral hepatitis using the knodell's liver histological activity index (r / . , p / . ). among the viral hepatitis group, the pathological distribution of the inducible nitric oxide synthase staining favored the periportal regions whereas less staining was observed in the bile duct and parenchyma regions. as nitric oxide mediated nitration of hepatocellular proteins is found to be elevated in the inflamed hepatic tissues and it well correlated with the severity of the disease, we suggest that inducible nitric oxide synthase can possibly have a critical role in the pathogenesis of chronic viral hepatitis. there were patients under observation who were divided into two groups, the first of patients (eight chronic virus hepatitis; three chronic virus hepatitis'/steatosis; four steatohepatitis; three chronic cryptogenic hepatitis; there were men and four women aged from to . the second group consisted of patients, eight with chronic virus hepatitis; four with steatohepatitis; six with chronic cryptogenic hepatitis. there were men and three women aged from to . diagnosis was confirmed with the help of clinical data, biochemical tests, serological markers, psr-diagnostics and ultrasound examination and computer tomography of the abdomen. in the first group of patients the treatment with ursofalk was administered at the dosage of mg/kg of body mass from month to years with improvement in general condition of the patients: heaviness, pain under the right rib, nausea and skin itch have disappeared. in all the cases, improvement in the biochemical blood analysis took place during treatment. the average index of alt activity was before */ . u/l, after */ . u/l and ast */ . and . u/l; alp */ . and . u/l; ggt */ . and . u/l; chol */ . and . mmol/l; tg */ . and . mmol/l. in the second group of patients the treatment was carried out with various hepatoprotectors during the courses from to months. before the average index of alt activity was . u/l, after */ . u/l; ast */ . and . u/l; alp */ . and . u/l; ggt */ . and . u/l; chol */ . and . mmol/l; tg */ . and . mmol/l. treatment of patients suffering from hepatitis of viral and other aetiologies with ursofalk produces a positive effect on both clinical symptomatic and biochemical indices. remission was more stable during a long period of taking the preparation. the hepatoprotective effect of ursofalk during the years was sustained for the whole of the period of the treatment. after stopping, an acute attack of cytolytic syndrome was observed. with other hepatoprotectors we did not get any improvement in clinical scene of the disease or in biochemical indices. shavlov nm, kletsky sk. minsk, belarus hsv- and - have possibility to damage different organs and systems. sometimes they cause damage of the liver, which resemble viral hepatitis. the etiology of such hepatitis may be confirmed only by results of liver biopsy. we have diagnosed cases of herpetic hepatitis: eight children and four adults. clinical course was different. in five cases the acute beginning took place: high temperature, the jaundice at the Á/ day (the level of bilirubin was Á/ mkmol, especially direct), cholestasis, the pain in upper right part of abdomen. the ascites was found in three patients with acute hepatitis during st week from the beginning of disease. in seven cases the beginning was gradual. the temperature was subfebrile, prolonged; malaise and moderate pain in upper part of abdomen were constant complaint. the jaundice was moderate; bilirubin increased until mkmol. the level of alt was moderately increased ( Á/ times). the blood analysis showed moderate leukocytosis with neutrophilia, and increased sre. the serological markers of hepatitis a, b, c, d were negative in all cases. hsv- and - were found in the blood. the diagnosis was defined by the results of hystochemical investigations, when the viruses were found in liver bioptat, and confirmed with the results of specific treatment. specific damage of liver cells was found: protein dystrophy and specific inclusions in cell nucleus. in all cases the treatment with acyclovir were given. the results we have observed during st week: the temperature became normal, the jaundice decreased and bilirubin was normal during Á/ days. in one case the recidive took place weeks later after treatment. the second course of acyclovir with intron a gave good results. nesic z a , delic d b , prostran m a , vuckovic s a , stojanovic r a . a department of pharmacology, school of medicine, university of belgrade, belgrade, yugoslavia , b clinical center of serbia, institute for infection and tropical disease, belgrade, yugoslavia the large number of unsolved cases of acute and chronic hepatitis has most probably the viral etiology. in mid s, two independent groups of authors reported a new human hepatotropic virus, with flavivirus like genomes, hepatitis g virus (hgv). the aim of this pilot study was to determine the prevalence of hepatitis g viral infection among patients at high risk of exposure to blood and blood products, as well as to evaluate if the risk of hgv infection was higher among them than in the general population. immunoenzyme test on microtitration plate for detection antibodies against hgv e antigen in plasma or sera (r&d systems, minneapolis, usa) was used for evidencing anti-hgv igg antibodies in sera. anti-hgv antibodies were detected in the control group (blood donors) in . % ( / ) patients. prevalence of anti-hgv antibodies among i.v. drug users was evidenced in . % ( // ), in hemophiliacs . % ( / ), in patients acquiring multiple blood transfusion . % ( / ), in hemodialyzed patients . % ( / ) and in patients with transplanted organs . % ( / ). our results suggest that patients exposed to blood or blood products have a higher risk of hgv infection than general population. evaluation of ortho total hcv core antigen assay in assessment and follow-up of patients treated for chronic hcv ps lunel f, veillon p, payan c. chu angers, laboratoire de bactério-virologie, angers, france an assay to quantitate 'total' hvc core antigen (hcv ag) in serum or plasma, may reflect viral load, has been developed by ortho-clinical diagnostics. methods: we evaluated hcv ag with two quantitative assays for hcv rna: bdna . (bayer) and monitor . (roche). we studied samples from untreated patients and from patients with chronic hcv treated with ifn or ifn/ribavirin. results: correlation of ag and quantitative assays was high (r / . for bdna . and . for monitor . ). no difference between the levels of rna and ag among hcv genotypes (r / . Á/ . ) was found. ag values, before treatment, were significantly lower in sustained responders (sr) than in other groups ( . log versus log, p b/ . ). in patients treated with ifn or combination therapy, we found very good correlation between decrease and negativation of ag and viral load: log iu/ml decline after m of interferon was significantly correlated with the negativation of hcv ag and sr. thirty-eight/ of sr had a rna load decrease !/ log iu/ml and / had a negativation of hcv ag after m . conclusion: total hcv core ag appears to be a new tool for monitoring patients with hcv infection. hepatitis c virus rna and hcv core antigen kinetics predict the efficiency of interferon-alfa and ribavirin therapy in naive patients infected by hcv genotype or ps fifty-five patients infected by genotype or were treated with a primary dose of (if hepatitis c virus (hcv) rna b/ meq/ml) or million units of interferon alfa- b (ifn) thrice weekly for months. ribavirin was added at month (m), until m if hcv rna was found positive after m of ifn. the viral kinetic was assessed during the follow up by serial measurements of hcv rna (bdna . and monitor . ) and using a new assay from ortho-clinical diagnostics which is able to quantify total hcv core antigen. sustained virologic response was observed in % of the patients ( / ). after month of ifn treatment, sustained responders had a fall of hcv rna and hcv core antigen higher than non-responders ( . / . log ui/ml versus . / . log ui/ml, p b/ . , for hcv rna) and ( . / . log (pg/ml)/ ) versus . / . log (pg/ml)/ ) p b/ . , for hcv core antigen). after month of ifn, the positive and negative predictive values of sustained response were, respectively, and . % for hcv rna negativation and . and . % for hcv antigenemia negativation. these results suggest that both kinetics of viral load and antigenemia are highly predictive of sustained response. theodorou m, petinelli i, pontikaki d, mela c, blana a, papanastasiou a, toliopoulos a, stavrakaki m, sagkana e. microbiology department, western attika general hospital, greece, egaleo, greece greece has accepted a big number of economic immigrants lately. we investigate the prevalence of hepatitis b/c as well as the epidemiological features that might influence the public health. . economic immigrants from: albania , eastern europe and from asiatic-african countries , visited our hospital to be checked in order to get a health certificate to obtain the green card. they where tested for hepatitis b/c. the serological markers were determined by immunoenzymatic method. all hbsag('/) and anti-hcv were further tested for hbv dna and hcv rna by competitive rt pcr. hcv rna('/) were genotyped by strip hybridization immunoassay. in albanians . % were hbsag('/), . % hbv dna('/), . % anti-hcv('/) and . % hcv rna('/). in east europeans . % were hbsag('/), . % hbv dna('/), . % anti-hcv('/) and . % hcv rna. in asians-africans, . % were hbsag('/) and % hbv dna('/). in pakistanis, . % were anti-hcv('/) and . % hcv rna('/). of the rest of asians-africans, . % were anti-hcv('/) and . % hcv rna('/). albanians: higher prevalence of hbv infection ( . %). greek blood donors: % pakistanis: hcv infection is % (predominance of a type), general greek population: %. public health services in greece and europe must take appropriate measures. el zawawy la a , mohamed on b , ali sm a , eissa me a , allam sr a . a faculty of medicine, parasitology, alexandria, egypt , b high institute of public health, microbiology, alexandria, egypt the purpose of the study was to investigate the influence of schistosomal suppression on the antibody response to hepatitis-b vaccine (hbv) and to study if the vaccine has any protective effect on experimental () infection. the results obtained revealed that infection reduced the serum antibody level against hbv. parasitological and histopathological findings showed significant protection against infection. the conclusion reached was; in order to reduce the incidence of virus-b infection especially in schistosomiasis endemic areas, public health officials should evaluate a policy for regulation of hbv booster vaccination to enhance the population immunity against hepatitis-b infection. cooper e, fisher t, shingadia d. newham general hospital, family clinic, london, uk sustained anti-retroviral combination chemotherapy requires excellent adherence to the regimen so as to suppress viral replication sufficiently to delay the emergence of resistance. if chemotherapy were taken to scale, e.g. in africa, erratic adherence might soon lead to multi-resistant circulating virus. we reviewed our experience in a well established london family clinic with a team including community nurses. we reviewed the records of the african immigrant children, aged Á/ , treated with anti-retrovirals exclusively at our centre throughout . whereas had undetectable hiv rna within the year, only four had undetectable rna throughout the year. four failed therapy through proven resistance mutations, but nine were considered through circumstantial evidence to have rising viral loads primarily because of poor adherence. three were known to have stopped taking drugs for extended periods. the three boys over years were unreliable in adherence, but the one girl in this age-group was fully adherent. our preliminary assessment is that for the children in our families, despite a team approach and home visits, nonadherence to haart may be twice as common as selection of a dominant viral mutant as a primary cause of failure to sustain viral suppression. quiros-roldan e, moretti f, castelli f, el-hamad i, carosi g. the prevalence of hiv related lipodystrophy-syndrome depending on the definition and severity of lipodystrophy ranges from to %. we have retrospectively reviewed the medical records of african patients followed. the characteristics are shown in the . % of africans had triglycerides !/ mg/ml and . % had cholesterol !/ mg/ml, none had both metabolic alterations. glycemia !/ mg/ml was observed in . % patients. it is interesting highlight that in any the africans morphological changes were noted and all of them showed weight stable. although the low prevalence of metabolic alterations may be attributed to the different ethnic alimentary behavior if self-body perception by african is not as accurate as by caucasian on the estimation of the body changes have to be investigated. alabaz d a , alhan e a , yaman a b , evliyaoglu n a , kocabas e a , aksaray n a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey hepatitis a virus (hav) infection is usually asymptomatic in children. however, it may occasionally cause a severe disease with high morbidity and mortality, and loss of school or business days. in a previous study, we have shown that every one of two to three school children from upper social classes living in adana carries high risk of hav infection. it is well known that maternally transmitted anti-hav antibodies interfere with hav vaccination. in an effort to determine the optimal age for hav vaccination, babies ( % girls and . % boys) born in our hospital were prospectively followed up at least months for the presence of maternal antibodies to hepatitis a (anti-hav igg). anti-hav igg titers were measured from the blood specimens obtained at birth from the mothers and from the offsprings at months, , , , , , , , and . the prevalence of positive anti-hav at birth ( %) was similar to those of hav seroprevalance studies carried out in adults in our area. the disappearance of antibodies occurred between the st and st month of life. the prevalence of anti-hav igg among children aged , , , , and months were , , , , and %, respectively. in light of these findings, we suggest that hepatitis a vaccination be given after months of age. earlier vaccination may be ineffective due to interference with maternally transmitted anti-hav antibodies. ghaderi b, alaghebandan r, rastegar lari a. department of microbiology, iran university, tehran, islamic republic of iran diarrhoea is a major public health problem in developing countries. amoebiasis is one of the most common causes of diarrhoea in iran as an endemic area for amoebiasis. little, however, is known about the extent of the condition in our society. the aim of this study is to determine socio-demographic and clinical characteristics of patients with intestinal amoebiasis. during july and august , we collected patients with diarrhoea among patients who visited at a referral hospital in shahriar area (in countryside of tehran), iran. thirty out of patients ( %) had intestinal amoebiasis and were followed up prospectively until the resolution of the illness. nineteen of ( . %) patients were male and the remaining of . % was female. the patients were aged Á/ with mean of . years. most of the patients ( %) were below years of age and the peak of occurrence was between the age of and years. watery diarrhoea with abdominal cramps was the main clinical feature. seventy percent of patients were resident in urban area and the remaining ( %) in rural area. average family income was low and all patients were in low socioeconomic level. water supplying system for all patients was pipeline water. low socioeconomic level associated with poor personal hygiene was the most important factor for highly prevalence of this problem in our society. also it seems that food plays important role in transmission of protozoa then water. the new strategy for allele identification of the genes coding for pertactin and pertussis toxin subunit s in bordetella pertussis ps bordetella pertussis strains demonstrate a significant polymorphism in toxin s subunit and pertactin, which are major protective antigens of the organism. monitoring the changes in prevalence of particular alleles of genes coding for these proteins in local b. pertussis populations is an essential issue in cases of the observed decrease of vaccination effectiveness. we have developed a new method for allele identification of these genes, which eliminates the necessity of dna sequencing. the approach is based on the identification of the number of repeats or the presence of specific nucleotides in the polymorphic regions or residues, respectively, of the genes and utilises products of their full or partial pcr amplification. the nucleotide heterogeneity in each polymorphic site is analysed either by the differential digestion of the amplicons or by the arms (amplification-refractory mutation system) methodology. numbers of repeats in particular regions of the genes are revealed by the size analysis of the adequate pcr products or their restriction fragments. in all cases the presence, size or pattern of dna molecules obtained is visualised by the agarose gel electrophoresis. the preliminary analysis of the recent and archival b. pertussis strains identified in poland was performed using the described approach. the presented strategy provides a much easier, faster and more cost-effective than dna sequencing mean to study the polymorphism of the major b. pertussis antigens. vaccination coverage and history of vaccine preventable infectious diseases among students in second year of medicine and pharmacy of tours university ps borderon jc a , hamed a b , ragot s b . a centre hospitalier universitaire, tours, france , b médecine préventive universitaire, tours, france the purpose of this study was to determine the level of infectious risk in students who will be exposed to patients. information was obtained by a questionnaire for each student, and by checking medical records for immunization coverage and vaccine preventable infectious diseases. answers could be specified for students, of whom females (f) and males (m). the number of non-immunized students was against diphtheria: two, tetanus: three, pertussis: four, poliomyelitis: two, hepatitis b: six, and hepatitis a: , respectively. among the students non-vaccinated against measles, (nine f and five m) had no history of that disease. among ( f and m) non-vaccinated against rubella, ( f and seven m) had no history of that disease, uncertain in seven others (six f and one m). the date of vaccination was often late regarding recommendations. fifteen students had no history of varicella. one student had not received bcg vaccination. fifty-eight students had received two, and three bcg vaccines. post-bcg tuberculin skin testing was missing after first bcg, second and third bcg. the date of the first tuberculin test was often one or several years after bcg vaccination. adverse effects of vaccination were rarely reported: two cases of fever (dt polio, measles); three cases of local reaction (dt polio, dtp polio). one case of contraindication for influenza vaccine: egg allergy. the survey shows failure in immunization coverage actually recommended in health care students. objectives: to present the morbidity of rabies and evaluate the efficiency of our prophylaxis scheme in lasi county. material and method: we made a retrospective study of the rabies cases in the patients admitted in our unit in a th-year period. we have analysed all the clinical, epidemiological and biological aspects. results: in a -year period, cases of rabies were admitted in the clinical infectious diseases hospital lasi. the highest incidence was for Á/ */eight cases ( %); the highest yearly cases were three cases in and . most of the patients were male ( %), came from suburban areas ( patients). eight cases occurred in may Á/june, wild animals were involved in half the cases (fox, wolf). for patients, no prophylaxis was performed and an incomplete course in four cases. the period of time to the appearance of the first symptom was Á/ days. the prophylaxis scheme led to a good protection. conclusions: in lasi county, rabies is a problem with a prevalence of . %/year. trends in the use of antimicrobials in riyadh in were analyzed. data was obtained from a survey of randomly selected families of school children aged Á/ years in a -month period in . one hundred and ninety-nine ( . %) students were on antibiotics in the month preceding the study; ( %) received antibiotics for the diagnosis of pharyngitis; ( %) students antibiotics were prescribed by a physician; and in ( %) the duration of antibiotics was less than week. this study shows a major problem in antibiotics prescription in our community and also the need to establish effective antibiotics policy in general practice to limit the potential emergence of drug resistance bacteria in the community. mir s a , cura a a , erdogan h a , guler s a , sengul gn a , koyu a a , ozinel ma b . a department of pediatrics, ege university medical faculty, izmir, turkey , b department of microbiology, ege university medical faculty, izmir, turkey antibiotic susceptibility spectrum of childhood urinary tract infection agents are geographical variation. the current antibiotic regimens and the selection of antibiotics for prophylaxis should be re-evaluated periodically. the objective of our study was to determine the local resistance rates to antibiotics and to give a direction for the selection of antibiotics in uti treatment. we evaluated urinary culture assays retrospectively, sent from pediatrics and pediatric surgery inpatient and outpatient clinics of our hospital during the last months, and investigated the isolated pathogens and the resistance rates to antibiotics. in addition, the data obtained were compared with of years ago. with respect to the resistance rates to antibiotics of uti pathogens, the resistance rates of e. coli for carbapenems, aminoglycosides and third generation cephalosporins were , and %, respectively as before, but the rates for ampicillin increased from to % and for tmp-smx it increased from to %. we concluded that the resistance profiles to antibiotics should be reviewed every years at least and thus the selection of proper antibiotics would lessen the morbidity as well as the medical expenses. chanturidze tk, tsiklauri r. ministry of labor, health and social affairs, public health, tbilisi, georgia purpose: since infectious diseases (id) are increasing in georgia. this study is aimed to reveal economic barriers of effective id control by assessing financial contribution to id from public and private sources, household's total spending on health and their capacity to pay. sources: ) national household expenditure and revenue survey. ) who fair financial contribution methodology. ) meta-analysis. results: . % of population leaves under the poverty level; % out of total household expenditure (average gel; us$ ) % is spent on food */non-subsistence income covers expenditures on goods and services including health; % of population refuses health services because of inability to pay; public spending on health comprises % of total health expenditures; public spending on id control is below gel per capita; almost all private spending goes to id treatment and equals to . gel per patient. conclusions: insufficient public spending on id control transfers the burden to the population with extremely low capacity to cover health expenses. refusal to utilize health services, and incomplete treatment and increases the threat of id spread and drug resistance. government should increase the allocations to id from public sources for effective id control in georgia. antimicrobial consumption trends in children's university hospital ps ratchina s a , averchenkova l b , jarkova l a . local surveillance of antimicrobial (am) consumption is essential to promote the rational use of this group of drugs. the purpose of this study was to analyze the trends of am use in the children university hospital in and . data on am usage were obtained from the hospital drugstore requests in the -beds multi-ward children university hospital. consumption was expressed as the number of ddd per bed-days (b Á/d). the total am consumption figures were similar in and ( . and . ddds/ b Á/d, respectively) with notable differences in am prescribing patterns. penicillin consumption increased from . to . ddds/ b Á/d mostly due to amoxicillin. the overall aminoglycoside usage remained comparable ( . vs. . ddds/ b Á/d) though amikacin has considerably replaced gentamicin. there was a sevenfold increase of ciprofloxacin ( . vs. . ddds/ b Á/d) along with the evident decrease of tetracycline and co-trimoxazole consumption found ( . vs. . ddds/ b Á/d and . vs. . ddds/ b Á/d, respectively). the tendency to prescribe more effective in respect of the local resistance data and/or more safe am was detected in comparing with that can be explained by the introduction of the local guidelines for infectious diseases management in . clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis */a pilot study ps the aim of this pilot study was to determine the efficacy and tolerability of clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis (ct). fifty-two patients older than years of age with diagnosed chronic chlamydial prostatitis were enrolled. the presence of ct in expressed prostatic secretion or urine specimen voided immediately after prostatic massage was confirmed by isolation on mccoy cells and lugol staining. the majority of patients suffered from suprapubic pain and pain in the groin. twelve patients had no clinical symptoms. according to rectal palpation, prostate gland was normal in patients, tender and soft in and firm in five patients. clarithromycin was administered orally mg twice daily for days. simultaneously the patients' partners received mg orally twice daily for days. clinical efficacy and tolerability of administered clarithromycin were evaluated Á/ days and Á/ weeks after the end of treatment. bactericidal efficacy of administered drug was evaluated Á/ weeks after the end of treatment. the eradication of ct was achieved in out of patients, while patients were clinically cured. two patients had nausea and elevated serum transaminases. in asymptomatic patients, the eradication of ct was achieved in of patients who reported no side effects. this pilot study has shown an excellent efficacy and tolerability of clarithromycin in the treatment of patients with chronic chlamydial prostatitis. women with diagnosis of urinary tract infections (uti) often demonstrate vaginal colonisation with alpha-haemolytic escherichia coli strains. in the present study we decided to evaluate a distribution of virulence genes encoding for cytotoxic necrotizing factor type (cnf ), p-fimbriae, s/f c-fimbriae aerobactin (aer), and afa genes in alpha-haemolytic e. coli strains isolated from gynaecological material in our region and to compare the detected sequences in clinical isolates of other diagnostic groups. of alpha-haemolytic e. coli strains, were isolated from urine, from gynaecological specimen, and were faecal strains. e. coli strains were tested for the production of haemolytic phenotype on blood agar plates. the amplification of virulence factors was performed by pcr according to previously described protocols (le bouguenec et al., ; blanco et al., ; and yamamoto et al., ) . we found that all gynaecological alphahaemolytic strains were positive for cnf , (p b/ . compared to % of urine strains, and p b/ . compared to % of faecal strains). similarly, sfa/foc specific dna sequences were found in % of gynaecological isolates (p / . compared to % of urine strains and p / . compared to % of faecal strains). from this point of view, the female genital tract seems to be a potential reservoir of these uropathogenic e. coli strains. azithromycin in the treatment of pelvic inflammatory disease caused by chlamydia trachomatis ps administered in hospitalized patients with chlamydial pid. the diagnosis was made prior to hospitalization. microbiological analysis of urine, blood and swab specimens collected from endocervix, vagina and urethra confirmed c. trachomatis to be the single suspected causative pathogen of pid. the presence of c. trachomatis in swab specimens from endocervix was examined by dnk/rnk hybridization. azithromycin was administered Á/ days after samples for microbiological analysis were collected in dose of )/ mg iv for days. clinical efficacy and tolerability of therapy were assessed Á/ days after the end of therapy and clinical and microbiological analysis Á/ weeks after completion of therapy. the eradication of c. trachomatis and normalization of gynecological findings were achieved in and disappearance of subjective symptoms in patients. no side effects and deviations from normal values in hematologic and biochemical blood parameters were recorded. this study showed high bactericidal efficacy, rapid clinical effect and good tolerability of once-daily administration of mg azithromycin for days in the treatment of patients with pid caused by c. trachomatis . altindis m a , cevrioglu s b , aktepe oc a , cetinkaya a . a kocatepe university school of medicine, microbiology, afyon, turkey , b kocatepe university school of medicine, obstetrics and gynecology, afyon, turkey diagnosis of the causative organism of the vaginitis is usually based on clinical criteria. a standardized, laboratory based and rapid diagnostic test for the identification of these organisms is desirable. to determine the laboratory method that best predicted the causative organism, we calculated the sensitivity, specificity and predictive value of positive and negative test for clinical criteria, an oligonucleotide probe test (affirm vpiii-bd usa) and compared them with the combination of positive vaginal culture and gram-stained vaginal smear. we evaluated consecutive women aged Á/ years, attending for vaginal discharge. vaginal swab specimens were used for culture of gardnerella vaginalis , trichomonas vaginalis and candida sp, preparation of a vaginal smear for gram-stain interpretation and wet mount evaluation and affirm test. affirm detected g. vaginalis in ( %), candida sp in three ( . %) women and no trichomoniasis case found by any methods. the sensitivity and negative predictive values of affirm test and clinical signs were same ( %) in identifying of bacterial vaginosis. however, affirm test was more specific ( vs %) and also has higher positive predictive value ( vs %) than clinical signs. we did not evaluate the results for patients with candidiasis because of less number. according to these results affirm-microbial identification tests are objective and specific for the rapid diagnosis of the bacterial vaginosis. comparison of efficacy of single dose of tinidazole with standard dose of metronidazole in giardia lamblia infection (preliminary report) ps fallah m a , moshtaghi aa b . a university of medical sciences, parasitology, hamadan, islamic republic of iran , b university of medical sciences, pediatrics, hamadan, islamic republic of iran objectives: giardia lamblia is the most common intestinal protozoa in developing countries. treatment of the infection with metronidazole, the drug of choice, requires a long course of therapy and produced some side effects. the object of this study is to determine efficacy and side effects of tinidazole in g. lamblia infection. this is a preliminary report of an ongoing trial. methods: a randomized controlled clinical trial was carried out and subjects with g. lamblia infection were treated with tinidazole or metronidazole. tinidazole mg/kg single dose and metronidazole mg/kg three times a day for days were given orally to and children, respectively. parasitological cure was documented when there were consecutive negative stool examinations at Á/ weeks after therapy. results: twenty-one of individuals treated with tinidazole and of children treated with metronidazole had parasitological cure. cure rates between two groups were not significant statistically. no major side effects were observed except one case in metronidazole group who had mild headache and abdominal pain for days. conclusions: we concluded, tinidazole at the dose has efficacy equal of metronidazole in the treatment of g. lamblia infection. because of single dose prescription, short course of therapy and good compliance of patients, this preparation is preferred to metronidazole in giardial infection. ebeid fa, seif el-din sh. theodor bilharz research institute, pharmacology, cairo, egypt b-carotein was given in different doses starting from . to mg/kg body weight (b.w.) for different groups of albino mice days before infection with s. mansoni . infection of animals was done by body immersion using egyptian strain of s. mansoni cercariae/mouse. forty-nine days after infection the animals were sacrificed and hepatic and mesenteric worms were extracted and determined. ova count in liver and intestinal tissue and the total number of worms/animals were also determined in experimental groups comparing with infected control animals. the results indicated marked decrease in number of worms and ova count in both liver and intestine comparing with control ones. this reduction increased significantly with increasing dose. it was concluded that b-carotein could be used as a prophylactic agent against s. mansoni infection. barisic z, babic-erceg a, borzic e, zoranic v, carev m, kaliterna v. department of microbiology, public health institute, split, croatia the aim of this study is to determine frequency of pseudomonas aeruginosa urinary tract infection (uti) in outpatient's population in south croatia and to suggest optimal antimicrobial treatment for these patients. during months long observation period, from total number of examined urine specimens, significant bacteriuria was found in specimens. p. aeruginosa was the sixth most common isolate, it was isolated from specimens ( . %). these specimens were taken from different patients. susceptibility testing was performed by disk diffusion method, and the following results were obtained: resistance to cefibuten occurred in . % patients, to norfoxacin in . %, to ciprofloxacin in . %, to gentamicin in . %, to netilmicin in . %, to amikacin in . %, to ceftazidime in . % and to imipenem in . % patients. p. aeruginosa strains showed better susceptibility to tested parenteral antibiotics than to antibiotics for oral use which complicated treatment in outpatients. the best susceptibility was shown to imipenem, but this drug is inappropriate for use in outpatients setting, so the best choice for treatment p. aeruginosa uti in our outpatients is treatment with ceftazidime, and the second choices are aminoglycoside drugs amikacin and netilmicin. silan l a , breza j b , krcmery v jr. c . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school, bratislava, slovakia , c department of pharmacology, st. elisabeth cancer institute, bratislava, slovakia we studied the clinical efficacy of oral treatment with ciprofloxacin/ cpf/ alone and combined with clarithromycin in patients with complicated urinary tract infection/cuti/ with or without an indwelling catherer. patients were randomly allocated to mg cpf/cpf group/ or to mg cpf plus mg cam/combination group/ for days. evaluation was done on day according to the criteria advocated by the japanese urinary tract infection committee. in patients with a urinary catherer, the combination achieved a higher complete bacterial elimination rate / %/ and clinical efficacy rate / %/ than cpf alone / and . %, respectively/. while no significant difference was found in the bacterial elimination rate between the two groups, the clinical effect of the combination / %/ was superior to that of cpf alone / %/ in patients with an indwelling catherer. the better clinical efficacy of the combination may partly be attributed to the antibiofilm effect of cam in the clinical setting. the results also indicated that difficulties still remain in the treatment of cuti in patients with an indwelling catherer. in conclusion, clinical study suggested that cam might have an inhibitory action on biofilm formation in the clinical setting. combination of cam with other appropriate antimicrobial agents may have a favorable effect on the treatment of cuti. vesicoureteral reflux and urinary tract infections */the management of primary vesico-eureteral reflux in children ps the children studied presented with primary vesicoureteral reflux at derer s universitz hospital in bratislava between and . seven hundred and sixty patients, boys and girls, suffering from primary vesicoureteral reflux in age from months to years were tested and systematically analyzed outcomes data for seven treatment alternatives. key outcomes identified were probability of reflux resolution, likelihood of developing pyelonephritis and scarring, and possibility of complications of medical and surgical treatment. available outcomes data on the various treatment alternatives were summarized and the relative probabilities of possible outcomes were compared for each alternative. conclusions: increased of urinary tract infection, vesicoureteral reflux nephropathy includes early diagnosis, appropriate evaluation, effective atb therapy, and surgery indicated. the main determinants of renal damage are bstruction, age, sex, predisposition on renal scarring, reflux grade and laterality, therapeutic delay, individual susceptibility, bacterial virulence and immunogenetic status. ) the one and only absolute indication for surgical management is failure of medical therapy to prevent chronic recurrent pyelonephritis, renal injury or other reflux complications and eliminations of the reflux condition will minimize their likelihood. genetically conditioned immunopathogenic mechanisms are involved in the pathogenesis of the chronic recurrent pyelonephritis in patient suffering from vur. for most children we recommended continuous antibiotic prophylaxis as initial treatment-medical therapy is based on the principle that reflux often resolves with time, and antibiotics maintain urine sterility and prevent infections while the patients awaits spontaneous resolution. ) vur predispose an individual to renal infection, the immunological and inflammatory reaction caused by a pyelonephritic infection may result in renal injury or scarring. silan l a , breza j b . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school of medicine, bratislava, slovakia elderly patients with uti are believed less likely to be cured by antimicrobial therapy than younger patients. the reasons for this poorer outcome have not yet been clarified. we have investigated the efficacy of antimicrobial therapy in elderly patients with complicated uti. five hundred patients, men and women, who had complicated uti/ symptomatic and symptomatic and were Á/ years of age, were treated with one of three different drugs, one was a never quinolone and two were oral cephems. multivariate logistic regression analysis of treatment outcome revealed that the clinical response was significantly related to general underlying diseases and diseases of the urinary tract, but not to age, symptomatic or asymptomatic uti, or infection site such as the kidney or bladder. we concluded that the clinical effectiveness of an antimicrobial agent was not directly related to age, and that urological examination for underlying disease and control of them is quite important for effective treatment and control of complicated utis, especially in elderly patients. the study on the frequency and antimicrobial resistance of escherichia (e ) coli in urine isolates of patients admitted to maribor teaching hospital in and ps rebersek gorisek j a , baklan z a , unuk s a , novak d b . a department for infectious diseases, teaching hospital maribor, maribor, slovenia , b department for microbiology, regional institute of public health maribor, maribor, slovenia purpose: the aim of the study was to determine the frequency and antimicrobial resistance of escherichia coli isolated from urine samples of patients admitted to maribor teaching hospital in and . the frequency and the antimicrobial resistance were compared between years and . methods: in the prospective study going on between and , all urine isolates from patients at maribor teaching hospital were collected and analysed. urine cultures were done using the modified sanford method. the susceptibility testing was performed by disk diffusion method according to nccls. results: in the year , urine isolates and in the year , urine isolates were analysed. e. coli represented . % of urine isolates in and . % of urine isolates in . e. coli resistance rates (%) to amoxycillin was . in the year and . in the year ; to amoxycillin/clavulate was . and . ; to cefalotin was . and . ; to cefaclor . and . ; to trimethoprim sulfamethoxazole was . and . ; to ciprofloxacin was . and . ; to gentamicin was . and . . conclusion: compared to , the frequency of e. coli isolated from urine samples is similar to that in the year . the resistance to amoxycillin, cefaclor and gentamicin is stable. the resistance to trimethoprim sulfamethoxazole and ciprofloxacin is increased and the resistance to amoxycillin/clavulate and cefalotin is decreased. prevalence of the resistance to metronidazole, furazolidone and nitrofurantoin in helicobacter pylori clinical strains ps de la obra sanz p a , roman jl a , lomas e a , villar h b , lopez-brea m a . a hospital de la princesa, microbiology, madrid, spain , b hospital de san agustin, microbiology, aviles, spain the objective of this study was to determine the prevalence of metronidazole, furazolidone and nitrofurantoin resistance in helicobacter pylori clinical isolates. methods: a total of strains of h. pylori were included in this study. all these were tested against metronidazole, and against furazolidone and nitrofurantoin by an agar dilution method. resistance was defined as: metronidazole, mic !/ mg/l; and mic !/ mg/l for furazolidone and nitrofurantoin. results: sixty-eight strains were resistant to metronidazole ( . %). the mic and mic values were and mg/l, respectively. three of strains ( . %) were furazolidone resistant (mic / mg/l), two of these strains were metronidazole resistant (mic / mg/l) and they had mic of mg/l for nitrofurantoin. the mic and the mic were . and . mg/l, respectively for furazolidone. only one of the strains ( . %) was nitrofurantoin resistant (mic mg/l), this strain was metronidazole resistant (mic mg/l) and had a mic / mg/l for furazolidone. the mic and the mic were . and mg/l, respectively for nitrofurantoin. conclusion: the low frequency of furazolidone and nitrofurantoin resistance, compared to metronidazole suggests that the furazolidone and the nitrofurantoin may be good alternatives to metronidazole for treatment of h. pylori infections. antimicrobial resistance of campylobacter isolated from human origins ps zhukhovitsky vg, drabkina iv. department of bacteriology, botkin hospital, moscow, russian federation the purpose of the study was to determine the antimicrobial resistance of thermophilic enteropathogenic campylobacter spp. (tec) isolated from human under acute diarrhoea in in moscow. among tec strains c. jejuni and five c. coli were identified. the antibiotic tested by disk diffusion test on mueller-hinton agar with sheep blood were ampicillin (a), amoxycillin/clavulanate (ac), imipenem (i), meropenem (m), erythromycin (e), clarithromycin (cl), tetracycline (t), doxycycline (d), gentamicin (g), azithromycin (az), chloramphenicol (ch), lincomycin (l), ciprofloxacin (c), nalidixic acid (na). the resistant rate of the tec isolates was highest for na ( %) followed by a ( %), t ( %), d ( %), n ( %) and cl ( %). a moderate resistance rate was obtained for a ( %), ch ( %), az ( %), ac ( %). none of the isolates demonstrated resistance to i, m and g and four of isolates ( %) were sensitive for all the antibiotics tested. mic to na was estimated as mg/l. among na resistant tec strains ( %) were identified as c. jejuni and one ( %) as c. coli . among c. jejuni and c. coli na resistant rate was and %, respectively. one na resistant c. coli and nine na resistant c. jejuni were resistant to ciprofloxacin. ring c, atanassova v. department of food hygiene and microbiology, school of veterinary medicine, hannover, germany aim of the study: poultry meat is known to be often contaminated with salmonella and other foodborne pathogens and thus has to be considered as a possible source for human infections. the aim of the study was to monitor the resistance of salmonella isolates from poultry meat of different european countries to various antibiotics. material and methods: from september to december a total of samples of frozen poultry meat from france, germany, italy, spain, the netherlands and portugal were examined for the prevalence of salmonella using classical cultural detection as well as rflp-pcr. all isolates were tested for their sensitivity towards ampicilline, kanamycine, ciprofloxacine, tetracycline, trimethoprim, sulfamethoxazole, nalidixic acid and erythromycine using standard procedures. results: from . % of all examined samples salmonella spp. were isolated. of these isolates . % were characterized as salmonella , . % as s. hadar and . % as s. typhimurium . nearly % of all isolates were resistant to erythromycin. resistance towards four or more isolates was observed in several cases. discussion: the consumption of poultry meat, if insufficiently prepared, has still to be considered as a major source for human infection with salmonella spp. the question arises whether the resistance of the isolates to various antibiotics is of clinical importance in the treatment of the patients. objective: to provide insight into the epidemiologic situation of salmonellosis for the nis area (the largest area in serbia, with inhabitants */ , ). methods: the material was processed at the institute for public health (epidemiology and microbiology divisions). isolation of microorganisms was performed on apparatus for rapid identification (vitek-biomerieux) and by applying elisa tests and classical microbiological methods. results: in the period Á/ , salmonella laboratory confirmed cases were reported. the greatest number of diseased in the Á/ years group. the most frequent isolated salmonellae were: s. enteritidis ( . %) and s. typhimurium ( . %), s. hadar , s. agona , s. virchow , s. infantis , s. derby , s. enteritidis showed the greatest sensitivity to antibiotics with the infrequent resistance to ampicillin and trimethoprim-sulfamethoxazole. s. typhimurium showed the greater resistance to the wide spectrum of antibiotics and some isolates were resistant to all antibiotics tested. the less common types of salmonella were sensitive to all antibiotics except trimethoprimsulfamethoxazole and ampicillin. conclusion: specific resistance to some antibiotics was related to serotypes. typhoid fever */retrospective study of cases in lebanon ps tohme a, abboud j, ghayad e. hôtel-dieu hospital, internal medicine, beirut, lebanon objectives: to present epidemiological and clinical features of typhoid fever in lebanon. methods: fifty-two patients were seen at hotel-dieu hospital of beirut between and . diagnostic criteria were positive blood culture for s. typhi or paratyphi and/or a somatic o agglutinin titer ]/ / as determined by the widal test with symptoms suggestive of typhoid fever. we also present an epidemiological study of cases registered by the ministry of health during the same period. results: among the cases, % of the patients' ages were between and years and % were less than years. the overall male to female ratio was . and % of cases were seen on january, february and % on august. among the patients, young adults were the most affected. average duration of symptoms before the diagnosis was / days. the main presenting symptoms were: fever ( %), diarrhoea ( %), abdominal pain ( %) and headache ( %). complications were noted in % of cases and digestive complications were the most prevalent. leucopenia was not a helpful diagnostic marker. s. typhi was the most frequent ( %) serotype identified. resistance to ampicilline was %, to cotrimoxazole and chloramphenicol % for each. the mortality rate was %. conclusion: typhoid fever is still an endemic disease in our country and the occurrence of resistant strains of s. typhi will favor ceftriaxone or fluoroquinolones in the treatment. maaloul i a , hammami b a , zambaa f a , elleuch r a , hammami a b , -ben jemaa m a . a chu hedi chaker, service des maladies infectieuses, sfax, tunisia , b chu habib bourguiba, laboratoire de microbiologie, sfax, tunisia although its not very frequent, the psoas abscess is not an exceptional entity. in order to specify its clinical, biological, radiological and evolutionary features, a retrospective study has been led in our service, on a period of years (january Á/december ). on the whole, cases have been listed. they were men and women. the age average was years (extreme Á/ years). the study did not find any underlying diseases, except diabetes mellitus for three patients. the clinical symptoms were dominated by fever with abdomino-lumbar aches ( cases), and psoitis (eight cases). biology showed an inflammatory syndrome in all cases and a hyperleucocytosis in cases. the diagnosis of psoas abscess, evoked on clinical data, has been confirmed by the imagery data: ultra-songraphy ( cases), ct scanning (six cases), magnetic resonance imaging (three cases). the tubercular etiology has been confirmed in six cases, among which two were associated to escherichia coli (one case) and to brucella melitensis (one case). the other etiologic agents were dominated by staphylococcus aureus (eight cases), b. melitensis (two cases), e. coli (one case), bacteroides fragilis (one case), streptococcus anginosus (one case), fusobacterium nucleaticum (one case) and candida glabrata (one case). all patients received an anti-infectious treatment adapted to the micro organism in question. a drainage of the abscess has been realized for patients (percutaneous: nine cases, surgical: six cases). the evolution was favourable for patients. however, two patients had a relapse after stopping the treatment. conclusion: the diagnosis of the psoas abscess, difficult on the clinical data, is based on the imagery techniques (us, ct, rmi). the percutaneous drainage guided by the imagery is recommended (in an etiological and therapeutic aim). associated to an adapted antibiotherapy, it allows to defer a surgical drainage. zezoski mbz, nikolova o, gavriloski pmg. medical center, infectious diseases, prilep, the former yugoslav republic, macedonia purpose: to make a list of the most frequent abdominal changes in patients with human brucellosis. materials and methods: there were new patients with human brucellosis, between and . diagnosis was made using standard clinical, biochemical and serological investigations (bab, wright, coomb's, rvk, -mercaptoethanol, elisa igm and igg), and specially ultrasound examination of the abdomen and retro peritoneum. results: weight loss is the most frequent change, presented in ( . %) patients. follow atypical abdominal pain in ( . %), vomiting in ( . %), diarrhea in ( . %), enlarged liver in ( . %), enlarged spleen in ( . %) and hepatic lesion with increased ast and alt in ( . %). conclusion: although frequent, abdominal changes seldom could be missed in patients with human brucellosis. we recommend routine ultrasound examination with standard biochemical test for liver function, due to avoid unnecessary complications. osteoarticular complications are common in brucellosis. the most common site of involvement is the sacroiliac joint. the osteoarticular complications such as, sacroiliitis and spondylitis are diagnosed with radiologically. in the present study, we aimed to determine the severity (grade) of sacroiliitis by using some laboratory parameters such as esr, crp and tube agg test. seventy-two ( male, female) patients with brucellosis were included in the study. osteoarticular involvement was present in patients. the most common osteoarticular finding was sacroiliitis in the patients ( %). twenty ( ) healthy subjects were formed the control group. there was statistically significant difference between patients and controls regarding esr, crp, and tube agg test (p / . , . , . , respectively). in addition, sacroiliitis has an effect on esr and crp. there was a positive correlation between the grade of sacroiliitis and the value of crp (p / . , r / . ). in conclusion, it has been suggested that, crp may be used as an auxiliary or a secondary parameter in grading sacroiliac joint involvement in brucellosis. magira ee a , papandreoy s a , gounaris t a , spirelis ma a , tasopoulos g a , anagnostopoulou m b , paniara o b , gounari p a , sioulal e a . a evagelismos, internal medicine, athens, greece , b evagelismos, micro- a -year-old greek farmer was admitted to the hospital because of painful scrotal swelling, hepatosplenomegaly, lumbar pain and lowgrade fever accompanied by profuse sweating. his life style included occupational animal exposure ingestion of raw milk and dairy products. the laboratory data were within the normal ranges. focal hypoechoic right testicular lesions, swelling of the concurrent epididimis along with an increase in the vascularity of the right testis were seen on an echo examination. these findings were consisting in unilateral epididimo-orchitis. a ct scan of the lumbar spine area showed a decrease of the signal intensity localized in the anterior aspect of l vertebral body at the diskovertebral junction involving the subchondrial parts of the l and s vertebrae standard tube agglutination test was positive for antibodies to brucella melitensis (titer !/ / ). cultures of blood specimens were positive for b. melitensis . the patient had been given to a combination of antibiotics with doxycycline, streptomycin and rifampin. a remarkable improvement of his clinical condition was showed weeks later. this case illustrates the following point: in areas in which brucellosis is endemic when scrotal abnormalities are seen the possibility of genitourinary tract complications of brucella should be considered. pappas ga a , akritidis nk b , mastora m b , tsianos e a . a university hospital, internal medicine, ioannina, greece , b 'g. hatzikosta ' hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and forms of complications associated with brucella infection. patients and methods: we studied the most recent patients, in all larger series approaching , diagnosed as suffering from brucellosis, and assessed the presence of signs and symptoms of arthritis and spondylitis, or other forms of bone involvement. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures or cultures from other media. results: osteoarticular complications were noted in patients, presenting with arthritis, and presenting with spondylitis. eight patients presented with genitourinary complications, either orcheoepididymitis (four patients), or hematuria resolving with treatment (four patients). meningitis was present in two patients. gastrointestinal complications (vomit and diarrhea) were present in three patients, while one patient presented with ascites. respiratory tract complications, in the form of pneumonia (four patients) or bronchitis (three patients) were noted in seven patients, while one patient with pneumonia exhibited pleural fluid. skin rashes, of macular type, were present in three patients. no patient presented with complications from the heart. hematologic complications were frequent, in the form of severe (one patient) or moderate (two patients) pancytopenia, isolated thrombocytopenia (three patients), or lymphocytosis (eight patients). akritidis nk a , pappas ga b , mastora m a . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and modes of bone and joint involvement in the course of brucellosis. patients and methods: we studied the most recent patients, in all larger series approaching , diagnosed with brucellosis, and assessed the presence of arthritis and spondylitis. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures. results: twenty-three patients exhibited a form of osteoarticular involvement. arthritis was present in patients, most often involving the knees, but also the hips, elbows, even smaller joints as intephalangeal joints of the hand. synovial fluid, when aspirated, was often characterised by an intense mononuclear infiltrate. spondylitis was present in patients, most often involving the lumbar spine, but also the thoracic spine. the characteristic erosion on the upper anterior crest of the vertebral body was visible in plain x-rays in three patients, while mri and bone scan were helpful in other cases. discussion: osteoarticular involvement in the course of brucellosis is the most common focal presentation of the disease. acute brucellosis is often accompanied by bone and joint ache, especially of the lumbar spine, still frank involvement in the form of arthritis and spondylitis is not rare. arthritis usually presents in the acute form of the disease, while spondylitis tends to be characteristic of a chronic form of the disease, often necessitating prolonged use of antibiotics. bosilkovski m, krteva l, caparoska s, grozdanovski k, sajn b. clinic for infectious diseases and febrile conditions, medical faculty, skopje, the former yugoslav republic, macedonia one hundred and twenty-six patients with brucellosis were studied prospectively. seventy-eight ( %) of them had osteoarticular involvement. peripheral arthritis in ( %) patients was the most frequent, followed by spondilitis in ( %), sacroiliitis in ( %), rarely bursitis, tendinitis and osteomyelitis. the overall male to female ratio was : . their average age was (sd ) years. direct contact with animals was the reason for acquisition of the illness in % of patients, in % alimentary or aerogenous route was incriminated, and in % the route of aqisition was unknown. the average duration of the symptoms from the onset to establishing the diagnosis was (sd ) days. the main presenting symptoms were joint pain ( %), sweating ( %), fatigue ( %) and fever ( %). hepatomegaly was present in %. in % of patients, involvement of some other system was evident. comparison with patients, who did not have osteoarticular illness, showed that patients with osteoarticular involvement had significantly more often joint pain, fatigue, weight loss and more prolonged duration of symptoms before the diagnosis was established. doxycycline and chloroquine as combination therapy for chronic q fever endocarditis ps calza l, attard l, manfredi r, chiodo f. division of infectious diseases, university of bologna, s. orsola hospital, bologna, italy introduction: endocarditis is the main clinical manifestation of chronic q fever, occurring in about Á/ % of all reported cases, and it is diagnosed almost exclusively in patients with either cardiovascular abnormalities or an immunocompromised condition. case report: a -year-old caucasian male patient with biological prosthetic aortic valve was first hospitalized because of an interstitial pneumonia. six months later, our patient was re-admitted owing to intermittent fever, chills and weight loss. echocardiographic study showed a small vegetation of mm in diameter on left cusp of aortic valve. serology for coxiella burnetii revealed a complement-fixing igg antibody titer to phase i antigen of more than : , consistent with chronic q fever endocarditis. antimicrobial therapy with i.v. doxycycline and oral chloroquine was started, leading to a clinical and echocardiographical recovery. therapy was continued by oral doxycycline and chloroquine, and the patient remained asymptomatic during a -year follow up. conclusion: the optimal treatment of q fever endocarditis has not been well established: the most effective antimicrobials are fluoroquinolones and rifampin, but chloramphenicol, doxycycline and trimethoprim are also useful. the role of chloroquine in combination with doxycycline seems to be promising, because chloroquine may increase the lysosomal ph, enhancing the doxycycline bactericidal activity. akritidis nk a , pappas ga b , mastora m a , liappis e a , tsianos e b . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scopes: to review the incidence and the forms of lower respiratory tract infection in patients suffering from q fever, and their clinical and radiological characteristics. patients and methods: twenty-seven patients diagnosed as suffering from q fever, were assessed for the presence of lower respiratory tract infection. the diagnosis was confirmed serologically. results: thirteen patients expressed lower respiratory tract pathology, as confirmed by clinical examination and chest x-ray. in of these patients the main cause of admission was respiratory tract symptoms, ranging from dry cough to hemoptysis. chest x-ray was pathological in patients: patients had lobar pneumonia, two of them multiple nodular opacities, and one of them bronchopneumonia. hepatitis was a common finding. all patients were treated with tetracycline. discussion: although coxiella burnetii infection is acquired via the respiratory tract, it is paradoxical that symptoms attributed to the lung are not invariably positive. q fever pneumonia is an atypical pneumonia that usually follows a benign course. diangostic suspicion is usually raised by the epidemiologic pattern and the accompanying mild hepatitis. pleural effusion is not a common finding. the usual radiologic appearance of q fever pneumonia is that of a lobar or segmental pneumonia. one important aspect of q fever pneumonia is its common presentation in the form of multiple nodular opacities often necessitating the exclusion of malignancy. ( ), culture ( ), and serology'/culture ( ). there were Á/ cases per year, mainly in october ( %). a history of exposition to hare was present in / ( %) and to marmot in / ( %). skinning ( / %), animal contact ( / %), bite ( / ) and wound during bait preparation with frozen meat for hunting ( / ) were noted. the initial clinical presentation was ulceroglandular ( %), glandular ( %) and pneumonic ( %). the involved nodes distribution was axillary ( / ), cubital/axillary ( / ) and cubital ( / ). median incubation period was days (range Á/ ); time to consultation days (range Á/ ), and time for effective treatment days (range Á/ ). an initial diagnosis of tularemia was made presumptively in %. effective antibiotic regimen used was aminoglycosides in % ( / ), and tetracyclines in % ( / ). note that intravenous netilmicin was used in cases. complication rate was % ( / ) with one death ( %), and was associated with delay in effective treatment ( !/ days of illness) (p b/ . ). conclusion: in our area tularemia occured mainly in male population, during autumn, with a short incubation period and history of hare contact. delay before appropriate treatment increased the complication rate. bompolaki i, doukakis s, triantafillidou d, polimili g, kastanakis m, nikiforakis k, vittorakis e, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece a severe frontal and/or retroorbital headache represents the most common neurologic manifestation of murine typhus. other neurologic manifestations as confusion, stupor, nuchal rigidity and in severe cases delirium, extreme agitation or coma appear less commonly. eightyfour patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi, were studied from our team. seventy-four patients ( %) presented headache and nine patients ( %) presented confusion. one patient ( . %) presented nuchal rigidity in combination with severe headache and confusion giving us the suspicion of meningitis. in this case a lumbar puncture was performed emergently and the cerebrospinal fluid (csf) was examined. the findings of csf were proteins: mg/dl, wbc: /ml and glucose: mg/dl and its culture was negative. all patients were treated with a specific anti-rickettsial treatment. the outcome of murine typhus was favorable for all patients ( %). no one patient presented neurologic sequelae. conjunctivitis usually accompany rickettsial diseases such as rocky mountain spotted fever, epidemic typhus and murine typhus. eightythree patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january and the first semester of . the clinical examination of these patients revealed the presence of conjunctivitis in / patients ( . %). in the same time these patients referred retroocular pain and mild photophobia. this study showed that in murine typhus the injection of conjunctivae is rather common. almost a quarter of the patients presented conjunctivitis despite the fact, that this ocular manifestation is less severe than in other typhus and spotted fevers. eighty-three patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january and the first semester of . three blood samples were obtained from each patient for the study of their hematological abnormalities. the first sample was obtained on admission, the second sample weeks after the first, the third sample, month after the second. on admission / patients ( %) presented anemia, / patients ( %) presented leukopenia and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. two weeks later anemia was presented in / patients ( %), / patients ( %) presented leukopenia, / patients ( %) presented leucocytosis and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. one month later / patients ( %) had anemia and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. our study showed that early thrombocytopenia and anemia are frequent in murine typhus and that white blood cells count is usually normal. renal function in murine typhus: a study of cases ps doukakis s, polimili g, triantafillidou d, kastanakis m, vittorakis e, palla k, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece the clinical course of murine typhus is usually uncomplicated and the mortality rate is low ( b/ %). advanced age and prolonged interval before administration of a specific anti-rickettsial treatment are correlated with severity of the disease. renal function in murine typhus is usually unaltered except in elderly patients with prolonged hypotension. eighty-three patients with compatible clinical status of murine typhus and high serological titres of antibodies against rickettsia typhi, were studied from our team, during a period of time between january and the first semester of . three blood samples were obtained from each patient for the study of their renal function. the first sample was obtained on admission, the second sample approximately weeks after the first, the third sample, taken from the half of the patients, was obtained one month after the second. on admission / patients ( . %) presented acute renal failure. the outcome of murine typhus was favourable for all patients ( %). the four patients who presented acute renal failure reversed after the administration of anti-rickettsial treatment and careful administration of fluids. in murine typhus the induction of hypovolaemia insufficiently corrected by normal homeostatic mechanisms may lead to prerenal azotaemia. in these cases the immediate onset of an antirickettsial treatment and the correction of hypovolaemia are essential for the rapid clinical improvement of the patient. experimental ocular toxoplasmosis: clinical, histopathological, immunological and therapeutic studies ps el zawawy lae a , hammoda na a , allam sr a , ali sm a , galal as b . a faculty of medicine, parasitology, alexandria, egypt , b faculty of medicine, ophthalmology, alexandria, egypt the purpose of the study was to investigate clinical, histopathological, immunological and therapeutic features of an experimental model of ocular toxoplasmosis in sensitized and non sensitized rabbits and to assess the influence of treatment by interleukin (il- ) on ocular lesions. the results obtained was that 'toxoplasma' retnochoroiditis developed in both groups of rabbits with more pronounced effect in non sensitized animals. administration of il- improved ocular lesions in both groups with more evident effect in sensitized rabbits. immunological findings were consistent with clinical and histopathological observations. the conclusion reached was that; ocular lesions were manifested in non sensitized rabbits more than in sensitized ones. il- revealed a significant impact on improving the host defense against toxoplasm infection in eye. immunotherapy with il- would open the way for a new range of treatment based on immunomodulation. express-diagnostic of streptococcus antigen for the adequate antibiotic therapy in patients with pharyngitis ps pertseva to, konopkina li, kireeva tv. dsma, internal medicine , dniepropetrovsk, ukraine the purpose of the study: evaluation of effectivness of streptococcus express-diagnostic for the adequate antibiotic therapy in patients with pharyngitis. results: we deal with clinical and microbiological comparison in patients with pharyngitis. using of streptococcus antigen express diagnostic in swabs from the backside of pharynx allowed to get positive results in the seven cases ( . %). following cultural study has confirmed these results. positive test was more probable in patients with pronounced fever (more than c), headache, weakness and in cases associated with chronic tonsillitis. isolated streptococcus pyogenes was susceptible to ampicillin, claritromicin, erytromicin, azytromicin, , clindamicin, ceftriaxon, levafloxacin, oxacillin, cefuroxim, roxytromicin. conclusion: using of the express diagnostic of streptococcus antigen allows to restrict groundless prescription of antibiotic therapy in patients with other types pharyngitis (i.e. viral, candidal etc.). alabaz d a , turgut m a , kocabas e a , tumgor g a , yaman a b , alhan e a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey chickenpox is a common viral infection that usually follows a benign, self limited course in healthy children. the most common complication in children with varicella is superimposed cutaneous infections with pyogenic bacteria (streptococcus pyogenes and staphylococcus aureus ). varicella gangrenosum, a necrotising soft tissue infection complicating the vesicular eruption of chickenpox, is rare. here we present three cases with necrotising soft tissue infections following chicken pox. these children were admitted because of common crusted lesions and necrotising soft tissue infection over the neck, the back, and the inguinal area. they all had the contact history and ensuing vesiculopapular rush. these infections were caused by group a streptococci in two cases, and s. aureus in one case. after instituting of appropriate antibiotic therapy, each patient underwent a surgical exploration with fasciotomies and debridement. widespread use of varicella vaccine may decrease invasive infections in children, adolescents, and adults, thus decreasing the burden the disease with its complications impose up on the family and the society . cefprozil is a second generation cephalosporin. the aim of this open, multicentre, non-comparative study was to investigate the efficacy and safety of cefprozil in the treatment of streptococcal tonsillopharyngitis. fifty-eight patients were clinically assessed for signs and symptoms of streptococcal infection. laboratory confirma-tion was sought using three tests; culture, rapid strepto test and estimation of antistreptolysin (aso). one or more tests were done in of the patients. treatment was for days, mg/kg per day in children, mg per day in adolescents. patients were again clinically assessed on the th Á/ th day. the results showed clinical success in patients ( . %) and in of the who had laboratory tests ( . %). two patients had treatment withdrawn because of nausea and abdominal pain ( . %). of the patients with laboratory tests at least one test was ositive. the most helpful was the strepto test giving the quickest positive result in % of those tested ( / ). culture was positive in % of those tested ( / ). the aso test was of limited value. in conclusion, cefprozil showed high clinical efficacy and safety in the treatment of streptococcal tonsillopharyngitis. tsiara s, militadou g, milionis c, elisaf m. internal medicine department, university of ioannina, ioannina, greece streptococcus group b agalactiae (gbs) is a rare pathogen for healthy male adults. we present an old man in whom (gbs) was isolated in blood cultures. case report: a -year-old man was admitted to the hospital in order to investigate osteolytic lesions in the lumbar spine. two weeks before, he experienced severe low back pain radiating to the right leg and fever arising to . c with chills and rigors. a gbs was isolated from blood cultures and treatment with penicillin was initiated. a spine ct scan revealed osteolysis in the t and s vertebrae and the patient transferred to us. he was a previously healthy man. he received only antihypertensive therapy. on admission he was afebrile with arthralgias and myalgias. on clinical examination there was tenderness on the right pleurospondylic angle radiating to the right leg. laboratory data on admission: hb: . g/dl, wbc: . )/ /l, esr: mm/h. biochemical values, serum protein electrophoresis, rectal examination and a prostatic specific antigen (psa) were normal. a bone marrow aspiration and biopsy were negative. a transoesophageal ultrasound revealed vegetation on the right cusp of the aortic valve, with low grade regurgitation. an mri of the lumbar spine revealed infectious myositis with concomitant osteomyelitis involving the l , l vertebrae without any evidence of osteolytic lesions. a thorough investigation did not revealed any underlying immunosuppressive disease. treatment with vancomycin and gentamycin iv was initiated and the patient discharged from the hospital in excellent health after weeks. discussion: gbs infections usually occur in neonates and pregnant, or in adults with underlying disease as diabetes mellitus or immunosuppression. the most common site of infection is soft tissue. we present this case because gbs infections are rare in the elderly in the absence of underlying disease. common sites of involvement are soft tissues, while bone, joints, and heart valves account to Á/ % of the involved organs. although our patient had more than one site of involvement he responded well to medical treatment without surgical debriment or heart valve replacement. objective: to evaluate the safety and efficacy of rhugm-csf in combination with broad-spectrum antibiotics for the treatment of ccnf. methods and results: a retrospective review of all patients (pts) with ccnf treated with antibiotics'/rhugm-csf at our hospital from january to december was performed. five patients were identified with the diagnosis of ccnf. ages ranged from to years; there were three women and two men. dental infection was the most common source of ccnf in %. one patient had acute tonsillitis leading to ccnf. all cases studied experienced infection of the neck with spread into the submandibular, submental, sublingual, retropharyngeal, and parapharyngeal spaces. all infections were polymicrobial. diabetes mellitus was a co-morbidity in one case. all pts were treated with dual antibiotic coverage (vancomycin'/meropenem), rhugm-csf ( mcg/kg/daily given s.c.) and aggressive wound care. rhugm-csf was given for Á/ days. in spite of the severity of the infection all pts recovered and do not experienced local or systemic complications. discussion: ccnf is a severe bacterial infection of the cervical fascia resulting in extensive fascial necrosis with widespread undermining of the surrounding tissues. prompt antibiotic therapy combined with rhugm-csf resulted in a % overall survival in our experience. to our knowledge, this is the first report describing the successful treatment of ccnf with use of broad-spectrum antibiotics combined with rhugm-csf. the following case adds to the clinical manifestations and course of meningococcal disease. a previously healthy -year-old girl presented acutely with high fever purpuric rash including conjuctival haemorrhages and hypotension. the child had also neck stiffness. a presumptive diagnosis of meningococcal septicaemia was made and treatment with penicilline, chloramphenicole, fluids and inotropes was initiated. laboratory investigations showed wbc: /ml, hb: . g/dl, hct: . %, esr: mm in the first hour, pt: s, aptt: s. neisseria meningitidis group b was isolated from the blood cultures. csf obtained after antibiotics were started did not grow n. meningitidis . the patient had an adverse outcome. she died after h of hospitalization. this patient developed a fulminating meningococcal septicaemia. shock is a clinical diagnosis arising from the failure of compensatory mechanisms that maintain perfusion of vital organs at the expense of non vital. septic shock results from loss of circulating plasma volume due to increased vascular permeability and maldistribution of intravascular volume. in young children there is a prevalence of serogroup b meningococcal disease which can be explained by the immaturity of the immune system and by the fact the group b capsule synthesis is known to inhibit alternative complement pathway activation. this case emphasizes the need for further protection against n. meningitidis group b. . results: forty-eight patients were included in this study. the etiological agents were: viral (n / , . %, mean ('x ) age / years), streptococcus pneumoniae (n / , . %,'x age / ), neisseria meningitidis (n / , . %, 'x age / ), s. viridans (n / , %, 'x age / ), p. multocida (n / , age ). the peak incidence of bacterial meningitis was in winter (pneumococcal %, meningococcal %, s. viridans %) .the cerebrospinal fluid (csf) findings in viral meningitis were 'x white cells / /mm , 'x pmn / %, 'x glucose csf/ serum . , 'x protein mg/dl and in bacterial meningitis were 'x white cells / /mm , 'x pmn / %, 'x glucose csf/ serum / . , 'x protein / mg/dl, gram stain was positive in %, culture was positive in %. all pneumococcal and meningococcal strains were susceptible to penicillin. the case fatality rates for pneumococcal and meningococcal meningitis were and . %, respectively. conclusions: the cases of bacterial meningitis were according to typical epidemiological features of age and season. the case fatality rate of pneumococcal meningitis appear to be high regardless of susceptibility to penicillin. none had received pneumococcal vaccine prior to becoming ill. diagnosis and therapy of meningococcal meningitis */trend and particularities of a 'romanian model' ps lintmaer i, moroti r, popescu a, popescu c. institute of infectious diseases matei bals , unit , bucharest, romania background: newer diagnosis methods and antimicrobials are expected to change the management of menigococcal meningitis (mm) and to improve its prognosis. objectives: to determine the changes in the diagnosis methods and therapy of mm patients in a infectious diseases hospital. to compare mm management in bucharest with literature data. methods: retrospective rewiew of mm in adult patients hospitalized over a -year period. our results were compared with other studies made in the s, taken from medline. results: there were episodes of mm during the study period ( episodes in Á/ and episodes in Á/ ) . we noticed a defined diagnosis increase and increased blood culture specificity. the antimicrobial monotherapy was maintained but penicilin was replaced by ceftriaxone. hhc was replaced by dexamethasone in pathogenic therapy. we noticed a shorter length of treatment and a reduced lethality. the most important differences between our results and other studies are: monotherapy regimens are less frequent and therapy lengths are longer; however, prognosis is similar. conclusions: the mm management has been modified in the last Á/ years: prognosis is improved, but the changes do not bring clear cost/ effective benefits. tiouiri th, kilani b, amari l, zouiten f, kanoun kf, ghbontini a, ben chaabene t. rabta hospital, infectious diseases, tunis, tunisia objectives: in order to study epidemiological, clinical and therapeutical characteristics of the infective endocarditis (ie). methods: we reviewed all the cases of ie fulfilling the duke criteria. data were collected during a -year period ( Á/ ) in the unit of infectious diseases. results: one hundred and eight cases were identified. the mean age was . years. sex ratio was . . eighty-five ie ( . %) occured in patients with native valve, and ie ( . %) with prosthetic valve. fever was the most common sign, % had a congestive heart failure, . % had cutaneous signs. the most common primary focus of ie was orthodontic. blood cultures were positive in % of cases. in one case, serological test identified rickettsia conori . streptococci and staphylococci were isolated in . and . %, respectively. echocardiography detected abnormalities in . % of cases. rheumatic heart disease was the most predisposing condition. empirical therapy was based on combination of b lactam with aminoglycoside. recovery was obtained for patients. cardiac surgery was performed in cases. overall mortality rate was . %. conclusion: ie affects young persons. prevention needs eradication of acute rheumatic arthritis. a major outbreak of legionnaires' disease in spain: diagnostics aspects ps guerrero c a , toldos cm a , yagü e g b , ramírez c a , rodríguez t a , -segovia m a . a hospital morales meseguer, servicio de microbiología, murcia, spain , b departamento de microbiología, facultad de medicina, universidad de murcia, murcia, spain objective: to evaluate the value of different methods (serological tests, culture of respiratory secretions, blood cultures and urinary antigen testing) for the diagnosis of legionella pneumophila pneumonia during an outbreak in spain. results: we have studied patients from a recent outbreak of legionellosis in murcia (spain). the diagnosis was achieved in patients. urinary antigens were positive in patients. in the patients with urinary antigen negative the serological response was demonstrated by indirect immunofluorescence (ifa) in patients. all blood cultures processed were negative. sputum samples were obtained from patients, of these l. pneumophila was isolated only in six patients. in all of them direct immunofluorescence test (dfa) was positive. conclusions: although the serological diagnosis was the most sensitive method the urinary antigen testing was of great value in the rapid diagnosis of the legionella's outbreak in murcia. the isolation of l. pneumophila by culture showed a poor sensitivity probably because of the low severity of the illness. purpose: to evaluate chloramphenicol for an initial empiric antibiotic treatment of purulent meningitis in adults. study group: one hundred and twenty patients hospitalized for the diagnosis purulent meningitis in the department in years Á/ , males and females, age range Á/ years, mean age . years. children up to years were not included. method: a retrospective analysis of the study group focused on antibiotic treatment both initial and changes during treatment. results: chloramphenicol was used as an initial antibiotic in ( %), rd generation cephalosporin in ( %), penicillin in ( %), ampicillin in five ( %) and other antibiotic in five ( %), respectively. during treatment chloramphenicol was switched for rd gen cephalosporin in seven of patients with streptococcus pneumoniae meningitis and in five of patients with meningitis of unknown etiology. the reason for the change was non-improving csf formula in three, persisting csf culture positivity in two and persisting coma in seven patients. conclusion: because of repeated necessity to switch chloramphenicol for rd gen cephalosporin during treatment of purulent meningitis of pneumococcal and unknown etiology the initial treatment strategy was changed in . third gen cephalosporin is now used as a first choice antibiotic, what is in consent with international recommendation of treatment. to evaluate and compare groups treated initially with chloramphenicol and with rd gen cephalosporin will need several more years. low prevalence of multi-drug resistant mycobacterium tuberculosis in jerez de la frontera-cadiz (spain) ps alados jc, aller ai, de miguel c, de francisco jl, calbo l. hospital del sas-jerez , microbiologia, jerez de la frontera, cadiz, spain introduction and aim: previous reports indicate that multi-drug resistance mycobacterium tuberculosis (mtb) is an worldwide problem. the aim of this study was to review the resistance of mtb to the first-line antimycobacterial agents in our area. material and methods: over a period of years ( Á/ ), strains of mtb isolated from non-treated patients with tuberculosis ( strain in , in , in and in ) were studied. these isolates were tested for in vitro drugs susceptibility to isoniacid-i, rifampicin-r, streptomycin-s and ethambutol-e using the bact/ alert method (organon teknica) as described by the manufacturer. results: our results showed that . % ( / ) strains were resistant to one or more drugs. single drug resistances were detected on nine strains to i ( . %), one to r ( . %), two to s ( . %), one to e ( . %). three mtb strains were resistant to more than one drug but only one was multi-drug resistant (i'/r).the incidence of i-resistant strains over the period fell from % in to . % in . conclusions: ( ) multi-drug resistance is not an important problem in our area. ( ) isoniacid resistance was declined to an admissible level. to investigate the anti-tuberculosis drug resistance pattern of pulmonary tuberculosis isolates in southern taiwan, an area with higher tuberculosis incidence and mortality than other regions of the island, we performed a hospital-based surveillance at a southern taiwan medical center from to . the combined drug resistance rates to at least one of five first-line agents was . %, and to both isoniazid and rifampin (multi-drug resistance, mdr) was . %, indicating high resistance rates compared with those reported in the who/iuatld global project and in northern taiwan. the resistance rates to two second-line drugs, cycloserine, and kanamycin, were . and . %, respectively. a significant decreasing trend in resistance rates to all drugs except streptomycin was observed during the -year period. though combined drug resistance rate may not be the most accurate tool as it includes previously treated cases which inflates the resistance rate, the observation of trends in the susceptibility of pulmonary tuberculosis in accompany with the increasing percentages of tuberculosis patients receiving complete treatment course and the decreasing percentages of cases lost of follow-up in kaohsiung after the institution of new governmental regulations for case management in suggest the usefulness of intervention programs. lipid profile in patients with multidrug resistant pulmonary tuberculosis ps extrapulmonary tuberculosis may sometimes present with confusing clinical manifestations. a -year-old female patient was admitted with a history of recurrent supra-sternal abscess for year. mri confirmed the presence of sternal osteomyelitis and an anterior mediastinal mass. the diagnosis of tuberculosis was proved by histologic examination and acid-fast stain. the patient was treated with first-line agents, which isoniazid, rifampin, pyrazinamide, and ethambutol. tobacco smoking as a factor of the decrease of chemotherapy effectiveness and of the development of the drug resistance in patients with pulmonary tuberculosis ps shprykov as a , zhadnov vz a , shprykova on b . a medical academy, department of tuberculosis and lung diseases, nyzhny novgorod, russian federation , b medical clinic for infectious # , laboratory of bacteriology, nyzhny novgorod, russian federation studies of the effect of smoking on the results of chemotherapy of patients with tuberculosis of lungs. intensive tobacco smoking slowed down clearance of positive sputum and of lung tissue destruction (in smokers . % and . vs. . % and . % in nonsmokers, p b/ . ). drug-resistant mtb strains have been found to be isolated more often in smokers */ . vs. . % in non-smokers, p b/ . . resistance to streptomycin and isoniazid prevailed, reaching in heavy smokers . and . %, respectively. resistance to rifampicin increased . times. the concentration of rifampicin in the blood serum of heavy smokers decreased in . times. clinical data are in complete correlation with the findings of our experiments: % of experimental cultures developed resistance to streptomycin, isoniazid and less to rifampicin in the study of drug sensitivity under the effect of tobacco smoke condensate. thus, our findings show the development of drug resistance in mtb under the effect of components of tobacco smoke and also showed less effectiveness in therapy. kilani kb, ammari la, tiouiri ht , ben chaabène tbc. rabta hospital, infectious diseases, tunis, tunisia guerrero c a actinomycosis is a chronic disease characterized by abcess formation, tissue fibrosis and draining sinuses. it is caused by anaerobic bacteria belonging to the genus actinomyces. thoracic actinomycosis may involve the lungs, pleura, mediastinum or chest wall. the authors present a case of pulmonary actinomycosis complicating a cervicofacial site. a -year-old man with a history of cervicofacial actinomycosis treated by penicillin g years ago was admitted because of right-sided chest pain for months before presentation, cough and fever. physical examination shows a painless indurated mass in the neck with multiple fistula of the sternum. chest radiograph and ct scan revealed a mass in the upper lobe of the right lung with an infiltrate of the upper lobe of the left one. magnetic resonance imaging confirms the previous lesions, with extending process to the sternum and right collar bone. bronchoscopy was performed while patient was on antimicrobial therapy. culture of bronchoalveolar lavage fluid was negative. transbronchial biopsy was not conclusive. fungal serologies were negative for aspergillosis, histoplasmosis, blastomycosis. bacterial examination of purulent drainage from sternal wound shows inclusion bodies identified as actinomyces. he was treated then with penicillin iv for months, than switched to doxycycline. after months of treatment, he is asymptomatic with radiological improvement. kanellopoulou m a , skarmoutsou n a , iglezos i b , mylona e a , martsoukou m a , apostolopoulou f b , papafrangas e a . a laboratory of clinical microbiology, sismanoglio general district hospital of attica, athens, greece , b nd department of pneumology, sismanoglio general district hospital of attica, athens, greece introduction: achromobacter xylosoxidans is a rare human pathogen. it is an important cause of bacteremia in patients with cardiac diseases, malignancies and immunosuppression. it has been recently recognized as an emerging microorganism in cystic fibrosis (cf), whose its pathogenic role is unknown. aim: to investigate the sensitivity to eleven different antibiotics of a. xylosoxidans strains isolated from adults with cf, during . methods: the susceptibility was tested by kirby bauer and microdilution methods (wider i, fransisco soria melguizo, s.a.), according to nccls recomendations. results: the resistance to antibiotics was as follows : gentamicin, tobramycin, aztreonam %, amikacin %, ceftazidime %, ticarcillin %, carbapenems, cotrimoxazole %, colistin % and piperacillin %. conclusions: ( ) a. xylosoxidans isolated from cf patients appeared resistant to the most usually tested antibacterial agents. ( ) colistin which is used as aerolized antibiotic for cf patients seems to be effective in the half of the isolated strains. ( ) piperacillin was the most active antibiotic against a. xylosoxidans . morris ka, perry jd, jain s, gould fk. microbiology department, freeman hospital, newcastle upon tyne, uk alafosfalin, l-alanyl-l- -aminoethylphosphonic acid, is an antibacterial peptide mimetic which inhibits peptidoglycan biosynthesis. we report the in-vitro activity of this compound in combination with ceftazidime, cefsulodin, fosfomycin, piperacillin/tazobactam, aztreonam, ciprofloxacin and timentin. drug combinations were evaluated against burkholderia cepacia strains, and pseudomonas aeruginosa strains isolated from patients with cystic fibrosis. for this purpose a chequerboard technique was adopted using doubling dilutions of each antibiotic incorporated into a highly defined agar medium free of antagonists. the minimum inhibitory concentrations (mics) and fractional inhibitory concentrations (fics) of all the antibiotic combinations were determined which revealed the antibiotic interaction occurring. alafosfalin in combination with ceftazidime, meropenem, piperacillin/tazobactam and timentin demonstrated the highest percentages of synergy in both b. cepacia and p. aeruginosa . synergy was shown to occur in , , and % of b. cepacia strains respectively, and in , , and % of p. aeruginosa strains. these four combinations were re-tested with all isolates and the results were shown to be reproducible. alafosfalin shows potential as a treatment for cystic fibrosis patients colonised with p. aeruginosa and/or b. cepacia , when applied in combination with these agents. community-acquired pneumonia */does its aetiology matter? ps lintmaer i, popescu a, popescu c. institute of infectious diseases matei bals, unit , bucharest, romania the aetiology of a pneumonia is not one of the criteria used to determine pneumonia's severity. however, it is accepted that identified based Á/based therapy is less expensive (and possibly more effective). objectives: our study aims were to: ( ) to evaluate the role of aetiology identification in pneumonia; ( ) to evaluate the first-line therapy in pneumonia. methods: we conducted a retrospective study in an infectious diseases hospital on patients with pneumonia. we excluded all the cases with nosocomial pneumonia. primary end-point was the day clinical failure (deaths, icu admission), secondary end-points were the average time of fever and length of stay and the antimicrobial regimen changes. results: causative agent identification rate was . %. the evolution was different for patients with identified aetiology compared with other patients in terms of: -day failures, length-of-stay and changes of the antimicrobial regimen. the patients with inadequate first-line therapy had a more severe course of illness with a greater rate of day clinical failure, longer fever and length-of-stay. conclusions: pneumonia's treatment was better for the patients with identified causative agent. that is why we should include aetiology among the pneumonia severity criteria, especially at an 'after -day therapy' re-evaluation. results: pneumomococcal aom was detected in children ( . %) and s.pn. was the only pathogen in . %. the resistance rates of the organism to antibiotics were as follows: penicillin . % (micb/ mg/ml; intermediately resistant . %, mic . mg/ml . %, and mic!/ mg/ml; highly resistant . %), erythromycin . %, clindamycin . %, cotrimoxazole . % and chloramphenicol . %. all isolates were uniformly susceptible to rifambicin and vancomycin. the large majority of pneumococcal isolates ( . %) had the mphenotype and the remaining strains ( . %) the constitutive mls phenotype. a various of serogroups were detected; the serogroup was the most predominant one ( . %), followed by serogroups ( . %), ( . %) and ( . %). the non-typable s.pn. strains compromised the . % of the strains. conclusions: high prevalence of resistance to penicillin, macrolides and cotrimoxazole in pneumococcal aom of childhood was recognized. a strategy for preventing aom caused by drug-resistant pneumococci is mandatory to start. material and methods: a total number of strains were examined. the sensitivity test was performed by kirby bauer, microdilution method (pasco, difco) according to nccls guidelines and by e -test. results: a percentage of . % of s. pneumoniae strains revealed high level resistance to penicillin (mic]/ mg/ml), while the % showed intermediate resistance (mic . Á/ mg/ml). the resistance to erythromycin and cotrimoxazole was . % (mic ]/ mg/ml) and . % (mic]/ / mg/ml), respectively. all strains were sensitive to cefotaxime (mic . mg/ml), vancomycin (mic / . mg/ml), meropenem (mic / . mg/ml) and levofloxacin (mic / mg/ml). conclusions: ( ) the prevalence of high resistance s. pneumoniae to penicillin seems to be low in examined strains ( . %). ( ) intermediate resistance to penicillin of s. pneumoniae isolates was high as expected ( %). ( ) most of the strains were sensitive to erythromycin ( . %) and cotrimozaxole ( . %). ( ) s. pneumoniae isolates were completely ( %) sensitive to levofloxacin, vancomycin and meropenem. beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy aim: of this study was a retrospective ( Á/ ) evaluation of the more effective and practical antibiotic treatment in ae-copd patients (pts) admitted to our unit. methods: before introducing any antimocrobial drug, sputum specimens were collected for microbiological purposes, while blood analysis, to monitor adverse systemic effects, were performed at the beginning and the end of treatment. antibiotic treatment ranged from to days according to four regimens: regimen a ( pts) /oral therapy only: . % with amc g b.i.d.; . % with cip mg b.i.d.; . % with dox mg u.i.d.; . % with lev mg u.i.d.; . % with cla mg b.i.d.; . % miscellaneous. regimen b ( pts) /sequential therapy (e.v. for days /oral): . % with amc g b.i.d.; . % with cla mg b.i.d. regimen c ( pts) /e.v. therapy only: same drugs. regimen d ( pts) /an association of two antibiotics. results: of evaluated pts, only ( . %) required a second regimen of treatment because of failure of the previous one: . % of regimen a; . % of regimen b; . % of regimen c, and % of regimen d. mild adverse effects were detected only in four pts. our results confirm that oral antibiotic treatment is practical, safe, and effective, and can be considered as the first line regimen also in hospitalized patients with ae-copd. becher g a , gillissen a a , rothe m b . a st. george medical center, robert-koch-hospital, leipzig, germany , b filt, lung and chest diagnostics ltd., berlin, germany patients with severe form of chronic obstructive pulmonary disease (copd) are prone by frequent exacerbations. bacterial infections are judged to cause at least half of exacerbations. haemophilus influenzae and streptococcus pneumoniae are the most frequent isolates, gramnegative bacilli account for the severe cases, aggravating the inflammatory process in the airways eventually leading to respiratory insufficiency. the aim of this ongoing placebo controlled, parallel group, mono center study trial is to evaluate beneficial effect of cefixim to reduce bacterial load and pulmonary inflammation in patients (n / ) with acute bacterial exacerbation of severe copd. thus, patients received in randomized fashion either cefixim ( mg/day) or placebo ( days). on days , , and breath condensate is collected using 'ecoscreen' (jaeger germany) for ltb -, il- -, nitrite-and ph-analysis. in parallel sputum gathered for detection of bacterial strains, and for ltb -, and il- quantification purposes. these data are compared to clinical outcome parameters such as lung function tests, radiographic findings, serum inflammatory markers and length of hospital stay. the preliminary data obtained confirm successful antibiotic therapy with oral cefixim in bacterial related acute exacerbations of copd is a useful approach to reduce bacterial load, and concomitantly lower inflammatory indices of the central and peripheral airways leading to clinical improvement of the patients. soriano garcia f a , fenoll a b , fernandez-roblas r a , coronel p c , gimeno m c , rodenas e c , garcia m a , granizo jj d . a fundacion jimenez diaz, microbiology, madrid, spain , b instituto de salud carlos iii, centro nacional microbiologia, majadahonda, spain , c tedec meiji farma, scientific, alcala de henares, spain , d fundacion jimenez diaz, epidemiology, madrid, spain purpose: to describe the susceptibility of streptococcus pneumoniae against cefditoren and other antimicrobials by serotype a multicenter study in south europe was carried out. a total of strains were collected between september and march from adult patients (more than y.o.) with respiratory tract infection (respiratory tract samples and blood cultures). all the isolates were sent to a central laboratory (fundació n jiménez díaz, madrid, spain) where susceptibility test was performed by broth microdilution (sensititre) following nccls recommendations. serotype was determined by quellung reaction and dot assay in carlos iii institute in strains. results: a total of strains ( . %) were not typable. the most prevalent serotypes were ( . %), ( . %), ( . %), ( . %), ( . %) and ( . %). two hundred and sixty-four strains were grouped in different serotypes. the proportion of susceptible strains by serotype to penicillin, erythromycin and levofloxacin were: serotype ( . , . , %); ( . , . , . %); ( . , . , . %); ( . , . , . %); ( . , . , . %); ( . , . , . %). the mic to cefditoren was / . (serotype ); . (serotype , and ) and mg/l (serotype and ). conclusions: the most prevalent serotype was . the susceptibility was higher in serotype than in serotypes and . community acquired pneumonia */a study among closed military community of young people ps martynova av, turkutyukov vb, vostrikova aa, andryukov bg. vladivostok state medical university, epidemiology, vladivostok, russian federation purpose: the etiology of pneumonia is still partly unknown. we should like to clear up an etiological role of respiratory pathogens in community-acquired pneumonia among youth. and we had chosen for it a model of a closed community both investigation of etiology of disease and for further investigation of mechanisms of transmission drug-resistant mechanisms. methods: we studied adults in age of Á/ from closed military collectives who presented to two public hospitals (one urban and one rural) with acute radiologically confirmed pneumonia during winter Á/ . we did blood and lung-aspirate cultures, mycobacterial cultures, serotype-specific pneumococcal antigen detection, and serology for viral and atypical agents. results: streptococcus pneumoniae is recognized as an important cause of community-acquired pneumonia, it probably accounts for % of cases of community-acquired pneumonia among youth. chlamydia pneumoniae and mycoplasma pneumoniae responsible for approximately % of cases. haemophilus influenzae caused . % sever cases of disease, % of all cases were due to moraxella catharralis . conclusion: pneumococcial infection accounted for % of the cases diagnosed. s. pneumoniae was the most common bacterial infective agent, with a low incidence of both m. pneumoniae and s. pneumoniae . other causative pathogens occurred only within groups of individuals with deficiency of immunological status. berezin en a , cardenuto md a , nobuko e b , guerra ml c , brandileone mc d . a santa casa, pediatrics, s. paulo, brazil , b santa casa, microbiology, s. paulo, brazil , c adolfo lutz, microbiology, s. paulo, brazil , d adolfo lutz, bacteriology, s. paulo, brazil to determine antimicrobial susceptibility of sp isolated from the upper respiratory tract, we collected np swab specimens from children, between months and years old. those children attended the outpatient clinic in s. paulo city, with diagnosis of bacterial infection requiring antibiotic therapy between march , to may , . penicillin susceptibility of isolates was determined by screening with oxacillin mcg disk and performing the minimal inhibitory concentration by the e -test. we performed also susceptibility test for amoxicillin and cefaclor. results: sp was recovered from np of children ( . %). the carriage of sp was more prevalent in children attending day care centers, children with young siblings at home, and children with tobacco users at home. the prevalence of penicillin non-susceptible strains was . % all of them with intermediate resistance. all the strains were susceptible to amoxicillin and . % were resistant to cefaclor. serotypes . b, f, n, , a and were the most common. these findings suggest that nasopharyngeal isolates of streptococcus pneumoniae from children with upper respiratory infections can be used to conduct surveillance for antimicrobial resistance in a defined geographic area. we were able to conclude also that penicillin intermediate resistent strains can be susceptible to amoxicillin. hinojosa rm a , saenz a a , collazo m a , echaniz g b . a universidad autonoma de nuevo leon, infectologia, monterrey, mexico , b instituto nacional de salud publica, epidemiologia, cuernavaca, mexico the emergence of penicillin-and multidrug-resistant pneumococcal strains has become a global concern, necessitating the identification of the epidemiological spread of such strains. material: ninety streptococcus pneumoniae clinical isolates were collected from march through march . typing was done by the capsular reaction with pooled, type, or group antisera. susceptibility testing to antimicrobials was done by the e -test and the disk diffusion method. results: only ( %) s. pneumoniae strains were classified by serotyping; the most frequent types were a/b, f, v, f and . the oxacillin screening test detected . % penicillin-resistant s. pneumoniae strains isolated from children and . % from adults. the susceptibility percentage of s. pneumoniae to ceftriaxone was % in both children and adults. s. pneumoniae isolates from children exhibit a susceptibility of % to azithromycin, while in adults % of the isolates were susceptible. for the rest of the antimicrobial agents, the susceptibility ranged from to %. s. pneumoniae had a lower susceptibility to ceftazidime and ciprofloxacin. conclusions: ceftriaxone and azithromycin had a good in-vitro activity against s. pneumoniae strains. but the percentage of penicillinresistant s. pneumoniae detected in this study is alarming, therefore we conclude that a continuous surveillance system is necessary in mexico. vertkine al, prokhorovitch ea, alexanyan la. a retrospective analyses of cases of ambulant pneumonia with fatal outcome was made. among the patients who died from ambulant pneumonia the prevailing age was over ( . %) and the prevailing sex was male ( . %). . % had pneumonia accompanied with some pathology: chronic lung disease ( . %), alcoholism ( . %), diabetes mellitus ( . %). . % of the patients had a big volume of lungs lesion */ . % of the patients suffered from bilobular pneumonia and . % */from trilobate pneumonia. in . % of the cases pneumonia was complicated with abscess formation and/or exudative pleurisy. we studied the antibiotics therapy used for the patients treatment. the change of antibiotics was made only in cases ( . %) whereas in the other cases no change of preparations was made though the signs of the therapy non-effectiveness were obvious. thus, the rational antibiotics therapy with the timely change of non-effective antibacterial drug is significantly important. while choosing the antibiotics, the patient's age, the accompanying diseases, the volume of the lungs lesion and complications which define pneumonia seriousness are to be taken into consideration. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to describe the management of lower respiratory tract infections (lrti) in healthy adults, by general practitioners (gp). methods: a questionnaire was sent to a representative national sample of gps. this questionnaire assessed their perception and management of lrti, the indication for antibiotics (ab) in a case of lrti in a healthy adult with no focal signs and no signs of severity, knowledge of the micro-organisms responsible for acute bronchitis and knowledge of the afssaps (french agency for the safety of health products) recommendations. results: three thousand seven hundred and thirty-eight gps, who reported seeing an average of patients per week, including . / . patients with lrti, returned the questionnaire. the main results are presented in the following for the majority of gps, the main objective of the visit is to determine the indication for antibiotics. according to gps, alp and whooping cough are rare, while atypical pneumonia is frequent. gps also declare that the diagnosis of alp is often easy right from the first visit, in contrast with that of atypical pneumonia. complementary investigations are not often requested. gps consider that they often delay prescription of antibiotics ( %) and declare that they tend to prescribe a macrolide as first-line treatment. finally, gps have a poor knowledge concerning the micro-organisms responsible for acute bronchitis and the majority of gps declare to be familiar with afssaps recommendations. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to study the management of one case of lower respiratory tract infection (lrti) in adults by general practitioners (gps). methods: prospective study conducted on a representative national sample of gps. each gp had to include the first healthy adult patient seen during the -week data collection period, either on a home visit or in the office for recent cough and acute fever !/ . c. clinical data, the diagnostic perception and the therapeutic approach to the patient were collected by means of a standardised questionnaire, distributed by abbott laboratories. results: three thousand seven hundred and thirty-eight general practitioners included patients. conclusions: during lrti in adults, gps observe few focal signs, confirming the marked predominance of bronchitis compared to pneumonia. in view of the frequency of the signs, the diagnosis of pneumonia appears to be overestimated. one-third of clinical situations were diagnosed as 'bacterial superinfection of acute bronchitis', despite it is not a recognised diagnostic entity. antibiotic prescription was immediate in % of cases and delayed in % of cases. this last point shows that clinical practice differs from the gp's perception of their described prescribing practice shown in a simultaneous survey ( % of gps declared that they prescribed antibiotics immediately, while % delayed this prescription). results: thirty-three patients ( men, mean age b years, mean apache ii score . &bdqup;b . ) during the years Á/ were prospectively studied. thirteen patients ( %) had no identifiable risk factor for severe cap. an etiologic factor was revealed in patients ( %). in of them this was achieved with noninvasive methods. psb cultures were taken from eight patients and were positive in only . the offending organisms included: streptococcus pneumoniae in six cases, gnb as the sole pathogen in six cases, haemophilus influenzae (with s. pneumoniae or klebsialla pneumoniae ) in two cases, s. aureus in two and legionella pneumophila in one patient. initial antibiotic regimen a combination of marcodile '/ rd gen cephalosporin /aminoglycoside was successful in patients who all survived and had to be changed empirically or according to culture results in patients who had a mortality of %. the overall mortality rate was %. the identification of the causative factor did not seem to have any impact on survival. conclusion: severe cap in our icu was most often caused by s. pneumoniae and gbn. the high mortality of this entity seems to be influenced by the immediate use of the appropriate antibiotic combination and not by the identification of the causative organism. this underscores the need for knowledge of topical microbiology which helps in designing an effective empirical initial antibiotic regimen. mily mn a , golubev sa b , lugovoy vy a , voronov gg b . a vitebsk emergency hospital, pharmacotherapy unit, vitebsk, belarus , b basic and clinical pharmacology department, vitebsk state medical university, vitebsk, belarus purpose: the study aims were to assess the spectrum and predictors of the antibiotic use during pneumonia management in a regional emergency hospital in belarus. patients, treatment and physicians characteristics of cases ( Á/ ) were collected and possible associations were examined with using defined daily doses methodology (ddd) and american thoracic society (ats) guidelines. results: the mean treatment duration was . / . days, the total antibiotic ddd/ bed-days was . . the ddd/ bed-days of the most used antibiotics were: penicillins . ; aminoglycosides . ; macrolides . ; cephalosporins . ; tetracyclines . . in manova certain ats categories were associated with ddd/day, but not with frequency of definite antibiotic use. ddd/day was higher in case of multilobar infiltrates ( conclusion: our study indicated the low rate of macrolides and cephalosporins using, and the high one of aminoglycosides. antibiotic prescriptions were associated with disease severity and physician personality rather then with empirical choice rules recommended. acute exacerbation of copd: most frequent infecting agents and their suspectability to the different types of penicillins. analysis of medical documentation ps pertseva to, bogatska ke, gashynova ky. dsma, internal medicine , dniepropetrovsk, ukraine number of copd cases has been increased in ukraine. treatment of acute exacerbation (ae) of copd is not always successful because of inadequate antibiotic therapy. the aim of study was to reveal most frequent infecting agents and their susceptibility to the different types of penicillins in patients with ae of copd. medical documentation of patients with ae of copd (type i) was studied. data of sputum analysis and susceptibility of isolated agents to penicillin, ampicillin, oxacillin and amoxicillin/clavulone acid were evaluated. there were patients with haemophilus influencae/parainfluencae ( . %), klebsiella pneumoniae ( %), staphylococcus aureus ( . %), pseudomonas fluorescens , pseudomonas putida , serratia marcescens , serratia liquefaciens ( . % each), streptococcus agalactiae , acinetobacter baumanii ( . % each) in samples of sputum. in . % cases there was mixt infection. . % had no any bacterial agents. only % of agents were susceptible to penicillin, % */to ampicillin, % */to oxacillin. however, % of microorganisms were susceptible to amoxicillin combined with clavulone acid. this study has shown that most frequent infecting agents caused ae of copd were gram-negative microorganisms and s. aureus . according to antibiogram the prescription of amoxicillin/clavulone acid is most expedient in this case. pertseva to, bogatska ke, konopkina li, kireeva tv, gashynova ky. dsma, internal medicine department, dniepropetrovsk, ukraine we examined patients ( men, mean age . / . years) with acute exacerbation of cob (type i). the most frequently isolated agents were haemophilus influenzae and parainfluenzae */eight patients, gram-negative rods in , staphylococcus aureus in two and mixed in six and one patient had no bacterial agents isolated in their sputum. high susceptibility to azithromycin was found in all cases of gram-positive agents and in h. influenzae and parainfluenzae . other gram-negative agents were resistant to this drug in vitro. however, treatment with mg/day during days was clinically effective in . % of cases. only . % of patients had a further acute exacerbation of cob. there were peculiarities of the infecting agents causing acute exacerbation of cob in this study: klebsiella pneumoniae and s. aureus were found more frequently than in other studies. high efficacy of surnamed in the treatment of acute exacerbation of cob was established in . %. . % had partial positive clinical effect after this therapy. there were no patients with adverse events. efficacy and tolerance of amoxicillin mg/kg bid versus amoxicillin mg/kg tid in the treatment of acute otitis media (aom) in children / years ps borek m a , guggenbichler jp b . a biochemie gmbh, international medical department, kundl, austria , b department of pediatrics, university of erlangen, erlangen, germany five hundred and sixteen patients (mean age / . years) with clinical and otoscopic diagnosis of aom were included in a randomized, double blind, multicentre study, and were treated days either with amox mg/kg bid or amox mg/kg tid. assessments were made during therapy (day Á/ ), after end of therapy (eot, day Á/ ) and follow up (fu, day Á/ ). the primary efficacy endpoint was the clinical response at eot defined as success (cure/improvement) or failure. both regimens were well tolerated; one or more drug-related adverse events (aes) were reported in . % ( / ) of bid patients and in . % ( / ) of tid patients. the most frequently reported drugrelated aes in each group were gastrointestinal symptoms (bid . % vs. tid . %), which were mainly of mild or moderate severity. both regimens were clinically equivalent. the higher cure rates in the bid group suggest a possible higher benefit from bid therapy in children / years. children attending family day-care (fdc) should be less exposed to upper respiratory tract infections than those in group day-care (gdc) and therefore to antibiotic treatment; fewer should thus carry resistant bacteria. to test this hypothesis, np carriage of sp and hi with reduced susceptibility to penicillin (pdsp and hi bl'/, respectively) was investigated among children in fdc (maximum three children) and in gdc ( Á/ children) in the alpes maritimes (france) between november and march . a two stage cluster sample of children attending gdc or fdc was selected. np samples were cultured for sp and hi. penicillin susceptibility was tested by disk diffusion and e -test, and b-lactamase production by api-nh † tests (biomerieux, lyon). two hundred and thirty-five children in fdc and in gdc aged Á/ months were sampled. age and sex distribution were similar in both groups. sp was isolated in children in fdc ( %), and in ( . %) children in gdc (p b/ Á/ ). proportions of pdsp were . and . %, respectively (p / . ). hi was present in . % of children in gdc vs. . % in fdc (p b/ . ). proportions of hi bl'/ were . % vs. . %, respectively (p / . ). antibiotic exposure during the previous months concerned . % of children in gdc vs. . % in fdc (p / . ). there was no correlation between antibiotic use and carriage of pdsp or hi b'/ strains. sp and hi carriage rates are significantly lower among children in fdc than in gdc. advising alternative types of daycare for children attending gdc should reduce exposure and thus limit the spread of resistant bacteria. however, the proportion of pdsp and hi bl'/ is similar in both groups and comparable patterns of antibiotic use are observed. continued efforts must concentrate on parental education and enforcement of recommendations for management of pediatric upper respiratory tract infections. during a period between january and august , invasive haemophilus influenzae (hi) isolates had been collected at the children's hospital of tunis. we used haemophilus test medium to test antibiotic susceptibility. the mic of beta-lactams was measured by e -test. beta-lactamase production was determined by using the cefinase test and biotyping by apinh. presence of capsular antigen was determined by using hi typing anti sera. hi strains were isolated from meningitidis ( ), bacteremia ( ) and arthritis ( ). all strains were serotype b and . % of them belonged to biotypes i and ii. amoxicillin resistance with beta-lactamase producing mechanism occured in . %. mic of beta-lactamase producing strains was vs . mg/l in non-producing one. there is no betalactamasenegative amoxicillin resistant among these invasive isolates. antibiotic resistance concerned chloramphenicol: . %, trimethoprim-sulfamethoxazole: . %, tetracycline: . % and kanamycin: . %. invasive hi infections in tunisian children's were always associated with type b strains. introduction of a hib vaccine programme in tunisia is recommended. the aim of this study was to analyse the clinical picture and treatment of neurological manifestations of neuroborreliosis in children. the study included children ( Á/ years) with neuroborreliosis diagnosed on the basis of the clinical and serologic criteria. symptoms of facial palsy occurred in six children symptoms of iii Á/vi cranial nerves palsy in three children, meningitis in four and paresthesias in three. symptoms of v or viii nerve palsy, mental disturbances, radiculoneuritis or cerebellitis were found in singular cases. all children received ceftriaxone intravenously Á/ weeks. total recovery was obtained in children following the first course of therapy. recovery following the second course of therapy (amoxicillin) occurred in one child with mental disorders and one with vi nerve palsy. improvement was achieved after the second course in the patient with radiculitis, however, muscular atrophy persisted. irreversible, unilateral deafness was found in a child with viii nerve palsy in spite of three courses of therapy applied. infection with borrelia burgdorferi in children causes a wide spectrum of neurological manifestations. facial palsy was the most common sign in our study. applying ceftriaxone in the treatment of neuroborreliosis is characterised by a good effectiveness. double infection by c. pneumoniae and m. pneumoniae as a cause of cystic changes in the lungs ps streharova a, moravcikova d. department of infectious diseases, university of trnava, trnava, slovakia chlamydia pneumoniae and mycoplasma pneumoniae are human respiratory pathogens manifested in early childhood. immunological disbalance could trigger autoaggressive diseases. the authors describe the case of a -year-old girl with development of multiorgan failure and septic state, which followed multiple cystic changes in the lungs. the girl did not have a diagnosis of cystic fibrosis. the authors consider that cystic changes are a consequence of double infection by c. and m. pneumoniae. dzarlieva m, momeva l, temelkovska g, balevska p, pejkovska m. medical centre, neonatology, bitola, the former yugoslav republic, macedonia unicef skopje has supported a nationwide safe motherhood needs assessment in representative samples of hospitals. eighteen of maternity wards and facilities renovated and certified as 'babyfriendly'. all mature newborns with successful adaptation to extra uterine life and satisfactory vital parameters are h during the day with their mothers at rooming-in system. aim: with rooming-in system we reached decreasing of incidence of neonatal infections. material and methods: history records of newborns from our department. for the period of months, neonates have been borne. with suspection of infection there */ babies ( . %). newborns borne through meconium stained liquid */ ( . %). results: microbiological findings: from blood culture */staphylococcus coagulaza negative from swabs */staphylococcus aureus , escherichia coli , staphylococcus epidermidis. conclusion: in , from all babies who had risk factors for infection in newborns ( %) we had positive findings and in year (before rooming-in sistem), in ( . %). after that period (with rooming-in) newborns ( . %). with rooming-in system we reached decreasing in incidence of neonatal infections by breaking the chain of infection */only mothers take care of their babies with help of the staff. newborns are in their micro environment, the same they will have at their home. with this practice we have also reduction of nosocomial infections. a study on antibiotic susceptibility and resistance factor transmissibility among antibiotic resistant salmonellae isolated from children affected to diarrhea ps sharifzadeh a. azad university of shahrekord, microbiology, shahrekord, islamic republic of iran in spite of happened drug resistance, antibacterial therapy still is the best route of treatment of salmonellosis in man and animals. in order to detection of dominants serotypes of salmonellae in children and detection of antibiotic susceptibility and r-factor transmissibility among those isolated salmonellae. this study was conducted on diarrheic stool samples were collected from children affected by diarrhea in ayatollah kashany hospital of shahrekord, during spring of to autumn of . after isolation and identification of salmonellae, seven serotypes were detected. one of those was s. typhi and another six serotypes were s. paratyphi b . in order to detection of antibiotic different antibiotic disks were used in disk diffusion method. best results were taken from ceftizoxim, cephtriaxon, cephazolin and chloramphenichol. the r-factor were transferred from isolated salmonellae to escherichia coli k in all of cases of resistance to penicillin and ampicillin. pharyngeal colonization by streptococcus pneumoniae and group a bhs were evaluated in randomly selected school children aged Á/ years in riyadh, saudi arabia. fifty-six children ( . %) had positive culture for either organisms of the isolates from school children, ( %) were s. pneumoniae , of them ( %) were penicillin-sensitive, three ( %) were penicillin-resistant, and two ( . %) were resistant to two antimicrobials. forty isolates of bhs ( %) were group a bhs. all isolates were penicillin and erythromycin sensitive. the carrier rate among school children for penicillin-resistance s. pneumoniae and resistance to two antimicrobials were ( . %) and ( %), respectively. the carrier rate of group a bhs was ( . %). riyadh has a low rate of antibiotic-resistant s. pneumoniae and a similar rate of group a bhs carriers among school children as that seen in temperate areas. boukadida j a , boukadida n b , hannechi n a , said h b , erraii s a , elmhabrech h a . a chu f. hached, laboratoire de microbiologie, sousse, tunisia , b c s base, sousse, tunisia the acute pharyngitis is a very frequent pathology in which group a streptococcus is the most incriminated bacteria. however, other non a b-haemolytic streptococcus (sbna) could be responsible. the aim of this work is to determine the part of each non a b hemolytic streptococci (sbna) in acute pharyngitis and the related antibiotics susceptibility pattern. the study was realized in sousse-tunisia (north africa) during months from may . the origin materials of isolates are throat swab (transystem venturi, copan, bovezzo). the mean age of patients is years with extremes Á/ years. the samples are cultured on blood agar plates in a delay of h maximum. identification was done to samples that have over than b-hemolytic colonies, groupage with pastorex strep. sanofi pasteur france, susceptibility pattern according to nccls norms, mic is determined by e -test. the control strain is s. pneumoniae atcc . twentyone clinical isolates of sbna are distinguished from clinical isolates of b-hemolytic streptococci recovered from patients with acute pharyngitis without symptoms of viruses' infections (tearing, corysa, sneeze). all b-hemolytic streptococcus represents . % of all collected samples. sbna were . % of the isolates. sbna were strains group g streptococci, seven strains group c streptococci and three strains group f streptococci. susceptibility pattern of each sbna to antimicrobial agents is as follow: group g streptococci: peni g / %, amoxicillin / %, pristinamycin %, clindamycin and erythromycin / . %, tetracyclin / . %, telithromycin . % and levofloxacin / . %. group c streptococci: peni g / %, amoxicillin / %, pristinamycin %, clindamycin / . %, erythromycin / . %, tetracyclin / . %, tel-ithromycin / . %, levofloxacin / %. group f streptococci-peni g / %, amoxicillin / %, clindamycin / . %, erythro / . %, tetracyclin / . %, telithromycin / %, levofloxacin / . %. all sbna have mic to penicillin g under . mg/l. according to available data, penicillin g and amoxicillin still the reference treatment of acute bacterial pharyngitis in spite of the new antibiotics introduction. berezin en a , quevedo sg b , nicolla l b , viegas d c , eizenberg b d , pedrosa f b , santos ag a . a santa casa, pediatrics, s. paulo, brazil , b elly lilly, scientific, s. paulo, brazil , c fac. abc, pediatrics, santo andre, brazil , d university hospital, pediatrics, s. paulo, brazil a multicenter, open label, prospective, randomized trial in which patients Á/ years of age with proven gabhs pharyngitis were randomized to receive either -day course of the broad spectrum oral cephalosporin, cefaclor or a -day course of amoxicillin. patients were included if they have signs and symptoms of streptococcal tonsillopharyngitis and a rapid streptococcal rapid test positive . patients were evaluated at days Á/ , Á/ and Á/ posttherapy. pharyngeal cultures were conducted at baseline and at follow-up visit ( Á/ days). we considered for bacteriologic eradication analysis only patients with positive culture to gabhs. there were patients with a rapid streptococcal rapid test. clinical success were achieved in around % of the patients. for evaluate eradication of the initial pathogen we considered patients of the amoxil arm and patients of the cefaclor arm. thirty-four of patients of the amoxil arm ( . %) and of ( %) patients of the cefaclor arm were considered bacteriological cured at the second culture performed at day Á/ . ten days of a penicillin or amoxicillin therapy may not be the best therapeutic choice for all pediatric patients. in developing countries where rheumatic fever is still an important problem to evaluate the bacterial eradication achieved with the different antibiotics may be important. prophylactic affect of saccharomyces boulardii for antibiotic-associated diarrhea in a paediatric age group ps erdeve o, tiras u, camurdan mo, tanyer g, dallar y. ankara education and research hospital, pediatrics, ankara, turkey the process resulting in antibiotic associated diarrhea is the alteration of enteric flora due to bacteria. saccharomyces boulardii is a yeast, isolated from cover of a kind of hazelnut and its usage became widespread recently. there is no enough study about s. boulardii activity at pediatric ages. we aimed to define s. boulardii activity on azithromycin and sulbactam-ampiciline, and associated diarrhea at pediatric age group. the . % of cases only with antibiotic usage developed diarrhea, whereas rate was . % for probiotic using group (p b/ . ). all of the clostridium difficile toxin a defined cases were from sulbactam-ampiciline using group. the rate of diarrhea for sulbactam-ampiciline using group was . %, while it was . % for group used s. boulardii as probiotic beside sulbactam-ampiciline (p b/ . ). it was observed that probiotic usage decreases diarrhea rate four times (p b/ . ). when age groups considered, the rate of sulbactamampiciline associated diarrhea increased at Á/ ages and s. boulardii effect on preventing diarrhea was significant at Á/ ages (p b/ . ). antibiotic associated diarrhea is a common clinical problem at pediatric age group. s. boulardii is a hope giving probiotic especially for sulbactam-ampiciline associated diarrhea. grey e a bilikova e a , hafed bm a , kovacicova g a , chovancova d b , huttova m b , krcmery v a . a school of health, trnava university, trnava, slovakia , b postgraduate academy of medicine, neonatal clinic, bratislava, slovakia the purpose of the study: the aim of the study was to find out, whether artificial abortion of a mother has impact on neonatal infection of her future baby. the results obtained: therefore, we compared neonates with infection, who were born to mothers with past history of abortion ( Á/ artificial abortions within years), with the babies of mothers, who have not experienced abortion before. control group consisted of neonates hospitalized at the same clinic, at the same time period. according to the analysis of risk factors for neonatal infections, it was found, that prematurity ( Á/ weeks of gestation) ( . vs. . %, p . ) and low birth weight- Á/ g ( . vs. . %, p . ) were significantly more frequently observed in babies of mothers with past history of abortion. drug abuse (heroin) ( . vs. . %, p . ) and nicotine use ( . vs. . %, p . ), were more significantly related to neonatal infections in babies of mothers with past history of abortion. etiological analysis showed that only candida albicans ( . vs. %, p . ) was significantly related to neonatal infections in mothers with past history of abortion. the conclusion reached: in conclusion, artificial abortion has not only direct impact to the health of the mother, but also on her next pregnancy. bacteraemia and patient mortality in a peadiatric intensive care unit ps armenian sh, singh j, arrieta ac. children's hospital of orange county, infectious disease, orange, usa purpose: this study will identify the factors that significantly contribute to mortality in patients with bloodstream infections (bsi) at a pediatric intensive care unit (picu). results: medical records of patients who were admitted to the picu and had a documented bsi were reviewed. there were separate episodes of bsi's, with nine patients having multiple bsi's during their hospital stay. a casecontrol model was used. cases were bsi's with eventual mortality (n / ; . %) and controls were those who survived bsi (n / ; . %). patients who died were older ( . vs. . years; pb/ . ), more likely to have a nosocomial bsi ( . vs. . %; pb/ . ), longer hospitalization prior to bsi ( . vs. . days; pb/ . ), and have a polymicrobial bsi ( . vs. . %; pb/ . ). infection related mortality (irm)-defined as death within days of bsi */was significantly higher in those receiving inadequate antibiotic treatment at the time of diagnosis of bsi ( . vs. . %; p b/ . ), as well as in those with gram negative bacteremia and/or fungemia ( vs. %; p b/ . ). logistic regression was used to adjust for potential confounding variables. conclusions: we found that being older, multiple organisms, and a longer hospitalization prior to the bsi were significantly associated with overall patient mortality. irm was significantly higher for those with inadequate initial antibiotic coverage and in those with gram negative bacteremia and/or fungemia. somer a a , gü r d a , diri s a , yalcýn i a , salman n a , ongen b b , gü rler n b . a department of pediatric infectious diseases, istanbul university istanbul medical faculty, istanbul, turkey , b department of microbiology and clinical microbiology, istanbul university istanbul medical faculty, istanbul, turkey teicoplanin is a glycopeptide antibiotic active against a broad range of gram-positive pathogens including methicillin-resistant staphylococci and offers the advantages of once daily administration, choice of administration route, lack of requirement for routine therapeutic drug monitoring and lower propensity to cause nephrotoxiticy and anaphylactoid-like reactions. in this study the efficacy and safety of teicoplanin were evaluated retrospectively in children with serious bacterial infection. sixty-three children ( girls, boys) aged between month and years were treated with teicoplanin (three loading dosages of mg/kg at h intervals, followed by a maintenance dosage of mg/kg/day). the infections treated were pleural empyema (n / ), joint and bone infections (n / ), septicemia (n / ), skin and soft tissue infections (n / ), and lung abscess (n / ). the pathogens isolated were staphylococcus aureus (n / , of which were methicillin resistant), coagulase-negative staphylococci (n / ), s. pneumoniae (n / ), and group a hemolytic streptococci (n / ). the duration of therapy ranged from to days (median days). clinical success (cure plus improvement) was achieved in . % of cases. no side effects attributable to teicoplanin therapy were encountered. teicoplanin appears to be an effective and well-tolerated treatment for serious gram-positive infections in children. the risk of infection is present in all children with acute leukemia. two hundred and sixty episodes of febrile neutropenia were analyzed in children (aged . Á/ years) with aml */ cases and all */ cases over years during cytostatic therapy (protocols: mbfm */ aml and mbfm */ all). a degree of fn occurred in % of cases in aml, in % */in all. the sites of infection were: blood'/ central venous catheter ( %) and respiratory tract ( %). pathogens isolated from blood were: gram-positive */cns */ %, streptococcus spp. */ . %, gram-negative */enterobacteria */ %, pseudomonas aeruginosa */ %, candida spp. */ . %, aspergillus spp. */ . %, other */ . %. for the treatment of fn we used empirical antibiotics regimens. clinical response was noticed: in st line of therapy */ cephalosporins Á/ generation used */ %, carbapenems used */ % as nd Á/ rd lines */vancomycin */ %, amphotericin b */ %. thirtytwo percent of children with aml and . % children with all died because of sepsis. conclusion: carbapenems are more active in the st line of antibiotics therapy both for patients with aml and all. vancomycin is useful in the nd line for patients with all. amphotericin b and vancomycin are useful in combination in the nd line for patients with aml. pavlenishvili ivp. medical academy, pediatrics, tbilisi, georgia our drug of choice in cases of complicated neonatal sepsis i. pavlenishvili, g, iobashvili tbilisi, medical academy georgian society of pediatrics chemotherapy (gspc) with collaborations of drug and therapeutic committee (dtc) of childers hospital 'republic' finished the observation study about nosocomial infections of neonatal icu in above mentioned hospital. study begins from november . during this period we observe cases of neonatal sepsis, most of them were due gram-negative bacteria. thirty-four cases of neonatal sepsis were cause different stains of escherichia coli , proteus spp. and acinetobacter . we notice that during last years the role of acinetobacter in etiology of neonatal sepsis and nosocomial complications of neonatal sepsis in our hospital is gradually increased. as our observation reveals most optimal in the case of complicated neonatal sepsis was (according criteria of dts) use of ceftasidime. almost in all cases we used ceftasidime with success. we have only one case of mortality. since the macrolide resistance (mr) in streptococcus pyogenes has been increasing in europe, we studied the incidence and genetic basis of mr in s. pyogenes in russia. s. pyogenes isolated at baseline and during follow-up visits from children with acute pharyngitis receiving penicillin or midecamycin ( -membered macrolide) were studied. mr was evaluated by agar dilution (nccls), resistance mechanisms */by pcr. a total of s. pyogenes were obtained at the baseline, ( %) of which were erythromycin-resistant: strains ( %) were erm (a)-positive, one */mef (a)-positive, and one was negative for all primers used. all erm (a)-strains were inducibly resistant to clindamycin and represented seven pfge profiles with one profile found in / strains. in three midecamycin treated patients mr was selected during the therapy (one strain had erm (a), one */mef (a), one */ unknown resistance determinant). mef (a)-strain obtained during follow-up visit had different pfge pattern with mef (a) strain isolated at baseline, while both the baseline and follow-up strains with unknown mechanism of resistance had unique pfge pattern. these data showed moderate incidence of erythromycin-resistant s. pyogenes in smolensk. ribosomal methylation (erm (a)) was the most common mechanism and though the polyclonal nature of mr was established, most erm (a)-strains belonged to only a few clones. bacillus cereus infections in traumatology-orthopaedy department: retrospective study and re-evaluation of healthcare practices ps bacillus cereus was cultured for patients from traumatology department who had developed postoperative wound infections between august and march . all patients presented inferior members open fractures, frequently contaminated with telluric material and requiring external fixators. genomic study of clinical isolates by pulsed field gel electrophoresis and random analysis polymorphic dna, allowed us to eliminate an outbreak. furthermore, the reduced delay in which patients developed the infection ( days'//- ) led us to re-evaluate the procotols used in our institution. indeed, all patients had received amoxycillin '/ clavulanate iv g for antibioprophylaxis during anesthetical induction then relayed per os for hours. because of the production of a potent beta-lactamase by the bacteria, this association could not be efficient. furthermore, accordingly to the afnor en norm, we have tested clinical isolates' sensitivity to the principal antiseptics used for antisepsis and disinfection (iodophors, chloride derivated and biguanidines) and observed a major resistance of all strains tested. even if these postoperative wound infections are considered as nosocomial because of the delay in appearance, we actually think that bacillus cereus were initially present in telluric material. this fact led us to propose a systematic screening for bacillus at admission for this type of wound and to administrate quinolones such as ciprofloxacin for prophylaxis. internal thiols and reactive oxygen species in the candidacidal activity exerted by a n-terminal peptide of human lactoferrin ps the purpose of the study: the emergence of candida albicans strains resistant to current antifungals points to the need for new antifungal agents, e. g. antimicrobial peptides. the results obtained : we report that hlf( - ), a synthetic nterminal peptide of human lactoferrin, displays excellent killing effect against fluconazole-resistant c. albicans and that sub-optimal concentrations of this peptide combined with fluconazole act synergistically. previous investigations revealed that hlf( - ) required an energized mitochondrion, atp release by candida , and ligation of atp receptors for its killing effect. we now report that reactive oxygen species (ros) are involved in the killing effect of hlf( - ). since internal thiols protect cells from oxidative damage, our observation that hlf( - ) caused a % reduction of internal thiols in candida is of interest. as expected, n-acetyl-l-cysteine (nac), which is a precursor of glutathione and a ros scavenger, inhibited the killing effect of hlf( - ). diamide, which oxidizes internal thiols, was candidacidal and hlf( - ) and diamide acted synergistically in killing c. albicans and ros production. moreover, the hlf( - )-induced activation of mitochondria was inhibited by nac, indicating that internal thiols/ros affect mitochondrial activity. the conclusion reached : the candidacidal activity of hlf( - ) involves ros production and reduction of internal thiols. objective : an audit was conducted to explore the observation of increasing resistance to ciprofloxacin in enterobacteriacea isolated from specimens from such patients in the hematology unit. results : enterobacteriacea isolates in specimens from such patients processed over the years to were examined. number of specimens per year was */ , , , and respectively and the annual percent ciprofloxacin resistant enterobacteriacea from these was %, %, %, % and %. conclusion : conclusion drawn from the audit was that rapid rise in ciprofloxacin resistance may possibly be attributed to the use of this fluoroquinolone as a single agent in dose of / mg [cut off patient weight kg] twice a day in neutropenic patients till neutrophils exceed /c.mm. subsequent to the audit, prophylaxis protocol was modified to use of oral colistin -mega units/twice a day in combination with ciprofloxacin / mg twice a day. a prospective audit is proposed to test the benefits of this combination. rhinocerebral zygomycosis: diagnostic dilemma for emergency physician: can the associated morbidity and mortality in this rare but deadly disease be reduced? ps samavedam s, guleri as. western infirmary, clinical microbiology, glasgow, united kingdom rhinocerebral zygomycosis [rcz] is a rare, invasive, rapidly progressive opportunistic infection caused by ubiquitous fungi of the order mucorales. it usually occurs in diabetics or immunocompromised patients. the emergency physician will typically see patients with rcz in its earliest stages masquerading as a variety of other less serious diseases. the key markers like necrotic patch on hard palate, nasal septum or turbinate, marked facial pain, and cellulites with marked eye and neurological signs may present late in disease. we report a case of rcz caused by rhizopus arrhizus [oryzae] in an year old woman with poorly controlled diabetes. she presented with a right-sided facial droop of short origin and being generally unwell. ct scan was non-conclusive and delayed presentation of key markers of rcz permitted disease to rapidly progress. despite an intensive antifungal therapy with ambisome and insulin sliding scale, patient rapidly succumbed within days. fine needle aspiration cytology is less invasive, easier and equally effective alternative to pre op biopsy. the key to successful reduction in morbidity and mortality associated with this rapidly fatal disease is -increasing awareness of the disease, an early diagnosis, correction of underlying metabolic derangement, prompt intensive antifungal therapy with amphotericin b and radical surgical debridement of the necrotic tissue. an 'optimal dosage' of ambisome requires discussion. clinical audit in the haematology ward of a tertiary care hospital: study of degree of correlation between bacteraemia and oro-pharyngeal screens in immunocompromised patients over five years and role of antibiotic prophylaxis ps guleri as, butcher i. western infirmary, clinical microbiology, glasgow, united kingdom introduction : a clinical audit was carried out over -years [ ] [ ] [ ] [ ] [ ] in the immunocompromised patients including neutropenic patients and bone marrow [autograft] transplant recipients in hematology ward of gartnaval general hospital, glasgow, a tertiary care center. objective : it was aimed to establish the degree of correlation between bacterial isolates in oro-pharyngeal screen during bacteraemia episodes and role of antibiotic prophylaxis. methods purpose : to assess whether antiretroviral therapy (art) intensification, gm-csf use and remune initiation before stopping art lead to viremia containment, and long periods off art. methods : ten adults with chronic hiv disease, hiv- rna levels (vl) b/ . log copies/ml and median cd '/ -t cell count of /ml were enrolled. after art intensification with ddi ( months) '/ hydroxyurea [hu]( mo.) '/ gm-csf ( mo.) and a remune dose, art was stopped but remune continued. art was resumed if rebound vl did not decrease to b/ . log in months or if cd '/ counts decreased to b/ . results : vl rebounded in all patients after stopping art, and developed an acute retroviral syndrome (ars). cd '/-t cells decreased, and cd '/ '/ increased ( -fold). after a median stoppage of weeks, art was resumed in patients and vl decreased to b/ . log, cd '/ counts were regained, il- and il- levels rose. at the nd interruption, / patients had a rebound, and / had a nd ars, but peak vl and loss of cd '/ were lower (p / . ). after . weeks off art, patients resumed therapy. the breadth and magnitude of hiv-specific activity increased and thymus size grew. the patients were off art for a median of . out of weeks. two of them are off art for and weeks respectively ( vlb/ log). conclusions : this approach led to a high ars incidence, long periods off art, increases in hiv-specific responses, il- and il- levels, and thymus size. urinary tract infection in hospitalized population is a significant problem. this is a study of catherized patients urinary infection in corfu hospital. in Á/ , urine cultures were sent to our bacteriology laboratory. of them were collected from the catheters. clinical data of age and type of disease were analysed. the samples were cultured on mc conkey agar-urotube and vitek cards. the organisms identified with vitek automatic system. the sensitivity was tested with vitek and kirby-bauer method. results: no growth of organisms in . %. positive cultures %. . % of the bacteria were gram((/) rods ( . % e. coli , pseudomonas spp. %), % were gram('/) cocci (enterococcus spp.) and % was candida spp. the resistance of e. coli was: % to ampicillin , % to co-trimoxazol and % to quinolons. e. faecalis was resistant % to vancomycin . conclusions : ( ) the possible interference of acetaminophen in the amoxicillin/ clavulanic acid (a/c) or erythromycin (ery) efficacy in the treatment of acute otitis media (aom), and its possible role in the evolution to otitis media with effusion (ome), were determined in a gerbil model. a f streptococcus pneumoniae strain exhibiting a mic of a/c and ery of / . and . mg/l, respectively, was used. both antibiotics were tested at . and mg/kg. acetaminophen at mg/kg was administered min before each antibiotic dose. antibiotic concentrations in serum and middle ear exudate were determined. both antibiotics significantly reduced the number of culture-positive ears and colony counts, with serum concentrations over the mic of the microorganism for ]/ % of the dosing interval. antibiotic concentrations in middle ear exudate were almost identical in animals receiving and not receiving acetaminophen. clinical and microbiological efficacy was correlated with antibiotic concentrations in middle ear exudate ]/ . times the mic of the microorganism, for both antibiotics. both antibiotics demonstrated efficacy in the treatment of pneumococcal aom, with the same rate of ome. acetaminophen, concomitantly administered, did not interfere the efficacy of the two antibiotics tested and did not prevent the evolution of aom to ome. parra a a , ponte c a , cenjor c b , garcía-olmos m a , giménez mj c , aguilar l c , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b fundación jiménez díaz, otorrinolaryngology, madrid, spain , c glaxosmithkline, medical department, madrid, spain a gerbil model of otitis media with effusion (ome) induced by haemophilus influenzae (amoxicillin/clavulanate-a/c-and erythromycin-ery-mics of / . and mg/l, respectively) was used to evaluate the efficacy of a/c ( / and / mg/kg) and ery ( and mg/kg). antibiotics were administered subcutaneously h post-middle ear inoculation, and continued t.i.d for h, with or without acetaminophen (ap), at mg/kg, administered min before each antibiotic dose. antibiotic concentrations in serum and middle ear (me) were measured by bioassay. me samples for colony counting were collected on day . a/c reduced (p / . ) positive me samples and colony counts versus untreated controls or ery: me positive cultures of % for controls, % for a/c , % for a/c , % for a/c '/ap, % for ery , % for ery and % for ery '/ap. this was due to a/c (but not ery) concentrations in me exceeding . times the mic despite the higher percentage of antibiotic penetration of ery versus a/c ( versus / %). animals receiving ap showed less polymorphonuclear cells and more bacteria in me than those receiving only antibiotics, suggesting that the anti-inflamatory drug diminish the phagocytes and therefore, the efficiency in bacterial clearance. amoxycillin treatment for acute otitis media caused by penicillinresistant streptococcus pneumoniae . a pharmacodynamic analysis pm parra a a , ponte c a , cenjor c b , garcía-calvo g a , giménez mj c , aguilar l c , soriano methods: a serotype f streptococcus pneumoniae strain exhibiting a mic of amoxycillin of mg/l was used in an experimental model performed in gerbils (meriones unguiculatus ) following previously described procedures. amoxycillin was tested at the following doses: . , . , . , . , . and mg/kg. amoxycillin concentrations in serum and middle ear exudate were determined after drug administration. results: doses of ]/ . mg/kg significantly reduced the number of culture-positive ears, colony counts and otorrhoea (p / . ) as compared with untreated controls or animals treated with doses lower than . mg/kg. doses of ]/ . mg/kg achieved antibiotic concentrations in the middle ear . Á/ . times higher than the mic of the infecting strain and serum concentrations over the mic for Á/ % of the dosing interval. conclusions: amoxycillin at doses achieving serum concentrations similar to those obtained in children after standard doses, obtained therapeutic and microbiological efficacy regardless the susceptibility of the infecting strain. better correlation was found between antibiotic efficacy and antibiotic concentrations in middle ear exudate than between efficacy and serum concentrations, which were suboptimal from the pharmacodynamic perspective. increasing prevalence of amoxycillin Á/clavulanate-resistance among e. coli strains in a hungarian university hospital pm veréb i, vígh a, urbán e, hajdú e, nagy e. department of clinical microbiology, university of szeged, szeged, hungary background: amoxicillin Á/clavulanate resistance (acr) is an emerging problem in escherichia coli as reported from different parts of europe. the aims of the present study were to evaluate statistically the prevalence of acr among e. coli isolates and to investigate the genetic background of the resistance. methods: all e. coli strains isolated between and were screened for acr by kirby Á/bauer disc diffusion method. the resistance to other beta-lactam Á/beta-lactamase inhibitor combinations and to different beta-lactam antibiotics were also tested. selected strains underwent determination of beta-lactamase activity. confirmatory tests for suspected extended spectrum beta-lactamase were performed. pcr testing for tem and shv genes were carried out on plasmids isolated from selected strains. results: in out of e. coli strains ( . %) were found to be resistant to amoxicillin clavulanate (amc). most of the resistant strains ( %) were obtained from the genitourinary tract and no acr isolate was found in blood cultures. in out of isolates ( %) proved to be acr and . % were isolated from blood cultures and . % from the genitourinary tract. thirty-five selected strains were further analysed. thirty-two were also resistant to (sam) and six were further resistant to tzp. quantitative beta-lactamase determination showed increased activity in strains which were partially susceptible to amc. the presence of esbl could be proved only in three acr isolates. this open parallel-group study compared the efficacy and tolerability of cef with cfix mg once daily in the treatment of community acquired uncomplicated uti. seventy-eight female patients were randomized to receive either oral cef or cfix for or days. the efficacy of treatment was evaluated by clinical response (by symptoms of uti dysuria frequency urgency suprapubian pain and by clinical signs) by bacteriologic response and health status measures at baseline and posttherapy. results: the clinical cure (complete resolution of symptoms and signs) rate for patients receiving cef was . % of the evaluable patients and . % of the patients receiving cfix. bacteriologic response (based on the results of urine cultures obtained posttherapy) the pathogen was eradicated in . % for cef . % for cfix. no drug related side effects have been reported in cef and side effects were experienced by . % of the patients receiving cfix. improvement in health status comparing visual scale scores baseline and poststudy to have detected a higher change in average score from to in cef, from to in cfix. wilcoxon improvement value was significant on the rd day of therapy in case of cef and on the th day of therapy in cfix group. in conclusion, the results of this study indicate that cef course is more effective than cfix in producing a favourable clinical outcome and achieving higher bacteriologic eradication rate, furthermore cef was better tolerated. cefepime is a fourth generation cephalosporine that has a broader spectrum of antibacterial activity than the third generation cepfalosporines and is more active in vitro against gram-positive aerobic bacteria. the purpose of this study was to measure cefepime concentrations in plasma, bile fluid and gall bladder tissue in patients undergoing cholecystectomy. thirty patients male, female, mean age: years had data acceptable for analysis and were included in this study. all patients received iv g of cefepime. several hours after administration and at different time intervals, during surgery, samples were obtained from plasma, bile fluid and gall bladder tissue concomitantly. antibiotic levels were measured by an agar diffusion method. the mean delta time was / min. the values for plasma, bile fluid and gall bladder tissue, were . / . , . / . and . / . mg/ml, respectively. the plasma/bile fluid ratio was . / . . there was a significant correlation between plasma and gall bladder tissue concentration (r / . , p / b/ . ). a correlation between bile fluid and plasma cefepime concentration was not observed. the minimum inhibitory concentration (mic) data from previous in vitro studies indicate that the cefepime concentration observed in plasma bile and tissue samples of this study would be adequate against typical biliary tract pathogens. furthermore, these cefepime concentrations correlated well with the favorable clinical outcome reported in previous clinical studies in biliary tract infections. there was also good correlation between delta time and plasma and tissue concentrations and if the dose were given closer to the time of surgery, cefepime concentration would be higher reducing the possibility of an infection. objectives: the use of antibiotics may lead to decreased colonization resistance and increased formation of resistant bacteria. present concept was developed to overcome these untoward effects. methods: b-lactamase of bacillus licheniformis was overproduced in bacillus subtilis . this targeted recombinant b-lactamase enzyme (trbl) was released in the small bowel from a controlled-release formulation. beagles (n / ) were treated bid with either mg/kg ampicillin (i.v.)'/placebo (p.o.), mg/kg ampicillin (i.v.)'/trbl (p.o.) or only placebo (i.v.'/p.o.). stool was collected at days and . samples were cultured for total and main groups of aerobic and anaerobic bacteria and yeast. temperature gradient gel electrophoresis (tgge) was used to separate the ribosomal rna genes. results: ampicillin'/placebo group had clearly decreased counts of both aerobic and anaerobic bacteria during the treatment, whereas those receiving trbl had only minor overall changes and some occasional changes by single species. intravenous ampicillin decreased the fecal similarity percentage to %. the similarity percentage during treatment with ampicillin'/trbl did not differ from that of placebo ( vs. %). conclusions: according to our results the trbl can maintain the large intestinal microflora almost unchanged. these results indicate that trbl is a promising novel approach for overcoming the ecological adverse effects on gut flora caused by b-lactam antibiotic agents. adamis since broad-spectrum â-lactams combined with amikacin are often applied for nosocomial infections, their pharmacokinetic interactions might be interesting. one gram of aztreonam and . g of amikacin were administered intravenously single and in combination in six healthy volunteers. blood samples were collected at regular time intervals and concentrations of antimicrobials were determined by a microbiological assay applying a strain developing resistance to single agent after serial passages. mean concentrations of amikacin in serum when administered alone and in combination with aztreonam were . and . , . and . , . and . , . and . , . and . and and . mg/ml immediately after and . , , , and h after infusion of antimicrobials. respective concentrations of aztreonam were . and . , . and . , . and . , . and . , . and . and . and . mg/ml. aucs for amikacin when administered alone and in combination with aztreonam were . / . and . / . mg h/l, respectively. respective auc for aztreonam were . / . and . / . mg h/l. it is concluded that the co-administration of aztreonam and amikacin results in earlier clearance of aztreonam and in higher levels of amikacin compared to the administration of each single antimicrobial. molecular modelling of b-lactams reveals the structural basis for their inhibition of penicillin-binding proteins, susceptibility to b-lactamases and oral bioavailability pm grail bm, gupta s, payne jw. school of biological sciences, university of wales, bangor, uk b-lactam antibiotics are peptide mimetics that act as suicide substrates for transpeptidase enzymes that cross link bacterial cellwall peptides. for the first time, the structural and electronic features needed for their recognition by transpeptidase have been fully described, using innovative molecular modelling techniques to compare the conformational forms adopted by cell-wall peptides and blactams. comparison of features in the backbone and c-terminal regions of conformers of active b-lactam antibiotics and model cellwall peptides, has allowed definition of the molecular recognition template required for substrate recognition by transpeptidase. these shared structural features allow both to act as substrates and to acylate the active-site serine. however, a significant difference in a critical backbone torsion between the two substrates, provides an explanation for the inability of the enzyme Á/antibiotic complex to undergo the deacylation step that causes inhibition of transpeptidase. on the other hand, b-lactamases appear to have evolved molecular mechanisms that facilitate the deacylation reaction through compensating for the altered structural orientations in b-lactams caused by the different backbone torsion. finally, analysis of the conformer repertoires of blactams for structural features required for substrate uptake by peptide transporters, provides insights into how their structures can be tailored for optimal oral absorption. antimicrobial susceptibility of proteus mirabilis clinical isolates producing extended spectrum beta-lactamases (esbls) pm objectives: the aim of the present study was to determine in vitro susceptibility to antimicrobials of proteus mirabilis isolated from urinary tract infections. methods: we studied the susceptibility profile of esbl positive p. mirabilis strains in three adopted children from india with age range from months to years. esbl was identified using the synergic effect of clavulanate with betalactams (ceftazidime and cefotaxime the in vivo efficacy of amoxicillin (amx) sub-therapeutic doses ( . mg/kg, t.i.d for h, achieving serum levels over the mic of only % of the dosing interval) and concomitant specific serotherapy (single intraperitoneal dose of / diluted hyperimmune serum (hs) obtained from mice immunized with the heat-inactivated strain) was assessed in a pneumococcal sepsis balb/c mouse model. mice (five mice/ treatment group) were intraperitoneally infected with . )/ cfu/ ml of a serotype b penicillin-resistant strain (mic of and mg/l for penicillin and amx, respectively). treatments started h after bacterial inoculation. study groups were: control (k; receiving nonimmune serum (nhs)), amx'/nhs, hs, and amx'/hs. survival rates (%) over time were: purpose of the study: the study was performed to determine the consumption of imipenem and resistance of gram-negative pathogens (pseudomonas aeruginosa , acinetobacter sp., klebsiella sp., escherichia coli , proteus mirabilis , serratia marcescens , enterobacter sp. ) to imipenem. gram-negative pathogens were isolated at the sestre milosrdnice university hospital from zagreb, croatia, in and . the imipenem sensitivity testing was performed by disk diffusion and e -test methods. the consumption of imipenem was expressed in ddd/ hospital days in the same periods. results obtained: imipenem resistance of acinetobacter sp. decreased significantly in the year (p / . ), especially in the first months (p / . ) when the lowest consumption of imipenem was recorded. imipenem resistance of other gram-negative pathogens did not decrease significantly. conclusion reached: comsumption of imipenem might lead to changes in resistance to imipenem among acinetobacter strains. vacheva-dobrevski rs, savov ez. military medical academy, clinical microbiology, sofia, bulgaria purpose: acinetobacter baumanii is becoming increasingly frequent nosocomial pathogen at our hospital, and beta-lactam resistant strains are on the increase, especially among icu isolates. to study the susceptibility of a. baumanii clinical isolates to beta-lactams and to determine the esbl-producing strains during , year. a total gram-negative nonfermenters (gnnf) isolates was investigated by semiautomated mini api system (bio merieux, france). eighty-four a. baumanii non-repeated isolates was studied for esbl-producing by double-disk synergy test (ddt) and atb-blse test (bio merieux, france). mics for beta-lactams were determined by e -test (ab biodisk, sweden). results: a. baumanii (n / ) showed a multidrug resistance. the isolates were resistant to cefotaxime ( %), cefoxitin ( %), ceftazidime ( %), amoxicillin/clavulanate ( %), piperacillin ( %), aztreonam ( %), imipenem ( %). the ( %) of investigated a. baumanii expressed esbl activity and originated more frequently from icu ( %). esbls producing strains were isolated from endotracheal aspirate ( %), surgery wounds ( %), blood culture ( . %). conclusions: in general resistance levels were higher in clinical isolates a. baumanii to beta-lactams. the ddt seems to be a practical method for esbl-screening; atb-blse method is more sensitive. our study display to be the first report of esbl-producing a. baumanii strains from our country. carbapenems seems to be the most active agents against a. baumanii . salmonella infantis , strain , was isolated from a newborn baby at wassila bourguiba maternity in tunis. it exhibited high resistance to penicillins, extended-spectrum cephalosporines (cefotaxime, ceftriaxone, ceftazidime, cefpirome) and aztreonam but remained susceptible to cefoxitine and imipenem. involvement and characterization of enzymatic mechanism in b-lactam resistance were investigated in strain . isoelectricfocusing revealed that this strain produced a b-lactamase of pi . this enzyme had a broad-substrate profile, hydrolyzing amoxicillin, ampicillin, ticarcillin, cephaloridine, cefuroxime, cefotaxime, ceftriaxone, cefpirome and ceftazidime. the highest specific activity was observed with ampicillin. cefotaxime was hydrolyzed the most efficiently of the extended-spectum cephalosporines. the pi extended-spectrum b-lactamase (esbl) was inhibited by clavulanic acid and sulbactam. no inhibition of the esbl was observed with mm edta. thus, no metal ion is involved in hydrolysis for this b-lactamase. resistance due to the production of the pi esbl was transferred with dna plasmid into escherichia coli . on the basis of substrate and inhibition profiles and isoelectric point, the pi esbl was not previously described in s. infantis in tunisia. the presence of such a resistance on a plasmid raises concer for rapid dissemination among bacteria and loss of effectiveness of blactams. poizot-martin i a , enel p b , benhaïm s c , vion-dury f c , dinh t c , drogoul mp c , gastaut ja c . a assistance publique hôpitaux de marseille, cisih sud, pr ja gastaut, marseille, france , b assistance publique hôpitaux de marseille, cellule santé publique dmi , marseille, france , c assistance publique hôpitaux de marseille, cisih sud, marseille, france objective: to assess liver biopsy (lb) practices in a cohort of co-infected hcv and hiv patients followed up in an hiv specialized medical unit. method: transversal study with questionnaire among patients in pre-therapeutic's evaluation with pcr'/ and without lb at months. results: among the patients, ( %) are lost of follow up, ( . %) have had lb, ( . %) have no lb. the characteristics of these patients are: median age / . / years; sex ratio / . , cdc-stage a / . % b / . % c / . %, undetectable viral load / . %, median cd / / , anti-retroviral therapy / . %, hcv-genotype / . %; a / . %; / . %. causes of non-made lb are: ( ) refusal from patients because of biopsy's fear / . %; ( ) contraindications because of hiv infection / . % (clinical events / . % which contraindicate anti-hcv treatment, grade iii thrombocytopenia / . % which contraindicate biopsy, non-adherence to previous hiv follow up / . %); ( ) other / . % (alcoholism / . %, psychiatric/depressive disorders / . %, decompensated cirrhosis / . %). drug use or methadone/buprenorphine treatment are not considered as contraindication. conclusion: one-third of patients are afraid of lb. alcoholism and psychiatric/depressive disorders are the principal contraindications to anti-hcv treatment. it seems important to improve information of patients about lb and to focus on alcohol and psychiatric/depressive disorders management in such population. kashiwagi kk a , furusyo nf a , nakashima hn a , kashiwagi sk b , hayashi jh a . a department of environmental medicine and infectious disease, kyushu university, fukuoka, japan , b national kyushu medical center, fukuoka, japan the purpose of the study: the aim of this prospective study was to explore the effect of htlv-i co-infection on the development of hcc among patients with chronic hcv viremia. a total of consecutive patients with chronic hcv viremia were studied and followed-up over a mean period of . years: ( . %) were infected with htlv-i infection and ( . %) were not. the results obtained the annual hcc development rate was . % in patients co-infected with hcv and htlv-i and . % in patients infected with hcv alone. hcc was significantly higher in ( . %) of the patients co-infected patients than in ( . %) of the patients infected with hcv alone (p b/ . , logrank test). in patients under the age of years, hcc development was significantly higher in seven ( . %) of patients co-infected with hcv and htlv-i than in eight ( . %) of patients with hcv alone (pb/ . , logrank test), whereas there was no significant difference in hcc development between patients over age with or without htlv-i infection ( ( . %) of and ( . %) of , respectively). the conclusion reached htlv-i infection accelerates the development of hcc in chronic hcv patients, especially among patients under the age of years. to analyze hbv genotype-related clinical differences among patients with chronic hbv infection, all patients were serially tested for serum alanine aminotransferase (alt) and hepatitis b e antigen (hbeag) and followed up for a mean . ( . ) year period. genotypes b and c were found in ( . %) and ( . %) of the patients, respectively. hbeag positivity and alt abnormality rates at the start of the observation period were significantly higher in genotype c patients ( . and . %) than in genotype b patients ( . and . %). the annual rate of spontaneous hbeag disappearance in genotype b patients was much higher than in genotype c patients ( . versus . %, respectively). patients with genotype c who were continuously hbeag negative from entry had significantly higher alt abnormality ( . %) than those with genotype b ( . %). interestingly, patients with genotype c who became hbeag negative by interferon treatment had high alt abnormality ( . %). all patients with alt abnormality were serum hbv dna positive. these findings indicate that hbv genotype c patients are more severe liver deterioration because of the delay of hbeag disappearance and continued hbv replication after hbeag disappearance. nossik nn a , nebolsin ve b , zheltukhina ga c , yevstigneeva rp c . a the d.i. ivanovsky institute of virology, viral reproduction, moscow, russian federation , b pparminterprisis co., chemistry, mocow, russian federation , c moscow state academy of fine chemical technology, piptide chemistry, moscow, russian federation objective: to study the effects of 'gamma'-l-glutamylhistamine (glu-ha) derivates on non-specific immunity ('alfa'-ifn, 'gamma'-ifn and nk cell activity) and antiviral activity on the experimental influenza and herpes virus infections in mice. the glu-ya and its derivate glu-ii were synthesized by peptide chemistry techniques. the glu-ha and glu-ii was administered i.p. . and . mg/kg before and after influenza virus (type a/aichi) and showed a protective effect even at the high infective dose ( ld ) */the rate of protection / Á/ / % in the positive/control group. they were not very effective in the protection of herpes simplex virus encephalitis in mice. the model of the physico-emotional stress in mice was used to investigate the ifn system and nk cell activity. the production of ifns and nk cell activity of splenocytes decreased in h after the stress and back to normal level in Á/ days. it was shown that glu-ha and glu-ii can protect or substantially prevent the decrease in nk cell activity and ifns synthesis in post-stress period (so normally did not induce the ifns' synthesis). conclusions: the glu-ha and glu-ii showed antiviral effect against influenza virus infection in mice. the immunomodulating activity and ability to normalize the ifn synthesis and nk cell activity depressed the post-stress period and probably play an essential role in the antiviral activity. no dose adjustment of an anti-influenza prodrug oseltamivir is required in patients with hepatic impairment pm oo c a , snell pr b , liu b a , martin d b , simkins t b , small i b , ward p b . a hoffmann-la roche inc., global development, nutley, usa , b roche products ltd., global development, welwyn garden city, uk background: oseltamivir (ose; ro - , tamiflu † ) is an oral ethyl ester prodrug of its active metabolite oseltamivir carboxylate (oc: ro - ), a potent and selective neuraminidase inhibitor of the influenza virus. the purpose of the study is to evaluate the need for ose dosage adjustment in hepatic impaired patients (hi). method: healthy volunteers (hv) versus hi (child-pugh score Á/ ) [matched on the basis of age ( / years), gender and weight ( / %)] were compared. each subject received mg ose. results: based on c max (ng/ml) and auc inf (ng h/ml) analysed using nominal times, ls mean ratios and % ci between hi and hv were similar. ose* values in hi were marginally elevated but not sufficiently to require dose adjustment. the aim of this study is to investigate the influence of molecular structure of macrocyclic pyridinophanes and their analogs on antiinfluenza and antiherpetic activity of these compounds. we used d-qsar approaches on the basis of simple representation of molecular structure. such representation for biologically active substances allows the description of the spatial structure of compounds with the complete stereochemical information. it determines spatial structures either promoting or interfering of the concrete biological activity. it is easy to realize the molecular design of compounds with the given level of activity with the help of the combinations of simplexes. statistic characteristics for qsar of partial least-squares models are satisfactory (r / . Á/ . ; cvr / . Á/ . ). the molecular fragments that increase the antiviral activity were defined and will be demonstrated. this information was used for design and directed synthesis of several novel antiviral agents with predicted high anti-influenza or antiherpetic activities. predicted activities were confirmed experimentally. d-qsar approaches are useful for development of antiviral compounds. this work was partially supported by intas foundation (grant intas - ). lozitsky vp. ukrainian mechnikov research anti-plague institute, chemotherapy, odessa, ukraine the purpose of this study was to research the anti-influenza activity of proteolytic inhibitor e-aca. it prevents the enhancement of proteolysis during the interaction of virions with cell membranes and decreases penetration of virions into cells. e-aca brings down proteolytic cleavage of ha-precursor to ha- and ha-polypeptides and reduces the infectious virus harvest. it shows the prophylactic and therapeutic action during the experimental influenza reducing the enhancement of alkaline proteases activity in lungs after infection. e-aca promotes the intensification of specific antibodies production and cell immunity, prevents vessels' permeability and hemorrhagic phenomena, decreases the destruction of bronchi's epithelium. it reduces the duration of intoxication, catarrhal instances and hyperthermia in sick children. e-aca improves the indexes of immunity, non-specific resistance and decreases the rate of bacterial complications. application of e-aca for treatment influenza and other arvi in children is recommended in ukraine on the base of results of our researches. the higher effects demonstrated as a result of combine usage of e-aca with specific ig, or deitiforin, or unithyol, or ribavirin. in our opinion, the study of effectiveness of e-aca combine application with inhibitors of influenza na is the perspective direction of anti-influenza researches development. we should we treat immediately all varicella patients with acyclovir if the patient is older than years pm during past years in institute for infectious and tropical diseases in belgrade, immunocompetent varicella patients were treated and cured. among them were older than years ( . %). x-rays were performed in all patients. diagnosis of pneumonia was made in patients ( . %), but in ( . %) patients older than years. varicella is a benign, self-limited disease, if it strikes early, i.e. preschool, school children and teenagers. at that time there is no need for specific therapy. but in neonates, immunocompetent adults and in all immunocompromised patients it can be difficult and life-threatening disease. in immunocompetent adult population pneumonia is a very serious, sometimes fatal complication. knowing the pathophysiology of primary varicella Á/zoster infection, specific therapy with acyclovir should be started immediately after making the diagnosis in patients older than years, without waiting for x-ray proof of pneumonia. brivudin compared to acyclovir for the treatment of herpes zoster: effects on acute disease and posttherapeutic pain pm objective: comparison of efficacy and safety of brivudin )/ mg and acyclovir )/ mg, both for days, in the treatment of herpes zoster. methods: randomised, double-blind study on immunocompetent patients ]/ years (brivudin: n / , acyclovir: n / ). a subgroup of patients ]/ years (brivudin: n / , acyclovir: n / ) was examined for the occurrence of posttherapeutic pain in a poststudy survey. posttherapeutic pain was defined as any zoster-associated pain, regardless of intensity, after the end of acute zoster. results: brivudin was superior to acyclovir in reducing time to last occurrence of new vesicles (rr(itt): . [ . Á/ . ], p / . ). the advantage of brivudin was more pronounced in patients ]/ years (rr(itt): . , [ . Á/ . ], p / . ). incidence of posttherapeutic pain was significantly lower with brivudin ( . %) than with acyclovir ( . %, p / . ). duration of pain was comparable in both treatment groups (rr: . , [ . Á/ . ], p / . ). potentially treatment-related adverse events occurred in . % of the brivudin recipients and in % of the acyclovir recipients. conclusions: brivudin mg once daily for days is superior to standard acyclovir in stopping viral replication in acute herpes zoster. in patients ]/ years, brivudin is more effective than acyclovir in reducing the risk of developing posttherapeutic pain. brivudin is as well tolerated as acyclovir. varadinova t a , genova p a , garcia-raso a b , terron a b , fiol j b , badenas f b . a laboratory of virology, sofia university, sofia, bulgaria , b chemistry, universitat de les balears, palma de mallorca, spain we have published that cu(ii) complexes of acyclovir (acv) are active against hsv infection (mbd, ) . here we present data on the activity of acv complexes of ni(ii), cd(ii), co(ii) and ag(i) against resistant to acv hsv strain r- in comparison with the effect against acv sensitive strain victoria. selectivity indexes (si) compared to that of acv were indicative for activity. the following data were obtained: (i) was times less selective inhibitor of strain r- than of strain victoria; (ii) under the action of [cd(acv)cl ], [ni(acv) (h o) ]cl ×/ acv and [ni(acv)(no )] ×/ h o was up to % higher than that in the control; (ii) was times less selective inhibitor than acv; (iv) si of was two times higher for strain r- and five times lower for strain victoria then that of acv. these data show that the selectivity of acv against resistant hsv strains can increase when acv is bond to a proper metal ion. methods: an antiviral activity against herpes simplex virus of the type i (hsv-i/leningrad/ / ) and variant hsv- (vvt/ / r) resistant to acyclovir was determined using commonly accepted method. viruses were grown on a continuous culture. maximal toxic dose was determined by the administration of compounds orally ( mg/kg) or intraabdominally ( mg/kg) to white mice that had mass Á/ g. condition of the animals was controlled during h. mice pneumonia model was used for the testing activity in vivo. results: derivatives tested have activity against hsv- and hsv- resistant to acyclovir. maximum protection of the cells up to % was reached at concentration of compounds Á/ mg/kg. tested compounds have low toxicity and animals did not die after intraabdominal and after per oral administration of the substances. using these compounds led to essential relief of diseases in animals. the number and square of virus specific areas of inflammation in lung was decreased to compare with control untreated group. tested compounds protected animals similar to acyclovir that was used as control. conclusion: derivatives of carboalkoxysulfanilic acids are active against hsv in vitro and in vivo and act on the acyclovir resistant variant viruses. markiewicz r a , szepietowski jc b . a jelfa s.a., medical department, jelenia gora, poland , b department of dermatology, university of medicine, wroclaw, poland background: denotivir is a -benzoamino- ?-chloro- -methyl- isothiazolecarboxanilide anti-inflammatory agent with antiviral and immunomodulatory activities. it possesses also mild antibacterial and antifungal action. the purpose of the study: the aim of this presentation is to give an overview of recent studies demonstrating denotivir efficacy in herpetic infections. the results obtained: in vitro studies revealed that denotivir in the doses below its cytotoxicity (about um) significantly inhibited (by Á/ %). herpes simplex virus (hsv)- and hsv- replication in fibroblast and kidney cell cultures. moreover, it was showed that denotivir in the dose of mg/ml markedly inactivated hsv- after min incubation in c. in giunea pigs research, % denotivir in % dmso appeared to be superior to % dmso alone and untreated groups in the therapy of animal skin infected by hsv- . there was no huge difference in eythema and oedema scorings between studied groups, however in the group treated with denotivir, in contrast to others, no vesicles developed. several clinical studies showed usefulness of denotivir in controlling herpetic infections in dermatology, ophthalmology and otolaryngology. in the majority of studies within few hours after the drug application itch and pain relief was noted and within Á/ days the vesicular lesions were dried up. the conclusion reached: in conclusion, denotivir is an effective antiherpetic agent. this study was conducted on cows affected with teat papillomatosis. in the first step, each cow was located on one of three groups. the first group contained four cows from to years that were treated with fig tree (ficus carica) latex. the second group contained four cows from . to years that were treated with a % solution of salicylic acid, and the third group contained four cows as control. in group one and two following treatment with fig tree latex and salicylic acid, superficial necrosis begun from day and all of the warts disappeared by day . in the control group, after day , there were no changes in number of lesions, but some of them were larger than first observation. on day , one of the marked warts disappeared and on day another wart was disappeared but six were present until day . comparison of effects of salicylic acid and fig latex showed similar effects in treatment of udder papillomatosis in cow. laboratory markers of skeletal muscle toxicity in hiv-infected patients: a cross-sectional case-control survey of frequency, potential correlation with antiretroviral therapy, clinical significance, and outcome pm manfredi r, motta r, patrono d, calza l, chiodo f, boni p. to assess skeletal muscle toxicity among Â/ hiv-infected outpatients (p), the p who had ]/ altered cpk assay ( !/ u/l) between may and november , were compared with p randomly selected among those who had ]/ laboratory exams in this -month interval, in a : case-control study. among the p with altered cpk levels only six were females, and received antiretrovirals. the overall frequency of altered cpk among all p who underwent ]/ laboratory workouts in months was . %. cpk alteration was transient in p, with values ranging from to (mean . / . ) u/l, but was recognized ]/ times in Á/ months in p ( %), of them showing concomitant high aldolase levels ( . Á/ . u/l). a myopathy or a rhabdomyolisis were recognized in four p only; a myositis was confirmed in one p by histopathology. in a multivariate logistic regression analysis, when excluding the unexpected prevalence of the male gender (p b/ . ), no significant difference emerged between p and controls as to age, risk for hiv infection, iv drug use, duration of hiv infection, prior anti-hiv therapy and its length, selected drug combinations, administered nucleoside analogues,hiv disease stage, mean cd '/ count and hiv viremia, signs and duration of lipodystrophy, increased glucose, triglyceride and cholesterol levels, and other therapies. muscle abnormalities, though frequently asymptomatic, are underestimated hiv disease complications, and the role of metabolic (i.e. mitochondrial) alterations, deserves investigation. poor efficacy of non-nucleoside reverse transcriptase inhibitor (nnrti)based salvage haart in hiv-infected patients heavily pre-treated with all other classes of antiretroviral compounds pm manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy poorly comparable literature series show conflicting results of nnrti-based rescue haart: Á/ % rate of virologic success. to assess the response to a Á/ -drug rescue haart including a nnrti, patients (p) treated with nucleoside analogues (na) and protease inhibitors (pi) for !/ and !/ months, respectively but naïve to nnrti, with a viremia !/ copies/ml, were prospectively followed during Á/ years, provided that they ensured a !/ % adherence. efavirenz was used in p, nevirapine in , and delavirdine in two. most p ( . %) had an early laboratory improvement, but mean peak viral load decrease was . log, and a significant reduction vs baseline (p b/ . ) lasted months only. a mean % increase of zenith cd count was obtained (p b/ . ), but only . % of p remained !/ cells/ml year after switch to nnrti. in a multivariate analysis, the concurrent introduction of novel pi(s) ( p) and/or different na(s) ( p) acted favorably until the th month of follow-up (p b/ . ), while genotypic mutations conferring nnrti cross-resistance, usually associated with a broad resistance profile, predicted failure in all p (p b/ . ), and the response did not vary according to duration and type of prior therapy, and selected nnrti. a deep salvage nnrtibased haart has a poor and transient virologic outcome also in nnrti-naïve p, while a more evident and sustained immunologic response is expected. p who can introduce novel pi/na and have no mutations impairing nnrti activity are entitled to a better outcome. fatal lactic acidosis without elevation of liver-enzymes during the treatment with stavudine, didanosine and efavirenz: a case report pm winzer r, langmann p, väth t, zilly m, klinker h. medizinische poliklinik der universität würzburg, schwerpunkt hepatologie/infektiologie, ürzburg, germany nucleoside reverse transcriptase inhibitors (nrtis) cause various side effects, many of which are thought to be due to their effects on mitochondria. a -year-old hiv positive (hiv rna: copies/ml, cd cell count: /ml), obese (body-mass-index: . ), therapy-naïve female patient, who after months of well tolerated and effective antiretroviral therapy (stavudine, didanosine, efavirenz), had slight gastrointestinal discomfort and suddenly developed a lactic acidosis (arterial-ph . [ . Á/ . she died days later despite intensive care (continuous venovenous haemodiafiltration, sodium-bicarbonate infusion, high doses of vitamins, respiration). the pathologic examination showed an enlarged liver ( g) with yellowish appearance and pasty consistency, which microscopically appeared as a massive macro-and microvesicular fatty degeneration, and only slight signs of terminal pancreatitis. this reported case gives evidence that a massive lactate acidosis may develop without previously disarranged laboratory parameters for liver or pancreatic function. a fatal outcome may evolve without further accompanying-illnesses. efficacy and tolerability of atorvastatin in the treatment of hypercholesterolemia in hiv-infected patients receiving haart pm calza l, manfredi r, chiodo f. division of infectious diseases, university of bologna, bologna, italy introduction: significant increases in plasma triglyceride and cholesterol levels have been reported in patients treated with haart, and prolonged metabolic imbalances could significantly act on the longterm prognosis and outcome of hiv-infected persons. patients and methods: fourteen hiv-infected patients on pi-based haart since at least months and presenting hypercholesterolemia ( !/ mg/dl) of at least -month duration and unresponsive to a hypolipidemic diet and physical exercise, have been treated with a single daily dose of atorvastatin ( mg) for months. results: one patient was ecluded from evaluation due to early dropout. ongoing antiretroviral treatment included ritonavir in four cases, indinavir in four, nelfinavir in three, and saquinavir hard-gel in two. at the close of -month follow-up of atorvastatin therapy, a decrease of total cholesterol level of . % versus respective baseline value was observed; eight out of patients reached normal values for cholesterol. mild gastroenteric symptoms were found in only one of the treated patients, while no skeletal muscle and liver toxicity has been observed. discussion: in our study, pharmacological treatment with atorvastatin proved certainly effective in the management of diet-resistant hypercholesterolemia, and was associated with a favourable tolerability and adherence profile. the effect of combination antiretroviral therapy regimens on hiv- proviral dna level in peripheral blood mononuclear cells (pbmcs) was examined in hiv- -positive patients, using endpoint dilution pcr and serially cloning and sequencing of the gag region of hiv- . the major clone was defined as the most numerous of analyzed clones, and observation periods ranged from to months (mean, . / . months). in five patients (one with primary-stage hiv- infection) receiving three antiretroviral drugs, hiv- rna levels reduced to undetectable (i.e. b/ copies/ml). hiv- proviral dna levels and the number of major clones reduced in four of these patients. hiv- rna levels reduced, but remained detectable, in five other patients. in the two remaining patients (both receiving two rather than three antiretroviral drugs) hiv- rna levels increased. these results suggested that the population of the major clones may be affected when hiv- rna levels reduce following combination regimens of antiretroviral therapy. saquinavir hard gel (shg) as a part of a spontaneous Á/ -month deintensification anti-hiv regimen following successful highly active antiretroviral therapy (haart) pm manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy the induction-maintenance concept was poorly studied in hiv'/ patients (p), and shg was never assessed after prolonged response to potent protease inhibitors (pi)-based haart. shg-naïve p who refused indinavir, ritonavir, or nelfinavir-based haart after achieving long-term viral suppression, and resorted to shg'/ nucleoside analogues (na), were followed prospectively. in . % of the p assessed for Á/ months, ]/ na was changed. prior haart was interrupted after . / . months, due to adverse events ( p), or p's request ( p), while a viremia b/ copies/ml was present since . / . months. a viremia of Á/ hiv-rna copies/ml was maintained in p ( . %), while a higher viral load occurred in p after . / . months, and was related to a pre-haart viremia !/ / ml, a more frequent !/ % recovery of cd count, mutations of codons Á/ , and failure to change na (p b/ . Á/ . ). a cd drop !/ %/ cells/ml was found after . / . months in only eight p, who also had virologic failure: immunologic deterioration was earlier and deeper when na were not changed (p b/ . ). all the p who introduced shg'/novel na after a successful !/ -month induction with a potent pi-based haart had a stable Á/ -month outcome. a suboptimal haart including the less effective but better tolerated shg may be effective for !/ year, especially when novel na are introduced, and specific mutations are absent. despite a lower potency, drugs with a good safety and compliance profile may be recovered for simplified regimens. objective: to evaluate efficacy of antiretroviral therapy (art) with two or three drugs in the nervous 'reservoir'. patients: thirteen acute neurological and art naive aids patients underwent a paired and simultaneous sample from plasma and cerebrospinal fluid (csf) for a quantitative detection of hiv- rna (amplicor roche) before art. all patients underwent a ct and/or mr of the brain to perform a diagnosis. all of them had an hiv related neurological acute inflammatory disease. after diagnosis all patients received art: / received two nrti and / received haart including two nrti and one protease inhibitor. all patients underwent a paired and simultaneous follow-up from plasma and csf during the nd month of treatment. results: in all patients baseline levels of hiv-rna were higher (p b/ . ) in the plasma (log . '/ . ) than in the csf (log . '/ . ). the / patients who received dual therapy had undetectable levels (cut-off copies/ml) of viral rna at the follow-up in csf, but not in plasma: three of these seven patients had a detectable plasma hiv- rna. all / patients with haart had undetectable hiv- rna both in plasma and in csf at the follow up. conclusions: dual nrti therapy is rapidly effective in csf (because of an high penetration of drugs through a more permeable blood Á/brain barrier and lower hiv rna baseline levels) but not in plasma. haart is rapidly and equally effective both in csf and plasma. there are no reports of fulminant and fatal hepatic failure after the start of highly active antiretroviral therapy (haart) in an hiv subject without chronic viral hepatitis. case report: a -year-old naive aids woman with clinical symptoms due by a pcp was observed. baseline alt was increased ( . m.n.v.) because of a mild hepatosteatosis and a silent cholelithiasis. serology for hbv, hdv and hcv was negative; igg anti-hav, anti-ebv, anti-cmv and anti-hsv were present. hiv- rna was . log , cd '/ count was /ml. during the pcp treatment with cotrimoxazole alt values increased ( !/ m.n.v.); nevertheless, she completed the treatment. liver enzymes returned to the pre-treatment values over several days. then she started haart with stavudine, lamivudine and efavirenz. after days the patient showed an efavirenz-related skin rush that resolved within days, without treatment discontinuation. fourteen days after the start of haart jaundice appeared. laboratory revealed severe alt increase ( !/ m.n.v.) and hyperbilirubinemia ( mg/dl) and she died because of an acute liver failure syndrome within few days. an haart efavirenzbased regimen can result highly hepatotoxic when given in presence of a hepatosteatosis, of a recent hepatotoxicity caused by a nonantiretroviral treatment and of a previous idiosyncratic reaction to efavirenz. our experience with bulgarian herbal extracts for improving the general condition of hiv-positive patients pm methods: we used a combination of bulgarian herbal extracts and treated six patients, divided in two groups: three with symptomatic and three with asymptomatic hiv-infection. the all three patients with asymptomatic hiv-infection were treated only with herbal extracts, another three patients with symptomatic hiv-infection were treated with combination of herbal extracts and anti-retroviral therapy. the general status of patients has been evaluated by both subjective and objective surveillance. the immunologic monitoring has been performed by absolute count of cd '/ lymphocytes. results: all patients have shown an obvious improvement in their general condition: high spirit and working capacity, good appetite and sleep, a restoration of body weight. the number of cd '/ lymphocytes has been lightly increased or constant. conclusion: the combination of bulgarian herbal extracts has shown significant positive effect on the general condition and improve the quality of life. antifungal activity of in vitro and in vivo combinations of voriconazole with -fluorocytosine and amphotericin b against candida and cryptococcus spp pm hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: the present study was designed to determine whether the activity of voriconazole (vor), a novel triazole, was reduced against candidal and cryptococcal infections by the addition of standard antifungal agents, amphotericin b (amb) and -fluorocytosine ( -fc). vor was tested in combination with standard antifungal agents both in vitro, using a checkerboard mic determination test, and in vivo in immune normal guinea pig models of fungal infections. the results indicate that the efficacy of vor against candida albicans and c. neoformans was not antagonised by amb or -fc in vitro. furthermore, in guinea pig models of systemic candidiasis and intracranial cryptococcosis, no antagonism was observed between the lower doses of vor and either amb or -fc on the basis of reductions in tissue fungal loads. at the highest doses of vor, both amb and -fc showed some antagonism, but the combinations were still effective in significantly reducing fungal tissue loads compared with vehicle-treated control animals. conclusion: these results suggest that vor may be used in combination with standard antifungal agents, and future studies to elucidate the clinical potential of vor combination therapies in the management of candida and cryptococcus infections are warranted. itraconazole in the treatment of pityriasis versicolor pm tiodorovic j, jovanovic d, binic i, nikolic lj. faculty of medicine, clinic of dermatovenerology, nis, serbia, yugoslavia a comparison of two short-term dose schedules with itraconazole was carried out in patients with pityriasis versicolor. the patients were divided in two groups. each group consisted of patients who completed the therapy and controls. the clinical diagnosis was confirmed mycologically, by direct microscopic examination. the first group received mg of itraconazole daily for days. the second group received mg daily for days. the patients were controlled clinically and mycologically and days after the initiation of treatment. erythema, scaling and pruritus was evaluated clinically. clinically and mycologically cured patients accepted as cured. the cure rate were % in the first group and % in the second group at day . the effects of these two groups are similar. none of the patients reported side-effects. fungal urinary infections: emerging species, antifungal susceptibility trends and antibody response pm badawi he a , kamel ai b , fam ns a , el-sayed me a , elian sae c . a theodor bilharz medical research institute (tbmri ), microbiology, giza, egypt , b theodor bilharz medical research institute (tbmri ), urosurgery, giza, egypt , c faculty of medicine, medical microbiology and immunology, cairo university, cairo, egypt objectives: to assess the role of candida species in patients with urinary tract infections (utis) with or without schistosomiasis and/or cancer bladder, to compare chromogenic; chromagar (cma), biggy agar; morphologic (corn meal, rice agar-tween ) media and biochemical candifast test for identification of candida species. susceptibility to antifungal agents using e -test and candifast and the performance of elisa test for detection of anticandida antibodies (igm and igg) in serum were evaluated. results: c. albicans was the most frequent ( . %) species responsible for fungal utis. however, non-albicans species, c. glabrata ( . %), c. tropicalis ( %) and c. krusei ( . %) were also isolated. rice agar-tween was found to be cheap, available and sufficient to make a final identification ( %). cma could not identify c. glabrata . biggy agar could not adequately differentiate candida species. candifast biochemical identification showed low sensitivity of . %. e -test on sabouraud dextrose agar (sda) is simple method for mics determination and could detect s-dd strains in case of azoles. conclusion: the emergence of non-albicans species such as c. glabrata , c. tropicalis and c. krusei have contributed to complicated utis. this necessitates accurate isolation and identification of candida to the species level. morphology on rice agar-tween and antifungal susceptibility using e -test on sda is a simle rapid scheme for routine identification of clinically important yeasts. purpose: classification of allergic fungal rhinosinusitis (afr) is based on the immunologic relationship of the host to the fungus. afr must be differentiated from other fungal rhinosinusitis infections, which include acute invasive, chronic invasive, fungal balls and saprophytic colonization. although many cases of fungal rhinosinusitis is caused by species of aspergillis , dermatiaceous moulds have become an emerging pathogen in immunocompetent individuals. results: our case study involved a year male suffering from facial pain, headache, postnasal drip and loss of smell. he was hiv negative and a nonsmoker. the following laboratory tests were performed: ige- . ( . Á/ . ) iu/ml iga- . ( . Á/ . ) g/l igm- . ( . Á/ . ) g/l allergen specific ige for alternaria */ . ( . Á/ . ) ku/l fbc-normal except eosinophils slightly raised . ( . Á/ . ))/ / l. ct scans indicated fungus proliferation, bone erosion and extension of disease into adjacent anatomic area. sinus tissue following debridement was sent for microscopy and culture. hyphae was microscopically observed and cultures yielded two dermatiaceous fungi, bipolaris spp and alternaria spp . conclusion: it is important to differentiate these two species from curvularia , helminthosporum , drechelria and exserohilum . knowledge of these dermatiaceous fungi is important in directing appropriate antifungal therapy and selecting the correct antigens for postsurgical immunotherapy after initial debridement and irrigation. antifungal activity of in vitro and in vivo combinations of voriconazole with -fluorocytosine and amphotericin b against aspergillus fumigatus pm hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: a key requisite for a new antifungal drug is to demonstrate that it is devoid of significant antagonism in combination with other agents. combinations of the new triazole, voriconazole (vor), and standard antifungal agents ( -fluorocytosine or amphotericin b; -fc or amb) were tested against aspergillus fumigatus in vitro and in guinea pig models of infections to confirm that antifungal activity was not antagonised by using combination therapies. vor was studied in combination with amb or -fc in vitro, using a checkerboard mic determination test, and in vivo, using immune normal and immunocompromised guinea pig models of systemic aspergillosis. results: the results indicate that the potency of vor was not antagonised by amb or -fc in vivo; indeed, at lower concentrations of vor, significant improvements in reducing fungal burden in both in vivo models were achieved by the addition of amb. in vitro, no antagonism was found between vor and amb, although -fc had a significant antagonistic effect on vor activity. conclusion: these results from in vitro and in vivo models of aspergillosis suggest that vor may be used in combination with standard antifungal agents and, therefore, justify further examinations of vor combination therapies in a clinical setting. in vitro activity of caspofungin compared to that of amphotericin b, fluconazole, and itraconazole against candida species pm arikan s, sancak b, hascelik g. department of microbiology and clinical microbiology, hacettepe university medical school, ankara, turkey purpose: to evaluate the in vitro activity of caspofungin against various candida spp. and particularly against isolates with decreased amphotericin b, fluconazole, and itraconazole susceptibilities. methods: susceptibility tests were done by nccls m a microdilution guidelines for clinical candida strains. the mics (mg/ml) were read at and h. results: caspofungin mics at h are shown in the table. mics at h were similar to h readings. expectedly, no evidence of crossresistance was detected between caspofungin and other drugs tested. caspofungin was similarly active against fluconazole-or itraconazolesusceptible and resistant isolates. conclusions: ( ) caspofungin is active in vitro against all candida spp. tested. ( ) caspofungin mics are slightly higher for c. parapsilosis compared to other species. ( ) its activity against fluconazole-and itraconazole-resistant isolates is noteworthy. ( ) validation of these data require clinical investigations. oropharyngeal microbiological samples of bmt patients were evaluated. weekly cultures (days (/ , and '/ ) revealed presence of fungi in patients ( . %): in four ( %) patients before bmt only, in ( %) after bmt only, and in ( . %) both before and after bmt. in one patient candida norvegensis was isolated from the throat, buccal and palatal surfaces. three c. albicans , two c. krusei , and three c. norvegensis from four patients were chosen to comper their antifungal sensitivities and extracellular virulence factors. using fungitest method we determined the sensitivities of these isolates to flucytosine, amphotericin b, miconazole, ketoconazole and fluconazole. the fluconazole sensitivities were also determined by the e -test. on the basis of the fungitest data the three c. norvegensis isolates were sensitive to flucytosine, amphotericin b, and also to ketoconazole. in case of fluconazole and miconazole they proved to be susceptible dependent upon dose. the mic fluconazole values determined by the e -test for the three c. norvegensis isolates were , ]/ and ]/ mg/ml, respectively. the oropharyngeal isolates of c. norvegensis produced high amounts of extracellular aspartic protease and phospholipase similarly to c. albicans strains. these enzymes may contribute to the pathogenesis of this new emerging candida species. in vitro activities of antifungal and antiseptic agents against rhodotorula sp pm preney l, théraud m, guiguen c, gangneux jp. laboratory parasitologie-mycologie, chu de rennes, france purpose of the study: rhodotorula species are common saprophyte yeasts widespread in nature. since the last years, they have been implicated in several severe infections, especially in immunocompromised patients, and various antifungal therapies were used. however, only limited data are available on the susceptibility of rhodotorula sp. to antifungal and antiseptic agents. material and methods: in this work, we evaluated the in vitro activities of eight antifungal agents against strains of rhodotorula ( strains of r. rubra and nine strains of r. glutinis using atb fungus system (biomerieux) and etest strips (ab-biodisk). beside, the effect of eight antiseptic agents was assessed on a suspension of r. rubra . the quantification of yeasts after exposure to antiseptic agents was performed by subculturings using a microtitration method in well plates. results and discussion: all strains tested were susceptible to amphotericin b, fc, and nystatin. twenty-nine out of strains were susceptible to ketoconazole, out of were intermediate to econazole. all strains were resistant to fluconazole (cmi!/ mg/ml) and itraconazole (cmi!/ mg/ml), and out of were resistant to miconazole, suggesting that antifungal therapy must be adapted when rhodotorula yeasts are implicated in invasive infection. beside, min exposure to sodium hypochlorite , chlorhexidine . % or ecodiol (isopropyl alcohol'/alkylamin) showed fungicidal activities. susceptibility testing of aspergillus fumigatus and emerging aspergillus pathogens by a modification of the nccls m -p method pm logotheti m a , kapsanaki-gotsi e b , velegraki a a , zagoura d b . a department of microbiology, mycology reference laboratory, medical school, university of athens, athens, greece , b biology department, section ecology and systematics, university of athens, athens, greece aspergillosis in high risk groups of patients is still associated with high mortality rate ( Á/ %). aspergillus fumigatus is the primary pathogen, while other opportunistic aspergillus species are emerging. amphotericin b (ab), itraconazole (it), voriconazole (vo) and terbinafine (te) minimum inhibitory concentrations (mic) were determined by modifying the nccls m -p microdilution method. stock drug solutions were prepared in rpmi , dimethyl sulfoxide (dmso), and polyethylene glycol (peg ). inocula, of the a. fumigatus group ( ), a. flavus group ( ) ( ) and the m -p quality control strains were prepared according to, and by modifying, the nccls guidelines. plates were incubated at and c and read at and h. peg effectively dissolved it and vo, while either dmso or peg dissolved te. low and c Á/ h ab, it, vo and te mics ( . Á/ . mg/l) were recorded. a. terreus ( ) and a. parasiticus ( ) were resistant to ab. certain clinical isolates demonstrate clinical and in vitro resistance. standardization of susceptibility testing would offer reliable assistance in selecting and monitoring antifungal therapy. otag f, aslan, g, ozturk c. microbiology department, faculty of medicine, mersin university, mersin, turkey rates of opportunistic fungal infections have risen markedly. because some of these species have potential resistance to antifungal agents, rapid presumptive species level identification is crucial in allowing for directed antifungal therapy. in this study, isolated yeasts from the clinical specimens were identified by atb id c (biomerieux, france). the number of identified yeasts were, respectively; ( %) candida albicans , six ( %) c. glabrata , five ( . %) c. tropicalis , three ( . %) c. parapsilosis , two ( . %) c. krusei , two ( . %) c. kefyr , one ( . %) c. guillermondii , one ( . %) c. dubliniensis . twenty-one of strains were investigated for antifungal sensitivities by atb fungus kit (biomerieux, france). the results are as follows: % sensitivity was detected to myconasol, % to flusitozin, nystatin and econasol, % to amphtericin b and ketokonazol. it is important to achieve empirik treatment of the opportunistic candida infections and the following of resistance to antifungals. shakhmatov da, strelchenco, ov. novosibirsk state medical academy, dermatovenerology, novosibirsk, russian federation at the present stage in russia with a background of a high case rate of syphilis, it becomes necessary to exclude biological false positive serological tests. because the serodiagnosis of syphilis has significant limitations, the direct detection of t. pallidum in suspect blood may serve as an alternate diagnostic strategy. polymerase chain reaction (pcr) has been the most widely used amplification method. the study of patients receiving examination related and treatment for syphilis in std clinic and persons directed from other hospitals where routine serologic examination revealed doubtful results. pcr reaction was carried out with nested primer pairs based on the dna sequence of the Á/ and Á/ kda gene of t. pallidum . pcr was utilized with whole blood. a complex of serological tests: fta-abs and tit was used as the &rdqup; gold standard''. as a result the sensitivity of pcr was . % and specificity was . %. selective comparison of pcr results with vdrl, the fta-abs and treponemal immobilisation test (tit) has shown concurrence . %. in conclusion, the preliminary results of pcr in whole blood in syphilis detection revealed its high sensitivity and specificity; possibility to obtain rapid results in unclear cases. chlamydia pneumoniae (cp) is an atypical pathogen whit intracellular location, whose eradication is very difficult. in the past years it has been objects of many studies that lead to the demonstration of a relationship between its presence and the development of widespread multifactorial pathologies such as atherosclerosis and asthma. the lack of its eradication can become an important clinical and social problem. the study objective is the comprehension of pathogen Á/host interaction mechanism, to characterize therapeutics protocols that cold lead to complete eradication of cp from organism. the research had been principally made on the studying the molecular mechanisms that are at the root of pathogen permanence inside host cell. using proliferation and apoptosis tests we underlined a different behaviour of infected cells towards control cells. in presence of p ( mg/ml), i.e. a peptide that can inhibit the proliferation and induce apoptosis in vitro inhibiting nf-kb, uninfected cells proliferation decreased of % in comparison whit the controls, while the one of infected decreased only of about %. moreover, using various apoptosis-inducers, the infected cells showing apoptosis were about % while the uninfected were about %. the caspace iii activity increased significantly in uninfected cells. in conclusion, cp could delay its elimination from the host inhibiting the apoptosis via nf-kb activation. hryniewiecki t a , gzyl a b , rawczynska-englert i a , a department of acquired valvular heart disease, national institute of cardiology, warsaw, poland , b department of sera and vaccines, national institute of hygiene, warsaw, poland infective endocarditis (ie) frequently causes problems in diagnosis, especially where blood cultures are negative and with fungal etiology (also as a fungal superinfection in bacterial ie). the purpose of the study: the purpose of the study was to evaluate the usefulness of broad-range fungal pcr in diagnosis of fungal superinfection of bacterial ie. twenty-five blood samples were taken for analysis from patients with infective endocarditis. ie was diagnosed according to duke criteria including positive blood cultures. suspicion of fungal superinfection was established on serological investigation in five patients, confirmed by blood culture in two patients. control group consisted of patients without infection. dna was isolated using the commercially available s.n.a.p. kit. amplification products were analyzed by gel electrophoresis stained with ethidium bromide. the results obtained: fungal dna was found in two patients with fungal superinfection of bacterial ie confirmed by culture. in the remaining patients with ie and controls no fungal dna was found. the conclusion reached: broad-range fungal pcr is a fast and inexpensive tool for the detection of fungal dna, but it is more prone to contamination than species-specific pcr. the method may be valuable in the identification of fungal superinfection of bacterial ie or diagnosis of fungal ie. rivanera d, lilli d, lozzi ma, piunno m, mancini c. microbiology, science and public health, rome, italy aim: the aim of this study was to evaluate the eia method for detection of antibody to ttv virus (ttv) and to investigate the anti-tt virus prevalence in patients with hepatitis b (hbv) virus, hepatitis c (hcv) virus, in group of 'high risk'subjects to hepatitis and in healthy subjects. the elisa methods (nuclear laser vienna lab) using ttv s and ns antigens: orf ( aa) and orf ( aa) was applied to detect anti-ttv; the serological screening was performed from samples to italian subjects. results: the positive rates of anti-ttv antibodies were . % in patients with hepatitis b Á/c and . % in 'high risk' hepatitis patients. the anti-ttv was also found in . % in healthy people. conclusions: the anti-ttv were detected in all groups studied, however, its positive rate was similar in patients with hepatitis b Á/c and in 'high risk' hepatitis respect to heathly people. our results shown that tt virus is frequent in italy both in patients infected by others transmitted viruses and in general population. the positivity found in healthy adults included in our studies suggests that the virus might be transmitted non-parenterally. the study of pattern of antibody to ttv may be an infectious marker of ttv similar to that of anti-hcv. a stress test on a miniaturized identification system designed for neisseria and haemophilus pm rich m a , bannatyne rm a , memish za b . a king fahad national guard hospital, division of microbiology, riyadh, saudi arabia , b king fahad national guard hospital, infection prevention and control, riyadh, saudi arabia we report an incident that occurred in our laboratory when the bbl crystal identification system for neisseria and haemophilus was used to identify a haemophilus-like-organism. the numerical profile generated was not in the system database. conventional biochemical tests subsequently revealed an identification of brucella melitensis , a common isolate in our area. as a result of this revelation we subjected this system to a mini 'stress-test' with a collection of isolates of b. melitensis . two numerical profiles were obtained, and , neither of which are listed in the system database. brucella species have been misidentified as moraxella species, moraxella phenylpyruvica , and as haemophilus influenzae biotype iv in various identification systems. two cases of laboratory-acquired brucellosis have been attributed to misidentification. to its credit the bbl crystal identification system for neisseria and haemophilus neither generates a profile number with a misidentified organism nor assigns a confidence level. instead it properly directs the user to resort to conventional methods to secure an identification. if further studies on additional brucella isolates and strains from different geographical sources confirm the unique biochemical profiles identified here, it may be worthwhile to incorporate these into the database where they would be of considerable assistance in areas where brucellosis is widespread. cloning and characterization of aflmp in aspergillus flavus pm chong tk, woo pcy, leung asp, yuen ky. the university of hong kong, microbiology, hong kong, hong kong purpose of the study: to clone and characterize an antigenic protein for serodiagnosis of infection caused by aspergillus flavus which is the commonest aspergillus species causing aspergilloma (ao) and invasive aspergillosis (ia) in asia. result obtained: we cloned the aflmp gene, which encodes the first antigenic cell wall protein in a. flavus . aflmp codes for a protein, aflmp p, of amino acid residues, with sequence features that are present in mp p and afmp p, the antigenic cell wall mannoprotein in penicillium marneffei and aspergillus fumigatus that we described previously. it contains a serine-and threonine-rich region for o glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. specific anti-aflmp p antibody was generated with recombinant aflmp p protein purified from escherichia coli to allow further characterization of aflmp p. indirect immunofluorescent staining indicated that aflmp p is present in the cell walls of the hyphae and conidia of a. flavus . furthermore, it was observed that patients with ao and ia due to a. flavus develop a specific antibody response against aflmp p. conclusion reached: this suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with ao or ia, and the protein may represent a good cell surface target for host humoral immunitiy. grape purpose: to investigate the basis for increasing resistance to trimethoprim and sulphamethoxazole. methods: pcr screening for integrons of clinical urinary tract isolates was performed. isolates were tested for resistance to antibiotics. integrons in isolates were sequenced. results: integrons of class were found in isolates and class integrons were found in . eight isolates in the study were resistant to five antibiotics or more and not shown to carry any integron. nineteen of isolates resistant to trimethoprim did not carry integrons. only one of these isolates was shown to carry sul and is thus probably also carrying an integron. none of the isolates were shown to carry dfr , one of five trimethoprim resistance genes known to exist outside integrons. three isolates were resistant to sulphonamides but were not shown to carry neither sul nor sul . only dfr and aad gene cassettes were found in the sequenced integrons. conclusions: resistance to trimethoprim in of trimethoprim resistant isolates is mediated by genes not detectable, as in the case with three sulphonamide resistant isolates. sequenced integrons that contain dfr genes do not carry any gene cassettes mediating resistance to modern antibiotics. unusual diagnosis tool for an unusual presentation of alveolar echinococcosis: report of two cases of local progression after an animal bite pm bardonnet k, bart jm, loiseau j, gérard a, estavoyer jm, heyd b, badet jm, dubiez a, piarroux r, bresson-hadni s. who collaborating centre for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the classical human contamination route for alveolar echinococcosis (ae) is ingestion of eggs. two exceptional human cases are reported with extensive local evolution of ae after a bite. case no. : between and , a patient underwent surgery seven times for a muscle growing tumour which developed after a bite. the diagnosis of muscle ae was assessed on histopathological examination. in , serological tests were in accordance with echinococcus sp infection. case no. : in , a man presented 'cat-scratch fever' with a right supraclavicular tumefaction following a cat bite. between and , five recurrences occurred. different surgical explorations indicated multiple abscesses of the cervical muscles. in , serological tests were in favour of echinococcus sp infection and the pathologist described a parasitic wall suggesting hydatidosis, but specific pcr from histological samples prompted the diagnosis of ae. conclusion: in these exceptional observations, the liver which is the most usual location of ae was lesion-free. the chronic inflammatory ae lesions have developed in the local lymphatic chain area of the bite site. to perform diagnosis in these very unusual forms of ae, it is necessary to add unusual tests such as specific pcr to classical tests. antibiotic resistance in foodborne salmonella is an emerging public health concern. integrons are now recognized as the main genetic vehicles of antibiotic resistance in gram-negative bacteria, including in salmonella . the purpose of the present study was to investigate the presence of class i integrons in resistant isolates of several serotypes of salmonella isolated from poultry products and to determine their association with multidrug-resistance phenotypes. a total of isolates of salmonella belonging to seven different serotypes were tested. the most frequent multiresistant phenotype, found alone or together with other resistances, was to streptomycin and tetracycline. all but seven were resistant to three or more antimicrobial agents, including quinolones and amoxicillin. pcr analysis with the ?cs and ?cs primers detected the presence of class i integrons of . kb in one isolate, with the multiresistant phenotype: amoxicillin, chloramphenicol, streptomycin, trimethoprim-sulphametoxazol and tetracycline. our findings suggest that the uncontrolled use of the antimicrobial agents in food animals may have contributed to the development of the pattern of resistance observed in salmonella isolates. also the presence of integrons in low prevalent human salmonella serotypes but associated with food animals underscores the public health problem of antibiotic resistance acquisition and spread. prevalence and antimicrobial resistance of campylobacter jejuni and c. coli isolated from broilers and pigs in france pm avrain l a , humbert f b , sanders p c , kempf i a . a afssa, umb, ploufragan, france , b afssa, hqpap, ploufragan, france , c afssa, lermvd, fougères, france in , caeca from standard, export or free-range broilers and in , fecal samples from pigs, were collected in french slaughterhouses. prevalence of campylobacter jejuni and c. coli strains was . % in standard, . % in export and % in free-range broilers. in standard and export productions, the most often isolated species was c. jejuni , whereas c. coli was predominant in free-range production. . % samples collected from pigs contained c. coli . the sensitivity of strains to ampicillin, nalidixic acid, enrofloxacin (broilers) or ciprofloxacin (pigs), tetracycline, erythromycin and gentamicin was tested by an agar dilution method. in broilers, the percentages of resistant strains were, respectively , , , , . and % for c. jejuni and , , , , and % for c. coli . in pigs the percentages of resistant c. coli were, respectively , , , , and %. in broiler production, significant differences between distributions of species or percentages of resistant strains were observed according to type of production or administrated antimicrobials. the enzyme dhps (dihydropteroate synthase) participates in the folate synthesis pathway, and is well recognized as the target for sulphonamides. the enzyme preceding dhps in this pathway, pppk (dihydropterin pyrophosphokinase), is another interesting candidate drug target. the metabolic role of pppk is to provide one of the substrates for dhps. earlier studies have suggested that pppk and dhps enzymes need to have physical contact with each other for full enzyme activity. studies of potential interactions between the enzymes have been initiated. so far, indication of a weak interaction has been detected in gelfiltration experiments and the two-hybrid system. to confirm these results, we are currently developing a method to study substrate channeling, as interfering with such interactions could lead to impaired growth and thus be used as inhibitory drugs. we have also cloned and sequenced the operons coding for the enzymes in the folate biosynthesis from different clinical isolates of streptococcus pyogenes . comparisons revealed some isolates with a mosaic structure in the operon, suggesting that horizontal transfer of genetic material has occurred. multi-resistance gene cluster on a plasmid in a clinical isolate of e. faecium pm werner g, hildebrandt b, klare i, witte w. department of nosocomial infections, robert koch institute, wernigerode branch, germany purpose: strain uw was isolated from an urine sample of a patient with a permanent catheter. the purpose of our study was to identify and localize the resistance determinants in this isolate. results: isolate uw was resistant to the following antibiotics: penicillin, ampicillin, gentamicin (high-level), streptomycin (highlevel), erythromycin, clindamycin, vancomycin, teicoplanin, ciprofloxacin, moxifloxacin, nourseothricin, rifampicin, and fusidic acid (lowlevel, mic / mg/l); but showed susceptibilities to oxytetracycline, phosphomycin, chloramphenicol, trimethoprim/sulfamethoxazol, linezolid, and quinupristin/dalfopristin. hybridization, pcr and sequencing experiments localized a cluster consisting of several resistance genes in a composite element on a plasmid. the cluster included genes and transposons tn (vana) Á/tn (ermb) Á/tn (aade Á/ sat Á/apha- ). the plasmid itself was not transferable in filter-matings into a fusidic acid high-level resistant enterococcus faecium recipient while selecting either for erythromycin or vancomycin resistances. however, after transposing a tn -related determinant into uw , determinants became mobilizable with the help of the conjugative transposon. transconjugants were, besides others, high-level resistant to fusidic acid, but susceptible to penicillin and ampicillin. pfge of transconjugants demonstrated a pattern almost identical to the recipient but clearly different from the donor. conclusion: resistance genes in e. faecium could be arranged in a cluster and are mobile via mobilizable/transferable plasmids. lilli d, rivanera d, barbacini ig, lozzi ma, mancini c. department of science and public health, university la sapienza, microbiology, rome, italy aim: hepatitis g virus (hgv), a new rna virus that is parenterally trasmitted has frequentley been found in patients with chronic hepatitis c infection but its role in chronic liver desease is unknown. the aim of this study was to determine the prevalence of hgv infection in patients infected with hcv. ninety-eight patients infected with hcv were evaluated for the presence of hgv rna. the hcv genotypes distribution was genotype b, genotype a, genotype a and four genotype c/ d. hcv rna and hgv rna were detected by rt-nested pcr. results: infection with hepatitis g virus was detected in ( . %) patients and ( . %) were hgv rna negative. none of our patients with genotypes a and c/ d results hgv rna positive. prevalence of hgv infection was % in patients infected with hcv genotype b and . % with genotype a. conclusions: infection with hgv occurred frequently ( . %) in this sample of patients with chronic hepatitis c. we observed a height prevalence of hcv/hgv coinfection in patients infected with hcv genotype a. this association with hcv genotype a was indipendent of the source of infection, infact some of our patients have not history of intravenous drug use. characterization of extended-spectrum beta-lactamase (esbl)mediated resistance in salmonella spp. from durban, south africa pm moodley p a , essack s b , gajee k a , sturm w a . a department of medical microbiology, nelson r. mandela school of medicine, school of infection, university of natal, durban, south africa , b school of pharmacy and pharmacology, university of durban westville, durban, south africa background: gastroenteritis is a common condition among the paediatric population presenting to king edward viii hospital in durban, south africa. from july , we noticed that the susceptibility of the salmonella spp. isolated from stool samples among these children were resistant to multiple antibiotics. aim: to characterize the phenotype of the resistance mechanisms involved. methods: minimum inhibitory concentrations (mics) of ampicillin, azithromycin, ciprofloxacin, cefepime, cefuroxime, cefotaxime, ceftazidime, ceftriaxone, cefoxitin, chloramphenicol, cotrimoxazole and gentamicin were determined by means of the agar dilution method. isolates were subjected to the e -test for extended-spectrum betalactamase (esbl) production. isoelectric focusing was performed as a preliminary step in enzyme characterization. results and conclusion: thirty isolates of multiresistant salmonella spp. were obtained. antibiogram typing revealed six different resistance phenotypes. all isolates depicted ceftazidime/ceftazidime Á/clavulanate ratio of !/ and were considered putative esbl-producers. isolates expressed Á/ beta-lactamases each with pi values ranging between and . indicative of tem-, shv-and/or ctx-m-related esbls. nine isolates expressed two beta-lactamases each and two isolates expressed three beta-lactamases each. there was evidence of the simultaneous expression of both tem-and shv-derived esbls as well as the simultaneous expression of multiple tem-or shv-derived esbls in single isolates, a phenomenon reported in esbl-positive klebsiella pneumoniae isolated at the same hospital. neutrophils exhibit reduced chemiluminescence response to serum opsonized klebsiella pneumoniae producing extended spectrum b-lactamases (esbl) pm objective: to investigate the ability of esbl and non-esblproducing klebsiella pneumoniae isolates treated with human serum to induce a chemiluminescence response in neutrophils. methods: oxidative burst induced by the interaction of esbl (n / ) and non-esbl-producing (n / ) klebsiella pneumoniae isolates with neutrophils from healthy individuals was monitored by measuring the chemiluminescence response (cl). pooled sera from healthy individuals served as source of complement for pretreatment of the bacteria. cl responses triggered by serum treated zymosan served as positive control. the serum opsonized klebsiella strains were arbitrarily graded as high (h) and low (l) inducers of cl when the cl response induced by the bacteria was cl b/ and !/ %, respectively, of that induced by opsonized zymosan. results: out of non-esbl-producing klebsiella isolates, . % induced high cl response in neutrophils whereas only % of esbl-producing klebsiella isolates did so (pb/ . ). conclusions: strains harboring the esbl plasmid were more virulent than non-esbl-producing strains by virtue of their higher tendency to escape serum-dependent recognition by neutrophils. osiris */an automated system for susceptibility testing in agar diffusion technique pm chegrani f, kolbert m, shah pm. universitätsklinik frankfurt, zentrum der inneren medizin med iii schwerpunkt infektiologie, frankfurt am main, germany objective: osiris measured zone sizes were compared to manually measured inhibition zones using round (rp) and mm mueller hinton square agarplates (sqp). variations of / mm in zone size measurements were defined as tolerable. 'very major errors' (vme) were defined as classification of a resistant organism as sensitive by osiris. thirty thousand two hundred and ninety-eight single measurements testing antibiotics on staphylococci and enterobacteriaceae were done according to the din recommendations. results: vancomycin, rifampicin, gentamicin gave the best results on rp with a concordance of , and %. vancomycin, rifampicin, teicoplanin performed best with , , % on sqp testing staphylococci . worst results on rp gave cefuroxim ( . %) and fosfomycin ( . %), on sqp fosfomycin ( . %), ofloxacin ( . %). for enterobacteriaceae amikacin ( %), gentamicin ( %), ciprofloxacin ( %) performed best on rp; worst nalidixinacid ( . %), piperacillin ( . %). concordance on sqp amikacin ( %), cefotaxim ( %), gentamicin ( %), nitrofurantoin ( . %), cotrimoxazol ( %). very major errors were seen in b/ % of all test performed. interpretation: osiris is a rapid and reliable system for susceptibility testing with round and square agarplates and has an excellent expert system. altindis m a , aktepe oc a , kocagoz t b . a kocatepe university school of medicine, microbiology, afyon, turkey , b diomed inc. tr, istanbul, turkey dio-bacit, in a two section plate, that contains % sheep blood agar on one side and sheep blood agar with bacitracin ( mg/ml) was compared for its efficiency in identification of group a beta hemolytic streptococcus (gabs) with other two different growth plates, one containing % sheep blood agar with bacitracin (b) and the other containing b-sxt. we used latex-agglutination for this comparision. throat specimens obtained from cases were inoculated to dio-bacit plates, first to one side with % sheep blood agar and to the other side with b. after an overnight inoculation at c, colonies with beta hemolysis an % sheep blood agar but no growth with b, were inoculated to % sheep blood agar again and antibiogram identification discs containing . u b and . and . mg sxt (oxoid, uk) were placed onto the plate and incubated overnight at c. after that, colonies with beta hemolysis were defined as b-sensitive while colonies resistant to sxt were defined to be gabs. all colonies are serologically classified by latex-agglutination (oxoid, uk). seventy-one ( . %) inoculations revealed growth of gabs at dio-bacit plates. after inoculating these colonies to % sheep blood agar, of them were found to be sensitive to b, while were found to be sensitive to b but resistant to sxt and of them were defined as gabs by latex test. when compared with latex-agglutination test, we found dio-bacit method's sensitivity and spesificity to be and . %, respectively. method: agar diffusion technique as recommended by din was used to determine izs (read using aura and manually) for staphylococci and enterobacteriaceae . variations in automated measured zone sizes of / mm to the manual readings were considered to be within acceptable range. results: six thousand and fifty-two zone sizes were determined for staphylococci and for enterobacteriaceae . mha displayed tendency to smaller zone sizes in automated readings than isa, as well in staphylococci and enterobacteriaceae . on the other side automated readings presented on isa more precise results than mha. overall less major discrepancies ( b/ mm) were found on isa. izs were generally smaller on mha. the tables below show differences in manually and automated measured zone sizes on different media and species. we discovered seven more cases of resistance in the case of metronidazole. we did not have such experience with clarithromycin. conclusion: our results show that e -test is comparable to ad for clarithromycin, but for metronidazole our findings confirm nccls recommendantion. classical ad is time consuming for every day use in the laboratory. the use of screening agar plate with mg/ml of metronidazole to detect possible resistance could be the solution. rokosz a a , sawicka-grzelak a a , meszaros j b , luczak m a . a department of medical microbiology, the university medical school, warsaw, poland , b department of bacteriology, state institute of hygiene, warsaw, poland purpose: to identify esbl-positive strains and to compare two methods applied for the detection of extended-spectrum beta-lactamases (esbls). methods: two hundred and sixty strains of gram-negative rods were cultured from clinical specimens from hospitalized patients. identification of strains was performed in the automatic atb system (biomerieux, france). these strains were identified as esbl-positive on the basis of the double-disc synergy test (ddst according to jarlier et al., ) results. all strains were also determined using a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: cpd (cefpodoxime) and cd (cefpodoxime/clavulanic acid) (oxoid, england). results: consistent results of two methods (ddst and dd) were obtained in the case of from among of examined strains ( . %). consistent results concerned out of strains of enteric rods ( . %) and only five from among other strains (mostly nonfermenting rods). conclusions: the novel method of esbl-producers detection (dd) is more objective and easier for interpretation than the double-disc synergy test (ddst). diagnostic disc test should be used as the basic one or to confirm the results of ddst in difficult cases. assessment of e -test for determining penicillin resistance in pneumococci pm sener b, yeniþehirli g, ercis s, hasçelik g. department of clinical microbiology, hacettepe university medical faculty, ankara, turkey there is a greater need for susceptibility testing methods that distinguish between susceptible and resistant pneumococci. an alternative method could be the e -test, which is compared with the reference agar dilution method in this study. penicillin susceptibility of a total of pneumococci was determined by e -test and agar dilution methods. streptococcus pneumoniae atcc and enterococcus faecalis atcc were used as controls. the results were given in the effect of anoxic conditions on the minimum inhibitory concentration of metronidazole in helicobacter pylori pm de la obra sanz p, lomas e, roman jl, alarcon t, lopez-brea m. the objective of this study was to determine the effect of incubation under anoxic conditions on the metronidazole resistance of helicobacter pylori . methods: a total of clinical isolates were used in this study. mics were determined by an agar dilution method using mueller-hinton agar plus % lysed horse blood. three plates series contained twofold dilutions of metronidazole from to . mg/l were prepared. the first one was incubated under microaerophilic conditions (oxoid) for days; second and third series were incubated anaerobically (anaerobic system, oxoid) for and h, respectively, and were then transferred to the microaerophilic enviroment up to complete days of incubation. results: with microaerophilic incubation, of strains were resistant (mic and mic were and , respectively). with h anaerobic preincubation, four of strains were resistant (mic and mic were . and , respectively). with h anaerobic preincubation, of strains was resistant (mic and mic were . and , respectively). conclusions: anaerobic preincubations causes an increase in sensitivity to metronidazole, the extent of which was dependent on the length of the anaerobic period. methods: the susceptibility to antibiotics was performed by microdilution method according to nccls guidelines. the production of b-lactamase was tested by nitrocefin sticks (oxoid). results: the mics /mics (mg/ml) appeared, respectively: ampicillin / , amoxicillin/clavulanic . / . , cefaclor / , ceftriazone . / . , erythromycin . / . , azithromycin / . / . , clarithromycin . / . , ciprofloxacin . / . , imipenem / . / . , tetracycline / . / / . , trimethoprim/sulfamethoxazole . / . . b-lactamase was detected in . % of the strains. conclusions: ( ) m. catarrhalis isolates were uniformly susceptible to all tested antimicrobials except ampicillin. ( ) the production of blactamase was responsible for ampicillin resistance ( . %). ( ) m. catarrhalis strains had almost the same behavior 'in vitro' to the tested microlides (erythromycin, azithromycin, clarythromycin). rokosz a, sawicka-grzelak a, luczak m. department of medical microbiology, the university medical school, warsaw, poland purpose: to isolate, identify and determine the drug-susceptibility of fungal strains cultured from fecal samples routinely submitted for detection of clostridium difficile and its toxins in cases of antibioticassociated diarrhea (aad). methods: one hundred fecal samples from hospitalized patients were examined (may Á/october ). c. difficile toxins a/b were detected directly in stools with c. difficile tox a/b ii test (techlab † , usa). fecal specimens were inoculated on ccca and candida id (biomerieux, france) media. c. difficile and fungi were identified with standard microbiological procedures. susceptibility of fungal strains to anti-fungal agents was determined (atb fungus, biomerieux, france). results: c. difficile toxins were detected in and c. difficile strains were isolated from of examined specimens. sixty-two fungal strains of genera were cultured from stool samples ( c. albicans isolates). massive fungal growths were observed on primary plates in all cases. fifty-five fungal strains were susceptible to nystatin, -to fluorocytosine, -to amphotericin b, -to ketoconazole, -to miconazole and -to econazole. conclusions: in some cases of antibiotic-associated diarrhea fungal strains are responsible for symptoms of this disease. certain persons having aad should be treated with anti-fungal agents. results: a total of isolates ( . %) were penicillin nonsusceptible (intermediate and resistant) and ( . %) were erythromycin non-susceptible. non-susceptibility to both antibiotics was found in ( . %). conclusions: if penicillin administration eliminates all penicillin susceptible strains, the prevalence of penicillin non-susceptible strains will increase as well as the erythromycin non-susceptible ones. this means that the proportion of erythromycin non-susceptible strains should increase from . to . %. at the same time, if erythromycin eliminate all susceptible strains to this antibiotic, the prevalence of penicillin non-susceptible strains would increase from the initial . to . %. these data can explain the co-selection results observed in different surveillance studies. antimicrobial susceptibility and capsular types/groups of streptococcus pneumoniae isolates causing pneumococcal diseases in bulgaria pm setchanova l a , gergova r a , ioneva m b , sredkova v c . a department of microbiology, medical university, sofia, bulgaria , b department of microbiology, ii city hospital-sofia, sofia, bulgaria , c department of microbiology, faculty of medicine, pleven, bulgaria a prospective study of pneumococcal infections was performed in cooperation with five clinical microbiology laboratories in bulgaria. mics values to antimicrobials and serotype/serogroup distribution were determined for strains of streptococcus pneumoniae . pneumococci were isolated from patients with systemic or respiratory infections. the incidence of penicillin g-intermediate and penicillin gresistant isolate was . and . %, respectively. the rates of resistance to other antimicrobials were: cefotaxime/ceftriaxone */ . %; erythromycin */ . %; clindamycin */ . %; tetracycline */ %; chloramphenicol */ . %; trimethoprim/sulfamothoxazole */ %; ciprofloxacin */ . %; rifampin */ . %. the s. pneumoniae isolates belonged to capsular types/groups. the most common serotypes/serogroups in bulgaria are , , , , , , we aimed to determine the pneumococcal antibiotic resistance rates and the serotypes of those resistant isolates in our hospital. the mic values of isolates (year Á/ ) were determined by agar dilution method. serotyping was performed by using pooled antisera of the pneumo-test. the results were as follows (n / ). objectives: to asses the antimicrobial susceptibility of clinical isolates of pseudomonas aeruginosa obtained from to and to monitor trends in antimicrobial resistance. methods: mics were determined by microdilution testing according to nccls. the antibiotics tested were: ceftazidime (caz), aztreonam (atm), imipenem (imp), gentamicin (cn), tobramycin (tb), amikacin (ak) and ciprofloxacin (cip). results: a total of isolates were included. urine was the most common site of isolation for outpatients isolates ( . %) while for hospitalized patients respiratory samples were the most frequent ( . %). susceptibility to antibiotics was: % caz, . % atm, % imp, . % cn, . % tb, . % ak and . % cip. comparison of susceptibility data through Á/ showed that the increase in the resistance rate was significative for caz ( . vs . %), atm ( . vs %), cn ( vs %), tb ( . vs . %), ak ( . vs . %) and cip ( . vs %). significant differences were found under the following circumstances: isolates from intensive care units and from inpatients were significatively more resistant to caz, atm and imp. isolates from respiratory samples were more resistant to caz and atm and isolates from urine samples were more resistant to cip. conclusions: although the antimicrobial susceptibility level has been decreasing p. aeruginosa isolates still show good susceptibility percentages for all antibiotics tested. antimicrobial susceptibility testing of clinical isolates of bordetella pertussis : report on isolates from rouen, france pm lemee l a , nouvellon m a , caron f b , lemeland jf a . a chu rouen, bacteriologie, rouen, france , b chu rouen, maladies infectieuses et tropicales, rouen, france reports of an increased clinical incidence of pertussis and the development of resistance by bordetella pertussis to erythromycin prompted the collection and antimicrobial susceptibility testing of recent clinical isolates from patients, who were hospitalized in rouen between and . mics of nine antimicrobial agents (erythromycin, josamycin, spiramycin, roxithromycin, ketolide hmr , cotrimoxazole, ciprofloxacin, rifampicin and amoxicillin) were measured by agar dilution method on mueller-hinton agar containing % horse blood. mbcs of erythromycin and rifampicin were also determined against four isolates of b. pertussis . all isolates were fully susceptible to the nine antimicrobial agents tested. mics (mcg/ml) were . for erythromycin, ketolide hmr and ciprofloxacin, . for josamycin, . for spiramycin, roxithromycin and rifampicin, . / . for cotrimoxazole, and for amoxicillin. mbcs (mcg/ml) were . Á/ . for eyrthromycin and . Á/ for rifampicin. in conclusion, our isolates of b. pertussis remain extremely susceptible to all antimicrobial agents tested, especially macrolides. no resistance was detected. finally, if erythromycin remains the molecule of choice, other macrolides (c and c ) also confirm their good in-vitro activity. in addition, the good in-vitro potency of rifampicin, together with its great diffusion within the respiratory tract, suggests that rifampicin has potential clinical efficacy in pertussis too. the emergence of streptococcus pneumoniae (sp) with diminished susceptibility to penicillin g (psdp) suggests the use of other antibiotics such as newer fluoroquinolones (fq). the resistance phenotypes of consecutive pneumococcal strains isolated from patients of four hospitals (observatoire régional des pneumocoques du nord-pas de calais) were studied: strains were susceptible to penicillin g and were psdp. reference strains provided from the centre national de réfeacute;rence des pneumocoques were added to the study. the activity of pefloxacin, ciprofloxacin, norfloxacin, sparfloxacin, levofloxacin and moxifloxacin was studied. reserpine was used to detect the efflux phenotype. methods used were performed according to the recommendations of the comité de l'antibiogramme de la société française de microbiologie. for each strain, the resistance phenotype to fq was deduced by comparison of mics or diameters obtained with those obtained with the reference strains of known phenotypes. fq resistance phenotypes were not correlated to blactam agent susceptibilities. wild type phenotype was observed among . and . % of the susceptible and psdp strains, respectively. a 'wild efflux' mechanism, deduced by addition of reserpine to norfloxacin, represented the predominant phenotype. it was detected among sp susceptible to penicillin g ( . %) as well as among psdp ( . %). the aim of this study was to determine macrolide resistance phenotypes of sp isolated in three french departments (alpes maritimes, doubs, nord) from nasopharyngeal aspirates of children aged months to years attending a dcc. a random sample of children attending randomly selected dccs was obtained during three periods (spring, autumn and winter ) in each department ( children attending dccs and children sampled). analysis of macrolide susceptibility of sp strains was performed using the ca-sfm method. out of strains, . % had decreased susceptibility to penicillin (spdp) and . % were resistant to erythromycin. the triple disk diffusion method (erythromycin (e), clindamycin (cl) and spiramycin) was used to determine resistance phenotypes. macrolide resistance is a well known phenomenon in france and is confirmed by our study. these results show that the constitutive phenotype is predominant as in other parts of europe and the frequency of efflux mechanism is lower than that observed in the usa and canada. developing antibiotic resistance surveillance of helicobacter pylori in england and wales pm elviss nc, owen rj. central public health laboratory, laboratory of enteric pathogens, london, uk purpose: helicobacter pylori antibiotic resistance is a key contributing factor in Â/ % of infected patients failing drug treatment. our aim was to survey rates of primary in-vitro resistance at different locations, and links to disease severity. antral gastric biopsies/cultures were received from phls in chelmsford, mid-essex ( isolates Á/ ); london ( isolates Á/ ); and bangor, north wales ( isolates Á/ ). susceptibilities to metronidazole (mtz), clarithromycin (cla), tetracycline (tet) and amoxicillin (amx) were tested by disc diffusion and also by e -test for cla and mtz. results: overall resistance rates ( isolates) were % for mtz and % for cla. all were susceptible to amx and tet. dual resistance rate was %. breakdown by location showed some marked differences. mtz resistance was highest in london ( %) compared to % in chelmsford and % in bangor. by contrast cla rates were % for london, and about % for bangor and chelmsford. in london, the majority of mtz resistant isolates were from non-uk borne individuals ( % non-uk vs % uk). comparison of duodenal ulcer-associated isolates with those from non-ulcer patients indicated similar rates of mtz resistance ( %). conclusion: resistance rates may vary significantly between locations depending on the local population with non-uk birth being a key risk factor for primary resistance with a mtz resistant strain. local resistance rates should be taken into account in test and treat strategies. potrykus j, benetkiewicz m, wegrzyn g. department of molecular biology, university of gdansk, gdansk, poland purpose of the study : because of their ability to extrude a wide range of compounds, multidrug efflux pumps have recently become an important issue in combating bacterial infections. acrab-tolc is the major efflux system of escherichia coli . we investigated the effect of acra on plasmid-borne and intrinsic chloramphenicol, tetracycline and ampicillin resistance. results and conclusions: recently, we reported a chloramphenicol sensitivity of e. coli mutant expressing cat , the chloramphenicol resistance gene. the strain was shown to bear a nonsense mutation in the acra gene. our studies indicate that this mutation is, at least in part, responsible for the observed chloramphenicol sensitivity phenotype. the mutation seems also to influence the strain's susceptibility to ampicillin and teta (c )-mediated (plasmid-borne) tetracycline resistance. although the teta(c) protein retained its biological function, there was a considerable growth impairment of the mutant strain when cultured in tetracycline containing medium. deletion of the acrab locus prevented any growth in the presence of tetracycline. upon the addition of ampicillin, the mutant underwent lysis more rapidly than the control strain. such was also observed in acrab deletion derivatives of other e. coli strains. we are trying to elucidate the role of the acra gene product in the phenomena described above. existence of efflux pumps in wild type isolates of drug-resistance bacteria pm raja ray rr. medical microbiology and parasitology, calcutta university, kolkata, india efflux pumps possessed by the bacterial cells of different kinds of bacteria had presented as a newer mode of drug resistance in many organisms. the capacity of bacterial cells to cause outward flow of noxious agents was known, however, for a considerable time with respect to tetracycline. recently, interest in the efflux pump system has brought to light some previously ill-understood mechanisms of drug resistance, involving noxious agents, toxins or poisons. we have found high level of resistance in pseudomonads towards cetrimide and other germicides for which no definite chromosomal/plasmid-mediated genes/mechanisms could be identified. likewise, occurrence of nonantibiotic sensitive vibrios, staphylococci and pseudomonads in the background of their high level of resistance to most of the common antibiotics suggest a mechanism of interference with the efflux pump, which accounts for such sensitivity in such cases. involvement of multiple resistance of marine isolates of v. parahaemolyticus to numerous clinically used antibiotics to which they have never been exposed also suggests a possible role of efflux pumps in determining such resistance */that these can simultaneously develop against multiple marine toxins/poisons and other noxious agents. interaction between oxacillin and glycopeptides in a teicoplanin-resistant mutant of staphylococcus epidermidis with reduced susceptibility to vancomycin pm greco aa, ben hassen a. laboratory service of national bone-marrow transplant center, tunis, tunisia we selected a laboratory-generated mutant of staphylococcus epidermidis capable of growing in the presence of mg/l of teicoplanin ( b tm ), from a methicillin-resistant (mic!/ mg/l), teicoplanin-sensitive (mic mg/l) and vancomycin-sensitive (mic mg/l) clinical isolate of s. epidermidis ( b so). in a previous work, we studied the different phenotypic characteristics acquired by the teicoplanin-resistant mutant b tm ( th interdisciplinary meeting on anti-infectious chemotherapy, december , poster sessions, /p ). in this work, we examined the interaction between oxacillin and glycopeptides against this teicoplanin-resistant mutant of s. epidermidis with reduced susceptibility to vancomycin. to study the combined antibiotic activity of oxacillin and glycopeptides, we used different methods: a modified disk diffusion test, the e -test, time-kill assays and population analysis profiles. the synergistic activity of glycopeptides in combination with oxacillin against the teicoplaninresistant mutant b tm was demonstrated with a bactericidal effect. no synergy was seen against the parental strain b so. moreover, the synergy between glycopeptides and oxacillin occurred with suppression of the subpopulation with the highest level of glycopeptides resistance. we concluded that combination of glycopeptides and oxacillin may be a possible alternative in the treatment of infections caused by methicillin-resistant, teicoplanin-resistant s. epidermidis . compositional changes in microcosm biofilms induced by application of minocycline: a preliminary study pm the aim of the study was to observe the effect of application of minocycline upon microcosm dental plaques. the plaques were cultivated in a constant departmenth film fermentor (cdff), which produces biofilms under conditions mimicking those present in vivo. the composition of the biofilms was determined by viable counting on selective and non-selective media. the proportion of antibiotic resistant genera within the biofilm was determined by viable counts utilising media containing minocycline ( mg/ml). before commencing antibiotic pulsing, the biofilms had a total viable anaerobic count of . )/ cfu per biofilm, with negligible ( cfu/biofilm) minocycline-resistant bacteria. however, h after introduction of the antibiotic, the total count had been reduced to . )/ cfu/biofilm whilst the number of minocycline-resistant bacteria had risen to . )/ cfu/biofilm. at the final sampling time point ( h) the total viable anaerobic count was . )/ cfu/biofilm whilst the number of minocycline-resistant bacteria was . )/ cfu/biofilm. hence, there is a very low basal level of inherent resistance to minocycline within microcosm dental plaques, but this increases considerably once the biofilms are exposed to minocycline. mechanism of resistance to aminoglycosides (amg) e. coli isolated from children with community-acquired urinary tract infections (cau-tis) pm methods: during the Á/ years nine centers took part in the study. the mics of antimicrobials were determined by the agar dilution method as described in the nccls guidelines. results: a total of consecutive urine isolates from children aged month to years with cauti were collected. the most frequently isolated species from children with cauti was e. coli ( . %), followed by klebsiella spp. ( . %) and proteus spp. ( . %). results of the in vitro susceptibility testing of e. coli to amg are shown in table below. resistance of the strains was conditioned on production of amg-modifying enzymes. there has been found following phenotypes among resistance strains: gentamicin Á/ tobramycin Á/netilmicin ( . % */aac( )-v and . % */aac( )-iv enzymes) and gentamicin Á/tobramycin ( . %, due to ant( ƒ) enzyme). conclusion: amikacin is most active amg against e. coli . resistance to gentamicin and netilmicin was mainly determined by production of aac( )-v enzyme. effect of b-lactamase inhibitors (b-l-i) on the evolution of resistance (r) to b-lactams (b-l) in gram the b-lactams antimicrobials still are the most frequently used. among the bacteria responsible of high resistance to b-lactams are gramnegative rods; its most frequent mechanism is the production of b-lactamase. the use of b-l-i has reversed partially this mechanism of resistance. we expect changes in the frequencies of r using b-lƒci after more than years. since , the venezuelan group of bacterial resistance, with health institution in the country; analyse and publish data on bacterial resistance of isolates from patients with bacterial infection coming from hospitals. it was used diffusion disk, according nccls. the software program whonet (world health organization net) was used. we follow the trends of r of gram-negative rods to b-l alone and with b-l-i during the decade Á/ . statistical analyses were made by evaluating the differences among percentages of resistance between the two series (p / . ). results and discussion: the difference in r between b-l and b-l/b-l-i are: ( ) piperacillin, piperacillin/tazobactam: between and % of r for most isolated, except for escherichia coli ( %) and serratia spp. ( %); ( ) ampicilin, ampicilin/sulbactam: between and %; ( ) cefoperazone, cefoperazone/sulbactam: between and %. how is expected gramnegative rods resistance to b-lactams with a b-l-i is lower than the b-lactam alone; furthermore the difference between both series, grows higher with time. these results are relevant and they were not expected, since b-l-i have been shown to be b-lactamase inductors. trends in the resistance (r) to b-lactams and others antimicrobials in p. aeruginosa in venezuelan medical centres. nosocomial (nos) and communitarian resistance pm in order to approach the infection produced by resistant bacteria, it is convenient to consider the hospital and the community as two separate ecosystems. the hospital ecosystem has special relevance in the infection and r of gramnegative aerobic bacilli. today, they are the main responsible of nos infection, with special reference to pseudomonas aeruginosa . infection by resistant bacteria is a world wide problem, specially related to nos. since , the venezuelan group of bacterial resistance, with health institution in the country; identify, analyse and publish data on bacterial r to antimicrobials: b-lactams, quinolones and aminoglicosides of isolates from patients with bacterial infection coming from hospitals and the community. it was used diffusion disk, according nccls. the software program whonet (world health organiza-tion net) was used. statistical significance (p / . ) was determined by application of the x technique. we show significant differences in the r of p. aeruginosa nos and communitarian (highest differences: piperacilina / %, tobra / %). we also established significant differences between the r arising in public hospitals and private hospitals (highest differences: ceftazidime / %, amika / %). we show the tendency in decreasing of frequency of r since ; this is more evident in private hospitals (b-lactam and aminoglicosides). new antimicrobials and new mechanism of action, and in the future the new technology will solve today's problem. however, the most important tools we have today are prevention and antimicrobials, and we must make them suitable. susceptibility to antibiotics of enterobacter cloacae and citrobacter freundii from drinking water pm quintera sm, sousa jc, peixe l. department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal the increased use of antimicrobials in farming, together with the practice of raw sewage discharge into receiving waters, has resulted in a significant increase in the number of antibiotic resistant bacteria present in aquatic environment. our objective was to determine the antimicrobial susceptibility, with focus on b-lactam resistance, among enterobacteriaceae strains isolated from raw drinking water samples. several isolates (n / ) of enterobacter cloacae and citrobacter freundii obtained from drinking waters were screened for antibiotic susceptibility patterns, using the agar diffusion technique, according to nccls's procedures. only % of e. cloacae strains, as well as % of c. freundii strains show resistance to amoxicillin and amoxicillin/ clavulanic acid. a reduced incidence of resistance to several others antibiotics was also observed. the obtained results suggest that strains isolated from raw drinking water have greater susceptibility to antimicrobial agents than pathogenic strains from hospital or outpatients infections. the 'natural' antimicrobial resistance phenotypes, usually described for c. freundii and e. cloacae , only seem to apply to strains isolated from human infections. notwithstanding the high susceptibility of the tested isolates to b-lactams, the role of environmental bacteria as a reservoir of resistance genes justify its periodical monitoring as a valid index for resistance spreading. a snapshot of the soil. using bacterial communities for tracing the evolution of metal-resistance pm quintera sm a , sousa jc a , peixe l a , monteiro nm b . a department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal , b department of zoology and anthropology, faculty of sciences, university of oporto, oporto, portugal it is well known that pathogenic bacteria, specially those resistant to antimicrobial agents and heavy metals poses public health risks of great concern, and its detection, namely in soils is generally related to pollution. in this study, the heavy metal resistance patterns of the microflora isolated from polluted (dump area) and unpolluted soil environments were examined. the plate growth covering percentage in the soil samples was determined using mueller-hinton plates supplemented with different heavy metal (al '/, cd '/, cu '/, pb '/, hg '/ and zn '/) concentrations. parallelly, using icp-aes, it was possible to ascertain the real heavy metal concentration for each soil sample. we found that the percentage of plate growth covering from the used samples was closely linked to the level of chemical pollution measured for each location. moreover, using anova, we found significant differences between locations. the dump site showed the highest tolerance to all the tested metals (newman Á/keuls test). this pattern of results was consistent when using the data from the icp-aes. furthermore, it was possible to observe that pseudomonas spp., with a relatively high mic for the studied metals, might become a relevant model for both public health issues and eco-toxicological studies. biochemical characteristics of environmental isolates of listeria monocytogenes pm moshtaghi h a , garg sr b , mandokhot uv b . a shahrekord university, food hygiene, shahrekord, islamic republic of iran , b haryana agricultural university, food hygiene, hisar, india purpose: the investigations were carried out to study the biochemical reactions of listeria monocytogenes isolated from different sources in the environment. results: a total of isolates of l. monocytogenes were obtained from samples of agricultural soil, faecal matter of animals and sewage. all the isolates were gram-positive, small rods, catalase positive, oxidase negative, motile with tumbling motility in hanging drop at Á/ c, aerobic, facultative anaerobic, fermentative and produced acid from glucose. all the isolates of l. monocytogenes were beta haemolytic and positive for camp reaction with staphylococcus aureus . all the isolates were negative for phenyl alanine deaminase, ornithine decarboxylase, lysine decarboxylase, malonate utilization and beta galactosidase tests. these were also negative for acid production from arabinose, d-xylose, mannitol, soluble starch and sucrose but acid was produced in rhamnose, salicin, and trehalose. hydrogen sulfide production was recorded in tripticase soy broth with lead acetate paper strips but negative with triple sugar iron agar. all the isolates were found to hydrolyse aesculin. out of isolates of l. monocytogenes only two produced acid from lactose. in serotyping all the isolates were serotype b. conclusion: we can conclude that l. monocytogenes serotype b at least in fermentation of lactose shows different reactions. methods: the samples were pre-enriched in bhi broth with and without vancomycin ( mg/l) and then plated onto m-enterococcus agar with and without antibiotics: vancomycin ( mg/l), gentamicin ( mg/l), kanamycin ( mg/l), and streptomycin ( mg/l). representative colonies of each morphology were isolated and identified as enterococcus sp as previous described. pcr was used to identify e. faecium and e. faecalis and to characterise vancomycin resistant genotype. api strep was also used in the identification. susceptibility testing to antibiotics was performed by an agar dilution method (nccls). results: three hundred and fifty-three enterococci were isolated from of a total of faecal samples ( %, n / / ). the majority of enterococci were identified as e. faecium , e. faecalis and enterococcus sp. resistance to almost all antibiotics studied was observed: vancomycin */ . %; teicoplanin */ . ; ampicillin */ . %; tetracyclin */ . ; erythromycin */ . %; ciprofloxacin */ . %; chloramphenicol */ . %; gentamicin */ . %; streptomycin */ . %; kanamycin */ . %; linezolid */ %. the vancomycin resistant enterococci presented a vana genotype. conclusion: resistance to several common antibiotics used in therapy was observed among enterococci isolated from healthy human from community. many of these isolates presented multi-resistance. of concern is the presence of vana genotype among these populations that may constitute a reservoir of vancomycin resistant genes. antimicrobial resistance in tetracycline-resistant oral bacteria pm mercury release from dental amalgam may select for mercuryresistant oral bacteria. mercury resistance is often associated with multiple antibiotic resistances. the aims of this study were to determine whether tetracycline-resistant oral bacteria from children with and without amalgam fillings were also resistant to: (a) mercury; and (b) multiple antibiotics. tetracycline-resistant organisms were isolated on iso-sensitest/blood agar containing tetracycline ( mg/ml). the mic of hgcl and several antibiotics were determined using agar dilution (bsac). one hundred and three organisms were isolated from patients without amalgam. ninety-one were streptococcus species, seven neisseria species, three veillonella dispar and two rothia species. fifty-seven percent exhibited resistance to at least one antibiotic, % were mercury-resistant, % were penicillin-resistant, % were ampicillin-resistant and % erythromycin-resistant. fiftytwo organisms were isolated from patients with amalgam. forty-five were streptococcus species, five neisseria species, one v. dispar and one staphylococcus aureus . sixty-three percent exhibited resistance to at least one antibiotic, % were mercury-resistant, % penicillinresistant, % were ampicillin-resistant and % showed erythromycin-resistance. statistically, the results showed that in tetracyclineresistant organisms, the presence of dental amalgam did not affect the level of resistance to mercury or to the antibiotics tested. conway-wallace hl a , mullany p a , bedi r b , wilson m a . a eastman dental institute, university college london, microbiology, london, uk , b eastman dental institute, university college london, transcultural oral health, london, uk the purpose of this study: to determine the prevalence of antibioticresistant oral bacteria in children who had not received antibiotics during the months prior to sampling. plaque samples were obtained from children aged Á/ years and plated onto media containing: penicillin, ampicillin, tetracycline, erythromycin and vancomycin. resistant isolates were enumerated, sub-cultured and frozen for subsequent identification. the process was repeated and months later. the results obtained: bacteria resistant to each of the antibiotics were present in all of the children at each sampling time (except in the case of ampicillin and penicillin at months). the proportion of antibiotic-resistant bacteria in the oral microflora ranged from ]/ . (erythromycin) to / . % (ampicillin). the proportions of bacteria resistant to a particular antibiotic remained reasonably constant over the -month sampling period. in only two cases (penicillin and ampicillin) was there a statistically significant change in the proportions of resistant bacteria at different time periods. the conclusion reached: the results of the study have revealed that bacteria resistant to a wide range of antibiotics may be isolated from children who have not been administered these agents during the months prior to sampling. furthermore, in many cases the proportion of bacteria resistant to a particular antibiotic remains constant over a -month period. antimicrobial use in the intensive care unit: results of a pharmacoepidemiological study in italy pm periti p. e.i.f.t. srl, firenze, italy a retrospective survey of antimicrobial chemotherapy use in intensive care units in italy was carried out in using a computerized questionnaire under the auspices of the journal of chemotherapy. of the icus contacted, . % replied, being mainly general or post-surgical and pediatric units having a mean of beds, nine doctors and nurses. the antimicrobial agents used in these wards were almost always polychemotherapy with prevalent use of beta-lactams, aminoglycosides and glycopeptides or as empirical treatment during the first h after hospital admission. the continual use of medium-high dose combinational antimicrobial chemotherapy was justified by microbiological testing, which revealed that more than one-third of bacterial pathogens were resistant. approximately, % of gram-positive bacteria were methicillin-resistant, whereas about % of gram-negative strains were resistant to at least one of the tested antibiotics. forty percent of the responding icus furnished microbiological testing data, of which three quarters indicated the incidence of chemoresistance of the isolated strains. fungal infections were less frequent than bacterial, the most commonly isolated agent being candida spp. in conclusion, the sample of icus examined showed adequate and reasonable use of antimicrobial agents, with heavy reliance on medium-high dose combination therapy due to the elevated incidence of resistant isolates found. plasma concentrations (p), urinary excretion (u) and bactericidal activity of gatifloxacin (gat) mg versus ciprofloxacin (cip) mg in healthy volunteers after a single oral dose pm boy d a , kinzig-schippers m b , sö rgel f b , well naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, ibmp, nürnberg-heroldsberg, germany twelve volunteers received a single oral dose of mg gat versus mg cip to assess p up to h, u (by hplc), and urinary bactericidal titers (ubt) in eight intervals up to h. the mean pmax of gat/cip was . / . mg. the ucum (mean) for gat/cip was . / . %. the ubts, i.e. the highest twofold dilution of urine still bactericidal, were determined for nine uropathogens and one reference strain */mics (mg/ml) (microdilution) for gat/cip: escherichia coli atcc ( . / . ); e. coli ( . / . ); klebsiella pneumoniae ( . / . ); proteus mirabilis ( . / . ); pseudomonas aeruginosa ( / . ); s. saprophyticus ( . / . ); two strains of s. aureus ( . / . ); two strains of e. faecalis ( . / and / ). the median ubts measured within the first h for gatifloxacin were between : and : for the five gram-negative strains (incl. p. aeruginosa ) and between : and : for the five gram-positive strains. the median ubts for ciprofloxacin were between : and : for the gramnegative strains (incl. p. aeruginosa ) and between : . and : for the five gram-positive strains. for the ubts up to h, gat was significantly superior to ciprofloxacin in all gram-positive strains, not different in the two e. coli strains, and inferior in the klebsiella , proteus and pseudomonas strains. for the ubts at Á/ h, gat was generally superior to cip, but showed no difference in the proteus and pseudomonas strains. gat showed overall comparable urinary bactericidal activity as cip. this is in agreement with a clinical study performed previously. malaria is one of the most prevalent endemic infectious disease affecting humans. in bichat hospital cases of malaria acute illness were reported during . among them, patients were hospitalised and intravenously treated by quinine. this retrospective study consisted of comparing the therapeutic drug monitoring (tdm) of quinine distinguishing, respectively and patients cured in infectious medical department (imd) and intensive care unit (icu) where a standardised quinine regimen was established ( and % malaria attacks, respectively). in icu, the treatment consisted of an infused loading dose mg/kg/ h of quinine diluted in % glucose followed by mg/kg/day. plasma quinine maximal concentrations were assessed after selective liquid Á/liquid extraction and spectrofluorometry detection. statistical analysis was performed using t -test. results showed that patients had comparable weight ( . / . and . / . kg) but quinine doses and plasma concentrations were significantly different in icu and imd, respectively ( . / . versus . / . mg/kg/day, pb/ . and . / . versus . / . mg/l, p b/ . ). in icu and imd, respectively: and % were in the therapeutic range ( Á/ mg/l) with and % below the requested therapeutic concentration ( mg/l) and and % above the limit of toxicity ( mg/l) conveying the importance of tdm in intravenous quinine treatment to avoid infra-therapeutic or toxic concentrations. simultaneous central nervous system distribution using microdialysis and pharmacokinetic Á/pharmacodynamic modelling of the electroencephalogram effect of norfloxacin in rats pm chenel m, marchand s, dupuis a, bouquet s, couet w. university of pharmacy, pharmacology, poitiers, france purpose: to investigate the epileptogenic activity of norfloxacin by a pharmacokinetic Á/pharmacodynamic (pk Á/pd) modelling approach and to assess the contribution of distributional processes across the blood Á/brain barrier (bbb) to the delayed effect. methods: rats (n / ) received an iv bolus dose of norfloxacin ( mg/kg). convulsant effect was quantified by electroencephalogram (eeg) recording during h post-dose. arterial blood samples were collected for drug assays in plasma. unbound norfloxacin concentrations were monitored in brain extracellular fluid (ecf) using microdialysis with in vivo calibration of the probes by retrodialysis with ciprofloxacin. results: the eeg effect reached its maximum between and min post-dose. a pk/pd effect compartment model was successfully fitted to these data. the relationship between effect and concentration at the effect site was best described by a spline function. norfloxacin concentrations in brain ecf were relatively low compared to plasma levels (ecf/plasma areas under curve (auc) ratio equal to . / . %), but central distribution was rapid. therefore, the effect versus brain ecf concentrations curves still exhibited a marked hysterisis. conclusion: the delay observed between plasma concentrations and norfloxacin convulsant effect cannot be explained by a slow distribution of norfloxacin across the bbb. pagoulatou a a , kanellakopoulou k b , vafiadou m a , kostakopoulos th c , kastriotis i b , giamarellou h c . a department of anesthesia, sismanoglio general hospital, greece , b th department of internal medicine, athens medical school, athens, greece , c department of urology, athens medical school, athens, greece csf kinetics of van and fu were studied in patients who underwent short urological surgery under spinal anesthesia. patients were excluded if they were already receiving an antibiotic or were suffering from renal and hepatic dysfunction. van was administered at g over h infusion. serum and csf samples were collected post-dose and the mean serum levels were as follows: min Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (six patients) and Á/ h: . mg/ml (seven patients). fu was administered at mg dose over h infusion. serum and csf samples were taken post-dose and the mean serum concentrations were found as follows: Á/ min: . mg/ml (six patients), min Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (five patients). in csf, both van and fu were undetectable. it is concluded that in the absence of meningeal inflammation van and fu do not penetrate (with the applied microbiological assay) the csf barrier. comparison of the pharmacology of intravenous and orally given moxifloxacin in an in-vitro model pm wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: the intravenous form (iv) of mg moxifloxacin (mox), one of the newer fluoroquinolones, has been recently approved by the fda. during the iv treatment higher peak serum concentrations are achieved in comparison to the oral administration (po) of the same dose. the antibacterial activity of fluoroquinolones is concentration dependent. we therefore simulated human pharmacokinetics of single po and iv dosages of mg mox in an in-vitro model using six different gram-negative and -positive pathogens to elucidate the different effect of these two dosing schedules. results: the comparison of the pharmacological parameter auc/ mic shows an increase ( table ) that could predict an enhanced antibacterial effect. however, the analysis of the killing curves with the following parameters, ka.max (maximal killing activity) and aac (area above the killing curve between and h), reveals no major difference between the po and iv dosage. conclusion: the serum concentration after oral administration is already sufficiently high to show the optimal bactericidal effect of mox that can only be slightly increased by higher peak concentrations and higher auc/mic ratios. thus the concentration dependence is not linear but ends already at concentrations achievable by oral dosing and documents that auc/mic calculations cannot easily be translated into dosing schedules. background : bacteria growing in vivo multiply much more slowly than in vitro. whether the bactericidal activity of quinolones may be affected by an increase in generation time (g) was studied in batch cultures. methods: by limiting the nutrient supply, generation times were lengthened from approximately . to . h up to . h. alternatively, the quinolones were added to the bacterial cultures during the lag-, exponential-and stationary phase. recent clinical isolates of escherichia coli were exposed to multiples of the mics of ciprofloxacin or norfloxacin. the 'killing rates' were calculated in analogy to the growth rate. results: the bactericidal activity of the quinolones tested against e. coli was minimally influenced by the reduced generation time. ciprofloxacin concentrations of ]/ )/mic eliminated the test strains within / h from the test system if added during the lag or exponential growth phase; four times higher concentrations were needed to reduce cfus by % within h, if added during the stationary phase. norfloxacin was significantly less active. conclusion: in contrast to norfloxacin, the bactericidal activity of ciprofloxacin is minimally affected by the generation time or growth phase of the bacteria. wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: moxifloxacin (mox) is one of the newer fluoroquinolones, now available for parenteral application. the pharmacology of an intravenous once-daily dose (od) of mg mox was determined with five gram-negative and -positive pathogens (streptococcus pyogenes , streptococcus pneumoniae , moraxella catarrhalis , escherichia coli , and klebsiella pneumoniae ). a twice-daily dose (bid) of mg mox was simulated with the gram-positive species in order to increase the bactericidal effect. results: to determine the efficacy, killing curves were analyzed, and following parameters were calculated: ka.max: maximal killing activity [log cfu]; ka. conclusion: an intravenous once-daily dose of mox is active against all tested pathogens. the gram-negative species are rapidly killed (ka. h similar to ka.max). there is no pronounced initial effect on the two gram-positive species but a general slow reduction in the viable cell count (ka.max is reached after h). the efficacy of mox (measured as aac and ka.max) on s. pyogenes and s. pneumoniae is to some extent increased after the second dose. however, the analysis of the killing curves reveals no major difference between od and bid. even the od nearly gives the maximal bacterical activity of mox against gram-positive pathogens. objectives: to evaluate the dose proportionality of amoxicillin and to compare the respective pk/pd parameters of two dosage regimens. methods: the dose proportionality of amoxicillin was evaluated using linear regression of mean auc -inf and c max data of different bioequivalence studies (n / volunteers) performed with formulations containing various amounts of amoxicillin alone or in the combination with clavulanic acid. the volunteers received a single oral dose in the range of Á/ mg. amoxicillin plasma concentrations were determined by hplc/uv or lc/ms/ms methods. time above mic (tmic) expressed in% of dosing interval was calculated with three target mic values ( . , . and . mg/l) for mg hourly and g -hourly dosage regimens. results: the absorption of amoxicillin (auc -inf ) showed a linear dependence with a correlation coefficient of . . the correlation coefficient of the linear regression for the cmax dependence on the actual dose was . . the respective tmic for both dosage regimens were very similar, with largely overlapping confidence intervals, supporting a pd breakpoint of mg/l for the g -hourly regimen (tmic ]/ mg/l: . %, % ci . , . %). conclusion: this analysis shows the dose proportionality of amoxicillin over the dosage range of Á/ mg and supports the pharmacodynamic rationale for a g bid dosage regimen. piperacillin/tazobactam concentration profile after high dose administration pattern in nosocomial pneumonias due to mecanical ventilation pm pedeboscq s a , gruson d b , bassoua v a , hilbert g b , pometan jp a . a st. andré hospital, pharmacy, bordeaux, france , b pellegrin hospital, reanimation, bordeaux, france the piperacillin (p)/tazobactam (t) antibacterial spectrum covers the largest part of bacteria responsible for pneumonias due to mechanical ventilation. but, due to important bacterial inoculum and pharmacokinetic parameter modifications in intensive care patients, high doses of beta-lactamines seem to be necessary to obtain antibiotic concentrations above suspected bacteria's mic (minimal inhibitory concentration) . this led us to compare, in patients with pneumonia due to mechanical ventilation, two intermittent administration patterns: g three times a day (usual pattern) versus g four times a day (high dose pattern). this study is carried out in collaboration with intensive care unit, bacteriological department and pharmacy where antibiotic concentrations are determined. twenty-three takings of blood are executed within a h period, in addition to two bronchial secretion samples. concerning p seric concentrations, the high dose pattern seems to be more adapted because of relatively high residual concentrations ( !/ mg/ml). three hours after each injection, t seric concentrations are lower than the mg/ml activity threshold. first and second day residual bronchial concentrations of p seem to be sufficient although t concentrations are below activity threshold. these results are to be correlated with the mic determined by the bacteriological department, and only this correlation will make us able to conclude the better efficacy of the high dose pattern in intensive care patients. anti-inflammatory drugs interference in absorption and tissue penetration of amoxycillin pm del fiol fs a , menon sz b , caramez th b , celotto tf b , lopes ras b . a university of sorocaba, pharmacology, sorocaba, brazil , b school of pharmacy, university of sorocaba, sorocaba, brazil antibiotics and anti-inflammatories are frequently associated in clinical practice. there is some concern about the quantity of antibiotic that reaches the infection sites, which may be reduced in the presence of an anti-inflammatory drug. the purpose of the present study was to analyse how steroids (dexamethasone (dexa)) and aines (celecoxib (cele)) influence on the penetration of amoxicillin to inflamed tissues. thirty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs to form granulomatous tissue. one week later the animals were divided into three groups. one group received only amox ( mg/kg), another received amox ( mg/kg) plus cele ( . mg/kg) and the last received amox ( mg/kg) plus dexa ( . mg/kg). one hour later the animals were sacrificed and the concentration of amoxicillin in the serum and tissue investigated. there was no difference among the groups in the quantity absorbed (amox / . / . mg/ml; amox'/cele / . / . mg/ml and amox'/dexa / . / . mg/ml). there was a reduction in the tissue concentration of amoxicillin (p b/ . tukey-kramer) for the group that received the drug with dexamethasone. for the other groups, there was no difference in the tissue concentration of amoxicillin. the results indicated that in inflamed tissue, a significant reduction of antibiotic penetration was induced by sinultaneous dexamethasone therapy. prediction of the optimal amoxicillin dose regimen based on coupling of pharmacokinetic data and bactericidal activity pm background: given its short half-life, amoxicillin (amx) should be administered at least three times a day to patients with acute exacerbations of chronic bronchitis, in order to achieve serum concentrations well above the mic of the responsible pathogen. however, several authors have recommended twice-daily administration of a higher dose for a shorter period. we assessed the relationship between amx sputum concentrations and antibacterial activity following two treatment schedules in healthy volunteers. subjects and methods: twelve healthy volunteers were randomized to receive amx for days at a dose of either g bd or mg bd. serum and sputum were collected every day, h after the morning administration, and again days after the last dose. amx concentrations were determined by hplc with fluorometric detection. sputum killing activity was determined against haemophilus influenzae , streptococcus pneumoniae and moraxella catarrhalis . results: mean serum concentrations measured h after the morning administration were . ( mg bd) and . mg/l ( g bd), and were above the mics of the three microrganisms. in contrast, sputum concentrations were always below . mg/l. in terms of sputum killing activity, g bd was more effective than mg bd against s. pneumoniae and m. catarrhalis , whereas no sputum samples were active on h. influenzae . conclusion: the optimal amoxicillin treatment schedule cannot be established on the basis of serum pharmacokinetics only. galmiche h, louchahi k, tod m, drugeon h, giroud jp, rouveix b. service de pharmacologie clinique, hopital cochin, paris, crepit , hopital avicenne, bobigny, service de microbiologie, hopital laennec, nantes, france background: cysteine-based mucolytics are commonly used in combination with antibiotics to treat patients with acute exacerbations of chronic bronchitis (aecb). they are also used to allow in vitro mic determination in sputum specimens. we conducted an in vitro and ex-vivo compatibility study designed to detect a possible interaction between mucolytics and antibiotics. methods: serial samples of bronchial secretions were collected from aecb patients and from healthy volunteers who received g of amoxicillin twice a day for days. two mucolytics were used to fluidify sputum specimens: , -dihydroxy- , -dithiolbutan (digest-eur † ) and acetylcysteine ( % solution). amoxicillin was assayed using a chromatographic system with fluorometric detection. each sample was also tested in a microbiological assay. results: amoxicillin could not be detected in the presence of the mucolytic agents. conclusions: this mucolytic Á/amoxicillin interaction may be explained by amoxicillin fixation to fluidified mucoproteins, and should be taken into account when assessing antibiotic efficacy in vivo. del fiol fs a , ferro c b , albuquerques et b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil physicians frequently recommend that macrolides should be administered with milk to decrease the discomfort they cause. thus the objective of this study was to verify the interference of milk in the absorption and distribution of erythromycin (eryt); clarithromycin (clar); roxithromycin (roxi) and azithromycin (azit). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs eryt, clar, roxi and azit with and without milk ( . ml/kg [ca'/'/] / . mg/ml). the animals were sacrificed and the serum and tissue concentration of the drugs was investigated. there was no reduction (pb/ . tukey-kramer) in the serum and tissue concentration in the presence of milk for azit and clar. there was a % reduction for roxi in the serum concentration in the presence of milk ( . / . and . / . mg/ml), but no alteration in the tissue concentration. there was a % reduction for eryt (p b/ . ), in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and a % reduction in the tissue concentration. the milk decreased the effectiveness of treatments with erythromycin and roxithromycin and the bioavailabilities of this macrolides were affected by co-administration with milk. del fiol fs a , souza gp b , duzzi mr b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil the degree to which tetracyclines are absorbed differs greatly. this absorption is impaired by the concurrent ingestion of divalent and trivalent cations. thus the objective of this study was to investigate the interference of milk in the absorption and distribution of tetracycline (tetr), oxytetracycline (oxyt), minocycline (mino) and doxycycline (doxy). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their back for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs: tetr; oxyt; mino; and doxy with and without milk ( . ml/kg [ca'/'/] / . mg/ml). the animals were sacrificed and the drug concentrations in the serum and tissue were determined. there was no reduction (pb/ . tukey-kramer) in the serum and tissue concentrations in the presence of milk for mino. there was a % reduction (p b/ . ) for doxy in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. for oxyt, there was a reduction of % (pb/ . ) in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. the tetr results show a . % reduction (p b/ . ) in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. milk decreased tetracycline bioavailability and effectiveness. isotopic studies with oxine labelled platelet. platelet kinetics in thrombocytopenic malaria patients pm introduction : thrombocytopenia is a common feature in human malaria ( ). excessive splenic platelet pooling has been suggested to play a role in uncomplicated cases of malaria, but a moderately shortened platelet life span during the period with decreasing parasitaemia seems the most plausible cause of the frequently observed thrombocytopenia ( , ). consumption coagulopathy, eventually manifested as disseminated intravascular coagulation, has been described in malaria ( ). in uncomplicated malaria, however disseminated intravascular coagulation is rarely found ( ). results: in malaria patients the sequestration was not different to normal. platelet half-life was reduced in patients with p. falciparum malaria to Á/ h (normal Â/ days). in one patient with p. vivax malaria platelet half live was . h. conclusion: no significant differences in the sequestration of platelets when compared to healthy individuals could be detected by in-labelled platelet scintigraphy. especially, no enhanced splenic sequestration, as previously expected, was the cause of the thrombocytopenia. therefore, other mechanisms than sequestration are responsible for the dramatically reduced life span of the platelets during acute malaria. zaharanka ag, rozhdestvensky da. vitebsk state medical university, vitebsk, belarus aim: the studying of chromatingeterogenic test (ct) results in sperm of subjects taking doxycycline (d) and some macrolides (erythromycine (e), jozamycine (j), and azytromycine (a)) in moderate therapeutic doses. methods: forty healthy volunteers ( Á/ years) were studied. daily dose of d was . ; e was administered in dose . four times per day days; j */ . before meals twice daily days; a */ . before meals once daily days. ct for evaluation of dna condition in human spermatozoids was performed before treatment (twice), on the th and th days of treatment, as well as after and months after treatment course completing. results: ct data analysis revealed that the mean amount of defective spermato-zoids before treatment was . '/ . %. by the th day of d treatment the index of de-natured dna was . '/ . % (pb/ . ), by the th day */ . '/ . % (pb/ . ). one and months after the d treatment course the amount of generative cells with denatured dna was . '/ . and . '/ . %, respectively (p b/ . in both case). under e treatment the amount of defective spermatozoids changed as . '/ . ( th day), . '/ . ( th day), . '/ . (after month), and . '/ . % (after months) (pb/ . in any case). under a using the ct results at the same control points were . '/ . , . '/ . , . '/ . , . '/ . % (pb/ . in any case); and under j treatment */ . '/ . , . '/ . , . '/ . , . '/ . %, respectively (p !/ . in any case). conclusions: the data obtained permit to conclude that d demonstrates the high level of toxicity to male generative cells. this effect preserves during months after the course of d treatment. objective: to study the effect of aggressive isolation and decontamination measures to control an outbreak of multi-resistant acinetobacter baumanii (mr-ab) in an icu. the outbreak: the index case was transferred from a mediterranean hospital, directly into an open-plan -bedded icu, with severe injuries to his head and thorax. he died shortly after admission. sputum, bronchoalveolar lavage fluid, blood cultures and a chest drain swab grew mr-ab, resistant to ampicillin, co-amoxiclav, aztreonam, amikacin, ceftazidime, cefotaxime, cefuroxime, ciprofloxacin, gentamicin, meropenem, piptazobactam, tobramycin and sensitive only to colistin. within days, mr-ab was isolated from two further icu patients. all isolates demonstrated identical antimicrobial susceptibility profiles. the icu was closed to admissions and thoroughly cleaned. all patients were isolated and their contacts screened. the icu was reopened, however, mr-ab was isolated from a fourth patient. this patient was isolated, the icu closed, for a second time, thoroughly cleaned, and all contacts isolated until discharge. all subsequent patients screened were negative for mr-ab conclusion: this illustrates the importance of aggressive isolation measures and thorough supervised cleaning in control of an outbreak, and the need to screen patients for resistant bacteria before admission to the intensive care unit in a general hospital. extended spectrum beta-lactamase-positive bacteria isolated in neonatal intensive care unit pm sandorcinova z a , siegfried l a , kmetova m a , viragova s b . a institute of medical microbiology, faculty of medicine, university of p.j. safarik, kosice, slovakia , b hospital, kosice-saca, neonatal intensive care unit, kosice, slovakia extended-spectrum beta-lactamases hydrolyse all penicilins, cephalosporins, including third-generation cephalosporins and aztreonam. esbl are predominantly produced by klebsiella spp. but may be presented in other enterobacteriaceae, too. the aim of present study was to investigate the occurrence of esbl-producing bacteria isolated from patients hospitalized at the neonatal intensive care units (icu). fifty escherichia coli and klebsiella spp. were isolated from rectum of patients hospitalized at the neonatal icu. the mics of antimicrobial agents were determined by the standard agar plate dilution method according to the nccls guidelines. for screening of esbl production we investigated strains showing reduced susceptibility (mic equal and/or more than ml/l) to at least one of third generation cephalosporins. esbl production was detected by double disk synergy test (ddst), e -test for esbl, and pcr employing specific primers for the presence of blashv and blatem genes. by using ddst and e -test, among e. coli isolates, an expression of esbl was detected in the three by the former method, while among klebsiella spp. isolates, a production of esbl was found in the two by the latter method. in esbl-positive e. coli strains the presence of blatem genes fragments was detected while in esbl-positive klebsiella spp. genes encoding for shv-type beta-lactamases were found. isolation of staphylococci from wound swabs and their susceptibility to antibiotics pm markov mij, shopovski e, despotovski v, angelevski a, nikolovski b. military hospital, microbiology, skopje, the former yugoslav republic, macedonia purpose: to determine percent of staphylococci from wound swabs and to establish their susceptibility to antibiotics. material and method: the wound swabs have been evaluated with standards microbiological techniques. bacteria have been identified with strips from the 'atb expression' system. the susceptibility testing has been performed with strips with dilution technique, read by the same system. results: during the last years ( Á/ ), a total of wound swabs have been evaluated in the military hospital in skopje. positive bacterial finding have been determined in ( %) swabs with isolated bacterial species, from which ( . ) were staphylococci: staphylococcus aureus ( . %) isolations ( methycillin resistant s. aureus ); s. epidermidis ( . %); s. haemolyticus ( . %); s. hominis ( . %); s. chromogenes and s. lugdunensis nine ( . %); s. intermedius , s. lentus , s. sciuri and s. warneri all with seven ( . %) isolations. the susceptibility of s. aureus was to: penicillin %, ampicillin %, amoksicillin/clavulonic acid %, ceftazidime %, gentamicin %, tetracycline's %, erythromycin %, lincomycin %, ciprofloksacin %, cotrimoxazole %, vancomycin %, fusidic acid %, cefixime %, and azitromicin %. conclusion: in our study most frequently isolated bacteria from the wound swabs were staphylococci, especially s. aureus . susceptibility, except for the penicillin ( %), was high to other antibiotics. the study went on from january to december . at maribor teaching hospital, staphylococcus aureus isolates were collected. in , ( %) and in , ( . %) were mrsa. mrsa were recovered from routine clinical material and from surveillance swabs (nose, throat, skin). for isolation, conventional culture media were used and for surveillance swabs mrsa-screening plate (manitol salt agar with % oxacillin) and trypticase soy broth with . % nacl were added. s. aureus was identified by catalase, dna-se and tube-coagulase test. antibiotic susceptibility was determined by the disk-diffusion method according to ncci guidelines. all mrsa isolates were tested for sensitivity to the following antimicrobials: gentamicin, netilmicin, ciprofloxacin, erythromycin, chloramphenicol, tetracycline, cotrimoxazol, vancomycin and clindamycin. it was found that all mrsa isolates were sensitive to vancomycin and partially or totally resistant to the rest. there were no important differences between the years and . our mrsa isolates were completely ( %) susceptible to vancomycin, but resistant to the other antimicrobials in use to some extent. although the monitoring of mrsa susceptibility to antimicrobials once a year did not show any important change in antimicrobial resistance, the periodical monitoring of mrsa susceptibility to antimicrobials and revaluation of current treatment regimens of mrsa infections is necessary. staphylococcus aureus strains with reduced susceptibility to vancomycin among clinical isolates in university hospital in warsaw pm the visa and especially h-visa are very difficult to be found in the routine laboratory. in our investigations we examined of s. aureus strains isolated and stored in our laboratory for several last years ( Á/ ) . most strains were isolated in , and some in . for all staphylococcal strains mrsa as well as mssa the mics of vancomycin were performed by the standard dilution method. among strains isolated in the last year three strains were recognized as visa (mic values were mg/l). the frequency of visa was . %. in the aim of founding the h-visa strains the population analysis was used. for this analysis all strains growing on the concentration mg/l of vancomycin from the inoculum were chosen. it was strains, but only of them were recognized as h-visa. the frequency of h-visa among investigated strains was about %. most but not all of the h-visa and all visa strains were methicillin resistant. in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci isolated from patients of surgical intensive care unit pm kucukates e, karayel n, kansiz e. institute of cardiology, university of istanbul, istanbul, turkey objectives: oxacillin-resistant staphylococci have emerged as a major infection control problem in our hospital. the aim of this study was to evaluate the in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci. material and methods: this study was performed between january and december , at university of istanbul, institute of cardiology. the antimicrobial susceptibilities of staphylococci isolates for vancomycin, teicoplanin and oxacillin have been investigated by e -test according to nccls guidelines. results: fifty-five ( . %) of clinical isolates were staphylococcus aureus . one hundred and twenty-four ( . %) of clinical isolates were coagulase negative staphylococci (cns). none of staphylococci isolates were resistant to vancomycin. but three of cns isolates were intermediate and six of cns isolates were resistant to teicoplanin. twenty-eight ( . %) of s. aureus were resistant to oxacillin. ninety ( . %) of cns isolates were also resistant to methicillin. conclusions: nosocomial staphylococcal infections, especially in intensive care units increase day by day. staphylococcal infections are a major problem in many hospitals. according to our experiences the rate of oxacillin resistant staphylococci isolates in our hospital has also increased. methicillin-resistant staphylococcus aureus (mrsa) is a clinically significant pathogen because mrsa is resistant to many kinds of antibiotics and causes nosocomial infections around the world. the antiseptics are used for prevention of infections by mrsa. antisepticresistant mrsa strains have been isolated from clinical specimens. antiseptic resistance genes confer resistance to many kinds of drugs structurally and the resistance mechanism is the energy-dependent drug efflux system. in addition, the fluoroquinolone (fq)-resistance gene, nora , confers also resistance to many kinds of antiseptics. we studied the relation of the susceptibility to antiseptics and fqs of mrsa strains isolated in japan. a total of strains of mrsa were isolated from hospitals in japan from to . acriflavin (af), acrinol, benzethonium chloride, benzalkonium chloride and chlorhexidine digluconate were used as the antiseptics. norfloxacin and sparfloxacin were also used in this experiment. the mic was determined by agar double-dilution method as recommended by the nccls. about % of mrsa showed resistance to af (mic: !/ ug/ml). no strain was resistant to a specific antiseptic. fq-susceptible strains were susceptible to all antiseptics. this study showed that antiseptic-resistant mrsa are widely spread at hospitals in japan. the drug of choice in treatment of serious infections caused by mrsa was still vancomycin, however sometimes failures were observed, especially in monotherapy. some conflicting are present in literature about an effect of combined action of vancomycin and betalactams. in the present work, the common effect of vancomycin and methicillin against chosen staphylococcus aureus strains was examined. the investigated strains were characterized as visa, h-visa and clones obtained from h-visa in population analysis. two methods were performed: e -tests with methicillin and vancomycin placed on the media supplemented with the second antibiotic and the chessboard micro-analysis with increasing concentrations of both antibiotics. the fic indexes were calculated for different combinations of concentrations. on the basis of the fic indexes it was shown that the simultaneous action of vancomycin and methicillin was synergistic in all examined strains visa and h-visa, but only in appropriate concentrations. in different combinations the observed effect was addition or indifference. antagonism was never observed. the synergistic effect was not observed in the case of standard s. aureus strain sensitive to methicillin. supplementation of media with % of nacl substantially decreased the observed effect. incidence of antibiotic resistance in staphylococcus aureus strains in hungary with special reference to mrsa pm ghidán Á , maró di c, csukás z, kamotsay k, szabó d, ostorházi e, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary between january and december , a total of staphylococcus aureus strains isolated from patients admitted to the clinics of the semmelweis university were examined for antibiotic sensitivity with the disc diffusion test. resistance to individual antimicrobials were as follows: penicillin %, oxacillin %, erythromycin %, ciprofloxacin %, amikacin %, netilmicin %, tobramycin %, gentamicin %, clindamycin %, mupirocin %, tetracyclines %, chloramphenicol %, teicoplanin % and vancomycin %. all mrsa were b-lactamase producer. they showed coresistance to erythromycin ( %), ciproflxacin ( %), amikacin ( %), netilmicin ( %) and mupirocin ( %). multiple resistant mrsa strains to mupirocin'/tetracyclines'/chloramphenicol amounted to . %. triple resistance to oxacillin'/ciprofloxacin'/netilmicin was %. the detection of meca gene by pcr in randomly chosen mrsa qualified with mg oxacillin disc resulted in only % meca positivity indicating that the traditional disc diffusion test overestimates the frequency of mrsa strains particularly in such an environment where the usage of penicillins and cephalosporins is so liberal as in hungary. consequently, the selective pressure for blactam-resistance and b-lactamase induction exists everywhere. this conclusion is coherent with the relatively low frequency of multiple resistant mrsa strains and urge the need of a routinely available genetic method to apply for mrsa detection. objectives: the main objectives of this study were to monitor antibiotic resistance, identify new/emerging resistance mechanisms at an early stage, prevent their dissemination, early detection and prevent the outbreaks. methods: our laboratory used antibiotic disc sensitivity testing methodology (nccls ) . zone sizes were measured objectively using a biomic automated radius zone reader. results: throughout years (january till december ) we have surveyed organisms collected from outpatient departments ( , . %), radio-oncology department ( , . %), medical department ( , . %), obg department ( , . %), surgical-oncology non icu department ( , . %), icu department ( , . %). from ( %) strains of enterobacteriaceae ( . %) were resistant to th generation fluoroquinolone and ( . %) strains were esbl positive. from ( %) strains of staphylococcus aureus were only five ( . %) strains resistant to methicilin (mrsa). we collected ( %) strains of enterococci, whereabout only two ( . %) were resistant to glycopeptides (vre). from ( %) strains of pesudomonas aeruginosa , ( . %) were resistant to aminoglycosides. conclusions: national restrictive antibiotic policy hand in hand with local hospital antibiotic policy and regular rotation of antibiotics used in prevention and treatment on all departments is leading in our case in positive situation in antibiotic resistance in comparing with other slovakian and european centers. antimicrobial resistance of nosocomial strains of staphylococcus aureus pm dekhnich av, stratchounski ls, edelstain ia, narezkina ad. institute of antimicrobial chemotherapy, smolensk, russian federation purpose: to determine the antimicrobial resistance of staphylococcus aureus causing nosocomial infections in smolensk regional hospital. results: a total of s. aureus strains isolated during Á/ were studied. antimicrobials tested included oxacillin (oxa), erythromycin (ery), clindamycin (cli), gentamicin (gen), vancomycin (van), linezolid (lnz), tetracycline (tet), chloramphenicol (chl), rifampicin (rif), fusidic acid (fus), trimethoprim/sulfamethoxazole (ts), ciprofloxacin (cip), mupirocin (mup), quinupristin/dalfopristin (qd). susceptibility testing and its interpretation were performed by agar dilution according to nccls guidelines where applicable. results are presented in the conclusions: the most active antimicrobials were vancomycin, linezolid, quinupristin/dalfopristin, fusidic acid, mupirocin, followed by co-trimoxazole, rifampicin. beta-lactams, macrolides, lincosamydes, tetracyclines and chloramphenicol should not be used for the treatment of nosocomial s. aureus infections. we investigated all staphylococcal infections within years among neonates hospitalized for infection in the neonatal icu in a tertiary neonatal referral center. univariate analysis, to assess risk factors for neonates infected with staphylococcus aureus ( ) vs. without s. aureus ( ) was performed. from the total number of cases, in cases s. aureus was isolated from various samples; in cases from blood cultures, in cases from urine, in cases from eye swabs and in cases from gastric content (no significant differences in comparison with control group). colonization with s. aureus , was a predictor of infection: nasal swabs, throat swabs, ear swabs, skin swabs and umbilical swabs were significantly more commonly observed in neonates infected with s. aureus , than with other infections. etiological analysis showed that co-pathogens escherichia coli and viridans streptococci were significantly more frequently associated with neonatal infection caused by s. aureus , in comparison to other organisms. according to localization of infection site, conjunctivitis and thrush stomatitis was the commonest s. aureus neonatal infections. outcome was similar to other infections and without any significant differences between both groups. mortality was similar to other infections, probably because: initial therapy in our centre contains an antistaphylococcaly active agent (cefuroxime or cefotaxime plus aminoglycosides). morozova ot, semina na. laboratory of hospital infections, central research institute of epidemiology, moscow, russian federation purpose is to study the role of enterococcus spp. in the aetiology of nosocomial infections among the patients of the childrens clinical hospital and susceptibility of these strains to antibiotics. methods: the strains of enterococci were isolated from patients with hospital infections in Á/ . results: the aetiological structure of enterococcus -infections showed the predominance of skin and soft tissue infections ( . %), urinary tract infections ( . %), bloodstream infection ( . %), pneumoniae ( . %), infection of central nervous system, gastrointestinal tract, eye, surgical wound infections were of rare incidence ( Á/ . %). various nosological forms of infections were caused more often by e. faecalis than e. faecium ( . , . %). the antibiotic resistance to ampicillin and other beta-lactams occured in % of e. faecium isolates, but all strains of e. faecalis were susceptible to these drugs. high-level gentamicin resistance demonstrated e. faecalis isolates */ . %, e. faecium */ %; and high-level streptomycin resistance showed e. faecalis */ . %, e. faecium */ . %. all the e. faecalis were active against fluoroquinolones, but e. faecium were resistant in . %. there were no vancomycin resistant enterococci. conclusion: e. faecalis predominated in the aetiological structure of nosocomial infections due to enterococcus spp. antibiotic resistance patterns for two species of enterococci were different, all the strans were susceptible to vancomycin. evaluation of antimicrobial resistance of enterococcus spp. experience of years ( Á/ ) pm lopez-barba j, jesus de la calle i, solino-ocañ a i, rodríguez-iglesias m, perez-ramos s. microbiology laboratory, puerto real university hospital, cádiz, spain objective: determination of quantitative changes in antimicrobial resistance of enterococcus isolated from clinically significant not urinary samples of patients remitted to the laboratory of microbiology during a years period ( Á/ ) . material: the period of the study was comprised between and . the samples has been processed for the isolation of enterococcus following conventional methods. were isolated enterococci strains. the identification and susceptibility to antibiotics have been performed in automated system microscan(c) dade-behring(c) through panels combo cgp. the data were processed by the statistical system statgraphics plus . . results: of the enterococcus , have been identified e. faecalis and e. faecium . the resistance is shown in table . conclusions: the resistance to va and tei of e. faecalis remains through last years ( Á/ %). the high resistance to erythromycin and tetracilin ( !/ %) and the resistance ( Á/ %) to quinolones, antibiotics all of them used in community-acquired infections justify the susceptibility testing to the clinical strains isolated of this group of microorganism. in e. faecium the antimicrobial resistances was high and increasingly to imipenem, meropenem, erythromycin and quinolones. characteristics of strains e. faecium colonizing the neutropenic patients pm abbassi ms, achour w, gréco a, ben hassen a. laboratory of bone marrow transplant center, tunis, tunisia digestive colonization by enterococcus faecium in the neutropenic patients under gut decontamination is important. seventeen multiresistant strains of e. faecium isolated from stools of seven neutropenic patients were the target of an epidemiological analysis through the determination of the mics of amoxicillin, gentamicin, vancomycin, the transferability gentamicin resistance to the recipient strain e. faecalis jh - by filter-mating assay, analysis of plasmid profiles of e. faecium -strain and of transconjugants and the amplification by pcr of the gene aac( ?)-aph( ??) coding for the bifunctional enzyme by using primer m who gives a fragment of kb. among the seventeen strains, eleven had the same antibiotype a , had a gentamicin mic!/ mg/l. the mic of the amoxicillin was of mg/l. all the strains were sensitive to vancomycin. ten strains harbored a plasmid of kb transfered at a frequency of . Á/ , also found in gentamicin-resistant transconjugants. however, strains belong to nine distinguished plasmids profiles. all high-level gentamicin resistant-strains had a positive pcr amplification of the aac ( ?)-aph ( ƒ) gene. the features of the studied strains establish their endogen origin, specific for every patient, sharing only high-level resistance to gentamicin. gut decontamination treatment with gentamicin enhance either the spread and the preservation of easy-transferable plasmid carrying genetic transposable element. frequency and antibiotic resistance of bacteria isolated from patients suffering infectious complications following the implantation of prosthetic devices pm kristó f k, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary for patients with indwelling joint prosthesis, early recognition and prompt therapy for infection in any location may be critical to reduce the risk of seeding the joint implant heamatogenously. a year period ( ) a total of swabs of aspiration from patients with infectious complications following the implantation of prosthetic devices were cultured. cultivation and identification of the strains were performed by conventional methods and by vitek system (biomȇrieux) and susceptibility testing by disc diffusion. potentional pathogens were recovered in cases ( . %). gram positive cocci, in particular staphylococcus spp. proved to be the most commonly isolated bacteria. coagulase-negative staphylococcus (cns) was isolated more frequently ( %), followed by s. aureus ( %), enterococcus faecalis and e. faecium ( %), gram-negatives ( %), anaerob isolates ( . %). resistance to individual antimicrobials of s. aureus and cns were as follows: methicillin and %, clindamycin . and . %, fluoroquinolones and %. mupirocin resistant strains of s. aureus were not found, while . % were among the cns strains. our results could be essential for the rational selection of treatment at our orthopedic wards. results: in the analysed period the percentage of isolated enterococcus sp. strains among all non-repetitive clinical isolates in , and was . , . and %, respectively. cultured strains were identified as e. faecalis, e. faecium, e. gallinarum and e. avium . the most prevalent was e. faecium strains, isolated with a frequency of , and %, respectively. vancomycin-resistant strains were all identified as e. faecium and in they comprised % of all isolates of this species. conclusions: ( ) the frequency of isolation of enterococcus sp. in blood cultures of haematological patients remained relatively stable in Á/ . ( ) the predominant enterococcal species isolated from these patients was e. faecium . ( ) in we recorded for the first time an emergence of vancomycin-resistant e. faecium , which comprised % of all isolates of this species. kalai s, ben hassen a, achour w, greco a. laboratory of microbiology, national bone marrow transplantation center, tunis, tunisia from april to june , non-repeated strains of pseudomonas aeruginosa were isolated from immunocompromised patients. thirty-six percent of strains were isolated from abscess, % from blood culture and % from urine. susceptibility to antibiotic was studied by the routine disk diffusion method (ca-sfm). mics were determined using agar dilution to antibiotics ( b-lactams, four aminosides and two fluoroquinolones). serotyping of the different trains was performed using antisera to the international antigenic typing systems serotypes. the study showed % of resistance to c cochin port royal hospital, service de gynecologie-obstetrique site st vincent-de-paul, paris, france , d cochin port royal hospital, cclin, paris, france aims: to determinate risk markers of an outbreak of postpartum endometritis due to group a streptococcus . design: a case-control study using data collected with a structured form. setting: the cases of postpartum endometritis were diagnosed in the department of obstetric of the paris hospital network during days (december Á/january ). the group of controls consisted of women delivered in the same department during the same period. participants: cases (n / ) and controls (n / ). findings: cases had smoked more often during pregnancy ( vs. %; p / . ) and received more often immunosuppressive treatment than controls ( vs. %; p / . ). instrumental delivery has been needed more often for cases than controls ( vs. %; p / . ). cases had been hospitalized after delivery in a ward z of the department more often than controls ( vs. %; p / . ). they had been examined after delivery more often by a midwife x ( vs. %; p / . ) and a nurse y has provided care to cases and not to controls ( vs. %; p / . ). conclusion: smoking, receiving immunosuppressive treatment during pregnancy, and instrumental delivery were significantly associated with postpartum endometritis (pb/ %). a midwife and a nurse might be involved in the transmission of the infection. petoukhova i a , sokolova e a , dmitrieva n a , nummaev b b . a laboratory microbiology, cancer research center of russia, moscow, russian federation , b department of oncogynecology, cancer research centre, moscow, russian federation the aim of the study was to assess efficacy of perioperative ap. total pts were included. two hundred and one pts with cervical cancer (cc) undergone extensive hysterectomy, pts with cancer of vulva (cv)-extensive vulvoectomy, pts with ovarian cancer (oc) Á/ extensive/combined operations. fifty-one pts (group ) received ap with amoxicillin/clavulanate (am/cl) . g iv min prior to operation, then . g iv thrice per day for Á/ days. fifty pts (group ) received cefotaxime (ctx) g iv min prior to operation, then g four times per day for Á/ days'/metronidazole (mtz) mg two times per day for the same period. one hundred and forty pts were retrospective control (they received ii Á/iii generation cephalosporins or linkosamides only after operation). the rate of swi in pts with cc, cv and oc was . , , %, respectively; dwi */ . , and %, respectively; uti */ , , . %, respectively. the ap with am/cl was more effective compared to ctx'/mtz (swi */ vs %, respectively, pb/ . , dwi */ . vs %, respectively, p b/ . ). the rate of postoperative uti was equivalent in two groups ( vs %, p /n.s.). thus, ap with am/cl is preferrable option in extensive operations in og pts. fifty-eight patients were randomized into two groups. group , a treatment consisting of patients and group , consisting of patients as a control. the patients were at the age of / . and had had different surgical interventions with general anaesthesia from to h and accompanying copd ( %). artificial pulmonary ventilation was used in cases. in the early post-surgery period the patients of group were administered inhalation therapy, including ipratopium of bromide in the combination with fenoterol (atrovent, berodual) and ambrocsol (lazolvan) through a nebulizer. the inhalation therapy was not administered to the patients of group . under the influence of the inhalation therapy pulmonary ventilation and respiratory metabolism was restored faster in all the resuscitation patients (in group */by the end of the first h, in group */on the rd Á/ th day). the percent rate of pef was . '/ . and . '/ . , respectively. artificial pulmonary ventilation ended in . and . h, respectively. the time of the patients' stay in the resuscitation department was . days in group and . days in group . by the end of the st week pneumonia developed in one patient from group and in eight patients in group . aerosol therapy application accelerates medication delivery to the respiratory tracts, increases the local activity and effects good prophylaxis for surgical hospital pneumonia. variation in ethiology of early and late onset ventilator associated pneumonia pm nikolopoulos j, daganou m, michailidou m, karabela e, kavada k, retsou s, antoniadou a, rasidakis a. department of respiratory abstracts s failure and icu, sotiria general and chest disease hospital, athens, greece purpose: to compare the distribution of causative microorganisms, their susceptibility to antibiotics and outcome of 'early' and 'late' vap in a greek icu. methods and results: retrospective study of mechanical ventilated (mv) patients (pts) with early and late vaps during a -month period. diagnosis of vap was made by clinical, radiographic criteria and quantitative cultures of bronchial secretions. vap was diagnosed in pts ( %) out of consecutive admissions in icu. all pts before the development of vap received antibiotics. three episodes of vap ( . %) were developed before the th day of mv (early vap) and were caused: ( ) by multi-resistant acinetobacter; and ( ) by antibiotic-susceptible pseudomonas aeroginosa . in this group one pt died from septic shock related to vap and two pts survived. fourteen pts ( . %) developed vap after the th day of mv (late vap). five cases were caused by multi-resistant p. aeroginosa , two cases by mrsa, two cases by multi-resistant acinetobacter, two cases by susceptible to antibiotics klebsiella pneumoniae , and three were polymicrobial and caused by multi-resistant microorganisms (mrsa and gnb). four pts died ( . %) from septic shock related to vap, five pts ( %) died because of another cause and five pts ( %) survived. conclusions: early and late onset episodes of vap were caused by 'potentially drug-resistant bacteria'. p. aeroginosa as a cause of early vap was susceptible. mortality attributed to early and late vap was similar. antibiotic prophylaxis in oncological and major reconstructive orthopaedic surgery pm de biase p, ciampalini l, astone a, capanna r. azienda ospedaliera careggi, oncological and reconstructive centre, aoc, florence, italy during last year patients scheduled for oncological surgery or major reconstructive procedures were randomised to either vancomycin or teicoplanin prophylaxis. prophylaxis was performed with either vancomycin g i.v. twice daily or teicoplanin once daily i.v. two hundred patients were included. four patients did not agree the study protocol and were excluded. we treated patients with teicoplanin and patients with vancomycin. out of the patients were operated for oncological disease, while the remaining underwent major orthopaedic procedures. we experienced cases of red man syndrome, and five cases of moderate hypotension. five patients had postoperative complications: two deep venous thrombosis, one pulmonary embolism, two postoperative haematoma. in five patients we observed a wound dehiscence; two of these patients showed clinical sign of ssi and microbiological examinations were positive for mrsa. one patient recovered from infection with medical therapy, while the other patient showed a local tumour recurrence and was amputated at the thigh. at last surgery infection was still present clinical and at microbiological examination. in conclusion we had an infection rate of . % which is comparable to the infection rate of a 'clean' surgery in patients with normal risk of infection. teicoplanin showed lower toxicity, has a longer half-life and has a simpler way of infusion and it is our current choice in high risk surgery. injuries with contaminated sharp articles in health care workers in general hospital celje, slovenia pm lesnicar g, sibanc b. department of infectious diseases, medical center celje, celje, slovenia in a prospective study carried out from january till june , we registered subcutaneous injuries with sharp objects, mostly in nurses and cleaning service workers. in % of cases the incident occurred outside the hospital, in persons who were not medical workers. in cases the injury causing object was a needle that had been used in known patients, of which were hepatitis b positive. fifty-five ( . %) of the injured health workers had been previously vaccinated against hepatitis b; the protective antibodies to hepatitis b in the blood were found in / ( . %) health workers only, while the tests for antibodies to hepatitis c and hiv were negative in all cases. following the incident, the majority of the injured persons, i.e. , were vaccinated against hepatitis b, while persons ( . %) also received passive prophylaxis with human immunoglobulin against hepatitis b. none of the injured persons have developed the disease or showed evidence of sero-conversion. in the year the general as well as specific preventive measures practised in our hospital became more rigorous. thus, approximately % of our health workers at risk have already been vaccinated against hepatitis b. to improve measures preventing dissemination of multidrug-resistant bacteria (mrb), a cross-sectional survey ( ) was conducted to analyse healthcare workers' (hcws) isolation precaution knowledge for mrb infection at investigation and outpatient departments excluding four declaring not to be involved in care to mrb patients (emergency, obstetrics, nuclear medicine, and bacteriology). two hundred and eight hcws answered ( % of the paramedical staff, % of the physicians). thirty-three percent of them reported they do not know frequently or always the patient mrb status. they ( %) wish mrb status to be mentioned on the test form or on the advice request letter. mrb patient visit or test was appropriately timed in % answers. gowns ( %) or masks ( %) use were not systematically reported. other hcws ( %) reported better isolation precaution knowledge than physicians ( %) and nurses ( %) than investigation assistants ( %). physicians declared lower compliance with use of gowns, gloves or draw-sheets than other hcws. they had also lower education in isolation precautions and were less interested in education programs. this study suggests the necessity to improve mbr infection information. physicians and investigation assistants seem to be insufficiently aware of hospital infection control. therefore, education strategies targeted at physicians and investigation assistants working at outpatient and investigation departments should be developed. outbreak of clostridium difficile -associated diarrhoea in infectious disease department: risk factors and hygiene measures assessment pm henoun loukili n, martin m, remy v, hansmann y, christmann d. hôpitaux universitaires de strasbourg, maladies infectieuses et tropicales, strasbourg, france (shock)'/ * (bedridden status)'/ * (age !/ years)'/ * (previous antibiotic treatment) and points (women) / * (shock)'/ * (bedridden status)'/ * (age !/ years)'/ * (immunosuppression). the vast majority of patients ( and % of males and females, respectively) could be classified in the subgroups with lower scores (six points or less) which had a very limited risk of death ( Á/ . and Á/ . % for men and women, respectively), whereas for patients in the highest score subgroup ( points or more), the risk was % for men and % for women. conclusion: risk stratification of patients with ap is possible from simple clinical variables available on admission. procalcitonin (pct) and c-reactive protein (crp) for differentiation of systemic inflammatory response syndrome (sirs), sepsis and severe sepsis pm lupse m a , ursu l b , slavcovici a a , carstina d a . a clinical department, teaching hospital of infectious diseases, cluj-napoca, romania , b laboratory department, teaching hospital of infectious diseases, cluj-napoca, romania objectives: to evaluate the value of pct in the differentiation of patients with sirs, sepsis, severe sepsis and bacteremia in comparison to crp. design: prospective study including patients who meet criteria for sirs, sepsis or severe sepsis (consensus conference of the accp/ sccm) admitted over -month period. patients and method: a total of patients were included: eight with sirs, with sepsis and with severe sepsis. sixteen from patients had bacteremia. pct and crp were evaluated in the first h after admission: pct by brahms pct-q test and crp by turbidimetric assay. the sensitivity, specificity, predictive value of different cutoff points for crp and pct were determined. results: with a cut off point of . ng/ml for pct and . mg/dl for crp sensitivity and specificity for sepsis were %, respectively % (ppv . , npv . ) and %, respectively % (ppv . , npv ). a cutoff point of ng/ml for pct accurately predict severe sepsis (sensitivity %, specificity %, ppv ). a pct level of at least ng/ml was a good predictor for bacteremia (sensitivity %, specificity %, ppv . and npv ). conclusion: pct is a good discriminating marker to characterize the level of inflammation caused by infection and can predict bacteremia . giamarellou h a , aoun a b , klastersky j b , anagnostopoulos n c , galani l c , grecka p c , panaretou e c , papageorgiou e c , repoussis p c , syrseloudis pct has been considered as a useful diagnostic marker in neutropenic patients with bacteremia and/or severe sepsis (giamarellos-bourboulis ej et al. clin infect dis ; : ) . in an attempt to define its value in the diagnosis of localized infections in neutropenic hosts, daily determinations of pct and of c-reactive protein (crp) were performed before and after the onset of fever in subjects male and female aged . / . years with various haematologic malignancies (aml , nhl , mds , all ) developing neutropenia ( b/ pmns/mm ) after chemotherapy. thirty-three patients were presented with fever of unknown origin (fuo) and with localized bacterial infections (lbi; pneumonia , acute pyelonephritis , soft tissue infections , acute pharyngitis ). pct was determined by an immunoluminometric assay and crp by nephelometry. it is concluded that febrile neutropenia followed by a localized bacterial infections is accompanied by significantly higher levels of pct than in case of fuo ( . / . vs . / . ng/ml). similar differences are not observed with crp, which lacks the appropriate specificity. our study included patients who were categorized as having proven ( ), probable ( ), or possible ( ) systemic fungosis according to eortc criteria; showed no sign of infection, and were used as controls. blood samples were received on the st, rd, and th day from the onset of signs of a fungal infection, and then twice a week. pct levels were determined by an immunochemioluminent assay, and candida and aspergillus antigen levels by elisa. in only five patients pct indicated early signs of infection, albeit at barely detectable limits. six patients, however, showed significantly increasing titres preceding time of death. positive antigens titres were observed only in patients who had proven or probable systemic fungosis. only half of the control group had negative antigen titres; a high rate of false negatives was also observed. both pct and antigens titres increased in parallel in / patients with unfavorable outcome. pct and antigens titres cannot reliably indicate early diagnosis of systemic fungal infections although may be used as a prognostic tool of severity. lactulose, a factor that decreases endotoxaemia, in obstructive jaundice? pm koutelidakis im a , papaziogas v a , makris i a , giamarellos-bourboulis ej b , giamarellou h b , papaziogas t a . a thessaloniki med school, nd surgical clinic, thessaloniki, greece , b th department internal medicine, athens medical school, athens, greece bacterial translocation is a process implicated in the pathogenesis of spontaneous peritonitis. in order to evaluate the impact of lactulose administration on systemic endotoxaemia, obstructive jaundice was induced in rabbits by common bile duct ligation. animals were divided into two groups, group a of five rabbits not receiving lactulose and group b of six rabbits, which received . ml/kg of lactulose orally by an oral catheter. blood was collected daily, before and after operation for a total duration of four days. samples were applied for culture and for determination of endotoxins (lps) by the lal qcl- assay. concentrations of lps (mean /sd) of group a were . / . , . / . , . / . and . / . eu/ml on the st, nd, rd and th day, respectively. respective concentrations of lps (mean /sd) of group b were . / . , . / . , . / . and . / . . all blood cultures were sterile in both groups. differences activity of linezolid against nosocomial strains of staphylococcus aureus in russia: results of multicentre study pm were included in the study. antimicrobial susceptibility testing was performed by agar dilution method in accordance with the nccls recommendations. all tested strains including mrsa strains ( . % of all strains) were found to be susceptible to linezolid with the mic ranged from . to mg/l. both mic and mic were mg/l. conclusions: linezolid had excellent in vitro activity that was not affected by resistance to other classes of antimicrobials susceptibility to antiseptics of mrsa isolated in japan during Á % to piperacillin, % to ceftazidime, % to cefepime, % to imipenem, % to amikacin and % to ciprofloxacin. fiftythree percent of p. aeruginosa strains were multiresistant ( strains) and were isolated in patients. wild phenotype to b-lactams was observed in % of strains. the most frequent b-lactams resistance phenotypes were: cephalosporinase over production ( %) and penicillinase ( %). imipenem, ceftazidime and piperacillin-tazobactam were the most active b-lactams (mic of . and mg/l, respectively) these results showed high rates of antibiotic resistance and predominance of o serotype in multiresistant strains compared to the o serotype in europe. infectious complications sustained by stenotrophomonas (xanthomonas ) maltophilia in hiv at present, very limited informations are available about s. maltophilia infections in the setting of hiv disease. patients and methods: a retrospective survey of clinical and microbiological records of hiv-infected patients referring to out tertiary care centre between and was performed, in order to identify all episodes of s. maltophilia infections, and analyze its epidemiological, clinical, and microbiological variables. results: sixty-one episodes of s. maltophilia infection were observed in patients: sepsis/bacteraemia in cases ( . %), lower airways infection in five, urinary tract infection in four, pharyngitis in two, lymphadenitis and liver abscess in one case each. forty-seven out of episodes of s. maltophilia infections ( %) occurred as nosocomial disease, generally in association with advanced immunodeficiency, neutropenia, instrumentation, and prior antimicrobial therapy. bacterial isolates showed an elevated resistance profile against many betalactam compounds, aztreonam, imipenem, and aminoglycosides. conclusion: s. maltophilia represents an emerging opportunistic pathogen in hiv-infected patients extended spectrum beta-lactamases producing germs in intensive care units pm university of medicine and pharmacy 'victor babes ', microbiology, timisoara, romania injury and complications lasted months, demanded for surgical procedures and total cost was comparison of different methods for detection of extended spectrum beta lactamases (esbls) and their genetic relatedness among enterobacteriaceae clinical isolates in a research medical institute pm methods: one hundred out of isolates that were screened positive for esbls were tested with double disk synergy test (ddst), three dimensional test (tdt), e -test-esbl and vitek-esbl test. pulsed field gel electrophoresis (pfge) analysis was applied to esbls; five klebsiella pneumoniae and eight escherichia coli . results: revealed the prevalence of esbls in . % of clinical isolates. the sensitivities of the ddst, tdt, e -test and vitek were , , . and . %, respectively. in the ddst, aztreonam was the most sensitive indicator ( . %). pfge demonstrated that % of k. pneumoniae were derived from a single clone whereas . % of e. coli isolates were derived from two different clones. non-clonal origin was demonstrated in % of k. pneumoniae and . % of e. coli . conclusion: there is an increased prevalence of esbls. the ddst is the most sensitive, practical and cost effective diagnostic method reliable for routine use in our laboratory. both clonal spread and plasmid dissemination contributed to the concurrent nosocomial outbreaks caused by esbl-producing k severe nosocomial infections due to stenotrophomonas maltophilia pm the teaching hospital of infectious diseases total og pts after extensive hysterectomy ( pts), extensive vulvoectomy ( pts) and extensive/combined operations for ovarian cancer ( pts) were analysed. ic developed in pts. one hundred and sixtyseven pts had no ic. twenty-eight of rf analysed were independent rf of ic. most important included: age !/ years (p / . ), grade Á/ obesity (p / . ), diabetes mellitus (p / . ), diagnosis of cervical cancer (p / . ), history of pre-cancer of vulva postpartum endometritis due to group a streptococcus : a case-control study pm unite operationnelle d 'hygiene all isolates were sensitive to vancomycin. among gram-negative bacteria klebsiella spp. was isolated in . % of cases, acinetobacter baumanii in . %, enterobacter spp. in . %, providencia spp. in . %. of the klebsiella spp. isolates % were resistant to amikacin, % to cephalosporins, % to piperacillin/ tazobactam. all were sensitive to imipenem. of the a. baumanii isolates % were resistant to amikacin, aztreonam, cefoperazone, cefotaxime, ceftriaxone, piperacillin; % to ampicillin-sulbactam, % to ceftazidime, and % sensitivity to imipenem antimicrobial activity of selected pharmacopoeial antiseptics analysed according to european standards pm european committee for standardisation approved several european standards (en), describing test methods establishing, whether an antiseptic has or does not have a bactericidal or fungicidal activity under the laboratory conditions defined by en. the aim of the study was to investigate, if some chemical compounds in concentrations recommended by polish pharmacopoeia for skin disinfection, comply european standards requirements. methods: basic bactericidal (en ) and fungicidal (en ) activity were investigated as well as bactericidal activity of products for hygienic and surgical handrub and hand wash used in human medicine (pren ). all methods and used neutralizers were validated. standard strains: staphylococcus aureus , pseudomonas aeruginosa , escherichia coli , e. hirae , candida albicans and a. niger were used, when en standards were evaluated. results: ethanol, izopropanol and n -propanol caused viable microbial count reduction required by ens in pharmacopoeial concentrations the purpose of the study: we collected bacteriological samples from adult and neonate patients who were admitted in intensive care units (icu) . the aim was to observe the colonization status with microbes that may have a nosocomial potential and to establish circulating phenotypes in icus. the results obtained from a total of samples strains of gram negative bacteria (enterobacteriaceae family) were isolated. fourteen strains showed extended spectrum beta-lactamases (esbl) phenotype (eight strains of klebsiella pneumoniae , three of escherichia coli , two of klebsiella ornithynolitica , one of klebsiella oxytoca ). we used both disc diffusion test (extended antibiotic susceptibility test and synergy test to visualize 'champagne stopper' pattern) and mini api † system.the conclusion reached: we put in evidence a massive colonization with germs that may have a nosocomial potential especially microbes that produce esbl ( . % from all enterobacteriaceae isolated) which implies a rational policy in prescribing antibiotics in hospitals from western part of romania.carbapenem activity against nosocomial gram-negative rods pm sawicka-grzelak a a , rokosz a a , meszaros j b , luczak m a . a department of medical microbiology, university medical school, warsaw, poland , b department of general and transplantation surgery, university medical school, warsaw, poland purpose: to determine a susceptibility of nosocomial gramnegative rods to carbapenems.methods: two hundred strains of gram-negative rods were cultured from clinical specimens from hospitalized patients (july Á/november ). identification of strains was performed in the automatic atb system (biomerieux, france). susceptibility of strains to carbapenems: imipenem and meropenem was determined with disc diffusion method according to nccls recommendations. esbl-producing strains were detected with double-disc synergy test (ddst according to jarlier et al., ) or a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd ) (oxoid, england) .results: one hundred and ten strains of enteric rods and strains of non-fermenting rods were cultured. twenty eight ( %) esblpositive strains were detected. carbapenems were active against % of enteric rods. the percentage of non-fermenting rods susceptible to imipenem was and to meropenem */ .conclusions: carbapenems: imipenem and meropenem demonstrated high activity against clinical strains of enteric rods. however, the antibiotics were less active against nosocomial strains of nonfermenting rods.inhaled antibiotics against multiresistant bacteria in bronchial secretions of icu patients: a preliminary report pm horianopoulou m a , kanellopoulou m b , paraskevopoulos i a , valakis k a , kyriakidis a a , lambropoulos s a . a intensive care unit, sismanoglio general hospital, athens, greece , b department of microbiology, sismanoglio general hospital, athens, greece purpose: the aim of this study was to assess the effectiveness of aerosolized ampicillin/sulbactam, ceftazidime and colistin, in icu patients with multiresistant acinetobacter baumannii or pseudomonas aeruginosa colonization of the respiratory tract.methods: fifty-three intubated, mechanically ventilated patients participated in the study. multiresistant a. baumannii , sensitive only to ampicillin/sulbactam, or p. aeruginosa , sensitive to ceftazidime or colistin, were isolated from the bronchial secretions ( Á/ cfu/ ml). all patients were subsequently treated with intravenous ampicillin/sulbactam, ceftazidime or colistin, whereas of them were also given the same antibiotic in aerosolized form.results: a decrease in the number of colonies by Á/ cfu/ml was observed, following Á/ days of combined treatment with both intravenous and inhaled antibiotic. none of the patients developed vap. in the patients who only received the antibiotic intravenously, the decrease ranged from zero to cfu/ml, after days of treatment. two of patients developed vap.conclusions: our results suggest that the administration of aerosolized antibiotics represents an effective means of preventing ventilator-associated pneumonia caused by a. baumanni and p. aeruginosa . introduction: pseudomonas aeruginosa is an important nosocomial pathogen. resistance to certain beta-lactam antimicrobial agents among p. aeruginosa is increasing. despite the development of new antibiotics multiresistant strains of p. aeruginosa represent an important therapeutic problem. the aim of this study was to investigate the activity of imipenem, amikacin, piperacillin, ciprofloxacin, ceftazidime, against clinical isolates of p. aeruginosa . methods: a total of isolates by tracheal aspiration from hospitalized patients, admitted to intensive care units were identified as p. aeruginosa using an algorithm that included: gram stain, pigment, oxidaze ('/ ,) and gram negative identifications microscan walkaway- (dade behring) were used according to the manufactures instructions. minium inhibitory concentrations were determined using walkaway, interpretation based on ncclsm -s , january ' .results: the respiratory tract was the single site of isolation for this study. the best activity was showed by imipenem %, followed by amikacin %, piperacillin, pip/tazobactam, ciprofloxacin had the same sensitivity %.conclusion: a high level resistance to antibiotics was observed to p. aeruginosa isolated from tracheal aspiration. carbapenems seem to be the most active against p. aeruginosa in this study. materials and methods: clinical samples were collected from patients admitted to this hospital. only one isolate per patient was included. antimicrobial susceptibility testing was performed as recommended by the nccls. all bacterial isolates were tested by dd and ad to provide a comparison of both test results. very major error was considered when the strains were resistant (r) by ad and susceptible (s) by dd and major error when s by ad and r by dd. categories of s and r were stablised using the breakpoints suggested by mensura ( ) . colistin r strains was typed by rep-pcr.results: among the strains included in this study ( . %) were s to colistin and five ( . %) were colstin r by ad, of this five colistin r strains four ( . %) were s to colistin by dd and one was r by both methods. all r isolates were similar by rep-pcr.conclusions: most of the ad colistin resistant strains were s when tested by dd indicating that this method is not useful to determine the resistance to colistin. rep-pcr patterns show that the spread of a colistin r clone seems to be involved. methods: gnb were isolated from per-operative biopsies (pob) and/ or from articular punction (ap). patients (pts) received cfp, g bid'/ ofl, mg tid or cip, mg bid intravenously for days, followed by a prolonged oral fq monotherapy. cure was defined as: resolution of all clinical signs of infection, normalization of the biological inflammatory profile at the end of treatment (eot) and absence of infection at the same site during the post-treatment followup period (ptfu).results: all of the studied patients [mean age / years] had hospital acquired bji. seventeen/ had an infected orthopedic device (prosthetic joints / , other orthopedic prosthetic devices / ). culture of pob and ap yielded to pseudomonas sp. ( ), enterobacter cloacae ( ), others ( ). vancomycin was added for six pts co-infected by gnb-mrsa. nineteen/ pts underwent a surgical intervention (debridement / , removal-replacement / , amputation / ). after ptfu period of months (range Á/ ), the overall success rate was / ( . %) without serious adverse events.conclusion: cfp Á/fq combination was safe and efficient in the treatment of hypercase gnb, bji.treatment of posttraumatic mrsa osteomyelitis of the femur with longterm cotrimoxazole */a case report pm the authors report a case of posttraumatic osteomyelitis of the femur caused by methicillin-resistant staphylococcus aureus (mrsa), following the shot injury. relapses of the infection occured in months interval and were treated by revision, debridement, lavage and vancomycin. because of laboratory signs of renal insufficiency vancomycin became contraindicated for treatment of the third relapse of infection and the different approach was employed: classic open treatment of bone infection sec. orr was combined with a long-term administration of high-dose cotrimoxazole. the patient was given cotrimoxazole mg daily divided in four doses ( mg/kg/ h) for months, then for gastrointestinal complaints with lowered dose of g daily for next months. the wound completely healed. during months after the final surgery there was no relaps of infection, but the atrophic pseudoarthrosis of the femur resulted. the patient can walk with a rigid orthesis and two crutches. the whole treatment of the objective: to present a variety of severe nosocomial infections due to stenotrophomonas maltophilia in patients hospitalized in tertiary medical units from cluj.results: during the last year nine strains of s. maltophilia obtained from patients with severe infections and hospitalized in different wards were isolated. all but one were considered nosocomial infections: four cases of pneumonia, one urinary tract infection, three cases of surgical wound infections and one case of endocarditis under surgical treatment. the cases of pneumonia were either primary occurring in a granulocytopenic patient with leukemia or secondary in patients that underwent surgical treatment. in the case of endocarditis the ethiology was established after surgery from the damaged valve in a negative hemoculture patient with a poor outcome under medical treatment. in all cases of surgical wound infection bacteremia occurred diagnosed on clinical basis in the presence of severe sepsis or hematogenous dissemination in the lung. the urinary tract infection occurred in a patient after urinary surgery and having a catheter in place. the immediate evolution was favorable in all cases but treatment was difficult due to the highly resistant strains and to underling diseases.conclusions: s. maltophilia should be considered in nosocomial severe infections and prophylaxis by interrupting environmental transmission has to be promoted. sawicka-grzelak a, rokosz a, luczak m. department of medical microbiology, university medical school, warsaw, poland purpose: to identify and determine the drug-susceptibility of esblpositive strains isolated from urine samples.methods: seven hundred and twelve strains of gram-negative rods were cultured from urine samples from hospitalized patients during months (july Á/november ). identification and susceptibility were performed in the automatic atb system (biomerieux, france) using id e, id gn and atb ur strips. esbl-activity was detected with double-disc synergy test (ddst according to jarlier et al., ) or using a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd ) (oxoid, england).results: five hundred and ninety-five strains ( . %) belonging to enterobacteriaceae family, strains ( . %) of non-fermenting rods and two strains ( . %) of other gram-negative rods were isolated. eighty-two esbl-producing strains ( . % of all strains) were detected. fifty-nine esbl-positive strains were susceptible to nitrofurantoin, -to norfloxacin and ciprofloxacin and -to fosfomycin.conclusions: esbl-positive strains were detected most frequently among enteric rods ( strains). nitrofurantoin and quinolones were the most active in vitro antibacterial agents against examined esblpositive uropathogens. results: the mechanisms of resistance were evaluated phenotypically using different aminoglycosides. a total of aminoglycoside resistant gram-negative strains were studied. one hundred fifty-eight strains were collected in Á/ , in and in . the resistant profiles were determined: enterobacteriaceae */ ; pseudomonas aeruginosa */ ; acinetobacter spp. */ . the most frequently phenotypes were gt (gentamicin, tobramycin) */ % and gtnet (gentamicin, tobramycin, netilmicin) */ %. the gt phenotype due to production ant( ƒ)-i enzyme, the gtnet Á/aac( )-v ( strains), aac( )-iv */one strain and aac( ƒ)-i */one strain. the resistance to amikacin in % strains was due to production aac( ?)-i ( %) and aph( ?)-vi ( %). the most of examined strains were simultaneously resistant to kanamycin and neomycin caused by production of aph( ?)-i ( %). only seven strains were resistant to all aminoglycosides due to impermeability of outer membrane. no substantial differences were observed between years.conclusions: the main mechanism of aminoglycoside resistance is fermentative modification. the high rate to gentamicin and tobramycin was due to production of ant( ƒ)-i and aac( )-v. amikacin and isepamicin were the most active aminoglycosides against gramnegative nosocomial isolates.the incidence of clostridium difficile associated diarrhoea (cdad) increased in our department from january to june .objective: to confirm the out break of cdad, to identify the risk factors and assess the effectiveness of the measures implemented for controlling this outbreak.methods: cdc definitions were used to identify the cases. the scope of the outbreak was defined. cdad incidences during the outbreak period and during the same period in were compared. risk factors (reduced mobility, antibiotic treatments . . .) were studied for patients whom length of hospital stay (lhs) was more than days. contact precautions and environmental cleaning with clona implemented were assessed.results: seventeen episodes of cdad were identified. sex ratio: . , mean age / . , mean lhs / days, mean delay for cdad occurring / days. one hundred and fifty-two patients involved in the study of risk factors. relative risk (r.r.) evaluated were: blactams (r.r. / . , ic %: . Á/ . ), reduced mobility (r.r. / . , ic %: . Á/ . ). incidence of cdad was less than two cases per days of hospitalisation after june .conclusion: we confirmed the outbreak of cdad in our department b-lactams and reduced mobility were identified as risk factors for cdad. measures implemented to control the outbreak were effectiveness.positive heart transport fluid cultures associated with severe infections in heart transplant recipients pm at the mount sinai hospital in new york city heart transplants were performed between and june . cultures were routinely performed on all heart transplant transport fluids. culture data was available for of these patients. in total / ( . %) were positive for bacteria, fungi or both. the organisms isolated included coagulase negative staphylococci ( ), pseudomonas aeruginosa ( ), staphylococcus aureus ( ), acinetobacter baumanii ( ), serratia mercescens ( ), enterobacter cloacae ( ), escherichia coli ( ), proteus mirabilus ( ), enterococcus faecalis ( ), viridans streptococci ( ), and fungi (aspergillus fumigatus ( ), penicillium species ( ), and rhodotorula rubra ( ). two heart transplant recipients had two organisms isolated from the transport fluid. isolation of resistant gram-negative bacilli in the transport fluid was associated with significant infection in / patients ( %) with the same organism. the observed infections were pneumonia secondary to e. cloacae , sternal wound infection secondary to p. aeruginosa , and bacteremia secondary to p. aeruginosa . it appears prudent to provide prophylaxis against resistant gram negative bacilli to prevent infections. zacharof ak, flevaris c, petrogianopoulos c, karachalios g, vroulis j, chartzoulakis g, drakogiorgos g, loizidou a, svoukas g. nd department of internal medicine, hellenic red cross hospital, athens, greece objective: we studied the trends of nosocomial bloodstream infection and calculated the population-attributable risk for death among hospitalized patients. methods: we perform a -year retrospective study for all patients (n / ), admitted to our department between and .results: between and , a total of patients developed episodes of nosocomial bloodstream infection. the crude infection rates increased linearly from . to . per discharges ( . Á/ . episodes per patient-days) during the -year study period. increases in the infection rates were due to gram-positive cocci, yeasts and essentially explained by infections caused by coagulasenegative staphylococci, staphylococcus aureus , enterococci, and candida species, respectively. although the crude mortality in patients with nosocomial bloodstream infections decreased from % in to % in , the in-hospital population-attributable mortality among infected patients increased from . deaths per discharges in to . per discharges in . the etiologic fraction or the proportion of deaths in patients with bloodstream infection to all deaths occurring in the hospital increased from . % in to . % in .conclusions: the incidence and the population-attributable risk for death among patients experiencing nosocomial bloodstream infections increased progressively during the last years in our department.ventilator-associated pneumonia before and after intensive care unit temporary closure pm results: we compared the incidence, causative organisms and mortality of vap in two different time periods. period june to december . period june to december . between those two periods the icu remained closed for months because of reconstruction works.period : sixty-seven consecutive patients (pts) were studied with bronchial secretions cultures at least days after mechanical ventilation (mv) initiation. the vap was diagnosed by clinical, radiological and microbiological criteria in pts ( %). causative organisms included: pseudomonas aeruginosa , acinetobacter , staphylococcus aureus , klebsiella pneum. , enterobacter . in three cases vap was proved polymicrobial. fourteen ( ) episodes of vap ( %) were developed after days mv (late vap) and were attributed to multiresistant microorganisms. mortality of vap was %.period : among consecutive pts, vap was diagnosed in ( %). causative organisms included: acinetobacter , p. aeruginosa , s. aureus , k. pneumoniae , escherichia coli . five ( ) cases were polymicrobial and cases were 'late vap' ( %). causative microorganisms had similar patterns of sensitivity to antibiotics (compared to period one). mortality of vap was %.conclusion: temporary icu closure had no significant influence on the incidence, distribution of causative organisms, their sensitivities to antibiotics and mortality of the vap. efstathiou sp a , pefanis av a , tsioulos di a , tsiakou ag a , zacharos id a , kanavaki s b , mountokalakis td a . a third university department of medicine, sotiria general hospital, athens, greece , b microbiology laboratory, sotiria general hospital, athens, greecepurpose: the aim of this study was to derive a scoring system for the prediction of outcome in adult patients with acute pyelonephritis (ap) severe enough to need hospitalization. therefore, the charts of patients ( men, median age years) were reviewed.results: logistic regression analysis identified in both sexes four independent correlates of in-hospital mortality, the coefficients of which divided by . and rounded to the nearest interval, resulted in the following integer-based scoring system: points (men) / * between concentrations of lps of the two groups were statistically significant on the nd and the th day (p b/ . ). it is concluded that the administration of lactulose may decrease systemic endotoxaemia in the field of obstructive jaundice.nosocomial infections: a prevalence study in the island of crete pm doukakis s a , tzimis l b , perogambrakis g a , kalloniatou m a , christodoulakis n a , evaggelopoulos a a , koutsoumba d a , kastanakis s a . a first medical department, 'saint george ' general hospital, chania, greece , b pharmacy department, 'saint george ' general hospital, chania, greeceprevalence surveillance is a rapid and inexpensive mode to estimate the problem of hospital-acquired infections (hais). to study the problem of nosocomial infections in our hospital, a prevalence study was made from our team in . the study included patients (the total number of hospitalized patients at the time of the study). from these patients were males ( . %) and females ( . %). one hundred and ninety-one patients ( . %) belonged in the groups of age between and years. fifty-seven patients had a urine catheter ( . %). one hundred and fifty-five/ patients ( %) received antibiotics and from these patients received one antibiotic and the remaining patients two or more. a nosocomial infection was found in patients and consequently the prevalence of hais was . %. among these, urinary tract infections were six ( . %), lower respiratory tract infections were three ( . %), surgical site infections were three ( . %), and bloodstream infections was one ( . %). the incidence of multiresistant bacteria was primarily enterococcus spp and secondary, pseudomonas aeruginosa , enterobacter spp, klebsiella pneumoniae , escherirchia coli , staphylococcus aureus , enterobacter cloacae . unfortunately prophylactic chemotherapy of long duration was found despite the suggestions of the infection control committee. regarding age the highest incidence of hais occurred in the third age group. bagirova ns, dmitrieva n. laboratory of microbiology, cancer research center of russia, moscow, russian federation objectives: to determine the pathogens and susceptibility to antimicrobials.methods: blood samples were collected from adult pts ( Á/ ). the bacteraemic episodes were classified according to the definitions of the cdc. laboratory detection of bacteraemia and fungaemia was performed according to cumitech b (blood cultures iii, ). susceptibility testing was performed by disk diffusion method (nccls).results: the total number of blood samples */ , -positive ( . % episodes of significant bacteraemias). bsi was confirmed microbiologically in of febrile pts ( . %). the most frequent pathogens were gram('/) cocci ( . %) (p b/ . ), gram((/) bacilli */ . %, fungi */ . %. coagulase-negative staphylococci (cns) represented . %, staphylococcus aureus . %, streptococcus spp. . %, enterococcus spp. . %, enterobacteriaceae . %, pseudomonas aeruginosa . %, other non-fermenting */ . %, yeast . %, mould . %, anaerobes . %. one hundred percent cns were resistant to penicillin, . % to oxacillin, . % to clindamycin, . % to cefazolin, . % to ceftazidime, . % to ciprofloxacin, . % to gentamicin, and no isolate was resistant to vancomycin. the predominant pathogens in all types of hm were gram('/) cocci (mainly cns). all gram('/) microorganisms were sensitive to vancomycin. katashinsky o a , opriatova t b , tchuev p c . a state clinical hospital, anaesteziology, odessa, ukraine , b state clinical hospital, bacteriology, odessa, ukraine , c medical university, anaesteziology, odessa, ukrainethis investigation was carried out in odessa state clinical hospital during Á/ . of the patients with post-operation complications, % of gram-negative cultures were sensitive to ceftazidime and % to amikacin. sixty-nine percent of gram-positive cultures were resistant to penicillin g, but they were sensitive to vancomycin and nitrofurantoin in and % of cases, respectively. sixty percent of isolates of pseudomonas aeruginosa were sensitive to ceftazidime and % to amikacin. rates of resistance to carbenicillin and gentamicin were and %, respectively. one hundred and fortythree isolates of escherichia coli were studied and % of them were resistant to ampicillin % to cephalothin and % to tetracycline. of the isolates tested against ciprofloxacin, all were sensitive. one hundred and eight isolates of staphylococcus aureus were studied and they were resistant only to penicillin g ( %). staphylococcus aureus was sensitive to erythromycin ( %), tetracycline ( %), oxacillin ( %) and vancomycin ( %). all isolates enterococcus faecalis were sensitive to nitrofurantoin and % to ciprofloxacin. the majority of s. aureus and e. faecalis isolates were susceptible to most other antibiotics, but the majority of e. coli isolates were resistant to the studied antibiotics expect ciprofloxacin.campylobacter foetus bacteraemia in an immunocompromised patient: a case report pm monno r a , ierardi e b , rendina m b , ceci g a , luzzi i c , de vito d a , rizzo g a , francavilla a b . a department of internal medicine and public health, university of bari, bari, italy , b department of emergency and organ transplantation, university of bari, bari, italy , c istituto superiore di sanità, rome, italy a -year-old woman was admitted for recurrent fever. the patient underwent a liver transplantation and splenectomy in . she had followed immunosuppressive therapy until when tacrolimus was added for chronic rejection. in non-hodgkin lymphoma was diagnosed and chemotherapy was started. six months later because of the presence of two metastatic encephalic foci affecting the optic chiasm, a new chemotherapy course was started with the regression of lesions. in january she was treated with steroid recycle and cyclosporine Á/azathioprine Á/prednisone reintroduction. fever occurred after months and cytomegalovirus (cmv) infection was diagnosed. treatment with ganciclovir was started with clinical remission. in november cmv infection recurred and blood cultures were positive for a bacterium that was identified as campylobacter fetus . the patient was successfully treated with intravenous ciprofloxacin. bacteremia frequently occurs in cancer patients. bacteremia due to c. fetus are rare, occurring mainly in immunocompromised patients. c. fetus expresses a proteinaceous surface layer that confers serum resistance. in our patients steroid and immunosuppression may have contributed to the development of lymphoma. all of these factors and chemotherapy have contributed to cmv infection and all have made the patient susceptible to bacteremia with this infrequently found bacterium. the clinical microbiologist should be aware of this infection in immunocompromised host. minenko sv, dmitrieva nv, sokolova en, ptushkin vv. bone marrow transplantation department, n.n. blokhin cancer research center, moscow, russian federation c'/a is the standard regimen as empirical therapy for febrile neutropenia (fn). activity of c against g'/ bacteria and g(/ bacteria, producing chromosomally-mediated b-lactamases (e.g. ampc) is suboptimal. cep is active against a broad range of g'/ and g(/, including ampc producing bacteria. the purpose of the study was to compare the efficacy of two regimens in the treatment of fn.methods: patients with fn received either cep ( g/ h) or c ( g/ h) plus amikacin ( mg/kg/day) or netilmicin ( mg/kg/day). data were collected prospectively.results: a total of pts with episodes ( / ) of fn were included. fifteen/ in cep group and / in c'/a group. the median duration of neutropenia grade , distribution of age, sex and underlying disease were comparable in both arms. mdi was in and %, cdi in and % fuo in and % in fep and c'/a groups correspondingly. response to the initial empirical regimen according to ihs criteria was in % of cep and . % of c'/a groups (p / . ). modification of therapy with a change of cep or c to carbapenem took place in and % of cep and c'/a groups (p / . ). no patient in either treatment group died due to the presenting infection. tolerability of cep was good and no laboratory abnormalities took place. transient elevation of serum creatinin level was observed in two patients c'/a group.conclusions: cep monotherapy is as effective as c'/a combination in the treatment of patients with fever and granulocytopenia purpose of the study: retrospectively, we analysed patients with intraabdominal infection for aetiology, risk factors, and outcome from hospitals in slovak republic within year (march Á/march ). results: in this group significantly more frequent were older patients ( !/ years) with cancer ( vs. %, p b/ . ). acinetobacter spp. ( vs. %, p b/ . ) and enterobacteriaceae ( vs. %, pb/ . ) were predictive for monoinfection. pre-treated patients with other antibiotics had inferior prognosis and more risk factors: permanent urinary catheter ( vs. %, pb/ . ), ventilation or intubation ( vs. %, pb/ . ) and polymicrobial infection ( vs. %, pb/ . ). the risk factors with poor prognosis were enterobacteriaceae ( vs. %, pb/ . ), diabetes mellitus as underlying disease ( vs. %, p b/ . ) and uraemia ( vs. %, p b/ . ). surprisingly, negative prognostic factor was also non-effective previous antibiotic therapy. failed patients died significantly more frequently patients due to underlying disease. cefoperazone/sulbactam was shown to be useful, effective and well tolerated also in one group of patients with % efficacy of treatment, and it belongs to a group of antibiotics suitable for treatment of nosocomial infections. key: cord- -yppi a authors: nan title: th european congress of pathology, ljubljana, slovenia, september - , date: - - journal: virchows arch doi: . /s - - - sha: doc_id: cord_uid: yppi a nan introduction formation of terminal villi with syncytiocapillar membranes is necessary for optimal maternal-foetal exchange and especially for oxygen diffusion. immature placentas with little number of syncytiocapillar membranes may worse foetal nutrition and gas exchange. the aim of this study was to estimate the role of villous tree immaturity in the development of intrauterine hypoxia. materials and methods placentas from singleton pregnancies were investigated. according to ultrasound and doppler velocime-try data, apgar score, placentas were divided into groups with chronic hypoxia ( ), iugr ( ), acute intrauterine hypoxia ( ), from normal pregnancies ( ). estimation of the degree of maturity according to gestational age was made on routine histological slides. vascular endothelial growth factor (vegf) and placental growth factor (plgf) concentration in placental tissue homogenates were investigated by elisa method in placentas. results the frequency of placental immaturity was -fold higher in placentas with hypoxia in compare with control group. preterm maturation of placental tree accompany with severe foetal hypoxia, a very high mortality and morbidity. concentration of vegf in placental tissue was elevated in placentas with terminal villi deficiency, and concentration of plgf was elevated in placentas with synchronous immaturity. conclusion due to importance of the placental maldevelopment it is necessary to devise methods of its early diagnostics and investigate mechanisms of villous tree development. determining of vegf and plgf may be useful for this purpose. molecular analysis of hereditary hemochromatosis g weirich, r langer, h nina, n jörg, h hoefler institute of pathology technische universität münchen, munich, germany hereditary hemochromatosis (hh) is an autosomal recessive disorder, common in individuals of northern european descent (prevalence close to : ) . hh is characterized by increased iron absorption and iron deposits in various organs. hh patients may develop liver and pancreas cirrhosis, primary liver cancer, diabetes mellitus, and cardiomyopathy. in the majority of cases, hh is caused by mutations of the hfe-gene on chromosome p. hfe encodes an mhc class-i gene, a putative negative regulator of iron uptake. three hfe mutation are common, c y, h d and s c. patients with a homozygous c y genotype most often endure a severe clinical course, whereas homozygotes for h d may develop mild forms of the disease. heterozygotes are usually symptom-free, but show elevated serum ferritin levels. it has been speculated that secondary iron overload (heavy alcohol, chronic hepatitis, ?-thalassemia) may be a critical co-factor in those heterozygotes that develop symptoms of hh. hh not associated with hfe mutations is much less common. within this heterogeneous group of non-hfe hh, three subtypes have been attributed to genetic alterations. juvenile hemochromatosis (hfe ) is a rare but severe disorder mapped to chromosome q. hfe has recently been mapped to the transferring receptor gene (tfr ) on chromosome q . hfe follows an autosomal dominant trait and is associated with mutations of the ferroportin gene (slc a ) on chromosome . except for juvenile hemochromatosis, hfe, hfe and hfe are amenable to mutation analysis. the advent of modern high throughput technologies such as denaturing high performance liquid chromatography (dhplc) has sparked clinical mutation screening programs, which also include screening of patients suspected to be affected by hh. in addition, dhplc-based mutation screening can also be performed using archival formalin-fixed, paraffin embedded tissues (ffpe). we present here results from hfe-analyses of ffpe from autopsy cases using dhplc and direct sequencing. introduction diseased tissues contain all the information on the genetic and epigenetic changes that caused the disease and determine its outcome (prognosis). recent developments in genome and proteome research offer the opportunity to unravel this information and provide new insight into the molecular pathogenesis of diseases. aims: we are establishing a unique human tissue resource for genome research based on a collection actually comprising . mio paraffin-embedded and frozen tissue samples stored at the temperature of liquid nitrogen. materials and methods this collection is being expanded by national and international interdisciplinary cooperations. blood samples, patient history, laboratory parameters, treatment and outcome information are included in addition to tissues. special emphasis is placed on quality control and compliance with ethical and legal criteria. all protocols follow established standard operating procedures in accordance with guidelines of the fda and recommendations of european countries. informed consent is obtained from patients and appropriate security measures guarantee patient confidentiality. results to make this resource available to a wider scientific community, we generate disease-specific gene libraries and produce tissue micro arrays. well defined expandable cell lines and nude mice xenografts are established for characterization of gene function. conclusion proper access to collections of diseased human tissues is currently a major limiting factor for genome research in medicine. there is an urgent need of tissue banks that meet highest quality and legal criteria. introduction neural cell adhesion molecule (ncam) is highly expressed on cells of the murine metanephrogenic mesenchyme at early stages of kidney development. during further development, ncam can still be detected on epithelial cells of comma and sshaped bodies as well as on interstitial mesenchymal cells. during maturation of the fetal kidney ncam gradually disappears from all epithelial structures and is confined to small areas of interstitial medullary cells. in the adult murine kidney ncam expression is virtually absent. materials and methods in the present study ncam expression was analyzed by immunohistochemistry and western blots in adult human kidneys, including healthy kidneys and kidneys with different glomerular diseases associated with incipient interstitial fibrosis (if). ncam positive cells were characterized by triple immunofluorescence staining using antibodies against human ncam combined with a panel of antibodies against neurofilaments, a smooth muscle actin (asma), hla class ii, cd and cd , and dapi for nuclei staining. digital pictures from every fluorescence channel were taken and superimposed for the specific staining using the analysis dokutm software from soft imaging systems. results in adult human kidneys, ncam expression was restricted to rare interstitial cells with a dendritic morphology, long cytoplasmatic extensions and elongated nuclei, which were localized predominantly in the outer medulla. in comparison to healthy kidneys, the number of ncam-positive interstitial cells increased in the initial phases of if in different forms of glomerulonephritis. western blot analysis of renal tissues with incipient if showed the exclusive expression of the kda isoform of ncam; other isoforms were not detectable. staining with antibodies against neurofilaments clearly differentiated nerve cells from ncam-positive interstitial cells, which are neurofilament negative. they are also asma negative, though the number of the asma+ cells increased at the early stages of if. however, some of the ncam-positive cells clearly express the stem cell markers cd and cd . conclusion these data indicate that a subpopulation of ncampositive interstitial cells may be renal progenitor cells, which can participate in the initial phases of if. introduction cell migration from the host to the transplanted organ results in chimerism ( ) , and seems to be associated with chronic transplant rejection ( , ). however, early detection of recipient cells might provide an important clue for anti-rejection therapy. materials and methods cases from sex-mismatched renal transplants (male kidney into female receiver) with consecutive biopsies (one, six, and twelve months) were analysed by fish and immunohistology (c-kit and smooth muscle actin). in additional cases of renal transplants each at day and twelve months after transplantation, glomeruli were microdissected and a rt-pcr for c-kit and ß-actin was performed. umbilical vein endothelial cells served as positive control. results glomeruli: transplant glomerulopathy shows increasing numbers of y-positive cells with progression of the sclerosing lesion. by lm -rt-pcr, increased c-kit transcription is observed compared to controls, while immunohistology is negative. arteries: recognition of vascular lesions as chronic transplant vasculopathy was easily possibly by fish, but not by light microscopy. tubuli/ interstitium: infiltrates of acute interstitial rejection change sizes from focal/large to disseminated/small over time. chimeric cells are seen in tubulitis but also in tubular epithelium with ischemic damage. conclusion transplant glomerulopathy is a lesion of chronic rejection associated with the appearance of c-kit positive, recipient cells. chimeric cells can be demonstrated in transplant vasculopathy and in damaged tubular epithelium, possibly homing to ischemic lesions as part of a repair process. . lancet ; : ; . liver transp ; : ; . transfusion ; : introduction the aim of our demonstration is to call attention to a unique pediatric case with as yet unknown ultrastructural changes in the glomerular basement membrane (gbm). case report the full-term female patient - g/ cm -has been developing well with the exception of early (intrauterine?) unilateral intraocular hemorrhage, which necessitated the enucleation of the bulbus at the age of month. nine months later, varicose enlargement of the left saphena vein was observed, and microscopic hematuria was detected. later selective proteinuria and mild hypertension was found. at the age of year an open renal biopsy was performed. light microscopy showed thick-walled and dilated capillaries of glomerular tufts. collagen iv staining demonstrated corrugated positivity in the gbm, also seen in col iv alpha chain. chains alpha and appeared normal. immunofluorescence gave only strong global igm positivity. electron microscopy showed peculiar mostly circular splitting of the gbm lamina densa with inclusions of small amorphous densities as well as of some remnants of cellular organelles. analogous changes were observed in adjacent arterioles. at present, the year-old child with signs of somatic underdevelopment is being treated for mild hyperazotemia, anemia and renal hypertension. her body shows some asymmetry, the skin is thin, pink, but no areas of hyperextensibility are seen as well as any hypermobility of joints. nmr investigation of the brain disclosed a slowly growing mass in the left thalamus. conclusion the classification of the case among connective tissue disorders is difficult, type iv of ehlers-danlos syndrome seems to be the closest. chronic renal thrombotic microangiopathy (tma) following radiation: study of cases introduction there is evidence for roles of the cip/kip family of cdki (p , p ) in mediating growth arrest in glomerular cells. mature podocytes, highly specialized cells with a complex cellular structure (interdigitating foot processes) leave cell cycle and may undergo nuclear division, but not cytokinesis, because this would involve the breakdown of the actin cytoskeleton. it has been recognized that mesangial cells proliferate and accumulate matrix in re-sponse to certain forms of injury. the aim of the current study was to elucidate the role of cdki expression in human glomerular diseases. material and methods the analysis was performed on groups of renal biopsies (every consisting of cases): minimal change disease (mcd), membranous glomerulonephritis (mgn), mesangioproliferative proliferative glomerulonephritis: iga (igan) and igm nephropathy (igmn). we examined the expression of p , p , ki and pcna by immunohistochemistry. adult nephrectomy specimens (n= ) without evidence of glomerular disease served as controls. morphometric analysis was additionally done in mcd, igan and igmn cases. color sampling was performed to extract "brown" nuclei stained for p and all nuclei of glomerular cells. then the number of segmented "brown" nuclei was calculated per area of glomerular profiles and related to the number of all nuclei. results immunostaining for p was abundant in normal podocytes and some mesangial cells. there was no evidence of p and proliferation markers ki and pcna in normal glomeruli. in mcd and mgn expression did not change. in contrast there was a marked decrease in staining intensity for p in mesangial cells in patients with igan and igmn. the ratio of stained for p nuclei was . % and . % for mcd and igan, igmn group, respectively (p= . ). interestingly also podocytes focally -in sclerotic portions of glomeruli -revealed a marked decrease of p . this loss of immunostaining occurred predominantly in mesangial cells and podocytes expressing ki and pcna. this was accompanied by the de novo nuclear immunostaining for p . conclusion the decrease in p level is required to induce podocyte and mesangial cell proliferation. also the increase of p was observed. thus changes in cell cycle regulatory molecules play central role in determining the renal response to injury and may underlie the development of progressive glomerulosclerosis. understanding mechanisms regulating cell proliferation may target future therapies on these molecules. comprised microhaematuric patients. the thinning was global in patients, and segmental in . the third cohort contained nonhaematuric patients who had a globally thinned gbm and focal proliferative lupus gn or minimal change np, respectively. in summary, cases of global tbmnp ( + nm; controls: + nm; p<. ), and cases of segmental tbmnp ( + nm; p= . ) were verified. conclusion tbmnp manifests with either a globally or a segmentally thinned gbm. tbmnp is not infrequently associated with other glomerular diseases. the clinical and morphologic data should be considered simultaneously when the diagnosis of tbmnp is made. supported by otka t- (budapest, hungary) grant to i.b. simultaneous quantitative gene expression analysis in human renal biopsies in patients with lupus nephritis (lgn) introduction inflammatory flares, enhanced proliferation and delayed apoptosis are important pathomechanisms in lgn. these are associated with increased expression of genes previously studied by semi-quantitative immunohistologic detection of an individual gene product. the aim of this study was to perform a simultaneous quantitative comparison of expression of genes tentatively relevant to pathogenesis of lgn. material and methods renal tissue samples, fresh frozen ( ) and formalin-fixed ( ), obtained by percutaneous biopsy of patients with lgn (who class ii, iii and iv) were used. formalinfixed zero transplant biopsies served as controls. from µm thick sections, glomeruli were laser-microdissected, rna was extracted and transcribed. a real-time, quantitative pcr analysis was performed using vcam- , fractalkine as markers of inflammation, il- , pdgf-ßr as markers of proliferation, bax, fas ligand and tnf-r as regulators of apoptosis and ß-actin as housekeeping gene. results frozen biopsies allowed detection of all genes; formalinfixed only of some. there was no correlation between levels of gene expression and age, sex, proteinuria or activity/chronicity indexes. who class ii cases showed increased levels of vcam- , pdgf-ßr and bax but not fractalkine as compared to controls. who class iii and iv cases showed higher expression of fractalkine, il- and pdgf-ßr than who class ii cases. in who class iii cases, il- , and fractalkine levels were clearly, while bax levels were slightly higher than in who class iv cases. conclusions . real-time quantitative pcr is useful for a simultaneous analysis of gene expression in frozen samples of human renal biopsies. . in this limited study, class ii lesions, as expected, showed lower expression of genes in comparison with classes iii and iv. however, class iii lesions seemed to exhibit higher expression levels of genes involved in proliferation, inflammatory cell infiltration and apoptosis regulation compared to class iv, possibly relating to a different pathogenesis of those two classes. haemorrhagic glomerulonephritis (gn) and massive pulmonary haemorrhage in goodpasture syndromeunique case report introduction diagnostic use of ttf- as an organ specific marker for thyroid and lung origin of carcinomas was documented mostly in tissue sections, while the information on ttf- in cytology samples is still insufficient. the aim of the study was to analyze the expression of ttf- by immunocytochemical detection in cytology samples. we obtained material by fine needle aspiration biopsies (fnab) of primary and metastatic neoplasms ( cases). first, methodological approach with ttf- was tested in thyroid tissue ( goiters, adenomas, carcinomas). afterwards, we analyzed primary and metastatic lung adenocarcinomas, primary and metastatic extrapulmonary adenocarcinomas, primary lung tumors and metastatic lung carcinomas of other types and metastatic carcinomas of different types. additionally, metastatic malignant melanomas were also included in the study. finally, we tested metastatic tumors of unknown origin. results all primary and / metastatic lung adenocarcinomas stained positive with ttf- . two of metastatic tumors of unknown origin stained positive, however primary tumor was not found. except for thyroid samples, normal lung, metastatic microcellular and macrocellular carcinoma of lung origin, none of other primary or metastatic tumors stained positive for ttf- . conclusion in fnab specimens, ttf- can confirm thyroid origin of carcinoma and more importantly, determine the lung origin of metastatic or primary adenocarcinoma. therefore, ttf- could be used for early identification of primary site (pulmonary vs. extrapulmonary) in fnab of metastatic adenocarcinomas skipping further expensive investigations. comparable to the digital radiology, recent significant developments in the microscopic slide scanning, data storage technology can contribute to the development of digital histology laboratory techniques. the aim of the study is to evaluate this technology in a routine setting. methods in the daily routine in a university pathology department to slides were prepared from to cases in a day. from the usual paraffin blocks µm sections were prepared and processed. the bar-coded slides were digitized and stored on a digital slide server. commercial short and long term storage may be used. the evaluation of the slides was done in local and remote access. for specific tissue types (colon, gastric) automated tissue component segmentation and analysis modules were developed. for the d reconstruction of serial sections a dedicated program was developed. results the daily prepared slides ( - pcs) could be scanned in the night. the virtual microscopy analysis yielded concordant results with the optical microscopy analysis. teleconsultation based on digital slides and virtual microscopy was applicable in selected cases. the automated algorithms could classify gastric biopsy specimen cases with high accuracy ( %). d reconstruction yielded a more detailed insight and diagnosis. reports including the low resolution digital slide image, high resolution selected field of views, alphabetical report were sent in e-mail to the referring specialists in higher safety and speed as compared to the traditional way. conclusions the digital histology laboratory technology improves the specialisation in pathology allowing remote access to selected tissue types, the reporting process' safety and efficiency. experiences in the production of digital slides by an automated high-resolution scanner system after automated slide preparation introduction this study aims to investigate the expression of ret/ptc and p in the local normal, benign and malignant thyroid lesions in order to shed light on the pathogenesis of papillary carcinoma and explain the high prevalence of this condition among the nodular hyperplasia (multinodular goiter) cases in the local population. archival blocks from follicular adenomas, nodular hyperplasia and papillary carcinoma cases were retrieved from husm pathology department files. they were studied by immunohistochemistry for the presence of ret/ptc and p mutant protein. normal tissues from cases served as controls. results . % normal thyroid tissue, % follicular adenomas, . % nodular hyperplasia cases and . % papillary carcinomas expressed ret/ptc mutation. ret/ptc expression in papillary carcinoma was not associated with coexistence of nodular hyperplasia lesion. p is expressed by % of papillary carcinoma. no association was found between p expression of nodular hyperplasia with or without coexisting papillary carcinoma. p , not ret/ptc was an excellent predictor of tumour lymph node metastasis and capsular invasion. it is also a significant prognosticator of survival outcome. conclusion ret/ptc mutation is highly prevalent in local papillary carcinoma, indicating a significant role in the pathogenesis of this tumour; with no apparent role in tumour behaviour and survival outcome. p on the other hand appear to be a significant factor in the latter events. the two genes appear to act in two different pathways; the former being the initiator, and the later perpetuator of papillary carcinoma. vascular invasion in pleomorphic adenoma of salivary gland: a possible consequence of fna biopsy introduction benign intravascular invasion in association with pleomorphic adenoma (pa) of salivary glands is uncommon. in none of the reported cases has it been associated with subsequent development of metastases. it may cause a diagnostic and therapeutic dilemma in salivary glands, which appears otherwise benign. no explanation of the etiopathogenesis has been offered in previously reported cases. aim: we suggest that pre-operative procedures undertaken such as fine needle aspiration (fna) of the salivary gland may cause iatrogenic seeding of tumour cells. materials and methods two cases of pa of the parotid gland showed vascular invasion. both presented with a palpable nodule in the parotid gland. one patient had two fnas the other one prior to surgery. both underwent superficial parotidectomy. results histologically both tumours were pas positive with little chondromyxoid component but both were rich in myoepithelial cells. clusters of myoepithelial and epithelial cells were seen within muscular and thin walled blood vessels, both outside the capsule and within the tumour. in spite of genuine vascular invasion there was no cytological evidence of atypia or malignancy. our speculation is that the previous fnas may have caused spillage of pa cells within vessels giving an impression of a potentially malignant tumour. conclusion we recorded two cases of pa with associated vascular invasion. we speculate that preceding operative procedures such as fna may be relevant to the pathogenesis. extra-cellular signal-regulated erk- /erk- pathway activation in human salivary gland mucoepidermoid carcinoma: association to aggressive tumor behaviour and tumor cell proliferation a handra-luca, a lesot, jc bertrand, p fouret université paris vi ufr pitié-salpętrire, paris, france information on oncogenetic events in salivary gland mucoepidermoid carcinoma is so far limited. activation of extracellular signalregulated kinases erk- and erk- is strongly correlated to cancer. using an antibody specific for phosphorylated (active) erk- /erk- , we examined human salivary gland mucoepidermoid carcinoma samples by immunohistochemistry. the comparison in paired tumor and normal tissue samples showed that phosphorylated erk- /erk- expression was higher in tumor cells as compared to surrounding normal salivary parenchyma. using a threshold below which > % of normal samples expressed phosphorylated erk- /erk- , we showed that ~ % of mucoepidermoid carcinomas expressed high levels of phosphorylated erk- /erk- . those tumours where erk- /erk- pathway was activated had a more aggressive tumor behaviour as compared to the group where this pathway was inactive. the association of erk- /erk- phosphorylation to a worse prognosis was independent of histological grade. erk- /erk- phosphorylation was associated with an increased tumor cell proliferative index. there was no relationship between erk- /erk- phosphorylation and her- /neu expression. in conclusion, erk- /erk- pathway is active in salivary gland mucoepidermoid carcinoma and this activation is associated to a more aggressive tumor behaviour and a higher proliferative activity. these data suggest that deregulation of erk- /erk- pathway contributes to mucoepidermoid carcinoma phenotype and, possibly, represent a target for new anticancer drugs. introduction adenocarcinoma of the nose and paranasal sinuses is rare but has been shown to occur with increased frequency in workers exposed to wood dust. histologically the tumours resemble adenocarcinoma of the gi tract and show the same spectrum of histological types and differentiation. therefore tumours at this site may present a diagnostic problem in distinguishing between primary and metastatic disease. immunostaining has shown colonic adenocarcinomas to express cytokeratin ck and carcinoembryonic antigen (cea) but not cytokeratin ck . lung and squamous carcinomas of the head and neck are usually ck positive and ck negative. this prompted assessment of the immunohistochemical expression of ck , ck and cea in a retrospective series of primary nasal adenocarcinomas. the aims of the study were to establish the pattern of expression of ck ck and cea in primary sinonasal adenocarcinoma and to compare it with that seen in colonic adenocarcinoma. materials and methods cases of primary sinonasal adenocarcinoma were available from archival files. these were reviewed and classified into histological subtypes including papillary, tubular, mucinous and signet ring. representative paraffin sections were immunostained for ck , ck and cea. results normal respiratory epithelium included in the sections was positive for ck and negative for ck and cea. / ( %)of tumours expressed ck . all of the tumours expressed ck and all but one of the tumours expressed cea, with showing only focal positivity. there were tumours designated mucinous or signet ring type only one of which expressed ck . conclusion primary sinonasal adenocarcinomas express ck and cea. there is variable expression of ck , which may help to distinguish between primary sinonasal adenocarcinoma, which are positive, and metastatic colonic adenocarcinoma of papillary and tubular subtype, which are negative. poorly differentiated mucinous tumours are indistinguishable by immunohistochemistry alone. provisional extracellular matrix -indicator of oral squamous cell carcinoma (oscc) progression and target for therapy introduction pleomorphic adenoma is the most common tumour of the parotid gland. histomorphologic features and peculiarly tumour capsule play a major role in recurrences of parotid pleomorphic adenomas. cytogenetically, pleomorphic adenomas are characterized by recurrent chromosome rearrangements. the aim of this study was to correlate cytogenetics and histopathology features in pleomorphic adenomas in order to define cytogenetical characteristics of tumours with high risk of recurrence. material and methods histological and cytogenetical studies were performed in patients with pleomorphic adenomas of the parotid gland treated in our institution during the period may -november . structural and numerical chromosomal abnormalities were: rearrangement of chromosome (breakpoint at q - ), rearrangement of chromosome (breakpoint at q ), translocation between chromosome and : t( ; )(q ;q ), hyperdiploidy. results the studied population was distributed into histological subtypes according to seifert classification. hypocellular (stroma rich) pleomorphic adenoma was reported in %, hypercellular pleomorphic adenoma was reported in % and "classic" in %. thirty patients ( %) had normal karyotype while ( %) had clonal chromosomal abnormalities. chromosome rearrangement tumours and hyperdiploid ones tend to be hypocellular. tumours showing chromosome abnormality tend to be hypercellular. conclusions as recurrences most likely appear in hypocellular tumours, patients with hyperdiploidy and chromosome abnormalities could have a greater risk of recurrence because of their histomorphological characteristics. because of the lack of patients, not sufficient statistical significance was demonstrated. introduction pleomorphic adenoma is the most common tumour of the parotid gland. the main issue of pleomorphic adenoma surgery is postoperative recurrence. histomorphologic features and peculiarly tumour capsule play a major role in recurrences of parotid pleomorphic adenomas. the aim of this retrospective study was to define histomorphological characteristics of pleomorphic adenoma for better understanding of recurrence processes. this work corresponds to the preliminary prospective study of a long term follow-up for detection of recurrence. material and methods histological study was performed after reviewing of slides originating from patients with pleomorphic adenomas of the parotid gland. these randomly selected patients underwent parotid surgery in our institution during the period may -november . results the studied population was distributed into histological subtypes according to seifert classification for better analysis. hypocellular (stroma rich) pleomorphic adenoma was reported in %, hypercellular pleomorphic adenoma was reported in % and "classic" in %. thinness of capsule was significantly related to hypocellularity. pseudopodias and satellite nodules were reported in % of this series. conclusions due to capsular characteristics, surgical exerecise should avoid dissection in the vicinity of the tumour to minimize the risk of surgical induced recurrence. according to these findings, enucleation surgery on a pleomorphic adenoma should not be performed anymore. these tumours should now be followed up in order to detect and define risk factors of recurrence. trend of nasopharyngeal cancer at the ibadan cancer registry ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) increase in the incidence of npc over the last here decades. overall, the mean age was . years (age range to years). the females had a mean age of . years (age range - years) while the mean age for the males was . years (range - years). the peak age group of incidence for the females was - while in the males it was the - age group with an almost an equal incidence in the preceding three decades. the ratio of regaud to schmincke type cancer reversed with increasing age amongst the females with the schmincke type being more common in the first decade but this was not reproduced in the males. conclusions there is a steady rise in the incidence of nasopharyngeal cancer over the last three decades. the two main histological types showed differential variation between the sexes suggesting a biological effect in manifestation of this disease. there is a need for health education and increased physician awareness at all levels of health care to stem this trend and especially to detect a role of epstein barr virus infection in aetiopathogenesis of the nasopharyngeal cancer. lactate dehydrogenase (ldh) in peripheral blood lymphocytes (pbl) of patients with solid head and neck tumours b jakovljevic lactate dehydrogenase(ldh)is intracellular enzyme, which take part in process of anaerobic glycolyse. it is located in all cells, but in higher amount in the stromal cells. ldh activity increased in the malignant cells, as well as in the peripheral blood lymphocytes (pbl) of patients suffered from head and neck tumours. the authors present results of their investigations in activity of intracellular ldh in pbl, spontaneous ldh releasing from pbl and correlation between spontaneous releasing of ldh and increasing of intracellular level of ldh in patients with solid head and neck tumours. spontaneous releasing of ldh in all the patients with solid head and neck tumours were to times higher then in the health controlling group. there are correlation between clinical stage of the tumours and spontaneous releasing of ldh. the authors concluded that measuring of level of spontaneous released ldh from pbl, can prove the presence of head and neck tumours in the very early stage. introduction squamous cell carcinoma (scc) constitutes more than % of the malignancies from the head and neck. thereafter, the survival of patients in stage iii-iv is between %- % at years. as most stromal types, head and neck squamous cell carcinomas (hnscc) harbour deregulation in the expression levels of many proteins that participate in critical biological pathways leading to cancer development. the aim of the study was to evaluate the correlation between several molecules implicated in cell cycle and apoptosis in an attempt to define their importance as prognostic indicators in hnscc. material and methods hnscc specimens from stage iii-iv patients were collected prior to chemotherapy. we constructed a tissue-microarray with paraffin-embedded tumours to analyse, by inmunohistochemistry, the expression levels of proteins implicated in cell cycle arrest and apoptosis (p , cyclin-d , cyclin-a, rb, hdm , topoisomerase, chk , cdc , survivin, bcl- , cdk- , cdk- , cyclin d , cyclin e, cyclin b , p , p and p ). results alterations in the expression levels of proteins that participate in the g /s checkpoint were frequent. rb and p proteins were absent in % and % of the tumours, respectively, whereas high expression of cyclin d , cdk- and cdk- was observed in %, % and % of the cases, respectively. on the other hand, des-regulation of markers implicated in the apoptosis were also evident as shown by over-expression of bcl- protein in % of the tumours. this last observation seems to be related to bad prognosis. conclusions definitive conclusions from our results concerning expression levels of each marker isolately, according to tumour location and in relation to survival are still being evaluated. p and mib expression in basaloid squamous cell carcinoma of the larynx inversely correlates with p expression patients, two showed an inverse correlation between p and ki- expression and p expression. conclusion an inverse correlation between p and ki- and p and p was demonstrated in cases. image analysis of dnac showed aneuploidy in all cases. the present study shows that overexpression of p , and ki- , together with a down-regulation of p kip correlates with biological aggressiveness and consequent shortened survival in bscc of the larynx. the dna status doesn't correlate with any of the analysed parameters since all tumours in our series displayed an aneuploid pattern. altogether patients were operated on at the department of transplantation and surgery of the semmelweis university in the past four years, because of clinical symptoms of hyperparathyroidism. chronically impaired renal function was found in cases. the removed parathyroid glands showed histologically hyperplasia in , adenoma in and carcinoma in cases. the oxyphil/chief cell ratio and occasional mitotic and apoptotic figures were determined. the oxyphil component predominated both the hyperplastic and tumorous lesions. apoptosis and mitosis were rarely seen in hyperplasias ( - %) more frequently in adenomas ( - %) whereas in carcinomas - % of the tumor cells showed signs of mitosis and - % were apoptotic. immunohistochemical detection of p , bcl and bax revealed diversity between the various lesions. cytoplasmic p positivity could be observed in the majority of adenomas and in some of the oxyphil cells of the hyperplasias. the carcinomas and about one sixth of the adenomas showed nuclear p positivity. bcl was detected in the cytoplasm of the tumor cells of adenomas and in lower extent in the oxyphil cells of hyperplasias. colocalistion of bcl and bax was found randomly in all types of lesions. no significant differences in the p , bcl and bax spectrum were found regarding the primary and secondary (i.e. renal failure) parathyroid alterations. the very low incidence of carcinomas, the low mitotic and apoptotic ratio in adenomas and hyperplasias point to the potent antiproliferative defense mechanism in the parathyroid cell-population. this may be also reflected in the cytoplasmic co-localisation of various gene products which regulate cell death and cell proliferation. introduction at present the significance of occult axillary lymph node metastasis (om) in early breast cancer patients is not known. the aim of the study was to evaluate the prognostic role of om in stage i breast cancer. materials and methods we studied patients with t breast carcinoma who underwent axillary dissection and originally had negative lymph nodes by routine microscopic examination. patients who developed distant metastases within years after surgery (m group) were compared to age-matched patients who were disease free for years (nm group). we examined lymph nodes by three deeper h&e levels and one section stained with immunohistochemistry for keratin. results we detected om in / patients. all had micrometastases (< . mm). om was identified in patients ( %) in the m group and in ( %) in the nm group (p= . ). micrometastases larger than . mm were x more common in the m group ( / vs. / , p= . ) . tumors in the m group were significantly more often of higher grade, had a higher mitotic index (mi) and showed lymphovascular invasion. the two groups did not differ in size, histologic type, estrogen receptor status or type of treatment. in multiple logistic regression, only high mi and presence of micrometastases larger than . mm showed an independent significant correlation with the occurrence of distant metastases. axillary micrometastases are associated with occurrence of distant metastases and are important prognostic factor in t breast cancer patients. this was not significant for om smaller than . mm. the presence or absence of myoepithelial (me) cells has been considered as an important feature in the differential diagnosis of papillary breast lesions. the aim of this study was to evaluate the usefulness of monoclonal antibodies: alpha-actin (smooth muscle actin) and s- protein, which are known as good markers of me cells in breast tissue. material and methods the distribution of me cells in formalinfixed paraffin-embedded tissue sections of intraductal papillomas and papillary carcinomas were immunohistochemicaly stained by abc method. results alpha-actin reacted strongly and consistently with the me cells in all papillomas. in papillary carcinomas there were no alpha-actin reactive me cells. rare isolated me cells were present in two cases, but they were inconspicuous and these reactions were also considered negative. the me cells failed to express s- protein in four papillomas, while majority were weak positive. all the papillary carcinomas were s- protein negative. conclusions consistency and uniformity of me cells reaction is important because benign and malignant papillary lesions often coexist in the same tissue section. although vascular smooth muscle in the papillary fronds and stromal myofibroblasts may mimic me cells, the number of alpha-actin immunoreactive cells is much smaller and their distribution more irregular. s- protein has broad spectrum reactivity and it is of little value in the differential diagnosis of papillary breast lesions. alpha-actin is more specific marker than s- protein for recognition of me cells in breast tissue. introduction the utilization of lymphatic mapping for breast carcinoma has made intraoperative evaluation of sentinel lymph node (sln) attractive, because axillary lymph node dissection can be performed during the initial surgery if sln is positive. the aim of our study was to evaluate the accuracy of intraoperative sln examination by imprint cytology (ic). material and methods slns from women were examined. for intraoperative cytological examination, touch imprints from bisected slns were prepared, air dried and stained with quick staining method (hemacolor-merck). slns were than formalinfixed and embedded in paraffin. histological sections were evaluated with h&e stained levels and cytokeratin immunohistochemistry (ck-ihc). results histological examination of the sln revealed ( , %) cases with metastatic deposits: ( . %) macrometastases, ( . %) micrometastases and ( . %) cases with isolated tumor cells or clusters (itc) detected by immunohistochemistry. sensitivity and specificity of intraoperative ic was % and %, respectively. ic was significantly more sensitive for detection of macrometastasis: they were detected in out of cases ( %). ic generally failed to detect micrometastases or itc: only one case of micrometatasis was cytologically suspicious for malignancy. out of histologically negative slns, one ic diagnosis was false positive. conclusions ic is a simple and quick method of intraoperative screening for the presence of sln macrometastases in patients with breast cancer. if sln micrometastases and itc are used to determine the need for further lymphadenectomy, more sensitive intraoperative methods, such as rapid ck-ihc on touch preparations, will be needed to avoid second operation. introduction fine-needle aspiration cytology (fna) is an established, highly accurate method for diagnosing breast lesions. however, in recent years there has been increased use of core biopsy (cb) as a diagnostic alternative. this study aimed at comparing the value of fna and cb in palpable and non palpable breast lesions. materials and methods the material consisted of fna samples performed in our institution between and . correspoding histology results from lesions including cb were available for comparison. quality assessment parameters were calculated using metodology detailed in the national health service breast screening programme guidelines. results high specificity ( %) and sensitivity ( %) and low inadequacy rates were found for cb. fna showed high sensitivity ( %). low specificity ( %) for fna due to inclusion of many cell-poor lesions was particularly seen in the submitted material sampled by physicians lacking experience with fna procedures. conclusion cb may be an alternative method for preoperative diagnosis when experienced cytopathologists are not available. cb is superior to fna in fibrotic and collagenous lesions such as lobular carcinoma and radial scar. fna is most accurate when immediate assessment by cytopathologist is performed for evaluation of material adequacy so that additional aspirations can be performed if necessary. atypical ductal hyperplasia (adh): practical relevance in diagnostics and therapy introduction in contrast to ductal hyperplasias, adh belongs to intraductal neoplasias. adh can be distinguished from non-high grade dcis (among other criteria) by its size. therefore, a question that is asked quite frequently is whether such small findings can be removed with sufficient security by minimal invasive biopsy (mib). we prospectively investigated: . relative frequency of adh without associated intraductal or invasive carcinoma . relevance of such small findings in mib specimens. materials and methods between between and between , breastlesions could be clarified by means of mib in the interdisciplinary team. excisions were carried out in all invasive carcinomas and all dcis as well as in all cases with minimal parts of intraductal nonhigh grade neoplasias (with the differential diagnosis adh vs. dcis). additional adhs were diagnosed in excision specimens as incidental findings associated with other targets (except for dcis and invasive carcinomas). the mib-and operation specimens have been examined prospectively by two team pathologists according to the criteria of page and tavassoli. results we diagnosed altogether adhs and dcis. the proportion of adh in all operated intraductal neoplasias (adh+dcis) was . % ( / ). findings with the differential diagnosis of adh vs. dcis could be found in cases of mibs ( , %).the following excision specimens showed in of cases ( %) non-high grade dcis and in of cases adh ( %). the other adh were coincidental findings in excision specimen of benign lesions. conclusions adh is a very rare finding. in adh-like findings in mib specimens even specialised breast-pathologists cannot exclude a non-high grade dcis with sufficient security when applying strictly the most common morphological definitions. the high rate of % of dcis in the specimen of the following operation highlights the indication for clarifying by excision (according to the european guidelines for quality assurance in mammography screening). multifocal stromal invasion in microinvasive squamous cell carcinoma of the cervix -how to measure and stage these lesions o reich, h pickel dpt. obstet/gynecol, university of graz, graz, austria objectives about % of microinvasive squamous cell carcinomas of the cervix have more than one invasive focus at time they first break through into the cervical stroma. it is unclear how the lateral extent of lesions with multiple invasive foci should be measured to distinguish figo stage a and a carcinomas from clinically occult stage ib disease. we believe that three presentations of multiple invasive foci should be distinguished. type i shows plump and coherent infiltration of the cervical stroma at different sites in one ore more step-serial sections. they originate from the squamous epithelium outside the transformation zone or from metaplastic cervical gland epithelium. all foci are contiguous with the epithelium. in this case, the largest lateral spread of each individual invasive focus should be measured. these measurements are added up to determine the total of horizontal spread. the normal tissue separating the different early invasive foci is not measured. in type ii the origin of invasion cannot be seen in all early invasive foci but the greatest lateral extension can be seen in one of the step-serial sections. lateral extension is defined as the maximum distance from one most lateral point of the border between the invasion and the adjacent tissue to the other. the lateral extension of type ii includes the normal tissue separating the early invasive foci. type iii shows the same pattern as type ii, but the maximum horizontal spread cannot be measured in a single step-serial section. in such lesions the width needs to be measured across all the step-serial sections involved and the second dimension must be calculated by knowing the distances between the sections. conclusions these growth pattern of stromal invasion should be considered when measuring the width of early invasive squamous cell carcinomas with multiple invasive foci. lesions with multiple invasive foci taken together measuring more than mm in lateral extension should be classified as stage ib. introduction cyclooxygenases regulate the production of prostaglandins and play a role in tumor development and progression. the aim of this study was to investigate the prognostic impact of expression of the cyclooxygenase isoforms, cox- and cox- in primary breast carcinomas as well as in primary ovarian carcinomas. materials and methods cox isoform expression was determined by immunohistochemistry. results expression of cox- was detected in % of ovarian carcinomas and was associated with reduced overall survival in univariate analysis (p= . ) as well as multivariate analysis ( . ). there was no correlation with other tumor parameters such as grade, stage or type. in contrast, cox- was no prognostic parameter in ovarian carcinoma. expression of cox- was detected in % of breast carcinomas and was significantly associated with several clinicopathological parameters, such as positive nodal status (p< . ), larger tumor size (p< . ), poor differentiation (p< . ), vascular invasion (p= . ) and negative estrogen receptor status (p= . ). in univariate survival analysis a significant association between elevated cox- expression and decreased disease-free survival (p= . ) as well as decreased overall survival (p= . ) was observed. in multivariate analysis expression of cox- was of borderline significance for disease-free survival (rr( %ci) . ( . - . ) ). conclusions our data suggest that increased expression of cox- may play a role in progression of primary breast carcinomas as well as ovarian carcinoma. it remains to be investigated if treatment with selective inhibitors of cox- may be an additional therapeutic option for patients with gynecological tumors. simple endometrial hyperplasia: bcl- , hldf expression and clinical behavior ii the endometrial hyperplastic processes is characterized by major variety of clinical outcomes. the main feature of "spontaneous endometrial hyperplasia" is complete normalization after total curettage (lindahl b., willen r. ) ; there is also proof of recurring hyperplasia. the purpose of present research was to carry out clinical and morphological correlations between various types of endometrial hyperplasia and expression of pro-(hldf) and antiapoptotic (bcl- ) proteins. human leukemia cell differentiation factor (hldf) with molecular mass . kd, having dna/rna hydrolyzing activity, was originally allocated from hl- cell line of promyelocyte leukemia medium, processed by retinoid acid or c -ceramid (kostanyan i.a. et al., ) . previously we have shown that hldf is a useful tool for apoptosis visualization in prostate (babichenko i.i. et al., ) . we performed analysis of patients with histologically documented simple endometrial hyperplasia without atypia: women ( %) had spontaneous recovery after total curettage, in ( %) patients endometrium recovered after gestagen treatment and in ( %) cases persistent recurring hyperplasic process was observed during - years. immunohistochemical research showed that in samples of recurring hyperplasia hldf was expressed in cytoplasm of glandular epithelium, while in sections with spontaneous or aftertreatment recovery, hldf was revealed as a thin line on the surface of glandular cells. bcl- protein was found only in the cytoplasm of glandular cells in all investigated groups. thus expression of the hldf factor in cytoplasm of glandular cells in endometrium allows revealing the disturbance of apoptotic processes in cells and predicting the subsequent recurrence of simple hyperplasia. surface epithelial changes in endometrial adenocarcinoma b djordjevic, d mihailovic, d dimov, l velickovic, v zivkovic, z mijovic institute of pathology, university of nis, nis, serbia and montenegro introduction epithelial changes on the surface of endometrial adenocarcinoma (eac) show greater maturation and better differentiation than the underlying adenocarcinoma. these changes are relatively common and can be misdiagnosed as benign surface changes on curettings, including endometrial metaplasias (em), papillary syncytial change (psc), or microglandular hyperplasia (mgh) . the aim of this study was to investigate surface epithelial changes in eac. material and methods cases with eac ( endometrioid, serous, clear cell and mixed types) were studied. results ( %) of the eac showed surface epithelial changes: four with microglandular pattern simulating mgh; with syncytial aggregates of eosinophilic cell, frequently with papillary or squamoid appearance, simulating em or psc; and with mixed patterns. surface epithelial change often demonstrated focal conjunction with a frankly malignant gland or a fibrovascular core and ranged from rare foci to diffuse surface involvement. cytologic atypia was less pronounced than that of the underlying eac and varied from mild to moderate. mitoses were very rare. in some scanty curetting specimens, distinguishing these surface epithelial changes from benign changes was difficult, as was a diagnosis of eac. conclusion in postmenopausal women with curettings showing features of em, mgh, or psc, caution should be exercised in making that diagnosis, particularly psc, in the absence of evidence of benign shedding, and any associated cytologic atypicality should raise the possibility of the underlying eac. prevalence of cervical neoplastic lesions in egypt: a national cervical cancer screening project. final report introduction reducing morbidity and mortality is one of the main objectives of the health and population policies in egypt. however, data from egyptian studies provide widely varying estimates of the prevalence of pre-malignant and malignant cervical lesions. the main goal was ( ) to define the prevalence of pre invasive and invasive cervical cancer among egyptian women, and ( ) to identify risk factors including hpvs and associated co-factors, as bacterial vaginosis, hsv, cmv, chlamydia, schistosomiasis. the study was a community based full-scale cross sectional, household survey that included women aged between and years conducted between and . however, women with complete data were included in the study ( %). the bethesda classification was used for cervical cytology. three percent of cases were unsatisfactory. normal, metaplastic, infectious, and neoplastic lesions represented . %, . %, . % and . %, respectively. the most common type of infection was bacterial ( . %). neoplastic conditions, were categorized into ascus and agcus ( . %), ascus ( . %), agcuc ( . %), lgsl . % ( . %), hsil ( . %) invasive squamous and invasive adenocarcinoma ( . % for each). colposcopic-guided biopsy was performed on cases representing . % of the epithelial abnormalities. for standardization, a score of ten points including koilocytic atypia ( ) , binucleation ( ), papillomatosis, dyskeratosis, acanthosis and basal cell hyperplasia ( point for each), was applied to qualify a lesion as hpv-related ( points out of ten). unsatisfactory biopsies were encountered in . %. normal biopsies including inflammatory and metaplastic changes and atypical metaplasia represented . % and . %repectively. cervical intraepithelial lesions (cin) ( . %), further categorized as cin i ( . %), cin ii ( . %), cin iii/cis ( . %) were identified. endocervical lesions represented . % and were divided into endocervical glandular dysplasia (egd) ( . %) and adenocarcinoma in situ (ais) ( . %). combined dysplasia was found in . %, while invasive squamous cell carcinoma (sqcc) and lymphoma accounted for . %of the biopsies. on histological basis, hpv was found in association with cin i, cin ii, cin iii/cis and sqcc in . %, . %, . % and % respectively. neither egd nor ais showed evidence of hpvs. in situ hybridization (ish) using the broad spectrum hpv probe recognizing hpvs , , , , , , , , and was tested on biopsy specimens starting from the atypical metaplastic changes and was found to be positive among . %, negative in . % and non significant in . %. interestingly, . % of atypical metaplastic changes were positive. further subtyping with probes / , / and / revealed positivity in . %, . %, and . % of cases respectively. noteworthy, multiple infections with hpv / and / ( cases) and hpv / and / ( cases) were identified. to examine the main determinants of cervical cancer, multiple regression analysis was conducted using high-grade lesions as the dependant variable and risk factors as independent variables. this showed that the only determinant was hpv infection. when the regression analysis was conducted without hpv as one of independent vari-ables, history of treatment from schistosomiasis was found to have or of . ( %ci . - . ) but the model was not statistically significant. conclusion in the egyptian population, the prevalence of the precursor lesions of the invasive carcinoma is % while invasive lesions account for . % of the population. the prevalence of hpv histologically is . % and by ish . %. however, detection of hpvs by ish in this study cannot be precise due to technical limitations and limited number of probes used. evaluation of hpvs using pcr technology will be subject to another study. possible role of irf- in the genesis of endometrial adenocarcinoma. a series of such tumours was investigated using immunohistochemical techniques. relatioships were sought with thymidine phosphorylase (tp), conventional histological features and prognosis. malignant endometria were, by and large, deprived of irf- , as of the cases ( . %) were negative for this antigen. only of the cases were expressing irf- in inner tumour areas. at the invading tumor front, however, irf- was depressed in tumour cells in % of cases. this phenomenon was independent of the extent of lymphocytic response, but it was a strongly associated with tp expression. tp up-regulation and host's lymphocytic response in the area were directly associated. irf- derepression by invading tumor cells was associated with poor prognosis. this prognostic parameter was independent of stage. it is concluded that down-regulation of irf- is a constant finding in endometrial tumorigenesis. however, derepression of irf- may occur in a subset of tumours, and this event is associated with tp up-regulation and aggressive tumor behaviour. morphometric study of gnrh analogue/ hmg/hcg effects on ultrastructure of human endometrial epithelium in the early luteal phase introduction there is a little information about the effects of ovulation induction drugs on morphometry of human endometrium especially at electron microscopic level. the aim of the present study was to determine the effects of long protocol (gnrh analogue/hmg/hcg) on the ultrastructure of human endometrial epithelium at lh+ . material and methods endometrial biopsies were obtained from fertile women (n= ) as well as infertile women (n= ) who had undergone this protocol at lh+ . quantitative and qualitative studies on endometrial epithelium were performed by transmission electron microscopy (tem) and morphometry and the results were statistically compared between the two groups. results qualitative results revealed presence of nuclear channel system (ncs), sub vacuole of glycogen and giant mitochondria (gm) in both groups. similarly, in quantitative analyses, the volume fractions (vv) of glycogen, mitochondria and rough endoplasmic reticulum to cell and the also the vv of euchromatin to nucleus were statistically not different between two groups (p> . ). conclusion these results suggest that ovulation induction by long protocol is not associated with retarded or advanced endometrial epithelial development. introduction historically the diagnosis of mitochondrial encephalomyopathies relied, on the presence of ragged red fibres (rrf) as well as finding ultrastructural abnormalities in mitochondria. currently accurate diagnosis requires a multidisciplinary approach but the usefulness of electron microscopy has been questioned. the aim of this study was to define the diagnostic role of electron microscopy in comparison to histology and histochemistry. patients and methods thirty-five patients who fulfilled a clinical phenotype for mitochondrial encephalomyopathies, were separated into two groups. the paediatric group, which included cases, with an age range of days to years old and the adult group which included cases, with an age range of to years old. all patients underwent a muscle biopsy which was examined by histology, histochemistry and electron microscopy. results in the paediatric group, cases exhibited rrfs, cases showed rrfe and cases had cox abnormalities. electron microscopy revealed the presence of mitochondrial abnormalities in cases and mitochondrial aggregates in others. in cases light microscopy was normal, but in of these electron microscopy was abnormal. in the adult group all patients, showed cox abnormalities, cases had rrfe and others had rrfs. electron microscopy revealed the presence of mitochondrial aggregates in cases and structural abnormalities in cases. conclusions electron microscopy examination is a useful independent diagnostic tool in the investigation of mitochondrial encephalomyopathies. it is particularly helpful, in the paediatric cases, where the classic light microscopical findings are often not well developed. introduction tumour-associated tissue eosinophils (tate) have been observed in human tumours and experimental tumour models, but the mechanism by which eosinophils induce tumour cell damage remains poorly understood. aims: electron microscopy is an essential technique in evaluating sublethal and lethal cellular injuries. therefore, gastric carcinomas were studied by light and electron microscopy, focusing on the relationship between eosinophils and tumour cells and on the nature of tumour cell death. material and methods fresh tumour tissue was obtained in the operating room for immediate fixation and processed for both light and electron microscopy examination from until . results a light microscopy review of cases of resected gastric carcinomas identified cases ( of intestinal-type and of diffusetype) with a massive tate. in cases of intestinal-type adenocarcinomas, eosinophils formed varied-sized aggregates in the tumour stroma. some were seen passing through the neoplastic epithelium, and others were found lying within neoplastic tubules. in cases of diffuse-type carcinoma, eosinophil clusters were seen to surround single tumour cells. with electron microscopy, performed in these cases, tumour cells in intimate contact with eosinophils revealed sublethal and lethal cellular injuries. in particular, sublethal injuries, such as cytoplasmic vacuoles and indiscernible cell membrane, occurred at the contact region. eosinophil "satellitosis" was associated with lethal damage of tumour cells including brush border disorganization, dilation of nuclear envelope, rough endoplasmic reticulum and golgi apparatus forming empty spaces or vacuoles, small condensed chromatin particles, irregular distribution of nuclear pores. these lethal injuries were compatible with non-apoptotic (non-lysosomal vesiculate) cell death. the adenocarcinoma cells, not contacted by eosinophils remained morphologically well preserved. conclusions these morphological data suggest the hypothesis of a direct relationship between eosinophil infiltration and induction of non-apoptotic tumour cell death in some cases of gastric adenocarcinoma. a deeper insight into the molecular mechanisms acting as cytokines that stimulate specific eosinophil functions may assist in the development of novel therapies to better treat human malignancies expression of p and c-erbb- oncoproteins in chronic schistosomal urinary bladder lesions o hammam, h el baz, m wishahi theodor bilharz research institute, giza, egypt this study was conducted in a trial to better understand the genetic mechanisms underlying the proliferative, premalignant and malignant changes frequently displayed in chronic schistosomal cystitis (ch.sch.c.) and schistosomal associated transitional cell carcinoma (sch.tcc) of the bladder. tissue exepression of p (tumor suppressor gene) and c-erbb- (oncogene) was assesed immunohistochemically in bladder urothelial specimens of a well characterized cohort of male egyptian patients ( ch.sch.c; sch.tcc and normal urothelial specimens as controls). both p and c-erbb- oncoproteins were not expressed neither in morphologically normal nor in the hyperplastic epithelium. overexpression of nuclear p and membranous c-erbb- was detected in % of ch.sch.c cases in whom premalignant changes (metaplasia and dysplasia) were displayed. while, in the sch.tcc cases they were detected in % and % of cases respectively. on stratifying these patients according to the well recognized prognostic parameters (histopathological grade, stage, tumor size, multifocality and growth pattern) p was positively correlated with all the forementioned parameters. in contrast c-erbb- was only correlated with tumor size and growth pattern. in conclusion, the results of the current study may suggest the early involvement of both p and c-erbb- in the initiation of the premalignant changes occuring with ch.sch.c and the progression of these changes to malignancy. moreover, the high incidence of p and c-erbb- expression among sch.tcc cases with poor prognostic parameters (higher grade, stage; bigger tumor size; tumor multifocality and nonpapillary growth pattern) may point out to the possibility of using these biomarkers as early predictive markers in ch.sch.cyst. cases displaying premalignant lesions and as poor prognostic indicators for sch.tcc. introduction cd is a neutral endopeptidase which participates in the peptide hidrolysis in the renal proximal tubules. its immunohistochemical detection has been considered useful in the differential diagnosis of renal carcinomas because of its expression in - % of clear cell renal and papillary renal cell carcinomas and its absence in chromophobe renal cell carcinoma (chrcc), a histotype positive for parvalbumin, a protein of the distal nephron. material and methods we studied immunohistochemically chrccs including four with aggressive features (two with sarcomatoid transformation, one with renal vein involvement and one with pancreatic metastasis), for cd and parvalbumin developing the reactions with different methods (abc, envision peroxidase detection and labeled polymer-ap dako envision systems) to prevent false-positive staining due to endogeneous biotin. western blotting was done to confirm the results. as control we stained clear renal cell carcinomas with both antibodies. results cd immunoreactivity was observed in ( %) primary chrccs, three with aggressive features, using all the developing systems. all chrccs were positive for parvalbumin, but not the sarcomatoid areas. western blotting confirmed the specificity of the immunoreactivities. conclusions ) about % of chrcc express cd ) cd is not specific for clear cell and papillary carcinomas of the kidney; ) a panel including both cd and parvalbumin is useful in the differential diagnosis between chrcc and clear renal cell carcinoma; ) three out of four tumors with aggressive features stained for cd , but further studies are needed to highligh the prognostic significance of cd in chrcc. introduction tenascin (tn) is an extracellular matrix glycoprotein, which is expressed during embryogenesis at the epithelial-stromal interface. tn is decreased in normal adult tissue but reexpressed in inflammatory, reparative and neoplastic processes. tn seems to be involved in neoangiogenesis, promotion of invasion and metastasis of malignant tumors. the aim of the study was to investigate the expression of tn in carcinoma in situ (cis), invasive and superficial papillary bladder cancer. material and methods cases of bladder cancer were selected ( who grade i, grade ii, grade iii tumors, cis). we investigated the different staining patterns of tn by immunohistochemistry using a monoclonal antibody against tn (t h ). results immunhistochemical evaluation revealed stromal and cytoplasmic staining. stromal staining was found at the borders of invasive tumors and in the lamina propria of most cis. tn staining of blood vessels was located at the tips of the papillae or in invasive tumor. cytoplasmic tn expression was present in all bladder cancer specimens. in cis positive cells were present in all cell layers or distributed in a pagetoid pattern while adjacent normal urothelium showed staining of superficial cells. in who grade i papillary tumors tn staining was located in superficial cells, in grade ii tumors scattered positive intermediate cells were present, while in grade iii tumors tn was expressed focally in all cell layers. most invasive tumor cell nests showed extensive tn staining. conclusion expression of tn in bladder cancer seems to be associated with dedifferentiation and invasive potential. introduction synchronous, antecedent or subsequent appearance of secondary primary malignancy (spm) in patient with primary renal cell carcinoma (prcc) is yet considered to be extremely rare. recent surveys tend to confirm at least their higher if not even significant incidence. this study was performed to determine the incidence of spm in the patients with prcc as well as its localization and time of appearance in relation to prcc. material and methods of patients who underwent surgery for renal malignancies at department of urology, sestre milosrdnice university hospital during the period from to , data of were available in croatian national cancer registry. there were patients with prcc ( males and female patients) ranging in age from - years (median . years). results and conclusion in our series there were ( . %) patients with spm. secondary malignancies in descending order of frequency were cancer of prostate ( . %), colon ( . %), lungs ( . %), bladder ( , %) and skin ( . %). there were . % antecedent, . % synchronous and , % subsequent spm. only one patient had more than one synchronous second malignancy (urinary bladder, prostate and colon). spm in patients with prcc are of great importance for both the clinician and pathologist. therefore, the clinician should maintain lifetime high-technique follow-up of the patients with prcc. the knowledge of such an existence should raise an interest in pathologist for elucidating etiologic factors. namely, it seems that the frequency of spm in patients with prcc is much higher that previously expected. introduction in our recent studies we showed increased oxidative stress with p gene alterations in urinary bladder lesions in people living in radio-contaminated areas of ukraine, after the chernobyl accident. the aim of the study was to examined whether base and nucleotide excision repair is activated in bladder urothelium by chronic low doses of ionizing radiation in male patients with benign prostate hyperplasia and females with chronic cystitis living more than years in cs contaminated areas. materials and methods bladder biopsies from patients were submitted for histological examination and biopsies from patients -for immunohistochemical study of -hydroxy- 'deoxyguanosine ( ohdg), -oxoguanine-dna-glycosylase (ogg - ), apurinicapyrimidinic endonuclease (ape ) and xeroderma pigmentosum a (xpa). results chronic proliferative atypical cystitis with multiple areas of dysplasia and carcinoma in situ was observed in % of patients from radio-contaminated areas with greatly elevated levels of ohdg, ogg - , ape and xpa in bladder urothelium. conclusion these findings support the hypothesis that significant activation of dna damage repair (base and nucleotide excision repair) is induced by long-term low dose of ionizing radiation. the levels of dna oxidative adducts pointing to mutagenic and carcinogenic potential were in line with the histopatologically diagnosed urothelial lesions. introduction urinary cytology (uc) and cystoscopy remain the standard methods for initial diagnosis and monitoring of recurrences in urothelial tumors. low sensivity of uc and invasiveness of cystoscopy led to develop different ancillary methods to improve sensitivity of uc. specific genetic alterations occurr in bladder cancers (bc), represented by aneuploidia of chromosome , , and loss of chromosome . the aim of the study was to evaluate the use of thin layer techniques applied to uc diagnosis associated with multicolour interphase fluorescence in situ hybridization (fish) for early identification of initial bc and recurrences. methods a total of voided urine specimens were collected, before resection of bladder cancer, from patient with previous bc and flogistic control patient. the samples were processed with thin prep (tp) monolayer technique and cytospin preparation (cs) and in cases with morphological alterations, was performed fish urovysion (vysis) assay, consisting of four probes ( , , and p ) . cytomorphology. results in general tp technique was better for cytomorphology, cleaner background little overlapping, with difficulties when only few urotelial cells are present and in cases of contamination with squamous cells. f i sh technique was positive in all carcinomas, with multiple gain of chromosomes , , , while none of negative controls was aneusomic. only five cases in group of patients in follow-up with minimal atipia, showed fish positity, with prevalence of loss of chromosome ( %) compared to gain of chromosomes ( %). conclusions these preliminary findings suggest that the association of tp preparation with fish anlysis can improve the efficience of uc to detect bc and predict its recurrence. chromophobe renal cell carcinoma: the variant with the best outcome? introduction few large series comparing conventional, papillary and chromophobe renal cell carcinomas (rcc) have been published. most have been heterogeneous, including rcc subgroups of different stage and/or grade. the purpose of this study was to examine the clinico-pathologic features of a series of chromophobe rccs comparing their outcome with that of conventional and papillary rccs in a grade and stage matched fashion. materials and methods the clinico-pathologic features of chromophobe rccs, papillary and conventional rccs were examined, focussing on the following parameters: patient age and sex, tumor size, stage ( ptnm), fuhrman grade, and histology. survival was analyzed using the kaplan meier method. the most important prognostic factors were identified using the cox model. results for patients with localized disease at diagnosis; median, -year, and -year disease specific survivals (dss) were similar for chromophobe ( mths, and -yr dss: %) and papillary ( mths, and -yr dss: . %) tumors. conventional rcc patients had a significantly worse outcome ( mths, -yr dss: . %; -yr dss: . %). the best outcome in low grade (g +g ), low stage (pt +pt ) tumors was observed in chromophobe rccs. in multivariate analysis, conventional (nonpapillary) rcc histology was the major independent adverse prognostic factor whether or not stage and grade were included in the cox models. conclusion chromophobe rcc is a rare rcc variant of good prognosis. this favorable prognosis is maintained when low grade (g +g ) / low stage (pt +pt ) tumors are specifically examined. introduction c-kit is a transmembrane tyrosine kinase receptor, expressed at high levels in hematopoetic stem cells, mast cells, melanocytic cells, germ cells and the interstitial cells of cajal. recently, it has been shown that for certain tumors (especially gastrointestinal stromal tumors and mast cell diseases) c-kit mutation or activation is a major pathogenetic event. only one previous study has been published regarding c-kit expression in normal renal tissue, and none regarding renal neoplasms. in this preliminary report of an ongoing study, we investigated the expression of c-kit protein via immunohistochemistry in various normal and neoplastic renal tissues. material and methods our material included eight fetal kidneys ( - weeks of gestation), seven normal adult kidneys, two cases of renal dysplasia, and renal tumors: renal cell carcinomas, two nephroblastomas, three oncocytomas, one mesoblastic nephroma and one angiomyolipoma. staining intensity was recorded in a three-grade scale. results all fetal kidneys showed cytoplasmic staining of proximal tubules (++). henle loops were also positive (++), especially in older embryos. cytoplasmic and membrane staining (++) of renal tubules was also observed in all normal adult kidneys. the two cases of renal dysplasia studied, had c-kit expression in their normal but not in their aberrant tubules. of the renal cell carcinomas, all conventional type cases (n= ), one sarcomatoid and one papillary type case were negative, while one chromophobe and one granular type were positive (++ and + respectively). all oncocytomas were positive (+++ to ++), and quite interestingly the mesoblastic nephroma was also positive. the angiomyolipoma and the two nephroblastomas were negative. conclusion we conclude that c-kit is expressed in normal fetal and adult renal tubules, as well as in a subset of renal tumors. the expression of c-kit in the latter group may prove to have diagnostic significance and therapeutic implications. background relapsed/refractory acute myeloid leukemia (aml) is characterized by a deteriorating clinical course and resistance to therapy. biological mechanisms responsible for resistance remain unclear, since consistent molecular characteristics of aml at relapse and refractory disease have not yet been reported. methods we explored paired bone marrow biopsy specimens of patients (at diagnosis and relapsed/refractory aml after high dose chemotherapy) in an immunohistochemical study using anti human ki- antigen (mib- ) antibody to determine the proliferation fraction of leukemic blasts. gene expression was analyzed in a different sample set ( at diagnosis and relapsed/ refractory aml) using a "relapse specific" cdna microarray covering genes, selected by literature and suppression subtractive hybridization. microarray results were validated using quantitative real time pcr, immunohistochemistry and immunoblotting. results recurrent and refractory aml blasts demonstrate a highly significantly increased (p< . ) proliferation fraction compared to primary diagnosis. mrna levels of proliferation markers cd and pcna were elevated . -fold and . -fold, respectively, in relapsed and refractory aml compared to diagnosis. enhanced activation of the raf/mek/erk mitogen-activated protein kinase cascade was indicated by increased mrnas of b-raf ( . -fold), cyclin d ( . -fold), mkp- ( . -fold), c-fos ( -fold) , and egr- ( . -fold). additional immunohistochemical and immunoblotting analyses demonstrated an elevation of biphosphorylated erk / protein in recurrent aml, confirming enhanced activation of the raf/mek/erk pathway. conclusion our results provide for the first time evidence that high dose chemotherapy selects or induces leukemic blasts with increased proliferative potential via enhanced activation of raf/ mek/erk cascade. introduction diffuse large b-cell lymphoma (dlbcl) is the most common of the non-hodgkin's lymphomas (nhl), comprising approximately % of newly diagnosed cases. in order to define the role of angiogenesis in dlbcl, mrna and protein expression of the two most important angiogenic growth factors, vegf and bfgf, and two vegf receptors, vegfr- /flt- and vegfr- /kdr were evaluated by real time quantitative pcr (rtq-pcr) and immunohistochemistry in dlbcl. methods we analyzed dlbcl cases, low-grade follicle center lymphomas (fls) and cases with chronic lymphocytic leukemia (cll) for expression of vegf-a, bfgf, vegfr- /flt- and vegfr- /kdr. we used a real-time reverse transcriptionpolymerase chain reaction (qrt-pcr) method to quantify mrna levels of these angiogenic markers using an abi qrt-pcr system. protein expression of these angiogenic markers was assessed by immunohistochemical staining. the results of the current study show that both the mrna and protein expression of vegf and bfgf varied considerably among dlbcl patients, which do not correlate to the microvessel density. vegf mrna expression showed significant correlation with bfgf mrna expression. the vegfr- /kdr mrna expression was higher than vegfr- /flt- mrna expression in dlbcl but their expressions were strongly correlated. histological staining of both vegf receptors in dlbcl sections showed strong staining of both receptors on the microvessel endothelial cells but not on the tumor cells. our data suggest that vegf plays a role in the pathophysiology of dlbcl however autocrine/paracrine stimulation by vegf does not seem to be relevant in dlbcl. the expression of vegf and its receptors raises the possibility of using angiogenesis inhibitors as a novel therapeutic strategy. introduction ecm regulates cellular differentiation, life span, migration and contributes to lymph node compartmentalisation. the aim of this study was analysis of occurrence and distribution of embryonal fibronectin (fn), tenascin (tn) isoforms and laminin chains (ln) in reactive lymph nodes (rln) and follicular lymphoma (fl). material and methods shock frozen tissue of rln and routine material of fl and rln. primary antibodies: all fn variants (ist- ), ed-a fn (ist ), (bc- ), all tn-c variants , tn-cl (bc- ), ln-alpha -( h ), ln-alpha -(bm ), ln-alpha -( c ), ln-beta -( e ), ln-beta -(c ), ln-gamma -( e ), ln-gamma -chain (gb and d b ). apaap-technique. results rln: all fn's, ed-a fn and tn-cl were demonstrated in lymphatic / blood vessels and as reticular depositions in the stroma; spot-like depositions in the follicle center and ribbon-like at the border follicle centre / mantle zone. ln-alpha , -alpha , -beta , -beta and -gamma -chains were positive mainly in lymphatic and blood vessels with depositions in the stroma. ln-gamma -chain was restricted to follicular dendritic cells and ribbon-like codeposited with tn-cl at the border follicle centre / mantle zone. fl: in contrast to normal follicles, a total loss of ln-gamma -chain is regular observed in neoplastic follicles. conclusions . a provisional ed-b fn / tn-cl / ln-gamma chain matrix membrane controls the traffic follicle centre/mantle zone. . in analogy to thymic t cell maturation, a role of ln-gamma -chain (ln- ) can be assumed for b cell maturation mediated by ln-gamma -chain (ln- ) positive follicular reticulum cells. . the loss of ln-gamma -chain indicates neoplastic follicular structures in follicular lymphoma. follicular lymphoma grading by mann and berard are predictive of overall survival and time to treatment failure, however, only grade has favorable os and ttf follicular lymphomas (fl) are a heterogeneous group of tumors, varying in clinical features, immunophenotype, and cytogenetics. who recommends grading of fls by using the method of mann and berard, which recognizes three grades. the aim of this study was to examine whether mann and berard's grading has prognostic significance in patients with follicular lymphoma who were not uniformly treated. samples from patients with follicular lymphoma with follow up ranging from - months (median months) were included in the study. forty-three patients ( %) achieved a complete response (cr), ( %) partial response (pr), while ( %) were considered non-responders during the follow up period of months. twenty-seven ( %) patients died, patients ( %) from dissemination, from transformation to other lymphoma type and ( , %) patients died from non-lymphoma related causes. os rate was significantly worse in patients with ann arbor stage iv than patients ann arbor stage i-iii (p= . ). patients with lower ipi score did significantly better than those with a higher ipi score for os (p< . ) and ttf (p< . ). grade fl patients had longer os (p= . ) than grade and patients. a trend for longer ttf was also found (p= . ). cox regression analysis showed that these results were independent of stage, but were not independent of ipi score. higher grade was also associated with the presence of b symptoms (p= . ) and larger size of the primary tumor (p= . ). follicular lymphomas (fl) are a heterogeneous group of tumors, varying in clinical features, immunophenotype, and cytogenetics. the aim of this study was to examine interrelationship and prognostic significance of bcl and cd protein expression for overall survival (os), and time to treatment failure (ttf). samples from patients with follicular lymphoma were evaluated by immunohistochemistry. bcl- and cd expression levels were determined semiquantitatively. since almost all cases expressed bcl- , fls were designated either "low expression level-group" (llg and +, patients) or "high expression level group" (hlg, + and +, patients) . based on cd expression, fls were designated either "negative" ( patients) or "positive" ( patients). patients with high levels of bcl- expression had favorable overall survival (os) (p= . ) and ttf (p= . ) compared to patients with low levels of bcl- expression. cox regression analysis showed that the results for os, but not ttf were independent of ipi score. patients with cd -negative follicular lymphomas also had worse os (p= . ) and ttf ( . ). our results suggest that expression of cd and strong expression of bcl- protein could be indicators of welldifferentiated follicular lymphoma since such cases were associated with lower histological grade and better clinical outcome. "borderline lesions" with clinicopathologic features in between lyp and alcls. lyp nature (neoplastic vs reactive) is matter of debate because of its benign clinical course and variable molecular findings (t-cellular clonality in less than % of cases). borderline lesions have a favourable outcome similar to lyp but, due to their rarity, limited data are available on their biologic features and clonal status. the aim of this study was to compare possible divergences in the search for clonality in the same individual skin lesions using respectively dna extracted from laser microdissected cd + cells and dna from whole paraffin sections. materials and methods lyp and borderline skin lesional biopsies were tested for tcr-gamma rearrangements by means of pcr on dna extracted respectively from whole-tissue and a pool ( - cells) of cd + cells microdissected using the zeiss-palm robot-microbeam system. results and conclusions pcr analysis of whole-tissue dna documented tcr-gamma monoclonal rearrangement in / cases ( / borderline, / lyp); tcr analysis on dna extracted from microdissected cd + cells showed monoclonal rearrangement in / cases ( / borderline, / lyp). we confirm that pcr analyses on dna extracted by microdissected cd + cells enhance the rate of monoclonality detection mainly in lyp lesions, in which the limited number of atypical cd + cells and predominant reactive cellular background may be responsible for negative or ambiguous signalling when dna extracted by the whole lesion is tested. malignant testicular lymphoma accompanied with complete av block -case report m a -year-old male patient was admitted, with the signs of complete av block and bradycardia accompanied by syncopes. a complete lab examination with us, ct and mri was done which all confirmed a massive tumorous infiltration into the root of mesenterium, presence of lymphadenomegaly around it, as well as nonhomogeneous and unclearly demarcated infiltrative lesion of myocardial septum. the patient completed his anamnesis by imparting that his right testicle was slightly enlarged and periodically painful. semicastration was indicated and the removed tissue was prepared for pathohistologic analysis. the testicle had only peripherally preserved normal tissue, whereas the tumor was clearly demarcated, . cm in diameter. five-micrometre-thick tissue sections from the paraffin-embedded tissue were stained with h&e. the cells were quite variable in their morpholoric features and size. the nuclei were generally hypochromatic, with irregular chromatin distribution. the nuclear morphologic properties varied and included pleomorphic mono-and multinucleated giant tumour cells with multiple nuclear lobulations. nucleoli were conspicuous. also, numerous benignappearing lymphocytes and plasmacytoid elements were scattered throughout the background. this histologic picture could correspond either to spermatocytic seminoma or lymphoma. since the patient needed an urgent therapy, histochemically (pas-) and immunohistochemically (lca +, cd +, cd +), the diagnosis of lymphoma was confirmed. the patient received ct (chop protocol), which resulted in a satisfactory clinical response. in this case, it is interesting to point to the most probable infiltration of the myocardium by the diagnosed lymphoma which elicited the cardiologic symptoms and from which the diagnosing of the patient's disease was to start. diffuse large b-cell lymphoma of bone: an analysis of differentiation-associated antigens with clinical correlation l de leval * , k braaten, m ancukiewicz, e kiggundu, t delaney, h mankin, n lee harris * department of pathology, university of liège, belgium, massachusetts general hospital, boston, ma twenty-nine patients with diffuse large b-cell lymphomas (dlbcl) presenting with bone involvement, including localized primary bone lymphomas (group i), multifocal primary bone lymphomas (group ii) and patients with extraskeletal disease at diagnosis (group iii) were studied. the tumors were subclassified according to the criteria of the who classification and evaluated by immunohistochemistry for expression of antigens associated with germinal center (gc) and non-gc stages of b-cell differentiation (bcl- , cd , mum- , vs c, cd , bcl- and cd ). the presence of a bcl- /igh gene rearrangement was investigated by pcr. all cases were characterized by similar clinicopathologic and morphologic features, and had similarly good overall outcome. the patients ( m, f, median y) had tumors in long bones ( ), axial skeleton ( ), and limb girdles ( ) and multiple sites ( ) . most tumors ( ) were centroblastic, with multilobated cells in cases. almost half of the tumors ( / , %) were bcl- +cd + («gclike»), / cases ( %) were bcl- +cd -(«indeterminate» phenotype) and / cases ( %) were cd -bcl- -(«post-gc like»). the «indeterminate» phenotype was seen only in primary bone lymphoma. mum- was frequently expressed in gc-like and non-gc-like categories. we found no evidence of plasmacytic differentiation by cd and vs c immunoreactivity was distinctly rare ( / cases). cd was detected in tumors, all cd -. bcl- was expressed by % of the tumors but only / cases tested had a bcl- /jh rearrangement by pcr. a survival analysis showed that gc-like tumors had a longer overall survival duration compared to non-gc-like tumors (p= . ). in conclusion, a "gc-like" immunophenotype, characterizes roughly half of large b-cell lymphomas of bone and is associated with an improved survival. ultrastructural characteristics of liver-associated lymphocytes in chronic delta hepatitis b aliev, s khodjaev institute of virology, tashkent, uzbekistan liver sinusoids contain a heterogenous population of lymphocytes including 'pit cells' discovered by wisse in . however, ultrastructural patterns of liver-associated lymphocytes (lal) in delta hepatitis are still not well identified. in present work we investigated lal in chronic delta hepatitis (chd) with electron microscopy. liver biopsies from with chd were fixed in , % glutar aldehyde, postfixed with % osmium teroxide and embedded in epon. ultrathin sections were stained with uranyl acetate and lead citrate. ultrastructural study revealed migration of lymphocytes between hepatocytes in chd. they have heterogenous population of lymphocytes varying in morphology. some of them were large granular lymphocytes and could be natural killer cells. some of lal could correspond to a t lymphocyte. in electron microscopic study of lal we also observed presence of plasma cells and the pattern of intracellular membrane systems common to cells that secrete protein: well developed granular endoplasmic reticulum, large juxtanuclear golgi complex. a centriole occupies a position just marginal to the golgi. the chromatin in the nucleus of these cells is characteristically condensed into large masses just within the envelope. our results demonstrate a heterogenity of lal in chronic delta hepatitis including the presence of b cells. hepatic steatosis in relation to serum apolipoprotein level in egyptian patients with hcv and hcv-associated hcc: histopathological, biochemical and clinico-sonographic study t aboushousha chronic hepatitis c is a known risk factor for cirrhosis and hepatocellular carcinoma (hcc). liver steatosis is common in hcv patients suggesting that specific mechanisms might be involved. the aim of our study was to assess variability in serum apolipoproteins in relation to hepatic steatosis in hcv patients of genotype with or without hcc. material included egyptian subjects; (hcv), (hcv-associated hcc), and controls. clinical examination for stigmata of chronic hepatic affection and ultrasonography for assessment of liver size, surface, echogenicity and the presence or absence of a focal lesion were done. histopathological examination of liver biopsies was done for assessment of hepatitis activity index (hai) and stage as well as the grade of hepatocytic steatosis (hs). biochemical study included liver function tests, lipid profile and assessment of apolipoproteins. results showed a significant decline in apo-a, apo-b, apo-cii, apo-ciii and apo-e lipoprotein in hcv and hcv-associated hcc groups compared to the control group. analysis of hai in hcv patients showed significant association of hs with periportal necrosis, but not portal inflammation or lobular degeneration. in patients with hcv associated hcc, hs was significantly associated with portal inflammation and lobular degeneration, suggesting a correlation between virus activity and hepatocytic neoplastic changes. we concluded that steatosis should be searched for in patients with chronic hcv, being an important cofactor in accelerating the development of hepatic fibrosis and in increasing necroinflammatory activity. a correct therapeutic approach to steatosis would slow down the evolution of chronic hepatitis. introduction nonalcoholic fatty liver disease (nafld) is defined as a constellation of clinical conditions characterized by predominantly macro vesicular steatosis. it can be extremely difficult to reach the diagnosis of nash in the absence of correct clinical data. exclusion of excess alcohol intake is notoriously difficult and requires a desialylated transferin (the method rarely present in laboratories). aim: histomorphologic investigation of liver biopsies from patients with mild laboratory test abnormalities and steatosis complicated by necroinflamatory changes. a pilot study of morphometric characteristics of lipid droplets in nash and alcoholic hepatitis were also performed. material and methods forty liver biopsies with steatosis and inflammation were examined using the method of dixon. ten of these biopsies were morphometrically analysed regarding the lipid droplet features. following morphometric parameters were estimated: ) diameter, ) perimeter and ) area. we used olympus micro image-image analysis software v. . . . the presence and distribution of mallory hyaline was the most variable parameter. morphometric analyses confirm a statistically significant differences for all parameters tested in nash and alcoholic hepatitis, and were more pronounced in nash. conclusion in the cases of fatty change with necro-inflammatory changes and pericellular fibrosis in liver biopsy and presence or absence of mallory hyaline, morphometric analysis of lipid droplets can help in reaching the diagnosis of nash, if testing of desialilated transferring is not possible. the raf/mek/erk (mapk) signal transduction cascade is an important mediator of a number of cellular fates including growth, proliferation and survival. the braf gene, one of the human isoforms of raf, is activated by oncogenic ras, leading to cooperative effects in cells responding to growth factor signals. this study was performed to elucidate a possible function of braf in liver tumours. methods mutations of braf and kras were evaluated in hepatocellular carcinomas (hcc) and in cholangiocarcinomas (cc) by direct dna sequencing analyses after microdissection. the presence of active intermediates of the mapk pathway was assessed immunohistochemically. the results obtained were correlated with histopathological variables and patient survival. results activating braf missense mutations were identified in / cc ( %) and in one case of tumour surrounding liver. kras mutations were found in out of ( %) cc examined and in two cases of tumour-surrounding non-neoplastic liver tissue. in hcc, neither braf nor kras mutations were detected. all cc with kras mutations had an intact braf gene. we failed to observe a correlation between braf or kras mutations and histopathological factors or prognosis of the patients. conclusions our data indicate, that braf gene mutations are a relatively common event in cc, but not in hcc. disruption of the raf/mek/erk (mapk) kinase pathway -either by ras or braf mutation -was detected in about % of all cc and is therefore one of the most frequent defects in cholangiocellular carcinogenesis. prognosis and treatment of cholangitis after cholecystectomy n akhtar, ae kuzmenko, vv khatsko, ga grintsov, am dudin, yg kolkin donetsk medical state university, donetsk, ukraine introduction the purpose of our research was to study prognostic tests of cholangitis after cholecystectomy (ce) for prophylaxis and optimal treatment. materials and methods for years patients with so called "postcholecystectomic syndrome" (pces) were treated, ( %) had a clinical picture of acute cholangitis (ac), which was shown by pains in the right hypochondrium, yellowness of skin and scleras, shivering, high hyperthermia and leukocytosis. patients ( women and men) were - years old, of them more than years. initial operation (ce) was made in various hospitals in terms from months to years ago. the diagnosis was verified by: clinical, laboratory and immunological researches, peroxidation of lipids (pol), ultrasonic research, computerized tomography, fibrogastroduodenoscopy, endoscopic retrograde pancreatocholangiography, transdermic and transhepatic cholangiography, fibrocholangiograpgy, bacteriological researches of bile and blood. results according to investigations, in patients the reason of infringement of bile flow was choledocholithiasis (in a combination with stenosis of big duodenal papilla (bdp) or without it, in -stenosis of bdp, in -cicatricial stricture of hepatocholedochus, in extended stenosis of bdp and a terminal part of the common bile duct (cbd). laboratory prognostic tests (risk factor) ac concern: hyperbilirubinemia, increase of activity of alanine transpeptidase, aspartate transpeptidase, hypoproteinemia, dysproteinemia, decrease of a level of protrombin lower than %, leukocytosis, anemia, increase of the contents of urea in blood, bacteriocholia, concentration of molecule of average weight were higher than . unit, decrease of level of vitamin e, high contents of immunoglobulin m on a background of a low parameter immunoglobulin g. conclusions at the presence of risk factors prognosis was frequently in contrary to expectations and mortality reached to - %. all patients with acute cholangitis after cholecystectomy represented a complicated problem and demanded a complex approach. revealed prognostic tests of the development of acute cholangitis are necessary for taking into account for its prophylaxis and optimum treatment. structural alterations in c-myc oncogene, copy number and expression have been implicated in the pathogenesis and progression of several human neoplastic diseases. however, the biologic significance of c-myc gene in human hepatocellular carcinoma (hcc) is unconfirmed. in the present study we correlated c-myc gene amplification and protein expression with the clinicopathologic features. in hcc cases c-myc amplification in tumour tissue was determined using a differential pcr, a procedure for evaluation of gene amplification in comparison with a dopamine d receptor gene. the c-myc gene was amplified in out of tumor specimens ( %). amplification of c-myc was more frequent in younger male patients with hbv infection and in less differentiated tumors. all cases demonstrated positive staining using anti-c-myc monoclonal antibody with increasing percentage of immunoreactive cells in less differentiated tumors. however, the high protein expression was not statistically correlated with c-myc amplification. histological sectioning of brush bristles allows improved diagnosis of biliary tract and bronchial lesions g bussolati, g accinelli, l macri dept. biomedical science and human oncology, turin, italy brushing of thin ducts such as those of the biliary tract, pancreas and bronchial tree is often used by endoscopists in order to obtain diagnostic material proving the nature of related lesion. the material is spread on slides. cytological examination folows. preservation of the material so obtained is often sub-obtimal, partly because of the presence of fluids such as bile affecting cell preservation, partly because spreading of delicate cells may result in crushing artifacts. to circumvent such problems, we devised an original procedure, where brushes were not smeared, but instead immediately embedded in immersed methanol. then the tube with alcool and brush was sent to the laboratory. in order to induce formation of a glue surrounding the brush, this was immersed while wet into egg albumen (a mixture : of egg albumen and glycerol), then returned to methanol. for paraffin embedding, the metal handling was cut away and the brush was introduced as such into a cassette. sections parallel to the long axis were cut until the metal wire was almost reached, then the block was rotated and new section was obtained from the opposite side. sections were stained in haematoxilin-eosin and with the papanicolau procedure. small fragments of the mucosa, of inflammatory cell aggregates or of related carcinomas were observed. these resulted optimally fixed and allowed a definite histological diagnosis which proved mandatory for proper therapy. advantages and prospects of this novel procedure will be illustrated and discussed. is telomerase catalytic subunit gene re-expression an early event in gallbladder carcinogenesis? were to determine whether a.) differences in the levels of htert exist among different grades of gallbladder epithelial abnormalities, and b.) htert gene re-expression has any role in gallbladder carcinogenesis. material and methods the expression of htert was analysed in gallbladder tissue samples: normal epithelia, reactive hyperplasias, low grade dysplasias, high grade dysplasias and in adenocarcinomas by immunohistochemistry. at least nuclei were quantified per slide and the number of positive signals per nucleus was expressed as a htert index. results the mean htert index increased progressively with the degree of gallbladder epithelial abnormalities: from . (standard deviation= . ) in normal epithelia, . (sd= . ) in hyperplastic epithelia, . (sd= . ) in low grade dysplasia, . (sd= . ) in high grade dysplasia to . (sd= . ) in adenocarcinoma. according to the htert expression, three groups of gallbladder epithelial changes were found: .) normal and hyperplastic gallbladder mucosa, . low grade dysplasia, and .) high grade dysplasia and adenocarcinoma of the gallbladder. the differences between the groups were statistically significant (p< . ). conclusions we believe that occasional presence of htert in normal and reactively hyperplastic gallbladder mucosa merely reflects its regenerative capacity. nevertheless, significantly higher htert indices in low grade as well as high grade dysplastic epithelium and gallbladder adenocarcinoma are most probably the consequence of htert re-expression -an early event in the multistep process of gallbladder carcinogenesis. spontaneous intestinal perforation vs. necrotizing enterocolitis: different entities clinically and pathologically g de hertogh, p van eyken, m miserez, h devlieger, k geboes k u leuven, leuven, belgium introduction necrotizing enterocolitis (nec) is the most common neonatal surgical emergency. however, during recent years an increasing number of laparotomies has been performed for spontaneous intestinal perforation (sip) in premature infants. the aims of this study were to compare the clinical and pathological features of sip and nec and to identify parameters useful to make the differential diagnosis. materials and methods we reviewed the clinical files and biopsies from infants indexed as "necrotizing enterocolitis" or "intestinal perforation" between and . results twenty-six patients had sip and had nec. patients with sip had lower gestational age and birth weight. they presented early with abdominal distension. frequent radiological signs were dilatation of bowel loops and pneumoperitoneum. perforation was in the small bowel in all cases. % survived. patients with nec presented later with more systemic symptoms. pneumatosis intestinalis was present in more than half. the colon was involved more frequently than the small bowel. % survived. cases of nec were characterized by necrosis, inflammation and reparative tissue changes with or without pneumatosis intestinalis. in contrast, the bowel in cases of sip was not necrotic and showed little inflammation. its most distinguishing characteristic was the presence of one or more defects in the internal layer of the muscularis propia. the myenteric nerve plexus and interstitial cells of cajal had developed normally. conclusions spontaneous intestinal perforation and necrotizing enterocolitis differ in their clinical and histopathological characteristics. in our cases, sip was always associated with segmental absence of the intestinal musculature. impact of epitope retrieval in detection of cd (c-kit) in gastrointestinal stromal tumors (gists) using manual and automated immunohistochemical (ihc) procedures introduction gastrointestinal stromal tumours are solid tumours associated with c-kit and pdgf-r expressions. the recent introduction of gleevec/glivec, an effective inhibitor of the activated c-kit and pdgf-r has stimulated interest in standardizing the immunohistochemical detection of c-kit in gist-tissues, assuring an appropriate selection of patients for therapy with gleevec. aims: the dakocytomation anti-human c-kit antibody, code a , was investigated in four different set-ups: ) without heat-induced epitope-retrieval (hier), ) with hier, ) preabsorbing the primary antibody with the synthetic peptide used for generation of the primary antibody and ) testing on fresh versus archival tissue sections. materials and methods tissue sections were obtained from different cases of gist, confirmed by morphology and ihccharacterisation (c-kit, cd , s , sma, desmin). the ihc was performed manually as well as on automated platforms including the techmate™ and the autostainer instruments. results the use of hier consistently displayed a stronger and more uniform staining pattern as compared to an ihc protocol without hier. in few cases the expression of cd was found faintly positive in ihc without hier. automated and manual ihc methods displayed similar staining patterns. the specificity of detecting cd in a protocol using hier was confirmed by preabsorption of the primary antibody with immunogen. storing of fresh cut sections for more than months demonstrated a decrease in ihc cd staining intensity. conclusion epitope retrieval enhances the cd staining intensity in gist-tissue. the specificity of the cd detection in gist's can be confirmed by complete blocking of the positive staining in a parallel set-up, using preabsorption of the antibody by a synthetic c-kit peptide. introduction - % of gastric cancer patients worldwide are under and the clinicopathological features are different from the older age group. here we characterise some of the genetic changes in cases of early onset gastric cancer (eogc). methods using dna isolated from microdissected material, a panel of polymorphic microsatellite markers near known or presumed tumour suppresser genes was used to evaluate loss of heterozygosity (loh). microsatellite instability (msi) status was ascertained using polymorphic microsatellite markers in cases of eogc and in an additional biopsy cases. corresponding immunohistochemistry was performed for p , smad , ecadherin, mlh , msh and msh . ebv status of the carcinomas was determined using eber -mrna in-situ hybridisation. results the most common losses were at loci adjacent to cdh ( %), tp ( - %), runx ( - %) and smad / dpc ( - %). less frequently, losses were detected at p , q , and loci adjacent to apc, p ink a, pten, and tff. no msi was detected in the cases examined. immunohistochemistry revealed abnormalities for e-cadherin in % and p in %. all tumours examined were ebv negative. conclusion common events in eocg are genetic alterations in cdh , tp and runx whereas other loh findings were infrequent. protein abnormalities of e-cadherin and p were commoner than losses near the corresponding gene. in contrast to gastric cancer in older patients, msi does not appear to occur in young patients. these findings confirm that unique genetic alterations lead to gastric cancer in the young and highlight the need for further investigation to elucidate the mechanisms by which they occur. introduction a place of foveolar hyperplasia (fh) in benign epithelial gastric polyps (begp) classification, as well as its role in a development of hyperplastic polyps (hp) is still a matter of controversy. aims: two questions are to be studied: . does fh, hp, adenoma (a) and carcinoma (ca) represent a continuum in terms of ki- proliferative activity ? . are there any differences of the means of ki- labelling index (li) in hyperplastic areas of hp when comparison is made between hps without other lesions (hp-nos) and with hps with different focal lesions such as intestinal metaplasia (hp-im), dysplasia (hp-d) and/or carcinoma (hp-ca)? materials and methods begp from patients were selected to perform ki- immunohistochemical analysis. there were fh, hp (including hp-nos and hp-im, hp-d and/or hp-ca) and a. as controls cases of normal mucosa (n), cases of regenerative fh (fh-r) and cases of ca were used. paraffin sections were studied immunohistochemically for ki- li with application of mib- antibody. the data were analysed using one-way anova models. results it has been found that the means of ki- li between all the groups studied differed significantly (p< . ). ki- li for fh was high ( . ) and did not differ significantly from fh-r ( . ), a ( . ), and areas of low grade dysplasia in hp-d ( . ), but was significantly higher from n ( . ) and hp ( . ). conclusions .we hypothesize that fh may probably represent an early event in begp formation. .there were no significant differences of ki- li results for hyperplastic areas in hp ( . ) independently if there were focal im, d and/or ca simultaneously, or not. introduction among the malignant tumors that occur in stomach, carcinoma is overwhelmingly the most important and common ( - %). the aims of this study were to report how we histologically evaluate gastric carcinoma (gc) on endobiopsies, using who and lauren classifications. additionally, we tried to detect the rhabdoid features in specimens with diffuse gc. the study included gastric endobiopsies, gc being diagnosed in cases ( %), mostly in men ( cases - %). endobiopsies, routinely embedded, were stained by hematoxylin-eosin, giemsa, pas and alcian blue (ph . ). immunohistochemical studies were performed using vimentin and cytokeratin antibodies for detection of rhabdoid features. results and conclusions the highest incidence of gc was in - -year-old aged patients ( %). using lauren classification, cases were categorised as diffuse type ( %), intestinal type ( %) and ( %) mixed type gc. each of these three types were also histologically analyzed according to who classification: intestinal type gc (formed by recognizable well/moderately differentiated glands) and diffuse type gc (consisting in poorly cohesive cells diffusely infiltrating the gastric wall with little or no gland formation). in diffuse type gc with rhabdoid features we evidentiated vimentin and cytokeratin in cells with an eccentric nucleus and a paranuclear inclusion. finally, we pointed out that is very easy to diagnose gc by lauren and who classifications, but is difficult to evidence the mixed type gc due to reduced size of endobiopsies. recognition of the rhabdoid phenotype in diffuse type gc is important because this feature is associated with poor prognosis and unresponsiveness to conventional therapy. gastrointestinal tract. the patient had a -year history of dyspepsia, diarrhorea, mild diabetes mellitus and cholelithiasis. one hour after the admission, he died in hypovolemic shock. taken tissues from all organs were fixed in % formaldehyde and embedded in paraffin. laboratory sections were stained with: he, ab-pas, van gieson, grimelius, masson's argentaffine reaction and bsp methods, using antibody to somatostatin. results cafe au lait spots, multiple skin neurofibromas, mental retardation, skeletal deformities and a variety of tumours arising in the brain (craniopharyngioma), follicular thyroid adenoma and duodenal somatostatinoma, were found during autopsy. duodenal bleeding ulcer of gigantic size, was covered with coagula. in the bottom of the duodenal ulcer was discovered duodenal carcinoid of the glandular architecture, intermingled with solid acinar and trabecular growth pattern; its cells contained somatostatin. marked increase in number and thickness of nerve fibres also was seen, leading to disordered motility and to striking hypertrophy of the muscularis propria. conclusion having in mind that the patient has the sister, and that this disease has hereditary character, we will invite the sister as soon as possible and she will be in the future the target of our investigation. increased expression of muc and muc and decreased expression of muc in colorectal adenoma-carcinoma spectrum: a tissue array study introduction mucin-type glycoproteins are widely expressed in the gastrointestinal tract. little is known concerning the relationship of altered mucin antigens in colorectal polyps. the aims of this study were to determine the different patterns of mucin distribution of muc , muc , muc ac and muc and compare their patterns of expression with respect to histopathologic criteria of malignant potential in colorectal carcinogenesis by tissue array using immunohistochemistry. peritoneal dialysis (pd) is an alternative to hemodialysis for the treatment of end-stage renal disease. the long term exposure to dialysis solutions causes progressive peritoneal fibrosis and functional decline of the peritoneum. the pathologic process that leads to fibrosis is not well understood and most studies have focused on mesothelial cells, probably because they are easy to obtain from peritoneal effluents. the aims of this study were to establish diagnostic markers of peritoneal fibrosis and pathogenic clues to explain the fibrogenic process. the study was focused on submesothelial fibroblasts from normal controls and pd patients. materials and methods parietal peritoneal biopsies were collected from three patient groups: normal controls (n= ), non-pd uremic patients (n= ) and uremic patients on pd (n= ). in order to study myofibroblastic differentiation and mesothelial-to-mesenchymal transition, alpha-smooth muscle actin, desmin, cytokeratins, and ecadherin were analyzed. we also evaluated cd expression by submesothelial fibroblasts. results fibroblasts from normal controls and non-pd, uremic patients showed no myofibroblastic nor mesothelial markers, but an intense expression of cd . the opposite immunophenotypic pattern was present during pd-related fibrosis. expression of mesothelial markers (cytokeratins and e-cadherin) by fibroblastlike cells and alpha-smooth muscle actin by mesothelial and modified mesothelial cells are evidence that mesothelial-to-mesenchymal transition occurs during pd. loss of cd expression was another helpful indicator of fibrosis. conclusion the immunophenotype of submesothelial fibroblasts varies depending on the existence of a fibrogenic condition. this information results useful for the differentiation between quiescent and activated fibroblasts. in addition, it shows interesting information regarding the origin of this cell population. immunohistochemical and genetic high-throughput analysis of gastrointestinal stromal tumors tract. they are characterized by diffuse immunohistochemical positivity for c-kit protein (cd ). the aims of this study were to assess the immunohistochemical and genotypic features of a large series of gist using tissue microarray technique. methods gist were retrieved from the files of dpt of pathology of our institutions and a tissue microarray (tma) was built. immunohistochemistry for cd , actin, desmin, s , nse, mib was performed. interfase fish analysis was carried out for the following genes: mdm , egfr, c-myc, ccd and her- . results % of the tumors resulted positive for cd , % for desmin, % for actin, % for s , % for nse, % for mib . a significant difference between gastric and non-gastric gist was observed for cd , desmin and s . age, size, necrosis and nse positivity significantly correlated with aggressive behavior of the tumors. moreover, an amplification of c-myc, mdm , egfr, ccd in tumors was found. the tumors were all but two borderline or high risk tumors. cases of coamplification were also detected in clinically malignant or histologically high risk tumors. conclusion useful parameters for prognostic evaluation of gist are age, size and necrosis. furthermore, the observed amplification of c-myc, mdm , egfr suggests a role of rb-ccd pathway in the acquisition of malignant phenotype in a subset of gist. digital slide and virtual microscopy based routine and telepathology evaluation of routine gastrointestinal biopsy specimen l berczi introduction the aim of the study was the evaluation of a recently developed digital slide and virtual microscope system, its comparison with optical microscopy on routine gastric biopsy specimen in local and remote access mode. materials and methods h/e stained routine gastric, duodenal and colon (altogether ) biopsy specimen were selected, scanned and evaluated by two specialists on an optical (om) and virtual microscope (vm). the overall concordance of vm to a consensus diagnosis (con) was . % and % of the om to con. the clinically important concordance was % and % respectively. the two methods showed concordance in % and clinically important concordance in . %. the reasons of the discordance was image quality (one case), interpretation difference ( cases). the overall hard disc place for a gastric biopsy was between to megabytes. the scanning time was between to minutes on a commercial microscope (axioplan , carl zeiss, germany) and to minutes on an automated slide scanner (hi-scope, dhistech ltd., budapest) conclusions remote evaluation of the digital slides through internet shows the advantages of the previously used static and dinamic telepathology methods. the digital slide and the virtual microscope are real alternative techniques in the computerization of the histology laboratory and in teleconsultation services. the expression pattern of the muc glycoproteins muc , muc and muc ac may be useful in classifying the various types of intraductal pancreatic neoplasms (ipmns). recent studies have revealed that muc is expressed in ductal pancreatic carcinomas (pdac) only. this study investigates whether muc is expressed in ipmns and in particular in those with invasive components. in addition, muc reactivity was studied in panins of both ipmn and pdac. methods paraffin blocks from resection specimens from pdacs and ipmns ( invasive) were investigated immunohistochemically ([ihc] antibody clone ig ) and by in-situ hybridization ([ish] digoxigenin labeled oligomer nucleotide specific for sequences corresponding to repeat domains of muc ). the normal pancreas is muc negative. of pdacs were positive for muc in both ihc and ish. only ipmns were muc positive, and these cases were of the oncocytic type. invasive ipmn components of the dac type ( / ) and one of muconodular type ( / ) showed muc expression at both the protein and the rna level. in ipmns associated panin lesions of all grades were negative, whereas in pdacs a few panin lesions were positive. conclusions muc neoexpression in the pancreas seems to be a feature of invasive ductal neoplasms (ipmn, pdac), with the exception of oncocytic ipmns. the aberrant expression, in particular the shift from the negative intraductal to the positive invasive component in ipmns, is so far unexplained, but it may be related to the genetic alterations known in ipmns and pdacs. introduction hypermethylation of cpg islands represents an important epigenetic mechanism for silencing of tumor suppressor genes during carcinogenesis. in this investigation, the prevalence of hypermethylation of the dna mismatch repair gene hmlh and of the recently identified hpp gene was investigated in adenocarcinomas of the upper gastrointestinal tract. materials and methods using methylation-specific real-time pcr and fluorescence-labeled dna-probes, hypermethylation of hmlh and hpp was investigated in barrett's-esophagusassociated adenocarcinomas, cardiac carcinomas and in gastric carcinomas. additionally, hmlh protein expression was investigated by means of immunohistochemistry. in a subset of cases the presence of dna-microsatellite instability was tested. hypermethylation of hmlh was found in % of barrett's carcinomas, % of cardiac carcinomas and in % of gastric carcinomas, whereas hypermethylation of hpp was found more frequently in all tumor types ( % vs. % vs. %). in gastric cancer, hpp hypermethylation was found more frequently in tumors with concomitant hmlh hypermethylation ( %), than in those without hmlh hypermethylation ( %, p= . ). complete loss of hmlh protein expression which was present in carcinomas ( cardiac and gastric) was invariably correlated with hmlh hypermethylation and with microsatellite instability. conclusion our data indicate that microsatellite instability and loss of the mismatch repair protein hmlh , which is mainly caused by hmlh gene hypermethylation, is more prevalent in the carcinogenesis of the stomach and the cardia than in the esophagus. moreover, in gastric cancer hmlh hypermethylation is correlated with hypermethylation of the hpp gene. antral g-cells in sporadic fundic gland polyps patients: an immunohistochemical study introduction malignant melanoma is an increasing neoplasm in the western world. advanced tumors gain metastatic potential and are responsible for high mortality rates. during tumor progression a downregulation of the transcription factor ap- a and the receptor thyrosine kinase c-kit was observed. however, mechanisms leading to a loss of both proteins are unknown. besides genetic changes post-transcriptional downregulation by caspase- and - is recently discussed. aims were: i) to investigate malignant melanoma for ap- a and c-kit genetically and by protein expression, ii) to study the implication of activated caspases. methods ssms, nms and metastases were analyzed by direct sequencing analysis for all exons of ap- a and c-kit genes and by immunohistochemistry for the respective proteins and cleaved (activated) caspase- and - . results four tumors showed point mutations in the activation motif of ap- a (exon ). no genetic alterations were detected in the c-kit sequence. immunohistochemistry revealed a progressive loss of dermal ap- a and c-kit expression when comparing ssms with nms and ms which was paralleled by a strong expression of caspase- in the primary melanomas and ms, the later were also characterized by caspase- expression. conclusion tumor progression in melanoma is characterized by a loss of ap- a and c-kit. caspase- probably contributes to this down-regulation, as it was recently shown in vitro. the activation of caspases in melanoma might not only induce apoptosis but could be substantial for tumor growth. introduction in cancer patients, tumor-expression of the angiogenic factor vascular endothelial growth factor-a (vegf-a) correlates with high tumor vascularity and increased metastatis, but how these latter two relate is unclear. we investigated the mechanisms of vegf-a -enhanced metastasis of the human melanoma cell line mel . methods stably transfected mel cells, expressing vegf-a or egfp, were injected into the internal carotid artery or tail vein of nude mice to establish brain or lung metastases (colonization models). subcutaneous tumors were grown to examine spontaneous metastasis to lung. tumors and metastases were examined by (immuno)-histochemistry. results in colonization models, vegf-a expression did not give tumor cells a colonization advantage. metastases originated predominantly by clonal expansion of single cells. in contrast, spontaneous metastasis from subcutaneous tumors was enhanced in tumors with vegf-a expression, this enhanced metastatic potential was induced by a distinct architectural transformation of the s.c. tumors: while mel -egfp and parental mel tumors showed extensive necrosis, mel -vegf-a tumors presented a micronodular growth pattern, with nodules protruding into sinusoidal dilated vasculature. these micronodules proved to be polyclonal, had tissue characteristics and caused metastatic embolism in the pulmonary artery with subsequent metastatic outgrowth. conclusion vegf-a may enhance spontaneous metastasis not simply by increased single cell shedding into the circulation by an increased tumor vascularity but by induction of a micronodular transformation of a tumor resulting in enhanced shedding of (polyclonal)tissue fragments into the circulation. this step of tumorintravasation is probably a rate limiting factor in development of metastasis. correction of cytogenetic disturbances in patients with relapsing genital herpes la lebedinskya, op abaza donetsk medical state university, donetsk, ukraine introduction serious forms of relapses of genital herpes (gh) are accompanied by disturbances of chromosomal apparatus of lymphoid cells (l.a. lebedinskya, ) . correction of the given infringements is the important problem of practical public health service. the aim of our research was development of a complex method of correction of cytogenetic infringements in patients with gh. materials and methods we had investigated and treated patients (complex treatment) with gh in the age of - years, with frequency of relapses of one time in . - months. the control group was made by healthy persons, same age group. cytogenetic disturbances in t-lymphocytes of peripheral blood surveyed determined by metaphase method. complex method of treatment of patients with gh included acyclovir mg times per day before clinical recovery and phytopreparation "bacplan", received by an original way from quercus cortex. phytopreparation "bacplan" shows immunomodulation, anti-inflammatory and antimutagen properties. preparation "bacplan" appointed per os . g per day within days. external - % solution of bacplan in the form of lotion on affected body parts time in day. results average frequency of cells with aberrations of chromosomes (ac) in t-lymphocytes of peripheral blood of patients with gh before treatment were much higher in comparison with parameters of control group (accordingly . ± . and . ± . % p< . ). in patients were marked disturbance of chromosomal and chrmotoid types. in . % cases aberration of chromosomes were registered in - chromosome pairs. after complex treatment of average frequency of cells with ac in patients has decreases to days to . ± . % (p< . ) and practically come nearer to control parameter ( . ± . %). conclusions thus, use of viral specific preparation acyclovir in combination with phytopreparation "bacplan" reduces frequency of structural infringements of chromosomes in t-lymphocytes of patients with gh. different expression patterns of s- versus s- b in primary skin melanoma j timar, n udvarhelyi, z orosz national institute of oncology, budapest, hungary differential diagnosis of melanoma involves immunohistochemistry where s- is a traditional marker which is recently completed by gp /hmb , mart- and tyrosinase. the s- family of ca-binding proteins comprises the a ( - ) and b isoforms. recent microarray analysis identified s- b as the melanocyte lineage marker. in this work we have compared the expression patterns of s- proteins using antibody recognizing both isoforms versus that which is specific for s- b in human skin primary melanoma samples. there were expression patterns observed: focal, herogenous and diffuse for both antibodies. the most frequent expression pattern was the diffuse one ( - %) while the least frequent was the focal pattern ( - %) in case of both antibodies. when the individual cases have been analysed for the expression patterns for the two antibody we found % discrepancy. in about half of these cases ( %) the discrepancy was minor (heterogenous versus diffuse staining pattern), but in almost a third of the cases ( %) it was found to be considerable (focal pattern versus heterogenous or diffuse). we have concluded that the expression patterns of s- and s- b proteins in human melanoma are frequently different. since gene expression studies identified s- b as linage marker, it is highly recommended to use monospecific antibody for routine diagnosis. furthermore, s- b is validated as sensitive serum marker for melanoma progression accordingly it is another indication for the use of anti-s- b antibody in the routine diagnosis. introduction primary cutaneous b-cell lymphomas represent subtypes of extranodal b-cell lymphomas with distinct clinicopathologic features. they account for approximately % of primary cutaneous lymphomas. the most common forms include follicle cell lymphoma, marginal zone lymphoma, immunocytoma, and diffuse large b-cell lymphoma. we report cases of primary cutaneous diffuse large b-cell lymphomas, one in the left gluteal region ( / cm in diameter), and the other in the left leg ( / cm in diameter), both appearing clinically ulcerated. materials and methods routine paraffin embedding and usual stainings, followed by immunohistochemistry were used. results microscopy evidentiated in the papillary and reticular dermis a diffuse pattern of growth of cells with a moderate degree of morphologic variation, with moderate, slightly basophilic cytoplasm and round-ovalar nuclei, with vesicular aspect owing to margination of chromatin at the nuclear membrane, - nucleoli, and frequent mitoses. gomory staining revealed a network of reticulin with reduced intervening spaces. immunohistochemistry evidentiated a diffuse positivity of vimentin and cd (l ), s positivity in few cells, desmin negativity, mic negativity, cd (berh /ki- ) negativity, actin negativity in tumor, but positivity in vessels, ema negativity and pcna positivity in almost % of the cells. conclusion final diagnoses of primary cutaneous diffuse large bcell lymphomas were considered correlating the clinical, morphologic, and immunophenotyping features. although prognosis of most primary cutaneous b-cell lymphomas is favorable, with -year survival rates over %, diffuse large b-cell lymphoma is more aggressive. we appreciated an unfavorable prognosis of both cases, but especially in the patient where the lymphoma occurred in the lower leg, as this location is associated with a more aggressive behaviour. in malignant melanoma cd protein expression is associated with metastatic tumors, higher clark levels and tumor thickness according to breslow n department of pathology, the norwegian radium hospital, norway cd antigen is a kd a cell surface zinc metalloendopeptidase and is expressed by a variety of normal and neoplastic lymphoid and non-lymphoid tissue including melanomas. kanitakis et al. (melanoma res ; : - ) have recently demonstrated that cd expression is significantly more common in metastatic than primary melanomas. we have evaluated biopsies from patients with primary and patients with metastatic melanoma. formalinfixed/paraffin-embedded tissues were studied by immunohistochemistry using mab c from novocastra laboratories and envision+® from dakocytomation. overall, cd was expressed in / samples, but the percentage of cd -positive cell was significantly higher in metastatic tumors (p= . ) and also in primary tumors with higher clark level (p= . ) and tumor thickness according to breslow (p= . ). even though the intensity of expression was associated with depth of invasion (clack levels p= . , breslow p= . ), there was no definitive difference between primary and metastatic tumors (p= . ). we conclude that cd expression in malignant melanoma is associated with tumor progression. sclerosing adenosis of the skin g sclerosing adenosis is a lesion that occurs in the breast; rare cases have also been reported in other organs such as prostate and skin. we report a case of sclerosing adenosis of the skin incidentally found at the periphery of a basal cell carcinoma. a -year-old man had a polypoid lesion of the scalp measuring . cm in diameter. the lesion was surgically removed and, at histology, it was composed of a nodular basal cell carcinoma. below the basal cell carcinoma, there was a well circumscribed but not encapsulated lesion constituted by small, packed glands separated by cellular stroma, reminiscent of sclerosing adenosis of the breast. some glands showed open lumina bordered by cells with eosinophilic, granular cytoplasm while other glands were arranged in solid clusters or elongated cords. two distinct layers of cells were seen bordering the glands: a luminal layer of cuboidal cells and an outer layer of basal cells with scanty cytoplasm and spindle nuclei. at immunohistochemistry the outer cell layer was positive for actin, calponin, caldesmon and keratin antibodies showing therefore myoepithelial cell differentiation. the luminal layer was positive with low weight keratin; moreover luminal cells were decorated by gcdfp- antibody, a marker of apocrine differentiation. to the best of our knowledge this is the first case of sclerosing adenosis of the skin showing apocrine metaplasia of luminal cells. the case here reported is an additional example of the existence of occasional similar lesions between skin and mammary gland. immunolocalization of human sperm protein in skin cancer introduction a variety of cancer	estis (c\t) antigens has been found to be expressed in healthy testis and in a wide range of human malignancies, including melanoma. the effectiveness of these antigens as target for active specific immunotherapy has been largely demonstrated. recently a new human c\t, called sperm protein (hsp ) has been investigated in multiple myeloma and ovarian cancer. the aim of this study was to investigate the expression of hsp in healthy skin and melanocytic skin lesions, in order to evaluate the usefulness of this antigen to develop effective specific immunotherapeutic procedures. methods the authors assessed the immunolocalization of hsp in paraffin embedded formalin fixed specimens of healthy skin (n= ) and tissues representing cutaneous melanocytic lesions [common nevi (n= ), spitz nevi (n= ), blue nevi (n= ), atypical nevi (n= ) and melanomas (n= )]. results hsp was not detected in healthy skin. it was recognized with a high frequency in cutaneous melanocytic lesions in some cells of the dermal stroma, often containing melanin, compatible with melanophages. a higher expression of hsp was observed in hyperpigmented nevi. conclusion the emergent complex function of hsp suggests the necessity of further studies to investigate the relation between melanophages and hsp . although hsp was not detected in healthy skin, its limited detection in resident melanophages recognized in cutaneous lesions, and not in tumoral cells precludes its applicability as target in skin cancer immunotherapy. introduction crimean congo haemorrhagic fever (cchf) is endemic in many countries in asia, africa and europe. the virus, which causes cchf, is a nirovirus from the bunyaviridae family. although primarily a zoonosis, sporadic cases and outbraces of cchf affect humans. case report a -year-old female patient was hospitalized due to sudden onset of fever, headache, vomiting and abdominal pain. during the next days, her condition became worse, she was unconscious, anuric, cyanotic with multiple edema on extremities, large vaginal coagulums and with appearance of haemorrhagic secretion in aspired fluid from the respiratory tract. three days after admission, due to sudden onset of cardiac arrest, patient died. pathologic findings: severe bleeding from the mucous membranes and internal organs were found, particularly emphasized in the liver, kidneys, heart, brain and other organs. widespread hepatocellular necrosis with focal microvesicular steatosis and multiple intracytoplasmatic microvesicular inclusions were the most striking morphological findings. diffuse necrosis of renal tissue was also found. recent haemorrhages in lungs, cardiac muscle, suprarenal gland, spleen, and gastric mucosa were found. lymphoid depletion was detected in lymph nodes. the rt-pcr method for detection cchf virus has been applied in analysing serum and tissue samples. serological testing and testing from paraffin embedded tissue were positive. conclusion autopsy pathologic findings of cchf include widespread haemorrhages of the skin, mucous membranes, pleura, peritoneum. necrosis is frequent in all organs and tissues, often of ishaemic nature. autopsy results of our patient showed similar pathologic findings and by pcr method we confirmed etiopathogenesis of this disease. the modern morphological characteristic of placenta in syphilis z parpiev , g reymnazarova , b magrupov dermatology and venerology, tashkent, uzbekistan first tashkent medical institute, tashkent, uzbekistan we investigated the placentas in three cases of intrauterine death caused by inherited syphilis. grossly, they were small and medium lobular, showed a marked sclerosis of large vessels and they looked white-muddy. the umbilical cord was also sclerotic and showed obliteration of vessels. their weight corresponded to term of gestation, whereas their area was smaller and their thickness greater than in normally proceeding pregnancy, but these differences were not significant (p> . ). results light microscopic investigation revealed avascularized stroma and sclerotic villi with vessels showing obliterating angiopathy. these changes were combined with processes of compensatory character, such as sharply extended subsyntitially located capillaries and large number of small vesels. conclusion in the morphological study of placenta, the attributes of damaged placental tissue and development of compensatory reactions directed towards the maintenance of necessary balance in the mother-placenta-fetus system were revealed. in placenta, contrary to the descriptions of the last years, we found no significant increase of its weight and change in this connection fetus-placenta of factor. the basic changes in placenta were characterized by the development of productive vasculitis with obliteration of the vascular lumina and presence of avascularized terminal villi. the thickening of vessel walls of the main villi as a result of proliferative endarteriitis can be considered as a characteristic attribute for syphilitic infection. the latent membrane protein (lmp ) of epstein-barr virus is one of two postulated viral oncogenic proteins. previous reports have suggested that sequence variations, and in particular a base pair deletion variant called cao, may define different disease populations. we developed a cocktail of rat monoclonal antibodies that were specific for the non-wild type lmp and compared the presence of the antibody staining with lmp dna sequence analysis on clinical samples of nasopharyngeal carcinoma, peripheral t-cell lymphoma, hodgkin's disease and lymphoblastoid cell lines from normal volunteers and patients with npc. results and conclusion our results demonstrate specificity of the monoclonal cocktail for detecting the wild-type lmp and the ability to sub-differentiate between the mediterranean type of lmp and the cao-lmp . visceral leishmaniasis primarily diagnosed in duodenal biopsy: report of three cases f pardal, a silva, v velasco, m teixeira serviço de anatomia patológica, braga, portugal introduction visceral leishmaniasis (kala-azar) is a zoonosis caused by a flagellate protozoan, of the genus leishmania, which is transmitted to man from reservoir animals (like a dog), by a vector of the genus phlebotomus. generally it affects the mononuclear phagocytic system of the bone marrow, liver and spleen. the gastrointestinal system is rarely involved. diagnosis of the infection depends on microscopic identification of leishmanias amastigotes in tissues or blood cultures or by serologic means. aims: visceral leishmaniasis is a rare disease in patients without immunodeficiency, the authors report three cases diagnosed primarily in duodenal and gastric biopsy from immunocompetent patients. materials and methods from the files of pathology department of s. marcos hospital five cases of visceral leishmaniasis have been collected in a years period, all from immunocompetent patients, namely without hiv infections. the authors describe three of the five cases, which were primarily diagnosed by gastro-duodenal biopsy. lately, was noted the presence of amastigotes in other tissues. after histologic diagnosis resulted a full clinic diagnosis of systemic leishmaniasis with advanced stage of disease. conclusions pathology department files reveal a significant percentage of visceral leishmaniasis diagnosed by gastroduodenal biopsy, three of five cases. thus, in no endemic countries for this opportunistic infection and in immunocompetent patients, gastrointestinal biopsy can, eventually be a rapid and sensitive method of diagnosis, since most of the patients present at an advanced stage of disease. impact of the sudden acute respiratory syndrome (sars) on the pathology laboratory: a singapore experience ar chang, a wee, sm chong, t ming pathology department, national university hospital, national university of singapore, singapore the sudden acute respiratory syndrome (sars) is a new global health problem that has received unprecedented attention. the predominant route of infection is by droplets and close contact with an infected person poses the highest risk of being infected with the sars virus. the majority of cases among health care workers have occurred in doctors and nurses who have cared for sars patients. pathologists and laboratory personnel are regarded as low risk for infection because they are infrequently in direct contact with patients. however, they receive specimens, including sputa, bronchiolo-alveolar lavage samples, excised tissues, blood and body fluids from infectious cases. two further possible sources of infection are during autopsies and the onsite processing of fine needle aspiration specimens for immediate diagnosis. not uncommonly, few clinical details are attached with a specimen to alert the laboratory so universal precaution must be in place and all specimens regarded as infectious. but with a new infection such as sars where information on its natural history, infectiousness and mode of transmission are still evolving, greater vigilance and a pro-active approach are needed. during the period march -may singapore was faced with a potentially serious sars epidemic and to minimize the chance of personnel in the pathology department, national university hospital being infected and thus compromise the diagnostic service a contingency plan was implemented. this report describes the impact of the plan on the department. sars is a global disease and pathologists in countries who have not encountered it may find the information useful for preparing their laboratory for the eventual arrival of sars. angiotropic large cell lymphoma of the central nervous system -report of four cases introduction angiotropic large cell lymphoma (alcl), or intravascular lymphoma, is a rare form of high grade non-hodgkin's lymphoma, predominantly of b-cell lineage. it is characterized by extensive proliferation of lymphoma cells within the lumina of small blood vessels particularly of the cns and skin with tendency to disseminate into multiple organs. because of nonspecific manifestations the clinical diagnosis of alcl is difficult as well as the morphological diagnosis, which may be problematic and subtile. methods we studied four cases of alcl with dominant neurologic symptoms in the form of vasculitis, dementia and cauda equina syndrome. the diagnosis was made by autopsy in three cases and by brain biopsy in one case. in one autopsy case, the alcl was associated with aids in a -years old hemophilic boy. results microscopically, in all cases, small blood vessels of the cns were distended by collections of large atypical lymphoid cells which obstructed the vascular lumina. tumor cells cytoplasm was amphophilic, the nuclei were large, irregular with prominent nucleoli. mitoses were numerous. in the vicinity, there were small zones of spongy edema, reactive gliosis and foci of total or selective necrosis. in an autopsy case with diagnosis 'vasculitis' (female -years) there was dissemination into multiple organs. immunohistochemistry shows strong positivity for cd and cd , and the negative reaction for cd , cd ro and factor viii-related antigen. based on the above findings the diagnosis of angiotropic large b-cell lymphoma was made. conclusion the antemortem diagnosis of alcl of the cns may be established by brain biopsy with application of immunohistochemistry. evaluation of cell death in animal model of brain contusion treated by hyperbaric oxygen therapy e vlodavsky, j soustiel rambam medical center, haifa, israel cerebral contusion (cc) remains the most important consequence of traumatic brain injury and has unexplained unique evolving clinical course.the aim of this study was to evaluate the extent of apoptosis in perilesional area of cc and to examine the effects of hypoxia and hyperbaric oxygenation therapy (hbot).the model of cc was based on the technique of cortical dynamic deformation (shreiber et al., ) on sprague-dawley rats. one group of animals afterwards was placed in closed chamber and exposed to mild hypoxemia, simulating posttraumatic hypoxemia. both groups of animals (hypoxia-exposed and non-exposed) were treated by hbot. brains were cut through produced lesion and paraffin sections were stained by tunel method and for active caspase- . apoptotic cells (ac) were counted in five successive perilesional layers of . mm each. it was shown that in the group of rats that were exposed to hypoxia, the width of perilesional area and the number of ac were significantly higher than in non-hypoxic animals, raising from . % to . % at . mm (p< . ).one treatment by hbot induced significant decrease both in the radius of perilesional area and the number of ac in both groups of animals (in hypoxia-exposed animals this effect was even more pronounced).the results of our study prove the role of perilesional area in the 'growth' of cc. hypoxemia caused profound increase in cell death extension. hbot shows marked positive effect in both hypoxic and nonhypoxic animals. we used quantative pathologic monitoring of number of ac to develop the optimal regimen of hbot in animal model of cc. introduction surgery for seizures consists of surgical procedures made in the central nervous system to treat drug-resistant epilepsy. different lesions may be the cause. aims: we review the cases diagnosed between the years and in our hospital. materials and methods the surgycal specimens were stained with hematoxilin-eosin and neural or glial immunostains after paraffin embeding. results the most frequent lesion is mesial temporal sclerosis ( - % of the cases). other causes are tumors; dysembrioplastic neuroepithelial tumor (dnt), neuronal tumors and glial tumors. less frequent lesions are phacomatosis, malformative lesions like cortical dysplasias or vascular malformations, vascular or trauma lesions and inflammatory or unspecified lesions. mesial temporal sclerosis consists of neuronal loss and gliosis in sectors ca , ca , ca and dentate gyrus. between and we diagnosed cases; were women and were males. dnt is a very infrequent, intracortical tumor composed of glia and neurons, usually associated with cortical dysplasia. in our experience we diagnosed cases; women and men. we have found other tumors associated with epilepsy; cases of ganglioglioma ( women and men), cases of gangliocytoma ( women and men), and astrocytic tumors ( protoplasmic astrocytomas, mixed astrocytoma and fibrillary astrocytomas). we also diagnosed other processes associated with epilepsy: cases of cortical dysplasia, of glial scar, of gliosis or unspecified changes and cases of vascular malformation. conclusion pathology associated with seizures is very diverse; a multidisciplinary and experienced team is needed. expression of sperm protein , a novel cancer-testis antigen in human nervous system tumours introduction human sperm protein (hsp ) is a highly conserved protein originally isolated from a rabbit epididymal sperm membrane and testis membrane pellet. recently, hsp has been included in the cancer/testis antigens (ct) family and showed to be expressed in multiple myeloma and ovarian cancer. the aim of the study was to investigate the immunolocalization of hsp in specimens of nervous system malignancies, in order to establish the usefulness of this antigen as target for tumour-vaccine strategies for nervous system tumours. methods the authors assessed the expression pattern of hsp in formalin-fixed and paraffin embedded surgical specimens of nervous system malignancies, including neuroectodermal primary tumors ( astrocytomas, glioblastomas multiforme, oligodendrogliomas, and ependymoma); meningeal tumours; and peripheral nerve sheath tumours ( schwannoma, and neurofibroma), using a standardized immunohistochemical procedure. results hsp was found to be expressed in a low number of tumoral cases (nearly %), with a major expression in astrocytomas histological group ( %). no case among peripheral nerve sheath tumours was found to be immunopositive for hsp . the expression pattern was found heterogeneous in all of the positive samples and it does not correlate with the degree of malignancy. conclusions the low frequency and the heterogeneous distribution of hsp suggest the useless of this antigen as unique target for immunotherapeutic strategies. however, the present study shows firstly the immunolocalization of hsp in some cells of nervous system tumors tissues. the emergent complex function of hsp renders necessary further studies to understand the link between positive-cells and this protein. in vitro fddnp labeling of pathological protein aggregates in the cns of selected "conformational" disease cases introduction a molecular imaging probe - ( -{ -[( -fluoroethyl) (methyl)amino]- -naphtyl}ethylidene)malononitrile (fddnp), is a highly hydrophobic, fluorescent substance, known to label senile plaques and neurofibrillary tangles (nft) in the brain of alzheimer disease (ad) patients. the aims of this study were to evaluate in vitro fddnp labeling of protein aggregates in selected "conformational diseases" of the brain and to determine if they fulfill the tinctorial and polarizing optics criteria for amyloid. material and methods brain sections of patients with confirmed ad, variant ad (vad), amyloid angiopathy (aa), sporadic creutzfeldt-jakob disease (cjd), variant cjd (vcjd), gerstmann-sträussler-scheinker syndrome (gss), pick disease (pid) and progressive supranuclear palsy (psp) were labeled with fddnp, congo red (cr) and pas. analysis was performed using fluorescent microscope (fddnp), and light microscope (cr and pas) with polarizing optics (cr). results in addition to senile plaques and nft in ad and vad fddnp reliably labeled: prion plaques in cjd, vcjd and gss, lewy bodies in vad and vascular amyloid in aa. it did not label pick bodies in pid, and globoid nft in psp. with the exception of fddnp positive hirano bodies in ad and lewy bodies in vad, cr and pas stained all the fddnp positive deposits. all cr positive aggregates also showed birefringence. conclusion in our study, fddnp labeled all protein aggregates that were shown to posses tinctorial and birefringent characteristics of amyloid, but also some that cannot be classified as amyloid. aside from its potential for in vivo diagnostics of some neurodegenerative diseases, fddnp may prove useful in labeling certain pathological protein aggregates in vitro. work supported by mŠzŠ grants - and l - . aim to investigate the development and clinical significance of vascularization in adenomatous alveolar hyperplasia (aah) and peripherally located adenocarcinomas of the lung. material and methods histological slides obtained from patients with aah, and from patients with potentially curatively operated lung adenocarcinomas including normal lung parenchyma are immunohistochemically stained with an anti cd antibody, and subject to quantitative image analysis. syntactic structure analysis measures the absolute and relative features of vessels including the vessel-associated cellular densities. these data are compared in between and associated with tumor volume, post surgical tnm stage, and the patients' survival. results aah displays an increased level of vascularization characterized by regular size and increased number of newly formed vessels. spatial agglutination (clusters formation) of vessels is common in aah. the tumor vascularization (volume fraction vv) amounts to % in aah and to % in lung carcinomas, and increases in advanced tumor stages (pt , pn ). aah displays the lowest, advanced tumor stages the highest numerical vascular density. minimum diameter and circumference of vessels maintain. the density of epithelial cells decreases with increasing distance from the vascular surface in both aah and adenocarcinomas. it is the highest in advanced tumor stages. according to multivariate statistical analysis the patients' survival is closely associated with the pn-stage, tumor volume, cell type and the numerical density of tumor cells within a distance < m to the nearest neighboring vessel. conclusion aah is characterized by probably non-reversible changes in vascular density and spatial vessel clustering. vascularization of pulmonary adenocarcinomas becomes altered in advanced tumor stages. of prognostic significance is the distribution of tumor cells in relation to the nearest neighboring vessel only. changes in vascularization probably play an important role in genesis and development of pulmonary adenocarcinomas. cellular and connective elements between bronchial glands in sarcoidosis: a quantitative analysis z kosjerina, v kosjerina ostric institute of lung diseases, sremska kamenica, serbia and montenegro objective objective of the study was to quantify the inflammatory and connective cells, as well as connective fibres between the bronchial glands in pulmonary sarcoidosis (s). material the study included the bronchobiopsy samples obtained from sarcoid patients. the control group (cg) included young healthy subjects who lost their lives in an accident, having the bronchus sampled at autopsy. the quantity of inflammatory and connective cells was measured by the stereometric numerical density method and the quantity of the connective fibres by the volume density method. results in the examined group the total quantity of all cellular elements between the bronchial glands in s was estimated to range from . /mm to . /mm or . /mm on the average. in the cg, the quantity ranged from . /mm to . /mm , or . /mm on the mean. besides cellular elements, connective fibres were also registered between the bronchial glands. in s, the connective fibres made % of the interstitium volume and only % of it in the cg. this difference is statistically significant. conclusion in sarcoidosis, the total quantity of all cellular elements between the bronchial glands exceeds their quantity in the cg for . times. the most numerous are lymphocytes in both groups, but in s their quantity is . times as high as their quantity in the cg. the quantity of the connective fibres is times as high as their quantity in cg. these differences are statistically significant. depending on the lung cancer stage and type vs. - % by conventional cytology) at an acceptably reduced specificity ( % vs. %, for aqc and conventional cytology, respectively). the role of the aqc cytology test in a comprehensive approach to detection of early (non-invasive and minimally invasive) lung cancers will also be discussed. introduction characterization of bronchial preneoplastic lesions is based upon cellular atypia and disturbed tissue architecture. depending on the individual experience, the grading of such lesions varies considerably. we evaluated immunohistochemical staining for p and mib- in regards of their value in routine diagnostics of bronchial biopsies. material and methods bronchial biopsies ranging from normal bronchial epithelium to invasive squamous cell carcinoma have been stained with antibodies against p and mib- . the biopsies have been divided into groups: squamous cell metaplasia with basal cell proliferation to low grade dysplasia, high grade dysplasia, and carcinoma in situ. for comparison, normal bronchial epithelium and invasive carcinoma have been included. in normal epithelium % of the cells showed positivity for p . with increasing degree of atypia, the number of posititive cells per nuclei increased to % in carcinoma in situ. almost linear progression has been found in the proliferation activity assessed by immunohistochemical staining with mib- . in normal epithelium the activity was < . %, and the number of positive cells increased to % in carcinoma in situ. in bronchial preneoplastic lesions an increasing accumulation of p and mib- can be found in accordance to the degree of atypia. this provides additional and valuable information to facilitate differentiation between low grade and high grade lesions. in regards of an increase in less invasive diagnostic and therapeutic methods, immunohistochemical staining with p and mib- adds valuable information for a correct grading of these lesions, and is therefore of considerable clinical importance. pitfalls in diagnosis and assessment of lymph node metastases in lung cancer -a morphometrical analysis of pulmonary and mediastinal lymphnodes t gumprich, k junker, km müller institut für pathologie, bochum, germany introduction besides histological subtype and tumor size, metastatic spread in pulmonary and mediastinal lymphnodes in lung cancer is also an important factor concerning therapeutic strategies. prior to resection of the primary tumor a thorough staging of pulmonary and mediastinal lymphnodes is performed using computer tomography -but lymphnodes will be judged by size only. in an autopsy study of ten cases with advanced lung cancer, lymphnodes have been dissected. lungs have been resected en bloc, and were cut in horizontal sections according to computer tomographic layers. lymphnodes have been measured and histologically examined. lymphnodes were judged as normal with a transversal diameter of not more than mm. in the histological examination metastases were found in % of normal-sized lymphnodes, while % of enlarged lymphnodes presented as negative. using other parameters did not yield better specifity or sensitivity. discussion: there is a wide range of what maybe the normal size of a lymphnode which is influenced especially by the study collective. we found a variety of size-influencing factors (pneumoconioses, silicosis, previous infections). furthermore, there are differences in size of lymphnodes in collectives from rural and industrial populations. the high number of metastases in normalsized lymphnodes is explained by the highly advanced stage of disease in our study collective. conclusions for imaging diagnostics, sufficient size parameters for lymphnodes are not existing. we are convinced that preoperative staging of lymphnode status is important. in many cases the radiological evaluation on the lymphnode stations is not sufficient to differentiate between reactive enlargement and metastatic involvement. mediastinoscopy is still needed in preoperative diagnostics, because it allows taking biopsies with following histological examination. expert system dedicated on diagnosis in thyroid pathology introduction encoutering diagnostically difficult cases, pathologists usually asks consultations from more experienced colleagues or uses materials in form of journal articles, textbooks, atlases etc. these sources can provide the information in a passive way. usage of expert systems (special computer programs) let pathologists obtain the information in an interactive way and have more accuracy in the diagnosis. our purpose was to create an expert system capable to improve histological diagnostics in area of thyroid tumors and tumor-like conditions. materials and methods materials for our work consisted of more than cases of thyroid tumors and tumor-like conditions (near pictures), representing thyroid diseases. every case was described by two experts according to histologic features and then entered in pc data base. results our computer program can act in two principal ways. these are ) diagnostics -expert system propely, ) learningvirtual textbook. ) using the expert system, pathologist enters new data in pc and marks so many features as he can. then he obtains the information about all the possibilities of probable diagnosis and some other results. entering more features can help to narrow the borders of probable diagnosis. ) applying our program as a virtual textbook, it is possible to learn better the known significance of pathological features, to take a look on the descriptions the experts made etc. the program can also function as a virtual trainer and examiner. conclusion our computer program increases the diagnostic quality in the area of thyroid pathology by means of high capability of information integration and improving pathologist's qualification during his learning. introduction xenin is a biologically active -amino-acid peptide extractable from the gastro-entero-pancreatic system. the aim of our study was to identify the unknown cellular source of xenin and to analyze the expression of xenin in neuroendocrine tumors. methods normal tissue of humans, rhesus monkeys and dogs and a large series of neuroendocrine foregut and midgut tumors were investigated by immunohistochemistry, confocal laser scanning microscopy, immuno-electronmicroscopy and high pressure liquid chromatography, assessed by radioimmunoassay. results in all three species, xenin was expressed in a subpopulation of gastric inhibitory (gip)-cells of the upper intestine. immunoelectronmicroscopy demonstrated presence of xenin in secretory granules ( ± nm diameter). xenin immunoreactivity and extractable xenin was found in of duodenal neuroendocrine tumors, including gastrinomas, somatostatinomas, non-functioning and enterochromaffin cell tumors. in contrast, xenin was absent from gastric, pancreatic, ileal and bronchial neuroendocrine tumors. conclusions the localization of xenin in ultrastructurally characterized secretory granules supports the concept that xenin represents a further endocrine regulatory peptide. the finding that xenin expression distinguishes between endocrine tumors arising in the duodenum from those of nonduodenal origin may be useful for topographical tumor classification and the differential diagnosis of neuroendocrine tumors of the upper gut. (supported by dfg) introduction the tall-cell variant (tcv) is known as a rare but aggressive form of papillary thyroid carcinoma (ptc) with increased incidence of local recurrence and mortality. it is defined as papillary thyroid carcinoma with at least % of specific cellular changes within the tumor cells and occurs in - % of all ptcs. materials and methods in our series of paraffin embedded and h&e stained tumor tissues, tall cell morphology was exactly defined for the first time and then cases of ptcs, including all stages and variants, were investigated concerning tall cell morphology and associated changes within the tumors. results in contrast to previous reports, cases ( , %) were classified as tcv in our series. taken together with minor tumor parts, cases ( , %) presented with tc morphology. these parts were in a high percentage associated with fibrosis and lymphocyte rich areas and showed a higher mitotic activity than usual ptcs. discussion the differences in the occurrence of tcv and tcmorphology between the presented series and previously reported cases might result from until now not clearly defined tall cell morphology as well as from similarities to ptcs, such as the oxyphilic variant, which is extremely rare in our series, and maybe also from often described squamous changes within ptcs. due to these data it is not clear which tumor parts have relevance for prognosis and which tumors should be treated more aggressively than others. in accordance with previous reports concerning the prognostical impact of this variant, we think that the occurrence of tc morphology plays a major role in both development of extrathyroidal growth and metastatic disease. myocardial extensions around venae cavae as a potential substrate of atrial fibrillation altered endothelial permeability secondary to ischemiareperfusion e stefanovic, v kanjuh, n tasic dedinje cardiovascular institute, belgrade, serbia and montenegro introduction during open heart surgery some damages to myocardial capillary endothelium occur, with the consequence to the regarding structure and function of cardiomyocytes as well. cardioplegia (cpl), long term hypoxia and reperfusion are strong contributing factors for these lesions. the aim of the study is to assess ultrastructural changes and permeability of endothelium of myocardial capillaries due to hypoxia and reperfusion during open heart surgery. the study included consecutive patients (pts) who underwent open heart surgery. samples were taken from right ventricle anterior wall and apcal region. biopsies were taken at the end of cardioplegic period, during ischemic period and during period of reperfusion. all patients were divided in two groups depending on duration of period of ischemia or cardioplegia-group (clp< min) and group (clp- - min). all samples were analyzed by electron microscope using quantative stereological analysis. volume fraction of the pinocyte vesicles (pv), mitochondria (m) and endoplasmatic reticulum (epr) were calculated. results endothelium ultrastructure analysis showed increase in permeability of the capillary wall. the worse damage was found after min. of cpl usage: edema of the endothelial cells, widening of the cell junction and other degenerative changes of cardiomyocytes. analyses of result confirmed that the more significant damages were found during reperfusion in group . conclusion our study showed significant changes in endothelial permeability and function after ischemia and reperfusion. disturbances of transendothelial transport may play certain role in the development of heart failure after open heart surgery. the authors compared the clinico-pathological and immunohistochemical features of acute postinfarction cardiac rupture and control (with near the same clinical age without rupture) cases. material and methods cryostat sections of both groups from infarction/rupture and controls were stained with oil red o, sudan black b (bdh chemicals) and were reacted to anti-c-reactive protein (crp), to complement c a and c b- (dakopatts). clinico-pathological findings myocardial rupture occured in elder women with relative smaller weight of heart, thrombosis of one main coronary artery, location in the anterior wall at the first infarction within the first week. microscopically the main observation is the large amount of granular lipid deposition in the area of myocardial rupture even in the cytoplasm of the necrotic myocardial fibers using oil red o and sudan black b staining. immunohistochemistry: the anti-crp and c b- antibodies showed intense staining around the area of the rupture. the inflammatory cells were neutrophils in high numbers in both group. eosinophils were represented in low percentage (below %). conclusion the postulation is, that the granular myocardial lipids in the area of infarction/rupture are phospholipids (mainly mitochondrial cardiolipin), which through binding crp induce pathologic complement activation. in experimental heart infarctions complement target (recombinant etc.) molecules could reduce the neutrophil chemotaxis to the area of necrosis and the size of the infarction. nuclear factor-κb regulates apoptosis of intimal smooth muscle cells introduction apoptosis contributes to reduce intimal smooth muscle cell (smc) hyperplasia following angioplasty or during atherogenesis. nuclear factor (nf)-kb is involved in the regulation of many smc functions. aim of the work was to verify the role of nf-kb in bax-dependent smc apoptotic cascade and its possible targeting for novel biologically based therapies of vascular hyperplasia. methods apoptotic proteins were evaluated by immunohistochemistry and western blotting and nf-kb using a anti-p (rela) antibody recognizing its active form. apoptosis was induced in rat aortic neointimal smcs after endothelial injury in vivo and in vitro by propyonil-l-carnitine (plc). in vivo, expression of constitutive nf-kb activity of smcs of neointima was higher compared to underlying and normal media. treatment with plc associated to reduced neointimal relative volume and increased apoptotic rate of intimal smcs compared to controls. increased apoptotic rate associated to bax overexpression and rela reduced immunopositivity. in vitro, plc reduced g to s phase progression of intimal but not of medial smcs. hoechst staining, flow cytometry and ligation-mediated pcr showed increased apoptotic rate of intimal cells treated with plc together with bax overexpression and the release of cytochrome c. sn , an inhibitor of nf-kb nuclear translocation, induced an increase of apoptotic rate in intimal but not in medial smc cultures, prevented the apoptotic effect of plc and upregulated the cytoplasmic bax expression and the release of cytochrome c. conclusions our studies with inhibitors of nf-kb strongly suggest that this nuclear factor plays a crucial role in the control of intimal smc apoptosis. alpha-actin isoform distribution in normal and failing human heart: a morphological, morphometrical and biochemical study introduction two sarcomeric actins, α-cardiac (α-ca) and αskeletal (α-ska), are coexpressed in adult myocardium and represent the preponderant thin filament actin isoforms of cardiomyocyte contractile units. in the human heart, the relative amount of α-ska mrna increases steadily during development and after birth and becomes the predominant isoform in adults but data in the literature concerning hypertrophy and failure are scarce and controversial. aim of the work was to determine how and where α-ska isoform accumulates during heart development, ageing and with disease. methods by using monospecific antibodies, we investigated by immunohistochemistry and western blotting the distribution of α-ska, α-ca and α-smooth muscle actin (α-sma) isoforms. results at weeks of fetal life, α-ska was localized in a small proportion of subendocardial and papillary muscle cardiomyocytes, whereas a diffuse α-ca staining was observed, associated with a focal expression of α-sma. in normal adult subjects, α-ska positive cardiomyocytes were distributed in a transmural gradient with the highest proportion located subendocardially. in myocardial hypertrophy and cardiomyopathies, the amount of α-ska was increased and a diffuse staining was seen in all layers of ventricular myocardium, with the exception of idiopathic dilated cardiomyopathies. cardiomyocytes were always negative for α-sma. as expected, fibroblasts in post-infarct scars expressed α-sma and tgf-β , but were negative for these proteins in interstitial fibrosis. conclusions changes in the distribution of actin isoforms in hypertrophy and failure provide new insight into the mechanisms by which the heart adapts to work overload and help to define their role in these pathological situations. introduction arrhythmogenic right ventricular dysplasia (arvd) has received much interest, since it may cause sudden cardiac death (scd). yet, pathogenesis remains controversial, not at least because adipose and fibroadipose types are considered different entities by some. the present study has looked into the histopathologic features of hearts with arvd with a particular eye for a clue to pathogenesis. clinical history review and heart macroscopic/microscopic examination (with histochemical/immunohistochemical techniques) were performed on patients dying suddenly with arvd. twenty-three, m/ f were young ( - years) and , m/ f adults ( - years). results )one young had family history of scd. )one adult had cardiovascular antecedents. )seven patients had died during exercise. )both young and adults presented adipose (n= vs ) and fibroadipose (n= vs ) types, whilst each variant basically showed similar overall architectural arrangements. ) occasional inflammatory infiltrates were present in fibroadipose, but not in adipose type. ) left ventricular involvement occurred in young and adult heart. ) coexistent left ventricular pathology was occasionally identified. conclusions ) arvd can be manifest in young and older persons, either as adipose or fibroadipose variants. ) both types are morphologically identical, except that some may have additional fibrosis, occasionally accompanied by small inflammatory aggregates. subtle differences hard to interpret as features distinguishing different entities. ) preferential and almost selective involvement of the right ventricular wall suggests a congenital nature of the disease, particularly since recent insights in right ventricular development have showed "regionalization" of genetic expression in the formation of cardiac chambers. fatty/fibrofatty tissues may appear ab initium through an alternative mesenchymal pathway. a new method of quantitative functional morphology for systemic examination of the cardiac muscle t ghevondyan , h guski , g avtandilov national institute of health, yerevan, armenia medical faculty of humboldt university of berlin, berlin, germany introduction determination of the vital capacities of heart muscle has special importance for cardiac surgery. morphological methods could be the most trustworthy in this respect. but it was not possible yet to extract this information from myocardial specimens. the aim of this study was to elaborate a method, which will be able to determine the volume of vital reserve of myocardium taking into account all known morphological changes and alterations. the investigation was carried out on heart muscle tissue of healthy people, who died accidentally and of patients who died from myocardial infarction. the myocardium of white rats (normal, physically trained, during pancreonecrotic shock) was used. methods of histology, electron microscopy, cytoenzymochemistry, morphometry, micromechanography, statistics and mathematical modelling were used. results most informative parameters were chosen for characterisation of the microcirculatory bed and of the paths of ultracirculation. the property of contractility has been taken as a systemforming criterion. a mathematical model was elaborated, which allows to unite the most important structural changes and alterations from tissue, cellular, ultracellular and macromolecular levels of the heart muscle. the role of endomyocardial biopsy in end-stage heart failure introduction endomyocardial biopsy is considered to be an important diagnostic procedure for the evaluation of patients with end-stage heart failure. the aim of this retrospective study was to evaluate the role of apoptosis, proliferation markers, volume density of interstitium, and myofibril volume fraction for the prognosis in patients with end-stage dilated cardiomyopathy (dcm). methods endomyocardial biopsy was performed during openheart surgery in pts with end-stage dcm. patients were divided into groups, one with shorter survival ( ± months, mean ± standard deviation) and another with survival of more than years after operation. the tunel method was used for the detection of apoptosis, and immunohistochemical methods were used for the evaluation of inhibitor of apoptosis (bcl- ), and proliferation markers (pcna, ki- ). results the increased percentage of apoptotic myocytes, decreased expression of bcl- , pcna and ki- antigen, but lower myofibril volume fraction and lower volume density of interstitium was found in the group with early mortality compared to that with longer survival. conclusion morphometrical analysis of the endomyocardial specimens of the patients with terminal heart failure due to dcm may enable us to get access to crucial information about the status of the myocardium, which may help us to predict the prognosis and optimal treatment of patients with end-stage heart failure due to dcm. introduction trafficking studies in bone marrow transplantation (bmt) more often use fluorescent reporter molecules like the green fluorescent protein (gfp). dendritic cells (dc) represent an interesting population for trafficking analysis, because they undergo steps of maturation, which seem to be related to their localization in the tissues. the aim of this study was detection of ex vivo expanded dcs, transduced to express gfp, in tissues of mice that had received an allogeneic bmt. material and methods tissues of recipient mice were pretreated by fixation in % formalin followed by % sucrose, before embedding in oct and freezing on dry ice. in addition tissues were also fresh frozen and exposed to formaldehyde vapor as cryostate sections. the gfp signals were validated using an anti gfp antibody. additional markers investigated were cd c, cd , cd , h- kb and h- kd. results vapor fixation of slides showed equal properties in containing gfp in comparison with the immersion method. gfp+ dcs were detected in small bowel, lymph nodes, thymus and spleen for up to days after i.v. injection. many of these gfp+ cells were positive for cd c. the gfp signals could be validated on fluorescence and light microscopic level using anti gfp antibodies. conclusions allogeneic dcs survive in bmt recipients for up to days. they migrate into small bowel, mesenterial lymph nodes, thymus and spleen without causing gvhd. the visualization of survival and migration in bmt settings will provide new insights into the immunological role of dcs in vivo and might contribute to the optimization of dc based treatment strategies. breast hamartoma: cytologic, pathologic and immunohistochemistry study of cases introduction breast hamartomas enter the differential diagnosis of breast masses, necessitating an accurate diagnosis. it is still not quite clear into which group of lesions these masses should be categorized. the aim of our study was to investigate the cytologic, histologic and immunohistochemical profile of breast hamartoma in order to obtain a detailed picture of this somewhat unusual lesion. materials and methods this study presents cytological, pathological and immunohistochemical analysis of patients with breast hamartoma. the immunohistochemical panel included: estrogen and progesterone receptors, her -neu protein, p- , ki- , ckmnf , vimentin, alpha-sma. results the patients ranged in age between and years. all patients presented with a palpable unilateral mass. the aspirates contained sheets of both bland ductal cells and lobular units. adipose tissue was present in varying amounts. typical macroscopical and microscopical features were noted. immunohistochemical studies showed estrogen and progesterone receptor positivity in epithelial cells as well as in the stromal cells, in all hamartomas. her -neu protein over expression was noted, p- expression was not observed. ki- showed a - % positivity in epithelial cells in most cases. conclusions the finding of intact lobular units and a relative paucity of stroma in an aspirate from a well-circumscribed breast lesion, may suggest the diagnosis of hamartoma. typical macroscopical and histological features of breast hamartoma were noted. the immunohistochemical profile appears to be similar to that of normal and fibrocystic breast tissue, raising the possibility of a spectrum of lesions. introduction ck-pattern in nos breast carcinoma has been studied extensively, including high molecular weight types, such as ck- , present in rare cases. published data on ck- -expression in less common subtypes of breast carcinoma are limited the aim of this study was to learn more about the immunohistochemical profile of ck- in cmf, a systematic analysis on ck- -reaction in a consecutive series of this variant of breast carcinoma was undertaken, including correlation with some traditionally recorded tumor characteristics. forty-three consecutive cases of cmf were examined for ck- . extent of staining was assessed semiquantitatively and reported as a percentage; its correlation to tumor size and receptor status was calculated. results sixteen ( , %) of the cases comprised ck- labelled tumor cells. though ck- + tumors were less commonly receptor+ and tended to be larger than ck- -cases, statistically significant differences were not obtained (p= . and p= . , respectively). conclusion no specific immunohistochemical profile typifying cmf has been identified. nevertheless, most cmf seem prone to exhibit immunophenotypic aberrations with higher incidence of vimentin+, c-erb-b +, and p- +, lower incidence of her + and er/pgr+. we here demonstrate that ck- -expression can be added to this list. though not statistically significant, a trend towards an inverse relation between basal cell characteristics and receptor expression and a positive correlation between ck- + and tumor size was demonstrated. introduction residual disease leads to most local recurrences, especially in patients treated with breast conserving therapy. this study was undertaken to identify whether assessment of excisional biopsy margins accurately predicts the presence or absence of residual tumor in the lumpectomy bed. material and method the margin status of consecutive lumpectomy specimens followed by re-excision, taken from an equal number of patients with primary breast carcinomas ( infiltrating, in situ), were evaluated microscopically and classified as 'positive' if the tumor was found at the inked margin, 'negative' if the entire tumor was found in > . cm from the inked margins, 'close' if it was within . cm of the inked margins, but did not transected them and 'indeterminate' if biopsy specimen was not inked or the tissue was fragmented. the tumor size and grade of the initial biopsy were also analyzed, as potential predictors for residual disease. results residual tumor was found in % of the patients ( / ): in %( / ) of the cases with negative, in %( / ) with positive, in % ( / ) with close and in . %( / ) with indeterminate margin status. in %( / ) of the 'positive margins' and in %( / ) of the 'close margins' cases no tumor was found in re-excised specimens. in %( / ) of the cases the residual disease was composed entirely of an in situ component of the same histologic type as the initial biopsy. no relationship was found between tumor size or grade and residual disease. conclusions for breast tumors histologically negative margins in biopsy specimens do not guarantee complete excision. 'close' margins may be considered almost equal to 'positive' ones. residual in situ carcinoma in the immediate vicinity of the previous biopsy site seems to represent the advanced edges of the tumor, apparently difficult to be excised completely and most likely responsible for the locally recurrent disease. introduction syringomatous adenoma of the breast is a benign lesion of the nipple, which may be confused with an infiltrating carcinoma. the presence of the myopithelial cells and the immunohistochemical profile is still controversy in the literature. we present the case of an years old patient, with an left nipple mass. materials and methods after excision, the excised tissue was fixed and paraffin embedded and h-e stained. we performed also immunohistochemical staining: with cytokeratin, actin, er, pr, p . the excised tumor measured cm, had ill-defined margins and yellow color.microscopically, the lesion consisted of tubules and strands, composed of small and uniform basophilic cells, diffusely infiltrating the stroma of the nipple. the tubules were lined by two layers of cells, had a comma-like appearance and their lumen was usually open and round. the tumor cells lack pleomorphism. squamous differentiation resulted in keratotic cysts. immunohistochemically, the luminal layer of epithelial tumor cells was cytokeratin-positive and the peripheral myoepithelial cells were actin-positive. the tumor cells were negative for er, pr, p . conclusion syringomatous adenoma of the breast is a benign rare lesion. the pathologist must not confuse it with an tubular carcinoma or with an adenosquamous carcinoma. we demonstrated in this case the presence of the myoepithelial cells. the role of smooth muscle myosin heavy chain in diagnosing sclerosing adenosis of the breast with an infiltrative pattern introduction sclerosing adenosis (sa) of the breast can produce an infiltrative pattern mimicking invasive carcinoma. immunohistochemical markers for myoepithelial cells, such as smooth muscle myosin heavy chain (smmhc), have been shown to stain basal myoepithelial cells in normal mammary glands and fibrocystic changes. absence of myoepithelial cells is associated with invasive carcinoma; however, we have observed occasional attenuation of smmhc staining in sa. studies evaluating the role of smmhc in sclerosing adenosis have not been performed. the intent of this study is to further evaluate the staining pattern of smmhc in sa with an infiltrative pattern. materials and methods twenty cases of sa with an infiltrative pattern were identified, h&e stained, and immunohistochemistry performed for smmhc (m , dako corp), µg/ml, as primary antibody, using a polymer based envision+ detection system (dako corp). normal-breast tissue positive controls and negative isotype matched igg controls were used. cases were then classified into one of three categories: positive (staining > % of myoepithelial cells), intermediate ( % - %), or attenuated (< %). results three of the first ten cases ( %) showed attenuated staining with smmhc. the remaining seven cases demonstrated positive staining of myoepithelial cells. all cases exhibited positive staining within normal lobules, serving as an internal control for each case. ten additional cases of sa have been identified and are being stained with smmhc for evaluation. conclusions preliminary findings show that smmhc yields inconsistent results when applied to sa with an infiltrative pattern. such lesions can have features similar to invasive carcinoma on h&e, and application of smmhc could produce misleading results. co-expression of erbb family members in human breast cancer: her- /neu is the preferred dimerization candidate in nodal-positive tumors introduction overexpression of members of the erbb receptor family has been asssociated with malignant transformation and the amplification of her- /neu in tumor tissue is now an established prognostic factor in breast cancer. in order to initiate signal transduction, erbb receptor monomers need to form homo-or hetero-dimers. the composition of these dimers is thought to greatly influence both quality and quantity of downstream signalling pathways, and to ultimately determine the elicited biological response. material and methods the protein expression pattern of all four erbb receptors egf-r, her- /neu, her- and her- , and expression of their putative ligands egf, tgf-a and hrg was investigated in women with invasive breast cancer by membrane isolation and western blot analysis. results the co-expression of all four erbb family members was detected in , % of cases, and of all of the three investigated ligands in , %. while we did not observe a correlation between egfr and her- /neu or her- protein expression, egfr and her- (p= . ), and her- and her- (p= . ) were clearly coexpressed. the strongest overall correlation, however, was found between her- /neu and her- (p= . ) and between her- /neu and her- (p= . ). this was particularly true in nodal-positive tumors (p< . and p= . , respectively) whereas in nodalnegative tumors the co-expression was either less significant (her- /neu and her- : p= . ) or not significant at all (her- /neu and her- ). the co-expression of egfr/her- was associated with the expression of all three ligands, whereas the her- /neu/her- was correlated with hrg (p= . ), thereby indicating a functional relation between specific receptor-dimer combinations and putative ligands. conclusion taken together, we have performed a comprehensive survey of erbb system expression in breast cancer, and have demonstrated the presence of a co-regulated receptor/ligand system in vivo. her- /neu appears to function as the preferred coexpression partner in nodal-positive tumors, thus rendering him the most likely dimerization candidate in malignant breast tumors. introduction members of the ap- family of transcription factors (c-jun, junb, jund, c-fos, fosb, fra- and fra- ) are able to form jun-jun or jun-fos dimers which bind to the regulatory sequences of target genes. in a prior study on mammary carcinomas, we found that high fra- and reduced fosb expression are associated with indicators of poor prognosis. as most of the proteases involved in degradation of the extracellular matrix and basal membrane (matrix-metalloproteinases, members of the upa/paisystem) are regulated by ap- complexes, we assumed that the expression of single ap- proteins might also be important for the process of invasion of breast cancer cells. method in order to analyze the influence of the single fos family members (c-fos, fosb, fra- and fra- ) on the invasive behavior of mammary cancer, we performed western blot analysis of mmp and mmp and of the members of the plasminogen activator system pai- , upar, and pai- -upa-complexes in breast cancer samples, and correlated the expression of these proteins with that of the ap- proteins. results interestingly, c-fos, fra- (phosphorylated form), and fra- correlated with mmp expression levels. high expression of fra- was also significantly associated with that of pai- and the sds-insoluble upa/pai- complex. on the other hand, the expression of fosb was significantly associated with high levels of mmp . conclusion our results indicate that a shift in ap- protein expression, especially high expression of fra- and fra- , might be involved in invasion of breast cancer cells. (supported by deutsche krebshilfe, no. - -ba ) immunohistochemical study of estrogen receptors and progesteron receptors in primary breast carcinomas and their axillary lymph node metastases nh aksoy, he pestereli, s karaveli akdeniz university school of medicine, department of pathology, antalya, turkey estrogen receptors (er) and progesteron receptors (pr) was investigated in primary breast carcinomas and their axillary lymph node metastases to determine if the malignant cells retained or changed this phenotypic feature during the metastatic process. the data were also compared with the tumour size, histologic type, grade and age. immunohistochemistry with the er (dako- d -n ) and pr (dako-pgr -n ) on formalin-fixed paraffin embedded tissue was used. the er status in primary tumours and lymph node metastases was concordant in patients ( , %) and discordant in ( , %). six of these eleven patients had er-positive primary tumours and er-negative lymph node metastases. the pr status in primary and lymph node metastases was concordant in patients ( , %) and discordant in patients ( , %). ten of these twenty-four patients had prnegative primary tumours and pr-positive lymph node metastases. we observed higher pr positivity in axillary lymph node metastases. the parameters, tumour histology, grade and age showed no statistically significant relation with receptor status. there was a significant correlation between the tumour size and the number of axillary lymh node metastases. the discordance of hormone receptors status between the primary tumour and lymph node metastases might be due to the heterogenity of tumour cells or loss of the phenotypic features of metastatic cells. breast carcinoma in a cancer hospital p arapantoni -dadioti , o tzaida , v leodara , g sotiropoulou , e trichia , pk sotirakoglou pathology department metaxas cancer hospital of piraeus, piraeus, greece department of mathematics and statitics agriculture university of athens, athens, greece a total of cases of infiltrating breast ca, diagnosed in our hospital during the last decade, was studied. the material concerned mastectomies with ispilateral lymph nodes dissection specimens. the patients were classified according the tnm staging system, based upon the primary tumor size (t), the presence of regional axillary nodal involvement (n) and distant metastases (m). this categorization revealed a preponderance of patients with t tumors. the overall decade rate for t , t and t tumors was , , %± , %, , %± , % and , %± , %, respectively. there was a statistically significant difference between the three groups (p< , ). the annual distribution of these groups revealed that since the mean group rate had a tendency to approach the corresponding decade rate, a finding suggesting some degree of stabilization of these proportions. on the other hand, the lymph node metastases were more common in the t tumors. the mean rate for t n+, t n+ and t n+ was , %± , %, , %± , % and , %±- , %, respectively; the difference is statistically significant for the t n+ and t n+ (p< , ) as well as for t n+ and t n+ (p< , ). the annual overall rate of patients with lymph nodes metastases, independently of t (tn+) was , %± , %. in conclusion, there is no tendency of early patient presentation and a more careful intimate medical care. the infiltrating breast cancer in our material, is represented mainly by thet tumors and the great majority of patients come to the hospital in an advanced stage regarding the t and n. introduction breast cancer is a genetically complex disease, which involves the accumulation of various structural and numerical aberrations. in the present study, we assessed the numerical status of chromosomes and x in primary invasive breast carcinomas in relation to clinicopathological parameters, patients' overall survival, and indices of cell growth (c-erbb- , topoisomerase iia) and survival (caspase- , bcl- ). material and methods the number of copies of both chromosomes was detected by interphase chromogenic in situ hybridization with pericentromeric probes on paraffin embedded sections. er, pr, cerbb- , topoiia, caspase- and bcl- expression was immunohistochemically detected (abc/hrp). the results were statistically assessed by univariate and multivariate analysis. results polysomy of chromosomes and x was detected as the predominant aberration ( . % and . % respectively). gain of chromosome copies was associated with high nuclear grade (p= . ), increased tumor size (p= . ), advanced stage (p= . ), the expression of topoiia proliferation index (p= . ), and worse overall survival by multivariate analysis (p= . ). the levels of chromosome x polysomy were elevated in ductal carcinomas of high histological grade (p= . ), in high nuclear grade tumors (p= . ), and were associated with the expression of topoiia (p= . ) and loss of caspase- protein (p= . ). in addition, gain of chromosome x copies was an independent predictor of impaired prognosis of ductal carcinomas (p= . ). conclusion polysomy of chromosomes and x was observed as the predominant alteration in phenotypically aggressive breast tumors, characterized by poor differentiation, increased growth potential and impaired prognosis, whereas gain of chromosome x in particular, is probably implicated in cell survival. expression of survivin, bcl- , p and bax in breast carcinoma and ductal intraepithelial neoplasia (din a) f kayaselcuk , tz nursal , a polat , t noyan , s yildirim , a tarim , g saydaoglu introduction survivin is a recently discovered member of the family of proteins that inhibits apoptosis. this anti-apoptotic compound can be detected in most types of cancer and expression is associated with a poor prognosis. we immunohistochemically investigated the expression of survivin in breast carcinomas and intraductal epithelial neoplasia of breast to determine whether expression of this protein is associated with clinicopathological parameters such as grade, stage, mitotic rate. the study involved patients with invasive ductal carcinoma (idc) and patients with intraductal epithelial neoplasia with mild, moderate and severe, florid ductal epithelial cell hyperplasia. survivin, bax, bcl- , and p monoclonal antibodies were administered by immunohistochemical method. findings were analyzed with statistical methods. results in of cases ( . %) of breast carcinomas and of cases ( . %) of intraductal epithelial neoplasia with mild, moderate and severe ductal epithelial cell hyperplasia stained positively for survivin. none of the histological parameters analyzed were significantly correlated with survivin expression in breast carcinomas. in the carcinoma cases, survivin expression was positively correlated expression of bcl- , but was not correlated with expression of p , bax, c-erbb- and estrogen, or progesterone receptors. some of the intraductal epithelial neoplasia cases with moderate or severe ductal epithelial hyperplasia stained positively for both survivin and p . the breast carcinomas exhibited significantly increased expression of survivin, p , and bcl- than the breast with intraductal epithelial neoplasia. survivin was not correlated with any of the clinicopathological parameters studied, however, it could be a useful tool in early carcinomas and florid, severe ductal epithelial hyperplasia. introduction certain non-malignant and pre-malignant lesions cause dilemmas for the most appropriate management following lncb. the aim of this study was to determine the accuracy of lncb in diagnosising the most frequent non-conclusive findings: benign papillary lesions (bpl), radial scars (rs), fibroepithelial lesions with stromal hypercellularity (flsh), atypical ductal hyperplasia (adh) and intraductal carcinoma (dcis). material and methods in a retrospective review of , imaging guided lncbs ( - ), we found: ( . %) bpl - with surgical correlation (sc) and with mammographic follow-up (mf); rs ( %) with sc in and mf in ; ( . %) flsh all with sc; ( . %) adh -with sc in , and ( . %) dciswith sc in . results ) bpl: patients without changes in mf. at surgery: bpl - with adh and invasive carcinomas ( with mammographic histologic discordance and diagnosed years post lncb). ) rs: without mammographical changes and rs ( with adh). ) flsh: at surgery, fibroadenomas, benign and malignant phyllodes tumours. ) adh: at surgery, ( . %) adh, ( %) dcis, ( . %) invasive carcinomas - of which microinvasive. ) dcis: at surgery, ( . %) dcis; ( . %) microinvasive, and ( . %) invasive carcinomas. conclusions ) diagnosis of bpl at lncb seems accurate when correlating with image findings. ) rs at lncb can be followed mammographically. ) flsh at lncb always requires surgical excision. ) adh and dcis diagnosed at lncb need complete removal of lesion, due to frequent underdiagnosis. introduction estrogen receptor beta (erb) is known to be present in breast tumors; its prognostic and pathophysiological roles, however, remain to be established. the aim of this study was to investigate erb protein expression in a well documented series of invasive breast carcinomas. materials and methods standard immunohistochemistry with a specific monoclonal antibody (clone ppg / , serotec) was performed on paraffin sections; % of strongly immunostained carcinoma cells was used as a cutoff point to classify tumors as erb-positive. statistical correlations were sought with clinicopathological variables (including hormone receptor status) and patients' disease-free and overall survival. cell proliferation was immunohistochemically assessed by topoisomerase iia (topoiia) index. statistical analysis was univariate and multivariate. results erb immunoreactivity was detected in the majority of specimens ( . %)and was positively linked with era immunoreactivity (p< . ) and increased topoiia index (p= . ). a significant favorable impact of erb immunopositivity emerged with regard to disease-free and overall survival in both univariate (p= . , p= . , respectively) and multivariate (p= . and p= . respectively) statistical analysis; progesterone receptor expression was also found to exert an independent favorable influence on patient's survival with less statistical significance though (p= . and p= . ). on the contrary, patients' survival was not significantly impacted by era status. conclusions because of the positive association between erb immunoreactivity and topoiia expression, the presence of erb in breast cancer cells may be considered an indication of increased proliferation. nevertheless, erb immunoreactivity emerges as a valuable, independent indicator of favorable prognosis. aims to determine diagnostic value of lncb in patients with ilc using ec and ck sse . retrospective review of cases diagnosed as ilc either at lncb or at surgical biopsy (sb), between and . immunohistochemical (ihq) study with ec and ck sse was performed at both lncb and sb. three immunoprofiles were established: ec-ck sse + (ihq of ilc); ec + ck sse -(ihq of ductal carcinoma -idc) and ec + ck sse + (ihq of mixed ilc-idc). initial diagnosis at lncb was correlated with final diagnosis (morphologically and immunoprofile). discordant cases were analysed in order to determine usefulness of ihq at lncb. results in lncbs initially diagnosed as: ) ilc ( patients), final diagnosis was ilc in cases (ec-ck sse +) and idc in case (ec+ ck sse -); ) invasive carcinoma probably lobular type (ilcp) ( patients), final diagnosis was ilc in cases (ec-ck sse +), idc in (ec+ ck sse -) and mixed ilc-idc in (ec+ ck sse +); ) idc ( patients), final diagnosis was ilc in all cases (ec-ck sse +), of them ilc pleomorphic-type. in all discordant cases, immunoprofile at lncb was concordant with final diagnosis conclusion % of patients diagnosed as ilcp at lncb corresponded finally to idc or mixed idc-ilc. . % of patients finally diagnosed as ilc were initially interpreted as idc at lncb. our findings suggest that ec and ck sse are of great value to help characterise breast carcinomas at lncb, and improve management of patients. background her- status became an important prognostic and predictive factor in breast carcinoma. there are two main techniques of evaluation her status: immunohistochemistry (ihc) for the protein expression and fluorescence in situ hybridisation (fish) for amplification of her gene. the aim of the study was to compare the results obtained by ihc and fish methods in determination of her status in breast cancer. materials and methods two hundred breast cancer specimens were examined. ihc and fish were performed in every case. ihc was performed with dako herceptest, fish with oncor-qbiogene reagents. ihc results were classed into groups, accordingly to the intensity of membrane staining ( , +, +, +). fish results were divided into three main categories: na -no amplification, la -low amplification and ha -high amplification. the number of copies of chromosome was also assessed. results over % of cases described by ihc as + exhibited also amplification of her /neu gene. remaining cases were positive with ihc, but presented no gene amplification. this might be due to the subjective assessment of the membrane staining or to the procedure of material fixation. another possibility is that overexpression of the protein was caused by mrna stability or disorders in receptor circulation. the majority of cases classed by ihc as + were also negative by fish (over %). nearly one fifth of ihc + were found to exhibit gene amplification. remaining cases showed no amplification of her /neu gene, combined with aneuploidy of chromosome . all cases described by ihc as / + showed no amplification. conclusions ihc is well-established method of assessing her status in breast cancer. nonetheless, group of cases described as + should be additionally examined using fish method because the results are more reliable. in order to improve accuracy and gain the highest quality of her status evaluation, in those cases both methods should be applied. dcis of the breast with excessive cancerization of the lobules simulating invasive carcinoma b nielsen institute of pathology, randers, denmark a -years old woman had a right sided mastectomy with axillary dissection performed, because of suspicious mammografic microcalcifications and finding of malignant cells by fine needle aspiration. grossly, an cm large suspicious area with accentuaded consistency and multiple pale-yellow flecks, representing comedonecrosis, were found. multiple sections representing the suspicious area showed dcis. the ducts were dilated and contained malignant cells and central necrosis. in areas classical cancerization of the lobules were present. furthermore, in multiple microscopic areas the stroma of the lobules were oedematous and infiltrated by lymphocytes. the acini were slightly dilated and covered by one or two layers of malignant epithelial cells, but the central lumen was preserved. the morphology greatly simulated invasive ductal carcinoma. however, by immunohistochemistry the myoepithelial cells were showed to be preserved and the lobular unit were well demarcated with preserved circumscription. the axillary fat contained lymph nodes all without metastasis. it was concluded that the alterations represented a variant of lobular cancerization, earlier described by tavassoli ( ) . spindle cell tumors of the breast are rare entities with difficult differential diagnosis. we report four spindle cell tumors: a metaplastic carcinoma, a fibromatosis, a malignant spindle cell myoepithelioma and an inflammatory pseudotumor. the metaplastic carcinoma occurred in a -year-old woman as a . x . x cm nodule with scarce bland clear cells proliferation and very important desmoplastic reaction; immunohistochemical stains revealed positivity for cytokeratins (mnf ) and vimentin and negativity for actin and desmin. the fibromatosis occurred in a year-old man as an x x cm tumor with bland spindle cell morphology; very little atypia and rare mitoses were identified and immunohistochemical positivity for vimentin was demonstrated. the malignant spindle cell myoepithelioma occurred in a -yearold woman as x x cm nodule with central necrosis; histopathologic examination revealed a malignant spindle cell proliferation with many mitoses and atypical cells; focal positivity for mnf , vimentin, actin and s was identified. the inflammatory pseudotumor occurred in a -year-old woman as a highly cellular spindle cell proliferation with fascicular pattern admixed with myxoid areas and inflammatory cells -type i-ii mammary inflammatory pseudotumor. one important criterion which should be kept in mind in the process of evaluation of a spindle cell tumor of the breast is that these tumors may have deceptive histopathologic appearance -some of them have bland looking morphology despite of their biological behavior (our example of metaplastic carcinoma) and others with worrisome morphology were diagnosed as benign or intermediate malignancy entities (inflammatory pseudotumor). introduction calcitonin receptor (ctr) belongs to the g proteincoupled receptors and it binds calcitonin (ct), which acts on bone and kidneys to maintain calcium homeostasis. however, both of ct and ctr have also been identified in a large number of tissue and cell, suggesting it might has any unrecognized roles. although ctr expression has been found in several breast cancer cell lines and in primary breast cancer, quantitative analysis of ctr expression in matched normal breast tissue and primary breast cancer has not been explored up to date. here we present the detection and quantitation of ctr gene expression in matched normal breast tissue and breast cancers, by using laser capture microdissection. materials and methods fourteen breast cancers and matched normal epithelium from the same patient were laser capture microdissected to obtain pure cell groups and to submit to quantitative analysis by using one step real time rt-pcr. results both normal breast epithelium and cancer cells expressed ctr. as compared with paired normal epithelium, decreased ctr expression was found in of cases breast cancer ( . %) while increased ctr expression in cases ( . %) and no change in cases ( . %). conclusion our investigation revealed that ctr expression was constantly expressed in normal breast tissue and breast cancers. to the best of our knowledge, this is the first report, which revealed that ctr rna expressed in normal breast epithelium. downregulation of ctr is a common phenomenon in the breast cancer, suggesting that it may have important roles on the development of breast cancer. effect of neoadjuvant chemotherapy on her status in patients with breast carcinoma s popovska, t betova medical university-pleven, pleven, bulgaria introduction neoadjuvant chemotherapy is the standard of care for patients with locally advanced breast cancer and is being evaluated in patients with earlier-stage operable disease. the effects of chemotherapy on biological markers such as an oncoprotein expression are not well known. the aim of the study was to determine whether her expression was modified by neoadjuvant chemotherapy. methods forty-nine patients with t - n - m breast cancer were treated in a prospective trial utilizing fec regimen. matched pairs of pre and post-chemo therapy breast tissue were evaluated immunohistochemically for her status using herceptest kit (dako). results her overexpression was observed in / patients ( %). the her status of ( . %) patients was unchanged after chemotherapy. modification was found in cases-decreasing in , increasing in and loss of expression in patient. her status was not changed significantly following primary chemotherapy. conclusions our results showed that, in most patients her status is stable parameter in patients with breast carcinoma after primary treatment with neoadjuvant chemotherapy. the microvessel density in female breast cancer among younger and older age groups introduction breast cancer remains one of the most important oncologic problems worldwide. it seems that breast cancer first detected by the age of - years follows more aggressive clinical course than tumors developing later. young females with breast cancer more frequently present with axillary lymph nodes metastases. expression of the estrogene receptors is less frequent, and the histological grade is usually higher among this group. the main purpose of our study was the evaluation, quantification, and comparison of the angiogenesis in breast cancer among young women, below the age of years and older patients, above years. material and methods tissue samples of breast cancer from patients aged years and less ( cases), and from patients older than ( cases) were studied. ten specimens of non-neoplastic breast tissue from the normal breast in the tumor vicinity (younger patients) served as a control. microvessels were visualized immunohistochemically with use of cd- antibody and counted within the areas of the highest density ("hot spots"). six separate high power fields (hpf), . mm each, were studied in each case. results and conclusion the average microvessels count per hpf in the breast cancer tissue in younger group was higher than in the older age group and non-neoplastic tissue. the differences were statistically significant. the increased density of proliferating microvessels seems to play a role in the aggressive clinical behavior. total frozen section diagnoses have been performed mainly on sentinel lymph node of the mammary gland.the aim of the study was to analyze our result, and estimate whether the telepathology is of practical use or not. material total cases including lymph nodes diagnosed using telepathology. results and conclusion we failed to find carcinoma metastasis in out of sentinel lymph nodes of breast. it was very minute metastasis, and was not contained in the transmitted pictures. our transmission speed is kb that takes about seconds for each picture. there exists a limitation about the transmittable pictures within a restricted time. improvement of transmission speed may solve this problem. in two cases, while telepathologically negative of sentinel node, we found metastasis in the other dissected nodes by permanent section examination. the method of sentinel node identification should be improved. there was a confounding of the specimens that occurred between two patients on the same day. it was a case that happened at the beginning of telepathology. since the surgeon and the technicians are far away from the pathologist, and communication is done by the telephone, it is important for each party to keep in close contact with each other. our result is fairly good considering the limitation of the telepathology. although some issues are still remaining, we conclude that telepathology is now of practical use. pigmented mammary paget disease in a year old man-a case report ar monsef esfahani hamedan university of medical sciences, hamedan, islamic republic of iran mammary paget disease results from intraductal mammary paget carcinoma that extends to the epidermis of the nipple and areola through a lactiferous duct or from invasive breast carcinoma that reaches the epidermis via direct extension from the dermis. pigmented mammary paget disease is a rare clinicopathologic variant of mammary paget disease. it may mimic malignant melanoma clinically and histophatologically. it seems that involvement of the dermo-epidermal junction by neoplastic cells of the mammary paget carcinoma seems to be a prerequisite for the development of the clinical pigmentation. we report a case of pigmented mammary paget disease. the patient was a -year-old male who complained of an erythematous, pigmented scaly plaque around his right nipple measuring x cm with mild fissuring of two year duration. the histopathology revealed pagetoid spread of large cells in the epidermis with pigmentation of basal cells and melanocytes. the ihc confirmed the mammary paget disease and excluded malignant melanoma and squamous cell carcinoma. frozen section evaluation of sentinel lymph nodes in breast cancer introduction the use of sentinel concept as the newest approach in surgical treatment of breast cancer, apart from tumorectomy, means the extirpation and analysis of one or more regional lymph nodes which receives lymph drainage from primary tumor (guardian lymph nodes -sentinel nodes). the presence or the absence of secondary deposit in them is considered the most important predictor of the positive secondary deposit status of the other axillary lymph nodes. depending on histological analysis of sentinel node, the operation is finished with or without the axillary lymph node dissection. materials and methods sentinel concept in surgical treatment of the breast cancer is applied only in the cases where the largest dimension of the tumor is not more than cm (pt ). marking of the sentinel node is conducted before the operation by a combined method, the usage of patent blau color and technetium m as a radio marker. sentinel lymph nodes identity, before sending to a pathohistological analysis, is checked by gamma detection probe. two groups of prospective tests (a and b) were formed with cases each. sentinel lymph nodes from group a in the first stage of the testing were treated by pathohistological method (one hematoxylin and eosin preparation) and then microscoped. the cases in which secondary deposit was not noted, were subjected to a serial cutting and immunohistochemical staining by the ema markers, specific for intraductal epithelium of a breast. sentinel nodes from group b were submitted fresh for frozen section investigation, where they were approached with a series of cuttings and immunohistochemical analysis by the ema markers. the nodes without secondary deposits were fixed in formalin, paraffin embedded and, with the application immunohistochemistry (ema), submitted to serial cutting (similar to the second phase in group a) as a precaution. results in the first stage paraffin samples (group a) were first examined by h&e method, and additionally with immunohistochemistry (using ema). with this additional examination secondary deposits were found in cases ( %). this can only prove already known fact about statistically significant higher sensitivity and specifity of immunohistochemistry compared to h&e. in the second stage (group b) new samples of sn, first investigated by frozen section investigation, were examined by immunohistochemichal method (using ema). in addition, these samples were serially cut and treated like paraffin samples by immunohistochemistry method. the results of these two analysis were identical. facts mentioned above show that sensitivity and specifity of the frozen section analysis using immunohistochemical method (ema) is % (high percentage is probably the consequence of a relatively small number of samples). discussion acquired data suggested that implementation of one of the used methods (serial frozen section analysis with immunohistochemistry) from the sentinel concept is an efficient histo-morphological method in the evaluation of sentinel lymph nodes that make valid intraoperative staging of breast carcinoma possible. the authors came to significant data in both groups of tests and determined sensitivity, particularity and the efficiency of the applied methods. conclusion application of the suggested method would spare a considerable number of patients with pt breast cancer of subsequent axillary lymph node dissection and thus reduce the number of postoperative complications and long term morbidity. introduction needle core biopsy (ncb) of palpable lesion and stereotactic biopsy (smb) of non-palpable mass are the major preoperative methods of breast tumor diagnosis. the aim of this study was to evaluate methods accuracy and to emphasize the main detectable prognostic factors. materials and methods core biopsies ( ncbs and smbs) were performed between january and december . in / cases, the correlation between biopsy results and subsequent surgical excision was available. results the lesion diameter was < cm (in % of the ncbs and % of the smbs). with smb, the average number of tissue core obtained per lesion was . at histology, in % of ncb and in % of smb diagnosis of malignancy was given. the dcis/idc ratio was / for ncb and / for smb, respectively. grade and type of the invasive and in situ carcinoma were determined in both biopsy and definitive excision, and the results compared. the agreement for overall grade was in % with smb and in % with ncb. at surgical excision, / dcis showed foci of idc. in % of smb cases no residual neoplastic tissue was found. conclusions the accuracy of both methods is very high. smb shows highest reliability and accuracy, because of the increased quantity of tissue obtained. the possibility to assess tumour size is limited. the preoperative assessment of the other prognostic factors (type and grade, tumour markers) are key determinants in planning the optimal breast lesions management. expression of mcp- mrna and macrophage infiltrates in invasive breast carcinoma introduction monocyte chemotactic protein- (mcp- ) has been detected in many human tumors; however, its chemoattractant role for macrophages in breast carcinomas is still questionable. the aim of this study was to determine the possible correlation between tumor-associated macrophages (tam) and the number of mcp- mrna in the invasive breast carcinoma. material and methods tissues obtained from surgical specimens of invasive breast carcinomas and cases of normal breast tissues were frozen in liquid nitrogen and kept at - o c until they were used for quantification of mcp- mrna by rt-pcr. the expression was calculated as a number of mcp- cdna copies per copies of g- -phd mrna. paraffin embedded sections from breast carcinomas were immunohistochemically processed for cd staining. positively stained tumor associated macrophages (tam) were counted on three "hot spots" (x ) and mean value was calculated. results the result of rt-pcr showed that in all cases of breast carcinomas ( / ) and the majority of normal breast tissues ( / ) the number of detected mcp- cdna copies was above the detection limit. furthermore, carcinomas showed higher levels of mcp- mrna ( . ± . ) then normal breast tissue ( . ± . ). the value of tam within a single carcinoma was very inconsistent ( . ± . ). statistical analysis did not find a significant correlation between mcp- expression and macrophage infiltrations (r= - , ) . conclusion mcp- is an important factor in the recruitment of macrophages. however, the results indicate that mcp- is probably not the only and/or crucial factor involved in macrophage attraction to tumor locus in breast carcinomas. comparison of mitotic index, growth fraction (evaluated by ki- ), er, pr, cathepsin d, p , and bcl- status between primary breast cancer and corresponding axillary metastatic sites introduction it has been postulated that the equilibrium of different clones within a tumor is eventually overcome by a biologically dominant one with enhanced metastatic potential. therefore, the metastatic tumor should be composed of more aggressive tumor cell clones than the primary one. we analyzed mitotic index, proliferation antigen ki- , estrogen receptors (er), progesterone receptors (pr), cathepsin d activity, protein p , and bcl- oncoprotein status in order to explore the assumed differences in the clonal composition between the primary breast cancer and axillary metastasis. materials and methods er, pr, p , bcl- , ki- , and cathepsin d statuses in patients with the primary ductal invasive carcinoma of the breast and at least one axillary metastatic lesion were analyzed by immunohistochemistry (ihc -dako lsab system). mitotic index was assessed by counting the number of mitoses in primary and metastatic lesions. statistical analysis included t-test for dependent samples, linear correlation analysis, and roc-curve based contingency table analysis. results we found higher mitotic index (p= . , r= . ), higher growth fraction (ki- , p= . , r= . ), and higher level of overexpression of bcl- (p= . , r= . ) in axillary metastatic lesion than in primary breast tumor. there were no significant differences in er (p= . , r= . ), pr (p= . , r= . ), cathepsin d (p= . , r=- . ), and p (p= . , r= . ) status. higher mitotic and ki- rates were associated with lower er and pr expression, whereas higher cathepsin d level was associated with lower bcl- expression, both in primary tumor and metastasis. between the paired primary tumor and axillary metastatic lesions, samples had concordant er, pr, p , and cathepsin d status when analyzed by ihc. tumor clone with enhanced metastatic potential might be characterized with higher mitotic index and overexpression of both ki- antigen and bcl- oncoprotein. basaloid breast carcinoma with pleomorphic lipoblastlike giant cells and neuroendocrine differentiation js reis- filho , liposarcomatous differentiation in breast neoplasms is a rather infrequent phenomenon and so far restricted to rare cases of malignant phyllodes tumour and true liposarcomas of the breast. the terms lipid-rich and lipid-secreting breast carcinomas encompass breast tumours in which neoplastic cells accumulate or secret lipid; however, lipoblasts are not usually seen. recently, we have observed a distinctive type of breast carcinoma in which pleomorphic lipoblast-like cells were seen admixed with the carcinomatous component. an -year-old female presented with a cm hard lump in the right breast with palpable nodes in the right axilla. mammography and ultrasound disclosed two irregular dense central masses in the right breast, measuring cm in aggregate, and an enlarged axillary node measuring approximately cm. a diagnosis of breast carcinoma was made and the patient received neoadjuvant tamoxifen despite the lack of estrogen receptor (er) in the primary tumour. four months later, her disease had progressed and she underwent right mastectomy with axillary dissection.. histologically, both the primary tumour and the / lymph node metastases were characterised by a high-grade carcinomatous component which imperceptibly merged with pleomorphic lipoblast-like cells. immunohistochemistry demonstrated epithelial differentiation in the carcinomatous and liposarcomatouslike components (epithelial membrane antigen, cytokeratins / , / , , and mnf ) . both components were also positive for vimentin, s- protein, and focally for chromogranin, gcdfp- , and e-cadherin. hormone receptors (estrogen, progesterone and androgen receptors), her /neu, p , smooth muscle actin, desmin, synaptophysin were negative. based on the histological and immunohistochemical profile, we believe that this case represents a breast carcinoma with basaloid phenotype and showing divergent differentiation (liposarcomatous and neuroendocrine), features that have to our knowledge not been documented to date. collagenous spherulosis arising in an adenomyoepithelioma of the breast: a hitherto unrecognised association js reis-filho , , lg fulforf , b crebassa , s charpentier , sr lakhani collagenous spherulosis (cs) is a benign epithelial and myoepithelial breast lesion characterised by the accumulation of basement membrane material in the form of eosinophilic or rarely basophilic spherules that exhibit concentric and radiating fibrillar patterns. cs is an incidental finding that usually occurs in association with benign proliferative and pre-invasive lesions. adenomyoepithelioma is a biphasic neoplasm of the breast, composed of both luminal epithelial and myoepithelial cells. despite the shared histogenesis, cs has never been described in association with adenomyoepithelioma. we report a case of a year-old female who presented with cs of the breast arising in an adenomyoepithelioma. the differential diagnosis included adenoid cystic carcinoma. immunohistochemistry with antibodies for cytokeratins (cks) , and / , smooth muscle actin (sma), calponin, smooth muscle myosin heavy chain (smmhc), p , collagen iv, oestrogen receptor (er), progesterone receptor (pgr), ki , and c-kit was performed. myoepithelial cells of the adenomyoepithelioma and the cells surrounding the collagen spherules of cs showed a similar profile (cks and / , sma, calponin, smmhc, p , and s reactivity), luminal cells of both lesions also showed overlapping immunophenotypes (ck , er and pgr positivity). collagen iv highlighted the spherules in cs. ki labelling indices were similar in both lesions. c-kit expression was restricted to the adenomyoepithelioma and no expression was observed in the cs. the association is important to recognise as it could potentially lead the unwary to a misdiagnosis of adenoid cystic carcinoma, cribriform carcinoma or cylindroma of the breast. introduction lymphoepithelioma of the nasopharynx is an undifferentiated carcinoma with a prominent lymphoid infiltrate. tumors, with histologic features similar to nasopharyngeal lymphoepithelioma in other locations are referred as lymphoepithelioma-like carcinoma.(lec). in differential diagnosis of this lesions, medullary carcinoma and lymphoma must be considered. in this study, we report a case of lec of the breast which is very rare tumor.. material and method a -year-old female was noted to have a lesion of the right breast on mammography. an excisional biopsy was performed and sections revealed a well-demarcated elastic mass of cm in diameter. routine histologic examinations and immunohistochemical analysis were performed and compared with reported cases in literature. results clinical features, macroscopic and microscopic findings, immunohistochemical analysis were consistent with lec of the breast. on immunostaining, the tumor cells showed strong staining for epithelial membrane antigen (ema) and low weight cytokeratin (lcw).lymphoid cells stained positively for leucocyte common antigen (lca).the tumor cells showed no staining for cd . staining for uchl - and l demonstrated that there were more t cells than b cells. the tumor cells were immunohistochemically negative for epstein-barr virus (ebv). introduction this retrospective study was performed to investigate the role of dna-ploidy and s-phase fraction in determining prognosis in advanced breast cancer. methods the study included female patients with breast cancer, investigated for the period - . the nuclear dna-content has been measured by cas- (beckton dickinson, image analysis) on intraoperative cytologic samples of the tumour (imprints). the tumours have been divided into diploid, tetraploid, aneuploid and hyperdiploid. results of all patients had aneuploid and multiploid histogram, whereas % of them had developed distant metastases. lymph metastases have been registered in % of the cases with diploid histogram. s-phase has been considerably higher in patients with aneuploid than those with diploid histogram. the survival of of all patients has been analyzed as to the type of their histogram and as to the presence of metastases in lymph nodes and distant organs. the results showed that breast cancer patients ( . %) with aneuploid histogram and metastases in lymph nodes had a survival of less than years, compared to the better survival rate of those patients with diploid histogram and no metastases in lymph nodes. the higher s-stage has correlated to the metastases in lymph nodes and distant locations. conclusion the analysis of all results proved that the dna-morphometry could successfully be used as an independent criterion for evaluation of the biologic behaviour of breast cancer tumours and their prognosis. hypercholesterolemia impairs angiogenesis in patients with breast carcinoma and therefore lowers the risk of metastasis bh introduction angiogenesis plays a key role in tumor progression and it is also necessary for the development of distant metastasis. previous experimental studies indicate that both diet-induced and genetic hypercholesterolemia (hch) impairs angiogenesis. our aim was to investigate the effect of hch on angiogenesis induced by breast carcinoma and to elucidate the possible mechanisms by which it inhibits the progression of the cancer. material and method fifty-one patients with invasive ductal carcinoma were included in the study. of cases had hch and had no hch. all patients were followed for a mean of months after radical mastectomy. the intratumoral microvessel density (mvd) was evaluated immunohistochemically using anti-cd antibody and correlated with clinicopathologic parameters. also the expression of bfgf and vegf were examined immunohistochemically and graded semiquantitatively. results mvd was significantly correlated with tumor grade, vegf and bfgf expression, recurrence, lymph node metastases (lnm), distant metastases (dm) and survival (p< . ). patients without hch had significantly higher mvd ( . . ) than those with hch ( ) (p< . ). in addition the risk of lnm and dm were found higher in cases without hch when compared to cases with hch (p< . ). both bfgf and vegf expression showed significant negative correlation with hch (p< . ). conclusion we suggest that, hch impairs angiogenesis by suppressing bfgf and vegf expression and therefore lowers the risk of metastases in cases with invasive breast carcinoma. the cd antibody (calla) is usually expressed in lymphoblastic acute leukemia and in follicular center cells of lymph nodes. it is also used in breast cancer cell culture to differentiate epithelial cells from myoepithelial cells. usually the smooth muscle actin (sma) is used to identify myoepithelial cells in breast lesions, but this antibody is also positive in blood vessels. this study compares the expression of cd and sma as a marker of myopepithelial cells in breast lesions. we have compared breast lesions, both benign and malignant ( adenosis, fibroadenomas, intracystic papillomas, hamartoma, gynecomastia, intraductal carcinomas, invasive ductal carcinomas, tubular carcinomas, mucinous carcinomas, invasive lobular carcinomas, medullary carcinomas, phyllodes tumors). the antibodies used have been cd ( c, : , dako) and sma ( a , : , biogenex) . results we observed cd expression in the cell membrane of myoepithelial cells and also in some stromal myofibroblastic cells. sma expression was observed in the cytoplasm of myoepithelial cells, in some stromal myofibroblastic cells and also in smooth muscle cells. proliferative breast lesions and benign tumors contain myoepithelial cells expressing cd and sma. in malignant lesions the myoepithelial cell component is lost. conclusions the cd is a myoepithelial cell marker and yields a good, reproducible staining in the studied cases. we think that the use of cd combined with sma is helpful in the differential diagnosis between benign and malignant lesions (epitheliosis vs intraductal carcinoma, tubular carcinoma vs atrophy, adenosis vs invasive ductal carcinoma, papilloma vs intracystic papillary carcinoma). introduction the identification of myoepithelial cells in breast lesions remains of diagnostic value for the discrimination between benign and malignant lesions. p is a p homologue, which is expressed in epithelial basal cells of different organs. the aim of the present study has been to investigate the immunohistochemical expression of p in breast lesions. materials and methods we examined immunohistochemically formalin-fixed, paraffin-embedded, archival breast samples, which consisted of benign lesions and malignancies ( in situ carcinomas, invasive ductal carcinomas, nst and invasive carcinomas special type). the monoclonal antibodies anti-p (biocare medical/menarini), anti-a-smooth muscle actin (biogenex) and anti-s- protein (biogenex) were used. results in all cases, p expression was nuclear. in all benign lesions, there was an almost continuous basal rim surrounding the epithelial structures, whereas it was present but discontinuous in the in situ carcinomas. all invasive ductal carcinomas, nst, were devoided of p staining. p immunoreactivity was noted in a minor fraction of neoplastic cells in / ( . %) in situ papillary carcinomas and in / ( . %) invasive papillary carcinomas. the stromal cells were negative to p . there were s- positive luminal epithelial cells in almost all cases, whereas many fibroblasts of the stroma were positive to smooth muscle actin. conclusion it appears that p is a more sensitive and specific myoepithelial marker than those currently used, with no staining of stromal myofibroblasts. the observed staining in scattered cells, only in papillary neoplasms, might be a clue to the speculated different histogenesis of this specific type of breast tumors. prediction of aggressive outcome in t n m breast cancer introduction small tumour size at the time of diagnosis is one of the most significant prognostic factor of favourable outcome in invasive breast cancer. however, unpredictable breast cancer recurrences and deaths also occur among the group of patients with t n m breast cancers. aims: we set out to identify the clinically useful prognostic features predicting unfavourable outcome in patients with t n m invasive breast cancer. the study comprises cases of invasive ductal or lobular carcinomas diagnosed and treated in turku university hospital and jyväskylä central hospital during the years - . twenty-seven patients had an aggressive disease with a recurrence or death of breast cancer. the prognostic factors of each of the patients with aggressive disease were compared with control patients matched by tumour size, age at diagnosis and histological type of tumour. the follow up time was - years. the clinicopathological prognostic factors of the patients were surveyed. results the prognostic features indicating unfavourable outcome were immunohistochemically detected high ki- expression, high p expression, low estrogen receptor expression and positive her /neu chromogen in-situ hybridization. the number of mitotic figures (mitotic activity index, mai), histological type of the tumour or number of studied axillary lymph nodes did not provide prognostic information. conclusion our study summarizes the immunohistochemical features and criteria predicting unfavourable outcome in t n m invasive breast cancer. detection of overexpression of c-erbb through immunohistochemical and amplification of her /neu established by fish in breast cancer. a comparative study introduction the prognostic and predictive implications of oncogene her /neu activation are very controversial, due to the extensive variation of results, mainly due to the different methods applied. activation of her in node positive patients as an independent prognostic factor has been established and correlated with tumour sensitivity to different therapeutic agents. the overexpression of this membrane receptor has converted it into a new therapeutic aim, requiring the application of a more suitable methodology for each case. objectives to evaluate the most suitable method (immunohistochemical vs. fish) to determine her activation and its correlation with other prognostic factors. material and methods a study of cases of invasive breast carcinoma. histological type: % infiltrating ductal carcinoma; age: - , hormonal receptors: . % (rh+), . % (rh-); histological grade (bloom-richardson mod): . % (g ), . % (g ), and . % (g ). immunohistochemistry was performed in patients with (herceptest dako), and with anti c-erbb (dako : ). scores were used for evaluation: absence of membrane staining ( ), faint and incomplete in > % of tumour cells ( +), weak to moderate in > % ( +), strong complete in > % ( +). fish study was performed on paraffin sections through path vision system with evaluation of nuclei. a good correlation between immunohistochemical overexpression and amplification by fish was observed, especially in cases evaluated as ( +) / ( %), and as ( ) / ( %). no amplification by fish occurred in . % of ( +) / and in . % of ( +) / . % of negative hormonal receptor tumours showed amplification, while only . % of positive receptor tumours amplified. none of the tumours classified as (g ) showed amplification. conclusions we emphasise the necessity to study gene amplification through fish in cases with weak immunohistochemical overexpression ( +). introduction cell cycle progression is governed by cell cycle regulators and inhibitors such as cyclin dependent kinases (cdks), p , p /waf and p /kip . the purpose of this study was to correlate the immunohistochemical expressions of p , p /waf , p /cip and p cdc , with its clinicopathologic prognostic parameters in human breast cancers. materials and methods paraffin-embedded tissue sections from patients with human breast carcinomas were examined for immunohistochemical staining. primary antibodies used in immunohistochemical staining were mouse monoclonal antibody to human p , p /waf , p /cip and p cdc . results the expression rates of p , p /waf and p cdc were . %, . % and . % in breast carcinomas, respectively. in normal breast tissues, p , p /waf and p cdc were not expressed. the expression rate of p /cip was . % in breast carcinomas and % in normal breast tissues. the correlation between the expression of p and p /waf was noted(p= . ). the expression of p /cip was correlated with that of progesteron receptor(pr) (p= . ). the expression of p /waf was correlated with that of estrogen receptor (er) (p= . ) and pr (p= . ). no correlation was demonstrated between survival in patients and expression of p , p /waf , p cdc and p /cip . conclusion the loss of normal cell growth cycle by abnormal cyclin dependent kinases, inhibitors and steroid hormones may play an important role in human breast carcinogenesis. the p dependent p pathway, p gene loss and cdc overexpression were important in development and progression of human breast cancer. withdrawn p- interobserver variability in determination of her protein overexpression by immunohistochemistry using herceptest assay - cases study material and method among cases evaluated in population screening project total cases from all centers were selected and presented to representatives of those centers. the immunohistochemically stained slides came from all laboratories. all ihcs were done using fda-approved dako herceptest. evaluation was performed without knowledge of the original results the results from each researcher were coded. the study lasted hours. the average concordance was nearly %, while the lowest concordance was and the highest - %. this wide range of the results might be due to several things. firstly, the experience in ihc evaluation varied among the participant of the study. secondly, the quality of the stains differed significantly. thirdly, the researchers worked under time pressure and the material was selected to include more cases considered 'difficult' ( + according to dako herceptest or technically inadequate). conclusions to achieve higher concordance among the obtained results, evaluation should be performed by the experienced pathologists. * -participants from: bialystok, bydgoszcz, gdansk, gliwice, kielce, krakow, lublin, lodz, olsztyn, poznan, szczecin, warsaw and wroclaw. the population screening project study was supported by roche-polska. a morphometric analysis of early invasive breast carcinoma using serial sectioning and d reconstruction technique introduction mechanism of early invasion of breast ductal carcinoma has not been completely investigated not only from the genetic point of view but also morphologically. one of the reason is that starting sites of invasion from ductal carcinoma in situ (dcis) might be extremely diminutive that routine histopathological examination should hardly reveal them usually. thus, we examined a large number of complete serial sections from breast carcinomas so as to find out destroyed sites of duct wall from which carcinoma cells have started on invasion. also we reconstructed d images from the sections so as to visualize the sites and to measure the size of those. materials and methods used were three surgical specimens of breast cancer having extensive intraductal components with multiple invasive foci. after formalin fixed and paraffin embedded, six blocks from three cases were selected and sectioned serially in µm thickness. total amount of the serial sections reached sheets (mean / block) indicating . µm in actual thickness. after he stained, invasive lesions connected with dcis were searched and the lesions were reconstructed as d images with a computer-assisted- d-reconstruction system. results although numerous sections containing multiple invasive foci were examined, areas which connected with dcis to invasive lesion through the destroyed site of duct wall were only three. the site were approximately µm, um and µm in diameter respectively, should be called as 'hole' of the duct wall. invasive lesions were extended along the ducts with the holes. conclusion the holes, starting sites of invasion, were apparently smaller than surrounded invasive lesion. histological and immunohistochemical profile of invasive pleomorphic lobular carcinoma y erhan introduction invasive pleomorphic lobular carcinoma (iplc), a distinct subtype of invasive lobular carcinoma (ilc), shares the typical histologic patterns of classical ilc, but the nuclei are more pleomorphic and increased mitotic activity is a prominent feature. we reviewed cases of iplc and compared them with cases of clssical ilc. immunohistochemical analysis was performed in all cases for estrogen (er) and progesterone receptors (pgr), p , c-erbb- , and ki . in this series, the average patient age was . years (age range, - years) for iplc and . years (age range - years) for ilc. tumor size ranged from to . cm (mean size . cm) for iplc and . to cm (mean size . cm) for ilc. axillary lymph node metastasis was detected in % of iplc, and % of ilc. fifty percent of iplc and % of ilc cases had extranodal extensions (p< . ). tumor cells were large with eccentric pleomorphic nuclei and eosinophilic granular cytoplasms. high mitotic activity was observed. immunohistochemically er and pgr positivity in iplc was % and %, and in ilc % and %, respectively. iplc cases showed %, and ilc cases % p positivity (p< . ). c-erbb- was detected in % of iplc, and % of ilc. ki mean rates were % and % for iplc and ilc. conclusion iplc exhibits distinct histologic fetures, higher expression of p protein and ki rate in comparison with classical ilc. the aggressive clinical course of patients with iplc is supported by higher rates of axillary lymph node metastasis and extranodal extensions. introduction c-kit is a proto-oncogene that codes for a transmembrane tyrosine kinase receptor: cd . once activated, c-kit propagates signalling events throughout the cell via multiple signal transduction pathways. it was described an activating mutation in exon of c-kit gene in gastrointestinal stromal tumours (gist), which can be blocked by using a tyrosine kinase inhibitor (sti ). phyllodes tumours (pt) are rare biphasic breast tumours that similarly to gists show a monoclonal stromal proliferation and high recurrence rates. aims: to study the immunohistochemistry expression and prevalence of activating ckit mutations in pt in order to explore whether this disease could be a potential target for treatment with sti . we analysed pt ( benign and malignant) by immunohistochemistry for c-kit expression and direct sequencing of exons , , and of c-kit gene for evaluate the mutational status. c-kit expression was detected in out cases ( / benign and / malignant). no mutations were found for exons , and . in exon we found the same silent alteration in out of cases. conclusions although c-kit expression is a frequent finding in pt of the breast, we did not find activate mutations similar to those described in gists. recently, the role of cyclin d expression in breast cancer had received attention. it causes the activation of cyclindependent kinases in g phase and phosphorilation of the retinoblastoma gene, required for entry of cell into s phase of the cell cycle. recent investigations confer the role of cyclin d once again in g phase. the aim of this study was to analyse the expression of cyclin d in human breast cancer cells and to compare its value with the estrogen receptors and histological parameters. material and methods immunohistochemical analysis for cyclin d and estrogen/progesteron receptors (er/pr) was performed on formalin-fixed infiltrating ductal breast carcinomas. scoring was done using semiquantitative system. the stainining score was ranged from - (called cyclin d -negative) and score larger or equal to (called cyclin d -positive). results according to the staining score % of the breast carcinomas were cyclin d positive, and those carcinomas were associated with higher nuclear and histological grade, while only % were er-positive. conclusion these reslults suggest that higher expression of the cyclin d is related with poor prognostic parameters. c-erb b overexpression in breast cancer with negative lymph nodes background it is known that c-erb b overexpression in advanced breast cancer represents a predictive factor of tumoral agressivity. the aim of this study was to assess the involvement of c-erb b and other prognostic factors expression (hormonal receptors, egfr, pcna) in breast cancer with negative lymph nodes. methods we investigated histopathologically and immunohistochemically selected cases with g ductal invasive carcinoma and no lymph nodes involvement, from breast cancers. we used a monoclonal antibody panel (estrogen and progesteron receptors, egfr, c-erb b , pcna) in formalin fixed paraffin embedded tissues. results we found a significant increased c-erbb overexpression associated with a low er and pgr expression and minimal microangiogenesis as expressed by egfr. the average of proliferative rate assessed by pcna was . %. conclusion immunohistochemical c-erbb overexpression in negative lymph nodes breast cancer, may suggest an early amplification of its encoding gene, which can predict an aggressive biological behaviour (invasion and metastasis), so that this type of patients would benefit of humanized anti-her /neu monoclonal antibody treatment. introduction aspiration cytology of the breast is one of the three components of the triple test for breast cancer diagnosis. ultrasound (us) guidance increases the complete sensitivity of this technique. the aim of this study was to compare the results between a group of mammary lesions aspirated with or without us using the european quality assurance guidance. material and method we analyzed mammary lesions by aspiration cytology with or without us guidance. one group was formed by breast aspirates from women older than fifty years old, included in an screening programme, with palpable and non palpable lesions, using us guidance. another group was formed by breast aspirates from outpatients origin of all ages only with palpable lesions using no us guidance. we analysed and compare the results between both groups using using the european quality assurance guidance for breast screening pathology. results in both groups the positive predictive value of the c results were % (false positive rate= %). the complete sensitivity of us guided aspirates were . % and for the non guided aspirates were %. we compare the rest of values and analyze our deviations from the standards. conclusion the quality of our breast aspiration practice is between the margins of the standards in both techniques, with an increment in the sensitivity with us guidance and a light loose of specificity in the screening group (probably because of patients age characteristics). ultrastructural study of intercellular junctions in metastatic and non-metastatic breast cancer introduction functional and morphologic characteristics of inter-cellular junctions are involved in tumor dissemination. the purpose of this study was to investigate whether the number of intercellular junctions in breast cancer is predictive for the presence of lymph node metastases. materials and methods infiltrating duct carcinomas were the subject of this study, of them without (g ) and with metastatic disease (g ) in axillary lymph nodes. the number of intercellular junctions was counted, and the length of the cell-to-cell contact surfaces was measured in randomly taken electron micrographs. the number of desmosomes/ µm of cell membrane apposition has been statistically compared between both groups. results cell junctions in tumors with lymph node metastases were usually smaller and poorly developed. there were no statistically significant differences in the number of intercellular junctions between both groups [g = %, ( , %; %). g = , % ( , %; %). p= , ]. conclusions although intercellular junctions are more rudimentary in tumors that metastasize, their number is not significantly reduced. therefore, it will be necessary to combine number, size and development of cell junctions in a single parameter to attempt prediction of tumor dissemination. supported in part by grant fis / . introduction numerous publications have been devoted to effects of myoepithelial cells on biology of breast cancer. the cells are thought to be able to inhibit invasion of neoplastic cells by production of inhibitors for serine proteases. some authors suggest that, acting in a paracrine way, myoepithelium can affect breast cancer cells. present study aimed at analysis of the relation between presence of myoepithelial cells (smooth muscle actin, sma) in the structure of a ductal breast cancer and expression of the recognized prognostic factors (er, pr, her- , tgf alpha, ki and p ) in the cancer cells. the studies were performed on samples of primary ductal breast cancers originating from females, including cases at g , cases at g and cases at g grade. immunocytochemical reactions were performed using antibodies directed to sma, er, pr, her- , tgf alpha, ki and p . the study showed that the highest content of myoepithelial cells was noted in tumours of g grade while the lowest content of the cells was observed in tumours of g grade (kruskal-wallis anova rank test, p= . ). in the entire group (g -g ) a positive correlation was detected between the presence of myoepithelial cells and intensity of her- expression (gamma correlation, p= . ) and a negative correlation with proliferation intensity measured by expression of ki antigen (gamma correlation, p= . ). no relation was observed between manifestation of myoepithelium and expression of the other studied variables. our studies confirmed the potential for paracrine communication between myoepithelial cells and cells of ductal breast cancer. immunohistochemical vegf expression in breast carcinomas. correlation with prognostic factors a matilla, l vicioso, l pérez, m Álvarez, i ramírez dpto. anat. patológica. universidad de málaga, malaga, spain introduction vegf (vascular endothelial growth factor) is considered as one of the most important angiogenic factors in breast cancer and its expression in tumor cells correlate with microvessel density. several studies have proposed that vegf may be a relevant prognostic factor. aim: to study the immunohistochemical expression of vegf in different histological types of breast cancer and its relation to established prognostic and predictive factors. we have analyzed carcinomas ( of invasive ductal type, lobular, mucinous, papillary and ductal in situ). immunohistochemical staining was performed on formalin fixed, paraffin sections, using an avidin-biotin immunoperoxidase technique. a rabbit polyclonal antibody for human vegf was used at a : dilution. immunoreactivity was scored from to +. results ( , %) of all invasive carcinomas showed vegf expression (ductal: %, mucinous: %). all but lobular carcinomas were negative or weakly positive. both papillary carcinomas showed strong positivity. staining in dcis was generally absent or weak. vegf positivity correlated inversely with nodal involvement (p= . ) and re status (p= . ). there were no correlation of vegf staining with tumor size, histological grade, p and c-erbb- . conclusion expression of vegf is characteristically weak or absent in lobular carcinomas and them no relevant as a prognostic factor in this type of carcinomas. as vegf staining correlate with absence of nodal involvement, its prognostic value may be mainly due to facilitate invasion of blood vessels, more than to lymphatic spread. introduction squamous carcinoma accounts for approximately % of all primary vulvar carcinomas. although the anatomic localization of this tumor allows early diagnosis and considerably easy surgical accessibility, the prognosis for these patients remains relatively grim. the explanation for this finding should be sought in the fact that most of the patients are diagnosed in the advanced clinical stage of the disease. since radical vulvectomy (a complex and debilitating procedure) is often a therapy of choice, the recent studies have been focused on determining reliable prognostic parameters that would allow individualized, and hopefully simpler therapeutic modalities to be applied in a certain group of patients with the same effectiveness. the aim of this study was to determine the most important clinical and histopathological parameters that influence the prognosis and outcome of patients with invasive squamous carcinoma of the vulva in order to offer them the most suitable form of therapy. moreover, it was designed to assess the clinical significance of dna content analysis by flow cytometry. patients and methods cases of squamous cell carcinoma of the vulva, diagnosed between . and . were selected. in order to assess the influence of the various prognostic factors on survival, the study included clinical and pathohistological parameters as well as the results of flow cytometric dna analysis of paraffinembedded tumor samples from all cases. clinicopathological parameters focused on the age of patients, clinical stage of the disease, menstrual status, diameter and localization of the tumor, histological grade, nuclear grade, combined histological and nuclear grade, depth of tumor invasion, tumor growth pattern, presence of giant cells in the tumor and the form of therapy, while the flow cytometric dna analysis comprised dna ploidy, proliferative activity and dna index. five-year survival of our selected population of patients was , +/- , %. the results of univariate statistical analysis confirmed that statistically significant prognostic parameters included the age of patients (p= , ), clinical stage of the disease (p= , ), histological grade (p= , ), nuclear grade (p< , ) and the depth of invasion (p< , ). on the other hand, the results of multivariate statistical analysis showed that only combined histological and nuclear grade (p= , ) and the depth of tumor invasion (p< , ) can be considered independent, statistically significant prognostic parameters. conclusion the age of patients at the time of diagnosis, clinical stage of the disease, histological grade and nuclear grade are considered to be the parameters of crucial relevance for the prognosis of patients with squamous cell carcinoma of the vulva. however, only combined histological and nuclear grade, as well as the depth of tumor invasion were proven to be independent statistically significant parameters relevant for the outcome of patients with this disease. can a pleomorphic vulvar leiomyoma be a tumor with local aggressive behavior? a case report vulvar leiomyoma is uncommon and its pleomorphic or bizarre form is especially rare. bizarre vaginal and vulvar (bartholin's gland area) leiomyomas were reported in only few cases. this tumor is hard to differentiate from leiomyosarcoma. the main criterion to make a distinction between them is the mitotic activity, which is less than per hpf in the former. case report a -year-old female was referred to the clinic for the evaluation of paravaginal tumor in distal vaginal third and right vulvar area with a history of month duration. she had no history of any earlier disease, and other examinations revealed normal findings. macropathology: the tumor was a large solid mass measuring x . cm in diameter. the tissue was yellow and seemed partly necrotic. micropathology tumor consisted mostly of spindle cells arranged in bundles with round or oval nuclei and large prominent nucleoli or showed hyperchromatism in places where there were cells with giant nuclei. cytoplasm was eosinophilic, elongated with small vacuole. there were areas where cells had two or more nuclei with the earlier mentioned polymorphism. areas of hyaline or myxoid degeneration of the stroma were found. mitoses were not found although the tumor was examined by three independent pathologists from different countries. the tumor margin looked infiltrative. the tumor was immunohistochemically positive for vimentin, smooth actin and desmin. cytokeratin mnf , ema, s- , cd- , and cd- were negative. conclusion we describe a vulvar smooth muscle tumor with marked polymorphism, without mitoses, with myxoid changes in the stroma and with infiltrative margins. because of all these characteristics the tumor was considered biologically aggressive and additional surgery with resection of the tumor site and regional lymphadenectomy was performed. three years after the last surgery the patient is alive and well. trophoblast apoptosis and proliferation in preeclamptic placentas d staribratova , z zaprianov , d dikov , v sarafian plovdiv medical university, plovdiv, bulgaria hospital center lagny introduction preeclampsia is a specific disorder of pregnancy with an increased risk for both neonatal and maternal mortality rates. it is associated with shallow invasion of the placental cytotrophoblast within uterus developing decidual vasculopathy and results in dereased blood flow. the aim of the present report is to determine whether hypoxia influences the rates of apoptosis and proliferation and to compare them with those of normal controls. materials and methods samples were taken from liveborn, singleton preeclamptic pregnancies and normal controls. we used routine light microscopy, immunohistochemical and quantitative studies for bcl- , and bax (to assess apoptosis) and pcna (to assess cellular proliferation). bcl- -and pcna-positive trophoblast cells were counted in a minimum of high-powered fields, of consecutive sections free from any infarction of at least paraffin blocks. results while increased apoptosis has been estimated in study groups, the rates of proliferation seems to be unaffected. placental pathologic features of preeclampsia d staribratova , z zaprianov plovdiv medical university, plovdiv, bulgaria introduction the clinical and placental features of preeclampsia are subject of an extensive study. clinical evidence suggests that this pregnancy specific disorder may be demographically heterogenous. our purpose is to record the range of the most common pathologic features in placental specimens from a gypsy cohort and to compare them with those in nonethnic bulgarian population. review of preeclamptic placentas from liveborn singleton deliveries of gypsy mothers and matching controls from bulgarian population revealed histologic lesions that were considered as belonging to one of the following groups: ( ) vascular lesions; ( ) acute or chronic inflammation and ( ) coagulation-related lesions. for each separate case, additional demographic and reproductive history was collected from the patients' charts. all placentas were studied by previously published standard protocols. nonparametric statistical analysis was applied. results in gypsy preeclamptic group, lesions related to coagulation ( % vs %) and vascular lesions ( % vs %) were more frequent. inflammatory features were equally distributed among the two groups ( %). introduction versican is a chondroitin sulfate proteoglycan, which belongs to the aggrecan gene family. it has not been studied in epithelial ovarian cancer before. the aim of this study was to clarify the relation between versican and hyaluronan in epithelial ovarian cancer and to find out if versican expression affects prognosis in these patients. materials and methods localization of versican was studied immunohistochemically in primary epithelial ovarian cancers and their metastases, as well as in normal ovary specimens. the staining was scored according to the percentage area of strong versican signal of total peri-and intratumoural stroma as low or high. epithelial staining of the tumours was scored as positive or negative. results low and high levels of strong stromal versican staining were observed in and carcinomas, respectively. a low level of strong stromal versican was found to be a significant marker for early figo stage and mucinous histology. stromal versican staining correlated significantly with stromal hyaluronan staining. cancer cell-associated versican positivity was seen in tumours and correlated with clear cell histology and early figo stage. the -year prognosis of patients decreased with increasing stromal versican levels for overall survival. the -year prognosis for recurrence-free survival was slightly better when cancer cells were positive for versican. in cox's multivariate analysis neither stromal nor cancer cell-associated versican had any prognostic value. conclusion enhanced stromal versican expression correlates with hyaluronan accumulation and is associated with unfavourable disease outcome, but it is not an independent prognostic marker in epithelial ovarian cancer. cytobrush-collected malignant cervical epithelial cells display a cancer-specific gene expression pattern in cdna array analysis g hudelist , introduction although it is well known that persistent high-risk hpv infections are the principal causative agents of cervical cancer, the discrepancy between high infection rates and the relatively low rates of high-risk hpv positive women who actually develop carcinoma of the cervix suggest that development of cervical cancer is a multistep process involving additional factors and mutational events. therefore, differentially expressed genes in normal and malignant cervical epithelial cells and the feasibility of gene expression profiling on cervical cells obtained by regular cervical swabs were investigated. material and methods cervical epithelial cells obtained by regular cytobrushes from women with high-risk hpv positive invasive cervical cancer and from hpv negative women without suspicious pap smears were pooled and subjected to cdna array analysis. dot blotting was then performed to detect the proteins which corresponded to the most highly up-regulated genes in the cdna array analysis. results mrna expression of c-erbb- , c-kit, vegfr- , n-myc, k-ras, cyclin-dependent kinase inhibitor (p ink a), cyclin d , nm -h , nm -h , c-met, kgf, kgfr, and stat was increased in malignant samples. several genes associated with antiapoptosis, such as bcl- like protein, cell structuring and/or cell attachment were also upregulated in tumoral cells. conversely, decreased gene expression was observed for members of the transforming growth factor receptor superfamily (tgf-beta), interleukin (il- ), insulin-like growth factor binding proteins (igfbps) and for members of the integrin family. conclusions we identified a mrna and protein expression pattern that might have an important role in the pathogenesis of invasive cervical carcinoma and appears to be specific for malignant cervical epithelium. we hypothesize that the presence of a cancer-specific gene-expression signature assessed by cdna array analysis or immunohistochemical techniques could eventually turn out to become an additional tool to evaluate the dignity of routinely obtained cervical swabs. prognostic features and molecular markers in ovarian borderline tumors e nistor , ad stanescu , cd vrabie , e ionica bucur hospital, bucharest, romania v. babes institute, bucharest, romania university of bucharest, bucharest, romania introduction ovarian cancer represent the fourth most common cancer in women. borderline ovarian serous tumors (bsot) affect women of childbearig age and their prognosis is outstanding. the aims of this study were to characterize the group of patients with bsot and evaluate the significance of various molecular markers expression versus serous papillary ovarian carcinomas (spoc) material and methods we analyzed a total of cases including: cystadenoma, borderline and cystadenocarcinoma. for the bsot the median age was years. eight women with bsot underwent radical surgery and two fertility sparing surgery. the recurrence of disease appeared in one case with fertility sparing surgery. all the cases were analyzed on haematoxilin-eosin stained and immunohistochemistry (the indirect tristadial streptavidin-biotin method) for estrogen receptor (er), progesteron receptor (pr), p protein (p ), proliferative activity (ki ) and carcinoembrionic antigen (cea). results the immunostaining demonstrated a significant increase in the expression of er ( %) and a decrease for p ( %) and ki ( %) in bsot vs spoc (er- %, p - % and ki - %). conclusions the main differences between bsot and spoc was regarding the expression of p and ki . there was no significant correlation between ki and the known histologic parameters of prognostic significance. transvaginal ultrasound and ca serum levels are the most effective diagnostic techniques for the follow up of patients treated conservatively for bsot. introduction gonadoblastoma with dysgerminoma or other germ cell tumor occurs almost exclusively in girls and young women with underlying gonadal disorder. the aim of this study was to review biopsy examinations of the tumours, stage, treatment strategy, follow up and outcome of five girls with gonadoblastoma and germ cell tumours. material and methods retrospective review of patients with gonadoblastoma and germ cell tumours was performed. the patients were , , , and years old. the first -year-old girl (karyotype, xx, normal ovary bilateral) with gonadoblastoma, dysgerminoma and choriocarcinoma had iiic stage disease. she was treated by chemotherapy with cisplatinum and radiotherapy. four patients had gonadoblastoma and dysgerminoma. the second -year-old girl (mixed gonadal dysgenesis, karyotype, , xy) had stage ib and was treated by chemotherapy with cisplatinum. two patients were treated with surgery only: year-old girl (streak gonads, karyotype, , xy) -stage ib and year-old girl (karyotype , xx, der / , xx, - ) -stage ia. the -year-old patient (karyotype, , xx, normal ovary bilateral) -stage ia disease, had adnexotomy only, but because of recurrence (stage iiic) months after the original diagnosis, she was treated by chemotherapy. results our review confirmed the original diagnosis of gonadoblastoma and dysgerminoma and in one case gonadoblastoma, dysgerminoma and choriocarcinoma. all patients were followed up for to months. only one patient, who was initially treated with surgery only (stage ia), had disease recurrence, but achieved durable remission with chemotherapy and further surgery. the latest examinations revealed that all patients were in good health. conclusions because the gonads are often replaced by a tumour, a diagnosis of the underlying abnormality may be impossible. gonadoblastoma is regarded as in situ malignancy, and there is a high possibility for development of invasive germ cell tumour such as dysgerminoma. young girls with unilateral encapsulated gonadoblastoma and dysgerminoma can be treated only by unilateral oophorectomy or salpingo-oophorectomy and subsequent careful follow up to prevent damage of contralateral ovary and preserve the fertility. the patients with gonadoblastoma and mixed germ cell tumor are treated in accordance with other tumour structures. the paper is sponsored by ministry of health of the czech republic (international grant agency) no - , cz introduction cadherins and their associated cytoplasmic protein catenins are cell adhesion glycoproteins that are altered in tumor progression the aim of this study is to reveal the altered expression of e-cadherin, p-cadherin, α-catenin and β-catenin in cervical squamous cell carcinoma progression from dysplastic lesions (low grade squamous intraepithelial lesion-l-sil, high grade squamous intraepithelial lesion-h-sil) to invasive squamous cell carcinoma. material and method immunohistochemical method was used in formalin-fixed, paraffin embedded representative tissue sections of l-sil (cin i), h-sil (cin ii-iii) and invasive squamous cell carcinomas. the presence of immunostaining and the pattern of distribution of e-cadherin, p-cadherin, α-catenin and β-catenin were analyzed. results e-cadherin was detected in . %, . %, . % of l-sil, h-sil and invasive squamous cell carcinomas respectively. the difference was statistically significant (p= . ). the expression of p-cadherin and α-catenin was reduced significantly from l-sil through invasive carcinoma (p= . for p-cadherin, p= . for α-catenin). β-catenin expression was revealed in all lesions with nearly the same frequency, . % l-sil, . % h-sil and . % invasive carcinomas. conclusion loss of adhesion proteins, e-cadherin, p-cadherin and α-catenin is observed in cervical squamous cell carcinoma progression. the presence of β-catenin in h-sil and invasive carcinomas might suggest its role in tumor progression by its effect in wnt signaling pathway in cervical carcinomas. introduction p, e-cadherin and their cytoplasmic domains alpha and beta-catenins are epithelial cell adhesion complexes. their alteration is important in tumor progression. beta catenin is also involved in wnt signaling cascade which regulates cell proliferation. we investigated the altered pattern of cadherin/catenin complex in cervical squamous cell carcinomas. we also tried to find out if there was a correlation between adhesion proteins and clinicopathologic variables (stage, depth of invasion, lymph node metastases) and hpv status. material and methods e-cadherin, p-cadherin, alpha catenin and beta catenin expression in squamous cell carcinomas were investigated by immunohistochemical method. the presence of hpv / infection was detected by in situ hybridization. results there were large cell keratinizing, nonkeratinizing and small cell subtypes of squamous cell carcinoma. lymph node metastases were found in carcinomas. hpv / infection was detected in of carcinomas, all were large cell keratinizing except one. no correlation was found between the hpv status and cadherin and catenin expression of the tumors. e-cadherin was negative in carcinomas, p-cadherin was positive in only carcinomas. strong cytoplasmic beta catenin positivity was revealed in carcinomas. alpha catenin reactivity was seen in of carcinomas. neither cadherins nor catenins expressions were correlated with other clinicopathologic variables. conclusion our results reveal that e-cadherin and p-cadherin expressions are lost in squamous cell carcinomas. however alpha and beta catenin immunopositivity in cervical squamous cell carcinomas might suggest catenins' role in tumor progression in a different way other than their activity in cell adhesion. prognostic significance of karyotype analysis in ovarian carcinomas e introduction traditional prognostic factors of ovarian carcinoma (oc) include disease stage, age, histologic grade and residual disease after surgery. at present it is quite difficult to predict the survival benefit of platinum analogues based chemotherapy. the aim of this study was to evaluate the clinicopathological / cytogenetic profile in a series of ocs and the reliability of karyotype in predicting benefit of chemotherapy and prognosis. materials and methods a series of ocs ( serous, undifferentiated, endometrioid, mucinous and one mixed carcinomas) was investigated for grade ( / : % vs / : %), stage (i/ii: % vs iii/iv: %), residual disease after surgery ( %), chemotherapy ( %), remission ( %) vs progression ( %) after chemotherapy and follow-up (mean: , range: - months). cytogenetics were performed on direct preparations using the qfq banding technique and describing abnormalities according to iscn. karyotypes were classified as diploid/near-diploid ( %) and complex (hyperdiploid, multiple cell lines, fragmented chromosomes) ( %). statistical analysis using chi-square and fisher test was done. statistical analysis demonstrated that low stage (p= . ) and low grade (p= . ) were predictors of good prognosis, while residual disease correlated with a poor outcome (p= . ). diploid/near-diploid karyotype correlated with a favourable prognosis (chi-square: . ). complex karyotypes appeared much more frequently associated with chemoresistance ( % vs %). conclusion our results confirm the correlation between grade, stage, residual disease and prognosis of ocs. in addition, these data suggest that a diploid/near-diploid karyotype might predict benefit of chemotherapy and favourable prognosis. introduction the aim of this study was to critically assess the progression-and recurrence rates of microinvasive (mic) stage ia cervical carcinoma, based on medline search, and to compare these data with our own experience on these tumours in ljubljana medical centre. in the published studies covering the years - , a total of . squamous cell mics were reported, including . stage ia lesions, . in stage ia , and cases of occult stage ib, classified according to figo or sgo classifications. our experience in ljubljana is based on the management of stage ia squamous cell carcinomas (scc), accumulated in the past years and early invasive adenocarcinomas (ac) collected in years. results in the reported . scc mic cases, lymphatic space involvement was recorded in ( %) cases, and in of those, the disease recurred, and progressed to death in patients. in series, mics were associated with ( . %) recurrences. in seven series, ( . %) cases of death due to cancer were reported. lymph node metastases were found in ( . %) cases. even when stage ia and ia carcinomas were treated with conservative surgical approach, very low risk of recurrence ( . %), lymph node disease ( . %), or death caused by cancer ( . %) was reported by these studies. most authors conclude that mic with the depth of invasion < mm, without confluent growth or lymphatic invasion can be safely treated with conization. during - lymphadenectomy was included in operations of mic in ljubljana. the pelvic lns were invariably free of cancer. the -year survival was %, while patients were lost to follow-up. in medline search . cases of stage ia acs were analysed in a -year period. lymph node metastasis were found in ( . %) of cases with lymphadenectomy, complicated with recurrent disease in ( . %) cases compared to the scc mic, the risk of recurrence in ac was considerably higher, . % and progression to death . % ( cases analysed by Östör, ). in our ac patients diagnosed in stage ia there was no recurrence or death of disease. conclusions according to the world literature and our own experience, the prognosis of mic of both squamous and glandular type is very favourable. this suggests that conservative management of stage ia cervical cancer is indicated and safe when strict evaluation of tumour extension and surgical margins of the cone are assured. agnor counts, histological and immunohistochemical characteristics of placenta in preterm delivery with live and dead newborns g burkadze, g turashvili, n meckhvarishvili tbilisi state medical university, tbilisi, georgia introduction the pathology of placenta constitutes one of the most poorly understood domains of general pathology. the significance of histological characteristics of preterm delivery is disputable. the aim of this study was to investigate histological and immunohistochemical characteristics and agnor counts of placenta in preterm delivery with alive and dead newborns. we studied placental tissues in preterm deliveries of live (first group) and dead (second group) newborns (n obstetrics clinic of tsmu, tsmu, - . paraffin sections were stained using h&e, agnor (bio-optica) and immunohistochemistry by ki- , cd , cd , bcl- (dako). results in the first group the number and area of chorionic villi was times lower, the number and area of villous vessels . times higher, the number of villous lymphocytes is . times higher, neutrophils . times higher, hofbauer cells . times lower, syncytiotrophoblasts . times lower (their area was the same), syncytial knots times lower (their area is times higher). the width of nitabuch layer was times greater, decidual tissue . times greater, the number of decidual cells was . times higher, decidual lymphocytes . times higher, neutrophils . times higher, macrophages . times lower, agnor(+) syncytiotrophoblastic cells . times lower, agnor granules did not differ, agnor(+) decidual cells times higher, agnor granules . times higher, ki- (+) syncytiotrophoblasts and cytotrophoblasts times higher. conclusion preterm delivery shows incoordination between proliferation and involution of placental tissue. in death of newborns, morphological changes reflect non-efficacy of adaptative-compensative reactions of chorion-decidual system. institute of pathology, charité university hospital, berlin, germany clontech laboratories inc., paolo alto, california, usa introduction the ubiquitously expressed actin-binding protein gelsolin is known to play a role in modulation of the actin network and regulation of cell motility and cell growth. decreased gelsolin expression occurs in transformed cells and in carcinomas of the colon, bladder, breast, lung and prostate. in the present study we analyzed expression of gelsolin in matched human normal and tumor tissues using a cancer profiling array. we found a decreased expression of gelsolin in cancer tissue of female reproductive organs including ovary. further we examined the expression of gelsolin in human benign and malignant ovarian tissues by immunohistochemistry, observing a diminished expression of gelsolin in borderline tumors and ovarian serous carcinomas compared to epithelium of normal ovaries and benign adenomas. in addition, low levels of gelsolin protein were observed in four of six ovarian carcinoma cell lines in contrast to immortalized ovarian surface epithelial cells. re-expression of gelsolin in the ovarian carcinoma cell lines oaw and es- resulted in a suppression of tumor cell survival in vitro. finally, we showed an up-regulation of gelsolin in ovarian cancer cell lines after inhibition of both dna methylation and histone deacetylation using -aza- 'deoxycytidine and trichostatin a, respectively. conclusion our results suggest that the gelsolin gene has a potential function as a class ii tumor suppressor gene in human ovarian epithelium. steroid cell tumor of the ovary: presentation of two cases i lekka, o tzaida, v leodara, a lountou, p arapantoni-dadioti, i iakovidou pathology department, metaxas cancer hospital of pireaus, piraus, greece two cases of steroid tumor of the ovary, diagnosed at our department during the last five years among a total of ovarian tumors, are presented. steroid cell tumors, previously classified as lipid or lipoid cell tumors are rare neoplasms of sex-cord-stromal origin derived in almost all cases from ovarian stromal lutein cells or from leydig cells and rarely from adrenal rest cells. they have been subclassified as steroid cell tumors not otherwise specified (nos), stromal luteomas and leydig cell tumors. in our report we present two cases of the nos subtype: one occurred in a -yearold woman and the other in a -year-old postmenopausal woman. both patients presented with a pelvic mass and signs of virilization (resulting effect of elevated serum testosterone). both tumors were composed of characteristic eosinophilic-granular or vacuolated (lipid rich) neoplastic cells lacking crystals of reinke and expressing the appropriate immunohistochemical phenotype. based on thorough pathological examination and evaluation of several findings such as tumor size, necrosis, haemorrhage, cellular atypia, mitotic activity, we classified the first case as a malignant and the second as a benign one. various aspects of the clinical and pathological presentation, differential diagnosis and treatment of these tumors are discussed. choriocarcinoma following term pregnancy uncommonly choriocarcinoma follows a normal pregnancy. the aim of this study was to stress the importance of a correct and rapid diagnosis which will allow a high rate of success in therapy for this neoplasm. material and methods histopathologic and immunohistochemical studies were performed to an endometrial curettage and a hysterectomy specimen. case report a -year-old female presented with uterine bleeding three months after a normal delivery from a term gestation in which placenta was not submitted for study. endometrial curettage was performed, and it showed a dimorphic population of atypical cytotrophoblast and syncytiotrophoblast cells. cytotrophoblast cells had pale cytoplasm with single nucleus, eosinophilic nucleoli and prominent mitotic figures. syncytiotrophoblast appeared as large multinucleated cells with polygonal basophilic cytoplasm. extensive necrotic and hemorrhagic areas were found. there was no presence of placental villi. neoplastic cells expressed strongly ß-hcg and cytokeratin. with a diagnosis of choriocarcinoma, hysterectomy was performed and showed areas of normal proliferative endometrium, and hemorrhagic areas with nests of trophoblastic neoplastic cells infiltrating among myometrial muscle. there was no involvement of cervix or adnexa. treatment was completed with chemotherapy, and one year after diagnosis the patient has not evidence of disease. conclusions the modern treatment with chemotherapeutic drugs is highly effective in complete cure of choriocarcinoma. therefore a quick histological diagnosis has a very important role to reduce the risk of metastasis disease, even avoiding surgery to preserve reproductive function. the mechanism of action and the histological features of uterine leiomyomas (ul) treated with gnrh are still a matter of debate. recent reports suggest that neovascularization induced by specific angiogenic molecules may play a role in the response to gnrh. also a reduced proliferative activity was observed and postulated as a possible histopathological feature of ul treated with gnrh. the aim of this study was to assess the possible role of angiogenesis and to quantify the percentage of proliferative cells in uterine leiomyomas in response to treatment with gnrh from premenopausic patients. material and methods a total of patients were studied, with two groups comprising samples each, including patients treated with gnrh and age-matched control patients. clinical (age, length of treatment, assessment of response to treatment by ultrasonography) and histopathological dates (microvessel density (vessels/mm ) and cellular proliferation assesed by ki- stain in cells were studied. the study group showed cd = , vessels/mm (mean) and ki- = , (mean). the control group showed cd = , vessels/mm (mean) and ki- = , . conclusion the leiomyomas treated with gnrh showed a) high number of microvessel density, b) low proliferative activity and c) significant reduction in size compared with patients without treatment. introduction evaluations of expression of apoptosis related oncogenes are being increasingly called upon in an attempt to better understand the carcinogenesis of cervical carcinoma and to provide possible prognostic information. the aim of this study was to analyze the expression of bcl- , p and mdm- oncoproteins and cellular proliferative marker ki- in early stage cervical carcinoma, with an emphasis on their association with human papillomavirus (hpv) infection, recurrence rate and lymph node status. material and methods using immunohistochemistry, radical hysterectomy specimens with cervical carcinoma (pt b /pt b ) were studied. evaluation of expression of p , bcl- , mdm- and ki- was performed in surface area, center and invasion front of the neoplasms. the hpv presence was determined by card in situ hybridization. results and conclusion in the invasion front bcl- was expressed in ( %), p in ( %) and mdm- in ( %) cases. hpv infection was detected in ( %) cases. carcinomas with a higher ki- labeling index were more frequently hpv positive than hpv negative ( . % vs . %, p< . ). no association was found between p , mdm- or ki- and either lymph node status or recurrence rate. negative staining for bcl- was associated only with the presence of lymph node metastasis ( % vs %, p= . ), and not with the recurrence rate. significant correlation among expression of bcl- , p , mdm- oncoprotein and ki- values was also observed. these results suggest that further study of a larger series is needed to confirm whether bcl- , either alone or in combined evaluation with other markers, could be a useful marker to identify more aggressive behavior in early stage cervical carcinoma. introduction during the past three decades, the incidence of cervical invasive primary adenocarcinoma (aca) has increased. at the same time, incidence of squamous cell carcinomas of the cervix decreased, due to early detection of their well documented precursors lesions. the aim of this study was to determine if dysplastic endocervical cells (ec) have neoplastic potential as a precursor lesions of adenocarcinoma in situ (ais) of the cervix. the malignant potential was determined by assessing the ploidy status, proliferative activity and c-myc expression in normal, dysplastic and malignant ec separately. patients and methods serial sections from patients with diagnosed ais or primary invasive aca were analyzed. one representative slide of cervix which showed normal, dysplastic and malignant endocervical glands simultaneously was chosen from each patient. determination of ploidy status and s-phase by flow cytometry and expression of c-myc oncoprotein by immunohistochemistry in normal, dysplastic and malignant ec was done. results the morphologicaly normal ec were diploid, with normal proliferative activity and no expression of c-myc oncoprotein. the dysplastic and malignant ec had a significant proportion of cells with aneuploidy, higher proliferative activity and c-myc oncogene overexpression. the χ test showed significant association of malignant ec with aneuploidy (p= . ) and high proliferative activity (p= . ). as one third of dysplastic ec are aneuploid, with high mitotic activity, they probably have malignant potential. dysplastic endocervical cells had statistically significant association with c-myc oncogene expression (p= . ). conclusion our results support the existence of pre-malignant glandular lesions while the immunohistochemical detection of cmyc oncoproteine could be helpful in detection of ec with malignant potential even without dysplastic morphologic changes. introduction the attempts to determine the prognostic significance of c-erbb- oncoprotein expression (ope) and its relation to human papillomavirus (hpv) infection in cervical cancer have yielded controversial. the aim of this study was to evaluate the patterns of c-erbb- ope in early stage cervical carcinoma and to assess its prognostic value by exploring its relationships to various clinicopathological characteristics, hpv status and recurrence rate. materials and methods radical hysterectomy specimens from cervical carcinoma patients (pt b / b ) were investigated immunohistochemically for c-erbb- presence. the c-erbb- score (range: - ) was determined in the surface area, center and invasion front of each carcinoma. card in situ hybridization was used for hpv detection. results and conclusion strong c-erbb- ope was detected in , , and cases in the invasion front, center and surface area of the tumor, respectively. there was a significant difference in positive staining rate of c-erbb- between squamous cell, mixed carcinomas and adenocarcinomas ( %, % vs %, p= . ). c-erbb- ope was significantly higher in carcinomas with abundant than in tumors with less abundant peri-tumoral lymphocytic infiltration ( . % vs . %, p= . ). in hpv positive carcinomas ( ), c-erbb- was detected more frequently in type / versus type / lesions ( % vs . %, p= . ). no association was found between c-erbb- expression and recurrence rate, lymph node metastasis or any other clinicopathological variable investigated (age, tumor diameter, depth of invasion, grade, vascular invasion). therefore, immunostaining for c-erbb- is unlikely to be of use as a prognostic indicator in early stage cervical carcinomas, while further study is warranted to examine relationships between hpv infection and c-erbb- ope. the predictive values of estrogen (er) and progesterone receptor (pgr) status and cell proliferation kinetics in cervical carcinomas are still unsettled. the purpose of this study was to clarify the associations among er/pgr status and ki- expression and to determine their relationship to human papillomavirus (hpv) infection, recurrence rate and other clinicopathologic parameters (age, tumor diameter, depth of invasion, histotype, grade, vascular involvement, inflammatory infiltrate, lymph node status) in early stage cervical carcinomas. materials and methods er, pgr and ki- immunostaining was performed in cervical carcinoma radical hysterectomy specimens (pt b /pt b ). er/pgr staining was scored in a semiquantitative fashion, while to evaluate the cell proliferation, the ki- labelling index (li) was assessed in the surface area, center and invasion front of each tumor. hpv status was determined by card in situ hybridization. results and conclusion er positivity was detected in ( %), while pgr positivity in ( %) carcinomas. er/pgr values were in correlation with ki- li in all three tumors' compartments (p< . ). in contrast to er/pgr status, ki- li was strongly associated with hpv infection (p< . ). no relationship was found between pgr or ki- immunoreactivity and either recurrence rate or any other clinicopathological variable investigated. nevertheless, reduced er expression was significantly associated with larger tumor diameter (p= . ) and poor differentiation (p= . ), as well as lymphovascular involvement (p= . ) and lymph node metastases (p= . ). these results suggest that er, pgr and ki- expression are closely related to neoplastic cell proliferation, probably induced by hpv infection. their determination may provide additional prognostic information in early stage cervical carcinomas. immunohistochemical assessment of heat shock protein expression in epithelial ovarian carcinomas: relationship with apoptotic markers as over-expression of bcl- and p purpose heat shock proteins (hsps) occupy a central role in the regulation of intracellular homeostasis and especially hsp and hsp are potent antiapoptotic proteins. overexpression of hsp protects cells from stress-induced apoptosis. the aim of this study was to determine the distrubition pattern of hsp -expression in ovarian carcinomas by immunohistochemistry and to evaluate its relationship with the prognostic parameters and over-expression of bcl- and p proteins. materials and methods thirty-five patients with ovarian carcinoma were included in the present study. expression of hsp was assessed by immunohistochemistry. the relationship between expression of these proteins and other prognostic markers such as stage was evaluated. results hsp was expressed in all of ovarian cancer cells with different intensity. the staining was localized in the cytoplasm and/or nuclei. there was a negative correlation between stage and both intensity of hsp positivity and bcl- positivity (respectively: r= - , ; p< . ; r= - . , p< . ). intensity of hsp positivity was statistically associated with bcl- positivity (r= . , p< . ). there were no significant associations between p positivity and bcl- positivity, intensity of hsp or stage of ovarian carcinoma. conclusions these data show that the intensity of hsp positivity was significantly stronger in early stage than in advanced stage ovarian carcinomas. hsp expression detected by immunohistochemistry was correlated with bcl- expression but not with p protein expression. background avb integrin and pinopodes have been proposed as new markers of endometrial receptivity. there is also a very limited experience on the responses of these markers to hormonal therapies. the aim of this study is to evaluate the effect different hormone replacement treatment on these markers. design we studied fertile controls and women with ovarian failure receiving hormone replacement therapy (estradiol valerate (ev) and vaginal progesterone (vp), group e+ep). those women were divided randomly into two subgroups of patients. group e+p received ev for days and vp alone for days. group e+p+ep was given e+ep therapy but including episodic vp on days and . all women underwent two endometrial biopsies during a single menstrual cycle (days + to + and four days later). avb integrin was detected in frozen tissue using the envision system. pinopode formation was evaluated in tissue fixed in glutaraldehide using a zeiss dsm a scanning electron microscopy. both avb integrin and pinopode formation were semiquantitatively evaluated. results patients in group e+p+ep showed markedly decidualized endometria in the midluteal biopsy and a significant increase of avb integrin expression (p= . for both parameters). no significant changes in pinopode formation were observed in midluteal biopsies. no differences were observed in late luteal biopsies. conclusions avb integrin expression and pinopode formation are signs of endometrial maturation related to progesterone action but their significance in terms of endometrial receptivity in the clinical setting remains to be shown. supported by grant pi (fondo de investigaciones sanitarias). gestational trophoblastic diseases (gtd) are extremely rare conditions, especially the ones associated with ectopic pregnancies (ep). case reports only three cases ( . %, / ) of gtd associated with ep were diagnosed at the department of histopathology and clinical cytology in the last years. the first patient, age underwent salpingectomy for clinical suspicion of tubal pregnancy, whereas in the second, age hysterectomy with bilateral salpingo-oophprectomy (hsop) was performed for a clinical diagnosis of ovarian endometriosis. the third patient, age underwent hsop for malignant ovarian tumour which metastasised to lungs. pathological findings: chorionic villi were found in the tubal lumen of the first patient, showing marked hydropic degeneration, reduced/absent vascularisation and excessive trophoblastic cell growth. diagnosis of partial hydatiform mole was established, and the patient remained under surveillance for the hcg level. atypical trophoblastic cells, which penetrated the full thickness of the tubal wall and invaded the ovarian blood vessels of the second patient, led to the diagnosis of an invasive mole. the third patient was diagnosed with choriocarcinoma due to the presence of a tumour consisting of malignant trophoblastic cells that infiltrated the right ovary, tube and uterine cornu. the last two patients received chemotherapy and have been well for and months, respectively. conclusion due to extremely low incidence of gtd associated with ep, these entities are rarely clinically recognised. therefore, a careful histopathologic examination of the adnexal masses is essential for the establishment of the correct diagnosis and further treatment of the patients. in an organized screening programme for cervix cancer the diagnosis atypia (ascus) has always been a problem. the subsequent histological examination shows different diagnoses, such as negative, inflammation, cin i-iii (sil) and carcinoma. in the county of funen, a well-organized screening programme has existed since .using the cytological diagnosis of atypia, we distinguish between atypia (asc-us) probably inflammatory and atypia (asc-h) probably dysplasia. in previous studies on papsmears we have shown histological follow-up diagnoses for atypia (asc-us): negative . % and cin i-iii . %, and for atypia (asc-h): negative . %, cin i-iii % and carcinoma , %. since june we have implementated liquid-based cytology (lbc) thinprep, cytyc. the aim of this investigation is to show the histological follow-up diagnoses of cytological atypia when using lbc-technique. the total number of lbc-samples in were examined. the recommandations for atypia are a repeated cervical sample, and for persistent atypia or atypia colposcopy, cervical biopsies and abrasion within months. in a total of , smears, including , lbcsamples, were examined. the diagnoses of the lbc-samples were: negative , %, inadequate , % (a reduction of % compared with pap-smears), atypia , % and cin i %. preliminary histological follow-up diagnoses for atypia and were: negative % and cin i-iii %. conclusion thinprep shows two significant advantages: a markedly reduced number of cytological atypia diagnoses ( %) compared with pap-smears and a higher diagnostic quality with fewer false positive diagnoses. introduction ovarian sertoli-leydig cell tumors (slct) are very rare sex cord-stromal tumors and among them, tumors with heterologous sarcomatous elements are exceptional. aims: a unilateral ovarian stage ic slct with heterologous mesenchymal elements is presented. materials and methods a -year-old woman, para , was admitted to hospital with severe abdominal pain. at laparotomy, resection of a large right ovarian cyst was carried out. histological examination revealed a slct, and subsequently surgical staging was performed. no adjuvant therapy was given. at years followup she was well and without signs of recurrence. routine staining of slides from paraffin blocks and immunohistochemistry (ihc) were available. results gross examination revealed a x x cm large multilocular ovarian cyst with smooth interlocular septa and outer surface. microscopy disclosed a slct of intermediate differentiation with a very few leydig cells. major areas consisted of a hypocellular stroma with edema, hemorrhage and necrosis. in a few sections small foci of cartilage were seen. in a number of sections groups of small cells with indistinct cytoplasm and hyperchromatic nuclei were present. mitotic and apoptotic figures were common. ihc revealed a positive reaction for desmin, myf and myogenin, indicating rhabdomyosarcoma of embryonal type. conclusion the most important prognostic factors of slct are stage and grade of differentiation. variants of slct with immature skeletal muscle are often fatal. ihc with myf or myogenin with a high sensitivity and specificity for rhabdomyosarcoma, combined with inhibin is very important in differing immature sertoli cells from rhabdomyoblasts, in order to diagnose the rhabdomyosarcoma and to grade the slct correctly. expression of the nerve growth factor (ngf) receptors trka and p shows biological diversity in ovarian, breast and mesothelial malignancy b davidson , r reich , p lazarovici , va florenes , m skrede , b risberg , c bedrossian , jm nesland the norwegian radium hospital, oslo, norway department of pharmacology, hebrew university, jerusalem. israel department of pathology, northwestern university, chicago, il, usa neurotrophins and their receptors are known to have a central role in biologic processes in the nervous system. however, recent studies have additionally highlighted the role of ngf and its receptors trka (inducer of proliferation) and p (inducer of apoptosis) in the development and progression of malignant tumors, including those of non-neural origin. we studied protein expression of p and activated phosphorylated trka (p-trka) in serous ovarian carcinomas ( effusions, solid tumors), breast carcinomas ( effusions, solid tumors) and malignant mesotheliomas (mm) ( effusions, biopsies) using immunohistochemistry and immunoblotting. p-trka was expressed in ovarian carcinoma cells in effusions in only / ( %) specimens, compared to / ( %) solid tumors, while the opposite was true for p . in contrast, p-trka membrane expression was higher in effusions (p= . ) of mm patients, while the opposite was true for p (p= . ). in addition, p-trka (but not p ) expression was significantly higher in peritoneal mm compared to their pleural counterparts ( / vs. / positive tumors; p= . ). in breast cancer, p-trka expression in effusions was significantly higher than in primary tumors (p< . ) and lymph node metastases (p= . ), while p expression was lower in both effusions (p= . ) and primary tumors (p= . ) compared to lymph node metastases. our results provide the first evidence regarding the role of nfg receptors in tumor progression through site-specific changes in expression. specifically, they may provide biologic evidence for the uniformly and rapidly fatal outcome of breast cancer patients with effusions and of mm patients. the significance of mast cells in the differential diagnosis of uterine smooth muscle tumors bh ozdemir aim to assess the role of the mean mast cell count in the differential diagnosis of uterine smooth muscle tumors. material and method twenty-cases of ordinary leiomyoma, cases of atypical leiomyoma, cases of leiomyosarcoma, cases of mitotically active leiomyoma and cases of cellular leiomyoma were included in the study. the morphological features and clinical presentation was evaluated retrospectively. toludine blue stain was used to highlight the mast cells in the endometrium, myometrium and smooth muscle tumors. at least high power fields were counted. results considerable variation in mast cell numbers was found among smooth muscle tumors (p< . ). the intratumoral mean mast cell count was found significantly higher in ordinary leiomyomas ( . ), when compared with other tumors, including atypical leiomyoma ( . . ), mitotically active leiomyoma ( . ), cellular leiomyoma ( . . ) and leiomyosarcoma ( . . ) (p< . ). no significant differences were found between these tumor groups in regards of the mean mast cell count of endometrium and myometrium adjacent to the tumor. in the endometrium, a drop in the number of mast cells has been demonstrated with advancing age, particularly after menopause (p< . ). in addition cases showed eosinophilia, cases showed basophilia and cases showed allergic reactions to different causes. a significant positive relationship was found between the intratumoral, myometrial mast cell number and basophilia. conclusion these data suggest that intratumoral mast cell quantitation may allow the differential diagnosis in uterine smooth muscle tumors. ovarian carcinoma is the most frequent cause of death in the world among gynaecologic malignancies. diagnosis is usually late, and the biological behaviour is highly variable, depending on several factors such as the histopathological grading, the local and/or peritoneal spread and the proliferation rate. we choose a list of genes involved in cell growth and proliferation (igf , igfbp , ctnnbeta , cdk- c, fgfr ). among these, so far, we analyzed igfbp gene and studied loss of heterozygosity (loh) in tumour tissue in order to identify potential relevant genes in ovarian cancer homeostasis. twenty cases of formalin fixed, paraffin embedded ovarian carcinomas from the archives of the department of pathology of padova university and five effusions (from peritoneal or pleural recurrences) of ovarian carcinomas, were selected for this study. genomic dna and tumour dna for each case were extracted from blood cell, tumour or not-cancer surgery biopsy. loh was searched using published single nucleotide polymorphism (snps) in igfbp gene. pcr product covering the snps from genomic and tumour dna were dhplc separated. three of the twenty patients whose tumour dna was obtained from paraffin material, were heterozygous at nucleotide c/a (dbsnp ref. n. ) and a/c (dbsnp ref. n. ) of the igfbp gene in genomic dna. analysis of tumour dna failed to reveal loh (preserved heterozygosity). in effusion cases, one of five patients showed heterozygosity at the same nucleotides. in these patients, loh was also not identified. our preliminary results suggest that dhplc analysis for loh is feasible, rapid and maybe of potential interest in the identification of genes involved in ovarian cancerogenesis. the progression of low grade squamous intraepithelial lesion (l-sil) to high grade squamous intraepithelial lesion (h-sil) occurs in around % of cases. there are no morphologic changes to predict this progression. aims: to study whether the ki immunostaining pattern and labeling present upon diagnosis of l-sil is able to predict the regression or persistence/progression of the lesion to h-sil of the cervix over a two year period following diagnosis. materials and methods cervical biopsies from patients diagnosed cytologically as cervical squamous intraepithelial lesion (sil) were used. biopsies were performed immediately after the cytological diagnosis resulting in l-sil and h-sil. ki immunostaining was performed on all cases and was classified as suprabasal immunostaining pattern (sbip) for cases without ki- immunoreactive cells in the upper two thirds of the epithelium thickness, or dispersed immunostaining pattern (dip) for cases with ki- immunoreactive cells in the upper two thirds of the epithelium thickness. nuclei in hpf including the total thickness of the epithelial layer of each sample were counted to determine the mean number of cells which reacted against ki- antibody. in situ hybridization was used to demonstrate the presence of hpv and its typing. chi square test, fisher's exact test and student t test were used for statistical analysis with significance set at p< . . results of the biopsies performed cases resulted in l-sil ( s-bip and dip patterns) and in h-sil. % of the l-sil cases that resulted in a s-bip pattern showed persistence/ progression of the lesions while % of the cases that resulted in a dip pattern showed persistence/progression of the lesions. the difference between these values where statistically significative (p< . ). regarding ki- labeling index (li) in l-sil cases, we found that % of the cases ( out of ) with a ki- li less than % persisted/progressed, while % of cases ( out of ) with ki- li over % persisted/progressed. in this case there was also a statistically significative difference (p= . ). we found no statistical differences (p= . ) regarding hpv "in situ" hybridization. % of the cases ( out of ) where hpv was detected by in situ hybridization persisted/progressed while % of the cases did not show hpv by hybridization. conclusions the l-sil cases which showed dip and high ki- li were significantly related with persistence or progression to h-sil of these lesions, while l-sil cases which showed sbip and low ki- were significantly related to regression of the lesions. therefore, we conclude that ki- immunostaining pattern may be of prognostic value to predict regression or persistence/progression of l-sil. introduction sclerosing stromal tumor (sst) of the ovary is a rare tumor. it was first described by chalvardjan and scully in as a distinct subtype in the thecoma-fibroma group. most ssts occur in young patients below the age of years. the most common clinical symptom in patients is menstrual irregularity. in differential diagnosis, thecoma, fibroma, and hemangiopericytoma must be considered. aim: in this report, cases of sst of the ovary were described and compared with other ovarian tumors. material and methods two cases of ovarian sst were examined. the first case occurred in a -year-old female. at operation, the patient was found to have the ovary replaced by a tumor mass measuring x x cm. the second case occurred in a -year-old woman and the tumor mass measured x x cm. the surgical material was fixed in % formaldehyde solution and prepared for paraffin sections. in addition to h&e staining, immunohistochemical analyses were performed. results the clinical features, gross features, light microscopic findings and immunohistochemical results were described and compared with literature. the findings were consistent with sst, which is a very rare tumor of the ovary. the tumor cells showed positive staining for vimentin and desmin. we discuss the results and differential diagnosis. unususal chorangioma of the placenta -a case report w pokieser , w umek , r jahn , w ulrich institut of pathology, lainz hospital, vienna, austria institut of pathology, department of obstetrics and gynecology, university of vienna, austria chorangiomas (ca) are common lesions, occurring in of placentas. it is not clear whether ca are hyperplasias, hamartomas, or true neoplasms. depending on their size and location, they may be devoid of symptoms or may cause difficulties for the infant and the mother. we describe a case of manual removal of the placenta in association with ca. on gross examination the tumor had a nodule-in-nodule appearance and was histologically composed of two well defined nodules. the larger, outer one had the appearance of an usual chorangioma; the inner nodule showed highly cellular vascular proliferation. this architecture might represent a morphological variant of the same disease. in the present case, the placenta had to be removed manually. it remains unclear if this was due to impaired contractility of the uterine wall or due to a lack of decidualisation, following a worse vascular support in the surrounding area of the ca. it can´t be excluded that placenta accreta was caused by strictures after cesarian section which had been performed at the first delivery two years ago. on the other hand a placenta with a nodule form of ca could be less elastic than a placenta with regular architecture. however manual removal of the placenta may be regarded as a further complication of ca's larger than cm in diameter. are muc and muc ac reliable markers in the differential diagnosis of primary and metastatic ovarian carcinomas? a ensari , d ergun , a yorganci , a sertcelik , f ortac , n kursun ankara university medical school, department of pathology, ankara, turkey ankara university medical school, department of gynaecological oncology, ankara, turkey numune hospital gynaecology department ankara university medical school, department of biostatistic, ankara, turkey introduction aberrant expression of mucin glycoproteins has been observed during malignant transformation of human ovarian epithelium. however, there is limited information regarding the expression of these mucin proteins in primary and metastatic ovarian carcinomas. the aim of this study was to examine immunohistochemical expression of muc and muc ac mucin proteins in primary and metastatic carcinomas of the ovary in order to assess their value in the differential diagnosis. materials and methods forty eight ovarian tumours comprising surface epithelial carcinomas ( endometrioid, clear cell, serous papillary and mucinous type) and metastatic carcinomas of gastrointestinal primaries were included in the study. the age of the patients, the histological type and grade of the tumours were evaluated in relation to the expression (percentage distribution and staining intensity) of muc and muc ac studied immunohistochemically. results muc was expressed in ( . %) primary ovarian tumours while muc ac was expressed in ( %) primary tumours in comparison to % expression of muc and . % expression of muc ac in metastatic ovarian tumours. the differences in muc expression between non-mucinous ( . ± . ) and metastatic tumours ( . ± . ) and metastatic and mucinous tumours ( . ± . ) were statistically significant (p< . ). the difference in muc ac expression was statistically significant (p< . ) between non-mucinous ( . ± . ) and mucinous tumours ( . ± . ). conclusion muc proves to be highly reliable in the differential diagnosis of primary and metastatic mucinous tumours of the ovary. deregulation of sfrp and sfrp rna-expression in endometrial stromal sarcoma introduction endometrial stromal sarcomas (ess) are very rare uterine tumours. whereas histology and immunohistochemistry of this tumour are well documented, little is known about molecular mechanisms involved. to characterize ess-altered genes, we generated a genome-wide library by ssh-hybridisation and used it to produce cdna arrays. particular emphasis was also placed on data obtained from qrt-pcr analyses. materials and methods using ssh-library a gene-bank with app. different clones was established. total rna was extracted from frozen tumour tissue and used for cdna hybridisation. qrt-pcr analyses were performed both on frozen and paraffin-embedded tissue samples. results after cdna-array analysis more than genes were selected, sequenced and rearranged to different functional groups. among the most strongly deregulated genes were secreted frizzledrelated protein- (sfrp ) and sfrp . these are putative genes that regulate wnt-signalling pathway and can influence different cell events including cell proliferation. compared to post-menopausal endometrium the expression of sfrp and sfrp was increased in low-grade ess, but decreased in aggressive ess form. these results were additionally verified on paraffin embedded tissue probes by qrt-pcr analysis. conclusion deregulation of sfrp and sfrp expression appears to be one of the crucial features of ess. whereas in low-grade ess these two genes are up-regulated, their down-regulation could be associated with high-grade form of ess. therefore a sfrp and sfrp may not only represent useful markers for early ess diagnosis but may also have prognostic impact and help to distinguish between low-and high-grade ess. (this work was supported by lore-saldow -research fund) introduction fgt-cs is a highly malignant neoplasm with poor response to current chemotherapy. the proto-oncogen c-kit encodes a transmembrane-tyrosine-kinase (t-k) receptor involved in cell diferentiation, that has been found to be expressed in certain solid tumors and may play a role in their tumorigenesis. a t-k inhibitor specific for c-kit (sti ) has been reported to have therapeutic effects in tumors expressing the aberrant forms or high quantities of target protein. contradictory results are reported in the literature about the expression of c-kit in cs. the aim of this study was to investigate the immunoreactivity for c-kit in cs. immunostaining with anti-c-kit (cd ) polyclonal antibody (dako) was evaluated in cs ( endometrial, ovarian, cervical and tubal). only cases with membrane staining were considered to be positive. results c-kit was expressed in cases: in cases only in the carcinomatous component ( endometrial, tubal), in case only in the sarcomatous component (endometrial) and in case in both components (ovarian). positivity was focal and less than % of cells in all cases. conclusion cs of the female genital tract rarely express c-kit. it is unlikely that patients with these tumors will benefit from treatment with the currently avalaible t-k inhibitors. uterine heterologous sarcoma with the component of osteosarcoma on the base of lipoleiomyoma -a case report uterine homologous and heterologous sarcomas are rare neoplasms that are usually misdiagnosed as a component of malignant mixed mullerian tumor (mmmt). we present a case of a -year-old woman who had a heterologous sarcoma with a component of osteosarcoma on the base of lipoleiomyoma, that can be differentiated from mmmt by the absence of epithelial component. in heterologous sarcomas, the other components of neoplasm are rhabdomyosarcoma, chondrosarcoma, osteosarcoma, liposarcoma and combined forms. these neoplasms are misdiagnosed as endometrial stromal sarcomas or as a component of mmmt. they are more agressive than mmmt. immunohistochemical detection of er-β in ovarian sex cord-stromal tumors introduction since the discovery of a second subtype of estrogen receptor, er-beta (er-β), in , approximately years after the cloning of er-α, several reports have been published regarding the tissue distribution of this receptor. in mammals so far studied er-β predominates in the ovary. high expression of er-β was observed by in situ hybridization or immunohistochemistry in granulosa cells of the ovarian follicles. the aim of the present study was to examine immunohistochemically the expression of er-β in ovarian sex cord-stromal tumors. materials and methods ten cases of ovarian granulosa-stromal cell tumors (five adult granulosa cell tumors, two juvenile granulosa cell tumors, and sclerosing stromal tumors) and two cases of sertoli-leydig cell tumor were immunostained for er-β (polyclonal erb , biogenex). representative sections from ten cases of ovarian carcinomas were immunostained for comparison. results positivity for er-β was observed in % to % of the nuclei in adult granulosa cell tumors and % to % of the nuclei in sclerosing stromal tumors. only - % of the nuclei from juvenile granulosa cell tumors were positive for er-β. in sertoli-leydig cell tumors positivity was more extensive in areas of diffuse growth. four cases of ovarian carcinomas exhibited positivity. conclusion positivity for er-β is a common finding in ovarian sex cord-stromal tumors. these findings might have therapeutic implications. adult fibrosarcoma of the vulva ol mejia mejia instituto nacional de cancerología, bucaramanga, colombia introduction sarcomas are rare and unusual lesions of the vulva. the aim of this study was to present a case with analysis of clinical, morphologic and immunohistochemical findings. materials and methods a -year-old female patient presented a rapidly growing of a mass in the pubis and left labium major of vulva of x x cm associated with a second satellite lesion ( months later) in the right inguinal area of x x cm. results histologic examination of both lesions disclosed a malignant neoplasm composed of uniform and orderly spindle cells with occasional epithelioid cells, oriented in curving or interlacing fascicles, forming a herringbone pattern with mitotic figures ( / hpf) and areas of necrosis and hemorrhage. the immunohistochemical profile showed strong reactivity for cd and vimentin, and focal and weak reactivity for smooth muscle actin. immunoexpression for s , nse, bcl- , hmb- , melana, ae ae and desmin was lacking. discussion and conclusions we present a case of a malignant mesenchymal tumor of vulva. due to histological features and behavior, pathologists must discard others spindle cell tumors, such as malignant melanoma, malignant peripheral nerve sheath tumor, monophasic synovial sarcoma or leiomyosarcoma. fibrosarcoma is a diagnosis of exclusion and a wide immunohistochemical panel is useful to make differential diagnosis. introduction serous tumors of borderline malignancy (stbm) of the fallopian tube are exceedingly rare. we report a case of stbm of fallopian tube in a -year woman. material and methods a -year old woman was found to have right adnexal mass on her routine gynaecologic examination. serum ca- was not elevated. at laparoscopy a swollen fallopian tube was removed. during the operation a fine needle aspiration was performed. the cytological findings of the adequate giemsa-stained smears were recorded. haematoxylin and eosinstained sections from the routinely fixed and paraffin embedded surgical material were histologically analysed. results a papillary tumor, with maximal diameter of cm in the dilated part of the fallopian tube toward the fimbrial end, was found. histologic features of the tumor were similar to the atypical proliferative serous tumor of the ovary. two years after the operation the patient is well, without evidence of the disease. conclusion to the best of our knowledge only four cases of stbm of fallopian tube have been reported. our case is unusual due to lack of any symptoms. recent studies have suggested that in the setting of an unknown primary, cdx- immunostaining may be used in identifying a metastatic carcinoma as colonic in origin. aim: we sought to evaluate the immunoreactivity of cdx in a series of primary and metastatic ovarian mucinous neoplasms. materials and methods cases of mucinous tumour of the ovary, including benign cystadenomas, borderline mucinous tumours and mucinous adenocarcinomas, were tested for cdx immunoreactivity. cases of serous and endometrioid lesions as well as cases of ovarian metastases from colonic adenocarcinoma were also evaluated. results cdx immunoreactivity was detected in / ( %) cases of primary ovarian mucinous tumour. particularly, / ( , %) cases of cystadenomas, / ( , %) cases of borderline tumours and / ( , %) cases of adenocarcinomas were immunostained. immunoreactivity was usually related to an intestinal morphology. no immunoreactivity was observed in serous or endometrioid lesions whereas all five cases of ovarian metastases from colonic adenocarcinoma were immunoreactive for cdx . conclusions cdx immunoreactivity may be detected in the majority of benign, borderline and malignant ovarian mucinous tumour whereas serous and endometrioid lesions are unstained for this marker. cdx immunoreactivity is not useful in distinguishing between primary and metastatic ovarian mucinous carcinomas whereas it may be useful in the distinction between endocervical and intestinal type of mucinous tumour of the ovary, which may have clinical relevance. an ovarian primary should be added to the list of cdx positive metastatic carcinoma in the setting of an unknown primary. expression of p ink a in serous lesions of the female genital tract c heffron introduction a spectrum of serous lesions including benign, borderline and malignant tumours and endosalpingiosis commonly arise in tissues of the female genital tract. p ink a is a tumour suppressor protein which functions as a cyclin dependent kinase inhibitor and plays a vital role in the cell cycle control at the g /s phase transition. although well recognized as a discriminator of benign and potentially malignant squamous and glandular cervical lesions, the expression of p in ovarian lesions and their mimics is less well established. the aim of this study was to evaluate p ink a expression in serous lesions of the female genital tract and to investigate its potential diagnostic utility. methods immunohistochemical analysis of p ink a (mouse monoclonal anti-p antibody) was performed on cases of serous epithelial lesions of the female genital tract (n= ) comprising endosalpingiosis, borderline serous cystadenomas of ovary and peritoneum, benign serous cystadenoma of ovary, serous adenocarcinoma of ovary and peritoneum and normal fallopian tube epithelium. results all cases of endosalpingiosis were positive (definite cytoplasmic staining with occasional nuclear positivity) for p ink a expression, as were all normal fallopian tube epithelium samples. varying degrees of p ink a expression were found in the serous neoplasms examined, with no clear pattern associated with any histological subtype. conclusion diagnostic difficulties may arise in the distinction between benign serous glandular lesions and more sinister potentially malignant serous neoplasms. in this study the pattern and degree of expression of p , although showing some differences, does not allow definitive classification of these lesions. epithelioid trophoblastic tumor [ett] is an unfrequent gestational trophoblastic disease [gtd], whose epithelial-like cells derive from chorionic-type intermediate trophoblast. ett mostly shows a benign behaviour, but metastases have also been reported, and pathologic prognosticators are not as yet recognized. the kinase-inhibitor p kip has been seen to be lacking in malignant gtds, while different growth factors, such as the c-kit receptor, have been related to growing trophoblasts of p -null embryos. since c-kit is said to promote trophoblast growth and invasiveness, this study was aimed at immunolocalizing p and c-kit in ett, as compared to a proliferation antigen. materials and methods -after a preterm birth of a living infant, a -year-old woman underwent hysterectomy for a postpartum metratonia. the patient had an antecedent miscarriage. a cervical ett was found, and immunohistology was performed on paraffin sections, using c-kit, p , and ki antibodies, following a microwave antigen-unmasking. positive reactions were recorded either as a focal or diffuse immunostaining. an escavated, hemorrhagic, endocervical lesion was found, which focally infiltrated the myometrium. light microscopy showed poorly limited nodules of epithelial-like cells, arranged in cords and nests, with interposed hyaline material, extensive hemorrhages, and necrotic areas. atypical mononucleate and syncytiotrophoblastic cells occasionally occurred. immunohistology revealed a diffuse presence of c-kit-expressing tumor cells. the p nuclear staining was focally observed to be negative or weak, and ki was totally unlabeled. in conclusion, this slowly-growing ett is lacking in p , but the c-kit over-expression substantiates trophoblastic survival and invasiveness. carcinosarcomas of the corpus uteri r balan, c amalinei, c cotutiu histology department, university of medicine and pharmacy, iasi, romania the aim of our study was to analyze the clinical and morphological characteristics of carcinosarcomas of the corpus uteri. carcinosarcomas of the corpus uteri were studied in postmenopausal women. paraffin-embedded material was stained with hematoxylin and eosin, trichromic, reticulin and pas staining. antibodies against ema, cytokeratins, vimentin, desmin, and pcna were also used. the size of tumor, depth of myometrial invasion, presence of vascular invasion, necroses, cellular atypia and differentiation, and mitotic activity were analyzed. all tumors were characterized by large size, foci of hemorrhage and necrosis. morphological and immunohistochemical analysis proved biphasic structure of the tumors. epithelial component was represented by adenocarcinomas with high ( cases) and moderate ( case) degree of differentiation. all presented homologous pattern -leiomyosarcomas. no metastasis was encountered at the time of diagnosis. as ema was intensely positive, both in malignant epithelium and in the majority of stromal component, we conclude that carcinosarcomas of the corpus uteri represent combination tumors with both elements originating from a single common stem cell. this conclusion was emphasized by the presence of pas positive residues in both components. knowledge evaluation among albanian women on risk factors of cervix cancer k kule , n filipi institute of public health, unit of cancer, department of epidemiology, tirana, albania university hospital 'mother tereza', tirana, albania aim this study evaluates the rate of awareness of women on risk factors and the preventive ways for the cervix cancer. methods the data were obtained through the questionnaire in a sample of women aged - years randomly selected throughout albania (random cluster sample). the questionnaire included questions on: social-demography, sexual behavior, women health, and sources of the health information. results only % of interviewed women were informed on paptest. in % of cases it was the doctor notified the women of the cluster for this screening as an examination of cervix cancer prevention. the pap-test examination had been performed by only % of the women who had prior knowledge on pap-test. in about % of cases the examination was performed for diagnostic reasons and not for prophylactic control reasons. in proportion to total only . % had performed the test, which presents a very problematic situation. among the women who performed pap-test, % were aware of the need of periodical check-up every - year, but % of them never performed it. the reason for not doing periodical screening seemed to be the daily routine and fear of the unknown events. % of women who performed pap-test were from the cities. only % of women use contraceptive methods of the family planning. about % of women had undergone at least one abortion. conclusions the study supports the concept that information on health services as well as health education of women at risk plays an important role in early detection and prevention of cervix cancer. expression of checkpoint proteins p , hus- and rad- and its relation with apoptosis in ovarian epithelial tumours introduction eukaryotic cells show surveillance mechanisms (checkpoints) of genomic integrity. an impaired checkpoint has been linked to genomic instability, and, in higher eukaryots has been associated to risk of cancer. the genes to be studied are involved in the mitotic checkpoint and dna damage checkpoint (like p , the tumour supressor gene frecuently mutated in human cancers). these proteins are also involved in the checkpoint responding to incomplete dna replication (replication checkpoint) termed the rad/hus group of proteins. failure of this checkpoint progress can result in genomic instability, and, in higher eukaryotes may lead to tumorigenesis or certain degenerative disease states. material and methods we have obtained antisera against recombinant hhus and hrad . for this we will clone and overexpress in bacteria and immunize animals to obtain antisera. we will carry out extensive analysis of the expression levels of several proteins by western blot analysis. we looked for possibles associations among hus- and rad- immunohistochemical expression and prognosis in ovarian carcinomas prior to chemotherapy. we studied, in addition to the levels of expression of the mentioned proteins, the localization and modifications of these proteins in epithelial ovarian tumours (benign, borderline and carcinomas) in order to establish the probable relationship between these proteins and ovarian carcinogenesis. results increased levels of checkpoint hus- protein was related to malignant transformation across all types of ovarian carcinomas (p< , ). similar results were observed with the expression of rad- protein, however was not statistically significant (p> , ). in univariate survival analysis, the expression of hus- and rad- protein were not significantly correlated with shorter survival and shorter free period of illness (p> , ). there were positive correlation between the apoptotic index, p and bax and the expression of checkpoint protein hus- (p< , ), but none relation was detected with rad- expression (p> , ). conclusions -the expression of hus- is higher in ovarian carcinomas and low in benign and borderline tumours. -the expression of hus- shows a positive correlation with the apoptotic index and with the pro-apoptotic factors (p and bax). -there was not statistical significant differences in the expression of hus- and rad- among the different histological types of ovarian tumours. -there was a positive correlation between the expression of hus- protein with other prognostic factors such as the figo stages, and the histological grade. unilateral gonadoblastoma associated with , xy pure gonadal dysgenesis (swyer's syndrome): a case report with numerous microcalcifications. the patient underwent bilateral salpingoophorectomy and total hysterectomy. the authors underline the importance of the correct diagnosis and the need of bilateral salpingoophorectomy for preventing the possible progression of the gonadoblastoma into malignant germinal cell tumor. histological and electron microscopic identification and analysis of uncommon particles in tissue after use of orthopaedic implants introduction deposits of solid products occur widely in tissue either from endogenous sources due to pathological changes, or from exogenous sources. after use of orthopaedic implants, particles produced by wear are commonly recognizable and identifiable in tissue by the surgical pathologist. foreign body related tissue re actions may contribute to aseptic loosening of implants, which is regarded as one of the most significant complications in orthopaedic surgery. in the present study we report on very uncommon particles in periprosthetic tissue after aseptic loosening of metallic implants. our study was performed to identify particles in periprosthetic tissue and to clarify whether they may contribute to the loss of implants. materials and methods periprosthetic tissue including joint capsule, acetabular and femoral interface membranes of cases of aseptic loosened cementless total hip replacements was histologically investigated. four cases with present uncommon particles were selected for electron microscope investigation and dispersive x-ray analysis. results in four cases the histological investigation revealed uncommon green to yellow coloured foreign bodies. by the use of electron scanning microscopy and energy dispersive x-ray analysis, these particles were identified as deposits rich in chromium, phosphorus and oxygen. this detection indicates the presence of solid corrosion products derived from implant surfaces. conclusion particles produced by wear and/or corrosion are associated with an alteration of the environment and the development of periprosthetic osteolysis which may result into the loss of the implant. the surgical pathologist should be aware that corrosion products may occur. their identification is helpful to determine implant failures. paratesticular lipoleiomyosarcoma: a case report l kachko, r shaco-levy, i sinelnikov, j goldstein department of diagnostic pathology, soroka medical center, beer-sheva, israel introduction leiomyomatous differentiation in liposarcoma is a rare phenomenon and may occur as a well differentiated liposar-coma with an intrinsic malignant smooth muscle component, so called " lipoleiomyosarcoma" or in the context of "dedifferentiated" liposarcoma. material and methods we have studied a paratesticular lipoleiomyosarcoma in a man years old. gross examination revealed tumor nodules dispersed between identifiable testis and epididymis, the largest of which measured x x cm. the specimen cut surface revealed smooth yellow tissue with distinct areas of gray myxomatous change. microscopically the specimen consisted of a well differentiated lipoma-like, sclerosing liposarcoma with mature adipocytes and fibrous septa containing spindled and floret-like cells. the smooth muscle accounted for about % of the total tumor mass and consisted of fascicles and bundles of spindle cells with very little atypia or pleomorphism. mitotic activity was moderate at most and focal in distribution. the cells colored well with immunohistochemical stain against sma. results and conclusions an extraordinary rare combination-type tumor is presented. only two previous examples of paratesticular lipoleiomyosarcoma have been published. the nature of combination-type tumors and dedifferentiated liposarcoma is addressed. the existence of two lines of tissue differentiation in the same tumor suggests the possibility of two separate tumors arising together or two lines of differentiation appearing from one pluripotent precursor cell. the differential diagnosis includes angiomyolipoma, which should be hmb positive. expression of estrogen receptor (er) and progesterone receptors (pr) in renal angiomyolipoma n savelov, g chemeris, i sokolova cancer research center, moscow, russian federation introduction angoimyolipomas are the most common mesenchymal tumours of kidney. they are more common in female. possibly estrogen and progesterone influence on tumours growth. we investigated the expression of estrogen receptors (er) and progesterone receptors (pr) in renal angiomyolipomas. materials and methods renal angiomyolipomas from patients were tested. the expression of er and pr was studied immunohistochemically. the expression of hmb- , alpha-smoothmuscle actin and vimentin was also studied in all cases. results most angiomyolipomas were of classic morphology with one exception. in that case, classical angiomyolipoma was combined with cystic hamartomatous lesions and involvement of regional lymph nodes. the vast majority of cells in the cases with typical morphology expressed alpha-smooth-muscle actin. expression of hmb- was diffuse or focal. in the case with cystic hamartomatous lesions, an inverse correlation of expression of hmb- and alpha-smooth-muscle actin was observed in both renal and extrarenal components. of the tumours, two tumours were only er positive and three tumours were er and pr positive. in four cases, focal immunoreactivity for er was mainly observed in the spindle shaped cells. in two cases, the immunoreactivity for pr was diffuse, but less than percent of cells were positive. er and pr positivity was diffuse, strong and widespread only in the case with cystic hamartomatous lesions. conclusion to our knowledge, estrogen and progesterone are not critical players in all angiomyolipoma growth, but only in the specific cases. myofibroblastic tumors are sometimes difficult to be distinguished from smooth muscle tumors. recently, cd , a cell surface metalloprotease, was shown to be present in myofibroblasts within the stroma of infiltrating cancers. to clarify the specificity of cd expression in myofibroblasts, we performed immunohistochemical studies on myofibroblastic and smooth muscle tumors. methods and materials consisted of myofibroblastic and smooth muscle lesions, ranging from reactive to benign and malignant tumors. all myofibroblastic lesions arose in soft tissue and smooth muscle tumors in soft tissue and organs. using paraffin sections, immunohistochemical staining for cd (clone: c , serotec) was carried out by an automated stainer (benchmark, ventana). results significant cd -expression was demonstrated only in malignant or borderline lesions, including malignant fibrous histiocytoma ( of cases), low grade myofibroblastic sarcoma ( of ), inflammatory myofibroblastic tumor ( of ), and leiomyosarcoma ( of ). however, no positive reaction was seen in reactive lesions and benign tumors, such as scar ( case), nodular fasciitis ( ), su-perficial fibromatosis ( ), desmoid ( ), leiomyoma ( ), vascular leiomyoma ( ), angiomyolipoma ( ), and glomus tumor ( ) . furthermore, the intensity of staining was almost parallel to the microscopic anaplasia. conclusion the present study demonstrated that cd expression is not specific to myofibroblasts, but seems to be related to the biologic aggressiveness of myofibroblastic and smooth muscle tumors. therefore, cd expression may be a potential predictor of malignancy in these tumors. correlation of metallothionein (mt) and antigen ki- expressions with grade of malignancy (g) in some soft tissue sarcomas dysplasia may be found in the bones during the childhood and adolescence. they can cause pathologic fractures and deformities of involved bone. rarely, some of them (fibrous dysplasia), undergo malignant transformation. the aim of this study is to analyse all the cases of tumor-like lesions of bones in children and adolescents, during the period time - . the study was performed at the institute for pathologic anatomy, school of medicine belgrade. the data source was the bone and joint tumor registry. the study was a retrospective analysis of patients who had tumor-like lesion before the age of . results solitary bone cyst was the most common lesion ( patients). the median age was years for fibrous dysplasia and solitary bone cyst, years for aneurysmal bone cyst and eosinophilic granuloma, and years for non-ossifying fibroma. male to female ratio was : . for fibrous dysplasia, : . for aneurysmal bone cyst, . : for solitary bone cyst, . : for eosinophilic granuloma and : for non-ossifying fibroma. the most common sites were femur for fibrous dysplasia and aneurysmal bone cyst, humerus for solitary bone cyst, tibia for non-ossifying fibroma and bones of scull and vertebrae for eosinophilic granuloma. the most frequent symptoms and signs were pain, pathologic fractures and swellings. conclusion the results of our study mostly correspond to those of the current literature. extraspinal myxopapillary ependymoma. case report and literature review introduction ependymomas are glial tumors that on rare occasions occur in subcutaneous locations dorsal to the sacrum and coccyx. case report a -year-old woman presented with a soft tissues tumor in the intergluteal region. mri revealed a cm large mass, with solid and cystic areas, located below the coccyx, without bone alteration. surgical excision was performed. pathological features grossly, the tumor was encapsulated and showed solid, spongy and myxoid areas with a peripheral hemorrhagic focus. histologically it consisted of medium-sized cuboidalcolumnar cells, with round-oval nuclei, granular cromatin and eosinophilic cytoplasm. tumor cells showed myxopapillary and cystic-papillary growth pattern or were arranged on fibrovascular stalks, with a central hyalinized wall vessel, surrounded by a single layer of cuboidal cells. tumoral necrosis and hemorrhage were observed. the peritumoral stroma was highly hyalinized. the immunohistochemical stains showed vimentin(+), s- (+), gfap(+) and keratins(-). discussion and conclusion extraspinal ependymoma (ee) is a rare tumor usually localized subcutaneously in sacrococcygeal region of young adults; most are of myxopapillary type. ee may arise from normal remnants of the neural tube or from abnormal remnants resulting from embryologic malformations. ee has a greater propensity to metastasize than its intraspinal counterpart, because of easier accessibility to lymph channels. in a study of cases, elson found a metastases incidence of % and no clinical or histologic data was found to predict the biological behavior of the tumor. the differential diagnosis must be done with choroid plexus papilloma, myxoid chondrosarcoma, chordoma, meningioma, adenoid cystic carcinoma and paraganglioma. introduction fungal infections of bone represent about , - , % of all osteomyelitis cases. the disease most often affecting the feet -mycetoma pedis, is also known as madura foot. in our geographic regions mycetoma is extremely rare. it is endemic in tropical and subtropical countries. case report we present a case of mycetoma pedis -madura foot. the patient was a years old woman. the clinical signs were pain, indurations and local redness with a long history of years. the surgical material was routinely stained with hematoxylin-eosin (he). pathologically, a granulomatous inflammation of the bone was confirmed. all pathological characteristic pointed to fungal infection in the form of mycetona pedis. special stains (pas, grocott's hexamine-silver and giemsa) for fungi were performed: and the diagnosis of a mycetoma was confirmed. for the definitive microbiological analysis tissue inoculation on the sabouraud dextrose agar laboratory media was done. pseudoallesheria boydiia sexual staging of monosporium apiospermum was isolated. after microbiological verification of fungal infection the patient was treated surgically. seven months after first operation the patient developed similar clinical signs again. the diagnostic procedure was repeated and mycetoma was confirmed again. the surgical therapy was performed as a therapy of choice because pseudoallesheria boydii does not react to treatment with antimycotic drugs. spindle cell lipoma is a rare entity with an uncertain histogenesis; although the current classification includes this tumor in the lipomatous neoplasms, the immunohistochemical and electron microscopic data suggest a much closer link to the fibrous solitary tumor. we report the case of a years woman with a / / / cm nodule in the scapular area; the histopathologic examination revealed a tumor proliferation consisting mainly of giant cells, most of them multinucleated with floret-like appearance. the stroma consisted of an abundant collagen deposition with rope-like fascicles and prominent myxoid changes, most striking in the periphery of the tumor. the tumor proliferation was contiguous with nearby adipose tissue with no capsule in-between. immuno-histochemical tests revealed diffuse positivity for cd ; pcna was positive in % of the cells. smooth muscle actin, desmin and s -protein were negative in tumor cells. the patient is well with no recurrence of the tumor one year after the resection. despite the highly pleomorphic histopathologic appearance the tumor is benign with an uneventful clinical behaviuor. expression of p protein in gct of bone with lung metastases introduction the giant cell tumor of bone (gct) is a local osteolytic tumor with variable degrees of aggressiveness. in rare cases pulmonary metastases can be observed. the stromal cells are the neoplastic components of the gct, and are probably responsible for biological behavior of the tumor. in the last years cases of gct with lung metastases were diagnosed at the institute of pathology, school of medicine, belgrade. both tumors displayed characteristic histological appearance of gct: a high number of osteoclast-like multinucleated giant cells and two mononuclear cell types. the first one resembles a monocyte, the second are fibroblast-like stromal cell. mononuclear cells had benign cytological characteristics without polymorphism and mitosis. to elucidate the mechanism of metastasing, we searched for the molecular abnormalities of p . we examined the expression of p in stromal cells immunohistochemically. unexpectedly, both tumors were p negative and we could not show correlation between p expression and metastasing of gct to the lung. conclusion although p positivity in other tumors is closely associated with aggressive biological behavior we could not confirm this association for gct. trichoblastic fibroma or primary soft tissue adamantinoma? introduction benign extragnathic tumors that may mimic the histological features of odontogenic ameloblastoma (and ameloblastic fibroma) include trichoblastic fibroma and primary osseous and soft tissue adamantinoma. trichoblastic fibroma is a rare ectomesenchymal tumor of hair germ involving most often the dermis and subcutis of the head and neck. the mean age at presentation is , years.the youngest case reported was that of a shoulder tumor in a year old female. its occurrence in the soft tissue of the upper arm has not been documented. primary soft tissue adamantinomas are extremely rare with isolated cases reported in the region of the jaw and the leg ie. close to sites where odontogenic and primary osseous adamantinomas occur, respectively. no cases involving the upper arm have been reported. we report a case of a benign appearing adamantinoid tumor involving the deep soft tissue of the upper arm. a year old black male presented with a slow growing mass involving the deep soft tissue of the left upper arm without associated cutaneous or osseous abnormalities. results the adamantinoid features in this benign appearing biphasic tumor included nests and acini of palisading columnar epithelium exhibiting reversed nuclear polarity, areas reminiscent of stellate reticulum and an intimately associated spindle cell proliferation. conclusion our favored diagnosis was a trichoblastic fibroma but we discuss the complete differential diagnosis of all tumors with adamantinoid features with respect to clinical, morphological and immunohistochemical characteristics. intraosseous lipoma -a case report intraosseous lipoma is a rare benign tumor of mature adipocytes arising in the medullary canal, most often diagnosed incidentally. most of these tumors occur in the long bones, with propensity for the proximal femur, fibula, and tibia. ribs, calvarium, sacrum, metatarsus and calcaneus are other frequent sites. we report a case of a -yearold female with a histologically verified solitary intraosseous lipoma involving the distal part of the right fibula. radiographically the tumor was a lytic benign-appearing lesion with sharply demarcated borders surrounded by a zone of sclerosis. computed tomographic scan showed the lesion to have features of adipose tissue. the examination was made on surgically removed distal part of the right fibula. for histological verification of intraosseous lipoma, resected bone was decalcified, embedded in paraffin and stained with standard staining with hematoxilin-eosin. histopathologic examination revealed mature adipose tissue with widely scattered trabeculae of bone and central calcifications. this case emphasizes the characteristics of intraosseous lipoma, and highlights the need for clinicopathologic and roentgenographic correlations. fasl, c-erbb , cyclin d , p and tgfb expression in osteosarcomas: evaluation of relationship with clinicopathologic parameters introduction fasl, c-erbb , cyclin d , p and tgfb are molecules which were shown to be related to prognosis of tumors. in this study expression of these molecules were evaluated in osteosarcoma (os) cases. the correlation between the expressions and age, gender of the patients, location, grade, and histologic subtype of tumors were investigated. material and method fasl, c-erbb , cyclin d , p and tgfb expressions were detected immunohistochemically. results thirty-six os cases ( osteoblastic, fibroblastic, five chondroblastic) were included in the study. twenty-two were males and were females. os were located in the extremities ( ), jaw ( ), pelvis ( ), vertebra ( ). twenty-five of were high grade and were low grade tumors. fasl and p were detected in . % of the cases, tgfb in . %, c-erbb in . %, and cyclin d in . %. there were statistically significant correlations between fasl and tumor grade (p: . ), fasl and tgfb (p= . ), fasl and c-erbb (p= . ), p and tgfb (p= . ). no correlation was found among other parameters. conclusion expression of these molecules and detection of correlation between some molecules show that pathogenesis of os is a complex mechanism regulated by several molecular factors. better insight into tumorigenesis may help to provide new therapeutic approaches and idntification of patients with better prognosis. a rotter , s kovac , v pisot , r trebse institute of pathology, medical faculty, university of ljubljana, ljubljana, slovenia valdoltra orthopedic hospital, ankaran, slovenia introduction granulation tissue with macrophages is usually found around loose total hip prosthesis (thp), usually explained with deposition of wear debris from thp. however, we often found a lot of hemosiderin (an insoluble form of storage iron) in macrophages. the aim of this study was to evaluate the amount of hemosiderin in granulation tissue around thp removed after relatively short period of duration, and compare it with an interface tissue around thp of very long duration. we assessed the interface tissue around the loosened thp of the longest duration described ever (judet's thp was removed after years) and compared it with the granulation tissue around thp of short duration (less than six years). at histology h&e, giemsa, and weigert-van gieson staining were used. for the demonstration of hemosiderin perls' reaction and for macrophages cd were used. we compared the results of histologic evaluations performed according to modified mirra's classification: semi quantitative assessment of neutrophils, mononuclear histiocytes, chronic inflammatory cells, giant-cells, metal particles, polymethacrylate globules, necrobiosis, bone chips, necrosis and necrotic debris. but, we also included the assessment of the intensity of hemosiderin deposition. results comparison of granulation tissues of short duration thp to that of -year duration revealed a surprising difference in the amount of hemosiderin particles with intensive intracellular hemosiderin deposition in mononuclear macrophages and giant cells in the first group. in the -year-old thp there was predominantly hypocellular tissue with very little amount of mononuclear macrophages containing slight amount of intracellular hemosiderin deposition and no multinucleated giant cells at all. conclusions we don't know why hemosiderin deposition is more evident in early loosening of thp. hemosiderin deposition may give rise to accumulation of macrophages, perhaps increasing the process of osteolysis and granulation tissue formation. but more 'aggressive' forms of loosening may also cause recurrent bleeding. whether the higher level of hemosiderin is the cause or the result of the loosening of thp is to be determined. the types of tumor and peritumor stroma and biological behavior of fibrohistiocytic tumors introduction tumor-host interaction induces different types of tumor and peritumor stroma which may reveal some aspects of tumor biology. the aim of this study was to establish a correlation between different types of tumor and peritumor stroma of fibrohistiocytic tumors (fh tumors) and their biological behavior. the presence of colagenous-reticular (cr), myxoid (myx) and mixed (mx) tumor and peritumor stroma was determineted on selected paraffin sections stained by masson-trichrome, silver impregnation, alcian blue with and without hialuronidase pretretment and pas reaction of fh tumors: benign (bfh), dermatofibrosarcoma protuberans (dfsp) and malignant (mfh), all graded in three degrees of malignancy. the collected data were statistically evaluated by means of student t proportion test. results the difference in presence of myx and mx tumor and peritumor stroma in relation to the cr type was statistically significant between mfh and bfh (p< , ) or dfsp (p< , ). with an increasing malignancy grade of mfh percentage of tumors with cr tumor stroma was decreasing and percentage of tumors with myx and mx tumor stroma was increasing. however, mfh of g and g malignancy grade did not differ in distribution of peritumor stroma types, but , % of mfhg had myx and mx peritumor stroma. conclusion while myx and mx types of tumor and peritumor stroma are associated with agressive, cr type of tumor and peritumor stroma is associated with benign and less agressive fh tumors. dynamic angiogenic models of human soft tissue tumors transplanted into immunodeficient nude mice introduction animal models were used to study the vascular neogenesis of tumors. we undertook experiments using human sarcomas transplanted into the subcutaneous pad of immunodeficient nude mice. the aim of this study was analysis of: the microvasculature present within the tumor before the transplant; the early stages of the neovascularization; and the types of vascular connections established between the host and the tumor graft. material and methods implants of . - . cm in size of a ewing's sarcoma (es), an osteosarcoma (os) and a fibrosarcoma (fs) were analyzed. the study was successively followed for , , hours, one, two, three and four weeks. he, pas and immunohistochemistry for angiogenic factors were complemented with electron microscopy. after - hours, the implants over-expressed several angiogenic factors. at this stage, the tumors initiated an active angiogenic remodeling in the surrounding stroma where capillary congestion and microhemorragies occurred. endothelial sprouting of the host capillaries was seen. vascular co-option between the endothelial cells, newly formed endothelial cells and the tumor cells was observed. no clear basal lamina was seen. tumor-endothelial cell mosaics and vascular mimicry were detected only in the es and os. conclusions vascular expression and neogenesis varies considerably from the fast es to the fs while the os reproduces patterns that are common in all three. based upon present experience it seems evident that each tumor shows a different type of neovascularization. these models provide excellent examples of all the systems that are involved in the recapillarization of a sarcoma. this work has been supported by grants fis / and fis / from the ministry of health, madrid, spain. prognostic significance of intratumoral neoangiogenesis in childhood osteosarcoma and ewing sarcoma d mikulic , i ilic , m coric , l batelja , j stepan , s seiwerth institute of pathology medical faculty, zagreb, croatia childrens hospital, zagreb, croatia introduction intratumoral neoangiogenesis quantified by microvessel density (mvd) has been shown to be a strong prognostic indicator in a number of malignant tumors, primarily carcinomas. its association with prognosis in bone sarcomas has been subject to less extensive research. the aim of our study was to investigate clinical significance of neoangiogenesis in osteosarcoma and ewing sarcoma. patients and methods a retrospective immunohistochemical study was performed. patients with osteosarcoma and patients with ewing sarcoma were included. sections from diagnostic biopsies were immunostained using factor viii and cd (dako) antibody and microvessels were counted at x magnification on three microscopic fields showing highest mvd per patient. mvd was correlated with overall and event-free survival by kaplan-meier and log-rank analysis. the difference between the mean microvessel counts in patients with or without metastasis and alive or dead was assessed by mann-whitney test. results in patients with osteosarcoma significant statistical difference was found as regards the death risk and risk of development of metastasis between patients with high (> vessels/field) and low (< vessels/field) microvessel counts (log-rank test p= . and p= . , respectively). in patients with ewing sarcoma a trend was noted for the patients with lower (< . vessels/field) microvessel counts to have better outcomes than patients with higher vessel counts, however, the difference was not statistically significant. conclusion these findings strongly suggest that neoangiogenesis quantified by microvessel density is predictive of metastasis and poor prognosis in osteosarcoma. valorization of its role in patients with ewing sarcoma awaits further studies. epithelioid sarcoma -clinicopathological findings in cases m demiryont epithelioid sarcoma is a soft tissue sarcoma of unknown histogenesis with a predilection of young adults and hand/forearm location.the tumor is often multinodular showing central necrosis. epithelioid sarcoma has a high potential of local recurrence and metastasis. six cases were presented. five patients were male with a mean age of . tumor locations were finger, hand and forearm. cytokeratin and vimentin were positive in all cases. four cases showed positivity for cd . desmin and cd were negative. ki- (mib- )proliferation index was - % in cases. initial surgery performed in all of the patients has been local excision with negative margins.three patients received radiotherapy or chemotherapy after the primary excision.except for one patient who was lost to follow-up, local recurrence was detected in all the patients in a period of months to years.three patients died with distant metastasis. epithelioid sarcoma can be easily identified with its typical features, but it can be confused with non-tumor lesions in patients having a long clinical history and a small tumor. radical surgery and regional lymph node dissection may bring benefit; the incidence of the lymph node metastasis is high. radiotherapy and chemotherapy can be added to therapy in the recurrent or metastatic state of the disease. introduction intramuscular hemangiomas are relatively uncommon but significant lesions, and their unorthodox location and infiltrative pattern often raise the question of malignancy to clinician and pathologist. we present the histomorphological and immunohistochemical data of a hemangioma in a -year-old man who presented with an enlarging painful soft tissue mass of his right upper leg. methods the complete local excision of tumor arising in the quadriceps muscle was done. sections of formalin-fixed, paraffinembedded tissue material were stained with he, van gieson, pas, ptah, gomori's reticulin and trichrome, and immunohistochemically for detection of factor viii-related antigen. results grossly, the lesion ( x , cm) was poorly circumscribed, reddish brown and partially haemorrhagic. microscopically, it was composed both of small, capillary-sized and large, cavernous vessels lined by endothelial cells and surrounded by connective and mature adipose tissue. the adipocytic component wasn't prominent. many cavernous spaces were separated by dense fibrous tissue and some of them were thrombosed. foci of hemosiderin deposits and moderate lymphocytic infiltration were also noted. the small vessels in some areas hadn't discernible lumina so that areas appeared solid. mitotic figures and intraluminal papillary tufting were not seen, but infiltration of skeletal muscle bundles and rarely perineural spaces were present. the muscle fibres showed atrophy, degenerative changes and signs of regeneration. the cells lining both cavernous and capillary-sized vessels showed expression of factor viii-related antigen. conclusions presented case of tumor arising in the quadriceps muscle show histomorphological and immunohistochemical features of mixed type hemangioma with pronounced infiltrative pattern. alveolar soft part sarcoma: a review of cases at national cancer institute from colombia ol mejia mejia instituto nacional de cancerología, colombia, bucaramanga, columbia aims to review the histology and immunohistochemistry of alveolar soft part sarcoma with comparison of clinical features. material and methods histology slides, paraffin blocks and files of patients were reviewed. results and discussion there were patients retrospectively analysed, female and male, between year and years old. the initial symptom was mass. the most frequent location was thigh ( %), followed by the leg, orbit, chest wall, trunk, arm and forearm. six patients had haematogenous metastases at the time of diagnosis (lung , brain , lung and bone ). histologically, all tumours were composed of nests of medium sized cells arranged in pseudo-alveolar or lobular patterns, separated by fibrovascular stroma. pas & pas diastase were positive in all cases, with rhomboid or rod-shaped crystals. immunohistochemical staining for myo d showed cytoplasmic positive in cases ( %), desmin in and smooth muscle actin in cases. conclusion alveolar soft part sarcoma is an uncommon neoplasm. the microscopic picture is a constant and typical feature of the lesion, and immunohistochemistry is a useful complementary method for diagnosis. a jiménez sánchez, em huertas valero, ja ramos Ńíguez, r baeza guixot, p sánchez cańizares hospital francesc de borja de gandia, gandia, spain introduction malignant epithelioid schwannoma is a rare variant of malignant nerve sheath tumor (mpnst) that often causes differential diagnostic problems with other neoplasm. material and methods a -year-old woman presented with a palpable tumor located in the supraclavicular region. the lesion was partially resected. grossly, the tumor was firm, gray to whitish and slighty nodular. standard histological procedure and inmunohistochemical study was performed. results microscopic examination revealed a solid proliferation of moderately large, rounded epithelioid cells, with pleomorphic nuclei, prominent nucleoli and atypical mitoses. in peripheral areas the tumor showed smaller cells with nuclei arranged in palisades and central necrosis. immunohistochemical studies showed vimentine, s- protein, and cytokeratine positivity and hmb- negativity. the histopathology and the immunohistochemical results were consistent with mpnst, but a ssx /syt gene fusion was detected by molecular analysis, a characteristic chromosomal change of synovial sarcoma (ss). conclusions spindle cell tumors with epithelioid areas can be difficult to diferentiate by histologic pattern alone. cytogenetic analysis and molecular studies may reveal the diagnosis by demonstrating a characteristic chromosomal abnormality or molecular rearrangement. ssx/syt gene fusion results from the t(x; )(p . ;q . ). the translocation fuses the syt gene from the chromosome long arm to either of highly homologous genes on the chromosome x short arm, ssx or ssx . the translocation t(x; ) is currently regarded as a specific molecular marker of ss. our case shows, that we might consider that ss may have a larger histologic spectrum than previously thought. a case of idiopathic retroperitoneal fibrosis (ormond's disease) and renal supracapsular liposarcoma t georgiev , e nakov , r gajdarski department of pathology, faculty of medicine, sofia, bulgaria department of pathology, military medical academy, sofia, bulgaria background idiopathic retroperitoneal fibrosis is an uncommon inflammatory disease characterized by proliferation and fibrosis of retroperitoneal tissue complicated by obstruction and encasement of retroperitoneal structures. case report we describe a -year-old female patient who underwent a gynecologic operation for left ovarian cyst in october . a periaortic mass alongside with peritoneal nodules were biopsied which revealed endometriosis, necrotic xantomatous nodules and ormond's disease. the last diagnosis was rejected and tumor misinterpreted as malignant fibrous histiocytoma. the course of the disease progressed and on computed tomography a left renal supracapsular tumor measuring / / cm together with a diffuse retroperitoneal fibrosis was demonstrated. in november , a tumor localized in the adipose capsule of the left kidney was removed together with the kidney weighing , kg. the kidney was markedly atrophic. a parallel excision of ten retroperitoneal tissue masses with a size up to / / cm was performed. our final histological diagnosis was highly differentiated liposarcoma. four months later the abdominal ct and the tumor markers were normal. conclusion the symptoms in our case were not specific for renoureteral obstruction and included abdominal pain and mass, weight loss and ascites during the year prior to second operation and finally urinary symptoms. we interpret our case as a slowly growing perirenal lipomatous tumor gradually compressing the kidney and causing slowly progressing retroperitoneal fibrosis. a rare case of thoracic angiomatosis in an adult and review of the literature n lapidus, d andrejev, a arro north-estonian regional hospital, tallinn, estonia introduction angiomatosis is a rare condition in which large segments of the body are involved by proliferating vessels. the majority of angiomatoses present during childhood or infancy. a diffuse angiomatosis affecting thoracic organs is a rare, frequently fatal disorder. the origin of the lesion is unknown. the lesion consists of thin-walled sometimes bloodless capillary blood vessels. the definite diagnosis is usually made by open lung biopsy or autopsy. in our paper we are presenting the rare case of angiomatosis of pleura and lung in -years old woman and the review of the literature. the patient was presented to the hospital with complaints to the pain in the right chest, dyspnea, weakness. the lesion manifested on chest radiographs and ct scans with unilateral pleural fluid and nodular pleural thickening. thoracocenthesis was performed and light brown fluid was drained. cytologically the effusion contained erythrocytes and lymphocytes. further ml more fluid was obtained. the openlung biopsy revealed the extensive diffuse angiomatosis affecting visceral and parietal pleura and subpleural lung tissue. the lesion consisted of bloodless, thin walled, endothelium channels. factor viii-related antigen and cd were the most reliable immunocytochemical markers in highlighting the endothelia. the palliative methods of treatment were not effective, and the patient was treated by total pleurectomy. conclusion diagnosis is based exclusively on histologic finding as the only possibility of diagnosis. open pleurectomy is the most succesful treatment in preventing reaccumulation of the effusion. background chronic inflammation with remodelling of the airway wall structures is an essential characteristic in asthma patients. lysosomal proteinases are involved in extracellular proteolysis, which leads to matrix changes of subepithelial connective tissue in the airway wall. the aim of the study was to localise the expression of different cysteine proteinases and their endogenous inhibitors in lung tissue of asthma patients. material and methods we included ten specimens of bronchial biopsies of asthma patients and lung tissue specimens of fatal asthma case. tissue specimens were fixed in formalin and embedded in paraffin. h&e slides were used for morphological examination. immunohistochemistry was performed on an automated stainer (benchmark, ventana, tuscon, az, usa). we used monoclonal antibodies to visualize cysteine proteinases (cathepsin b, h, l and s) and their inhibitors (stefin a and b, cystatin c) (krka, ljubljana, slovenia). results cathepsin b showed the most intense and wide distributed positive reaction. it was localized in the respiratory epithelium, macrophages, lymphocytes, mucous glands and smooth muscle cells. cathepsin h showed intense reaction localized in the respiratory epithelium, macrophages, lymphocytes, mucous glands, but not in the smooth muscle cells. cathepsins l and s showed positive reaction only in macrophages. the positive reaction of all three inhibitors was weaker compared to cysteine proteinases; it was observed in the respiratory epithelium, macrophages and mucous glands. conclusion these results suggest involvement of cysteine proteinases and their endogenous inhibitors in asthma, showing distinct immunoreactivity and different distributions in the lung tissue of asthma patients. since the inhibitor expression is less intense we can speculate that remodelling of the extracellular matrix may be also a consequence of imbalance between cysteine proteinases and their inhibitors. the correlation between aberrant connexin mrna expression induced by promoter methylation and nodal micrometastasis in non-small cell lung cancer jt introduction reduced connexin (cx) gene expression has been shown in most of lung tumors and lung cancer cell lines. although aberrant cx gene expression was linked with lung tumorigenesis, our understanding to the mechanism was still limited. we hypothesized that the evidence of aberrant cx gene expression was gradually intensified from adjacent normal lung tissues surrounding tumors towards to tumor tissues. materials and methods in this study, lung tumors and adjacent normal tissues were collected to examine cx mrna expression by reverse transcription-polymerase chain reaction (rt-pcr). results our data showed that cx mrna expression in adjacent normal lung tissue was significantly correlated with nodal involvement (p = . ), but the similar trend was not observed in tumor tissues. to verify whether lack of cx mrna expression was resulted from promoter methylation, pcr-based methylation assay was performed for cx promoter methylation analysis. high frequency of promoter methylation was observed in cx mrna negative patients ( of , . %) compared with cx mrna positive patients ( of , . %, p < . ). to elucidate whether aberrant cx gene expression is originated from adjacent normal lung tissues, lung tumors and each of five adjacent normal tissues with various distances from tumor tissues were collected to examine cx mrna and protein expression by rt-pcr and western blot, respectively. as the results shown, cx mrna and protein expressions were gradually decreased from adjacent normal lung tissues to tumor tissues with a positive correlation to the distance from tumor tissues. gel shift assay data also revealed that shifted band binding with ap was only observed in adjacent normal tissues which were far from the tumor tissues. conclusion these results indicate that promoter methylation may interfere with ap binding to promoter to cause aberrant cx gene expression. thus, cx mrna in adjacent normal tissue surrounding lung tumor simply detected by rt-pcr may be act as a molecular marker of nodal micrometastasis in nsclc. introduction an adenoma-carcinoma sequence is recognised in peripheral lung. atypical adenomatous hyperplasia (aah) and bronchioloalveolar carcinoma (bac) are the pre-invasive precursors of invasive adenocarcinoma, which frequently has a 'residual' peripheral bac component. the biology of this neoplastic progression is poorly understood. the aim of this study was to examine the expression of the tumour suppressor gene fhit, cd v , e-cadherin, beta-catenin, mmp- , timp- , and the cell cycle marker mcm at different stages in the proposed sequence. materials and methods sections from formalin-fixed, waxembedded tissue blocks of aah, bac and invasive adenocarcinomas, stained immunohistochemically for the above molecules, were scored semiquantitatively. in invasive tumours, any bac component was scored separately. for mcm a labelling index was determined as the ratio of immunopositive to total cells counted. results fhit protein is abundant until the disease becomes stromally invasive (p< . ). cd v levels are high in aah and bac, but fall in invasive disease (p= . ). e-cadherin and beta-catenin levels rise between aah and bac (p< . ), and remain well expressed thereafter. mmp- and timp- expression is relatively low in aah, rises in bac but falls when tumour becomes stromally invasive (p < . ). the bac component of invasive tumours expresses some molecules in a manner similar to pure bac but mirrors expression in invasive tumour for others. mcm counts increase from . %(low grade aah) to . % in invasive adenocarcinoma (p= . or less) conclusion in peripheral adenocarcinogenesis, loss of fhit is a relatively late event, while cd v loss occurs earlier. fhit loss has potential as a surrogate marker of invasion. e-cadherin and beta-catenin expression probably reflects changes in lesion architecture. mmp- /timp- may be implicated in bac but down-regulated as stromal invasion develops. increasing cell cycle entry is consistent with neoplastic progression. immunohistochemical study of squamous and adenosquamous cancer of lungs g methods using formalin-fixed and paraffin-embedded tissues, we examined by immunohistochemistry tenascine, collagen iv, cd , mmp , mmp , timp , timp , cd , bcl , p , pcna, ki , cyclin d in cases of lung carcinoma - squamous and adenosquamous with (first group) and without (second group) metastases. results the positive staining with tenascine was observed in all cases of squamous and adenosquamous carcinoma, but the most strong expression was found in extracellular matrix and tumor cells of carcinomas of first group. the overexpression of mmp and mmp was found in cases with metastases in tumor cells and stromal cells. the reaction was localised on periferical zone of tumor complexes. at the same time reaction with timp was low in tumor of second group and very low -in first group, reaction with timp was moderate in metastatic tumor and low -in second group. overexpression pcna and ki changed from % to % in group and from % to % in group. the increase expression of cyclin d was only in adenosquamous lung carcinoma. the overexpression of p was found only in metastatic tumors. conclusion there are some difference between squamous and adenosquamous lung carcinomas in expression of proliferation markers, and cyclin d , but no difference in expression of metalloproteinases and their inhibitors. we found more prominent differences in groups of metastatic and nonmetastatic tumors. lack of her- /neu amplification or overexpression in malignant pleural mesothelioma (mpm) introduction the her- /neu receptor, a member of the epidermal growth factor receptor (egfr) family, represents a novel therapeutic target for the newly marketed recombinant anti-her monoclonal antibody herceptin®. mpm mortality continues to rise steeply ( % to % per year) with no known curative modality. approximately % of mpms have high levels of expression of egfr. two recent studies have reported expression of her- /neu in mpm but there was no analysis of overexpression or amplification. in this study, the amplification/overexpression of her- /neu gene has been evaluated in a series of consecutive mpm biopsy specimens. materials and methods the tumour specimens had been formalin-fixed and paraffin-embedded. immunohistochemistry (ihc) for her- /neu overexpression was performed using the dako herceptest kit. only membrane staining intensity and pattern were evaluated using a to + scale with + being the lower limit for a positive test. amplification of the her- /neu gene was tested with the fluorescent in-situ hybridisation (fish) method (ventana inform her- /neu probe) in out of the cases. cases were considered as amplified by fish when the mean number of fluorescent signals per nucleus was greater than four. results of the mpms evaluated for the overexpression of the her- /neu receptor, all scored on the ihc test and all cases additionally assayed by fish were negative. conclusions although the sample size was limited, these findings suggest that mpm do not overexpress or amplify her- /neu, which therefore should not be considered a prognostic and predictive marker in mpm. (this work was funded by the alessandria branch of the italian league against cancer). morphological changes of bronchial glands at the localisation of preceding biopsy introduction at the location of preceding biopsy, in sero-mucous bronchial glands, there are certain changes of regenerative type. hyper-plastic and meta-plastic squamous epithelia partially or wholly fills the acinus and duct of secretory glands. aims: regenerative atypical characteristics found in repeated materials can be misinterpreted as epidermoid or muco-epidermoid carcinoma. material and methods the research used materials made by re-biopsy of bronchia, with regeneratory changes in bronchial glands. presence of morphological parameters was examined semiquantitatively, and then processed with methods of descriptive statistics. results in . % lobular architectonics of the lesion was preserved; fibrin exudates were present in . % cases; granulocytes in stroma were present in . % cases; granulation tissue in . % cases; planocellular metaplasia of duct and acinus was present in every single case. in . % cases mucin-secreting cells were found in squamous epithelia. the squamous cells were diskeratotic in % cases; with polymorph nuclei in . % cases; hyper-chromatic in %; multi-nuclear in . %; with visible nucleolus in . %, and with mitotic activity in . % cases. in . % cases the squamous cells were located in the fibrous tissue. mucus in interstitium was present in . % cases; cystoid formations were present in . % cases. the changes are built of solid material, undifferentiated and/or squamous cells located in the granular tissue or in the fibrin. infiltrates of granulocytes are found in fibrin, stroma, and squamous epithelia. epithelial cells in regeneration show atypical cell characteristics in small percentage and therefore are hard to differentiate from carcinoma. immunohistochemical expression of her- /neu in patients with lung carcinoma g petrusevska , b ilievska, s banev, s smickova institute of pathology, skopje, republic of macedonia the her- protein or p her is a membrane receptor with tyrosine kinase activity encoded by he /neu gene. overexpression of her- /neu has been observed in many human cancer, including lung cancer. there are different reports about the expression of this protein in different lung cancer. the aim of the study is to determine the expression of her- protein in the spectrum of lung cancer (adenocarcinoma, squamous cell carcinoma and small cell carcinoma). the study population consisted of two groups: patients that have undergone to surgical treatment and patients that have undergone to fiber-optic bronchoscopy and biopsy for primary diagnosis.tissue specimens were neutral formaline fixed and paraffin embedded. standard histochemical and immunohistochemical staining were used for diagnosis. expression of her- /neu protein was determined by immunohistochemical staining with hercep testtm (dako). the results were graded - as negative and - as positive. overall incidence of her- /neu overexpression was , % ( of ). higher incidence was found in patients with adenocarcinoma , % ( of ); squamous cell carcinoma , % ( of ) and small cell carcinoma % ( of ). no statistical significant difference was seen with age and gender. her- /neu overexpression was more relevant for patients with advanced tumor: all patients with squamous cell carcinoma were in stage b and , and % with adenocarcinoma were in stage a and b. these results are satisfactory and encourage us to continue this work in the follow-up study to evaluate her- /neu role as predictive and prognostic factor for patients with lung cancer. we applied immunohistochemistry on six neuroendocrine lung carcinomas using moabs specific for extracellular matrix proteins and integrins; moreover transwell test migration assays evaluated the rate of transendothelial migration of ae- neuroendocrine carcinoma cell line throught a monolayer of eahy endothelial cell line expressing laminin-α -chain to establish wheather this protein may favour in vivo vascular invasion and metastasis. results %, %, %, %, % and % of vessels were respectivelly distributed within the parenchyma of these tumors. studies on contignous sections demonstrated that parenchymal vessels were positive for all ecmps, but laminin-α -chain, which was respectively expressed in %, %, %, %, % and % of parenchymal and in %, %, %, %, % and % of stromal vessels of these tumors. in all cases about % of neoplastic cells were α positive. preliminary results of transwell test migration assay indicate that the pretreatment of the ae- cell suspension with anti laminin-α -chain and anti α -integrin moabs reduced their migration through the eahy monolayer. conclusions as known α -integrin is a receptor of laminin-α chain, therefore the up-regulation of this protein in a large number of vessels during tumor angiogenesis may favour metastasis increasing neoplastic cell adhesiveness to newly formed vessels. brain death as a cause of primary lung graft disfunction in an isolated rabbit lung model ma introduction early lung dysfunction observed after lung transplantation has been attributed to an increase of vascular permability. factors involved in this mechanism are unknown. our hypothesis was that brain death (bd) is a preconditioning factor that induces primary lung graft dysfunction. methods twenty-four rabbits were randomized to control (crl) or induced bd (increasing intracraneal pressure above systolic pressure for minutes) groups and were ventilated in volume controlled mode (tidal volume ml/kg, positive end expiratory pressure (peep) cmh o and respiratory rate (rr) to obtain normocapnia) for minutes. isolated lungs were perfused with ml/min (constant flow) and ventilated in pressure controlled mode (peack pressure cmh o, peep cmh o and rr ) for minutes. pulmonary edema and lung injury were assessed by weight gain, change in ultrafiltration coefficient before and after ventilation; and extent of hemorrhage (scored by histology: intensity of alveolar hemorrhage, percentage of alveoli with hemorrhage and number of vessels with perivascular hemorrhage) mean values ( samples per lung) of the indices of hemorrhage showed above were scored ( to ) and added to obtain a score of overall lung hemorrhage (range to ) for each lunch. data are expressed as mean and % confidence interval and were compared with paired t test. conclusions under these experimental conditions bd contributes to pulmonary edema and lung hemorrhage. expression of cyclooxygenase- in nonsmall cell lung carcinomas n akyürek introduction cyclooxygenase (cox) is an inducible enzyme that catalyses the conversion of arachidonic acid to prostaglandins and thromboxanes. cox- is induced by cytokines and growth factors and has been shown to be important in carcinogenesis. the aim of this study was to investigate the expression of cox- and clinicopathologic factors in nonsmall cell lung carcinomas (nsclc) with regard to survival times. materials and methods seventy-seven nsclc specimens were evaluated for cox- expression by immunohistochemical method. staining patterns were assessed semiquantitatively and correlated with tumor histologic type, tnm stage and prognosis. chi-square and kaplan-meier survival analysis was performed. results cox- was not expressed in the normal bronchial epithelium. cox- expression was detected in ( . %) nsclc. expression of cox- was significantly higher in adenocarcinomas than in other histologic subtypes (p= . ). there was no significant relationship found between cox- expression and age, gender, tnm stage, and prognosis. conclusion this study suggests that an increase in cox- expression may be associated with the development of adenocarcinomas. but, prognostic significance of cox- expression is controversial. prognostic significance of cyclin-dependent kinase inhibitor p and bcl- protein in nonsmall cell lung cancer introduction the expression of p , which is known a cyclindependent kinase inhibitor, has considerable value for the prognosis of cancer patients. bcl- gen product (bcl- protein) is implicated in oncogenesis by its ability to prolong cell death through the inhibition of apoptosis. we studied the expresssion of p and bcl- protein to investigate their association between clinicopathologic factors and prognosis in nonsmall cell lung cancer (nsclc) materials and methods patients with nsclc were included in our study.there were patients( men, women), patients had adenocarcinoma (ac) and had squamous cell carcinoma (scc).tumors were examined immunohistochemically with p and bcl- protein expression, using formalin-fixed, parafinembedded tissue. for the evaluation of p expression, the number of nuclear staining was counted and showed as a percentage.bcl- protein was primarily identified in the cytoplasm of tumor cells. statistically, correlation between immunoreactivity and the clinicopathologic factors were evaluated. results p immunoreactivity was observed in ( %) of cases. there were ( %) scc, ( %) ac. statistically, these findings were found significant (p= , ). percentage of p positive nuclei, the average being %. positive bcl- immunostaining was found in ( , %) of cases. cases ( , %) of scc and ( %) of the ac were positive. these findings were not found significant (p= , ) conclusions there is no correlation between p and bcl- expression and clinical parameters, clinical stage, pathologic grade and prognosis. our study suggest that p immunreactivity in nsclc may provide prognostic information. pulmonary hamartoma -report of four cases introduction neuroblastoma is a malignant tumor of early childhood and is very rare in adults, thus we report this case. case report we describe a case of a -year-old man, never before notably ill. basic laboratory values were not significantly anomalous except of mild anaemia and high erythrocyte sedimentation rate. an x-ray scan showed a spherical opacity cm in diameter in the top right pulmonary field paramediastinally. ct displayed a low-density tumor x , x , cm in the apex of the right hemithorax. the tumor was well circumscribed and seemed to be in a subpleural localisation. it was subsequently punctioned with chiba and core needle under ct control. cytological and histological material did not contain any malignant cells. on mr scan there was a well encapsulated extrapulmonary tumor without destruction of ribs, probably of benign origin. tumor extirpation was indicated. the lesion was growing out of spinal chord. it was completely removed. grossly, the tumor looked like an old hematoma. histologically, it had the typical appearance of neuroblastoma -malignant tumor which is found in more than % of cases in patients under years of age. a scintigrafical examination and trephine biopsy showed massive bone marrow infiltration by neuroblastoma. conclusion a -year-old man was confirmed to have a typical early childhood tumor -neuroblastoma in a subpleural localisation. despite the poor prognosis of this tumor in adults and a wide bone marrow infiltration, the patient is well and without signs of disease after surgical resection and two cycles of chemotherapy over months after the initial diagnosis. measurements of dna amount and nuclear size by image cytometry: are these two features sufficient for diagnosis of early lung cancer? background lung cancer represents % of all cancers, but is responsible for nearly % of cancer related deaths due to the fact that it is most often detected in a very late stage. it has been proposed that selected nuclear features could be used to assist lung cancer detection in an early, operable stage. it was shown that both dna amount (dna ploidy) and nuclear size are key features for positive diagnosis in oral, laryngeal, bronchial, and gastric epithelial tumors. we used image cytometry to assess the potential of this approach using nuclei of epithelial lung cells from sputum samples. experimental design sputum samples were obtained by induction method, using hyperosmolar salinic solution, from lung cancer patients and high risk individuals. the cells were fixed and after breaking mucosa with mechanical and chemical treatment, they were cyto-centrifuged onto the microscopy slides. cell nuclei were stained by a dna specific and stoichiometric (feulgen-thionin) stain and dna amount (ploidy) and nuclear size of (on average) cell nuclei from each sample were measured by a high resolution image cytometer. the results showed that there were no statistical differences between the dna ploidy values between control and cancer cases, as very few cancerous cells were found in the sputa of lung cancer patients. interestingly, there was a measurable increase in the mean nuclear area of diploid cells from cancer patients in comparison to control cases. conclusions despite the slight difference in the size distribution the nuclear size feature alone does not provide effective discrimination between the negative and positive populations. pulmonary blastoma: report of cases d yilmazbayhan, mg gulluoglu istanbul university, istanbul faculty of medicine, department of pathology, istanbul, turkey introduction pulmonary blastomas (pb) are rare lung tumors having unique histomorphological features. although they were once regarded in the same group of lung neoplasms with well differentiated fetal adenocarcinomas, recently the two tumors were seperated from each other. we aim to discuss the morphological features and the changes of morphology on recurrences of pulmonary blastomas. we present ten cases of pb, six of which were biphasic, two were pleuropulmonary, and two were cystic. biphasic pb patients were five males and one female, with an age range of - . two female patients with pleuropulmonary blastoma were and years, and cystic pb patients were and years old female and male children, respectively. results one of our most striking histomorphological findings was that four of the biphasic pbs accommodate mature epithelium, not fetal epithelium. one of the other two cases presented with a fetal adenocarcinoma and the tumor recurred as a biphasic pb. one of the cystic pb, which arised in a background of congenital bullous emphysema, recurred with pleomorphic cells forming solid groups. the other cystic pb was diagnosed as congenital cystic adenomatoid malformation. conclusion because of the changing morphological features and composition of epithelial component, classification of the pulmonary blastomas should be reviewed. (romano et al., faseb j. : , ). aim of our study was to investigate in vivo expression of -lo in human mesothelioma. materials and methods immunohistochemical analyses were conducted in five micron sections from human pleural mesothelioma ( epithelial, biphasic and one fibrous type). -lo expression was evaluated using a -lo polyclonal antibody. results immunohistochemical staining was observed in all cases. the staining pattern was cytoplasmic in cases ( epithelial, biphasic and fibrous type), both nuclear and membranous in one case of the epithelial type, both nuclear and cytoplasmic in cases ( epithelial and biphasic type), nuclear, cytoplasmic and membranous in two cases of the epithelial type. conclusion these results are consistent with our in vitro data showing -lo expression in mm cells and strongly suggest that -lo may play a relevant role in the pathobiology of malignant pleural mesothelioma. the present study is scheduled to analyze the intratumoral expression of vascular endothelial growth factor (vegf) in human lung cancer and to determine the relation with microvessel density (mvd), e-cadherin expression and clinicopathological parameters. a -years old male presented with chest discomfort, dyspnea, weakness. heart function tests were normal. ct examination revealed a well demarcated mass in the anterior-superior mediastinum as well as pleural and pericardial effusions. there was no clinical/radiographic evidence of tumor elsewhere. a surgical excision of the mass was performed. grossly, the mass was round, well demarcated, measured x . x cm. the cut surface showed solid whitish or reddish with yellowish foci areas and cystic areas filled with mucinous fluid. histologically, the tumor consisted of sheets and nests of atypical squamoid cells intermingled with gland-like lumens. areas of clear cells were also present. in the tumor stroma, abundant amounts of mucin, foci of necrosis, lymphatic invasion, intense inflammation and cholesterol granulomas were observed. in some areas, the tumor cells were seen in contact to the lining of the cystic cavities. the latter were lined by a single layer of cuboidal or flattened or stratified epithelium with morphological features of squamous cells. at the periphery of the tumor, rare lymphoid follicles, residual thymic elements or degenerative hassall's corpuscles were seen. these histologic features were more consistent with a mucoepidermoid carcinoma arising in association with a thymic cyst. the patient died two months after chemotherapy administration. mucoepidermoid carcinoma of thymus is a rare entity and, to our knowledge, very few cases have been described in the international literature. however, in case of absence of metastatic disease or other common primary tumors of thymus, the diagnosis of a mucoepidermoid carcinoma should be taken into consideration. tissue array for immunohistochemical profiling of intrathoracic localized fibrous tumors ultrastructure of bronchial mucosa in chronic obstructive bronchitis n zoirova, s ariphkhanova uzbek institute of medical rehabilitation and physical therapy, tashkent, uzbekistan the purpose of this investigation was to determine ultrastructural peculiarity of bronchial mucosa in the patients with chronic obstructive bronchitis (cob). the study was performed in bronchial biopsy samples obtained from patients with cob. material was fixed in , % glutaraldehyde, postfixed with % osmium tetroxide and embedded in epon. ultrathin sections were stained with uranyl acetate and lead citrate. morphological three types of cob have been reported, including chronic catarrhal, catarrhal sclerotic and sclerotic forms. catarrhal cob was characterized with significant increasing of goblet cell count as compared to the bronchial mucosa in controls. hyperplasia of endoplasmic reticulum and golgi complex was observed. in the goblet cells, numerous secretory droplets were crowded together where they have been deposited from the surrounding cytoplasm. in ciliated cells, few cilia were seen in longitudinal section extending from the apical pole of epithelial cells, fragmentation of the endoplasmic reticulum and appearance of autophagosome were detected. in other types of cob, number of cell rows was decreased with different secretory activity of goblet cells. interepithelial migration of lymphocytes with the damage of the basement membrane was described. our findings demonstrated more aggressive changes of bronchial mucosa in sclerotic cob. chest trauma-induced rupture of benign mediastinal teratoma followed by a possible implantation of the tumor into the parietal pleura: a case report introduction mediastinal teratomas rarely rupture. an unusual case of chest trauma-induced rupture of benign mediastinal teratoma followed by a possible implantation of the tumor into the parietal pleura is reported. case presentation in a -year old woman, a pre-employment plain chest radiograph and ct of the chest revealed a large mediastinal tumor. two smaller tumors were located on the left parietal pleura. on operation completely obliterated left pleural space and three well-encapsulated tumors were found. microscope examination revealed three well-differentiated teratomas with no evidence of malignancy. three years before the detection of the tumor the patient had a blunt chest trauma, with a left-sided chest pain and shortness of breath, which gradually disappeared. at the time the plain chest radiograph was analyzed as normal. retrospectively an unusual mediastinal line, adjacent to the aortic arch, was disclosed. this line could be due to a small mass in the mediastinum. the left diaphragm was elevated, and a small leftsided pleural effusion could have been present. we suspect that teratomas located on the parietal pleura were caused by implantation of the tumor cells after the rupture of the mediastinal teratoma into the pleural space during blunt chest trauma. conclusion the chest trauma seen in presented case, could be at least a trigger, if not the only cause of the mediastinal teratoma rupture. the rupture was probably followed by both, obliteration of pleural cavity and possible implantation of benign tumor into the parietal pleura. the introduction malignant mesothelioma is an aggressive tumor with adverse prognosis (median survival - months) both in treated and untreated patients. in fact, only few patients survive more than two years. the identification of prognostic factors is critical to evaluate life-expectancy and crucial to approach novel therapeutical strategies. the study was performed to evaluate the use of mib- and p kip antigens as prognostic indicators of survival in malignant mesotheliomas. materials and methods a series of surgical biopsies of patients with known follow-up were immunohistochemically examined. the results were compared with survival data and correlated with clinical and histological parameters. for immuno-histochemistry, sections were stained for mib- and p kip antigens using abc method. number of positively stained nuclei was evaluated in consecutive high magnification fields ( neoplastic cells were considered the minimum number to include the case in the study). results mib- , which is largely expressed in sarcomatoid mm (p= . ), is more represented in patients with shorter (< months) then in subjects with medium ( <months< , p< . ) and longer (> months, p< . ) survival. on the other hand, p is more expressed ( ± . ) in patients with longer (p< . ) then in subjects with shorter survival. pearson's analysis showed no significant correlation between mib- expression and p , but this was predictive of survival in the entire population (r= . , p< . ). conclusion the findings suggests that p and mib- expression, although reliable attribute of mm, represent independent markers of prognosis in malignant mesothelioma. familial clustering of malignant mesothelioma without direct contact to asbestos. a genetic study introduction very few papers dealt with clustering of familial malignant mesothelioma (mm). most of these, who developed mesotheliomas, presented work or domestic history of exposure to asbestos, but only in very few of them asbestos did not demonstrated any association. the aim of this study was to contribute to the hypothesis of a genetic predisposition in the pathogenesis of the mesothelioma. methods we report two cases of pleural malignant mesothelioma that affected two young sisters as part of a nine-member family with enviromental exposure to asbestos. they were affected by marfan's syndrome and both developed pleural malignant deciduoid mesothelioma with long survival. we have performed comparative genomic hybridization (cgh) analyses of tumor samples to identify critical chromosomal regions involved in the onset of mm. we found similar chromosomal changes in both cases and in particular we found losses at q -q and q . -q chromosome. conclusions early onset s. and same genetic impairments in the two sisters suggest that genetic factors may induce the onset of this cancer in those subjects who are exposed even to small environmental amounts of asbestos. however, the specific chromosomal changes we found might provide evidence for locations of relevant suppressor genes of familial mm. video assisted versus needle biopsy of the pleura in patients with pleural effusion results correlation was found between p- -posivity tracheal mucosa and tnm classification system: p- index: control group= %, group t = %, t = %, t = %. conclusion the opinion that the presence of p- oncoprotein reflects mutation of the p- gene is commonly accepted. other data published evaluating the relationships between p- and tnm-stage, g-stage, tumor size and site, age and sex, did not reveal any dependencies, although some studies have suggested such correlations. our analysis had shown a correlation between tnm stage and p- index of tracheal mucosa, but is significant only between control group and t , and control group and t . authors expect such results that show the correlation between prognostic factors in laryngeal malignant epithelium and tracheal epithelium. undifferentiated nasopharyngeal carcinoma (lymphoepithelioma). report of four cases v blažičević, m mrčela, Ž kotromanović clinical hospital, osijek, croatia background nonglandular nasopharyngeal carcinomas are much more common in some areas of the orient than the other parts of the world. they are subtyped as keratinizing squamous cell carcinoma (type ), nonkeratinizing carcinoma (type ) and undifferentiated carcinoma (lymphoepithelioma). we present on four undifferentiated nasopharyngeal carcinomas and detail clinical and histopathological characterisation of these rare cases. methods all our patients were male, white and - years old. initial complaints were various: hearing loss, epistaxis, headache, and dysphagia. examination of the nasopharynx showed us entirely normal features, or fullness, surface granularity, or obvious carcinoma. but, all our patients had cervical lymph nodes metastases, two of them bilateral. involved lymph nodes were typically posterior of the sternocleidomastoid at the level of the angle of the jaw. results histologically, lesions show cytologically uniform cells with ovoid vesicular nuclei prominent nucleoli and indistinct cell borders resulting in a syncytial pattern. an inflammatory infiltrate rich in lymphocytes and occasionally containing prominent eosinophils is usually a component of lymphoepithelioma, but diagnosis is based solely on the nature of the neoplastic epithelial component. neoplastic cells are negative for lca, but positive for cytokeratin and ema. conclusion undifferentiated nasopharyngeal carcinoma (lymphoepithelioma) is very rare. about % to % of patients are initially seen with cervical lymph node metastases from an occult primary. such lesions may be different or impossible to distinguish from lymphoma on haematoxylin and eosin stained sections, particularly when the lesion appears as a lymph node metastasis from an occult primary. ancillary methods like immunohistochemistry and molecular techniques or detection of serum antibodies against viral proteins may be necessary. "atypical" pleomorphic adenoma of the parotid gland introduction pleomorphic adenoma is a benign, epithelial tumor, composed of cells that demonstrate both epithelial and mesenchymal differentiation, with the absence of abnormal cytomorphologic features. we present the case of -years old white women, with a mass localized in the superficial, lower part of the right parotid gland. on clinical examination, the lesion was nodular, painless swelling, of smooth surface, measuring x mm in diameter. the tumour was excised, fixed in formaline, embedded in paraffin and the sections were stained with he, pas and alcian blue. the tumour was composed of both epithelial and mesenchymal-like tissue, with a complete capsule at the periphery. there was no neoplastic tissue within or extending through the fibrous capsule. on the other hand, the epithelial component expressed mild, limited, abnormal cytomorphologic features, such as enlarged, pleomorphic and hyperchromatic nuclei. conclusions presence of prominent cytomorphologic atypias and capsular involvement in a pleomorphic adenoma causes concern about malignant transformation, but a lesion with mild or focally limited cytological atypias is designated as atypical pleomorphic adenoma and indicates an increase likelihood of malignant transformation if the tumor recurs. gene expression profiles were determined in fresh-frozen samples of mucoepidermoid carcinoma (mec), acinic cell carcinoma (acc), salivary duct carcinoma (sdc), and adenoid cystic carcinoma (adcc), using cdna microarrays containing cdna fragments of cancer-related human genes. the validity of findings was checked by means of real-time reverse-transcript polymerase chain reaction studies. seventy-five genes were found to be more than . times overexpressed and less than . times underexpressed than in samples of normal parotid gland. five genes, namely fibronectin (fn ), tissue metalloproteinase inhibitor (timp ), biglycan (bgn), tenascin-c (hxb), and insulin-like growth-factor-binding protein (igfbp ) were overexpressed in all of the types of carcinoma studied. sixteen genes, including mothers against decapentaplegic homolog (madh /smad ) and prostate-specific membrane antigen (psm/psa) were underexpressed in all types of carcinoma studied. average-linkage hierarchical clustering undertaken in relation to selected genes showed that high-and low-grade mecs formed one group of tumours, acc/sdc another. significance analysis of microarray findings identified genes related to the mec group, associated with the acc/sdc group. our results indicate genes that may be involved in carcinogenesis in the salivary glands. gene expression in normal and malignant salivary tissues was also found to be similar in several respects to gene expression in normal and malignant pancreatic and prostate tissues. rare tumours in the sinonasal tract p nagy, f salamon national medical center, dept. of pathology, budapest, hungary introduction sinonasal neoplasms include numerous groups of different histogenetic types. the epithelial tumours have the highest prevalence among them in the daily routine, but we can encounter such lesions, which may cause diagnostic difficulties. the purpose of this study is to determine the least frequent tumours in our sinonasal biopsy files. we collected those kinds of tumours which occurred only once in our series. we reviewed our archive material of a year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) five cases were selected. the histologic slides were re-examined. he, special stains, immunohistochemistry and electronmicroscopy were applied. the cases in the order of their occurrence: . fibrous solitary tumour, we supported our original opinion by cd immunostaining performed retrospectively. . chordoma, in addition to the morphologic features the s and cytokeratin co expression were pathognomic. . pnet, in this problematic case finally the cd positivity proved to be diagnostic. . intestinal-type adenocarcinoma, the mucocele-like appearance and the highly differentiated colonic type epithelial structures reminiscent of adenoma caused our dilemmas. . meningeoma, the immunohistochemical results were not convincing. we established the diagnosis by means of electronmicroscopy. conclusions the incidence of sinonasal neoplasms is low. the recognition of the unusual lesions in this region is a challenge for the pathologist. ancillary techniques may be necessary in the diagnostic process. squamous cell carcinoma: diagnostically problematic variants f salamon, p nagy national medical center, dept. of pathology, budapest, hungary the diagnosis of squamous cell carcinoma (scc) is seldom problematic. although it may cause difficulties for the pathologist to verify its uncommon varieties. the aim of this study was to distinguish the rare variants of scc from the conventional ones in our laryngeal tumour file and to present the most instructive cases. material and methods we reviewed our archive material of a year period ( ) ( ) ( ) ( ) ( ) . four cases were selected. the histologic slides of the preoperative biopsies and the surgical specimens were re-examined as well. he, special stains, immunohistochemistry and electronmicroscopy were applied. results . " carcinosarcoma" with rhabdomyosarcomatous differentiation. this case represents a unique example of the spindle cell carcinoma category. . verrucous carcinoma with verrucous hyperplasia. the interpretation of the latter term is a complicated issue. by virtue of our patient's follow-up we encountered this dilemma. . papillary squamous cell carcinoma (pscc). this -year long case history is a well-documented instance of a papilloma-papilloma with dysplasia-carcinoma sequence. . adenosquamous carcinoma. it was not easy to justify the clinical suspicion of malignancy preoperatively. furthermore we faced a differential diagnostic problem when we evaluated the removed larynx. conclusions in all the cases we managed to establish the definite diagnosis only by the thorough examination of the surgical specimens. additionally we found special features as far as the morphology and histogenesis are concerned. so we can confirm the subheading of the corresponding afip fascicle for this tumour group: diagnostically problematic variants. intraocular leiomyoma: case report introduction intraocular leiomyomas are rare tumours probably arising from the ciliary body smooth muscle. they represent a diagnostic dilemma and must be differentiated from neural tumours and, more importantly, from amelanotic melanoma. they occur more frequently in women, usually between the third and sixth decades of life. case report we report the case of a year-old woman with gradual loss of vision in her right eye. slit-lamp examination and binocular ophthalmoscopy showed a large and pigmented ciliary body mass located in the inferior quadrants. a and b ultrasound examination revealed a multicavitary tumor with low internal reflectivity measuring x . x . mm. enucleation of the affected eye was decided. histopathological examination showed a solid neoplasm composed of interlacing and tightly packet of spindleshaped cells with little intervening collagen. nuclei were oval with blunted ends with no significant atypia. mitotic activity was negligible. immunohistochemical stains for smooth muscle actin, hhf- , vimentin and desmin were positive. no stain was noted for s- protein, ae -ae cytokeratin, cam . cytokeratin, hmb- , glial fibrillary acid protein, neurofilaments, neuron-specific enolase and synaptophysin. final diagnosis was leiomyoma and the patient is free of disease two years after surgery. conclusions intraocular leiomyomas are of clinical concern since they are difficult to differentiate from more aggressive neoplasms, especially amelanotic melanoma. in this particular case, the situation was even more complicated because the lesion was clinically pigmented. once removed, the diagnosis was not difficult and was based on conventional histopathological examination along with confirmatory immunohistochemistry. results twenty-six of cases were male and were female. follow-up was available for cases and ranged between to months. eight cases died and had recurrences. the recurrent tumour histology changed from type to in cases, remained unchanged in . none of the clinical parameters were correlated with the survival. the type of therapy seemed to have no impact on survival. according to who classification cases were type , were type and were type and it showed correlation with survival (p= . ). lymphocytic infiltration, which, was added to who system, were seen by cologne classification in of tumours, but did not have an impact on survival. cellular morphology and atypia suggested by hsu's classification had no significance as well, although most of the tumours were classified as type a (% , ). ebv infection was found to be present in of cases. however no correlation with other parameters was found. conclusion in accordance to literature we concluded that who classification is the only parameter that has an impact on survival in npc. vascular endothelial growth factor expression and microvessel count in laryngeal squamous cell carcinoma -related antigen. paraffin embedded tumour specimens were stained with vegf and f-viii using immunohistochemical methods. vegf staining was evaluated as negative (<% of tumour cells) or positive (>% of tumour cells). staining intensity was graded mild, moderate and severe. for evaluation of f-viii staining, average counts of the three most vascular areas on an x field were recorded. statistically correlation between immunoreactivity and the clinicopathologic factors of laryngeal carcinomas was evaluated. results vegf expression in the tumour cells was found positive in all cases (% ). staining intensity was detected as severe of cases, moderate of cases, mild of cases. in evaluation of f-viii staining, average mvc of all cases was found , . statistically, no association was observed between the vegf expression, f-viii of carcinoma cells and histologic grade, tnm stage, tumour size and tumour site. association was observed between the vegf expression and mvc (p= , ). in additional, significant correlation was observed between the vegf percentage of positive tumour cells and staining intensity (p= , ). conclusion our data suggest that in laryngeal squamous cell carcinomas, vegf expression and mvc of carcinoma cells are not prognostic markers. acinic five cases of acinic cell adenocarcinoma, diagnosed in our laboratory during a period of years, are presented. four of them concerned salivary gland tumours of the parotid gland, the main site of their development, and the fifth was found as an endobronchial mass. the patients ranged from to years of age and the greatest stromal diameter varied from , to , cm. microscopically all neoplasms had the typical morphological features consistent with acinic cell adenocarcinoma. this neoplasm has distinct clinicopathological features and a rather indolent biological course, in spite of its malignant potential, responsible for the prevalence of the term carcinoma instead of "stromal". a broad histo-and immunohistochemical investigation was performed confirming the diagnosis. a review of the available reference is reported. relationship of p oncoprotein expression with histological grade and outcome of patients with oral squamous cell carcinoma introduction an alteration in the p tumour suppressor gene is the most frequent genetic abnormality in human cancers. overexpression of the p oncoprotein and histological differentiation were investigated in routinely formalin-fixed and paraffin embedded squamous cell carcinomas of the oral cavity. p oncoprotein was detected in all specimens with the monoclonal antibody do- . p oncoprotein expression was scored in the following way: score < %, score - %, score > % positive cells. results and conclusions using histological differentiation, all specimens are divided in three groups: group i -well-differentiated ( cases), group ii -moderately differentiated ( cases) and group iii -poorly differentiated carcinomas ( cases). in the first group % specimens were negative, %highly positive for the p oncoprotein. in the second group, positive detection of the p oncoprotein was observed as low in , % of cases, moderately in % and highly positive in , %. in the third group % expressed low positivity for the p oncoprotein and % of the cases had moderate and high positivity respectively. patients' survival was monitored for a five years. in the group with the highestp oncoprotein expression , % of the patients survived years or longer and , % survived less than years. mutation in the p tumour suppressor gene is highest in the well-differentiated squamous cell carcinomas, and its expression decreases in poorly differentiated carcinomas. immunohistochemical prognostic factors in laryngeal squamous cell carcinoma introduction the molecular mechanisms of growth and progression of laryngeal carcinoma are not studied well. the aim of the present study was to investigate the prognostic significance of over expressions of p protein, cyclin d , c-erbb , and markers of cellular proliferation (ki- and pcna). materials and methods primary tumours were studied on a series of patients. all patients were treated between the years and . the expression of p was analysed with monoclonal do antibody, cyclin d (clone dcs- ), c-erbb (polyclonal antibody hercep test), proliferate activity with ki- (clone mib- ) and pcna (clone pc ) results overexpression of the p protein was found in % of patients, cyclin d in %, and c-erbb -in % of patients with laryngeal squamous cell carcinomas. the reverse relationship of cyclin d with -year survival of patients (p< . ) was revealed. overexpression of the p protein was correlated with lymph node metastasis (p< . ). the value of pcna index in patients with metastasis is significantly higher than those in patients without metastasis (p< . ). the pcna index was correlated with the stages of the tnm. high level of the ki- index and the pcna index really decreases -year survival in patients. no significant correlations were found between positive p , cyclin d , c-erbb , ki- and pcna immunostaining and anatomical localization and histological grade. conclusions immunohistological examination of c-erbb is not a valuable prognostic factor in laryngeal carcinoma. high proliferation index (ki- and pcna) and expression of cyclin d correlate with worse prognosis of the disease. overexpression of the p protein is correlated with progression of laryngeal cancer. introduction warthin's tumor (wt) is a well-recognized benign neoplasm of salivary gland. granulomatous inflammation occurring in wt is a rare phenomenon. it has been suggested that prior fine needle aspiration (fna) biopsy might be the triggering factor. mycobacterial infection has also been reported as a cause of granulomatous change seen in wt. we report six cases of wt with granulomatous inflammation to analyse the possible relationship to prior fna. materials and methods a computerized search of pathology file of chang gung memorial hospital yielded cases of wt from to . granulomatous inflammation was described in six cases ( . %). fna cytology was performed in two cases before surgical excision. clinical history, histology and cytology slides were reviewed. ziel-neelsen stain was performed. results all tumours occurred in parotid gland. there were men and women. their age ranged from to yrs (mean ) and the tumor size ranged from to cm (mean ) in maximal dimen-sion. all six cases showed typical features of wt accompanied by multiple granulomas composed of epitheloid histiocytes and multinucleated langhans giant cells with or without necrosis. no acid-fast bacilli were found in any of these cases. history of prior fna was identified in two cases. both specimens also showed granulomas and features of wt. conclusions the pathogenesis of granulomatous inflammation in wt remains speculative. prior fna has been suggested to be the cause. however, this hypothesis cannot be supported by our observation of granulomas in the cytology specimens of two of our cases. it is most likely an immunologic reaction to the tumor, but a ziehl-neelsen stain should be performed to exclude the possibility of mycobacterial infection. malignant peripheral nerve sheath tumor masquerading as an allergic nasal polyp introduction approximately % of all malignant peripheral nerve sheath tumours (mpnst's) occur in the head and neck. of these, % are sporadic. the mean age at presentation is years. sinonasal tumours are extremely rare and whilst they share the morphology of mpnst's at other locations, they tend to be of low grade at this site. we describe a unique case of a high grade mpnst clinically simulating an allergic nasal polyp in an otherwise healthy child without identifiable risk factors. methods a year old black boy presented with an obstructive nasal mass, which was first felt months earlier. the clinical diagnosis was an "allergic polyp" and the mass was excised without antecedent roentgenographic investigations. results the x x cm tumor was unencapsulated but circumscribed, lobulated and friable. histologically, predominantly highgrade sarcomatoid and epithelioid areas were seen. however, differentiating features of peripheral nerve sheath tumor were also found. these included alternating hypercellular and hypocellular areas, palisading nuclei, perithelial accentuation of cells and serpentine, buckled nuclei. in low-grade areas, axons were demonstrated by silver staining. of the broad panel of immunohistochemical stains done, only s protein positivity was observed. the latter was uncharacteristically prominent. conclusion the young age of the patient, the tumor location and clinical presentation as an allergic polyp, the predominantly high grade microscopic appearance and the strong s protein positivity of this mpnst are unusual and hence worthy of documentation. follicular dendritic sarcoma in a black teenager -report of a south african case and literature review t n c) . histologically, the tumor cells were large with a low nuclear:cytoplasmic ratio, vesicular chromatin, and prominent nucleoli. the cells were arranged in organoid and trabecular patterns in a background of extensive necrosis and mitotic figures. ultrasound biopsy of the liver revealed metastatic disease. the patient died within weeks of surgery. the cause of death was unknown. no post-mortem examination was conducted. immunohistochemical expression of antikeratin ae /ae , chromogranin, and synaptophysin was evaluated. electron microscopy was performed. the tumor cells stained with anti-keratin ae /ae , chromogranin, and synaptophysin. electron microscopy revealed dense core granules. these findings, along with the morphologic criteria, fit with a lcnec. conclusion lcnec is a rare tumor in the larynx. recognition at this site is essential so that proper patient management can be initiated. analysis of blood serum lymphocytic phenotypes in chronic hepatitis c infected patients with advanced fibrosis and periodontium diseases introduction few studies focus on periodontium diseases in the course of chronic viral hepatitis c. the aim of this study was analysis of the relationship between the stage of periodontium disease and a number of lymphocytic phenotypes in blood serum of patients infected with chronic hepatitis c and advanced fibrosis. the study included patients (mean age of , years), whose blood serum revealed the presence of hcv-rna, demonstrated with rt-pcr method, while biopsies helped to reveal chronic hepatitis with septal fibrosis or cirrhosis. periodontium inflammation was assessed according to the following criteria: inflammation limbus, swelling, gingival pocket depth, presence of supragingival and subgingival calculus and gomphiasis. each criterion was assigned or point, cumulatively from to points. flow cytometry analysis included monoclonal antibodies: cd , cd , cd , cd /cd /cd , cd provided by becton-dickinson. analysis of morphological parameters and fluorescence was performed with cellquest computer software. results all patients with advanced liver fibrosis were found to show signs of advanced periodontum diseases (mean cumulative value of , points). the diseases most frequently encountered included: bleeding and calculus (n= ), deep gingival pockets (n= ) and gomphiasis (n= ). the degree of periodontal disease depended on the total number of t lymphocytes (p< , ) and on the number of t helper lymphocytes (p< , ). no significant dependencies were found between the degree of a periodontal disease and nk cells, b-lymphocytes or t suppressor. conclusion degree of periodontum disease in patients infected with hepatitis c is dependent on low values of t and t helper lymphocytes in blood serum. adenomatous ductal proliferative foci accompanied the warthin tumour in parotid gland t ohuchi , adenomatous ductal proliferation (adp) and intercalated duct hyperplasia (idh) which are two new similar disease concepts in the field of salivary gland have been reported recently focusing on the relationship of precursor lesion to some salivary gland tumour, differential diagnosis, and possibility of dedifferentiated lesion. we report here a case of warthin tumour accompanied by the foci of the adenomatous ductal proliferation in the parotid gland and these epithelial foci exhibited the histological and immunohistochemical findings resembling tubular type of basal cell adenoma. a -yearold female received the resection of warthin tumour in the parotid gland, which was diagnosed as a benign parotid gland tumour. macroscopically, this tumour showed two small legions in the parotid area, x mm and x mm in sizes respectively. the bigger legion was a typical warthin tumour consisted of papillary epithelial projections into cystic spaces associated with variable number of lymphocytes. papillary projections were lined by eosinophilic two cell layers having luminal columnar cells and basal polygonal to triangular shaped cells. on the other hand, histologically, the smaller legion resembled tubular type of basal cell adenoma. this lesion showed ductal proliferation with some admixed acinar cell complexes having a fibrous capsule in its half area. ductal proliferation consisted of bilayered structure (inner luminal cells were strongly positive for cytokeratin and weakly positive for s- protein). some stromal areas around the ductal components appeared to be cellular with spindle shaped cells strongly positive for s- protein immunohistochemistry. introduction malignant rhabdoid tumour of the oral cavity is very rare. to our knowledge, there has been one reported case of malignant rhabdoid tumour of the tongue so far. patient and methods a case of an -year-old female with malignant rhabdoid tumour of the tongue is presented. a patient presented with the induration of the anterior third of the tongue of months duration. the surface was bulging, berry-shaped and bleeding, suggestive of an animators lesion. the radial excision of the tongue was performed. results histopathological analysis revealed a solid tumour measuring cm in the largest diameter. the tumour was composed of epithelioid, eosinophilic cells with "rhabdoid" features; some of these cells contained rounded eosinophilic cytoplasmic inclusions that displaced the nuclei to the periphery. most of the cells showed strong diffusely distributed vimentin immunoreactivity and focal epithelial membrane antigen and keratin immunoreactivity. angioinvasion was present at the periphery of the tumour. following pathological report re-excision of the tongue was done and the patient received chemotherapy. seven month after the diagnosis, respectively, the patient showed no signs of tumour recurrence or metastases. conclusion although there has been a contradiction whether malignant rhabdoid tumour represents a distinctive clinicopathological entity or a phenotypic pattern shared by heterogeneous neoplasms, most of the authors support the diagnosis of "rhabdoid tumour" because the morphological pattern is attended by aggressive biological behaviour. a case of sarcomatoid salivary duct carcinoma ex pleomorphic adenoma in buccal mucosal region results and conclusion correlation of the presence of merkel and langerhans cells as well as the extent of mononuclear cellular infiltrate in the perimatrix according to the degree of ossicular defect and the destruction of the bony walls were made. we found that these cells are predominantly seen in the cholesteatoma with aggressive and destructive growth. they were seen in the less degree in the skin. the role of high-resolution flow cytometry in the grading of mucoepidermoid carcinomas malignancy with poor prognosis, but occasionally arises in the various mucosa of general site including nasal or paranasal sinuses and oral cavity. it shows wide variety in cellular morphology and histological architecture and, therefore, can be easily disregarded as other neoplasm in such instance above. we describe herein a series of malignant melanoma arising in the head and neck mucosa, which diagnosis was corrected in our review, and discuss its histological and immunohistochemical characteristic as well as important clues for diagnosis. materials and methods six cases were selected from the pathology files of kurume university hospital. histological sections were routinely processed, stained with h&e, and observed with a light microscope. immunohistochemistry and special stains were done as needed. among these, two arose in ethmoid sinus and the others in nasal cavity, inferior nasal choncha, tongue and palate, respectively. age ranged between and years old (average . ). the tumours showed undifferentiated morphology with anaplastic or sarcomatoid appearance and cellular population included ovoid, polygonal and spindle cells, occasionally intermingled with those with rhabdoid or plasmacytoid features. immunoreactivity for hmb , melan-a and cd , know as melanoma-related markers, considerably varied for each case, while that for s protein was relatively stable and useful for initial screening. melanin deposition was easily passed by, especially if it appeared in the stroma rather than neoplastic cells themselves, but quite important as well as intraepithelial spread of atypical melanocytes. if resected together, also mucosal tissue should be examined carefully. tenascin expression in salivary gland tumors: an immunohistochemical study in cases introduction tenascin is an extracellular matrix glycoprotein, which interacts with other extracellular matrix proteins and cells. it is reported that tenascin expression is detected in different localizations of normal salivary gland tissue and in most of salivary gland tumours. it is accepted as an indicator of invading potential in a report. the aim of this study is to detect whether tenascin expression patterns relate with type and malignancy of salivary gland tumours. material and method cases of salivary gland tumours: warthin's stromal, mixed stromal, myoepithelioma, basal cell adenoma, basal cell adenocarcinoma, acinic cell carcinoma, adenoid cystic carcinoma, malign mixed stromal were included in our study. antibody anti-tenascin was applied to the paraffin sections. results tenascin expression was observed in all tumours. most invasive expression was observed in mixed tumours, generally in extra cellular matrix localization and in the pattern of coarse clusters. warthin's tumours demonstrated dominantly epithelial tenascin expression, sometimes in globular pattern. in malignant salivary gland tumours tenascin expression is relatively rare compared with benign ones. conclusion our findings suggest that tenascin expression may show variations in patterns and localizations in different kind of salivary gland tumours. tenascin expression is not a feature of invasive potential but may be responsible of epithelial mesenchymal interactions, stromal growth, cell differentiation and production of mixoid and chondroid matrix. links between nucleolar and extracellular matrix proteins: tenascin and ki- in vocal cord biopsy m krstulja department of pathology, medical faculty of rijeka, rijeka, croatia introduction it is postulated that cancerogenesis goes through interchange of differentiation and proliferative processes. the aim of this study was to investigate morphogenesis and proliferation in the epithelial compartment of hyperplastic, dysplastic and neoplastic vocal cord mucosa with simultaneous expression of tenascin and ki- . materials and methods vocal cord biopsies were analysed: invasive squamous cell carcinomas, in situ squamous cell carcinomas, hyperkeratoses with low dysplastic change, papillomas and nodules with hyperkeratosis. immunohistochemistry was performed for each marker separately as well as simultaneously for both markers. results tenascin was expressed in differentiated and neoplastic tissues, decorating the membrane/cytoplasm of cells in carcinoma and dysplastic mucosa, but only the intercellular epithelial junctions in papilloma and hyperkeratosis. ki- , a nucleolar protein, decorated nuclei, nucleoli and chromosomes of cells engaged in mitosis in homotypic and heterotypic bioptic tissue. a double staining for tenascin and ki- revealed synchronous decoration of cytoplasms and nuclei in invasive squamous cell carcinoma ( / ), in cells of in situ squamous cell carcinoma ( / ) and occasionally in cells of dysplastic hyperkeratosis ( / ). simultaneous staining was not found in homotypic tissue. neoplastic cells in mitosis had tenascin stained membranes/cytoplasms or were tenascin negative. in dysplastic hyperkeratosis basal cells in mitosis had rarely tenascin positive membranes ( biopsy only). conclusion the observation that tenascin (a marker of morphogenesis) and ki- (a marker of proliferation) express simultaneously in neoplastic epithelial cells makes a difference regarding homotypic proliferation. introduction anaplastic thyroid carcinoma with an osteoclastoma like component is a rare type of thyroid neoplasm. the origin of the osteoclastoma like element has been a source of controversy, with conflicting theories emerging from the literature, some supportive of an epithelial origin with others favouring macrophage/ histiocytic lineage. ret/ptc is a fusion transcript associated with papillary thyroid carcinoma but not previously described in a tumour of this type. our aim was to examine more closely the osteoclastoma like component and assess the histogenesis of this tumour. materials and methods electron microscopy, histochemistry and a broad immunohistochemical panel were performed. in addition pure populations of each constituent cell type comprising the tumour were isolated using laser capture microdissection. after rna extraction, each of these was examined by rtpcr for chimeric transcripts of ret/ptc- and ret/ptc- . at this stage of our study, due to small differences in each epulis type, no far-reaching conclusions can be drawn. further studies of cd and mdm- protein in the same group of patients seem to be justified since in the authors' opinion, they will explain biological behaviour of that type of tumours. conclusions great proliferation activity of the cells in all types of epulides analysed with pcna, with low ki- and lack of p activity, was observed. adenoid cystic carcinoma and polimorphous low grade adenocarcinoma of salivary glands. a morphometric study c giardina results nuclei of plga resulted significantly larger and with a more regular shape than nuclei of acc. multivariate analysis discriminated the % of nuclei by utilizing variables: nuclear area and variables obtained by the nuclear asymmetry evaluation. conclusion morphocytological differences could have a discriminating power between plga and acc, they seem to be more important than architectural differences and could be related to their different biological behaviour. oral synovial sarcoma -a report of two cases and a review of the literature introduction the head and neck region constitutes the second most common site of involvement for synovial sarcomas, accounting for up to % of all cases. intra-oral synovial sarcomas are rare, with only cases having been reported. the aim of this study is to report additional cases of intra-oral synovial sarcoma, which occurred in the floor of the mouth and retromolar areas respectively in middle-aged black males and to review the clinico-pathologic features of all previously reported intra-oral cases. the current cases were immunohistochemically stained with ema, cd , bcl- , vimentin, cam . , mnf , ck , s- protein, hba , cd and calretenin; and subjected to ultrastructural studies. the literature was reviewed over a period of years, examining a total of cases of oral synovial sarcoma. the intra-oral cases differ from lesions occurring in other sites only in respect of a more marked male predilection, and a generally painless presentation. this is the first report demonstrating calretinin immunopositivity in intra-oral synovial sarcomas. even though in the oral cavity possible earlier detection, easy accessibility and small size renders them more amenable to surgical excision, their biological behaviour remains aggressive with a poor long-term prognosis. conclusion this study clearly demonstrates the histomorphologic, immunohistochemical and cytogenetic similarity of intra-oral synovial sarcomas to that of synovial sarcomas in general. the unusual intra-oral location often results in these lesions being misdiagnosed. these tumours should be considered, as both primary and metastatic tumours, in the differential diagnosis of intra-oral spindle cell malignancies. thirty nine years long follow up of the patient with dermatofibrosarcoma protuberans background dermatofibrosarcoma protuberans (dfsp) is an infiltrative, aggressive, nodular stromal with a great potential to relapse, that appears early in the life, in % of cases at the head and neck region. during last ten years there were five patients with the dfsp at the clinic for maxillofacial surgery of the military medical academy that makes . % of all cases with mesenchimal malignancy. here we present the most extreme case of the dfsp. case report s.s. was years old when she injured a small subcutaneous stromal at the right side of her forehead. ten years later it was cm wide and excised for the first time, as well as two years later when the relapse of the same size, this time involving the bone, was removed and the patient went through radiation therapy. after four corrective operations in a meantime, at her age of and the relapse cm wide from the right frontal region was removed. three years later, at her age of , stromal sized cm was excised from her right buccal mucosa. two years later, at her age of , local relapse was widespread into the right parotid gland, superior eyelid and frontotemporoparietal region including dura. two years after extensive ablative surgery and delicate reconstruction with scary compromised local cutaneous flaps, the relapse from the previously hardly mutilated right forehead region together with dura was removed. six years later, at her age of , the right orbit was exenterated together with excision of adjacent temporoparietal tissues. one year later the local relapses from the right infraorbital and buccal region were removed. two years after another stromal cm sized from the periorbital region was removed. after two years, at her age of , the lesion from her cheek was removed. it has been her tenth, but obviously not the last surgery. almost one year later there were no signs of local relapse or regional metastases at her routine postoperative check up. in spite of such a great mutilation of face and psychological disturbances connected with it, this is a case of successful surgical management of the dfsp. few times some differential diagnostic dilemmas appeared, but careful and detailed revision of all specimens definitely confirmed the dfsp. multiple and multifocal malignancy -a case report background it is well known that one either benign or malignant stromal can appear multifocally, as well as the fact that histologically different tumours can exist coincidentally. there is just a few papers concerning on how many different concomitant tumours can appear in the human body. this is a case of years old male with several tumours of different malignancy degree and morphologically benign or semimalignant cutaneous lesions. case report during previous years three times he underwent surgical excision of epitheliomas of the scalp, body and limbs. the third one has been removal of the epithelioma of the right frontoparietal region. at his age of because of the stromal sized cm in the right parotid salivary gland, followed by paralysis of the frontal and paresis of buccal branches, along with laboratory finding of . leukocytes, right-sided radical parotidectomy and suprahyoid dissection of the neck has been performed. pathohistologic analysis confirmed malignant mixed stromal of the parotid salivary gland. postoperative irradiation therapy has been applied in fraction and total dose of gy. nine months later the patient has been operated again because of an exulcerated, cm diameter cutaneous lesion of interscapular region, which has proved to be the infiltrative squamous carcinoma g- grade. at the same time the chronic lymphoproliferative disease in the form of chronic lymphocytic leucosis a grade has been diagnosed (although the reactivity following the neoplastic process could not be excluded), but specific haematological treatment has not been indicated. nine months later nine cutaneous facial lesions have been removed: malignant mixed stromal of the left auricle, senile keratosis from the left temporoparietal region, chronic dermatitis from the forehead, squamous carcinoma g- and seborrhoic keratosis one inch inferiorly, two lesions, sebaceous adenomas from the right zygomatic region, corneal squamous carcinoma g- from the right parietal region, baseocellular carcinoma from the left one and adenoid type of baseocellular carcinoma from the right supraclavicular region. the patient has been operated without complications and came five times for monthly control in a good general condition, without relapse, regional or distant metastases. later on we ascertained that the patient suddenly died during a sleep seven months after surgery. conclusion pathohistologic examination of removed tissues and revision of the former specimens undoubtedly excluded the spreading of the one and same stromal in different regions, but proved multifocal appearance of tumours of the same embryologic -ectodermal origin in very different structures. melanoma of the oral cavity -a case report background melanoma is a capricious malignancy followed by reputation of aggressive growth and dissemination, able to originate from any tissue containing melanin. in less than % it appears at mucous membranes. here we present a case of such localization. case report during last ten years there were patients with primary cutaneous head and neck melanoma who underwent surgery because of secondary deposits at the neck, but only one patient, r.m., years old (pale skin, dark hair and eyes, never working outside), who appeared four years ago with inoperable intraoral melanoma involving tongue, base of mouth, mucosa of the left cheek, retromolar region, tonsillar fossa and soft palate, including a single pendular mass from the left palatoglossal arc that almost completely narrowed his isthmus faucium. the first pigmentation he noticed eight months before his first admittance suffering of disturbed swallowing for few weeks. although there was no sign of regional or distant metastases, only the last-mentioned mass has been removed and pathohistologically verified as melanoma. patient was able to breathe normally, and continuing with his previous way of life. he received dtic during the first four postoperative months. one year later, smaller mass from the same location has been removed again. routine laboratory analyses, radiographic examination of the lungs, ultrasonographic examination of the neck and abdomen never revealed any changes. the third reduction under the same circumstances took place one year later and has been followed with minimal doses of adjuvant immunotherapy. conclusion this is a rare case of such a large, inoperable intraoral melanoma that has been three times reduced at the place of its exophytic growth into throat, otherwise demonstrating no signs of regional or distant spreading, as well as any disturbances in patient's general condition during last four years. introduction recent studies have shown that stromal reaction in cancer has an important diagnostic and prognostic significance. cd -positive stromal cells and myofibroblasts may play an important role in host response to cancer. the aim of this study was to analyse the expression and significance of cd , alfa-smooth muscle actin (sma), and tgfbeta in epithelial hyperplastic lesions (ehl) and squamous carcinoma (sc) of the larynx, and tried to reveal the mechanism of myofibroblast formation in sc. we investigated samples of resected larynxes with sc, laryngeal biopsies of ehl and sc, and samples of normal laryngeal mucosa. immunohistochemistry was performed with antibodies against sma, cd , cd , tgfbeta and tgfbeta receptor. the expression of tgfbeta mrna was detected by rna in situ hybridisation. results stroma of normal mucosa and ehl contained scattered cd -positive cells, but there were no sma-positive myofibroblasts. stroma of sc contained sma-positive myofibroblasts but there were no cd -positive stromal cells. in normal mucosa and ehl, tgfbeta and its receptor were present in epithelial cells and scattered stromal and inflammatory cells. in sc, tgfbeta , its receptor and tgfbeta mrna were found in stromal cells, inflammatory cells and some stromal myofibroblasts. conclusions disappearance of cd -positive cells and appearance of sma-positive myofibroblasts in the stroma is associated with transformation of laryngeal ehl to sc. this pattern of stromal reaction is limited to invasive carcinoma and should be regarded as an additional marker of invasion. our results also support the hypothesis based on experimental studies that tgfbeta may be one of the mechanisms of transformation of stromal cells to myofibroblasts in sc. introduction loss of heterozygosity (loh) on short arm of chromosome p is frequently found in squamous cell carcinoma of larynx and hypopharynx (lhscc). this is a region where potential stromal suppressor gene, frequently changed in pulmonary cancer, the fhit gene, is located. we wanted to determine whether there is presence of molecular and/or immunohistochemical changes in fhit gene in patients with lhscc. materials and methods dna was isolated from stromal and adjacent normal tissue using standard procedure. non-isotopic loh analysis was performed with micro satellite markers d s , d s , d s , d s , d s . formalin fixed tissue samples were stained immunohistochemically with antibody against fhit protein and reactions scored according to % of nuclear positivity in stromal cells. results loh at p was detected in ( . %) and microsatellite instability (msi) in ( . %) tumours, which were mostly moderately differentiated ( . %). introduction permeation of fixatives into tissue for optimal dna preservation for molecular pathology studies is related to tissue type and fixative but also to glycosaminoglycan content of the ground substance. this may alter with age, warranting further study. the aim of this study was to assess nuclear dna preservation related to age and fixation agent. materials and methods fresh standardised testicular tissue samples from dogs - months (i), months - years (ii) and years and over (iii) were used for immediate dna extraction (t ), extraction after hours (t ) and after days (t ) of immersion in formaldehyde-based and formaldehyde-free, commercially available fixatives. dna extraction used standard protocols, commercially available kits (qiaamp minikit, qiagen, the netherlands), standard primers for the betaglobin gene preserved between species (myo /my , digene/abbot, usa) and quantitative pcr (realtime light cycler, roche, amplification cycles). in formaldehyde free fixatives, initial loss of extractable dna in t samples of young (i) animals was % as compared to % in older (ii+iii) animals (p< . , student t-test). the same, with a significantly poorer result (p< . ) was found in formaldehyde based fixation, with loss of % in young (i) as compared to % (p< . ) in older (ii+iii) animals. at days, further loss to % of t values occurred in all age groups (p< . ), with formaldehyde free fixatives retaining the proportional age associated difference. in formaldehyde based fixatives at t , loss to % of t values was found (p< . ), again without statistically significant differences between age groups. the g a polymorphism of ccnd correlates with susceptibility and prognosis of breast cancer in taiwan cp yu a frequent g a polymorphism of ccnd gene located on the splice donor site on exon -intron boundary is implicated in influencing cyclin d mrna splicing and production of two distinct mrna transcripts. although cyclin d is believed to perform an important role in cell cycle progression, this g a single nucleotide polymorphism (snp) seems to be a critical modulator of its functions. it has been reported that the prognostic significance and mrna splicing regulatory effect of g a varies with tumor type and ethnic diversity. to explore the association between the ccnd g a polymorphism and the breast cancer susceptibility as well as other prognostic parameters, we employed infiltrating ductal carcinoma (idc) cases and native controls in this study. there is obviously correlation between the genotype distribution of these two groups (p= . ), and the aa genotype reveals a higher association and risk than the ag+gg genotypes (p= . with c test and or . , % ci . - . , p= . ). moreover, there is no statistical significance found between genotype or allele frequencies and tumor stage, grade, nodal involvement, or patient'¦s age. however, the trend tests of ccnd g a genotype distribution or allele frequencies to the increasing of tumor grade or tumor stage are significantly different when comparing the a vs. g allele (p= . in both tumor grade and stage) or aa vs. ag+gg genotype groups (p= . and . in tumor grade and stage, respectively) but no significance when comparing gg vs. ag+aa or ag vs. aa+gg genotypes. these results suggest that ccnd g a polymorphism correlates with idc susceptibility, and a allele reveals an increased risk in carcinogenesis of idc and worse prognosis. the new hope*-fixative as an enhancement in molecular pathology e during the last years the growing demand for the detection of prognostic and diagnostic biomarkers has been slowed down to a large degree by the use of formalin and its influence onto antigenic structures and nucleic acids, although other reasons also exist. with regard to morphology a limitation in the possibilities, compared to frozen sections, takes place. with the introduction of the hopefixation to date both has become possible; substantially enlarged possibilities concerning the detection of dna, rna and proteins together with a formalin-like morphology. this will be demonstrated by presenting several examples like preservation of nucleic acids (dna, rna), the detection of those by pcr and rt-pcr, in situ hybridization and the preservation and delectability of proteins by immunohistochemistry. we conclude that hope-fixation opens up new possibilities, especially within the growing number of diagnostic and prognostic biomarkers. *hepes-glutamic-acid-buffer-organic-solvent-protection-effect introduction high-risk human papillomaviruses (hpv) represent a major risk factor for the development of cervical cancer. quantitative real-time pcr for viral load determination of hpv in archival cervical cancer specimens could be of clinical and prognostic relevance. the aim: we investigate the prognostic value of hpv status and viral load. furthermore, we compared the applicability and sensitivity of three molecular hpv-detection methods. materials and methods all examinations were carried out from formalin-fixed, paraffin-embedded tissue of cervical carcinomas. hpv status was detected by signal amplified chromogenic in situ hybridization (sa-cish), conventional polymerase chain reaction (pcr), and detection and additional quantitation was performed using real-time pcr. results patients with low viral load of hpv in stage i carcinoma had a significantly better prognosis than hpv -negative patients (p= . ). additionally, high viral load of hpv in patients indicating a higher stage carcinoma strongly tended to benefit prognosis compared to hpv-negative patients. our evaluation of the molecular hpv-detection methods showed a higher sensitivity of real-time pcr and conventional pcr compared to sa-cish. conclusion it appears that cervical cancer patients can be distinguished in a hpv -negative subgroup revealing a worse prognosis, and a lower-stage subgroup with low viral load, respectively a higher-stage subgroup with high viral load, both indicating a better survival, maybe due to an altered immune status caused by hpvinfection. these findings are crucial to accord each patient the individual therapy, and point out the importance of quantifying viral load and the necessity of real time-pcr in hpv-diagnosis. laser capture microdissection in -d coculture models as a novel tool to study tumor-stroma-interactions introduction cellular adhesion, migration and invasion are essential processes in tumor progression and do include actions by malignant cells as well as by the stromal microenvironment. in vitro, these interactions can be studied in coculture models. in conventional coculture systems, the different cell types are grown together in glass-slide based chambers with medium. in situ analysis can be easily performed, however, for mutational or gene expression analyses the cell compartments have to be dissociated and sorted. we present a novel technique for co-culturing fibroblasts and carcinoma cells and separating them without cross-contamination. methods our method is based on cell cocultivation on a . µm thin membrane followed by laser microdissection of tumor and stroma cells. for identifying the tumor cell compartment, immunolabeling for the laminin gamma chain was performed, a marker that is expressed only in epithelial tumor cells. dna and rna was isolated from the microdissected cells. rna quality was tested by rt-pcr for a housekeeping gene transcript and for the laminin gamma chain gene transcript. results co-cultivation on the membrane did not influence growth behaviour and cell morphology. the rna quality from the microdissected co-cultured and immunostained cells could be successfully proved by rt-pcr. laminin cdna was amplificable only in tumor cells and not in the co-cultivated fibroblasts indicating no cell-cross-contamination during microdissection. conclusion our technique provides a suitable tool to study tumor -stroma interactions at the dna or rna level for expression profiling and genetic analyses. p expression in prostate cancer and its correlation with microvessel density i pavlenko, a matsionis rostov regional bureau of pathology, rostov on don, russian federation introduction angiogenesis is important in the development and progression of all cancer types, including prostate adenocarcinoma (pca). increased vascularization may be influenced by p status. the aim of this study was to evaluate mvd in prostate cancer and its relationship with p nuclear accumulation. methods paraffin-embedded tissue from patients with well-, moderately and poorly differentiated prostate carcinoma was examined immunohistochemically using monoclonal antibodies against cd , vegf and p (clone do- ). a modified semiquantitative weidner score and computerized image analysis was used to assess mvd. results expression of immunohistochemically detected p protein was found in of cases ( %). p nuclear accumulation was observed in two different groups of cancers -poorly differentiated pca with high vegf expression and high mvd, with high level of serum psa ( cases) and moderately differentiated pca without significant microvessels with low serum psa ( cases). we suppose p mutation in the first group of patients which leads to increased angiogenesis as the result of loss of wild-type p induced anti-angiogenic control. in our opinion p detection in the second group of p + patients is the result of overexpression due to stabilization but not mutation. conclusion p overexpression is observed due to either mutations or protein stabilization. increased angiogenesis seems to be influenced by p mutations, while low mvd is observed in cancers with non-mutated p . immunohistochemistry alone is not able to detect p mutations, additional techniques are need for this purpose, especially in patients with p nuclear accumulation. malignancy-associated x chromosome loh in foregut endocrine neoplasms: further evidences in lung tumors introduction association of x chromosome allelic losses (loh) with tumor malignancy has been found in foregut-derived gastropancreatic endocrine neoplasms, but not in midgut carcinoids. also lung endocrine tumors originate from the embryological foregut. they are classified into "typical" carcinoids (tc), "atypical" carcinoids (ac), large cell neuroendocrine carcinomas (lcnec) and small cell lung carcinomas (sclc), in order of increasing malignancy the aim of this study was to investigate x chromosome loh and its association with malignancy in a series of endocrine tumors of the lung. materials and methods dna extracted from tcs, acs and lcnecs from female patients was pcr-amplified using fluorescently-labeled primers for microsatellite markers spanning the whole x chromosome. for loh evaluation, amplimers were analysed in an automatic dna sequencer (ceq , beckman coulter). all samples were formalin-fixed, paraffin-embedded and dna from tumoral and non-tumoral adjacent tissue was available results allelic losses were absent in tcs, while loh on x chromosome was found in of acs ( %) and in of lcnecs ( %). allelic losses were generally wide, involving all or most informative markers investigated conclusions allelic losses on x chromosome, absent in benign "typical" carcinoids, progressively increased their frequency from intermediate-grade "atypical" carcinoids to high-grade lcnecs. therefore the observation that x chromosome loh is associated with malignancy, already demonstrated in gastro-pancreatic endocrine tumors, is extended to another group of foregut-derived endocrine neoplasms, the lung endocrine tumors we investigated formalin-fixed, paraffin-embedded tissue samples. from each paraffin-block four times one to two µm sections in three steps and two µm sections were cut and put to . ml reactiontubes. one tube of the thin sections and the thick sections were exposed to heat/cold shocktreatment, one tube had a proteinase-k treatment for only three hours instead of overnight, and the thin cuts in another tube were treated with a riboliser. dna amount and purity measured by spectrophotometry after extraction. pcr was performed with routinely used srdna-primers. results over all we obtained best results with cutting up thin sections and proteinase-k treatment overnight. this tissue yielded the best purification, the highest dna amounts and strongest bands in agarose gelelectrophoresis. second best were thick and thin sections with heat/cold shock-treatment and proteinase-k treatment overnight. conclusion the mechanical opening of the mycobacterial wall using a standard microtome is a feasable way to obtain higher dna-amounts -purity and -complexity resulting in stronger signals following pcr. impact of tumour suppressor gene pten on cell cycle and apoptosis in prostate cancer cell lines introduction the progression of prostate cancer from androgenresponsive to an androgen-unresponsive status remains the greatest problem in the treatment of this disease. aims: to study the relationship between telomerase activity and gene expression after bicalutamide treatment in prostate cancer cells. material and methods androgen-responsive (lncap) and androgen-unresponsive (du- ) prostate cancer cell lines treated with µm bicalutamide for hours were analysed by affymetrix hg-focus array chips and their expression pofile was compared with control. the decrease of telomerase activity which was associated with increase of p waf /cip and decrease of dyskerin and hsp were found in lncap cells only. dna damage-responsive genes were upregulated in both cell lines. in lncap cells, the increase of p waf /cip was associated with induction of p -dependent gene expression (gadd α, bax, mdm- and trail-r ). we have also found induced transcription of pro-apoptotic genes represented by bnip -like, bcl- , cbx and tradd and the decrease of anti-apoptotic protein, survivin. in du- cells, we observed an increase of mt p , trail-r , gadd , gadd α expression together with a decrease in expression of survivin and the pro-apoptotic bnip gene. conclusion the decrease of telomerase acivity in lncap cells after bicalutamide treatment could be explained by changes in expression of p waf /cip , dyskerin and hsp . bicalutamide upregulates dna-damage genes in androgen-responsive as well as androgen-unresponsive prostate cancer cell lines. this work was supported in part by msm and nc/ - . introduction bicalutamide, an androgen antagonist used in the prostate cancer treatment, has a significant cytostatic effect on cancer cells. the aim of this study was to analyse the changes of bcl- family members after bicalutamide treatment in androgen-responsive (lncap) and androgen-unresponsive (du- ) prostate cancer cells. comparing the toxicity and potential carcinogenicity of two antiestrogens. a short-term study with microarray analysis in female rat liver introduction tamoxifen and toremifene are triphynylethyle antiestrogens used in the treatment of breast cancer. tamoxifen is genotoxic hepatocarcinogen in the rat, whereas toremifene is not. in humans, long-term tamoxifen treatment causes an increased risk of endometrial cancer. the major difference between the genotoxicity of the two drugs is the formation of liver dna-adducts in tamoxifen-treated rats. differential gene expression in rat liver after administration of antiestrogens was studied by microarray analysis. materials and methods equimolar doses of antiestrogens were given p.o. to six week old female sprague-dawley rats for days. total rna was isolated using trizol reagent. equal amounts of total rna from four animals in the same group was pooled and then purified from mg total rna using mg oligo(dt)cellulose. our cdna array contained approximately cdna clones selected from the tigr rat gene index. the samples were hybridized to rat cdna array scanned with a gms scanner (afflymetrix). image analysis was performed using the genepix pro software. normalization was performed using lowess normalization. the results from rat cdna array were evaluated according to 'significance analysis of microarrays' (sam). results sam plot for tamoxifen vs. control revealed significant genes from which were positive significant (up-regulated, red) q < %, . particularly the nadph-cytochrome p oxidoreductase and microsomal epoxide hydrolase were upregulated genes which are apparently related to tamoxifen-induced carcinogenesis. sam plot for toremifene vs. control revealed significant genes from which were positive significant q < %, . conclusion microarray technology gives important information of the toxicity as well as carcinogenicity of antiestrogens in experimental conditions. the ret proto-oncogene tyrosine kinase receptor is activated by a ligand complex comprising glial cell line-derived neurotrophic factor (gdnf) and gdnf family receptor alpha- (gdfralpha ). they are expressed in multiple organs during development and also in some types of human tumors in adults. the aim of this study is to investigate ret, gdnf and gdfralpha expression in normal tissue and tumors derived from the gastrointestinal tract, circumstance that we have not found it published in the literature. material and methods ret, gdnf and gfralpha expression was analyzed in samples of fresh frozen and formalin-fixed tissues from tumoral zone, and adjacent non-affected tissue of biopsies from patients with gastrointestinal cancer using rt-pcr and immunohistochemical methods. results presence of ret, gdnf and gfralpha transcripts in most of normal tissues was confirmed by rt-pcr. immunohistochemical study showed positive staining in citoplasms of glandular epithelium of the mucosa, ganglionar cells and muscular fibers (only gdnf) of esophagus, stomach, duodenum, colon and rectum but not in jejunum and ileum. gastric oxyntic cells were negative. sixty four gastrointestinal tumors ( adenocarcinomas and cases of gist) were also studied. by immunohistochemistry, % of cases showed citoplasmic immunoreactivity for gdnf; % for ret and % for gfralpha . these findings were confirmed by rt-pcr. there was not association with microscopic type, size, grade or duke stage. our study shows ret, gdnf and gfralpha positive protein and mrna expression in normal tissues and in the majority of tumors derived from the gastrointestinal tract. there is no association with microscopic type, size, grade or lymph node metastasis. this expression gives no prognostic information in the clinical management after two years follow-up of our patients. expression analysis of ret and its gdnf/ gfralpha ligand complex in breast cancer introduction the ret proto-oncogene tyrosine kinase receptor is activated by a ligand complex comprising glial cell line-derived neurotrophic factor (gdnf) and gdnf family receptor alpha (gfralpha ). they are expressed in multiple organs during development and also in some types of human tumors in adults. in this study we evaluated the expression of ret, gdnf and gfralpha in normal mammary tissue and different types of breast cancer. material and methods ret, gdnf and gfralpha expression was analyzed in samples of fresh frozen and formalin-fixed tissues from tumoral zone, and adjacent non-affected tissue of mastectomies from patients with different types of breast cancer ( dic, lic, medullary carcinoma, mucinous carcimoma, papillary carcinoma and fibrosarcoma) using rt-pcr and immunohistochemical methods. results normal breast ductal epithelium express ret, gdnf, and gfralpha . by immunohistochemistry, , % of breast tumors showed citoplasmic immunoreactivity for ret, , % for gdnf and , % for gfralpha . rt-pcr confirmed mrna expression in , % of cases for gdnf, , % for ret and , % for gfral-pha . by chi-square analysis, ret expression was associated with less tendency to the regional lymph nodes metastases (p< , ) and low grade differentiation (p< , ). there was not association with microscopic type, size or stage. conclusions we found that ret and its gdnf/gfralpha ligand complex are expressed in normal breast tissues and in a high percentage of different types of breast tumors using immunohistochemistry and rt-pcr. univariant analysis also revealed that only ret expression was associated with less tendency to the lymph nodes metastases and low grade dics (highly differentiated tumors). the short time follow-up of our patients (less than two years) gave no information about clinical outcome. telomerase activity in chronic b-lymphocytic leukemia r telomerase, the enzyme that elongates telomeres, contains an rna template complementary to ttaggg repeats that permits de novo synthesis of telomeric dna onto chromosomal telomeric ends. recent findings have suggested that activation of telomerase is one of the most common and fundamental steps in cancer genesis, although reactivation or telomerase up-regulation alone might be insufficient for cells to proliferate indefinitely. telomerase expression seems to be concomitant with the attainment of immortality of cancer cells, but there are contradictory reports about telomerase activity in early and late b-cll stages. in order to investigate the telomerase activity in patients with cll and its correlation with the clinical stage, frozen blood lymphocytes samples from patients with cll ( males, females), and age matched controls ( males, females) were investigated for telomerase activity using the 'telomerase pcr elisa-plus kit' from roche, based on the telomeric repeat amplification protocol. binet and rai stages were evaluated at the time of sample collection, and re-evaluated after - months. results were analyzed with a commercial statistic software package. the study showed that peripheral lymphocytes telomerase activity from b-cll patients is significantly higher ( , times; p< , ) compared to normal lymphocytes. the age and sex of patients does not influence the telomerase activity, while it is age dependent in healthy controls (pearson correlation - , ; p< , ). the study also showed that telomerase activity is increased more significantly in advanced stages of the disease, and correlates with the disease course. introduction deltan-p isoforms may act as oncogenes, owing to their ability to bind to p -reporter genes without inciting their transcription, thus blocking the p -driven cell cycle arrest and apoptosis. a novel mechanism, linking p and wnt pathways has recently been proposed. briefly, in vitro studies have suggested that deltan-p may block the phosphorylation of beta-catenin, leading to its nuclear accumulation and triggering beta-catenin-responsive transcription of genes related with proliferation and oncogenic biological behaviour. to test this new mechanism, the authors evaluated the co-expression of deltan-p and beta-catenin in a large cohort of human neoplasms. methods two sections of tarp- multi-tumour tissue microarray, composed of normal tissue cores and human neoplasms (breast (n= ), colon (n= ), lung (n= ), prostate (n= ) and ovary (n= ) neoplasms, melanoma (n= ), and glioblastoma (n= )) were subjected to immunohistochemistry with deltan-p and beta-catenin monoclonal antibodies. p nuclear expression and b-catenin membranous, cytoplasmic, membranous+ cytoplasmic, and nuclear localisation were independently evaluated by three of the authors. results p expression and beta-catenin nuclear localisation were found in . % and % of squamous cell carcinomas, . % and % of breast carcinomas, . % and % of lung adenocarcinomas, . % and . % colon adenocarcinomas, % and . % of prostate adenocarcinomas, . % and % of ovary carcinomas, . % and . % of malignant melanomas, and . % and . % of glioblastomas, respectively. no statistically significant association between p and nuclear beta-catenin expression was found for all tumours. conclusions at variance with squamous cell carcinoma cell lines, p -driven nuclear accumulation of beta-catenin is an unusual phenomenon in human neoplasms. caution should be exercised when translating the results of studies performed on cell lines to human neoplasms. four gist had no identificable kit sequence alteration in exon . mutations in c-kit seemed to be associated with favorable response to sti- (p= , ). conclusion kit mutations in exon are common in diseminated gastrointestinal stromal tumors and appear to be associated with response to sti- . the use of macro-array to study the effect of intestinal trefoil factor on gene expression in caco- derived enterocytes intestinal trefoil factor (itf) is a member of the trefoil family of peptides. it increases resistance to apoptosis, enhances mucosal healing and restitution in vivo and promotes migration of intestinal epithelial cells in vitro. the aim of this study was to investigate the influence of itf on gene expression in intestinal epithelial cells. the human colon carcinoma cell line caco- develops structural and functional characteristics of normal enterocytes when kept in culture. enterocyte-like caco- cells were treated with recombinant itf during hours. rna was isolated and labelled cdna was synthesised. the cdna probe was hybridised to the human unigene set colony filters (rzpd). after several washing steps, the amount of signal was measured using a phospo-imager. spot intensities were measured and signal quality was evaluated. using a fold increase or decrease as a cut off, comparison of the gene expression between un-stimulated caco- cells and cells stimulated with itf using the macro-array technique revealed a few hundred genes that were potentially differentially expressed. our data show that the macro-array is a powerful technique to screen for differen-tial gene expression as a result of trefoil administration, helping to study signalling cascades. however, it is necessary that the huge amount of data obtained is filtered through repeated hybridisations, only to retain the relevant changes and these data must be confirmed using other methods like quantitative real-time pcr and eventually studies at protein level. introduction melanoma therapy still remains without notable success of any pharmacological agent. gastric pentadecapeptide bpc is a peptide with various pharmacological effects on different organ systems including wound healing and inflammation. its possible effect on cell growth and differentiation was not investigated on human cancer cell lines. human melanoma cells were maintained in rpmi- medium supplemented with % fbs and antibiotics. cells were grown to confluency in humidified incubator ( % air atmosphere, %.co ) at °c. upon reaching confluency, cells were maintained in chemically defined medium (cdm). bpc was added in the culture medium in the various concentrations ( pg. and ng, respectively) for a period of hours. cell morphology was observed daily under light microscope and the photomicrographs were taken. after harvesting cells were prepared in etoh for flow cytometry analysis. results microscopy did not reveal any changes upon treatment with bpc after h comparing to controls. flow cytometry showed % of cells (p> . ) were in g -g phase. control cells had total s-phase fraction of . %. however, upon introduction of bpc , in concentration of pg/ml, the percentage of cells in sphase was decreased by % compared to controls. moreover, the concentration of ng/ml bpc lowered total s-phase fraction by % comparing to controls and by % comparing to bpc in picogram values (p> . ). conclusion we assume that gastric pentadecapeptide bpc could act as a inhibitory agent during g -s transition state of cell cycle thereby preventing dna synthesis and cell growth. introduction histones play an important role in the structural and functional properties of chromatin. their protective role against radiation-induced damage of dna is well established. the aim of this study was to evaluate relation between expression of histones genes and radiosensitivity of brain gliomas in vitro. five human glioma cell lines obtained from dsmz (germany) were irradiated with gy and gy doses (co- ) and further grown for , and hrs. the expression of genes coding for histones h , h a, h b, h , h was measured with real-time quantitative rt-pcr assay (taqman). radiosensitivity of the cell lines was assessed with cytokinesis-block micronucleus assay. cell lines were regarded as radioresistant and radiosensitive at micronuclei per binucleate cell ratio (mn/bnc) < . , and > , respectively, as measured over a dose range of - gy. results the global number of copies of histone mrna did not differ in both groups. in radiosensitive cell lines (gamg, -mg-ba) expression of h b was highest in / cases, while in radioresistant (gos- , -mg-ba, u- -mg) h predominated in / cases. in both groups significant decrease of global histone genes expression after gy dose was found. relative increase of h and h b fractions after irradiation was observed. conclusion overall expression of histone genes does not seem to influence radiosensitivity of glioma cells. probably, more important is the profile of expression, e.g. differences between h and h b genes. this profile may be a useful adjunctive criterion in the choice of treatment protocol for brain glioma. introduction the detection of breast cancer (bc) micrometastasis in effusions is difficult since the malignant cells are rare and spread amongst the normal population. rt-pcr for human mammaglobin (hmam) has been described as a new, sensitive method for use in bc cell research. some authors have proposed using this assay for screening bc effusions for bc micrometastatic disease. the aim of this study was to investigate the possible application of rt-pcr for hmam for assessing bc cells in effusions and compare this methodology with cytological examination. the study was performed on fluid specimens ( bc, other types of cancer and without known carcinoma). the samples were analyzed by staining with ee, papanicolau technique, and by nested rt-pcr for hmam. results / ( %) cases of bc pathology were positive using nested rt-pcr for hmam. all cytology positives were also positive by nested rt-pcr. in contrast, bc cases that were positive in the molecular test were negative according to the staining assay. hmam was also detected in / ( %) specimens of other types of cancer and only / ( . %) samples of non-neoplastic origin. conclusion nested rt-pcr for hmam was more sensitive than cytology in determining bc micrometastasis in effusions. positivity was not restricted to samples from bc patients as specimens from other tumors and only . % of patients without cancer were also positive. this test could be helpful in investigating neoplasticity in effusions. introduction hepatic ischemia-reperfusion injury results from implementation of pringle's manoeuvre to prevent blood losses in patients undergoing liver segment resection. reoxygenation after the ischemic period leads to failure of hepatic microcirculation, leucocyte invasion and kupffer-cell activation. subsequent production of inflammatory cytokines and ros (reactive oxidative species) are triggered, thus causing oxidative stress and damage of liver parenchyma. the goal of our study is to evaluate markers associated with the severity of this particular type of oxidative stress at the gene expression level. we have analyzed liver tissue biopsies at different timepoints obtained from patients undergoing liver segment resection. in each case, the first sample is taken before clamping, the second sample is taken after minutes of ischemia, and the third sample is taken after minutes of ongoing reperfusion. we compared mrna levels of several genes (sqstm , hspa a, gpx , sod , hmox) in every set of samples by quantitative rt-pcr analysis. significant elevation of hspa a at timepoint was detected in of cases. this finding suggests that hspa a may represent a useful marker for oxidative injury of hepatic tissue. at the same time, we show that the detection of changes in gene expression within minutes of reperfusion is possible by quantitative rt-pcr from a single biopsy. we hypothesize that individual differences in gene expression may reflect severity of the inflicted ischemia reperfusion injury as well as patient prognosis. the clinical significance of our preliminary results is currently under investigation. automated tissue microarray evaluation and normalization using a dna scanner oridis-biomed gmbh, graz, austria institute of pathology, graz, austria introduction tissue microarray (tma) immunohistochemistry is gaining importance in drug target validation. hundreds of tissue samples on a single slide enable simultaneous immunohistochemical assessment of protein expression. correlation of expression levels of the target antigen with clinical parameters can facilitate determination of disease relevance and therapeutic potential. the aim of this study was to extend the scope of this technology, we developed a robot for automated tma production. we further assessed the use of a dna microarray two color laser scanner for automated analysis of tma immunohistochemistry and the use of an internal reference antigen for standardization to the tissue content. the tma robot was developed in cooperation with an engineering company. a breast carcinoma tma was stained with an estrogen receptor (er) antibody. lung carci-nomas tmas were double stained with combinations of rabbit and mouse antibodies. immunoreactivity was detected with fluorescently labeled secondary antibodies. the slides were evaluated using a cdna array scanner and software. fluorescence intensity for er ihc on a breast carcinoma tma correlated positively with independent assessment of the diagnostic er immunoreactivity score, suggesting that ihc quantitation can be achieved with the dna array scanner. double ihc on lung tmas demonstrated that one antigen reference can normalize tumor marker ihc signals for the cellular content of tma cores. ihc signal normalized in this fashion correlated with clinical parameters in agreement with published results. manifestation of metallothionein (mt) correlates rather with cell differentiation than with intensity of proliferation. introduction metallothioneins (mt) are proteins which are supposed to play role in the cell cycle. numerous publications describe positive correlation between intensity of mt expression and proliferative potential of cells. our study aimed at examining the relationship between mt expression and intensity of proliferation, measured using ki antigen. the studies were performed on samples of ductal breast carcinomas of g , g or g grade of differentiation as well as on samples of normal epithelium and planoepithelial carcinoma of uterine cervix. paraffin sections of the examined cases served to perform immunocytochemical reactions using mouse monoclonal antibodies to mt and ki . double staining reactions were also performed using the same antibodies. the performed studies showed that mt expression in breast cancers of g grade was significantly lower than in cancers of g or g grades (p= . ). in the case of ki , the least intense expression characterized cancers of g grade and the most intense reaction was noted in cancers of g grade (p= . ). no relation between mt and ki expressions was observed in g and g groups of breast cancers (p> . ) while in the g group as well as in the entire group of breast cancers (g to g ) a positive correlation between the two expressions was noted (p< . ). analysis of reactions in the non-keratinising stratified flat epithelium and in the planoepithelial cancers of uterine cervix showed no correlation between expressions of mt and ki . however, mt expression was detected only in the least differentiated cells of the epithelium. conclusion our showed that mt expression tends to correlate more with cell differentiation extent than with proliferative potential of the cells. is the activation of akt a prognostic parameter in colorectal cancer? akt (protein-kinase b, pkb) is a serine-threonine kinase that is activated mainly by insulin, igf- receptor and erbb family receptor signaling in a pi -kinase dependent manner, and is implicated in tumor proliferation in breast, ovarian, pancreatic and gastrointestinal cancers. the phosphorylation of its substrate proteins result in increased growth and decreased susceptibility to apoptosis. the activation of the pi k-akt pathway is downregulated by pten phosphatase, and mutations or a loss of pten activity results in a high constitutive activation of the kinase. the aim of the present study is to investigate the levels of the activation of the pi k-akt pathway in a series of colorectal carcinomas and to evaluate its role on tumor resistance to chemotherapy. we achieved surgical colorectal carcinoma specimens at pt stage. all patients received at least two chemotherapy lines based on platinum and cpt- after surgery. levels of activation of akt were tested by immunohistochemistry in primary tumors using a polyclonal antibody that recognizes the phosphorylated akt at ser . in the series, ( . %) showed staining for phosphoakt in tumor cells. phos-phoakt was detected in tumor as clusters of cells related to necrotic foci and differentiated areas and correlated with less response to chemotherapy. these results point out that . activated akt is often overexpressed in human colorectal carcinomas and .activated akt can be a parameter of chemotherapy resistance in colorectal cancer. mutational analyses of the ret proto-oncogene in slovenian men families introduction alport syndrome (as) is a progressive inherited nephropathy, characterized by irregular thinning, thickening and splitting of the glomerular basement membrane (gbm), associated with hearing loss and ocular symptoms. as is caused by col a mutations in its x-linked form and by col a and col a mutations in its autosomal recessive form. the mutations in col a and col a have been reported in familial benign hematuria (bfh). aims: we screened col a , col a and col a genes in slovenian families with x-linked as and in slovenian families diagnosed with bfh to determine pathogenic mutations, to correlate them with clinical features and to confirm or provide precise diagnosis. materials and methods genomic dna was isolated from blood. screening for mutation was done by the optimised ssca after pcr amplification of each exon of col a , col a and col a and changed samples were sequenced. we found six different mutations in col a gene in as suspected patients. one of the mutations was present also in three families with bfh. in col a gene three and in col a four mutations, all in heterozygous state, were identified only in patients with bfh. eleven of the mutations are new and private. four rare variants of unknown pathogenesis and many polymorphisms were found in the col a and col a genes. a mutation was detected in % of as patients and in % patients diagnosed with bfh conclusions our study broadened the spectrum of mutations in col a , col a and col a and demonstrated the involvement of the col a and col a genes in the pathogenesis of bfh. introduction prion diseases are a group of fatal neurodegenerative disorders. the commonest human prion disease is creutzfeldt-jakob disease (cjd). sporadic cjd is genetically characterized by a lack of pathogenic mutations in prnp gene but methionine/ valine polymorphism at codon determines the susceptibility to sporadic and other non-genetic forms of cjd. homozygosity for either methionine or valine increases the risk for acquiring cjd. aims: we searched for changes in prnp gene of healthy populations and scjd cases and compared genotypes of codon in order to prove differences among populations and non-familiar origin of scjd cases and also to confirm the influence of codon homozygosity on the disease development. material and methods blood samples from six populations (slovenian, afro-american, pima indian, cheyenne indian, finnish, german) and brain or blood samples from scjd slovenian cases were collected and dna was extracted. pcr-ssca was performed and changed samples were sequenced. introduction tumour suppressor gene rassf encodes different isoforms, derived from alternative splicing and differential promoter usage. the major transcripts a and c are expressed in all normal tissues, but transcript a is missing all cancer tissues. loss of expression is mainly corellated with methylation of the cpg island promoter sequences of rassf a but mutations in exons have also been found. we examined the status of promoter methylation and potential changes in the coding region of the rassf a gene in colon cancer tumours. materials and methods isolated dna from tumours of colon cancer was used for exon amplification of rassf a gene. for identification of mutations and polymorphisms ssca was used. abnormal patterns on ssca plates were sequenced. for determination of methylation status of the cpg islands we used cobra method. results cobra analysis revealed the hypermethylation of cpg islands. sequencing analysis of exons showed different nucleotide changes. we identified c>a mutation (r s), g>c mutation (d h), g>t mutation (a s), g>a mutation (r h), g>a mutation (r q) and c>a mutation (l i). r s, d h, r h and l i mutations have not been published yet. conclusions these results indicate that hypermethylation of the promoter of rassf a gene in colon tumours and missense mutations in protein itself could be involved in tumorigenicity of colon cancer. introduction pineal lesions are rare and comprise , - % of all intracranial tumors. various histological types of tumors arise in the pineal region. the aim of this study was to determinate histological types, immunohistochemical expression, age and sex distribution of lesions in pineal region. twenty-four expansive lesions in the pineal region, obtained between and , were analyzed. formalin-fixed, paraffin embedded surgical specimens were stained routinely and immunohistochemically for panel of antibodies; synaptophysin, neuron specific enolase, neurofilament, chromogranin a, glial fibrillary acid protein, cytokeratin, carcinoembrional antigen, β-hcg, human placental antigen, α-fetoprotein and ki- . results histology revealed six germinomas, seven pineocytomas, three pineoblastomas, five glial cysts and one ganglioglioma, pilocytic astrocytoma and epidermoid cyst. the study group consisted of females ( , %) and males ( , %) ranged from to years of age. all germinomas were in male patient while glial cysts were diagnosed in female patients. slightly female predominance was observed in pineal parenchymal tumors. immunoreactivity for neuron specific enolase, synaptophysin, neurofilament and for chromogranin a was positive in the pineocytomas, and partially expressed in pineoblastomas. the mean ki- index in pineoblastomas and germinomas were significantly higher than in pineocytomas. germinomas showed focal immunoreactivity for β-hcg, human placental antigen, α-fetoprotein and low immunoreactivity for cytokeratin and carcinoembrional antigen. conclusion the most common tumors are pineal parenchymal tumors and germ cell tumors. glial pineal cysts are also common. occasionally other types of tumor are found. immunohistochemistry is an important method in differential diagnosis of pineal tumors. immunohistochemical study of the metastatic brain tumors with accent of the role of thyroid-transcription factor- introduction the exact diagnosis of the metastatic brain tumors /mbt/ is challenge for the surgical pathologist especially in a case of unrecognized primary site. the aim of this study was to determine reliable immunohistochemical panel based on the histological features of the mbt. materials and methods sixty five cases with solitary mbt operated in neurosurgical department of the hospital were examined from the time period - ; thirty nine were of unknown primary origin ( %). immunohistochemical studies were performed using antibodies to ck-s, ema, nse, synaptophysin, psa, vimentin, s- protein, hmb , ttf- and gfap. results diagnosis of the mbt of unknown origin were as follow: squamous cell carcinoma - cases from total , small-cell undifferentiated - from , large cell undifferentiated - from , adenocarcinoma - from , adenosquamous - from , clear cell - from , papillary carcinoma - from , tubular - from , spindle cell - from , unclassified - from . the primary site of adenocarcinoma, undifferentiated small and large cell and clear cell carcinoma was established by using ttf- (positive in from lung cancer, . %), neuroendocrine markers, ck- and psa. after the investigation only three tumors were of unknown origin ( . %). the origin of the rest unknown before studies of mbt, confirmed with radiographic and endoscopic data were: lung - from total, breast - from , kidney - from , skin- from , thyroid gland - from , stomach - from , prostate - from . the colon, testis and ovary, at one case each, were known before operation. conclusions immunohistochemistry remains important and still very helpful method in determining primary origin of the mbt generally in undifferentiated neoplasmes and adenocarcinomas. among the most reliable tumor markers should be included ttf- , for its frequent expression in the lung cancer, predominant among mbt. brain calcification simulating brain tumour. a case report a shmeleva, s romodanov neurosurgery institute, academy of medical science of ukraine, kiev, ukraine objectives the aim of this study was to describe the plural calcifications in the brain. the patient was a -year-old man. he had a severe headache and disorientation, he felt weakness in the left arm. the patient was mentally handicap. st scan showed a tumour located in the deep parts of the right temporal lobe. with a working diagnosis of tumour, the patient underwent the operation. the surgical specimens were prepared by standard histological techiques. results macroscopically, the lesion appeared as a stone. a histological examination demonstrated a massive deposits of calcium in the parenchyma of the brain. calcification developed as small granules in the vessels and calcospherites of various dimensions were formed by confluence. there were zones of haemorrhage, neuronal loss, edema and proliferation of astrocytes. conclusions this patient is an interesting clinical case of dementia characterized pathologically by diffuse calcification. carcinoid tumour metastatic to the orbital muscle. a case report introduction optical contrast is sensitive to monitor dynamic and functional changes, such as blood concentration, tissue oxygenation, and cellular swelling. optical imaging has been widely employed in clinical applications including the investigations of functional neuroanatomy and neuroactivity. due to the overwhelming scatter of light during the propagation inside biological tissues, past attempts to visualize brain architectures non-invasively with the optical contrast have been severely restricted by the unsatisfactory spatial resolution. the aim of this study was to investigate the feasibility of a novel non-invasive transcranial imaging of the structure and function of brain in vivo accomplished with laserinduced photoacoustic tomography (pat). material and methods pat visualized the tissue structures in rat/mouse brain based on the high sensitive intrinsic optical contrast while utilizing the diffraction-limited high spatial resolution of ultrasound. results two-dimensional (or three-dimensional) rat (or mouse) brain structures, with and without lesions, were imaged clearly. in response to whisker stimulation, functional cerebral hemodynamic changes in cortical blood vessels around the whisker-barrel cortex were also mapped successfully. pat imaged hyperoxia-and hypoxia-induced cerebral hemodynamic changes as well. conclusions this study demonstrates that (i) structural pat of soft tissues lesions in the brain presents high intrinsic contrast, (ii) functional pat based on intrinsic optical signals can map the hemodynamic changes in the cerebral cortex and (iii) in contrast to other previously used optical methods, this technique enables completely non-invasive neuroimaging with high spatial resolution transcranially. this novel functional neuroimaging technique, will help to advance the research in neurophysiology, neuropathology, and neurotherapy significantly. microscopic disorders of cortical development of brain and the etiopathogenetic relevance of their detection in patients with temporal lobe epilepsy due to hippocampal sclerosis introduction hippocampal sclerosis (hs) represents a common structural basis of temporal lobe epilepsy (tle). however, the etiological factors and mechanisms leading to its development still remain unexplained. the aim of this study was to identify the disturbed neuronal migration and differentiation (nmd) in the temporal lobe resected for hs and to evaluate its potential role in the individual pathogenesis of hs. we present neuropathological findings in the resected hippocampus and the pole of the temporal lobe in patients with hippocampal sclerosis with an attention paid to the histopathological identification of defects of nmd in the temporal lobe. the morphological findings were correlated with the results of clinical examinations and the patients´ histories. results 'initial precipitating injuries' that are thought to cause the development of hs (febrile seizures in early childhood, head injury or meningoencephalitis) were present in the history of patients. in the remaining cases, no predisposing factors were found. defects of nmd were observed in cases; in three of these, no predisposing factors were identified in the patients´ histories. we suggest that in these cases, hs arises due to previously undetected disorders of cortical development. a latent neocortical malformation may also contribute to the development of hs in patients with an initial precipitating injury in anamnesis. conclusion histopathological examination of resected epileptic brain tissue can provide insights into the individual pathogenesis of epileptic disorders, especially by the detection of microscopic disorders of cortical development. supported by: gacr / /d , vz fnm , vz msmt and introduction cerebellar liponeurocytoma is a newly recognized rare clinicopathological entity of the posterior fossa, with advanced neuronal/neurocytic and lipomatous differentiation. the term 'cerebellar liponeurocytoma' was recently adopted by the who group to replace several other different terms used up to now. this tumour shows many morphological similarities to medulloblastoma and neurocytoma, but according to available literature has more favorable prognosis then typical medulloblastoma. we report a case of cerebellar liponeurocytoma in a -years-old woman who developed typical symptoms of a posterior fossa tumor. computed tomography (ct) scan disclosed a heterogeneous tumor mass in the left cerebellar hemisphere. the lesion was totally resected. methods the surgical specimen was rinsed in saline solution and fixed in % paraformaldehyde in phosphate buffer at ph . for hours, embedded in paraffin and prepared for histological and immunostaining. results microscopically, the tumor was composed of small less differenerentiated, round or oval closely packed medulloblastomalike cells and vacuolated cells with macrovesicular lipid accumulations giving in some large areas an appearance indistinguishable from mature fat cells. glial and neuronal differentiation was histopathologically and immunohistochemically (gfap, nse, nf) noted. these cellular components were intermixed in different proportions without distinct separation. many tumor cells of all types showed single or multiple smaller lipid vacuoles in their cytoplasm. conclusion our case provides evidence that mature adipose-like tissue in these tumors appears as result of progressive lipidization of neuroectodermal tumour cells rather than as result of adipose metaplasia. introduction determination of proteins taking place in the control of proliferation in normal cells helps a better understanding of cellular transformation and proliferation mechanisms. object although they do not arise from a tissue normally thought to be a target tissue for progesterone, meningiomas show a number of epydemiologic and clinical features which suggest that females sex hormones can play a role in their development (higher incidence of meningiomas in woman, rapid progression of symptomas and icreasing in size during the pregnancy and during the luteal phase of the menstrual cycle, as well as significant association of meningiomas with obesity and breast carcinomas). the aim of our study was to investigate the progesterone receptor contents of bening meningiomas (who gr. i), which is completly excised and evaluated possible relationship between receptor content and sex and age of the patients, histological tipe of meningiomas and localisation of tumor. material and methods tissue samples, fixed in formalin, and embedded in paraffin were evaluated immunohistochemically for progesterone receptors using specific monoclinal antibodies. the correlation between number of positively nuclei and progesteron status was determined by the analitic statistical test mann-whitney. % of the meningiomas tested were positive for progesterone receptors. progesterone receptors in the benign meningiomas in the male patients was significantly higher when compared to the female patients (p< , ). neonatal intractable seizures revealing diffuse and bilateral cortical dysplasia. a case report s bellefqih , c rambaud , t lacaze , jj hauw , g lyon , d seilhean groupe hospitalier pitié-salpétrire, paris, france hôpital antoine-béclre, paris, france cerebral cortical dysplasia is generally focal and sometimes related to tuberous sclerosis. it is believed to result from disrupted neuronal migration during cortical development. we report an unusual case of neonatal diffuse and bilateral cortical dysplasia revealed by refractory epilepsy. a male infant, born at weeks' gestational age (ga) without familial history of neurologic disease, presented at birth a convulsive encephalopathy, associated with severe hypoxia with pulmonary hypertension and iterative hypoglycemia. a foetal goitre was attributed to hyperdose therapy for maternal graves' disease between and ga. the patient died at weeks of life from respiratory failure after intensive care. all the iso-and allocortical areas examined were normal at gross examination, but microscopically dysplastic. there was disorganization of the laminar architecture and presence of large multipolar neurones diffusely distributed over the cortex. they showed positive immunoreactivity with neurofilaments and synaptophysin, and were negative with gfap and vimentin. numerous ectopic neurons were found in the white matter. in addition, c shaped thickened inferior olive was noted. the cerebellum, including dentate nuclei, and other brain stem nuclei were normal. focal cortical dysplasia lesions are localized malformation of the isocortex with positive gfap and vimentin in dysplastic neurons. in this case, the dysplastic lesions were diffuse and astrocytes were normal. no genetic investigation was available, but the negative immunostaining of large cells by gfap does not favour the diagnosis of tuberous sclerosis. the pathogenesis of this exceptional disease remains obscure but the responsibility of the foetal thyroid disorder cannot be excluded. structural genomic abnormalities of chromosomes and in myxopapillary ependymomas in an attempt to clarify the chromosomal status of these tumors and identify commonly aberrant regions in the genome we have combined molecular/cyto/genetic methods to study genomic abnormalities in mpe. comparative genomic hybridization analysis of / tumors identified concurrent gain on chromosomes and as the most frequent finding. the majority of the tumors were also studied using microsatellite analysis ( markers for chromosome and markers for chromosomes ) and interphase-fish with centromeric probes for chromosomes and . our combined results were consistent with polysomy and in cases, polysomy either or with partial gain in the other chromosome in cases, tetrasomy or partial gain either or with complex rearrangement in the other chromosome in cases. in the remaining cases allelic imbalance alone of either chromosome or was identified. other numerical abnormalities observed included gain of chromosomes , , , , , , , q, and x and loss of chromosomes and . the combination of gains of genetic material from chromosomes and seems to be common in mpe and may represent genomic events of importance in the development of the tumor. nonsymptomatic nontumor cysts of the pineal gland: frequency and morphological characteristics the effect of bcl- protein and ki- expression on the prognosis of medulloblastomas introduction medulloblastoma is the most common primary central nervous system tumor in childhood and accounts - % of all intracranial neoplasms in children. aim: in this study the correlation between the patients survival and prognostic factors (ki- and bcl- ) and the tumor size in ct scan were evaluated. material and method selected cases were analyzed. immunohistochemical staining was applied as polyclonal antibody for ki- (dako-a- ) and monoclonal antibody for bcl- (bcl- / /d novo castra) on paraffin blocks.fisher's qhisquare, correlation and mann-whitney-u tests were used for the statistical analyses. results the ages of these patients were ranged between - (mean . ).the size of the tumoral mass varied between to centimeter. patients were alive without illness. patients were lost due to tumor. we could not found a statistical revelance between neither immunohistochemical staining (bcl- , ki- ) nor tumor size on survival rates. conclusion due to radiotherapy and chemotherapy following surgery, the survival rates of medulloblastoma has increased despite the agresiveness of the tumor. there are reports showing no correlation between expression of the bcl- with the outcome of patients. further investigation about the correlation of the prognostic factors with the outcome is required. introduction results of glioma treatment are still unsatisfactory. an accurate histopathological study supplemented with examination of tumour parameters, like proliferation potential and apoptosis may be invaluable in designing an individualised therapy. the aim of this study was to analyze clinical data and histo-clinical correlations in recurrent brain tumours of astrocytic origin. one hundred surgically removed supratentorial gliomas were studied (mean age: . yrs). histological grade was determined according to who classification and the daumas-duport scale (st.anne/mayo). proliferation activity was measured using the pcna index and agnor count. the expression of bcl- protein was examined with immunohistochemistry. results and conclusion the initial symptoms of glioma were headaches and vomiting ( %), paresis ( %), epilepsy ( %) and aphasia ( %). the tumour location was predominantly in the left hemisphere, in the temporal ( %), parietal ( %) and frontal ( %) lobes. the pcna index correlated with who grade (giiindex %, giii - - % and giv - %). for grades i, ii, iii and iv progression free survival (pfs) was: . , . , . and . months respectively. the agnor count did not correlate with who grade, daumas-duport grade or pfs. bcl- expression was observed in single cells in gi and gii tumors, in % and % of cells in giii and giv, respectively. the daumas-duport scale is simple and useful in prognosing. bcl- expression does not correlate in a linear manner with tumour grade. as gliomas are extremely heterogeneous, for better characterization of the clinicopathologic correlations new prognostic factors are needed. introduction schilder's type of myelinoclastic sclerosis is rare variant of multiple sclerosis in children and young adults. usually, brain lesions occur in the white matter like solitary, or less often multifocal, acute plaques ranged in diameter more then cm. case report the -year-old female patient was referred to our hospital with progressive right-sided hemiparesis, motor dysphasia and frontal lobe syndrome. symptoms started days earlier with acute headache and no febrile state was reported. mri of the head revealed two oval lesions with hyperintensity in t weighted images of left frontal and parietal lobe. the first diagnostic impression was of glioblastoma multiforme. results from standard blood and urine tests, tumor markers and cytological analysis of bone marrow were normal. analysis of csf and culturing csf and serum for bacterial, viral, protozoan and fungal infections were negative. analysis of csf revealed increased immunoglobulins, ocb and increased intratecal synthesis of igg. a stereotactic biopsy was performed one month after first symptoms. neuropathology findings. the biopsy after neurosurgical treatment yielded five needle cores of grayish cerebral tissue. hematoxylin and eosin-stained sections of paraffin embedded tissue showed demyelinating white cerebral tissue with relatively sharply defined margins. lesion was infiltrated by macrophages (cd immunopositive cells and luxol fast blue negative) and large reactive astrocytes with ki and pcna nuclear positivity. around blood vessels were scattered perivascular cd and cd lymphocyte. silver impregnation and nf immunostaining showed that the axons within the plaque are attenuated but not destroyed. conclusion clinical and pathological findings suggested a demyelinating disease destroyed huge parts of white matter in before health mid-aged women, without previous infection or vaccination. accordingly we diagnosed schilder's type of ms. stereotactically guided brain biopsy: six years' experience of pathologic interpretations from turkey e erden , a okcu heper , a savas ankara university, school of medicine, pathology department, ankara, turkey ankara university, school of medicine, neurosurgery department, ankara, turkey introduction stereotactic biopsy is a widely used procedure in brain lesions. the aim of this study was to analyse the reasons of the discordance between the results of the intraoperative crush preparations (icp) and final permanent paraffine preparations (fppp) of stereotactically guided brain biopsies (sgbb) in our hospital, and find out the clinical benefits of this procedure. total ct and/or mri guided brain biopsies performed between - were analysed retrospectively. icp and fppp preparations were evaluated and discordant cases were detected. then we evaluated the reasons of discrepancies. results the correct diagnosis was achieved by stereotactic biopsy in . % of the patients. of them were diagnosed as neoplasia and were diagnosed as non-neoplastic pathology. there was discordance between icp results and fppp diagnosis in . % of the stereotactically diagnosed patients. the patients diagnosed as neoplasia were treated by rt and/or kt and the non-neoplastic pathologies were taken in suitable treatment and follow-up scheme. conclusion to improve the diagnostic accuracy of the sgbb, the following items should be taken into consideration: the quality of the icp with giemsa staining is very important. in the biopsies from the lesions, which are suspected as neoplasia having, microscopic features such as gliosis, edema, hemorrhage must be interpreted as non-diagnostic. the number of the biopsies must be increased in the presence of necrosis. epitheloid and pleomorphic cellular groups can mimic benign and malignant pathologies. interpretation of the icp and the fppp should be done by the same pathologist. intraneural capillary haemangioma of spinal nerve root. case report p xirou , s barbanis , i efstratiou , p sevastiadou , i tsitouridis papageorgiou general hospital, department of pathology, thessaloniki, greece papageorgiou general hospital, department of radiology, thessaloniki, greece intraneural haemangiomas of the spinal nerve roots are rare. the few cases reported in the literature are usually of the capillary and less often of the cavernous type. these lesions occur in adults, mainly males and the majority of them are located in the cauda equina. a case of intraneural capillary haemangioma involving one lumbar nerve root is reported. the patient, a year old male, presented with a -year history of severe intermittent low back pain. magnetic resonance imaging detected an intradural, extramedullar, space occupying mass at the level of l -l , causing displacement of cauda equina roots. laminectomy and complete removal of the lesion were performed without neurological problems. on gross examination the resected specimen was a redbrown, well demarcated, lobular mass, measuring . cm in greatest diameter. remnants of a nerve root were recognized at the periphery. histological examination showed that the lesion was located within the endoneurium. it consisted of multiple small dilated capillaries, often showing incomplete lobular pattern, dispersed within normal nerve fascicles. the endothelial lining showed neither nuclear atypia nor mitotic activity. in the substrate mild edema and focal inflammatory lympocytic infiltrations were found. haemangiomas of the spinal nerve roots often pose a challenging diagnostic problem, and knowledge of their existence is relevant since they can mimic other more frequent tumors of the region. introduction sensorimotoric polyneuropathy has rarely been reported as a presenting paraneoplastic neurological disorder associated with anti-hu antibodies. electrophysiological studies suggest a dysfunction of axon-schwann cells, but the exact pathological mechanisms remain unclear. case report a -year old woman with short history of weight loss and parastesias in the legs was admitted to the hospital. triparesis and dyspnea were found. mri scan showed leukencephalopathy, eeg revealed diffuse encephalopathy. sensorimotoric axonal polyneuropathy was present in emg, and small lesion in the lung was found on the ct scan. anti-hu antibodies were present in the serum and in the cerebrspinal-fluid. autopsy revealed macrocellular adenocarcinoma of the lung with neuroendocrine differentiation and sensorimotoric neuronopathy/ encephalomyelitis. methods the brain, spinal cord, sensory ganglia and a part of sural nerve were fixed in formalin, paraffin embedded and stained with h&e. other part of sural nerve was prepared for immunofluorescence, semi-thin and ultra-thin sections. results axonal degeneration without any inflammatory reaction and a very few signs of regeneration were found in the sural nerve. the inflammatory destruction of spinal ganglia neurons, axonal degeneration in dorsal columns and motor neurons inflammatory destruction in the spinal cord were found. neuronal degeneration, glial proliferation, perivascular inflammatory infiltrates and some cd + lymphocytes surrounding single neurons were a common finding in the brain. conclusions this study shows that inflammatory destruction of the spinal ganglia neurons and motor neurons in the spinal cord was the main cause of clinically rapidly progressive sensorimotoric polyneuropathy in the anti-hu positive patient with macro cellular lung carcinoma with neurendocrine differentiation. isolated extranodal spinal rosai-dorfman disease. a case report ai freitas rc silva l costa, v velasco, p monteiro, m teixeira, f pardal de oliveira hospital de sao marcos-serviço de anatomia patológica, braga, portugal introduction sinus histiocytosis with massive lymphadenopathy was first described in , by rosai and dorfman, as an idiopathic histiocytic disorder, which typically involves lymph nodes. lately, involvement of many other organ systems has been documented, either alone or in association with nodal manifestations. nowadays, rosai-dorfman disease is a wellrecognised clinico-pathologic entity. extranodal disease has been reported in % of the reported cases, occasionally representing the first and exclusive manifestation of the disease. although central nervous system involvement has been well documented, the spinal canal is affected only exceptionally. we report a case of an isolated extranodal spinal rosai-dorfman disease with review of the literature. the authors describe a old woman with a history of spastic hemiparesis in which radiology disclosed a dural-based intramedullary spinal cord lesion at c level. the authors describe the histologic and immunohistologic features of the lesion and discuss the differential diagnosis. conclusion isolated cns rosai-dorfman disease without contemporary lymph node involvement can be a diagnostic challenge. pathologists must be aware of this disease in order to make a correct diagnosis and avoid diagnosis of malignant histiocytic lesions. ezrin immunoreactivity: is it a usefull marker for the prognosis and grading of astrocytoma? ie gurer , ga gökhan , t uçar akdeniz university scholl of medicine department of pathology, antalya, turkey akdeniz university scholl of medicine department of neurosurgery, antalya, turkey introduction the actin-binding protein ezrin is supposed to increase the invasiveness of the malignant cells. in this study, the value of ezrin immunoreactivity (ir) was invastigated as a grading and prognostic parameter in astrocytomas. material and method ezrin ir was studied in low grade and high grade astrocytomas. the mean age of the patients was . . monoclonal ezrin antibody (ms- -r neomarkers) was applied on the sections of the paraffin-embedded tissues. immunostaining intensity of ezrin was estimated as semiquantitatively. when no staining was visible the score of , mild staining scored as , moderate , and severe staining scored as . also positive stained areas were evaluated as percantage ( %, - %, above % staining areas). statistically man-whitney u, kruskal wallis and spearman tests were used. results there was no correlation between prognosis and neither the grade nor the staining intensity of ezrin ir. conclusion there are controversial reports especially about the prognostic value of ezrin ir in different types of tumors. there is one report about the association of ezrin ir with the increasing malignancy of astrocytic tumors. results of our study revealed no evidence of correlation between prognosis and ezrin ir for grading of astrocytomas. we conclude that ezrin is not a usefull factor for the prognosis and grading in astrocytomas. introduction hereditary cystatin c amyloid angiopathy (hccaa), an autosomal hereditary disease uniquely found in iceland, usually leads to early death of young adults from cns vascular complications following a disease progress of several years. histopathological characteristics are cystatin c amyloid deposits in meningocerebral blood vessels, narrowing, splitting of media, segmental fibrinoid necrosis and microaneurysms of small arteries and arterioles. associated changes: hemorrhages, infarctions and reparative reactions depending on length of the disease course. this study is a part of an investigation mapping the distribution of cystatin c immunopositive deposits outside the cns in hccaa. material and methods formalin fixed, paraffin embedded autopsy material from patients, females and males, was studied by conventional light microscopy (he, congo red; thioflavin s) and immunohistochemistry (avidin-biotin-peroxidase) with cystatin c antibodies (abc-complex kit from vector). for electron microscopy and specific identification of cystatin c deposits specimens were fixed in macdowell's fixative and embedded in l.r. white resin and immunogold labelled (colloidal gold labelled protein a. sigma). results and conclusion cystatin c immunoreactive deposits were found in most organ systems outside cns. the deposits were primarily located in the adventitia and perivascular tissue of small blood and lymphatic vessels and vascular smooth muscle; in subepithelial connective tissue of intestinal surface epithelium and glandular epithelium; in peripheral nerves. cystatin c immunoreactive blood vessels were not obviously damaged as those in the cns possibly reflecting environmental factors different in these two separate locations not yet identified and characterized. details of cystatin c deposition in the vascular walls will be demonstrated. neurological disorders are among the most prominent clinical manifestations of antiphospholipid syndrome (aps). they are predominantly related to thrombo-occlusive vascular events in central nervous system, vasculitis playing little or no role. case report a -year-old female with a history of seven spontaneous abortions presented with sudden onset of pain and numbness in her right and later left foot followed by pruritic rash over her hands and back and livedo reticularis around knees. emg showed signs of axson injury in multiple nerves. laboratory tests showed lymphopenia and antiphospholipid antibodies of igg class in medium titer at the begining and in low titer three months later. anca, ana, ena, anti-dna were negative, platelet count, complement, lupus anticoagulant and tumor markers were normal. sural nerve biopsy revealed active necrotizing arteritis of small epineural arteries. intimal circular fibrinoid necrosis was accompanied by pyknosis, karyorexis and myofibroblast proliferation in media. inflammatory infiltration by lymphocytes, macrophages, plasma cells and segmented leucocytes was present in intima, but was most prominent in adventitia. thrombosis in organization was observed. vasculitis was present also in arterioles and venules. immunofluorescence showed scarce focal granular deposits of igm, iga class, and complement components c and c q in media of small epineural arteries, together with abundant fibrin deposition. it has been suggested that aps can not be the cause of vasculitis and that vasculitis in aps is associated with an independent underlying disease, such as systemic lupus erythematosus. except for suspected aps, our patient did not fulfill the criteria for any systemic autoimmune disease that could present with vasculitis. immunoautoradiographic characterization of serotonin transporter in human pineal gland m the mechanism that regulates human pineal gland melatonin synthesis is not completely clear. in fact, several studies performed on animals demonstrated the possible role of serotonin and the involvement of its receptors -ht( c) and -ht( a) in the modulation of this process even if is not clear how it is realized. the aim of our study is to verify the presence of serotonin transporter (sert) in the pinealocytes, a molecule involved in the reuptake of -ht from synaptic space, and to represent the action site of i.r.s. antidepressant drugs. we tested with immunoautoradiographic method (iarg) frozen slices conserved at - c o , derived from twenty cadaveric epiphysis ( men, women) by utilizing a mouse monoclonal anti-sert antibody followed by a secondary biotinilated anti-mouse igg antibody, consecutively linked with an avidine/[ h]-biotine complex for a precise localization of the researched molecule. the tritium sensitive films were exposited to treated slices in special cassetes. after thirty days of exposure at - c o , the films have been developed and fixed to obtain auto radiographic images in which the radioactivity distribution followed a continuum pattern in sequential slices of each subject. it was the expression of sert presence in epiphysis. this is the first study that demonstrate the presence of sert in human epiphysis. we suppose that serotonin in human is not only a melatonin precursor synthesized in the pinealocytes but it is involved in the neuroendocrine regulatory mechanism of the gland. loss of heterozygosity (loh) studies allow to identify sites harbouring tumor suppressor genes involved in tumor initiation and progression. this study was performed to establish the frequency of loh on chromosome q in glioblastoma. cases of glioblastoma were examined ( women, men; age: - years). two cases were secondary glioblastoma. in all cases the representative neoplastic tissue and normal brain tissue were cut out with lancet from respective paraffin blocks. dna was isolated with proteinase k with phenol/ chloroform extraction and isopropanol precipitation. polymorphic markers were localized close to three supressor genes pten (d s , d s ), lgi (d s ), dmbt (d s ) which are known to be lost or mutated in glioblastoma. the fith marker was d s q . . fragments were pcr-amplified and analysed using automated sequencer abi . signals from neoplastic and normal tissue were calculated. result under , was scored as loh. in cases at least one marker from chromosome q was lost. cases with loh were men, both secondary glioblastomas showed deletion in q. the mean patients' age with and without qloh was similar. cases showed loh only in pten locus. in cases deletions were stated in two or more loci, what might suggest loss of longer part of chromosome . cases with loh in q revealed additional changes in different chromosomes: loh in p ( cases), p ( ), q ( ), p ( ), q ( ), q ( ), q ( ), p ( ). our results are similar to the other studies, showing changes in q in about % of glioblastoma. histopathological changes of the skin in systemic lupus erythematosus ta vervekina, ba magrupov, gsh shamukhitdinova tashkent, uzbekistan introduction morphologic examination of the skin is of great importance for the diagnosis of various nosologic forms of rheumatic diseases. the aim of the present study was to evaluate histopathologic changes in skin biopsies of patients with systemic lupus erythematosus (sle). material and methods skin biopsies of patients ( women and men; age range from to years) with preliminary diagnosis of sle, based on clinical data and laboratory analyses, were studied with the purpose to specify the differential diagnosis. biosy tissue specimens were fixed in % neutral formalin, embedded in paraffin, and serial sections were stained with haematoxilin-eosin, picrofuxin, and the combined rhitter-olesson staining. after treatment of the specimens with . % trypsin, direct immunofluorescence technique was carried out for detection of immunoglobulins g and m at the dermo-epidermal junction (lupus band-test). results our study established the presence of hyperceratosis with formation of keratic clogs, epidermal atrophy with vacuolization of basal cells, vasculitis, eosinophilic degeneration of collagen and an atrophy of appendages. specific immunostaining for immunoglobulins g and m at the dermo-epidermal junction was observed in all except in cases. we did not observe inflammatory cell infiltrates in dermis in any case. conclusion the pathogenesis of skin histologic lesions in sle is most likely associated with immunological reactions and therapy carried out earlier. structural peculiarities of different nevuses and their prognostic value e stupina first tashkent medical institute, tashkent, uzbekistan for the time being the causes of skin melanoma have not been clearly understood. development of skin melanoma was related to preceding pigment formations in % to % of patients. among a variety of investigation methods histologic one is basic to establish the final diagnostic. complex morphologic study was carried out on skin biopsies with different types of pigment nevuses and melanomas. of them biopsies were selected for the thorough pathomorphologic investigation. light microscopy, electron microscopy and morphometric method were used. serial sections for light microscopy were stained with hemotoxylin and eosine, picrofuchsin-fuchselin by van-gizon, combined staining by riter and oleson. the findings have shown that the rate of pigment skin neoplasms of all the biopsy material of a specialized institution came to , % among neoplastic and , % among non-neoplastic skin lesions. pigment nevuses more frequently occurred in females aged to years applied for medical aid, while melanoma in ones above years. intradermal nevus was diagnosed in , % of cases, its proliferative activity was expressed in % of patients. in , % of patients skin melanona was assigned to - th grade of invasion according clark's classification. each form of pigment nevus had morphometric peculiarities, significantly differing in dimensions of the cell, nucleus and their ratio. the most reliable parameter of malignization was the length of the nucleus and cell axial section circle. of frequently occurred nevuses intraepidermal (borderline) one was more predisposed to malignization which was evident from significant increase of all morphometric parameters. introduction cutaneous ciliated cysts are a rare type of skin cyst, usually presenting themselves in women between the nd and th decades of life. their origin is unknown and has been related to müllerian remnants based on similarities of the epithelium to the fallopian tube, but the occurrence of cases in men has given rise to the possibility of a sweat gland origin. case report a year-old woman complained of an asymptomatic, longstanding, lesion located in the skin of the thigh. once removed it was seen to correspond to cyst with clear fluid inside. histopathological examination revealed a dermal cyst with ciliated cuboidal epithelium, usually double layered, but with some pseudostratified areas. occasionally, papillary projections were seen in the lumen. there was no evidence of cellular atypia or mitotic activity. immunohistochemical staining with oestrogen and progesterone receptors was strongly positive throughout the lesion. the clinical and histopathological findings fit in with the diagnosis of a typical cutaneous ciliated cyst of the lower limbs (cutaneous ciliated cyst). this is a rare cyst usually arising in the lower limbs of young women. its origin is unknown and the demostration of sex-hormone receptors has been used to favour the müllerian origin. nevertheless, some cases have been reported in men, which has diverted attention towards sweat glands as a possible origin of the cyst. the real incidence of the sex-hormone receptor if unknown, since it has not been clear in most published cases. introduction p protein is essential for the regulation of cell proliferation and its aberrant accumulation is usually seen in malignant tumors but, also, occurs in squamous epithelium of inflammatory skin diseases characterized by hyperproliferation. the aim of this study is to elucidate the role of the p tumorsuppressor protein in the pathogenesis of different hyperproliferative, non-malignant and malignant skin diseases, and association between p overexpression and cell proliferation. we also investigated the influence of ageing on p and ki- protein expression. material and methods hundred and fifty skin specimens, consisting cases of each, normal skin (ns), psoriatic skin (ps), keratoacanthomas (ka), basal cell carcinomas (bcc), squamous cell carcinomas (scc) were examined immunohistochemicaly to assess p and ki- protein expression. results p immunostaining of ns, ps, ka, bcc and scc was positive in . %, . %, . %, % and . % cases, respectively. p and ki- positive cells were present in basal (ns) and suprabasal layers (ps), and not only in cancer nests of ka, bcc and scc, but also in dysplastic and even morphologically normal epidermis adjoining cancers. the positivity of p and ki- protein differed significantly among the groups, with no differences in p expression between ns and ps, and in ki- expression between ps and ka. within all groups, there was a significant correlation between p and ki- protein expression. localisation and age were significantly related to p and ki- expression in all groups, except for localisation in ps, and age in bcc. conclusions our findings suggest that p overexpression occurs widely in neoplastic and non-neoplastic skin lesions. it is associated with the cell proliferation in normal, as well as, in changed epithelium. p accumulation is an age related process and significantly related to sun exposure, especially in ns and ps, as well as in ka and scc. we also suggest that p overexpression begins in the early stage of carcinogenesis, before the appearance of malignancies in skin, and may be a useful predictor for the detection of nonmelanocytic skin cancer. background maspin, a member of the serine proteinase inhibitor (serpin) family, has been recently pointed to as a tumor suppressor gene and angiogenic inhibitor. objectives of this study were to investigate role of maspin and beta-catenin in basal cell carcinoma (bcc). we studied immunohistochemically their expression in basal cell carcinomas together with trichoepitheliomas (te) and normal skin controls. microvessel count (mvc) was also done using fviii-related antigen. we examined the correlation of nuclear and/or cytoplasmic maspin expression with histological type, age, gender, invasion of safety margin, as well as stromal reaction (lymphocytic or fibroblastic. all primary antibodies (maspin, beta catenin and fviii-related antigen) as well as detection kit (abc peroxidase/dab quick test), were purchased from novocastra labs.uk. maspin expression was strongly cytoplasmic and membranously expressed in te and down-regulated in nodular bcc. nuclear maspin expression was found in . % of our cases. it was statistically positively correlated with invasion of safety margin (p= . ) and with infiltrative type (p= . ) and negatively correlated with a mvc > (p= . ). cytoplasmic maspin was correlated to a strong fibroblastic stroma (p= . ). beta-catenin expression was found in both te and bcc with the same intensity and was only correlated to younger age(p= . ). conclusion cytoplasmic maspin expression could differentiate between te and nodular bcc nuclear maspin expression could be related to aggressive behaviour of bcc and is realted to lower mvc (< ). introduction trichilemmal carcinoma (tc) is rare skin tumour occurring in the sun-exposed areas (largely on the face or ears) of the elderly. clinically, it may be mistaken for a squamous cell carcinoma, basal cell carcinoma, nodular melanoma or keratoacanthoma. although the histological picture suggests a high-grade malignant neoplasm, tc has an indolent course, and conservative surgical excision with clear margins is curative. aim: histological and histochemical examination of rare cases of tc of the skin. materials and methods we report two patients (males) with tc on the ears, presented as slow-growing nodules. tumour biopsy specimens were routinely fixed and processed. deparaffinized sections were stained by he, pas, pas-d. results histologically tumour cells showed a solid and a lobular growth patterns in continuity with the epidermis, with foci of pilartype keratinisation and peripheral palisading. tumour nests were mostly composed of large atypical cells with clear cytoplasm containing pas-positive, and diastase sensitive materials. these cases were treated only with surgical excision, and there has been no evidence since of local recurrence or metastasis. conclusion we believe that careful histopathological examination of pillar appendage tumours will help to classify the wide range of clinically and histologically different tumours and to establish the exact diagnosis. introduction pigmented cutaneous metastases from breast carcinoma are uncommon and may simulate malignant melanoma both clinically and morphologically. we report a case of a -year-old female who developed multiple cutaneous pigmented metastases twelve years after radical mastectomy for left breast carcinoma. materials and methods hematoxylin-eosin and immunohistochemically (streptavidin-biotin method) stained slides from three different regions of the chest skin and from the scalp plaque. results numerous darkly pigmented plaques and nodules have been developed over a -month period on the back of the chest spreading anteriorly to the right subcostal region together with large, focally eroded scalp plaque. histopathological examination revealed full thickness dermal infiltration and superficial subcutaneous fat involvement with cords and nests of neoplastic epithelial cells showing ductal structures and intracytoplasmic lumina in some areas. irregular nests and single tumor cells at epidermodermal junction were accompanied with melanophages in papillary dermis. immunohistochemically all tumor cells were cytokeratin positive and cytokeratin negative while epidermal and most superficial dermal nests showed hmb- and melan-a reactivity as well. scalp metastasis displayed less pronounced epidermotropism and only focal, single cell hmb- and melan-a positivity. conclusion epidermotropic breast carcinoma metastases may mimic malignant melanoma not just clinically and morphologically but also immunohistochemically and that might be related to the location of metastatic lesions. immunolocalization of lactoferrin in pigmented lesions of the skin g barresi, g giuffre, f fedele, m grosso, v barresi, g tuccari department of human pathology, university of messina, messina, italy lactoferrin (lf) expression has been immunohistochemically investigated in formalin-fixed paraffin-embedded bioptic skin samples ( nevi, melanomas, vulgaris or seborrhoic pigmented warts, pigmented basal cell carcinoma; specimens of normal skin were utilized as control. on µm thick sections, depigmentation and antigen retrieval procedures were performed. lf immunoreactivity was revealed by a rabbit anti-human lactoferrin (dako, denmark, : ), incubating all sections at c overnight; successively, bridging antibody and pap complex for min at room temperature (rt) were applied. for the demonstration of peroxidase activity, the sections were incubated with - ' diaminobenzidine tetrahydrochloride-h o substrate solution for min at rt in darkness; slides were then slightly counterstained with mayer's haematoxylin. quantification of lf immunoreactivity was performed using the intensity-distribution (id) score based on both the percentage and the staining intensity of positive neoplastic cells. an evident cytoplasmic immunoreactivity for lf was encountered in melanocytes, either in nevi or in melanomas; no ap-preciable differences were found among junctional, intradermic or compound nevi as well as between spindle or epitheliod cells melanoma. basal cell carcinomas were always unstained, even if some degree of positivity was found in areas which showed squamous differentiation; sheets and whorls of keratin also exhibited a strong immunoreactivity, when encountered in warts. if the lf immunoexpression could represent the result of an endogenous synthesis by cells in order to have greater availability of iron for their metabolism or to modulate an unspecific inflammatory or anti-oxidant response remains to be fully understood. cutaneous melanomas were detected at an increasing rate worldwide. even thought many patients are diagnosed at an early stage, the death rate continues to rise due to the increasing incidence of more advanced lesions. the aim of this study is to detect in cases of malignant melanomas. materials and methods analysis were performed in paraffinembedded cases of cutaneous malignant melanomas ( cases in situ and cases invasive melanomas). apoptotic index (ai) was determined using the in situ end-labeling technique(tunel), who quantify apoptotic cell death at single cell level and tissues. discussion and conclusion ai was high in the cases of in situ melanomas, but in invasive melanomas (nodular melanomas, superficial spreading melanomas with vertical growth phases, acral lentiginous melanomas etc) ai was decreased in tumor cells. we considered that usually apoptotic cells represent ~ % of tumor cells. at cases of cutaneous malignant melanomas in which ai was high ( %), frequently at in situ melanomas, the prognostic is good, when in the others cases in which apoptosis is missing or is very rare in cells ( %), the prognostic is reserved. this nonmorphologic technique to evaluate the prognosis of melanoma must be regarded as investigational and cannot yet be recommended for routine application. all necessary pathologic data, such as tumor location, size, depth, ulceration, mitotic activity and growth phase, can be obtained by examination of standard microscopic preparations of malignant melanoma as stained with hematoxylin and eosin. introduction small vessel vasculitis is a group of disorders of unknown etiology, possibly associated with connective tissue disease (ctd). the aim of this study is to find correlation between clinical, morphological and immunological changes in the skin and muscle biopsies of the patients with ctd, where small vessel vasculitis is the main finding. we analyzed skin and muscle biopsies from patients with ctd: dermatomyositis, systemic sclerosis, syndroma raynaud, vasculitis, sle, polyarteritis nodosa. were females, and males. the age of the patients was between - . standard histological and histochemical stainings (he, pas, van gieson-elastica) were done. small vessel vasculitis was found throughout skin and muscle biopsies, characterized with thickening of the basal lamina, tiny fibrinoid necrosis (if present), swollen endothelial cells and perivascular edema with lymfocytes. the muscles were necrotic with regeneration and fibrosis and reduction in the intramuscular capillaries. the changes were positive on pas and elastic tissue staining. direct immunofluorescence technique was done with antihuman igg, igm and iga antibodies. granular or linear immunofluorescent deposits were found at the dermo-epidermal junction zone and in the vessel walls. antinuclear antibodies were done by indirect immunofluorescence technique using serum from same patients of which % were positive. lymphoid cells around the vessels and in healthy muscle fibers were positive for markers for t cells (cd ro and cd ). conclusion there is correlation between morphological and immunohistochemical changes. the presence of immunologic deposits in the vessels suggests possible autoimmune etiology. cutaneous angiomyolipoma introduction angiomyolipoma is a benign tumor almost exclusively seen in the kidney. cutaneous angiomyolipoma (aml) is a very rare benign vascular tumor that consists of a proliferation of blood vessels, smooth muscle bundles, and mature fat tissue. aml occurs more often in adult male patients and is often seen in acral locations. case report a -year-old man who had a -year history of a painless, solitary, noninvasive nodule on the right auricle lobule. the patient had no signs of tuberous sclerosis. material and methods the lesion was cm in diameter and clinically appeared to be a lipoma. the lesion was excised. skin specimens was fixed in % buffered formalin, routinely processed, and embedded in paraffin. it was examined by routine light microscopy with histochemical and immunohistochemical stains. results on sections, the lesion was a well-circumscribed nodule in the dermis. there was fibrous pseudocapsule around it. the nodule was composed of an intimate mixture of mature adipose tissue, blood vessels, and bundles of smooth muscle cells. combination of different types and sizes of vessels were particularly evident. cellular pleomorphism, mitotic figures, and mucoid degeneration were absent. reactivity for hmb- was negative. the histopathological diagnosis was angiomyolipoma. conclusion amls are very rare at extrarenal locations. in , argenyi presented the first report of cutaneous angiomyolipoma in the upper helix of the ear. the reported lesions were located at acral sites. as most cases of angiomyolipoma were long-standing lesions located at acral sites, and therefore exposed to external forces, some angiomyolipoma could be considered to be a degenerated with replacement by fatty tissues. the primary b-cell cutaneous lymphoma is a low frequent disease in a general hospital. its nodal equivalent has very different behaviour and prognosis. aims: to study the incidence, behaviour and follow up of the b-cell cutaneous lymphomas in our hospital over a period of years. material and methods in a total of cases between and we have cases of b-cell cutaneous lymphoma. the skin biopsies were diagnosed acording eortc classification. we did histological and immunohistochemistry studies. these included cd , cd , cd , cd , cd , bcl- and ki . results eith cases of were salt and cases were folicular lymphomas. the ages are between and and is no sex predominance. salt lymphomas were located two in the face and six in the trunk. all the cases of folicular lymphoma were in the face. the immunohistochemistry study in salt lymphoma showed positivity for cd and cd and negative for cd , cd and cd . the expression of ki was very low. the reactive foliculars centers were demostrated with the negativity of bcl- . the folicular lymphomas showed positivity for cd and cd and the number of positive cells for ki was bigger. conclusions in a general hospital in our country the incidence of b-cell cutaneous lymphoma is very low. the majority of the cases arrive with surgical excision, that is a good therapeutic approach. the general pathologist must be aware with reactive limphoid infiltrates because the majority of the cutaneous lymphomas are low grade neoplasies. background cd expression is a distinct feature of both b and t cell activation. the presence of cd + large atypical lymphoma cells in a cutaneous infiltrate has been considered a characteristic feature of lymphomatoid papulosis/cutaneous cd +. recently, however, cd expression has also been described in some nonneoplastic infiltrates of de skin. design twenty skin biopsy specimens from patients with scabies were evaluated. of these, the lesions had been present for more than months in cases. the diagnosis of scabies was based on the presence of characteristic clinical features and the demostrations of sarcoptes scabiei mite. for each biopsy, a panel of histopathological and inmmunophenotypic features was independently assesed by two pathologists. cd , cd , cd , cd and s- protein expression was evaluated in each case. results cd + cells, either in isolation or in small clusters, were seen in biopsy specimens ( / ; %). cd + cell clusters were located in the papillary and superficial reticular dermis and tended to show a perivascular pattern. all biopsy specimens obtained from long-standing (> months) scabies lesions demonstrated the presence of cd + cells. in contrast, no cd expression was observed in lesions of less than months duration. cd + large cells can be detected in skin inflammatory lymphoid infiltrates associated with scabies, particularly in long-standing (> months) lesions. cutaneous cd expresion, therefore, is not an exclusive feature of neoplastic lymphoid proliferations. probably as the result of persistent antigenic stimulation, cd expression can also be detected in activated lymphoid cells of nonneoplastic infiltrates associated with insect bites, viral and bacterial infections and peculiar drug reactions. introduction malignant transformation of melanocytes is a multistep process characterized by distinct histopathological stages. differentiation between naevi, especially dysplastic naevi and melanoma, can sometimes be difficult to assess by conventional histopathological analysis. the aim of this study was to analyse the differences in proliferative activity and apoptosis between intradermal naevi (in), dysplastic naevi (dn) and cutaneous melanoma (cm). materials and methods fifteen in, dn and cm (thickness less than . mm) were immunohistochemically stained for ki- which was used as a proliferative marker, whereas bcl- and p were used, respectively, as an anti-apoptotic and apoptotic marker. statistical analysis showed a signifficant difference in proliferative activity and the overexpression of p (p < . ), while observed differences in bcl- expression were not statistically significant between these three groups. conclusion proliferative and antiapoptotic activity showed a progressive growth from benign to malignant melanocytic lesions. the strongest expression of p in dn suggests that its overexpression could be the first step in the loss of cell cycle regulation and possible malignant transformation of melanocytes. introduction it is now known that myofibroblastic transdifferentiation in glomerulus takes place during mesangial activation. material and methods we analyzed functional disorders (filtration, concentration insufficiency) and kidney biopsies of patients aged from to years with various forms of glomerulonephritis. we used standard histological, immunohistochemical, immunofluorescence methods as well as electron microscopy in our research. for studying myofibroblastic transdifferentiation we stained tissue immunohistochemically for alpha-smooth muscle actin (alpha-sma) and then carried out a quantitative analysis of the results. results seventy patients had myofibroblasts in kidneys. we found out that level of alpha-sma expression correlates with decrease of glomerular filtration. while disorders of concentration a lot of myofibroblasts appear in kidney interstitium. but we have not found any correlation between clinical forms of glomerulonephritis and those cell growth we had seen. and we should mention that for patients without glomerular or interstitial fibrosis we didn't reveal any changes in cells phenotype. conclusion so we conclude that myofibroblastic transdifferentiation must be considered as an early symptom of kidney sclerosis and can be used as a prognostic criterion. cc tsai , hc hsu dept. of pathology, far eastern memorial hospital, taipei, taiwan, republic of china dept. of pathology, national taiwan university hospital, taipei, taiwan, republic of china introduction to early detect renal and metabolic diseases, a mass urine screen in elementary and high school students was started in in taiwan. we retrospectively reviewed the nephropathologic features to elucidate its value. materials and methods one hundred students, boys and girls, who were found to have hematuria and/or proteinuria by this urine screen program and received renal biopsy, formed the basis of this study. the glomerular pathology was divided according to disease severity into two groups. results group i diseases ( %) showed minor histologic alterations, including minimal glomerular change in cases and mesangial cell proliferation in . among them, children had electron microscopic examination, ( %) had thin glomerular basement membrane (tgbm) disease. group ii diseases ( %) had more severe glomerular changes, including iga nephropathy in cases, membranous nephropathy (mn) in , lupus nephritis in , focal sclerosis in , chronic glomerulonephritis in , and type i and type ii membranoproliferative glomerulonephritis in one case each. these findings confirm that glomerular diseases often progress silently. we found a high frequency ( %) of iga nephropathy and mn in group ii diseases and a close association of mn with hepatitis b virus infection. conclusion we conclude that mass urine screen in school children and renal biopsy are very useful for the early detection of the progressing glomerular diseases and provide great opportunity for early therapeutic intervention and long-term follow-up to better understand the natural courses of glomerular diseases, including tgbm disease. introduction the purpose of the study was to establish a correlation between morphometric kidney biopsy parameters and routine laboratory findings in patients with nephrotic syndrome. material and methods kidney biopsies of patients with nephrotic syndrome were studied using standard techniques of paraffin sections stained with haematoxylin and eosin, picrofuxin van gieson and pas reaction. direct immunofluorescence on snap frozen sections was performed using commercially available antibodies against iga, igm and igg. of the patients, ( . %) were men and ( . %) women, with an average age of . ± . years. biopsy study revealed different types of chronic glomerulonephritis of various incidence: membranous gn ( ), mesangioproliferative gn ( ), mesangiocapillary gn ( ), focal segmental glomerulosclerosis ( ) and terminal sclerosing gn ( ) . morphometric analysis using the semi-automatic image analyser »integral- mt« included cross-section surface areas of the glomeruli and glomerular capillaries, the numerical density of total number of cells per glomerulus, numerical density of mesangial cells, cross-section surface area and numerical cells density of the proximal and distal tubules and cross-section surface area of the peritubular capillaries. routine clinical laboratory analysis included the level of proteinuria, serum creatinine and creatinine clearance, erythrocyturia, leukocyturia and cylinders in urine. a correlation of clinical laboratory parameters with mathematical morphometric models was established for each type of gn associated with nephrotic syndrome. the role of proliferation, growth arrest and apoptosis in the carcinogenesis and prognosis of renal cell carcinoma ga gökhan , g karpuzoglu , t köksal akdeniz university school of medicine department of pathology, antalya, turkey akdeniz university school of medicine department of urology, antalya, turkey introduction nuclear grade and stage are insufficient to predict the clinical behaviour of renal cell carcinoma (rcc). therefore additional prognostic factors are needed. in this study we investigated the role of proliferation, growth arrest and apoptosis in the carcinogenesis and prognosis of rcc using ki , cyclin a, bax, p and bcl- . material and methods formalin-fixed paraffin embedded tissue blocks from patients were studied. cases were divided into low/high grade and low/high stage. apoptotic index (ai) was evaluated as morphologically in h&e stained sections. proliferation index (pi) was detected by using ki and cyclin a immunostaining. ai, pi, p , bcl- and bax expressions in tumor tissue (tt) and in adjacent non-tumoral tissue (ant) and associations with clinical and histopathological parameters were analysed statistically. results the mean indices for apoptosis, ki and cyclin a were: . ± . ( - ), . ± . ( - ) and . ± . ( - ) in tt as well as . ± . ( - ), and . ± . ( - ) in ant, respectively. the number of cases in which the expression of p , bax and bcl- were detected was: ( . %), ( . %), ( , %) in tt, and ( %), ( , %), ( %) in ant, respectively. statistically, there was a significant relationship between the values of tt and ant for ai but not for the pi and the other parameters. only ai and pi significantly correlated with the grade. conclusion in this study the role of pi as a prognostic indicator in rcc was again confirmed. our observations suggest that apoptosis, cyclin a, bcl- and bax may have roles in the carcinogenesis of rcc and that ai may be used as a prognostic indicator. introduction isolated microscopic hematuria (imh) in children always raises the question of the necessity of renal biopsy. many authors consider imh as minor abnormality where glomerular changes are not expected, but general agreement has not yet been achieved. the aim of this study was to evaluate contribution of renal biopsy to diagnosis of glomerular disease in imh. material and methods biopsy was performed in children with imh of proven glomerular origin. mean duration of imh prior to biopsy was . years. biopsy specimens were examined by light (lm), immunofluorescence (if) and electron microscopy (em). results patients ( . %) were found to have glomerular abnormalities. on lm patients had normal glomeruli (ng), mesangial proliferative glomerulonephritis (mepgn), focal segmental glomerulosclerosis (fgs) focal glomerulonephritis (fgn) and membranoproliferative glomerulonephritis (mpgn). if revealed cases as iga nephropathy. em discovered gbm changes consistent with alport syndrome in patients and diffuse thinning of gbm in . in children, subepithelial hump-like deposits were found and considered as the sign of acute postinfectious glomerulonephritis in resolution. one of cases of mpgn showed to be type ii (ddd). on follow-up, of children with alport-like gbm changes developed full clinical picture of the syndrome. further surveillance is needed to confirm the significance of em findings in others. conclusion in children with imh renal biopsy is justified and should always be analyzed by light, immunofluorescence and especially electron microscopy, because em was decisive for final diagnosis in about % of all cases. mesoblastic nephroma of adult. report of a case introduction mesoblastic nephroma is a very uncommon tumor in adults, similar to the congenital mesoblastic nephroma of infancy, first described by bolande et al. microscopically the tumor consist in uniform spindle cells proliferations with numerous vascular spaces, nest of glomeruli, renal tubules that often cystic and dysplastic and lack of nuclear anaplasia or mitotic activity. case report a -year-old woman was admitted to the emergency services for acute abdominal pain in september . she was diagnosed with acute pancreatitis. in cat (without urologic symptoms) a solid mass in the upper pole of the right kidney was founded. with a clinical diagnosis of renal cell carcinoma a radical nefrectomy was performed. follow-up was without event and had no recurrence years afterwards. the right kidney weighed grs. and was unremarkable except for a cms-diameter circumscribed, yellow-tan, solid tumor in the upper pole. microscopic ex-amination revealed a tumor composed of spindle cells, mostly smooth-muscle cells and fibroblast, with numerous vascular structures and some areas of renal tubules with clear cytoplasm. in some places there was atrophic tubules. no mitotic figures were identified. immunohistochemistry findings: the majority of spindle cells were positive for actin, desmin and smooth-muscle actin, and vimentin. and the tubular epithelium stain for ema and keratins. conclusion a combination of histological and immunohistochemical findings is useful to distinguish mesoblastic nephroma from other tumor of the kidney with spindle cells. introduction hyperparathyroidism occurs in patients with chronic renal failure and is usually associated with multiglandular parathyroid hyperplasia. clonal analysis has suggested that in renal hyperparathyroidism glands initially grow diffusely and polyclonally. mechanism of monoclonal proliferation observed with appearance of nodular hyperplasia is not well understood. it might be induced by certain genetic disorders such as overexpression of prad/cyclin d induced by dna rearrangement of the parathyroid hormone gene. the aim of this study was to analyse the expression of cyclin d in normal and hyperplastic, diffuse and nodular parathyroid glands in order to ascertain the possible difference. materials and methods hyperplastic parathyroid glands, from uremic patients who underwent surgery during the period - , were analysed. immunohistochemistry for cyclin d protein was performed on normal parathyroid glands and selected hyperplastic, of diffuse and of nodular type. overexpression of cyclin d was considered to be present if % or more of the sample showed positive nuclear staining. results in normal glands, the mean value of nuclear positivity was . %. overexpression for cyclin d was present in / ( %) diffuse and / ( %) nodular hyperplastic glands. no significant difference was found. conclusion in comparison to normal, hyperplastic glands (nodular as well as diffuse) overexpress cyclin d protein. these results indicate that overexpression of cyclin d is not limited only to neoplastic proliferation, as the most studies showed, respectively this genetic abnormality is not the only responsible mechanism for the monoclonal proliferation. proteins of extracellluar matrix as predictors of glomerulonephritis outcome s Čužić , m Šćukanec-Špoljar , b jelaković , z sonicki , d kuzmanić , m laganović pliva inc., pharmaceutical industry, zagreb, croatia klinički bolnički centar "zagreb", croatia Škola narodnog zdravlja "andrija Štampar", zagreb, croatia introduction chronic renal insufficiency develops only in those cases of glomerulonephritis in which the lumina of postglomerular capillaries in the renal cortex are narrowed due to expansion of the extracellular matrix (ecm). aim of the study was to compare the composition of interstitial ecm proteins with clinical data in various forms of primary glomerulonephritis (gn). material and methods we examined renal biopsies from gn patients, while normal renal tissue was obtained from nephrectomy due to small renal tumour. specimens fixed in b and/or dubosque fixative were analysed by immunohistochemistry (appap-method) using a monoclonal antibodies against tenascin (tn), fibronectin (fn), collagen i, iii and iv (coi, coiii, coiv) (dako). the same fixatives were used for control renal tissue. evaluation of fibrin was performed on frozen sections by immunofluorescence. clinical data were: proteinuria and serum creatinine level, obtained by standard laboratory methods, immunotherapy and ace-genotype. results fifty patients had proteinuria at time of biopsy and of them continued to have it during the follow-up period. patients with persistent proteinuria had more often interstitial fibrosis with elevated content of coi and coiv. only % of patients who responded well to immunotherapy had deposits of fibrin. there were patients with elevated creatinine at biopsy with similar composition of ecm as patients with proteinuria. majority of patients with interstitial fibrosis had ace-genotype ii although this genotype wasn't the most common in the cohort. conclusion our study showed that persistent proteinuria could be linked to interstitial fibrosis and fibrin deposits. patients with ii ace-genotype are more prone to develop interstitial fibrosis. introduction haemorrhagic fever with renal syndrome (hfrs) in slovenia is caused by two hantavirus agents -puumala and dobrava serotypes. there is still a lack of clinical and pathological information, especially about the dobrava virus associated disease. the aim of this study was to analyse and compare the clinical course, outcome and pathological features of hfrs caused by these two virus serotypes. the clinical course of patients ( women) aged ± years (range - ) treated in the nephrological department for serologically confirmed hantavirus associated hfrs was analysed, a serological diagnosis was made using immunofluorescence test and enzyme-linked immunosorbent assay. kidney biopsy specimens of patients were analysed by light microscopy, immunofluorescence and electron microscopic techniques. a semiquantitative scale for gradation of cumulative tubulointerstitial lesions and interstitial haemorrhagic lesions was applied (scores - ). results seventeen patients ( . %) had puumala and ( . %) dobrava virus infection. all patients were febrile and had proteinuria, and other clinical features were: nausea and vomiting ( % of patients), oliguria ( %), abdominal pain ( %), headache ( %), haemorrhages ( %), myalgias ( %), diarrhea ( %), neurological and ophthalmological abnormalities ( %), hypotension ( %). pancreatitis ( %) and elevated transaminases ( %) were observed only in patients with the dobrava serotype. none of the patients in this study died and there was none with permanent haemodialysis dependence. comparison of clinical and pathological features of dobrava and puumala diseases is given in the a need for haemodialysis and the creatinine level at discharge were not associated with the age of patients. those patients who needed haemodialysis, had clinical haemorrhages or had elevated creatinine at discharge, also had higher haemorrhagic and cumulative tubulointerstitial lesion scores, but differences were not significant. elevated serum creatinine above basal levels was present at discharge in patients ( %), range - µmol/l and some patients remained with chronic renal insufficiency. conclusion our results show that dobrava virus infection is associated not only with a significant mortality, comparable to hantaan virus infection, but also with a worse clinical course of renal disease than puumala. the histopathology of the kidney in dobrava and puumala is characterised by haemorrhagic necrotising tubulointerstitial nephritis involving particularly the outer medulla, with quantitative and not qualitative differences between these two infections. renal parenchymal malakoplakia after kidney-pancreas transplantation. case report introduction malakoplakia is an unusual chronic inflammatory disease rarely involving parenchyma of the transplanted kidney. it represents an unusual host response, probably due to leucocyte bactericidal defects to an infecting agent, usually gram-negative organisms, of which escherichia coli is the commonest. malakoplakia in renal transplant is very rare (to date only six cases have been reported) and is associated with a high rate of graft loss and significant mortality. case report we report a -year-old female recipient of kidneypancreas allografts immunosupressed with tacrolimus (prograf, x mg) and mycophenolate mofetil (cellcept, x mg). the patient had suffered from recurrent urinary tract infections. three years after transplantation she was admitted to the hospital with decreased kidney graft function. percutaneous kidney needle biopsy was performed and diagnosis of malakoplakia was established. histologically, on the inflammatory background many mi-chaelis and gutmann bodies were present and confirmed ultrastructurally. the patient was treated with antibiotics and simultaneously immunosuppression (tacrolimus) was reduced. gradually, her renal function improved (with serum creatinine of umol/l). for the entire following year, the patient has been monitored regularly and her renal and pancreas function are stable and both her allografts remain in situ. morphometric study of transbronchial biopsies in pulmonary transplantation e conclusion in our series of tbb for transplant pathology, the aa and nv are the most important morphometric parameters of the biopsy to reach the diagnosis of ar. conversely, the nf does not show significant statistical difference between diagnostic biopsies and nd biopsies. the importance of glomerular deposits of von willebrand factor in human renal allografts bh ozdemir aim to evaluate the role and prognostic importance of glomerular endothelial cell alteration in human renal allografts. material and methods we examined patients with renal transplantation among whom showed acute rejection (ar) and chronic rejection (cr). biopsies were immunostained for von willebrand factor (vwf), fibronectin (fn) and cd . glomerular staining for vwf and fn was graded in a semiquantitative manner using the - + scale. results in the cr group there was markedly increased expression of vwf and fn in the glomeruli, whereas only of ar cases showed intense (grade ) glomerular vwf (gvwf). a significant difference was found between patients with ar and cr with regard to gvwf (p< . ). a positive correlation between the degree of gvwf and early interstitial fibrosis was found in cases with ar in follow-up biopsies (p< . ). the presence of transplant glomerulopathy was found significantly earlier in cases with grade gvwf in ar group (p< . ). we found strong correlation of both intraglomerular fn expression and macrophage infiltration with the degree of gvwf both in ar and cr groups (p< . ). the outcome of grafts that showed intense (grade ) gvwf was significantly worse than the outcome of those grafts with grade and glomerular vwf during the follow-up (p< . ). conclusion increased gvwf deposition in cases with ar is in risk of early transplant glomerulopathy, early interstitial fibrosis and early graft loss. in conclusion, it may be beneficial to use new therapeutic approaches such as anticoagulant medicine for the treatment of ar and cr in future. introduction acute renal allograft rejection is the most important threat to graft survival, and must be diagnosed and treated as early as possible. clinical, radiological and histopathological confirmation of the diagnosis is essential. several factors are being investigated as potential diagnostic markers for acute rejection (ar), and one of these is the hepatocyte growth factor (hgf). the aim of this study was to investigate the presence/density of hgf in core needle biopsies from renal allografts with ar using immunohistochemical staining. the study involved cases of kidney recipients whose allografts were investigated for an initial ar episode without any other pathology. all the patients received cyclosporine-a anti-rejection treatment. each archived slide was reexamined according to the modified banff classification, and patients were categorized as type i ar (group ; n= ), type ii ar (group ; n= ) and type iii ar (group ; n= ). normal histopathological findings in graft biopsies were obtained as controls (group ; n= ). the clinical findings of patients were reviewed from archived records. the sections were stained immunohistochemically with monoclonal anti-hgf antibody. the mesenchymal cells (interstitial macrophages and glomerular endothelium) that showed cytoplasmic positivity were then counted using ocular micrometer. the results were statistically analyzed using mann-whitney u test. results on analysis of the groups' mean cell count, the only significant finding was higher concentration of hgf in the ar patient group than in the control group (p< . ). concerning all other parameters studied, the only significant differences were higher blood urea nitrogen and creatinine levels in group compared to group (p < . ). conclusion our results showed that presence/density of hgf increases in favor of acute rejection and accompanies the histopathological findings. therefore we suggest that density of hgf, excreted in renal allograft tissue, may be a useful marker for supporting ar diagnosis. graft biopsies after renal transplantation in children: differences between fk and cya immunosuppression materials and methods biopsies were examined. besides light microscopy (lm) and electron microscopy (em), igg, iga, igm, fibrinogen, c and c d were detected by immunofluorescence (if). immunohistochemistry (ihc) was performed using cd , cd ro, cd , and cd antibodies to study the presence of different types of cells in acute rejection. banff's classification was used to evaluate the samples. results graft rejection was found in ( . %) biopsies -hyperacute rejection (har) ( . %), borderline changes (bch) ( . %), acute rejection (ar) ( %), and chronic rejection (chr) ( . %). cya and fk nefrotoxicity (tox) was present in ( . %) biopsies. c d ( biopsies from patients) showed linear positivity of peritubular and glomerular capillaries in patients, with har and ar. these three grafts failed and the same pattern was seen in the excised grafts. in all ar cases, ihc proved the prevalence of cd ro+ cells in rejection infiltrates, including tubulitis. the rejection infiltrate contained from % to % of monocytes-macrophages. em showed disjunction and tubular epithelia regression in tubulitis. ar and tox findings in patients receiving immunosuppression with fk were compared with a group given cya. no significant differences in the rate of toxic manifestation were found between the groups of patients on fk and those on cya. more frequent findings of toxicity were noted in fk patientsb iopsies (p= . ). ar was found more frequently both in the patients (p= . ), and in the biopsies (p= . ) of cya-treated subjects. conclusion fk seems to be more potent suppressant than cya in spite of its persistent toxic effect. the paper is sponsored by ministry of health of the czech republic (international grant agency) no: ne/ - . introduction bk polyomavirus causes a variety of infections in the urinary tract, involving transitional and renal tubular epithelium in immune suppressed patients, including transplant recipients. it can cause irreversible loss of graft function. we report a case of polyomavirus infection in a transplant recipient diagnosed by renal biopsy, urinary cytology and electron microscopy of the urine. patient and results a -year-old female patient underwent renal transplantation, and received immunosuppressive treatment with tacrolimus, mmf and prednisolone. serum creatinine level increased and hydronephrosis was observed in ultrasonography in the rd month. a renal biopsy was taken. no glomerular or vascular changes were observed. some tubular epithelial cells displayed pleomorphism, hyperchromatism with chromatin irregularities and clumping, and intranuclear inclusion bodies. no immunostaining was detected with anti-cmv, hsv and ebv antibodies. cytopathic tubular lesions in biopsy, many decoy cells in urinary cytology and intranuclear viral particles in electron microscopy of the urinary sediment proved the bk-polyomavirus infection. as the high serum creatinine levels persisted, despite alternative immunosupressive therapies, two more renal biopsies were taken, and acute rejection banff grade ib and iia was diagnosed respectively. the patient is still under a novel antiviral treatment as the focal tubular cytopathic changes were present in the latter biopsy. serum creatinine levels were still high, even though urinary cytology is normal. conclusion as we have seen in this case, the differential diagnosis between rejection and infection in renal transplant biopsies is critical and complicated for both clinicians and pathologists, as well as patients. introduction the analyses of serums or tissue specimens from the patients with autoimmune diseases are dispersed between microplate reader elisa techniques and immunofluorescence (if) microscopy techniques. the number and the complexity of if morphological parameters are becoming greater, as well as their association with some of the clinical conditions. the quantity of the morphological signs and the notice capability exceed the possibilities of the experts who are not morphologists. the aim of this paper is to present compilation of if morphological slides and their association with the clinical conditions, indicating the real position of this kind of diagnostics. material and methods more than serums and tissue specimens from different locations were used as study material. the methods used were indirect iif microscopy with commercial and own antigens, microplate reader elisa techniques (mpr-elisa) and direct if microscopy. the study showed that the morphology of antinuclear and anticytoplasmic antibodies, using hep- kits was easily examined, wider, and associations with some clinical conditions could be easily done. the antigens for mpr-elisa techniques derived from recombinated genetic technology, unlike those produced from extraction showed better correlation with indirect if microscopy. the own antigens originating from the domestic animals and human cadavers, used for indirect if microscopy in bullous diseases, had the same value as the commercial ones. conclusion in order to emphasize the importance of this paper, we can conclude that, undeservingly, very few papers from this area are being presented in the pathological congresses and are left to other specialities. the gastrointestinal stromal tumors (gist) are rare tumors. there have been few factors identified that predict survival and biological behavior. this study analizes the experience in two university hospitals. forty nine patients with gist were identified from the tumor registry of both institutions. a retrospective review of patient clinic data, tumor and treatment variable was done. expression of p and cellular proliferation antigens (ki /pcna) was also analyzed with overall survival as the main outcome variable. statistical analysis was performed by log rank test and cox regression model. significance was defined: p< . . as results we found that median age was years (range - years). twentyfive patients were women ( %) and were men ( %). hispanics were predominant race with patients ( %) followed by caucasians with patients ( %) and african american patients ( %). the stomach was the most common site of presentation ( %) followed by small bowel ( %). mean tumor size was cm (range - cm). complete resection was performed in patients ( . %). actuarial -year survival was . % with a median follow-up of months . univariable analysis identified overexpresion of p (p= . ), cellular progression antigens (ki /pcna) (p= . ), high grade (p= . ), tumor size bigger than cm (p= . ), and incomplete resection (p= . ), as significant negative factors, hispanic race (p= . ) and good performance status (p= . ) were significantly associated with prolonged survival. on multivariable analysis, overexpression of p was the only independent negative factor. in conclusion, p is the most significant negative prognostic factor for survival in gist. introduction granulomatous gastritis (gg) is a poorly understood entity, reportedly identified in less than % of all gastric biopsies. a precise definition of granulomas has rarely been given, in particular mg, well-described in intestinal disorders, have not been focused on in gastric lesions. aims: we summarize our experience with gg, including mg, and record prevalence as well as associated background morphology. materials and methods among gastric biopsies from . patients ( . gastroscopies), gg was diagnosed in patients. groups of histiocytes producing conspicuous alteration of gastric architecture were classified as well-defined granuloma (wdg); subtle collections of histiocytes failing to distort surrounding glands were labelled mg. the background morphology and presence of microorganisms were recorded. results the distribution of types of granulomas were as follows: mg only patients, mg and wdg patients, wdg only patients. the background morphology of cases with only mg/wdg with/ without mg included chronic gastritis (n= / ); chronic active gastritis (n= / ); ulcus (n= / ); lymphocytic gastritis (n= / ); carcinoma (n= / ); otherwise normal morphology (n= / ); h. pylori (n= / ). conclusion in concert with previous reports, we identified gg in less than % of all gastric biopsies. since the identification of mg requires assiduous histologic examination, such subtle features may be overlooked; hence our calculated prevalence of gg is a minimun value. the functional relation between mg and wdg is uncertain. however, their coexistence in a proportion of gg could support a precursor function of mg in the development of wdg. additionally, the background pathology of biopsies with mg and wdg were comparable. intravascular signet ring-like cells cd /lysozyme positive in gastric ulcer due to chronic use of nsaid, diagnostic implications in endoscopic biopsy and surgical management of an unusual invasive cancer mimicker j nuncio, j arista-nasr, b martinez pathology department, instituto nacional de ciencias medicas y nutricion salvador zubiran, mexico city, mexico a -year old man treated with naproxen during two years was admitted because of hypovolemia and peritoneal irritation. a panendoscopic study was performed and an ulcer localized at the large curvature of the stomach was disclosed. the microscopic examination of the endoscopic biopsy reveled invasive intravascular and stromal neoplastic signet-ring cells. because of the subsequent poorly differentiated carcinoma diagnosis, a gastrectomy was performed. in the specimen the ulcer showed necrosis, edema, fibrosis, chronic inflammatory infiltrate with lymphocytes and plasma cells. additionally atypical cells with irregular and hyperchromatic nuclei or vacuolated cytoplasm were seen in the lamina propia and infiltrating the muscular layers; isolated signet-ring-like cells were also seen. there was no involvement in regional lymph nodes. histochemical study with periodic acid-schiff, mucicarmin, and colloidal stains revealed mucosubstances in these cells. a poorly differentiated carcinoma was initially diagnosed. however the immunohistochemical studies were positive for histiocytic markers (cd- , s- protein and lysozyme) and negative for epithelial markers (cytokeratin, and ema). the positivity mucus stains in the histiocytes could be explained in this case by phagocytosis of mucous substances released from broken hyperplastic glands in the vicinity of the ulcer. to our knowledge, atypical histiocytic infiltration in gastric ulcers has not been previously described; thus, it should be included in the group of gastric carcinoma mimickers; with this basis, the pathologist must to be careful in the assessment of vascular permeation or stromal infiltration by neoplastic cells overall in endoscopic specimens. ileal mucosa changes after total colectomy in patients with juvenile polyposis and familial adenomatous polyposis a tertytchnyi , a talalaev , d sotnikov , v lukin russian state medical university, moscow, russian federation centre of children's health, russian academy of medical science, moscow, russian federation introduction we studied follow-up ileal mucosa biopsies from patients with juvenile polyposis and patients with familial adenomatous polyposis who had been previously underwent total colectomy with mucosal proctoectomy with the creation of straight ileoanal anastomosis. patients' age ranged from to years (mean . years). the follow-up ranged from months to years (mean . months). aim of this study was to identify the long-term changes in ileal mucosa after total colectomy in patients with polyposis coli syndromes. methods the biopsy specimens have been taken from the posterior wall of terminal ileum. paraffin sections were stained with h&e, van gieson, periodic acid schiff (pas) and gomori's aldehyde fuchsin -alcian-blue (gaf-ab) stains. six biopsies were subjected to electron microscopy analysis. results on histological examination ileal mucosa revealed focal shortening of villi and lengthened crypts. the villi were lined by increased number of goblet cells. mucin histochemical studies demonstrated partial conversion of the epithelium to a colonic sulfomucin mucin profile by positive gaf-ab staining in cytoplasm of goblet cells. only out of patients had increased number of mononuclear cells in lamina propria. the electron microscopy examination revealed shortening and rarification of microvilli of absorptive enterocytes. conclusion follow-up biopsies show incomplete and focal neocolonic transformation of ileal mucosa. nevertheless, in most of the cases ileal mucosa preserved its architectural and histochemical characteristics. we failed to identify evidences of chronic inflammation that made us suggest that chronic terminal ileitis is not a common long-term event in patients with polyposis coli syndromes after total colectomy. eosinophilic gastroenteritis with pleural outflow and ascites. a case report introduction in our region eosinophilic gastroenteritis is a very rare disease. etiology is unknown. case report a -year-old female patient was admitted to the clinic due to slight cough, right side pleural outflow and ascites, without other symptoms. gynaecological ultrasound, ultrasound of the upper abdomen, ct refer to ascites besides normal findings of abdominal and pelvic organs. laboratory examinations were normal except peripheral blood eosinophilia and increased values of ca . cytological findings of pleural outflow and ascites revealed a great number of eosinophilic granulocytes but without malignant cells. patohistological findings during explorative laparotomy resected parts of both ovaries, a lymph node, a hyperplastic part of pylorus, a knot from a small bowel designated as a tumor, and an appendix were taken out for a patohistological examimation. both ovaries reveal normal ovarial parenhima. a regional lymph node showed reactive changes with hyperplastic follicles and prominent germinative centers. the main histological finding reveals pyloric stromal edema and a dense infiltrate of eosinophilic granulocytes throughout the muscle layer as well as eosinophils situated in perivascular spaces which is accompanied with swelling of endothelium of capillary blood vessels. eosinophilic granulocytes were found in the smooth muscle layer of intestine as well as in the subserosal tissue. lumen of the appendix was obliterated and the muscle layer and the subserosal tissue contained eosinophils. conclusion from our review it is visible that eosinophilic gastroenteritis can represent a great diagnostic problem especially when it is accompanied with pleural outflow and ascites and increasing values of tumor markers. that can possibly mislead a diagnostic procedure to the neoplastic rather than the immunological or allergic disease. three years after surgery the patient is well. expression of tnf-alpha and il- in patients with chronic pancreatitis n shirinskaya , v akhmedov emergency hospital, omsk, russian federation omsk medical academy, omsk, russian federation introduction to study the serial levels of tnf-alpha and il- in patients with chronic alcoholic and chronic relapsing pancreatitis. methods consecutive patients with chronic pancreatitis admitted to the emergency hospital ( patients with alcoholic pancreatitis and -with chronic relapsing pancreatitis) were studied. serum levels of tnf-alpha and il- , were determined on and days after admission and in remission by immunoenzyme multiplied. results in group of the patients with chronic alcoholic pancreatitis on day one after admission the high levels of tnf-alpha ( , ± , pg/ml) and il- ( , ± , pg/ml) were marked with the subsequent consecutive decrease of these values for days (tnfalpha- , ± , pg/ml, il- - , ± , pg/ml) and in the period of remission (tnf-alpha- , ± , pg/ml, il- - , ± , pg/ml). in patients with chronic relapsing pancreatitis the low levels of cytokines on days one after admission (tnf-alpha- , ± , pg/ml, il- - , ± , pg/ml) were marked with the deferred peak of values on day (tnf-alpha- , ± , pg/ml, il- - , ± , pg/ml), and decrease in the period of remission ± , pg/ml, pg/ml) . conclusion the deferred peak of cytokines levels (on days) in patients with chronic relapsing pancreatitis can testify indirectly about more expressed cytokines disturbance. the majority of glomus tumors are benign neoplasms that occur in the dermis of extremities. however, rare case have been reported in the visceral locations, most often in the stomach. case report the tumor occurred in a -year-old male who presented ulcer-like pain. ct scan diagnosed a submucosal tumor. there was no endoscopic biopsy specimen. a wedge resection of stomach was performed with a frozen section study that conclude to a carcinoid tumor glomus tumor was confirmed by further study of multiple sections of a surgical specimen and by immunhistochemical study. conclusion correct identification of this essentially benign, very rare gastric tumor can prevent unnecessary radical operative procedures. composite carcinoid-adenocarcinoma of the ileum associated with transitional cell carcinoma of the urinary bladder i venizelos , d sioutopoulou , z tatsiou department of histopathology, athenes, greece it is well known that sometimes carcinoid tumor is associated with other tumors. approximately % of carcinoid tumors in the small intestine are associated with non-carcinoid neoplasms, most frequently adenocarcinomas of the gastrointestinal tract. the concurrent occurrence of carcinoid and other tumors in various organs is extremely rare. here, we present a case of a -year-old male who was admitted to our hospital with a -month history of haematuria. on cystoscopy a large tumor of the urinary bladder has found. a total cystectomy was performed. during the operation, while exploring the abdomen, a tumor with maximum diameter cm was found in the terminal ileum, from which a biopsy was taken. microscopically, the bladder tumor was a papillary transitional cell carcinoma grade ii and the tumor of the ileum fulfilled the criteria for a composite carcinoid adenocarcinoma. the carcinoid component was positive for chromogranin and nse and negative for ema, cea and ca - whereas the adenocarcinoma component was positive for ema, cea and ca - and negative for chromogranine and nse. -months after the operation there is no metastasis radiologically and the patient is in a good condition. due to the small number of tumors reported as composite carcinoid-adenocarcinoma there is need for larger number of similar cases as well as long term follow-up to determine their natural history and prognostic significance. we describe the first reported case of composite carcinoid-adenocarcinoma of the ileum associated with carcinoma of the urinary bladder. mutational analysis and expression of cdx homeobox gene in cases of colorectal carcinomas e pilozzi, m rapazzotti onelli, c fidler, v ziparo, js wainscoat, l ruco istopatologia ospedale sant'andrea facolta, roma, italy introduction cdx is a transcription factor specifically expressed in normal intestinal epithelium. it plays a role in the regulation of cell proliferation and differentiation. experimental evidences suggest that it could have a pro-oncogenic effect since cdx overexpression up-regulates bcl- and reduces p ; it is up-regulated by ras activation and its ectopic expression has been reported in intestinal metaplasia, in carcinomas of the stomach and in barrett's esophagus. despite these observations the role of cdx in colorectal carcinogenesis is not yet fully understood. in fact it has been shown to be down-expressed in colon cancers at mrna and protein level. the aim of the study was to investigate the presence of cdx gene mutations. materials and methods twenty cases of sporadic colon carcinomas were collected. total rna and dna from neoplastic and normal tissues were extracted. cdna was synthesized and used as template in rt-pcr using oligonucleotide for cdx and â-actin. cdx three exons were pcr amplified and directly sequenced. results we found lower expression of cdx in tumour samples in four out of ten cases. sequencing showed a transversion g>c ( / cases) of exon and a t>c transition in exon ( / cases) that, being present in the corresponding normal tissue, were regarded as polymorphisms. no base substitution was observed in exon three. conclusions our results showed that cdx expression is reduced in colon carcinoma. however cdx was not found to be mutated suggesting that alterations, other than base substitution, may affect cdx gene in colon cancer. we are now looking at cpg islands methylation in the same cases since methylation could be a likely explanation of the low expression of cdx . aim this study investigates tgfbeta , tgfbeta and tgfbeta proteins expression in patients with colorectal carcinoma and evaluates their correlation with classic prognostic markers and patients' survival. design the study comprised patients with colorectal carcinoma. according to astler-coller system, tumors were of stage a, -b, -c and -d, whereas tumors were low-grade and high-grade of malignancy. on paraffin sections the streptavidin-biotin technique, using antibodies to tgfbeta , tgfbeta and tgfbeta (santacruz, usa) was employed. staining results followed morphometric analysis and were correlated with clinicopathologic parameters. results tgfbeta protein was expressed in / ( %) carcinomas whereas tgfbeta and tgfbeta proteins were detected in all tumors examined. normal colonic mucosal epithelial cells expressed less tgfbeta (p< . compared to neoplastic cells) and less tgfbeta (p> . compared to neoplastic cells), but not at all tgfbeta . statistical analysis revealed higher expression of tgfbeta in low grade carcinomas (p= . ) and higher tgfbeta presence in advanced stage tumors (p= . ). tgfbeta expression was related with higher disease free survival and higher total survival (p< . respectively). tgfbeta presence was correlated with worse prognosis (p< . ). cox analysis revealed that besides tumor grade and stage, tgfbeta expression constituted independent prognostic factor. conclusions this study shows that in cases of colon adenocarcinoma there is different expression of tgfbeta , tgfbeta and tgfbeta . tgfbeta may be implicated in the pathogenesis of these tumors since it is expressed only within neoplastic and not normal cells. tgfbeta is related with higher disease free survival, higher total survival and constitutes an independent prognostic factor. in the late stages, tgfbeta seems to be involved in tumor progression and it is related with worse prognosis. cdx is useful in distinguishing primary colorectal adenocarcinoma form pulmonary, mammary and pancreatic adenocarcinomas metastatic to the colorectum g groisman, a meir, m amar hillel yaffe medical center, hadera, israel background the large bowel may be the site of metastasis from a number of tumors including pulmonary, mammary and pancreatic adenocarcinomas. the histological distinction of primary colorectal adenocarcinoma from these metastatic lesions may be difficult. the aim of this study was to evaluate the utility of the intestinal marker cdx in distinguishing primary colorectal adenocarcinoma from pulmonary, mammary, and pancreatic adenocarcinomas metastatic to the large bowel. design routinely processed mucosal biopsies of adenocarcinomas involving the colorectum [ primary, metastatic (lung , breast , pancreas )] and control cases of primary lung, breast, and lung adenocarcinomas were immunohistochemically stained for cdx (biogenex, san ramon, ca, usa). the intensity and extent of the staining were recorded semi-quantitatively. results cdx diffussely and strongly stained the normal colorectal epithelium as well as all cases of primary colorectal adenocarcinoma. in contrast, all cases of metastatic lung, breast and pancreas adenocarcinoma as well as all control cases were cdx negative. conclusions cdx immunostaining is useful in discriminating primary colorectal adenocarcinomas from pulmonary, mammary and pancreatic adenocarcinomas metastatic to the large bowel. we recommend its use as a component of any antibody panel put together to discriminate between primary and secondary colorectal tumors. infiltrative and proliferative activity in central versus peripheral parts of colorectal cancer introduction growth of primary and metastatic tumour is accompanied by formation of stroma composed of connective tissue and new blood vessels. colorectal cancer is a malignancy characterised by marked desmoplastic reaction both in primary tumour and in its secondary foci (metastases). presented report was aimed to describe quantitatively microvessel density in primary tumour and lymph node metastases from this neoplasm. materials and methods study material included cases of colorectal cancer with metastases to the regional lymph nodes. investigated group comprised women and men (aged - yrs); tumours were t and four were t according to tnm classification. microvessels were highlighted using monoclonal anti-cd antibodies. vessel counts were performed in five fields of each primary and metastatic focus (x ; . mm ). numbers of blood vessels in colorectal tumour were counted in areas free from prominent necrotic foci and non-specific granulation tissue due to process of reparation. vessel density in nodal metastases was determined in fields occupied by neoplastic cells forming their own connective tissue stroma. results in the primary tumour group mean vessel density was . vessel/mm (median value . ; range - ). in metastatic foci the same parameter was . vessel/mm (median value . ; range - ); no statistically significant difference was detected. only slight correlation was found between the microvessel densities in primary and metastatic tumours (r= . ; p= . ). conclusion presented results seem to prove the similar pattern of neovascularisation in both groups, whereas marked differences in this process can be observed between subsequent analysed cases. early diffuse-type gastric carcinoma, especially signet ring cell carcinoma, often shows the superficially spreading growth pattern, and one of the unsolved questions concerning the histogenesis of this type of carcinoma is whether it is monoclonal or multiclonal in origin. we have performed human androgen receptor gene assay (humara assay) based on the random inactivation phenomenon of x chromosomes in female and reported that most of this type of carcinomas were of monoclonal origin ( / cases). however, this method demonstrated only probability of monoclonality in each lesion. recently, % of diffuse-type gastric carcinomas have revealed mutations of the e-cadherin gene at the early stage of their progression, and in-frame skipping of exon has been seen frequently. thus, we performed an immunohistochemical analysis of early signet ring cell carcinomas using e-cad delta - , which is a specific antibody against a peptide spanning the fusion junction region between exons and of mutant ecadherin. e-cad delta - specifically reacts with tumor cells expressing e-cadherin mrna lacking exon . seven of cases were positive for this antibody. in each positive case, the paraffin blocks of the tissue slices that included the whole tumor were analyzed using e-cad delta - . although some signet ring cells with abundant mucin in the upper and/or lower layers of a layered structure did not react with this antibody, tumor cells were confirmed to express e-cadherin mrna lacking exon- by reverse transcriptase-polymerase chain reaction with paraffin-embedded formalin-fixed tissues. these findings suggest that they are of monoclonal origin ( / cases) and support our previous data using humara assay. e tsikou-papafragou , t papadaki , m tsamouri , e nikolaou , c barbatis korgialenion-benakion hospital, athenes, greece evangelismos hospital, athenes, greece introduction malt lymphoma is the commonest primary gi lymphoid malignancy but for correct diagnosis and classification, immunohistochemical and molecular analysis have been proven necessary. the aim of this study was to demonstrate the value of current methodology for diagnosing gimls by presenting four unusual cases. materials case . a years old male with common variable immunodeficiency. an obstructive ileal tumor cms was resected and multiple biopsies were taken along the gi tract from nodular areas. case . female years old with generalised lymphadenopathy, lung shadows, dyspepsia and duodenal nodularity. case . female age underwent gastrectomy with initial diagnosis of high grade b lymphoma. case . female age , had gastrectomy/ splenectomy for relapsed gastric lymhpoma which had been diagnosed as malt type. methods immunophenotypical analysis for detection of cd , cd a, cd , bcl- pr, bcl- pr, cd , cyclin d , cd , cd , cd ro, cd , ki- and genotypic analysis for the clonal igh, tcva genes (case ) and the igh/bcl- rearrangement (case ). results per case: . atypical burkitt lymphoma with diffuse lymphoid hyperplasia of the gi tract (cd / / a+, bcl- +/ -, ki %). . generalised follicular b lymphoma, grade according to the who classification with involvement of the duodenum. . diffuse large b cell lymphoma with aberrant cd ro expression. rearrangement of the igh genes and not of the tcva gene. . cd -mantle cell lymphoma with anaplastic foci in the spleen. clonal rearrangements of the igh and concomitant igh/bcl- gene rearrangements involving tonsils, stomach, spleen and abdominal lymph nodes. conclusion all cases represent unusual types of gimls easily misclassified or graded without thorough immunophenotypic and molecular analysis. the incidence of gastric cardiac adenocarcinoma has increased in the last decades. gaining insight in the pathogenesis of this lesion is hampered by the limited knowledge of the origin and histology of cardiac mucosa (cm). currently, the location, extent and even the existence of cm are controversial. aims: we studied the development of the gastro-oesophageal junction (goj) in embryos, fetuses and infants to clarify whether cm is a normal structure at birth and where it is located. materials and methods twenty-one autopsy cases were evaluated. age range: weeks gestational age (ga) - months. the distal oesophagus and proximal part of the stomach were embedded entirely. serial sections were stained with haematoxylineosin and alcian blue/periodic acid-schiff. the following parameters were measured: length of abdominal oesophagus; length of columnar-lined oesophagus; length of cm; distance from cm to angle of his. results cm was present in all evaluated sections. its mean length varied throughout gestation. a maximum value was reached at a ga of weeks ( , mm). after term delivery, it was very short ( , - , mm). cm was proximal to, or straddled the angle of his in all cases. during gestation, the mucin staining pattern of the cm was to a high degree similar to that of the developing pyloric mucosa. conclusions cm develops during pregnancy and is present at birth as a normal structure. if the angle of his is taken as a landmark for the goj, cm is located in the distal oesophagus. gastric mucosa-associated lymphoid tissue (malt) and non-malt lymphomas: clinicopathological study of cases s taban, a dema, n tudose university of medicine and pharmacy, timisoara, romania introduction histologic features of low-grade b-cell malt lymphoma of the stomach have been well described in recent years, but its relationship with the more common large b-cell gastric lymphoma has not been clarified. the authors investigate clinicopathological differences among low-grade and high-grade gastric lymphomas. methods clinical and histopathological aspects of consecutive gastric lymphoma cases found in gastrectomy specimens were studied: cases of low-grade malt lymphoma (lg malt lymphoma), cases of high-grade malt lymphoma appearing in low-grade malt lymphoma (hg/lg malt lymphoma), cases of high-grade malt lymphoma (hg malt lymphoma) and cases of diffuse large cell lymphoma (dlcl). results mean age were as follow: lg malt lymphoma- , years; hg/lg malt lymphoma- , years; hg malt lymphoma- , years; dlcl- , years. there was a male predominance of malt lymphoma patients (male to female ratio- / ). macroscopically, the most common pattern of lg malt lymphomas was the superficial spreading type with or without ulceration, whereas hg malt lymphomas and dlcl exhibited a solitary tumor-forming lesion. b-cell immunophenotype was confirmed in cases, only one case of dlcl was a t-cell lymphoma. conclusions in our study, the prevalence of high-grade malt and non-malt lymphomas was , %. our results suggests that some gastric lymphomas with high malignancy can arise through blastic transformation of the lg malt lymphomas, this process(change) seems to take about ten years at least. background the recently described serrated adenocarcinoma accounts at least . % of colorectal malignancy. it develops from serrated polyps. dna microsatellite instability (msi) is more common in serrated adenocarcinomas than in conventional adenocarcinomas, but the frequency of msi-low and msi-high in these tumours is unknown. materials and methods an unselected series of serrated adenocarcinomas were included. msi status was evaluated using panel of five markers d s , d s and d s ) . a tumour was considered to present msi-high if two or more markers showed msi, msi-low with one marker, and mss when none of the markers showed msi. results dna msi was observed in . % ( / ) of serrated adenocarcinomas, . % ( / ) being msi-high and . % ( / ) msi-low. msi status showed no association with gender, age, location, size, dukes stage, grade or mucinocity. msi-positive serrated adenocarcinomas tended to have better prognosis ( -year survival . %) than mss cancers ( -year survival . %), but this was not statistically significant (p= . , log-rank). conclusions serrated adenocarcinomas show prevalence of msilow higher than that reported for colorectal cancers ( to %), supporting idea that serrated carcinomas are formed through a pathway differing from other carcinonomas. absence of any association of msi and clinicopathological features could be due to homogeneity of these neoplasms although a more extensive material is necessary to show more conlusively absence of any association. a trend for better prognosis in msi -positive serrated adenocarcinomas probably reflects the biological behaviour of msi observed earlier in colorectal carcinomas in general. thyroid hormone receptor beta- expression in colorectal cancer is associated with polypoid growth type and k-ras mutations t hörkkö, tj karttunen, p jernvall, mj mäkinen department of pathology, university of oulu, finland background the action of thyroid hormones is mediated via their receptors (tra , tra , trb and trb ). trb expression is abnormal in cancer cells in vitro and in some malignant neoplasms, but the role trb in colorectal carcinogenesis is unknown. materials and methods trb expression pattern was evaluated by immunohistochemistry in operated colorectal cancers and correlated with k-ras mutations and clinicopathological features. results in normal mucosa, cytoplasmic trb immunoreactivity was seen mainly in the superficial parts, while nuclear reactivity was most prominent in the cryptal epithelium. in tumours, nuclear expression was observed in % of cases and cytoplasmic staining in %, both mostly co-existing. cytoplasmic staining for trb was more common in polypoid ( %; / ) than in flat carcinomas ( %; / ; p= . ). k-ras mutations were more common in tumours with cytoplasmic expression of trb ( . %; / ) than in those without ( %; / ). in lymph node metastases, occurrence of nuclear staining of trb was more pronounced than in the primary lesion (p= . , wilcoxon). conclusions expression of trb is present in normal colorectal mucosa and is mostly retained in carcinomas, suggesting that thyroid hormones and their receptors may be involved in the physiological regulation of normal colorectal epithelium and in the pathogenesis of colorectal cancer. association of expression with k-ras mutations indicate occurrence of functional linkage between trb and ras signaling. observations of a high expression in lymph node metastases and in polypoid cancers offer new clues to factors affecting metastatic cascade and growth pattern of colorectal cancer. gastric mucosal calcinosis in chronic renal failure patients materials and methods a review of the records for crf patients who underwent endoscopic biopsy revealed gmc (patient group) and non-gmc (control group) cases. for each subject we recorded the number and sites of biopsies, and reassessed for inflammatory activity, mucosal atrophy, intestinal metaplasia, lymphoid aggregates, helicobacter pylori, amyloid deposition, and gastritis type. age, sex, cause of renal failure, time from renal failure diagnosis to biopsy, type of dialysis, indications for endoscopy, saca use, and serum levels of calcium, phosphorus, calciumxphosphorus product and parathyroid hormone were also recorded. the gmc cases were classified according to pattern of deposition (nodular, granular, mixed) and disease severity (mild, moderate, severe). results the incidence of gmc was . %. in gmc group there was male preponderance ( %). compared to the control group, the gmc group had a significantly higher number of corpus biopsies; a significantly lower number of antral biopsies; a significantly lower frequency of mucosal atrophy; and a significantly lower parathyroid hormone level (p< . for all). none of the gmc patients had used saca. conclusion unlike previous reports, these results show that gmc is not linked with saca use or female gender in crf patients. there was also no correlation with mucosal injury. these findings suggest that gmc is probably a metastatic process, not a dystrophic one. the value of histology in diagnosis of inflammatory bowel disease (ibd) v tzioufa, g karayannopoulou, i venizelos, a asimaki, e vrettou, c kalekou, t kehaya, a kriaka, e nenopoulou, k patsiaoura aristotle university of thessaloniki, greece in patiens with colitis colorectal biopsy is helpful in the differential diagnosis between ibd and other forms of colitis and also between ulcerative colitis (uc) and crohn's disease (cd). however the histological features in biopsy specimens are not always sufficient for a definitive diagnosis and there is need to establish more sensitive and specific criteria for the distinction between uc and cd. a retrospective blind evaluation of biopsy specimens from patients with the endoscopical diagnosis of colitis was undertaken in order to assess the value of histology in the diagnosis of ibd and the degree of inter-observer agreement. twelve experienced pathologists participated in the study. twenty histopathological features according to a modified version of the british society of pathology guidelines for the evaluation of colorectal biopsies in suspected ibd were studied and the results were statistically analysed. the clinical diagnosis after follow up was uc in cases, probably uc in cases, ibd cases, cd cases and nonspecific colitis cases. higher than % agreement (by all or participating pathologists) on the diagnosis of uc based only on histologic parameters was reached in of the cases for which clinical follow up proved the diagnosis of uc. unanimous agreement on the diagnosis of non-specific colitis was reached in of the cases. our results indicate that although detailed assessment of a combination of histopathological features and the experience of histopathologist are important for the diagnosis of ibd, the final diagnosis depends on combination of histopathology with the clinical and endoscopical evaluation. s taban, a dema, n tudose university of medicine and pharmacy, timisoara, romania aim the authors presented two cases of polipoid tumor lesions (gastric and anal) with pseudomalignant changes. multiple sections from the stomach and anal mucosa were analysed by light microscopic and immunohistochemical methods. in the gastric hyperplastic polyp, there was surface ulceration with granulation tissue and acute and chronic inflammation. within the stroma were numerous atypical cells with bizarre cytomorphology and atypical mitoses. the second case was a fibroepithelial polyp of the anus with collagenous stroma covered by squamous epithelium. stromal cells with two or more nuclei were found and showed atypical nuclear features. in both cases, mast cells were frequently observed and sometimes intimately related to the stromal cells. immunohistochemical study revealed that the stromal cells stained positive for vimentine and negative for actine, desmin, cytokeratin, s- protein and leukocyte common antigen, so considered to be fibroblastic cells. clinical follow-up was available. none of the patients followed developed metastases or recurrences. prognostic value of nm and c-erbb- expression in colorectal cancer v delektorskaya, a perevoschikov, ne kushlinsky cancer research centre, moscow, russian federation increased expression of genetic markers is observed in a variety of malignancies, including colorectal cancer (crc). overexpression of these markers in the primary tumor is known to predict outcome after colonic resection. in this study we aimed to evaluate the expression level of the nm and c-erbb- proteins in crc and its correlation to prognosis and liver metastasis. protein expression was examined immunohistochemically in formalin-fixed, paraffinembedded tissue from colorectal patients. crc showed a high level of nm protein expression in % of primary tumors and % of liver metastasis. expression of c-erbb- was observed in % of primary carcinomas and % of distant metastasis. the positive expression of protein markers in primary lesion was significantly higer in the cases with liver metastasis than in those without metastasis. no significant relationship was observed between nm and c-erbb- expression and other clinicopathological parameters. these results suggest that nm and c-erbb- expression play an important role in the progression of crc and could provide additional information for the development of liver secondaries. introduction sporadic gastric carcinomas (gcs) with high level microsatellite instability (msi+) tend to be poorly differentiated adenocarcinomas with abundant lymphocytic infiltration and are associated with a relatively good prognosis. the aim of the study was to define whether the nature and the activation status of lymphocytes infiltrating neoplastic epithelial cells (iels)of msi+ gcs could be involved in an increased apoptosis of tumor cells and could be consistent with a tumor-specific immune response. materials and methods tumor cells apoptosis and immunophenotype of iels were investigated by immunohistochemistry in msi+ gcs. for comparison, a series of msi-gcs with similar histological features was investigated, including cases, which were positive for epstein barr virus infection (ebv+). results msi+ and msi-/ebv+ gcs displayed a significant higher mean number of cytotoxic iels than msi-/ebv-tumors (cd : . and . versus . ; cd : . and . versus . ; tia- : . and . versus . ; p< . ). in addition the activated iels were more frequent in msi+ than in msi-/ebv-gcs (mean granzyme b immunoreactive iels: . versus . ; perforin . versus . ; p< . ). the percentage of apoptotic tumor cells, evaluated with the m cytodeath antibody, was higher in both msi+ and msi-/ebv+ gcs than in msi-/ebv-gcs ( . % and . % versus . %; p< . ). the lymphoid infiltration in msi+ gcs was significantly higher than that of msi-gcs (except for ebv+ gcs) and this might explain the increased apoptotic index and the relatively better prognosis of these tumors. introduction goblet cell carcinoid (gcc), also known as mucinous carcinoid or adenocarcinoid, is a rare tumor of appendix, which exhibits both endocrine and adenocarcinoma differentiation. different mucin phenotypes are associated with neoplastic transformation in epithelial tissue in various organ systems. muc apomucin is a mucin gene product specific to intestinal goblet cells. muc is suggestive to inhibit cell-to cell adhesion. in the present study, we retrospectivelly studied the clinicopathological features and immunohistochemical expression of muc , muc and muc a in gcc. materials and methods three appendiceal gcc were retrieved from our pathology files from to . medical records and histologic slides were reviewed. the expression of muc , muc and muc a was assessed by immunohistochemistry. results all three cases were males and the age ranged from , and yrs. two patients presented with acute appendicitis. the other one had ruptured diverticulitis and renal cell carcinoma. two patients had appendectomy, and one patient had right hemicolectomy and right radical nephrectomy. histologic examination showed infiltrative small nests and glands of tumor cells with goblet cells and mucinous cytoplasm. perineural invasion was noted in one case. the tumors had invaded through the appendiceal wall to subserosa in two cases and to adjacent cecum in one case. the tumor cells are positive staining for synaptophysin. there was strong and diffuse reactivity in the tumor cells with muc , muc and muc a antibodies in all three cases. follow-up ranged from month, months, and months. all were alive and without recurrence. conclusions these observations suggest that there is no differential expression of muc , muc and muc a in gcc and both muc and muc may play a role in cancer cell growth and invasion. gastric parietal cell carcinoma: report of three cases ml gomez-dorronsoro, r beloqui, i amat, p de llano, a cordoba, b larrinaga, e borobio hospital de navarra, pamplona, spain gastric parietal cell carcinoma is an infrequent newly recognised variant of gastric carcinoma with only cases published in the literature. the present study describes the findings in three cases of gastric parietal cell carcinoma consisting of parietal cells. we have used light and electron microscopy, and histochemical stains. we have studied three cases, two men and one woman, aged between and years. the tumour size ranged from , to , cm. the surgical specimens were stained with hematoxylin-eosin, alcian blue and ptah. small pieces of formalin fixed tumor tissue were collected in one case, and fresh tissue fixed in glutaraldehyde in another both for electron microscopy. involvement of the gastric wall was observed in all tumours. the tumour pattern was characterised by an arrangement of tumour cells in sheets, elongated cords or in diffuse poorly cohesive sheets interspersed with a lymphocytic infiltrate. tubular differentiation was only focally present in one of the cases. all the cases were composed by a large number of parietal-like cells with abundant eosinophilic cytoplasm, with a mixture of signet ring cells population in one of the cases. alcian blue stain was negative throughout the tumour, whilst ptah stained many tumour cells. ultrastructurally the tumour cells were characterised by abundant mitochondria, tubulovesicles, intracellular canaliculi and intercellular lumina filled with microvilli. the three tumours presented are primary gastric carcinoma with cytological and ultraestructural patterns of parietal cell differentiation. they represent a morphologically distinct type of carcinoma, which has been said to have a favourable prognosis. aim the aim of study is to investigate do really cd + cells have cytotoxic or suppressor influence upon gastric epithelium. material and methods we have examined biopsies from patients with peptic ulcer and -with gastritis only. samples were formalin fixed, paraffin-embedded, stained with h/e, toluidineblue, gimsa stain, pas reaction. comparative immunohistochemical analysis of cd + lymphocytes, proliferative markers: mib- and pcna were performed. we evaluated the amount of cd + cells in mm and the proliferative activity of epithelium per nuclei. biopsies were examined electronmicroscopically for the detection of cell-to-cell interactions and subcellular alterations of gastric epithelium. results distribution, amount and characteristics of cd + cells are different in mucosa around ulcer and in gastritis (g.) cases without ulcer. in active g. around ulcer cd + cell are , ; without ulcer - , , but in inactive g.- , . in helicobacter pylori-infected subjects cd + are located in addition at the lymphatic follicles. we have not proved any rise of apoptosis, exfoliation or ultrastructural injuries of epithelial cells in the areas of collections of tlymphocytes of this subtype. in sites of increased amount of cd + cells there is high proliferation activity of epithelium ( , - , %) due to the broadening of labeled nuclei zone with mib- antibody. conclusions the amount of cd +cells correlates with activity of gastritis as this kd glycoprotein realize molecular reactions between mediators or against h.pylori, food and other antigens in lamina propria, but it has no external direct domain upon gastric epithelium, capillaries or glandulocytes. the incidence of gastric cancer decreases worldwide. despite declining (due to intestinal type), incidence still remains the second cause of death amongst all malignancies worldwide. diffuse type of gastric cancer shows constant incidence. therapy and prognosis directly correlate with extent of the disease and reliable prognostic factor is still lacking. the aim of the study is to establish a value of two morphometric methods -nuclear area and microvessel density and their influence to the survival of the patients with advanced gastric cancer. patients who had undergone gastric resections for gastric cancer were analysed in this study. we estimate size of the tumor, histologic type (according to lauren's classification), stage, nuclear area (median value of nuclear areas in cancer cells measured with computerised nuclear morphometry) and microvessel density (median value of f viii-positive microvessels in five x power field) for each case. statistical analysis was performed and p-values lower than , were considered significant. nuclear area shows statistical significant correlation with size of the tumor, tumor stage, tnm classification and metastases in regional lymph nodes, microvessel density does not. in univariate analysis to survival, size of the tumor, tumor stage and tnm classification have p< , . nuclear area only in intestinal type of gastric cancer correlates with survival (p< , ). in multivariate analysis, size of the tumor, tumor stage and tnm classification have an influence on survival. reliable prognostic factors in patients with gastric cancer are still stage and size of the tumor. diffuse type of gastric cancer is still an enygma. spreading and metastazing of intestinal type of gastric cancer could be predicted using a nuclear-area method as a support in therapy planning and prognosis. the role of microvessel density in prognosis is unreliable. f iordanidis, e vrettou, e nenopoulou, p hytiroglou, c papadimitriou pathology dept. medical school. aristotle univ., thessaloniki, greece introduction the kit receptor normally becomes dimerized and its tyrosine kinase phosphorylated (activated) upon the ligand binding, then enabling it to phosphorylate other proteins in the signal transduction pathway that ultimately carry the proliferation signal into the nucleus. the aim of this study was the evaluation of ki- (mib- ), proliferative cell index, in correlation to c-kit stain intensity, histological grade and immunophenotype in gists. we studied a series of gists (c-kit positive) in correlation to age, anatomic location, size, morphological features, mitosis and necrosis. also we stained with vimentin, cd- , smooth muscle actin (sma), s- protein and ki- with abc method on paraffin sections. results the average age of the patients was years (ranging from to ), included male and female patients. were located in the stomach, in the small intestine, in the colon and in the esophagus. microscopically the tumors showed a fusiform cytology and of them showed a mixed fusiform-epitheloid cytology. the average tumor size was , ± , cm (range , - cm). we found necrosis in tumor with size> cm. the tumor cells expressed vim %, cd %, sma %, s- % and ki- ranging from - %. the most gists showed c-kit positivity in % of tumor cells with a small minority showed more focal staining in as few as % to % of tumor cells. the stain intenisty was mild to moderate. conclusion in our study, the ki- expression was associated with tumor size and not with c-kit staining profile, other immunophenotypic features or histological parameters. expression of pcna and ki- protein in colorectal carcinomas. correlation with tumor grade v zivkovic, a nagorni, v katic, j gligorijevic, b djordjevic, m milentijevic, z mijovic institute of pathology, medical school, nis, serbia and montenegro introduction the prognostic significance of pcna and ki- proliferation markers in colorectal carcinomas is debatable. therefore, we evaluated immunoreactivity of the tumor for pcna and ki- antigen in colorectal carcinomas with different histological grades. methods the paraffin embedded tissues were immunostained for pcna and ki- antigen by employing streptavidin-biotin(lsab) method. results pcna was positive in % of carcinomas and localized in the nuclei of malignant cells.immnunoreaction for ki- was demonstrated in all carcinomas and mainly and strongly expressed in the nucleoli.positive staining for pcna and ki- correlated positively with increasing grade. the labelling index was significantly higher in the poorly differentiated carcinomas than in the well differentiated tumors. conclusion the expression of pcna and ki- proliferation markers clearly shows differences between colorectal carcinomas of varying differentiation grades and it is useful as a diagnostic and prognostic parametar. introduction collision tumours have been thought to arise from the accidental meeting and interpenetration of two independent tumours. here we present three cases of gastroesophageal junction tumours that could fill these criteria. we employ molecular and genetic techniques to ascertain the clonality of these tumours in order to determine whether they represent true collision tumours. methods p immunohistochemistry was performed, and p sequence analysis was carried out on rt-pcr material derived from fresh frozen tissue. mutations were subsequently confirmed in paraffin embedded material. standard and laser microdissection techniques provided tumour-enriched samples from which dna was isolated. analysis of loss of heterozygosity was performed using the microsatellite markers d s , d s , d s , d s , d s , d d , d s , tp , p alu and bat . results p immunohistochemistry was positive for both tumours in each case. all three tumours possessed a p point mutation that was identical in both components. polymorphic microsatellite analysis confirmed shared losses of heterozygosity with an identical pattern of retention and loss at six loci in each component. genetic differences were also observed between tumour components. conclusion a comparison of immunohistochemistry, p sequence and the pattern of loh for a spectrum of polymorphic microsatellite markers for three possible collision tumours suggests that both components are derived from a single precursor cell that undergoes divergent differentiation in the evolution of the tumour. the evidence presented here refutes the "collision hypothesis" and introduces a new histopathological entity of a cardia tumour with distinct diffuse signet ring cell and squamous components. a nasierowska-guttmejer, w michej department of pathology, cancer centre, warsaw, poland introduction gists are the most common mesenchymal tumors of the gastrointestinal tract. they are histologically heterogenous group and a practical diagnostic criteria for gists is c-kit (cd ) expression by immunohistochemistry. the aim of this study was to present morphological pattern of radically operated and disseminated gists. material and methods material included gists treated between and in cancer center, warsaw. thirty pts. had been radically operated of gastric gists and pts. -intestinal gists. fifty three pts. had peritoneal and liver dissemination. the histopathological diagnosis based on the type of cells, mitotic activity and tumor size. the immunohistochemical expression of cd (dako), cd (dako), sma (dako) and s p (dako) were performed in each of the case. results . microscopic spectrum. majority ( %) of cases had heterogenous pattern-spindle and epithelioid cells with high mitotic activity-more than mitoses per hpf. the intestinal and disseminated tumors had generally spindle cell histology with variable myxoid matrix or extracellular collagen globules reffered to as skeinoid fibers. the majority of gastric gists were composed of epithelioid, polygonal cells with perinuclear vacuolization. .the immunohistochemical analysis revealed strong and diffuse cd expression in of cases, whereas cd was expressed in cases. four cases were negative for cd and positive for cd . a minority of the tumors expressed smooth muscle actin which was focal. conclusions the heterogenous histologic pattern of gists cd (+) correlated with dissemination, diameter over cm and more than mitoses per hpf. carcinosarcoma of the esophagus: two cases of the unusual neoplasm m malinowska, a nasierowska-guttmejer department of pathology, institute of oncology, warsaw, poland introduction esophageal carcinosarcoma comprise approximately - % of all esophageal tumors. this is the first published report in poland of the unusual malignant neoplasm consisted of both carcinomatous and sarcomatous components. materials and methods two patients with esophageal tumors were presented. they were analyzed using endoscopic, histological and immunohistochemical procedures. an endoscopic examination showed polypoid tumors in the lower esophagus. esophagogatrectomy with lymph node dissection was performed. the resected specimens were fixed in % formalin and paraffin embedded tissue sections were stained with routine hematoxylin and eosin. immunohistochemical analysis for vimentin, cytokeratin, p and mib were performed. results histologically, the neoplasms consisted of two components, squamous cell carcinoma and sarcomatous components. in case , the sarcomatous component was with no specific differentiation and in case resembled malignant fibrous histiocytoma. a gradual transition between two components was found. in case regional introduction the cyclin-dependent kinase inhibitor p is a critical downstream effector in the p pathway and this protein acts as tumor suppressor in a negative cell cycle regulation. aims: we investigated the prognostic influence of p waf /cip reactivity in gastric carcinomas in relation to the p expression. materials and methods the expression of p waf /cip and p protein was revealed immunohistochemically in gastric carcinomas. staining patterns were assessed semiquantitatively and correlated with clinicopathologic variables and survival times. median follow-up time was months (range - months). chisquare and kaplan-meier survival analysis were performed as statistical analysis. results the p waf /cip protein was detected in ( %) and p protein in ( %) of cases. p waf /cip expression was significantly higher in stages i and ii carcinomas than in stages iii and iv carcinomas (p= . ). in tumors without lymph node metastasis, p waf /cip expression was significantly more frequent in tumors with lymph node metastasis (p= . ). low p waf /cip expression significantly associated with p protein expression (p= . ). the survival rate of patients with p waf /cip -positive tumors was significantly better than those without p waf /cip expression (p= . ). in patients with p positive tumors showed significantly poorer prognosis than in patients with p negative tumors. conclusion the status of p waf /cip expression may have prognostic value in gastric carcinomas and evaluation of the expression of both p and p waf /cip might provide more prognostic information. introduction the normal motility of the gastrointestinal tract depends on the enteric nervous system, the smooth muscle layers, and the interstitial cells of cajal (iccs). the aim of this study was to investigate both the distribution of interstitial cells of cajal and the pathohistology of the enteric nervous system in colonic inertia. materials and methods large bowels were obtained from patients with colonic inertia. the patients have been treated surgically with total abdominal colectomy and ileorectal anastomosis. control large bowel specimens were collected from patients with non-obstructing neoplasia. colonic specimens were investigated with monoclonal anti-neurofilament antibody and cd antibody and compared with those of control patients. the analysis of iccs included registration of the number and process length of the myenteric and muscular iccs. in the patients with colonic inertia the myenteric plexus showed moderate or severe hypoganglionosis compared with the control group. the apparently normal axon bundles in the myenteric plexus stained markedly less than normal ones with monoclonal antibody. myenteric iccs were evident as single cells or cell clusters closely related to the small myentric ganglia. the iccs did not form the typical networks seen in the normal bowel. muscular iccs were markedly reduced in number compared to the number seen in normal bowels and were mainly expressed at the innermost layer of the circular muscle layer. conclusion the enteric nervous system and iccs are altered in colonic inertia and may play a crucial role in the pathophysiology of colorectal motility disorders. immunohistochemical detection of the hmlh and hmsh proteins in hereditary and sporadic colon cancer tissues germline mutations of hmlh and hmsh mismatch repair genes (mmr) are associated with hereditary nonpolyposis colon cancer (hnpcc). somatic impairment of these genes can be detected in sporadic colon cancers (cc). all these tumours are characterised by microsatellite instability (msi). the aim of this study was to correlate the results of immunohistochemical detection of hmlh and hmsh protein with results of mutational and msi analyses. patients and methods there were included: cc samples from hmlh /hmsh germline mutation carriers, cc samples from non-carriers, and cc samples from patients not subjected to mutational analysis. immunohistochemistry was performed using mouse monoclonal antibodies (anti-hmlh , clone g - ; anti-hmsh , clone g - , pharmingen, usa). msi was analyzed at abi prism genetic analyzer, mutational analysis by dna sequencing. results loss of nuclear protein expression was detected in all tumours from mutation carriers, all of these exhibiting a high grade of msi (msi-h), and in of msi-h tumours from the other patients. none of the tumours with stable microsatellites or a low level of msi exhibited a nuclear protein loss. immunohistochemical analysis was able to detect msi-h tumours with a % specificity and a % sensitivity. conclusion immunohistochemical detection of hmlh and hmsh proteins is a specific method of detection of tumours with mmr gene inactivation and might be used as a screening method of hnpcc. the msi-h cases with retained nuclear protein expression might express non-functional protein or carry another mmr gene defect. suppported by nc - . study of mucin changes in colorectal adenomacarcinoma sequence m danciu , ms mihailovici , m stan , a trifan , c sfarti , s khder , am gruia university of medicine and pharmacy, iasi, romania institute of gastroenterology and hepathology, iasi, romania introduction mucins are known to have a protective role for the colorectal epithelium against extrinsic noxious factors (including carcinogens), changes of its characteristics being an early process in the adenoma-carcinoma sequence. aims: quantitative and qualitative evaluation of mucin changes in hyperplastic polyps (hp), adenomatous polyps (ap) with different degrees of dysplasia and adenocarcinomas (ac) of colon. material and methods we selected colorectal endobiopsies ( hp, ap and ac). thin serial sections were stained with hematoxylin-eozin, van gieson and alcian blue (ab) ph (for strongly sulphured mucins) and ph . (for acid mucins: weakly sulphured mucins, hyaluronic acid and sialomucins). observations were made only on intracellular mucins. intensity of the stain was analyzed and compared with non-tumoral (normal) adjacent mucosa. results and discussions normal mucosa and hp showed a preponderance of sulphomucins (ab ph ) comparing with sialic acid (ab ph . ). in ap (especially in those harboring high-grade dysplasia) we detected sialomucins in . % cases and the depletion of sulphomucins ( . %). in ac sialomucins were identified in . % cases, while sulphomucins only in . % cases. significant depletion of sulphomucins occurs in ap with moderate and severe dysplasia and in ac, but it was also observed in ap with mild dysplasia. this suggests the possibility that without a proper mucin secretion consisting mainly in strongly sulphured mucins, colorectal epithelium is more vulnerable to environmental carcinogens. histological this study is a continuum of the analysis until . we analyzed biopsy and operative materials from the stomach and small bowel. we used the following histochemical stainings: he, van gieson, reticulin, as well as immunohistochemical stainings with the following antibodies: ckws, lca, s , actin, desmin, nse, chromogranin, synaptophysin, vimentin. results and conclusion the analyzed group of cases consists of patients of age between - years with male predominance ( : ). four of the analyzed ten cases were located in the stomach and in the small bowel. cases were benign, histologically composed of bundles of spindle cells with slightly pleomorphic nuclei and eosinophilic granular cytoplasm, with hyalinised collagen between the cells. the mitoses were either rare or absent. immunohistochemical analyses were most frequently positive for s , actin, desmin and synaptophysin. three of the cases were ranked as malignant, using the following diagnostic criteria: size of the neoplasm, existence of metastases and histological appearance with anaplastic spindle cells arranged in bundles and whorls with high mitotic index ( mitoses per hpf) and marked positivity for actin, desmin and vimentin. what is the relation of age, gender and histological types to the subsite location of colon carcinoma? introduction characteristics that determinate the anatomic site within the colon where the carcinoma is most likely to occur would be very useful in choosing optimal factories for screening of colon cancer /cc/. aims: to study any association of gender, age or histological types with cc in the left or in the right colon. materials and methods retrospectivitaly are studied cases of cc in nmtb between and .the age and the gender of patients were determined. localization, histological type and tnm staging of the cancer are identified on the histological slides. results cases of cc at the age between - years old are diagnosed. gender, age and histological type were independently significantly associated with the frequency of the development of right and left colon carcinoma. the gender distribution of cc is in benefit of male independently of sub site location as they are suffered of cc in the younger age in comparison with the female. independently age, gender and localization prevails the histological type adenocarcinoma with different morphological and functional differentiation. one cc in the left colon is developed on the background of fap. solitary are the cases with congenital fap and lynch ii. in the most cases cc was diagnosed in advanced stage. solitary is the cases with tis and t . conclusion we consider: /cc is more frequently in the male colon; /in cc predominate histological type of adenocarcinoma; /cc is developed in the middle and in the age advanced in years; / cases with multicentrical cc with metachronic carcinoma are solitary. introduction cathepsin b (cb) and l (cl) are cysteine peptidases with capacity of degrading extracellular matrix. cb expression in colorectal cancer (crc) has been related with tumor progression. there are no data about the cl immunohistochemical expression. the aim of this study was to analyse the expression of both cb/l in crc and to relate these findings with clinicopathological data. material and methods epithelial and stromal immunohistochemical expression of cb and cl was studied in and samples respectively. cb was analysed in adenomas. global and local infiltrating edge data were recorded. clinico-pathological parameters were correlated with the immunohistochemistry. results cb/l expression in normal epithelium was apical, with scarce macrophages in the lamina propria. an increase of both cb/l was observed in tumoral stroma, with higher cb at the infiltrating edge. the both global and deep stromal cb increased according to the t (p< , ). neoplastic epithelial cb revealed a basal stain whereas cl was occasional. epithelial cb tended to show high expression in t (like high grade dysplasia) and low expression in t that increased progressively in t - (p= , ) . no differences were evidenced either as a function of n state, gender, site, differentiation or histological type. conclusions cb/l immunoexpression in colon have a distinctive pattern which is altered in carcinoma. whereas increased cb stromal expression ise related with local progression, epithelial positivity seems to play an important role in the first steps of carcinoma. high stromal cl isassociated with carcinoma but cl epithelial expression seems not to be contributory. introduction new approach on estrogen and progesteron receptors of the gastric signet ring cell carcinoma explains its histogenesis and poor prognosis. the pointed out facts are the reason for this study. material and methods the material was obtained from surgically resected stomach, together with ovaries. after two months from gastrectomy right mastectomy has been done. the specimens were fixed in % formaldehyde and embedded in paraffin. the sections were stained with he, pas, hid-ab ph= , methods and abc using estrogen and progesteron antibodies (dako). results histologically, mucin-producing signet ring carcinoma that diffusely infiltrates the antral wall and the ovaries known as krukenberg tumor, has been found. histochemically, type b signet ring cell carcinoma was discovered: cells contained blue positive (sialo) mucin. ductal type of breast carcinoma with lymph node metastases also was found. immunocytochemically, estrogen and progesteron positive receptors were discovered in cancer cells of the stomach, breast and ovaries. introduction primary gastric low-grade marginal zone b cell lymphoma of malt type is a distinct disease entity with a characteristic histological presentation and clinical behavior. the development of gastric malt lymphoma is dependent of h.pylori infection. many reports have stated that the cure of h.pylori infection can induce a complete remission of low-grade gastric malt lymphoma. we performed a prospective study to evaluate the outcome of patients with low-grade gastric malt lymphoma and the histological characteristics before and after therapy. we prospectively enrolled patients with h. pylori positive low-grade gastric malt lymphoma (stage i e except one patient in stage ii e). paraffin sections were stained with: he, giemsa, pas and immunohistochemical lab methods. the patients were examined by endoscopic ultrasound before and after eradication therapy. results endoscopically, enlarged gastric folds, erosions or ulcerations, hyperaemia or intragastric nodularity, with preferential localization in the antrum, were described. histologically, the neoplastic cells infiltrated between pre-existing lymphoid follicles, then colonized the lymphoid follicles. the resemblance of these cells to the centrocyte has led to the term "centrocyte-like (ccl)" cell-neoplastic component of malt lymphoma. in some cases the cells had monocytoid appearance, but plasma cell differentiation also was prominent. typical lesion for malt lymphoma -"lymphoepithelial lesion", was frequent, representing infiltration of the glandular epithelium by clusters of neoplastic lymphoid cells with associated destruction of gland architecture and morphological changes within the epithelial cells, including increased eosinophilia. after the eradication of h.pylori, the patients were regularly followed-up with endoscopic and histological assessment. patients achieved complete remission within weeks, one had partial gastrectomy induced by ulcer with severe dysplasia, but with regression of malt lymphoma (within weeks), but the remission of the patient with stage ii e was achieved within months. lamina propria appeared "empty" with gland loss. scattered lymphocytes and plasma cells were seen within the lamina propria and there were nodular collections of small lymphocytes. lymphoepithelial lesions were scanty or absent. conclusions the time taken for achieve remission of gastric malt lymphoma in these patients varied from to months. the only significant prognostic variables were stage and tumorgrade. comparative evaluation of the prognostic value of muc and muc antigens in colorectal adenocarcinoma introduction during the last decade, information accumulated regarding the experssion of human mucins. muc belongs to the group of membrane bound mucins. it is assumed to play various roles in tumor immunology. muc is marker of goblet cell or mucinous differentation of adenocarcinoma. the significance of muc and muc as prognostic markers in colorectal adenocarcinoma with mucinous differentation and mucinous adenocarcinomas was studied because previous investigations revealed inconsistent results. materials and methods tissues from operated patients with mucinous or mucinous differentiation of colorectal adenocarcinomas were investigated. all specimens were classified according to who and tnm classification, including lymph node status and distant metastases at the time of the surgical intervention. formalin-fixed paraffin-embedded colorectal cancer tissues were cut ( µm) and deparaffinized acording to standard histological techniques. classical he, histochemical pas and hid-ab ph= , and immunohistochemical bsp techniques were applied. the obtained results were evaluated by the x test. results the muc immunoreactivity showed a strong correlation with tumours progression (as suggested by advancing tnm stages) and also de-differentiation (as reflected by tumorous grading). localization and histological grading were also statistically signifficant. on the other hand, muc did not show any association with the data concerning prognosis. conclusions according to our data, only muc presents as an independent prognostic factor of colorectal adenocarcinoma. muc should therefore be considered as a potential target of immunotherapeutic strategies. sporadic form of colon cancer of a -year-old boy i stojanovic, v katic, j gligorijevic, k katic, m andjelkovic-matic institute of pathology, medical faculty of nis university, nis, serbia and montenegro introduction sporadic form of colon cancer is one of the most frequent cancers in adults, but very rare in children; thus we report this case. case report we describe the case of a -year-old boy who had the agressive sporadic coecal cancer, surrounded by nodular lymphoid hyperplasia.the boy underwent to partial colectomy cum omentectomy during the night with clinical diagnosis "acute abdomen". the surgical-pathological stage was: stage iii (t nlmo). the patient received postoperative therapy, but he died with liver metastases eight months from surgery. materials and methods formalin-fixed paraffin-embedded coecal cancer and surrounding nodular tissue were cut and deparaffinized according to standard histological techniques. he, pas, hid-ab ph , and immunohistochemical lab techniques were applied. results fungous advanced carcinoma, mm in diameter, induced stenosis and clinical symptoms of occlusion. histologically, it was poorly differentiated adenocarcinoma with high mitotic index(high activity of ki- ) and neuroendocrine differentiation; cancer cells were diffusely positive for p protein. immunological reaction to muc , muc ab and muc , as well as to b lymphocytes, was negative. microscopic examination of surrounding malt showed large, confluent lymphoid follicles without germinal centres within lamina propria, causing nodular appearance of the overlying mucosa. b lymphocytes were markedly depressed in this nonhereditary form of colon cancer. conclusions the authors have suggested that antibody deficiency, environmental, endocrine and genetic factors cooperated in the development of this nonhereditary coecal carcinoma. the histological findings in the gastroesophageal junction of fetuses introduction the histopathological concept of the gastroesophageal junction has been changed by american pathologists providing the evidence that the gastric cardia is not a physiological structure. instead, it develops through glandular metaplasia of the squamous epithelium of the distal esophagus due to gastroesophageal reflux. methods, results and conclusion the development of the esophageal and gastric mucosa in the gastroesophageal junction was studied in fetuses of - weeks of the gestational age. during the th - th week, the esophageal multilayered epithelium was covered by a continuous layer of columnar mucous ciliated cells which were present only focally till the th week and disappeared later. before the th week, the gastric mucosa was formed by pits only. the glands started as proliferating tubules in the basal parts of the pits in the th week. further, they differentiated into oxyntic glands. the mucosa of the corpus was fully developed in the th week. the cardiac mucosa was absent in all the fetuses examined between the th and st week of gestation. this supports the view that the gastric cardiac mucosa is not a physiological structure but that it results from glandular metaplasia of the distal esophageal mucosa due to gastroesophageal reflux. mr jalali nadoushan , a dievan beigi , n fallah , b mofid shahed university, tehran, islamic republic of iran shahid beheshti university of medical sciences, tehran, islamic republic of iran introduction there is not enough studies about relationship between colon cancer and abo and rh blood groups in compare to gastric cancer. therefore in this study we will examine abo and rh blood groups distribution and correlation of them with pathologic stage of colon adenocarcinoma. methods data on age, sex, abo and rh blood types and pathologic stage (wall thickness involvement and involvement of lymph nodes) of the patients with colon adenocarcinoma; were under surgical operation, were collected from three tehran hospitals. and also data on abo and rh blood groups from healthy persons from above hospitals collected by randomized selecting as control group. results the distribution of abo blood groups in patients was different from control group; however, blood group a in patients was . % higher than the corresponding control. there was no significant association between abo blood groups and pathologic stage of colon adenocarcinoma. the distribution of rh in patients was likely equal with control group; although, there was a significant relationship between rh (+) and wall thickness involvement (p< . ) and involvement of lymph nodes (p< . ). conclusion -abo blood group a is more common in patients with colon adenocarcinoma. -rh(+) has correlation with poor prognosis in colon adenocarcinoma. introduction vascular endothelial growth factor (vegf) is a cytokine involved in tumor angiogenesis. compelling data implicate that deregulation of p protein function may be associated with increased neovascularization and aggressive tumor growth. the aim was to investigate the prognostic significance of vegf overexpression and microvessel count (mvc) at the deepest invasive site in colorectal carcinoma tissue as well as their relationship with p protein expression in tumor tissue. material and methods surgical specimens of operated colorectal carcinomas were studied by immunohistochemical methods for detection of vegf and p protein in tumor tissue and cd positive microvessels. median folow up was . month (range, - months). results vegf expression and nuclear p protein overexpression were found in , % and % of colorectal carcinoma. vegf and p protein positive status was identical in . % of tumors. the dukes stage c and high mvc were significantly more frequent in vegf positive tumors ( . % and . % respectively) comparing with vegf negative tumors ( . % for both dukes stage c and high mvc). hypervascularity significantly correlated with advanced dukes stages, but not with the p protein expression. survival analysis showed that vegf and p protein expression as well as hypervascularity of colorectal carcinomas correlated with poor survival (p= . ). conclusion vegf is an important angiogenic factor in colorectal carcinoma, and its expression being dependent on p protein expression. our data suggest that combined analysis of vegf, p protein and mvc may be useful for predicting the more aggressive biological behaviour of colorectal carcinoma. occult involvement of lymph nodes in the splenic hilus and along the splenic artery in patients with proximal gastric carcinoma m bjelovic introduction the aim of this study was to find out the incidence of occult metastatic involvement in lymph nodes in the splenic hilus (lymph node group according to jrsgc) and along the splenic artery (lymph node group according to jrsgc) as well as to identify pathological features that might be prognostic factors associated with involvement of these lymph nodes in the patients with proximal gastric carcinoma. material and methods total of patients with advanced proximal gastric carcinoma and up to positive perigastric lymph nodes (t /t n category) have been studied in the period between and . histopathological features (tumor size, macroscopic and histologic type, grade of differentiation, depth of penetration, invasion of intramural lymphatics and veins) as well as lymph node involvement have been routinely examined according to tnm and jrsgc systems. detection of microcarcinosis (occult lymph node involvement) in lymph nodes groups and has been performed by multiple serial sectioning and using immunohistochemistry (monoclonal antibodies against cytokeratins and , lsab+ technique) and classified according to modified kikuchi criteria. results occult lymph node involvement in lymph node group has been observed in patients ( , %). three of them ( , %) had only single cell micrometastases (g type of microcarcinosis) and patients ( , %) had micrometastatic deposits greater than microns (g type of microcarcinosis). occult lymph node involvement in lymph node group has been observed in patients ( , %) expressing single cell micrometastases in one case and micrometastatic cell clustering (up to microns in diameter) in another case. conclusion occult involvement of lymph node groups and could be expected in lymph node positive patients with more than three positive perigastric lymph nodes. in the patients with advanced proximal gastric carcinoma examined pathological tumor characteristics could not be associated with the involvement of lymph node groups and . lymph node micrometastases in patients with gastric carcinoma: immunohistochemical evaluation introduction for patients who underwent curative resection for gastric carcinoma, lymph node status seems to be a major prognostic factor of disease recurrence. contrary to histologically overt lymph node metastasis, the biological significance and prognostic relevance of micrometastatic lymph node involvement are still controversial. we examined frequency and distribution of micrometastases in lymph nodes obtained from patients with advanced gastric carcinoma ( , lymph nodes per patient) without primarily histologically overt lymph node metastases. identification of lymph node micrometases was performed using multiple serial sectioning and immunohistochemical detection of cytokeratin and positive cells. the results were correlated to tumoral histological type, growth pattern, depth of penetration and vascular invasion. results the overall detection rate of micrometastases showed their occurence in more than half of examined patients ( / or , %) and in , % of examined lymph nodes ( / ). most of them were so-called single cell micrometastases ( , %), followed by cluster-type of micrometastases up to microns ( , %) and few greater than microns ( , %). the incidence of micrometastases was , % ( / ) in lauren`s diffuse type of carcinoma contrary to % ( / ) in lauren`s intestinal type of carcinoma. in addition, there was no significant difference in occurence of micrometastases in cases with and without penetration of muscular layer, contrary to cases with serosal tumoral involvement and obvious vascular invasion. conclusion detection of micrometastases might influence tumour stage migration to tnm stage iii in further % of examined patients with primarily stage ii determined diseases. the frequency of micrometastases seems to be closely correlated to poorly differentiated carcinomas, especially lauren`s diffuse type of carcinoma and higher pt stages, including serosal involvement. however, possible independent prognostic significance is still to be elucidated after survival analysis of larger series of these patientss ubgroups. introduction gastrointestinal stromal tumors (gists) are the most common mesenchymal tumors of the gastrointestinal tract with the stomach as the most common site. the term 'gist' is preferentially used for the tumors that express cd and cd /c-kit and the latter has been suggested as a specific marker for gists. as gists constitute a group of phenotypically heterogenous mesenchymal neoplasms with uncertain biological behaviour, we reevaluated their histogenetic differentiation and proliferative status. material and methods we reexamined clinically, histologically and immunohistochemically cases of paraffin-embedded gists noted in the tumor registry of clinical centre of serbia between and , and especially in regard to their proliferation, measured by mitotic index and immunohistochemically by ki- and pcna expression scores. in addition, evaluation of malignancy of gists was based on mitotic count greater than / hpf and/or tumor size greater than cm and extra-gastrointestinal spread. results most of gastric gists were composed of spindle cells ( %), epithelioid cells ( %) or both cell types ( %), and nearly % showed obvious clinical malignant behavior. immunohistochemical examination showed strong cd expression in %, cd in , % (two of cases exlusively cd positive) in nonmyogenic and non-neurogenic tumors. in addition, in gists with cd expression, focal expression of s- was found in , %, focal sma in % and desmin in , % of cases. ki- and pcna indices were correlated to mitotic index and other criteria of malignancy. conclusion phenotypic differentiation of gists toward smooth muscle were found to be correlated with proliferative indicators of poor prognosis more consistently than those toward partial/focal neurogenic differentitation or uncommited type. deep topographic compartments of colorectal adenocarcinomas: down-regulation of mlh /msh expression results in lack of physiologic cell kinetic balance background topographic tumor cell selection is not well characterized in colo-rectal adenocarcinomas (crc). the contribution of mlh- /msh- expression to cell kinetics by topographic compartments remains unknown in sporadic crc. design we selected ( low-grade, high-grade) consecutive sporadic crc, including stage i, stage ii, and stage iii. mitotic figure counting, ki- index, g +m phase fraction, in situ end labeling (isel) of dna fragments, and mlh- /msh- immunoexpression were scored by topographic tumor compartment (above muscularis propria vs. muscularis propria). statistical correlation between proliferation and apoptosis variables was performed in each topographic compartment. variables were studied by topographic compartments and considered statistically significant if p< . . physiologic correlations were preserved only in the superficial compartment for mitotic figures, ki- expression, isel index, and mlh /msh expression. g +m phase fraction correlated with mlh- expression only in superficial compartments (p= . ) and msh- expression only in deep compartments (p= . ). conclusions superficial compartments of sporadic crc are characterized by high cellular turnover and maintained cell kinetic balance. mlh- and msh- expression in sporadic crc is inefficient (no g +m phase fraction differences by topographic compartments) and is dissociated (only one gene product correlates with g +m), eventually resulting in mutation accumulation and cell progression. gastro the biologic behaviour of gists has been difficult to predict based on the original criteria proposed in the early s. many isolated histological features, such as site, size, extent of bowel-wall involvement, necrosis, cellularity, mitotic counts, atypia, etc., had to be evaluated and integrated into a single and reproducible result that had also to be as accurately predictive as possible. therefore numerous studies have been performed in order to reach the ideal combination of factors that may predict these tumors correctly. gists were diagnosed either originally or retrospectively after reviewing diagnoses of git spindle cell tumors, examined in our pathology department between the years - . all tumors were defined as gists only when displaying c-kit (cd ) immunopositivity. additional immunohistochemistry was performed to corroborate the diagnosis. we reclassified the tumors according to different proposed classifications and correlated the results with the clinical followup available for these patients. the followup periods ranged between - months for gastric gists (mean- . months, median- . months) and - months for patients with small-bowel gists (mean- . months, median- months). while the median disease-free survival was . months for patients with malignant gastric gists, it was only months for patients with small-bowel malignant gists. the results of the study show that all classifications stratified the patients in a very similar manner although some were more accurate than the others and more user-friendly and therefore probably also more reproducible. the relative high rate of deaths occuring in the smallbowel gist group ( %) as compared to the . % death rate in the gastric gist patients, strengthens the supporters of different criteria to be applied to the gists arising in these different sites. another important issue that has imerged in our study was the much higher incidence ( %) of malignant gists developing in the younger age group ( - years), compared to . % malignant gists in the age group of above years, making this feature to be probably of major significance. it should therefore be considered whether age should be a built-in criterion when evaluating the biological potential of a gist. introduction gastric ulcer disease is the most important disease of the digestive tract becouse this illnes have big frequecy and circle gastric ulcus presents many histologic features characteristics which may be precancerous lesions. gastritis is lesion which is always associated with gastric ulcer. epithelial metaplasias are transformation of one kind of epithelium to other type epithelium and circle gastric ulcus it is very frequency changes of epithelium. the aim of this study was to to find frequency of appearance epithelial metaplasias circle gastric ulcus, types of metaplasias and grade of gastritis. also we searched changes of epithelial mucins. we had citochemistry examination endocrine g, ec and d cells and we wanted to describe immunohistochemicaly feature characteristics in different kinds of epithelial metaplasias. material and methods for examination we use biopsy material:surgery and endoscopic of patients with gastric peptic ulcer. we use standard he method for identification micromorpholic changes; ab-pas (ph , ) and hid-ab method for illustration biochemistry alteration about mucins;grimelius ( ) and masson citochemistry methods; immunocytochemistry avidin-biotin peroxidasa (abc) with antiserotonin, antigastrin, antisomatostatin. results we found cases completly mature intestinal metaplasias (im), cases incompletly im; case imature im and case imature im with displasia. also we detected composition completely and incompletly im in cases, composition incompletly and pyloric metaplasia in cases.we find pyloric metaplasias in cases, colons metaplasia in cases. composition colons and incompletly mature im were detected in cases;composition pyloric and incompletly mature im in case.summary the bigest frequency of appearance metaplastic alteration were in older patients.histologicaly claracteristics metaplasias were imitation some segments of digestive tract. metaplasias include many biochemicaly alterations and it can be present with changes in secretion of mucins.gastric metaplasia is dynamic process with changes both in epithelial and endocrine cells. conclusion metaplasias in gastric mucosa circle gastric ulcer is sign very important alteration.standardisation of research this alteration may be very important guider about prognosis for patients with gaster ulcer disease. choice of immunohistochemical method for detection of defect mismatch repair system c fenger, o nielsen, tp hansen department of pathology, odense, denmark colorectal cancer (hnpcc) and in % of sporadic colorectal cancers, and in the latter the defect is associated with a better prognosis but posssibly also a higher risk for metachronous cancer and a different response to adjuvant chemotherapy. screening for such defects can be done by testing for microsatellite instability, but immunohistochemistry gives similar results with less costs. the aim of this study was to select the optimal antibodies and methods for immunhistochemical detection of proteins involved in the mmr system. material and methods twelve antibodies ( against hmlh , against hmsh , against hmsh and against pms ) were tested using different dilutions and hier-techniques on a material consisting of paraffin embedded tumours from patients with well defined mutations and from controls. the results were evaluated semiquantitatively. the following clones, dilutions and pretreatment regimens were selected: introduction colon's metaplasia may be variant of intestinal metaplasia in the gastric mucosa circle gastric ulcer.it is important precancerous stadium because % colon's metaplasias of gastric mucous membrane transverse in gastric carcinoma. aims: we want to find frequency of appearance colon's metaplasia in gaster mucosa with ulcer disease.also we want to describe histologicaly, histochemicaly and immunohistochemicaly feature characteristics colon's metaplasia in gastric mucosa. materials and methods for examination we use biopsy's matherial:surgery's and endoscopic of patients with gastric peptic ulcer. we use standard he method for identification micromorfologic changes; ab-pas (ph , ) for illustration biochemistry alteration about mucins;grimelius citochemistry method;imunocytochemistry avidin-biotin peroxydasa with antiserotonin, antigastrin and antistomatostatin. results we find two cases so-called colon's metaplasia gastric mucous membrane. it was males in vi decade of life.both of them were detected near parts with intestinal metaplasia and atrophic gastritis. histology's design metaplastic epithelium appear big changes than normal gastric mucosa watched on cellular and tissue level;it was alike epithelium of colon. there was winding and arborescent cryptas, and wasn't find villosity. cells in cryptas were voluminous and they group. predominant componentof cells were goblet cells. cylindric cells also was alike goblet cells. we didn't see paneth's cells. histochemycaly we detected sufficiently sulphomucins and less sialomucins. g cells and ecl cells were insufficient; ec cells were less multiplied. conclusions colon's metaplasia is sign of big intracellular alteration and we can detect this with changes in secretion of mucin. colon's metaplasia gastric epithelium is dynamic process with changes both in epithel and endocrine cells. upregulated expression of relp, a reg-like protein, in inflammatory, metaplastic and neoplastic gastrointestinal mucosa lc andersson we screened expressed sequence tag (est) databases to detect genes specifically expressed in the gastrointestinal tract. we identified a gene, called relp (regenerating protein (reg) family-related protein). the relp cdna encodes a amino-acid preprotein with a -amino-acid signal peptide. the amino acid sequence of relp is - % identical and - % similar to the previously characterized members of the reg protein family. the reg proteins, which all belong to the family of c-type lectins, have been implicated in cell proliferation, migration and differentiation in the gastro-intestinal tissues. the relp gene spans . kilobases and maps to chromosome p - . , whereas the genes of the previously known reg proteins cluster on chromosome . dot-blot hybridisation revealed expression of relp mrna mainly in the gastrointestinal tract, but also in prostate, and testes. immunohistochemical staining demonstrated expression of relp in mucosal neuroendocrine cells of in the small intestine and in the parietal cells of normal gastric mucosa. regenerating borders of gastric ulcera, the goblet cells of inflammatory mucosa in ulcerative colitis and crohns disesase, showed a strongly upregulated expression of relp. areas of intestinal metaplasia in the stomach and esophagus also displayed a robust content of relp. more-over, relp was strongly expressed in mucocellular and mucinous tumors of the gastrointestinal tract. the orthologous gene for mouse relp was also cloned. conclusions relp represents a novel marker of inflammatory, metaplastic and neoplastic changes in the gi-tract. current approach to the biopsy diagnosis of gastrointestinal stromal tumors (gists) -is it possible to predict clinical behaviour? introduction gists are the most common gi mesenchymal tumors showing cd positivity. although according to accepted criteria none of them are 'benign', their metastatic risk depends on the tumor size (ts) and mitotic count (mc). aim: reclassification of a series of gists using morphological and immunohistochemical analysis of paraffin-embedded material and evaluation of the relevant clinical data. material and methods patients with gists fulfilled the criteria of fletcher et al. ( ) . in addition to histological stainings, primary antibodies against vimentin, desmin, actins, s- protein and cd (all dakocytomation) and cd (immunotech) were used for the immunoanalysis. results majority of the tumors was localized in the stomach (n= ) and small intestine (n= ), one in the colon and two were extragastrointestinal. in relation to the accepted mc and ts criterias, of cases were of low and one of very low risk category. tumors belonged to intermediate risk category, mostly due to ts in contrast to low mc. the inclusion of cases into high risk category was based predominantly on the mc (> / hpf and ts - cm /n= / and > cm /n= /; in the last one it was > - / hpf in contrast to ts < cm). conclusion a prediction of clinical behaviour of gists requires a correct histopatological analysis related to initial staging data. although the categorisation of the gists into different risk categories uses both criteria of ts and mc, the significance of the mitotic activity evaluation in our set of cases seems to be higher. multiple gastric composite carcinomas (adenocarcinoma and endocrine carcinoma) with adenosquamous differentiation y kapran , n akbas , m aksoy , f dizdaroglu university of istanbul, istanbul medical faculty, pathology department, istanbul, turkey university of istanbul, istanbul medical faculty, general surgery department, istanbul, turkey a case of multiple gastric composite carcinomas composed of endocrine carcinoma and adenocarcinoma with adenosquamous differentiation is presented. tumours showed a composite architecture both within the site of origin and in lymph node metastasis. a year-old woman had a diagnostic upper gastrointestinal system endoscopic evaluation for her long term dyspeptic complaints. after determination of two concurrent tumours in the stomach she underwent total gastrectomy operation. gross examination of the stomach revealed a polypoid lesion in the anterior wall and an ulcerous lesion in the posterior wall of the corpus. also there were multiple minute nodular lesions both in the corpus and fundus. histopathological examination of the polypoid tumour revealed a well-differentiated tubular adenocarcinoma admixed with an endocrine carcinoma proved immunohisto-chemically. ulcerous lesion was composed of a poorly-differentiated adenocarcinoma showing adenosquamous differentiation admixed with an endocrine carcinoma. other nodular lesions were composed of endocrine tumours having different levels of invasion in the wall of stomach. non-tumourous mucosa harboured atrophic gastritis. this is a unique case of multiple composite carcinomas devoloped in the setting of atrophic gastritis worth to discuss. introduction one of the most common malignant soft tissue tumours in adults is malignant fibrous histiocytoma. these tumours are accepted as an aggressive tumour and mainly treated surgically. primary and metastatic malignant fibrous histiocytomas of the gastrointestinal tract are rarily described. our case is a -year-old woman who had a retroperitoneal malignant fibrous histiocytoma that had metastased to the lung after months following an adequate initial treatment. four months later, because of having recurrent attacks of vomiting and nausea, gastroscopic examination was performed. multiple nodular lesions in her stomach were seen and microscopic examination of these lesions revealed spindle-shaped, pleomorphic tumour cells. the positive immunoreactivity for anti-vimentin and anti-cd justified the diagnosis of malignant fibrous histiocytoma's metastases. total gastrectomy was performed following that diagnosis and she died after month due to renal failure. conclusions this case is presented because of the rarity of distant metastases of malignant fibrous histiocytoma to the stomach and to discuss the entities that should be considered in the differential diagnosis of these tumours. role of p ink a in gastrointestinal stromal tumours introduction the classification based on c-kit/cd expression reordered the formerly rather confuse category of gist. p ink a (p ) is a tumour suppressor gene of the cyclin d-cdk , /ink /rb/e f pathway, altered in > % human neoplasms. it is located on chromosome p , a region consistently involved in dna losses found in malignant gists. aims: we studied the role of p in gists. materials and methods cd -immunoreactive mesenchymal tumours with morphological and clinical features consistent with gist were studied, excluding smooth muscle (desmin+, cd -) and schwannian (s- +, cd -) neoplasms. cases ( gastric, small intestinal and colorectal; males, females; age at diagnosis: - years; follow-up: - -mean: . -months) were considered. immunohistochemistry, rna extraction with semiquantitative rt-pcr and methylation specific pcr (msp) were used. kaplan-meier method followed by log-rank test, and univariate and multivariate analyses with a proportional hazards model were performed (malignancy criteria adopted for uncensored events: recurrence, invasion of adjacent organs, peritoneal dissemination, metastases and death of disease). results a tumoral cell fraction with low/absent p immunoreactivity > % was associated with malignancy; semiquantitative rt-pcr confirmed this result. by msp, cases (all underexpressing p , six malignant) showed p promoter methylation. low/absent p immunoreactivity, together with size, cellularity, mitotic count and presence of coagulative necrosis resulted associated with malignancy by univariate analysis, with the former factor being the only prognostic one selected by multivariate analysis. conclusions p downregulation is implied in gist progression; p promoter methylation is one of its causes; p assessment opens possible perspectives in gist prognostication. introduction regenerative abilities of gastric mucosa glandular epithelium cells are the main mechanism which condition its right function both within the range of gastric mucosa integrity and normal activity of its epithelial cellular elements. the aim of the work was to evaluate dependence between proliferative activity of gastric mucosa glandular epithelium and sex, age, chronic inflammation type and helicobacter colonization. the examinations were conducted on paraffin blocks from gastric oligobiopsies taken from women (of average age- ) and men (of average age- ).to the examinations were rated only cases where oligobiopsies were taken from at least two gastric prepyloric parts (antrum) and corpus with recognized unspecific chronic gastric mucosa inflammation. chronic gastritis was graded according to modified whitehead's classification. helicobacter pylori (hp) was evaluated according to the sydney system. examination of the cells with pcna proliferative antigen expression were conducted separately for foveolar area glands and for glands below gastric mucosa foveolar area. all the measurements were performed separately for gastric mucosa prepyloric part and gastric corpus. in tissue sections, in which were carried out proper immunocytochemical reactions, measurements of the pcna positive cells number in circular, oblique short, oblique long and oblong gastric glands sections were conducted five times. the statistic analysis also included age, presence of hp colonization, gastric mucosa inflammation type and the area where an oligobiopsy section was collected. results no significant differences were found in proliferative activity depending on the place of collecting a section, age and hp colonization, while a higher proliferative activity was found in superficial chronic gastritis, particularly in gastric mucosa prepyloric part. conclusions . chronic superficial inflammation coexists with significantly higher proliferative activity of gastric mucosa glandular epithelium, particularly with connection to its prepyloric part. . changes in gastric mucosa glandular epithelium proliferative activity do not depend on its age and histotopography and hp colonization. introduction chromogranin-a-plus (cha) and d cells exist almost in the whole alimentary canal and in pancreas. these cells have an influence on secretory functions and normal gastric mucosa histostructure. up to now investigations confirmed participation of these cells in e.g hcl and gastrin secretion regulation and in some of the proliferative states of gastric mucosa. the aim of the study was to estimate participation of cha and d cells in microscopic superficial (gchs) and deep (gch) gastric mucosa inflammation's indices. material and methods paraffin blocks from gastric oligobiopsies was the material which was collected from women (of average age- ) and men (of average age- ). to the examinations were classified only the cases where oligobiopsies were taken from at least two areas of gastric prepyloric part (antrum) and corpus, with diagnosed unspecific chronic gastric mucosa inflammation. chronic inflammations were graded according to modified whitehead's classification. helicobacter pylori (hp)was evaluated according to the sydney system. neuroendocrine cells of gastric pylori were counted after proper immunohistochemical identification in circular, oblique short, oblique long and oblong gastric glands sections. all measurements were conducted separately for gastric mucosa prepyloric part and gastric corpus. the results of quantitative researches were analysed with the use of statistic methods. in gastric mucosa antrum there were twice more cha and d cells in comparison with corpus. gastric mucosa inflammation type did not have a significant influence on cells localization in gastric mucosa. the number of cha cells diminished in gchs gastric antrum among patients who were over years old, whereas the same number increased in corpus inflammations of this type. the increase of cha cells number was found in gchs gastric antrum with hp colonization. in these cases d cells number diminished. conclusions . in gchs gastric antrum with hp colonization cha cells number significantly increases while d cells number decreases. . quantitative changes of cha and d cells run differently in gchs and gch both in prepyloric part and in gastric corpus. response to preoperative chemoradiation on rectal adenocarcinoma: histopathological effects and relationship with level of p and pcna expression introduction adjuvant chemoradiotherapy delivered before surgery has been used to improve control and to reduce the likelihood of local recurrence in selected patients with rectal cancer. although the status of p gene and the expression of pcna has been widely studied in these tumors, little is known about their value to predict the responsiveness of rectal cancer to chemoradiation. the aim of this study was to to assess effects of preoperative chemoradiation in advanced rectal adenocarcinoma. we examined the histopathologic effects of chemoradiotherapy on rectal tumors and correlated the efficacy of treatment with the level of p and pcna proteins expression in pretreatment biopsies. results three patients showed no primary tumor in the resection specimen. another eight carcinomas showed a good response to chemoradiation with only small nests of viable tumor cells. grouping these two categories, we considered that the tumor response had been total or subtotal in cases ( %). nuclear accumulation of p protein was detected in ( %) specimens. thirty five tumors showed a high pcna index ( %). no obvious relationship seemed to exist between pretreatment, p immunostaining and response to chemoradiotherapy. tumors with high pcna index were more likely to respond to chemoradiation: / ( %) versus / ( %)(p= . ). conclusion knowledge of proliferative activity of rectal cancer, as determined by pcna immunostaining, should be useful in predicting the likelihood of response to preoperative chemoradiation. introduction mitotic count and tumor size are the main predictors of outcome in gastrointestinal stromal tumors (gist). however, they are not always useful in individual cases. the aim of the present study has been to assess whether mitotic counts performed using phosphorylated histone (phh ) antibody, expressed from the early steps of mitosis, could improve the prediction of prognosis in these tumors. materials and methods sections from gist were stained with phh antibody (upstate biotechnology, lake placid, ny; : , dilution). mitoses were counted per high power fields ( x, olympus bx). in addition, the h&e-based mitotic counts and tumor size were obtained from the pathology reports. patient outcome was recorded as the endpoint. conclusion in the present series, phh -based mitotic counts allow a better prediction of prognosis than h&e-based counts or tumor size. thus, phh antibody could be applied in the pathological classification and grading of these tumors, and in the more precise definition of cases with higher risk of progression. immunohistochemical expression of cyclooxygenase- (cox- ) in human colorectal cancer introduction recent studies have implicated metabolites of arachidonic acid, such as prostaglandin e( ) in colorectal carcinogenesis. cox- , a key enzyme in arachidonic acid metabolism, is present in the majority of colorectal cancer (crc). its overexpression seems to be associated with worse prognosis. the aim of this study was to examine the relationship between the expression of cox- in human crc and the clinicopathological characteristics of the tumor. materials and methods paraffin embedded tissue samples from patients (m: , f: , median age: yrs) with crc (tnm staging: i -iv), who underwent radical surgical treatment in thessaloniki cancer hospital "theagenio" from / / until / / , were evaluated. tumor sections were stained for cox- using a rabbit polyclonal antibody against human cox- and the staining was graded on a scale - , using mononuclear cells as reference. results cox- expression was found in tumor epithelial cells in . % of crc ( / cases) with diffuse mainly, cytoplasmic staining and in inflammatory cells, vascular endothelium and fibroblasts. low cox- expression (grade - ) was found in . %, high cox- expression (grade - ) in . % of cases and the remaining were negative. there was no correlation between clinicopathological characteristics of patients and intensity of cox- expression, although there was a slightly better overall survival rate in low expression group of patients (p:ns) . conclusions this small study shows that cox- protein is overexpressed in most cancer cells in colorectal carcinomas and may be related to overall survival. the expression of bcl family proteins in b-chronic lymphocytic leukemia introduction b-chronic lymphocytic leukemia (b-cll) is a neoplastic disease caused primarily by defects in the apoptosis mechanism. apoptosis is the process of programmed cell death or cell suicide. the bcl- proteins are a family of proteins involved in the response to apoptosis. some of these proteins (such as bcl- and bcl-xl) are anti-apoptotic, while others (such as bad or bax) are pro-apoptotic. materials and methods our study includes peripheral blood specimens from patients with b-cll and healthy individuals (as a control group). using western blot analysis, we examined the levels of bcl- , bcl-xl, bax and bad protein expression. the level of bcl (p= , x - ), bax (p= , ) and bad (p= , ) proteins were significantly increased in all analyzed patients while the level of bcl xl protein (p= , ) showed no difference between patients and healthy individuals. differences between the levels of analysed protein expression in group of patients had no statistical significance. conclusion increased level of expression bcl , bax and bad protein represents the most striking feature of b -cll cells. the variations in expression of only one protein of the bcl family can not represent the prognostic factorr in b-cll. introduction kikuchi -fujimoto disease (kfd) is a distinctive type of necrotizing lymphadenitis with unknown cause, has a selflimited clinical course and affects usually the cervical lymph nodes of young women. although kfd occurs most often in young women, only two cases have been reported during pregnancy. in one of these cases kfd developed initially years before. case report we report a case of a -year-old pregnant woman on her th week of gestation who was admitted with a history of swelling in the right side of the neck and fever of days duration. on physical examination a cm cervical lymph node was palpable. microscopically, the excised lymph node showed large areas of necrosis and extensive karyorrhexis. no neutrophils or plasma cells or eosinophils were identified. it was found an admixture of nonphagocytic histiocytes and t-lymphocytes in the necrotizing area. there was a predominance of cd + cells over cd + cells. the patient received no treatment and months later had no symptoms. later she delivered a healthy boy. five years ago the patient presented similar clinical history and histological diagnosis of the excised cervical lymph node. we present the second reported case of recurrent kfd during pregnancy and the third case of kfd developing during pregnancy. conclusion from our case and the other two reported cases it seems that if kfd occurs in a pregnant woman there is no need to terminate the pregnancy as there was no effect on both mother and fetus. monitoring of patients with follicular lymphoma carrying t( ; )(q ;q ) by real time quantitative pcr introduction follicular lymphomas (fl) are characterized by t( ; )(q ;q ) in a majority of patients. the identification of the translocation is utilized to monitor the disease behavior, and, specifically, to detect minimal residual disease (mrd). qualitative pcr is routinely used but without specification of the tumor cell quantity. real time quantitative pcr (rq-pcr) may be employed to address this issue. aims: rq-pcr was introduced to monitor mrd in patients with fl bearing the t( ; )(q ;q ). materials and methods samples of primary tumors, bone marrow and peripheral blood (bm/pb) specimens were taken in the course of the disease and were used for the t( ; )(q ;q ) assessment. for translocation screening, fish on interphasic nuclei was applied. qualitative pcr was used to detect the break in the major breakpoint region (mbr) of the bcl- gene ( q ). relative quantity of cells bearing mbr t( ; )(q ;q ) was performed by the rq-pcr. fifteen samples of primary tumors and bm/pb in duplicates were investigated with appropriate controls. the relative quantity of cells with t( ; )(q ;q ) was significantly higher in the primary tumors than in the bm/pb samples. the changes of the bm/pb infiltration were found during the course of the disease and included shifts toward the tumor progression or disappearance of the tumor cells. rq-pcr is a technique suitable for monitoring the disease response to the therapy or the disease progression and it is superior to morphology and immunohistochemistry. supported by: iga-mzcr-nc/ - , research project fn-motol/ . phosphatase activity dependent apoptotic effect of transforming growth factor beta in ht human lymphoma cells introduction transforming growth factor beta (tgfb ), a multifunctional cytokine, has antiproliferative and/or proapoptotic effect on lymphoid cells. the continuously proliferating lymphoma cells could be resistant to the endogenously produced tgfb . aims: to study the responsiveness of lymphoma cells using exogenously activated tgfb . the apoptotic effect and depolarization of the mitochondria was detected by flow cytometry, the different gene expression was studied by rt-pcr. the amount, the activity (western blot) and the localization (confocal microscopy) of the signal elements (smads, phoshpo-smad , erk / , jnk, p mapk) and other proapoptotic and survival proteins (bcl- , blc-xl, bid, bax, bad, nfkb, phospho-ikb) were also studied. the role of pp a phosphatase, mek kinase and caspase activity was estimated by different inhibitors (endhothal, okadaic acid, pd , z-vad-fmk, z-ietd-fmk, z-lehd-fmk) in vitro. results ht b cell lymphoma cells lost their sensitivity to the antiproliferative action of exogenous tgfb , but the cells were killed by apoptosis. smad phosphorylation, nuclear localization and the increased expression of tgfb induced early gene (tieg) proved the rapid activation of smad signal. the role of pp a phosphatase activation followed by decreased expression of active phospho-jnk and phospho-erk / was supported by the antiapo-ptotic effect of pp a inhibitors and the proapoptotic effect of specific mek inhibitor. the apoptosis was induced primarily through the mitochondrial pathway. it seems that both main iniciator caspases (caspase and ) took part in the apoptotic response. conclusion these data suggest that exogenous tgfb had double but interrelated actions on ht cells: a) suppression the survival signals produced by protein kinases, which b) allowed the smads to induce apoptosis. therefore, it is possible that the lost sensitivity of malignant lymphoid cells to proapoptotic regulators (such as tgfb ) could be reactivated especially by lowering the survival threshold. the work was supported by otka (t ), ett ( / ett ( / , / , fkfp ( / ) and békésy foundation ( / ). clinicopathological study of chromosome abnormality related hematological disorders introduction cytogenetic abnormalities sometimes determine the subtype of hematological disorders and are common in myelodysplastic syndromes (mds). although chromosome abnormality is frequently observed in mds, the specific characters of this type are not determined. in this study, we examined the clinicopathological characters of hematological disorders associated with chromosome abnormality. aim of this study is to assess whether these disorders are classified in special subtype or not. we selected hematological disorder cases with chromosome abnormality from the bone marrow files of the department of pathology, nagoya university hospital. clot sections of aspirated bone marrow were examined histopathologically. immunohistochemical study was performed using paraffin embedded tissues. results patients, males and femals respectively, with monosomy , with der ( ; ) and with other abnormalities were studied.the median age of the patients was . years (range, - years). pancytopenia was observed in , bicytopenia in and monocytopenia in cases. past history of aplastic anemia was in cases. histopathologically, they were cases of mds, of aml, in all and of others. hypoplastic mds (< % cellularity) were found in cases, of ra, of raeb, of raeb-t and case of mds with fibrosis. all cases of acute leukemia had dysplastic multi-lineage features other than blasts. histopathological characteristics were weak erythroid dysplasia, increased micromegakaryocytes and hypocellular tendency. these characteristcs were common in patients with monosomy and other abnormalities. concusion hematological disorders with chromosome abnormalities share similar clinicopathological characteristics. expression of topoisomerase i and topoisomerase ii alpha as an independent prognostic factor and evaluation of the overall survival in nhl e catalano cooper hospital/university medical center, camden, usa introduction topoisomerase ii alpha and topoisomerase i (topo i and ii alpha) are the key targets for some anti-cancerous drugs that inhibit these enzymes. the purpose of this paper is to examine the expression of topo i and topo ii alpha in different types and stages of nhl's the response of treatment with the overall survival. materials and methods immunohistochemistry techniques with anti-topo i and topo ii alpha were used. the results were evaluated with a semi-quantitative method. results and conclusion cases were studied with topo i and topo ii alpha. we include % of follicular lymphomas, % diffuse, % follicular and diffuse and high grade %. the stage of these tumors were stage i, %; stage ii, %; stage iii %; stage iv, %. the results show a higher intensity expression of the topo ii alpha, and topo i indices, in the higher grade nhl. there appears to be a similar expression between the intermedium and indolent nhl. the survival indicates that the large cell nhl's show a higher response rate and better survival than the mixed or small cell type. the expression of these two enzymes may be useful for selecting the appropriate chemotherapy in nhl. our data, although small, suggests an excellent correlation in response to treatment and longer survival in the nhl that have a high expression of topo i and topo ii alpha. mantle cell lymphoma (mcl): ki- antigen and chromosome ploidy in an estimate of the tumor progression introduction mcl belongs to prognostically unfavorable lymphomas. however, the behavior may vary and it is difficult to predict it. a more aggressive course tends to be associated with blastic and anaplastic subtypes. numerical chromosomal changes and proliferative activity may be of importance to improve the prediction. aims: risk of the tumor progression with the assessment of ki- antigen-positive cell count and ploidy status of chromosome . materials and methods patients with mcl characterized by t( ; )(q ;q ) and by overexpression of cyclin d . the proliferative activity was evaluated immunohistochemically (ihc) by double staining for ki- antigen and cd (to rule out counting admixed ki- positive t-lymphocytes). ploidy status of chromosome was evaluated by interphasic fish. results and conclusion trisomy and polysomy of chromosome ( patients) was associated with blastic/anaplastic forms of mcl with the same frequency as with the common variant. a finding of > % ki- and cd positive cells was associated with the blastic variant of the disease - patients. of them died of the disease progression, one of unrelated cause and one is alive with the disease. of the remaining patients in whom the follow up was available, died of the tumor, and the proliferative activity, measured by counting the ki- positive cells was < %. the assessment of proliferative activity with immunohistochemical demonstration of ki- antigen seems to be useful in prediction of clinical behavior of mcl supported by iga-mzcr-nc/ - , research project fn-motol/ . morphological criteria for differentiation of lymphocyte predominance hodgkin's lymphoma from lymphocyterich classical hodgkin's lymphoma aims to reveal morphological features of differential diagnosis between lymphocyte rich classical hodgkin's lymphoma (lrchl) and nodular lymphoid predominance (nlphl). retrospective analysis of lymph node biopsies (age - ) with histological diagnosis of "nodular paragranuloma" or "hodgkin's disease, lymphocytic and histiocytic variant". immunological data were available in all cases. all patients presented with the early stages of disease. we used the following morphological criteria: growth patterns (nodular, nodulardiffuse or diffuse), distribution and cytomorphology of tumor cells ("popcorn", lacunar, classical, mummified cells), cellular composition of reactive background, presence or absence of secondary and atrophic follicles. the results of morphological algorithm were compared with immunology data in every case. results among biopsies, cases ( %) were classified as nlphl. cases were attributed to lrchl, including cases which were characterized by interfollicular sheets of cells resembling lacunar cells ("cellular phase of nodular sclerosis"), cases had rare classical cells within expanded mantle zone ("follicular variant" of hodgkin's disease). all cases lrchl exhibited secondary follicles. there were no cases of lrchl with "pure" diffuse growth type; both nlphl and lrchl had nodular or nodular and diffuse pattern. cases corresponded morphologically to nlphl or lrchl and this division was confirmed with immunological analysis.. conclusion we proposed the algorithm of morphological criteria for differentiation of nlphl from lrchl bone marrow sinusoids in acute lymphoblastic leukemia (all) v rugal russian institute hematology, st.petersburg, russian federation introduction bone marrow sinusoids regulate the development of hemopoietic precursors and migration of stem cells and mature blood cells in blood stream. the purpose of the investigation was to study endothelium of bone marrow sinusoids in disordered hemopoiesis in all. pretreatment bone marrow trephine biopsy sections from patients with all were studied using morphometric and ultrastructural methods. results development of myelosuppression in all was accompanied by considerable changes in the morpho-functional status of sinusoid endothelium. most vessels became thinned, endothelial cells were flattened. in such vessels cells of endothelium came apart with formation of gaps between cells. the basement membrane was interrupted. however, blast migration continued to be transendothelial. there were vessels in which the structure of endothelial cells was identical to that of blast cells. there were no signs of endo-and exocytosis in such cells. intranuclear virus-like inclusions were found in endothelial cells of sinusoids of patients with all. the above data suggest the involvement of endothelial cells in the disordered haemopoiesis in all. defects of the bone marrow sinusoids in leukemia can be the cause of the early release of blasts into circulation. blast transformation of endothelial cells does not contradict the suggestion about existence of a common cell precursors of the endothelial and hemopoietic cells. complex diagnosis of follicular lymphomas e toth, e csernak, z melegh, n udvarhelyi, t schneider, a rosta, z szentirmay national institute of oncology, budapest, hungary introduction the second most common type of non-hodgkin lymphomas is follicular lymphoma (fl). moreover, follicular lymphomas are the most common type of the low grade lymphomas. in hungary they comprise about - % of all the lymphomas but its occurrence shows increasing tendency. our aim was to survey and revise fls diagnosed in the last years in the national institute of oncology. we studied the diagnostic relevance of histology, immunohistochemistry and the detection of immunoglobulin heavy chain (igh) and bcl- gene rearrangements. materials and methods we surveyed cases which were diagnosed as follicular or centrocytic, centroblastic lymphoma before. after the histological re-examination immunohistochemistry (cd , cd , bcl- , cd , bcl- , cd , p , cyclin d and ki- ) was performed on each case. we studied the igh and bcl- (mbr) gene rearrangement both with conventional and real-time pcr methods from the paraffin embedded blocks. the classification was made according to the new who classification. results after the revision of the cases we found follicular, diffuse large b-cell, mantle cell and marginal zone lymphoma, paragranulomas and follicular hyperplasias. the grade of the fls showed relation to the expression of different antigens. cd and bcl- expression decreased with the grade. p expression was very rare and occurred only in grade cases. the proliferation index with ki- showed good correlation with the grade of fls. the igh showed types of pattern: monoclonal, polyclonal and a sharp line in a polyclonal background. grade and grade - fls were monoclonal almost in the same ratio. we could detect bcl- gene rearrangement in % of the fls ( % grade - , % grade ). all the grade cases which showed bcl- gene rearrangement belonged to grade a group while all the grade b cases were negative. conclusion in agreement with others we found that grade , and a fls showed similar morphological, immunohistochemical and genetical features while gr b fls are more similar to diffuse large b-cell lymphomas. considering the diagnostic relevance of the different methods we can conclude that histology alone is not sufficient. % of our cases were solvable with the help of immunohistochemistry and in % of the cases the diagnosis based on the molecular pathological results. in the remaining % even with the help of all diagnostic tools no unambiguous diagnosis could be reached. the bcl- and p oncoproteins play important roles in carcinogenesis. mutation of the p gene is considered the most common genetic aberration in many cancers including hematologic malignancies. ki- , a proliferation antigen, evaluates the proliferative activity of a lesion. angiogenesis is also critical for tumor proliferation and has a prognostic value. the vascular density of tumor directly correlates with poor outcome in many malignancies. the aim of this study was to investigate correlation between ki- , p and bcl- immunostaining in b cell lymphomas and to identify any relation between these markers and microvessel density (mvd) and histopathological parameters including stage and tumor grade. materials and methods low grade and high grade non-hodgkin lymphomas (nhl) were immunohistocemically stained with p , ki- , bcl- and factor viii. the results were analysed statistically. results there was no correlation between stage and ki- li, mvd, p and bcl- expression. the cases which showed immunoreactivity for bcl- had decreased mvd counts compared with the cases negative for bcl- . p immunoreactivity was correlated with mvd. microvessel counts were significantly higher in patients with high grade nhls. statistical analysis showed association between ki- li, p , bcl- expression, mvd and histological grade. conclusions the levels of ki- , bcl- and p expression and microvessel counts were closely related with the grade of nhls. our findings suggest that apoptosis, angiogenesis and proliferative activity can be used as prognostic indicators in nhls. diffuse large b-cell lymphoma of the maxillary sinus complicated with blastomycotic leptomeningitis sinus; however such a lesion is more likely to arise from an infraorbital lymph node, part of the facial lymph node group. case presentation we present a patient with diagnosed diffuse large b cell lymphoma of the maxillary sinus as well as involvement of the infraorbital region, who suffered from infective complications. -years old male was admitted at the clinic of hematology because of tumor mass in nasal cavity. ct-scan showed tumor mass filling right maxillary sinus. surgical biopsy was done. histochemical and immunohisto-chemical findings suggested a diagnosis of diffuse large b cell lymphoma. other clinical investigations showed i-e clinical stage. treatment with four cycles of chop and mab-thera followed. after that adjuvant radiotherapy was delivered. because of increased body temperature and meningeal symptoms, this therapy was interrupted. microbiological investigations showed positive hemoculture for escherichia coli and staphylococcus aureus, and positive finding of blastomyces in cerebrospinal fluid, while serological analyses revealed positivity for cmv and ebv. beside severe antibiotic, antimycotic and antiviral treatment, the patient suffered from sepsis with main features of suppurative leptomeningitis and acute hepatitis. conclusion this case implies the conclusion that the clinicians should have in mind the possible presence of immune disregulation in patients with nhl, that could complicate the administered chemotherapy and radiotherapy. aim the aim of this study is to determine the most frequent pattern of involvement by nhl of head and neck and to correlate it with clinical outcome of the disease. material and methods a retrospective review was performed on patients with nhl during five years period. paraffin sections stained with standard histochemical and immunohistochemical stainings were histologically analyzed for classification of the nhl accordingly to the who classification of lymphoid neoplasms. clinical histories were used for clinical follow-up. results in fifty cases out of patients with nhl the disease was primary localized in the head and neck region. male-female ratio was , . twenty four cases were primarily nodal, primarily extranodal and had extranodal/nodal expression of the disease. diffuse large b cell lymphoma comprised % of the group: nodal, extranodal and extranodal/nodal. three of these cases were cd + and alk- -. six cases ( %) with b-cll nhl had nodal manifestation. malt-zone lymphoma ( %) showed extranodal involvement in cases and extranodal/nodal in cases. b-lymphoblastic lymphoma ( %) showed cases with nodal, one extranodal and one extranodal/nodal involvement. two cases with burkitt's lymphoma ( ) had typical mandibular localization. follicular lymphoma comprised %, with two nodal and two extranodal involvement. t cell nhl-s comprised % of the group with three nodal, one extranodal and one extranodal/nodal involvement. there was no significant predilection for clinical expression of the nhl of head and neck, and no difference between the mean survival of the groups for these parameter. expression of the bcl- family proteins, caspase (cpp ) and poly adp-ribose polymerase (parp- ) in multiple myelomas v zolota , m melachrinou , i xagoraris , a mouzaki , p aroukatos , d koumoundourou , ds bonikos dept. pathology, univ. patras med. school, patras, greece dept. laboratory hematology and transfusion medicine, univ. patras med. school, patras, greece introduction multiple myeloma (mm) is a malignancy of the bone marrow characterized by the clonal expansion of plasma cells with low proliferative activity. aberrant expression of genes regulating apoptosis/survival seems to be essential in the development of multiple myeloma. the aim of this study was to investigate the immunohistochemical expression apoptosisregulating proteins in bone marrow of patients with initially presented multiple myeloma. methods the expression of bcl- , mcl- , bak, bax, cpp and parp was examined on paraffin sections of bone marrow biopsies obtained from equal number of patients with newly diagnosed mm, using commercially available monoclonal and polyclonal antibodies and lsab technique. specimens containing > % immunostained tumor cells were considered positive. results bcl- , bak and bax positive immunostaining was detected in %, % and % of cases respectively. most positive tumors showed high percentage of tumor cells immunoreactive for the above proteins: > % for bcl- , > % for bak and > % for bax. the mean value for bcl- /bax ratio was . , and the mean bcl- /bak ratio was . . mcl- expression was detected in a small percentage of tumors ( %). cytoplasmic expression of cpp was observed in , % of cases and the number of positive cells was low. nuclear expression of parp was seen in , % of cases and no association was found with the other apoptosis-related antigens, bcl- /bax or bcl- /bak ratio. conclusion the above data demonstrate an overexpression of bcl- , bax and bak in myeloma plasma cells and an increased bcl- /bax ratio. cpp does not seem to mediate any apoptotic event in mms. the role of overexpression of parp remains to be elucidated. immunoexpression of bcl- family members, p , caspase (cpp ), and poly adp-ribose polymerase (parp- ) in diffuse large b-cell lymphomas introduction diffuse large b-cell lymphomas (dlbcl) represent a biologically and clinically heterogeneous group. although these tumors are curable with chemotherapy, their response to treatment varies. apoptosis-related proteins may play an important role in the chemosensitivity or chemoresistance of tumors. the aim of this study was to investigate if the expression of apoptosis-regulating proteins in dlbcl could be indicative of tumor chemosensitivity. methods formalin-fixed, paraffin-embedded tissue from cases of dlbcl [ nodal, extranodal, splenic ( centroblastic, immunoblastic, burkitt's-like, anaplastic, tcrbcl, primary mediastinal)] were immunostained with monoclonal [bcl- (dako), mcl- , cpp (neomarkers), p (biogenex)] and polyclonal [bax, bak (dako), parp (neomarkers)] antibodies using lsab method. specimens containing > % immunostained tumor cells were considered positive. clinical data were available for cases ( chemosensitive, chemoresistant). results bcl- , mcl- , bax, bak, p , cpp and parp positive immunostaining was detected in . %, . %, . %, %, . %, . % and . % of cases, respectively. mcl- , bax, bak and parp were overexpressed in > % of tumor cells in most cases. there was a significant association between the expression of bcl- and mcl- (p = . ). a higher immunoexpression of bcl- ( . % vs . %), mcl- ( . % vs %), bax ( . % vs %), p ( . % vs %) and cpp ( . % vs %) was observed in chemoresistant cases. parr expression was higher in chemosensitive cases ( . % vs . %). in the p -negative group of cases ( cases) those with a strong ( +) parp expression showed a significant better response to treatment (p = . ). a significant chemosensitivity was also detected in the group ( cases) with strong ( +) bax and weak (+) or negative (-) p immunostaining (p = . ). conclusion apoptosis-related proteins, though overexpressed in dlbcl, were not associated with response to treatment. a p (-)/parp( +) or p (+ or -)/bax( +) tumor status may reflect sensitivity to chemotherapy. introduction non-hodgkin lymphomas (nhl) are a heterogenous group of lymphoid malignancies classified according to their morphological, immunophenotypical and genotypical characteristics as well as their clinical behaviour. however, the progression of these diseases may be influenced by various others factors such as apoptosis, proliferation and angiogenesis. aim: a possible correlation between bcl- expression, apoptosis, disease outcome and overall survival in patients with nhl was analysed. material and methods nhl devided in low-(n= ) and highgrade (n= ) were immunohistochemicaly analysed for bcl- expression, while low-and high-grade nhl were procesed for terminate tdt mediated dutp nick end labeling (tunel) method in order to determine the apoptotic index of tumor cells. results were obtained by image analyser and statistically processed. results the expression of bcl- protein, as an antiapoptotic marker was significantly higher in low-compared to high-grade nhl, p= . , whereas the apoptotic index obtained with the tunel method demonstrated inverse results, p< . . in highgrade nhl, partial remission was observed in patients whose tumors expressed significantly higher values of bcl- , (p= . ), while complete remission was observed in a group with a lower expression of bcl- . furthermore, patients with lower bcl- expression showed longer overall survival p= . , whereas patients with a higher apoptotic index showed shorter survival, p< . . in both low and high-grade groups of nhl, bcl- has been found as an independent predictor of patients disease relaps, p< . . conclusion the expression of bcl- is a strong independent predictor of disease progression in nhl patients. the heterogeneity of anaplastic large cell lymphomas (alcl) o mederle, m raica dept. of histology and cytology, university of medicine and pharmacy, timisoara, romania introduction the aim of this study was to investigate the expression of cd and the distribution of the reticular network on the alcl. material and methods twelve cases of lymphomas were analyzed using morphologic and immunophenotypic features. the patients ( male and female) were from to years. five patients presented with generalized lymphadenopathy and seven with involvement of extranodal sites. alcl was composed of large cells that grow in a diffuse pattern and involve the lymph node sinuses. the tumor cells have an abundant eosinophilic or pale cytoplasm. the nuclei were irregular with indentation, multilobated, round to oval. multinucleated cells with wreath-like configurated nuclei were commonly seen. alcl cells may closely resemble cells of malignant histiocytosis. additional features include reed-sternberg-like cells, reactive infiltrate, partial lymph node involvement with localization of tumor cells in the paracortical region resembling hodgkin's diseases. tumor cells are cd +. the regular expression of cd antigen in both carcinoma and alcl (both are anaplastic large cell tumors) has important implication for the differential diagnosis of these tumors. leukocyte common antigen (cd ) positivity excludes an embryonal or any other non-hematopoietic tumor, but its absence does not rule out alcl (a significant portion of alcl are cd negative). antibodies to cytokeratin label embryonal carcinoma but not alcl. thus, if cytokeratin is expressed in an anaplastic large cell tumor simultaneously with cd antigen, it is most likely to be an embryonal carcinoma conclusion this study confirms the view that alcl are heterogeneous group of lymphomas and the cd is a useful marker for the diagnosis. leiomyosarcoma of the meckel's diverticulum. we describe a -years-old male developing a well differentiated leiomyosarcoma of the meckel's diverticulum synchronously with b-cll. the patient suffering disuria and malaise was admitted at the clinic for surgery. clinical examinations showed leucocytosis ( . x /l) with lymphocytosis ( %), anemia (hgb = g/l) and thrombocytosis ( x /l). ct-scan found a tumor mass near the urinary bladder and enlarged spleen. the fnab from the tumor mass revealed neoplastic tissue composed of ribbons of spindle-shaped cells with hyperchromic and pleomorphic nuclei and variable amount of cytoplasm, surrounded by a fine reticulin network. mitoses were rare ( - on hpmf). immunohistochemistry showed positivity for sm-actin and desmin. s- , cd and cd were negative. a leiomyosarcoma was diagnosed. three weeks after the biopsy a large tumor mass ( g) from the vicinity of the urinary bladder was extracted, together with incidental meckel's diverticulum. at the same time a splenectomy was performed because of splenomegaly ( g). pathohistologic examination of the tumor mass confirmed the previous diagnosis, and the pcna proliferative index was estimated to - %. the analysis of the meckel's diverticulum revealed a disturbance of the muscular layer structure caused by a tumor proliferation with the same histological features, infiltrating into the serosa. the pathohistologic examination of the extracted spleen showed partially presence of b-cll infiltration (monoclonal positivity for lambda-light chains, cd , cd , cd , cd ). follow-up shows that the patient is in binet a stage, and still in a good shape without any further treatment. it remains unclear which of the neoplasms developed first. introduction synchronous composite low grade b-cell lymphomas involving the same site is rare and few cases have been reported. we describe three cases of composite lymphoma. material and methods histological, immunohistochemical and molecular study. results the three neoplasms showed different morphological and phenotypic areas: case . years-old female with large splenomegaly. the splenectomy showed a blastoid mantle cell lymphoma (mcl) with nodular pattern, positive to cd , cd a, cd , cd and cyclin d , and a splenic marginal zone b-cell lymphoma cd , cd a and igd. the mcl progressed, with liver and bone marrow involvement, treatment failure and death months after diagnosis. case . years-old female with cervical and inguinal lymphadenopathy. a nodular mcl cd , cd and cyclin-d positive, p negative, was observed. the internodular areas demonstrated a small lymphocytic lymphoma (sll) expressing cd , cd a, cd , cd , and p . progression to peripheral blood involvement of sll, and gastric and nodal relapses of mcl two and four years after diagnosis. case . years-old female with nodal follicular lymphoma expressing cd , cd , bcl- , igd-kappa restriction. the internodular areas showed a lymphocytic lymphoma with plasmacelular differentiation, cd , cd a, cd , igd, and bcl- positive. igh showed a biallelic arrangement, and bcl- /jh rearrangement was obtained, suggesting the presence of two different clones. alive with disease after first chemotherapy cycle. conclusion morphology and immunophenotypic profiles may allow the identification of each component in composite lymphoma. molecular studies can recognize different clones originating the neoplastic compartments. introduction aitl accounts for about % of peripheral t-cell lymphomas (ptcl), presents with advanced stages at diagnosis and follows aggressive clinical course. in - % of cases oligoclonal or monoclonal b-cell populations are detected, usually associated with ebv infection. we describe three cases of aitl with clonal b-cell lymphoma non-related to ebv infection. histological, immunophenotypical, including ebv (lmp- , ebers), and molecular study. results three cases of aitl in three female, , and yearold patients were diagnosed. all of them showed atypical lymphocytes with aberrant t-cell phenotype (cd /cd + with loss of cd ), associated with vascular and dendritic proliferation, the latter showing t-cells cd +. in addition, a population of b-cells with plasmacelular differentiation and immunoglobulin light chain restriction ( kappa/ lambda) was identified. ebv was not demonstrated in these populations. tcrgamma and igh clonality was observed in two cases. the first patient presented isolated nodal aitl involvement, and she is in complete remission after chemotherapy six months after diagnosis. the second patient presented nodal aitl and developed skin involvement two months after the diagnosis. the third patient presented with an atypical ganglionar t-cell proliferation, developing cutaneous ptcl with associated clonal b-cell proliferation one year later and ganglionar ptcl with ailt features two years after the first biopsy. the three biopsies showed identical tcrgamma gene rearrangements in this case. conclusion aitl can show different morphological patterns. alternative mechanisms non-related to ebv infection may be involved in the development of b-cell lymphomas in aitl. introduction histopathological confirmation of clinical suspicion of sarcoidosis is based on finding of non-caseating granulomas in biopsy material, usually in prescalene lymph nodes. reactive sinus histiocytosis seen in relation to various inflammatory and noninflammatory diseases can mimic pregranulomatous phase of sarcoidosis. differentiation of sinus histiocytosis based on histopathological features alone can be impossible. aim of this study is immunohistochemical determination of lymph node cellular response in granulomatous sarcoidosis, pregranulomatous phase of sarcoidosis and reactive sinus histiocytosis using panel of antibodies. materials and methods patient groups under study, each contained patients include those with clinical and histopathological picture of sarcoidosis; the group of patients with clinical picture of sarcoidosis and histologically detectable sinus histiocytosis without granuloma formation and finally, the group free of clinical suspicion of sarcoidosis with 'reactive' sinus histiocytois in lymph nodes. lymph node biopsy tissue processed routinely and stained immunohistochemically using triple-layer apaap protocol with purified antibodies sp and sp from mycobacterium tuberculosis and monoclonal antibodies cd , cd , cd , cd , cd . intensity of immunostaining assessed semiquantitatively by two independent observers using the scale from to + results an increased cd :cd ratio, moderate increase of immunopositivity of cd and slight decrease of immunopositivity of cd , cd , and sp noted in psh when compared with rsh. the most notable difference between studied groups is the intensity of immunostaining with sp and cd antibodies. conclusion both immunostainings were more intense in lymph nodes with sarcoid granulomas and pregranulomatous sinus histiocytosis and can be used in differentiation of these lesions. haemophagocytic syndrome followed by a large b-cell lymphoma showing only bone marrow involvement: case report haemophagocytic syndrome (hps) is a rare disease characterized by generalized histiocytic proliferation showing phagocytosis of hematopoietic cells. it occurs mainly in adults associated either with an acute viral infection, or with non-hodgkin's lymphomas. the latter are usually t-or nk-cell lymphomas. the present case concerns a patient with hps followed by a large b-cell lymphoma showing only bone marrow involvement. this constellation is extremely rare in western countries and has been reported mainly in asia. the patient was a year old male admitted for persistent fever. peripheral blood examination showed pancytopenia. bone marrow biopsy showed haemophagocytosis by proliferating macrophages often situated into dilated marrow sinusoids. no viral infection was detected. the patient was treated with haemopoietic growth factors and showed improvement. after months his blood cell count began to fall again and a second bone marrow biopsy showed, apart from haemophagocytosis, a focal, mainly intrasinusoidal, infiltration by large b-cell lymphoma cells immunopositive for cd and cd ra. epstein-barr virus rna was not detected. dissemination of lymphoma to other sites was not apparent. the patient was treated with cycles of chop and mab-thera and showed remission. some authors believe that these rare cases represent a peculiar "asian" variant of intravascular lymphoma characterized by hps and early involvement of bone marrow without dissemination to other organs. this variant has a poor prognosis and might constitute a distinct biological and clinical disease entity which merits separate consideration because of the problems posed in the initial diagnosis and therapeutic approaches. introduction mycosis fungoides is a mature t-cell lymphoma presenting in the skin and characterised by epidermal and dermal infiltration of small to medium size t-cell with cerebriform nuclei. it is a rare disease wich accounts for no more than , % of all non hodgkin lymphomas. case report a year-old man was refered to the clinic of hematology because of the nodular red dermal tumours on the trunk. these lesions persisted for one year. the blood and bone marrow had not been involved. lymph nodes were normal. the patient had the so called d' emblee lesions, presenting with skin tumours to cm of diameter. the initial diagnostic lesions were rare limited patches and plaques. extracutaneous dissemination did not occur. formaldehyde fixed and paraffin embedded histological sections, taken from surgically resected skin tumours, were stained with he, giemsa, trichrome and immunohistochemical stains. morphology: epidermotropic infiltrates consisted of medium size cells with cerebriform nuclei. a minority of larger cells with similar nuceli and with many mitotic figures also were present. so called pautrier microabscesses consisted of aggregates of cerebriform cells in the epidermis (highly caracteristic for mycosis fungoides) were numerous. in the dermis, infiltrates were band like and diffuse, associated with inflammatory infiltrate consisting of small lymphocytes and eosinophils. conclusions in spite of the presence of bad prognostic factors (very old patients, extent of disease and tumorous form) he had a very indolent course of the disease. the role of the observed inflammatory small lymphocytes and eosinophils is probably very important for the excellent prognosis of mycosis fungoides in this patient. low expression of p protein combined with altered p and rb/p expression status is associated with increased expression of cyclin a and cyclin b in diffuse large b-cell lymphomas introduction reduced p expression combined with alterations in rb or p status, may have cooperative effects resulting in uncontrolled tumor cell proliferation and aggressive malignancies in experimental mouse models and in humans. the aim of this study was to investigate p expression in relation to a) the expression status of p , rb, p , and b) proliferation profile (ki , cyclin a, cyclin b ). expression of cell cycle regulating proteins was investigated immunohistichemically, in cases of de novo diffuse large b-cell lymphomas (dlbcl). results p expression was low/null in large tumor cells in / and intermediate/high in / cases. increased expression of p was observed in / cases. decreased expression of rb/p was found in / and / cases, respectively. low/null p expression was correlated to increased p expression (p= . ). there was a correlation between increased p and decreased rb/p expression levels (p= . ). these findings suggest a tendency for concurrent alterations of cell cycle regulators p , p and rb/p in dlbcl, which might result in impaired tumor growth control. indeed, combined p /p /rb/p expression status analysis, with respect to proliferation profile showed that: ) three alterations in the combined p /p /rb/p status were correlated with increased expression of cyclin b (p= . ) and ) two or three alterations were correlated with increased expression of cyclin a (p= . ). conclusions these findings suggest combined impairment of a complex cell-cycle control network involving the cdk inhibitor p , as well as p and rb pathways, which exerts a cooperative effect resulting in enhanced tumor cell proliferation. introduction the aim of this study was to examine a) cyclins d , e and d expression in relation to other proliferative features (ki , cyclin a and cyclin b ), apoptosis status and p , rb, p , p expression and b) whether distinct clusters of proliferation and apoptosis could be identified in dlbcl. materials and method immunohistochemical expression of cell cycle regulating proteins were studed in seventy-nine cases of de novo diffuse large b-cell lymphomas (dlbcl). results overexpression of cyclins d , e was found in / ( %) and / ( %) cases, respectively, whereas cyclin d was not detected. in / cases, overexpression of cyclins d and e was mutually exclusive, proposing different underlying pathways that induce deregulated expression of these cyclins. in / cases, cyclin d overexpression was equally exclusive with rb/p aberrant expression status, supporting an oncogenic role for cyclin d and suggesting that pathogenetic effect of cyclin d overexpression occurs through perturbation of rb pathway. combined alterations of p and rb/p /cyclin d expression profile were significantly correlated to higher mean values of cyclins a (p= . ) and b (p= . ) indicating that concurrent impairment of p and rb pathways induces increased tumor cell proliferation. cluster analysis of apoptosis and proliferation status permitted separation of dlbcl into distinct groups with low and high apoptotic activity, as well as into groups with low, intermediate and high proliferative activity. conclusions in the present study, identification of separate groups with respect to proliferation and apoptosis profile indicates that groups with discrete cellular kinetic properties can be defined in the dlbcl population. immunotopographical distribution of protein networks involved in cell-cycle and/or apoptosis regulation is useful in thymic histophysiology understanding. the aim of this study was to examine a) p and rb growth control pathways, b) proliferation, c) apoptosis in relation with age. materials and methods p , p , rb, p , ki , p , bcl , bax, bak, cyclin d , cyclin a, and cyclin b expression, and apoptotic index (ai) were investigated in adult, adolescent, infant and newborn normal thymuses, with immunohistochemistry. results and conclusions rb, ki , cyclins a and b expression was elevated in cortex with a decreased tendency toward medulla. apoptotic cells were mainly detected in cortex/corticomedullary junction, but rarely in hassall's corpuscles. mean values of ki and cyclins a, b immunoreactivity were . %, . %, . % (newborns), . %, . %, . % (infants), . %, . %, . % (adolescents), . %, . %, . % (adults), respectively. mean values of ai were . %, . %, . %, . %, respectively. most of hassall's corpuscles were p -positive. p expression paralleled aging, suggesting a role of p in cellular senescence. p was detectable within medulla, but not in subcapsular thymocytes expressions of ki , cyclin a and cyclin b were inversely related to p . p and p showed primarily subcapsular cortical epithelial cell localization. our findings suggest: a) thymocyte apoptosis is mainly p -independent, b) p function as negative cell cycle regulator seems redundant to other negative regulators (p / ), and c) p expression is chiefly p -independent. hassall's corpuscles were p -positive. bcl was principally detected in medullary thymocytes. expression of bax/bak was widely distributed, suggesting an advanced role in thymic apoptosis, compared to bcl . paraffin-embedded bone marrow biopsies of patients were examined by a standard panel approach and evaluated semiquantitativelly. the course of the disease was recognized by evaluation of instrumentalized questionnaires. results a/ mds with less than % of blasts (n= ) /either of myelo-(n= ), megaloblastic (n= ) or bilinear profile (n= )/ -aml evolution appeared in % of patients, b/ mds with more than % of myeloblasts (n= ) -aml diagnosis was established in % of cases. conclusion although the therapeutical aml managment requires 'clinical verification', the identification of the mds blastic proliferation higher than % is of a great clinical importance. however, the clinical manifestation of aml may be influenced by a delay of the increase of blasts in the peripheral blood. introduction there is increasing evidence that bcl and cd expression may be related to apoptosis and cell cycle progression. materials and methods to examine immunohistochemically the expression profile of bcl and cd in cases of de novo diffuse large b-cell lymphomas, in relation to a) the apoptotic index (ai) b) the proliferation-associated proteins ki , cyclins a and b c) the expression of bcl , p , rb, p , p . results and conclusions expression of bcl , cd and bcl was detected in / ( %), / ( %) and / ( %) cases, respectively. the bcl /cd patterns were as follows: bcl +/ cd + ( cases, %), bcl +/cd -( cases, %), bcl -/cd -( cases, %) and bcl -/cd + ( cases, %). significantly positive correlations were found between bcl /ki (r= . , p= . ), bcl /cyclin a (r= . , p= . ), bcl /ai (r= . , p= . ), cd /ki (r= . , p= . ) and cd /ai (r= . , p= . ). high expression of bcl was correlated with null/low bcl expression (hi-square-test, p= . ). these findings indicate that increased expression of bcl and cd is associated with increased apoptosis and proliferation in diffuse large b-cell lymphomas. association between increased bcl expression and enhanced apoptosis might be due to null/low bcl immunoreactivity, since previous in vitro data revealed that bcl overexpression induces apoptosis accompanied by bcl and bcl-xl downregulation. significant correlation was found between increased ai and bcl +/cd + pattern (t-test: p= . , mann-whitney test: p= . ). this result combined with positive correlation between ai and bcl and cd immunostaining may be in accordance to previous studies showing that expression of these proteins has favorable effects on the clinical outcome of these neoplasms. primary non-hodgkin lymphomas of bone: morphologycal and immunohistocehmical aspects introduction primary non-hodgkin lymphoma (nhl) of bone (plb) is a rare clinic-pathologic entity that accounts for approximately % of all malignant bone tumors and - % of all extranodal nhl. we analyzed morphological and immunohistocemical aspects of plb presented as solitary localized bone tumor. methods paraffin embedded biopsy specimens of all patients were diagnosed by the histomorphology and by immunohistochemistry using a wide panel of monoclonal antibodies (ema, lca, nse, tdt, cd , hla-dr, cd a, cd , cd , surface and cytoplasmic ig, cd , cd , cd , cd , cd , cd , cd , bcl , bcl and ki- ). the median age of our pts. was (from to ) with slight female predominance. various sites were involved: tumors were in tubular long bones, in pelvic bones and in spine with infiltration of soft tissue and without bone marrow involvement. all cases were classified as diffuse large b-cell lymphoma (dlbcl) according to morphology and immunophenotype. patohistological analyses showed markedly polymorphous cellular infiltrate with large cells with complex nuclear contours and multilobated nuclei, together with centroblasts-like cells and immunoblasts. all tumors were associated with some degree of fibrosis. immunologic studies showed strong expression of pan-b markers and hla-dr, weak expression of cd and cig (mostly igm) in half of tumors. bcl was positive in all cases as well as bcl . the plasmacytic differentiation was observed in / of cases. the proliferative fraction was high, > % cells in all patients. conclusion our analysis revealed that all plb were of dlbcl type with characteristic morphology and immunophenotype. further study will show the outcome of patients with plb, as well as the validity of various prognostic parameters. peripheral t-cell lymphoma with perifollicular growth pattern expressing cd inflammatory cells. ptcls with nodular or perifollicular growth patterns are very rare. only cases have been reported in the literature. we report an unusual case of ptcl showing perifollicular growth pattern with expression of cd and bcl- . the patient was a -year-old male who presented with a generalized lymphadenopathy, fever, and weight loss. hepatosplenomegaly and polyclonal gammopathy were not identified. cervical lymph node biopsy was performed. the histology showed nodular effacement of nodal architecture with proliferation of atypical lymphoid cells which mainly infiltrate perifollicular area. interfollicular area also showed proliferation of atypical lymphoid cells and high endothelial venules with a few immunoblasts and eosinophils. neoplastic cells were medium-sized, with irregular nuclei and clear cytoplasm. immunohistochemically, the neoplastic cells were cd +cd +cd +cd -cd +cd -cd -bcl- +bcl- +, demonstrating helper/inducer t-cell origin. the cd was strongly expressed mainly in the perifollicular tumor cells, whereas the normal germinal center (gc) cells showed down-regulation of cd . the distribution of cd + perifollicular tumor cells was closely related with cd + follicular dentritic cells (fdcs), suggesting cd up-regulation might be induced by the proliferating fdcs. tcr-gamma gene rearrangement using pcr-sscp was monoclonal. this case could be classified as ptcl, unspecified, and should be discriminated from other lymphomas, including marginal zone b-cell lymphoma and follicular lymphoma. further studies are necessary to clarify the distinction of this tumor from angioimmunoblastic t-cell lymphoma. introduction cd + anaplastic large cell lymphomas (alcls) frequently involve extranodal sites, mainly skin and gastrointestinal tract, but primary bone alcls are exceedingly rare. the aim of this study was to detail the morphofunctional features and outcome of cases of primary bone cd + alcls. materials and methods all the patients (m:f : , median age years, range: - ), but (with simultaneous occurring skin lesions) presented with bone confined lymphoma (osteolytic lesions of jaw, rib, sternum, iliac and pelvic bones). biopsies from all cases were analysed. results and conclusion according to the who lymphoma classification, all cases were cd + alcls ( / common type, / small cell predominant variant). lymphoma population showed a t ( / ) or "null" ( / ) phenotype, expressed cytotoxic associated molecules (tia , granzyme b and/or perforin); cd /alk was positive in / cases, whereas / expressed cd . tcr-gamma clonal rearrangement was detected in / cases. all patients received polychemotherapy; at last follow up they all are alive, in complete remission (mean: months, range: - ). in our cases, the morphofunctional features of the lymphoma population were similar to their nodal counterparts, including cd expression in the younger patients. on such bases, the hypothesis must be considered that primary bone cd + alcls represent the first extranodal site of presentation of a systemic disease. in contrast with similar reported series, our patients run a favourable prognosis and in the single positive case, cd expression does not negatively influence outcome. however, larger series of similar cases must be analysed before definitive conclusions can be drawn. introduction follicular dendritic cells (fdc) form a meshwork in the lymphoid follicle and take part in the regulation of the germinal centre reactions. fdc also interact with the neoplastic lymphoid proliferations. in b-cell chronic lymphocytic leukaemia/small lymphocytic lymphoma (b-cll/sll), fdc structures are reduced. the aim of this study was to evaluate the presence and patterns of follicular dendritic cells b-cll/sll using several anti-fdc antibodies. materials and methods formalin-fixed paraffin-embedded lymph nodes diagnosed as b-cll/sll according to the who criteria were studied using cd , cd , and cna. antibodies after microwave oven antigen retrieval results in cases ( %) fdc meshworks were identified in none of the reactions. in specimens ( %), fdc meshworks were exceptionally rare as follicle remnants at the lymph node periphery or as small irregular clusters not reminding of the normal follicle meshwork. in cases ( %) fdc formed either frequent small irregular clusters, or rings at the follicle periphery, or regular follicular meshworks. reticular structures in a given case were most frequent in cna. stain, scarcer in anti-cd , and anti-cd stains. the majority of cases showing fdc was obtained by cna. ( cases), cd ( cases) and cd ( cases). conclusion fdc structures were absent or scarce in up to % of b-cll/sll cases which can be used as an auxilliary diagnostic criterion. the positive cases showed small irregular clusters, rings at the follicle periphery, or normal follicular meshworks. best results were obtained with cna. antibody. this may be due to better antigen preservation or to non-specifity. introduction myelodysplastic syndromes (mds) are clonal haematopoietic stem diseases characterised by dysplasia and ineffective haematopoiesis. the dysplasia may be often associated with an increased number of myeloblasts in peripheral blood and bone marrow, which represents an important prognostic factor. immunohistochemical detection of blast cells in bone marrow histological sections using cd antigen and its correlation with smear blast count and mds classification are not well established. we report a quantitative study of cd blast cells in mds and acute myeloid leukaemia with multilineage dysplasia (amlmd), and analyse the relationship with both cytologic smear and clinical diagnosis. we have studied bone marrow biopsies diagnosed of mds ( with refractory cytopenia with multilineage dysplasia (rcmd), with refractory anemia with excess blast (raeb) type i and with raeb type ii) and with amlmd. we added controls and calculated the cd blast cells mean per high power field (hpf). cytologic smears were reviewed. results the results were: controls showed - blasts/ hpf, rcmd - / hpf, raeb i - / hpf, raeb ii - / hpf and amlmd - / hpf. conclusions -cd + blast cells density is a good parameter in the histological diagnosis of mds and has a close correlation (p< , ) with smear count results -cd does not distinguish between rcmd and raeb i. -more than blast cells/ hpf raise the diagnosis of raeb ii. more than blast cells/ hpf raise the diagnosis of amlmd. the extrapolation of these features to "dry tap" aspiration remains to be established. inroduction bone marrow angiogenesis has been recently implicated in the pathophysiology and course of various haematological malignancies. little is known, however, about the significance of this phenomenon in hairy cell leukaemia (hcl). methods we evaluated various morphometric characteristics of microvessels, highlighted by means of anti-cd immunohistochemistry, in the bone marrow of patients with typical hcl, before and after treatment with interferon-α. overall bone marrows from hcl patients and twenty control bone marrows were examined. microvessel density (mvd) and several size-and shape-related parameters were quantitated in the region of most intense vascularization using image analysis. results mvd, size-related parameters and the percentage of branching microvessels were higher in hcl than in controls and in partial/non-responders in comparison to complete responders. acvievement of complete response was accompanied by smaller calibre microvessels. interferon-α induced a decrease in mvd and branching values in cases with diffuse marrow involvement. in univariate analysis, progression-free survival was adversely affected by mvd and branching. multivariate analysis indicated that factors related to the size of the microvessels and mvd/ branching independently affected progression-free survival. mvd/ branching was also related to the likelihood of complete response. conclusions our data suggest that the generation of bone marrow microvessels connotes an increased risk for progression and interferon-α treatment failure in hcl. furthermore the prognostic significance of angiogenesis requires the concomitant assessment of mvd, the size of microvessels and the complexity of the microvascular network. key words: hairy cell leukaemia, bone marrow, angiogenesis, morphometry introduction inflammatory pseudotumors are uncommon and enigmatic benign lesions which may develop at several anatomical sites and recent studies demonstrated the frequent presence of epstein-barr virus (ebv) in them. the occurence of inflammatory pseudotumor in the spleen is rare. a case of inflammatory pseudotumor of the spleen is presented and its relationship to epstein-barr virus (ebv) is examined. material and method the case was a -year-old woman in whom a solitary splenic mass was discovered as part of the workup of idiopathic thrombocytopenic purpura. splenectomy was performed. results grossly, the removed spleen, weighing g, contained a tan-white, circumscribed mass, measuring . x x cm. microscopically, the mass was composed of a heterogeneous cellular population of acute and chronic inflammatory cells. plasma cells predominated, but lymphocytes, neutrophils, eosinophils, interlacing fibroblast-like spindle cells and rare multinuclear giant cells were found. amorphous, acidophilic material was present focally with the plasma cell proliferation. plasma cells presented light chains polyclonality. lymphoid cells were mature and mainly of t-lineage. spindle cells exhibited histiocyte phenotype (cd +++). immunohistochemical stain for ebv was negative. conclusions inflammatory pseudotumor of the spleen is a benign tumorous lesion that has been described in only a few cases in the world literature (< cases). its etiology and pathogenesis is unknown and association ebv is not always present. recognition of this rare entity is important, as the clinical manifestations and imaging features could be indistinguishable from a lymphoproliferative disorder or another malignancy of the spleen. introduction the diagnosis of nodal non-hodgkin's lymphoma (nhl) in montenegro has been done only in clinical center of montenegro -incidence of diagnosed nhl can be considered as the incidence in epidemiological sense.the use of monoclonal antibodies since , has improved our ability to diagnose and to treat nhl. aims: by revision of immunohistologically diagnosed cases of nhl between - , we wanted to estimate (a) the frequency of particular clinico-pathological types of nhl, (b) hypothesis about geo-socio-economic influences on patterns of nhl, and (c) the influence of immunophenotypisation of nhl on speciffic chemo-and immunotherapy. materials and methods nhls were diagnosed with the use of he, giemsa, mgp, pas, gomori, lca, bcl , tdt, cd , cd , cd , cd a, cd , cd , and ema stained slides and classified according to the working formulation criteria. results between ' and ' we diagnosed nhl. low, intermediate and high grade malignancy comprised , %, , % and , % respectively. the highest incidence was estimated in : , / . the nhls of fcc origin constitute , % of all our nhls. in % of patients ( ) low grade malignant nhls were highly cd +. nine patients recieved mabthera+chop/or +cop therapy during the last three years.all patients have been in remision. conclusion the estimated pattern of nhl -the most frequent nhl derivated from fcc-is comparable with geographic and socio-economic description of montenegro as ''underdeveloped, ecological, farming european country''.the high frequency of cd + and initial therapeutical results indicate future prospective patho-clinical studies of this issue. i oehri, m baumann, g cathomas kantonales institute for pathology, liestal, switzerland background the multicentric castleman's disease (mcd) is a lymphoproliferative disease with increased risk for non-hodgkin lymphoma. in aids patients, mcd is usually associated with human herpesvirus (hhv ), the infectious agent of kaposi's sarcoma but in contrast to kaposi's sarcoma, hhv infected cells in mcd show evidence of lytic viral infection. similar to other immunosuppression-associated lymphoproliferative diseases it has been suggested that anti-cd antibodies may be useful in the therapy of these lesions. aim of the present study was the analysis of cd expression in hhv associated mcd and their association with lytic viral infection. material and methods formalin fixed, paraffin embeded lymph nodes were analyzed by a double immunofluorescence assay using antibodies against viral proteins and cd . hhv proteins which were analyzed include the latent viral protein lana (orf ) and the viral interleukin (vil- ) and the orf , both associated with lytic hhv infection. results eleven lymph nodes of seven hiv-infected patients with mcd were analyzed. in the mantle zone of the lymphoid follicle, , %, , % and % of cells show positivity for lana, vil- and orf , respectively (p < , ). by double immunofluorescence, % of hhv infected cells showed co-expression of lana/vil- and lana/vorf and % showed co-expression of lana/vil- , respectively. in addition, there was statistical significant correlation between vil- and lana as well as vil- and vorf (p < , ). of the hhv infected cells, , % showed cd expression and no difference between the three viral proteins analyzed were observed ( , %, , %, , % of lana, vil- and vorf , respectively). conclusion the present data support the concept that hhv runs, in contrast to kaposi's sarcoma, through a lytic viral infection in lymph nodes of patients with mcd. however, only a small subset of infected cells show co-expression of cd , independent of the state of viral replication. this small number of infected cells expressing cd indicate little effect of anti-cd therapy. based on the evidence of lytic hhv infection, however, anti-viral drugs inhibiting hhv replication may be more efficient. anaplastic large cell lymphoma -pathological and immunohistochemicals aspects the who classification of nonhodgkin lymphoma (nhl) included also the cathegory of t and null anaplastic large-cell lymphoma (alcl). alcl is a very rare nhl (about %), with the systemic-aggressive variant, and the cutaneous variant. the presence of the t( ; ) and the formation of alk protein identified one subgroup of alcl with a good prognosis. the aim of our study is to analyze the histopathological and immunohistochemical characters of the alcl diagnosed in our department in the last years. cases of t and null alcl (m/f= / , more half of them younger of years) were clinical and immunohistochemical analyzed (by abc indirect methods, with a large panel of monoclonal antibodies). the pattern of lymph node infiltration was diffuse ( cases), parafollicular ( cases) and only with sinus infiltration - case. the pathological subtypes were common ( cases), lymphohistiocytic ( cases), small cell variant ( case), sarcomatous ( case), eosinophils-rich ( case) and neutrophil-rich ( case). immunohistochemically, the malignant cells presented with t-fenotype ( cases) and null ( cases); case (m, y) presents histiocyte markers (cd +cd +ema+alk+). all cases of alcl were cd +, but only cases were ema+ ( cases were cd ro+cd +alk-ema-). / of cases were alk+ ( -t and -null), with a nuclear and a membranous pattern. cases were bnh + ( cases alk+). only case presented cd expression(cd +ema+alk+bnh +). of the cases with t fenotype expressed cd markers (both were cd +cd -). the pcna and ki proliferation markers were positive in a very large percent of the malignant cells (between and %). numerous reactive t-lymphocytes presented a helper fenotype (cd >cd ), and leu- + cells were very rare. the null and t-cell alcl pose numerous problems of differential diagnosis and immunohistochemicals methods are very useful for diagnosis and prognosis of these nhl. myeloid sarcoma - cases analysis f vasilescu , c dobrea , , e niculescu-mizil , a colita , l caban , r stoia , d ostroveanu , a iofciulescu , d terzea , c iosif 'v babes' national institute, bucharest, romania dpt of haematology, 'fundeni' clinical institute, bucharest, romania myeloid (granulocytic) sarcoma (ms) -also known as "chloroma", is a localized tumor of primitive myeloid cells, that can present before, simultaneously with, or after the diagnosis of acute myeloid leukaemia (aml). rarely, ms may represent the first sign of blastic transformation of myelodysplastic syndrome (mds) or chronic myeloproliferative disorders (cmpd). the aim of this study is to present our experience in the diagnosis of ms. in the last years, we diagnosed cases of ms (m/f= / , between and year-old, with , years median). a large panel of monoclonal antibodies (lymphoid and myeloid cell markers) was used for the diagnosis of ms and for the differential diagnosis with nonhodgkin lymphomas (nhl). the tumor localizations were: lymph nodes ( cases, including a bulky inguinal tumor), lymph node and skin ( case), paravertebral ( cases), testis ( case). in our cases, patients presented with ms before the aml diagnosis (f, y, paravertebral ms, months before aml-m , and m, y, years after nhl diagnosis, presented with an inguinal lymph node ms, months before aml-m ). in cases, ms was diagnosed simultaneously with aml (aml-m ; aml-m ; aml-m ), and in cases after aml (m, y, ms with testis localization, years after aml-m ; f, y, lymph node and skin ms, years afteraml-m ). in one case (f, y), years after successful treatment of an acute lymphoblastic leukaemia (all), the patient presented with a lymph node ms, considered as a blastic transformation of a chronic myelo-monocytic leukaemia (cmml). the diagnosis of ms may be very difficult if this tumor precedes the myeloid proliferative disorders, and the differential diagnosis with nhl may pose a lot of problems. introduction b-pll is an infrequent disease, considered by the who lymphoma classification as an aggressive condition and characterized by the presence of b symptoms, massive splenomegaly without significant lymphadenopaty and peripheral lymphocytosis with more than % prolymphocytes. herein we report cases of b-pll to show the wide clinical and genotypic spectrum of this disease. case reports patient . a -year old male with a history of petechiae and ecchymoses of year evolution. a cbc showed significant lymphocytosis with > % prolymphocytes. physical examination revealed an enlarged cervical lymph node and splenomegaly. lymph node biopsy was diagnosed as infiltration by b-pll (cd +, cd a+, igm+, cd -, cd -, cd -and cyclin d -). a bone marrow biopsy showed infiltration by b-pll. cytogenetic analysis of the bone marrow showed a burkitt's variant translocation t( ; ) (p ;q ) molecular analysis of the lymph node showed igh rearrangement and germline bcl- and bcl- by pcr. he was treated with four cycles of fludarabine ( mg/m ) and rituximab ( mg/m ) and is in clinical and cytogenetic remission at year of follow-up. patient . a -year old female with a month history of multiple cervical lymph node enlargement and fever. physical examination was normal and she had no lymphocytosis on the cbc. lymph node biopsy showed infiltration by a non-hodgkin's lymphoma with a prolymphocytic morphology (cd +, cd a+, igm+, cd -, cd -, cd -and cyclin d -). bone marrow biopsy showed focal infiltration by b-pll. cytogenetic analysis of the bone marrow revealed a normal female cariotype and normal fish patterns were found for the following probes: chormosome centromere, q (c-myc), chromosome centromere, q (d s ), q . (igh) and p . (p ). she was initially treated with chop and then switched to fludarabine having achieved a good clinical response after one cycle each. discussion. the association between b-pll and translocation t( ; ) (p ;q ) has been reported in very few cases, however direct involvement of c-myc alterations affecting specifically q has been proposed as an important factor in the pathogenesis of b-pll. in patient , the observed genetic alterations could be of significance to define the genetic spectrum of b-pll. case corresponds to a b-cell prolymphocytic lymphoproliferation with isolated lymph node involvement and no leukemic component. to our knowledge this presentation has not been reported previously and it is not considered in who classification. morphological and immunohistochemical characteristics of this case are fully compatible with a b-pll. even though it has been considered that some cases of b-pll could in fact represent a morphological variant of mantle cell lymphoma, in this case the negativity for cd and cyclin d partially rule out this possibility. bcell prolymphocytic lymphoproliferations constitute an heterogeneous group of disorders in which c-myc rearrengement can be present. in this group of neoplasia therefore, the nodal involvement can be either predominant or the only clinical manifestation, thus we propose to consider the term b-cell prolymphocytic lymphoma/ leukemia instead of b-pll to denote these disorders. background alcl is a particular type of t-cell non-hodgkin's lymphoma originally characterized by a peculiar morphology and the consistent expression of cd . this entity is frequently associated with the translocation t( ; ) (p ;q ) or some of its variants resulting in the overexpression of the chimeric protein npm/alk (alk+), even though in the current classification alk-negative cases are also considered within this group. ebv is classically and constantly associated with certain specific types of lymphoma but its association with alcl is not considered a characteristic feature of this disease. the aim of this study was to investigate the frequency of the alcl-ebv association in a population of adult mexican patients. results eighteen patients with a morphological and immunohistochemical diagnosis of alcl with nodal involvement were included. immunohistochemical and ish analysis was performed for cd , cd , cd ro, cd rb, cd , cd , cd , ema, lmp- , alk.and eber . mean age was years and the male-tofemale ratio was . : . immunohistochemistry and in situ hybridization results are summarized in the next table. all the so considered ebv positive cases expressed lmp- and eber . conclusion in contrast to the information available in the literature we found a high incidence of the alcl-ebv association / ( %). the high incidence of alk-/ebv+ cases / ( %), with morphological and immunophenotypical characteristics of alcl gives rise to the possibility that this is a different subset of t-cell lymphomas with anaplastic morphology in which ebv plays a pivotal roll in the oncogenesis and evolution. these disorders have been considered so far to be alcl /alklymphomas. the high incidence of ebv infection in mexico leads to the development of peculiar lymphoid proliferations as well as associations (ebv/alcl) different from those considered to be frequent in world literature. lazzi s , bellan c , de falco g , schuerfeld k , micheli p , bartolommei s , pileri s cdk is a member of the cdc -like family of kinases. its cyclin partners are members of the family of cyclin t (t , t a and t b) and cyclin k. cdk /cyclin t complex is very important in the differentiation program of several cell types, controlling specific differentiative pathways. limited data are available regarding the expression of cdk /cyclin t in haematopoietic and lymphoid tissues. the aim of this paper is to analyze the expression of cdk /cyclin t complex in lymphoid tissue, in order to assess its role in b and t cell differentiation and lymphomagenesis. in reactive lymph nodes cdk /cyclin t expression was found by immunohistochemistry in germinal center and in b and t cell blasts in interfollicular areas, suggesting a role for cdk in the activation and differentiation of b and t cells. the staining of cdk and cy-clint complex in malignant lymphomas seems to reflect their normal counterpart, as it is highly expressed in lymphomas derived from germinal center cells, such as follicular lymphomas and classical hodgkin's lymphomas and from activated t cells, (i.e. anaplastic large cell lymphomas). diffuse large b-cell and burkitt's lymphomas showed a wide range of values. no expression of cdk and cyclin t was detected in marginal zone and mantle cells lymphomas. however, at rna level we found an imbalance in cdk /cyclin t ratio in follicular lymphoma and diffuse large b cell lymphomas with germinal center phenotype, and in the cell lines of classical hodgkin's lymphomas, burkitt's lymphomas and anaplastic large cell lymphoma, in comparison with reactive lymph nodes. here we report cdk /cyclin t complex deregulation in neoplastic conditions for the first time, representing a step towards better understanding of through which molecular mechanism cdk /cyclin t complex acts on the activation and differentiation program of lymphoid cells and the role of its deregulation in malignant transformation. introduction hairy cell leukaemia (hcl) is a chronic lymphoproliferative disease characterized by the presence of neoplastic b cells with hairy cytoplasmic projections in spleen, bone marrow and peripheral blood. complete or near-complete remission can be achieved by recent chemotherapy protocols. these treatment methods are quite different from the chemotherapy protocols used for the treatment of other lymphoproliferative diseases. an absolute and correct diagnosis, i.e. detection of hcl cells by specific methods, is necessary for the achievement of complete or near-complete remission by special chemotherapy programs. detecting intracytoplasmic tartrate resistant acid phosphatase (trap) enzyme activity in the peripheral blood and bone marrow aspirates is a classical, simple, sensitive and quite specific method for the detection of hcl cells and in the diagnosis of hcl. however, this method is insufficient in some cases. for example, the most of the hcl patients are leukopenic and there are a few number of neoplastic cells in their peripheric blood. an adequate number of neoplastic cells cannot be obtained by bone marrow aspiration because of the increase in reticulin fibbers of the bone marrow. in such cases, bone marrow trephine biopsy is almost the only and the most important method in diagnosis and follow-up of this disease. specific detection of trap enzyme activity in bone marrow trephine biopsy materials is not possible because of the effect of tissue fixation and processing on these materials. in conclusion, a new method for detecting trap enzyme activity in bone marrow trephine biopsy materials and for conforming the diagnosis of hcl is needed. a recently developed anti-trap antibody has been started to be used as an immunohistechemical marker in the diagnosis of hcl. the aim of this study was to investigate the specificity and the sensitivity of anti-trap antibody immunoreactivity in bone marrow trephine biopsy materials in the diagnosis of hcl. material and methods we divided the cases into two groups; a study and control group. the study group included the cases diagnosed as hcl by clinical, haematological and histopathological investigations. the cases in the control group were the cases characterised by non-hcl neoplastic or reactive lymphoid infiltrations, which raised the suspicion of hcl in the differentia l diagnosis. immunohistochemical staining techniques were applied to paraffin block sections of biopsy materials with anti-trap antibody. results and conclusion we found that there is a statistically highly significant difference between trap immunoreactivities of the study and the control groups, and hcl can be diagnosed by trap immunoreactivity in the bone marrow trephine biopsy materials with the specificity of , % and with the sensitivity of %. cathepsin l participation in collagen degradation during the recovery from experimental hepatic fibrosis v ryvnyak introduction cathepsin l is one of the most important lysosomale cysteine proteinase, indispensable for proteolitic processes. the enzyme is capable to degrade the main components of the intercellular matrix. aims: . to determine the participation of cathepsin l in collagen degradation in liver and to reveal the intracellular and/or extracellular localization of the enzyme activity. . to determine which cells of fibrotic liver are sources of cathepsin l. methods biochemically cathepsin l activity and hidroxyproline level was determined in normal, ccl -induced fibrotic rat liver and at , , , , days after discontinuation of treatment. the activity of cathepsin l in fibrotic rat liver at the th and st days was investigated electron-histochemically. cbz-phe-arg- mbna (bachem) served as the substratum. results biochemically in fibrotic liver an increase of cathepsin l activity as compared with normal liver was revealed. during fibrosis regression the maximums of enzymatic activity at the th day ( %) and at the st day ( %) after cessation of ccl -treatment were fixed. at the th day of recovery some diminution of cathepsin l activity was noted. at the th day after toxin abolition enzyme activity was close to control. a close correlation between cathepsin l activity and liver hidroxyproline level was found. electron-histochemically reaction product was revealed in the kupffer cells and fibroblast phagolysosomes containing fragments of collagen fibrils. we also found extracellular localization of cathepsin l in both terms of investigation. the reaction product was noted on the hepatocyte, kupffer cell and fibroblast plasmalemma and on adjacent collagen fibrils. conclusions cathepsin l is directly implicated in collagen resorbtion during liver fibrosis regression. detected extracellular activity of cathepsin l suggests that in addition to the intracellular proteolysis, cathepsin l is secreted by hepatocytes, macrophages and fibroblasts in intercellular space and can take part in extracel-lular collagen degradation. ryvnyak victor laboratory of morphology "nicolae testemitanu" state university of medicine and pharmacy bd. stefan cel mare, kishinev, md- moldova victorrivneac@yahoo.com introduction the solitary necrotic nodule (nns) of the liver is an uncommon lesion with a round appearance near the surface of the liver, with a dense collar of hyalinised collagen surrounding a necrotic core. case reports we present three cases of nns. two patients underwent surgery to remove liver metastasis. we suggest a relationship with the previous hepatic surgery and with the microwave tissue coagulator. the other case have an history of abdominal traumatism by a seizure. the radiological image of nns was misinterpreted as a tumor and the diagnosis by fnac or frozen section could not exclude a metastasis. the potential risk of a malignant secondary tumour existed, and that was why limited liver resection was recommended. several pathogenetic mechanisms have been avocated (infection, parasitic, traumatic, ischaemic, post chirurgical, iatrogenic, etc.) , all of them having an identical morfological appearance. conclusion nns is a rare non-tumorous lesion that might be misinterpreted as a tumor. the correct diagnosis can only be histological. it is important to differentiate the nns from metastases due to the great influence on the treatment. the significance of morphometrical analysis in staging of fibrosis in liver biopsies from patients with chronic hepatitis c a matsionis , e romanova , y kirichenko , y ambalov , p povilaitite rostov regional bureau of pathology, rostov on don, russian federation rostov central hospital, rostov on don, russian federation introduction prognosis of the course of chronic hepatitis c (chc) depends on fibrosis stage and its progression temps in the liver. semi quantitative methods are currently regarded as a routine diagnostic procedure. but it is obvious that these methods are subjective. morphometrical analysis with calculation of fibrosis index (fi) is an alternative method of evaluation. the aim of the study was to compare data obtained after morphometrical analysis of collagen formation to semi quantitative methods results and find out accordance with fibrosis stage. materials and methods liver biopsies from chc patients were used. we applied v.j.desmet ( ) semi quantitative method and software sigmascan pro . for morphometrical analysis of sections. all sections were stained by masson trichrome method for the detection of collagen. fibrosis index (fi) was calculated as collagen area divided all section area. results , % of patients had clinically significant level of liver fibrosis (desmet f , f , f ). f desmet stage was corresponding fi , - , , f desmet (moderate fibrosis) -fi , - , and high level of fibrosis (f and f ) -if , - , . / cases ( , %) showed differences between semi quantitative and morphometrical results. four patients ( , %) without fibrosis by semi quantitative methods showed if corresponding moderate fibrosis (f ). conclusion our results suggest that morphometrical analysis is beneficial in the evaluation of fibrosis stage and should be used for prognosis and monitoring the course of chronic viral hepatitis. introduction hepatic mucinous cystadenomas with mesenchymal stroma have been recognized as rare neoplasms that occur only in females and have recently been postulated as being hormonally dependent. because of the similarity of these neoplasms to ovarian mucinous cystadenomas, we compared the immunohistochemical profiles of these neoplasms arising in two different organs. the aim of this study was to establish whether these two tumors were phenotypically similar. materials and methods we employed the following immunohistochemical stains, estrogen receptor, progesterone receptor, inhibin, ck , ck and alcian blue/ periodic acid-schiff, to cases of ovarian mucinous cystadenomas and to cases of hepatic mucinous cystadenomas with mesenchymal stroma. results both the ovarian and hepatic neoplasms showed similar results with all of these stains. conclusions phenotypically, we have been unable to establish any difference between these two neoplasms. there appear to be two possible explanations for the origins of these tumors: one is that of a displaced ovarian anlage in the developing liver and the other would be that some of the primitive hepatic stem cells are capable of acquiring ovarian characteristics assessment of periodontum condition in hepatitis c infected patients with rapid and slow dynamics of advanced fibrosis p radlowski aim analysis of the relationship between the stage of periodontium disease and the extent and dynamics of fibrosis in patients infected with chronic hepatitis c. the study included patients whose blood serum revealed the presence of hcv-rna via rt-pcr, while biopsies showed chronic inflammation and either septal fibrosis, or liver cirrhosis. fibrosis was evaluated according to scheuer's point scale, with azan-staining method and the authors' own modifications. fibrosis dynamics (fu/y) was evaluated as a quotient of the number of points assessing fibrosis and the number of years of the disease. periodontitis was determined by the following criteria: inflammation limbus, gum swelling, gingival pockets depth, presence of supragingival and subgingival calculus and gomphiasis. each criterion was assigned either or point within the cumulative scale of - points. results all patients with advanced liver fibrosis were found to show signs of periodontosis. the patients whose periodontium condition scored points with the mean value of fu/y amounting to , , while those who scored points showed mean values of fyu/y equalling , . the group of patients with high values of periodontium disease showed significantly higher values of periodontum disease stage (most frequently assessed at points) compared with the group of nine patients with low fu/y values (mean value = p < , ). periodontium score of the patients with cirrhosis and bridge fibrosis showed similar values. conclusion the degree of periodontal disease was found to be dependent on the pace of fibrosis dynamics. molecular evidence for a toxic memory effect in chronic liver disease c stumptner, k wagner, h denk, k zatloukal institute of pathology, university of graz, graz, austria introduction alcoholic liver disease shows a toxic memory effect: patients with a history of alcoholic hepatitis show even after prolonged withdrawal from alcohol rapid reappearance of alcoholic hepatitis upon reexposure to alcohol. this type of liver injury can be mimicked in a mouse model by feeding , . after months of ddc intoxication mouse livers revealed ballooned hepatocytes containing numerous mallory bodies. within four weeks of recovery from intoxication most mallory bodies disappear but rapidly reappear within three days of reintoxication indicating the existence of a toxic memory effect. the aim of this study was to investigate the molecular basis of the toxic memory effect. materials and methods cdna-libraries were generated from normal and ddc-intoxicated mouse livers by subtractive suppressive hybridization. these gene libraries were used to produce cdna-arrays for expression profiling. rna was isolated from mouse livers at different stages of intoxication, recovery and reintoxication and hybridized to the cdna-arrays. expression profiles were analysed by genesight-and imagene software. results and conclusion results obtained demonstrated several groups of genes differently expressed in short-term intoxicated naive and reintoxicated mice. these genes comprised cyp a as well as genes involved in the cellular response to oxidative stress and methionine metabolism. the sustained elevated level of cyp a during recovery and the enhanced induction upon reintoxication could explain why certain drugs lead to enhanced liver toxicity upon repeated administration even after long drug-free intervals. introduction tgf-beta, fas, fasl molecules take part in the regulation of apoptosis. fas is a cell surface receptor present in most of the cells. fasl can be secreted from the surface of the cells and when it binds to fas, they activate the caspases to trigger the apoptosis. although the mechanism of tgf-beta is not clear yet, it plays a role in tumorigenesis. tgf-alpha may indicate inhibitory or stimulatory functions in conjunct with the stage of the tumors and cooperation with oncogenes and growth factors. we aimed to evaluate the correlation among the expressions of fas, fasl, tgf-beta molecules in hcc. material and method in this study fifty cases diagnosed as hepatocellular carcinoma (hcc) between the - years were included. tgf-beta , fas, fasl expressions were evaluated immunohistochemically. results fas, fasl expressions were observed together in % of tissue samples with hcc and % of the nontumoral liver tissue samples. it was noticed that fas and fasl staining were more densely in the lymphocytes of the tumorous areas and neighboring normal tissues than the control group. there was no statistically significant correlation between fas, fasl, tgf-b staining except the positive correlation between the tgf-beta and fas. the poor differentiated cases weakly expressed fas, fasl and tgf-beta . where as the majority of the cases stained cytoplasmic positive, some cases stained either cytoplasmic or membraneous positive. conclusion in hcc, tumor cells use variable pathways for apoptosis. the lymphocytes near the tumorous tissue have an important role in this process. dynamics of liver histology in patients with chronic hepatitis c after interferon-alpha and ribavirin therapy introduction interferon-alpha therapy for chronic hepatitis c improves hepatic inflammation and may change the evolution of disease to cirrhosis and hepatocellular carcinoma. accurate assessment of liver fibrosis is necessary for morphologic diagnostics. the aim of this study was to evaluate the effect of interferon-alpha and ribavirin on liver histology; to compare the semi-quantitative (according to ishak) and quantitative methods for evaluation of fibrosis. materials and methods twenty-two patients ( males, females at mean age years) with chronic hepatitis c were studied. within to months from the pre-therapy biopsy and at least months after the end of treatment patients were followed with second liver biopsy. histological activity in paired liver specimens was assessed by knodell modified score. fibrosis was evaluated according to ishak staging system. in addition, fibrosis was quantified in masson trichrome stained sections using morphometric image analysis. interferon-alpha (mean dose me) and ribavirin were administered for to months. patients were classified as complete sustained responders (n= ), relapsed after therapy cessation (n= ) and "non-responders" (n= ). results after treatment reduction of the histological activity score (at least one grade) was observed in patients ( %). lobular and periportal necroinflammatory lesions were decreased in both responders and non-responders (p< . ). after therapy, dynamics the fibrosis degree and calculated fibrosis ratio were not significant. fibrosis stage was improved in only sustained responders ( %); remained unchanged in patients, and progressed in patients ( %). conclusions improvement of liver histology after interferon-alpha and ribavirin treatment was constricted to the patients who achieved hepatitis c virus eradication. morphometry was the informative method for evaluation of fibrosis dynamics in addition to fibrosis assessment with semi-quantitative classification systems. introduction liver biopsy is one of the diagnostic procedures for neonatal hepatitis (nh)and intrahepatic bile duct paucity (ihbp). in this study we aimed to evaluate the prognostic benefits of quantitative analysis of histopathological changes in nh and ihbp. material and methods we studied liver biopsies in which were nh and were ihbp. the relationships between histopathological and prognostic parameters were analyzed. hepatic histopathological changes such as portal and periportal fibrosis, portal and lobular inflammation, bridging necrosis with fibrosis, pericellular fibrosis, giant cell transformation and extramedullary hematopoesis were evaluated and according to their absence ( ) or presence ( ) a histopathological score was assessed. the sum of these data was accepted as total pathological injury score. clinical prognostic parameters consisted of the patients height, presence or absence of hepatosplenomegaly and ascites, serum levels of alt, albumin, bilirubin, and the value of protrombine time. the sum of these data was accepted as the clinical score. the patiens were also divided into two groups whose clinical scores were < as good prognosis and ≥ as bad prognosis. for statistical analysis pearson's chi-square, mann-whitney-u and roc curve tests were used. results we found a statistically significant negative relation between prognosis and total pathological injury score (p= . ), so as pericellular fibrosis (p= . ). conclusion during the interpretation of liver biopsies of nh and ihbp, scoring of histopathological changes should be done in order to assess the clinical prognostic outcome. moreover, the presence of pericellular fibrosis should be evaluated and reported. immunohistochemical analysis of hepatocellular carcinoma and intrahepatic cholangiocellularcarcinoma using markers supposed to indicate respective levels of diffentiation of the hepatic cell lineage k ishida, m endoh, j takeyama, k sakamoto, m watanabe, t moriya, h sasano department of pathology, tohoku university hospital, sendai, japan it is widely recognized that there are diagnostically problematic cases among primary hepatic tumors. they cause difficulties in distinguishing between hepatocellular carcinoma (hcc) and intrahepatic cholangiocellularcarcinoma (cc). meanwhile, recent progression of study on hepatocyte-diffrentiation has revealed that both mature hepatocyte and biliary epithelial cell should originate from the common cell lineage called as hepatic-progenitor-cell (hpc), bipolar-cell (bc) and/or intermediate-hepatocyte-like-cells. thus, we performed immunohistochemical analysis for hcc and cc using markers supposed to indicate respective levels of differentiation of the hepatic cell lineage, in order to identify the origin of the tumors particular in the diagnostically problematic cases. materials and method surgical specimens of cases of hcc and cases of cc were used. ordinary immunohistochemical staining was performed with following antibodies; hepatocyte monoclonal antigen (hep), c-kit, ck , ck and ck . the results were compared from the view of the histopathological findings and immunohistochemical reactions. results rate of positive (> %) and focal positive (> %) were hep; hcc %, cc %, c-kit; hcc %, cc % and ck ; hcc %, cc; %. ck and ck were similar as previously reported. in addition noteworthily, double positive for hep and ckit was observed on two cases of cc having apparent glandular formation. conclusions ) hcc had a lower positive rate for hep compared with previous reports. ) cc with positivity for hep might exist. ) according to the c-kit staining pattern, we considered that there might be a peculiar variant of tumor cells in cc differentiating both hepatocyte and biliary epithelial cell. introduction proteus anguinus, the sole species of the european cave salamander and the remarkable cave dweller of underground waters in the karst of slovenia is well adapted to poor and discontinuous food supplies. remarkable accumulation of lipid droplets and glycogen deposits in the liver beside slow metabolism allow proteus to survive for long periods without food. the aim of this study was to establish morphological changes in the liver during starvation with particular emphasis on mobilization of the liver energy reserves. material and methods the liver was examined after food deprivation periods of one, four and eighteen months, respectively using light and transmission electron microscopy. glycogen and proteins concentrations were determined spectrofotometrically. total fat in the liver was estimated by folch method. results intense depletion of glycogen was present during first month of starvation. the glycogen wasn't formed in clumps typical for normal liver tissue. after eighteen months of fasting glycogen was re-synthesized, dense glycogen deposits appeared. lipid droplets were less condensed. numerous peroxisomes formed clusters with small and round mitochondria. particular mitochondria increased in size and lost cristae. lysosomes and autophagic vacuoles were numerous, too. dilated intercellular space included electron dense granular material. conclusions carbohydrate and lipid reserves in the liver of proteus serve for metabolic needs during food deprivation. while in many other vertebrates fasting causes depletion of liver carbohydrates, these reserves are maintained in large amounts in proteus. results of our study confirm that a detailed morphological analysis of the liver could be a relevant informative method to prove metabolic adaptability of this organ. evaluation of t lymphocyte subpopulations in chronic hepatitis-relationship with histological lesions ci iosif, f vasilescu, g butur, dc terzea, c dobrea, f andrei, c ardeleanu 'victor babes' national institute, bucharest, romania background intrahepatic t lymphocytes are believed to be involved in immunopathogenesis of chronic viral hepatitis. the aim of this study was to assess the correlation between morphological lesions and t lymphocyte subpopulations in liver and peripheral blood (pb) of patients with chronic hepatitis. methods and results liver biopsy specimens from patients serologically confirmed ( hbv and hcv) were studied by histopathological and immunohistochemical methods. t cell subtypes (cd , cd , cd ) were tested by using monoclonal antibodies on tissue samples and by flow cytometry in pb. results in pb, % of patients have t lymphocyte cd + decreased and % of them have augmented values of t lymphocytes cd +. in liver biopsy the prevalent subpopulation were cd + cell, being approximately twice higher than cd + cells. the observed histological lesions were quantified by ishak score as moder-ate\severe chronic hepatitis. conclusions decreased subpopulation of cd + t lymphocyte might contribute to the severity of liver disease. relatively more frequent cell type was cd positive t cell, having a significant association with a higher inflammatory activity in both types, b and c virus hepatitis. the role of survivin in regulation of proliferative and apoptotic activity of hepatocellular carcinoma introduction imbalance between proliferation and apoptosis is an important factor in progression of hepatocellular carcinoma (hcc). aims: the percentage of proliferative (pi) and apoptotic cells (ai) in hcc compared to non-tumor liver was investigated. the role of unprocessed as well as active form of caspase- , which is an important apoptotic protein was of great interest for this study. expression of survivin, an inhibitor of apoptosis was investigated, too. material and methods twenty patients with hcc were included in the study. two samples were obtained from each patient, one of hcc and one of the surrounding non-tumor liver. mib- antibody was used to determine the proliferative activity. immunohistochemical staining for the detection of active form of caspase- was used to determine the percentage of apoptotic cells and for detection of procaspase- and survivin expression, as well. results ai was higher in hcc ( , ± , ) compared to nontumor liver ( , ± , ), but the proliferative activity in hcc was much higher than apoptotic activity. % of hcc showed positive staining for pro-caspase . positive correlation between expression of pro-caspase and active form of caspase proved to be statistically significant. % of hccs were survivin positive. stronger expression of survivin was detected in hcc compared to non-tumor liver. positive correlation between survivin expression and the grade of tumor diferentiatiation as well as a significant association between survivin expression and both ai and pi was also detected. conclusion our results indicate that survivin promoting cell proliferation in hcc has an important role in hcc progression. the histopathological effects of offloading on liver of rat embryo a khaki , aa khaki , j solimany rad faculty of veterinary medicine of tabriz islamic azad university, tabriz, islamic republic of iran faculty of medicine science.tabriz medical science university, tabriz, islamic republic of iran introduction ofloxacin is an antibiotic from fluroqinolne with a wide range and widely used in various infection diseases. since there is little information about ofloxacin side effect on liver of rat embryo. this preliminary study was planed to see what kind of changes occurs in development of liver in rat embryo. the aim of present study was to determine the histopathological effects of offloading on liver of rat embryo. material and methods fourty nine wistar rats were selected and randomly divided into two groups; control (n= ) and test (n= ). the test group has been received mg/kg (po) ofloxacin daily during pregnancy. however, the control group just received plate. after delivery, liver tissue of neonatal in both groups were taken and prepared for light microscopy. staining method was h&e. results microscopic study of liver tissue specimens showed that in the test group hepatocytes were vesiculated. degeneration and vacuolated pycnotic nuclei were seen when compared to control group. weight of neonatal in test group was reduced when it was compared with the control group (p< . ). conclusion since ofloxacin had side effects on liver of rat embryo, it is not suggested to be used during pregnancy in human. introduction while the underlying etiology of hcc has been well characterized the molecular mechanisms of hepatocarcinogenesis are poorly understood. the extremely poor prognosis of hcc and the limited value of the current standard clinical markers underscore the need for improvent of diagnosis and treatment of this deadly disease. aims: we performed gene expression profiling using cdna microarrays in order to identify novel molecular markers and therapeutic targets for the treatment of hcc. material and methods we used the method of subtractive suppressive hybridization (ssh) to generate cdna libraries that are highly enriched with clones representing genes both up-and downregulated in the diseased liver tissue. the expression pattern of clones representing ~ genes was analyzed in surgically resected hepatocellular carcinomas, focal nodular hyperplasias, one adenoma, and cirrhotic livers by competetive hybridization on custom made cdna microarrays using a pool of non-neoplastic human liver as reference. the expression data were verified independently by quantitative rt-pcr (taqman). results we found genes which were consistently deregulated in more than one third of the hccs under investigation. of these, genes had been previously implicated in hcc, whereas known genes as well as novel genes (with unknown function) had not yet been linked with liver cancer. conclusions the present study revealed a number of genes specifically deregulated in hepatocelluar carcinoma. future work will aim to evaluate the medical relevance of the encoded proteins which will ultimately help to identify potential new markers for better diagnosis/ prognosis of hcc and new targets for therapeutic intervention. introduction actinomycosis is a suppurative and granulomatous chronic infectious disease caused by actinomyces sp. and most commonly affecting the cervicofacial area. aim: to study the clinical characteristics of patients with actinomycosis, with regard to clinical history, presentation, method of diagnosis and comparing to the literature. patients and methods from january to july , a retrospective review was performed on all cases of histologically proven actinomycosis. results ten patients were studied, men and women, - years old. two patients had a history of surgical procedures and/or dental manipulations. two patients had a long history of diabetes mellitus. locations were the liver (patient n ), the feet articulation (patient n ), the lung (patient n ), the jugular lymph node (patient n ), the skin (patients n and ), mandibular (patient n ), the psoas muscle (patient n ), the appendix (patient n ) and the large bowel (patient n ). three patients showed a tumoral presentation (patients n , and ) and two patients had a tuberculosis disease presentation (patients n and n ). the patient n had a clinical presentation of an inflammatory large bowel disease. one patient was diagnosed by cultures positive to a. israelii on microbiologic study and the others patients by surgical biopsy and detection of an actinomycosic granuloma. special stains were used in all cases. three patients received the classic initial regimen of iv. conclusion actinomycosis is an uncommon disease in our country. in these cases gastrointestinal locations were predominant. two uncommon locations were present. there was no iud associated infection. tuberculosis endemic in our country can mislead the diagnosis. further autopsy hazards: mycobacterium tuberculosis in the autopsy room r flavin, s o briain st. james hospital/tcd, dublin, ireland introduction tuberculosis has been the major occupationally acquired disease in the autopsy room. the risk of encountering tuber-culosis at autopsy has fallen with the decreased incidence of the disease and greater emphasis has been placed recently on the hazards of viral diseases. however, tuberculosis may still be found at autopsy. the aim of this study was to assess the magnitude of exposure to mycobacterium tuberculosis at autopsy in a large general hospital setting. methods retrospective search of the autopsy records from - . the patient record and histological slides were reviewed. the quantity of mycobacteria was assessed from the ziehl-neelson stains as scanty, moderate or abundant. results cases of active mycobacterium tuberculosis were identified in the -year period in which autopsies were performed ( per autopsies) of which cases were unsuspected ( %). these included cases of miliary tuberculosis and with pulmonary infection. cases contained abundant organisms. patients tended to be middle aged and male with complex clinical histories. patients were dead on arrival at hospital. conclusion the risk of unexpectedly encountering tuberculosis at autopsy continues and has implications for the health of autopsy room staff, autopsy room ventilation and design, protective equipment and for the involvement of the public health service. introduction the course of chronic hepatitis c virus infection is difficult to predict. although the disease is frequently asymptomatic, the development of fibrosis and cirrhosis are the major complications. among patients suffering from chronic hepatitis c (chc), special group are young drug abusers. immunological toxicity of narcotics modulates the total immunologic response to the viral infection with characteristic morphologic expression of this disease. the aim of this study was to asses which pathological features are associated with liver fibrogenesis and are predictive to fibrosis in chc drug abusers. methods eighty liver biopsies with chc from drug abusers ( actively consumers; free from drugs - months before liver biopsy and free for more than one year before performing liver biopsy) stained by he, trichrom masson, perls and reticulin were analyzed semi quantitatively using standard questioner for determination of patohistological features for histological activity index. results of fibrosis were correlated to all of them. results the fibrosis was , and in this group of young patients (mean age , years). there were no correlation between any of the patohistological parameters and fibrosis, even for lobular necrosis. conclusion histomorphological changes and the extent of fibrosis are in correlation to the duration of drug consumption. morphological expression of immune toxicity of narcotic drugs in chc patients make this disease special entity to which it is not possible to apply standard histopatologic parameters, nor the parameters predicting liver fibrosis. introduction epithelial tumors of the conjunctiva are rare diseases in europe. in sub-saharan countries, an increasing incidence was observed in connection with the hiv epidemic. the definite causes of these tumors are not known. aims: based on tumor material from africa, we search for possible cancerogenic agents and its pathogenetical interaction in conjunctival carcinoma. materials and methods conjunctival specimens of patients from three african centers with the following histological lesions were examined: papilloma ( case), pinguecula ( ), conjunctival intraepithelial neoplasia ( cin i, cin ii, cin iii), squamous cell carcinoma (scc, cases). immunohistological reactions for p and p were done. of tested patients were hiv positive. results and conclusion of the benign and precancerous lesions were associated with histological signs of a solar damage. of the cases showed immunohistologically an overexpression of p , among them all scc. this finding might indicate a p mutation as mostly seen in solar induced skin cancer too. % of the cases with cin iii and scc had an overexpression of p . corresponding findings are known from transformed or neoplastic cervical epithelium in strong correlation with an active hpv (type , ) oncogene expression. no significant differences occurred between hiv positive and hiv negative cases in comparison to the frequency of different histological lesions as well as the p and p expression. the average manifestation age for cin iii and scc in hiv positive cases ( resp. years) was years lower than that of hiv negative cases ( resp. years). the results suggest a multifactorial tumorgenesis (uv radiation, oncogenic viruses, hiv infection) for the conjunctival carcinoma in africa. introduction botryomycosis is an enigmatic pyogranulomatous infection, which may be caused by a variety of non-filamentous bacteria including staphylococcus aureus and pseudomonas aeruginosa. while the exact pathogenesis remains elusive, it is acknowledged that the interrelationship of host and agent factors is fundamental. we describe the clinicopathological features of a rare and particularly mutilating case of delayed onset post traumatic craniofacial botryomycosis. methods an immunocompetent -year-old black male presented with multiple sinuses draining granules involving the frontal, parietal, orbital and maxillary areas. nodulo-fibrous thickening was also present. this had evolved over a three-year period. apart from the history of a head injury requiring cranioplasty years earlier, no other predisposing factors were found. results the diagnostic features of clumped bacteria (cloxacillin sensitive staphylococcus aureus), splendore-hoeppli phenomenon, granulomata and intense fibrosis were confirmed histologically. there was no response to intravenous cloxacillin and the patient died before surgical debridement could be done. conclusion this case report serves to highlight several points. firstly, it illustrates that when considering the diagnosis of post traumatic botryomycosis, the time interval between the traumatic event and the onset of clinical features may span decades. we submit that this is due either to the small size of the bacterial load or their low virulence. secondly, it is our contention that the poor response to antibiotics is due to the severe, impenetrable fibrosis. finally, the destructive nature of the disease underscores the urgency in diagnosis and treatment. tuberculous meningitis in adulthood introduction tuberculous meningitis is a very frequent clinical manifestation of postprimary tuberculosis in childhood as the result of hematogenous dissemination of a pulmonary lesion; in adult patients tuberculous meningitis occurs less frequently, mainly associated with extrapulmonary disease, consequent to the rupture of a subependymal tubercle. case reports we report cases of tuberculous meningitis in adulthood diagnosed by autopsy examination -median age , yrs -in an autopsy series of tuberculous patients in years. all but one of the patients had disseminated disease (lung, lymph nodes, liver, spleen and kidneys). none of the patients presented subependymal tubercles. one patient had dual mycobacterial infection -miliary tuberculosis due to mycobacterium tuberculosis in all the extracranial affected organs and tuberculous meningitis with mycobacterium avium-intracellulare (mac). cerebrospinal fluid examination revealed high cellularity and high albumin concentration in all cases; glucose levels were low, except in one case with normal glucose content -the mac infected patient. one patient had associated systemic lupus erythematosus (sle) and low cd + cells count. the histopathologic appearance consisted of a granulomatous meningitis with giant cells and epithelioids in three cases and polymorphous inflammatory infiltrate including numerous positive bacilli in ziehl-neelsen stain in mac infected patient. conclusion the overall appearance suggested a hematogenous spread of the infection, the immune status correlating with the occurrence of the meningeal disease; in our series we had an immunodepressed patient (sle) and another patient with miliary tuberculosis and superimposed mac infection. also, mac meningitis had a different clinical and histopathologic appearance. introduction clinical presentation and lesion morphology are often suggestive, but not conclusive for mycobacterial (mb) infections. rapid and accurate specific diagnosis is mandatory since appropriate therapy can be chosen only on the basis of the precise identification of the causative agent. one of the most promising tools for rapid and sensitive identification of mb species is represented by amplification of specific sequences of the s rrna mb specific gene on the fragmented dna (max bp) obtained from fixed tissues. methods dna was extracted from cases selected from the archives of the department of pathology, university of padova, with histopathologic features of granulomas, caseous necrosis, and langhans-type giant cells. ziehl-neelsen staining for acid-fast bacilli and pcr/direct sequencing of a bp variable region of the mb s rrna gene were performed. h rv genome was employed as positive control. results mb specific sequences have been identified in of samples analyzed ( %). in two dna samples ( %) an inserted dna product was detected and it is currently under sequencing, while in one sample ( %) no evidence of mb infection has been gained. agreement with ziehl-neelsen was present in cases ( %). conclusion our study suggests that pcr is a rapid, sensitive method for the diagnosis of mb in routinely processed formalinfixed, paraffin-embedded histological specimens and is highly recommendable in cases of chronic inflammation without definite evidence of granulomatous inflammation. introduction cladosporium bantianum is a pigmented highly neurotropic fungus which can be identified easily in tissue sections due to their golden brown colour. cerebral lesions caused by this fungus are rare. case reports we present six cases diagnosed at our institute over a period of yrs. . there were males and female. all patients belonged to the second to fourth decade and had presented with symptoms of space occupying lesion. radiological investigations revealed well circumscribed lesions with ring enhancement. the smallest lesion measured . x . cm and largest measured x . x cm. only two of the six cases had detectable predisposing debilitating illness. one yr. old male had past history of liver tuberculosis and had taken anti-tuberculosis treatment and a yr. old female had systemic lupus erythematosus and was on immunosuppressants. we received four surgically excised specimens and two autopsy specimens of the entire brain. histopathology revealed necrotic areas, micro-abscesses and mainly foreign body type of giant cells. lying within the giant cells as well as freely within the tissue were numerous golden brown to deep brown spherical forms of cladosporium bantianum measuring - microns in diameter and their hyphal forms measuring - microns in diameter, - microns in length with septae and occasional branching. these were confirmed by gomori's methenamine silver stain for fungus and species identification was carried out by culture studies. all the four patients with surgical excision showed recurrence within - months. three succumbed to the disease and one is still alive. conclusion cladosporium bantianum can affect both healthy as well as immunocompromised individuals. it has a fatal outcome. cases are being presented because of the relative rarity. necrobiotic changes of skeletal muscle simulating myxoid sarcoma. report of a case with immunohistochemical and electron microscopic studies kw min , is seo deaciness hospital, oklahoma city, usa wishard memorial hospital, oklahoma city, usa background skeletal muscles are known to undergo bizarre reactive changes in response to traumatic injuries and may produce pseudosarcomatous lesions which may give diagnostic challenges to pathologists. such lesions usually produce proliferative myositis in which atypical spindle cells of fibroblastic and myofibroblastic nature predominate and may simulate the appearance of fibrosarcomas and myofibrosarcomas. however with proper setting of previous trauma history, their true nature can be easily appreciated. we are reporting an unusual case of myxoid cellular lesion found in the abdominal rectus muscle involved in a post hysterectomy abdominal hernia which simulated the appearance of an extraskeletal myxoid chondrosarcoma (emc). its true reactive nature was recognized by electron microscopic (em) study. design light brownish gelatinous material aggregating . x . x . cm was found around the sheath of rectus abdominis muscle of a year old female during a repair surgery of posthyterectomy ventral hernia. the tissue was fixed in buffered formalin and a representative portion of the tissue was fixed in phosphate buffered . % glutaraldehyde and post fixed in osmium with en bloc staining with uranyl acetate. epon embedded thin sections were stained with uranyl acetate and lead citrate. for immunochemistry, paraffin sections were stained for s protein, myosin, and actin by routine abc-peroxidase techniques. results on routine examination, the lesion was characterized by loosely arranged spindly to epithelioid cells in bundles with abundant myxoid stroma. the lesional cells varied in size with frequent bizarre hyperchromatic nuclei. mitosis was rare. the cells showed weakly positive diffuse cytoplasmic stain for all of above antibodies. with the initial impression of emc, an expert opinion confirmed the diagnosis of grade ii emc. however, em revealed the lesional cells to be consistent with skeletal muscle cells having of varying stages of degeneration and regeneration. no ultrastructural characteristics seen in the tumor cells in emc were present. the attending physician was advised of careful follow-up and close observation without any additional treatment. the patient remains with no evidence of disease by year follow-up in spite of incomplete excision of the lesion. discussion and conclusion the myxoid gelatinous material found in the abdominal rectus muscle presented herein showed histopathologic characteristics of emc, however, electron microscopic studies revealed the lesion to be necrobiotic process of the rectus muscle. evidence of no disease in years of follow-up with incomplete excision supports the conclusion the lesion was not malignant as em study indicated. this case exemplifies another variety of pseudosarcomatous reaction of skeletal muscles important in the differential diagnosis in the pathologic evaluation of cellular myxoid lesions. peritoneal psammocarcinoma. a case report with ultrastructural study is seo background psammocarcinoma (pc) is a very rare variant of low grade serous tumors that appears to arise from the peritoneum and ovaries. this tumor has been defined as a serous neoplasm displaying ) destructive invasion, ) no more than moderate nuclear atypia, ) no areas of solid epithelial proliferation except for occasional nests no more than cells in diameter, and ) at least % of papillae or nests are associated with or completely obscured by psammoma bodies. pc shares some clinicopathologic features with serous tumor of low grade malignant potential (lmp), but may have been under-recognized at surgery and gross examination. the present case was incidentally found during a routine hysterectomy procedure for leiomyomas. the pc involved the omentum, peritoneum and diaphragm. we report, to our best knowledge, the first electon microscopic (em) findings of pc. design tumor tissue was fixed in buffered formalin for routine examination. in addition, a representative tissue was fixed in phosphate buffered . % glutaraldehyde and post fixed in osmium with en bloc staining with uranyl acetate for em study. results this tumor met the diagnostic criteria of psammocarcinoma. there were numerous psammoma bodies with or without nests of epithelial proliferation with minimal nuclear atypia. more than % of papillae were either associated with or completely replaced by psammoma bodies. multifocal involvement was noted on the surfaces of both ovaries and fallopian tubes. ultrastructurally, the tumor cells showed markedly villous surfaces with abundant long slender villi. their length/diameter ratio (ldr) was with some variation of their diameter. the cytoplasm contained abundant cytoplasmic organelles and intermediate filaments. intracytoplasmic lumens were absent. decalcified psammoma bodies resembled cut surfaces of geode. discussion and conclusion our case met the proposed criteria for this tumor that arose de novo in peritoneum. ultrastructurally, this tumor shared some findings with serous tumors (junctional complexes, irregular microvilli), but differed by the lack of prominent desmosomes and frequent intercellular lumens. pc differed from mesothelioma in that significantly greater microvillus ldr, prominent desmosomes, tomofilaments, intracytoplasmic lumens, external lamina and glycogen were rountine findings in the latter. our findings justify classification of pc in the group of serous epithelial tumors arising from the coelomic mesothelium. application of electron-and immunoelectron microscopy in the differential diagnosis of pituitary adenomas m maksymowicz, w olszewski department of pathology, institute of oncology, warsaw, poland aim evaluation of practical implementation of electron microscopic techniques in diagnosis of pituitary adenomas. material and methods material consists of consecutive cases of surgically resected pituitary tumors. in pituitary adenomas diagnosed clinically, there were patients with symptoms of acromegaly, patients with hyperprolactinemia, cases of cushing's syndrome and case of tsh hypersecretion. there were clinically nonfunctioning tumors. all cases were diagnosed by histological examination and immunohistochemical identification of known pituitary hormones (growth hormone, prolactin, acth, tsh, fsh, lh and alpha-subunit). the cases evaluated at light microscopy level as pituitary adenomas were examined with electron microscopy and immunoelectron microscopy using a postembedding immunogold technique. results immunohistochemical investigation showed that about , % of specimens of pituitary adenomas contained more than one pituitary hormone. additionally, immunopositivity was discovered in % of tumors from patients with clinical and biological signs of silent pituitary adenoma. ultrastructural analysis provided proper diagnosis of selected cases, e.g. silent corticotroph adenoma or distinction between densely-and sparsely granulated growth hormone secreting adenomas. double labeling immunoelectron microscopy using gold particles of two different sizes greatly facilitated the distinction among mixed growth hormone/ prolactin, mammosomatotroph and acidophil stem cell adenomas. conclusions electron microscopy analysis was valuable to provide a useful informations regarding the diagnosis. our results confirmed practical value of who recommendation for five-tier evaluation of pituitary adenomas (clinical, neuroimaging, histological, immunohistochemical and ultrastructural) . on the basis of our results and the published data, diagnostic postoperative algorithm for proper pathologic management has been proposed. apoptosis, sarcomere disruption and myofibrilar degeneration in skeletal muscles of stable copd patients: an ultrastructural study md ferrer introduction in patients with chronic obstructive pulmonary disease (copd), the diaphragm muscle shows signs of injury, even during clinical stability. in addition, these patients have a markedly increased susceptibility to additional diaphragm injury following inspiratory loading. we have hypothesized that, if sytemic factors play a role in diaphragm injury, limb muscles may also be affected. the purpose of this study is to investigate whether apoptosis occurs in the diaphragm and quadriceps of copd patients, and to evaluate fiber injury in non-respiratory muscles. methods samples from both diaphragm and quadriceps muscles were obtained in patients undergoing thoracotomy for a lowstage lung neoplasm (ln) and stratified into two cohorts according to pulmonary function: ln+copd (n= ) and non-copd patients (ln+non-copd, n= ). in addition, samples form quadriceps muscle in healthy age-matched volunteers were also included (control group). all samples were processed for ultrastructural analysis. the proportion (%) of normal nuclei (nn) and those showing early (ean) and advanced (aan) signs of apoptosis were determined in all samples. digital images of longitudinal fiber sections ( x) were used to determine the area fraction (%) of abnormal myofibrilar structures (aab). results the level of apoptotic signs in the diaphragm of both copd and non-copd groups was minimal and very similar to that of control limb muscle. in contrast, quadriceps muscle of both copd and non-copd groups showed a two-and three-fold increase respectively in the apoptotic nuclei when compared to controls, mainly due to ean. apoptotic bodies were not found. in control quadriceps aab was , ± , %, mostly due to sarcomere disruption, while quadriceps in copd patients was associated with an increased aab ( ± %, p< , ) including myofibrilar disorganization and degeneration. conclusions in patients with copd, the diaphragm is not altered by apoptotic phenomena. muscle remodeling through apoptotic mechanisms as well as through structural changes, such as sarcomere disruption and myofibrilar degeneration, seem to be enhanced in the quadriceps muscle of these patients, regardless of the severity of pulmonary function impairment, suggesting that additional factors such as the degree of physical inactivity either related to the disease or to the preoperative status could also be involved. background renal chromophylic (papillary) carcinoma (cprc) has a propensity for storing hemosiderin in tumor cells. by transmission electron microscopy (tem), some tumors show a prominent lysosomal component, while in other cases, cells contain mostly isometric lipid vacuoles. it has been proposed that the latter would merely represent a papillary variety of clear cell renal cell carcinoma (ccrc). the aim of this study has been to analyze the lysosomal content in chromophylic (papillary) renal cell carcinoma by both immunohistochemical study of cd expression and tem, and to compare these results with those of conventional ccrc. design cases of cprc (n= ) and ccrc (n= ) are the subject of the study. the presence and relative amount of lysosomes and isometric fat vacuoles was investigated by tem (n= ). immunostaining with cd antibody (pg-m , dako, denmark) was performed in representative sections. the presence, distribution and percentage of cd staining in tumor cells was compared with tem data. results expression of cd was observed in all cases of cprc. the mean percentage of positive cells was % ( - %). distribution of positive cells was diffuse in % of cases. of the tem cases, contained abundant fat vacuoles, but only had extensive isometric vacuolation by light microscopy. electrondense lysosomes were less abundant than fat in cases, of which had massive cd expression. detailed examination of fat vacuoles in these cases suggested they were membrane-bound and of probable lysosomal origin. cd expression was also observed in cases of ccrc, all of them with focal distribution, involving a mean of % tumor cells ( - %). these differences were statistically significant (p< . ). conclusions diffuse cd expression is typical of cprc and may be a useful auxiliary diagnostic criterion. the isometric fat vacuoles in cprc are often membrane-bound and of probable lysosomal origin. these findings support the notion that the lipidcontaining, vacuolated variety of cprc is different from ccrc. introduction any lesion in prostate is followed by different proliferative activity of cells. one of the markers, which relate to proliferative activity, is ploidy. it can be determined by cytometry, morphometry or by other methods. aims: study of dna ploidy status in different lesions of prostate by morphometry. materials and methods samples from patients with atrophy, benign prostatic hyperplasia (bph), squamous cell metaplasia (scm), atypical adenomatous hyperplasia (aah), basal cell hyperplasia (bch), prostatic intraepithelial neoplasia (pin) and prostatic carcinoma were processed in formalin, dehydratated in graded alcohols, embedded in paraffin, stained by feulgen-stain. integral optical density was assessed by morphometry on image analysis system. integral optical density of lymphocyte was counted as c ploidy. integral optical density at different prostatic lesions was divided on c. results average ploidy at atrophy was . , bph - . , scm - . , bch - . , aah - . , low grade pin was . . average ploidy at high grade pin was . , high grade carcinoma - . , moderate grade carcinoma - . , low grade carcinoma - . . conclusion increase of ploidy status from atrophy to bph, scm, bch, aah, pin and to carcinoma was observed. regarding to ploidy lesions may be divided in benign and neoplastic processes, with cutoff point between them as . . pseudohyperplastic prostatic adenocarcinoma in transurethral resection of the prostate j arista-nasr, b martinez, j nuncio instituto nacional de ciencias medicas y nutricion salvador zubiran, mexico city, mexico background the pseudohyperplastic prostatic adenocarcinoma (phpa) is a recently described variant of adenocarcinoma, studied in needle biopsies and prostatectomy specimens. phpa is characterized by malignant glands that simulate benign hyperplastic glands of variable size with complex architecture, papillary enfolding, luminal undulations branching or cystic dilations, columnar cells with macronuclei and nucleoli enlargement. the aim of this study was to define the frequency and microscopic aspect of phpa in specimens originated in transuretral prostatectomy (tup). specimens of transurethral prostatectomy were analized from the surgical pathology department at incmynsz. cases were initially diagnosed as benign glandular hyperplasia (bgh) and as conventional adenocarcinoma (cc) results cases ( . %) with phpa were identified in specimens diagnosed as bhg. these specimens demonstrated phpa in two microscopic areas of mm. both patients are well and free of disease and years after diagnosis. the review of cases diagnosed as cc, three specimens had microscopic segments of phpa. in the outcome the patients developed early bone metastasis. the histopathology study revealed papillary projections, nuclear enlargement, crawled glands, macronucleoli ( / ); cystic glandular dilatation, straight luminal borders, pink amorphous secretions ( / ); nuclear hypercromasia and transition to small cell carcinoma ( / ) . other changes observed were corpora amylacea, double nucleoli and mitoses. conclusions the phpa is a rare neoplasm observed in tup specimens and is observed in only few areas. the false negative frequency in bgh is . %, and % in usual adenocarcinoma. the outcome for these cases is related to the main conventional adenocarcinoma component. vascular endothelial growth factor: an important angiogenic mediator in chronic urinary bladder lesions o hammam, h el baz, a el baz theodor bilharz research institute, giza, egypt vascular endothelial growth factor (vegf) is an overriding growth factor mediating tumor anigogenesis. this study was performed to assess vegf levels of expression in both serum and tissue in benign, premalignant and malignant urinary bladder lesions with or without schistosomal infestation in a trail to study the angiogenic phenotype of these lesions and to address any possible role of vegf as a prognostic biologic marker of bladder cancer. also the impact of schistosomal infestation on the vegf level of expression was assessed.the study included patients: chronic cystitis cases ( schistosomal and non-schistosomal), cases with schistosomal premalignant lesions (metaplasia, dysplasia and leukoplakia) and cases with bladder carcinoma ( schistosomal sqcc, schistosomal tcc and non-schistosomal tcc). eight cases with normal urothelium served as controls. vegf serum concentration was measured by the enzyme immunoassay (elisa) while, its tissue expression was detected by immunohistochemical staining.. significantly higher levels of vegf where detected in the premalignant and malignant cases in tissue (p< . & p< . respectively) and in serum (p< . ) compared to controls. also an aberrant increase in vegf serum and tissue levels was recorded in malignant cases compared to those with chronic cystitis (p< . & . respectively). schistosomal infestation was found to have a positive impact on vegf tissue and serum levels. tissue vegf positive rates was % in all the premalignant and malignant schistosomal associated cases. on stratifying malignant cases according to their histopathological grade and stage, an evident stepladder increase in vegf serum and tissue levels was recorded. vegf serum levels were well correlated with their corresponding tissue level which may indicate that vegf serum levels are reflecting intratumoral angiogenic status. although both vegf serum and tissue levels were positively correlated with tumor histopathological grade and stage, yet this correlation was more evident with the tissue expression (r= . , r= . , p< . in tissue, while r= . , r= . , p< . , p= . in serum with grade and stage respectively). in conclusion, our data indicate that vegf expression level in both serum and tissue may be used as a valuable angiogenic marker for prediction of prognosis. this may help in selecting patients for more intensive surgical or chemotherapeutic approaches. serum vegf concentration may be used as a non-invasive prognostic parameter especially in high risk patients who need continuous follow up. microvessel density in urothelial tumors of the upper urinary tract m raica , c suciu , f baderca , r minciu 'victor babes' university of medicine, department of histology&cytology, timsoara, romania 'victor babes' university of medicine, department of urology, timsoara, romania background angiogenesis in tumors of the urinary bladder is relatively well studied, in spite of some conflicting results and usually correlates with bad prognosis. few data are available about microvessel density (mvd) in urothelial tumors of the upper urinary tract (uut). our purpose was to investigate the mvd in patients with urothelial tumors of the uut and to check for its relationships with stage, grade, and survival. patients, material, and methods there were investigated patients with urothelial carcinoma of the uut. routine-stained slides were reviewed by two independent pathologists for the final diagnosis, t stage, and grade. only cases with invasive and mixed papillary-invasive tumors were included. two cases had lymph node metastasis. additional slides were stained for fviii, cd , and cd . mvd was estimated using the hot-spot method and mean value, calculated on consecutive fields from the tumor area and from the deeper connective tissue (x ). all cases had a follow-up of minimum years. results best results were obtained with cd and cd . in the tumor area, mean values of mvd were . for t , . for t , and for t . the hot-spot method consists of choosing the field with maximum density of blood vessels, and results showed for t , . for t , and . for t . the comparison between mvd and g showed a significant increase from g to g in the mean values and maximal density as well: . / for g , . / . for g , and . / . for g . no relationship was found between mvd and n positive cases. nine patients with high mvd died within years. the extra-tumor mvd had no prognostic significance. conclusions our results show a strong correlation between tumor areas mvd, t, g, and survival. mvd had no value as a predictor of lymph node metastasis. is there any prognostic significance for her protein in urothelial tumors of the upper urinary tract? of her protein in urothelial tumours of the upper urinary tract, and its relationships with the stage and grade. the final purpose was to conclude on the prognostic value of her protein in patients with tumours of upper urinary tract. patients, material and methods there were investigated cases with tumours of the upper urinary tract. routine he stained slides were reviewed for the pathologic diagnosis and grade. additional slides were stained with her- /neu (dako herceptest). interpretation of results was based on the score system (from to + ) already used in breast cancer. results obtained with her- /neu were compared with stage and grade. results eight from cases were positive for her protein ( . %). no relationship was found between her overexpression and 'g'. all but one case with g had a + score for herceptest. conclusions the overexpression of her protein does not correlate with tumour stage, and has no predictive value for lymph nodes metastasis. it strongly correlates with 'g'. introduction some authors consider postatrophic hyperplasia (pah), a glandular hyperplasia that mimics some patterns of prostate carcinoma (pca), without any correlation with neoplastic and preneoplastic condition of the prostate. the association of pah with pca recently suggested (mucci et al., ; tsujimoto et al., ) induced us to analyze the role of pah as precursor of pca. material and methods formalin fixed and paraffin embedded archival sections ( selected patients undergoing radical prostatectomy -mean age = . yr) were studied. twenty cases ( with pah and without pah) were selected for each group: a) bph (benign prostatic hyperplasia), b) pca-ghs< (gleason histological score) and c) pca-ghs=> . slides were immunocytochemically treated with psa, ße , cd , vimentin, cd , ar, nse, chromogranin a, pgp . , p , bcl , ki /mib , incubated with streptavidin-biotin complex (sbc) and revealed by diaminobenzidine (dab). immunohistochemical investigations revealed the presence of basal cells ( ße ) only in pah. the nuclear cell cycle proliferation (mib /ki ) and p mutation were quite similar in number of positive nuclei between pah and pca-ghs< . the chromogranin a were positive in pah, randomly in pca-ghs< , and in nests in pca-ghs=> . vimentin stained nests in pah, whereas were negative in pca. the bcl were positive in pah and negative in pca-ghs< . conclusion these findings suggest a strong overlapping of cell cycle nuclear proliferation and mutation markers, supporting a possible role of pah as a precursor of pca. eosinophilic cystitis: clinico-morphologic aspects d staribratova , d staikov , d dikov , v belovejdov , d staikov plovdiv medical university, plovdiv, bulgaria hopsital centre -lagny, paris, france collaborative study called for review of archived urinary bladder biopsy specimens with inflammatory lesions for the period - . features consistent with the diagnosis of eosinophilic cystitis were identified in cases, thus positioning it at th place with an incidence of , % from all inflammatory lesions. the distribution of clinical signs manifested in these patients often deceptive of interstitial cystitis or tumor processes is as follows: polakisuria, hematuria, suprapubical pains are evident in / , %/ patients; / , %/ presented with acute obstructive pyelonephritis as a result of bladder wall remodeling and case / , %/ revealed combined features of gastroeneteritis and cystitis. inflammed bladder wall and reduced bladder volume is estimated in / , %/ cases, ulcerations in / , %/ papillomatous and solid pseudotumoral lesions are / , %/ and / , %/ respectively. as all clinical signs are nonspecific, the only way to set a proper diagnosis is through bladder biopsy. morphologic confirmation is based on the standard protocols. histochemical (positive ziehl-neelsen stain) and ultrastructural identification of charcot-leyden's crystals are evident in some cases. adenocarcinoma of the male urethra in association with nephrogenic metaplasia f hostalet , je hernández , ja ruiz , a romero department of pathology, 'vega baja' hospital, orihuela, spain. department of urology, 'vega baja' hospital, orihuela, spain. we describe a case of adenocarcinoma of the urethra associated with so-called nephrogenic metaplasia in a year-old male patient that showed a polypoid tumor of the proximal penile urethra. histopathology examination of the biopsy specimen revealed two distinct patterns. the first showed a tumor composed of papillary fronds and glands lined by columnar epithelial cells with eosinophilic and clear cytoplasm, and contained atypical nuclei with frequent mitotic figures. this tumor was interpreted as adenocarcinoma. the second pattern was different, it consisted of small tubules some with mild cystic changes lying in an edematous stroma. the tubules were lined by a single layer of flattened cells with faintly eosinophilic cytoplasm and bland nuclei, mitotic figures were not identified. this area was interpreted as nephrogenic metaplasia and showed a direct transition into the adenocarcinoma. the immunohistochemical study revealed the presence of low-molecular-weight and some high-molecularweight keratins in both types of lesions. the adenocarcinoma cells also showed nuclear staining for p , in contrast, the cells of nephrogenic metaplasia were negative. the nephrogenic metaplasia as well as the adenocarcinoma cells were not stained with psa and pap. the histogenesis of adenocarcinoma of the lower urinary tract remains unclear. the association and direct transition of the nephrogenic metaplasia into the adenocarcinoma in this case suggest the proposed histogenetic pathway that the urethral adenocarcinoma may arise by malignant transformation of nephrogenic metaplasia. introduction benign prostatic hyperplasia (bph) is associated with a shift in sex hormone balance towards an increase in the oestrogen:androgen ratio. this is accompanied by elevated levels of oestrogen receptor alpha (er), predominantly in the stroma. recently, we described a simple, in vitro model to create moderate upregulation of er in human, prostatic stromal cells. the aim of this study was to investigate the hypothesis that in bph upregulated er and the action of androgens differentially regulate expression of several stromal growth factors. materials and methods eight human, prostatic stromal cell strains were subjected to a procedure to upregulate er alpha by exposing them to micro mol beta-estradiol for days, followed by passage and growth in the absence of steroids. four cell strains received instead n mol of dihydrotestosterone for hr. immunoexpressions of er alpha, ar, and six growth factors (fgf- , fgf- , igf- , tgf-beta , ngf and enos) were quantified by flow cytometry. results expression of er was significantly increased above control ( out of cell strains). expressions of all six growth factors were elevated but not significantly so. however, there was a significant positive correlation between the change in er (expressed as percentage of control) and the change in fgf- and fgf- . exposure of four cell strains to dihydrotestosterone reduced the expression of er and all six growth factors in comparison with oestrogen-treated cells. this reduction was significant for er and fgf- . conclusion upregulated er in prostatic stroma have a greater modulating effect on synthesis of certain growth factors than the direct action of androgens and hence may play a major role in the development of bph. the diagnosis of prostatic adenocarcinoma in needle core biopsy is based on major and supportive criteria. one of the supportive criteria is the presence of retraction clefting around neoplastic glands. the aim of our study was to analyze needle core prostatic biopsies in order to determine the frequency of periacinar clefting spaces in tumors and non-tumorous prostatic tissue. materials and methods we analyzed a consecutive series of prostatic cancer cases diagnosed by needle core biopsy to determine the frequency and extent of periacinar clefting. all tumors consisted of at least neoplastic glands. specimens were fixed in % buffered formaldehyde, embedded in paraffin, cut at µm thickness and routinely stained with hematoxylin and eosin. clefting was analyzed on neoplastic and normal glands in three different high power fields. results one third or more of analyzed glands with clefts affecting more than % of circumference were significantly more common in tumors ( . %) than in benign glands ( %). more strict criterion that designated as positive cases with at least % of neoplastic glands (fifteen out of thirty) with clefts that affected more than % of circumference revealed clefts in the malignant cases only ( . %) but not in benign cases ( %). fifteen or more glands with clefts regardless of their extension were also more prominent in malignant cases ( . %) than in benign cases ( . %). conclusion we conclude that retraction clefting represents a reliable criterion for diagnosis of prostatic adenocarcinoma. analyze the causes of ureteral obstruction in a series of patients managed at the department of urology, timisoara county hospital between and . there were selected only those particular cases of ureteral obstruction, others than cases with lithiasis, stenosis of the ureteropelvic junction and transitional cell carcinoma. an additional case was send for evaluation from another institution was also included in the study. for three of the selected cases we performed additional histochemical (alcian-blue-pas) and immunohistochemical study (anti-ck, anti-psa, anti-vimentin, anti-smooth muscle actin, abc and lsab system, visualization with dab). results we identified cases of ureteral obstruction due to unusual pathological lesions. they occurred in men and women. the mean age of the study group was years (range and years). there were malignant and benign lesions that produced ureteral obstruction. in cases, the compression was extrinsic and only in cases the obstruction was intrinsec. the site of ureteral obstruction was in the lower ureter in cases, in the lower and the middle ureter in patients and in the upper ureter in cases. the pathological lesions were: ureteritis cystica - cases, nephrogenic adenoma of the ureter - case, retroperitoneal fibrosis - case, reproperitoneal sarcoma - case, pelvic endometriosis - case, prostate cancer metastasis - case, signet ring cell carcinoma of the colon invading the ureter - case. conclusions after discarding the more common causes of extrinsic or intrinsic ureteral obstruction, the proliferativemetaplastic lesions of the urothelium, the retroperitoneal fibrosis and sarcoma, the endometriosis and metastatic/invasive carcinoma should be considered in the diagnosis. immunohistochemical study of androgen receptor and tgf alpha in human benign and malignant prostate a petrescu , g berdan , c ardeleanu , v jinga , s persu prof. dr. th. burghele hospital, bucharest, romania victor babes institute, bucharest, romania aims that androgen receptor(ar)and tgf alpha might be prognostic of hormonal response and biological behaviour in prostate carcinoma. methods ar and tgf alpha were studied in paraffin sections from men ( treated for prostate cancer and for bph by open, transurethral prostatectomy or prostate biopsy) no patient had undergone previous treatment for the prostate disease. the sections were stained with he to establish morphological characteristics (gleason score) and then were studied with indirect immunoperoxidase method applying monoclonal antibodies to ar(f . . biogenex) and antitgf alpha (clone , biogenex). results were semiquantified using histoscore.nuclei were graded for intensity of ar staining ( -absent, -weak, -moderate or strong) and the percentage of these intensity levels and a total intensity score were determined. we compared the ar and tgf alpha characteristics of prostate cancer (pca) with bph. results increasing gleason grade was correlated with decreasing intensity score and greater heterogeneity of ar staining and increased (+++) tgf alpha immunoreactivity. conclusions our data indicate that ar and tgf alpha content could be useful predictors of endocrine response and biologocal behaviour of prostate cancer. and ductus deferens samples), patients in group b were orchiectomised for the diagnosis of prostate cancer, teratocarcinoma of the testis and/or epididymal cyst (normal epididymal and ductus deferens samples) and subjects in group c were treated by radical prostatectomy or cystectomy for prostate or urinary bladder cancer ( samples of seminal vesicles without inflammatory changes). results hpv dna was detected in ( %) of patients in group a (hpv types , , , , , ) , in ( . %) patient in group b(at present unclassified type) and in ( . %) patients in group c(hpv types , and ).neither koilocytes nor dysplastic changes were observed. conclusion our data confirmed the presence of hpv, both lowand high-risk types, in the male genital tract. they support the idea of a male reservoir of hpv infection. spermatogenetic function of the testis in patients of different varicocele grades É magyar , e erdei , i lellei national medical center, pathology, budapest, hungary national medical center, andrology, budapest, hungary introduction varicocele is one of the main reasons of male infertility. surgical repair may improve sperm quality. the aim of this study is to investigate a grading system of varicocele and its correlation with the histological appearance using a multiple biopsy method. materials and methods bilateral double testicular biopsies of young infertile grade - varicocele patients were examined histologically. spermatozoa numbers counted in ten round shaped tubules, severity of atrophy and presence of maturation arrest was recorded. distribution pattern of observed spermatozoa numbers was evaluated. results grade and cases showed normal distribution. in grade cases five peaks were found indicating group inhomogenity. lower grade varicoceles presented with milder histological changes (mild atrophy or normal spermatogenesis) while in higher grade varicoceles more marked alterations (moderate-marked atrophy and/or maturation arrest) were seen. conclusion grade varicocele cases represent an inhomogeneous group, which should be divided into more categories. modification of the grading system is required. spermatogenetic activity of varicocelic patients cannot be predicted from clinico-radiological examinations so before art a diagnostic testicular biopsy is recommended. synchronous double urogenital cancer -a case report s we present a case of synchronous double urogenital cancer, comprised of invasive transitional cell carcinoma of the urinary bladder and adenocarcinoma of the prostate. cystoprostatectomy was performed in a -year-old male patient with previous cytological diagnosis of transitional cell carcinoma of the urinary bladder and no evidence of prostatic malignancy on preoperative clinical investigations. gross analysis of the urinary bladder revealed a cauliflower tumor mass filling the entire lumen and infiltrating the wall of the bladder. the size of the prostate was slightly increased. histological analysis of the urinary bladder specimens showed poorly differentiated transitional cell carcinoma, with only a few foci of preserved papillary architecture, infiltrating deeply into the muscle layer of the bladder wall. the specimens taken from the prostate gland for the purpose of staging of the neoplasm excluded the existence of infiltration of the prostatic urethra with the previously described neoplasm, but foci of moderately differentiated adenocarcinoma (gleason score ) of the prostate were discovered in its vicinity. the subsequent extensive sampling of the prostate showed organ confined adenocarcinoma. the review of the literature showed that the reported prevalence of synchronous existence of double primary malignant neoplasms of the urinary tract (transitional cell carcinoma of the bladder and prostatic adenocarcinoma) varies between % and %. the prevalence is noted to rise with increasing age. this leads to a conclusion that during standard cystoprostatectomy analysis, extensive sampling of the prostate is needed, even in the absence of macroscopic changes and preoperative clinical data suggesting prostatic neoplasm. multiple synchronous renal cell tumors d passetchnik rostov state medical university, rostov on don, russian federation rostov regional hospital , rostov on don, russian federation introduction the use of the nephron-sparing surgery demands detection of multiple renal cell tumors. materials we performed a retrospective analysis of nephrectomies and autopsies performed in our clinic. results of renal cell tumors ( , %) were multifocal. most of the patients were men. twelve cases were present where the histological subtype of the largest tumor was clear cell. satellite tumors were clear cell carcinoma ( ), papillary adenoma ( ), angiomyolipoma ( ), renomedullary fibroma ( ), bellini duct carcinoma ( ) . six multiple renal cell tumors were found in t , two in t , four in t a stage of the primary tumors. four patients had bilateral neoplasms. in thirteen cases only multiple papillary adenomas and carcinomas were found. usually tumors were no more than cm in diameter. in six cases multiple tumors originated on a background of nephrosclerosis of various genesis (chronic pyelonephritis, gouty nephropathy, amyloidosis). conclusion when conservative surgery of kidney cancer is performed, it should be remembered that % of patients with renal cell tumor have multiple neoplasms, which may cause local recurrence. the origin of multiple renal cell tumors may be bound up with hereditary predisposition or nephrosclerosis creating multifocal tumor field. introduction prostate cancer is a leading cause of cancer-related deaths in men and is the most common malignancy in american males. the heterogeneity of prostate cancer defines the current clinical challenges. one challenge is to identify in early, still potentially curable, stages of prostate cancer, molecular markers that predict aggressive or metastatic behavior. prostate cancer is characterized, like many other cancers, by molecular and genetics aberrations involving chromosome . parkin, mapped at the long arm of chromosome , is a amino acid protein with a molecular mass of kda, an ubiquitin-like domain at its amino terminus and two ring finger motifs flanking a cysteine-rich region. our aim was to investigated whether parkin alterations play a role in development and progression of prostate carcinoma materials and methods we examined parkin expression, by western blot analysis in eight prostatic carcinomas derived cell lines as well as in normal prostate, and in three prostatic intraepithelial neoplasias (pin) and in twenty primary prostatic carcinomas by immunohistochemistry. results parkin protein was undetectable in six out of eight prostate cancer cell lines tested and barely detectable in the remaining two whereas normal prostate showed a substantial expression. preliminary results using immunohistochemistry showed that parkin expression was absent in pin lesions, and in the prostatic carcinomas analyzed. conclusion our findings suggest that parkin might be a useful tool to detect early alteration in prostate cancer. the kdr/flk- receptor of vascular endothelial growth factor in urothelial bladder cancer: relation with clinicopathologic parameters and patients' survival introduction the impact of different angiogenic inducers and inhibitors in the switch of angiogenesis in urothelial bladder cancer, is not clarified. kdr/flk- is a receptor-tyrosine kinase that binds specifically vascular endothelial growth factor (vegf) on vascular endothelium, mediating its full biologic spectrum. although kdr/flk- expression was at first reported to be restricted to the classic endothelium there is increasing evidence that kdr/flk- is also expressed by normal and tumor cells other than endothelial. aims: as data about kdr/flk- protein in urothelial bladder cancer is very limited, we investigated its expression in a series of urothelial carcinomas in relation with clinicopathologic parameters and patients' survival. moreover, prompted by studies suggesting that kdr/flk- may have broader functions apart from angiogenesis we evaluated kdr/flk- expression in relation with markers of cellular proliferation and apoptosis (ki- , p , bcl- ). materials and methods immunohistochemistry (abc-hrp) was performed for the detection of kdr/flk- (mouse/a- , sc- , santa cruz), ki- , p and bcl- using monoclonal and polyclonal antibodies. statistical analysis was univariate (pearson's x , mann-whitney test, log-rank test) and multivariate (cox's model). results kdr/flk- expression was observed in the cytoplasm of cancerous cells in / cases. no significant associations were observed between kdr/flk- expression and clinicopathologic parameters, ki- , p and bcl- expressions. on the other hand, widespread kdr/flk- expression in more than % of cancerous cells, was associated with patients' increased survival, in univariate and multivariate analysis (p= . , p= . , respectively). conclusion although non endothelial kdr/flk- expression has not yet been elucidated, its association with better patients' survival may be related with the failure of non-endothelial kdr/flk- to mediate angiogenic and mitogenic effects, that has been reported in some previous in vitro studies. timp- protein expression in relation with cellular proliferation, apoptosis and survival of urothelial bladder cancer patients introduction the role of tissue inhibitors of matrix metalloproteinase (timps) in cancer progression remains a controversial matter since apart from the well established inhibitory action of timps on mmps, several new functions are now attributing to timps such as growth promoting activity, involvement in apoptosis control and in angiogenesis. the aim of this study was to elucidate the role of timp- in urothelial bladder cancer, we investigated its expression in a series of urothelial carcinomas in relation to clinicopathological parameters, patients' survival and the expression of the ki- proliferation index. materials and methods immunohistochemistry (abc-hrp) was performed for the detection of timp- and ki- proteins, using monoclonal and polyclonal antibodies. statistical analysis was univariate and multivariate. results timp- stromal cell expression was observed in / cases and associated significantly with urothelial carcinomas of high histologic grade (p< . ) and advanced stage (p= . ) as well as with poor patients' survival (p= . ). timp- expression in cancerous cells was found in / cases. timp- either stromal or cancerous cell expression correlated significantly with ki- expression (p= . , p= . respectively). conclusion the association of timp- with ki- supports the growth promoting activity of timp- . moreover, timp- expression in stromal cells may be a valuable marker of adverse prognosis in urothelial cancer patients. gleason score is important predictor for differentiation and biological potential of prostate cancer. nodenegative (p n ) prostate cancer is the best result after radical prostatectomy which is indicated in patients with localised prostate cancer. the aim of the study was to analyze gleason score in patients with nodal metastases of prostate cancer (pc). material and methods we analyzed cases of radical prostatectomies due to localised prostate cancer in the period - in clinic of urology in clinical centre of serbia, belgrade. nodal metastases (stage of disease), average age of the patients, grade and gleason score of tumors and premalignant lesions were analysed. immunohistochemical staining was performed and pathological analyzed. we used statistical analysis anova and h test. results the average age of the patients was . years (range - , pick - ). most cases ( %, p< . ) were in pt a n m , in pt b n m ( . %), in pt bn m ( . %), in pt bn m ( . %), in pt an m ( . %). positive nodal status was present in cases: ( %) in pt bn m -iliac (right , left ), obturatory (right l, left ) and cases in pt bn m -iliac left and obturatory ( right and left). we found gleason score in cases ( . %) in pt bn m versus cases ( . %) without nodal metastases. gleason score was found in cases ( . %) in pt bn m versus one case without nodal metastases (p> . ). gleason score was present in . %, in . %, in . %, in . %, in . %. grade tumors were found in cases ( %), grade in ( %), and grade in ( %). hg pin was present in cases ( . %), lg pin in ( . %). conclusion gleason score differ, but not significantly in prostate cancer with nodal metastases. pelvic lymphadenectomy is necessary for staging purposes in prostate cancer. radical prostatectomy is the most adequate method in surgical treatment of localised prostate cancer. immunohistochemical and molecular evaluation of c-erbb gene amplification and c-erbb protein overexpression in prostate cancer the amplification of oncogene c-erbb and overexpression of c-erbb protein found in some human cancers is associated with more aggressive development of tumor. the previous data suggested an independent role of c-erbb protein as a prognostic factor in human breast cancer. however, the role of c-erbb gene and its protein product in prostate cancer cells still remain unclear. the aim of our study is to show what is the role of c-erbb protein in prostate cancer and to examine association between c-erbb gene amplification and its protein product expression in the histologicaly graded prostate tumor. we have analyzed c-erbb gene amplification by rt pcr and c-erbb protein expression by immunohistochemistry in cases of paraffin embedded slides. the tumors were divided according to who by mostofi classification: cases of g -well differentiated cancer; cases of g -moderately differentiated cancer; and cases of g poorly differentiate cancer. these groups are equivalent to - , - and - gleason classification respectively. results the correlation between c-erbb protein expression and well, moderately and poorly differentiated cancer respectively existed (p< . ). there was no significant correlation between c-erbb gene amplification and c-erbb protein expression in all examined groups. our results suggest that the c-erbb protein expression is an independent factor on the c-erbb gene amplification in prostate cancer cells. it is possible that there are other ways, except gene amplification, for overexpression of c-erbb protein in prostate cancer. comparison of needle biopsy and surgical specimen in prostatic carcinoma s nordling, j vasama department of pathology, university of helsinki, helsinki, finland in finland carcinoma of the prostate is the most common form of cancer in men. the number of radical prostatectomies is increasing in most countries. the radical prostatectomy is normally preceeded by a needle biopsy. at the university central hospital of helsinki radical prostatectomies were performed between and . in patients, the preoperative needle biopsy was available. in many cases the preoperative psa value was also available. the mean psa value dropped from to less than ng/ml. the percentage of seminal vesicle invasion dropped from to . except for the earlier years, the men gleason score was about point higher in the radical prostatectomy specimen than in the biopsy. the mean gleason score has increased, while the stage and psa values have decreased indicating that the criteria for the gleason grading has changed ovewr the years. this makes the comparison of treatment results very difficult. introduction distinguishing atypical adenomatous hyperplasia (aah) from small foci of prostatic adenocarcinoma in needle biopsies of the prostate often presents as a challenge for pathologists. the aim of the study was to establish characteristic immunohistochemical patterns in benign and malignant prostatic lesions so they can be used to classify foci of aah as benign or malignant. materials and methods using immunohistochemistry, prostatic needle biopsies from forty-five patients were evaluated for cytokeratin , p and alpha-methylacyl-coa racemase (p s). twenty-five of these biopsies were benign and twenty contained prostatic adenocarcinoma. results % of the benign prostatic biopsies were positive for cytokeratin and p and % were negative for p s. only one case of benign prostatic tissue was focally positive for p s. when evaluating cases with adenocarcinoma % were positive for p s and % were negative for cytokeratin and p . p s immunostain showed consistently positive staining in areas of low and intermediate grade adenocarcinoma and areas with high grade pin. high-grade prostatic adenocarcinoma was frequently negative for p s. sensitivity and specificity for cytokeratin /p were % and % while sensitivity and specificity for p s were % and %. conclusion cytokeratin was as sensitive and as specific as p and can be used interchangeably in cases of aah. the combination of the two antibodies did not improve sensitivity or specificity. a combination of negative cytokeratin or p with positive p s may be helpful in positively identifying prostatic carcinoma in cases of aah. myofibroblastic stromal reaction in prostatic adenocarcinoma introduction reactive stromal changes that occur in different human cancers probably are involved in local tumor spreading and progression. it seems that myofibroblast plays very important role in stromal reaction to certain types of human cancer. we have analyzed prostatic carcinomas in radical prostatectomy specimens to determine the proportion of myofibroblasts around normal and neoplastic acini. myofibroblast showed positive staining for alpha-smooth muscle actin (sma) and vimentin. positive cells were counted in the hot spots areas under high power magnification (hpf). results were expressed as negative (-) for no staining, (+) for positive staining in up to % of stromal cells, (++) in - % of cells and (+++) for more than % of positive cells. stromal blood vessels were counted in ten hpf. the degree of desmoplasia was also analyzed by mallory staining and expressed semiquantitatively as weak, moderate and strong. results and conclusions highly increased number of myofibroblast was observed around neoplastic acini in ( . %) cancer cases, moderate in ( . %) and sparse in ( . %) cases while there were occasional myofibroblastic cells adjacent to normal acini. there was a positive correlation between the degree of stromal desmoplasia and the proportion of sma and vimentin positive cells in adjacent stroma. the number of vessels in the adjacent stroma was also significantly increased in comparison with normal glands (p< . ). it seems that myofibroblasts are present in the stroma and may play a significant role in the desmoplastic stromal reaction in prostatic adenocarcinoma. results among the srcc, ( %) showed no epithelial differentiation, and in the other cases the epithelial component was conventional rcc in cases ( %), papillary ccr in ( %), and chromophobe rcc in ( %). there was no sex predominance and the average patient age at the time of diagnose was years. sixteen cases ( %) presented an advanced stage at the time of diagnose, and ( %) died of disease. the immunohistochemical study showed that cases were positive for c-kit ( . %), diffusely (> % of the sarcomatoid cells positive), moderately ( - % of the cells), and focally (< % of the cells). conclusion the high expression of c-kit in srcc in our series and the existence of target therapy against cells expressing this marker (imatinib (sti- , gleevec, novartis, basel, switzerland) suggests its potential use in the clinical management of these patients in order to improve their prognosis, especially in disseminated cases. does hpv play a role in the development of bladder tcc? a comparison of pcr and immunohistochemical detection methods the role of human papilloma virus (hpv) in the development of bladder carcinomas remains controversial. varying techniques have been used to identify this virus in transitional cell carcinoma (tcc). here we used two different methods to detect the presence of mucosal hpv in tcc samples. the first, immunohistochemistry, involved using a polyclonal antibody that reacts with the l capsid protein of most known papilloma viruses. the second method is used on a general primer-mediated polymerase chain reaction (pcr) assay. this involves using type-specific oligonucleotide probes to identify different mucosal hpv genotypes from a bp pcr product from the l region of the hpv genome. the two methods produced contrasting results, % of the samples were positive for hpv antigen and % were positive for hpv dna. the reason for such conflicting results is that polyclonal antibodies are not specific and are likely to cross react with other antigens. pcr is specific, more sensitive, and it is widely used to detect hpv in a variety of clinical samples. the results obtained in our study implies that hpv does not play a role in the development of bladder carcinoma, and pcr remains to be the best method to detect hpv in tcc. does tgf-α prevent carcinoma spreading? o gladskikh introduction most human carcinomas including prostate cancer are associated with stromal reaction typified by changes in growth factor expression and over-accumulation of extracellular matrix. our study deals with the differential tgf-α production in neoplastic focuses and in adjacent paraneoplastic stroma in prostate. materials and methods tissue samples from radical prostatectomies were examined. to verify malignant material or benign the immunohistochemical staining against high molecular weight cytokeratin was performed on sections and in heterogeneous primary cultures of the same specimens. the immunohistochemical identification of tgf-α was also performed. the elisa technique for quantitative analysis of tgf-α soluble form in culture supernatants was applied. results majority of selected cancer cases were histologically estimated as small-sized glands with large desmoplastic fibromuscular stroma. the immunohistochemical staining for tgf-α in malignant focuses was much more significant than in neighbouring benign areas on sections in contrast to, the cultures obtained from neoplastic or paraneoplastic portions of prostate tissue which stained positively for tgf-α without any difference. considering that immunohistochemistry shows only latent tgf-α we evaluated its bioactive form in culture supernatans. we found dramatically high supernatant levels of active tgf-α in benign cultures compared to the cultures from the malignant areas. conclusion our data demonstrate that desmoplastic alterations in benign areas adjacent to malignant glands was associated with elevated expression of tgf-α active form. so significant alteration in factor production and accompanied tissue remodeling can be considered as a protective reaction of surrounding stroma against tumor invasion. introduction primitive neuroectodermal tumors (pnets) represent a family of neoplasms, of presumed neuroectodermal origin, that mostly presenting as bone or soft tissue masses in the trunk or axial skeleton in adolescents and young adults. the aim of this study was to report a case of pnet arising from kidney which is a rare localization. methods a year-old-man presented with abdominal pain and abdominal mass. the imaging methods revealed a mass nearly replacing the right kidney and he underwent a right nephrectomy. on gross examination the tumor measured cmx cm with cystic areas and necrosis. microscopically, it was a highly cellular neoplasm of consisting sheets of small-round-to-oval cells with irregular nuclei. immunohistochemical stains were positive for vimentin and cd . microscopic and immunohistochemical findings were compatible with pnet. conclusion although rare, pnet must be included in the differential diagnosis of renal tumors especially in children and young adults. we present a case of renal pnet as an unusual case and discuss the differential diagnosis pathologic stage of urinary bladder cancer at presentation in a non-selected patient population in a high-incidence region of the united states introduction most studies of pathologic stage in bladder cancer (ca) are retrospective, using selected, usually hospital-based, patient populations. the aim of this study was to determine the pathologic stage of bladder ca at initial presentation in a nonselected patient population in a high-incidence region of the northeastern united states. materials and methods all patients newly presenting with bladder ca in the state of new hampshire (u.s.a.) from july to december were identified through the state tumor registry. slides were classified according to who and isup criteria. results of cases, were diagnosed as non-malignant or technically inevaluable, leaving ca cases. ( . %) were carcinoma-in-situ (stage tis). ( . %) were papillary, of which ( . %) were low-grade and ( . %) high-grade. of the low-grade cases, only ( . %) were invasive, all limited to lamina propria (stage t ). of the high-grade cases, ( . %) were noninvasive (stage ta), ( . %) invaded lamina propria (stage t ), and ( . %) invaded muscle (stage ). cases ( . %) had no papillary component and all were invasive; ( . %) were stage t and ( . %) were stage t . cases ( . %) were nonurothelial, including squamous cell ca, small cell ca, and adenoca; of these presented as stage t and as stage t . overall, cases ( . %) were invasive (at least t ) at the time of presentation. this population-based series establishes the distribution of bladder tumor stage at initial presentation and documents a smaller percentage of invasive cancers than most hospital-based series the relationship between mitotic index and nucleolar organizer region with histologic grade in transitional cell carcinoma of urinary bladder mr jalali nadoushan, t rezaei nour, n fallah, sk foroutan shahed university, tehran islamic republic of iran background and object transitional cell carcinoma (tcc) of urinary bladder is the second most common cancer of the genitourinary tract, that the prognosis and treatment is related to several parameters such as proliferative cell markers. in this study relationship between two proliferative cell markers: -mitotic index(mi)& -agnor and tumor grade were studied. methods and materials in a cross sectional descriptive study paraffin blocks of patients with tcc of the urinary bladder in the period from to from pathology department of shahid mostafa khomeini hospital, were obtained and from each one micron sections were provided. tumor grade on basis of who/isup grading system, was determined and number of mitosis on tumor cells were counted. in the other section, after one-step silver nitrate staining, the number of nor also on tumoral cells were counted and the mean of it in each cell were determined. results the high grade tumors possessed more agnor per nucleus than did the low grade ones. significant relationship between tumor grade and mi (p= . , rs= . ) and agnor (p= . , rs= . ), and also between mi and agnor (p= . , rs= . ) were found. introduction the human epidermal growth factor receptor (her ) is overexpressed not only in breast, but in a range of other tumor types including, ovarian, salivary gland, endometrial, pancreatic, non-small cell lung (nsclc) and bladder cancer. however, non-standardisation in her status evaluation or population selection has generated conflicting results among the incidences of her expression in individual tumor types and bladder cancer. the aim of the present study was to evaluate her overexpression and gene amplification with standardized routinely performed assays (dako herceptest, fish-vysis, cish) on a selected population of poorly differentiated urothelial carcinomas of the bladder. materials and methods we analyzed formalin-fixed, paraffin embedded tissue samples of urothelial carcinomas of the bladder at g /pt - as well as cases at g / ptis ( )and pta- ( ) using herceptest to verify the her -status. staining and scoring was performed according to the manufacturers instructions. according to diagnosis of her , all herceptest positive ( +) tissue samples were used for further evaluation with fluorescence in situ hybridisation (fish; vysis) and chromogenic in situ hybridisation (cish) to approve the expected gene amplification. this procedure is in agreement with the standardized guidelines for breast cancer. results ( %) cases were considered as her positive ( + and +) in the primary tumor and therefrom cases ( %) + positive and cases ( %) + positive. out of the cases with + positivity, only two cases showed a fish/cish positivity. no polysomy for chromosome was detected. our results indicate that her may not only be a useful prognostic parameter in poorly differentiated invasive bladder cancer, but accurate determination of overexpression and verification of gene amplification may also have therapeutic importance. the prognostic importance of tumor volume (tv), nontumor volume (ntv), tv/ntv, fibronectin distribution, β-hcg and psa expression in localized prostatic adenocarcinoma bh ozdemir, an sar, fp uyar, b demirhan, a dirim baskent university, faculty of medicine, departments of pathology and urology, ankara, turkey aim to assess the importance of fibronectin (fn) distribution, β-hcg and psa expression in localized prostatic adenocarcinomas with regard to tumor volume (tv), non-tumoral volume (ntv), tv/ntv and with other clinicopathological prognostic parameters. material and method fifty-one radical prostatectomies were whole mounted, serially sectioned and examined for tv, ntv, tv/ntv, capsular invasion, vascular invasion, seminal vesicle invasion and gleason score. the fn distribution, β-hcg and psa expression were examined immunohistochemically. a fn-positive staining was defined as a constant diffuse, or pericellular demarcation of fn-positive fibers surrounding tumor cells at the invasive border. in lack of such staining pattern, fn-negative staining was recorded. results both tv and tv/ntv showed very strong positive correlation with grade, capsular invasion, vascular invasion, seminal vesicle invasion, β-hcg and psa expression (p< . ). β-hcg was detected only in cases ( . %). a significant relationship was found between β-hcg expression and grade, capsular invasion, vascular invasion, seminal vesicle invasion, psa expression (p< . ). the fn-positive staining reaction was significantly associated with lower incidence of capsular, and seminal vesicle invasion (p< . ). tv and grade was found higher in tumors with negative fn staining (p< . ). psa expression showed significant relationship with grade, vascular and capsular invasion (p< . ). ntv statistically showed no relationship with all parameters. conclusion these data suggest that, tv, tv/ntv, fn distribution, β-hcg and psa expression in prostatic adenocarcinoma have strong prognostic importance irrespective of gleason score. the fn distribution could be important for the invasion of the tumor cells during cancer progression. these may allow stratification of patients to type of treatment and may allow selection of expectant management for men with localized prostatic adenocarcinoma. neuroendocrine differentiation in prostate cancer: correlation with biochemical failure after radical prostatectomy s cerovic introduction focal neuroendocrine differenatiation (ned) is a commom feature of prostate cancer (pc), occuring in %- % tumors studied. published results of the evaluation of ned as a prognostic factor in pc after radical prostatectomy (rp) have been contradictory. the aim of this study was to correlate ned in pc in relation to biochemical failure after rp. materials and methods radical prostatectomy specimens from pc patients without preoperative hormonal or radiation therapy were followed for to months. neuroendocrine (ne) cells were identified using a panel of immunohistochemical ne markers: chromogranin a, serotonin, and neuron-specific enolase. ned was scored as ne-negative ( to +) or ne-positive ( to +) per hpf. biochemical progression (bp) was defined as a prostate-specific antigen (psa) level greater than . ng/ml in two consecutive measurements. kaplan-meier method was used to estimate bp-free survival time for each risk group. the cox proportional hazards regression analysis was the model for bp time using pathological and clinical variables. results the total of ( %) patients developed bp after rp. the average time for bp was months. the preoperative psa serum level range < ng/ml in % of the patients. postoperative staging confirmed that ( %) of patients had pt stage and ( %) of patients had pt stage. ned demonstrated in the ( %) pc patient with bp. the average time for bp was months. tumor volume (p< . ), pathological stage (p < . ), tertiary gleason grade (p= . ) and nedf (p< . ) significantly affect the level of postoperative psa progression. conclusion the expression of ned in pc correlates with tumor volume and tertiary gleason grade and represents an independent prognostic factor of bp after rp. expression of vascular endothelial growth factor (vegf) and correlation with microvessel density in benign hyperplasia and prostate carcinoma nj aim the present study is scheduled to evaluate the levels of vegf expression in benign prostatic hyperplasia (bph) and prostate carcinoma and to correlate them with angiogenesis. material and method formalin-fixed, paraffin-embedded tissue sections from patients who underwent radical prostatectomy or transurethral resection of the prostate were studied. our material consisted of cases of bph and cases of prostate carcinoma. microvessel density (mvd), as determined by cd , and expression of vegf were investigated by immunohistochemistry using the monoclonal antibodies cd (dako) and vegf (neomarkers), respectively. results expression of vegf was detected in / ( , %) cases of bph and in all ( %) invasive prostate carcinomas. cytoplasmic immunoreactivity for vegf was detected in epithelial and stromal cells. vegf expression was stronger in carcinomas, when compared to bph (p< . ), while it was minimal to absent in normal adjacent prostate tissue. a positive correlation between vegf expression and gleason score was found (p< . ). microvessel density (mvd) was higher in bph and prostate carcinomas, when compared to normal adjacent prostate tissue. a statistically significant association was noted between vegf and mvd expression (p< . ). our results indicate that generation of new capillary blood vessels begins in bph and keeps progressing in invasive prostate carcinoma. vegf seems to be a key functional regulator of angiogenesis in the prostate gland. the relationship between histologic grade and abo blood groups in transitional cell carcinoma of the urinary bladder b mofid , mr jalali nadoushan shahid beheshti university of medical sciences, tehran, islamic republic of iran shahed university, tehran, islamic republic of iran introduction according to the statistics of , the urinary bladder cancer is the fifth common cancer in iran. the majority of urinary bladder tumors are transitional cell carcinomas. different factors are effective in the prognosis of these patients. one of these factors is the histologic grade. another factor is to determine the abo blood groups and the relationship of it with the histologic grade. thus, this study is conducted to shed some lights on this issue. materials and methods a cross sectional study was conducted in patients with urinary bladder transitional cell carcinoma during - . results of patients, were female ( . %) and were male( . %). the mean age of patients was . +/- . there was no significant statistical relationship between of histologic grade and abo blood groups. there was no significant differences between the abo blood groups frequency in patients with urinary bladder transitional cell carcinoma and general population. conclusion although the existing differences between asian, european and american studies might be due to racial effects, it is better to carry out more studies in this respect in order to comment in this area with more confidence and certainty. introduction a variable degree of mucin secretion may be present in prostatic carcinoma (pc), but true mucinous tumors are rare. prostatic mucinous carcinoma (pmc) is a variant of high-grade adenocarcinoma, with a . % rate of prostate-specific antigen (psa) elevation and a similar rate of response to endocrine therapy. the aim of this study was to report on a case of pmc with signet ring cells treated with radical prostatectomy (rp). materials and methods a -year-old man presented with acute urinary retention and long history of difficult voiding. the serum psa was . ng/ml. the transperineal biopsy of the prostate gland was done and the histology showed a poorly differentiated pc with large lakes of extracellular mucins. isotope bone scan was normal and a ct-scan did not show evidence of extracapsular spread of the tumour. radical prostatectomy was performed. patient died seven month after rp. results histopathologically, over % of the prostate samples were infiltrated by a poorly differentiated pc with mucinous and signet ring patterns. mucin secretion is evident as large pools of mucin and periodic-acid-schiff-positive diastase-resistant intraluminal and citoplasmic globules. immunohistochemical staining for psa and prostate-specific-acid phosphatase was positive, and negative for carcinoembryonic antigen and cytokeratin . conclusion the diagnosis of pmc with signet ring cells requires exclusion of an extra-prostatic origin, mainly within digestive tract. since only small number of mpc cases treated with rp have been reported, treatment and prognosis of this tumour remain unclear. introduction cd is a well-known marker for hematolymphoid neoplasms and is expressed as well in various nonhematopoietic tissues and solid tumors. a systematic study of cd expression in tgcts, however, is lacking. the aim of this study was to investigate the immunoreactivity for cd in the different patterns of tgcts. material and methods immunostaining with mab against cd (clone c ) in unselected cases of primary ( cases: classical seminoma (cs); pure embryonal carcinoma (ec); mixed-non-seminomas; spermatocytic seminoma (ss) and metastatic ( cases) tgcts. in mixed tumors, the different histological patterns were evaluated separately. results cd was expressed in ( %) out of cases in the cs component, in ( %) of teratomas, in ( %) of endodermal sinus tumors (est), in ( %) of cases of choriocarcinoma (chc) or syncytiotrophoblastic giant cells (stgc) and in ( %) of ec. cd was focally positive in ( %) of cases of intratubular germ cell neoplasia (itgcn) and was negative in one ss. conclusions except for ec and ss, cd is expressed in the majority of histological types of primary and metastatic tgcts. cd is useful to highlight minute foci of est, chc or stgc in ec. a tgct is to be taken into account in the differential diagnosis of a cd positive metastatic tumor of unknown origin. further studies are needed to evaluate the eventual significance of cd as a prognostic factor or as a potential therapeutic target in tgcts. introduction it is generally accepted that the prognosis of renal cell carcinoma (rcc) differs according to the histological type, and, whatever the histological type, the tumor stage and nuclear grade are considered the main prognostic factors. however, in many cases of conventional renal cell carcinomas (crcc), these parameters are insufficient to predict the clinical behaviour of these tumors. therefore, several studies have focused on the evaluation of new markers. the cd proteins form a broadly distributed family of cell surface adhesion molecules involved in cell-cell and cell-matrix interactions. de novo expression of cd s and its variant isoforms has been associated with aggressive behaviour in various tumours. the aim of this study was to analyse the prognostic value of cd s and cd v in renal cell carcinoma. materials and methods the expression of cd s and cd v was immunohistochemically evaluated on rcc: were crcc, papillary, cromophobe and collecting duct carcinoma. the expression was compared to usual clinicopathological parameters. results in normal renal tissue no staining was observed. the cd s molecule was upregulated in crcc ( %), papillary, one cromophobe and collecting duct carcinoma. only tumors were focally cd v positive. the statistical analysis showed the correlation between cd s expression and fuhrman nuclear grade (p< , ) as well as ki proliferative index (p< , ). conclusion the expression of cd s molecule appears to mediate a more agressive phenotype to rcc. amplification of cerbb expression in bone metastasis of prostatic cancer: a pilot immunohistochemical and cytogenetic study introduction her- -neu expression is reported to increase in androgen-independent prostatic cancer. consequently, it has been suggested that trastuzumab, an antibody targeting the extracellular domain of her- , could be used in this setting. the possibility of assessing her- expression by immunohistochemistry could be used as a rationale for the treatment of advanced cases with this drug. the aim of this study was to assess whether her- expression is increased in bone metastases of prostatic cancer compared to the respective primitive tumors, and to determine whether this overexpression could be related to gene amplification. materials and methods in this pilot study, primary tumor needle biopsies and bone metastases from patients diagnosed with prostatic cancer were immunostained wit c-erb-b antibody (dako, glostrup, denmark), and the results scored using a threetiered system. in addition, in a bone marrow sample from one of the cases, her gene amplification was investigated using fish. results there was a significant increase in her- expression in four cases, with the same level of expression in primary and metastatic tumors in the remaining three patients. in addition, fish showed no amplification of her gene. conclusion her- overexpression is not a constant event in prostate cancer metastatic to bone. preliminary evidence suggests that in cases with overexpression, it is not related to gene amplification. a larger study will be required, however, to definitively determine the real therapeutic meaning of these findings. introduction sgs are known to occur in association with a variety of hematologic and solid neoplasms. concerning rcc-associated sgs, only isolated case reports have been published. the aim of this study was to disclose the prevalence and clinical significance of sgs in a series of rccs from a single institution. clinicopathologic review of consecutive cases of rcc surgically treated at the hub between january- and march- . immunohistochemical staining with cd , panb and pant antibodies. results sgs were seen in cases, both within the tumor and/or in the nontumor tissue nearby. patients were females and males aged to (median ) years. tumors were conventional clear cell rccs and chromophobe rccs. there were pt , one pt and one pt b. two tumors were fuhrman grade and were grade . in each case, the lymphocytes in the sgs were predominantly t cells. one patient was already known to have a crest syndrome. in another one sarcoidosis was subsequently diagnosed. in the remaining no systemic disease has been found to date. conclusions sgs are found in about . % of rccs. morphology and immunohistochemistry allow no distinction between local sarcoid-like reactions and sarcoidosis. the clinical profile of the patients and the pathologic features of the tumors are not different from those of rccs unassociated with sgs. the finding of tumorassociated sgs must be reported to the clinicians in order to rule out a systemic granulomatous disease, thus avoiding misinterpretation of clinical symptoms or overstaging of tumors. metanephric adenoma in the kidney coexistent with paraganglioma in the bladder: a case report s seckin, i paker ankara numune teaching and education hospital, department of pathology, ankara, turkey we report a case of metanephric adenoma in the kidney and paraganglioma in the bladder in a year-old woman. the patient presented with hematuria. abdominal usg and bt revealed a mass in the lower pole of the kidney and in the posterior wall of the bladder. left nephrectomy and transurethral resection of the mass in the bladder were performed. the tumor in the kidney which measured x x cm. had a yellow to white cut-surface and welldefined margin. histological examination demonstrated a monomorphous population of small cells with bland cytology arranged generally in tubular and occasionally in papillary structures. no atypia and mitotic activity were present. immunohistochemically tumor was diffusely positive for ema, focally positive for pancytokeratin and negative for vimentin. the tumor in the bladder was characterized by well-defined nests of cuboidal cells with granular cytoplasm which were seperated by highly vascularized fibrous septa. sustentacular cells were demonstrated with anti-s protein immunohistochemically. we didn't encounter the coexistence of these two tumors in the literature. we think that it is a coinsidence. co-occurrence of extra-adrenal myelolipoma, renal cell carcinoma and thymoma -a case report k steplewska, d sabat, w zajecki department of pathomorphology, medical university of silesia, zabrze, poland a case of never reported in literature of co-occurrence of a very rare tumour -extra-adrenal myelolipoma (eaml) with two other neoplasms, was presented in the report. -year man after a short hospital stay due to heart ischaemic disease died with the symptoms of acute circulatory insufficiency. an autopsy, which confirmed clinical diagnosis, was performed. a tumour of the size x x cm, which compressed the heart and aorta, was found during the mediastinum autopsy. the tumour was covered with a sack, lobular, gray and beige at the cross-section with blood extravasation, necrosis foci and calcifications (histopathologic result -medullary thymoma). the compressed heart was moved to the middle, right side of the chest. in the adipose capsule of the left kidney, near the lower pole, an encapsulated solid fatty tumour of the size x x cm was found. the tumour was connected to the kidney. histopathologic result -myelolipoma. the tumour was composed of fat cells with normal marrow hematopoietic elements including megakaryocytes. the third tumour, cm of diameter, was found on the front surface of the right kidney (histopathologic result -multilocular cystic clear cell, renal cell carcinoma). none of the tumours was diagnosed during the patient's life. signet ring cell carcinoma of the prostate: pathologic and immunohistochemical analysis of two cases m kowanski, t zabkowski military medical institiute, warsaw, poland introduction signet ring cell carcinoma of the prostate is extremely rare and was first described by giltman in . to date, there have been cases reported in the literature. we evaluated two cases formaline fixed paraffine embedded tissue by histochemical (mucicarmine, alcian blue) and immunohistochemical methods (psa, psap) to confirm our histological diagnosis. results both our cases were high grade adenocarcinomas gleason score ( + ) and were positive for psa and psap. one case contain a small amount of acid mucin, but both were negative for neutral mucin. the combined results from our and from previously reported cases: psa positive in . % ( / ), mucicarmine stain positive in . % ( / ) and alcian blue stain positive in . % ( / ) of cases. conclusion signet ring cell carcinoma is variant of high grade adenocarcinoma of the prostate but results of histochemical and immunohistochemical staining are variable. the isup classification of urothelial tumors was followed by a modified who classification in . the purpose of this study has been to assess the value of both systems in the prediction of progression, in a large series of cases with detailed follow-up. material and methods "superficial" (pta + pt ) urinary bladder tumors are the subject of this work. all tumors were reassessed strictly adhering to published criteria. progression (increased grade or stage) was used as the end-point. results with a relative risk (rr) of for g , progression rr for g was . ( . - . ) and for g . ( . - . ) (p< . ). with a risk of for pta, rr for pt was . ( . - . ) and for pt . ( . - . ) (p< . ). when combining grade and stage, rr for pta (g + ) was . ( . - . ) and for pt (g + ) was . ( . - . ) (p< . ). conclusions strictly adhering to the who grading and staging criteria, both systems result in a very efficient prediction of progression of urothelial carcinoma. grouping g and g into a single high grade category does not improve these results. "superficial" infiltration (pt ) introduces a relevant risk of progression when restrictively identified, thus suggesting that pt should not be lumped with pta for therapeutic purposes. neuroendocrine expression in prostate cancer l velickovic , v katic , v savic , b djordjevic , s zivkovic institute of pathology, nis, serbia and montenegro military hospital, department of surgery, nis, serbia and montenegro introduction presence of neuroendocrine (ne) cells that are suggested to be important regulators of cell growth and differentiation represents uncommon finding in benign and neoplastic prostate epithelium. in order to correlate cellular expression with histological differentiation in benign epithelium and primary prostate cancer, expression of chromogranin a (cga) had been determined. materials and methods he and immunohistochemical analysis with antibody against cga was performed in order to determine the coexistence of ne differentiation in tissues of twelve patients with prostatic adenocarcinoma (pc). results ne differentiation was found in ( , %) tumors. among them, ( , %) had cga positive cells scattered or focally distributed, ( %) intermediated and intensive. there was a significant correlation between the extent of ne features and gleason score (p< , ). these data support the concept that ne differentiation in human prostate cancer has a positive correlation with histological differentiation, i.e. patients with gleason score > had higher cellular expression of cga. the p retinoblatoma gene protein and p expression in transitional cell tumors s ramón y cajal , c corbacho , v fernández, j guinea, c parada, r sánchez hospital vall d´hebron, madrid, spain clínica puerta de hierro. madrid, spain background the retinoblastoma family of proteins (p , p and p ) are implicated in cell cycle regulation, differentiation and apoptosis. p (retinoblastoma supressor gene) alterations seem to be correlated with worse prognosis in urothelial neoplasms, and the status of the other two proteins has been less studied. p mutations are usually associated with intranuclear accumulation of p protein, and have also been correlated with worse prognosis in urothelial neoplasms. design formalin-fixed, paraffin-embedded archival tissue from low-grade and high-grade transitional tumors was immunostained with polyclonal and monoclonal antibodies against the retinoblastoma family of proteins, and with monoclonal antibody against p protein. the avidin-biotin-peroxidase (abc) method was utilized with every antibody (dako, clone rb for p ; santa cruz, clone sc- for p ; santa cruz, clone sc- for p and dako, clone do- for p ). immunostaining was scored quantitatively by pathologists, following previously described criteria, to study p , p and p . nuclear and/or cytoplasmic location was determined by pathologists to study p . results p expression: among low-grade tumors, ( . %) showed nuclear positivity and ( . %) showed a granular cytoplasmic pattern of staining. among high-grade tumors, ( . %) showed nuclear expression and ( . %) showed a granular pattern of staining. p expression: among low-grade tumors, ( . %) showed positivity and among high-grade tumors, ( . %) stained positively. we found no stadistically significative differences regarding p and p immunohistochemical stainings. conclusion due to granular cytoplasmic pattern of staining for p is more frequent among high-grade transitional neoplasms, we propose that p granular cytoplasmic immunohistochemical pattern of staining may be considered a marker of tumor progression in transitional cell tumors. the prognostic value of p cytoplasmic positivity among low grade cases, as well as the prognosis of tumors with p overexpression and p granular cytoplasmic pattern will be discussed. are there any relationship between bellini duct carcinoma and urothelial cell carcinoma? ( ) aexudative -at , productive -at , and mixed -at ., from them ( ) infiltrative -at and destructive -at patients, aexudative type of inflammation was also diagnosed at patients with nonspecific defeat of prostata. patomorfological data's not always can help in diagnostic of tuberculosis of prostate it ties with patomorfose of tuberculosis under influence of treatment. cancerogenesis in prostate: ploidometric differential diagnostics avtandilov background the problem of unification in diagnostics of various prostate tumours and tumour-like lesions has gained a special significance in the modern oncology. aim: use the ploidometry research for studying the epithelial cell clones in the prostate, forming the different stages of cancerogenesis. methods computer ploidometry test was carried out for nuclei of epithelial cells in biopsy specimen of the prostate, taken from patients in different stages of cancerogenesis. histological preparations of micron thickness, stained by feulgen's technique, were subject of the research. nuclei's ploidy was defined by means of the computer image analyzer "imager-ch" ("avtan-san" software version). the integral brightness of nuclei of small lymphocytes, found in histological sections, was tested to define the "tissue ploidy standard" ( c), which, in its turn, was used for obtaining the data on ploidy of epithelial cell nuclei of the prostate gland. inter-phase cell nuclei of clones with normal and hyperplastic prostate gland, areas of prostate intraepithelial neoplasia (pin-i-low grade -and pin-ii-high grade) and adenocarcinoma in different stages of dedifferentiation (well, moderate, poorly) were the subjects of investigation. the research determined that while the cancerogenesis progressed, the proliferative activity (indirect type information) of prostate gland cells and the ploidy of nuclei naturally increased. introduction calcitriol and its analogues affect function of several normal and neoplastic cells, including cells of medullary thyroid carcinoma originating from parafollicular cells. in the cells expression of calcitonin gene takes place, yielding due to alternative splicing calcitonin (ct) and calcitonin gene related peptide (cgrp). the aim of this study was examination of effects of calcitriol and of its two analogues, pri- and pri- on proliferation of two cell lines in vitro and on expression of calcitonin gene. the studies were performed on mtc - (rat cell line) and tt (human cell line) cells originating from medullary thyroid carcinomas. the cells were cultured with calcitriol or its analogues at (- ) to (- )m for days. the estimated variables included: cell number, proliferation-associated antigens (ki- , pcna), ct and cgrp levels in medium (ria), amounts of the two hormones and of their mrnas in the cells (immunocytochemistry or hydridocytochemistry accompanied by image analysis). results calcitriol and its analogues exerted low antiproliferative effect, more evident in mtc than in tt cells. pri- affected production and secretion of the two hormones. evident correlation was noted in tt cells, positive for ct and negative one for cgrp. in mtc cells, the effect was slightly less intense. pri- exerted a similar effect in the two cell lines but only at higher concentrations of the analogue. on the other hand, calcitriol manifested its activity only in mtc cells. effects of studied substances were also examined on cgrp/ct ratio of secreted hormones, which reflected corresponding ratio of alternative splicing of ct gene transcript. in tt cells the ratio decreased in presence of either analogue and in mtc cells the effect was observed following culture with calcitriol and the two analogues. conclusions calcitriol and its analogues exert a weak antiproliferative effect and an effect on alternative splicing of calcitonin gene. allelotyping analysis of gep endocrine tumors mixed medullary-papillary carcinomas are rare neoplasms and must be distinguished from medullary carcinoma with entrapped follicles, papillary variant of medullary carcinoma and solid growth pattern of sclerosing variant of papillary carcinoma. these mixed tumors could arise from uncommited stem cells; it is also possible that these neoplasms could be collision tumors. the aim of our study was to show that these mixed tumors have some particular features. we studied cases of thyroid carcinoma and for diagnosis we used immunohistochemical analyses for thyroglobulin, calcitonin, cea, chromogranin and cytokeratin . we found cases of mixed medullary-papillary carcinoma. these mixed tumors are not so frequent but the admixture of papillary and medullary components confer a different prognosis and a potential response to radioactive iodine treatment. results only four hereditary ( / ) tumours showed genomic imbalances. mtc appeared as sporadic forms. in of the sporadic tumours we detected dna sequence copy number changes (mean: per tumour, range: - ). the minimal common regions for the most frequent gains were narrowed down to q -q , p , and q ( percent), q -q , p , and q ( percent), p -p , p . -p , and q ( percent), and p pter, q -q , and p . -p ( percent). eight high-level amplifications were observed in four samples. p was affected twice. the minimal common regions for the most frequent losses were observed for p ( percent), and q ( percent). conclusion only limited genomic alterations could be detected by cgh for hereditary mtc, which share a germline mutation of the ret proto-oncogene as initial step of cancerization. additional genetic alterations, which are responsible for the progression of these tumours, are not identified yet. in contrast, sporadic mtc revealed a recurrent pattern of genomic imbalances. our data are in accordance in part with the few cytogenetic data, which are reported in the literature and can help to rule out parts of the genome on which further studies should focus on. immunohistochemical analysis of tumor infiltrating immunocytes in thyroid tumors v zolota, p aroukatos, s sidira, d koumoundourou, d papachristou, cd scopa dept. pathology, univ. patras med. school, patras, greece introduction the presence of host inflammatory cells within or at the periphery of human tumors has long been recognized and is commonly viewed as representing a host tumor immune response. in the thyroid gland, the occurrence of chronic lymphocytic thyroiditis (clt) in thyroid cancers has been reported and considered by some authors as a favorable prognostic sign. the inflammatory cell population in consecutive thyroid tumors ( papillary, occult, follicular, myeloid carcinomas and follicular adenomas), accompanied with chronic non-specific thyroiditis, were characterized imunohistochemically, using a panel of monoclonal antibodies against t-cells (anti-cd , anti-cd , anti-cd ), b-cells (anti-cd ), macrophages (anti-cd ) and dendritic cells (anti-s- ). the extent of lymphocytic infiltration was graded as: +(subtle), ++(moderate) and +++(intense and diffuse lymphoid aggregates). results were similar in cell block, although involucrine was negative in three pc. expression of mib- and p is variably detected in benign and malignant proliferative lesions. interestingly, galectin- , ck- and involucrin are invariably detected in papillary carcinomas, although heterogeneous positive expression was observed into the same lesion. conclusions our findings show that galectin- (p< . ), ck- (p< . ) and involucrin(p< . ) are useful to identify papillary carcinoma from other thyroid lesions; however, heterogeneous positive pattern expression is not useful for preoperative diagnostic of conventional cytology. introduction pheochromocytomas are rare neuroendocrine tumors that are highly vascular. their malignancy is extremely difficult to estimate on the basis of histopathological features. vascular endothelial growth factor receptor (vegfr- ) is a marker for lymphatic endothelium. its ligands vegf-c and vegf-d are expressed in many neuroendocrine tissues. our aim was to investigate the expression and the potential role of vegf-c, vegf-d, and vegfr- in various pheochromocytomas. materials and methods thirty-three pheochromocytomas, six of them metastasized, were studied using immunohistochemistry. thirteen tumors exhibited histologically suspicious features, such as necrosis, mitosis count over / hpf, vascular or capsular invasion, but they not metastasized, and were thus called borderline tumors. results and conclusions vegf-c immunohistochemical positivity was found in most pheochromocytomas. the expression of vegf-c was heterogenous and varied also within different tumor groups (benign, borderline, and malignant tumors). however, it tended to be stronger in extraadrenally located tumors. vegf-d positivity was also found in most tumors studied ( / ). interestingly, all malignant tumors (n = ) were totally negative for vegf-d. vegfr- expression was found in out of tumors, but the staining was mostly weak. the expression of vegfr- did not seem to correlate with the expression of vegf-c or vegf-d. in conclusion, vegf-c, vegf-d, and vegfr- are expressed in various pheochromocytomas, however, the diagnostic value of these markers needs further investigations. mixed corticomedullary tumor of the adrenal gland. a case report corticomedullary mixed tumors of the adrenal gland are quite rare, with only seven well-documented cases reported in the literature. we report the light microscope and immunohistochemical features of one case of this rare tumor. the patient is a -year-old woman who presented with hypertension, hirsutism, gain weight and ammenorhoea of -month duration. the surgically resected specimen consisted of a well-circumscribed, single adrenal mass, weighing , gr, and mm in diameter, surrounded by a rim of uninvolved adrenal cortical tissue. paraffin-embedded cut sections were stained by both hematoxylin-eosin and pas.the expression of inhibin, melan a, chromogranin a, synaptophysin, s- protein and acth was examined using the pap method, with appropriate antibodies being applied. the tumor was composed of cords and nodules of adrenal cortical cells intimately admixed with pheochromocytes. immunohistochemical investigations highlighted these two cellular components. the adrenal cortical cells revealed inhibin and melan a immunopositivity, whereas the pheochromocytes were strongly reactive with chromogranin a, synaptophysin, and acth, and the sustentacular cells with s- protein. thus, we report one additional case of mixed corticomedullary tumor of the adrenal gland. introduction the study of apoptotic markers seems to be important in understanding the pathogenesis of chronic inflammatory processes of the thyroid gland. aim: intensity determinations of bcl- and fas immunohistochemical reactions with the quantitative assessment of bcl- and fas gene expressions in inflammatory affected thyroid glands. the specimens were taken from women aged to . the type of inflammation and the intensity of lymphocytic infiltration were assessed according to the mizukami and the waterhouse and doniach classifications, respectively. the expression of bcl- and fas antigens was determined immunohistochemically. moreover, bcl- and fas genes were quantitatively analysed by rt-pcr assay using taqman technology. results intensity of immunostaining for bcl- antigen in patients with chronic thyroiditis was stronger than in patients without lymphocytic infiltration. an increase in the intensity of lymphocytic infiltration did not affect bcl- mrna levels. fas antigen got stained in subjects with mixed inflammation; a presence of oxyphilic cells enhanced the stain reaction. in the case of oxyphilic thyroiditis, the changes in fas antigen were accompanied by a concomitant increase of fas mrna levels. small differences, between various samples of the same specimen, in bcl- and fas mrna and protein levels were found in patients with focal thyroiditis. these differences were related to the intensity of lymphocytic infiltration. conclusions the thyroid gland is characterised by a high degree of antiapoptotic activity. each type of thyroiditis has its own characteristic pattern of bcl- and fas expressed as mrna and as protein. the use of galectin- and cytokeratin in differential diagnosis of thyroid tumors with follicular growth pattern a ryska aim to study the use of galectin- (gal) and cytokeratin (ck ) in differential diagnostics of thyroid tumors with follicular growth pattern. material and methods a series of thyroid lesions ( follicular adenomas (fa), follicular carcinomas (fc), follicular variants of papillary carcinoma (fvpc)) was studied retrospectively by immunohistochemistry using monoclonal antibodies. results the staining intensity of either antibody was evaluated as positive or negative using different thresholds ( , and % of positive cells). generally, the intensity of staining of gal was much higher than that of ck . using % threshold, / fa showed positivity of gal and none was positive in ck . in fc, / tumors were gal positive, / were also ck positive. among fvpc, all cases were gal positive and / were also ck positive. using % threshold, only / fa was gal positive, none was ck positive. among fc, however, all tumors were both gal and ck negative. on the other hand, / of fvpc were gal positive and / were ck positive. the sensitivity, specificity, negative and positive predictive value of gal were . ; . ; . ; . for % threshold and . ; . ; . ; . for % threshold, respectively. for ck it was . ; . ; . ; . for % threshold and . ; . ; . ; . for % threshold, respectively. conclusions the use of gal and ck may be helpful in differentiating fvpc from benign lesions. however, the use of these markers in differential diagnostics of fc from fa is of little value. moreover, due to low intensity of staining, its use in preoperative diagnostics (e.g. in fnac cytoblock specimens) seems to be questionable. adrenal cortical adenoma in association with cushing's syndrome -case report introduction morphological feature of adrenal cortical adenoma by itself doesn't allow reliable distinction between tumors associated with any particular endocrine syndrome. without being aware of the clinical setting and/or endocrinological data, it may be impossible to distinguish them from an incidental nonhyperfunctional macronodule. the aim of this investigation was to represent the only case of adrenal cortical adenoma in association with cushing's syndrome in our material. we have analyzed the only case of adrenal cortical adenoma with cushing's syndrome according to its morphological changes, laboratory data, ct-scan as well as mri of adrenal gland. results gross examination showed a small ovoid adenoma. on section, it appeared yellow to golden yellow with dark brown foci, blood and degenerative changes. microscopically, tumor contained cells with pale, lipid-rich cytoplasm, compact eosinophilic cytoplasm, arranged in trabeculae, short blunt cords, as well as in rounded nests. nuclei were somewhat vesicular with a single small dot-like darkly stained nucleolus. discussion and conclusion our data indicate that being aware of characteristic histologic feature in terms of architectural pattern and cell type for cortical adenoma associated with cushing's syndrome, this could be the purpose of more comprehensive discussion. correlation of histopathology and sequential analysis of gene expression in follicular thyroid neoplasms sj diaz-cano , , s arif , a blanes barts and the london school of medicine, university of london, uk university of malaga school of medicine, malaga, spain background the expression profile and diagnostic relevance of nuclear features, inflammation, and stromal changes have not been systematically evaluated in follicular thyroid carcinomas (ftc). design we selected ft neoplasms ( adenomas, minimally invasive carcinomas, widely invasive carcinomas, and anaplastic carcinomas), classified using standard criteria. histological variables including anisokaryosis, chromatin pattern, nucleolus, nuclear pleomorphism, nuclear/cytoplasmic ratio, necrosis, stromal changes, and tumor interstitial lymphocytes (til) were studied by histological diagnosis (significant if p< . ). oligo(dt)-primed double strand cdna was produced from total rna, and libraries were produced from normal thyroids and ftc. sequential analysis of gene expression (sage) was performed (ftc/control expression factor≥ , significance≤ . ). results til were absent in / neoplasms. minimally invasive carcinomas were distinguished from adenomas by conspicuous nucleolus, increased nuclear/cytoplasmic ratio, and coexistence of apoptosis and myxoid changes in adenomas. the most specific variables of high-grade carcinoma were coagulative necrosis, and nuclear hyperchromatism and pleomorphism. ftc sage showed ubiquitin c and sequestosome down-regulation, and tumor protein translationally controlled and malate dehydrogenase up-regulation. this sage profile correlated negatively with proinflammatory molecules expression (interleukin- beta, chemokine xc-receptor , interferon-induced transmembrane protein , protein tyrosine phosphatase c), brca , and protein phosphatase . the correlation was positive with proliferation markers (myb, mitogenactivated protein kinase , cyclin-dependent kinase , brca /cdkn a interacting protein, and ras oncogene family). conclusions proliferation up-regulation and apoptosis downregulation results in prominent nucleolus and high nuclear/ cytoplasmic ratio for low-grade carcinomas and hyperchromatism and nuclear pleomorphism for high-grade carcinomas. tumor down-regulation of proinflammatory molecules explains til absence. papillary carcinoma of the thyroid with bone formation. a case report introduction papillary carcinoma is by far the most common type of thyroid malignancy. however, ossification in papillary carcinoma is very rare. a case of papillary carcinoma of the thyroid metastatic to cervical and supraclavicular lymph nodes with associated bone formation and extensive calcification is presented. case report a year-old male with a history of gastric carcinoma was admitted to hospital presenting with lumbal pain. a computed tomography scan revealed a calcified nodule, measuring in mm diameter in the thyroid. total thyroidectomy and modified neck dissection were performed. the thyroidectomy specimen showed a well circumscribed, calcified, rock-hard nodule measuring x x cm. histologic examination of the hard nodule after decalcification showed spicules of bone formation and papillary carcinoma. foci of metastatic papillary carcinoma with bone formation were also seen in the cervical and supraclavicular lymph nodes. there was also bone marrow formation both in the thyroid nodule and in the metastatic lymph nodes. immunohistochemically, the tumour cells were positive for thyroglobulin. conclusion ossification in papillary carcinoma of the thyroid is an unusual finding. the mechanism of bone formation is not clear. the presence of ossification both in primary and metastatic tumoral masses suggests that ossification is not a dystrophic process in the preexisting benign thyroid tissue but somehow related to the papillary carcinoma in the present case. introduction c-kit gene encodes for a transmembrane tyrosine kinase receptor which is expressed in a variety of human normal tissues and malignancies, being small cell lung carcinoma one of the most frequent among the latter group. the search for c-kit expression in human tumors bears clinical implications since several tyrosine kinase inhibitors have been proposed in the treatment of c-kit positive tumors. the aim of our study was to investigate c-kit protein expression in a series of merkel cell carcinomas (mcc) and of other extrapulmonary poorly differentiated neuroendocrine carcinomas (pdnecs). merkel cell carcinomas (mcc) of the skin (from patients, of them with follow up information available), and cases of extrapulmonary pdnecs from different sites (bladder - cases, colon - cases, gallbladder and pancreas - cases, parotid - case) were analyzed by means of immunohistochemistry for c-kit protein expression. only a membranous staining was considered specific, and scored according to the percentage of positive cells. results c-kit protein was demonstrated in / ( %) mcc, and in / ( %) of other pdnecs. in mcc, a correlation, slightly below statistical significance (p= . ), was observed between the presence of c-kit expression and clinically aggressive behaviour. conclusion our data indicate that c-kit expression may play a role in the growth regulation of extrapulmonary pdnecs. therefore this group of tumors may be considered as a possible target for tyrosine kinase inhibitors therapy. carcinoid tumors in the thyroid gland. primary or metastatic? report of two cases m volante , m bongiovanni , p filosso , g bussolati , m papotti dept. biomedical science and human oncology university of turin, turin, italy dept. thoracic surgery university of turin, turin, italy introduction the occurrence of well differentiated endocrine tumors (carcinoid) in the thyroid gland is unfrequent and may represent both a differential diagnostic problem with medullary carcinoma, and a potential pitfall in the searching for its primary or metastatic origin. our aim was to determine the differential morphological and immunohistochemical profile of two cases of carcinoid tumors localized in the thyroid -one metastatic from the lung, and the other of unknown origin. materials and methods the first collected case had a clinical history of an atypical lung carcinoid diagnosed two years before, while the other lacked a clinical, instrumental and laboratory history suggestive of a possible primary carcinoid elsewhere. paraffin embedded material was histologically reviewed and analyzed by immunohistochemistry for thyroglobulin, calcitonin, cea, chromogranin a, s- , ttf , and ki- . results the first case (f/ ) presented as multiple nodules in both lobes, composed of poligonal eosinophilic cells arranged in the typical organoid pattern. foci of necrosis were present. the morphological features overlapped with those of the primary lung tumor. the second case (m/ ) presented as a single nodule in the left lobe, morphologically resembling typical carcinoid without necrosis. both cases were negative for thyroglobulin, calcitonin, s- and cea, positive for chromogranin a and ttf , and low proliferating (ki- < % in both). conclusion our data suggest that, even in the absence of a clinically detected primary tumor, the occurrence of a carcinoid tumor in the thyroid gland should be firstly considered metastatic in origin, the lung being the major candidate for the primary localization (as determined by ttf immunostaining). telomerase expression and proliferation activity in gastroenteropancreatic tumours s vural, y kapran, m gulluoglu, u cevikbas, f dizdaroglu university of istanbul, istanbul medical faculty, department of pathology, istanbul, turkey intruduction gastroenteropancreatic endocrine tumours have a variable prognosis and the prediction of biological behavior based on histopathology is not always possible. telomerase is a reverse transcriptase that synthesis telomere dna to stabilize telomere length.telomerase activity is increased in different human cancers. the aim of this study was to determine whether the expression of telomerase (htert) correlates with the histological typing of endocrine tumours and ki- proliferation index. material and methods formalin-fixed, paraffin-embedded tissue sections from gastroenteropancreatic endocrine tumours and normal pancreas were stained immunohistochemically for human telomerase reverse transcriptase (htert) and ki- antigen. results cases were diagnosed as well-differentiated endocrine tumour, were well-differentiated endocrine carcinoma and were poorly-differentiated endocrine carcinoma according to who classification of endocrine tumours. the percentage and intensity of positive neoplastic cells were assessed for each case. expression of htert was observed in one well-differentiated endocrine tumour ( %), in well-differentiated endocrine carcinomas ( . %) and in poorly-differentiated endocrine carcinomas ( %). there was a significant correlation between the expression of htert and histopathological diagnosis (p< . ). also the correlation of htert expression and ki- index was statistically significant (p< . ). conclusion immunohistochemical detection of htert expression in gastroenteropancreatic endocrine tumours may be a helpful tool in addition to morphological findings and ki- index in predicting the malignant potential of these tumours. proliferation, p expression and apoptosis in pituitary adenomas: relationship to tumor behaviour introduction pituitary adenomas are known to vary considerably from the stand point of their intrinsic aggresiveness that might imply a different therapeutical approach. the aim of this study was to estimate the relationship between proliferative activity (ki ), p expression, apoptosis and pituitary tumor behaviour. materials and methods immunohistochemical analysis of ki and p expression were performed in paraffin-embeded pituitary adenomas: functioning; non-functioning; invasive adenomas. apoptotic index (ai) was determined in adenomas by the in situ end-labeling technique (tunel). results ki expression in all adenomas was . % (significantly higher in functioning comparatively with non-functioning pituitary adenomas - . ± . % versus . ± . %). we noticed a positive correlation between the proliferative activity and the tumoral invasiveness (ki labeling index . ± . % in invasive and . ± . % in non-invasive adenoma). p nuclear positivity was identified in % of the invasive adenomas and only in % noninvasive adenomas, with a positive correlation between p and proliferative activity (ki : . ± . in p positive versus . ± . % in p negative tumors). apoptosis was found in % of tumors, higher in some of the invasive ones, with a positive correlation between the proliferation rates and apoptotic indices (ai: . ± . in total versus . ± . in invasive pituitary adenomas). also, some differences were recorded in functioning and non-functioning adenomas (ai: . ± . versus . ± . ). conclusions the data suggest that the assessment of proliferation, p expression and apoptosis might be used to evaluate tumor behaviour and susceptibility of adenomas to therapy. differentially expression of nesp in langerhans islets of the human pancreas and overexpression in endocrine pancreatic tumors nodules, while others recommend to do it only if clinical changes are present. this study was done to determinate the usefulness of systematic repetition of fna in patients with benign cytology result in the initial fna. materials and methods data were collected from patients referred to endocrinology service for evaluation of thyroid nodular disease between and . all patients underwent repeated fna of thyroid nodule. age of patients was . + . years, . % were females, % had uninodular goiters, nodule size was . + . cm. time elapsed between both fna was . + . months. results at second fna, aspirations had benign cytology, were non diagnostic, suggestive of follicular neoplasm and had malignant cytology results. excluding non-diagnostic aspirations, we get % of discordant results. uninodular goiters as compared to multinodular have a higher rate of discordant results. we did not find other variables between groups of concordant and discordant results. twenty-two patients with benign cytology underwent thyroid surgery, without any histological malignant result. conclusion -uninodular goiters with an initial benign cytology have a significant rate of discordant results after repetition of fna. however, in % of these cases the first diagnosis was based on limited material. -our results show that cases of thyroid carcinoma out of patients with nodular goiter were misdiagnosed by initial fna cytology. therefore we recommend systematic repetition of fna cytology in all patients with uninodular goiter. immunocytochemistry in fna biopsy material objective assessing diagnostic value of immunocytochemical methods in destained cytology smear specimens prepared from fine needle aspiration (fna) biopsy material. material and methods investigated material comprised samples from fna biopsies that were diagnosed in - . alcohol-fixed cytology smears were typically stained with hematoxylin-eosin. for immunocytochemical studies, preparations were first destained using alcohol series. following this, chosen antibodies were used either separately, or as a panel ckmnf, ema, lca, tg, ct, hmb- , s- , chr, nse, er, pr, cd (dako lsab system, peroxidase). results immunocytochemical staining was helpful in % of cases. initial diagnosis was confirmed in cases. immunocytochemical staining was important particularly in confirming diagnosis of poorly differentiated neoplasms. it was also helpful in identifying the primary site of metastatic carcinoma in fna cases. in ( %) cases diagnosis based on the investigated method failed. precise diagnosis was not possible either due to negative staining with single marker/panel or when only single cells were stained positive. conclusion immunocytochemical methods in destained smears allow basic as well as discriminating diagnosis even in cases of poor availability of cytologic material. fine needle aspiration of lesions in the thyroid bed. e chmielik, b szczesniak-klusek, d ponikiewska, e stobiecka, d lange oncology center, department of pathology, gliwice, poland introduction clinical suspition of recurrent thyroid carcinoma based on ultrasound examination of thyroid bed with correlation to increased thyreoglobulin level or calcitonin level and/or radiomarker uptake on scintigraphy can be supported or dismissed by fine needle aspiration biopsy (fnab). the aim of this study was assesment of specificity and possitive predictive value of ultrasound-guided fnab of the lesions in the thyroid bed after total thyroidectomy for diagnosis of recurrent thyroid carcinoma. material and methods ultrasound guided fnab of thyroid bed lesions of diameter . - . cm performed by pathologist in patiens after thyroidectomy. immunohistochemistry stain for calcitonin was performed using dako antibody in cases. the recurrent papillary carcinoma was diagnosed in ( . %) patients, the recurrent follicular carcinoma in ( . %) patients, hürtle cell carcinoma in ( . %) patients and medullary carcinoma in ( . %) patients (immunocytochemistry stain for calcitonin confirmed diagnosis in patients). pathologic correlation was able in cases: postoperative examinations confirmed recurrent carcinoma, and cytologic diagnoses were falsenegative. in the remaining patients granulation, purulent inflammation and benign lesions were diagnosed. in ( . %) cases material was nondiagnostic. specifity of the method was . %, possitive predictive value . %. conclusions ultrasound-guided fine needle aspiration biopsy performed by pathologist is valuable and specific method in the diagnosis of recurrent thyroid carcinoma in thyroid bed. introduction fine-needle aspiration (fna) is one of the most useful methods in the diagnosis of benign goiter. in consideration of the goiter; it is the most frequent lesion in the thyroid tissue, significance of fna as a diagnostic method become greater. material and methods in cases diagnosed as goiter we analyzed: ) sex distribution, ) age distribution, ) correlation between clinical and cytological diagnoses and ) goiter classification in accordance with cytological characteristics. results goiter was five times more frequent in females than males ( women, men). the majority of the patients were between and . the youngest was , and the oldest years old. almost % of cytologically diagnosed goiters had the same clinical diagnosis, in only . % of all patients tumor was suspected clinically. we confirmed high correlation between clinical and cytological diagnosis. according to the cytological characteristics all goiters were classified in categories: goiters with and without degenerative changes. hi-square test confirmed statistically significant difference in the presentation of such goiters (hi-square test= . ; p< . ). fna permits a substantial reduction or even elimination of other diagnostic procedures, such as imaging, with consequent saving in time and money. furthermore, benign cystic lesion may be cured by fna (a diagnostic and unexpected therapeutic value). samples, furthermore it is difficult to predict at patient's bedside which ancillary methods, if any, will be necessary. the aim of this study was to demonstrate the value of a universal short-term storage cell medium in processing fnab samples for different ancillary methods. materials and methods after diagnostic smear preparation, the needle and syringe were washed out with the short-term storage cell medium in fnab samples, obtained from to . microscopic smear analysis determined the choice of ancillary method used. cell suspensions were then processed as required for each individual ancillary method. results we used ( %) fnab samples stored in the medium for ancillary methods, while samples ( %) were discarded due to the clear morphology-based diagnosis or sample inadequacy. immunocytochemistry was performed on samples. for various membrane, cytoplasmic and nuclear antigens, adequate immunoreaction without background was obtained in samples stored in the medium overnight ( ) or over the weekend ( ). immunophenotyping by flow cytometry was performed in fnab samples where up to lymphoma antigens per sample were successfully determined when the samples were stored in the medium overnight. high quality flow cytometric dna histograms were obtained from all fnab samples suspended in the short-term storage cell medium. conclusion the short-term storage cell medium that we have been using, allows effective and easy processing of fnab samples for several ancillary diagnostic methods. it preserves cell morphology, various membrane, cytoplasmic and nuclear antigens as well as dna content. fine needle aspiration cytology of ovarian lesions introduction an improved understanding of the pathogenesis of cervical hpv-induced lesions has significantly changed the approach to diagnosis and treatment. the pap smear is still the most effective cervical cancer screening test. however, molecular testing for the presence of high risk hpv-dna increases the ability to identify women with an increased risk for cancer and true cancer precursors. the aim of the present study was to evaluate high risk hpv-dna test systems in cervical cytological material and the correlation with pap smear cytology. materials and methods we analysed routinely processed cytological cervical specimen. material was taken consecutively for pap smear cytology and for hybrid capture (hc) hybridisation assay (digene). the cytological report showed mild to severe dysplasia ( cases were scored as pap i/ii). after performing the hc-assay, dna was extracted from the same sample. hpv-dna detection with conventional pcr using consensus primer (my /my ), followed by subtyping of hpv and was carried out. from the specimens showing dysplasia ( . %), koilocytosis has been observed in % of pap smear cytology. hpv-high risk positivity with the hc hybridisation assay was detected in % and % showed positivity for hpv-dna content with conventional pcr. subtyping of hpv-positive cases resulted in hpv- positivity in %, . were positive for hpv and . % showed a coexistence of hpv and . conclusion our results indicate that hpv-detection with highly sensitive and widely used molecular methods show a good concordance with cytological findings. fine needle aspiration findings of two different types of gastrointestinal stromal tumor mg gulluoglu , d yilmazbayhan , y kapran , k acarli , f dizdaroglu istanbul university, faculty of medicine, department of pathology, istanbul, turkey istanbul university, faculty of medicine, department of general surgery, istanbul, turkey introduction gastrointestinal stromal tumors (gist) are the most common mesenchymal tumors of the gastrointestinal tract, derived from interstitial cells of cajal. the cytologic features of these neoplasms in fine needle aspiration (fna) have been described in several articles. aims: cytologic and histologic features of two gist cases one of which presented with liver metastasis and other with an intrathoracic mass mimicking lung carcinoma radiologically are presented comparatively. sixty year-old male patient with a previous history of gastric gist has had an fna from a liver mass. in the cytologic examination, epithelioid cells with round-oval nuclei, large cytoplasms and indistint cytoplasmic borders, forming three dimentional groups with thin fibrovascular stroma were observed. the other patient was a seventy two year-old female who had an fna from an intrathoracic mass. branching fascicles of spindle cells with indistinct cytoplasmic borders in a necrotic background were observed in the cytologic examination. mesenchymal origin was verified by vimentin immunopositivity. by detailed computerized tomographic evaluation, it was revealed that the mass originated from the gastroesophageal junction. both tumors were resected and histologically examined. cytologic and histologic findings correlated including immunocytochemical and immunohistochemical results which were positive cd and cd , and negative s and smooth muscle aktin immunoreactions. conclusion as they share common histomorphological features with other types of mesenchymal tumors of the gastrointestinal tract, cd immunopositivity is the only diagnostic marker to differentiate these tumors from other tumors of mesenchymal origin. cytologic diagnosis also requires detection of this marker in the neoplastic cells. peritoneal fluid cytology of sarcomas k zachou , e tzartza , a orologa , a skenderi , k patsiaoura , e kostopoulou , k boni hippokratio hospital of thessaloniki dept. cytopathology, thessaloniki, greece hippokratio hospital of thessaloniki dept. pathology, thessaloniki, greece material and methods peritoneal fluid smears of female patients of all ages, were studied. results cases were cytologically diagnosed as malignant ( , %), the majority of which was characterized as adenocarcinomas. histological diagnosis was available for cases. sarcomatous component was reported in only cases, cases were malignant mixed müllerian tumors (mmmt), one was leiomyosarcoma, one rhabdomyosarcoma and one liposarcoma. the cytological findings in the equivalent cases were « abnormal» neoplastic cells in isolation, with enlarged and hyperchromatic nuclei. adenocarcinoma cells were identified in peritoneal fluid smears of patients with mmmt. conclusion cytological diagnosis of sarcoma is rare. even when these «abnormal» cells are found, it is very difficult to give a definite diagnosis of sarcoma, based directly on the cytomorphological characteristics of peritoneal smears. however, such a possibility should be kept in mind by the cytopathologist to avoid missing the diagnosis. r bokun, z tatomirovic, v skuletic institute of pathology, military medical academy, belgrade, serbia and montenegro purpose the aim of this study was to show that imprint cytology is a valuable method in the evaluation of bone tumors as well as to determine its diagnostic accuracy in primary and secondary bone tumors. materials and methods from thirthy three surgical specimens of bone tumor imprints were made, air-dried and stained by may-grunwald-giemsa method. the cytologist gave detailed description of the smears, concluded whether the lesion was malignant and suggested a final diagnosis if it was possible. results were compared with the corresponding histopathologic diagnoses. results out of cases with primary malignant bone tumors (chondrosarcoma , ewing sarcoma , ameloblastoma , synoviosarcoma , malignant osteoclastoma , malignant fibrous histiocytoma , malignant schwannoma , leiomyosarcoma ) the cytologist recognized malignancy in of them and in suggested the final diagnosis. in patients with metastases (squamous cell carcinoma , anaplastic carcinoma , malignant melanoma ), the cytologic and histopathologic diagnoses correlated in all cases. conclusion cytologic analysis of the imprints of bone tumors is valuable as early orientation for the clinicians since the final histopathologic diagnosis needs time because of the decalcination procedure. the correlation of cytologic and histopathologic diagnoses was better for the secondary bone tumors because cytologist was more familiar with their morphology. the value of combined fine needle aspiration and needle core biopsy in diagnostic assessment of pancreatic tumors p sevastiadou , p xirou , s barbanis , i efstratiou , e athanasiou , i tsitouridis papageorgiou general hospital, department of cytology, thessaloniki, greece- papageorgiou general hospitaldepartment of cytology, thessaloniki, greece papageorgiou general hospital, department of radiology, thessaloniki, greece introduction due to the anatomical location of the pancreas, it is often difficult to obtain sufficient diagnostic material from pancreatic lesions. the aim of this study is to present the results and our experience from ct guided fine needle aspiration (fna) and needle core biopsy (ncb) from pancreatic tumors. materials and methods fna using gauge needles and ncb using and gauge needles were performed in patients with pancreatic tumors. both procedures were done under computerized tomography guidance, without complications. twenty one tumors involved the body and the tail of the pancreas results the diagnosed cases were adenocarcinomas, neuroendocrine tumors, lymphoma and pseudocysts. diagnosis was reached in cases by both fna and ncb and in cases by ncb only. other cases were "diagnosed" as suspicious for malignancy by fna only. finally in cases no diagnosis was reached by both methods, due to insufficient material. conclusion according to our experience, we believe that the combination of fine needle aspiration with needle core biopsy of pancreatic tumors under ct guidance improves the diagnostic accuracy. value of fine needle aspiration cytology in subtyping mucinous breast carcinoma background subtyping of mucinous breast carcinoma into pure and mixed type has prognostic implications. aims of the study were to evaluate a) the usefulness of fine needle aspiration biopsy (fnab) in differentiation between pure and mixed mucinous carcinomas, and b) to correlate key cytological features with survival. material and methods we reviewed fnab of women with mucinous breast carcinoma treated at the institute of oncology from to . key cytological features analysed were cellularity, amount of mucin, presence of capillaries, cell pattern, cytoplasmic characteristics and nuclear pleomorphism. type of mucinous carcinoma was determined histologically after resection of the tumour. association of cytological features with cancer specific survival was evaluated by univariate and multivariate analysis. results histological analysis revealed cases ( %) of pure and cases of mixed mucinous carcinoma. nuclear pleomorphism and amount of mucin in fnab differed significantly between the two subtypes of mucinous carcinoma. whereas cellularity of cytological samples, presence of capillaries, cell pattern and cytoplasmic characteristics were not related to prognosis, nuclear pleomorphism and absence of mucin in cytological samples were associated with poorer cancer specific survival. furthermore, the absence of mucin in fnab samples retained independent prognostic significance also in multivariate analysis. conclusions higher grade of nuclear pleomorphism and small amount or even absence of mucin in fnab favor the diagnosis of mixed mucinous cell carcinoma and are features associated with poorer prognosis. cardiac lipoma in a mexican patient -multidisciplinary approach in a right atrium tumor j nuncio , g najera-hernandez , g garcia-ramos pathology department, instituto nacional de ciencias medicas y nutricion salvador zubiran, mexico city, mexico internal medicine and neurology departments, fundacion medica sur, mexico city mexico primary tumors of the heart are rare. lipomas account for approximately % of all neoplasms of the heart and represent % of benign cardiac tumors. symptoms may be produced by large lipomas, but more often the patients are asymptomatic and the diagnosis is made by chance. recently, the ready availability of high-definition noninvasive imaging, such as echocardiography, computed tomography, and magnetic resonance image, has allowed easier diagnosis. therefore, more cases have been reported. to date up to cases of lipoma have been described. this case describes the multidisciplinary approach to the diagnosis of this rare entity with the use of open heart biopsy from right atrium, to sustain the correct diagnosis. after exhaustive internet-based search, this is the first case reported in latin-american literature, a condition not previously informed from in vivo patient in mexico. the cardiac hydatid cysts are rare cases, representing , - % from all human hydatid cysts. they are commonly located in the left or right ventricle and exceptionally in the interventricular septum. because the symptoms and clinical signs of the cardiac hydatid cysts are non-specific, the diagnosis may be difficult. material and methods we report an intramyocardial hydatid cyst, diagnosed at cardiology center iasi, with an early history of atypical chest pain, the death occuring through anaphylactic shock, secondary to the hydatid cyst endocavitary rupture. the study was made on necropsiy specimens, and the diagnosis was established by using routine morphological techniques. results the gross examination evidentiated an hydatid cyst of about cm in diameter, communicating through a small orifice with the left ventricular cavity and through a slit-like fissure with the right ventricular chamber. the systemic embolism was revealed, only grossly, by the presence of small "hydatid daughters" at the level of the ascending aorta and the pulmonary embolism was confirmed only histologically, by the presence of anhiste membrane emboli in the peripheral pulmonary vessels. the study revealed the morphological aspect defining hydatid cyst, emphasising the necessity of an early diagnosis due to a high risk of death through complications. a case report of arrhytmogenic right ventricular dysplasia -cause of sudden death c borza, d butcovan, c grigoriu, v astarastoaie university of medicine and pharmacy iasi, romania arrhytmogenic right ventricular dysplasia (arvd) is a new form of cardiomyopathy probable more frequent than commonly reported. arvd is a heart muscle disorder of unknown cause that is characterized clinically by an electrical instability associated with secondary ventricular arrhythmia and pathologically by fibrofatty replacement of the right ventricular myocardium. it is a rare but important cause of sudden unexpected cardiac death in young, otherwise healthy persons, especially those that are exercise related. we report a case of cardiac sudden death in a years old man without personal and familial cardiac history. the study was made on a necropsic biopsy specimens from the medico-legal laboratory. the diagnosis was made using routine histological techniques. grossly, we revealed a fibro-fatty right and left ventricular wall myocardial replacement and a focal white thickening of the left ventricular endocardium. histologically, we evidentiated a fibro-fatty myocardial replacement of the right and left ventricle suggesting an arrhythmogenic ventricular dysplasia of cardiomyopathic type and an endomyocardial fibrosis supported by the presence of a subendocardial fibrosis, affecting internal one third of the myocardium. there were no evidence of coronarian and valvular lesions and hypertensive cardiac changes, as well. the study revealed the morphological features defining a fibro-fatty myocardial replacement and an endomyocardial fibrosis evidentiating the case particular features, as well. registered. repeated biopsy revealed ongoing mc in / pts, latehealing mc in / pts, focal-healing mc in / pts, healed mc in / pts, and dilated cardiomyopathy in / pts. amount of cellular infiltrate, degenerative and/or necrotic changes, and fibrosis was reduced in , , and pts, respectively, remained the same in , , and pts, respectively, and increased in , , and pts, respectively (p< . , between groups). the significant reduction was achived in the amount of cellular infiltrate and degenerative and/or necrotic changes, contrary to the amount of fibrosis. applied immunomodulatory therapy made substancial reduction of inflammation with "relative" increase of fibrosis. conclusion interferon-α, as main immunomodulatory agent, has favourable effects on myocardial histology and contractility in patients with biopsy-proven myocarditis. morphological changes in heart died of a heart attack of a myocardium v ubaydullaeva the centre of emergency medical aid, tashkent, uzbekistan diseases cvs take conducting place among the most actual problems of modern medicine. from them it is possible to count the main thing ihd. ihd -the special form of pathology cvs including group of diseases which, according to definition the cart, are treated as follows: «dysfunction sharp or chronic, arising owing to relative or absolute reduction of supply of a myocardium by arterial blood». despite of significant number of the morphological researches devoted ihd, morphogenesis separate displays of it, in particular, ami, and also mechanisms of indemnification and decompensation of hearts are investigated insufficiently. in connection with, the purpose of the present research definition morphofunctional conditions of various departments of heart was above-stated at diagnostics of an intimate ischemia. material and methods for achievement of an object in view researches of hearts died of a heart attack of a myocardium have been lead (have been carried out), and also have been investigated morphometric parameters, such as: weight of a cardiac muscle, pure weight of heart, weight right and left ventricles, an index right and left ventricles, an index of blood supply of a myocardium. all received data have been processed statistically on personal computer with use of standard software package windows excel. results summarizing macro-and the microscopic changes which have been found out by us in a cardiac muscle, it is possible to make the conclusion that the long reorganization occuring in a cardiac muscle in conditions increasing hypoxy, results in an exhaustion processes, to use of all possible resources cardiomyocytes and causes development decompensation. introduction cardiac myxoma, the most common primary tumor of the heart, has a varying clinical presentation and immunohistochemical profile. an abundant mucopolysaccharidic matrix exists, including mucin, within the cardiac myxoma. this study elucidates the clinical presentation and expression of mucin genes in cardiac myxoma. materials and methods from december to february, , the retrospective study included consecutive patients with cardiac myxoma underwent surgical excision. detailed clinical parameters were reviewed as well. mucin genes, additionally, muc , muc and muc ac, have been studied by immunohistochemical method. the expression was scored from to . results ( %) women and ( %) men with a mean age of +/- years comprised the control group. the presentation included asymptomatic ( %), dyspnea ( %), stroke ( %), chest pain ( %), fever ( %), syncope ( %) and tricuspid regurgitation ( %). we divided these patients into two groups; group a contained the myxoma located in the left atrium, group b included three ( %) recurrent myxomas and ( %) myxomas other than in left atrium. they did not differ with respect to many pathological scores, such as vascular proliferation, inflammation, cellularity, and hyaline. however, although less calcification and thrombosis occurred, more congestion was found in group b (p < . ). moreover, muc / ac had a higher expression than muc (p < . ). interestingly, the group a displayed a stronger mucin expression than group b did. conclusion preliminary results demonstrate invaluable and different pathological patterns, including mucin genes, between the myxoma coming from the left atrium and the other sites. introduction nowadays understanding of atherogenesis, russell ross' response-to-injury paradigm, considers atherosclerotic lesions as "a protective, inflammatory-fibroproliferative response" to harmful stimuli. among these stimuli are the major risk factors, which contribute for atherosclerotic cardiovascular complications. arterial blood flow impairment is associated with the progression of intimal atherosclerotic lesions. that is why most of the research work is concentrated upon intima, while adventitia is neglected. several lines of evidence clearly show that adventitia is not an innocent bystander in atherogenesis. the aim of the present study is to evaluate morphologically adventitial changes in atherosclerotic arteries. the cellular components and adventitial thickness are followed in postmortem human coronary arteries. results mononuclear cellular infiltration (lymphocytes, mast cells), neoangiogenesis and collagen overproduction are established in this part of adventitia, that corresponds to atherosclerotic lesion. morphometric analysis reveals increased adventitia/media thickness ratio. the adventitial thickening represents an example of negative vascular remodeling, because of hampering the compensatory enlargement of the affected artery. on the other hand the cellular processes involved in adventitial thickening could be responsible for atherosclerotic lesion initiation. we need further arguments into the cell biology of atherosclerosis, viewed as a vascular healing process, involving also the adventitia, to provide new insights into atherogenesis and alternative therapeutical strategies respectively. study on postmortal autolytic changes of the myocardial blood vessels. experimental study l kostic-banovic , v katic , r karadzic , j stojanovic , g ilic , m zdravkovic , a antovic department of forensic medicine nis, nis, serbia and montenegro department of pathology nis, nis, serbia and montenegro introduction modern forensic medicine practice has not yet been given the methods of investigation that would enable answering with sufficient accuracy to the question of death occurrence time. present work is an attempt to make contribution to that very complex and significant problem in forensic medicine. the investigation has been done on wister rats. the sacrifice was exerted by strangling. to respond on questions how both temperature of microenvironment and passed time influence on coronary arteries, the experimental animals were divided into groups: i ( c), ii ( c) and iii ( c). every group had subgroups, depending of passed time from the sacrifice, beginning of hour, through , , , , , and up to hours. control animals were autopsied and myocardial sections were taken immediately after sacrificed animals. formaldehyde fixed and paraffin embedded blocks were cut and stained by he, van gieson and gomori methods. results in groups ii and iii almost all blood vessels of heart muscle had fragmented walls in the animals examined upon hours after sacrifice. the vessels were almost normal in the group i also up to hours after sacrifice. basal membranes of blood vessels were observed upon hours after sacrifice at the temperature of c. fragmented basal membranes in ii and iii group were found, in the form of spider's web residues. total hemolysis of erythrocytes was upon hour after sacrifice in iii temperature group. however, contours of erythrocytes could be recognized upon hours after sacrifice in i experimental group. conclusion autolysis of myocardial vessels is influenced by two factors: time interval from the death and micro environmental temperature. the temperature exerted more profound influence on autolysis than time interval. the observed autolytic changes could be used in the expertise of the time of death occurrence. introduction heart transplantation remains the only therapeutic option for pediatric patients (pts) with severe, end stage myocarditis. the aim of this study was to determine the presence of viral genome in native heart and the frequency of viral recurrence after heart transplant in pts with myocarditis. material and methods from march to november , heart transplants were performed in children ( month to years m/ f) at our institution. histology/immunohistochemistry was carried out in all native hearts; among inflammatory cardiomyopathy, cases were active myocarditis and dilated cardiomyopathy/ chronic myocarditis. foci of active myocarditis were also found in one case of hypertrophic/restrictive cardiomyopathy and one case of congenital heart disease respectively. retrospective pcr (polymerase chain reaction) and sequencing analysis for detection of cardiotropic viral genomes was performed in native hearts ( %) and in all serial post-transplant biopsies from viral pcr positive cases. results pcr amplified a viral product in native hearts ( %). pcr analysis on postransplant serial biopsies disclosed the same viral infection in of ( %). early enteroviral recurrent myocarditis with identical genotype was demonstrated in cases at month after transplant. fatal outcome occurred in / pts with viral recurrent myocarditis when prompt molecular diagnosis was not available; misdiagnosis of acute rejection in histological monitoring may have concurred in the fatal outcome. conclusion viral myocarditis frequently relapses after heart transplantation in children and is a risk of fatal outcome. molecular study in native hearts is mandatory to improve diagnostic accuracy allowing to a more strict surveillance after transplantation. sudden cardiac death induced by acute viral myocarditis introduction most commonly the sudden cardiac death (scd) is defined as unexpected death from cardiac causes early after of without the onset of symptoms. data suggested that scd is a multifactorial catastrophe involving in most instances myocardial lesions that we confirm by this report. materials and methods five cases were autopsied in institute for forensic medicine because they have died from scd. the mean age was , years, including one boy ( years) and one girl ( years). all specimens were fixed in % formalin. paraffin sections were cut and stained by: he, van gieson and ab-pas, ph= , . results clinical symptoms lasted short time: fatigue, dyspnea, palpitations and precordial discomfort, accompanied by fever. macroscopically, the hearts were normal or enlarged, with dilatation of chambers. the ventricular myocardium was typically flabby and mottled by either pale foci or minute hemorrhagic lesions. histological finding: myocarditis was characterized by an interstitial mononuclear predominantly lymphocytic inflammatory infiltrate and by injury of focal necrosis to myocytes adjacent to the inflammatory cells; focal aggregates of macrophages and microhaemorrhages, often surrounding the conduction system, were also observed. scattered incipient fibrosis inside the areas with disappeared myocytes was one of the features of myocarditis. conclusions the ultimate mechanism of death in viral myocarditis was fatal arrhythmia induced by inflammatory mononuclear infiltrates of the conduction system. surgical pathology of primary cardiac and pericardial tumors in the paediatric age background primary cardiac and pericardial tumors (cpt) are rare at all ages and even less common in infancy and adolescence. aim of this study is to assess the prevalence and pathologic features of paediatric cpt consecutively examined in a single institution. among surgically removed cpt collected at our cardiovascular pathology unit in the time interval - , ( %) occurred in the paediatric age (< yrs). we retrospectively analysed them by histotype, age and clinical presentation, location, type of surgery and outcome. they were male and female pts, age ranging from days to years (median months). the majority of cpt were diagnosed before the age of year ( %), including prenatal diagnosis by foetal echocardiography. in the remaining pts, clinical presentation of cpt was congestive heart failure in , cardiac murmur in , systemic embolism in , and constitutional symptoms in , whereas four were completely asymptomatic and the mass was an incidental finding at echocardiography ( ) or during cardiac surgery for congenital heart disease ( ). pct were located most frequently in the right ventricle ( ), followed by right atrium ( ), left atrium ( ), left ventricle ( ), tricuspid valve ( ), pericardium ( ) and ventricular septum ( ) . all had surgical resection except one who underwent cardiac transplantation. as far as histotype, myxoma was the most frequent ( ), followed by rhabdomyoma ( ), fibroma ( ), angioma ( ), pericardial teratoma ( ) and hematic cyst ( ). no recurrence was found at echo during follow-up and only one pt died three years after cardiac transplantation due to cerebral malignancy. with the improvement of cardiac imaging techniques, cpt have increasingly been detected even early during life. despite the benign histotype, cpt carry the risk of heamodinamic, embolic and arrhythmic complications. prompt surgical resection is indicated in all symptomatic pts and in those with tumour related obstruction. among surgically resected primary cpt in the paediatric age rhabdomyoma is less frequent than expected since it often does not require surgical resection due to its potential spontaneous regression. in vivo histologic diagnosis of primary malignant cardiac and pericardial tumors c basso, m valente, g thiene institute of pathology, padua, italy background primary cardiac and pericardial tumors (cpt) are rare and even less common are malignant ones. aim of our study was to assess the prevalence and histotype of malignant forms among a consecutive series of primary cpt examined in a single institution. in the time interval - among consecutive pts who had an in vivo diagnosis of primary cpt, either by surgical pathology and/or endomyocardial biopsy investigation, ( . %) had a malignant form. they were female and male pts, age ranging from to yrs (mean ± ). besides gross and histologic examination, tumour histotype was defined by immunohistochemistry using a large panel of antibodies and by transmission electron microscopy. primary malignant cpt comprised extracardiac ( pericardial mesothelioma and pulmonary artery sarcoma) and intracardiac masses. the latter group consisted of angiosarcoma of the right atrium +/-right ventricle ( ), undifferentiated sarcoma of the left atrium ( ), fibrosarcoma of the right atrium ( ), malignant fibrous histiocytoma of the left atrium ( ), b cell lymphoma of the right atrium ( ) and leiomyosarcoma of the right ventricle ( ) . in four ( %) a preoperative histologic diagnosis was achieved by transvenous endomyocardial biopsy (all right-sided). four malignant primary cpt, all located in the left atrium, had a preoperative misdiagnosis of myxoma. surgical resection was performed in all but two pts. all pts died with a maximum survival time of months. conclusions primary malignant cpt are rare and represent . % of all primary cpt in our series. the prognosis is poor and generally measured in months independently from the tumour histotype and from surgical resection and chemotherapy. endomyocardial biopsy is a safe procedure to achieve an in vivo diagnosis in the setting of right intracavitary masses. introduction primary sarcomas of the aorta are extremely rare neoplasms, described in occasional single case reports and antemortem diagnosis is difficult because of their polymorphous presentation. the tumours are classified into intimal and mural types according to their location and clinical behaviour. we describe a case of intimal sarcoma arisen in the thoracic aorta of a -years-old woman with multiple embolic abdominal metastases (spleen, kidney, adrenal). case report in november the patient was hospitalized for the embolic occlusion of the left popliteal artery. the recent clinical history revealed irritative cough, dispnea and fever. the laboratory tests reported anemia (hemoglobin: . mg/dl) and the ves was . electrolities, liver function tests and fibrinogen were within normal limits. the antiphospholipid antibody and lupus anticoagulant were negative. a ct-scan showed an intraluminal thrombus-like mass in the thoracic aorta and multiple geographic infarctions of the spleen suggestive for a primary aortic tumor. she died before undergoing to surgery, for left ventricular failure. results post-mortem examination showed mild atherosclerosis and a mass with grayish-yellow, solid, rubbery and mixoid aspects, attached to the wall of the aorta, just after the origin of the left subclavian artery. histologic sections disclosed a spindle-shaped sarcoma cells with pleomorphic nuclei in a myxoid stroma; necrosis was abundant. at immunohistochemistry tumor cells resulted positive for vimentin, actin and cytokeratins. conclusion the preoperative diagnosis of sarcoma of the aorta is possible but it should be supported with imaging techniques; the definitive diagnosis need to be confirmed histologically and immunohistochemically. arterial allografts cryopreservation antigenicity in pigs m santos background coronary surgery requires vascular grafts. donor fresh arterial grafts are useless because of rejection vasculopathy. cryopreservation can be a good way for banking purposes and may limit rejection. the aim of this study was to assess the utility of arterial cryopreservation in increasing the quality of grafts, in an animal model in pigs. material and methods small arteries, less than mm in diameters were implanted in pigs. three groups were done: group (n= ): fresh autograft (au) and fresh allograft (al) one in each side; group (n= ): cryopreservated au and cryopreservated al; group (n= ): fresh al and cryopreservated al. all animals got aspirin for three months and group also cyclosporine a. the implanted grafts were embedded in paraffin and microscopically studied with hematoxilin/eosin and elastic stains for morphology and morphometrics. a basic immunohistochemical study with cd and mac was also performed. results the patency rate of cryopreservated al and au was %, as it was in the fresh au. the inflammatory reaction, evaluated by cd and mac was present in all al arteries, and absent in au cases. in group , the infiltration was lower in cryopreservated, than in the fresh ones. conclusions in our series of pig's arteries, cryopreservation increases vascular patency and the inflammatory response is lower than in fresh arteries. this fact is independent of the immunosupressive therapy. introduction glandular cardiac myxomas are very rare tumors of uncertain histogenesis, displaying glandular structures within otherwise typical myxomas. origin of glands has been attributed to epithelial differentiation of a cardiomyogenic precursor cell or to entrapped embryonal rests. we studied four cases of glandular myxomas ( sporadic and one familial) to define the immunophenotypic profile of the glandular elements. materials and methods histology and immunohistochemistry were performed on serial sections. broad spectrum cytokeratin, cytokeratin (ck ), cytokeratin (ck ), cea, ema, chromogranin a, calretinin, thyroid transcription factor- (ttf- ), ki- and p were investigated. results three patients were males, aged , , , respectively and the fourth a y.o. woman. the glands were either scattered within myxomatous stroma ( cases) or in groups. in the latter case the glands featured villous projections, irregular profile, active inflammation or focal low grade dysplasia, and showed expression of acidic and neutral mucins (mostly sialomucins). cytokeratin and co-expression was found, similarly to foregut derivatives. there were scattered chromogranin-positive neuroendocrine cells in one case and ki- immunostaining showed a very low proliferative activity. there was no reactivity for ttf- , calretinin or p . none of the patients had adenocarcinoma or other neoplasm, either at diagnosis or at - years follow-up. conclusions our findings indicate that glandular component of cardiac myxoma is morphologically heterogenous. the isolated glands suggest a possible divergent (epithelial) differentiation of myxoma cells, but other findings point to the possibility of entrapped foregut rests with mature neuroendocrine and mucous cell populations. work partially supported by grants from the italian ministry of education (rome, to mp) and the associazione italiana per la ricerca sul cancro (milan). infection and immune inflammation peculiarities in atherosclerosis i zota, e foca, e melnic pathology department, the state university of medicine and pharmacy n.testemitanu, chisinau, republic of moldova introduction during the last decade the basic postulate of atherosclerosis etiology and pathogenesis has changed. this review presents a spectrum of data regarding the link between infection and immune inflammation development in atherosclerosis. material and methods this study has been carried out on the human aorta, coronary heart vessels collected from early performed autopsies and on the carotid arteries gathered during surgical vessels remodeling of persons. the patients with cardiovascular insufficiency on atherosclerosis background from to years were taken into study group. chlamydia pneumoniae was detected by routine microscopy on shtive fixed material, and by immunohistochemical reaction performed on cryophilic specimens marked with fits antibodies. hsp- and tnf-α implication have been studied trough immunohistochemical reaction using monoclonal antibodies. this material was fixed in % formaldechyde solution. results chlamydia pneumoniae has been detected in endothelial cells, macrophages, smooth muscle cells of fatty streaks and fibrous plaques. its detection incidence decreases in advanced arterial lesions. we also determined that chlamydia pneumoniae infection is capable to induce immune reaction against hsp- which can be considered like a target in autoimmune reaction. they produce an cytotoxic effect on the endothelial cells followed by permeability disturbances, faciliting the atherosclerotic lesions progress. among the inflammation mediators in increased quantity tnf-α have been detected being localized on the surface and in depth of atherosclerotic plaque around the foam cells. conclusions our outcomes demonstrate the toxic and infectious factors existence which can be considerated decisive in atherosclerosis pathogenesis. this data give us the reason to elaborate other diagnosis and treatment strategy. introduction primary tumours of the heart are rare, the majority are benign, myxomas representing - %. hemangiomas are extremely rare, with an incidence of . %- . %. they usually occur in adults, mostly are an incidental finding. we present a man with asymptomatic cavernous hemangioma of the left atrium. case report transthoracic echocardiogram of a -year-old man with arterial hypertension, otherwise healthy, with normal ecg and chest x-ray, revealed a wide based isoechogenic tumour, measuring . x . cm, located in the moderately enlarged left atrium, attached to interatrial septum and anterior wall, adjacent to mitral valve annulus, sparing oval fossa. it showed minimal mobility but did not cause any obstruction of the blood flow or mitral regurgitation. macroscopically, surgically removed tissue was spongy, partly haemorrhagic. microscopically, there were cavernous venous structures, filled with blood and focally thrombosed. focally, a few smaller capillary channels were found. there was no endothelial atypia and no papillary hyperplasia. discussion and conclusion histologically, hemangioma of the heart has two major differential diagnostic possibilities. in case of a myxoid background, it is possible to be misdiagnosed as myxoma, but in hemangioma there are no myxoma cells and no ring structures. grossly, opposite to myxomas, hemangiomas are rarely attached to the oval fossa. papillary hyperplasia, which is reactive endothelial proliferation in hemangioma, may lead to misdiagnosis of angiosarcoma. angiosarcoma can be excluded only when the whole tumour is examined and no marked endothelial atypia, cellular areas of spindle cells with poorly formed vascular channels, and necrosis are found. compression, obstruction, effusion and bleeding are responsible for clinical picture. symptoms depend also on the location, size and invasiveness. the natural history is unpredictable, for this reason surgical intervention is mandatory. introduction the limb-body wall complex is a congenital syndrome, which includes limb defects, abdominoschisis associated with facial and neural tube malformations. some authors believe that the limb-body wall disruption is separate from amniotic band syndrome. others suggest that both belong to a wider spectrum, with a possible overlap between these two categories. in the present study, we describe a case of lbwc and based on our findings we suggest a new pathogenic mechanism. case report a years-old cap-verdean female gave birth at weeks to a pair of twins. one foetus showed multiple malformations incompatible with life. physical examination showed partial lower extremity amputations. a large omphalocele was also noted. there was a large lumbosacral meningocele and the head showed micrognathia and high-arched palate. there was right hydronephrosis secondary to right ureteral obstruction and cloacal anomaly. the lungs were hypoplastic. the histology of the internal organs was characteristic for the developmental stage and cytogenetic testing revealed a normal male karyotype. discussion and conclusion we believe, that early amniotic bands may lead to vascular insults, proximally and distally by altered haemodynamics. the lower limbs were partly amputated as a result of the continued growth associated with altered vascular supply. the same mechanism may explain the other findings as well. vascular disruption by virtue of altered haemodynamic state may give rise to thromboemboli, which may affect the growth of external/internal structures, distant from the site of disruption. however, this vascular lesion was masked by continued growth of the foetus. therefore, the abs may precede and may cause the lbwc. two cases of pleuropulmonary blastoma k vuopala , h tuominen , r jauhola , p valmari , r herva lapland central hospital, rovaniemi, finland oulu university hospital, oulu, finland pleuro-pulmonary blastoma (ppb) is a malignant tumour of childhood. the most common clinical sign is respiratory difficulty. ppb appears as a pulmonary and/or pleural-based mass and is characterized histologically by a primitive, variably mixed, blastematous and sarcomatous appearance. no metastases have been reported in neonatal cases. we describe two cases that presented ppb. the first was a term male infant of a g p mother. the pregnancy was uneventful. after normal delivery, a male infant with severe respiratory distress was born. by ultrasound examination, a thoracic mass were identified. there was no response to the intensive care. the infant died at the age of . hours. at autopsy, a tumour of g, measuring x cm was found in the left lung. in addition, there was a smaller mass, of cm diameter in the mediastinum. micro-scopically, the tumour masses consisted of ppb. the second case was a -year-old female with persistent fever and respiratory difficulties. in ct scan, the lower lobe of the left lung was replaced with a tumour mass. during operation, the tumour was seen to be infiltrating to the mediastinum and was partially resected, followed by intense chemotherapy. histologically, the diagnosis of ppb was given. the child has shown no sign of the disease during follow-up of six months. the ppb is regarded as the pulmonary dysontogenetic analogue of wilms' tumour, neuroblastoma, and hepatoblastoma. the first case showed its highly aggressive nature, with mediastinal metastases found at birth. causes of intrauterine foetal death associated with placental pathologic features v blažičević , v Štitić , m mrčela clinical hospital osijek, croatia general hospital karlovac, croatia introduction improvements in antenatal and intrapartum care have reduced perinatal mortality, but the rapid advances in intensive neonatal care have had a dramatic effect in reducing neonatal deaths so that stillbirths have become relatively more important. the aim of this study was to identify the relationship between placental lesions and stillbirth. materials and methods we analysed the causes of intrauterine foetal death after the th week of pregnancy during the years - at the departments of obstetrics and gynaecology of the clinical hospital osijek and general hospital karlovac. a complete autopsy was made on each stillborn, including investigation of the placenta, cord and foetal membranes. histological specimens were studied from all internal organs of the foetus and from the placenta, cord and membranes. haematoxylin and eosin stain is used in routine causes, pas staining to assess thickening of the basement membrane and trichrome staining to assess stromal fibrosis of the villi when it was necessary. results there were stillbirths who died before labour - placentas of them were examined and weren't. abnormalities were found in ( . %) placentas and 'normal' placentas were ( . %). autopsy findings in stillborn foetuses: asphixia ( . %), maceration only ( . %), major malformations ( . %), vitium cordis ( . %), anencephalia ( . %), others ( . %). placental pathologic findings: infections ( . %), single chronic and acute infarction ( . %), multiple chronic and acute infarctions ( . %), placental failure for other reasons ( . %), chronic ischemic change ( . %), infections associated with circulatory disturbances ( . %), retroplacental haematoma ( . %), massive subchorial thrombosis ( . %), chorangiosis ( . %), umbilical cord tumour ( . %), intervillous thrombus ( . %), chorionic cysts ( . %). conclusion the most of women with a stillborn infant were quite healthy and did not reveal any risk factors when assessed on clinical grounds. there were patients in whom evident foetal growth retardation had been overlooked at the maternal health centre before the death of the foetus. the importance of routine ultrasound screening for detection of unrecognised risk patients and placentas is emphasized. introduction microscopic diagnosis of oligobiopsy material taken during endoscopic examination plays an important role in diagnosing syndromes of absorption disorders, especially in case of celiac disease. the aim of the study was a morphologic evaluation of the small intestine mucosa and comparison of the used from many years shiner/shmerling classification with a new modified marsh classification. materials and methods during the study, bioptates if small intestine evaluated in the chair and department of pathomorphology in zabrze in the years - were analysed again. out of the bioptates were with i of villous atrophy, with ii, with iii and with iv. study material was from the paediatric hospitals in zabrze and bytom. during routine histopathologic diagnosing, villus atrophy was evaluated in the shiner/ shmerling scale. presently, the marsh scale is being introduced into histopathologic diagnosing. it takes into consideration not only features of villus atrophy and glandular crypts proliferation but also intraepithelial lymphocyte infiltrations. another evaluation of villus atrophy was performed according to the modified marsh classification. the received results are presented in the modified marsh classification: type - ( . %), type - ( . %), type - ( . %), type a - ( . %), type b - ( . %) and type c - ( . %). our study allowed to compare two classifications used in the evaluation of the atrophic and proliferous processes and to select from the shiner/shmerling degree i a group of cases (type and according to marsh), which had no equivalents in the so far used scale. moreover, it was shown that the sub-groups of the type of destructive changes did not correspond fully with the so far used shiner's degrees ii-iv. it enables fast comparison of the atrophy degree with the results received before the introduction of the new modified classification. moreover, a routine application of precise descriptions of histopathologic results of small intestine mucosa in syndromes of absorption disorders facilitates retrospective selection, from the so far used shiner's degree i, of the cases corresponding with marshal types and . conclusions application of the modified marshal classification for an evaluation of the small intestine mucosa in celiac disease enables accurate presentation of atrophic and proliferous changes including selection of type and , which have no equivalents in the so far used shiner/shmerling classification. embryonal rhabdomyosarcoma: a case report s kocmanovska -petreska, b ilievski, p cvetkovski, l spasevska, s kostadinova -kunovska, s duganovska institute of pathology, faculty of medicine, skopje rhabdomyosarcoma is an aggressive malignant skeletal muscle neoplasm arising from embryonal mesenchyme. the head and neck region is the most common site for this tumour in children. neonatal presentation of this tumour is rare. we present an autopsy case of a female newborn having a large tumour mass ( x x cm) in the frontal region, covering the entire upper half of the face. similar subcutaneous noduli with diameter , cm, cm and cm were found on the left cheek, and another with diameter , cm, , cm, cm and cm on the right cheek. the histological examination showed a tumour composed of large spindle cells with myoblastic features and small round to elongated undifferentiated cells. associated with the spindle cells is various numbers of large round or globular cells with hyperchromatic nuclei surrounded by acidophilic granular or homogeneous cytoplasm. cross-striations were found in the spindle cells. imunohistochemically tumour cells were positive for desmin and vimentin. the final diagnosis was embryonal rhabdomyosarcoma. immunohistochemical staining helped to make a proper diagnosis. introduction apoptotic cell death represents an important mechanism for the precise regulation of cell numbers in normal tissues. various apoptosis-associated regulatory proteins may contribute to the rate of apoptosis in neoplasia. the present study analyses the inter-relationship between the expression of the pro-apoptotic bclx (s/l) protein and histological type and stage of wilms tumour. materials and methods bcl-x (s/l) expression was investigated using an indirect immunoperoxidase technique, applying antibodies to bcl-x (s/l) protein. correlation of semi quantitatively scored bcl-x (s/l) levels with histological type and tumour stage was performed for cases of wilms tumours ( classical and anaplastic). results the expression of bcl-x (s/l) protein was present in most cases of wilms tumour ( %). generally, bcl-x (s/l) was expressed in the blastemal and epithelial components of wilms tumour. in most cases, the expression of bcl-x (s/l) was moderate. blastemal bcl-x (s/l) expression decreased with tumour stage. there was no correlation between histological type and expression of bcl-x (s/l) protein. diffuse expression of bcl-x (s/l) protein was observed in cases of anaplastic wilms tumour. these two cases were stage i and of unfavourable histology. in cases of wilms tumour, no immunohistochemical staining was observed. so, these results cannot indicate whether absence of bcl-x (s/l) could play a role in the development of anaplasia. conclusion according to clinical follow-up, the results of our morphologic and immunohistochemical analysis suggest that decrease of the blastemal bcl-x (s/l) protein could be associated with the capacity of wilms tumour for local and distant spread. expression of alpha, beta and gama catenins in wilms tumour s radojevic background loss of catenins is suggested to promote tumour invasion and distant metastasis in tumour development. the aim of this study was to determine the expression of catenins in wilms tumour and their relationship with histological type and stage of wilms tumour. materials and methods catenins expression was investigated using an indirect immunoperoxidase technique, applying antibodies to alpha, beta and gama catenins. correlation of semiquantitatively scored adhesion molecule levels with histological type and tumour stage was performed on cases of wilms tumours ( classical and anaplastic). results beta and gama catenins were present in most cases ( % and % respectively) and were usually coexpressed. anaplastic type of wilms tumour displayed higher beta catenin and lower gama catenin level in comparison with other types. nuclear positivity, in each case, was found to be very strong, but was usually present only in % of tumour cells. beta and gama catenin expression was not correlated with stage of wilms tumour. alpha catenin was present in % cases of wilms tumour. stage iii displayed higher alpha-catenin levels than stage i/ii, suggesting that impairment of their function might exist without actual loss from tumour cells. conclusion our results suggest that the level of catenin expression is associated with histological type of wilms tumour, regardless of tumour stage. observed nuclear expression of beta catenin indicates that the activation of wnt-signaling pathway is crucial step in wilms tumour genesis. comparison of apoptosis and fas, fas-l and bcl- expression in wilms' tumor m ergin, a uguz, s erdogan, g gonlusen, n tunali cukurova university school of medicine dept. of pathology, adana, turkey background apoptosis is a complex process involving many genes that play role as antiapoptotic or apoptotic factors. it controls normal homeostasis of tissues. expression of these genes vary from one type of tumour to another type. the aim of this study is to evaluate the expression of bcl- , fas and fas-l and apoptosis in wilms' tumour (wt). method thirty paraffin embedded tissue specimens were included. immunohistochemical study was performed for bcl- , fas and fas-l and in-situ end-labelling technique (isel) for apoptosis. results the expression of these proteins varied among the tumours. there was no statistically significant correlation between the expressions of bcl- , fas, fas-l and. apoptotic index. conclusion apoptosis is an important mechanism in the growth of the tumours. we suggest that not only bcl- , fas and fas-l play role as regulators of apoptosis in wt but also other apoptotic and antiapoptotic genes involve this process. further investigations are necessary to understand the molecular mechanism of the progression of wt. uncommon tumours of the neuraxis in childhood j vila-torres hospital universitari sant joan de déu. department of pathology, barcelona, spain case a year-old girl presented with seizures and weakness in the left part of de body of -year duration. ct scan showed a mass that filled the right ventricle, with a strong mass effect, peritumoural oedema, midline shifting and slight ventricular enlargement. a partial excision was carried out and the pathological diagnosis was that of choroid plexus carcinoma. the patient died six months later due to adjuvant therapy side effects. in the last who classififcation of brain tumours, grade iii choroid plexus carcinoma is defined as a choroid plexus tumour showing features of malignancy: nuclear pleomorphism, frequent mitosis, high nuclear/cytoplasmic ratio, increased cellular, density, blurring of the papillary pattern, poorly structured sheets of tumour cells, necrotic areas, and often diffuse brain invasion. all those tumours classically displaying a papillary pattern must be considered in the differential diagnosis. case a month-old child presented with respiratory distress since months old. on rx and ct scan, a tumour in the left hemithorax was seen, with right shifting of the mediastinum. the tumour was partially removed. pathological diagnosis was that of spinal canal invasion by a soft tissue lipoblastoma. eight months after surgery the tumour relapsed, infiltrating the epidural spinal canal from c to d . the tumour was fully excised by means of microsurgery techniques, without neurological deficits. this case is an example of invasion of the vertebral canal by a soft tissue tumour previously removed. lipoblastoma is a rare fatty tumour occurring in children in the extremities or less commonly in the trunk or retroperitoneum. the tumour is benign but may recur. grossly, lipoblastoma showed a distinct lobulated pattern of yellow-grey myxoid tissue, histologically composed of highly vascular fat in a myxoid stroma resembling embryonic white fat. differential diagnosis, based on clinical-pathological grounds, include fibrolipoma and myxoid liposarcoma. case a year-old boy presented with partial complex seizures for year duration. nmr showed a discrete hypodense corticalsubcortical lesion in the left frontal lobe. neither oedema nor mass affect was detected on ct scan. the tumour was completely removed by surgery and the pathological diagnosis was that of dysembryoplastic neuroepithelial tumor. this is an intracortical grade i tumour composed of oligodendrocyte-like cells. macroscopically, is a nodular outward expansion of the superficial cortex. histologically, a low-power view of a perpendicular section of the cortical surface shows the characteristic pattern of multiple intracortical nodules and internodular tissue, both containing mucin. also seen, is a specific glioneuronal component of vertical columns of neu-rites transversing the cortex, sheathed by oligodendrocyte-like cells accompanied by mucin and with 'floating neurons' (cortical ganglion cells seen in individual small pools of mucin). case a year-old boy presented with diplopia and proptosis. the nmr showed a hypodense, diffuse lesion in the left temporal and parietal lobes that produced a slight collapse of the left lateral ventricle. two weeks later the patient developed symptoms of time and space cognitive disturbances, language difficulty and seizures. a biopsy was done and the pathological diagnosis was that of gliomatosis cerebri. this is a rare primary brain tumour characterized by a diffuse proliferation of neoplastic glial cells that typically involve multiple brain areas with preservation of brain structures. the diagnosis depends on both histologic and radiologic features. on nmr there is a uni or bilateral diffuse involvement of the white matter extending to the cortex with poor mass effect. microscopically, there is a proliferation of monomorphous astrocytes, grade ii or iii, infiltrating the brain parenchyma freely. some undifferentiated cells show 'naked' nuclei while others look like 'rod' structures. in cases of low cellularity and cytologically bland astrocytes, it may be extremely difficult to make the differential diagnosis with reactive gliosis. gc is associated with a poor prognosis. introduction production of materials for dental implants is of great social importance and is related to the progress in material technology. main directions of development in this discipline concern an accurate choice of implant material in order to fix an implant in a bone tissue firmly and to decrease tissue reactions. new direction of research on implant materials is powder metallurgy. in comparison with the so far used technologies, it has many advantages, such as homogenous fine grained material structure, possibility to make composites including biologically active implants, and also ability to overgrow the implant with bone tissue. aim of the study was morphological analysis of healing of dental alveolus canals filled with metal implants. evaluation of tissue reactions in bones and surrounding soft tissue from several types of metal implants. material and method the experimental study was performed on mixed breed rabbits of - g weight. after premedication with atropine with relanium, surgical procedure was performed in each animal. one of the incisors was extracted and after the canal was formed, metal implant was inserted. the first group (d ) consisted of animals whose dental alveolus canals were filled with cylinder of x mm made of pressed co-cr-mo alloy covered with calcium triphosphate. the second group (d ) consisted of animals whose dental alveolus was filled with implants of cylindrical shape of a size x mm made of pressed co-cr-mo alloy. the third group (d ) consisted of animals whose dental alveolus canal were filled with a titanium screw of a size x mm. the control group (k) consisted of animals whose dental alveolus canal was left only with blood clot. the postoperative wounds were sutured with dexon thread. the clinical and observations and histopathological introduction correct reconstruction of bone defects is a serious problem in orthopedics and in maxillo-facial surgery. hence, numerous experiments and clinical studies on a usage of natural and artificial materials being a scaffold enhancing a reconstruction of bone tissue (i.e. carbon fibre, hydroxyapatyte granulate). deproteinized bovine bone is a new material. it was elaborated in at the mineralogy faculty, mining and steel academy in kraków. the technology of natural animal bone tissue enables its use in filling bone defects, accelerates reconstruction of bone tissue and minimizes unfavorable tissue reactions through total immersion into the reconstructed bone. it gives no incorrect immunological response of the body to the implanted xenogenic tissue. the aim of the study is to evaluate healing processes of the defects made in rat femoral bones and filled with deproteinized bovine bone and to compared it with spontaneous healing. material and method the experiment was conducted on wistar rats divided into groups ( rats per group). the surgically made bone defects were filled with the study material. the animals, whose defects filled with blood clots spontaneously, made up a control group. the healing process in bone tissue was examined clinically and morphologically after , , , , and days after the experiment. results in histopathological estimation, the healing speed of the defect in femoral bone filled with blood clot (control group) was compared to the healing of the defect filled with deproteinized bovine bone (study group). no inflammatory reaction to the implant was observed. moreover, acceleration of the healing of the defect filled with deproteinized bovine tissue was observed. bovine bone was, in the study group, totally immersed in the body's bone tissue, i.e. was surrounded by the arisen on its surface sponge bone. in the last periods of the observation, on the borders of the observed synostoses no osteoblast cell activity was observed and normal bone marrow was found between osseous trabeculae. conclusions osseous wounds filled with deproteinized bovine bone heal without any inflammatory complications. no negative systemic reactions proves that the applied material was properly prepared for implantation. histopathologic studies showed that deproteinized bovine bone accelerates regeneration process of a damaged bone tissue. withdrawn p- the effect of specific inhibitors of caspases on apoptosis of thymocytes induced with paclitaxel although caspases are involved in molecular mechanisms of the execution phase of apoptosis, the precise mechanisms of their involvement in paclitaxel induced apoptosis are still not identified. the aim of this study was to investigate and compare the effects of inhibitors of caspases ( mmol/l) on apoptosis of rat thymocytes induced by paclitaxel ( mmol/l) and colchicine ( mmol/l). the effect of z vad and specific inhibitors of caspases (caspase , and ) after , and hours of incubation of thymocytes with paclitaxel and colchicine were analyzed. after incubation cells were fixed, processed for lm and em, and the percentage of apoptotic cells (apoptotic index) was determined. general inhibitor of caspases zvad and inhibitor of caspase led to a significant decrease of apoptotic index after hours of incubation with paclitaxel (from . % to . % and . %). inhibitor of caspase and caspase lowered the apoptotic index after hours incubation with paclitaxel from . % to . % and . % respectively. inhibitor of caspase did not affect the apoptotic index in controls, while the other used inhibitors reduced it. inhibitors of caspase , and and zvad had similar effects on apoptosis of thymocytes induced by colchicine. these results show that apoptosis induced by paclitaxel is caspase dependent, and that it, due to the effect of inhibitor of caspase , probably involves mitochondria. caspase activity might also be involved in mechanisms of induction of apoptosis of thymocytes by paclitaxel. effects of concurrent administration of lithium and gentamicin on the histoembryogenesis of rat fetus during the developing periods this study was planned to investigate the effects of gentamicin and littium on rat histoembryogenesis and to determine the effects, if any, of lithium as a potent gentamicin inhibitor. the rats were exposed subcutaneously to -mg/kg gentamicin on days - of gestation. lithium administered orally at doses mg/l on days - of gestation. fetuses were collected on day and compared with those of the untreated, and saline treated control as well as with those of the gentamicin and lithium treated groups. all control and cases embryos groups were killed and their tissues were obtained. then prepared for paraffin block, sectioning, h-e staining and light microscopic studies. we fond that gentamicin caused growth retardation versus lithium which dosn't did so. lithium has a state like control but no wary exactly. furthermore, concurrent administration of gentamicin and lithium caused regularity in histoembryogenesis cycle and the normal growth beside the lithium or gentamicin. of course the recent result is not exactly comparable to those of controls. t- toxin, a solitary hypergranular mcs were situated on the external wall of blood vessels. however, on days , and the number of perivascular mcs was increased. more than percent of these mcs showed degranulation. conclusion our results suggest that perivascular localisation and later degranulation of mcs may play an importantant role during the acute reaction in the heart of rats poisoned by t- toxin. effects of bromadiolone on rodent liver: pathohistological findings o- , o- takeda k, p- takeyama j, p- talalaev a, p- talan-hranilovic j, p- tammi m, p- tammi r, p- tanase c, p- tanaskovic italy author index the numbers are the abstract numbers p- o- gruia am o- , p- o- , p- sever m, p- sever-prebilic m skender gazibara m, o- , o- , m, p- p- , p- , p- , p- , p- , p- , p- srebotnik kirbis i, o- arrhythmogenic right ventricle dysplasia and sudden cardiac death. controversial aspects p- basal cell characteristics in breast carcinoma with medullary features (cmf) acknowledgement: dr chu is supported by nhri-ex - sc, nhri-ex - sc and bmrp . all chosen antibodies gave a specific and reproducible staining. there were no false positive or negative results. the panel can be recommended for screening for defects in the mmr-system. immunoexpression introduction several cytological features are taken into account in infiltrating duct carcinoma of the breast. the aim of this study has been to assess the prognostic value of the nucleolar area by means of ultrastructural morphometry. materials and methods forty-six cases of ductal carcinoma of the breast were included in the current study. all biopsies were processed for electron microscopy and electron micrographs were taken at x , magnification. nucleolar area measurements were done using sigmascanpro software and statistical analyses were performed. nucleolar area measures were categorized in terciles. a kaplan-meier estimation was performed to assess those measures related with disease-free interval. measures that reached statistical significance were dichotomized at the cut point with maximum likehood in a cox regression model and then were included in a multivariate analysis. results statistically significant differences were found in terciles per area (p= . ) and per variation coefficient (p= . ). in terms of nucleolar area measurements, any given nucleolar area with a standard deviation . , showed a relative risk for recurrence of . ( . - . ) (p= . ).conclusions ultrastructural morphometry shows that nucleolar features, particularly those related to size heterogeneity, have a significant impact on prognosis. determination of ploidy in different lesions of prostate a ibraeva , m alchinbaev , a seitaliyeva scientific center of urology, department of pathology, almaty, kazahstan scientific center of urology, almaty, kazahstan immunohistochemical correlation between pattern of postatrophic hyperplasia and carcinoma of the prostate ar botticelli penile leiomyosarcoma-morphological and immunohistochemical study g berdan, a petrescu, c ardeleanu prof. dr. th.burghele hospital, bucharest, romania introduction our case is about a years old male patient who was admitted with the diagnosis of a penile tumor, reason why a total penectomy was performed. material and methods fragments of the tumor and penile tissue were fixed in formalin %, embedded in paraffin and sections were stained with he and vg. immunohistochemical tests have been done: actin, vimentin, s , ae -ae , hmb , pcna. results on macroscopic examination, the tumor was bulky, ulcerative nodular, occupying the glans and the distal part of the shaft; it was gray white coloured, with hemoragic areas. microscopic examination revealed a tumorous proliferation of elongated cells with moderate nuclear pleomorphism and mitosis (typical and atypical), organised in a fascicular pattern, well vascularised; the tumor was superficially ulcerated and invaded the corpora cavernosa. the tumor was diffusely positive for actin and vimentin, focally positive for s , negative for ae -ae and hmb , and pcna was %. conclusions the morphological appearance and immunhistochemical results led us to the diagnosis of penile leiomyosarcoma, moderately differentiated. do human papillomaviruses infect proximal parts of the male genital tract? introduction human papillomaviruses (hpv) play an essential role in the pathogenesis of squamous cell carcinoma of the uterine cervix. the male urogenital tract is considered to be a reservoir of hpv infection. our previous finding of hpv in the squamous metaplastic epithelium with dysplasia in the epididymis prompted us to start a study focused on the presence of hpv in the male genital tract. aims: verifying the presence of hpv in the epididymis, ductus deferens and seminal vesicles. materials and methods samples from three groups (a, b, c) of patients were analysed by means of nested pcr for the presence of hpv dna extracted from a paraffin-embedded tissue sections. types of hpv were determined by dna nucleotide sequencing of the pcr products. group a consisted of patients (mean age , age range - , except for one patients years old), group b consisted of patients (mean age , age range - , except for two patients and years old) and group c consisted of patients (mean age , age range - ). patients in group a were surgically treated for non-tuberculous epididymitis ( epididymal introduction glucagonoma is an alpha-cell pancreatic neoplasm with incidence of one in million; the malignancy rate is unknown. the clinical, pathologic, immunohistochemistry and electron microscopy characteristics are described in a mexican cohort of patients. material and methods we reviewed the pathology archives of incmynsz and hocmnsxxi for pancreatic tumors with neuroendrocrine morphology and expression of immunohistochemistry markers for neuroendocrine and glucagon-related peptides. complete clinical history and follow-up were achieved. results six cases of glucagonoma were obtained. electron microscopy was available in two cases. three man and three women with median age of . years, were admitted with glucagonoma syndrome complaints. the tumor localization was: head of pancreas ( / ) and tail ( / ), with average size of . cm. two cases showed hepatic metastasis. the histopathologic study was constant with contrasting architectural, cytologic and mitotic count in all the cases. expression of neuroendocrine markers was observed in all cases, with positive glucagon stain, and negative for secretion markers (insulin, pp, serotonin, calcitonin). in electron microscopy, dense core granules were identified in two cases. discussion there are cases of glucagonoma described in english literature; no one is originated from latin american population. in this series, the neoplasms have equal prevalence for female and male patients, all from elderly group. the morphologic feature extremely varies in each tumor, the malignancy criteria were correlated in two cases with vascular invasion and this coincides with metastasis. a peculiar finding are the only expression of glucagon in tumoral cells, and two malignant cases with liver metastasis. estrogens regulate the development, maturation, and function of various organs, including the thymus. therefore it is suggested that estrogens play important roles in the development of human thymoma. however there is no study about influences of sex steroids to human thymoma. in this study, we examined the immunohistochemical localization of sex steroid receptors for estrogen (er)α and erβ, progesterone (pr)-a and pr-b, and androgen (ar) in human thymoma (n= ), and correlated these findings with various clinicopathological parameters. we used reverse transcription polymerase chain reaction (rt-pcr) and realtime pcr to further study the expression of these receptors in thymoma cases. results immunoreactivity for all sex steroid receptors was detected in the nuclei of thymoma epithelial cells. the percentage of immuno-positive cases and h-score values for each receptor (mean±sd) were: erα, . % and . ± . ; erβ, . % and . ± . ; pr-a, . % and . ± . ; pr-b, . % and . ± . , and ar, . % and . ± . , respectively. results of real-time pcr were consistent with those of immunohistochemistry especially results for erα, pr-b, and ar. a significant positive correlation was detected between immunoreactivity for erα and pr-b. erα immunoreactivity was inversely correlated with tumor size, clinical stage, who classification and ki- labeling index. in addition, the status of erα immunoreactivity was significantly associated with a better clinical outcome in thymoma patients. conclusions sex hormones receptors express in human thymoma, and especially erα may be related to sex, clinical stage and pathological cell type. galectin- immunodetection using a biotin-free system in a series of oxyphilic (hurthle) cell tumors of the thyroid m volante , f bozzalla , r depompa , c pecchioni , a bartolazzi , m papotti dept. biomedical science and human oncology university of turin, turin, italy s. andrea hospital, university of rome, rome, italy introduction galectin- was reported to distinguish follicular and papillary carcinomas of the thyroid from adenomas, but controversial results were obtained in oxyphilic cell tumors, possibly related to endogenous biotin reactivity. the aim of this study was to assess the role of galectin- immunodetection in a large series of benign and malignant oxyphilic tumors. materials and methods galectin- expression was retrospectively analysed in thyroid oxyphilic (hurthle) cell tumors, including adenomas [oa], carcinomas [oc], oxyphilic variant of papillary carcinoma [optc] and in control cases of goiter with oxyphilic changes and hashimoto's thyroiditis. a purified galectin- monoclonal antibody and a biotin-free immunoperoxidase procedure were used in paraffin embedded specimens. results of ocs were focally ( cases) or diffusely ( cases) positive. / optc were focally ( cases) or diffusely ( cases) reactive. overall, malignant oxyphilic tumors were positive in % ( / ). oa had a focal reactivity in % ( / ) of cases, with a preferential subcapsular immunostaining. sensitivity and specificity were % and %, respectively. control oxyphilic goiters were negative ( / ), as opposed to hashimoto's thyroiditis (oxyphilic cells positive in / cases). conclusion the role of galectin- immunoreactivity in the differential diagnosis of benign and malignant follicular lesions is confirmed also in oxyphilic cell tumors. the focal immunoreactivity observed in adenomas may be probably related to a true expression of galectin- , as confirmed by mrna expression by means of in situ hybridisation, and could be related to early malignant changes in tumors still lacking the currently accepted morphological signs of malignancy. introduction fine-needle aspiration (fna) is a very useful method for diagnosing various lesion in the thyroid gland, among them hashimoto's thyroiditis (ht). although many methods may be used, fna is a necessary diagnostic procedure nowadays in order to confirm the diagnoisis of ht. material and methods durind a period of years citologically aspirates were citologically analyzed, stained with he and mgg. the main cytological guidelines in the diagnostic procedure were: . lymphocytic cellularity of smears, . oncocytic follicular cells, . a lot of cellular debris. results from aspirates analyzed ( , %) were diagnosed as ht. there was a high statistical significance in sex and ages for the genesis of ht (χ = , ; p= , ). we analyzed correlation between clinical and cytological diagnosis: in cases initial clinical diagnosis was "thyroiditis", and among them ( %) were cytologically confirmed. from patients with an fna diagnosis of ht the same diagnosis was susspected clinically in cases ( %). several authors noted a high incidence of thyroid carcinoma in patients with ht. we researched association of ht and thyroid tumors, and we did not confirm that ht is a premalignant lesion (χ = , , p< , ). conclusion ht and its association with other lesions could be clearly diagnosed with fna, and we recommend fna as a first line investigation in ht. colposcopic and cytologic findings in hpv infection of the uterine cervix r zivadinovic, v lilic, b djordjevic, s petric gak nis, university clinical center, nis, serbia and montenegro introduction uterine cervical carcinoma is a sexually transmitted disease. hpv infection, especialy types , and oncogene is directly associated with occurrence of preinvasive and invasive changes in the uterine cervix ranging from - %. cyto-pathologic effect of this virus presence in a cell is called koilocytosis and occurs by the effect of fragment e on cytokeratin building protein.koss observed these changes back in , describing them as irregular cavities in cell protoplasm, but only later these cytologic findings were related to hpv infection. in our retrospective study we analyzed colposcopic and cytologic findings of examinees in which the diagnose of hpv infection was established by biopsy. material and methods the mean age of examinees was , , and in regards to parity and menarche there was no statistically significant difference between experimental and control group, since coefficient t was lower than , p< , . therefore we concluded that the sample was representative. results sensitivity of cytology in detection of hpv infection of the uterine cervix was , % and the specificity was %. positive predictive value was % and negative %. the most frequent colposcopic findings in the uterine cervix in case of hpv infection were aceto-white epithel in % and mosaic in %. other colposcopic findings were significantly lower (punctate %, leucoplakia , %). in examinees with colposcopically diagnosed hpv infection in external genitalia, the infection was also present in the uterine cervix in , % which was statistically significant for x = , >(x ( ; , )= , ). conclusion based on the results presented above, we concluded that sensitivity of cytology in the detection of hpv infection of the uterine cervix was low and that it was necessary to combine that method with colposcopy and directed biopsy. in case of colposcopically diagnosed condylomas of the external genitalia and presence of pathologic colposcopic findings of the uterine cervix (aceto-white epithel, mosaic), the presence of hpv infection in the uterine cervix can be expected with high probability. fine needle aspirate of papillary thyroid carcinomas in correlation with histological diagnosis material and methods cases of thyroid aspirates were studied in correlation with histological findings. the patients were - years old (mean age , ). cytological diagnosis was made according to morphological criteria such as arrangement of cells, nuclear atypia and special features (colloid, giant cells, psammoma bodies). cytological diagnosis was in agreement with the histological diagnosis in cases ( , %). cases were follicular adenomas and one hürthle cell tumor. in cases of disagreement between cytological and histological diagnosis the cytomorphological findings were analysed. conclusion cytological evaluation of papillary thyroid carcinomas is of high diagnostic accuracy, although overlapping of cytological criteria between the various pathological entities may lead to false evaluation. the value of universal short-term storage cell medium in processing fnab samples for ancillary diagnostic methods introduction the application of ancillary methods to fnab samples is mainly impaired by a limited quantity of cells in the introduction changes in the collagenous matrix may participate in the pathogenesis and progression of human aortic valve stenosis (as). we investigated the expression, localization, rate of synthesis and composition of type i and iii collagens in as. methods type i and iii collagen mrna expression and the immunohistochemical localization of the collagen antigens were studied from as and normal aortic valves. the concentrations of various type i (pinp, picp, ictp and synthetic peptide sp ) and iii (piiinp and iiintp) collagen antigens were measured by radioimmnoassays in soluble tissue extracts and trypsin solubilized calcified and noncalcified matrices of as samples, and healthy aortic valves of different ages. results the synthesis of type i collagen, localized in the myofibro-blasts adjacent to calcified nodules, was - -fold higher in as compared to controls. the proportion of collagen in total protein fraction was % in the healthy valves, % in the noncalcified matrix and % in the calcified matrix of as valves. in calcified valves the ictp content was -fold higher than in the age matched controls and -fold higher than the young control group. in the controls, ictp (r=- . , p< . ) and iiintp (r=- . , p< . ) decreased with age, whereas sp /ictp ratio increased (r= . , p< . ). conclusions type i collagen synthesis was increased in as. localization of this collagen adjacent to calcified nodules and the profile of the c-telopeptide cross-linking indicate that the ectopic aortic valve calcification may have similarities to bone matrix mineralization.introduction the objective of this study was to determine the potential value of digital thermography (dt) for the assessment of micro-and macroangiopathies of lower limbs in diabetes mellitus (dm). we have studied patients (pts) with dm ( male). pts had dm type i, pts had dm type ii, with age varying from to years. thermograms were acquired using an original pc-interfaced thermograph, averaged and reformatted to x -bit images. analysis included filtering, background cutoff, obtaining x-and y-axis profiles and averaged temperature curves (atc), determination of local maximums and minimums of the atcs, definition of anatomic regions, building regions of interest (roi). results following thermogram patterns were found: . asymmetric unilateral hypothermia of st toe (in % of dm i cases; probably indicating microangiopathy); . bilateral symmetrical hypothermia of both feet at the level of distal phalanges (in % of dm i cases); . unilateral hypothermia of foot ('thermo-amputation'); . unilateral hyperthermia of foot and tibial region; . focal hyperthermic regions (diameter = .. cm; indicating potential subsequent ulceration). atcs and symmetric left-right roi comparisons helped distinguishing between macro-and microvascular disease, between neuro-and angiopathy in dm, evaluating therapy effectiveness. dt was most valuable in surgical cases, permitting ( ) to locate the optimum level of amputation and ( ) to detect early the inflammation. conclusion dt results correlate well with pathomorphology, and depend upon dm type, dm duration, and presence of local inflammation. immunomodulatory treatment improves myocardial histopathology and function in patients with biopsyproven myocarditis aim the aim of this study was to determine the impact of immunomodulatory treatment (interferon-α) on myocardial histopathology and contractility in patients with biopsy-proven myocarditis (mc). methods a total of consecutive pts ( / male, mean age + years, baseline ef= . + . %) underwent endomyocardial biopsy which established diagnosis (using modified dallas classification) of mc (active mc in / pts, focal-active mc in / pts, borderline mc in / pts, and healing mc in / pts). all patients received interferon-α (sc application, miu, time pw, for months) on top of conventional triple therapy for heart failure. rebiopsy was performed in all patients after the termination of interferon-α treatment, and both baseline ef and repeated biopsies were analyzed. semiquantitatively we comapred the amount of cellular infiltrate, degenerative and/or necrotic changes, and interstitial fibrosis. repeated biopsies were than classified as improved, no change and/or deteriorated, for each histopathological variable (by pathologists, in a blinded manner). results by the end of interferon-α treatment ef improved significantly ( + . % vs. . + . %), and no deaths were introduction tumor necrosis factor (tnf)-alpha has been recognized as important physio-pathogenic factor in the initiation and continuation of inflammatory cardiomyopathies. experimental and preliminary human studies have demonstrated that tnf-alpha plays a crucial role in enteroviral-induced myocarditis. the aim of this study was to investigate the expression of tnf-alpha and both its receptors (tnfri and tnfrii) in the endomyocardial biopsies of patients (pts) with viral and non-viral myocarditis. myocardial expression of tnf-alpha was then correlated with different clinical and pathologic findings. material and methods tnf-alpha expression was investigated in endomyocardial biopsies obtained from pts with myocarditis and from control subjects by using reverse transcriptase (rt)polymerase chain reaction (pcr) and immunohistochemistry. viral etiology was diagnosed by pcr in cases. immunohistochemistry was also used to analyse both tnf-alpha receptors (ri and ii). score - was used to grade morphological findings. tnf-alpha mrna and tnf-alpha protein was significantly more present in viral myocarditis than in non-viral myocarditis ( / vs / , p= . ). remarkable immunostaining was observed for both receptors, particularly for tnfri. histological analysis revealed that myocardial necrosis (score . vs . p= . ) and cellular infiltration (score . vs . p= . ) were more prominent in tnf-alpha positive cases. among tnfalpha positive cases, tnf-alpha mrnas were greater in pts with more impaired cardiac function. conclusion our findings indicate that the expression of tnfalpha plays an important role in the pathogenesis of viral myocarditis of any etiology and may influence severity of cardiac dysfunction. cytokine effects are more strictly linked to overexpression of tnfri. recurrence of viral myocarditis after cardiac transplantation: a retrospective study in a pediatric population introduction the objectives of this study was tot verify comparatively clinical age reported antecedents and the morphological aspect of the intestinal mucosa in patients sensitive to gluten, hospitalised at iomc (alfred russescu hospital) in the last years. celiac disease is an inflammation most frequently affecting the proximal small intestine, depending on the presence of gluten in the diet, whose pathogenesis seems to be immunological in nature. methods cases were divided in three groups following clinical manifestations types at hospitalisation time: typical digestive, untypical digestive and extra digestive manifestations. intestinal biopsies, made with crosby probe, in children aged between . and years (one single case was diagnosed as late as at the age of ), regardless of gender. then we analysed morphologically (he usual and pas histochemical staining) and immunohistochemically (lymphocytes b, t with possible subtypes). the lesions counted according to the marsh score were at the first biopsy: - marsh i infiltrating lesions (normal mucosa architecture, villi epithelium infiltrated with lymphocytes; lymphocytosis score: lymphocytes / enterocytes); - marsh ii infiltrating lesions (almost normal villi, enlarged crypts with immature epithelium and infiltrated with lymphocytes); - marsh iiia destructive lesions (partial villi atrophy (va) shortened, flattened villi, slight infiltration with intraepithelial lymphocytes and hyperplasic crypts); - marsh iiib destructive lesions (subtotal va clearly atrophied villi, still recognizable, widened hyperplasic crypts containing immature epithelial cells with a higher growth rate and influx of inflammatory cells); - marsh ilic hyperplasic lesions (total va completely absent villi, with severe atrophy, atrophied, hyperplastic glands and infiltrating lesions). the immunohistochemical tests have indicated the prevalence of t lymphocytes (uchl , cd , cd , cd , gama-delta) both in the luminal epithelium with various degrees of aggression in lamina propria and also spread in stroma. b lymphocytes (l ) are distributed prevalently nodular in stroma. conclusion in conclusion, it is cd t cells that are present in particular in the control of the gluten immune response in patients with marsh i and marsh iii lesions. clinical morphological analysis of early forms of polyarteritis nodosa h mazur- introduction polyarteritis nodosa is a necrotic vasculitis of small and medium size arteries of not fully recognized aetiology. however, it occurs mostly in adults, it can also occur in children. the cutaneous form is one of the types of polyarteritis nodosa. it occurs in children very rarely. polyarteritis nodosa is diagnosed on the basis of histopathologic results and clinical criteria established by the america college of rheumatology. the aim of the study was to present cases of polyarteritis nodosa in children. case reports three cases of cutaneous polyarteritis nodosa in year old girls and case of general form of p.n. in a -year old boy at the beginning a tumour of pancreas head was suggested in the latter case. the final diagnoses in all cases were defined on the basis of histopathologic results the morphologic studies showed: skin and subcutaneous tissue specimens of mm diameter and mm thick were evaluated. epidermis and corium were of normal structure. abundant inflammatory infiltration consisting of neutral and acidophilic granulocytes, lymphocytes and plasmatic cells were observed on the boarder of corium and subcutaneous tissue. the infiltration was situated mainly in arterial walls and surrounding fibrous and fatty connective tissue. different degrees of change advancement were observed in the walls of the vessels. some changes were characteristic for an early stage of a disease and were demonstrated as swelling of the vessel wall and surrounding tissues (degeneration stage). in the oedematous wall fibrin concrement and only a few inflammatory cells were present. other cells included abundant transmural inflammatory infiltrations (inflammatory stage). vessel endothelium underwent desquamation and thrombi begun to appear in the arterial lumen. only in few vessels, the process of considerable advancement of changes was observed -a repair process with consecutive fibrosis and thickening of arterial wall with considerable narrowing of its lumen. the described children were treated successfully with corticosteroid therapy and antistreptococcal prophylaxis with long-term remission. comparative morphologic analysis of the small intestine mucosa in a course of coeliac disease p- effects of sildenafile on ipsilateral testis damage following testicular torsion in rats g kale , ih gol , d orhan , n kale hacettepe university, medical school, ankara, turkey gazi university, medical school, ankara, turkey introduction testicular torsion results in damage of the gonad and it is a surgical emergency. testicular torsion evokes infertility as a result of ischemia. the aims of this study were to determine the effects of a vasodilatator agent sildenafile on the testicular damage following testicular torsion. materials and methods forty-two rats included to the study were divided into groups: group ; control, group ; sham, group : torsion (detorsion is performed hours after torsion and orchiectomy is done at the th hour), group ; torsion, detorsion and sildenafile ( mg/kg), group ; torsion and sildenafile ( mg/kg), group ; sildenafile ( mg/kg) without torsion, group ; sildenafile ( mg/kg) without torsion. testicular specimens were examined histopathologically for johnsen tubular score and tubular diameter. the differences of these parameters between groups were evaluated statistically. results johnsen tubular score and tubular diameters were fond to be significantly different in control and torsion groups (p< . ). johnsen tubular score and tubular diameter were significantly higher in the torsion groups which were given sildenafile, and these parameters were again higher in the group which have taken sildenafile at a higher dose (p< . ). conclusion it is concluded that, by inhibiting cyclic guanosin monophosphate specific phosphodiesterase and resulting in vasodialatation, sildenafile reduces the testicular damage caused by torsion (ischemia). introduction autopsy studies in adult patients who had died of sepsis revealed a profound, progressive, apoptosis-induced loss of cells of the adaptive immune system (hotchkiss et al, new engl j med, ; : - ) . in neonatal sepsis and chorioamnionitis, thymus modifications and shrinkage have been exhaustively described (toti et al, hum pathology ; : - ), while morphology of the spleen has not. the aim of this study was to describe the morphology of the spleen in neonates with early onset sepsis and with chorioamnionitis. material and methods the study population has been described previously (toti et al, hum pathology ; : - . ten preterm or full-term neonates who died because of proven sepsis within hours after birth (mean gestational age±sd, . ± . weeks); foetuses spontaneously aborted ( ± . weeks) because of extensive ascending chorioamnionitis. controls included foetuses with induced termination of pregnancy ( ± . weeks) and babies who died suddenly in the perinatal period, without evidence of chorioamnionitis. spleen cell populations were studied by means of immunohistochemistry. results early onset sepsis in the neonates occurred with severe spleen depletion, involving both b and t lymphocytes. in particular, cd +, cd r + and cd + cells were dramatically diminished in the spleen of neonates with sepsis, in comparison to controls. foetuses with chorioamnionitis also showed spleen cell depletion. conclusion these results indicate that preterm and term neonates show an inflammatory reaction similar to that of adult patients, and that chorioamnionitis is associated with an unspecific inflammatory response comparable to that of sepsis. introduction hemophagocytic lymphohistiocytosis (hlh) is a disorder of an immunological disturbance with proliferation of cells of the mononuclear phagocyte system, with the histiocyte as a central cell. it includes two different conditions: primary or familial hlh, an autosomal recessive disorder in which most patients develop the disease very early in life, and secondary hlh associated with neoplastic, autoimmune and a variety of infectious diseases, occurring as sporadic cases throughout life period. clinically the disease is characterised by the occurrence of a hemophagocytic syndrome of prolonged fever and splenomegaly with laboratory signs of cytopenias, lipid, coagulation and liver abnormalities. hemophagocytosis is readily identified in bone marrow, spleen or involved lymph nodes. central nervous system spread of the disease may be present early, but is frequently seen later during the course of the disease. the aim of this study was to presentat an autopsy case of a months old boy. case report signs of hlh were present right after birth. he was treated by the treatment protocol hlh- . the disease later progressed with cns involvement. a complementary bone marrow donor was not found in time. tissue samples collected at the autopsy were proceeded with standard histological and immunohistochemical methods. results severe bleeding in the right lung with other signs of coagulopathy were found as the main cause of death. hepatosplenomegaly was a prominent feature. we detected hemophagocytosis in the bone marrow, liver and central nervous system. conclusions the diagnosis of hlh must be based on clinical, laboratory and cyto/histological findings. the pathogenesis of familial hlh is still not completely understood. introduction a changeable number of goblet cells in intestine epithelium is rarely a matter of consideration among other morphological factors of small intestine mucous membrane atrophy in celiac disease. it is also not evaluated in the current classifications of goblet cell condition in celiac disease. the aim of the study was to evaluate a number of goblet cells in small intestine epithelium with reference to the degree of atrophy in celiac disease. morphological analysis of bioptates of small intestine mucous membrane, taken for a routine histopathologic diagnosis from patients aged from to years, was performed. the patients were diagnosed and treated in paediatric university hospitals in zabrze and bytom in the years - . specimens were stained with haematoxylin and eosin (h.e.) and alcian blue-pas (ab-pas). villous atrophy was evaluated according to the modified marshal scale. results the following types were defined: type - cases, type - cases, type - cases, type a - cases, type b - cases, type c - cases. then, a number of goblet cells per enterocytes of small intestine epithelium of each oligobioptate were calculated. the received results were evaluated statistically with u mann-whitney test. the average number of goblet cells in small intestine epithelium was calculated in relation to the degree of atrophy with the modified marsh scale: type - . (+/- . ), type - . (+/- . ), type - . +/- . ), type a - . (+/- . ), type b - . (+/- . ), type c - . (+/- , ). the analysis used significant relations between an average number of goblet cells in small intestine epithelium and a degree of mucous membrane atrophy in celiac disease. moreover, a correlation according to rang spearmann test showed a coefficient r= . for p< . . conclusions significant relation between a number of goblet cells in epithelium and a degree of small intestine mucous atrophy in celiac disease. it allows an introduction of a new objective quantitive parameter in the evaluation of atrophic lesions. introduction microscopic evaluation plays an important role in diagnosing small intestine inflammatory diseases. defining a degree of villous and brush border atrophy, enterocytes changes, elongation of glandular crypts allows a correct evaluation of treatment clinical effects and child's body response to the applied selective diet. development of immunomorphological studies contributed to confirmation of the autoimmunological basis of several diseases and of the influence of exogenous factors on their development. due to the new approach, evaluation of the changeable character of inflammatory infiltrations including mast cells and marking activity of enteroendocrinal cells have become very important diagnostic elements. the aim of the study is to examine a changeable intensity of infiltrations from mast cells of small intestine mucosa in children in the course of celiac disease. material and method oligobioptates of small intestine mucosa taken in endoscopy from patients (age - years) at the department of pathomorphology in zabrze in the years - . the patients were diagnosed and/or hospitalised in paediatric university hospitals in zabrze and bytom. a degree of villus atrophy was evaluated according to the modified marsh classification. tissue material in paraffin cubes from the routine oligobiopsy of the patients with celiac disease was qualified for immunohistochemical study. immunohistochemical reactions revealed a presence of brown dyed mass cells in intestine mucosa. the cells were evaluated by counting their number in high power fields (magnification x) in each oligobioptate. the received results were calculated statistically with mann-whitney u test. results mean number of mass cells in intestine mucosa was calculated in relation to the degree of villus atrophy according to the modified marsh classification: type (status normalis) - . (+/- . ), type - . (+/- . ), type - . (+/- . ), type a - . (+/- . ), type b - . (+/- . ), type c - . (+/- . ). the analysis showed significant relation between a number of mass cells and a degree of mucosa atrophy in a course of celiac disease. moreover, a correlation after the rang spearman test showed a coefficient r= . for p< . . conclusions significant relation between a number of mass cells and a degree of small intestine mucosa atrophy in a course of celiac disease was shown. defining (marking) a number of mass cells in small intestine mucosa in a course of celiac disease allows an introduction of quantitive method of atrophic changes evaluation as a new objective parameter, especially in cases difficult to diagnose. morphological analysis of tissue reactions in mandible after dental implants of the pressed cocrmo alloy introduction schistosomiasis mansoni infection increases the level of prostaglandins and leukotrienes that are secreted mainly by macrophages within the granulomas. these mediators have a pronounced role in mediation and modulation of inflammation. most non steroidal anti-inflammatory drugs (nsaids) act mainly by inhibiting the cyclooxygenase pathway of arachidonic acid metabolism, thereby diverting it towards the lipoxygenase pathway. these leukotrienes are important in maintenance of acute and chronic inflammatory condition. methods in present study, we used mofebutazone (cylcooxygenase and lipoxygenase inhibitor) and tenoxicam (cylcooxygenase inhibitor) either alone or in combination with praziquantel (antibilharzial drug) in mice infected with schistosomiasis mansoni. we study their effects on liver pathology as pcna (proliferating cell nuclear antigen), granuloma diameters, percent of degenerated ova and type of granuloma after & weeks after infection. results shown that infection increased the percent of positive nuclei in granuloma and in hepatocytes to be about % & % at weeks and these percentages were reduced to be about % & % at weeks after infection respectively. treatment with mofebutazone and tenoxicam produced a reduction in pcna to be about % & % in granuloma and % & % in hepatocytes at weeks and more reduction occurred after weeks after infection. also, mofebutazone tended to produce more protection to isolated hepatocytes (% viability of hepatocyte) than tenoxicam in schistosomiasis mansoni infected mice. combined therapy with praziquantel produced more reduction in pcna and more protection to hepatocytes. it is evident from the present study that, mofebutazone when combined with praziquantel has shown good tolerability more than tenoxicam which was associated with an improvement in pcna, granuloma diameter, % viability of isolated hepatocytes, liver function tests, a fact that would seen to advocate the use of mofebutazone in bilharzial patient. patterns of injury in car and motorcycle accidents jm suárez peńaranda, c bellido, r rico, mt alvarez iglesias, m rodríguez calvo, i muńoz, l concheiro institute of legal medicine, santiago de compostela, spain introduction the role of the forensic pathologist in road-traffic accidents is to detect atypical injuries that could suggest an alternative cause of death, demonstrate natural diseases capable of creating sudden incapacity, and to collect samples for toxicological examinations. the main objective of this work is to study the different patterns of injuries in car and motorcycle fatalities. material and methods we have studied the autopsy reports of deaths related to road traffic accidents ( involving cars and motorcycles). a complete autopsy was performed in every case including toxicological investigation. results the most common cause of death was head injury, responsible for % and . % of deaths in car and motorcycle accidents, respectively. cranial base fractures were more commonly found in passengers rather than in drivers, both for cars and motorcycles ( % vs. % and . % vs. %, respectively). the distribution of cranial vault fractures was similar with percentages of and . for car and motorcycle drivers, versus and per cent for the respective passengers. nevertheless, brain injury was even more frequent in car accidents, accounting for % of the cases, and in motorcycle fatalities, where every single case showed some degree of brain injury. conclusion our results confirm the importance of head injury in road-traffic accidents, especially when motorcycles are involved, being the most common cause of death. although all motorcycle victims wore a helmet at the time of the accident, it did not seem to provide an effective protection, probably because of the high speeds involved. the incidence of cranial fractures and brain injury is higher for passengers than for drivers, probably due to the lack of attention to the traffic of these latter, and the fact that drivers are usually holding the handlebar or wheel at the moment of the accident. heat-induced epitope retrieval in immunohistochemistry c espersen, b crillesen, b jepsen, jw hansen, th veie, h winther, j askaa department of immunohistochemistry, dakocytomation a/s, glostrup, denmark introduction heat-induced epitope retrieval (hier) has dramatically changed the applicability of immunohistochemistry in routine pathology. hier has increased the number of primary antibodies useful on formalin-fixed paraffin-embedded material and in general improved the histochemical staining. the aim of this study was to compare various buffers for hier on a broad range of primary antibodies. materials and methods hier using four different buffers (citrate ph ; tris/edta ph ; dakocytomation target retrieval solution, high ph, code no. s and dakocytomation target retrieval solution ph , code no s ) for pre-treatment of formalin-fixed paraffin-embedded material has been compared using more than different primary antibodies and the envision+tm visualisation system.results hier with citrate buffer retrieve epitopes recognised by the majority of the tested antibodies. however, in several cases the epitopes could not be detected using this method but were retrieved using tris/edta and/or target retrieval solution, code no. s . also retrieval by means of the target retrieval solution, high ph can be used however, the morphology quite often was partly destroyed. furthermore, some antibodies could only be used after enzymatic pre-treatment whereas hier abolished the histochemical staining. conclusion although hier using citrate ph buffer is a standardised and widely used technique, it is in numerous cases sub-optimal or even obliterate the immunohistochemical staining. in general more epitopes can be retrieved using tris/edta ph or dakocytomation target retrieval solution. bone morphology and calcemia in ovariectomised (ovx) wistar rats l markovic postmenopausal oestrogen deficiency results in bone loss (osteoporosis) in humans and experimental animals. wistar rats provide a useful experimental model of post-menopausal osteoporosis. to demonstrate the histomorphometric and histological changes of bone weeks after bilateral ovariectomy in rats and to investigate the impacts of different hormone replacement therapies on the bone. specimens of proximal femur were embedded undecacified for histomorphometric analysis and of distal femoral metaphysis were procured for pathologic examinations. three weeks after ovx the femoral bmd, mean cortical thickness decreased significantly while the number of osteoclasts increased significantly as compared with sham group. the trabeculae became thinner and irregular which changed the bone microstructure in three dimensions. after treatment with different preparations, the above parameters restored to various extents to the sham operation levels. bilateral ovariectomy induced increased osteoclast activity and bone turnover, therefore caused accelerated bone loss. treatment with combined sex hormones preparation could inhibit bone absorption and stimulate bone formation. this investigation was supported by mstd of srbia. vilnius pathological anatomy in the first half of the th century d sabat department of pathomorphology, medical university of silesia, zabrze, polandthe beginnings of pathological anatomy as a separate medical subject were connected in the area of the republic of poland and lithuania with the vilnius university. the first post mortem was done in vilnius by a priest, stefan bisio only in year. at the end of the th century, jakub briotet, a surgeon and anatomist, created a modest anatomical surgery (study). in , jan piotr frank and his son józef arrived to vilnius and became professors of the university. jan piotr frank became the head of the teaching hospital and józef frank the head of the chair of pathology. in november , he started lecturing for students, which he recalled in his "memoirs": several days after my father, i also started a course with a dissertation "de meliori pathologiam tradendi modo" in this speech i put the main stress on a significance of the explanations at the patient's bed-side and of a good knowledge of pathological anatomy… when in , józef frank became the head of the teaching hospital, he founded the first anatomo-pathological study (surgery). most of the samples kept in spirit in the frank's study were eaten by the starving french soldiers during the retreat of napoleon's army. besides frank's study at the vilnius university at the chair of anatomy, there still existed the study founded by briotet. in , the emperor alexander i destined the ruins of the spaska's orthodox church for an anatomical theater. after seven-year redecorations, the building was opened in . besides the theatre itself, dissection room and veterinary institute, it also included a zoological, veterinary and anatomical museum. the growing anatomical museum had, in , preparations including anatomopathological preparations. after closing down the vilnius university and foundation of the academy of medicine and surgery, pathological anatomy was made an individual subject of study. the first lecturer of pathological anatomy in vilnius was ludwik siewruk. he held the classes till and then he took charge of the chair of anatomy at the moscow university. his successor was jan leonow who continued his work until the tsar's authorities closed down the academy of medicine and surgery in . after the academy had been closed down, all the exhibits of the vilnius anatomical museum, including anatomopathological preparations were moved to the anatomical museum of kiev university. few of them remained in the vilnius medical society. introduction t- toxin, a mycotoxin produced by fusarium fungi, is a well-known cardiotoxin that cause myocardial injury, capillary damage, haemorrhage and focal polymorphonuclear cell infiltration. mast cell (mc) is one of the the earliest multifunctional effector cells of the immune system and important constituent of microvasulature in the heart. aim: we wanted to investigate appearance and localisation of mast cells in the heart of t- toxin-poisoned rats. materials and methods adult female wistar rats were poisoned with a single injection of ld- of t- toxin ( . mg/kg sc). rats were divided into two groups: ( ) control group and ( ) t- toxin group. t- toxin was produced in laboratory conditions from fusarium sporotrichoides fungi. animals were sacrificed after the end of the day , , and of the study. cardiac mcs were counted in whole visual fields, magnified by -fold on paraffin section stained with haematoxylin and eosin (he) and giemsa methods. results in the heart of rats treated with t- toxin, blood vessels were congested with thickned walls, and a large number of mononuclear cells was present nearby. hours after administration of introduction bromadiolone is one of the most commonly used rodenticides. this coumarin anticoagulant blocks multiple steps in the coagulation cascade. it is absorbed through the gastrointestinal tract and accumulated in the liver, mainly. the aim of this study was to investigate pathohistological alterations in the liver after oral application of bromadiolone in rodents. materials and methods adult wistar rats and swiss mice, of both sexes, were fed with a ready-to-use baits of low bromadiolone concentration ( . %), for first three consecutive days. animals were sacrificed every day during the one week period, starting with h after the beginning of the experiment. liver paraffin sections were stained by haematoxillin and eosin (he) method. results the pathological alterations detected in all animals ranged from diffuse degeneration to a focal necrosis of hepatocytes and massive circulatory changes. parenchymal degeneration, diffuse oedema and hyperemia were predominant in the liver of animals sacrificed on the first three days of experiment. dissolution of cytoplasm with nuclear pleomorphism in round or ovoid hepatocytes were seen. thickening of the blood vessels with necrosis of endothelial cells were particularly prominent from the fourth to seventh day of experiment. the most interesting finding is the presence of massive hemorrhages with a focal mononuclear cell infiltrations. the majority of hepatocytes were irregular, with macronodular fat transformation of cytoplasm. these changes were the most intensive in male mice on the day of the study. conclusion multiple oral ingestion of bromadiolone induced prominent pathohistological alterations in rodent liver, especially mice. mobile telepathology -a new intringuing tool s seiwerth, l batelja institute of pathology medical faculty, zagreb, croatiathe mobility of information, patient data and images introduced by telemedicine/ telepathology systems designated a completely new viewpoint in pathology. mostly two areas of diagnostic work are so far covered by telepathology service -second opinion/expert consultation and frozen section diagnosis. frozen section diagnosis, is still a very important issue. smaller hospitals, with no sufficient material for a resident pathologist often could benefit by introduction of telepathology. as in many of these hospitals surgery requiring frozen section diagnosis is not performed on a daily basis the telepathology system is not in full use, producing an unfavorable cost-benefit ratio. also establishing a fully equipped frozen-sections lab yields additional costs and personnel cutting frozen sections only occasionally often produce suboptimal. in this the introduction of a mobile telepathology unit could solve some very important problems. the unit should include a fully equipped mobile frozen sections lab including a macro stand and a microscope with ccd tv color camera and a notebook (laptop) computer. the telepathology software used must be stabile and versatile like the pharos/issa system (vamstech, croatia) used by us. by such a system and synchronization of operation schedules hospitals in a radius of up to km around the referral center could be covered without exceeding standard working hours. the issa-server based system option alerting the consultant and referring pathologist by sms that a consultation case is in his mailbox at the server ads additional time effectiveness. a novel high throughput marker validation tool j korbelik, j matisic, c macaulay b.c. cancer agency, vancouver, canada high throughput genetic and expression screening is generating many possible diagnostic markers and therapeutic targets for wide variety of clinical conditions. the rapid validation of these markers is a challenging process. tissue microarrays can be used for the evaluation of many of these markers. however, they have drawbacks and unresolved issues, which are result of the sampling and sectioning problems. we introduce a novel microarray technique based on cell suspensions. multiple slides can be made, all of which are equally representative of the initial samples. a manually operated mechanical device was designed that can deposit distinct spots on the glass slide. each spot of cells deposited in this manner may correspond to unique source. controlling the number of cells per spot their uniformity and size can be achieved by modifying the viscosity of the cell solution or precisely regulating amount of fluid deposited. a fully automated analysis of quantitatively stained microarray samples has been performed to quantify the number of cells per spot, the size of spots, and how reproducible these parameters are. cellular material used in the experimental arrays constructed to date has been acquired from cell lines and liquid prepared lung and cervical specimens. a variety of molecular markers can be evaluated, with minimal cost. fish probes have been tested on cytology microarrays constructed from different lung cell lines. using cell suspensions generated by the dissociation of tissue would enable the creation of more representative samples for clinical marker/target validation or evaluation. key: cord- -rz r sj authors: nan title: abstracts for the th annual meeting of the japan neuroscience society (neuroscience ) date: - - journal: neuroscience research doi: . /j.neures. . . sha: doc_id: cord_uid: rz r sj nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns . sl - - frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl - - neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif a/kif bbeta), nmda type(kif ) and ampa type(kif s) glutamate receptors and mrnas(kif s) such as camkii ␤ mrna and arc mrna with a large rna-transporting protein complex containing at least rna related proteins such as hn rnp-u, staufen, and fmrps. kif a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award - what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in - months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v ). after complete ablation of v , the monkeys recover performance of visually guided saccades toward the blind field in months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi , kakenhi sy - - - distinct roles of two -pass transmembrane cadherins in neurite growth control tadashi uemura , , yasuyuki shima , , keisuke sehara , manabu nakayama , shinya kawaguchi , mikio hoshino , yoichi nabeshima , tomoo hirano , graduate school of biostudies, kyoto university, kyoto, japan; school of science, japan; kazusa dna research center at chiba, japan; graduate school of science, japan; graduate school of medicine, japan; crest, jst, japan drosophila flamingo (fmi) and mammalian celsr - , which are pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian -pass transmembrane cadherins celsr and celsr are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr enhanced neurite growth whereas celsr suppressed it. our result suggested that celsr had a stronger activity as g-protein coupled receptor than celsr did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of -pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin- is preferentially localized in axons; and nectin- , in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin- and dendritic nectin- is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi sy - - - lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa , fumikazu suto nagoya university, nagoya, japan; institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema a is expressed in ca pyramidal cells and principally suppresses invasion of mossy fibers to ca . second, the repulsive signal of sema a is mediated by plexin-a expressing in mossy fibers. third, the repulsive activities of sema a are attenuated by plexin-a in the proximal dendritic segments of ca pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on ( ) working memory maintenance and manipulation, ( ) rule representation and task-switching, and ( ) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy - - - towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy - - - neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of − genes on chromosome q . , has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi ( ) sy - - - a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and k songbird cdna array. we used the arrays to reveal a set of genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. - - - stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi sy - - - role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii ␤ acts as an actin stabilizing molecule to maintain spine structure. camkii ␤ is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii ␤ oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii ␤ oligomer slows down actin dynamics and camkii ␤ is enriched in spines by actin polymerization. the suppression of endogenous camkii ␤ alters spine shape to filopodia-like structures. these experiments suggest that camkii ␤ plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii ␤ detaches from f-actin in an activity dependent manner. we will discuss how camkii ␤ maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using -photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., ) . furthermore, in response to single spine stimulation using -photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration ( ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy - - - basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼ hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy - - - coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f- fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad- found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca + sensing protein was activated. synchronies of sv fusion are maintained by a ca + sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy - - - sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim . thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka , takeshi baba , sumiko mochida , yoshimi takai department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca + -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab system, munc- , munc- , doc- , and tomosyn. we have previously reported that tomosyn, a syntaxin- -binding protein, works as a molecular clamp that controls free syntaxin- availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin- , resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin- , which increases the affinity of syntaxin- for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca + sensor for neurotransmitter release. however, the physiological contributions of its ca + -binding domains (c a and c b) are still unclear. we have studied the roles of aspartate (asp) residues in the c b ca + -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell , p. ) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca + binding to the c b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca + binding to the c b because mutation in the second asp inhibits ca + binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin- (nt- ) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt- and its low-affinity receptor p genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c t in the noncoding region, bdnf-linked polymorphic region, and val met), three polymorphisms of the nt- gene (g- a, microsatellite in intron , and gly- glu) and a missene polymorphism of the p gene (ser leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val met) in the bdnf gene resulting in a prodomain substitution at position from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns sy - - - processing of bdnf and brain disorders masami kojima , aist, osaka, japan; sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p neurotrophin receptor (p ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); (t/g) and (t/g) of the human bdnf gene, causing amino acid substitution (r m and r l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy - - - pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣ leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣ , the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv- vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il ss+fgf- , and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi ( ) sy - - - hgf gene therapy for the treatment of spinal cord injury masaya nakamura , akio iwanami , kazuya kitamura , , yoshiaki toyama , hideyuki okano department of ophthalmology surgery, keio university, tokyo, japan; department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th level in adult rat days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. or weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap +fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., ) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., ) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., . nature neuroscience. nakamura et al., . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first - months after stroke, suggesting the presence of a critical period. in the cat, illert et al. ( ) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c -c segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. ( ) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c -c propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. ( ) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca + responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l cam(l ) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l . therefore, these syndromes have been reclassified as l syndrome. we performed a nation-wide l gene analysis and identified li gene mutations in families with l syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l -mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l syndrome. research funds: kakenhi ( ), ( ) sy - - - rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein gene (mecp ) located at xq is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like gene (cdkl ) manifest as atypical rtt. the mutations of mecp are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in -ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase- are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp , suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca , and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣ or ␤-adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣ and ␤-adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context - days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr , nr a, b, c), glycine transporter , and eaac , by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase ␣ -subunit (pde c), a component of cone-type pde. since genetic mutations of pde c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde in visual functions. shinichi nakagawa , masatoshi takeichi , , fumi kubo , nakagawa initiative research unit, riken, wako, japan; riken cdb, kobe, japan; department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi ( ) sy - - - identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea- to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea- during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea- double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea- -positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea- have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx and crx play critical roles in retinal photoreceptor development. while otx is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi ( , , ) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf found in the mesoderm. we find that although fgf can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf and is actually inhibited by fgf . for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy - - - how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel , encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein e was identified as the gene responsible for this behavioral difference among the species. obp e forms a gene cluster with obp d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp d/e region obtained from species phylogenetically located between d. melanogaster and d. pseudoobscula. sy - - - an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi , , akinori nishi , , toshihiko shiroishi , , tsuyoshi koide , sokendai, kanagawa, japan; national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c bl/ (b ) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b and msm, and those strains have the same genetic background as b except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b - msm, which have msm chromosome , showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi ( . ) sy - - - cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome (capsq ) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv ) was impaired in kjr neurons in contrast to that of c bl/ j mice. by searching the candidate genes, eh domain-containing four (ehd ), a trp-binding scaffold protein encoding gene was found. ehd binds to c-terminal of trpv . three mutations were found in ehd of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv . this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy - - - comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than different strains of mutant mice with the battery. surprisingly, more than % of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi ( , , , ) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least genes and loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt f caused by prx, and scan caused by tdp . sy - - - gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm , who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm /gd synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, ) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa ) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi ( ) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo- is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo- mediates the inhibition of axonal growth as a component of the ngr /p /lingo- and ngr /troy/lingo- signaling complex. inhibition of endogenous lingo- by soluble lingo- or dominant negative lingo- can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo- treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo- treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo- mediates the inhibition of differentiation and myelination. loss of lingo- function using dominant negative lingo- , lingo- rnai, or soluble lingo- or lingo- knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo- led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo- in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc - a sy - - - neuronal avalanches reveal neuronal wirings of layer / cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi ( ) sy - - - spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi , jean-pascal pfister , kazuyuki aihara , , wulfram gerstner department of complexity science and engineering, university of tokyo, japan; school of computer and communication science & bmi, epfl, japan; aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows j and sci. res. on priority areas from mext of japan, and swiss natl. sci. found. - / sy - - - timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as - ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi , ikue otaki , saya suzuki , yasunobu yasoshima , kazuto kobayashi laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin- receptor ␣ subunit (hil- r␣) was expressed under the control of dopamine ␤-hydroxylase gene promoter. anti-hil- r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. - - - integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy - - - molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj- is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are ( ) leukemia in patients exposed with atomic bomb during the iind world war, and ( ) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, ( ) many genetic/familial diseases show clinical symptoms at the middle or old age. ( ) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. ( ) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp , which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼ drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc /+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi k/akt signaling, which results in decreased gsk activity, has also been shown to ameliorate ami. both pka and gsk phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk at young ages, declining igf levels during aging may increase gsk activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk causes neuronal dysfunction during normal aging. research funds: kakenhi sy - - - molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, - - tsukiji, chuo-ku, tokyo - , japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy - - - identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue , department of geriatric medicine, university of tokyo hospital, tokyo, japan; research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of - - sigma, a negative cell cycle regulator that causes g arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka , hossein esteky , kiani roozbeh , tadashi sugihara , gang wang riken brain science institute, wako, saitama, japan; institute for studies in theoretical physics and mathematics, tehran, iran; kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of inferotemporal cells to object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to • rotation. research funds: kakenhi alexander thiele , gene stoner , louise s. delicato , mark roberts university of newcastle upon tyne, uk; the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy - - - context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy - - - effects of task demands on color processing in area te of the monkey hidehiko komatsu , , kowa koida national institute for physiological sciences, okazaki, japan; sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development . monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception - . here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy - - - dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui , hirotaka sakamoto , katsunori sasahara , hanako shikimi , kazuyoshi ukena , mitsuhiro kawata laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi ( and to kt) sy - - - roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er-␤ genes have revealed that activation of er-␣ and er-␤ differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er-␤ in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er-␤ knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi ( , ) sy - - - sex steroid receptor function in sexual behavior shigeaki kato , , takashi sato , takahiro matsumoto , imcb, university of tokyo, tokyo, japan; erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., ) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., ) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., ) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., . pnas usa , . sato, t., et al., . pnas, usa , . shiina, h., et al., research funds: probrain sy - - - annexin : a mediator of cell-cell communication in the neuroendocrine system julia buckingham , helen christian , john morris imperial college london, uk; department of human anatomy and genetics, university of oxford, uk annexin (anxa ) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca . the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori , yuki takayanagi , masahide yoshida , yoshiyuki kasahara , masaki kawamata graduate school of agricultural science, tohoku university, sendai, japan; department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) ( ) sy - - - cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f ); f activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m ). local field potential activity in f and m is also tuned to grasp, and there is strong beta coherence between f and m , indicating reciprocal transmission of information. this is also seen in synaptic responses of m neurones to stimulation of f (and vice-versa). single pulse stimulation in f strongly modulates corticospinal outputs from m through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy - - - where tactile signals are ordered in time shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., . s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy - - - decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo , biology/cns, california institute of technology, pasadena, ca, usa; jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. -channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha ( ) ( ) ( ) ( ) ( ) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy - - - why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust ( ) sy - - - recognizing self actions through externalized eyes atsushi iriki , symbolic cognitive development, riken brain science institute, saitama, japan; cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut , existing mostly in cortical pyramidal cells, and vglut , found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut -expressing terminals (n = ), but were found in- % of spines innervated also by vglut -expressing terminals (n = ). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy - - - gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼ -fold decrease in the potassium-chloride co-transporter (kcc ) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer / pyramidal cells in visual cortex share common excitatory inputs from layer and from within layer / , when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer / cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi ( , ) sy - - - local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer of rat visual cortex and layer / of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer , axondescending cells receive excitatory input predominantly from layer / while neurogliaform cells receive stronger input from deeper layers. layer / neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer / , while the cr− martinotti cells also receive strong excitation from layer . irregular-spiking basket cells also receive strong excitatory input from layers / and , but they often have a gap at the top of layer , with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers / and , and only weak input from deeper layers. research funds: nih sy - - - physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers ( - ) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r ey , mcknight neuroscience of brain disorders award sy - - - axon guidance mediated by localized ca + signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca + signals. its turning direction depends on the occurrence of ca + -induced ca + release (cicr) through the ryanodine receptor type (ryr ). the activity of ryr is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr , that serves as a key regulator of axon guidance. attractive ca + signals facilitate intracellular membrane transport to the leading front and subsequent vamp -mediated exocytosis on the side with elevated ca + . in contrast, repulsive ca + signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp -mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy - - - molecular mechanisms for signaling through plexin-a hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema a acts as a guidance cue for axons through a receptor complex comprising neuropilin- as the ligand-binding subunit and plexin-a as the signal-transducing subunit. the ferm domain-containing gef, farp , associates directly with plexin-a . sema a induces the dissociation of farp from plexin-a , resulting in activation of farp 's rac gef activity, rnd recruitment to plexin-a and down regulation of r-ras. simultaneously, the ferm domain of farp sequesters pipki␥ from talin, thereby inhibiting its kinase activity. these activities are necessary for sema a-mediated repulsion of outgrowing axons. plexin-a also functions as a ligand binding receptor of a transmembrane semaphorin, sema d and contributes to cardiac morphogenesis. sema d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a forms a receptor complex with off-track in the ventricle or with vegf receptor type in the conotruncal segment, which are responsible for the effects of sema d on the respective regions. research funds: crest sy - - - axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins a (sema a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema a stimulated the transport of neuropilin- ::egfp, plexin-a ::egfp, and fyn::egfp, which are the components of sema a signaling, but not neuropilin- ::egfp. interestingly, sema a accelerated the anterograde transport of trka::egfp. these facts suggest that sema a selectively facilitates the transport of its own signaling components and that sema a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein- (crmp- ) is enriched in growing axon, and play a crucial role in axon specification. crmp- interacts with tubulin dimers to promote microtubule-assembly, and binds to sra- , an effector of rac to regulate wave-dependent reorganization of actin filaments. crmp- links kinesin- to tubulin dimmers and sra- , and participates in the kinesin- -dependent transport of tubulin dimmers and the sra- /wave complex to growing axons. we also found that the par- /par- complex and the ras/pi -kinase/akt/gsk- b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk- b and inactivates its kinase activity downstream of ras/pi -kinase, thereby increasing non-phosphorylated active crmp- which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy - - - regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki , , takashi abe , , , shigeyuki kan , , satoru miyauchi , national institute of information and communications technology, kobe, japan; japan society for the promotion of science, tokyo, japan; graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; kyushu institute of technology, kitakyushu, japan; crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o -and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p x receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p x receptors produce a reduction of the neuropathic pain behaviour ( . nature , - ) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p x causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury ( ( . nature , ( - . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi ( ) sy - - - pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk , which labeled with carbon- can be used for positron emission tomography (pet). using c-(r)-pk pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy - - - application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy - - - brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality ( . neuropsychobiology , ), aging ( . neuroimage , , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, ) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia ( . biol. psychiatry , ; . neuroimage , ) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every . s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy - - - clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy - - - measurement of language related brain activities during recovery from aphasia eiju watanabe , yumiko muroi , chizuru nakajima department of neurosurgery, jichi medical university, tochigi, japan; department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy - - - neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy - - - neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy - - - neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy - - - bridging neuroscience and public: neuroethics in cultural contexts osamu sakura , interfaculty initiative in information studies, university of tokyo, tokyo, japan; research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura , , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira , argye hillis showa university school of medicine, japan; crest, jst, saitama, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi ( ), crest sy - - - impairment of surface perception in patients with ventral occipital damages shinichi koyama , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , masato taira , argye hillis showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient was a -year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory ) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient was a -year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy - - - how do pictorial cues influence d information processing in the parietal association cortex? masato taira , arish, nihon university, tokyo, japan; department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for d perception. basically, it is thought that the parietal association cortex processes d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence d information processing in the parietal association cortex. research funds: kakenhi sy - - - case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy - - - neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v + for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi ( ) sy - - - neural correlates of visual attention argye hillis , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira johns hopkins university, usa; showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp gene, encoding methyl-cpg binding protein , cause rtt. mecp acts as a transcriptional silencer. abnormal expression of some genes due to mecp dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp -downstream genes. using mecp -null mice (mecp −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp directly regulates expression of insulin-like growth factor binding protein (igfbp ) gene in human and mouse brains. pathological features of mecp −/y have the similarity of igfbp transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp due to lack of mecp may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements- or l (line- ) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute % of the mammalian genome with l elements alone representing %. the fact that l can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy - - - notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima , jun kohyma , tetsuya taga , masakazu namihira grad. sch. biol. sci., naist, ikoma, japan; inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat . a cpg dinucleotide in the stat recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat binding element in the gfap gene promoter. sy - - - neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. ( ) which type of brain infarction will be a future target of this therapeutic approach? ( ) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? ( ) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy - - - gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that ( ) motor function may recover after minor injury to the primary motor cortex, ( ) this recovery is, at least in part, associated with reorganization of cortical motor representation, ( ) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and ( ) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy - - - neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji , toshiya sato , mitsunori yamada department of neurology, the university of tokyo, tokyo, japan; center for bioresource-based researches, japan; department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with cag repeats. the q mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy - - - control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi , sy - - - mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima , dept. of anat., keio univ. sch. of med., tokyo, japan; inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day . mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca + signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca + elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca + and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp , rho gtpase, mapk and pi k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy - - - membranes, water and diffusion denis j. le bihan shfj/cea, france among einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy - - - diffusion tensor fiber tractography using a tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded -t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on -t tractography compared to . t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using -t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy - - - anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to % of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor ( - mg/kg) into male wistar rats (from days to weeks of age) significantly caused hyperactivity at - weeks old. immunohistochemical analyses of the brain tissues at weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy - - - non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about - meters from a base station. another remarkable observation in our studies is the fact that a significant (p < . ) neuronal damage is seen in rat brains days after a hour exposure to gsm at sar values , and mw/kg. we have followed up this observation in a study where animals were sacrificed and days respectively after an exposure for hours to gsm mobile phone electromagnetic fields at sar values , , , . and (controls) mw/kg. significant neuronal damage is seen after days and albumin leakage after . our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after and days. sy - - - the mitochondrial toxin -nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs , france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin -nitropropionic acid ( np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of np toxicity and show that np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by np. in summary, the np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of -ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at days of age showed behavioral hyperactivity at - weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of -ohda-induced neurotoxicity, using pc cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of -ohda. we also characterized the oxidation of cellular proteins by -ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by -ohda, ros and p-quinone. sy - - - environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥ subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥ subunit-containing receptors. disruption of the direct interaction between prip and the ␤ subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥ subunit-containing receptors in gh and hek cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥ subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi ( ) sy - - - involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca + rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k a at nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k a application was started soon after hfs or when k a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi ( ) sy - - - autocrine mglur activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur -gq/ transduction pathway as well as being initiated by direct mglur activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy - - - regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca + -activated protease calpain or by disturbance of association of gaba a receptor ␥ subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy - - - contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy - - - detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai , , susumu takahashi department of psychology, kyoto university, kyoto, japan; crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl- s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi ( ) sy - - - impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto - , japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy - - - tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad the amount of the tonic inhibition is reduced in ca hippocampal interneurons, while unchanged in pyramidal cells. sy - - - modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca region yoshihiko yamazaki , yasukazu hozumi , kenya kaneko , satoshi fujii , hiroshi kato dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca region. research funds: kakenhi ( ) sy - - - carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of m carbachol induces beta oscillations which occur intermittently with the interval of - s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is . ± . hz (mean ± s.e.m.). the oscillations are induced via muscarinic m , , receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations ( - hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that ( ) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, ( ) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca and ca neurons discharged during both up and down states, ( ) gamma and fast (ripple) oscillations were observed in hippocampal ca area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: ( ) most of the mprf neurons were active during rem sleep; ( ) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; ( ) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi ( ) sy - - - clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) : h and ld : h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per and per rhythms suggest that light-on resets per rhythms in both light conditions, while per rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld : . the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per and per showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy - - - genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified genes that fluctuated between day and night in the livers, lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose- -phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy - - - neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy - - - a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e) . to e . , the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy - - - live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi ( ) sy - - - visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol , , -trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy - - - rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦ (glur␦ ) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦ into glur␦ -null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦ lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi sy - - - gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi ( ) sy - - - physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi ( ) sy - - - deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid- th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy - - - firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no. ) sy - - - characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama , department of neurological surgery, nihon university school of medicine, tokyo, japan; division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate ( . ± . hz, n = ) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca + -atpase isoform that is located on the th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that ( ) d -type dopamine receptors are downregulated presynaptically in the striatum, and ( ) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy - - - metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr ( . nature , ) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat ( . endocrinology , ) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca + imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi sy - - - metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy - - - molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi , sy - - - thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp c is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t and reverse t , but the transport activity of t is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp a , the transport activities of t and t are similar, is also expressed. the uptake of t and t by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t and t . research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy - - - alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi , masaki takahashi , yasuhiro yoshikawa , tomoko tashiro department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to -hydroxy- , , , , pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to -hydroxy- , , , , , , -heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites ( . dev. brain res. , ) . koibuchi et al. ( ) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. ( ) detected some pcb and oh-pcb congeners in human csf, of which oh-cb is the highest concentration. to determine its effects on developing neurons, we examined oh-cb in rodent cerebellar culture cells. interestingly, oh-cb promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or -htp to examine the relevance of serotoninergic function. the treatment of -htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., ) . reference kawano et al., . j. neurosci. res. , - . research funds: kakenhi os a- - neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama , kazumi takahashi , yukihiko kayama cluster of science and technology, fukushima university, fukushima, japan; department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic ( ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣ receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os a- - correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi , , kazuyoshi hirano , , yukimasa muraishi , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan; secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l ) and grey matter volume among students aged - , who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l . os a- - grammar center activation in honorification judgment of japanese sentences kanako momo , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f op/f t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os a- - top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui , , , kimitaka kaga , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, tokyo, japan; dept. of otolaryngology, univ. of tokyo school of medicine, japan; crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m ) (neurosci. res. , s ). in a subsequent study, we confirmed that m was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m elicited by the original songs and that by the modified ones. however, while m without the deviation showed no significant effect of lateralization, m with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os a- - cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os a- - hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima , , naoki fukui , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, komaba, japan; crest, jst; dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os a- - individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext ( ), jsps research fellowship ( ) os a- - brain activity of happy facial recognition in mother-daughter relationship jun shinozaki , nobukatsu sawamoto , toshiya murai , takashi hanakawa , hidenao fukuyama hbrc, kyoto univ. grad. sch. of med. kyoto, japan; neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os a- - anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto , , atsushi maki , , kuniyoshi l. sakai , advanced research laboratory, hitachi, ltd., tokyo, japan; crest, japan science and technology agency, saitama, japan; dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f t/f o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f t and f o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (− , , ) , (− , , ) , and (− , , − ) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os a- - effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada , sunao iwaki , mitsuo tonoike aist, osaka, japan; chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = ), and with those from an inner semantic category (middle, d = ), while others were not changed (normal, d = ). peak amplitudes of waveforms of the root mean square values on the components of ms (f ( / ) = . , p = . ) and ms (f ( / ) = . , p = . ) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the and ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os p- - mechanisms of p y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono , xiaoming zhu , takashi tominaga , fumihito saitow , shiro konishi , waseda-olympus bioscience research institute, singapore, singapore; department of neurophysiology, tokushima bunri university, kagawa, japan; department of pharmacology, nippon medical school, tokyo, japan postsynaptic p y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and - min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were ± . % and ± . % of the controls, respectively. pretreatment with the protein kinase a inhibitor h- , but not the calmodulin kinase ii inhibitor kn- , completely abolished the atp-induced ipsc enhancement. os p- - activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca + permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca + influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein (arip ). activin-induced nmdar activation persists more than h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os p- - roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr phosphorylation following ltp induction in rat hippocampal ca region. like camkii activation following ltp (fukunaga et al., ) , the increased camki-thr phosphorylation remained elevated at least for min after ltp induction. the increased camki-thr phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto- significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing ( ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing ( ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi ( ) os p- - mouse brains deficient in neuronal pdgf receptor-␤ develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr-␤ subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-␤. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr-␤ expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr-␤ expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p ) rats that had received a single brdu injection at p were substituted with the corresponding region of entorhino-hippocampal slices from p rats. after five days in vitro, newborn gcs, detected by brdu and prox , migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os p- - cdk is required for neuroblast migration in the adult mouse brain yuki hirota , , , toshio ohshima , takuji iwasato , ashok b. kulkarni , hideyuki okano , , kazunobu sawamoto , , bridgestone lab. keio univ., tokyo, japan; dept. physiol., keio univ., tokyo, japan; dev. neurobiol. riken, tokyo, japan; behavioral gen. riken, tokyo, japan; nih, bethesda, usa; sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase (cdk ) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk mice to emx cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk has a cell autonomous function in neuroblast migration in the adult brain. os p- - colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi , wu sheng-xi , yuchio yanagawa , , kunihiko obata , nobuaki tamamaki kumamoto univ., kumamoto, japan; fourth military medical univ., xi'an, people's republic of china; gunma univ., maebashi, japan; sokendai, hayama, japan; riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng , plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad -gfp knock-in mouse neocortex at e -p by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s -␤ expression in the gabaergic neuron progenitors. os p- - role of ␤-catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi , , , masanori sakaguchi , toru yamashita , , , yuko fujita , yukiko gotoh , arturo alvarez-buylla , takeshi kawase , hideyuki okano , kazunobu sawamoto , neurosurgery, keio univ. sch. med., tokyo, japan; bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; physiol., keio univ. sch. med., tokyo, japan; inst. mol. cell biosciences, univ. tokyo, tokyo, japan; neurosurgery, ucsf, san francisco, usa; neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of ␤-catenin signaling in the adult mouse svz. ␤-catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated ␤-catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that ␤-catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt b. to examine the molecular interactions between c-tsk and wnt b, we co-electroporated them into the optic vesicle at stage - chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) in developing cortical neurons. an expression vector of gfp-tagged hdac was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac was primarily localized in nuclear until week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac contributes to neuronal differentiation in cortical development. research funds: kakenhi ( ) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., ) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum . ns) was measured. in high-voltage electron microscopy (hitachi m, nips, , gap junctions (average size . m long) were present in contacts. in confocal laser-scanning imaging, connexin localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be /m and a single channel conductance is ps, the conductance of each junction would be ns. the presence of seven junctions between a pair will lead to estimate a total junction of ns. the measured conductance could allow to estimate a fraction of open channels as . %. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi ( ), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os p- - multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer stimuli and propagated horizontally in the layer / . in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os p- - presence of two phases in the sensitive period of orientation plasticity shigeru tanaka , toshiki tani , kazunori o'hashi , , jerome ribot brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after weeks of goggle rearing starting around the age of weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os p- - residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida , , , tadashi isa , , dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; sch. life sci., grad. univ. adv. stud., hayama, japan; crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield ( - % correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v lesion is unlike normal, near-threshold vision. research funds: kakenhi , kakenhi and crest, jst os p- - comparison of the angle representation in macaque visual areas v and v minami ito , , hidehiko komatsu , national institute for physiological sciences, okazaki, japan; the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v neurons has angle selectivity. here, we studied the angle selectivity of area v , which is the major source of inputs to area v . we conducted single-unit recordings from the superficial layer of area v , while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v neurons responded mainly to sharp angles ( • ), straight lines ( • ) or right corners ( • ), but not to intermediate angles ( • or • angle width). this contrasted with area v , where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v . os p- - firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os p- - integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v , we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os p- - probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner , , pei sun , keiji tanaka , david j. heeger , kang cheng department of psychology and center for neural science, new york university, usa; laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri ( . mm × . mm × mm voxels) in human v . we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t *-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih ( f ey - ) os p- - relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations ( - ms; isi = s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase ( - ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase ( - ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼ % of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os p- - the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe , kazuto terai , osamu hieda , shigeru kinoshita dept. of ophthal., kyoto second red cross hospital, kyoto, japan; dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from . to . . the logmar average was . . eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was . , improved from . . the residual five eyes showed less or no improvement. the change of logmar average was from . to . . conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os p- - the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada , hideomi tanaka , , satomi nakayama , miki iwasaki , , hitoshi okamoto , laboratory for developmental gene regulation, bsi, riken, japan; crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) and migrate tangentially to r near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr and frizzled a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os p- - disrupted-in-schizophrenia (disc ) regulates the transport of the nudel/lis complex to axons via direct interaction with kinesin- shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc . disc is reported to interact with nudel, which forms a complex with lis . although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc remain largely unknown. here we report that disc interacted with kinesin- , a motor protein of anterograde axonal transport. kinesin- interacted with the nudel/lis complex through disc , and these molecules accumulated at the distal part of axons. the knockdown of disc by rnai of disc induced the delocalization of nudel and lis from the axons and reduced axonal growth. the knockdown of kinesin- induced the delocalization of disc from the axons. taken together, these results indicate that disc links kinesin- to the nudel/lis complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os p- - role of a novel collapsin response mediator protein- interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein (slp ) can mediate the vesicle transport. slp is known to associate with rab . we found that slp associates with collapsin response mediator protein- (crmp- ), which is a key regulator of axon formation. slp could form the trimeric complex with rab b and crmp- , and also associate with kinesin- through crmp- . slp is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp functions as a mediator of recruitment of certain receptor depending on crmp- and kinesin- . os p- - absolute quantification of mdr a, mrp , mrp and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie , , yuki katsukura , , sumio ohtsuki , , xiao-kun cai , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr a, mrp , mrp , and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between and fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr a, mrp , bcrp were . , . and . fmol/g, respectively, while it of mrp was under detection limit of standard curve. this quantitative profile suggests that mrp and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr a. os p- - dominant expression of claudin- in highly purified brain capillary endothelial cells sumio ohtsuki , , hirofumi yamaguchi , saori sato , tmoko asashima , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam- ) and magnetic cell sorting. mrna expression of caludin- - was measured by real-time rt-pcr. claudin- showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin- and - were . % and . % of that of claudin- . claudin- mrna was concentrated in the purified bcecs, while claudin- and - mrna in the purified bcecs were lower than that in the whole brain. rat claudin- mrna was also concentrated in rat brain capillary fraction, but claudin- mrna did not. these results suggest that claudin- is a dominant tjs protein in bcecs, and expression of claudin- and - , which was reported as tj protein in bcecs, are not restricted in bcecs. os p- - effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad , a. pauzi m. yusof , p.d. mourad , m. bainbridge , s. ab ghani universiti sains malaysia; university of washington, seattle, usa; brody school of medicine, east carolina university, nc, usa h o is the main peroxides produced in mammalian cells that consume o . the main source of h o in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h o towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h o microsensor, we investigated the level of h o in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h o level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h o was injected into the brain. fatality occurs when the injected h o exceeds m. these finding showed that the altered paracellular permeability in the presence of h o is caused by a series of events that happen one after another. research funds: short term grants pkimia and pfar-masi os p- - somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t - ) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t - spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os p- - responses of renal sympathetic nerve activity and sodium excretion to days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions ( , , , meq./l nacl) over days. the sodium loading with meq./l nacl suppressed rsna significantly and then it gradually recovered while either meq./l nacl or meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os p- - cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku , haruko masumiya , yasumasa okada dept. physiol., hyogo col. med., nishinomiya, japan; dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c ventral root (c vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: ( ) a continuous column extending from the para-facial region to the pre-bötzinger complex, and ( ) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os p- - slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca + -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi , os p- - the effect of music to sex hormones of elderly person hajime fukui , kumiko toyoshima , kiyoto kuda , katsuhiko iguchi nara univ. of edu., nara, japan; grad. school of human sciences, osaka univ. japan; nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a -kda protein band in csf of % (n = ) of confirmed and % (n = ) of suspected tbm patients out of tbm patients. the -kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv c (ag a) and rv c (ag b) and one host derived protein as the components of the tbm specific -kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q , vitamin d and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os p- - involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh , boon chock , dennis l. murphy , chuang c. chiueh dept. applied pharmacol., univ. toayama, toyama, japan; lab. bioch., nhlbi, nih, md, usa; lab. clin. sci., nimh, nih, md, usa; cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk / and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy y cells. consistently, trx increased the expression of mnsod and bcl- supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl- proteins for protecting against mpp + -induced neurotoxicity. os p- - pgd induces neuronal apoptosis via d- , -pgj tatsurou yagami , noboru okamura , toshiyuki sakaeda facul. health care sci., himeji dokkyo univ., himeji, japan; kobe univ. grad. sch. med., japan prostaglandin d (pgd ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd induced neuronal apoptosis in rat cortical cultures. however, a pgd receptor blocker did not suppress neurotoxicity of pgd . little pgd receptor was detected, suggesting an involvement of pgd metabolites in the apoptosis. among pgd metabolites, -deoxy- , -prostaglandin j ( d- , -pgj ) caused neuronal apoptosis most potently and rapidly. although d- , -pgj is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that d- , -pgj induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ h] d- , -pgj (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd induced apoptosis via d- , -pgj in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the d- , -pgj -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c to t ). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi os p- - compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi ( ) os p- - timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae , xiaofeng lu , , yusuke uchida , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of targets following a gosignal given at a random timing between and ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: ( ) those that showed buildup activity during the delay period until the go-signal, and ( ) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, - h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (> ms) than larvae (> hpf, ms). m-cell activity monitored with confocal ca + imaging during the tail flip (> hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (< %) after hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca + release through ryanodine receptors, triggered by glutamate receptor-mediated ca + influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os p- - distribution of voltage-gated calcium channel ␣ ␦- mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka - , japan the ␣ ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ ␦- to ␣ ␦- . the distributions of ␣ ␦- , - , - mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ ␦- remains to be determined. we cloned ␣ ␦- cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ ␦- mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ ␦- mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ ␦- subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os p- - hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb ) depressed the excitation, while bicuculline depressed the inhibition. h or h histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os a- - activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c bl/ mice. by recording field epsp (fepsp) along m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at - div, then the amplitudes of fepsps in the ventral side began to decrease at - div, and dominated in the dorsal area at div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at - div but the axons terminals in the ventral area were eliminated until div. this synapse elimination from the ventral side was blocked by apv application from div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until div. the number of those ventral axons is reduced before div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during - div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os a- - transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os a- - segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa in r is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa function resulted in selective loss of eph expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa dependent cues are required to determine the territory for whisker representation in r and the assembly of a somatosensory circuit. os a- - the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p and those in the venrtolateral (vl) area were eliminated from p to p and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p , reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os a- - proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi ( ), kakenhi ( ) os a- - the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m , s , sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms ( , , hz, stimulations) was compared in dp patients. the pain relief was evaluated with visual analogue scale. high frequency ( , hz) rtms of m was the only effective stimulation for treating intractable pain in of patients ( %). the pain relief continued for h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m . os a- - involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p x r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,␤-meatp (p x , , / rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,␤-meatp with ppads (p x , , , , / , / r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p x rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p x r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the ␤-endorphin (␤-ep)-ir was decreased days after snl. furthermore, the subpopulations of ␤-ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os a- - the cantor-like patterns in rat hippocampal ca pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after days of age, before which they had been reared with song tutors from birth. when the noise stopped at - days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization ( days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, ) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, ) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada ( ) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca , may be encoded in ca in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca , using -compartment model of neuron, and we found the cantor coding of information of time series in the model ca . it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in patients with chronic ischemia due to diverse vascular occlusive diseases (n = ) and moyamoya disease (n = ). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os a- - hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata , , heng zhao , gary steinberg department of neurology, brain research institute, niigata university, niigata, japan; department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia ( • c) reduced infarct size by %. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < . ). p-␦pkc (t ) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s ) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, -vessle occlusion was induced. fluo- /am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu- , yokogawa, japan) equipped with × objective lens] was inserted into the brain. -vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo- increased up to %, and persistently increased up to % during -min reperfusion, indicating the long-lasting ca + increase in the ca region. in contrast, in the frontal cortex, -min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca + in situ. research funds: kakenhi # , # os a- - reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto , h. watanabe , y. kumon , t. ohnishi , chi ii , y. imai , j. tanaka dept. neurosurgery, ehime university, japan; dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b and cd b recognized with ox antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba + mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba + amoeboid-shaped mg started to accumulate days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os a- - insulin regulates the hepatic clearance of amyloid ␤ peptide tetsuya terasaki , , chihiro tamaki , sumio ohtsuki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the liver is the major organ that eliminates amyloid ␤-peptide (a␤) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein (lrp- ) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a␤ . insulin infusion into the rat portal vein increased lrp- expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a␤( - ), which reached . -fold greater uptake than non-treated control after min treatment. increase of the hepatic uptake of a␤( - ) by insulin was concentration dependent (ec = pm), and was completely suppressed by rap ( m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp- to the plasma membrane of hepatocytes, leading to increase of a␤ hepatic clearance from the circulation. research funds: sorst, jst os a- - mr images of intra-arterially administered microglia surrounding ␤-amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a␤-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used n astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of n cells was inhibited by an inhibitor of ip receptors, -aminoethoxydiphenyl borate ( -apb) or an endoplasmic reticulum ca + -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca + was inhibited by -apb and cpa. moreover, removal of external ca + accelerated the reversal of thrombin effects. these results suggest that refilling of ca + store by ca + entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age and a -year-clinical course. the brain showed severe atrophy ( g). microscopic examination disclosed that all telencephalic neocortices had nft of more than and sp of more than . all limbic cortices and nuclei had nft of more than and sp of more than . although there was no sp, various numbers of nft were observed in the following structures: claustrum , caudate , globus pallidus , hypothalamus , meynert's nucleus , thalamus , substantia nigra , central gray , locus ceruleus , purkinje cells , posterior root ganglion , adrenal medulla . this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os a- - transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv was expressed in soma, dendrites and synapses in the neurons. since trpv was firstly cloned as an osmotically activated channel, we hypothesized trpv might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv -deficient neurons at • c, and found rmp in wild type was more depolarized by approximately mv than rmp in trpv -deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv -deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv is involved in regulation of both neural activity and spine motility in hippocampus. os p- - a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at locations per ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by -photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os p- - d -like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a receptors (adora ) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora in the ng decreased by > % of that in the non-treated ng, being not accompanied by a change in mrna level for a a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase- activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf blockers such as brefeldin a, exo , and arf n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf and subsequent pld may regulate the k + -current response induced by da. os p- - p gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa , toshihiko kuriu , ayako m. watabe , toshiya manabe , shigeo okabe , tadashi yamamoto div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; dept. of cell biol., tokyo medical and dental univ., tokyo, japan; div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p gap, which interacts with the nr b subunit of nmdar. within neurons, p gap was highly concentrated in the postsynaptic density and co-localized with nr b and an actin-binding protein, cortactin. p gap promoted gtp hydrolysis of cdc and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p gap. when over-expressed in dissociated neuron, p gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os p- - non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os p- - spatial frequency tuning of disparity-selective neurons in macaque v hironori kumano , seiji tanabe , ichiro fujita grad. sch. of engineering science, osaka univ., osaka, japan; grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional ( -d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using -d filtered noise stereograms. the disparity tuning of most v neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os p- - predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v neurons. the results suggest that v neurons are involved in mediating stereoacuity. os p- - the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field -d ternary dynamic random noise, generally refreshed every - ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v /v neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v , and especially areas beyond. research funds: mext( ), jsps( ), coe os p- - analysis of center-surround organization of v neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area (v ) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of cells analyzed, % showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ± degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os p- - spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki , izumi ohzawa , grad. school of eng. sci., osaka univ., japan; grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with × dots was presented over an area to times larger than the rf of a complex cell. for each dot location, a nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of nd-order kernels at all locations. nd-order kernels had roughly comparable extent as the rf, and contained . subregions on average (n = ). among complex cells, whose rf envelopes were elongated, cells exhibited the horizontal elongation. research funds: mext( ), jsps( ), coe os p- - orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito , osamu sadakane , masahiro okamoto , hironobu osaki , hiromichi sato grad. sch. med., osaka univ., osaka, japan; grad. sch. front. biosci., osaka univ., osaka, japan; med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about % lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that % of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os p- - temporal dynamics of suppressive receptive field surround in cat v satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v ), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes ( ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (< ms) but not in middle ( - ms) and late responses ( - ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from . deg (early response) to deg (middle response) and . deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi ( ) os p- - spatial-frequency dependent surround suppression in cat v ayako ishikawa , satoshi shimegi , hiroyuki kida , hiromichi sato grad. sch. front. biosci., osaka univ., osaka, japan; grad. sch. med., osaka univ., japan; grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v . we used a stationary flash (duration, ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus ( ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = ) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os p- - contrast-dependency of spatial summation property in cat v and lgn masahiro okamoto , tomoyuki naito , osamu sadakane , hiromichi sato grad. sch. front. biosci., osaka univ., japan; grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v ) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by . and . times in v and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v , the strength of surround suppression did not affected by stimulus contrast on average. we conclude that ) rog model describes the surround suppression better than dog model both in v and lgn, ) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida , satoshi shimegi , ayako ishikawa , hiroshi sakamoto , hiromichi sato grad. sch. eng. sci., osaka univ., japan; grad. sch. med. sci., osaka univ., japan; grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v ), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into flanks ( • step), and stationary stimulated with an annulus, oppositely-faced flanks ( -fk) or a flank ( -fk) stimulus. the durations of stimulus presentation were ms for crf and ms for srf stimulation. localized srf stimulation with either -fk or -fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for -fk stimulation throughout the crf response. however, -fk stimulation of end position to crf had strong and long-lasting suppression on responses during - ms after onset. os p- - temporal-frequency dependency of receptive field size and surround suppression in lgn and v osamu sadakane , tomoyuki naito , hironobu osaki , masahiro okamoto , hiromichi sato grad. sch. med., osaka univ., japan; med. sch., osaka univ., japan; grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v ) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v neurons, suggesting that the subcortical tf tuning property contributes to that in v . ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. ; yang and maunsell ) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using . t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around %) was smaller than that of bold (> %), the temporal profile showed a constant precedence of the diffusion signal by . s. os p- - new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os p- - spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international - system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad ( . ), approaching unity (∼ . ) for oh, and in between for control ( . ). statistical analysis was performed and significant differences were observed for these groups (p < . ). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: ( ) resting, ( ) staring at a standing human object, ( ) thinking of standing, ( ) standing with eyes open, ( ) standing with eyes closed. regional cbf data analyzed by spm were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os p- - potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono , , eiji shigetomi , kiyoharu inoue , fusao kato dept. neurol., jikei univ., sch. med., tokyo, japan; lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os p- - effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan , -methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin ( -ht) release and cause degeneration of -htergic nerve terminals via -ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the -htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on -ht release and -htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant -htergic neurons around the raphe nuclei. treatment with a -htergic neurotoxin , -dihydroxytryptamine dramatically reduced the tissue contents of -ht and its metabolite, which was blocked by a selective -ht reuptake inhibitor. mdma and meth ( . - m) increased -ht release, and reduced the tissue contents of -ht and its metabolite at higher doses. the mesencephalic slice culture including the -htergic raphe nuclei may be useful to examine the mechanisms underlying -htergic neurotoxic effect of mdma in vitro. os p- - studies on drug dependence (rept. ): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os p- - prostaglandin d is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd by hpgds and that astrocytes expressed both dp and dp receptors and were activated by pgd . we found that gliosis and demyelination were well suppressed in hpgds-or dp -null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd is a key molecule of neuroinflammation involved in the demyelination. research funds: , os p- - on a sodium channel distribution enabling high frequency signal processing go ashida , , kousuke abe , kazuo funabiki grad. sch. medicine, kyoto univ., kyoto, japan; grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below khz, while that in the node does up to khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated nl cells from owls and achieved intracellular recordings in of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = ± mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, vision impaired ( blind and low vision) and sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os p- - autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada , runa araya , naoto kitamura , yuji mishinsa yamada unit, riken bsi, saitama, japan; nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba -positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os p- - fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita , , hideomi tanaka , , sachiko tsuruoka , hironori wada , , hitoshi okamoto , riken bsi, wako, japan; jst crest, kawaguchi, japan; riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose , dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi ( ) os p- - initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd -homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin (fas ) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas genetically expressed on a muscle, we found fas accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas is crucial for the postsynaptic localization of fas . in addition, postsynaptic localization of a scaffolding protein dlg, psd -homolog, and glutamate receptors was impaired in fas mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor a (mef a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef a that is sumoylated at lys promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef a at ser and thereby promoted a switch from sumoylation to acetylation at lys , leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns , ag ps a-a characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., ) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., . mol. brain res., , - . research funds: kakenhi ( ) ps a-a the distribution of snap- protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap- derived from exon splicing are expressed in brain. we generated two specific antibodies for snap- a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap- a and b during early postnatal period. snap- b was low at the birth and increased remarkably thereafter. by the contrast, snap- a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap- b but not snap- a was recovered in raft-enriched fractions of triton x- -treated lp membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap- b was widely distributed throughout brain, whereas, snap- a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap- protein are regulated differently in isoformspecific manners, and snap- a and snap- b play different functional roles in brain. ps a-a erc(elks/rab ip /cast) regulates syaptic short-term plasticity by recruiting bmunc - to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain ( . mol. brain res. , - ) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt- , are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt- , the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt- (-/-) mice, and a novel antagonist of glial glutamate transporters, ( s, s)- -[ -( -methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately %) of functional transporters, glast and/or glt- , is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat- gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat- mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat- vglut. a mutation in avr- glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr- functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps a-a biochemical characterisation of the vesicular glutamate transporter stephan schenck, shigeo takamori department of neurology and neurological science, st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut and vglut are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut and vglut has been found. to further characterise the specific properties of the transporters we make use of the vglut -ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut by yeast two-hybrid screening and found endophilin a . as expected for the interacting molecule to vglut , endophilin a was typically localized to vglut -positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in ∼ min, followed by spontaneously recovered after min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) was demonstrated in the lactate medium. results suggested that increased mct expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu , kazuhito tomizawa , shuang liang , iori ohmori , teiichi nishiki , kohji takei , hideki matsui dept. of physiol., okayama univ., okayama, japan; dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca + -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos- cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately ( th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm - and fm - , and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca + channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca + channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca + influx and epscs, suggesting that exocytotic machinery downstream of ca + influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca + or g-proteins. ps a-b spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca + channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap- subunit can directly bind with synprint site. this interaction was ca + -dependent, being weaker at concentrations higher than nm. in contrast, the interaction of synaptotagmin with synprint was optimal at m ca + , being weaker at lower or higher concentrations. the binding domain of synprint for ap- and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca + channel synprint site makes ca + -dependent interactions with ap- and synaptotagmin thereby contributing to vesicular endocytosis. ps a-b acl- , an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl- ˜ , and generated their deletion mutants. the mutants of acl- gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat- mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl- expression, suggesting that certain phospholipid species produced by acl- are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku , hussam jourdi , akiyoshi kakita , tadasato nagano , hitoshi takahashi , nobuyuki takei , hiroyuki nawa dept. mol. neurobiol., brain res. inst., niigata univ., japan; brain resource center, brain res. inst., niigata univ., japan; dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb ) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip and sap . moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima , eriko miura , keiko matsuda , tetsuro kondo , , masahiko watanabe , michisuke yuzaki dept. physiol., sch. med., keio univ., tokyo, japan; dept. anatomy, hokkaido univ., sch. med., sapporo, japan; mol. neurophysiol., aist, tsukuba, japan cbln is a member of the c q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln - , are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln , cbln and cbln were secreted as glycoproteins, whereas cbln was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd- and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by ␤-catenin. s-scam forms a ternary complex with neuroligin and psd- . more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd- at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf /ra (ra ), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca +independent homophilic trans-cell adhesion activity. ra participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra -/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra interacted with par- being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par- was colocalized in the cell adherent region of p embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra +/+ testis, whereas it was undetectable in the spermatid of the ra −/− testis. ra and jam-c were localized around the head portion of spermatid and ra deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra and par- . research funds: izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd- clusters in cultured hippocampal interneurons. higher density of dendritic psd- clusters was observed in interneurons at div. at div, this difference was less prominent, mainly due to > -fold increase of psds in excitatory neurons. psd- -gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd- -gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps a-c two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in - div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, ; shigetomi & kato, ) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites ( s, -micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi ( ) ps a-c ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of d-and d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps a-c integrative spike dynamics of rat ca neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur connects with syntenin at the dendrites and mglur with pick at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur that was known to be ectopicly expressed in the dendrites of mglur -deficient on cone bipolar cells. we observed no pick but only syntenin at the mglur -deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur immunopositivity. these findings suggest that mglur connects with syntenin in place of mglur that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca + -permeable channels. trpv , expressed in sensory neurons, is activated by noxious heat above • c, whereas trpv , expressed in keratinocytes, is sensitive to moderate temperatures (> • c). here we examined the role of trpv and in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to • c did not induce the change in bt, desensitization of trpv with capsaicin resulted in the increase in bt. on the other hand, in trpv -knockout mouse, moderate thermal radiation (> • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv and respectively. research funds: kakenhi ( ) ps a-c generation and biochemical analysis of a glur␣ knockout mouse hirotsugu azechi , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan glur␣ (glur ) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣ , we generated a conditional glur␣ knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra f using c bl/ (b ) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra f was used as a target of glur␣ line. by crossing gra f and tlcn-cre that expressed cre in germ line cells, glur␣ null ko mice were produced, but most of them died within days after birth. to overcome the lethality, the glur␣ mutation was transferred onto b / or b /cd- genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣ in the distribution of functional ampa receptors in vivo. ps a-c gisp: a novel brain specific protein that binds to the gaba b subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b and gaba b from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b protein and also promotes gaba b surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b /gaba b complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b -binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut - ) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut and vglut seem to participate in this glutamate accumulation. in the present study, we found that vglut mrna is also expressed in the adult rat pinealocytes. vglut immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut -ir or vglut -ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito , , , kenji nakamura , toshikazu kakizaki , , satoe ebihara , masakazu uematsu , shigeo takamori , minesuke yokoyama , shiro konishi , masayoshi mishina , , jun-ichi miyazaki , kunihiko obata , yuchio yanagawa , gunma univ., maebashi, japan; sokendai, hayama, japan; sorst, kawaguchi, japan; mitsubishi kagaku inst. life sci., machida, japan; kumamoto univ., kumamoto, japan; toyohashi univ. tech., toyohashi, japan; tokyo med. den. univ., tokyo, japan; univ. tokyo, tokyo, japan; osaka univ., suita, japan; riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi ( ) ps a-c postnatal changes in the colocalization of vglut and vglut immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura , , akiya watakabe , hiroyuki hioki , fumino fujiyama , yasuyo tanaka , tetsuo yamamori , takeshi kaneko , dept. morphol brain sci., grad. sch. med., kyoto univ., japan; crest, jst, japan; div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut) and vglut accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut and vglut in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p . the cc was higher in area s than areas m and area v throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r and r . the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin ( -hydroxytryptamine, -ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several -ht receptor subtypes, -ht transporter ( -htt) and vesicular monoamine transporter (vmat ) in primary cultured mouse osteoblasts and mc t -e osteoblastic cells. sustained exposure to fluoxetine, a selective -ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc t -e . these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦ glutamate receptor (glur␦ ). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦ were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho j, was caused by a new type of intragenic deletion of the grid gene, which was indeed translated as glur␦ lacking -amino acids in the n-terminus. mutant glur␦ proteins were retained in the soma of purkinje cells and degraded. as a result, ho j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦ signaling and cerebellar functions. research funds: kakenhi ( ) ps a-d role of the dry motif in melanin-concentrating hormone receptor in signaling yumiko saito , yoshimi aizaki , mituse nakano , kei maruyama dept. pharamacol., saitama med. sch., saitama, japan; international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp , arg and tyr in the dry of rat melanin-concentrating hormone receptor (mch r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a r and p y r electrophysiologically using xenopus oocyte expression system. when a r and p y r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/ pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps a-d dual signaling via metabotropic glutamate receptor ␣ is regulated by a cytoskeletal protein . g michihiro tateyama , , yoshihiro kubo , department of biophysics and neurology, nips, aichi, japan; sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor ␣ (mglur ) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur are differentially regulated by different types of ligands, glutamate and gd + . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur . these proteins, such as homer and . g, are also known to change the membrane expression of and modulate the function of mglur . here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur . interestingly, the functional couplings of mglur to gq and gs pathways were altered by co-expression of . g, but not by homer. deletion of the c-terminal tail abolished the effect of . g, indicating that the interaction of . g with the c-terminal tail of mglur regulates the multi path signaling. ps a-d modulation of the eaac -mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto , , saori akiduki , age dimension research center, aist, ibaraki, japan; graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein - (gtrap - ), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier (eaac ). it contains two potential pkc phosphorylation motifs at positions - and - . however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac -mediated glutamate transport activity in c bu- cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine into alanine (s a) or at serine into alanine (s a). as compared with wt, s a had no inhibitory effect on glutamate transport activity under exposure to nm pma, and had increased glutamate transport activity under normal condition. by contrast, s a had the same glutamate transport activity as that of wt. thus, the eaac -mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine in addicsin. kaori akashi , manabu abe , toshikazu kakizaki , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, japan; sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur␤ - (glur - ) and glur␥ - (ka - ) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur␤ ko mice from c bl/ es cell line. the glur␤ ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥ and glur␥ proteins were significantly decreased in the crude fraction of ca region of glur␤ ko. furthermore, subcellular localizations of both subunits were also changed in glur␤ ko. these results suggested that native kainate receptors might function as heteromeric channels (glur␤/␥) and the glur␤ subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps a-d sema d/plexin-b activates gsk- ␤ via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin d (sema d) receptor plexin-b induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b -mediated r-ras gap activity, leading to growth cone collapse. sema d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk- ␤. ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk- ␤ antagonist suppressed the sema d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b -induced dephosphorylation of akt and gsk- ␤. plexin-a also induced dephosphorylation of akt and gsk- ␤ through its r-ras gap activity. thus, we conclude that plexin-b dephosphorylates akt and gsk- ␤ through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur . hops was co-immunoprecipitated with grip (glutamate receptor interacting protein ) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur via grip binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc- . a neural amino acid transporter snat /ata also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc- immunoreactivity (asc- -ir) was detected in dendrites and somata of pyramidal neurons. the snat -ir was widely detected in neurons, whose intracellular localization was similar to that of asc- -ir. deferent from asc- -ir, snat -ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc- and snat to the neuronal uptake of l-serine. snat might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps a-d neuronal glutamate transporter eaat controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake , si-young song , shiro konishi , keiji imoto natl. inst. physiol. sci. (nips), okazaki, japan; mitsubishi kagaku inst life sci, tokyo, japan; tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat /glt- blocker threo- -methylglutamate, but not the glt- blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat was abundant. the results suggest that eaat plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat expression in postsynaptic pcs. chitoshi takayama , yoshiro inoue department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k- cl co-transporter (nkcc ) and k-cl co-transporter (kcc ). in this study, we focused on kcc , which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc with special reference to the neuronal development in the cerebellum. kcc was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc . the kcc -protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc -expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima , hisashi mori molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d -like and d -like according to their pharmacological properties. the d -like receptors consist of d and d receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop ( i loop) domain of d r fused with egfp effectively reduce camp production mediated by d and d receptors. interestingly, we also identified that the i loop domain of d r interfere with gs coupled beta adrenergic receptor signaling. these results suggest that the third intracellular loop of the d receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d -like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama , yoichi ogawa , hiroki yoshino , masayuki yamashita , toshifumi kishimto dept. psychiatr.; dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca pyramidal cells. da ( m) profoundly decreased gaba a receptor-mediated postsynaptic currents to % in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to % in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d -like receptor, since skf , a selective agonist for pi-linked d -like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d -like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina , jinko konno , sachine yoshida , hideki ohmomo , hitoshi kawano , fumihiro shutoh , haruo nogami , setsuji hisano graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut or - mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day (e ), vglut immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e , vglut -ir was seen as puncta close to l -ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l or tag -ir in the imz, whereas vglut -ir was first observed in the sp and upper imz where l -ir existed. these results show that vglut ir corticofugal fibers appose to elongating vglut -ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps a-d developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca regions michie kon , yoichi avil , hiroshi tsubokawa , dept. of information engineering, tohoku univ., sendai, japan; grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine ( . - mg/kg) produced a dose-dependent cpp. using a -day schedule of conditioning, it was found that intra-ca administration of nmda receptor antagonist, mk- ( and g/rat) significantly attenuated the morphine ( . mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda ( . , . and g/rat) significantly potentiated the morphine ( . mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk- receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura , kouichi nakamura , , hiroyuki hioki , masanori uemura , takeshi kaneko , dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d receptor are expressed in striatonigral neurons and d receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d , d , d , d , d ). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p - ) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since ( ) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, ( ) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, ( ) depolarizing agent released tpa from isolated neurosecretosomes, ( ) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u st, galnac - st, d st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda , maki ishii , isao nagata , yumiko shimazaki dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo- epitopes, glca( s)␤ - galnac( s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa , mineko kengaku , nobuaki maeda dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka , tohru marunouchi division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp or sulffp appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of -o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of -hydroxytryptoamine ( -ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day or rat cerebral cortex was cultured in the presence of -ht, na or da. after days, we analyzed dendrite formation using anti-map antibody. after - days, we analyzed synaptogenesis with anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( - nm), na ( - nm) or da ( - nm) increased dendritic length of pyramidal neurons. -ht ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by -ht a receptor, ␣ a receptor and d receptor, while inhibited by -ht a and ␤receptors. in addition, synaptogenesis was promoted by -ht a and -ht c receptors, whereas inhibited by -ht a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in t cell, singar associated with p and p , the subunits of pi kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps a-e lrfn , a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd- naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd- , a postsynaptic scaffold protein. we examined the physical interaction between lrfn and psd- by immunocrecipitation and pull-down assay, since lrfn contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn associated with psd- /nmda receptor complex in the brain extracts and lrfn directly bound to psd- via its pdz domain-binding motif. in this study, we suggest that lrfn may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt - ). among the prmt proteins, the prmt has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt in the rat brain. the prmt was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt in the brain, we tried to identify novel interacting proteins with the prmt in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps a-e proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi , , michihiro igarashi , div mol cell biol, grad. sch. med dent sci; trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi ; project-promoting grant from niigata univ ps a-e netrin- is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda , keisuke watanabe , kazuhiro ikenaka , katsuhiko ono , hiroyuki yaginuma dept. anat., fukushima med univ. sch. of med., fukushima, japan; div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin- is known to be a chemorepellent for a subset of axons, the role of netrin- for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin- in this repulsion. in the mouse embryo at e , netrin- is expressed in the floor plate and the dermamyotome, and the netrin- receptor unc c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin- repelled chick e drg axons. moreover, using function-blocking antibody against netrin- , we revealed the fact that netrin- plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin- to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p neonates, but the ventral neurons became unresponsive after p . the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe , hirohide takebayashi , , kazuhiro ikenaka , katsuhiko ono div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; dev stem cell biol. program, ucsf, usa netrin- is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin- also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin- −/− background, using olig -lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig +/lacz ;netrin- −/− mice, ventral migration of olig cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig -positive cells. these results suggest that netrin- exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g and netrin-g are vertebrate-specific membrane-anchored members of the unc- /netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g and netrin-g proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g and netrin-g differentially bind to lrrcontaining proteins, ngl- and a related molecule nag , in vitro. ngl- and nag in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g and netrin-g , respectively. furthermore, in netrin-g and netrin-g deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang , ryuichi sakai , teiichi furuichi lab. molecular neurogenesis, brain science institute of riken, saitama, japan; division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p -p , stimulation of parallel fibers produces excitation of purkinje cells lasting for - ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p -p . since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p and that they then locate in purkinje cells, in cell body on p and in primary dendrites on p -p . the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya- and mmahya- ) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya- protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya- is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya- for the early cerebellum development. toshitaka morishima , erina fukushi , kazuto kobayashi , naohiro hozumi , sachiko yoshida toyohashi university of technology, toyohashi, japan; honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto , hideyuki dekimoto , tomiyoshi setsu , masahiko watanabe , mikio hoshino , yo-ichi nabeshima , toshio terashima dept. of anat., kobe univ. grad. sch. of med., kobe, japan; dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida , tomoko nishiyama , hitoshi tatsumi , masahiro sokabe , department of physiology, nagoya university graduate school of medicine, nagoya, japan; icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately h and the elongation started at approximately h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca . once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca , which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) or e rat. dendritic outgrowth of pyramidal neurons was analyzed after days using anti-map antibody. synapse formation was analyzed after - weeks, using anti-psd- and anti-synaptophysin antibodies. in the presence of cgrp ( - nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of hydroxytryptoamine ( -ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day rat hippocampus was cultured in the presence of -ht or na. after days, we analyzed formation of dendrites using anti-map antibody. after days, we analyzed formation of synapses using anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( nm) or na ( nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites -ht ( - nm) and na ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ a receptor promotes dendritic outgrowth, while ␤ receptor suppress dendritic outgrowth and branching. in addition, -ht a receptor and ␣ a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di- anepeq) and the imaging system (micam ). in dscam-ko mice, the optical signal which precedes c inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps a-f hippocampal cells cultured on d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after weeks in vitro, neurons formed a dense layer, > . mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, ( ) collagenasespecific inhibitor did not affect cell depth, ( ) limiting gliosis by antimitotics reduced the thickness of the tgl by %, ( ), neither glial nor neuronal cell body were found in the tgl by gfap/map detection, ( ) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca + by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama , kazuto kujira , tetsuya kawabe , takao ito , yoshihiro tsuruo department of cell biology and anatomy, wakayama medical university, wakayama, japan; department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) ( ) ps a-f metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama , hitoo nishino , takehiro yahata nagoya city university graduate school of medical sciences, nagoya, japan; nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit , anand goswami , nobuyuki nukina , nihar ranjan jana national brain research centre, india; laboratory for structural neuropathology, riken brain science institute, - hirosawa, wakoshi, saitama - , japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin- for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc . finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of , ests ( , non-redundant sequences). of them matched known genes of rodents in the genbank databases, but remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of , , or weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps a-f transforming growth factor-␤ in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor-␤ (tgf-␤) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-␤. these results led us to postulate that tgf-␤ in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf-␤ in the brain using intracisternal administration of anti-tgf-␤ antibody or sb- , an inhibitor of the type tgf-␤ receptor (t␤r ). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf-␤ has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the t␤r signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps ( h restraint; min forced-swimming; ether anesthesia) then they were put in untouchable situation for days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c bl/ j mice. pups were separated from mothers daily ( h) on postnatal days through . starting at weeks of age they were tested for ( ) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; ( ) responses to social stimuli in social investigation (sit) and social preference tests; and ( ) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of db (so), or were forced to swim (sw). these manipulations were performed for min once a day at weeks after birth (from postnatal days to ) or weeks after birth (from postnatal day to ). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of weeks. it was found that the spatial learning activity of both so and sw mice manipulated weeks after birth was impaired as compared to no mice. so mice manipulated weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo , yukiko yamada , mitsuo iinuma , yasuo tamura , fumihiko iwaku , kazuko watanabe , minoru onozuka dept. oral anat., asahi univ. sch. dent., japan; dept. ped. dent., asahi univ. sch. dent.; dept. physiol., gifu univ. sch. med., japan; dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara , yayoi kitamura , junichi tanaka , yukio hattori , yasushi hayashi department of human nutrition, notre dame seishin university, okayama, japan; department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c bl/ j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a -min interval, to measure the basal temperature (t ) and stress-enhanced temperature (t ), respectively. the difference ( t = t − t ) gives the sih. in control mice, t was approximately • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t . furthermore, spermidine and spermine also lowered t and t at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox- and cox- . it has been known that, under some stressful conditions, cox- is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for h and subjected them to the elevated plus maze task h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox- by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps a-g imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang , takesi sakurai , yasuitirou fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. pharmacol., univ. tsukuba, japan; dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of - weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between and weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between and weeks of age, and in the hippocampus at the age of weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi # ps a-g a systematic analysis of genetic factors associated with behavioral diversity between msm and c bl/ koide tsuyoshi , , aki takahashi , , toshihiko shiroishi , , akinori nishi mgrl, national institute of genetics, mishima, japan; sokendai, hayama, japan; mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c bl/ . in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b - cmsm, which carries chromosome of msm, showed extreme behavioral differences from c bl/ . the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga , yu kajiyama , shigenobu shibata , hiroshi kunugi department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e ) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e ) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps a-g synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi ( ) ps a-g involvement of nr b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary , takanobu nakazawa , yuji kiyama , toshiya manabe , tadashi yamamoto div. of oncology, ims, univ. of tokyo, tokyo, japan; div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr b is tyrosine-phosphorylated, with tyr- being its major phosphorylation site. to investigate the role of tyr- phosphorylation, we generated mice with a tyr phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr- and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr b is involved in the control of emotional responses by regulating crf content. ps a-g increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca + signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for weeks, locomotor activity and longinterval ( , and ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui , , yosky kataoka , , yasuhisa tamura , yasuyoshi watanabe , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at - times/min), inhibition periods (less than times/min) were often observed h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns- , a selective cox- inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps a-g impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima , naoya aoki , andras csillag biology, hokkaido univ., sapporo, japan; agriculture, nagoya univ., nagoya, japan; anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task , chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to s. in task , chicks chose between small reward located at cm and large reward at − cm, where cues signaled the distance of invisible food. task was similar to the task , except that cues signaled the food quantity. in task , total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi ( , ) ps a-g analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe , which had a missense mutation in the casy- gene. several casy- deletion mutants also showed learning defects. casy- has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x l/mint . however, after dissociation of x l, alcadein is susceptible to cleavage by protease(s). we found that casy- was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy- can also be proteolytically cleaved. ps a-g insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama , atsushi kuhara , akiko mohri , , kotaro kimura , , masatoshi okumura , masahiro tomioka , yuichi iino , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; mol. genet. res. lab., univ. of tokyo, japan; inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins- encoding insulin homologue caused defective associative learning, mutations in daf- and age- encoding the homologues of insulin receptor and pi -kinase, respectively, suppressed the defect of ins- , and the mutation in daf- encoding forkhead transcriptional factor caused the learning defect. this suggests that ins- antagonizes daf- insulin-like signaling for associative learning. interestingly, age- animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age- in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins- . we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps a-g analysis of ttx- : novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx- (nj ) and ttx- (nj ) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx- encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx- is similar to ric- , identified in c. elegans at first and conserved among several species (halevi et al., (halevi et al., , . ric- is thought to be required for the maturation of acetylcoline receptor (halevi et al., ) , so ttx- may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps a-g analysis of aho- mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio , akiko mohri , , eiji kodama , atsushi kuhara , mizuho koike , kotaro kimura , , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho- (nj ) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., ) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho- gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho- is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho- gene further. we have demonstrated aversive conditioning in lymnaea using mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b , located in the buccal ganglion. an associative memory, lasting h, was produced with pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura , dai hatakeyama , tetsuro horikoshi , etsuro ito , manabu sakakibara lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno , minoru saitoe , , tim tully tokyo metropolitan institute for neuroscience, tokyo, japan; department of biology, tokyo metropolitan university, japan; cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi ke zhang , jian z. guo , ai k. guo , institute of neuroscience, chinese academy of sciences, china; institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal /uas-shi ts system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (< min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari , , kazuo okanoya , , lab. for biolinguistics, riken-bsi, wako, japan; grad. sch. of sci. and tech., chiba university, chiba, japan; presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day , developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps a-h facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi ps a-h the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment , rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of . m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment , rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi ( ) ps a-h transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps a-h gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c bl/ mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over %, while those in the mscand ptx-injected mice decreased to %. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦ that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps a-h post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao , , takehito okamoto , fumihiro shutoh , lab for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan; dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of h daily training for week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi ( ) ps a-h modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri , , takehito okamoto , shin-ichi nishimura , fumihiro shutoh , , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; univ of the air, chiba, japan; dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., ) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps a-h influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho , , t kobayashi , , e hori , , k umeno , , t ono , h nishijo , system emotional science, univ. of toyama, toyama, japan; crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of place cells recorded, activity of was significantly modulated by the car speed and/or distance between the car and rat; , and cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a . - . mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas and , contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca + but is blocked by mg + in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg + binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l is a component of the ubiquitin system. uch-l is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl . gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca . in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps a-i learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage ). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band ( - hz) was strong at stage and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps a-i longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about . mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal ubiquitously expresses in brain. bmal functions by forming a heterodimer with either clock or npas , which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal function in a forebrain. using a dominant negative mutant of bmal (bmal r a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., ), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal r a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn ( , , or db; intermittent white noise). db bgn induced significant decreases in number of error and time to goal in maze compared with and db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn , , anucha pongpanparadorn , wasana pratchayasakul , anchalee pongchaidacha , nipon chattipakorn fac. dent. cmu, chiang mai, thailand; cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were ± , ± and ± in the groups administered , and mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was . ± . , . ± . and . ± . for , and mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a -h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a -h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses ( , and min). percentage inhibition of cache activity following a single tde injection at doses and mg/kg significantly decreased at and min after tde injection, but not at min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣ and ␣ -nachr and kinases involved in pi k-akt pathway, and increase the levels of phosphorylated akt and bcl- . these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi k-akt pathway via ␣ and ␣ -nachrs. ps a-i arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae , yoshiyuki ishikura , shigeaki fujikawa , yoshinobu kiso , manabu sakakibara lab. neurobiol. engr., tokai univ., numazu, japan; inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in -month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps a-i thimet oligopeptidase co-exists in gfap-and cd b-positive glia in rat pc/rsc treated with mk- takeshi kato , mohammad arif , michiyuki yamada , toshiyuki chikuma , md. mahiuddin ahmed lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; grad. sch. integr. sci., yokohama city univ., yokohama, japan; dept. hygien. chem., showa pharmaceut. univ., machida, japan; dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep . ) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk- activated gfapand cd b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) day after the treatment. mk- also increased ep . and prolyl oligopeptidase. immunohistochem data showed that ep . co-localized with gfap and cd b positive glial cells. since mk- causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep . in the pc/rsc. these data suggest that ep . in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps a-i effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) and in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) and , code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps and ps with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps a-i effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloid␤proteins. in vitro, we examined the effects of various form of a␤peptide on the proliferation and differentiation of nsc from svz of -week-old adult mice. in this study, a␤ peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a␤ peptide. we found that treatment of nsc with oligomeric form of a␤ peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a␤ peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a␤ clearance is noted. high mobility group box protein- (hmgb ) is a non-histone chromosomal protein. here, hmgb was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb immunoreactivity was observed around dying neurons in the kainic acid-and a␤ - (a␤ )-injected rat hippocampi. hmgb was not co-localized with a␤ in transgenic mice which show massive a␤ production without neuronal loss. furthermore, co-injection of hmgb delayed the clearance of a␤ and accelerated neurodegeneration in a␤ -injected rats. these results suggest that hmgb released from dying neurons may inhibit microglial a␤ clearance and enhance the neurotoxicity of a␤. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps a-i x -like protein regulates metabolism of app in the mouse brain yoshitake sano , , tadashi nakaya , shigeyoshi itohara , toshiharu suzuki riken bsi, saitama, japan; hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a␤) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x -like protein (x l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells ( . j. biol. chem. , . j. biol. chem. , ) . there is no evidence, however, that endogenous x l suppresses app metabolism and a␤ generation in vivo. to examine the physiologic role of x l in app metabolism in the brain, we generated x l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the ␤-site and a␤, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x l suppresses the production of a␤ by inhibiting ␤secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin- protein by d-electrophoresis and tof-mass delineate that mutant at and htt proteins similarly reduce transcriptional factor x and x . immunoprecipitation and pull-down assays support interaction between polyq and factor x and x . immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x and x into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x and x ameliorates poly-q pathology in htt-/at -expressing neurons and transgenic drosophila. these results suggest that factor x and x are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps a-j ba - was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, rats/group (g). group received e at . g/kg per day; groups and received ycj at ml, and ml/kg day, respectively, once everyday. group received ycj ml/kg plus e at . g/kg day twice a week, all for weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e in group was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti-␤-amyloid - antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer and layer of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group was lesser than ovx and control groups. amyloid deposition was correlated with e serum at r = − . . ps a-j correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki , shigeo kinomura , kazunori sato , shinya uchida , ryoi goto , kentaro inoue , ichiro tsuji , hiroyuki arai , ryuta kawashima , hiroshi fukuda department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; tohoku univ. grad. school of med., sendai, japan; niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h -kenko- ), (h -choju- , h -choju- ) ps a-j effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook , d.g. goodenowe , y. yamazaki , j. flax phenomenome discoveries inc., saskatoon, canada; precisionmed inc., san diego, ca, usa dementia affects about % of the population over the age of and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an -metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the -metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin- (chr ). in this study, we quantitatively investigated the rapidness of the light-gated current of chr expressed in pc cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about -fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori , shigeki ohta , kenji hamada , naofumi yamada-okabe , yonehiro kanemura , hideyuki okano , yutaka kawakami , masahiro toda , neuroimmnology research group, keio univ., tokyo, japan; chugai co. ltd., kanagawa, japan; inst. cli. res., onh, japan; physiology, keio univ., tokyo, japan; cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day were dissociated. after the cells were treated with nm estradiol- beta (e ) or ethanol for days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis ( d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than spots were detected from d dige. compared with ethanol treatment, e increased the expression of spots in the hypothalamic cells and spots in the cortical cells (p < . , difference > . ). proteomics analysis showed different effects of e for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp client protein family. -allylamino- -demethoxygeldanamycin ( -aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. -aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of -aag in a mouse model of sbma. administration of -aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of -aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp function by -aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately - % of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps a-k selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura , yoshiyuki ishii , kazuto kobayashi , yasufumi sato , keiichi itoi lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; dept. mol. genet., fukushima med. univ., japan; inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a , a and a ) and dopaminergic (a , a and a ) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg b has been shown to play an important role in the processing of lc , a mammalian homologue of yeast atg , but the tissue distribution of atg b remains unknown. to understand the role of atg b in rat tissue cells, we prepared an antibody to atg b and pc cells in which atg b expression was knocked down by rnai. in rnaitreated pc cells where atg b expression was % of that in the wild-type pc cells, the expression of cytosolic lc -i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc to lc -i. moreover, the expression of atg b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg b was intense in neurons, especially in the cerebellum. these results suggest that atg b plays a major role in the processing of lc , while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps a-k spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev n strain into the central nervous system was examined after its subcutaneous inoculation ( pfu) in the rat hind footpad. on day post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki , hiroshi kameda , hisashi nakamura , taro okunomiya , koji ohira , kouichi nakamura , , takahiro furuta , takeshi kaneko , dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn , rta , mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii ␤ receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣ and gly␤ immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr increased the surface expression of glur in cos cells compared with grip/glur or ␦-catenin/glur transfection. this study points to ␦-catenin as a regulator of glur receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca area in wild type (wt) and gad knockout (ko) mice. this parameter was twice lower in gad ko mice (wt: . ± . pa/pf, n = ; gad ko: . ± . pa/pf, n = ; p = . ). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba ( m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad ko animals (wt: . ± . ms, n = ; gad ko: . ± . ms, n = ; p = . ). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p to p ) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur but not by mglur because it was antagonized by the mglur antagonist ly but not by the mglur antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps p-a involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita , michiko ohtomi cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d antagonist sch into pfc by using a microdialysis method impaired md-pfc ltp but the d antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by - %). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer / pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya - , japan we showed in previous study that somatic inhibitory synapses of neocortical layer pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate ( nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr in the regulatory mechanisms for ltp in the amygdala, we utilized ntr -knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr -ko mice than in wild-type mice. the bla-ltp in ntr -ko mice was attenuated by sulpiride, a d receptor antagonist. these results suggest that d -ntr interaction regulates the extent of ltp in the mouse la-bla synapses. ps p-a facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over -day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca + sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca + at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin- , and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps p-a inhibitory modulation of the hippocampal ca transmission and plasticity by glucagon-like peptide- jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode- (glp- ) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp- (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp- effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp- on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices ( m) were prepared from to days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca in the whole-cell mode using glass microelectrodes (resistance: - m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp- ( nm- m) inhibited spontaneous excitatory postsynaptic current. glp- ( nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp- may play an inhibitory role in the dg-ca transmission. ps p-b quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp- and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a , a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, hz for s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, hz for s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of m apv, m bicuculline, but not m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at − mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine ( - m) or capsaicin ( . - m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin ( m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv ), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji , akira komatu , akinao nose dept. phys., univ. tokyo, tokyo, japan; dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca + -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr * flies did not show mg + blockade, and dnr * flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf- , and notch. we examined how mg + blockade of dnr links to these transcription factors. research funds: kakenhi ps p-b response properties of wind-sensitive giant interneurons in the th-instar nymphs of the cricket tetsuya matsuura , masamichi kanou dept. of welfare eng., iwate univ., morioka, japan; dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) - , - , - and - in the th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the response magnitudes of gi - reached a plateau at a stimulus velocity of mm/s in most stimulus directions. the response magnitudes of gis - and - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi - , the ipsilateralfront and contralateral-rear in gi - , the ipsilateral-rear in gi - and the ipsilateral-front in gi - , designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca . serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca + , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca region. we found that ptp induced by the -hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca + channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca + channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma , kunihiko kaneko , , makoto asashima , grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca + concentration ([ca + ] i ) may change because of nonsynaptic communication. we loaded pc cells with a ca + indicator, indo- am, and the [ca + ] i of more than , cells was measured using flowcytometry. the [ca + ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca + ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca + -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss from human and mouse cns. in mouse cns, four isoforms (types - ) were expressed. subcellular localization analysis revealed that type (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc and nb a derived from neuronal cell express only type , whereas os and kt- derived from astrocyte express both type and type . interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os and kt- . we analyzed promoter region of m-spinesin gene, and identified that -flanking region from − to − bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme -phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca + signals, such as intra-and intercellular ca + waves; however, their physiological roles remain elusive. the overexpression of an ip -hydrolyzing enzyme, ip -phosphatase, suppressed the spontaneous ca + signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca + -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca + -signal-deficient astrocytes. these results indicate that spontaneous ca + signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai , noriko osumi , tohoku univ. sch. med., japan; crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax in postnatal mouse brain and found that pax was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax heterozygote mutant (sey/+) mice at weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, and h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., ) . we previously reported a unique expression of dim ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., ) . we show here that mab dim also recognizes adult neural stem cells and glial cells at postnatal period. dim , brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad / / (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk . lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in s and s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in -ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after h ( and %) and h ( and %) exposure to − and − m -ohda respectively. it was accompanied by morphological changes and induction of gfap, s- and ox . -ohda ( − m, h) was found to cause a significant impairment in h glutamic acid uptake ( %) and gsh levels ( %). further neurons (in coculture with -ohda pre exposed glial cells) on exposure to -ohda ( − m), showed loss of th expression and significant neuronal cell death ( %). the results of the present study suggest that -ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at day after injection, then started to repair at days and returned to the normal levels at weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at ∼ days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng ) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba + cells accumulated in stab wounds and ischemic lesions were found to express ng , of which molecular weight of its core protein was higher by kda than that of ng expressed in contralateral brain region. this was due to the lack of shedding of ng in the brain lesions. we found that iba + cells accumulated in stab wounds and ischemic core lesion, most of which were ng +/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd and von willebrand factor. ng + mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng + mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from < to m . most of the fibers were myelinated and counts produced a mean of , ( , - , ). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from . to . m (from fetal age to years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p - . costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca , ca pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps p-d impact of s b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani , akiko seto-ohshima , shigeyoshi itohara , hajime hirase neuronal circuit mechanisms research group, japan; lab. for behavioral genetics, bsi, riken, wako, japan s b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s b, we have recorded hippocampal spontaneous activities from urethane anesthetized s b ko and wt mice. typical eeg patterns including theta ( - hz) and sharp wave associated fast ripple ( - hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca radiatum, where ictal event was characterized by hyper-synchronous gamma band ( - hz) activity. while both populations developed ictal event within min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s b related pathways could potentially be activated. yoshiko takagishi , erina okabe , xiaoyang sun , sen-ichi oda , yoshiharu murata riem, nagoya univ, nagoya, japan; grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps p-d regulation of hippocampal neurocircuit activity by glutamate transporter glt- noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt- , the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap ) and the glt- blocker (dhk) with the signal subtraction, we could visualize the activity of glt- as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt- by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt- function in the ca region was elicited by much lower concentration of dhk than that in the ca region. ps p-d monoclonal antibody rip specifically recognizes , -cyclic nucleotide -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg- cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was , -cyclic nucleotide phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg- cells. moreover, the same localization was also observed in rat cnp transfected hek t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca + signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca + signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca + signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki , , hisatsugu koshimizu , megumi kashihara , tomoko hara , masami kojima , research institute for cell engineering; sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that -day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p ) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p expressed in the neural progenitor cells at embryonic days (e ) mice brain. to ascertain the function of p on these cells, we treated the cultured e cells with the selective chemical inhibitor for p (sb ) for days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit , takashi yamazaki , , takeshi oya , yoko ishii , masakiyo sasahara pathology ii, university of toyama, toyama, japan; oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on to days after injury, and activation of pdgfrs, mapks, akt and p were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from to days after injury. p-tyr was highly detected from to days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to days after injury and then gradually decreased. activation of akt and p continuously increased from to day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln is a cerebellum-specific protein structurally related to the c q and tumor necrosis factor families. recently, we have shown that cbln is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to -untranslated region (utr) of bdnf mrna to investigate the effect of the utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps p-e bmp expression in the adult rat brain bone morphogenetic protein- (bmp ) is a member of the transforming growth factor ␤ (tgf-␤) superfamily and plays important roles in multiple biological event. although bmp expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp expression in the adult rat cns using immunohistochemistry. bmp is intensely expressed in most neurons and their dendrites. in addition, intense bmp expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp protein. these data indicate that bmp is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp plays pivotal roles also in the adult brain. ps p-e hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl- protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl- expression, decreasing the level of bax expression in c bl/ mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase . here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of -methyl- -phenyl- , , , tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda , yoko tohyama , shinichi kohsaka , tadashi kurihara , kazuyuki nakajima , soka university, hachioji, tokyo, japan; dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt- , and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf␤ among tgf␤ family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba , chiaki nakagawa , masako tsuzuki , motoko matsuda , satoshi ichisaka , yoshio hata inst. dev. res., aichi human ser. ctr., kasugai, japan; div. neurobiol., tottori univ., sch. med., yonago, japan; div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats h after beginning an -h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin- (nt- ) in selected brain regions from -day-old male rats and compared to those from adults. the changes in levels of bdnf and nt- were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci ( kdyne, ih injury device) at t / and were assigned to three groups; a (placebo), b (mp treated; mg/kg i.v. immediately after injury), c (g-csf treated; g/kg i.v. for days after injury). animals were assessed with the bbb locomotor rating scale for w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi ( ) ps p-e glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons ( div) exposed to glutamate for min at m. glutamate exposure led to a decrease in mrna of nd and nd , which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least - h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk- . these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj- is enhanced by oxidative stresses. although exact functional significance of dj- has still unknown, it is thus proposed that dj- is protective against neural damage under oxidative stresses. in this study, we tested expression of dj- in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj- was markedly increased in the molecular layer of the dentate gyrus on days and post tmt injection. on day post tmt injection, enhanced expression of dj- was observed in the stratum lucidum of the ca . in glutathione-depleted mice, tmt was more effective in enhancing expression of dj- , compared with that in untreated mice. furthermore, double staining of dj- and gfap demonstrated that most of cells highly immunoreactive to dj- were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj- is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt ( . mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus days later, with visually apparent recovery of the granule cell layer days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone ( . - mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of . mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb ( g/ml) on the pc cells was elicited through apoptotic cell death. activation of caspase- / was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin ( nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase- / activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc cells. ps p-e kinesin superfamily protein (kif ) regulates activity-dependent neuronal survival by suppressing parp- enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein (kif ), a microtubule-based molecular motor. the c-terminal domain of kif is a module that suppresses the activity of poly (adp-ribose) polymerase- (parp- ), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif from parp- , resulting in upregulation of parp- activity, which supports neuron survival. after dissociation from parp- , kif enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. research funds: ps p-e expression of hsp , apg- , and apg- in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt ( . mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp , apg- , and apg- in the granule cell layer of the dentate gyrus, but not in ca -ca pyramidal cell layer, h to days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp highly expressed in survival neurons in the culture, apg- and apg- highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day . thirty gray of x-irradiations were performed on the cultured cells at or days in vitro (div). then the cells were fixed at h after the irradiation with dapi. at -div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at -div. this indicates that the radiosensitivity of -div immature neurons is higher than that of -div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦ glutamate receptor (glur␦ ), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦ is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca + , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin- and found a novel gene down-regulated in response to mutant ataxin- in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca molecular pathology. ps p-f permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca + -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca + -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways. interestingly the up-regulation of bcl- , a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat probe. scat is an indicator protein for caspase- activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase- cleavage sequence. using this probe, we could monitor the activation of caspase- at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt ( . mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex days later. in cortical neurons, tmt exposure for h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases and , but not caspase , were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride ( . mg/kg, i.p.) in -( w) and -week-old ( w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in w mice. by contrast, slight, diffuse neuronal damage was found in the ca and ca subfields and dentate gyrus of w mice. the hippocampal putrescine level was elevated markedly in w mice on tmt administration, whereas a minor putrescine increase was detected in w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps p-f establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai , toshinori hongo , kimisato naito , shigeto sasaki department of physiology, kyorin university, school of medicine, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for . - . s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of . s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in volunteers. experiment was performed by three tasks, control task is a forearm rotation, task is simulation of tools-manipulation, task is execution of tools-manipulation. we found different brain regions by this experiment. task to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at khz, to record simultaneously and independently the • horizontal saccades of each eye for subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr␤ is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi ( ) ps p-f dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼ hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from healthy subjects, high and low scorers (n = each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band ( - hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a -t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = . s) with online monitoring of meps. the intensity of tms pulses was varied from % to % at a % step (frequency at ∼ . hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above % stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = . , p = . ). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps p-f shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda , , yukihiro gomi , itsuki imai , nobuaki shimoda , , hiroyuki kato , international university of health and welfare, ohtawara, japan; crest, jst, kawaguchi, japan; nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels ( channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of s task and s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky , , rieko osu , hiroshi imamizu , mitsuo kawato , computational brain project, icorp, jst, japan; atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed hz isometric force pulses to seven targets separated by • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a d strain gauge. twenty repetitions of each condition were performed in s blocks (tr = s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only . % (chance: . %), errors were distributed normally about the target such that . % (chance: . %) of the data was classified correctly to within • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from • c, fish showed repetition of short time swimming in the range . - • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in - days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps p-f to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu , ken-ich morishige , jun nakanishi , , hiroyuki miyamoto , mitsuo kawato atr computational neuroscience labs, kyoto, japan; kyushu institute of technology, japan; jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p pups of the surviving cases increased greatly between p ( % of the control value) and p ( %), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p , dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p and p pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p to p . we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully ( • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative , , and days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps p-g role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa , tatsumi nagahama dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps p-g neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto , sayaka hihara , kazuo okanoya , atsushi iriki lab for symbolic cognitive development, bsi, riken, japan; lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about . • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps p-g prostaglandin e -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, - - toyama, shinjuku, - , japan unilateral microinjection of pge into the region around the rostroventral wall of the third ventricle (av v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of - pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide ( . mm, nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge injection site was examined. this treatment alone had no effect on the o consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge -induced responses were blocked min after the pretreatment with bicuculline, and recovered at ∼ min. pretreatment with vehicle saline had no effect on the pge -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge -induced thermogenesis. tetsufumi ito , hiroyuki hioki , kouichi nakamura , takeshi kaneko , yoshiaki nojyo dept. anat., univ. of fukui, fukui, japan; dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad -ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad -positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad . we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad -ir. after cutting t -t vrs and drs, almost all gad -ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama , ning ma , reiji semba , satoru naruse dept. of anat. ii, mie univ. school of med., tsu, japan; inst. for developmental research, kasugai, japan; dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin (aqp ) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp -immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp -ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp -ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp -ir fibers were found both in the myenteric and submucosal plexi. many aqp -ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp -ir fibers penetrated into the muscle layers. these results suggest that aqp -ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi ( ) ps p-g abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days - . the increase in the volume and frequency setting of the artificial ventilator (fio = %, fico = %) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad -gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = ) in medullary transverse slices. about % of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about % of gabaergic neurons recorded in the superficial rvm were co inhibitory, and all of the remaining neurons were co insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with and mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with mv forehead spla, in comparison to subgroups mv forehead spla. such law was common for both for sg and for cg. in two subgroups of and mv nasal zone spla in sg the greater rt gain after il was in mv nasal spla subgroup, and for cg in mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin ( ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via ht receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c bl/ n) were pretreated with pcpa ( . g/ ml/kg body weight) or saline intraperitoneally for consecutive days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o inhalation was changed to stepwise , and % co in o inhalation every min. hypercapniainduced increases in tidal volume and minute ventilation during % co inhalation were reduced by pcpa pretreatment; these results suggest that ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a . % gosha-jinki-gan diet group. after weeks, continuous cystometry with physiological saline or . % acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with . % acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps p-g differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to db white-noise for min. db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase (gad ), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were . on the small neurons, . on the medium-sized neurons, . on the large neurons, and . on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about % of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large ( . m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps p-h neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c nerve and facial nerve activities. addition of mm k + to the standard superfusate decreased burst rate of c activity. addition of or mm k + caused initial inhibition of c burst and subsequent high frequency c burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi ( ) ps p-h regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp ( m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at − mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin receptor (nk r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps p-h effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min- cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm- , shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda , usa) at the right middle finger from volunteers ( . ± . years). mild thermal stimuli ( ± • or ± • ) were administered every min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm- . we examined the effects of color environments on cognitive function in healthy subjects and patients after traumatic head injury using p components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p latency in the red environment was significantly shorter in controls than in patients. the p amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps p-h neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi , , mikinobu takeuchi , , takahiro omote , naoyuki yosimura , etsuro hori , , kazuo sasaki , taketoshi ono , , hisao nishijo , system emotional science, univ. toyama, toyama, japan; crest, japan science and technology agency, japan; bio-information engineering, univ. toyama, toyama, japan; molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems ( channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate ( ) human facial stimuli with different gaze directions and ( ) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi , sachiko koyama , ayumu matani , andrzej cichocki riken, wako, japan; hokkaido university, sapporo, japan; university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: ( ) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. ( ) the functions were averaged across subjects. ( ) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. ( ) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi ( ) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps p-h the reflection of category perception of sound in the auditory evoked n m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for different fundamental frequencies (f ) and different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n m source strength for nonlinear sounds was significantly larger than that for linear sounds. n m peak latency only for vocal sounds was not affected by f . these results suggest that perceptual categorization was reflected in n m source strength and location (linear or nonlinear), and in n m latency (vocal or nonvocal). sunao iwaki , hiroko kou , kouichi sutani , mitsuo tonoike national institute of advanced industrial science and technology, osaka, japan; chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p , related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p components, suggested the involvement of frontal and anterior cingulate cortex in the early p component (p a) and the contribution of the parietal and temporal regions to the later component (p b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l and l , respectively). we compared the brain activity of chinese learners of japanese as l with that of japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm . while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l and l . neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a . t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya , mie matsui , , tsuneyuki kobayashi , , hisao nishijo , , michio suzuki , , yasuhiro kawasaki , , masayoshi kurachi , , jun nakazawa , kyo noguchi , hikaru seto neuropsychiatry, univ. toyama, toyama, japan; crest, japan science and technology corporation, japan; psychology, univ. toyama, toyama, japan; system emotional science, univ. toyama, toyama, japan; neuropsychiatry, univ. toyama, toyama, japan; developmental psychology, univ. chiba, chiba, japan; radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura , atsushi sekiguchi , keisuke wakusawa , , yuko sassa , , hyeonjeong jeong , , kaoru horie , , shigeru sato , , ryuta kawashima , miyagi university of education, sendai, japan; niche, tohoku univ., japan; dep. pediatrics, tohoku univ. school of medicine, japan; ristex, jst, japan; gsics, tohoku univ., japan; lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain , , points or lose , , points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps p-h brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara , masato taira , , toshio iijima , ken-ichiro tsutsui div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; arish, nihon university, tokyo, japan; appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either yen or a gain or loss of , , or yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps p-i neural activation during experience-based reasoning chisato suzuki , , takashi tsukiura , hiroko mochizuki-kawai , yayoi shigemune , , toshio iijima neurosci. res. inst., aist, japan; div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps p-i the difference of brain activity elicited by different styles of art hiromi yamamura , yasuyuki kowatari , , shigeru yamane , miyuki yamamoto , comprehensive human sciences, university of tsukuba, tsukuba, japan; system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm . while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba , ba ). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j- ) ps p-i effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were - aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi , ps p-i the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo , michio nomura , jun kawaguchi ntt commun. sci. labs., ntt corp., atsugi, japan; dept. psychol., tokai women's univ., kakamigahara, japan; dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo , , , yulri nonaka , katsumi mizuno , kazuo okanoya , riken, bsi, biolinguistics, saitama, japan; chiba university, chiba, japan; jsps, japan; department of pediatrics, showa university, tokyo, japan; presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for neonates within days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three ( ) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around - ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism ( : - : ) and agematched typically developing children ( : - : ) were instructed to name pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = . ± . , loudness = . ± . ) than in the children with autism (duration = . ± . , loudness = . ± . ) (p < . ). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at . /s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser ( nm, mw) to the muscle surface ( . mm ) significantly delayed its attenuation (p < . ). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < . ). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps p-i nedl , a novel e ubiquitin ligase for dishevelled- , targets mutant superoxide dismutase- and interacts with p yuanyuan li , , , kou miyazaki , toshinori ozaki , akira nakagawara division of biochemistry, chiba cancer center research institute, chiba, japan; production technology development center, the furukawa electric co., ltd., ichihara, japan; hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e ubiquitin ligase gene termed nedl . previous study has shown that nedl is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled- , a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl for uniquitination and proteasome-mediated degradation. on the other hand, nedl bound and ubiquitinated mutant (but not wild-type) sod in a mutant sod type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl physically bound p , and induced apoptosis in a p -dpendent manner. taken together, our results suggest that nedl may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod and p . spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc interacting protein), u-box type e ubiquitin ligase, has been shown to interact with hsp or hsp and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca + current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox- inhibitor, rofecoxib in pentylenetetrazol (ptz, mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the th day of ptz treatment. chronic treatment with rofecoxib ( . and . mg/kg, p.o.) for days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced ( mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin ( and mg/kg, p.o.), naproxen ( and mg/kg, p.o.), nimesulide ( - mg/kg, p.o.) or rofecoxib ( - mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib ( mg/kg) or nimesulide ( mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox- inhibitors. cox- inhibitors also antagonized the effect of flumazenil ( mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps p-j cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps p-j a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus ( mg/ min). after min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that -oxoguanine ( -oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that -oxog accumulated greatly in mtdna of the ca microglia. wild-type and mth -null mice, the latter lacking an ability to hydrolyze -oxo-dgtp and -oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca neuron loss after kainate administration, however, levels of -oxog accumulated in mtdna and cellular rna in the ca microglia were significantly increased in mth null mice in comparison to wild-type mice. we thus demonstrated that mth efficiently suppresses the accumulation of -oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps p-j transcription factor nrf regulates brain response to kainate-induced excitotoxicity yukihiko dan , kosuke kajitani , noriko yutsudo , ken itoh , masayuki yamamoto , yusaku nakabeppu kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e related factor (nrf ) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf has been reported to increase neuronal death under increased oxidative stress, nrf seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca subregion of hippocampus. to unveil the functional significance of nrf in the brain, we compared seizure responses between wild-type and nrf -null mice after systemic kainate administration. we found that nrf -null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps p-j synaptic plasticity and -aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine ( -ap) at the concentration below . mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the -ap induced discharges in ca region was studied. ten m -ap induced the epileptic discharges with the frequency of . ± . hz (mean ± s.e.m.; n = ) and the amplitude of . ± . mv. when ap- , an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap- was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of -ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps a-a influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno , hugh p.c. robinson engineering science, osaka university, osaka, japan; university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣ a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the -day-old rat pup spinal subarachnoid space at the rate of l/ . min. the rats were sacrificed h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣ a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps a-a on the membrane potential profile of ca pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga , , yoko tominaga dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; lab. for dynamics of emergent intelligence, riken bsi, hirosawa - , wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps a-a overexpression of inwardly rectifying k + channel . in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) . have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca pyramidal neurons. then, egfp and kir . expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer / neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types and of ryanodine receptors (ryr and ryr ) in the cerebellum of a ca + channel ␣ a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr but not ryr in the rmn cerebellum: a less ryr mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr -mediated ca + release, contributing altered ca + homeostasis in the rmn purkinje cells. ps a-a dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than s. d receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi , , hiroyoshi miyakawa , masashi inoue , masato okada , tokyo institute of technology, japan; brain science institute, riken, japan; tokyo university of pharmacy and life science, japan; the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai , maya yamazaki , , mika tsujita , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan we previously reported that stargazin family (␥ , ␥ , ␥ , and ␥ ) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., ) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥ subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥ subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day , while the reduction was not so significant in adult brain. these results suggested that ␥ might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph . ) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop ). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop -transfected cells. mvsop induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn + sensitivity. neutralization of a positive charge in the s -like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh ). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca region. in the pyramidal layer, the amplitudes of the responses to the -hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the - -hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels (asic ) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic -expressing cells in rat central nervous system (e -p ) using immunohistochemical techniques. asic positive fiber first appeared in brain stem and spinal cord at e - stage. asic -expresseing cells appeared in white matter of brain stem and spinal cord at e stage. in early postnatal stages asic expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval ( st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long st isi by whole-cell recordings in rat slices. application of m apamin, an sk-type ca + -activated k + (kca) channel blocker, shortened the st isi and decreased the amplitude of the slow ahp. the shortening of the st isi was observed when membrane potentials were depolarized. moreover, application of m mibefradil, a t-type ca + channel blocker, shortened the st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca + influx through t-type ca + channels. research funds: kakenhi (c) ( ) jafar vatanparast , , mahyar janahmadi , houri sepehri , ali haeri-rohani , ali reza asgari neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px ( . m, within min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca + spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca + dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm ). repeated menthol application induced trpm desensitization. this desensitization was depended on extracellular ca + , indicating that involvement with ca + -dependent kinase. pkc activator (pma) desensitized trpm and go (pkc inhibitor) abolished pma-induced trpm desensitization. pma similarly desensitized wild type trpm and mutant trpm , in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm . as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm causes pkc to desensitize trpm itself. yosuke sawada , hiroshi hosokawa , kiyoshi matsumura , shigeo kobayashi dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa is activated by pungent compounds stimuli. if trpa responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa is activated by cooling or not is still controversial. here, we investigated that trpa -expressing hek cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was . ± . • c. in inside-out singlechannel recording, cooling activated trpa directly. single channel conductance was . ± . ps. single channel currents showed inword rectification. in conclusion, trpa is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn - ) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn - demonstrated that samples from the nerve and the heart showed an hcn band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn , hcn and hcn could not be found in those samples. when an hcn channel blocker, zd was applied on the stimulus portion of sciatic nerve and the nerve was elicited at . /s by a duration of ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < . ). it is suggested that hcn channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir ) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir . and kir . channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps a-b proliferation of rat c glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c ), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control ( mm) to or mm. an increase in [na + ] i was revealed by an image processor in c cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr- , which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr- level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma ( - m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma ( m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi ( ) il-sung jang , in-sun choi , eun-ju park , jin-wha cho , man-gee lee , byung-ju choi kyungpook national university, school of dentistry, south korea; kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (< m), but decreased those induced by gaba at higher concentrations (> m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav . -deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav . -deficient mice as negative controls to examine nav . distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav . expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav . distribution, in the present study, higher magnification analysis revealed that nav . is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav . antibodies used in these previous studies, none of which utilized nav . -deficient mice. based on our findings, we propose that nav . might be involved in propagating action potential to presynapses. keiji miura , , masato okada , , , shun-ichi amari department of physics, kyoto university, kyoto, japan; "intelligent cooperation and control", presto, jst, japan; department of complexity science and engineering, university of tokyo, chiba, japan; brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. and ) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [ - c] glucose intravenously ( g/kg) or h after the intraventricular injection of colchicine ( g/ l) and the amino acid fractions were extracted from the brains at , or min after the glucose injection. the amount of [ - c] glucose in the cerebra was increased, however, the c incorporation into glutamine, glutamate, gaba and aspartate from [ - c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a ␤ alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes or hes gene, phes -and phes -egfp transgenic (tg) mice were generated (ohtsuka et al., ) . in each transgenic mouse, a promoter of hes or hes gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes or hes regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day (e ) to postnatal day (p ). in the phes -egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e to p . in the phes -egfp tg mouse inner ear, gfp-ir cells were observed from e . to p . gfp-ir cells in phes -gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., . mol. cell neurosci. ps a-c expression of zfh- in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine , kenji nakamura , motoya katsuki , tetsuo yamamori national institute for basic biology, okazaki, japan; mitsubishi kagaku institute of life science, machida, japan zfh- is a transcription factor containing three homeodomains and zn fingers and expressed in differentiating neurons. we have reported that the level of zfh- mrna is negatively regulated by antisense transcripts of the zfh- gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh- antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh- mrna starts to be expressed. recently, we have raised an antibody against mouse zfh- and examined the expression profile of the zfh- protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh- protein was not detected even when the zfh- mrna was already expressed. this observation together with other data suggested that the zfh- protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue , maya ota , katsuhiko mikoshiba , jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan; laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic - ) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic /zic compound mutant mice. mice carrying homozygous zic mutant allele together with zic null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic -or zic -single mutant. these observations indicate that zic , in cooperation with zic , have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp , a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp transcripts are first detected at embryonic day in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day - embryos, rp transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e , rp transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c hc-type zinc finger motifs. using p cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf- and nzf- respectively. nzf- null mice are born alive, but die within min after birth with cyanosis. on the other hand, nzf- null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf- and nzf- by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , ) . in the present study, to examine the role of runx in the development of drg in more detail, we examined the development of drg neurons in runx -deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx −/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days . and . . shin hisahara , , susumu chiba , hiroyuki matsumoto , yoshiyuki horio department of pharmacology, sapporo medical university, sapporo, japan; department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt is still unclear. recent studies indicated that sirt interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes promoter. we performed overexpression of sirt and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt and n-cor in the sirt with n-cor. sirt and n-cor suppressed hes transcription by notch-icd in the luciferase assay. hes transcription was suppressed in overexpression of sirt and n-cor, suggesting that interaction between sirt and n-cor represses hes transcription. consistent with this, chip assays revealed that not only n-cor but also sirt bind to the promoter of hes gene. taken together, these results indicate that sirt and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes promoter site and repressing hes transcription. yasushi maruyama , mitsuhiko kurusu , masataka okabe , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, japan; natl. inst. genetics, mishima, japan; inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf-␤ and bmp signaling. we reported previously that runx mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., ) . furthermore, we announced previously that runx mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi , hirohide takebayashi , takayuki manabe , kazuhiro ikenaka , akio wanaka dept. anatomy, nara med. univ., kashihara, nara, japan; division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng proteoglycan and olig at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig lineage cells in the forebrain is largely unknown. here we examined fates of olig expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig -creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) . or . , when most of olig + cells are observed in the basal forebrain. the olig + cells at e . gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig + cells at e . generated more glial cells than neurons. these results indicate that olig lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi , , masato yano , , satoru hayashi , , hirotaka j. okano , , hideyuki okano , dept. physiol., keio univ. sch. med, tokyo, japan; sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p , p and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf and nf , binding partner of nf , respectively. in mouse embryonic brains, we found that nf / expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf / formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er and brn ) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi ( ) ps a-d protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-␤iii tubulin immunoreactivity increased through the re-cultivation for - weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-␤iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng + cells in the neocortex are the opcs. we investigated about subtypes of ng + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp and wnt a, a significant proportion of these neural cells become math + . the induced math + cells mitotically active and express markers characteristic of granule cells precursors (pax , zic , and zipro ). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l + /calbindin-d k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf rather than by wnt a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p cip , p kip and p kip mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p kip mrna was shown. transfection of p kip into npcs induced activation of neurod promoter and increase of number of ␤tublin iii-positive cells. treatment with deferoxamine to npcs from e . rat midbrain and hb .f cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p kip elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g /s phase did not activate mapk and pi -k signaling. yuichi tanaka , yusuke tozuka , dai muramatsu , kin-ichi nakashima , tatsuhiro hisatsune departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng + and olig + cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj- + or map- + neuronal cells by adding retinoic acid or bdnf. more than % of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi , michiko imanishi , teiko kuroda , masao tachibana , masahiko takada department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv . b and kv . were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv . in the frog retina and by kv . b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv . b. thus, the differential expression of kv channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou , , kazunori yamamoto , masayuki hara , akinori homma , akimichi kaneko , masahiro yamada tokyo metropolitan univ., hino, tokyo, japan; astellas pharma. inc., osaka, japan; sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko ( ) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by -hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as . ± . ph unit by mm k + . acidification of pho was suppressed by . m bafilomycin a , a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps a-e analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar -channel field potential recording system in slice preparations obtained from to days old mice. stimulation at ssc induced negative field potential with short latency and short duration ( - ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing m bicuculline, the same stimulus induced a large negative field response with long duration ( - ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya , ken tsutsui , kimiko tsutsui dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap- binding, and the c-terminal sh domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe- , also expresses amph ir. arpe- thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe- cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura , satoshi ichisaka , chihiro hayashi , hirotoshi maki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v ) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi ( ) ps a-e rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut) and vglut , respectively. a complementary distribution of vglut and vglut was established by postnatal day (p) and both signal intensities increased further by p - (peak cp). the immunoreactivity for vglut decreased around layer iv after brief monocular deprivation ( dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area , with a small projection to layer vi of areas and a. cells in layer vi of area projected to dlgn. in addition, cells in layer v of area projected to dlgn, which is not observed in normal rats. in area a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of a. cells in layers v and vi of area a projected to lp. smaller number of cells in layer v of area also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps a-e activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida , takemasa satoh , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v ) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v to the lgn. we focused on the density of type metabotropic glutamate receptor ␣ (mglur ␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur ␣ in the lgn decreased after elimination of v , showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur ␣ signal in the lgn of young rats. yu morishima , hiroshi sakamoto , takahumi akasaki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., japan; div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, ( ) how rapidly the reverse od shift proceeds and ( ) whether the shift is induced only in young animals. we infused the cortex with muscimol in -week-old kittens and in adults. the reverse od shift was observed after days md, but not significant after days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps a-e experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai , nafiseh atapour , john renger , john roder , peter seeburg , takao hensch neuronal circuit dev., riken, bsi, wako, japan; mount sinai hospital, toronto, canada; max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur , ) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation ( d md) rapidly alters both the phosphorylation state and surface expression of glur in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as d md eventually led to full od shifts. in glur ko mice, even d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx promotor and dlx / enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca and revealed a nonpyramidal neuron projecting to ca and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita , takao oishi , motoharu hayashi div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than % of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area ( . %) than in other areas, such as the prefrontal ( . %), primary motor ( . %), somatosensory ( . %) and secondary visual areas ( . %), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than % of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe , guy n. elston , , tomofumi oga , hiroshi tamura , , ichiro fujita , osaka univ., japan; univ. queensland, australia; crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v ). pyramidal cells in prefrontal cortex, such as area , have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v (n = ), area te (n = ), and area (n = ) of a -week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area , followed by area te, and v . thus, considerable heterogeneity in pyramidal cell structure already exists -weeks after birth. hiroaki matsushita , mahito ohkuma , masami watanabe , ei-ichi miyachi department of physiology, fujita health university, aichi, japan; department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura- based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the % of axotomized rgcs. bath application of m carbachol, an ach receptor agonist, rose [ca + ] i in % of normal rgcs. although the % of rgcs responded to carbachol at days after axotomy, no responsive rgcs appeared during - days. ach responsiveness recovered in axonal regenerated rgcs ( %). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi ( to em, to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., ) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor ( hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames ( ms) or only one frame ( ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps a-e motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima , , tohru kiryu , kazuhiko ukai , takeshiko bando div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura , kenichiro miura , masakatsu taki , hiromitsu tabata , naoko inaba , kenji kawano , frederick a. miles grad. schl. med., kyoto univ., kyoto, japan; lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn ) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency f and f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps ( hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the f forwards and the f backwards. the contrast of the f was fixed at , , or %, while the contrast of the f was varied from one-fourth to four times the contrast of the f. when the contrast of the f ( f) was less than about half that of the f ( f), the f ( f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < . ). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito , yaoxing chen , yukiko tanada dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; china agricul. univ., beijing, china twenty white-leghorn chicks (p - ) were perfused with % paraformaldehyde through the heart under deep anesthesia of nembutal ( mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, ). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to in tec-rgcs, in tha-rgcs, and in hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers - ). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie , kenji yuda , eiichi okawa , katsuyoshi maruyama , hiroshi uozato , hiroko horie , satomi nakajima , kenkichi tanioka , yuji ohkawa , tomoki matsubara , wolfram tetzlaff advanced res. centr. biological sci., waseda univ., tokyo, japan; technomaster co. ltd., yokohama, japan; kikuna yuda eye clinic, yokohama, japan; healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; nhk sci. technical res. labo., tokyo, japan; icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between and cm toward and away in a high speed (top speed: mm/s) from the subjects. the device significantly improved visual performances in over % of the school-aged children with myopia and % of adults with presbyopia. hiroyuki miyamoto , toral s. surti , takao k hensch laboratory for neuronal circuit development, riken brain science institute, wako, japan; san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v ) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v . here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa , miquel bosch , shuzo sakata , yoshio sakurai , tetsuo yamamori division of brain biology, national institute for basic biology, okazaki, japan; mit, ma, usa; rutgers university, nj, usa; department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., ) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki , youichi miyawaki , tsunehiro takeda department of complexity science and engineering, university of tokyo, chiba, japan; mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly ( ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = - ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa ( and ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p , n and area under curve of a late positive potential expanding from to ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse , ayumu matani , , tomoe hayakawa , , norio fujimaki university of tokyo, kashiwa, japan; nict, kobe, japan; teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p amplitude becomes. this suggested that a factor of the variability of the p amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. ) hitoshi sasaki , takuya ishida , masayoshi todorokihara , junichiro miyachi , tahei kitamura , ryozo aoki dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama , nobuo usui , izuru nose , , masato taira , department of applied system neuroscience, nihon university school of medical science, tokyo; faculty of human science, bunkyo university, saitama, japan; arish, nihon university, tokyo, japan when an object is moving, perception of its d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps a-f local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a ) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., ) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., ) . moreover, aaf has dense synaptic connections within the area (budinger et al., ) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were -ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima , yasuo takeda , yasushi shimoda , kazutada watanabe department of bioengineering, nagaoka university of technology, nagaoka, japan; department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb- that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb- expression is prominent in the auditory system. nb- knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb- is indispensable for the function of auditory system. here we report the detailed analysis of the nb- localization using anti-nb- monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb- was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb- immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb- regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation ( khz octave band, db, h), and then subjected to electrophoretic mobility shift assay to determine activator protein- (ap- ) dna binding. ap- binding was increased - h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea h later. taken together, ap- may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c bl/ mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli ( - khz for ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by khz were different from those elicited by or khz. however, the areas activated by khz were also activated by the mixture of plus khz but not by that of plus khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps a-f functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by - %, whereas the inhibition of dc resulted in reduction only by - %. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh- . a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato , , kazuo okanoya laboratory for biolinguistics, riken bsi, wako, saitama; graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, ) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented - times at a fixed interval, through a speaker at - db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (< hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (> hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki , , , , , hokkaido university, sapporo, japan; hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan; kobe shoin women's university, kobe, japan; riken, wako, japan; sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children ( - years old, n = ) and adults ( - years old, n = ). there were six gap conditions with durations of , , , , and ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n and p complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps a-f an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua , tamaki miyamoto , atsushi terao , sachiko koyama , takashi omori , harumitsu murohashi , shinya kuriki jst, saitama, japan; hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba ), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation ( subjects, p < . corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi , yuko toyosawa , fumiya takeuchi , michinao matsui , shinya kuriki research institute for elecronic science, hokkaido university, sappro, japan; jst, saitama, japan; hokkaido university, school of medicine, japan; kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = ). four synthesized vowels were used as stimuli (stimulus duration ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used ( / ms). for the native vowels, an early sf ( - ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf ( - ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi ( ) ps a-f spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto , , junsei horikawa department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh ) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli ( - khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe , go ashida , , kazuo funabiki graduate school of informatics, kyoto university, kyoto, japan; hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi , shigenori takebayashi , masahiko bundo , masazumi fujii , toshihiko wakabayashi , jun yoshida , hiroyuki fujisawa , kazunori ando , kazumasa hayasaka department of radiology, national hospital for geriatric medicine, obu, japan; department of neurosurgery, national hospital for geriatric medicine, obu, japan; department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using . tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in out of patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = ) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, ) . surprisingly, subjects tended to misidentify the second toe as the third ( . %), and the third toe as the fourth ( . %), while the reverse misidentification rarely occurred (third as second, . %; fourth as third, . %). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps a-g effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi , , shunjiro moizumi , ayami okuzumi , humine saito , shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, jst, saitama, japan morrone et al. ( ) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade ( • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps a-g function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda , , miki taoka , department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas b and of four conscious macaque monkeys was studied by recording single-neuron activities. a total of electrode penetrations were made in areas b and . in penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining penetrations, however, % of adjacent pairs ( / ) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas b and might be responsible for forming composite rfs of area neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi ( , ) miki taoka , michio tanaka , hisayuki ojima , atsushi iriki secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them ( % contra-, % bi-and % ipsilateral hand movements), we could determine receptive fields for only neurons ( %). most of activities ( neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta , , kouichi nakamura , takeshi kaneko department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata , , shanlin chen , hiroki kitaura , masaharu kudoh , minoru shibata , katsuei shibuki department of neurophysiology, brain research institute, niigata university; department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane ( . g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation ( hz for s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi , nanae fukushima , tetsuhiro fukuyama , akira kakegawa , tetsuji moriizumi department of anatomy, shinshu university school of medicine, matsumoto, japan; department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima , takeshi kawano , hidekuni takao , , , kazuaki sawada , , , hidekazu kaneko , , makoto ishida , , department electrical & electronic engineering, toyohashi university of technology, aichi, japan; issrc, toyohashi university of technology, aichi, japan; aist, hsbe, ibaraki, japan; jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, m in diameter, m in length; array: -m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = . ). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi ( ), the st century coe program "intelligent human sensing" ps a-g a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko , hiroshi tamura , shinya s. suzuki , takahiro kawashima aist, hsbe, ibaraki, japan; laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over h. seiji matsuda , takehiro terashita , tetsuya shimokawa , kyoujy miyawaki , yuji miguchi , takuya doihara , jie chen , shuang-yan gao , chun-yu li , min wang , zhong wang , bing xue , naoto kobayashi , , kazuhiro shigemoto department anatomy, ehime university of medicine, ehime, japan; medical education c, ehime university of medicine, ehime, japan; department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than . m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat , whereas other genes known to be regulated by stat were not detected. stat is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat and stat phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto , susumu hyodo , makoto kawasaki , minori shibata , takeshi saito , hiroaki fujihara , takashi higuchi , yoshio takei , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; department of integrative physiology, university of fukui, japan adrenomedullin (am ) (identical to intermedin) belongs to the super family of am. centrally administered am and am activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am was stronger than those of am and the effects of am on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am would have unknown receptor except cgrp and am receptor. arata oh-nishi , makoto saji , taku uchida , sen-ichi furudate , nobuyuki suzuki division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, ) . here we report that the density and morphology of spines of pyramidal neurons in ca region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca , both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day - , the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day . the staining of somatostain mrna in individual neurons was diminished when examined on day or , albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps a-g short chain sugar acid, -buten- -olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki , tatsushi onaka , hirofumi hashimoto , hiroaki fujihara , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; department of physiology, jichi medical school, tochigi, japan -buten- -olide ( -b o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of -b o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at - min after intraperitoneal (i.p.) administration of -b o ( mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei min after i.p. administration of -b o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of -b o. these results suggest that peripherally administered high dose of -b o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps a-g estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto , , shinpei higo , , yasushi hojo , , kouhei nakajima , , hironori nakanishi , , hirotaka ishii , , suguru kawato , department of biophysics & life science, university of tokyo, tokyo, japan; crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps a-g gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from − to - mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than - mv and most of them were ∼− mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr , galr and galr , in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr selective agonist m induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr . hiroaki fujihara , tomoki fujio , david murphy , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking % saline for days in , , and months old tg rats. in and months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats ( and months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps a-h prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera , hiroaki fujihara , hirofumi hashimoto , makoto kawasaki , tatsushi onaka , takakazu oka , sadatoshi tsuji , yoichi ueta department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; department of physiology, school of medicine, university of occupational and environmental health, japan; department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin , afsaneh eliassi , nasser naghdi , ali ghanbari ghsemi; pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above ( . mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from min up to the end of experiments ( min). at the peak acid response, the vmh microinjection skf ( . , ) significantly reduced the amount of gas (p < . ). there was no any effect by microinjected sch ( . ) into the vmh. injecting skf into the vmh, min after sch , had no effect on gas in compare with control. but, the acid suppressant effect of skf was completely removed by peripheral injection of sch (p < . ). our results show that the vmh d dopamine receptors have regulatory mechanisms of gas by interaction with h receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p y purinoceptors (p y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p y r agonists increased [ca ++ ] i in son, with p y r agonist being the most effective. the possibility that p y r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p y r antagonist, mrs . atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs ( m) in mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs . furthermore, combined treatment with mrs and ppads ( m, a p x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p y r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps a-h analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii , , yasuhiro sonoki , , aizo furukawa , , yasushi hojo , tetsuya kimoto , , suguru kawato , department of biophysics and life science, university of tokyo, tokyo, japan; crest, jst, japan; kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day (pd ) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd to pd with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai , , gen murakami , shirou kominami , john h morrison , william g.m janssen , tetsuya kimoto , , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo - , japan; crest project, jst, japan; faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima , japan; kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc- , mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca , ca , and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud , song t. yao , jing qiu , julian fr paton , david murphy university of bristol, hw-line, uk; department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic ( d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase , has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt - . delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt - cells cultured in dmem supplemented with % fbs for days. bk current was increased by addition of ␤-estradiol (e ) in culture medium in a physiological concentration range. this action of e was blocked by ici- , , a potent estrogen receptor (er) antagonist. we further examined whether e acted through er ␣ or er ␤ by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e but ppt had no effect. e had no effect on the i k . these results indicate that e increases the bk current by activating er␤ without affecting the i k . research funds: kakenhi , ps a-h myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi , haruko kumanogoh , shun nakamura , seiji miyata , shohei maekawa department of biosystems science, kobe-university, kobe, japan; division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, kda protein, was identified as myelin protein zero (p ) from mass analysis and this result was confirmed by western blotting that a specific antibody to kda band reacted to an authentic p prepared from rat sciatic nerve myelin. p is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan ␤-estradiol (e ) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e (hojo et al., ) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca , ca and dg with a custom made multi-electrode measuring system (med ). ltd was enhanced by e dose-dependently in ca , ca and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito , takushi x. watanabe , tomoko urabe , hirofumi hashimoto , hiroaki fujihara , yukio hirata , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; peptide institute, inc., osaka, japan; department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin-␤ was newly discovered as a bioactive endogenous peptide. low concentration of salusin-␤ stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin-␤ coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin-␤ in body fluid homeostasis, we examined the effects of salt loding for days on salusin-␤-like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin-␤-li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin-␤ may play a role of body fluid balance by regulating avp release. naoyuki yamamoto , hao-gang xue , yuji ishikawa , yoshitaka oka , hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan; national institute of radiological sciences, chiba, japan; department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki , eiji munetsuna , asuka kamogawa , suguru kawato , shiro kominami graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p arom in the hippocampus. method: the cultured hippocampus slices from days male rat were treated with - nm of tbt for h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p arom was determined by quantification of [ h]estradiol from [ h]testosterone. result: forty-eight hours treatment of hippocampal slices with nm tbt induced increases in mrna contents of p arom, and with nm tbt induced that of ␤-hsd. estradiol content was increased by the treatment with nm tbt, but not affected by nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of , , and ng in nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent h. the ng dose of bicuculline methoiodide had minimum effect whereas and ng produced arousal. maximal wakefulness was observed at dose of ng with no further increase in wakefulness at higher dose of ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe , hiroyuki tsuda department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee ). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, ( antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at h after from hibernation onset compared with before hibernation. in addition, a localized ␤-endorphin-like immunoreactivity (␤-end ir) was observed in neuronal perikarya in arc at h after from hibernation onset. although ␤-end ir in arc got weak, the ␤-end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the ␤-endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via -opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d is a potent somnogenic substance, and isomerized from pgh through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl decreased rapid eye movement (rem) and non-rem sleep for h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl administrations. these findings indicate that pgd plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd involved in sleep-wake regulation. under baseline conditions, h r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (< s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., ) . ps a-h dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = nm). the scn neurons responded to the light in intensities above photons/cm /s and were activated maximally at around photons/cm /s, they were about . log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca + in scn neurons. since cytosolic ca + regulates diverse cellular systems, we have hypothesized that the cytosolic ca + rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca + rhythms, mouse bmal and its dominant negative sequence (mbmf r ) are transfected into the organotypic culture of scn with a yellow cameleon ca + sensor by the gene gun. the results demonstrated that over-expression of bmal or mbmf r significantly inhibited the circadian ca + rhythms and thus we concluded that the native bmal rhythm is essential for cellular output processes of the murine clock system. ps a-h the activation of ␣ adrenergic receptor increases the frequency of carbachol-induced ␤ oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses ␤ rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce ␤ oscillation in rat hippocampal slices like ␤ rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced ␤ oscillation in ca region of rat hippocampal slices. carbachol ( m) induced ␤ oscillation with the frequency and the amplitude of . ± . hz, and . ± . mv, respectively (mean ± s.e.m.; n = ). epinephrine ( m) significantly increased the frequency of . ± . hz (**p < . ), not change the amplitude. clonidine ( m), an ␣ receptor agonist, alone significantly increased the frequency at the concentration of m (*p < . ). yohimbine, an ␣ receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal ␤ rhythm via ␣ receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart . mm from each other in the hippocampal ca region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency - ms), oscillatory ( - hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties ( / slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca region. ryozo aoki , hiroshi wake , hitoshi sasaki , kiyokazu agata dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as /f, ranging from − to + hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida , hiroki sato , , naoki tanaka , atsushi maki , japan science and technology agency, crest, saitama, japan; advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from to hz) decreases and the sleep spindle power (from to hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from to s in (i), and from to s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim , kazuki nakajima , yutaka oomura , kazuo sasaki div. of bio-information eng., univ. of toyama, toyama, japan; dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca + exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng , wei zhang , akira nakamura , masashi yanagisawa , yasuichiro fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. autonom. physiol., chiba univ., japan; dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb- (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb- . icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co -sensitivity at least during waking periods. research funds: kakenhi , junko hara , taizo matsuki , katsutoshi goto , masashi yanagisawa , takeshi sakurai , department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; yanagisawa orphan receptor project, erato, jst, tokyo, japan; howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps a-i prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto , kazuhiko kubo , shuji aou dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. ) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. , s.a.) shuji aou , tetsuya fujimoto , yumi ichihara , kimiya narikiyo , toru ishidao , hajime hori , yukiko fueta dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan -bromopropane ( -bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to -bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to ppm of -bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. -bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that -bp is the potential candidate of endocrine disruptors which affect brain development. ( ) ps a-i changes in hippocampal excitability of rats prenatally exposed to -bromopropane yukiko fueta , toru ishidao , susumu ueno , yasuhiro yoshida , hajime hori department of environmental management, school of health sciences; department of pharmacology; department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to -bromopropane ( -bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to -bp ( ppm) during gestational day - ( h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca at the pnd - . conversely, s/r curves of fepsp as well as ps in the ca were inhibited at the age of - weeks. our results suggest that -bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca of the offspring during development. research funds: grant-in-aid for exploratory research ( ) ps a-i effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura , , kyoko itoh , takeshi yaoi , tohru sugimoto , shinji fushiki dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously g/kg of bpa daily from embryonic day (e ). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e . and e . , whereas those cells increased in the cortical plate at e . , as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant keiko ikemoto , teruko uwano , hisao nishijo , taketoshi ono , masayuki ito , ikuko nagatsu , katsuji nishi , shin-ichi niwa dept. neuropsychiat., fukushima med. univ. sch. med.; toyama med. pharm. univ.; faculty med., mie univ., mie, japan; fujita health univ. sch. med., toyoake, japan; dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day and after fertilization. pups were perfused at postnatal day . in the frontal cortex, the number of largesized (more than m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c /a catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps a-i developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi , , , kaori morohoshi , , rie yanagisawa , erina saita , gen watanabe , , masatoshi morita , kazuyoshi taya , , hirohisa takano , , toshiyuki himi , , hideki imai , dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; res. inst. pharmaceut. sci., musashino univ., tokyo, japan; nation. inst. for environ. stud., ibaraki, japan; grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; wildlife rescue veterinarian associ., tokyo, japan; facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; the united grad. sch. veterinary sci., gifu univ., gifu, japan; div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor ␤, which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps a-i the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic ( -ht) systems in stressinduced anxiety, we measured brain da and -ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of -ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and -ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although -ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl - ) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl and nl in autistic disorders. nls have a relative identical structure (∼ %); nl and nl localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl seems express in the olfactory glia, but nl distribution is unknown. here we have generated antibody against human nl , and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl is important for neuroendocrine function. nl cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl were rough and thicker than those of nl or nl transformants. it suggested the unique activity of nl for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting - % of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below . here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura , daisuke horiuchi , tomoyuki miyashita , minoru saitoe , hitoshi okazawa department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein- (pqbp- ) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp- disease that pqbp- mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp- in xlmr pathology, we produced two kinds of flies, human pqbp- overexpression flies (hpqbp flies) and drosophila pqbp- knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp- in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun , sentaro takahashi , yoshihisa kubota , rui zhang , chun cui , yoshihiro fukui natl. inst. radiol. sci., chiba, japan; sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr subunits paired with two nr subunits. nr and nr (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr /nr a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr /nr b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca pyramidal neurons from mice (at weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca region after ischemia. in blood, corticosterone levels increased biphasically at min and h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca region. by days after ischemia, mr and gr were not significantly altered in the ca region. from days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca region, and at days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase- and midkine in the mca area. stroke was induced by a -min mca occlusion using an intraluminal filament. rats were exercised for min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase- expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps a-j reversed astrocytic glutamate transporter glt- crucial to the ca + paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca + paradox" is the phenomenon whereby the intracellular concentration of ca + paradoxically increases during reperfusion with normal ca + -containing media after brief exposure to a low ca + solution. the present study aims to characterize the ca + paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca + solution for min significantly injured only neurons after reperfusion with a normal ca + medium for h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt- . the present findings suggested that ca + paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt- during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c t, which results in pro ser. the second patient carried the missense mutation c g, which results in arg pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps a-j mci - , a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol kinase madinyet niyaz , tadahiro numakawa , yoshinori matsuki , emi kumamaru , yuki yagazaki , harumi kitazawa , hiroshi kunugi , motoshige kudo pathology department of tokyo medical university, tokyo, japan; department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci - , a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci - prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci - exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways and that the mci - -dependent survival effect was blocked by the inhibitors, u (an mapk pathway inhibitor) or ly (a pi k pathway inhibitor). these results suggested that mci - exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru , noriko yoshimura , tetsuro sakamoto , takaaki kitano , naoko nisimaru , isao yokoi dept. physiol., sch. med., oita univ., japan; med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by p-nuclear magnetic resonance ( • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with mm ep (ep- ), acsf replaced by acsf with mm ep after ischemia (ep- ), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep- than in control (p < . ). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) # from mext to t.k. hideaki tamai , kuniko shimazaki , norimasa seo department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc ), yintang (ex-hn ) and sanyinjiao (sp ), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through -bromo- ,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc , ex-hn and sp . the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago , keiko takahashi , shigeo nakamura , akemichi baba , toshio matsuda laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug ␤-cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara , tatsuya mishima , takefumi kofuji , kimio akagawa department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc- /syntaxin a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc- /syntaxin a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc- /syntaxin a knock-out mouse will be discussed. research funds: kakenhi ( ) ps a-k effects of local administration of the gaba agonists into the hippocampus ca area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka , hiroko miyakubo , junichi tanaka , yasushi hayashi , yukio hattori , masahiko nomura department of curriculum, teaching and memory, naruto university of education, tokushima, japan; department of human nutrition, notre dame seishin university, okayama, japan; department of physiology, saitama medical school, saitama, japan in fischer male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite -hydroxytryptamine ( -hiaa) concentration in the ca area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad- . fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka , sho koyasu , akinori nishi , tomoyuki furuyashiki , toshiyuki matsuoka , shuh narumiya department of pharmacology, university of kyoto, kyoto, japan; department of physiology, university of kurume, kurume, japan prostaglandins e (pge ) exert their actions in various organs through specific receptor, ep to . the previous study suggests that ep modulates da system. to investigate the roles of ep in da system, we examined ep ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼ % in both wild-type and ep ko mice. however, increase of locomotor activity in ep ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d receptor (d r) signaling, we tested the density of d r and d r signaling with phosphorylation of darpp- . there were no differences in d r binding. d r signaling was significantly attenuated in the striatal slices from ep ko mice. the effect of d r agonist on locomotor activity was also attenuated in ep ko mice. these results indicate that pge has enhancing effects on locomotor activity via ep by potentiating the d r signaling. central serotonin ( -ht) function has been implicated in impulsivity. the present study examined rats with -ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of -ht and its metabolites ( -hiaa) in the brain. for both tasks, significant negative correlations between learning performance and -ht and -hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and -ht and -hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of -ht roles on selective indices of impulsivity. yuki sato , , , tatsushi onaka , norimasa seo , eiji kobayashi dept. anesthesiol., jichi med. univ., tochigi, japan; dept. physiol., jichi med. univ., tochigi, japan; div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p - ; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p ; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io␤) by p . within the vestibular nucleus of p - rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p in the vestibular nucleus as fuzziness about neuronal cell bodies. from p onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p . during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p . with ul, the pn network on the lesioned side remained compact until days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca region of rat hippocampal slices, hz/ pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at hz. ikuko yao , hiroshi takagi , hiroshi ageta , tomoaki kahyo , ken hatanaka , kaoru inokuchi , mitsutoshi setou , mitsubishi kagaku institute of life sciences, tokyo, japan; university of tokyo, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps p-a presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., mv, s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca + currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca + channel knockout mice. we conclude that the p/q-type specific ca + channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi , koji ikegami , ken hatanaka , , , yoko fujiwara-tsukamoto , mineo matsumoto , ikuko yao , mitsutoshi setou , , mitsubishi kagaku institute of life sciences, japan; presto, japan; school of pharmaceutical sciences, the university of tokyo, japan; okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa (−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki , shingo kimura , reiko fujita , shuji watanabe , kazuhiko sasaki dept. of physiol., sch. of med., iwate medical univ., morioka, japan; dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c , a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦ glutamate receptor (␦ r) plays a crucial role in cerebellar functions. although ␦ r has a putative channel pore domain, and ␦ r displayed ca + -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦ r functions as a channel. here we introduced a ␦ r transgene, which had a mutation (gln arg) in the putative channel pore conserved in ca + -permeable glutamate receptors, into ␦ −/− mice. surprisingly, a mutant ␦ r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦ −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca + influx through ␦ r is not required for its function in the cerebellum in vivo, and that wild-type ␦ r may not function as a ca + -permeable ion channel. research funds: kakenhi ( ) and takeda science foundation ps p-a distribution of tarp - on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya , mika tsujita , maya yamazaki , etsuko kushiya , manabu abe , kaori akashi , masanobu kano , haruyuki kamiya , kenji sakimura , masahiko watanabe department of anatomy, hokkaido university school of medicine, sapporo, japan; department of cellular neurobiology, brain research institute, niigata, japan; department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the - is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized - on excitatory synaptic and extrasynaptic membrane. in - -ko mice, ampars were reduced in hippocampal homogenates ( % of control) and psd fraction ( %). immunogold labeling also exhibited reduction of extrasynpatic ( %) and synaptic ( %) ampars in ca pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites ( %) and spines ( %). ampar-mediated responses were reduced at ca synapses ( %). therefore, - is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa , yugo fukazawa , elek molnar , masahiko watanabe , ryuichi shigemoto , div. cerebral structure, nips, okazaki, japan; mrc, univ. of bristol, bristol, uk; hokkaido univ., sapporo, japan; sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed . to times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed . to times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda , , , hitoshi kita dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; japan society for promotion of science, tokyo, japan; dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp abolished the sipsp. an mglur antagonist, but not an mglur antagonist, partially blocked the sdepo. in addition, cgp enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur -mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps p-a essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto , yoshikazu isomura , , michiko imanishi , tomoki fukai , masahiko takada system neurosci., tokyo met. inst. neurosci., tokyo, japan; neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for > s after the induction. gaba amediated cl-influx lasted for . s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura , yuko sekino , , toshiya manabe , division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < ), using electrophysiological methods and optical imaging. in immature animals (p ), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate (<p ) with a significant change in the paired-pulse ratio. the presynaptic effect of baclofen was gradually decreased with development (p - ). in situ hybridization and immunohistochemistry experiment revealed gaba b r expression in mntb neurons was also gradually decreased. the property of mntb-lso synapses in gaba b r knock out mouse remained immature at p - compared with control mouse. thus, gaba b receptor seems to play an important role in development of synaptic property. pyramidal cells in the hippocampal ca area receive gabaergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (ais). here, we used the sdsdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣ , ␣ , and ␤ / subunits quantitatively on distinct subcellular compartments of pyramidal cells. labelings for these subunits were accumulated over clusters of intramembrane particles (imp) on the protoplasmic face (p-face) of the plasma membrane, indicating that many imp clusters of a certain size represent gabaergic synapses. the synaptic labelling densities for gaba-a receptor subunits were not significantly different on the tested subcellular compartments. double labelling experiments showed that the majority of synapses contains ␣ , ␣ , and ␤ / subunits. in the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. therefore, we investigated the effects of the ␣ -or ␣ -adrenoceptor agonist on firing rates of presynaptic interneurons (ins). presynaptic cell-attached recordings showed that spontaneous activity of gabaergic ins was enhanced by the ␣ -adrenoceptor agonist phenylephrine, while reduced by the ␣ -adrenoceptor agonist clonidine. immunohistochemical studies showed that ␣ -adrenoceptors were expressed in the molecular layer and the purkinje cell (pc) layer, while ␣ -adrenoceptors were observed in cell bodies of pcs and ins. these results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of ins via ␣ -adrenoceptors, which were located in the presynaptic in soma. thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of gabaergic inputs from ins to pcs. research funds: kakenhi ( ) ps p-b involvement of basal pkc and erk / activities in constitutiveinternalization of ampa receptors in cerebellar purkinje cells tetsuya tatsukawa , takahiko chimura , azumi matsumoto , kazuhiko yamaguchi lab. for motor learning control, riken, bsi, japan; lab. for memory & learning, japan ampa receptor (ampar) internalization provides a mechanism for cerebellar ltd, which is the underlying basis of motor learning and motor coordination. however, the relationship between cerebellar ltd and constitutive ampar internalization in parallel fiber (pf)-purkinje cell (pc) synapses remains unclarified. in the present study, we demonstrated that tetanus toxin (tetx, a blocker of exocytosis mediated by vamp) infusion into pcs caused the rundown of pf-epsc amplitude through the constitutive ampar internalization, and then addressed question whether intracellular signals involved in cerebellar ltd induction modify the constitutive ampar internalization at pf-pc synapses using electrophysiological and immunocytochemical techniques. our results suggest that the regulation of actin polymerization is involved in the surface expression of ampars and the surface expressing ampars are constitutively internalized depending on both basal pkc and mek-erk / activities at pf-pc synapses. taisuke miyazaki , kohichi tanaka , masahiko watanabe department of anatomy, school of medicine, hokkaido university, sapporo, japan; department of molecular neuroscience, tokyo medical and dental university, tokyo, japan glutamate released to the synaptic cleft has to be removed by glutamate transporter. in the cerebellum, glutamate transporter glast is richly expressed in bergmann glial cells (bg) that enwrap synapses, dendrites and somata of purkinje cells (pcs). in this study, anatomical analysis was applied to glast-deficient mice. in the mutant mice, lamellate processes from bg fibers became atrophic, resulting in incomplete sealing of synaptic cleft. furthermore, the distribution of climbing fiber (cf) terminals was decreased proximally. by anterogradely labeling, multiple cf innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. these results suggest that glast is essential for complete ensheathment of pc synapses and for the establishment of cf mono-innervation by suppressing local ectopic innervation between nearby pc dendrites. miwako yamasaki , , kouichi hashimoto , taisuke miyazaki , masahiko watanabe , masanobu kano dept. of anatomy, grad. sch. of med., hokkaido univ., sapporo, japan; dept. of cellular neuroscience, grad. sch. of med., osaka univ., suita, japan the ␥ isoform of protein kinase c (pkc␥) is highly expressed in cerebellar purkinje cells (pcs), and its deficiency impairs the late phase of climbing fiber (cf) synapse elimination. in the present study, we analyzed multiple cf innervation in pkc␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of cfs. in general, cf innervation regressed proximally in the pkc␥-deficient mouse. in some mutant pcs, a considerable part of the proximal dendrites lost association with cf terminals. in the majority of multiply innervated pcs, single main cf innervated broad region of proximal dendrites, whereas surplus cfs innervated the somata and basal portion of the proximal dendrites. moreover, immunoelectron microscopy revealed synaptic contact of cf terminals to somatic spines. these results suggest that pkc␥ is crucial for strengthening innervation by a main cf and elimination of surplus cfs from proximal somatodendritc compartments of pcs. keiji imoto , , takashi kodama , , ryuichi shigemoto , , , yugo fukazawa , , yasuo mori division of neural signaling, nips, okazaki, japan; school of life science, graduate university for advanced studies (sokendai), okazaki, japan; division of cerebral structure, nips, okazaki, japan; sorst, japan science and technology agency, kawaguchi, japan; department of synthetic chemistry and biological chemistry, kyoto university, kyoto, japan a murine ataxic mutant, rocker, has a point mutation in the ca v . (p/q-type) ␣ subunit gene and shows the mildest phenotype among the series of ca v . mutants. although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-purkinje cell synapses was considerably impaired. we demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of ampa receptors in psd, whereas the presynaptic function remained normal. we also found the dendrites of purkinje cells showed poor arborization in rocker mice. these lines of evidence indicate that ca v . exerts its strong influence on postsynaptic maturation and dendritic structure. bai-chuang shyu, chia-ming lee, wei-chih chang institute of biomedical sciences, academia sinica, taipei, taiwan roc the aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the anterior cingulate cortex (acc) using a novel mouse brain slice preparation protocol. the mono-and poly synaptic responses in the acc were excited by direct thalamic activation. the synaptic transmission involves both ampa and nmda glutamate receptors. several thalamus-evoked responses were identified: the early negative component (n ) emerged in layer vi near cingulum. subsequently a positive component (p) appeared in layer vi. the second negative component (n ) became apparent in layers ii/iii and v. then the activities spread downward to layer vi and developed as n component in a medial-to-lateral direction. we confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. the thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the acc and this process was modulated extensively by glutamatergic synapses. ps p-b target-dependent extracellular ca-dependence of the short-term plasticity of the solitary complex synapses in the rat lab. neurophysiol., dept. neurosci., jikei univ. sch. med., tokyo, japan the discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (sc) forms the primary filter of the visceral information. such frequency filtering of the synaptic transmissions from the solitary tract (ts) to neurons in the sc depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (ppr). here we analyzed the mechanism underlying distinct pprs among distinct types of synapses. in synapses from ts to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, ppr significantly increased from . ± . to . ± . (n = ; mean ± s.d.) by reducing [ca + ] o from to . mm, whereas that in low-pass synapses from ts to the neurons in dorsal motor nucleus of the vagus was affected only slightly. these results suggest that the synaptic frequency filtering in the sc is under distinct control depending on the function of the postsynaptic target systems. ps p-b response of spinal monosynaptic reflex to high frequency stimulation in newborn rat the susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. in an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (msrs) evoked by dorsal root stimulation, were mediated by both nmda and non-nmda glutamate receptors. msrs were constant in amplitude at / s, and were depressed completely by cnqx. when stimulus rate was increased to /s, msr amplitudes were greatly reduced initially, and recovered later. this recovery of msr was eliminated by apv. non-nmda component of msrs was eliminated completely at /s, but appeared in constant amplitudes in the presence of cyclothiazide. from these results, non-nmda glutamate receptor mediated almost most of msrs at / s, but not at /s due to desensitization. in contrast, nmda glutamate receptor does not mediate msrs, but activated at /sec, in place of non-nmda receptor. in conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission. kenichi ono , keisuke shiba , ken nakazawa , ichiro shimoyama department of otolaryngology, chiba university, chiba, japan; department of otolaryngology, kimitsu central hospital, chiba, japan; department of integrative neurophysiology, chiba university, chiba, japan; section for human neurophysiology, research center for frontier medical engineering, chiba university, chiba, japan we determined synaptic source of the respiratory-related activity of laryngeal motoneurons (lms) using spike-triggered averaging of lm membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. in inspiratory (i) lms, monosynaptic epsps were evoked by spikes of i neurons with augmenting firing patterns, and monosynaptic ipsps were evoked by spikes of expiratory (e) neurons with decrementing firing patterns and of i neurons with decrementing firing patterns. in elms, monosynaptic ipsps were evoked by spikes of i neurons with decrementing firing patterns and of e neurons with augmenting firing patterns. we conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of lm membrane potentials. ps p-b in-vivo gene transfer of exogenous protein to the primary afferent neurons chiaki yamada , , eiji shigetomi , , fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan; dept. basic biol. sci., kyoritsu univ. of pharm., tokyo, japan; jsps, japan in order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. in the anesthetized young wistar rats, we injected pcaggs-egfp plasmid vector (through courtesy of drs. j. miyazaki and k. nakajima) into the nodose ganglion (ng) at the neck and transferred it by electroporation. two days after transfection, the ng was extirpated and fixed to observe egfp signals. a large portion of somata in the ng as well as the centrally and peripherally projecting afferent fibers expressed high levels of egfp with few damaged cells. this technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain. research funds: kakenhi ( , ) ps p-b laminar sources of synaptic input to layer neurons in rat visual cortex takuma mori, edward m. callaway salk institute, usa layer of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. very little is known about connectivity of these cells with cells in other cortical layers. we examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer neurons of rat visual cortex, using scanning laser photostimulation. we find that axon descending cells get excitatory synaptic inputs primarily from layer / . neurogliaform cells receive more excitatory inputs from layers and . the dominant inhibitory synaptic inputs onto both types of cells originate from layer / . these neurons also get inhibitory inputs from layers and - . the presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer are highly integrative. takako nishi , tsukasa gotow , shiro nakagawa ins. nat. sci., senshu univ., kawasaki, japan; dept. neurol., kagoshima univ. grad. sch. med.-den. sci., kagoshima, japan four extra-ocular photoreceptors, the photoresponsive neurons, a-p- , es- , ip- and ip- which respond directly to light, exist in the abdominal ganglion of the mollusc onchidium. a-p- /es- of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other ip- /ip- . the present study was conducted to examine the functional significance of the above ip- /ip- , having a peak sensitivity at nm light. the body wall of the animal transmitted enough nm to hyperpolarize ip- /ip- in the ganglion covered by the body wall. ip- /ip- were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. ip- /ip- were not coupled to a-p- /es- one another. finally, ip- /ip- , the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera. ps p-c cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken tatsuo sato, iwao fukui, harunori ohmori department of physiology, faculty of medicine, kyoto university interaural level difference (ild) and timing difference (itd) are the major two cues to localize the sound source, and are processed separately. although the details of itd processing are well investigated, not much is known about ild. in the barn owl, it is proposed that ild is calculated in lldp (posterior part of the dorsal lateral lemniscal nucleus). we confirmed already that in vivo lldp calculates ild in the chicken as well. in this work using the patch clamp recording with the brainstem slice preparation, we found that lldp neurons fire tonically under the regulation of two k + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. we are intending to clarify the cellular mechanism of ild calculation as the interaction of both excitatory and inhibitory synaptic inputs. to understand the functional organization of the cns, it is essential to know how sensory information is processed within the cns. we have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. in this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. stimulation applied to the olfactory nerve elicited epsp-related optical signals in the olfactory bulb from the -day old embryonic stage. the epsp was mediated by glutamate, and nmda-and non-nmda-receptor components were identified. in more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. the results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. yoko momose-sato, katsushige sato dept. physiol., tokyo med. dent. univ. sch. med., tokyo, japan in an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around e . it is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. we found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. at early stages of development (e -e ), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. as development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. the oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. we examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. akiyoshi shimada , nyberg tobias , nahoko kasai , yuriko furukawa , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, ntt corporation, kanagawa, japan; sorst, jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; department of genetic and behavioral neuroscience, graduate school of medicine, gunma university, gunma, japan in the immature stage of cerebral cortex, gaba a receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. cortical neurons of glutamic acid decarboxylase -green fluorescence protein (gad -gfp) knock-in mice at postnatal day - were cultured on a microelectrode array. spontaneous firing of cortical neurons appeared within the first days in vitro (div). when a gaba a receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least div: increase, decrease, disappearance and no change. these results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of gaba a receptors. ps p-c pathway specific signal modulation by purinergic receptors in the parallel processing system makoto kaneda , toshiyuki ishii , , yasuhide shigematsu , toshihiko hosoya , yukio shimoda dept. physiol., keio univ. sch. med., tokyo, japan; bsi, riken, wako, japan; mri, tokyo womens' medical univ., tokyo, japan; dept. biomolecular science, univ. toho, funabashi, japan the retina divides visual information into on-and off signals, and processes them with independent subsets of neurons. so far no difference between these pathways has been found for neurotransmitter functions. we have previously found that p x -purinoceptors are selectively present on the off-type, but not on the on-type cholinergic amacrine cells of the mouse retina. we therefore asked if atp has different functions in the two pathways. as expected, only the off-type, but not the on-type, cholinergic amacrine cells responded to atp. this response was mediated by the p x -purinoceptors. furthermore, a p x-purinergic receptor antagonist activated the tonic phase firing of the off ganglion cells, but not of the on cells. thus atp has off-pathway specific modulatory functions mediated by p x-purinergic receptors. bhupesh mehta, gulnaz begum, preeti g. joshi department of biophysics, national institute of mental health and neuro science, bangalore, india cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic ca + . these oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. the synchronized ca + oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. these spontaneous oscillations are inhibited by activation of purinergic receptors. we observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists atp and utp, but an enhanced oscillatory pattern was observed when the action of mesatp was observed. the inhibition of mesatp response by p y receptor specific antagonist mrs indicates that different subtypes of purinergic receptors play different role in the regulation of synchronized ca + oscillations. data will be presented on the p receptor mediated regulation of synchronized ca + oscillations in cultured hippocampal neurons. mahomi kuroiwa, akinori nishi department of pharmacology, kurume university school of medicine, kurume, japan presence of additional d -like dopamine receptors that selectively couple to gq/phospholipase c (plc) has been proposed. in recent studies, skf was shown to selectively stimulate plc-linked d -like dopamine receptors. darpp- is a phosphoprotein that regulates the efficacy of d receptor signaling. activation of d receptors that couple to golf/pka signaling leads to darpp- phosphorylation at thr . we investigated the effect of skf on darpp- phosphorylation at thr by using mouse neostriatal slices. treatment of neostriatal slices with skf increased thr phosphorylation. the effect of skf was enhanced by a plc inhibitor, u . in contrast, the effect of skf was partially blocked by a d receptor antagonist, sch , and the sch -insensitive increase in thr phosphorylation was completely abolished by an adenosine a a receptor antagonist, zm . thus, analysis of darpp- phosphorylation revealed that skf activates at least three signaling cascades in the neostriatum: ( ) plc, ( ) d receptor/golf/pka, ( ) adenosine a a receptors. ps p-c thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus naoshi fujiwara division of medical technology, niigata university school of health sciences, japan effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. cortical and hippocampal slices of the c bl mouse were dyed with a voltage-sensitive dye rh . in cortical slices, electrical single-pulse stimulation to the layer v evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers ii-iii and widely expanded in these layers. the excitation propagation in the layers ii-iii was significantly inhibited by thiamylal ( mol/l). in hippocampal slices, excitation propagation elicited by the same stimulation to the ca stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. on the other hand, an ampa/kainate receptor antagonist cnqx ( mol/l) inhibited excitation propagation in both cortical and hippocampal slices. the results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus. research funds: grant-in-aid from jsps no. ps p-c brain-derived neurotrophic factor regulating ampa receptor translocation to postsynaptic density through ip r and trpc calcium signaling hiroko nakata, shun nakamura national institute of neuroscience, tokyo, japan the change in the number of postsynaptic ampa-type glutamatergic receptors (ampars) by neuronal activity is implicated in the excitatory synaptic plasticity. here, we showed that bdnf induced ampar translocation to postsynaptic site of the dissociated cortical pyramidal neurons. using calcium imaging, we located the spines that were activated by bdnf. with thus identified spines, we analyzed the ampar subunit glur localization to postsynaptic density (psd) by immunostaining. we found that bdnf increased the ratio of surface glur at psd within min. bdnf selectively translocated ampar, but not nmdar. the essential signaling was ca + transients evoked by the activation of trkb/plc␥ pathway. both the intracellular ca + rise, that is, ca + release from ip r and ca + influx through storeoperated cation channel trpc contributed to the increase of the surface expression of glur at psd. these results suggest an important role of bdnf in the regulation of ampar trafficking by spatial and temporal ca + signaling regulation. research funds: health sciences research grant of nano- hiroyuki konishi , kazuhiko namikawa , , keiji shikata , yuji kobatake , taro tachibana , hiroshi kiyama dept. anat. & neurobiol., osaka city univ., grad. sch. med., osaka, japan; dept. anat., asahikawa med. col., hokkaido, japan; dept. bioeng., osaka city univ., grad. sch. eng., osaka, japan activation of akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. in this study, we attempted to identify novel substrates for akt by using an antibody, which recognized phosphorylated consensus motif by akt. using pc cells, which overexpressed constitutively active akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. we then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. these results suggest that peripherin is a novel substrate for akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. the truncated trkb receptor, t , is involved in the control of cell morphology via the regulation of rho proteins via rho gdi . however, it is unclear whether t regulates the downstream of rho signaling and the actin cytoskeleton. in this study, we investigated this question using c rat glioma cells, which express t endogenously. rho gdi was dissociated from t in a bdnf-dependent manner, which also causes decreases in the activities of rho-signaling molecules such as rhoa, rock, pak and erk. moreover, bdnf resulted in the disappearance of stress fibers in the cells treated with lpa, an activator of rhoa, and in morphological changes in cells. furthermore, a competitive assay with cfp fusion proteins of t -specific sequences reduced the effects of bdnf. these results suggest that t regulates the rho signaling pathways and the actin cytoskeleton. anjana kalita eukaryotic gene expression laboratory, national institute of immunology, new delhi, india jak and stat are activated in response to many cytokines and growth factors. this pathway has been primarily studied in non-neuronal origin cells. it has been shown that stat activated cellular transformation, occurs through transcription regulation of specific genes like c-myc, cyclin d , bcl . the present study was undertaken to identify target genes of jak/stat pathway in response to tnf-alpha & igf- in neuronal origin cells. we also looked at the mechanism of activation of these target genes. we saw that jak/stat pathway was involved in upregulation of cyclind in response to igf- both at protein & rna levels but no change was observed in c-myc, bcl levels. interestingly, mapk-erk, which is also known to activate cd in other systems, had no effect on the cd level in neuronal cells. on the other hand, pi kinase not only activated jak/stat pathway but also affected on cyclind level. thus it appears that jak/stat is activated by pi k, which in turn upregulates cyclind level. pi k also activated erk without changing cd level, thus we show that jak/stat pathway is important for survival of neurons, activated by pi k. cd is a one of the major downstream targets of jak/stat. hiroko inoue , haruhisa kawasaki , ritsuko fujii , tohru yoshioka , kazunori sango , toshihiko kadoya , hidenori horie sch. of sci. and eng., waseda univ., tokyo, japan; advanced res. center for biol. sci., waseda univ., tokyo, japan; tokyo metropolitan inst. for neurosci., tokyo, japan; pharmaceut. res. lab. kirin brewery co., ltd., gumma, japan oxidized galectin- (gal- /ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. but, the mechanism how gal- /ox stimulates macrophages remains to be clarified. in the present study with rt-pcr and western blotting, we searched for the molecules contributing to the mechanism. rt-pcr analysis with cultured rat peritoneal macrophages revealed that the application of gal- /ox enhanced mrna expression of nerve regeneration-related factors such as il- , il- ␤. however, those of gdnf, ngf, bdnf, nt- , nt- , igf-i, and igf-ii scarcely detected in control condition were not changed by the application of gal- /ox. since il - , il- , and lif can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with gal- /ox may enhance nerve regeneration after nerve injury. kennichi katou, taku iwamoto, satoshi kida dep. of bioscience, tokyo univ. of agriculture, japan ␣camkii has been known to play essential roles in synaptic plasticity. in response to increase in ca + concentration, ␣camkii is activated by the interaction with ca + /cam. prolonged increase in ca + level leads to further activation of ␣camkii through autophosphorylation at t . to understand the molecular dynamics of ␣camkii activation in vivo, we have tried to visualize and monitor the ␣camkii activity in various types of cells using fret. in hela cells, increase in ca + level induces continuous interaction between ␣camkii and cam and conformational change of ␣camkii, indicating that the conformational change of ␣camkii mainly reflects the interaction with cam. in sh-sy y cells, the increase in ca + level induces only transient interaction between ␣camkii and cam but the conformational change of ␣camkii is maintained following the termination of the interaction with cam, suggesting that conformational change of ␣camkii is induced by the interaction with cam and then maintained by autophosphorylation. thus, mechanisms for regulation of ␣camkii activation is cell-type dependent. akifumi kamata, yusuke takeuchi, kohji fukunaga dept. pharmacol., grad. sch. pharmaceu. sci., tohoku univ., sendai, japan ca + /calmodulin-dependent protein kinase ii (camkii) is highly expressed in brain including dopaminergic neurons, however, the role of camkii in the dopaminergic neurons remains to be unclear. camkii consist of four isoforms, ␣, ␤, ␥ and ␦, and differential isoforms are implicated in the different neural functions. we have examined the isoforms of camkii expressed in rat substantia nigra (sn). rt-pcr and immunoblot analyses revealed that the ␥ and ␦ isoform mrnas including ␦ isoform, a nuclear isoform of camkii, were predominantly expressed in sn. the immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the sn dopaminergic neurons, and immunoreactivity against anti-camkii␦ - antibody was detected in both nucleus and cytoplasm. furthermore, we defined expression of bdnf mrnas having the exon ii and iv in sn. taken together with our previous observation, the camkii␦ isoform was involved in the expression of bdnf in sn dopaminergic neurons. ps p-d the role of ca + /calmodulin-dependent protein kinase ii in neuronal functions revealed by inactivated camkii␣ knock-in mouse yoko yamagata , , hiroyuki sakagami , keiji imoto , , kunihiko obata , yuchio yanagawa okazaki, japan; sokendai, okazaki, japan; tohoku univ., sendai, japan; riken, wako, japan; gunma univ., maebashi, japan ca + /calmodulin-dependent protein kinase ii (camkii) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. we engineered knock-in mice with the inactivated ␣ subunit of camkii by replacing k with r to study functional significance of camkii activity in intact animals. as expected, camkii activity was selectively reduced, while camkii subunit protein levels were comparable to those of wild type controls in homozygous mutants. in situ hybridization revealed normal expression patterns of camkii subunit mrnas, including dendritic localization of camkii␣ mrna. further analyses of these mice will be presented in the meeting. research funds: a grant-in-aid for scientific research from japan society for the promotion of science (kakenhi , # ) ps p-d characterization of ca + -dependent membrane binding proteins of rat brain shohei maekawa , katsutoshi taguchi , haruko kumanogoh , shun nakamura division of biosystems science, grad. sch. of sci/tech, kobe-u kobe - , japan; division of biochemistry and cellular biology, national inst. neurosci. kodaira, tokyo - , japan ca + -dependent membrane binding proteins were purified through a ca + -dependent membrane binding and egta-induced membrane dissociation protocol from a crude triton solubilized membrane fraction. molecular characterization of these ca + -dependent membrane binding proteins through lc-ms/ms identified annexin vii, annexin xi, and copin isoforms, in addition to annexin vi and neurocalcin␣. since the membrane fraction contains phosphatidylethanolamine (pe), cholesterol, and phosphatidylcholine (pc), the membrane binding activity of annexin vi, a major protein in this fraction, was studied using artificial liposomes. annexin vi showed a pe-dependent, but not cholesterol-dependent liposome binding activity. immunostaining of annexin vi in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. these results suggest the participation of annexin vi in the membrane cycling at the presynaptic region. ps p-d generation of dominant negative mutants of transcription factor crest takuzou simo, kenichi kato, hiroshi hosoda, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan transcription factor calcium-responsive transactivator (crest) has been shown to be required for dendritic growth of neuron. previous studies have shown that crest contains two functional domains; multifunctional domain (mfd; aa ∼ ) required for transactivation, nuclear localization and homodimerization and c-terminal transactivation domain (ctd; aa ∼ ). to further understand the function of crest on brain function, we tried to generate the dominant negative mutant of crest to use mouse genetics study. indeed, we generated fusion proteins of gal -dbd with mutants of crest; crest c ( ∼ ) lacking c-terminal transactivation domain, mfd and mfd-nls that is a fusion protein of mfd with nuclear localization signal (nls). from the experiments using gal one hybrid system, we found that mfd-nls shows the strong dominant negative effects on transcriptional activation by crest. ps p-d characterization of iq-arfgef, a novel exchange factor for arf , in mouse brain hiroyuki sakagami, hisatake kondo department of cell biology, tohoku university graduate school of medicine, sendai, japan adp-ribosylation factor (arf ) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. to understand its neuronal functions, we have focused on guanine nucleotide exchange factors (gef) for arf . in this study, we determined the entire sequence of a partially identified cdna (mkiaa ) encoding a novel sec domain. the deduced protein contained coiled coil, iq, sec , plekstrin homology domains. interestingly, it contained a type i pdz domain-binding motif at its cterminal tail. arf pull-down assay demonstrated its ability to active arf more selectively than arf . thus, we named this protein as iq-arfgef. in situ hybridization analysis demonstrated the preferential expression of iq-arfgef in the forebrain and cerebellar granule cells. furthermore, iq-arfgef mrna was localized not only in the hippocampal neuronal layers but also in their dendritic fields. our present findings suggested that iq-arfgef may play important roles in dendrites through activation of arf and interaction with various pdz proteins. eriko miura , masahiro fukaya , tokiharu sato , kazushi sugihara , masahide asano , katsuji yoshioka , masahiko watanabe dept. anat., hokkaido univ. sch. med., sapporo, japan; dept. mol. cell. biol., cancer res. inst., kanazawa univ., kanazawa, japan; adv. sci. res. center, kanazawa univ., kanazawa, japan the c-jun n-terminal kinase (jnk) is one of the major mitogenactivated protein kinases (mapks), and jsap (or jip ) is a jnkassociated scaffold that controls the specificity and efficiency of jnk signaling cascades. here we studied its expression and distribution in mouse brains. jsap mrna was expressed in developing and adult brains, showing spatial patterns similar to jnk ∼ mrnas. in embryos, jsap immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. in adults, jsap was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with ser and cell membrane. this wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to jnk mapk pathway and other intracellular machineries. yasukazu hozumi, kaoru goto department of anatomy and cell biology, yamagata university school of medicine, japan diacylglycerol kinase (dgk) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. to address the functional implications of dgk isozymes in the brain, we have raised specific antibodies against the isozymes. by immunoblot analysis, we found that the immunoreactive bands for dgk␤, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. immunohistochemical examination on rat brain revealed that dgk␤ was detected as dot-like structure in the cell membrane, and dgk␥ as round-shaped structure in the juxtanuclear region of neurons. dgk was detected in the perinuclear region and in the dendrites of purkinje cells. these data show that dgk isozymes localize differentially to the internal membrane system in neurons, suggesting that dgk isozymes play unique roles in distinct internal membrane system. kotaro hattori , shigeo uchino , tomoko isosaka , , shinichi kohsaka , takeshi yagi , shigeki yuasa dept. ultrastractural res., nat. inst. neurosci., ncnp, kodaira, japan; dep. neurochem., nat. inst. neurosci., ncnp, kodaira, japan; kokoro biology group, fbs, osaka univ., suita, japan recently, we have found that fyn-mediated tyrosine-phosphorylation of nmda-r subunits is required for haloperidol-induced catalepsy. haloperidol induced catalepsy in the control mice, but such response was significantly reduced in fyn-deficient mice. fyn activation and enhanced tyrosine-phosphorylation of the nmda-r nr b subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in fyn-deficient mice. both nr b phosphorylation and the nmda-induced calcium responses increased after dopamine d -r blockade in cultured striatal neurons of the control, but not in fyn-deficient neurons. accordingly, d -r antagonist activates striatal fyn and the subsequent tyrosine phosphorylation of nr b alters striatal neuronal activity, thereby inducing catalepsy. we also report further proteomics analysis on this molecular pathway. hiroaki okuda, shingo miyata, tsuya taneda, atsushi yamaguchi, shinsuke matsuzaki, natsuko kumamoto, masaya tohyama department of anatomy & neuroscience, graduate school of medicine, osaka university, osaka, japan the schizophrenia is a well-known debilitating psychiatric disorder. recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including dysbindin (dtnbp : dystrobrevin binding protein ) at p . . however it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. since, there is an important another factor, an environmental factor, for the schizophrenia. although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the n-terminal region of dys-bindin as a bait. as a result, we detected several transcriptional factors. thus, we further examine that these interactions regulate the expression level of particular genes in the neurons. ryota adachi , naoko oda , ryuzo shingai , st coe program, iwate university, morioka, japan; department of welfare engineering, faculty of engineering, iwate university, morioka, japan the response to environmental stimuli is important to live for organisms. the soil nematode caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. c. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. the sensory neurons detected these stimuli are in the head region. the neural network and genes required in each neuron are well known. however, it is under investigation about the integration of chemical and thermal stimuli. to understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. wild type worms cultivated at • c showed decreased chemotaxis response to water-soluble chemicals and alcohol. alteration of assay temperature did not affect response to cl − but that to na + . to discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay. takashi takekawa , masaki nomura , , toshio aoyagi , tomoki fukai riken brain science institute, saitama, japan; graduate school of informatics, kyoto university, kyoto, japan; crest, japan science and technology agency, kawaguchi, japan the neuron model proposed by izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. however, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. in fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. here, we study the phase responses of the izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks. honghai cui, hirotaka sakamoto, mitsuhiro kawata department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan the amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. in the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (sps) paradigm as ptsd (post-traumatic stress disorder) model. rats were perfused days after sps, and intracellular injections of lucifer yellow were carried out in neurons of central (cea) and basolateral amygdala (bla). at the cellular level, we observed a significant increase of dendritic arborization in bla pyramidal neurons but no effect on the cea neurons. these results suggest that sps-induced structural plasticity of the bla provides a cellular substrate for affective disorders that are triggered in ptsd. takahiko kawasaki , , tatsumi hirata division of brain function, national institute of genetics, sok-endai, mishima, japan; prest, kawaguchi, japan in macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (mob) and the accessory olfactory bulb (aob), respectively. the secondary projections from the mob and aob first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. therefore, we investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of mob and aob neurons. ps p-d an essential role for click-iii/camki␥ in regulation of neurite extension natsumi ishihara, sayaka takemoto-kimura, hiroyuki okuno, haruhiko bito dept. of neurochem., univ. of tokyo, tokyo, japan click-iii/camki␥, a membrane-anchored camk, is a novel member of the camki family that acts downstream of camkk. in order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged click-iii was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. phenotypic studies in pc- cells expressing various mutants of camkk and click-iii revealed that depolarization-induced activation of camkk-click-iii signaling is sufficient to robustly induce neuritogenesis. interestingly, click-iii localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. furthermore, lipidification mutants of click-iii were impaired in their ability to facilitate activity-dependent neuritogenesis. our results thus indicate a novel role of click-iii in activity-dependent neurite formation and extension. takuro maruyama , masahiro matsuura , nobuhiko yamamoto grad. sch. of frontier biosci., osaka univ., suita japan; riso kagaku corp., tsukuba japan during development, thalamocortical (tc) axons invade into the cortex and stop growing in layer . to study tc axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. the dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered fc-tagged extracellular domains of several candidate molecules. after four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-a , sema a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. a tendency for tc axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. these results suggest that ephrin-a , sema a and kit ligand may contribute to regulating tc axonal growth in the developing cortex. research funds: kakenhi , kakenhi ps p-e expression pattern of the guidance molecules and their receptors during gnrh neuron migration from the nose to the ventral forebrain shizuko murakami , katsuhiko ono dept. anat., juntendo univ. sch. of med., tokyo, japan; div. of neurobiol. and bioinfo., nat. ins. for physiol. sci., okazaki, japan using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of gnrh neurons of chick embryos. netrin- expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. a few gnrh neurons expressed netrin- receptor neogenin. slit- was expressed in the forebrain, but its receptor robo- was not expressed in gnrh neurons. semaphorin (sema) a expression was detected in the olfactory-forebrain region. within the forebrain, sema a was absent in the restricted region of the medial forebrain where gnrh neurons migrated in association with a subset of olfactory fibers. at the end of this pathway, sema a expression was observed in the caudal forebrain, and then gnrh neurons changed their course ventrally. many gnrh neurons expressed sema a receptor neuropilin- , suggesting that sema a may act as a repellent during the migration of gnrh neurons. kazuto fujishima , , qing-hua jin , junko kurisu , tomoo hirano , mineko kengaku riken, bsi, lab. for neural cell polarity, saitama, japan; department of biophysics, graduate school of science, kyoto university, kyoto, japan during development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. dendrite morphology is also regulated by cell-cell contacts with neighboring cells. recent studies have implicated notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. we previously showed that delta/notch-like egf-related receptor (dner), neuron-specific transmembrane protein, acts as a notch ligand and mediates neuron-glia interaction during cerebellar development (eiraku et al., ) . dner is expressed in the dendrite of developing cortical pyramidal neurons. here, we analyzed the role of dner in the development of pyramidal neuron dendrites. we quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and dner knock out mice. reelin signal controls neuronal migration in the developing brain. because the binding of reelin to apoer /vldlr induces dab phosphorylation, dab is a critical component of reelin signal. recent studies suggested that reelin signal is involved not only in neuronal migration but in other aspects of neural development. we investigated a possible function of the reelin signal in the filopodia and neurite formation. administration of reelin protein increased number of filopodia in a lesser extent than bdnf treatment. pp suppressed effects of reelin and bdnf on filopodia formation, suggesting that both signal pathways involve src kinase function. in spite of these similarities of reelin and bdnf functions in the filopodia formation, bdnf did not phosphorylate dab . reelin treatment of the neurons derived from dab deficient mice did not induce significant increase in the number of filopodia. these results suggest that the filopodia and neurite formation involves the reelin-dab signal pathways. hidenobu mizuno , , tomoo hirano , , yoshiaki tagawa , dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan; crest, jst, kawaguchi, japan the left and right hemispheres are interconnected via the corpus callosum. in mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area / border of the other hemisphere, where they terminate in layers / and . we used in utero electroporation-mediated gene transfer of gfp to assess distribution of callosal projections during development. gfp-labeled callosal axons arrived at the / border and started to invade the cortical plate around p . they generated exuberant axonal arborizations by p then underwent remodeling to form the adult pattern by p . neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. exogenous expression of a potassium channel kir . in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers / . area-specific targeting to the / border was not affected. results suggest that certain stages of callosal axon development require neuronal activity in cortex. ps p-e identification of a novel tetraspan membrane protein, nplp, that is up-regulated after the facial nerve axotomy chihiro akazawa, yoh sasaki, masato hoshi, yasuko nakamura, shinichi kohsaka department of neurochemistry, national institute of neuroscience, ncnp, japan facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. to identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cdnas expressed in the injured facial nucleus. we identified a novel putative tetraspan protein designated as neuronal pmp like protein (nplp) that has a distant homology to pmp /claudin family proteins. the mrna expression of nplp was detected on the cell bodies of neurons meanwhile mrna of pmp resided on the cell bodies of schwann cells. the nplp mrna level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. nplp has a large extracellular domain that may interact with other membrane molecules. overexpression of nplp showed the microspike formation in pc cells. our results suggest that nplp plays an important role in the regeneration of injured motor neurons. shinsuke shibata , shin-ichi sakakibara , hirotaka j okano , hideyuki okano , dept. physiol., keio univ., sch. med., japan; dept. histol. neurobiol., dokkyo med. univ., japan ; crest-jst, japan musashi (msi) is an evolutionarily conserved rna-binding protein. drosophila-msi regulates asymmetric cell division in neural development. in mammalian cns we identified two members, msi and msi , which are co-expressed in neural precursor cells including neural stem cells, suggesting msi proteins regulate the immaturity of cns stem cells. to reveal the roles of msi family in vivo, we generated msi and msi deficient mice. msi −/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. msi −/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of pns. msi −/− msi −/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. in vitro and in vivo analysis show msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian cns and pns development. youhei koshimizu , satoshi yamagoe , kazuo suzuki , michiko ohtomi department of biomolecular science, graduate school of science, toho university, chiba, japan; department of bioactive molecules, national institute of infections diseases, tokyo, japan we have previously suggested that lect (leukocyte cell-derived chemotaxin ) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. however, the mechanisms of these regulations have not yet been clarified. in this study, we carried out multi-immunostaining against lect and tau or map in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of lect in neurons. at the early stages in cultured neurons, lect was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. in tissues of adult mouse brain, lect was localized to soma, axons and dendrites in neurons. these results suggest the possibility that lect influences the extension of dendrites and axons from the early stage of neuritic development. furthermore, it is thought that lect may regulate the dendritic and axonal morphology in mature neurons. mitsuru noda , , takahiro moriya , hideyuki terazono , suguru kudo , masahiro yamaguchi , kenji yasuda , izumi nagata , kazuyuki shinohara div. neurobiol. & behav., nagasaki univ. grad. sch. biomed. sci., nagasaki, japan; dpt. neurosurg. radiol. nagasaki univ. nagasaki, japan; dpt. life sci., grad. sch. arts & sci., univ. tokyo, tokyo, japan; dpt. physiol., grad. sch. med., univ. tokyo, tokyo, japan; special division for human life technology, national institute of advanced industrial science and technology (aist), japan neural stem cells (nscs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of bdnf. the individual nscs from e . nestin-promoter gfp transgenic mice were placed into pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of bdnf. we observed that a part of nscs exhibited neuron-like morphology and extended some neurites. the application of bdnf increased the rate of neurite outgrowth. thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of bdnf. ayumi kyuka, ryoichi yoshimura, yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan in order to investigate the relationship between neurite outgrowth and target cells, ng cells were co-cultured with vascular smooth muscle cells (sm- ). sm- cells promoted the neurite outgrowth, but repelled the contact of their growth cones. the dna microarray analysis indicated that neuropilin- mrna was specifically up regulated in ng cells. but the western blotting analysis did not show the significant increase of neuropilin- . sema a, a candidate of ligands for neuropilin- , was found in ng cells rather than in sm- cells. the immunocytochemical analysis revealed that neuropilin- was increased in the surface of ng cells co-cultured with sm- cells or cultured with the conditioned medium of sm- cells. these results suggest that neuropilin- may be transferred from cytoplasmic pool to surface of ng cells by some soluble factors released by sm- cells. we will discuss about kinetics of neuropilin and chemorepellents. yoshikuni edagawa , j. nakanishi , h. hamada , y. yokomachi , k. yamaguchi , n. takeda inst. biomed. eng., waseda univ., tokyo, japan; riken, japan; kanagawa univ., japan we show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of uv light. this molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. for the neurite outgrowth, pc cell was recruited as a model of neuron. by irradiating uv light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ecm. to draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. ngf stimulated each pc cell to extend a neurite along the narrow path: the elongation required both photo-activated path and ngf. the successive irradiation in front of developing neurite achieved bending or branching of the neurite. these results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system. tetsuhiro kakimoto, hironori katoh, manabu negishi lab. of molecular neurobiology, grad. sch. of biostudies, kyoto univ., kyoto, japan n-wasp is important for actin polymerization and is strongly expressed in brain. toca- was shown to be required for cdc to activate n-wasp in vitro, although its physiological role is unknown. here we studied neural function of toca- . toca- is strongly expressed in developing brain. knockdown of toca- in pc cells significantly enhances neurite elongation. consistently, overexpression of toca- suppresses neurite elongation through its amino terminus including the f-bar/efc domain, which induces plasma membrane invagination. in addition, knockdown of n-wasp also enhances neurite elongation in pc cells, in clear contrast to the previous report using dominant negative mutants. on the other hand, knockdown of toca- in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of n-wasp enhances both axon elongation and branching. these results suggest that a vesicle trafficking regulating protein toca- regulates different aspects of neuronal morphology from n-wasp. research funds: kakenhi ( ) ps p-e pragmin, a novel effector of rnd gtpase, stimulates rhoa activity and regulates neurite outgrowth hironori katoh, hiroko tanaka, manabu negishi kyoto university, japan the rho family small gtpase rnd is specifically expressed in brain. although rnd and rnd display an antagonistic action for rhoa, signaling pathways of rnd are not well known. here we have performed a yeast two-hybrid screen using rnd as bait and identified a novel rnd -effector protein, pragmin (pragma of rnd ). pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. in in vitro and in vivo binding assays, pragmin specifically bound to rnd among the rho family gtpases in a gtp-dependent manner. rnd -bound pragmin significantly stimulated rhoa activity and induced cell contraction through rhoa-and rho-kinase-dependent pathway in hela cells. in pc cells, expression of pragmin inhibited the ngf-induced neurite outgrowth in response to rnd , and knockdown of pragmin by a pragmin-specific small interfering rna enhanced the neurite elongation. therefore, these results suggest that rnd controls neurite outgrowth by regulating rhoa activity through pragmin. ps p-e regulation of growth cone motility by collapsin response mediator protein- (crmp- ) masumi iketani , yanai shigeki , fumio nakamura , yoshio goshima , , kohtaro takei , dept. of mol. pharmacol. & neurobiol., yokohama city univ. grad. sch. of med., yokohama, japan; crest, japan sci. & tech. agency, kawaguchi, japan nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. collapsin response mediator protein- (crmp- ) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. the local functions of crmp- within growth cones, however, remains unclear. chromophoreassisted laser inactivation (cali) that can inactivate selectively a target protein with high spatial and temporal resolutions. acute localized loss of function of crmp- in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. asymmetric inactivation of crmp- in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. these findings suggest that a spatial distribution of crmp- activity within the growth cone can regulate growth cone motility and direct neurite outgrowth. research funds: crest, jst ps p-e eph receptors are negatively controlled by protein tyrosine phosphatase receptor type o takafumi shintani , , masaru ihara , , hiraki sakuta , , hiroo takahashi , , ikuko watakabe , masaharu noda , division of molecular neurobiology, national institute for basic biology, okazaki, japan; crest, jst, kawaguchi, japan eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (ptps) responsible for the negative regulation of eph receptors have not been elucidated. here, we identified protein tyrosine phosphatase receptor type o (ptpro) as a specific ptp that efficiently dephosphorylates both epha and ephb receptors as substrates. using the retinotectal projection system, we show that ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections. ps p-e neogenin acts as a displacement factor that releases rho from rho-gdi katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan repulsive gudiance molecule (rgma) is a potent inhibitor of axon regeneration in the adult central nervous system. rgma inhibits mammalian cns neurite outgrowth by a mechanism dependent on activation of the rho/rho-kinase pathway. here we show that direct interaction of the rho gdp dissociation inhibitor (rho-gdi) with neogenin which is a receptor of rgma initiates the activation of rhoa, and this interaction between neogenin and rho-gdi is strengthened by rgma. we also found that neogenin facilitates the release of prenylated rhoa from rho-gdi. thus, understanding of the molecular mechanisms of rgma may be useful for developing methods to overcome the inhibitory signals. nobuyuki fukushima, masumi nakashima, ryou tokunaga, ryutaro moriyama department of life science, kinki university, higashiosaka, japan lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through g protein-coupled lpa receptors, including lpa and lpa . here we examine how lpa , third lpa receptor subtype, is involved in neuronal development by using pheochromocytoma (pc ) cells. pc cells expressed low levels of endogenous lpa mrna. when pc cells were infected with retrovirus expressing lpa and treated with nerve growth factor under serum-free conditions for days, neurite formation was observed in % of total infected cells, which was similar to that in control virus-infected pc cells. however, the mean length of neurites was increased in pc cells expressing lpa , compared with that in control pc cells. interestingly, multiple neurite sprouts were frequently observed in lpa-treated lpa -expressing cells. indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. these results indicated that lpa was involved in neuritogenesis and branch formation. masao sakurai , tomoko aoki , kyoko ishikawa , shingo yoshikawa , john b. thomas , chihiro hama cdb, riken, hyogo, japan; salk institute, usa in drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (al). each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (orns) and projection neurons (pns). in an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified wnt as a regulatory factor for glomerular patterning. in the wnt mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by wnt expression in orns. previous studies in embryos have shown that wnt repels the axons expressing derailed (drl). in the als of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the wnt mutant. this and other genetic data suggest that drl is an antagonist for wnt signaling. we propose that wnt signal transmitted from orns to pns provide one of mechanisms for glomerular patterning in drosophila. ps p-e a homeodomain transcription factor, homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the drosophila brain mai ando, yoko totani, katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan the precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. we are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. marcm mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. currently, we are looking for genes that are either downstream or work cooperatively with hth. based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons. ps p-e differential microarray analysis of mushroom body transcripts using chemical ablation ayako ino , masatomo kobayasi , taiki nakajima , lydia michaut , indrayani ghangrekar , kuniaki takahashi , ryu ueda , kaoru saigo , walter j. gehring , katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan; biozentrum, univ. of basel, switzerland; genet. strains res. ctr., natl. inst. genet.; dept. biophys. biochem. grad. sch. sci., univ. tokyo, japan mushroom bodies (mbs) are the centers for olfactory associative learning in the drosophila brain. we have surveyed mb transcripts using a drosophila whole-genome microarray and identified , genes that exhibited reduced expression levels with pharmacological mb ablation. among the identified genes, we have further selected genes that exhibited a most consistent reduction of expression levels in the ablated brains. in situ hybridization analyses confirmed preferential mb expression patterns for the majority of the identified genes. moreover, we also demonstrate that rnai of many of the identified genes causes mild to strong mb defects. these results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the mb neurons in brain development and plasticity. mikiko inaki , yoshie suzuki , hiroyuki aburatani , akinao nose dept. phys., univ. tokyo, tokyo, japan; rcast, univ. tokyo, tokyo, japan in drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. to understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. we focused on two neighboring muscles, and , which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. by comparing gene expression data from the two muscles, we identified ∼ genes with more than two-fold difference in expression level. for candidate genes out of ( %), the differential expression was confirmed by in situ hybridization and/or quantitative pcr. we focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. ectopic expression and/or knockdown of some of them altered the target specificity of muscles or , implicating them in neuronal target recognition. makoto aoki, hiroshi segawa, mayumi naito, hitoshi okamoto laboratory for developmental gene regulation, brain science institute, riken, saitama, japan the sensory neurons have two axons, the central and peripheral axon. previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the lim domains of islet- . to find the molecules which regulate the formation of peripheral axons of sensory neurons in the islet- signaling cascade, we have created cdna library from trigeminal sensory neurons of zebrafish larvae. most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. most of them show cns or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the lim domains of islet- . loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry. hiroshi yamamoto, kiyokazu agata development of biophysics, graduate school of science, kyoto university, kyoto, japan highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. they act through receptors of the deleted in colorecal cancer (dcc) and unc receptor families. freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. the planarian central nerve system (cns) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. we have identified eight netrin related genes from planarians, dugesiajaponica, including four different netrin homologues and three unc homologuesand one dcc homologue. we analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted rnai experiments to investigating their functions in the process of regeneration of visual neurons. here we will summarize these results and speculate their molecular actions during eye regeneration in planarians. ps p-e analysis of the significance and mechanism of neurite elimination using c. elegans as a model yu hayashi , takaaki hirotsu , eriko kage , hideaki takeuchi , hirofumi kunitomo , yuichi iino , takeo kubo dept. biol. sci., grad. sch. sci., univ. tokyo, tokyo, japan; dept. biol., fac. sci., kyushu univ. grad. sch., japan; mol. genet. res. lab., univ. tokyo, japan during brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. we previously showed that elimination of neurites occurs in the nematode c. elegans and that a novel transcription factor mbr- is involved in this process. in the present study, we attempted to clarify the significance of this phenomenon. the mbr- mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. by executing cell-specific rescue experiments, we suggest that mbr- functions in a set of interneurons involved in olfactory processing. we are now examining whether neurite elimination occurs in these neurons using gfp reporters. we expect that our research provides a useful model for genetic dissection of neurite elimination. taro asakura , ken-ichi ogura , yoshio goshima , dept. of mol. pharmacol. and neurobiol., yokohama city univ., grad. sch. of med., yokohama, japan; crest, japan sci. and tech. agency, kawaguchi, japan netrin is an evolutionary conserved axon guidance molecule. in c. elegans, netrin/unc- is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. one of the neurons guided by netrin/unc- is the hsn neuron. the hsn neuron of c. elegans has interesting axon guidance. netrin/unc- is required for the first ventral growth of the hsn neurons. however, what molecules participate in the axon guidance is largely unknown. in this study, we found that the vulval precursor cells strongly expressed netrin/unc- during the axonal guidance of the hsn neurons. silencing of netrin/unc- only at the vulval precursor cells resulted in abnormal axon guidance of hsn. in addition, expression of netrin/unc- only at the vulval precursor cells in unc- null mutants partially restored the hsn guidance defects. these results suggest that netrin/unc- expressed in vulval precursor cells plays essential roles on axon guidance of the hsn neuron. tomomi sato, takanori hamaoka, hidenori aizawa, hitoshi okamoto brain science institute, riken, saitama, japan the optic tectum is a visual center in vertebrates. it receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. here, we established a stable transgenic line tg(brn a-hsp :gfp) in zebrafish to visualize the retinotectal and the tectobulbar projections. to explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxp and gal /uas systems. using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. in addition, we showed that ephrinb a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere . these results suggest a neuroanatomical and a molecular basis for the motor map in the tectum. research funds: grant-in-aid for young scientists (b) masahiko taniguchi , yoshinori mikami , tomoyuki masuda , tomoyuki yoshida , naoto matsuda , masayoshi mishina , takao shimizu department of biochemistry, cancer research institute, sapporo medical university, sapporo, japan; department of molecular neurobiology and pharmacology, graduate school of medicine, university of tokyo, japan; department of anatomy, fukushima medical university, japan; dapartment of biochemistry and molecular biology, faculty of medicine, university of tokyo, japan semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. here we report the identification of zebrafish semaphorin d (sema d). the orf of sema d is bp ( aa). zebrafish sema d protein showed a . % identity with mouse sema d protein. to clarify the expression pattern of sema d, in situ hybridization was performed. in the embryos, sema d is expressed in the lens and hindbrain. we also found that sema d has the repulsive activity. these results indicate that sema d repels specific types of the axons and functions in the nervous system development. research funds: kakenhi on priority areas from the mext ( ) mayumi yamada , shohei maekawa , seiji miyata department of applied biology, kyoto institute of technology, kyoto, japan; division of bioscience, graduate school of science and technology, kobe university, kobe, japan obcam belongs to the immunoglobulin superfamily cell adhesion molecule (cam) and was synapse-specific cam in cortical and hippocampal neurons in vivo. however, it is uncertain how obcam is involved in synaptic functions. in this study, we showed that obcam was highly concentrated on spines of cultured mature neurons. the inhibition of obcam function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. the suppression of obcam mrna expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. in contrast, overexpression of obcam augmented the formation of synapses. moreover, obcam was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. these results suggest that obcam, a spine-specific cam, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity. ps p-f semaphorin d inhibits ␤ integrin activity through the r-rasgap activity of plexin-b izumi oinuma, hironori katoh, manabu negishi lab. mol. neurobiol., grad. sch. biost., kyoto univ., kyoto, japan plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. we have recently reported that the semaphorin d (sema d) receptor, plexin-b functions as a gap for r-ras, a member of ras family gtpases implicated in regulation of integrin activity and cell migration. here we characterized the role of r-ras downstream of sema d/plexin-b in cell migration. activation of plexin-b by sema d suppressed the ecm-dependent r-ras activation, r-ras-mediated pi -k activation, and ␤ integrin activation through its r-ras gap domain, leading to inhibition of cell migration. in addition, inactivation of r-ras by overexpression of the r-ras-specific gap or knockdown of r-ras by rna interference was sufficient for suppressing ␤ integrin activation and cell migration in response to the ecm stimulation. thus, we conclude that r-ras activity is critical for ecm-mediated ␤ integrin activation and cell migration, and that inactivation of r-ras by sema d/plexin-b -mediated r-ras gap activity control cell migration by modulating the activity of ␤ integrins. shun hamada , emi fukuda , , shota katori , , takeshi yagi , dept. nutrition health sci., fukuoka women's univ., fukuoka, japan; grad. sch. frontier biosci., osaka univ., osaka japan, crest, jst, japan cnr/protocadherin (pcdh)␣ is a subfamily of the clustered pcdhs that comprise > cadherin-related molecules and are encoded by three gene clusters (␣, ␤ and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga is expressed by layer / neurons throughout the neocortex at p mice, but is absent in adults. between e . and late p , stages that span the generation and radial migration of layer / neurons, mdga is expressed in patterns consistent with its expression by migrating layer / neurons, suggesting a role for mdga in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e . to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day , but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps p-f analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono , yoshiyuki kuroda , fumiko matsui , yoshihito tokita , keiko nakanishi , michiru ida , masahito ikawa , masaru okabe , katsuhiko ono , atsuhiko oohira institute for developmental research, aichi human service ctr., kasugai, japan; research institute for microbial diseases, osaka university, suita, japan; national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi ( ) ps p-f phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata , tatsuhisa watanabe , ikuko suzuki , junichi kitagawa , akiko ogawa , kenro kanda , kazunao kuramoto dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats ( - months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura , koichi obata , masafumi sakagami , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk , mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days and after cfa injection. furthermore, time course of phosphorylated-erk level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk in drg neurons may contribute to the development of inflammatory pain. ps p-f activation of erk in drg neurons contributes to acute pain toshiyuki mizushima , , koichi obata , takashi mashimo , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p in dorsal root ganglion (drg) neurons. however, there has been no study examining erk (also known as big mapk or bmk ) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk in small-to-medium diameter sensory neurons with a peak at min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and spinal cord and participate in pain-related behaviors in the l spinal nerve ligation (snl) model. l snl induced an increase in the phosphorylation of erk not only in the injured l drg, but also in the spared l drg at day after surgery. furthermore, l snl induced a striking increase in erk phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai , minoru asada , naoki seno , hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan; pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣- , which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps p-f interleukin- ␤ enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda , jun kadoi , msanori nasu , masayuki takahashi , shigeji matsumoto dep. physiol and res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il- ␤ paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il- ␤-immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il- ␤ induced depolarization in inflamed rats was larger than that in control rats. il- ␤ application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il- ␤ paracrine mechanism following peripheral inflammation. junichi kitagawa , mamoru takeda , jun kadoi , yoshiyuki tsuboi , shigeji matsumoto , koichi iwata dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day after ion treatment and the threshold decrement was lasting more than day . single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day - after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for min. at , , , and h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at h, peaked at h and then decreased. intense gfap immunoreactivities were also detected at and h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for min at m/min. after the running, rats were applied the weight on the gastrocnemius muscles for min, and then spinal cord and soleus muscles were removed from the rats. productions of il- beta, il- and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at h post-running, number of fos-positive neurons was increased, peaked at h and then decreased to control level at h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo , yusuke sakai , aya masawaki , takashi shimoda , masayuki moritani , motohide takemura dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep receptor (ep r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca + ] i ) in a ptx-sensitive manner in cho cells expressing canine ep r and mouse bk b receptor (b r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk ( nm) was applied twice with a -min interval to the cells expressing b r, the second [ca + ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h- ( m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk ( pm) treatment for h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca + ] i was at least in part mediated by pka. ps p-f contribution of peripheral ht a or ht receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral ht a or ht /r in male rats with tmj inflammation. here we tested if these two ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c . fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for days compared to naive rats. we tested if local ht a or ht /r antagonist affects fos expression in both groups. the number of fos-li in the vc/c but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral ht a and ht /rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of -ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼ % of sg neurons examined, -ht induced an outward current. the outward current was mimicked and suppressed by a -ht a agonist and -ht a antagonist, respectively. in ∼ % of sg neurons, -ht evoked an inward current which was mimicked by a -ht agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that -ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of -ht a and -ht receptors, respectively. the reciprocal postsynaptic actions of -ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by -ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a␤ fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic of bupivacaine and r-bupivacaine were almost equal on a␤, a␦ and c neurons. however, ic of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a␤ neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps p-g nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi ( ) and km ( ) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about % of neurons examined, melittin ( m) superfused for min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at mv in the presence of bicuculline. this action was visible about min after the beginning of its superfusion and subsided within min after washout. these melittin actions were reduced in extent by a pla inhibitor -bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi ( ) ps p-g presynaptic p y receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied -methylthio adp ( mesadp, m), a p y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about % of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by mesadp with no change in the amplitude. the mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs ( m), a selective p y receptor antagonist. these results indicate that the activation of presynaptic p y receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p y receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi ( ), the japanese health sciences foundation (kh ) ps p-g presynaptic enhancement by proteinaseactivated receptor- agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)- but not par- agonist (each m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par- mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par- agonist (sfllrn, m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at - mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par- antagonist (yfllrnp, m). these results indicate that the activation of par- s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par- activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi ( ) ps p-g effect of tramadol metabolite m on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m , which is one of its metabolites, at mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at − mv. this action was not seen in the presence of a -opioid receptor antagonist ctap ( m). m also reduced the peak amplitudes of epscs which were monosynaptically evoked at − mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk- or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk- injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk- . these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps p-g studies on pain control system (rept. ): changes in phosphorylation of nr b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for weeks. under these conditions, the immunoreactivities of phosphorylated-ser- nr b (p-ser- nr b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr- nr b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps p-g prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, hz, a, s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by -ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps p-g the nav . sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav . , a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav . . four weeks post-sci, nav . protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav . caused a significant reduction in nav . expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda , hiroyuki ichikawa , ryuji terayama , tomosada sugimoto department of oral maxillofacial rehabilitation, okayama university, okayama, japan; department of oral function and anatomy, okayama university, okayama, japan ib -sap is a neurotoxin designed for targeting primary nociceptors with ib binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib -sap ( . l of . % solution for each ganglion) directly into the th and th lumbar (l and ) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib -binding. after counterstain, the cell body size of neurons were measured. ib -sap reduced the total number of drg neurons in l and ganglia combined by % ( ± on untreated side versus ± on treated side). small neurons (< m ) were reduced by % whereas large ones (≥ m ) were not affected. ib -binding neurons were mostly small (≥ %) and were reduced by %. the number of small neurons, that were not stained for ib -binding, increased by % ( ± versus ± ). schuichi koizumi , kaoru nasu-tada , makoto tsuda , emiko kunifusa , , kazuhide inoue div. pharmacol., natl. inst. hlth. sci., tokyo, japan; dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p x receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p x receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p x receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p x receptor in microglia, leading to neuropathic pain. research funds: mf , kakenhi ( ) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a␤-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi ps p-g lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at mm decreased the protein levels of myelin basic protein (mbp) at h. this action was significantly inhibited by botulinum neurotoxin/c (bont/c ). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp , mag, p in cultured fibers. the maximal decrease was observed all at as early as h after the addition of lpa. bont/c and y abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at and , , , , days after operation. pre-emptive co-administration of mk- and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk- decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama , , naomi akiyama , mikie hirata , takayuki nakagawa , shuji kaneko , masabumi minami dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a ␤-adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for h per day between postnatal day and . visceral sensitivities were assessed in stomach and colon at weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research ps p-g change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered to days after exercise in rats ( w old). c-fos expression in the superficial dorsal horn increased in l spinal segment when the edl muscle was compressed days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats , ( - ) and ( - ) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in w old rats, but not significant. after lec, the threshold started to decrease day after lec in all three age groups. it returned to the pre-lec level days after lec in and w old rats only. recovery of w old rats delayed up to days after lec. increased c-fos expression in the superficial dorsal horn was observed in l as well as in l in w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama , masaharu akita kanagawa life-science res., japan; kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day to of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days - of age, they did not show the first estrus before days of age with mean age of . ± . day. in contrast, most of normal mouse igg-treated controls showed the first estrus by days of age with mean age of . ± . day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss- gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days of age (d ), and first estrus on around d . brain tissues were obtained on d , , , and . kiss- mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d to and was kept at a high level thereafter. gpr mrna expression in the medial preoptic area increased significantly from d to . metastin-immunoreactive cells were not found on d but were apparent in the arc-me on d onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito , sei-etsu fujiwara , kenjiro konno , takashi yamaguchi , tetsu nemoto , etsuko kasuya , ryosuke sakumoto anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; grad. sch. sci. & eng., yamagata univ., yonezawa, japan; sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps p-g hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi , teruko uwano , , anh tran , , eturou hori , , taketoshi ono , , hisao nishijo system emotional science, univ. of toyama, japan; integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of hf neurons, responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue , atsu aiba , kaoru inokuchi mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; grad. sch. med., kobe univ., kobe, japan vesl- s/homer- a and vesl- l/homer- c are splicing isoforms encoded by the vesl- gene. vesl proteins bind and regulate mglur / , ip receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl- s is upregulated by tetanic stimulation that elicits l-ltp. vesl- s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl- s gene-targeting mice (ko) and examined whether vesl- s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl- s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta , r. migishima , s. kida , k. inokuchi , mitsubishi kagaku inst. life sci (mitils), japan, tokyo univ. agricul., japan; crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin ␤a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps p-h sex differences in the effects of chronic estrogen treatment on fear conditioning in c bl/ j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c bl/ j mice were implanted with a silastic capsule containing , , or g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps p-h analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps p-h analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps p-h analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor (cb ) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb and lvgccs, we also found that cb is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda , takahiro furuta , kouichi nakamura , , takeshi kaneko , ps p-h difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama , tomo unzai , kouichi nakamura , , sakashi nomura , takeshi kaneko , department of morphological brain science, kyoto university, kyoto, japan; department of physical therapy, kyoto university, kyoto, japan; crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut) , thalamostriatal inputs, were less dense in patch region, whereas vglut -positive corticostriatal inputs were evenly distributed. quantitative analysis revealed % of vglut positive synapses in patch region were formed with spines, whereas % in matrix region were made with dendritic shafts. in contrast, the targets of vglut -positive inputs were mainly spines in both regions. moreover, vglut -positive axospinous synapses in patch region were larger than vglut -positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi ( , , , ( ), ) ps p-h single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai , fumino fujiyama , takeshi kaneko , department of morphological brain science, university of kyoto, kyoto, japan; crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi ( , , , ( ) , ) ps p-h lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui , kayo nishizawa , nobuyuki kai , kazuto kobayashi , dept. of psychology, fukushima univ., japan; dept. mol. genet., fukushima medi. univ., japan; crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of -ohda injections into the nucleus accumbens (acb) in c bl/ j mice. all mice were trained to press the lever for a food pellet using a fixed ratio (fr ) schedule. the mice were injected with ascorbate vehicle or -ohda into the acb, and then tested post-surgically using the fr schedule again. the -ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area , it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area , suggesting different functional roles played by each subdivision of area in spatial memory and behavior. eiichi jodo , yoshiaki suzuki , tadahiro katayama , ken-yo hoshino , yukihiko kayama dep. of physiol., fukushima med. univ., fukushima, japan; dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, %) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about . s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in % out of vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only % out of it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., ) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., ) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., ) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of sessions in phase , although they learned easily the four problems in phases and . our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto , noriyuki higo , munetaka shidara neuroscience research inst., aist, tsukuba, japan; grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d receptor study using antisense cdna showed that d receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d receptor antipeptide antiserum. d receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area of the perirhinal cortex than in area teav. the differential localization between area and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano , natsuko yoshida , kiyohisa natsume kyushu kyoritsu university, fukuoka, japan; kyushu institute of technology, fukuoka, japan; kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f and f was the highest and suggests that the theta rhythm at f and f may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after -month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi ( ) ps p-h neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe , sho yagishita , hideyuki kikyo , department of physiology, the university of tokyo school of medicine, tokyo, japan; department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a . t scanner system was used (te = ms; tr = s; acquisition time = . s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong , motoaki sugiura , yuko sassa , keisuke wakusawa , , kaoru horie , , shigeru sato , , ryuta kawashima , gsics, tohoku university, sendai, japan; niche, tohoku university, japan; miyagi university of education, sendai, japan; department of pediatrics, school of medicine, tohoku university, japan; the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz , christie l. sahley , kenneth j. muller physio. & biophys., univ. of miami, miami, fl, usa; biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps p-h sensory/motor modules regulating the development of peer social relationship mamiko koshiba , , shun nakamura jst, crest, japan; ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata , minoru hattori department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) -s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi -s procedure over days. the -s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the -s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi -s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps p-i the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka , , ritz oshio , , norihiro sadato , , manabu honda , nips, okazaki, japan; jsps, tokyo, japan; nagoya univ., nagoya, japan; ristex, jst, tokyo, japan; sorst, jst, tokyo, japan; ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps p-i sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi ( ) ps p-i selective manipulation of working memory through d and d receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d and d receptors. the computer simulation with this model shows that d receptor activation mainly affect working memory activity itself, while d receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d and d receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or mg/kg mam on the th day of gestation. the activity of male and female offspring was measured for each h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every min interval for h under min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca pyramidal layer and ectopic neuron mass). ps p-i long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur . hamid gholamipour , shirin babri , khameneh saied department of physiology, university of tabriz, tabriz, iran; university of tabriz, iran; university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca region of hippocampus on memory formation. fifty male rats were divided into five groups. ( ) control ( ) sham operation ( ) test ( ) diabetic/saline ( ) diabetic/insulin. groups and were made diabetic by treatment with stz ( mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < . ). compared to sham group diabetic/insulin group showed increased latency (p < . ) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca + /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk␤ is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk␤ null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk␤, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of per , in india. we studied the park polymorphism in north indian population and parkin expression in early parkinson's disease (n = ) and sporadic parkinson's disease (n = ). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon- , and in early pd and exon- and in sporadic pd, heterozygous mutations in exon and in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul , kittisak sripanichkulchai , david finkelstein faculty of medicine, srinakharinwirot university, thailand; faculty of medicine, khon kaen university, thailand; university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c bl/ mice were pretreated with beta-estradiol (e ), injected with mptp on the day and brains were collected on day . e pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps p-i effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki , fumihiko yoshino , masaichi chang-il lee dept. of food sci. and nutrition, showa women's univ., tokyo, japan; clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi ( ) ps p-i recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi , kaori sawada , haruo okado , atsushi nambu , masahiko takada tokyo met. inst. neurosci., fuchu, tokyo, japan; natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype- raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type- ) of raav (raav ) carrying αs cdna (raav -␣s), and a cocktail of raav -␣s and raav carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav -␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with ␤-band local field potentials (␤-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged - s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of . - (i), . - (ii), . - (iii), and . - (iv). the ␤waves were rated with total periods (%) of - hz wavelets in -s sample records. rigidity was re-scored after the surgeries. the vl ␤-waves were rated - % in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about % in group iv. the pvp ␤-waves were - %, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp ␤-waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana , , hirokazu iwamuro , , masahiko takada , atsushi nambu , div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; sokendai; dept. neurosurg., univ. tokyo, tokyo, japan; dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd expression on microglia and also enhanced the mt -mmp expression to shed off cd at the cell surface and degrade surrounding ecm to open the migratory way. a t mutant ␣-synuclein showed greater level of cd shedding and cell migration. extracellular treated ␣-synuclein also increased cd and mt -mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant ( grant ( - ps p-j serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai , hiromasa yamada , yoshinari aimi , ikuko nagatsu department of anatomy, shiga university of medical science, otsu, japan; fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba -and tb distribution in microglia were immunohistochemically investigated at h and days after twice mptp-treatments (one treatment comprised of intraperitoneal injections of mg/kg mptp at h interval) to c bl/ and balb/c at months (mo) interval. the recognizable change of iba -and tb -distibution in microglia of both mice strains was observed even mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps p-j activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada , ryohei hishida , yoko hirata , kenji ono , hiromi suzuki , shin-ichi muramatsu , imaharu nakano , kunihiro tsuchida , toshiharu nagatsu , , makoto sawada school of medicine, fujita health university, aichi, japan; division of neurology, jichi medical university, japan; department of biomolecular science, gifu university, japan; research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps p-j phosphorylation of erp in adult rat brain with neonatal -ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of -ohda at (p ) and days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p , tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the -ohda lesioned rats at p , and it was identified as disulfide-isomerase a precursor (erp ) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi ( or nm, h) was applied to cultures at days in vitro and succeeding changes of da neurons were investigated up to days. more than % of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps p-j specific expression of proapoptotic factor pag on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p -activated gene (pag ), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag in the internal capsule of l-dopatreated and constitutive expression in the smi- -immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps p-j an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang , , yuzuru imai , haruhisa inoue , , ayane kataoka , sachiko iita , nobuyuki nukina , ryosuke takahashi , neurology, university of kyoto, kyoto, japan; bsi, riken, saitama, japan since loss of parkin e activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., ) have found that application of an antisense oligodeoxynucleotide specific for nr have successfully knockdown the expression of nr gene expression in the striatum of -hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr was re-addressed using a small interfering rna (sirna) specific for nr . in pc cells, reductions of nr proteins after a single application of nr sirna were found by western blot experiments. and after one single application of nr sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr immunofluorescence were found in the striatum after the sirna treatments. lai et al., . neurochem. int. , - . research funds: faculty research grant, frg/ - /ii- , hong kong baptist university ps p-j homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy ( . - mol in l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on th day. animals with mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps p-j ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca takahiro seki , takayuki shimahara , naoko adachi , naoaki saito , norio sakai dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type (sca ), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca . however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca + homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki , yo sasaki department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase (nmnat ) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at min, but next increased at h and returned to basal at h after cocaine challenge. actin showed a similar pattern but was maintained at high level at h. both psd- and glur were increased between h and h like actin. by contrast, some proteins such as drebrin were decreased after the peak at h. interestingly, the s proteasome subunit demonstrated a dramatic upregulation at h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps p-k effects of mdma and -meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, , methylenedioxymethamphetamine (mdma) and -methoxy-n,ndiisopropyltryptamine ( -meo-dipt), on sert and dat were examined. as previously reported, mdma ( . - m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, -meo-dipt ( . - nm) evoked an outward current response in sertexpressing cells similarly to that of selective -ht reuptake inhibitors. no current response was observed when -meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that -meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka , nobue kitanaka , tomohiro tatsuta , , yoshio morita , motohiko takemura department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine ( mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of . mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of and mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth ( . mg/kg, i.p.), pretreatment with subchronic clorgyline ( . - mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura , nobue kitanaka , tomohiro tatsuta , , yoshio morita , junichi kitanaka department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline ( . - mg/kg, i.p.) min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum min after drug challenge. these results suggested that the inhibitory effect of lobeline ( . - mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with mg/kg meth and locomotor activity measured daily for days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, nmol ufp- (a nop receptor antagonist) and mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp- strongly suppressed locomotor activity. thus, it was unclear if ufp- suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp- co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine ( - mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine ( . - mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine ( , . and mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine ( mg/kg, i.p.) caused more than % mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine ( mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps p-d regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: ( ) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis (ca ) were increased after fs. the rule of this kind of changing was of region-and time-specific. ( ) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi , , tomoaki shirao dept of neurobiol & behav.; ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only . ± . % of drebrin in the spine is stable, with a turnover time of . ± . min. blockade of ampar by m cnqx reduces the population of stable drebrin ( . ± . %), and has no effect on a turnover time. on the other hand, blockade of nmdar by m ap has no effect on the population of stable drebrin, whereas shortens a turnover time ( . ± . min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu , yoshito saitoh , kaoru inokuchi , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki , , daisuke okada , kaoru inokuchi , , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; yokohama natl. univ. environment information sci., kanagawa, japan; crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto , mitsutoshi setou , , kaoru inokuchi laboratory for molecular gerontology, mitils, japan; laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps a-a an activity that traps vesl- s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl- s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at m, but not nm. because nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps a-a acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosening, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca region in rat hippocampal slices. an application of . dheas for min to the slice augmented instantly the epsp, which was terminated within min. however, even h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosensing, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan to know whether ␤-estradiol (e ) can protect ca neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion ( vo) for min, and the effect of e against this ischemic injury was examined. the electrophysiological properties of ca -ca synapses were examined by a real-time optical recording method days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e ( mg/kg) h before vo was able to protect ca neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt ( mg/kg) produced a similar protective effect, but the estrogen receptor-␤ agonist dpn ( mg/kg) did not. above results suggest that e can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps a-a non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima , mari ogiue-ikeda , yuki oishi , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er␤ hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca , ca , and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er␤ hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps a-a morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki , gen murakami , , tetsuya kimoto , , suguru kawato , college of humanities and sciences, nihon university, tokyo, japan; department of biophysics and life sciences, university of tokyo, tokyo, japan; crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of nm dex induced rapid modulation of the density and morphology of dendritic spines in ca pyramidal neurons within h. the total spine density increased from . spines/m to . spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura , yuji ikegaya , sadao shiosaka division of structural cell biology, naist, nara, japan; laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: st century coe research ps a-a actin organizations within single dendritic spines in ca pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with ␤-actin in rat ca pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of . min and min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds ( . - . um/min) and lengths ( . - . um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori , katsushige sato , kohichi tanaka , hiroko hamazaki mol. neurosci., tmdu, tokyo, japan; physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at or days post-hatching (p or p ), and at p . this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p , but they lose this ability at p . in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps a-b effect of estrogen on hippocampus in male and female mice takanori sugawara , shinji hayashi , victoria luine graduated school of integrated sience, yokohama city university, yokohama, japan; department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received ␤-estradiol (e ) or oil-vehicle injections at adult before golgi impregnation. spine densities m of apical dendrites of the pyramidal neurons in the hippocampus ca region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto , , takafumi inoue , , akinori kuruma , katsuhiko mikoshiba , , lab. developmental neurobiology, brain science inst., riken, saitama, japan; div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca + reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p mapk and c-jun nh -terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u , a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb and sp , antagonists for p mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps a-b regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey , chou-chi li , yumiko motoyama , junko yamaoka , masao ito riken brain sci. inst., japan; national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur . glur phospho-ser promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur -vcp interaction. yuichi kitagawa , , shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp- and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps a-b long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from to days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over days old, when song learning is nearly complete, but ltd remained possible in birds over days old who had been isolated from song tutors, and thus retained the capacity for learning. ps a-b involvement of ca + -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that exposures to glu of cultured rat hippocampal slices at h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur and glur , showed that the number of glur -positive puncta increased transiently after the repeated glu exposures, whereas the number of glur -positive puncta increased gradually and persistently. jstx (a ca + -permeable ampar blocker) suppressed fepsp amplitude recorded at ca -ca synapses by - % in the period corresponding to the transient increase of glur -positive puncta. this transient increase should represent the delivery of ca + -permeable (glur -lacking/glur -including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca + -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka - , japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd times ( h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of -ht ( m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the -ht b receptor agonist cp mimicked the presynaptic inhibitory effect of -ht. -ht b receptor antagonist nas- reversed the -ht inhibitory effect, indicating that the -ht induced inhibitory effect occurs by mediating -ht b receptors. the presynaptic inhibitory effect of -ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, -ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this -ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that -ht, upon activating -ht b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku , masato ano , yuki hashimotodani , tadasato nagano , masanobu kano dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, -ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a , a a, a b and a ). adenosine a and a receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a and a receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a agonist (cpa) and a agonist ( -cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and -cl-ib-meca as reported previously. although ca currents were decreased by -cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of -cl-ib-meca. at administration of cpa and -cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a receptor may inhibit adenosine a receptor throughout a intracellular pathway in neurons. ps a-c influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa , j. yamada , k. inoue , y. yanagawa , a. fukuda , dept. physiol., hamamatsu univ. sch. med., japan; dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad -gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa , hiroyuki hioki , kouichi nakamura , , hisashi nakamura , takeshi kaneko , dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter (vglut )-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut -positive cells showed immunoreactivity for -hydroxytryptamine ( ht), a significant proportion ( . %) of vglut -postive cells was ht-negative. in this study, triple immunofluorescence staining was performed for vglut , ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad ) and tyrosine hydroxylase (th). our results showed that all of the vglut -positive/ ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut positive/ ht-negative neurons didn't show any immunoreactivities for gad nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut -positive neurons in these nuclei. research funds: kakenhi , , ps a-c cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig , mika ushimaru , yoshiyuki kubota , akiya watakabe , tetsuo yamamori , yuchio yanagawa , yasuo kawaguchi div. cerebral circuitry, nips, okazaki, japan; div. brain biology, nibb, okazaki, japan; dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin ( -ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, % of the cs were inhibited by -ht through -ht a r and % were excited through -ht a r. however, % of the fs cells were inhibited and % excited. these results show that cs cells are more inhibited by -ht than fs. the expression of -htr was confirmed by in situ hybridization. research funds: jsps pe and ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v , teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer but also in layer and/or . in each of the cases, about - gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad , somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine ( ) their other features, ( ) possible species variability, ( ) their functional significance. supported by riken bsi. withdrawn ps a-c regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge , , susanna b. park , greg j. stuart , yasuo kawaguchi national institute for physiological sciences, japan; div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure ( m) inhibited layer pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers / were generally less responsive to ach, but ach inhibited layer cells in visual cortex. prefrontal layer pyramidal neurons were more responsive to ach than were layer cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations ( mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in % of non-fs interneurons ( of cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki , , kenzi saito , , yuchio yanagawa , department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; sorst, jst, kawaguchi, japan; sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad -deficient mouse fetus. ps a-c gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal to day in egl. its release, especially from premigratory zone, was inhibited by ni + , but cd + couldn't. we suggest that gaba excitation induces granule cell migration. ps a-c gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi , hiroaki wake , junichi nabekura , osamu takahashi department of histology, kanagawa dental college, yokosuka, japan; national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata , kunihiko obata , jonathan davies , mark bellingham , peter g. noakes lab. for memory & learning, riken-bsi, wako, japan; obata res. unit, riken-bsi, wako, japan; sch. biomed. sci., univ. queensland, queensland, , australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., ) . we investigated the involvement of gaba alone in the mn death using gad -deficient mice, in which cerebral gaba is reduced to less than % of the wild-type. mn numbers at embryonic day (e) were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e embryos and subjected to electrical recording from the c and c ventral roots and also gaba measurement. in gad -deficient embryos, increase in number of brachial mns ( %) and decrease in both spontaneous discharges in the c , c roots and gaba content (less than %) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣ ␤ ␥ and ␣ ␤ ␥ combinations ( % reduction) but had no effect at ␣ ␤ ␥ or ␣ ␤ ␥ . in slice recordings dmcm blocked ipscs by % in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥ subunits whereas in the central amygdala some inputs contain ␥ while others contain ␥ subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of ␤-adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps a-d hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane , takashi iwai , yasuo nagai , junichiro oka , hideo kimura dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h s. we examined the effect of h s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na s), a donor of h s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na s. these observations suggest that h s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by ( l) lidocaein ( %) min before training. animals were allowed to self administer morphine ( mg/kg per inf.) ten consecutive daily -h session. during all morphine self administration session lever pressing was measured. our results show that: ( ) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. ( ) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca + wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of ␤ estradiol from glucocorticoid activity yuki oishi , suguru kawato department of physics, graduate school of science, university of tokyo, tokyo, japan; graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand, ␤ estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as ␤-amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor. ␤ estradiol rescued this reduction via estrogen receptor ␣ and ␤. so we found that ␤ estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps a-d the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex ( . mg/ g b.w., sc) or sesame oil (vehicle control) everyday for - days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf- mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of ␤-estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf- , estrogen receptors ␣ and ␤, and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf- and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and ␤ in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps a-d mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of -hydroxydopamine. ps a-d the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba , kimi watanabe , munekazu komada institute for developmental research, aichi human service center, aichi, japan; graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida , , asif k mustafa , kenji hashimoto , kumiko fujii , , kazuhisa maeda , , hiroshi ujike , richard l. huganir , solomon h. snyder , akira sawa dept. of systems biology, obi, suita, japan; depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; chiba univ. forensic mental health, chiba, japan; dept. of psychiat, shiga univ. med. sci., shiga, japan; div. of neuropsychiat, tottori univ., yonago, japan; dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick knockout mice were significantly lower than those of their wildtype littermates at age of p , but not in adults, suggesting regulation of pick on sr at developing stage. in case-control association study, we observed an association of the pick gene with sz, which is more prominent in disorganized sz. our findings suggest that pick contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps a-d epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe , , werner kilb , ileana l. hanganu , taizhe qian , daiichiro nakahara , atsuo fukuda , heiko j. luhmann dept. of physiol., hamamatsu, japan; inst. of physiol., mainz, germany; dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca region of an intact preparation. mm taurine blocked epileptiform activity induced by mg + free acsf and m -ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps a-d responses of pge in undifferentiated and differentiated ng - cells kayoko matsushima , takashi imanishi , akinori kawaguchi , tetsuyuki wada , shigeru yoshida , seiji ichida school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of sci. & eng., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan our previous findings showed that -ht-and bk-induced [ca + ] i increases were enlarged in differentiated ng - cells. for the next stage, we investigated the effect of pge , an inflammatory mediator for -ht and bk, on the cells. ng - cells were loaded with fura- /am, and the change in [ca + ] i was monitored by an image processor. the results showed: ( ) pge -induced response was decreased when ng - cells were differentiated by bt camp, ( ) − m ah and sc irreversibly inhibited pge -induced response by about % and %, respectively, while − m ah and sulprostone had no effect, and ( ) pge -induced response was abolished under ca +free conditions in about % of both ng - cells. these results indicate that the response to pge , via ep and ep receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from -to -day-old rats. bath application of serotonin ( -ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during -ht application. under the current-clamp recording, -ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that -ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps a-d searching for endogenous ligands of trace amine receptors in mammals ( ) akira komatsu , airi yamaguchi , noriko makikusa , osamu koizumi dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by % dms, post-fixed by % formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps a-d effects of mg + on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa , , nahoko kasai , akiyoshi shimada , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, kanagawa, japan; sorst/jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg + plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca + concentration are largely affected by mg + removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg + removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg + concentration and their actions on neural activity using a mg + indicator, kmg- -am together with fluo -am. we observed different effects of the mg + removal on gabaergic and non-gabaergic neurons by using gad -gfp knock-in mice. research funds: jst/sorst ps a-e transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p -p . zn + cortical neurons were also labeled, intrinsic and extrinsic, from p . unlike in the adult, these were in layer , instead of layers , , and . at p , neurons occurred in layers , , and and, in some areas, layer . at p , zn + neurons first appeared in layer ; and at p , there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. ps a-e systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano , , ryohei kanzaki , kei ito , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi , , taizo nakazato , shigeru kitazawa neurol, juntendo univ., tokyo, japan; physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, ) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns ). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse ( to mv for da; to mv for ht) was applied at hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps a-e time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui , , yuko sekino , , tomoaki sirao dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; div. of neural network, inst. med. sci. univ. of tokyo, tokyo; crest, jst, kawaguchi, japan; jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at days in vitro (div), and analyzed the dynamic localization of gfp-da at div. glutamate stimulation started gfp-da translocating within s and completed in min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps a-e distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa , jun shiota , hiroyuki tsutsumishita , hiroyuki sakagami , masaaki tsuda , akiko tabuchi dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr- , c-fos,␤-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi ( ) to a.t. shoko shimizu , shinsuke matsuzaki , tsuyoshi hattori , ko miyoshi , masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia (disc ) was identified as a novel gene disrupted by a ( ; ) (q . ;q . ) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc at centrosome and residues - of disc (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc to target to the centrosome. furthermore, we have shown that inhibition of the disc -kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc is important to the disc -kendrin interaction and that a truncated form of disc lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca + or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca + and cl − imaging. we found that the relatively low density a glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca + oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps a-e involvement of ca + influx in the unpolarized non-vesicular release of fgf- hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf- lacking a conventional signal peptide sequence. we found that fgf- is co-released with s a , a ca + binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf- is co-released with s a from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf- and s a required ca + . the overexpression of s a - mutant lacking an ability of interaction with fgf- inhibited the their release, suggesting that s a is a cargo molecule. the release of fgf- upon either stimulation was abolished by voltage-dependent n-type ca + channel blocker. these findings suggest that ca + influx may be involved in the unpolarized non-vesicular release of fgf- . in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that -ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca + ] i ) in pc cells. however, it is unclear how [ca + ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca + ] i on actin dynamics in pc cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a . treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to -ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp is endogenously expressed in pc cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp in actin dynamics in growth cones and found that knockdown of clp expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp deletion mutants that both of pdz and lim domains are necessary for the proper function of clp . ps a-e screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura , tsuyoshi kobayashi , yuchio yanagawa , kunihiko obata , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; grad. sch. of medicine, gunma univ., maebashi, japan; bsi, riken, wako, japan; sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the clones tested, two genes, neuronatin and seizure related gene (sez- ) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu , , tomoko matsumoto , , sakae mikami , takashi nagasawa , fujio murakami , grad. sch. of frontier biosci., osaka univ., japan; sorst, jst, japan; inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf- and its receptor cxcr in the precerebellar migration. we show that cxcr is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr and sdf- knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps a-e involvement of an immunoglobulin superfamily molecule, neph /mkirre in the migration of precerebellar neurons kazuhiko nishida , , kazuhide nakayama , saori yoshimura , , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph , neph /mkirre, and neph , only neph /mkirre was strongly expressed in pcns. expression of neph /mkirre was detected from e . when pcns migrate tangentially. the expression level became weaker at p , when pcns stop the radial migration, raising the possibility that neph /mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph /mkirre in the migration of pcns. hiroki umeshima , , toshio ohshima , tomoo hirano , mineko kengaku lab. for neural cell polarity, riken bsi, wako, japan; department of biophysics, kyoto university, kyoto, japan; lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk (cdk -dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin -gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk -dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito , , takahiko kawasaki , , tatsumi hirata , division of brain function, national institute of genetics, mishima, shizuoka, japan; department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc functions with unc- to regulate axonal transport in drosophila hiroaki mochizuki , hirofumi toda , , emiko suzuki , joseph gindhart , toshifumi tomoda , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; gene net. lab., natl. inst. genet. mishima; beckman res. inst., city of hope, ca, usa; dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc , which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc is essential for neural development in drosophila. loss of function of drosophila unc results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc genetically interacts with unc- , an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc mutants, unc- was separated from synaptotagmin vesicles. these results suggest that unc coordinates kinesin-cargo interaction via unc- to regulate dynamic axonal transport. ps a-e change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi , daisuke takahashi life science inst. sophia university, tokyo, japan; waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi ( ) and kakenhi on priority areas ( ) ps a-e generation and analysis of region-specific rac -deficient mice hidetoshi kassai , masahiro fukaya , eriko miura , mizuho sakahara , masahiko watanabe , , atsu aiba div. cell biol., kobe univ. grad. sch. med., kobe, japan; div. physiol. sci., hokkaido univ. grad. sch. med., japan rac is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac in the cns is poorly understood because of the embryonic lethality of rac knockout mice. in this study, we generated and analyzed region-specific rac -deficient mice (emx -rac ko mice) by the cre-loxp system, in which a promoter for emx homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx -rac ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx -rac ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map b containing nterminal amino acids promoted neuronal death. to reveal the mechanism of map b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map b by two-hybrid system. alpha-tubulin was found to interact with map b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map b n-terminal has not yet been reported. beta-tubulin was also found to interact with map b n-terminal. when ␤ tubulin was divided in fragment at between amino acid and , there was no interaction between ␤ tubulin fragments and map b n-terminal. interaction needs the continuous region over aa and . there were much proportion of round formed cos cells in n-terminal containing map b transfected cells than in n-terminal lacking map b transfected cells. there might be some interference in interaction between map b and tubulin in cells express map b containing n-terminal. otone endo , , masaaki mizuno , yasukazu kajita , jun yoshida department of neurosurgery, ja kainan hospital, aichi, japan; department of neurosurgery, nagoya university, nagoya, japan; department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta , masae yaguchi , yumi matsuzaki , yoshiaki toyama , yutaka kawakami , hideyuki okano , masahiro toda , neuroimmunology research group, keio univ., tokyo, japan; physiology, keio univ., tokyo, japan; orthopaedic surgery, keio univ., tokyo, japan; institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd c + dcs, cd ␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd ␣ + dcs compared to cd c + dcs. these results suggest that cd ␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps a-e von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo , hiroshi kanno , takaakira yokoyama , shuichi nakano , naoki sugimoto , nahoko kobayashi , tetsuhiko yoshida , isao yamamoto dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; fiber and dept. of chemistry, konan university, kobe, japan; toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly %, and peptide with the elongin c binding site (residues - ) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps a-e the effect of pueraria mirifica on erk / and s- following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e ) on the expression of erk / and s- following sciatic nerve crush and transection in rats. protein levels of perk / and s- in distal segments of nerve at day were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e , caused a significant decrease in the expression of perk / levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk / phosphorylation. following treatments, levels of s- were significantly decreased both in crushed and transected nerves with respect to control group at p < . . this estrogenic effect was blocked by ici , . because of their structural similarity to e , pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day . this study suggested that pm as well as g could enhance nerve regeneration like e by interfering with the injury-induced erk signaling pathway. yasuhiro kato , takafumi suzuki , kunihiko mabuchi department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf- and stat , which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p - wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y , a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin ( ht) positive cells increased in number progressively from day to weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and ht cells surrounded the axons and glia. thus, ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda , yukio someya , ryo kadota , chikato mannoji , tomohiro miyashita , atsushi murata , masashi yamazaki department of orthopaedic surgery, togane hospital, department of ortopaedic surgery, graduate school of medicine, chiba university, japan; division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt ( . mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), -bromo- -deoxyuridine- -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps a-f early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski , hanna kozlowska , marcin jurga , aleksandra habich , elzbieta wanacka , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed h, h, h and days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue , , and days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext , one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican- and syndecan- were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe , hiroe sagawa , masahiro ichikawa , yoshihito tokita dept. perinatol., int. dev. res., kasugai, japan; dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y , can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for d. after fixation, the neurites were stained with anti-tuj antibody to obtain number and length of tuj neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day , wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of -days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of -days-old embryonic mice by culturing in dmem/f medium for days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of - div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for - div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for days. dha increased the number of tuj (+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi ( ) ps a-f interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps a-f effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the d structure of surrounding stimuli defined by binocular disparity. ps a-f rotational motion aftereffect in positive direction for -dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for -dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known dimensional mae. this finding suggests that the visual system has a mechanism that detect -dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j takanori uka, ryo sasaki department of physiology , juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch ( deg) of a center/surround display presented degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always percent. for each of subjects, we calculated direction discrimination thresholds (at % correct) at each surround size. consistent with crowding, thresholds increased when surround size was . and degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was and degrees, relative to degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps a-f generation of receptive fields in higher visual areas based on v columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v and v , respond to stimuli with complex shapes. how do these neurons integrate signals from v ? in particular, does the well-known columnar organization of v play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about % of model neurons exhibit responses similar to those of v and v neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a -d surface slant is perceived (blakemore, ) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa ) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext( ), jsps( ), coe ps a-f consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi , kota sasaki , izumi ohzawa , grad. school of frontier biosci., osaka univ., japan; grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and -d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by -d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext( ), jsps( ), coe ps a-f firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate , , hiroshi tamura , , ichiro fujita , graduate school of frontier biosciences, osaka university, osaka, japan; jsps, japan; crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi - ps a-f spatial-frequency dependency of receptive field size and surround suppression in lgn and v hironobu osaki , tomoyuki naito , osamu sadakane , masahiro okamoto , hiromichi sato , med. sch., osaka univ.; grad. sch. med., osaka univ.; grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v ), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v (n = ) and lateral geniculate nucleus (lgn) (n = ) in anesthetized cats. first, we found that v neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura , akichika mikami , kazuo itoh department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v a, we injected a bi-directional tracer biotinylated dextran amine into the v a. our results indicated that the v a has connections with the occipital, parietal and temporal cortices. the v a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v a has commissural connections with the v , the v a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v . these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps a-g activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera ( fps), and were also monitored with a d or d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity - ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps a-g timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki , , arman abrahamyan , catherine stevens , andreas a. ioannides , brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately ms (n ) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto , shoutaro akaho , kenji fujikumi , yasuko sugase-miyamoto , masato okada aist, ibaraki, japan; ism, tokyo, japan; university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of neurons for visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps a-g relationship between color and shape selectivity in area teo of the monkey masaharu yasuda , , hidehiko komatsu , national institute for physiological science, okazaki, japan; sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps a-g neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a -choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was . , a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across - simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant ( ) ps a-g behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki , atsuko komori , tomonori manabe , toshihiko hosoya , hiroshi sagara , emiko suzuki , hitoshi okamoto , ichiro masai masai initiative research unit, riken, wako, japan; riken bsi, wako, japan; ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde c activity is important for the sensitivity of vision. ps a-g localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: - years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from to years of age, and showed a decline and lack of ir in older retinas ( - years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from to years of age, and absent at years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps a-g temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa , norio fujimaki , toshihide imaruoka nict, kobe, japan; kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak ( , ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity ( , ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano , , derek arnold , alan johnston , tsunehiro takeda univ. tokyo, chiba, japan; ntt cs lab., kanagawa, japan; univ. sydney, sydney, australia; ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, ) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around hz and matched with the alpha frequency of meg. as hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps a-g the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya , , kei ito , , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps a-g dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi , , toshiki tani , shigeru tanaka , graduate school of life science & systems engineering, kyushu institute of technology, japan; laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of weeks. we also found that when the goggles were removed after or weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, ) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than . hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps a-g retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., . neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada , yasuhiro enami , hiroshi jouhou , takehiko saito , kaj djupsund tokyo metropol. univ., hino, tokyo; astellas pharma. inc., osaka, japan; suny upstate med. univ., center for vision and ophthal., ny, usa; univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph only . units from the baseline of . , we found a remarkable reduction of the conduction velocity by - %, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps a-g analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi ( ) ps a-g stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of % ( of ) of neuron pairs (inter-neuronal distance > m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in % ( / ) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii , , toshihiko hosoya bsi, riken, japan; dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v ). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v . this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v neurons. masayoshi tsuruoka , masako maeda , bunsho hayashi , ikuko nagasawa , tomio inoue dept. physiol. showa univ. sch. dent. tokyo, japan; dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t -l dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention ( mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil ( %, ml). the increased emg activity significantly reduced following bilateral l -s ventral rhizotomies. a baseline emg did not significantly alter when the l -s ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps a-g hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba ) was significantly greater (p < . , uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < . , uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm agonist (l-menthol: . - mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = ) or boo rat (n = ). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm on the urothelium cell of the urinary bladder. ps a-g caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito , a.d. craig dept. physiol, shimane univ. sch. med., izumo, japan; atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol ) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns ps a-g ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps a-h fasting induced long-chain fatty acid receptor gpr expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor (gpr ) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of h fasting on gpr expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. h fasting induced gpr mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr -like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr at least fasting period. ps a-h ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that hydroxybutylate ( hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls ( v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of v respond to the change in hb. change in the intracellular calcium concentration ([ca + ] i ) in vitro was measured in dispersed ependymocytes taken from the v in rats. the present results showed that the [ca + ] i increased in response to hb, but the increase was blocked by ␣-cyano- -hydroxycinnamic acid, which is a monocarboxylate transporter (mct ) inhibitor. immunohistochemistry showed that mct -immunoreactivities were located on the v ependymocytes. these results indicate that the ependymocytes may sense hb through a mct -dependent mechanism. research funds: kakenhi ps a-h comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin ( . mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with -hydroxydopamine ( -ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at weeks after -ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at mm; anteroposterior, + mm; lateral and − and − mm; dorsoventral from bregma at weeks after -ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better -ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: ( ) retrieving the food with a rake (a motortool), ( ) retrieving the hidden food with a mirror-attached rake, ( ) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, ( ) moving a mirror hung along the rail by hand to find the food, ( ) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada , , shigehiro miyachi , michiko imanishi , masato taira , masahiko takada div. applied system neurosci., nihon univ. sch. med., tokyo, japan; dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area ( m), dorsal area ( d), and ventral area ( v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from m, d, and v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that m, v, and d receive multisynaptic projections from the entorhinal cortex, area , and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps a-h new neural activities of reward anticipation and task errors h. ogawa , h. ifuku , t. nakamura , s. hirata kumamoto kinoh hosp, kumamoto, japan; fac educ, kumamoto univ., kumamoto, japan; nat kikuchi hosp, kumamoto, japan; dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, & - ) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca s after led offset at correct trials. relations between cues and responses were reversed. of reward-related neurons found, . % showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = ) only at correct trial, i-type (n = ) at around possible reward onset only at incorrect trials, and c-i type (n = ) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai , takayuki hosokawa , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps a-h neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue ), and centrally allocated one of the objects presented in cue (cue ). the frequency of which object was presented as cue , i.e., task context, was manipulated across blocks. we focused on cue period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps a-h anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki , barry j. richmond , munetaka shidara , grad. sch. of tsukuba univ., ibaraki, japan; neurosci. ri., aist, tsukuba, japan; lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in conditions: ( ) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], ( ) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition / neurons responded in all trials. in the certain condition / neurons responded in the rewarded trials only. of these showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas ), aist masamichi sakagami , , kosuke sawa , xiaochuan pan , bsrc, tamagawa university, tokyo, japan; senshu university, kanagawa, japan; presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a -b -c and a -b -c . after monkeys could acquire the sequences, new pairs of stimuli (i.e., d and d , e and e , etc) were introduced to associated with b or b (d -b , d -b , etc). the asymmetric reward rule was instructed by pairing c (c or c ) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a and a , which were associated with c and c in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi ( ), hsfp, presto, jst ps a-h reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan , , kosuke sawa , , masamichi sakagami , bsrc, research institute, tamagawa university, japan; presto, jst, japan; department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a -b -c and a -b -c . the asymmetric reward rule was instructed by pairing c (c or c ) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of neurons from the lateral pfc, % showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about % of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps a-h neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima , hajime mushiake , jun tanji department of physiology, tohoku university school of medicine, sendai, japan; brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the -sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps a-h neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. ) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = ) preferentially when the instruction was associated with small reward. the instructions activated groups of putamen neurons preferring small reward-(n = ), large reward-action (n = ) and both types of action (n = ). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by - and - ms, respectively. putamen neuron activation lead that of cm neurons by - ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi ( ) ps a-h encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t ), second (t ) and third (t ) choices with reward probabilities of about , and %, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r , %). most da neurons ( / ) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t to t and to t trials, then decreased in r trial. in another task with two repetition trials (r and r , %), magnitude of start-cue responses decreased gradually from t to r and to r trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi ( ) ps a-h striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada , , hitoshi inokawa , minoru kimura dept. of physiol. kyoto prefect univ. med., kyoto, japan; jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded striatal projection neurons from a monkey. after depressing a start button, the monkey chose of target buttons with correct rates at st, nd, rd and repetition trials of , , and %, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice ( / ) rather than to upcoming choice of target ( / ). during the target choice, another subset of neurons showed firings selective to choosing particular target ( / ) rather than to expected reward probability ( / ). after the target choices, another group of neurons fired related to expected reward ( / ) rather than to chosen action ( / ). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi ( ), jsps fellows ps a-h encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose of target buttons at average correct rates of (first), (second), (third) and % (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans ( / ) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi ( ) ps a-i representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda , k. samejima , k. doya , m. kimura dept. physiol., kyoto pref. univ. med.; brain sci. res. center, tamagawa univ.; irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of , or %. a linear regression of neuronal discharge rates showed: neurons encoded reward values of either action during delay period before go signal, with most ( %) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = ), while other neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi ( ) ps a-i delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c ), first delay (d ), second cue (c ), second delay (d ), and response periods. activity during the d and d periods was analyzed in this study. firing rates during the d period linearly depended on c durations. on the other hand, d period activity depended on trial types (ls and sl), but not on the variety of c durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi ( ) ps a-i neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku , ryoi tamura , teruko uwano , taketoshi ono dept. integrative neurosci., univ. toyama, toyama, japan; dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi ( ) ps a-i reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori , mikio inagaki , wu lisa , taijiro doi , eishi hirasaki , hiroo kumakura , ichiro fujita osaka univ., japan; massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from and takeda science foundation ps a-i response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, ( ) performance of visually guided sex discrimination task and ( ) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) ( ) (sa) and coe program in kit from the mext ps a-i activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa , satoshi katai , masanori saruwatari , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro ) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto , tetsuya iidaka department of psychology, nagoya university, nagoya, japan; department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: - hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the - ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the - ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included images ( objects × views). novel objects were generated by deforming a prototype in four directions. for deg-interval object sets, views were obtained by rotating each object with the interval of deg, deg-interval set and deg-interval set were with deg and deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across deg separated views was significantly higher in the trials with deg-interval sets than those with deg-interval sets. similarly, the difference was also found in the performances across deg separated views between those with deg-interval sets and deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps a-i brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps a-i obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps a-i sirna-induced nr knockdown causes hypofunction of nmda-r and cognitive deficit m. saji , , t. utida , a. ohnishi , k. noda , m. ogata , h. akita , n. suzuki , physiol, health sci. sch. kitasato univ., sagamihara, japan; brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr using hvj-envelope vectors. we confirmed that marked down-regulation ( %) of nr expression occurred only in the hippocampus among various brain regions - days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr knockdown, the nr down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr knockdown, the nr down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka , , hiroshi ageta , ikuko yao , kaoru inokuchi , yutaka kirino , mitsutoshi setou , graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; mitsubishi kagaku institute for life sciences, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps a-i decreased interneurons in the pax mutant mouse limbic system hasumi haba , tadashi nomura , yoshinobu hara , , noriko osumi , div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at weeks, but not at weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp ) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine ( . mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: , ps a-i gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba , , satoko hattori , hiroaki hori , tetsuo nakabayashi , hiroshi kunugi , ryota hashimoto department of mental disorder research, national institute of neuroscience; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of patients with schizophrenia and age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: , ps a-i genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto , hiroko noguchi , hiroaki hori , tetsuo nakabayashi , satoko hattori , sachie chiba , seiichi harada , osamu saito , hiroshi kunugi department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; musashi hospital, ncnp, kodaira, japan; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in healthy volunteers and patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: , ps a-i detection of f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura , nicola bright , toshio yanagida , masatoshi takeda , paul grasby department of physiology, osaka university, japan; department of psychiatry, osaka university, japan; cyclotron unit, imperial college, hammersmith hospital, uk we used f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and f-dopa scans were obtained from a database of scans published in mcgowan et al. , archives general psychiatry. the schizophrenic patients all met dsm-iv criteria on medication and healthy volunteers were compared. we attempted to improve the quality of the f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > . ). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p component of the visual eventrelated potential in patients with schizophrenia and healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p amplitude between the faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area between the faces in controls, while in patients, there was no significant activity difference between the faces. stimulation with crying face induced higher activities in the and right areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps a-j inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura , shoji tanaka , koki yamashita , motoichiro kato , haruo kashima department of neuropsychiatry, school of medicine, keio university; department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps a-j pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc , a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as -ht are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in of the sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps a-j alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta , ryo yamamoto , shigeki sugiura , kaoru inokuchi , nobuo kato dept. integrat. brain sci., grad. sch. med., kyoto univ.; nara med. univ.; mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer a/vesl- s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer a protein by diffusion from patch pipettes. homer a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer a may be a biological mediator of ecs effects. masanori kasai , nozomi miyagi , norio kawashiro , daisuke torizuka dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant ( . mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the nd, th, th, st, th and th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc ( . ± . g/dl, n = ) on the the day was significantly lower than that in intact rats ( . ± . g/dl, n = ). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps a-j molecular cloning of a novel candidate for ethanol-responsive genes, yy ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy ap (yy -associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy -binding and transactivation domains previously demonstrated in yy ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy genes was demonstrated in the same neurons. analogous to yy ap as a co-activator of transcription factor yy , it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps a-j excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr b-selective nmda antagonists ifenprodil ( and m) and ro - ( . and m) markedly inhibited degeneration of orexin neurons induced by h application of nmda ( m) or qa ( . mm). we also show that stimulation of gaba a receptors by muscimol ( and m) or isoguvacine ( and m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba ( m) plus a gaba uptake blocker nipecotic acid ( mm) was abolished by a gaba a antagonist picrotoxin ( m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh ). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps a-j a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi , osamu fukayama , takashi sato , takafumi suzuki , kunihiko mabuchi , dept. biomed. eng., univ. tokyo, tokyo, japan; dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps a-j on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki , yasuhiko jimbo , kenji yasuda department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are khz in ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita , tetsu okumura , kazuo okanoya , jun tani lab. for behavior & dynamic cognition, riken-bsi, japan; lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps a-k local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura , yuichi yamashita , kazuo okanoya , jun tani behav & dynamic cognition, riken-bsi, saitama, japan; biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata , shinya ishii , munetaka shidara dept. of e&e engineering, oita univ., oita, japan; grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [ ] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki , shinya yamamoto , sunao uchida , shigeru kitazawa , faculty of hum sci., waseda univ., tokorozawa, japan; neurosci. res. inst, aist, tsukuba, japan; faculty of sport sci., waseda univ., tokorozawa, japan; dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ± ms, s.d. = ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi ( ) ps a-k single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama , fumiko sato , ken nakazawa , kenichi ono chiba university, japan; field of home economics, faculty of education, chiba univ., japan; department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm- , shi-madzu) over the frontal area ( channels) for volunteers ( - y). thirty tasks were presented visually every s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was − . +/− . between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi , , kosuke hamaguchi , masato okada , , department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; jst, japan; riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi ( ) and ( ) ps a-k auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa , , sachiko koyama , , takashi omori , takashi morotomi research institue for electronic science, hokkaido university, sapporo, japan; japan science and technology agency, saitama, japan; graduate school of information science and technology, hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = ). the erps were elicited by tones ( hz) with different durations ( , , , , , ms) and gaps ( , , , , , ms) embedded in a continuous pure tone ( hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for and ms were high both for the tones ( ms, %; ms, %) and the gaps ( ms, %; ms, %). ps a-k determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps a-k theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to % till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, , -diphenyl- , , -hexatriene, and -[ (trimethylammonio)phenyl]- -phenyl- , , -hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp expression correlates with wnts/bmps in the cortical hem, sp with fgfs in the cop, and sp with shh in the ventral midline and mge. by using in utero electroporation, we show that sp regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp and fgf exhibit reciprocal induction, indicating that sp is a positive feedback regulator of fgf . surprisingly, though, ectopic expression of both sp and its dominant active form shift cortical areas in the opposite manner to fgf , suggesting that sp activates additional targets that overcome fgf function. our results indicate that fgf is an additional target of sp , showing effect on patterning similar to sp . these findings indicate that sp balance the proper cortical arealization through fgf and fgf . research funds: nihr ns ps p-c fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira , kotaro hattori , takeshi yagi , shigeki yuasa dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi , kazuko keino-masu , tatsuyuki ohto , satoshi kunita , satoru takahashi , masayuki masu dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp -deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp /sulfatase- and sulffp /sulfatase- , which have hs -o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp and sulffp . here we report the isolation and the characterization of the third homologue, sulffp . sulffp has about % and % overall amino acid homology with sulffp and sulffp , respectively. at h postfertilization, sulffp is expressed in the ventral region of spinal cord, whereas sulffp is expressed only in the floor plate and sulffp is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp was shown to mediate the repulsive effect of sema a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp , , , and are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps p-c developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa , n. yamamoto , m. yoshimoto , t. yasuda , k. maruyama , t. kage , h. takeda , h. ito nat. inst. rad. sci., chiba, japan; nippon med. sch., japan; tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax -or dlx -positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er homologue (zfer ), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is % identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta , , hiroo takahashi , , takafumi shintani , , kazuma etani , masaharu noda , div. of mol. neurobiol., nibb, okazaki, japan; crest, jst, japan in the developing chick retina, the expression of bmp is relieved by that of bmp at around e with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina , are expressed in a double-gradient fashion from e on like ventroptin and bmp . when bmp expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp knockdown and ephrina -misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp to bmp thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en , wnt b and pcp /l gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and di- asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and di- asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or di- asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru , , takahisa taguchi aist, ikeda, japan; presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a -dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps p-d laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps p-d synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki , toshio aoyagi department of physics, kyoto university, kyoto, japan; graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf- mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into weekold wistar strain rats in several doses followed by successive injections of brdu for the next days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps p-d the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato , james e. goldman division of pharmacology, national institute of health sciences, tokyo, japan; department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p rat svz, sagital sections of forebrain were made at p and cultured for days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣ integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps p-d localization of neurotrophin receptors trka in pc cells: d reconstruction analysis of membrane proteins tomoki nishida , hiroshi jinnai , tatuo arii , akio takaoka , ryoichi yoshimura , yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan; department of polymer science and engineering, kyoto institute of technology, kyoto, japan; national institute for physiological sciences, myodaiji, okazaki, japan; osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin- in pc cells, analyzed by high-voltage electron microscopy, and reconstructed d structure of their subcellular distribution by imod. our results indicated that localization of caveolin- , known as an integral membrane protein of caveolae, was never found in the invagination structure in pc cells, but trka and caveolin- immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd , a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/␤-catenin pathway. ccd mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd in the wnt-mediated neuronal development. there are three subtypes of mouse ccd gene products, ccd a, ccd b and ccd c, which are generated by different promotor usage. mouse ccd a as well as zebrafish ccd a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi ( , ) ps p-d analysis of a role of r-spondin on proliferation of the cortical neuroepithelium yumiko hatanaka , masahiro yamaguchi , fujio murakami , masayuki masu grad. school of comprehensive human sci., univ. of tsukuba, japan; grad. school of med., univ. of tokyo; grad. school of frontier biosci., osaka univ r-spondin (rspo ) is a secreted activator of wnt/␤-catenin signaling (kazanskaya et al. ) . rspo is expressed in the developing medial cerebral wall and transgenic mice expressing rspo in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. ) . since wnt a has a role for expansion of caudomedial cortical progenitor cells (lee et al. ) , these findings lead us to the idea that rspo may synergistically promote proliferation of cortical neuroepithlial cells together with wnt a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a ␤-catenin/tcf reporter gene into these cells of embryonic day . mouse. an application of wnt a on these cells increased level of the reporter expression, and an addition of rspo further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt a and rspo directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il- ␤, il- , lif, gdnf) or lowered o ( . %), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells ( . -fold) as compared to normal o . cytokine cocktail significantly increased th (+) cells ( . -fold) compared to nontreated control. treatment of lif and il- ␤ to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps p-d structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui , terukazu nogi , mitsuharu hattori , kenji iwasaki , , junichi takagi research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer and vldlr), resulting in tyrosine phosphorylation of dab . in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic d reconstruction of four-domain reelin fragment (i.e. r - ) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r - ) was capable of binding to the receptor (apoer ), and was also able to induce tyrosine phospholylation of dab in primary neuronal culture. ps p-d effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax and pax compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps p-d neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res : , ) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi- k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps p-d fabp is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa , miho matsumata , , yuji owada , shigeki yuasa , noriko osumi , natl. inst. of neurosci., ncnp, tokyo, japan; tohoku univ. sch. of med., sendai, japan; crest, jst pax transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp , a downstream of pax , regulating maintenance of embryonic neural stem/progenitor cells (arai et al., ) . fabp was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). % of fabp -expressing cells co-expressed gfap (a marker for early progenitors), and % of them co-expressed psa-ncam (a marker for late progenitors). fabp expression was also overlapped with pax , and expression of fabp was down-regulated in the dg of pax deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp knockout mice. taking all together, it is concluded that fabp is required for maintenance of neural stem/progenitor cells in dg. ps p-d involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day - . sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani , , shiki okamoto , masato imada , kouichi itoh , atsushi irie , hitoshi sakuraba , hideo kubo department of molecular biologu, ohu univ., koriyama, japan; dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; dept. anatomy, nihon univ. shl. med., tokyo, japan; dept. mol. pharma., univ. tokushima bunri, sanuki, japan; dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam- shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam- is possible. in the present, we show that the isolated randam- high+ cells enrich nscs. the randam- high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam- low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam- is a usefule marker for the isolation of nscs by facs. yasuharu takamori , yasuhisa tamura , , yosky kataoka , , yilong cui , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b and b are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b (++), b (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b (+++), b (+), and mature neurons possess lamin a/c (++), b (+), b (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka , yuichi tanaka , tatsuhiro hisatsune department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps p-e new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba , , hideo namiki , tatsunori seki dept. of anat, juntendo univ. sch. of med., tokyo, japan; integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p . three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., ) . the dividing pattern is currently under investigation. akiya watakabe , noritaka ichinohe , sonoko ohsawa , tsutomu hashikawa , kathleen s. rockland , tetsuo yamamori div. of brain biol, nibb, okazaki, japan; lab. for cortical organization and systematics, bsi, riken, wako, japan, lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer neurons in several areas of monkey neocortex. we previously reported that nurr , ctgf and sema e mrnas are specifically expressed in subsets of layer neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer . by double ish, layer neurons in monkeys are roughly divisible into cck(+) and sema e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi ps p-e rbp-j regulates the cortical laminar formation kenji tanigaki , kazue muraki , norio yamamoto , tasuku honjo shiga medical center, research institute, shiga, japan; department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps p-e search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi , tomoko ikawa , yuya kasukawa , hironori ueda , , kazuki kurimoto , michinori saitou , fumio matsuzaki , lab. for asymmetric cell division, cdb, riken, kobe, japan; functional genomics subunit, cdb, riken, kobe, japan; lab. for systems biology, cdb, riken, kobe, japan; lab. for mammalian germ cell biology, cdb, riken, kobe, japan; crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi ( ) ryosuke tatsuno , tomoaki sai , , masahiro otsu , kuniko akama , takashi nakayama , tosifusa toda grad. sch. of sci. and tech., chiba univ., chiba, japan; lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; dept. orthop. surg., jikei univ. sch. med., tokyo. japan; dept. biochem., yokohama city univ. sch. med., yokohama, japan; proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using -d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps p-e identification of se , a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se in developing and matured cns. teiichi furuichi , akira sto , , yukiko sekine , noriyuki morita , tetsushi sadakata , satoshi shoji , jin-hong huang , toshio kojima laboratory for molecular neurogenesis, riken brain science institute, japan; comparative systems biology team, riken genome sciences center, yokohama - , japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki , makoto mizuno , kazuhisa sakai , yoshimoto kiyohara , kazuhiko yamaguchi , tsutomu hashikawa , hiroyuki nawa institute of biomedical engineering, waseda university, tokyo, japan; department of molecular neurobiology, brain research institute, niigata university, niigata, japan; laboratory for memory and learning, bsi, riken, saitama, japan; laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg knock out mice (nrg type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps p-e psd- protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama , , satoshi wakisaka , shiho honma , akira ito , , kei azuma , , mikihiko kogo department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd- implicates in formation and maturation of excitatory synapses. psd- regulates the localization of the nmda receptor by means of binding with nr . rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd- protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd- expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf . in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro-␤-erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps p-e ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps p-e comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi , kenji mizutani , hiroto ogawa , kohji hotta , kotaro oka ctr. for biosci. and info, keio univ., yokohama, japan; neuro, karolinska inst, stockholm, sweden; bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion ( % or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps p-e three dimensional ( d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in monkeys. of a total of p-cells that were examined during both frontal and vergence-pursuit, % discharged for both, % only for vergence, and % only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had d pursuit signals. majority ( %) of these p-cells discharged before onset of vergence eye movements with the typical lead time of ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi ( ) ps p-e information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey , rr aya takemura , yumi murata , , kenji kawano , neurosci. res. insti, aist, tsukuba, japan; dept. integ brain sci., grad. sch. med., kyoto univ., japan; grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid ( mg/ml, - l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps p-e the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara , kenji kunita , kaoru maeda , takeo kiyota department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for ms with -ms intervals. the oscillation trial ( . -hz frequency and . -cm amplitude) ( - s) was repeated times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the th trial. a significant correlation was shown between th- st trial differences in mean speed and vep amplitude (r = . ). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps p-e primary motor cortex contributes to generating manual following response toshitaka kimura , naoki saijo , hiroaki gomi , ntt cs labs, kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m ), that ( ) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and ( ) intracortical inhibition within m assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m . such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in radial directions applied during reaching. the data show that the direction of the initial phase ( - ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase ( - ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps p-e spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps p-f influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka , takashi obu , kazuyuki kanosue asmew, waseda university, saitama, japan; graduate school of human sciences, waseda university, saitama, japan; faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = ) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at . , . , and . hz. over % success rate was observed in all tasks except oppo ( - %). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps p-f simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu , tetsuro muraoka , kazuyuki kanosue , , faculty of human sciences, waseda university, saitama, japan; faculty of sport sciences, waseda university, saitama, japan; asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at , . , and . hz. they were asked to perform tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps p-f activities of erector spinae muscles during jaw clenching in man kayoko yasunaga , , tadachika yabushita , kazuo toda , kunimichi soma orthodontic science, tokyo med. & dent. univ., tokyo, japan; div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada , sachiko motoyama , futoshi mori , shigemi mori department of veterinary physiology, yamaguchi university, yamaguchi, japan; national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for - weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps p-f neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima , futoshi mori , akira murata , masahiko inase dept. of physiol., kinki univ. schl. of med., osakasayama, japan; dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi ( ) ps p-f activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara , , nobuhiko hatanaka , , masahiko takada , atsushi nambu , school of life science, the graduate university for advanced studies, japan; division of system neurophysiology, national institute for physiological sciences, japan; tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu , sei-ichi sasaki , yuichiro cho , kenji sato anatomy and physiological science, tokyo medical and dental university, tokyo, japan; department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s and s spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl -s spinal segments which contain afferent fibers of eas muscles. after weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for consecutive days. we compared adaptation in day and that in day . the gain decrease for the first saccades in day ( . ± . ) was larger than that in day ( . ± . ) (p = . , n = , paired-t test). the rate of adaptation in day ( . ± . × − /sac) was higher than that in day ( . ± . × − /sac) (p = . ). the overall gain change ( saccades) in day ( . ± . ) was larger than that in day ( . ± . ) (p = . ). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi ( ) ps p-f asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v . the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = ) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in % of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida , xiaofeng lu , , shogo ohmae , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi ps p-f frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps p-f projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki , , kei ito , , dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; center for bioinform., imcb, univ. of tokyo, japan; bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening , strains, we identified about candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps p-f immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura , yoshitaka ohtubo , takashi kumazawa , kiyonori yoshii graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi ( ) and the st coe program (center # ) granted by mext of japan ps p-f inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa , atsushi kuhara , hitoshi inada , eiji kodama , takafumi mizuno , ikue mori , lab. of mol. neurobiol., nagoya univ., japan; institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx- gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx- mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx- mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps p-f postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut and - ) during rat ob development from postnatal day (p ) to p by in situ hybridization and immunohistochemistry. at p vglut immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut mrna signals were detectable in the mitral cell layer from p to p . in contrast, vglut ir was prominent in the glomerulus at all days examined, and only at p and p in mitral cells. despite mitral vglut ir disappeared at p , the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps p-f v r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito , , masumi ichikawa , yuji mori , kimiko hagino-yamagishi tokyo metrop. inst. med. sci., tokyo, japan; the univ. of tokyo, tokyo, japan, tokyo metrop. inst. neurosci., tokyo, japan in rodent, v r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v r gene, which is expressed in the olfactory epithelium (oe). to examine when the v rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified v r sequences. these v rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps p-f analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t r /t r heterodimer plays a role as a sweet taste receptor. but, mice lacking t r showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t r -independent sweetener binding site also exist in mice. in this study, to predict binding sites on t r /t r and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [( ) sucrose, glcose, fructose, ( ) saccharin, aspartame, acesulfame-k, glycine, ( ) d-phenylalanine, ( ) d-tryptophan, ( ) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t r and t r , respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu , sachiko saito , yuko murata , ding ming , tatsu kobayakawa , robert f. margolskee , yuzo ninomiya sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; saito sachiko taste and smell research institution, ibaraki, japan; national res. institute of fisheries sci., kanagawa, japan; dept. of physiol. & biophys., mount sinai sch. med., new york, usa; national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t r receptors which coupled with ggustducin. ps p-f newborn infant body odor attenuates their mother's postpartum moods shota nishitani , mayumi kokuryo , tsunetake miyamura , kazuyuki shinohara div. neurobiol. & behav., nagasaki university, japan; obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps p-f human prefrontal activity in taste encoding: an fnirs study masako okamoto , mari matsunami , haruka dan , tomoko kohata , kaoru kohyama , ippeita dan national food research institute, tsukuba, japan; nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = ) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes ( subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes ( subfamilies) were expressed in the dorso-medial zone (zone ) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone . these results indicate that there is a clear boundary between zone and non-zone areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi ( ) ps p-g taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to ( ) best-taste profiles and ( ) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi ( ) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼ members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi ( ) ps p-g identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka , kazuhisa sakai , tsutomu hashikawa , kensaku mori , masahiro yamaguchi dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps p-g nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi ( ), kakenhi ( ) ps p-g integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than % of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi ( ) ps p-g odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi ( gs ) ps p-g odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa , takaaki sato , akira shimizu , ken-ichiro tsutsui , toshio iijima div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range ( hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps p-g chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when m lysine was spotted on ammonium chloride background, . - . m and . m background did not influence lysine chemotaxis, while . m background augmented and . - . m background suppressed the chemotaxis. in contrast, when . m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps p-g glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu , , , takeshi ishihara , eisuke nishida , yuichi iino dept. biol., fac. sci., kyushu univ., japan; mol. genet. res. lab., univ. of tokyo, japan; grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr- , a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr- appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr- . in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr- . these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr- localization in aiy neurons. kohei ueno , yoshiaki kidokoro dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼ %), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal /uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps p-g spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki , graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps p-g medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka , yoshinori sahara , shinichiro horie , masumi ichikawa , shun nakamura st. marianna univ. schl. med., kawasaki, japan; ntl. inst. neurosci., ncnp, tokyo, japan; tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps p-g synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi ( ) ps p-g rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii , , ken yoshikawa , yuki takatori , koichiro kawashima dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca + signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca + signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m , m , m and m machr subtypes, and ␣ , ␣ , ␣ , ␣ , ␣ , ␣ and ␤ nachr subunits. transfection of anti-m , anti-m and anti-␣ small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca + signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m or anti-m sirna. ca + signals evoked by nicotine were reduced by anti-␣ sirna. these findings indicate that m , m machr and ␣ nachr subtypes play major roles in ca + signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi ( ) ps p-g is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto , sachiho kubo , seiji matsuda , naoki maruyama dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, ) research funds: kakenhi ( ) ps p-g dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day . electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day . analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c from kansai medical university ps p-h unusually folded sod species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno , yumiko simazaki , fuminori saitoh , ryosuke takahashi , toshiyuki araki national institute of neuroscience (ncnp), tokyo, japan; dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod protein is thought to be responsible for the selective loss of motoneurons in sod -related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod species are not yet clarified. since we have detected accumulation of unusual sod species in motoneuronal axons from g a sod -tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod species. immunoprecipitation analyses revealed specific interaction of unusual sod species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod -tg compared with wt sod -tg mice. these data strongly suggest that the toxic property of unusual sod proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c ( ) ps p-h relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately % of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day . the mice started to show ncl symptoms on p , and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from % to % of that in the wild-type mice. research funds: kakenhi ( ) ps p-h neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura , katsumi higaki , junichiro matsuda , yoshiyuki suzuki , eiji nanba division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; national institute of biomedical innovation, osaka, japan; clinical research center, international university of health and welfare, tochigi, japan g m -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal ␤-galactosidase, excessive lysosomal accumulation of gm is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm accumulation of lysosome and pm and the membrane fluidity was also reduced. gm -bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m -gangliosidosis. katsuya inoue , , katsuaki endo , takamitsu fujikawa , seijyun fukuda , tatsuo nakamura department of physical therapy, university of aino, osaka, japan; institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after min occulusion. ps p-h development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando , yuya kawanaka , minoru saito , hiroaki mochizuki , ken honjo , hirofumi toda , , toshifumi tomoda , akira sawa , katsuo furukubo-tokunaga grad. school of life & envir. sci., univ. tsukuba, japan; molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; beckman res. inst., city of hope. california, usa; dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia- (disc ) gene, originally identified at the breakpoint of a chromosome ( ; ) (q . ; q . ) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc functions in vivo, we expressed disc in drosophila and examined developmental and behavioral phenotypes. overexpression of disc resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps p-h effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake , ayumi kamo , kenji minato , hitoshi haruna , hiritsugu fukuda , yuji murata , takayoshi hosono department of bomedical engineering, osaka electro-communication university, japan; graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of of -day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing % oxygen at • c for min. after the insult, of the rats were put in a box at • c for h (hypothermia, h-group). the other rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps p-h tau hyperphosphorylation in ts cje, a partial trisomy mouse model for down syndrome ebrahim abdul , a. shimohata , w. yu , m. yamaguchi , m. murayama , d. chui , t. akagi , t. takeuchi , k. amano , h.s. karthik , t. hashikawa , h. sago , c.j. epstein , a. takashima , k. yamakawa research scientist; lab. for neural arch.; lab. for alzheimers disease; div. of fetal med. ncchd; ucsf, usa although down syndrome (ds) or trisomy is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts cje, a ds mouse model partially trisomic for chromosome , shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome q , has about genes. importantly, sod and app, which may contribute to the ds phenotype, are excluded from the ts cje trisomic segment. here we report that ts cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk ␤ and jnk/sapk activities without alterations in a␤pp metabolism. our results suggest that genes on the trisomic ts cje segment other than app and sod can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: ps p-h increased oxidative stress and mitochondrial dysfunction in ts cje, a down syndrome mouse model atsushi shimohata , ebrahim a. s. , m. yamaguchi , w. yu , h. sago , c.j. epstein , k. yamakawa lab. for neurogenetics, riken-bsi, japan; div. of fetal med. ncchd, japan; dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome (hsa ) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome extending from sod to znf , containing genes syntenic with hsa . since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts cje brains. collectively, our results implicate dosage imbalanced genes other than sod and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts cje ds mental retardation-like phenotype. ps p-h polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan , , kouko tatsumi , takayuki manabe , takahira yamauchi , , eri makinodan , juro shimoda , toshifumi kishimoto , akio wanaka department of psychiatry, nara medical university, kashihara, japan; department of nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded ( q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. genes, encompassing around % of the drosophila genome, were screened, resulting in the identification of candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps p-h analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. ( ) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the , ,-cyclic nucleotide -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps p-h dysregulation of sodium channel ␤ subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel ␤ (␤ ) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified ␤ as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in ␤ started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of ␤ expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of ␤ gene in pc cells. forskolin, an activator of the camp/pka pathway, did not affect b promoter activity, indicating that ␤ is not camp-responsive gene. these findings strongly suggest that sodium channel ␤ subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps p-h repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki , yuji takahashi , jun goto , mutsuo oyake , toshiya sato , shoji tsuji department of neurology, the university of tokyo, tokyo, japan; department of neurology, brain research institute, niigata university, niigata, japan; center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of (q ), (q ), (q ), or (q ). in the accelerating rotarod ( w), the latencies of q , q , q and q were %, %, % and %, respectively. in the open field, moving distances of q , q , and q were decreased to %, %, and %, respectively, while that of q was increased to %. home cage activity was decreased depending on the repeat length. the q mice, however, showed increased ratios of the activity during the light time to that during the total day at weeks ( %) and weeks ( %), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps p-h the mice lacking schnurri- show multiple behavioral abnormalities related to psychiatric disorders keizo takao , nobuyuki yamasaki , keiko toyama , tsuyoshi takagi , shunsuke ishii , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; riken, tsukuba, japan schnurri- (shn- ) is a zinc finger transcription factor, a mouse homologue of human hiv-ep , that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn- is known to play important roles in the mammalian immune systems. however, the role of shn- in the central nervous system (cns) is still unknown. to investigate the functional significance of shn- in mammalian brain, we analyzed the shn- knockout (ko) mice using a comprehensive behavioral test battery. shn- ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn- mice. our results demonstrate a critical role of shn- in cns and suggest that shn- ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca + /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi ( , , , ) , jst bird ps p-h effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c bl/ j, c bl/ n, c bl/ c, svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than mice (, including wild type and mutant mice from strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behaivoral analysis of ryanodine receptor type knockout mouse suzuko ohsako , koichi tanda , , nobuyuki yamasaki , keiko toyama , hiroshi takeshima , tsuyoshi miyakawa kyoto university graduate school of medicine, kyoto, japan; dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca + signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr deficiency, we subjected ryr knockoout mice to a battery of behavioral tests. ryr knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr in regulating general locomotor activity and social behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama , koichi tanda , , nobuyuki yamasaki , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi ( , , , ) , jst bird ps p-h primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi , keiko shimizu grad. sch. human and environmental std, kyoto univ., kyoto, japan; primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected -ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. -ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in -ohda injected monkeys. these results suggest that monkeys injected -ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi ( ) ps p-i training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata , , , noriyuki higo , , takao oishi , , , akiko yamashita , keiji matsuda , motoharu hayashi neurosci. res. inst, aist, tsukuba, japan; grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; crest, jst, kawaguchi, japan; dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext ( ) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about , g animals (dba/ j × enu-treated c bl/ j) for home-cage activity, open-field activity, and passive avoidance response, and obtained lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in of mutant lines. hyperactivity was predominant phenotype, and of mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps p-i ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., ) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg -knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., ) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc , and found that in our ncl model mice, positive signals for ubiquitin and lc were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)( ) and creative scientific research ( gs ) ps p-i activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama , eiichi jodo , yoshiaki suzuki , ken-yo hoshino , yukihiko kayama dept. of physiology, fukushima medical university, fukushima, japan; dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch , mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps p-i increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara , hiroyuki sakagami , hisatake kondoh , kohji fukunaga department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps p-i severity of audiogenic seizures is influenced by multiple factors in vlgr -mutated mice hideshi yagi , , makoto sato , division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr -mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c bl/ backcrossed vlgr -mutated mice and /svs backcrossed vlgr -mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr -mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps p-i reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota , ken kadota , department of bioenvironmental medicine, university of chiba, chiba, japan; chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of mg/kg of map i.p. for days (d d ) and then withdrawn from the drug for days (wd wd ). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg ) and mfc. the decrease in the density of pac was first observed in cg and then in mfc. the reduction began on d and advanced to higher levels on d and subsequently wd . these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg and mfc, particularly in layers ii and iii. ps p-i up-regulation of ␤ -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of ␤ -adrenergic receptor (␤ -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of ␤ -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons. ␤ -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of ␤ -ar-immunoreactive cells were labeled with ki- , a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of ␤ -ar-immunoreactive cells also exhibited fgf- -ir. these findings suggest that ␤ -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf- . ps p-i effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita , norihiro muraoka , hiroshi hasuo , takashi akasu , minoru shigemori dept. of physiology, kurume univ. sch. of med., kurume, japan; dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh ). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity ( - v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was - v. the amplitude of slow peak in fpi group was about % larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps p-i comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca + imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca + imaging. the slices ( m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of ml/min. green autofluorescence (> nm) of the slices illuminated by blue light ( - nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca + imaging was also performed in the slices. slices were incubated in acsf medium containing m of fluo / am for h at • c. the ca + image was recorded with an excitation wavelength of - nmand an emission wave length of > nm. the autofluorescence and ca + responses wereobserved in slices perfused with l-glutamate ( mm). takuya hayashi , hiroshi sato , shinichi abe , takashi hanakawa , hiroshi watabe , hidenao fukuyama , babak aldekani , hidehiro iida department of investigative radiology, national cardiovascular center research institute, osaka, japan; human brain research center, kyoto university, kyoto, japan; nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area corresponded to that revealed by the tracer (mncl ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h -kokoro- ps p-i optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata , , yasutake mori , , tsuya taneda , , hiroaki okuda , , masaya tohyama , department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps p-i a model selection of glm applied to fmri data using aic jobu watanabe , fumikazu miwakeichi , andreas galka , , ryuta kawashima , tohru ozaki , sunao uchida , institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; department of medical system engineering, faculty of engineering, chiba university, chiba, japan; institute for statistical mathematics, tokyo, japan; institute of experimental and applied physics, university of kiel, keil, germany; new industry creation hatchery center, tohoku university, sendai, japan; faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for - min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps p-j flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata - , japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps p-j effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune , , takashi tsukiura , hiroko mochizuki-kawai , chisato suzuki , , toshio iijima neurosci. res. inst., aist, tsukuba, japan; div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps p-j different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno , kenji kansaku , yu aramaki , mitsuo kawato atr cns, kyoto, japan; institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = ) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, or yen is provided with a probability of . , but only with . if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < . ) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps p-j brain networks for communicative speech production: feeling inference and speech content production yuko sassa , , motoaki sugiura , hyeonjeong jeong , , keisuke wakusawa , , kaoru horie , shigeru sato , ryuta kawashima niche, tohoku university, sendai, japan; ristex, jst, tokyo, japan; miyagi university of education, sendai, japan; gsics, tohoku university, sendai, japan; department of pediatrics, tohoku university, sendai, japan; the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the st coe program ps p-j the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi , yasuhisa fujibayashi , yoshiharu yonekura , tatsuya asai department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps p-j imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai , kuniyoshi shinya , tetsuo araki , masahiro kusakabe , yasuhisa fujibayashi , yoshiharu yonekura department of nuclear power and energy safety engineering, university of fukui, fukui, japan; department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura , , winfried denk , michael hausser department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; university college london, london, uk; max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps p-k analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka , yasuhiro tanaka , takeshi kaneko , dept. of morphological brain science, kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad -gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after h incubation in nmdg-based solution than in nacl-based solution. cutting at • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at • c. the viability after h incubation was better kept at • c than at or • c. we thus recommend that slices be cut at • c in na-free solution, and incubated at • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi ( , , ) ps p-k contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh , yoshichika yoshioka faculty of engineering, iwate university, morioka, japan; iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal ( . ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals ( . , . , and . ppm). a phantom solution mimicking the normal condition (gsh:gssg = : ) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = : : : ), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was . mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area ) and the right superior and middle frontal gyri (brodmann area ) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps p-k cortical commissural connection in macaque and human callosum using diffusion mri rishu piao , takuya hayashi , hiroshi sato , shinichi abe , , takashi hanakawa , hidenao fukuyama , hidehiro iida national cardiovascular center, osaka, japan; human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps p-k optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong , niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the -d neural cortical activity using the -d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps a-c sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp ) nobuyuki karasawa , kazuko watanabe , keiki yamada seijoh universitry, tokai, japan, dept. physio., sch. med., gifu univ., gifu, japan, dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, , -diamino- -hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp ) mice. time course of immunoreactive intensity for serotonergic ( -ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that -ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and -ht neurons. katsuya yamada , , , yoshihiro matsumura , takashi miki , makoto wakui ␣-smooth muscle actin + arterioles were fewer in kir . (−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir . (−/−) barrel cortex than in wild-type one for short ( s, hz) but not long ( s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps a-h learning to use sensory-tools by japanese monkeys yumiko yamazaki , hiromi namba support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps p-h mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h receptor (h r) in cognition in physiological and pathological conditions by using h r mutant (h −/−) mice. in normal condition, several behavioral studies indicated h −/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h −/− mice. these results indicate h receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h −/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h −/− mice. mutation of h receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h r impairs cognition in normal conditions, whereas h r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi ( ; ) ps a-h -ht a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura , , hirohito-m. kondo , makio kashino , department of psychology, tokai women's university, kakamigahara, japan; ntt communication science laboratories, ntt corporation, atsugi, japan; shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of -ht neurotransmission. we examined whether this -ht a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the -ht a receptor gene polymorphism in impulsive behavior (nomura et al., ) , together with the present findings, this observation indicates the involvement of -ht a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps a-h role of cortical thin arterioles in neurovascular coupling; analyses of kir . -containing atpsensitive potassium channel-deficient mice ps p-g rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal , chinmay k. mukhopadhyay , birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india; special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv- and the direct infection of hsv- are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv- jrfl-infected macrophages or with hsv- . the generation of primary neurospheres did not suppressed by hiv- -infection or by hsv- infection at no more that moi . in the secondary neurospheres, cd + neural stem cells were intact in these infections, although beta- -tubulin + cells were decreased in hiv- infection and intact in hsv- -infection. in the differentiation assay, neun + nfp + neurons in hiv- -infection and gfap + s + astrocytes in hsv- infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv- infection and in hsv- infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv- and hsv- infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi grant-in-aid for young scientists(b) ps p-g effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga , , taeko yamauchi , toshihiro konagaya , hideki ohira department of psychology, nagoya university, japan; department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka , shen jin-song , saburo saito , shigeki yuasa dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori , tomoko yamamoto , kiyoshi matsumura , hiroshi hosokawa , shigeo kobayashi dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e (pge ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge but also induce an enzyme cyclooxygenase- (cox- ) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox- , and whether cox- is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox- was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox- specific inhibitor, ns- . these results indicate that aa itself or its metabolites induces cox- that accelerates the formation of pge from aa, and, hence, enhances fever. mitsunari abe , tatsuya mima , shinichi urayama , toshihiko aso , nobukatsu sawamoto , hidenao fukuyama human brain research center, kyoto university graduate school of medicine, japan; nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at . hz ( min; % of the rest motor threshold) applied over the left primary motor area (m ). we scanned sets of dwi (before, and min, min and min after rtms) using -t mr scanner, and calculated apparent diffusion coefficient (adc). in out of subjects, the adc decreased (mean . × − /mm s − ) in the left m just after the rtms, which recovered at min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v and v a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v a located more dorsal has a larger receptive field size then v . both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v and v a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v and v a.ps p-i measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro -channel magnetometer consisting of a × matrix of superconducting quantum interference device (squid) with its integrated base of . × . mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with - ms peak by the white led flashing stimulation and the aef wave with - ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of bits was used, along with a "blue dye" (rh ) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were . - . % of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto , yoko hoshi , chie sato , susumu terakawa tokyo institute of psychiatry, tokyo, japan; photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals ( and nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around -ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps p-j multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh , a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a -s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba ), superior occipital gyrus around the parieto-occipital sulcus (ba ) and superior parietal lobule (ba ). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps p-j influence of task difficulty during meter inspection: an fmri study naoki miura , , makoto takahashi , jobu watanabe , , shinya uchida , , shigeru sato , kaoru horie , masaharu kitamura , toshio wakabayashi , katsuki nakamura , , ryuta kawashima crest, jst, kawaguchi, japan; niche, tohoku univ. sendai, japan; graduate school of engineering, tohoku univ. sendai, japan; bme institute, waseda univ., tokyo, japan; idac, tohoku univ., sendai, japan; lbc research center, tohoku univ., sendai, japan; department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps p-j neural connectivity among brain areas related to language function shinya uchida , , , naoki miura , , jobu watanabe , shigeo kinomura , kazunori sato , yasuyuki taki , kentaro inoue , ryoi goto , ai fukushima , kaoru horie , shigeru sato , katsuki nakamura , hiroshi fukuda , ryuta kawashima department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; niche, tohoku university, sendai, japan; national institute of neuroscience, ncnp, kodaira, japan; japan science and technology agency, kawaguchi, japan; bme institute, asmew, waseda university, tokyo, japan; graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than % of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa , , motoaki sugiura , yuko sassa , , hyeonjeong jeong , , kaoru horie , shigeru sato , hiroyuki yokoyama , kazuie inuma , ryuta kawashima niche, tohoku univ., sendai, japan; department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; miyagi univ. of education, sendai, japan; ristex, jst, tohoku univ., sendai, japan; gsics, tohoku univ., sendai, japan; lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda , yasumasa okamoto , kazuhiro shishida , akiko kinoshita , shigeru toki , kazutaka ueda , hidehisa yamashita , shigeto yamawaki department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada , , , kenji yoshimi , , noriyuki higo , yong-ri ren , hideki mochizuki , yoshikuni mizuno , shigeru kitazawa , , dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; crest, jst, tokyo, japan; neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba- positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma − . mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections. key: cord- -kt gt t authors: nan title: poster session abstracts date: - - journal: pediatr pulmonol doi: . /ppul. sha: doc_id: cord_uid: kt gt t nan causing mutation is ∆f , making studies of nbd essential in understanding cftr function. crystal structures have been solved of wild-type (wt) and mutant cftr nbd , including variants containing ∆f (lewis et al. embo j. ; lewis et al. j. biol. chem. ; thibodeau et al. nat. struct. mol. biol. ) . cftr nbd contains a regulatory insertion (ri) and a regulatory extension (re) that contain pka phosphorylation sites. although the nbd core is very similar in the various structures, the ri and re in some structures of human wt nbd differ by °rotations, indicating that they are mobile. we have used nmr spectroscopy to study wt and ∆f nbd in the phosphorylated (phospho) and non-phosphorylated (non-phospho) states, and bound to different regions of cftr. despite their similar structures, differences are observed in the nmr spectra of wt and ∆f nbd . these changes, possibly due to altered dynamics or interactions of the ri and re with the nbd core, will be discussed. the inherent stability and function of nbd could be affected by these changes, accounting for part of the ∆f defect. different interactions between wt and ∆f nbd with the icls could also affect channel function. to identify residues comprising the icls, we generated a homology model of cftr based on the crystal structure of sav in which nbd and nbd form a productive dimer (dawson and locher. nature. ) . our data on a peptide corresponding to the first intracellular loop (icl ) indicate that binding to nbd requires phosphorylation of the ri. our homology model shows that the icl binding site is buried when the ri is bound to the nbd core. nmr data comparing phospho and non-phospho nbd indicates that phosphorylation of the ri and re disrupts their interactions with the nbd core, likely exposing the site for icl binding. the structure of the molybdate transporter modb c , with the nbds in an open conformation, shows icl binding is cognate nbd at a different interface (hollenstein et al. nature. ) . icl in modb c interacts with the nbd near y , the analogous residue to f in cftr. we will show binding data of icl with wt and ∆f nbd in different states and also present resonance assignments of nbd to map the icl interaction sites. since the sav and modb c structures were solved in different states, the different icl interactions may represent structural changes during the reaction cycle. the ∆f mutation may thus affect intramolecular associations in certain conformations, which would account for another part of the ∆f defect. probing differences in interactions of wt and ∆f nbd is critical for understanding the molecular basis of normal cftr function and of the altered cftr function in cystic fibrosis. shsps target ∆f cftr for erad via the sumo pathway ahner, a. ; brodsky, j.l. ; frizzell, r.a. . cell biology and physiology, university of pittsburgh, pittsburgh, pa, usa; . biological sciences, university of pittsburgh, pittsburgh, pa, usa the most frequent disease-causing mutation in cystic fibrosis (cf) is a deletion of phenylalanine at position (df ) in the first nucleotide binding domain of cftr. essentially all of the mutant protein, as well as % of wild type (wt) cftr, is retained in the er and targeted for erassociated degradation (erad) by the s proteasome. key mediators of cftr folding and degradation are molecular chaperones, which help protein substrates fold, but can target them for degradation if folding efficiency is compromised. to identify factors modulating erad of cftr we performed a microarray analysis, screening for transcription profile differences between yeast expressing cftr and control strains, and identified enhanced transcript levels for the small heat shock protein hsp in yeast expressing cftr. we then demonstrated that cftr degradation was blocked in strains lacking the genes encoding the two yeast shsps, hsp and hsp ( ) . to examine whether shsps regulate cftr biogenesis in mammalian cells, we asked which of the human shsp homologues are endogenously co-expressed with cftr using rt-pcr and western blot analysis. we detected shsps, including alphaa-crystallin and hsp , in calu- and primary hbe cells. co-expression of alphaacrystallin or hsp together with wt or df -cftr in hek cells selectively decreased the steady state levels of df -cftr. pulse-chase experiments showed that the rate of df -cftr degradation was enhanced when either shsp was over-expressed, but wt cftr biogenesis was unchanged. co-immuno-precipitation experiments in hek cells revealed that alphaa-crystallin and hsp interacted preferentially with df -cftr. thus far, our results suggest that shsps selectively increase df -cftr's accessibility to proteasome-mediated degradation pathways. previously, hsp was reported to interact with ubc , the sumo (small ubiquitin-like modifier) conjugating enzyme ( ) . sumo is covalently linked to its substrates, often at sumoylation consensus sites; similarly to ubiquitin, this occurs through a series of thiol transfer reactions. we confirmed this interaction by co-immuno-precipitation and found that ubc as well as the sumo specific protease, senp , are expressed in airway epithelial cells. over-expression of ubc decreased, while over-expression of senp increased df -cftr protein levels. pulse-chase experiments indicated that these enzymes regulate selectively the degradation of df -cftr, similarly to the shsps. preliminary studies in vitro and in vivo indicate that cftr, and particularly purified nbd , is sumoylated and that hsp facilitates this process. mutational analysis of the sumoylation consensus sites within cftr suggests a cycle of sumo modification that facilitates wt cftr biosynthesis but targets df -cftr for degradation, probably because the balance between sumo addition and removal is impaired by an excessive interaction of shsps with the mutant protein. [supported by grants from the nih and the cystic young, a. physiology and biophysics, chicago medical school, north chicago, il, usa efficient retrieval of wtcftr from the cell surface is mediated by components of the clathrin mediated endocytic pathway; however many aspects of cftr endocytosis remain to be elucidated. we have previously shown that endocytosis of wtcftr is dependent upon recognition of a tyrosinebased motif in the carboxyl tail of cftr by the ap endocytic adaptor complex. in contrast to wtcftr, less is known about the mechanisms whereby mutant cftr undergoes internalization. according to one model, df cftr undergoes accelerated endocytosis compared to wtcftr, whereas others have argued that endocytic rates of df cftr are comparable to those of wild-type cftr. to further define the function of proteins involved in the initial steps of wt and mutant cftr endocytosis, we investigated the role of the gtpase dynamin. dynamin has been proposed to play a key role in endocytosis by facilitating the scission of nascent clathrin coated vesicles from the cell surface. until recently, very few tools were available to modulate clathrin mediated events. we took advantage of dynasore, a newly discovered small molecule, non-competitive inhibitor of the gtpase dynamin. dynasore acts as a potent, rapid and reversible inhibitor of endocytic pathways known to depend upon dynamin, by blocking coated vesicle formation within seconds of dynasore addition. endocytosis of wtcftr was inhibited rapidly and efficiently upon addition of dynasore. furthermore, within minutes of dynasore washout, endocytosis of wtcftr resumed completely. also upon dynamin washout, normal trafficking and recycling of wtcftr was also observed. temperature correction of df causes expression of cftr at the cell surface, however such 'rescued' df cftr is not stabilized upon returning cells to c. application of dynasore prevented such rapid loss of mutant cftr from the cell surface. since maintenance of mutant cftr at the cell surface is critical for effective therapeutic intervention in patients with cf, our results provide insight into additional potential targets for pharmacological manipulation for the treatment of cf. richardson, j.m.; thibodeau, p.h.; watson, j.; thomas, p.j. physiology, ut southwestern medical center, dallas, tx, usa protein misfolding is the basis for a multitude of human diseases; however, the mechanisms underlying misfolding are not well understood. most cases of cystic fibrosis are associated with mutations-including the most common, deletion of phenylalanine (∆f ) in nbd -that interfere with the folding of the cystic fibrosis transmembrane conductance regulator (cftr). the resulting loss of functional cftr causes the disease. thus, elucidating how ∆f , affects the folding of cftr is critical to understanding the pathology of the disease. cftr is composed of five domains: two integral membrane transmembrane domains (tmds), a regulatory domain (r), and two nucleotide binding domains (nbds). ∆f occurs in the n-terminal nbd . both the murine wildtype and ∆f nbd can be expressed in bacteria and purified to near homogeneity. while the soluble expression yield of ∆f is lower than the wild type under identical conditions, ∆f achieves a native conformation similar to wild type as monitored by a variety of hydrodynamic and spectroscopic characteristics such as analytical size exclusion chromatography, circular dichroism, and fluorescence. recently, a non-native, but folded, species has been detected under mildly denaturing conditions. far-uv cd reveals a time and temperature dependent conversion from a mixed alpha/beta native conformation to a less helical non-native conformation, while fluorescence measurements reveal a parallel blue shift in the peak emission intensity from to nm. this non-native species is in a more open conformation as determined by both limited proteolysis and the change in retention time on analytical size exclusion chromatography. the conversion to this state is inhibited by the native state ligand (atp) and by the presence of the second site suppressors (g e, r m, and r k). notably, the ∆f nbd protein populates the non-native state under milder conditions than the wild type nbd . these studies reveal the properties of the native state and its conversion to a partially folded state that is affected by the ∆f mutation. this data highlight the proximal affect of the disease causing mutation and provide a means of assessing strategies designed to correct the defect. this work is supported by the cystic fibrosis foundation through a postdoctoral training grant and by the nih we have previously demonstrated that the stability of cftr is negatively modulated by the cftr interacting protein, cal. cal competes with nherf in binding to the pdz motif at the c-terminus of cftr and directs cftr trafficking to the lysosome. recently, silencing of cal or overexpression of nherf has been shown to restore chloride transport activity in ∆f -cftr cell lines. to identify the molecular machineries involved in cal dependent degradation of cftr, we investigated the role of the cal interacting snare protein syntaxin (stx ). stx protein expression and function were manipulated by sirna silencing, over-expression, and a dominant-negative mutant. we also performed a functional study of chloride transport activity in cystic fibrosis bronchial epithelial cell line cfbe o-stably expressing either wt-cftr or ∆f -cftr. the endogenous stx and cal protein expressions were knocked down by sirna techniques in multiple human cell lines including hek , hela cell stably expressing ∆f -cftr and cfbe o-stably expressing either wt-cftr or ∆f -cftr. in hek cells, knockdown of either stx or cal augments the expression of transfected gfp-tagged cftr (gfp-cftr), which is consistent with the observation that overexpression of either stx or cal enhances the degradation of cftr. stx interaction with cal was confirmed by a co-immunoprecipitation assay. because of this interaction, we hypothesize that stx negatively regulates cftr protein stability by interacting with cal which in turn binds to the c-ter-minal pdz motif of cftr. consistent with his hypothesis, stx knockdown has no effect on protein levels of a cftr mutant lacking the pdz motif (gfp-cftr∆trl). more importantly, cal knockdown eliminates the inhibitory effect of stx overexpression on gfp-cftr. a cell surface biotinylation assay and confocal microscopy showed significant increases in plasma membrane expression of cftr in stx knockdown cells without gross changes in the expression pattern. furthermore, consistent with its trans-golgi localization, stx knockdown has no effect on gfp-∆f -cftr expression. however, ∆f -cftr increases significantly in stx knockdown, temperature-rescued cells. to assess the effect of stx in bronchial epithelial cells, stx was knocked down in cfbe o-stably expressing wt-cftr. consistent with the observations in hek and hela cells, the expression of untagged wt-cftr is also augmented. knockdown of stx increases cftr expression in a does-dependent manner, reciprocal to the expression level of stx , without changing the level of expression of cal or gapdh. furthermore, stx knockdown lead to a does-dependent increase in cftr mediated short-circuit current that is stimulated by forskolin and inhibited by cftr chloride channel inhibitor cftrinh- . more importantly, in temperaturerescued cfbe o-stably expressing ∆f -cftr, stx knockdown tripled the cftr mediated short-circuit chloride current. these results not only delineate the effect of stx on post-golgi trafficking of cftr and its dependence on cal-mediated pdz-based interaction, but also points to it as a potential therapeutic target in conjunction with the er-quality control based rescue of ∆f -cftr. supported by the cystic fibrosis foundation and the national institutes of health. seavilleklein, g. ; evagelidis, a. ; amer, n. ; chappe, f. ; hanrahan, j. ; chappe, v. . physiology & biophysics, dalhousie university, halifax, ns, canada; . physiology, mcgill university, montreal, qc, canada cftr channel activity is regulated by phosphorylation and de-phosphorylation of the r-domain. direct pkc phosphorylation at specific sites in the r domain and nbd is essential and modulates cftr activation by pka. however, the molecular mechanism by which phosphorylation of the r domain induces channel activity remains unknown. we have reported biochemical evidence for the role of phosphorylation in domain-domain association using a deltar-split construct encoding the front (tmd -nbd ) and back (nbd -tmd ) halves of cftr as separate polypeptides ( ) . in agreement with the inhibitory role of the r domain, iodide effluxes and patch-clamp experiments confirmed that deltar-split channels were functional and constitutively active without camp stimulation. co-transfecting a plasmid that directs expression of the r domain restored the pka-dependence of re-assembled channels. co-immunoprecipitations of the halves of the deltar-split with a gst-r domain fusion protein in vitro or with the cotransfected r domain in bhk cells revealed that pka phosphorylation enhances the r domain binding to the rest of the channel. we have now investigated the effect of pkc phosphorylation on the r domain binding. our data demonstrate that pkc phosphorylation also induces a strong binding of gst-r domain in vitro or co-transfected r domain in vivo when bhk cells are stimulated with pma. interestingly, the combination of pkc+pka phosphorylation induced a stronger binding than pka alone. mutation of all consensus pkc sites on the r domain to alanine ( ca) eliminated specific pkc phosphorylation but did not alter pka phosphorylation in gst-rd. western blotting and confocal microscopy experiments confirmed that expression levels of all domains of the split- ca/rd are unchanged compared to split-rd and co-localize at the plasma membrane of bhk cells. basal activity of the split- ca/rd was similar to that of the split-rd in iodide efflux experiments. however, activation by camp+ibmx was delayed and significantly reduced ( . % of split-rd and . % of wt-cftr activation). these results confirm the functional re-assembly of the ca-rd with the front and back halves of the channel and the inhibitory role of the r domain. in co-immunoprecipitation experiments, the same level of interaction was observed between the ca/rd and rd with the split halves suggesting that basal, inhibitory interaction of the r domain is independent of pkc phosphorylation. however, ca/rd binding with the split channel was not increased by pkc or pka stimulation. we conclude that pkc phosphorylation is essential for pka-dependent binding of the r domain to the rest of the channel, and that the combined effect of these kinases further strengthens the r domain interaction. these structural changes are consistent with the essential role of pkc sites for cftr activation by pka as previously reported ( ) . supported by cihr, nshrf, dmrf & ccff. g.seavilleklein was supported by nserc. ( ) the in vivo folding mechanism of multidomain membrane proteins, including the cystic fibrosis transmembrane conductance regulator (cftr), is poorly understood. cftr, a member of the abc transporter superfamily, contains two symmetrical halves, each consisting of a membrane spanning domain (msd and msd ) and a cytosolic nucleotide binding domains (nbd and nbd ), connected by the regulatory (r) domain. to address whether cftr follows the archetypical domain-wise folding of multidomain soluble polypeptides and/or the cooperative domain folding mechanism, first we determined domain combinations that are necessary and sufficient to escape the endoplasmic reticulum (er) quality control. one-, twoor three-domain assemblies failed to be processed efficiently in cells, measured by biochemical and morphological assays. the smallest folding unit that escaped the er quality control was composed of a four-domain assembly, containing msd , nbd , r or msd as linear or split polypeptide. as a second approach we showed that cf-causing point mutations in the msd , nbd or msd provoked the er retention of the full-length cftr with severe conformational defect in the nbd , measured by limited proteolysis and immunoblotting, using domain spacific antibodies. the posttranslational folding kinetics of the four-domain folding unit was comparable to that of cftr, suggesting that cooperative domain assembly is essential for the channel biogenesis. this mechanism provides an explanation for the processing defect caused by numerous cf mutations and outlines a possible paradigm for cftr folding in living cells. cftr in post-golgi compartments. our results, jointly, uncover the role of n-glycans as a critical structural determinant of cftr conformational maturation/stability in a chaperone-independent manner. karamyshev, a.l. ; patrick, a.e. ; johnson, a.e. ; thomas, p.j. . ut southwestern medical center at dallas, dallas, tx, usa; . texas a&m university, college station, tx, usa cystic fibrosis (cf) is caused by mutations in cftr (cystic fibrosis transmembrane conductance regulator). cftr, a member of the abc transporter family, is a multispanning membrane protein critical to chloride and water movement in secretory epithelia. over one thousand mutations, in all parts of the cftr sequence, have been identified, many of which disrupt the function of the protein to cause the disease. some of the disease-associated mutations in the n-terminal portion of cftr were predicted to alter cotranslational interactions with factors involved in the membrane integration of cftr required for its proper function. to test this hypothesis, a photoreactive probe was incorporated into different positions of the n-terminal portion of cftr by introducing amber stop codons and translating these messages in vitro in the presence of the amber suppressor trna n ε -( -azido- nitrobenzoyl)-lys-trna amb . ribosome-associated nascent chains of a specific length containing the modified amino acid at a defined position were produced using truncated mrnas. some of these cftr nascent chains photocrosslinked with proteins of approximately and kda molecular mass. crosslinking to the ~ kda protein was observed only after the tm span had emerged from the ribosomal tunnel. moreover, truncation analysis demonstrated that tm is necessary for this interaction. in evaluating a range of nascent chains, the extents of photocrosslinking to the ~ kda and kda proteins often varied inversely. immunochemical and knockdown/depletion studies indicate the ~ kda protein is srp , a subunit of the signal recognition particle involved in targeting ribosome nascent chain complexes to the translocon in the endoplasmic reticulum membrane. highlighting the functional relevance of this interaction, in cells with reduced srp levels, nonglycosylated forms of cftr accumulate and total cftr levels decrease. notably, some cf-causing mutations in tm also reduce crosslinking to srp. these data reveal that mutational inhibition of nascent cftr association with srp likely contributes to or mediates some types of cf. aleksandrov, l.; aleksandrov, a.a.; riordan, j.r. the initial reversible steps of atp binding to the first (nbd ) and second (nbd ) nucleotide binding sites of cftr are divalent cation independent and dependent, respectively (aleksandrov et al, j biol chem , - , ) . subsequent to the initial binding, either mg or mn drive rapid hydrolysis at the second site while promoting trapping of the nucleotide at the first site. this occlusion at the first site of functional wildtype cftr is somewhat similar to that which occurs when the catalytic glutamates in both of the hydrolytic sites of p-glycoprotein are mutated. this occluded state has been proposed to be the result of dimerization of the two nbds and represent a transient intermediate formed during atp hydrolysis (tombline and senior, j bioenerg biomembr , - , ) . to test the possible relevance of this interpretation to cftr, we have now characterized the process by which nbd occludes [ p]- n atp and [ p]- n adp. we have now found that only n atp and not n adp can be bound initially at nbd in the absence of mg. consistent with this mg requirement for the ndp binding, n adp or adp is able to compete for the n atp binding site only when the divalent ion is present. despite the lack of a requirement for mg for atp binding, retention of the nucleotide triphosphate at the binding site at °c was dependent on the cation. however, at reduced temperature ( °c), n atp remains locked in the binding pocket with virtually no reduction over a hour period even in the absence of mg. these apparently paradoxical temperature effects hint that different molecular events can contribute to the occlusion of nucleotide at nbd . that the occlusion process occurs exclusively at nbd and does not depend on its interaction with nbd , was shown by experiments in which identical behavior was exhibited by ∆nbd constructs of cftr. thus, nucleotide trapping by wild-type cftr, unlike that by mutant p-glycoprotein, is accomplished by events at a single domain (nbd ), rather than by dimerization of the two nbds. (supported by the nih and cff). cftr consists of five domains or regions, two regions that span the cell membrane (the tmds), two cytoplasmic nucleotide-binding domains (the nbds), and a regulatory (r) domain. the r domain is unique to cftr: without phosphorylation of this domain, the cftr chloride channel does not open. the tmds and the nbds of cftr are similar to those found in many atp-binding cassette proteins, the majority of which are transporters. the domains are fused in a single polypeptide chain, linked in the sequence: tmd , nbd , r domain, tmd , nbd . the first nbd is the site of the location of the mutation that is responsible for the development of cystic fibrosis in the majority of human patients (loss of phenylalanine ). although there is a structure for nbd , we are still lacking a structure for the entire cftr protein. we have employed electron microscopy to provide low-to medium-resolution structures for the complete cftr protein which was expressed in bhk cells and purified by affinity chromatography. using single particle analysis combined with site-specific labelling using . nm diameter ni-nta nanogold, we have identified the locations of nbd and the region around residue of the r-domain within the low resolution d structure. by a process of elimination, this has also allowed the identification of nbd and the region occupied by the two tmds. these low resolution studies have now enabled us to interpret medium resolution d structural data obtained for cftr. progress in obtaining two-dimensional arrays (crystals a single molecule thick) should allow higher resolution structural data to be obtained for the entire cftr protein -sufficient for the identification of the transmembrane alpha helices. these data should allow a much clearer picture of the transmembrane channel formed by cftr and will inform molecular modelling approaches aimed at the development of novel drugs for the treatment of cystic fibrosis. we explored the gating process by introducing cysteines along the entire lengths of various transmembrane domains and extra-cellular loops, and studying their ability to chemically modified. we study the changes in their reactivity in closed and open channel states to characterize the changes in transmembrane domain conformations during gating. using membrane impermeant mts-reagents, we have characterized the conformational changes in tm -ecl , ecl -tm , ecl -tm , and ecl -tm of cftr. our results suggest that atp binding induces modest changes in the transmembrane domains and extracellular loops. channel opening involves a rearrangement of the tm helices leading to decreased hydration of residues near the extracellular end. this conformational change is either a part of, or allosterically coupled to the gating mechanism. physiological role of nherf in the regulation of cftr raghuram, v.; yang, y.; yaemsiri, s. laboratory of kidney and electrolyte metabolism, nhlbi/nih, bethesda, md, usa in the apical membrane of epithelial cells, cftr seems to exist within a multiprotein complex, in which its activity is regulated by interactions with other proteins. the c-terminus of cftr contains a pdz-binding motif, which binds to several pdz domain-containing proteins, including members of na+/h+ exchanger regulatory factor (nherf) family, nherf /ebp , nherf /e karp, cap /nherf , nherf , and cal/pist/gopc. pdz domain proteins act as scaffolds to assemble signaling proteins to form signaling complexes. these signaling complexes are central to achieve specificity and efficiency of signal transduction. nherf and nherf have a c-terminal erm domain, which interacts with cytoskeleton-associated erm proteins. this interaction may anchor cftr to the actin-cytoskeleton and restrict its movement within the membrane. in addition, several functional roles have been proposed for cftr-pdz domain interactions, including as an adapter to promote efficient pka phosphorylation of cftr, apical targeting and recycling of cftr, regulation of single-channel activity, organization of β-adrenergic, and lysophosphatidic acid- receptor signaling complex, and trafficking of romk channels. our current understandings of the role of cftr-pdz interactions are mostly obtained from heterologous expression studies. very few studies have addressed the role of pdz domain proteins in native epithelial cells or in organ physiology. we have used rna interference techniques to produce a knockout or knockdown (kd) calu- cell lines using a lenti-viral vector for stable suppression. using four different shrnas, we have generated four stable nherf -kd cell lines with varying levels of nherf suppression. in these cells, nherf expression ranged from - % of calu- nt cells (control cells). in nherf -kd cells, steady-state cftr protein level was greatly reduced, whereas its apical localization was unaffected. we also found structural defects in the apical microvilli. in polarized nherf -kd epithelia, membrane-permeable camp analogs were unable to stimulate cftr mediated short-circuit currents. however, forskolin induced response was nearly normal despite reduced cftr expression. examinations of βadrenergic-, and adenosine-receptor signaling pathways suggest that nherf plays a unique role in organizing the adenosine receptor signaling pathway. abnormal retention of the df cftr mutated protein in lung epithelial cells underlies the pathology in a large proportion of individuals with cystic fibrosis. a drug discovery alliance between cfft and biofocus dpi was initiated with the aim to identify novel genes which upon shrna-mediated knockdown were able to efficiently restore df cftr activity. a high-throughput assay based on an yfp halide reporter was developed in a human cystic fibrosis bronchial epithelial cell line (cfbe o-), whereby the rate of halide influx directly correlated to df cftr activity. biofocus dpi's proprietary adenoviral shrna libraries totalling , viruses were screened in this functional assay and yielded hits. these hits were repropogated to generate new adenoviral stocks and rescreened at three concentrations on the yfp halide reporter assay. genes were eliminated due to potential cytotoxic effects, and hit calling was performed on the remaining hits. a total of duplicate hits were confirmed ( %). of the confirmed hits had more than one shrnas which targeted the respective gene. confirmed hits were stronger in restoring df cftr function than the most potent positive control: syntaxin- . in order to elucidate which of these hits induce halide influx via direct rescue and activation of df cftr, the yfp halide reporter assay was performed in the presence or absence of the cftrinh . these cftr dependent hits will move forward into subsequent validation experiments whereby hits will be categorised into potentiators and correctors. the final goal of this program is to prioritize targets for entry into drug discovery. restoring expression and/or function of mutated cftr provides a means to treat cystic fibrosis. biofocus dpi -a galapagos company -in collaboration with the cystic fibrosis foundation therapeutics (cfft) has established a drug target discovery project that will enable the selection of proteins that rescue mutated cftr. by screening biofocus dpi's proprietary adenoviral shrna libraries in a high-throughput primary functional assay for cftr activity in human cells, we have identified and confirmed hits out of , that entered the primary screen. to further validate these proteins as potential drug targets, we have set up a series of experiments and assays focused on the rescue of cftr expression and its maturation by knock-down of specific targets. ) in order to establish whether the knock-down of a specific target was able to promote df -cftr plasma membrane localization, we performed immunofluorescence stainings of df -cftr in a patient-derived human cell line carrying the df -cftr mutation (cfbe o-) grown in air-liquid interface culture. a transduction protocol to deliver shrna adenovirus to this polarized airway cell model system has been optimized and preliminary results showed increased levels of expression of df -cftr at the plasma membrane level upon knock-down of syntaxin- , our positive control. experiments performed at οc also demonstrated redistribution of df -cftr to the apical plasma membrane at low temperature. we will show the results of all the shrna hits in this assay. ) changes in the post-translational maturation levels of df -cftr upon knock-down of specific proteins were evaluated by determining the accumulation of band b and c by using a western blot approach. in this way, we can select targets able to promote post-translational maturation of df -cftr and/or its escape from er in the cfbe o-cell line. accumulation of band b and c indicates maturation of cftr and will be considered as a beneficial effect. preliminary results show increased accumulation of band b upon knock-down of specific targets and overexpression of wild-type cftr induces band c accumulation. experiments performed at οc also demonstrated increased band b expression. we will show the effect of all the shrna hits in this assay on band b and c expression. we believe that the results obtained by the combination of those two approaches will guide us in obtaining very strong drug targets for entry into a drug discovery phase. because gene therapy as a corrective measure for cf is possible in the foreseeable future, it is important to understand whether the global effects of gene introduction will have significant detrimental impact on cell function. to address this question, global gene mrna expression profiles were created with the affymetrix u a & b chip sets. wild type cftr (n= ), ∆f (n= ) and g d (n= ) were transfected in separate ib cell cultures using lipofectamine ™. control expression profiles were generated from untreated ib cells; one from this experimental set and five from previous control experiments. differential gene expression was determined by anova using a % false discovery rate threshold. genespring was used for data analysis and visualization. genego was used to determine which functional categories were over represented in the list and further assisted in interpretation of the results. rt-pcr was used to confirm differential expression of selected genes and examine the response in more detail. all transfected constructs produced very similar gene expression profiles quite distinct from the untransfected cells. probe sets representing uniquely named genes were significantly different between transfected and untransfected cells. cftr mrna expression in all forms was increased an average of -fold. transfection of ib - cells resulted in upregulation of a variety of genes involved in several basic processes: inflammation, protein folding and cytoskeleton framework regulation. genego canonical pathways with a significantly enriched number of genes from the data set involved glucocorticoid signaling and regulation, cytoskeleton remodeling and adhesion plus a variety of other signaling pathways including il , chemokines, g-protein and mapk cascades. cell processes overrepresented in the list were predominately associated with protein folding, inflammatory responses and cytoskeleton regulation. geneontology categories over represented in the list were regulation of signal transduction, receptor mediated endocytosis and protein folding. the proinflammatory interleukins il and il were upregulated . fold and fold respectively. the genes most upregulated, samd ( -fold) and oasl ( fold), are not well annotated. as controls, rt-pcr analysis of cells treated with either lipofectamine ™ alone or any form of cftr dna alone did not show upregulation of il , il , samd or oasl. in cells transfected with lipofectamine containing dna, upregulation of il (n= ), oasl (n= ) and samd (n= ) was confirmed by rt-pcr. oasl is known to interact with methyl cpg-binding protein , a dna transcription repressor acting at methylated promoter regions. thus it is possible that the global effects noted here leading to dramatic upregulation of two novel genes oasl and smad could be previously unrecognized consequences of gene therapies. cystic fibrosis (cf) is a disease which is caused by mutations of the cf gene, which codes for the cftr chloride channel. the ∆f -cftr mutation results in a mutant protein that undergoes transcription but fails to localize to the apical membrane, where it normally functions as a camp-regulated clchannel. however, clchannel activity can be detected at the plasma membrane when ∆f -cftr is overexpressed, synthesized at a reduced temperature, or in the presence of chemical chaperones. thus, strategies that facilitate the translocation of ∆f -cftr to the plasma membrane by increasing cftr transcription may be beneficial for the treat-ment of cf. previous studies performed using fluorescence halide efflux measurements and short-circuit current voltage clamp have shown that treatment with pparγ (peroxisome proliferator activated receptor gamma) agonists, such as pioglitazone and fll (fmoc-l-leucine), resulted in an increased biosynthesis and trafficking of ∆f -cftr to the cell surface. this effect was at least partially due to increased ∆f -cftr expression. further studies were designed to investigate the effect of pparγ agonists on cftr promoter activity. two firefly luciferase-conjugated cftr promoter plasmids, containing either . kb or . kb fragments, were transfected together with a control plasmid containing a renilla luciferase in human alveolar basal epithelial (a ) cells. the ratio of the firefly to renilla luminescence was used to measure cftr promoter activity. pparγ agonists increased cftr expression acting both on . kb and . kb promoter fragments. these results were confirmed in quantitative rt-pcr studies. transcription factor binding site analysis using current online programs and matrices showed three putative ppre (peroxisome proliferator response element) consensus sequences in the cftr promoter. the interaction of these sites with pparγ was shown using emsa (electrophoretic mobility shift assay). interestingly, none of these consensus sequences appear in the . kb construct, which still maintained a significant pparγ activation response. therefore, we hypothesized that a novel pparγ agonist effector site(s) is involved in transcription activation of cftr. to characterize this novel site(s), a series of deletion constructs to the promoter region were created, using mutation-introduced restriction sites in the original promoter construct. the new constructs ranged from . kb to base pairs upstream of the transcription start site. in preliminary studies we found that at least one previously uncharacterized pparγ effector site was present in the . kb promoter fragment that regulated cftr expression. additional experiments were performed using emsa to determine the proteins and cofactors which controlled the optimal conditions for cftr expression. the results of this study show that pparγ agonists, currently prescribed to treat hyperglycemia and improve peripheral insulin resistance, may also have additional benefits for the treatment of cf. our studies also show that pioglitazone and other pparγ agonists, by increasing cftr expression, are potent activators of clflux in epithelial cells expressing ∆f -cftr. this work was supported by grant from the canadian cystic fibrosis foundation decreased by µm gsno. the effect of gsno on hop expression was post-transcriptional. in cfbe o-cells transfected with sirna duplex specific for hop ( hr), hop expression was modestly inhibited; however, gsno dramatically augmented the inhibition. we next showed that knockdown of endogenous hop by sirna increased the steady-state levels of immature and mature forms of cftr. though overexpression of hop did not significantly affect cftr expression (immature and mature forms increased . ± . and . ± . fold respectively), in the presence of µm gsno, expression of both immature and mature cftr forms significantly increased in the hop overexpressing cells ( . ± . and . ± . fold respectively). next, we treated cfbe o-cells with a pulse of gsno ( µm; hr); isolated cytosol, er and golgi fractions; immunoprecipitated cftr from these fractions; and studied whether hop, co-immunoprecipitated with cftr, was s-nitrosylated (using a biotin substitution technique followed by streptavadin isolation). we found that sno-modified hop coimmunoprecipitated with ∆f cftr in the cytosol and er at baseline; and that gsno decreased the association between sno-modified hop and ∆f cftr. we speculate that the effect of gsno to decrease hop-cftr association may ) result from increased hop s-nitrosylation; and ) be permissive for ∆f cftr maturation. these data may be of relevance to the development of no donor-based therapies for cf. supported by cf foundation and the nih. mouse models for cystic fibrosis (cf) provide new possibilities to study disease pathogenesis, to correlate genotype and phenotype, and to test novel cf therapies. we use the cftr tm eu mice homozygous for the f del mutation in a congenic fvb background as a tool in pre-clinical testing of therapeutic strategies aimed at the correction of the f del folding and trafficking defect. these mice show characteristic cf ion transport abnormalities such as reduced camp-and cgmp activated intestinal chloride secretion and an increase of the amiloride-sensitive sodium absorption in nasal epithelium. we developed an ex vivo assay for the rescue studies of the f del trafficking defect in murine intestine by proteasome inhibitors based on periodical measurements of camp-activated transepithlial chloride currents in the ussing chamber. muscle-stripped ileum was incubated at c in william's e glutamax medium supplemented with insulin ( µg/ml) and dexamethasone ( µg/ml), in the presence of vehicle or proteasome inhibitor. a partial gain of chloride secretory function was achieved by incubating the tissue for - h at c in the presence of the proteasome inhibitors (pi) epoxomicin ( µm), mg ( µm), nip-(leu) -vinyl and adalys(bio)ahx l vs (both µm) and bortezomib ( nm). exposure of the tissue for h at c to alln ( µm) revealed a functional recovery up to wt levels. immunohistochemical and western blot analysis of all pitreated tissues showed the appearance of cftr positive crypt cells and mature band c cftr protein. rescue by alln remained detectable following inhibition of the er to golgi transport by brefeldin a. our ex vivo experiments demonstrate that proteasome inhibitors can enhance the amount of mature, functionally active murine f cftr mutant protein in intact tissue by preventing proteolytic degradation and increasing its expression at the cell surface. in vivo treatment of homozygous f del mice with bortezomib at a dose of . mg/kg/day for days resulted in a high morbidity. administration of a lower dose ( . mg/kg/day ) was well tolerated and no mortality or significant weight loss could be observed. as outcome parameters for the in vivo treatment with pis we used a salivary secretion assay and bioelectric measurements of intestinal and nasal epithelium in the ussing chamber. there was no difference in isoproterenol-induced salivary gland secretion or and in cftr-mediated intestinal chloride currents between control -and bortezomib-treated animals. however we found a reduction ( . fold) of the amiloride-sensitive short circuit current (isc) in the nasal epithelium of bortezomib-treated mice, indicating a moderate correction of the enacmediated sodium hyper absorption, that was not paralleled by a gain of the forskolin-mediated transepithelial chloride secretion. current in vivo experiments focus on the effects of pis upon short term ( h- h) rather than chronic treatment, to allow a direct comparison with the ex vivo treatment data. supported by grants from the cf foundation, bethesda, the sophia foundation and the dutch stomach-liver-intestine foundation. rotterdam, netherlands; . physiology and biophysics, university of alabama birmingham, birmingham, al, usa; . physiology, school of medical sciences, university of bristol, bristol, united kingdom recent progress in the development of small molecule correctors and potentiators capable of restoring cftr function have increased the need for pre-clinical test models including cultured airway epithelial cells from human cf patients as well as cf mouse models. the validity of cf mice as a surrogate for human cf patients depends on the assumption that human and mouse cftr, despite their limited sequence homology ( %) and differences in open probability (p o ) and channel subconductance state, behave similarly in their response to pharmacological correctors and potentiators. to verify this assumption, we used the iodide efflux technique to compare the cftr chloride channel activity in (i) cho cells stably transfected with mouse f del-cftr, (ii) nih- t cells stably expressing human f del-cftr, and (iii) their wt-counterparts. forskolin ( µm)-stimulated iodide efflux was very low in both mouse and human f del cftr cells ( . respectively % of wt values) and was stimulated to a similar extent ( . and . fold) by the cftr potentiator genistein ( µm). preincubation for h at c, or at c in the presence of the vertex corrector cfcor- ( µm) caused a - fold increase in forskolin-activated i-efflux in both cell types that was further enhanced by . - fold upon addition of genistein. functional correction was paralleled by the appearance of mature, complex-glycosylated mouse or human cftr on western blots. surprisingly, two other, structurally unrelated compounds known to correct the gating defect in hcftr, i.e. the vertex potentiator cfpot- ( µm) and the uncharged nppb analogue nppb-am ( µm; cf. wang w et al. ; j biol chem : ) mimicked the effect of genistein on hf del-cftr but failed to potentiate mf del-cftr in the t cells. both cfpot- and nppb-am likewise failed to potentiate forskolin-stimulated, cftr-mediated transepithelial chloride currents ex vivo in muscle-stripped ileal mucosa from homozygous f del-cftr tm eu mice (congenic fvb) mounted in ussing chambers under conditions in which genistein ( µm) enhanced the current response by - fold. in contrast, preincubation of the tissue in william's e glutamax medium for h at c, or for h at c in the presence of cfcor- ( µm) enhanced the forskolin+genisteininduced transepithelial clsecretion up to - % of the response in cftr+/+ littermates, and was associated with the appearance of band c on western blots. these results indicate that mouse and human f del-cftr ( ) respond to low temperature incubation or to a small molecule corrector with a similar gain in surface expression and function, supporting the use of cf mouse models for in vivo tests of cftr correctors; ( ) respond differently to various classes of cftr potentiators, in line with their pronounced differences in gating behaviour, and emphasizing the specificity of cftr-potentiator interaction. further studies focussing on human/mouse cftr chimera and aimed to identify the domain(s) accounting for the differential response to the cftr potentiators are in progress. cell apoptosis is a highly regulated process which is central to the maintenance of pulmonary vascular homeostasis. recent investigations implicated cftr in the regulation of intracellular sphingolipid levels. our laboratory demonstrated that an imbalance in endothelial sphingolipid levels triggers apoptotic cell death. we therefore hypothesized that a dysregulated cftr function in human lung endothelial cells inhibits programmed cell death in response to stress. methods: patch clamp analysis was used to verify the expression of cftr on human lung endothelial cells and the effect of specific inhibitors. we inhibited cftr with dpc, cftr inh - , and nppb or specific sirna followed by treatment with prototypical stressors that induce endothelial cell apoptosis, staurosporine, h o , or tnf-α. apoptosis was quantified by caspase- activity or annexin/ propidium iodide staining. lipid extraction was performed by a modified bligh and dyer method and intracellular sphingolipids were measured by tandem mass spectrometry. results: patch clamp analysis confirmed functional cftr expression on endothelial cells and a complete inhibitory effect of dpc and nppb but no effect of dids. as expected, treatment with staurosporine, a general protein kinase inhibitor and h o , as an oxidative stress stimulus resulted in significant endothelial cell apoptosis. similarly, tnf-α treatment ( ng/ml) in combination with cycloheximide caused activation of caspase- associated with a significant upregulation of the pro-apoptotic sphingolipid ceramide. knock-down with specific cftr sirna was validated with western blot and whole cell patch clamp analysis. dpc, nppb, and cftr inh - consistently and significantly inhibited apoptosis in human lung and primary mouse lung endothelial cells. caspase- activity was decreased by - % (% decrease in caspase units/µg/min) in hlmvec treated with h o after pretreatment with dpc ( %), cftr inh - ( %), and nppb ( %). however, we observed that dids, a non-specific chloride channel inhibitor also inhibited staurosporine induced caspase- activation ( %), suggesting the possible involvement of other chloride channels. in conclusion, our data suggest a role of cftr in modulating endothelial cell apoptosis in response to various stresses. the absence of functional cftr impaired stress-induced apoptosis, a process which may be mediated via alterations in sphingolipid trafficking. this abnormal cftr function in cf leading to aberrant cellular responses to stress may perpetuate an activated, pro-inflammatory phenotype of the lung vasculature. in turn, aberrant vascular activation may have a significant impact to the abnormal airway remodeling and persistence of a chronic inflammatory state characteristic of cf. supported by: cystic fibrosis foundation clinical fellowship training grant hegedus, t. , ; serohijos, a.w. , ; dokholyan, n.v. ; he, l. , ; riordan, j.r. , . department of biochemistry and biophysics, unc at chapel hill, chapel hill, nc, usa; . cystic fibrosis treatment and research center, unc at chapel hill, chapel hill, nc, usa; . department of physics and astronomy, unc at chapel hill, chapel hill, nc, usa cftr (cystic fibrosis transmembrane conductance regulator) is a camp dependent chloride channel. the phosphorylation of its amino acid r domain by protein kinase a (pka) is obligatory for channel gating under normal conditions. r domain contains multiple pka phosphorylation sites, which participate in activation, but no specific site is essential for channel function. in spite of numerous studies of the role of r domain in cftr regulation, the mechanism is largely unknown. one reason for this is the lack of information on r domain structure and its interactions with other parts of cftr. it has been shown that r domain lacks well-defined secondary structural elements and is intrinsically disordered. in this study, we used computational methods to explore the structure and function of the disordered r domain. our results show a specific disorder-order pattern in r domain, which is conserved among species even though the primary structure is not conserved. this result implies a role of the disorder-order pattern in r domain function and provides a basis of a novel working model. to gain some insight into the three dimensional structure of r domain, we per-formed ab initio folding of r-domain using discrete molecular dynamics with an all-atom force field. as disordered domains can not be characterized with a single structure, an ensemble was generated by selection of structures with the lowest energy from a large number of folding simulations. these structures were clustered to determine the dominant r domain conformations. they reveal features of secondary and tertiary structure, the positions of phosphorylation sites in d, and residue contacts within the r region that might be important for structure and function. to computationally confirm the mechanism proposed in our working model, an ensemble of phosphorylated r domain structures was also produced and characterized. supported by nih (r dk ), cff (dokhol i ), and novartis ( ). physiology system that utilises the planar patch clamp technique (finkel et al ) . this format utilises perforated patch clamp recording of wells in parallel, permitting the generation of up to data points per day. we have employed this system to develop assays to detect modulators of mutant cftr. human cftr-g d and df , over expressed in bhk and cho cells respectively, were cultured to approximately % confluence before harvesting. df cells were temperature corrected at °c for hours prior to experimentation to facilitate channel trafficking to the plasma membrane. cells were placed on the quattro system and whole cell chloride currents measured using asymmetric chloride selective solutions such that the chloride reversal potential was significantly different from that of leak currents ( mv). currents were assessed using a voltage ramp from - to + mv applied prior and min subsequent to compound addition. to validate the assays we used the prototypical cftr potentiator, genistein, which was added simultaneously to cells under study with µm forskolin. after assessing the effects of genistein, the specific cftr inhibitor glyh , was added at µm to confirm the currents were mediated via activation of cftr. the ec values determined for genistein were . ± . µm (mean ± s.e.m, n= ) for df and . ± . µm (mean ± s.e.m, n= ) for g d. these values agree with literature values (bulteau-pignoux et al ) . two alternative potentiators, phenylglycine- (pg- ) and vrt- were also tested in the presence of µm forskolin and were active against both df and g d. the ec values for pg- were . ± . µm (mean ± s.e.m, n= ) for df and . ± . µm (mean ± s.e.m, n= ) for g d. the ec values for vrt- were . ± . µm (mean ± s.d, n= ) for df and . µm (mean, n= ) for g d. these values agree with published data from ion transport studies (pedemonte et al ; van goor et al ) . in summary the ionworks quattro can be used to identify compounds which modulate the activity of cftr. this assay has a number of advantages over other formats in that it is direct, sensitive and employs voltage control of the cells under study. these assays can be used for primary screening of small to medium compound sets or as a secondary screen to triage the output from higher throughput indirect formats such as membrane potential. references it is very well agreed that ∆f cftr is recognized as a misfolded protein by the er quality control and targeted for degradation by the proteasome. during the course of experiments for aav gene therapy for cf we created an aav / vector expressing a truncated cftr insert (∆ ) driven by a chicken beta actin promoter (raav-cb-∆ -cftr). ∆ -cftr contains the normal coding sequence of wt cftr except that it is missing the first amino acids. we observed that cos cells transiently transfected with raav-cb-∆ cftr express cftr at significantly lower levels as compared to cells similarly transfected with a wt-cftr construct. to examine why, cells were transfected with raav-cb-∆ -cftr and treated for hours with µm of proteasome inhibitor, mg . in the cells treated with mg , ∆ -cftr protein expression was significantly increased. because lysosomal inhibitors did not have a significant effect, the data suggest that a large frac-tion of ∆ -cftr protein is degraded in the proteasome. to evaluate the rate of degradation, cells were treated with cycloheximide. time course experiments showed that ∆ is more rapidly degraded than ∆f -cftr. importantly, in cos cells cotransfected with ∆f and ∆ -cftr, there was a significant increase in the mature c-band of ∆f cftr compared to cells transfected with ∆f alone. this suggests that ∆ -cftr enhanced the maturation of ∆f from b to c bands. enhanced maturation of cftr from b band to c band also occurred when ∆ -cftr was transfected into hela cells stably expressing ∆ cftr (hela ∆ ) and cfbe o-cells stably expressing ∆ (cfbe o-∆ ). a number of proteins participate in the er to determine the fate of cftr. ∆f -cftr is known to associate with retrograde translocation protein complex on the er membrane containing vcp (valosin-containing protein) and derlin . vcp is also found in aggresomes containing ∆f -cftr in association with cytosolic histone deacetylase, hdac . we hypothesized that since ∆ is more efficiently degraded as compared to ∆f -cftr it may have higher affinity with vcp protein complex. we verified that ∆ -cftr interacts more readily with vcp and also has a higher degree of association with hdac as compared to ∆f -cftr. our data suggest that ∆ is more readily degraded by the proteasome as compared to ∆f -cftr and lacks biosynthetic arrest in the er. ∆ -cftr binds more readily to vcp and associates avidly with hdac , two outcomes that are expected to enhance proteasomal degradation. by engaging key quality control proteins, ∆ -cftr allows more ∆f -cftr to mature. our data suggest that truncated forms of cftr (∆ ) may be useful for cf gene therapy by affecting the maturation of endogenous cftr. ∆f , the most common disease causing cftr mutation, has impaired conformational maturation during its biosynthesis, and thus is unable to exit the endoplasmic reticulum (er). a well-defined di-acidic er exit code has been identified within the first nucleotide binding domain (nbd ) of wildtype cftr (jcb : - , ) , the disruption of which prevents cftr from efficiently exiting the er. this is consistent with the role of the diacidic code in the copii-mediated cargo concentration process. however, it is not known whether other types of er-to-golgi trafficking signals play a role in cftr trafficking in the early secretory pathway. even less is known about the trafficking signal(s) that are responsible for the rescue of ∆f cftr by a number of means. to achieve a better understanding of the trafficking signals dictating the export of cftr from the er, we constructed a series of cftr mutants to probe the potential functional targeting signals within both wild-type and ∆f cftr. it has been shown that nbd is not required for the export of wild-type cftr from the er. we further found that an internal deletion mutant lacking membrane spanning domain (msd ) and nbd -r but retaining the n-terminal cytoplasmic region, msd and nbd does not have sufficient signal to allow er export, suggesting that the signal(s) that are responsible for cftr export largely reside within the nbd -r. unlike ∆f cftr, the di-acidic code mutants are not temperature-sensitive. dissecting the di-acidic code revealed that both acidic residues contribute to er export, and that simultaneous substitution of both leads to a much decreased export of cftr. we found that the temperature-dependent export of ∆f relies on the presence of the di-acidic code within the nbd , suggesting that this code has a functional role in the temperature-dependent rescue of ∆f cftr. in our effort to further examine the role of the di-acidic code in the rescue of ∆f cftr by second site mutations, we found that ∆f cftr rescue by the simultaneous disruption of two of the arginine-framed tripeptides (afts) is also dependent upon the di-acidic code. surprisingly, we found that while nbd is not necessary for the export of wild-type cftr, it is required for the aft-mediated rescue of ∆f cftr, suggesting a role for cytoplasmic inter-domain interactions in the aft-mediated rescue of ∆f cftr. a detailed biochemical analysis of these mutants provides valuable insights into the roles of different cftr "trafficking" motifs in the er-to-golgi transport of cftr, the specific conformational defects of ∆f cftr and the potential molecular mechanism underlying the rescue of ∆f cftr. supported by cff. sonawane, n.d. ; zhao, d. ; galietta, l. ; zegarra-moran, o. ; verkman, a. . medicine, university of california san francisco, san francisco, ca, usa; . genetica molecolare, istituto gianina gaslini, genova, italy cftr inhibitors are predicted to prevent intestinal fluid secretion in enterotoxin-mediated secretory diarrheas such as cholera. cftr inhibitors that are not absorbed across the intestinal wall are attractive for diarrhea therapy because they may provide safe oral therapy. we previously discovered low affinity glycine hydrazide (glyh, ic ~ µm) cftr inhibitors that block cftr at its external pore (muanprasat et al., j. gen. physiol. ; : - ) . in order to increase inhibitor potency and prevent washout during severe secretory diarrhea, we synthesized a series of glycocalyx interactive cftr inhibitors containing a malonic acid hydrazide (malh) cftr pore blocking moiety linked to a lectin (sonawane et al., gastroenterology ; : - . lectin conjugation improved cftr inhibitory potency by ~ -fold (ic to nm). high-affinity cftr inhibition was abolished by malh-lectin heat denaturation, protease digestion, or competition by mannose or unconjugated lectin. fluorescently labeled malh-lectin remained membrane-bound for > hours after washout, whereas washout occurred in a few minutes without the lectin. malh-lectins blocked cholera toxin-induced intestinal fluid secretion in closed intestinal loops in mice with ec - pmol, and greatly reduced mortality in a suckling mouse model of cholera. we recently synthesized mono-and divalent cftr inhibitors consisting of malh coupled via a disulfonic stilbene linker to flexible mono and bifunctional polyethyleneglycols (pegs) of molecular size . , . , , , , , , and kda, with calculated solution lengths of - nm, with the larger size pegs potentially spanning cftr dimers or inducing their formation. ic for inhibition of cftr chloride current was - µm for monovalent malh-pegs, but substantially lower and size-dependent for divalent malh-pegs, decreasing from . µm to nm with increasing peg size. the mechanisms responsible for the improved and size-dependent potency of divalent malh-pegs were studied by whole cell, single-channel patch-clamp and by functional analysis of multivalent malh-conjugated dextrans and asymmetric divalent pegs. whole-cell experiments revealed reversible voltage-dependent block of cftr currents, with outward (positive) currents being more strongly blocked. in outside-out patch-clamp experiments, inhibitors caused a reduction of the mean open time. for malh-peg kda-malh ( µm) , the mean open time decreased from ± to ± ms. the effect on currentvoltage relationship and channel kinetics are consistent with a mechanism involving occlusion of the cftr pore from the extracellular side. luminally added divalent malh-pegs blocked by > % cholera toxin-induced fluid secretion in mouse intestinal loops with ic s of < pmol/loop, and greatly reduced mortality in a suckling mouse model of cholera. nonabsorbable, multivalent cftr inhibitor-macromolecule conjugates may be useful as anti-secretory agents in the treatment of enterotoxin-mediated diarrheas. supported by cff and nih. activation of the cystic fibrosis transmembrane conductance regulator (cftr) clchannel is primarily controlled by pka-dependent phosphory-lation of the r domain. once the r domain is phosphorylated, atp binding and enzymatic activity at two nucleotide-binding domains (nbds) open and close the cftr clchannel. we hypothesized that r domain phosphorylation regulates cftr activity by modulating atp interactions within two nbds. to test this hypothesis, we studied the activity of wild-type cftr and five variants with deletions of portions of the r domain between residues and . to alter atp-dependent channel gating, we tested three cftr stimulators, pyrophosphate (pp i , mm), 'deoxy-atp ( 'datp, mm) and caatp ( mm) . consistent with previous studies, pp i , 'datp and caatp all increased current of wild-type cftr about two-fold. each stimulator produced a similar increase in the r domain variants. the similar effects of stimulators on the channel activities of wild-type cftr and r domain variants suggest that the r domain does not have a major role in regulating atp-dependent channel gating. the r domain deletions did not alter the single-channel current amplitude of the r domain variants. however, r domain variants missing the sequence between residues and markedly reduced the open state probability, suggesting that this region is required for normal gating. these data suggest that the r domain does not control cftr activity by modulating atp interactions with nbd binding sites. instead, we speculate that the c-terminal part of r domain might participate in the channel-gating machinery downstream of atp regulation. supported by the cystic fibrosis foundation and howard hughes medical institute. haggie, p.m.; verkman, a. medicine and physiology, cvri, u.c.s.f., san francisco, ca, usa it was recently reported that phagolysosomes of alveolar macrophages from cf mice acidify in a cftr-dependent manner and that defective phagolysosomal acidification impairs bactericidal activity (di et al., nat. cell biol. , : - ) . these findings suggested a unifying hypothesis for cf disease progression: defective phagolysosomal acidification in cf macrophages permits the initiation and promotes progression of bacterial infection in the lungs. to reassess the central finding of that study we measured phagolysosomal ph using a fluorescent ph indicator containing oregon green® (pka ~ . ) and tetramethylrhodamine covalently bound to zymosan. phagolysosomal ph was insensitive to cftr inhibition ( µm cftrinh- ) in j macrophages (ph . ± . vs. . ± . ), alveolar macrophages from mouse lung (ph . ± . vs. . ± . ), and alveolar macrophages from human lung (ph . ± . vs. . ± . ). phagolysosomal ph in alveolar macrophages from ∆f cf mice was not significantly different from that in alveolar macrophages from wild-type mice (ph . ± . vs. . ± . ). to account for their finding of defective phagolysosomal acidification in alveolar macrophages, di et al. reported that lysosomal acidification was cftr-dependent and that fusion of lysosomes to phagosomes (at ~ min after phagocytic uptake) was responsible for phagosomal acidification. we measured lysosomal acidification in j macrophages using a dextran-conjugate containing oregon green® and tetramethylrhodamine and found that acidification was not impaired by cftr inhibition (ph . ± . vs. . ± . ) . we also measured the kinetics of phagosomal acidification using a zymosan-conjugate containing -(and- )-carboxyfluorescein (pka ~ . ) and tetramethylrhodamine. phagosomal acidification in j macrophages and murine alveolar macrophages began within min of phagocytosis and reached steady-state by - min, in agreement with prior data in murine peritoneal and bone marrow-derived macrophages. acidification kinetics in j macrophages was not altered by cftr inhibition, nor was acidification kinetics different in wild-type vs. cf alveolar macrophages. our findings do not support the conclusion that phagolysosomal acidification in alveolar macrophages is cftr-dependent, nor that it is impaired in cf. the mechanism of phagosomal acidification proposed by di et al. is not in accord with our data or precedents in the literature. because phagolysosomal acidification is central to the proposed mechanism linking defective cftr chloride channel function with cf lung disease, our results do not support the involvement of cftr in defective macrophage function in the pathogenesis of cf lung disease. supported by cff and nih. pared to that of wild-type cftr. the hsp family of molecular chaperones play important roles in the protein quality control process within the er. hsp atpase activity is regulated by multiple co-chaperones such as hsp , bag- and hspbp . hsp , a high-molecular-weight member of the hsp family was recently shown to display nucleotide exchange activity for hsp s in vitro. furthermore, hsp was identified as a component of a cftr-associated multiple protein complex using a global proteomic approach (cell : - , ) . in an attempt to explore the role of hsp in cftr conformational maturation in the er, we over-expressed the co-chaperone in hek cells and quantitatively analyzed its effect on the maturation and degradation of cftr. consistent with its role as a nucleotide exchange factor for hsp s, over-expression of hsp inhibits the er export of wild-type cftr and promotes its degradation. however, in striking contrast, over-expressing hsp stabilizes ∆f cftr and promotes its er export at reduced temperature. this effect is less pronounced at physiological temperature. the apparently opposite effects of hsp on wild-type and ∆f cftr maturation and quality control, suggests distinct conformational maturation pathways for the two cftr molecules, and reveals a specific role for hsp in regulating ∆f cftr refolding. such conclusion is reinforced by rnai experiments and further supported by quantitative co-immunoprecipitation. rnai-mediated down-regulation of hsp expression destabilizes ∆f cftr and reduces its export at reduced temperature, and hsp shows more extensive association with the er form of ∆f cftr than that of wild-type cftr. further analysis revealed that hsp not only modulates the chaperone activities of hsp s but also alters their steady state levels within the cell, creating secondary effects on cftr maturation and quality control. further studies are necessary to achieve a better understanding of the machinery, pathway and mechanism of ∆f cftr conformational maturation at reduced as well as physiological temperatures, and this in turn will provide critical insights and key factors that are of potential value to the rescue of the trafficking defect of ∆f cftr. farinha, c.m. , ; pissarra, l.s. ; amaral, m.d. , . department of chemistry and biochemistry, faculty of sciences, university of lisboa, lisboa, portugal; . ctr hum genet, nat inst health, lisboa, portugal the most frequent mutation in the cystic fibrosis (cf) gene, f del, causes retention of its protein product, f del-cf transmembrane conductance regulator (cftr) in the endoplasmic reticulum (er) as a core-glycosylated intermediate that is rapidly degraded. therefore, most f del-cftr fails to traffic to the plasma membrane, where wild-type (wt) cftr normally functions as a chloride (cl-) channel. retention, however, is not due to lack of function, since the mutant still retains some function if it reaches the membrane. instead, it results from misfolding which is recognized by the er quality control (erqc) in which many intervenients, including molecular chaperones, participate. identification of molecular partners involved in the disposal of f del-cftr to the proteasome is therefore crutial. it was recently shown that casein kinase ii (ck ), a pleiotropic constitutively active protein kinase involved in several processes, such as neoplasia, cell survival and viral infections, binds wt-cftr near the f residue, phosphorylating its first nucleotide binding domain (nbd ) at s [ ] . interestingly, deletion of f abrogates this interaction, which is the first described f del-dependent protein-protein interaction. our aim here was to identify whether ck interaction affects the early steps of cftr biogenesis, turnover and processing. to this end, novel bhk cells were produced which stably express wt-or f del-cftr in which the consensus residue s was substituted by either a neutral (alanine -s a) or an acidic residue (aspartate -s d). initial biochemical analyses of these cell lines revealed that: ) cftr proteins bearing d or a at position are processed; ) despite producing equivalent levels of cftr transcripts, s a expressing lines consistently show lower levels of cftr protein. metabolic labelling and pulse-chase experiments followed by cftr immunoprecipitation were also performed in these lines. after quantification of bands b (immature form) and c (mature form) of cftr, these preliminary results show that substitution of s (by a or d) does not affect the turnover or processing of either wt-or f del-cftr. the effect of ck inhibition on the turnover and processing of cftr was also studied, by incubating cells with µm of the ck inhibitor tetrabromobenzotriazole (tbb) for min. pulse-chase experiments under these conditions show that: ) the steady-state levels of both wt-and f del-cftr are reduced; ) the turnover of wt-cftr (but not of f del-cftr) is increased; and ) processing of wt-cftr is decreased. such data are consistent with a putative stabilizing role for ck upon wt-cftr [ ] . however, in our preliminary results this effect does not appear to be dependent on residue (nor on the putative charge added by the kinase on this residue), thus suggesting that this effect may be indirect. further studies are underway to identify the mechanism by which ck affects the turnover and processing of cftr. . treharne et al ( ) pissarra, l.s. ; xu, z. ; farinha, c.m. , ; sheppard, d.n. ; amaral, m.d. , . department of chemistry and biochemistry, faculty of sciences, university of lisboa, lisboa, portugal; . dep physiology, univ bristol, school med sciences, bristol, united kingdom; . ctr hum genet, nat inst health, lisboa, portugal g e and rk (the simultaneous mutation of four arginine-framed tripeptides (afts): r k, r k, r k and r k) are second site mutations that rescue the processing and function of f del-cftr [ , ] . these revertant mutations rescue f del-cftr from retention within the endoplasmic reticulum by distinct mechanisms: g e likely alters the conformation of the first nucleotide-binding domain (nbd ), whereas rk plausibly allows f del-cftr to escape er retention/retrieval mediated by afts [ ] . both g e and one of the afts (r k) lie close to the residue g , where a common cf-causing mutation g d occurs, generating a correctly localized clchannel with a severe gating defect. our aim here was to assess whether the revertants g e and rk influence the folding, processing and gating behaviour of the g d mutation, by employing biochemical and functional approaches. to test this idea, we introduced the g d mutation into cftr cdnas containing either g e or rk in the pnut vector and stably expressed these constructs in bhk cells. preliminary results from biochemical studies indicate that the mature fully-glycosylated form of cftr protein (band c) of both g e-g d-cftr and rk-g d-cftr were present at similar levels to those of g d-cftr. analysis of cftr-mediated iodide efflux from these cells revealed that g e is unable to rescue the functional defect of g d. however, rk generated an efflux of iodide larger than that elicited by cells expressing g d, albeit smaller than that of cells expressing wild-type (wt) cftr. consistent with these data neither rk nor g e rescued the defective channel gating of g d-cftr. however, both revertants caused a small increase in g d-cftr activity by attenuating the prolonged interburst interval of g d. we conclude that f del and g d disrupt cftr channel gating by distinct mechanisms. altogether, our data suggest that at least when in cis with the g d mutation, the afts (together or individually) might have a direct effect on cftr channel gating. this raises the possibility that rk, in addition to its well-described effect on trafficking, may act on cftr structure and/or folding, as previously suggested for r k [ ] . work supported by grant pocti/sau/mmo/ / and pluriannual funding of cigmh (fct, portugal) and the uk cf trust. l. pissarra was a recipient of bd/ / doctoral fellowship (fct, portugal) . [ ] carvalho ac, gansheroff lj, teem jl ( ) j biol chem , - . [ ] chang xb, cui l, hou yx, jensen tj, aleksandrov aa, mengos a, riordan jr ( ) mol cell , [ ] [ ] [ ] [ ] [ ] [ ] [ ] roxo-rosa m, xu z, schmidt a, neto m, cai z, soares cm, sheppard dn, amaral md ( ) proc natl acad sci usa , - . [ ] teem jl, carson mr, welsh mj ( ) improves ∆f -cftr intracellular trafficking in cf epithelial cells, although the mechanism by which this occurs is not clear. to identify gene products with atlered abundance in response to pba, we performed a differential display rt-pcr screen on rna isolated from ib - cf epithelial cells treated with pba for - hours. we isolated a cdna encoding stip- , a putative human stat- (signal transducer and activator of transcription- ) interacting protein and confirmed that pba causes transiently increased stip- mrna and protein abundance after hours of exposure. stip- was originally described as a scaffold protein that is required for ligand-dependent activation of stat- . stip- is also identical to elongator protein (elp- ), a subunit of the multicomponent elongator complex that stimulates rna polymerase ii activity. interestingly, recent data suggests that elongator may also regulate polarized secretion (rahl, et al. ( ) , mol. cell, : - ). we therefore hypothesized that stip- /elp- would regulate ∆f -cftr intracellular trafficking. in ib - cells, stip- /elp- associates with ∆f -cftr in coimmunoprecipitation experiments after pba treatment. in ib - cells overexpressing stip- /elp- , immunofluorescence experiments suggested that ∆f -cftr trafficked to the plasma membrane even in the absence of pba treatment. stip- /elp co-localized with markers of the golgi ( kda golgi protein) and exocytic vesicles (snap- ) when overexpressed in these cells. in contrast, overexpression of a deletion mutant of stip- /elp- lacking the wd domain, or stat- binding region, blocks the improvement of ∆f -cftr trafficking in response to pba in immunofluorescence and surface biotinylation experiments. this stip- /elp- deletion mutant did not co-localize with golgi or exocytic vesicle markers. furthermore, immunofluorescence experiments also suggested that pba causes colocalization of stip- /elp- and elp- , another member of the elongator complex that is often defective in familial dysautonomia. these data are consistent with stip- /elp- regulating ∆f -cftr intracellular trafficking in response to pba. this regulation may occur in the context of pba stimulating assembly of elongator, which in turn may promote trafficking of exocytic vesicles carrying ∆f -cftr to the plasma membrane. supported by grants from niddk. fold [ , ] . however, functional studies demonstrate significant differences in the gating behaviour of hcftr and mcftr [ ] . a powerful approach to investigate cftr structure and function is to examine interspecies differences and identify regions of conservation and divergence. to understand the structural basis for the functional differences between hcftr and mcftr, we generated hmcftr chimeras containing mcftr domains on an hcftr backbone. for this purpose, we replaced all or part of nbd , nbd or the rdomain of hcftr with the equivalent regions of mcftr and investigated their biochemical and functional properties. the in vivo folding of these hmcftr chimeric proteins was indirectly evaluated from their maturation status, after their stable expression in novel bhk cell lines. like wt-hcftr, most chimeric proteins were processed within the cell. however, two chimeras failed to mature: clone b (mnbd , amino acid (aa) residues - ) and clone c (mnbd , aa - ). we compared the murine sequence of these two chimeras with that of hcftr to determine the respective physico-chemical distances (pcds) of their aa changes. changes with higher pcd values were selected and in vitro mutagenesis performed to introduce these aa alterations into hcftr cdna. for clone b the selected changes were: e q, e q (pcd = ); s t (pcd = ); k q (pcd = ), i t (pcd = ) and k e (pcd = ). for clone c the changes were: p t (pcd = ), k q (pcd = ), y n (pcd = ), s k (pcd = ), c y (pcd = ), d g (pcd = ) and e d (pcd = ). biochemical analyses of these mutants stably expressed in bhk cells revealed that for clone b, e q, s t, k q and i t were processed, whereas k e was not. for clone c, p t, k q, y n, d g and e d were processed. iodide efflux showed that s t, k q, i t, e d and d g were functional, but not k e. we consider that with additional functional analyses, our approach will identify critical residues responsible for conformational changes and hence, functional differences between hcftr and mcftr. work supported by the bbsrc and grant pocti/mgi/ / (fct, portugal) and pluriannual funding of cigmh (fct, portugal) . ac dapaula is a recipient of phd fellowship sfrh/ bd/ / (fct, portugal we recently reported that the upr decreases endogenous wild-type (wt) cftr expression. as a follow up of these studies, we investigated the role of the folding deficient, ∆f cftr on upr induction and identified a mechanism by which the upr decreases cftr expression. for these studies, we developed a cell line expressing recombinant ∆f cftr on the endogenous wt background (calu- ∆f) and established individual clones with different ratios of endogenous (wt) to recombinant (∆f cftr) expression. two clones which express : (calu- ∆fc ) and : (calu- ∆fc ) ratios of wt to ∆f cftr and cfpac- cells expressing endogenous ∆f cftr were tested as models. the upr was constitutively active in calu- ∆fc cells only. in calu- ∆fc induction of ∆f cftr expression resulted in upr activation, indicating that high expression of ∆f cftr is required for upr induction. furthermore, following pharmacological induction of the upr, endogenous cftr mrna decreased to undetectable amounts both in calu- ∆f and cfpac- cells. the decrease in cftr mrna levels was not the result of shortened mrna half-life. in contrast, using a human cftr promoter driven reporter (pcftr-pluc), we demonstrate suppression of the cftr promoter when the upr is activated. considering that correction of ∆f cftr is the main therapeutic approach for cf, it is important that ∆f cftr expressed at low levels, such as in vivo, does not activate the upr. however, our results also reveal that ∆f cftr correctors have to be tested for upr activation, since transcriptional inhibition in this setting may contribute to inefficient rescue in native cells or in vivo. genistein, a naturally occurring tryrosine kinase inhibitor, at low concentrations ( µm) stimulates wt -cftr-mediated chloride secretion in a variety of cell and tissue types by an adenosine ', '-cyclic monophosphate (camp)-independent pathway. genistein has been shown to potentiate the channel function of ∆f -cftr and g d-cftr channels by prolonging channel open time (hwanget al., am j physiol cell physiol : c -c , ; illeck et al. am j physiol cell physiol : c -c , ) patch clamp studies have provided indirect evidence that genistein may bind directly to cftr and potentiate channel open probability by modifying the function of the nucleotide binding domains (weinreich et al., pflügers arch : - , ) . the goal of the present study was to determine whether genistein activated purified and reconstituted cftr protein as an initial step toward defining its molecular mechanism of action. the effect of genistein on purified and reconstituted wt-cftr was first studied following its reconstitution into planar lipid bilayers. in the presence of µm atp, phosphorylated cftr exhibited a low open probability (po= . ). the addition of µm genistein, caused a significant increase to the open probability of cftr (po= . ). using an electrogenic flux assay on a population of purified and phosphorylated cftr molecules, we determined that µm genistein caused a potentiation ( % increase) of chloride channel activity in the presence of µm atp. these results suggest that this macroscopic assay of purified cftr function is sufficiently sensitive to detect the effect of this potentiator. currently, the consequences of genistein on the channel activity and atpase activity of purified reconstituted ∆f -cftr and g d-cftr are being assessed. preliminary results suggest that in the presence of low atp concentrations ( - µm), µm genistein inhibits the atpase activity of ∆f -cftr, suggesting that it interacts directly with the nucleotide binding domains to alter their function. previous studies indicated an acute coordination between the activities of cystic fibrosis transmembrane conductance regulator (cftr) chloride channel and the amiloride sensitive epithelial sodium channel (enac) so that both these channels are either activated or deactivated in a synchronous fashion in the human sweat duct (reddy et.al, ) . however, the mechanisms responsible for such cooperativity between these ion channels are unknown. previous studies indicated that: cytoplasmic ph controls cftr activity through effects on phosphorylation (reddy et.al. ) . at ph . , cftr chloride conductance was reduced to ± ms/cm , but incresed to ± ms/cm at ph . (n=number of ducts= ). enac channel activity is also a function of cytosolic ph in heterologous expression systems (chalfant et.al, ) . the objective of this study was to test the hypothesis that the cytosolic ph may mediate the cooperative effects that occur between cftr and enac. we used basolaterally a-toxin permeabilized apical membrane preparations of native human sweat duct which expresses enac and cftr robustly as an experimental system. we showed that while luminal ph had no effect, changes in cytosolic ph acutely affected enac activity. that is, acidic ph inhibited, while basic ph activated enac activity. alkalinizing cytosolic ph from . to . increased enac conductance (genac) by ± . ms/cm (n= ). ph regulation of enac activity appears to be independent of cftr and endogenous kinase activities because basic ph stimulated enac: a.) after deactivating cftr by removing camp and atp in normal ducts, b.) in the absence of cftr in cf ducts, and c.) after blocking endogenous kinase activity with the non-specific kinase inhibitor, staurosporine. na + /h + exchanger (nhe) may mediate changes in cytosolic ph as a function of intracellular na + activity. nhe mrna is expressed in the sweat duct and cytosolic ph responds to changes in na + gradients across the basolateral membrane. conceivably, when transport conditions are favorable and intracellular na + is low, alkaline ph would allow enac and cftr to cooperatively admit na + and clthrough the apical membrane. when conditions are unfavorable and intracellular na + is excessive, acid ph would limit na + and clentry to protect the cell from disruptive changes in cell volume. thus, changes in cytosolic ph may play a crucial role in coordinating the activities of enac and cftr during transepithelial salt transport. acknowledgements: kirk taylor and sucharitha madireddi for expert technical assistance. funded by nih-ro de , nih-ro hl and the nancy olmsted trust. the cystic fibrosis transmembrane conductance regulator (cftr) is an anion channel that is normally expressed in the plasma membrane but is mislocalized in cf. to monitor trafficking we inserted super ecliptic phluorin, a ph-sensitive variant of green fluorescent protein (gfp) (miesenbock g, de angelis da, rothman je ( ) nature , - ) into the fourth extracellular loop of cftr. this construct, which we call cftr-phluor, has weak fluorescence intracellularly that increases ~ -fold when it is inserted into the plasma membrane and the phlour becomes exposed to more alkaline extracellular ph . thus cftr-phluor should be less fluorescent than cftr with the normal gfp fused to the n or c termini when situated in intracellular vesicles. despite the insertion of an entire gfp in the middle of the channel protein, cftr-phluor trafficked to the membrane and patch clamp studies revealed little change in unitary conductance or open probability compared to wild-type cftr. cftr-phluor enabled visualization of vesicle insertion events at the plasma membrane by total internal reflection fluorescence (tirf) microscopy. faintly fluorescent vesicles containing cftr were observed under the plasma membrane and eventually fusing with it to generate a burst of fluorescence. phluorin inserted at the same position in ∆f -cftr, the predominant mutant in cf, was retained in the er where the phluor remained partially quenched. when the folding defect was corrected by culturing cells at °c or treating them with known correctors, ∆f -phluor trafficking was partially restored and total cell fluorescence increased. this enabled a quantitative analysis of ∆f -cftr correction based on the increase in fluorescence in individual cells by flow cytometry. cftr-phluor may be most useful for microscope-based high-content screening studies of cftr and ∆f -cftr trafficking. support: the breathe program, canadian cf foundation, canadian institutes of health research and cf foundation therapeutics (usa). tukaye, d.n.; guggino, w.b. physiology, johns hopkins university, baltimore, md, usa type iii secretion system (t ss) toxins of p. aeruginosa are important virulence factors in p. aeruginosa infections. one of the t ss toxins, exos, has been shown to facilitate uptake and invasion of p. aeruginosa at airway surface epithelium. in mouse models of pseudomonas pneumonia, infection with p. aeruginosa exos+ strains caused increased levels of fluid in lungs as determined at autopsy in contrast to infection with p. aeruginosa exosstrains. the exact molecular mechanism underlying these observations is not known. exos is a bifunctional protein with gtpase activating protein (gap) activity at the n terminal domain and adp ribosyl transferase (adprt) activity at the c terminal domain. we found that exos-gap activity increases total cellular levels of mature (c band) wild type (wt) cftr in cfbe o-cells as measured by western blot analysis. this increase was mediated by decreased delivery of wt cftr for lysosomal degradation. in contrast, exos-gap failed to increase total levels of ∆ cftr (b band). interestingly, exos-gap increased total levels of ∆ cftr, bands c and b, following rescue with -phenylbutyrate. this indicates that targets of exos-gap exist in cftr trafficking beyond the er degradation pathway. exos-gap also brought about a corresponding increase in surface levels of mature wt cftr as measured by surface biotinylation. in conclusion, we have shown that p. aeruginosa t ss exos-gap, upregulates total and surface levels of mature wt cftr by modulating cftr trafficking beyond er, most likely by decreasing lysosomal degradation. the decrease in lysosomal degradation could be occurring in part by inactivation of small molecular weight g-proteins involved in delivery of wt cftr to lysosomes. increases in total and hence surface levels of cftr could in part explain increased amounts of fluids seen in p. aeruginosa pneumonia mouse model treated with exos+ strains. the post-maturational trafficking and localization of cftr is regulated by a wide variety of proteins. among the most prominent are several pdz domain proteins that bind to the c-terminal residues of cftr. in particular, two of these proteins, nherf and cal, have been shown to mediate opposing effects on the apical membrane levels of the disease-associated ∆f -cftr mutant, by controlling the balance between endocytic recycling and lysosomal degradation. in particular, whereas overexpression of nherf rescues ∆f -cftr at the cell surface, overexpression of cal has the opposite effect on cftr. cal-mediated downregulation of cftr requires a functional pdz binding site. we have now shown that suppression of endogenous cal cooperates with temperature rescue to stabilize functional ∆f -cftr channels at the apical membrane in polarized bronchial epithelial cells. this work supports the hypothesis that the cal pdz binding site is a therapeutic target for treatment of cystic fibrosis. a more direct test of this hypothesis will require competitive inhibitors that can efficiently block the cal:cftr interaction. in addition, given the large-number of protein-protein interactions formed by both the cal pdz domain and the cftr c-terminus, these inhibitors should ideally exhibit bidirectional selectivity, neither disrupting the favorable nherf :cftr interaction, nor strongly displacing other membrane proteins that interact with cal. using a fluorescence polarization assay and peptide-array technology, we have now identified a high-affinity, cal-selective peptide inhibitor. compared to cftr, this sequence binds cal more tightly, but nherf more weakly. furthermore, we have shown that the cal:cftr interaction is the weakest among the known cal-binding proteins, and that cftr should thus be susceptible to selective displacement from the cal binding site. finally, we have designed cell-permeable peptides that allow us to test the hypotheses (a) that such compounds will specifically disrupt the cal:cftr interaction and (b) that such targeted stereochemical inhibition will stabilize the functional cell-surface expression of ∆f -cftr in airway epithelial cells. the pathways for the endoplasmic reticulum associated protein degradation (erad) of misfolded proteins in the mammalian cells are incompletely understood. we investigated the role of molecular chaperones in erad for the cystic fibrosis transmembrane conductance regulator (cftr) and its common folding mutant, ∆f cftr. we found that hsp and its cochaperone hdj- interacted significantly with ∆f cftr and wt-cftr in airway epithelial cells. hsp interacted with the immature form of cftr while hdj- associated with both immature form and ubiquitylated cftr. structure-function studies showed that hdj- recognized ubiquitylated cftr via its zn-binding domain. immunoprecipitation and gst-fusion protein pulldown experiments revealed that hdj- interacted with ubiquitin. in steady-state, over-expression of hdj- elicited increasing both immature and mature forms of wt-cftr, but it resulted in decreasing immature form of ∆f cftr. pulse-chase studies showed that co-expression of hdj- promoted ∆f cftr degradation, reducing its half-life from to min. in contrast, hdj- expression did not significantly affect wt-cftr biogenesis. these data are consistent with our finding that hdj- shows a selective physical interaction with ∆f cftr, which translates into a functional discrimination between the mutant and its wt counterpart. the hpd motif within the j-domain of hdj- , which is necessary for the co-chaperone to bind hsp and stimulate its atpase activity, was required for hdj- mediated ∆f cftr degradation. mutation of the hpd motif retarded the degradation of ∆f cftr and increased the steady-state levels of ∆f cftr -fold. a role for hsp in hdj- mediated ∆f cftr degradation was tested in which hsp knockdown increased ∆f cftr expression - fold. hjd- regulates ∆f cftr maturation and knockdown of endogenous hdj- promoted ∆f cftr and its camp-dependent anion conductance in airway epithelial cells. in contrast, we were unable to detect maturation of ∆f cftr in hsp knockdown experiments, indicating that the maturation of ∆f cftr mediated by reduced hdj- expression may be independent of hsp function. taken together, our data demonstrate that hdj- is a molecular sensor that can detect differences in the folding related to the ∆f cftr maturation. these data also highlight a novel pathway for hdj- linked, ubiquitin-dependent degradation of ∆f cftr. reduction of hdj- expression or its association with ∆f cftr promotes maturation of this mutant and therefore represents a potential therapeutic approach for cystic fibrosis. [supported by nih and cf foundation] how the loss of cftr function results in cholesterol accumulation within the cell is currently unknown. cftr activation is driven by the camp and we propose that cf cells respond to the loss of cftr activity by increasing the camp pathway in order to increase cftr expression. to test this hypothesis, epithelial cells were treated with the phosphodiesterase- (pde ) and pde inhibitors milrinone and rolipram, respectively. inhibition of pde function with rolipram in wildtype cells leads to peri-nuclear free cholesterol accumulation identical to what is observed in cf epithelial cells as viewed by filipin staining, strongly implicating a camp-dependent pathway in the regulation of cholesterol trafficking. the pde -selective inhibitor milrinone had no effect on cholesterol trafficking suggesting specificity for the pde pathway. our preliminary data support this hypothesis in that both cf-model hteo-pcepr cells and mouse nasal epithelium (mne) from cftr -/-mice exhibit reduced protein expression of the campspecific phosphodiesterases pde compared to wt controls. the proposed consequence of a chronic amplification in camp signaling is altered cholesterol transport regulation. to address this potential role of pde in cholesterol trafficking, free cholesterol was visualized using filipin staining in wild type cells that were treated with rolipram, a pde -specific inhibitor. conversely, treatment of cf-model pcepr cells with the pka inhibitor rp-camps significantly improves cholesterol processing, further pointing to the involvement of the camp pathway. we propose that the camp pathway influences cholesterol processing through the regulation of β-arrestin- (βarr ) according to the premise that chronic increase in the camp pathway would initiate an elevation in βarr expression. βarr is an important regulator of adrenergic receptor recycling and organelle trafficking. because βarr is pivotal in regulating endocytotic recycling pathways, it could also impact cholesterol processing. this predicted increase in βarr protein expression is observed in both cf-model pcepr cells and cftr -/-mne compared to respective controls. over expression of βarr in wt epithelial cells leads to cf-like peri-nuclear cholesterol accumulation further implicating a role for βarr in the development of this phenotype. altered cholesterol trafficking in cftr would lead one to expect different βarr localization throughout the cell. further understanding of the implications of altered camp signaling and its relationship to aberrant cholesterol accumu-rationale: we recently reported that vcp (valosin containing protein) physically interacts with gp /amfr (autocrine motility factor receptor) to couple retrograde translocation of ∆f -cftr to proteasomal degradation (vij et al. jbc ) . recent studies have revealed an alternative system to the proteasome for degradation of polyubiquitinated misfolded proteins termed the aggresome. histone deacetylase- (hdac ) is a unique cytoplasmic deacetylase capable of interacting with ubiquitin and mediating the accumulation of ∆f -cftr in aggresome bodies. hypothesis: vcp competes with hdac to dissociate ∆f -cftr perinuclear aggregates. methods: ib - (∆f /w x) cf bronchial epithelial cells were transiently transfected with ∆f -cftr and vcp-gfp constructs and treated with a hdac inhibitor (tubacin), proteasome inhibitor (ps- or mg- ), lysosome inhibitor (baflomycin a ) or nil-tubacin (control) for , or hrs. the effect of these inhibitors on ∆f -cftr and vcp:∆f -cftr interactions were quantified by immunoprecipitating these protein complexes followed by immunoblotting with vcp, hdac or clathrin antibody. ∆f -cftr levels were measured by metabolic labeling using tran- s-label ( µci/ml) for a min pulse and hrs chase. the subsequent effects of these inhibitors on vcp localization was detected by fluorescence microscopy of gfp moiety. results: the co-immunoprecipitation experiments showed that hdac inhibition by µm tubacin ( hrs) promotes vcp:∆f -cftr and prevents hdac :∆f -cftr interaction. the proteasome inhibition (mg µm) resulted in maximal hdac :∆f -cftr at hrs with minimal at hrs while vcp:∆f -cftr increased overtime ( to hrs). adding tubacin ( µm; hrs) reversed the proteasomal inhibitor effects. in pulse-chase experiments, mg- increased the accumulation of cftr bform and poly-ubiquitinated-∆f -cftr as compared to the untreated control. tubacin suppressed the levels of mg- induced poly-ubiquinated-∆f -cftr as well as ∆f -cftr b-form. the vcp localization by microscopy showed the accumulation of vcp-gfp in perinuclear aggregates in the presence of proteasome inhibitor (ps- µm). the µm tubacin blocked these ps- induced perinuclear aggregates. we observed that vcp is associated with endocytic protein-complexes containing clathrin in the presence of ps- indicating cytosolic re-localization of vcp. we further confirmed the cytosolic re-localization of vcp-gfp in the presence of lysosome inhibitor (bafilomycin a µm) by fluorescent microscopy. conclusion: we found that a small molecule inhibitor of aggresome function prevents hdac :∆f -cftr interaction, promotes vcp:∆f -cftr interaction, and suppresses the accumulation of poly-ubiquinated-∆f -cftr. wang, y. ; jiang, y. ; zhu, n. ; feng, x. ; yang, h. , ; ma, t. , . membrane channel research laboratory, northeast normal university, changchun, china; . biopharmaceutical center, liaoning normal university, dalian, china deletion of the codon encoding the phenylalanine residue at position (∆f ) in the cystic fibrosis transmembrane conductance regulator (cftr) is the most common mutation causing cystic fibrosis (cf). the ∆f mutation results in a cftr protein with impaired folding, trafficking and gating in human and rodents. the consequences of ∆f -cftr mutation in other species have not been studied. the purpose of the present study was to characterize the ∆f mutant of porcine cftr in cell culture model. cdna sequence encoding full-length porcine cftr (pcftr) was cloned from the lung by rt-pcr using primers designed according to porcine genomic sequences in two bac clones (genbank accession no. ac and ac ) containing all exons of the porcine cftr. pcftr encodes a -amino-acid protein that shows . % identity to human cftr. the phenylalanine residue at position is conserved in pcftr. functional analysis of pcftr stably expressed in fisher rat thyroid (frt) epithelial cells by short-circuit current assays indicated a campactivated cl-channel similar to human cftr except that the sensitivity of pcftr to the specific cftr inhibitor cftrinh- is -fold lower than that of human cftr. a ∆f mutation of porcine cftr was generated by site-directed mutagenesis. surprisingly, ∆f -pcftr expressed as a func-tional chloride channel activated by forskolin in stably transfected frt cells. western blot analysis of cos and bhk cells transiently transfected with cmyc-tagged ∆f -pcftr indicated a protein pattern identical to that of wildtype pcftr. immunofluorescence analysis of the transiently transfected cells demonstrated similar plasma membrane expression pattern of ∆f -pcftr and wildtype pcftr. these results clearly demonstrated unimpaired cellular processing of ∆f -pcftr. however, the sensitivity of ∆f -pcftr to forskolin activation (ic ~ . µm) was dramatically lower than that of wildtype pcftr (ic ~ . µm), indicating a defect of channel gating by pka phosphorylation. the present study provided the first evidence that the ∆f mutation in mammalian cftr does not result in impaired cellular processing that is closely associated with cf phenotype. we have optimized airway epithelial cell spheroid cultures for functional measurements of cftr chloride conductance in order to screen smallmolecule activators and inhibitors. spheroids, which are sealed spheroidal monolayers of airway surface epithelial cells with diameter - micron, were generated within to days after nasal brushing and suspension culture. yields of up to spheroids were obtained from nasal brushing of a non-cf or cf subject. approximately , spheroids could be prepared from one million freshly isolated human bronchial epithelial cells. we compared several electrophysiological and fluorescence methods to assay cftr function in spheroids. whole-cell current was measured in single spheroids following pipette immobilization and micropipette puncture of the solutionexposed apical cell membrane. large, forskolin-stimulated whole-cell chloride currents were measured in non-cf spheroids. as an independent approach, transepithelial potential difference was measured by micropipette insertion into the spheroid lumen. for fluorescence detection, cell cytoplasm was stained with the halide indicators mqae or lzq. fluorescently stained spheroids were immobilized on a coated coverglass or in a custom microfluidic chamber. halide flux across the apical surface was measured in response to chloride-nitrate or iodide-chloride solution exchange, in the absence and presence of camp activators. the fluorescence assay is suitable for medium-throughput screening of ∆f correctors and potentiators, with definitive verification of cftr chloride channel function by whole-cell current analysis. an airway spheroid cultures provide a powerful approach to prioritize the efficacy of ∆f correctors because of their rapid generation without prolonged culture, and their suitability for quantitative analysis of cftr function. supported by cff and nih. background and aims: cftr has been shown to be expressed and functional in ventricular cardiomyocytes. however, to date, no physiological role for cftr in the heart has been established. the aim of this study was to determine if cftr plays a role in the regulation of cardiomyocyte contraction rate. methods: cardiomyocyte contraction rate was assessed using the neonatal mouse beating assay. ventricular myocytes were isolated from neonatal mice and cultured for - days until they formed a functional in sitium. contraction rates were captured by video imaging and analyzed by metamorph in the presence and absence of beta-adrenergic receptor stimulation by isoproteronol ( µm). assessment of cftr activity was determined by pharmacological inhibition by glibenclamide ( µm), dpc ( µm), and cftr inh- ( and µm). the role of k atp channels was assessed by -hd ( µm). results: baseline contraction rate of vehicletreated (dmso or h o) cardiomyocytes was unchanged. addition of isoproteronol caused a significant increase in contraction rate, which was sustained for at least minutes. in contrast, application of glibenclamide to beating ventricular myocytes significantly, but transiently, inhibited cardiomyocyte contraction rate, with recovery occurring within minutes. glibenclamide did not affect the initial increase in contraction rate caused by isoproteronol stimulation, but inhibited sustained increases in isoproteronol-stimulated contraction rate. to determine if this was an effect of k atp channel inhibition we examined the effect of -hd on contraction rate. in contrast to glibenclamide, -hd had no inhibitory affect on basal or isoproteronol-stimulated contraction rate, indicating glibenclamide's inhibitory effect was due to its inhibition of cftr. to further verify this, we examined the effect of two other cftr inhibitors, dpc and cftr inh- on ventricular cardiomyocyte contraction rate. both dpc and cftr inh- elicited similar responses as glibenclamide, with a quick, but transient inhibition of baseline contraction rate and inhibition of sustained isoproteronol-stimulated increases in cardiomyocyte beating. summary and conclusions: using neonatal murine ventricular myocyte cultures inhibition of cftr leads to a significant, but transient inhibition of baseline ventricular cardiomyocyte contraction rate. additionally, while inhibition of cftr did not affect initial increases in beta-adrenergic receptor-stimulated contraction rate, cftr inhibition did prevent sustained increases in contraction rate. the data suggest that cftr plays a role in modulating ventricular cardiomyocyte contraction rate during resting and adrenergic-stimulated conditions. drevillon, l. , ; tanguy, g. , ; hinzpeter, a. , ; arous, n. , ; goossens, m. , ; fanen, p. , . génétique, inserm u. , créteil, france; . faculté de médecine, université paris , créteil, france cystic fibrosis is mainly caused by mutations that interfere with the biosynthetic folding of the cftr protein. the aim of this study was to find cellular proteins capable of interacting with cftr and modifying its processing or its trafficking. we have used a genetic screen in yeast to identify such proteins and identified commd that preferentially interacted with the third cytoplasmic loop of cftr. commd is a regulator of copper transporter atp b in hepatocyte and sodium epithelial channel enac, but its exact biochemical function and physiological relevance remain elusive. here, we report that first, commd interacted with wild-type and f del-cftr in epithelial cells and second, commd subcellular distribution was not only both nuclear and cytoplasmic, but also in cytoplasmic vesicular compartments. we wondered if commd was involved in the processing and/or the trafficking of cftr protein in epithelial cells. we demonstrated that commd was not involved in cftr processing but that cell surface expression of wild-type cftr at the plasma membrane was cut by half when commd expression was % reduced by rna interference. we drew the conclusion that commd is a major component of cftr trafficking and/or recycling at the plasma membrane, precise characterization of commd in these vesicular compartments is under process to decipher its function in cftr trafficking. commd has been labelled as the prototype of a newly described protein family that plays a role in inhibiting nf-kappab signalling. moreover, subcellular distribution of commd was different in cf and non cf cells, nuclear form of commd being increased in f del cells compared to wild-type cells. thus, this opens up a new area of investigation about commd nuclear function. finally, these data indicate that commd is involved in multiple cellular processes of outstanding interest in cf pathophysiology. understanding how its modulation modifies transepithelial transport and inflammation in cf versus non cf cells should give new therapeutic clues to reduce exaggerated inflammation and improve fluid secretion in cf patients. supported ; fanen, p. ; galietta, l.j. . lab. of molecular genetics, istituto giannina gaslini, genova, italy; . inserm u. , creteil, france class iii cystic fibrosis mutations cause reduced cltransport by impairing the process of cftr channel opening. typical class iii mutations include g d, g d, and f del, all of them affecting nucleotide binding domains. it has been reported that mutations residing in other regions of the cftr protein may also reduce channel activity. we investigated the extent of loss of function caused by such mutations, in particular by missense mutations localized in cftr intracellular loops, and the sensitivity to pharmacological stimulation with cftr potentiators. to determine cftr activity, plasmids carrying the coding sequence for wild type or mutant cftr were co-transfected in cos- cells together with the halide-sensitive yellow fluorescent protein yfp-h q/i l. the rate of cftr-dependent anion transport was measured after short-term stimulation with forskolin alone or in combination with potentiators (felodipine, phenylglycine pg- , or sulfonamide sf- ). processing of cftr protein was assessed by examining glycosylation status of cftr by immunoprecipitation experiments. when stimulated with forskolin alone, cftr mutants showed the following anion transport compared to the wild type protein: i t ( %), i v ( %), q k ( %), e k ( %), g r ( %), d h ( %). interestingly, all mutants having a reduced anion transport responded to stimulation with potentiators pg- , sf- , and felodipine ( µm) with an increase in anion transport that approached the level of normal cftr protein. the correction of defective cftr activity by potentiators was confirmed in frt cells expressing e k and d h mutants using short-circuit current measurements. our results indicate that some missense cf mutations, like e k and g r, really cause a severe reduction in cftr activity. others, like d h, cause only a partial loss of function which may explain their association with a milder form of the disease. conversely, some other amino acid changes (with the extreme example of i v that seems to cause a gain of function) do not impair cftr activity and therefore may represent polymorphisms and not cf-causing mutations. interestingly, all mutants having a mild or more severe anion transport defect were sensitive to potentiators. this finding indicates that cftr potentiators have a wide efficacy on many class iii mutants and therefore may represent a promising therapeutic strategy for a significant number of cf patients. supported by cfft and telethon-italy. cftr, a cl-channel membrane protein, is a member of the atp-binding cassette (abc) super-family. mutations in cftr result in a misfolded protein which fails to mature, leading to impaired flow of cl-ions across the membrane of the epithelial cells lining the airway, and to cf. the most common mutation leading to cf is df . currently available structural information relevant to cftr modeling includes low resolution structures of cftr ( Å) and p-gp ( Å) and high resolution structures of other abc transporters as well as nucleotide binding domains (nbd) . of particular relevance is the newly published structure of the bacterial multidrug abc transporter, sav , whose topology can serve as a starting point for cftr modeling. as a first step towards the development of cf therapeutics, epix has undertaken the modeling of the d structure of cftr (excluding the r domain) in its conducting (i.e., open-channel, nbd-dimer) state, using our proprietary membrane-protein modeling technology (predicttm) in combination with other modeling tools. to date, we have modeled the cytosolic part of the receptor, namely the nbds:icls construct and work is ongoing on modeling the transmembrane (msd :msd ) region. the now available partial model has been scanned for putative binding sites for df -cftr correctors/potentiators. several potential sites have been located, and epix has initiated its in silico screening technology in order to identify potential drug candidates. this work is supported by the cfft. the deletion of a phenylalanine residue at position (f del) in the first nucleotide-binding domain (nbd ) of cftr is the principal cause of cystic fibrosis (cf). the altered interaction of f del cftr with endoplasmic reticulum (er) quality control proteins, primarily chaperones, promotes its proteasomal degradation. however, it is believed that crucial cftr-interacting proteins (cips) remain unknown [ ] . moreover, there is little information currently available on the strength of cip-cftr interactions. our goals here are two-fold: ) to quantify cftr-cip interactions; and ) to isolate and characterise unidentified cips. to address our first goal, we used surface plasmon resonance (spr; biacore tm ) to quantify real-time binding of hsc (a well-known cip) to bacterially expressed wt-and f del-nbd . hsc /hsp was covalently immobilised onto the surface of carboxymethyl dextran (cm ) sensor chips ( µm) and the real-time binding of purified nbd and control proteins quantified. in control experiments, anti-hsc antibody b ( nm; against residues - ) bound specifically to immobilised hsc with high affinity (k d , . ± . nm; n = ) whereas bovine serum albumin ( µm) did not interact (n = ). given the difficulties associated with expression of hnbd carrying the f del mutation [ ] it was not used in our spr analyses. instead, we used purified murine (m) nbd to quantify the impact of f del on the strength of the hsc -nbd binding. in the presence of low concentrations of atp (≤ um), the affinity of nbd binding to immobilised hsc was strengthened when f was deleted (wt, k d app , . ± . µm; f del, k d app , . ± . µm; n = ; p < . ). interestingly, raising the concentration of mgatp weakened the binding of nbd ( . µm) to hsc (wt, k i , µm atp; n = ). furthermore, deletion of f dramatically increased the concentration of mgatp required to destabilise the nbd -hsc interaction (f del, k i , µm atp; n = ). in summary, we used spr to demonstrate that: (i) nbd of cftr binds specifically to hsc , and (ii) the f del mutation enhances the association of nbd with hsc . we are presently investigating the effect of co-chaperones and small molecules on the interaction of hsc/hsp with wt-and f del-nbd . to identify novel cips, purified nbd was immobilised onto metalaffinity resin and used to capture cips from epithelial cell lysates. by this approach, cips were captured from human respiratory cell (calu- ) lysates and analysed by d-electrophoresis. relevant protein spots so far identified by mass spectrometry include: ) raichu x (thr/ser kinase); ) profilin isoform b; ) annexin a ; ) ifapsoriasin (intermediate filament-associated ca + regulatory protein); ) mgc (member of the er reticulon family). current analyses are underway to determine their functional roles in cftr trafficking and function. [ ] amaral ( billet, a. ; melin, p. ; norez, c. ; bilan, f. ; vandebrouck, c. ; mettey, y. ; becq, f. . physiologie, cnrs umr , poitiers, france; . gmas umr , poitiers, france; . faculté medecine et pharmacie, poitiers, france in order to gain a better insight into the structure-activity relationship (sar) of cftr protein, we have functionally characterized a cystic fibrosis mutation: the cftr-g d, identified in patients with oligospermia (vankeerberghen et al., ) . we examined cftr chloride channel activity by patch-clamp analysis, in whole cell configuration, using cos- cells transiently transfected with gfp-cftr-wt or with the mutant gfp-cftr-g d. cftr channels were activated by µm forskolin (fsk), and, after stable activation µm cftrinh- was added. as gfp-cftr-wt, g d chloride channels elicit a time and voltage independent current in presence of µm fsk. gfp-cftr-g d current density at + mv ( . ± . pa/pf) is . fold less than gfp-cftr-wt current density at + mv ( . ± . pa/pf). g d mediated currents are blocked by µm of the specific cftr-inhibitor, cftrinh- (ma et al., ) . as expected, gfp-cftr-wt channels are stimulated by µm of the potent activator -butyl- -hydroxy- -chlorobenzo[c]quinolizinium chloride (mpb- , derand et al., ) with a current density at + mv of . ± . pa/pf. interestingly, no activation of g d channels is recorded in presence of mpb- : difference between currents recorded in basal condition or in presence of mpb- is not significant. using iodide efflux experiments, we showed that cftr-g d mutant does not respond to several benzo[c]quinolizinium derivatives. however, the mutant channel can be activated by forskolin, ibmx and genistein. thus, the mechanisms of activation by xanthine and isoflavone are not affected by the mutation. these results show that: ( ) cftr-g d channels are functional: activation by camp pathway, inhibition by cftrinh- . ( ) camp chloride currents elicited by cftr-g d protein are weaker than cftr-wt. ( ) cftr-g d channels are refractory to benzo[c]quinolizinium activation. the replacement of the glycine by a negative charged amino acid in position affects cftr chloride channel activity and prevents activation by benzo [c] quinolizinium. this amino acid g , localized in the interface of nbd and r domain between two β sheets (callebaut et al., ) seems to have a crucial role in pharmacological activation of cftr by mpbs. further experiments will be performed to evaluate the role of the charge and of the nature of the amino acid at position in cftr protein. supported by vaincre la mucoviscidose and cnrs. cysteine string protein (csp) is a j-domain containing protein that regulates the extent of wild type cftr maturation. over-expression of csp blocks the formation of mature, glycosylated (band c) cftr and increases the level of immature (band b) cftr, whereas knockdown of csp elicits a marked increase in cftr maturation (zhang et al., j. biol. chem. : , ) . thus, csp appears to block the exit of cftr from the er. when the er exit of cftr is blocked by a different mechanism, i.e. the expression of mutant sar -gtp (sar h g interferes with the formation of copii vesicles), the steady-state level of immature cftr is two-fold greater than that resulting from csp over-expression. this finding suggests that csp, in addition to blocking er exit of cftr, may facilitate the degradation of the immature protein. in agreement with this concept, csp expression increased cftr ubiquitylation. a mutation within the conserved hpd motif of the j-domain (h q) disrupts the ability of csp to interact with and stimulate the atpase activity of hsp . csp h q does not block cftr maturation (above ref); in addition, the level of immature cftr was ~ % higher when h q csp was over-expressed than the level observed with mutant sar . this finding suggests that the pro-degradative effect of csp on immature cftr requires hsp binding/activation. chip (carboxy terminus of hsp -interacting protein) is an e ubiquitin protein ligase that can target wt and ∆f cftr for proteasome-mediated degradation (meacham et al., nature cell biol. : , ) . we sought to determine weather chip was involved in the csp-mediated degradation of cftr. indeed, co-immunoprecipitation (co-ip) experiments showed that chip associated with csp. also, csp h q, which has reduced interaction with hsp , showed a similar level of chip interaction by co-ip, suggesting that their association did not depend on hsp as a linker between the two proteins. a csp mutant that is truncated after the cysteine string domain, which is its site of membrane attachment, blocked cftr maturation as did wt csp; however, this mutant did not decrease cftr band b levels like wt csp did. in addition, the csp mutant missing its cterminus did not associate with chip. these findings further implicate chip in the csp-mediated degradation of immature cftr, and they suggest that the c-terminus of csp is a chip binding domain. when a dominant-negative mutant of chip, which lacks the u-box needed for chip-mediated ubiquitylation (chip∆ub), was co-expressed with cftr, csp was no longer able to facilitate the degradation of cftr in the er. these data show that it is possible to separate the effect of csp on cftr maturation from its ability to mediate the degradation of immature cftr. thus, csp blocks the exit of cftr from the er, and this requires a j-domain mediated activation of hsp but not the csp c-terminus. the action of csp on cftr degradation requires both a functional j-domain and the csp c-terminus, and this may require a trimeric complex involving csp, hsp and chip. [ the cystic fibrosis transmembrane conductance regulator (cftr) represents the main clchannel in the apical membrane of epithelial cells for camp-dependent clsecretion. mutations of this channel causes cystic fibrosis disease ; thus discovery of pharmacological activators of cftr is crucial to design future medicament for protein therapy. recently, we reported on the synthesis and screening of a small library of -phenylpyrrolo [ , b] pyrazines (named rp derivatives) evaluated as activators of wild-type cftr, g d-cftr and f del-cftr clchannels (noël et al, ) . this preliminary structure-activity relationship study identified -hydroxyphenyl and -n-butyl as determinants required for activation of cftr (rp- and rp- ). here we studied structure-function relationship of more than compounds prepared by chemical synthesis, and the subsequent activation of cftr channels. within the -phenylpyrrolo [ , -b] pyrazines family, we observed by iodide efflux technique that rp- bearing a hydroxyphenyl substituant is more potent (ec = nm) than rp- having -hydroxyphenyl substituant (ec = nm). by whole-cell patch clamp recording analysis, we confirmed that nanomolar concentrations of rp- activate linear chloride current in cho cells stably transfected with human wild-type cftr. this current was inhibited by µm of cftr inh - . we also found significant stimulation of short circuit current (i sc ) by rp- (ec = nm) on colon of cftr +/+ but not of cftr -/mice mounted in ussing chamber. stimulation of i sc by rp derivatives was inhibited by glibenclamide. the structural analogue , was less potent (ec = nm). as for rp- compound, we found that the -nbutyl chain is crucial for rp- and rp- activity, and that the -hydroxyphenyl compounds without -n-butyl chain are unactive on cftr. in this study, we showed that the presence of an hydroxyl group at position , or of the pyrrolopyrazine cycle determined the highest activity on cftr. the most potent compound is the -n-butyl- -( -hydroxyphenyl) hpyrrolo [ , b] pyrazine . the potency of these agents indicates that compounds in this class may be of therapeutic benefit in cftr-related diseases, including cystic fibrosis. the most common mutation causing cystic fibrosis (cf) is the deletion of f in the cftr gene, which results in inefficient trafficking of the cftr protein from the endoplasmic reticulum to the plasma membrane. the surface expression of the cftr is usually quantified biochemically with western blot or functionally with electrophysiological assays. however, these methods are labor intensive. we explore using fluorescence-based techniques for monitoring cftr surface expression. wild-type cftr was tagged at the amino terminus with green fluorescent polypeptides (gfp-cftr). the gfp-cftr was either transiently or stably expressed in chinese hamster ovary (cho) cells. the cells were then treated with a red fluorophore that specifically labels plasma membranes. the fluorescent images were acquired with an olympus laser scanning confocal microscope. two image channels were taken simultaneously. the green channel was excited with a nm laser, and signals within - nm were collected. the red channel was excited with a nm laser, and signals within - nm were collected. for a single focal plane, each image was composed of * pixels. threshold values were given to both green and red channels. pixels with an intensity value above the threshold were marked as green/red pixels. the green pixels in the first channel represent the signal of gfp-cftr, and the red pixels in the second channel represent the location of the plasma membrane. thus, pixels that are both green in the first channel and red in the second channel are the cftr surface expression, and denoted as yellow pixels. the percentage of successfully trafficked cftr is thus quantitatively estimated by the ratio of yellow to green pixels in the single plane. alternatively, a stack of images from the same cell at different depth was collected and analyzed. the total amount and surface portion of cftr expression are then determined by summing the correspondent pixels. the percentage of cftr surface expression was then calculated and will be verified both biochemically and functionally. the approach is modified to study deltaf cftr trafficking. we anticipate that the method can be used to screen cftr correctors from a small or intermediate sized chemical database. supported by cfft and nih. the most common mutation of the cystic fibrosis transmembrane conductance regulator (cftr) gene (deletion of phe- (∆f ) in the first nucleotide binding domain (nbd )) causes retention of the ∆f protein in the endoplasmic reticulum (er). ∆f mutation prevents conformational maturation of cftr protein without altering profoundly the local structure of nbd , but possibly by disrupting the interaction between nbd and nbd . however, the individual role of nbd and nbd in biogenesis, folding, maturation and membrane stability of a full length cftr is still under debate. using splicing by overlap extension method (soe by pcr), we generated six recombinant cftr proteins with inverted or suppressed nbds. the native boundary domains of nbd and nbd were respected for all mutants (riordan j.r. et al. ) and their c-terminal tails preserved. the constructs were transiently or stably expressed in cos- and bhk cells respectively. immmunodetection and immunolocalization assays confirmed that deletion of nbd domain (n -cftr) did not alter the trafficking or the plasma membrane expression of cftr. both processes were abolished in the presence of ∆f mutation. however, using whole cell patch-clamp configuration, we did not detect any camp-stimulated clcurrent. pulse-chase experiments established that the turnover of n -cftr is - fold faster (t / ~ h) than its wild type counterpart (t / ~ h). when we replaced nbd with nbd ( n -cftr), we could detect a faint plasma membrane expression associated with a weak cl-channel activation. more surprisingly, the t / of n -cftr coreglycosylated form was around h. when nbd was deleted (n -cftr) or substituted with nbd domain ( n -cftr), the expression and functional pattern were similar to cftr ∆f . furthermore, we showed that the n -cftr folding was not attenuated as demonstrated by protease susceptibility and sdsresistant thermoaggregation tendency, supporting the notion that n -cftr membrane stability requires nbd -nbd interaction. altogether, our results suggest that nbd domain is essential to form a foldable cftr that satisfies er quality control (erqc) but correct inter-domain assembly is mandatory for its stability at plasma membrane. the results are also in agreement with previous suggestion that ∆f mutation alters the interactions between nbds and transmembrane domains of cftr. supported . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm , inserm, paris, france; . inserm u , inserm, paris, france the physiological role of cftr in renal epithelium is not known. in the proximal tubule, cftr co-localizes with the sodium-phosphate cotransporter npt a, a protein involved in phosphate (pi) reabsorption. the aim of our work is to determine if there is a functional interaction between cftr and npt a. to this end, the arnm coding for cftr and the arn coding for npt a (bearing a myc tag in c-ter) were injected in xenopus laevis oocytes. electrophysiological or biochemical experiments were performed days after arns injection. currents induced by mm pi (ipi) were measured in voltage-clamped (- mv) oocytes that expressed npt a alone, or in oocytes co-expressing cftr + npt a. for immunoprecipitation experiments, - cftrab and myc ab were used. results ipi was significantly reduced in oocytes expressing cftr + npt a compared to that measured in oocytes expressing npt a alone. using a two-bath electrodes configuration during voltage-clamping did not change this observation. this result suggests that when expressed in oocytes, cftr is in functional interaction with npt a. kinetic analysis (ipi as a function of pi concentration) of npt a showed a reduction in vmax but not in phosphate km. this suggests that npt a membrane expression and/or activity is altered when cftr is co-expressed. co-immunoprecipitation experiments performed on protein lysate derived from oocytes co-expressing cftr and npt a suggested an interaction between the transporters. using a pka activating cocktail as a pkc activator induced cftr-mediated currents in cftr+npt a expressing oocytes. in these oocytes, ipi is decreased by pkc activation (as expected from npt a regulation) but is increased shortly after exposure to the pka activating cocktail. the increase of ipi observed under this condition is at variance to that expected from classical npt a regulation. in oocytes expressing npt a alone, both pka activation and pkc activation reduced ipi. these observations suggest that in oocytes, cftr expression modifies npt a regulation. our study suggests that, after expression in xenopus laevis oocytes, cftr interacts functionally with npt a, and modifies its regulation. an interaction is also suggested by the co-immunoprecipitation of cftr and npt a. these results cannot discriminate between a direct or an indirect (via a third protein) interaction. to determine if an intracellular chloride concentration change may participate to the functional interaction, we are now measuring in cftr+npt a expressing oocytes intracellular chloride activity (using chloride-sensitive microelectrodes) during the use of the pka activating cocktail, and measuring ipi when pka is stimulated in the presence of a specific cftr inhibitor. cystic fibrosis (cf), the most commonly inherited lethal pulmonary disorder in caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (cftr). to date, more than mutations were identified in cftr and were associated with clinical disease. a phenylalanine deletion at position accounts for % of cf genotypes in caucasian populations, and determines cftr misfolding and degradation by proteasome. consequently, limited cftr abundance leads to multiorgan disease, affecting the lung, pancreas, gut, liver, sweat glands and the reproductive organs. the past several years have witnessed an explosion of information regarding the identification and elucidation of molecules and pathways that are regulated by cftr or that regulate cftr activity. genomics and proteomics technologies now offer the opportunity to examine global alterations in the mrna and protein expression patterns of cf cells and tissues to elucidate the pathways linking defective cftr to clinical disease. here we describe systems biology methods that integrate heterogeneous datasets, including protein-protein interaction networks, gene expression and mass-spectrometry profiles, and mutation and genetic variation information, in order to identify the regulatory circuits and active subnetworks that are responsible for the progression and the severity of cf disease. the study will focus on correlations that define the key events in the cftr folding and trafficking pathways that lead to pathogenesis and dysfunctions, and could provide insights and targets for intervention and therapy. the number of cftr cl-channels in the plasma membrane, and thus the transepithelial cl-secretion is controlled, in part, by the endocytic trafficking of cftr. rab gtpases regulate the endocytic trafficking by acting as molecular switches that cycle between the gdp-bound (i.e. inactive) and the gtp-bound (i.e. active) state, associate with target membranes, and recruit downstream effectors. rab facilitates cftr endocytosis and rab a facilitates the recycling of internalized cftr to the plasma membrane. furthermore, rab and rab facilitate cftr trafficking to the lysosome. the mechanism of sorting internalized cftr for either recycling to the plasma membrane or degradation in the lysosome is not well understood. recent evidence demonstrates that protein sorting occurs in rab specific sorting endosomes. presence of cftr in rab compartment has been previously demonstrated in human airway epithelial cells by electron microscopy. yet, the role of rab in cftr trafficking has not been determined in these cells. rab did not control cftr trafficking in fibroblasts (bhk cells). however, it negatively regulated the plasma membrane expression of cftr in the colorectal cells . the goal of this study was to elucidate the role of rab in the endocytic trafficking of cftr in polarized human airway epithelial cells (calu- cells and cfbe o-cells stably expressing wt-cftr or deltaf -cftr). endogenous rab coimmunoprecipitated with cftr in calu- cell and cfbe o-cells. to determine if rab plays a role in cftr trafficking, cfbe o-cells were transfected with either the flag-tagged, dominant negative (dn) rab mutant deficient in gtp binding (gdp-locked; flag-rab -n i) or with a non-specific cdna control (gfp). if rab sorts internalized cftr to the recycling pathway, the dn rab should inhibit recycling, and, thus, should decrease the plasma membrane expression of cftr. if, on the other hand, rab sorts internalized cftr for degradation the dn rab should inhibit cftr degradation, increase cftr recycling and thus, should increase the expression of cftr in the plasma membrane. the dn rab increased cftr expression in the plasma membrane as determined by cell surface biotinylation. these data are consistent with the role of rab in sorting internalized cftr for degradation. similar to overexpression of the dn rab , silencing endogenous rab with double stranded small interfering rna specific for the human rab sequence increased the plasma membrane expression of cftr and increased the cftr mediated cl-secretion across polarized cfbe o-cells in ussing chamber experiments. the plasma membrane half-life of deltaf -cftr is decreased compared to wt-cftr. however, after silencing rab , the plasma membrane half-life of deltaf -cftr was similar to that of wt-cftr, as determined by cftr disappearance from the plasma membrane following incubation with cycloheximide. our data are consistent with the role of rab in sorting the internalized cftr for degradation and are consistent with the changing paradigm for the role of rab in protein trafficking (supported by the nih grant p rr and the cff swiate qo). the conformational changes underlying activation of phosphorylated cftr by atp remain unclear. in the present study we assessed the utility of labeling endogenous cysteine(s) in cftr using an environmentally-sensitive fluorescent probe alexa- in monitoring such changes in structure. these studies were performed using purified and functionally reconstituted wild-type cftr. we labeled pka-phosphorylated cftr with µm alexa- maleimide prior to reconstitution in phospholipids liposomes. changes in fluorescence intensity were monitored in a suspension of proteoliposomes following addition of and mm mg-atp or mm mg-amp-pnp in order to evaluate nucleotide dependent changes in conformation. in order to identify the labeled cysteines, the protein was subjected trypsin-mediated proteolysis and analysis by mass spectrometry. we have shown that cftr is labeled using alexa- maleimide. a significant increase in fluorescence emission occurred in alexa- labeled cftr of ± (au) relative to empty liposomes one minute after the addition of activating nucleotide, mm mg-atp (n = ). the non-hydrolyzable analogue, mm mg-amp-pnp, failed to cause an increase in fluorescence of alexa- -labeled cftr. however, the higher concentration of mm mg-amp-pnp evoked an increase in fluorescence similar in magnitude to that of mm mg-atp suggesting that the fluorescence response reflects structural changes relating to atp binding to cftr. analysis by mass spectrometry identified the labeled residue as c . this cysteine resides at the junction between nbd and the "r" domain in a region which has been suggested to be highly dynamic on the basis of multiple crystal structures. our studies suggest that this flexible region around c of cftr undergoes conformational change upon mg-atp binding such that it moves the alexa fluorophore into a relatively more polar environment. this finding presents a first case where conformational changes in the cftr nbds induced by atp binding could lead to changes in the conformation of the r domain. further, these studies suggest that fluorescence methods can be used to probe dynamic conformational changes in purified reconstituted cftr. acknowledgements cystic fibrosis is caused by mutations in the apical chloride channel cftr with % of patients carrying at least one allele of the ∆f mutation. this mutant form of cftr is characterized by a trafficking defect in which it fails to exit the endoplasmic reticulum (er). we have previously reported that ∆f cftr is found in complex with hsp and its co-chaperones in cell extracts suggesting that either this mutant form of the protein accumulates in on-pathway folding intermediates which result in its accumulation in the er or that these intermediates include inhibitory factors leading to retention of ∆f cftr in this compartment. we have examined the role of one of these hsp co-chaperones, fk binding protein (fkbp ), in the life cycle of cftr. fkbp is a unique member of the immunophilin family of peptidyl-prolyl isomerases (ppiases) which mediate the cis/trans interconversion of peptidyl-prolyl bonds, which is thought to represent a rate limiting step in protein folding. in order to establish a functional role for fkbp in cftr stability and trafficking, we modulated the expression level of this protein in human bronchial epithelial cells endogenously expressing either wild type or ∆f cftr. a knockdown of fkbp by sirna resulted in a destabilization of both wild type and ∆f cftr as well as a concomitant loss in channel activity of wild type and temperature corrected ∆f cftr. conversely, over expression of fkbp resulted in a stabilization of the er glycoform of ∆f cftr in both mammalian and yeast systems. these data suggest that fkbp is an essential component of the hsp -mediated folding machinery for both wild type and ∆f cftr, which supports the hypothesis that ∆f cftr accumulates in the er in an on-pathway folding intermediate. dmh cftr folding involves a series of sequential, although potentially overlapping steps that involve i) formation, orientation and integration of (tms), ii) helical packing, iii) folding of cytosolic and extracytosolic domains, and iv) establishing proper domain-domain contacts. this process begins cotranslationally and is facilitated by the sec er translocation machinery and a diverse group of lumenal and cytosolic chaperones. the most common inherited cftr mutation, deltaf , disrupts one or more early steps along this folding pathway. a major obstacle in understanding how deltaf causes cf is that the native folding environment, comprised of lipids, cytosolic and er proteins, is not amenable to traditional biochemical and biophysical approaches. to overcome this problem we are developing a flu-orescence based analytical approach that enables one to directly access nascent ribosome-attached folding intermediates at virtually any stage of synthesis. the strategy requires incorporation of fluorescent probes into the substrate protein, which provide a highly selective means to monitor structural features at sub-nanomolar concentrations in highly complex biological mixtures. polypeptides are synthesized in vitro from truncated mrna templates to generate uniform cohorts of ribosome-bound nascent chains. progressive stages of folding can then be evaluated using mrna templates of different length. as proof-of-principle we have shown that fully synthesized gfp variants can be readily trapped in an unfolded state and complete their folding only after the entire nascent peptide has exited the large ribosomal subunit. this approach enabled us to measure real-time folding kinetics of extended peptide domains following synchronous ribosome release and compare fluorophore maturation of four fluorescent protein variants with different spectral properties cfp, gfp, venus and mcherry. to extend this approach to cftr and other general protein substrates, we are employing fluorescence resonance energy transfer (fret) using "donor" and "acceptor" probes incorporated simultaneously into the substrate protein. an in frame n-terminal fusion to cfp provides the "donor" fluorophore, while the "acceptor" fluorophore is incorporated at an engineered stop codon using a synthetic modified aminoacyl-suppressor trna. we are currently developing a 'molecular ruler' to correlate the efficiency of energy transfer with changes in the relative distance between probes in ribosome-bound and ribosome-released cftr folding intermediates. fret-based experiments are underway to evaluate folding of both n-terminal ( - aa) and nbd ( - aa) domains. these studies will provide new insight into the mechanism of cotranslational cftr domain folding and provide a potential means to identify agents that correct the deltaf folding defect. relationships between hypoxia, ado release, and prostenoid production in ariway cells are not defined. in the present studies, we examined mechanisms governing hypoxia-induced production of prostenoids in cfbe o-and calu- monolayers. using elisa-based detection of prostenoid and leukotriene production, both calu- and cfbe omonolayers treated with ado ( µm) or arachidonic acid (aa, µm, the parent molecule of prostenoids and leukotrienes) for hrs led to increases in pge levels ( - ± - pg/mg of total proteins vs controls ± pg/mg in calu- cells, - ± - pg/mg vs controls ± pg/mg in cfbe o-cells, p< . in both airway cell types compared with control). in contrast, ltb and ltc release were not stimulated by both maneuvers. we next used sirna techniques to knockdown a b adenosine receptor expression, and demonstrated durable knockdown of a b ar expression to~ % of scrambled sirna-treated controls following lipid-based transfection with sirna directed against the a b ar in cfbe o-cells. following sirna knockdown of a b ar expression, cells were exposed to hypoxic conditions (mucosal volume = ml in -well plate), normoxic conditions (mucosal volume = . ml, fio = . ), and ado ( µm). hypoxic stress led to high levels of mucosal pge production ( ± . pg/mg vs controls ± . pg/mg, p< . ) that was sensitive to a b ar knockdown ( ± . pg/mg, p< . ). exogenous ado also stimulated pge production, but this effect was small relative to hypoxia. we next examined basolateral effects of ado and aa on transmucosal clsecretion in cfbe o-and calu- cells grown as monolayers on permeable supports and conducted ussing chamber analysis under voltage clamp conditions. addition of ado and aa to the basolateral membrane ( µm) activated transmucosal clsecretion [ . ± . µa/cm (ado) and . ± . µa/cm (aa) for calu- cells; and . ± . µa/cm (ado) and . ± . µa/cm (aa) for cfbe o-cells expressing wtcftr] that was sensitive to basolateral blockade with barium cl -( mm). isc by either agonist was stimulat-ed in the presence and absence of apical glybenclamide ( µm), adenosine deaminase (ada - u/ml) and hexokinase (hexo - u/ml), confirming that the effects were specific for the basolateral membrane and could be accomplished with or without cftr activity. these results demonstrate that hypoxia is sufficient to stimulate pge production through a b ars, and that both ado and aa activate basolateral k + channels to promote transepithelial clsecretion. the findings further support a model in which ado (and prostenoids) regulate cltransport through effects on both the apical and the basolateral membrane, and elucidate molecules that couple the two membranes as part of the normal clsecretory response. hypoxia would be predicted to increase local prostenoid production through ado release and stimulation of a b ars, and further highlight a unifying model by which local hypoxia promotes inflammation in airway epithelia. supported by the nih and cff. low levels of tissue oxygen -observed in lung diseases such as cystic fibrosis -initiate a signaling cascade resulting in altered transcription of genes possessing a hypoxia response element (hre). we recently used gene chip mrna array, quantitative rt-pcr, protein analysis, and functional (short circuit current) assays to show that cftr expression is strongly repressed by hypoxia in vitro and in vivo. among several thousand human mrnas suppressed by low ambient oxygen, cftr was among the most strongly inhibited (by - fold) in a cell model system. however, cftr and the vast majority of other repressed genes lack a traditional hre. because hypoxia inducible factor (hif) acts primarily as a transcriptional activator, the mechanisms underlying hypoxia mediated suppression of mammalian genes are not well understood. we therefore tested the hypothesis that micrornas play a central role during transcriptional regulation of genes such as cftr. micrornas are evolutionarily conserved, short noncoding rna sequences believed important for repression of gene expression. some reports have estimated that at least % of protein-coding genes are regulated by micrornas. thousands of micrornas have been predicted in the human genome by available algorithms (e.g. palgrade, mirscan, and promir) and several hundred experimentally validated. the cftr gene contains predicted target sites for micrornas in the 'utr, including hsa-mir- (nucleotides - of the cftr 'utr), hsa-mir- (nt - ) and hsa-mir- (nt - ). however, no study to date has examined the effect of micrornas on cftr message levels, or the influence of micrornas during the cftr response to environmental perturbations such as hypoxia. we therefore performed a global, genome-wide analyses of both mrnas and micrornas expressed during hypoxia (using air/liquid or liquid/liquid interface culture systems) in ht (colonic epithelial) cells that robustly express cftr. we found that approximately % of micrornas were significantly altered by hypoxia, as judged by mirna profiling (ambion mirvana™ bioarray v , asuragen microrna expression profiling service). comparison of these hypoxia-related micrornas with expression profiles of their predicted targets indicated a much lower level of correlation than has been previously hypothesized by others. we show that none of the micrornas predicted to regulate cftr are altered in ht (in response to hypoxia). these findings are therefore consistent with the notion that highly expressed genes in a particular tissue type are not co-expressed with their predicted regulatory micrornas. our data also indicate that micrornas do not mediate the hypoxic repression of cftr via the ' utr. supported by nih and cff. . physiology, mcgill university, montreal, qc, canada; . biochemistry, mcgill university, montreal, qc, canada the ∆f mutation impairs cftr maturation and trafficking to the plasma membrane and results in a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. using our highthroughput trafficking assay, we previously showed that the quinoline km , a structural analogue of sildenafil, corrects the ∆f -cftr processing defect and leads to a significant increase in cftr surface expression after h treatment with µm and after h treatment with nm according to detection of mature cftr in western-blots. additional studies have now been carried out with other preparations to assess the functionality of ∆f -cftr following rescue by km treatment. different time and concentration exposures were tested on the cftr function using iodide efflux assays with both bhk and human bronchial epithelial cells over-expressing ∆f -cftr. exposure to µm km for h partially restored iodide efflux responses to µm forskolin + µm genistein in both cell line. after h incubation, rescue of the mutant protein in bhk cells still required µm, whereas nm was sufficient in the human cell line cfbe. iodide efflux assays were performed at different times following km wash out to functionally assess the stability of the rescued mutant protein. camp-stimulated iodide efflux was still detectable h after removing km , but responsiveness was lost after h in both cell types. electrophysiological studies were performed to examine the channel activity of ∆f -cftr rescued by km treatment. incubation with µm km for h increased camp-responsive ∆f -cftr current density in hek whole-cell patches ~ -fold. the conductance was time-and voltage-independent, anion selective, and strongly inhibited by glibenclamide. incubating polarized cfbe monolayers with µm km increased the forskolin + genistein-stimulated short-circuit current after h, and this response was inhibited by cftrinh ( µm). permeabilization of the basolateral membrane with nystatin confirmed the apical location of the forskolin + genistein-stimulation. ussing chamber experiments also revealed trafficking correction in ileum that had been excised from cftrtm eu mice homozygous for ∆f . when the ileum was pre-incubated with µm km for - h, the forskolin + genistein-stimulation of short-circuit current was significantly increased when compared to untreated intestine. these results confirm partial correction of ∆f -cftr by km in unpolarized cells and in human polarized epithelial cell monolayers and ex-vivo intestine isolated from ∆f -cftr mice. this work was supported by the breathe program funded by the ccff and cihr, cihr, cfft, and génome québec. primary sequence and overall structure of human cftr overlaps significantly with murine, rabbit and other homologues, a feature that has limited generation of conformation specific reagents to address cftr folding. because regions of ∆f cftr exposed extracellularly may exhibit aberrant topology (jbc : - ), probes that identify cftr from the extracellular surface could be useful to ) verify plasma membrane localization of ∆f cftr, ) discriminate wild-type from mutant protein, and ) provide a means of determining whether small molecule "correctors" of the ∆f processing lead to a native (wildtype) cftr configuration. our laboratories are investigating phage-display techniques suitable for detecting properly folded extracellular domains of cftr. two large phage libraries encoding or amino acid sequences (approximately phage per library) were panned against extracellular regions of cftr in hela cells. following incubation in blocking solution containing pbs and % bsa at °c, unbound fraction was incubated with recombinant helas expressing wild-type or temperature corrected ∆f cftr (protein expression verified biochemically and functionally). bound bacteriophage was dislodged after multiple washings by exposure to an acidic elution buffer and amplified to increase copy number (five rounds of antigen selection for each library, eur j biochem : - ). supernatants from microtiter wells expressing phage were analyzed for binding to parental (no cftr), wt or ∆f cftr expressing hela cells. using this method we obtained enrichment of phage at the surface of cells expressing wt or ∆f cftr. an example using a -amino acid sequence ( phage) with specificity for ∆f cftr (representative of four repeat experiments) is shown in a binding assay with hela cells/condition. reagents such as these can be further optimized by constructing second generation libraries using core recognition sequences (derived from the initial screen) and surrounding the core epitopes with random amino acid diversity. these experiments are intended to identify peptides with high binding affinity (nm binding constants) that recognize native cftr epitopes exposed extracellularly. because there is evidence that low temperature corrected ∆f cftr in the plasma membrane is misfolded, these probes may ultimately distinguish wild-type cftr from the surface targeted ∆f mutant and be useful as drug discovery reagents. supported by cff and nih. bridges, r.; nagubadi, s.; thakerar, a.; jia, y.; bradbury, n.a. physiology and biophysics, rosalind franklin university of medicine and science, north chicago, il, usa the goal of this project was to compare several reported correctors of ∆f -cftr biosynthesis for their efficacy on chloride channel activity under a standard set of experimental conditions. fisher rat thyroid (frt) cells were transfected with ∆f -cftr and a stable cell line selected with g . cells were grown on transwell filters and studied under short circuit current (i sc ) conditions in ussing chambers. i sc measurements were performed at °c. the basolateral membrane was permeabilized with α toxin and chloride currents were measured with a serosal to mucosal chloride gradient. atp ( mm) and gtp ( µm) were added to the serosal bath. cftr channel activity was stimulated with camp ( µm), ibmx ( µm) and genistein ( µm). cells were treated with correctors or vehicle (dmso) at and hours before the i sc measurements. a subgroup of cells were temperature corrected at °c for hours for comparison. because only modest currents were observed even in the temperature corrected cells ( - µa/cm ) cells were treated for hours with mm sodium butyrate and nm dexamethasone to enhance the observed currents. under these conditions temperature corrected cells displayed stimulated chloride currents of ~ µa/cm that were - -fold greater than cells maintained at °c. correctors c (n- ' ]bithiazolyl- '-yl]-benzamide) and c ( -( h-benzomidazol- -ylsulfanylmethyl)- -( -methoxy- -methyl-quinazolin- -ylamino)pyrimidin- -ol) caused a modest rescue of ∆f -cftr chloride currents compared to cells maintained at °c (c , . -fold; c , . -fold) but far below the effect of temperature correction. rescue by c was concentration dependent with a significant observable effect at nm and a maximum effect at µm. rescue by c was observed at only µm, lower concentrations had no effect and higher concentrations caused an inhibition of the stimulated chloride currents. compounds c -piperazin- -yl]-ethyl}- -piperidin- -yl-quinazoline) ( to µm), c ( -cyclohexyloxy- -{ - [ -( - methoxy-benzensulfonyl)-piperazin- yl]-ethyl}-quinazoline) ( to µm) and phenylbutyrate ( mm) did not significantly increase the stimulated chloride currents. all four compounds c -c caused significant decreases in the transepithelial resistances at the higher concentrations ( - µm) suggesting some degree of cytotoxicity. our results indicate only c and c demonstrate efficacy in frt cells and that these effects are modest compared to temperature correction. supported by the cystic fibrosis foundation. cftr contains two membrane-spanning domains (msd), two nucleotide-binding domains (nbd), and a regulatory domain that require proper assembly for chloride channel activity. the most prevalent disease causing mutation, cftr∆f , arises from deletion of phenylalanine in the nbd domain. the cftr∆f mutant is translated and inserted into the endoplasmic reticulum (er) membrane, but is unable to attain its native state and accumulates in a kinetically trapped conformation that is degraded by the ubiquitin proteasome system ( ) . the nature of the misfolded cftr∆f intermediate that is selected for premature degradation is not clear and is a topic of great interest ( ) . recent work in the cyr lab indicate that defects in cftr and cftr∆f folding are sensed by two different e ubiquitin ligase complexes that are located in the er membrane and cytosol ( ) . the membrane-associated derlin- /rma e complex appears to recognize early folding defects of cftr that may involve assembly of msd into a complex with the amino-terminal regions of cftr. in contrast, the cytosolic hsc /chip e complex appears to sense folding defects that occur after synthesis of the nbd domain. misfolded cftrs recognized at either checkpoint are retained and degraded via the proteasomal pathway. the presence of dual protein quality control (qc) checkpoints suggests a mechanism by which the folding status of cftrs membrane and cytosolic domains are sequentially monitored prior to its escape from the er. currently little is known about how these erqc complexes sense misfolded substrates. small-molecule chemical correctors have recently been identified which rescue the folding defect of cftr∆f in model cell culture systems. in the presence of these chemical correctors a subset of cftr∆f is able to localize to the cell surface and retain channel function. however, how the chemical correctors rescue cftr∆f folding is not clear. we have examined the extent to which the molecules correct folding defects that are recognized by the derlin- /rma or hsc /chip qc checkpoints. in addition, because it is unknown at what point of cftr biogenesis that the chemical correctors act to facilitate folding, we have determined how the chemical correctors affect the folding of different biogenic intermediates. our results indicate that the correctors act in a post-translational manner to influence assembly of cftr sub-domains. interactions with members of the qc pathway have also been used to monitor the folding and assembly process of cftr in the presence of chemical correctors. ultimately the results from these studies will enhance our knowledge of how chemical correctors permit passage of the cftr mutants past different erqc checkpoints. supported by -cystic fibrosis foundation combination therapy has proven successful in treating a wide variety of human diseases, including cancer, infectious disease, and diabetes. historically, combination treatments have been discovered through trial and error using drugs with proven disease modifying effects. we have developed a combination high-throughput screening (chts™) technology to systematically and efficiently find synergistic combinations that target multiple pathways underlying disease biology, and that may be developed into new therapeutics. we are applying chts technology to discover and develop combination therapeutics for the treatment of cystic fibrosis. we have optimized two high throughput screening assays for identifying correctors and potentiators of the cftr (cystic fibrosis transmembrane protein regulator) ∆f mutation. one assay uses the flipr membrane potential dye (fmp) and the other uses genetically modified yellow fluorescent protein yfp h q/i l to measure changes in halide flux through ∆f cftr. both assays were optimized and validated on the flipr tetra using the panel of cf correctors, potentiators and inhibitors provided by the cf foundation. using our automated chts, we systematically evaluate pairwise combinations of a library of ~ , approved drugs and other bioactive molecules to identify novel disease-relevant biological interactions. candidate single agents and combinations are tested in secondary assays and prioritized for testing in animal efficacy models. combination therapies can act on multiple pathways in a coordinated way. chts may prove to be a useful tool for identifying combination therapeutic candidates with novel multi-target mechanisms and a way to enhance co-therapy regimens for the treatment of cystic fibrosis. supported the highly variable clinical phenotypes of cf airway disease suggest that a number of genetic factors, other than cftr, play a role in its pathophysiology. some of these modifier genes are expected to play a role in the endoplasmic reticulum quality control (erqc), since the major defect caused by f del is misfolding, retention and degradation of the mutant protein by this cellular surveillance system. the nematode caenorhabditis elegans is an excellent multicellular genetic model, which has been successfully used in studies of human diseases. its ~ , -genes genome has been fully sequenced, it has short life-span ( - weeks) and life cycle (~ . days) and it is easily cultured and amenable for gene disruption, both by knockout or rnai. our goal is to generate a c. elegans model for the cftr folding defect, so as to take advantage of this model for the identification of genes involved in cftr folding and/or degradation. to this end, and because no cftr orthologue has been described in c. elegans, we constructed two previously described human p-gp-/cftr chimeras (p-gp/wt-cftr and p-gp/f del-cftr) [ , ] to be used as an in vivo folding substrate in this organism. the two chimeric cdnas and intact human p-gp cdna were cloned into the c. elegans ubiquitous expression vector ps and injected into mutant nematode strains for multidrug resistance genes [ ] . the effect on the nematodes phenotype was evaluated by an assay of heavy metal sensitivity ( . mm arsenite), as described [ ] . our quantitative results show that the p-gp/wt-cftr chimera increases the resistance to arsenite when injected into the pgp- /pgp- c. elegans double mutant, whereas p-gp/f del-cftr causes no effect. these preliminary results indicate it is possible to generate two distinct nematode phenotypes caused by each of the transgenic chimeras (p-gp/wt-cftr and pgp/f del-cftr). they also suggest that the same folding defect impairing f del-cftr function in cf may be responsible for the loss of heavy metal resistance function by the p-gp/f del-cftr chimera. these chimeras are currently under test in pgp- /pgp- double and in pgp- /pgp- /mrp- triple c. elegans mutants. analysis of these strains by rt-pcr showed that the mrnas from the two p-gp/cftr chimeras have low expression levels. this led us to produce more robust constructs, including: ) to test chimera expression under the c. elegans endogenous pgp- or intestinal-specific (potent) promoters; and ) to develop p-gp chimeras with a full cftr-nbd . these c. elegans models will be used in genome-wide rnai screens to identify genes involved in the erqc of the p-gp/cftr chimeras. work channel gating of cftr is regulated by atp binding and hydrolysis at the nbds and by pka phosphorylation at multiple sites, primarily within the intrinsically disordered regulatory (r) region. phosphorylation at multiple sites is required for full activation of channel function, but no one specific phosphorylation site is required. the phospho-regulation of r region interactions with nbd likely contributes to the regulation of cftr channel function by modulating r region interactions with nbd at the nbd /nbd dimerization interface, consistent with nbd crystal structures that include the regulatory extension (re) comprising the first residues of the r region ( ). this regulation likely occurs via a dynamic complex where multiple segments of nonphosphorylated r region with αhelical propensity transiently engage nbd and are released. pka phosphorylation disrupts r region α-helical propensity and decreases binding of each interacting segment ( ) . regulation of the ∆f cftr by pka is altered, with the rate of channel activation by pka -fold slower than for wild-type ( ). we hypothesize that the dynamic interactions between the r region and nbd are affected by the ∆f mutation. to characterize the differences in r region binding between wild-type and ∆f nbd at residue-level resolution, we use nuclear magnetic resonance (nmr) techniques. experiments are performed using either r region or nbd samples labeled with nmr-active nuclei and monitoring their structural changes upon the addition of unlabeled binding partner, allowing us to confirm r region binding to nbd at the nbd /nbd dimerization interface and to identify other potential interaction surfaces on both the r region and nbd . together, these experiments will allow us to characterize the dynamic associations between the r region and nbd in order to better understand the molecular basis for the pathogenesis associated with ∆f cftr. funded by the canadian institutes for health research, the canadian cystic fibrosis foundation, and the us cystic fibrosis foundation . lewis, h.a., et al, embo j. , - ( ) . . baker, j.m.r., hudson, r.p., kanelis, v., choy, w-y, thibodeau, p.h., thomas, p.j., and forman-kay, j.d. submitted. the ∆f -cftr mutation, the most common gene mutation in cystic fibrosis (cf), results in diminished plasma membrane expression of cftr, leading to loss of functional cftr and altered mucociliary clearance. this impairment promotes chronic infection of cf patients by the opportunistic pathogen, pseudomonas aeruginosa. we previously reported that a secreted protein from p. aeruginosa (cftr inhibitory factor (cif)) reduces the wt-cftr and ∆f -cftr-mediated chloride secretion and the plasma membrane expression of cftr by decreasing endocytic recycling of cftr. the aim of the current study was to investigate the mechanism by which cif is secreted by p. aeruginosa and delivered to human airway epithelial cells (cfbe o-). in this study, we show that cif is packaged in outer membrane vesicles (omv) for secretion from p. aeruginosa. interestingly, cif secretion via omv was increased when the bacteria were exposed to airway epithelial cells. changes in cif protein level or bacterial growth did not account for the increase in cif secretion. purification and application of p. aeruginosa omv to airway epithelial cells caused a decrease in plasma membrane expression of cftr. the decreased plasma membrane cftr expression was followed by increased ubiquitination and lysosomal degradation of cftr. cif delivery to the airway epithelial cells via omv showed a more robust decrease in plasma membrane cftr expression, in comparison with cif delivery as purified protein. to investigate the mechanisms whereby cif enters epithelial cells, optiprep gradient fractionation experiments were conducted in polarized airway epithelial cells. in airway epithelial cells treated with p. aeruginosa omv, cif was associated with airway cell lipid rafts. pharmacological inhibition of lipid raft formation with filipin iii blocked the decrease in cftr plasma membrane expression upon treatment with omv. these studies demonstrate that cif is released by p. aeruginosa via omv and enters the airway epithelial cells through lipid rafts. upon entry into the epithelial cell, cif increases the ubiquitination and degradation of cftr and redirects cftr trafficking from the recycling pathway to the lysosomal-degradative pathway. these data suggest that chronic infection of p. aeruginosa in the cf lung may reduce the efficacy of therapeutics developed to increase plasma membrane expression of the mutant ∆f -cftr (supported by the nih ro hl - and t -dk - ). . physiology, dartmouth medical school, hanover, nh, usa; . microbiology and immunology, dartmouth medical school, hanover, nh, usa we have identified and cloned a secreted protein from pseudomonas aeruginosa (p. aeruginosa pa and clinical isolates), designated cftr inhibitory factor (cif). cif reduces the apical membrane expression of cftr and inhibits the cftr-mediated chloride ion secretion by human airway epithelial cells expressing wt-cftr and df -cftr. cif does not have general effects on protein trafficking, as the localization and expression of gp , na + -k + -atpase, and the transferrin receptor were not affected. cftr is a member of the atp-binding cassette (abc) transporter superfamily. abc proteins are atp-dependent transporters involved in exporting a wide variety of cytotoxic agents across the plasma membrane. p-glycoprotein (pgp; abcb , mdr gene product), also an abc transporter, is one of the major drug-efflux pumps expressed in normal tissues, as well as in many human cancers. over-expression of pgp results in reduced intracellular drug concentration and reduced cytotoxicity, conferring multi-drug resistance (mdr) to cancer cells. the aim of the current study was to examine whether cif also affects pgp expression in the plasma membrane, which could be exploited as a potential therapeutic strategy for restoring the sensitivity to pgp-transported drugs in cancer chemotherapy. cif significantly reduced the apical expression of pgp in mdck cells stably transfected with gfp-tagged pgp (mdck-gfp-mdr cells) and in caco- and calu- cells, which express endogenous pgp in the apical plasma membrane. the drug sensitivity of mdck-gfp-mdr cells to doxorubicin, a pgp substrate, was evaluated in the absence and presence of cif by measuring the ec . cif reduced the cytotoxicity of doxorubicin by -fold. neither cif nor vehicle alone had a cytotoxic effect. the drug sensitivity of the parental cell line, mdck-c , that expresses -fold less pgp than the transfected mdck cells, to doxorubicin was also increased by -fold. by contrast, cif had no effect on the ec values of cisplatin (a substrate of mrp ) and etoposide (a substrate of mrp ), suggesting that the alteration of the sensitivity to doxorubicin was due to decreased apical expression of pgp, but not other abc transporters. these results suggest that although cif may be an obstacle to therapeutic attempts to restore the apical expression of cftr in the cf lung, it could be useful for the development of a novel class of inhibitors of pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs (supported by the nih ( ro hl )). hamai, h. ; keyserman, f. ; worgall, t.s. , . pathology, columbia university, new york, ny, usa; . pediatrics, columbia university, new york, ny, usa cystic fibrosis (cf) is characterized by an inflammatory state and susceptibility to chronic lung infections. a common clinical finding is dyslipidemia characterized by altered plasma free fatty acid patterns, low plasma hdl levels and increased phospholipase a (pla ) activity. it is not clear how cftr mutations relate to lipid abnormalities. it was shown that defective cftr is associated with decreased uptake of sphingolipids (sl). sl homeostasis is tightly regulated and metabolites are relevant to lipid metabolism and cf. sl synthesis (sls) correlates with activity of srebp, a key transcription factor of lipid metabolism; ceramide- -phosphate, a sl metabolite, is a regulator of pla that is highly activated in cf. we investigated the hypothesis that sls is increased in cf. experiments were carried out in airway epithelial cell lines that express defective cftr (c / ib ), no cftr ( hbe sense / antisense) and overexpress cftr (a cells infected with adcftr or adcontrol). sls was assessed using radioactive tracers h-serine (de-novo sls) and h-sphinganine (recycling pathways). ceramide and sphingomyelin mass were determined enzymatically. neutral and alkaline sphingomyelinase activity was assessed using h-sphingomyelin as substrate. srebp mediated gene transcription was assessed using sre-promoter assays. cholesterol synthesis was evaluated by incorporation of h-acetic and h-mevalonic acid. cholesterol mass was determined by gas-chromatography. abca- expression was determined by western blot analysis. pla activity was measured using fluorescent phospholipid substrates. sls was post-transcriptionally increased in cells expressing defective or no cftr through the de-novo pathway ( % ± % for ib ; %± % for hbe-antisense; p< . ) and the recycling pathways ( % ± % or ib ; % ± % for hbe-antisense; p< . ). ceramide mass was increased by % (± %) in hbe antisense, % ± % in ib cells. sphingomyelin mass was increased -fold in ib , up to -fold in hbe antisense cells. neutral and alkaline sphingomyelinase activity was increased up to -fold in both cell lines. sre-mediated gene expression was increased up to -fold in ib and by % in hbe-antisense cells. overexpression of cftr in a decreased sls and srebp activity up to %. free cholesterol synthesis was increased by % in ib and by % in hbe antisense. expression of the cholesterol efflux receptor abca- was decreased in ib and hbe antisense compared to controls. sl mass, sre-mediated gene transcription and pla activity were decreased following incubation with inhibitors of sls in ib and hbe antisense cells. conclusion: defective cftr or lack of cftr expression correlates with ) increased sls synthesis and sl mass; ) increased sremediated gene transcription and free cholesterol synthesis; ) decreased expression of the abca- cholesterol efflux receptor. inhibition of sls decreases sre-mediated gene transcription and pla activity. dysfunctional sls is a newly recognized pathway associated with defective cftr and a possible therapeutic target in cf. the ability to make accurate, reproducible measures of mucociliary and cough clearance (mcc/cc) in cf patients is critical to assessment of new therapies designed to improve mcc/cc function, and thus decrease pulmonary infections and decline in lung function. for example, using mcc/cc methods developed in our laboratory, we showed that -week treatment with aerosolized hypertonic ( %) saline (hs) led to a sustained increase in mcc and improved lung function in cf patients (donaldson et al, n eng j med ; : ) . the mcc/cc technique involves patient inhalation of radiolabeled particles followed by gamma camera scanning to determine the rate of particle movement from the lungs. while a few cf centers have shown the capability to measure mcc/cc, the techniques across these centers are not standardized, making comparison of results difficult. it is vital to develop standard, simplified techniques for these measures to make larger cf patient populations available for such studies. to standardize a radiolabeled particle inhalation technique that can be easily used by other cf centers, we have compared three protocols in healthy and cf subjects. protocol # was identical to that used in recent studies, and incorporates a devilbiss jet nebulizer to generate µm mmad (gsd = . ), aqueous droplets containing suspended tc m-sulfur colloid particles. tidal volume and inhaled/exhaled flow rates were guided by a volume signal displayed on an oscilloscope. protocol # was further standardized and simplified to allow use at other centers. this protocol utilized the same nebulizer, but triggered by a commercial dosimeter (spira electro , finland) during inhalation as the subject breathed in time to a metronome ( breaths/min) at flow rate of . l/sec. subjects controlled inspiratory flow rate by feedback from a digital flow readout on the dosimeter. repeat measurements of mcc/cc were made in healthy subjects and mild cf patients to compare mcc/cc variables following radioaerosol inhalation using protocols # and # . there were no differences in particle deposition (i.e. central to peripheral ratio, or "c/p"), or clearance of particles through minutes and hours in either subject group. these data suggest that the new methodology (protocol # ) will be useful and easily employed by other cf centers to test effectiveness of new therapies for cf. the third protocol (# ) was then tested in five of the healthy subjects. this protocol used a nebulizer that generated very large droplets ( . um mmad, gsd = . ) and subjects inhaled at very slow flow rates (~ ml/sec). it was designed to provide greater bronchial airway deposition and thus an "improved signal" for mcc/cc evaluation. indeed, this methodology produced a larger average c/p and significantly greater particle clearance from the lung compared to the other standardized methodology (protocol # ) (c/p = . vs. . ; min clearance = % vs. %; hr clearance = % vs. %, respectively). interestingly, the greater fraction of clearance between min and hr may also reflect heightened ability to assess clearance from smaller airways, which could be particularly important in cf. protocol # will be tested further in cf patients. supported by cf foundation. previous clinical trials in cystic fibrosis (cf) indicate that anti-inflammatory therapy probably will not result in improvement in lung function, but will slow the rate of decline. this imposes constraints on study design for new anti-inflammatory agents, requiring that they use many patients over long periods. it is highly desirable to design a strategy for evaluation of antiinflammatory agents that will allow for the selection of only the most promising agents for phase iii trials. sputum induction (si) samples lower respiratory tract secretions and permits measurement of inflammatory markers. the purpose of this study was to assess the measurement of inflammatory markers in induced sputum as one such strategy by using ibuprofen as the test agent because it has demonstrated clinical benefit in cf. if changes in inflammatory markers are detectable in ibuprofen treated patients, then si might serve as a quick, noninvasive method by which to select the most promising anti-inflammatory agents for further study. methods: in this twoarm (ibuprofen and no treatment), open-label, parallel group study, cf patients >/= years of age with mild to moderate lung disease were screened at sites. si was performed on days , , and . patients in the ibuprofen group received drug on days - . subjects met eligibility criteria. were randomized to ibuprofen and to no treatment. subjects ( ibuprofen and no treatment) completed the study through day and comprise the intent-to-treat efficacy population. a maximum of subjects ( ibuprofen and no treatment) and a minimum of subjects ( ibuprofen and no treatment) across all markers had acceptable inflammatory marker values at both days and . within group and between group comparisons were made. results: with respect to the within group comparisons (before and after ibuprofen), most inflammatory markers slightly decreased in the ibuprofen group and increased in the no treatment group. for ibuprofen patients, mean changes were most apparent for il- (- . log pg/ml, p= . ) and % neutrophils (pmns) (- %, p= . ). with respect to the between group comparisons (ibuprofen vs. no treatment), differences were strongest for il- ( . log pg/ml, p= . ), percent pmns ( %, p= . ), and absolute pmn count ( . cells/ml, p= . ). fourteen days after discontinuing ibuprofen, inflammatory mediators tended to increase. there was no difference in aes between the two groups, and si was generally safe. conclusions: . overall, si is well-tolerated in patients >/= years, . a -week treatment period may not be long enough to study anti-inflammatory drugs, . with respect to changes in inflammatory markers associated with anti-inflammatory therapy, the effect size is small and the variance of the effect is small in clinically stable patients. . measuring inflammatory markers in induced sputum has potential as a screening tool for studying anti-inflammatory drugs in cf, . further studies of si in cf are warranted. supported by cystic fibrosis foundation therapeutics, inc. in preparation for our gene therapy clinical trial programme we are currently assessing a number of sputum biomarkers including viscosity and elasticity, total solids and dna content and hr sputum weight. we tracked and correlated these biomarkers in cf patients ( years and over) during a course of iv antibiotics (ab) by collecting samples on several occasions [visit (v) : at the start of ab treatment, v : at the end of ab treatment (generally after weeks) and interim periods]. to ensure adequate reproducibility of the results viscosity/elasticity measurements were carried out in triplicate using a csl rheometer which required a comparatively large volume ( mls) of spontaneously expectorated sputum. because of this requirement paired samples could only be obtained from approximately % of the patients. there was no change in viscosity/elasticity (n= ), solid or dna (n= ) content when comparing samples at the beginning and end of iv ab. in contrast, hr sputum weight was significantly (p< . ) lower at the end of the ab treatment (visit : . ± . g, visit : . ± . g, n= ). there was a strong and significant correlation between solid content and viscosity/elasticity (r= . , p< . ) and a modest correlation between hr sputum weight and % predicted fev (r=- . , p< . ) and hr sputum weight and patient scored symptom severity (r= . , p< . ). surprisingly, sputum dna content did not correlate with viscosity/elasticity, despite being generally thought of as a contributor to viscosity, which may in part be related to the assay not being able to discriminate between free and cell-enclosed genomic dna.in summary, after a course of iv antibiotics which lead to significant subjective and objective (fev ) improvement the overall quantity of expectorated sputum was significantly reduced but, based on analysis available to date, none of the other parameters (viscosity, elasticity, solid and dna content) changed significantly. considering the difficulties we encountered in collecting sufficient sputum during this period of an exacerbation, sputum viscosity/elasticity measurements may not be feasible parameters to measure in a gene therapy trial to which stable patients re likely to be recruited. this study was funded by the cf trust. chronic neutrophilic inflammation is a feature of cystic fibrotic (cf)related inflammation and serves as a significant contributor to morbidity and mortality associated with the condition. recently, our group has described a novel collagen-derived neutrophil chemoattractant, proline-glycine-proline (pgp), in a murine model of lipopolysaccharide-induced inflammation. the purpose of this study was to explore both the presence of this peptide in sputum from cf patients and the determination of a proteolytic system involved in pgp generation. we show that lower airway secretions from cf subjects have -fold increase in pgp levels compared to normal controls and that cf sputum (p< . ) is able to generate pgp from intact type i and ii collagen -fold above that seen from normal control sputum ex vivo (p< . ). we further demonstrate that production of pgp is dependent on two specific matrix metalloproteases (mmps), mmp- and mmp- . finally, we show that the generation of pgp can be significantly inhibited ( - % inhibition) by the use of either mmp- or mmp- specific inhibitors (p< . versus no inhibitor); a nonspecific mmp inhibitor (doxycycline) also demonstrates significant attenuation of pgp production. the determination of the requirements of these proteases in pgp generation allows for the identification of logical targets for disease-modifying therapeutics in cf. . cfrc, uab, birmingham, al, usa; . cfrc, university of north carolina, chapel hill, nc, usa cystic fibrosis (cf) lung disease is characterized by chronic neutrophilic inflammation. the discrete collagen breakdown product prolineglycine-proline (pgp) is a potent neutrophil chemoattractant thought to be generated by a specific proteolytic cascade present during cf pulmonary exacerbation (nat med ( ): , ) . high-mobility group box (hmgb ) is a potent inflammatory mediator found in sepsis, rheumatoid arthritis, and other inflammatory diseases characterized by neutrophilic inflammation. the role of pgp and hmgb as pmn attractants in cf is unknown. in this study we utilized specimens derived both from humans and scnn b-transgenic mice (βenac mice) in which overexpression of the βenac subunit in the airways leads to sodium hyperabsorption and airway surface liquid depletion, mimicking the cf muco-obstructive phenotype. pgp was quantified using tandem ms. neutrophil chemotaxis was measured in vitro following preincuabtion of sputum or balf with anti-hmgb neutralizing antibody. murine balf was assayed for hmgb by western blot and elisa. human sputum was similarly evaluated after normalizing for total protein concentration. in vivo hmgb activity was measured after intratracheal instillation in wt mice. βenac mice had % higher cell counts and a greater percentage of pmns ( . % vs. . %) in bal samples than wild-type (wt) littermates (p< . , n= /genotype). pgp was elevated in balf of scnn b animals ( . vs. below limits of detection in wt controls, p< . , n= ), increased in serum from cf subjects ( vs. pg/ml normal controls, p< . , n= ), and detected at high levels in cf sputum ( , pg/ml). balf screened for hmgb by western blot revealed elevated levels in βenac mice (p< . by densitometry, n= /genotype); elisa confirmed % greater hmgb concentration ( . vs. . ng/ml, p< . ). hmgb was also detected by western blot in human cf sputum ( of ) and at higher levels than secretions from normal subjects (p< . by densitometry; n= cf, control). purified hmgb induced dose-dependent pmn chemotaxis in vitro (peak - ng/ml, p< . , n= ) and cf sputum caused potent chemotaxis ( -fold over control, p< . , n= ) that was inhibited by preincubation with hmgb antibody (p< . ). balf from βenac mice (but not wt controls) was also chemotactic ( -fold greater than media control, p< . , n= ) and inhibited by anti-hmgb antibody (p< . ). exogenous hmgb ( ng) administered intratracheally to lps resistant mice (c hej, toll- receptor mutant) and balb/c mice caused pmn influx in balf at hours (c hej: . x pmns after hmgb vs. . x after vehicle, p< . ; balb/c: . x vs. . x , p< . ), and generation of pgp ( . vs. . pg/ml in c hej, p< . , n= ) . in summary, pgp and hmgb are elevated in balf of βenac mice that present with cf-like lung pathology; in cf airway secretions, hmgb is present in vivo at concentrations that induce neutrophil chemotaxis in vitro and pgp production in vivo, and is inhibited by blocking antibody. the role of pgp and hmgb in cf deserve further evaluation as they may be potential therapeutic targets of dysregulated inflammation. esther, c.r. ; jasin, h.m. ; collins, l.p. ; boysen, g. ; boucher, r.c. . pediatric pulmonology, university of north carolina at chapel hill, chapel hill, nc, usa; . cf research center, univeristy of north carolina at chapel hill, chapel hill, nc, usa; . center for environmental health and susecptibility, univeristy of north carolina at chapel hill, chapel hill, nc, usa biomarkers of airway inflammation are needed in cystic fibrosis (cf) to aid clinical management and assess the efficacy of novel therapies. we describe a simple and non-invasive method to measure airway purines, signaling molecules previously shown to be biomarkers of neutrophilic airway inflammation. airway secretions were obtained using exhaled breath condensate (ebc) collection, a technique that was easily performed in the outpatient setting on children as young as three years. the purines adenosine and amp in ebc were measured using ultra-sensitive liquid chromatography/tandem mass spectrometry (lc/ms/ms). in addition, we used lc/ms/ms to simultaneously measure urea as a dilution marker to control for the known dilutional variability of airway secretions within ebc. detection of adenonosine, amp, and urea was optimized using positive mode ms with selected reaction monitoring, and stable isotope dilution was utilized to improve quantification. detection was linear with concentration over four orders of magnitude, with coefficients of variation < %. the method was sensitive, with limits of detection for adenosine and amp (~ . nm) and urea (~ . µm), below expected ebc concentrations. applying the methods to ebc demonstrated that detection of purines and urea was reliable, with intraclass correlation coefficients greater than . between duplicate measures (n= ). we also measured purines and urea in ebc samples collected prospectively from children with cf (n= ) and healthy controls (n= ) during regular clinic visits. all samples were lyophilized and reconstituted to increase concentration -fold. adenosine, amp, and urea were detected in all samples. neither adenosine nor amp levels differed between groups, but urea was significantly lower in cf ( . ± . µm) versus healthy control ( . ± . µm, p= . ), suggesting that airway secretions were more dilute in cf samples. when dilutional variability was controlled using purine to urea ratios, the amp to urea ratio was found to be elevated in subjects with cf ( . ± . ) compared to healthy controls ( . ± . , p= . ), consistent with previous findings. the amp to adenosine ratio was also elevated in cf ( . ± . ) compared to control ( . ± . ), although the difference did not reach statistical significance in this small sample set (p= . ). experiments are ongoing to increase sample size and assess ebc purine levels before and after treatment of a cf exacerbation. these results demonstrate that lc/ms/ms analysis of ebc provides a non-invasive method to measure purine biomarkers of cf airway inflammation. given the flexibility of ms, this methodology could also prove applicable for study of other small molecule biomarkers. importantly, we also show that dilution of airway secretions in ebc may be altered in cf, and lc/ms/ms can be used to control for this variability through measurement of urea as a dilution marker. introduction: mucociliary clearance and antimicrobial peptide activity may be affected by airway surface liquid (asl) ph, which is regulated by epithelial ion transport. asl ph is abnormal in disease states in which il- a is elevated, including cystic fibrosis (cf), and the il- a receptor is expressed at the basolateral surface of bronchial epithelium. therefore, we investigated the effects of il- a on vectorial ion transport in well-differentiated hbe cells. methods: hbe cells were grown with an apical air interface and incubated with il- a at or ng/ml in the basolateral medium for hours prior to being studied with standard short-circuit current (isc) techniques. results: il- a treated cells had a minimal increase in resting and amiloride-sensitive isc compared to control cells, and had a dosedependent, statistically significant increase in forskolin-stimulated isc. at a dose of ng/ml, il- a doubled forskolin-stimulated isc ( . ± . µa/cm for control vs. . µa/cm for il- a). the increased isc was not bumetanide-sensitive, but was sensitive to acetazolamide and dnds, and was not present in hco --free solutions, suggesting it was due to hco secretion. to investigate whether this hco secretion was cl --dependent, we studied hbe cells in cl --free solutions, and there was no difference between untreated and il- a treated hbe cells. to test the hypothesis that il- a promoted cl -/hco exchange, we performed experiments in hbe cells mounted in cl --free solutions, and after addition of amiloride and forskolin, mm nacl was added to the mucosal bath and mm nagluconate was added to the serosal bath. under these conditions, one would predict a decrease in isc due to mucosal to serosal clmovement down its electrochemical gradient. in untreated hbe cells, we saw a decrease in isc followed by a recovery. in il- a treated cells there was a sustained increase in isc, suggesting that movement of cldown its electrochemical gradient resulted in exchange of clfor an anion at the apical membrane. in the absence of hco -, both untreated and il- a treated cells responded to claddition to the mucosal bath with decreases in isc. to assess the cftr dependence of the il- a-induced isc, we stimulated primary cf hbe cells treated with il- a. in cf cells, there was a minimal increase in amiloride-sensitive isc ( . ± . µa/cm for control vs. ± . µa/cm for il- a ) and in forskolin-stimulated isc ( . ± . µa/cm for control vs. . ± . µa/cm for il- a ). conclusion: the proinflammatory cytokine il- a induces cl -/hco exchange in hbe cells. notably, the observed cl -/hco exchange appears to be cftr-independent. however, cftr activation is required for maximum cl -/hco exchange as demonstrated by the smaller isc generated in cf cells. these data suggest that the cytokine milieu of the airway epithelium can alter ion transport and potentially asl physiology. furthermore, they suggest that cftr may interact with or regulate novel proteins in the presence of inflammation. supported by cff and nih periciliary fluid balance is maintained by the coordination of sodium and chloride (cl-) channels in the apical membranes of the respiratory epithelia. in the absence of the cystic fibrosis transmembrane conductance regulator (cftr), cl-secretion is diminished and sodium reabsorption becomes exaggerated. activation of non-cftr-dependent cl-channels can provide an alternate pathway for cl-secretion in the airways and gastrointestinal tract. the ph and voltage-dependent type- cl-channel (clc- ) is expressed in airway epithelial cell luminal membranes. we hypothesize that topically applied clc- agonists may restore cl-secretion in cystic fibrosis (cf) murine airways. using in vivo nasal potential difference measurements, we quantified clc- -mediated cl-transport in both cf and wild-type mice during nasal perfusion with lubiprostone (a prostone compound and specific clc- agonist; sucampo, pharmaceuticals, inc., bethesda md) or vehicle control. wild-type (c bl , n = ) and cf knock-out (cfko; n= (jax stock# )(jackson laboratories, bar harbor, me) mice were sedated and intubated to protect the lower airways from aspiration of perfusate. nasal and subcutaneous bridges were connected to fluid-filled silver chloride electrodes. baseline (in ringer's), amiloride-inhibited, low cl-(cl-free, gluconate-substituted ringer's with amiloride), and low cl-plus lubiprostone (with increasing concentrations of lubiprostone or vehicle) mice were perfused and potential differences were measured with a high impedance voltmeter (world precision instruments, sarasota fl) and continuously recorded. a clear dose-response relationship was detected in both wild-type and cf mice. at µm lubiprostone, wild-type mice showed hyperpolarization of - . ± . mv and cf mice responded with - . ± . mv hyperpolarization. a paired t-test of low cl-perfusion and lubiprostone perfusions revealed significant (p< . ) differences in both genotypes. five clc- cfko mice were similarly tested and showed no response to lubiprostone (+ . ± / mv). cftr inhibitor- ( µmol) (calbiochem, san diego ca) added to the low cl-perfusion in additional wild-type mice eliminated the low cl-response but did not abolish the lubiprostone response, confirming that clc- is present and independent of cftr regulation. we conclude that direct application of a clc- agonist in the cf murine upper airways restores near normal levels of cl-secretion. cftr is an apical membrane chloride channel whose activity is required to maintain airway surface liquid (asl) volume and efficient mucociliary clearance. cftr is activated by camp dependent stimulation of protein kinase a. however, studies have suggested that the actin cytoskeleton may also be required for regulation of cftr, by acting as a structural element in second messenger compartmentalization ( ) . to determine whether an intact cytoskeleton is required for asl volume homeostasis, adenosine (ado), atp, or isoproterenol (iso) (all at µm) were added apically to human bronchial epithelial cultures (hbec's) before or after the cytoskeleton had been disrupted by cytochalasin d exposure ( µm for h). in control cultures ado, atp and iso all resulted in a significant increase in asl height (∆asl) within min (∆asl; ado, . ± . µm; atp, . ± . µm; iso, . ± . µm). however, in cytochalasin d exposed cultures ado was without effect while atp and iso were still able to evoke an increase in height (∆asl; ado, - . ± . µm; atp, . ± . µm; iso, . ± . µm). however, we showed that a b receptor function was not compromised by cytoskeleton disruption as camp levels were seen to increase both after addition of adenosine and adenosine in the presence of cytochalasin d. thus, the effect of cytochalasin d on asl height appeared specific for adenosine-mediated cl-secretion. to further test the effect of cytochalasin d on the relationship between adenosine and cftr, we designed a fluorescent resonance energy transfer (fret) pair of cfp-cftr (labeled at the n-terminus) and yfp-a b receptor (labeled at the c-terminus). addition of adenosine ( µm) increased fret by . times (% efficiency; control, . ± . %, ado, . ± . %) compared to the vehicle control (n= ). cytochalasin d exposure ( µm for h) completely inhibited the increase in fret that was observed after adenosine addition (p< . ). in contrast, cytochalasin d had no effect on fret between cftr and the β adrenoceptor. to determine whether cytochalasin d treatment ( µm for h) had any direct effect on cftr protein levels and localization we used a cell line stably expressed with an exotope-tagged cftr (hb cells) ( ) . cftr protein levels were found to be significantly reduced as compared to nontreated controls by both western blot analysis and chemiluminescence detection (n= ). cells were also stained with an antibody against the external cftr exotope and imaged by confocal microscopy and a significant decrease in staining at the plasma membrane was observed after cytochalasin d treatment (n= ). we conclude that cytochalasin d exposure decreases cftr protein levels and specifically inhibits adenosine-mediated regulation of cftr activity in the airways, leading to disruption of asl homeostasis and inhibition of mucociliary clearance. numerous studies have reported evidence that cftr is expressed in the apical membrane of serous cells in the airway submucosal glands. thus, it has been reasoned that ( ) cftr plays some role in normal anion and fluid secretion by airway glands and ( ) loss of this channel's function in cystic fibrosis (cf) airways plays some role in the etiology of cf lung disease. a recent study by joo et al. (jbc : - , ) indicates that cf airway glands lose the ability to secrete fluid when treated with forskolin or vip but maintain the ability to secrete when treated with muscarinic agonists. this result suggests that vip and muscarinic agonists mediate secretion by cftr-dependent and cftr-independent pathways, respectively. the present study was undertaken to explore the dynamics of fluid secretion by individual submucosal glands to determine if further differences in their responses to these two agonists could be distinguished. pig tracheas were obtained from a local slaughterhouse. after removal of the cartilage, tissues were mounted in a warm ( °c) observation chamber that permitted exposure of the submucosa to krebs solution. the mucosal surfaces of the airways were dried with a stream of warm dry air and then layered with mineral oil. liquid secreted from the gland duct openings formed spherical aqueous droplets within the oil layer, and their volumes were estimated from spatial dimensions taken from sequential digital images. liquid secretion rates were estimated for individual glands present within a . mm region of interest. rates were determined for two experimental protocols. for one group of tissues, secretion rates were determined for a control period, followed by a period of exposure to µm vip, and then a period of exposure to both vip and µm acetylcholine (ach). in the second group, the order of vip and ach treatment was reversed. these agonist concentrations were expected to produce near maximum rates of secretion. when the agonists were applied first, secretion rates were significantly (p< . ) increased from the control period by both ach ( . ± . nl/min to . ± . nl/min) and vip ( . ± . nl/min to . ± . nl/min). the secretion rates for individual glands were highly variable with both agonists (ach: . - . nl/min; vip: . - . nl/min). when ach application followed vip treatment, the mean secretion rate was significantly increased further to . ± . nl/min; however, when vip application followed ach treatment, the secretion rate was only marginally and insignificantly increased to . ± . nl/min. we conclude that both ach and vip are efficacious secretagogues for porcine airway glands and that the rate of secretion from individual glands to either agonist is very heterogenous. because vip has no effect following stimulation by ach, we speculate that ach induces secretion by activating all transporters relevant to vip-induced secretion including cftr. however, ach must also activate channels and/or transporters not induced by vip since its effects are additive to vip. this most likely includes activation of a non-cftr anion channel. ( serous cells are thought to serve as the principal anion and fluidsecreting cells of airway submucosal glands. mucous cells primarily secrete gel-forming mucins. these respective physiological roles for these two cell types imply that the mucous cells, which secrete the thick mucus gel, should lie downstream of the serous cells so that their watery secretions can flush the mucins out of the ducts and onto the airway surface. early studies indicated that such arrangements existed within glands but reports of cell distributions have been descriptive rather than quantitative. because of resurgent interest in serous cell function, we revisited this issue to formally document the distribution of these cell types within the secretory tubules of the submucosal glands as well as at the surfaces of the glands, where serous cells are most accessible for study. a piece of porcine lung, containing a mm diameter bronchus, was treated with formalin fixative, and consecutive µm sections were taken. the apices of virtually all gland epithelial cells, except the cells lining the collecting and ciliated ducts, stain positive for pas indicating that these cells are either serous or mucous cells. however, pas staining does not allow distinctions to be made between these two cell types. consequently, all slide sections were stained with hematoxylin and eosin. with these stains, mucous cells were identified as cells containing lucent granules in their apices whereas serous cells were considered to be cells where the apical cytoplasm stained a uniform dark reddish color. first, the distribution of serous and mucous cells in the secretory tubules were determined. the acini of individual tubules were located in the slide sections, and cells were identified and counted in consecutive sections that approached the collecting ducts until the tubules were no longer distinguishable as discrete tubes. secretory tubules from individual glands were studied. in the acini, serous cells accounted for . ± . % of total cells whereas mucous cells accounted for . ± . % of total cells (remaining cells were not distinguishable as either cell type). there was a significant (p<. ) negative correlation of serous cell numbers with distance from the acini so that at µm from the acinus serous cells accounted for only . ± . % of total cells. there was a coincident significant positive correlation with mucous cell numbers so that at µm from the acini mucous cells accounted for . ± . % of total cells. next, we examined the cell distribution at the adventitial and mucosal surfaces and at the lateral margins of the glands. serous cells accounted for . %, . %, and . % of the total cells at these respective locales. mucous cells represented . %, . %, and . % of total cells in these same respective regions. we conclude that serous cells are by far the dominant cell type in secretory tubules of porcine submucosal glands. mucous cells are rare in acini but increase in frequency with distance from the acini. the outer surfaces of the glands are dominated by serous cells as well. at the lateral margin of the glands, serous cells outnumber mucous cells by -to- . (supported by nih hl ) . many of the membrane transporters that participate in this process have been identified; however, the identity of the class of kchannels that maintains resting membrane potential and/or cell membrane polarization during the secretion process remains poorly characterized. liquid secretion by porcine submucosal glands is insensitive to numerous k + channel blockers including ba + , tetraethylammonium, apamin, charybdotoxin, iberiotoxin, clotrimazole, penitrem a, -aminopyridine, and quinidine. recently, a new class of k + channels, the "tandem-pore" or k p channels, have been described. potassium channels of this class are insensitive to many traditional k + channel inhibitors but are blocked by local anesthetics, such as lidocaine and bupivacaine. additionally, many k p channels are sensitive to changes in extracellular ph. in the present study, we considered whether k p channels might participate in the liquid secretion response to acetylcholine (ach). intrapulmonary bronchi, excised intact from the lungs of pigs, were cleared of accessible luminal liquid, cannulated, and treated with µm ach to induce secretion. paired airways were pretreated for h with mm bupivacaine. bupivacaine blocked . ± . % of the liquid secretion response to ach. the ic for the bupivacaine inhibition was approximately µm. the inhibitory effect of bupivacaine was not due to nonspecific toxicity since tissues exposed to bupivacaine for h and then washed with fresh inhibitor-free buffer recovered to % the control achinduced secretory rate. the bupivacaine effect was not due to inhibition of voltage-gated na + channels in intrinsic neurons since µm tetrodotoxin did not inhibit ach-induced secretion. no significant effect was seen in the secretion rate when the extracellular solution ph was lowered from . ± . (normal hco --buffered krebs gassed with % co ) to . ± . by gassing with % co gas (balance o ). while we recognize the relative nonspecificity of this agent, the inhibitory effects of bupivacaine on liquid secretion by porcine bronchi are consistent with k p channel participation in this secretory response. the failure to demonstrate inhibition with acidification of extracellular solution could signify a k p channel subtype that is insensitive to low extracellular ph. ( . novartis, horsham, united kingdom; . rosalind franklin university, chicago, il, usa; . mount sinai medical center, miami, fl, usa; . genomics institute of the novartis foundation, la jolla, ca, usa enac activity in the human airway epithelium has been reported to be partially-sensitive to the broad spectrum trypsin-like protease inhibitors aprotinin and placental bikunin (bridges et al., am j physiol :l - ; donaldson et al., jbc : - ) . a low molecular weight inhibitor of the airway channel activating protease (cap) would represent a potential therapeutic approach to attenuating enac function in cystic fibrosis (cf) lung disease. the aims of the current study were to: ( ) further characterise the in vitro efficacy of cap inhibitors in human bronchial epithelial cells (hbes), ( ) to evaluate the relevance of a cap mechanism to the regulation of enac function in vivo, and ( ) to establish whether the inhibition of the airway cap can modulate mucociliary clearance in vivo. primary cultures of hbes (normal and cf), cultured under air-liquid interface conditions, demonstrated an amiloride-sensitive short circuit current (isc), that was sensitive to aprotinin but that was insensitive to inhibition by sbti, α -anti-trypsin and α -pdx. the amiloride sensitive isc was also attenuated by the low molecular weight cap inhibitor nvp-qau (hbc ) with an ic value of approximately nm. nvp-qau caused a time dependent inhibition of the isc with a t ⁄ of approximately min and this effect could be reversed by the addition of excess trypsin. in vivo, aprotinin attenuated the guinea pig tracheal potential difference (tpd) from - . ± . mv to - . ± . mv (n= - ) with an ed value of pmoles/kg, measured at hours following intra-tracheal instillation. the tpd was unaffected by either sbti or α -anti-trypsin at doses up to pmoles/kg. the combination dosing of an enac blocker with aprotinin did not attenuate the tpd beyond the effect observed with either agent alone, consistent with aprotinin attenuating enac function in this model system. intra-tracheal instillation of nvp-qau attenuated the tpd from - . ± . mv to - . mv with an ed value of µg/kg (n= - ). in the sheep, the administration of nvp-qau into the airways by aerosolisation of a mg/ml solution resulted in a - fold enhancement of the rate of clearance of mtc sulfur colloid from the lungs compared with the vehicle control. these studies indicate that the cap mechanism of enac regulation can translate through in vitro human assays, into in vivo models. cap inhibitors can attenuate tpd in the guinea pig in vivo and enhance the rate of mucociliary clearance in the sheep, thereby representing an approach to the therapeutic regulation of enac function in cf lung disease. cotton, c. pediatrics, physiology and biophysics, case western reserve university, cleveland, oh, usa cystic fibrosis is caused by mutations in the gene that encodes cftr, a camp-activated apical membrane anion channel. loss of cftr-mediated anion conductance is a primary defect in cf but secondary defects such a sodium hyperabsorption are also implicated in cf pathophysiology. it is generally accepted that mucociliary clearance is compromised in cf airways due to reduced fluid secretion and/or increased fluid absorption. the recent generation of a transgenic mouse model with enac overexpression that exhibits cf-like lung disease highlights the importance of sodium hyperabsorption. therapies designed to reduce mucus production and viscosity, increase lumenal water content, stimulate fluid secretion, and inhibit fluid absorption are under development for treatment of cf airways. although controversial, several recent studies have demonstrated that serca pump inhibitors partially correct the trafficking defect associated with delta f mutant cftr. the goal of this work is to determine if serca pump inhibitors affect enac-mediated sodium absorption. a renal collecting duct epithelial cell line and primary cultures of well-differentiated human tracheal epithelial cells (air/liquid interface culture) were used for these studies. amiloride-sensitive short circuit current was determined as a measure of enac-mediated sodium absorption. quantitative rt-pcr was used to evaluate expression levels for each of the subunits of enac (alpha, beta, and gamma). epithelial monolayers were treated for - hours with serca pump inhibitors (thapsigargin, - nm; dbhq, - um; and curcumin, - um). treatment with serca pump inhibitors decreased amiloride-sensitive short circuit current by - %. in contrast, campstimulated short circuit current was not reduced by treatment with serca pump inhibitors. the steady-state levels of alpha, beta, and gamma enac were reduced by - % in renal epithelial monolayers treated with thapsigargin whereas beta and gamma enac were reduced by - % with no significant change in alpha enac in airway epithelial cells. the results of these studies demonstrate that sustained depletion of endoplasmic reticulum calcium stores and/or elevation of intracellular calcium by inhibition of serca pump activity reduces enac mrna expression and enac-mediated sodium absorption. thus, inhibition of serca pump activity in airway epithelial cells of cf patients that carry the delta f mutation may provide dual benefit by promoting delivery of mutant cftr to the membrane . pediatrics, national jewish medical and research center, denver, co, usa; . integrated department of immunology, national jewish medical and research center, denver, co, usa; . department of medicine, cystic fibrosis/pulmonary research and treatment center, the university of north carolina at chapel hill, chapel hill, nc, usa increased cytosolic calcium ([ca + ] i ) initiated by the release of stored ca + from the endoplasmic reticulum (er) is a key signal to elicit a wide range of essential cellular responses, including secretory functions of the respiratory epithelium that are modified in cf. serca pumps are responsible for (re)filling the er ca + stores, and serca blockers such as thapsigargin are very potent and commonly used pharmacological triggers of ca + -signals. modulation of the activity of sercas can profoundly affect ca + homeostasis. although defective calcium homeostasis is a characteristic of several pulmonary diseases including cf, the role of serca is unknown. lung tissue samples (bronchus and distal lungs) from normal (n= ) and cf subjects (n= ) were evaluated by immunohistochemistry. serca expression was decreased in the bronchial and bronchiolar epithelia of cf. non-cf and cf bronchial epithelial cell line pairs including calu- and jme/cf , c- and ib - , hbe o-( hbe), cfbe o-(cf o-) and cfbe o-(cf o-) were also probed. given certain limitations of such cells, several consistent findings still emerged. a % and % decrease in serca expression was observed in cf o-and cf ocells as compared to hbe cells. immunocytochemical studies in these cells confirmed that the serca was localized in the er and that the decreased serca expression was not associated with decreased er content. reduced serca expression and activity ( . ± . vs . ± . and . ± . pmol/min/mg protein in hbe vs. cf o-and cf ocells respectively) was observed in the purified er membranes from cf cell lines. northern blot analysis revealed a parallel reduced mrna expression as well. decreased serca was accompanied by increase in the low affinity isoform serca ( . ± . vs. . ± . and . ± . arbitrary serca intensity/b-actin in respectively) . we have also evaluated a limited number of primary airway epithelial cells isolated from lung samples of normal and cf subjects for serca expression. serca expression in polarized tracheobronchial epithelial cell lysates from cf subjects was decreased by about % as compared to those from normal subjects ( . ± . vs. . ± . arbitrary serca intensity/b-actin units in cf and normal respectively, n= ). expression of serca could also be suppressed by inhibiting cftr with cftr inh in normal human bronchial epithelial cells ( various studies indicate that the airway surface fluid possess an antibacterial system based on the combined action of lactoperoxidase, h o , and scn -(thiocyanate). the enzyme lactoperoxidase (secreted by submucosal glands) utilizes h o and scnto generate oscn -(hypothiocyanite) a molecule with antimicrobial activity. h o is produced by dual oxidases expressed on the apical membrane of airway epithelial cells whereas scnis transported across the epithelium through anion transporters and channels. in particular, scntransport seems to occur through cftr and other ca +dependent clchannels. therefore, reduced scntransport in the airways of cystic fibrosis patients may contribute to impaired antimicrobial activity. in a recent study, we have found that cytokines, in particular il- , cause a strong increase in the ability of cultured bronchial epithelia to transport scnfrom the basolateral to the apical membrane. this effect is mediated by upregulation of ca + -dependent clchannels and of the slc a (pendrin) anion transporter. these findings suggest that under proinflammatory conditions the activity of the scn -/h o /lactoperoxidase system is potentiated. we evaluated the antimicrobial activity of airway surface by seeding bacteria on the apical membrane of human bronchial epithelia grown with an air liquid interface on a porous membrane. an inoculum corresponding to , cfu of s. aureus was added to the cells in µl of saline solution with and without lactoperoxidase ( . µg/ml). experiments were performed in the presence or absence of scn -( µm) in the basolateral solution. bacteria were recovered after four hours and plated on agar plates for colony counting. our preliminary results show that simple addition of lactoperoxidase to the apical surface decreases bacterial survival. in addition, bacterial killing is strongly enhanced by prestimulation of cells for hours with il- ( ng/ml). the effect of lactoperoxidase is dependent on h o as it is prevented by addition of catalase and is absent in cell-free experiments when h o is omitted from the reaction mixture. surprisingly, the presence of scnin the basolateral compartment did not increase bacterial killing and, in some cases, appeared to generate a protective effect. our results suggest that lactoperoxidase, in the presence of h o , is an effective antimicrobial molecule. the contribution of scnand the mechanism of the potentiation caused by cytokines is less clear and requires further investigation. a possibility is that, in the absence of scn -, lactoperoxidase generates an oxidant molecule that is more toxic to bacteria than oscn -. elucidation of this mechanism and comparison between cf and non-cf epithelia is in progress to assess the role of cftr and of other anion channels. supported by cfft and telethon-italy. cf patients become infected with pseudomonas aeruginosa, which release flagellin into the airway surface liquid to activate toll-like receptor and proinflammatory signaling. flagellin has been shown to inhibit na absorption by airway epithelia. we tested flagellin on cl secretion and proinflammatory signaling (nf-κb activation) by calu cells, a cftrexpressing, serous-like airway gland cell line. calu cells were grown on filters and either mounted in ussing chambers (clamped to zero mv) in the presence of a serosa-to-mucosa gradient of [cl] for measurements of transepithelial cl secretion (i cl ) or treated with an adenovirus expressing nf-κb-controlled luciferase to assay nf-κb activation. flagellin ( - g/ml) on either the apical or basolateral surface of cells increased apparent anion secretion that began in - mins and increased over - mins by - µa/cm . this i cl was blocked by glibenclamide and glyh , indicating that it resulted at least in part from activation of cftr. flagellin also stimulated fluid secretion by intact human tracheal glands. flagellin activated nf-κb (luciferase assays) in both calu- cells and in the cf cell line cf , while i cl was stimulated in calu but not in cf cells. flagellin-stimulated i cl in calu- cells was blocked % by sb (a p mapk blocker) and by a similar amount by wortmannin (pi kinase blocker). interleukin β and the tlr -agonist pam cys also activated i cl . the effect of flagellin was not due to increases in cytosolic [ca + ] (ca i ) because flagellin did not alter ca i . in contrast to the slow effects of flagellin, pam cys and il β, atp rapidly increased i cl (within secs, up to µa/cm ) followed by slower decrease to steady i cl = - µa/cm that had a similar time signature as the increases in ca i . forskolin (to increase cytosolic [cyclic amp]) increased i cl within mins to a steady value = - µa/cm . flagellin had small or no effects on i cl following maximal stimulation with either atp or forskolin. atp and forskolin on their own had no effect on nf-κb. flagellin increased phosphorylation of p mapk and of akt (down-stream kinase phosphorylated by pi k) with a time course similar to the increase in i cl . flagellin-activated nf-κb was reduced by roughly % by either wortmannin or sb . these results indicated that tlr agonists and inflammatory cytokines stimulate both nf-κb and proinflammatory processes and also cftr-dependent cl and fluid secretion by airway gland cells. these responses are mediated in part by activation of both p mapk and pi kinase. flagellin-tlr -activated increases in cftr-mediated cl secretion and reduction in enac-mediated na absorption will increase fluid secretion into the airways, which may facilitate bacterial removal by the mucociliary escalator and thereby reduce the proinflammatory stimulus. in cf it is expected that bacteria will activate tlr signaling to trigger innate immune responses, but cl and water secretion and the resulting "bacterial flush," will be missing, leading to sustained inflammation. ( , ) . treatment with hs improved several measures of lung function including mucociliary clearance. in one study amiloride was seen to have a negative impact on the benefit of hs ( ) . the long duration of action of hs and the paradoxical effect of amiloride are surprising and require further investigation. aims: our goal was to study the effect of exposure to a hypertonic challenge (hc) on amiloride sensitive na+ transport (ina) in primary cultures of human bronchial epithelial (hbe) cells from non-cf and cf patients. methods: hbe cells were grown at an air liquid interface and studied under short circuit current conditions. cells were challenged with hs by exchange of the apical or basolateral baths. in some experiments the timedependent effect of a small ( µl) isosmotic or hs volume at the airway surface was tested on ina and apical osmolality. results: application of hypertonic nacl and mannitol solutions from either the mucosal or serosal sides of the epithelium inhibited ina. the degree of inhibition was a saturable function of the imposed hypertonicity with an ic of (mannitol) and (nacl) mosmol/kg h o and a maximal inhibition of %. the inclusion of amiloride ( - µm) did not affect the hc-induced reduction in ina. the inhibition in ina by hc was rapid in onset and accompanied by a fall in the transepithelial resistance and the total transepithelial capacitance as expected for cell shrinkage. the inhibition in ina was only partially reversible (~ %) after returning to isotonic solutions for min. exposure of the airway surface to hypertonicity ( mosmol/kg h o) inhibited ina by - % at min of exposure. as osmotically-driven water moved from the serosal to the mucosal side, the apical volume increased concomitant with a decrease in its osmolality and a recovery of ina. the osmolality declined exponentially and reached the isosmotic value after ~ hrs. the recovery of ina lagged behind the recovery of apical osmolality by , and % at . , and hrs of incubation, respectively. after hrs of airway hc exposure, the osmolality and ina recovered completely. our results demonstrate that hc causes a rapid, dramatic and prolonged decrease in na+ absorption and that continuous presence of amiloride had no effect on the inhibition of ina or the recovery of apical osmolality. hc also causes the epithelium to shrink. no appreciable differences were observed between cf and non-cf hbe cells. conclusions: we propose cell shrinkage together with the continued influx of na+ increases intracellular na+ ([na]i) and lead to an inhibition of na+ transport. elevated [na] i is a known inhibitor of epithelial na+ channels ( ). the sustained nature of the inhibition in na+ transport to a hc may help explain the longer than expected duration of action of hs in the clinical trials ( , ) . explanation of the paradoxical effects of amiloride observed in a clinical trial ( ) requires further investigation. references: . n engl j med : - , ; . n engl j med : - , ; . physiol rev : - , reduced airway surface liquid (asl) volume resulting from enac mediated na+ hyperabsorption and cftr-mediated hyposecretion plays a critical role in cf lung disease pathogenesis. however, the mechanisms involved in enac/cftr regulation are poorly understood. using human bronchial epithelial cultures (hbecs) under thick film conditions, i.e. in the ussing chamber with the asl washed away, adenosine (ado) directly stimulates cl-secretion. however, in a cl-free environment, ado has no effect on the amiloride-sensitive current, which is mediated by enac. mean change in i sc following µm ado to the apical chamber was . µa/cm from baseline compared to vehicle alone (- . µa/cm ;ns;n= ) in hbecs. mean delta amiloride ( µm) was - . and - . µa/cm respectively (ns; n= each). in contrast, under thin film conditions we have previously shown that ado stimulates sustained asl secretion via activation of cftr in the presence of the protease-inhibitor aprotinin, whereas trypsin pre-treatment abolished this secretion, likely by activating enac and abolishing the electrical driving force for cl-secretion . further, ado-mediated asl secretion is significantly increased if hbecs (thin film conditions) are left with intact asl for h, an action that is abolished by acutely washing the apical surface . thus, we hypothesised that a soluble enac inhibitor was secreted into the asl under thin film conditions, which could accumulate sufficiently to inhibit enac and provide the necessary driving force for cftr-mediated cl-secretion after ado-addition. to search for such an inhibitor, we incubated asl with trypsin-coated beads and identified bound proteins by mass spectrometry (albumin-coated beads were used as a control to exclude non-specific binding). the major identified protein was plunc, a protein that is secreted into the asl both in vivo and in vitro which has no current known function. secretion of plunc into hbec asl was confirmed by western blot. to test whether plunc could alter asl volume homeostasis, we made an anti-plunc shrna retroviral construct which we used to infect hbecs. unlike the control anti-luciferase shrna infected hbecs, anti-plunc shrna-infected hbecs exhibited > % knockdown of plunc and a failure to regulate asl volume (ctrl, . ± . µm; ± . µm; n= ) . when co-expressed with enac in oocytes, plunc inhibited enac currents by % (n= ). cftr function in contrast was unaffected by plunc expression (n= ). thus,it is likely that ado stimulates cftr mediated cl-secretion but is not involved in enac regulation. however, experiments were performed in a very simplified environment i.e. static cultures with little mucosal atp or mucus and these findings will need to be expanded and further evaluated in native tissues. we conclude that plunc, rather than ado may be the soluble mediator in the asl which regulates enac function by reducing its activity. . tarran, r. et al., j. gen. physiol., . ( ) airway surface liquid (asl) absorption is mediated by epithelial na + channels (enac), which establish an osmotic gradient that drives fluid absorption. we and others have recently reported that a protease / anti-protease balance regulates enac in normal human bronchial epithelial cells (hbe) and provides a mechanism for auto-regulation of asl volume. in cf, this balance is disturbed, leading to constitutive proteolytic activation of enac and the pathological na + hyper-absorption characteristic of the disease. to determine if channel activating protease expression is regulated by changes in asl volume and is altered in cf, we examined prostasin expression in control and cf hbe under basal and asl volume expansion conditions using western blotting. prostasin migrates as kda and kda bands in apically biotinylated proteins, apical secretions, and whole cell lysates of primary hbe. following apical aprotinin exposure, only the kda prostasin molecular species was present in the biotinylated proteins, suggesting that the proteolytic conversion of prostasin zymogen to active enzyme occurs on the cell surface. following asl volume expansion, cell surface prostasin expression increased by > % (p= . , n= tissue donors, > cultures from each). as our recent studies indicated that increased proteolytic activation of enac occurs in cf airway epithelia, we next compared prostasin expression in cf and control hbe cells. prostasin expression in the apical biotinylate of cf hbe was . fold greater than in control hbe (p= . , n= tissue donors). furthermore, the ratio of the kda (active enzyme) to kda (zymogen) prostasin molecular species was . fold greater in cf (p= . ), indicating that increased activation of prostasin occurs in cf airway epithelium. we next determined whether increased prostasin activation in cf may reflect the deficiency of a protease inhibitor. serpin e , also known as protease nexin- (pn- ), forms an ~ kda complex with prostasin and permanently inactivates its protease activity. while no significant difference in pn- expression was observed between control and cf hbe, pn- was found to contribute to prostasin regulation by (i) forming an inactive prostasin complex, (ii) inhibiting the amiloride-sensitive short-circuit current across hbe, and (iii) preventing conversion of prostasin zymogen to active enzyme. these findings demonstrate that cellular mechanisms coordinate prostasin expression and activity with asl volume. accordingly, at times when the asl volume is high, prostasin expression increases, and this presumably augments na + and asl absorption to absorb the excess luminal fluid. these regulatory mechanisms govern the apical membrane expression and processing of zymogen to active enzyme. because prostasin is incapable of autocatalysis, these findings support the existence of a proteolytic cascade that controls enac activity during asl volume homeostasis, perhaps reflecting the recently reported matriptase-prostasin cascade. furthermore, the increased expression of prostasin in cf suggests that abnormal regulation of prostasin contributes to na + hyper-absorption in cf airways. supported by the nih, cff, and ala. we report that annexin (anx )-s a forms a functional camp/pka/calcineurin (can)-dependent complex with cftr. cell stimulation with forskolin/ibmx significantly increases the amount of anx -s a that reciprocally co-immunoprecipitates with cell surface cftr and can a. pre-inhibition of the cells with pka or can inhibitors attenuates the interaction. furthermore, we find that the acetylated peptide (stvheilcklsleg, ac - ), but not the non-acetylated equivalent n - , corresponding to the s a binding site on anx , disrupts the anx -s a /cftr complex. analysis of dids and cftr inh -sensitive currents, taken as indication of the outwardly rectifying cl-channels (orcc) and cftr-mediated currents, respectively, showed that ac - , but not n - , inhibits both the camp/pka-dependent orcc and cftr activities. can inhibitors (cypermethrin, cyclosporin a) discriminated between orcc/cftr by inhibiting the cftr inh , but not the dids, sensitive currents, by more than %. furthermore, peptide ac - inhibited acetylcholine-induced short-circuit current measured across a sheet of intact intestinal biopsy. our data suggests that the anx -s a /cftr complex is important for cftr function across epithelia. the content and water (h o) mobility in airway epithelium is tighly coupled to airway function. in cystic fibrosis (cf), h o epithelial permeability is reduced in airways. the demand of noninvasively imaging techniques with high spatial resolution potential is rising because such imaging tools would expedite anatomical and functional phenotyping in the genetically altered mice. magnetic resonance microscopy (mrm) is a noninvasive, inherently three-dimensional ( d) imaging technique capable of visualizing anatomical structures in the mouse and allows for interpretation of complex spatial relationships between substructures and h o. in this study, we explore different mr contrast parameters and signal-to-noise ratios at a µm pixel size to characterize microstructure and h o mobility in ex vivo trachea of cf transmembrane conductance regulator (cftr)-deficient (cftr knockout, cftr tm unc ) mice and their aged-matched wt littermates. this study is performed using a bruker mrm system at . tesla. we demonstrate for the first time the ability of d-mrm to map the h o content and mobility in trachea epithelium. from the d-mrm videoimages, differential h o content was visualized in different levels of trachea in wt and cf mice. t mrm images depicting the h o rotational mobility which is related to environmental viscosity of trachea epithelium will be also shown. finally, this d-mrm imaging method is a valuable method for measuring h o content and permeability in airways and can serve for assessing the effects of drugs on h o mobility in cf airways. supported by grants from inserm, upmc-paris , cnrs, and the french cystic fibrosis association (vlm). cystic fibrosis (cf) is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (cftr) protein resulting in progressive lung oxidative damage. in this study, we evaluated the role of cftr in the control of ubiquitin-proteasome activity (ups system) and the nf-κb / iκb-αsignaling after lung oxidative stress. we exposed cftr deficient (cftr-/-) and wild type mice for h to hyperoxia-mediated oxidative stress. cftr deficient mice exhibited significantly higher lung proteasomal activity than cftr+/+ animals after oxidative stress. this was accompagnied by a strong reduction of lung caspase- activity and an absence of degradation of nf-κb inhibitor iκb-α. in vitro, human cftr-deficient lung cells also exhibited higher proteasomal activity and a lack of increased nf-κbdependent transcriptional activity compared to cftr-sufficient lung cells after oxidative stress. furthermore, inhibition of the proteasomal activity by mg in cftr-deficient lung cells restored the nf-κb/iκb-α signaling to that of cftr-sufficient lung cells. inhibition of caspase- by z-dqmd in cftr-sufficient lung cells mimicked the response profile of increased proteasomal degradation and lowered nf-κb transcriptional activity of cftr-deficient lung cells when exposed to oxidative stress. all together, these results suggest that cftr is a crucial molecule in regulating proteasomal degradation and nf-κb activity in lung epithelium under oxidative stress. staphylococcus aureus, one of the major pathogen involved in airway infections, releases in the airway lumen virulence factors that may impair the airway epithelial functionality. to date, the effect of s. aureus virulence factors on the loss of electrolyte homeostasis of the airway epithelium has not been investigated. we have previously shown that the combination of a corticosteroid and a long-acting beta agonist attenuated the airway epithelial cell inflammatory response induced by s. aureus virulence factors. the aim of the present work was to investigate the effect of s. aureus virulence factors on the airway epithelial tightness and on the chloride efflux of airway epithelial cells incubated or not with salmeterol hydroxynaphthoate × - m and fluticasone propionate × - m (sm/fp). the airway epithelial tightness was assessed by immunocytochemistry, western blotting and transepithelial resistance measurement. the chloride efflux was evaluated by dynamic imaging using the -methoxy-n-( -sulfopropyl) quinolinium (spq) fluorescent probe. s. aureus (strain - ) virulence factors were obtained by growing bacteria at × cfu/ml for h at °c. the bacteria supernatant containing s. aureus soluble virulence factors was then centrifuged, filtered and diluted at % in cell culture medium (a concentration that did not alter cell viability). human airway epithelial cells (mm ) were incubated with % s. aureus virulence factors for h and then co-incubated or not with sm/fp for h. we observed that the incubation with % s. aureus virulence factors alone did not significantly alter the epithelial integrity assessed by the expression of tight junction proteins and transepithelial resistance measurement. however, when the cells were incubated with sm/fp alone or with % s. aureus virulence factors plus sm/fp, the expression of tight junction proteins was significantly increased (p< . ) as compared to cells incubated with s. aureus virulence factors. interestingly, the chloride efflux in airway epithelial cells was significantly decreased in a time-dependent way by s. aureus virulence factors ( fold decrease after a h incubation). the co-incubation of airway epithelial cells with s. aureus virulence factors and sm/fp prevented the s. aureus virulence factorsdependent chloride efflux decrease. our results demonstrate that bacterial virulence factors induce the loss of the electrolyte homeostasis and suggest that the treatment by the combination of a corticosteroid and a long-acting beta agonist may preserve the airway epithelial functionality. supported by association vaincre la mucoviscidose and glaxo-smithkline nilsson, h.e. ; dragomir, a. ; ahlander, a. ; johannesson, m. ; roomans, g.m. . medical cell biology, uppsala university, uppsala, sweden; . women's and children's health, uppsala university, uppsala, sweden inhalation of hyperosmotic solutions (salt, mannitol) has been used in the treatment of patients with cystic fibrosis or asthma, but the mechanism behind the effect of hyperosmotic solutions on mucociliary clearance (mcc) is unclear. one explanation has been suggested, namely that hypertonic solutions open tight junctions, which may lead to increased water transport followed by an increased airway surface liquid (asl) volume. furthermore, the role in cftr-mediated hco -conductance in regulating asl ph has led us to investigate if ph changes may have an effect on the tightness of tight junctions. the effect of osmolarity was investigated on the hbe o-cell line by the addition of nacl, nabr, licl, mannitol or xylitol ( - mosm). the effect of ph was investigated on the hbe o-, calu- and t cell lines as well as the cystic fibrosis cell line cfbe o-. transepithelial resistance was measured as indicator of the tightness of the cultures. cell-cell contacts and morphology were investigated by immuno-fluorescence and by transmission electron microscopy, with lanthanum nitrate added to the luminal side of the epithelium to investigate tight junction permeability. the electrolyte solutions caused a significant decrease in transepithelial resistance from mosm on, when the hyperosmolar exposure was gradually increased from to mosm, whereas the non-electrolyte solutions caused a decrease in transepithelial resistance from mosm on. immunofluorescence micrographs showed weaker staining for the proteins zo- , claudin- in treated samples compared to the control. the ultrastructure revealed an increased number of open tight junctions as well as a disturbed morphology with increasing osmolarity, and with electrolyte solutions opening a larger proportion of tight junctions than non-electrolyte solutions. it was noted that during the exposure of the cultured cells to the hyperosmolar solutions, the ph of the medium increased from . to . . hbe o-cells exposed to both a rise in ph and hyperosmotic stress showed an overall lower teer and a significantly reduced ability to recover from stress compared to cultures at a ph hold constant at . . without exposure to hyperosmolar solutions, a rise in ph caused a significant decrease in transepithelial resistance in hbe o-cells, calu- and t cell lines but not in the cfbe o-cell lines, where the reaction was significantly less and delayed. in conclusion, hyperosmolar solutions caused a reversible opening of the tj in hbe o-cell cultures with electrolytes having stronger effects than non-electrolytes, where one of the effects on mcc may be due to increased water transport across the leaky paracellular space. an increase in ph caused a significant decrease in teer in the healthy cell lines compared to the cfbe o-cell line. we speculated that an impaired alkalinisation of the apical fluid due to a defective cftr also will cause the tight junctions to react different to other external stimuli, such as osmolarity, compared to healthy cells, which is also indicated by preliminary experiments on the effect of nacl on teer in cfbe o-cultures. extracellular nucleotides regulate surfactant secretion in alveoli and mucociliary clearance in airway epithelia, but the mechanism(s) of their release and their regulatory pathways remain incompletely understood. previously, we showed that hypotonic swelling of a epithelial cells induces ca + -dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. in this study, we examined sources of intracellular ca + ([ca + ] i ) elevation evoked by acute % hypotonic stress and the role of autocrine purinergic signaling in ca + -dependent atp release. we found that atp release does not directly involve ca + influx from extracellular spaces, but depends entirely on ca + mobilization from intracellular stores. the [ca + ] i response consisted of slowly-rising elevation representing mobilization from thapsigargin (tg)-insensitive stores and a superimposed rapid spike due to ca + release from tg-sensitive endoplasmic reticulum (er) stores. the latter could be abolished by hydrolysis of extracellular tri-and di-nucleotides with apyrase; blocking p y /p y receptors of a cells with suramin; blocking udp receptors (p y ) by ppads; emptying tg-sensitive stores downstream with µm tg or mm caffeine in ca + -free extracellular solution; or blocking the ca + -release inositol , , -triphosphate (ip ) receptor channel of the er with µm aminoethyl diphenylborinate. these results demonstrate that the rapid [ca + ] i spike results from the autocrine stimulation of ip )/ca + -coupled p y /p y receptors, which accounts for ~ % of total ca + -dependent atp release evoked by hypotonic shock. our study reveals a novel paradigm in which atp release is amplified by the synergistic autocrine/paracrine action of co-released uridine and adenosine nucleotides. we suggest that a similar mechanism of purinergic signal propagation operates in other cell types. (this study was supported in part by the canadian institutes of health research and the canadian cystic fibrosis foundation (ccff). s.t. was the recipient of a ccff studentship). the maintenance of a thin liquid layer on the airways and alveoli surfaces is essential for normal lung physiology.yet, the mechanism sensing the height of the layer remains obscure. in this study, we examined atp secretion from human lung a epithelial cell monolayers mounted in a closed, flowthrough chamber ( . mm height) and found that passage of an air bubble over the monolayer caused transient (< . min) but significant atp release ( to , pmoles/ cells). air bubble-induced atp release was reduced by~ % from cells loaded with the intracellular ca + chelator bapta-am, and was completely abolished in n-methylmalemide-treated ( mm) cells, suggesting the involvement of ca + -dependent exocytosis. fura- intracellular ca + ([ca + ] i ) imaging experiments revealed transient [ca + ] i elevation during the passage of an air bubble over the cell monolayer, but the [ca + ] i response did not involve non-specific ca + influx from the extracellular space, e.g. due to cell damage, since similar responses were observed in ca + -free extracellular solution. ethidium bromide staining did not disclose any cell damage in these experiments. confocal fluorescence microscopy study showed reversible cell deformation (flattening) of % to % in height in the monolayer part in contact with the air bubble and confirmed that cell integrity was entirely preserved. these experiments demonstrate that in close proximity to the air-liquid interface (i.e. between the air bubble and the wet cell surface), surface tension forces are transmitted directly on cells, causing their mechanical deformation, elevation of [ca + ] i and subsequent atp release. we propose that a similar mechanism may operate in vivo in the airways, where surface tension forces acting directly on exposed epithelial cell surfaces may provide a fail-proof mechanism to protect proper airway surface liquid volume via mechanosensitive, ca + -dependent atp release and purinergic modulation of fluid secretion. this mechanism may be defective in cystic fibrosis due to excessive mucus layer covering the airways. introduction : cystic fibrosis is a genetic disease that reflects the consequences of mutations in the cystic fibrosis transmembrane conductance reg-ulator (cftr) gene, affecting anionic transport in epithelia. slc family members are anionic transporters involved in cl-and hco -absorption or secretion in epithelia. in addition, the activation of some slc family members by cftr has been demonstrated (nature cell biology, , : - ) . slc a is a poorly characterized member of this family, being the solely expressed in lung. putative interaction domains with cftr are also present in the slc a protein. in this study, we have investigated the transport properties of slc a (human) to determine the functional and pharmacological characteristics of this transporter. methods : to this aim, electrophysiological studies (two-electrode voltage clamp or current clamp methods and intracellular ph measurements using ph sensitive microelectrode) were performed in xenopus laevis oocytes expressing slc a proteins. complementary ( cl) transport studies were also undertaken. results : the protein expression results in the appearance of an anionic current showing a linear current/voltage relationship. cl influxes experiments confirmed the induced cl-permeability following slc a protein expression. the sequences of conductivity, cl->i-> no -≥ gluconate > so -and selectivity (px/pcl), i-> cl-= no -> gluconate > so -are found. the hco -conductance mediated by the slc a protein expression is low. using co /hco -containing ringer solutions, no intracellular phi changes were detectable in conditions (low chloride in external medium) favoring the cl-/hco -exchange whereas phi changes (alkalinization) were observed with the expression of the ae exchanger. however, phi changes could be detected in conditions largely favoring the driving force for hco -entry. dids and ns inhibited slc a associated currents. the specific cftr inhibitor (cftrinh ) or glybenclamide had little effect. elevation of intracellular camp (a cftr activator) was also ineffective while maneuvers increasing intracellular calcium blocked the slc a associated currents. conclusions : our study demonstrates that slc a presents an electrogenic anion conductance (characteristics of an anionic channel) when expressed in xenopus oocytes (however a transporter functioning as a ncl-/hco -exchanger can not be excluded). this function (channel) has also been described for another member of this family, slc a (j biol. chem, , : - ) . the physiological role of slc a , present in the bronchial cells of airway epithelia and its potential interaction with cftr has to be precised in situ or in oocytes and in mammal expression systems. medicine, women's & children's hospital, nth adelaide, sa, australia; . cf research & treatment center, university of nth carolina at chapel hill, chapel hill, nc, usa; [ ] [ ] [ ] [ ] [ ] [ ] jasri, hyogo, japan; . physics and synchrotron science, monash university, clayton, vic, australia; . paediatrics, university of adelaide, adelaide, sa, australia non-invasive imaging of lung (e.g. hrct, mri, pet) is a valuable modality for detection and monitoring of the effects of cf in human airways, but resolution is limited; e.g. airway hrct detects airways no smaller than ~ . mm dia. detection of wall or lumen changes in smaller airways, or the detection and monitoring of induced airway disease in live rodent models, demands significantly greater resolution coupled with rapid image capture. we report substantially increased airway resolution is achievable using synchrotron phase-contrast x-ray and can produce three-dimensional reconstructions encompassing the smallest mouse lung airways. nembutal-anaesthetised c bl/ mice (~ gm) were imaged at the spring- synchrotron in hyogo, japan. -d phase-contrast airway images were obtained on ccd detectors ( . µm pixels) with a cm phase-contrast propagation distance at kev; nose and trachea were imaged at or sec intervals ( - ms exposures) over mins. mice killed with nembutal overdose were imaged in axially aligned segments to obtain dimensional ct data voume of approximately x x mm with x x um voxels of nose, trachea, and lung (hammamatsu ccd detector). volume renderings were produced using volview or osirix software. live, -d nasal or tracheal imaging revealed airway-surface activity in some live mice consistent with dynamic mucociliary clearance activity. individual ct slices revealed the fine detail in the mouse lung, with dynamic ("fly through") sequences of lung ct slices permitting visual identification and tracking of lung tree branching from trachea (~ mm dia) into small airways (approx um dia). adjustment of x-ray contrast, opacity, and range in these volume reconstructions permitted selective display of the mouse lung conducting-airway tree and airway surfaces. selected regions of the lung could be examined statically, or in rotation through different orientations in high-resolution -d. synchrotron phase contrast x-ray provides a new option for non-invasive imaging of intact mouse lung, at a resolution that permits examination of small conducting airways in mice. combined with volume-reconstruction software these ultract images can provide a powerful option for understanding the structure-function relationships produced by airway disease. ultra-ct -d studies are underway to compare lung airways at high resoltuion in normal and transgenic mice having altered airway pathophysiology. the epithelial sodium channel, enac, has a vital role in the function of the pulmonary epithelia and significantly contributes to the pathophysiology of the cf airway. thus, strategies to repair mutant cftr dysfunction must also consider the influence of such repair on enac functional expression. the ∆f trafficking repair agent -phenylbutyrate modulates the expression of the kda molecular chaperones hsc and hsp in cf epithelial cells. we therefore assessed the role of these chaperones in the regulation of enac trafficking. in xenopus oocytes, we previously observed that overexpression of hsc inhibits murine enac (menac) functional and surface expression. in contrast, hsp can either enhance or inhibit menac functional and surface expression depending on the extent of overexpression [goldfarb et al. ( ), proc. natl. acad. sci. : - ]. here, we tested the hypothesis that these differential effects of hsc and hsp on menac expression also occur in epithelial cells. mdck cell lines with stable expression of α-ha, β-v , γ-myc-menac and tetracyclineinducible expression of either hsc , atpase-deficient hsc , hsp or atpase-deficient hsp containing myc/his epitope-tags were selected. all epitopes were fused to the respective c-termini. these cells were grown as high-resistance (> Ω/cm ) monolayers on permeable supports. doxycycline-induced overexpression of hsc decreased amiloride-sensitive i sc and the whole cell content of α-haand β-v -menac. in contrast, lower amounts of tetracycline-induced hsp overexpression increased amiloridesensitive i sc and whole cell α-haand β-v -menac expression; these effects were absent at higher levels of hsp overexpression. these data are consistent with our previous data in xenopus oocytes. interestingly, the atpase-deficient chaperones had the opposite effect on menac functional expression. modest doxycycline-induced expression of atpase-deficient hsc increased amiloride-sensitive i sc in these cells, while modest overexpression of atpase-deficient hsp decreased amiloride-sensitive i sc . these effects may result from "dominant negative" interference with hsc and hsp function, respectively. these data are consistent with hsc and hsp having different effects on menac functional expression in epithelial cells, and that these effects are dependent upon the atpase activity of the respective chaperones. supported by grants from niddk. we have studied survival, airway epithelia bioelectrics and lung pathology of mice over-expressing various combinations of the epithelial na + channel (enac) subunits (α, β and γ, genes scnn a, scnn b and scnn g). we generated double-transgenic mice and crossed them with single-transgenic mice to obtain litters comprising all possible genotypes. survival analysis revealed that overexpression of βenac in combination with either αenac or γenac significantly reduced survival in comparison to wild-type (wt) littermates. strikingly, at days of age, all genotypes were represented according to the expected mendelian proportion, except for the triple transgenic αβγenac, which was significantly under represented. in utero studies are ongoing to understand if over-expression of αβγ affects the fetuses or the newborn pups during the first hours post-partum. due to the high mortality of mice over-expressing βenac subunit combinations, we studied tracheas from pups at days of age. as previously reported, mice overexpressing β, but not the α or γ enac subunits, exhibit airways hyperabsorption of na + and lung pathology (mall et all ) . the βenac tracheas exhibited na + absorption [as measured by the change in short circuit current (isc) in response to amiloride in the ussing chamber] that was ~ . fold greater (∆isc . ± . µa×cm - , n= , means±sem) than wt ( . ± . µa×cm - , n= ). over-expressing αβ or αγ enac subunits resulted in rates of na + absorption that did not differ significantly from those of βenac or γenac subunit alone, respectively. however over-expressing the β and γ enac subunits together resulted in an amiloride sensitive isc that was ~ . fold greater (∆isc . ± . µa×cm - , n= ) than wt. we also studied the tracheal bioelectrics of the few day-old αβγenac pups available. tracheas from the αbγ pups exhibited na + absorption that was ~ . fold greater (∆isc ± . µa×cm - , n= ) than wt. due to the small sample size, we cannot determine if this response is significantly different from the response of the βγenac pups. analysis of lung pathology in day-old pups revealed that all the combinations that exhibited increased airway na + absorption and decreased survival in comparison to wt littermates, e.g. αβ, βγ and αβγ, also presented with alveolar space enlargement, maybe due to postobstructive air trapping, and airway epithelia necrotic degeneration associated with ab-pas negative bronchial obstruction. from these data, it appears that the rate of airway na + absorption negatively correlates with the survival of the pups. thus, it is likely that the mucus and airway surface liquid is more dehydrated as a function of the rates of na + absorption (aβγ > βγ > αβ, β > wt) which produces gradual failure to survive due to asphyxia secondary to airway obstruction. supported by nih scor p hl choi, j. , ; joo, n. ; wu, j. ; krouse, m. ; wine, j.j. . cystic fibrosis research laboratory, , stanford, ca, usa; . otorhinolaryngology, yonsei university, seoul, south korea submucosal glands, which produce most airway mucus, are mainly controlled by parasympathetic pathways that stimulate glands via airway intrinsic neurons distributed along the airway walls. the intrinsic neurons can be stimulated via axon reflexes from receptors in the mucosa. in previous experiments, it was shown that capsaicin applied to the mucosa stimulated gland secretion in the upper tracheas of wt but not cftr -/-mice (ianowski et al., j physiol, , , ) . it seemed important to assess a possible role of capsaicin-sensitive pathways in pigs and humans. we obtained pig tracheas following acute experiments carried out for other reasons, and human airways following lung transplants from donor tracheas and from the excised lungs of the transplant recipients. secretion from individual submucosal glands was quantified optically by time-lapse digital imaging of the growth of spherical bubbles of mucus in an oil layer in humans, commercial chili oil ( µm dispersed in µm mineral oil) stimulated mucus secretion from submucosal glands from both donors (n = glands from subjects, . ± . nl/min/gland) and disease control subjects (n = glands from subjects, . ± . nl/min/gland). however, there were no responses to chili oil in submucosal glands from cf subjects (n = glands from subjects, . ± . nl/min/gland). in pigs, we determined concentrationresponse relations for gland secretion in response to purified capsaicin (sigma). the threshold was ~ µm and the ec was . ± . µm. the response to µm capsaicin was partially blocked by high dose ttx (> µm). as in humans, the capsaicin response required cftr, because it was blocked ~ % by cftrinh- , (n = glands, from pigs, p < . ). cftrinh- is presently the most specific inhibitor of cftr that can be used with glands, where we do not have access to the apical membrane. in ferrets, intrinsic airway neurons express acetylcholine, vip and sp, often colocalized within the same neurons (dey et al., am j respir cell mol biol, , , ) . in pigs, all three of these transmitter systems appear to be involved in gland secretion to capsaicin, because the response was partially blocked by the nk- receptor blocker l (oxalate salt, µm), the vip receptor inhibitor l k ( µm), and the muscarinic receptor blocker atropine ( µm); each of these blocked to % of the response (n = to glands from to pigs). indeed, most of the local pathways are probably damaged, which may account for the relatively small responses to strong stimulation. relatively few experiments have been carried out in intact airways with uninterrupted innervation and circulation, but in those experiments the glands were highly responsive to modest mucosal stimuli (reviewed in wine, auto neurosci, , , ) . hence, we hypothesize that ( ) reflex stimulation of glands plays an important role in lung innate defenses; ( ) defects in these responses is an important reason that cf airways are prone to infection, and ( ) local reflexes may assume greater importance for maintaining the mucosal defenses of transplanted lungs. supported by niddk ro - (jjw), cff and cfri. most airway mucus arises from submucosal glands, which express cftr in their ciliated ducts and in serous cells. the airway glands secrete to agonists that either increase intracellular ca + (e.g. acetylcholine) or camp (e.g. vip).substance p(sp) also stimulates airway gland liquid secretion in pig (trout l et al., am j physiol lung cell mol physiol. , ,:l ) , suggesting that sensory afferents or local neurons that express sp play a role in stimulating submucosal glands. we used pharmacological methods to dissect the secretory mechanism of sp-induced gland secretion in comparison with responses to carbachol. tracheas were obtained from pigs after acute surgeries carried out for other reasons. the ventral mucosa with underlying glands was dissected from the cartilage, pinned mucosal side up at the gas/bath interface of a physiological chamber, and covered with oil. secretions from individual glands could be visualized as spherical bubbles in the oil, and secretion rates determined by optical monitoring of bubble diameters. concentration-response relations for gland secretion were determined for sp ( - bubbles at each concentration from pigs). the threshold was ~ nm for sp and the ec was . ± . µm. the maximum secretion rate to sp was . ± . nl/min.gland, which is only ~ / of the maximal secretion rate to carbachol ( . ± . nl/min.gland). the inhibition profile for responses to sp ( µm) was quite different from those to carbachol ( nm, a value chose to mimic the secretion rate to µm sp). gland secretory responses to µm sp ( . ± . nl/min.gland, glands from pigs) were strongly inhibited by µm cftrinh- ( . ± . nl/min.gland, glands from pigs, p< . ), and by µm clotrimazole ( . ± . nl/min.gland, glands from pigs), whereas µm niflumic acid did not inhibit ( . ± . nl/min.gland, glands from pigs). in contrast, secretory response to nm carbachol ( . ± . nl/min.gland, glands from pigs) were only weakly inhibited by cftrinh- ( . ± . nl/min.gland, glands from pigs, p< . ), and by clotrimazole ( . ± . nl/min.gland, glands from pigs, p> . ), but niflumic acid, which was ineffective with sp, inhibited the carbachol response ( . ± . nl/min.gland, glands from pigs, p< . ). gland secretion to sp depends at least partially on intracellular ca + release, because the response was partially inhibited by µm bapta-am ( . ± . nl/min.gland, glands from pigs, p< . ). there was no additional effect of sp on top of µm carbachol, but a subthreshold level of sp ( nm) showed synergic response ( . ± . nl/min.gland, glands from pigs) with subthreshold level of vip ( nm). we are presently testing how sp activates cftr-dependent mucus secretion. in initial experiments to determine if pkc was being recruited, however the pkc inhibitor (gf , sigma) did not suppress sp-induced mucus secretion. in contrast, the nkcc inhibitor bumetanide ( µm) markedly suppressed secretion to sp ( . ± . nl/min.gland, glands from pigs, p< . ). supported by niddk ro - (jjw), cff and cfri. mucus obstruction of airways is considered the most vicious agent of morbidity and mortality in cystic fibrosis (cf). to provide optimal defense of the small airways the volume/thickness of airway surface fluid must be controlled according to physiological demand. to date we have no clear understandings of how these fluids are maintained in a steady state between two pathological extremes (too little or too much) in native small airways. earlier studies have attempted to measure the ion transport properties of small airways of sheep (al-bazzaz et. al. ) and pigs (ballard et. al. ), but the complicated branching structure of small airways may have compromised electrical signals. to avoid dissection trauma wang et. al. ( ) examined the electrophysiological properties of undissected intact small airways from pigs in vitro by microperfusing bronchioles (diam. - mm) embedded in the lung parenchyma; however, these studies were limited to luminal manipulations and transbronchiolar electrical potentials only. to more rigorously define small airway properties, we designed a special micro ussing chamber ( area ≈ mm ). we excised and opened small airways (diam. - mm) of pig lung that we mounted as a flat sheet over a pvc supporting filter ( - µm holes). both sides of the tissue could then be bathed with different solutions at °c. the luminal side was isolated by pressing a pipette (≈ mm diam., tip fire polished) on to the apical surface of the tissue until it sealed electrically. constant current pulses ( . - . µa) were passed across the tissue. transepithelial potential (tep) and resistance in bilateral mm nacl ringers were, - . ± . mv and . ± . Ω.cm (mean ± se) respectively. when mm naglu ringers replaced the luminal solution, tep increased significantly to - . ± . mv (n= , p< . ) and resistance increased to . ± . Ω.cm (n= , p< . . adding forkskolin (fsk, µm) plus ibmx ( . mm) hyperpolarized the tep to - . ± . mv (n= , p< . ), but decreased the resistance to . ± . Ω.cm (n= , p< . ). luminal amiloride ( µm) depolarized the tep to - . ± . mv (n= , p< . ) but significantly increased resistance to . ± . Ω.cm (n= , p< . ). these results show that this system supports measurements of ion transport properties of airways smaller than mm diameter where cf lung pathology is thought to originate. these airway epithelia express a very high constitutively active clconductance and are apparently capable of secretory as well as absorptive functions. amiloride sensitive na + conductance (≈ ms/cm ) was not significantly affected by fsk added to activate secretion. the observation that stimulation with fsk did not significantly suppress na + conductance (≈ ms/cm ) (p= . ) may suggest that separate groups of reabsorptive and secretory cells comprise small airway epithelia. mucins are class of proteins uniquely characterized by large glycosylated domains. these proteins likely play a key role in cystic fibrosis (cf) lung disease as major constituents of mucus. in airways of both humans and mice, the gel-forming secreted mucins are represented by muc ac and muc b, and the transmembrane mucins are represented primarily by muc , muc , and muc . we have been interested in the role of the transmembrane mucins in airways and have previously demonstrated that lack of muc in a knock-out mouse model (provided by s. gendler) shows increased susceptibility to adenoviral-mediated gene transfer (stonebraker, j. virology, ) . to expand upon these findings, mice deficient for muc were developed using embryonic stem cell technology by deleting exon of the mouse muc gene, which contains the atg start codon and signal peptide. rna analysis reveals loss of exon in homozygous mutant mice and immunohistochemistry using a vntr antibody indicated a loss of full length muc protein. muc homozygous mice are viable and do not develop any spontaneous, readily detectable disease phenotype. although the muc deficient mice do have reduced fertility consisting of both reduced litter size and number, all major tissue systems where the muc protein is easily detected, including the eye, respiratory tract, intestine, and reproductive tracts, appear normal in year old mice after histological analyses. in contrast to the results we reported in the muc mice, the muc deficient mice do not show an increase in adenoviral mediated gene transfer, suggesting that muc is not involved in the barrier aspect of the airway glycocalxy, at least for adenoviral infection. interestingly, quantitative real-time pcr analysis revealed up-regulation of muc protein in the trachea of the muc deficient mice, suggesting that muc may be able to compensate for loss of muc . the expression of muc in human ciliated cells as revealed by recent antibody studies prompted us to evaluate the potential role of muc in ciliary function. ciliary beat frequency (cbf) was monitored in a shear chamber using microscopy image analysis. cbf under baseline conditions for this study (low shear stress) from muc deficient trachea (c bl/ j congenic n ) was significantly lower than that for littermate controls ( . vs. . , sem ± . , p = . ). application of shear resulted in increased cbf in both groups, although the wt response to shear was smaller in magnitude. thus, muc may have a role in supporting basal cbf and may make cbf less vulnerable to stimulation by shear, perhaps by reducing the inherent friction of ciliary motion. this finding underscores the usefulness of this model for dissecting out the normal roles that the transmembrane mucins play in airway biology. supported by nih (hl ) and cff (r -cr ). enac (joo et al. jbc , and jbc , ) . in a search for a model to test the hypothesis we found that ussing chamber short-circuit current(i sc ) measurements with freshly obtained airway mucosa provided useful information about enac activity in response to various drug treatments. in these studies we observed an as yet unidentified pathway that regulates surface enac activity. airway mucosa from sheep tracheas displays a large, amiloride/benzamil-sensitive i sc prior to stimulation. when stimulated with basolateral carbachol, the enac i sc was almost abolished, as indicated by a sustained decrease ( . ± . %, n= ) in i sc after an initial increase and left a small effect to subsequent amiloride or benzamil. in contrast, when carbachol was given after benzamil, it again produced a peak response followed by a sustained increase (~ %, n= ) in the i sc . enac inhibition by carbachol was dose dependent with a minimal sensitivity between . ~ µm and was slowly reversible. apical µm atp also inhibited enac i sc by . ± . % (n= ) and carbachol on top of atp further reduced the enac i sc by . ± . % (n= ). however, u/ml of apyrase pretreatment, in attempt to breakdown atp, failed to inhibit carbachol-induced enac inhibition, indicating that atp release is not on obligatory component of carbachol inhibition of enac. carbachol inhibition of enac could result from a direct action of carbachol on the surface epithelia, by activation of airway intrinsic neurons, or by activation of the glands, which were intact in these preparations and are strongly stimulated by carbachol. rabbit tracheas, which lack airway submucosal glands, displayed large enac currents and these were not inhibited by cholinergic stimulation (n= ). carbachol-induced enac inhibition, like gland stimulation but unlike stimulation of neurons, was mediated by muscarinic receptors since it was eliminated by atropine but not by the nicotinic receptor antagonist, hexomethonium bromide (n= ). however, inhibition of gland secretion with hepes/bumetanide did not eliminate the ability of carbachol to inhibit enac. the effects of carbachol on surface na + transport in our study are consistent with previous observations in microperfused sheep bronchiole preparations (al-bazzaz, ajp-lung , ). although we do not yet know the mechanism, it would be physiologically efficient if parasympathetic, cholinergic activation of airway gland secretion was accompanied by enac inhibition, which would minimize the fluid absorption by surface epithelia and increase clearance. it will be important to determine if cholinergic inhibition of enac is present in human tissues, and if so, if it is defective in cf. supported by cff and nih. introduction: lung disease accounts for more than % of the current morbidity and mortality associated with cf. the majority of research aiming to elucidate the reason(s) of severe lung disease has focused on airway surface liquid (asl) layer homeostasis or composition. the cftr gene encodes a camp-regulated clchannel located on the apical side of the epithelium and is instrumental in maintaining a hydrated asl layer and promoting effective mucociliary clearance (mcc). in patients with class i or class ii cftr genetic mutations, normal cftr clchannel activity in the epithelium is impaired resulting in na + hyperabsorption and consequently in the generation of a thick and static overlaying asl layer creating an environment that favors progressively worsening bacterial infections. in this study we investigated the potential contributory role of altered respiratory cilia function to inefficient mcc in cf airways allowing for bacterial infestations. our ex vivo model system was the nasal airways of the cftr gene knockout (cftr tm unc ) mouse in which key aspects of the phenotype of human cf lung disease, such as dehydrated asl layer and goblet hyperplasia, are observed. methods and results: ciliary beat frequency (cbf) was measured using a dual temperature controlled perfusion chamber, differential interference contrast microscopy and high speed digital video analysis system. we evaluated the cbf of nasal septa explants from homozygote cf (affected) and non-affected littermate wild type (wt) and heterozygote mice congenic on the c bl/ background. the basal cbf of homozygote cf mice ( ± hz, n= ) was significantly lower than that of wt mice ( ± hz, n= ) and also heterozygote mice ( ± hz, n= ) (p< . , anova, snk test). to determine whether the dehydrated asl layer was still present on the surface of the cf mouse nasal airway epithelium and as such impeding normal cilial beating, we fixed nasal septa (n= ) of cf and wt mice immediately following isolation using the pfc/oso fixation method to preserve the fragile asl layer and compared the height of the asl layer to nasal septa (n= ) from cf and wt mice fixed with pfc/oso immediately following cbf analysis. electron microscopy analysis revealed that although the asl layer was present at the time of isolation, there was no asl layer following the perfusion of solutions necessary for the cbf measurements. this finding demonstrated that the activity of cilia was not impaired in the cf nasal airway epithelium due to the presence of a dehydrated asl layer. cbf frequency was also significantly reduced in three different codes of mouse ciliated nasal airway epithelial cultures derived from affected cf mice compared to those derived from non-affected wt mice. conclusion: we show that the absence of the cftr gene product in the cf knockout mouse nasal airway epithelium is associated with significantly decreased basal cbf compared to the non-affected wt and heterozygote mice. this observation potentially provides an additional mechanism as to why lung disease persists in human cf lungs. ml and mba contributed equally. clinical studies have linked increased sputum and peripheral blood neutrophil mpo activity with increased airflow obstruction in cystic fibrosis (cf) patients of the same age, gender, airway bacterial flora, and cftr genotype. variations in the tgf-β gene associated with increased tgf-β production have been linked to worse airflow obstruction in cf patients of similar age, gender and cftr genotype. we hypothesized that in the presence of mpo, tgf-β production would increase in airway epithelial cells. we obtained normal human airway epithelial cells (hae) and cultured them in trans-wells. tgf-β mrna was measured by pcr. tgf-β protein was detected by immunofluorescence staining and immunoprecipitation. we found increased tgf-β mrna after hae were exposed to mpo (at activities found in cf sputum) normalized to mrna of constitutive beta actin. under identical conditions, tgf-β protein expression was increased in hae. we conclude that neutrophil mpo present on the apical surface of cultured hae induces transcription and protein synthesis of tgf-β . these results suggest that mpo induction of airway epithelial tgf-β is one molecular system linking the chronic neutrophilic endobronchitis seen in cf and subsequent airway wall fibrosis. are more oxidatively stressed than normal. as oxidative stress (os) is a potent activator of nf-κb, these data supported our hypothesis that cftr dysfunction causes alterations in the expression and/or modification of redox related proteins thereby enhancing activation of nf-κb. delineation of this interaction will produce good therapeutic targets. in this report we examine the role of os in cf inflammation. methods: to address our hypothesis, we used both in vitro and in vivo models of cf. our airway epithelial cell models are the hteo-pcep and pcep-r cell pair; and the hbeo-sense (s) and antisense (as) cell pair. additionally, we examined whole lungs from - week old b . s -cftr tm mrc mice (r h mutants back-crossed into the c bl j background). proteins were prepared from homogenates of whole samples, run on -d gels, and their densities compared. the gel spots of differentially expressed proteins were excised, subjected to in-gel trypsin digestion, and identified by lc ms/ms. for biochemical analysis, we tested for the levels of h o , glutathione, and lipid peroxidation. we correlated these data with measurements of nf-κb activity (elisa and cytokine production), and the activity of nrf- , an antioxidant (ao) response pathway transcription factor. we studied cultured cells in the presence and absence of inflammatory stimulation (tnf-α/il- β), and compared non-stimulated cf mouse lungs to lungs from normal litter mates. results: our proteomic analysis revealed that in the absence of inflammatory stimulation, a paradoxical decrease in ao proteins exists in cf cells by or more fold compared to matched pairs. this is despite a significant increase in intracellular os, which we confirmed by different biochemical measures. activity of nrf- was decreased by ~ % in cf cells vs. normal. the data predict an increase in h o , which is a potent activator of nf-κb, and we confirmed both a significant elevation in h o (up - fold in cf) and a related significant increase in nf-κb activity, as assessed by elisa for nuclear p , and il- and il- production. when we analyzed whole lungs from cf mice for antioxidant protein levels, we similarly observed decreases vs. normal littermates. when cells were stimulated, differences in protein expression and oxidative stress between cf and normal were further enhanced, corresponding to an excessive activation of nf-κb and increased production of il- and il- . treatment with the antioxidants nac or selenium decreased the activation of nf-κb to normal levels. members of the clc family of proteins form either voltage-gated chloride channels or cl -/h + exchangers; nine subtypes are found in mammals. among these, clc- is one of the most ubiquitous variants, which is expressed in several of the types of epithelial cells that also express the cystic fibrosis transmembrane conductance regulator (cftr). it has been postulated that clc- may be a suitable alternative chloride pathway in these cells; however the contribution of clc- to chloride transport in these cells is poorly understood. this is partly due to the lack of appropriate pharmacological tools that are capable of specifically inhibiting clc- . we describe here the isolation, from venom of the scorpion leiurus quinquestriatus hebraeus, of the first peptide inhibitor of any clc channel. this toxin, which we have named gatx , specifically inhibits the clc- channel. gatx bears primary sequence identical to that of a toxin thought to serve as a k + channel inhibitor, although no molecular target for this toxin was previously known. a homology model of gatx reveals that it adopts a similar fold to other scorpion toxins. gatx was prepared via solid-phase chemical synthesis, and the synthetic toxin inhibits clc- with higher affinity than any other inhibitor of clc- or any other chloride channel. the k d for gatx mediated inhibition of clc- in multichannel patches is only pm at v m = - mv, and pm at - mv. single channel experiments show that gatx does not alter single channel amplitude, but may alter channel gating. these experiments provide the basis for developing gatx into a useful tool that can be used to define the role of clc- in airway and intestinal epithelial cells. furthermore, the high affinity and specificity of inhibition suggest that gatx interacts intimately with the channel, and thus may the exploitation of alternative chloride channels to bypass the defect in cftr-mediated chloride secretion is an established therapeutic strategy in cf. in search of such potential therapeutic targets, we assessed camp/pkadependent chloride secretion across excised nasal epithelium of cftr null mice and wildtype controls, by measuring short-circuit currents (isc) in an ussing chamber set-up. all experiments were performed in the presence of amiloride to block the contribution of epithelial sodium channels to the isc. in cftr null mice of mixed background (c bl/ ; ), the camp agonist forskolin activated a bumetanide-sensitive rise in isc that was comparable in magnitude to the cftr-mediated isc response of littermate controls ( ± vs. ± µa/cm , respectively). the forskolin-induced isc in these null mice was inhibited by the general chloride channel inhibitor dpc, but appeared insensitive to the cftr inhibitors cftrinh- and glibenclamide, the orcc and calcium-activated chloride channel inhibitor dids, and the clc- inhibitors cadmium and pre-activated omeprazol. moreover, the isc response to forskolin greatly exceeded the response to the calciumlinked agonists ionomycin and carbachol. intriguingly, in nasal epithelium of cftr null mice with a different genetic background (fvb), no such forskolin-inducible secretory pathway was evident (cftr-/-: ± vs. cftr+/+: ± µa/cm ). when, in nasal epithelium of cftr null mice (c bl/ ; ), the basolateral membrane electrical resistance was negated by nystatin treatment, and in the presence of a transepithelial chloride gradient, forskolin failed to elicit a rise in isc, suggesting that camp/pka signaling targets a basolaterally located ion transport system. indeed, in intact epithelium, addition of chromanol b, a selective inhibitor of camp/pka-activated potassium channels, to the serosal bath, blocked the forskolin-induced rise in isc. we tentatively conclude that the hyperpolarization of nasal epithelial cells, which ensues from camp/pka-induced potassium efflux across the basolateral membrane, drives chloride exit across the apical membrane via a constitutively active anion conductive pathway that is absent from fvb cftr null mice. rt-pcr and immunostaining demonstrated that, among the candidate anion channels that fit the above pharmacological profile, clc-ka, but not clc-kb, was expressed in nasal epithelium of both cftr null mice and controls (c bl/ ; ), together with the accessory protein barttin. importantly, immunostaining suggested a strong increase in clc-k protein abundance in the cftr null mice, as compared to wildtype animals. on the basis of these findings, we postulate that clc-ka channels may serve as cftr bypass channels in nasal epithelium of cftr null mice that, albeit indirectly, are responsive to camp/pka signaling. future studies aim to characterize clc-ka and barttin in the airways from cf patients and controls, and to identify the post-transcriptional mechanism resulting in the up-regulation of clc-ka protein in cftr null mice. in the disease cystic fibrosis (cf), the most common mutation f del results in endoplasmic reticulum retention of misfolded cf gene proteins (cftr). the endoplasmic reticulum (er) f del-cftr protein retention is dependent upon chaperone proteins, many of which require ca + for optimal activity. an increase in cytosolic free ca + concentration is dependent on the activity of ion channels in the membrane of er and on extracellular ca + influx. this influx, named capacitative ca + entry (cce), induced by calcium store depletion, represents the major ca + influx mechanism in cells (putney et al., ) . during the cce, ca + influx can be mediated by one or several of the isoforms of transient receptor potential canonical channels (trpc) (putney et al., ) . aims & methods: in the present work, we studied the cce in human cf tracheal gland serous cfkm cells compared to non cf human tracheal mm cells. for this, we characterized the molecular identity of trpc channels responsible of cce by rt pcr technique and we compared global ca + influx in cf and non cf cells by using fluo -am probes. moreover, we measured the single channel activity of plasma membrane trpc channels by cellattached patch-clamp configuration in cf and non cf cells. we also studied the cce in cf cells after correction of the abnormal f del-cftr trafficking by miglustat ( h at µm of n-butyldeoxynojirimycin, nb-dnj) (norez et al., ) and low temperature ( h at °c) (denning et al., ) . results: we detected by rt-pcr technique the presence of trpc and trpc in both cell lines and we observed that cce was increased -fold in cf-km cells compare to non-cf mm cells. following stimulation by histamine, we recorded elementary ca + currents with similar amplitude ( . pa at - mv) and conductance ( ps) whatever the cells tested (cf-km or mm cells). however, we observed a -fold increase of the npo (number of channels in each patch × open probability of single channel) in cf cells compare to non-cf cells. in f del-cftr corrected-cf cells, the ca + influx stimulated by the ca + stores depletion was reduced to a similar level to non cf mm cells. the permanent contact of the airway epithelium with the external milieu induces frequent injuries caused by inhaled pathogens and particles. regeneration of the wounded airway epithelium needs to respect its prior structure in order to restore its defence functions. the underlying molecular mechanisms associated with the regeneration of the human airway epithelium are poorly described. the aim of our study was to determine, using a global expression approach, the transcriptional profile of human airway cells during the epithelial regeneration process. human airway epithelial cells collected from nasal polyps were seeded on type iv collagen-coated porous membranes, cultured up to confluence in liquid-liquid(ll) conditions, then put at the air-liquid interface (ali) allowing epithelial mucociliary differentiation. rna were extracted from epithelial cells at % confluence in ll conditions (epithelial cell proliferation and migration stage, step i), after days in ali conditions (beginning of epithelial cell differentiation, step ii), when first ciliated cells appeared (step iii) and when cultures were completely differentiated (step iv). our results demonstrated that between step i and step ii, genes were significantly modulated (ratio r<- or r> , p< . ), among them were activated and were repressed. between step ii and step iii, transcripts were up-regulated (tubulin, dynein, stath, microtubule associated proteins…) and only one gene (fatty acid desaturase- ) was repressed. finally, between step iii and step iv, except stath which was~ -fold down-regulated, we observed only few genes differentially and slightly modulated. all along the regeneration process, between step i and step iv, genes were significantly modulated with % of activated and % of repressed genes, among them a family of genes encoding proteins involved in the ciliary differentiation (foxj , tektin, dynein, tubulin, …), extracellular matrix proteins (collagen, laminin, …) and inflammatory-related genes like cytokines, growth factors and their receptors (ccl , vegfc, csf , il , il ra …). by elucidating the specific transcriptome involved in each step of the human airway epithelium regeneration, our study could allow to better understand the different cellular and molecular mechanisms, as well as the chronological events involved in these processes, and will further be applied to the study of the regeneration process in cystic fibrosis airway epithelium. supported by french association vaincre la mucoviscidose. electrolytes may be transported either through cells, the transcellular pathway, or between cells, the paracellular pathway. most research has focused on the channels and transporters of the transcellular pathway, while much less is known about paracellular transport. paracellular transport is regulated by tight junctions. claudins, the main component of tight junctions, are necessary and sufficient for tight junction formation and are important in establishing ion selectivity. the goal of this study was to further investigate the role of tight junctions in human airway epithelia. real-time pcr identified claudins , , , , , and in primary human airway epithelia. immunocytochemistry localized claudins , , , and to tight junctions. to measure the relative ion selectivity of tight junctions, we studied well-differentiated human airway epithelia in ussing chambers. to minimize transcellular transport, we used cf epithelia, which lack cftr and blocked enac with amiloride. we found the pna/pcl is . ± . , suggesting tight juctions are more permeable to cations than anions. the relative cation selectivity was na+ > k+ > cs+ > tma+ > mg + and the relative anion selectivity was cl-> br-≥ i-> no -> gluconate-. we compared these in vitro results to in vivo studies in people with cf and measured nasal voltage in the presence of amiloride. we found the pna/pcl is . ± . . tight junctions were slightly more selective for cations than anions in vitro and in vivo. to determine the effect of specific claudins on the selectivity of the paracellular pathway, primary human airway epithelia were infected with adenovirus expressing claudin or gfp. epithelia overexpressing claudin had a higher transepithelial resistance (ter) and the pna/pcl was . . however, infection efficiency was only %. this lead us to investigate if transformed human airway epithelial cells lines may be a better model to study permeability. cufi cells also express claudins , , , , , and . furthermore, like the primary human airway, cufi cells are more selective for cations; pna/pcl = . ± . . future studies will investigate the effect of claudin expression on the paracellular permeability of these cells. the results will help identify the properties of a pathway critical to epithelial transport and barrier functions. altered adenosine (ado) signaling is associated with chronic lung disease and increased asl ado levels are thought to drive inflammation by stimulation of a b receptors (a b-r) in airway epithelia, which can activate nfκb. when measured by lavage, asl ado is ≤ nm. due to its relatively low affinity to ado, a b-r are thought to be inactive under normal conditions (ec is ~ . µm), and only stimulated during chronic lung disease. however, we have previously demonstrated that the ado-receptor (ado-r) antagonist -spt inhibits asl volume regulation, and that a b-r is in close proximity to cftr. thus, we hypothesized that the asl ado concentration close to the apical membrane is higher than measured by asl lavage in bulk solution. we tested this hypothesis by looking for ado-r expression and function in well-differentiated human bronchial epithelia cultures (hbecs) and in vivo. by pcr analysis, we found that a b-r was the only ado-r expressed in hbecs. we then measured asl height over h ± ado-r antagonists by xz confocal microcopy when asl height was preset to ~ µm at t= . at µm, caffeine is a non-specific ado-r antagonist, which inhibited asl volume regulation ( h asl height was . µm vs. . µm for controls, n= ). similar inhibition was observed with alloxazine, which is relatively specific for a b-r (n= ). in contrast, dpcpx, zm & mrs , which inhibit a , a a & a -r respectively had no effect on asl volume regulation. asl height regulation was also ablated by the addition of adenosine deaminase (ada) to the asl (n = ). we next attempted to recapitulate the changes in asl height seen under control conditions, i.e. increase from to µm over the initial h followed by a plateau (the remainder of the h) by adding the non-hydrolyzable ado analogue neca to the asl in the presence of ada. nm neca was insufficient to regulate asl volume and height remained at µm over h. in contrast, addition of µm neca to the asl resulted asl volume regulation that was indistinguishable from control conditions (n= ), suggesting that near-membrane asl is much higher than has been measured by lavage. to confirm that a b-r stimulates cftr chloride secretion, we measured transepithelial potential differences of hbecs mounted in ussing chambers. a dose-dependent increase in cl-secretion was observed by ado addition with an ec of . +/- . µm, similar to the concentration of neca required to regulate asl height. to demonstrate which ado-r are expressed in human airways in vivo, we used laser capture to obtain ciliated cells from frozen tissue sections of excised donor bronchi. relative expression of a b-r by qpcr was found to be . +/- . fold higher than the a , a a-r, or a receptors (n= ). to confirm that ado-r activate cftr in vivo, we measured nasal potential differences. we found that ado activated chloride secretion at similar levels to isoproterenol. the effect of ado was inhibited by pre-exposure to µm caffeine (ado, . +/- . mv; ado + caffeine, . +/- . mv). we conclude that a b-r has an important role in asl homeostasis through regulation of cftr and it is active in human airway epithelia under normal physiological conditions. through osmotic forces, hypertonic saline (hs) may increase the volume of cf airway surface liquid, restore mucus clearance, and improve lung function. the na + channel antagonist amiloride is predicted to increase the duration of action of hs. however, patients taking amiloride with hs showed less clinical benefits than patients taking placebo with hs. in vitro data suggested that amiloride inhibits transepithelial water flux (pf) in cystic fibrosis airway epithelia. we have extended this study by looking at the effects of amiloride on aquaporins in a heterologous expression system (mdck cells) and have used immunofluorescence to determine which aquaporins are expressed in human airways in vivo and in vitro. inhibition of enac by aprotinin pretreatment had no effect on hs-induced pf (n= ), suggesting that amiloride did not exert its actions on pf by inhibiting enac. however, since hs-induced pf was also inhibited with hgcl , we speculated that amiloride directly blocked an aquaporin. to test this hypothesis, we measured the rate of cell shrinkage of wild-type mdck cells, and those stably-expressing aqp or , or expressing aqp (after h exposure to a mildly hypertonic media; mosm). following mucosal exposure to a mosm hypertonic ringer solution, mdck cultures grown to confluency on permeable filters shrunk by % after s (n = ), as measured by xz confocal microscopy following staining with calcein-am. in contrast mdck/aqp or mdck/aqp cultures shrunk by % over the same period and mdck/aqp cultures shrunk by % (all n= ). a min pretreatment with µm amiloride abolished hypertonic ringer-induced cell shrinkage in mdck/aqp cultures ( % at s; n= ) but had no affect on cell shrinkage in aqp or aqp expressing cultures. amiloride is fluorescent and can be imaged in epithelia by xz confocal scanning under uv excitation. amiloride was not internalized immediately (< s) before hypertonic ringer addition and did not block aqp -mediated cell shrinkage ( % at s; n = ). however, a min pretreatment with amiloride caused it be internalized and at this time aqp -induced cell shrinkage was blocked, suggesting that amiloride blocks aqp intracellularly. to confirm that aqp is present in airway epithelia, we performed immunostaining with an aqp antibody and found that aqp is highly expressed at the apical membrane of ciliated cells in human bronchial tissue and well-differentiated bronchial cultures. we conclude that amiloride blocks aqp , which is abundantly expressed in the superficial airway epithelia. supported we have previously observed that cftr, annexin (a ) and cytosolic phospholipase a (cpla ) are partially recruited in detergent insoluble microdomains (dim) upon proinflammatory stimulation. this is prevented by specific inhibition of cftr. a participates in the regulation of inflammation by inhibiting cpla activity. we have also shown that a expression is decreased in cftr-/-mouse tissues. we postulate that cftr, a and cpla participate in the regulation of inflammation by their dynamic interaction within dim. in this study we aimed to (i) demonstrate and characterize this interaction, and (ii) identify other potential partners of this complex in dim. by coimmunoprecipitation, we examined the interaction between cftr, a and cpla in human pulmonary epithelial cells calu . to search for additional partners we developed a new proteomic approach based on double sds-page (dsds-page), which is compatible with membrane protein analysis. calu cells and human epithelial bronchial cfbe cells (expressing either wt or f delcftr) were subjected or not to proinflammatory conditions (tnfα). dim were isolated by density gradient and analyzed by dsds-page. cftr immunoprecipitated with a and cpla in calu cells. dsds-page analyses showed significant differential expression of cytokeratin , actin and protein disulfide isomerase (pdi, involved in protein folding) in dim between control and proinflammatory conditions in calu cells, and between wt and f del cfbe cells, as determined by mass spectrometry. in conclusion, cftr, a and cpla interact in basal conditions. proinflammatory treatment causes differential expression of cytoskeletal proteins and pdi depending on the expression of normal and mutated cftr. this work suggests that cftr could participate in the regulation of inflammation within a complex with a and cpla . cytoskeletal proteins and pdi could be involved in the dynamics of this complex. this could be related to the abnormal inflammatory response characteristic of cf. we acute and chronic inflammation, common features of cf airways disease, have been linked to higher atp release and changes in atp metabolic patterns in vivo, e.g., in induced sputum and exhaled breath condensate from cf patients and bronchoalveolar lavage fluid from inflamed lobes (esther, ped pulm sppl , ) . we, therefore, investigated the contribution of airway epithelia per se to the raised atp concentrations on cf airway surface during inflammation. when exposed to supernatant of mucopurulent material (smm) from cf airways for h, or infected with respiratory syncytial virus (rsv) h prior to the study, basal atp release from well-differentiated primary human bronchial epithelial (hbe) cultures did not differ from that of control cultures. however, basal atp release was increased weeks after rsv infection when inflammation had waned (as indexed by il- secretion). the increase in basal atp release correlated with increased basal udp-glucose and mucin release, and goblet cell metaplasia. these data indicate that the increased basal atp release following rsv infection reflected increased goblet cell secretion. a different pattern was observed for stimulated atp release. hbes exposed to smm for h, or infected with rsv h prior, released - times more atp following hypotonic challenge than control cultures (okada, ped pulm sppl, ) . the increase in peak atp concentrations correlated with the degree of inflammation as indexed by il- secretion and the increase in intracellular calcium (ca + i ) mobilization in response to hypotonic challenge, but not with the goblet cell number. ca + i mobilization in response to hypotonic challenge in control hbes was almost completely due to extracellular ca + i influx which was inhibited by antagonists of a transient receptor potential v (trpv ) ca + channel. in contrast, an additional component of atp-induced ca + i release from endoplasmic reticulum (er) was observed in smm-treated hbes. the increase in hypotonicity-induced atp release in smm-treated hbes compared to control hbes was almost completely sensitive to chelation of ca + i by bapta ( µm; h), or inhibition of exocytosis by brefeldin a ( µm; . h), monensin ( µm; h) or n-ethylmaleimide ( mm; min), whereas these reagents were ineffective against hypotonicity-induced atp release from control hbes. these data suggest a positive feedback loop between the increased atp release linked to ca + i -dependent exocytosis and the increase in atp-induced ca + i mobolization linked to increased er ca + i stores (ribeiro, jbc , ) in inflamed hbes. in conclusion, we propose that basal and hypotonicity-induced atp release from hbes are regulated in distinct mechanisms during inflammation. basal atp release reflects continuous exocytosis processes independent of ca + i mobilization and differs as a function of cell type (ciliated vs goblet cell), whereas acute inflammation upregulates hypotonicity-induced atp release via increased ca + i mobilization and ca + i -dependent exocytosis. we conclude that airway epithelia exhibit multiple mechanisms of increasing atp release into the lumen in response to inflammation. supported by cff grant okada i to sfo and grants from nih. the main biological role of vitamin d is in control of bone and mineral metabolism, but it has also been discovered to have many immune system functions. vitamin d, a fat soluble vitamin, is poorly absorbed in cf leading to low serum levels. we sought to investigate the involvement of vitamin d in the control of inflammation in a cf model. methods cf tracheal epithelial cells (cfte) were pretreated with vitamin d ( , [oh] d ) or control and exposed to p aeruginosa lipopolysaccharide (lps) for hrs before collecting the cell supernatants. cytokine levels in the supernatants were assessed by cytokine array and subsequent elisas specific for il- , il- and mcp- . vitamin d was measured in the plasma of children with cystic fibrosis aged months to years. neutrophil numbers, neutrophil elastase (ne) activity and il- concentrations were measured in the bronchoalveolar lavage (bal) of these children. children with cystic fibrosis were deemed to be non-colonised, intermittently colonised or chronically colonised with p aeruginosa on the basis of the leeds criteria. pretreatment with vitamin d significantly reduced secretion of il- , il- and mcp- from cftes after challenge with p aeruginosa lps in a dose dependant manner. plasma vitamin d levels were highest in the noncolonised group and lowest in the chronically colonised group with intermediate levels in the intermittently colonised group. bal neutrophil counts, ne activity and il- concentration showed a reciprocal pattern. conclusions vitamin d acts on cf lung epithelial cells to protect against excessive cytokine production in response to lps. levels of vitamin d are inversely related to both p aeruginosa colonisation status and bal markers of inflammation. low levels of vitamin d may contribute to advancing lung disease in children with cystic fibrosis. more aggressive correction of vitamin d deficiency may have a role in protecting against excessive inflammatory response to infection. sleeping in a mist tent has been used as a treatment for cystic fibrosis (cf) patients, in order to hydrate the viscous mucus and make it easier to be removed by coughing (matthews et al., ) . however, the efficiency of the method has been questioned, and its use was largely discontinued. with a new method to measure the ion content of human nasal fluid the effect of sleeping in a moisture tent on the ion content of nasal airway surface liquid (asl) in cf-patients and healthy controls was determined. the cf-patients and healthy controls spent a night ( h) in a mist tent, and samples of the nasal asl were taken before the experiment, after the period in the tent, and then at each hour during h after the persons had left the tent. samples of nasal fluid were collected on sephadex g- beads that had been mounted on strips of filter paper, which were then put into the nasal cavity of the cf patients or controls for - min. the strips were removed, and the beads were isolated, dried, and analyzed by x-ray microanalysis (vanthanouvong et al., ) . the concentration of na, cl, and k in the nasal fluid of cf patients was , , and mm, respectively, before the patient entered the tent, significantly higher than the levels in the nasal fluid of the controls (na mm, cl mm, and k mm). during the period in the tent, the ion content decreased, to levels of na mm, cl mm, and k mm (cfpatients) and na mm, cl mm, and k mm (controls) immediately after leaving the tent. after leaving the mist tent the ion levels in the nasal fluid increased, reaching after h values of na mm, cl mm, and k mm (cf-patients) and na mm, cl mm and k mm (controls), which was for both groups higher than before entering the tent. no major changes in the ion content of the asl occurred after h. the salt content of the asl may be relevant in cf, since the asl is known to contain anti-bacterial proteins, defensins, which are sensitive to the salt concentration (bals et al., ) . hence, the higher salt concentra-tions in the asl of cf-patients may have negative consequences for the anti-bacterial defense system in the lungs, and conversely, the decrease in ion concentrations, caused by spending time in a mist tent, may have positive effects. (we have previously shown that none of our patients was chronically colonized with pseudomonas aeruginosa while regularly sleeping in mist tents.) however, with currently used procedures, the effect of sleeping in a mist tent on the ion content of the nasal asl is short-lived. bals background: antibody microarrays for clinical applications have been anticipated for more than a decade but the technology has only recently become sufficiently mature for reproducible, robust detection of low abundance proteins. the advantage of this new technology is the high throughput, parallel detection of identified, known, low abundance proteins. antibody microarray results are usually given in terms of ratios between two samples (e.g., "experiment" and "control"). however, given the printing and calibration issues inherent with antibody microarrays, this ratio'ing approach precludes statistically valid inter-array comparisons of the individual protein concentrations. we have therefore developed and implemented a different calibration strategy using a benchmark mixture that is applied to every array as an internal standard, thus controlling for any differences in antibody activities as well as printing imperfections. the amount of protein bound to each spot is determined relative to the respective protein in the internal standard. since all samples in the study are compared to the same internal standard, this semi-quantitative approach permits the comparison of multiple samples. all data for the same antibody on multiple arrays can then be used to calculate the average and standard deviation for a given population (such as patients or control groups) in a parallel fashion. methods: cf lung epithelial cultured cells ib - and the cftrrepaired ib - /s were biosynthetically labeled with a hour pulse of [s]methionine, and then chased with cold methionine for , or hours. we then isolated total protein from each sample, and labeled the proteins with cy . labeled proteins were applied to clontech® antibody arrays ( antibodies printed in duplicates) according to the manufacturer's protocols. the internal standard, consisting of a mixture of cell cultures and tissue extracts known to contain the cognate protein for each antibody on the array, was labeled with cy . the data were then used to compare the cf and the corrected cells in terms of amount, biosynthesis rate and degradation half-life (t / ) for each one of the features on the array. data analysis: for each spot on the array the ratio of cy /cy (after background subtraction) was calculated, normalized and stored for further analysis. for "pulse-chase" experiments the ratio of [s] counts to cy at each time point gives a measure of the fraction of newly synthesized (radiolabeled) to total protein (dye labeled) bound to each given antibody on the array. the addition of the cy labeled internal standard enables the calibration of these totals, allowing the comparison all the spots for any given antibody on multiple arrays. furthermore, the internal standard calibration makes it possible to compare not only t / values but also to calculate the relative degradation rates in different cell lines even when the amounts of that protein are different in the "control" and "experiment" cells. the new approach quantitatively identifies and subjects to analysis the effect of the cftr mutation on protein biosynthesis and degradation of the cellular signaling proteome. supported by nih no -hv- (hbp) and ro - (hbp) in cf patients, defective apical chloride secretion, due to lack of functional cftr, leads to dehydration of airway surface liquid (asl) and mucus plugging. this defect is likely ameliorated, in part, by an alternative route of chloride conductance, the calcium activated chloride channel (cacc) . female gender appears to be associated with more rapid decline of lung function and earlier death in cf. this suggests that gender differences in hormonal environments might influence asl height regulation, thus modifying the pulmonary phenotype. we hypothesized that changes in estrogen concentrations might alter asl hydration by affecting cacc. consistent with this, we have shown that estrogen attenuates increases in intracellular calcium and asl height that follow purinergic receptor activation in primary cultures of human bronchial epithelial cells (hbecs, see accompanying abstract by hengrui sun et al). here, we report correlations between estrogen levels and nasal ion transport in vivo and further probe hormonal effects on ion transport and asl height regulation in vitro. in vivo studies: in healthy, spontaneously menstruating females, nasal potential difference (npd) was measured (both nostrils) on two occasions during a single monthly cycle. based on a history of the most recent menstrual cycles, we measured npd on a day when estrogen levels were predicted to be low (generally - days following onset of menses) and subsequently on the day when the pre-ovulatory estrogen surge was predicted. the recording electrode was positioned at the location where maximal stable basal pd was detected and changes in pd recorded continuously during sequential perfusion with ] ringer's solution, ] ringer's solution containing amiloride (to inhibit sodium absorption via enac), ] modified (low chloride) ringer's containing amiloride and finally ] a similar solution also containing utp (to activate cacc). as predicted, blood estrogen levels were higher on the "pre-ovulatory" day ( . ± . vs . ± . nm, p< . ). utp-stimulated change in pd was significantly lower on the study day when estrogen levels were elevated ( . ± . vs . ± . mvs, p< . ). further, the amiloride-sensitive component of basal pd was higher on this day ( . ± . vs . ± . mvs, p< . ). the change in pd in response to perfusion with a low chloride solution was not different. in vitro studies: expression of estrogen receptor (er) α (but not β) was confirmed in non-cf and cf hbecs by real time pcr. erα was detected in hbecs from males and female donors. in cf and non-cf hbecs mounted in modified ussing chambers, exposure to nm e was associated with a significant (~ %) reduction in utp-stimulated short circuit current. e inhibited the atp-stimulated increase in asl height in male and female hbecs and this asl regulatory phenotype was not reproduced by testosterone or progesterone. in conclusion, estrogen inhibits utp-activated chloride secretion in vivo and in vitro suggesting that elevated pre-ovulatory estrogen levels may further impair mucociliary clearance in females with cf. all data mean±sem. supported by cff leroy matthews award [ ] . data demonstrating an upregulation of caccs when cftr is absent or defective [ , ] have triggered further studies of these channels and prompted further elucidation of the functional (and possibly also physical) interactions between these two channels. although searched for long, the molecular identity of caccs is still under debate. the role of bestrophin family proteins as putative candidates for caccs is discussed controversially. bestrophin (best- ) has been proposed to form ca + activated clchannels in epithelial cells. moreover, caccs have been shown to support cell proliferation, namely in the development of cancer [ ] . here, our goal is two-fold: ) to investigate the correlation between expression levels of best- and epithelial cell proliferation; and ) to pursue a search for interacting protein partners of best- to better understand the role and function of bestrophin(s) in epithelial cells. to study the correlation between best- expression and cell proliferation, we analyzed two populations of the t colonic carcinoma cell line with very different proliferation rates: the original (t -slow) and the spontaneously transformed t cells (t -fast). we observed that t -slow cells have a small proliferation rate and express low amounts of best- , while t -fast cells express high levels of this protein. best- is spontaneously active in t -fast cells. best- -rnai inhibited ca + activated clconductance and cell proliferation, therefore establishing a novel role of bestrophins in cell proliferation, which may be of relevance for the regeneration of the epithelia in cf and also for alternative therapies for cf. for the second objective, two putative cytoplasmic domains of best , polyhistidine -tagged (phis) n-term (aa - , best -n) and c-term (aa - , best -c), were cloned into the pet-sumo bacterial expression vector. these domains were then immobilized onto metal-affinity resin to capture interacting proteins from human t whole cell and sub-cellular lysates. protein-containing fractions recovered from best -n/c-coated and blank resins were subjected to d-gel and protein identification. it is expected that functional characterization of the interaction between the captured proteins and best- will give new insights into the biological role, plausibly of relevance to cf. work supported by dfg-sfb -a grant (germany) and pluriannual funding of cigmh (feder-eu and fct, portugal extracellular nucleotides acting on epithelial cell surface purinergic receptors regulate the mucociliary clearance process that removes noxious materials from the lung. however, how epithelial cells release nucleotides into the airways remains largely unknown. cftr has been suggested to mediate atp release in airway epithelia, but conclusive demonstration remains elusive. calu- is a human airway derived cell line often described as a model of airway gland serous cells. at least % of cells in a monolayer expressed high levels of cftr protein and activity at the apical membrane. atp, adenosine, and udp-glucose, the naturally occurring agonists for the p y , a b, and p y receptors, respectively, were quantified in airway surface liquid with nanomolar sensitivity using radiolabeling and fluorescence hplc-based techniques, and real-time atp measurements. atp (and udp-glucose) was found to be released constitutively and selectively onto the mucosal surface of calu- cells. however, the specific cftr inhibitor ( µm, - min) failed to decrease atp concentration in the mucosal bath of resting calu- cells. forskolin ( µm, min) stimulated cftr channel activity, but did not affect basal levels of atp (or udpglucose) release. in contrast, elevation of intracellular calcium using ionomycin ( - µm, - min) significantly increased atp (and udp-glucose) release into the mucosal (but not serosal) bath of calu- cells. calciummediated nucleotide release enhancement was not abolished by cftr inhibitor . maneuvers like pre-incubation in nominally free-calcium solution, bapta ( µm, min), or cytochalasin d ( µm, min) to disrupt actin cytoskeleton, which inhibited regulated exocytosis in calu- cells, also reduced calcium-mediated increase in nucleotide release. calu- monolayers presented a fraction of cells (< %) exhibiting negligible cftr immunostaining signal, but displaying airway goblet cell mucin granules. remarkably, stimulation of atp and udp-sugar release with ionomycin resulted in concomitant enhanced exocytotic secretion of muc ac. these results suggest that cftr is not involved in regulation of atp release in airway epithelial cells. more likely, nucleotides and nucleotide-sugars residing in the biosynthetic pathway involved in modification of apically targeted glycoconjugates (e.g. mucins) are released to the extracellular environment via calcium-triggered exocytosis. supported although the regulation of na+ transport is relatively well described in normal and cf airway epithelial cells, the impact of inflammation and infection, two major components of cf lung disease, on na+ transport has not been studied extensively. significant amounts of evidence suggest that bacterial by-products may influence na+ transport in airway epithelium. furthermore, the secretion of exoenzyme s by pseudomonas aeruginosa is enhanced during an exacerbation of lung disease in cystic fibrosis. in the past few years, our laboratory has shown that exposition of lung epithelial cells to p. aeruginosa lps leads to a rapid decrease in activity and expression of enac channel. the aim of our study was to evaluate the mechanism involved in the modulation of enac expression and activity by lps. primary culture of lung epithelial cells grown on filters were treated with lps ( µg/ml) for different time ( min, , , h). at each time point, the total and amiloride-sensitive short circuit current (isc) was determined in ussing chamber. in parallel, the cell proteins were collected to investigate by western blot the signalling pathways involved in this response to lps. a hours lps treatment leads to a significant decrease in alpha and gamma enac mrna to % and % of control respectively. similarly, the amiloridesensitive current was decreased by % of the current in untreated cells after a min exposure to lps. by western blot, we demonstrated the strong implication of the pi k/akt pathway in response to lps in the regulation enac mrna and the potential implication of tyrosine/kinase receptors signalling pathway through mtor in the regulation of enac channel activity. in conclusion, we demonstrate that inflammatory molecules can modulate the expression and function of the na+ transport mechanism in lung epithelial cells and could contribute to the modulation of airway surface fluid in the chronic infectious process associated with cf. serous acinar cells are thought to be crucial for secretion of airway surface liquid by submucosal glands in large airways, but the molecular mechanisms of serous cell ion and fluid transport are not well understood. to address this, we isolated murine nasal serous acinar cells and studied them using simultaneous dic and quantitative fluorescence microscopy to track changes in cell volume and intracellular concentrations of ions involved in fluid secretion ([cl -] i ; spq) and its regulation ([ca + ] i ; fura- ). previous results indicated that serous acinar cell volume changes were indicative of solute efflux (shrinking) and solute influx (swelling), reflecting the secretory state of the cell. acinar cells stimulated with µm carbachol (cch) exhibited a rapid increase in [ca + ] i and subsequent ~ % cell shrinkage due to efflux of ~ % of cell clcontent and parallel loss of k + and osmotically obliged h o. because murine serous cells are sites of apical cftr expression in the airway, we tested the requirement for cftr in cch/ca +activated shrinkage/clefflux. acinar cells isolated from either cftr tm unc-/mice or wt cells pre-treated with cftr inh exhibited identical rates and magnitudes of ca + -induced cell shrinkage and clloss compared to wt control cells. resting [cl -] i was also identical in wt and cftr tm unc-/serous acinar cells ( ± and ± mm, respectively), suggesting similar levels of resting clconductance. thus, murine serous acinar cells can secrete kcl/h o by ca + -dependent mechanisms in the absence of functional cftr, in agreement with studies of intact glands suggesting cch-induced glandular fluid secretion is cftr-independent. wt serous acinar cells were stimulated with camp-agonists, including µm forskolin, µm isoproterenol, µm adenosine, and µm vip. however, no camp-induced shrinkage was observed, even in the presence of a cocktail of inhibitors to block solute uptake. to determine if cftr activation could enhance cch/ca + -induced cell shrinkage at a sub-optimal [cch], serous acinar cells were pre-treated with forskolin followed by stimulation with µm cch in the continued presence of forskolin. however, similar rates and magnitudes of cell shrinkage were observed in forskolin-treated and control cells. these results suggest that the magnitude of the cftr clconductance in murine serous acinar cells is much smaller than the ca + -activated clconductance, below the resolution of these optical assays. more sensitive electrophysiological approaches are being used to determine the molecular identity of the non-cftr clconductance(s) and the role of cftr in murine serous acinar cell ion transport. females with cf exhibit more rapid decline in fev than males, which reduces their life span. normal airway epithelia regulate airway surface liquid (asl) volume and mucus transport by secretion through both cftr and the ca +-activated cl-channel (cacc), and inhibition of either of these pathways in isolation does not abolish mucus transport. however, a loss of both pathways is predicted to result in mucus plugging which contributes to lung disease. despite a loss of cftr, cacc is functional in cf airways due to mechanical stimulation of atp release and activation of the p y pathway which serves to raise intracellular ca +. we hypothesized that e may inhibit cacc, leaving females with cf more prone to lung disease than males due to a greater depletion of asl volume. using confocal microscopy, we found that acute pre-exposure with e inhibited atp induced asl secretion in normal and cf human bronchial epithelial cultures (hbecs) from both male and female donors. the ic for e -inhibition of atp-induced asl secretion was . nm. to better understand how estrogen receptors (ers) interact with the p y pathway, we transiently transfected er-α or er-β linked to an orange fluorescent protein in an er null bhk cell line. with the co-transfection of ha tagged p y -r, we observed that e addition did not induce internalization of p y -r, neither did it alter the internalization induced by atp. we then measured total cell inositol phosphates (ip) in cf airway cells +/-e pretreatment. cell ip levels were not different between groups (n= ), suggesting that the effect of e is downstream of ip production. the investigation of the changes of the intracellular ca + (ca +i) by recording fura- emission ratio over time in er-α or er-β expressed cells provided clues. after nm e exposure, the fura- emission ratio in untransfected cells (n= ) or er-β transfected cells (n= ) increased by . following um atp addition. this increase was significantly inhibited by~ % in er-α transfected cells (n= ), providing good evidence that er-α is respon-sible for inhibiting the p y pathway. we next measured ca +i in normal hbecs. while acute nm e addition had no affect on basal ca +i levels, which were around nm, this maneuver reduced the ca +i response to nm following atp addition (both n= ), suggesting that e addition altered ca + signaling in response to atp. thapsigargin induces ca +i release from the endoplasmic reticulum, but the magnitude of ca +i release after thapsigargin addition was not altered by e pretreatment, indicating that e works further up stream to affect the p y signaling pathway. together, these results suggest that e directly interacts with the p y signaling pathway through er-α by altering ca +i. we propose that this interaction leaves females more vulnerable to infections due to a reduced asl volume during periods of high e , such as occur prior to ovulation. the nasal potential difference (or npd) is a sensitive, real time bioelectric assay of cftr-dependent and independent ion transport that is a commonly used study endpoint in early clinical stages of drug development. technical limitations of the assay must be controlled to facilitate its use in multicenter trials and limited intra-site variability. two forms of equipment are commonly employed to evaluate nasal potential difference in humans: silver-silver chloride electrodes (agcl) with saline bridges or calomel electrodes with agar bridges; however, these techniques have never been directly compared. we repeatedly evaluated (within days) the basal potential difference of five normal subjects using the two available techniques, then compared variance and repeatability of the measures. basal pd was measured during perfusion of ringer's solution, and measured at . , . , . , . , and . cm within the inferior meatus, as indicated in the cf-tdn standard operating procedures. mean basal pd were within . mv using the two techniques, with very similar coefficients of variation (cvar; . vs. . in agcl vs. calomel, respectively). maximum basal pds were also similar between the two methodologies (mean difference . mv; cvar . and . , respectively). the correlation between first and second measures of mean pd was highly reproducible, and similar between the two techniques (r= . vs. . in agcl vs. calomel, respectively); however, when each individual basal pd measure was compared, correlation between first and second pd using the agcl device appeared superior (r= . vs. . respectively; n= measures per device). comparison of values obtained in cf subjects are currently in progress and will also be reported. next, we examined diagnostic npd tracings in cf subjects for whether the effect of amiloride might influence values obtained in the contralateral nare. an effect of cross-contamination by amiloride or other agent (gene therapy vectors or other nasal administered compounds, for example) could influence accurate measure of npd values. when basal pds were performed in both nostrils prior to amiloride exposure in either nostril, mean basal pd between nares remained stable (< mv, p=ns for change). however, if the basal pd in one nostril was measured after amiloride exposure in the contralateral side, mean pd depolarized by mv (from - to - mv; p< . ), indicating the presence of a 'cross-nostril' amiloride effect that might potentially affect interpretation of this endpoint if not performed in a sequence designed to avoid amiloride exposure. in summary, we show that calomel and agcl electrodes perform similarly in repeated measures from the same normal subjects, and that improper npd sequence can adversely affect basal pd measures. other potential impediments to the pd measurement (e.g. solution temperature, tonicity, ionic content, and sources of electrical offset) will also be discussed. these findings are relevant to the optimizing the use of the npd assay in clinical trials. supported by nih, cff, and cfft. the gene defective in patients with cystic fibrosis (cf) is the cystic fibrosis transmembrane conductance regulator (cftr), a camp-activated chloride channel in the apical membrane of epithelia, but alteration of expression and function of many genes undoubtedly contributes to the pleiotropic manifestations of cf. a hallmark of cf in airway epithelia is the hyperabsorption of sodium through the epithelial sodium channel (enac) in the apical membrane followed by extrusion through the na,k-atpase in the basolateral membrane. this results in airway surface liquid (asl) dehydration, mucus buildup and bacterial infection characteristically observed in the lungs of cf patients. previous studies have shown that the na,k-atpase is increased in cf airway epithelia, consistent with increased sodium transport along the enac/na,k-atpase axis. we have previously demonstrated that fxyd , a member of a small family of proteins known to regulate the na,k-atpase, is increased in s x cf mouse lung and nasal epithelia and in primary human tracheal epithelial cells (hte) after treatment with an inhibitor of cftr ( ). recent evidence has indicated that enac activation occurs through modification of the serine protease-protease inhibitor balance in asl. thus, we hypothesized that enac-mediated increases in sodium absorption may be partially responsible for the increase in fxyd expression observed in cf airway epithelia. we now report that increasing asl volume on hte cells upregulated fxyd expression (p< . ) after hours, an effect that was further increased after trypsin addition and abrogated by the serine-protease inhibitor aprotinin. treatment of hte cells with nystatin also increased fxyd expression measured by quantitative rt-pcr (p< . ). based on these results and the observation that mice overexpressing enac beta-subunit exhibit cystic fibrosis-like lung pathophysiology, we examined fxyd expression in the lungs of mice overexpressing the scnn b-transgene. contrary to our expectations, fxyd lung expression was decreased as assessed by quantitative rt-pcr and immunoblot analysis (p< . ), indicating fxyd may be alternatively regulated in epithelia under conditions of acute versus chronic sodium hyperabsorption. therefore, we measured fxyd expression in an epithelial cell culture model using an inducible, tri-cistronic vector capable of expressing enac alpha, beta and gamma subunits. we determined that fxyd expression is decreased after chronic enac induction. we conclude that fxyd expression is coordinately regulated based on acute versus chronic enac-mediated sodium absorption and suggest that fxyd is increased in cystic fibrosis airway epithelia as a result of an imbalance between increased inflammatory signaling and altered ion transport. picher, m.; van heusden, c. cystic fibrosis center, university of north carolina, chapel hill, nc, usa airway infection by the respiratory syncytial virus (rsv) is considered an important cause of pulmonary exacerbation and hospitalization in cystic fibrosis (cf) children. this disease is characterized by functional mutations in the cf transmembrane regulator (cftr). the inability of cftr -/-mice to clear rsv is associated with exaggerated inflammatory responses. a relationship was recently established between airway inflammation and chronically-elevated airway adenosine. this signaling molecule regulates key aspects of lung defenses, including bacterial clearance and inflammatory cells. high adenosine levels measured in bronchoalveolar lavage fluid of asthmatic or cf patients cause airway inflammation, remodeling and fibrosis in animal models. the objective of this study was to investigate the longterm effects of rsv infection on adenosine regulation and inflammatory responses in human airway epithelia. methods. polarized primary cultures of bronchial epithelial cells from healthy subjects and cf patients were exposed to vehicle, uv-inactivated or active green fluorescent protein (gfp)-rsv. the cultures were monitored days for infection (fluorescence microscopy), adenosine levels (fluorescence chromatography), the release of cytokines regulating adenosine tnfα) and enzyme activities (ecto '-nucleotidase [ecto '-nt: amp Ç adenosine] and adenosine deaminase [ada : adenosine Ç inosine]). results. (a) both normal and cf cultures exhibited detectable rsv infection over days, with peak density on day - . (b) we demonstrate that rsv enhances the release of all five cytokines from normal cultures over days. in contrast, cf cultures responded to rsv by a transient decrease in il- β, il- and tnfα (peak on day ), but a sustained decrease in il- , and a sustained increase in il- , secretion. (c) on normal epithelia, rsv transiently stimulated ecto '-nt (peak on day ) but caused a sustained increase in ada activity, resulting in chronically-low adenosine levels. in cf, rsv raised adenosine levels by the combined effects of a sustained increase in ecto '-nt activity and the lack ada response. (d) chronically-elevated adenosine generated by ada inhibition [erythro -( -hydroxy- -nonyl)adenine] stimulated the secretion of all cytokines, except il- . conclusion. these data support a complex relationship between adenosine and cytokine regulation in human airway epithelia. the il- -adenosine amplification pathways identified in rsv-infected cf epithelia are in agreement with animal models and their key role in chronic airway inflammation/remodeling. on the other hand, the data also suggest that ada activity is influenced by il- , known to stimulate the expression of a major protein anchoring soluble ada to epithelial surfaces: cd . altogether, this study suggests that an rsv infection in cf children may accelerate the progression of the disease by setting the stage for airway adenosine-cytokine amplification pathways. supported by the cystic fibrosis foundation (picher g ). cystic fibrosis (cf) airway submucosal glands are defective in mucus secretion rate (jayaraman, pnas, , , ; joo, j biol chem, , , ; joo, pediatr. pulmonol., , suppl. , ; wine, proc ats, , , ; thiagarajah, faseb j, , , ; song, ajp cell, ; salinas, faseb j, , , ) . one hypothesis is that loss of cftr results in reduced secretion of electrolytes leading to underhydration of mucins, thereby altering the mucus properties. it has been generally believed that the cf mucus in response to cholinergic agonists is thicker and more viscous. however, comparisons of pure gland mucus are difficult because of variability in gland secretion rates and a decline in mucus solids with repeated stimulations (wu, pediatr. pulmonol. , suppl. , ) . in the present study, we directly compared the solid content of airway submucosal gland mucus between control and cf individuals. human donor tracheas (hn) or disease control bronchi (dc) were dissected and mounted as previously described but without mineral oil. tissue was kept warmed and humidified in a chamber with two layers of saturated degree c / % co /o water vapor. the mucosal surface was blotted dry and coated with a thin layer of dimethylpolysiloxane to prevent re-absorption by the surface epithelium. the tissue surface was covered with a cover slip, leaving minimal air space between it and the surface to further reduce evaporation. a µl droplet of % nacl was placed at the side of every tissue as a standard to assess evaporation and measurement error. any basal secretions were removed prior to stimulation with µm carbachol. mucus secretion from individual glands was pooled using forceps and then drawn into a constant bore microcapillary (drummond) that had been alcohol-cleaned, dried, and weighed. stimulation time was allowed to vary so that at least µl of mucus was collected. after mucus collection, both the standard and the bath solution were refreshed. mucus filled capillaries were weighed with a mettler microbalance before and after overnight degree c oven drying to obtain nonvolatile solid content. the average solids measured in six repeated measures of % salt solutions was ± . % with no trend over time. results for hn (n= ) and dc (n= ) did not differ and were pooled as 'control'. the initial control secretion yielded . ± . % solids (mean ± sem, n= subjects) and required ± min to produce adequate mucus. the initial cf secretion gave . ± . % solids (n= ) in ± min. the second control secretion yielded . ± . % solids (n= ) and required ± min, whereas the second cf secretion gave . ± . % solids (n= ) and required ± min. in summary, cf gland mucus solids content was % higher than control following the first stimulation (p = . ). after the second stimulation, cf solids content was % higher than the control (p = . ). solids contents of controls observed in our experiment are much higher than was found for mucus collected from airway lumens after hr stimulation (c. %, trout, am j physiol, , , l ) . supported by niddk ro - (jjw) and cff. in patients with cystic fibrosis (cf) as well as in some animal models of cf lung disease, higher levels of prostaglandin e (pge ) have been detected compared to control subjects. it is not clear whether this excess of pge contributes to the airway pathology of cf, or whether it may serve a protective function against airway constriction. inhaled pge has been tried in asthma and may be of benefit as a bronchodilator. interestingly, pge has shown benefit in aspirin-sensitive asthmatics, a population that shares some features with the cf airway phenotype including nasal polyposis. the mechanism of airway relaxation to pge is mediated primarily by ep receptors signaling through camp. in cf, beta-adrenergic bronchodilators are commonly used, but are often only partially effective at reversing bronchospasm. beta-agonists and pge both signal through g-proteins coupled to camp and pka signaling, thus pge may offer another treatment when beta-agonists are ineffective at reversing bronchospasm in cf. in mouse tracheas, the beta-agonist isoproterenol (iso) will partially relax a pre-contracted airway, but ± % of contractile force remains (n= ). in contrast to the limited relaxation with iso, constricted mouse airways demonstrate significantly greater relaxation to pge , with only ± % of force remaining (n= , p< . compared to iso). this difference in relaxation responses suggests different intracellular signals are activated by each agonist despite both signaling through pka. to investigate phosphorylation events in response to iso or pge , we developed a new method for perfusing isolated mouse lungs. using this technique, tissues were perfused with buffered solution containing p for radiolabeling. lungs were then stimulated with either iso or pge , and phosphoproteins were compared from each condition. radiolabeling was effective under all three conditions. targets were chosen from each group (unstimulated, iso, or pge treated) that showed phosphorylation or dephosphorylation, and proteins were identified by mass spectrometry. over differentially phosphorylated proteins were identified, and two of these (rho gdi and inositol-requiring protein ) were selected as candidates to be tested in the context of relaxation to pge compared to iso. by identifying signaling targets involved in airway relaxation to pge , it may be possible to activate beneficial airway relaxation responses without the limitations seen in iso relaxation and without activating pge responses other than relaxation that may link pge to the airway pathology seen in cf. the parental ∆f /∆f cf bronchial epithelial cell line (cfbe o-) was compared to clones of cfbe o-complemented with . kb wild type (wt) or . kb ∆f cftr cdna in terms of their ion transport characteristics. the transepithelial resistance (rt) (~ (cm ) observed in the parental cfbe o-was maintained in the complemented cells. two clones complemented with wt cftr cdna (c - . wt and c - . wt) demonstrated rt ~ Ωcm , while another clone (c - . ∆f) complemented with the ∆f cftr cdna showed an rt of ~ Ωcm . two stable clones expressing wt cftr were selected based on electrophysiological characteristics and stability of cftr expression. forskolin-stimulated cl secretion in clone c - . wt was on average . ± . µa/cm and . ± . µa/cm in clone c - . wt. furthermore small camp-stimulated transepithelial current of . ± . µa/cm was observed in clone c - . ∆f indicating that ∆f cftr can traffic to the plasma membrane if present at high enough levels at physiological temperatures. the role that cftr plays in ca-activated cl conductance was investigated following treatment of the cells with atp in parental cfbe o-and wtcftr . -cfbe o-monolayers. in symmetrical cl-containing solutions, mucosal application of atp ( µm) elicited a small, transient cl secretory response in the parental cfbe ocells ( . ± . µa/cm ), while the magnitude of the atp-stimulated cl current was markedly enhanced in cftr-corrected cfbe o-cells with peak currents at . ± . µa/cm . recordings done in presence of a serosal-tomucosal cl gradient to increase the driving force for cl secretion showed a transient atp-dependent activation of cl currents in parental cfbe ocells with peak cl currents of . ± . µa/cm (n= ). cftr corrected cfbe o-cells showed a sustained activation of cl currents of similar magnitude ( . ± . µa/cm ; n= ). atp-stimulated cl currents were effectively blocked by the cftr inhibitor glyh ( µm). histochemical analysis showed prominent epithelial characteristics in all clones. these observations indicate that i) the apical driving force is limiting for atp-stimulated cl secretion and that ii) cftr participates in the cl secretory response to atp. furthermore, the findings suggest that these novel clonal isolates of the cfbe o-bronchial epithelial cell line can be useful for the investigation of cf therapies. this work was supported by grants from the cff, cfri, nih, elizabeth nash foundation, and pacfi. the lactoperoxidase (lpo) system functions in normal airways to prevent infection and may be compromised in cystic fibrosis (cf) due to defective transport of its substrate thiocyanate. a computational model of normal airway surface liquid suggests that hydrogen peroxide levels regulate lpo system activity and that the lower thiocyanate in cf airways may result in elevated levels of hydrogen peroxide that is likely produced by duox the major nadph oxidase in airway epithelial cells. to investigate the regulation of duox by bacterial products, passage normal human airway epithelial cells were grown and redifferentiated at the air liquid interface and treated for , or h with either pseudomonas aeruginosa flagellin or lps, applied apically. treated and mock treated cultures were analyzed for levels of duox and lpo mrna and protein, and for hydrogen peroxide production. quantitative pcr was performed using taqman kits, protein was measured by western blots, and hydrogen peroxide was quantified using amplex red-hrp coupled fluorescence. the data showed the duox and lpo mrna but not duox mrna, were significantly upregulated at all measured time points following apical treatment of the cultures with purified flagellin compared to mock treated. at h duox was increased . +/- . fold and lpo was increased . +/- . fold (means +/-s.e.m., n = lungs, - cultures of each lung). muc ac mrna was also upregulated by flagellin treatment ( . +/- . fold, n = lungs, - cultures of each lung). apical treatment with lps had no effect on any tested mrna and induced minimal il- secretion in these cultures. western blots using anti-duox antibodies (courtesy of f. miot, brussels, belgium) showed corresponding increases in duox protein following flagellin treatments. hydrogen peroxide production by flagellin treated cultures was increased in proportion to increases in duox mrna. application of flagellin alone to the apical surface of previously untreated cultures did not increase hydrogen peroxide production. thus, pseudomonas flagellin up-regulates duox and results in higher activity in normal human airway epithelial cells perhaps serving to increase innate host defense activity of the lpo system. chloride ion in phagolysosomes is an essential substrate for neutrophils to produce hypochlorous acid (hocl), an important bactericidal agent. we have previously shown that cftr was present in the phagolysosomal membrane of neutrophils and the cftr defect led to deficient chlorination of ingested pseudomonas aeruginosa (pa) (painter et al, biochemistry, : - , ) . these results suggested that chloride accessibility to the phagolysosomal lumen might be impaired in cf neutrophils thereby affecting neutrophil-mediated bacterial killing. to understand the role of chloride ion in the process, we first assessed the effect of extracellular chloride concentration on pa killing by normal neutrophils under iso-osmotic conditions. surprisingly, bacterial killing was strongly dependent on extracellular chloride concentration. the initial rate of killing of pa was~ fold less in a chloride-free medium than a physiological chloride medium. killing efficiency increased as the chloride concentration was raised in a dosedependent fashion and plateaued at~ mm chloride concentration. to determine what percent of the chloride-dependent killing was oxidantdependent, we applied the cftr inhibitor glyh- , the myeloperoxidase (mpo) inhibitor aminobenzoic acid hydrazide (abah) and the nadph-oxidase inhibitor diphenylene iodium chloride (dpi), respectively to bacterial killing assays. the results showed that the killing of pa could be divided into three discernible components: ( ) a component accounting for about - % of the total killing rate that was inhibited by glyh- , abah or dpi; ( ) a second component amounting to~ - % that was chloride-dependent and partially sensitive to glyh- and; ( ) a third accounting for~ % of the total killing activity that was not sensitive to oxidant inhibitors, or extracellular chloride concentration. dpi and abah had no effect on killing of pa in the absence of extracellular chloride suggesting that most of the oxidant-mediated killing activity toward pa was due to hocl. none of the inhibitors had any significant effect on phagocytosis. finally, the rate of pa killing by cf neutrophils was found to be significantly lower ( - %) than that seen for normal neutrophils and was comparable to the effect of glyh- on killing of pa by normal neutrophils. the data suggest that cftr plays a significant role in killing of pa by normal neutrophils and, when defective as in cf, may compromise the ability of neutrophils to efficiently kill pa. this research was supported by a cff grant to gw. %. the cf group demonstrated a significantly higher frequency of pseudomonas respiratory infections than the non-cf group. interestingly, no significant differences were detected in any infections from other systems including blood, sinuses, skin, wounds, oral cavity, bowel, eyes, peritoneal cavity, and urinary tract. moreover, the cf lung-transplant patients had significantly less time free from pseudomonas infections in the transplanted lungs. conclusion normal lungs transplanted into cf patients had significantly higher susceptibility to pseudomonas infections than those transplanted into non-cf patients, which strongly suggests that defective host defense mechanism(s) beyond the respiratory system contribute to cf lung pathogenesis. pyocyanin (n-methyl- -hydroxyphenazine, pcn) is produced by pseudomonas aeruginosa and is found in pulmonary secretions of infected cf patients at concentrations up to µm. pcn is a redox-active compound that generates superoxide and hydrogen peroxide in the presence of cellular reductants. since cftr was reported to be modulated by h o , we hypothesized that pcn, through its ability to redox cycle and to produce h o , would affect cftr-mediated cl transport. we therefore tested pcn and h o for effects on cltransport (transepithelial short-circuit currents in ussing chambers measured with serosal-to-mucosal clgradient) and cytosolic redox potential (imaging microscopy on cells transfected with a redox sensitive gfp, rogfp ) using cftr-corrected cf bronchial epithelial cells (wtcftr . . under resting conditions, acute exposure of the apical epithelial surface to µm pcn elicited a small clsecretory response ( . ± . µa/cm , n= ) that was completely blocked by the cftr inhibitor glyh ( µm). in contrast, in presence of the camp-elevating agonist forskolin, pcn caused a time-dependent decline of forskolin-stimulated cftr clcurrents by %. exposure to h o ( µm) elicited a more pronounced clsecretory response than pcn in resting cells and h o -stimulated clcurrents peaked at . ± . µa/cm (n= ). h o was less effective than pcn at inhibiting forskolin-stimulated cftr clcurrents and currents declined by %. both pcn and h o oxidized the cytosolic redox potential from a resting value of - mv by similar amounts ( ± mv for pcn vs. ± mv for h o ), but rates of oxidation were faster for h o ( . ± . mv/min) than for pcn ( . ± . mv/min). inhibition of camp-stimulated cftr clcurrents by oxidation was a linear function of the cytosolic redox potential, but pcn ( µa×cm - ×mv - ) was more potent than h o ( . µa×cm - ×mv - ). cf bronchial epithelial cells homozygous for ∆f -cftr did not show a clsecretory response to pcn or h o . these data suggest that cftr-corrected cf bronchial epithelial cells respond acutely to oxidative stress by turning on cftr activity probably as part of the innate host defense response and this mechanism is absent in cf airways. furthermore, prolonged exposure to pcn and h o is detrimental for the proper function of the camp-regulated cftr clconductance. we conclude that oxidative stress in p. aeruginosa-infected airways is a key factor that needs to be considered for successfully correcting cftr function in cf airways. supported by nih (nccam p at ), cff (fischer g ) the lung is continually exposed to environmental agents and pathogens. the lung epithelial lining fluid (elf) is first to encounter these agents and gsh is a major antioxidant found in this apical fluid. the cystic fibrosis transmembrane conductance regulator protein (cftr) is the only known gsh transporter that maintains lung elf gsh levels. cystic fibrosis (cf) patient have low levels of gsh in their elf and have copious and viscous mucus. hypertonic saline is used in cf patient to help clear mucous and improve lung function however the mechanism(s) by which it does so are poorly understood. the purpose of these studies was to examine whether hypertonic ( . %) saline can modulate apical gsh levels and protect lung epithelial cells against the oxidant, t-butyl hydroperoxide (t-booh). we used human lung epithelial cell lines sufficient (c ) and deficient (ib ) in cftr. the cftr deficient ib cells have % lower basal levels of apical gsh as compare to cftr sufficient c cells. cells were exposed separately and in combination to % saline, and t-booh ( um) for hours. the cftr deficient ib cells were much more sensitive to t-booh-mediated oxidative injury as measured by lactate dehydrogenase (ldh) release. hypertonic saline exposure was associated with an increase in apical gsh levels in both cftr sufficient and deficient cells and decreased t-boohmediated oxidative injury in both cells lines. hypertonic saline increased gsh in the apical compartment, which appeared to be largely cftr mediated. we examined this issue in mice given a clinically used dosing regimen of % hypertonic saline nebulized twice daily and examined changes in elf gsh levels hours after last treatment. mice receiving the hypertonic saline treatment had significantly higher gsh levels in their elf than untreated controls. this data suggests that cftr and gsh adaptive responses play an important role in lung's reaction to oxidants. we propose that factors, which interfere with the lung's capability to mount and maintain an adequate adaptive apical gsh response, may compromise and sensitize the lung to oxidative injury. (supported in part from nih grant hl ). the absence of functional cftr in airway epithelia of cf patients leads to abnormal airway surface liquid, which favors chronic bacterial infection. this chronic infection in cf lungs is associated with an exaggerated inflammatory response characterized by abnormal cytokines secretion and massive lung neutrophils infiltration. we have previous recapitulated the abnormal inflammatory response in cftr-/-(cf) mice by repeated administration of pseudomonas aeruginosa lipopolysaccharide (lps) (ped. pulmonology, supp. , ) . we demonstrated that the bronchoalveolar lavage (bal) fluid of cf and heterozygous (het, cftr+/-) littermate control mice when compared to the bal from wt mice shows ) higher numbers of neutrophils and ) higher concentrations of cytokines (il- alpha, il- , kc, mcp- , gm-csf, m-csf) as assessed using multiplex analysis of selected cytokines. of note, airway epithelial cells from cf patients are known to have elevated secretion of some of these cytokines. however to date, the contribution of immune cells to these abnormal cytokine levels has not been well characterized. this study aims to investigate the contribution of cf macrophages in this altered cytokine profile/response. we examined cytokine secretion from two different macrophage populations: alveolar macrophages (am) obtained from the bal fluid and bone marrow (bm)-derived macrophages differentiated in presence of m-csf. both macrophage populations were cultured in the presence or absence of lps, and the supernatants were tested for the concentration of the cytokines that we had shown to be abnormal in vivo. these macrophage populations were examined from cf, het and wt mice. in absence of lps exposure, bm-derived macrophages from cf mice have lower levels of il- and kc compared to wt and het mice. in contrast, am of cf mice have higher levels of il- and kc than am of wt mice. this difference may be due to am's prior exposure to antigens in vivo whereas the bm-derived macrophages are a truly naïve population. interestingly, after lps stimulation, il- a, il- , mcp- , kc, g-csf and gm-csf are higher in cf macrophages compare to wt and het mice suggesting that cf affected macrophages display an altered inflammatory response when compared to wt and het. the abnormal production of these cytokines was also detected at the transcriptional level using quantitative rt-pcr analysis. in conclusion, our data support the hypothesis that cftr-null macrophages may have an impaired cytokine profile at baseline as well as after stimulation. furthermore, cftr deficient macrophages may contribute directly to the abnormal inflammatory response in cf. (supported by cff, nih, niddk) cystic fibrosis lung disease is associated with an excess of free neutrophil elastase (ne) in the airway arising from persistent neutrophil inflammation. dx- is a potent small protein inhibitor of neutrophil elastase that could be a useful therapy for diseases involving neutrophilic inflammation such as cystic fibrosis and copd. dx- effectively inhibits ne activity in cf sputum sol (ic = pm). however the physical barriers posed by cf whole sputum may prevent dx- accessing and inhibiting ne ensconced within the mucus. the mucolytic dnase acts by cleaving extracellular dna in mucus thereby reducing viscosity. however dna binds and inactivates ne and therefore dnase treatment of mucus results in an acute increase in active ne. the lipid portion of pulmonary surfactant acts as a lubricant and our group has previously shown that bovine lung extract surfactant (bles) increases the fluidity of whole sputum. ( aim we aimed to show that, by reducing the surface tension of sputum, bles would enhance the anti-elastase effect of dx- in whole sputum. methods whole sputum ( . g per well in a -well plate) was pre-incubated with µl saline as control, µl dnase ( µg/ml) or µl bovine lung extract surfactant (bles, µg/ml) for minutes at °c followed by addition of µl dx- ( µm or µm) for h at °c. the contents of each well were aspirated, added to ml nacl ( . %) and centrifuged at , g for min. the resulting sputum sol was assayed for ne activity using the colorimetric substrate n-meosuc-aapv-pna. results ne activity in whole sputum was significantly inhibited by µm dx- (p= . ) and µm dx- (p= . ). µm dx- inhibited significantly more ne when sputum was pre-treated with bles (p= . ) treatment of sputum with dnase caused a significant increase in ne activity compared to untreated sputum (p= . ) whereas treatment of sputum with bles did not have this effect. conclusion dx- effectively inhibits elastase in cf sol. pre-treatment of cf whole sputum with bles enhanced the anti-elastase effect of dx- and treatment of sputum with bles alone did not cause an increase in ne activity in contrast to treatment with dnase. dx- inhibitory capacity in whole sputum could be improved by co-treatment with surfactant which increases fluidity of sputum and could allow greater access of drugs to sputum components. bovine lung extract surfactant, bles™ was a kind gift from bles biochemicals inc., on, canada. dx- was a kind gift from dyax corp, ma who also funded part of this research. table : results are summarised as neutrophil elastase activity µg/ml and show the mean and sem of n= experiments. cystic fibrosis lung disease is associated with an excess of free neutrophil elastase (ne) in the airway arising from persistent neutrophil inflammation. dx- is a potent small protein inhibitor of neutrophil elastase that could be a useful therapy for diseases involving neutrophilic inflammation such as cystic fibrosis and copd. dx- effectively inhibits ne activity in cf sputum sol (ic = pm). however the physical barriers posed by cf whole sputum may prevent dx- accessing and inhibiting ne ensconced within the mucus. the mucolytic dnase acts by cleaving extracellular dna in mucus thereby reducing viscosity. however dna binds and inactivates ne and therefore dnase treatment of mucus results in an acute increase in active ne. pulmonary surfactant acts as a lubricant and our group has previously shown that bovine lung extract surfactant (bles™) increases the fluidity of whole sputum. ( we aimed to show that, by reducing the surface tension of sputum, bles™ would enhance the anti-elastase effect of dx- in whole sputum. methods whole sputum ( . g per well in a -well plate) was pre-incubated with µl saline as control, µl dnase ( µg/ml) or µl bovine lung extract surfactant (bles™, µg/ml) for minutes at °c followed by addition of µl dx- ( µm or µm) for h at °c. the contents of each well were aspirated, added to ml nacl ( . %) and centrifuged at , g for min. the resulting sputum sol was assayed for ne activity using the colorimetric substrate n-meosuc-aapv-pna. results ne activity in whole sputum was significantly inhibited by µm dx- (p= . ) and µm dx- (p= . ). µm dx- inhibited significantly more ne when sputum was pre-treated with bles™ (p= . ) treatment of sputum with dnase caused a significant increase in ne activity compared to untreated sputum (p= . ) whereas treatment of sputum with bles did not have this effect. conclusion dx- effectively inhibits elastase in cf sol. pre-treatment of cf whole sputum with bles™ enhanced the anti-elastase effect of dx- and treatment of sputum with bles alone did not cause an increase in ne activity in contrast to treatment with dnase. dx- inhibitory capacity in whole sputum could be improved by co-treatment with surfactant which increases fluidity of sputum and could allow greater access of drugs to sputum components. we would like to thank prof. f. possmayer, on, canada and bles biochemicals inc., on, canada for the bovine lung extract surfactant, bles™. dx- was a kind gift from dyax corp, ma who also funded part of this research. table results are summarised as neutrophil elastase activity µg/ml and show the mean and sem of n= experiments. cf patients infected with p. aeruginosa (pa) have increased levels of proinflammatory cytokines, such as tnf-α, il- β, il- , and il- ; but have low levels of the anti-inflammatory cytokine, il- in their airways. the proinflammatory mediators in the cf airway recruit a large number of polymorphonuclear neukocytes (pmns), which release high levels of neutrophil elastase and human neutrophil peptides and damage the lungs. the intense neutrophil-dominated inflammatory response associated with chronic pa infection results in progressive airway obstruction, the cause of death of over three-quarters of cf patients. recent studies highlight the significance of sphingolipid metabolism in cell growth, differentiation, membrane traffic, apoptosis, senescence, and immune regulation. acid sphingomyelinase (asm) plays a critical role in sphingolipid metabolism by hydrolyzing sphingomyelin to ceramide. ceramide self-associates to form ceramide-enriched membrane platforms(also called lipid rafts). these platforms have been shown to be involved in the internalization of pa; induction of apoptosis and immune regulation. our in vitro and in vivo studies of asm confirm that there is a loss of induction of asm after pa infection in both cf bronchial epithelial cells and cftr knock out mice as compared with controls. in vitro, we examined the asm activity in human bronchial epithelial cells with normal cftr expression (s cells) and with mutant cftr expression (ib cells ) after pa infection at , , , , , and min. asm activity was up-regulated in s cells following pa infection after min and kept increasing until min, while asm induction was lost in ib cells. asm protein and ceramide levels also increased in s cells following pa infection compared with ib cells. in our in vivo studies, cftr knock out mice and control c bl/ mice were both administered various doses of pa orally ( x cfu to x cfu) . six hours after pa infection, lung homogenates from the c bl/ mice indicated increasing asm protein and ceramide production, which corresponded to the dose of pa given. in contrast, asm protein and total ceramide production remained at baseline levels in the lung homogenates of cf mice regardless of the dose of pa given. rna interference (sirna) assays were performed by transfecting a bp pre-designed sirna specific for human asm in s cells. these cells were then infected with pa for hrs to see if they had similar il- level compared with infected ib cells. we demonstrated a significantly elevated il- expression in s cells tranfected with asm sirna compare with s cells transfected with a scramble sirna (p< . ). our results suggest that the loss of asm induction, caused by mutant cftr, is involved in pa clearance and contributes to the exaggerated proinflammatory cytokine response in cf airway. we are conducting research on defining the roles of asm and the sphingolipids in pa infection; cell signaling; as well as cytokine induction, we are also looking at the therapeutic potential of aav.asm vectors in pa-infected cf bronchial epithelial cells and cftr knock out mice. work supported by nhlbi introduction: new therapies are critically needed to treat cystic fibrosis (cf) lung disease, which is characterized by chronic infection and inflammation. novel therapies to target airway inflammation are likely to improve the clinical outcome in cf. as yet there is no consensus regarding the molecular pathway(s) mediating inflammation in the cf lung. objective: to establish whether toll-like receptor (tlr) signalling is the key molecular pathway responsible for the inflammation seen in cf lung disease and to determine whether inhibitors of tlr signalling can reduce this damaging inflammatory response. methods and results: to quantify the airway inflammatory response, we used established cf and cf-corrected lung epithelial cells (ib - and c ). since artefacts may alter the inflammatory phenotype of cell lines, we validated all findings with fresh peripheral blood mononuclear cells (pbmcs) from cf patients (n= ) and healthy controls. we measured the inflammatory response (il , il , tnfα) of these cells following stimulation with: (i) cf pathogens (p. aeurginosa, b. multivorans, b. cenocepacia); (ii) purified tlr ligands. both cf respiratory epithelial cells and cf pbmcs had a greatly increased inflammatory response compared to matched controls when stimulated with whole bacteria and purified tlr ligands (p< . ). interestingly, the cf airway cells responded most vigorously to the tlr ligand, flagellin (p< . ). to investigate tlr as a potential anti-inflammatory target, we found that tlr mrna (p< . ) and protein (p< . ) were expressed at a higher level in cf compared to cf-corrected lung epithelial cells. finally, to determine the contribution of tlr activation to cf lung inflammation we exposed the airway cells to wild-type (pak & pao ) and isogenic flagellin-deficient strains of p. aeruginosa (pak∆flic, pao ∆flic, pao ∆flge & pao ∆flim) . strikingly, the absence of the tlr -flagellin interaction significantly reduced the proinflammatory response of cf respiratory epithelial cells to p. aeruginosa (p< . ). we show that tlr-mediated innate immune responsiveness is increased in both cf respiratory epithelial cells and fresh blood cells from cf patients. moreover, inhibition of the tlr -flagellin interaction markedly reduced proinflammatory response of cf respiratory epithelial cells following exposure to predominant cf pathogen p. aeruginosa. these data suggest that tlr activation may represent a novel anti-inflammatory target for improving cf lung disease. supported by the canadian cf foundation. cf airway cells (ib - ) were stimulated with wild-type (pak) and flagellin-deficient p. aeruginosa (pak∆flic). introduction: genetic polymorphisms for tgf-β have recently been identified as a significant modifier of cf lung disease severity. however, at present little is known about protein measurements of tgf-β in bronchoalveolar lavage fluid (balf) or serum obtained from pediatric cf patients during an acute respiratory exacerbation. hypothesis: tgf-β in cf balf and serum is increased in association with markers of cf lung disease severity, and is decreased after completion of iv antibiotic (iv abx) therapy. study design and methods: in a descriptive, prospective study of children with cf presenting for hospital admission to treat a pulmonary exacerbation, balf specimens were obtained from patients undergoing clinically-indicated flexible bronchoscopy on admission. balf was immediately analyzed for total and differential cell counts with the cell-free balf supernatant stored at - °c. serum specimens were obtained at the initiation and conclusion of iv abx and similarly stored at - °c. measurement of total tgf-β utilized a commercial elisa kit. t-test statistical analysis assumed significance at p<. . results: balf was obtained from cf patients with a mean age of . ± . years (range - years). three balf cultures were pseudomonas aeruginosa positive (psa+). balf tgf-β levels ranged from - pg/ml (mean ± . pg/ml) with the highest tgf-β measured in a patient with abpa and culture positive for m. abscessus. in balf, higher neutrophil counts (greater than the median, . x pmn/ml) were associated with increased balf tgf-β ( ± . pg/ml vs. ± . pg/ml, p<. ). serum tgf-β was collected in cf patients with a mean age of . ± . years (range - years), four of whom were psa+. serum tgf-β ranged from . - . ng/ml (mean . ± . ng/ml). in both balf and serum, neither patient age nor psa culture status was associated with increased tgf-β at admission. however, previous hospitalization in the last months was significantly associated with an elevated admission tgf-β in both balf ( ± . pg/ml vs. ± . pg/ml, p<. ) and serum ( . ± . ng/ml vs. . ± . ng/ml, p<. ). after completing the clinically-indicated course of iv abx, / psa-cf patients had a reduction in serum tgf-β (mean . ± . ng/ml to . ± . ng/ml, p=ns). in contrast, / psa+ cf patients had an increase in serum tgf-β (mean . ± . ng/ml to . ± . ng/ml, p=ns) after completing iv abx therapy. conclusions: tgf-β is measurable in balf and serum from pediatric cf patients and varies in association with clinical parameters. these preliminary results support our hypothesis that increased tgf-β may be associated with more severe lung disease as evidenced by ) association of increased tgf-β in balf and serum obtained from cf patients with previous hospitalization in the last months, and ) association of increased tgf-β in balf with increased cellular inflammation. the direction of change in serum tgf-β after iv abx therapy may be influenced by the presence or absence of pseudomonas infection. acknowledgments: supported by cff (harris ao). paula murphy and cassidy henegar provided technical support. background: cystic fibrosis is one of the leading genetic causes of end stage lung disease for which the treatment is lung transplantation. however, mortality due to chronic rejection known as obliterative bronchiolitis (ob) remains high due to small airway obliteration. ob is associated with increased levels of matrix metalloproteinase- (mmp- ), an interstitial collagenase, expressed by polymorphonuclear cells (pmns). pmns are among the first inflammatory cells to be detected in ob but very little is known about the role of pmn mmp- or the mechanisms used by pmns to migrate through extracellular collagen matrices. objective: to identify the molecular mechanism for neutrophil recruitment into the airway. we hypothesize that mmp- promotes accumulation of pmns in ob lesions by promoting their migration through the extracellular matrix protein barriers by its pericellular collagenase activity. method: the heterotopic airway transplant model was used to study the role of neutrophil mmp- in promoting pmn migration through extracellular matrices. mhc-disparate balb/c tracheas were subcutaneously transplanted into c bl/ wild-type (wt) or mmp- deficient (mmp- -/-) mice. to further dissect the mechanism of pmn migration in vitro their migration through collagen gels was studied. collagen gels were made in transwells using rat-tail collagen providing a highly cross-linked thick collagen barrier. bone marrow derived pmns from wt or mmp -/-mice were fluorescently labeled and stimulated to induce the surface expression of mmp- . pmns were placed on thick collagen gels in traswells. the transwells had polycarbonate membranes at the bottom of the gels. the pore size of the membranes did not allow pmns to pass through into the lower chambers that had flmp a chemo attractant. at hours and hours the gels were fixed and the polycarbonate membrane at the bottom of the collagen gel was gently peeled off. pmns on the membrane were counted under a fluorescent microscope. results: significantly decreased migration of pmns into the airway lumen was noted in the mmp- deficient mice (p< . ) weeks post-transplantation compared to wt mice in the heterotopic airway transplant model. in addition there was more than a three-fold increase in the pmns outside the lumen in the extracellular collagen matix (p< . ). wt-pmns were able to penetrate collagen barriers in greater numbers both at and hours compared to mmp -/-pmns in vitro (p < . for hours and . for hours). in addition, mmp inhibitor gm -treated wt cells demonstrated decreased migration through collagen gels ( hours p< . and hours p< . ). conclusions: mmp- -/-mice has significantly less pmns in the airway lumen due to decreased migration through collagen matrices providing protection against ob. this has great significance for conditions such as cystic fibrosis, lung transplant rejection (obliterative bronchiolitis) and other inflammatory diseases mediated by pmns. mmp- blockade can potentially be a novel therapeutic target to decrease neutrophil efflux into the airways and thus reduce airway inflammation. background: our laboratory has demonstrated alterations in cf-related regulation of cholesterol processing. hydroxycholesterols (oxysterols) are oxygenated derivatives of cholesterol formed from autoxidation of cholesterol and from de novo synthesis. oxysterols are known to be key regulators of cholesterol metabolism and cellular signaling. the role of oxysterols in the cellular pathways involved in cholesterol processing in cystic fibrosis has not been elucidated. based on our previous findings of increased de novo cholesterol synthesis in cf tissues, we hypothesize that oxysterol production in cf would be positively impacted. increased oxysterol production may lead to abnormalities in cellular signaling associated with oxidative stress and may influence the inflammatory response in cf. the goals of this research are to investigate the effect of oxysterols on inflammation in cf and to determine if there is a difference in oxysterol production in cf compared to wild type controls. results: the goal of these studies is to determine if cf cells respond uniquely to oxysterols compared to control cells. initial studies demonstrate that cf-model /hteo-pcepr cells failed to respond to -oh cholesterol ( -oh) with regards to nfκb activation. control /hteo-pcep cells, however, exhibited a significant -fold increase in nfκb activity upon -oh stimulation ( . -fold +/- . increase from il- , tnf alpha alone). however, il- promoter activation in response to -oh was not significantly altered in either cf or wt cells ( . -fold +/- . (cf) increase from il- , tnf alpha alone, . -fold +/- . (wt) increase from il- , tnf alpha alone). in order to verify il- promoter results, il- protein production was measured directly in response to pseudomonas aeruginosa. il- protein level in response to -oh was increased in cf cells ( . -fold) and decreased in wt cells ( . -fold), although not significantly in either group. oxysterol content and de novo synthesis are currently being examined in vivo in cf and wild type mouse models. mice lacking cftr on a c bl/ background and wild type controls were obtained from our animal core and the amount of oxysterol present in these mouse models is currently being studied. conclusion: these findings suggest that endogenous oxysterol production or other components of oxidative stress are pre-stimulating nfκb activation via the oxysterol-sensitive pathway. interestingly, although oxysterols robustly activate nfκb in wt cells, oxysterol-dependent pathways are not involved in il- production. these data suggest that il- is not a primary cause for increased oxysterol mediated inflammation. elucidating the role of oxysterols in cf will be an important contribution to our understanding of the inflammatory pathways in cf and will contribute to the growing body of evidence for aberrant cholesterol regulation in cf. this work is supported by grants from the cff and the nhlbi. cystic fibrosis (cf) is a common genetic disorder caused by a mutation in the cftr, a chloride transporter of the abc transporter family. innate immunity, the first line in host defense against infectious agents, is abnormal in cf patients. cf patients are commonly colonized with bacterial pathogens, which is the major clinical problem of this disease. despite a marked difference in the clinical outcome amongst cf patients infected with pseudomonas aeruginosa and burkholderia cepacia, the mechanisms under-lying these differences remain unclear. understanding the innate immune response, specifically the role of cftr, in p. aeruginosa and b. cepacia infections will lead to new insights into mechanisms that limit these infections. our objective is to develop a cf model in the simple organism, the worm, c.elegans, and to test the role of the cftr orthologue, mrp- , in host defense. using this model, we have compared the survival of c. elegans between virulent p.aeruginosa (pa ) and b.cepacia (bc). our experiments have demonstrated that, when compared to growth on their natural food source, e.coli op , worms fed on pa and bc die prematurely. specifically, worms survive up to hours on op , whereas pa fed worms die with in hours. death on pa also requires active infection, as heat inactivation of these bacteria destroys their killing capacity. two bc strains have been tested, a virulent strain ( ) and an avirulent strain ( ). worms infected with bc show an intermediate phenotype, surviving approximately hours. our future work will define whether synergy occurs during coinfection with p.aeruginosa and b.cepacia, as would be suggested by clinical data. in addition, we will define the transcriptional changes induced in c.elegans fed on pa and bc, and use these data to identify common and pathogen specific responses to these infections. finally, mrp- knock-down worms will be generated using rna interference, and the role of the cftr orthologue in infection will be tested. in conclusion, c.elegans offers a genetically tractable model organism in which the innate immune response to pseudomonas aeruginosa and burkholderia cepacia can be studied. in addition, this simple model system will allow us to define the role of the cftr orthologue in host defense. supported serine proteases released from neutrophils are considered central to the pathogenesis of cf lung disease, and have been obvious therapeutic targets. although intracellular serine proteases are important in host defense and bacterial killing, extracellular enzymes may contribute to bacterial persistence and promote airway inflammation. neutrophil elastase (ne) digests key opsonins present in the lung and has been shown to disrupt phagocytosis, allowing the bacteria to persist in the face of established pulmonary inflammation. in addition we have found that other proteases, like cathepsin g (cg), an abundant serine protease found in human and murine neutrophils, may also have other, critical roles in development of cf lung disease. indeed, using murine models of endobronchial inflammation, cg inhibits airway defenses and interferes with the host's ability to clear pseudomonas aeruginosa from the lung with effects quite distinct from ne. to test the hypothesis that differences in bacterial killing are due to defects in innate defenses created by excess, unopposed cg at the apical surface of the airway epithelium, we have examined profiles of proteins secreted into the airway lumen by epithelial cells, which are necessary for p. aeruginosa clearance and susceptible to cg proteolysis. analysis of lavage fluid utilizing -d page indicated a total of spots which were increased in pooled lavage fluid from infected cg-deficient mice as compared to wild-type littermates. tandem mass spectrometry analysis identified several novel proteins that were cleaved by cg, including inter-alpha inhibitor protein, selenium-binding protein, sec -like protein, beta- -glycoprotein, and annexin a . another protein candidate, serum amyloid p component (sap), is a novel opsonin and may be involved in airway defenses. immunoblot analy-sis demonstrates that cg cleaves sap. these results suggest that cg, like ne, plays an important role in the pathogenesis of lung diseases. we have previously demonstrated in ∆f -cf mice that in vivo treatment with azithromycin (azm) attenuates cellular infiltration in baseline and lipopolysaccharide (lps)-induced inflammation, and inhibits proinflammatory cytokine release in induced inflammatory condition (legssyer et al, ) . this study aimed at investigating macrophages isolated from wild type (wt) or ∆f -cf mice (van doorninck et al, ) and the effect of azm on these cells. purified peritoneal and alveolar macrophages were stimulated with lps (p. aeruginosa, . µg/ml) plus ifnγ ( . µg/ml), with or without azm ( µg/ml). macrophage inflammatory status was investigated by assessing different pro-and anti-inflammatory mediators at mrna level h after stimulation. pro and anti-inflammatory markers seemed to be reduced in naive alveolar cf macrophages. by contrast, a trend to overexpression of these markers was seen in naive peritoneal cf cells. pro-inflammatory status, as assessed by il- β, was induced in stimulated alveolar and peritoneal cf macrophages and in naive peritoneal cf cells. moreover, nos- was found to be overexpressed in stimulated alveolar cf macrophages while il- was downregulated. azm significantly reduced il- β expression in stimulated alveolar macrophages. in conclusion, peritoneal and alveolar macrophages exhibit distinct phenotype. a pro-inflammatory status was more prominent in stimulated cf alveolar and peritoneal cells. interestingly, azm modulates il- β overexpression only in cf stimulated alveolar macrophages, supporting the antiinflammatory activity of this macrolide and identifying, at least partly, alveolar macrophages as possible target cells for its effects. supported it has been recently reported by perez and coll. that the inhibition of function of w/t cftr produces an inflammatory profile that resembles that observed in cf patients [am j physiol lung cell mol physiol, ] , whereas we demonstrated that correction of f del cftr function with mpb- down modulates the pseudomonas aeruginosa (p.aeruginosa) dependent expression of the pro-inflammatory mediators il- and icam- in cf bronchial cells [am. j. resp. cell. mol biology, ] . since both evidence support a direct link between cftr function and inflammatory response in respiratory epithelial cells, we extended our investigation to other f del cftr correctors, such as miglustat [norez et al, febs letters, ] , which is an approved drug for gaucher disease, in comparison with an isomer without any correcting effect, namely nb-dgj. human bronchial ib - (cftr f del/w x), cufi- (cftr f del/f del) and nuli- (cftr w/t) cells were exposed to the laboratory strain of p.aeruginosa (pao ) or tnf-alpha or il- beta and the transcription of icam- and il- were quantitated by real time rt pcr. cftr function was assayed by single-cell fluorescence imaging, using the potential-sensitive probe disbac [renier et al, hum. gene ther. ]. analysis of binding of nf-kb and ap- transcription factors to labelled dna target was performed with electrophoretic mobility shift assay (emsa) [borgatti et al, j. biol. chem. ]. miglustat significantly reduced the expression of il- by % in ib - and by % in cufi- cells and of icam- by % in ib - and by % in cufi- cells, upon infection by pao . in parallel, correction of f del cftr function was observed after miglustat treatment in both ib - and cufi- cells. miglustat had no major effects on overall binding activity of transcription factors nf-kb and ap- , activated by pao in ib - cells. the inflammatory response induced by tnf-alpha or il- beta was also significantly reduced in ib - and cufi- cells treated with miglustat. nb-dgj, which is not a corrector of function of f del cftr, down modulated the induction of il- and icam- in ib - and cufi- cells. in addition, both miglustat and nb-dgj reduced the inflammatory response to pao in non cf nuli- cells. in conclusion, miglustat and nb-dgj have an anti-inflammatory effect in bronchial cells independently of the correction of f del cftr, by interfering with the pro-inflammatory signaling downstream the receptors for pathogens and pro-inflammatory cytokines. since miglustat is already approved for the treatment of gaucher disease and other glycosphingolipidoses, it may be an interesting candidate to ameliorate lung inflammation in cf patients. background: the primary cause of mortality and morbidity in cystic fibrosis (cf) is lung disease, which is characterized as a chronic cycle of infection and inflammation dominated by a neutrophilic infiltrate and increased levels of circulating mediators such as il- . a wide variation in the rate of progression of lung disease is observed in cf. hypothesis: we postulate the presence of a brisk systemic inflammatory response in cf patients and that this systemic response reflects the magnitude and progression of lung disease. methods: lung function and clinical data were obtained from cf patients treated and followed in the adult cf clinic, st paul's hospital. forced expiratory volume at s (fev ) and forced vital capacity (fvc) values dating back to were used in regression equations to group patients into quartiles based on their decline in predicted fev %. rate of decline in lung function was compared with clinical characteristics and various systemic inflammatory biomarkers, including wbc, band cell count, crp, il- β, il- , il- , mcp- , and gm-csf results: the quartiles exemplified significantly different rates of decline in fev /fvc% values. subjects with a rapid decline in lung function were younger and were colonized/infected more commonly with methicillinresistant staphylococcus aureus (mrsa) (p< . ). in addition, altered calcium, magnesium, and albumin levels were found in subjects with increased rates of decline in lung function (p< . ). preliminary results show that patients with cf have significantly elevated levels of wbc ( . e /l), crp ( . e ng/ml), and il- ( . pg/ml) in serum when compared to control subjects (p< . ). there were trends in relationships between levels of il- , il- and mcp- and rates of decline in lung function, however none reached statistical significance. conclusion: there is a variable rate in the decline in lung function among cf patients and one factor may be a heterogeneous systemic inflammatory response. circulating pro-inflammatory mediators may not only impact progression of lung disease, but could also be novel biomarkers to monitor and assess disease severity in cystic fibrosis. nutritional status in addition effects the rate of decline in lung function perhaps by modifying the inflammatory response. this research is supported by the bc lung association. overexpression of the epithelial na + channel β subunit (protein = βenac, gene = scnn b) in transgenic mice results in cf-like lung pathology, characterized by neonatal mortality, mucus obstruction and airway inflammation (mall et al., nat. med. ) . breeding βenac mice into gene-deleted mice enables quick and efficient determination of the specific pathways relevant to the development of lung pathology. tnfα is a pleiotropic pro-inflammatory cytokine released by many different cell types, including t cells, macrophages, granulocytes, and epithelial cells. tnfα levels are significantly elevated in bronchoalveolar lavage (bal) from βenac mice in comparison to wild-type (wt) littermates. we crossbred βenac mice (heterozygous, inbred line c - ) with tnfα knockout mice, generating four types of mice: wt, tnfα +/-; wt, tnfα -/-; βenac, tnfα +/-; and βenac, tnfα -/-. all mice had comparable, high survival, ranging between - %. the lack of tnfα did not prevent neutrophil and eosinophil infiltration and did not significantly modify the lung pathology characteristic of βenac mice, namely mucus plugs, mucous secretory cell hyperplasia, airway inflammation and emphysema. the absence of tnfα in bal from βenac, tnfα -/-mice, in comparison to βenac, tnfα +/mice, was confirmed by luminex cytokine assay. the levels of kc, a potent neutrophil chemoattractant, were significantly elevated in bal fluid from βenac mice, regardless of the presence or absence of tnfα. in wt mice, no granulocytic infiltrate or morphologic changes were observed and tnfα and kc were undetectable. similarly, the pathologic changes in βenac mice were not mitigated by crossbreeding to knockout mice for tnfα receptor (tnfαr ), the major mediator of biologic responses classically attributed to tnfα. these results suggest that tnfα per se does not have a critical pro-inflammatory role in the development of inflammation in the βenac mouse model. the production of alternative cytokines may compensate for the loss of tnfα bioactivity in the tnfα and tnfαr -/-mice. since tnfα has been suggested to play a role in the regulation of enac, ussing chamber studies are underway to test whether the bioelectric features of tracheobronchial epithelia from βenac mice are altered by the absence of tnfα in vivo. these data suggest that a tnfα-independent signaling cascade links airway surface dehydration to airway inflammation in the βenac mouse model. innovative pharmacological approaches to control the excessive neutrophil infiltrates into the bronchial lumen of cf patients are thought to be beneficial to reduce the extensive airway tissue damage. the activation of expression of proinflammatory genes by p.aeruginosa with bronchial epithelial cells is a central mechanism to be targeted with novel therapies. medicinal plants from the socalled traditional asian medicine are attracting a growing interest because of their potential safety, already tested in large scale applications in human diseases. however, due to the presence of different active principles in each plant extract, whose multifunctional effects may even result contradictory, understanding the effect of each component is mandatory to pursue selective and reproducible applications. a panel of medicinal plant extracts have been firstly screened for their capacity to interfere in the binding of nuclear transcription factor proteins (tf) with dna consensus sequences identified in the promoters of the pro-inflammatory genes, thus for their potential inhibitory action on gene expression. extracts from several medicinal plants have been screened for their ability to interfere with the tf nf-kb, ap- and creb, which are induced by p.aeruginosa, and some of them, such as emblica officinalis (eo), aegle marmelos (am), polyalthia longifolia (pl), have been shown to inhibit tf/dna interactions, opening the possibility of potential applications to downregulate expression of pro-inflammatory genes. extracts from eo, am, pl were tested in ib - cf bronchial cells exposed to the p.aeruginosa laboratory strain pao . eo, am and pl strongly inhibited the pao -dependent transcription of il- in ib - cells. pyrogallol, one active principle of eo, was tested in ib - cells, where it inhibited the transcription of the neutrophil chemokines il- , gro-alpha and gamma, of the intercellular adhesion molecule icam- and the pro-inflammatory cytokine interleukin , similarly to the whole eo extract, whereas a second active principle from eo, namely hydroxy-isoquinoline, had no effect. similar results were obtained with eo and pyrogallol in the monocyte-derived macrophage cell line thp- exposed to pao . in conclusion, extracts from plants of the traditional medicine can inhibit expression of pro-inflammatory genes and screening active principles purified from medicinal plants could result useful to identify safe and innovative pharmaceutical molecules to control lung inflammation in the lung of cf patients. supported by italian cystic fibrosis research foundation and by fondazione cariverona -bando -malattie rare e della povertà. massive infiltrates of neutrophils in the mucosal wall and lumen of the conductive airways of cf patients contribute to the progressive lung func-tion decline by releasing different proteases responsible for the progressive airway tissue damage. bacterial products and pathogens themselves within the mucopurulent material of the airway surface fluid induce the activation of transcription factors such as nf-kappab, ap- , sp , nf-il , nf-at, elk- , creb resulting in expression of chemo/cytokine genes driving the recruitment of leukocytes inside the bronchial lumen. to find new treatment options focused on the reduction of neutrophil chemotaxis, without abolishing the immune response against pathogens, we are exploring the transcription factor (tf) "decoy" strategy, in which oligodeoxynucleotides (odn) mimicking the consensus sequences for the tfs proteins identified in the promoters of different chemo/cytokines are delivered inside the cell in order to interfere with gene transcription. cf bronchial epithelial cells ib - have been exposed to the (i)p.aeruginosa(/i) strain pao . a clear pao -dependent activation of tfs such as nf-κb, sp , ap- , nf-at, nf-il was confirmed by electrophoretic mobility shift assay (emsa). in parallel, transcription of genes involved in innate immune response has been quantified by real-time rt pcr. transcription factor "decoy" odns directed against the consensus sequences identified in the promoters of different genes have been designed and validated by testing their interference in tf protein/dna binding assays. transfection of ib - cells with hiv- ltr and igk chain nf-κb odn "decoys" complexed with lipofectamine, performed hrs before challenge with pao , was shown previously to inhibit strongly pao -dependent transcription of il- but not gro, il- β, il- and icam- . therefore other tf "decoy" odns have been also tested: a) odn for nf-κb from il- promoter inhibited il- , gro-gamma and il- by %, b) odn for sp from hiv- genome inhibited il- by %, c) odn for ap- from il- promoter inhibited both il- and gro-gamma by %. a tf "decoy" molecule designed as peptide-dna chimera mimicking the consensus sequence of hiv ltr nf-κb strongly and selectively inhibited il- transcription. in conclusion, transcription of chemo/cytokines induced by (i)p.aeruginosa(/i) in cf bronchial epithelial cells (i)in vitro(/i) can be inhibited with different efficiency and selectivity by tf "decoy" molecules. these results provide useful hints for a gene-targeted anti-inflammatory approach and add further information on the regulation of expression of pro-inflammatory genes induced by (i)p.aeruginosa(/i) in bronchial epithelial cells. supported by italian cystic fibrosis research foundation and by fondazione cariverona -bando -malattie rare e della povertà. background: cf is characterized by hypersecretion of the pro-inflammatory cytokine il- from airway epithelial cells. however the mechanism by which il- gene expression is dysregulated in cf is not known. the expression of cytokine and chemokine genes is known to be regulated at multiple mechanistic steps including transcription, mrna decay, translation, and various post-translational steps. sequence-specific mrna degradation is now recognized to be an important site controlling the expression of several chemokine mrnas. this selective behavior is conferred by cisacting elements in the mrna composed of au-rich sequences (ares). the regulatory function of ares is thought to be mediated via rna-binding proteins that specifically recognize the are motifs. the steps required for mrna decay is comprised of deadenylation, decapping and body decay. deadenylation or removal of the poly (a) tail by poly(a)-specific ribonuclease (parn) appears to be the first and perhaps the rate-limiting step. this is accompanied by decapping or enzymatic removal of the ' methylate guanosine cap. subsequently, exonuclease activities in the ' to ' or ' to ' direction predominate to degrade the remaining mrna. hypothesis: we have hypothesized that defects in transcription as well as modulation of the post-transcriptional stability of il- mrna might contribute to hyper-production of il- protein in the cystic fibrosis. methods: il- mrna stability was assessed in cystic fibrosis ib - lung epithelial cells and in aav-cftr-repaired ib - /s cells, by measuring residual il- mrna levels at various intervals of time after addition of actinomycin-d to the culture. protein s extracts, prepared from the cf cell line as well as the cftr-corrected cell line, were analyzed by western blot. the expression levels of the various factors known to participate in are-mediated mrna decay, including ttp (an are-binding protein), parn and the exosome were compared in the two sets of s extracts. results: we find that the levels of il- mrna in the cf cells are greater than the levels found in the cftr-repaired cells. in addition, we found that the rate of decay of il- mrna in cf cells was significantly less than that in the repaired cell line. the levels of ttp, parn and exosome proteins were significantly reduced in the cf cells compared to the cftrrepaired controls. however, expression of ttp in ib - cells causes significant destabilization of il- mrna. conclusion: we conclude that the high levels of il- protein expression in cf lung epithelial cells can be partly due to enhanced stability of the il- mrna. consistently, the actual levels of il- mrna are higher in the cf cells compared to controls. understanding the mechanism by which ttp promotes enhanced destabilization of il- mrna in cf cells may be important for developing novel therapeutic targets to alleviate the pulmonary pathophysiology of this disease manifested by hyper-secretion of il- protein. understanding the mechanism of airway remodeling could lead to the identification of novel therapeutic targets for the prevention of irreversible lung damage in cf. we have investigated transcriptional responses to epithelial injury in a mouse model of cf that was developed in our laboratory (f del, cftr tm eur fvb backcross f ). to induce transient epithelial lung injury, homozygous normal and mutant age matched littermates were treated in parallel with naphthalene ( mg/kg ip) or carrier (corn oil) as control (n= x ). naphthalene causes an almost complete selective ablation of clara cells overnight, followed by migration of resistant ciliated cells and rapid proliferation of undifferentiated progenitor cells. two and seven days after treatment, lungs were collected for histological and transcriptome analysis. based on a previous affymetrix microarray analysis (n= ), a quantitative pcr array was designed containing genes differentially expressed after naphthalene injury. as expected, naphthalene injury results in a transient increase of cell proliferation markers (mki , cdc , cdc a etc), and a strong ( %) reduction of clara cell markers (cc /scgb a ; claudin /cldn ; cyp f ). in addition, we have now identified twenty-five genes that are increased (p< . ) two-to fifty-fold after naphthalene injury in both normal and mutant mice. this includes known and novel markers of epithelial tissue injury such as timp , retlna, mmp , serpin n, and lipocalin. in particular, a group of egf receptor agonists is strongly induced: amphiregulin (areg, fold), epiregulin (ereg fold) and heparin binding egf ( fold). amphiregulin mrna can be detected by in situ hybridization only in airway epithelial cells after injury. these egfr agonists are involved in stimulating epithelial repair, but can also activate cells in the underlying mesenchyma. indeed, we observe a substantial increase of major extracellular matrix mrna's two days after injury (co a : -fold, col a : -fold, elastin: -fold). whereas expression levels were substantially reduced in normal animals seven days after injury, in cf mutant animals two to three fold higher levels were observed for these three genes at day seven (p< . ). conclusions. we have designed and validated a gene array that can be used to study distal lung injury and repair. egfr agonists produced by epithelial cells are the most prominent growth factors after injury, involved in both epithelial repair and extracellular matrix production by mesenchymal cells. ecm production in cf mouse lung is sustained compared to normal mice, suggesting an inherent tendency towards fibrosis in cf lungs. our data suggest that the mesenchymal egf receptor and regulation of its agonists are important future targets of novel therapeutic strategies. supported by eec th fw projects eurocarecf, improved precision the mucosa of the proximal airways defends itself and the lower airways from inhaled irritants, allergens, and microbial and viral infections by several mechanisms. sensory nerves monitor the luminal microenvironment and trigger reflexes in the central nervous system (cns) that alter breathing, induce cough and stimulate mucus secretion when challenged with noxious stimuli. sensory nerves also release the tachykinins substance p (sp), neurokinin a and calcitonin gene-related peptide through axon reflexes in neighboring tissues, and these locally released tachykinins stimulate mucus secretion by binding to neurokinin receptors on submucosal glands. recently we reported that local fluid secretory responses to noxious stimuli are dependent on the clchannel cftr in mouse airway submucosal glands and are defective in glands from cftr knockout mice (ianowski et al., j physiol, , , ) . we have now tested the effects of sp directly and examined the possible role of cftr in mediating these responses using tracheas from congenic wild-type and cftr knockout mice (cftr m unc /cftr m unc ) that had been bred onto c bl/ j or balb/c genetic backgrounds. we compared single gland secretion rates using optical methods as described previously. the cftr genotype of each mouse was assessed by using pcr to amplify genomic dna from tail clippings obtained at age days. after the cftr knockout mice were weaned, intestinal obstruction was minimized by supplementing their water with peglite. capsaicinoids (chili pepper oil) increased fluid secretion from glands of wild-type mice from ± pl/min (n= tracheas, glands) to ± pl/min (n= tracheas, glands, anova p< . , tukey-kramer multiple comparison test p< . ). this response was abolished by exposing the basolateral surface of the tracheas to l- , ( µmol/l), a known sp (nk- ) receptor antagonist ( ± pl/min, not different from control, tukey-kramer multiple comparison test p> . ). secretion was stimulated from ± pl/min to ± pl/min (n= tracheas, glands, student's t-test p< . ) by the direct application of sp, and this response was strongly inhibited by pre-incubation with the cftr inhibitor cftrinh (saturating concentration, nominal µmol/l; n= tracheas, glands). finally, submucosal glands from cftr knockout mice failed to secrete when exposed to sp ( µmol/l) whereas wild-type littermates were responsive. these results indicate that sp mediates local responses to capsaicinoids through a cftr-dependent mechanism. loss of this local regulation in cf may contribute to the susceptibility of cf airways. support: canadian cf foundation, canadian institutes of health research, nih (dk ), and the cff (usa). chronically infected/inflamed cf human bronchial epithelia (hbe), or normal hbe exposed to supernatant from mucopurulent material (smm) from human cf airways, exhibit expansion of the endoplasmic reticulum (er) ca + stores and amplification of ca + -mediated inflammatory responses. we have shown that infection/inflammation of hbe triggers an unfolded protein response (upr) coupled to mrna splicing of x-box binding protein- (xbp- ). spliced xbp- (xbp- s) is a transcription factor that promotes er expansion to augment the protein folding capacity during increased protein synthesis. because we have shown that hbe inflammation couples to increases in protein synthesis, we hypothesized that hbe infection/inflammation-induced er ca + store expansion is mediated by xbp- s. to test this hypothesis, we constructed four retrovirally-tranduced stable hbe o-cell lines containing empty vector (ev), xbp- unspliced (xbp- u), xbp- s, or dominant negative xbp- (dn-xbp- ). cells were grown to confluence under an air-liquid interface and er ca + store expansion and il- secretion studied in the absence or following hr mucosal pbs or smm exposure. consistent with our hypothesis, expression of xbp- s in the absence of stimulation induced er expansion and increased mucosal utp-sensitive er ca + stores ( to confirm that these effects were mediated by upr activation, cells were transfected with a upr response element luciferase reporter plasmid, and luciferase activity was measured in the absence or presence of the upr inducer tunicamycin (tm). cells expressing xbp- s exhibited higher baseline luciferase activity than cells expressing ev or xbp- u, whereas luciferase activity was decreased in the dn-xbp- expressing cells. furthermore, while tm increased luciferase activity in cultures expressing ev or xbp- u, its effect was blocked in dn-xbp- expressing cells. these findings suggest that xbp- s is the major trigger of er ca + store expansion, which mediates the amplified ca + -dependent inflammatory response in infected/inflamed airway epithelia. although it remains to be established whether upr-dependent xbp- s is a beneficial or a maladaptive response in infected/inflamed airways, therapies aimed at manipulating this upr pathway may be beneficial for patients with chronic inflammatory lung diseases. luminal exposure of well-differentiated normal human bronchial epithelia (hbe) to supernatant from mucopurulent material (smm) from human cf airways increases the secretion of inflammatory mediators and triggers an unfolded protein response (upr) mediated by the mrna splicing of the x-box binding protein- (xbp- ). spliced xbp- (xbp- s) is a transcription factor that expands the endoplasmic reticulum (er) and protein secretory pathway during er stress induced by accumulation of unfolded proteins due to increases in protein synthesis. our previous studies in primary cultures of hbe and hbe o-cells stably expressing empty vector, inactive unspliced xbp- (xbp- u), xbp- s or a dominant negative xbp- (dn-xbp- ) suggest that induction of xbp- s by smm exposure promotes er ca + store expansion, which mediates a ca + -dependent hyperinflammatory response. the present studies were designed to determine if pseudomonas aeruginosa pak strain (p.a.) would reproduce the effects of smm in both in vitro and, importantly, in vivo models. for in vitro studies, hbe were exposed to broth (tsb) or to a % p.a. extract for hr, and inflammation, xbp- s, and er ca + store expansion investigated by il- secretion, southern blots and calreticulin expression, respectively. similar to smm, p.a. induced a . fold increase in il- secretion coupled to a fold increase in xbp- s and larger er ca + stores as compared with tsbexposed hbe. these data demonstrate the link between p.a. infection, upr activation and xbp- s in vitro. we next tested the relevance of these findings in vivo. wild-type mice airways were challenged with pbs or cfu of p.a., and airway epithelial er density was assessed by calreticulin staining hr later. in comparison with pbs challenges, p.a.-challenged mice exhibited airway epithelial er expansion, which correlated with the degree of airway inflammation, based on the presence of inflammatory cells. to test whether this p.a.-induced er expansion was linked to xbp- s, we utilized "er stress activated indicator" (erai) mice expressing a fusion protein consisting of xbp- u and the fluorescent protein venus. in erai mice, upr activation-dependent xbp- splicing leads to venus expression; hence, venus fluorescence is an index of xbp- s. consistent with our hypothesis that p.a. infection-triggered inflammation would induce upr-dependent xbp- s, venus fluorescence, after hr challenge with cfu of p.a., was increased in inflamed as compared with non-inflamed airways. these findings suggest that ) airway epithelia respond to bacterial infection-induced inflammation by up-regulating the er ca + stores and ) activation of the xbp- s pathway by bacterial infection may be relevant to airway inflammatory responses in vivo. funded by the cff. we have shown that luminal exposure of well-differentiated primary cultures of normal human bronchial epithelia (hbe) to supernatant from mucopurulent material (smm) from human cf airways increases total cellular protein synthesis, which reflects the increased secretion of inflammatory factors induced by the infectious and inflammatory process. in the present studies, we first investigated whether these hbe responses to smm were linked with an increased metabolic rate by measuring lactate accumulation into the serosal media. smm increased lactate production, and this effect was maximal within hrs ( . + . vs. . + . , and . + . vs. . + . mmol/l in and hr pbs vs. smm, respectively; n= - ), suggesting that the increase in protein synthesis couples to a hyper-metabolic state in infected/inflamed hbe. in agreement with this notion, or hr smm, as compared with pbs exposure, induced the expression of genes associated with amino acid transport and metabolism (n= ). in addition, or hr smm exposure up-regulated genes involved in oxidative stress (n= ). we hypothesized that these hbe responses were linked to an unfolded protein response (upr) mediated by activation of the pkr-like er kinase/pancreatic eif α kinase (perk)-induced activating transcription factor (atf ), since this pathway has been shown to confer protection against amino acid loss and oxidative stress in other cells. we first tested whether smm induced activation of perk/atf in hbe by performing western blot analyzes of the components of this pathway. twenty-four hr smm exposure induced perk activation, as indexed by phosphorylation of perk, in comparison with pbs-exposed hbe. on the other hand, total perk protein levels were unchanged in smm-treated hbe. phosphorylation of eif α, the downstream effector of perk, and increased atf protein levels, which depend on the phosphorylated status of eif α, provided additional evidence that the perk/atf pathway was activated by smm. these data are consistent with the hypothesis that induction of atf is triggered by upr activation resulting from increased synthesis of inflammatory factors. we next utilized rna microarrays to test whether atf target genes were induced by smm. six or hr smm exposure induced atf target genes (e.g., ero , an oxido-reductase that provides protection against the accumulation of endogenous peroxides during er stress; stanniocalcin , whose expression is associated with anti-apoptotic functions; and heme oxygenase ). these findings suggest that ) activation of the upr-dependent atf pathway is a compensatory component of the airway epithelial adaptive response to luminal infection/inflammation, and ) activation of the atf pathway protects against inflammation-induced amino acid loss and oxidative stress by up-regulating genes involved in amino acid transport/metabolism and oxidative stress responses. unraveling the functions of atf should help determine if therapies targeted to manipulate pathway activity would be likely to improve lung function in patients with cf or other chronic inflammatory airway diseases. funded by the cff. miller, t.j.; perez, a.; qian, y.; davis, p. pediatrics, case western reserve university, cleveland, oh, usa fxyd is a cell surface protein originally identified in a screen for molecular markers of tumorigenesis. increased fxyd expression was found in tumors from stomach, thyroid, colon, pancreatic, breast and lung cancers and correlated with down-regulation of e-cadherin and poor patient prognosis. recent studies have shown that overexpression of fxyd promotes cell motility, decreases cell-cell attachment and increases tumor metastasis. fxyd , also known as dysadherin, is a member of small family of proteins known to regulate the na,k-atpase. we now report that fxyd is upregulated in cystic fibrosis (cf) airway epithelia and modulates wound healing. we show by immunohistochemistry and immunoblot analyses that fxyd is increased in the lungs of s x cf mice, and demonstrate an almost -fold increase in fxyd expression in the nasal epithelia of cf mice compared to wild-type littermates (p< . ). furthermore, we show that fxyd is upregulated in nasal scrapings from human cf patients compared to controls (p< . ). immunofluorescence data show that flag-tagged fxyd co-localizes with the na,k-atpase in epithelial cells, suggesting that fxyd , similar to other members of the fxyd family, may regulate na,k-atpase function. it has previously been shown that expression and localization of the na,k-atpase is required for efficient polarization and suppression of cell motility in epithelial cells. the recurrent remodeling of pulmonary epithelium as a result of bacterial infection in cf requires that airway epithelial cells polarize and migrate to wound sites in order to maintain lung integrity. thus we hypothesized that fxyd may be involved in wound healing after infection. laser-capture microdissection and microarray analysis of murine lung epithelia after hours treatment with p. aeruginosa indicated a significant, -fold increase in expression of fxyd that was confirmed by immunoblot analysis. others have shown that fxyd may mediate expression of mcp- , a critical determinant in monocyte recruitment, through activation of the nf-kb pathway. treatment of human tracheal epithelial (hte) cells with a cftr inhibitor ( ) confirmed that loss of cftr function correlated with increased fxyd expression by quantitative rt-pcr (p< . ), an effect that was abrogated with treatment of pdtc, an inhibitor of nf-kb (p< . ). we speculated that fxyd -induced increases in cell motility may be due in part to phosphorylation at serine . in a murine airway epithelial cell wound healing model, serine to alanine (s a) mutations at serine inhibited wound healing compared to wild type fxyd overexpression, whereas aspartic acid (s d) mutations increased wound healing (p< . ). immunoblot and immunofluorescence analyses of these mutants suggest phosphorylation at ser regulates membrane localization. we conclude that fxyd is increased in cystic fibrosis epithelia due to increased inflammatory mediators and suggest that fxyd may modulate airway epithelia wound healing after infection with p. aeruginosa through phosphorylation at ser . the inflammatory response to bacterial infection in the cf airway is exaggerated compared to normal, leading to the accumulation of millions of necrotic neutrophils. extracellular neutrophil elastase (ne) activity in the cf airway not only compromises innate defences by cleaving opsonins and reducing ciliary activity, but amplifies the inflammatory response by stimulating expression of the neutrophil chemoattractant il- and increases mucus production, in addition to degrading the tissue matrix leading to fatal bronchiectasis. ne therefore represents an important target for the development of new therapies. however, this strategy requires consideration of the normal physiological function of this enzyme, since previous studies in knock-out mice indicated an essential role for ne in cell migration, bacterial phagocytosis and killing. our approach was to use the intracellular neutrophil elastase inhibitor gw a to 'knock-out' ne activity in neutrophils in normal human blood and test the function of isolated cells in chemotaxis, bacterial phagocytosis and killing assays. whole normal human blood was incubated with gw a, or pbs control, for h at °c. neutrophils were isolated by sedimentation of red blood cells (rbc) on dextran , purification on lymphoprep and hypotonic lysis to remove contaminating rbc. chemotaxis towards il- was measured using a modified micro-boyden chamber. phagocytosis was assayed by the depletion of staphylococcus aureus (sa), pseudomonas aeruginosa (pa) and e. coli (ec) in supernatants following culture in a : ratio with pmn for h at °c. supernatants were diluted and remaining organisms were plated on agar and grown overnight to count viable colonies. bacterial killing was assayed by incubating bacteria and cells in a : ratio for minutes to allow phagocytosis, washing off remaining organisms, and incubating for h at °c. neutrophils were lysed with water and lysates plated on agar to test for bacterial growth overnight. results; gw a inhibited intracellular ne dose-dependently and at µm . ± . % ne activity remained (n= ). there was no significant effect of µm gw a on neutrophil chemotaxis, bacterial phagocytosis or killing of any organism compared to pbs-treated controls. phagocytosis data is shown in the table. thus, in the absence of ne activity, human neutrophils remain wellequipped with other defence molecules including myeloperoxidase and defensins to successfully maintain the role of the neutrophil in innate immunity. however, mouse neutrophils which lack defensins require ne activity for optimal intracellular bacterial killing, and mice are not a perfect model for studies of human infection. the development of novel inhibitors of ne to treat lung disease in cf therefore remains an important goal. supported by the cf trust of great britain. background: sphingolipid signalling may differ between individuals with cf and healthy controls. the response to bacterial inflammation is different, and uptake and inactivation of sphingosine- -phosphate, an intracel-lular pro-inflammatory mediator, is reduced in cf cells. it may therefore continue to act on g-protein coupled receptors in the plasma membrane. (boujaoude et al, j biol chem ) . furthermore, ceramide originating from basolateral sphingomyelin hinders augmentation of cftr-mediated anion conductance across the apical membrane, resulting in reduction of transepithelial airway anion secretion (ito et al, bbrc ) . aim of study: to determine if there is a difference in the levels of alkaline, neutral or acid sphingomyelinase (smase), or in the levels of neutral or acid ceramidase, in the intestinal or bronchial mucosa and some other tissues, between wildtype, homozygous (+/+) and heterozygous (+/-) delta-f cftr mice. methods: enzyme activities (duan and nilsson meth enzymol ) were determined in intestine (and content) divided into four regions, liver, lungs, kidney and spleen from deltaf -cftr mice (+/+) and controls (wildtype, +/-). results: there was an increased amount of neutral ceramidase in spleens from deltaf -cftr mice (+/+) in comparison to control mice (p= . ). no other significant differences were seen. conclusion: delta-f mutation did not influence the levels of alkaline smase and neutral ceramidase acting as ectoenzymes, or the levels of intracellular smases and ceramidases, which may all generate bioactive sphingolipid metabolites in intestine and lungs. the implications of the increased level of neutral ceramidase in spleen are not known. in children with cystic fibrosis (cf) there is a clear correlation between the development of chronic p aeruginosa infection and acceleration in the decline of lung function. when chronically present, p aeruginosa takes on a mucoid phenotype and is impossible to eradicate. prior to this, when colonisation is intermittent, it is possible to eradicate it with aggressive antibiotic regimes. we sought to examine the degree of inflammation and innate defence status in the lungs of children with cystic fibrosis in various stages of colonisation by looking at a range of proteases, innate defence proteins and markers of inflammation in broncho alveolar lavage (bal). children with cf were allocated to one of three groups in relation to p aeruginosa infection; chronically colonised, intermittently colonised and non-colonised, on the basis of the leeds criteria. bal was collected as per ers guidelines as part of each patient's routine clinical care. bal was collected from control patients undergoing elective non-pulmonary surgical procedures. differential cell counts in bal were performed manually. secretory leukocyte protease inhibitor (slpi), elafin, alpha- antitrypsin (a at) and lactoferrin concentrations were measured by elisa. neutrophil elastase activity and cathepsin activity were assayed by colorometric activity assays. fifty two patients were included in the study ranging in age from months to years ( chronic, intermittent, non colonised and controls). neutrophil counts, neutrophil elastase activity and cathepsin activity were markedly increased in children chronically colonised with p aeruginosa compared to those in the intermittent and non colonised groups. in contrast, levels of the antiproteases slpi and a at and the antimicrobial peptides elafin and lactoferrin were highest in the control group and decreased as colonisation progressed, with levels in the chronically colonised group markedly lower than those with intermittent colonisation. this study demonstrates that in children with chronic p aeruginosa colonisation, there is a marked decrease in antiproteases and antimicrobial factors and a marked increase in protease activity and neutrophil influx in comparison with those who are non-colonised or intermittently colonised. these findings underline the importance of careful microbiological surveillance and early aggressive treatment of p aeruginosa infection in children with cf in order to avoid chronic colonisation. background : abnormal bronchial angiogenesis is responsible for hemoptysis in cystic fibrosis (cf). expression of vegf-a in airway epithelium induces bronchial angiogenesis in animal models. we have recently found that vegf-a and egf receptors (egfr) are increased in the airway epithelium of subjects with advanced cf lung disease. aims: to examine the effects of pa bacterial products and egfr inhibition on vegf synthesis in airway epithelium. methods: culture of non cf (nci-h ) and cf (cfte o-) human airway epithelial cell lines. stimulation with pa lipopolysaccharide (lps). assessment of vegf mrna and protein by rt-pcr and elisa. use of chemical inhibitors, blocking antibodies and sirna. results: pa lps increased vegf gene expression and protein production time-and dose-dependently in both cells lines. using chemical inhibitors, we show that egfr and erk / activation are required for lpsinduced vegf production. using blocking antibodies to egfr and its ligands, we show that tgf-alpha-dependent egfr activation mediates pa lps-induced vegf gene and protein synthesis. using pharmacological inhibitors (an ros scavenger and an nadph oxidase inhibitor) and using small interfering rna of dual oxidase (duox) and tnf-alpha converting enzyme (tace),we show that lps-induced vegf upregulation is dependent on duox -mediated ros release and tace activation. thus, pa products induce vegf synthesis in airway epithelium via a duox -ros-tace-tgf-alpha-egfr-erk / cascade. conclusions: these results describe a novel pathway by which bacterial products induce angiogenic signaling in cf and non cf airway epithelium. background: unlike bronchoalveolar lavage (bal), the airway mucosa has been under-investigated in cystic fibrosis (cf), despite the fact that irreversible airway wall changes (bronchiectasis) are a feature of endstage disease. cf is characterized by a neutrophil-dominated inflammation in bal, but little is known about the pattern of inflammation in the airway mucosa, especially in children with relatively early stage disease. we aimed to assess whether the pattern of inflammation seen in cf bal was also found in the airway mucosa in cf children. methods: to date, endobronchial biopsies and bal from children ( - years) with cf and control children ( - years) without lower respiratory disease have been assessed. bal cell differential was assessed on may-grünwald-stained cytospins. endobronchial biopsies were stained for neutrophils (neutrophil elastase, ne), t-(cd ) and b-(cd ) lymphocytes, eosinophils (eg ), and macrophages (cd ). area profile counts of immunopositive cells in subepithelial tissue were performed by investigators blinded to disease group. results: all cell types were increased in cf bal compared to controls. cf bal was characterized by an abundance of neutrophils ( x /ml vs. x /ml in controls, p< . ) with moderate numbers of lymphocytes ( x /ml vs. x /ml in controls, p< . ). in contrast, cf subepithelial tissue was characterized by a lymphocytic infiltrate ( cells/mm vs. cells/mm , p< . ) with only very few neutrophils ( cells/mm vs. cell/mm , p< . ). the lymphocytic infiltrate in cf consisted mainly of t lymphocytes ( %). eosinophil counts in subepithelial tissue did not differ between cf and controls. for all cell types, there was no correlation between counts in bal and counts in subepithelial tissue. conclusions: in contrast to the neutrophil-dominated inflammation in the airway lumen, cf is characterized by a lymphocytic inflammation in the airway mucosa. the lymphocytic infiltrate consists mainly of t lymphocytes, the pathophysiological function of which may be important and is being investigated in future work. support: ers long-term fellowship and swiss national foundation grant to nr the epithelium serves as a barrier to the penetration of foreign antigens, particles, and infectious agents across the airway. the integrity of this barrier is dependent, in part, upon the apical junctional complex consisting of the tight junction (tj) and the adherens junction. alterations in tj permeability have been linked to the pathogenesis of inflammatory bowel disease and this increased intestinal permeability may actually precede the onset of chronic inflammation. in cystic fibrosis (cf), the airway lumen is filled with high concentrations of inflammatory cells, bacteria, and inflammatory mediators. since tj barrier function can be significantly reduced by inflammatory mediators, we hypothesized that measures that enhance airway tj barrier function will decrease airway responses to the continuous presence of inflammatory mediators in the lumen. to test this hypothesis, we examined the relationship between lung inflammation and epithelial permeability in vivo using a lipopolysaccharide (lps) model of lung inflammation. pseudomonas aeruginosa lps was instilled intratracheally into the lungs of c bl/ mice which were then euthanized at , and hrs. lung inflammation was assessed by total cell counts using a hemacytometer and differential counts by wrights staining of cytospin preparations of the bronchoalveolar lavage fluid (balf). lps increased total cell counts and neutrophil concentrations that peaked at hours after lps administration, compared to saline controls. measurements of the proinflammatory murine cytokine kc in balf, by a cytokine antibody bead technique, showed an increase in murine kc at hr following lps administration, which correlated with the substantial increase in neutrophil concentration. changes in lung permeability with inflammation were assessed by elisa measurements of the levels of serum protein murine albumin in balf. correlating with changes in cellular inflammation and murine kc levels, albumin concentration peaked at hr after lps administration. this increase was subsequently resolved, consistent with the restoration of barrier function. an examination of frozen sections of lung from lps-treated animals showed a redistribution of the tight junction protein zo- consistent with the disruption of barrier function. since the p map kinase signaling pathway has been implicated in lps-induced airway inflammation, and an inhibitor of this kinase, sb has been shown to reduce this inflammation, the effect of this inhibitor on barrier function is being investigated. in initial studies, sb appears to reduce total and neutrophil cell counts by % in vivo. the effect of sb on murine albumin concentrations in balf and on tj protein localization is currently being evaluated. a reduction in these parameters will be used as indices of improved barrier function with sb . these studies will determine whether a reduction in lung inflammation correlates with a restoration of barrier function. the degree of protection provided by the p map kinase inhibitor sb could have important implications for inflammatory lung diseases such as cf. background: chronic pulmonary inflammation in cf is characterized by a robust neutrophil response associated with airway damage and failure to eliminate the pathogen, p. aeruginosa (pa). pa is a highly adaptable opportunist which quickly develops resistance to antimicrobials. thus, the development of specific immunotherapy targeting the neutrophil recruitment without ablating the host's immune response to infection or promoting pathogen resistance would be ideal. our group has identified il- as a prime target for the development of immunotherapy due to the central role that the il- /il- proinflammatory axis plays in neutrophil recruitment. however il- does not mediate the early neutrophil recruitment seen in response to infection. hypothesis: il- , acting synergistically with il- , is critical to early neutrophil recruitment during pulmonary pa infection. the primary effector cells are the il- -producing antigen presenting cells: alveolar macrophages (ams) and myeloid dendritic cells (dcs). methods: wt and il- -deficient mice were infected with pa at x cfu/ ul by intratracheal (it) inoculation for hours. bal inflammatory cell counts, and cytokines and chemokines were measured. am and dc cultures were infected for hours in vitro and supernatant cytokines/chemokines were measured by luminex and elisa; il- levels were measured by taqman. these studies were designed to elucidate the role of il- in the early neutrophil peak and define am-and dc-mediated cytokine/chemokine production. recombinant murine il- , il- , and il- + il- were instilled via it into wt and il- -deficient mice. bal inflammatory cell counts and cytokines/chemokines were measured at hours. these studies were designed to elucidate the role of il- and il- in the early neutrophil peak and define am-and dc-mediated cytokine and chemokine production. results: at hours post-infection, il- deficient mice had significantly lower percent neutrophils (p< . ) and lower mip α, kc, and il- (p< . ) in the bal. il- was undetectable. there was no significant difference in bacterial load that could account for these cytokine/chemokine differences. infected wt ams elaborated significantly more mip a, gm-csf, mcp- , il- , g-csf, ip- , kc and il- than the il- -deficient ams (p< . ) and the response was inoculum-dependent (p< . ). dcs elaborated no il- and exhibited il- -dependent differences in mip- α and mcp- production (p< . ). in vivo studies of il- and il- effect demonstrated a synergistic increase in bal neutrophil recruitment (p< . ) and cytokine and chemokine induction (p< . ). conclusion: the first wave of neutrophil recruitment seen during pa infection is il- -dependent and il- -independent. ams and dcs are critical to il- and il- indcution of this neutrophil recruitment. these studies identify il- as a key mediator of neutrophil recruitment in the early stages of infection as well as the proximate mediator in the il- /il- pro-inflammatory axis and suggest il- as a potential target for anti-inflammatory therapy in the treatment of pa pulmonary infection. supported by the cystic fibrosis foundation, american lung association and the nih the airways are under constant assault from air-borne pathogenic material. despite the intake of up to , bacteria per hour, the airways are sterile below the larynx in healthy individuals. the task of maintaining this sterility falls to the airway surface liquid layer (asl), the protective twophased system consisting of the viscoelastic mucus layer and the periciliary layer (pcl) through which cilia beat, sweeping the mucus layer away from the lungs. the mucus layer, which is responsible for trapping pathogenic material, is comprised of mucins (high molecular weight glycoproteins), cellular debris, dna, neurtifils, and more than other proteins. this chemically heterogeneous mixture forms a viscoelastic gel that is thick enough to trap pathogens of various sizes and surface chemistries, while not sticking to the underling cellular / cilia layer, allowing the transport of trapped pathogenic materials away from the lungs. the performance of this trapping / transportation system is defined by the rheological properties of the mucus layer and the force imparted on the mucus. therefore, understanding how mucins and other chemical components of the mucus layer interact with each other to form a successful mucus gel (i.e. one that is cleared from the airways) is crucial to understanding airway defense. here we present the results of physical and chemical composition studies of sputum samples collected from patients with chronic obstructive pulmonary disease (copd) and cystic fibrosis (cf). the rheological properties of each sample was assayed using parallel plate rheolometry, probing the materials non-linear viscoelastic properties such as viscosity, elasticity, and yield stress. the physical properties of the sample are then correlated to the sample's chemical properties such as percents solids (divided between salts, proteins, and mucins), as well as the relative concentrations of the key airway mucins muc b and muc ac. our results indicate that the physical properties of sputum are not well predicted from the total amount of biosolids in a given sputum sample, but by the relative concentrations of muc b and muc ac and the interactions of these molecules with themselves and the other proteins present. further, we establish that the heterogeneous physical properties within a given sputum sample correlate to differences in the muc b and muc ac concentrations. the gene modifier study (gms) was established as an effort to identify potential genetic modifiers of cystic fibrosis pulmonary disease and survival. during the course of this study over delta f homozygous cf patients classified as having mild lung disease, severe lung disease, or increased survival, have submitted both clinical data and blood samples for single nucleotide polymorphism (snp) analysis. the original snp analyses have shown a significant association between variants in the endothelin receptor a (ednra) gene, and cf survival, most markedly in female cf patients. sixteen additional snps within and around the ednra gene have now been genotyped, and have implicated the ' and ' untranslated regions of the gene as having the most significant association with pulmonary disease in females (p< . ), suggesting quantitative differences as a possible mechanism for the association with pulmonary phenotype. we are in the process of saturating the ' and ' regions of ednra with an additional snps to further delineate the genetic association. ednra binds endothelin- (et- ) in airway smooth muscle cells, causing increased cell proliferation, smooth muscle contraction, and stimulation of inflammatory molecules. because each of these effects is known to be deleterious to the cf lung, we hypothesize that the ednra variants found more commonly in "severe" cf females ("severe" alleles) are marking increased ednra expression compared to alleles found more commonly in "mild" cf females ("mild" allelels). because the genetic association was strongest in cf females, we used the matinspector software to analyze kb of ednra promoter sequence and found several putative binding sites for both estrogen and progesterone. we then used a brdu assay to measure cell proliferation after stimulation with both estrogen and progesterone. these experiments showed that the asm cells with the "severe" ' ednra genotype proliferated at levels approximately twice that of the asm cells with the "mild" ' ednra genotype following stimulation with either estrogen or progesterone. using a single base extension protocol and quantitative pcr on human airway smooth muscle cells, we were also able to compare ednra expression from the "severe" allele, and the "mild" allele. these comparisons of ednra expression demonstrate that expression levels appear to be approximately % higher from alleles found more frequently in the "severe" cf females. in addition, preliminary data suggest that stimulating the asm cells with estrogen increases ednra expression by approximately fold, and like the cell proliferation experiments, these increases are most pronounced in cell lines with the "severe" genotypes. these data suggest that the "severe" genotypes are marking alleles with increased expression, perhaps due to estrogen binding, that leads to increased et- functional effects that over time are deleterious to the cf lung. cf patients do present with variable spectra of lung disease, of which infections are most life-threatening. β-defensins have an antimicrobial activity against a broad spectrum of microorganisms and are chemotactic agents for cells of the adaptive immune system, and therefore assist in combating these infections. β-defensins - are part of a repeat region. this repeat region is polymorphic between individuals and therefore the dosage of these defensin genes/proteins varies. we developed a real time pcr assay to quantify the number of βdefensin repeats in this region. appropriate controls are needed for an accurate quantitative assay. therefore we made concatemeric constructs with copy of defb and a particular number of defb copies, which ranged from to copies. using these controls as standards, the number of defensin repeats could be accurately determined in dna samples. we then tested f del homozygous cf patients from belgian ( patients), czech ( patients) and south-italian ( patients) origin. the diploid number of repeats varied between and . for each patient group, a higher number of repeats was found in the group of patients with milder disease (fev > %) compared to the group of patients with more severe disease (fev < %) (student t test, p-values of . , . and . respectively). moreover, in our cohort of belgian cf patients, cf patients of years or older have a significant higher number of repeats than the cf patient group below (p-value . ). to evaluate this at the functional level, we cultured nasal epithelial cells from individuals with a low number of repeats (i.e. or repeats) and individuals with a high number of repeats (i.e. repeats). the cells where grown in air liquid interface cultures. after differentiation, the cells were stimulated with ng tnfα. in cells with a high number of repeats, defb expression, as measured by the extent of transcription, was strongly upregulated by tnfα. in cells with a low number of repeats, defb was not upregulated (p-value = . ). we also tested the antimicrobial activity of epithelial cells. we challenged epithelial cells from individuals ( repeats) with a laboratory strain (pa ) of pseudomonas aeruginosa and a clinical isolate ( - cfu), either in combination with tnfα or without tnfα. after h, surviving bacteria were counted by a plating out method. cells that were stimulated with tnfα h prior to the bacterial challenges were more bactericidal. the clinical strain was more vulnerable to the surface liquid than the laboratory strain. in epithelial cells from individuals having a low number of repeats, these effects were very variable from individual to individual. in summary, the β-defensin region is a modulator of cystic fibrosis lung disease. the pro-inflammatory response in cultured epithelium cells strongly correlates with the number of β-defensin repeats. cells with a higher number of repeats respond to tnfα treatment, which in turn results in a better antimicrobial activity of the surface liquid. rationale: studies of affected twins and siblings demonstrate that modifier genes are major contributors to variation in cystic fibrosis (cf) lung disease severity. we performed genome wide linkage analysis to identify regions likely to contain modifier genes affecting severity of cf lung disease. methods: individuals with cf from families were analyzed. pulmonary function data were collected from patient chart review and were supplemented with data from the us cystic fibrosis foundation patient registry. to minimize environmental variation, only data obtained while subjects were living with an affected twin or sibling were analyzed. the pulmonary phenotypes were defined using the best cf-specific percentile for fev (kulich, et al) within the last year of available pft data as a crosssectional measure (maxfev cf%) and using two longitudinal measures: the lifetime average cf-specific % for fev (avgfev cf%) and the estimated percent-predicted fev at age (estfev %pred@ yrs, schluchter, et al). longitudinal measures were derived from a minimum of years of pft data. short tandem repeat markers (strs) were typed in all affected individuals and their parents (marshfield genotyping center: markers or decode genotyping center: markers). two-point and multipoint linkage analyses were performed using sequential oligogenic linkage analysis routines (solar). results: patients represented the spectrum of lung disease severity, with maxfev cf%'s ranging from to , mean . ± . and avgfev cf% ranging from . to . , mean . ± . . the maxfev cf% was predictive of avgfev cf% (r= . , p< . ) for the individuals for whom both measures were available. the two longitudinal measures were also highly correlated (r= . , p< . ). linkage was found at chromosome for all three phenotype definitions. peak multipoint lod scores on chromosome occurred at cm for maxfev cf% and avgfev cf% (lod . and lod . , respectively) and at cm for estfev %pred@ yrs (lod . ). single point lod scores on chromosome peaked at marker aaat ( . for maxfev cf%, . for avgfev cf%, and . for est-fev %pred@ yrs). the region of linkage encompasses approximately megabases near the telomere of chromosome q. conclusions: chromosome appears to contain one or more genetic modifiers of cf lung disease severity. supported by the nhlbi, cff and genome canada through the ogi. cystic fibrosis-related diabetes (cfrd) is the most common extrapulmonary complication of cf and is an increasingly important contributor to morbidity and mortality as cf patients live longer. while pancreatic fibrosis and loss of exocrine and endocrine tissue are common in cf, - % of cf adults develop defects in insulin secretion and accumulation of islet amyloid polypeptide, features typical of type diabetes (t dm) in the general population. to test whether modifier genes play a role in cfrd, we compared concordance rates for cfrd in pairs of monozygous (mz) twins, sets of dizygous (dz) twins, and sets of or more siblings ( individuals with cf). criteria for defining cfrd included physician diagnosis, treatment with insulin/oral agent, and episodes of glucose ≥ mg/dl. mz twins were highly concordant for cfrd ( of pairs, %). the young age of dz twin recruits precluded analysis of this group in isolation ( of pairs were concordant). twelve of ( %) sibling pairs were concordant for cfrd. with heritability defined as: h = *(mz concordance -dz concordance), and including siblings as a proxy for dz twins, heritability is estimated as ~ . . the same results were obtained considering only same-sex dz twins and siblings, correcting for differences in age and duration of clinical follow-up, or restricting analysis to ∆f homozygotes. these data support a significant role for one or more modifier genes in development of cfrd. we then tested whether cfrd correlated with a strong family history of adult-onset diabetes (at least first-degree or second-degree relatives on the same side of the family). of those reporting family history of diabetes, of had cfrd, compared to of with no family history (or= . [ . - . ]; p= . ). this correlation persisted after adjusting for age, sex, and pancreatic insufficiency (or= . ; p= . ). thus, family history of diabetes correlated with increased risk of cfrd. we then tested whether variants in tcf l , a transcription factor in the wnt signaling pathway, that have been reproducibly associated with t dm in the general population were associated with cfrd in our study subjects. genotyping of four single nucleotide polymorphisms associated with t dm (rs , rs , rs , rs , here termed snpa-d) and transmission disequilibrium testing (tdt) of parent-parent-child trios revealed significant overtransmission for snpb ( : , p= . ) and snpc ( : , p= . ), and possible overtransmission for snpa ( : , p= . ) and snpd ( : , p= . ). in every case, the tcf l allele overtransmitted to patients with cfrd is the same allele that confers increased risk for t dm. furthermore, individuals with cfrd who were homozygous for risk alleles were diagnosed at a significantly earlier age (average . vs. . ; p= . ). these data support a key role for modifier genes in development of cf-related diabetes, and demonstrate that cfrd and type diabetes may share disease mechanisms such as alterations in wnt signaling. supported by nih dk , hd , dk and hl , and cf foundation grant cuttin p . cystic fibrosis (cf) phenotypes and survival are highly variable among df homozygous patients, pointing to the existence of modifier genes and/or environmental factors that contribute to this disease. studies to identify genetic modifiers of cf are being carried out using dna from homozygous df cf patients. important clinical features, such as severity of lung disease, liver disease and meconium ileus (mi) status, are well defined. tgfβ has been previously identified as a modifier of cf lung disease (drumm et al., nejm, ( ) : [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) , but it does not explain all of the genetic heterogeneity in this population. current evidence suggests that mucus is involved in the progression of cf, making the muc genes prime candidates as modifiers of lung disease and/or other phenotypes. methods: our approach to evaluate muc genes utilizes both variable number tandem repeat (vntr) polymorphisms and single nucleotide polymorphisms (snps). vntr polymorphisms in the muc and muc ac genes (n= and n= patients, respectively) were detected by southern blotting under conditions fully optimized to maximize the allele size resolution. to minimize gel to gel variation, a genomic dna mixture with the most common muc and muc ac alleles was used as internal markers. accuracy and reproducibility were evaluated by duplicating the southern on the critical dna samples for muc and muc ac genes (n= and n= patients, respectively). fifty snps in muc ac, muc b, muc , muc and muc genes are being tested using illumina technology in cf patients. results: preliminary analysis suggests there are significant differences between cf patients with "severe" and "mild" lung disease for both muc and muc ac allele distribution, which is mainly driven by the male population; exhaustive statistical data analysis still is underway. the vntr data also suggest significant association between the larger muc allele size and cf patients with mi. the ongoing vntr analysis will be complemented by muc gene snps being genotyped. conclusions: initial results indicate that we can reproducibly characterize muc and muc ac vntr alleles. additional characterization of muc and muc ac vntr alleles, coupled to snp data, will allow us to better define the significance of muc gene variations as modifiers of different cf phenotypes. supported by cff perezv g (jpv), cff knowle a (mk), cff r -cr (wko), nih rr , r hl , and cff drumm a . reporting for the gene modifier study group (mrk). we have previously reported that βenac transgenic mice, which overexpress the beta subunit of the amiloride sensitive sodium channel (scnn b) specifically in the airways, share common features with cf, including increased enac activity, reduced airway surface liquid, mucus accumulation and obstruction, inflammation, and death. interestingly, analysis of this model also suggested the existence of potential genetic modifiers of phenotype severity, and we speculated that identification of these modifiers would provide novel insights into disease phenotype. to establish a set of reagents that could be used to uncover genetic modifiers, we have bred βenac transgenic mice from two independent founder lines ( and , b :c background) onto several strains of inbred mice, including c bl/ n, c h/hen, balb/cj, fvb/j, and /svj. these studies revealed dramatic phenotypic differences as measured by survival ( to %) among strains and between lines. all lines thus far tested show ~ - fold increases in amiloride sensitive short-circuit current as measured by ussing chambers in the trachea. complete phenotypic characterization of c bl/ n line at backcross generation reveals high survival ( ± % in comparison to ± % of the mixed b :c background), yet the mice maintain the pulmonary features of the originally reported mice, including increased mucus plugging, mucous cell hyperplasia, neutrophilic and eosinophilic inflammation peaking at early timepoints ( days - weeks). lymphocytic nodules, which are not commonly seen in - weeks-old animals with mixed strain background, are a common feature in the c bl/ n congenic line. emphysema and early airway epithelial cell necrosis, two phenotypes initially not strongly associated with transgene expression, are also observed. line on balb/cj and c h/hen backgrounds has reduced survival compared to the c bl/ n background, and generation backcross lines are now being evaluated in these two strains for other phenotypic characteristics. analysis of backcross data from line , which has low survival on all genetic backgrounds tested to date, including c bl/ n, suggested that the transgene may have integrated onto a c h locus with a dominant negative effect on survival. genetic analysis using genome-wide snp genotyping revealed a region of chromosome linked to the transgene in line (lod score > . ). further analysis of this region is underway. in summary, backcrossing onto different genetic backgrounds is revealing genetic modifiers for phenotypes in the βenac overexpressing model. characterization of these phenotypic and genetic differences should provide clues about the mechanisms relevant to disease development. furthermore, inbred lines with variable phenotype will likely be an important reagent for the cf community as the utility of this model is evaluated in future studies. supported by nih (scor p hl ) and cff (mall go, oneal go). introduction: new york state screens newborns with immunoreactive trypsinogen levels within the top % of all infants, for common cf gene mutations. infants found to be heterozygote carriers are referred to a cf center to determine sweat chloride concentration. the proposed abnormal sweat chloride value for this group of newborns is ≥ mmol/l, ≥ standard deviation (sd) above the mean (farrell, ) . this retrospective study reports on the mean sweat chloride value + sd in cf heterozygote newborns who have been referred for evaluation to suny upstate medical university cystic fibrosis center, and regarding the genotype of these infants with abnormal sweat chloride levels. method: from october, to december, , infants were referred for positive cf screening, and of these ( %) were identified as heterozygote carriers by the screening program. at our center, these patients underwent pilocarpine iontophoresis, followed by collection of sweat (≥ µl) in macroduct ® coils. sweat testing was performed successfully in of the ( %) patients. the patients' age (mean ± sd) at the time of sweat testing was . ± . days. for infants with an initial sweat chloride level ≥ mmol/l, the sweat test was repeated within a week and a complete gene sequencing was requested (quest laboratory, ca). results: the sweat chloride level (mean ± sd) in newborns who were heterozygous for a cf mutation (excluding those who were found to have an additional mutation or deletion) was . ± . mmol/l (n = ). the mean + sd was mmol/l, which defined our minimal value for an abnormal test. eleven infants had sweat chloride values of - mmol/l (table) ; of them ( %) were subsequently diagnosed with cf by complete gene sequencing. four of the remaining patients ( %) had ∆f mutation coupled to the t variant on the opposite chromosome (sweat chloride levels, - mmol/l). conclusions: the reference range for sweat chloride in cf heterozygote infants appears to be significantly lower for some centers than previously reported. thus, each cf center should consider evaluating the cutoff values for the test at their site. moreover, the t polymorphism may account for sweat chloride elevations in heterozygote infants. method: we reviewed the charts of all patients with cf who were referred as a result of the new york state newborn screening program to the suny upstate medical university cf center from october, through april, . we included the patients who were identified by the state as heterozygote carriers of one of common cf gene mutations, and whose second mutation was identified only after complete cf gene sequencing. results: seven of the patients ( %) met inclusion criteria. six of the patients ( %) were compound heterozygous for a novel or a rare cf gene mutation. one patient was compound heterozygous for a large deletion in the cf gene. the genotype and clinical status of the patients are shown in table. all patients have been pancreatic sufficient to date. conclusions:the clinical effect of compound heterozygosity as a result of novel or rare cf gene mutations appears to be mild in early childhood. prior to newborn screening (with the exception of the patient with cftr deletion), these patients may not have been diagnosed with cf in the first few years of life. these patients may have subclinical airway inflammation and thus benefit from early treatment. patients with cf manifest symptoms in the pancreas, respiratory tract, male reproductive tract and sweat gland due to mutations in cftr. patients with non-classic cf have disease in a subset of these organ systems. most non-classic cf patients have two disease-causing mutations in cftr and at least one mutation permits residual cftr function. a subset of non-classic cf patients have only one cf-causing mutation after screening for a panel of common cf-causing mutations or following mutation scanning of the coding region of cftr. these patients present a diagnostic dilemma and a challenge for genetic counseling. we evaluated cf patients with only one cf-causing mutation identified after a screen of cftr mutations ( patients) or scanning of the coding region of cftr ( patients). nine of these patients, including one set of siblings, have non-classic cf with borderline or elevated sweat [cl -] plus lung disease (table) . one patient is pancreatic insufficient and has classic cf. many of these patients have features which are consistent with cftr dysfunction including a cf-like nasal potential difference (npd),p. aeruginosa infection, or congenital bilateral absence of the vas deferens (cbavd), suggesting that they have a second cftr mutation. mutations that are not detected by screening methods include insertions or deletions, mutations outside of the cftr coding region that affect rna splicing or expression, or mutations in the coding region of cftr that were missed by screening methods. to exclude the third possiblity, dna sequencing of the exons and flanking introns of cftr was performed. a second mutation was identified in the coding region of cftr in of the patients; had screening for known cftr mutations (genzyme), while the remaining had comprehensive scanning of the coding region of cftr by modified tgge (ambry). each of the mutations identified by sequencing has been previously described in patients with cf and is predicted to cause cftr dysfunction. these results reaffirm that patients with one cftr mutation who have biochemical and clinical features of cf are likely to have a second mutation in the coding region of cftr. thus, we suggest sequencing cftr in patients with only one mutation after mutation screening before employing other more complex genotyping methods (insertion/deletion or rna analysis). diagnostic criteria for confirming cf in symptomatic individuals includes two positive sweat tests or two known disease-causing cftr mutations. accurate sweat testing is performed at accredited cf centers, while cftr testing is available through national and specialty genetic labs. most labs offer analysis of a basic panel of cftr mutations as recommended by the american college of medical genetics (acmg), while specialty laboratories may offer an expanded panel or full sequencing/scanning. we report on one center's use of these methodologies to confirm the diagnosis of cf presenting in adulthood. the charts of patients diagnosed with cf at 〉 years of age were reviewed. were sweat tested. all were genotyped. results: twelve ( %) pts had two positive sweat tests (> mmol/l). an additional five ( %) had at least one borderline result ( - mmol/l). two pts had negative results ( - mmol/l). one refused sweat testing because dna analysis through the acmg panel had confirmed the diagnosis prior to initial consultation. genotyping results for the pts are summarized in the table below. rare mutations identifiable only through gene sequencing accounted for / alleles ( %) in our population. importantly, among this group, only one pt had a negative sweat test, a suggestive mmol/l. two pts with positive sweat tests and clinical symptoms failed to reveal any cf mutations after sequencing. we continue to follow pts without genotypic confirmation, based on their clinical presentation and sweat chloride levels, and have recommended additional evaluation, including nasal potential difference studies in our series of adult-diagnosed patients, sweat test results were positive, borderline, or suggestive in all cases tested. sweat testing costs $ -$ and results are ready in a day. genotyping costs $ -$ and takes several weeks. we acknowledge that circumstances may arise where reliable sweat testing is not conveniently available; but in our series, genotyping with the acmg panel would have diagnosed % of pts; using an expanded panel would have diagnosed %. genotyping is an important tool for genetic counseling, determination of eligibility for research studies, furthering knowledge of cftr dysfunction and cf pathophysiology, and for confirming a cf diagnosis after borderline or suggestive sweat test results. based on our findings and the dramatic difference in cost, we conclude that sweat testing should remain the first approach in the diagnostic workup of adult patients with a clinical presentation suggestive of cf. in colorado, infants with cf (non-meconium ileus) have been diagnosed with cf by a two tiered immunoreactive trypsinogen (irt/irt) based newborn screening approach. the irt/irt algorithm has been recently adopted by other screening programs with two mandatory screening tests. while most infants in colorado have been successfully identified, the program has had a missed case rate of approximately %. the more common approach to cf newborn screening is the irt/dna method in which the blood spot of infants with an initial elevated irt is tested for the most common cf mutations. the initial irt cutoff is lower in the irt/dna programs than in the irt/irt programs, resulting in a lower missed case rate. the considerable number of carriers identified through the irt/dna approach puts a significant burden on the genetic counseling community, as carriers are identified at a rate of / - / of positive irts we propose an irt/irt/dna newborn screening algorithm that will maximize sensitivity and specificity while minimizing the number of identified carriers. using new database technologies in the newborn screening lab we will be able to identify those infants with an elevated first irt (> ng/ml, approximately th percentile). all infants with an irt > ng/ml will have a repeat irt on their second state mandated blood-spot. if the second screen is also elevated (> ng/ml), the blood spot will be tested using a panel of mutations, including mutations specific to the hispanic community. infants with one or two cftr mutations will have a sweat test to confirm the diagnosis, or rule out cf. we compared the projected statistics of our current method irt/irt to the new irt/irt/dna method, and to irt/dna is presented in the table, based on , births per year in colorado. four infants ( . %) identified under the current irt/irt protocol would not have been identified by the mutation panel proposed in the new algorithm, out of genotyped, non-meconium ileus infants. three of these missed cases are hispanic. two would be identified using an extreme irt cutoff of the . th percentile ( ng/ml). the projected missed case rate would be < . % ( . - . %, % ci), using the irt/irt/dna algorithm, with carriers identified, maximizing both sensitivity and specificity. this algorithm may provide a better alternative to the irt/irt screening methods in states with two mandatory screening tests, and has advantages over both the irt/irt and irt/dna methods. newborn screening for cystic fibrosis (cf) is rapidly expanding and has been implemented in at least states. although most newborn screening assays are done using biochemical testing, many laboratories screening for cf include both biochemical and molecular testing of multiple alleles in the cystic fibrosis transmembrane conductance regulator gene. in response to the growing need for proficiency testing (pt) materials for molecular testing, the centers for disease control's newborn screening quality assurance program (nsqap) in collaboration with the university of wisconsin school of medicine and public health, the johns hopkins hospital, and case western reserve university, created a repository of dried-blood spot specimens with known mutations in the cftr gene to be used in a pt program. twenty milliliters of blood was collected voluntarily from adult donors with cf and sent to the nsqap laboratory. each specimen was adjusted to a hematocrit of % before being spotted onto whatman paper ( µl per spot), dried, and stored at - °c with desiccant. proficiency testing (pt) panels consisted of to blind-coded specimens from adult donors. the panels were sent quarterly to laboratories worldwide that test specimens for cf using molecular methods. laboratories were asked to report the genotype, method used, and the presumptive clinical assessment of each specimen. twenty-two laboratories participated during both quarters and , . the laboratories used different methods ranging from in-house assays to commercially available kits. most reporting laboratories tested the following alleles -∆f , g x, and g d. another twelve alleles were detected by most participants. nine more alleles were common among commercially available kits. laboratories were evaluated based on the clinical assessments. mutations that were not detected by a particular method were not evaluated. overall, the laboratories performed well. data compiled from both quarters demonstrated that there was incorrect clinical assessment and amplification failures. developing a pt program for dna-based testing is complicated by the number of methods and different alleles each laboratory chooses to test. though molecular testing for cf may be complex, pt monitors the laboratory's ability to test multiple alleles, including uncommon alleles, the limitations of various assays, and the different algorithms used for screening. the repository will also allow storage and access to rare specimens that may be useful for future research but are not readily available. who did could not reproduce. genetic counseling focused on a patient's parents, who were counseled about their recurrence risk at the time of the child's diagnosis. today, cf is a disease of adulthood. in , > % of cf patients in the us were > ; by it will be > %. together with advances in assisted reproductive technology (art), reproduction and recurrence risk are now important issues for adolescent and young adult cf patients. methods: a item questionnaire was developed from the results of prior semi-structured interviews with cf patients age - years. knowledge based questions (medical issues, inheritance, and reproductive options/risks) as well as communication patterns (preferred resources for learning about cf and preferred people with whom to talk about reproductive issues) were addressed. recruited from the uab cf clinic population, patients age - (mean ), male ( %), female ( %), completed the questionnaire. results: regarding autosomal recessive inheritance of cf, only % knew that two carriers have a % chance of having a child with cf, and % knew that two carriers have a % chance of having a child who is a carrier. however, % knew that two carriers could have a child who did not have cf, and % knew that two carriers could have a child who did not carry cf. on their own reproductive risks, % knew that a cf patient had a % chance of having a child with cf if their partner was not a carrier, but only % knew that all their children would be carriers even if their partner was not a carrier. in the scenario of a cf patient with a cf carrier partner, % knew that a child had a % chance of having cf, and % knew that a child had a % chance of being a cf carrier. most patients knew about their reproductive potential, as % responded that cf patients are able to have children. however, when asked about whether the chance for having children was different for males and females with cf, % answered that it was more difficult for males, % that it was more difficult for females, and % answered "not sure." while % reported that they knew that there were options for male cf patients who wanted to have children, only % knew of art. conclusions: despite widespread availability, the lack of knowledge of adolescents and young adults with cf about the genetics of their disease continues. furthermore, these patients are unaware of both modern technologies that could enable them to have biological children and the risk of those children having cf. this study illustrates the changing needs of patient education as medical knowledge progresses. cf patients would benefit from further genetic knowledge and counseling to enable them to make informed decisions about reproduction as they mature into adulthood. center at the university of minnesota is one of three sites in the state providing confirmatory testing and follow up services for newborns identified by screening. while cf nbs identifies children with cf, most of the infants with positive screening results are carriers. our goal is to provide genetic counseling to every cf nbs patient seen at our center, and we believe that a protocol incorporating genetic counseling in the initial care plan for infants both with and without cf is imperative. the literature has shown that families who obtain genetic counseling through the cf nbs process recall genetic information more easily and accurately and are more likely to have testing to determine parental carrier status. for parents of a child that is determined to be a cf carrier, it is especially important to find the optimal method and timing of genetic counseling as many of these families are from several hours away and therefore less likely to return to clinic. to provide genetic information and emotional support for the families of infants screening positive on cf nbs, our center has a genetic counselor who serves as the cf nbs coordinator and clinical contact for the family. this allows many opportunities to speak with the genetic counselor and ask questions, as well as learn about their child's diagnosis or carrier status and the subsequent carrier testing recommendations for the infant's parents and families. to assess the success and impact of the minnesota cystic fibrosis center's nbs follow-up program and the incorporation of genetic counseling, an anonymous questionnaire was developed for parents of infants who were seen at our center due to a positive cf nbs result. questionnaires were mailed to parents of all infants seen at our center for a sweat test and genetic counseling due to a positive cystic fibrosis newborn screening result. as of the last mailing, this totals families. two questionnaires were returned due to incorrect address and questionnaires were returned answered, indicating a response rate of % ( / ). responses overwhelmingly indicated that parents were satisfied with our center's algorithm for the cf nbs follow-up program and found the information and support provided through genetic counseling to be a useful and recommended portion of the program. as cf nbs continues, it is critical that we learn about the patient's experience with genetic counseling and the nbs program, as well as identify areas needing improvement. genetic counseling is vital to the comprehensive success of our center's program, and we will report on the responses gathered from the families identified through cf nbs this first year, as well as discuss the lessons learned from setting up such projects on a state-wide basis. background & aims: aberrant splicing and nonsense mediated decay (nmd) lead to dysfunctional mrnas by skipping exons and to a reduced number of functional mrna respectively. both mechanisms have a strong quantitative aspect and may determine whether a cf patient develops a classic or atypical disease phenotype. in order to approach these highly important questions we wanted to establish a new quantitative real-time pcr based assay which allows allele specific quantification on cdna level. using this assay we like to determine the exact proportions of the f del and non-f del cftr mrna in cf patients compound heterozygous for the f del mutation (for example in cf patients carrying the f del and a nonsense mutation such as the r x). material & methods: materials: we used genomic dna (gdna) and total rna (extracted from white blood cells and nasal epithelial cells respectively) from cf patients with compound heterozygosity for the f del mutation, homozygosity for f del mutation and from healthy individuals (controls). methods: the lasq (ligation dependent allele specific quantification) assay comprises reactions: . reverse transcription of cftr mrna into cdna using gene specific primers (all rna specific). . overnight hybridization ( - h) of the cftr cdna with either the f del specific or the wt specific oligo probe pair provided by jan schouten (mrc holland). . ligation of the hybridized oligo pairs using the ligase enzyme from mrc holland. . quantitative real-time pcr of the allele specific ligation products on the lightcycler (roche). results: in order to establish the lasq assay we first validated it using gdna instead of cdna as template. mixing experiments were performed to verify the accuracy of the assay. in brief, gdna (c= mg/l) of a f del homozygous and a f del compound heterozygous cf patient were mixed in such a manner that . , . amplification products of the f del and the wt allele (both bp long) were analyzed by gel electrophoresis (page) and direct sequencing (abi ) to control specificity. our results using gdna and cdna showed that there occurs unspecific hybridization/ ligation for both probe pairs. the proportion of unspecific amplification products varies between . and . and increases the lower the initial number of templates is. however, the specificity of this assay can be significantly improved by increasing the hybridization temperature and/or decreasing the ligation time. conclusion: although some minor limitations concerning allele specificity the lasq assay has been proven to be an accurate, reliable and reproducible method for allele specific quantification and may be applied for several important questions in cystic fibrosis such as the exact determination of the amount of nmd of cftr mrna containing a premature termination codon (ptc) or the allele specific determination of aberrant splicing of cftr mrna . background & aims: as clinical presentation varies significantly among cf patients with the same genotype, e.g. in the f del homozygous, it is evident that factors in addition to the cftr genotype such as modifier genes, are involved in determining disease severity. however, only one of several previously postulated modifier genes, the tgfβ gene, could recently be confirmed in a large association study. hence the identification of new modifier genes is a very important task in order to find new explanations for the heterogeneity of pulmonary disease in cf patients. we decided to search for new potential modifier genes applying a quantitative proteomic approach comparing the proteomes of a wild type ( hbe o-) and a f del homozygous bronchial epithelial cell line (cfbe o-). the main goal of this study is the identification of up or down regulated proteins in the cfbe cell line which may act as modifiers of cf disease. material & methods: materials: we used two bronchial epithelial cell lines, e.g. a wild type ( hbe o-) and a f del homozygous (cfbe o-) cell line which we obtained from dr. gruenert (california, usa). additionally, we also used nasal cells from f del homozygous cf patients obtained either from nasal brushings or nasal polyps. methods: proteome analysis was performed by making d-gels using high sensitive staining protocols (ruthenium and deep purple). quantitative analysis was accomplished applying the powerful dige (difference in gel electrophoresis) method. for each dige experiment we made gels whereby each cell line was twice labelled with cy and cy (= technical replicates). finally, identification of the protein spots was done by the use of a maldi-tof mass spectrometer. results: in a first step we established the proteomes of the two bronchial epithelial cell lines. we were able to optimize protein extraction and d gel electrophoresis in such a manner that the proteomes of the two cell-lines looked very similarly and the assignment of spots could easily be done. protein spots from both cell lines were analyzed using our mass spectrometry (maldi-tof ms) and allowed the identification of more than different proteins so far. in a next step we quantitatively compared the proteome of the two cell lines using the d-dige method leading to the identification of proteins which are down regulated and proteins which are up regulated at least twofold in the cfbe cell line. out of the aforementioned differently expressed proteins could already be identified because they were among the previously determined proteins. while glutathione s-transferase p and protein s -a (s calcium binding protein) were down regulated ( . and . -fold respectively), superoxide dismutase was up regulated ( . -fold) in the cfbe cell line. conclusion: comparative quantitative proteomics using the dige method is a promising tool in search of potential new modifier genes which may unravel one of the key problems in cf: the large heterogeneity of pulmonary disease in f del homozygous patients. background: cystic fibrosis is one of the most common autosomal recessive disorders among caucasians, and manifests a wide range of disease severity. although this range of disease expression can be attributed, in part, to specific mutations within the cftr gene, much of this variability has not been adequately explained. from the gene modifier study (gms-a multicenter study of , cf patients), ten genes were tested as potential modifiers of cf, and the tgfβ codon cc genotype was associated with lung disease severity. objective: to test whether the adverse codon cc genotype is associated with higher circulating levels of tgfβ , compared to the tt genotype in cf patients and healthy controls. if true, then a link will be established between genotype, disease severity, and circulating levels of tgfβ , and have implications for novel treatment of cf patients. methods: the study includes clinically stable cf patients and healthy control subjects equally distributed between the cc and tt genotypes. the cf patients enrolled are age and older, of both genders, and all ethnicities that fulfilled the standard diagnostic criteria for cf, using the genetic information from the gms. healthy controls are age and older, caucasian males and females, obtained from the environmental polymorphism registry (epr), a dna registry of , self-reported normal volunteers. we genotyped blood samples from the epr to define healthy control subjects for each of the cc and tt cohorts. subjects have a blood draw of ml. blood is divided into tubes and each tube is used to measure a different parameter: cbc with differential; tgfβ levels in platelet poor plasma by quantikine human tgfβ elisa kit; tgfβ levels in the buffy coatwhich includes platelets; and tgfβ mrna levels in lymphocytes from the buffy coat, using real-time pcr roche light cycler. cbc with differential is performed to quantitate lymphocytes and platelets in order to reference tgfβ protein and rna levels to the number of circulating blood cells. analyses will include graphical comparisons between the two groups, chi square analysis, student's two-sample t test, and one way analysis of variance (anova). results: pilot studies show that elisa tgfβ measurements are reproducible and mrna levels can be quantified. we have currently enrolled cc genotype and tt genotype of the cf subjects and cc genotype and tt genotype of the healthy controls. blood samples have been collected and processed. conclusion: genetic variants that predispose to more severe cf disease are potential targets for new therapies. we are testing the hypothesis that the adverse (codon ) cc genotype is likely to reflect increased transcription and/ or tgfβ protein synthesis/ secretion. if true, "anti-tgfβ " therapies could provide a novel therapy in cf. *reporting for the gene modifier study group. supported by cff knowle a , cff drumm a , gcrc rr , nih r hl . aim: centralized periodic evaluations of data from the screening laboratories and cf centers by afdphe (french association for screening and prevention of infant handicaps), were analysed to optimize the efficiency of the program. methods: the strategy combined d irt assay/dna analysis (kit elucigen cf- arms) /d -fail-safe irt. revised irt-cut off levels were decided in order to maintain the percentage of positive screens around a . % target. a questionnaire yearly sent to the cf centers collected the cf false negative cases. results: from to december, , cf cases were detected through nbs ( screened infants). the i period (p) (n= ); d -irt: µg/l and d -irt: µg/l showed a) . % infant above the d cut-off, generating an excess of costly dna tests b) % had a d -irt above the cut-off leading to a very high number of st (n= ) with an extremely low rate of cf (n= ). by increasing slightly both d /d irt cutoff levels ( µg/l, µg/l) during the ii p (n= ) a) the number of positive screens decreased to . % b) . % of infants with elevated d -irt had to be referred for st (n= ) with cf diagnosis. since the risk to had true cf remained very low among infants with no detected mutations, during the iii p (n= ), d -irt concerned only the ones with d -irt> µg/l and the percentage of infants requiring a st was reduced to . % (n= , cf). the incidence of cf detected during these p did not vary significantly ( / - / - / ). another point of concern was the false negative cohort; with a follow-up period over months, cf were detected on clinical symptoms ( . %) at a mean age of months. only were directly related to the modifications of the strategy. conclusion: centralisation of data made possible changes in the flow charts of the screening strategy to limit the number of false positive cases without significant alteration of the global performance of the program. there were three infants of mixed aa/caucasian origin diagnosed with cf, all had ∆ /genotype on screening. mutation data on the additional aa cf patients followed at the cf centers in new york was collected. six patients have not been genotyped. there were patients who were homozygous for ∆ , patients had only one mutation identified and were not found to have any cf mutations. ∆ ( %) and + g>a( %) were the most common mutations. there are five cf patients (including infants diagnosed by nbs) with mixed racial origin who are not included in this analysis. mutations results: the median (range) mbl plasma level was . ( . - . ) µg/ml in cf patients, compared to . ( . - . ) µg/ml in controls. mbl genotype frequencies were similar in patients and controls. lung function level was not correlated with mbl genotype or plasma level. the frequency of colonization with pseudomonas aeruginosa was % in mbldeficient (xa/o and o/o genotype) children with cf and % in mbl-sufficient (a/a and ya/o genotypes) children (p= . ). we found a trend of a decreased age of first onset of colonization with staphylococcus aureus, haemophilus influenza and pseudomonas aeruginosa in mbl-deficient cf patients (p= . , p= . , and p= . , respectively). conclusions: mbl-deficiency was associated with an increased frequency of pseudomonas aeruginosa colonization in children. mbl deficiency was not associated with lung function deterioration. in a larger cohort we hope to confirm that mbl deficiency influences age at onset of bacterial colonization in cf patients. acknowledgments although there is some evidence that cftr gene mutations may be associated with respiratory diseases, little is known about the relationship between cftr gene mutations and idiopathic bronchiectasis (ngiam et al. ) . we have recently showed that a rare allele (- g>a) in the minimal cftr promoter, previously reported in patients with idiopathic bronchiectasis ( using supershift assays, we demonstrated that these transcription factors bind cftr promoter in vitro. a functional analysis, by using co-transfection assays with expression vectors of each transcription factor in pulmonary epithelial cells, showed that nrf , irf significantly decrease cftr expression, whereas irf and sp increase it. in an attempt to further elucidate the mechanisms involving these factors in the cftr transcriptional regulation, for instance, to determine whether these factors could interact together in order to regulate cftr transcription, we started several experiments such as co-immnoprecipitation, multiple co-transfection and rnai. taken together, these data evidence that the variant - a in cftr promoter should be considered as an important risk factor in bronchiectasis pathogenesis. furthermore, we have identified a novel regulatory complex on the minimal cftr promoter, which will enlighten the understanding of the transcriptional regulation of the cftr gene. a better knowledge of cftr cis-and trans-acting elements will allow to consider new approaches to modulate and/or control more specifically cftr expression. this work is supported in part by the association vaincre la mucoviscidose. (nat genet, ) . a recent study reports strong genetic influences for mi (blackman et al., gastroenterology, ) , but it did not replicate the modifier locus on chr. q . inconsistency of results may reflect the variability of data reporting and different classifications of mi (i.e. surgically or medically treated). we tested the accuracy of reporting of mi on case report forms (crfs), as compared to primary source documents. methods: the crf for the gms has a checklist for past medical history. for example, we requested "yes" or "no" for mi (an obstruction of the terminal ileum at birth), but did not require source documents or identification of the type of treatment. to evaluate crf reporting of mi, we requested source documents for patients with reported mi, including a surgical or medical treatment report. if a written report from the time of birth was unavailable, a clinic note, detailing mi at birth, treatment, and evidence of a surgical, abdominal scar (if applicable), was required. verbal confirmation by the patient and evidence of a surgical scar was also accepted, if no written documentation was available. results: on crfs, of patients ( %) were initially reported to have mi. to date, source documentation has been obtained for patients, and of those ( %) have been confirmed to have mi ( % by written report, % by verbal report). of the with confirmed mi, ( %) had surgery, and ( %) had medical treatment. there were false reports of mi ( . %). mi could not be confirmed or refuted for patients ( . %) because of insufficient information (n= ) or confounding circumstances (n= ). to date, documentation for patients who were reported to have no mi has been obtained, and no false negatives have been found. additional documentation for mi from other sites is expected. conclusion: at least %, and perhaps as many as %, of the reports of mi on crfs were inaccurate. gene modifier studies must include rigorous documentation of mi to ensure an accurate correlation between phenotype and genotype. studies should also characterize different classifications (i.e. surgical vs. medical treatment) to assist the detection of gene modifiers of mi. reporting for the gene modifier study group (mrk); supported by cff knowle a , cff drumm a , nih r dk , nih r hl , and gcrc rr . any mutation that disrupts or diminishes the efficiency of the splicing process will have an impact on disease manifestation. in the cystic fibrosis (cf) transmembrane conductance regulator (cftr) gene more than , mutations were identified, most of them being disease-causing [ ] . among these,~ % are classified as missense and about % are classified as splicing mutations, given that they disrupt the consensus splice sites [ ] . however, the splicing mutation concept is evolving, as it nowadays also includes mutations other than just those within or close to the consensus splice sites, rendering prediction of mutation consequences a hard task. nevertheless, it is still very important for the clinical settings to determine the functional effect of gene mutations. our aim here was to study the effect of i v, a rare cftr missense mutation in nbd (exon ), directly in native tissues of a cf patient bearing f del in the other allele, so as to gain insight on how it influence the disease outcome. to look for a possible effect at the rna level, total rna was extracted from native nasal cells and colonic tissue, and cdna producd using random primers. rt-pcr amplification was performed in the region spanning exons - , being one of the primers fluorescently labelled. the products were analysed as described before [ ] . the i v( a>g) mutation which creates both a novel acceptor and a novel donor, was found here to cause alternative spliced cftr transcripts lacking the last nucleotides of exon , thus showing that only the novel donor is used in vivo. moreover, our data show that no normal (only alternatively spliced) transcripts result from this allele. we have also analysed the i v mutation at the protein level by producing a stable bhk cell line expressing the i v-cftr, after generating the respective mutant cdna construct by site-direct mutagenesis. protein expression and function was determined by immunoblot and iodide efflux assay, respectively. results show that i v-cftr protein is processed and functional. however, given the above-described absence of normally spliced mrna coding for this cftr variant, we have to conclude that this protein is not produced in vivo. indeed, since only the mrna coding for cftr lacking the last six aminoacids of exon was detected, we are currently characterizing in vitro the proprerties of this truncated protein. altogether, our data clearly demonstrate that the functional effect of this mutation is not due to the amino acid change but to abnormal splicing. in conclusion, characterization of the consequences of mutations in native affected tissues is important, not just because this provides unexpected information about the mechanisms underlying the basic defect but also for disease diagnosis and prognosis. (darrah et al, nacfc ) . the purpose of this study was to examine the influence of common genetic variation in the endothelin pathway on the cf phenotype. methods: patients were recruited from the cf clinics in dublin, belfast and seattle. serial clinical data were abstracted from medical charts and local clinical databases. studentized residuals of maximum fev , after adjusting for age, gender and height, was the phenotype of interest. twentyone maximally informative tagsnps were identified in the edn , edn , ednra and ednrb genes using the niehs environmental genome project and were genotyped using the illumina beadarray system. genotype and haplotype analysis were carried out using helixtree genetic analysis software. results: clinical and genetic data were available on cf patients ( from seattle and from dublin/belfast). in the combined cohorts, tagsnps in the ednra gene were significantly associated with differences in cf lung disease severity (p= . ). this was observed in both the irish cohort (p= . ) and the seattle cohort (p= . ). the effects were independent of gender and cftr genotype. four common haplotypes (haplotype frequency> %) were identified in the ednra gene. there was significant association between ednra haplotypes and cf lung disease severity (table ). there was no association between genetic variants in ednrb, edn and edn and cf lung disease severity. conclusions: the ednra gene is a genetic modifier of the cf phenotype. our findings were seen in two independent cohorts and verify existing associations found in other populations. the endothelin pathway may be a novel therapeutic target for the treatment of cf lung disease. background: cystic fibrosis (cf) is a recessive "monogenetic" disorder, but there is heterogeneity of lung disease severity and survival reflecting environment and non-cftr genetic modifiers. the unc/cwru multisite gene modifier study (gms) identified patients who were "severe" or "mild" as teenagers or young adults and pertinent cross-sectional data was collected; however, we did not collect all pertinent information about early (age < years) clinical features. objective: we sought to retrospectively evaluate the early clinical features of "severe" and "young mild" patients enrolled in the gms. methods: we obtained all cff registry data available on patients. there were "severe" (worst th percentile of birth cohort, age range - ) and "mild" (best th percentile, age range - ). initial analyses focused on cross-sectional plots of patient age versus multiple clinical features, including age at diagnosis, hospitalizations, cdc height and weight percentiles, presence/absence of ps. aeruginosa from respiratory cultures, and fev (% pred). results: there were . and . years of cff registry data per patient for "severes" and "milds," respectively. preliminary results indicate "severe" patients were diagnosed earlier in life than "mild" patients (mean: . vs. . years, p= . ). this difference is greater after omitting patients who had meconium ileus (diagnosis: . vs . years, "severes" and "milds" respectively, p= . ). between and years of age, "severe" patients were hospitalized more frequently than "mild" patients and, from age onwards, the disparity in frequency of hospitalization increased. "severes" and "milds" had similar cdc height percentiles (~ th percentile) until age , at which point percentiles increased more for "mild" versus "severe" patients. by age , "severe" patients already had lower cdc weight percentiles than "milds" ( nd versus st percentile) and this disparity increased throughout adolescence. from age to , "severe" patients had a - fold higher prevalence of ps. aeruginosa than "mild" patients. as early as years of age, fev (% pred) was ~ points lower in "severes" as compared to "milds." however, some "severe" patients had normal lung function at ages - and overlapped with mild patients; thus phenotyping of lung severity for young patients is not optimal. all of these results were similar for males and females. summary: retrospective analysis of the cff registry data indicates that patients classified later in life as being "severe" experience a worse course of disease from early in life. understanding the early clinical course may prove helpful in defining surrogate phenotypes for modifier studies, and help define appropriate therapeutic targets. cystic fibrosis is mainly caused by small molecular defects of the cftr gene; despite the genotype is defined in the majority of patients, a number of cf cases still remain uncharacterised. the cf mutation database lists more than large rearrangements that may escape detection using pcrbase techniques. the innogenetics assay, the dhplc and sequencing screening showed a mutation detection rate of . % in our population. we report here the results of mlpa screening for ctfr gene rearrangements, performed on the unidentified alleles of our cf patients. our sample population consists of unrelated italian cf patients (for a total of alleles), followed at cf centres of lombardia region. mlpa analysis was performed in patients who still had one or two unidentified alleles after extensive analysis of cftr gene. all patients studied had classical clinical cf symptoms. subjects presented with persistent or recurrent respiratory symptoms, failure to thrive, salt loss syndrome and gastro-intestinal findings. we characterized different deletions and a new duplication (dup promoter-ex. ). thus, . % ( / ) tested alleles had a large gene rearrangement. the deletion of exons - ( / ) was the most frequent in our cohort. all patients had positive sweat chloride values (above meq/l), except the patient carrying duplication who has borderline sweat chloride value. out of patients, ( %) had fecal elastase levels consistent with a preserved pancreatic function: of these patients, had mild mutation, had severe, and had unknown mutation. six patients present liver involvement. the results of the present study could indicate that compound heterozygosity for large rearrangements in cftr gene, is strongly associated with severe pancreatic disease as mutations in classes i, ii, and/or iii. l f is a missense substitution which changes from leucine to phenylalanine at position , resulting from a g/c transition at position in exon a of the cftr gene. it has been described in patients with disseminated bronchiectasis, recurrent idiopathic pancreatitis, sarcoidosis, newborns with hypertripsinemia,. recently derichs et al. reported one healthy -year-old girl homozygous for l f; therefore the pathogenic role of the variant is still unclear. in this study we present subjects compound heterozygotes with l f. no other mutations have been identified after molecular analysis performed using sequencing analysis of the whole coding region of cftr gene and mlpa technique in order to search for gene rearrangements.none of them presented ivs t allele. three of them had a severe mutation in trans (r x, aa>g, and n k), while the other three had a mild mutation on the complementary allele (d h, r q, and r w). individuals with a severe mutation in trans presented a remarkably different clinical picture compared to those with a mild mutation in trans. the formers were diagnosed at a mean age of . years, had an average sweat chloride of . meq/l; besides, all of them have respiratory symptoms, staphilococcus aureus in sputum cultures (one had pseudomonas aeruginosa as well), and in two the chest x-rays was abnormal. the three l f/ps mutation individuals were diagnosed at a mean age of . years, had an average sweat chloride of . meq/l; besides none of them had respiratory symptoms, abnormal chest-xrays, or positive sputum cultures, but two had a history of pancreatitis. these data seem to suggest that l f cannot in any case be considered a neutral polymorphism. the variability of its clinical expression seems to be influenced by the mutation in trans. further studies are needed in order to support our results. expression of the cystic fibrosis transmembrane conductance regulator (cftr) is tightly regulated both spatially and temporally, yet the molecular mechanism of this regulation is not well understood. because no tissue-specific regulatory elements were recognized in the basal cftr promoter, the crucial cis-regulatory elements are likely to be located elsewhere within the cftr locus. a number of the non-coding regions of the cftr gene were found by our laboratory to contain dnase i hypersensitive sites (dhs), suggesting the presence of regulatory elements at these sites. studies presented here investigate the role of an intron dhs (dhs ) in the tissue-specific regulation of cftr gene transcription. the elucidation of molecular mechanisms underlying the temporal and spatial expression of cftr may aid in developing more specific targeted therapies for cystic fibrosis (cf). footprinting analysis of cftr intron revealed a protected region within the core of dhs at + kb. in silico analysis of this sequence uncovered a number of transcription factors binding motifs, including a consensus binding sequence for hepatocyte nuclear factor (hnf ). we have previously identified a role for this factor in the regulation of cftr expression. results of electrophoretic mobility shift assays (emsa) demonstrate that hnf α specifically binds to the motif in cftr intron in vitro. in addition, chromatin immunoprecipitation (chip) analysis of cells expressing both cftr and hnf α factor shows that this factor binds to the intron site in vivo. when cloned as an enhancer, the dhs element was found to augment minimal cftr promoter activity in a luciferase reporter based assay. this increase in luciferase activity was abolished when two nucleotides within the core hnf binding site were mutated, suggesting a functional role for hnf α in cftr gene transcription. further experiments are underway to determine whether additional transcription factors can be recruited to the core of the intron dhs regulatory element and can interact with hnf and the cftr basal promoter to modulate tissue-specific cftr gene expression. the promoter of the cftr (cystic fibrosis transmembrane conductance regulator) gene is not solely responsible for its complex pattern of expression. to identify potential regulatory elements for cftr we previously mapped dnase i hypersensitive sites (dhs) across kb spanning the locus. in addition to intronic dhs, a number of sites were observed that flank the cftr gene. we hypothesized that these may include insulator elements that establish the chromatin expression domain, within which the cftr gene is regulated. insulators are elements that shield against the effects of regulatory elements from adjacent genes, and they may also block silencing of integrated transgenes. using a well-established insulator assay, in which dna regions of interest are assayed for their ability to interfere with communication between a chicken β-globin enhancer and a neomycin resistance gene, we demonstrated that two dhs, at - . kb from the cftr translational start site, and at + . kb from the ' end of the gene, exhibit enhancer blocking activity comparable to known insulator elements. electrophoretic mobility shift assays (emsa), demonstrated in vitro binding of the well-characterized insulator protein ctcf (ccctc-binding factor) at the - . kb dhs. this was confirmed in vivo in both cftr-expressing and non-expressing cell types using chromatin immunoprecipitation (chip). in contrast, although the + . kb dhs did not bind ctcf, we obtained in vitro evidence for the interaction of other factors that may be involved in insulator activity. furthermore, chip analysis of histone modifications across the cftr locus revealed striking differences between the - . kb and + . kb dhs, again suggesting mechanistic differences between these elements. the characterization of insulator elements flanking the cftr locus may be of direct practical relevance in the design of vectors for effective cf gene therapy. one of the problems encountered in gene therapy protocols is the relatively rapid loss of expression from the cftr cdna once it is introduced into mammalian cells. incorporation of the - . kb and + . aminoglycosides, particularly tobramycin, are primary antibiotics used to treat the airway infections in cystic fibrosis patients. lifetime, systemic exposure to these antibiotics is significant and can be associated with significant nephro-and ototoxicity. these toxicities have the potential to significantly reduce the quality of life in the aging adult cf population. we previously reported an incidence of aminoglycoside ototoxicity of greater than % with sensitive audiometric studies (i.e. pure tone audiometry and distortion product otoacoustic emissions). these studies indicated considerable variability in ototoxicity in patients with similar systemic aminoglycoside exposure. the literature suggests that genetic variability in mitochondrial dna can partially explain these differences. single nucleotide polymorphisms (snps) in the mitochondrial srrna gene are associated with aminoglycoside ototoxicity. we genotyped unselected adult cf patients for four of the mitochondrial s-rrna polymorphisms associated with aminoglycoside ototoxicity by direct sequencing of dna harvested from peripheral blood. four patients exhibited polymorphisms in this gene. two patients possessed the a g transition, while another patient each revealed a polymorphism at a g and c t transitions. the patient with the a g polymorphism died prior to audiometric testing. both patients with the a g had audiometric studies consistent with moderate to severe ototoxicity. while the patient with the c t had mild aminoglycoside ototoxicity. all four patients had severe airway obstruction and at least one full course of parenteral tobramycin at mg/kg/d. there was no history of toxic serum levels aminoglycosides during this therapy. both patients with the a g polymorphism had a family history consistent with the expected maternal inheritance pattern associated with the mitochondrial s-rrna gene. we found no evidence of the delt polymorphism in this population. these studies indicate a higher than expected frequency ( %) of mitochondrial s rrna polymorphisms associated with aminoglycoside ototoxicity. these studies demonstrate that genetic screen-ing provides valuable susceptibility information and may change clinical decision-making. support: rising hope foundation/national cf foundation. cystic fibrosis is a genetic autosomal recessive disease that is caused by deleterious mutations in the cftr gene. in its most severe form, cf results in abnormal sweat electrolytes, sino-pulmonary disease, male infertility, and pancreatic exocrine insufficiency. in fact, cf is the most frequent cause of pancreatic insufficiency in humans. carriers of cf (i.e., heterozygotes) do not express any of these classic symptoms. recent studies indicate that significant numbers of non-cf patients diagnosed with congenital bilateral absence of the vas deferens (cbavd) or idiopathic chronic pancreatitis (icp) are compound heterozygotes and carry two cftr mutations of varying severity. in dogs, especially german shepherds and rough-coated collies, exocrine pancreatic insufficiency (epi) has been observed with symptoms similar to those seen in human patients. epi in dogs is most often caused by pancreatic acinar atrophy (paa), a degenerative disease of the exocrine pancreas that has been shown to be inherited as an autosomal recessive trait. the locus responsible for canine paa has not been identified to date. we considered the possibility that cftr mutations might be responsible for this disease. a dog that had been diagnosed with paa and one that was a known carrier of paa were screened for cftr mutations. our samples also included dogs diagnosed with idiopathic pancreatitis as well as healthy dogs. we have established protocols for detecting putative mutations in the canine cftr gene using temporal temperature gradient gel electrophoresis (ttge). in the dog with paa and in the carrier of paa, our screening methodology identified mutations in of the amplicons that span the exons of the cftr gene. dna sequencing revealed silent mutations in exons , and . in exons (l ) and (l ), single nucleotide polymorphisms converted ctt codons into ctc codons, both leucine codons. in exon (p ) another single nucleotide polymorphism converted a ccc proline codon into a cct proline codon. the other identified mutations (amplicons , and , , , , , and ) were found in intronic sequences and have no predicted effect on cftr expression or function. based on these findings, we conclude that cftr mutations are not responsible for paa and the epi that it causes in dogs. hypothesis: specific polymorphisms in genes of the innate immunity may modify the severity and progression of cf pulmonary disease and influence pseudomonas aeruginosa colonisation. methods: single nucleotide polymorphisms (snps) analysis of multiple genes contributing to the innate immunity ( (mbl ), masp (mbl associated serine protease) Ç , fcn (ficolin) Ç , lbp (lipopolysaccharide -binding protein), cd , tlr (toll-like receptor Ç ) ) was performed in cf patients (age - years). spirometric data and bacterial colonisation status with pseudomonas aeruginosa were collected retrospectively. a decline in fev of % over a years period and a percent predicted fev value of % were used to discriminate mild from more severe affected adult cf patients (≥ years). the frequency of single and combined snps in well-defined subgroups of the cf population was compared. results: in adult cf patients with a mean fev lower than %, the combinations of snps of cd (promoter) with tlr (promoter), fcn with tlr (exon ), and masp with tlr (promoter) were more common than in adult cf patients with mean fev > % (odds ratio (or) , ( % confidential interval (ci) : , - , ) , or , ( % ci: , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) and or , ( % ci: , - , ) respectively). the frequency of combination snps of masp with tlr and masp with tlr was significantly higher in cf adults with more than % decline in fev compared to cf adults with less than % decline in fev (or , ( % ci: , - , ) and or , ( % ci: , - , ) respectively). however, the single snps of masp , tlr , tlr were not more frequently found. in pseudomonas aeruginosa colonised cf patients, an increased frequency of combined snps of tlr (exon ) with tlr (exon ), lbp (exon ) with tlr (exon ) and mbl with tlr (exon ) (or , ( % ci: , - , ), or , ( % ci: , - , ), or , ( % ci: , - , ) respectively) was found. in contrast, for single snp tlr (exon ) no statistically significant differences were found between the groups (or : , ( % ci: , - , )). in general, the odds ratios for the combined snps were higher than for the single snps and remained significant when the different groups were subdivided according to cftr genotype. conclusion: certain combinations of snps of genes of the innate immunity are more frequently found in cf patients with a lower fev , a stronger decline in pulmonary function and pseudomonas aeruginosa colonisation. however, for the single snps this trend was less pronounced or absent. congenital bilateral absence of the vas deferens (cbavd) is a rare condition associated with mutations in cftr. cbavd lacks extra-genital organ system abnormalities; it has been viewed as a mild (variant) form of cf. the disease serves as an excellent model for studying the clinical and experimental ramifications of marginal cftr activity, and the functional thresholds necessary to confer a cf-related phenotype. recently, drumm et al. (nejm, ) found that the codon cc genotype of tgfβ- is associated with more severe pulmonary disease, particularly in individuals homozygous for ∆f cftr. tgfβ is also known to play a critical role during normal development and differentiation of the human vas deferens (han-nema et al., horm res, ) . because % of cbavd subjects carry at least one defective cftr allele (but millions of males worldwide are heterozygous for cftr mutations without developing cbavd), the present study was designed to test whether tgfβ polymorphism might serve as a genetic modifier underlying isolated vas deferens loss in the setting of cbavd. we sequenced the ' end of the tgfβ gene in eighty cbavd individuals possessing at least one cftr mutation, and compared genotype frequency with results previously published from demographically similar controls. when cc frequency in cbavd subjects was compared to pooled estimates from earlier trials, no significant differences were seen using a non-dominant model. compared to frequencies reported by arkwright et al. and garrote et al., a greater number of homozygous cc subjects ( . % vs. . %, p< . ) was observed. our preliminary results therefore indicate that if tgfβ- codon polymorphism is associated with cbavd penetrance, it does not represent the major modifier of disease phenotype. in order to evaluate this possibility further, we plan analysis of a larger (matched) control set of cbavd and non-cbavd individuals, studies of tgfβ- activity in vas deferens epithelial cells with and without cftr, and experiments to investigate the effects of tgfβ on cftr biogenesis and function. supported by nih and cff. newborn screening (nbs) for cf has been successful in identifying cf infants, particularly those who have positive sweat tests immediately following the positive screening result. tracking by personnel at the central nbs program facilitates resolution of diagnostic status because cf nbs presents a challenge to cf center (cfc) systems and primary care providers when apparently asymptomatic infants have inadequate (quantity not sufficient (qns)), borderline or indeterminate sweat tests. short-term follow up is more problematic when the screening algorithm requests earlier sweat tests and defines a lower cutoff for indeterminate sweat test results than is used for older infants, children and adults. the massachusetts (ma) cf nbs workgroup chose a low cutoff for indeterminate results of meq/l up to months of age, reverting to the accepted meq/l cutoff thereafter. follow up by the central nbs program identified infants whose diagnostic status remained unresolved more than weeks after a qns or borderline sweat test and eliminated the need for individual cfcs to follow infants tested at more than one cfc. the program sent informational letters and a reminder recommendation to finalize the diagnosis to primary care providers of infants with unresolved diagnoses. of the cf infants identified by ma cf nbs, had an initial sweat result that was indeterminate and another had an initial result that was qns. the central screening program has the capacity to track infant and child outcomes across cfcs; for example, we have been tracking growth outcomes of all cf infants identified by ma cf nbs, despite of the cf infants having moved from one cfc to another during their first years of life. tracking and outcomes data will be presented. when more than one cfc operates within a screening program, centralized tracking ensures capture of quality data. despite several educational campaigns cf is often not diagnosed in early infancy in poland (the mean age of diagnosis months, median is months). after several years of discussions,in september a neonatal screening for cf supported by the ministery of health was started as the third obligatory neonatal screening in poland. more than , neonates were screened in the first seven months using the existing screening infrastructure and spare blood spots after pku and congenital hypothyreosis screening. the protocol was two stage irt/dna (covering the most common mutations in the polish population). the final diagnosis was based on sweat tests (conventional pilocarpine-iontophoresis and conductometric nanoduct) as well as clinical examination. values of chloride in sweat > mmol/l and nacl > mmol/l were considered diagnostic for cf. children were confirmed as cf. although delta f is the most common mutation in the polish population, only two infants were delta f homozygotes, six were delta f heterozygotes and the remaining had different mutations ( children had + kbc>). half of the diagnosed group was still pancreatic sufficient. the children had confirmed cf diagnosis at the mean age of weeks. only children were free from any radiological changes in lungs. the most common pathogen in this group was staphylococcus aureus. at the first visit information for parents about cf was given along with physiotherapy and dietary education. anthropometric measurements, airways bacteriology and elastase - in stool were performed. psychologist consultations were available. neonatal screening for cf enabled earlier diagnosis and the introduction of complex therapy in comparision with symptomatic screening. objectives: the clinical course of cystic fibrosis (cf) varies widely among patients carrying the same cftr gene mutations supporting that additional genetic modifiers could affect the cf phenotype. as inflammation is a central contributor to the pathogenesis of cf lung disease, genes involved in the inflammatory response are potential modifier candidate genes. methods: we examined the influence of polymorphisms in genes involved in the inflammatory response (tnf, il- β, il- receptor antagonist (rn), il- , il- , il- and tgf-β ), on disease progression in a group of caucasian children with cf. the genotypes were tested for an association with changes in lung function tests, pseudomonas aeruginosa colonization and nutritional status by multivariable analysis. results: we found a significant association between tgf-β + t/c variants and decline in lung function measured as the forced expiratory volume in one second (fev ) and the forced vital capacity (fvc) (p= . and . respectively). il- gene polymorphisms (- a/t, + g/t and + t/c) were associated with the occurrence of pseudomonas aeruginosa colonization (p< . ). conclusions: this study suggest that il- variants may influence pseudomonas aeruginosa colonization in cf, however this need further confirmation. moreover, we provide additional support to the results of other trials and strongly suggest an association between tgf-β variants and lung disease phenotype in cf. if such a role of tgf-β is confirmed by functional studies, it may have important clinical impact for the identification of patients at risk to develop more severe respiratory manifestations and for the development of new therapeutic strategies aimed at adequately balancing tgf-β production and action introduction: cystic fibrosis (cf) is the most common monogenic disease in caucasian population, its frequency is about one in live born. the estimated frequency in hispanic population is about one in , with differences in mutation frequency especially for low frequent alleles. heterogeneity in pulmonary manifestations cannot be explained by the cftr mutation genotype. recent studies are focused in the study of modifier genes. these are genes different from the primary cftr gene that could influence the fq phenotype, acting trough mechanisms like inflammation, repair and remodeling, protease-anti protease balance, innate immune response, etc. this work shows some advances in cf diagnosis and modifier genes analysis in mexican population. methods: we tested the effectiveness of innolipa cftr kit (innogenetics) in clinically diagnosed cf patients. thirty six frequent mutations in cftr gene were analyzed by pcr and reverse hybridization with allele specific oligonucleotides probes in dna samples extracted from blood or oral swabs. for frequency analysis of modifier genes we tested about seventy dna samples from a dna bank of patients previously tested positive for cftr mutation. the polymorphism analysis of modifier genes was made by pcr-rflp. for alpha antitrypsin (aat), z and s alleles were analyzed according stanford and cols. ( ) . for tumor necrosis factor alpha (tnfα) - polymorphism in the promoter region was analyzed according chen and cols. ( ) . results: mutation was detected in in of patients ( % of effectiveness). df allele frequency was % (caucasian - %) followed by + g-a, s n, g x, g e and delt ( %). % of the mutant alleles remain undetected. preliminary results in modifier genes show low frequency for aat mutant alleles ( . % for aat s and z alelle respectively) and . % for tnf- . conclusions: df alelle frequency was %, and we found the low fequency alleles s n and + g-a. frequency of modifier genes mutant alleles in cf patients are low. this work is in progress of selection and recruitment of patients and controls, as well as standardization of the other genes modifiers, along with some other biomarkers that will allow to understand the physiopathology of cf and to define a possible genetic risk profile of severe pulmonary disease. over putative cystic fibrosis (cf)-causing mutations have been reported to the cf mutation database and almost half (~ ) are rare missense mutations that are predicted to substitute a single amino acid. to determine if any of these rare missense mutations cause cf by altering localiza-tion of cftr, we examined mutations in the cytosolic loops of cftr since localization signals have been identified in cytosolic loops of other ion channels. amino acids alignments comparing human cftr sequence to that of species revealed that cytosolic loop (cl ) was the most conserved loop by identity. furthermore, cl had the most naturally observed mutations: of the amino acids were found to be mutated in cf patients and sites had multiple missense mutations at the same amino acid. in addition, different substitutions of the same cl amino acid have been associated with different disease consequences, such as the missense mutations in the arginine at codon . patients with the r p mutation have pancreatic insufficient (pi)-cf, those with r w mutation have pancreatic sufficient (ps)-cf or cbavd, while patients with r q primarily had pi-cf. to determine whether the r mutations cause disease by affecting cftr localization, we used the flp-in system to create stable polarized mdck-gfp-cftr cells to produce isogenic clones with cftr expressed from the same integration site. confocal microscopy studies of stable mdck-frt-gfp-cftr-r mutant cell lines revealed that cftr-r p was cytoplasmic, cftr-r w was apical and cytoplasmic, and cftr-r q was apical. to confirm these observations, quantitative biotinylation studies were performed. biotinylation assays of mdck cells stably expressing cftr-r p confirmed that this mutant was absent from the apical surface, cftr-r w had a low level of fully glycosylated protein at the apical membrane, while cftr-r q had fully glycosylated protein at the apical membrane comparable to wild-type cftr. thus, cftr-r p and cftr-r w displayed properties in polarized cells that were distinctly different from wild-type cftr consistent with their associated cf phenotypes. however, the profile of cftr-r q (apical localization and cl-channel function (seibert et al. , mickle et al. ) appears inconsistent with a pi-cf phenotype. indeed, re-analysis of cftr genes bearing r q revealed that of carried a second mutation in cis (s x). in nine of these patients in whom cftr genotype and pancreatic status is known, presence of the nonsense mutation s x is associated with the pi-cf phenotype. since nonsense mutations are known to cause severe gene dysfunction by promoting decay of their rna transcript, we concluded that the s x mutation rather than r q is responsible for the pi-cf phenotype. the finding of s x explained the otherwise enigmatic functional studies of r q and clinical observations in patients bearing this mutation. this study also demonstrates that stable expression of cftr mutants in polarized mdck cells using the flp-in system provides a useful screen for evaluating the disease potential of rare missense mutations. the clinical course of cf is characterized by recurrent pulmonary infections and chronic inflammation. in cf patients, up-regulation of toll-like receptor- (tlr ) gene in airway epithelial cells is believed to enhance proinflammatory responses towards bacterial tlr ligands. we have recently shown that decreased methylation (demethylation) of the tlr promoter is responsible for cf-related up-regulation of tlr in bronchial epithelial cells (shuto. t. et al., faseb j, ) . however, the molecular mechanisms responsible for dna demethylation-dependent tlr gene upregulation in cf cells remain unknown. here, we identified minimum region of the tlr promoter critical for its expression at - /+ , which contains one putative binding site for sp . sp inhibitor mithramycin a treatment reduced tlr promoter activity and its expression in cf bronchial epithelial cells (cfbe o-), suggesting the importance of this sp site for the tlr gene regulation. moreover, bisulfite sequence analysis revealed the hypomethylation of adjacent this sp binding motif within tlr promoter in cfbe o-cells, implying the cf-specific demethylation of adjacent sp binding motif. although mithramycin a rarely affected basal expression of tlr gene in hbe o-cells (non-cf bronchial epithelial cells), mithramycin a inhibited tlr expression in -azacytidine (dna-demethylating agent)-treated hbe o-cells. taken together, our results suggest that sp is crucial for dna demethylation-induced gene upregulation of tlr in cf bronchial epithelial cells. mucus hypersecretion due to goblet cell metaplasia is a critical feature of cf lung disease, affecting both mucociliary clearance and drug delivery. various studies in cf lungs and cf primary cultures have shown increased expression of muc ac, the main gel-forming mucin produced by goblet cells; however, little is known about the role of muc ac in the progression of lung disease. to determine whether overexpression of muc ac alone is sufficient to induce lung pathology, we generated a mouse model overexpressing muc ac. the entire muc ac cdna (minus % of the predicted vntr sequence, final size ~ kb) was cloned and tagged with an internal gfp epitope at a site close to the vntr to preserve the integrity of mucin domains. gfp-muc ac was linked to the rat ccsp promoter to drive specific airway expression, primarily in clara cells since these cells are capable of secreting mucins. blastocyst injections were used to generate transgenic founder lines in c bl/ background. although muc ac-gfp mrnas were expressed in transgenic mice, lung histology did not show significant increases in inflammation, ab/pas-positive cells, or luminal secretion compared to wild-type littermates. gfp fluorescence from freshly excised lung was weak but immunohistochemistry showed that the distribution of gfp correlated with the expected expression pattern, with the majority of positive cells localized to the airways and few positive cells in the alveolar space. at higher magnification, gfp-positive granules were observed in dome-shaped cells that were also positive with cc antibody, suggesting proper muc ac-gfp packaging in clara cells. bronchoalveolar lavage samples separated by agarose gel western blots and probed with gfp antibody confirmed that each line secreted muc ac-gfp, with a migration pattern comparable to a mucin. to further characterize the secreted transgenic mucin, a pool of lavage samples was separated into supernatant and pellet. the pellet was resolubilized in m guanidine. both elements were passed through a s size-exclusion column. analysis by light scattering and refractometry to determine molecular weight and concentration revealed that the solubilized pellet contained an enormous but compact complex ( x g/mol and nm radius of gyration). gfp-rich fractions were pas-positive, indicating proper glycosylation of the transgenic muc ac-gfp. cscl density gradient studies revealed that the gfp-rich fractions had a high molecular weight but did not appear to be a prominent part of the megacomplex retrieved in lavages, unlike other tested mucins. in conclusion, we showed that the overexpressed muc ac-gfp was a high-molecular-weight protein that was glycosylated, packaged in clara cells and formed multimers. lack of discernable phenotype in the transgenic lungs suggests that mucin overproduction alone is not sufficient to trigger goblet cell metaplasia and mucus accumulation, supporting the protective role of mucins. currently we are crossing the muc ac-gfp with the scnn b mouse model to study muc ac-gfp under dehydrated conditions and we are conducting allergen-challenges in the transgenic mice. results of such studies will provide novel insights into the role of secreted muc ac in development of lung disease in these models. supported by grants from the cf foundation. the major characteristic in cystic fibrosis (cf) is acquirement of chronic lung infections with p. aeruginosa. once established the chronic infection can only be suppressed, not eliminated. the cf patients harbour one single strain of p. aeruginosa which only rarely is replaced by other strains. during the chronic lung infection there is a year long mutual impact of the bactaria on the host, and of the host response and antibiotics on the bacteria, resulting in a continous adaptation of the bactaria in the lungs of the cf patients with selection for different clones. in contrast, mouse models of the lung infection in cf is limited to two weeks! therefore, we introduced a new strategy infecting different groups of balb/c mice with pulsed field gel electrophoresis-(pfge) identical clones isolated from one cf patient during -years of p. aeruginosa lung infection. ) infecting the mice with non-mucoid isolate showed that the early isolates were significantly better in establishing an infection with reduced clearance of the bacteria from the lungs (p< . ), increased dissimination of the macroscopic pathology (p< . ) and a more acute type and a higher degree of inflammation (p< . ) as compared to mice infected with a late isolate. in addition, the pulmonary cytokine response was comparable with observations in cf where gm-csf and il- correlated to a milder disease, whereas g-csf, mip- (il- analog), and il- b correlated to severe disease. ) infecting the mice with the mucoid pfge-identical isolates from the same cf patient revealed a signicantly higher mortality in mice infected with the late isolates (p< . ). in addition, the pulmonary g-csf and mip- response increased in the groups of mice that were infected with the late isolates as compared to the early isolate (p< . ). moreover, the g-csf and mip- increased during the first three days of infection with the late mucoid isolates (p< . ), indicating an impairment of the host response in controlling the lung infection. ) when tobramycin treatment was initiated h after infection of mice with a mucoid isolate, the number of bacteria were cleared or reduced to a significantly lower level (p< . ), and a significant decrease of both g-csf and mip- at day one, two and three was observed (p< . ). in contrast, when treatment was initiated after h the number of bacteria did not change significantly, and the reduction in inflammatory cytokine response became significant for both late isolates only at day three of treatment (p< . ). moreover, cytokine levels were significantly lower in the h as compared to the h group (p< . ) confirming the importance of early and aggressive antibiotic treatment in cf. non-mucoid isolates were reduced during the first three days of infection independent of treatment. in conclusion, we have implemented a clinical important cf-time perspective (years) in experimental p. aeruginosa lung infection, useful for patho-physiological and treatment studies. cystic fibrosis (cf) is a complex disease that affects multiple organs and results in a wide range of phenotypes in humans. mouse models of cf display many of the phenotypes observed in human patients and provide necessary tools to dissect this multifaceted disease. however, due to the involvement of cftr in various organ systems, the ability to discern the contribution of each organ system to each phenotype of cf is unknown. for example, we have found that mouse growth is dramatically reduced compared to wildtype, and mechanisms ranging from neuroendocrine disruption, intestinal malabsorption and cacchexia have been proposed. we have found that cf mice consume more calories per gram body weight than non-cf mice and intestinal absorption of dietary lipids is similar between the two. cf animals absorb % and non-cf % (p > . ). to identify the source of the growth deficit, it would be advantageous to restrict expression of cftr in specific tissues to determine each tissue's contribution to the phenotype. to accomplish this, we have created a conditional, null murine cftr allele. a conditional null allele is one in which the gene of interest is induced to be non-functional in either a specific tissue or cell type, or at a specific developmental time. examination of cftr absence on a tissue-by-tissue basis will greatly facilitate our understanding of the disease. the conditional cftr allele was created by "floxing" exon of cftr, known to be critical for function, with flanking loxp sites that allow for the deletion of exon when bacterial recombinase cre is present. to date, mice stably transmitting the floxed allele have been created. these founder mice have been crossed with mice expressing cre from the promoter of the villin gene, which restricts transcription of cre to intestinal epithelium. first generation animals show appropriate recombination and deletion of exon in the gut. however, these first generation animals are heterozygous for the floxed allele. we are currently backcrossing these animals with the founders to generate animals with homozygous deletion of cftr in the intestinal epithelium. similar crosses are planned to target other tissues. this new animal model of cf will be available to the cf research community to further our understanding of cf pathophysiology. this work was supported by a grant from the cf foundation increased enac-mediated na+ absorption is a hallmark of cystic fibrosis (cf) airways. we demonstrated that mimicking na+ hyperabsorption by overexpression of βenac in mouse airways results in airway surface liquid (asl) depletion and reduced mucus clearance causing a spontaneous cflike lung disease with high pulmonary mortality, and airway mucus obstruction, mucous cell metaplasia and chronic airway inflammation in adult survivors (mall et al., nature med. : , ) . the aim of this study was to identify the initiating lesions and investigate the natural history of lung disease caused by asl depletion in βenac overexpressing (βenac-tg) mice. to achieve this goal, we performed longitudinal studies on lung morphology, airway mucus obstruction, bronchoalveolar lavage inflammatory cell counts, expression levels of mucins (muc ac, muc b, muc ) and proinflammatory cytokines (tnfα, kc, ifnγ, il- , eotaxin- ), lung volume, lung mechanics, and airway na+ transport in neonatal to adult βenac-tg mice and wild-type littermate controls. we show that airway mucus obstruction in βenac-tg mice originated in the trachea in the absence of mucous cell metaplasia in the first days of life, and was associated with hypoxic degeneration and necrosis of airway epithelium, and death. in surviving βenac-tg mice, mucus obstruction extended into the lungs and was accompanied by secondary mucous cell metaplasia, increased expression of muc ac, muc b and muc , and airway inflammation with transient increases in macrophages and tnfα, in eosinophils and il- , and persistent increases in neutrophils and kc in the lung. βenac-tg mice also developed emphysema with increased lung volumes, distal airspace enlargement, and increased lung compliance. several disease surrogate markers waned in parallel with βenac transcript levels and airway na+ absorption in adult βenac-tg mice indicating that the level of enac overexpression was critical in determining disease severity. in summary, our results provide several novel insights into the in vivo pathogenesis of lung disease caused by asl depletion. we show that (i) asl depletion is sufficient to initiate severe airway mucus obstruction in the absence of mucous cell metaplasia and/or mucus hypersecretion; and (ii) airway na+ hyperabsorption and mucus plugging-induced hypoxia is associated with airway epithelial necrosis constituting a mechanism that initiates airway inflammation in the absence of infection. further, our results point to a novel link between airway na+ hyperabsorption, eosinophilic inflammation, and emphysema. the high frequency of allergic airway inflammation and emphysema in cf patients warrants further studies of the mechanisms that link asl depletion with these associated pathologies. the balance of airway surface liquid (asl) at a height compatible with efficient clearance of mucus is a process regulated by two main pathways: the secretion of clvia cftr, and absorption of na + via the epithelial na + channel (enac). to study this interplay, an immortal cell line capable of growing at an air liquid interface and having properties of human bronchiolar epithelium would be desirable for its simplicity in preparation and ease of molecular manipulation. an immortal human bronchial adenocarcinoma cell line (hbac) was infected with a lcfsn retrovirus carrying either cftr cdna (hbac cftr ) or the blank vector (hbac mock ). - day monolayers, grown on transwell clear membranes, were mounted in ussing chambers and studied under open-circuit conditions. to study regulation of clsecretion, the equivalent short circuit current was calculated in the presence of amiloride (apical, µm). in hbac mock cells, forskolin ( µm, basolateral) had no effect upon i sc . in contrast, hbac cftr cells demonstrated a robust increase in i sc ; a rapid increase to a peak (∆~ µa cm ), followed by relaxation to a stable and sustained plateau. genistein ( µm) also stimulated i sc in hbac cftr (∆i sc µa cm ) but not hbac mock cells. amiloride resistant basal current was consistently higher, by - µa cm , in hbac cftr versus hbac mock cells, presumably reflecting low basal cftr activity. we next examined the ability of endogenous receptors present in hbacs to regulate transfected cftr. stimulation of i sc in response to apical adenosine ( µm) in hbac cftr was qualitatively and quantatively similar to the forskolin response. while the above experiments provided evidence to suggest that hbac cftr cells possess the bioelectric capability of sustaining anion transport via nucleoside-stimulated cftr activity, it does not guarantee a resultant vectorial flow of fluid. in the final series of experiments, we examined whether adenosine was indeed able to stimulate secretion across the hbac-cftr monolayer by assaying asl height. monolayers were loaded apically with fitc-labeled dextran ( µl), and the majority of dye was then aspirated. monolayers were then allowed to equilibrate over a hour period, and asl measured by xz scanning on a leica confocal microscope. ~ µm adenosine and ~ µm benzamil was then added apically in pfc and asl height measured after and minutes. in both hbac mock and hbac-cftr cells, the basal asl height was ~ µm. over minutes asl height of hbac mock cells did not change in response to adenosine. in contrast, adenosine stimulated hbac cftr cells demonstrated a doubling of asl height (to µm) over the same period. this response is consistent with the prolonged increase in i sc observed upon stimulation with adenosine. in summary, the hbac model which displays only absorptive currents endogenously, can be transformed into a cell line in which cftr-mediated secretion can be stimulated and sustained by endogenous receptor systems present in the apical membrane. this new model will allow us to study cftr-mediated fluid transport in a defined manipulatable system. despite the identification of the cftr channel more than years ago, many questions remain about its regulation and in vivo function. in order to better understand how cftr functions in vivo, numerous studies have searched for and identified potential interacting partners but to date, there is little evidence linking any of the known cftr interactors to its activity. during a forward genetic screen in zebrafish, aiming to identify genes regulating endodermal organ development, we identified a mutant, which we named baobab (bao), after the african tree that accumulates water during the wet season, which exhibits a dramatically enlarged fluid-filled gut tube. most interestingly, we have shown that various cftr inhibitors reduced the appearance of enlarged guts in bao mutant embryos, suggesting that the fluid accumulation phenotype resulted from an increase in cftr activity. to test whether increased cftr activity resulted from changes in cftr protein levels or localization we raised antibodies against zebrafish cftr. western-blot and immunolocalization studies revealed that the channel was expressed and localized as in wild type in bao mutant embryos indicating that the increased cftr function most likely resulted from the activation of the channel. these data, together with the recessive character of the mutation, suggest that the bao protein is a negative regulator of cftr activity. we have positionally cloned the bao locus and isolated the affected gene. bao encodes a cytosolic protein that has not been previously shown to regulate cftr. in agreement with the pharmacological data pointing to a gut-specific function of bao, in situ hybridization studies showed that bao is higly expressed in gut. we are currently investigating how the bao protein regulates cftr function. in addition, we are also investigating the function of cftr during zebrafish development. these studies lay the groundwork to take advantage of the zebrafish system to further investigate cftr biology. we establish a genetic model system for studying the regulation of the cftr channel that will contribute to the understanding of the pathophysiology of cystic fibrosis and several intestinal secretory conditions. acknowledegements: this work was supported by an embo long-term postdoctoral fellowship to mb and by nih grants to d.y.r.s.. we thank a. verkman for providing cftr inhibitors and k. brand for technical assistance. the trachea of the adult cf mouse (cftr tm unc or cftr tm kth ) expresses little cftr and exhibits neither a defect in camp-mediated clsecretion nor hyperabsorption of na + , both signature abnormalities in human cf airways. we generated a mouse exhibiting hyperabsorption of na + in the airways by over-expressing the β subunit of the epithelial na + channel (βenac, gene scnn b). βenac mice exhibit airway pathology (decreased mucociliary transport, mucus plugging, and airway inflammation) similar to human cf airways. to determine whether inactivation of cftr in βenac mice results in a more severe phenotype, we generated a cf/βenac mouse by crossing the ∆f (c bl/ n) mouse (cf mouse) with the βenac mouse (c bl/ n). survival analysis showed a drastic reduction in survival for cf/βenac mice in comparison to cf mice or βenac mice. we studied the airway bioelectrics of day-old pups of each genotype. the basal short circuit current (i sc ) was ~ fold greater in wild-type (wt) ( ± µa . cm - ; mean ± sem, n= ) vs. cf ( . µa . cm - , n= ) neonatal tracheas. as previously reported, the basal i sc in βenac preps was significantly elevated ( . ± µa . cm - , n= ) vs. wt tracheas. however, the basal i sc in the cf/βenac tracheas ( . ± µa . cm - , n= ) was intermediate between the cf and the βenac tracheas. the amiloride-sensitive i sc was elevated similarly in the tracheas of the βenac and cf/βenac pups and was ~ x greater than that exhibited by the wt or cf tracheas. the post-amiloride i sc was strikingly reduced in both cf and cf/βenac tracheas compared to the wt and βenac tracheas. we suggest that the post-amiloride i sc reflects constitutive cftr-mediated clsecretion, which may help hydrate airways in the neonate. unlike the tracheas of adult cf mice, neonatal cf tracheas exhibited the classic defect in camp-mediated clsecretion, with the forskolin response in the cf and cf/βenac tracheas being reduced ~ fold compared to the response in the wt or βenac preps. the response to utp (ca ++ -mediated clsecretion) was similar in the four genotypes. absence of cftr-mediated clsecretion does not appear to cause airway pathology in neonatal cf mice, but when this defect is coupled with na + hyperabsorption, it results in a significant increase in neonatal mortality. due to lack of cftr-mediated clsecretion, cf/βenac mice appear to experience greater mucus adhesion, producing death due to asphyxiation. in addition, the tracheas of cf mice (especially congenic c bl/ n) often appear constricted or collapsed due to a cartilaginous defect resulting in increased compliance. this anatomical defect does not appear to be detrimental to the cf mouse. however, in cf/βenac pups, that accumulate thick, sticky mucus in their tracheas, this tracheal constriction may contribute to their asphyxiation. these results demonstrate the importance of both abnormal na + absorption and decreased clsecretion in the pathogenesis of cf lung disease. supported by nih scor p hl . recently, we reported that species-specific differences in the airway biology of raav transduction exist between mice and human (am j respir cell mol biol, , : - ) . these differences significantly interfere with the use of mice as surrogate models for human lung gene therapy with certain raav serotypes. to survey which species might be an optimal animal model for raav preclinical testing, we utilized differentiated airway epithelia grown at an air-liquid interface (ali) from five species (human, non-human primate [nhp] , pig, ferret, and mouse) to test the efficiency of transduction with three serotypes of raav (type , , and ). our results demonstrated significant species-specific differences in raav transduction biology in airway epithelia. the serotype preferences of raav transduction from the apical membrane in human, ferret, and pig epithelia was raav >raav ≈raav , different from that seen in mouse epithelia (raav >>raav >>raav ). surprisingly, the profile of raav transduction with these three serotypes in nhp rhesus monkey airway epithelia (raav ≈raav >>raav ) was less similar to human than pig and ferret. additionally, raav lacked a bias in polarity of transduction in human airway epithelia, however, in nhp epithelia, raav transduced the basolateral membrane -fold more effectively than the apical membrane. furthermore, differences in the properties of raav receptors between these species was also seen. using neuraminidase treatment of the apical surface of ali cultures, we tested whether n-linked sialic acids were potential receptors for raav binding and/or transduction. these studies demonstrated removal of n-linked sialic acids inhibited raav transduction of human, nhp, and mouse airway epithelia, but not ferret and pig. however, raav binding in this setting did not correlate with changes in transduction; neuraminidase treatment dramatically reduced raav binding to nhp epithelia, but enhanced binding to human epithelia (~ fold). these findings suggest viral binding to airway epithelial cells may not be a major barrier for raav transduction. evidence that proteasome-modulating agents enhancing raav transduction - logs for all serotypes and species tested, suggest that the ubiquitin/proteasome pathway represents a common intracellular block in raav transduction of airway epithelia. these findings support the notion that viral interactions at the surface of airway epithelial cells (with co-receptors and receptors) influence intracellular processing aav virions that can alter the efficiency of productive transduction. enac surface density in cortical collecting duct (ccd) epithelial cells is established by camp/pka regulated insertion and retrieval of the channel at the apical membrane, together with the recycling of internalized enac back to the apical surface (butterworth et al. ( ) journal of general physiology : - ). ubiquitination by the nedd - ligase elicits endocytosis of surface enac, which may lead to its degradation. however, enac recycling to the apical surface requires ubiquitin removal by a deubiquitinating enzyme (dub). using a chemical probe approach, we identified ubiquitin carboxy-terminal hydrolase (uch-l ) as the predominant dub in vesicular endocytic and recycling compartments in mouse ccd epithelia. specific inhibition of uch-l ( µm of , , , -tetrachloroindan- , -dione) in filter-cultured ccd cells resulted in a rapid decline in enac currents, which was accelerated by forskolin stimulation, suggesting that uch is responsible for recycling enac to the apical surface. knockdown of uch-l significantly reduced both basal and camp-stimulated enac currents (from . ± . µa/cm in control sirna treated to . ± . µa/cm in uch-l knockdown cells). to determine whether the same dub was responsible for the regulation of enac in human bronchial epithelial (hbe) airway cells, differentiated cultures derived from cf and non-cf lung tissue were treated with the uch inhibitor. to address the possibility that protease cleaved enac may be regulated differently from unprocessed channels, differentiated hbe cultures were either maintained at air/liquid interface or were submerged under liquid/liquid conditions to alter the cleavage state of enac, as we have previously described (myerburg et al. ( ) journal of biological chemistry : - ). in all cases, no significant difference in enac-mediated na+ transport was observed between control and uch-l inhibited conditions. these data are in sharp contrast with the mechanisms of enac regulation observed in the ccd model system, and they support our previous findings that enac turnover and mode of regulation in hbe are distinct from the recycling mechanisms defined in the ccd epithelia (butterworth et al. primary human tracheobronchial epithelial (htbe) cells cultured at an air-liquid interface (ali) regulate ion transport, mucin secretion, and cilia beating to recapitulate directional mucus clearance, a key innate defense mechanism that fails in cystic fibrosis (cf). thus, ali htbe cell cultures are crucial for studying cf pathogenesis and for developing/validating novel therapies. different research centers use alternative protocols that enable reproducible creation of ali htbe cell cultures. however, limited primary htbe cell availability has driven the creation of cognate cell lines, accomplished mainly by the introduction of viral oncogenes. viral oncogenes disrupt normal cell physiology causing cellular hyperplasia, abundant apoptosis, genomic instability and, sometimes, poor polarization/differentiation, rendering the cells less useful than primary cells for assessing cftr function. furthermore, genetically unstable cell lines will continuously acquire changes independent of cftr genotype. bmi- is a polycomb group protein that controls cell cycle and cell identity via epigenetic regulation of chromatin, maintaining stem cells by suppressing expression of p , an inhibitor of cyclin dependent kinases. bmi- expression has been used to create cell lines that recapitulate normal cell structure and function better than viral oncogene-immortalized cell lines. using hiv lentiviral vectors, we introduced bmi- and the catalytic subunit of telomerase to enhance the growth of different ∆f homozygous cf and non-cf primary human airway epithelial cell preparations. all new cell lines grew for at least population doublings, while their normal counterparts senesced prior to a maximum of doublings. at passage - , the new cell lines had a diploid karyotype compared to grossly abnormal chromosomes in cells immortalized by viral oncogenes. ussing chamber analysis of ali cultures at passage (p) - revealed variable transepithelial resistances among the cell lines ranging from -> Ω*cm , but short circuit current (isc) responses stimulated by forskolin and inhibited by cftr were true to the cell's genotype and comparable to early passage primary cells, except for one non-cf cell line with relatively low cftr isc's at p . amiloride sensitive and utp-stimulated isc's were present but were more variable in magnitude in comparison to the currents observed in low passage primary cells. ali cultures exhibited a pseudostratified morphology with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells and occasional ciliated cells. all cf and non-cf cell lines in culture produced similar levels of il- at baseline and cf and non-cf cells equally increased il- secretion in response to il- β, tnfα and the tolllike receptor agonist, pam cys. these novel cell lines will help fill gaps currently hindering cf research and therapeutic development. supported by cystic fibrosis foundation grant randel g . we recently considered that domestic dogs could represent a large natural reservoir to search for cftr mutations and, ultimately, to identify and breed animals with cf traits. the canine genome has been completely sequenced, and the canine cftr gene, found on chromosome , consists of exons spread over kb. an animal model that faithfully expresses human cf lung pathology is critical for the development of effective treatments for this most lethal aspect of cf disease. while transgenic mouse models of cf replicate numerous aspects of cf disease, they unfortunately do not develop the characteristic lung pathology including mucus plugging of airways, chronic bacterial infections, and bronchiectasis. the reason for this is not completely understood but it is likely related to significant differences in lung morphology between these species. to date, no non-murine genetic model of cf has been developed, and naturally-occurring, cf-causing mutations have not been identified in any non-human animal species. temporal temperature gradient gel electrophoresis (ttge), developed as a screening tool to identify sequence variations and mutations in the human cftr gene, was used to screen dogs for cftr mutations. dna was purified from whole blood received from veterinary clinics treating dogs for a variety of ailments. we have performed ttge analysis on dna from dogs, which includes dogs with pancreatitis and dogs with bronchiec-tasis, and have found hundreds of potential mutations amongst the thousands of amplicons tested. ttge can detect single base pair changes within a fragment of dna. as the temperature increases, the migrating dna becomes partially melted, creating single stranded regions (internal bubbles and branched ends) that decrease its mobility through the gel. the dna fragments have distinct melting profiles that are sequence dependent; therefore, changes in sequence of even a point mutation can alter the profile and result in a change in the rate of migration through the gel that can distinguish wild type from even a homozygous mutant. thus far exonic mutations have been sequenced but none have caused changes to cftr at the amino acid level (i.e., the mutations have all been silent). we conclude that ttge is a sensitive and effective method for screening large canine populations for cftr mutations. ( introduction: the search for "correctors" of defective protein processing of ∆f cftr has led to the development of high throughput screening assays to find compounds that promote the surface activity of the mutant protein. by necessity, primary screening assays typically use cell lines expressing recombinant ∆f cftr. however, "hits" identified in primary screening require scrutiny for false positives whose activity is either dependent on the ∆f cftr expression system or insufficiently robust to correct processing in the complex physiological setting of a native epithelium. to this end, we have developed a secondary functional assay that measures the shortcircuit current (isc) responses in organ cultures of gallbladder mucosa from ∆f cftr mice (cftr tm kth ). previous studies have shown that murine ∆f cftr recapitulates the protein processing defect including the capability to increase processing when mucosa is incubated at reduced temperature ( °c) (j. clin. invest. : invest. : , . gallbladder mucosa was chosen for this assay because the murine gallbladder can be removed aseptically for organ culture, the epithelium is essentially a monolayer, and the mucosal wall is transparent which facilitates morphological assessment. methods: using either wild-type (wt), ∆f cftr (df) mutant or cftr knockout (cftr ko) mice, excised gallbladders were opened longitudinally, rinsed and positioned on loose nylon mesh. the preparation was mounted between two halves of a polycarbonate culture cup with . mm aperture and placed in submersion culture for - days at either °c or °c using supplemented ham's f medium mixed : with nih t fibroblast-conditioned dmem containing % calf serum. after incubation, the culture cups were placed in custom designed ussing chambers for measurement of isc responses to a cocktail of µm forskolin and µm ibmx (camp isc) at °c. contribution of calcium-activated clchannels to the camp isc was eliminated by addition of the distilbene dids ( µm) to the luminal bath. results: the mean ∆camp isc responses (in µa/cm ) after two days incubation were as follows: wt at °c = . ± . (n = ); df at °c = . ± . (n = ); df at °c = . ± . (n = ); and cftr ko at °c = . ± . (n = ). in a pairwise comparison, the wt ( °c) was significantly greater than the df ( °c) and cftr ko ( °c) but not significantly different from df ( °c). conclusions: incubation of ∆f cftr mutant murine gallbladder mucosa at °c for two days in organ culture restored approximately % of the cftr-dependent isc responses to camp stimulation. the temperature-dependent effect is consistent with correction of murine ∆f cftr processing, indicating a physiological screening assay that is useful for preclinical testing of candidate drugs targeted at correcting the protein processing defect of ∆f cftr. supported by cfft and nih. the lack of a large animal model that replicates the lung disease found in humans with cystic fibrosis is a major impediment to understanding disease pathogenesis and the development of effective therapies. although cystic fibrosis is a multi-system disease, airway infection and inflammation currently cause most of the morbidity and mortality in patients. several homozygote null and missense cftr mutations have been developed in mice, but they do not exhibit the lung phenotype observed in humans. in contrast to mice, the structure and physiology of porcine lung and airways closely resemble those of humans. therefore, the goal of this project is to develop a swine model for cystic fibrosis by combining gene targeting and nuclear transfer to produce a pig with a cftr null genotype. we employed homologous recombination to disrupt exon of pig cftr with a neomycin resistance cassette. we generated primary male fetal fibroblasts that were positive for cftr-targeting at one allele as detected by multiple pcr screens. genomic southern blot analysis confirmed proper gene targeting and revealed most clones were free of random integration events. these cells were used for somatic cell nuclear transfer that resulted in the birth of ten cftr null heterozygote piglets. subsequent breeding of these male pigs to wild-type females has yielded at least female cftr null heterozygotes. breeding male and female heterozygotes will provide cftrnull homozygotes. our recent published and preliminary studies demonstrate that mouse slc a , a newly discovered member of slc family of anion exchangers, is expressed on the apical membrane of tracheal epithelial cells and gastric surface epithelial cells and in the tubulovesicle membranes of gastric parietal cells. the functional properties of mouse slc a were examined in xenopus oocytes and in stably transfected cultured cells. in xenopus oocytes injected with the slc a crna and studied with the voltage and/or ph sensitive microelectrodes, the rank order for anion selectivity at + mv was cl->i-> no ->gluconate > sulfate, and that of the selectivity (px/pcl) was i-> cl-> no -= gluconate > sulfate. inhibitor profile studies demonstrated that nppb at µm inhibited the current at min by % (p< . compared to control period, n= ). the effect of nppb was partly reversible. dids at µm had no significant effect on the slc a -mediated current (p> . compared to control period, n= ). niflumic acid at µm inhibited the current at min by ~ % (p< . compared to control period, n= ) which was partly reversible. cftr inh at µm inhibited the current at min by ~ % (p< . compared to control period, n= ) which was reversible. clamping currents in a mm k-gluconate medium and using mm k-cl or k-bicarbonate revealed very low bicarbonate conductance for slc a compared to that of cl-. no significant phi changes were observed in oocytes when favoring cl-exit by substituting perfusate cl-with gluconate in the presence of co /hco -. conversely we observed significant alkalinization in ae -expressing oocytes in the same experimental solutions. interestingly, in hek cells stably transfected with the mouse slc a , a significant intracellular alkalinization was observed upon switching to a chloride-free perfusate. the alkalization in slc a -expressing cells was inhibited by ~ % in the presence of µm dids. baseline intracellular ph was significantly increased in slc a expressing hek cells vs. non transfected cells. however, baseline phi was comparable in control and slc a -expressing cells in the presence of µm eipa, indicating the activation of endogenous nhe by slc a . in conclusion, slc a displays characteristics of an anionic channel in xenopus oocytes, with low bicarbonate permeability. in mammalian expression systems, slc a can also function in cl-/hco -exchange mode with moderate sensitivity to inhibition by dids. we propose that slc a plays an important role in anion (chloride) secretion and/or ph regulation in tracheal and gastric epithelial cells. no cell lines exist at present that robustly express endogenous wt and ∆f cftr on the same genetic background. zinc finger nucleases (zfns) allow the introduction of specific genetic changes at loci within the human genome (nature : [ ] [ ] [ ] [ ] [ ] [ ] ) . we have been using this technology in an attempt to generate human cells carrying the major cf-causing genotype -∆f -at the endogenous cftr locus. such lines could be expected to provide better understanding of the ∆f maturational processing defect, as well as new model systems for cell based assays of ∆f repair. we performed a bioinformatic analysis of cftr exon , and designed multiple distinct zfns against the ∆f dna sequence. all of these nucleases were assembled, cloned into mammalian expression vectors, and assayed for dna binding affinity and specificity. the zfns were then assayed for endogenous cftr locus editing in calu- and ht- cells using a novel, massively parallel methodology applied for the detection of genome editing events. the effort demonstrated that specific zfn pairs successfully bind to, and cleave, within exon of the endogenous cftr locus in both cell types. we will describe experiments that yielded optimized zfns for the cftr locus, and a robust viral platform to transiently deliver genome editing reagents to the human pulmonary cell line, calu- . supported by cff. the localization of slc anion transporters, as well as their function vary among members. some (slc a , slc a , slc a , slc a ) are widespread, implying specific physiological function in diverse tissues. others are expressed only in a single epithelia, e.g., slc a (prestin) in the inner ear. we have previously found that slc a is a highly electrogenic cl --hco exchanger with a large anion conductance. to determine the possible physiologic roles of slc a , we developed a chicken antibody (designed against sulfate transporter and anti-sigma domain, stas, of slc a ) to localize the protein. to test the antibody specificity for functional protein, we inject xenopus oocytes with crna for slc a , slc a or slc a and documented appropriate ph and membrane potential changes. only oocytes functionally expressing slc a were stained using our primary slc a antibody. further, specific staining was eliminated when either pre-immune serum or blocking peptide was used. slc a was only found in epithelia. in the respiratory system, slc a was in the apical membrane of nasal epithelial cells and tracheal submucosal glands. membranes of calu- cells, derived from human tracheal submucosal glands, stained indicating that our antibody also recognizes the human slc a protein. slc a is found in the proximal parts of the gastrointestinal tract (esophagus, stomach), with lesser amounts in the small intestine and colon. however, slc a is not present in liver. slc a is present in ducts of secretory epithelia such as salivary glands and pancreas. in the urinary tract, slc a is localized to the renal proximal tubule apical membrane but not in urinary bladder. in the reproductive system, we found slc a in epithelia of the uterus and seminal vesicles but not the vas deference or the prostate. finally, slc a is found on the eye surface, i.e., corneal epithelial cells. together, these results and the varied slc a physiology suggest that several epithelial function models will need to be re-evaluated to incorporate an electrogenic cl --hco exchanger and/or another apical anion channel. (support: romero- g , sindic- f ). a tgfβ- polymorphism modifies severity of cystic fibrosis lung disease in the setting of ∆f cftr homozygosity (drumm et al., nejm, ) . the mechanism(s) underlying this observation, and whether elevated or repressed tgfβ are responsible, are not known. in addition, the pathogenic role of tgfβ in other cf organs and in murine models of the disease has not been well studied. because the tgfβ codon cc genotype is associated with worsened cf pulmonary prognosis, and since high levels of tgfβ are also known to suppress cftr mrna expression and apical cftr protein expression in colonic epithelia (howe et al., exp cell res, ) , we developed a mouse model to test tgfβ/cftr interactions that may directly influence cf phenotype. we bred ∆f heterozygous mice (cftr tm kth , ∆f cftr) against the tgfβ receptor-deficient murine strain (mtrii ). mtrii mice express a dominant-negative mutant form of the tgfβ type ii receptor which lacks the intracellular (kinase) receptor domain. the receptor binds tgfβ, but fails to transmit a tgfβ dependent signal. the tgfβ receptor transgene was inserted downstream of a metallothionein-derived promoter and expressed in numerous murine tissues following induction with mm znso in drinking water. an antibody raised against active tgfβ (lc- - - , gift of dr. kathy flanders, national cancer institute) was used to establish tgfβ in murine tissues including lung and intestinal tract of the mtrii strain. rt-pcr confirmed high level transgene expression similar to gapdh internal control. nasal potential difference measurements in cftr +/+ mice expressing the tgfβ transgene indicated intact camp dependent cltransport following induction of the dominant-negative receptor. homozygous cf animals deficient for tgfβ signaling have recently been obtained and exhibit the expected litter sizes and distribution of the ∆f allele. studies of cf homozygous mice following induction with znso are in progress, and will be used to determine whether a failure to signal through tgfβ in vivo ) influences the severity of cf intestinal disease, ) alters airway or intestinal bioelectric findings characteristic of cf, or ) changes survival, histopathology or other features of the disease. these experiments are intended to provide an in vivo means of investigating tgfβ signaling and resultant cf modifier effects in vivo. supported by nih and cff. objective: to determine if the abpa phenotype observed in cftr mice was preventable by corrective gene replacement, we delivered aav cftr to the lung. in addition to further assess whether cftr-/-lymphocytes alone where responsible for the phenotype, we performed adoptive transfers of cftr-/-lymphocytes into cftr+/+ mice and proceeded to expose the mice to the abpa model. design/methods: cftr-/-mice underwent it administration of raav cftr or raav gfp. all mice were then sensitized with ug of crude af antigen via intraperitoneal route. non-injected mice were sensitized without prior virus exposure. challenges were performed on all mice, using an af solution delivered by aerosol inhalation in a closed chamber on consecutive days. serum ige levels were then obtained, as well as bronchoalveolar lavage fluid for differential cell counts, histologic and cytokine analysis. adoptive transfer experiments were done using spleens cells isolated from af sensitized delaf -/-and f +/+ mice and transfer into c /bl _rag-/-mice. after weeks post transfer c /bl _rag-/-mice with f -/-and f +/+ lymphocytes were then challenged with af. results: following challenge with af, raav -cftr mice had significantly lower total serum ige levels ( , ) as compared to raav -gfp controls ( , ) (p= . ). analysis of af-specific ige also revealed a two-fold reduction in the mice receiving the corrective gene therapy. curiously, gene transfer to the lung lowered the secretion of inflammatory cytokines from activated splenocytes of raav -cftr when compared to raav -gfp controls. these results suggest that correction of immune cells in the lung that traffic to the spleen may be partially responsible for the attenuation of the phenotype. in addition the transfer experiments showed that the hyper-ige syndrome can be partially transferred into b rag-/-by f -/-splenocytes. conclusions: correction of cftr deficiency by aav lung transfer attenuates the inflammatory phenotype associated with abpa. epithelial cells lining the vas deferens express cyclooxygenase (cox) when exposed to testosterone in vivo, although no functional link to ion transport has been established. we hypothesized that testosterone modulates cftr-mediated anion secretion across vas deferens epithelia that is stimulated via cox-dependent pathways. vas deferens epithelial cells were isolated from human and porcine tissues and cultured as monolayers on permeable supports until assayed in modified ussing chambers. rna was isolated concurrently for semi-quantitative gene expression analysis. bradykinin (bk) caused an increase in anion secretion (measured as short circuit current) and this effect was inhibited by indomethacin, which indicates cox dependency. testosterone treated monolayers exhibited a bkinduced response that is % greater than paired vehicle treated monolayers. qrt-pcr revealed that cox- mrna is times more abundant than cox- mrna in cultured vas deferens epithelial cells. the effects of bk on ion transport are inhibited by the b receptor antagonist hoe while the b receptor agonist des-arg -bk produced no effect. prostaglandin e and selective agonists of the ep ( -oh-pge ) and ep (butaprost) receptors produced concentration dependent increases in short circuit current whereas sulprostone, an agonist of ep and ep receptors, had no effect. taken together, these results suggest that testosterone plays a key permissive role for bk-stimulated anion secretion by inducing cox- expression. bradykinin acts at the b receptor to increase cox- activity and the synthesis of prostaglandins that ultimately bind to ep receptors to promote cftr-dependent hco -and cl-secretion that would be expected to modify the environment for sperm. the results further suggest that developmentally associated increases in testosterone levels during gestation and puberty will produce physiological changes in vas deferens luminal environment that are likely compromised in cystic fibrosis patients. supported by cystic fibrosis foundation schult po and ksu cobre nih rr- . background: recent studies in cystic fibrosis (cf) suggest a prominent t h profile; a facet that may interfere with normal bacterial clearance. this phenotype appears to increase secondary to infection with aspergillus fumigatus or pseudomonas aeruginosa and also, may interfere with macrophage activation. these same characteristics are present in cftr-deficient mice (cftr s x -/-fabp-hcftr (+/+), hereafter designated cf mice). as a study of pathogenesis of cf related diabetes (cfrd), we previously observed increased il- and splenocyte proliferation in cf mice subject to streptozotocin-induced hyperglycemia. herein, we hypothesized that differences in glucose concentration would increase t h profiles of splenocytes in vitro. methods: cf mice and c bl/ j mice (b mice; n= /group) were sacrificed at wk of age. splenocytes were collected and cultured at the following glucose concentrations: , , and mm. each mouse and glucose concentration was stimulated with cona, lps and cd /cd . supernatants were collected at hr and measured by luminex analysis. statistical analysis was performed by a one-way anova. results: stimulation by cona produced increased levels of il- , il- , tnf-a, gm-csf and il- in splenocytes of cf mice (p< . for all). stimulation by lps produced decreased concentrations of il- in cf mice(p< . ). stimulation of cf mice by cd /cd produced decreased concentrations of il- (p< . ). interestingly, no difference was observed in cytokine elaboration as a function of glucose concentration. data are presented in table as fold increase of cytokine concentration for cf mice compared to b mice. discussion: profound differences in cytokine production were measured in stimulated splenocyte responses of cf in comparison to b mice. the profiles did support the notion for a prominent t h response, with the potential to interference of normal macrophage activation. in addition, differences in cytokine production profiles appear related to the nature of adaptive and innate stimuli provided (cona vs. lps). the absence of influence by glucose concentration in vitro suggests that local/immediate concentration of glucose is not relevant to prolonged expression of cytokine responses to stimuli. further studies on the impact of chronic hyperglycemia and its relation to immune response are necessary. an improved understanding of these mechanisms holds hope for a better overall outcome in patients with cf and cfrd. studies from different laboratories to characterize ∆f cftr clchannel activity have yielded disparate results, likely due to variations in technique, cell type, or method of analysis. in the present study, we compared ∆f cftr activation in two well characterized polarizing cell models. fisher rat thyroid (frt) cells have been used in high-throughput drug screening to identify ∆f cftr active agents, whereas cfbe ocells have been transduced to provide robust ∆f cftr expression in an airway epithelial background. ∆f cftr biogenesis following low temperature correction was compared, indicating qualitatively similar levels of cftr transcripts and band b and c cftr. in paired studies in ussing chambers, distinct differences in isc were seen following exposure to cftr activating stimuli. in frt cell monolayers grown at °c, forskolin ( µm) or genistein ( µm) were strong stimuli of isc, producing mean isc of . +/- . (forskolin) and . +/- . (genistein) µa/cm . regardless of exposure sequence, both agents induced equivalent changes in maximal current ( % of . µa/cm by forskolin when administered prior to genistein; % of . µa/cm by forskolin in converse order). growth at °c for hours increased currents approximately . fold, but relative contributions of the two stimuli were similar to cells grown at °c ( % of total current stimulated by forskolin). in cfbe o -∆f cells grown at °c, forskolin-dependant currents were minimal and genistein-stimulated currents small ( . +/- . µa/cm , compared to zero current in parental, nontransduced cfbe ocells, p< . ). in temperature-corrected ∆f cfbe ocells, forskolin was a poor stimulus of cl-transport relative to genistein, contributing % to the total current produced by both agents (p< . ). genistein-stimulated currents did not require pre-treatment with forskolin to produce maximal effects ( . µa/cm with genistein alone vs. . µa/cm after forskolin and genistein), and forskolin did not cause further isc when added after genistein ( µa/cm ). levels of camp did not account for these differences, since forskolin markedly increased camp in both models. to determine whether excessive phosphodiesterase (pde) or phosphatase (pp) activity limited ∆f cftr activation by forskolin in the cfbe o -∆f cells, monolayers were pretreated with the nonspecific pde inhibitor papaverine ( µm) or the pp inhibitor endothall ( µm), each sufficient to activate isc in cfbe oexpressing wtcftr (papverine: . vs. . µa/cm with vehicle, p< . ; endothal: . vs. . ua/cm , p= . ). neither agent restored isc in ∆f cfbe oby forskolin ( . mv difference, p=ns). these results indicate that activity of ∆f cftr in polarizing monolayers is exquisitely dependant on the model chosen for study. substantial differences in ∆f cftr behavior in two commonly used cell models, and suggest that the failure of camp to activate ∆f cftr in cfbe ocells is due to cell-specific factors that may ) influence ∆f cftr folding and tertiary structure, or ) favor interactions of ∆f cftr with inhibitory binding partners that limit activation by camp. adverse immune responses to viral vectors or vector-encoded proteins may hinder the practical application of gene therapy. to date, little effort has been devoted to determining the immunological consequences of topically delivered lentiviral vectors to respiratory epithelia. previously, we demonstrated that an integrating feline immunodeficiency virus-based lentiviral vector pseudotyped with a baculoviral envelope glycoprotein (gp -fiv) readily transduced differentiated airway epithelia in vitro when applied to the apical surface. furthermore, using a luciferase reporter gene and bioluminescence imaging, we observed in vivo gene transfer to murine nasal epithelia following a single application of gp -fiv. longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for greater than months without significant decline. by histological analysis, surface epithelial cells were transduced following a single nasal application of gp -fiv expressing beta-galactosidase. importantly, we observed that consecutive doses of gp -fiv delivered over consecutive days greatly increased the number of beta-galactosidase positive epithelial cells. using quantitative bioluminescent imaging, we observed that repeated doses given over consecutive days resulted in a linear additive increase in gene expression. in addition, we performed studies investigating gene transfer efficacy and immune responses following lentiviral vector readministration with greater intervening time periods. in control mice, we observed that giving nasally instilled doses ( weeks apart) of an e /e deleted ad vector resulted in significantly attenuated expression after the second dose. the attenuated expression correlated to the production of neutralizing antibodies. in contrast, using the same delivery protocol, doses of gp -fiv resulted in gene transfer without loss of expression of the forth dose. no significant production of neutralizing antibodies was observed following the lentiviral delivery protocol. we further report the additive increase in reporter gene expression following doses of lentiviral vector delivered over consecutive weeks ( dose/week), as well as the lack of production of systemic and local neutralizing antibodies. gp -fiv efficiently transduces and persistently expresses a transgene in respiratory epithelia and has the potential for repeat administration without eliciting adverse immune responses. these data have important implications for the application of lentiviral gene therapy to pulmonary diseases. uniform delivery of gene therapy vectors to all lung lobes is important for cf gene therapy. however, this important objective has not been achieved in large animals. to address this obstacle, we have developed an approach to specifically deliver vector into all lung lobes. in this strategy, an intracorporeal nebulizing catheter (aero-probe) is inserted into a bronchoscope to permit visual targeted aerosolization of vector specifically into each lung lobe. using this approach, ml of . % lpc (to transiently open tight junctions) containing x vp of hdad-k lacz was sequentially aerosolized into each of the six major lung lobes of a baboon. aerosolization was triggered by the ventilator upon inspiration at . ml/pulse at a rate of to pulses/min. halfway through the procedure a very slight, transient and fluctuating decrease in oxygen saturation of no more than % was noted that did not warrant supplemental oxygen. the entire procedure was otherwise uneventful and well tolerated with no clinical manifestations of toxicity. no changes in chest x-rays taken at h, h and at necropsy h post-vector were observed compared to baseline. x-gal staining of the lungs at h revealed extensive transduction of the epithelium in the large and small airways in all six major targeted lung lobes. x-gal histochemistry revealed substantial transduction that was exclusively restricted to the airway epithelial cells and submucosal glands, the target cells for cf gene therapy. assessment of the proximal major bronchi from each lung lobe revealed that~ % of the airway epithelial cells were transduced in the left upper lobe (lul), ≥ % were transduced in the lml, lll, rul, rml and rll, and~ % were transduced in the accessory lobe. approximately % of the airway epithelial cells in the tracheal sections examined were transduced. we also investigated the duration of pulmonary transgene expression. in these studies, we aerosolized a hdad expressing the baboon α-fetoprotein from the k promoter into the lungs of baboons. by measuring serum bafp levels, we found that pulmonary transgene expression from transduced airway epithelial cells can be detected for at least days post-vector. these results demonstrate for the first time that exceedingly high levels of transduction of the airway epithelial cells and submucosal glands throughout all lung lobes in a large animal can be achieved with negligible toxicity resulting in long term trangene expression and should pave the way towards successful clinical cf gene therapy. inefficient gene transfer after repeat administration of most viral vectors has limited their suitability for cf gene therapy. however, lentiviral vectors are reported to be less immunogenic. this property combined with their capacity to integrate into the genome of transduced cells and, therefore, allowing gene expression for the life-time of the cell (approximately months for airway epithelial cells and theoretically unlimited for stem cells) justify further investigation of these vectors for cf gene therapy. to ensure high transduction efficiency in the lung without the need for any pre-conditioning strategies we pseudotyped a simian immunodeficiency virus (sivagm)-based vector with the sendai virus f and hn proteins (f/hn-siv-gfp) and transduced mouse nasal epithelium ( x e tu/mouse). at this titre transduction efficiency along the nasal septum was up to % when measured and days after transduction (n= /time-point). the vast majority (approx. %) of gfp positive cells across all time-points were respiratory epithelial cells followed by neuronal and sustenticular cells (approx. %) in the olfactory epithelium. interestingly, we observed two types of duration patterns of gfp expression in mouse nose. in some mice the number of gfp positive cells decreased over time (day : ± cells/section, n= , day : ± cells/section, n= ). however, in other mice strong gfp expression persisted for up to days (day : ± cells/section, n= ), significantly longer than the proposed life-span of airway epithelial cells. in order to support the idea of f/hn-siv integration into nasal respiratory stem/progenitor cells we artificially induced cell division after siv vector transduction by damaging the nasal tissue with the detergent (polidocanol), which strips of the epithelium within a few hours, while retaining the basal cell layer. the epithelium completely regenerates over the course of a few days. seven and days after the vector transduction ( x e tu/mouse, n= ) the nasal tissue was perfused with % polidocanol ( µl/mouse) and gene expression was analyzed weeks after the last detergent treatment. gfp expression was detectable in various cell types of the regenerating epithelium including ciliated and basal cells. importantly, gfp-expressing cells were more clustered after polidocanol treatment, possibly indicating origination from a common progenitor. these data suggest that, f/hn-siv integration may have occurred into differentiated airway epithelial cells as well as into stem/progenitor cells involved in the regeneration of the airway epithelium. introduction: hypersecretion of mucus is a major cause of airway obstruction in cystic fibrosis, asthma and chronic bronchitis. though mucus is a complex, non-homogeneous mixture of secretions, one group of its constituents, the mucous glycoproteins or mucins, contributes greatly to the obstruction associated with airway diseases. currently, there are a limited number of therapeutic agents available for controlling mucin overproduction and hypersecretion. the objective of this study is to develop a sensitive, cost-effective technique for reliably quantifying total mucin concentration in a robust mucin-secreting cell line for use in high throughput screening of small molecule compounds with potential to inhibit mucin secretion. methods: an enzyme-linked lectin assay (ella) was adapted using a lectin from helix pomatia (hpa), which binds specifically to n-acetylgalactosamine residues. bovine sub-maxillary mucin was used for test development and later, as a mucin standard. the test sample was "sandwiched" between hpa pre-coated onto -well microtiter plates and biotinylated hpa lectin. the plate was developed with a fluorescent hrp substrate, and measured at nm using a standard fluorescence plate reader. using the ella, a number of carcinoma (hm , a , ls , nci-h ) and immortalized lung epithelial ( hbe, cfsme) cell lines were tested to determine their responsiveness to various secretagogues. preliminary experiments indicated that the nci-h cells, derived from a human mucoepidermoid lung carcinoma, were most suitable for adaptation to high throughput studies. nci-h cells were seeded into -plates at a density sufficient to produce confluence in approximately hours. then, individual wells were incubated for hour in either control or secretagogue-containing media. supernatants were collected and mucin concentration of individual wells was determined using the ella. results: optimization of the ella provided a sensitive calibration curve from ng/ml to . mg/ml. mucin secretory ratios (sr) were calculated by comparing mucin release from test and control wells. significant (p ≤ . ) secretory ratios were obtained following incubation with utp at µm ( . ± . ), vip at nm ( . ± . ), carbachol at . mm ( . ± . ), interleukin- β at ng/ml ( . ± . ) and heparin-binding egf-like growth factor at ng/ml ( . ± . ); all data expressed as sr ± sem, n = . histamine at µm did not result in significant mucin secretion. conclusion: a relatively simple and economical assay has been developed for high throughput screening of small molecules for detecting their effects on mucin secretion. cystic fibrosis (cf) is the most common lethal genetic disease in north america, where it occurs with a frequency of one in ~ live births. it is caused by mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene, the most common being the deletion of phenylalanine (delf ). the delf mutation causes a folding defect that inhibits maturation and trafficking to the plasma membrane, however the mutant still functions as a chloride ion channel when delivered to the plasma membrane, and partial rescue ( - %) may be sufficient to alleviate disease symptoms. thus cftr is an attractive target for the development of pharmaceutical therapies. we recently described a novel, cell-based assay for protein trafficking which directly monitors expression of delf cftr on the surface of baby hamster kidney (bhk) cells and is suitable for high throughput screening of small molecule libraries. in this study we have extended the approach to natural products by studying extracts derived from marine sponges that were collected from the coasts of indonesia and papua new guinea. sponges are a particularly rich source of novel bioactives and secondary metabolites. iterative use of our high throughput assay and chemical deconvolution of active fractions yielded four alkaloids jmcg , jmcg , jmcg and jmcg . these compounds caused a detectable increase in delf cftr trafficking to the plasma membrane within h, and after exposure to pm for h all four increased surface delf cftr expression - % and enhanced camp-stimulated halide conductance. we tested a series of analogues of the alkaloids and found three others jmcg , jmcg and jmcg that also caused trafficking correction and functional ion channels in the plasma membrane. these results were confirmed in functional studies of the human bronchial epithelial cell-line cfbe stably overexpressing delf cftr. these compounds increased surface delf cftr by - % within h when treated with nm. these results identify a promising group of delf cftr trafficking correctors that are now being actively pursued as potential pharmacotherapeutics for cystic fibrosis along with other hits from compound library screening campaigns. work supported as part of the breathe program jointly funded by the ccff and cihr, and grants from cystic fibrosis foundation therapeutics, (cfft). kim chiaw, p. , ; bear, c.e. background/rationale: biosynthetic misprocessing of the major cf mutant ∆f -cftr has been proposed to result from the disruption in domain-domain interactions (du et al. nature struct and mol biol, ) that may lead to the exposure of di-arginine (rxr) retention/retrieval motifs. simultaneous site directed mutagenesis of all four rxr motifs to rxk or kxr motifs overcame retention and promoted cell surface expression of ∆f -cftr (chang et al. mol cell, ) . we hypothesize that it is possible to compete inhibitory interactions with rxr motifs exposed in ∆f -cftr by delivering synthetically derived cftr peptides bearing one or a combination of rxr motifs (cf-rxr), each conjugated to cell penetrating peptides (cpp) to permit intracellular delivery. results: confocal immunofluorescence microscopy confirms that alexa- -tagged cpp-cf-rxr peptides can be taken up rapidly (within min) by bhk cells stably expressing recombinant human ∆f -cftr. fluorophore-conjugated peptides appear to be concentrated in vesicular compartments, consistent with the current hypothesis that cpp-peptides enter cells into endosomal vesicles via macropinocytosis from which efflux into the cytosol occurs (wadia et al. nature med, ) . we determined that treatment of cell cultures with cpp-cf-rxr peptide at a final concentration [ µm] for minutes is effective in inducing cell surface functional expression of human ∆f -cftr in bhk cells as observed by a . -fold increase in cyclic amp-mediated iodide efflux over control ∆f -cftr (n= ; p < . ). this rescue is specific to the cf-rxr peptide as controls (cpp alone or peptides bearing a kxk motif in place of rxr) show no functional rescue of ∆f -cftr (n= ; p > . ). preliminary cyclic ampmediated iodide efflux results also indicate that ∆f -cftr functional rescue by cpp-cf-rxr peptide is as effective as temperature ( o c) rescue. although we hypothesize that the peptide acts to facilitate trafficking of the major mutant to the cell surface, immunoblotting studies fail to show a clear conversion of the er-form (band b) to the fully processed, band c form of the protein. currently, we are developing more sensitive assays of cell surface expression as well as evaluating the possible efficacy of this peptide in potentiating function of ∆f -cftr. discussion: future efforts include investigating the relative efficacy of peptides corresponding to the four different rxr motifs in rescuing ∆f -cftr trafficking. the promising candidate described above is currently being evaluated with respect to functional rescue of the major mutant in primary human bronchial epithelial cells obtained from patients homozygous for ∆f -cftr (joseph zabner and phil karp, university of iowa) and in cftr-∆f mice. these studies may provide the basis for future pre-clinical studies of peptide based therapies of cystic fibrosis (supported by ccff (breathe program grant) and cihr -(strategic training program in the structural biology of membrane proteins linked to disease)). several promising methods of promoting ∆f cftr escape from er degradation increase cftr chloride channel activity at the cell surface. however, increasing data from a number of laboratories indicate that in both epithelial and non-epithelial cell lines expressing ∆f cftr, the rescued chloride channel is unstable at the cell surface compared to wild-type cftr. small molecule correctors have therefore become a widely used approach to enhance ∆f cftr function at the cell surface. in most cases, the exact mechanism of these promising compounds has not been established. in the present study, we use stably transfected, polarized cfbe o-cells to elucidate the effect of two of these compounds, cfcor- and corr- a, on the surface stability of wild type and low temperature rescued ∆f cftr. specifically, we cultured cells expressing ∆f cftr at c for hours to establish a large pool of surface-expressed ∆f cftr; we then monitored cftr internalization rates and half-life with and without small molecule treatment using surface biotinylation-based assays, performed as described previously (jurkuvenaite et al. j. biol. chem. : , ) . as a control, internalization and half-life of transferrin receptor was also followed. our results indicate that both cfcor- and corr- a slow ∆f cftr endocytosis from ~ %/ . min to ~ %/ . min, and this is similar to wild type cftr internalization rates. neither wild type cftr nor transferrin receptor internalization rates were affected by corrector treatment, suggesting that the effects of the correctors were specific for ∆f cftr. in the surface half-life studies, both correctors doubled ∆f cftr halflife from to hours, but they did not correct the half-life to wild type levels (~ h). again, the correctors had no effect on either wild type cftr or transferrin receptor surface half-lives. our results suggest that small molecule correctors may not only rescue cftr from erad during biogenesis, but can contribute to the stabilization of ∆f cftr at the cell surface, and this effect seems to be ∆f cftr-specific. the ∆f mutation in cftr causes defects in chloride channel gating and cellular processing. cell cultures models suggest the need for both a potentiator and corrector to restore ∆f -cftr channel function and processing, respectively. previously, we identified several classes of potentiators, including 'phenylglycines', which probably bind to cftr at the cell surface and restore normal channel gating (pedemonte et al., mol. pharm. ; : - . we also identified several classes of correctors, including 'bisaminomethylbithiazoles', which may bind to ∆f -cftr in the er and partially restore its folding and plasma membrane targeting (pedemonte, et al., j. clin. invest. ; : - . these compounds were identified from screening a diverse collection of , small molecules; the most active potentiator was phenylglycine pg { -[( - h-indol- -ylacetyl)-methyl-amino]-n-( -isopropylphenyl)- -phenyl-acetamide} and the most potent corrector was the bisaminomethylbithiazole corr- a {n- ( -[ -chloro- -methoxyphenylamino]- '-methyl- , '-bithiazol- 'yl) benzamide}. here we synthesized and characterized a hybrid molecule (corr- a-linker-pg ) containing both corrector and potentiator fragments. based on sar data of potentiators and correctors, a hybrid molecule was designed that incorporated an enzymatically hydrolysable linker to generate and deliver the potentiator and corrector-linker fragments. using synthetic organic chemistry we synthesized a hybrid molecule containing a pg -oh moiety and a corr- a-linker-cooh moiety, linked with an ethylene glycol spacer through an ester bond. the potentiator and corrector fragments (after cleavage) had low micromolar potency or better. however, the intact hybrid molecule was inactive, likely because of its large size ( daltons) and hence poor cell penetration. as proof-of-principle evidence that the hybrid molecule can be hydrolyzed to the active fragments, treatment with carbonic anhydrase or rodent stool specimens gave the appropriate potentiator and corrector-linker fragments identified by lcms. smallmolecule corrector-potentiator hybrids have potential utility as single drug therapy for cf caused by the ∆f mutation. supported by cff and nih. cftr is a chloride channel gated by atp binding and hydrolysis at its two nucleotide binding domains (nbd and nbd ). the two nbds may dimerize in a head-to-tail configuration, as observed in other abc transporters, forming two atp binding pockets (abp and abp ), with the atp molecules buried at the dimer interface. abp , formed by the walker a and b motifs of nbd and the signature sequence of nbd , was proposed as the site critical for the atp-dependent opening of the cftr channel, while abp (consisting of the walker a and b motifs of nbd and the signature sequence of nbd ) may contribute to the stabilization of the open channel conformation. g d, the third most common cf-associated mutation, is characterized by a very low open probability, and patients carrying this mutation present a severe phenotype. this mutation is located in the signature sequence of nbd (and thus in abp ). we have recently characterized this mutant channel and found that its low activity is atp-independent. this behavior corroborates with the idea that the occupancy of abp by atp is crucial for catalyzing the opening of the channel. interestingly, we now found that a high affinity atp analog, n -( -phenylethyl)-atp (p-atp), increases g d currents primarily by increasing the open time of the channel. this effect of p-atp is reduced when p-atp was applied together with atp, suggesting a competition between atp and p-atp for a common binding site. introducing the mutation y g (located at abp ), which reduces by more than -fold the atp-binding affinity in wild-type channels, does not alter the effect of p-atp in the g d/y g mutant. in contrast, when we introduce the mutation w g (located at abp ) in the g d background, the effect of p-atp is reduced remarkably, suggesting that abp is the binding site where p-atp binds to increase the activity of g d channels. these new results further confirm the idea that nucleotide binding at abp could stabilize the open channel conformation. since the competition experiments show that atp and p-atp are readily exchanged in the binding site, we conclude that the atp molecule is not occluded in abp , at least under the g d background. our observation that p-atp enhances the g d activity by binding at abp implicates that abp can potentially be a target for drugs to bind and increase the channel activity. supported by cff grant to s. bompadre and nih grants to t.-c. hwang (hl r , dk r ) and s. bompadre (dk k ). van goor, f.; hadida, s.; grootenhuis, p. vertex pharmaceuticals, san diego, ca, usa several cf-causing mutations, including ∆f , g d, and r h, cause gating defects in cftr that decrease the open probability (po) of the channel, resulting in decreased cl-secretion across epithelia of multiple organs, including the lung. a therapeutic strategy for the treatment of cf is to develop pharmacological potentiators that increase po leading to increased fluid transport in affected tissues. vx- was identified through cell-based screening and chemistry optimization as a potent potentiator of cftr, including ∆f -, g d-, and r h-cftr. in single-channel recordings of cftr expressed in fisher rat thyroid cells, vx- increased the po of ∆f -cftr from . ± . to . ± . . the po of g d increased from . ± . - . ± . . these levels are similar to the po of wild-type cftr, indicating that vx- restores normal gating at the single channel level. to assess the potency and efficacy of vx- in native airway cells, cl-secretion was monitored in ussing chamber studies using human bronchial epithelia (hbe) cultured from the bronchi of cf and non-cf donors. in the absence of the potentiator vx- , cftr-mediated clsecretion in ∆f /∆f or g d/∆f -hbe reached a maximum amplitude of . ± . and . ± . ua/cm , respectively. this is ~ % of that observed in wild-type-hbe ( ± ua/cm ) and consistent with the low level of residual cftr activity reported in some individuals with severe cf disease. in ∆f /∆f -hbe, vx- caused a -fold increase (peak response; . ± . ua/cm ) in cftr mediated cl-secretion with an ec of ± nm, whereas in g d/∆f -hbe, cl-secretion increased fold (peak response; ± ua/cm ) with an ec of ± nm. the improved efficacy observed in g d/∆f -hbe is consistent with a higher cell surface density of g d-cftr compared to ∆f -cftr, which has defective trafficking to the cell membrane. vx- was found to be highly selective for cftr and to have good oral bioavailability in multiple species with a half-life of - hours. the biological and pharmacokinetic data support the clinical development of vx- for the treatment of cystic fibrosis. we would like to acknowledge the cfft for their support and dr. joseph pilewski for providing cf hbe. the pari eflow ® rapid nebulizer is a member of the "soft mist" inhaler class for the administration of aerosolized antibiotics that eliminates the need for a compressor. objectives: to compare the pharmacokinetics and safety of tobramycin inhalation solution (tobi ® ) delivered via the pari eflow ® rapid electronic nebulizer vs the pari lc ® plus jet nebulizer with compressor. methods: we compared tobramycin levels in sputum and serum, incidence of bronchospasm and frequency of adverse events (ae) of mg tobi, administered twice-daily for weeks from these two delivery systems in a randomized, crossover study in cystic fibrosis patients. blood and sputum samples were collected over a dosing interval after the first and last dose on each device (days and ). tobramycin serum and sputum area under the concentration-time curves (auc) were derived and the therapeutic ratio (mean sputum auc / mean serum auc) calculated. pulmonary function tests were performed before and min after nebulization on days and , and ae were recorded. results: the total nebulization time was reduced by half for the pari eflow rapid vs pari lc plus ( . ± . min vs . ± . min [p< . ] on day , and . ± . vs . ± . [p< . ] on day , respectively). tobramycin predose serum levels > µg/ml or cmax > µg/ml, predefined as a potential for increased risk of systemic toxicity, were not exceeded in any patient during use of either nebulizer. as tabulated below, mean systemic exposure to tobramycin from the pari eflow rapid on day was slightly lower, by %, whereas mean sputum exposure was higher, by %, compared with the pari lc plus. consequently, the therapeutic ratio was nearly -fold higher for the pari eflow rapid due mainly to higher sputum concentrations. adverse events occurred in patients using the pari lc plus nebulizer (primarily headache and abdominal pain) and in patients using the pari eflow rapid nebulizer (primarily headache, cough, dyspnea). on day , no clinically relevant bronchospasm (defined as ≥ % decrease in fev ) was observed for either treatment. the mean percentage change in fev at mins from predose on day was - . % for the lc plus and - . % for the eflow rapid. conclusions: tobi delivered via the pari eflow rapid electronic nebulizer required shorter delivery times with similar safety and a higher therapeutic ratio (sputum/serum exposure) compared with the pari lc plus jet nebulizer. sponsored by novartis pharma ag which acknowledges support from pari gmbh introduction alpha- antitrypsin (aat) is a protein protecting lung tissues by inhibition of neutrophil elastase. the function of neutrophil elastase is to digest bacteria and other foreign objects in the lungs. in a person deficient of aat, the neutrophil elastase acts uncontrolled, destroying healthy tissue. the result of the damage is emphysema, which progresses if not treated. patients show a specific impaired breathing pattern of short inhalation followed by prolonged exhalation. current therapy consists of weekly i.v. infusion of aat of about - mg/kg body weight. by this method only about % of the administered dose is estimated to reach the lungs. since aat can currently be derived only from human blood serum by an expensive purification process, the worldwide supplies are limited and do not allow to treat all patients. an improvement of the delivery efficiency is highly desirable. inhaled treatment would offer a targeted therapy by delivering aat directly to the lungs and allow for a better use of the limited drug. the eflow ® , a novel electronic nebulizer, is more efficient than conventional nebulizers. it operates by means of a perforated vibrating membrane, applying no or only little stress to the nebulized substances. hence it is well suited for the pulmonary delivery of proteins. it has been shown previously, that a % aat solution can be nebulized by eflow ® without a loss of biological activity. we investigated two eflow ® configurations comparing the in-vitro delivered dose for different breathing patterns. method a highly purified % aat preparation (kamada ltd, rehovot, israel) was aerosolized with the eflow ® electronic nebulizer. initial studies were conducted using the l configuration of eflow ® . delivery performance is compared to a specially designed xl configuration, with increased aerosol chamber volume. aerosol delivery efficiency was determined by breath simulation using an emphysema breathing pattern (tidal volume ml, breaths/min, inh:exh ratio = : . ) generated by a pari compas™ breath simulator. as reference, a standardized regular breathing pattern (tidal volume ml, breaths/min, inh:exh = : ) was also investigated. the respirable fraction (drug in droplets < and . µm) was determined using the andersen cascade impactor at . l/min. samples were analysed for aat activity by an elastase inhibition assay. when the eflow ® l configuration was investigated for the standardized breathing pattern, a delivered dose (dd) of ± % of the loaded dose ( mg) was found. this value was reduced to ± % when the emphysema pattern was applied. with the new eflow ® xl configuration an improved dd of ± % was obtained. furthermore, when the emphysema pattern was applied, the dd did not drop strongly, but remained at values above %. with % of aerosol droplets in the respirable size range (< µm) and % < . µm, a high % of aat can be expected to reach both, the central and peripheral lungs of patients. conclusion inhaled aat has the potential to significantly improve the efficiency of aat replacement therapy. a customized eflow ® electronic nebulizer produced delivered doses > % even when breathing patterns of patients with impaired breathing capabilities were mimicked. the formulation is optimized for rapid aerosol administration through use of higher drug concentrations and is taste-masked. this study was conducted to evaluate the aerosol performance of novel higher dosed levofloxacin inhalation solutions in a customized eflow ® electronic nebulizer. methods two concentrations of mp- ( mg/ml and mg/ml of levofloxacin) were nebulized at fill volumes of and ml using the eflow ® l nebulizer configuration. the in-vitro delivered dose (dd) was determined by breath simulation using a standardized breathing pattern (tidal volume ml, breaths/min, inh:exh ratio = : ) generated by a pari compas™ breath simulator. the geometric droplet size distribution was determined by laser diffraction (ld). the aerodynamic droplet size distribution was determined using the andersen cascade impactor (aci). the invitro respirable dose (rd) was calculated by multiplying the respirable fraction (rf, %droplets < µm) derived from the cascade impaction experiment and the dd derived from breath simulation. nebulization time was determined by the electronic shut-off of the eflow ® control unit. all tests were conducted for three devices in duplicate, each (n= ). the eflow ® electronic nebulizer delivered mp- at both concentrations and fill volumes with equal efficiency, obtaining in-vitro dds of around ± %. the average nebulization times were between and minutes for the ml fill volume and between and minutes for the ml fill volume and were also independent of the concentration in the investigated range. determination of the mass median diameter (mmd) by ld resulted in values between . and . µm. values of the mass median aerodynamic diameter (mmad) obtained by cascade impaction were similar (between . and . µm). the average respirable fraction was in the range between % and % for both experimental methods. conclusion the eflow ® electronic nebulizer can be customized to deliver high doses of mp- . the short nebulization times achieved are patient friendly and should support high patient compliance. mp- , with the potential for once daily administration, provides significant advantages over currently avail-able aerosol antibiotics. clinical evaluation of mp- delivered by the eflow ® nebulizer in cf patients is in progress. lavange, l. ; engels, j. ; accurso, f.j. . university of north carolina, chapel hill, nc, usa; . inspire pharmaceuticals, inc., durham, nc, usa; . university of colorado, denver, co, usa introduction: percent change from baseline in a continuous outcome variable, such as lung function, is a useful descriptive measure in therapeutic clinical trials. while often favored by clinicians as the primary efficacy measure, the use of percent change as the basis for statistical comparisons raises a number of issues. defined as *(post-pre)/pre, where pre and post represent baseline and follow-up values, respectively, percent change is a ratio of random variables and as such, does not follow a normal distribution. analyzing percent change with standard methods can result in inefficiences and may be inappropriate without a suitable transformation (e.g., logarithmic). furthermore, the treatment effect estimated on the transformed scale is difficult to interpret. the purpose of this abstract is to describe an alternative method for analyzing percent change and illustrate its utility in cf clinical trials. methods: probability plots are generated to compare the distributions of percent change in fev from baseline to study endpoint among treatment groups. the plots are similar to kaplan-meier curves, with percent change on the horizontal axis in lieu of survival time. the percentage of patients on the vertical axis depicts the cumulative percentage of patients who had a percent change at least as great as the corresponding value on the horizontal axis. a log-rank test statistic provides an overall test for any difference in distributions among treatment groups. the methods are illustrated using data from a -day, phase , multicenter, randomized, double-blind, placebo-controlled clinical trial of denufosol in cf patients with a screening fev ≥ % predicted. results: a probability plot of denufosol (active doses combined, n= ) vs. placebo (n= ) is shown below. more denufosol patients ( %) experienced improvement compared to placebo ( %). the log rank test, adjusted for study site, showed that the overall distributions of percent change in fev between denufosol and placebo were significantly different (p-value= . ). conclusions: the proposed methods provide a convenient means for assessing differences in percent change and may be useful in cf clinical trials. comparisons of both a descriptive and inferential nature can be made with minimal assumptions, thereby avoiding the pitfalls in analyzing percent change with standard techniques. the methods are easy to implement with existing software packages (e.g., sas). trial supported by inspire pharmaceuticals, inc. and cf foundation therapeutics, inc. chen, x. , ; davis, p. compacted dna nanoparticles formulated with pegylated polylysine and plasmid dna transfect airway cells in vivo at high efficiency, so they have great potential for gene therapy. the mechanism by which dna nanoparticles enter the cell and get expressed is not well understood. we previously showed that rhodamine labeled dna nanoparticles enter primary human tracheal epithelial (hte) cells within min, and accumulate in the nucleus in hr, where the transgene can be expressed. dna nanoparticles do not enter via clathrin-mediated endocytosis (cme), which leads to the degradation of internalized material in lysosomes. further characterization of the kinetics and trafficking pathway may allow us to improve the formulation of the nanoparticles and facilitate its gene delivery. as they do in hte cells, rhodamine labeled dna nanoparticles enter hela and hbeo-, an immortalized human bronchial epithelial cell line, within min, and by - hr accumulate in the nucleus, especially the nucleolus, where they colocalize with nucleolin. expression of the gfp reporter gene in the nanoparticles was observed at hr, which confirms the functionality of the labeled dna nanoparticles. cellular entry is energy dependent, as little or no intracellular rhodamine was observed at °c by hr. we also applied rhodamine labeled nanoparticles and biotin-conjugated transferrin, a marker for cme, simultaneously. little colocalization was observed during a min to hr time course, so the nanoparticles do not follow this pathway. we then found a direct and strong relationship of cell surface nucleolin and the ability of the cells to take up and express dna nanoparticles. nucleolin directly binds to dna nanoparticles with kd= . nm. manipulations of cell surface nucleolin in hela cells affect transfection of dna nanoparticles with a positive correlation. we therefore costained nucleolin in both hela and hbeo-cells treated with the rhodamine labeled dna nanoparticle. the rhodamine fluorescence colocalizes with nucleolin both in the cytoplasm at early time points and in the nucleus/nucleolus at hr and hr, which further supports that nucleolin might be associated with the nanoparticles during their trafficking. to confirm this association, we performed uptake experiments with ms- , a mouse monoclonal antibody against nucleolin, alone or with rhodamine labeled nanoparticles over a min to hr time course. the ms- antibody enters hela cells at similar kinetics to the nanoparticles. at min, we observed substantial amount of cytoplasmic staining, while nuclear staining tends to increase by time until hr. it has little or no colocalization with transferrin in the cytoplasm, which suggests that this antibody also enters the cell via a pathway other than cme. in contrast, it showed extensive colocalization with dna nanoparticles in both cytoplasm and nucleus at all time points, which suggests that cell surface nucleolin and dna nanoparticles are associated or at least in close vicinity during their trafficking inside the cell. therefore, we suggest that dna nanoparticles enter the cell by binding to cell surface nucleolin and enter the cell via the same pathway as nucleolin antibody. chen, x. , ; davis, p. dna nanoparticles are non-viral gene delivery vectors in clinical trial for treating genetic disorders including cystic fibrosis. we previously discovered that cell surface nucleolin serves as a receptor for the dna nanoparticles, and is important for their gene delivery efficiency. as nucleolin has no signal sequence or membrane-spanning domain, it is not clear how nucleolin are expressed on the outer surface of plasma membrane or what signals increase its surface delivery. we initially observed that the transfection efficiency of dna nanoparticles in hela cell decreases by . % following hr serum-free medium treatment, which reduces cell surface nucleolin by . % as determined by cell surface biotinylation and streptavidin bead pulldown. since serum depletion inhibits cell proliferation and affects cell cycle progression, we examined the level of cell surface nucleolin at different stages of cell cycle. hela cells were arrested at s phase by high concentration of thymidine, and allowed to progress synchronously through cell cycle after removing this block. surprisingly, we observed substantial increase of cell surface nucleolin at the onset of m phase, about hours after release of thymidine block. it has been reported that nucleolin is phosphorylated by a cell cycle dependent kinase (cdk) cdc , and has eight consecutive cdk phosphorylation sites. therefore it is appealing to speculate that phosphorylation by cdc might increase the targeting of nucleolin to the cell surface. we then determined the potential export signal in nucleolin by serial deletions. deletion of c-terminal aa of nucleolin, including the c-terminal glycine/arginine rich (gar) domain and the four rna recognition motifs (rrm) does not affect its arrival at the cell surface. the n-terminal aa of nucleolin is sufficient to target a gfp protein to the cell surface. in contrast, when we further delete the cdk phosphorylation sites from aa to , cell surface expression of nucleolin is significantly diminished. furthermore, nucleolin lacking the n-terminal aa is not present on the cell surface. therefore, phosphorylation on the cdk sites may serve as a signal to enhance the cell surface expression of nucleolin. there has been recent interest in dry powder inhaled mannitol as a therapeutic agent in patients with cystic fibrosis (cf). it is has been shown to increase mucociliary clearance (mcc) by airway rehydration. whilst there has been one short term clinical trial of mannitol in adult subjects [ ] , to date no studies have been conducted looking at its potential as a therapy in children with cf. it could be argued that children may have the potential to benefit most from a therapeutic agent that acts relatively proximally in the cf pathogenic pathway. the aim of this study was to determine acute tolerability of inhaled mannitol in children with cf. we recruited children (aged to years) with cf. inclusion criteria were either rhdnase treatment or an fev > % and < % of mean predicted normal value. bronchial provocation challenges with incrementally increasing doses ( , , , , , , mg) of dry powder mannitol were carried out. subjects were pre-treated with bronchodilator minutes prior to the challenge ( mcg of salbutamol or mg of terbutaline). fev was measured following each dose increase up to a maximum cumulative dose of mg. oxygen saturation monitoring was carried out throughout. a positive challenge was defined by a drop in fev of > % from baseline. these subjects received bronchodilator treatment and had spirometry repeated at minute intervals until fev returned to within % of baseline. those children with a negative challenge had spirometry repeated minutes post-completion of the challenge results mean baseline fev was % predicted ( - , sd . ). / subjects ( %) had a positive challenge. mean pd (dose of mannitol required to cause a % reduction in fev ) was mg ( - , sd ). the mean time to complete the challenge was minutes ( - , sd . ) for negative challenges and minutes ( - , sd . ) to pd for those subjects with a positive challenge. we found no association between a positive challenge and age, sex, weight, height, baseline fev , pseudomonas aeruginosa colonization, bronchodilator reversibility, previous aspergillus fumigatus sen-sitization, atopy or corticosteroid use. there was a non-significant trend for lower fef - (means . versus . ; p= . ) and higher prevalence of aspergillus fumigatus cultured in sputum at baseline ( / versus / children; p= . ) amongst those children who went on to have a positive challenge. there was no significant drop in oxygen saturation in either group. although cough was common during the challenge, other adverse events were infrequent. we find that % of children with cf could not tolerate inhaled mannitol as compared with % of adult subjects as reported in the previous study [ ] . we could not identify factors predictive of a positive mannitol challenge in these patients. the most common cause of cystic fibrosis (cf) is the deletion of phenylalanine (∆f ) in the cf transmembrane conductance regulator (cftr) chloride channel [ ] . a major problem with ∆f cftr is that the protein is defective in folding so that little mature protein is delivered to the cell surface [ ] . expression of ∆f cftr in the presence of small molecules known as correctors or pharmacological chaperones can increase the level of mature protein [ ] [ ] [ ] [ ] . unfortunately, the efficiency of correctorinduced maturation of ∆f cftr is low and other approaches are needed to increase the therapeutic value of correctors. we postulated that expression of ∆f cftr in the presence of multiple correctors that bound to different sites in the protein may have an additive effect on maturation. in support of this mechanism, we found that expression of p-glycoprotein processing mutants (cftr's sister protein) in the presence of two compounds that bind to different sites (rhodamine b and hoechst ) had an additive effect on maturation. therefore we tested whether expression of ∆f cftr in the presence of combinations of three different classes of corrector molecules would increase its maturation efficiency. it was found that the combination of the quinazoline vrt- together with the thiazole corr- b or bisaminomethylbithiazole corr- a doubled the steady-state maturation efficiency of ∆f cftr (about % of total cftr was mature protein) compared to expression in the presence of a single compound. the additive effect of the correctors on ∆f cftr maturation suggests that they may directly interact at different sites of the protein. the use of multiple correctors has the potential to increase the therapeutic value of pharmacological chaperones. pharmacol. , - . the cftr protein is expressed at the surface of the airway epithelium, where it plays a critical role in maintaining airway hydration by secretion of chloride. for gene therapy of cf lung disease to be successful, it is critical that the endogenous transgene be expressed in the correct cell type and at levels sufficient to restore normal function. the expression of high levels of cftr has resulted in cftr mediated chloride secretion in a wide range of experimental systems. however, the cell type or types that need to be corrected and the level of cftr expression required to ameliorate the pulmonary manifestations of cf are not yet clear. because cftr has been localized to the ciliated cells of human airway epithelial cells, we have been investigating the use of ciliated cell-specific promoters to improve the efficiency and safety of gene therapy for cf and other airway diseases. in previous studies a fragment of the human foxj promoter was shown to target transgene expression specifically to ciliated cells. however, expression of cftr from this promoter in transgenic cf mice did not significantly improve the cf phenotype, as measured by the nasal potential difference (pd) technique (ostrowski et al, ) . this may be due to the inability of this promoter to correct the olfactory epithelium, which comprises approximately % of the mouse nasal cavity. in cf mice, the olfactory epithelium exhibits sodium hyperabsorption and an absence of cftr mediated chloride secretion, similar to the respiratory epithelium (grubb et al, ) . in this work, we used a fragment of the ciliated cell-specific promoter foxj to drive cftr expression specifically in human ciliated cells. replication deficient adenovirus expressing either egfp or cftr from the foxj promoter were used to transduce well-differentiated cultures of human cf cells following treatment with c to disrupt tight-junctions. cultures were studied hours after treatment, and those treated with ad-foxj /egfp showed strong expression of egfp that was dependant on ciliated cell differentiation. rna analysis demonstrated strong expression of cftr from the foxj promoter, and western blotting demonstrated levels of protein that were much greater than the level in normal airway cells (> -fold). immuno-localization of cftr with specific antibodies resulted in a strong signal at the apical membrane of ciliated cells. cultures treated with ad-foxj /cftr demonstrated a significant increase in forskolin-stimulated short circuit current (isc; . +/- . µa/cm , mean +/-sem, n= ), that was approximately -fold greater than the response in ad-foxj /egfp treated cultures ( . +/- . µa/cm , n= ). the increase in isc was blocked by inh , an inhibitor of cftr mediated secretion. under these conditions, ad-foxj /cftr restored approximately % of the forskolin response of normal human airway cells ( . +/- . µa/cm , n= ). because patients expressing low levels of normal cftr mrna ( - %) have mild disease symptoms, these studies demonstrate that the incorporation of the ciliated cell-specific foxj promoter into gene therapy vectors may be useful for treatment of cf. supported by nhlbi ro hl and the cff. there is a compelling need for safe and effective ai therapy for cf lung disease. low-dose methotrexate (mtx) has been used to treat inflammatory diseases and its use has been reported in cf. encouraging results previously described regarding mtx in cf prompted this study. the study objective was to determine if mtx safely reduces inflammation in the airways of cf patients. methods: this was a single-center, open label study of mtx in stable cf patients with mild to moderate lung disease. baseline levels of neutrophils, free elastase, pro-inflammatory cytokines, and bacteria were determined from an induced sputum specimen. subjects received mg/m /week of oral mtx (single dose). after weeks of treatment, a second induced sputum specimen was obtained for the same inflammatory indices. within subject comparisons (end of treatment vs baseline) were performed for the following primary endpoints: total white cell and neutrophil counts, percent neutrophils, and concentrations of free elastase, il- , il- , tnf-α, and il- β. sputum quantitative microbiology was obtained at baseline and end of therapy. routine laboratories and spirometry were performed monthly. pharmacokinetic testing was performed on the final day of treatment. results: thirteen subjects were screened with started on mtx. six completed the protocol and withdrew early secondary to adverse events (gastrointestinal [gi] complaints and pulmonary exacerbation). five of subjects completing the protocol had declines in pulmonary function: mean change in fvc was - . l (range - . to - . ); mean change in fev was - . l (range - . to - . ); change in fef - was . l (range - . to . ). mean change in weight was - . pounds (range - to . ). mean change in free elastase was . µg/ml (range - . to . ; sd . ). mean change in il- was pg/ml (range - to ; sd ). similar results were obtained for the other cytokines. analysis of cytology specimens is pending. esr and crp did not show significant changes. two subjects completing the protocol had significant gi side effects, including requiring admission for severe abdominal discomfort. quantitative microbiology specimens revealed an increase in colony counts in subjects, decreases in , and mixed results in the th. other safety laboratories were not remarkable. pharmacokinetic studies are pending. complete statistical analysis and safety assessment are also underway. conclusions: the small sample size of this study precludes definitive conclusions regarding the safety or efficacy of mtx. however, the data suggest that ) induced sputum inflammatory indices can be used to assess an ai in clinically stable patients, and ) mtx therapy may not be beneficial, and may be difficult to tolerate as along-term ai therapy for cf lung disease. additional analyses of cell counts and microbiology from this study are ongoing, and may provide additional insight into the effect of mtx in the cf lung. sponsored by the cf foundation , a cellular component of many gram negative bacteria (e.g. pseudomonas aeruginosa), is a common airway stimulus. dampening the inflammatory response in cf reduces the progressive decline in lung function, so anti-inflammatory agents have become both a cornerstone of cf clinical care and a focus of therapeutics research development. reactive oxygen species [ros] can play a role in proinflammatory signaling, including the activation of nuclear factor κb [nfκb] . research has demonstrated increased oxidative stress in cf epithelial cells, potentially highlighting one of the mechanisms responsible for the exuberant inflammation observed. we have previously shown that cddo, a novel anti-inflammatory agent, significantly limits nfκb activation in cftr deficient cell culture models at nanomolar concentrations. using cf mice, we have also shown that intra-tracheal cddo limits neutrophil accumulation and the concentrations of proinflammatory cytokines and chemokines in bronchoalveolar lavage [bal] fluid in response to lps. the synthetic triterpenoids have been shown to activate the nrf transcription factor, thereby inducing several genes involved in redox balance. we now present data demonstrating that cddo may inhibit inflammation in models of cf pulmonary disease by reducing the oxidative stress within cells. methods: b . s -cftr tm mrc cf mice received µl of µm cddo in pbs with %dmso or vehicle (control) daily. drug was administered intratracheally with an atomizer (penncentury) for three days before stimulation. in separate experiments, all mice received µg lps or free pseudomonas aeruginosa intratracheally. mice were sacrificed by co asphyxiation and cardiac puncture hours after stimulation and bal, serum and lung tissue were obtained. the lungs were incubated in lysis buffer containing protease inhibitors and immediately frozen. upon thawing, the tissue was homogenized and whole lung protein extracted. one mg of lung protein from each experimental animal was separated by two dimensional gel electrophoresis. gels were then imaged and analyzed with pdquest to identify protein spots that were differentially expressed in drugtreated mice compared with controls. spots of interest were excised from the gels, trypsin digested and analyzed by liquid chromatography/tandem mass spectroscopy. results: over proteins with greater than -fold differences in expression were identified. we categorized proteins appearing in repeated analyses and identified several affecting intracellular redox regulation; including glutathione s-transferase mu and , peroxiredoxin and , enolase (alpha non-neuron), contrapsin, alpha- antiproteinase and cu/zn superoxide dismutase. these proteins were expressed at significantly greater concentration in the lungs of mice treated with cddo rather than vehicle. conclusion: cddo exhibits anti-inflammatory effects in mouse models of cf pulmonary disease and one potential mechanism for this effect may be the upregulation of reducing proteins to combat oxidative stress. methods: lpc ( ul of . %, . % or . % (w/v) in pbs) was administered (trans-orally via a cannula) to trachea of c l/b mice. in sheep ( of completed) we targeted delivery of . ml of . % lpc dissolved in pbs to the main bronchus of the right lung at branch in a month old sheep (bronchoscope, via tracheostomy). one hour later ul (mice) or . ml (sheep) of a lv-lacz vector ( . x tu/ml) was administered in the same manner. one week post-exposure lungs were inflation-fixed in situ, removed, stained using x-gal, sectioned, and counterstained with saf-o or h/e. results: no lacz gene expression (blue cells) was found in lungs of control (pbs-treated) mice or in the left (untreated side) of sheep lung. blue cells were found in scattered punctuate groups, or in lines of stained cells in mice and sheep. mice given the highest lpc dose ( . %) had extensive gene transfer in larynx, trachea, carina, and in large, middle and small airways of most lobes of the lung, reaching % airway perimeter cell transduction in one animal. with decreasing lpc dose the distribution and number of blues cells was reduced, but transduced epithelial cells including nonciliated columnar cells, ciliated cells, and basal cells were seen in all cases. in sheep, blue cells appeared in the right main bronchus between branch and branch , and in the branch airway, and with highest expression found near branch and . cross-sections revealed that primarily ciliated columnar cells and basal cells (and no goblet cells or macrophages) were transduced. conclusions: lv gene transfer into mouse or sheep lung can be enhanced by pretreatment with lpc. in mice this early data suggests an lpc dose-dependency. for sheep, we await further results to confirm the encouraging finding in this first study; however, it does confirm that gene transfer into the airways of large animals with a lung size similar to humans can also be achieved using lpc pre-treatment and a vsv-g pseudotyped lentivirus. the transduction of both ciliated and basal lung epithelial cells in-vivo in both models is an encouraging funding for the development and understanding of our continuing efforts to produce life-long airway gene transfer suitable for a cf airway gene therapy. supported by: nh&mrc, usa cff, sa channel crf, philanthropic donations. increased airway na+ absorption is a characteristic abnormality in cystic fibrosis (cf), and is thought to play a key role in the pathogenesis of cf lung disease. we have previously demonstrated that mimicking na+ hyperabsorption by overexpression of βenac in mouse airways results in airway surface liquid (asl) volume depletion and reduced mucus clearance causing a spontaneous cf-like lung disease with high pulmonary mortality, and airway mucus plugging, mucous cell metaplasia and chronic airway inflammation in surviving βenac-transgenic (βenac-tg) mice (mall et al., nature med. : , ). in the present study, we used βenac-tg mice to test if inhibition of increased airway na+ absorption by the classic enac blocker amiloride has therapeutic effects on cf-like lung disease in vivo. specifically, we determined the effects of 'early' and 'late' enac blocker intervention on mortality, airway mucus obstruction and inflammation by starting intrapulmonary amiloride therapy in βenac-tg mice either at birth, i.e. prior to the onset of lung disease, or after mucus obstruction and inflammation had established. to achieve this goal, newborn, day, or week old βenac-tg mice and wild-type littermate controls were treated by intranasal instillation of amiloride ( mmol/l; µl/g body weight, tid) or vehicle (h o) alone for a period of days. initial deposition studies showed that this treatment protocol resulted in pulmonary amiloride concentrations sufficient for inhibition of enac. during amiloride therapy, growth and survival were monitored, and any loss in body volume due to diuretic side effects was substituted by subcutaneous injection of isotonic saline (nacl . %). after the day treatment period, mice were euthanized, bronchoalveolar lavage (bal) was performed to determine inflammatory cell counts, and lungs were processed for histology and morphometry to quantitate airway mucus obstruction and mucous cell metaplasia. we show that amiloride significantly reduced pulmonary mortality by~ % (p < . ), when therapy was started in newborn βenac-tg mice. further, early amiloride treatment significantly reduced airway mucus content (p < . ), mucous cell numbers (p < . ), and bal eosinophils (p < . ) compared to vehicle treated βenac-tg mice. in contrast, amiloride therapy had no benefits on airway mucus obstruction, mucous cell metaplasia or inflammation, if treatment was started in day, or week old βenac-tg mice with established lung disease. taken together, our results are consistent with previous human studies where amiloride lacked therapeutic benefits in cf patients with established lung disease, and demonstrate for the first time that early inhibition of na+ hyperabsorption is an effective therapy for cf-like lung disease in vivo. these results warrant an evaluation of more potent and longer acting amiloride derivatives in chronic lung disease in mice, and a clinical evaluation of preventive enac blocker therapy in human trials. supported by cff (mall g ) and ec (mext- - objective: hypertonic saline aerosol delivered intranasally is currently being studied to enhance mucociliary clearance. there is evidence of patients' reluctance to use concentrations above % due to potential discomfort. our study was performed to determine the short term tolerance of . % and % hypertonic saline versus normal saline delivered intranasally via a nebulizer/compressor system (pari sinustartm, pari respiratory equipment, midlothian, va). methods: using the sinustartm nasal aerosol delivery system, we administered concentrations of saline solution, ( . %, . %, %) to healthy, adult volunteers for minutes each. a washout period of minutes between treatments allowed volunteers to wipe their nose and cleanse their mouth with water. a question self-administered questionnaire was completed following each treatment using a point scale ( =high tolerability; =low tolerability). burning sensation, cold, cough, throat irritation, runny nose, and overall comfort were measured. the order of treatments was randomized and volunteers were blinded to the concentration. data: for all measurements . % and % were equally well tolerated compared to normal saline with burning, cough, and throat irritation measuring . or below. no variable measured more than . . out of volunteers stated they would continue this treatment on a regular basis. conclusions: our study indicates that during the time of treatment nasal aerosol delivery of hypertonic saline up to % is well tolerated in healthy adults. there appears to be no issue of discomfort associated with hypertonic saline that would prevent nasal aerosol treatment compliance. our overall research goal is to discover new drugs for clinical treatment of cystic fibrosis (cf) that will correct the biochemical defect in the predominant cf mutation, the ∆f form of the cystic fibrosis transmembrane conductance regulator (cftr) protein, which accounts for over % of all cf cases. this mutation leads to a misfolded protein which is rapidly degraded as well as changes in its function and half-life at the cell surface. however, it has been proposed that only a fraction of the normal surface expression level is needed to provide a clinically significant impact on the disease. thus, from a pharmacological standpoint, strategies that can partially but effectively correct these defects would be expected to have a clear clinical benefit to these cf patients, and these strategies may also be applicable to other cftr mutations. we previously demonstrated that several members of a particular class of related drugs, the anthracyclines, anthraquinones, and anthracenediones, significantly increased cftr cell surface expression and function in cell culture. in particular, we demonstrated that a non-cytotoxic concentration of doxorubicin (dox), a model anthracycline drug, significantly increased total cellular and membrane-associated cftr protein levels and cftr-associated chloride currents in human colon cancer t cells, and also caused a two-fold increase in ∆f cftr-associated chloride current in a canine mdck-c -derived cell line that expresses a stably transfected copy of human ∆f cftr. our previous studies also demonstrated that dox is able to impart structural integrity to ∆f cftr, increasing its half-life and decreasing its proteolytic sensitivity, which is indicative of efficient folding. additionally, two other structurally related drugs, i.e., the anthracycline, daunorubicin, and the anthraquinone, mitoxantrone, were shown to have similar effects on ∆f cftr expression. in the current work, we have extended these effects to two other related aza-anthracenedione molecules, bbr (pixantrone) which is a cancer chemotherapy drug with lower non-target toxicity than dox and which is in phase iii clinical trials, and a structurally related analog, bbr , which is essentially non-cytotoxic with a -fold lower cytotoxic potency than bbr . both compounds increased cftr-associated chloride currents to a similar extent as dox in cfbe human airway epithelial cells expressing ∆f cftr, as measured by ussing chamber experiments. the ability of the non-cytotoxic analog bbr to do so is particularly important, since it indicates that correction of the ∆f cftr defect is not directly tied to the toxicity of this class of compounds per se but rather is a result of other structural features. thus, it is likely that this and/or other non-toxic analogs in this chemical class can be developed that have potential as clinically useful agents for treatment of cf in patients. supported by a cystic fibrosis foundation (cff) grant to jwh and rm, and by the cff-supported dartmouth cf program. cystic fibrosis (cf) is caused by mutations in cystic fibrosis transmembrane conductance regulator (cftr). cftr is not only an epithelial chloride channel, but it also regulates other transporter and ion channels, including epithelial sodium channels (enac) and aquaporin water channels. in patients with cf, absent or dysfunctional cftr results in abnormal electrolyte and fluid content on the epithelial surface. treatments intended to normalize ion transport in cf airways through non-cftr dependent pathways represent attractive approaches to alleviate the underlying pathologic defect. calcium (ca +)-activated cl-channels have been proposed as rescue channel for the cyclic amp-dependent cftr cl-channels, offering a target for cf pharmacotherapy. increases in cytosolic ca + concentration activate epithelial cl-channels, but inhibit epithelial na+ channels, which is also beneficial in correcting the hyperabsorption of na+ in cf. herein we developed a high throughput screening (hts) assay for screening compound libraries with the intention of finding compounds which can increase intracellular ca +. in the pilot primary screening, we screened a mssp library which contains known bioactive compounds and natural products. compounds were classified as the real hits after the secondary screening validation with the hit percentage . %. we have found that the ec of these compounds are less than um in dose-response studies and none of those compounds were toxic to the cells. we have further discovered that those compounds stimulate cl-secretion through activating ca + dependent cl-channels in cf and non-cf human airway epithelial cells in short-circuit current experiments. in summary, our data suggest that modulation of intracellular ca + is a target for cf therapy. the compounds identified by our study will provide possible therapeutic leads in the treatment of cf. non-viral gene delivery particles with various synthetic polymer coatings have been developed for cystic fibrosis (cf). intracellular trafficking and in vivo tissue distribution of these particles must be carefully monitored to guide rational design of efficient delivery system. fluorescent semiconductor quantum dots (qds) allow sensitive, long-term and multi-target imaging in cellular environment, and so are a promising tool in tracing gene delivery materials in vitro and in vivo. however, application has been limited by the lack of efficient and reproducible techniques of qd bioconjugation. here, we describe a method of labeling dna nanoparticles with tunable qds that may enable us to study gene delivery in vitro and in vivo. highly fluorescent zns-coated cdse qds were first synthesized and encapsulated in amine-containing phospholipid micelles. the non-viral vector system we used is based on a polymer backbone consisting of lysines with a cysteine on the n-terminus (ck ) which is conjugated to polyethylene glycol (peg). this polymer vector condenses dna plasmids into stable nanoparticles that efficiently transfect airway epithelial cells in vivo. to introduce the aminecontaining qds, we utilized a hetero-bifunctional peg with an nhs ester at one end, which was first reacted with the amine groups of the qds and a maleimide group at the other end which was subsequently reacted with the thiol group in ck . excess reagents were removed from the final conjugates qd-peg-ck by filtration. conjugation was first confirmed by . % agarose gel electrophoresis. free qds were positively charged and migrated toward the cathode. pegylated qds migrated significantly slower than free qds, and qd-peg-ck conjugates migrated at still a different rate. to further demonstrate conjugation, we biotin-labeled the ck and assessed the binding of qd-peg-ck -biotin to avidin coated agarose beads by monitoring the yield of fluorescent beads. qds, pegylated qds, and physical mixture of qds and ck -biotin were also incubated with the agarose beads as controls. fluorescent beads were detected only when qd-peg-ck -biotin was present. to directly evaluate the ability of qd-peg-ck to bind double stranded dna, we also immobilized double stranded dna fragments to agarose beads. qd-peg-ck , but not unconjugated qd bound to the immobilized dna fragments as determined by the yield of fluorescent beads. qd-peg-ck was then used to compact luciferase reporter plasmids. electron microscopy images of the compacted dna showed that qds were integrated into some dna nanoparticles. we transfected hela cells with qd-labeled and unlabeled dna nanoparticles. luciferase assay and toxicity assay indicated that qd-labeled dna nanoparticles transfected hela cells, though with less reporter gene expression than unlabeled nanoparticles and more toxicity than uncompacted qd. in conclusion, we have developed a method to conjugate qd to dna nanoparticles. improvement of the quality and biocompatibility of the qd may be required for further in vitro and in vivo studies. this system is also versatile. other less toxic optical makers can be readily tested. this study was supported by dk and the cff. in cystic fibrosis (cf) the epithelial sodium channel (enac) hyperactivity plays a role in the pathogenesis of chronic lung disease. the missing enac regulation by the cf transmembrane conductance regulator (cftr) causes increased absorption of sodium ions and fluid across airway epithelia leading to the depletion of the perciliary liquid layer and to the consequent inhibition of mucus clearance. we developed a hiv-based lentiviral (lv) vector containing a sirna cassette to efficiently knockdown the expression and activity of enac in human respiratory cells. background: sildenafil has been implicated in the relocation of cystic fibrosis transmembrane conductance regulator (cftr) protein. the effect was observed in vitro and in the presence of doses roughly times larger than those commonly used for treating erectile dysfunction. aim: to evaluate in vivo therapeutic efficiency of clinical doses of sildenafil and vardenafil, two approved type v phosphodiesterase inhibitors, for correcting chloride transport defect in ∆f mice. methods: we measured transepithelial potential difference in vivo across the nasal mucosa as a readout for sodium and chloride conductance. the effect of a single intraperitoneal injection of sildenafil ( . mg/kg) or vardenafil ( . mg/kg) was investigated in df /df and normal homozygous mice. results: in df /df mice, chloride conductance, evaluated by perfusing the nasal mucosa with a chloride-free solution in the presence of amiloride and with forskolin, was corrected h after sildenafil administration. a more prolonged effect, persisting at least for h, was observed with vardenafil. the forskolin response was increased after sildenafil and vardenafil in both normal and df mutant animals. no effect on sodium conductance was detected in any group of animals. conclusion: our results provide preclinical evidence of effectiveness of both drugs for correcting chloride transport defects in the cf. acknowledgments: this work was supported by grants from the french cf association, vaincre la mucoviscidose and by an educational grant from pfizer belgium. there is a strong interest in developing small molecules able to correct the phenotypic effects of cystic fibrosis (cf) mutations. many mutations (e.g. ∆f ) impair the function of cftr protein, by altering the protein targeting to the plasma membrane and/or by causing an abnormally low open channel probability. drug-like organic compounds may restore the correct membrane localization ("correctors") or increase channel activity ("potentiators") of mutant cftr. in the last years, various research teams have identified molecules with activity as correctors (corr- a, vrt- , miglustat, curcumin) or as potentiators (tetrahydrobenzothiophenes, phenylglycines, sulfonamides, vrt- , , -dihydropyridines). however, these compounds have been tested using different assays and the results are sometimes controversial, with marked differences in declared efficacy and potency of compounds. we have tested a panel of correctors and potentiators to directly compare their effects under the same experimental conditions. for this purpose, we have used the functional assay based on the halide-sensitive eyfp-h q/i l to measure ∆f -cftr activity in frt and a cells. the assay for potentiators consisted in stimulation with the test compound ( . - µm) plus forskolin (on cells previously incubated at °c for hours). the assay for correctors consisted in hours incubation of ∆f cells with test compounds and then determination of cftr activity in the presence of forskolin plus genistein ( µm). our results indicate that all potentiators are active in our assay with a comparable maximal effect but with values of potency that vary significantly among compounds. the potency order measured in frt cells was: dhp- = pg- (ka~ nm) > sf- (ka = nm) > dhp- = dhp- (ka~ nm) > act- b (ka = nm) > felodipine (ka = nm) > vrt- (ka = . µm) > genistein (ka = . µm). a similar order of potency was found also in a cells expressing ∆f . the activity of correctors showed a more marked dependence on cell line. while the potency was comparable between frt and a cells, the maximal effects showed clear differences. in frt cells, corr- a, corr- b and corr- c generated a maximal effect that was . - -fold higher than that obtained by incubating the cells at low temperature. conversely, vrt- and vrt- were less effective (maximal correction equivalent to - % of low temperature rescue). in a cells, all compounds were instead less effective than low temperature, with vrt- being the molecule eliciting the highest activity ( - % of low temperature). our results indicate the , -dihydropyridine dhp- and the phenylglycine pg- among the most potent activators of the mutant cftr channel. the similar behavior of potentiators in two different cell lines is consistent with the assumption that all potentiators act with a similar mechanism, by interacting with the cftr protein itself. in contrast, the cell line dependence of correctors suggests that they act with indirect mechanisms, possibly by interacting with proteins involved in cftr biosynthesis and trafficking. supported by cfft and telethon-italy. we also thank cfft and rfums for providing chemical compounds. lung damage in cystic fibrosis (cf) patients is determined by mucus accumulation, pseudomonas aeruginosa infection and chronic inflammation. extracellular gsh is a scavenger of free radicals produced by neutrophils in inflamed tissues. glutathione transferases (gst) are a superfamily of dimeric proteins which conjugate glutathione to a wide range of substrates including oxidants and are involved the synthesis of leukotriens. clinical beneficial effects have been reported in cf patients following treatment with the macrolide azythromicin (azm); anti-inflammatory properties have been proposed as possible mechanism. the aim of this study is to investigate the regulation of the gstt and gstm activity and expression by azm. reductions of about % and % on gst enzymatic activity were detected in ib - and cfsmeo-cells respectively. gsts mrna expression in cf airway epithelial cell lines was analysed by quantitative pcr (qpcr). the level of gstt and gstm basal expression in cf cells ib - was significantly higher than in isogenic non-cf cells c . we found statistically significant decreases of gstt and gstm mrna of about % and % respectively in ib - cells after treatment with azm for hours, restoring the levels observed in c cells. in cfsmeo-cells after exposure to azm we observed % and % reductions in gstt and gstm mrna respectively. the macrolide jm, known to lack clinical anti-inflammatory properties, had no significant effects on gstt and gstm mrna expression in all cell lines. furthermore, azm did not alter the mrna expression levels of gstp , a glutathione-s-transferase not differentially expressed in cf and isogenic non-cf cells. decreased expression of % and % of gstt protein has been detected by immunoblotting in ib - and cfsmeo-cells, respectively, following treatment with azm. in the same conditions we found a drastic reduction of protein level of gstm in both cf cell lines. finally, gsts activity and the expression of gstt and gstm proteins in cf cells, were reduced approximately to the same level detected after treatment with interleukin (il- ), an anti-inflammatory cytokine, shedding light on a possible correlation between gsts inhibition and antiinflammatory properties of azm. the effects of azm described in this study suggest that downregulation of gstt and gstm expression may result in increased availability of intracellular gsh making cf cells less susceptible to oxidative stress induced by chronic inflammation. inhibition of gstt and gstm might provide a therapeutic approach for limiting the effects of inflammation critical for lung damage in cf patients. this study is supported by italian cf research foundation; comitato di vicenza-associazione veneta per la lotta contro la fibrosi cistica; azienda ospedaliera verona, italy. tradtrantip, l.; padmawar, p.; yangthara, b.; verkman, a. activation of cftr chloride conductance by gpcrs involves camp elevation and pka-mediated cftr phosphorylation. we developed a 'pathway screen' in which cftr-mediated iodide influx is used as a read-out of gpcr activation. the cell-based fluorescence assay utilizes multiply transfected epithelial cells expressing wildtype cftr, yfp-h q/i l and a specified gpcr. we recently used this assay to identify a new class of nanomolar-affinity, -aryl- -benzoyl- hydroxy- -( -arylethyl)- h-pyrrol- -one vasopressin- receptor antagonists (yangthara et al., mol. pharm. , in press ). additional screening of , diverse small molecules yielded novel chemical classes of inhibitors of cftr activation. the potential molecular targets of pathway inhibitors include the gpcr, gs or gi proteins, adenylyl cyclase, phosphodiesterase, pka and cftr. a series of target identification studies was done to classify the new pathway inhibitors, which involved the use of agonists acting at different sites in the activation pathway, and specific site-of-action assays. the pathway screen yielded new small-molecule cftr inhibitors that are unrelated to thiazolidinone and glycine hydrazide inhibitors. one interesting class of pathway inhibitors, thiophenecarboxylates, represent the first small-molecule phosphodiesterase activators, which strongly reduce camp and cgmp concentration in many cell types. the best thiophenecarboxylate greatly reduced intestinal fluid secretion in closed loop mouse models of cholera and travelers' diarrhea, and slowed cyst growth in a model of polycystic kidney disease. other pathway inhibitors, which are potential effectors of g-proteins and pka, are under evaluation. the gpcr-linked cftr pathway screen developed here is useful for high-throughput parallel identification of small-molecule inhibitors of multiple targets in the cftr activation pathway. potential uses of the inhibitors identified here include therapy of secretory diarrheas, polycystic kidney disease, and cyclic nucleotide-dependent tumor growth, as well as pharmacological creation of cf-phenotype in ex vivo human tissues and animal models. supported by cff and nih. inhibitors of intestinal caccs are predicted to have anti-secretory effects in certain diarrheas, and activators of airway and intestinal caccs are of potential use in cystic fibrosis therapy (activation of 'alternative' chloride channels). the purpose of this study was to identify small-molecule cacc inhibitors and activators that target caccs directly, rather than ubiquitous upstream processes such as calcium signaling. we screened a collection of , chemically diverse small molecules using a novel high-throughput screening assay involving lentiviral introduction of a yfp-based halide sensor in cacc-natively expressing human epithelial cells. cacc inhibitors were identified from iodide influx following cacc simulation by carbachol/atp. we identified five classes of cacc inhibitors with micromolar potency, including tetrahydro-cyclopentaquinolines, and -aryl- -(trifluoromethyl)-pyrazoles each of which was unrelated to known transport modulators. two classes of compounds inhibited calcium-activated halide flux following stimulation by multiple types of agonists, including thapsigargin and calcium ionophores, and by patch-clamp analysis appear to target cacc directly. structure-activity analysis of analogs of 'hits' yielded compounds with improved potency, which have been resynthesized and characterized for use in assays of antidiarrheal effiacy in rodent models of viral and drug-induced secretory diarrheas. screening for caccselective activators that act in a sustained manner (non-transiently) was accomplished using a similar cell-based fluorescence assay, but instead testing for increased halide influx. several classes of putative cacc activators with micromolar-potency were identified in screening of , small molecules, whose mechanism-of-action and specificity are under investigation. small-molecule modulators of cacc function that target caccs directly have potential clinical applications, and may be useful in defining the physiological roles and molecular identity of caccs. supported by cff and nih. introduction: inhaled hypertonic saline 〈hs〉improves lung function and decreases pulmonary exacerbations in older children and adults with cf. initiating therapeutic interventions in the youngest patients, particularly those that target the underlying cf defect of airway surface liquid volume depletion, has potential to preserve lung function and improve prognosis. subbarao et al performed baseline, post-albuterol and post-hs lung function testing in infants using the raised volume rapid thoracoabdominal compression technique 〈rvrtc〉and demonstrated no significant drop in lung function 〈 pediatric pulmonology, 〉. however, performing three sets of rvrtc maneuvers under the same sedation could prove difficult. before conducting a therapeutic trial of hs in this population, a simplified protocol must be possible at multiple centers. we sought to evaluate a simplified approach as well as to analyze changes in lung function and clinical findings after acute administration of hs. methods: in this ongoing study, clinically stable children with cf between the ages of months and years inhale . mg of albuterol prior to sedation with chloral hydrate. rvrtc and plethysmography are then performed before and after inhalation of ml of ‰ hs. fvc, forced expiratory volume in . seconds 〈fev . 〉, fef - , frc, rv/tlc ratio, respiratory rate, oxygen saturation, and chest exam findings are recorded. predefined stopping criteria include a % drop in fev . or in oxygen saturation to below ‰. results: six subjects 〈mean age . ± . years〉 have completed the protocol with ‰ hs. comparison of post-albuterol lung function to that obtained minutes after ‰ hs revealed no changes in mean fvc 〈 vs ml; p= . 〉, mean fev . 〈 vs ml; p= . 〉, mean fef - 〈 vs ml/sec; p= . 〉, frc 〈measured after each inhaled therapy in of subjects; vs ml; p= . 〉, or rv/tlc 〈measured after each inhaled therapy in of subjects; . vs . ; p= . 〉. respiratory rate, oxygen saturation and chest exam were unchanged. conclusions: results from this study demonstrate that a two-step protocol may used to evaluate the safety of hs. based on these findings, acute administration of ‰ hs is safe in children ages months to years with cf. despite the known improvement in mucociliary clearance, preliminary findings demonstrate a lack of an immediate response in lung volume measures. given the demonstrated benefits in older children and adults, a multicenter therapeutic trial of hs is warranted. supported by the cystic fibrosis foundation. methods and results: we have characterized aav serotypes - in addition to twenty novel vectors isolated from human or macaque tissues to transduce the murine airway epithelium in vivo. vectors [ e+ genome copies (gc)/mouse] expressing α- -antitrypsin (aat) and βgalactosidase (β-gal) were co-instilled into the mouse lung or nose. transgene expression levels were monitored by assaying aat concentration in serum as well as the number and cell-types positive for (β-gal) expression in lung and nasal airways. of all vectors tested aav and aav were the two most efficient vectors in conducting airways. when these aav vectors ( e+ gc) were subsequently evaluated on human ciliated airway epithelial cultures (haec), in contrast to our findings in mouse airways, aav failed to transduce haec, whereas aav resulted in~ % of the haec expressing transgene. since aav was the most efficient vector in mouse and human airway epithelium we performed structure-function analyses of the aav vector capsid and found two atypical capsid residues that were unique in otherwise conserved positions (f , k ). to generate a potentially fitter vector, residue f was mutated to its conserved state. residue k was found to confer lung tropism and was left unchanged. the resulting vector, aav . , transduced mouse lung and nasal airway with greater efficiency than all aav vectors tested. the increased transduction efficiency of this vector was also observed (~ %) in haec derived from six different human subjects. to continue our preclinical studies in a more relevant model, aav / . expressing egfp was tested in ciliated cultures derived from macaque airways and showed - % of cells expressing egfp one week after inoculation with e+ gc. confocal microscopy revealed that aav . targeted a significant number of ciliated cells: the airway cell-types that likely require cftr expression in cf patients. aav . expressing rhesus α-fetoprotein (rhafp) was then inoculated in the nasal airways of a rhesus macaque and transduction evaluated by monitoring concentration of rhafp in the nasal lavage fluid. we have found that rhafp expression remained high (~ ng/mg) and stable for at least days. conclusion: the enhanced transduction efficiency of aav / . vector in human and macaque airway cultures and its ability to stably transduce the nasal airway of a rhesus macaque in vivo demonstrates that aav / . is a good candidate gene transfer agent for the efficient expression of cftr in human cf airway epithelium. submitted for presentation at the american society of gene therapy. supported by gsk, cff, cfpo , p , mtcc. pharmacological correction of ∆f cftr cellular processing is a potential therapeutic strategy for cystic fibrosis. recent high-throughput screening has identified synthetic small molecules, such as bisaminomethylbithiazoles (corr- ), which partially restore chloride permeability in ∆f mutant cells. the purpose of this study was to examine the utility of natural compounds (chinese medicinal herbs) as ∆f correctors. a herbal compound fraction library was constructed from herbs most frequently used in traditional chinese medicine (tcm) that are believed to contain therapeutic compounds for a broad spectrum of human diseases including lung disease. for construction of the tcm fraction library, crude herbal extracts were first prepared by % ethanol extraction on soxhlet reflux apparatus followed by automated fractionation by preparative hplc. eighty fractions were collected from each of the herbs. each fraction contained to (average . ) individual compounds as determined by analytical hplc. collected fractions were dried and milligram of the material was dissolved in µl dmso to generate mg/ml solutions in -well plates. each -well plate contained fractions from one herb. high-throughout screening was done using the frt cell-based fluorescence assay developed previously (j. clin. invest. : - , . of , fractions screened, active fractions from herbs were identified, with positive fractions verified in secondary screening. the positive fraction did not increase halide transport in control non-transfected cells, and halide transport in ∆f -corrected cells was fully abolished by cftrinh- . we have fractionated some of the most active fractions by preparative hplc to identify which compound(s) conferred activity. for example, in one fraction there were single compounds, of which conferred corrector activity with ic s < µm and efficacy comparable to that of low temperature rescue. these results demonstrate the feasibility of ∆f -cftr corrector discovery from natural compounds. further fractionation, characterization and structure determination are in progress. the unexpectedly high 'hit'rate for the natural compounds suggests their further exploration in cf therapy. cystic fibrosis (cf) is the most common genetic disease affecting the caucasian population, with an incidence of approximately one in three thousand births. cf transpires as a result from a mutation in the cystic fibrosis transmembrane conductance regulator protein (cftr), which regulates ion transport across epithelial membranes. subsequently, patients afflicted with cf have an abnormally excessive incidence of chronic lung infection, with organisms such as pseudomonas aeruginosa. because cystic fibrosis is characterized by chronic bacterial infections, excessive neutrophil recruitment to the lungs, and a coinciding increase in pro-inflammatory cytokine production and nuclear factor-kappa b (nf-κb) activation, we hypothesized that exogenous addition of the nf-κb inhibitor iκbα might ameliorate this phenotype. we cloned the human iκbα gene as well as a mutated iκbα gene into plasmids with chicken-beta actin hybrid promoters. we then tested the new plasmids, paav .cb-hiκbα and paav .cb-hiκbαm, in vitro in the presence and absence of pseudomonas aeruginosa infection in the ib - and s cell lines. both plasmids produce iκbα at high levels as shown by enzyme linked immunosorbant assays (elisas). we also show that paav .cb-hiκbα transfected ib - cells, after infection with pseudomonas aeruginosa, express significantly reduced levels of interleukin (il)- β ( fold, p< . ), il- ( fold, p< . ), and tnf ( fold, p= . ) as well as nf-κb activation ( fold, p< . ) compared to p. aeruginosa-infected ib - controls as determined by human cytokine and nf-κb phosphoprotein bio-plex assays; cytokine expression and nf-κb activation levels in infected paav .cb-hiκbα transfected ib - cells were between levels found in infected ib - and s- cells, excluding il- levels which were below s- levels of expression. flavonoids are among the most potent cftr modulators known. equol [ hydroxy- -( '-hydroxyphenyl)-chroman] is a product of intestinal metabolism of dietary isoflavones such as daidzein, and has been of recent interest in studies of cancer, cardiovascular risk, and neurologic disease. equol is metabolically stable, and % circulates in the free (non-protein bound) form, which is considerably greater than the proportion of free daidzein ( %). structural differences such as modification of ring c (e.g. saturation at c- / , and absence of carbonyl group at c- ) and an absent hydroxyl at position c- distinguish equol from compounds previously reported to modulate cftr activity. in prior work by our center, we showed that equol activates wt and ∆f cftr in membrane patches excised from bhk cells and in cfbe o-monolayers studied in ussing chambers. activation by equol occurred only after r-domain phosphorylation in wt and ∆f cftr constructs, but was independent of rdomain phosphorylation in ∆r cftr, indicating activity may be related to dimerization of the nbds or other domain-domain interaction. because a molecule that alters nbd interactions might have effects on the aberrant processing of ∆f cftr, we screened equol and other flavonoids by preincubating compounds with cfbe o-cells expressing ∆f cftr for hours, and then tested for rescue of cftr dependent cl-channel activity after exchange of media solution. we found that preincubation with equol ( - µm) induced rescue of short-circuit current compared to vehicle treated cells ( vs. µa/cm , p< . , n= ). we then evaluated hour preincubation with equol ( um) for biochemical evidence of cftr processing correction in ∆f cfbe o-cells grown in polarizing conditions. immunoprecipitation and in vitro phosphorylation demonstrated minimal formation of band c compared to vehicle treated cells, but adaptation of a more sensitive avidin label/biotinylation assay specific for surface localized cftr revealed clear evidence that equol preincubation led to cftr at the plasma membrane. next, we examined equol in hela cells stably transduced with ∆f cftr. preincubation of equol led to dose-dependent increases in halide transport measured by fluorescence-based halide efflux (spq) after stimulation by genistein ( . and . fluorescence slope (∆%/sec) with equol and µm respectively, compared to . ∆%/sec in cells pretreated with vehicle alone; p< . , n= - cells/condition). immunohistochemical staining of ∆f hela cells for cftr with - c-terminus antibody showed rescue of surface localized protein with equol ( µm) preincubation for hours, while vehicle treated cells showed only perinuclear staining. in summary, we show functional, biochemical, and immunohistochemical evidence that the naturally occurring flavonoid equol corrects the ∆f processing defect in two model systems. a naturally occurring agent that both activates and corrects ∆f cftr deserves further exploration as a potential cf therapeutic, and may lead to new insights regarding domain-domain interactions that influence the activation and biogenesis of the mutant channel. supported by nih and cff. mote ∆f cftr maturation has been identified. although several small molecule agents have been described that overcome ∆f cftr processing defects in specific cellular models, few studies have directly compared the activity of temperature corrected and chemically corrected ∆f cftr in polarized cell systems. in the present study, we examined chemical and temperature corrected activity of ∆f cftr. correctors included all members of the cfft modulator library (c , c , c , and c ; rosalind franklin university, chicago, il). in a screen using ∆f cfbe omonolayers, maximal corrector activity across the two model systems exhibited a rank order of c >> c > c = c , using forskolin ( µm) and genistein ( µm) stimulated isc as a sensitive test for ∆f cftr activity at the plasma membrane. no change in isc was observed in matched control (parental) cells lacking ∆f cftr expression. based on these results, we further defined the activity of c in ∆f cfbe oand frt model systems, including dose/response and time dependence for peak isc rescue. in cfbe o-cells, exposure to µm c for hrs produced maximal reproducible correction of ∆f cftr processing, with loss of activity following prolonged or high concentration exposure; in frt cells, peak effects were seen at hours. ∆f cftr activity following small molecule treatment qualitatively mirrored temperature correction ( °c growth for - hrs) in both cell types. maximal currents produced by stimulation with forskolin ( um) and genistein ( µm) in ∆f frt monolayers following c pretreatment were % of that produced by temperature correction (p< . ). forskolin was responsible for % of maximal current in frt ∆f cells following chemical correction and % of maximal currents following temperature correction. in ∆f cfbe o-cells, maximal currents following chemical correction were % of that produced by temperature correction (p< . ). forskolin was responsible for . % of maximal currents in cfbe o -∆f cells following chemical correction and . % of maximal currents following temperature correction. these results illustrate dose and time response with a small molecule corrector in two polarizing epithelial model systems, and provide reassurance that observations based upon ∆f cftr following low temperature incubation are relevant to functional analysis after chemical correction. similarity in activation properties between chemical and low temperature correction suggest it is unlikely that the two maneuvers result in ∆f cftr with significantly different structural properties. the studies also indicate fundamental differences in ∆f cftr behavior in frt compared to cfbe ocells, and emphasize the importance of identifying an agent that can restore camp dependent regulation to bronchial epithelial cell types. supported by the nih, cff and cfft. participants: n= patients ( randomised to azm and to placebo) who had successfully completed a course of intravenous antipseudomonal antibiotics immediately before the trial (mean age: . years; mean fev : % of predicted). measurements and results: after treatment (mean dose of . mg/kg body weight once a week) pulmonary function declined in both groups compared to baseline (i.e. after cessation of iv antibiotics). the azithromycin group had signifcantly better results regarding the mean changes in serum crp (azm: + . mg/l, placebo: + . mg/l, p= . ), lipopolysaccharide binding protein in serum, lbp (azm: + . µg/ml, placebo: + . µg/ml, p= . ), serum interleukin- (azm: - . pg/ml, placebo: + . pg/ml, p= . ) and alginate in sputum (azm: + µg/ml, placebo: + µg/ml, p= . ). quality of life (german version of the cfq) showed significantly better results after azm in adolescents and adults. azithromycin was well tolerated with no increase in treatment-related adverse events. conclusion: once-weekly azithromycin ameliorated inflammatory reactions and improved quality of life. a decline of pulmonary function after cessation of intravenous antibiotics could not be prevented, however. this study has been sponsored pfizer gmbh, germany this open-label, multicenter study was conducted in the usa and australia to evaluate the clinical responsiveness of a patient-reported outcome measure, the cfq-r respiratory scale, by determining the minimal clinically important difference (mcid) following a -day course of tobramycin inhalation solution (tis). cf patients (n= [≥ to < yrs, n= ; ≥ yrs, n= ]) with pseudomonas aeruginosa and clinical symptoms predictive of a pulmonary exacerbation (increased cough, increased sputum /chest congestion, decreased exercise tolerance or decreased appetite) were enrolled. three efficacy measures were included: )change in forced expiratory volume in second (fev ) from baseline (day ) to end of treatment (day ) or end of study (day ); )one question about change in respiratory function (days and ; global rating of change questionnaire, respiratory domain, grcq rd; =no change; =maximal improvement or worsening);and )change in cfq r-respiratory scale (day , ). average change from baseline fev (mean [standard deviation, sd]) was . % ( . ) at day and . % ( . ) at day . based on grcq-rd at day , patients ( %) reported no change in respiratory symptoms (score < . ), ( %) a minimal change (≥ . to < . ), ( %) a moderate change (≥ . to < . ), and ( %) a large change (≥ . ). mean (sd) change from baseline cfq-r respiratory was . ( . ) at day and . ( . ) at day . at day , change in cfq-r was moderately correlated with change in fev and with grcq-rd; the correlation was stronger for patients with baseline fev < % of predicted fev values (see table) . mean change from baseline on the cfq-r respiratory scale was . at day for patients with grcq-rd scores indicating minimal change in symptoms (≥ . to < . ; n= ); this provided an estimate of the mcid for the cfq-r respiratory scale for those in exacerbation. this mcid value was consistent with estimates from distribution-based methods( ⁄ sd and standard error of measurement). the cfq-r was responsive to changes in pulmonary symptoms in patients in mild exacerbation following tis treatment; the mcid in this population was . points. responses on the cfq-r-respiratory scale were moderately correlated with changes in fev . funded by gilead sciences, inc. this was an open-label, multicenter study conducted in the usa. we determined the minimal clinically important difference (mcid) for the cfq-r, respiratory scale following a -day course of tobramycin inhalation solution (tis) in patients with cf and chronic pa infection (n= , children [< yrs]). patients had received ≥ courses of tis (mean = . ) within the previous year, however their respiratory symptoms were stable at study entry, with forced expiratory volume in second (fev ) between % to % of predicted values. efficacy measures included: ) percent change in fev (l) from baseline (day ) to treatment end (day ); ) a single question about change in respiratory function (day ; global rating of change questionnaire; respiratory domain, grcq-rd; =no change, =maximal improvement or worsening); ) change in cfq-r-respiratory scale (day to ); and ) change in log pa colony-forming unit (cfu) density in sputum (day to ). the mcid for cfq-r was estimated using three methods: ) change in cfq-r (day to ) for the patient subset with minimal change in respiratory function, as determined by the grcq-rd at day ; ) cfq-r standard error of measurement (sem) from a validation sample), and ) ⁄ standard deviation (sd) of the cfq-r respiratory scale scores. at day , change from baseline fev (mean [sd]) was . % ( . ), change from baseline cfq-r was - . ( . ), and change in log pa cfus was - . ( . ) . based on the grcq-rd at day , patients ( %) reported no change in respiratory symptoms (score < . ), ( %) a minimal change (≥ . to < . ), ( %) a moderate change (≥ . to < . ), and ( %) a large change (≥ . ). pearson r-values for the correlation of change in the cfq-r-respiratory scale and change in fev , log cfus and grcq-rd were . , . , and . , respectively. estimates of the mcid for cfq-r-rd ranged from . to . for adults/adolescent patients (table) . in patients with cf who had no immediate need for antipseudomonal therapy at study entry, the cfq-r-respiratory scale appeared responsive to changes in patient disease perception following days of tis treatment; the mcid for cfq-r was approximately points for the adolescent/adult patient population. funded by gilead sciences, inc. ex vivo chloride secretion measurements (intestinal current measurement, icm) in cf patients have been established over the past years to study the cftr-basic defect in more functional detail. modified micro-ussingchambers are used to registrate the transepithelial short-circuit current (isc) in freshly obtained human rectal suction biopsies as a measure of ion transport after stimulation with secretagogues. hereby, the cftr clchannel, its amount of residual function in cf and alternative cl-channels can be investigated by a standardised protocol.in the course of the development of cftr pharmacotherapeutics as well as agents activating alternative cl-channels, icm may function as an useful outcome parameter in preclinical and clinical trials. it is easy to perform repeatedly at all patients ages and comprises the safety advantages of an ex vivo method which is relevant especially for early study phases. aim of this study was to describe reference values and quantify the intraindividual variability of different icm parameters. methods: a total of n= rectal biopsies from n= individuals; with pancreatic insufficient (pi)-cf (n= ; mean age . years), pancreatic sufficient (ps)-cf (n= ; . years), excluded cf by icm diagnostics (n= ; . years) and healthy control (n= ; . years) were included into analysis. for calculation of intraindividual variability, - biopsies/patient were compared with respect to basal tissue resistance (rt basal), basal open circuit potential difference (pd basal), basal short circuit current (isc basal) and the isc responses to stimulation with carbachol ( - mol/l, serosal), -bromocyclic monophosphate (camp) ( - mol/l, mucosal+serosal) + forskoline ( - mol/l, serosal) and histamine ( x - mol/l, serosal).results:we determined icm reference values for the groups of pi-cf (isc basal . ± . µa/cm , ∆isc carbachol . ± . µa/cm ,∆isc camp/forskoline . ± . µa/cm ,∆isc histamine - . ± . µa/cm );ps-cf (isc basal . ± . µa/cm ,∆isc carbachol . ± . µa/cm ,∆isc camp/forskoline . ± . µa/cm ,∆isc histamine . ± . µa/cm ),and healthy control (isc basal . ± . µa/cm , ∆isc carbachol . ± . µa/cm , ∆isc camp/forskoline . ± . µa/cm , ∆isc histamine . ± . µa/cm ). for the total cohort, mean coefficients of variation were: rt basal %, pd basal %, isc basal %, ∆isc carbachol %, ∆isc camp/forskoline %, ∆isc histamine %. conclusion:this first comprehensive analysis of the intraindividual variability of icm basal tissue and cl-secretion parameters provides the basis for the method as an useful outcome measure for future clinical trials aiming to rescue the cftr basic defect. possible effects of pharmacological therapeutics in cf relevant human epithelia have to be adequately interpreted with respect to subject variability and laboratories reference data. ex vivo cl-secretion measurements have the potential of being an essential step in the evaluation process of cftr-correcting/potentiating agents on their way from laboratory screening to the application in human cf tissue without any risk of toxicity. center, placebo-controlled, double-blinded pilot study we assessed safety and tolerability of . mg/d moli versus placebo (normal saline) administered by inhalation (pari lc plus) once daily for days. patients included were ≥ years of age in phase i and ≥ years of age in phase ii, with a fev > % predicted and stable lung disease. overall, subjects received moli and placebo. exclusion criteria included abpa, b. cepacia infection and severe liver disease. the study involved clinic visits over a period of weeks to assess adverse events, spirometry, pulse oximetry and quality of life. a total of adverse events (aes) were observed in subjects ( ae in subjects receiving moli ), with only (productive cough) in the moli group being of severe intensity. the most frequent aes related to the study medication were (productive) cough ( x) and dry throat/throat irritation ( x), and most of these resolved within hour after inhalation. in the moli group no significant ae, defined as a decline of fev ≥ % from baseline accompanied by symptoms, a decrease in oxygen saturation to < % or a fall of % from baseline requiring therapeutic intervention, or a change in safety parameters judged to be clinically significant was observed. this trial was not primarily designed to show efficacy; however, the median change in fev from day to day was - % in the placebo group, and + % in the moli group, and this difference was significant (wilcoxon test, p= . ). similarly, there was a significant difference between the median change in fef - % from day to day in the placebo group as compared to the moli group (- % vs. + %, p= . ). no significant changes were observed for the other study days or for fvc and pulse oximetry. moli was well tolerated in this trial, with the observed aes generally being mild and of short duration. these encouraging explorative results are currently being further evaluated in explorative and confirmatory trials. introduction: results from published data elucidate that microbes found in the upper respiratory tract are similar or the same as those found in the lower airways of cf patients. inhaled, aerosolized drug delivery to the lower respiratory tract is an established treatment route. however, drug delivery systems capable of depositing drug to the paranasal cavities are not yet established and require evidence of deposition and efficacy. pari developed the vibrent™ paranasal drug delivery system to enable the aerosol and drug to penetrate into the nose and sinuses. objectives: this study was conducted to demonstrate that the pari vibrent™ pulsating drug delivery system is capable of ventilating the human paranasal sinuses of healthy volunteers. methods: mkr-gas was continuously ventilated through the nasal tract of three healthy non-smokers in front of a single-head gamma camera (diacam, siemens, germany), using the pari vibent™ pulsating drug delivery system. the nebulizer was coupled to the right nostril and a flow resistor to an output tube was inserted into the left nostril. during ventilation with the krgas (about sec) the subject closed their soft palate to transmit the pulsation and to prevent penetration into the lower respiratory tract. the gas supply of the vibrent™ was directly taken from the mkr-gas generator output channel. kr-gas ventilation imaging was performed with and without pulsation. serial images were recorded with anterior and lateral views. additionally, mri (magnetic resonance imaging) lateral slices of the subjects' head were recorded. the gamma camera images were superimposed to the mri images by adjusting the spatial resolution. with no pulsation from the vibrent™ no ventilation of sinus cavity was visualized by gamma camera images and radioactivity was detected in the nose only. when pulsation was added the maxillary sinuses can be visualized in the gamma camera images of all volunteers. conclusions: without pulsation no ventilation was observed. gas penetration to the paranasal sinuses can be demonstrated using the pulsating action of the pari vibrent™, potentially enabling drug delivery via aerosols. this confirms results of in vitro studies using a cast model. mkr-gas ventilation of the nasal cavities during sec breath holding in front of a planar gamma camera head (anterior) using the pari vibrent without (w/o, left image) and with (w, right image) the pulsation system. the delivery and the exhaust tubing of the kr-gas are shown together with the outline of the head, obtained from mri pictures. introduction: pediatric patients with cf were previously studied in clinical trial studies of denufosol, a novel selective p y agonist that enhances ciliary beat frequency and activates chloride secretion to hydrate the airways in the lung. pediatric patients are often discouraged from participation in clinical trials until later stages of drug development. aims: in order to evaluate the safety experience with denufosol in this population, we have retrospectively examined integrated data for pediatric cf patients with mild to moderate pulmonary disease that participated in three phase studies. demographic and baseline characteristics in addition to safety and tolerability results for cf patients aged - years old are reported. methods: three phase , multicenter, randomized, double-blind, placebo-controlled, parallel group studies were conducted. patients were randomized to receive either denufosol ( , or mg) or placebo (normal saline) tid for days by inhalation. only study included denufosol mg, while all studies included denufosol mg and mg. the fev predicted normal required to be eligible was > % (study ); %- %, inclusive (study ); > % (study ). all three studies included a one-week pre-randomization period during which reproducibility of fev (l) ± % was required in order to be randomized to double-blind study medication. patients were allowed to use bronchodilators, dornase alfa and corticosteroids in studies , and . patients were allowed to use oral antibiotics including macrolides and inhaled tobramycin solution in studies and . results: a total of cf patients - years old were randomized and dosed in three -day studies. eighty-one received denufosol (active doses combined) and received placebo. demographics were similar for all treatment groups -denufosol pediatric patients had a mean (sd) age of . (± . ) years old compared to placebo pediatric patients who had a mean (sd) age of . (± . ) years. denufosoltreated patients were % male and placebo-treated patients were % male. the mean (sd) percent predicted fev at baseline was similar between treatment groups [ . % (± . ) and . % (± . ) for denufosol and placebo, respectively]. the overall incidence of treatment emergent adverse events (ae) was similar between treatment groups ( % denufosol, % placebo). the most common ae was cough, reported by % and % of patients that received denufosol and placebo, respectively. seven percent of denufosol and % of placebo patients prematurely discontinued from the study due to aes. there were no differences in compliance with administration of study drug ( % in patients given denufosol and % in patients given placebo). conclusion: doses of denufosol up to mg given tid for days were well tolerated in pediatric cf patients - years old. these data demonstrate that inclusion of patients - years old is feasible regardless of administration of denufosol or placebo. a longer term phase study of denufosol in cf patients > years old in north america is currently ongoing. acknowledgements: this research was funded by inspire and the cystic fibrosis foundation. objective: pulmonary delivery of anti-infectives provides the potential to attain pk-pd indices exceeding those which can be achieved with systemic dosing. mp- is a novel formulation of levofloxacin that enables delivery of high concentrations over a short period, and provides taste masking. the objective of this study was to: i) determine the safety of aerosol doses of mp- and, ii) determine pharmacokinetics of levofloxacin in serum, urine, and sputum following aerosol doses of mp- using the pari eflow nebulizer in normal healthy volunteers (nhv) and patients with cystic fibrosis (cf). methods: nhv and patients with stable cf were enrolled in a single within-subject ascending dose study of dose levels (loaded doses of , , and mg) of inhalational mp- (levofloxacin solution for inhalation) or placebo. study participants were monitored for safety and changes in pulmonary function. serum, urine, and sputum (cf patients only) samples were collected at various times following the dose and assayed for levofloxacin concentration using hplc. in addition, each participant received a single iv dose of levofloxacin to determine the systemic bioavailability following aerosol mp- . noncompartmental and compartmental methods were used to determine serum, sputum and urinary pharmacokinetic parameters. pharmacokinetic deconvolution methods were used to determine the amount of the dose remaining in the lung over time. results: this study is ongoing. dosing in nhvs ( active mp- , placebo) has been completed. there were no serious adverse effects, and no significant changes in pulmonary function were noted. preliminary pharmacokinetic data from nhvs show a proportional increase in serum levofloxacin concentrations with increasing aerosol mp- dose; serum aucs were . , . , . , and . mg-h/l for the iv dose, low, mid, and high aerosol doses, respectively. deconvolution of the serum levofloxacin concentrations from aerosol mp- and iv dosing shows absorption of drug from lung over time. conclusion: preliminary results show that mp- is well tolerated following aerosol administration in normal volunteers. serum concentration data following aerosol dosing suggests that absorption of levofloxacin into the systemic circulation is a major route of elimination from the lung. studies in cf patients are in progress and will be presented. methods: a mg/kg aerosol dose of mp- was given using a microspray aerosol generation device. when placed just above the tracheal bifurcation, and activated, it delivers a bolus aerosol dose to the lung. a mg/kg intravenous (iv) dose of lvx was given as a slow bolus into the lateral tail vein. plasma lvx was determined in all rats up to sacrifice at , , , or hours, at which time rats were humanely euthanized and bronchialalveolar lavage (bal) performed. fluids were analyzed for lvx concentration using an hplc/ms method. results: the plasma lvx versus time profiles of both iv and aerosol doses were best described by a two compartment model. the plasma auc after an iv dose of levofloxacin and an aerosol dose mp- were similar ( . mg᭹hr/l vs. . mg᭹hr/l, respectively) suggesting near % bioavailability from the lung. after an aerosol dose, the mean residence time (mrt) was prolonged when compared to the intravenous dose ( . vs. . hours). this delay in absorption was associated with an increase in bal lvx auc - h in bal ( . mg᭹hr/l vs. . mg᭹hr/l for iv vs. aerosol dosing, respectively). conclusion: these data show that levofloxacin is highly available to plasma following a single microspray aerosol dose of mp- . the aerosol dose does produce a slightly longer mean residence time in plasma, suggesting delayed, but complete absorption from the lung; this delay in absorption was associated with increase in bal aucs. these data suggest that high concentrations of lvx can be attained in lung fluids following an aerosol dose of mp- . auc: area under the time-concentration curve; cl: clearance; cmax: maximum concentration; f: bioavailability; mrt: mean residence time. methods: animals were assigned to one of five exposure groups for all studies. for the -day toxicology studies, animals were exposed for up to four hours daily for twenty-eight consecutive days, followed by a -day recovery period. respiratory function and cardiovascular safety were conducted on the first and last day of the -day study in dogs. rats were exposed to , , or mg/kg/day, and dogs were exposed to , , or mg/kg/day. at the conclusion of the study, necropsy was performed and all respiratory tissues were harvested, weighed, and underwent gross and microscopic examination. a separate dog respiratory safety study was conducted in which dogs were exposed to , , or mg/kg. results: four week repeat dose inhalational exposure of mp- at target doses up to mg/kg/day in dogs and mg/kg/day in rats was not associated with any test article-related changes in any respiratory tissues. aerosol administration of mp- at doses of up to mg/kg in dogs was found to have no acute effects on minute volume, tidal volume, respiratory rate, or ecg. conclusion: local effects due to the inhalational administration of levofloxacin formulated as mp- were not observed in either rats or dogs. these data suggest that the risk of respiratory toxicity from nebulized doses of mp- is low. methods: bacteria were grown overnight in mueller-hinton broth (mhb) at °c and then sub-cultured into fresh mhb and allowed to reach log phase ( hours). female swiss mice were infected under anesthesia by intratracheal instillation of . ml of a x cfu/ml bacterial suspension. ip doses were selected to provide lvx exposures (as auc) comparable to that obtained with systemic dosing regimens. for the p. aeruginosa infection, treatment was initiated by either the ip or aerosol route hours post-infection. mice were euthanized and hours after the start of treatment, lungs harvested, and bacterial counts in lung determined. results: lvx plasma pharmacokinetic profiles were nearly identical following intraperitoneal or aerosol dosing. the geometric mean log cfu/lung pair (sd) for the p. aeruginosa are shown in the table below: in both the k. pneumoniae and p. aeruginosa lung infection studies, aerosol administration was more effective than systemic administration; for k. pneumoniae, the extent of bacterial killing at hrs was ca. . log cfu greater with aerosol mp- than with systemic lvx. conclusion: aerosol administration of mp- produces a greater extent of bacterial killing than systemic dosing of lvx in mouse models of pneumonia due to k. pneumoniae and p. aeruginosa. the majority of morbidity and mortality in cystic fibrosis patients is caused by chronic and persistent lung infections especially with pseudomonas aeruginosa. since galactosyl ceramide had been previously shown to be involved in pseudomonas internalization, ceramide levels in the plasma of cf patients were assessed and compared to healthy volunteers using hplc followed by mass spectrometry. the results demonstrated that cf patients displayed significantly lower levels of several ceramide species. also, cftr-knockout mice displayed diminished ceramide levels in cf related organs (lung, pancreas, and ileum) and plasma compared to wildtype controls. treatment with a semi-synthetic retinoid (fenretinide), which was previously reported to induce ceramide in neuroblastoma cell lines, was able to increase ceramide concentrations in cf related organs in cftr-knockout mice to the levels of wildtype mice. treatment also dramatically improved the ability of cftr-knockout mice to control pseudomonas infection. following infection with pseudomonas-impregnated agar beads, fenretinide treated cftr-knockout mice were able to clear bacterial infection as efficiently as wildtype mice. overall, these findings not only documented a novel deficiency of ceramide in cf patients but also demonstrated a pharmacological means to correct this defect in cftr-knockout mice. our data provides a strong rationale for clinical intervention that may benefit cystic fibrosis patients suffering from cf lung disease. recent reports show that adult bone marrow-derived stem cells can localize to and acquire phenotypic and functional markers of lung epithelium. these findings raise the novel possibility of stem cell therapy for multitude of lung diseases. however, only small numbers of adult marrowderived stem cells localize to lung and it is not clear whether these cells will be clinically useful. we investigated whether mesenchymal stem cells (mscs) obtained from human cord blood might have increased potential to participate in structural lung remodeling. cord blood was obtained from normal deliveries at the university of vermont. mononuclear cells were isolated and plastic adherent cells were expanded and characterized as mscs according to international society for stem cells research (isscr) criteria. following systemic ( x cells/mouse by tail vein) administration to sublethally irradiated ( . gy) immunodeficient (nod/scid) mice, lungs harvested day, weeks, or months later demonstrated small numbers of human b -microglobulin positive cells in the airway epithelium at all time points. small number of cells was found also stain positive for pancytokeratin (pan-ck) and rarely, we identified cells of b -microglobulin+/pan-ck+ in the airway epithelium of these mice after weeks. we are currently characterizing the phenotype of these cells with ccsp and cftr but these data suggest that cb-mscs may be a potential alternative source of stem cells for use in lung remodeling. high-throughput screening (hts) and other drug development programs have identified cftr activators and potentiators that require secondary evaluation and mechanistic confirmation. we recently evaluated the cfft modulator library (rosalind franklin university, chicago, il) and found some, but not all, cftr potentiators induced potent phosphorylation of the regulatory-domain (r-d), conventionally viewed as the first step in cftr activation. to assess whether this observation has functional significance regarding ion channel activation by these agents, we evaluated cftr potentiators in cfbe o-and fisher rat thyroid (frt) cells stabily transduced with ∆f cftr. cells were studied in modified ussing chambers under control conditions and after correction of ∆f cftr misprocessing using either low temperature ( οc x hrs) or pre-incubation with the chemical corrector c . total currents were determined following potentiator (at reported ec ), forskolin ( µm), and genistein ( µm) stimulation. in temperature corrected ∆f cfbe ocells, potentiator (p , cfpot- ), an agent that does not induce r-d phosphyorylation, caused modest activation when acutely administered ( µm) ( . vs . µa/cm in vehicle treated cells, p < . ). importantly, this agent potentiated forskolin mediated short-circuit current (isc: . vs . µa/cm , p < . ). forskolin accounted for % (vs. % with vehicle) of total current, demonstrating potent rescue of camp dependent cftr activity, an effect not previously reported in this cell type (p< . ). in contrast, two cftr potentiators that induce r-d phosphorylation, p (uccf- , a benzoflavone intermediate, µm) and p (uccf- , an isoxazole, µm) induced modest direct activation of cftr ( . and . µa/cm respectively, p= . and p< . ), but no evidence of forskolin potentiation (p : . µa/cm , % of total stimulated current p=ns; p : . µa/cm , %, p=ns). in ∆f frt cells grown at low temperate, p elicited strong potentiation of forskolin mediated currents ( . vs. . µa/cm with vehicle, p < . ), and modest direct cftr activation ( . vs. . µa/cm , p < . ). p and p were limited to minimal (not significant) activation and no evidence of forskolin potentiation. in cfbe oand frt cells pretreated with c (to chemically correct ∆f cftr processing), p again potentiated forskolin mediated current ( . vs. . µa/cm with vehicle, p< . in the frt model) but did not directly activate i sc . findings with p and p were otherwise as seen with low temperature corrected cells. in summary, in ∆f cftr polarized epithelia, p both activates and potentiates cftr activity (with potentiation being the predominant effect, a unique observation in cfbe ocells), while p and p confer activation of cftr without forskolin potentiation. given that p and p induce r-d phosphorylation while p does not, our findings suggest that agents that do not confer phosphorylation of the r-d may be better suited to rescue the endogenous camp mediated component of i sc . screening of potential therapeutic agents for effects on r-d phosphorylation may help predict utility at restoring ∆f chloride channel activity. supported by nih and cff. activation of cftr is conventionally viewed as a two step process: pka-regulated phosphorylation of multiple sites within the regulatory domain (r-domain), followed by atp dependent gating mediated by binding sites at the interface of the two nucleotide binding domains (nbds). cftr 'potentiators', small organic molecules that overcome mutant cftr gating defects at the cell surface, have been proposed as therapies for cf. although a number of these agents are advancing to the clinical testing phase, their mechanism(s) of action are not well understood. as a step towards better characterizing cftr potentiators available through the cfft modulator library or other resources, we are developing standardized biochemical and functional assays to evaluate the r-domain during cftr activation in living cells. we have previously described a gel-shift method by which phosphorylation of isolated r-domain (residues - ) can be monitored. using this method, we have confirmed that one potentiator, p (cfpot- ), does not induce phosphorylation of the r-domain ( % of forskolin response, n= , p = ns). unexpectedly, two potentiators, p (uccf- , a benzoflavone intermediate) and p (uccf- , an isoxazole), robustly confer phosphorylation of the r-domain (p : % of forskolin response, n= , p = . , p : % of forskolin response, n= , p = . ). we found the phosphorylation conferred by either p or p could be inhibited with the pka inhibitor h ( µm). maximal stimulation of phosphorylation occurs within minutes, indicating time dependence similar to forskolin. importantly, neither p nor p increased total cellular camp, a finding confirmed by a number of other laboratories. the results may therefore implicate compartmental inhibition of cftr-associated phosphotases (eg. pp a) or phosphodiestereases (eg. pde ) as an underlying mechanism by which isoxazole or certain flavone-derivative compounds stimulate cftr. to further test this hypothesis, p , p and p are being examined for effects on ∆r-cftr chloride channel activity. these studies provide a means by which novel cftr potentiators can be biochemically categorized based on r-domain phosphorylation, a measure of the first step of cftr activation. compounds working through distinct mechanisms may have particular relevance to certain cftr mutations, and could provide synergy in the clinical setting. supported by nih and cff. mucociliary clearance (mcc) is an innate defense mechanism that protects the lungs from bacteria and viruses. mcc requires maintenance of a thin layer of airway surface liquid (asl) to eliminate inhaled particles. the asl volume is tightly regulated by a balance of ion and water transport across the airway epithelia. in cf, the loss of cftr clsecretion coupled with unregulated na + absorption via enac results in asl volume depletion and impaired mcc. although significant progress has been made in the identification of the basic disease mechanism in cf, therapeutic approaches that address abnormal cftr biogenesis are not currently available. the extracellular nucleotides atp and utp are important mediators of asl volume and mcc. in the airways, secreted atp acts on the g-protein coupled p y receptors to fine-tune mcc via the regulation of apical ion transport, ciliary beating, and mucin secretion. we have previously demonstrated that atp controls asl volume by inhibiting absorption through enac and increasing secretion through apical membrane chloride channels. p y receptor agonists are good candidates to treat cf. however, the rapid hydrolysis of atp and utp on the airway surface of cf patients limits the effectiveness of this approach. consequently, inspire pharmaceuticals has developed di-nucleotide molecules which retain the ability to activate p y receptors, but are more resistant to hydrolysis by ectonucleotidases. denufosol, inspire's lead compound for cf treatment, potently activates p y receptors to stimulate cl -/water secretion, ciliary beating, and mucin release in epithelial tissues. however, the stability and potency of denufosol has not been determined using human bronchial epithelial (hbe) cells. in primary cultures of hbes, denufosol was significantly more stable than utp. under thin-film conditions, the initial hydrolysis rate of a therapeutic concentration of denufosol ( mm) was . nmol × min - × cm - with a half-life of approximately hour. by comparison, denufosol is more than times more stable than utp on the mucosal surface of hbes. importantly, the increased stability of denufosol translated into an increase in efficacy for this compound in vitro. equipotent concentrations of denufosol and utp significantly increased asl height. however, denufosol produced maximum asl height increases that were both greater ( . % versus . % maximum increase over basal asl for denufosol and utp, respectively) and longer-lasting ( . % versus . % increase over basal asl following a minute application of denufosol and utp, respectively) compared to utp. furthermore, the asl height increases are specific for denufosol as the addition of apyrase blunts the response to utp, but not denufosol. our data demonstrate that denufosol is more stable than utp on the mucosal surface of human airway epithelia, which results in larger and more sustained increases in asl volume. recent phase ii clinical trials show that administration of denufosol over days was well tolerated and associated with improved lung function in mild cf patients. taken together, these data suggest that denufosol is a promising candidate molecule for cf therapeutics. mucociliary clearance (mcc) is the primary airway host defense against chronic exposure to infectious and noxious agents. mcc is dependent upon ciliary beating and the volume and composition of the airway surface liquid (asl). asl volume is regulated via isotonic fluid transport which is dominated by na + absorption in the superficial epithelium. in cf, unregulated na + absorption through enac drives asl volume depletion and a subsequent decline in mcc. the accumulation of mucus in the airways of cf patients supports persistent and life-threatening bacterial infection. increasing airway surface hydration represents a promising therapeutic approach for treating cf. in vivo, aerosolized osmotic agents such as hypertonic saline (hs) improve mcc and lung function in cf patients. however, the benefits derived from hs treatments are transient, as nacl is relatively rapidly absorbed by airway epithelia. previously, donaldson et al. (nejm ) tested the hypothesis that blocking na + absorption with the enac inhibitor amiloride would increase the efficacy and extend the benefit of hs. surprisingly, amiloride blunted the response to hs, which resulted from a previously unrecognized property of amiloride to inhibit aquaporin-mediated water transport. while potent inhibitors of enac would likely be of great benefit to the cf treatment milieu, the compounds then available were not adequate for this purpose. parion sciences has developed novel -substituted pyrazinoylguanidine compounds that selectively inhibit enac and are > -fold more potent than amiloride. in the present study, we evaluated the ability of two compounds, - and , to increase asl volume under thin film conditions. in primary cultures of human bronchial epithelial cells (hbes), - alone produced a small, but significant increase in asl height. in cf cultures exposed to phasic motion that simulates the shear stress generated by normal tidal breathing, - alone increased asl heights by . %. additionally, we tested the effects of - pre-addition in combination with % hs. as expected, hs alone produced a rapid and substantial increase in asl height ( . % by minutes) which declined to % bỹ hour post-treatment. pre-treatment of cultures with - prior to hs was both more potent ( . % by minutes) and longer-lasting ( % initial response at ~ hours) than hs alone. on normal hbes cultures, the compound alone increased the basal asl height by . % following a hour exposure. similar to - , in combination with hs likewise extended the longevity of the hs effect (t / ~ h versus ~ h for + hs and hs, respectively). by blocking na + absorption, the parion compounds alone increased asl volume in both normal and cf hbes. strikingly, when used in combination with hs, the parion compounds enhance and sustain the increase in asl volume associated with hs alone. our data demonstrate that the parion compounds alone are sufficient to increase basal asl volume. furthermore, these data provide a proof-of-concept that combinational therapies utilizing osmotic agents and compounds that regulate ion transport will provide therapeutic benefits to cf patients. methods: a total of patients (with fev ≥ % of predicted) received mg of arikace by inhalation for days. drug was administered using the pari lc star nebulizer. laboratory parameters, adverse events and pulmonary function tests were collected for all study subjects in order to determine safety and tolerability of arikace™. sputum samples were collected to determine changes in bacterial density, and amikacin pharmacokinetic parameters were assessed at selected time points in urine, serum and sputum specimens. change in fev , fev % predicted, fef - % and fvc relative to baseline and change in log cfu on days and were assessed. results: on day , and , the observed change for fef - % was . (p < . ), . (p = . ) and . l/sec (p = . ), respectively. on day and , the observed change for fev was . (p = . ) and . l (p = . ), respectively, and was . (p < . ) and . l/sec (p = . ) for fev % predicted. significant relationships (p ≤ . ) between log cfu and serum auc:mic ratio, and between changes in log cfu and fev , fev % predicted and fvc were identified. treatment was safe and well tolerated with the most frequent adverse events being dyspnea and headache of mild to moderate severity. conclusion: inhaled arikace™ mg/ml was well tolerated and in select patients improved pulmonary function. together these clinically relevant changes from baseline likely represent drug effect and warrant further development of liposomal amikacin for inhalation in patients with cf. allergic bronchopulmonary aspergillosis (abpa) is a disease caused by hypersensitivity to aspergillus fumigatus (af). the prevalence of abpa is approximately %- % of patients with cf and can contribute to worsening of their pulmonary disease. the treatment of abpa consists of high-dose oral corticosteroids for many months and may include also antifungal antibiotic such as itraconazole. the clinical effectiveness of corticosteroids is usually shown by an improvement in clinical symptoms and radiological parameters, as well as a reduction in total ige. the prolonged period of systemic steroids may cause significant side effects such as cushingoid appearance, hypertension, glucose intolerance and more. over the past years, we have used pulse high-dose methylprednisolone in patients with cf. method:all patients were diagnosed as suffering from abpa by the standard criteria as the may have mucoid impaction or central bronchiectasis on chest radiography, an elevated ige (> iu/ml), the presence of specific ige anti af and an elevated eosinophil count. the patient may have a positive skin prick test to af allergen. they were treated once a month with iv pulse high-dose methylprednisolone ( mg/kg/a day) for days once a month for several months until the total ige decreased to normal values. three patients were treated also with itraconazole. in patients itraconazole was discontinues due to side effects. results: we treated patients age - years old ( m/ f). four patient are pancreatic insufficient and pancreatic sufficient. ige was +/- ( - ) at the time of diagnosis and decrease to +/- ( - ). fev increased from +/- to +/- . the patients also gained weight and improved their chest x-ray finding. side effect were minor and mainly during the treatment days and resolved - days after each treatment and included malaise during the infusions, glucose intolerance on infusion days in one case, and hypertension in another case. conclusion: iv pulse steroid treatment should be considered in abpa as it was found effective with fewer side effects that should be expected from prolonged oral corticosteroid treatment. lung pathology in individuals with cystic fibrosis (cf) is linked to sodium hyper-absorption. the defective regulation of the epithelial sodium channel enac is thought to be a major contributor to reduced airway surface liquid (asl) volume and impaired mucociliary clearance of the airways. thus, strategies designed to inhibit enac function may result in clinical benefit. rna interference (rnai), mediated by short, double-stranded rna molecules, can be used to target complementary rna sequences for degradation via the rna induced silencing complex (risc). we investigated the possibility of using rnai to reduce expression of enac in the mouse lung, as proof of principle for a strategy to reduce sodium hyperabsorption in the cf lung. potent sirna molecules capable of efficient knockdown of enac alpha, beta and gamma subunits were identified in an optimised cell culture system utilising mouse kidney m cells ( , cells per -well; pmol sirna complexed with lipofectamine ; hour harvest) in which enac expression was quantified using real-time rt-pcr. approximately % knockdown of each enac subunit was observed with the most potent sirna molecules in this system. subsequently, sirna molecules were delivered to the lungs of female balb/c mice via hydrodynamic tail vein injection ( µg naked sirna, n= - ). after hours, lungs were harvested, rna extracted and enac mrna measured using real-time taqman pcr. whereas, enac subunit mrna levels in mice treated with a negative control sirna were not different from untreated mice (p < . ), delivery of enac alpha and beta-specific sirna molecules resulted in a reduction to . ± . % or . ± . % of the expression levels observed in untreated mice, respectively (p < . , mann-whitney u). interestingly, despite efficient knockdown in cell culture, in vivo treatment with the most potent enac gamma-specific sirna molecule led to no reduction in enac gamma expression compared with untreated controls (p > . ). these data show proof of principle that enac expression can be reduced in the lung using sirna. further work is needed to assess the functional consequences of inhibiting enac in the lung. gene therapy for cystic fibrosis lung disease will likely require longterm transgene expression; however, in the lung, many gene transfer agents have resulted in only transient gene expression. previously we have shown that the choice of enhancer/promoter elements has a strong influence on the duration of reporter gene expression following delivery of non-viral vectors to the mouse lung (gill et al., , gene ther, , . however, it is also possible that other elements of plasmid vector design play an important role. using a clinically relevant mouse lung aerosol model, we have studied the effect of varying the plasmid cpg-dinucleotide content on the duration of expression from plasmids that had an identical enhancer/promoter sequence. firstly we constructed a cpg-free plasmid vector containing a synthetic cpg-free luciferase gene under the transcriptional control of the human cmv enhancer/ef α promoter. secondly, we constructed a similar vector containing an identical enhancer and promoter but with a small number of cpg motifs, in the luciferase gene ( cpgs) and in the backbone sequence ( cpgs). we then investigated the level and duration of transgene expression following aerosol delivery of these plasmids complexed with the genzyme lipid gl to the lungs of balb/c mice ( . mg/ml pdna, mm gl , ml, pari lc+ nebuliser). total lung extracts were assayed for luciferase activity at days , , , & (n= per time-point). the cpgcontaining plasmid initially directed approximately -fold higher levels of reporter gene expression than the cpg-free plasmid (p< . both days and post-delivery). however, while expression from the cpg-containing plasmid subsequently fell slowly to background levels, expression from the cpg-free plasmid increased -fold to day , and remained at the peak levels observed with the cpg-containing plasmid until the end of the experiment at day (cpg-free plasmid -fold, -fold and -fold higher expression at days , and post-delivery respectively; p< . for each). these data suggest that the ability of a promoter/enhancer sequence to direct long-term expression in the mouse lung is dependent by the sequence of the plasmid backbone. one explanation for this observation is that a specific, but as yet unidentified, sequence exists in the cpg containing backbone that triggers transcriptional silencing. alternatively, the well-described host inflammatory response to cpg-containing dna, may be involved and the cpg content of the vector could be responsible for these differences. we are currently evaluating the potency and duration of effect of cftr expressing, cpgfree, plasmids in late-stage pre-clinical studies prior to the initiation of a further round of clinical studies of non-viral gene transfer in cf patients. background: novel approaches to cf therapy by improving/restoring cftr chloride channel function using gene therapeutic tools or small molecules are currently undergoing phase i and phase ii clinical trials. there remains a great need for accurate and practical methods of measuring cftr function in vivo to act as primary outcome measures of efficacy. we propose that an in vivo assay measuring cftr function in sweat glands offers several potential advantages over the nasal potential difference test. objective: to develop a reliable test of sweat gland function that is capable of measuring the range of cftr function in vivo. methods: we are performing repeated measures of sweat gland function in healthy controls (n= ), obligate heterozygotes (n= ), pancreatic sufficient (cfps, n= ) and insufficient cf patients (cfpi, n= ). sweat secretion is stimulated by iontophoresis ( µa/cm ) with % pilocarpine. to elucidate the most sensitive and discriminatory parameter of sweat gland function within this study group we are performing simultaneous measurements of: a) the transglandular potential difference of a stimulated skin area using an ecg electrode (espd) and b) single sweat gland potential differences in single sweat glands (spd). using a wescor® collector cup we also measure: c) sweat secretion rate and (d) sweat chloride concentration (sweat cl-). results: we report interim results of our ongoing study (table) . sweat rate was not different between the groups, but a gender difference was observed (mean±sd; male: . ± . µl/min/cm ; female: . ± . µl/min/cm , p< . ) as previously described. our preliminary data show that espd as well as sweat cl-allow good discrimination among healthy controls, cfps and cfpi (p< . and p< . respectively). spd show similar trends to espd but with greater overlap (differences not significant). conclusion: these encouraging preliminary results, particularly the espd method and sweat cl-following stimulation with a lower iontophoretic current, justify further efforts to complete enrolment of subjects and to further refine technical challenges such as minimizing the effect liquid junction potentials across the ecg electrode. results will also be compared to the classical sweat test using higher iontophoresis current and npd measurements in these patients. this study is supported by the ccff and genome canada. ino- , a prodrug, has been demonstrated to inhibit nasal potential difference (pd)in human cf nasal airway epithelia and cf mice and this effect is more potent with repeated dosing. as such it is being developed as potential therapeutic for cystic fibrosis. ino- cell entry is facilitated by its hydrophobic propionoxy(methyl)ester protecting groups which can be hydrolyzed by intracellular carboxyesterases after the prodrug enters the cell. once the protecting groups are removed, the drug (ino- ) with the ether-linked octyl group is expected to be more slowly metabolized. however, the precise kinetics of the uptake of ino- , its conversion to its active metabolite, and methods. confluent cultures of hela or t cells were incubated for varying periods of time ranging from min to hrs with medium containing [ h] ino- ( x cpm/ml) and µm ino- cold carrier to ascertain the rate of uptake. in pulse chase experiments after cultures were incubated for hours, they were washed and incubated with media without radiolabel for varying periods of time. after the indicated times, the media was removed and the cells were washed, harvested, extracted and the aqueous and organic extracts subjected to sax and reversed phase hplc respectively. results ino- , a prodrug, has been demonstrated to inhibit nasal pd in human cf nasal airway epithelia and cf mice and this effect is more potent with repeated dosing. ino- cell entry is facilitated by its hydrophobic propionoxy(methyl)ester protecting groups which can be hydrolyzed by intracellular carboxyesterases after the prodrug enters the cell. once the protecting groups are removed, the drug (ino- ) with the ether-linked octyl group is expected to be more slowly metabolized. however, it is not known whether the mucus present in cf airways could impede its access to airway epithelia or whether ino- would cross the epithelial layer to enter the bloodstream. here, in order to address these issues we measured its uptake into human tracheal epithelial sheets in the presence and absence of mucus and compared its transepithelial permeation to mannitol. we studied the uptake and transepithelial fluxes of [ h]ino- in primary cultures of human tracheal epithelium with transepithelial resistance of from to ohms.cm . the mucosal surface of some tissues was rinsed three times with pbs to remove mucus for comparison with control cells which were not rinsed. elisa for human airway mucins performed on the rinsings confirmed that three rinses sufficed to remove the mucosal mucous blanket. the development of long-term, safe and efficacious gene therapies for lung disease will require efficient gene delivery to stem cells which produce the differentiated progeny of affected epithelial tissues. recombinant lentiviral vectors are being considered for gene therapy applications for the lung because they can efficiently transduce a wide variety of stem and progenitor cell types. however, it is of concern that strong constitutive viral promoters have produced oncogenesis in x-scid patient recipients of retroviral vectors in part due to the disregulation of proto-oncogenes by the strong viral promoter/enhancer located near the integration site. we propose that regulation of transgene expression to the tissue or cell type of interest will enhance the safety of gene therapy for respiratory disease. in this study we have investigated the potential for respiratory specific transgene expression from integrated lentiproviruses in cell lines in vitro and in mice in vivo. the lentiviral vectors constructed for this study contained regulatory elements predicted to produce lung specific transgene expression: the surfactant protein c promoter (spc) for alveolar epithelial type ii cell (aecii) expression, the clara cell kd protein (cc ) for clara cell expression in the airway, and the jaagskiete sheep retrovirus (jsrv) promoter for expression in both cell types. transgene expression from the spc and cc vectors was restricted to aecii and clara cell lines respectively, while expression from the jsrv vector was observed in multiple respiratory and non-respiratory cell types. following intra-tracheal delivery of lentivector supernatant to mice, transgene expression was observed in aecii from the spc lentivector (n= mice), and in clara cells from the cc promoted lentivector (n= mice). transgene expression was not detected in non-respiratory tissues following intravenous delivery of cc and spc lentiviral vectors to neonatal murine recipients (n= and n= respectively). in summary, incorporation of genomic regulatory elements from the spc and cc genes resulted in respiratory specific transgene expression in vitro and in vivo. these vectors will provide a useful tool for the study of lung biology and the development of gene therapies for lung disorders such as cf. cystic fibrosis (cf) is associated with chronic pulmonary inflammation and progressive lung dysfunction, associated with the formation of oxidants derived from neutrophil myeloperoxidase (mpo). indeed, respiratory tract levels of mpo are extensive, and have been found to correlate with decreases in respiratory parameters or disease severity in cf. mpo-derived oxidants thus may play a role in perpetuating the progressive lung dysfunction associated with cf. based upon these premises, we reasoned that mpo could represent a novel therapeutic target for the treatment of inflammatory diseases such as cf. herein we report that substituted urea and thiourea compounds are potent inhibitors of mpo. utilizing tetramethylbenzidine (tmb) as a classical heme peroxidase substrate, we screened a large number of structurally varied urea and thiourea derivatives. alkyl, cycloalkyl, and aromatic ureas and thioureas all proved to be effective mpo inhibitors, however, with a high degree of variability in potency. of those compounds examined, substituted phenylthioureas proved to be the most potent mpo inhibitors. various ortho-, meta-, and para-substituted phenylthioureas (ptus) were then assessed for inhibitor potency. the most potent inhibitor of mpo was -chlorophenylthiourea (ic = nm, ki = nm). the potency of ortho-ptus as mpo inhibitors showed a linear relationship with ortho sigma substituent constants (reflecting resonance, steric and inductive effects). moreover, cyclic voltametry experiments with ortho-substituted ptu compounds revealed a linear relationship between inhibitor potency and thiourea oxidation potential. this correlation is indicative of a substituent effect on the relative ease of sulfur oxidation within the thiourea moiety. this finding suggests that the redox-active sulfur plays a crucial role in mpo inhibition, perhaps via a direct interaction with the heme of mpo. thioureas proved to also inhibit the chlorinating activity of mpo, whereas sustituted ureas were not effective in this regard. both ureas and thioureas were also effective inhibitors of dityrosine formation by mpo. these findings will help facilitate the rational design and optimization of mpo inhibitors with even greater potency, which could serve as novel therapeutic agents for the treatment of cf. gene therapy is the transfer of a normal copy of the cftr gene and should, in theory, provide a long-term cure for cf. the development of prenatal screening programs for cystic fibrosis has provided an opportunity to identify cf patients in utero, and may provide an opportunity to treat the disease before birth, prior to the onset of disease symptoms. the long-term goal of our study is to investigate the therapeutic potential of delivering the normal cftr gene to epithelial stem cells in fetal cf mice by in utero administration of recombinant lentiviral vectors carrying the normal cftr gene. this would provide us with the unique opportunity to "correct" the chloride channel defect in the epithelial stem cells, which give rise to the epithelial cells in the tissues affected by cf at a very early stage. the aim of the current study is to optimize the in utero gene delivery of recombinant lentiviral vectors, carrying an egfp reporter gene to epithelial stem cells in fetal mice. mice have been used for these studies, as mouse strains with a variety of cf mutations are available. in all studies, the uterus was exteriorized following a mid-line laparotomy, and the injections were made directly into the amniotic fluid of individual fetuses. in the first studies, the survival of pregnant females and pups following saline injection at e . (n= ) and e . (n= ) was evaluated. all five females survived the procedure and an average of % ( / ) of the pups present at the time of injection were born alive. this experiment demonstrated that the surgical procedures were reasonably safe. in the next experiments, we injected . ul lentiviral supernatant (~ . x iu/pup) into the amniotic fluid of each pup and evaluated the effect of the gestational age pups on transduction efficiency. the pregnant mice were allowed to carry the fetuses to term. at p , p and p , - pups of each litter were sacrificed and skin, trachea, lung, stomach and intestines were harvested for analysis of gene transfer and egfp transgene expression. all but one pup ( / ) from the e . time point aborted prior to parturition. from the e . and e . injections, / and / pups were born naturally, respectively. at e . and e . , proviral sequences were observed in the skin, trachea and stomach, although the level of transduction varied among individual fetuses and tissues. immunofluorescence analysis on the skin of one mouse with a high proviral copy number demonstrated that some of the egfp positive cells were also positive for vimentin or f / , markers of mesenchymal cells and macrophages, respectively but negative for pancytokeratin. our preliminary studies suggest that in utero gene therapy with lentiviral gene delivery may be safe, however ongoing studies will provide more in depth analysis on safety and feasibility of delivering the normal cftr cdna to epithelial stem cells in the tissues affected by cf in utero. future studies will investigate the therapeutic potential of delivering a lentiviral vector containing the cftr gene to fetal pups in a murine model of cf. supported background & objectives: cystic fibrosis is characterized by progressive loss of lung function resulting from chronic bacterial infection and concomitant airway inflammation. while a number of bacteria are known pathogens in cf, mounting evidence suggests that the lungs of cf patients harbor numerous bacterial species, and that interactions within this ecosystem may affect outcomes. however, the composition of this community is not well characterized. we therefore initiated work to profile the bacterial diversity of the lungs of cf patients with end-stage disease by s rrna gene sequencing. methods: genomic dna was purified from snap-frozen explanted lung tissue of randomly selected cf patients undergoing transplantation in toronto between and . bacterial s rrna genes were amplified from each sample using broad-spectrum pcr primers, and pcr products used to generate s rdna libraries for each patient. s rdna profiles for each patient were generated by automated dna sequencing of up to randomly selected clones per library, and tentative species assignments made by comparison to known s rrna gene sequences using phylogenetically-based analyses. results: sequence analysis of s rdna sequences from this preliminary set of patients (avg. = sequences/patient; range = - ) demonstrates a range of complexity of the microflora in cf lungs, with some patients showing a single dominant organism and others showing multiple co-existing species. in total, over species have been identified to date in this set of patients. these include species identified previously by clinical microbiology, including pseudomonas aeruginosa (detected in / patients), and burkholderia species (detected in / patients known clinically to be infected). numerous organisms not previously reported in the lung were also identified, including several from the taxonomic order burkholderiales. interestingly, while organisms commonly associated with cf were detected in patients transplanted throughout the year, a cluster of four plant-associated species demonstrated striking seasonal variation in rates of detection. conclusions: the lungs of cf lung transplant patients harbor a diverse bacterial community. sequencing of s rrna genes is a powerful method to profile this diversity. correlation of these profiles with clinical information may help identify new patterns of infection and important interactions between species in this community. introduction and aims: pulmonary infection, primarily with pseudomonas aeruginosa, is the leading cause of morbidity and mortality in cystic fibrosis (cf) patients. we have demonstrated that anaerobic bacteria, belonging primarily to the genera prevotella, veillonella, propionibacterium and actinomyces, can be cultured from the sputum of cf patients. the aim of this study was to determine the antibiotic susceptibility of these anaerobic isolates grown planktonically and as biofilms. methods: the planktonic susceptibility of anaerobic isolates to antibiotics used in the treatment of cf (meropenem, piperacillin/tazobactam) as well as to antibiotics traditionally used to treat anaerobic infection (clindamycin, metronidazole and ampicillin) was examined using e-test® strips. selected anaerobic isolates were grown as biofilms for or hours in well trays prior to challenge with antibiotic. bacterial biofilm formation was determined using crystal violet. results: the planktonic susceptibility of anaerobic isolates ( prevotella, veillonella, propionibacterium, actinomyces and additional isolates) comprising different genera was determined. although all of the anaerobic isolates examined were sensitive to meropenem when grown planktonically, high levels of resistance to the anti-anaerobic drugs, metronidazole and clindamycin were observed. examining susceptibility by genera, the veillonella were the most resistant to piperacillin/tazobactam and the propionibacterium the most resistant to metronidazole. furthermore, several isolates, including of the prevotella isolates examined, were found to be resistant to multiple antibiotics. the susceptibility of anaerobic biofilms to antibiotics was found to be isolate and antibiotic dependent. generally antibiotic treatment of hour old biofilms was often able to eradicate biomass and prevent biofilm formation; however, antibiotic treatment after hours of biofilm formation with up to x the mic of an antibiotic often failed to eradicate the biofilm. conclusion: these results suggest that alternative antibiotic treatment regimes may be necessary to treat cf pulmonary infection if anaerobes are present. these results also suggest, based on in vitro susceptibility, that meropenem and not metronidazole or clindamycin would be the most effective antibiotic in treating any anaerobes present in cf pulmonary infection. early eradication protocols for the first appearance of pseudomonas aeruginosa (psa) have become standard practice in many cystic fibrosis (cf) clinics. the details of such protocols often vary significantly between clinics, making it important to compare efficacy from one approach to another. however, comparison of reported results is currently very difficult. this is not only because of variations in completeness of reporting, but especially because calculation of average time to regrowth cannot by definition include patients who remain free of psa regrowth at the time the "average" is calculated (nor those in whom a re-appearance of psa is found to be due to a different strain). such results would be better expressed using "time to event" (tte) statistics (analogous to "life tables") and different protocols can be better compared using this methodology. we report for the first time, results from our psa early aggressive eradication protocol for the period - , expressed using tte statistical analysis. children are seen in clinic on average times yearly for full assessment, and at each clinic visit sputum or pharyngeal cultures obtained by the physiotherapist. standard treatment for "first growth" pseudomonas aeruginosa consists of weeks of intravenous antibiotics (piperacillin mg/kg/day plus tobramycin mg/kg/day) followed by weeks of oral ciprofloxacin ( - mg/kg/day) and inhaled colymycin ( mg bid) for months. a total of treatment courses have been completed for the period - with a % clearance rate, defined as or more consecutive negative cultures for pseudomonas aeruginosa over months. since the initiation of this protocol, the percentage of patients colonized with psa in our cf pediatric clinic has declined from % in , down to % in . subsequent regrowth of psa occurred in / of patients. treatment courses may then be repeated a second, third, or fourth time. to determine whether these repeat psa isolates are newly acquired or identical to the previous isolate, rapd typing of these isolates is routinely assessed. a total of isolates have been rapd tested and have identified the same strain as the previous isolate in ( %) and a different strain in ( %)treatment courses. the average time to regrowth for those who had a recurrence of psa following this treatment protocol was months, but this does not include those patients who have had no further growth of psa for periods up to years. to reflect the results for all patients, including those who have not as yet had further regrowth of psa, we have constructed tte curves. these measures can be used to compare results using our protocol with results from other approaches to clearing pseudomonas aeruginosa in cf. retsch-bogart, g.z. inhaled antibiotics have been part of the therapeutic armamentarium for patients with cf for decades. we studied the effect of inhaled aztreonam on respiratory symptoms in cf in a phase , double blind, placebo (pl) controlled trial of azli, a novel formulation of aztreonam, which enrolled patients with cf from centers in the us, canada, australia, and new zealand. all patients have completed study participation. inclusion criteria included age ≥ years, pa in sputum or throat swab, fev ≥ % to ≤ % predicted, and no use of anti-pseudomonal antibiotics in the previous days. following a day screening period, patients were treated for days with either azli mg or pl. study drug was administered tid using the pari eflow® electronic nebulizer after pre-treatment with bronchodilator. concomitant standard cf therapies were allowed, with the exception of anti-pseudomonal antibiotics, azithromycin and hypertonic saline. the primary endpoint was change from baseline to day in scores from the cystic fibrosis questionnaire-revised (cfq-r) respiratory domain, a validated patient-reported measure of respiratory symptoms. other efficacy measures included change in pulmonary function (fev , fvc, and fef - ), number of hospital days and number of courses of iv, oral or inhaled anti-pseudomonal antibiotics. microbiological endpoints included change in pa bacterial density in sputum, change in susceptibility of pa to aztreonam, and emergence or disappearance of other pathogens. safety evaluations included adverse events, airway reactivity (defined as acute decrease in fev ≥ % at minutes after dosing) and clinical chemistry and hematology. complete efficacy and safety results from this trial will be presented. salam, a.p.; orchard, c.; wee, a.; hodson, m. introduction: oral ciprofloxacin is often used for pseudomonas aeruginosa (psa) infections in patients with cystic fibrosis (cf) not requiring intravenous antibiotics. oral chloramphenicol is commonly used at the royal brompton hospital (rbh) as an alternative to ciprofloxacin, but less so in other cf centres due to concerns about aplastic anaemia and uncertainty regarding effectiveness. this study aimed to address three questions: ) what proportion of psa is sensitive to ciprofloxacin and/or chloramphenicol? ) were there any adverse haematological effects with the use of oral chloramphenicol at rbh? ) how effective is chloramphenicol in comparison to ciprofloxacin? study design: we carried out a retrospective review from the rbh cf database ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) together with analysis of patients' notes. methods: ) psa sensitivities to ciprofloxacin and chloramphenicol were recorded from sputum samples taken in late . ) all blood dyscrasias in patients who had received chloramphenicol from to were reviewed. ) the database was searched for patients who had received a course of either oral chloramphenicol or ciprofloxacin from to . patients from each group were randomly selected. fev , fvc and oxygen saturations pre and post treatment completion were analysed, as were markers of symptom improvement. results: ) sputum samples from patients grew psa. some were sensitive to only one antibiotic, and there were some cross-sensitivities, but in total . % were sensitive to chloramphenicol and . % to ciprofloxacin. ) patients received oral chloramphenicol and oral ciprofloxacin from - . multiple courses were often given, but usually no more frequent than monthly with chloramphenicol. over years, cases of blood dyscrasia were recorded, none due to chloramphenicol, and all recovered. ) in the ciprofloxacin group: the mean oxygen saturations improved by . % (p . ), the mean fev improved by . % (p . ), and the mean fvc by . % (p . ). in the chloramphenicol group: the mean oxygen saturations improved by . % (p . ). ), the mean fev improved by % (p . ) and the mean fvc improved by . % (p . ). % of patients in the chloramphenicol group and % of patients in the ciprofloxacin group reported symptom improvement. a significant proportion of psa is sensitive to chloramphenicol and in over years there have been no documented cases of related aplastic anaemia. clinical improvements with choramphenicol comparable to ciprofloxacin were also recorded. our data shows chloramphenicol is safe and may be the oral antibiotic of choice in ciprofloxacin allergy or psa resistance to ciprofloxacin. a randomised prospective study is needed to further evaluate the efficacy of chloramphenicol in comparison to ciprofloxacin. pseudomonas aeruginosa lung infections are one of the main factors causing disease in cf patients. at present the generally recommended treatment is repeated courses of antibiotics. this has resulted in fewer infections and a markedly improved prognosis for the patients. but it has also severe drawbacks such as antibiotic resistance, disturbed normal flora and allergenicity. antibiotic resistance is an emerging problem world wide and alternatives are urgently needed. specific chicken antibodies, anti-pseudomonas igy, have potential both as an alternative and a complement to antibiotics (pediatr pulmonol ; : ) . anti-pseudomonas igy prevents p. aeruginosa from adhesion to epithelial cells and has affinity for flagellin in vitro. humans do not produce anti-igy antibodies and oral administration of igy is generally regarded as safe. the risk that bacteria should develop resistance to igy is extremely low. in total, cf patients have received antipseudomonas igy for up to years. since november, the drug is given to a group of cf patients (at present patients) on individual permissions granted by the swedish mpa and reimbursed by the swedish government. in our first study about the effect of igy there were . positive cultures/ months, compared to . / months in a control group. up to december , , nearly five years later, the effect is maintained with . / months for the whole study period. we are at present compiling the data from the control group during the same period. only two siblings in the igy-treated group have been chronically colonized (with an identical p. aeruginosa strain), which still is non-mucoid after . and . years. during the whole treatment period there have been no cultures positive for mucoid p. aeruginosa or b. cepacia and other pathogens (s. maltophilia, a. xyloxidans, atypical mycobacteria and a. fumigatus) have only appeared sporadically -possibly due to the relatively low use of antibiotics. all patients have preserved pulmonary functions and nutritional status. there have been no adverse events. thus treatment with anti-pseudomonas igy has diminished the number of positive cultures and delayed the onset of chronic infection. the need for antibiotics is reduced as well as their adverse side effects. in conclusion, igy is an important complement to antibiotics for prevention of p. aeruginosa infections in cf. introduction and aims: we have previously shown by culture that the lungs of clinically stable cystic fibrosis (cf) patients are not only colonised by commonly recognised aerobic bacteria, such as pseudomonas aeruginosa, but also by a range of potentially pathogenic anaerobic species. the aim of this study was to determine whether anaerobes are also present in the sputum of cf patients with an acute exacerbation of pulmonary infection. methods: sputum samples were collected and processed using strict anaerobic bacteriological techniques, prior to commencing and at the end of antibiotic therapy, from adult cf patients admitted for treatment of an acute exacerbation of pulmonary infection. bacteria within the samples were detected by plating on selective agars, quantified by total viable count and identified by pcr and sequencing of s ribosomal rna genes. as a control induced sputum samples were collected, using hypertonic saline, from healthy volunteers who did not have cf and processed using similar methods. results: anaerobes from a range of species including prevotella, veillonella, propionibacterium, actinomyces and gemella were detected in high numbers (up to x cfu/g of sputum) from all patients prior to commencing antibiotic therapy with the predominant primary pathogens (p. aeruginosa or b. cepacia complex) detected in similar numbers. anaerobic bacteria were also detected in sputum samples at the end of antibiotic treatment and for / ( %) patients they were present in lower numbers than detected before antibiotic treatment. moreover, in / ( %) patients there was greater than a one log reduction in the total viable count of anaerobes. similar data for detection of the predominant aerobic pathogens was also collected for of these patients. p. aeruginosa or b. cepacia complex was present in lower numbers following antibiotic treatment in / ( %) patients and in / ( %) patients there was more than a one log reduction in the total viable count. anaerobes were not detected in the induced sputum samples from volunteers and in the remaining samples they were present in much smaller numbers, ranging from to cfu/g of induced sputum, than detected in cf sputum. the most commonly isolated bacteria from the induced sputum samples were the actinomyces and streptococcus, with prevotella cultured from only samples. no veillonella and propionibacterium species were isolated from the induced sputum samples and combined with the low frequency of isolation of prevotella species, these results strongly suggests that the prevotella, veillonella and propionibacterium species recovered from cf sputum samples are not oral contaminants. conclusion: these results indicate that anaerobes are present in large numbers within the cf lung during an acute exacerbation of pulmonary infection. their presence could be of important clinical relevance to cf patients as they may contribute significantly to the inflammatory process. airway infection and inflammation cause the majority of morbidity and mortality in cystic fibrosis (cf). the microbiology of cf is complex. not only are the routinely identified pathogenic bacteria present, increasingly a number of other bacteria are associated with pulmonary disease. in addition, the normal microbiota likely contribute to airway disease in cf via intracellular signaling, but remains largely unstudied. the application of culture independent methods for bacterial identification in cf airway secretions provides for the first time the ability to characterize the bacterial communities in the cf airway comprehensively and efficiently. here we present the results of a two-year study of cf airway samples to describe the bacterial communities present by ribosomal rna sequences. we have examined airway specimens from subjects ( cf, controls). these results provide a unique perspective on cf airway microbiology. bacterial communities identified in sputum were more complex than communities identified in bronchoalveolar lavage fluid (balf). however, pulmonary status determined the level of complexity, with higher complexity observed during stable pulmonary function. we identified a collection of anaerobic bacteria that were present at high levels during pulmonary exacerbation in a subset of cf subjects. detection rates ranged from - % of cf subjects examined depending on the specific organism (table ). these organisms of interest are suspected to be involved in pulmonary exacerbation especially when cultures for standard cf pathogens are negative (~ % in our center). preliminary data will be presented from quantitative real-time pcr experiments to track these organisms. we also will present the application of new meth-ods for community comparisons. this analysis provides the ability to look at how clinical information from individuals with cf corresponds to the microbial communities that occur in the airway. overall, the results point to a much richer bacterial community associated with the cf airway than previously thought. the results also point to new opportunities to understand the differences within the cf population, and thus potentially improve individual patient outcomes. cystic fibrosis (cf) patients are characterized by persistent microbial colonization of the airways and recurrent pulmonary infection. however, due to the domination of bacterial communities present in cf sputum by a small number of species (typically pseudomonas aeruginosa), culture-independent approaches to characterize the depth of bacterial diversity in cf sputum have yielded limited information. here we describe application of a novel microarray, the s rrna phylochip to comprehensively describe the bacterial diversity present in temporal sputum samples of cf patients who experienced periods of exacerbation (defined as hospitalization) and remission. in addition, these samples permitted assessment of bacterial community dynamism during periods of antimicrobial administration in these patients. phylochip analysis identified bacterial taxa present across these patient samples, including a large number of known human pathogens. antimicrobial administration caused a profound decrease in bacterial diversity in all patients examined. despite this, a sizeable fraction of each community remained stable or proliferated during the period of antimicrobial administration, suggesting many of the bacterial taxa present were resistant to the antimicrobials administered. taxa exhibiting this behavior included p. aeruginosa, arcobacter cryaerophilus, streptococcus constellatus and burkholderia mallei amongst other pathogens. these results suggest that complex polymicrobial communities exist in cf sputum that exhibit significant antimicrobial driven dynamism and consist of multiple antimicrobial resistant pathogens. future studies will focus on whole community gene expression to determine which virulence systems are activated in response to bacterial community perturbations and contribute to pathogenic processes in cf airways. the lungs of chronically infected cystic fibrosis (cf) patients have been intensively studied. it is well documented that pseudomonas aeruginosa is the predominant pathogen in cf (høiby , baurnfeind et al. , koch , and that p. aeruginosa accumulates in heaps (aggregates), detected in cf sputum (høiby ) and intraluminal in the lung (baltimore et al. ) . the bacterial density is highest in the bronchi (potts et al ) and these p. aeruginosa microcolonies are embedded in a matrix (lam et al. ) . the morbidity of the infection is due to an extensive and ongoing pmn response which apparently does not eradicate the bacteria, instead leading to slow degradation of the lung. in addition, even highest deliverable doses of antibiotics fail to clear the bacteria completely, though survival has significantly increased the last years due to aggressive anti p. aeruginosa therapy (koch and høiby ) the present study was performed to investigate the significance of the aggressive therapy for the distribution of p. aeruginosa and how p. aeruginosa is organized and persists in the cf lung. the material used was: autopsies from dead short term colonized cf patients (n= ) obtained before today's aggressive antibiotic treatment , and explanted lungs from long term p. aeruginosa infected cf patients (n= ) (i.v. antibiotics every rd month since + inhalation of colistin daily since . histological sections of the lungs were investigated using he stain, gram stain, alcian blue, antibodies against alginate and p. aeruginosa specific pna-fish combined with bacuni pna-fish and with dapi as counter stains. due to the high specificity of the pna-fish probes employed in this study, our results provide strong evidence for that before the aggressive antibiotic therapy, p. aeruginosa infected and destroyed the cf lung due to fast spreading into the respiratory zone. today antibiotics suppress but can not eradicate the bacteria from the conductive zone, whereas the remaining respiratory zone is protected from massive biofilm infection for prolonged time. the conductive zone serves as a reservoir; here the bacteria are organized in microcolonies embedded in puss, a trait which is independent on the time course of the infection and amounts of antibiotics. these microcolonies consisted solely of p. aeruginosa. in addition, we found no bacteria adhering to the epithelial tissue. the pus consisted mainly of leukocytes, surrounding the microcolonies. a smear of dna and dead leukocytes were detected just around the microcolonies, possible due to the quorum sensing dependent rhamnolipid killing recently described by us (jensen et al. ) . we conclude that p. aeruginosa persists in the cf lung due to its ability to create microcolonies i.e. biofilms. within these biofilms the bacteria are protected against antibiotics and the host defence. respiratory viral infections, including influenza (flu) and respiratory syncytial virus (rsv) contribute to the morbidity of children with cf. additional human respiratory viruses have been identified in children such as human metapneumovirus (hmpv) and coronavirus (cov), but these have not been examined in cf patients. in our laboratory, pcr assays have been developed to identify both traditional and newer respiratory viruses in healthy and immunocompromised children. these assays are usually performed on nasal swabs or nasal wash samples. however, oropharyngeal (op) and sputum samples are routinely obtained in cf for bacterial culture. the goals of this trial are to determine whether these available samples can be used for viral pcr studies and to examine the epidemiology of respiratory viruses in cf. for this single center observational trial, cf patients, ages . to . yrs (mean . yrs), have been recruited. subjects provide op and nasopharyngeal (np) samples at quarterly clinic visits and with acute pulmonary exacerbations requiring hospitalization; sputum is also collected if patients can expectorate. np and op samples are pooled when both are available. viruses assayed are flu a and b, rsv, parainfluenza (piv) , , and , hmpv, cov, adenovirus (adv), and rhinovirus (rhv). subjects are being followed for two respiratory viral seasons. we report here the methodology of viral pcr detection in cf sputum, as well as the results of viral detection during the first respiratory viral season in the seattle community. the mean length of time subjects were followed was . yrs (median: . yrs, range: . - . yrs). overall, samples ( % of the samples analyzed) were positive. twenty-four subjects ( . %) had at least one viral infection detected and ( . %) had two viruses at different visits. both sputum and pooled op/np samples were available at visits and were concordant in % of cases. among the discordant cases, it was more common for sputum to be positive and op/np to be negative ( . %) than the converse ( . %). the most frequently identified viruses were rhv ( isolations); piv and cov were the next most prevalent with and isolations, respectively. in conclusion, sputum viral pcr shows promise for the diagnosis of both traditional and novel viral infections in cf. as part of ongoing clinical trials in cf, non-fermenting gram negative bacilli (nfgnr) identified at site laboratories are often sent to the tdn core microbiology laboratory for confirmation of identification. among nfgnr from cf patients, p. aeruginosa are the most frequently isolated and also are acknowledged to be the easiest to identify. however, in a survey of "p. aeruginosa" isolates sent to the laboratory in the past year, pcr identified ( %) as alternate species. these included: non-aeruginosa pseudomonads, stenotrophomonas maltophilia, achromobacter xylosoxidans, ralstonia spp., alcaligenes spp., brevidimonas spp., and bordetella bronchiseptica. identification of these nfgnr can be difficult; however, p. aeruginosa is an important isolate for the clinical microbiology laboratories in all cf centers to be able to identify. many methods are available including conventional biochemical testing and rapid identification kits. in the tdn core laboratory we previously demonstrated the importance of combining phenotypic identification (morphology plus a short set of biochemical tests) and genotypic identification ( s rdna sequencing of v and/or pcr using three primer sets) and developed an algorithm for testing. in this study, we examined all of the cf cultures performed in the clinical microbiology laboratory at chrmc over the past two years to determine the number of isolates for which it was necessary to perform genotypic identification in nfgnr. among this collection of nfgnr from clinical cultures sent to the microbiology laboratory from the seattle children's hospital cf center, pcr identified isolates as burkholderia cepacia complex. next most commonly identified were a. xylosoxidans ( ), p. aeruginosa ( ) and non-aeruginosa pseudomonas spp. ( ). three isolates remained unidentified, even with pcr sequencing of the v determinant. the clinical microbiology laboratory at chrmc has served as the core microbiology laboratory for tdn clinical trials for years and thousands of cf samples are processed annually. the result is that there is increased experience with cf isolates among the laboratory personnel. however, even with experienced technologists, a significant number of nfgnr required genotypic identification in the past two years. thus, it is important for clinical microbiology laboratories with less experience in the identification of cf nfgnr to utilize additional molecular techniques or send problematic isolates to a reference laboratory. based on these results we propose an algorithm for the most efficient means of identifying nfgnr from cf samples using a combination of tests including phenotypic (colony morphology, biochemical testing) and genotypic identification (pcr, sequencing). background: the epidemiology of s. aureus has been changing in both cf and non-cf patients. in non-cf patients, methicillin-resistant s. aureus (mrsa) has traditionally caused infections in patients with risk factors such as hospitalization, surgery, and anterior nares colonization. recently, community-acquired (ca-) mrsa infections have increased among patients without known risks. in cf, colonization of the anterior nares with methicillin-susceptible s. aureus (mssa) is associated with transmission between patients and household members. the prevalence of respiratory tract colonization/ infection with mrsa has increased in cf, but little is known about risk factors for mrsa, including possible transmission among families, and if strains are ca-or healthcare-acquired. methods: this is a case-control, multicenter study to investigate potential risk factors for mrsa among children with cf - years of age. subjects are enrolled from cf centers in the new york metropolitan area that have a mrsa prevalence ranging from % to %. cases are children with a positive respiratory tract culture for mrsa within the past years. controls ( age and center matched per case) are negative for mrsa. the aims of the study are: [ ] to determine the point prevalence of anterior nares colonization with s. aureus in children with cf and their household members; [ ] to assess potential risk factors for mrsa by administering a survey to the parents of subjects inquiring about factors such as crowding, pets, and participation in contact sports and by reviewing the medical record for antibiotic use, hospitalization, and surgery; [ ] to determine potential transmission of s. aureus within families and centers by assessing the molecular epidemiology of strains using pulsed field gel electrophoresis and meca type of mrsa isolates. results: to date, cf subjects ( cases, controls), mean age . years, and household members ( to per subject) have been enrolled. preliminary data demonstrate positive anterior nares cultures among cases, controls, and household members to be / ( %), / ( %), and / ( %), respectively. of the s. aureus isolates, were mssa and were mrsa ( cases, household members of cases, and household members of controls). during the past months, household members have been hospitalized and one has stayed overnight in a nursing home; had staphylococcal infections and had skin infections. data collection and molecular analysis are ongoing. conclusions: we expect this study to provide insight into risk factors for respiratory tract colonization/ infection with mrsa, the role of ca-mrsa in cf, and potential strategies to prevent mrsa. aims: . revise infection control guidelines for the institution . establish outpatient infection control policies consistent with those used for hospital inpatients . cohort young and recently diagnosed cf patients without pseudomonas or mrsa to clinic days separate from colonized patients . establish a protocol for eradication of mrsa in newly colonized patients. methods: working with representatives from hospital epidemiology, the cf center, inpatient units, outpatient clinics, respiratory therapy, and cf families, a uniform infection control policy for cf patients in all settings (inpatient, outpatient, home care) was drafted and revised. arrangements were made to cohort newly diagnosed patients and children < years free of pseudomonas and mrsa to separate clinic days from colonized patients. review of the mrsa positive patients in our pediatric population, for whom data was available, showed median age of acquisition of years (range mo to yrs). for patients the first culture obtained at diagnosis or transfer from another center was mrsa positive ( mo, yr, yr). an eradication protocol for newly acquired mrsa has been developed involving treatment with oral and topical antibiotics and phisohex or clorox washes for days, plus changing disposable respiratory care equipment at the beginning and end of the treatment period. surveillance cultures of nose, groin, and airway or sputum are done after completion of the treatment protocol and re-treatment initiated if still positive for mrsa. conclusion: using a three-tiered approach of infection control guidelines, cohorting, and mrsa eradication protocol we expect to decrease our overall rate of mrsa and protect our newly diagnosed and young cf patients from acquisition. objective: studies have shown that respiratory pathogens can be transmitted within cf centers. in a recent cross-sectional study at cf centers, we assessed the rate of bacterial shedding by cf patients during office visits and noted that % of patients carried respiratory flora on their hands. the current study was performed to assess the effectiveness of alcohol-based rubs on hand carriage of respiratory pathogens by patients during the course of office visits. methods: four bacterial organisms were chosen for study: pseudomonas aeruginosa (pa), staphylococcus aureus (sa) [including methicillin-resistant (mrsa) strains], stenotrophomonas maltophilia (sm) and burkholderia cepacia complex (bcc). at the beginning of clinic visits, hands of study patients were cultured using the "glove-juice" technique (gjt) [ ] . hand hygiene was then performed using an alcohol-based rub. at the end of the visit, hands were again sampled using the gjt. samples were sent to the microbiology laboratory at dartmouth-hitchcock medical center for culture and identification of study organisms. hand and respiratory tract isolates were compared using pulsed field gel electrophoresis (pfge). recovery experiments were also performed to assess the sensitivity of the gjt. results: samples were collected from encounters ( adult; pediatric). median encounter time was min (range . - . min). recovery experiments determined that the level of detection for each study organism using the gjt was ~ colony forming units. confirmation of matches between hand and respiratory isolates was determined by pfge. the number of patients with study organisms recovered from the respiratory tract was: pa, sa, mrsa, sm, and bcc. the overall hand contamination rate with a matched respiratory tract isolate at either culturing time was % ( % ci, . - . %). before use of the alcohol rub, hand contamination was . % ( % ci, . - . %). in those patients with hand contamination by respiratory pathogens prior to alcohol rub, . % ( % ci, - %) had negative cultures at the end of the clinic visit. however, there was strong evidence of hand contamination during the encounter, despite hand hygiene, as the overall rate at the end of visits was . % ( % ci, . - . %). there was a trend toward increased hand contamination in those suffering from pulmonary exacerbations versus clinically stable patients ( . % [ % ci, . - %] vs. . % [ % ci, . - %], p= . ). conclusions: contamination of patient hands by respiratory tract pathogens is observed in the outpatient setting in approximately % of patients. a single application of an alcohol-based hand rub, while safe and inexpensive, has limited antiseptic effectiveness during the full course of a typical cf clinic visit. repeated use of alcohol-based hand rubs during outpatient encounters would likely be more effective. these data support recent recommendations in the cf foundation consensus guidelines for infection control [ ] (nthi) is the most common initial bacterium infecting the airways in cystic fibrosis (cf). nthi infection usually precedes pseudomonas aeruginosa infection in many chronic lung diseases, including cf, emphysema, diffuse panbronchiolitis, and immotile cilia syndrome. it is hypothesized that nthi acts as a gateway organism paving the way for subsequent infection with p. aeruginosa, however, the mechanism by which this occurs remains poorly understood. we used a novel co-culture model of persistent nthi biofilm infection on airway epithelial cultures to study epithelial immune responses following nthi biofilm infections. objective: we hypothesized that prolonged exposure to nthi biofilms causes airway epithelia to become tolerant to inflammatory stimuli and show subsequent decreased innate immune responses. methods: nthi were inoculated onto the apical surface of calu air-liquid interface epithelia and grown in co-culture. rna from sets of paired airway epithelial cultures at , , and hours both with and without nthi inoculation was isolated and hybridized on custom affymetrix chips. data were normalized with rma and data biases were removed using the mixed model anova method. differentially expressed gene analyses were performed using anova, and pathways analyses were performed using gene set enrichment analysis (gsea). to test the hypothesis that prolonged nthi infection induces tolerance, epithelial cultures were pre-treated with either days of nthi or days of pbs before stimulation with pbs, il β ( ng/ml) or th cfus of live p. aeruginosa before subsequent measurement of epithelial responses. results: both rma and gsea analyses of microarray data showed many innate immune and pro-inflammatory responses at early timepoints that largely returned to baseline after days of co-culture, despite an increasing apical nthi biofilm infection. stimulation with il β or p. aeruginosa after days of nthi infection resulted in significantly decreased il production compared to uninfected controls. nthi treated cultures subsequently infected with p. aeruginosa also displayed increased tight junction integrity and resistance to cell culture death. conclusions: persistent nthi infections on airway epithelia resulted in increased tolerance to bacteria and inflammatory stimuli and increased resistance to bacterial toxicity. airway epithelial tolerance may contribute to a failure to clear subsequent bacterial infections. these phenomena would help to explain how early infections with nthi may promote chronic bacterial infections and p. aeruginosa acquisition in cf and other nthi related airway diseases. the colonization of cf airways by p. aeruginosa leads to intractable and persistent lung infections that resist permanent eradication by antibiotics. the lack of efficacy of current therapies is believed to be due to the formation of p. aeruginosa antibiotic resistant biofilms in the cf airways. however, little is known about biofilm formation on human airway epithelial cells. thus, we designed a continuous flow, live cell imaging system to grow p. aeruginosa biofilms on glass or on the apical surface of polarized human cells. cfbe ohuman airway epithelial cells homozygous for the ∆f mutation (cfbe), and cfbe o-cells complemented with wt-cftr (cfbe+wt-cftr) were grown as polarized monolayers at an air-liquid interface and gfp-labeled p. aeruginosa were applied to the apical surface of the cells. after hours biofilms did not form on glass but large bacterial macrocolonies developed on cfbe cells. surprisingly, biofilms also developed on cfbe+wt-cftr cells: however, p. aeruginosa biomass on cfbe+wt-cftr cells was significantly reduced compared to cfbe cells ( . ± . µm /µm vs . ± . µm /µm ). the minimal bactericidal concentration for tobramycin was > , µg/ml for p. aeruginosa grown on cfbe and cfbe+wt-cftr cells. these results explain why tobramycin given clinically, which is~ , µg/ml in bronchoalveolar fluid collected from cf patients, fails to eradicate the persistent infection by p. aeruginosa. p. aeruginosa also formed biofilms on human bronchial epithelial cells, which produce mucus. indeed, biomass was similar on cfbe+wt-cftr cells, which do not produce mucus, and human bronchial epithelial cells. to determine if human airway epithelial cells secrete factors that facilitate biofilm formation, conditioned medium was collected from cfbe and cfbe+wt-cftr cells and applied to p. aeruginosa grown on glass. under these conditions, biofilm formation was dramatically facilitated, with a biomass of . ± . µm /µm when p. aeruginosa was grown in conditioned medium collected from cfbe+wt-cftr cells and . ± . µm /µm when grown in conditioned medium collected from cfbe cells. the secreted factor(s) were greater than kda, as assessed by size exclusion. our results suggest that human airway epithelial cells secrete factor(s) that facilitate biofilm formation by p. aeruginosa, in the presence and absence of mucus, and that secretions from cf cells are more effective in promoting biofilms than secretions from non-cf cells. the ability of non-cf cells to facilitate biofilm formation appears to contradict the observation that non-cf lungs do not harbor p. aeruginosa. we propose that the innate immune response in the non-cf lung effectively eliminates p. aeruginosa from the lung, thus, biofilms do not form even thought the airway epithelial cells secrete factors that have the potential to facilitate biofilm formation. however, in cf when mucociliary clearance is compromised, p. aeruginosa accumulates in the lung and secretions by cf airway epithelial cells greatly enhances the formation of drug resistant biofilms (supported by the nih (ro ai , ro -hl- , p -rr , t -dk- ), and the cff (stanto ro, anders fo). ma, l. ; parsek, m.r. ; wozniak, d.j. . microbiology and immunology, wake forest university health science, winstonsalem, nc, usa; . university of washington, seattle, nc, usa bacteria in natural, industrial and clinical settings live in surface-associated communities termed biofilms, which are the source of persistent infection and resistance to antimicrobial treatment. biofilm development is initiated by the attachment of planktonic cells to a surface, followed by formation of microcolnies, and finally disperses swimming cells from microcolnies to occupy a new surface. to maintain the community structure, bacteria in a biofilm are usually enmeshed in an extracellular polymeric matrix, which consists of nucleic acids, proteins, and polysaccharides. exopolysaccharides (eps) have been known as a component of the biofilm matrix for years. however, little is know about how the eps matrix forms and develops and whether an eps can form a matrix independently. in the present report, we use lectins that specifically detect the mannose or galactose structure in pseudomonas aeruginosa psl eps. this technique allows us to visualize psl eps on the bacteria cell surface and in the biofilm matrix. our results indicate that psl eps is likely anchored on the cell surface in a helical pattern and clearly forms a matrix, which holds bacteria in the biofilm and on the surface. in a flat multiple-layer biofilm, psl eps is equally distributed in the entire biofilm. however, microcolonies reveal peripheral staining of psl eps with minimal staining of matrix in the center of the microcolonies. instead, this region has swimming cells indicating a biofilm development stage prior to dispersion. more strikingly, this area also has concentrated dead cells and/or extracellular dna, which fills up the viod spaces in the lower center of microcolonies at the stage prior to dispersion. these data provides a plausible mechanism for how p. aeruginosa sacrifices a portion of cells to make void spaces and free another portion of cells for future dispersion. in addition, our data also show that psl eps matrix and dna matrix are not overlapping, which suggests that these two components of the matrix work coordinately to encase bacteria in the biofilm. finally we show that the psl eps matrix is present in biofilms formed by mucoid p. aeruginosa, and formation of the psl eps matrix is independent of the production of alginate and pel, two eps that also contribute to p. aeruginosa biofilm formation. overall, our data indicates that psl eps functions as a primary scaffold, holding biofilm cells together in the matrix. moreover, our data along with published literature suggest a possible model for how p. aeruginosa biofilms persist in cystic fibrosis patients. . clinical microbiology, rigshospitalet, copenhagen, denmark; . biocentrum, technical university of denmark, kgs. lyngby, denmark; . department of paediatrics, copenhagen cystic fibrosis centre, rigshospitalet, copenhagen, denmark pseudomonas aeruginosa predominates chronic lung infections in patients with cystic fibrosis (cf). a hallmark of chronic p. aeruginosa lung infections in cf patients is the presence of mucoid p. aeruginosa biofilms surrounded by numerous polymorphonuclear leukocytes (pmns) in the lower airways. how p. aeruginosa escapes the bactericidal pmns is not fully understood. several virulence factors of p. aeruginosa is controlled by quorum sensing (qs); a density dependent mode of inter-bacterial communication based on signal transmitter molecules. active qs is present during chronic p. aeruginosa lung infections in cf patients and we have previously demonstrated a qs-regulated tolerance of biofilm bacteria to the antimicrobial properties of the pmns. the precise qs-regulated effect on the pmns is, however, unknown. in the present study, further elaboration using flow cytometry and microscopy revealed that qs-competent p. aeruginosa (pao and ∆rhli lasi complemented with c -hsl and -oxo-c -hsl) induces rapid necrosis of the pmns. this mechanism was also observed in in vitro biofilms and in mouse lungs infected with p. aeruginosa embedded in alginate. by using hplc fractionation the toxicity could be ascribed to a single substance. using lc-ms and d nmr this substance was identified as rhamnolipid b. in accordance, concentrations of rhamnolipid exceeding µg/ml were found in the toxic supernatants from wild-type p. aeruginosa (pao ) whereas non-toxic supernatants from qs-deficient knock-out mutants (∆rhlr lasr and ∆rhli lasi) and from ∆rhla mutants contained low amounts of rhamnolipids approaching µg/ml. our results demonstrate the potential of the qs system to facilitate infections with p. aeruginosa by utilizing rhamnolipid to disable a major first line of defense of the host -the pmns. furthermore, our study emphasizes the inhibition of qs as a target for the treatment of infections with p. aeruginosa. carlsson, m. , ; pettersson, a. ; andersson, c. ; wieslander, j. ; eriksson, l. ; segelmark, m. ; hellmark, t. . dept of microbiology, immunology, and glycobiology, university of lund, lund, sweden; . department of nephrology, lund university, lund, sweden; . heart lung division, the cystic fibrosis center, lund university hospital, lund, sweden; . wieslab analys ab, lund, sweden the clinical consequence of chronic pseudomonas (p) aeruginosa colonization in cystic fibrosis (cf) varies between individuals for unknown reasons. auto-antibodies against bactericidal/ permeability increasing protein (bpi-anca) are associated with poor prognosis in cf. we hypothesize that there is a correlation between the presence of bpi-anca, the biological properties of the colonizing bacteria and the clinical conditions of the hosts. we have compared isolates of p aeruginosa from two groups of cf patients: one with positive serum levels of bpi-anca and deteriorating lung disease, and one with negative bpi-anca levels and stable clinical conditions. epithelial cells (a ) and isolated polymorphonuclear granulocytes (pmns) were stimulated with the clinical isolates and cell death was analyzed with flow cytometry. interleukin- (il- ) released into the supernatant was measured by elisa. we found that the anca associated strains in most cases showed a pyocyanin negative phenotype. these strains also induced less inflammatory response than the non-anca associated strains as shown by the number of necrotic cells and il- release, yet elevated compared to control. we conclude that colonization with strains of p aeruginosa that induce a weak inflammatory response is associated with unfavourable outcome in cf. we speculate that inadequate control of pathogen proliferation through an insufficient inflammatory response results in a slowly increasing number of bacteria and accumulation of dying pmns in the airways, contributing to the progressive lung disease seen in many cf patients. . pulmonary critical care, northwestern university, chicago, il, usa; . microbiology/immunology, northwestern university, chicago, il, usa; . pulmonary medicine, children's memorial hospital, chicago, il, usa; . pediatrics, children's memorial hospital, chicago, il, usa purpose: most cystic fibrosis (cf) patients are infected with pseudomonas aeruginosa (pa) and have progressive loss of lung function. among individual patients, however, there are marked differences in rates of lung function deterioration. although the reasons for this variability are not completely understood, it is likely that microbiological factors play a role. type iii secretion in pseudomonas aeruginosa (pa) isolates from non-cf patients have been associated with poorer outcome. the aim of this study was to determine whether there is an association between type iii secretion properties and deterioration in lung function in cf. methods: we prospectively enrolled cf children and adults over years. demographics, clinical characteristics, spirometry and a respiratory culture were obtained at the first visit. the age at time of first postive pa culture was also determined. subsequently, spirometry and respiratory cultures were obtained and clinical characteristics recorded every months until december . from each sputum culture individual isolates were selected for evaluation and type iii secretion was evaluated for each isolate by western blot analysis. the children were subcohorted into those newly infected ( st positive pa culture) or those chronically infected (minimum sputum pa (+) > year duration). results: there were patients evaluated, of which were adults and were children. ninety percent of our population cohort was caucasian and % were female. overall % of the patients were on tobi and % on macrolides chronically. the mean age at time of a first pa positive culture was years for the chronically infected subjects(adults and children) and years for the st time infected subcohort. a total of sputum samples were collected for the current study. the prevalence of type iii secretion properties for the overall population was . %. foe adults, of ( . %) pa isolates were type iii secretion positive compared to of isolates ( . %) in chronically infected children and of isolates( . %) from newly infected children. at study entry the mean fev % predicted was +/- for the whole cohort. for the adult subcohort the fev % predicted was % +/- %, for chronically infected children it was % +/- % and newly infected children is was %+/- %. the average annual rate of fev % change was - . % for adults, - . % for chronically infected children and + . for newly infected children. analysis on the relationship between type iii secretion pa isolates and fev % decline is ongoing and will be presented at nacf. conclusion: prevalence of type iii secretion in pa isolates from cf patients decreases with age. there is a slower decline in fev % in chronically pa infected adults compared to chronically pa infected children. the relationship of type iii secretion to fev % change and pulmonary exacerbations will be presented at nacf. a limited number of bacterial species -in example pseudomonas aeruginosa, staphylococcus aureus, burkholderia cepacia complex, stenotrophomonas maltophilia -is typically found in the cf lung. these facultative anaerobic bacteria exhibit the capability to switch from aerobic to anaerobic metabolism, which enables them to survive in the hypoxic environment in the cf lung [ ] . absence of oxygen would also favour the growth of fastidious anaerobes and, indeed, sequencing of s rrna resulted in the detection of strict anaerobic species in cf sputum [ ] . thus, we identified and quantified strict anaerobes in cf sputum using conventional anaerobic microbiological methods, examined the recovery rate of identical species in sputum samples from the same patients, and determined the antibiotic susceptibilities. sputum samples were collected from cf children and cf adults (mean age . ± . yrs). samples were incubated aerobically on columbia agar, supplemented with % sheep blood, and anaerobically on brain heart infusion agar and schaedler agar supplemented with % mutton blood, for up to days. bacteria were identified (rapid ana ii identification system, remel, lenexa, ks) and cfu's were determined by dilution plate counting. fastidious anaerobes ( strains) were submitted to e-test ® susceptibility testing (ab biodisk, solna, sweden) using the anaerobically active antibiotics ceftazidime, clindamycin, meropenem, metronidazole, and piperacillin/tazobactam. in . % of the patients, the following strict anaerobes were detected with a mean of . x cfu/ml (range . x to . x cfu/ml): peptostreptococcus spp., clostridium spp., actinomyces spp., prevotella spp., wolinella spp., propionibacterium spp., streptococcus spp., lactobacillus spp., gemella spp., bacteroides spp. and eubacterium spp., whereas p. aeruginosa, s. aureus and b. cepacia revealed . x cfu/ml (range . x to . x cfu/ml). in sputum samples contaminated with p. aeruginosa together with strict anaerobes . x ± . x cfu/ml p. aeruginosa were counted, whereas in samples with p. aeruginosa without strict anaerobes only . x ± . x cfu/ml were found (p= . ). for s. aureus, no significant difference was observed. identical strict anaerobic species were detected in out of patients with two or more repeated sputum samples ( %). e-test® sensitivity testing for strict anaerobes yielded high sensitivity for meropenem (only . % resistant strains), piperacillin/tazobactam ( . %), and clindamycin ( . %), but not ceftazidime ( . %) or metronidazole ( . %). high numbers of strict anaerobes are present in the majority of cf patients. possibly, strict anaerobes promote growth of p. aeruginosa but not s. aureus. the high persistence of identical anaerobic strains reflects chronic lung infection and may be caused by their increased resistence against standard antibiotics such as ceftazidime. [ in sputum in the cf lungs bacteria such as pseudomonas aeruginosa have to metabolize anaerobically [ ] . for anaerobic energy generation, p. aeruginosa can use nitrate and arginine, but also pyruvate which is produced from glucose via anaerobic glycolysis [ ] and can be metabolized to lactate and vice versa. in order to investigate if p. aeruginosa may benefit from externally produced lactate, we measured the concentration of lactate in p. aeruginosa, staphylococcus aureus, burkholderia cenocepacia and polymorphonuclear neutrophils in vitro and in cf sputum. in sputum samples of cf patients and in neutrophils ( x /ml) from healthy donors lactate concentrations were determined. in addition, p. aeruginosa (starting with x cfu/ml in tryptone soy broth), s. aureus ( x cfu/ml), and b. cenocepacia ( x cfu/ml)were grown aerobically ( through hrs) and anaerobically ( through days). l-lactate was measured spectrophotometrically (detection limit: . mmol/l), and total lactate gaschromatographically. aerobic and anaerobic gene expression of p. aeruginosa strain pao was determined using affymetrix® microarrays. lactate concentrations in cf sputum amounted to . ± . mmol/l (range . to . mmol/l). concentrations were similar in sputum samples colonized with p. aeruginosa, s. aureus ( . ± . vs. . ± . mmol/l, p= . ) and b. cenocepacia ( . mmol/l). neutrophils produced . mmol/l. in all samples exclusively l-lactate was found. during in vitro experiments, p. aeruginosa did not generate any lactate at all, neither aerobically nor anaerobically. in contrast, anaerobically grown s. aureus produced up to . mmol/l lactate, and b. cenocepacia up to . mmol/l. a p. aeruginosa suspension [ x cfu/ml] spiked with mmol/l l-lactate did not change its concentration, indicating that p. aeruginosa does not metabolize lactate. similar results were obtained in our gene chip experiments: after three days of anaerobic growth, the genes encoding for the lactate dehydrogenases were downregulated (pa ldha - . fold, pa llda - . fold) or unchanged (pa lldd . fold). in contrast, the genes encoding for pyruvate decomposition to acetyl coa (pa and pa , both encoding for pyruvate dehygrogenase e components) were upregulated by and fold, respectively. we could demonstrate that p. aeruginosa does not benefit from externally produced lactate. we confirmed the important role of pyruvate metabolism for anaerobic p. aeruginosa energy generation. whether lactate production of neutrophils, s. aureus or b. cenocepacia contributes to cf lung pathophysiology still remains to be investigated. references: [ ] macleod, d. ; barker, l. ; gurgel, j. ; kenney, t. ; burns, j. ; baker, w. . gilead sciences, inc., seattle, wa, usa; . university of washington, seattle, wa, usa antibiotic resistance may severely limit therapeutic options in individuals with cystic fibrosis (cf) or bronchiectasis. because of frequent antibiotic treatment courses, resistance continues to emerge, even to newer agents. treatment with multiple antibiotics in a single aerosol formulation may be a promising approach to slow development of resistance. fosfomycin is a phosphonic acid antibiotic that is bactericidal against both gram positive and gram negative organisms. fosfomycin inhibits the first committed step in the synthesis of peptidoglycan, suggesting cross resistance to other cell wall acting antibiotics will not occur. the aminoglycoside tobramycin is one of the most commonly used antimicrobials in cf, with potent activity against gram negative bacteria and the majority of staphylococcus aureus isolates. a : (wt/wt) fixed combination of fosfomycin:tobramycin (gs- / ) was used to determine the in vitro susceptibilities of a panel of respiratory pathogens: cf pseudomonas aeruginosa ( ), s. aureus ( ), haemophilus influenzae ( ), stenotrophomonas maltophilia ( ) and burkholderia cepacia complex ( ), including minimal inhibitory concentration (mic) and time-kill experiments in the absence and presence of % porcine mucin. synergy was evaluated using the checkerboard method, and spontaneous resistance mutation frequencies were determined in antibiotic-containing agar ( x, x and x mic). in vivo drug efficacy was examined using a rat agar bead pneumonia model of either p. aeruginosa or s. aureus. all experiments compared gs- / to fosfomycin and tobramycin as single agents. gs- / had a lower mic than tobramycin for the s. aureus strains, % of which were methicillin resistant (mrsa). for p. aeruginosa, gs- / had a lower mic and mic than fosfomycin alone, but tobramycin was more active than either. for h. influenzae and s. maltophilia, gs- / , fosfomycin and tobramycin had similar mic and mic . b. cepacia complex were resistant to all three drugs. results in the presence of mucin were similar. time-kill studies showed a more rapid and prolonged killing of s. aureus and p. aeruginosa by gs- / compared with either agent alone at the same drug concentrations. gs- / was bactericidal and exhibited concentration-dependent killing. synergy studies showed no antagonism between fosfomycin and tobramycin, and the majority of p. aeruginosa and all of the s. aureus tested demonstrated indifference for the combination. at x mic concentrations the mutation frequency of gs- / was at least - logs lower than tobramycin and - logs lower than fosfomycin alone for s. aureus. for p. aeruginosa the mutation frequency of gs- / was - logs lower than fosfomycin and - logs lower than tobramycin. in the rat pneumonia model, gs- / and tobramycin alone demonstrated bactericidal killing of p. aeruginosa; both were more active than fosfomycin alone. in vivo killing of s. aureus by gs- / was also demonstrated. gs- / appears to have advantages over single agents for the treatment of both gram positive and gram negative bacterial lung infections in cf and bronchiectasis. britton, l.j. antibiotic resistance is becoming a major problem in the treatment of pulmonary exacerbations in cystic fibrosis (cf). organ-isms that are resistant to multiple antibiotics infect the airways of an estimated - % of adults with cf. as a result of the growing resistance in cf patients, many centers have been performing synergy testing of sputum cultures in addition to the conventional culture and sensitivity testing. the purpose of our investigation was to determine if antibiotic synergy studies and the use of synergistic combinations of antibiotics improve therapeutic outcomes in cystic fibrosis patients with acute pulmonary exacerbations. methods: this study is a retrospective chart review of cf patients who had antibiotic synergy testing performed while hospitalized for respiratory tract infections. eligibility criteria included cf patients hospitalized with respiratory tract infections from to . laboratory data from each hospital admission was reviewed for synergy among those antibiotics commonly used against pseudomonas aeruginosa. a review of medical charts ascertained each patient's pulmonary function (determined by fev before and after antibiotic therapy), weight z-score, organism cultured from sputum sample, time to next hospital admission, and the antibiotic(s) actually prescribed. primary endpoints were determined to be the change in fev and the time to next admission. results: four hundred seventy-five hospital admissions were analyzed. a total of cystic fibrosis patients, age birth to years, were included in the study. patients receiving antibiotic synergy experienced a significant decrease in mean time to next admission ( days with synergy vs. days without synergy, p= . ). no significant difference was found in the change in fev before or after antibiotic therapy, with an increase of . % with synergy vs. . % without synergy (p= . ). patients infected with non-mucoid p. aeruginosa experienced days to the next hospital admission, while patients infected with mucoid p. aeruginosa experienced days to the next hospital admission (p = . ). no statistically significant difference was observed between the synergy and non-synergy groups in regards to nutritional status and lung function prior to antibiotic therapy. speculations: antibiotic resistance in cystic fibrosis patients increases the morbidity and mortality caused by this disorder with each exacerbation the patient experiences. synergistic antibiotic therapies should improve patient outcomes through more efficient bacterial eradication and increased time to next hospital admission; however, we were unable to substantiate this assumption based on the results from this study. synergistic antibiotics did not show an improvement in the therapeutic outcomes of days to next admission or change in fev . background: expectorated sputum (es) technique is currently the most frequently used method for routine assessment of lower airway infection in patients with cystic fibrosis (cf). induced sputum (is) using hypertonic saline (hs) has been successfully used in cf patients unable to produce sputum spontaneously, but only limited data are available comparing the diagnostic yield of expectorated versus induced sputum in cf patients. while ultrasonic nebuliser have been used in the majority of studies, new high output jet nebulisers may offer a suitable alternative technique to induce sputum expectoration. aim: to assess the feasibility of sputum induction using a pari e-flow nebulizer and to compare the diagnostic yield of is and es in children with cf . methods: this is a preliminary report of an ongoing study in routine clinical care in sputum producing children with cf. es is being obtained before sputum induction. subsequently, sputum induction is performed using stepwise inhalation of nebulized ml of %, % and % hypertonic saline with an e-flow nebulizer (pari, starnberg, germany). lung function is assessed by portable spirometry before the procedure and after inhalation of each saline concentration. results: so far cf patients ( females) with a mean age of . years (range to years) and fev between - % predicted (mean %) have been included in the study. all subjects provided es samples and all produced sputum after induction. discrepancies in cultures between es and is samples were seen in cases ( . %). in cases additional cf pathogens were found in is samples, whereas in cases es yielded additional organisms compared to is. the number of distinct pathogens was similar in the remaining patient, but different bacteria were found with the techniques. in cases p. aeruginosa was detected only with one of the techniques ( es versus is). the spectrum of side effects was similar to previous reports using other nebulizer systems, with throat irritation being the most common adverse event of the is technique. all patients who had symptoms during the procedure became asymptomatic at the time of discharge from the clinic - minutes after the procedure. patients did not finish all cycles of sputum induction procedure due to symptoms of shortness of breath and/or drop in fev > % from baseline (in patients); all reversed after salbutamol inhalation. vomiting occurred in one patient. patients refused to complete the procedure due to unpleasant taste and/or throat irritation. conclusion: these preliminary results show discrepancies between expectorated sputum and induced sputum cultures in a significant proportion of cf patients. this may be explained by the previously described regional differences in lower airway infection in cf airways rather than by a higher diagnostic yield of one of the techniques. the results also demonstrate that the e-flow system is an efficient and safe device for sputum induction in sputum producing children with cf. seidler, m.j.; salvenmoser, s.; müller, f.c. dept. pediatrics iii, university heidelberg, pediatric pulmonology, cystic fibrosis centre & infectious diseases, heidelberg, germany background: the preferred growth form of bacteria is a biofilm. s. aureus, h. influenzae, and p. aeruginosa can produce an extracellular matrix (ecm) with implications in cystic fibrosis (cf) lung disease. the biofilm can protect against host defenses and antimicrobials. a. fumigatus is a frequent colonizer of the cf respiratory tract and can cause allergic bronchopulmonary aspergillosis (abpa). while antifungals in vitro are active against a. fumigatus, in vivo antifungal therapy is often complicated or resistance is observable. the aim of this study was to investigate the ability of a. fumigatus to form a biofilm-like matrix in vitro on polystyrene (ps), human bronchial epithelia cells ( hbe) and human bronchial epithelia cells with f del/f del (cfbe o-). methods: a. fumigatus atcc # was incubated in rpmi at different ph and concentrations of fbs. temperature, production time and different flow conditions were varied on ps, hbe and cfbe o-. dry weight measurement and antifungal drug susceptibility testing was performed. scanning electron microscopy (sem) and confocal scanning laser microscopy (cslm) images were analyzed. results: the thickest biofilm was produced on ps with rpmi (+ % fbs, ph= . ) at °c for h slightly rocking. biofilm dry weight on ps was . mg after h and . mg after h. the dry weight of produced biofilm exceeded . mg on hbe and . mg on cfbe o-cells after h of biofilm production. there was no significant difference in dry weight increase between the cell lines and ps. planktonic a. fumigatus was susceptible to itraconazole ( . µg/ml), voriconazole ( . µg/ml) and amphotericin b ( µg/ml). aspergillus in biofilm was resistant against all drugs (> µg/ml). the sem pictures displayed a network of hyphal structures and matrix at h. characteristic flow channels were observed at h. cslm images displayed conidia and hyphal structures embedded in matrix formations. a-alexafluor dyed polysaccharides of the cell wall and of the ecm in the biofilm. three dimensional constructs of the cslm pictures displayed biofilm on hbe and proofed viability of the cells after h co-incubation. differences in biofilm production between hbe and cfbe o-were not significant. conclusions: a. fumigatus is able to form a biofilm structure in vitro on ps, hbe and cfbe o-. a biofilm-like matrix produced by a. fumigatus was evidenced by dry weight measurement, sem, cslm and antifungal drug resist-ance in comparison to planktonic cells. potential clinical implications of a. fumigatus biofilm formation in vivo require further attention and investigations. etherington, c. ; peckham, d. ; conway, s. ; hall, m. ; denton, m. . seacroft hospital, regional adult cf unit, leeds, united kingdom; . microbiology department, leeds teaching hospitals, leeds, united kingdom susceptibility testing results are not predictive of clinical response to antibiotic therapy in chronic pseudomonas aeruginosa infections in cystic fibrosis. we assessed the impact of reducing the number of routine susceptibility tests performed on clinical outcome in these cases. in june we introduced a protocol of limiting susceptibility tests to p. aeruginosa isolates obtained from respiratory samples taken at the commencement of antibiotic therapy, when there was evidence of clinical failure, or routinely if not tested in the previous three months. at all other times, isolates were identified and reported as normal but p. aeruginosa isolates were not subjected to susceptibility tests. between st june and th november p. aeruginosa, was isolated on at least one occasion from patients attending our adult cf unit. in this six month period we reduced the number of susceptibility tests by % (from a projected , tests on samples to an actual tests on samples). this resulted in projected savings of $ , in consumables and hours (costed at $ , ) of laboratory staff time per annum, a total saving of $ , (£ , ) per annum. we assessed the response to intravenous antibiotic treatment between the study period in and the same period in . no significant differences in median change of fev , fvc, crp, white cell count, weight, or duration of intravenous antibiotics were observed. for cf units sending regular, routine sputum samples, a reduction in the number of susceptibility tests performed in cases of chronic p. aeruginosa can be carried out without impacting on clinical outcomes. we report our preliminary results for a total of strains obtained from patients ( patients harbored more than strains). these patients were classified in groups of respiratory insufficiency according to their fev : severe in patients (fev < %), moderate in patients (fev : - %) and mild in patients (fev > %). the clonal distribution was analyzed for the different strains of sa ( to ) isolated from patients sputum. these strains were analyzed for their antibiotic susceptibility and typed by pulsed-field electrophoresis gel (pfge) after smai digestion of chromosomal dna. thirty seven patients ( %) were colonized with mrsa. nine patients were both colonized with mrsa and mssa. among mrsa strains, / ( %) were also resistant to more than three other antibiotic family. strains harboring minor differences in the banding pattern (> % similarity as assessed by the dice coefficient) were considered clonal. our results show that % of the patients were colonized with a single persistent strain during the year of follow-up. consecutive isolates with different pfge profiles were obtained from only / patients ( %). pfge analysis revealed that mrsa isolated from patients were grouped in clusters. these results revealed a possible clonal relationship between mrsa isolated from different patients with cf. we did not find any difference in the distribution of sa strains in our cf patients among the groups of respiratory insufficiency. the study is ongoing in our adult cf population and in necker pediatric cf centre in paris. giusti, r. ; furfaro, s. . pediatrics, long island college hospital, brooklyn, ny, usa; . research, lumina fund, new york, ny, usa palivizumab(synagis) is a humanized monoclonal antibody to rsv. the redbook acknowledges that some patients with cystic fibrosis may be at increased risk of rsv infection but that there is insufficient data to determine the effectiveness of this therapy. the objective of this study was to assess: )practice patterns of cf physicians in the us and canada )the severity of rsv disease in cf infants during the past rsv season. )if there is a standard of care concerning the use of synagis for cf infants. methods a questionnaire was developed using a web-based commercial vendor. an embedded web link was distributed via an e-mail sent to all us and canadian pediatric cf center directors. respondents clicked on a link to respond to the survey and results were automatically tabulated in real-time. completed responses were received from center directors ( us and canadian) for a response rate of % in us and % in canada. most responders ( %) have prescribed synagis for infants with cf in the first rsv season, however only % routinely prescribed synagis for all infants with cf. only % expressed having had difficulty in obtaining insurance approval for this medication. many physicians indicated that synagis was frequently prescribed by the general pediatrician and that infants living at long distances from a cf center may be hospitalized at local hospitals. these issues may affect the accuracy of the data and result in an underestimate of actual synagis prescription and hospitalization rates. there were infants diagnosed with cf in the past year and of these infants were reported as symptomatic. there were cf infants ( %) reported as having a documented rsv infection and ( %) of these infants had received synagis. there were infants ( %) with documented rsv infection who responded to outpatient management.there were no deaths but patients were hospitalized and of these had received synagis. of the infants that required admission to an icu had received synagis. of the patients who were noted as having persistent chest x-ray changes had received treatment with synagis. there were infants with persistent wheezing and of these infants had been treated with synagis. conclusions despite the limitations of a retrospective survey, this data demonstrates that rsv can cause significant and prolonged pulmonary disease and is a significant precipitating factor resulting in hospitalization of cf infants. the data also notes that many cf infants infected during the past rsv season have a mild illness and respond to outpatient management. a surprising finding of this survey is that infants at cf care centers where synagis was prescribed for all cf infants continued to have significant rsv related hospitalizations, persistent wheezing or prolonged chest x-ray abnormalities. there are different opinions among cf physicians concerning the routine use of synagis and currently the data suggests that there is not a standard of care concerning the prophylaxis of cf infants with synagis. this results of this survey should encourage physicians to prospectively study the efficacy of synagis prophylaxis in preventing hospitalization, persistent wheezing and chest x-ray abnormalities in cf infants. milani, a. ; cisbani, g. ; macchi, r. ; vidal-aroca, f. ; bertoni, g. . biomolecular sciences and biotechnology, university of milan, milano, italy; . basilea pharmaceutica ltd., basel, switzerland with ever increasing frequency, we now observe several examples of bacteria being resistant to every clinically available drug. therefore this urgently calls for the development of novel and improved antibiotics that may escape the extant mechanisms of bacterial resistance. one recent and promising development of the genome-wide search for target functions for antibiotics led to the identification of essential genes of pathogens by interfering antisense rnas. the first step is the construction of shotgun antisense libraries (sals) as follows. genomic dna is extracted from the bacterial strain of interest, fragmented by shearing into short pieces of dna, blunt-ended and cloned in an expression vector under the control of a regulatable promoter. the library is then reintroduced into the cognate bacterial strain and screened by replica plating colonies both in the presence and the absence of an inducer of the vector promoter. by this method, insert sequencing of clones showing conditional growth phenotypes is expected to lead to the identification of essential genes that can be silenced via antisense rna activity. we adopted this technology in order to generate a panel of essential functions of the cystic fibrosis-related opportunistic pathogen pseudomonas aeruginosa. so far, we tuned the protocol for sal generation in p. aeruginosa and identified a number of sequences conferring different levels of growth inhibition we are now characterizing these putative antisense rnas in order to define the minimal sequence able to cause the toxic effect and, on the other hand, to understand the cellular role of their targets. pseudomonas aeruginosa (pa) is an extremely versatile microbe with a vast array of pathogenic and metabolic mechanisms that allow it to form persistent infections in select patient populations, especially patients with cystic fibrosis. an ineffective immune response is considered partially to blame for failure of pa eradication. we have discovered that some strains of pa express peptidylarginine deiminase (padi) activity. padi is an enzyme that post-translationally modifies peptidylarginine to peptidylcitrulline with ammonia as a byproduct. our lab has shown that human padi can modulate the immune system through downregulation of tlr and ikk-gamma signaling. characterization of the pa padi will offer insights into a completely novel method of immune modulation by pseudomonas aeruginosa. using a widely published colorimetric assay for padi activity we have found the specific activity of crude pa cell lysates is very low. however this is similar to the only other known prokaryotic padi described in porphyromonas gingivalis. a protein homology search with the porphyromonas padi has revealed the likely genetic locus of pseudomonas padi in a . kb operon that appears in the genome of the pathogenic pseudomonas isolate pa . this operon appears to contain two candidate genes for padi activity based on conserved motif searches (padi and padi ). both have been cloned into expression vectors, partially purified by affinity tag technology and tested in the colorimetric padi assay. curiously padi autocitrullinates itself while padi has not shown activity. reaction conditions and substrate specificities for pa-padi are not like either the human or porphyromonas padi. furthermore analysis of up to clinically diverse strains has demonstrated % carry the gene for padi . this work is only the second description of padi activity in any prokaryote. pa padi could clearly have a dramatic impact on the local inflammatory milieu if it can access the same targets as human padi. the description of this activity in pseudomonas will advance our knowledge of the human-pathogen relationship and give insight into new therapies. which functions by translocating toxins into the cytoplasm of host cells. these toxins cause disease by damaging the surrounding host tissue, promoting dissemination of the organism and paralyzing the phagocytic mechanism of macrophages. pcrv is a factor required for the translocation of the toxins. the bases of these studies were to evaluate pcrv as a protective antigen in "p. aeruginosa" pneumonia and cldc as a vaccine adjuvant. methods: mice were vaccinated , , or times with the pcrv antigen combined with either aluminum hydroxide (alum) or cldc by various routes of administration. efficacy of vaccination was evaluated by challenge with "p. aeruginosa" and evaluation of survival and/or measurement of various parameters associated with lung injury. results: increase in median survival time was highly significant when cldc/pcrv was compared with cldc or pcrv alone. following subcutaneous administrations cldc/pcrv showed an increase in median survival time ( hours versus hours) and overall survival benefit following intraperitoneal administrations ( % versus %). mice with anti-pcrv antibody levels above µg/ml were significantly protected. conclusion: the investigators establish the efficacy of cldc/pcrv vaccines via several parenteral routes of administration compared to no treatment as well as cldc and pcrv-only controls. differences were demonstrated between performance of cldc/pcrv and alum/pcrv in measures of lung injury, median survival, and overall survival. the results correlated with antibody levels and histological examination of the lung tissues. importantly, these studies indicate that protection can be achieved against "p. aeruginosa" infection by targeting an antigen associated with the type iii secretion system. background: there has been a recent increase in the number of reported cases of acute renal failure (arf) in cystic fibrosis (cf). our group have undertaken a national survey, which measured the incidence risk of arf in cf patients at between . and . cases / , cf patients / year. we have now conducted a case control study to determine which factors which are associated with an increased risk of arf. methods: in our initial survey we confirmed cases of arf, in cf patients from uk cf centres, presenting between & . using the uk cf database, we identified sex and age (within months) matched controls. informed consent was sought from the control patients, or their parents, for access to the case notes and clinical data were extracted. analysis of risk factors was by conditional logistic regression, using stata (version ) and by fisher's exact test. results: there were cases of arf ( male, median age y, range m- y) and controls ( male, y, m- y). in the group of patients with arf, / had received an aminoglycoside at the time of their episode of arf or in the preceding week, compared with only of the controls for the same time period (p< . ). the median number of days of aminoglycoside in the year prior to the index case developing acute renal failure was ( - ) for cases and ( - ) for controls. conditional logistic regression showed that the odds ratio for arf per each day of aminoglycoside was . ( % ci . to . , p= . it is well-known that pa senses the environment and changes its phenotype. for instance, it produces greater amounts of the extracellular polysaccharide alginate in the cf lung, characterized by a microaerobic environment. little is known about the changes in protein secretion induced by oxygen limitation in pao , the proto-typical pa laboratory strain. no data are available on this regard about pa clinical strains. our work was aimed to study the differential regulation of proteins secreted by pa strains grown in microaerobic or aerobic conditions. a pa clinical isolate and pao were grown overnight in aerobiosis and in microaerophilic conditions. the supernatants were collected and proteomic analysis was carried over by two-dimensional capillary chromatography -tandem mass spectrometry (mauri et al., faseb j ) to evaluate the differential protein expression. in the pa clinical isolate, we identified proteins down-regulated and proteins upregulated in aerobic conditions in comparison with microaerophilic culture while in pao proteins were down-regulated and were up-regulated. proteins were down-regulated and up-regulated both in the clinical and in the laboratory strain. these proteins can mediate different biological functions since they are enzymes, heat shock proteins, chaperones, proteins involved in adaptation, motility and in the transport of small molecules. among all these proteins, as the alkaline metalloproteinase is associated with tissue invasion not only by causing rupture of epithelial tight-junctions but also by degrading several chemokines, we decided to validate its up-regulation observed in aerobic conditions by zymography. we found that the proteolytic activity of the supernatants of pa grown in aerobic conditions was higher than in microaerophilic culture both for the laboratory and clinical strain, indicating the functional relevance of data obtained by proteomic analysis. the identification of proteins differentially regulated in aerobiosis and oxygen limitated conditions in pa laboratory and clinical strains might be helpful for the knowledge of the mechanisms of colonization and lung damage due to pa in cf patients. for instance, the validation of the upregulation of the alkaline metalloproteinase in aerobic condition may shed light on these mechanisms of cf lung disease. further studies are in progress to evaluate the function of other bacterial exoproducts regulated in this model and to extend the analysis to other clinical strains. supported by the italian cystic fibrosis research foundation (ffc-grant# / ), comitato di vicenza dell'associazione veneta per la lotta contro la fibrosi cistica and azienda ospedaliera di verona, italy. cystic fibrosis (cf) sufferers are subject to repeated lung infections most commonly with the bacterium pseudomonas aeruginosa. in spite of antibiotic treatment p. aeruginosa tends to become established giving rise to persistent chronic infection. in the lung environment, the bacterium grows as a highly structured biofilm consisting of a complex community of cells embedded within a self-secreted polysaccharide matrix. investigations of p. aeruginosa biofilm growth using model strains have elucidated mechanisms which appear to govern the biofilm life-cycle. our research aims to test whether observation of these mechanisms in planktonic culture can be related to the efficiency of biofilm formation. biofilm initiation has been linked to cell motility and in particular to the presence of flagella and pili, which are thought to be important for cell attachment and the formation of microcolonies. on testing a large, genetically diverse group of clinical p. aeruginosa isolates retrieved from the lungs of cf patients we found no definitive correlation between the degree of motility of an isolate in planktonic culture and its ability to form a biofilm in vitro. the development of biofilm architecture has been demonstrated in model systems to be coordinated by the production and secretion of n-acylhomoserine lactone (ahl) quorum sensing molecules. however among the clinical isolates tested we observed no obvious correlation between the amount of ahls produced in planktonic culture and the extent of biofilm formation. overall we have found observation of phenotypic characteristics in planktonic culture to be poor predictors of efficient biofilm formation. a proteomics approach was adopted to provide further insight into the physiology of biofilm growth of p. aeruginosa isolates by comparison with planktonic growth of the same isolates. two genetically unrelated clinical isolates, demonstrated as being capable of efficient in vitro biofilm formation, were selected from our culture collection on the basis of their diverse phenotypic characteristics when cultured planktonically. one displays both twitching and swimming motilities, is mucoid and expresses ahls, while the other is non-motile, non-mucoid and no ahls have been detected in planktonic culture. we have developed a simple flow-through bioreactor to provide sufficient quantities of biofilm for proteomic analysis. gel-based and gel-free techniques were employed to study protein expression patterns for both biofilm and planktonic cultures of each isolate by mass spectrometry. proteins specific for each growth phase could be detected and may prove suitable biomarkers for monitoring the physiological status of biofilm forming strains when a larger bank of clinical isolates are examined. liposomal amikacin (arikace tm ) is a liposome-encapsulated form of amikacin that is formulated to treat chronic p. aeruginosa infections in cystic fibrosis patients. these liposomes carry a zwitterionic surface charge and are composed of lipids found naturally within the lung. a key aspect of the activity of the formulation is the ability to penetrate to the sites of pseudomonas biofilm-like growth in the lung. experiments were designed to investigate the penetration of liposomes into p. aeruginosa biofilms and in vitro activity. methods and results: model liposomes of the same size and lipid composition as liposomal amikacin (arikace tm ) were prepared with membrane-associated or encapsulated fluorescent labels, a hydrophobic carbocyanine dye and calcein, respectively. a mucoid strain of pseudomonas aeruginosa (pa ) was used to establish biofilms in rectangular optical grade glass flow cells. biofilms were observed after four days of growth by confocal laser scanning microscopy using a focal plane set to view within the biofilm cluster or outside as a control. time dependent accumulation of fluorescent liposomes within the biofilms was measured by the spatial distribution of fluorescence intensity in regions within or outside of the biofilm. images indicated significant penetration of liposomes into the interior of biofilms under these conditions. the rate of penetration was considerably slower than typical rates for small molecules, consistent with the size of the liposomes. liposome concentrations were higher near the periphery than the interior. however, even the interior concentration was at least as high as the concentration of liposomes in the fluid outside of the biofilm, suggesting some binding or trapping of the liposomes within the biofilm. penetration of liposomes was observed under flow or static conditions. in a "washout" experiment, where medium is passed through the biofilms previously treated with liposomes, a significant portion of the liposomes remained associated with the biofilms for an extended period of time. the penetration of liposomes was reflected in the observation of killing of bacteria in colonies in the interior of agar beads. exposure of these cultures to liposomal amikacin resulted in a large reduction of viable bacteria throughout the beads as monitored by a fluorescent dna content assay. similar colony forming unit reductions in animal models (to be shown in other poster presentations) suggest that these principles also operate in vivo. liposomes similar to liposomal amikacin (arikace tm ) readily penetrate into biofilms of pseudomonas aeruginosa and may even have enhanced binding to biofilms. this binding along with localized release can explain the substantial efficacy observed in animal models. coates, a.l. adherence to recommended therapy in cf has always been a challenge, in part, due to the time demands of the daily therapy. while twice daily inhaled tobramycin for those infected with pseudomonas aeruginosa (pa) has become an accepted standard of care, as much as minutes a day may be consumed inhaling mg in ml of tobramycin (tobi ® ) from the pari lc plus ® breath enhanced jet nebulizer. the purpose of this study was to determine if equivalent levels of pulmonary deposition could be achieved in a much shorter time period using . ml of a more concentrated ( mg/ml) tobramycin solution delivered by a perforated vibrating membrane nebulizer (eflow ® membrane configuration l) both, developed by pari pharma; germany. methods with a goal to study children and adults, to date, the subjects are children years and older and adult males, all with an fev > % predicted, with stable cf. all were receiving inhaled tobramycin for positive sputum cultures of pa. following pretreatment with albuterol, they inhaled both preparations on two occasions with m tc-dtpa added to the tobramycin in the nuclear medicine facility. in vitro preliminary work demonstrated that the radiolabel tracked with both formulations of tobramycin. deposition was measured by a gamma camera taking both tissue attenuation and mucociliary clearance during nebulization into account (pediatric pulmonol suppl : a ; ) . in order to have a continuous rate of deposition, the pari lc plus ® was run for a timed minutes and then both the total deposition and time of nebulization "scaled up" from in vitro testing when the nebulizer was run to dryness. this was done by multiplying the deposition by the total output when run to dryness divided by the total output in minutes. (blood samples were taken for quantification of tobramycin in the serum but not yet analysed). the rate of output per minute was calculated from the minute run and the total time was total output from in vitro testing divided by the rate of output. the eflow ® pro-vides a continuous output and stops automatically at dryness. quality assurance was the agreement between total radioactivity pre nebulization (in the nebulizer) and post which included the subject, the nebulizer, the connectors and the expiratory filter. the pari lc plus ® delivered . ± . mg in . ± . minutes compared to . ± . mg in . ± . minutes for the eflow ® . only the time of delivery was significantly different with p< . (paired t-test). tolerability of the treatment was comparable for both inhalation regimes, but the shorter treatment was preferred by all patients. these results demonstrate the possibility of delivering equivalent levels of tobramycin in much shorter periods of time into the lungs of cf patients when using eflow ® , a very efficient electronic nebulizer. this time saving may improve adherence to recommended therapy. (pediatrics ; : ) . in order to properly interpret op cultures from nbs infants, especially those with non-classic cf mutations, we need to know the op flora of non-cf infants. we obtained op cultures from healthy infants under yr of age. op specimens were plated on standard cf culture media. exclusion criteria included a first degree relative with cf, respiratory illness at the time of culture, or positive newborn screen for cf. data on cigarette smoke exposure, animal exposure, and exposure to hot tubs/swimming pools was collected. samples have been collected to date. in healthy, non-cf infants, the most common finding is non-specific mixed gram negative and positive growth. however, infants have grown pa (ages months, months, and months). many infants have grown multiple organisms. the following bacteria have been found in (n) number of infants: s. aureus ( ), e. coli ( ), e. cloacae ( ) , h. flu ( ), klebsiella ( ), pseudomonas aeruginosa ( ), h. parainflu ( ), unidentified non-lactose fermenting ( ), other ( ). data on infants including correlation to environmental factors will be presented. conclusion: non-cf infants commonly have s. aureus and many gram negative organisms including pa in their oropharynx. these results may have some bearing on interpetting colonization and clearance of pa in infants identified through nbs and in epic study participants. # ) ). identification of smg by sputum cultivation represents a significant challenge because the organisms are phenotypically diverse, grow poorly on routine culture media, and are very difficult to discriminate from other members of the oropharyngeal flora. we have developed a solid media for the selective isolation of smg from sputum. the value of the media is highlighted by the identification of smg as the quantitatively dominant organism in sputum samples of three cf patients admitted to hospital for an acute pulmonary exacerbation. in all three, the smg species failed to be identified on routine or selective media currently described for the culture of cf-specific sputum pathogens. antibiotic treatment directed against the smg correlated with clinical resolution of acute symptoms as well the reduction of smg on daily serial sputum cultures during hospitalization. this novel selective media makes use of antibiotics (colistin, sulfadiazine and oxolinic acid) inhibitory to the growth of principal cf pathogens and much of the usual oropharyngeal flora. smg agar utilizes a colorimetric indicator to uniquely identify smg colonies. the sensitivity and specificity of the selective media has been evaluated by molecular methods using terminal restriction fragment length polymorphism analysis. smg organisms do not respond well to anti-pseudomonal therapy, therefore proper detection and culture-directed antibiotic therapy is paramount. we believe that smg represent significant respiratory pathogens in cf, and because of the inability to effectively culture and identify smg they have largely gone unrecognized. pseudomonas aeruginosa releases substantial amounts of the blue antibiotic pigment pyocyanin. in presence of a reductant (such as nadph), pyocyanin redox-cycles and generates superoxide and h o . in infected cf airways, pyocyanin concentrations can reach high micromolar levels and contribute to oxidative stress of the airways. the structural basis of the pyocyanin molecule that underlies the redox-cycling with reductants of the airways is not clear. we therefore investigated i) the ability of physiologically or pharmacologically relevant reductants of the airways to support the redox-cycle activity of pyocyanin, and ii) the molecular features of pyocyanin that support redox cycling. dose-and time-dependent h o production by pyocyanin was measured by amplex red oxidation in presence of horseradish peroxidase. rates of h o production by µm pyocyanin in presence of µm reductant were: nadph ( pmole/min) > l-ascorbate ( pmole/min) > reduced glutathione ( pmole/min) > α-tocopherol ( pmole/min). in contrast, lipoic acid, genistein, or resveratrol did not significantly support pyocyanin-mediated h o production. in absence of a reductant, pyocyanin showed no measurable formation of h o . to identify the structural characteristics of the pyocyanin molecule that allow for its redox-cycling activity we synthesized a number of new pyocyanins containing electron-donating or electron-withdrawing substituents. functional assays were performed in presence of l-ascorbate as reductant. molecular substituents that donated electrons to the positively charged core of pyocyanin, either by hyperconjugative or resonance effects, reduced the h o output of the corresponding pyocyanin. in contrast, a closely related analog ( -hydroxyphenazine- , dioxide) showed significantly increased activity ( . x compared to pyocyanin) suggesting that the electron-withdrawing effect of the n-oxide functionality led to an increase in the redox-cycle activity. these data indicate that the functional characteristics of pyocyanin as a redox-cycling compound are governed by its positively charged core. in the airways, pyocyanin is predicted to utilize several reductants that are present in the airway surface liquid or intracellularly, thus contributing to cf airway disease. because pyocyanin utilizes a variety of reductants, it appears prudent to test whether inhaled small-molecular cf therapeutics support pyocyanin function. supported by nih (hl- , p at ), cfri, philip morris usa inc and philip morris international, and cff (fischer g ). taccetti, g.; braggion, c.; ravenni, n.; zavataro, l.; neri, a.; festini, f.; campana, s. meyer hospital, university of florence, cf center of tuscany, florence, italy for practical purposes, after early eradication treatment, at least three consecutive negative respiratory cultures over a -month period would indicate that the organism has been eradicated (cf trust guidelines). this recommendation is based on opinion/clinical experience of respected authorities in the absence of directly applicable studies of good quality. aims: using molecular biological techniques, we evaluated whether this -month interval is really trustworthy for distinguishing between regrowth of the same strain, suppressed but not eradicated by treatment, and new pseudomonas aeruginosa (pa) colonization. patients and methods: cystic fibrosis (cf) patients were treated with oral ciprofloxacin and nebulized colistin at detection of pa. all pa colonization episodes were recorded in an appropriate database. molecular study of each bacterial isolate from each colonization episode was performed with the rapd-pcr. results: between between - of patients in follow-up in our center had repeated pa colonization. the patients' mean age at first pa colonization was ± . months. a total of episodes was observed (mean of . episodes per patient, median of , range - ). molecular typing on strains indicated that ( . %) were a different genotype from later colonization episodes while ( . %) isolates had the same genotype as those of the preceding episode. the same genotype as preceding colonization was observed in ( . %) of isolates from patients in which a successive colonization was verified in less than months, and was verified in ( . %) of strains in patients having a successive colonization in over months (or= . ). colonization was due to a genotypically diverse strain in % of cases where colonization occurred within months of eradication. during the observation period ( %) of patients acquired chronic pa infection. conclusion: re-colonization by pa following eradication therapy is mostly ( %) caused by strains with a different genotype, suggesting acquisition from an external source. a short pa-free period is mainly due to transient suppression of pa growth, and true eradication followed by acquisition of a new pa genotype occurred in most cases only after a pa-free period longer than months. those patients with a pa-free interval of less than months had a six-times higher chance that the pa was not eradicated compared to those with a germ-free interval of over months. this evidence demonstrates that the definition of successful eradication should be reconsidered, taking into account additional parameters such as molecular analysis. cystic fibrosis (cf) patients appear to have an increased risk of urolithiasis. while a number of possible explanations for this have been proposed and investigated, no definitive mechanism has yet been demonstrated. as cf patients frequently get respiratory tract infections they regularly receive ciprofloxacin treatment, often given in doses well in excess of conventional prescription regimes. there are occasional reports of ciprofloxacin crystals urine and stones in the urinary tract. here we investigate the hypothesis that ciprofloxacin excreted in urine might act as a promoter of crystallisation of calcium or magnesium salts and thereby increase the risk of kidney stone disease. the effect of ciprofloxacin was tested in artificial urine (au). in vitro crystallisation was tested using a well plate turbidity method, to identify a metastable limit of oxalate concentration (ml) and a growth and nucleation parameter, the turbidity rate index (tri). the nucleation ph of urine was examined by tritrating oxalate free au through a ph range of . to . and monitoring the solution/suspension turbidity. in au at ph . , ciprofloxacin, at , , or mg/l, had no detectable effect on initiation of calcium oxalate crystallisation (ml) or its progress (tri) (n= for each concentration). when au with mm ca, mm mg and mm po was titrated there were two distinct nucleation points; a slow event starting at about ph . and a much faster event at about ph . . omitting ca or mg confirmed that the first event was due to calcium phosphate precipitation and the second to struvite. including ciprofloxacin at , or mg/l did not alter these nucleation ph values, but the magnitude of the turbidity rise showed that the ciprofloxacin co-precipitated with the struvite. ciprofloxacin at mg/l and without ca or mg began to precipitate at ph . and could be held in solution until ph . when ca and mg were included. even at high concentrations, ciprofloxacin does not influence calcium oxalate crystallisation. nor does it promote calcium phosphate or struvite precipitation; on the contrary, while the calcium and magnesium remain in solution, they help to prevent precipitation of the ciprofloxacin itself. urinary ciprofloxacin does not appear to act as a stone or crystal promoter. pseudomonas aeruginosa is a significant cause of mortality in cystic fibrosis (cf) sufferers. cf patients were thought to acquire p. aeruginosa from the environment; however genotyping over recent years has revealed clonal strains in sputa from cf patients in the uk, australia, and canada that are transmitted person to person or from a common source. one clonal strain, australian epidemic strain- (aes- ), (formerly melbourne epidemic strain, m or pi) currently infects up to % of patients in five cf clinics on the eastern seaboard of australia. most cf clonal strains have been associated with increased virulence not fully explained by greater antibiotic resistance. both genotypic and phenotypic differences have been postulated as important in enabling transmission of clonal strains. in order to compare the expression profile of aes- to the type strain p. aeruginosa pao , the cf research group at the university of sydney compared the genome expression data of four clonal aes- isolates and pao , when grown as planktonic and as -hr biofilm cultures. in aes- , a set of significantly differentially expressed genes (all downregulated) were identified, including the quorum sensing genes lasa, lasb and rhll. in contrast, both upregulated and downregulated genes were differentially expressed in pao biofilm compared to pao planktonic culture. expression data was validated using quantitative real-time pcr. to compare biofilm growth at the phenotypic level, the four clonal strains and pao were grown as -hr biofilms in a double-blind study, and the size of ten randomly selected biofilms per isolate, stained with syto® green fluorescent stain was measured. at hr, the biofilms formed by aes- isolates were significantly larger (ca. -fold) than pao (average size: ± µm vs ± µm )(p< . ). the average thickness of three biofilms per isolate, measured by confocal microscopy, showed aes- biofilms to be approximately . -fold thicker than those formed by pao ( . ± . µm vs . ± . µm). the general gene downregulation observed in aes- biofilms suggests an adaptation to the cf host, while a larger biofilm would provide for more effective bacterial dispersal. thus the transmissibility of aes- may be linked to enhanced biofilm formation upon colonisation. background -objective: whilst influence induced by bacterial colonization in cystic fibrosis (cf) is established, risk induced by fungal colonization is less defined. prevalence of other species than aspergillus sp. or candida sp., and factors associated with fungal presence are also poorly documented. our preliminary study aimed to determine which fungal species were present in sputum collected from adult cf patients, and which factors were associated with fungal presence. methods: in a monocenter, transversal prospective study, cf adult patients were included to determine fungal presence in sputum using semi selective growing media. clinical parameters (shwachman score, respiratory function, nutritional status, gastro-oesophageal reflux, pancreatic insufficiency and diabetes); therapeutics used (including oral or intravenous antibiotics, systemic or inhaled corticosteroids or bronchodilatators, antifungal treatments); microbiological data of bacterial colonization and environmental parameters (potted plants or domestic animals presence) were determined for each patient. correlation between fungal, clinical, environmental, therapeutic or microbiological data was evaluated by mann-whitney non parametric u test. results: patients ( %) presented with fungal presence in sputum. % presented with yeasts species, % with moulds. aspergillus fumigatus and candida albicans were the predominant species in moulds and yeasts respectively, but less common mould species such as exophiala dermatidis or paecilomyces variotii were also recovered. factors associated with fungal presence were pancreatic insufficiency (p= , ); malnutrition (p= , ), bacterial colonization and inhaled corticosteroids. candida albicans was correlated with more severe shwachman score (p= , ), bacterial colonization (p= , ), notably with pseudomonas aeruginosa (p= , ) , and intravenous antibiotics use (p= , ). moulds species were significantly associated with inhaled corticosteroids (p= , ). antifungal use was associated with frequent resistance to azoles treatments ( resistant isolates out of patients treated). conclusion: fungal presence in cf appears frequent. some species could have been previously overlooked due to diagnosis difficulties. the effect of corticosteroids on moulds species, already found in other pathologies, appears important in cf. influence of fungal presence on cf course needs prospective studies, in order to establish if patients could benefit from antifungal treatments or preventive measures. dren on a ventilator without a previous diagnosis of cystic fibrosis is unknown. the aim of our study was to investigate the prevalence of these microorganisms in routine sputum cultures in young children on a ventilator in a pediatric intensive care unit (picu). methods: from all ventilated children aged - years admitted from - , sputum culture results obtained from tracheal aspirates within the first week of admission were retrospectively analysed. three patient subgroups were identified: respiratory failure due to pulmonary disease (group ), ventilation after elective surgical procedures (group ) and other ventilated children (group ). children with a previous intensive care admission or cf were excluded. the cf database was checked ( ) to identify any children with a new diagnosis of cf that were included in the study. results: . % of all ventilated children had a positive sputum culture with one of the "cf-bacteria" s. aureus, h. influenzae and p. aeruginosa these were found mainly in group (see table) . / of these children were admitted for treatment of respiratory syncytial virus (rsv) bronchiolitis. a sweat test was performed in children, all admitted with pulmonary disease and because of co-existing clinical signs or symptoms. one sweat test was positive and subsequently cf was diagnosed in this child. no other children out of the study group have since then been diagnosed with cf. as the population in the northern part of the netherlands is very stable, it is unlikely a diagnosis of cf has been missed. conclusion: s. aureus and h. influenzae are cultured frequently in ventilated children without cf, especially when ventilated because of pulmonary disease. the opportunistic pathogen p. aeruginosa causes both acute and chronic human infections. the balance between systemic infection and mortality or chronic persistence and morbidity depends on complex relationships in which the immunological status and genetic potential of the host but also the bacterial biodiversity are determinant factors. p. aeruginosa extensive genetic adaptation and microevolution have been repeatedly observed in chronic infections of cf patients in contrast to what it is documented in acute infections. whether these p. aeruginosa clonal variants differ in their pathogenic potential is not yet known. a total of clinical p. aeruginosa isolates from six cf patients which carried unique clonal lineage from the onset of colonization over - years were selected (bragonzi et al, ; montanari et al, ) . five p. aeruginosa environmental strains, which represent the source of acquisition for cf patients, and two laboratory strains pao and pa were also used as references. multiple genotypic analysis of sequential p. aeruginosa isolates which included pfge, atchip and multilocus snps showed intraclonal diversity with genome rearrangements, variations in pathogenic islands and acquisition of prothoadaptive mutations in the muc genes. the largest divergence was observed between the completely sequenced reference strains pao and pa , the latter represented in our panel by three cf isolates. p. aeruginosa virulence has been assessed by monitoring its capacity to induce bacteremia and to establish chronic infection in a murine model (bragonzi et al, ) . overall, p. aeruginosa environmental strains increased five times the risk of bacteremia when compared to clinical strains (test of proportion: p< . ). the high risk of mortality was also evidenced for p. aeruginosa cf strains isolated at the onset of the infection when compared with those isolated after years of colonization (p= . ) indicating that environmental strains or newly acquired strains were similar in their virulence. p. aeruginosa clinical strains isolated after years of colonization increased chronic persistence and reduced the risk of bacteremia (p= . ) when compared to strains isolated at the onset of the infection. the strain pa was found to be lethal in contrast to p. aeruginosa clonal strains of clinical origin that established chronic persistent infection in the murine lung. furthermore, our data showed that adaptive traits commonly associated with the chronic p. aeruginosa infections in cf patients, such as transition to the mucoid phenotype, did not confer a selective advantage to bacterial cells in colonizing the murine lung (p= . ). these results suggest that p. aeruginosa pathogenicity is independent of the strain's genotype but rather genetic adaptation to the cf airways plays an important role in the development of persistence and in the resistance to host defences. supported by telethon and italian cf research foundation. beringer, p. the combination of antipseudomonal beta-lactam and aminoglycoside antibiotics is frequently prescribed during acute pulmonary exacerbations. since the introduction of the beta-lactam compounds there has been numerous reports citing altered pharmacokinetics of several compounds (but not all) within the beta-lactam class. recently it has been suggested that substrate specificity for the renal transporter pgp may explain the variability in renal drug clearance observed in patients with cf. pgp is structurally related to cftr and evidence suggests it serves a complementary role in modulating alternative chloride channel function. in a cftr knockout model mdr expression was reported to be increased nearly four-fold. in an effort to elucidate the potential contribution of pgp to the renal clearance of drugs in patients with cf, we conducted a controlled clinical trial comparing the pharmacokinetics of fexofenadine (fx) in patients with cf and age matched healthy volunteers (hv). fexofenadine is not significantly metabolized and is a relatively specific substrate for the membrane transporters pgp and oatp. probenecid (pb) is a selective inhibitor of oatp and was used pharmacologically to block the activity of this transporter in-vivo. our hypothesis is that if pgp is upregulated within the renal tubules of patients with cf we would expect to see an enhanced renal clearance of fexofenadine in these patients when compared with control subjects. methods: (n= cf, hv) subjects underwent this prospective controlled study which fx was received alone or in combination with probenecid (pb). iothalamate was given each day to measure glomerular filtration rate (gfr). blood and urine samples were obtained at specified times over hours each study day for determination of fexofenadine and iothalamate concentrations. fexofenadine and iothalamate concentrations were assayed using liquid chromatography-tandem mass spectrometry and hplc-uv respectively. pharmacokinetic analysis was performed using a -compartment cumulative urinary excretion model with the adapt ii software. differences between groups were determined using a paired t-test or mann whitney-u. results: patients were included, cf patients were slightly older and had lower body mass index when compared to hv, (mean±sd, ± . vs. . ± years p= . , . ± . vs. . ± . p= . respectively) but did not differ in gfr ( ± . vs. ± ml/min- . m p= . respectively). no significant differences were found between cf and hv in clr of fx alone and fx in combination with pb clr ( . ± vs. . ± . ml/min- . m p= . and . ± vs . ± . ml/min- . m p= . respectively). conclusion: the results of this study indicate the enhanced clearance of certain antibiotics previously reported in patients with cf do not appear to be due to upregulation of renal tubular pgp or increased gfr. maceachran, d.p. department of microbiology and immunology, dartmouth medical school, hanover, nh, usa p. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (cf) patients. shortly after birth cf patients are colonized by p. aeruginosa, which leads to a lifelong chronic infection. the mechanisms behind p. aeruginosa colonization of the cf lung are still poorly understood, however several secreted toxins have been associated with this phenomenon. cf is characterized by a loss of mucocilliary clearance, a key component of innate immunity in the lung, and an increase in sputum viscosity as a result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (cftr). we have previously shown that the novel p. aeruginosa secreted protein cif is capable of reducing apical membrane expression of cftr. we have begun to characterize cif expression and have identified a key regulator of cif transcription. mutations affecting the divergently transcribed putative tetr family repressor encoded by the pa gene result in a significant increase in cif expression. furthermore, we have demonstrated that the pa gene product is capable of binding the promoter region located immediately upstream of the cif-containing operon. this work has been supported by the nih t -dk - . ryall, b. ; davies, j.c. , ; wilson, r. ; shoemark, a. ; williams, h.d. . division of biology, imperial college london, london, united kingdom; . department of gene therapy, imperial college london, london, united kingdom; . host defence unit, royal brompton hospital, london, united kingdom; . paediatric respiratory medicine, royal brompton hospital, london, united kingdom pseudomonas aeruginosa is the most important respiratory pathogen in patients with cystic fibrosis (cf) and non-cf bronchiectasis. the factors which enable p. aeruginosa to establish chronic, debilitating respiratory infections are unclear. however p. aeruginosa is one of a limited number of bacteria that is able to synthesise hydrogen cyanide, a potent inhibitor of cellular respiration. this study examined whether hydrogen cyanide is produced by p. aeruginosa in cf and non-cf bronchiectasis airway infection. cyanide concentration was measured in fresh sputum from cf and non-cf bronchiectasis patients with and without p. aeruginosa lung infection using a cyanide ion sensing electrode. cyanide was detected in sputum from / cf and non-cf bronchiectasis patients with current p. aeruginosa infection, whereas it was not detected in any of the patients without this organism (p< . ). maximum lev-els were µm (mean ±se: ± . µm), which compares with levels of above µm which would be considered toxic in blood. concurrent lung function data were available on all p. aeruginosainfected cf patients; the group with measurable sputum cyanide (n= ) was not different from those without (n= ) on the basis of age, gender or p. aeruginosa phenotype (mucoid/ non-mucoid). however, those with detectable cyanide had significantly poorer lung function than those without (fev % predicted: . ± . % versus . ± . %, p< . ; fvc% predicted: . ± . % versus . ± . , p< . ). we have shown that cyanide is detectable at clinically significant levels in sputum from cf and non-cf bronchiectasis patients infected with p. aeruginosa and is associated with significantly impaired lung function. we have recently reported a dose regimen for once-daily tobramycin dosing in patients with cystic fibrosis which was based on a retrospective study (lam w et al., j antimicrob chemother ) . we present here an interim analysis of an ongoing prospective study to evaluate the proposed dosing in paediatric patients with cystic fibrosis. as part of this protocol pharmacokinetic parameters were determined in cystic fibrosis (cf) patients receiving intravenous antibiotic therapy for a pulmonary exacerbation. serum creatinine, audiometric testing, respiratory cultures and fev were assessed at baseline and after a week treatment period. serum tobramycin concentrations were interpreted by clinical pharmacists performing therapeutic drug monitoring (tdm) to optimize dosing. serum tobramycin levels were drawn after the first dose, repeated with each dose adjustment and weekly thereafter. the proposed sample size of this study is patients; an interim analysis was performed to identify trends for pharmacokinetic and safety outcomes from patients admitted from november through april . seven ( . %) patients achieved the target c max of - mg/l, ( . %) patients remained under the target range and patient was above the target range with the st dose. with nd, rd, and th tdm performed for either routine weekly levels or dose adjustment levels, , , and patients met the target c max respectively. the percentage of patients who achieved target c max increased to . % with the rd set of tdm levels and . % with the th set of tdm levels (p-value< . ). there were no significant changes in k e , t / and v d /kg for each patient from st to nd set of tdm levels over time. compared to the previous retrospective study the mean v d /kg was significantly higher ( . l/kg versus . l/kg, p= . ) and the mean t / was significantly shorter ( . hrs versus . hrs; p< . ). none of the patients' serum creatinine increased ≥ % from baseline. baseline audiometric tests were within normal limits in ( . %) patients. p. aeruginosa was isolated from respiratory specimens of patients on initial culture. of the patients with follow-up cultures, patients grew p. aeruginosa sensitive to tobramycin, demonstrating no decrease in sensitivity. our preliminary results suggest the pharmacokinetic data differ from the expected profile suggested by the retrospective study. all study patients treated according to the guidelines were clinically well after their course of therapy and did not experience clinically significant toxicity from once-daily tobramycin dosing. the once-daily tobramycin dosing will continue to be evaluated until the proposed sample size is achieved for final analysis. anderson, g.g.; o'toole, g.a. dartmouth medical school, hanover, nh, usa pseudomonas aeruginosa chronically colonizes the lungs of individuals with cystic fibrosis (cf). evidence suggests that this infection progresses through distinct phases, wherein initial colonizing strains undergo a switch to mucoidy that corresponds to formation of bacterial microcolonies in the airways and life-long persistence. currently, the environmental signals and mechanisms regulating conversion from the acute, highly virulent form to a less virulent biofilm state remain obscure. to investigate these issues, we developed a tissue culture model system for growth of p. aeruginosa biofilms on cf-derived human airway epithelial cells in vitro. biofilms grown on these cells appear similar to those found in cf airways as well as to abiotic biofilms. eventually, epithelial cells were killed by the p. aeruginosa biofilms. however, we discovered that addition of the antibiotic tobramycin preserved the monolayer integrity for at least hours. bacteria were not completely eliminated by this treatment, suggesting that tobramycin influenced the virulence of the biofilm bacteria. microarray analysis of cf cell grown biofilms treated with tobramycin revealed marked alterations in transcriptional profiles compared to untreated controls. two tobramycin-induced genes were identified from this screen which, when deleted, exerted altered virulence phenotypes on airway epithelial cells in culture. quantification of cytotoxicity of these mutants revealed either increased or decreased ability to kill the epithelial cells. complementation with full-length copies of these genes restored wild-type function. these studies suggest a complex relationship between acute and chronic forms of p. aeruginosa, and that antibiotic treatment may influence transitions between these modes. it is this interplay that may determine the virulence status of the microorganism upon initial infection as well as during cf exacerbations. neville, m.; sardaryan, g.; ejaz, s.; scotto, a.w.; gupta, r. transave, inc., monmouth junction, nj, usa purpose: during the course of treatment, patients with cystic fibrosis and chronic pseudomonas aeruginosa infections in their lungs may be treated with nebulized tobramycin (tobi®). reduction in treatment times or dosing frequency may improve compliance and outcome. nebulized arikace™ may offer advantages to tobramycin by producing sustained lung levels of drug, reducing dosing frequency and increasing antimicrobial efficacy. herein are presented biodistribution data and efficacy data in a chronic rat model of p aeruginosa comparing arikace™, soluble amikacin and tobramycin. method: female rats (charles river) received (~ mg / kg) of arikace™, soluble amikacin and tobramycin by inhalation. test articles were aerosolized in a pari lc star nebulizer at psi using devilbiss compressors (sunrise medical) for min. - rats were euthanized at time and hr post inhalation by co asphyxiation. blood was collected by a cardiac puncture. serum was collected from coagulated blood by centrifugation and stored at oc until analysis for drug content. in addition lungs, kidneys, intestinal contents, urine and brains were harvested. tissues and biological samples were homogenized in saline and antibiotic concentrations determined by tdx analyzer (abbott). chronic lung infections were produced in rats by intratracheal instillation of agar beads containing p. aeruginosa. three days post instillation, rats (n= /group) were treated with aerosolized saline or once daily with arikace™, or twice daily with tobramycin (total daily dose = mg /kg) for days. rats were euthanized days post final dose and the pseudomonas cfu counts per lung were determined. results: the biodistribution and pharmacokinetics of aerosolized arikace™ was significantly different from that of amikacin and tobramycin. the primary difference was the amount of antibiotic retained in the lungs. rats that inhaled soluble amikacin cleared approximately % of the amikacin from the lungs after hr with concomitant % increase in drug levels in the urine. rats that inhaled arikace™ cleared only % of the deposited amikacin from the lungs after hrs. this initial clearance of drug most likely reflected the amikacin released during nebulization. extended clearance of arikace™ from the lungs was measured by recovery of amikacin in the urine at later time points. in a separate longer-term study comparing arikace™ and tobramycin, auc ( - hr)for arikace™ in the lung was ug*hr/g while only ug*hr/ g for tobramycin which demonstrates a -fold increase in retention of antibiotic in the lung with arikace™. eradication of p. aeruginosa from the lungs of rats with arikace™ was equal to tobramycin (given equivalent daily doses). however, arikace™ reduced the number of cfu in the lungs with a single daily exposure to the same degree as inhalation of tobramycin twice a day. conclusions: inhalation of arikace™ achieved higher concentrations and increased retention time of the antibiotic in the lungs of rats when compared to inhaled amikacin or tobramycin. it is expected that these properties of arikace™ will reduce dosing frequency and improve antimicrobial efficacy in patients. nielsen, x.c. ; johansen, u.r. ; nørregaard, l. ; vandamme, p. ; høiby, n. . clinical microbiology, rigshospitalet, copenhagen, denmark; . laboratory for microbiology, university of ghent, ghent, belgium background: the burhkolderia cepacia complex (bcc) is a diverse group of bacteria with at least genomovars (gv) or species. accurate species identification is necessary for better understanding of the epidemiology and pathogenesis of bcc. species identification based on phenotypic characters is difficult when it is based on few if any differences. crossed immunoelectrophoresis (cie) has been performed routinely to identify all burkholderia and other difficult isolates in our department (høiby et al.: "taxonomic application of crossed immunoelectrophoresis." int. j. syst. bact. ( ): - .) . in this study, we compared the results obtained by cie with those obtained by the standard molecular biological method using reca gene pcr and rflp to identify bcc at the species level (golden standard). methods: between and , burkholdreia isolates were recovered from patients in the two danish cystic fibrosis (cf) centres. . cie method: immunological cross-reactivity between antigens from the reference strains and the strains isolated from our cf patients were compared by employing cie and rabbit standard-antibodies (purified igg) raised against water-soluble antigens from reference strains of the bcc. species identification was made based on matching coefficient (mc). . reca gene-based identification: bcc specific primers were used to amplify reca gene. species-specific pcr is then performed using b. multivorans specific primers to identify the b. multivorans gv ii. rflp patterns were generated for those non-multivorans bcc by digesting the bcc-specific pcr products with haeiii. the patterns were then compared with the patterns generated from reference strains in the database. pulsed field gel electrophoresis (pfge) genotyping was performed for all strains to detect patient-topatient transmission. results: / isolates were bcc-specific pcr positive. species-specific pcr identified isolates as b. multivorans (gv ii). the other three isolates were identified as b. cenocepacia (gv iii) based on rflp patterns. two isolates with negative bcc-specific pcr reactions were identified as b. gladioli by cie (confirmed by reference laboratory in belgium). results from cie showed that strains from bcc were immunologically closely related with only a few non-cross-reactive antigens. compared with the standard reca gene-based method, cie method resulted in correct identifications in isolates ( . %), misidentifications in isolates ( . %) and uncertain identifications in isolates ( . %). genotyping by pfge showed unique patterns for all isolates except for those from two sisters. this suggested that patient-to-patient transmission of bcc among the danish cf patients has remained very rare. conclusions: reca gene-based pcr and rflp is a quick and reliable method for identification of bcc at the species level. cie proved useful in initial classification of bcc strains, but additional information is obtained by the reca gene-based species identification and pfge method. background: in childhood, the cystic fibrosis airway is characterized by persistent inflammation with high quantities of neutrophils. in this setting, recurrent exposure to low numbers of pseudomonas aeruginosa occurs routinely. chronic infection by p. aeruginosa is associated with a significant increase in morbidity and mortality in cf, and is believed to occur by formation of a biofilm in the cf airway. previously, we have shown a significant neutrophil dependent enhancement of p. aeruginosa biofilm density that is greatest for low initial concentrations of p. aeruginosa. biofilm enhancement is facilitated via neutrophil-derived dna and f-actin, which form a framework that p. aeruginosa can exploit for growth. dna and factin polymerize via positively charged molecules such as histones. p. aeruginosa then associates with these polymers, which results in increased early biofilm density, displaying an increase of over fold at hours compared to p. aeruginosa in the absence of neutrophils. hypothesis: compounds that disperse dna and/or f-actin can disrupt neutrophil-enhanced biofilms. methods: poly(aspartic acid), a negatively charged amino acid chain with the capacity to disrupt f-actin, was examined singly and in combination with dnase and/or antibiotics (ciprofloxacin and tobramycin). the nunc-tsp system was used to allow for high throughput assessment of the density of biofilm formation in the presence of neutrophils and the effect of various agents in disrupting the biofilm. the biofilm assay is independent of cellular settling, as the reactor is vertically suspended in the culture. p. aeruginosa strains tested were pao , and two isogenic cf isolates recovered from an initial infection (early), and following established infection ~ years later (late). biofilms were formed by incubation of p. aeruginosa with human neutrophils for to hours in rpmi with % heat inactivated platelet poor plasma. results: antibiotics were incapable of disrupting biofilms. poly(asp) significantly disrupted neutrophil-induced biofilms. in the presence of neutrophil products this action is sensitive to proteolytic degradation, but can be protected by the presence of protease inhibitors. dnase also disrupts a biofilm formed in the presence of human neutrophils. the combination of poly(asp) + dnase resulted in an increase in biofilm disruption for a hour biofilm. biofilms allowed to form for hours were more resistant to disruption by dnase and poly(asp) as single agents, but the combination of both demonstrated a synergistic effect. similar disruption of biofilms were observed when pao was compared to early and late cf strains. conclusions: pseudomonas aeruginosa biofilms formed in the presence of neutrophils in vitro can be disrupted by agents that disperse the f-actin and dna framework. support: cff, max and yetta karasik foundation cystic fibrosis (cf) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. the black yeast, exophiala dermatitidis (synonym wangiella dermatitidis) has been found with increasing frequency in sputum specimens of cf patients, with reported isolation rates ranging from between . and . %. at present, no species-specific diagnostic pcr exists to aid with the clinical laboratory detection and identification of this organism. a novel species-specific pcr-based assay was developed for the detection of e. dermatitidis, based on employment of rdna operons and interspacer (its) regions between these rdna operons. two novel primers, (designated exdf & exdr) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of exophiala, as well as with other commonly described yeasts and filamentous fungi within cf sputum, including candida, aspergillus and scedosporium. pcr employing exdf (forward primer [ -,mer], '-ccg cct att cag gtc c- ') and exdr (reverse primer [ -mer], '-tct ctc cca ctc ccg c- '), was employed and optimized on extracted genomic dna from a well characterized culture of e. dermatitidis, as well as with high quality genomic dna template from a further unrelated fungi, including candida albicans, c. dubliniensis, c. parapsilosis, c. glabrata, scedosporium apiospermum, penicillium sp., aspergillus fumigatus, aspergillus versicolor, pichia guilliermondii, rhodotorula sp., trichosporon sp., aureobasidium pullulans, fusarium sp., mucor hiemalis, bionectria ochroleuca, gibberella pulicaris. results demonstrated that only dna from e. dermatitidis gave an amplification product of the expected size, whilst none of the other fungi were amplifiable. subsequent direct employment of this primer pair detected this yeast in the sputum of / ( %) adult cf patients, employing a nested pcr approach with panfungal outer flanking primers of the its -its region. these two patients were the only patients who were previously shown to have a cultural history of e. dermititidis from their sputum. e. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan-and multiresistant bacterial pathogens from sputum, namely pseudomonas aeruginosa and burkholderia cepacia complex organisms, hence this species-specific pcr assay may help detect this organism from cf sputum more specifically and rapidly. overall, employment of this novel assay may help in the understanding of the occurrence, aetiology and epidemiology of exophiala dermatitidis, as an emerging fungal agent in patients with cf. cystic fibrosis patients are particularly susceptible to infection by strains of pseudomonas aeruginosa and burkholderia cepacia complex. since , the pseudomonas genome database has been a resource for peer-reviewed, continually-updated annotations for the pseudomonas aeruginosa pao reference strain's genome and, more recently, comparative analyses to several closely-related pseudomonas species. in order to facilitate better cross-strain and cross-species genome comparisons, we have developed or are incorporating methods to improve the identification of orthologs (genes diverged due to speciation) and identify genes undergoing unusual selection. our analysis of several completely sequenced pseudomonas genomes has revealed that approximately % of ortholog predictions by the classic "reciprocal best blast hit method" are likely incorrect. we have also performed an analysis of unusually large intergenic regions in p. aeruginosa pao that appear to be essential (according to saturation transposon mutagenesis) or involved in virulence (according to signature tagged mutagenesis). we identified at least new putative protein-coding regions and non-coding rnas that were not previously annotated and may play critical roles in microbial pathogenesis or viability. we are also focusing our attention on facilitating better cross-strain comparisons between recently sequenced burkholderia cepacia complex genomes through development of a new online burkholderia genome database. in addition to improved ortholog prediction, this new database will provide access to a very flexible boolean search feature that allows researchers to search and compare annotations within or between the genomes of burkholderia strains, returning annotations from multiple genomes suitable for simultaneous viewing and downstream analyses. we have also included new, very accurate protein subcellular localization predictions for the deduced proteome from each of these genomes -predictions that can aid the identification of new cell surface or secreted therapeutic targets or vaccine candidates. further comparative analyses with other newly-sequenced, related strains should provide insight into strain-specific features that may be exploited to better understand virulence and antimicrobial resistance exhibited by cf-relevant pathogens. funding provided by cystic fibrosis foundation, therapeutics (usa). popova, a.p.; verma, r.; zanni, r.l.; sembrano, e. pediatrics, monmouth medical center, long brnach, nj, usa background: cystic fibrosis (cf) is a chronic, progressive, genetic disease leading to poor lung function, chronic bronchial infection with certain bacteria and recurrent pulmonary exacerbations. despite evidence of inflammation within the airway, it is unclear which circulating inflammatory biomarker best reflects the airway inflammation and lung function in cf. objective: to determine the utility of c-reactive protein (crp) value in evaluating patients with cystic fibrosis pulmonary exacerbation. design: a retrospective chart review was carried out on patients followed at the cystic fibrosis center at the children's hospital. information about serum crp values, pulmonary function test results(pft), presence of pulmonary exacerbation as documented in the chart, sputum culture results, current medications, as well as demographic data, genetic compositions and weight percentiles between january and march was collected from the charts and was analyzed. results: of patients whose charts were reviewed, were excluded because they underwent lung transplant, were excluded because they had other mild infection, not diagnosed as pulmonary exacerbation. for patients crp was not measured and they were also excluded from the analysis. total of patients were included in the study. for patients information about crp was obtained on more than one occasion. the patients were divided into groups based on the presence of pulmonary exacerbation and elevation of crp values -in the group with pulmonary exacerbation, patients had elevated crp and had normal crp and in the nonexacerbation group had elevated crp and had normal. fisher's exact test (two -tail) was applied (p= . ) and confirmed statistically significant elevation of crp in patients with pulmonary exacerbation of cystic fibrosis. the means and standard deviations of the crp values for the patients without pulmonary exacerbation and the patients with pulmonary exacerbation were calculated and analyzed using independent group t-test (two tail). mean crp value for the exacerbation group was . with standard deviation of . and mean crp value for the non-exacerbation group of . with a standard deviation of . . statistically significant difference between the two means was noted (p= . ). conclusion: serum crp values in patients with cf pulmonary exacerbation were significantly higher than in patients without pulmonary exacerbation. serum crp may be a useful marker of pulmonary exacerbation in children. in the absence of pulmonary exacerbation, cf is not associated with elevated crp values in children. the findings of this study could aid in identifying patients with pulmonary exacerbation earlier and initiate appropriate therapy sooner. further studies are needed to determine whether crp values are associated with the severity of the underlying disease, to what extent the pft are affected, the age of the patient, the type of bacterial pathogen, and genetic factors. background pulmonary infections with nontuberculous mycobacteria (ntm) are commonly associated with structural lung diseases or airways clearance disorders such as cystic fibrosis and primary ciliary dyskinesia. we follow approximately individuals with ntm pulmonary disease who share common clinical features but have no identifiable systemic immune defect or clearly defined predisposing conditions. the pulmonary disease and clinical presentation suggest these individuals may have disorders of ion transport or ciliary function. while some cftr mutations and ciliary abnormalities are difficult to detect through standard genetic and ultrastructural analyses, in vivo physiologic tests of ion transport and measures of ciliary function may allow us to identify ion transport abnormalities in subjects with no identified cftr abnormalities, determine the functional significance of novel cftr mutations, and distinguish disorders of ciliary function from other airways clearance disorders. methods we evaluated the medical and family histories, chest and sinus ct's, sputum cultures, cftr genotypes, and sweat chloride levels of individuals with nontuberculous mycobacterial pulmonary disease and individuals with chronic airways disease and bronchiectasis. we systematically tested the pulmonary function (pft), nasal nitric oxide production (nno), and nasal potential difference (npd) of each subject. nasal mucosal scrape biopsies were performed and sent to unc-chapel hill for assessment of ciliary ultrastructure as part of the genetic diseases of mucociliary clearance consortium. pulmonary function, nno, and npd were measured on healthy controls. results all subjects had sino-pulmonary symptoms consistent with those associated with cf and pcd. subjects had low nno ( . ± . nl/min) consistent with levels reported in subjects with pcd (< nl/min, noone ). one subject with variant cf (∆f /r h) displayed low basal pd (- . mv), elevated na+ absorption ( . mv), and no cftrmediated cl-conductance (- . mv). subjects with cftr mutations, subject with isolated elevated sweat chloride ( mmol/l), and subject with isolated borderline sweat chloride ( mmol/l) displayed normal basal potential difference (- . ± . mv) and na+ absorption ( . ± . mv) but decreased cftr-mediated cl-conductance (- . ± . mv) relative to controls (- . ± . mv). conclusions ntm patients heterozygous for cftr-mutations or with elevated sweat chloride (and no identified mutations with coding region sequencing of cftr) may have detectable subtle differences in cftr clconductance, while measurement of nno may identify disease associated with ciliary abnormalities. these findings suggest subtle abnormalities in airways clearance may predispose to airway infection with environmental organisms such as ntm. support: intramural funds, national institute of allergy and infectious diseases muhlebach, m. ; goodrich, j. ; sutton-shields, t.n. ; wedd, j.p. ; henegar, c. ; miller, m.b. ; gilligan, p.h. . dept. of pediatrics, unc chapel hill, chapel hill, nc, usa; . university of north carolina hospitals, chapel hill, nc, usa; . school of public health, university of north carolina hospitals, chapel hill, nc, usa; . dept. pathology and laboratory medicine, university of north carolina hospitals, chapel hill, nc, usa introduction: proportion of methicillin resistant (mrsa) vs. susceptible sa infections increased from % in to % in in us hospitals. similarly, the prevalence of mrsa in the community has risen in recent years. these community acquired (ca) mrsa isolates are genotypically different, have different antibiotic susceptibility, and often carry the pvl virulence gene. this virulence gene is associated with more invasive, mostly skin infections in healthy individuals. reports on impact of mrsa in cf show conflicting data. , . the aim of this study was to determine the prevalence of ca-mrsa strains at our pediatric and adult cf center and to assess the impact of these infections on clinical outcome. methods: all mrsa isolates recovered from routine clinical cultures, were prospectively collected and analyzed for months ( / - / ). using molecular assays to determine the pvl status and to identify the sccmec (staphylococcal cassette chromosome) type, the organisms were classified as ca or ha. classification as ca mrsa was based on presence of pvl and sccmec iv type. clinical outcomes included lung function measurements in patients reliably performing spirometry and nutritional parameters (bmi%). patients who had undergone lung transplant were excluded as their lung functions vary depending on transplant outcome. results: of the patients identified in this month period, % had ha and % ca mrsa. neither current age (mean . yrs. in ha vs. . years in ca) nor age at acquisition of mrsa ( . vs. years) was significantly different. sixty-eight % of all mrsa infected patients had chronic p. aeruginosa infection, ( % in the ha and % in ca, relative risk . of having ha in presence of chronic p. aeruginosa infection). crosssectional comparison of patients infected with ca vs. ha acquired strains showed no difference in nutritional status (bmi vs. %). lung function (fev and fef - ) was not different in ca vs. ha patients, neither when including all mrsa infected patients or those who had no concomitant infection with other organisms (n= ). we will present further comparisons to age and gender matched children with ossa infection in the final report. conclusion: comparison to ossa in matched patients will assess clinical outcomes of infections with mrsa. initial data collected at our center did not show a significant difference in outcomes between patients infected with ca vs. ha mrsa. a multicenter study would most efficiently assess the impact on clinical outcomes and determine regional differences of mrsa patterns (ca vs. ha) in different cf centers. ref : the stringent response is a global regulatory mechanism used by bacteria to adapt to nutrient limitation and other environmental stresses, and is mediated by the signaling molecules (p)ppgpp (phosphorylated guanosine nucleotides). increases in (p)ppgpp repress metabolic processes and growth, and regulate genes involved in long-term stress and starvation survival in several species ((i)e. coli, m. tuberculosis(i)), bacterial multicellular behavior ((i)myxococcus xanthus(i)), and pathogenesis ((i)p. aeruginosa(i)). while many studies have investigated bacterial functions (such as attachment and motility) involved in biofilm development, less is known about the physiological adaptation occurring in biofilm growth. when cells grow to high densities entrapped in a polymer matrix, they must adapt to heterogeneous micro-environments where gradients in nutrients, oxygen and metabolic waste exist. nutrient may thus become depleted as they are consumed by overlying cells, and some biofilm subpopulations are likely starved. we hypothesized that the stringent response mediates adaptation to nutrient starvation in (i)p. aeruginosa(i) and is important for biofilm formation. we tested a (i)rela(i) deletion mutant unable to synthesize (p)ppgpp in response to amino acid starvation, and its isogenic wildtype parent pao . we measured the starvation survival of planktonic bacteria in phosphate buffered saline and observed that the (i)rela(i) has a to fold greater loss in viability compared to wild type after h. we also tested biofilm formation in a static biofilm assay using polystyrene peg lids and estimated biomass accumulation by crystal violet staining. in this system, the (i)rela(i) accumulates times less biomass than wild type. in a flow-cell reactor system, the wildtype strain forms complex mushroom-like biofilm structures, but the (i)rela(i) mutant forms flat, thin biofilms. we determined cell attachment to polystyrene by crystal violet staining, and to glass surfaces by direct visualization under microscopy. the attachment to both surfaces is similar in the (i)rela(i) and wildtype strains, and the strains have identical planktonic growth rates. our findings suggest that the stringent response is involved in starvation survival, a physiological condition relevant to growth in biofilm. furthermore, (i)rela(i) inactivation impairs biofilm formation, suggesting that the stringent response may be an important stress adaptation in biofilm. belfast, united kingdom; . northern ireland public health laboratory, belfast city hospital, belfast, united kingdom; . northern ireland regional adult cf unit, belfast city hospital, belfast, united kingdom; . department of respiratory medicine, queen's university, belfast, united kingdom pseudomonas aeruginosa (pa) is a clinically significant bacterial pathogen responsible for increased morbidity and mortality in patients with cystic fibrosis. small non-coding micro(mi)rna species (generally ~ nts or less) in prokaryotes are involved in numerous cellular processes as is the case with eukaryotes. as in eukaryotes, these mirnas act by base pairing with target mrnas imposing translational and stability changes of mrnas and thus culminating in the control and regulation of target mrnas, crucial for bacterial stress responses and virulence to changing environments surrounding the infected zone or host cell (e.g. human airway epithelial cells). we recruited a commercial kit (biodynamics dynaexpress mirna cloning kit av, tokyo, japan) and isolated mirnas from a variety of pseudomonas pathogens from plant, soil / agricultural environment (pseudomonas syringae -ps), type strain (pseudomonas aeruginosa -pa) and a collection of clinical isolates of pseudomonas aeruginosa (cfpa) from adult cf patients, who were chronically infected with p. aeruginosa. the results on the mirna clones obtained exhibited a wide variation in the occurrence of mirnas in this bacterium. the putative mirnas obtained, cfpa in particular, yielded small non-coding rnas of sizes from as little as nts to well over nts. an initial search using the bioinformatic tool srnapredict (www.waldorlab/tufts.edu) has not as yet, fully revealed the genomic annotations for these novel srna sequences in the inter-generic regions of the pathogenic pseudomonad group of bacteria. we present our data on these unknown cellular mirnas cloned from cfpa isolates from cf patients and discuss their significance as novel regulators to bacterial stress responses and in the context of the bacterium's involvement as the predominant pulmonary pathogen, associated with cystic fibrosis. airway function is diminished in infants with cf diagnosed clinically but whether this is true for those identified by newborn screening remains unclear. we investigated whether lung function is diminished in infants with cf diagnosed by newborn screening and if this occurs in association with pulmonary infection. methods:lung function was measured in asymptomatic infants with cf following sedation with chloral hydrate ( - mg/kg) using the raised volume rapid thoracic compression technique at an inflation pressure of cmh . we measured forced vital capacity (fvc), forced expired volume after . seconds (fev . ) and forced expired flow at % expired fvc (fef ) which were then expressed as z scores. broncho-alveolar lavage (bal) was performed under general anesthesia within forty-eight hours following infant lung function testing. three separate aliquots of ml/kg sterile saline were instilled into the right middle lobe and a further aliquot in the left lingula and the bal fluid analyzed for bacterial, fungal and viral pathogens using routine culture techniques. results: bal was successful in all infants studied aged . to . yrs (median . yrs) and lung function in infants. median (range) z scores for fvc, fev . and fef were - . (- . to . ), - . (- . to . ) and - . (- . to . ) respectively. these decrements in lung function were all significant (p< . ). bal cultures were positive in ( %) infants of whom ( %) had evidence of infection with s. aureus and ( %) with p. aeruginosa. in infants there was no growth in bal or growth only of mixed oral flora. viruses were not identified in any infants. there was no association between any of the lung function parameters and pulmonary infection detected by bal. lung function was successful in a subgroup of of infants who were less than . yrs (median age . yrs) when tested. diminished airway function was identified in this younger subgroup even though only one infant had evidence of pulmonary infection with respiratory organisms in bal. in this younger subgroup median fev . z score was - . which was significantly below that predicted ( % ci: - . ,- . and p= . ). conclusions: airway function is diminished in infants with cf diagnosed by newborn screening and occurs irrespective of infection identified by bal. lung function is abnormal in very young infants before pulmonary symptoms develop and when no evidence of pulmonary infection can be detected in bal. inhaled high-dose tobramycin appears to transiently clear p. aeruginosa (pa) from airway secretions in young children with cf, though inflammation may not be markedly reduced (am j resp crit care med ; : ) . because inhaled antibiotics may not reach some areas of infection, we hypothesized that intravenous (i.v.) antibiotics may be more effective than inhaled high-dose tobramycin for reducing lower airways inflammation in cf children with early pa infection. study design: we initiated a single-center, randomized, prospective study comparing the effects of two antibiotic treatment regimens on bronchoalveolar lavage fluid (balf) inflammatory markers. clinically stable cf children with a recent isolate of pa from surveillance cultures are randomized to receive weeks of tobi® (group ) or weeks of i.v. ceftazidime and tobramycin (group ). balf is obtained just before treatment, and repeated - weeks after completion of treatment. the primary study outcome is change in balf % neutrophils (pmn). multiple secondary outcomes are assessed including quantitative bacterial cultures and cytokines in balf. this report summarizes interim analysis at the halfway point of the study. results: to date, subjects have enrolled. five subjects ( in group , in group ) dropped out after the initial bronchoscopy due to a decision by the primary cf physician to use other treatments based on balf culture results ( ) , or because of the development of a new respiratory illness before the second bronchoscopy ( ). a total of adverse events ( significant, unrelated to protocol) were reported in subjects, none resulting in a change of protocol request by the irb or data safety monitor. of the subjects completing the protocol to date, were in group , and were in group . the groups were well matched in terms of age, pa in initial balf, and initial balf %pmn. a majority of subjects in each group were children with their first isolation of pa on surveillance cultures. there was a tendency for group , but not group , to have mild reductions in balf %pmn (- ± %) after treatment. interim efficacy analysis did not suggest a significant difference between the treatment groups at this stage. similar changes were seen in group for pmn/ml (- ± x /ml) and several cytokines including gm-csf (- ± pg/ml) and mcp- (- ± pg/ml). two group subjects were unable to have stable i.v. access established and were treated with oral ciprofloxacin and inhaled tobi® for weeks as alternative systemic antibiotics, per protocol. dropping these subjects from group data did not change data trends. conclusions: the protocol appears to have adequate safety. parents of children with first-time pa infection may be more likely to consent to treatment randomization, than those with repeated pa infection. interim data analysis suggests that continuation of the study toward the original sample estimate is appropriate. acknowledgements: supported by cff(noah a ) and nih (gcrc r ). we thank tracy callahan rn, lupe haynes rrt, justin hubbard, paula murphy, cassidy henegar, nancy lee rn, benjamin butler rn, and sheree berckmans rn for logistical and technical support. methods: our local cf database was searched for patients born - . patients were included in the analysis if they were followed in our cf center before the age of . patients were separated into two birth cohorts, born - (cohort ) and - (cohort ). yearly peak fev % predicted from ages - were obtained using knudson reference equations. yearly peak bmi% and weight-for-age% at each age were also obtained. type tests of fixed effects and repeated measures analyses were used to assess mean peak bmi%, mean peak weight-for-age%, mean peak fev % predicted at first pft, mean peak fev % predicted from ages - , and mean rate of change (slope analysis) in peak fev % predicted from ages - . chi-square test for association was used to analyze differences in demographic parameters. wilcoxon rank-sum test was used to assess differences in age at diagnosis. wilcoxon non-parametric test was used to analyze differences in mean clinic visits per year. chi-square testing with a two sided alpha of p < . was used to determine significance. results: cf patients were included in the analysis. there were no significant differences between birth cohorts and for age of diagnosis (p= . ), sex (p= . ), race (p= . ), genotype (p= . ), pancreatic insufficiency (p= . ), or the mean number of clinic visits per year from ages of - (p= . ). there was a significant difference in the mean number of clinic visits per year from ages of - (p=. ; see table ) . birth cohort had a significant effect on the rate of decline in lung function from ages - . birth cohort and age had significant effects on the mean weight-for-age%, mean fev % predicted at first pft, and mean fev % predicted from ages - . sex had a trend for significant effects on lung function parameters. adjusted for age and sex, there were significant differences between birth cohorts and in mean bmi% and mean weight-for-age% at ages and , mean fev % predicted at first pft, mean fev % predicted from ages - , and in the mean rate of decline in fev % predicted (see table ). conclusions: improvements in lung function and nutritional outcomes in our cf center are associated with an increase in the mean number of clinical visits per year for cf patients ages - . the frequency of clinical follow-up is a marker of cf care that may have positive impacts on cf outcomes. background: current management of cf airway disease includes the use of key pharmacological therapies designed to combat the chronic cycle of obstruction, infection, and inflammation. randomized controlled trials have demonstrated that maintenance therapy with azithromycin (azm), dornase alfa (dnase), and tobramycin for inhalation (tob) each improves pulmonary function and reduces the frequency of exacerbations in patients with p. aeruginosa. the purpose of this study was to determine if cf patients in an uncontrolled environment (clinical setting), experience a reduced rate of pulmonary function decline when receiving azm + dnase + tob compared to those not on all three therapies. methods: adult cf patients were selected based on positive sputum cultures for p. aeruginosa, pulmonary function test results (fev % predicted) for the past year, and receiving one of the key pharmacological therapies. in addition data was collected on cfrd, depression, medication adherence, airway clearance, and nutritional status. patients were stratified according to baseline pulmonary function: mild (fev ), moderate (fev of - %), and severe (fev < %). patients were then sub-grouped as to whether they were receiving all or ≤ of the key therapies. the annual rate of decline in % predicted fev was determined using linear regression on non-exacerbation data and was categorized as stable (< . % decline), intermediate ( . - . %) , or rapidly declining (> . %). time to intermediate or rapid decline in pulmonary function was determined using cox proportional hazard modeling. results: patients ( males/ females, median age years) were included in this study; with mild, with moderate, and with severe pulmonary disease. overall, the mild and moderate groups experienced median declines of . % and . %, while the severe group experienced a . % improvement in fev % predicted respectively. the use of regimens containing azm therapy significantly delayed time to intermediate or rapid decline in lung function in the mild group (median days vs. . days, hr= . , p= . ). similarly, regimens containing dnase + azm + tob significantly delayed time to intermediate or rapid decline in lung function in the mild group (median days vs. days, hr= . , p= . ). no significant differences were seen in the moderate or severe group for patients receiving all or individual therapies. con-clusion: administration of pharmacological regimens containing dnase + azm + tob, or azm + tob or azm + dnase significantly delay progression of pulmonary disease in cf patients with mild lung disease. sawhney, v. ; arthur, b. ; seltzer, r. ; kraynack, n.c. . internal medicine, akron general medical center, akron, oh, usa; . pediatrics, akron children's hospital, akron, oh, usa; . biostatistics, neoucom, rootstown, oh, usa; . pulmonology, akron children's hospital, akron, oh, usa early and efficient diagnosis of pulmonary exacerbations (pex) in patients with cystic fibrosis (cf) is crucial in the management of cf. in oct , we implemented a pulmonary exacerbation scoring (pes) system to uniformly and consistently identify pex in cf patients as part of a quality improvement project at the cf center at akron children's hospital. we have previously reported improved pulmonary function outcomes in pediatric patients at our center after implementation of the pes. we now describe the impact of the pes on clinical practice in the management of pex and pulmonary function in our adult cf patients. the pes was developed by our multidisciplinary cf team after an extensive literature review and was implemented in oct . the pes has been previously described and consists of clinical questions divided into three domains: systemic and pulmonary symptoms and signs, and objective measurements. these elements are scored individually and a cumulative pes of ≥ (range - ) is considered a pex and treatment with antibiotics is recommended. the course and choice of antibiotic regimen is left to the physician's discretion. we examined median percent predicted fev decline from quarterly percent predicted fev measurements obtained from port cf. we calculated this for all the patients seen in our adult cf clinic (age> ) from oct till sep during which the pes was in use. we compared this rate of decline to median percent predicted fev decline for the same cohort of patients over the preceding two years (oct till sep , during which the pes was not in use. the percent utilization of the pes was calculated individually for all adult cf care providers. the influence of individual components of pes in decision to treat was also studied. we found no significant difference between the rates of decline in percent predicted fev during the pre-pes and post-pes periods (mean = -. , sd = . vs mean = -. , sd = . , p= . ) as measured by paired samples t-test. logistic regression analysis was done to evaluate the relative importance of each pes component in predicting decision to treat a pex. we found that changes in fev (or= . ), cough (or= . ), sputum (or= . ), and chest examination (or= . ) are most likely to affect the decision to treat where as dyspnea (or= . ) has the little effect on decision to treat. we found a high degree of correlation between adult provider use of the pes and an independent chart reviewer (correlation coefficient . - . ; p< . ). in contrast to cf patients ages - years, we found no significant effect of use of the pes on pulmonary function in our adult population, despite the high frequency of use by our adult providers. it is unclear why this is the case given the robust effect of the pes on our pediatric population. this study suggests that a simplified score that includes fev , cough, sputum, and chest exam may be useful in identifying a pex in adult cf patients. special thanks to tasha capozzi for her help with chart review. this project was supported by the akron children's hospital foundation. objective chronic pulmonary infection with pseudomonas aeruginosa is the main cause for morbidity and mortality in cystic fibrosis (cf) patients. tobramycin and colistin, widely used inhaled antibiotics in cf patients, were found to affect elastase activity in vitro. we showed recently that cxcr on neutrophils mediates bacterial killing, but is cleaved in cf airways by elastase. therefore, we examined the effects of inhalation with tobramycin and colistin on cxcr expression and antibacterial capacities by airway neutrophils in cf patients in vivo. methods cxcr expression was quantified by flow cytometry on neutrophils in peripheral blood and induced sputum of cf patients without inhalation therapy, cf patients with tobramycin and cf patients with colistin inhalation therapy. the longitudinal effect of a weeks tobramycin or colistin inhalation period on cxcr expression, free elastase levels and bacterial killing by airway neutrophils was assessed. results inhalation with tobramycin increased, while inhalation with colistin decreased cxcr expression on neutrophils in induced sputum of cf patients. neutrophils isolated from sputum of cf patients after tobramycin therapy had higher, whereas patients after colistin therapy had lower bacterial killing capacity and α-defensin release as compared to neutrophils before antibiotic inhalation therapy. conclusions tobramycin and colistin differentially modulate the pulmonary host defense in cf patients in vivo via cxcr on neutrophils. these findings may have clinical implications when considering antibiotic treatment in cf patients. sharp, j. ; sheehan, d. ; ren, c.l. . pediatrics, women & children's hospital, buffalo, ny, usa; . pediatrics, university of rochester, rochester, ny, usa background: airway infection and inflammation occur early in infants with cf. the raised volume/rapid thoracoabdominal (rv/rtc) technique is more sensitive than older infant pulmonary function test (ipft) methods in detecting airflow obstruction in these patients. however, there are limited data regarding lung function measured by rv/rtc in infants with cf nbs. we hypothesized that abnormalities of lung function measured by rv/rtc are present in cf nbs infants. to test this hypothesis, we reviewed the ipft data from cf nbs infants followed at our cf centers. methods: this study was a retrospective chart review. we identified infants with cf nbs followed at our cf centers who had had at least ipft performed in the first year of life. ipfts were performed using the collins ipl. forced expiratory flows were measured using rv/rtc as described by feher et al (j appl physiol : , and fractional lung volumes were measured by whole body plethysmography (bp) as described by castile, et al (ped pulm : , ) . data from the cf infants were compared to normal data reported by jones, et al (ajrccm : , ) for rv/rtc and castile, et al for bp. results: we reviewed data from infants. their mean age was . m (range= - ). pancreatic insufficiency was present in ( %) infants, and ( . %) had been treated for pseudomonas infection. their pft findings are summarized in the table. fvc and fev . were not significantly different from the normal predicted values. however, the mean fef and fef - z scores were significantly below the mean of normal infants (p= . and . respectively). gas trapping was present, as evidenced by significantly increased frc, rv, and rv/tlc (p< . for all measures). six infants ( %) had an fef z score of < - . , and ( %) had an frc > % predicted. conclusions: infants with cf nbs have abnormal lung function early in life. our results suggest that pfts in cf nbs infants can be useful in detecting early changes in lung function. we speculate that early therapeutic interventions may improve lung function in this group of patients. table. pft findings in infants with cf nbs. forced expiratory flows are reported as z scores, while fractional lung volumes are reported as % predicted. objective: clinicians use pfts to assess effectiveness of therapy in hospitalized cf patients. however, the variability of multiple pft efforts has not been described in this group of patients. previous study of within day variation of pfts has been reported in healthy adults and children (enright, , and nickerson, ) and clinically stable outpatients with cf (nickerson, , and cooper, ) . nickerson and cooper found coefficient of variations (cv) for fvc of . - %, fev of . - . %, and fef - of . - . % for adults and children with cf. no one has studied the within day variation of pfts in cf patients during pulmonary exacerbations. our objective was to determine the within day variation of pfts during hospitalizations for cf pulmonary exacerbations. we hypothesized that within day variation would improve during treatment. methods: we retrospectively reviewed pft data for all patients admitted to childrens' hospital and regional medical center for a cf pulmonary exacerbation in . patients performed pfts under the direction of experienced pulmonary function technicians. the test was complete when the patient performed efforts that met ats/ers criteria for acceptability. if the patient was unable to produce efforts because of coughing, efforts were accepted if the fvc and fev were within % or ml of each other. for analysis, we included only those pfts: ( ) with at least - flow-volume curves that fulfilled ats/ers criteria, and ( ) were performed within days of hospital admission or discharge. for patients with multiple admissions, we used pft data for their first admission only. for patients with admission and discharge pfts available, we compared the coefficient of variation (cv) for pfts (fvc, fev , ). there were at least days between admission and discharge pfts. the wilcoxon rank-order test was used to compare the cv for admission and discharge pfts. data: sixty-one patients were admitted times in . excluding repeat hospital admissions, patients had individual pft trials available at the time of admission and patients at the time of discharge. seven admission and discharge pfts included only trials. three patients at admission, and patients at discharge, were unable to produce pft curves that met ats/ers criteria, despite previously having done so when well. nine patients had paired admission and discharge pfts from the same hospitalization. the cv for fvc at discharge was significantly better than at admission, p = . (see table) . conclusions: within day variation of pfts during hospitalizations for cf pulmonary exacerbations is comparable to published outpatient data. cv for fvc improves significantly during exacerbation. changes in fvc > %, fev > %, and fef - > % can be considered a significant spirometric response to therapy in hospitalized cf patients. background: lung clearance index (lci), a measure of ventilation heterogeneity, can be calculated from multiple breath washouts of inert gas. in cross sectional studies it is a more sensitive measure of early airway dysfunction in cf than spirometry alone. in normal subjects, the technique is reproducible, with a narrow normal range of . - . . we hypothesized that it may be helpful in detecting change longitudinally, and may have value in assessing novel therapeutic interventions such as the upcoming gene therapy by the uk cfgt consortium. methods: patients aged years and over, presenting with an exacerbation requiring intravenous antibiotics, were recruited. lci was assessed in triplicate by multiple breath washout of . % sulphur hexafluoride (sf ), using a novel gas analyser (innocor, denmark) and custom-built software within the first hours of starting treatment. the test was repeated at the end of parenteral antibiotic therapy and again - weeks later when clinically stable. patients were recruited at three sites and assessed using standardised equipment and a data analysis protocol. results: paired lci measurements were available in patients, mean age (range - ) years, male. in a further patients, technically acceptable washouts could not be obtained at one or other time point. mean (sd) lci improved from . ( . ) to . ( . ), p< . . in / patients there was an improvement in lci of > % after antibiotics. patients showed a > % deterioration in lci. mean fev was significantly greater after treatment ( . v . l/s, p< . , n= ). there was a weak correlation between percent change in fev and lci (r = . , p< . ). patients also had lci measured at the follow up. mean (sd) lci at this visit was . ( . ), p< . vs visit , no significant difference vs visit . conclusions: lci can be used to demonstrate changes in the lung after antibiotic therapy. this is the first study in cf to demonstrate a lon-gitudinal improvement after intervention in a marker of ventilation heterogeneity, and offers different but complimentary information to spirometry. the higher sensitivity of lci may make possible measurement of improvement in mild patients after novel therapies such as gene therapy. this work was funded by the cystic fibrosis trust. aim: inhaled hypertonic saline positively affects sputum production in patients with cystic fibrosis (cf) by improving mucociliary clearance. this study was conducted to compare the aerosol delivery performance of various nebulizers upon aerosolization of hypertonic saline (hyper-sal™) . % and %, available as preservative-free, sterile ml solution in single dose, patient friendly blow-fill seal vials. method: ml hypertonic saline (hyper-sal™) % was nebulized by three jet nebulizers the pari lc plus®, lc star® and hudson rci micro mist as well as the eflow® for hypertonic saline (hs) generating aerosols via a perforated vibrating membrane. hs . % was characterized in the eflow®hs. droplet size distribution patterns were assessed by laser diffraction at a flow rate of l/min and aerosol delivery performance by breath simulation tests mimicking a sinusoidal breathing pattern. the delivered dose (dd) was determined by gravimetric analysis of the inhalation filters. inhalation and exhalation filters were changed after min and the drug delivery rate (ddr) was calculated as nacl found on inhalation filters per min. respirable drug delivery rate (rddr; ddr x respirable fraction < µm) indicates how much nacl can reach the lungs per minute. results: aerosol characteristics of the different nebulizers after aerosolization of ml hs % are summarized in the table. data for hs . % nebulized by the eflow®hs were as follows: dd= . %, ddr= . %, nebulization time = . min and comparable to data for hs %. the lower recoveries for the jet nebulizers are probably associated with increased evaporation and longer nebulization times. conclusion: about - % of the charged sodium chloride from ml hs was delivered and % - % of these droplets were in a respirable size range < µm resulting in vitro respirable doses of about % - % of the label claim. ddr and rddr are about -to -fold higher for eflow®hs indicating a much higher delivery efficiency compared to conventional jet nebulizers. further studies will be needed to demonstrate that about ml hs may be sufficient to deliver a comparable dose to the lungs using an eflow®hs compared with jet nebulizers. this bears the potential to further reduce nebulization time and increase acceptance by patients. objectives: individuals with cf frequently have complicated and time consuming treatment regimens consisting of inhaled antibiotics, mucolytics, and airway clearance. patients and clinicians may have difficulty determining which specific therapies are effective for any individual patient or whether a particular treatment is causing adverse effects. this study assessed the utility of home spirometry for providing an objective measure of pulmonary status in between scheduled clinic visits. methods: consecutive adults with cf were recruited at a single academic medical center and were asked to measure fev twice daily for six months using the piko- meter (nspire health™, llc). subjects agreed to participate and completed the study. subjects were given diaries to record changes in respiratory symptoms and changes in medications during the study period. subjects were evaluated in clinic at least every three months and fev values obtained in the pulmonary function lab were compared to home fev from the same day using linear regression and bland-altman analyses. graphical displays of home fev measures were visually inspected for changing trends and these were compared with medication changes. results: the mean fev measured at home was . ± . l. the mean fev in the pulmonary lab was . ± . l. the home fev was significantly correlated with the clinic value from the same day, r = . , p< . . fev measures fell within the limits of agreement (± sd) by bland-altman plot. in of subjects ( . %) there was at least one sustained change in fev that correlated with either stopping or starting a particular treatment. the figure shows two significant changes in fev in one patient related to medication changes. there is an increase in fev at the initiation of aerosolized % saline and a fall in fev that corresponded to inhaled tobramycin use. the improvement on hypertonic saline increased this patient's enthusiasm for this treatment. conclusions: home fev monitoring is feasible and appears to provide accurate measurement of pulmonary function. it can provide clinicians with objective data to tailor treatments without necessitating a clinic visit. feedback from home monitoring may improve medication adherence and may allow treatments to be individually tailored based on patient responses to the medications. . respiratory unit, royal hospital for sick children, glasgow, united kingdom; . diagnostic imaging, nhs greater glasgow & clyde, glasgow, united kingdom; . medical statistics, royal hospital for sick children, glasgow, united kingdom; . medical physics department, gartnavel hospital, glasgow, united kingdom background: there have been calls for caution regarding the suggested use of regular chest computed tomography (ct) scans for monitoring disease progression in cf . consideration of the associations of higher ionizing radiation exposure must be made before regular chest cts are performed on the wider cf population. aim: we tested the hypothesis that higher ionizing radiation exposure from chest radiographs (cxr) and chest cts would be associated with markers of severity including gender, genotype (class i/ii or iii-v), height sds, weight sds, bmi, fev sds, length of hospital admission (days), scottish index of multiple deprivation (simd) scores and pa/bc microbiological infection (chronic or intermittent colonisation with pseudomonas aeruginosa and/or burkholderia complex). methods: effective doses of ionizing radiation (msv) from cxrs and chest cts (including hr scans) were determined for all cf children at our centre between st january and th july ( weeks). the most recent anthropometric and lung function data to july were used. simd scores report a data zone that encompasses ~ individuals and comprises indicators in six domains (current income, employment, health, education, housing and access). results: cf patients ( male: female) underwent radiation exposure procedures (mean of . procedures per patient per year). the average effective doses were calculated for of the procedures ( . %) including cxrs and chest cts. lung function data were available on patients ( %). univariate analysis revealed that higher ionizing radiation exposure was significantly associated with fev sds (p< . ), length of stay (p< . ) and pa/bc infection (p< . ). the regression line indicates that for each drop of two fev sds, each child receives an extra msv in radiation exposure from cxrs and chest cts alone. similarly, each child receives an extra msv in radiation exposure from cxrs and chest cts for every days of hospital admission. multivariate analysis with backward selection demonstrated that length of hospital admission (p< . ) and pa/bc infection (p= . ) were associated with higher ionizing radiation exposure. discussion: in cf, higher ionizing radiation exposure from cxrs and chest cts in childhood are associated with poor lung function, pa/bc infection and length of hospital admission but not other indicators of severity such as gender, severe genotype, anthropometric measures or socio-economic status. using effective doses of ionizing radiation as an outcome marker in cf is an alternative means of assessing the impact of the disease and its management on the child. background: exhaled volatile compounds have attracted attention as potential non-invasive biomarkers of chronic lung disease. early sustained airway inflammation and bacterial infection are characteristic features of pulmonary involvement in cystic fibrosis (cf). the cellular response is dominated by neutrophils, which have been shown to generate oxygen-and halogen-derived radicals as part of their host defense armamentarium. aims: to gather pilot data on the pulmonary output or uptake of selected organic trace gases in cf patients and healthy controls; and to explore the impact of pulmonary exacerbations and antibiotic treatment. methods: samples of ambient air and exhaled breath were collected from children, adolescents and young adults with cf and from healthy controls. the patients' age ranged from - years and their fev between - % predicted, were pseudomonas positive. twelve patients were studied at the start of i.v. antibiotic treatment for a pulmonary exacerbation; repeated sampling after the course was performed in subjects. all samples were analyzed on a customized gas chromatography system with flame ionization and electron capture detectors. after determining the mixing ratios of a variety of volatile organic compounds, alveolar gradients were calculated from the corresponding exhaled and ambient values. results: compared to healthy control subjects, cf patients showed a significantly lower respiratory output of methanol, ethanol, acetaldehyde and dimethylsulfide but a higher net release of benzene, methylchloride, trichloromethane and trichloroethene. these differences were most pronounced in patients with acute exacerbations. a relevant scatter and overlap between groups was noted for ethane, pentane, acetone, isoprene, toluene, and tetrachloromethane. after antibiotic treatment, the output of benzene, methylchloride and trichloromethane decreased by - %. conclusion: although important pathways remain incompletely understood, the local biochemical milieu in cf lungs may be informative and accessible through non-invasive breath testing. in this context, the diagnostic potential of exhaled organic traces gases (especially oxygenated, halogenated and aromatic compounds) should be further explored. supported by: german research foundation (dfg ba / - ). toy, e. ; weiner, j. ; sacco, p. ; duh, m.s. . health economics, novartis pharmaceuticals, east hanover, nj, usa; . analysis group, inc., boston, ma, usa objective: to review up-to-date economic outcomes data in patients with cystic fibrosis (cf), and in particular, costs related to respiratory infection by pseudomonas aeruginosa (pa), which is the leading cause of morbidity and mortality in cf patients. methods: a systematic search of the medline database from - was conducted to identify major and review articles that contained the terms "cystic fibrosis" and "cost" and which were published in english language journals. selected conference abstracts were searched, and additional articles were identified through bibliographies of retrieved articles. recent articles that contained economic data on antibiotic and mucolytic therapies were selected for in-depth review. cost estimates were converted to us dollars for comparability. results: approximately articles fulfilled the initial search criteria, and were selected for in-depth review. articles were divided into four categories: economic impact of inhaled tobramycin ( articles), the effect of home-vs. hos-pital-based antibiotic therapies for pulmonary exacerbations ( articles), economic impact of recombinant human deoxyribonuclease (rhdnase) ( articles), and cost-of-illness for cf ( articles). inhaled tobramycin (actual or recommended dose of mg/ ml) has been associated with reductions in health care costs; these cost savings offset between % and % of the cost of this therapy. antibiotic therapy for pulmonary exacerbations generally resulted in lower health care costs when administered in a home setting rather than a hospital setting. use of rhdnase led to reduction in health care costs, with higher reductions observed in patients with higher levels of use; the cost savings offset between % and % of the drug cost. cost-of-illness studies have been conducted in different countries; the economic estimates varied widely across studies due to differences in treatment patterns, health systems, methodologies, and patient populations. most cost-of-illness studies were retrospective observational studies from the perspective of a hospital or third-party payer, and the cost of cf ranged from $ , to $ , per patient per year. the largest cost categories included hospitalizations, out-patient visits, rhdnase and antibiotics, while disease severity and presence of pa infection were common determinants of cost. cost-of-illness studies have underestimated societal costs because they have rarely considered patient time costs and none have considered indirect costs (e.g., burden on lost productivity or informal caregivers). conclusions: studies show that inhaled tobramycin and rhdnase can partially offset medical costs; home-based iv antibiotic therapy is likely to reduce costs; and direct costs associated with cf can be high but vary widely across countries and analytical methodologies. areas for future research include direct comparisons of inhaled antibiotic therapies, examination of the relationship between treatment adherence and economic outcomes, and estimation of societal cost-of illness. this study was sponsored by novartis pharmaceuticals corporation, east hanover, nj. background: sputum interleukin (il ) and myeloperoxidase (mpo) are advocated as markers for infective exacerbations in cf. we have previously shown that the neutrophil protein, calprotectin, is a sputum biomarker of disease activity in cross sectional and longitudinal studies. in the present study we evaluate longitudinal measurement of sputum and serum calprotectin during infective exacerbation. methods: sputum and venous blood samples were taken at the beginning (visit ) and end of a course of iv antibiotics (visit ), and at time of clinical stability following the infective episode (visit ). processed sputum supernatant was analysed by elisa for calprotectin, il and mpo. serum calprotectin was assayed by elisa. results: paired sputum samples samples were available for visits and and paired samples for visits and . all data are presented as mean(sem). sputum calprotectin decreased from ( ) to ( ) µg/ml, p< . , from visit to . there was a non-significant increase to ( ) µg/ml at visit . sputum il did not change from visit to [ . ( . ) ( . ) ]. serum calprotectin decreased from ( . ) to . ( . ) ng/ml, p< . (n= paired samples) from visit to . at visit there was an increase to ( ) ng/ml, p< . (n= ). discussion: calprotectin appears a valuable marker both in sputum and serum for tracking changes in lung inflammation during an exacerbation of cf and may be more sensitive to change than il or mpo. on stopping antibiotic therapy sputum and serum calprotectin increase implying that this marker may be sensitive to small sub-clinical changes in lung inflammation. these data suggest that sputum and serum calprotectin might be employed to assess responses to drug therapies in cf such as gene therapy. this research was funded by the cf trust uk. chronic lung disorders are usually associated with a hypoxia driven increase in red cell mass. however, patients with cystic fibrosis often have normal or decreased haemoglobin levels. the present prospective observational study in cystic fibrosis (cf) patients was performed to determine which factors were involved in alterations in the hematopoetic response to corresponding arterial oxygen pressure. sixty adult patients (age - ) with stable cf were included. they all had vitamin a, d, e and k but no vitamin b supplementation. patients were on oral fe + ( mg/day). resting arterial blood gases, lung function, complete blood counts, parameters of iron status, crp, sputum microbiology and serum erythropoietin were measured at recruitment and after and months. patients had varying degrees of pulmonary functional impairment and % were hypoxemic (arterial oxygen pressure < mm hg). low-grade systemic inflammation (crp > . mg/dl) was present in % of the patients, who all had bacterial colonization. none of the patient had erythrocytosis and patients had anemia . there was no significant difference in iron status between patients with or without chronic iron supplementation and erythropoietin levels were normal. during the months observation period no significant changes occurred. the patients exhibited an impaired erythropoietic response to hypoxemia with normal or low hematocrit in spite of chronic lung disease. linear multivariate regression analysis (table) revealed crp and iron levels but neither iron substitution, nor erythropoietin levels nor lung function parameters as independent determinant of haemoglobin levels. cf may be associated with anemia of variable severity as expression of the chronic inflammation present in these patients. the therapeutic consequences are to treat the underlying inflammation rather than to supplement iron. variables included in initial model: background improvement in the treatment and survival of females with cf has led to many leading a normal adult lifestyle, and some can now expect to raise a family. however, pregnancy can have a profound impact on wellbeing, and caring for a young child can affect compliance with cf treatment. to look at this further, we surveyed the experience of pregnancy and motherhood in cf women attending our large adult cf centre ( patients). methods we reviewed the records of cf women who chose to remain pregnant ( babies [ male] from pregnancies), looking at changes in lung function, bmi, respiratory exacerbations during pregnancy, clinic attendance, and oral and iv antibiotic therapy (home and inpatient care) during pregnancy and also in the first year post delivery. we devised a questionnaire to examine how patients coped with pregnancy and the demands of motherhood, focussing on changes in compliance with therapy, worries regarding the effect of cf treatments on the baby, and support given by the cf team. results at conception mean fev was % predicted (range to ) and mean bmi (range to ).there were no miscarriages or neonatal mortalities and all children remain well, but the sickest mother died at years. mean fev following pregnancy was % [range to ] (p= . ), with mother losing % by one year post delivery. at one year, bmi was unchanged (mean , range to ). rates of respiratory exacerbations and iv antibiotics were similar pre and during pregnancy, although during the first year post delivery mothers avoided hospital based treatment preferring home care. six mothers' clinic attendances increased during and post pregnancy ( attended outpatients times in the first year post delivery). nine mothers worried about the effects of cf medication on their unborn child: the remaining mother was in her nd pregnancy. all managed their health differently during pregnancy: whilst increased compliance, worked harder to stay healthy, and increased clinic attendances; avoided clinic, postponed treatment, avoided any medication in the first trimester, and reduced oral antibiotic use to prevent harm to their unborn child. following delivery, mothers spent less time on their own health due to motherhood demands, and missed treatments during the first year: missed all except iv antibiotics. seven mothers ceased chest clearance and nebuliser therapy, and whilst missed oral therapies only avoided iv therapy. one mother who returned to work early felt this had contributed to her post natal depression. nine mothers indicated that cf team support was adequate, but the remaining mother felt that she should have been offered more frequent home visits post delivery due to lack of family support. conclusions the experience of pregnancy in our centre has positive outcomes for most mothers. however, compliance with treatment was affected and a number of mothers lost significant pulmonary function due to pregnancy. our study has shown that the demands of motherhood especially in the first year challenge the management of their own health. we offer a proactive service to women considering pregnancy, with early participation of the multidisciplinary team, and have increased home visit frequency to during the first year post-delivery. geller, d.e.; kesser, k.c. research, nemours children's clinic, orlando, fl, usa background: alpha- antiprotease (α- ap) is being developed as an inhaled drug in cf to inactivate excess elastase in the lungs. it is estimated that tens of milligrams of α- ap need to be deposited in the lungs to be effective. delivering a high dose with conventional jet nebulizers may be too time-consuming. we speculated that slowing and prolonging inspiration could optimize α- ap delivery. the i-neb® adaptive aerosol delivery (aad® system, respironics) is a portable, electronic, battery-powered, mesh nebulizer that uses the aad algorithms to deliver drug in the first portion of inspiration. interchangeable mouthpieces allow the i-neb to be used in a conventional tidal breathing mode (tbm) or in the target inhalation mode (tim) that guides the patient to inhale very slowly. the low residual volume after nebulization reduces drug waste and optimizes efficiency. we compared delivery efficiency of α- ap in vitro with the i-neb in the tbm vs. tim modes. methods: we studied new i-neb devices using tbm (tidal volume [vt] . l, respiratory rate [rr]= , i-time= seconds), tim- (vt . l, rr= , i-time= sec), and tim- (vt . l, rr= , i-time= sec) using a breath simulator. nebulizers were filled with . ml α- ap ( mg/ml). nebulized drug was captured on a filter and measured via spectrophotometer (inhaled dose), and particle size was measured with an insitec laser system. results: median particle sizes for the modes were similar ( . - . µm) . the dose emitted from the i-neb was very high, and similar between modes ( - % of nominal dose). predicted lung doses for these experiments were calculated from a previous scintigraphy study (nikander ) that showed % of the emitted dose deposited with tbm and % with tim. predicted lung dose of α- ap (as % of nominal) and delivery times were tbm: . % in . min, tim- : . % in . min, and tim- : . % in . min. discussion: the i-neb has a very high predicted lung dose with both tbm and tim. however, tim reduces the time of administration to as little as / that of tidal breathing. if larger predicted lung dose is necessary to see a clinical effect, either the initial drug volume or drug concentration may be increased. we conclude that slow, deep, controlled inspirations using i-neb is a very efficient method to deliver α- ap, and is faster than tidal breathing. we also speculate that tim has the additional potential advantage of better distribution of drug in the lungs because of slower flow rate and particle velocity. objective: early inflammation was observed in infants with cf regardless the occurrence of bacterial infection. lung function (lf) is a tool to assess bronchial inflammation consequences. few data are available on the pronostic value of impaired lf in infancy. methods: in infants, airflow obstruction (t ptef/te , curve shapes, tidal volume, functional residual capacity (frc, nitrogen washout technique) were assessed with the sensor medics and interpreted with stocks reference values. at y, fev and pulmonary flows (spiro , nova medical sc, zapletal reference values) and frc he , were measured. symptomatic children were defined when cough, wheeze or rattle were recurrent. all symptomatic infants have been treated with inhaled steroids as any infantile wheezer. results: screened infants ( ∆f /∆f , ∆f /other, other/other) were investigated at mean age mo and years. ( %) had respiratory symptoms (rs); the mean tidal volume was normal ( . ml/kg), ( %) had a marked obstructive curve shape, ( %) a decreased t ptef/te , and ( %) hyperinflation. then, ( %) had normal lf, ( %) ao, ( %) hyperinflation and ( %) both. at age , % had rs, and lf with mild alteration as mean flows ≥ %, and frc he = . %. accurate analyses showed that ( %) had normal lf, ( %) ao and ( %) small airway diseases (only distal ao and/or hyperinflation). frc in infancy and at age were correlated (r= . ; p= . ). conversely, no correlation between ao parameters in infancy and at age was found. the lf alteration was independent of the genotype, bacterial colonization and recurrent symptoms at both ages. conclusion: ao in infancy is frequent as found in half of the screened cf infants. the lack of correlation with the ao observed at age suggested that ao in infants might be due to a non specific inflammation (i.e viral), transient and then relevant from a treatment. hyperinflation, although mild, is conversely an early marker of the cf disease as persistent between infancy and age . we describe the efficacy of a novel system using an electronic real-time distant home monitoring of symptoms and spirometry in cf patients with the aim of early detection of p exs patients and methods: this is a month prospective observational study. all patients were provided with a set consisted of a mobile phone-palm pc (the x daii, o -uk) and with a spirometer (vitalograph) with an attachment to the pc. the pc contained a purpose-built software on which cf symptoms and spirometric values were recorded once daily. these were then sent on a real time to a computer server where the data were plotted on a time-magnitude graph. the site was accessed by a password-permitted cf physicians. patients were asked to score four symptoms (cough, sputum, breathlessness and fatigue) once a day and to record at least three attempts of spirometry. these were accepted by the software only if they met the ats criteria. preset criteria for p exs were stipulated. when the recordings met the criteria for p exs patients were invited to come to the cf clinic for review and were managed by either watchful waiting, oral or iv antibiotics. a prodrome phase is deemed to be present when a patient had an increase in one or more symptom for one week prior to the p ex. twenty one evaluable patients were included in the study. in ( %) there were an increase in one or more than one recorded symptoms throughout the study. a total of p exs median . per patient were detected within the months study period. in ( %) p exs, symptom increase and/or decline in fev met the preset criteria for p ex and in cases patients presented despite that symptom changes were not sufficient to meet the pre-set definition of p ex. prodromal phase prior to p exs were seen in ( %) patients. adherence and technical issues were the main two problems throughout the study. distant daily monitoring of patients symptom is easy and sensitive. it can be used to monitor symptoms and early detect p exs. the vast majority of our patients have an increase in their symptoms even when they perceive their disease to be stable. a prodormal phase preceded p ex is seen in the majority of patients. the pre-set criteria for defining p ex may have been too stringent. the research was funded by o telecommunication, uk the monitoring system used in the study. a combination of a mobile phone-palm computer (x daii, o uk) and a spirometer (vitalograph uk) .. the aim of this research was to examine whether systemic inflammation is present in adult patients with cf during their stable status. patients and methods: blood samples were obtained from a total of cf patients female, mean age , range( - ) males mean . range - and from control subjects, female, mean age . , range( - ) males,mean age . yrs, range( - ) all cf patients were included when they were at least weeks away from any pulmonary exacerbations. all control subjects were not known to have any pulmonary or systemic disease. venous blood samples were taken and spun at rpm.separated sera was aliquted and frozen at - degrees c. they were analysed in batches for cytokines il- b,il- ,il- , il- , ilp ,and tnf-alpa using a cba human inflammatory kit(bd bioscience). whitney-u test was used to compare values of serum cytokines between stable cf patients and control subjects.. serum il- and il- were raised in cf patients compared to control subjects. the mean value for il- was . pg/ml (sd . ) for patients and . pg/ml (sd . ) for controls, p< . . similarly, the mean value of serum il was . pg/ml (sd . ) in cf patients compared to . pg/ml (sd . ) in the control group, p< . (figure below). there was no difference between cf patients and control with regards to serum il- b, il- and il- p . conclusion: serum il- and il- were raised in cf patients compared to an agematched control subjects. inflammatory markers were raised in the sera of cf patients even during disease stability. even during disease stability, the lungs remain an inflamed organ which could explain the decline in lung function tests and the countinued daily systemic symptoms experienced by patients with cf. serum il and il in cf patients with stable disease compared to the control group (please see abstract body) we have shown that even during disease stability, an increase in inflammatory markers are present in the sera of cf patients compared to an agematched control group. this longitudinal study examines changes in serum inflammatory markers during p exs. we investigated cf patients female, mean age , range - years. their mean baseline fev was . % of predicted (range . - %). a baseline venous blood sample was withdrawn and sera were stored at - degree c from patients during disease stability. out of those there experienced p exs. blood samples were obtained at the beginning and at the end of the p exs.the following inflammatory markers were analysed: white blood counts, c -reactive protein (crp), il- , il- , il- , il- b, il- p and tnf-α. patients recorded their symptoms and their fev on a daily basis on an electronic diary and the data were beamed daily to a research centre where data were analysed. p ex was defined as ). an increase in two symptoms for successive days, or ). an increase in symptoms and % reduced fev from baseline over successive days or ). % decline in fev over successive days or ).when a patient felt that he/she was going through a p ex. paired t tests were used to analyse changes in inflammatory markers. there was no change in the number of circulating neutrophils, lymphocytes or eosinophiles at the start compared with the end of p ex. there was an increase in mean crp values at the start of p ex and reduction at the end of p ex, but this did not attain statistical significance. mean (sd) of serum il increased from . pg/ml ( . ) at baseline to . pg/ml ( . ) at the start of p ex (p= . ) and returned to base line . pg/ml ( . ) at the end of p exs (p= . compared to values at the start of p ex). (figure ) there were no changes in the values of serum il- , il- , il- b, il p or tnf-α at the start and at the end of p exs. serum il increased at the start of p ex and returned at the baseline at the end of p ex. crp was the closest inflammatory marker to follow that pattern. none of the other inflammatory markers changed with the diagnosis of p ex's. this and other studies call for the price of analysis of il- to be reduced to become available in routine clinical practice. background. abpa can be a therapeutic challenge in children with cf. aim was to describe the tolerability of voriconazole and nebulised liposomal amphotericin in the treatment of abpa in children with cf. methods. we performed a retrospective case note review of voriconazole and nebulised liposomal amphotericin use, in the treatment of abpa in children with cf, at our centre. results. children diagnosed with abpa according to the current criteria, failed to improve despite previous treatment with itraconazole and steroids, and received voriconazole as a second line agent. the dose of voriconazole was as recommended by the manufacturer's summary of product characteristics and the duration of treatment was two weeks. significant side effects noted in children. observed side effects included visual hallucinations (n= ), photopsia (n= ), sleep disturbances (n= ), severe headache (n= ), influenza like symptoms (n= ), gastrointestinal side effects (n= ) and elevation of alkaline phosphatase (n= ). of these children, one child tolerated voriconazole when concomitant treatment with omeprazole was withdrawn. a second course of voriconazole, given at a lower dose and without the loading dose was tolerated by two children. voriconazole blood levels were not measured in any patient. we used nebulised liposomal amphotericin in of these children. one child received amphotericin as a second line agent when voriconazole had to be stopped because of significant side effects. children had active abpa despite treatment with first line (itraconazole and steroids) and second line (voriconazole) agents and received amphotericin as a third line agent. amphotericin was used at a starting dose of mg nebulised twice daily, with weekly increments as tolerated, up to a maximum dose of mg twice daily for four weeks. the starting dose and all subsequent increments in dose were supervised. all children received salbutamol immediately before nebulised amphotericin. the standard formulation available for intravenous infusion was made up into single doses for nebulisation by the pharmacy. this was stored at c and diluted with water for injection for use with the nebuliser. the only side effect, experienced by all children was a dry irritant cough at the start of nebulisation that settled soon without need for discontinuation of the medication. improvement in clinical symptoms, lung function and total serum ige was noted in all children. the improvement in fev and fvc, expressed as mean (range) of percentage predicted were and respectively. the mean (range) fall in serum total ige and aspergillus specific ige were ku/l ( - ) and mg/l ( - ) respectively. conclusion. voriconazole use was associated with significant side effects. avoidance of drugs that may potentially result in interaction is important. omitting a loading dose and using a lower maintenance dose may help selected patients tolerate voriconazole. nebulised liposomal amphotericin was tolerated by our children with cf and abpa who did not tolerate or responded poorly to voriconazole. recent studies have shown that high resolution computed tomography (hrct) is a more accurate method of evaluating early lung disease in cystic fibrosis (cf) than pulmonary function tests (pfts) or chest x-rays. since young children can not perform the breathing maneuvers required for high quality hrct, lung volume control is necessary. positive pressure facemask ventilation using chloral hydrate allows full inspiration and end exhalation images, but requires special expertise. controlled ventilation can also be produced with general anesthesia (ga). ga has not been reported in children undergoing hrct for cf research. the advantages of ga over sedation include rapid onset, faster recovery and reliability of scan completion. the undesirable effects of sedation are unpredictability and the higher failure rates. additionally, failed sedation contributed significantly to parental dissatisfaction. (malviya, et al., ) . the primary outcome of our study was to examine the safety of using ga for hrct scans and the quality of images obtained. a secondary outcome was parental satisfaction. methods: participants were recruited from a larger nih/cffti funded multi-site randomized clinical trial focusing on behavior and nutrition treatment to improve growth in preschoolers with cf. hrct in subjects to years old (mean age ± months) was performed with ga. parents accompanied the child during induction and left after the child was asleep prior to the intravenous (iv) being placed. after induction of anesthesia, a proseal®, specialized laryngeal mask airway (lma), was placed which allowed ventilation with higher pressures and the ability to suction the stomach. volumetric thin section ct scans were obtained. ga using inhalation (sevoflurane) and/or iv (propofol) agents was used and out of families who were eligible ( % recruitment rate) participated in the sub-study. results: the quality of the hrct scans in all subjects were optimal. there were no anesthetic complications upon emergence and none of the patients experienced any side effects after follow-up phone calls at hours. parents also expressed that the clinical interpretation of the hrct scan provided beneficial information about their child's current lung disease status. the mean time from completion of scan to discharge was . ± minutes. conclusions: the use of ga in radiology departments is becoming more common in pediatric centers. our initial findings of this study demonstrate that ga can be safely administered for children with cf. in addition to very good quality scans being obtained, parental satisfaction was also noted. the use of the proseal® lma for hrct may improve the image quality and aid in the diagnosis of early lung disease in preschoolers with cf. supported by nih grant r dk and a cf foundation clinical research grant. whey protein is rich in sulfhydryl (sh) groups and is recognized for its ability to increase glutathione and reduce oxidative stress. hyperbaric pressure treatment of whey protein increases its digestibility, promotes the release of novel peptides for absorption, increases intracellular glutathione in healthy subjects (zavorsky et al, int j vit nutr res, ) , and reduces in vitro production of il- (vilela et al, inter immnuopharmacol, ) . we present here the initial results of a pilot study of supplementation with pressurized whey in cf patients. children and adults with cf were supplemented with hyperbaric whey protein for month ( gm/day in patients less than years of age, gm/day in older patients). anthropometric measures, pulmonary function, serum c-reactive protein (crp) and plasma lipid peroxides were measured before and after supplementation. the results of the first patients to have completed the study are reported, with further data to be presented. in / subjects weight increased and in / subjects lean body mass increased. there were no significant changes in fev or fev/fvc ratio. in / patients the crp fell ( / by more than %) and in / the crp increased. in the remaining two patients, the crp was undetectable at baseline and did not change at follow-up. plasma lipid peroxides decreased in the three patients in whom it could be measured ( / to undetectable levels). these preliminary results suggest that supplementation with pressurized whey protein can reduce systemic inflammation in cf patients and has the potential to beneficially modulate the inflammatory response in cf patients. supported by the breathe initiative of the canadian cf foundation with pseudomonas aeruginosa. soon after its introduction, the mesh technology based pari eflow® nebulizer gained preference over jet nebulization in the cf community because of its short nebulization time, portability and noiseless operation. concerns were raised about the durability of the device due to expected vulnerability of the mesh. the aim of this study was to compare the particle size distributions in the aerosol, delivered doses and output rates from the eflow and the pari lc plus®(lc+)with paritur-boboy compressor before and after use in daily practice. lc+ nebulizers in use by the adult patients taking part in the study were taken in for testing and new eflows, cleaning instructions and a disinfection device were provided. after months use ( cycles of x doses tsi: mg/ ml), the eflows were collected by the local cf center. particle size distributions were measured at a constant flow rate of l/min with a laser diffraction apparatus. because of uncertainty regarding the cleaning procedure after last use, additional e-flows were collected from the same patients after another period of months use. conclusions: new eflows produce slightly larger droplets in a narrower size range than new lc+ nebulizers. after use in daily practice, droplet size distribution changes for both devices. output rate (delivered dose per minute) decreases for both devices. for used eflows delivered dose is lower than for used lc+ nebulizers, because the eflow is programmed to switch off after min, even when nebulization is still incomplete ( out of tested eflows). this may result in a considerable reduction of delivered dose. however, when a mesh is replaced, performance is the same as that of a new device, suggesting that the changes in performance originate in the vibrating mesh. for daily practice these results indicate: ( ) the performance of both nebulizers changes during use but the changes are smaller for eflow than for lc+ ( ) changes in droplet size distribution are relatively small for both devices;in contrast with output rate and delivered dose ( ) proper functioning of eflow can be improved by timely replacement of the mesh, ultimately when eflow switches off after minutes. without replacement, the delivered dose is unpredictable. mean values for size distribution, delivered dose, nebulization time and output rate (x is volume median diameter) backgrounds: lung surfactant protein a (sp-a) belongs to the family of collagen containing, c-type lectins, called collectins. sp-a plays a critical role in the innate immune defense of the lung. sp-a binds calcium dependent to carbohydrate structures on pathogens via its carbohydrate-binding domain (crd). cystic fibrosis (cf) lung disease is characterized by chronic inflammatory and proteolytic processes in the airways, leading to destruction of the lungs and early death. aims: we hypothesized that sp-a might have a reduced function in cf. this might result from sp-a degradation by neutrophil proteases, an altered macromolecular organization of sp-a, or inhibition of crd-binding by airway components. methods: we studied bronchoalveolar lavage fluid (balf) from clinically stable patients with cf and from children suffering from bronchitis. balf samples were purified by carbohydrate-binding assay on fucose attached to sepharose resin with and without calcium. in the absence of calcium sp-a cannot bind via its crd. each fraction was analysed for sp-a and igg by immunoblotting. macromolecular organization of sp-a was determined by gel chromatography. results: only % of total sp-a of cf balf bound to the fucose column, whereas % bound from the bronchitis balf. the structural organization of the sp-a oligomers did not differ between the non-binding and the binding fractions of both groups. of the cf balf had sp-a at kd that did not bind to fucose. these balfs had a high percentage of neutrophils (> %). in vitro human leucocyte elastase decreased sp-a to less than % of its initial levels and sp-a fragments at kd were detected. conclusions: neutrophil proteases degrade sp-a in cf lungs. the carbohydrate dependent binding of sp-a is diminished due to a reduced function of the crd. these alterations contribute to the reduced ability in cf to remove specific pathogens from the lungs. chiron, r. , ; murris-espin, m. , ; crampette, l. , ; varrin, m. ; didier, a. , ; wallaert, b. , ; chanez, p. , ; leroy, s. , . crcm, chu, montpellier, france; . crcm, chu, toulouse, france; . orl, chu, montpellier, france; . iurc, chu, montpellier, france; . crcm, chu, lille, france; montpellier, france in cystic fibrosis (cf) patients, upper airways involvement is frequent but its impact and relationships on the course and severity of the disease has been rarely investigated. objective: to relate upper airways involvement with lower airways parameters and quality of life in cf patients. methods: clinical and radiological prospective study of subjects with cf at french cf centres. clinical rhino-sinusitis (rs) was defined by the presence of more than episodes of nasal symptoms during the last year, current nasal obstruction or rhinorrhea. nasal investigations included: anterior rhinoscopy, sinus ct scan analysis and rs quality of life questionnaire. lower airways were assessed using a thoracic ct scan (bhalla score), spirometry and sputum culture and a cf quality of life questionnaire (cfq +). results: ( f/ m) patients aged . ( - ) years old were enrolled. endoscopy demonstrated polyposis in % and nasal hyper secretions in . % of the patients. spirometry, bhalla score, sputum colonization did not discriminate patients with (n= ) or without rs (n= ), and regardless the presence of polyps. cfq + was significantly lower in patients with of rs (p= . ). conclusions: rs involvement altered quality of life in cf patients, although, they do not have a more severe pulmonary disease. a specific attention and treatment to rs should be paid in cf patients. the impact on different outcomes deserves to be examined in longitudinal studies. as part of the uk cystic fibrosis gene therapy consortium "tracking study", ebc was measured (ecoscreen, jaeger) in adult and paediatric patients presenting with an infective exacerbation requiring iv antibiotics. exacerbation was defined by pre-determined criteria. ebc was repeated at the end of parenteral antibiotic therapy and again - weeks later. patients were recruited at three sites and assessed using standardised equipment. samples were obtained according to ers guidelines. exhaled breath ph was assayed (shindengen ph meter, camlab, uk) immediately after collection without a de-aeration step. results: paired ebc measurements at the start and end of antibiotic therapy were available in patients. mean age was yrs (range - ). data from patients were excluded because at least one ph measurement was > , suggesting salivary contamination. for the remaining patients mean ebc (sd) ph increased from . ( . ) to . ( . ), p< . . of these patients, also had measurements available at a third visit, - weeks after completion of antibiotics. were excluded because of salivary contamination. no difference in mean(sd) ebc ph was found ( . ( . ) vs. . ( . )) conclusions: ebc ph increases after treatment with antibiotics, and may offer a non-invasive means of assessing airways inflammation in chest exacerbations. further work is required to follow the longitudinal change in ebc ph and other inflammatory markers in clinically stable patients, as ebc may be a useful tool in assessing response to novel treatments, such as gene therapy. this work was funded by the cystic fibrosis trust. background: children with cystic fibrosis (cf) undergo multiple procedures exposing them to ionizing radiation in hospital. some specialist centres have advocated regular chest computed tomography (ct) as a means of better quantifying lung pathology. the clinical benefit of this approach is unproven and previous calculations of risk have failed to account for other ionizing radiation exposure procedures . aim: to quantify the exposure of ionizing radiation in the pediatric cf population of a tertiary level children's hospital. to determine the potential difference in exposure of introducing biannual chest ct scans. methods: effective doses of ionizing radiation from chest (cxr) and abdominal (axr) radiographs, fluoroscopy and cts (sinuses and chest including hr scans) were determined for cf children at our centre between st january and th july ( weeks). the maximal effect was tested i.e. introduction of biannual chest ct scans from age years abolishing the requirement for cxrs and the assumption was made that axrs, sinus cts and fluoroscopy rates were unchanged (median exposure values calculated for age groups -< year, -< year, -< year, -< year, -< year, > year) . results: cf patients (n= , gender ratio : ) underwent a mean of . ionizing radiation exposure procedures per patient over the study period (total of procedures). the average effective ionizing radiation doses (msv) were calculated for ( . %) procedures. each cf child received a mean (median) effective radiation dose of . ( . ) msv/year with a range of . - . msv/year. the reason for the skewed distribution was identified by analysing the proportions of the procedures to the total effective dose in this population: fluoroscopy . %, chest ct . %, sinus ct . %, cxrs . %, axrs . % and hrct . %. it was calculated that these patients undergo . cxrs per patient per year. we calculated that an average patient diagnosed by newborn screening could expect to receive . msv from ionizing radiation procedures between birth and age . biannual ct chest scans in addition to median exposures for each age group from axrs, sinus cts and fluoroscopy would potentially result in . msv by age , an fold increase. discussion: there is a in lifetime risk of developing a solid or haematological cancer with each exposure to msv of ionizing radiation . although fluoroscopy forms the most significant contribution to a pediatric cf patient's ionizing radiation exposure in our centre, introducing biannual chest cts would markedly increase exposure. as cf survival improves, cf physicians must be cognisant of the potential health cost of survival. references : previous studies have used generic measures of hrqol, which are not sensitive to important changes that may occur as a result of treatment. this study assesses the impact of iv antibiotic treatment for a pulmonary exacerbation on both generic and disease-specific hrqol of children and adolescents with cf. method: participants included patients (m age = . years) from the cincinnati children's hospital and university of florida cf centers. fiftyfour percent of the sample were male. patients completed two hrqol measures, the pedsql™ (generic) and cfq-r child or teen/adult version (cf-specific), which have excellent reliability and validity. these measures were completed within hours of hospital admission (pre) and hours of discontinuation of iv antibiotics (post). note that approximately % of patients went home on iv antibiotics and % remained in the hospital for approximately weeks. results: significant improvements were found in pulmonary functioning (fev % predicted) pre to post iv antibiotic treatment (paired t ( ) = - . , p < . ), with an average of % increase in fev % predicted. paired t-tests were conducted to examine changes in hrqol scores, using holm's procedure to correct for multiple comparisons. significant improvements were found on the cfq-r respiratory (paired t ( ) = - . , p < . ) and weight scales (paired t ( ) = - . , p < . ). trends were also noted for improved emotional functioning (paired t ( ) = - . , p = . ) and vitality (paired t ( ) = - . , p = . ), and worse treatment burden (paired t ( ) = . , p = . ). on the pedsql, only emotional functioning improved significantly (paired t ( ) = - . , p < . ). conclusions: results of this study confirm the effectiveness of iv antibiotics for the treatment of pulmonary exacerbations, with significant improvements found for both pulmonary functioning and hrqol. these results also highlighted the importance of using a disease-specific hrqol instrument, which proved to be more sensitive than the generic measure. on the cfq-r, significant improvements were observed in respiratory symptoms and weight, aspects of functioning that are not assessed on generic measures. fukushima, l.k.; hsu, e.; woo, m.s. division of pediatric pulmonology, childrens hospital los angeles, los angeles, ca, usa do hispanic and caucasian cf patients share the same frequency of pulmonary exacerbations? increased frequency of pulmonary exacerbations is linked to increased risk for cf morbidity and mortality. we have previously reported that pediatric hispanic cf patients have greater mortality than our caucasian cf patients who attend the same accredited cf center. we performed a retrospective review of cf pulmonary exacerbations (defined as physician prescription of intravenous or oral antibiotic therapy) in our cf patients who routinely attended the cf clinic during the period of january , through december , . data collected included demographics, number of intravenous antibiotic courses and number of oral antibiotic courses. unpaired student's t-test was used to compare age of the groups and chi-square was used to compare use of intravenous and oral antibiotics between hispanic and caucasian cf patients as well as between males and females. during the study period, there was a total of cf patients ( males: females; mean age . ± . years). of the patients, ( %) were classified as hispanic ( males: females; mean age . ± . years) and ( %)were classified as caucasian ( males: females; mean age . ± . years; p= . , ns). hispanic cf patients ( patients had episodes; males: females) had significantly greater number of pulmonary exacerbations (received treatment with intravenous and/or oral antibiotics) than caucasian cf patients ( patients had episodes; p = . ). hispanic cf patients who required intravenous antibiotic therapy were significantly younger ( . ± . years vs . ± . years; p< . ) than the caucasian cf patients who were treated with intravenous antibiotics. there was no significant difference in age between hispanic and caucasian cf patients who received oral antibiotic courses ( . ± . years vs . ± . years; p= . , ns). gender did not have a significant impact on pulmonary exacerbation risk in our population (hispanic iv use by gender p= . ; hispanic oral use by gender p= . ; caucasian iv use by gender p= . ; caucasian oral use by gender p= . ). we conclude that hispanic cf patients had increased incidence of pulmonary exacerbations than caucasian cf patients who attend the same cf clinic. hispanic patients who were treated with intravenous antibiotics were younger than the caucasian cf patients. we speculate that hispanic ethnicity has a major impact on the incidence of pulmonary exacerbations. other factors, such as modifier genes, environmental exposures, or inflammatory responses, may play a role in the increased risk of pulmonary exacerbations in the hispanic cf population. introduction: one of the main goals of the multidisciplinary team of the cf association of minas gerais, brazil is to find strategies to preserve lung tissue from the bacterial exacerbation in cystic fibrosis patients. objective: to investigate clues on onset of bacterial exacerbation. to our knowledge, no studies were found regarding the identification of the early warning signs of bacterial exacerbation with cf patients. we wanted to know whether cf patients present early warning signs before onset of fever and/or breathing difficulties. methods: telephone interviews were carried out with the cf patients registered in the association, by a physiotherapist academic who did not know the patients and just ask two questions. the first question was about the use of antibiotics during the year of and the second was related to early signs before the onset of fever. results: from the patients, only patients replied. patients who were using antibiotics therapy during and had fever: / patients who were using antibiotics and did not have fever: / patients who had fever and did not use antibiotics: / patients who did not have fever and did not use antibiotics: / the results revealed the following warning signs and also that some patients had more than one sign. tiredness, quietness, slowness ( / ); increased cough ( / ); lack of appetite ( / ); sticky sputum ( / ); sleepiness ( / ); headache ( / ); increased excitement, nervousness ( / ); crybaby ( / ); dry mouth ( / ); clammy ( / ); difficult to walk ( / ); gastric upset ( / ); itchy throat ( / ); abdominal pain ( / ) ; itchy eyes ( / ); breathing difficulties ( / ); tremulous ( / ); paleness ( / ); burning ear ( / ); chest pain ( / ); hissing ( / ) ; and syncope( / ). conclusions: it is helpful to know these early warning sings. we assume that the earlier the diagnosis of the exacerbation is made, the easier it is to resolve the problem and the less destruction of the lung tissues. reference: lung line, national jewish center for immunology and respiratory medicine . background: cystic fibrosis patients are experiencing increasing survival and are being treated with more chronic therapies. how these changes are reflected in day to day clinical symptoms has not been evaluated. objective: we examined data from the epidemiologic study of cystic fibrosis, a large longitudinal observational study, to characterize the change in respiratory symptoms over time. for each year between and data from patients with at least one visit recorded as occurring while clinically stable were included. data from all visits, including sick as well as stable, were used for each year in which at least one clinically stable visit was recorded. patients were separated into age groups less than , to , to , and or older. note that in data collection changed, potentially confounding the results. results: the average number of patients per year was . the percent of patients with no reported cough progressively increased over time (table) . results were similar for pateints with no reported sputum production at clinically stable visits. when sick visits were added there were similar results. the frequency of cough reported at greater than percent of visits progressively decreased over time (table) . again the results for sputum production were similar. these findings were seen in all age groups although older patients are more likely to experience these symptoms. conclusions: these data suggest that cf patients are experiencing fewer respiratory symptoms of cough and sputum production over the last decade. this improvement has occurred in patients with either intermittent or chronic symptoms. how clinical care has impacted on these symptoms has yet to be determined. also, more people are being diagnosed with cf at an earlier age or with milder disease, potentially resulting in the entire cf population having fewer symptoms. lung disease in children with cf begins in early childhood, with intermittent and then chronic infection, associated with an exaggerated inflammatory response. the extent to which inflammation contributes to bronchiectasis has not yet been identified. the aim of this study was examine relationships between inflammation and early lung damage methods: twenty eight children with cf were assessed at diagnosis and annually thereafter with a bronchoalveolar lavage (bal). bal fluid was used to assess micro-organisms and inflammation, including total and differential cell counts. a three slice hrct technique (inspiration and expiration) was performed under general anesthesia at age - y, immediately preceding the annual bal. hrct scans were scored by an independent and experienced radiologist using a modification of the brody score. results. twelve children ( %) had evidence of bronchiectasis on hrct. inflammation present in bal at months of age predicted bronchiectasis at - y. total cell count at mo, predominantly neutrophils, was higher in those who developed bronchiectasis compared to those who did not ( . vs . x cells/ml bal; p= . ). the total number of infections detected in bal was not related to bronchectasis. however, children with bronchiectasis had a higher incidence of pseudomonas aeruginosa within the first years of life ( % vs. %). conclusions: bronchiectasis is evident within the first five years of life and appears to be associated with an exaggerated inflammatory response and the early acquisition of pseudomonas aeruginosa. supported by: cystic fibrosis foundation (usa), national health and medical research council (australia). fortunately the survival of patients with cystic fibrosis has improved remarkably over the last few decades. many patients can life quite "normal". therefore family planning becomes more important. several reports docu-mented the good maternal and fetal outcome in pregnant women with cystic fibrosis. besides the data from the french cf registry no longterm data about the impact of paternity of male patients with cystic fibrosis are available until now. in a retrospective analysis we gathered data about all our male patients who became father while being followed-up in our cystic fibrosis center for adults. from male patients, patients became father between and . patient no. became father twice (second time father of twins); patients no. became also father of twins .we looked at fev % year prior to childbirth, perinatal and and years after the childbirth. for the fev % one year prior to birth as well as for fev % and years after birth, we calculated the difference to the fev % at time of birth. between and , male adult patients with cystic fibrosis became fathers of a total of children, all by the assistance of reproductive techniques. their fev % are shown in table . although the male patients are not directly influenced by a pregenancy (as the pregnant women are by growth of the uterus), there seems to be more than ususal differences in this lung function parameter. there were also more fluctuations in fev per year. the biggest differences in fev % were seen year after birth of their children. further investigations in a larger group (in cooperation with other centers) are planned; especially with a focus on the frequency of infections and quality of life compared to male patients without children. intravenous (i.v) aminoglycosides are widely used in cystic fibrosis (cf) patients as treatment for pulmonary exacerbations. in an effort to reduce undesiderable effects of these antibiotics it is recommended to measure drug serum levels. recent experience suggests that tobramycin (to) may be administered as a single daily dose with equal efficacy as multiple doses but less risk of nephrotoxicity. aim: we evaluated the efficacy of once-daily i.v to treatment to improve pulmonary function expressed as forced expiratory volume in one second (fev ) in cf patients with pulmonary exacerbations compared to the same group of patients who were previously treated with to in multiple daily i.v doses, as historic control. we evaluated baseline and peak drug serum levels of to administered once daily compared to multiple dose levels. methods: all patients were treated with i.v to in combination with a beta-lactam antibiotic for two weeks. respiratory exacerbation was defined according to cff criteria. all cf patients were colonized by non-fermentative gram-negative bacteria. serum concentrations of to were measured (fluorescent polarisation immunoassays) before the fifth infusion (basal) and minutes after the fifth infusion (peak). reference to blood levels were considered to be ≤ mg/l (basal) and < mg/l (peak) for multiple daily doses, and ≤ mg/l (basal) and - mg/l (peak) for single daily dose. treatment efficacy was evaluated on the basis of improved clinical condition and increased fev values compared to the beginning of therapy. results: patients with respiratory exacerbation were evaluated, with a mean (± ds) age of . years (± . ; median . , range . - . ) . out of patients were chronically colonized by pseudomonas aeruginosa. the total mean (± sd) daily dose of to administered in multiple doses was (± . ) mg whereas the mean single dose was . (± . ) mg. of those patients given multiple doses of to, only one patient ( . %) had basal to values outside the range and patients ( . %) had peak values outside the range. of those patients given a single daily dose, no patients had basal to values outside the range whereas out of ( %) had peak values of less than mg/l and patients out of ( %) had peaks between and mg/l. the mean (± sd) increase in fev was . % (± . ) in patients treated with a single dose and % (± . ) in those treated with multiple doses. the mean (± sd) period between fev measurements before and after exacerbation was . days (± . , median , range - ) in case of monodose therapy, and . (± . , median , range - ) in case of therapy with multiple doses. conclusions: both single and multiple-dose i.v to therapy caused a comparable fev increase and were effective in resolving the respiratory exacerbation. although clinical improvement was observed, the mean i.v to dose of mg/day in patients treated with once-daily i.v to determined peak values between and mg/l in % of subjects. intravenously administered aminoglycosides are efficacious for the treatment of pulmonary infections of cystic fibrosis (cf) patients, with the principal adverse effects of these molecules being acoustic nerve damage and nephropathy. damage to the eighth cranial nerve varies from . to % in cf patients treated with multiple daily doses of aminoglycosides. recent experience suggests that tobramycin can be administered as a single daily dose, resulting in less nephrotoxicity but equal efficacy. patients and methods: we evaluated the prevalence of oto-and nephrotoxicity due to aminoglycosides administered to cf patients followed at the tuscan regional center where we are currently following patients. cochlear damage was evaluated using tonal audiometry (amplifon amplaid s). renal damage was evaluated by measuring patients' creatinine clearance. results: ( %) patients ( males and females) between the ages of and (mean age . years, median , sd ) were tested with audiometry. all these patients had been repeatedly treated with multiple daily doses of intravenous aminoglycosides. ( . %) ( males and females) out of patients between and years (mean age . , median . , sd . ) had their creatinine clearance measured. we found that patients ( . %) had an abnormal audiometry, typically attributable to aminoglycoside ototoxicity (high frequency deficit). two patients required a hearing aid. one patient with normal cochlear function had labyrinth deficit. one out of patients tested had pathological creatinine clearance values. conclusions: our data indicate that ( . %) of our patients had damage to the eighth cranial nerve due to repeated multiple daily doses of aminoglycosides. we recommend that cf patients under aminoglycoside therapy be routinely given audiometric and creatinine clearance exams in an attempt to reduce the incidence of undesirable side effects of these drugs. davies, j. , ; voase, n. , ; dewar, m. , ; mullard, k. , ; gammie, f. , ; saunders, c. , ; horsley, a. , ; gray, r. , ; macleod, k. , ; somerton, l. , ; higgins, t. , ; donovan, j. , ; cornish, n. , ; hansell, d. , ; aziz, z. , ; ashby, d. ; geddes, d. , ; greening, a. , ; cunningham, s. , ; innes, a. , ; alton, e. , . gene therapy, imperial college, london, united kingdom; . western general hospital, edinburgh, united kingdom; . royal hospital for sick children, edinburgh, united kingdom; . royal brompton hospital, london, united kingdom; . queen mary, university of london, london, united kingdom; . uk cf gene therapy consortium, edinburgh, london, oxford, united kingdom the improving clinical status of patients with cf and the slow rate of decline of conventional outcome measures such as fev necessitate the development of clinically-relevant surrogate outcome assays for use in clinical trials. in our forthcoming clinical trial of cftr gene therapy, the uk cf gene therapy consortium will use both established, clinically-available assays and more novel measures designed specifically for this purpose. both groups of assays are being subjected to rigorous testing prior to use to establish their utility as disease biomarkers. in this study, we examined their performance in the context of an infective exacerbation treated with iv antibiotics. this abstract will report the response of established, clinically-available assays; available data from novel assays will be reported separately. children ( years and above) and adults with cf were recruited from three centres at the time of a clinician-defined infective exacerbation requiring iv antibiotic treatment. exclusion criteria included fev < % predicted and requirement for supplemental oxygen. a panel of assays was performed at the start and end of treatment, which was most commonly days. data are presented as mean (sd) or median (range) and differences compared with either parametric or non-parametric paired analysis as appropriate. clinical assays included spirometry, sputum microbiology, sputum cell count and differential, serum inflammatory markers (crp, white blood cell (wbc) count,) and chest ct. patients also completed a symptom score sheet. to date, patients (mean age . [ . ] years, male) have paired data available from the start and end of the course of ivs. at baseline, were infected with p. aeruginosa, with b. cepacia complex organisms and with s. aureus ( mrsa). significant changes from baseline were observed in fev ( . [ . ] the clinical response to any novel intervention, for example, cftr gene therapy is difficult to predict. prior testing of experimental assays in a study such as this provides data on the variability of the measurements within the disease population and the degree of change observed with an intervention known to lead to clinical benefit. this should aid the design of rational, powered clinical trials. funded by the cf trust influenza can worsen cf and lead to serious complications and mortality. influenza vaccine is safe and effective in cf patients. in france, annual influenza vaccination is recommended for all cf patients aged over months and for healthcare workers in regular and prolonged contact with high-risk patients. objective: to estimate the influenza vaccination coverage rate for - season in cf patients and healthcare workers in cf care centres of the great south region of france. methods: a multicentric observational study between february and september was conducted. healthcare workers were contacted by telephone and were questioned about their - influenza vaccination status, and the reason for not getting vaccinated. for patients aged over months whose vaccination booklet was available, the physician filled in a written questionnaire. results: a total of professionals were interviewed. the survey included . % of doctors, . % of nurses, . % of dieticians, . % of physiotherapists, . % of social workers and . % of psychologists. the overall influenza vc rate was . % and varied considerably according to profession: . % of doctors, . % of physiotherapists, . % of dieticians, % of psychologists, . % of nurses and . % of social workers. the overall influenza vc rate in cf patients was . % ( . % in children (age < years old) versus . % in adults; p< . ). receiving a voucher for free vaccination from the national insurance increased the influenza vc rate. for healthcare workers, the main reason for non vaccination was that flu was considered as a benign disease (useless vaccine); for patients, it was a lack of time. conclusion methods: adult patients with cf were studied. patients completed a standardised questionnaire. the questionnaire identified which changes and duration of symptoms would cause them to contact the cf team and/or change treatment. results: all patients would contact the cf team for haemoptysis but % would wait for more than hours and % would wait for more than hours before doing so. up to % of patients would wait a week or more before contacting the cf team because of sputum purulence. % of patients would not contact the cf team if they developed new chest pain. conclusion: there appears to be a disconnect between recognizing a change in symptoms and the length of time before acting on the change. this has major implications for the delivery of cf care. supported by cystic fibrosis association of ireland. aim: to determine the efficacy of standardized desensitization protocols in treating antibiotic allergies in adults with cystic fibrosis (cf). background: dependence on high dose and multiple combination intravenous (iv) antibiotics to treat pulmonary exacerbations in adult cf patients has resulted in an increased frequency of allergic reactions when compared with the general population. ( , ) strategies to address these allergies are essential to maintain effective antibiotic treatment in this population. antibiotic desensitization is the process by which one induces immune tolerance by incremental exposure to the antibiotic, which may induce stabilization of mast cells. the mechanism remains undetermined but may be mediated via ige or memory t-cells. prior to the study period, standardized antibiotic desensitization protocols were developed by an allergist/immunologist based on published reports and were approved through the hospital's pharmacy and therapeutics committee. methods: the toronto cf database was accessed to generate a list of hospital admissions for iv antibiotics during the -year study period ( ) ( ) ( ) ( ) ( ) ( ) . patients who underwent desensitization were identified and each of their case notes underwent retrospective analysis. data were collected with respect to age, organism requiring antibiotic therapy, antibiotic allergy requiring desensitization, reactions during desensitization, reactions post desensitization, treatments required to treat these reactions, and the completeness of antibiotic therapy post desensitization. statistical analyses were performed using graph pad prism®. results: during the study period the cf unit dispensed and initiated approximately courses of iv antibiotics. a total of desensitizations were performed in patients ( female). although some patients were desensitized to only one drug or to the same drug more than once, there were patient-drug combinations with a median of (+/= . ) unique combinations per patient. of the desensitizations protocols initiated, patients developed documented reaction to the antibiotic during desensitization however only desensitization protocol was not completed due to an adverse event. there were no episodes of anaphylaxis either during or after desensitization. a total of patients developed documented reactions during the subsequent antibiotic course - required termination of the antibiotic and completed the course of iv antibiotics with management of allergic type reactions with antihistamines. conclusions: standardized desensitization protocols were initiated in patients during the study period. the success rate for desensitizations in these patients was % however when the data is analyzed for true allergic phenomenon, the success rate rises to %. this demonstrates the efficacy of these standardized protocols in the treatment of ige-mediated allergic reactions to antibiotics in the adult cf patient. references: . burrows, j., toon, m., bell, s., ( ) . antibiotic desensitization in adults with cystic fibrosis. respirology, : - . . ramesh, s. ( ) . antibiotic hypersensitivity in patients with cf. clin rev allerg immun, : - . surgery for symptomatic disease. there is a long-held suspicion that poorly-controlled chronic sinusitis negatively impacts the respiratory status of cf patients, but whether sinus surgery improves the clinical course of cf lung disease is unclear. aim: assess the impact of functional endoscopic sinus surgery (fess) on respiratory exacerbation rate and lung function in adult cf patients methods: this is a single-center, retrospective study based on chart review. during the study period, - , adult patients underwent separate fess procedures. this abstract contains results so far from the analysis of patients that underwent a total of surgeries. primary outcomes were -year change pre to post fess in pulmonary function parameters and pulmonary exacerbation rate. results: at the time of surgery, the mean fev was % of predicted (ppd), the mean fvc . ppd, and the median age was . years (range . - . ). . % of patients were female. the median number of iv antibiotic courses for respiratory exacerbations in the months prior to fess was (range - ) compared to (range - ) in the year after surgery (p = . ). further,there were fewer days on intravenous (iv) antibiotics for respiratory exacerbations in the months prior to fess than in the month post-operative period (median vs. days), but this difference was not statistically significant (p = . ). the use of oral antibiotics for respiratory flares was comparable in the months before and after fess (median vs. days, respectively, p = . ). the median number of hospital admissions and median number of days hospitalized for respiratory exacerbations did not differ in the months pre vs. post-fess (p = . and p = . , respectively). the mean fev and fvc in the months pre and post surgery did not differ significantly ( . vs. . ppd, p = . , and . vs. . ppd, p = . ). the linear rate of change in fev and fvc did not differ significantly in the months prior to and following fess (-. l/month vs. +. l/month, p = . and -. l/month vs. +. l/month, p = . , respectively). there were no significant differences (p > . ) for any of the above comparisons when the analysis was limited to months before and after fess. there were no significant differences (p > . ) in antibiotic use and hospitalizations when the analysis was limited to the surgeries in which patients were experiencing both respiratory and sinus symptoms. this latter group had lower fev and fvc ppd in the month post-fess period ( . vs. . ppd, p = . , and . vs. . ppd, p = . , respectively). however, the rate of decline in fev and fvc in the months before and after surgery did not differ significantly ( p = . and p = . , respectively). conclusion: fess did not significantly affect the rate of respiratory exacerbations or rate of decline in pulmonary function in adults with cystic fibrosis. cumulative decline in lung function as measured by fev in cystic fibrosis (cf) pulmonary exacerbation is a major cause of cf-related morbidity and mortality. high-dose extended interval aminoglycoside (hdei ag) use may be more effective in improving lung function than traditional multiple daily dosing in cf-related pulmonary exacerbation. intermittent dosing of beta-lactam antibiotics are often used with hdei ag in patients with cf pulmonary exacerbation, which does not optimize betalactam pharmacodynamics. the primary purpose of this study is to determine the effect of hdei ag plus continuous infusion beta-lactam (cibl) on patients' return to best baseline fev from the previous months. this study was conducted at university of kentucky healthcare. this was a concurrent observational trial with patients serving as their own historic controls via review of the medical record. all pediatric and adult patients with cf respiratory disease hospitalized with an acute pulmonary exacerbation between november , and may , , with pseudomonas aeruginosa in their respiratory culture and were followed for at least one year were included. excluded were patients with known hearing disability or renal insufficiency or an inability to reliably perform spirometry (ie.less than six years old). patients served as their own historic controls via conventional treatment (control) versus hdei ag plus cibl (treatment). the primary endpoint was to determine the percent of patient courses that return to best baseline fev in the last twelve months after treatment with hdei ag plus cibl. secondary endpoints included determination of patient courses that returned to mean baseline fev in the last twelve months after study treatments, and description of beta-lactam dosing characteristics. thirteen patients were included in the pilot analysis which totaled patient courses (mean . courses per patient). average patient age was ± . years with a mean fev of %; . % were female. after hdei ag plus cibl no patients showed a return to their best baseline fev in the previous months with the exception of one patient whose value was unchanged. average overall percent change in best baseline fev group was - . ± . at follow up. % of patient courses exhibited a return to their mean baseline fev . average overall percent change in mean fev baseline was . ± . at follow up. five of the ( . %) study patients who had serum beta-lactam concentrations obtained were able to achieve at least four times the mic of the target organism. more data will be collected on subsequent courses and follow up lung function. our preliminary data suggest that our patients lost % of their best fev with an acute exacerbation and returned to % above mean baseline after treatment with hdei ag and cibl therapy. this evidence suggests that this might be an alternative to intermittent beta-lactam therapy. follow up data on remaining patients may provide sufficient evidence to validate this hypothesis. respiratory exacerbation in cystic fibrosis patients is characterized by increased sputum that may become more purulent. the detection of the exacerbation is based mainly on clinical subjective parameters. fev measurements are accepted as gold standard to be used for the life-time of the patient but do not adapt fast enough after resolution of an exacerbation. we compared parameters that are affected by the uneven distribution of ventilation in the lung due to increase sputum. methods: a zen body plethysmograph was used to measure tlc, fev and dlco (co/ch mixture). tlc was also calculated by the volume of ch , the angle of the slope of phase iii was calculated in cf patients at the beginning of the respiratory exacerbation and after weeks of antibiotic treatment. patients were - years old ( m/ f). results: fev changed by . + . % at the end of the antibiotic treatment, the tlc(pleth) however was stable and changed by only . + . %. the rv/tlc decreased by . + . % and the tlc(gas) was increased by + . %. the difference between tlc (pleth) and tlc (gas) before and after the antibiotic treatment decreased from . + . % to . + . %. conclusion: the changes in the parameters affected by the unevenness of distribution of ventilation (m/p reflecting the increased sputum in the airway) are more pronounced than fev and may be used as a more sensitive parameter for assessing the course of respiratory exacerbation. fibreoptic bronchoscopy was performed in children aged - years: with cf, with non-cf bronchiectasis (bx), with asthma, and control children without lower respiratory disease. endobronchial biopsies were taken and stained with haematoxylin and eosin. asm content, myocyte number and size were quantified using stereology. results: the volume fraction of asm in subepithelial tissue (median; iqr) was . ( . - . ) in controls. by comparison, it was . ( . - . , p< . ) in cf, . ( . - . , p< . ) in bx, and . ( . - . , p< . ) in asthma. myocyte number (cells per mm of reticular basement membrane) was ( - ) in controls, ( - , ns) in cf, ( - , ns) in bx, and ( - , p< . ) in asthma. myocyte size (um , mean; sd) was ( ) in controls, ( , p< . ) in cf, ( , p< . ) in bx, and ( , p< . ) in asthma. in cf, the volume fraction of asm in subepithelial tissue was related to myocyte number (r= . , p= . ), but not to myocyte size. conclusions: asm remodeling occurs in cf children. this, however, is not disease-specific and is also found in non-cf bronchiectasis and in asthma. both myocyte hypertrophy and hyperplasia contribute to increase in asm in cf. support: ers long-term fellowship and swiss national foundation grant to nr purpose: ct scans are increasingly being ordered on a routine basis in patients with cystic fibrosis. while prior studies have investigated their clinical utility in children, there is limited and conflicting information on their utility in adults. the purpose of this study was to determine the relationship between clinical disease severity as assessed by spirometry and ct findings as well as initial ct score ability to predict change in lung function in a nonselected group of adult cf patients. methods: a retrospective review of cf patients (mean age . +/- . , male %) who had undergone a routine chest ct was performed. the ct scans were scored using the brody method by a blinded reader with the degree of bronchiectasis, mucous plugging, peribronchial thickening, parenchymal disease, and air trapping assessed individually and a composite score generated. the ct metrics were first compared with spirometric test that were temporally related to the date of the ct scan, and then with future spirometric tests. results: for the cohort the patients had a wide range of severity of disease with the average fev % . +/- . , and the average fvc% was . +/- . . no significant correlation was found between initial fev % and total ct score ( . p value . ), or initial fvc% and total ct score (- . p value . ). for ct metrics and future spirometry, the average time from ct to pft's was days +/- with median of days. predictors for a significant decline in pft's were a decreasing bmi and male sex (p-values . and . respectively). further, for patients with a low fev % (< % at baseline), the fvc% change over time was associated with total ct score, male gender, and bmi (p-values . , . , and . respectively) conclusions: in adult cf patients, ct scan findings did not correlate with lung function. however in longitudinal analysis, bmi and gender had an effect on fvc change. for patients with low lung function, total ct score did have an association with change in fvc along with bmi and gender. pulmonary exacerbations (pes) are episodes of worsening of respiratory symptoms that commonly occur in patients with cystic fibrosis (cf). in clinical trials of new therapies in cf, pulmonary exacerbation is widely used as a primary or secondary endpoint. despite its importance, no single clearly agreed upon definition exists. some are based on patient reported symptoms while others require hospitalization or antibiotic prescription. use of different definitions makes it difficult to compare the results across studies and also to plan future studies. moreover, the rate of pes (proportion of patients with at least one exacerbation over a given time-period) is affected by baseline patient characteristics such as age, fev and medication use at the start of the clinical trial; factors which also contribute to the difficulty in comparing trials. using data from the cf therapeutics development network (tdn) data bank, we examined several pe definitions and baseline characteristics for their effect on pe rates. we find that pulmonary exacerbation rates vary substantially depending on both the pe definition and the baseline characteristics. for example, in one study, the proportion of subjects hospitalized and/or prescribed antibiotics during the month treatment period was %, while the proportion who met a symptoms based criteria for pe during the same period was %. we also examined the implications of varying control group exacerbation rates on sample size requirements for clinical trials and find that the control group pe rate has a large impact. for illustration, we assume a two group study with two-sided significance level . and % power. if the control group rate is %, then approximately subjects per group are required to detect a relative rate reduction of % (corresponding to a treatment group rate of %). however, if the control group rate is %, then only approximately subjects per group are required to detect the same relative rate reduction of % (corresponding to a treatment group rate of %). thus, a pe definition that selects for a greater pe rate in the control group will require fewer study subjects to detect the same relative rate reduction given that all other variables remain equal. sample size requirements can be further reduced by analyzing the number of events or the time to first event instead of the proportion of subjects who have at least one pe during a given time period. supported by cystic fibrosis foundation therapeutics, inc. (ram-sey y ), the national center for research resources (ncrr mo -rr ) and csl behring. briglia, h.; hilliard, j.; chmiel, j.f.; krenicky, j.; konstan, m.w. pediatrics, case western reserve university, cleveland, oh, usa cystic fibrosis (cf) is characterized by a vicious cycle of obstruction, infection and inflammation. the inflammatory response, which is profound and excessive relative to the burden of bacteria, is characterized by a massive neutrophil (pmn) influx. cf patients often experience intermittent declines in lung function associated with increases in both bacterial burden and pmn influx. these pulmonary exacerbations require treatment with antibiotics. there are substantive issues in the identification of pulmonary exacerbations and the assessment of therapies. a marker which indicates the inflammatory state of the lung would be useful to identify infective/inflammatory exacerbations, as opposed to worsening due to pulmonary vascular disease, or simply upper airway infection (cold), and might guide therapy for intensity and duration. g-csf and gro-α are cytokines produced in the lung by a variety of cells, including macrophages and epithelial cells. g-csf is involved in the proliferation, differentiation and activation of pmn precursors, while gro-α has known chemotactic and activating effects on pmns. transfer of these cytokines through the basolateral membrane into the bloodstream results in increased pmn production and activation in the bone marrow. the plasma concentrations of g-csf and gro-α might serve as potential biomarkers for a pulmonary exacerbation. in this pilot study we measured g-csf and gro-α in the plasma of healthy volunteers (n= ), clinically stable cf patients (n= ) and cf patients experiencing a pulmonary exacerbation, both before (n= ) and following (n= ) - days of antibiotic (abx) therapy. cytokines were measured using commercially available elisa kits (r&d systems, minneapolis, mn) . t-tests were performed on natural log transformed cytokine data (sigmastat v . , systat software inc., san jose, ca). ᭹ the lack of a significant difference between g-csf and gro-α plasma levels in healthy and clinically stable cf patients suggests that these cytokines are not indicators of chronic inflammation. ᭹ both cytokines are increased during pulmonary exacerbations in patients with cf and decrease to baseline values following antibiotic therapy, suggesting that these cytokines might serve as potential biomarkers for cf pulmonary exacerbations. ᭹ although not statistically significant, there was a trend towards an inverse correlation between both cytokines and fev during pulmonary exacerbations in this small study (data not shown). ᭹ further studies are warranted to increase our understanding of g-csf and gro-α as potential biomarkers of pulmonary exacerbation in cystic fibrosis. this work was supported in part by the cystic fibrosis foundation through an institutional research development grant. pletcher, s.d. ; koff, j.l. ; kleinhenz, m. . otolaryngology, university of california, san francisco, san francisco, ca, usa; . medicine, university of california, san francisco, san francisco, ca, usa introduction: while sinus disease is common in adult patients with cystic fibrosis (cf), the severity of sinus symptoms and relationship of sinus disease to other manifestations of cf are relatively unknown. objectives: to evaluate the severity of sinus symptoms in adult patients with cf and correlate these findings with other cf manifestations. methods: twenty-six consecutive adult patients with cystic fibrosis were surveyed during routine clinic visits for sinus specific and overall quality of life outcomes using both the sino-nasal outcome test (snot- ) and the cystic fibrosis questionaire (cfqr). analysis of snot- and cfqr scores were compared to %fev and sputum culture growth of pseudomonas for all patients. results: snot- scores ranged from . to . with a mean of . on a - point scale. for the purpose of data analysis, the patients were divided into two patient cohorts: group with minimal sinus symptoms (snot- < . ) and group with mild to moderate sinus symptoms (snot- > . ). the mean %fev for group was significantly higher than the mean %fev for group ( . % vs. . % respectively, p< . ). patients in group were more likely to have sputum culture growth of pseudomonas although this trend did not reach statistical significance ( % vs %, p= . ). cfqr scores differed significantly among the two groups with group patients reporting more disability in the physical, role, vitality, body, eating, health, respiratory, weight, and digestion domains but not in the emotion, social, and treatment domains. conclusions: this cohort of adult cf patients show minimal to moderate symptoms of sinonasal disability. those patients with mild to moderate sinonasal symptoms have decreased pulmonary function and decreased overall quality of life compared to cf patients with minimal sinus symptoms. , . internal medicine, university of iowa hospital and clinics, iowa city, ia, usa; . translational lung imaging program, university of iowa hospitals and clinics, iowa city, ia, usa; . pulmonary and critical care medicine, mount sinai medical center, miami beach, fl, usa mucociliary transport is an important component of airway host defense against inhaled particles and microbial pathogens. mucociliary clearance is hypothesized to be defective in cystic fibrosis allowing for airway colonization and infection. current methods for measuring mucociliary transport and clearance lack the ability to measure mucociliary transport in specific proximal and distal airway segments. using a -slice high resolution ct scanner (somatom , siemens), we are developing a method to track radiopaque particle movement in the swine airway. swine were induced with ketamine/acepromazine, intubated, and then anesthesia maintained with propofol infusions. animals spontaneously breathed humidified air with the endotracheal tube cuff deflated. radiopaque teflon/bismuth trioxide disks ( mm diameter, . mm thick) were instilled via a catheter into the airways. serial ct images ( . mm slice thickness and slice increment of . mm) were obtained every minutes for a total of minutes. airway tree segmentation was performed using pulmonary workstation . (vida diagnostics, iowa city, ia). this program allows for fully automated airway tree segmentation. baseline mucociliary transport rates ( -dimensional) were . ± . mm/min. following aerosol delivery of human sputum leukocyte elastase ( mu), mucociliary transport was markedly diminished ( . ± . mm/min) at minutes. thus far, we are able to measure mucociliary transport down to the rd generation airways. future studies include more distal deposition and measurement of particle movement. in summary, this novel method measures mucociliary transport in defined airways and will allow us to examine the effect of altered airway epithelial function on mucociliary transport. lung transplant. purpose: to design a ct scoring system to quantify structural abnormalities on ct scans from cf patients with severe advanced lung disease (sald) and to investigate the correlation between ct scores and survival. materials and methods: ct scans of cf patients screened for lung transplant between and were collected from transplant centres. all scans were scored with the brody ii scoring system. to design a new scoring system sensitive for the specific changes in sald, a panel of experts reviewed a random set of ct scans on eligible items to be used in a pilot analysis. the resulting sald scoring system consisted of four items: bulla/cysts; areas with consolidation/mucous; areas with hypo perfusion/air trapping and hyper/normal perfusion. in each ct slice ( mm spacing) and for each of the items the surface area of corresponding lung tissue involved was estimated on a - % scale. total surface area for the items and each slice added up to %. the final sald score was a mean volume estimate of abnormal and normal lung tissue. results: pilot analysis of a set of ct scans showed a a spectrum of abnormalities, ranging from predominantly bronchiectasis to predominantly mosaic perfusion. a moderate correlation was found between the sald components inflammation and hypo perfusion/ air trapping and brody score (p= . , r= . and p= . , r= . ). the inter-observer variability of both scoring systems was comparable. (ri ranging from . to . ) analysis of the complete cohort with the brody ii score showed a significant correlation with survival with a hazard ratio of . ( % ci of . to . ) for dying on the waiting list for every ten point increase in brody ii score. (p= . ). the brody component scores for bronchiectasis, airway wall thickening and parenchyma showed a similar correlation with hazard ratios ( % ci's) of respectively . ( . - . ), . ( . - . ) and . ( . - . ) for every ten point increase in score. (p= . , . and . ). the preliminary results with the brody score suggest that it is useful to include ct information in prediction models. it is likely that the predictive value of the ct can for survival can be further improved using the dedicated sald-ct score. scoring of the remaining scans according to the sald system is ongoing and will be completed shortly. snell, g. . physiotherapy, the alfred, melbourne, vic, australia; . allergy, immunology and respiratory medicine, the alfred, melbourne, vic, australia; . epidemiology and preventative medicine, monash university, melbourne, vic, australia; . radiology, the alfred, melbourne, vic, australia background despite the widespread use of airway clearance (ac) techniques to clear excessive secretions and improve lung function, little is known about their efficacy following lung transplant (lt). this study aimed to compare the effects of two ac strategies (proactive vs reactive) on a range of clinical outcomes following lt. methods a prospective randomized trial design with stratification for suppurative pre-lt disease was used. consecutive patients at month post lt were eligible for inclusion. subjects were excluded if medically unstable, ventilator dependent or had a contraindication to performing positive expiratory pressure (pep) therapy. patients performed ac using pep either twice daily (proactive strategy) or only in the presence chest infection (reactive strategy). lung function (fev and fvc), chest radiography (brasfield score), exercise capacity ( minute walk) and bronchoscopic airway characteristics (anastomotic healing, patency and secretions) were assessed at , and months post lt. patient adherence and satisfaction were measured. results of consecutive patients, ( in each group) were recruited and completed the study. both groups improved lung function (fev ± % to ± % p< . ; fvc ± % to ± % p< . ), brasfield scores ( . ± . to . ± . p< . ) and minute walk ( ± m to ± m p< . ) over the study period. no significant differences between groups for any outcome were found. the vast majority had fully healed, % patent anastomoses without secretions at months. there were no significant differences between airway characteristics and incidence of chest infection. adherence to both strategies was high ( % proactive, % reactive). conclusion in the absence of significant differences in outcomes, it is recommended that ac only be performed in the presence of chest infection based on greater treatment cost and treatment time required by a proactive strategy. further studies in those with reduced anastomotic patency and in those with recurrent chest infections later post-transplant are warranted. supported by an alfred trusts grant. munro, p.e. ; button, b. , ; bailey, m. ; holland, a. , ; snell, g. . physiotherapy, the alfred, prahran, vic, australia; . allergy, immunology and respiratory medicine, the alfred, melbourne, vic, australia; . epidemiology and preventative medicine, monash university, melbourne, vic, australia; . school of physiotherapy, la trobe university, melbourne, vic, australia background the optimal duration and structure of pulmonary rehabilitation (pr) for lung transplant (lt) recipients is not known. this study aimed to describe changes in functional outcomes in lt recipients who participated in a post lt pr program at the alfred, melbourne, australia. methods prospective, repeated measures design. functional exercise capacity (six minute walk test- mwt), lung function (fev , fvc) and quality of life (sf ) were assessed at , and months following lt. following discharge, all subjects attended a hour supervised outpatient exercise training class days per week until weeks post lt and education sessions. patients with post-operative complications were excluded. data were analysed using descriptive statistics and anova with repeated measures. results consecutive lt recipients from sept to mar were assessed for inclusion. ( % male) subjects, mean age ± yrs were recruited and completed the study. % had undergone bilateral lt. % had cystic fibrosis, % chronic obstructive pulmonary disease. significant improvements were demonstrated in mwt ( ± m to ± m, p< . ), fev ( ± % to ± %, p< . ), fvc ( ± % to ± %, p< . ) and all quality of life domains, p< . . the greatest changes in mwt and lung function were seen between and months. conclusion functional exercise capacity, lung function and quality of life improved significantly over the first months in lt recipients who participated in pr at our institution. these data will allow benchmarking with other centres and program structures. supported by an alfred trusts grant. does gender play a role in perception of cf quality of life domains before and after lung transplantation? the cf quality of life (cfq-r) for patients м years of age is a disease-specific instrument. previous studies using this instrument have reported that cf females have higher scores in weight and body perception than cf males. to determine the impact of lung transplantation on gender-based perception of quality of life, we reviewed the cfq-r on all cf patients м years of age and who underwent successful lung transplantation at our center. cfq-r was administered - months prior to transplant surgery and then months after lung transplantation while the patients were in the outpatient clinic setting. domains measured: physical, role, vitality, emotion, social, body image, eating, treatment burden, health status, weight, respiratory symptoms, and digestion. the cfq-r responses were computer-scored and domain scores were then generated (score - , =better). during the ⁄ year study period, cf patients underwent successful lung transplantation ( males: females; mean age . ± . years). prior to lung transplantation, female cf patients had lower physical domain scores but higher scores in health status perceptions than the male cf lung transplant candidates (see graph). all patients had marked improvement in all domains months after lung transplantation (see graph). after transplant surgery, cf males had higher scores than cf females in emotion and in eating domains. scores for body image domain were similar for both genders before and -months after lung transplantation. we conclude that although there were differences in physical and health domain perceptions prior to lung transplantation, both male and female pediatric cf patients had marked improvement in all domains after lung transplantation. however, female cf lung transplant recipients had lower emotion and eating domain scores after transplantation. we speculate that lower scores in these domains may reflect increased post-traumatic stress and depression in female lung transplant recipients. further study on cf quality of life in pediatric lung transplant recipients is on-going. background cf liver disease (cfld) with severe cirrhosis develops in - % of patients, usually within the first decades of life. most patients with cfld suffer from complications of portal hypertension but hepatocellular function usually remains intact for many years, even decades. patients with cfld referred for lung transplantation (tx) may be offered lung tx, lung-liver tx or be excluded from lung tx. however, variceal hemorrhage due to portal hypertension can be managed by banding, sclerotherapy or shunt procedures without liver tx. the aim of this study was to examine outcome of cfld patients who underwent lung tx and the subsequent progression of cfld, particularly with the use of potentially hepatotoxic immunosuppressive drugs. we conducted a retrospective cohort study to compare cf patients undergoing lung tx with and without cfld. b. cepacia-negative cf patients undergoing lung tx at toronto general hospital from - were eligible for inclusion. liver cirrhosis was defined by histology, esophageal varices on endoscopy or imaging evidence of splenomegaly. each patient with cfld was matched for age and year of tx with cf patients without cfld. demographic data, survival and liver function tests (lfts)(ast, alt, alp, inr, albumin, protein at week , month , , , , , , , , and post tx) were obtained by chart review. results of the groups were compared using unpaired t-test (parametric data) or mann-whitney u test (non-parametric data). results b. cepacia-negative cf patients underwent lung tx over this period, ( . %) having cfld with cirrhosis. data from the cfld patients vs. matched cf patients without cfld were analyzed. no significant difference was found between the groups in pre-tx age, gender, bmi, pi status, cfrd status, fev % predicted, -minute walk distance or lfts. in the cfld group albumin was lower week post-tx ( . + . g/l vs. . + . g/l, p< . ), alp was higher month post lung tx ( . vs. . , p< . ) and alt was higher months post-tx ( . + . u/l vs. . + . u/l, p< . ). there was no significant difference in the other blood tests. azathioprine was changed to mycophenolate mofetil in cfld patients due to liver test abnormalities. cfld patient developed hepatic encephalopathy and ascites years post lung tx and is being assessed for liver tx. no difference was seen in the number of episodes of acute rejection or post-tx survival ( . % vs. . % alive at years). discussion cfld patients undergoing lung tx did not have a worse prognosis than patients without cfld. transient elevation of lfts were seen in the immediate post-tx period but settled either spontaneously or after discontinuation of azathioprine. we conclude that selected patients with cirrhosis due to cfld may be safely offered lung tx without concomitant liver tx. infectious mononucleosis, commonly called mononucleosis, or "mono," is an illness caused by the epstein-barr virus. mononucleosis has been nicknamed the "kissing disease" because the epstein-barr virus commonly is transmitted in saliva during kissing. however, sneezes and coughs also can transmit the virus occasionally. the epstein-barr virus (ebv) permanently infects more than % of the people on earth, but it causes symptomatic mononucleosis only in a small number. in developed nations, such as the united states, mononucleosis most often occurs between the ages of and . unfortunately ebv can not only infect, but can also transform, b cells after transplant and may lead to lymphoma with a resulting ~ % mortality rate. the source of ebv is presumed to be passenger lymphocytes in the donor tissue and those at greatest risk are patients who are ebv naive before transplant. since most cf patients with endstage lung disease in the developed world are now being referred for transplant given the success of this intervention, we reviewed our entire lung transplant database to determine the nature of ebv infection and the likelihood of developing lymphoma following lung transplantation. of first-time lung transplants, ( %) had cf. ebv serology was not available in ( cf, others) before transplant. of ( %) cf patients were ebv seronegative before transplant as compared to of ( %) patients with other end stage lung diseases (composed mostly of copd and ipf) in the control population (chi square p < . ). the cf ebv seronegative cohort was (sd ) years old on average, an age when most seroconversion has already occurred in our society. of the cf ebv seronegatives, survived beyond months and were thus at risk for post transplant lymphoma. of these , developed lymphoma (incidence = %) in comparison to of the ebv seronegative controls (incidence = %, p = ns). all lymphomas were stage iv at presentation and the majority ( of ) arose in the first post transplant year. the cf ebv seronegatives who developed lymphoma did not differ from those who did not with regard to levels of immunosuppression (cyclosporine, methylprednisolone or prednisone) or doses of antiviral therapy (i.e., ganciclovir which is active against both cmv and ebv). in addition, the cessation of anti-lymphocyte antibody induction therapy in the summer of , somewhat surprisingly, had no impact on the development of lymphoma. two cases of lymphoma developed in cf ebv seropositives (incidence = . %) and no cases of lymphoma developed in the control ebv seropositives (p = ns). thus ebv seronegativity raises the lymphoma risk fold in cf patients (p < . ). two of the ( %) cases of lymphoma resulted in death (one cf and one other) and one cf patient probably died of complications related to chemotherapy even though he was tumor free at that time. lymphoma outcome was not stage or clonality dependent. in conclusion, cf lung transplant recipients are at an increased risk for lymphoma largely due to their lack of exposure to ebv before transplant. kissing more before transplant may lower the risk of lymphoma afterward. university hospital uzb, brussels, belgium; . chest medicine, erasme university hospital, brussels, belgium; . dermatology, erasme university hospital, brussels, belgium; . gynaecology, erasme university hospital, brussels, belgium; . gastroenterology, erasme university hospital, brussels, belgium; . pathology, erasme university hospital, brussels, belgium hpv infection is an underestimated phenomenon in organ transplantation (tx) recipients, being unable during immunosuppression (is) to mount an adequate anti-viral immune response and risking genital/anal warts (condylomata acuminata) as well as pre-cancerous/cancerous lesions. we retrospectively assessed the incidence/treatment of genital/anal hpv-associated lesions in cystic fibrosis (cf) lung tx recipients. the files of all tx patients transplanted in the ulb center between and / ( men/ women, median age ± . , range - yrs) were reviewed. maintenance is consisted in chronic triple therapy combining a calcineurin inhibitor, a cell cycle inhibitor and corticosteroids. median survival was . ± . months (range - ). ten of sexually active patients ( %) who survived ≥ months post-tx developed hpv-associated genital/anal lesions: men ( - yrs) and women ( - yrs). genital/anal hpv-pcr proven condylomata were diagnosed between - months in the men and between - months post-tx in the women. all underwent local treatment (cryotherapy ± laser ± topical imiquimod), / patients underwent multiple treatments under general anesthesia. recurrence of the condylomata was high. one male patient presented high-grade anal dysplasia and women moderate to high-grade cervical dysplasia; underwent conization and one complete hysterectomy after conizations. one of the females presented both condylomata and a high grade cervical dysplasia. these retrospective data in a cf population indicate that ) hpv infection may cause significant morbidity in young subjects with chronic disease needing organ tx. it may even compromise life expectancy after tx. ) data on the effectiveness of treatment strategies -including topical immunomodulators -have to be collected ) the potentially protective effects of the now available hpv vaccination in females and males? with chronic disease at risk for future organ tx, when administrated before sexual activity and before tx, should be rapidly evaluated in a multi-center effort. ballmann, m. ; pfister, e. ; schlueter, k. ; becker, t. ; melter, m. ; junge, s. . pediatric pulmonology and neonatology, medical school, hannover, germany; . pediatric nephrology and hepatology, medical school, hannover, germany; . visceral and transplant surgury, medical school, hannover, germany liver disease is an important comortality and comorbidity in cf patients even in the pediatric age group. liver transplantation (ltx) is an accepted option in end stage liver disease. nevertheless the clinical outcome in cf patients is still under discussion. here we reported the clinical outcome months after isolated liver tx in pediatric cf-patients.since n= ltx were performed in children (male n= , female n= ; age (mean±sd) , ± years) . immunosuppressive therapy was done with ciclosporin ± basilixumab and steroid. we followed pulmonary function, cn-score, nutritional status (bmi z-score) and body com-position, airway cultures and igg.results:all were still alive months after ltx. before ltx all patients had a mild to moderate pulmonary disease: fvc(%pred) , ± ( , %- ), fev (%pred) , ± , ( - ), mef (%pred) , ± , ( - ) , cn-score ± , points. nutritional status: bmi(mean) , ± , % (range: , %- , %, z-score: , ± , ), upper arm circumference(cm)(mean±sd) , ± , , triceps skin fat fold(cm) , ± , . body composition: , ± , % of bodyweight were fat. one year after ltx all patients were still in a stable pulmonary situation: fvc(%pred) , ± (range: , %- %), fev (%pred) , ± , (range: %- %), mef (%pred) , ± , (range: , %- %), cn-score , ± , points. there weren't any significant changes in the airway microbiology under immunosuppressive therapy (steroid, ciclosporin±basiliximab), the serum igg levels declined significant from , ± , g/l to , ± , /l(p< . ). the growth over the year was , ± cm and the increase of weight come to , ± , kg, while the bmi didn't increase in this first year after transplantation. certainly we found an increase of the body fat mass (to , ± , % of body weight), of the upper arm circumference (to ± , cm) und of the triceps skin fat fold (to , ± , cm) .conclusion:liver transplantation is an effective therapy even in children with cf related liver disease and stabilise pulmonary function and improves nutritional status in patients with cf and mild or moderate pulmonary involvement before ltx. (neglia jp et al., n engl j med ; : - ) , and colon cancer has been described in young adults with cf (chaun h et al., can j gastroenterol ; : - ) . three of lung transplant (ltx) recipients from our center developed colon cancer after successful bilateral lung transplant for end-stage lung disease. data from these individuals are given in table below . an additional recipients with cystic fibrosis were screened via colonoscopy. colonic polyps were detected in and included lesions up to cm in diameter. five years after a colonoscopy that had shown a mm polyp, one patient underwent a nd colonoscopy that revealed multiple polyps, including a cm diameter sessile polyp in the sigmoid colon. colonic malignancies appear to arise from mucosal polyps, and screening via colonoscopy can detect and remove premalignant lesions. lung transplant recipients have an increased risk of gi malignancies and tumor surveillance is impaired by post-transplant immunosuppression, colonoscopic screening should be considered for transplant recipients with cystic fibrosis. additionally, colonic malignancy should be considered as a potential cause of unexplained gastrointestinal symptoms such as constipation. nossent, g. ; kastelijn, e. ; teding van berkhout, f. ; zanen, p. ; van den bosch, j. ; van de graaf, e. . respiratory dis, university medical centre, utrecht, netherlands; . respiratory diseases, st. antonius hospital, nieuwegein, netherlands objective: in the netherlands lung allocation is based on waiting time. to prioritize patients with a poor prognosis and to reduce waiting list mortality the high urgency (hu) status was introduced in . to be considered for hu status there has to be a limited life expectancy on clinical judgement and all three transplantation centers have to audit this decision. the aim of this study is to analyze the potential effect of las on patients with cystic fibrosis (cf) who became hu or died on the waiting list. it would be expected that patients who became hu had a higher las. methods: from till in all cf patients placed on the hu waiting list, the median las ( th, th) was calculated at the moment of listing and at the moment the patients became hu. also for the cf patients who died on the waiting list the mean las (sd) was calculated at the moment of listing. results: from the patients on the waiting list patients had cf. of these patient were placed on hu list and died on the waiting list. in the cf patients that became hu the median las on the moment of listing was . ( . ; . ) . at the moment they were placed on the hu waiting list the median las was . ( . ; . )(p= . ) . the median las of the patients who died on the waitinglist was . ( . ) . this las did not differ significantly from the las of listing of the patients that became hu (p= . ). conclusion: the las does not detect the deterioration in patients with cf as diagnosed by clinical judgement. this may be due to the exclusion of specific prognostic factors of cf in the calculation of the las. besides that, the las does not differentiate cf patients on the waiting list with a high chance of mortality. introduction: lutx recipients have one of the highest rates of ia in solid organ transplantation causing high morbidity and mortality rates. nowadays antifungal prophylaxis is extensively used but clinical trials are rare. methods: all patients who received lutx in our hospital were included in this retrospective study. aspergillus airway colonization was defined as aspergillus cultured twice from airway specimens in the absence of ia or tracheobronchitis (tb). definition of ia and tb was according to literature. after the death of cf-patients due to ia, a prophylaxis protocol was introduced containing avoidance of use of the cell saver during the operation in pretx colonized patients to avoid hematological fungal spreading and targeted prophylaxis with itraconazole or voriconazole in all pre-and posttx colonized patients during months and inhaled amphotericin during hospital admission. results: a total of lutx were performed (in patients) ( % cf patients) from - - until - - . patients ( . %) were colonized with aspergillus pretx and patients posttx. only patients were colonized before and after tx. before introduction of the protocol patients ( / = . %) ( % cf patients) developed an aspergillus infection: ( . %) patients developed a. fumigatus tb and ( . %) patient suffered from ia: cerebral a. fumigatus infection (proven ia). two patients, both cf patients, ( / = %) died due toaspergillus infection (cerebral and tb).after introduction of the protocol only one a. fumigatus tb occurred ( / = . %) but no invasive fungal infection. conclusions: the rate of fungal infections in our lutx patients was comparable with data from literature. since introduction of azole prophylaxis protocol and avoidance of use of cellsaver no ia occurred. fischer, a. ; müllinger, b. ; arendt, t. ; bernhardt, t. ; hannah, k. ; scheuch, g. . activaero gmbh, gemuenden, germany; . biological products, bayer healthcare, leverkusen, germany; . activaero gmbh, gauting, germany; . talecris biotherapeutics, research triangle park, nc, usa background patient compliance during a study is an important factor in view of assessing the clinical effect of a treatment. this is especially true when patients administer the drug at home. usually, patient records, counting of returned doses or mechanical counters are used to track compliance, which may be biased by the study subjects. this report is about an aerosol study in cf which used a device for controlled breathing (akita® inhalation system, activaero, germany). the device works with a patient-individual smart card that records every single breath during a treatment including a date/time stamp in an encrypted manner. each patient's inhalation protocol can be displayed by loading the smart card into the proprietary "compliance manager" software. methods for this report, the data set of a recent controlled study (griese et al., ) was analyzed. cf-patients were instructed to inhale with the akita inhalation system at home for days. after the study, the smart cards were returned to activaero, and the inhalation records on the chip were analysed. we analysed the compliance of patients who participated at least days ( out of patients, others deemed to be drop-outs . most of the patients showed a dsc which is lower than the tdc, indicating that they had missing treatment days, which were compensated by additional inhalations on other days. we found patients with a dsc more than % lower than their tdc (max difference: . %). conclusion this aerosol study with home treatment demonstrated a high compliance rate for tdc and dsc. this information is more reliable for compliance than study participation in days alone. for future studies it is recommended to define in the protocol not only a minimum of study participation in days but also a minimum in tdc and dsc. in addition the compliance thresholds may be defined with regard to the drug's pharmacokinetic profile. in general, the inhalation record of the akita smart card provides an unbiased view of the inhalations treatments during a study especially when the subjects perform the inhalations at home. compliance calculations as shown above may be performed and linked to other outcomes of a study for validation. compliance data like these may also be used routinely by the treating physician in order to guide and supervise his patients. introduction: adaptive aerosol delivery (the i-neb ® aad ® system) has been developed to deliver precise, reproducible doses of aerosol into inhalation. i-neb utilizes a medication chamber, which incorporates a metering chamber to deliver a preset (metered) dose of aerosol. inhalation of hypertonic ( %) saline has been shown to aid mucociliary clearance in patients with cystic fibrosis. [ ] we performed an in vitro test to determine the emitted, delivered and residual doses of hypertonic saline from the i-neb aad system with . ml metering chamber, and a conventional jet nebulizer (lc plus ® ) with a ml fill. the results were then entered into a model to predict the likely lung dose from the in vitro tests, in order to determine the equivalent i-neb dose to the ml conventional jet nebulizer fill. methods:an i-neb device configured to operate at power level (of ) was fitted with a metering chamber designed to deliver a preset dose of . ml. the i-neb was weighed, loaded with ml of % saline and run on the cen simulated breathing pattern. this process was repeated using an lc plus nebulizer loaded with ml of % saline. aerosol was collected on a filter placed between a harvard pump and the device. emitted dose and residual dose were determined by gravimetric output, and delivered dose was determined by ion analysis. all tests were conducted in triplicate. results: as shown in table , the emitted dose approximated the delivered dose for i-neb, whereas for lc plus the emitted dose was approximately twice the delivered dose, due to the wastage of aerosol upon simulated exhalation. as i-neb has a delivered dose of mg, and i-neb has been shown to deposit . % of the dose in the lung, [ ] this would result in a lung dose of mg. the pari lc plus has a higher delivered dose, but the overall lung deposition is only % of the fill volume, i.e. mg. [ ] conclusion: the results of this test suggest that two treatments would be required in order to deliver an equivalent lung dose of hypertonic saline from an i-neb device fitted with a . ml metering chamber, compared with a ml fill in an lc plus jet nebulizer. references introduction: inhaled colistimethate sodium can be used for the eradication of pseudomonas aeruginosa in patients with cystic fibrosis. the tonicity of colistimethate sodium inhalation solution has been linked to the occurrence of bronchospasm in some patients. it has been suggested that this bronchospasm can be avoided by using an isotonic solution of colistimethate sodium. [ ] this can be complicated when using conventional jet nebulizers, since evaporation during nebulization tends to change the tonicity of the solution remaining in the nebulizer. however, because very little evaporation occurs within the i-neb medication chamber during nebulization, the tonicity of the loaded solution at the beginning and end of nebulization remains almost constant. aim: we investigated the effects of various saline concentrations upon the tonicity of two concentrations of colistimethate sodium solution ( . miu/ml and miu/ml). methods: diluent containing sodium chloride concentrations of: % (water for injection), . %, . %, . %, and . % (normal saline) were used to reconstitute vials of colistimethate sodium (promixin ® , profile pharma, uk). each vial was reconstituted with either or ml of diluent to make up miu/ml or . miu/ml colistimethate sodium solution, respectively. the tonicity of each diluent concentration/colistimethate sodium concentration combination was made up and tested in triplicate using an advanced micro osmometer (advanced instruments, norwood, usa). the results were plotted on a graph along with a line of best fit, to determine the diluent concentration that gave a mean tonicity closest to that of an isotonic solution. results: tonicity increased linearly with increasing concentrations of saline solution for both miu/ml and . miu/ml concentrations. the tonicity of the miu/ml solution was approximately mosm higher than the . miu/ml solution across the range of saline concentrations tested. the best fit line passed through the isotonic solution point ( mosm) closest to the . % saline concentration point. conclusions: the diluent that produces the closest approximation of isotonicity for the mean of . miu/ml and miu/ml colistimethate sodium concentrations was . % saline. references . transave, inc., monmouth junction, nj, usa; . activaero, gmbh, gauting, germany arikace™ is an inhalation formulation of a liposomal amikacin suspension that is designed to treat chronic pseudomonas aeruginosa infections in cystic fibrosis patients. liposome encapsulation of amikacin reduces nonspecific binding of this cationic aminoglycoside drug to the negatively charged mucus and biofilm surfaces and allows penetration and delivery of packets of highly concentrated drug to the otherwise protected bacteria. as nebulized and delivered to the lungs, arikace™ comprises % liposomal amikacin and % 'free' amikacin that is not entrapped by liposomes; free drug is produced by liposome leakage during nebulization. this profile provides an initial high peak concentration of amikacin followed by a sustained level as drug leaks from the liposomes. nebulized arikace™ with this profile was evaluated previously in human clinical studies using the lc star® nebulizer. although the . µm liposomes of arikace™ are efficiently loaded with drug, the expected effective dose in humans will require relatively large volumes of drug solution to be administered. to minimize treatment time and improve patient convenience we sought to find a nebulizer that would efficiently deposit drug and produce aerosol at a high output rate, while still producing the same level of free drug during nebulization. methods: nebulization of liposomal amikacin was compared using several nebulizers, including jet nebulizers: lc star® and lc sprint® (both from pari), and electronic mesh nebulizers: microair® ne u v (omron), aeronebgo® (nektar), and eflow® (pari). additionally, meshes of different pore sizes were examined for the microair® and eflow® devices. output rates were measured by gravimetric differences. droplet size distribution was assessed by a laser light scattering method and by cascade impaction; mass median aerodynamic diameters (mmad), geometric standard deviation (gsd), and fine particle fraction (fpf) (i.e., % droplet mass < µm). amikacin association with the liposomes was measured using a centrifugation-filtration assay. results: in terms of output rate, the order of performance was eflow® (~ . g/min) > lc sprint® > lc star® > aeronebgo® > microair®. while the aeronebgo® and microair® mesh nebulizers performed well with saline, their performance declined dramatically when nebulizing the liposomal amikacin solution, and, in fact, the microair device clogged. in additional studies with the eflow® where different meshes were compared, the l mesh was selected as it provided ideal aerosol properties (mmad = . µm, gsd = . , fpf > %) with a ratio of entrapped to free amikacin of approximately %/ %. importantly, from in vitro breath simulation studies it is estimated that about twice of the nominal drug dose is expected to deposit in the lungs compared to the lc star®. conclusions: for nebulization of liposomal amikacin, the eflow® configured with a l mesh provided optimum aerosol characteristics with a high output rate and ideal size for distribution throughout the lung. the eflow® reduces dose administration time not only because of its faster output rate but also because its higher deposition factor greatly reduces the amount of drug needed. zeman, k.l.; bennett, w.d. cemalb, university of north carolina, chapel hill, nc, usa many inhaled, therapeutic drugs have their site of action in the conducting airways, but may deposit elsewhere in the respiratory tract, resulting in unwanted side effects and waste. the quantity and location of particle deposition in the respiratory tract depends on both the particles' aerodynamic size and breathing pattern. in subjects with normal lung function, we evaluated three methodologies for their ability to deliver radiolabeled particles ( mtc-labeled sulfur colloid) to the conducting airways: . inhalation of µm mmad particles (devilbiss jet nebulizer) using a resting tidal volume and flow (mean . l and . lps); . inhalation from the same nebulizer using a very large inspiratory capacity volume and higher flow rate (mean . l and . lps); and . inhalation of larger . µm mmad (heart jet nebulizer) particles with a large tidal volume and very low flow rate (mean . l and . lps). gamma scintigraphy gave an estimate of % conducting airway deposition (%cad) by multiplying the percent of activity in the lungs immediately post-deposition relative to the total deposition (i.e. lung+mouth +esophagus+stomach) times the percent of activity cleared from the lungs over hours. %cad was %, %, and % for the three methods, respectively, significantly greater for the large particle, very slow inhalation technique when compared to the other two methods. the amount deposited in the mouth and larynx was %, and %. in addition, we evaluated protocols and above in a preliminary group of cf patients. for protocol , mean inhaled volume and rate was . l and . lps. mean inhaled volume and flow were . l and . lps for protocol . the %cad for the two protocols and were % and %, respectively. the amount deposited in the mouth and larynx was % and %. higher therapeutic value of a medication delivered to the airways where the primary defect is associated with cf disease, and with fewer losses to the extrathoracic airways, can be obtained by using a "very large particle/slow inhaled flow" routine when compared to normal or higher flow breathing associated with typical nebulizers. supported by cf foundation. positive effects of physical conditioning in cystic fibrosis (cf) have been reported for quality of life (qol) in addition to effectis on fitness and lung functions. the objective of this study was to identify the effects of different modes of training on qol in cf. cf-patients (age - yr., fev > %pred.) were randomized into groups: germany: unsupervised homebased training, hours per week, free choice of training mode and means (ut, n= ) and controls (con , n= ); switzerland: aerobic training (at, n= ) or weight training (wt, n= ) in a fitness center, x min per week, or controls (con , n= ). subjects in the training groups were asked to train for months. at study entry and after and months, qol was assessed by questionnaire and maximal physical working capacity (pwc, %pred) was determined during cycle ergometry (godfrey protocol). changes from baseline values were calculated and anova for repeated measures was used to test for differences among groups. weight training resulted in a significant decrease in qol (generic dimension) at and months compared with all other training modes including controls. there was no significant difference between at or ut and controls in qol during the program. however, there was a positive association between the change in pwc and the change in qol (r= . , p< . ) in the entire sample. in conclusion, physical training may not always result in an increase in ql in cf. possibly, the decrease in qol in the wt group may have resulted from tiredness induced by the training. thus, a different outcome might have been found with less intense training. positive effects may be achieved by improving aerobic fitness. supported introduction: exercise and exercise training programs are felt to be important to improve aerobic exercise capacity, muscle strength, lung function, and quality of life in cf-patients. recent studies in children and adults with cf have demonstrated an increase of motor performance after exercise training. less is known about trainability of motor performance in younger children with cf. the aim of this study was to assess the effects of a training program on motor performance in preschool children with cf. methods: preschool children with cf ( female and male patients, mean age . yrs; range - yrs, fev %pred. , ± . rage from . - . ) participated. at baseline (t ) and at the end of the training program (t ) the "motorik test (mot)" was used to assess motor performance and to evaluate training effects. components of motor performance tested with the mot are balance, agility and flexibility, strength, coordination, fine motor skills, reactivity, accuracy of movement). lung function in patients with cf was measured by spirometric techniques. the duration of training was - weeks ( times/week, minutes per session) with different kinds of sport activities. results: at t , strength and balance had increased significantly (p< . ). no other components of the mot increased, but the increases in strength and balance were enough to cause a significant (p< . ) increase overall motor performance, as indicated by an increase of . % in mq. the mq is the sum of all test-items of the mot. no correlation was found between lung function and motor performance. discussion: to our knowledge this was the first study that examined the effects of an exercise training program on motor performance in preschool children with cf. main findings of this study were that a training program of - week of duration improved some aspects of motor performance. the improvements may be explained by different kinds of sports activities during training when compared with activities before training. classification of motor performance of the tested children was normal (mq = . ± . ). this could be the reason that only some aspects of motor performance increased. parents of the children were asked about habitual physical activity at home. all children were physically active and some of the children participated in organized sports. a recent study showed that with an increase in age lower motor performance in children and adolescent with cf was seen compared to healthy children. we speculate that time spent in physical activity in preschool children with cf is comparable to healthy children. conclusion: an exercise program offered to preschool-children with cf may lead to an improvement in motor performance. pre-school children with cf have normal motor performance. to prevent a decline of motor performance, exercise programs should be available for preschool children. gruber, w. ; braumann, k.m. ; orenstein, d.m. ; huels, g. . dept. for sports and exercise, clinic sattelduene for children and adolescent, nebel/amrum, germany; . institute for sport and exercise medicine, university of hamburg, hamburg, germany; . school of medicine, university of pittsburgh, pittsburgh, pa, usa; . dept. of medicine, clinic sattelduene for children and adolescent, nebel/amrum, germany introduction: a decline in physical activity with age is one of the main problems in patients with cf. as a consequence, lower exercise capacity and a more rapid decline in lung function may be seen in those patients who are physical inactive. furthermore, motor performance is lower in children and adolescents with cf compared to healthy children of the same age. little attention has been paid to younger children with cf and motor performance. the aim of the study was to compare motor performance in preschool-children with cf with healthy children of the same age. methods: children with cf (mean age . yrs; range - yrs) and healthy children (mean age . yrs; range - yrs) participated. the "motorik test" (mot) was used to assess motor performance. the mot consisted of test-items which evaluated seven different components of motor performance (balance, agility and flexibility, strength, coordination, fine motor skills, reactivity, accuracy of movement). lung function in patients with cf was measured by spirometric techniques results: results of the mot showed no difference either for single testitems or for parameter "motorische quotient (mq)" (p> . ). this parameter classified motor performance of all aspects tested with the mot. classification of children with cf and of healthy children was normal. mean value for parameter mq (cf: . ± . vs. healthy . ± . ) was slight higher in patients with cf than in healthy children (p> . ). differences between healthy and cf for different components of motor performance were not significant (p< . ) discussion: the mot is a reliable and valid test method in germany to determine motor performance in younger children. to our knowledge this was the first study to examine motor performance in preschool-children with cf. furthermore, we compared patients with cf and healthy children. we observed no differences between groups for motor performance. in older children with cf, lower motor performance has previously been found compared to healthy children of the same age. we do not have any information about the level of habitual activity in tested healthy children. we therefore speculate that time spent in physical activity in cf is similar to that of healthy children at this age. a recent study showed a decline in physical activity in cf when age and problems during physical activity increased. we think that motor performance is normal in children with cf up to an age of yrs. when age increased, time spent in physical activity decreases (treatment, school) and therefore motor performance decreases. conclusion: motor performance is normal in preschool-children with cf compared to healthy children. to prevent a decline or to maintain motor performance it is recommended to motivate cf-children from early infancy to participate in sports activities or other physical activities to maintain or to increase their physical fitness. . respiratory medicine, the hospital for sick children, toronto, on, canada; . respirology, st. michael's hospital, toronto, on, canada; . respiratory medicine, montreal children's hospital, montreal, qc, canada purpose: the habitual activity estimation scale (haes) questionnaire has been shown to be a feasible tool to measure physical activity however the reliability has yet to be determined in cf populations. we therefore assessed the reliability and validity of the haes questionnaire in paediatric and adult cystic fibrosis (cf) populations. methods: fourteen ( male, female) patients aged . + . yrs with cystic fibrosis from the cf clinics at the hospital for sick children and st. michael's hospital participated in this study. participants were clinically stable at the time of the study (fev > % predicted) and participating in their 'habitual' physical activity. to assess test-retest reliability, patients completed each of the haes (schneiderman-walker et al., j pediatr. ; ( ): - .), and a validated -day activity diary (bratteby et al., eur j clin nutr. ; ( ) : - ), and wore an actigraphtm tri-axial accelerometer for two consecutive weeks. validity was assessed by comparing the activity results of each of the three instruments over a single week time period. results: intra-class correlation coefficient estimates of reliability for the haes, diary and accelerometer were . (p< . ), . (p< . ), . (p< . ), respectively. validity was estimated by comparing the results from each instrument using a one-way anova with block design. there were no overall differences among the participants' activity results as estimated by the haes, diary and accelerometer. furthermore, there were no significant differences between activity measures among the instruments when broken into morning, afternoon, or evening periods, or between measures from weekday or weekend days. while there were no significant differences among the instruments when recording 'very active' intensity levels, they did detect differences during the inactive / somewhat inactive (p = . ) and somewhat active (p = . ) intensity levels. conclusion: the findings of this study suggest that the haes questionnaire is a reliable and valid instrument that can be used to assess activity levels in patients with cystic fibrosis. in agreement with others, our patients found it easiest to recall and record accurately those periods in which they are participating in vigorous activity. acknowledgement: this research was supported by the canadian cystic fibrosis foundation. . respiratory medicine, hospital for sick children, toronto, on, canada; . respiratory medicine, montreal children's hospital, montreal, qc, canada; . respirology, st. michael's hospital, toronto, on, canada objective: previous work showed a relationship between habitual physical activity (hpa) and decline of lung function (fev ) in cf females (sneiderman-walker, j peds : , ) . this study only included a small number of patients and the follow-up period was limited to months. the purpose of this study was to evaluate daily activity levels and decline in fev in a larger cohort over a longer period of time. methods: a total of (n= female) patients with cf (age - yrs) were studied over a six year period at the hospital for sick children, st. michael's hospital and montreal children's hospital. at each quarterly cf clinic visit, patients performed pulmonary function tests to determine fev and completed the habitual activity estimation scale (haes). weekday total activity (wdta), expressed as hours/day (h/day), was calculated from the sum of 'very active' (wdva) and 'somewhat active' (wdsa) categories as previously described. a repeated measures linear regression was performed to evaluate the relationship of fev and hpa over time. results: at baseline, mean fev was % predicted (range - % pred). mean baseline wdta was . h/d (wdva = . h/d and wdsa = . h/d) similar to that previously reported. subjects were divided into high and low activity groups based on the group median wdta. those in the low activity group had a significantly (p= . ) steeper rate of decline (- . % predicted per year) compared to those in the high activity group (- . % predicted per year) . unlike in the in the previous study this difference was observed in males as well as females. conclusions: six year follow-up of this patient cohort has shown that higher activity levels are clearly associated with a slower rate of decline of fev . we are currently evaluating whether strategies aimed at increasing physical activity will have an impact on lung function decline in cf patients. background: knowledge of exercise principles, benefits, and techniques has not been assessed in children with cystic fibrosis (cf) or their parents. regular exercise is a recommended cf treatment regimen that is often not followed, but one which might improve cardiovascular fitness, lung function, sputum clearance, and health-related quality of life. further, aerobic fitness, measured as peak oxygen uptake (vo ), is positively related to survival in cf. construction of a reliable and valid measure of exercise knowledge is necessary to assess level of knowledge and study its relationship to exercise behavior. the aim of this study was to develop and test a measure of exercise knowledge for pediatric patients with cf and their parents. methods: we constructed a -item exercise knowledge test (ekt) and then examined its psychometric properties. a comprehensive review of the exercise literature guided the formulation of objectives and development of items. to establish content validity, a panel of experts (cf physician, two exercise physiologists, and a clinical cf nurse) evaluated the objectives and test items for relevance, clarity, and accuracy. after feedback from the panel, we revised the ekt through an iterative process. in consultation with a reading level expert, we wrote the ekt items so that the test could be administered to children and adults. a measurement and evaluation expert also reviewed the test, and additional revisions were made. during routine clinic visits, the ekt was administered to subjects with cf and parents. the cf subjects included boys and girls, age to years, fev range: - % predicted. the parents had an average age of years and most (n= ) were female. results: the ekt includes items, true-false and multiple choice. it is self-administered, yields a single summed score, and has a flesch-kincaid rd grade reading level. the means were . + . ( % correct) and . + . ( % correct) for the cf subjects and parents, respectively; % of the parents scored between and . internal consistency reliability estimates were . for the children and . for the parents, reflecting both the multidimensionality of the material and the lack of variability in the parent scores. item difficulty ranged from . to . for the cf subjects and from . to . for the parents. in both groups, the discrimination values were positive for all items with difficulty values less than . . conclusions: we were able to develop a measure of exercise knowledge with an acceptable reading level for children with cf and their parents. this sample of cf subjects and parents showed substantial exercise knowledge. the ekt can be a useful tool to identify gaps in exercise knowledge in cf patients and their parents, develop educational programs to increase knowledge, and examine how knowledge relates to exercise behavior in children with cf. supported by cystic fibrosis foundation therapeutics, inc. purpose: although physical activity is routinely recommended for adolescents with cystic fibrosis, past research suggests that many patients do not regularly participate in physical activity. there may be a variety of reasons, both related to disease and otherwise, for this inconsistent participation. in this study, qualitative methods were used to describe factors that facilitated or served as barriers to physical activity in adolescents with cf and compared these factors to those in a healthy peer group. methods: ten subjects with cf and ten subjects without cf aged - volunteered for this study. each subject participated in two, open-ended semi-structured interviews with - weeks between each interview. subjects were questioned about their current and past physical activity as well as the benefits and barriers to participation. the adolescents with cf were also asked about their perceptions of physical activity on their own health and other adolescents with cf. interviews were transcribed and coded by three investigator groups. analysis was conducted using the grounded theory approach at two points in the study, following each interview and again at the completion of all interviews. at the first point, individual interviews were coded using line-by-line coding, and similar information was grouped into categories. in the second interview, categories were confirmed and subjects were also asked to elaborate or clarify points from the first interview. interviews were continued until themes reached a saturation point. upon completion of all interviews, data for each group were organized by question. data categories appearing throughout the interviews became major themes describing facilitators and barriers to exercise. the major themes were identified by the three researcher groups and then through collaborative group analysis. results: the central theme for both groups was perceived importance of physical activity for health benefits. for the cf group, facilitators and barriers were both psychological and physical. physical benefits were categorized as either relating to their general health (such as feeling energized) or as benefiting their disease (such as improving breathing). in the non-cf group, facilitators included social factors in addition to general health-related and psychological benefits. in the non-cf group, barriers were categorized as either internal or external, with the internal barriers being similar to the cf group (physical and psychological), however, the non-cf participants also indicated external barriers (such as time) not articulated by the cf group. conclusions: in general, we found very similar facilitators and barriers to physical activity in adolescents with cf and those without. however, the adolescents with cf strongly indicated that physical activity would have positive effects on their physical health. further work is needed to determine ways to accentuate facilitators and remove barriers to improve physical activity levels in adolescents with cf. this project was supported by a research grant from the cardiovascular & pulmonary section, apta. lloyd, e. ; dodd, m. . paediatric cystic fibrosis service, central manchester and manchester children's university hospital, manchester, united kingdom; . adult cystic fibrosis centre, wythenshawe hospital, university hospital of south manchester nhs foundation trust, manchester, united kingdom background: training in the skills of communication and self care are considered important components of transitional care . physiotherapists work to involve families and young people to encourage this independence. the cf manager training programme (cfmtp) is a recognised tool for measuring age appropriate tasks in cf care. aim: the aim of the study was to survey the current levels of autonomy in all patients with cystic fibrosis in the north west of england from birth to years to provide a baseline measure for encouraging autonomy in preparation for transition. method: three areas of the cfmtp were selected for the survey: physiotherapy (p), inhalation therapy ( it) and communication (c) for the age groups: - . , . - , - , - , - , - , - , - , - ,and + years. physiotherapists from all the north west cf clinics were invited to assess all the patients under their care. data collected were age, gender and whether the patients skills fell "below", "same" or "above" the age appropriate self management tasks as described in the cfmtp. 'not applicable' was indicated where the skills did not apply. results: / clinics responded. out of a total of patients in the nw were assessed, three patients were excluded. population by clinic ranged from - . population by age category: ( - . years) ( . - years) ( - years) ( - years) ( - years) ( - years) ( - years) ( - years) ( - years and ( + years). / ( %) were male. for p (n= ), it (n= ), c (n= ), %, %, % = below, %, %, % = same, %, %, % = above respectively. there were no gender differences. conclusions: overall scores revealed that / rd of patients were gaining age appropriate independence in all areas. there was the least autonomy with it. it was rare for a child to be setting up and cleaning equipment regularly at - years, but this continued throughout the adolescent years. overall % were 'below' for p and it; this was highest after the age of years. autonomy with physiotherapy appeared slow to develop. some categories in cfmtp were different from our practice for e.g. patients are not invited to be seen alone in our clinics until years v - years and this may account for the lower scores in communication below this age. this study has highlighted that some of our patients are not fully independent in the time up to transfer. we need to encourage independence from an early age with all aspects of care and integrate the process into our routine clinical practice. refs mcdonagh and viner bmj ; : - , . parents' guide to the cf manager training programme. children's hospital of eastern ontario planner, s. ; morrison, l. . physiotherapy, gartnavel general hospital, glasgow, united kingdom; cystic fibrosis (cf) causes a deterioration in lung function and exercise tolerance. assessing exercise capacity is therefore essential in order to monitor changes in disease severity. the -minute walk test ( mwt) and minute step test ( mst) are validated and widely used outcome measures for identifying exercise capacity in patients with cf. exercise testing is part of annual review of all patients in the west of scotland. previous studies identified that the mst was a sensitive measure of exercise tolerance, but did not successfully challenge patients with mild or moderate lung disease as defined by fev(sub) (/sub)> % (morrison ) . further analysis of the mst, where step height was increased to inches did not show any difference in sao(sub) (/sub). decline or rate of perceived exertion (rpe) in patients with mild disease. the chester step test (cst) was developed to assess aerobic fitness which is used in the uk cardiac population for exercise prescription (sykes ) . it is a minute progressive, sub-maximal test with a variable step height ( - inches) , making it suitable for those with a wide range of exercise capacities. in addition, cst results extrapolate to a mets value (oxygen utilisation: met = rate of oxygen utilisation at rest) as an outcome measure which can facilitate exercise prescription based on established values for physical activities. we aim to determine whether the cst is a more sensitive measure of exercise capacity than the mwt or mst when considering decrease in sao(sub) (/sub) and rpe. patients ( aged - mean fev(sub) (/sub) . l) with mild-moderate cf performed the cst and mwt and results were correlated with fev(sub) (/sub). decrease in sao(sub) (/sub), increase in hr and rpe scores were also compared between the cst, mwt and recent mst data. patients were exercised to a symptom limited maximum which differed from the cardiac cst where patients are exercised to % of maximal hr. the cst appears to be more challenging as only patients completed it, all with an fev(sub) (/sub)of > % predicted, whereas in the mst and the mwt all patients completed the test. mets levels for the cst were weakly correlated with fev(sub) (/sub)(r= . ) and the mwt (r= . ) . this could be explained by the narrow range of mets data available or the size of the sample population. there were significantly greater rises in hr (p= . , p= . ), decrease in sao(sub) (/sub) (p= . , p= . ) and rpe (p= . , p= . ) , between the cst, mst and mwt respectively, suggesting that the cst is a more sensitive measure of cardiorespiratory effort. conclusions the cst appears to be a more challenging exercise test for those with mild to moderate disease and therefore may be a better reflection of exercise capabilities. cst results are useful for prescribing exercise and monitoring programmes appropriately for this patient group. further study could determine whether the cst is also suitable for those with severe disease. lee, a.l. ; button, b. ; holdsworth, m. ; holland, a. . physiotherapy, the alfred, melbourne, vic, australia; . latrobe university, melbourne, vic, australia musculoskeletal pain is prevalent in cystic fibrosis (cf). while the chest and back are frequently reported regions of acute or chronic pain, it is unclear if pain in these areas is related to disease severity or respiratory symptoms. manual therapy and postural advice has been shown to alleviate pain, but the effects of a combined approach of musculoskeletal physiotherapy and massage has not been studied. the aim of this study was to identify the primary regions of musculoskeletal pain, the relationship between pain severity and respiratory symptoms and to examine the effect of musculoskeletal physiotherapy techniques and soft tissue therapy, including remedial massage on pain and ease of breathing (eob) in adults with cf. one hundred and twenty-nine adults with cf ( with acute exacerbation, post lung transplant) aged ± years (mean ±sd) with fev(sub) (/sub) of ± % participated in this study. following assessment of primary pain regions, each subject underwent a single treatment session including spinal joint / intercostal mobilisation and soft tissue therapy. pain and eob on a visual analogue scale were measured before and after treatment. changes were compared using paired-samples t-tests. pain was frequently reported in the thoracic spine region ( % of subjects), followed by the shoulder region ( %), cervical spine region ( %) and chest wall region ( %). equivalent ratings of pain and eob prior to treatment were reported for all regions of pain. eob rating prior to treatment were worse in those with low bmi (r=- . , p= . ) and low fev(sub) (/sub) (r=- . , p= . ). a single treatment session was associated with reduction in pain ( %ci . to . , p< . ) and improvement in eob ( %ci . to . , p< . ), irrespective of clinical status. improvement in pain was equivalent for all primary pain regions. however, greater improvement in eob was evident in subjects with shoulder pain compared to other regions ( %ci - . to - . , p= . ). a combination of musculoskeletal physiotherapy techniques and soft tissue therapy reduces musculoskeletal pain and improves eob in adults with stable cf, during an acute exacerbation and post lung transplantation. service cystic fibrosis is characterized by an excessive production of airway secretions responsible for bronchial obstruction and recurrent infections of the respiratory tract. the transport by ciliary activity and cough may be dependent on the unsteady rheological properties of the respiratory mucus such as thixotropy and shear-thinning properties which correspond to a decrease of viscosity with time or flow rate, respectively. we have previously shown that respiratory mucus with high shear-thinning and thixotropic properties is better transported by the cough mechanism (zahm et al, eur. respir. j., ) . to promote mucus clearance and to finally expectorate the mucus by a cough manoeuvre, an innovative device, the airhelp, has been developed and aimed to improve chest physiotherapy effectiveness. during a prolonged expiratory manoeuvre, the airhelp delivers negative pressure variations (maximal amplitude: - kpa) at an oscillatory frequency of hz. the aim of the present work was to test the effect of the airhelp device on the in vitro clearance of mucus by airflow. a plexiglass tube of mm in diameter and mm in length has been connected by one part on the airhelp and by the other part on a l thank mimicking the lung compliance. a µl drop of mucus simulant at different concentrations (viscogum at . % or . % with actigum at . % to . %) or of respiratory mucus, was deposited within the plastic tube and the distance travelled by the sample under a sec-airflow was measured. twelve respiratory mucus samples collected from cf patients were analyzed and the viscosity of the samples was measured before and after the airhelp experiment. we observed a significant (p < . ) and negative relationship (r = - . ) between the viscosity of mucus simulants and oscillatory airflow transport: the lower the viscosity, the higher the mucus transport by oscillatory airflow. using cf respiratory mucus samples, we observed that without the oscillatory airflow, no transport occurred, whereas when the samples were submitted to the oscillatory airflow, a significant increase in mucus transportability was measured. in addition, the viscosity of the cf respiratory mucus samples was significantly decreased after oscillatory airflow treatment ( ± pa.s to ± pa.s, p < . ). these results demonstrate that the mucus transport efficiency by an oscillatory airflow is dependent on mucus viscosity and that the improvement in mucus transport is related to the thixotropic and shear thinning properties of the airway mucus. we conclude that the airhelp device may improve chest physiotherapy effectiveness. potter, e. ; nufer, j. ; cullina, j. , ; quinton, h. ; jain, m. , ; mccolley, s.a. , . northwestern university feinberg school of medicine, chicago, il, usa; . children's memorial hospital, chicago, il, usa; lebanon, nh, usa; . northwestern memorial hospital, chicago, il, usa socioeconomic status (ses) significantly impacts health outcomes in cystic fibrosis (cf). the cystic fibrosis foundation (cff) is currently using median income estimated by zip code from the us census of the population for ses risk adjustment of data reported publicly from the cff patient registry. median income estimated by zip code is an "ecologic" variable that may misclassify patient and family income. in order to more fully assess the impact of ses on cf health outcomes, the cff began to ask centers to collect data on household income directly from patients and families. these data were captured for . % of patients and families in and . % in in the national registry. we report a method of collecting ses data from children and adults implemented at the children's memorial hospital and northwestern university cystic fibrosis center, and compare directly collected income to income based on zip code. methods: a short survey with the questions on household composition, income, and education level from the cff patient registry was developed. a letter from the center director and adult program director was mailed to patients and families informing them of the survey's purpose and confidentiality measures, i.e. responses would be seen only by the individual entering the data and identified by cff id number. the survey was distributed to cf patients and families in clinic by clerical staff. completed surveys were placed in an envelope and sealed by the patient or family. both directly collected income and income from zip code was classified into groups (<$ k, - k, - k, - k, - k, - k, - k, - k, and > k) . percent concordance and spearman's rank correlation were calculated. data were used for patients who provided data in ; otherwise, data were used. each patient's data was used only once in the analysis. results: unique patients were identified, pediatric and adult patients. eighteen percent of pediatric and % of adult patients preferred not to answer income questions or did not complete the survey. the mean income by zip code was $ k for patients who did not report income data and $ k for the entire group. for the responses, reported income data was moderately correlated with income by zip code (rho= . , p< . ). concordance between the categories was %. the correlation was stronger for pediatric patients (rho= . , p< . ) than for adults (rho= . , p= . ). re-classifying income as "low", "medium" and "high" (<$ k, $ - k, >$ ) led to a concordance of %. conclusion: survey methods that protect patient and family confidentiality result in good return rates of full ses data from patients and families. zip code derived median income shows moderate concordance with reported income. comparing the associations of the forms of income data with important cf outcomes needs to be done in a larger data set. we acknowledge the cystic fibrosis foundation, gerald o'connor, kathryn sabadosa, and participating patients and families. the patient registry provided a unique opportunity to assess changes in the epidemiology of respiratory pathogens in cf. while the incidence of p. aeruginosa remained stable, the prevalence decreased, suggestive of the impact of antibiotic eradication strategies. in contrast, the increasing incidence and prevalence of s. aureus and mrsa may reflect improved microbiological surveillance and laboratory techniques. the trends noted for b. cepacia complex may reflect successful implementation of infection control strategies. future studies are needed to better define the association between these observed trends and improving care for cf patients. vanderwyden, a. , ; drumm, m.l. , ; schluchter, m. . pediatrics, case western reserve university, cleveland, oh, usa; . epidemiology and biostatistics, case western reserve university, cleveland, oh, usa; . genetics, case western reserve university, cleveland, oh, usa in the genetic modifier study (gms) of lung disease of cystic fibrosis (cf), patients, homozygous for the ∆f mutation, were classified as having either severe or mild lung disease, as defined by the lowest or highest quartile of forced expiratory volume in one second (fev ) and a linear model was fit from these patients' longitudinal data (drumm , schluchter . while these models successfully dichotomized patients for the association study, we assessed whether a single slope model could be improved. here we have begun to compare models with one, two and three slopes, each representing different age ranges, for their use in this type of association study. as an example, interleukin- (il- ), a neutrophil chemoattractant, associates with the severe lung inflammation seen in cf patients. we have observed the tt genotype of the il- snp rs , correlated with severe pulmonary function in the gms sample of patients, relative to the aa and at genotypes of this snp. in a replicate study of patients, with cftr genotypes associated with exocrine insuffiency, longitudinal linear mixed models were used to further characterize the decline in lung function over time as associated with the il- snp, rs . three linear regression models of fev % predicted of patients were fit over age and compared with likelihood ratio tests. the three models differed with respect to the number of nodes. the first model had a single slope while the second and the third were piecewise linear models, with nodes at age , and ages and , respectively. age was chosen as the first node, as by this age most cf patients have reached puberty, an event believed to coincide with changes in pulmonary function. the second node, age , was arbitrarily chosen as a point in the third decade, as survivor effect becomes a significant issue during this period. when compared to the single slope and two slope models, the three slope model with nodes at age and age better explained the pulmonary function of this study (p< . , - loglikelihood chi-squared tests). when the explanatory variable, il- rs genotype, was added, the single slope model and the three slope model were again compared. the three slope with nodes at age and age again resulted in a better fit of fev % predicted over age as correlated with rs genotype (p< . , - loglikelihood chi-squared test). estimates of fev % predicted at ages , , , and for each rs genotype (aa, at/ta, or tt) were determined using the three slope model (p< . for all estimates). other explanatory variables, like gender and survival (p< . ), were added to further elucidate the relation between il- genotype and lung disease phenotype. we propose to use this piecewise three slope model with nodes at age and age to look at the pulmonary function of patients in the cf foundation registry data to better explain the decline in fev % predicted over time seen in cf patients. (supported by hl- and grants from the cf foundation) stephenson, a.l. purpose: the purpose of this study is to determine the annual incidence of hospitalizations for individuals with cf living in ontario and to examine predictors of hospitalization, specifically gender. methods: this is a retrospective cohort study from to , using newly linked clinical and administrative databases. the canadian cf foundation patient data registry (cpdr), contains detailed clinical information on all cf patients receiving care at one of accredited cf centres across canada. the institute for clinical evaluative sciences (ices), holds linked administrative databases containing information on all publicly reimbursed health care services delivered in ontario. canadian institute for health information (cihi) discharge abstracts database (dad), ontario health insurance plan (ohip), and homecare databases were used for this study. the cpdr was probabilistically linked (automatch software) to the ices administrative database using name, gender and date of birth. all individuals were then linked to the cihi dad, ohip, and homecare databases using unique numeric identifiers. with these databases, longitudinal records for each individual were created. cihi-dad, ohip, and homecare claims data were used to calculate the number of hospitalizations per year for pulmonary infections. males and females were analyzed separately to elucidate any gender disparities. other predictor variables include age, geographical location, number of clinic visits, socioeconomic status (income quintile) as well as clinical variables such as pulmonary function tests, nutritional status, diabetes mellitus, sputum bacteriology and pancreatic status. results: the cpdr contained data on individuals with cf followed within canada between - . of those individuals, could be found within the ices administrative databases using probabilistic linkage. of those individuals matched to the administrative databases, ( . % female) had hospitalizations. a total of , ohip claims were made during this -year period which represents any encounter between an individual with cf and a physician in ontario. multivariable analysis of hospitalization predictors is in progress. zhang, z.; lai, h. uw-madison, madison, wi, usa the cff practice guidelines recommend adjusting for genetic potential when evaluating height status in children with cf. however, calculation of adjusted height percentile is not recommended due to the complexity of method involved in such calculation. instead, a simple method to estimate a target height based on mid parental height was recommended. however, this target height includes a -cm confidence interval, which spans most of the channels on the growth chart. therefore, a child's unadjusted height may be considerably below the target height, yet remains above the lower bound of the target height range. in addition, there is a paucity of data on the associations between adjusted height to lung disease outcomes to justify whether adjustment for genetic potential is necessary. the objective of this study is to compare two methods of incorporating genetic potential in identifying short stature, namely, the cff's target method and the method developed by himes et al (pediatr ; : - ) , which applies positive adjustments to children with short parents and negative adjustments to those with tall parents. data from - cff registry were utilized. a total of children born between - who had complete height data from age - years and their parental height data were analyzed. short stature was defined by unadjusted height < th percentile, unadjusted height below the lower bound of cff's target height, or himes' adjusted height < th percentile. our results showed that adjusted height percentiles are consistently lower than unadjusted height percentiles, with an average difference of . z-score unit between ages - years and . z-score unit between ages - . proportionately more children were classified as short stature based on himes method compared to unadjusted height ( % vs. %, p = . ). in contrast, proportionately fewer children were below the lower bound of cff's target height ( %) compared to unadjusted height < th percentile ( %), p < . . among children with average parents, unadjusted and adjusted height percentiles were similar. however, among children with short parents (< th percentile), the percentage children with of short stature decreased from % with unadjusted height to % with himes' adjusted height and % with cff's target height method. among children with tall parents (> th percentile), the percentage of short stature increased from % with unadjusted height to % with himes method and % with cff's target height method. taken together, these results demonstrate that, without incorporating genetic height potential, - times more cf children who have short parents would be classified as short stature, while times fewer cf children who have tall parents would be classified as short stature. further analyses show that, compared to unadjusted height percentile, himes adjusted height percentile is more sensitive to, and has stronger association with, percent predicted fev- . in conclusion, our findings provide evidence that genetic potential should be incorporated in evaluating short statue in cf children, particularly for children with short or tall parents. the cff's target height method and himes method differ significantly in identifying short stature in cf children with short parents. cohort study of individuals in the cff patient registry from - . all individuals years of age or older and diagnosed before age were included. the new, persistent mrsa infection cohort was defined by having at least two cultures negative for mrsa over a two-year lead-in period followed by at least two mrsa positive cultures. individuals with only one mrsa culture (transient infection) were excluded. we developed multiple linear regression models using generalized estimating equations to assess the effect of mrsa on fev %predicted (%fev ) and rate of change of %fev . results: during the study period , individuals cultured mrsa. of these, , ( %) cultured mrsa only once (transient infection) and were excluded from subsequent analysis. , individuals met the criteria for new and persistent mrsa infection. , met the criteria for never having mrsa resulting in a total study cohort of , . participants were followed for a median of years. the median time to first isolation of mrsa in the persistent mrsa cohort was . years and the mean number of positive mrsa cultures per patient was . . a comparison of baseline characteristics between the persistent mrsa and never mrsa cohorts revealed the mrsa cohort to be younger ( . vs . years p< . ), equivalent in lung function (%fev . % vs . % p=ns), and with higher likelihood of methicillin sensitive staph aureus colonization ( % vs. . % p< . ). . years into the study period (the median time of acquisition for new mrsa), the mrsa cohort had a lower %fev ( . % vs . % p< . ), and averaged twice as many hospital admissions per year ( . vs . p< . ) and home iv courses per year ( . vs . p< . ). in an unadjusted analysis, the presence of mrsa was associated with a mean %fev that was . % lower than the mrsa cohort ( %ci . %- . %). however, after adjusting for sex, age, pancreatic status, p. aeruginosa, and b. cenocepacia, the presence of mrsa was associated with a mean %fev . % lower ( %ci . %- . %). most importantly, an analysis investigating the interaction between mrsa and time, after adjusting for confounders, indicated no statistically nor clinically meaningful difference in the mean rate of lung function decline between the two groups. conclusions: approximately one-third of patients who culture mrsa do so only once and do not subsequently develop persistent mrsa infection. mrsa infection occurs in a group with more severe structural lung disease as measured by fev , and frequent hospitalizations and iv antibiotics are strongly associated with mrsa acquisition. after adjusting for confounders, mrsa infection is not statistically significantly associated with a more rapid decline in %fev over time. simmonds, n.j. , ; macneill, s. ; cullinan, p. , ; hodson, m.e. , . department of cystic fibrosis, royal brompton hospital, london, united kingdom; . national heart and lung institute, imperial college, london, united kingdom introduction: disentangling the influence different factors have on long-term survival in cf is complex. there are a myriad of influences (including environmental, healthcare-related, psychological and socioeconomic) which may be important, as there is generally a poor genotype-phenotype correlation. the aim of this case-control study was to identify these factors. the cf database at the royal brompton hospital was used to identify long-term survivors. they were classified as cases and were patients who had reached years of age (without transplantation). each case was agematched with at least one control who died of cf (or a cf-related condition) or was transplanted before reaching years of age. late diagnosis patients were excluded. conditional logistic regression models were used to identify potential influences on survival. results: cases and controls were analysed, producing matched pairs. % of the patients were pancreatic insufficient. of the many factors investigated, those resulting in increased probabilities of survival included: increased body mass index (or= . , % ci . - . ), fev (or per % increase= . , ) at transfer to the adult clinic (after adjusting for age and sex) and the use of oral antibiotics before attending the adult clinic (or= . , . - . ) after adjusting for age at first attendance. factors associated with a reduced probability of survival included: cf diagnosis < years old (or= . , . - . ); initial presentation of chest symptoms or malabsorption (or= . , . - . and or= . , . - . , respectively); referral from a paediatric clinic in a deprived area (or= . , . - . ); pneumothorax before transfer to the adult clinic (or= . , . - . ) after adjusting for age at first attendance; and s. aureus or p. aeruginosa colonisation before years of age (or= . , . - . and . , . - . , respectively). factors not influencing survival included: sex, h. influenzae colonisation before years of age, development of diabetes before years of age, major haemoptysis before transfer to the adult clinic, parents' occupation, number of siblings (cf and non-cf) and school achievements. in a very carefully matched study we failed to identify major non-clinical determinants of long-term survival in early diagnosis cf. findings suggest that the majority of significant factors were directly related to optimal physical parameters, the use of oral antibiotics, avoiding bacterial colonisation and a low pneumothorax rate. adler, a. , ; bilton, d. ; haworth, c. ; gunn, e. ; shine, b. . addenbrooke's cambridge university hospitals, cambridge, united kingdom; . papworth hospital, papworth everard, united kingdom; . oxford centre for diabetes and endocrinology, oxford, united kingdom background and aims: although the prevalence of diabetes in patients with cf (cfrd) is high, few prospective studies exist, and even for genet-ic factors, cross-sectional studies may bias the magnitude of associations. this study sought to identify risk factors for cfrd from a predominantly white cohort of individuals with cf in the united kingdom. methods: , individuals aged - were identified from the uk cf database, a registry of patients coordinated by the uk cystic fibrosis trust with data from over specialists centres from england, scotland and wales, countries which provide medical care free of charge. of the patients, , had no diabetes at baseline ( - ) , and also had at least one follow-up annual visit. diabetes was defined as either a physician diagnosis of diabetes, a blood glucose of > . mmol/l two hours following an oral glucose tolerance test, or treatment with insulin or oral hypoglycemic drugs. follow-up was calculated as time from baseline to the first detection of new diabetes or censoring. risk factors included clinical, genetic (functional classes i,ii,iii vs iv,v), and anthropometric characteristics measured at baseline. survival analyses were limited to patients with complete data. cox proportional hazards modeling was used to estimate the magnitude of the association between potential risk factors and incident diabetes. one-way interactions with sex were tested. results: the median age of entry into the cohort was years and bmi was . kg/m . % were male, and % were white. patients developed diabetes during , person-years of follow-up (mean . years); the incidence was . % per year. patients who developed diabetes were more likely in univariate analyses to be older, have a high body mass index (bmi), bacterial pulmonary infections, liver and pulmonary function abnormalities, poor oxygen saturation, a history of organ transplantation, supplemental feeds, and take pancreatic enzymes or bile acids. in addition, patients with class iv,v genotypes were less likely to develop diabetes relative to patients with other genotypes, as were patients screened for cf at birth. not associated with incident diabetes was age at diagnosis of cf and ethnicity. in multivariate analyses (n= , ) age (hazard ratio . per year, % ci . - . ), female sex ( . ( % ci . - . ), % predicted forced expiratory volume in second ( . , . - . per % decrement), dose of pancreatic enzyme replacement ( . , . - . ), above vs below median dosage, use of bile acid supplements ( . , . - . ) , and genotype class i,ii, iii ( . , . - . ) relative to class iv/v. these associations were not confounded by bmi or history of transplantation. there were no significant interactions between sex and other risk factors. conclusions: the study confirms the high incidence of cfrd, and shows that patients with class iv/v genotypes are less likely to develop diabetes independent of other cf-related complications. females are disproportionately at risk for diabetes which is not explained by a higher prevalence of known risk factors nor by effect modification. cystic fibrosis children are at increased risk for hearing disorders due aminoglycoside exposure, mucopurulent upper respiratory tract infections, and increased inflammatory responses. pure tone audiometry is the standard instrument used to determine hearing loss. however, it can be expensive and time-consuming to use audiometry for routine hearing screening. hearing questionnaires have been used as a screening tool to assess possible hearing loss in otherwise healthy populations. what are the results of hearing questionnaires administered to pediatric cf patients? does cf patient age or ethnicity increase the likelihood of hearing loss as determined by a hearing questionnaire? a hearing questionnaire (english and spanish versions) was administered to pediatric cf patients over a week period at our accredited cf center. data collected included demographic information. inclusion criteria: age м years and in baseline health status. possible hearing loss was defined as at least "yes" responses (> ) to the quesions administered to the patients. fisher's exact test was used to perform the analyses. a total of cf patients were seen in the cf clinic during the study period. cf patients completed the hearing questionnaire. mean age was . ± . years (median . years). there were males and females; were classified as hispanic and were classified as caucasian. patients were older than . years (the median age) and patients were less than . years. patients who were older than age . years had a score of > and patients < age . years had scores > (p= . , ns). thus, of cf patients ( %) were classified as having possible hearing loss as per their questionnaire scores. of hispanic cf patients had hearing scores > and of caucasian patients had hearing score > (p= . , ns) . we found that a significant proportion ( %) of our cf children may require formal hearing evaluation. we speculate that hearing questionnaires may be useful to screen for hearing loss in the cf patient population. hispanic cf patients have increased morbidity and mortality risks compared to caucasian cf patients who attend our accredited cystic fibrosis center in southern california. is the increased morbidity/mortality risk due to decreased access to outpatient cf care? we performed a retrospective review of all patient visits to our cf clinic between january , through december , . data collected included demographic information (age, ethnicity, gender) , and the number of clinic visits during the study period. data was analyzed by unpaired student's t-test as well as by chi-square. during the study period, a total of cf patients ( males: females; mean age . ± . years; range months to years) were routinely followed in our cf clinic. these patients had a total of outpatient visits to the cf clinic ( . clinic visits/patient over the year study period). there were hispanic cf patients ( males: females; mean age . ± . years) and caucasian cf patients ( males: females; mean age . ± . years; p= . , ns). there was no difference in gender distribution between the group (p= . , ns). hispanic patients had a greater number of cf clinic visits ( . visits/patient) as compared to caucasian patients ( . vists/patient; p= . ) during the same period. we conclude that the increased morbidity and mortality of hispanic cf patients in california cannot be attributed to barriers to outpatient care. in fact, hispanic cf patients were seen more frequently in cf clinic compared to caucasian cf patients who attend the same clinic. we speculate that hispanic cf patients are seen more frequently in cf clinic due to increased severity of cf disease manifestations. duguépéroux, i. ; scotet, v. ; audrézet, m. , ; blayau, m. ; parent, p. ; journel, h. ; boisseau, p. ; férec, c. , . inserm u , brest, france; . dep. of genetics, brest, france; . dep. of genetics, rennes, france; . unit of medical genetics, brest, france; . unit of medical genetics, vannes, france; . dep. of genetics, nantes, france aim: the aim of this study was to describe -year experience of prenatal diagnosis (pd) for cf in brittany (western france) and to assess its impact on incidence. method: we registered, by the genetic laboratories of our region, all the pds performed in women living in brittany over the period - . we described the number of pds made for each reason (way by which the one-in-four risk was identified: previous affected child, family testing, echogenic bowel, etc). we reported the proportion of cf-affected fetuses and of consecutive terminations, and assessed the incidence modification due to pd. results: over the -year period, a total of pds were performed in couples living in brittany. most of them were done in couples already having cf child(ren) (n= , . %). extended testing in families -a practice largely proposed in brittany -led to the identification of new onein-four risk couples among the relatives of cf patients who opted for pd times over the study period ( . %). the other pds were made in couples without previous history of cf. the one-in-four risk was mainly identified following the detection of an echogenic bowel during pregnancy ultrasound examination ( pds were done directly following the positive ultrasound ( . %), whereas others were done for subsequent pregnancies ( . %)). the other pds were consecutive to the detection of an heterozygote through newborn screening (n= , . %) or for an other reasons (n= , . %). over the study period, the number of pds per couple varied between one and five, the mean being . . overall, a total of cf fetuses were identified, among whom were terminated ( . %). the inclusion in the incidence calculation of these pregnancy terminations led that to an incidence modification of . % over the study period. conclusion: this study shows that pd for cf is commonly used in brittany and highlights the impact of family testing and of routine ultrasound examination of pregnancies in that region. supported context: hispanics with cystic fibrosis (cf) in the united states experience an % increased annual risk of death from cf compared to non-hispanic patients. when adjusted for socioeconomic status this disparity persists. studies characterizing this at-risk population do not exist. objective: to characterize the center-reported us hispanic population with cystic fibrosis through a cystic fibrosis foundation (cff) patient registry analysis and to elucidate factors present within this hispanic population that may affect outcomes. design: retrospective cross sectional study of the cff patient registry. patients: all , patients in the cff patient registry were included in the analysis. there were no exclusion criteria. the hispanic population is defined as those who were entered by their respective care center as hispanic ethnicity regardless of race. main study measures: prior to analysis, study measures were selected to demographically and genetically characterize the hispanic cf population. genotype, state of residence, mean age and mean age of diagnosis were obtained in the entire population. maternal education (for patients < years of age) and insurance status data were captured as indicators of socioeconomic status. complication rates, fev percent predicted (under years of age) and absolute fev (over years old of age) were obtained as measures of clinical status. t-tests and logistic regression analyses were used to compare measures between hispanic and non-hispanic groups. odds ratios (or) and % confidence intervals are reported. results: center-reported hispanic patients with cf were identified. over % of hispanic patients resided in california, florida, texas or new york. the most common genotype was delta f homozygous, accounting for % of the population. mean age was . years +/- . years for hispanic patients and . years +/- . years for non-hispanic patients. hispanic patients were diagnosed at an earlier age ( . years vs. . years, p= . ). they were more likely to have mothers with education at less than a high school level (or . ( . , . ) ) and have government insurance (or . ( . , . ) ). complication patterns differed between the two groups; non-hispanics were more likely to have complications reported that were related to depression (or . ( . , . ) ), bone health (or . ( . , . ) ) and cf related diabetes (or . ( . , . )) compared to hispanics. hispanics however, were more likely to have complications reported from cf liver disease (or . ( . , . ) ). pulmonary function, as measured by fev percent predicted in children (p< . ) and absolute fev for adults (p= . ) were lower for hispanic patients. conclusions: differences exist between hispanics and non-hispanics with cf with respect to maternal education, insurance status, complications rates and pulmonary function. the lower reported rates of depression, bone complications and cf related diabetes may represent ascertainment bias. further study is needed into the etiology of health outcome disparities in this population, and to design interventions for this high-risk population. mastella, g.; baldo, e.; forneris, m.; furnari, m.; lucidi, v.; manunza, d.; marinelli, i.; messore, b.; neri, a.; raia, v.; salvatore, d.; buzzetti, r.; cairo group, italian cf research foundation, verona, italy with our work we reviewed the international scientific literature coming from cf registries worldwide. our aims were: to verify what has been produced in scientific literature thanks to the material derived from fc registries and to see which clinical problems have been tackled and which clinical questions were answered correctly and exhaustively. a search in medline and embase produced articles (our strategy was "cystic fibrosis"[mesh] and ("registry"[mesh]) or registr$) updated to the / / ). out of these articles have been selected by two independent assessors on the basis of their pertinence (primary studies that used data taken from a formally established registry, at least national, to test some hypothesis of research). each article has been examined with the help of a pre-defined form that in particular evaluated skills of different registry data, selected clinical characteristics of the study populations and statistical methods. more than one half of cases data came from usa-cff foundation patient registry, the remaining coming from canadian, uk, french, italian, german, and other european registries. the main focuses of the articles were included in the following topics: ) incidence/prevalence of the disease and survival ( studies); ) neonatal screening, growth and nutrition ( ); ) genotype/phenotype correlation/ different ethnical groups and twin/brothers ( ); ) complications and outcomes (abpa, diabetes, nasal polyposis, pregnancy and paternity, etc) ( ); ) microbiology ( ). our assessment scores were good/excellent for / studies for the relevance of the problem addressed; / for the usefulness of the outcomes and / for the usefulness of the implications for research from the italian registry. thirty-two articles gave a significant contribution to the analysis of the studied problem, but of them left a partial uncertainty, while left complete uncertainty. cf registries are a very important source of data to provide a powerful tool for clinical and research questions. however, further comparable studies have to be planned to assess registry data as a platform for improvement in clinical practice. vandenbranden, s.l. ; mcmullen, a. ; konstan, m. ; morgan, w. ; wagener, j. ; schechter, m. ; watts, k. , ; mccolley, s. , . children's memorial medical center, chicago, il, usa; . emory university, atlanta, ga, usa; . university of arizona, tuscon, az, usa; . case western reserve university, cleveland, oh, usa; . northwestern university feinberg school of medicine, chicago, il, usa; . university of colorado, denver, co, usa; . university of rochester, rochester, ny, usa background: although life expectancy in cf has dramatically improved over the last years, most of the improvement is due to improved survival during early childhood. adolescence and young adulthood continue to be a time of worsening pulmonary disease and mortality. study objective: data from a large longitudinal observational study, the epidemiologic study of cystic fibrosis (escf), was examined to characterize lung function decline in subjects with cf during the periods preceding and after the age of . design: escf data from individuals with cf collected during the period of - were analyzed. inclusion criteria required individuals to have a minimum of encounters in each of the . year time periods before and after the age of . the cohort was strati-fied by disease severity based on the best fev at the th year (+/- months). one hundred thirty seven subjects had severe lung disease (fev < % predicted), had moderate-severe lung disease (fev -< % predicted), had mild lung disease (fev -< % predicted), and had very mild disease (fev > % predicted) the study compared the annualized rate of fev decline during the adolescent period, defined as ages - . years, and in the young adult period defined as . - years. results: in the entire cohort, the annualized rate of decline was less in the young adult period than in the adolescent period (p< . ). the most dramatic improvement in slope of decline was seen in the severe group, and significant improvements were also noted in the moderate group (p< . ). the mild group demonstrated insignificant change; the very mild group was the only group that demonstrated a more negative annualized rate of the decline in young adulthood than in adolescence (p= . ). conclusions: these data suggest that, overall, young adults have a slower rate of decline in fev in the period after age . this "stabilizing" of lung function is most prominent in those individuals with moderate-to-severe lung disease. only those with very mild lung disease have a slightly more rapid rate of decline between the ages of and . overall there is less variation in the rate of fev decline across disease strata after the th birthday. further analysis is underway to better understand the factors leading to these findings. . department of paediatrics, university of florence, cystic fibrosis centre, florence, italy; . italian cystic fibrosis microbiology group, florence, italy; . institute of medical microbiology and hygiene, tübingen, germany mrsa is now a worldwide public health problem, due to the increasing rate of infection in several settings as well as in cf patients. mrsa was first recognized as being acquired from hospitalized patients (ha-mrsa), but the onset of mrsa infection outside the hospital setting, in communityassociated strains (ca-mrsa), has recently been described with increasing frequency. ha-mrsa are known to be responsible for infections in hospitalized patients, although highly virulent ca-mrsa are increasingly reported worldwide as the cause of severe outbreaks, replacing ha-mrsa strains in nosocomial infections. little evidence is available about the characterization (sccmec type) and epidemiology of community-acquired mrsa (ca-mrsa) or hospital-acquired mrsa (ha-mrsa), transmissibility, antibiotic susceptibility and virulence in the cf population. the present multicenter study investigated the susceptibility pattern, epidemiology, and sccmec type of mrsa strains isolated from nine italian cf centers. the susceptibility pattern was determined by the disk diffusion method and pulsed field gel electrophoresis (pfge) was performed for epidemiological purposes. the sccmec type in order to identify hospital-and community-acquired mrsa strains (ha-mrsa and ca-mrsa respectively) was determined with molecular methods, and the presence of gene encoding panton-valentine leukocidin (pvl) was tested. during the study period, mrsa strains were isolated from out of ( . %) patients attending these cf centers. of the sccmec type representing ha-mrsa, our data showed an high prevalence of sccmec i ( . %) while the prevalence of sccmec ii, (reported as the most represented among ha-mrsa) was only . %. furthermore a high prevalence ( . %) was found of sccmec iv (suggestive of ca-mrsa strains). epidemiological analysis showed that ( . %) out of analyzed mrsa strains belong to the same pfge clone shared among six centers, belonging to the sccmec type iv, suggestive of ca-mrsa isolates. twenty-four ( . %) out of these strains were sccmec type iv, indicative of ca-mrsa, as suggested by the good susceptibility rate to trimethoprim-sulfamethoxazole and rifampin. surprisingly all the isolates belonging to this epidemic clone were negative for the pvl genes. these results show that ca-mrsa is now spreading in the cf population, documenting the first epidemic of ca-mrsa in cf patients who are considered at risk for ha-mrsa acquisition due to frequent hospitalizations. these data show a peculiar picture of mrsa epidemiology indicating that further studies are required to clarify the role of ca-mrsa in global epidemiology, their pathogenicity and clinical impact on cf patients. we thank the fondazione per la ricerca sulla fibrosi cistica-onlus for its grant (ffc# ) . there are concerns regarding an increased risk of cancer in patients with cystic fibrosis (cf). this study aimed to review the occurrence of malignant disease in a large population of adult patients with cf. the case notes of patients attending the leeds adult cf unit were reviewed. demographics and data regarding a diagnosis of malignant disease were recorded. a total of eight patients ( male, female) were diagnosed with a malignant condition over the year period. the median (range) age of diagnosis of a malignant condition was . years ( - years). five patients were diagnosed with malignancy post-transplantation. within this group of patients there were two cases of lymphoproliferative disease, one case of basal cell carcinoma, one case of liver cancer and one case of small cell lung cancer related to the donor lung. the median (range) time to diagnosis post-transplantation was two years ( - years) with a median (range) age of years ( - years). three patients were diagnosed with a malignant condition without solid organ transplantation. this group consisted of one case of pancreatic carcinoma, one case of oesophageal carcinoma and one case of bowel carcinoma. the median age of diagnosis in this group of patients was years ( - years) . prior to transplantation we found all malignant diagnoses were related to the gastrointestinal tract. post-transplantation we found a wider variety of malignant diagnoses, with lymphoproliferative disease being most common. awareness of the increased risk of malignant disease is important and appropriate investigations should be undertaken particularly in patients with atypical symptoms. background: patient outcomes can be improved by implementing evidence-based guidelines for initiating therapies. in our cf center, practitioners have traditionally prescribed medications without a centralized database to screen patients for evidence-based criteria. goals: the primary goal of this project was to increase cf practitioner, family and patient awareness of evidence-based medication guidelines to facilitate discussions regarding therapies. the secondary goal of this quality improvement (qi) project was to determine if cf practitioner prescribing patterns would be altered by increasing awareness. methods: the cf steering committee at cincinnati children's hospital medical center (cchmc) identified oral azithromycin, inhaled tobramycin, and dornase alpha as target medications for this qi project. an evidence-based medicine committee was formed, including a parent advocate, to direct the project. a consensus was reached among cf practitioners for when to initiate therapy discussions. dornase alpha was recommended for cf patients ages six and older. inhaled tobramycin was recommended for cf patients ages six and older with chronic pseudomonas (defined as positive cultures in past months). oral azithromycin was recommended for cf patients ages six and older, with chronic pseudomonas, and a weight of kilograms or more. the cf database was screened to determine each patient's medication eligibilities. eligibilities were reviewed weekly at our cf chart conference. medication specific family and patient handouts were developed, reviewed by the parent advocate, and given to eligible families and patients on arrival to clinic. data was collected to document the number of discussions facilitated at clinic visits, the decisions regarding medications, and the number of new treatment prescriptions that were facilitated. after seven weeks of the initiative, statistical analysis was conducted using mcnemar's test for differences. a chi-square test with a two sided alpha of p < . was used to determine significance. results: of the cf patients in our center were eligible for pulmonary medications using our evidence-based criteria. there were individual prescribing opportunities in these patients (taking each medication for each patient as a unique prescribing opportunity). at the start of the project, . % ( / ) of the prescribing opportunities were fulfilled. % ( / ) of patients were prescribed all of their recommended medications. during the first seven weeks of the project, cf practitioners discussed initiating new pulmonary medications with cf families and patients. the proportion of prescribing opportunities fulfilled increased from . % ( / ) to . % ( / ) (p value < . ; % ci . %- . %). the proportion of patients prescribed all of their recommended medications increased from % ( / ) to . % ( / ) (p value < . ; % ci . %- . %). conclusions: increasing awareness of evidence-based pulmonary medication recommendations with cf practitioners, families and patients facilitates therapy discussions, increases the proportion of therapies prescribed and increases the proportion of patients on all recommended therapies. purpose: our purpose was to optimize lung function (fev ) by consistently and aggressively diagnosing and treating pulmonary exacerbations (pex) in all cf patients at our center. it was important to work on this because our families wanted standardized care and we felt our patients' fev 's were suboptimal. methods: our core llc team included two parents of cf patients and one adult patient. we met weekly, received coaching every other week, and learned and applied quality improvement techniques. physicians began monthly meetings, and monthly meetings of the entire cf team continued. the fab met every other month. we identified our specific aims, and approached them systematically. the providers, starting with criteria used by several authors, agreed upon the definition of pex and created a pulmonary exacerbation score sheet (pexss). we selected a score of as the threshold for defining a pex. after using the pexss for three months, all providers reviewed the individual pexsss and validated our original choice of . run charts were produced and reviewed periodically to display each provider's percent of pexsss used. when the use of the pexss was not standardized after a year, it was reprinted on bright yellow paper to make it stand out. to educate the families and patients about the early symptoms of pex, a refrigerator magnet was designed and distributed that included all pex criteria. results: we standardized the use of our pexss by all providers on all patients seen in cf clinic with ongoing measurement of its use and regular feedback. many patients and families gained greater knowledge about what a pex was, and the importance of early recognition and treatment. the cf nurses note that ) many patients call with milder pex symptoms than they did before, ) providers treat pex earlier, ) many patients call with their pex score as well as their symptoms. we hospitalized more patients for pex after this project began, with an average of patients/year in the years prior, and in . the median fev % predicted of our - conclusions: participating in the llc changed the culture and functioning of our cf team. limitations to our study include: ) fev change may not have been due solely to this project, ) the groups who's fev 's were averaged over the various years are different. we will continue to optimize and standardize ) prevention of pex, ) inpatient care, ) care of pex in cf providers' private offices, and ) follow up of patients with pex. patient adherence to airway clearance techniques (act) in the treatment of cystic fibrosis is known to be sub-optimal resulting in deterioration of lung function. the global aim of our project was to improve median fev . the specific aim was to improve adherence to act. we believed that these goals could be acheived by implementing a program which included re-education of airway clearance techniques (react) for our cystic fibrosis patients. methods: the initial phase of this project began with the administration of an anonymous survey to patients/families. this survey was designed to assess act practices, knowledge of the rationale for performing act and barriers that prevent patients/families from adhereing to act. the next step included an in-clinic questionnaire and patient/family demonstration of current aerosol therapy and act. based on individual results patients were identified as: adherent with correct technique, adherent with incorrect technique or non-adherent. we defined adherent as correct performance of act one to two times daily as prescribed. then all patients entered the react program which included a standardized tutorial on proper act and correction of techniques if necessary. in the non-adherent group barriers to performing act were discussed and problem solving techniques were used to overcome barriers. the patients in the non-adherent group were re-evaluated in clinic monthly while the adherent patients returned on an every two month schedule. results: analysis of our initial survey revealed that while the majority of patients/families reported performing act daily, the duration of treatments was less then medically prescribed. many patients/families reported barriers which decreased adherence to therapy. to date % ( / ) of our patients have completed the in-clinic questionnaire and entered the react program. the results of these evaluations and patient demonstrations revealed: % ( / ) were adherent with correct technique, % ( / ) were adherent with incorrect technique and % ( / ) were non-adherent. in the six months since the implementation of the react program, patients/families are self-reporting an increased adherence to act and the median fev for our center has increased form . % ( cff registry data, ages - years) to . % (cff registry, first quarter, , ages - years). conclusion: while adherence to act in our center was poor, we were able to improve adherence and ultimately improve the median fev by implementing a program of re-education for act (react). we believe re-assessment of adherence to therapy and reviewing correct technique is necessary at every clinic visit. continuous quality improvement (cqi) techniques have recently been employed in medicine to improve quality of care. although there is evidence that cqi improves efficiency in hospitals, little is known about effects of cqi on improving patient outcomes. we have previously reported creation of a unique nutritional risk pathway (nrp) to care for cystic fibrosis (cf) patients who are at nutritional risk. the goal of this study is to assess perceived impact and objective clinical efficiency of our nrp. patient data was recorded from all children attending the cf clinic over an -month observation period (between sep and mar ). bmi percentiles were assessed and nutritional risk zones -green: acceptable (bmi > %ile), yellow: at risk (bmi - %ile) or red: at urgent risk (bmi < %ile) -assigned at each visit. the visit intervals were every to weeks based on severity of nutritional risk. patients that remained in the red zone for an average of to months with no improvement were considered for gastrostomy tube (gt) placement. the cf dietitian updated the data weekly and e-mailed quarterly to cf physicians for review of their patients' status. patient/family perceptions and understanding of the nrp was assessed by questionnaire. of the children in the study, ( %) were identified to be at nutritional risk and were enrolled into the nrp: in red and in yellow zones; ( %) male, ( %) female; ( %) age < yrs, ( %) age ≥ yrs. over the -month observation period, ( %) showed improvement ( moved from red to yellow, from red to green and from yellow to green zones). thirteen ( %) females improved, whereas ( %) males improved. in the under yrs age group, ( %) improved compared to ( %) in the ≥ yrs age group. as a result of participation in the nrp, ( %) children had gt placed: six of the showed improvement with four moving from the red to green zone. from the survey responders, % stated they understood the meaning of the cf nrp. all responders were extremely to somewhat motivated to focus on their child's nutrition when asked to return for growth monitoring or follow-up visits. having specific goals for weight gain, calorie requirements and bmi were considered extremely helpful by % of families. when gt placement for their child was recommended, % felt they lacked all the necessary information to make a decision. our new cf nutrition education booklet was not received by % of families. some of the physicians who received the quarterly nutritional patient data provided positive feedback and found this information beneficial for patient care. nutritional cqi in our cf center utilizing a specific nrp and monitoring of nutritional risk status resulted in improved patient outcomes. overall perceptions of our nrp were positive from both patients and cf physicians. these results suggest that this cqi strategy improved clinical outcomes in our patient population. patients with cf require frequent healthcare visits to optimize growth, initiate early interventions and delay progression of lung disease. the cff recommends evaluation at a cf center at least quarterly. as part of a llciv quality improvement (qi) initiative, our cf team set a goal to increase the percent of cf patients attending clinic or more times a year to % or greater by developing strategies to monitor and improve adherence rates. methods: we reviewed cf patients who attended clinic times or less in to determine patterns of non-adherence in regards to age, gender, distance from center, season, physician or insurer. a patient survey was used to assess clinic attendance barriers from a patient/family perspective. a brainstorming session identified other barriers. a fishbone diagram was drafted into barrier categories: external, internal, communication, and patient/family perceptions. after reviewing all barriers, we concluded the most effective improvements could be made to internal clinic barriers. clinic processes were then assessed including making of appointments, reminder letters/calls, a time cycle analysis, and number of cf appointments available per patient per physician. our family advisory board participated by focusing on the importance of or more clinic visits in their newsletter and by assignment of a member to the llciv team. we discovered a lack of a standardized process to follow-up (fu) patients who "did not keep appointments" (dnka). a spreadsheet was created to monitor physician dnka rates. a primary fu strategy was devised to identify patients who dnka through daily auto-emails to the cf office assistant and utilization of a rescheduling decision tree for reappointment of these patients by our appointment center. two secondary fu strategies were developed. the first involves the clinical coordinator printing a monthly report off port cf entitled "patients due a visit" then emailing this list to the cf team to contact and reappoint. the second involves the cf social worker tracking patients seen per quarter on a spreadsheet and emailing the spreadsheet quarterly to the cf team as well as posting on our datawall. results: in , and , %, %, and % of our patients attended clinic or more times, respectively. in in , and , %, %, and % of our patients were at nutritional risk, respectively. in and , mean fev % predicted was . % and . %, respectively. conclusion: the number of cf patients attending clinic quarterly at our center increased by implementing processes to track dnka patients and reappoint them to clinic promptly. however, some of the early improvement in clinic attendance was partially due to an increased awareness of our qi efforts by our clinic staff, cf team and patients/families. we speculate that improvements in nutritional status and lung function are at least partially related to more frequent clinic attendance by our patients. continued monitoring of the number of dnka patients and patients who attend clinic quarterly is required to sustain these gains in improved patient outcomes. data when we first measured patient/family satisfaction in , parents and children were given questionnaires to measure their satisfaction with services provided at the clinic. at that time, responses in our survey were very positive. with regards to our new model we received very positive feedback, encouraging us to continue with the model. parents and children liked the elimination of duplication in questions and they also like having the whole team involved in the meeting and problem-solving. final results for our second satisfaction questionnaire will not be available until june of this year as the questionnaires are being given at clinics over a month period this spring. the children complete a questionnaire with the help of their parents if needed and the parents also complete a survey. results to date are very positive for both the parents and the children. of the children who have completed the survey to date, all stated they would like to continue to meet with the group as a whole as opposed to meeting individually with team members. of the parents surveyed to date, all are either very satisfied or satisfied with the group format. conclusion: we have not yet completed the survey but results to date clearly show that both the patients and their families are happy with the round table model. results will be compiled early this summer and would be ready for the poster and/or presentation in the fall. the cff clinical care guidelines recommend routine clinic visits for assessments, interventions, monitoring, education and counseling every months or more often as indicated. adherence to these guidelines is important in ensuring better clinical outcomes. in reviewing our cf center data from the cff registry report, . % of pediatric patients ( - yrs.) and . % of adults ( yrs. and older) adhered to the guidelines. national goals were not provided then; instead data was given for the top ten cf centers ( . % and . % for pediatric and adult patients respectively) as well as the national rates ( . % and . % for pediatric and adult patients respectively). our cf care center acknowledged the need to improve adherence to these guidelines and at the beginning of the year , embarked on a quality improvement project to address this issue. following the pdsa (plan-do-study-act) steps, we agreed on the objective of increasing the percentage of pediatric and adult patients adhering to the guidelines by at least % and at the same time, to exceed the national average in year or by the end of the year . the physicians and social worker obtained feedback from patients and/or families during a clinic visit regarding barriers in complying with scheduled clinic visits. suggestions were elicited to overcome these barriers. the team members then identified barriers in meeting the guidelines and implemented measures to improve compliance with clinical care guidelines. among these measures were sharing the information about clinical care guidelines and importance of adherence through our cf newsletter and at every clinic visit, reminder calls to patients or caregivers at least days prior to the visit, appointments for the next visit given prior to discharge from the clinic, and same day calls to patients who failed to keep their appointment for rescheduling. our social worker identified patients with difficulties or problems in consistently keeping appointments and helped address these problems. progress made in this endeavor was shared with parents and caregivers during the "parents' night" held in the fall of . indeed, with consistent implementation of these measures, we have seen improvement in the percentage of patients adhering to guidelines. in our cf care center data, . % of pediatric patients and . % of adult patients adhered to recommended clinical care guidelines, approximating and in fact for the adults, exceeding, our target of % increase in the percentage ( % and % increase respectively for pediatric and adult patients). these data were better than the national average for ( . % and . % for pediatric and adult patients respectively) but for pediatric patients, still % below the national goal of %. adults approximated the national goal of %. we are committed to continually engage in the process of quality improvement in further improving adherence to clinical care guidelines and to hold on to the gains. weight z-scores and height z-scores were monitored prospectively for all infants, children, and adolescents at a pediatric outpatient cf clinic during an ongoing, multidisciplinary quality improvement (qi) initiative. all cf team members participated in this qi project which emphasized the importance of good nutrition and adequate growth for optimal lung health. specific strategies for improvement included education to families at each visit with a written nutrition action plan, increased emphasis on liquid supplements and enteral feeds as needed, and a consist message from all cf clinic staff in encouraging attention to nutrition. the study objective was to determine if multidisciplinary qi interventions to improve nutritional status had any effect on weight z-scores and/or height z-scores over a -year period. the null hypothesis was that z-scores for weight and height would either not improve or worsen. inclusion criterion for this study was attendance at cf oupatient clinic during the first months of the qi initiative (time ) with a subsequent clinic visit years later (time ). patients not attending clinic at both time and time were excluded. a cohort of patients ( male, female) met the inclusion criterion. the mean time to time interval was . ± . years (range . to . years) with mean patient age at time of . ± . years (range . to . years). pancreatic enzyme replacement therapy was used for ( . %) of the cohort. paired sample t-tests indicated significant improvements for weight z-score (p <. , t ( ) = . ) and height z-score (p <. , t ( ) = . ) after the -year intervention. at time , children ( . %) were underweight or less than th percentile while children ( . %) were normal-weight, at or more than th percentile for age. mean differences in both weight and height z-scores in the initially underweight patients were greater than those for the normal-weight patients over the year interval. no significant differences were found for the initially normal weight-for-age children between time and time for weight z-score ( p = . ) or height z-score (p =. ). children who were not underweight at time grew normally, without acceleration over the yr intervention. therefore, we conclude that a longterm multidisciplinary qi project to enhance nutritional status resulted in significantly improved weight z-scores and height z-scores for those individuals who were initially underweight for age. the most common cause of morbidity and mortality for patients with cystic fibrosis (cf) is pulmonary exacerbations. these exacerbations lead to hospital admissions and treatment with intravenous (iv) antibiotics. all cf patients at our facility are admitted to a teaching service which includes house officers and students. standardized order forms are used to improve accuracy and timeliness of medications and therapies. the efficiency of this process was brought into question in / , when several parents complained about a delay in the receipt of first dose iv antibiotics after hospital admission. the objectives of this study were to determine what were the actual delivery times of the iv antibiotics to the patients (both retrospectively and prospectively), and areas where improvements could be made. our predetermined target time was < hours. two analyses were performed in order to answer the objectives. the first analysis was done in a retrospective fashion. this analysis included cf patients admitted from / / - / / . a total of patients were included. total median time from admission to receipt of first dose of iv antibiotics was analyzed. a second collection of blinded prospective data was performed between / / - / / . only the pulmonologist, pharmacist collecting data, and pharmacy clinical manager were aware of the data collection. a total of patients were included in the prospective project. each part of the process model were recorded and analyzed so that areas of improvement could be realized. the median time from hospital admission to first dose of antibiotics was hours (range - hours) (retrospective cohort). the prospective cohort median time from hospital admission to first dose of antibiotics was hours (range min- hrs). the median time from admission to order entry by pharmacy: hrs, min (range min- hrs, min). the median time from the medication order being written by medical practitioner to order entry by pharmacy: minutes (range min- hrs, min). the median turn-around time by the hospital pharmacy of order entry until reaching the nursing unit: min (range min- min). the median time required by the nursing staff to administer the antibiotic: hrs, minutes (range min- hrs). this project was initiated in response to parental concerns. from the results of the retrospective and prospective analyses, it was determined that a delay is occurring in the receipt of iv antibiotics, which translates to hours in the hospital without therapy. this is both inconvenient and uncomfortable to the family, and wasteful of medical resources. the findings of this project were presented to the pharmacy and therapeutic committee of the hospital, to the nursing directors, and to the parents of cystic fibrosis patients. the delay is greater than expected before this project was initiated. as a result of these findings a "fast track" medication order set was developed to expedite initiation of inpatient therapy. four key areas included in this order set are sputum, iv access, diet, and iv antibiotics. we speculate that time to first drug delivery will decrease and patient satisfaction with improve. the pediatric intermountain cystic fibrosis center (imcfc) provides multidisciplinary cystic fibrosis (cf) care to pediatric patients from utah and parts of idaho, wyoming, and nevada. historically the imcfc has embraced early intervention. as new therapies have been added to the cf armamentarium it has become necessary to have a method of keeping track of which patients are candidates for various therapies and also which medications have been prescribed, utilized or even should be discontinued. in order to address this quality issue, the imcfc decided to develop and implement the use of a medication tracker (mt). the imcfc decided to focus on key therapies which were felt to be at risk for variability in prescriptive practice. the five therapies included were: pulmozyme<®>, tobi<™>, hypertonic saline(hs), azithromycin and asthma medications (inhaled corticosteroids and/or leukotriene receptor antagonists). ibuprofen was intially included and later removed from the mt. prior to implementing the mt, the imcfc met to review literature, medication trackers from other centers, and develop consensus among team members. six versions of the mt have been developed between / / - / / . each mt has been tested in a pdsa cycle and revised. since initating the mt, patients have been seen at least once. there have been mt encounters. the mt is used only after the first visit to the imcfc and reflects changes to established cf care. there have been a total of changes in prescribed therapies in different patients( % of patients seen). a minority of patients were receiving therapies that did not meet our pre-determined consensus for prescription. the most common therapies in this category were tobi™ and azithromycin. primary reasons cited for prescribing were: chronic suppressive antibiotic use, pseudomonas positive and < years old, and significant changes on chest imaging. reasons patients were not prescribed clinically indicated therapies were: patient refusal, participation in clinical trials, cost, and lack of compliance with other therapies. changes in asthma therapy included the discontinuation of medication in encounters and the initiation of therapy in . the use of a specific cf patient mt allows consistent practice in selection of therapies. it can be used by all team members and helps to identify discrepancies in therapy. assuring all patients receive information and are considered for new therapies is increasingly important as our treatment options expand. the mt also highlights the need to consider discontinuing therapies. we speculate the mt will become an increasingly valuable tool over time and improve patient care. rationale: recent reports document increasing prevalence of antimicrobial resistance across a range of pathogens in patients with cf. the increase in multidrug resistance of these organisms complicates the therapeutic management of these patients. objective: to describe patterns of antibiotic use among physicians treating cf patients. methods: for the original purpose of evaluating site characteristics that act as effect modifiers on the relative efficacy or cost of novel therapies, we administered a survey to acquire information on practice patterns and characteristics of physicians participating in a clinical study of an investigational drug for patients with cystic fibrosis. chi-square and spearman rank test were used to evaluate associations between physician characteristics and patterns of reported antibiotic use. results: fifty-nine physicians, representing study sites, completed the survey. ninety percent reported a medical specialty of pulmonology and % reported practicing at a teaching hospital. on average, respondents reported that about half of their practice consisted of patients with mild cf (defined by fev ≥ % of predicted normal.). forty-two percent of respondents reported prescribing oral antibiotics and % of respondents reported prescribing inhaled antibiotics for maintenance therapy for at least % of their mild cf patients. eight-five percent and % of respondents reported prescribing iv antibiotics for pulmonary exacerbation and pulmonary 'clean out', respectively, with about a third of these cases being prescribed for outpatient iv use. physicians that prescribed iv antibiotics for pulmonary clean out were more likely to pre-scribe oral (p= . ) and inhaled antibiotics (p=. ) for maintenance therapy than physicians that did not prescribe iv antibiotics for pulmonary clean out. conclusions: antibiotic use for maintenance therapy and iv antibiotic use for pulmonary exacerbation and pulmonary clean out acted as 'complements' rather than 'substitutes' as there was a group of physicians who reported less use of antibiotics. our findings warrant further research as well as additional exploratory analyses with a larger and broader sample to examine associations between physician characteristics and patterns of antibiotic use. understanding the relationship between physician characteristics and antibiotic use has the potential to influence the approach to management of cf patients in an environment of increasing antimicrobial resistance. introduction: hypertonic saline (hs) has been shown to increase mucus clearance and improve lung function in cf, and is now increasingly being used as part of routine therapy. one of the goals of our cf center quality improvement (qi) initiative is to increase use of hs among all eligible patients. objectives: to assess adherence to treatment with hs, parental opinions on treatment, perception of side effects, and the effect of initiating hs on the use of other nebulized medications in pediatric cf patients started on hs therapy during . methods: patients under years of age were identified as initiating hs therapy during . a telephone survey of their parents using a -item multiple-option questionnaire was administered in april as part of our cf center qi initiative. results: parents ( %) completed the survey: ( %) patients were still using hs. of those still reporting use of hs, ( %) were using it twice a day; ( %) once a day; and less often than daily. among the patients who discontinued hs, ( %) stopped following recommendations from their pulmonologist; ( %) stopped due to side effects; ( %) felt hs took too long to administer; and ( %) felt hs solution was too difficult to mix. the most common side effect reported was cough in patients ( %); ( %) reported no side effects. the majority of patients ( %) spent - minutes per day on hs therapy. thirteen ( %) patients discontinued another nebulized medication when they initiated hs; ( %) of those stopped pulmozyme. reasons for stopping pulmozyme included: time to nebulize both medications was too long ( %), perception that hs was working better for their symptoms ( %); and doctor's instructions ( %). overall, ( %) parents felt hs improved their child's symptoms; ( %) saw no difference; and ( %) believed hs made symptoms worse. there was a significant difference in the proportion of patients that reported improvement of their symptoms in the group using hs twice a day ( / , %) when compared to those using it once a day ( / , %) (p= . ). discussion: the majority of patients continued to report the use of hs therapy several months after our initial intervention, with the most parents reporting improvement in their child's symptoms. physician recommendation was the most common reason cited for discontinuation of hs therapy. further study of physician opinions on hs therapy, particularly the timing of initiation and discontinuation, is warranted. one-half of patients reported using hs only once a day; however those reporting twice daily use were more likely to report subjective improvement of symptoms. since treatment complexity in cf increasing, determining the effectiveness of once daily hs therapy, either alone, in place of, or in combination with other therapies such as pulmozyme is essential. long-term follow up of these hs-initiated patients will focus on effects on exacerbations, pulmonary function, and hospitalizations. the uab/children's hospital (chs) cf center participated in learning and leadership collaborative ii. as part of the qi journey, the chs cf qi team thoroughly evaluated the current system of care in an effort to identify areas of needed improvement. one area assessed was clinic attendance. it was discovered that % of patients failed to attend clinic appointments every week. according to cff care guidelines, patients with cf should be seen a minimum of times each year at the cf center. the cff has also found that those centers that see their patients more frequently have improved outcomes for fev and nutritional status. aim to decrease the number of missed appointments at the chs cf clinic. the chs cf center administered a qi survey to identify the reasons patients and families were missing appointments. this survey was a item questionnaire that evaluated: reasons for missed appointments, if the families rescheduled missed appointments, difficulties with rescheduling, the need for reminder calls, distance traveled to the cf center, and insurance status. results completed surveys were collected. % of the families reported medicaid as their insurance provider. % of families live more than miles away from the cf center. % of the families confirmed that they had missed an appointment in cf clinic over the last months. the reasons for the missed appointments were widely variable; however, . % of those who missed appointments reported that they did not have access to transportation to get to the appointment. overall, families did not report difficulties in rescheduling appointments. % of those that missed appointments identified medicaid as their insurance which can be correlated with lower income families and . % of those that missed lived greater than miles away from the cf center. % of families reported that a reminder call would be helpful for them. interventions after evaluation of the survey data, the center quickly initiated reminder calls for cf clinics. a student employed by the cf center made these telephone calls to the patients scheduled for clinic every week. the majority of the time the student caller left a message for the family. with the implementation of this simple intervention, missed appointments have decreased from % to % which is a % improvement rate. reminder calls are now a routine part of the cf center's system of care. other ongoing interventions include increased involvement of primary care doctors for patients lost to follow up for more than months and a system for quickly rescheduling patients. next steps in addition to reminder calls, the cf team will now determine where patients who routinely miss appointments live. if there are pockets of patients in common areas of the state, the team will attempt to maximize services for those patients such as linking them with community transportation resources, community health advocates, or providing satellite clinics. this will be accomplished through geomapping of the alabama cf population based on zip code. we designed a scoring system to uniformly identify patients experiencing a pulmonary exacerbation. the elements of this pul-monary exacerbation score (pes) have been previously described. at the time this project began the median fev % predicted of the cf patients at our center - years old was below the national average ( cf registry data). our hypothesis was that greater uniformity of identification of pulmonary exacerbations, would improve pulmonary function in the pediatric age group at our center. this abstract describes continued improvement in the pulmonary function of this age group at our center now that the pes has become standardized in our clinical practice. methods: beginning in october , a pes was calculated for all patients presenting to our center age - years of age. any patient with a pes of > was defined as having a pulmonary exacerbation, and treatment with antibiotics was recommended. the patient's cf physician dictated the ultimate decision for treatment and specific course of treatment. median fev of all patients age - years was calculated quarterly to determine the effect of the pes use on pulmonary function. in october the pes was incorporated into our cf clinic medical record and its use was standardized. the use of the pes, the adherence to the recommendation for treatment, and quarterly fev of the population has continued to be monitored. results: from / to / (the duration of the original qi project), patient visits occurred, with a pes calculated on (utilization of . %). patients were treated for a pulmonary exacerbation of the patients with a pes of > (adherence of . %). . % of exacerbations were treated with oral and/or inhaled antibiotics, and . % were treated with intravenous antibiotics. since standardization of the pes in / , visits have occured. use of the score has decreased slightly ( . % utilization), however adherence to the score has increased ( . % of pes > have been treated). use of oral vs. iv antibiotics has been similar ( . % oral vs. . % iv). the median fev of our patient population has improved from an average of . % predicted during the months before implementing the pes to an average of . % predicted during the ensuing months of the original qi project (a . % improvement). since standardization of the pes in / , the average median fev of the population has continued to increase to . % predicted (an . % increase from the baseline period). this improvement in median fev is reflected in our cf registry data. summary: implementation of a pulmonary exacerbation score, designed to encourage uniform identification of pulmonary exacerbations in patients with cf, is associated with an improvement in median fev in children - years of age. standardization of the pes has resulted in continued improvement in pulmonary function of this population at our cf center. this work has been supported in part by the cff and akron children's hospital. our cf family council parent and patient advisory group has been involved in the development and implementation of this project. utilizing data from the cff patient registry, we determined that our center has had nutritional outcomes consistently below the national average for pediatric and near the average for our adult patients. as a result, we developed the global aim to improve and maintain optimal growth and nutrition in all patients at our cf center. our specific aims included: )to record growth parameters at all outpatient visits; )to identify patients at nutrition risk, categorizing them based on anthropometric data; ) to develop and implement a specific nutrition plan based on nutritional category; and ) to educate staff, patients,and families on the benefits of normal growth and nutrition. we surveyed our patients to determine their overall attitudes toward nutrition and nutritional care received at our center. we discovered that many patients and families did not feel that nutrition was addressed at every cf visit nor that they had a nutritional plan at each encounter. this led to the development of an intake form/home-going sheet for our patients to complete that included data elements necessary for assessment of nutrition and that clearly outlined a nutrition action plan. a standardized nutritional assessment score (nas) was devised that incorporated weight, height/length, bmi and weight for length percentiles as well as weight loss, failure to gain or maintain, and failure to remain in the expected growth channel. the nas was developed after examining the medical literature, reviewing work done by other qi teams, and acquiring input from our pediatric gastroenterologist and endocrinologist. we then developed nutritional algorithms to address nutritional intake, malabsorption, evaluation for cfrd, and short stature. these four algorithms were used to develop a nutrition action plan for each individual. beginning in march , a nutritional category (optimal/acceptable, concerning, at risk or failure) was assigned to each patient at each visit based on their nas. an individualized nutrition action plan was developed with each patient. at the time of project implementation, patients age - at our center had a median bmi %ile of . % with . % of our patients with a bmi < th%ile. our patients > years had a median bmi of . with . % having a bmi< th%ile. after one month of implementation of the nas, % of our patients had been categorized with % of our patients having individual nutrition action plan developed. to track our progress, we will monitor median bmi, % of patients with bmi < %ile, and % of our patients in each nutritional category quarterly using portcf. our goal is to have all our patients assessed and a nutrition action plan implemented by the end of . we have developed a nutritional assessment score and nutrition treatment algorithms for pediatric and adult patients with cf as part of a qi initiative to improve nutritional status of patients at our center. the project has been supported, in part, by akron children's hospital. we are indebted to lori lundquist, a parent of two of our cf patients, who has been an integral part of our nutriton qi team. in germany there are about , cf patients who are usually treated in special centres by a team of trained and experienced health care professionals. the result of this structured approach to patient care, including frequent clinical assessments, monitoring, and aggressive interventions is an improved mean survival and a higher quality of life of pediatric and adult cf patients. however, a recent survey showed that german reimbursement schemes cover only about % of resources used within these centres. objective to assess and evaluate direct costs of outpatient treatment of cf patients in germany. a micro-costing approach was used to record resource use data directly within representative cf centres. results of the evaluation may be used to initiate and support discussions between health care providers and insurances about adequate reimbursement schemes in germany. outpatient care was evaluated in seven different centres for pediatric and adult cf patients. patient reported outcomes, clinical patient data, resource utilisation, and physician related time consumption for treatment was recorded for every patient during one representative month in . cost data for staff, materials, medical equipment, rooms, etc. were assessed within the respective centres. data for cf patients were collected. about half of them were children and adolescents, and half of them were adults. a comparison of the resource uses to the actual remuneration of these services indicates that only about % of the costs are covered by the reimbursement system. correlation analysis identified significant cost drivers like the age of the patient, comorbidities like pancreatic insufficiency and hepatobiliary complications, lung function (% fev ), and the presence of certain pathogens in the lungs. as the actual reimbursement covers only about % of the resource usage costs in germany, the existence of the cf centres and sufficient treatment for cf patients is uncertain in the future. in order to ensure the existence of the centres it would be necessary to agree to a cost-covering reimbursement at minimum. this may be based on a lump-sum payment that could be differentiated for pediatric and adult centres or according to comorbidities or clinical parameters. the data calculated in this study could be used to trigger and support discussions between health care providers and insurances about a new cost-covering reimbursement system for the out-patient treatment of cf patients in germany. the impact of the new us cystic fibrosis foundation (cff) nutrition practice guidelines, i.e., discontinuing the use of percentage of ideal body weight (%ibw) to define "nutritional failure" and setting body mass index (bmi) below the th percentile to define "nutritional risk", on evaluating nutritional care practices is unclear. methods: data from children reported to the cff patient registry were analyzed to compare the rates of malnutrition among cf centers. results: nationally, eliminating %ibw < % as a criterion for underweight resulted in a . % reduction (from . % to . %) in "nutritional failure" rate. misclassification of "nutritional failure" by %ibw < % ranged . - . % among centers and was greater for centers having a larger proportion of tall patients. one-third of centers switched to a different tertile ranking, after correcting for misclassification by %ibw. use of bmi < th percentile led to the classification of . % of patients as "nutritional risk". more than half of the centers had different tertile rankings on "nutritional risk" compared to "nutritional failure". a total of . % ( - . % among centers) of patients who had height < th percentile but bmi ≥ percentile were not considered at nutritional risk. the cystic fibrosis questionnaire-revised (cfqr) is a disease specific quality of life measure that is currently undergoing clinical validation and may prove to be a useful adjunct to provider assessments. we proposed the routine use of the cfqr in a busy adult cystic fibrosis (cf) clinic would assist in identifying patients in need of limited clinic resources. this performance improvement project focused on new and infrequently-seen patients (i.e. fewer than three outpatient clinic visits/year) to determine the frequency of patients with domain scores that fall more than one standard deviation below our clinic mean. methods: the cfqr is routinely self-administered to all adult cf patients in clinic at least once every year. these assessments occur after vital signs are obtained and prior to any other clinical assessments. patients were deemed to be at baseline if there were no changes made to their pharmacologic or chest physiotherapy regimens. the mean and standard deviation of each domain of baseline cfqr assessments were determined. the frequency of rarely seen and new patients with domain scores less than one standard deviation below the mean was determined. results ( ). of these baseline assessments, forty five were obtained from patients who were rarely seen or new to the clinic. . % (n= ) of these patients had one or more domain scores falling more than one standard deviation below the mean. . % (n= ) of these patients had two or more domain scores below these cutoffs. % (n= ) of the rarely evaluated patients had domain composite scores (i.e. the sum of the individual domains) that fell below the standard deviation cutoff. the most common outlying domains in these infrequently seen or new patients were body ( ), weight ( ), physical ( ), health perceptions ( ) and the composite domain ( ). conclusions: the cfqr is easy to administer and score during routine adult cf clinical visits. as many as % of our infrequently evaluated patients had multiple scores that were more than one standard deviation below our clinic mean. we conclude that the cfqr is a useful adjunctive measure to provider assessments and can help focus limited personnel resources. aminoglycoside antibiotics are known to be toxic to the inner ear. however, they continue to have a leading role in the treatment of certain infections and in the treatment of pulmonary exacerbations in patients having cystic fibrosis (cf). while there is ample evidence in the literature of ototoxicity, and there are established protocols for monitoring the effects of toxic agents on hearing, less is known about the prevalence of vestibular toxicity among patients who are exposed, and there are no commonly accepted protocols for monitoring vestibular system function. oscillopsia and unsteadiness when standing and walking are the two most commonly reported and disturbing symptoms associated with bilateral loss of vestibular system function. oscillopsia is the perception that viewed stationary objects or surroundings move coincident with head movement. when it is severe, oscillopsia can prevent an individual from having clear vision with even the slightest head movement. the unsteadiness that accompanies vestibular loss can range from mild to disabling. because of the referral of a number of patients to the vestibular testing center (vtc) at the university of michigan in whom severe bilateral vestibular loss was evident secondary to apparent aminoglycoside toxicity and our concern about quality of life issues, we initiated a quality improvement clinical protocol with our cf patients. vestibular system function and hearing are evaluated at the time of admission for pulmonary exacerbations and coincident with the three month follow-up visit with the pulmonologist. our goal is to understand the incidence of ototoxicity in this patient population, and to determine how best to measure incremental changes over time. the ultimate desired outcome is to investigate the efficacy of protective agents for limiting the toxic impact of these drugs on hearing and vestibular function. to date, we have completed vestibular testing on patients with cf ranging in age from - years, some of whom were referred prior to the initiation of this monitoring program. of the sample, individuals have completely normal vestibular test results, and of the had no prior aminoglycoside exposure. of the remaining patients, have bilateral vestibular loss ranging from mild to severe, two have evidence of a unilateral vestibular loss, and each of the others has non-lateralizing evidence of vestibular involvement. we have also documented change in the three individuals we have seen for repeat testing. specifically, repeat testing has shown incremental decline in vestibular function with repeated exposure to aminoglycosides. interestingly, only individuals in the group have documented hearing loss, and although most report that they do not and have never had any problems with dizziness or balance, many patients have evidence of oscillopsia or abnormal postural control test results. it is clear from our limited data that it is important to monitor vestibular system function whenever potentially toxic agents are used. while monitoring hearing is also warranted, our data suggest that a monitoring program that includes only hearing is insufficient. background: the correlation between bmi, lung function, and longterm outcomes in cystic fibrosis patients has been well documented. based on this data the cf foundation (cff) has established adult patient care guidelines for bmi of or greater for women and or greater for men. in our cf center implementation of nutrition therapies in cf adult patients has been difficult due to lack of acceptance of proposed interventions. due to the importance of optimal nutrition to attain the cff recommendation for bmi, university of cincinnati adult cf center implemented a weight management quality improvement protocol to improve the nutritional status of patients below the cff recommended bmi. our aim is to improve to the nutritional outcomes in our center through patients' acceptance of our nutrition interventions. strategies: as part of the weight management protocol we developed a nutrition action plan, nutrition algorithm, and weight management education record and learning log. the action plan took a systematic approach to weight management, classifying patients into different risk categories based on bmi. patients were categorized as mild nutritional insufficiency (bmi - ), moderate nutritional insufficiency , and severe nutritional insufficiency . each category required a specific action to address weight loss or inability to gain weight. areas of focus for the action plan include; intake assessment through computerized dietary analysis, assessment of pancreatic function, pancreatic enzyme replacement therapy (pert) regimen assessment, hour stool collection for malabsorption, assessment of glycemic control, assessment for gerd, and enteral/parental feeding. the dietitian completed a chart review and nutrition assessment with the patients and classified them into one of the three nutritional risk categories. eleven patients with a bmi below were initially identified. nine ( %) of the eleven patients with a bmi of or less have been approached with the protocol and algorithm. patients completed a three day food journal and oral intake was assessed via computerized dietary analysis. oral enteral supplements were recommended - times per day. g-tube placement was discussed and an educational material on tube feeding was distributed. the protocol and algorithm guided identification and assessment of contributing factors to weight loss. results: of eighty patients seen in the adult center since the beginning of october, thirty-seven of the patients ( %) have a bmi below and eleven patients ( %) have a bmi below . out of the nine patients approached with the protocol % (n= ) completed and returned the three day food journal. of the patients that completed their food journal % (n= ) agreed to a peg placement, one of the patients is still contemplating peg placement while sorting through some psychosocial issues, and the other patient has had a differential diagnosis that might negate the necessity for a peg placement. conclusion: we developed a systematic approach to weight management within our adult cf program. this systematic approach to weight management, which includes patient involvement and specific education, has improved the overall acceptance of nutrition interventions in our adult population. the inpatient care of adults with cystic fibrosis can be a challenging given complicated medical regimens, specialized respiratory care orders, and unique dietary requirements. a large proportion of these patients are admitted to academic centers with rotating house staff that may have never treated an adult with cystic fibrosis. to improve the quality of care of our adult patients we have implemented a standardized electronic order set, a recurring educational program, and a "pocket card" to help the house staff. the adult cystic fibrosis program at vanderbilt cares for over patients, with - inpatient admissions per month. vanderbilt's inpatient order system is completely electronic. we have devised an order set based on our agreed standard of care for inpatients with pneumonia. the order set guides the house staff through most aspects of inpatient care including culturing, antibiotic choice and dosing, dietary consultation, nutritional recommendations, and respiratory care orders. this order set not only educates the house staff on our standard of care but also provides direct access to each order they may wish to enter. we have reiterated this educational process with a pocket sized handout that guides them through the typical issues in adults with cf as well as provide phone numbers to members of the cf team. these resources are presented in short talk given at the beginning of each month to incoming house staff. a preliminary data analysis suggests that over % of all adults with cf are admitted using some component of this order set. an informal survey of the house staff also concludes that the pocket card and lectures have been helpful. all three aspects of this quality improvement have been implemented since december of . we plan to formally test the impact of this intervention when a meaningful number of house staff have been through the service. * background: malnutrition is a common complication of cystic fibrosis (cf). the median bmi percentile in cf patients between the ages of and years is . % but ranges between various centers from . to . percent. previous research has shown a trend toward improvement of pulmonary disease and a significant improvement in weight, height, and bmi z-scores after nutritional supplementation via a gastrostomy tube ; furthermore, fev is positively correlated with bmi percentile. higher weight at three years of age predicts better pulmonary function at six years of age. the effect of gastrostomy tube feedings on pulmonary exacerbation frequency has not been well defined. hypothesis: the incidence of pulmonary exacerbations in children will decrease after the initiation of supplemental gastrostomy feedings. methods: a retrospective chart review of all pediatric patients seen at the cmh cf center, and who received gastrostomy tubes between and was performed. data was evaluated in six month time intervals for two years prior and two years after gastrostomy tube placement. we assessed weight, height and bmi percentiles, fev percent predicted, number of courses of exacerbation therapy (intravenous and oral) and microbiologic data. results: we identified patients, females and males, with ages ranging from months to years of age ( . years mean) at time of gastrostomy tube insertion. all patients had increases in their weight-forage percentile (mean of all patients of . percentile pre to . percentile post, p< . ), height-for-age percentile ( . percentile pre to . percentile post, p< . ), and bmi percentiles ( . percentile pre to . percentile post, p< . ). the mean fev percent predicted declined overall from . % to . % ( . percent predicted per year) over four years in patients. there was a trend of exacerbation reduction after gastrostomy tube placement from to total courses (p= . ). this decline in exacerbations after gastrostomy tube placement was most dramatic in those patients growing pseudomonas aeruginosa without other organisms, with a decline from total courses to total courses in patients (p= . ). discussion: weight, height and bmi percentile improved after initiation of gastrostomy tube feedings. lung function (fev % predicted) decreased at the rate predicted in spite of gastrostomy feedings. there is a trend toward decrease in exacerbation frequency after improved nutrition with gastrostomy tube feedings, especially in patients infected with pseudomonas aeruginosa. limitations of this study include the small sample size, relatively short follow-up time, and the retrospective nature of the study which did not allow assessment of adherence. conclusion: improved nutrition through gastrostomy tube placement may decrease the frequency of pulmonary exacerbations. evidence of an association of improved pulmonary function with improved nutritional status has motivated aggressive nutritional intervention. percutaneous endoscopic gastrostomies (pegs) are employed increasingly in cystic fibrosis to optimize nutritional status. we reviewed our center data over the past years to assess changes in our peg practice and effects on nutritional and pulmonary outcomes. we hypothesized that our change in practice to earlier peg intervention improved patient's nutritional and pulmonary outcomes. we conducted a case controlled retrospective study of pediatric cf patients spanning the past years divided into two periods: p (peg placed before ) and p (peg placed or later) compared with their respective age/sex matched controls. we compared peg vs. control cf patients with respect to age, nutritional and pulmonary status. we compared weight, height and bmi directly preceding peg placement and at approximately one-year post peg. we compared available data for best yearly fev and fvc before peg and at , and -years post-peg. we then compared data between the two time periods. results: peg patients in p (n= ; mean age . + . yrs.) had lower bmi ( z = - . v. - . , p< . ) pre-peg placement compared to controls. by year post peg, the bmi normalized and equaled controls (z=- . v. - . , p= . ). peg patients' fev %ile prior to placement was lower than controls ( v. , p= . ) as was their fvc %ile ( v. , p= . ) and remained lower at year post-peg. following peg placement, absolute bmi increased ( . kg/m , p= . ), as did bmi z-score ( . , p= . ). we did not see significant changes in pulmonary function tests (pft). peg patients in p , (n= ; mean age . + . yrs.) also had lower bmi (z= - . v. - . , p= . ) pre-peg than controls. at year post-peg, the peg patients still had a significantly lower bmi (z = - . v. - . , p= . ). fev was lower in peg patients compared to controls in pre-, year, and -year post-peg data (p< . ). variability in pfts and small sample size limited our analysis. pre-peg weight, height, bmi, fev and fvc were lower in p patients compared to the younger p patients. at -year post-peg, similar bmi zscores (- . , p= . ) was obtained between the two periods. variability in pfts among the patients within each period prevented valid comparisons, although p tended to have better pft values. conclusions: peg placement was effective at improving nutritional status in patients with cystic fibrosis. placement in younger patients with better pre-peg nutritional and pulmonary status allowed more complete nutritional recovery. we speculate that improved height z-scores with apparently stable fev is consistent with increased pulmonary growth. demonstration of an attenuated rate of pft decline to correlate with this earlier intervention will require a larger population and longer observation period. a multi-center longitudinal outcome study would help determine optimal timing and criteria for peg intervention. . pediatric gastroenterology and hepatology, university medical center groningen, groningen, netherlands; . biochemistery, erasmus medical center, rotterdam, netherlands background: ursodeoxycholic acid (udca) treatment is frequently applied for cystic fibrosis-related liver disease (cfld). it has been hypothesized that udca is beneficial through its choleretic activity, i.e. its capacity to induce bile flow. the hepatic expression of the cftr protein is restricted to the cholangiocytes lining the bile ducts. cholangiocytic cftr is involved in the generation of ductular bile flow. it is not known to what extent the choleretic activity of bile salt treatment depends on expression of competent cftr protein, and whether or not udca differs in this respect from other bile salts. we evaluated the role of cftr in the acute and chronic choleretic effect of bile salt treatment, through comparative studies in cftr-null, homozygous f del, and corresponding wild type control (wt) mice. methods: bile flow was determined after gallbladder cannulation. bile flow during the first minutes after acute interruption of the enterohepatic circulation was regarded as basal bile flow. after min, taurocholic acid (tca) or tauroursodeoxycholic acid (tudca) were iv administered in stepwise increasing dosages, up to nmol/min/ g bw, to cftr-null mice and wt littermates fed a standard chow diet. other cftr-null, homozygous f del mice and their respective wt littermates were fed either the standard chow, or the same diet supplemented with cholic acid (ca, . wt%) for weeks. finally, cftr-null mice and wt littermates were fed the standard chow or the same diet supplemented with ursodeoxycholic acid (udca, . wt%) for weeks. : upon a regular chow diet, the basal bile flow was similar in cftr-null and homozygous f del mice, compared to their respective wt littermates (cftr-null, . ± . vs. . ± . ; and, f del(∆/∆), . ± . vs. . ± . µl/min. g bw, resp.; ns). iv administration of tca or tudca to cftr-null mice and wt littermates increased bile flow to similar extents. dietary ca treatment increased basal bile flow significantly less in cftr-null and homozygous f del mice than in their respective wt littermates (cftr-null, . ± . vs. . ± . , p< . ; and, f del(∆/∆), . ± . vs. . ± . µl/min. g bw, p= . ; resp.). interestingly, dietary udca treatment increased basal bile flow more profoundly than ca treatment, and to similar levels in cftr-null mice and wt littermates (+~ %, . ± . vs. . ± . µl/min/ g bw, resp.; ns). conclusion: upon chronic treatment, udca is highly choleretic in mice, independently of the presence of functional cftr. the independence of functional cftr is udca specific, since the choleretic activity of chronic ca treatment is diminished in cftr-null and homozygous f del mice. we speculate that this specific, cftr-independent choleretic effect of chronic udca treatment could be therapeutically important for cystic fibrosis. most cystic fibrosis (cf) patients have mild to moderate focal portal tract changes (ductal obstruction and cholangitis) and hepatosteatosis. etiology of severe liver disease (ld)with cirrhosis and portal hypertension which occurs in - % of cf patients is unknown. the c bl/ j congenic cf mice develop progressive cf-like ld. as a useful therapeutic model, we have shown that dietary addition of docosahexaenoic acid (dha) significantly ameliorates inflammation but has little effect on ductal obstruction (am j physiol gastrointest liver physiol : g -g , ) . we hypothesized that treatment with udca,a hydrophilic bile acid will ameliorate portal tract ductal obstruction and have an additive effect with dha in preventing ld. methods: thirty-day old wild-type and cf littermates are fed peptamen and either olive oil or dha, with and without . % udca ( groups, mice/group). mice are killed following days of treatment and bile salts, serum liver enzymes and serum and tissue lipids analyzed. h&e stained liver tissues are coded and assessed blindly by an expert hepatopathologist (jp) for evidence of hepatosteatosis, inflammation, bile duct obstruction, fibrosis and other signs of liver pathology. an arbitrary scale of to is used, with representing no pathology and being the most severe. we present interim statistical analysis performed for pathology results only for untreated mice (wt, n= ; cf, n= ); treated mice with dha, (wt, n= ; cf, n= ), treated mice with udca (wt, n= ; cf, n= ) and treated mice with udca and dha (wt, n= ; cf, n= ). results: mean pathology scores from groups of mice are pooled to examine the effects of genotype, treatment and diet (table) . at present,the bile,lipid and liver biochemical test data are insufficient to perform statistical analysis. udca-treated mice have significantly increased bile duct obstructed scores compared to the untreated mice which subanalyzed (not shown) reveals increased bile duct obstruction in the wild-type littermates and no effect in cf mice. hepatosteatosis and inflammatory scores in the untreated mice (fed olive oil) are increased compared to the mice treated with dha. summary: we anticipate completing and performing final analysis within six months. however, these preliminary data confirm that dha reduces inflammation in c bl/ j mice livers and suggest that udca therapy has an adverse effect in wt mice and is not effective in ameliorating liver disease in cf mice. udca with dha therapy does not appear to have an additive effect in ameliorating cfld in this mouse model. supported by axcan pharma inc. background the cf mouse intestine has an innate response associated with small intestinal bacterial overgrowth (sibo). a previous affymetrix genechip analysis of the cf mouse intestine showed altered expression of several eicosanoid metabolic genes. eicosanoids are biologically active arachidonic acid metabolites with a variety of pro-and anti-inflammatory actions as well as effects on mucus production, electrolyte transport, and gastrointestinal motility, all of which are of potential importance to the cf phenotype of the intestine. methods wild type (wt) and cf mouse (cftr tm unc ) littermates congenic on the c bl/ j background were fed an elemental liquid diet (peptamen). intestinal rna was isolated for quantitative rt-pcr of expression levels of genes involved in eicosanoid metabolism to confirm and extend the genechip analysis. various eicosanoids were measured in small intestinal lavage fluid using specific enzyme immunoassays. results we confirmed microarray results by qrt-pcr that the following genes were differentially expressed in the cf small intestine relative to wt: cyp a (cytochrome p a ; -hete synthesis; % of wt); ltb dh (leukotriene b dehydrogenase; metabolizes prostaglandins and leukotrienes; % of wt); cyp c (cytochrome p c ; -hete synthesis; % of wt); ltc s (leukotriene c synthase; ltc synthesis; % of wt); hpgd (hydroxyprostaglandin dehydrogenase ; metabolizes prostaglandins; % of wt); and pla g (group phospholipase a ; potentiates arachidonic acid generation; % of wt). non-significant or less than -fold changes in gene expression were measured for: cyclooxygenase (cox) genes, pge synthase, pgi synthase, and -and -lipoxygenases. as measured by their stable metabolic products, pge and pgf α were significantly increased in the cf mouse intestine ( % and % of wt, respectively). the following eicosanoids were significantly decreased in the cf mice: -hete ( % of wt), -hete ( % of wt) and -hete ( % of wt). there were not significant changes in the levels of pgi , pgd , ltb , cys-lts, or lxa . the changed levels of eicosanoids are generally consistent with the differential gene expression. conclusions there are significant changes in expression of eicosanoid metabolic genes and certain eicosanoids in the cf mouse small intestine. the increase in pla g and decrease in cyp expression levels are expected to make more arachidonic acid available for eicosanoids except hetes. decreases in degradative genes (hpgd, ltb dh) will prolong availability of prostaglandins. altered levels of eicosanoids are expected to play important roles in the pathophysiology of the cf intestine. elevated pge may contribute to increased mucus secretion which is characteristic of cf. pgf α and pge regulate intestinal motility and small intestinal transit is slower in cf patients and in cf mice. the hetes are mostly known for their effects on vascular tone and electrolyte transport in the kidney but are less-well characterized in the intestine. their dramatic decrease in the cf intestine may affect blood flow, electrolyte transport, intestinal motility, or other gi functions. the specific effects of the altered eicosanoid metabolism in cf remain to be explored. supported by cff grant delisl g . for the scandinavian study consortium. background: progressive pulmonary disease, correlating with igg levels, is a major cause of morbidity and mortality in cf patients. recently, immunosuppressive effects of vitamin d and a potential role of vitamin d deficiency in the pathophysiology of autoimmune diseases have been described. we investigated whether vitamin d deficiency could contribute to the chronic proinflammatory state characterizing cf and the higher occurrence of immune-hyperreactivity-related conditions in cf patients. methods: multiple linear regression analysis, taking the confounding factor of age into account, was used to evaluate the relationship between cross-sectionally determined vitamin d variables and: lung function parameters, inflammatory parameters and immune hyperreactivity markers in patients followed at the cf centers in sweden, norway and denmark. vitamin d intake was based on a -day precoded dietary food record, calculated in the national food databases. findings: in the population of all patients included in the study (n= ), blood vitamin d positively correlated with fev (p< , ) as well as with fvc (p< , ). negative correlation was found between blood vitamin d and igg (p< , ), supplemented vitamin d per kg bw and igg (p< , ), total vitamin d intake per kg bw and igg (p< , ). additionally, lower blood vitamin d levels were related to both presence of pathological ogtt (p< , ) and occurrence of cfrdm (p< , ). correspondingly, food vitamin d (p< , ), food vitamin d per kg bw (p< , ), supplemented vitamin d (p< , ), supplemented vitamin d per kg bw (p< , ), total vitamin d intake (p< , ) and total vitamin d intake per kg bw (p< , ) all correlated negatively with hba c values. most of the studied vitamin d variables were lower in patients with endomysial antibodies positivity (n= ) and higher in patients with abpa (n= ) than in the rest of cf population studied (ns). interestingly, in children (n= ) blood vitamin d correlated negatively with transglutaminase levels (p< , ) and there was also a significant negative correlation between hba c and vitamin d intake. surprisingly, in adult cf patients (n= ) iga positively correlated with total vitamin d intake (p< . ) and total vitamin d intake per kg bw (p< . ). in the population of stockholm cf-center patients (n= ), where it was possible to allow for the influence of both age and compliance, negative correlation between blood vitamin d and igg (p< . ) was found. after allowing for age, compliance showed significant correlation only with igg (p< . ) and esr (p< . ), and with none of the rest of the studied variables. conclusions: vitamin d deficiency might contribute to the continuous shift towards inflammation as well as to the higher prevalence of dm and celiac disease in cf patients. new recommendations for vitamin d supplementation and monitoring of blood vitamin d levels are needed, in order to improve immunological balance and decrease the need for anti-inflammatory therapy in cf patients. supported by swedish heart lung foundation, stiftelsen frimurare-barnhuset i stockholm, norwegian and swedish cystic fibrosis associations, and by an unrestricted grant from solvay pharma. von drygalski, a.; biller, j.a. medical college of wisconsin, milwaukee, wi, usa anemia is associated with increased morbidity and mortality in other chronic diseases, but little is known about anemia in cf. recognition and correction of anemia in cf might lead to better outcomes. chronic inflammation, iron deficiency and malabsorption may be important mechanisms contributing to the anemia in cf patients. we hypothesized that anemia might be correlated with poor lung function. methods: clinical charts and portcf.org visit logs of patients of all ages ( m to yrs) with cf were reviewed for as many years as charts permitted (range - yrs). the following data were extracted: cbc, iron studies, pulmonary function tests (pfts), vitamins a, d, and e levels, creatinine, pertinent medical history, and current medications. anemia was defined by age-and gender-specific world health organization (who) criteria. patients were considered anemic if low hemoglobin(hb) was present on two separate occasions at least two months apart, or average annual hb met who criteria. pfts in patients (all > yrs) included forced expiratory volume (fev ) and forced vital capacity (fvc) as percent predicted of normal and were considered a reflection of patient performance. the most representative annual pft from at least annual tests was chosen for analysis since acute infection adversely influences daily performance. results: of patients were anemic (prevalence %). prevalence of anemia increased with age from . % in patients < age to % in patients > age and was significantly higher in patients with vitamin deficiencies. % vitamin-deficient patients vs. . % non-vitamin deficient patients were anemic (p= . ). mean hb was . mg/dl (range . - . ). roughly two thirds of patients had moderate and severe anemia (hb < mg/dl) with no gender difference in regard to prevalence or severity identified. complete iron studies were available in / patients. were identified as iron deficiency anemia (ida), as anemia of chronic illness(aci), and as combined; others had renal failure. in patients, iron studies were incomplete, but renal failure, hemoptysis, hematochezia and solid organ transplants were contributing factors in half. pfts obtained in patients ≥ yrs were compared in anemic and non-anemic patients. mean fev and fvc at all ages were statistically significantly poorer in the anemic patients (p < . ). complete iron studies were also available in non-anemic patients with impaired lung function. / patients had ferritins < ng/ml suggesting relative iron deficiency. conclusions: the prevalence of anemia in cf patients is high and increases with age. in patients for whom iron studies were available, ida was the prominent underlying mechanism, followed by aci or a combination of both. in addition, a group of non-anemic patients with poor lung function had relative iron deficiency. the strikingly significant association between anemia and poor pulmonary function as well as the higher prevalence of anemia in vitamin deficient patients uncover anemia as significant co-morbidity in cf. identification of iron deficiency in anemic as well as in non-anemic patients with poor lung function could be important since iron supplementation might allow hb levels to increase as an appropriate response to hypoxia. oral administration of dha to a cf mouse model corrected the lipid imbalance and reversed certain pathological manifestations in tissues affected by cf. the aim of this study was to investigate the metabolism of la through the n- pathway, and to determine the effect of dha supplementation on la metabolism in cultured cells with a cf phenotype. methods: sense (wt) and antisense (cf) cftr hbe cells were cultured in mem containing % horse serum. cells were supplemented with various concentrations of la (ranging from to µm) and dha ( , , and µm) for one week. cellular lipids were extracted from confluent monolayers, and fatty acids were methylated and analyzed by gc-ms. metabolism through the n- pathway was studied by incubating the cells with . µm [ c] la for hours, and quantitating its downstream metabolites by hplc. results: the levels of la and dha were significantly decreased in cf cells compared to wt cells (la in wt: . ± . , cf: . ± . %, p< . ; dha in wt: . ± . , cf: . ± . %, p< . , mean ± sem for each). la supplementation resulted in an increase in la and aa levels in both wt and cf cells. at most la concentrations tested, la levels were significantly lower and aa levels were significantly higher in cf cells, indicating an increased production of aa from la in cf cells. the study of [ c] la metabolites showed an increased production of multiple downstream fatty acids including : n- ( . fold), aa ( . fold), and : n- ( . fold) in cf cells compared to wt cells, further substantiating an increased metabolism through the n- pathway. supplementing hbe cells with dha for week resulted in a significant increase in la levels and a corresponding decrease in aa levels in wt and cf cells. when supplemental dha was added in combination with la, dha inhibited the la-derived increase in aa levels in cf cells and normalized them to the wt values. the formation of [ c] la metabolites was inhibited after -hour supplementation with dha. this inhibitory effect of dha was greatest on aa production, and it was more prominent in cf than in wt cells (wt: . fold, cf: . fold decrease in aa production). conclusions: decreased la levels in cf are at least in part related to increased la metabolism through the n- pathway. the fact that dha supplementation decreased the flux from la to fatty acid metabolites in the n- pathway, especially to aa, may partially explain dha's mechanism of therapeutic benefit. these data would also suggest that dha combined with la in the diet may normalize fatty acid metabolism in cf patients. background: decreased levels of linoleic acid (la) and docosahexaenoic acid (dha) have been found in the blood and tissues of cf patients. studies on cultured cf cells and cftr-/-mice have indicated that decreased la levels in cf might be related to its increased metabolism through the n- pathway. other potential mechanisms of the observed decreased la and dha levels in cf could be related to altered cellular uptake of these two essential fatty acids. the goal of this study was to investigate the uptake of la and dha into cultured cf cells, and their distribution among the major lipid classes. methods: sense (wt) and antisense (cf) cftr hbe cells were cultured in mem containing % horse serum. the uptake of la and dha into the cells was studied by incubating the cells with . µm [ c] la or [ c] dha for hour and hours, followed by measurement of radioactivity in the supernatant and cell lysate. lipid fractions from the cells (total phospholipids, triglycerides, cholesteryl esters, and free fatty acids) were separated by thin layer chromatography (tlc), and the fatty acid composition was determined by gc-ms after methylation. incorporation of la and dha into cellular lipid fractions was determined by incubating the cells with . µm [ c] la or . µm [ c] dha for hour and hours, followed by tlc separation of lipids and measurement of radioactivity associated with the various fractions. (all data are mean±sem). results: the levels of la and dha were significantly decreased in cf cells compared to wt cells (la in wt: . ± . , cf: . ± . %, p< . ; dha in wt: . ± . , cf: . ± . %, p< . ). fatty acid composition analysis of lipid fractions indicated that la levels were significantly decreased in the phospholipid and triglyceride fractions of cf cells ( . % and . % of the wt values, respectively), and dha levels were significantly decreased in the phospholipid fraction of cf cells ( . % of the wt values). the uptake of la was significantly higher into cf cells compared to wt cells at hour (wt: . ± . , cf: . ± . dpm/µg protein, p< . ) and hours (wt: . ± . , cf: . ± . dpm/µg protein, p< . ). the uptake of dha was also significantly higher in cf cells compared to wt cells at hour (wt: . ± . , cf: . ± . dpm/µg protein, p< . ) and hours (wt: . ± . , cf: . ± . dpm/µg protein, p< . ). incorporation of la and dha was increased in the phospholipid, cholesteryl ester, and free fatty acid fractions of cf cells at hours. dha, but not la, incorporation was significantly increased in the triglyceride fraction of cf cells. conclusions: low la and dha levels in cf cells are associated with a decrease in cellular lipid fractions, the largest of which is in total phospholipids. there is an increased uptake of la and dha into cf cells, and into most lipid fractions of these cells. the decreased levels of la and dha in cf cells, despite their increased cellular uptake and esterification in lipid classes, indicate that there is increased mobilization of these fatty acids in cultured cf cells. perez, a.; issler, a.; davis, p.b. pediatrics, cwru, cleveland, oh, usa even though lung disease continues to be the primary cause of death in cystic fibrosis (cf), the considerable increase in life expectancy of the cf patient over the last decades, has made management of the diverse gastrointestinal and nutritional complications in the adult cf increasingly important. malnutrition and fatty acid deficiency has been a hallmark of cf and its impact in long-term lung health has been established. data obtained by us using affymetrix mg-u av arrays from ileum and liver of pristine -week-old male mice bearing the y x cf mutation and their normal littermates, indicates that there is a profound disturbance in genes associated with lipid metabolism, many of which are regulated by the peroxisome proliferator-activated receptor-gamma (pparγ), a ligand-regulated transcription factor that regulates lipid metabolism and possess anti-inflammatory properties. we hypothesize that lack of cftr function results in altered expression of pparγ regulated genes in the gut and liver, leading to the fatty acid metabolism abnormalities observed in cf. pparγ mrna and protein levels were measured in ileum, colon, liver, and fat of week old female mice bearing the cf mutation s x and its wt littermates (c bl/ j background), and fabpcftr mice (gut corrected, mixed background) after the administration of ethanol or pioglitazone (a pparγ agonist, mg/kg) by gavage every h for days, sacrificed h after last dose. highest pparγ mrna levels were found in fat, followed by colon, liver, and lowest in ileum. at basal state, mrna levels were higher only in fabp ileum vs. wt and fabp colon vs. cf. after treatment, higher expression was seen in cf and fabp ileums and cf liver vs. controls and wt treated mice. however, there were striking differences at the protein level, especially in the colon. at basal state, pparγ was localized, mainly at the surface of the epithelia in wt, in crypts in cf, and mainly in crypts but extending towards the surface in fabp mice. upon treatment, pparγ location in wt colon moved almost exclusively to the crypts, and in the cf and fabp mice, although expression remained mainly in crypts, pparγ could now be seen at the surface. changes in pparγ protein were also seen in ileum and especially liver, between wt and cf, at basal and upon treatment, but changes were subtle and will require more sensitive methods to further examine them. we also examined mrna and protein levels for several genes involved in fat processing that could be related directly or indirectly to pparγ. mrna and protein levels for aqp and cubilin, and protein levels for megalin, apoa- , and apob- were reduced in the ileum of cf mice compared to wt. our data indicates a pparγ dysfunction in the gastrointestinal system of cf mice, which could be responsible for the gastrointestinal manifestations observed in cf. understanding the association between pparγ, cftr, and fat processing may help us to develop new therapies directed towards improving the nutritional status of cf patients that, in the long run, will influence their lung health. cf is characterized by low linoleic acid (la) and docosahexaenoic acid (dha) levels. although this could be due to a primary abnormality in fatty acid biosynthesis, this could also be explained by alterations in phosphatidylcholine (pc) formation. pc are formed by two pathways. the most utilized is the de novo conversion of choline to pc via the cdp-choline pathway, which preferentially forms pc containing saturated and monounsaturated fatty acids. in the other pathway phosphatidylethanolamine (pe) is converted to pc through three methylation steps utilizing the enzyme phosphatidylethanolamine n-methyltransferase (pemt). this pathway preferentially forms pc containing polyunsaturated fatty acids. thus decreased pemt activity would be predicted to result in decreased dha and la levels. this is supported by results seen in pemt knockout mice (watkins et al, j nutrition, ) showing decreased dha and la levels, and variable to increased levels of arachidonic acid (aa). furthermore, innis et al have shown that cf patients have decreased choline levels making them more dependent on the pemt pathway. we hypothesized that alterations in fatty acid levels in cf may be due at least in part to decreased pemt activity. methods: cftr -/mice (cf) and wt littermates were fed with either a diet containing choline (control) or, in order to mimic choline status in humans, an otherwise matched choline deficient (cd) liquid diet for days. choline levels were measured using a kit. livers were perfused and microsomes prepared. to measure pemt activity, microsomes were incubated with radioactive s-adenosylmethionine in the presence of dimethyl pe. the pc product was extracted and radioactivity measured. fatty acids from liver and pancreas were analyzed by gc/ms. results: there was no difference in choline levels in serum or liver comparing wt and cf mice. there was no difference in pemt activity in livers from cf mice compared to wt on control diet. cf mice on cd diet had decreased pemt activity in the liver compared to wt mice on cd diet (wt: ± ; cf: ± cpm/mg protein/min; p< . conclusion: induction of choline deficiency in cf mice to mimic the situation in cf patients, leads to decreased pemt activity. this resulted in low la levels compared to wt mice in both the liver and pancreas and indicates that the fatty acid abnormality in cf is at least in part due to defective pc metabolism through the pemt pathway. ( ). urolithiasis appears to be more common in adult cystic fibrosis (cf) patients than in the general population. a variety of urinary findings have been observed which could account for this, including low volumes, raised oxalate and low citrate, but not all the reports are consistent. most cf patients have pancreatic insufficiency and a plausible explanation for an elevated oxalate excretion in these patients is malabsorbed fat in the intestinal tract sequestering calcium and permitting more oxalate to be absorbed from their food. if this were the mechanism for the elevated stone risk in cf patients then we would expect to observe differences in stone incidence and oxalate excretion between cf patients with exocrine pancreatic sufficiency (ps) and pancreatic insufficiency (pi). methods diagnosis of cf is by clinical and genetic criteria and ps or pi status is based on faecal elastase measurement. patients and healthy volunteers (hv) provided h urines for analysis of volume, ph, ca, mg, na, k, po , oxalate, citrate, creatinine, urea. supersaturations were calculated using equil . there is a history of renal stone disease in out of cf patients attending our unit. patients have pi and include of the stone formers. the remaining stone former is amongst the ps patients. no significant differences in urine chemistry or derived supersaturations were found between the cf patients without a history of stones that were ps (n= ) or pi (n= ). nor were any such differences found when stone forming cf patients were included (ps, n= ; pi, n= ) or when the healthy volunteers were included in the ps group (ps, n= ; pi, n= ). amongst the cf patients, only citrate and creatinine excretions were significantly different between stone formers (n= ) and non stone formers (n= ) (medians (mmol/ h), citrate, . vs. . , p= . ; creatinine, . vs. . , p= . ) . the difference in creatinine excretion can be accounted for by the difference in body mass of these two groups (median (kg) . vs. . , p= . ) compared to the hv, the cf patients with a history of stones had significantly lower citrate excretion and higher calcium oxalate supersaturation (medians, citrate (mmol/ h), . vs. . , p= . ; . vs. . , p= . ) conclusions there is no evidence to suggest that the prevalence of stones is different in the ps and pi groups nor could we detect any difference in excretion of oxalate or any other urine variable related to pancreatic sufficiency. comparing those with and without stones, the only consistent finding was a lower citrate excretion by stone formers. between the hv and stone formers this was also associated with a significantly reduced calcium oxalate supersaturation. our evidence does not support the hypothesis that fat malabsorption by the majority of cf patients contributes to an elevated risk of renal stone disease. we undertook assessment of the use of a hypopharyngeal sensor for detection of laryngopharyngeal reflux in infants and children. gastric reflux in the airway, or supraesophageal reflux, commonly takes a gaseous form that cannot easily be measured using conventional technology. the miniaturized ph sensor at the tip of the dx-ph probe is the only sensor able to measure ph in the airway. fifteen infants and children referred to the pediatric pulmonary center for assessment of chronic cough, hoarse voice, or uncontrolled asthma unresponsive to usual medical intervention underwent - hour to placement of the dx-ph probe in supplementation of radiologic assessment of gastresophageal reflux. the probe was placed transnasally and visualzed in the hypopharynx with the red light at the tip. drops in ph < were correlated with food and symptom diaries. positive results were found in / patients, leading to surgical intervention in three patients. one patient with cystic fibrosis age months with failure to thrive underwent study for symptoms of cough. upper gi demonstrated no gross gastroesophageal reflux or anatomic obstruction.gastric emptying scan was mildly prolonged. outpatient supraesophageal ph probe demonstrated drops to ph< associated with respiratory symptoms. patient underwent nissen and g tube placement with resolution at six week follow-up.the patient returned at age six months with onset cough and underwent repeat study. there were no drops in ph and the cough responded to oral steroid burst. the device was tolerated in / patients with early removal in one young adult with endstage neuromuscular disease and in an infant with "colic" due to a hysterical parental reaction.as a result of our experience we have incorporated the use of supraesophageal ph monitoring into our practice, with particular attention to infants with persistent asthma and children with histories suggestive of gastric asthma. we have now incorporated it into our vivometrics lifeshirt, an ambulatory physiologic monitoring device which will allow us to examine supraesophageal ph and its relationship to sleep apnea, cough, tachycardia, tachypnea, and hypoxemia. aim: monitoring and adjusting dose requirements of pancreatic enzyme replacement therapy (pert) are an integral part of the dietetic assessment of patients with cf. we investigated characteristics of enzyme use in our adult clinic population and determine the extent to which inappropriate enzyme use contributed to poor nutritional and clinical state. method: information was collected using an annonymous self-administered questionnaire developed to measure patient practice, knowledge and beliefs on pert. exclusion criteria included pancreatic sufficiency, < units lipase/kg/d, and fev < %. results: out of potential clinic population of patients completed the questionnaire ( - y, % male, fev - %). % of participants reported to never miss enzymes with meals; this was considerably lower for snacks ( %). those patients who omit enzymes with meals also missed enzymes with snacks (r = %, p< . ). a more appropriate use of pert was observed in patients with lower as opposed to higher bmi. despite intensive dietetic input % of patients missed pert with foods containing fat and % took pert inappropriately with food and drink that did not contain fat. the results identified potentially better practices for measuring pert behaviour and knowledge: ) taking pert with all meals, ) taking pert with majority of snacks ) swallowing capsules intact rather than splitting them open, ) carrying enzymes around with them and ) the ability to titrate pert in accordance to the fat content of food. patients were scored on the basis of the above criteria to differentiate between prudent enzyme users and those who are compromising therapy. in conjunction with their nutritional status score (bmi and gastrointestinal symptoms) risk for intervention can be assessed. discussion:underweight patients have more optimal enzyme use, suggesting greater dietetic involvement in these patients. schall et al also found this to be the case in children. the findings emphasised the need for targeted and effective input in patients where problems are less obvious. the questionnaire has been a useful research tool, and has been adapted as a screening tool for dietitians to gain a subjective perspective of patient's enzyme management and identify patients who need support. the combination of patient's pert usage and their nutritional status could help capture and identify risk objectively and quickly and allows resources to be allocated most effectively. schall . ), nutritional failure, measured as bmi< , (p> . ), or lung transplant status (p> . ) between patients with and without colonic wall redundancy. there was no significant difference in cftr function, per sweat chloride values, between groups (p> . ). conclusion: we report a previously undescribed colonic wall abnormality seen on ct in patients with cystic fibrosis: proximal colonic wall redundancy was found in % of adults with cystic fibrosis, but not in children, appeared to be chronic, and was associated with non-∆f cftr gene mutations, particularly the g x mutation. recognition of the ct appearances will prevent erroneous diagnosis of acute colonic disease in this patient population, and stimulate investigation of a biologic basis for this finding as a recognizable feature of adult cystic fibrosis. intro: infants and children with cystic fibrosis, both pancreatic sufficient and insufficient, are at risk for developing hyponatremia due to an excess loss of salt though skin during sweating. these children should receive daily supplemental sodium. children without cf require - meq/kg of sodium daily; and one could argue children with cf need more. a historical recommendation is / tsp table salt daily for infants; this contains to meq of sodium based on the density of the table salt. parents may measure the salt inaccurately, and it may be difficult logistically to distribute the salt throughout the day. if supplementation is given as sodium chloride solutions distributed through pharmacies, as suggested in a recent consensus conference, co-pays or full payments may be required. our aim is to provide a simple, inexpensive, and more accessible method for accurately giving sodium supplementation. do pre-packaged salt packets contain a precise amount of sodium and thus could be recommended to fulfill the daily requirement of sodium in patients with cf? method: pre-packaged table salt packets were collected with permission from four national fast food restaurants and one commercial salt producer. contents (sodium chloride) of ten packets from each source were weighed individually. measurements were made using a scientific scale with the ability to measure up to / , of a gram. weights of sodium chloride from individual packets were converted to meq sodium. means, ranges and standard deviations were calculated for the data using microsoft office excel ™ program. see table conclusions: there is some variability in the content of pre-packaged salt packets, both between and within brands, though some brands have less variance than others. measurements showed that packages contained mean values of approximately to meq of sodium. there is a range in the sodium dose recommendations, and spot urine sodium can be used to determine if patients are sodium depleted. pre-packaged salt packets lack the precision of pharmacological dosing but are inexpensive, practical, and supply a reasonably predictable dose of sodium. background: ten to forty percent of people with cf have vitamin d deficiency secondary to pancreatic insufficiency. obtaining optimal vitamin d levels ( -ohd) has been a challenge. for vitamin d deficient individuals, current supplementation strategies ( , iu weekly for months) have been mostly unsuccessful. objectives: to identify individuals at risk for vitamin d deficiency and to evaluate the efficacy of a week repletion course of high dose ergocalciferol in children and young adults with cf. methods: as part of a quality improvement initiative, a prospective cohort study was performed from january to april . phase i included querying our cf practice database to identify patients who were due for annual routine blood tests or who had recently documented levels of -ohd less than ng/ml. in phase ii, , iu of daily ergocalciferol was prescribed for a day period as part of routine inpatient or outpatient care. during phase iii, a post treatment -ohd level was obtained. baseline subject characteristics were obtained at entry and included age, gender, pubertal status, pancreatic status, and fev %. post -ohd levels were classified as sub therapeutic (< ng/ml), therapeutic ( - ), high therapeutic ( - ), or potentially toxic (> ). a paired t test was performed to evaluate pre and post intervention differences. the impact of age, gender, and lung function on the response to vitamin d supplementation were also analyzed. all values are expressed as mean ± standard deviation. results: eighteen individuals with cf participated in the study. the mean age was years with a range of to years. . % were male, and % were pancreatic insufficient requiring pancreatic enzyme replacement. fev % was . ± . %. all participants had -ohd levels less than ng/dl pre-treatment. -ohd levels increased from . ± . to . ± . (p< . ). of the participants ( . %) became therapeutic over the two week interval. an increase of . ± . (ng/ml) or % was seen in the two week period. no correlation was seen on the extent of increase in -ohd and baseline lung function. pre and peri pubertal individuals had a greater increase in -ohd levels than post pubertal individuals ( . ± . vs . ± . , p< . ) although the magnitude of change did not reach significance. the degree of response also appeared related to age (r=- . ). no impact of gender was seen. seven individuals achieved normal therapeutic values while were in the high therapeutic range. no participants had toxic levels. conclusions: the results of this study demonstrate that high dosing of ergocalciferol over a day period is an effective strategy in achieving therapeutic levels of vitamin d. it is unclear whether a pubertal effect on the degree of response exists or if this response is merely age related. further research is needed to evaluate whether this strategy is able to maintain therapeutic levels after completion of the intervention. additional studies that monitor compliance and evaluate responders and non-responders are needed. foundation consensus committee established guidelines for optimizing bone health in individuals with cf, including screening for vitamin d insufficiency and a treatment protocol for achieving and maintaining normal serum vitamin d concentrations. however, since the guidelines were published, there has been additional research assessing various vitamin d treatment regimens and their effect on serum vitamin d concentrations. in addition, vitamin d assay variability has recently been well documented. the objective of this study was to evaluate variability of vitamin d surveillance methods as well as treatment protocols used to treat low vitamin d concentrations at cf centers. methods: a survey was created via survey monkey, and was sent to the cf nutrition listserv, which includes over cf healthcare providers. spss version . was used for statistical analysis. results: fifty-nine listserv members responded to the survey. the majority ( %) of respondents measured oh vitamin d concentrations, and % considered patients to be vitamin d deficient when serum concentrations were below ng/ml. however, % were monitoring only , oh vitamin d or a combination of both concentrations. there was considerable variability in vitamin d supplementation protocols for treating low oh vitamin d concentrations. only % were following the consensus guidelines of , units of ergocalciferol once/week for ages through adults. one third indicated that they followed consensus guidelines for patients less than age ; however nearly half of the remaining two thirds were not sure what is prescribed or have not observed low vitamin d concentrations in this age group. fifty-two percent of respondents were aware of vitamin d assay variability. despite this knowledge, the majority were unaware of the type of vitamin d assay used to measure serum vitamin d concentrations at their institution. conclusions: there is considerable variability in the measurement and treatment of vitamin d concentrations across cf centers despite guidelines provided via a cff consensus report. the consensus guidelines for measuring vitamin d concentrations and treatment of vitamin d insufficiency should be revisited based on recent studies and expert opinion. children with cystic fibrosis (cf) who suffer from sub-optimal growth are offered overnight enteral feeding via gastrostomy tube. this study aimed to assess the nutritional impact of overnight feeding over a year period using a retrospective study design. data, including height, weight, fev , pancreatic function were collated for all patients with cf who had a gastrostomy tube placed in the past years. data were collected at baseline and then every months for years post-gastrostomy insertion. data for height, weight and body mass index (bmi) were converted into z scores using centre for disease control reference ranges and the lms method ( , ) . a total of patients ( . % of the clinic population) were identified as having a gastrostomy tube. two were no longer receiving feeds and there were incomplete data for subjects. on placement of the tubes, there were substantial deficits in nutritional status with mean z scores for height - . (sd . ), weight - . (sd . ) and bmi - . (sd . ), with compromised lung function (mean fev %, range - ). using a repeated measures anova with fev as a co-variate, there were no significant differences in weight, height or bmi from baseline to years (at years post placement: mean z scores for height - . (sd . ), weight - . (sd . ) and bmi - . (sd . ). fev remained stable over time. these results indicate that gastrostomy feeding can potentially halt the decline in nutritional status that is a feature of cf. however, patient expectations that over night enteral feeding will lead to an increase in nutritional status need to be sensitively managed. background : cystic fibrosis (cf) is the most common life threatening autosomal recessive disorder in caucasians.following a landmark paper by corrie et al a high fat,high calorie diet has promoted a normal growth pattern.improved nutritional status together with prevention and early treatment of respiratory infections has contributed to improved survival. objective: the aim of this study was to assess the relationship between lean body mass and disease severity in children with cf. methods: the nutritional status and body composition of children with cf was measured using a harpenden stadiometer, calibrated electronic scales and a ge lunar prodigy densitometer. the following indices were calculated; body mass index (bmi), fat mass (expressed as logarithm % total body fat (ln%tbf) and fat free mass (ffm). body composition data were expressed as z-scores using dutch reference values. correlations were performed between indices of nutrition (bmi, height, ffm, tbf) z scores and markers of disease severity (percent predicted fev and shwachman kulczycki (sk) scores).the nutritional status of these children was also compared with healthy controls. statistical analyses were performed using microsoft excel . results: there was a weak but significant correlation between sk scores and height z score and ffm (r = . , p< . & r = . , p= < . respectively). there was also a weak but significant correlation between % predicted fev and bmi and ffm (r= . , p < . , r= . , p< . ). height and weight z scores were significantly lower in children with cf (- . and - . respectively) than in control subjects ( . and . respectively) with p < . in both groups. conclusion: this study demonstrates that there is an association between indices of nutrition and disease severity in children with cystic fibrosis. muscle mass (ffm), assessed by dual energy x-ray absorptiometry (dxa) correlates with lung function and with sk scores. the strongest correlation between markers of disease severity (sk scores and % predicted fev ) was with ffm. ffm may be expected to be associated with respiratory function tests.however,sk scores are a more general assessment of well being and less obviously directly related to muscle mass. ffm and growth may reflect long term nutrition and health in contrast to bmi and ln%tbf which may reflect acute deterioration or short term nutritional intervention. therefore ffm may prove to be a useful tool to assess quality of nutrition and may be predictive of respiratory and general decline. body composition assessment has an important role in chronic conditions such as cystic fibrosis, in order to identify nutritional depletion and evaluate nutritional interventions. it is important that reliable and accurate methods are used. the usefulness of non-invasive methods, such as skinfold thickness measurements (sft) to measure change in body composition has not been evaluated in cf. this study aimed to compare changes in fat-free mass (ffm) and fat mass measured using sft and dual-energy x-ray absorptiometry (dxa) in adults with cf. methods: adults with cf ( % male, % pancreatic insufficient, mean age at baseline . (sd . ) years, mean fev at baseline . (sd . ) % predicted) were studied. they underwent body composition assessment using dxa and sft, at baseline and a mean of . (sd . ) years later. durnin and womersley equations were used to estimate ffm and fat mass from sft. estimates of change in ffm and fat mass obtained using sft were compared with dxa using paired t-tests, univariate analysis and bland and altman analysis. results: at baseline, mean ffm was . (sd . ) kg; mean fat mass was . (sd . ) kg and mean bmi was . (sd . ) kg/m . mean ffm, fat mass and bmi were not significantly different at follow-up, indicating no change in nutritional status for the population overall. individual change in ffm ranged from - . to + . kg, while change in fat mass ranged from - . to + . kg. the table shows mean change (∆) in ffm and fat mass by each method, correlations (r ) between sft and dxa, and the % limits of agreements (loa) between sft and dxa for change in ffm and fat mass. mean change in ffm and fat mass estimated using sft did not differ significantly from dxa. mean bias between the methods was small, and overall correlations between sft and dxa were strong for changes in both ffm, suggesting good agreement for the overall population. however the % loa between the two methods were wide for change in both ffm and fat mass. this suggests that sft will not accurately predict changes in body composition in all cf patients. conclusion: sft measurements do not accurately estimate change in ffm or fat mass over time in individual adult cf patients compared with dxa. body composition changes measured using sft tended to more closely reflect changes detected by dxa in male patients compared with females. caution should be exercised when interpreting the results of serial measurements of body composition using sft in individuals with cf. analysed were significantly correlated with age-cml or srage levels (age, gender, bmi, presence of cf-related diabetes mellitus (cfrd) or hba c level). conclusions: serum levels of ages are elevated in adults with cf. elevation is not restricted to those with cfrd. the levels of srage reflect ability to respond to this pathway and are associated with poorer lung function. modification of the diet in cf may reduce this mediator and may have the potential to modify lung and renal injury. mouse models of cf generated by expression of improperly processed cftr (∆f ) or lacking cftr expression (ko) demonstrate selective induction of sult e in the liver (falany et al., biochem. j. : , ) . sult e is responsible for the sulfation and inactivation of β-estradiol (e ) at physiological concentrations. the increase in sult e expression is specific to the hepatocyte, whereas cftr is selectively expressed in cholangiocytes. the induction of sult e activity is associated with changes in levels of e regulated proteins in cftr (-/-) liver (li and falany, j cystic fibrosis : , ) . increased sult e activity is correlated with low body weight and decreased igf- message levels in livers of cftr(-/-). the mechanism for the induction of sult e in hepatocytes by cholanigiocytes lacking cftr expression, and the mechanism for the inhibition of igf- expression by increasing sult e expression was investigated in human cells. human mmnk- cholangiocytes in which cftr expression was inhibited by short interfering rna (sirna) induced sult e expression in human hepg hepatocytes when cocultured in a permeable membrane separated system. sult a expression was not altered in this model. the data suggests that loss of cftr function in cholangiocytes is capable of selectively modulating sult e expression in hepatocytes. to investigate the ability of increased sult e activity to modulate expression of igf- , hepg cells were stably transformed with sult e /pcdna to activity levels intermediate to the levels observed in cftr(-/-) mice. the increased sult e activity was associated with decreased igf- message expression in the hepg cells. since a major pathway for igf- regulation involves growth hormone (gh) stimulation of stat b phosphorylation, the effects of e and increased sult e activity on gh stimulated stat b phosphorylation were examined. stat b activation was identified using immunoblot analysis with a rabbit anti-tyrosine phosphorylated-stat b antibody. treatment of control hepg with nm e prior to the addition of gh increases stat b phosphorylation. in hepg cells expressing increased sult e activity, the stimulation of stat b phosphorylation by nm e was significantly decreased. e had an apparent rapid action on stat b phosphorylation that is not attenuated by the estrogen receptor antagonist, ici , . e was effective at increasing stat b phosphorylation when applied to hepg cells - min before gh. increased stat b phosphorylation was observed in control hepg cells at nm e although -fold higher e concentrations were required in the sult e -hepg cells consistent with the increased e sulfation. no differences were observed in total stat b in any of the studies. this study demonstrates that human cholangiocytes with low levels of functional cftr are capable of inducing sult e expression in human hepatocytes and the increase in e sulfation inhibits the gh stimulation of igf- expression via a decrease in stat b phosphorylation. supported by grants from the cystic fibrosis research, inc. and nih (gm ). williams, j.e. ; benden, c. ; jaffe, a. ; suri, r. ; wells, j.c. ; fewtrell, m.s. . mrc childhood nutrition unit, institute of child health, london, united kingdom; . portex respiratory medicine unit, great ormond street hospital, london, united kingdom background: patients with cystic fibrosis (cf) are at high risk of malnutrition. to our knowledge, no study so far has employed a reference fourcomponent model(sup) (/sup)( cm) to assess body composition (bc) in cf children, which allows accurate evaluation of both fat mass (fm) and the components of fat-free mass (ffm; mineral, protein, water) and most studies have been cross sectional. methods: cf subjects, aged - yrs, were compared with age-and sex-matched healthy controls for assessment of bc using a reference cm. comparison between groups was performed using paired t-tests and general linear models to adjust for age, height and pubertal status. in addition fm index (fmi; fm/height ) and ffmi (ffm/height ) standard deviation scores (sds) were calculated using measurements performed in a reference population of healthy children aged to yrs. repeat measurements were made in children with cf ( boys) after yrs and the change in fmi sds and ffmi sds investigated. results: for the initial measurement at baseline, boys with cf (n= ) were significantly shorter (mean (se) height sds: - . ( . , p< . ) compared to uk reference data; girls with cf were lighter, bmi sds (- . ( . ), p< . ). at baseline, comparison of cf children with pair-matched controls indicated that there was no difference in the boys but cf girls had less fm (cf minus control) (- . ( . )kg, p< . ) and mineral mass (- . ( . )kg, p< . ) after adjustment for age, height and puberty. fmi sds adjusted for age and puberty was also significantly lower in girls with cf (- . ( . ), p< . ) whereas ffmi sds was not. for comparison with the large reference population there was no difference in the boys at either time point for fmi sds and ffmi sds (paired t-test). for girls however, there was a significant difference at both baseline fmi sds (- . ( . ), p< . ), ffmi sds (- . ( . ), p< . ) and yrs fmi sds (- . ( . ), p< . ) and ffmi sds (- . ( . ), p< . ). paired t-test analysis between baseline and yr followup measurements indicated that fmi sds and ffmi sds were not significantly different at each point in time in either boys and girls. conclusion: at baseline, the bc of cf boys appears to be within normal range, but the cf girls already have lower fm and mineral than their healthy pair matched controls, even when adjustment for size and puberty is made. follow-up measures in cf children indicated that these descriptions of nutritional status of cf boys and girls remained consistent over the following years, with no evidence of any further deterioration in girls. however, nutritional surveillance is important in both sexes in cf at around puberty to address potential deterioration and this study suggests that the cm is a useful tool for this purpose. fuller background: although lung function (lf) in children with cystic fibrosis (cf) has been reported to be related to their nutritional status, particularly body fat, the latter has usually been assumed from anthropometric measurements and not measured using an appropriate reference method. habal, h. ; al-turkmani, m. ; freedman, s. ; laposata, m. . pathology, massachusetts general hospital and harvard medical school, boston, ma, usa; . medicine, bidmc and harvard medical school, boston, ma, usa background: increased dietary intake of fatty acids is recommended for cf patients. it is unknown if this results in increased incorporation of all fatty acids-saturated, monounsaturated, and polyunsaturated-into cells with a cf phenotype to a greater extent than into non-cf cells. docosahexaenoic acid (dha) supplementation of cftr-/-mice has been shown to reverse the pathologic cf phenotype. aim: to investigate whether the uptake of multiple predominant fatty acids and the release of membrane-bound arachidonic acid (aa) are altered in cultured epithelial cells with a cf phenotype; in addition, to determine whether fatty acid uptake is selectively inhibited by dha supplementation. methods: sense (wt) and antisense (cf) cftr hbe cells were cultured in mem containing % horse serum. aa release from membrane phospholipids (pls) was studied by incubating the cells with µm [ c] aa for min, and then with serum free medium containing . % fatty acid free human serum albumin in the presence or absence of µm adenosine for min, followed by measurement of radioactivity in the supernatant. the uptake of fatty acids into the cells was studied by incubating the cells for different time points with . µm [ c] linoleic acid (la), [ c] aa, [ c] dha, [ c] palmitic acid or [ c] oleic acid, in the presence and absence of µm or µm dha, followed by measurement of radioactivity in the cell lysate. results: basal and adenosine-stimulated aa release from membrane pls was significantly higher in cf cells compared to wt cells (basal wt: . ± . , cf: . ± . dpm/ cells, p< . ; stimulated wt: . ± . , cf: . ± . dpm/ cells, p< . ; mean±sem for each). the uptake of la, aa, dha, palmitic acid and oleic acid were significantly higher into cf cells compared to wt cells. dha significantly decreased the elevated uptake of these fatty acids into cf cells, but not into wt cells. data are shown in the table below. conclusion: in this cell culture model, these data demonstrate that aa uptake and release are increased in cf cells, which may suggest elevated mobilization of this fatty acid by cf cells. our data also show that cftr dysfunction leads to increased uptake of all tested fatty acids into the cells. downregulation of this increased uptake may be one mechanism by which dha exerts its therapeutic benefits. low bmi in cf correlates directly with worsening lung function and is an independent risk factor for mortality. the causes for this decreased bmi are incompletely understood but could be related to levels of the orexigenic peptide ghrelin, and to levels of leptin, an anorexigen. studies assessing plasma levels of leptin in cf have shown contradictory results, while the data on ghrelin in cf are scarce. we assessed both leptin and ghrelin plasma levels in mild (fev > % predicted), moderate (fev % to % predicted), and severe (fev < % predicted) adult cf patients. we compared these levels in cf subjects to age matched controls with normal bmi (range . to . kg/m ). we conclude that leptin levels are decreased, while ghrelin levels are increased only in cf patients with severe disease and low bmi. plasma levels of ghrelin and leptin did not differ from normals in those with mild and moderate disease. gastroesophageal reflux (ger) is common in cf before and after lung transplant (ltx). laparascopic fundoplication (lapf) is used when patients fail on conservative medical therapy. aims: to review longer term clinical outcomes of lapf in patients with cf in terms of lung function, body mass index (bmi), reflux symptoms, patient satisfaction with surgery and complications. method: retrospective review of patient records and patient questionnaire to gather relevant information. results:thirty-two patients with cf underwent lap.f recently; transplanted (ltxgroup), female (mean age [ - ]) years, and not transplanted (cf group), female ( ). the median time between ltx & lapf was ( ; ) days, range - . endoscopy was undertaken in patients prior to surgery and all underwent hr ph monitoring. complete (nissen) lapf was undertaken in % patients and a partial (toupet) in the other %. total median time in the operating theatre was ( - ) minutes. the average length of stay in hospital (in days) in the ltxgroup: pre-op. was . ; post-op. was . days; and in the cf group was: . and . respectively. lung function (fev and fvc in percent predicted) and bmi were measured at minus(-) , plus (+) , + and + days from lapf. patient satisfaction with lapf was a mean of % in the ltx group and % in the cf group and improvement in reflux symptoms of % in both groups. nocturnal cough reduced markedly in the cf group and there was an overall improvement in quality of life in both groups. complications consisted of port site hernias, failed lapf with re-operation months later; esophageal dilatations and patient with lower esophageal sphincter hypotonia. there were deaths on day , and post fundoplication. one was in a non-transplanted patient with recurrent episodes of hemoptysis. she succumbed to a catastrophic hemoptysis episode not related to lapf. the other were in transplanted patients. conclusions: in this relatively small series of patients there was a small but significant drop in fev in the ltx group days after lapf, but not in the cf group who remained stable throughout the review period. there was a significant drop in bmi in both groups after lapf, but the cf group returned to pre lapf values at + days. patient satisfaction with the procedure was high. fundoplication is an expensibe complicated procedure not without risk. a long term prospective study is warranted. historically this phenomenon has been attributed to intestinal malabsorption and cachexia as a consequence of chronic lung infection. however, cf mice exhibit similar growth and weight deficits but are not afflicted with malabsorption or lung infection, suggesting other mechanisms are participating as well. we hypothesize that small size is in part a result of increased β-oxidation in adipose stores. to test this we utilized a mouse model to study lipid metabolism. our study includes female cf mice with a null mutation in the cftr gene. these mice are congenic on the c bl/ j background as well as age matched with their controls (wt) to - days. liver, adipose and skeletal muscle (calf) were dissected from wt and cf mice in a fed state. tissues were immediately flash frozen in liquid nitrogen and used for rna and protein lysate harvest. quantitative pcr (qpcr) was performed using primers for the lipogenic isoform of acetyl-coa carboxylase α (acc α) in the liver and adipocytes, whereas the non-lipogenic isoform acc β was assessed in the skeletal muscle. primers for acyl coa oxidase (aox), carnitine palmitoyltransferase (cpt ) and long acyl-coa dehydrogenase (lcad) were used as gene markers for β-oxidation steps in the peroxisome, carnitine shuttle and mitochondrion, respectively. western blots were performed to detect acc (active) and phospho-acc (inactive) forms. the results show that there is a decrease in the total amount of acc protein in the fat as well as the skeletal muscle of the cf mice and the ratio of acc:phospho-acc is substantially lower in the cf tissues. phosphorylation and/or decreased expression of acc results in decreased expression of malonyl coa, leading to a cascade of events that increase β-oxidation. in addition, increased expression of aox and lcad, two downstream β-oxidation genes, is also observed. hyperactivity of the β-oxidation pathway could account for the decreased adipose stores seen in cf mice compared to the wt. such patterns of expression are reminiscent of starvation. caloric intake and intestinal absorption of dietary lipid were measured and cf animals consumed approximately % the calories of wt animals (per gram body weight) and lipid absorption was not significantly different ( % for cf, % for wt), indicating the cf animals have sufficient access to nutrition. we will continue to investigate fatty acid synthesis and degradation pathways during both the fed and fasted states to delineate the mechanism. this research was supported by grant hl . effect of gender on dha levels in p and rbc with lower values for male cf patients. acknowledgments: this work was supported by the belgian cf association (ablm), the french cf association (vlm) and by a grant from frs (ucl). data are expressed as means ± sd. * and indicate a significant difference with controls and cf ps patients respectively, p< . . background: deficit of antioxidant systems and increased oxidative stress have been demonstrated in cf patients with respect to healthy controls. the origin of this imbalance is known to be multifactorial. objective: the aim of our study was to assess changes in antioxidant systems and oxidative stress parameters in cf pediatric patients in presence or absence of pancreatic insufficiency material and methods: we recruited patients ( with pancreatic insufficiency pi and with pancreatic sufficiency ps) attending the cystic fibrosis centre of turin ( ) ( ) . during the annual routine control visit were performed antropometric measurements, pulmonary function tests (fev ), and were collected fasting blood samples and last days food record. vitamin e, a, c, selenium, glutathione (gsh, gshpx, gssg), dehydroascorbic acid, pcr were assessed. data were analysed using the two sample t-test with equal variances. results: there are not statistically significant differences between two groups for sex, age and fev . / of pi patients and / of ps patients took oral vitamin supplementation. serum levels of alpha-tocopherol and beta-carotene resulted within the local reference range either in pi or ps patients showing no significative differences. the mean serum levels were respectively suboptimal and optimal for vitamin e and a (biesalsky ' ). pi and ps patients did not show significant intergroup differences of water soluble and enzymatic antioxidants levels as well. serum levels of vitamin c, selenium, erythrocyte reduced glutathione (gsh) and glutathione peroxidase activity (gshpx) resulted within the local reference range. plasma vitamin c concentrations resulted slightly inferior to the optimal values (biesalsky ' ) in both groups. oxidative stress parameters (erythrocyte glutathione disulfide-gssg, dehydroascorbic acid) and inflammatory parameters (pcr) did not differ significantly, presenting values within the local reference range, in both groups. nutritional data revealed significantly reduced zscore for weight (p< . ) and height (p< . ) in pi patients vs ps patients. our data did not evidence significant differences in total energy (kcal/kg) and macronutrients (% of total kcal) intake in both pi and ps patients. dietary intake of antioxidant vitamins resulted slightly superior the rda (larn- ) presenting no significant intergroup differences. conclusions: this study shows that pancreatic insufficiency do not influence oxidant/antioxidant balance in cf patients at least in paediatric age. until innovative supplementation guidelines will be proposed for application, nutritional education and monitoring and correct dietary habits are useful to optimize antioxidant status in cf patients. there are more than cf mutations that are responsible for the spectrum of disease severity in cystic fibrosis (cf). the most common, f del mutation, is associated with pancreatic insufficiency (pi), and a classic phenotype. . yet even within a group of f del homozygotes, there is a small minority of patients ( . %) who are not taking pancreatic supplements, and are therefore presumably pancreatic sufficient (ps) (data from * cff registry). this observation prompted us to look more closely at enzyme use in cf patients with non-f del homozygous genotype. we reviewed enzyme use in cf patients who were fully genotyped and whose enzyme use was documented in the * cf registry. heterozygotes with one or two non-f del mutations were included in this analysis. using the assumption that the milder mutation is dominant, if the patient was ps and heterozygous with one f del mutation, the second mutation was classified as ps; if the person was pi, the second mutation was classified as pi. we also reviewed the pancreatic status and genotype of patients participating in the wisconsin newborn screening study. in this randomized control trial (rct), pancreatic status was determined by hour fecal fat analysis. enzyme use was extracted from the database for the year . there were patients in the cff registry with a total of different mutations. twenty-one of these mutations were associated with pi. this finding correlated well with the per cent of patients who were on enzyme supplements (range of per cent patients on enzymes by mutation - %). however when we looked at the six mutations associated with ps, the distribution of patients on enzymes vs. not, was much closer. see table below. possible reasons for enzyme supplement use by patients with ps mutations include a) treatment for recurrent pancreatitis, b)the fact that this is a cross-sectional analysis and patient may be on enzymes temporarily, c) patient may truly be pi, d) overuse of enzymes due to unreliable test to measure pancreatic status in patient, e) patients who are on enzymes are older, and have changed from ps to pi phenotype. this analysis suggests a need for better assessment of pancreatic status in patients with cf. * this analysis was originally done by request for the ecfs conference at garda on cftr genetics, and focused on the suggested " main mutations", and will be updated with the cff registry data. (supported by nih grants dk , dk , mo rr , mo rr ) number of patients in rct with each mutation: , , , , , respectively. dutta, a. ; woo, k. ; fitz, j.g. ; feranchak, a.p. . department of pediatrics, ut southwestern medical center/children's medical center, dallas, tx, usa; . medicine, ut southwestern, dallas, tx, usa extracellular atp is an important signaling molecule contributing to bile formation by the liver through binding biliary epithelial cell (cholangiocyte) membrane p receptors and stimulating cl-efflux. importantly, atp appears to work through cl-channels unrelated to cftr, suggesting it may be a potential way to modulate bile flow in cf liver disease. however, the signaling pathways linking receptor binding to channel activation in cholangiocytes are unknown. consequently, the aim of the present study was to identify the pathways responsible for atp-stimulated increases in [ca +]i and membrane clpermeability in a human biliary epithelial cell model. studies were performed in mz-cha- human biliary cells; [ca +]i was measured by fura- and membrane cl-currents by patch-clamp techniques. results: exposure of cells to atp ( µm) resulted in a rapid increase in [ca +]i to . ± . nm (n= ) which was abolished by prior depletion of intracellular ca + by thapsigargin ( . ± . nm; n= ), but unaffected by removal of extracellular ca + (egta, . ± . nm; n= ). in parallel studies, atp ( µm) increased current density from - . ± pa/pf to - . ± . pa/pf (n= ) . currents reversed at mv (ecl-), were outwardly rectifying, and were inhibited by the cl-channel inhibitor nppb (- . ± . pa/pf, n= ), but not the cftr inhibitor cftrinh . atp failed to activate currents after depletion of intracellular ca + stores by bapta-am (- . ± . pa/pf, n= ). the p y receptor antagonist suramin inhibited atp-stimulated increases in [ca +]i ( . ± . nm; n= ) and cl-channel activity (- . ± . pa/pf; n= ). however, the p x receptor antagonist brilliant blue g did not affect the magnitude of atp-stimulated cl-currents (- . ± . pa/pf, n= ). together these findings suggest that atp activates cl-currents primarily through p y, but not p x, receptors in human biliary cells. since p y are g-proteincoupled receptors and modulate [ca +]i by phospholipase c (plc) generation of inositol , , -trisphosphate (ip ), a series of experiments were designed to directly assess the effects of ip receptor inhibition or stimulation on atpstimulated cl-currents. first, the plc inhibitor, u blocked atp-stimulated cl-currents ( . ± . pa/pf; n= ). second, the specific ip receptor blocker -apb, inhibited both the atp-stimulated increase in [ca +]i ( . ± . nm, n= ) as well as membrane cl-currents (- . ± . pa/pf, n= ). lastly, intracellular dialysis with purified ip during whole-cell patch clamp activated cl-currents with identical properties to those activated by atp (- . ± . pa/pf, n= ). together these studies demonstrate that extracellular atp stimulates ca +-activated cl-channels in cholangiocytes primarily through p y receptor binding and plc/ip -dependent release of intracellular ca + stores. thus, the p y-ip receptor signaling complex represents a potential target to increase cholangiocyte secretion, and hence augment bile flow, in the treatment of the cholestatic liver disease associated with cf. supported by grants from the nih niddk and the cff to a. feranchak. background: supplemental pep therapy is used to treat cf patients with epi to assist with digestion, food absorption. this open-label, multipledose, single-treatment, multicenter study evaluated efficacy and safety of eur- for malabsorption treatment in young patients with cf and epi. eur- is a novel, zero-overfill, highly-stable formulation of porcinederived pancreatic enzymes, and was specifically developed for use in very young children allowing the product to be sprinkled on food. methods: eligible patients were < years with cf and epi, in acceptable nutritional status, needing peps and clinically stable. patients consumed a standard cf recommended diet, could not take drugs affecting gastric ph or motility. the trial involved a -day dose stabilization period and a -day treatment period. patients switched from their baseline enzyme treatment without washout. the optimal dose of eur- , determined during dose stabilization, was used during treatment. primary endpoint compared malabsorption of fat after eur- vs. previous treatment. clinical symptoms were also evaluated, as was physicians' and parents' judgment on the control of malabsorption signs and symptoms. safety measures included aes, physical exams, vital signs and changes in clinical laboratory findings, including cholesterol levels and fat-soluble vitamins. results: nineteen patients ( male) enrolled and completed all study phases. mean age was . years (range - ). percentage of responders (patients without steatorrhea (< % fecal fat content) and without signs and symptoms of malabsorption after and weeks of treatment) was . at baseline, . (p= . vs. base) after stabilization and . (p = . vs. base) at study end. frequency and oily stools showed a significant decrease vs. baseline at study end. the mean number of stools per day was . stools at screening and . stools during treatment (p< . ). the mean proportion of stool samples with visible oil or grease at screening was . % and . % during treatment (p= . ). a statistically significant improvement was seen in the incidence of moderate bloating. physicians characterized % of patients as "improved" in the control of epi signs and symptoms vs. previous therapies and % as "unchanged" at the end of the treatment phase, while parent assessments were % "improved" and % "unchanged." vitamin k absorption (measured through pivka) improved with eur- treatment over prior pep treatment measured at baseline. eur- was safe and well tolerated, with minimal aes. no aes led to discontinuation of study drug interruption. no incidences of uric acid toxicity or fibrosing colonopathy following eur- treatment. no trends were seen in lab parameters, physical examination, or vital signs. conclusion: eur- is effective, safe, well-tolerated in the treatment of epi in young cf patients. treatment controlled malabsorption and clinical symptoms of epi in young patients with cf. safety and efficacy of eur- in this trial is consistent with the pivotal phase iii trial. there is an association between the presence of cf and alterations in fatty acid metabolism, consisting of a decrease in linoleate ( : n- ). more recently, a deficiency of docosahexaenoate ( : n- , dha) has arisen as a prominent fatty acid abnormality in cf. an established breeding colony of exon cftr knockout (cftr-/-, cf, n= ) and wild type (wt, n= ) mice was used for this study. all mice were weaned at days of age and then raised on peptamen and water until days of age, and then continued for days with ml/day of peptamen to homogenize the nutritional status. after this period, some mice (n= per group) were subjected to dha treatment ( mg/day) for one week. after sacrifice, pancreatic tissue was collected, homogenized and extracted. lipid classes were separated by sequential elution in aminopropyl columns. the eluted fractions were transmethylated and injected into a hewlett-packard a gas chromatograph with a flame ionization detector for quantitative analysis. the vast majority of fatty acids were mainly distributed in phospholipids, except those with -carbons. wt and cf mice were studied a gender gap in the survival of cystic fibrosis (cf) is well-documented. however, little is known whether there is a gender différence in pubertal growth pattern in cf children. delayed and attenuated pubertal growth are commonly observed in adolescents with cf. however, characterizing height velocity (hv) pattern is very challenging because of errors associated with calculating hv, e.g. interpolation and extrapolation between heights measured in irregular intervals, as well as difficulties in determining the location and the magnitude of peak height velocity (phv). in this study, we applied a new statistical method, namely, the semi-parametric shape-invariant model, to characterize the hv pattern of cf children. this method is based on the assumption that all individuals' growth curves have a common shape. therefore, this method attempts to shift and stretch (squeeze) individual's growth data until they can be modeled by the common shape curve. the semi-parametric shape-invariant model has the advantage of fitting measured heights directly, thereby eliminating the errors associated with calculating hv. the study population included cf children who had height measurements between ages to years documented in the - cff registry. because fitting each individual height curve is computing-intensive, boys and girls were randomly chosen for this preliminary analysis. our results showed that girls exhibited a typical growth deceleration during late preadolescence, followed by accelerated hv and a single phv during adolescence (figure) . unexpectedly, cf boys exhibited a notable hv peak during late preadolescence in addition to the typical adolescent phv (figure) . overall, the mean magnitude and age of phv was . cm at . years for boys and . cm at . years for girls. when compared to phv of non-cf children based on tanner's reference (pediatr. , : ) , cf boys showed a greater delay in the age of phv while cf girls showed a greater attenuation in the magnitude of phv. further analyses are being performed to characterize the hv patterns of all children to confirm these results. (supported by nih-dk ). anelli, m.; foresti, r.; peloso, l.; ortenzi, g. medical affairs, eurand, pessano con bo, italy background: given their inherent instability, the dose units of all pancreatic enzyme preparations (peps) have always been "overfilled" to compensate for enzyme degradation over time and assure at least % of the label lipase content at the end of their shelf life. this overfill issue was first examined by whitehead who reported overfills ranging from to % after analyzing several commercially available peps (whitehead am. pharm weekbl sci. ) . the usp presently allows for pep overfills up to %. thus, the actual active lipase content of a pep capsule labelled at , iu might vary from , to , units according to its "freshness." as a result, patients taking peps receive a product with variable potency, which can lead to efficacy issues, increased pill burden, unnecessary drug interactions, and product switching. the fda has also noted the potential safety risk posed by overfilling and, in a recently published guidance, it imposed a zero overfill requirement for all peps to be marketed in the usa after april . methods: over a period of two years, we ran a series of tests similar to those conducted by whitehead to evaluate overfill on commercial peps currently available in the u.s. and europe. results: no finished product was formulated to % of the labelclaimed lipase enzyme activity. overfill on commercially available peps ranged from % to %, with a median value of %. the peps analyzed were to months old on average; therefore, the actual amount of overfill is likely underestimated. conclusion: our findings confirm those reported earlier by whitehead and demonstrate that none of the currently available peps complies with the zero overfill requirement of the fda guidance. these findings highlight a potential cause of suboptimal therapy with currently available peps. references: -whitehead am. study to compare the enzyme activity, acid resistance and dissolution characteristics of currently available pancreatic enzyme preparations. pharm weekbl sci. feb ; ( ): - . -food and drug administration. guidance for industry: exocrine pancreatic insufficiency drug products -submitting ndas. april . heubi, j. ; boas, s.r. ; blake, k. ; nasr, s.z. ; woo, m.s. ; graff, g.r. ; hardy, k.a. ; amaro-galvez, r. ; latino, m. ; lee, c. . children 's hospital medical center, cincinnati, oh, usa; . chicago cf care, glenview, il, usa; . nemours, jacksonville, fl, usa; . michigan u, ann arbor, mi, usa; . children's h, los angeles, ca, usa; . penn state h, hershey, pa, usa; . children's h, oakland, ca, usa; . texas u, tyler, tx, usa; . eurand, milan, italy; . eurand, vandalia, oh, usa background: supplemental pep therapy helps prevent maldigestion and malabsorption in cf patients. eur- is a novel, zero-overfill, highly-stable formulation of porcine-derived pancreatic enzymes developed to treat epi in cf patients. this phase iii, randomized, doubleblind, placebo-controlled, two-treatment, crossover, multicenter trial evaluated the efficacy and safety of eur- to treat epi in cf patients. methods: patients with confirmed cf and epi, age ≥ years, good nutritional status and weight ≤ kg were enrolled. no drugs affecting gastric ph or motility were allowed. after open-label dose titration, patients were randomized to receive eur- or placebo over a week-long period. following another open-label normalization, all patients were crossed over to the alternative treatment arm. primary endpoint was change in coefficient of fat absorption (cfa) following oral administration of eur- vs. placebo. secondary endpoints were change in coefficient of nitrogen absorption (cna), cholesterol, fat soluble vitamins, body weight, bmi and epi symptoms following oral administration of eur- vs. placebo. safety endpoints included aes, clinical laboratory parameters, physical exams and vital signs. results: thirty-four patients were enrolled ( female); were evaluated. mean age was . years (range - ). eur- treatment was associated with statistically significant (p< . ) increases in both cfa and cna vs. placebo. eur- treatment was associated with decreases in pivka ii (an indicator of vitamin k status). epi symptoms consistently improved during eur- treatment, including a statistically significant reduction in stool frequency and fewer soft and watery stools. treatment with eur- improved the signs and symptoms of malabsorption even in the small subset of patients with cfa values greater than % under placebo treatment. there were no notable changes in serum lipids, vitamin a and e values, mean weight or bmi in patients who received eur- . eur- was safe and well-tolerated. there were no unexpected or significant differences in the frequency or type of aes between eur- and placebo, and no trends were seen in lab parameters or vital signs. no patient discontinued from the study due to an ae. two serious aes were considered unrelated to the study drug and both resolved. conclusion: in this randomized, double blind, placebo-controlled study, eur- was safe, well tolerated and effective in the treatment of cf patients with epi, with clinically and statistically significant improvements in cfa, cna, and epi signs and symptoms in the absence of any concomitant treatment affecting gastrointestinal motility and ph. the therapeutic benefit of eur- was seen even in patients with very high cfa values (> %). the relative infrequency of cystic fibrosis related diabetes (cfrd), until recently, means little is known about hypoglycaemia ('hypos') associated with its treatment. insulin is frequently taken by those who have cfrd, for whom the risk of hypoglycaemia may be high due to dramatic changes in insulin sensitivity that can occur with pulmonary exacerbations and a decreased ability to secrete glucagon. a number of studies involving people with type diabetes mellitus (t dm) have reported on their experiences of hypoglycaemia, but whether results from such investigations apply to cfrd is unclear. an exploratory investigation was conducted to compare the type, frequency and severity of hypoglycaemic symptoms experienced by patients who had cfrd with patients who had t dm. method a cross sectional study was conducted, involving adults ( - years) with t dm or cfrd, recruited from two hospitals in england. a questionnaire, sent to patients with t dm and with cfrd, included investigator-developed items about knowledge and frequency of hypoglycaemia and the edinburgh hypoglycaemia scale (ehs), a standardised measure that assesses autonomic (e.g. sweating, hunger) and neuroglycopenic (e.g. confusion, poor co-ordination) symptoms associated with hypoglycaemia. questionnaire data were entered into spss for analysis. comparisons were conducted using t-tests, mann whitney tests or chi square tests as appropriate. results questionnaires were returned by patients with t dm and with cfrd. five of those with t dm were over years of age and, therefore, excluded from analysis. almost all participants had experienced a 'hypo' ( %). comparisons between the two groups are reported in table . conclusion patients with cfrd reported fewer and tended to have less severe episodes of hypoglycaemia compared to those with t dm. fewer neuroglycopenic symptoms in the former could be related to their shorter duration of diabetes or to how cfrd was managed; patients with cfrd were given shorter acting insulin, making them less prone to 'hypos' during the night, and had a high sugar diet to ensure weight maintenance. differences between the groups could also be related to specific characteristics of these two forms of diabetes. table : data from questionnaire insulin resistance. the purpose of this study was to investigate the relationship between pem and the risk of developing glucose intolerance and/or cfrd and the effect on lung function in children ≤ years of age whose history suggests pem. patients whose diagnosis of cf was suggested by failure to thrive(ftt) and whose growth parameters remain < % for weight and height for age were identified as having incurred pem. a retrospective analysis of our cf registry database was performed. cf patients ≤ years old were identified. of patients( . %) had cfrd. ( . %) of these plotted < % for weight and height (pem)and ( . %) were diagnosed with cfrd. further review of the patients showed whose diagnosis of cf was suggested by ftt and ( . %) of those had cfrd. statistical analysis did not show a clinical significance, but a strong relationship is implied. pulmonary function, fev %, was significantly lower in the pem/cfrd patients (mean fev . % ± . %) than the pem only group, (mean fev . % ± . %)(p= . ). this review provides further support that patients who have pem are at increased risk of developing cfrd. a significant risk was not established; however a strong association is suggested. assessment of a larger cohort of patients may show a stronger relationship. findings from this study would suggest that further research into the physiological relationship between pem and cfrd should be initiated. cfrd screening should be considered at a younger age than the current cff recommended age of > years. diagnosing cfrd/glucose intolerance early may contribute to improve growth parameters, preserve lung function, and improve survival. based on this review, our center is initiating cfrd screening for all patients ≥ years, and considers screening for all pem patients ≥ years. background: patients with cf are thought to have a rapid postprandial rise in plasma glucose which may be followed by a delayed and prolonged insulin response and hypoglycemia. we sought to estimate the prevalance, cumulative one year incidence, and factors associated with hypoglycemia during oral glucose tolerance testing in non-diabetic adults with cf. we also compared the prevalence of hypoglycemia to a geographically matched cohort of young adults without cf. methods: we performed a prospective study of individuals aged over years with cf followed in clinic providing population-based, specialized care in cambridgeshire, england. we excluded patients with previously-diagnosed diabetes or taking anti-diabetic medications. we evaluated individuals who underwent g hour oral glucose tolerance testing (ogtt) in . of these, individuals had neither new diabetes nor hypoglycemia in and were retested using ogtt in . we testing the association between biochemical and anthropometric factors measured in with the prevalence of hypoglycemia in , and incident hypoglycemia in . biochemical hypoglycemia was defined as a blood glucose between < . mmol/l ( mg/dl), but greater or equal to . mmol/l ( mg/dl) and severe hypoglycemia as a value lower than . mmol/l on either the fasting or post glucose challenge value. for comparison, we used data from the cambridgeshire-based young ely study of individuals age - years who underwent g ogtt testing in - . results: in , in patients with cf, the prevalence of fasting hypoglycemia was . %. no patients had fasting severe hypoglycemia. following glucose challenge, . % and . % of patients had glucose values consistent with hypoglycemia and severe hypoglycemia respectively. there were no diferrences in age, hepatic, pulmonary or renal function, but patients with hypoglycemia at two hours were five times more like to be male (odds ratio . , % ci . - . ) and more likely to have a higher bmi. the prevalence of a hours value of < . mmol/l in patients with cf at . % ( / ) was not different than in controls . % ( / ). of patients who did not have hypoglycemic readings in , at one year, patient developed fasting hypoglycemia and individuals developed post-challenge hypoglycemic for a cumulative incidence . %. of these . % ( ) had values in the range of severe hypoglycemia. there were no differences between age, sex, bmi, liver, renal, or pulmonary function in those who did and did not develop hypoglycemia. conclusions: hypoglycemia appears common in patients with cf but is also common in a healthy population. male sex and bmi were associated with post load hypoglycemia in patients with cf in cross-sectional analyses only. further research needs to assess whether these low blood glucose values are associated with symptoms. it is well recognised that cystic fibrosis related diabetes (cfrd) is a poor prognostic indicator in cf, and therefore early recognition is imperative to allow effective treatment in an attempt to prevent the associated decline in pulmonary function and nutritional status. although the oral glucose tolerance test (ogtt) is the accepted method of detecting diabetes mellitus in non cf individuals, glucose intolerance and lack of insulin can be variable in cf patients such that the reliability of ogtt in the diagnosis of cfrd has been questioned. under these circumstances, other methods of monitoring glucose intolerance, such as serial post prandial glucose monitoring (sgm), may be more appropriate. to look at this further, we prospectively compared ogtt with sgm in a group of adult cf patients. fourteen patients with no previous history of cfrd ( ], df homozygous, male) admitted for acute pulmonary exacerbations were evaluated. all were exocrine pancreatic insufficient, and was on long term oral steroids. eleven patients received mg/day oral prednisolone in addition to iv antibiotic therapy for the duration of their admission. ogtt was performed in the standard fashion (ingestion of . g glucose/kg body weight [maximum g] within minutes, after an overnight fast). sgm was performed hours post prandially and before bedtime over the period of the admission. the local ethics committee approved the study and informed consent was obtained from all patients. results sgm revealed elevated postprandial blood glucose values in all patients. there were episodes (mean per patient [ to ]) with glucose > . mmol/l (frank diabetes, who criteria) and episodes (mean per patient [ to ] ) with glucose . to . mmol/l (impaired glucose tolerance, who criteria). however, ogtt revealed impaired glucose tolerance in only one patient ( hour glucose value . mmol/l). in the remaining patients, ogtt revealed normal hour glucose values (mean . mmol/l [range . to . ]). fasting plasma glucose values were also normal (mean . mmol/l [ . to . ]) in all patients. we have shown a high prevalence of hyperglycaemia in our adult patients admitted for pulmonary exacerbations that was only detected using serial glucose monitoring. this study suggests that sgm may be more sensitive at detecting early cfrd than the ogtt. further work needs to be carried out to look at the best methods of diagnosing cfrd, to facilitate early treatment and thereby improve the prognosis. the diagnosis of cystic fibrosis related diabetes (cfrd) is associated with a decline in pulmonary function and nutritional state and is a poor prognostic indicator: the risk of its development increases with age. furthermore, the development of cfrd is due to a gradual loss of insulin production, and the disease may therefore be preceded by a period of glucose intolerance. despite this, the incidence of hyperglycaemia in cf patients is unknown. to study this further, we monitored the blood glucose profiles of adult cf patients admitted for the treatment of a pulmonary exacerbation. we looked at consecutive cf patient admissions for iv antibiotic therapy to our large adult unit. of these, had established cfrd and were excluded. the remaining patients (mean age years [range to ], male) formed the study population. none were on long term oral steroids, but received mg/day prednisolone as part of their routine exacerbation treatment. all patients underwent blood glucose measurement hours after each meal and before bedtime as part of a standard assessment protocol in our clinic. thirty patients ( %, / on steroids) demonstrated post prandial hyperglycaemia: exhibited values between . and . mmol/l (impaired glucose tolerance, who criteria) and > . mmol/l (frank diabetes, who criteria). there were no significant differences in age, genotype, fev , fvc, bmi, or duration of hospital stay between the two groups. of the hyperglycaemic patients, ( %) had abnormal glucose levels detected before bed time, significantly more than hours post breakfast ( %, p< . ), post lunch ( %, p< . ), but not post dinner ( %, p= . ). this study has shown a high prevalence of hyperglycaemia in adult cf patients treated for a pulmonary exacerbation who were not otherwise known to suffer from cfrd. nearly all our patients demonstrated glucose intolerance later in the day, when the recommended screening test for cfrd (the fasting oral glucose tolerance challenge) cannot be used. post prandial glucose monitoring of such patients during times of stress may therefore be advisable, allowing the introduction of insulin treatment at an earlier stage in the disease process. whittaker, l.a.; tilluckdharry, l.; christian, r. medicine, university of vermont, burlington, vt, usa objective: bone disease is a recognized complication of cystic fibrosis (cf). there are currently no guidelines for bone mineral density (bmd) testing or tools for fracture risk assessment in cf adults. we piloted a survey designed to identify cf-specific risk factors for low bone density and fracture. methods: a question survey was completed by patients prior to measurement of bmd at the hip, spine and radius by densitometry (dxa). the survey was comprised of questions on calcium and vitamin d intake, exercise, family history, medications, reproductive health, ultraviolet exposure, cf related diabetes (cfrd), weight loss, severity of cf lung disease and quality of life. z scores (bmd adjusted for age and gender) were used so that male and female data could be analyzed collectively. patient perceived health (pph) was rated by four questions and totaled for the pph score, which ranged from - with the highest score indicating the best health. functional status (fs) was rated by three questions that reflect the degree to which cf interferes with school, work or play. the total fs score ranged from - , with the highest score representing the least interference with life. questions on pph and fs were derived from a validated clinical assessment tool, the cf questionnaire (cfq) ( ) . results: all cf adult patients (n= , age and older) were offered participation in the study. dxa of total hip and lumbar spine was performed on patients ( %); ( %) of these patients also completed the survey. % of patients had low bone density on dxa (z score <- ). % of male cf adults had low bone density vs % of females. fracture was reported by % of patients who completed the survey ( % female and % male). lower pph score was associated with lower z score at the hip and spine. lower fs score was associated with lower z score at the hip and spine. low body mass index was associated with low z scores at both hip and spine. patients with bmi below kg/m were most likely to have osteopenia. conclusions: osteopenia and history of fracture were common in our adult cf population regardless of gender. bmi, pph and fs predicted bone density. risk stratification by a cf-specific bone health survey may guide bone density screening strategies for cf adults. prospective studies in cf adults are needed to determine whether a cf-specific survey can predict fracture risk as well as bone density. references : as life expectancy in cystic fibrosis (cf) increase, osteoporosis has become more prevalent. vitamin d status is one of many factors that contribute to optimal bone health. previous data has shown, the majority of cf patients are deficient in vitamin d despite being prescribed daily cf specific vitamin therapy. previous research has also shown that the current recommendations for vitamin d supplementation and repletion provided by the cystic fibrosis foundation bone health consensus ( ) are not sufficient to achieve optimal vitamin d levels in the majority of patients. moreover, the recommendations fail to sustain levels in the optimal range for bone health. purpose: we demonstrate a successful supplementation algorithm for individualized high dose vitamin d with continued high dose maintenance therapy which achieves and sustains appropriate levels. methods: all patients followed at the pediatric center are prescribed routine cf vitamin supplementation. vitamin d levels are obtained as part of routine clinical care via mass spectroscopy. if serum vitamin d level is < ng/dl, vitamin d therapy with ergocalciferol is initiated one time per week. patients < years of age receive iu/ dose and > years of age receive iu/dose. at the next routine quarterly visit, vitamin d levels are re-assessed via mass spectroscopy to determine efficacy of once weekly supplementation. if levels are > ng/dl, patients continue high dose supplementation time a week as maintenance therapy. however, if levels remain < ng/dl, the ergocalciferol is increased by time a week in month increments until adequate levels are obtained. once adequate serum vitamin d levels are obtained, patients remain on the level of supplementation needed to achieve levels > ng/dl as maintenance therapy. all patients are instructed to take vitamin d and their cf vitamins with enzymes and food to optimize absorption. results: after implementation of the algorithm, . % of patients, with varying levels of supplementation, had vitamin d levels > ng/dl (mean= . ng/dl). of the patients that achieved goal vitamin d levels, % were able to achieve and sustain adequate vitamin d levels without additional vitamin d supplementation while on standard cf vitamins (mean vitamin d level = . ng/dl). the remaining patients required chronic maintenance vitamin d: % required vitamin d supplementation once per week with a resulting mean vitamin d level of . ng/dl, . % required maintenance vitamin d supplementation twice per week with a resulting mean vitamin d level of . ng/dl, and, . % required vitamin d supplementation or more times per week with a resulting mean vitamin d level of . ng/dl. another % of patients take vitamin d supplements other than ergocalciferol and were not included in this study. conclusions: individualized vitamin d repletion and maintenance therapy as outlined in our algorithm is a successful mechanism to obtain optimal vitamin d levels in patients with cf. inhaled tobramycin ( %). sixty-two percent reported using at least different types of oral medications on that day including pancreatic enzymes ( %), oral antibiotics ( %), anti-reflux medication ( %), azithromycin ( %), anti-histamine ( %), and a decongestant ( %). twenty percent reported taking at least one medication for pain. forty-nine percent reported performing airway clearance at least twice during the day with % using a vest, % performing standard chest physiotherapy, and % using a flutter or acapella device. there was no difference in the reported number of medications or the reported time needed to complete therapies based on respondent age. although respondents with severe lung disease (fev < % predicted) reported a higher median number of therapies ( vs. , p= . ), the reported time for completing therapies was not associated with fev (p= . ). conclusion: daily treatment burden and complexity in all adults with cf is high, and is only marginally increased in those with worse pulmonary function. given this already high load of daily therapies, efforts to assess the treatment burden of new cf therapies are warranted. the impact of this high treatment burden on overall health related quality of life for adults with cf needs to be assessed. objective: the cystic fibrosis foundation (cff) has a network of over accredited care centers throughout the united states. data is collected from these care centers to help better understand the disease and describe changes in survival, standards of care, and health outcomes. when the cff was founded in few children survived to school age. now the predicted survival extends beyond the mid 's (cff, ) . with this change has come the need for age appropriate care in many areas including reproduction. the adult cystic fibrosis center at morristown memorial hospital presently follows adults. of those, women( %) with cf have had children affected with cystic fibrosis. each woman was diagnosed after her child. the cf data registry does not have any means of identifying if a patient has an affected child. as the average age of survival of cf patients has increased, so has the likelihood of cf patients having cf offspring. what is the estimated incidence of cf patients having cf affected children attending accredited cf centers in the united states? method: questionnaires were sent to care centers and satellite centers via standard mail or facsimile based on information from the cff directory. one month after the last questionnaire was mailed, the cf foundation sent out a reminder email to the nurse coordinators. questions asked were as follows: ) name of center ) do you presently care for any patients with cf who have given birth to, or fathered a cf affected child? ) how many patients at your center have had cf affected children? ) was the parent diagnosed before or after pregnancy was confirmed? ) if parent's diagnosis came after conception, was this after child was diagnosed? ) was the parent's diagnosis a result of prenatal testing? results: a total of questionnaires were mailed or faxed. this netted a result of responses. after email reminder, an additional questionnaires were returned for a total of responses, an % response rate. name of center was checked to remove duplicate information. the number of parents with cystic fibrosis who have had children with cf numbered . of those, ( %) were diagnosed before pregnancy; ( %) were diagnosed after pregnancy. of those diagnosed after pregnancy, the majority ( %) were diagnosed as a result of their child's diagnosis. only ( %) were diagnosed as a result of prenatal testing. conclusion: based on the responses from accredited cf care centers, patients with cf have children also affected with this disorder. prior to conceiving their child, % knew that they had cf. having cf did not appear to be a deterrent to delivering cf offspring. this study did not look at the age of the parent or child; they may have conceived their child before genetic screening was common for spouses. as the median survival of this population increases, the incidence of cf adults with cf children may also increase. the cff may choose to add a question to the cf data registry, identifying cf patients with cf offspring. the potential physical and psychosocial impact can then be tracked. anderson, a.; popli, k.; stewart, j.; heslop, k.; gascoigne, a.; bourke, s. adult cystic fibrosis centre and fertility centre, royal victoria infirmary, newcastle upon tyne, united kingdom as the outcome of patients with cf improves fertility issues become increasingly important. almost all men with cf are infertile because of azoospermia due to congenital absence of the vas deferens. these men can attain genetic paternity by sperm aspiration from the epididymis or testis with intracytoplasmic sperm injection (icsi), but few men with cf seek fertility treatment to assess their knowledge and attitude towards fertility issues a questionnaire was sent to men with cf: ( %) responded; ( %) declined to give information and ( %) replied in full. overall % thought that nearly all men with cf were infertile, % did not know how many were affected, and % could describe the exact nature of infertility. only % knew that treatment for infertility was available: % of these thought it was rarely successful and % were unsure of success rates. although % had thought of having children at some stage, only % had sought investigation of infertility. two men had children, one by icsi and one by donor insemination. overall % feared that the child could have cf and % worried that their health would affect their ability to function as a parent. in terms of receiving advice % thought that this should be given by a fertility specialist, % by a cf physician and % by a primary care physician. with regard to fertility treatment % thought that decisions should be made by the patients and their partners, % by the patient alone, and % by a doctor. the reluctance of men with cf to seek fertility treatment appears to be due to many factors including lack of knowledge of available treatments, fears about the child inheriting cf, and concerns that their health would adversely affect their role as a parent. in addiition to information provided by the cf team advice from a specialist in fertility treatment may help to improve the provision of reproductive counselling to these patients. nash, e.f. , ; coonar, a.s. ; stephenson, a.l. , ; delgado, d.h. ; singer, l.g. , ; tullis, e. , ; chaparro-mutis, c. studies have shown varying degrees of right ventricular (rv) dysfunction in adult cf patients with severe lung disease. left ventricular (lv) dysfunction is much less common with conflicting reports as to its prevalence. pulmonary hypertension (ph) is commonly seen, the severity correlating with fev . one group reported that ph was more prevalent in b. cepaciacolonized patients, albeit in a small sample. the aim of this study was to determine the true prevalence of ph and cardiac dysfunction in cf patients with severe lung disease. we carried out a retrospective cohort study of all adult cf patients referred to the lung transplant program at toronto general hospital from - . adult patients referred with non-cf bronchiectasis were included for comparison. demographic data, lung function, -minute walk, micro-biology, doppler echocardiography and resting and stress radionuclide ventriculography (muga) results were analyzed by students t-test (parametric) or mann whitney u test (non-parametric). results adult cf patients ( % non-b. cepacia, % b. cepacia) and non-cf bronchiectasis patients were included. data from these groups are summarized in the table. we found higher minute walk and bmi values and a higher percentage of males in the b. cepacia compared to non-b. cepacia group. ph was present in % of the cf patients in whom pasp could be obtained. b. cepacia patients had lower first pass rvef compared to non-b. cepacia patients. % of the b. cepacia patients had an abnormal lvef response to exercise (defined as < % increase on exercise) compared to % of the non-b. cepacia patients. we found a high prevalence of ph as well as rv and lv dysfunction in cf patients with severe lung disease. b. cepacia-colonized patients had worse rv function compared to non-b. cepacia-colonized patients on first pass radionuclide ventriculography. *non-cf bronchiectasis group older than cf groups (p< . ) b. cepacia cf group higher than non-b. cepacia cf group (p< . ) b. cepacia cf group lower than non-b. cepacia cf group (p<. ) perobelli, s. ; dorazio, c. ; assael, b. ; tamanini, a. ; castellani, c. . cystic fibrosis center, verona, italy; . molecular biology laboratory, verona, italy cf neonatal screeening (nbs) can identify neonates with elevated irt, normal or borderline sweat chloride concentrations, and one cf mutation plus an additional cftr sequence variant, or, alternatively, two cftr sequence variants. the combination of hypertrypsinogenemia and two cftr gene defects is consistent with the presence of a cftr-related disorder (cftr-rd). although these neonates are usually healthy at diagnosis, the long-term phenotypical consequences may be highly variable. such variability makes impossible to predict the clinical outcome, and provide satisfactory genetic counselling for the family. the diagnosis of such a vague condition could potentially cause considerable family stress. this study aimed at assessing how parents of infants diagnosed with a cftr-rd through nbs perceive their children's health. a questionnaire for parents was designed, consisting of three sections. the first section asked about information given to the family in the neonatal period, and about emotional reactions to the cftr-rd communication of diagnosis. the second section investigated the present perception of the child's health, and the parents level of anxiety/concern. the third section focused on the impact of the diagnosis on family planning, on relationships inside the couple and with the child, and on emotional distress; this section was concerned also with the use of carrier testing in relatives. parents were also asked to fill in a standardized form on child behaviour (achenbach child behaviour check-list). the questionnaire was distributed to two populations, the parents of children diagnosed with cftr-rd through nbs (group a), and the parents of children diagnosed with cf through nbs as the life expectancy of children with cf increases new issues arise that have not been dealt with in the past. in the past,issues related to the support and care of a young adult with cf were rarely raised because only small numbers of children with cf lived into adulthood. however, with an increasing young adult population, young adults without family support have surfaced in significant numbers and their needs should be identified and understood by the cf center care team. these young adults face the same range of issues others with cf face related to health insurance, government benefits, housing, education and employment. however, they do not have a family to help them wiht support, health insurance benefits and care needs. method in october , the cf legal information hotline ("hotline") began to provide free and confidential information to people with cf, family members and healthcare providers in the u.s. the hotline is staffed by attorneys, beth sufian and james passamano(bs and jp) and is accessed by dialing a toll free number or by e-mail. information about the hotline is available at cf care centers, in cf publications and on the web. information regarding the content of each call is recorded on an intake form and includes caller name, address, phone number, age of person with cf, cf center attended, and information related to the caller's question, attempts made to access legal information prior to calling the hotline, relation of access to information to access to care. calls and e-mails are tracked by seven major categories which are divided into subcategories. if a caller has more than one question, the call is tracked using the caller's primary issue. results from november to april the cf legal information hotline has received calls from young adults with cf who do not have family support and have issues related to obtaining health insur-ance, government benefits, educational oppornties or employment opportunities. as well as issues related to lack of support for care. the hotline has also received calls from cf care centers team members with questions related to the needs of young adults without family support. over an eight year period the cf hotline received _______calls. the categories for the calls were: social security benefits ( , ) , health insurance ( , ) , educational ( ), employment ( ), long term disability benefits ( ), family law ( ) and miscellaneous legal issues ( ). discussion people with cf face obstacles to care if they are unaware of their legal rights especially in the areas of health insurance and government benefits. healthcare providers are busy addressing the medical needs of patients and are often not well equipped to provide detailed information about the legal rights of their patients. the hotline provides important information to both patients and healthcare providers that can optimize both patient care and outcomes. access to legal information can result in individuals obtaining health insurance, medicaid, medicare or other state coverage that in turn results in better access to treatment. legal information can also lead to important modifications in the educational or employment environment for the individual with cf. these modifications can be critical in allowing those individuals access to important care and treatments. providing information that can help individuals obtain social security or long term disability benefits can result in a monthly benefit and health insurance that then allows the individual to devote more time to their healthcare. many people with cf and healthcare providers lack information about the legal rights of individuals with cf. understanding the legal rights of people with cf can reduce obstacles to obtaining care, optimize education and employment, and increase access to retirement benefits. further work needs to be done to identify ways to make individuals with cf and healthcare providers aware of the hotline. the cf hotline provides a unique service to cf patients and healthcare providers by providing information regarding the legal rights of people with cf. identification of ways to assist young adults without family support should be identified and cf center xcare teams shoudl be given the tools to allow them to help their patients access government assistance and other programs that can help make sure that the young adults have acces to care and support they need to live with cf. the dvd gives healthcare staff, patients and families a rare glimpse of ten couples with cystic fibrosis (cf) interacting at a cf care center-sponsored 'couples support group'. they discuss the impact this forum has in their daily lives for gainingo information about how cf affects their lives as individuals, and as couples, and how the 'non-cf partner' learns from others. o skills at networking and personal coping strategies. o reflections on 'family life and cf', and o observations on ways cf care centers can join them in greater health partnership. lastly, this dvd provides a 'starter kit' for ways to establish such a group. one of the realities of a life-threatening illness can be that of social isolation, and of separation from others who share the same disease. having the opportunity of participate in a support group can significantly reduce that sense of isolation and give the participants an ability to share experiences and give support to others with this disease. cystic fibrosis as an illness has growing ranks of 'survivors' who live longer, fuller lives, thanks to the advances made on multiple-research fronts. historically, adults with cystic fibrosis have not had much occasion to share person-to-person experiences within the structure of their cf care centers. justifiably, with the potential for cross-infection, the cystic fibrosis foundation has urged caution for the cf population in having face-to-face interactions. in , our cf care center recognized this need and sponsoredwith guidance from staff facilitators-an on-going adult support group. prior to the formation of the group pre-and on-site group ic protocols were developed. at the pre-group stage, the physician initially certifies that the patient is free from aggressive pathogens. each time the patient rsvp's permission to attend a group session, the physician must 'recertify' this status. on-site protocols emphasize a number of ic precautions to minimize crossinfection. the center recognized that by developing such a support group, 'patient and family centered care' could be enhanced. a goal in the group's formation was to create 'mutually beneficial partnerships among patients, families, and healthcare providers'. group structure/successes to date: the group is limited to participants. two cf care center staff act as facilitators. this is a support, not a therapy group. compliance with therapies is reported to have increased as one outcome of this forum. formal topics are purposefully not predetermined; the group's success is predicated on the personal responsibility of each participant to bring topics/issues from their daily life experiences. the group has gained identity and trust and has evolved over three years into two separate groups: an individual cf group and a couples cf group. ownership of these groups by those who attend is apparent. both the care center and the couples support group are forums for learning 'how to live with cf'. future goals will call for using identified quality of life tools to measure psychosocial and emotional growth in individuals through their participation over time in the group. sawicki, g. ; asher, d. ; dill, e. ; sellers, d. ; robinson, w. . children's hospital, boston, ma, usa; . education development center, newton, ma, usa background: advance care planning (acp) is an important tool to promote alignment of the patient's care with his or her needs, goals and values, and is particularly useful if the patient's ability to make decision becomes compromised. the purpose of acp is to develop a treatment plan promoting a high quality of life for those near the end of life. such planning is especially appropriate in persons with life limiting disease. minimal research exists on the advance care planning process of adults with cf.<p>methods: in the fifth survey wave of the project on adult care in cystic fibrosis (pac-cf), an ongoing longitudinal panel study of cf adults from cf centers in the us, participants were asked to report on their opinions and experiences with advance care planning. the survey asked about opinions on advance care planning documents, communication about advance care planning with family and clinicians and whether a directive had been completed. clinical data were obtained from the participating centers. preliminary bivariate analyses were conducted to examine factors associated with completing an advance care directive.<p>results: / ( %) surveys were completed. the mean age was ± years, % were female, and the mean fev (% predicted) was ± . % reported that they had spoken to someone about the care they would want if they became too ill to make their own decisions in the future, % had thought about whom they would want as their healthcare proxy, and % reported having specific wishes about the types of medical treatment they want or would not want if they became too ill to make decisions for themselves in the future. however, only % reported completing a healthcare proxy form, living will or any other type of written instructions concerning advance care directives. % of respondents reported feeling comfortable talking to their clinician about acp. % of subjects said that their cf clinicians have asked them about acp, and only % reported that they have discussed acp with their cf clinician. the following characteristics were significantly related (p<. ) to completion of an advance care directive: female gender ( % vs. %), age (mean years vs. years), having a college degree ( % vs. %), being married currently or in the past ( % vs. %), having children ( % vs. %), having diabetes ( % vs. %), having specific wishes about treatment decisions ( % vs. %), and reporting that a clinician had discussed acp with them ( % vs. %).<p>discussion: though the majority of cf adults report thinking about, communicating with family and deciding on their advance care wishes, only a minority report completing any legal documentation supporting their decision. additionally, very few cf adults report being asked about acp by their clinicians and even fewer report discussing acp with them. formulating specific wishes and discussing acp with a clinician are strongly associated with completing an advance care directive, suggesting that if clinicians were more active in talking to their patients about acp, patients are much more likely to complete advance care directives. efforts to improve clinician communication with cf adults around the issues of advance care planning are needed. objectives: caregiver involvement in children's cf treatments fosters better adherence and improved health outcomes. several studies have suggested that parents of children with cf report elevated symptoms of depression (quittner et al., ) , however, there is no data on the effects of caregiver depression on adherence. adherence to treatment is difficult to measure in patients with cf, and prior research has relied primarily on self-report, which is likely to be inflated. the purpose of this study was to examine the effects of caregiver depression on adherence to enzymes using both parent-reported and electronically monitored enzyme adherence over a three-month period. in addition, we examined the relationship between depression, rates of enzyme adherence and short-term changes in weight. methods: as part of a larger intervention study at cf centers, children with cf ages to and their parents were recruited. parents completed a standardized measure of depression (cesd) at enrollment and were provided with mems caps that recorded the date and time of each bottle opening. three months later, the electronic data from the pill caps was downloaded and parents completed a structured interview reporting their child's adherence to all components of the cf treatment regimen. only the section addressing enzyme adherence was included in this analysis. standard health outcomes, such as weight and height, were also assessed at enrollment and months later. results: preliminary analyses indicated that caregivers reported elevated levels of depression, with % scoring in the clinical range. rates of adherence to enzymes were poor ( % at home and % at school). caregiver depression was negatively associated with adherence, with depressed caregivers demonstrating lower rates of adherence ( percentage points). adherence to enzymes was associated with changes in weight, with a % adherence translating into percentile points of weight gain. final analyses will also include parent-reported adherence to enzymes. conclusions: a substantial number of parents scored in the clinical range on a depression screening measure. in addition, depression was associated with poorer adherence. rates of adherence to enzymes were surprisingly low, both at home and at school. furthermore, poor adherence was associated with a decrease in weight three months later. caregiver depression appears to be under-diagnosed and these results suggest that screening and intervention may be warranted. adherence to enzymes should also be targeted in clinical interventions. funding was provided by nih grant #hl of non-compliance with medical treatment and greater behavioral and emotional distress. adherence is particularly problematic during adolescence, however, few family-based interventions have been developed to target adherence behaviors in this population. the goal of this study was to compare the effects of behavioral-family systems therapy (bfst), an empirically-supported treatment, to both family education (fe) and standard care (sc). methods: as part of a larger intervention study, adolescents with cf ages to and their parents completed the conflict behavior questionnaire (cbq) and the parent-adolescent relationship questionnaire (parq) developed by robin and foster ( ) . adolescents completed these measures for each parent and both parents, if available, completed them with respect to their teen. the problem-solving, communication, and beliefs subscales from the parq were administered. participants were then randomly assigned to one of three treatment arms: bfst, fe or sc. families in the bfst group received ten -minute sessions over a month period, including family problem-solving, communication skills training, and cognitive restructuring. families assigned to the fe group received ten minute psychoeducational sessions over months aimed at increasing knowledge about cf and its management. those in the sc group received their usual care at the cf center. the cbq and parq were completed pre, post and , and months following the intervention. results: adolescents reported that the bfst intervention, relative to the other two, improved their communication with their primary caregivers (p < . ) and caregivers improved their communication with their adolescents (p < . ). in addition, caregivers reported improved problem-solving with their adolescents (p < . ). on the cbq, bfst reduced both adolescent and primary caregiver conflict and fe reduced caregiver conflict. no significant improvements were found for those in sc. conclusions: preliminary findings suggest that bfst is effective in improving communication skills and reducing conflict in adolescents with cf and their caregivers. future analyses will evaluate the effectiveness of bfst and fe on adherence and other family functioning measures over the course of the study. funding was provided by nih (hl # ) background: cases of diabetes among people with cystic fibrosis (cf) have increased as life expectancy for these patients improves, yet the impact of this additional illness on daily functioning is under-researched. to explore this issue, a study was conducted comparing the views of patients with either cystic fibrosis related diabetes (cfrd) or type diabetes mellitus (t dm) about being diagnosed and living with diabetes. methods: qualitative research was used because the study was concerned with understanding diabetes from patients' perspectives. purposive sampling was employed to achieve maximum variation in terms of duration and type of diabetes experienced by participants. data were collected via semi-structured interviews, all of which were taped and transcribed verbatim. recruitment continued until no new ideas or insights emerged from additional participants. a framework approach to analysis was adopted. this involved coding and summarising interview data into charts to explore and develop main themes. initial analysis was undertaken by two researchers (st and cd) and amended after comments from the remaining authors. results: interviews, carried out with cfrd and similarly aged t dm patients, lasted for an average of minutes (range - minutes). the following themes were derived from the data collected: evolving vs fracturing identity; diabetes in context; self-management motivators. for patients with cfrd, diagnosis represented a progression in their health status, which called on them to adapt existing treatment regimens to accommodate this additional condition. in contrast, interviewees with t dm had to re-evaluate their previous sense of self as 'healthy' and adjust to manag-ing a long-term complaint. these individuals were more likely to talk about diabetes in relation to a range of competing commitments (e.g. work or family related) and to describe feeling psychologically low due to diabetes compared to patients with cfrd, the latter depicting demands from their primary illness (cf) as a major obstacle to caring for diabetes. participants with cfrd recalled feeling lucky when told they would not face strict dietary restraints, which they associated with other forms of diabetes, and seemed less concerned about diabetic complications than those with t dm. for interviewees with t dm, a desire to reduce future health risks motivated their self-management efforts, whilst those with cfrd were driven by the negative effect poor control of diabetes had on their chest and weight. conclusions: both sets of interviewees found diabetes time consuming and, on occasions, frustrating to accommodate into daily life. findings from the study act as a reminder that patients manage their condition in the real world, against a plethora of other demands on their time and energy. in the narratives of participants with cfrd, these demands were often related to the existence of their primary illness. ing the course of development. the purpose of this study is to examine rates of medication adherence across a wide age span using an objective measure of adherence and to compare adherence to health outcomes. methods: patients with cf age years or older who are prescribed azithromycin, colistin, hypertonic saline, pulmozyme, and/or tobi for at least one year are eligible. recruitment is ongoing. the previous year's medication refill records were requested from participant-identified pharmacies. a medication possession ratio (mpr) was calculated for each drug and was defined as the sum of all days of medication supply received during the months divided by the number of days the medication was prescribed. values were truncated to % and averaged across all medications to obtain a composite score. medical records were abstracted to identify the prescribed drug regimen and health outcomes (e.g., lung function, exacerbations, bmi) over the same time period. results: thus far, patients have joined the study ( % participation rate), resulting in pharmacy records requested ( unique pharmacies). of the first participants with complete pharmacy data, the mean age was . years (sd= . ; range= - ) and % were female. the table presents mpr data for each drug and composite score overall and stratified by child, adolescent, and adult participants. the complexity of the drug regimen and age were significantly correlated with the composite score (rho= -. and rho= -. respectively; p<. ). future analyses will compare the composite score and each drug's mpr with health outcomes. con-clusions: participants had suboptimal medication adherence similar to that reported in the literature. as mpr provides the maximal possible level of adherence, actual adherence may be even lower. poor adherence spanned the age groups and was associated with regimen complexity. children had the highest adherence, but also the least complicated drug regimen. these results suggest that obtaining pharmacy records is a viable means to objectively assessing medication adherence. moreover, results suggest that interventions targeting medication adherence may be a strategy for improving health outcomes, particularly for adolescents and adults. * fewer than participants within this cell (cf) whose exchange capacities for oxygen and carbon dioxide are already diminished. lack of rem and delta sleep have significant implications for the daytime functioning of pediatric patients with cf, potentially leading to impaired memory and attention which would reduce their ability to complete typical activities, such as school work and disease-specific tasks related to self care and adherence. based on these clinic findings, the authors are beginning a prospective study on the effects of sleep on neuropsychological functioning, well-being and compliance in pediatric patients with cf. future research should examine the relationship between decreased slow wave, rem sleep and psychosocial outcomes, including attention, memory, and health-related quality of life. there are diagnostic challenges related to non-classic cf (knowles and durie, ) particularly patients presenting in adolescence or adulthood with obstructive azoospermia, chronic sinopulmonary disease and chronic or acute, recurrent pancreatitis. practitioners are often unable to provide clarity to patients about diagnosis, disease course, and prognosis. potential harms and benefits of relaying a clear or unclear diagnosis may be psychological rather than physical and have long-term implications for mental and social well-being. the aim of the current study is to assess the psychological impact of diagnostic information for adults presenting with a cf phenotype, be it confirmatory (cf or most likely not cf) or unclear. method: we administered self report measures pertaining to psychological state, cognitive appraisal and uncertainty in a welldefined cohort of adult patients. presenting symptoms of patients include chronic sinopulmonary disease, obstructive azoospermia, pancreatitis, and those presenting with more than more cf phenotype. patients completed the self-report measures on two occasions: at the time of diagnostic testing and months after being counseled and notified of the diagnostic results. at the time of the initial assessment, the subjects were unaware of the test results and had not been seen by a cf physician. results: we provide interim observations on patients (sinopulmonary (n= ), obstructive azoospermia (n= ), pancreatitis (n= ), and multiple phenotypes (n= ) who completed the initial assessment. at the time of diagnostic testing, the level of depression, hostility, anxiety and interpersonal sensitivity were elevated in each group (t score > ); male patients reported significantly greater depression, anxiety and interpersonal sensitivity than female patients. compared to published normative data of patients with mixed chronic illnesses, patients presenting with sinopulmonary or pancreatic disorders reported significantly more uncertainty(p < . ). to date, of these patients have completed the measures months after receiving the diagnostic information. preliminary review of results shows that the level of depression, hostility, anxiety and interpersonal sensitivity remained elevated while the degree of uncertainty decreased. among the patients who completed assessment at months, patients were told that it was unlikely that they had cf, were told that they had mild cf, while were given an unclear diagnosis. those who were told that they did not have cf reported more uncertainty than those who were told they had cf (p < . ). detailed analyses related to outcome diagnosis will be conducted at the time of study completion (october ) . when this study is complete, the information will assist in our understanding of the psychological impact of a genetic diagnosis at an older age, identify issues confronting those individuals and help to establish appropriate paradigms for delivering complex information about genetic diseases. supported by grants from niddk [scor] and the canadian cf foundation. ( ). in accordance with these guidelines, we report our experience in using our nutrition action plan (nap) in patients with suboptimal nutrition. objective: to determine if implementing our nap would improve the bmi of these patients. methods: our nap is a written contract between the patient, caregiver and cf team. for clarity of nutritional goals, the nap is based on the colors of a traffic light. the red zone indicates a bmi less than the th percentile and poor nutritional status. the yellow zone indicates a bmi between the th and th percentile and fair nutritional status. the green zone indicates a bmi greater or equal to the th percentile and desired nutritional status. within each color zone, specific recommendations to improve nutritional status are listed. the ultimate goal for each patient is reaching the green zone. the caregiver, patient and nutritionist review the current nutritional status and a mutually accepted weight gain goal is established. the goal is recorded on the nap which is then signed by the nutritionist and patient. this contract establishes a commitment to achieve the goal by the next clinic visit. a reward system is implemented in conjunction with the nap. for patients reaching their goal, a previously agreed upon prize is given. patients achieving some weight gain but falling short of their goal receive a gift such as candy or small toys that serve as an incentive for continued weight gain. patients chosen for inclusion into this study demonstrated a bmi less than the th percentile; those on growth hormone, appetite stimulants or chronic systemic steroids were excluded. results: twenty-one patients, male and female, with a mean age of . years (range to years) were included in this study. fifteen patients demonstrated an improvement in their bmi with use of the nap: improved by - %; improved by - %; improved by - %; improved by - %; improved by - %. six participants had a negative change in bmi which we attribute to family upheaval, missed appointments with the cf team, exacerbations of disease and non-adherence. conclusion: we intervened in patients with a bmi less than the th percentile using a nap and a reward system. the results of this pilot study suggest that our nap and reward system may be of benefit in achieving positive gains in bmi. reference: ( ) previously it was rare for women with severe cf to undertake pregnancy and doctors tended to advise against pregnancy if the forced expiratory volume in one second (fev ) was less than %. the attitudes of doctors and patients are changing and many women living their lives with cf nowadays chose to undertake pregnancy despite the severity of their health problems. over the last years females (age range - years) have attended the newcastle adult cf centre. there have been a total of pregnancies involving women with live births ( ectopic). a further women had termination of unwanted pregnancy. mean age at the time of pregnancy was years. we compared an age-matched cohort of never pregnant women to see if there were differences in key variables making successful pregnancy more likely. the mean fev was % (range - %) and % (range - %) of predicted in the pregnant and non-pregnant group respectively (p= . ); rates of chronic pseudomonas aeuriginosa infection were ( %) and ( %) respectively (p= . ). cases of burkholderia cenocepacia were identified in the non pregnant group. there were no cases in the pregnant group (p= . ); ( %) had diabetes with patients having cf related diabetes prior to pregnancy and patients developing gestational diabetes; this compared with ( %) of non pregnant women (p= . ); there was impaired nutritional status within both groups with a mean bmi of . pre pregnancy (range - ) and . in the non pregnant group (range - ) (p= . ); ( %) and ( %) patients had had previous gastrostomy feeding (p= . ). pancreatic insufficiency was present in ( %) and ( %) patients in respective groups (p= . ). no child had cf but one child had complex congenital heart disease. there were no maternal deaths but patients subsequently underwent lung transplantation and patients died leaving young children (ages and years). patients in the non-pregnant group have also died. conclusion the profile of women with cf undertaking pregnancy is now similar to the overall cf population. the woman's decision to undertake pregnancy is often not directly related to the severity of her disease. amongst those who successfully completed pregnancy maternal and fetal outcomes are generally favorable. objectives: health-related quality of life (hrqol) instruments provide important information on disease progression and are increasingly used as patient-reported outcomes (pros) for behavioral and pharmacological trials (goss & quittner, in press) . recent advances in medical care have improved the long-term stability of pulmonary functioning, which has made it more difficult to detect small improvements in fev % predicted. new outcome measures are needed; the cystic fibrosis questionnaire-revised (cfq-r; quittner et al., ) , a disease-specific hrqol measure, has shown promise in this regard. the purpose of this study was to examine the longitudinal relationships between fev % predicted, weight percentiles, and cfq-r scores for adolescents with cf. methods: as part of a larger study evaluating adherence interventions, adolescents with cf, ages to , and their parents were enrolled at six centers. parents reported on their adolescents using the cfq-parent version, and adolescents completed the cfq-teen/adult version as a selfreport. measures were completed pre and post treatment and at , , and month follow-up visits. spirometry and weight data were collected at these same time points. results: empirical bayes estimates were used to examine change over time. preliminary results indicated that adolescents' scores on the cfq-r respiratory scale were significantly and positively correlated with changes in pulmonary functioning. parents' scores on the cfq-r vitality scale were also correlated with changes in pulmonary functioning, with higher cfq-r vitality scores associated with better lung function. in addition, parents' scores on the cfq-r weight scale were significantly and positively correlated with changes in adolescents' weight. conclusions: these results support the validity and sensitivity of the cfq-r to changes in both pulmonary functioning and weight for adolescents with cf. additional analyses will be conducted to determine the relative stability of hrqol scores over time and to examine other cfq-r scales that may reflect positive changes in adolescent functioning (e.g., reduced treatment burden background: development of cf specific patient symptom tools as outcome measures is a critical step toward the development of potential new therapies for cf. we have developed a novel instrument to assess respiratory symptoms in cf patients. methods: we conducted in-depth qualitative interviews using the day reconstruction method and cognitive debriefing interviews ( adults, youth, and parents) at two cf programs, the university of washington and children's hospital and regional medical center. interviews were conducted until no new symptoms were mentioned by interviewees (i.e., data saturation). the interviews were audio-recorded, transcribed, coded and analyzed for themes. results: six pulmonary symptoms were identified in the interviews: cough, sputum production, wheeze, chest tightness, difficulty breathing/shortness of breath and fever. in addition, the most commonly cited activity and emotional impacts identified in the interviews were also included on the questionnaire. these emotions included: frustration, sadness/depression, irritability, worry, difficulty sleeping, and activities included time spent sitting or lying down, reduction of usual activities, and missing school or work. in all, eight symptom items were selected for inclusion on the self-administered questionnaire (see table) . as a result of the cognitive debriefing interviews, we changed initial language and adjusted the response options on some of the items in order to create greater distinctions between them. no important issues were felt to be omitted by patients who were interviewed. conclusions: using qualitative inductive methodology, we have created a novel patient reported outcome measure for cf. further study will be required to assess validation of the instrument. or more signs and symptoms occurred for more than two days, antibiotics were ordered with physician input and phone follow-up in days. if symptoms occurred less than two days, chest clearance was increased with phone follow-up in hours. the initial algorithm (ia) was piloted by nurses on patient calls on the emr and reviewed for adherence. subsequently, the cf phone note was revised, expanding the symptoms to include gi issues, last visit date and last hospitalization. the revised algorithm (ra) was used for a one month trial period with the entire nursing staff. a total of patient calls relating to pulmonary exacerbation were again reviewed for adherence to the algorithm. results: the review of phone notes following the ia found ( %) of the phone notes to be adherent to the assessment and treatment plan on the algorithm. several exceptions included calls received for gi issues, medications, complaints of general pediatric symptoms, and lab results. the phone follow-up within - hrs was also difficult to accomplish secondary to nursing responsibilities and difficulty locating the patient. the review of calls following the ra found ( %) notes were adherent to the algorithm. discussion: the revised algorithm and cf phone note for pulmonary exacerbation improved consistency in nursing assessment of signs and symptoms, evaluation of the current plan of care, and triggered appropriate interventions based on symptoms and length of illness. this improvement was particularly pronounced among the part-time rns who found it easier to obtain a more accurate history of patient's illnesses without asking for assistance from other cf team members. since the algorithm and cf phone note are newly implemented, tracking for consistency with the cf phone note will continue, revisions will be made as needed, and phone notes for conditions other than pulmonary exacerbation may be developed. methods: in , helen devos children's hospital integrated research coordinators (rc) from the spectrum health research department into the cf care team. this model incorporates two key aspects: . a shared resource of experienced, centrally educated and trained research coordinators (rc) with three rc's having a primary cf focus . incorporation of rc's into helen devos cf care centers' multidisciplinary team for weekly contact with patients. using the cf registry consent as a starting point of research discussion with the patients and families, the rc's present new and upcoming opportunities and answer questions about research. with attendance at local and national cf meetings, rc's have expanded their cf knowledge, networked with other rc's and explored new research trials. our site also integrated education about various aspects of research into both the clinical staff and cf family education meetings. a review of the research database was completed to assess our cf center's research program. results: over the years reviewed, participation in clinical research trials has seen significant growth and patient participation has exceeded patients enrolled into clinical studies. conclusion: utilizing a team of dedicated rc's from the research department has proven to be effective in advancing our cf center's clinical research program. discussion: to meet the growing demand of patients needed to participate in clinical trials, cf centers will need to provide research education to both the patients and families and the clinical team, improve on skills needed to identify and enroll our patients into clinical trials and include the rc's into the multi-disciplinary cf patient care team. centralized research represents an excellent model for cf centers that want to excel in clinical research. integration of rc's into the cf care team, along with education initiatives for both clinical staff and patients and families has led to successful participation and enrollment in clinical trials. further studies need to be done to better identify barriers that patients and families have with participation in research. the goal of "cystic fibrosis transition care" is to ensure that youth with cystic fibrosis (cf) are adequately prepared to participate in the management of their health condition into adulthood and as they graduate to the adult health care system. in canada, it is unknown if, or to what extent, cf transition care is practiced in individual clinics. we therefore surveyed all canadian pediatric cf clinic coordinators (n= )using a brief standardized questionnaire; surveys ( %) were completed and returned for analysis. findings: all responding clinics transfer their pediatric patients to a distinct adult cf clinic, however almost half of these share one or more team members between the two clinics. transition care is recognized and practiced by % of responding clinics: % follow a formal transition program, and % follow an "informal program". "informal" practices vary widely from one clinic to another. formal programs were created by individual clinics, and share the common properties of being based on current research, having set goals, and utilizing a tool to document the process. the average age of transfer to the adult clinic is years. most clinics have "rare exceptions only" to delaying the age of transfer, the most commonly cited reason being "very ill/palliative patient". a small number of clinics also cited "intellectually challenged patient" and "reluctant patient" as reasons to delay transfer. only % of pediatric clinics hold a formal "transition" or "graduation" clinic, which allows adult team members to be introduced to youth before the actual transfer of care takes place. conclusions: although sharing team members between pediatric and adult clinics may ease the change from pediatric to adult care, it can also create barriers to the transition process. therefore, it is encouraging to learn that the majority of canadian pediatric cf clinics view adult care as distinct from pediatric care, and that there is a consistent age of transfer across the country. transition preparedness is also recognized as an important component of care, as evidenced by the number of clinics following a formal or informal transition process. it remains undetermined, however, whether in general there is adequate preparation for adult care. with only % of clinics following a formal transition process (and % not following any type of transition program), further assessment seems warranted and may reveal inadequacies requiring remediation. future direction: to strengthen cf transition care in canada, a "patient readiness to transition" questionnaire has been developed and will be administered to canadian cf patients transferring to adult cf clinics in . analysis of this questionnaire will provide insight into how well canadian cf clinics are preparing their youth for transfer to adult life, and may provide directions for further improvement. nearly all females with cf now survive into adulthood, and advice regarding pregnancy and contraception is becoming increasingly important as part of their sexual health education. we set out to identify the level of knowledge of these issues in patients attending our large adult cf unit. using a structured questionnaire, we surveyed consecutive cf females (age range to years) attending routine cf clinics. we asked about knowledge and usage of contraception, and issues relating to pregnancy and its possible effect in cf, ensuring the opportunity for a one to one discussion with the individual on completion of the questionnaire. only ( %) claimed to have had previous advice relating to contraception and ( %) pregnancy education. of these, ( %) stated that this was from adult cf nurse specialists, ( %) from adult cf physicians and the remainder from other sources (general medical practitioners, written literature, and the internet). only patients ( %) recalled that they had been given information in the paediatric sector before transition. the majority of all advice was given verbally. of the ( %) who were using some form of contraception ( [ %] condoms, [ %] combined pill, [ %] depot injection, [ %] a coil, and [ %] the mini pill); ( %) had experienced problems, and in some cases patients had been misinformed about the reliability of their contraceptive choice. as regards pregnancy, ( %) had undergone previous termination and ( %) already had children. thirty six ( %) had considered becoming pregnant but ( %) said they would discuss this with a member of the cf team beforehand: ( %) were aware this might impact on their health. of these, ( %) believed a reduction in lung capacity to be the biggest possible problem. other problems identified were alteration of medication, tiredness, and weight loss or gain. overall, ( %) felt they did not have enough information regarding pregnancy in cf and ( %) that insufficient contraceptive advice was given, expressing a wish for further knowledge. conclusion this survey has shown that a significant number of adult females with cf require further education about contraception and pregnancy. in some cases the advice they had already been given may not have been appropriate for patients with cf. furthermore, few patients appeared to have had effective counselling in the paediatric sector, despite the risk of pregnancy. we are working with patients and paediatric colleagues to improve the education in this important area for our women with cf. the state of minnesota added cystic fibrosis to the newborn screening panel on march , . one-year follow-up to date reveals infants have screened positive, of the screen positive, have been diagnosed with cystic fibrosis, infants are pending sweat chloride tests and evaluation. of this population infants are being followed by the minnesota cf center. the university has a comprehensive, interdisciplinary team available to provide care to infants, children and adults with cystic fibrosis. in response to newborn screening for cystic fibrosis, the center has implemented a prophylactic care program to include clinical care through early intervention and education within the first two weeks of life. benefits of collaborative care have been previously identified in the literature, showing improved quality of care with increased patient satisfaction, lower mortality, and improved outcomes. infants who are identified by newborn screening and receive earlier treatment have the potential for improved physical health and development. the inclusion of comprehensive genetic consultation as an integral component of cf education, allows for informed reproductive decisions. the newborn screening intervention plan is supervised by a nurse practitioner and consists of initial evaluation, initial therapy implementation, and a teaching/consultation schedule, which covers a minimum of four appointments scheduled over weeks. follow-up visits are scheduled within hours of referral and then at one week, days and weeks post referral. the schedule is adjusted based on infant acuity and family needs. initial evaluation consists of the confirmatory sweat chloride testing and/or genetic mutation analysis, serum laboratories, chest x-ray, stool for fecal elastase, and nasopharyngeal culture. general, supportive teaching with evidence from the literature is provided. preliminary results to date demonstrate that our infants have had no pulmonary exacerbations, no pulmonary hospitalizations with pulmonary functions obtained in over half of the infants. nasopharyngeal cultures obtained at the time of ascertainment demonstrate newborn screening patients have positive cultures for staphylococcus aureus, haemophilus influenzae, escherichia coli, streptococcus pneumoniae, klebsiella pneumoniae, acinetobacter junii and alcaligenes faecalis. one infant was culture positive for pseudomonas aeruginosa at the first out-patient clinic visit. this demonstrates colonization of organisms prior to clinic exposure. through minnesota's early intervention and education plan, the families will be more adherent to prescribed care, which will result in improved cf infant outcomes as monitored by number of exacerbations, pulmonary functions and potential complications. a detailed outline of medical intervention, education, and outcomes will be presented. cf-pediatric centre we developed a malabsorption blood test (mbt) using odd-chain length fatty acids, pentadecanoic acid (pa) and heptadecanoic acid (ha), to assess fat absorption in subjects with cf. pa is a free fatty acid, while ha requires hydrolysis of triheptadecanoic acid (tha) by pancreatic enzymes for absorption. objective: to determine the mbt reproducibility in healthy subjects and subjects with cf. methods: subjects with cf ingested a liquid test meal including fats (pa and tha) on occasions at least days apart. for each subject, a standard dose of pert ( , lipase units) or the subject's usual dose (if higher) was given with each test meal. serum was analyzed for pa and ha levels at baseline and then hourly for hours. a non-compartmental pharmacokinetic analysis was performed using pa and ha concentration-time data from subjects with cf. c max was directly observed from the individual subject profiles, and auc was calculated using the linear trapezoid rule. summary statistics (mean±s.d.) were calculated for all parameters. reproducibility of the mbt was also assessed in healthy adult controls tested on occasions. between subject (bsv, %) and within subject variability (wsv, %) was calculated for pa, ha and the ha/pa ratio. results: bsv (%) and wsv (%) of pa and ha absorption are presented in the table for subjects with cf (age . ± . y, females) and healthy adult subjects (age . ± . y, females). as expected, the mbt had greater reproducibility in healthy controls than in subjects with cf. in healthy controls, the reproducibility for pa and ha absorption was comparable; there is less wsv than bsv, and the ha/pa ratio has the best reproducibility. in subjects with cf, pa and the ha/pa ratio show less wsv than ha. conclusion: wider variability in fat absorption in subjects with cf reflects the complex interactions of the biliary, pancreatic and intestinal factors background: the international, multi-center gene modifier study of cystic fibrosis liver disease (cfld) initially enrolled patients with severe liver disease (i.e. cirrhosis and portal hypertension). for the initial population despite severe liver disease with portal hypertension, many of these patients had normal liver biochemical function tests and/or inr ( - %), depending on the test. the mean age of enrollment into the study was years, with the mean age of cfld diagnosis of years. candidate gene testing revealed an increased prevalence of the alpha- antitrypsin z allele in cfld patients, particularly in females, as compared to cf patients (> yrs.) without cfld, and an association with tgfβ variants (- and codon ) in males smoothing reference centile curves: the lms method and penalized likelihood hepb) and inactivated hepatitis a (hepa) vaccines in cystic fibrosis (cf) patients chronic liver disease (cld) remains the second mortality cause in cf. we evaluated the immunogenicity of hepa-and hepb-vaccines in cf-patients because it is described lower in patients with cld. patients and methods: blood samples of cf-patients aged . - . y (mean . y) were tested twice (time interval y) by chemiluminescent microparticle immunoassay to asses the presence of hbs-ab and ha-igg, and their vaccination status was recorded. seronegative patients for hbs-ab and/or ha-igg were vaccinated in between tests with engerix®, havrix® or twinrix®. results: / ( . %) patients tested positive for hbs-ab. / cld ( steatosis and cirrhosis). / tested positive at first but negative at present data are incomplete for hbs-ab and ha-igg for and patients. conclusions: . immunogenicity of hepa-and hepb-vaccines is comparable in cf-patients and healthy subjects. . cf-patients are at risk for cld and seroconversion must be checked after vaccination. . vaccination records are important as numbers of antibodies may decline beneath the detection limit in time methods: lf was assessed in children with cf, aged - yrs, using forced expiratory volume (fev(sub) (/sub)% predicted). body composition was measured using a reference four-component model(sup) (/sup) ( cm) allowing accurate evaluation of both fat mass (fm) and the components of fat-free mass dual-energy xray absorptiometry (dxa; lunar prodigy, g.e. medical systems) was used to measure fat, lean (non mineral) and bone mass and skin-fold thicknesses (bicep, tricep, sub-scapular) were measured. strength of relationship was assessed using pearson's correlation coefficient (r) and significant body composition components were fitted into a regression model. difference in fev(sub) (/sub) between the sexes was assessed with an independent t test. results: boys with cf did not differ from the reference population conclusion: our results confirm that body fat, but not ffm or bone mass, are related to the severity of impaired lf in children with cf. it is likely that the significant association between fev(sub) (/sub)and fat in girls but not boys reflects the poor body composition of the girls with cf. longitudinal follow-up of these children should indicate whether this sex difference persists after puberty. given the fact that prognosis is worse in girls the sex difference we've identified merits more attention. fuller nj et al. four-component model for the assessment of body composition in humans: comparison with alternative methods and evaluation of the density and hydration of fat-free mass conclusions: these results show that pancreatic status plays an important role in relation with pufa status in cf patients and particularly in (the initial fatty acids in the n- and n- pathways) in neutral lipids, nonesterified fatty acids, and phospholipids this may, in some way, account for the fact that : n- is more readily metabolized, and therefore depleted, in cf animals than in wt mice. conclusion: dha supplementation increased the localization does an integrated clinical and nutritional approach prevent this case control study examines the impact of clinical approach on pre and post-cfrd clinical course. patients with cfrd (mean age . + . yrs) matched to cf controls ( . + . yrs) for age, sex and pseudomonal status had parameters of clinical status and nutritional intervention recorded annually from six years pre diagnosis of cfrd to two years post. weight and body mass index (bmi) were lower at all time points to diagnosis of cfrd (ns) but were stable as a % of control values intravenous antibiotic treatment intensified, peaking at one year post diagnosis . days/yr (cfrd) v . (cf) ns but there was no difference in nebulised antibiotic use. an aggressive clinical approach prevents nutritional decline and delays respiratory decline until the year preceding diagnosis of cfrd data show mean (sd) or % , ; battezzati with advancing age insulin secretory defects and insulin resistance cause glucose intolerance and diabetes in an increasing proportion of cystic fibrosis (cf) patients. prediction of diabetes development in cf is made difficult by unique features: many patients are normoglycemic or even hypoglycemic after overnight fast, and there are repeated changes of glucose tolerance status from normal to diabetes and vice-versa, for many years and for unclear reasons. aim of the study was to detect predictive factors of definitive cf related diabetes (cfrd) development in patients undergoing oral glucose tolerance test (ogtt) evaluations routinarily. methods. starting from , all patients followed at the cf center in milan aged > years and without established cfrd undergo ogtt yearly. among those who received their first ogtt between and , had developed definitive diabetes by µu/ml, and . ± . vs . ± . ng/ml). glucose (p= . ) and insulin aucs (p= . ) were the most important predictive variables, respectively directly and inversely related to cfrd development (pseudo r for the model: . , p< . ). glycated hemoglobin and baseline glucose concentrations were directly related with outcome at univariate analysis whereas c-peptide concentrations were inversely related. in contrast, no relationship emerged between insulin-sensitivity indexes and outcome. anthropometric (weight, height z scores, bmi) and pulmonary function indexes were also unrelated. conclusions. insulin secretory defects are an important determinant of subsequent cfrd developement response rate was / in group a and / in group b. group a included males and females, mean age was months, sd . ; group b included males and females, mean age months, sd . . followgroup b parents do ("poor-very poor" health % vs %, p< . ), but over time health perception in parents of children with cf improves, and the gap vanishes ("poor-very poor" health in last year % in group b). post-diagnosis anxiety, depressive symptoms, sleep disturbances, mood changes, and nervousness are less frequently reported in group a ( % vs % p< . ), and the diagnosis was considered to affect the parent-child relationship more in group b than in a (p< . ). the two groups did not differ in the assessment scores of internalizing, externalizing and total behavioural/emotional problems. however, group b performed worse than the general control population average (mean . sd . vs mean sd . , p< . ). thirty-five% of parents in group a and % in group b changed their family planning projects following the diagnosis physical health and anxiety symptoms: does monitoring mediate the relation? results: physical health, monitoring, and anxiety symptoms were related. children who perceived themselves to be relatively healthy were less likely to have an information processing style characterized by high levels of monitoring (r = -. , p < . ), and they reported fewer trait anxiety symptoms (r = -. , p < . ). monitoring, as hypothesized, was associated with increased trait anxiety (r = . , p = . ). low bmi, a potential indicator of poor nutritional status and physical health among youth with cf, also was related to higher monitoring (r = -. , p = . ) and trait anxiety (r = -. , p = . ). tests of mediation indicate that monitoring partially mediated the relation between poor health and trait anxiety for both child report of physical health (goodman test: z = . , p = . ) and for bmi (goodman test: z = . , p = . ), but not when parental report or fev were used to assess youth's physical health, or when parental report of youth's internalizing symptoms was used as the outcome. conclusion: youth who tend to generally scan for and are attentive to changes in their environment, which may include their internal physical environment, were found to report more anxiety. moreover, this monitoring style appears to partially mediate the relation between physical health and anxiety among youth with cf. if monitoring is found to be a risk factor leading to an increase in future anxiety symptoms among youth with cf data was analysed using a thematic analysis: "framework" results: young people say that befriending is fun, offers opportunities for new experiences, a confidant outside the family, and gives them a boost psychologically. carers see befriending as confidence building for their children, providing time out for themselves, and helping with the big questions. befrienders see their role as mentoring, broadening young people's horizons and providing a safe place physically and emotionally. challenges include: forming and ending relationships, having multiple befrienders, ongoing support and training for befrienders, maintaining boundaries, sibling rivalry, and cost. conclusions: befriending is a new innovation in cf, and has the potential to make a difference to young people's lives. careful planning at the outset, ongoing support for befrienders and regular evaluation are essential factors in ensuring its success good practice in befriending services for people with learning disabilities qualitative research practice sage london ଙ in addition, there was high use of non-cf vitamins (n= ) and protein shakes (n= ) which were not included as a cam therapies. when prayer was excluded (which was used by % of respondents), . % of the patients are still using some form of cam. the most commonly used cam therapies in cf were relaxation therapies (n= ), massage therapy (n= ), chiropractor care (n= ), herb/plant product therapies (n= ), homeopathy (n= ), and yoga (n= ). an analysis of the relaxation therapies revealed that deep breathing exercises (n= ) were the most common and frequently combined with other relaxation techniques: meditation (n= ), progressive relaxation (n= ) and guided imagery (n= ). the most common herb/plant products used were: echinacea (n= ), garlic (n= ), selenium (n= ), ragweed or chamomile (n= ) and ginseng (n= ). the use of possible cf specific cam therapies were fish oils/omega fatty acids (n= ), glutathione (n= ), docosahexaenoic acid (n= ) and curcumin (n= ). in conclusion, cam is widely used by the cf population with "prayer for health" being the most common modality, however, many patients are utilizing multiple cam therapies (data not presented) within the framework of a weighted satisfaction model of quality of life, we investigated the importance ratings of adolescent and adult patients regarding disease-specific aspects of living with cf. method: outpatients (aged - years, m= . , sd= . ; fev - %, m= . , sd= . ) repeatedly filled in the cf-specific module of the questions on life satisfaction (flz-cf) to measure satisfaction with nine cf-specific aspects of life in relation to the subjective importance of each life domain. a ranking list of the most important life domains across the study group was determined, and intra-individual changes of the importance scores were analysed. associations of importance ratings with changes of pulmonary functioning (fev %) were examined. results: the most important aspects (% of "very important" or "extremely important" answers) are sleep ( . %), integration of therapy into daily routine ( . %), breathing ( . %), gastrointestinal functioning ( . %), and eating ( . %), less important is understanding by others ( %) , ; durieu, i. , . département d'information médicale, hospices civils de lyon dr. von hauner children`s hospital helios hospital, e.v. behring, berlin, germany supported by the cystic fibrosis foundation leroy matthews physician scientist award, the national heart, lung, and blood institute cystic fibrosis foundation therapeutics inc. symptoms patient-reported respiratory symptoms in cystic fibrosis: initial validation children's hospital and regional medical center the first author is supported by a second year clinical fellowship from razvi, s. ; quittell, l.m. ; sewall, a. ; marshall, b.c. ; saiman, l. . pediatric pulmonary medicine, columbia university, new york, ny, usa; . cystic fibrosis foundation, bethesda, md, usa significance: significant improvements have been made in diagnostic and therapeutic strategies for cf patients in recent decades. we hypothesized that these changes could potentially impact cf respiratory microbiology. thus, we examined longitudinal trends in the annual incidence and prevalence of cf respiratory pathogens from to .methods: cf foundation patient registry data, estimated to include % of the cf population in the u.s., was utilized in this analysis. patients were included if results from at least one respiratory culture were in the registry from january to december . patients were excluded after organ transplantation. to avoid misclassification of incident cases, a retrospective review of registry data from to was performed to establish the culture status of included patients prior to . thus, incident cases were subjects with first detection of a given pathogen in a given year. prevalent cases were defined as subjects with at least one positive respiratory culture for a given pathogen in a given year. all submitted culture results were included.results: the number of cf patients submitting registry data increased from , in to , in . the proportion of subjects meeting inclusion criteria remained relatively constant ( % to % per year). the median age of the study cohort increased from . years in to . years in . during the study period, the incidence of hemophilus influenzae remained stable ( . % to . %) as did the prevalence ( . % to . %). the incidence of pseudomonas aeruginosa ranged from . % in to a peak of . % in . the prevalence of p. aeruginosa declined from . % in to . % in . there is a trend for both an increasing incidence and prevalence of staphylococcus aureus; the incidence increased from . % in to . % in and the prevalence increased from . % in to . % in . the age specific prevalence of s. aureus remained highest in children aged - years. the incidence of methicillin-resistant s. aureus (mrsa) increased from . % in to . % in , with a parallel increase in prevalence from . % to . %. the highest prevalence of mrsa was noted in subjects years of age and older. the incidence of burkholderia cepacia complex decreased from . % in to . % in , while the prevalence remained relatively stable (range . % to . %). both the incidence and prevalence of stenotrophomonas maltophilia increased (incidence: . % to . %, prevalence: . % to . %).this retrospective study aims at assessing the respiratory function and the cystic fibrosis (cf) co-morbid conditions according to the patients' nutritional status.method: data were collected from the french observatory (onm) (n= ) (vlm, ined). the last year data for weight (w), height (h), fev , fvc were extracted from the database (exclusion criteria: transplan-tation); were also analysed the genotype, pancreatic enzymes consumption, pseudomonas aeruginosa (pa) colonization, cirrhosis, diabetes, pulmonary transplantation and mortality (sas . ). we used international age-adjusted standards for bmi z-score for children (c) and bmi for adults (over years) (a) (cole tj bmj ) to define underw (uw), normal w (nw), overw (ow) and obesity (ob) .results: data are available for patients in the c cohort and for patients in the a cohort ( %). nutritional status subgroups prevalence (%) is: . , . , . and . in c and . , . , . and . in a for respectively uw, nw, ow and ob. mean age in a is significantly increasing with bmi (p<. ). frequency of del/ del in c and a is lower in ow/ob (p<. ) as well as the use of pancreatic enzymes (p<. ). ow/ob patients have the best fev and fvc values whatever the gender and the age, with significantly less pa colonization in a (p<. ). we could identify a positive correlation between the pulmonary function and bmi. the cf co-morbid conditions demonstrated a lower prevalence of diabetes ( / ) and cirrhosis ( / ) in ow/ob. none of the ow/ob c or a are on a transplant waiting list versus . % in uw/nw (n= / ) and none died versus % in uw/nw (n= / ).conclusion: the observed prevalence of ow and ob in cf is respectively . and % whereas the french obepi study collected and %. our results suggest that increased bmi is associated with better fev , fvc, lower prevalence of pa, cirrhosis and diabetes. the potential risks of chronically high bmi have not been studied in this population yet, but justify further investigations in this longer life expectancy cohort. materials and methods: we retrospectively identified abdominal ct scans of consecutive patients ( females, males, mean age of years) with cystic fibrosis and a control group of consecutive patients ( females, males, mean age of years) scanned as potential renal donors. three readers reviewed all scans and recorded the presence and location of colonic wall redundancy, and the wall thickness of the ascending, transverse, and descending colon. clinical information on cystic fibrosis patients, including cftr gene mutations, was queried from our cystic fibrosis patient registry database. additionally, medical records of all cystic fibrosis patients were reviewed to determine the indication for abdominal ct. results: colonic wall redundancy was seen exclusively in patients with cystic fibrosis, and was noted in of patients each for reviewers , , and (p< . ). colonic wall redundancy was seen in of adult patients with cystic fibrosis ( %), but was not seen in any children age or younger (p< . ). excellent agreement was found for the ct identification of colonic wall redundancy among readers (kappa= . , p< . ). cystic fibrosis patients with colonic wall redundancy had significantly thicker ascending colonic walls (mean . mm) vs. those without wall redundancy (mean . mm) or controls (mean . mm), (p= . ). three patients with colonic wall redundancy had follow-up ct, and all showed temporal stability (mean of months). among adult cystic fibrosis patients, cftr gene mutations were available for of patients with and of without colonic wall redundancy. while the common ∆f mutation was the predominant mutant allele among patients with normal colons at ct (only of patients, or %, had an identified mutation other than ∆f on either allele), a higher prevalence of less common non-∆f mutations was seen patients with colonic wall redundancy ( of patients, or %, p< . ). the g x mutation was seen exclusively in patients with colonic wall redundancy ( of patients, or %, p= . ). there was no significant difference in the proportion of patients with abdominal pain (> . ), pancreatic insufficiency (p> . ), diabetes mellitus (p> . ), history of meconium ileus background: airway inflammation in cf is associated with marked remodelling and bronchiectasis. pro-inflammatory mediators such as advanced glycation end-products (ages) and the soluble receptor for age (srage) may perpetuate this response in the lung and in other organs such as the kidney . the total body burden of ages reflects exogenous sources from the diet, endogenous production by the body, tissue degradation and renal clearance, which may be reduced in renal impairment. the accumulation of endogenous age is accelerated in conditions of high oxidative stress and inflammation, and ages have been implicated in the pathogenesis of diabetic nephropathy. the burden and significance of ages in cf has not been determined, but if elevated, dietary modification of ages may represent a novel anti-inflammatory approach for cf. the aim of this study was to determine serum levels of age and srage, and identify clinical correlates of age and srage levels.methods: adults with cf (n= , males, % pancreatic insufficient, mean age . ± . years (range to years), mean fev %predicted . ± . %predicted (range - )) and healthy adults (n= , male, mean age . ± . years (range - years)) were studied. cf participants provided - serum samples each over a six month period, while healthy controls provided a single sample. serum was analysed for levels of advanced glycation end-product (age-cml) and soluble receptor for age (srage) (elisa). for each cf participant, levels in the multiple samples were averaged, and the median for the study sample reported. clinical data, including bmi, presence of cf-related diabetes mellitus (cfrd) and serum hba c level were collected from the medical record. mann-whitney tests were used to compare cf and control levels, while spearman rank correlations were used to identify clinical correlates of age and srage.results: prevalence of cfrd was . %. mean bmi was . ± . kg/m and mean hba c level was . ± . %. median (iqr) levels for age-cml were ( , ) background: osteopenia is diagnosed in cystic fibrosis (cf) using dual-energy x-ray absorptiometry (dxa). areal bmd from dxa is subject to error when bones are smaller in volume than reference standards (t-scores). normalization of bone size by use of zscores in cf is controversial and not widely utilized, thus comparing to larger bone areas. reports in cftr-deficient mice (cf mice) reveal osteopenia when measured by dxa. we hypothesized that use of pqct, which eliminates bias of size, would more accurately analyze bmd.methods: femurs were collected at necropsy from cf mice and c bl/ j (b ) mice at wks, and cf mice and b mice at wks (all female) for pqct. time points were chosen to coincide with pre-pubertal and adult ages for comparison to human disease. total mineral, trabecular and cortical densities were measured. student's t-test was used to detect significant differences (p< . ).results: femur measurements from both cf mice and b mice are listed as mean ± sd in the table (below). length was greater in b mice compared to cf mice at and wks. total area at the metaphysis and diaphysis were greater in b mice at and wks. data are consistent with larger bones in b mice. total mineral density , trabecular density and cortical density , however, were greater in cf mice compared to b ( & wks, wks, wks).discussion: our study demonstrates greater bmd in cf mice when volumetric data are analyzed and size differences are accounted for by use of pqct. these findings persisted at adult age, suggesting a normal deposition of bone. in clinical management, dxa imaging is readily available compared to pqct, and can be used as a predictor of bmd and fracture risk. correct reference ranges must be utilized to minimize erroneous values secondary to size. z-scores allow correction for differences in bone area and size, even in adults. more studies into understanding bone mineral deficit and tools of measurement are needed in cf. meanwhile, bone measurements by radiographic imaging must be taken in context of overall health, pubertal progression, and size of individuals. gainesville, fl, usa; . nemours children's clinic, orlando, fl, usa; . suny upstate medical university, syracuse, ny, usa; . case western reserve university, cleveland, oh, usa; . genentech, san fancisco, ca, usa background: greater growth rates, bmi and fat-free mass are associated with improved lung function in individuals with cf (pedreira, et al. pediatr pulmonol. ) . current treatments for weight gain have focused on nutritional supplements and appetite stimulation, and not underlying issues of catabolism or chronic disease. we hypothesized that anabolic effects of gh would not only improve weight and height, but lbm as well.methods: sixty-seven prepubertal children with cf and height less than or equal to the th percentile were randomized to daily gh (n= ) or observation (n= ) for a period of year, followed by an off-treatment observation period of months. children randomized to gh received somatropin injections daily at . mg/kg/wk. height and weight were measured every three months. height was evaluated as height standard deviation score (sds) to control for differences in age and sex. lbm was measured by dexa scan at time zero, at and months, and at the end of the -month observation period. in addition, for lbm, change from baseline was calculated for subjects for whom the same dexa equipment was used at both time points. the preliminary results of the first months of the study are presented here.results: data for subjects in the observation arm and subjects in the gh arm, who completed months, are available. as shown in the table below (listed as mean ± sd), gain in height, weight and lbm were significantly greater in the gh treated group than in the observation group over the month period.discussion: gh improves growth in prepubertal children with cf as measured by height sds. in addition, gh significantly improves weight gain and this gain is, in part a result of the significantly greater increase in lbm in the gh-treated group than in the observation group. the relationship between the improvement in lbm and other outcomes in cf deserves further exploration. the national cystic fibrosis(cf) registry database notes cystic fibrosis related diabetes mellitus(cfrd) as a complication in . % of cf patients at all ages. cfrd is associated with poorer nutrition and increased pulmonary morbidity. the cause of cfrd is not completely known but insulin resistance may be associated with fibrotic damage to pancreatic islet cells due to chronic inflammation. other studies suggest protein energy malnutrition(pem) in early life leads to impairment of insulin secretion and cystic fibrosis-related diabetes (cfrd) accounts for increased morbidity and mortality in patients with cf and occurs in approximately % of patients by the age of years. cfrd net (network for epidemiology and trials) is a consortium of four large uk cf centres caring for adult patients with cf. the aim of this group is to undertake research into the diagnosis, investigation and management of cfrd. in this abstract, we estimate the prevalence of diabetes in a screened population of adults with cf. methods annual review data were collected on attending patients with cf aged and above during . g ogtts were performed after fasting for at least hours on patients without known diabetes. each ogtt was categorised as either normal ( hour glucose . - . mmol/l), impaired ( hour glucose ≥ . mmol/l and < . mmol/l) or diabetic ( hour glucose ≥ . mmol/l). all patients with a diabetic ogtt were followed up with serial bm monitoring to determine whether or not they had cfrd. fasting plasma glucose (fpg) was considered to be elevated ≥ . mmol/l, isolated impaired fasting glucose (igf) was defined as a value between . mmol/l and . mmol/l and hypoglycaemia was defined as a blood glucose < . mmol/l. in addition, we obtained information on age, sex and prescription of anti-diabetic medications subsequent to ogtt. in three of four centres, hba c was routinely performed on all patients. of patients (median age years, % male), ( %) had established diabetes and were therefore excluded from further screening. of the remaining patients, ( %) underwent formal ogtt testing. in this latter group ( %) had a diabetic ogtt, ( %) an impaired ogtt and ( %) had evidence of reactive hypoglycaemia at hours. of the patients with a diabetic ogtt, ( %) only had an abnormal hour value, ( %) only had an elevated fpg and (< %) had both. all patients with diabetic ogtts underwent blood glucose monitoring and ( %) went on to treatment with hypoglycaemic agents in the calendar year. none of the patients with an isolated elevated fpg had diabetes.hba cs were available in of newly diagnosed patients with diabetes (in the centres which performed hba c). in this group median hba c was . %. this study confirms the high prevalence of diabetes among screened patients and the growing burden of diabetes management in adult cystic fibrosis clinics. it further highlights the importance of performing screening for diabetes in this population; the majority of patients were identified on the basis of abnormal hour values. despite this only half of all suitable patients underwent ogtts in the four centres committed to screening. these patients merit further study, as previous work suggests that the decline in clinical status occurs several years before diabetes becomes apparent. cystic fibrosis related diabetes (cfrd) occurs in up to % of patients with cystic fibrosis (cf), the incidence rising with increasing age. the likelihood of developing long-term complications secondary to diabetes increases with poor glycaemic control and duration of diabetes. as survival has improved for people with cf those who develop cfrd may live with diabetes for several years. aim: to establish the frequency of diabetic complications in patients with cystic fibrosis related diabetes.method : patients with cfrd attending the adult cf service at the royal brompton hospital between april -march were screened for diabetic complications and cardiovascular risk factors. a total of patients (male/female: / ) including post transplant patients were screened. mean age was . years ( - ), mean hba c . % ( . - . % ) and average duration of diabetes was . years (< - years).as immunosuppressive therapy can also cause many of the complications associated with diabetes the results are presented separately for nontransplant and post transplant patients (table ) . patients in total had retinopathy: background retinopathy, proliferative retinopathy undergoing laser therapy and maculopathy. the average duration of diabetes in those with background retinopathy was . years with only patient having diabetes for > years. the patient with proliferative retinopathy (non-transplant) had been diabetic for years. a raised creatinine level was identified in all transplanted patients with microalbuminuria but none of the non-transplanted patients. patient (nontransplant) had macroalbuminuria. post transplant patients were on treatment for hypertension and a further patients ( post transplant and nontransplant) had elevated blood pressure (> / ) at screening requiring follow-up. none of the patients had evidence of cardiovascular disease or stroke.conclusion: macrovascular complications were not seen. microvascular complications occurred but were less common than the reported incidence in type and type diabetes. this may reflect the relatively short duration of diabetes (mean . years) of patients in this study. a further study comparing cfrd patients with a non-cf diabetic control group of similar duration of diabetes is warranted. cystic fibrosis (cf) is a disease that leads to serious disturbances in nutritional status and bone calcification. comparison of two methods for assessment of bone mineralization: dexa and hand radiograms in diagnosing osteopenia or osteoporosis. study was performed in a group of cf patients ( f, m), aged - yrs. nutritional status was assessed using bmi, cole's index and bmc. radiograms of non-dominant hand were assessed according to normalized optical density comparing to aluminum standard. bone density was also assessed using dexa. for statistical analysis, backward stepwise binary logistic regression (wald's test) was used. analysis of data revealed that using bmc, cole's index and hand radiograms markers we can diagnose bone mass disturbances (z score < sd) with precision up to . % comparing to dexa. sensitivity and specificity of this method was respectively . % and . %. false negative results were obtained in patients and false positive were also in patients. hand radiograms method could be an alternative for dexa in screening of bone density disturbances in cf patients. the study was partly supported by grant of ministry of science and higher education no p e .in a very recent publication( ) a high rate of fasting ( %) and reactive hypoglycaemia ( %) was described in a group of n= cf patients older than years who received an oral glucose tolerance test. reactive hypoglycaemia was related to the minute glucose concentration in the ogt test. we were surprised by the high percentage of asymptomatic hypoglycaemic situations in cf patients. as a part of a prospective intervention study in cf patients with cfrd, we screened more than patients years or older with cf for cfrd. in this multi-centres population ogt was performed in the morning after overnight fasting according to who standards. using the same definition for fasting hypoglycaemia (glucose < mg/dl; . mmol/l) and reactive hypoglycaemia (glucose < mg/dl; . mmol/l at minute during ogt test)as in the other study we calculated the percentage of cf patients with fasting or reactive hypoglycaemia in our cohort. results: ogt were done in patients with cf. age(mean±sd) , ± , years; bmi z-score - . ± , ;height z-score - , ± , and weight z-score - , ± , . ogt was categorised according to ada criteria. normal ogt (n= all ; fasting hypoglycaemia (fh) n= ( , %), reactive hypoglycaemia (rh)n= ( , %),ifg (n= all, fh n= , rh n= ( , %), igt(n= ,fh n= ( , %), rh n= ),fgt (n= all, fh n= ,rh n= ) and diabetes mellitus (n= all, fh n= ( , %)). fh was observed in ( , %) and rh in ( , %) out of ogt tests. there were no difference related to age, bmi z-score, height z-score and weight z-score comparing those patients with fh or rh to those without fh or rh in all categories of ogt tests.in this large multi-centres cohort of cf patients we were neither able to confirm the high percentage of fasting nor of reactive asymptomatic hypoglycaemia which was reported recently on a small group of cf patients. nutritional status measured by bmi z-scores, height and weight z-scores were unaffected by hypoglycaemia in our cohort as noticed also in the small group. as the ogt tests in the other study were done in a single centre it might by that a centre specific situation influenced the frequency of hypoglycaemia in that small group. we conclude that large numbers of investigations might be needed to come up with firm conclusions related to frequency of specific aspects of glucose disturbance in cf patients.fh does not exclude diabetes mellitus or igt. as survival from cf improves, surveillance to identify and treat complications associated with longevity is an important component of management. renal disease has been reported in adults with cf. risk factors may include cf-related diabetes mellitus (cfrd) and use of nephrotoxic medications. diabetic nephropathy has been observed in the absence of cfrd , highlighting the need for screening for renal impairment. this study aimed to measure renal function in a sample of adults with cf, and to compare estimated glomerular filtration rate (egfr) and urinary creatinine clearance (urcrcl) as markers of renal function.methods: adults with cf aged years or over (n= , % male, % pancreatic insufficient, mean age . ± . years) underwent screening for renal impairment. of these patients, ( males) had cfrd, and ( males) had no history or clinical features of cfrd. hour urine collections were analysed for urcrcl, with renal impairment being defined as < ml/min. serum creatinine level was used to calculate egfr (mdrd formula), which was classified renal function as normal or mildly impaired (> ml/min); moderately impaired ( - ml/min) or severely impaired (< ml/min). sensitivity of egfr for identification of renal function was calculated using urinary creatinine clearance as the reference method. unpaired t-tests were used to compared cfrd with non-cfrd patients.results: mean urcrcl was ± ml/min, and eight patients ( . %) had renal impairment (< ml/min). of these, only had egfr suggestive of renal impairment. the sensitivity of egfr for identifying renal impairment in this sample was %. a further patients had moderately impaired egfr, but normal urcrcl. five of the patients with impaired urcrcl had cfrd. age, gender, cfrd, fev %predicted and hba c level did not correlate with urcrcl. the table compares cfrd patients with non-cfrd patients. hba c level was higher in cfrd patients. there were no other significant differences in clinical or renal function parameters.conclusion: renal impairment, is common in the adult cf population and is not confined to patients with cfrd. screening using egfr is poorly predictive of impaired urcrcl in this population. these results suggest that surveillance to monitor renal function is indicated in the adult cf population, including in patients without cfrd, and that determination of urcrcl should be included in the screening process. cystic fibrosis (cf) patients suffer from pancreatic insufficiency resulting in malabsorption of fat soluble vitamins including vitamin d. many fac-tors contribute to low bone mass and fractures in patients with cf; however, chronic vitamin d deficiency plays a major role. the prevalence of vitamin d deficiency has been reported as high as % in some specialized care centers. in addition, reports of occult vertebral fractures have been reported as high as %. we sought to determine the prevalence of vitamin d deficiency (defined as -hydroxyvitamin d ( (oh)d) < ng/ml) and of vertebral fractures at our cf center. we obtained irb approval to review the records of all patients seen at our cf center during [ ] [ ] . we collected information related to bone health including (oh)d, bone mineral density and lateral spine or chest x-rays to examine for the presence of a vertebral fracture. we reviewed the records of subjects who were seen at our center during the study period. the mean age of the subjects was ± years. subjects had a mean bmi of . ± and an fev % predicted of . ± %. the percentage of subjects having an annual (oh)d level checked was only % and % in and , respectively. the mean (oh)d was . ± and . ± ng/ml in and . the prevalence of vitamin deficiency was % and % in and . about one quarter of the subjects had bone mineral density testing with half of the tests showing osteopenia or osteoporosis. twenty-seven percent of subjects had vertebral abnormalites detected on lateral chest x-ray. we sought to determine whether any factors were associated with vitamin d deficiency. we found that taking a multivitamin did not significantly protect against vitamin d deficiency. however, not taking a supplement containing vitamin d other than a multivitamin was associated with a % risk of vitamin d deficiency (p= . ). subjects having (oh)d levels determined in the winter or spring was associated with a % risk of vitamin d deficiency (p= . ). in summary, we found annual testing for vitamin d status was inadequate in our cf center and that when (oh)d level was determined, over % of subjects were vitamin d deficient. nearly one quarter of our adult patients already had evidence of vertebral compression fracture seen in lateral chest x-ray. we urge greater screening for vitamin d deficiency in the cf population. effective protocols to prevent and/or treat vitamin d deficiency are urgently needed in the cf population. improved vitamin d status in cf patients is one factor that may reduce the high prevalence of vetebral fractures. support for this study was provided by proctor and gamble pharmaceuticals background: more cf patients are surviving into adulthood, in part due to the increasing use of new therapies and more aggressive management of chronic respiratory and gi disease. as a result, the recommended daily treatment regimens for most cf adults are both complex and time consuming. objective: to assess the self-reported daily treatment burden of cf therapies in a cohort of adults with cf. methods: in the sixth survey wave of the project on adult care in cf (pac-cf), an ongoing longitudinal panel study of adults with cf from us cf centers, respondents were asked to report the type of medications, inhaled therapies, and airway clearance therapies they used during the day prior to completing the survey, as well as estimate the time generally required to complete each type of therapy.results: of the respondents (response rate %), the median number of therapies reported was (iqr - ) and the median reported amount of time usually spent on treatments was minutes per day (iqr - minutes). forty-eight percent reported using or more different inhaled therapies or using inhaled therapies or more times during that one day. the most commonly reported inhaled therapies were a bronchodilator ( %), pulmozyme ( %), an inhaled steroid ( %), hypertonic saline ( %), and objective: caring for a child with cf is stressful and often leads to adverse effects for both children and caregivers. support from family and friends can help reduce the adverse effects of chronic stress, however, there is little research documenting types of support provision as well as sources of support. moreover, social support may be related to important health outcomes. this study described the types of support provided to caregivers of children with cf and examined relationships between support and health outcomes.method: as part of a larger intervention study to improve adherence to medical regimens, children with cf ages to and their caregivers were enrolled across cf centers in florida: university of florida, nemours children's clinic, and all children's hospital. caregivers completed the norbeck social support questionnaire (nssq; norbeck, ) in addition to other measures at baseline and follow up. this questionnaire provides information about the number and type of people providing support, as well as its type and quality.results: caregivers received the majority of support from their families, spouses and friends and characterized these relationships as longer in duration, with more frequent provision of support. other individuals who provided support included the cf team, religious leaders, counselors or psychologists, and work associates. overall, caregivers rated family members and spouses as providing the greatest amount (i.e., "quite a bit") of emotional support. spouses received the highest ratings for tangible support; they were perceived as providing "quite a bit" of support in comparison to families (i.e., moderate) and friends (i.e., a little). in terms of size of the support network, caregivers in this study had similar networks to those of a normative sample of healthy adults. in addition, there was no difference in amount of emotional or tangible support received by these caregivers. preliminary baseline results indicated that social support was positively related to both adherence, children's growth (i.e., height and weight percentile), and family income.discussion: caregivers of young children with cf reported similar support networks as other healthy adults. however, there was variability in the amount of support provided by source, with spouses providing the greatest amount. social support appeared to be a protective factor, and lack of support was related to negative health consequences for their children. more attention should be focused on the potentially beneficial effects of social support.funding was provided by nih grant # befriending is reported to be valued by those who have been befriended, offering opportunities for social activities and new experiences and can impact positively on self confidence and self esteem . whilst government policy supports befriending, few schemes collect evidence to demonstrate the effectiveness of these services . young people with cf may suffer social isolation due to chronic illness and treatment demands and a befriend-ing service was offered to those considered socially vulnerable in the edinburgh area.this project aims to evaluate the effectiveness of this befriending service on young people with cf and their carers. methods liverpool, united kingdom; . child mental health, university of liverpool, liverpool, united kingdom; . child health, university of liverpool, liverpool, united kingdom; . psychology, university of miami, miami, fl, usa; . psychology, birkbeck college, university of london, london, united kingdom; . mathematics and statistics, university of lancaster, lancaster, united kingdom; . psychiatry, university of manchester, manchester, united kingdom introduction: existing treatments for cystic fibrosis (cf) are time-consuming and labour intensive and biomedical advances are likely to lead to further novel interventions. there is concern amongst clinicians that a high care burden associated with these interventions may compromise the wellbeing of caregivers, reduce adherence to the treatment protocol and increase the likelihood of inadvertent errors in the delivery of interventions. at present, there is no measure of 'treatment burden' and therefore no way to assess the impact of new and increasingly more complex interventions conducted by lay caregivers at home. furthermore, trials are hampered by the lack of measurable patient-reported outcomes beyond a broad quality of life assessment. to address this gap we have developed a measure of treatment burden for cf using qualitative methods (focus groups, action research, in-depth interviews) with participating parents and a working group of cf team professional staff. this measure addresses the time, effort, meaning and ease of management for caregivers of children with cf up to years and post the first year following a diagnosis. methods: we report here the pilot phase of the validation of this instrument, which involved (i) cognitive interviewing with n= caregivers; and, (ii) n= caregivers completing the clcf instrument together with a quality of life measure at time points. this yielded data for stability, reliability and coherence of the hypothesized constructs. results: the clcf takes minutes to complete. face validity and acceptability are established. for the first pilot sample the age of the child ranged from . - . years. treatments took, on average minutes per day to complete and an average of minutes of this was devoted to administering enzymes particularly for carers of infants. a prominent concern amongst these parents was their child's height and weight and their efforts were directed at optimising growth. for the child however, management of nebulised medications and physiotherapy were more frequently flagged as a concern. these data suggest this is a reliable, coherent and useful, although complex tool. conclusion: the caregiver challenge for cf cannot be understood simply in terms of time and effort involved in delivery of treatments. it also involves contextualising interventions for their meaning at multiple levels of explanation for the individual concerned. structural equation modelling would be an appropriate way to proceed with the main validation when such complex relationships occur between latent variables and would serve as a confirmatory factor analysis for the hypothesised relationships. sponsored by: royal liverpool children's nhs trust and university of liverpool. funded by: national institute for health research (nihr) research for patient benefit programme.introduction: the french cf practice recommendations, published at the end of in parallel with the creation of cf reference centres, advise that each patient should be seen at least every months at a cystic fibrosis reference centre.objective: to investigate the impact of these recommendations on the effectiveness of the follow-up of patients at the four reference centres in the rhône-alpes region.methods: all patients with cystic fibrosis attending one of the four cf centres between and were retrospectively included. the total number of visits was recorded for each patient and each year of the study period. to determine the evolution slope for each patient followed for at least years, a negative binomial regression of number of visits versus time was carried out (confirmed with a repeated model). to estimate the impact of the recommendations, the analysis was restricted to patients with at least two visits before and after and a second model was adjusted with a new intercept in to estimate the change in slope as of this point.results: a total of patients were included in the cohort. the average number of visits per patient rose from . in to . in (p< . ). the proportion of patients with at least visits per year increased from % to %. the negative binomial regression for the patients having had at least years follow-up confirmed this trend with an average slope of + . (sd= . ). a total of patients were evaluable for the change in slope. no significant change in trend was observed in : only % of patients had a higher rate of growth (this change was significant for only subjects). at the last follow-up visit, patients with increasing rates of number of visits were older ( yrs vs. yrs, p< . ), had lower %fev ( vs. , p= . ), had a similar average number of visits before ( . vs. . , p= . ), and a similar weight-for-age z-score (- . vs. - . , p= . ) .conclusion: the number of visits per patient is regularly increasing. since the publication of the practice recommendations in , the growth has tended to slow down: clinicians were already convinced by the need for closer follow-up and had begun to increase the rate of visits. methods: patients > years who presented for routine healthcare voluntarily completed four surveys: ) the revised eating attitudes test (eat), validated for cf patients by abbott and colleagues, where higher scores reflect worse attitudes towards eating; ) the rosenberg self-esteem scale (rse), where higher scores reflect better self-esteem; ) the body image scale (bis), developed for cf patients by wenninger and colleagues, where higher scores reflect better body image; ) the cystic fibrosis questionnaire (cfq), developed by quittner and colleagues, where higher scores reflect better hrqol. also, fev %, body mass index (bmi), and pancreatic sufficiency or insufficiency (based on the need for pancreatic enzymes) was recorded. regression analyses controlling for age, gender and bmi were used to examine the associations between the surveys. results: this study included patients with % males and a mean age of years. the eat was negatively associated with the rse (p= . , adjusted r = . ), bis (p= . , adjusted r = . ) and cfq (p= . , adjusted r = . ). the rse was positively associated with the cfq (p= . , adjusted r = . ). the bis was positively associated with the rse (p= . , adjusted r = . ) and cfq (p= . , adjusted r = . ). neither bmi nor pancreatic function was associated with the surveys (p=ns). fev % was positively associated with the cfq (p= . , adjusted r = . ) but was not associated with the other surveys.conclusions: more negative attitudes towards eating predict worse selfesteem and body image, while more positive body image predicts better self-esteem. also, attitudes towards eating, self-esteem and body image are significant predictors of hrqol with body image being the most important predictor.clinical importance: hrqol is an important clinical outcome measure in cf. clinicians need to be sensitive to attitudes towards eating, self-esteem and body image in cf patients, because they are important predictors of hrqol. riekert, k.a. ; mogayzel, p.j. ; bilderback, a. ; hale, w. ; boyle, m.p. . medicine, johns hopkins university, baltimore, md, usa; . pediatrics, johns hopkins university, baltimore, md, usa background: existing research suggests that self-reported adherence to all aspects of the regimen is likely suboptimal and objective measures suggest even poorer adherence. there is little empirical data on how much adherence is necessary to achieve desired health outcomes. moreover, there is limited data on how adherence changes dur-objective: cf may be associated with pain attributed to several etiologies. this study evaluates the prevalence of pain symptoms in adult cf patients and the influence on patients` lives.methods: patients of adult cf centers in germany completed a validated, self report questionnaire during a routine clinic visit assessing characteristics of chronic pain (prevalence, duration, location, quality and intensity of pain symptoms). furthermore, the impact of pain on different aspects of life was explored using a numeric scale from to with being the worst. every-day-life was divided into categories: duties at home, recreation, social activities, occupation, sexual life, autonomy, vital activities and cf-therapy. the average intensity of pain within the last weeks was correlated to fev , bmi and age.results: patients ( male) completed the questionnaire. the age was - years (mean . ± . ). patients ( %) aged - years (mean . ± . )reported pain within the last months. bmi was . - . kg/m (mean . ± . ), fev - % (mean . ± . ). if asked for pain within the last weeks ( %) reported painful episodes lasting from - days with a mean of . days (± . ). most patients described pain occurring at more than one site with the head being the most localized site, followed by chest and abdomen. concerning the quality of pain . % characterized their pain episodes as attacks whereas . % reported them as continious, . % had continious pain combined with pain attacks. patients desribed the intensity of pain as . (± . ) on a scale from to with being the most severe pain. the category of life negatively influenced most of all by pain episodes was recreation, followed by occupation and duties at home. female patients were more limited in their acitivites by pain symptoms than male patients with the highest difference being reported in sexual life. there was no correlation of the average intensity of pain within the last weeks to fev , bmi or age.conclusion: the prelevance of pain in cf is often underestimated. painful episodes can be the cause of worsening the quality of life for adult cf patients. assessment of pain should be routinely performed as part of care in cf centers. objective. sleep has been examined in a number of pediatric conditions, with impaired sleep resulting in worse neurobehavioral functioning (beebe, ) . recent research has shown that slow wave sleep is critical for memory (marshall, helgadottir, molle, & born, ) . to the authors' knowledge, no studies have examined slow wave and rapid-eye-movement (rem) sleep in pediatric patients with cystic fibrosis. methods. retrospective medical record review revealed two patients who underwent clinically indicated polysomnography (psg) who reported feeling tired and lacking energy. patients were females with cystic fibrosis, ages and years. results. psg revealed an increase in stage sleep for both patients, resulting in increased risk and observed hypopneic episodes. patient -a experienced . % in stage , . % in stage , and % in stages and combined. this patient also had no rem sleep. patient -b experienced % in stage , . % in stage , and % in delta wave sleep, with no rem sleep. respiratory disturbance index (rdi) for patients a and b were calculated at . and . , respectively. respiratory effort related arousal (rera) contributed to rdi by . and . points in patients a and b, respectively. in addition, endtidal co for patient a and b were maxed at mm hg and mmhg, respectively. their epworth sleepiness scales were scored at and for patients a and b, respectively. conclusions. structure of sleep was abnormal in both patients with decreased slow wave (delta) sleep in patient b and lack of delta sleep in patient a. neither patient experienced rem sleep. compensatory increased stage (nrem) sleep could cause respiratory related abnormalities, such as hypopnea and apnea in patients with cystic fibrosis background: understanding and advancing the application of tools to measure patients' symptoms is critical to advancing our evaluation of potential new therapies used to treat cf. we performed initial evaluation of the measurement properties of a cf-specific respiratory symptom daily questionnaire. methods: we planned to enroll cf subjects, stratified by age, at three cf programs, the university of washington medical center and children's hospital and regional medical center in seattle and mary bridge children's hospital in tacoma in a prospective assessment of a novel cf specific respiratory questionnaire. cf subjects years and older were eligible for enrollment. patients (parents for children under the age of ) completed a daily symptom questionnaire during two -day periods of clinical stability and one -day period when patients were ill for a total of days. the ill state began when patients/parents sought medical attention for respiratory symptoms. the questionnaires were completed using a secure web-based application or via paper for those patients without access to the internet. two health related quality of life measures (generic and cf specific) were completed by the subjects at the end of each -day period to assess the relationship between the novel questionnaire and health related quality of life. patients also used pedometers during the well and ill states to assess relationship between symptoms and activity level.results: cf subjects have been enrolled to date. at the time of this interim analysis, patients had completed questionnaires. of this total, only individual questionnaire entries have been missed in patients (< % of possible questionnaires). interim review of cross-sectional data suggests clear differences in symptom reporting between the ill and well periods. examples include % ( / ) noted difficulty breathing and % ( / ) noted tightness in the chest in the preceding hours during the initial well period compared to % ( / ) noting difficulty breathing and % ( / ) noting tightness in the chest during the ill period.conclusions: interim evaluation of this novel instrument demonstrates feasibility of deploying this instrument via the internet with an extremely high completion rate. using a cross-sectional analysis, the instrument can discriminate between the ill and well state. further data will be presented regarding within patient variability and discrimination of the instrument.supported by the cystic fibrosis foundation leroy matthews physician scientist award, the national heart, lung, and blood institute (hl - ), national institute of health (rr- - ), cystic fibrosis foundation therapeutics inc. background: improved communication in today's health care delivery system is a critically important aspect of patient care. nurses are in an effective position to improve communication due to their close interaction with patients. as a result, they are able to identify potential problems, communicate them to the healthcare team and improve patient care and satisfaction.hypothesis: improving communication between the patient/family and the health care team will improve patient/family satisfaction.method: as a member hospital partnering with the institute for healthcare improvement's (ihi) initiative, transforming care at the bedside (tcab), the children's hospital of philadelphia (chop) introduced the use of daily patient goal sheets (dpgs) as a vehicle to improve communication between members of the healthcare team, patients and families and the dpgs was refined to meet the needs of an inpatient medical unit to which cf patients are admitted. using rapid plan-do-study-act (pdsa) cycles, a multidisciplinary team engaged in small tests of change in different stages. initially, patient goals and discharge criteria generated in daily rounds were posted in each room on easel paper. then large "whiteboards" were placed in each patient room for documenting patient goals, discharge criteria, names of the care team members and questions from patients and families. as a final step in the process, we shifted the health care team rounds to the patient's bedside and implemented bedside shift to shift nurse report and safety checks. patient satisfaction was continuously monitored over this process using the press ganey survey.results: over an eight month period, the press ganey survey results for the unit showed an average increase of %. we attribute this increase to better information sharing among patient, family and hospital staff regarding the plan of care for the patient's hospitalization. the health care team also had an improved dialog with the patients/families. conclusion: patient/family and healthcare team communication and satisfaction were improved using an organized and stepwise pdsa process to develop daily patient goal sheets on a medical unit. background: the uab/chs pediatric cf center provides care for approximately patients. the pulmonary division has full-time rns and part-time rns who rotate phone triage for all calls (including cf) of patients seen by the faculty physicians. historically, patient calls received during office hours are taken by the receptionist and put into the emr, then returned by the rn on call. the rn contacts one of the staff md's, gives report of the patient's condition,treatment orders are received, and the patient notified. there are several issues with this phone triage system, which include inconsistencies in rn and md response as well as differences in documentation. global aim: to improve the consistency among rns in phone triage of sick cf patients.method: an algorithm for pulmonary exacerbation phone triage was developed by the cf center director and lead rns to improve consistency in symptom assessment and treatment plan. a specific list of systemic and pulmonary signs and symptoms was formed including length of illness. if three objective: patients with cf often require intravenous antibiotics for treatment of pulmonary exacerbations. patients often receive peripherally inserted central catheter (picc) lines or totally implantable venous access devices (tivads) for venous access. few studies have examined complications of tivad implantation and little published data exists concerning picc line complications in cf patients. this study sought to assess the complication rates of both tivad and picc lines as well as to identify possible risk factors for developing complications.methods: this retrospective study included patients from cf centers in northern new england. data was obtained from each patient's local medical record, port cf, and patient interviews. demographic data was recorded for all cf patients between / / - / / . for each tivad or picc line, the following information was recorded: type of line placed, history of prior line placement during the study period, patient age, history of use for blood draws, method of line flushing, and status of the line at the end of the study (if still in use). complications were defined as catheter occlusion, vascular thrombosis/stenosis, infection, or other local inflammatory reactions.results: data was collected for pediatric and adult cf patients during the defined study period. seventy-three tivads and picc lines were placed during the study period in patients. the tivad and picc line complication rates were % ( / ) and . % ( / ), respectively. in pediatric patients, % ( of ) tivads and . % ( / ) picc lines had a complication. of the tivad complications, three were systemic infections and three were catheter occlusions. of the picc line complications, there were two venous thromboses, one line occlusion, two systemic infections, and nine minor incidents of localized phlebitis. in adults, complications were recorded in % ( / ) of tivads and . % ( / ) of picc lines. of the tivad complications, there were five systemic infections, seven catheter occlusions, and four venous thromboses. of the picc line complications, there were three venous thromboses, three line occlusions, and four minor incidents of localized phlebitis. all adult patients who developed a deep venous thrombosis(dvt) associated with tivad implantation were homozygous for the deltaf mutation. the presence of diabetes or burkholderia cepacia complex(bcc) lung infection was associated with dvt with odds ratios of . ( % ci . - . ) and . ( %ci . - . ), respectively. conclusions: complications of picc lines were uncommon and usually minor. the rate of tivad complication observed was more common over the lifetime of the catheter and was similar to previously published reports. we identified potential risk factors for the development of dvt associated with tivads and picc lines, specifically cf related diabetes, bcc infection, and homozygous deltaf genotype. the mechanism by which these factors are associated with catheter complications is unclear and warrants further investigation. key: cord- -oecpqf j authors: nan title: aspho abstracts date: - - journal: pediatr blood cancer doi: . /pbc. sha: doc_id: cord_uid: oecpqf j nan myelodysplastic syndrome (mds) and frequently arise in the context of inherited bone marrow failure (bmf) syndromes, such as shwachman diamond syndrome (sds). monosomy /del( q) is associated with high grade mds and propensity to progress to acute myelogenous leukemia, a major cause of morbidity and mortality for patients with inherited bmf. development of non-transplant strategies to treat bone marrow failure without simultaneously stimulating outgrowth of malignant clones remains a major challenge. objectives: the aim of this study is to investigate the molecular consequences of del( q) in the context of bmf with the goal of developing more effective treatments. design/method: to study the biological and molecular consequences of monosomy/del( q) in bmf, induced pluripotent stem cells were generated from sds patients (sds-ipsc) . a deletion of the mds-associated region of the long arm of chromosome was then introduced using a previously published modified cre-lox approach. results: the sds ipsc phenocopied bone marrow failure with slow proliferation and impaired hematopoietic differentiation. we next explored whether deletion of q conferred a relative fitness advantage within the context of bone marrow failure. proliferation of the sds-del( q) ipscs was reduced below that of both the isogenic sds ipscs and normal controls without an increase in cell death. sds-del( q) demonstrated reduced hematopoietic differentiation compared with isogenic sds cells. these data demonstrate that deletion of q fails to confer a relative growth advantage relative to isogenic sds ipscs and results in further impairment of hematopoiesis. to gain insight into the mechanisms of del q-associated clonal evolution in sds, we performed rna sequencing (rnaseq) of sds+/-del( q) ipsc. expression of tgf pathways and their downstream targets were reduced in sds-del( q) ipscs compared to isogenic sds ipsc. single cell rnaseq analysis of primary sds bone marrow cells confirmed that the tgf pathway is hyperactivated in sds. western blot analysis showed increased phospho-smad levels in sds ipscs compared to sds-del( q) and normal controls, while total levels of smad were unchanged. pharmacological targeting of tfg with small molecule inhibitors resulted in selective improvement of sds hematopoietic colony formation and myeloid differentiation without stimulating outgrowth of the isogenic sds-del( q) cells or normal controls. these results demonstrate that del( q) reverses the tgf pathway hyperactivation of sds. furthermore, inhibition of tgf selectively rescues hematopoiesis in sds but not in isogenic del q cells, suggesting a potential strategy to treat bone marrow failure without stimulating del q clonal outgrowth. background: standard therapy of medulloblastoma consists of treatment with alkylating agents and radiation after surgical resection. although a statistically significant increase in survival is reported with this regimen, / rd recur and become resistant this class of agents ultimately leading to mortality. large numbers of somatic mutations were observed in recurrent medulloblastoma (rm) after alkylating agent and radiation treatment. high mutation rates in tumors can have twofold effect; ) a large number of non-synonymous mutations that have no role as drivers can still cause functional tumor antigens increasing the neoantigen burden and immunogenicity. moreover, ) such tumors can gain mutations in canonical or non-canonical dna repair pathways leading to a gain in the number of mutations as seen in case of glioblastoma, this can lead to even higher accelerated mutational rate. evidences suggest that high mutational load can cause higher neoantigen burden thereby making the tumor more susceptible to immune checkpoint inhibition. we propose that post therapy recurrent medulloblastoma gain mutational signature and immunophenotype of malignancies demonstrating clinical response to immune checkpoint therapy. objectives: ) rm has molecular signatures identical to tumors with high immunogenicity and clinical response to immune check point inhibition. ) rm has the immune inflammatory phenotype; harboring high percentage of tumor infiltrating lymphocytes (tils), macrophages and monocytes. design/method: to test our hypothesis, we downloaded the raw bam files of previously published data from international cancer genome consortium (icgc) . this set of about matched primaries and recurrent medulloblastoma cases forms our discovery cohort. we have called somatic variants using the gatk pipeline by the broad institute. to validate our key findings, we have procured human medulloblastoma specimens and are conducting whole exome sequencing. the primary assays utilized to assess immunogenicity are immunohistochemical (ihc) staining of formalin fixed and embedded recurrent medulloblastoma tissue to identify tils, tumor associated macrophages and other markers. mg/m had dlts of dyspnea (grade )/hypoxia (grade ) but no dlts were observed in any other cohort. adverse events were generally mild to moderate, consistent with the safety profile observed in adults. across the desc cohorts, plasma concentrations were dose-proportional and steady state concentrations were lower on day vs. day . mean systemic exposure in the mg/m cohort was ∼ -fold greater compared with the adult rp d of mg bid. a pk:pd relationship between tazemetostat exposure and h k me levels in peripheral blood monocytes and granulocytes was observed in the desc phase. consistent and significant post-dose reductions in h k me occurred at doses ≥ mg/m . further analysis of twelve patients treated at the rp d confirmed that h k me inhibition was maximally inhibited. doses - mg/m showed confirmed objective responses (cr/pr) per recist/rano in patients with es (n = ), chordoma (n = ), and atrt (n = ). background: previous studies established that the platelet/ fibrin(ogen) axis promotes metastatic potential by impeding the clearance of newly formed micrometastases by natural killer (nk) cells. however, multiple important questions remain, including the potential of fibrin(ogen) to promote metastasis through interactions with cells other than platelets (e.g., inflammatory cells), and the fundamental question of whether fibrin polymerization is required for metastasis. objectives: determine the role of fibrin polymerization and fibrin(ogen) engagement of integrins iib and m in metastasis. design/method: we performed experimental and spontaneous metastasis assays in immunocompetent mice carrying specific fibrinogen structure/function alterations. results: expression of a mutant fibrinogen lacking the binding motif for the leukocyte integrin m (fib - a) significantly decreased metastatic potential relative to wildtype fibrinogen, suggesting a role for fibrin(ogen)inflammatory cell interactions mediated by m in metastasis. to directly determine the importance of thrombinmediated fibrin polymerization in metastasis, we analyzed metastatic potential in fibaek mice, which carry a form of fibrinogen essentially "locked" in the soluble state due to a mutation in the a chain thrombin cleavage site. metastatic potential in fibaek mice was diminished relative to control mice, speaking to the importance of thrombin-mediated fibrin polymerization in the metastatic process. however, the fibaek mice retained significant metastatic potential relative to complete fibrinogen deficiency, indicating that fibrinogen monomer retains significant prometastatic properties. in order to better define the role of fibrin(ogen)-platelet interactions in metastasis, we compared metastatic potential in control and fib Δ mice, carrying a form of fibrinogen lacking the chain binding motif for the platelet integrin iib . surprisingly, this mutation had no impact on metastatic potential. together, these studies suggest fibrinogen plays a multifaceted role in metastasis. fibrin(ogen)-leukocyte interactions mediated by m appear to have a role in metastasis. previous studies showed that macrophages promote the metastatic potential of circulating tumor cells, which may represent at least one important m expressing cell type whose prometastatic behavior is influenced by fibrin(ogen) interactions. these studies show that thrombin-mediated fibrin polymerization promotes metastasis, but soluble fibrinogen retains some significant prometastatic capacity. surprisingly, loss of the fibrinogen chain iib binding motif had no impact on metastasis. given the established importance of platelets in metastasis, these findings suggest that fibrin (ogen) is capable of platelet stabilization through mechanism(s) independent of this iib binding motif. platelets may bind polymerized fibrin at other sites, and/or fibrin interactions with other matrix proteins capable of binding iib are sufficient to support platelet functions required for metastasis. the role of platelets in hemostasis and thrombosis is well defined, but it is becoming increasingly evident that platelets also assist in host defense and inflammation. platelets participate in the innate immune system through direct antimicrobial activity and interactions with effector cells (chapman , garlanda , kapur ). in the adaptive immune system, platelets recruit and costimulate t-cells, and promote b-cell differentiation and antibody class switching (kapur , morrell ). the question remains: which mechanisms influence platelet immune function and are they developmentally regulated? preliminary studies in the palis lab have revealed significant dif-ferences in embryonic versus adult platelet gene expression, including regulators of immune and inflammatory responses such as beta -microglobulin (b m) and major histocompatibility complex class i (mhc ). mhc is expressed on all cell surfaces except red blood cells and its molecular chaperone b m is a marker of inflammation highly expressed in platelet alpha granules (zufferey ). preliminary data from the morrell lab reveals a mass release of b m during platelet activation, which drives monocyte differentiation to an inflammatory phenotype through tgfb receptor signaling. we therefore sought to determine whether developmental changes in platelet b m expression mediate differences in platelet-mediated monocyte activation. with trilineage hematopoiesis with a predominance of early myeloid precursors, with full maturation. microarray, elane and sbds sequencing and deletion/duplication analyses were negative. immunologic evaluation was significant for agammaglobulinemia and an absence of memory (cd +cd +) b cells. a gene primary immunodeficiency panel revealed two variants of unknown significance-c. g>a and c. g>t in dnmt b; one previously reported in association with icf . parental testing demonstrated parental heterozygosity. centromeric instability was confirmed in mitogen stimulated lymphocytes showing characteristic, multibranched chromosomes containing at least arms of chromosome and joined near the centromere. decondensation of the qh and qh regions and triradial configuration of chromosome was noted, and a diagnosis of icf syndrome was made. the patient was started on monthly intravenous immunoglobulin (ivig). prophylaxis for pneumocystis jiroveci pneumonia and respiratory syncytial virus was initiated. a / matched sibling hsct is being planned. demonstrated the diagnosis of high grade osteosarcoma. the patient was started on multi-agent chemotherapy with planned a whole femur prosthesis at time of local control. cases of osteosarcoma have been described in the literature in patients with nf (median age; years, range - years) with slightly male predominance ( cases). the femur was the most common site of involvement ( cases). four patients died of metastatic disease despite surgery and multi-agent chemotherapy. conclusion: nf represents a major risk factor for development of malignancy and uncommonly osteosarcoma in adolescents and adults. we report a rare case of an extensive involvement of osteosarcoma of the left femur in a child with known diagnosis nf . this presentation should alert the pediatric oncologists to monitor for bone tumors in patients with nf by physical exam and detailed medical history. hasbro children's hospital, providence, rhode island, united states background: dysautonomia is a paraneoplastic syndrome most commonly described in adult malignancies. despite current therapies aimed at symptoms management, it is often debilitating. we present a case of a -year-old girl who initially presented with autonomic dysfunction and was subsequently found to have hodgkin lymphoma. objectives: describe hodgkin lymphoma presenting with dysautonomia and discuss symptom management with rituximab design/method: case report a year-old-girl presented with severe symptoms of orthostatic hypotension necessitating prone positioning to prevent syncopal episodes. additionally, she reported anhidrosis, xerostomia, urinary retention, and constipation. she had unmanageable peripheral neuropathic pain despite multiple analgesia medications. initially, it was suspected that her symptoms were caused by an atypical presentation of guillain-barre syndrome. she was treated with intravenous immunoglobulin g, without response. due to a suspicion of a paraneoplastic syndrome a positron emission test/cat scan (pet/ct) was performed and revealed widespread fdg-avid nodal and splenic disease. pathology from a thoracoscopic biopsy of a mediastinal lymph node demonstrated classical hodgkin lymphoma. she was classified as stage ivb. a paraneoplastic panel obtained during the first cycle of chemotherapy revealed elevated anti-amphiphysin antibodies and glutamic acid decarboxylase (gad) antibodies. therapy was initiated with abe-pc (doxorubicin, bleomycin, etoposide, prednisone, cyclophosphamide) ; vincristine was held given her significant neuropathy. due to persistence of autonomic symptoms following her first cycle and presence of antiamphiphysin and gad antibodies, rituximab was incorporated into her treatment. following two cycles abe-pc, she had a rapid early response by fdg-pet/ct. she completed an additional three cycles of abd-pc. end of therapy imaging demonstrated complete response with a single persistent mildly fdg-pet avid lymph node (deauville ) and her antibodies were negative. she continues treatment of maintenance rituximab with significant improvement, but not resolution, of her orthostatic hypotension. at this time, the patient can ambulate with assistance. constipation and urinary retention have fully resolved and, her peripheral neuropathy, xerostomia, anhidrosis have improved. conclusion: this is rare case of a pediatric hodgkin lymphoma patient developing dysautonomia associated with antiamphiphysin and glutamic acid decarboxylase antibodies and subsequently managed with chemotherapy and rituximab. clinicians should be suspicious of a paraneoplastic syndrome when a neurologic disorder fails to improve with standard treatment. results: labs obtained at an outside hospital one month prior to presentation showed absolute neutrophil count (anc) and hemoglobin . g/dl. she presented to our institution with days of fever, hepatomegaly cm below costal margin, a white plaque on her tongue, and circumferential perianal ulceration. labs were significant for anc and hemoglobin . g/dl. anti-granulocyte antibody testing was positive. bone marrow biopsy showed arrest of neutrophil maturation. after initiation of filgrastim ( . mcg/kg/day), her anc increased to > and repeat bone marrow biopsy demonstrated left shifted myelopoiesis. biopsy of her oral lesion demonstrated invasive actinomyces prompting a prolonged course of antibiotics. biopsies of her oral and anal lesions were reported as myeloid sarcoma without mll rearrangement. chemotherapy was not initiated due to complete resolution of both lesions within weeks of initiating filgrastim and appropriate antibiotic coverage. she has not developed any further lesions concerning for malignancy. testing for common genes associated with severe congenital neutropenia and autoimmune lymphoproliferative syndrome was negative. her immunoglobulin levels and the measurement of age-appropriate vaccine responses were normal. after her lymphocyte subpopulation analysis indicated a selective deficiency in cd positive t-lymphocytes (absolute cd cell count ), the severe combined immunodeficiency panel from genedx showed compound heterozygous mutations in results: a male infant was born with a large thigh mass. the child was clinically well aside from restricted movement of affected leg. mri showed mass expanding into pelvis without other lesions. an interventional-radiology guided core biopsy of the mass was reported as high-grade spindle cell sarcoma without etv rearrangement. surgery was deferred because of concern that it would result in excessive morbidity. the mass was treated with vincristine and dactinomycin per infantile fibrosarcoma protocols. after months of therapy, no significant change in size of the mass was noted on physical exam or imaging. repeat biopsy was obtained to confirm diagnosis and allow for expanded tumor testing. this biopsy showed triphasic distribution of adipose, fibrous and mesenchymal tissue consistent with fhi with rare sarcomatous foci. additional chemotherapy was deferred and the child was followed clinically. his tumor has remained approximately the same size and still unresectable. next generation sequencing of tumor utilizing panel based technology revealed braf-erc fusion consistent with braf activating mutation. this mutation was confirmed by fluorescent in situ hybridization (fish) probe for braf. braf and mek inhibitors have been pursued as treatments to decrease size of tumor and allow for resection. conclusion: braf mutations have been characterized in a variety of malignancies. inhibition of braf and downstream signaling components has produced promising results in a variety of patients. this is the first case report of a braf mutation in a fhi. although management of fhi is typically surgical, this does suggest a potential therapeutic target and may allow for improved surgical outcomes especially in cases where up-front surgery would result in unacceptable morbidity. genetic sequencing of fhi and other rare tumors is an important tool and has the potential to identify mutations amenable to targeted therapies. background: icf is a rare autosomal recessive disorder characterized by hypo-or agammaglobulinemia and often opportunistic infections suggesting t-cell dysfunction. it is further categorized into subtypes - based on mutations in dna methylation. mutations in the helicase-lymphoid specific (hells) gene, which is required for t-cell proliferation and participates in de novo dna methylation, are characteristic of icf type (icf ). of approximately reported cases of icf, less than percent are characterized as icf . while malignancy has been reported in icf (angiosarcoma, acute lymphoblastic leukemia), and icf (hodgkin lymphoma), here we describe the diagnosis and management of an icf patient with neuroblastoma and neutropenia, which has not been previously described. objectives: describe a novel phenotype and mutation of icf and its management to further expand our understanding of this disease. results: a month ex- week premature male with bronchopulmonary disease and failure to thrive presented with acute respiratory failure in the setting of recent viral bronchiolitis with associated chronic diarrhea. he was subsequently diagnosed with multiple infections including pjp pneumonia, norovirus, parainfluenza, rhinovirus, and pseudemonal cellulitis. he presented with profound neutropenia and agammaglobulinemia with presence of b and t cells on lymphocyte phenotyping. ct revealed a paraspinal mass that was mibg-avid on further study, strongly suggesting neuroblastoma. bone marrow was normocellular and negative for malignancy, however revealed marked granulocytic hypoplasia and maturation arrest concerning for severe congenital or, less likely, immune-mediated neutropenia. metastatic workup was negative. whole exome sequencing revealed a homozygous variant of unknown significance (c. t>c) in the hells gene, portending a working diagnosis of icf syndrome. immunoglobulin supplementation, pentamidine prophylaxis, and g-csf were initiated. he was able discontinue g-csf after months of treatment. his neuroblastoma, initially categorized as l , met criteria for observation. however, followup mri revealed interval growth nearing the spinal canal. he underwent tumor resection, confirming mycn non-amplified, favorable histology neuroblastoma. after infectious prophylaxis and immunologic support were initiated, he incurred two other hospitalizations, the first for g-tube cellulitis and the second for parainfluenza respiratory illness. he now has stable neutrophil counts off g-csf and remains in remission from neuroblastoma. current plan is to proceed with bone marrow transplantation for immunodeficiency. conclusion: icf has not previously been described with neutropenia or neuroblastoma. this report not only describes a novel mutation and phenotype of icf and the management thereof, but also reveals the potential curative role of bone marrow transplantation in such disease. staten island university hospital -northwell health, staten island, new york, united states background: desmoid tumors are rare tumors that arise from highly differentiated fibroblasts. they occur in isolation or as part of the disease spectrum of familial adenomatous polyposis (fap) . fap mutations between codons - typically correlate with increased extraintestinal disease such as desmoid tumors and upper gastrointestinal polyps. we describe a patient with a large intra-abdominal desmoid tumor who is heterozygous for a c. c>t (p.arg cys) apc gene mutation. we are not aware of any other patients reported with this germline apc mutation presenting with a desmoid tumor. objectives: to discuss a novel apc mutation and the presentation of a rare case. design/method: review of clinical presentation, genetic analysis and management of a rare tumor. a -year-old female with no significant medical history presented with abdominal asymmetry and intermittent pain. she reported urinary urgency, shortness of breath, early satiety, decreased appetite and a -pound weight loss over the course of months. ct scan of the abdomen demonstrated a × cm abdominal tumor abutting the local organs but no presence of bowel obstruction. a biopsy revealed a spindle cell neoplasm favoring fibromatosis. there was no known family history of fap, colon cancer, or desmoid tumors. apc gene mutation analysis demonstrated a c. c>t (p.arg cys) heterozygous gene variant. due to size and location of the tumor, it was initially deemed unresectable. the patient was started on a course of monthly liposomal doxorubicin. she tolerated the initial cycles well and interval ct after cycles of chemotherapy revealed a % decrease in tumor volume. variability exists in phenotypic presentation with regards to the location of the afp mutation locus. while fap mutations associated with desmoid tumors typically have changes in the - codon region, our patient presented with a heterozygous mutation resulting in a missense mutation at codon . due to the change in polarity and size, the mutation is not considered to be of conservative nature. we are only aware of one other report of this mutation, which occurred in an individual with a personal and family history of colon cancer. we are not aware of any patients with desmoid tumors who also have this germline apc gene mutation. our case report highlights an apc gene mutation that is not well-described; we are not aware of any other cases of this mutation reported in patients with desmoid tumors. future evaluation and tracking of this mutation may lead to the determination of further clinical significance. background: over time, advanced care planning for location of death has been associated with increased deaths at home rather than in the hospital. in some cases, however, complex management and symptom control can prevent families from achieving their goal of keeping their child out of the hospital and at home at the end of life. ascites is a sequelae of many conditions including malignancy that might lead to significant morbidity. increasingly, interventional procedures are being utilized. peritoneovenous "denver" shunts are placed internally with one end in the peritoneal space and the other buried within a major vessel such as the svc. a one-way valve and pump buried under the skin allows the patient to pump fluid from the peritoneal to the vascular space. the shunt is used frequently in adults, but has not seen much use in pediatric oncology patients. objectives: to describe a case of a terminally ill patient with refractory wilms tumor with ivc involvement who received symptomatic relief with denver shunt placement. results: an -year-old female was diagnosed with relapsed, refractory, metastatic wilms tumor with pulmonary and hepatic involvement, with tumor extension to the hepatic veins and ivc. multiple chemotherapeutic regimens and palliative radiation to the ivc were administered, but her disease continued to progress, leading to pressure on the portal vein and portal hypertension. the resulting ascites was causing the patient significant pain and was difficult to manage. the patient's code status was changed to dnr/dni after discussion with her mother, who identified a desire to have the child die at home as comfortably as possible. a peritoneovenous shunt was placed in order to control the patient's pain and avoid frequent medical procedures and therapies. despite initial anxiety, the patient was able to utilize the pump and achieve significant improvement in her ascites and pain. she was able to spend the remaining six weeks of her life at home. ascites is a common phenomenon of end stage disease. peritoneovenous shunts are a treatment modality that may be considered to allow for pain control at the end of life for pediatric oncology patients with ascites. the procedure is relatively low risk, allows for self-control of the pump to maintain comfort, and is easy enough to use by the patient or family. background: extraneural metastases (enm) from pediatric glioblastoma multiforme (gbm) are rare, with an estimated frequency of . %. etiologic factors include multiple neurosurgical procedures and sarcomatous dedifferentiation. their occurrence can seriously affect the patient's quality of life and survival. while enms have been well documented in adults, pediatric cases have not been previously summarized. a year old male with a cerebral gbm developed extension of disease outside of the neuraxis approximately months post initial presentation and at the time of disease progression. metastases included exracranial temporal lesions, cervical and mediastinal lymph nodes and s of s bilateral lung nodules. a large pleural-based soft tissue metastatic focus was identified on imaging when the patient presented with respiratory distress secondary to a right tension pneumothorax, which was recognized and managed promptly. we summarize the main reported cases in literature to better define risk factors for and evaluate the proposed mechanisms underlying these systemic metastases. design/method: we performed a literature review on the pubmed database using the terms gbm and enm. patients under years of age who met the weiss criteria for the diagnosis of enm from primary cns tumors were included. results: our patient fulfilled two of the three weiss criteria with confirmed gbm at the primary site with all enm in the temporal soft tissue and cervical lymph nodes displaying histopathologic features similar to the primary cns tumor. the intrathoracic adenopathy and lung nodules detected upon chest imaging during workup for respiratory distress were assumed to represent additional metastatic foci. our literature review identified pediatric patients with enm from gbm with a median age of years (range . - years) and a slight female predominance ( % females vs. % males). the most common sites of metastases reported were pleura/lungs, bones, lymph nodes and liver. in of patients, metastases were associated with csf shunting. conclusion: pediatric oncologists should have an increased index of suspicion when caring for patients with gbm, particularly those who have undergone shunting procedures and present with systemic symptoms including bony pain, respiratory changes, transaminitis or cytopenias which should prompt timely investigation for enm. although enm of cns tumors carry very poor prognosis, their diagnosis has potential therapeutic importance because treatment of metastatic lesions may alleviate symptoms and improve the quality of life. additional studies may be warranted to evaluate the incidence of enm that can provide valuable insight into the pathogenesis and biology of high-grade gliomas. nicklaus children's hospital, miami, florida, united states background: sinusoidal obstruction syndrome (sos) has been reported in patients undergoing intensive chemotherapy and as a complication post-hematopoietic stem cell transplan-tation. sos may be complicated by portal hypertension, hepatorenal disease or multi-organ failure. however, despite treatment, there may be further potential complications that can be anticipated in patients with history sos. we report two patients with history of sos presented with post-procedural bleeding after gastric tube placement. we believe that their presentations may be associated to their previous diagnosis of sos. design/method: pubmed search was done with search for terminology including "sinusoidal obstruction syndrome" "defibrotide", and "bleeding". papers relevant to our cases were selected for literature review. results: case : a year-old female with history of desmosplastic medulloblastoma status-post resection and intensive chemotherapy was diagnosed with sos one month after her second part of planned tandem transplant. she was managed with paracentesis and defibrotide. due to malnourishment, patient had a gastric tube placement months after she completed therapy and had an episode of upper gastrointestinal bleeding postoperatively from the g tube site. case : similarly, a year-old male diagnosed with anaplastic medulloblastoma status post resection and adjuvant multiagent chemotherapy. his treatment course was complicated with sos after the second cycle of induction chemotherapy which responded to -day course of defibrotide. likewise, the patient had a major bleeding event from the g-tube site approximately two months after sos diagnosis. defibrotide was discontinued in both cases before g-tube placement. both patients had no previous history of bleeding disorders or relevant family history. in addition, comprehensive laboratory evaluations were within normal limits before both procedures. in sos, there is blockage of fluid out of the liver that leads to congestion, ascites, ischemia of the liver, and post-sinusoidal portal hypertension. two related causes of sos should be considered as an explanation for g-tube bleeding. similar patients should have close monitoring postoperatively or if possible surgical intervention should be delayed until the sos process has been evolved. nicklaus children's hospital, miami, florida, united states background: the development of treatment related acute myeloid leukemia (t-aml) and myelodysplastic syndromes (t-mds) is a potential complication after cytotoxic chemotherapy or radiation therapy. the incidence of development of t-aml/t-mds varies from - % depending on the treatment regimen used. cutaneous myeloid sarcoma (ms) is a common presentation of extramedullary leukemia and usually occurs in the setting of aml. we report a rare case of cutaneous ms in an adolescent female after successful treatment for ovarian yolk sac tumor (yst) stage i with bep (bleomycin, etoposide and cisplatin) therapy. the ms was managed only with biopsy and close observation. design/method: a pubmed search was conducted for queries including t-aml/t-mds, cytotoxic agents, cutaneous myeloid sarcoma, regression. relevant papers were selected for literature review. a year-old female was diagnosed with a left ovarian yolk sac tumor, for which she underwent left salpingooophorectomy and successfully completed cycles of bep over months. during routine follow-up months after initiation of treatment for ovarian yst, she was noted to have a small, non-tender, indurated nodule on the left side of her upper back approximately cm in diameter. punch biopsy of the skin nodule was performed and pathology was positive for cutaneous myeloid sarcoma. at the time of next follow-up less than one month later, the skin lesion had resolved. two subsequent bone marrow aspirates were performed one month apart and were negative for leukemic involvement or mds. examinations and work-up including whole body pet with ct scan were negative for evidence of disease. although cutaneous ms can be regarded as the herald of systemic myeloid disease rather than a localized process, our patient was monitored periodically with physical exam and laboratory evaluations. she remains free of disease more than four years after the presentation of cutaneous ms without any further treatment. spontaneous regression ms has been previously reported. the authors would like to stress that a conservative approach with close observation could be an option in cutaneous ms even with history of chemotherapy exposure. nesreen ali, iman sidhom, sonia soliman, sherine salem national cancer institute, cairouniversity, egypt children cancer hospital egypt, egypt background: acute leukemia is the commonest malignancy in childhood. the coincidental occurrence of leukemia with hemophilia is extremely rare. hemophilia is a congenital rare x linked bleeding disorder. the main complication of the two diseases is bleeding diathesis which may be lifethreatening due to many factors, deficiency of coagulation factors in hemophilic patients, thrombocytopenia from disease and chemotherapy in leukemic patients, certain cytotoxic drugs such as asparaginase which may result in coagulation disorders and infection which may lead to disseminated intravascular coagulation. objectives: reporting such a case is imperative to set up treatment guidelines for prevention of bleeding and to optimize the therapeutic approach for these patients. design/method: seventeen years old boy, presented to children cancer hospital egypt in june with pallor and multiple ecchymoses.he was diagnosed with precursor b acute lymphoblastic leukemia, cerebrospinal fluid (csf) was free, the chromosomal analysis revealed hypodiploidy , xy. he had moderate type of hemophilia a since birth, factor viii level was . % at time of diagnosis, coagulation profile revealed prolonged partial thromboplastin time (normal - ), factor viii was low %, prothrombin concentration and prothrombin time were normal % and seconds, virology screening for hepatitis b core igg/igm, hbs ag, hiv and hc igg /igm were negative.the patient started induction total xv sjcrh protocol, factor viii unit/kg was given at presentation before doing bone marrow aspiration(bma), csf and as a prophylactic before intramuscular asparaginase injection, intrathecal and bma. it was given immediately within hours before the procedures and platelets transfusion was given regularly to maintain platelets count about , . the minimal residual disease by flow cytometry was . % and . % at d and d induction. results: our patient received his induction and reintensification chemotherapy without any major bleeding event which reveals the success of our guidelines for the prevention of bleeding. he developed very early relapse at w maintenance by the same clone. he received salvage chemotherapy but didn't achieve remission and died out of disease and resistant clone. the development of leukemia on top of hemophilia is a major problem. bleeding complication during chemotherapy can be prevented by regular prophylactic factor viii and platelets concentrate transfusion with good supportive care. life threating bleeding complication may be correlated with the severity of hemophilia. we need to collect data about the biology of leukemic cells, complications, and cause of death to optimize care for these patients. background: mucoepidermoid carcinoma (mec) is a rare malignancy that arises from exocrine glands in the upper aerodigestive tract and tracheobronchial tree. conventionally, mec diagnosis is based on histology, with prognosis based on the extent of resection and detection of metastases. mec is characterized by a translocation of chromosomes q and p resulting in a fusion between the mect and maml genes, that occurs in - % of cases. this fusion transcript has been recognized to have a favorable impact on disease features and prognosis of mec. however, recent studies indicate that high grade mec can have mect -maml fusion positivity and multiple other genomic imbalances that have not been studied in much detail. owing to the rarity of mec tumors, more definitive data related to the clinical and prognostic significance of these molecular markers are limited. objectives: . identify the presence or absence of mect -maml fusion in the tissue of our patient. . analyze the incidence of the fusion in mec cases in children and young adults retrieved from the iowa cancer registry. . determine if fusion status correlates with clinical, pathological and outcome data in our cohort. design/method: we describe the case of a year-old caucasian male who presented with recurrent pneumonia, persistent cough and radiographic evidence of right lobar collapse. bronchoscopy revealed an endobronchial lesion and the patient underwent right upper lobe sleeve resection. pathology report was consistent with low grade muco-epidermoid carcinoma. we retrieved archived formalin-fixed paraffinembedded (ffpe) specimens of pediatric and young adult mec cases (ages - ) reported in iowa from - using the iowa cancer registry. testing for the mect -maml fusion in the index case and ffpe specimens will be done using a custom-designed laboratory validated next generation sequencing (ngs) assay with the ability to detect novel fusion partners. clinical, pathological and outcome data (age, sex, tumor site, tumor size, nodal metastases, clinical stage, histologic grade, treatment and follow up) will be analyzed to correlate with fusion status. the mect -maml fusion tested positive in our index patient. we will obtain irb approval to test for the fusion in the archived ffpe specimens and correlate clinical, pathological and outcome data. conclusion: mect -maml fusion is a frequent event in mec that has prognostic and potential therapeutic applications in adults. the results of this study may enlighten the clinical management of mec in children and young adults. children 's mercy hospital, kansas city, missouri, united states background: mutations in the samd gene are associated with a rare syndrome comprising of myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (mirage syndrome). diagnosis is made through exome sequencing. in the largest reported case series, of eleven patients diagnosed with mirage syndrome, two developed loss of chromosome . given the potent growth restricting activity of samd mutants, the loss of chromosome is considered the first documentation of adaptation by aneuploidy mechanisms in humans and led to myelodysplastic syndrome (mds), with deaths occurring from related complications at and years of age. objectives: to report a case of mirage syndrome with congenital thrombocytopenia progressing to bone marrow failure, managed uniquely with bone marrow transplantation. results: male born at weeks gestation with prenatal diagnosis of iugr, two vessel cord, oligohydramnios was found to have ambiguous genitalia, adrenal insufficiency, partial panhypopituitarism and congenital thrombocytopenia with bone marrow showing absence of megakaryocytic precursors. severe thrombocytopenia was present from birth. bone marrow evaluation demonstrated a hypocellular marrow with markedly reduced megakaryocytic and myeloid precursors and no evidence of myelodysplasia. he required gastric tube placement for failure to thrive, had a laryngeal cleft repaired and developed focal segmental glomerulosclerosis. mpl gene testing for congenital amegakaryocytic thrombocytopenia was negative. testing for fanconi anemia, shwachman-diamond syndrome and dyskeratosis congenita was also negative. approximately % of cells had loss of heterozygosity on chromosome q. exome sequencing showed that he is heterozygous for a de novo gain of function variant, c. g>a (p.arg gln), identified in the samd gene, confirmed by sanger sequencing and consistent with a diagnosis of mirage syndrome. at years of age, he developed pancytopenia requiring frequent transfusions with platelets and packed red blood cells. he underwent a successful bone marrow transplant at years of age without significant complications, and remains transfusion independent without cytopenias greater than months from bone marrow transplantation. conclusion: it is imperative to pursue work up for persistent congenital thrombocytopenia in a stepwise multidisciplinary manner. to the best of our knowledge, this is the first case of mirage syndrome associated bone marrow failure treated with bone marrow transplant. due to the individual rarity of mirage syndrome and pediatric myelodysplastic syndrome, it is important to maintain an index of suspicion given their association and explore bone marrow transplant as a therapeutic option. results: the patient demonstrated disease regression, initially, and continued without disease progression for months. the regimen has been well tolerated with only minimal side effects of dry skin (ctcae grade ) and a transient episode of brief erythrodysesthesia (ctacae grade ) that resolved spontaneously. the combination of sorafenib and capecitabine was effective and well tolerated in this adolescent patient with fl-hcc. our observations, although in a single patient, lend support for further testing of this novel oral chemotherapy regimen in patients with fl-hcc, a disease for which there is no effective standard chemotherapy approach. background: epstein-barr virus (ebv) is a ubiquitous virus associated with a broad range of malignancies due to its oncogenic potential. history of organ or bone marrow transplantation, immunosuppressive therapy, and primary or acquired immunodeficiency syndromes increases the risk of ebvassociated tumors. epstein-barr virus associated smooth muscle tumors (ebv-smt) are unique and rare neoplasms typically discovered in immunocompromised patients. most information related to pathogenesis and therapeutic options is limited to case reports and case series of adult patients. there are several gene expression pathways that ebv utilizes, the most notable of which is the mammalian target of rapamycin (mtor) pathway. the mtor pathway performs a key role through integrating various cell growth signals and factors to regulate protein synthesis and metabolism related to smooth muscle proliferation. sirolimus is an immune modulating therapy that targets the mtor pathway to block activation of lymphocytes. objectives: several case reports have demonstrated shortterm clinical remission of ebv-smt in adult patients with the use of sirolimus. we report the first case of long-term background: bilateral neuroblastoma is characterized as neuroblastoma arising in both adrenal glands, a rare presentation with little data on its genetic make-up. a two-monthold patient was diagnosed with bilateral neuroblastoma in our clinic. her risk assignment was based on biopsy of the left adrenal lesion, which showed mycn amplification, an unfavorable genetic marker. treatment regimen was intensified accordingly and after courses of chemotherapy tumors were excised. patient went on to receive a stem cell transplant and immunotherapy. with no knowledge of genetic similarity between the two tumors it is unclear whether biopsy of the right lesion would have yielded similar results or whether bilateral biopsies are needed for risk assessment of bilateral neuroblastoma. objectives: utilize whole exome sequencing (wes) to characterize the genomic signature of bilateral adrenal neuroblastomas excised following chemotherapy treatment. design/method: paraffin-embedded samples from left (l) and right (r) tumors underwent wes at the broad institute. we analyzed resulting data including somatic variant calls, indel mutations, and copy number variants (cnvs) using ingenuity software to evaluate and compare differences between the two tumor samples. preliminary analysis of the data shows important descriptive information on the two tumor samples. out of somatic mutations in the r tumor cells and mutations in the l tumor cells, only two common somatic mutations were present. out of cnv calls in the r tumor and in the l tumor, cnvs were common between the two tumors, or % of each tumor's cnv calls. there was a fold higher frequency in gains versus losses. the median size of the common cnvs was , (range to , , bp). cancerrelated genes with increased copy numbers included transcription factors, receptors for signal transduction pathways, and histone methylation proteins. conclusion: preliminary analysis of the wes results of the two adrenal tumors show some genomic divergence. because the tumor tissue was exposed to chemotherapy prior to excision it is difficult to determine whether genomic divergence is a result of independently originated tumors or subsequent adaptation to chemotherapy of a clonal cell population. the high number of common cnvs in the two tumors points to a common cell of origin, however the low number of common somatic mutations does not fit that picture. a future study to help elucidate the question will be wes of the original biopsy tissue to provide information on tumor mutations prior to the effects of chemotherapy. baylor college of medicine, houston, texas, united states background: although there has been significant improvement in the overall survival rates of children with cancer many children will still die from their illness or complications secondary to treatment. research surrounding the deaths of children who succumb to their disease is warranted to ensure we are providing the best care possible for these patients. objectives: this case series aims to explore pediatric cancer deaths by focusing on perhaps the most extreme cases of high intensity end of life care. we explore those patients whom we know are dying or our very likely to die as evidence by their do not resuscitate (dnr) orders. in all of these cases despite the patients very grim prognosis, their great likelihood of death and limitations placed of resuscitation methods all patients continued end of life care in the pediatric intensive care unit (picu). the primary medical records of all children with a cancer diagnosis who died between february , and january , in the picu with a dnr order seven days or earlier prior to death. each medical history included disease-directed treatment history and response with particular attention to the events surrounding the terminal admission. results: eight patients met criteria for this study representing . % of all cancer patients who died during this time period and . % of those who died in the icu. the average time between dnr and death is . days ( days - days). the average length of terminal admission was . days ( day - days). the average time between diagnosis and dnr is . months ( months - months). the average time between diagnosis and death is . months ( months - months). conclusion: these cases highlight the journey that patients, families and providers endure leading up to death. medical care is complex, there are very few absolutes that are encountered when caring for patients and decisions around limiting or withdrawing medical care are made in a context of the prior journey. . these cases help to understand the complexity of death and how two seemingly opposite ideals can be congruent in the event of an anticipated death. most of these cases show the need for improved anticipatory guidance surrounding death and greater consideration for de-escalation of care when death is expected. the hospital for sick children, toronto, ontario, canada background: rhabdomyosarcoma (rms) is the most common soft tissue sarcoma in children, with embryonal (erms) and alveolar (arms) representing the most common subtypes. arms tumors are associated with inferior outcome when compared to erms, and they are characterized in about % of the cases by a t ( ; ) or t( ; ) chromosomal translocation with creation of a pax -foxo or pax -foxo fusion gene, respectively. it is increasingly clear that the pax-foxo fusion status is an important poor prognostic factor, thus the histological classification tends to be replaced by the fusion status, particularly in terms of risk stratifica-tion in contrast to arms, there are no recurrent chromosome alterations in erms; however, there are multiple numerical chromosome changes that are frequent in these tumours: gain of chromosome , , and have been found in to % of emrs karyotypes. moreover, erms tumors show frequently allelic loss, the .p . chromosomal region being the most frequently involved. recently, novel gene fusions have been described also in erms tumours. these fusions involved mainly the ncoa and or the vggl genes. the rearrangement partners are variable, and include, i.e. pax ( q ), srf ( q ) and tead ( p ). objectives: to present a patient who died as a consequence of brain metastases while on therapy in the setting of an foxo negative rms and the identification of a new translocation t( ; )(q ;q ). design/method: case report and retrospective review of the literature. we report a case of pelvic embryonal rhabdomyosarcoma in a -month old boy. he was treated as per cog arst intermediate risk group, but unfortunately was found to have a large cerebellar tumour during the course of his chemotherapy treatment and he subsequently passed away. a novel translocation between chromosomes and was observed in of metaphase cells by g-band analysis in the autopsy sample of the brain lesion. breakpoints of the translocation were estimated to be at q and q . there were no additional clonal chromosome abnormalities in the tumour cells. conclusion: erms tumors with fusion genes involved have been exclusively described in patients less than months of age; they seem to be associated with spindle cell histology and, a favorable outcome. in our patient, a novel ( ; ) translocation was found and clinically, the patient had a dismal outcome. further studies are indicated to inquire whether this finding is of significance in term of prognosis for these patients. children 's national medical center, washington, district of columbia, united states background: iatrogenic immunodeficiency-associated lymphoproliferative disorders (lpds) are a group of lymphoid s of s proliferations or lymphomas that are well known to be associated with an immunosuppressed state. these disorders most commonly occur following hematopoietic or solid organ transplantation (called post-transplant lymphoproliferative disorders or ptld), but cases have also been described during the treatment of autoimmune and rheumatologic disorders by immunosuppressive and immunomodulatory medications. these disorders are strongly associated with infection by the epstein-barr virus (ebv) as a result of impaired immune function in the immunosuppressed state. while this phenomenon has been well documented in autoimmune conditions, cases affecting pediatric patients while on antileukemia chemotherapy are lacking. background: atypical teratoid/rhabdoid tumor (at/rt) of the central nervous system (cns) in children younger than years old has a prevalence of % to % and accounts for . % of all pediatric cns tumors. only - % of patients have leptomeningeal dissemination. rhabdomyosarcoma is the most common soft tissue tumor in childhood, but represent only - % of all pediatric cancers. rarely, it can metastasize or even directly extend into the cns, but typically, cases of cns involvement arise either from parameningeal areas or other primary sites. primary spinal or meningeal rhabdomyosarcoma is extremely rare. objectives: our objective is to describe two unique cns malignancies presenting as rare, primary leptomeningeal disease. design/method: case a -month-old female presented with vomiting, fatigue and listlessness, despite a normal head ct and brain mri. csf showed hypoglycorrhachia and mild pleocytosis. ceftriaxone was started, but she developed nuchal rigidity and cranial nerve vii palsy. repeat brain mri showed evolving leptomeningeal enhancement concerning for meningitis. she gradually developed worsening opisthotonus and ultimately a brain biopsy of the temporal lobe was consistent with at/rt. case a -year-old male presented with new generalized tonic-clonic seizure activity and intermittent headaches with photophobia, phonophobia, and vomiting. brain mri was significant for enhancement of interpenducular and suprasellar cisterns extending to the optic nerves and chiasm most consistent with meningitis. neurosurgery ultimately placed a lumbar drain for hydrocephalus, and a tissue biopsy demonstrated primary meningeal rhabdomyosacroma. results: in case , our patient's temporal lobe biopsy demonstrated grade iv malignant tumor cells consistent with atypical teratoid/rhabdoid tumor. fish demonstrated a homozygous deletion of smarcb ( q . ). she was started on chemotherapy per the dana farber at/rt protocol but ultimately was discharged home on hospice. in case , our patient's lumbar arachnoid biopsy demonstrated cellular tumor consistent with group iiia embryonal rhabdomyosarcoma. immunostaining was positive for cd , desmin, myogenin, and myo-d with neural markers ema and gfap highlighting the meninges but without a neural component to the tumor. he completed craniospinal radiation to gy total with lumbar boost to . gy total. he is currently receiving chemotherapy per arst protocol. conclusion: these two cases are particularly instructive because of their similar initial presentations and neuroimaging, but with very different and unique diagnoses. university of iowa, iowa city, iowa, united states background: ebf -pdgfrb fusion causes ph-like b-cell acute lymphoblastic leukemia (b-all), which has a philadelphia positive phenotype without the bcr-abl translocation. this is one of several mutations associated with ph-like b-all and leads to downstream overexpression of tyrosine kinase. ebf -pdgfrb fusion accounts for about % of children with ph-like b-all. patients with ph-like b-all previously had poorer outcomes with conventional chemotherapy. the addition of tyrosine kinase inhibitors (tki), like imatinib, has improved the outcome for many patients predicted to have tki sensitive mutations. objectives: to review clinical characteristics and outcomes of two cases of ph-like b-all at the university of iowa stead family children's hospital and to compare these outcomes to similar cases reported in the literature. design/method: a retrospective chart review was performed for two cases of ph-like b-all diagnosed and treated at the university of iowa stead family children's hospital. results: both patients were males diagnosed at years of age with high wbc count ( , and , ) and positive for ebf -pdgfrb gene fusion. patient (pt ) was cns b at presentation while patient (pt ) was cns negative; neither had testicular involvement. both started treatment according to cog protocol aall . peripheral blasts cleared by induction day for pt and induction day for pt . at end of induction, pt had m bone marrow and pt had m bone marrow but mrd %. dasatinib was started induction day for pt and induction day for pt . pt was still not in remission at end of consolidation; bone marrow cell culture for tki resistance showed best response to dasatinib. pt proceeded to anti-cd car t-cell therapy followed by tbi-based matched unrelated donor bone marrow transplant. pt had negative mrd at the end of consolidation and continues chemotherapy according to aall , dasatinib arm. both patients are currently clinically well. our patients had the same tyrosine kinase gene fusion and similar initial clinical courses. while both patients had persistent disease at end of induction, pt had almost % blasts while pt had significant reduction of disease burden before starting tki. pt showed good response with the addition of dasatinib while pt did not. these findings suggest that response to conventional chemotherapy may potentiate the effect of tki and may predict overall outcome. there are likely additional factors which must be taken into account when determining response to tki for patients with ph-like b-all which have not yet been identified. background: medulloblastoma is the most common malignant brain tumor of childhood. classically, medulloblastoma presents as a well-defined mass lesion in the cerebellum, with a high rate of metastatic dissemination. primary leptomeningeal medulloblastoma (plmb) is an exceedingly rare type of medulloblastoma presentation with a dismal prognosis in which patients present with isolated leptomeningeal disease without an associated mass. to our knowledge, only three pediatric and three adult cases of plmb (ages - years) have been reported, all of which died within months of diagnosis. this is the first case of plmb to report a molecular classification. objectives: to report the case of a pediatric patient with plmb in which histopathologic and molecular characterization was performed and to describe the patient's treatment and clinical course. design/method: retrospective review of the patient's electronic medical record and review of the literature. a -year-old boy presented with headache, vomiting, diplopia, and fatigue. physical examination revealed upward gaze palsy, left-sided extremity and facial weakness, and ataxia. magnetic resonance imaging (mri) of the brain revealed diffuse cerebellar leptomeningeal enhancement and edema without an identifiable mass and moderate hydrocephalus. mri of the spine and cerebral spinal fluid analysis were normal. a diagnosis of cerebellitis was rendered, and the patient underwent placement of a ventriculoperitoneal shunt. an extensive infectious, neurologic, rheumatologic, and oncologic workup did not identify an etiology. empiric antibiotics, high-dose steroids, and intravenous immunoglobulin therapy yielded minimal improvement. two months later, repeat mri of the brain performed for declining mental status demonstrated progressive thickening of cerebellar leptomeningeal disease. a suboccipital craniectomy with decompression and cerebellar biopsy were performed. pathologic examination revealed a diagnosis of plmb, classic histology, non-wnt/non-shh, without gain/amplification of myc/mycn, and p wild type pattern. craniospinal radiation to cgy with a cgy boost to the posterior fossa was delivered with concurrent carboplatin/vincristine over six weeks. two months following chemoradiation, mri of s of s the brain demonstrates significantly reduced pathological leptomeningeal enhancement of the cerebellum, and the patient is awaiting initiation of systemic chemotherapy while recovering from a surgical wound infection. conclusion: plmb is extremely rare but should be considered in patients with cerebellitis and diffuse leptomeningeal involvement who are refractory to medical management or in whom an etiology has not been identified. cerebellar biopsy is recommended early to enable timely treatment and improved outcomes. molecular classification should be performed in cases of plmb to further characterize this disease, inform treatment decisions, and improve clinical outcomes. background: primary intracerebral osteosarcoma is extremely rare and limited to case reports. ptpn gain of function is associated with noonan syndrome, which has increased risk of multiple cancer types including brain tumors, but osteosarcoma has never been described. ptpn mutations have been reported in many cancers as both oncogenes and tumor suppressors, however no ptpn mutations have been described in osteosarcoma. pdgfr-a is a growth factor receptor whose activation is implicated in several malignancies. pdgfr-a and ptpn concurrent mutations are described in glioblastoma. there is no known link between holoprosencephaly, noonan syndrome, and osteosarcoma. we report a case of multifocal intracerebral osteosarcoma in a child with lobar holoprosencephaly and chronic subdural hemorrhage and discuss the genetic changes found in the tumor. design/method: a seven-year-old caucasian female, with a known diagnosis of lobar holoprosencephaly, chronic subdural hemorrhage and well controlled seizure disorder presented with status epilepticus shortly after completing antibiotic therapy for infection of subdural hematoma. mri showed diffuse dural thickening with mass lesions in the frontal lobe, temporal lobe, and the parasagittal region, the largest of which was contiguous with the subdural space but none of the lesions were associated with bone on mri or by direct neurosurgical visualization. tissue obtained for concern for recurrent infec-tion resulted in a diagnosis of high grade osteosarcoma. dna analysis was performed to help guide treatment choice. results: standard metastatic work-up was negative for skeletal primary tumor or metastatic lesions outside of the brain. she was treated with high dose methotrexate for two cycles per modified aost . despite maximal supportive care, she quickly developed rapid tumor growth as well as intratumoral hemorrhage with resultant herniation and death from respiratory failure just three months after diagnosis. tumor gene sequencing discovered three mutations with described roles in cancer: pdgfra d >vr, kdm a loss of exons - , and ptpn a v. conclusion: to our knowledge, primary multifocal extraosseus intracerebral osteosarcoma has not been previously described. despite known cns penetration of high dose methotrexate, this tumor proved resistant and aggressive. holoprosencephaly is associated with a multitude of known genetic drivers, but none are found in this case. furthermore, the genetic changes in this tumor are not typical for osteosarcoma. pdgfr-a over-expression is described in osteosarcoma, but is not clearly correlated with worse overall survival. further research is required to determine the role of ptpn in osteosarcoma. background: anaplastic lymphoma kinase (alk) encodes a receptor tyrosine kinase whose activation induces pathways associated with cell proliferation, angiogenesis, and cell survival. alk rearrangements are rare in neuroblastoma, while alk mutations and gene amplification occur more frequently. alk mutations have been found to be associated with increased alk protein expression that is associated with a worse prognosis. alk is commonly mutated in neuroblastoma at three hotspots (f , r , and f ). the eml -alk rearrangement has mostly been associated with lung adenocarcinomas, with only a few cases of non-lung cancers found. it has never been reported in neuroblastoma. multimodal therapy and to report the successful management of treatment related iron overload. results: a -year old male presented with abdominal swelling and ct showed a right kidney mass and bilateral lung nodules. he underwent right radical nephrectomy with lymph node sampling. pathology was reviewed centrally and revealed wilms tumor with diffuse anaplasia with rhabdomyosarcoma arising within the stromal component and of nodes positive. he received adjuvant intensive chemotherapy and radiation to the hemiabdomen and whole lungs. the -week chemotherapy regimen was vincristine, doxorubicin, cyclophosphamide (per cog arst ) alternating with carboplatin and etoposide (per cog aren revised uh- ). treatment was complicated by multiple episodes of fever and neutropenia and anorexia requiring g-tube placement. post-therapy, he had persistent neutropenia and thrombocytopenia without related complications. every months for evaluations he underwent a bone marrow which revealed normocellular marrow with maturing trilineage hematopoiesis. evaluation for a bone marrow failure syndrome was unrevealing. starting at months into therapy and all posttherapy imaging showed splenomegaly. he received units of packed red blood cells through the duration of therapy. he was diagnosed with iron overload based on serum ferritin and imaging, including t *mri. he received therapeutic phlebotomy for years with normalization of serum iron studies, t * of the heart, and liver iron concentration. he is more than years from completing therapy with no evidence of recurrent disease. asymptomatic cytopenias persist and he has no evidence of iron overload. conclusion: though a rare development, clonal sarcomatous transformation can occur in wilms tumor. our patient's tumor was successfully treated with intensive multimodal therapy targeting the diffusely anaplastic wilms and the rhabdomyosarcomatous component. treatment-related iron overload in a pediatric patient with a solid tumor was successfully treated with phlebotomy. consideration should be given to screen patients with solid tumors who receive multiple packed red cell transfusions for iron overload at the completion of cancer therapy. primary children's hospital, university of utah, salt lake city, utah, united states background: malignant solid tumors are less frequently encountered in infants. primitive myxoid mesenchymal tumors of infancy (pmmti) are a myofibroblastic malignancy and cases are rarely reported in the literature. cure is achieved in the majority of cases with surgical resection, however treatment for unresectable cases remains an enigma. recently published literature postulates that the newly discovered bcor duplication found in pmmti is tumorigenic via an epigenetic pathway. this molecular signature resembles that of clear cell sarcoma of the kidney (ccsk) and the growing number of bcor mutated sarcomas. a similar chemotherapeutic backbone and local control used for ccsk, has been proposed for the unresectable subset of pmmti. utilizing this approach a month-old with relapsed disease has remained disease free for months. however, given the rarity of this disease and the lack of published literature, there is no known standard of care treatment for unresectable and/or recurrent ppmti. we report a case of unresectable recurrent pmmti, a rare infant tumor, with less than cases reported. design/method: medical record, radiological studies, pathology and literature was reviewed. results: our patient is a now month-old female who presented with constipation and lower extremity weakness in the first weeks of life. an mri demonstrated a large lumbar epidural mass with spinal cord impingement. given prolonged (> days) neurological symptoms and location, emergent chemotherapy was initiated. biopsy showed a bcor positive, primitive myxoid mesenchymal tumor of infancy (pmmti). she was treated with ifosfamide, carboplatin and etoposide, and demonstrated clinical and radiographic response. we gave two additional cycles of cyclophosphamide, carboplatin and etoposide until surgical resection was feasible followed by two post-surgical cycles of chemotherapy. unfortunately, four month post-therapy mri demonstrated two new lesions; an unresectable paraspinal soft tissue mass and a left iliopsoas groove mass. given bcor association and reported successful therapy with vincristine, doxorubicin, cyclophosphamide alternating with ifosfamide and etoposide, we elected to incorporate vinca-alkaloid and anthracycline into her regimen. she is being treated with vdc/ie with plan for radiation consolidation. conclusion: pmmti is a locally aggressive tumor, for which surgical resection is curative. for those not amendable to resection, best care practices are still being determined. we report a case of pmmti initially responsive to chemotherapy, but not curative. this is the second case to conclusively demonstrate chemo-responsiveness. bcor mutation seems to be a common feature of this cancer; its role in the pathogenesis and as a target is an area of investigation. medical college of wisconsin, milwaukee, wisconsin, united states background: atypical teratoid/rhabdoid tumors (atrt) are central nervous system (cns) tumors that most commonly occur in very young children. there is no widely accepted standard of care for atrt patients, and while survival rates are improving they are historically poor. patients with metastatic disease to the spine at diagnosis have a worse prognosis, and for patients > years old, the presence of metastatic disease often results in the use of craniospinal radiation. the importance of correctly identifying metastatic disease at diagnosis aids in decision making and can have both prognostic and therapeutic implications. mr imaging at diagnosis is used to identify metastatic disease; however, here we present a case of diffuse leptomeningeal enhancement that spontaneously resolved after resection of a primary supratentorial atrt. objectives: to describe the resolution of diffuse leptomeningeal enhancement after resection of a primary atrt tumor in a -month-old prior to any adjuvant therapy. results: a -month-old male presented with a month history of vomiting and weight loss, regression of gross motor developmental milestones, and left hemiparesis. a brain mri demonstrated a × . × . cm solid and cystic right atrial mass with diffusion restriction and post-contrast enhancement. smooth diffuse enhancement was noted along the surface of the brainstem and within the interpeduncular fossa. a spine mri demonstrated diffuse circumferential post-contrast enhancement along the surface of the entire spinal cord. the patient underwent a successful near total surgical resection of the primary mass. pathology confirmed the loss of ini- staining in tumor cells, consistent with a diagnosis of atrt. no immediate adjuvant radiation or chemotherapy was given. repeat imaging was completed days after resection. brain mr demonstrated expected post-operative changes within the surgical cavity without definitive residual mass or leptomeningeal enhancement. spine mr demonstrated complete resolution of the previously seen circumferential enhance-ment along the entire spinal cord. csf evaluation at that time was negative for tumor cells. after recovery from surgery, chemotherapy treatment was initiated. conclusion: leptomeningeal enhancement at the time of diagnosis of atrt has historically been considered clear evidence of metastatic disease. this case raises questions about the previously accepted etiology of these imaging changes and suggests that widespread leptomeningeal enhancement should be carefully interpreted in future patients with similar imaging findings. in this setting, clinicians should consider repeat imaging following primary surgical resection in order to provide appropriate prognostic information and inform therapeutic decisions. poster # primary ewings sarcoma of cervical cord mimicking cauda equina syndrome sucharita bhaumik, joshua chan nyu winthrop hospital, mineola, new york, united states background: ewing's sarcoma (es) is a malignant primary bone tumor usually involving long bones. primary es of spine is quite uncommon ( . %) and its location in the cervical spine is even more rare. cauda equina syndrome (ces) is symptoms due to damage to the bundle of nerves below the end of the spinal cord known as the cauda equina (low back pain, radiating shooting pain down the legs, paraplegia, and loss of bowel or bladder control). it often occurs with lesions of lumbosacral spine. treatment with high-dose steroids may provide pain relief and improved neurologic function (by reducing edema) while awaiting diagnostic studies objectives: to demonstrate an unusual clinical presentation and emergent management of cervical es presenting with ces like symptoms. : year old male presented with a left sided posterior neck mass. soon after, he developed weakness of left arm, urinary and stool retention and inability to walk or bear weight in both legs. on physical exam a left tempero-occipital × cm fixed, non-tender, non-fluctuant mass was noted as well as motor and sensory impairment of left upper extremity, bilateral spastic paraplegia and loss of sphincter control. mri cervical spine showed a left cervical tumor with moth eaten appearance involving the vertebral bodies of c -c , adjacent muscles, displacing vital structures of the neck and compressing the cervical spinal cord. the thoracic and lumbosacral spine had no disease involvement. due to rapidly worsening spinal cord compression he was emergently treated with high dose steroids. he gained back all function in his extremities and regained bowel and bladder control. this eliminated need for urgent neurosurgical intervention. results: biopsy of the neck mass showed small blue round cells consistent with es with ewsr gene rearrangement. staging work up revealed no additional metastatic involvement. he then initiated treatment for localized es with systemic chemotherapy and radiotherapy and has had excellent response to treatment so far. conclusion: this is the first known case of non metastatic primary cervical es mimicking ces where an acutely enlarging mass presented with rapidly progressive neurologic deficits due to compression of anterior spinothalamic tract. in these unusual presentations of ces without lumbodorsal involvement it is important to consider cervical lesions. early rapid steroid initiation should be considered while awaiting biopsy results to prevent worsening cord compression followed by es focused treatments. this increases the chance of a successful outcome. the initial improvement with steroids may confuse the tumor with being a lymphoma children 's mercy hospital, kansas city, missouri, united states background: von willebrand disease (vwd) is a relatively common bleeding disorder with a high degree of genotypic and phenotypic variation. bleeding is usually mucocutaneous but can be severe and include muscle and joint bleeds especially in type vwd patients. most common bleeding management consists of desmopressin, anti-fibrinolytics, and/or plasma-derived antihemophilic factor/von willebrand factor (ahf/vwf) complex. a recombinant vwf has become available in the last few years. anaphylaxis and inhibitor development in vwd are rare. objectives: to describe the rare clinical manifestation of anaphylaxis to factor concentrate in a patient with severe type vwd. results: a -year-old female with severe type vwd [baseline vwag %, activity < %, factor viii (fviii) %] originally presented with heavy menstrual bleeding (hmb) leading to anemia requiring blood transfusion. she underwent placement of a levonorgestrel-releasing intrauterine device (lngiud) and began norethindrone. her hmb continued despite the lngiud and an increase in norethindrone dosing. plasma derived ahf/vwf complex was administered, which she had previously received. following the infusion, the patient developed anaphylaxis with hives, wheezing, tachycardia, and itching requiring doses of diphenhydramine and dose of hydrocortisone with resolution of symptoms. subsequently, she received recombinant vwf without incident. however, due to her low fviii level, she also required treatment with a full length recombinant fviii product. she again developed hives and itching after this infusion. she has since received recombinant vwf with recombinant fviii/fc fusion protein without further allergic reaction. there was no evidence of an inhibitor with her most recent post-infusion vwf level was %, factor viii %. conclusion: anaphylaxis to plasma derived factor products has been documented far less frequently within the vwd population compared to those with hemophilia and is typically seen in those with large gene deletions, usually with type disease. therefore, similar type vwd patients with severe disease may benefit from gene sequencing. it is unclear in this patient's case to which aspect of her treatment she is allergic, as she reacted to plasma-derived ahf/vwf and full length recombinant fviii, but not recombinant vwf or recombinant fviii/fc fusion protein. we hypothesize that she may be allergic to an epitope in the fviii b domain, or that the presence of fc fusion may have had a protective effect. further investigation including genetic analysis is planned. nodules. biopsies were consistent with neuroendocrine carcinoma, large cell type (g ). next generation sequencing revealed a khdrbs -braf fusion. he received conventional cytotoxic chemotherapy regimens both with cisplatin/doxorubicin, capecitabine/temozolomide, and doxorubicin/etoposide, but achieved a minimal response followed by rapid disease progression, massive ascites, and renal failure secondary to bilateral ureteral obstruction. results: based on his prior genomic testing, therapy with single agent mek inhibitor (trametinib) was initiated. this produced a rapid, dramatic response with greatly reduced disease burden at all sites, resolution of ascites and return to completely normal activity within months. this response lasted for approximately months before the tumor again progressed. further therapy with an erk inhibitor was ineffective, and the patient expired from progressive disease. located on the chromosome q , the braf oncogene, as part of the ras/mapk pathway, is involved in cellular proliferation, differentiation, migration, and apoptosis. braf mutations are recognized in a wide range of adult malignancies: thyroid cancers, non-small cell lung cancer, cholangiocarcinoma, ovarian cancers, and multiple myeloma. braf mutations have also been described in adult neuroendocrine carcinoma of the colon. trametinib is a highly specific inhibitor of mek /mek , a downstream mediator in the braf pathway. it has demonstrated activity in a number of tumors including advanced melanoma and gliomas. trametinib was chosen for this patient based on his atypical braf fusion. we believe this is the first documented case of its successful use in neuroendocrine carcinoma in the pediatric population. conclusion: this case demonstrates the presence of braf fusion in a case of pediatric neuroendocrine carcinoma and significant response to single agent mek inhibition in this context. this cases raises the question as to whether the combination of a targeted inhibitor, in addition to either conventional chemotherapy or other braf inhibitors, might offer a better approach to therapy than current treatment options. albany medical center, albany, new york, united states background: warm autoimmune hemolytic anemia (waiha) is characterized by autoantibody, and occasional complement binding of protein antigens, on the surface of red blood cells at temperatures ≥ oc resulting in targeted destruction. we describe the case of a year old male with a history of evan's syndrome, poor immune response to vaccines and lymphoid hyperplasia, presenting with altered mental status and severe anemia, found to have a warm igg pan agglutinin with evidence of both intra and extravascular hemolysis. his course was complicated by respiratory failure requiring intubation, pulmonary emboli, enterococcus bacteremia and hypertension. he received multiple transfusions with only transient increases in hemoglobin. the aiha was refractory to multiple rounds of treatment with high dose steroids, ivig, rituximab, cyclophosphamide, bortezomib, plasma exchange and mycophenolate mofetil (mmf). objectives: given the refractory nature of our patient's aiha the decision was made to trial eculizumab, a monoclonal antibody targeting c complement, preventing its cleavage and activation, and shown to be effective in treatment of atypical hemolytic uremic syndrome and hemolysis due to an igm cold agglutinin. prior to eculizumab infusion, ch and sc b- assays were significantly elevated. design/method: the patient was given two doses of eculizimab days apart. results: his hemoglobin steadily rose independent of red cell transfusions with a corresponding decrease in reticulocyte count, ldh and ch levels. the patient has remained stable with a normal hemoglobin ( - g/dl) on maintenance steroids and mmf. although we cannot definitively conclude that eculizumab directly caused his recovery, the clinical course post-eculizumab suggests this may be an efficacious treatment for aiha. genetic testing showed monoallelic frameshift mutation of the nfkb gene and monoallelic missense mutation of the dock gene. given the role of nfkb in both immunodeficiency and autoimmunity, it is thought that the patient's phenotype is due to nfkb haploinsufficiency and he is currently considering hematopoietic stem cell transplant. st. joseph's regional medical center, paterson, new jersey, united states background: heterozygous -thalassemia typically manifests as thalassemia minor, characterized by mild microcytic hypochromic anemia with minimal clinical ramifications. coinheritance of -globin gene triplication has been reported to exacerbate the clinical and hematological phenotype ofthalassemia trait, due to increase in the alpha/non-alpha-chain imbalance. reported phenotypes range from asymptomatic thalassemia minor to moderate thalassemia intermedia, usually diagnosed in adulthood without transfusion dependence. this combination has been described in mediterranean, european and asian populations, but rarely reported in hispanics. objectives: to report two cases of unusually severethalassemia intermedia in hispanic patients with heterozygosity for triplicated -globin gene and a ( )-thalassemia allele. results: case : sixteen-month-old male of mexican descent presented with persistent microcytic anemia and jaundice. peripheral smear showed nucleated rbcs with basophilic stippling and target cells. hemoglobin electrophoresis revealed: hba- %, hbf- %, hba - . %. -globin gene testing revealed heterozygosity for ( ) mutation (ivsi-i, g→a). given the unusually severe anemia, -gene testing was performed which showed -globin gene(anti . ) triplication ( / ). at four years, he had splenomegaly and bilateral maxillary prominence. head ct showed irregular contour of the parieto-occipital region due to medullary expansion. due to significant persistent anemia ( - g/dl) and progressive bony deformities of the skull, patient began chronic transfusions at age eight after family declined splenectomy.case : fifteen-year-old female, of peruvian and honduran descent, presented for evaluation prior to cholecystectomy for gallstones and recurrent ruq pain. father had known thalassemia trait. her hb was . g/dl with hypochromia, microcytosis, and target cells. electrophoresis indicated -thalassemia trait (hba- %, hba - . %, hbf- . %), confirmed by gene testing (heterozygous for a ( ) mutation in codon c>t). given jaundice and gallstones, -globin gene analysis was ordered showing triplication ( / ). ruq pain resolved post-cholecystectomy, but she developed persistent painful splenomegaly. she began hydroxyurea to increase gamma-globin production and decrease excess alpha chains, but it was discontinued due to hematological toxicity. due to recurrent luq pain and progressive splenomegaly, she underwent laparoscopic splenectomy at age with resolution of symptoms and improved hemoglobin. conclusion: -globin gene testing should be considered in -thalassemia carriers with an atypical clinical presentation including hispanic patients. the wide variability in the phenotypic expression of (anti . ) mutation andthalassemia trait suggest interplay of other genetic factors which remain undefined. the clinically significant presentation amongst certain subjects, as in our two cases, makes it imperative to identify these factors to aid in phenotype prediction and genetic counseling. ashley bonheur, shivakumar subramaniyam, jogarao vedula, sucharita bhaumik nyu winthrop hospital, mineola, new york, united states background: wilms tumor (wt) is one of the most common solid malignant neoplasms in children. a diverse range of genes and mechanisms are implicated in wt pathogenesis. predisposing syndromes result from a disruption of wt gene, crucial for renal and gonadal embryogenesis. another gene is wt gene locus at p , an area of imprinting. the p tumor suppressor gene on chromosome p . is seen in patients with anaplastic histology. in addition to these genes, whole and partial chromosome gains of q, , q, , , & and losses of p, p, q, q, as well as loss of heterozygosity (loh) are commonly seen. some genetic markers appear to be predictive of outcome and are now incorporated into the assigning of risk-directed therapy. patients with loh at chromosome p and q are treated with more intensive chemotherapy, as they have been associated with increased risk of relapse and mortality. objectives: to describe a new complex translocation involving chromosome , , and in a case of pediatric wt. design/method: a four-year old female presented with abdominal pain and emesis. on exam, patient had a firm and large abdominal mass. radiologic studies revealed a complex lobulated right renal mass. right radical nephrectomy was performed. histopathologic studies showed wt with triphasic histologic features with blastema predominance, invasion of the lymphovascular and perinephric adipose tissues, perinephric lymph node involvement and no anaplasia. chest ct scan showed bilateral lung metastases. tumor cytogenetics showed an abnormal karyotype, a complex translocation of , , and . the rearrangement occurred due to translocation between chromosomal bands q and q , with an insertion of q - on the q region. pcr based genotyping using microsatellite markers additionally identified loh for chromosome p and q . the patient was treated for high risk stage iv wilms tumor with favorable histology and received intensive chemotherapy and radiation therapy to the flank and the lungs. she is now in remission months after, with no evidence of recurrence on surveillance scans. complex translocations associated with wt have not been rigorously studied. a question for further study is whether there is any relationship between recurrence potential with a complex translocation compared to common chromosomal abnormalities. further knowledge of the molecular pathology and genetic changes in wt will help the development of new targeted therapies, as well as new biomarkers to aid diagnosis, risk stratification, and monitoring of treatment and relapse. results: a week-old girl was referred for evaluation of an abnormal newborn screen. mother was a known carrier of hb khartoum trait while father was a known carrier of thalassemia trait. patient's hemoglobin quantification performed by capillary zone electrophoresis showed hbf %, hb variant %, and no detectable hba. the hb variant ran in the d zone, a pattern consistent with mother's hb. alkaline agarose gel electrophoresis banding pattern showed f/s. acid agarose gel electrophoresis pattern showed v/f. later testing revealed abnormal isopropanol stability with + precipitation at minutes. this electrophoresis pattern is consistent with the pattern previously reported of hb khartoum. clinically, the patient is a healthy, active child whom we have followed for two years. she has not had any significant anemia outside of her physiologic nadir. she has not had any hemolytic episodes, and her bilirubin levels have always been within the normal range conclusion: to the best of our knowledge, this is the only reported case of hb khartoum/ thalassemia. the proline to arginine substitution of hb khartoum introduces a charged group on the chain at the site of contact. the resulting unstable chains can dissociate into monomers and favor the formation of methemoglobin, leading to hemoglobin instability. we had wondered if this unstable hemoglobin might result in clinical hemolysis when challenged with oxidative stress, such as in periods of infection. however, in the two years we have followed this patient, she has never had a hemolytic episode. at two years of age, she has hbf . %, hb khartoum . %, and hba . %. whether hbf elevation is protective from oxidative stress remains to be determined as we continue to follow this child. university of puerto rico -medical science campus, san juan, puerto rico, united states background: gm gangliosidosis is a lysosomal disorder caused by -galactosidase deficiency due to mutations in the glb gene. it is a rare autosomal recessive neurodegenerative disorder with an incidence of about : , - : , live births worldwide. this neurological disorder has three clinical forms. gm type , or infantile form is characterized by psychomotor regression by the age of months, visceromegaly (hepatosplenomegaly), macular cherry red spot, facial and skeletal abnormalities, seizures, and profound intellectual disability. we present a -year-old female with gm type and acute lymphocytic leukemia (all). design/method: she was diagnosed with gm type at the st months of age and family history was remarkable for an older sister with gm type . diagnostic studies reveal homozygous exon of the glb gene for a sequence variant defined as c. c>t, predicted to an amino acid substitution p.aarg cs. results: patient presented to our hospital with petechiae in lower extremities, pallor and intermittent tracheal bleeding. physical examination shows a hemodynamically stable girl that is chronically ill dependent of mechanical ventilation, severe mental retardation and scatter petechiae at upper and lower extremities. laboratory workup revealed severe normocytic anemia (hgb: . g/dl) with immature peripheral cells and thrombocytopenia ( × /l). serum chemistry revealed increase ldh ( u/l), increase hepatic enzymes (ast: u/l), normal uric acid level. there was no evidence coagulopathy. chest x ray was unremarkable except for evidence of chronic pulmonary illness. abdominal sonogram hepatosplenomegaly. during hospitalization, bone marrow aspirate and biopsy was performed which was diagnostic of b cell acute lymphoblastic leukemia (all) with . % lymphoblast and orderly myeloid/erythroid maturation. flow cytometry: % b lymphoblast with aberrant phenotype c/w b-acute lymphoblastic leukemia. karyotype revealed hyperdiploid female of favorable prognosis. cytogenetic by fish: hyperdiploid all with extra copies of runx and igh (no bcr-abl translocation). family was oriented about the new diagnosis and the dismal prognosis in conjunction to her primary condition. parents agree on no chemotherapy treatment for all with only supportive treatment. to this date, there is no evidence in literature that has previously described association of gm and leukemia. life expectancy of patient's primary condition is null therefore, correlation with leukemia might not be a coincidental finding. this patient opens the possibility of malignancy as part of gm type thus, malignancy diagnosis should be considered as part of their medical lifetime course. university of south florida, tampa, florida, united states background: hematological manifestations related to hiv infection are not uncommon, with thrombocytopenia having an estimated prevalence of - %. the pathophysiology is likely multifactorial. studies suggest that the primary mechanism may be immunologic resulting in accelerated platelet destruction. additional theories suggest that infection of megakaryocytes may also play a role causing inadequate platelet production. treatment of hiv-related thrombocytopenia is challenging. first-line treatments include initiation and optimization of antiretroviral therapies, immunoglobulin (ivig), and glucocorticoids. however, this approach is not effective in all patients and second line treatment options are less well studied, particularly in the pediatric population. objectives: we aim to present and discuss the case of a year old patient with perinatally acquired hiv- infection and persistent thrombocytopenia who, after failing first line therapies, showed normalization of platelet count on the novel thrombopoietin receptor agonist, eltrombopag. design/method: a retrospective chart review of the case patient's medical record was conducted. additionally, a thorough literature review was performed on this topic including the pathophysiology of hiv related thrombocytopenia and its treatment modalities. the patient required monthly ivig infusions for about year, but did not show a sustained response, often with platelet count dropping to less than , in between infusions. after initiation of mg eltrombopag daily the patient showed a sustained increase in platelet count (range , - , ). during a brief week lapse in eltrombopag treatment his platelet count dropped to , . upon re-initiation of therapy his count increased to , . the patient has remained asymptomatic, off of ivig for over one year, with undetectable hiv viral load and greater than cd t cell counts. no side effects or grade laboratory abnormalities were reported. conclusion: treatment of hiv-related thrombocytopenia can be challenging. first line therapies, including ivig and glucocorticoids, are not effective in all patients. several other treatment modalities have been utilized, including anti-d immunoglobulin, dapsone, danazol, interferon alfa, vincristine, thrombopoetic growth factors including romiplostim and eltrombopag, or splenectomy, but these are less well studied. this represents the first reported case of a pediatric patient with hiv who showed a positive response to eltrombopag with a sustained improvement in platelet count and no adverse effects from treatment. eltrombopag may be a safe alternative to first line therapies in those patients with hiv and refractory thrombocytopenia, however additional studies are needed. university of illinois college of medicine at peoria, peoria, illinois, united states background: achromobacter xylosoxidans is a gram negative rod with peritrichous flagella which causes rare opportunistic infections most commonly encountered by immunocompromised patients. it is primarily associated with uncomplicated bacteremia, cather-associated infections, and pneumonia. most reports of bacteremia associated with a. xylosoxidans are nosocomial, associated with neoplasm, and occurring mainly in adults. most reported infections with a. xylosoxidans in children are associated with cystic fibrosis. there are very few reported cases of septic shock from a. xylosoxidans bacteremia and pneumonia in the pediatric oncology population. objectives: to describe a rare case of a. xylosoxidans septic shock in a pediatric patient with relapsed neuroblastoma results: a -year old boy with history of stage iv highrisk neuroblastoma underwent standard frontline therapy with chemotherapy, hematopoietic stem cell transplant, radiation therapy, and immunotherapy, followed by a dfmo trial for maintenance. his -month follow-up scans demonstrated relapse and he was subsequently treated with additional chemotherapy, surgical resection, and mibg therapy, crizotinib for an eml -alk fusion and finally ifosfamide, carboplatin and etoposide (ice). he developed neutropenic fevers and was started on cefepime, vancomycin and fluconazole. blood cultures were initially negative. on the th day of fever, his previously scheduled pet scan was performed during hospitalization and showed new pulmonary opacities. he did not have respiratory symptoms, but therapy was escalated to meropenem, vancomycin and amphotericin. emergent bronchoscopy was performed the same day, with all bacterial and fungal cultures remaining negative. overnight, he developed tachypnea and saturations in the upper s, requiring nasal cannula. ir-guided lung biopsy was performed the next day, a flexible bronchoscopy was done to remove blood clots in the airway, the patient was placed on a ventilator, femoral lines were placed, granulocytes ordered and pressors were started for deterioration to presumed septic shock. arterial and femoral lines were placed but patient continued to have hemodynamic instability on multiple pressors. the following day, blood and respiratory cultures returned positive for results: at days after the start of iti, the inhibitor was < . bu and continued undetectable months after initiation of iti therapy. in this patient, iti with high-dose plasma-derived factor viii and von willebrand factor (vwf) complex was well tolerated and effective. genetic analysis confirmed a large factor viii gene duplication of exons to . we believe our patient developed inhibitor so quickly ( exposure days) due to the possibility of this mutation causing a frameshift that introduces a premature termination codon. this might be functionally similar to a deletion in the factor viii gene which poses the highest risk for inhibitor development in patients with severe hemophilia a. this variant has only been identified previously in two unrelated patients diagnosed with severe hemophilia a. this duplication is not listed in dbsnp variant database, nor observed in the general population database. our case proves the effectiveness of this method for patients with severe hemophilia a and an inhibitor. it also shows that more research is needed to identify patients at risk for inhibitor development. background: mercaptopurine ( -mp) is a prodrug that is a core component of maintenance chemotherapy for patients with a diagnosis of acute lymphoblastic leukemia (all). suppression of the neutrophil count is used to demonstrate adequate dosing of -mp during this phase of therapy. bone marrow suppression is mediated by the active metabolite -thioguanine ( -tgn), whereas the metabolite -methylmercaptopurine nucleotides ( -mmpn) has been shown to cause hepatotoxicity. allopurinol has been used infrequently in all maintenance therapy in the setting of skewed metabolism when adequate myelosuppression is difficult to achieve due to excessive hepatic toxicity. when given in combination with allopurinol a reduced dose of -mp may result in both increased -tgn levels and decreased -mmpn levels. objectives: describe the characteristics and clinical course of patients treated with allopurinol and reduced dose -mp during maintenance chemotherapy for all. we performed a retrospective chart review of patients at aflac cancer and blood disorders center of children's healthcare of atlanta with new diagnoses of b or t-cell all who received allopurinol during maintenance chemotherapy. we identified eleven patients with b-cell or tcell all who received allopurinol adjunctive therapy during maintenance chemotherapy at a single institution between - . these patients received adjunctive allopurinol for - weeks (median weeks) with reduced -mp ( - % of full dose). all ten patients with genetic testing for thiopurine s-methyltransferase (tpmt) had wildtype genotype associated with normal enzyme levels. indications for allopurinol use were most commonly unfavorable -mp metabolite levels, transaminitis (n = ), pancreatitis (n = ) and hyperbilirubinemia (n = ). favorable metabolite shift was achieved in all patients. liver enzymes improved in of patients with transaminitis after initiation of allopurinol/reduced -mp. three patients who experienced pancreatitis during maintenance did not have recurrence after initiation of allopurinol ( of these patients previously reported). six patients developed pancytopenia while on allopurinol, and two of those patients developed pancytopenia severe enough to require allopurinol cessation. four patients developed isolated anemia (hgb < . g/dl) without thrombocytopenia or severe neutropenia. no patient has experienced a recurrence of leukemia. overall, treatment with allopurinol and reduced dose -mp was successful in producing a favorable -mp metabolite distribution and reducing toxicity. therapy was generally tolerated; however a major and notable side effect was pancytopenia, in two cases severe enough to stop allopurinol treatment. anemia may be more prominent with allopurinol usage. allopurinol effect is variable among individual patients despite normal tpmt genotypes. baylor college of medicine, houston, texas, united states background: congenital sideroblastic anemia, b-cell immunodeficiency, periodic fevers and developmental delay syndrome (sifd) is a rare inherited sideroblastic anemia syndrome, first described in with clinically similar cases. genetic variations of trnt were identified as causative. objectives: to present an unusual presentation of a patient with sifd complicated by diagnosis of concomitant alpha thalassemia trait. design/method: retrospective chart review. a five month old male infant was referred to our hematology center for evaluation of elevated hemoglobin barts identified on newborn screen. despite numerous attempts, blood work was unable to be collected. at seven months of age he had microcytic anemia (hemoglobin . g/dl, mean corpuscular volume fl) more severe than what would be expected with alpha thalassemia trait. no variant hemoglobin was identified with isoelectric focusing or high performance liquid chromatography. by nine months of age he developed growth failure, intermittent emesis with fevers, developmental delays (predominantly gross motor), hearing loss, a disproportionally large head and coarse, thinning hair. over the next ten months, he was seen by numerous specialists for seemingly unconnected problems including sensorineural hearing loss, elevated liver enzymes and growth hormone deficiency. alpha globin analysis revealed deletion of two alpha globin genes. at months of age, he was admitted with one week of fevers, jaundice, and emesis. peripheral blood smear showed microcytic hypochromic anemia with marked anisopoikilocytosis including target cells, elliptocytes, tear drops, spherocytes, poikilocytes, marked polychromasia, and coarse basophilic stippling. given the inconsistency of his laboratory findings with the diagnosis of alpha thalassemia trait and clinical syndromic findings, bone marrow biopsy was performed which revealed rare ringed sideroblasts. one month later whole exome sequencing revealed trnt splicing variant c. - c>g and novel missense variant c. a>t consistent with sifd. hemoglobin barts on newborn screen with moderate to severe microcytic anemia directed initial diagnostic work-up towards variant alpha thalassemia. as additional medical conditions developed the focus shifted to a unifying syndrome. compared to previously described cases, our patient was diagnosed at an older age, presented with anemia rather than episodes of febrile illnesses, and had rare sideroblasts on bone marrow examination. diagnosis in this case led to identification of the novel c. a>t variant in his sister who had similar, but milder, features. sifd is a rare disease with variable phenotypic severity making diagnosis challenging without high index of suspicion which is crucial for appropriate management. wiseman, blood, . chakraborty, blood, background: cholelithiasis is uncommon in childhood. cholelithiasis is known to occur more frequently in children with predispositions, including female sex, obesity, parenteral nutrition, previous abdominal surgery, use of oral contraceptives, family history of gallstones, chronic hemolytic anemias, hepatobiliary disease, or exposure to specific drugs. although there have been occasional case reports linking cholelithiasis to childhood leukemia or leukemia therapy, the prevalence and risk factors of cholelithiasis in patients with childhood leukemia remain unclear. objectives: to estimate the prevalence of cholelithiasis in patients diagnosed with childhood acute lymphoblastic leukemia (all), and to evaluate possible risk factors for the development of cholelithiasis in patients with childhood all. we performed a computer-assisted review of the electronic medical records of patients diagnosed for b or t-cell all at children's healthcare of atlanta in the period from to . patients with diagnoses of cholelithiasis, cholecystitis or who had a cholecystectomy were identified. possible risk factors of age, sex, bmi, history of abdominal surgery and parenteral nutrition use were abstracted. patients with underlying chronic hemolytic anemia or pre-existing gallbladder disease were excluded. results: seventeen cases of cholelithiasis and cases of cholecystitis without documented cholelithiasis were identified. among patients with cholelithiasis, were female. median age at diagnosis of cholelithiasis was . (range . - . ) years. seven patients had no symptoms referable to cholelithiasis at the time of diagnosis. the median age of leukemia diagnosis among these patients was . (range . - . ) years. the median interval from diagnosis of leukemia to gallbladder disease was . years. four patients had bmi over the th percentile for age. two patients had a prior history of intraabdominal surgery. no patient received oral contraceptive pills. six patients received parenteral nutrition for more than days. there was no documented family history of cholelithiasis. seven patients did not receive any cholelithiasis directed therapy. two patients were managed with medical management only, with endoscopic retrograde cholangiopancreatogram with stone extraction, and with cholecystectomy. our study estimates the prevalence of cholelithiasis in childhood lymphoblastic leukemia to be . %, higher than the reported prevalence in the general pediatric population of . - . %. although our cohort size is small, it appears that all therapy and supportive care modalities associated with all are likely to play a larger role in the development of cholelithiasis than known predisposing factors in the general population. further studies are warranted. background: an uncommon side effect of intravenous immunoglobulin (ivig) administration is clinically apparent, sometimes severe hemolysis. we describe a severe case of coombs-positive hemolytic anemia secondary to ivig administration. ivig is a blood derivative manufactured from pools of , to , individual plasma donations. ivig is not abo-type restricted, so anti-a, anti-b and anti-a,b isoagglutinins are detectable. objectives: to describe a rare but serious type of transfusion reaction leading to gross hemolysis after ivig administration. results: a -year-old male with a past medical history of obstructive sleep apnea and obesity was admitted to the pediatric intensive care unit for adenoviral pneumonia and subsequent respiratory failure requiring mechanical ventilation. he had a complex hospital course with many complications including acute respiratory distress syndrome (ards), septic-shock, and coombs-positive hemolytic anemia. the patient was treated with commercial ivig (baxter/baxalta) -mg/kg daily for five days. he had two isolated episodes of severe hemolysis in relation to ivig administration requiring multiple transfusions of packed red blood cells (prbc). examination of pre-transfusion peripheral blood smear showed spherocytosis with rouleaux formation and large clumped rbc aggregates. the patient's blood type was classified as blood group a, rh-negative and his initial prbc transfusions were of this type. subsequently, the patient's coombs test was found to be positive using polyspecific and anti-igg typing sera. the patient's antibody screen against reagent group o screening cells was negative ruling out autoimmune hemolytic anemia. however, type specific anti-a antibodies were detected in his plasma as well as the acid eludate prepared from the coombs-positive red blood cells. it was concluded that the patient's hemolysis was due to anti-a antibodies presumed to arise from ivig. the patient's rbc transfusions were changed to o-negative blood and the hemolytic process resolved. the patient ultimately died due to complications of ards. although hemolysis is a known side effect of ivig, it is rarely considered when deciding to administer ivig. in addition, it has rarely been described in the pediatric population. ivig is used in the treatment of a growing number of medical conditions. due to the critical nature of many of these patients, hemolysis secondary to ivig may not be considered and continued blood transfusions with the patient's specific blood type may be used. it is crucial to remember that severe hemolysis can occur from ivig, and the importance of transfusing with blood group o, rh-negative blood when applicable. university of maryland medical center, children's hospital, baltimore, maryland, united states background: coagulopathy is a well-described complication of acute promyelocytic leukemia (apml), and remains a leading cause in induction failure. with treatment, coagulopathy associated with apml has been shown to rapidly improve. multiple organ dysfunction syndrome (mods) in apml, including acute respiratory distress syndrome (ards), has been associated with infection, traumatic injury, malignant infiltration, and cytokine release syndrome. when mechanical ventilation is no longer sufficient, extracorporeal membrane oxygenation (ecmo) can be considered; however, coagulopathy, severe end-organ damage, and malignancy are all relative contraindications to initiation of treatment. we report the case of a -year-old female presenting in respiratory failure, disseminated intravascular coagulopathy (dic), with intracranial hemorrhage, and mods, diagnosed with apml, successfully treated with ecmo therapy. design/method: retrospective case analysis and literature review. our patient, a -year-old female was admitted in respiratory failure and altered mental status, following a fall shortly prior to presentation. initial laboratory values were notable for pancytopenia, dic, and acute renal failure. a non-contrast head ct showed left temporal lobe intraparenchymal hemorrhage. she was diagnosed with apml by peripheral smear, later confirmed by fish for t( : ), and was started immediately on high-risk induction chemotherapy as per cog protocol aaml , including all-trans retinoic acid, arsenic trioxide, idarubicin, and dexamethasone. cvvhd was required for acute renal failure. despite maximal respiratory support, she remained hypoxemic, with oxygenation index of , pao /fio ratio of . ecmo was initiated hours after start of induction, hours after admission. coagulopathy resolved on day of induction, ecmo was discontinued after days, mechanical ventilation and cvvhd were stopped after days and she continued to improve, eventually achieving remission with few neurologic side effects. despite relative contraindications to ecmo, this patient was successfully treated with ecmo without significant neurologic side effects. the correction of her coagulopathy was multifactorial: ) restoration of adequate oxygen delivery via ecmo improving endothelial function; ) successful organ support to allow sufficient response to induction chemotherapy with atra leading to the terminal differentiation of leukemic blasts; ) complement and contact system activation through contact with ecmo circuitry. this case illustrates that ecmo can still be considered in patients despite coagulopathy and end organ damage. sinai hospital of baltimore, baltimore, maryland, united states background: primary polycythemia vera is an extremely rare diagnosis in the pediatric patient and is defined by a marked elevation of red blood cells due to erythropoietin-independent mechanisms. presentations of this disorder range from the asymptomatic person to severe thrombotic events, such as budd-chiari syndrome or cerebrovascular stroke. mutations in the jak gene are found in adult and pediatric patients with polycythemia vera; however, the jak v f mutation is less commonly identified in pediatric patients. we describe an otherwise healthy -year-old female who presented with a significantly elevated total erythrocyte count, hemoglobin, and platelets, incidentally discovered upon routine annual blood work obtained by her pediatrician. design/method: this is a report and discussion of a rare case. demonstrated cellular marrow with trilineage hematopoiesis and no dysplasia. cytogenetics were not assessed. his hemoglobin and platelet count recovered but leukopenia and neutropenia persisted. follow-up evaluation at three months revealed fevers, ongoing cytopenias, a one-month of a nodular skin rash on the trunk and extremities resembling erythema nodosum, and hepatitis (peak alt and ast of , and , , respectively). following clinical evaluation, a skin biopsy was performed and was remarkable for atypical lymphocytes within the subcutis with t-cell markers, a high ki- , and positive tia- , perforin, and -f immunoperoxidase stains. negative stains for cd , cd , and ebv were noted. these results are consistent with sptcl. additional evaluation did not support a diagnosis of hlh. a staging evaluation was performed. pet-ct showed widespread hypermetabolic subcutaneous activity in the legs, trunk and skull and diffuse marrow hyperplasia. bone marrow demonstrated involvement with precursor b-cell acute lymphoblastic leukemia, with a mll gene rearranagement. his skin biopsy was retrospectively stained with tdt, cd , pax- , cd a, and cd with negative results, and a blood smear taken at the time of the skin biopsy did not demonstrate leukemic cells. conclusion: this is the first report of a patient with sptcl having a synchronous malignancy. the patient is doing well, currently in the maintenance phase of treatment for his all, and his skin disease has resolved on pet-ct. while it is possible that his presentation was a function of chance, the possibility of an underlying immune dysfunction or cancer predisposition warrants further investigation. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hereditary xerocytosis (hx) is a rare red blood cell (rbc) dehydration disorder, characterized by variable hemolysis and propensity to iron overload. hx is often misdiagnosed as hereditary spherocytosis (hs). while splenectomy is curative for hs, it is relatively contraindicated in hx due to a substantial thromboembolism risk, signifying the importance of delineating these diseases. blood smear abnormalities are variable and often insufficient to make an accurate diagnosis. osmotic-gradient ektacytometry and genetic confirmation are critical in distinguishing these overlapping disorders. objectives: describe a family with hx, initially misdiagnosed as hs. discuss the importance of distinguishing these disorders and the utility of ektacytometry in making this distinction. design/method: a -year-old caucasian male was diagnosed with hs after presenting with prolonged neonatal jaundice starting on the first day of life. he described mild scleral icterus and history of intermittent jaundice and dark urine, without need for transfusions. his father, paternal uncle and paternal grandmother were all diagnosed with hs during childhood and underwent cholecystectomy. additionally, his father underwent splenectomy for abdominal pain. the child's blood counts revealed compensated anemia (hb . gm/dl) and reticulocytosis (arc × /mcl) with increased mcv ( . fl) and mchc ( . gm/dl). blood smear showed increased polychromasia and poikilocytosis with rare spherocytes and few stomatocytes. while the child had normal ferritin, his father had iron overload (ferritin ng/ml) despite no prior transfusions. osmotic-gradient ektacytometry profile of the child and father's rbcs showed a characteristic left-shifted, bell-shaped curve with decreased omin and ohyp, diagnostic of hx. the family is currently undergoing genetic studies. despite clinical similarities between hs and hx, distinguishing these diseases has significant management implications. hx is a disorder of rbc permeability, causing shortened rbc survival. stomatocytes on blood smear can raise suspicion for hx, but are insufficient to make an accurate diagnosis. identifying characteristic biomechanical membrane properties using osmotic-gradient ektacytometry is the gold standard for clinical diagnosis, which can then be confirmed by molecular studies. hs and hx can be easily and reliably distinguished using ektacytometry, as both disorders have very distinctive curves representing different rbc deformability patterns. after hx diagnosis was made, we counseled the family against splenectomy, as the risk of thromboembolism is significantly increased in hx compared to hs, and the father was diagnosed with iron overload. conclusion: hx is commonly misdiagnosed as hs. this case highlights the importance of making this distinction, and the utility of osmotic-gradient ektacytometry in reliably distinguishing these conditions. penn state health children's hospital, hershey, pennsylvania, united states background: relapsed acute myeloid leukemia (aml) presenting as an isolated central nervous system myeloid sarcoma (cns ms) is very rare and its treatment is not well-defined. thiotepa, vinorelbine, topotecan and clofarabine (tvtc) has been successful for re-induction therapy to induce remission prior to hematopoietic stem cell transplant (hsct). objectives: to describe our experience in utilizing tvtc therapy in two children with no extramedullary disease at initial diagnosis who presented with relapsed aml as intracranial myeloid sarcomas. results: case : month-old female was diagnosed with flt negative aml and completed treatment per the children's oncology group (cog) aaml study on the low risk arm without bortezomib. cerebral spinal fluid (csf) negative at diagnosis. fish testing positive for tcf gene deletion of unknown significance. mrd was undetectable after induction i and remained undetectable after each cycle. nine months off therapy, recurrent headaches prompted mri imaging which revealed two posterior fossa masses. csf and bone marrow testing were negative. stereotactic biopsy of the larger mass confirmed recurrence of aml. patient underwent two cycles of tvtc with a total of seven doses of intrathecal cytarabine with almost near resolution of the cns ms. completed cranial radiation and proceeded to allogeneic stem cell transplant with unrelated cord marrow donor and is disease free at approximately day + .case : year-old female diagnosed with flt and mll negative aml and completed treatment per cog aaml study on the low risk arm without bortezomib. csf negative at diagnosis. mrd was undetectable after induction i and completed therapy without complications. two months off therapy, a retrospective analysis of her diagnostic bone marrow by the cytogenetic laboratory to test a new panel identifying novel q partners revealed a cryptic insertional : (mllt /mll(kmt a) translocation. at four months off therapy, acute mental status changes prompted mri imaging which revealed two intracranial ms and lumbar spine involvement. resection of the larger lesion for symptomatic relief confirmed the mllt /mll(kmt a) fusion. csf positive for blasts and marrow negative for relapsed disease. patient completed two cycles of tvtc with a total of seven doses of it cytarabine with near resolution of cns disease (only mm contrast enhancement in the medulla). she received craniospinal radiation and is awaiting improvement in her cardiac function before proceeding to hsct. conclusion: tvtc is a successful reinduction regimen for relapsed aml with cns ms prior to hsct. background: acute severe anemia can be a life-threatening medical condition. the differential is quite broad for possible etiologies of acute severe anemia, including autoimmune hemolytic anemia (aiha) and atypical hemolytic uremic syndrome (ahus). autoimmune hemolytic anemia is an antibody-mediated process that targets the protein antigens located on the surface of red blood cells. treatment options for aiha include corticosteroids, with up to % of patients being responsive, with some requiring splenectomy. atypical hemolytic uremic syndrome is a medical urgency, defined as the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. the etiology is usually due to genetic causes, or less commonly, due to autoantibodies or idiopathic reasons. prognosis is very poor. objectives: differentiating between autoimmune hemolytic anemia and atypical hemolytic uremic syndrome can be a time-sensitive diagnostic dilemma while the patient is in critical condition, but this important delineation can vastly alter therapeutic options. design/method: here we discuss two cases highlighting the diagnostic workup involved in differentiating between atypical hemolytic uremic syndrome and autoimmune hemolytic anemia. patient a is a -year-old male who presented in extremis with severe anemia, uremic encephalopathy, and severe acute renal injury requiring hemodialysis and multiple blood transfusions. patient b is a -month-old male, who also presented in extremis with respiratory failure secondary to adenovirus/rhinovirus/enterovirus, with acute progressive renal failure and microangiopathic hemolytic anemia, requiring hemodialysis and cardiorespiratory support. : patient a underwent a full hematologic and infectious disease workup. subsequent laboratory studies confirmed enteropathogenic e.coli (epec) in the patient's stool; blood cultures remained negative. renal biopsy results were consistent pigment nephropathy. bloodwork indicated positive direct coombs. patient a was ultimately treated with steroids mg/kg/day, with significant improvement. patient b also included a full hematologic work-up, including adamts activity and ahus genetic panel, as well as full infectious disease work-up. subsequent laboratory test-ing revealed blood cultures growing streptococcus pneumoniae, with adamts activity at % (adult ref range: >/ = %), and normal complement levels. imaging findings also supported diagnosis of ahus. the management of a critically ill patient with acute severe anemia requires a thorough hematologic and infectious disease work-up. while molecular and genetic are helpful in definitive diagnosis of ahus, the utility of such results is limited by time. overlapping clinical presentation of a patient in extremis due to acute severe hemolytic anemia with progressive renal failure presents a rather broad differential, with time-sensitive treatment and prognostic implications. the favorable response to steroids delineates aiha from hus. background: d- -hydroxyglutaric aciduria (d- -hga) is a rare metabolic disorder characterized by developmental delay, hypotonia, and bi-allelic mutations in d- hydroxyglutarate dehydrogenase (d hgdh) or isocitrate dehydrogenase (idh ). metaphyseal chondromatosis with d- -hydroxyglutaric aciduria (mc-hga) is a type of d- -hga that has been previously reported in seven patients (omim ; pmid ), three of whom had somatic mosaicism for r variants in isocitrate dehydrogenase (idh ). we describe a -year-old boy with mc-hga who subsequently developed acute myeloid leukemia (aml) and was found to have a r variant in idh in a leukemic bone marrow sample. we report the first case of aml with this metabolic disorder. design/method: a -year-old hispanic boy presented with short stature, developmental delay, abnormal skin pigmentation, and unilateral congenital cataract. workup revealed multiple skeletal enchondromatosis and elevated urine d- -hydroxyglutaric acid levels. he was diagnosed with mc-hga. no pathogenic variants in d hgdh, idh and idh were identified in peripheral blood. germline testing with biopsies of skin lesions was declined by the family. two years later, he presented with streptococcal sepsis and pancytopenia. blasts were noted on peripheral smear. bone marrow morphology was consistent with acute myelomonocytic leukemia (∼ % blasts). chromosome analysis showed normal xy, and molecular testing by pyrosequencing idh and idh revealed a r c variant in idh ( % mosaicism). the patient is being treated as per the cog study aaml . end of induction i bone marrow aspirate was hemodiluted, but there was no obvious residual disease by flow cytometry ( . - . % sensitivity) or morphology. the previously identified idh variant was no longer detectable (limit of detection < %). although targeted therapy for aml with idh mutation is currently in phase i clinical trials in adults, there is no safety or efficacy data for using idh inhibitors in children. treatment with ivosidenib is therefore not currently an option for our patient. conclusion: this is the first case of aml reported with this rare metabolic disorder. somatic r variants in idh have been identified in three other mc-hga cases. this same mutation leads to the accumulation of d- -hydroxyglutarate in gliomas and aml. without any confirmed germline mutation or somatic mosaicism testing of multiple specimen sources, we can only speculate that the patient has an underlying somatic idh mutation associated with mc-hga which subsequently led to leukemogenesis. we present the first case of this association, to increase index of suspicion for development of aml in children with metabolic disorders associated with variants in idh . background: congenital combined deficiency of the vitamin k-dependent coagulating factors (vkcfd) is a rare heterogeneous autosomal recessive bleeding disorder. vkcfd is caused by mutations in the genes of either gamma-glutamyl carboxylase (ggcx) or vitamin k epoxide reductase complex (vkorc), which are responsible for the gammacarboxylation of vitamin k dependent proteins (vkdps) allowing for their activation. the clinical presentation ranges from no bleeding to intracranial hemorrhage. to date, vkcfd has been reported in few patients worldwide. objectives: we report a case of a girl with novel homozygous mutation of the ggcx gene, highlighting her clinical and biochemical characteristics with a review of the literature. a -month-old girl of consanguineous emirati parents, presented to our hospital with a history of bleeding from puncture site after receiving her second-month vaccine. that was associated with episodes of mild mucosal bleeding. review of systems was negative for jaundice, steatorrhea and failure to thrive and physical exam was unremarkable. investigations revealed markedly prolonged pt and aptt with high inr. fibrinogen, hemoglobin and platelets were always normal. activities of vitamin k-dependent factors including fii, fvii, fix, fx, protein c and s were all low. a measurement of proteins induced by vitamin k absence (pivka-ii) was done and came very high. this was associated with a mild elevation in liver enzymes but normal liver function test. the picture was supporting vitamin k deficiency, and as a result, she was started on oral vitamin k supplements of mg/day. she responded partially to vitamin k and required higher doses to stabilize her inr. after excluding acquired causes and due to her requirement of high doses of vitamin k, a mutation in either ggcx or vkorc genes was suspected. genetic analysis was conducted for her which revealed a novel missense homozygous mutation in the ggcx gene (c. a>t) confirming the diagnosis of combined deficiency of vitamin k-dependent clotting factors type . the asymptomatic parents were both heterozygous for the same mutation. results: she is currently stable on mg/day of vitamin k supplements. conclusion: vkcfd is a rare bleeding disorder with an overall good prognosis due to the availability of several effective therapeutic options. the function of the mutated gene is unknown. our patient demonstrated a partial response to vitamin k supplements suggesting presence of a residual carboxylation capacity and a possible role of this gene in the enzymesubstrate interactions. university of alabama at birmingham, birmingham, alabama, united states s of s background: gata is a zinc finger transcription factor that plays a critical role in the regulation of hematopoiesis and lymphatic angiogenesis. mutations leading to gata deficiency (gd) have been linked to a variety of clinical conditions. patients with gd have a striking predisposition to develop myelodysplastic syndrome (mds), acute myeloid leukemia (aml), or chronic myelomonocytic leukemia (cmml). acute lymphoblastic leukemia (all) has not been associated with gd, although the association of bcell all and gd has been previously reported. objectives: to describe a unique association of gata deficiency and t-cell all in a young child. results: an -year-old female presented with a one-week history of fever and malaise. she had a significant past medical history of verruca plantaris and self-resolving leukopenia associated with febrile illnesses. significant family history included sister with neutropenia and human papilloma virus (hpv) infection, and mother with neutropenia, monocytopenia, atypical mycobacterial infections, and hpv infection. peripheral blood revealed hemoglobin . g/dl, hematocrit . %, platelets , /ul, and white blood cell , /ul (neutrophils /ul, lymphocytes /ul, monocytes /ul). patient underwent a bone marrow biopsy demonstrating lymphoblast infiltration. flow cytometry analysis demonstrated monoclonal lymphoid blast population that co-expressed cd , cd , cd , nuclear tdt, cd , however, lacked expression of cd , cd , cd , cd , hla-dr, or myeloperoxidase. findings were consistent with tcell all with aberrant myeloid markers. cytogenetics analysis revealed ,xx,dic( ; )(p . ;p . ). patient began treatment as per children's oncology group aall and achieved remission at the end of induction. course of therapy was complicated by episodes of fever, reciprocating junctional tachycardia, asparaginase-associated thrombosis, viral meningitis, recurrent episodes of verruca plantaris, and resistant streptococcus pneumoniae or haemophilus parainfluenza infections causing chronic cough. later, she was also found to have low igm levels; after completion of therapy, she developed monocytopenia. lymphocyte subset panel revealed absent b cells, decreased number of natural killer (nk) cells, and cd /cd inversion. further work-up included gata sequence analysis that showed heterozygous nonsense mutation (c. c > t/c; reference nm_ ) likely resulting in gata haploinsufficiency. patient continues to be in remission, is receiving monthly immunoglobulin replacement and is on azithromycin for atypical mycobacterial prophylaxis. surveillance bone marrow biopsies have shown no evidence of mds or leukemia, however, have demonstrated persistent hypocellularity. the possibility of undergoing an allogeneic bone marrow transplant is actively being discussed given its curative potential. clinicians should be aware that t-cell all may be associated with gata deficiency. cincinnati children's hospital medical center, cincinnati, ohio, united states background: treatment for severe hemophilia a is centered on factor viii (fviii) replacement therapy. development of an alloantibody (inhibitor) against fviii is a significant treatment complication occurring in as many as - % of patients. high titer inhibitors render treatment with factor viii ineffective, necessitating the use of bypass agents that may not achieve hemostasis with the same efficacy. considering the substantial ramifications of inhibitor development on treatment, eradication of inhibitors is of great importance to achieve adequate hemostasis in this patient population. desensitization by immune tolerance induction (iti) is the primary method of inhibitor elimination. however, not all patients respond to iti. immunomodulation may be considered as the next line of therapy, although controversy remains in regards to agent selection and use. objectives: there is incomplete data on the use of immunomodulation therapy for inhibitor eradication in severe hemophilia a. we present a case of a pediatric patient with severe hemophilia a and high titer inhibitor who failed initial iti therapy to better illustrate potential treatment options for the future. design/method: a retrospective chart review was performed on a patient with severe hemophilia a at cincinnati children's hospital medical center. results: an -year-old caucasian male with severe hemophilia a secondary to intron inversion, was initially diagnosed following extensive bleeding after circumcision at birth. he was identified as having an inhibitor ( bethesda units (bu)) at months of age after exposure days of treatment. he failed multiple attempts of iti, with recombinant and plasma-derived (pd) fviii. he was advanced to immunomodulation therapy in combination with pdfviii, however demonstrated anaphylaxis to rituximab and ofatumumab. he underwent tolerization to rituximab, and received a six month course with a partial response (nadir of . bu). months following last dose of rituximab, a rising inhibitor titer ( . bu) was found. mycophenolate mofetil (mmf) was initiated with subsequent inhibitor stabilization and a decreasing titer ( . bu) over the course of the following year. mmf has been well tolerated without major side effects or infection throughout therapy. conclusion: development of an inhibitor against fviii is a considerable complication in patients with severe hemophilia a. use of immunomodulatory therapies following iti failure remains controversial. mmf has not been well studied in this patient population. we report a case of a patient who is being successfully treated with mmf with minimal side effects. further prospective studies should be considered to further define the role of mmf immunomodulation therapy. background: down syndrome (ds) children with aml (ds aml) have higher cure rates than their non-ds counterparts. outcomes for refractory/relapsed cases, however, remain dismal. somatic mutations of the gene encoding the transcription factor gata in ds aml patients are responsible for the observed hypersensitivity of ds aml blasts to cytosine arabinoside (ara-c). in view of excellent survival rates (approaching %) of ds aml patients, the ongoing children's oncology group (cog) aaml study seeks to determine the feasibility of treating standard risk (minimal residual disease/mrd negative) ds aml patients using a reduced dose ( -fold decrease) ara-c backbone. although results from japanese trials with this approach are promising, north american and european data are conflicting. although chromosome rearrangements in ds aml do not appear to carry the same adverse prognostic significance as in non-ds aml, monosomy in ds aml patients has been associated with a moderately worse outcome. isochromosome q, however, is rare and has only been reported in previous cases of ds aml. objectives: to report our institutional experience of very early relapse involving cases of ds aml patients treated per the reduced dose ara-c arm ( . g/m ) of the aaml study. design/method: we hereby report the disease course and cytogenetics of the above ds aml patients. : patient is a month old caucasian female who had gata mutation negative aml. patient is a -year old caucasian male whose chromosomal analysis revealed isochromosome q ( copies of the long arm of chromosome ). both patients achieved negative mrd (< . %) after induction i chemotherapy with thioguanine, low-dose ara-c and daunorubicin and proceeded per the reduced dose ara-c arm of aaml . patient relapsed immediately after completion of chemotherapy. salvage chemotherapy with mitoxantrone/high dose ara-c (hidac) failed to induce a second remission and the patient subsequently died of disease. patient relapsed within months from end of therapy. the patient underwent salvage chemotherapy utilizing a hidac backbone and remains in disease remission. the noted very early relapse following a reduced dose ara-c regimen in our above ds aml children suggests that testing for gata mutation and chromosome rearrangements may play a useful role in the development of future risk-stratified treatment strategies for ds aml. university of rochester, rochester, new york, united states background: in developed countries in the st century, severe nutritional deficiency is not an often considered differential diagnosis of unexplained childhood anemia. aside from iron deficiency anemia, vitamin deficiency severe enough to impact hematopoiesis is uncommon in the general pediatric population. here we present the unique case of a -monthold infant who presented with intermittent emesis, failure to thrive (ftt), developmental delay, macrocytic anemia, and neutropenia which was initially concerning for a congenital bone marrow failure syndrome. instead, she was discovered to have an underlying, potentially familial deficiency of b . objectives: . to describe the unique case of an infant with b deficiency. . to outline the importance of including b deficiency in the differential diagnosis of unexplained megaloblastic anemia in children. a -month-old exclusively breastfed infant presented for gastroenterology evaluation due to persistent emesis and poor weight gain over the course of months. her history was notable for delayed developmental s of s milestones and hypoactivity. marked pallor prompted hematologic evaluation, which revealed concern for macrocytic anemia (hemoglobin . g/dl, mcv ), reticulocytopenia ( . × ^ / l), and neutropenia (anc . × ^ /l). an otherwise reassuring physical examination and laboratory evaluation was notable only for the discovery of an undetectable b level and marked hyperhomocysteinemia ( mol/l). her hemoglobin (hgb) continued to decline (to . g/dl) over the first few days after presentation, and she required red blood cell (rbc) transfusion. within only a few days of initiation, daily cyanocobalamin injections resulted in a robust reticulocytosis response, improved hgb, immediate normalization in the neutrophil count, and resolution of hyperhomocysteinemia. additional history and laboratory evaluation from the patient's mother revealed a concurrent, asymptomatic maternal b deficiency as well as a history of a need for b supplementation in the maternal grandfather, raising concern for an inherited etiology. despite the rarity of vitamin-deficient hematologic abnormalities in the general pediatric population, b deficiency should be considered as a potential cause of an otherwise unexplained megaloblastic anemia, especially in the setting of concurrent ftt and neurodevelopmental delay. a detailed family history should be obtained in such cases and may have helped to prevent this patient's clinical sequelae had the deficiency been discovered sooner. our patient has experienced a favorable clinical response to b supplementation, attesting to the importance of vitamin b in early childhood growth and development. background: peg-asparaginase is universally utilized in the treatment of pediatric acute lymphoblastic leukemia (all). despite its high efficacy in this disease, it is associated with hypersensitivity and allergy in - % of patients. protracted anaphylaxis has been described in circumstances such as severe food allergy with ongoing allergen exposure; however, it has not yet been described in relation to peg-asparaginase. we describe the first reported case of protracted anaphylaxis after peg-asparaginase administration, provide guidance as to time course and management of protracted anaphylaxis, as well as evidence that erwinia asparaginase may be safely administered even in this high risk population. objectives: to provide guidance regarding the duration, course and management of protracted, severe anaphylaxis after peg-asparaginase therapy. a year old male with very high risk all presented for consolidation therapy with peg-asparaginase (intramuscular) and vincristine. one hour after administration, he developed generalized hives and angioedema, for which he was given diphenhydramine. he then quickly developed progressive hives, angioedema, subjective throat and chest tightness, and wheezing. he was treated with diphenhydramine, epinephrine, albuterol, and methylprednisolone with resolution of symptoms. one hour later, symptoms recurred and the patient became hypotensive; he was retreated with methylprednisolone and epinephrine, and was transferred to the pediatric intensive care unit (picu). in the picu, he was placed on an epinephrine drip, and continued on methylprednisolone, diphenhydramine, cetirizine, albuterol, and ranitidine. the epinephrine drip was successfully discontinued after hours, and his other medications were gradually weaned over the course of two weeks. of note, the patient did have st segment changes in his electrocardiogram during the first hours of anaphylaxis. these were associated with normal ventricular function as per echocardiogram, and resolved within one week. this patient has subsequently tolerated multiple doses of erwinia asparaginase (intramuscular) without premedication. this patient was acutely managed in the pediatric intensive care unit with steroids, anti-histamines, and continuous infusion epinephrine. symptoms consistent with severe anaphylaxis including hives, angioedema, throat and chest tightness, wheezing, and hypotension persisted for a total of four days before finally resolving. he has thus far tolerated multiple doses of erwinia asparaginase without any symptoms of allergy, hypersensitivity, or anaphylaxis. protracted severe anaphylaxis after peg-asparaginase therapy can be successfully managed with multi-agent therapy, including antihistamines, steroids, and continuous infusion epinephrine. re-challenge with an alternate form of asparaginase may be tolerated, even in a patient with protracted anaphylaxis to peg-asparaginase. ucsf benioff children's hospital oakland, oakland, california, united states background: vincristine (vcr) is widely used in pediatric cancers. unlike most cytotoxic agents, hematopoietic toxicity is uncommon. vcr-induced anemia has been observed but its mechanism has not been well studied. vinca alkaloid-induced membrane changes were seen in early studies of hereditary spherocytosis (hs) and anecdotal cases suggest vcr may increase hemolysis in such patients. here we describe a case involving severe vcr-induced anemia in a patient with hs and an explanation as to the mechanism. objectives: to describe the mechanism of vcr-induced anemia in hs. design/method: case report. a year-old female with hs was diagnosed with t-lymphoblastic lymphoma. she had required packed red blood cell (prbc) transfusions as a neonate and thereafter had done well without episodes of acute hemolysis or aplasia. complete blood counts (cbc's) demonstrated a compensated hemolysis, and she did not require further transfusions until she commenced chemotherapy. by the start of maintenance she had received many more prbc transfusions than the average patient. intermittent drops in hemoglobin (hb) did not correlate with any particular agent, and she had stable, mild splenomegaly. a clear pattern emerged during maintenance. her hb was - g/dl at monthly clinic visits, when she received vcr, intermittent intrathecal methotrexate, and corticosteroids. within - days, her hb dropped to . ± . g/dl, and reticulocyte count decreased from . to . ± . %. transfusion at day corrected hb, and the reticulocytes and hb returned to baseline. white blood cell and platelet counts did not change after vcr. blood samples from pre, immediately post, and days post vcr were analyzed and rbc characteristics and markers of hemolysis were not significantly different. ektacytometry showed identical curves, indicating no change in rbc deformability. in vitro incubation of patient blood samples with vcr also did not affect the osmotic deformability, confirming that a change in rbc rigidity was unlikely the reason for the drop in hb. these data indicate that a dysregulation of erythropoiesis was responsible for the anemia after vcr, rather than damage of peripheral rbc's. in most patients, maintenance therapy for lymphoblastic lymphoma does not cause severe anemia, likely because a temporary reduction in erythropoiesis in patients with a normal rbc survival and low reticulocyte count is not noticed. however, in a patient with decreased rbc survival and a brisk reticulocytosis, a disruption in rbc generation is more apparent. in conclusion, vcr administration to patients with an rbc disorder warrants close observation for potentially severe vcr-induced anemia. background: the addition of tyrosine kinase inhibitors (tki) to conventional chemotherapy has improved outcomes for pediatric patients with philadelphia chromosome-positive (ph+) acute lymphoblastic leukemia (all), however there remains an increased risk of relapse compared to other types of childhood all. typically, in relapsed disease the philadelphia chromosome persists and several mechanisms of resistance involving acquired mutations of the bcr-abl chimeric oncoprotein have been reported. objectives: describe a unique case of a pediatric patient with ph+ b-precursor all relapsing with b-precursor all without the philadelphia chromosome. results: an -year-old boy was diagnosed with ph+ bprecursor all with the presence of the t( ; )/bcr-abl translocation by cytogenetics and fluorescence in situ hybridization (fish), respectively. additional abnormalities included gains of runx and loss of one copy of etv . a remission bone marrow with negative minimal residual disease (mrd) was achieved at the end of induction with dasatinib and the esphall chemotherapy backbone. duration of tki therapy was two years post diagnosis. nearly one year after the completion of therapy, cytopenias prompted a bone marrow investigation. relapsed b-precursor all was established by immunophenotyping, however fish analysis did not identify the bcr-abl rearrangement. moreover, quantitative reverse transcriptase pcr was negative for the bcr-abl fusion transcript. again fish analysis of the bone marrow revealed multiple additional copies of runx and mono-allelic loss of etv , similar to the initial diagnostic sample. the patient was re-induced per aall anticipating a ph+ all relapse. however, with confirmation of the loss of the ph+ clone, tki therapy was not re-initiated. due to positive mrd of . % at the end of re-induction therapy, the patient was salvaged with blinatumomab therapy and subsequently underwent an allogenic stem cell transplant with a sibling donor. conclusion: this is the first known report of a pediatric patient with ph+ b-precursor all who developed recurrent b-precursor all without the philadelphia chromosome. the persistent findings of gain of runx and loss of etv makes it unlikely that a second unrelated b-precursor all developed following successful treatment of the original disease. this case highlights the possibility of a genetically distinct subclone present at the onset of disease that shared abnormalities of runx and etv but did not contain the philadelphia chromosome. nevertheless, the subclone harbored leukemogenic potential in the absence bcr-abl expression. it is plausible that the predominant clone present at diagnosis was effectively treated with dasatinib and extinguished, but the bcr-abl -negative clone persisted in the face of tki therapy. background: ligneous conjunctivitis is a rare form of pseudomembranous conjunctivitis that develops specifically in patients with type plasminogen deficiency. lack of plasmin activity in those patients result in defective fibrinolysis and formation of fibrin-rich membranous material/ masses that develops on the palpebral conjunctiva as well as other sites in the body.current management involve surgical excision of the masses that is usually complicated by multiple recurrences. recently, use of topical plasminogen concentrates helped delaying recurrence, but currently, those concentrates are not commercially available. we report on a -year-old omani girl, with hypoplasminogenemia who required optimization of plasminogen level at the time of surgery to delay/ prevent recurrence. objectives: case report on the peri-operative use of ffp versus cryopricipitate transfusion as an alternative replacement of plasminogen during surgical excision of ligneous conjunctivitis. design/method: pharmacokinetic study was performed to assess plasminogen recovery after ffp ( ml/kg) and precipitate ( bag/ kg) transfusion results: plasminogen levels remained subnormal after either ffp or cryoprecipitate administration. with ffp, the maximum concentration reached was almost % of normal. although half-life of plasminogen is known to be - . days, the patient seemed to have a high catabolic rate after receiv-ing cryoprecipitate, with plasminogen levels reaching basal levels within hours. because of the better recovery profile with ffp, we opted to give ffp before and after surgery. peri-operative management included ffp transfusion at ml/kg/ hours one day before and for days post operatively, followed by ml/kg once daily from day - , then ml/kg on th post-operative day. topical treatment was initiated using antibiotic and steroids ed on the day of surgery, followed by heparin ed on the second day. on follow up, she used topical heparin, cyclosporine, prednisolone, and topical lubricant eye drops for variable duration. clinical picture remained stable for almost year post operatively, when she started to develop recurrence of ligneous lesions again. background: ponatinib (inclusig®, ariad pharmaceutical) is a rd generation multi-targeted tyrosine kinase inhibitor (tki) approved for treatment of adults with chronic myeloid leukemia (cml) and philadelphia chromosomepositive acute lymphoblastic leukemia (ph+ all) resistant to or intolerant of other tkis. ponatinib has numerous drug-drug interactions and a black box warning for associated serious adverse vascular events and hepatotoxicity. for this reason, ponatinib use has been confined to specific high-risk populations. however, in patients who prove refractory to other therapies, the potential benefits of ponatinib may outweigh risks. to date, ponatinib has not been studied in the pediatric/adolescent and young adult (aya) population. furthermore, literature describing the use of ponatinib alone or in combination with other agents in pediatric oncology patients is scarce. objectives: to describe a single institutional experience using ponatinib in the pediatric patients with ph+ all. design/method: two cases of ponatinib use in pediatric ph+ patients resistant to other tkis were identified at our institution and are described. peripheral blood samples obtained from both patients identified bcr-abl p fusion transcripts and sanger sequencing was used to identify resistant mutations. results: our first case is a -year-old female who received upfront multi-agent chemotherapy plus dasatinib for ph+ all. relapse was confirmed on end-of-therapy bone marrow evaluation, thus bcr-abl mutation testing was performed and revealed a t i mutation. ponatinib was initiated then discontinued after one week due to clinically significant fluid retention with peripheral edema and bilateral pleural/pericardial effusions. the second case is a lateadolescent female with ph+ all who relapsed -years after stem cell transplant (sct). following relapse, tki therapy included both imatinib and dasatinib. due to persistence of bcr-abl fusion transcript despite tki therapy she was switched to ponatinib. shortly following initiation of ponatinib she developed a diffuse, maculopapular rash, which persisted despite dose reduction, resulting in ultimate discontinuation of the drug. bcr-abl mutation testing identified f l and f v resistance-conferring mutations. to date, there is scant existing literature detailing the use of ponatinib in pediatric patients. appropriate dosing is undefined and side effect profile not well described, particularly when used concurrently with other chemotherapeutic agents. thus, this case series reporting the response to and toxicity of ponatinib in pediatric ph+ all patients has important clinical implications. additionally, this is the first report of a pediatric ph+ all patient with documented t i mutation underscoring the importance of bcr-abl mutational testing, particularly at the time of relapse. cooper university hospital, camden, new jersey, united states background: myh -related disorder is a rare autosomal dominant disease, encompassing several subtypes: may hegglin anomaly, epstein syndrome, fechtner's syndrome, and sebastian syndrome. heterozygous mutations are seen in the gene encoding non-muscle myosin heavy chain iia (nmmhc-iia) which is involved in cell motility as well as functions to maintain cellular shape and integrity. the presentation of myh -rd is mainly characterized by macrothrombocytopenia, but various related expressions exist: nephritis often leading to renal failure, cataracts and sensorineural deafness ( ). a -year-old girl with history of extensive dental caries, hyperactivity, and speech delay due to suspected hearing loss was incidentally found to have thrombocytopenia at the time of genetic evaluation. she did not have any bruising or excessive bleeding. she did not respond to observation, immunoglobulins, or steroid therapy. her platelet count remained persistently low ( - k/ul). she underwent extensive evaluation to rule out platelet disorder vs. coagulation defect. her peripheral smear showed enlarged platelets by giemsa stain but no inclusion bodies were noted in granulocytes. her platelet aggregation and platelet surface glycoprotein by flow cytometry were negative. her coagulation profile was also normal. objectives: this case report summarizes the complexity in diagnosing myh -rd in a pediatric patient. design/method: since a unifying diagnosis for her clinical presentation was not apparent, whole exome sequencing (wes) was undertaken. results: wes revealed the r c heterozygous pathogenic variant, located in exon in the myh gene. myh gene alteration explained the patient's clinical features of macrothrombocytopenia and hearing loss. this mutation was paternally inherited, and her father demonstrates mosaicism. he was asymptomatic with normal platelet count but his morphology showed enlarged platelets with no inclusion bodies in granulocytes. when dealing with patients who have mild or no symptoms of bleeding diathesis but evidence of persistent macrothrombocytopenia, considering a platelet disorder belonging to myh -rd can help delineate certain predisposing syndromes and guide clinical management. patients are likely to benefit from early genetic testing while receiving supportive therapy. wes can highlight syndromes and provide information on recurrence risk for families. the renal and hearing abnormalities are indistinguishable between epstein and fechtner's syndromes, but the pathogenic variants differ ( ). the genotype-phenotype correlation implies that our patient may have either syndrome, although clinical features compatible with nephritis have yet to manifest. patients should be monitored closely for long-term progression of myh disease, and treatments should be initiated accordingly. we present an -year old female evaluated by genetics at birth due to prenatal microcephaly. chromosomes and microarray were normal. at age she developed standard risk pre-b-cell acute lymphoblastic leukemia (all). she completed treatment in and has been doing well in the interim, remaining in complete clinical remission. during and after treatment she exhibited developmental delay and neurocognitive deficits. at age her height and weight were at or below the th centile and head circumference was below the nd centile (approximately standard deviations below the mean and corresponding to the th centile for a -month-old girl). bone age was appropriate. she had a distinctive triangular face with micrognathia and a pointed nose resembling a seckel-like syndrome. the patient also had clinodactyly of the th toes, zygodactylous triradius involving the nd and rd left toes, tendency to sydney line in the right palm and a radial loop in the left middle finger. the patient's unique clinical presentation prompted a more thorough genetic evaluation, which led to a novel finding we feel is clinically significant with regard to the development of malignancy. design/method: whole exome sequencing (wes) was performed on the patient as well as her biological parents (trio). a de novo heterozygous mutation in the gene pcdh with potential relation to the phenotype was discovered. this c. dupa variant causes a frameshift starting with codon asparagine , changing this amino acid to a lysine residue and creating a premature stop codon at position of the new reading frame denoted p.asn lysfsx . this variant is predicted to cause loss of normal protein function via protein truncation or nonsense-mediated mrna decay. conclusion: pcdh is a member of the protocadherins family which is important in cell-to-cell adhesion and synaptic function in the central nervous system and is highly expressed in areas of the brain involved in higher cortical function and speech. aberrant expression of protocadherins has been associated with the development of malignancies in many organ systems. with regards to leukemia, the methylation status of this gene at diagnosis has been implicated in the prognosis of all and could be used as a biomarker to predict relapse. this patient's de novo mutation and clinical presentation are unique to what has been previously presented in the literature. we feel that this mutation is a clinically significant finding that may shed light on the role of this gene in the development of hematopoeitic malignancies. background: acquired hemophilia a (aha) is an uncommon and potentially life-threatening hemorrhagic disease characterized by sudden onset of bleeding in patients with neither personal nor family history of bleeding dyscrasia. it is usually seen in adults with autoimmune diseases, solid tumors, lymphoproliferative diseases, pregnancy or during the postpartum period; occurrence in the pediatric population has rarely been reported. we report a case of an otherwise healthy teenager who was found to have aha when he presented with acute onset of atraumatic soft tissue hematoma. results: a -year old male of middle eastern descent with history of congenital absence of the right external ear, but otherwise in good general health, presented to our emergency department with a three day history of progressive worsening of right lower leg pain, swelling, and paresthesia, without preceding history of trauma. evaluation by the pediatric orthopedics service documented significantly elevated compartment pressures, necessitating immediate four-compartment fasciotomy. pre-operative labs were significant for prolonged activated partial thromboplastin time (aptt) of . ( . - . ) seconds with normal prothrombin time (pt) and international normalized ratio (inr). ptt did not correct on mixing studies, suggesting the presence of a circulating anticoagulant. factors xii and xi were in the normal range; factor ix was elevated, ( - ). factor viii level was % and fviii inhibitor level was . bethesda units (< . ), confirming the diagnosis of aha. work up for autoimmune disease was negative. his bleeding and surgical hemostasis were managed with recombinant factor vii (novoseven) mcg/kg every hours for hours post operatively, with gradual interval prolongation. factor viii antibody eradication was managed with prednisone mg/kg/day. factor viii and inhibitor levels normalized by day of hospitalization. recombinant factor vii was discontinued; steroids were gradually tapered and discontinued at discharge (hospital day ). conclusion: acquired hemophilia is likely an underdiagnosed condition in pediatrics. while it is typically seen in adults with underlying autoimmune disease, solid tumors, lymphoproliferative disease, or during pregnancy or the postpartum period, pediatric cases may have no identifiable etiology. this case highlights the importance of considering this diagnosis in any patient with unexplained bleeding regardless of their age, so as to intervene early and prevent adverse consequences. university of oklahoma, oklahoma city, oklahoma, united states background: myeloid neoplasms associated with eosinophilia is a rare subtype of chronic leukemia characterized by clonal eosinophilia. the true incidence is unknown due to its rarity and possible classification as idiopathic hypereosinophilia syndrome. the most common chromosomal aberrations involve platelet-derived growth factor receptors (pdgfrs). we report one such rare case in a pediatric patient. most of the pediatric management of this entity is derived from adult case reports and case series. objectives: to describe a case of chronic leukemia presenting as eosinophilia results: a previously healthy year old caucasian male presented with a several week history of migrating joint pain, splenomegaly, and abnormal blood counts with leukocytosis, thrombocytopenia and absolute eosinophilia. white blood cell differential showed myeloid precursors suggestive of chronic myeloid leukemia. bone marrow evaluation showed % blasts and % eosinophils. bcr-abl testing was negative, ruling out cml. fish analysis for eosinophilic clonality revealed deletion of chic gene, resulting in fip l /pdgfra fusion gene, diagnostic for myeloid neoplasm with eosinophilia associated with pdgfr abnormalities. treatment was started with tyrosine kinase inhibitor (tki), imatinib mg daily. within months, fish analysis for fusion gene was negative. after approximately months of daily imatinib, he was switched to maintenance dose of mg weekly. he is approximately months since diagnosis and doing well on maintenance imatinib. in , the who revised its classification of some chronic eosinophilic leukemias to myeloid and lymphoid neoplasms associated with eosinophilia and rearrangement of pdgfra, pdgfrb, fgfr . the most common abnormality is the fip l /pdgfra fusion gene. other less common abnormalities include fusion genes kif b-pdgfra and etv -pdgfrb and point mutations in pdgfra . some features of chronic eosinophilic leukemia include absolute eosinophilia, splenomegaly, elevated vitamin b and tryptase levels, and organ damage from eosinophil infiltrates and cytokine release. patients with rearrangements or mutations involving pdgfra are usually very responsive to imatinib. starting doses have not been well studied or established. experts recommend co-administration of corticosteroids during the first few days of imatinib therapy in patients with a history of cardiac involvement and/or elevated serum troponin levels to prevent myocardial necrosis, a rare complication of imatinib therapy in eosinophilic patients. fortunately our patient did not have cardiac involvement and to date has not exhibited signs of chronic tki toxicity. conclusion: myeloid neoplasms with eosinophilia constitute a rare form of chronic leukemias. they are often associated with pdgfr abnormalities and are usually very responsive to tyrosine kinase inhibitor therapy. walter reed national military medical center, bethesda, maryland, united states background: germline samd l mutation is a rare cause of constitutional bone marrow failure with a unique propensity for clonal evolution to monosomy and mds. objectives: previous case series have demonstrated diverse clinical outcomes in patients with a germline samd l mutation. our case presents a novel samd l mutation (p.val leu). additionally, the case highlights the challenges in clinical decision making for a patient with a gene mutation that is known for clonal evolution towards monosomy with risk of progression to myeloid malignancy, but also known for self-correction through uniparental disomy or inactivating mutations which results in disease remission. design/method: a retrospective chart review and review of the literature was performed. dna was isolated from peripheral blood and used for whole exome sequencing. a peripheral blood sample from the patient's mother and father showed no samd l mutation. skin biopsies of the patient and parents were evaluated for uniparental disomy or new mutations. to determine the pathogenicity of this novel mutation, the specific samd l mutant dna was transfected into the human embryonic kidney cell line to assess its role in inhibiting cell proliferation. our patient presented at months of age with pancytopenia and hypocellular bone marrow in the setting of s of s sepsis. he had evidence of dysfunctional immune activation with hemophagocytosis and elevated soluble il with simultaneous severe hypogammaglobulinemia. analysis of the peripheral blood showed no increase in chromosomal breakage, normal telomere length, and normal flow cytometry. gene testing for primary hemophagocytic lymphohistiocytosis and inherited bone marrow failure were negative. after the patient recovered from his presenting illness, a repeat bone marrow biopsy demonstrated improved cellularity with myelodysplasia and cytogenetics significant for monsomy .whole exome testing demonstrated a novel samd l mutation. the patient continued to require intermittent ivig and failed to demonstrate appropriate leukocytosis with intermittent infections. on repeat bone marrow evaluation over the course of months, the patient demonstrated no evidence of evolution towards self-correction and had a persistent monosomy clone. the patient is scheduled to undergo a matched unrelated donor bone marrow transplant. our case highlights the unique clinical picture associated with constitutional marrow failure and clonal evolution secondary to a novel samd l mutation which is thought to cause pancytopenia by inhibiting cellular proliferation and often results in the development of monosomy which rescues hematopoiesis but with a risk for malignancy. background: notable labs developed a flow cytometricbased assay with a custom robotic platform to test fdaapproved drugs for anti-cancer activity against individual patient's tumor cells. this personalized assay is a potential method for identifying novel agents and drug combinations to treat aml patients who have failed standard therapies. objectives: to present the case of a teen who underwent successful treatment of relapsed aml post-sct with bortezomib, panobinostat, and dexamethasone-a regimen selected based upon results of notable lab testing. results: a -year-old male with m -aml had an isolated bone marrow relapse months after completion of scheduled therapy. at relapse, his aml was flt -itd positive. he achieved a second remission with negative mrd and underwent matched sibling donor bmt after busulfan/cyclophosphamide conditioning. bma performed on day + was mrd positive ( . %). repeat bma done on day + showed . % mrd. he started sorafenib on day + . he received donor lymphocyte infusion (dli) on day + , then received cycles of azacitadine (aza) followed by dli. marrow mrd by flow after sorafenib alone, sorafenib with dli, and sorafenib with aza/dli were %, . %, and . %, respectively. treatment was complicated by varicella meningitis, grade i skin agvhd, febrile neutropenia and c. difficile colitis, and metapneumovirus pneumonia. despite extremely low levels of leukemia (marrow mrd . %), notable lab testing performed on the patient's leukemia cells from marrow collected after aza/dli/sorafenib revealed sensitivity of his leukemic blasts to a combination of bortezomib, panobinostat, and dexamethasone. because of prolonged cytopenias, multiple infectious complications, and persistently positive mrd, he discontinued aza/dli/sorafenib and on day + started bortezomib . mg/m iv on days , , , and ; panobinostat mg po on days , , , , , ; and dexamethasone mg po on days , , , , , , , and . chemotherapy cycle started days later. he tolerated treatment without side effects and with resolution of rash and cytopenias. he achieved full donor chimerism, negative flt -itd, and complete remission by morphology and flow after two cycles. notable lab testing is a powerful tool for evaluating the sensitivity of small populations of leukemic blasts to novel drug therapy. results from notable lab testing may serve as a useful guide for treatment selection after failure of standard aml therapy. this patient achieved morphologic and mrd remission post-sct with bortezomib, panobinostat, and dexamethasone-a regimen predicted to be efficacious based upon notable lab results. maria ahmad-nabi, christine knoll, sanjay shah, esteban gomez, lori wagner phoenix children's hospital, phoenix, arizona, united states background: development of inhibitors in patients with factor ix deficiency (fixd) is a well-recognized complication occurring in - % of patients. within this subset a small percentage can develop anaphylaxis to factor. desensitization with cyclophosphamide, an alkylating agent used in the management of various oncologic malignancies, and reported for use in factor viii desensitization has been previously unreported for use in desensitization in patients with fixd. rituximab, an anti-cd antibody, however has been used. objectives: to induce immune tolerance (it) in patients with inhibitors to factor ix with either novel or under reported methods using cyclophosphamide and/or rituximab. we report a case series of patients at phoenix children's hospital with fixd who achieved it with cyclophosphamide and/or rituximab. results: patient one was a year old male with severe fixd, who at the time of desensitization had inhibitor levels of bu. he was desensitized with cyclophosphamide, then admitted for infusion of recombinant factor ix. he experienced a few minor symptoms of intolerance including an urticarial rash which was self-limited, and hemarthrosis of the right elbow on day which responded to novo . he tolerated the remainder of his infusion without issues. he continued recombinant factor ix daily, and returned to clinic for monthly cyclophosphamide for months. he did develop urticaria with hemarthrosis and spontaneous muscle bleeds which were tempered with zantac, zyrtec, solumedrol, and benadryl. he remained without a recurrence of inhibitors, however did have intermittent hemarthrosis of his ankles thereafter requiring prophylactic twice daily dosing recombinant factor ix. patient two was a year old male with severe fixd and a family history of anaphylaxis to factor causing early death in all male relatives with the disease. he had never received factor ix and did not have a detectable inhibitor prior to desensitization. he successfully underwent desensitization to recombinant factor ix with rituximab in the icu, and returned to clinic for weekly infusions x . he experienced no adverse reactions concerning for anaphylaxis. he continued to tolerate factor ix products without evidence of intolerance, development of inhibitors, and continues on as prophylactic dosing of recombinant factor ix every other day. our experience at a single institution proves cyclophosphamide as a novel agent for inducing it in those with fixd and anaphylaxis. it also provides further evidence that rituximab can desensitize patients with severe fixd. differences include longer duration for cyclophosphamide therapy ( months vs month). background: cartilage-hair hypoplasia (chh) is an autosomal recessive chondrodysplasia associated with defective cell-mediated immunity caused by mutations in the ribonuclease mitochondrial rna processing (rmrp) gene. cancer incidence is -fold higher in patients with chh than in the general population, especially non-hodgkin lymphoma. the use of rituximab, an anti cd antibody, results in decreased host b-cell number and impaired humoral function for - months. the safety of rituximab in pediatric patients with cancer and immunodeficiency is not well documented. a diagnosis of underlying immunodeficiency may discourage physicians from using rituximab due to the risk of severe bacterial infection or viral re-activation. objectives: to report a case of burkitt lymphoma in a young adult female with chh and defective cellular immunity successfully treated with rituximab. results: an -year old amish female with disproportionate short stature presented to our center for management of stage iv biopsy proven burkitt lymphoma with myc rearrangement. she had presented a week earlier with cervical, occipital, and submandibular lymphadenopathy, splenomegaly; fevers, night sweats, and weight loss for - weeks. on exam, her height was three feet associated with brachydactyly, mild bowing of the legs, normal size head without frontal bossing, fine and sparse hair. she had normal intelligence. her pattern of dysmorphisms was suggestive of chh (genetic testing not performed at time of diagnosis). pet-ct scan showed stage iv disease with involvement of cervical lymph nodes, spleen, iliac bone and bone marrow. treatment with standardintensity fab/lmb therapy (group c) with the addition of rituximab was initiated. she had an incomplete response to cop (∼ % reduction of tumoral masses) but achieved complete remission after copadam . her course was complicated with severe varicella zoster but she completed therapy and remains in complete disease remission for months after treatment completion. genetic testing subsequently performed proved homozygosity for chh with a n. a>g variant. she had no other opportunistic infections during or after therapy. conclusion: the use of rituximab was both safe and beneficial in our patient despite defective cell mediated immunity secondary to chh suggesting that rituximab may be safe to use in patients with cellular immune deficiencies. background: hemophilia a and b are bleeding disorders characterized by deficiency in factor viii or ix, respectively. spontaneous or provoked hemarthrosis is a known complication of hemophilia. repetitive episodes of hemarthrosis can lead to debilitating hemophilic arthropathy. lyme disease is a tick-born infection which is endemic to increasing parts of the united states. chronic lyme disease, the phase in which lyme arthritis typically develops, occurs months to years after initial infection and is characterized by swelling of one or more large joints generally in the absence of systemic symptoms. objectives: review cases of hemophilia a and b patients with episodes of provoked hemarthrosis refractory to intensive recombinant factor replacement therapy found to have concurrent lyme arthritis. design/method: we report two clinical cases and review relevant literature. results: first, we report a year-old male with moderate hemophilia a with a provoked knee hemarthrosis which failed to improve despite months of intense factor replacement therapy requiring multiple hospitalizations. factor replacement regimens included twice daily standard half-life recombinant factor viii products or daily to every other day extended half-life recombinant factor viii products with trough levels aimed as high as - %. factor viii pk studies were obtained for dosing, to confirm adherence, and to evaluate for subclinical inhibitors (inhibitor testing was negative). given protracted symptoms additional workup for hemarthrosis was pursed. lyme titers were positive for ( )igg, though negative for igm. he was treated with days of doxycycline during which time hemarthrosis greatly improved on examination and imaging, and he was able to recover function through physical therapy. second, we report a year-old male with moderate hemophilia b who required multiple hospital admissions for a provoked knee hemarthro-sis with no improvement in symptoms despite weeks of daily or twice daily factor replacement with standard halflife recombinant factor ix products aiming for % correction. we performed inhibitor testing (which was negative) and pk studies to assess for non-detectable inhibitors, dosing and adherence. lyme testing was positive for ( )igg, though negative for igm. he was treated with amoxicillin for days during which time hemarthrosis significantly improved on examination and imaging. diagnosis and follow-up imaging studies for both patients included mri and serial bedside ultrasounds performed as per uc san diego school of medicine mskus guidelines. background: relapse/refractory aml following allogeneic hematopoietic stem cell transplant (hsct) holds a high mortality rate. current relapse/refractory therapy modalities for younger patients may include re-induction with a clofarabinebased regimen followed by second allogeneic hsct. even for patients who undergo second hsct, the five-year survival rate is dismal. new therapies, including small molecule inhibitors, are being studied in the post-hsct relapse setting or those unfit for hsct with promising results. venetoclax is a small molecule inhibitor that has received breakthrough designation for aml treatment in elderly patients objectives: to report a young adult aml patient with relapse post hsct who was successfully re-induced with topotecan, vinorelbine, thiotepa, clofarabine (tvtc) and has sustained remission with venetoclax maintenance therapy. this approach appears to be unique in terms of reported literature. results: our patient is now a -year-old female noted to have mll rearranged aml at initial diagnosis when she was years old. she underwent chemotherapy consisting of cytarabine/daunorubicin according to standard + . due to persistent disease, she was re-induced with g-csf, clofarabine, and high-dose cytarabine (gclac) which put her in cr. her course was complicated by sepsis, colitis, gastrointestinal bleed, deep venous thrombosis, and transfusionassociated circulatory overload. given her co-morbidities, she received another cycle of clofarabine/cytarabine, and then proceeded to reduced intensity allogeneic hsct, according to bmt ctn . the patient tolerated hsct well and experienced no transplant-related complications, including no acute or chronic gvhd. unfortunately, she relapsed about month's post-hsct. initial salvage therapy consisted of another course of g-clac, but due to persistent disease the decision was made to re-induce her with topotecan, vinorelbine, thiotepa, and clofarabine (tvtc). during this time however, she was found to have extensive infection with a fusarium species requiring a course of anti-fungal therapy. bone marrow evaluation showed no residual disease with an mrd of < . %. once the absolute neutrophil count recovered, the patient was started on single-agent venetoclax for maintenance therapy, which has been well-tolerated. she remains in morphologic remission for over months. we describe herein a young adult with multiply relapsed aml wherein tvtc re-induction, followed by maintenance with venetoclax were safely used in the post-hsct setting. venetoclax therapy in the relapsed aml setting warrants further study. background: vitamin b deficiency is uncommon in children in developed countries, especially in the absence of risk factors like malabsorption or inadequate dietary intake. it often presents with non-specific symptoms and signs and can elude diagnosis. the recognition and treatment of vitamin b deficiency is critical as it can lead to bone marrow failure as well as severe neurological and developmental problems in children. to increase index of suspicion of vitamin b deficiency anemia in children. we report a rare case of vita-min b deficiency anemia in a child who presented with a severe macrocytic anemia, with signs of hemolysis and concern of malignancy. design/method: an almost three-year-old previously healthy girl presented with a few day history of fever, emesis, fatigue and pallor. she had no dysmorphic features, hepatosplenomegaly or lymphadenopathy on exam, growth and development were normal. laboratory findings showed severe macrocytic anemia (hemoglobin . grams/dl; mcv . fl) with reticulocytopenia. signs of intravascular hemolysis were present with elevated lactate dehydrogenase ( , units/l) and haptoglobin below assay limit. immune-mediated hemolysis was ruled out. initial picture of a hemolytic anemia was compounded by other findings of moderate neutropenia, mild thrombocytopenia and peripheral smear showing occasional blasts. further workup was done with a broad differential diagnosis that included leukemias, hemolytic anemias, bone marrow failure syndromes, and specific deficiencies. results: workup revealed abnormally low vitamin b levels along with significantly elevated homocysteine and methylmalonic acid levels indicating functional vitamin b deficiency. bone marrow evaluation showed megaloblastic anemia and dyserythropoiesis consistent with vitamin b deficiency, and ruled out leukemia. vitamin b deficiency can cause a hemolytic anemia like picture secondary to intramedullary hemolysis due to ineffective erythropoiesis. myeloid precursors are also affected which can lead to neutropenia, thrombocytopenia, and abnormal peripheral blood cells. in our patient, initial symptomatic anemia was treated with blood transfusion, followed by intramuscular vitamin b injections with normalizing lab values. so far, workup for an etiology for vitamin b deficiency is negative except for an equivocal range of anti-parietal cell antibodies raising concerns for pernicious anemia; however it is rare in this age group. another rare condition is an inborn error of the cobalamin transporter. she is currently on oral vitamin b supplementation and further workup will be planned based on response. conclusion: this case highlights the importance of early consideration and thorough evaluation of vitamin b deficiency in children with unclear etiology of anemia, so that prompt treatment can be initiated. memorial hospital/ university of miami, miami, florida, united states background: despite great success in the treatment of acute lymphoblastic leukemia (all), the outcomes for patients with relapsed all remain poor. prognostic indicators include timing and site of relapse. blinatumomab, is the first agent in its class that simultaneously binds cd -positive cytotoxic t cells to cd -positive b cells resulting in lysis of malignant cells. however, mechanisms of leukemia resistance to blinatumomab are unclear. objectives: to describe a case with multiple sites of extramedullary (em) relapse during blinatumomab therapy. results: a -year-old hispanic male with philadelphia positive, cd -positive b-precursor cell all refractory to chemotherapy, had failed a bone marrow (bm) and was placed on blinatumomab and imatinib. he achieved minimal residual disease (mrd)-negative systemic remission, but during his fifth cycle developed bilateral periorbital masses. biopsies confirmed cd -negative isolated em relapsed disease, which was treated with radiation therapy (rt). there was notable resolution of em disease and he continued systemic therapy. subsequently, he presented with a painful left scapular swelling. imaging showed muscle and lung parenchymal em relapse with cd -positivity confirmed on histology. he continued on blinatumomab with localized rt while awaiting car-t cell therapy. his bm mrd remained negative until he developed systemic mrd-positivity with cd -positive blasts following the sixth cycle. primary resistance to blinatumomab is poorly understood. it is proposed that expansion of cd -negative clones or downregulation of cd following blinatumomab may play a role. this was observed in our patient's periorbital relapse; but subsequent em and systemic relapses were cd -positive, consistent with the co-existence of multiple clones in relapsed all. it has also been postulated that em relapse could be linked to the failure of blinatumomab or t cells to migrate to em sites of disease or drug inactivation by the microenvironment. the second em relapse in our patient, with cd -positive disease suggests this as a possible mechanism of relapse. this was reported in patients with cd positive non-hodgkin lymphoma (nhl), and higher doses of blinatumomab however, have shown promising results in this population. despite blinatumomab's effectiveness in inducing remissions in patients with refractory/relapsed all, it appears to have limitations in patients with em disease. these may arise either from the multiclonality associated with relapsed all or due to the emergence of resistance to blinatumomab, including failure to migrate to em sites. background: cyclic neutropenia is a rare hereditary disorder, characterized by recurrent neutropenia, cycling at about week intervals, with variable associated symptoms including oral ulcers and fever. there are reported cases of cyclic neutropenia associated with chronic inflammation leading to development of reactive aa amyloidosis. one patient also presented with amyloid goiter. we report a new case of cyclic neutropenia with associated renal and thyroid amyloid. design/method: a -year-old female presented with a month history of thyromegaly, and recurrent aphthous ulcers associated with fevers. laboratory workup showed severe neutropenia, anemia, azotemia, and abnormal thyroid function, with an absolute neutrophil count - / l, hemoglobin - . g/dl, serum creatinine - . mg/dl, and uric acid - . mg/dl. thyroid stimulating hormone was elevated - . iu/ml, and normal free t . urinalysis showed + protein, + blood, and - urine red blood cells/hpf. chest radiograph showed mild narrowing of the trachea from thyroid compression. bone marrow biopsy showed a hypocellular marrow, with tri-lineage hematopoiesis, left shifted myeloid maturation with very rare mature neutrophils. both renal biopsy and thyroid fine needle aspiration revealed abundant amyloid. of note, her father had aa amyloidosis, resulting in end-stage renal disease (esrd) requiring hemodialysis, and recurrent aphthous ulcers. the family history suggested a familial predisposition. genetic testing revealed a pathogenic elane c. a>t gene mutation with autosomal dominant inheritance confirming the diagnosis of cyclic neutropenia. we treated our patient with daily granulocyte colony stimulating factor to reduce the burden of chronic inflammation induced by cyclic neutropenia, and to preserve renal and other end organ function affected by further amyloid deposition. results: proband with elane gene mutation positive cyclic neutropenia, amyloidosis of thyroid and kidney, with a positive paternal history of aa amyloidosis resulting in esrd. cyclic neutropenia may result in chronic inflammatory states leading to secondary amyloidosis. university of kentucky, lexington, kentucky, united states background: overall survival of burkitt lymphoma (bl), regardless of stage, is greater than % in the pediatric population when treated with multi-agent chemotherapy. adenovirus is a common, usually self-limited infection within the pediatric population; however, findings can vary within an immunocompromised host. hepatitis is a rare complication, with very few reports of radiologic findings in this patient population. we discuss a three year old male with history of bl who presented with clinical and radiographic evidence of relapse but was found to have adenovirus hepatitis. design/method: a case report of a patient with bl in complete remission after completion of standard of care chemotherapy, who presented with return of high fever, elevated ldh, transaminitis and hepatic lesions. we describe the hepatic imaging and pathology consistent with adenovirus hepatitis in this immunocompromised host. our patient presented at three years old with a six week history of worsening abdominal pain and fevers. he was found to have a right sided pleural effusion, multiple lesions of the liver, and diffuse abdominal lymphadenopathy; biopsy of lymph tissue was consistent with bl. he completed therapy per anhl arm b and was in a complete remission at the end of planned therapy. one month after completion of therapy, he returned with high fever, abdominal pain and transaminitis, similar to his initial presentation. ct scan showed multiple hypodense discrete lesions throughout the liver and re-accumulation of right sided pleural effusion. ldh peaked at u/l (uln u/l). uric acid remained within normal limits. bilirubin peaked at . mg/dl, conjugated . mg/dl. liver biopsy was performed, showing smudgy nuclei with immunohistochemical staining positive for adenovirus. there was no evidence of lymphomatous involvement. resolution of hepatic lesions and transaminitis, with normalization of ldh and fever, occurred with symptomatic treatment alone. adenovirus is known to cause systemic disease in immunocompromised patients and rarely hepatitis. no pediatric patients with discrete hepatic lesions secondary to adenovirus have been reported in the literature. three cases of discrete hepatic lesions have been reported in adult immunocompromised patients, two with fatal fulminant liver failure and one who required cidofovir. this case demonstrates that a common pediatric viral infection can present with lesions concerning for metastatic disease in a pediatric lymphoma patient. prompt diagnosis is vital in the management of these patients when recurrent lymphoma is in the differential. background: heparin induced thrombocytopenia (hit) is an immunologic process in which antibodies bind a heparin complex and cause a paradoxical hypercoagulable state. ramifications of this process may include a multitude of thrombotic events and bleeding complications secondary to platelet consumption. in our patient, hit manifested as increased bruising, an acute decrease in platelet count, and continual clotting of her crrt circuit. hit, although rare in pediatrics, should be included in the differential for children with thrombocytopenia who have received heparin products. to present a unique case report of a critically ill pediatric patient who developed hit in the presence of multiorgan system failure and to discuss the challenges encountered with identification of an alternative anti-coagulant. results: a yo obese, caucasian female child presented to our facility with bilateral pulmonary emboli (of unclear etiology). initially, she was started on a continuous heparin infusion, but was transitioned to enoxaparin within days without issue. five days after enoxaparin was initiated, the patient developed acute kidney injury (evidenced by increasing creatinine) attributable to her biventricular heart failure. due to her need for continuous renal replacement therapy (crrt), she was transitioned back to a continuous heparin infusion. whereas her initial platelet count on transition was normal, she developed severe thrombocytopenia ( , ul) within hours. due to intermediate risk but low suspicion for hit, pf antibodies were sent which were positive. after much discussion, she was transitioned to an argatroban infusion which was titrated according to ptt levels. within hours, her platelet count normalized. at discharge, she was prescribed apixaban for anti-coagulant management. conclusion: hit is an uncommon presentation in the pediatric population. given its rarity, there is often a delay in diagnosis which increases risk of complications such as bleeding, stroke, and limb ischemia. even if the diagnosis is suspected or proven, there may be challenges in initiating alternative agents as limited data exists on pediatric options. as argatroban remains the treatment of choice for patients with hit, experience in pediatric patients is limited, and dosing recommendations have been extrapolated from adult studies. anecdotal data exists for use of bivalirudin in children, although studies, primarily, focus on use in specific cardiac cases. in our patient's case, choice was further complicated by renal failure. this case study highlights the need for further research regarding the identification of a secondary anti-coagulant agent for use in pediatric patients with hit. background: subcutaneous panniculitis-like t-cell lymphoma (sptl) is a rare form of non-hodgkin's lymphoma characterized by infiltration of cytotoxic t-cells into subcutaneous tissue. sptl occurs in both adults and children and can present in both patient populations as either alpha/beta or gamma/delta subtypes. patients with the gamma-delta phenotype have an overall poorer survival, although the exact etiology is unclear. interestingly, both subtypes of sptl can present with secondary hemophagocytic lymphohistiocytosis (hlh), and this is associated with a worse prognosis. currently, there are no standardized treatment protocols for sptl, and clinical management includes watchful waiting, corticosteroids/immunosuppression, chemotherapy, and stem cell transplant. the primary objective was to compare how two patients with the same diagnosis responded acutely to therapy. we performed a retrospective chart review of two pediatric patients at our institution who were diagnosed with alpha/beta sptl and secondary hlh. we examined each presentation, treatment course, and outcome. we then completed a brief review of the current literature describing treatment of and outcomes for sptl with secondary hlh. results: these two patients presented in a similar manner with signs and symptoms of hlh. each was then subse-quently diagnosed with alpha/beta sptl after biopsy of cutaneous nodules and each had diffuse disease, as measured by pet. however, they demonstrated vastly different acute responses to therapy. one patient was pre-treated with systemic glucocorticoids before receiving definitive chemotherapy and tolerated therapy well as an outpatient. the other patient started systemic chemotherapy without steroid pretreatment and developed severe cytokine storm characterized by hypotension, cardiac dysfunction, multi-organ failure and cytokine elevation. both patients achieved complete remission (cr) after treatment with chop chemotherapy and remain disease-free - months off therapy. in patients presenting with sptl and secondary hlh, we propose that initial treatment with antiinflammatory or anti-cytokine therapy can decrease, or even prevent, the possibility of life threatening cytokine release as a result of cytotoxic chemotherapy. background: congenital dyserythropoietic anemia type ii (cda ii) is a rare autosomal recessive disorder, rarely presenting in the neonatal period. iron overload often occurs as a late sequela of ineffective erythropoiesis and intramedullary hemolysis. objectives: to report the novel use of iron chelation in an infant with cda ii associated with severe iron overload. the patient is a -month-old, former -week infant with prenatal non-immune hydrops and transfusion-dependent fetal anemia who presented with persistent anemia, reticulocytopenia, hyperbilirubinemia, liver dysfunction, and hyperferritinemia. his initial ferritin was . ng/ml, tibc ug/dl, and transferrin mg/dl. his bone marrow biopsy showed trilineage hematopoiesis and erythroid dyspoiesis characterized by binucleation of late-stage precursors. genetic testing revealed a compound heterozygous missense mutation and splice site mutation in the sec b gene, confirming the diagnosis of cda ii. initial liver biopsy revealed mild portal fibrous expansion, and abundant hepatic iron deposition. his ferritin continued to increase, peaking at , ng/ml, along with liver enzymes peaking at an alanine aminotransferase (alt) of u/l and aspartate aminotransferase (ast) of u/l. ferriscan showed an elevated estimated liver concentration of . mg/g dry tissue. repeat liver biopsy months later showed giant cell hepatitis with worsening mild portal fibrosis and hemosiderosis. additionally, tissue liver iron concentration was mcg/g dry weight. cardiac t * mri revealed mild cardiac iron deposition. given his significant degree of iron overload, deferoxamine was used to reduce hemosiderosis and liver morbidity in preparation for bone marrow transplantation. the patient received deferoxamine mg/kg/day iv x days/week for three months, without any clinically significant adverse events. blood counts and hepatic and renal function were monitored weekly without any abnormalities. growth parameters and liver enzymes significantly improved while receiving chelation therapy. as a noninvasive, cost-effective method, serum ferritin levels were monitored monthly to gauge response to treatment. despite receiving blood transfusions every - weeks, serum ferritin decreased to ng/ml and liver enzymes decreased to alt u/l and ast u/l prior to bone marrow transplantation. we report the use of deferoxamine in a patient with cda ii less than years of age, for treatment of iron overload. our patient tolerated deferoxamine well without significant adverse events or organ toxicity. deferoxamine may be a well-tolerated method of reducing iron burden in young patients with iron-loading pathologies. background: low grade gliomas with kiaa- -braf fusions typically have a favorable prognosis with infrequent rates of high grade transformation, low rates of metastasis and even lower rates of extra cns metastasis. while highgrade transformation has been reported for tumors with braf v e mutations and cdkn a deletions, it has not been pre-viously reported in gliomas with kiaa- -braf fusions. while there are case reports of high-grade cns malignancies metastasizing through a ventriculo-peritoneal (vp) shunt, low-grade gliomas metastasizing in this manner are extremely rare. objectives: to describe a unique case of peritoneal tumor dissemination of a braf fusion positive high grade neuroepithelial tumor in a child with a vp shunt placed for multifocal braf fusion positive low grade astrocytomas results: an eight-year-old male was initially diagnosed with multifocal low-grade astrocytomas of the hypothalamus and c -c spinal cord. initial testing revealed the kiaa- -braf fusion, but no cdkn a or braf v e mutation. initial surgical management included a vp shunt and resection of the cervical spinal lesion. he received vincristine and carboplatin, followed by transition to vinblastine given new thoracic metastatic lesions after months of therapy. at months after diagnosis, scans were concerning for diffuse leptomeningeal progressive disease and new intracranial lesions, necessitating craniospinal radiation. following a near cr, he presented months later with acute onset of abdominal pain. a ct scan revealed peri-renal and perirectal soft tissue masses, confirmed by exploratory laparotomy to be peritoneal tumor dissemination of high grade neuroepithelial tumor. a kiaa -braf fusion was noted and confirmed by rt-pcr, identical to that seen in the original cns tumors. additional findings included deletion of chromosome p (without q loss) and heterozygous and homozygous deletion of cdkn a found by fish. brisk mitotic activity justified a high-grade designation. salvage chemotherapy consisted of cycles of ice with subsequent resolution of pet-avid disease and only minimal peri-nephric tissue remaining. given the favorable response, surgical resection and multiple tissue biopsies were performed which documented no residual active disease. the shunt was revised and he started trametinib for maintenance. we present a unique case of peritoneal dissemination of high grade neuroepitheial tumors with the same kiaa- -braf fusion as multifocal low grade astrocytomas in a child with a vp shunt. this raises suspicion for tumor metastasis and transformation to a higher grade malignancy versus two distinct diseases, which may be indicative of an underlying cancer predisposition. texas children's hospital, houston, texas, united states background: polycythemia is a common referral to hematology. it is important to evaluate for a high oxygen affinity hemoglobinopathy, ensuring appropriate testing is performed for early diagnosis and avoidance of additional tests and procedures. a year old mexican female presented with an elevated hemoglobin and hematocrit, symptoms of plethora of her hands and feet, chest pain, palpitations, and fatigue. further confounding the picture, she also had significant menorrhagia and iron deficiency. she was diagnosed with the rare high oxygen affinity hemoglobin new mexico variant, only previously described once in the literature in a year old black boy. objectives: the patient initially presented at age with a hemoglobin of . g/dl and a hematocrit of . %. initial work up consisted of a hemoglobin electrophoresis which diagnosed sickle cell trait, a co-oximetry panel which was normal, and erythropoietin level of mu/ml, also normal. she was then lost to follow up and re-referred at age . she is a competitive basketball athlete, and at that time, she presented with a hemoglobin of . g/dl, and hematocrit of %. erythropoietin level continued to be normal at mu/ml. design/method: cardiology was consulted regarding chest pain and palpitations with a normal evaluation. chest x-ray was also normal. a bone marrow aspirate and biopsy was performed with results significant for mild erythroid hyperplasia and mild reticulin fibrosis. jak mutation, von hippel lindau, bpgm, and hereditary erythrocytosis mutations including phd , hif a, and epor mutation analysis were sent, all of which were normal. testing to mayo clinic for p rbc oxygen dissociation returned low at mmhg ( - mmhg normal range) and subsequently a hemoglobin electrophoresis identified a hemoglobin variant leading to beta globin gene sequencing. results: patient found to be heterozygous for hemoglobin new mexico, with . % hb new mexico and . % hba, and . % hba . there was no evidence of hbs. when evaluating patients with polycythemia, maintaining a high index of suspicion for high affinity hemoglobinopathies may eliminate further unnecessary and invasive testing for patients. caution should be used when using hemoglobin electrophoresis testing since hb new mexico is known to migrate similarly to hbs on hplc with minimal change that may not be detected in regular laboratories. most high affinity hemoglobinopathies are reported to not have significant symptoms. in this case, our patient complains of fatigue, occasional palpitations and plethora of hands and feet. we will need to further follow this patient for possible attributable symptomatology. divya keerthy, simone chang, warren alperstein, patricia delgado, claudia rojas, ofelia alvarez, matteo trucco university of miami jackson memorial hospital, miami, florida, united states background: improved technology is enabling detection of previously unidentified translocations and mutations in otherwise unclassified sarcomas. one such mutation is the bcl- co-repressor -internal tandem duplication (bcor-itd) allowing for the new classification of bcor positive undifferentiated round cell sarcomas (urcs). this sarcoma has a similar appearance to clear cell sarcoma of the kidney (ccsk), potentially representing an extra-renal manifestation of this tumor, but their clinical pathologic features are not identical. objectives: this case highlights how recombinant polymerase chain reaction (rt-pcr) and bcor immunohistochemical staining can ease the diagnosis of this rare sarcoma. results: a month-old female presented for right sided pre-septal cellulitis and a temporal subcutaneous mass. the detection of multiple other subcutaneous nodules on exam raised the concern for malignancy and she was admitted for evaluation. she had two subcutaneous masses on her abdomen, with more cutaneous masses on her legs, back, shoulder, cheek and submandibular areas. she lacked spontaneous lower limb movement and had bilateral clonus. imaging confirmed multiple masses throughout the body including paravertebral area from t to l , bilateral adrenal glands, left kidney and muscles of upper and lower extremities. initial differential included neuroblastoma, infantile myofibromatosis, rhabdomyosarcoma or atypical presentation of a renal tumor. however, synaptophysin and chromogranin stains were negative. with standard immunohistochemistry, the tumor could be only broadly classified as "undifferentiated sarcoma" maintaining the diagnostic challenge. using rt-pcr in the setting of a morphologically primitive round cell neoplasm with strong bcor expression, two external institutes simultaneously diagnosed the tumor as bcor-urcs. the primary lesion is unknown but potentially may have arose from the kidney. bcor-urcs has a heterogeneous histology with tumor cells appearing monomorphic in nests of - cells separated by septa with uniform nuclei. there is frequently an "orphan annie eye" appearance and sparse cytoplasm to the cells. diagnosis cannot be made solely on evaluation of this nonspecific histology. rt-pcr uses the genetic abnormality in undifferentiated sarcomas to narrow the differential and bcor immunohistochemical staining provides further context. bcor has significant diagnostic value given its sensitivity and specificity in urcs. another potential marker includes ywhae-nutm b fusions, which occur in smaller subset of cases, but requires further study. rt-pcr has helped further classify tumors leading to the diagnosis of a rare undifferentiated sarcoma with bcor overexpression. while this technology is beneficial, its availability is limited. if accessibility improves, earlier identification and treatment may be possible maximizing the chance for a positive outcome. background: hematohidrosis is a rare condition that mimics bleeding disorders. cases present with oozing blood tinged fluid from various sites like eyes, ears, nose, skin, etc. reported causes of this condition were stress or fear, physical activity, psychological disorders. the condition is self-limited and don't affect the general condition of the patients, but it may contributes to psychosocial problems and may increases their stress and anxiety. so this condition needs to be promptly treated. to test the response of this disease and the associated headache to propranolol treatment. design/method: our case female patient years old st offspring of non consanguineous marriage, was admitted with recurrent episodes of oozing blood tinged fluid from eyes, ears and nose months before admission, about . - ml from each orifice, lasted - minutes and subsided spontaneously. it could involve the sites simultaneously or - sites. the number of attacks was - times per day then gradually increased to - times per day. later on the patient developed a bleeding attack from umbilicus. these attacks were aggravated by stress and physical activity and decreased with rest and sleep. the condition was associated with severe headache involving the whole head, throbbing in nature of gradual onset, increased by physical activity and relieved by analgesics. the condition was not associated with vomiting, blurring or diminution of vision, ocular pain, eye discoloration. no earache, tinnitus or diminution of hearing. there was no other form of discharge from eyes, ears or nose. no history of ecchymotic patches, bleeding from other orifices or blood product transfusion. no history of trauma, drug intake, fever or rash. no symptoms of other system affection. past history of recurrent attacks of epistaxis and two operations were done that passed without remarkable bleeding. no similar condition in the family physical examination was free, no evidence of psychological problems. complete blood count, coagulation profile, platelets function, factor and c.t brain were normal. oozing fluid from the patient was analyzed showed the same components as blood. results: our case started oral propranolol . mg/kg/day based on its use in similar cases in literature. the frequency of attacks and headache reduced then stopped after months of treatment and didn't recur after stoppage of propranolol. propranolol can treat this condition successfully. further investigations are needed to determine the link between this condition and severe headache our case was suffering from. background: wilms tumor is the most common renal solid tumors of childhood and is derived from primitive metanephric cells located in the kidney. primary extra-renal wilms tumors (erwt) are extremely rare, estimated to comprise . - % of all wilms tumors. despite similar histologic appearance intrarenal and erwts differ in embryologic tissues of origin. erwts arise from the more primitive mesonephric or pronephric origin and, therefore, can develop anywhere along the craniocaudal migration pathway of these primitive tissues, most often retroperitoneal, inguinal/genital, lumbosacral/pelvic and mediastinal. these tumors are typically staged and treated per national wilms tumor study (nwts) guidelines, and, by definition, are stage ii or greater due to location beyond the kidney borders. based on the cases reported in the literature, outcomes for erwt are comparable to renal wilms tumors with an % local recurrence rate and an % two-year event-free survival. we report the first case of a stage iii testicular extrarenal wilms tumor in an -month-old male with an intrabdominal undescended testis who underwent complete surgical excision followed by chemotherapy and inguinal radiation. results: a full term -month old male underwent orchipexy for an undescended left testicle. the testicle was noted to be grossly abnormal with a pea-sized thickened tissue adherent to the upper pole and a separate mass outside of the scrotum on the superior epididymis. both masses were removed, and s of s pathology demonstrated wilms tumor with favorable histology and negative margins. ct imaging of the chest, abdomen and pelvis were negative for a primary renal tumor, local residual disease, pathologic lymph node enlargement or distant metastases. the tumor was classified per nwts as stage iii due to tumor removal in multiple pieces. the patient completed dd- a treatment with vincristine, doxorubicin and dactinomycin per aren with cgy left inguinal radiation. he is currently months off therapy without clinical or radiographic evidence of recurrent disease. primary erwt is an extremely rare malignant neoplasm associated with challenges in diagnosis, staging and treatment. based on the cases reported in the literature, outcomes are similar to that of intrarenal wilms tumor. there are four pediatric paratesticular wilms tumors reported in the literature and, to the best of our knowledge, this is the first case of stage iii testicular wilms tumor successfully treated with dd- a chemotherapy and radiation. in erwt, nwts guidelines for staging and treatment should be applied with evaluation of both kidneys to exclude an intrarenal primary tumor. background: patient is a yo f, with esrd secondary to atypical hus versus ttp, who presented with thrombotic microangiopathy, aki, thrombocytopenia and anemia after a living unrelated donor kidney transplant. patient initially had downtrending creatinine. on post-op day , hematology was consulted for an increasing ldh and drop in platelets. peripheral smear was notable for an absence of schistocytes. yet, biopsy of the kidney revealed microthrombi. the patient was diagnosed with a thrombotic microangiopathy. plasmapharesis was initiated on day # , at which time ms r was noted to have significantly elevated creatinine. plasmapharesis did not yield any correction in labs and significant bruising developed. patient was started on eculizimab; plasmapharesis was stopped. shortly after, creatinine, anemia and thrombocytopenia corrected to levels at which she was discharged. overall, patient was found to have progressive anemia, thrombocytopenia, an increasing creatinine and ldh ( s) concerning for atypical hus, despite absence of schistocytes on peripheral smear. she responded well to eculizimab, with correction of hematologic changes during induction. she was discharged on eculizimab and continued to respond with normalizing platelet counts and hemoglobin. the differential in light of patient's thrombotic microangiopathy and thrombocytope-nia also included ttp. yet, adamts remained normal. dic was unlikely given normal fibrinogen level and d-dimer. objectives: presentations of atypical hus vs ttp. discuss eculizumab as a treatment of atypical hus. highlight atypical presentations of illness in transplant patients. results: despite absence of schistocytes by smear, pt was diagnosed with atypical hus based on presentation and after failing plasmapharesis, she responded well to eculizumab. though her presentation was abnormal, her response to this antibody that blocks the complement cascade suggests that she was experiencing a complement-mediated process. there are rare documented cases in the literature of atypical hus without schistocytes. hemolytic uremic syndrome (hus) is characterized by hemolytic anemia, thrombocytopenia and acute kidney injury. atypical hus is a diagnosis of exclusion, not due common etiologies such as shiga toxin. among atypical causes are complement-mediated forms, caused by an antibody to complement factor. in addition to plasmapharesis, renal transplant and supportive care, the mainstay of treatment for atypical hus is eculizumab (an antibody that blocks the complement terminal cascade). this case describes a patient unique in that, she was diagnosed with atypical hus without any schistocytes by smear. secondly, she responded to eculizumab, with unremarkable gene studies. finally, this case highlights that transplant patients often have unique presentations. nicklaus children's hospital, miami, florida, united states background: synovial sarcoma is a spindle cell tumor categorized as a soft tissue sarcoma. the chromosomal translocation t(x; ) leading to the ss -ssx fusion protein is unique to this sarcoma. it is a slow growing tumor with common recurrences and often, at presentation, with evidence of metastatic disease. if resection is not feasible, then neoadjuvant with adjuvant chemotherapy is recommended. metastasis carries an unfavorable prognosis given synovial sarcoma historically does not respond well to chemotherapy. trabectedin is a well-tolerated alkylating agent currently indicated for the treatment of liposarcoma and leiomyosarcoma. we present a -year-old male with metastatic synovial sarcoma to the lungs that progressed and was refractory to chemotherapy. he was administered trabectedin as a form of palliative chemotherapy, with significant clinical and radiographic response. design/method: pubmed search was done with search for terminology including "synovial sarcoma" and "trabectedin". papers relevant to our case were selected for literature review. a -year-old male patient presented with a large right axillary mass. initial imaging showed a heterogeneous multiseptated mass invading the subscapularis and teres major muscles along with innumerable lung nodules. biopsy confirmed diagnosis of monophasic synovial sarcoma. the patient was started on protocol arst with ifosfomide, mesna, doxorubicin. he completed cycles followed by radical resection and sessions of radiation. due to progression of disease multiple chemotherapy regimens were tried including topotecan and cyclophosphamide, protocol advl with lorvotuzumab, and pazopanib. imaging of the chest continued to show significant progression of metastasis. the patient's clinical status deteriorated with worsening respiratory status, requiring l of oxygen therapy, and inability to ambulate. he was started on trabectedin . mg/m for palliative care. after cycles of treatment patient was no longer requiring oxygen and was ambulating without assistance. radiological imaging showed significant reduction in number and size of lung nodules. trabectedin is a recently approved alkylating agent for the management of sarcomas resistant to first line treatment. response in synovial sarcoma is scarcely documented in the pediatric population. epidemiology places the most common age group in the young adults and children. our case opens the doors to further consideration of the use of trabectedin in the pediatric patient with metastatic synovial sarcoma. background: gata is an x-linked gene that plays critical role in hematopoiesis. mutations of gata gene can be associated to various blood disorders including diamond blackfan anemia, cytopenia, congenital dyserythropoietc anemia and acute megakaryoblastic leukemia. we report a patient with macrocytic anemia and platelet dysfunction who carries a novel gata mutation that has not been reported. results: a now -month-old male with complex medical history including prematurity at weeks, dysmorphic features, global developmental delay, hyperinsulinism, hypogonadotropic hypogonadism, growth hormone deficiency, micropenis, failure to thrive, patent ductus arteriosus status post ligation, and severe hypotonia, was referred to hematology at months old for resolved, transient thrombocytopenia and macrocytic anemia since month of age. chromosomal microarray showed chromosome deletion of q . , which is the rps gene. he doesn't have a family history of diamond blackfan anemia (dba), despite mom having the same rps mutation. he was then diagnosed with dba. his lab workup showed mild macrocytic anemia (hgb . g/dl, mcv fl), normal to inappropriately low reticulocyte count, normal white blood cell and platelet counts, hgf %, erythroid ada . eu/gm hgb (elevated). he has abnormal pfa- , with prolonged closure time of both adp and epinephrine. he had low von willebrand antigen and ristocetin cofactor activity. he has severe pancreatic insufficiency. bone marrow biopsy showed normocellular marrow with trilineage hematopoietic maturation, without ringed sideroblasts. since mother has the same rps gene mutation, maternal labs were done and showed no evidence of macroytosis or anemia. the diagnosis of dba was questioned. whole exome sequencing did not identify any pathogenic sequence changes in the coding regions of rps gene, but detected a gata mutation r w, which was reported variant of uncertain significance. his mother shares the same mutation and is asymptomatic, but she may not be affected since gata iis xlinked. his father doesn't harbor the gata mutation. conclusion: gata gene encodes zinc finger dna binding hematopoietic transcription factor, which is important during erythroid differentiation. gata mutation r w has not been reported in literature and is a novel variant of gata mutation, which might be contributing to this patient's clinical picture. further studies are warranted to confirm gata mutation r w to be a pathogenic sequence change. alexander boucher, tomoyuki mizuno, alexander vinks, greg tiao, stuart goldstein, james geller cincinnati children's hospital medical center, cincinnati, ohio, united states background: hepatoblastoma (hb), the most common pediatric primary hepatic malignancy, can be associated with specific congenital syndromes. recently, chronic kidney disease and genitourinary anomalies have been linked to hb. cisplatin is a key chemotherapeutic agent in treating hb but its renal clearance and toxicity profile can limit its use for those with end-stage renal disease (esrd). objectives: using an institutional case series, we present data using cisplatin for hb in dialysis-dependent esrd and define recommended dosing for future use. design/method: a chart review of patients with concurrent hb and esrd on dialysis treated with cisplatin at our institution was undertaken. demographic data, diagnostic history, tumor pathology, alpha fetoprotein (afp), hearing assessments, dosing schema, treatment outcomes, and therapyrelated toxicities were reviewed. total cisplatin levels were collected at time points within days after each infusion. free cisplatin levels were also collected for infusions, as were dialysate cisplatin levels. pk parameters were generated using bayesian estimation with a published population pk model as a priori information. results: three patients meeting these criteria were identified. each had "low risk" (non-metastatic resectable) disease at presentation and underwent upfront resections. all had congenital renal anomalies with esrd prior to their hb diagnosis. all cisplatin infusions were given over hours, followed hours later by hemodialysis. patients and received cisplatin at % of children's oncology group's ahep weight-based dosing ( . mg/kg). patient received % of ahep body surface area-based dosing ( mg/m ) during cycle but required a second dose reduction ( mg/m ) for cycle due to prolonged cisplatin exposure (total area under the curve mg⋅h/l; average for all seven evaluable cycles mg⋅h/l) and early sensorineural hearing loss at - hz. no other hearing loss in any patient was identified; mild toxicities also included grade - emesis and grade neutropenia and thrombocytopenia. the median (range) of clearance, volume of distribution at steady-state, and elimination half-life at terminal phase for total platinum were . ( . - . ) l/hour/ kg, . ( . - . ) l/ kg and ( - ) hours, respectively. patients and received cycles with rapid afp normalization. patient required an additional cycles, for a likely second primary hb year after initial therapy. cisplatin can be used successfully in pediatric patients with esrd on hemodialysis to treat hb with minimal morbidity using % standard mg/kg-based dosing ( . mg/kg), achieving pharmacologically appropriate cisplatin exposures. background: treatment for immune thrombocytopenia (itp) has been grouped into rescue and maintenance therapy and often is reserved for patients with bleeding, severe thrombocytopenia, or for improvement in quality of life. splenectomy is considered one of the more invasive but definitive treatments with success rates of - %. treatment of itp can be more difficult in the setting of previous treatment with immune modulation or when the patient is immunocompromised and not a candidate for splenectomy. objectives: present an interesting case of a patient with an autoimmune disease that presented with severe thrombocytopenia, un-responsive to rescue therapy, and requiring emergent splenectomy in the setting of acute intracranial hemorrhage (ich). a year old female with a history of juvenile dermatomyositis presented with a fine purpuric rash on her extremities, wet purpura, and a platelet count of k/ l. bone marrow evaluation at that time was consistent with itp. she was on cyclosporine and plaquenil for dermatomyositis. platelets failed to increase after three doses of intravenous immunoglobulin and high dose steroids. following a two week course of oral prednisone and eltrombopag, she presented with persistent severe thrombocytopenia of k/ l, anemia of . g/dl, and a lower gi bleed. she was started on amicar, novo-seven, rituximab, and given platelet transfusions with no improvement in bleeding. subsequently, she developed a subdural hematoma with midline shift. surgery performed an emergent open splenectomy with concurrent continuous platelet transfusion. results: she was monitored closely post operatively and, due to ich, transfused to maintain platelets greater than k/ l. by week post-op she had normal platelet counts off transfusions. all medications were stopped within three days of discharge. she represented eight days later with abdominal pain and thrombocytosis and was found to have a portal vein, splenic vein and mesenteric vein thrombosis. she was started on lovenox therapy and admitted for monitoring due to her history of ich. it is unknown whether our patient's underlying immune dysregulation and history of treatment with immunosuppressive medications may have contributed to her unresponsiveness to multiple therapeutic agents. in addition, her significant bleeding did not allow us to fully evaluate her response to second tier therapy. this adds to the scarcity of literature of itp response in pediatric patients with autoimmune disease, and may support more aggressive therapy upfront in these patients. background: multivisceral organ transplantation involves concurrent transplantation of the stomach, pancreas, liver, and intestine with splenectomy, and has been classically used in the pediatric population for infants with intestinal failure from disorders affecting foregut integrity. while there is some data demonstrating its efficacy in adults with low-grade abdominal malignancies, it has not been traditionally used for hepatocellular carcinoma treatment. to describe a unique pediatric case of multivisceral organ transplantation as definitive therapy for refractory fibrolamellar hepatocellular carcinoma in an adolescent male. a year old male presents with a history of fibrolamellar hepatocellular carcinoma, tumor invasion of the portal vein, severe portal hypertension complicated by bleeding esophageal varices and hypersplenism. he had two treatments with yttrium- radioembolization, without significant response. he completed six cycles of traditional chemotherapy in combination with sorafenib with resolution of petavidity, but minimal decrease in tumor size and continued portal hypertension. since his disease remained relatively stable for over years, he was evaluated and listed for multivisceral organ transplantation. at approximately years and months after diagnosis, he underwent en bloc liver, pancreas, stomach, small bowel, and colon transplant with splenectomy. a single lymph node was positive for malignancy at the time of resection. in addition to expected post-transplant complications, he also developed skin only acute graft versus host disease at weeks after transplant, treated successfully with a thymoglobulin course. he clinically improved and was back to his baseline activity level, on full oral feedings within months post-transplantation. at three and six month post-transplantation, there is no concern for relapsed hepatocellular carcinoma on comprehensive imaging and evaluation. he is maintained on protocol immunosuppression and posttransplant support. we present the first known case of successful multivisceral organ transplantation in the treatment of refractory pediatric fibrolamellar hepatocellular carcinoma. background: hematohidrosis is a rare disorder that presents with spontaneous excretion of whole blood from intact skin or mucosa. diagnosis is based on clinical observation of the occurrence with the proven presence of erythrocytes and other blood components, without other abnormalities to account for the phenomenon. the existing literature is scarce and consists of primarily case studies. most reports describe bleeding from facial sites around the eyes, ears, and nose. the available literature suggests anxiety and physical or emotional stress reactions as the most common inciting events. little evidence exists regarding the ideal therapeutic approach, however propranolol has been used successfully to reduce bleeding frequency and severity in multiple case reports. a specific genetic etiology has not been elucidated, and no familial cases have previously been reported. we present a pair of half-siblings, both of whom presented with spontaneous cutaneous and mucosal bleeding before two years of age, and report on preliminary results of propranolol therapy. tanzania. at months of age, he became ill and developed spontaneous bleeding from his ears, nose, and scalp. he continued to have frequent bleeding episodes, usually related to illness or physical distress. a bleeding diathesis work-up was unremarkable, however some episodes were severe enough to require transfusions. the patient was subsequently diagnosed with hiv and hepatitis b, presumably acquired via unscreened blood product transfusions. patient b is an infant female born to the same mother as patient a, with a different father. she was healthy until two months of age when she developed spontaneous bleeding from the hairline, eyelids, ears and genital/rectal area. bleeding episodes were nearly always associated with irritability and crying. extensive coagulation workup was unremarkable. results: propranolol therapy was started in both patients, titrated to a goal of mg/kg/day. in both patients, the frequency and duration of bleeding episodes significantly improved. patient b continues to have milder occasional bleeding episodes from her eyes, ears and scalp but has significantly less discomfort and irritability during the episodes. conclusion: to our knowledge, there are no prior reports involving two related patients with hematohidrosis. this case series suggests that there may be a genetic predisposition which has yet to be identified. propranolol has shown effectiveness in reducing symptom frequency and severity. background: gliomas are the most common central nervous system tumors in children. they are classified into different grades based on genotype (idh, braf, tsc, etc.). lowgrade gliomas such as oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas are classified as grades i and ii. of the molecular level alterations this case report focuses on the braf v e mutation. braf is a member of the raf family of serine/threonine protein kinases and it plays an important role in cell survival, proliferation and terminal differentiation. objectives: here we discuss two cases where dabrafenib, a braf kinase inhibitor, was utilized in the management of gliomas. the cases focus on the use of dabrafenib late versus early in disease course. design/method: patient jl is a year old female who was diagnosed with a low-grade glioneuronal tumor (c -t with a metastatic lesion to the brain) in . jl was treated with chemotherapy, radiation, and surgical resection. despite treatment, the patient's disease progressed. she developed lower extremity dysfunction, urinary incontinence, poor truncal control, and hydrocephalus. dabrafenib was started after the braf v e mutation was confirmed. patient lg is a year old female who presented in november with left facial and upper extremity weakness. ct and mri scans demonstrated a mixed solid and cystic lesion extending from the optic chiasm and hypothalamus to the right thalamus and posterior basal ganglia with additional involvement of the right cerebral peduncle. neurosurgical intervention was undertaken and dabrafenib was started after the braf v e mutation was confirmed. results: patient jl's mri scans have demonstrated improvement of the spine with diminished areas of enhancement along thecal margins, decreased volume and enhancement within the trigeminal plate cistern and resolution of ependymal enhancement within the right ventricle. the patient's most recent mri exhibits no disease progression in head or spine. jl has shown improvement clinically since starting dabrafenib. patient lg has shown improvement in strength and recent mri of the brain has shown resolution of enhancement along surgical resection margins, decreased hyperintensity along the inferomedial aspect of the right basal ganglia and no new enhancements. conclusion: low grade gliomas can alter a person's quality of life and even lead to life threatening complications. often the standard chemotherapy, radiation and surgery don't prevent these complications. genetic analysis can help clinicians target therapy towards certain mutations such as braf v e. dabrafenib has shown to decrease tumor burden, early utilization as therapy can help prevent morbidity and mortality. children's hospital of pittsburgh of upmc, pittsburgh, pennsylvania, united states background: copper is an essential cofactor in enzymatic reactions essential to proper hematologic, skeletal, neurologic and vascular function. copper requirements in children over the age of are mg/day, which is readily acquired in a typical diet. copper deficiency is known to occur in patients with the rare x-linked mutation and in older individuals with gastrointestinal bypass surgery; however, it is rarely reported in other conditions. objectives: to highlight individuals with autism spectrum disorders or developmental delay with a limited dietary repertoire are at risk for copper deficiency, thus a high index of suspicion must exist in order to diagnose the disorder. design/method: a y/o boy with a prior diagnosis of global developmental delay and oral aversion presented with slowly progressive fatigue, weakness, gait instability, and weight loss. his longstanding feeding difficulties were refractory to intensive feeding programs. his daily diet consisted of - oz of milk and - individual servings of butterscotch pudding ( - calories/day, . mg iron/day). initial complete blood count demonstrated white blood cell count of . , absolute neutrophil count of , hemoglobin of . , mean corpuscular volume of < , reticulocyte count of . , platelet count of . review of his peripheral blood smear revealed microcytic, hypochromic red cells without marked fragmentation, anisopoikilocytosis and ringed sideroblasts; there were no morphologic abnormalities of his leukocytes or platelets. iron studies demonstrated ferritin of , total iron binding capacity of , and % iron saturation. he had no evidence of b , folate deficiency or blood loss. additional evaluation revealed a serum copper level of (range - ), and cerulosplasmin of . (range - ). results: once a diagnosis of copper deficiency was made, the patient promptly began a course of parenteral copper repletion. he received iv copper mcg/kg/day x days then weekly intravenous infusions. given his malnutrition, a gtube was placed to begin oral copper repletion and enteral nutrition. within weeks his copper level improved as well as his blood counts. unfortunately, although his blood counts and copper levels normalized, his neurologic status remains below his old baseline although, he has made gains in his gross and fine motor abilities. conclusion: acquired copper deficiency in the pediatric population is a rare event but given the hematologic and neurologic consequences, prompt recognition and treatment is important. this patient's clinical course demonstrates the need to have a high index of suspicion of concomitant nutritional deficiencies other than those routinely evaluated such as iron, b and folate. background: lymphoepithelioma-like thymic carcinoma (lelc) is a rare, aggressive neoplasm with a high rate of invasion, metastasis and recurrence. there are no known curative therapies for metastatic lelc. we report the case of a -year-old male who presented with metastatic ebv positive lelc. sites of disease included a large primary anterior mediastinal mass and metastases to hilar lymph nodes, lungs and liver. he was initially treated with cisplatin and fluorouracil followed by mediastinal radiation. he had a partial response to therapy but his end of therapy scans showed disease progression in lungs, liver, and hilar, supraclavicular and axillary lymph nodes. objectives: molecularly targeted therapies tailored to the patient's genetic profile offer a novel approach to obtain improved survival outcomes. design/method: the patient enrolled on a precision medicine trial, nmtrc : molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancer (nct ). in this study, tumor/normal whole exome sequencing and tumor rna sequencing were performed and a molecular report detailing the results of genomic and gene expression analysis was generated. a treatment plan was designed within a molecular tumor board comprising oncologists, pharmacists, genomicists, and molecular biologists with domain expertise. results: exome sequencing revealed somatic coding point mutations and no structural mutations (focal copy number changes or translocations). candidate somatic driver mutations included tp s x and r w as well as kit n k. both genes have been previously implicated in thymic carcinoma. rna expression analysis demonstrated aberrant activation of biological pathways, including overexpression of kit, hdac , and , tyms, and dhfr. the molecular tumor board selected the combination of pemetrexed ( mg/m ) on day of a day cycle, imatinib ( mg daily), and vorinostat ( mg days - , - , and - ) . on day of cycle , he was admitted with a herpes zoster infection and imatinib was discontinued in order to reduce risk of herpes zoster recurrence. imaging after cycles showed a complete metabolic response on f- fdg pet and a partial response by ct size criteria. as of december , the patient had received cycles of pemetrexed and vorinostat. scans in december showed an increase in the size and metabolic activity of two right lower lobe pulmonary nodules. there were no new sites of disease and imatinib was re-started. background: systemic lupus erythematosus (sle) is a chronic autoimmune disease that affects multiple organ systems and is associated with many different autoantibodies. patients can present with vague constitutional symptoms including fever, rash, fatigue, and weight loss. some of the various hematologic manifestations of sle include anemia of chronic disease, leukopenia, autoimmune hemolytic anemia (aiha), and idiopathic thrombocytopenic purpura (itp). these can be the presenting signs of sle. evans syndrome (es), a disease characterized by itp and aiha, is a rare hematologic manifestation of sle. neurofibromatosis (nf ) is a relatively common neurocutaneous disorder. these patients are at risk of developing benign and malignant tumors. its association with autoimmune disorders, including sle, remains rare. objectives: there are few cases in the literature that have patients with the combination of sle and nf . this is the only case that has a patient with sle, nf , and es. results: a -year-old caucasian female presents with two months of vaginal bleeding, weight loss and petechiae. her exam is remarkable for petechiae and café au lait macules. laboratory findings show severe anemia and thrombocytopenia. she receives blood and platelet transfusions during stabilization, and a bone marrow aspirate is performed to rule out a malignancy which is negative. based on the presence of thrombocytopenia and a positive coombs test, an autoimmune process such as es is considered. screening tests for sle reveal positive antinuclear and anti-double stranded dna antibodies as well as low complement. she receives intravenous immunoglobulin and methylprednisolone and eventually her vaginal bleeding slows and her counts recover. she begins sle therapy with hydroxychloroquine and azathioprine. due to the presence of café au lait macules on her exam, a genetics evaluation is performed and the patient is also diagnosed with nf . to date, there are seven cases of sle with nf reported in the literature, only two of which are pediatric cases. there are no reports of the combination of sle, nf , and es. conclusion: es is a rare hematologic manifestation of sle but can be the initial presentation of this disease. one large study estimates % of childhood-onset sle cases are observed to have es. screening for sle should be considered in all es patients even in the absence of typical clinical findings. association of nf and sle has been rarely described. whether this association reflects a causal relationship or is coincidental needs more investigation. (lube, ped blood & cancer, ) . university of california san diego, la jolla, california, united states background: high grade glioma (hgg) has poor outcomes in adults and children. extraneural metastases are very rare in hgg, and poorly characterized with only a few small case series in adults and only isolated case reports in pediatrics. no genomic data has previously been published for any children with hgg who develop extraneural metastases. objectives: our objective is to describe the natural history of two children with hgg and bony extraneural metastases, comparing their clinical characteristics as well as whole exome sequencing data for both tumors. this information would suggest similar patients should be monitored closely for extraneural metastasis and may benefit from more systemic therapy. design/method: we present a case series of two patients who presented with hgg and had development of bony metastases less than six months after initial diagnosis. both patients had molecular profiling with whole exome sequencing (wes). the first patient was an -year-old male with a tumor found in the left lateral ventricle invading into the fornices, hypothalamus, and left midbrain, who had subtotal resection. bony metastasis were found at . months after diagnosis, and he died months after diagnosis. he initially received radiation, followed by nivolimuab. the second was a -year-old female with a tectal/pineal tumor and multiple spinal cord metastases, who had subtotal resection. she developed bony metastasis at . months after diagnosis and died months after diagnosis. her histologic diagnosis was pineoblastoma, revised to hgg after whole exome sequencing. she received craniospinal radiation followed by chemotherapy per acns (cisplatin, vincristine, and cyclophosphamide) for cycles. when she failed to respond satisfactorily to this therapy, wes of tumor was performed and the findings were consistent with hgg. treatment was transitioned to temodar and lomustine after hgg diagnosis was given. she had ongoing progressive disease despite this therapy as well as trials of nivolumab, everolimus, and vorinostat. neither patient had extraneural metastasis at presentation. in both tumors, whole exome sequencing identified the h f a k m mutation. both tumors also had additional known mutations associated with hgg but no other overlapping mutations. this case series represents the first description of the genetic alterations of pediatric hgg patients who developed extraneural metastases. while h f a k m is a common mutation in pediatric midline hgg, especially dipg, and is associated with more aggressive disease, there has not been an association with extraneural metastasis prior to this series. background: deferiprone-induced agranulocytosis is a well -known albeit rare side effect of the drug. incidence of agranulocytosis varies from . - . %, while milder neutropenia is reported in . % of patients treated with deferiprone. deferasirox is unknown to cause such a complication. clinical trials and post marketing side effect monitoring studied possible correlations between different risk factors and development of agranulocytosis. unfortunately, no studies directly addressed a special risk in a community with background of ethnic neutropenia, like oman. objectives: to report on the incidence of neutropenia among omani children with b thalassemia using different iron chelators design/method: a retrospective study conducted on patients < year-old with b thalassemia treated with different iron chelators. electronic patients records were reviewed to detect episodes of neutropenia either mild (anc . -< . /cmm), moderate (anc . -< ), severe < . , or agranulocytosis anc = ). data were collected including sex, age, personal or family history of ethnic neutropenia, iron chelating agent, infective complications, management and outcome. detailed clinical, laboratory ± radiological information were reported for patients who developed life-threatening agranulocytosis. among young patients with b thalassemia, treated between - in squh, neutropenia, was reported in patients ( . %).severe neutropenia was encountered on occasions in patients ( / : . %) ( on deferiprone including episodes of agranulocytosis, on defersirox, on combined chelation, and off chelation). moderate neutropenia was encountered in patients ( / : . %), on occasions: deferiprone ( ), deferasirox ( ), combined chelation ( ), and episodes off chelation. mild neutropenia was more prevalent, encountered in patients ( . %) on occasions ( on deferiprone, on defersirox, on combined chelation, and off chelation) of patients exposed to deferiprone, patients had neutropenia ( %), higher than previously reported. deferiproneinduced agranulocytosis was encountered in patients ( / = . %). three of them had life threatening complications. one patient developed pneumonia complicated by rupture of pulmonary artery aneurysm-massive hemoptysis, who recovered fully after catheter embolization. the second had facial cellulitis and treatment with gcsf was complicated by frequent ventricular extrasystoles. the third had sepsis, disseminated herpes simplex and required admission to icu for inotropic support. in a community with background ethnic neutropenia, neutropenia is more common to be encountered among thalassemic patients, both on and off chelation therapy. careful monitoring of anc and rational choice/modification of chelating agents is required for optimal management of iron overload and to avoid life threatening complications. objectives: this case control study aimed to evaluate the systolic and diastolic cardiac function in groups of children with ti: non transfused group and a group that received early regular blood transfusion comparing them to healthy controls. design/method: thirteen regularly transfused patients with ti with a mean age of . + . years were compared with eight patients who are non-transfused or minimally transfused (< rbcs transfusion/year); mean age . + . years and healthy controls with a mean age of . ± . years. clinical parameters and standard echocardiographic and tissue doppler imaging (tdi) were compared. results: young non-transfused ti patients had a statistically significant higher peak late diastolic velocity of the left ventricular inflow doppler, a mitral valve a wave duration over the pulmonary vein a wave duration ratio and the pulmonary s of s vein s/d velocities ratio compared to the transfused group with p values of . , . , . respectively. in addition, they have a lower e/a ratio of the mitral valve inflow and a larger left atrial to aortic diameter ratio compared to the control group with p values of . and . respectively. the diameters of the right and left outflow tract were significantly larger in the non transfused group with a trend to have a higher cardiac index compare to the transfused group. systolic function was similar in the studied groups and none of the patients had evidence of pulmonary hypertension. young patients with ti who are receiving early regular blood transfusion have normal systolic function. diastolic function assessment revealed indicators of an abnormal relaxation of the left ventricle in the non transfused group which indicate diastolic dysfunction. the abnormalities affected multiple diastolic function parameters which give an indication that the changes are clinically significant. a statistically significant increase in the diameters of the outflow tracts are likely attributed to high cardiac output status in nontransfused ti patients as they had a trend to have a higher cardiac index. these findings support the early commencing of regular blood transfusion therapy for ti patients to prevent serious cardiac complications in adult life. background: in the -week sustain study, crizanlizumab . mg/kg significantly reduced the frequency of scpcs versus placebo ( . vs . , p = . ) and increased the time to first on-treatment scpc ( . vs . months, p = . ) in patients with sickle cell disease (scd). to evaluate time to first scpc in sustain study subgroups and the likelihood of not experiencing scpc for the duration of the trial using post hoc analyses. design/method: sustain was a randomized, double-blind, placebo-controlled, phase study (nct ). inclusion criteria were: scd patients aged - years; - scpcs in previous months; concomitant hydroxyurea use permitted if ≥ months and stable dose for ≥ months. patients were randomized : : to receive intravenous crizanlizumab . mg/kg, . mg/kg, or placebo. study treatments were administered on days and , then every weeks to week , with the final assessment at week . median time to first scpc after first dose was summarized for crizanlizumab . mg/kg or placebo in these subgroups: - or - scpcs in previous months; scd genotype; and hydroxyurea use at baseline. hazard ratios (hrs) for crizanlizumab . mg/kg versus placebo were calculated based on cox regression analysis, with treatment as a covariate. descriptive statistics were used to summarize the frequency of patients who were scpc event-free for the duration of the study by prior scpc events, scd genotype, and hydroxyurea use at baseline. : patients received crizanlizumab . mg/kg and received placebo. there was a meaningful delay in time to first scpc with crizanlizumab . mg/kg versus placebo observed in the entire study population. the effect was present in both scpc subgroups, and the largest treatment difference was observed in hbss scd versus other genotypes ( . vs . months; hr: . ). in patients taking hydroxyurea who experienced - scpcs in the previous year, time to first onstudy scpc was longer with crizanlizumab . mg/kg versus placebo ( . vs . months; hr: . ). a greater proportion of patients treated with crizanlizumab . mg/kg were scpc event-free versus placebo in each of the analyzed subgroups. one third of patients who were taking hydroxyurea and treated with crizanlizumab . mg/kg were scpc event-free during the study versus . % with placebo, possibly suggesting an additive effect. with crizanlizumab . mg/kg, there was a clinically meaningful delay in time to first scpc and an increased likelihood of being scpc-free versus placebo in all subgroups investigated. cincinnati children's hospital medical center, cincinnati, ohio, united states background: shwachman-diamond syndrome (sds) is an inherited marrow failure syndrome associated with increased risk of myelodysplasia (mds) and acute myeloid leukemia (aml). objectives: this multi-institutional retrospective study investigated clinical features, treatment, and outcomes of sds patients who developed mds or aml by central pathology review. design/method: nine individuals presented with aml ( male, female), mds-eb / ( males, females, with mds ( male and female), and one male with isolated persistent somatic tp mutation. one mds-eb and mds patient progressed to aml. median age (years) at diagnosis of mds was (range . - ), mds-eb / was (range . - ) and aml was . (range . - ). complex cytogenetics were noted in / aml cases, with one having normal cytogenetics. complex clonal cytogenetic abnormalities were noted in of mds-eb /eb patients and clonal abnormalities in of mds patients. follow up was available for aml patients; are deceased. received chemotherapy with intent to proceed to hematopoietic stem cell transplant (hsct). four failed to achieve remission and died with disease without proceeding to transplant. one patient proceeded to hsct without prior chemotherapy. four of six transplanted subjects died with relapsed disease. treatment related mortality was largely infectious or gvhd. the sole surviving aml patient had normal cytogenetics, achieved remission with chemotherapy and underwent hscts with separate stem cell infusions due to two primary graft failures. he remains alive in remission more than years after diagnosis. of the mds-eb / patients, underwent ric hsct, three of whom are alive, one died of infection. the fifth patient has stable disease on continued decitabine monotherapy for . years. of mds patients with treatment data, had upfront hsct therapy, upfront chemotherapy and had no therapy. three patients required ≥ hscts all due to graft failure. follow up is available for , of whom are deceased, with relapsed disease. treatment related mortality was largely infectious or graft failure. one individual died of hepatic failure unrelated to mds. seven mds patients are alive in remission. in summary, prognosis is poor for patients with sds who develop aml due to resistant disease and treatment-related complications. better markers for risk stratification are needed to identify patients who would benefit from early transplant. novel therapeutic strategies are urgently needed to improve outcomes of sds patients with mds or aml. background: unlike primary myelofibrosis (pmf) in adults, which is associated with somatic mutations in jak , mpl, or calr, myelofibrosis in children is rare and the underlying genetic mechanisms remain elusive. here we describe families with autosomal recessive congenital macrothrombocytopenia with focal myelofibrosis (cmtfm) due to germline mutations in the megakaryocyte-specific immune receptor tyrosine-based inhibitory motif (itim) receptor g b-b. objectives: to characterize the clinical phenotype, histological features and identify the causative gene for cmtfm. we performed affymetrix snp . genotyping on the index family to identify shared regions of homozygosity by descent. whole exome sequencing (ws) was performed on all three pedigrees to identify potentially causative mutations. we studied affected children from families, with macrothrombocytopenia, anemia, mild leukocytosis and a distinctive pattern of bone marrow (bm) fibrosis centered around clusters of atypical megakaryocytes. affected children had mild to moderate bleeding symptoms and required platelet and red cell transfusions. none showed evidence of extramedullary hematopoiesis, and all were negative for mutations in jak , mpl, and calr. snp genotyping identified multiple statistically non-significant genomic loci, including the region of the major histocompatibility locus (mhc) on chromosome p (lod = . ). we focused on this region because affected individuals in two families shared a common homozygous human leukocyte antigen (hla) type and had congenital adrenal hyperplasia (cah) due to -hydroxylase (cyp a ) mutation; the cyp a and hla loci are located at p . and p . - p . . wes revealed homozygous frameshift mutations in the megakaryocyte and platelet inhibitory receptor g b-b, encoded within the candidate linkage region. we identified two distinct g b-b frameshift mutations (c. _ + dup; p. fs and c. inst; p. fs) in individuals within these three families. no other mutations that segregated with the phenotype were identified. to validate g b-b as a potential disease-causing gene, we evaluated g b-b expression in bm biopsy specimens from affected patient and control samples by immunohistochemical staining using a monoclonal antibody. g b-b was strongly s of s and selectively expressed in megakaryocytes of control samples, but completely absent in clinically affected individuals. a murine knockout that lacks g b-b has a strikingly similar phenotype with macrothrombocytopenia, myelofibrosis and aberrant platelet production and function, further affirming the causality of g b-b mutations. we showed that autosomal recessive loss-offunction mutations in g b-b cause cmtfm, uncovering the molecular basis of this rare disease. loss of g b-b-dependent inhibition of megakaryocyte activation likely underlies the distinctive focal myelofibrotic phenotype and might be important in other forms of marrow fibrosis. cardinal glennon, saint louis, missouri, united states background: intrauterine transfusion is the method of choice for management of fetal anemia due to red blood cell alloimmunization. despite the decrease in prevalence of anemia due to rhesus d alloimmunization with prophylactic administration of anti-rhd immunoglobulin in rh d negative patients, maternal red red blood cell alloimmunization with other type of red blood cell antigens remains an important cause of fetal anemia. newborn who received intrauterine transfusion for hemolytic disease may have prolonged postnatal transfusion requirement. objectives: -to evaluate clinical outcome of fetuses and newborns who received intrauterine transfusions. -to determine the need of packed red blood cell transfusions until months of age. we conducted a retrospective case series study of all intrauterine transfusions due to anemia secondary to red blood cell alloimmunization performed in our regional center ssm in st louis missouri, between april and january . we evaluated the indications, diagnosis, gestational age, and frequency of intrauterine transfusions, along with the infant's gestational age at birth, duration of admission, timing of blood transfusion and monitoring of hemoglobin. results: intrauterine transfusions were performed in patients. the most common causes of alloimmunization were due to d antibodies (n = , %) and kell antibodies (n = , . %). the median gestational age of the first intrauterine transfusion was . weeks, and the median pre-transfusion hemoglobin was . g/dl. the gestational age at the first intrauterine transfusions was found to be significantly correlated with the number of postnatal transfusions (r = . . p = . ). the median gestational age at birth was found to be weeks ( . - . weeks), with a hemoglobin of . ( . - . ). in our population, patients ( %) received postnatal transfusions, of which were during the first weeks of life, and close monitoring follow up with a hematologist was established in patients at their discharge from the nursery/nicu. one neonatal death occurred and severe morbidity due to severe anemia occurred in one infant. despite the continuing risk factor for persistent anemia, only patients had follow up hemoglobin monitored by their primary care provider. conclusion: infants with anemia due to red blood cell alloimmunization treated with intrauterine transfusion should be monitored closely via regular complete blood count for persistent anemia due to suppression of fetal erythropoiesis. sebastian hesse, piotr grabowski, juri rappsilber, christoph klein dr. von hauner childen's hospital, lmu university hospital, munich, munich, germany background: neutrophil granulocytes are the most abundant leukocytes in the peripheral blood. validated diagnostic options for these cells are limited, leaving many patients with functional neutrophil defects without a defined diagnosis. objectives: here we evaluate proteomics as a new diagnostic tool to investigate defects of neutrophil granulocytes. we analyzed neutrophil granulocytes from children with severe congenital neutropenia (scn) associated with elane mutations, children with chronic granulomatous disease (cgd) with cyba ( ) or cybb ( ) mutations and children with leukocyte adhesion deficiency (lad) due to itgb mutations. in addition we collected samples of children with genetically undetermined neutrophil defects. neutrophils from healthy individuals served as controls. cells were isolated from fresh venous blood using negative selection (purity > %). whole cell proteome analysis was done by data-independent acquisition. showed a correlation coefficient of ∼ . . principal component analysis demonstrated unequivocal separation of the proteome of healthy and diseased cells. differential expression analysis showed minimal proteome aberrations in lad with deficiency in cell surface receptors and upregulation of alpl (total downregulated proteins: / total upregulated proteins: ). analysis of neutrophils from cgd patients also showed limited proteome aberration. cyba and cybb were both diminished independent of genotype, whereas protein clusters around a stat / centered network were increased (total down: / up: ). neutrophils with elane mutations showed the gravest proteome disturbance (total down: / up: ) with an upregulated translational apparatus (srpdependent ribosomes and protein folding complexes) and increased mitochondrial proteins. proteins of each granule subset were dysregulated and metabolic pathways upregulated. a detailed analysis of the proteome from patients with genetically undefined diseases is currently ongoing. one patient with clinical phenotype of cgd was found to have no mutations of nadph oxidase members in whole exome sequencing but critically low levels of ncf on protein level. heterozygosity mapping showed autozytocity in the ncf region warranting current efforts to sequence promoters and intronic regions of the gene. mass spectrometry based proteomics promises exciting new insights into monogenic disease of neutrophil granulocytes and may offer new diagnostic options, in particular in synergy with genome sequencing. by virtue of our international care-for-rare alliance, open to new partners, we hope that our proteome focus may lead to better delineation of as yet unknown disease of neutrophil granulocytes. background: warm autoimmune hemolytic anemia (aiha) is an igg mediated disease. although it can be post-viral, it is often idiopathic and can also be a forme fruste for malignancy or an autoimmune disease. initial management includes steroids. it often relapses on steroid wean and can be refractory to the use of second line treatment such as rituximab. objectives: abatacept (ctla- -ig fusion protein, ctla- mimetic) has been used to ameliorate autoimmune manifestation associated with ctla- haploinsufficiency. we used abatacept as a novel therapeutic agent to manage patients with refractory aiha. design/method: a retrospective case series of two patients at phoenix children's hospital with severe refractory aiha. results: patient , a previously healthy year old female, presented with weeks of icterus, fatigue, and hemoglobinuria. spleen was enlarged cm below the costal margin. laboratory evaluation demonstrated: hemoglobin . g/dl, mild leukopenia /microliter, platelets , /microliter, reticulocytosis . %, positive direct coombs' test, mycoplasma igm and igg positive. bone marrow evaluation showed a hypercellular marrow. she continued to need packed red blood cell (prbc) transfusions despite receiving high dose steroids, ivig and rituximab from may-july . in august, she started sirolimus decreasing her transfusion requirement. after starting abatacept ( mg/kg/dose bi-monthly for three doses and then monthly) in october, she maintained hemoglobin of - g/dl without transfusion. patient , a previously healthy month old male, presented with one week of progressive fatigue, jaundice, and poor feeding. splenomegaly was absent. laboratory evaluation revealed hemoglobin . g/dl, leukocytosis , /microliter, platelets , /microliter, reticulocytosis . %, negative direct coombs' test, and non-specific reactivity on antibody screen. evaluation for inherited hemolytic anemia including a next generation sequencing panel was negative. further evaluation by blood bank showed + positive coombs' for c d due to a warm antibody. cold agglutinin disease was ruled out. bone marrow evaluation was normal. he received high dose ivig as a steroid sparing agent but continued to require prbc transfusions weekly. when prednisone did not seem to slow down hemolysis, treatment with abatacept was initiated and he has not required transfusions for two months. steroids are being weaned. we present successful treatment of two refractory aiha cases with abatacept. patient is steroid and transfusion free and continues on monthly abatacept and sirolimus. patient is also transfusion free and continues on a steroid taper. ctla- is crucial for suppressive function of treg cells. abatacept by binding to cd / seems to enhance treg activity ameliorating autoimmune hemolysis. children's minnesota, minneapolis, minnesota, united states s of s background: transfusional iron overload is common in patients receiving chronic red cell transfusions. as a result, iron chelation is required to minimize toxicity from iron overload. chelation with a single agent can be inadequate at controlling or reducing iron burden. when combination therapy is required deferoxamine may be added to oral chelation. deferoxamine is generally given subcutaneous over - hours for - days a week at - mg/kg/day. many patients struggle to remain compliant with this schedule which has prompted trials of intravenous high-dose (hd) deferoxamine. prior reports of short-term hd deferoxamine have shown minimal side effects however, prolonged use of hd deferoxamine has known toxicity. when compliance is a concern, our center has used hd deferoxamine infusions at mg/kg/hr x hours every to weeks. objectives: evaluate the safety and efficacy of hd deferoxamine at our institution to help guide future therapy. design/method: a retrospective review was completed of patients previously treated with hd deferoxamine between april and september at children's minnesota. final sample included patients ages to years with underlying diagnosis of thalassemia ( ) and diamond-blackfan anemia ( ). deferoxamine infusions were given for hours every - days with a mean length of treatment of days. results: all patients were on combination therapy with deferasirox, however deferasirox was held during deferoxamine infusion. mean pre-deferoxamine liver iron concentration (lic) was . mg/g and mean post lic was . mg/g (p = . ). ferritin mean pre-deferoxamine was ng/ml compared with mean post ng/ml (p = . ). two patients had possible allergy, leading to deferoxamine discontinuation. one patient developed hives, eye swelling and cough while the other had emesis and cough. another patient experienced facial nerve palsy of unclear etiology, which did not recur with resumption of deferoxamine. no respiratory complications were seen. results showed significant decrease in iron burden following combination therapy with high dose deferoxamine and deferasirox. no significant pulmonary, liver, renal, vision, or hearing toxicities were observed. three patients reported reactions to deferoxamine infusions. however, one of these was able to successfully continue deferoxamine without further incident. short-term, hd deferoxamine was effective at reducing lic in combination with oral chelation but requires further evaluation to assess for potential increased risk of toxicity. short-term hd deferoxamine may be considered in the setting of poor compliance of subcutaneous administration or inadequate chelation with single agent therapy. further studies are needed to clarify ideal dosing, timing and risk of toxicity. background: immune thrombocytopenia (itp) is the most common cause of symptomatic thrombocytopenia in childhood but remains a diagnosis of exclusion warranting further evaluation if atypical findings are present. two male children ( months and years old) with newly diagnosed immune thrombocytopenia (itp) were found on initial evaluation to have persistent elevations of lactate dehydrogenase (ldh), alanine aminotransferase (alt), and aspartate aminotransferase (ast). these serum enzyme abnormalities cannot be attributed to itp. in the setting of thrombocytopenia, elevated transaminases and ldh create diagnostic complexity for the hematology/oncology provider as their elevation raises concern for malignancy, hemolytic disease, and other systemic diseases. to raise awareness about an unexpected pattern of duchenne muscular dystrophy in patients undergoing evaluation for itp. to expand the differential of a hematologist/oncologist when abnormal labs support a nonhematologic diagnosis design/method: this case-series of two patients with their clinical and laboratory findings were discovered with retrospective chart review. results: after a thorough evaluation for hemolytic anemias, liver disease and infectious etiologies was negative, bone marrow and liver biopsies were considered. eventually, both children were found to have severely elevated serum creatine kinase (ck). skeletal muscle has the highest concentration of ck of any tissue. thus, significant ck elevation is almost exclusively attributable to muscle injury and is the most sensitive and specific enzyme for diagnosis of muscle disease. referral to a neuromuscular specialist and further genetic testing confirmed the diagnosis of duchenne muscular dystrophy in both children allowing initiation of appropriate interventions. to date, there is no clear genetic predisposition to itp in patients with muscular dystrophy although further investigation may be needed. hematology/oncology providers should consider obtaining a serum ck to rule out muscle disease in any male child with unexplained elevations of serum ldh and/or aminotransferases, as it provides an easy and inexpensive, non-invasive approach to screening. additionally, clinical history and physical examination can aid in the diagnosis of muscular dystrophy, with gross motor delay, abnormal muscle bulk, gower's sign, and proximal muscle weakness all possible findings. objectives: to identify the range of cbcs in patients with ds without infections, hematologic or immune disorders and to create more accurate reference ranges for total white blood count; hemoglobin; hematocrit; mcv; platelet count and absolute neutrophils (anc), lymphocytes, monocytes, eosinophils, and basophils. design/method: a retrospective investigation of healthy pediatric patients with ds who received a cbc between and as part of their medical care at a single, large, pediatric teaching hospital. the study group consisted of children with ds (male = , . %; mean age = . years, sd = . ) at time of blood draw. initially children were reviewed for possible participation in the study; however, patients were excluded due to not meeting the study's inclusion criteria. descriptive statistics were performed on demographic and clinical characteristics. kruskal-wallis h tests, anova, and t-tests were run to determine the significant associations between independent means. results: a significant difference in absolute neutrophils between racial groups, f( , . ) = . , p = . , was observed. there was an increase in anc from . +/- . with african americans to . +/- . in the other racial groups and to . +/- . with caucasians. differences were also found in anc in hispanics/latinos versus non-hispanic/latinos. the results were higher in non-hispanics and latinos, a significant difference of -. ( % ci, -. to -. ), t( ) = . , p = . . preliminary kruskal-wallis h tests run determined that there were significant differences between age groups for total white blood cell, hemoglobin, hematocrit, platelets, lymphocytes and anc. further studies are being run to evaluate in which age groups these differences lie and create reference ranges by age, race and sex. conclusion: among patients with ds, there are differences between racial groups and age groups. this data has been compared to previously established reference ranges for cbcs, but we are currently establishing healthy cbc controls which we will use to validate the reference ranges. these ranges will be published to help guide providers in workup and management of patients with ds. background: transfusion is a critical part of the care provided in the neonatal intensive care unit, but it is not without risks. low birth weight and premature infants can become anaemic from an immature haematopoietic system and frequent phlebotomy. these infants often receive multiple red blood cell transfusions. identifying infants more likely to require such intervention is important in ensuring the appropriate usage of this scarce resource. to determine whether birth weight, gestational age, gender, length of stay and mode of delivery can predict red cell concentrate (rcc) transfusion, units required, donor exposure and time to exposure. design/method: a retrospective chart review of all infants born below weeks gestation and/or birth weight less than , g who received a red blood cell transfusion between july and july in the cork university maternity hospital neonatal unit. results: infants met the inclusion criteria, ( . %) received a rcc transfusion. our study showed lower gestational age (p< . ) and lower birth weight (p< . ) infants are more likely to be transfused. donor exposure increases with a lower birth weight (p = . ). multivariate analysis showed infants with a lower gestational age (or - . per day; p< . ); lower birth weight (or - . per g; p< . ) and a longer length of stay (or . per day; p< . ) are more likely to receive a higher number of rcc transfusions. the time to first rcc transfusion is shorter in those with lower birth weight (or . per g; p< . ) and lower gestational age (or . per day; p< . ). gender and mode of delivery were not found to be predictors of red blood cell transfusion in this study. conclusion: low birth weight and premature infants are more likely to receive a rcc transfusion during admission to the neonatal unit. our study highlights predictors of rcc transfusion, donor exposure and time to transfusion. these can be used in identifying at risk infants, counselling parents and in anticipating transfusion requirements. emily southard, r. grant rowe, david williams, akiko shimamura, taizo nakano children's hospital colorado, aurora, colorado, united states background: the mecom locus encodes transcription factors that regulate hematopoietic stem cell self-renewal and maintenance. overexpression of mecom has been noted in - % of acute myeloid leukemia, several solid tumors, and denotes a poor prognosis. mutations that reduce mecom expression or that disrupt protein function, however, have been implicated in the development of bone marrow failure (bmf) through undefined pathways. an association between mecom mutations and radioulnar synostosis with amegakaryocytic thrombocytopenia (rusat) syndrome has been reported, however further characterization of this phenotype has yet to be explored. to characterize the phenotypic spectrum of a cohort of pediatric patients with novel mecom mutations. we performed a retrospective review of five patients with mecom mutations who were referred to hematology at children's hospital colorado or boston children's hospital. clinical, laboratory, and genetic data was collected on subjects and available family members. results: four of subjects were identified in infancy presenting with congenital cytopenias or physical dysmorphisms that prompted broad genetic screening. platforms for genetic detection included microarray, targeted genetic panels, and whole exome sequencing. three of subjects with cytopenias presented with congenital thrombocytopenia, of whom rapidly progressed to severe aplastic anemia. four of subjects presented with congenital anomalies, of whom demonstrated radioulnar synostosis. additional dysmorphic features identified include craniofacial (low set ears x ), cardiac (pda x , vsd x , aortic root dilation x ), pulmonary (pulmonary hypertension x , arteriovenous malformations x ), and developmental delay. one subject presented at age years with acute pancytopenia, hypocellular marrow, no dysmorphisms, and a mecom variant of unknown signif-icance. the identified mecom mutations include one . mb deletion involving several genes including mecom, one variant affecting a splice acceptor consensus sequence predicted to disrupt splicing, and three novel missense mutations, tyr cys, arg thr, and tyr cys, all of which were absent from public databases and were predicted in silico to be deleterious. we describe the phenotypic spectrum of patients with novel mecom variants. a subset of patients lacked radio-ulnar synostosis and had presence of additional systemic anomalies, demonstrating a varied clinical phenotype that is not isolated to rusat syndrome. a centralized publically accessible database to share clinically annotated mecom variants, together with analysis by experts in mecom function would advance our understanding of the clinical interpretation of mecom variants. mecom should be considered in the differential diagnosis of bone marrow failure and we advocate for the inclusion of mecom in targeted sequencing panels. cairo university, cairo, egypt background: beta thalassemia is regarded as a serious public health problem in the mediterranean region, southeast asia, and the middle east. however, very few studies have been conducted to assess the quality of life (qol) among thalassemia major patients. objectives: to assess the quality of life among b-thalassemia major patients using short form (sf)- questionnaire and to determine the factors associated with their quality of life. design/method: a cross-sectional study was conducted among thalassemia major patients who were attending the hematology outpatient clinic at cairo university hospital, during the study period. data were collected between october and march . the quality of life was assessed for patients aged ≥ years. the mean age of the studied group was . ± . years. the majority ( . %) had one monthly blood transfusion. the mean total score of sf- was . ± . . general health perception domain was the most affected domain with mean score, while vitality was the least affected one. there was no statistically significant difference between males and females regarding different quality domains except for vitality where the mean score was significantly higher in males than females (p = . ). age at onset of disease, and at first blood transfusion were the most documented factors positively correlated with the quality of life among the enrolled thalassemia patients. conclusion: the quality of life in thalassemia major patient was found to be compromised. all thalassemia patients should undergo assessment of the quality of life so that interventions focusing on the affected domains can be implemented. background: international adoption of children with special needs has become more prevalent in recent years leading to tremendous growth in the number of u.s. thalassemia patients adopted from foreign countries. currently % of the , thalassemia patients registered in the cooley's anemia foundation (caf) patient database have been adopted from foreign countries, primarily china. as this population continues to grow, further information is needed in order to provide these families with best supportive care. the primary goal of this study is to characterize the socio-demographics and health statuses of adopted children with thalassemia and their families. a secondary goal is to describe adoptive families' motivations, experiences, challenges, and support resources. design/method: a redcap survey was accessed by families of adopted children with thalassemia through the caf website and caf social media from january to august . following a four-question screen, eligible subjects were directed to complete an adoption questionnaire. families who had at least one adopted child with thalassemia receiving care at a participating thalassemia treatment center or hematology office in the u.s. were considered eligible. descriptive statistics were analyzed using sas . . respondents who were ineligible or who provided incomplete data were removed from the dataset prior to analysis. of survey respondents, qualified and completed the survey. these households had adopted a total of children with thalassemia ( . % male), most from china ( . %), where they had been living in orphanages ( . %). legal guardians identified primarily as christian ( . %). the majority had completed post-secondary education ( . %) with reported household incomes greater than $ , ( . %). most adoptive families were connected to an adoption group or community including online groups, local support groups, and adoption networks ( . %). commonly cited challenges were: ) volume of frequent medical appointments, ) insufficient support from their local care centers, and ) financial burdens. the reality of care for the population of adopted patients with thalassemia in the u.s does not seem to match the expectations set by their providers. we are hopeful this data will be used to assist adoptive families navigating the complexities of thalassemia care. the findings suggest that this population would benefit from additional outreach, education, guidance, and advocacy resources -especially in the early stages of adoption and during initiation of post-adoption medical care. background: in many higher-income countries, thalassemia major has become a chronic disorder; many outcomes are different in emerging countries with more limited resources. most analyzes of health-related quality of life (qofl) in thalassemia have been conducted in high-income settings. objectives: to assess the impact of health status on qofl in thalassemia patients in an emerging country. we assessed qofl in randomly-selected patients ( thalassemia major; with hemoglobin e thalassemia; five thalassemia intermedia) at the national thalassemia center in kurunegala, sri lanka where approximately patients are managed. treatment is free, but compared to north america/europe, access to tertiary staff and other resources are limited. overall, control of body iron as estimated by serum ferritin concentration (mean± sem, ± g/l) was not optimal in many patients. to understand the impact of health status on qofl, we used the sf v health survey, analyzing scores of physical function, pain, general health, social functioning, emotional and mental health, to generate overall physical and mental component scores. results: compared to reports from higher-income countries (american journal of hematology ; : - ), physical function scores (mean±sd, . ± . ) were similar in sri lankan patients; indeed, in three categories (physical role, social function, emotional role), sri lankan scores were slightly higher. by contrast, compared to scores from higherincome settings, those estimating bodily pain, general health, and mental health were significantly lower, resulting overall in a significantly lower physical component score in sri lankan patients. male sri lankan patients reported higher scores than females, and somewhat surprisingly, in four categories (physical function, physical role, social function and emotional role) reported higher scores than those obtained in higher-income settings. lower scores in physical functioning, leading to an overall lower physical component score, were recorded by females. patients with hemoglobin e thalassemia reported generally poorer qofl than those with thalassemia major. the lack of differences in qofl in patients with "high" and "low" hemoglobins was likely related to low pre-transfusion hbs (mean±sem, . ± . g/dl) in nearly all patients. these early data in a small cohort of thalassemia patients in an emerging setting suggest that in many patients bodily pain, reduced mental health, and poorer views of general health affect overall qofl. prospective studies in larger cohorts including evaluation of adequacy of transfusions and chelation therapy, complications, and overall accessibility of care may guide approaches to improve qofl in lower-income settings of thalassemia care. geetanjali bora, anand prakash dubey, tarun sekhri, mammen puliyel, aparna roy maulana azad medical college, new delhi, delhi, india background: in the last two decades, the presence of osteopenia has been described in optimally treated patients with transfusion dependent thalassemia, the pathogenesis of which seems to differ from osteopenia in non-transfused patients. the prevalence rate of low bone mineral density (bmd) in pediatric population is highly variable amongst studies done worldwide. furthermore, the role of metabolic and endocrine factors in determining bone mass in this population is not well understood. objectives: to assess bmd in subjects with transfusion dependent beta thalassemia by dual-energy-x rayabsorptiometry and find its co-relation with clinical, biochemical and hematological parameters. design/method: this is a comparative cross-sectional study and includes patients with transfusion dependent beta thalassemia between ages to years enrolled from a thalassemia day care center in the year - . at the time of enrollment age, sex, bmi z scores, pubertal staging, duration and type of chelation therapy were noted. enrolled subjects were scanned for bmd at lumbar spine l - and left femoral neck using dexa scan. the bmd was expressed in mean values and z scores. age, bmi, ethnicity and gender matched historic controls were used to generate z scores. ml of pre transfusion fasting venous blood samples were obtained to test for serum calcium, phosphate, alkaline phosphatase, pth, thyroid function panel, serum ferritin and serum igf- levels. mean values for pretransfusion hemoglobin and serum ferritin over last months were calculated. results: total no of subjects , median age . years, male ( %), female ( %), ethnicity % asian, bmi < rd centile ( %), pre pubertal %, all receiving transfusion and chelation therapy. prevalence of low (z score < - sd) and very low (< - . sd) bmd was %, % at l -l respectively and %, % at left femoral neck respectively. there was trend of lower bmd z scores with advancing age. statistically significant co-relation (p value < . ) was found between low bmd and low mean pretransfusion hemoglobin, serum phosphate, igf - and vitamin d levels conclusion: a sizable proportion of children and adolescents with transfusion dependent thalassemia have suboptimal bone mineral density and this decline may start as early as - years of age despite being on transfusion regimen highlighting the importance of yearly dexa screening and optimization of pre-transfusion hemoglobin, vitamin d and igf levels. vanderbilt university medical center, nashville, tennessee, united states background: it is well described that iron deficiency anemia (ida) can co-present with thrombocytosis or thrombocytopenia, though cases of thrombocytopenia are less frequent than thrombocytosis. prior reports of thrombocytopenia have included adult and pediatric patients with menorrhagia ( - ), menorrhagia due to uterine fibroids ( ), or other gynecologic abnormalities ( ). our cases highlight the pattern of ida, thrombocytopenia, and menorrhagia in the setting of significant menstrual clotting without observed gynecologic abnormalities in african-american adolescents. objectives: to describe the clinical course of three adolescent females with severe ida, menorrhagia, and thrombocytopenia. results: our cases included three female african-american patients ages - who presented with severe anemia and concurrent thrombocytopenia in the setting of menorrhagia. all three patients reported heavy and prolonged menstrual cycle bleeding with significant clots. two of the three were admitted for transfusions at presentation and noted to have significant menstrual bleeding with continued blood loss requiring additional transfusions until bleeding was controlled with estrogen therapy. these two patients were evaluated with pelvic ultrasounds revealing a prominent endometrium in both patients and hyperechoic material consistent with a clot in one patient. average hemoglobin on presentation was . gm/dl ( . - . ), average platelet count was , /mcl ( , - , ), and average mcv was ( - ). all had severe iron deficiency with an average ferritin of ng/ml ( - ) subsequently treated with oral iron. one patient had a prior history of ida that required transfusion and had subsequent normalization of her complete blood count. two patients had subsequent thrombocytosis before normalization of their platelet counts. two patients received platelet transfusions: one due to recent neurosurgical intervention with a higher goal platelet count and the other to help control menstrual bleeding after a nadir platelet count of , . a review of the clinical history and red cell indices pointed to ida and ongoing blood loss from menorrhagia as the reason for the bicytopenias. the thrombocytopenia in these cases may have been exacerbated by consumption of platelets in the significant clots all three patients reported. it is reasonable to treat with iron supplementation and supportive care which may include transfusions or management of menorrhagia with oral contraceptives or other hormonal methods. background: sickle cell disease is one of the most common inherited red blood cell disorders, yet many are not aware of their carrier status. the american college of obstetricians and gynecologists' guidelines recommend that pregnant women of african, mediterranean and southeast asian descent be screened for hemoglobinopathies with a cbc and hemoglobin electrophoresis . however, adherence to this practice and frequency of improper screening with sickledex is unknown. proper screening and counseling can impact families' knowledge, allowing for establishing relationships with pediatric hematology providers earlier. objectives: we sought to assess prenatal hemoglobinopathy screening practice patterns and methods of obstetrics & gynecology (obgyn) and family medicine providers in the nyc regional area. design/method: a cross-sectional electronic survey was administered to obgyn and family medicine practitioners from four nyc institutions. questions focused on prenatal hemoglobinopathy screening practices using case scenarios with variations on parental trait status and ethnicities. chisquare analyses were used to compare the two provider groups on categorical variables. there were total responses; surveys were complete, of which were obgyn and family medicine providers. respondents were mainly from academic medical centers, with the majority being faculty ( % of the obgyns and % of family medicine). no significant difference was found in frequencies of screening patients with a positive family history of a hemoglobinopathy. when asked about screening practices for patients without a personal/family history of a hemoglobinopathy, % of obgyns versus % of family medicine providers "always" screened for hemoglobinpathies (p = . ). when analyzed by ethnic background, there were significant differences by group in screening patients of white ( % vs %), black ( % vs %), mediterranean ( % vs %), and asian descent ( % vs %) (p≤ . for all). however, in cases where the hemoglobinopathy carrier status of both parents was known, there was no difference in screening with a hemoglobin electrophoresis. furthermore, > % of all respondents use sickledex for screening in the case scenarios. conclusion: this pilot survey highlights a difference in the methods and likelihood of prenatal hemoglobinopathy screening based on the type of prenatal care provider. screening differences can lead to variations in prenatal guidance, diagnostic procedures, informed decision-making and knowledge of families referred to pediatric hematology clinics. this is the first study analyzing prenatal screening for hemoglobinopathies in obgyn and family medicine. improving prenatal screening practices by collaborating with hematologists may decrease health care disparities and allow for earlier relationship building with pediatric hematology. . acog, opinion# , poster # hermansky-pudlak syndrome: spectrum in oman background: hermansky-pudlak syndrome (hps) is a rare autosomal recessive disorder, characterized by the triad of oculocutaneous albinism, a hemorrhagic diathesis resulting from storage pool-deficient platelets, and accumulation of ceroid/lipofuscin-like material in various tissues. before , nine different types of hermansky-pudlak syndrome were identified, which can be distinguished by their signs and symptoms and underlying genetic cause. in , a tenth type was defined based on mutations in the ap d gene. hps type is characterized in addition by severe neutropenia and recurrent sinopulmonary infection. the disease is more common in puerto rico, and this is the first report from oman. to describe the clinical, laboratory and genetic characteristics of hps sub-types in oman, including the first cases of hps type . design/method: this is a retrospective study, including cases with hps that had been suspected clinically and confirmed through genetic mutation analysis. clinical data included sex, age at presentation, initial clinical presentation (skin, eyes, development, neurological involvement, bleeding tendency, recurrent infections) and course of disease. laboratory data (complete blood counts, platelet and absolute neutrophil counts, coagulation screening, platelet function tests by platelet function analyzer, and platelet aggregation studies using different agonist had been recorded. pcr and next generation sequencing for genetic confirmation by testing mutations in hps , ap b , hps , hps , hps , hps , dtnbp ,, bloc s , bloc s genes had been done. results: seven omani cases with hps have been identified ( males and females). their age ranged between (at birth) to years. two patients had hps type , patient had type , while the other cases had hps type . no other sub-types were encountered in oman. all patients were products of consanguineous marriage. one patient had adrenal hge, while the others had mild hemorrhagic phenotype, characterized by recurrent bruising and mild epistaxis. laboratory testing confirmed variable platelet aggregation defects with different platelet agonists. all patients had characteristic hypopigmentation, iris transillumination, nystagmus, and foveal hypoplasia. both patients with hps type had the same homozygous mutation in the ap b gene (c. _ delta), and presented with severe neutropenia. early diagnosis and initiation of gcsf on one of them improved outcome and prevented the development of complications. late diagnosis in the other patient resulted in the development of bronchiectasis as a result of recurrent sinopulmonary infections. background: sickle cell disease (scd), a genetic disorder characterized by defective sickle hemoglobin (hbs), triggers red blood cell sickling, hemolysis, vaso-occlusion, and inflammation. ischemic injury from scd starts in infancy and accumulates over a lifetime, causing pain, fatigue, and progressive end-organ damage that culminates in early mortality. voxelotor (gbt ) is an oral, once-daily therapy that modulates hemoglobin's oxygen affinity, thereby inhibiting hemoglobin polymerization. objectives: to assess the safety, pharmacokinetics, and efficacy of voxelotor in pediatric patients with scd. design/method: this ongoing study is being conducted in parts: part a: a single dose of voxelotor mg in pediatric and adolescent patients; part b: multiple doses of voxelotor mg/d or mg/d for weeks in adolescents. part b's primary objective is to assess the effect of voxelotor on modifying anemia. secondary objectives include measuring other markers of disease modification, such as hemolysis; daily scd symptoms, using a patient-reported outcome (pro) measure; and safety. results: as of november , , patients ( females) had received voxelotor mg and patients ( females) had received voxelotor for ≥ weeks. the median age for the patients was years, % were receiving hydroxyurea (hu), and % had ≥ painful crises in the past year. data for hemolysis measures are available for patients who received voxelotor for weeks. six of the patients achieved a hemoglobin (hb) response of > g/dl increase. laboratory markers of hemolysis improved concordantly; the median reductions in reticulocytes and indirect bilirubin were % and %, respectively. ten of patients showed reduction in total symptom scores (tss) at week , with a % median reduction in tss from baseline. there were no treatmentrelated serious adverse events (aes) or drug discontinuations due to aes. voxelotor mg for weeks in adolescents with scd, the majority receiving hu, demonstrated consistent, sustained efficacy on hb levels and measures of hemolysis; > % of patients showed a > g/dl improvement in hb. improvement in tss in mildly symptomatic patients suggests that the pro is sensitive to treatment effect and supports use in the ongoing hope phase study. voxelotor's reassuring safety profile is consistent with results in adults. these interim results support ongoing clinical evaluation of voxelotor as a potential disease-modifying therapy for adults and children with scd. supported by global blood therapeutics. background: acute kidney injury (aki) is a common complication in sickle cell disease (scd), and a potential risk factor for sickle nephropathy. aki is associated with acute decline in hemoglobin (hb) during vaso-occlusive pain crisis and acute chest syndrome (acs). it is unclear which pathologic factor plays a stronger role in aki development during hb drop: increase in free heme during vaso-occlusive events secondary to hemolysis or hb decline itself. objectives: to investigate if hb decline alone is associated with aki, we tested if the renal function of patients with scd worsened during parvovirus b -induced transient aplastic crisis (tac), in the absence of accentuated hemolysis. design/method: with irb approval, a retrospective study of patients who had laboratory confirmed parvovirus-b was conducted. serum creatinine (scr), both during and within months from the tac event, was collected. comparisons of the clinical and laboratory characteristics were analyzed using the wilcoxon test for continuous variables. aki was defined as an increase in scr by ≥ . mg/dl or a % increase in scr from baseline. to evaluate differences in change in hb on aki risk, changes in scr during tac were compared to those during pain crisis or acs admissions by fitting a generalized linear mixed model for binary outcome. a comparative sample of acs events and vaso-occlusive pain crisis were used to estimate rates of aki according to hb levels. results: three ( %) of the patients with scd developed aki during tac. no association was identified between change in hb from baseline to tac event (p = . ). no cases of aki were identified until hb decreased < . g/dl or the change in hb was ≥ . g/dl from baseline. next, we developed a model to evaluate the impact of change in hb from baseline for patients admitted with tac, pain crisis or acs on aki. with a g/dl decrease in admission hb from baseline, patients with tac had a % probability of developing aki, while acute chest syndrome and pain crisis would have a % and % probability, respectively. our data suggest that aki is still prevalent during parvovirus b -induced tac. however, the risk of aki during a tac event is and times lower than that from severe anemia induced by acute chest syndrome and vasoocclusive pain events, respectively. hemolysis-induced anemia during scd crisis appears to have a more significant role in the development of aki as compared to agenerative anemia. background: the natural history of hemoglobin e beta thalassemia (hbethal), the commonest form of severe beta thalassemia worldwide, has been examined in very few longterm studies. previously, we reported findings in hbethal patients in sri lanka. objectives: to evaluate longterm requirements for transfusion and splenectomy, complications and death in hbethal patients. design/method: all available patients were reviewed - times annually over years. results: patients ( %) died, aged (mean ± sem) . ± . years; the (known) causes commonly included iron overload ( ) and infection ( ); patients surviving patients are aged . ± . years. of patients originally classified by severity (group the mildest, and group the most severe, phenotypes), ( %) were assessed as mild (groups and ), of whom transfusions had been discontinued in . ultimately, / ( %) resumed transfusions, often following shifts to increasingly severe phenotypes including increasing intolerance to anemia. age at resumption of transfusions (following a transfusion-free interval of . ± . years) was . ± . years; in the more severe groups and , regular transfusions were stopped in / patients and resumed in / ( %), at younger ages ( . ± . years) and after shorter transfusion-free periods ( . ± . years) than in "milder" patients. mid-parental height (mph) was ultimately achieved in %. patients ( %) were splenectomized; updated analysis of responses to splenectomy (originally "group " patients), showed that splenectomy (at . ± . years) was followed by an extended, but impermanent, transfusion-free interval ( . ± . years); % patients resumed transfusions, usually related to exercise intolerance or poor growth. in groups and , complications of anemia and ineffective erythropoiesis, including leg ulcers (in % and %) and gallstones ( % and %), were more frequent than in groups and ; fractures were observed ( - %) across all groups, except for regularly-transfused group patients ( %). pulmonary artery pressures > mm were recorded in % patients. evaluation of patients with hbethal requires observations over years, without which definition of patients as "mild" or "severe" may be misleading. while in many patients transfusions may be withheld or reduced in frequency, troublesome complications may surface with advancing age even in "milder" patients. although individual consideration of transfusion requirements is critical, the availability of effective chelation, where this can be provided without prohibitive cost, may alter the balance of risks and benefits of regular transfusions in hbethal. (premawardhena a. lancet ). background: social determinants of health (sdh) are environmental and socioeconomic factors, such as access to food and housing that affect health outcomes. pediatricians are increasingly screening for sdh as part of primary care visits, however less is known about screening for sdh in pediatric hematology. evidence suggests that sdh play a role in disease severity for children with scd, who face significant socio-economic and racial disparities. the goal of our quality improvement (qi) project was to increase the percentage of patients with scd who were connected to community resources for unmet social needs. design/method: we based our intervention on the successful implementation of wecare in our institution's pediatric primary care clinic. eligible patients were identified at the start of each clinic session. on arrival the parent was given a self-reported screening tool for six sdh (childcare, education, employment, food, utilities and housing). results were entered in the electronic health record by the physician or social worker who then printed a pre-existing resource list for patients with a positive screen. we used a series of plan-do-study-act (pdsa) cycles to study tests of change. we tracked process measures (percentage of patients screened, percentage of patients with an unmet social need who received a resource sheet), outcome measures (percentage of patients with an unmet social need who connected with a community resource) and balancing measures (staff, patient and provider satisfaction). run charts were reviewed weekly and then monthly to inform further tests of change. examples of pdsa cycles include who gave the paper survey to patients (social worker or physician versus medical assistant) and length of time between surveys ( to months). results: between august and december screening rates improved from % to %. of the patients screened, % report at least one unmet social need; of those % received a targeted list of community resources in the first month of the project, and % in the fifth month. finally, % of patients reached by phone had connected with a community resource within weeks of the clinic visit. we have successfully implemented universal screening for sdh for patients with scd in our urban pediatric hematology clinic without requiring extra staff. next steps include further pdsa cycles to connect more patients to appropriate resources, and tracking improvement in health care utilization outcomes from addressing sdh in this vulnerable patient population. background: the clinical manifestations of sickle cell disease (scd), chronic hemolytic anemia, and vaso-occlusion occur as a direct result of sickle hemoglobin (hbs) polymerization. voxelotor (gbt ) is a first-in-class, oral, oncedaily investigational agent designed to modulate hemoglobin's oxygen affinity in a targeted approach to inhibit hbs polymerization. objectives: to examine the pharmacokinetics (pk), safety, and dosing of voxelotor in children (aged - years) and adolescents (aged - years) with scd from part a of the gbt - study. design/method: gbt - is an ongoing, open-label, phase a study in patients aged - years with scd (sickle cell anemia or sickle beta zero thalassemia). part a of this study (the focus of this abstract) is examining pk of singledose ( mg) voxelotor. pk samples to measure whole blood and plasma voxelotor concentrations were collected up to days following single-dose administration. separate population pk (ppk) models were developed to describe the concentration versus time profiles of voxelotor in whole blood and plasma using nonlinear mixed effects modeling (non-mem, version . ). ppk modeling and physiologically based pk (pbpk) modeling were used to simulate voxelotor pk parameters and support dose selection for future evaluation in younger children. : part a included adolescents ( females; median age years [range - ]) and children ( females; median age . years [range - ]). mean weight was . kg (range - kg) and . kg (range - kg) in adolescents and children, respectively. voxelotor was well tolerated with no drugrelated grade ≥ adverse events (ae) or serious aes. a compartment model with first-order absorption best described the pk of voxelotor (and was the same model structure used for adults with scd). voxelotor pk exposures in adolescents were comparable to those observed in adults, but higher exposures were observed in children. ppk and pbpk modeling support the use of a weight-based dosing strategy in younger children (aged < years) in future trials. adult voxelotor doses can be used in adolescents. however, based on higher pk exposures, a lower weight-based dosing strategy is recommended in children. ppk and pbpk modeling provides an innovative approach to minimize experimental dosing in children and accelerate dose selection of voxelotor in ongoing and future clinical studies. this abstract is supported by global blood therapeutics. background: hydroxyurea (hu) reduces rates of acute complications, and improves long term outcomes in patients with sickle cell disease (scd) and is now fda approved for children. through previous work we have increased the number of eligible patients on hu in our clinic, however accessing a compounding pharmacy remained a significant barrier to hu adherence for infants and children who cannot swallow capsules. objectives: the objective of our quality improvement project was to improve adherence to hu among pediatric patients with scd at our urban safety net hospital by addressing barriers to obtaining liquid hu. design/method: to begin we met with the leadership of our outpatient pharmacy which offers mail order delivery. however, like most retail pharmacies, they do not have the necessary protective equipment to compound liquid hu. through a series of discussions, we began a unique partnership with our institution's inpatient chemotherapy pharmacy who compounds the liquid hu and delivers it to the outpatient s of s pharmacy, who then dispenses liquid hu to families. using a series of plan-do-study-act (pdsa) cycles we tracked adherence by calculating the medication possession ratio (mpr), defined as the percentage of days in a given period of time that each patient had their medication on hand. the mpr for liquid hu mpr among enrolled patients was tracked by pharmacy staff and reviewed monthly. additional pdsa cycles included adding automatic refills and reminder calls by pharmacy staff and improving communication about delivery. we also tracked patient satisfaction. results: between march and december , a total of thirty pediatric patients were enrolled in our program for on-site compounding and free mail order delivery of liquid hu. mpr for liquid hu is currently . % among enrolled patients, significantly higher than the mpr of % reported in the literature, and has risen steadily since the beginning of the project. families are highly satisfied with the program, specifically appreciating the convenience of mail order delivery, saving on delivery fees, and reminder calls when refills were due. by compounding and dispensing liquid hu directly from our institution's outpatient pharmacy we have significantly improved adherence to this hu therapy in our high-risk population. next steps include analysis of change in clinical outcomes for patients enrolled in this program. as adherence to hydroxyurea is associated with decreased acute care utilization and cost, programs such as ours could play a crucial role in reducing the excessive costs and ed utilization among this patient population. background: experience with the iron-chelator deferasirox is reported widely in higher-income settings. by contrast, real-life experiences in emerging countries are infrequently reported. objectives: to evaluate, in a non-trial setting, the real-life response to deferasirox in an emerging country. design/method: in sri lanka's national thalassemia center which manages patients without tertiary staff, quantitative evaluations of body iron or estimates of extra-hepatic iron, the records of patients who began deferasirox in / were retrospectively reviewed. results: baseline assessments (mean±sem) indicated substantial iron loading [serum ferritin (sf) , ± ug/l; serum alt ± . u/l (normal ≤ u/l)]. deferasirox was introduced at low doses ( . ± . mg/kg/day); many patients started at < mg/kg and, after months, doses remained ≤ mg/kg/day in % patients. after months, sf in % patients remained > , ug/l; only by months had (mean) sf declined to < , ug/l ( ± ; p< . ). similarly, mean alt normalized (to ± u/l) only by months. death and complications were not systematically recorded by staff who had been charged, without provision of additional resources, with the introduction of this new drug in hundreds of patients. these results contrast to those in sri lanka's tertiary thalassemia center where, in patients following the introduction of deferasirox ± . mg/kg/day, sf declined rapidly, even in relatively less ironloaded patients (from , ± to , ± g/l after months; p = . ). these findings underscore the importance, during the implementation of new drug regimens in lowerincome centers with marginal resources, for investments in methods to quantitate body iron burden, hands-on educational initiatives to guide day-to-day management by competent but non-expert staff, and data systems to record efficacy, effectiveness, toxicity and compliance. such investment is critical to optimising therapy and improving complications in thalassemia patients worldwide: even in sri lanka, where resources directed to thalassemia management are greater than in most of asia, results in the oldest living cohort (born - ) indicate under-treatment [elevated iron burdens (sf , ± ug/l) and high prevalences of diabetes ( %) and hypothyroidism ( %)]. even in a younger cohort (born - ) which has benefitted from improved treatments, the prevalence of many complications exceeds those reported from high-income settings. over the next decade, and two decades after the who declaration that the impact of thalassemia on global mortality and morbidity is underrecognized, increased investments by governmental and nongovernmental sources will be necessary to improve outcomes for asian patients with thalassemia. background: a major barrier to success in hydroxyurea (hu) treatment of patients with sickle cell disease (scd) is non-adherence. objectives: to optimize hu adherence in patients with scd. design/method: a care model was designed by the sickle cell (qi) team at children's hospital to improve hu adherence among scd patients. the original model included bimonthly family phone contact, monthly dispensing pharmacy phone contact and lab monitoring. adherence measures included obtaining hu from pharmacy monthly, completion of monthly labs, hb f percentage and mcv, and mtd achievement. from / - / , several pdsa cycles refined our care model. a one-year follow-up survey gathered feedback on the care model. the first-year data involved ∼ patients. the biggest improvements resulted from making pharmacy calls before patient/family calls, shipping liquid hu to outlying patients, and tracking call time/content. the qi goal was % hu adherence by / . the % baseline adherence rate increased to % by / , and has remained in that range. the completion rate of patient/parent phone calls increased from % the first month to % at six months. pharmacy prescription pick-up has increased from % to % per month. lack of liquid hu availability was overcome by shipping the medication to the patient's home. parental hesitance to share information by phone, especially with qi team members with whom they had no established relationship, was overcome by having the longtime sickle cell nurse do many of the early calls. however, survey feedback showed families became comfortable with several clinic personnel calling. the calls gave families the opportunity to ask questions about their child and/or get additional information about scd. the calls also provided an opportunity for seasonal flu shot or tcd testing reminders. the surveys gave information on the optimal time of day to reach each family, providing individualization and further increasing the percentage of completed calls. two families surveyed said they no longer needed two calls a month because they were now able to remember to pick up hu, administer it, and get labs on their own. this qi project has not only improved hu adherence, but also fostered health education/counseling, increased patient/parent satisfaction, and enhanced service utilization. medical team member and patient/family comments demonstrate that it has helped build relationships and trust between families and the medical care system. based on survey feedback, we will further individualize care to increase adherence rate and sustain improvements. cincinnati children's hospital medical center, cincinnati, ohio, united states background: the thalassemias are a heterogeneous group of genetic blood disorders caused by mutations that decrease or eliminate the synthesis of the -and/or -globin subunits of hemoglobin. the phenotype of thalassemia depends on the interaction of the -and -globin gene clusters, because both loci determine the -/ -chain balance. for example, a -thalassemia phenotype can be more severe than expected when coinherited with -globin gene triplication (copy number gain), which exacerbates the -/ -globin imbalance. objectives: describe four individuals with an incorrect diagnosis of -thalassemia trait who were later properly diagnosed by comprehensive genetic testing to have -thalassemia intermedia caused by heterozygous -thalassemia mutations coinherited with triplicated -globin loci. design/method: sequence analysis of the -globin (hba /hba ) and -globin (hbb) genes, and copy number variation analysis of the -and -globin gene clusters by multiplex ligand-dependent probe amplification. results: four unrelated individuals of northern european ancestry were evaluated for signs and symptoms not explained by a diagnosis of -thalassemia trait (previously made by a pediatric hematologist), including growth delay, splenomegaly, moderate anemia, marked elevation of hemoglobin f, thalassemic facies, reticulocytosis, and/or indirect hyperbilirubinemia. genetic testing revealed that all were heterozygous ( / ) for the same, single -globin mutation [hbb.c. c>t (p.q *)] and also heterozygous for an -globin triplication ( / anti- . ). their previous diagnoses of thalassemia trait had been made by complete blood counts, hemoglobin electrophoresis, and/or sequence analysis of the -globin genes only. these individuals' phenotypes ranged from moderate anemia only to multiple stigmata of thalassemia, demonstrating the phenotypic variation of a thalassemia genotype. correct diagnosis was made at an average age of . years. a trial of chronic transfusions was initiated for one patient for growth failure. all were educated about the potential for exacerbations of anemia, gallstones, osteoporosis, and iron overload (even without transfusions). parental genetic testing was recommended to assess reproductive risk, because inheritance of this complex genotype can be apparently autosomal dominant. conclusion: heterozygosity for a -thalassemia mutation does not necessarily indicate -thalassemia minor or "trait". when coinherited with -globin gene triplication, a symptomatic form of -thalassemia can occur. correct and timely diagnosis of thalassemia requires careful consideration of the degree of anemia and examination for organomegaly, bony changes, and jaundice. sequence analysis and copy number variation analysis of both the -and -globin gene clusters is key. hematologists need to be aware of this diagnostic possibility and how to test for it to prevent inaccurate or delayed diagnosis. background: the burden of healthcare costs for sickle cell disease (scd) is nationally estimated at over $ billion. the major components of these costs are inpatient and emergency center (ec) visits, many of which are potentially avoidable. in several chronic conditions, a subset of patients account for most of the avoidable encounters. identifying these patients is the first step in targeted care delivery. objectives: to measure and analyze scd patient utilization patterns in the ec and inpatient at texas children's hospital (tch). we identified all individuals under years old with any encounter at tch associated with an international classification of disease (icd)- or code for scd, including hgb ss, hgb sc, and hgb s/beta thalassemia. for each patient, we identified all inpatient and ec encounters in the days prior to their most recent encounter. finally, each encounter was classified as associated with pain, acute chest syndrome (acs), or "other" using an algorithm of discharge diagnosis codes and pharmaceutical delivery. the total number of scd-associated ec and inpatient encounters over the prior year was calculated for each patient. we stratified each patient according to their utilization patterns: low ( - encounters), intermediate ( - encounters), and high (≥ encounters). we identified unique patients with scd that had at least one encounter from july until june . there were , scd-related encounters in the days prior to their most recent encounter. most ( %, n = ) patients exhibited low-utilization patterns and % (n = ) were intermediate. finally, a small subset ( %, n = ) demonstrated high-utilization patterns and accounted for % of all encounters. high-utilization was associated with older age and public payment mechanisms. pain encounters were predominantly in pre-adolescents and teenagers with high-and intermediate-utilization patterns. acs was most frequent in pre-teens and younger teens in the intermediate-utilization group. finally, the youngest-aged high and intermediate users presented for other reasons such as febrile episodes and splenic sequestration. our findings reflect national trends in that a significant portion of encounters are attributed to a small subset of patients exhibiting a high-or "super-" utilization pattern. at our institution, scd super-utilization is associated with older age and pain. we also identified a group of infants and toddlers with frequent encounters for fever. to comprehensively address this burden, it will be important to design interventions targeted toward age and specific medical needs. background: background: the rarity of diamond blackfan anemia (dba) has hindered describing the spectrum of disease, identifying predictive correlations, and guiding datadriven recommendations. long-term toxicities from steroid or transfusion therapy that start in childhood remain the major clinical problems in patients with dba who do not receive stem cell transplant. objectives: objective: to define the dba patient population at st. jude children's research hospital including treatment responses and toxicities to help inform recommendations on treatment and monitoring. design/method: method: medical records were reviewed for all patients with dba treated at st. jude between and for diagnostic testing, treatment types and regimens, and outcomes. two-sample t-test or wilcoxon rank sum test was used to compare continuous variables in two groups depending on the normality of the data tested by shapiro-wilk test. results: a total of patients with dba were identified with a median age of . years (range months - years) at last follow up. a ribosomal protein gene mutation was identified in / patients ( %) with an rps mutation / ( %). thirteen different congenital malformations were described in / patients ( %). fourteen of twenty ( %) patients treated with corticosteroids had an initial response and of those achieved full remission. three patients became steroid-refractory and were unable to wean to an acceptable dose. five of twenty patients continue on lower-dose steroids. five patients currently require no therapy. univariate analysis revealed no statistically significant genetic predictors of response or remission, however, / rpl patients responded to steroids with / ( %) in long-term remission. ten patients are maintained on chronic transfusions and have undergone successful hematopoietic stem cell transplant. nineteen of treated patients ( %) had a treatment-related toxicity. patients on steroids were more likely to have short stature than patients on transfusions or in remission (p = . ). severe bone mineral density deficit occurred in / ( %) patients, in before age years. eight patients had hepatic iron overload, in one documented by age years. other severe toxicities included restrictive cardiomyopathy from iron overload, pathologic fracture, diabetes mellitus, and premature ovarian failure in one patient each. this genotypically and phenotypically heterogeneous dba cohort had a high rate of treatment-related toxicities, notably growth retardation, bone density loss, and hepatic iron overload even in very young children. these findings underscore the need for early standardized monitoring. background: patients with sickle cell disease (scd) face worsening morbidity and mortality between ages and , when they must transition from pediatric to adult healthcare.( ) an effective curriculum addressing disease knowledge, educational and vocational skills, self-efficacy, and social supports is critical to a successful transition. traditional didactic approaches have not led to durable knowledge retention. ( ) technology-based methods have been attempted, but the best educational approach remains unknown. objectives: . to understand how adolescent and young adult (aya) patients with scd view existing transition education. . to include patient preferences in improving our transition curriculum. we developed a qualitative survey to assess patient views of existing approaches for learning about scd and their opinions about preferred transition topics. thirty patients with scd aged to years old were recruited between january and december . responses were managed using redcap electronic data tools hosted at the university of rochester.( , ) qualitative and quantitative data analyses were performed, including independent t-testing to compare responses between age groups. results: approximately % of subjects were under years of age, while % were or older. seventy-one percent had a computer, and . % had a cell phone, with most reporting daily use. subjects reported greatest satisfaction with learning from their doctor during clinic visits ( . % agree or strongly agree) and websites on a cell phone ( . % agree or strongly agree); the least popular methods were online chat rooms and microsoft® powerpoint presentations. satisfaction was similar across age groups. recommended transition topics were viewed positively, with subjects ranking highest understanding their bloodwork ( . % agree or strongly agree) and understanding laws protecting students with chronic disease ( . % agree or strongly agree). older subjects ( - years old) agreed more strongly with learning about opioid addiction and understanding differences between adult and pediatric doctors than did younger subjects ( - years old) (p < . ). this pilot study was successful in helping us to understand the educational needs of aya patients with scd. preliminary data underscore the importance of education provided by the pediatric hematologist. our results also suggest that the optimal use of technology-based methods requires further investigation and that tailoring transition education by age group may be useful. background: similar to patients with transfusion-dependent beta-thalassemias (tdt-beta), survivors of hemoglobin barts hydrops fetalis (homozygous alpha- -thalassemia, tdtalpha) will require lifelong transfusions of erythrocytes. we have previously shown that a transfusion strategy that is based on the guidelines developed for tdt-beta (conventional transfusion) is suboptimal for these patients owing to the differences in the pathophysiology of anemia in the two conditions: in tdt-alpha, conventional transfusion strategy will lead to a gradual increase in non-functional hbh with subsequent tissue hypoxia and hemolysis. an aggressive transfusion strategy that was based on reduction of hbh and increase in "functional" hemoglobin level resulted in improvement of tissue oxygenation and reduction of hemolysis but was associated with significant increase in transfusional iron burden [amid et al, blood ] . objectives: to define the optimal chronic blood transfusion targets for hbh% and functional hemoglobin in patients with tdt-alpha. design/method: following research ethics board approval, longitudinal data of patients with tdt-alpha ( males, median age . ( . - . ) were retrospectively collected. variables of interest included total pre-transfusion hemoglobin, hbh%, and "functional" hemoglobin [measured as total hemoglobin x ( -hbh/ )]. outcome variables were lactate dehydrogenase (ldh, marker of hemolysis), and soluble transferrin receptor (str, marker of erythropoiesis). hemoglobin analysis was done using high-performance liquid chromatography and capillary zone electrophoresis. we examined the association of "functional" hemoglobin with str, and hbh% with ldh, using repeated-measures anova to adjust for the effect of multiple testing. we constructed receiver operating characteristic curve and calculated the area under the curve to define the best cut-off values for variables of interests. there was a strong association between functional hb and str, as well as hbh and ldh. the optimal cut-off for "functional" hemoglobin that was associated with str < . mg/l was g/l (auc = . , sensitivity and specificity of . % and % respectively). the optimal cut-off for hbh to supress ldh to < u/l was % (auc = . , sensitivity and specificity of . % and % respectively). the optimal pre-transfusion hbh% for reduction of hemolysis was % and the optimal "functional" hemoglobin to adequately supress erythropoiesis was g/l. to meet these hbh% and functional hb targets by simple blood transfusions, patients with tdt-alpha would require a hypertransfusion regimen with a minimum pre-transfusion total hb of g/l and consequently high transfusional iron burden. an alternative approach using exchange transfusion to reduce hbh% and improve functional hemoglobin would be associated with less volume of transfusion and potentially better long-term outcome. hospital sacre coeur, milot, haiti background: initial results of work developing a pediatric sickle cell disease (scd) clinic at the hôpital sacré coeur (hsc) in milot, northern haiti were presented at aspho . the purpose of this clinic is for a pediatrician with a special interest in scd to provide scd care, advising on trait and managing disease with penicillin prophylaxis (pcn) and hydroxyurea therapy (hu) for select patients. this clinic was started in collaboration with a us based hematologist and support from yale-new haven hospital. objectives: to describe the success and challenges of providing pcn and hu in the scd clinic at hsc through a review of patient records. design/method: since this clinic's inception, a database of patients, with basic clinical information has been kept and made accessible, through 'drop-box', to the us hematologist. the records of those that presented to the clinic were reviewed. the hemoglobin diagnosis was made either by clinical history and sickle cell prep or by hemoglobin electrophoresis through alpha laboratory, port-au-prince, haiti. results: ninety-nine individuals were seen in the first years of the program. fifty-six underwent a hemoglobin electrophoresis. of these , are ≤ years old. thirty-two were started on pcn vk, of which / ( %) were ≤ years old. eleven patients were started hu therapy. all patients on hu have shown progressive increases in hemoglobin. there have been no clinical complications of hu therapy. none of the patients taking hu have required hospitalization or transfusion in . three patients (not on hu) were hospitalized in for complications of scd (osteomyelitis, pain). in , with less than half the numbers in the program, there were admissions for severe anemia, pain, stroke and splenic sequestration. with ongoing external support and a local reputation for excellence in sickle cell care, the clinic at hsc has been able to expand services and improve the health of a growing number of patients with scd. early data suggests that pcn and hu therapies are helping to reduce complications and improve quality of life. challenges to date have included lack of funding for transportation to clinics, for hospitalizations and to cover the cost of electrophoreses. at the same time as continuing providing excellent care and gathering data, it is crucial to explore opportunities for collaboration and cooperation in ways that will assure that the clinic can become independently sustainable while continuing to improve the quality of life for the individuals it serves. background: ykl- is an inflammatory glycoprotein expressed by infiltrating macrophages in various inflammatory conditions. it has been found to be elevated in patients with different pathological conditions like acute and chronic inflammations, increased remodeling of the extracellular matrix (ecm), development of fibrosis and cancer. several studies have found elevated ykl- concentrations in sera of patients with liver diseases such as hepatic fibrosis by hepatitis c virus. it has been suggested that ykl- concentrations reflect the degree of liver fibrosis. to evaluate serum ykl- levels in patients with -thalassemia and its relation to viral hepatitis, liver stiffness as assessed by transient elastography (fibroscan, fs) and hepatic iron concentration. design/method: a prospective study included patients with -tm ( males and females) with mean age . ± . years (range: - years). serum ferritin level, liver enzymes (alt and ast), hbs ag, anti hcv ab and serum ykl- using elisa kit were evaluated. all patients were subjected to liver mri t * to detect liver iron content by the sequence and transient elastography (fibroscan, fs) to assess degree of liver stiffness. results: mean fibroscan value was ( . ± . ) kpa with a median . (range . to ) kpa. ( %) patients were categorized as f - and ( %) were stage f - , ( %) patients had severe fibrosis. their median serum ferritin was ng∖ml, with ( %) patients had values exceeding g/l. median cardiac t * was . with patients had values below ms, and the median lic was . mg/g dw with patients showed readings above mg/g dw. nyl- was evaluated as a marker of inflammation and liver fibrosis and showed mean value . (± . ) pg/ml, and range from to pg/ml. mean ykl- was significantly higher among males (p = . ), patients on chelation therapy (p = . ), patients on dfs (p≤ . ), in those with abnormal liver enzymes, splenectomised patients, patients with hbv sero-positivity, those with moderate elevation of t * and patients with high grades of liver fibrosis (p< . ). ykl- showed positive correlation with the rate of transfusion, lic, ferritin, alt and ast but negative correlation with weight, height and t *. roc curve analysis revealed that the cutoff value of ykl- at pg/ml could differentiate -tm patients with and without viral hepatitis with . % sensitivity and specificity of . %, area under the curve (auc) . , positive predictive value . and negative predictive value . (p< . ). roc curve analysis revealed that the cutoff value of ykl- at pg/ml could detect -tm patients with liver cirrhosis with . % sensitivity and specificity of . %, area under the curve (auc) . , positive predictive value . and negative predictive value . (p< . ). conclusion: serum ykl- levels are elevated in patients with -thalassemia and can detect patients with active viral hepatitis and liver stiffness. background: the most common splenic complication in pediatric patients with sickle cell disease (scd) is acute splenic sequestration (ass), which has often been managed with splenectomy. although splenectomy has been a treatment of choice for years, long-term vascular complications have not been thoroughly evaluated. pulmonary hypertension (phtn) is a severe complication of scd. in adults with scd, phtn has been associated with a -month mortality rate of approximately %. it has been reported that splenectomized patients with hemolytic disorders are at even greater risk of phtn. several medications exist to treat phtn, but with few studies of their efficacy or toxicities in patients with scd. additionally, these patients are often treated with either chronic prbc transfusions or hydroxyurea (hu) to raise hemoglobin, reduce hemolysis, and prevent vaso-occlusive events. objectives: to evaluate effect of chronic prbc or hu vs. no intervention, on tricuspid regurgitant jet velocities (trv) in pediatric patients with scd and history of splenectomy. design/method: retrospective chart review of splenectomized patients with hbss followed at marian anderson center at st. christopher's hospital for children, philadelphia, between and . we analyzed trvs ( hu, prbc, and from control group receiving neither treatment) from patients ( hu, prbc, neither). mean age at echo was . +/- . . data was analyzed with linear correlations and analysis of variance (anova), including the post hoc test of least significant difference (lsd) for all pairs of treatment groups. results: trv was not significantly correlated with age at time of assessment or with time between splenectomy and trv. univariate anova among groups yielded trv means of: . +/- . cm/s (hu), . +/- . (prbc), . +/- . (neither). we found a notable difference as the mean of the hu group was almost cm/s lower than the others, but no overall statistically significant association for any of the groups exists. however, when we performed post hoc tests to adjust for multiple comparisons and looked at all pairings within the anova, we found that the lsd between the hu and the prbc groups was statistically significant (p = . ), and that a trend exists between the hu group and the neither treatment group (p = . ). our data suggests that treatment with hu is correlated with a reduction in trv in pediatric patients with scd who underwent splenectomy. given these promising results, we believe our data warrants further study with larger treatment groups. nancy olivieri, gaurav sharma, susmita nath, rajib de, tuphan kanti dolai, prakas kumar mandal, abhijit phukan, amir sabouhanian, robert yamashita, angela allen, david weatherall, prantar chakrabarti background: hemoglobin e thalassemia (hbethal), which accounts for % of all severe beta thalassemia worldwide, has an estimated prevalence of . / , in west bengal, from which little information about clinical findings has been reported. objectives: to document clinical and laboratory findings in patients with hbethal, ultimately to improve resources for clinical management. design/method: we reviewed records from: a database recording patient names; clinic charts; "special" charts containing additional details; and, in transfused patients, transfusion day-care records. additionally, because in india's public hospitals original lab/imaging reports are commonly retained at home, % of families were interviewed to provide additional information. we excluded records of patients aged < years and patients aged < years who had not been reviewed since . results: while at least one visit had been recorded in , hbethal patients at nrs hospital, most patients are not regularly reviewed there. we examined charts [ ( %) aged ≥ years; ( %) aged - years; % male], representing approximately % of regularly-reviewed patients. most families ( . %) reported monthly incomes (< , indian rupees), below the monthly cost of living ( , rupees) in kolkata. mean (±sem) hemoglobin was . ± . g/dl. % patients were receiving eight or more transfusions per year; from , % had been treated with deferasirox, . ± . mg/kg/day. iron control estimated by serum ferritin concentration ( . ± g/l) was highly variable. a total of % patients were splenectomized. a substantial obstacle to documenting complications was the lack of recording, in any of the five sources, of many relevant parameters: for example, the status of sexual maturation (normal, delayed, or absent) was documented in less than %, and measurements of fasting blood glucose in less than %, of records. where recorded, complication rates were high: delayed/abnormal sexual maturation was recorded in % patients aged > years; in the patients aged > years and those aged - years, respectively, hypothyroidism was recorded in % and %, and elevated serum alt in % and %. in most evaluable patients > years, height was measured between the rd- th percentiles. cardiac findings, rarely documented, included pulmonary hypertension and reduced left ventricular ejection fractions in a few patients. despite dedicated attention to many aspects of thalassemia care, insufficient documentation limited a clear understanding of the current morbidity in hbethal patients. investment in personnel and technology will be critical to record relevant information, ultimately to improve clinical management, over the next decade. children's hospital of richmond at vcu health, richmond, virginia, united states background: sepsis is a common cause of death in children with sickle cell disease (scd). recommendations for care of fever in children with scd include immediate medical evaluation including blood culture and initiation of broad-spectrum antibiotic therapy. the increasing availability of pcr-based respiratory pathogen panels (rpp) provide the opportunity to rapidly identify viral causes of fever. the role for rpps in identifying the source of fever in children with scd and how it affects provider practice is not well studied. ( ) to determine the epidemiology of respiratory virus-associated fever in children with scd and ( ) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all children with scd seen in our emergency department (ed) with temperature ≥ . oc at home or in the ed from january , , through september , , as well as, all febrile children for whom rpps were sent since the introduction of rpps april . we reviewed the results of blood cultures, rpps, chest radiographs, and ed notes and discharge summaries to identify sources of infections. independent t test and chi-square analysis were used as appropriate to compare results using spss©. overall, the rate of bacteremia was %. there were no cases of bacteremia among children with positive rpps. % of children with negative rpps had true bacteremia. a positive rpp did not reduce the likelihood of bacteremia (p . ). patients with bacteremia had higher presenting temperatures than those without bacteremia ( . oc vs . oc, p . ). the most common rpp findings were rhinovirus/enterovirus ( %), human metapneumovirus ( %), and influenza a ( %). sending an rpp did not affect admission rate ( % and % respectively, p . ); however, likelihood of admission was lower in patients with positive rpps ( % vs %, or . [ . - . ], p . ). length of stay (los) was shorter in patients for whom an rpp was not sent ( . vs . days, p . ). as previously reported, bacteremia in febrile children with scd is very low, but remains a serious concern, particularly in the setting of high fever (> oc). a positive rpp did not reduce the odds of bacteremia, but did have a sta-tistically significant impact on both admission rate and los. more work is needed to understand how rpp results impact provider decision-making and care for children with scd. cincinnati children's hospital medical center, cincinnati, ohio, united states background: diffuse myocardial fibrosis is a common, if not defining, feature of the heart in sickle cell anemia (sca) that is strongly associated with diastolic dysfunction. we found diffuse myocardial fibrosis in every patient in a sca cohort (n = ) ranging in age from to years (niss ). the treatment and prevention of this complication of sca has not been studied before. objectives: because diffuse myocardial fibrosis must begin in early childhood, we hypothesized that early initiation and uninterrupted use of disease-modifying therapy for sca can prevent it. design/method: we use cardiac magnetic resonance imaging (cmr) to measure the myocardial extracellular volume fraction (ecv) to quantify diffuse myocardial fibrosis in individuals with sca who have been treated, uninterrupted, with hydroxyurea or chronic transfusion therapy since ≤ years of age. two comparison groups were used: individuals with sca who have not been treated with disease-modifying therapy since ≤ years of age (n = ) and controls without sca (n = ). results: we studied individuals ( m/ f) with a mean age of . years (range - ). mean age at the start of diseasemodifying therapy was . ± . years (range - ). only had evidence of mild diffuse myocardial fibrosis (ecv . ); the other had no detectable diffuse fibrosis (all had ecv < . , the upper limit of normal). mean ecv was . ± . , which was significantly lower than the ecv of individuals with sca who have not received early uninterrupted therapy ( . ± . ; p = . ) and not statistically different from normal controls ( . ± . ; p = . ). none had macroscopic fibrosis by late gadolinium enhancement or evidence of myocardial hemosiderosis by t * imaging. no patient had diastolic dysfunction by echocardiographic classification, right heart catheterization, or both. disease-modifying therapy for sca can prevent diffuse myocardial fibrosis, and possibly diastolic dysfunction, if started in early childhood. prospective trials of disease-modifying and anti-fibrotic therapy are planned to prevent diffuse myocardial fibrosis, which can be monitored noninvasively by cmr, and improve outcomes in sca. (niss, blood, ) . background: a statewide sickle cell surveillance system (sscss) was developed with the goal of determining the prevalence of sickle cell disease (scd) in indiana and the level of care that patients receive throughout the state. persons with scd are at high risk of infection, especially with encapsulated organisms, as well as at increased complications from influenza. utilizing sscss data, the relationship between vaccination status and mortality was explored. to determine if vaccination status is associated with mortality in persons with scd. the project was granted a waiver of consent by the st. vincent irb. death certificates were obtained to identify cause of death. deceased patients (cases) were matched by age, gender, and sickle genotype to living patients (controls). vaccination data were collected from the medical record and the children and hoosier immunization registry program (chirp) through the date of death for each case. cases and controls were assigned a point for completion of the pneumococcus, meningococcus and haemophilus influenza type b (hib) vaccine series and one point if the influenza vaccine was given within a year prior to death of the cases [max vaccine status score (vss): ]. total points were compared between the cases and controls. two tailed t-tests to compare means of continuous data and wilcoxon signed-rank test to compare ordinal data. one thousand forty-eight individuals were included in the sscss. six hundred and seven ( . %) were seen at one institution and included in this analysis (mean age = years). thirty-three of the ( . %) were deceased at the time of analysis. six point one ( . )% of controls and . % of cases received a vss of . the mean vss for cases was . ± . and . ± . for controls. thirty point three ( . ) % of controls had a vss of one or more, compared to % of cases (p = . ). patients who died of infection [streptococ-cus (n = ), pseudomonas (n = ) and unidentified organisms (n = )] were not up to date on vaccination against encapsulated organisms, but two had received the influenza vaccine in the year prior to death. in this sample, mortality occurred exclusively among adult patients, which is consistent with current patterns in developed countries. among these adults, vss and mortality rates were not related. limitations to the study include small sample size and potential incompleteness of vaccine records. vaccination rates and other standard of care indicators should be explored in a larger cohort of patients to determine associations with mortality. background: sickle cell disease (scd) is a genetic disorder resulting in acute and chronic complications, including delayed puberty. delayed puberty can have adverse physical and psychosocial effects on affected children and families. there are no published reports from ghana on pubertal timing in children with scd. the aim of this cross-sectional study was to describe pubertal changes in children with scd at korle bu teaching hospital (kbth), accra, and compare these findings to those in a control group without scd. design/method: children with scd and children with hb aa, ages - years, were consecutively recruited and matched for age, sex and socioeconomic status. investigator-administered questionnaires were used to obtain demographic data for all participants and information on menarche (girls only). pubertal status was assessed by physical examination using tanner staging. testicular volumes were determined in boys using a prader orchidometer. body mass index (bmi) and socioeconomic status (ses) of participants were analyzed to determine if there were any associations with tanner stage. of the with scd, ( . %) were hb ss and ( . %) hb sc. females comprised . % (cases and controls). mean age at onset of breast development was significantly delayed in girls with scd ( . ± . years) compared to controls ( . ± . years) but there was no significant age difference at onset of pubic hair development. mean age at menarche was significantly delayed in girls with hb ss ( . ± . years) and hb sc ( . ± . years), compared to those with hb aa ( . ± . years). in boys, the mean ages at onset of puberty were significantly delayed in those with scd ( . ± . years, for genital development and . ± . years, for pubic hair development), compared to those without scd ( . ± . years and . ± . years, respectively). mean testicular volumes were significantly lower in cases compared to controls, across all age ranges (p< . ). mean bmi in both cases and controls were similar at onset of breast development in girls. however, in boys with and without scd, mean bmi values were significantly different at pubertal onset. in univariate analysis, ses was not associated with tanner stage for both genital and breast development. mean ages at pubertal onset were significantly delayed in children with scd. longitudinal studies are needed to further characterize any associations with bmi and determine potentially modifiable risk factors affecting pubertal onset in scd. background: sickle-cell disease (scd) is a life-threatening genetic disorder associated with multiple chronic and acute complications. specific monitoring and treatment for children is a major part of the medical focus, but there remains a lack of real-world evidence of the disease burden and practice patterns among the pediatric scd population. objectives: to examine the clinical burden and management of scd among pediatric patients. design/method: a retrospective claims study was conducted using the medicaid analytic extracts database from jan - dec . pediatric patients (aged < years) with scd were identified using icd- -cm diagnosis codes ( . - . , . - . ). the first observed scd diagnosis during the identification period was designated as the index date. patients were required to have continuous medical and pharmacy benefits for at least months pre-and months post-index period. patient data were assessed until the earliest occurrence of the following events: disenrollment, death, or the end of the study period. patient demographic and baseline clinical characteristics, clinical outcomes (mortality, incidence of pain crisis, complications), scd management, and healthcare utilization were examined. all variables were analyzed descriptively. results: a total of , patients met the study inclusion criteria, with a mean age of . years. most patients were black ( . %) and had a charlson comorbidity index score of ( . %). mortality during follow-up was . in personyears, and the event rate of pain crisis in the inpatient setting was . in person-years. the three most common complications after pain crisis (highest rates in person-years) were fever ( . ), infectious and parasitic diseases ( . ), and asthma ( . ). rates of life-threatening complications were also examined in person-years, including acute chest syndrome ( . ), stroke ( . ), splenic sequestration ( . ), pulmonary hypertension ( . ), and pulmonary embolism ( . ). . % of patients were prescribed antibiotics during the one-year post-index period. other frequent medications utilized among children were folic acid ( . %), nonsteroidal anti-inflammatory drugs ( . %), opioids ( . %), and hydroxyurea ( . %). . % of patients had a blood transfusion within one year post-index date. patients had frequent health care utilizations in the inpatient ( visit), emergency room ( visits), office ( visits), and pharmacy ( visits) settings during the one-year follow-up period. pediatric scd patients are burdened with a high rate of complications including pain crisis. in addition, patients utilized a substantial amount of health care resources including outpatient office care and acute care visits. background: novel use of hydroxyurea in an african region with malaria (noharm, nct ) is a randomized controlled trial of hydroxyurea for very young children with sickle cell anemia living in uganda. during year , study participants received blinded study treatment of hydroxyurea or placebo; those receiving hydroxyurea had no increased risk of malaria, but had both laboratory and clinical benefits. during year , all study participants received openlabel hydroxyurea treatment. to assess the effects of open-label hydroxyurea treatment in a very young population of children with sickle s of s cell anemia living in uganda. study endpoints included the rates and severity of malaria infections, clinical sickle-related events, and laboratory effects. design/method: all children in the noharm trial were enrolled at mulago hospital sickle cell clinic in kampala uganda. during year , all children received open-label fixeddose hydroxyurea ( mg/kg/day) for months, after previously receiving either hydroxyurea or placebo for months. results: a total of children entered year of the noharm trial and received fixed-dose hydroxyurea, including males and females, at an average age of . ± . years. among children previously on placebo, there were malaria events in children, including with severity grade ≥ , and three deaths (two acute chest syndrome, one sepsis). clinical adverse event rates dropped from . to . per patient year, and hospitalizations were reduced from to . expected hematological benefits of increased hemoglobin, mcv, and fetal hemoglobin, along with decreased neutrophils and reticulocytes, were rapidly achieved. laboratory adverse events were infrequent at . events per patient-year, and only half of those were dose-limiting hematological toxicities. among children previously on hydroxyurea, there were malaria events in children, including with severity grade ≥ , and two deaths (one acute chest syndrome, one sepsis). clinical adverse event rates and hospitalizations were maintained at low rates, the hematological benefits of hydroxyurea continued throughout the extended treatment period, and dose-limiting toxicities remained infrequent. fixed-dose hydroxyurea treatment of young children with sickle cell anemia living in uganda is associated with no increased risk for malaria. clinical and laboratory benefits occur, including children previously on placebo who crossed-over to hydroxyurea treatment. future studies should focus on the optimal dosing and monitoring strategies, in an effort to determine the overall feasibility and safety of introducing hydroxyurea therapy across sub-saharan africa. background: acute chest syndrome (acs) is the second most common cause of hospitalization in patients with sickle cell disease and is a leading cause of morbidity and mortality. in mid- , an algorithm was implemented at cohen children's medical center to initiate transfusions within four hours of diagnosis of acs in order to improve patient outcomes. objectives: the aim of this project was to analyze the effect of early blood transfusion on the outcomes of patients with acs. we focused on the number of total transfusions, need for exchange transfusion, need for intensive care unit (icu) stay, and length of hospitalization. design/method: a retrospective chart review was completed on patients admitted to ccmc with a primary diagnosis of sickle cell disease and a secondary diagnosis of either acs or pneumonia during the years of - . data from the three years directly prior to implementation of the algorithm was compared to data from the three years directly after implementation of the algorithm. a total of patients were analyzed, of which belonged to the pre-algorithm group and to the postalgorithm group. patients from the post-algorithm group had a higher incidence of transfusions ( % with a mean transfusion number of . pre versus % with a mean of . post) as well as exchange transfusion ( % pre versus % post). the post-algorithm group had a shorter overall length of stay (mean of . days pre versus . days post). while the overall percentage of patients requiring an icu admission was similar in each group ( % pre versus % post), the post-protocol group had a lower likelihood of requiring an icu admission for reasons outside of line placement for exchange transfusion, most commonly for icu-level respiratory support ( % pre versus % post). despite a higher total number of transfusions, early recognition and transfusion for acs can lead to decreased lengths of hospitalization as well as decreased need for icu-level respiratory support. further studies comparing different center's clinical practice guidelines are necessary to improve the standard of care. background: novel use of hydroxyurea in an african region with malaria (noharm) was the first placebocontrolled randomized clinical trial of hydroxyurea in sub-saharan africa. in noharm, young children with sca received either hydroxyurea or placebo during year , followed by open-label hydroxyurea for all study participants during year . an ancillary noharm project was designed to determine if hydroxyurea treatment lowers transcranial doppler (tcd) velocities and possibly reduces stroke risk in this very young cohort. objectives: to perform tcd screening on the noharm cohort, measuring the time-averaged mean velocity (tamv) at the end of both year and year . we hypothesized that the maximum tamv would be lower for noharm study participants receiving hydroxyurea compared to those receiving placebo, and that key clinical and laboratory parameters would also influence tcd velocities. design/method: all children enrolled in noharm were eligible to undergo tcd examination at two study time points: month - when they were completing the blinded treatment phase, and again at month - at the end of the open-label treatment phase. tcd measurements included tamv readings from the main intracranial arteries: middle cerebral artery, distal internal carotid artery, and bifurcation on tcd. all tcd examinations were scored and classified as normal (less than cm/sec), conditional ( - cm/sec) or abnormal (greater than or equal to cm/sec), with higher scores correlating to greater risk of stroke. results: at the end of year , tcd exams were conducted of which were suitable for analysis ( hydroxyurea, placebo). based on the maximum tamv, the median velocity was cm/sec (iqr - ) for children on hydroxyurea and cm/sec (iqr - ) on placebo, p = . . maximum tamv values had negative correlations with hemoglobin concentration (- . ), fetal hemoglobin (- . ), and oxygen saturation (- . ); positive correlations were noted with age ( . ) and absolute neutrophil count ( . ). at the end of year , tcd exams were conducted and all were suitable for analysis; the median velocity was cm/sec on open-label hydroxyurea treatment, regardless of previous blinded treatment. all correlations with tamv were maintained except for age. conclusion: compared to placebo, hydroxyurea treatment for young children with sca living in uganda was associated with lower tcd velocities, which have been correlated in other studies with lower risk of primary stroke. tcd velocities were correlated with hematological and clinical parameters that can be improved by hydroxyurea therapy. children's hospital of richmond at virginia commonwealth university, richmond, virginia, united states background: acute chest syndrome (acs), defined by respiratory symptoms and a new pulmonary infiltrate, is a serious complication of sickle cell disease (scd). acs can occur during hospitalization for non-pulmonary conditions, such as a vaso-occlusive crisis or after surgery. nih clinical practice guidelines encourage incentive spirometry (is) which decreases the incidence of acs. it is additionally widely accepted that early, frequent ambulation in post-operative and pneumonia patients decreases the length of stay (los). to decrease acs events in children with scd at our children's hospital, we aimed for is use in % of ageappropriate pediatric sickle cell admissions. design/method: a multidisciplinary team examined inpatient acs prevention practices, including is, at children's hospital of richmond. key drivers were identified, including educational awareness of patients and healthcare staff, order placement, and documentation. we aimed for all scd patients ≥ months of age hospitalized with any admission diagnosis to participate in is with the use of a traditional incentive spirometer or similar age-and ability-appropriate devices (e.g. positive expiratory pressure devices, bubbles, and pinwheels). we secondarily aimed to increase activity events, specifically ambulation and out of bed time. educational and outreach tools included patient informational brochure and incentive program, and staff informational sessions and reference materials at workstations. a disease-specific order set was implemented including desired is and activity orders. data were collected prospectively may through november , during which pdsa cycles were conducted. admissions during the corresponding months of the previous year were reviewed for comparison. independent t-test analysis was performed using graftpad prism statistical analysis software. results: improvements reaching statistical significance included increase in is order placement from % to % of admissions (p < . ), and admissions with documented is use increased from % to % (p < . ). los decreased from a mean of . days to . days (p . ). post-admission development of acs also decreased from % to % of admissions, but did not reach statistical significance (p . ). there was an additional increase in appropriate activity order placement and documentation of activity events. conclusion: improving education and outreach to patients and staff, including implementation of a disease-specific order set, can improve is use and activity events. the decline seen in incidence of acs development during hospitalization, though not statistically significant, and the decreased los are encouraging, and efforts continue to improve on these trends. background: painful vaso-occlusive crises (voc) are a frequent and debilitating complication of sickle cell disease (scd) and are thought to occur due to progressive blockage of the microvasculature with rigid sickle shaped red blood cells. any trigger that decreases the microvascular blood flow (mbf) can promote entrapment of sickled cells in the microvasculature and progression to voc. exposure to cold wind and changes in weather are common triggers of voc and are associated with increased frequency of hospitalizations for pain in patients with scd. there is limited experimental data on the physiologic effects of these factors on peripheral perfusion in scd. to study the effect of graded thermal stimuli on the peripheral mbf in scd. design/method: scd and control (healthy or sickle trait) subjects aging to years were exposed to their individual threshold temperatures for heat and cold detection, heat and cold pain via tsa-ii thermode that was placed on the thenar eminence. mbf was measured on the contralateral thumb using photo-plethysmography (ppg). the vasoconstriction response within the complex ppg signal was detected using cross-correlation technique. mean mbf was derived from the ppg amplitude during each of these stimuli and compared to baseline mbf. cross correlation analysis showed that cold pain caused significant vasoconstriction response in % of the subjects, followed by heat pain ( %), cold detection ( %) and heat detection ( %).there was a significant drop in the mbf during cold pain (p < . ), heat pain (p < . ), heat detection (p = . ) and cold detection (p = . ) when compared to baseline mbf, with cold pain causing the greatest drop in mbf. thermal sensitivity and mbf responses were comparable between scd and controls. conclusion: exposure to graded thermal stimuli causes a progressive drop in mbf with exposure to cold pain eliciting the strongest vasoconstriction response. vasoconstriction occurred in the contralateral hand at an average of seconds after the stimuli, suggesting a neurally mediated mechanism. although there was no significant difference in vasoconstriction responses between scd and controls, the drop in mbf in patients with sickle cell disease can increase the likelihood of entrapment of the sickled red blood cells, leading to vaso-occlusion. these findings are consistent with extensive reports in literature that exposure to cold weather is associated with a higher frequency of voc. this suggests that neurally mediated vasoconstriction is likely an important factor in the pathophysiology behind cold exposure leading to voc in scd. background: vaso-occlusive crisis (voc) is a major cause of hospital admissions in children with sickle cell disease (scd). although the use of clinical biomarkers in voc has been studied, especially with regards to acute chest syndrome (acs), there is less data regarding overall voc severity prediction. in addition new biomarkers such as platelet to lymphocyte ratio (plr), neutrophil to lymphocyte ratio (nlr), and lymphocyte to monocyte ratio (lmr) have been little studied with regards to scd. objectives: to identify whether admission laboratory values, changes from well baseline laboratory values, and new biomarkers such as plr, nlr, and lmr could predict severity of vaso-occlusive crisis in children with sickle cell disease admitted with voc. design/method: this was a retrospective single center observational study of admissions of voc in children aged - years with hbss or hbs-b thal from september to november excluding those on hyper-transfusion protocol or having an admission diagnosis of acs. univariate analysis was done using student's t-test, mann-whitney non parametric test, or fischer's exact test as appropriate depending on the distribution between admission laboratory data of complete blood count (cbc), reticulocyte count, comprehensive metabolic panel, lactate dehydrogenase (ldh), change from well baseline cbc values within months previously, plr, nlr, lmr, and the development of complicated voc. complicated voc was defined as the development of secondary acute chest syndrome, prolonged admission duration > days ( hours), requirement of blood transfusion, and readmission within days. results: a total of admissions were studied. fifty-nine ( . %) were female. of the , ( . %) were complicated with no significant differences in sex (p . ) or age (p . ). univariate analysis revealed significant elevations in total bilirubin (p . ), ldh (p . ), and platelet count (p . ) in those with complicated voc. there is also significant difference in the percentage change of platelet count from baseline with greater decline in uncomplicated voc (p . ). there were no significant differences in plr (p . ), nlr (p . ), or lmr (p . ). conclusion: elevations in total bilirubin, ldh, and platelet count in admission laboratory values are associated with developing complicated voc. in addition, those with complicated voc present with significantly less decline in platelet count from baseline well cbc. plr, nlr, and lmr do not seem to be useful predictive biomarkers for severity of voc. background: sickle cell disease (scd) causes health problems of varying frequency and severity. the only validated biomarker for children with scd is transcranial doppler. if reliable predictors existed for scd severity, children with scd could be treated according to risk category. many patients with scd face psychosocial or economic hardships, but these factors have not been evaluated as risk markers for medical or functional severity of scd. objectives: the goal of this project was to develop and stratify a preliminary list of psychosocial risk factors for health outcomes that could be used as scd severity predictors. st. vincent institutional review board. a list of potential psychosocial risk factors for adverse health outcomes was compiled based on assessment materials utilized by the sickle safe program (indiana's hemoglobinopathy newborn screening follow-up program). this list of items was distributed to child abuse prevention ( ) and scd ( ) experts, who ranked each item on a likert scale of (least important) to (most important). mean scores were calculated using spss version ; assessments were retrospectively analyzed to determine psychosocial risk factor frequency. risk factors occurring in ≥ % of homes were considered high frequency events. overall, there was high agreement among experts on the risk factors that were considered the most important predictors of severe scd outcomes. the risk factor with the highest frequency ( %) was eligibility for public assistance programs. fifteen risk factors were rated ≥ by the experts. four ( . %) were high frequency events occurring in ≥ % of homes: a child with hbss or hbs thalassemia not taking hydroxyurea ( %); parent report that they had treated a fever (> ®f) at home in the past months ( %); tobacco use by someone in the household ( %); and the family reporting significant psychosocial stressors in the past year ( %). tobacco use in the home was significantly correlated with several other risk factors (smoking during pregnancy [r = . ], other health concerns in the child [r = . ], and child having health insurance [r = - . ]), suggesting that it is part of a constellation of health risk. in general, the risk factors that were rated as most important for health outcomes occurred less frequently in the sample. this study represents important progress toward identifying a group of psychosocial risk factors for scd severity, which is a necessary first step for future investigation of empirical relationships between candidate risk factors and scd outcomes. unitversity of cartagena, cartagena, bolivar, colombia s of s background: sickle cell disease is an autosomal recessive disorder characterized by a mutation in the -globin chain, which produces hbs. acute and chronic complications as aplastic crisis, acute chest syndrome, priapism, stroke, leg ulcers and primary/secondary prevention of stroke can be treated with simple transfusion or exchange transfusion. the latter offers advantages as lower iron overload, post-treatment hbs goal control, lower viscosity and improved microvascular circulation. but it is not a widely-used option because is associated with technical difficulties. objectives: standardization of a new partial exchange transfusion protocol in a group of patients with sickle cell disease, within the framework of a chronic transfusion program. design/method: this is a prospective descriptive study, which included patients under years with sickle cell disease ( hbss, hbs-tal), with indication of partial exchange transfusion in a chronic transfusion program, according to the institutional protocol; patients who fulfilled the inclusion criteria were enrolled in the study between february and december . a registry of the medical and technical complications was made in each of the procedures. a database was constructed in excel, and the graph-pad prism® version oc software was used for statistical analysis. the sequence is as follows: isovolemic phlebotomy and transfusion of packed red cells. depending of the recent hemoglobin level ( hrs), we do the phlebotomy there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg; isovolemic solution (ns , %) there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg and packed red cell transfusion there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg. the safety of this exchange transfusion protocol was analyzed in patients with sickle cell disease ( procedures). there were no differences in the sex distribution, and the median age was years. % of the population was homozygous. the indication of transfusion was . %( / ) primary stroke prevention, . %( / ) secondary stroke prevention and . %( / ) was other reason. a low percentage of complications was found ( . %); of which, those of medical origin (hypotension and nausea/vomiting) were only presented in . % of the total procedures. the standardization of this protocol was safe and its use could be extended to other low-income centers that treat patients with sickle cell disease that need chronic transfusion program including patient with hemoglobin level until gr/dl. we suggest do studies for measure the security and efficacy of this protocol in patients with acute complications. background: clinical trials that aim to achieve pain reduction have challenges achieving clinical endpoints as pain has no quantifiable biomarkers and may be unrelated to scd. furthermore, the threshold of seeking medical care differs between patients and vocs that occur at home are missed. we present a non-interventional, longitudinal study to identify vocs in patients with scd. objectives: to examine the longitudinal relationship between pros and biomarkers in subjects with scd before, during, and after a self-reported voc event, in order to build a model of in-home and clinical voc and to collect longitudinal pros and biomarker data from subjects that span voc events in the home, clinic and the hospital. design/method: longitudinal measures of pain, fatigue, function, activity, and biomarkers from scd patients in steady state and voc were studied over a six month period. patients self-reported pain, fatigue, function, and medication use using a novel epro tool. voc was reported in real-time, triggering a mobile phlebotomy team. blood was collected sequentially after self-reported voc (at home or hospital). blood samples were drawn two days after resolution of voc, as reported by the patient. during non-voc periods, blood was drawn every weeks to establish a baseline. biomarkers included leukocyte-platelet aggregates and circulating microparticles, cell and soluble adhesion molecules, cytokines, inflammatory mediators and coagulation factors. patients wore an actigraphy device to track sleep and activity and rest. results: twenty-seven of thirty-five patients experienced a total of days with voc > hr, of which only days resulted in healthcare utilization. voc days had significantly higher pain and fatigue scores. voc days were associated with significantly decreased functional scores, with significantly greater decreases during vocs requiring medical contact compared to at-home vocs. different activity profiles were identified for non-voc, at-home voc and medical contact voc days by actigraphy monitoring. at-home voc days exhibited increased daytime resting compared to non-voc days. medical contact vocs had decreased average and peak activity, and increased daytime resting compared to non-voc days. a sleep fragmentation index trended up for both at-home ( %) and medical contact voc days ( %). significant changes during voc days were observed in: c-reactive protein ( % increase), nucleated rbc ( % increase), monocyte-platelet aggregates ( % increase) and neutrophil-platelet aggregates ( % increase), interleukin- ( % increase), interleukin- ( % increase) and tnfalpha ( % increase). the identification and assessment of at-home vocs through use of epros, actigraphy and biomarkers is feasible as demonstrated by this innovative at-home study design. background: risk-stratifying sickle cell disease (scd) patients and demonstrating response to disease-modifying therapies is challenging due to the phenotypical heterogeneity of scd. a pathogenic role for procoagulant von willebrand factor (vwf) via excess vwf high molecular weight multimers (hmwm) has been proposed, with variable reports of increased vwf and hmwm in crisis vs. steady-state in adults, but less so for vwf in children with scd. moreover, vwf and multimers have not been studied in sickle trait. objectives: our pilot study evaluated the potential for vwf antigen (vwf:ag) and hmwm on densitometric tracings to serve as biomarkers for disease severity or treatment response in children and young adults with scd compared to sickle trait (hbas) siblings. design/method: we evaluated vwf:ag, vwf multimers and retrospective clinical data from hbss, hbsc and hbas subjects at steady state. one hbsc subject also had a crisis sample. median scd age was years ( . - . years). % were female. scd severity was judged by annual vasoocclusive and acute chest events, or stroke/elevated tcd. eight of ( hbss and hbsc) took hydroxyurea. four hbss subjects had severe scd, all of whom were chronically transfused. results: mean vwf:ag (normal - iu/dl) was higher for hbss ( +/- . ) and severe hbss ( +/- . ) compared to hbsc ( +/- . , p = . and . , respectively); however, lacked statistical significance when compared to hbas ( +/- . , p = . and . , respectively). vwf:ag was elevated in / ( %) steady-state, including / ( %) with "severe" disease on chronic transfusion and / ( %) taking hydroxyurea, in hbsc crisis but no hbsc / ( %) at baseline. vwf:ag was high in / ( %) hbas siblings. four ( %) had increased hmwm at baseline: hbss/severe disease/chronic transfusion, hbss/hydroxyurea and hbsc untreated. hmwm were increased only during vaso-occlusive crisis in hydroxyureatreated hbsc subject. no ultra-large hmwm were observed. in this preliminary study, in young scd subjects, vwf:ag trended higher in hbss vs. hbsc and in severe hbss participants at a single time-point, but serial evaluations at baseline, in crisis and with optimized diseasemodifying therapy are needed to determine the potential of vwf:ag and hmwm as biomarkers for severity or treatment response. surprisingly, vwf:ag was high in some sickle trait subjects. since hbas is associated with some health challenges such as increased thrombosis risk, further examination of vwf and endothelial dysfunction in sickle trait may provide novel insights into its role as a biomarker. background: the national heart lung & blood institute(nhlbi) guidelines for acute management of voe recommends rapid evaluation and treatment of pain, including administration of a parenteral opioid within -minutes of triage or -minutes from registration, pain reassessment & repeat opioid delivery within - -minutes. inf use has been increasing in peds due to its rapid onset and ease of administration. objectives: to evaluate ped utilization of inf & its effect on intravenous (iv) opioid administration and pain control for the treatment of voe. design/method: a retrospective review of emr was performed on children with scd± years presenting to a ped with voe (pain scores on a - scale) from jan-june . variables studied were median time (iqr, %ci) from ped arrival to first-parenteral-opioid-administration, time-to-first-iv-opioid, first & final pain score, disposition and readmission rate. time-to-first-iv-opioid was also compared to historical data (jan-dec ,n = ) prior to inf protocol initiation. . additionally, % patients received iv opioids within minutes of ed arrival in the inf+iv opioid vs. % in the iv opioids alone group (p< . ). no differences in -hour-returnrates were found in any of the groups, including inf alone group. conclusion: use of inf in the ped for voe is an excellent strategy to shorten time-to-first-parenteral-opioidadministration, improve pain scores & improve adherence to the nhlbi guidelines. however we had distinct unexpected findings: ( ) delays in iv opioid delivery after inf use & ( ) inf alone appeared to provide sufficient pain control without iv opioids for disposition home in % of voe patients. whether the latter reflects insufficient pain management or that there is a milder subgroup for whom inf alone is sufficient, requires further investigation. this study illustrates our experience with a ped-based inf protocol in terms of unanticipated delays in iv opioids and also discharges after inf alone. efforts are underway to further improve use of inf in voe management. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: folate supplementation is commonly included as standard management in patients with sickle cell disease. however, clear evidence supporting the clinical benefits of this practice is lacking. a single study demonstrated improvement on the occurrence of repeat dactylitis at a higher dose of folic acid. to compare clinical outcomes in pediatric patients with sickle cell disease treated with folate supplementation versus those who were not. design/method: this study was a retrospective chart review that included patients to years old with sickle cell disease type ss and s followed at st. christopher's hospital for children. data collected included information about folate supplementation, red cell indices and the presence or absence of clinical outcomes including vaso-occlusive crisis requiring hospitalization in the last six months, acute chest syndrome, infections, asthma, sleep apnea, nephropathy, cerebral vascular disease, stroke and avascular necrosis. analysis of variance (anova) was used to evaluate mean differences between age, number of infections, number of voc events, hemoglobin, reticulocyte count, and mean corpuscular volumes. additionally, chi square analysis was implemented to evaluate differences in folate and non-folate groups for left ventricular remodeling (lvr), sickle cell nephropathy, asthma, obstructive sleep apnea (osa), nocturnal hypoxia, and avascular necrosis (avn). mean differences between the folate and non-folate groups were compared for patients on and off hydroxyurea therapy. one hundred and seven patients met inclusion criteria following review of clinical data. of the patients included in the study, patients were found to be taking folate ( %), while patients were not ( %). statistical analysis showed that there were no significant differences in the incidence of clinical outcomes between patients on folate versus those who were not on folate. of the patients who were not on hydroxyurea, hemoglobin levels were significantly higher in patients on folate versus those who were not (p = . ), but not significantly different for the patients on hydroxyurea. this study suggests that folate supplementation makes no significant impact on the red blood cell indices of anemia nor on the incidence of adverse clinical outcomes in children with sickle cell disease. however, a larger prospective study is needed to guide future considerations for folate supplementation in sickle cell patients in the clinical setting. background: tanzania ranks rd globally for the number of infants born annually with sickle cell disease (scd) but lacks a national newborn screening program. the prevalence of sickle cell trait (sct) and scd is highest in the northwestern regions around lake victoria served by bugando medical centre (bmc) a teaching and consultancy hospital in mwanza. bmc also houses the hiv early infant diagnosis (eid) laboratory that tests dried blood spots (dbs) from hivexposed infants. dbs can be tested for hiv and then retested for sickle cell trait and disease. to determine the prevalence of sickle trait and disease by region and district in northwestern tanzania using existing public health infrastructure. secondary objectives explored associations between sct, scd, malaria and hiv. design/method: the tanzania sickle surveillance study (ts ) is a prospective year-long cross-sectional study of hivexposed infants born in northwestern tanzania, whose dbs collected by the eid program are tested at bmc and available for further testing of sct and scd. samples from children ≤ months of age were tested by isoelectric focusing (ief) and scored independently by two tanzanian staff as normal, sct, scd, variant, or uninterpretable. dbs samples scored as disease or variant were repeated. over the course of months, ief gels have been run. a total of , dbs samples have been scored, including , from children less than -months old. the overall prevalence of sct is . % and the prevalence of scd is . %, along with . % hemoglobin variants. quality of the laboratory results is extremely high, with only . % dbs samples yielding an uninterpretable result. geospatial mapping of the first , samples revealed a regional scd prevalence ranging from . % up to . % among the regions served by bmc. the prevalence of sct and scd is very high in northwestern tanzania. geospatial mapping will identify high prevalence areas where targeted newborn screening can be started using existing public health infrastructure with minimal start-up cost and training. further data will enhance the accuracy of the map to the district level. background: pediatric patients with sickle cell disease (scd) could develop obstructive, restrictive or mixed abnormalities of pulmonary function (pf). several publications report progressive worsening of pf over time, which could lead to severe morbidity in adult patients with sickle cell disease. in adults with sickle cell anemia up to - % of mortality is related to lung disease. early intervention aimed at improvement of lung function could significantly decrease morbidity and possibly improve life expectancy. among disease modifying approaches commonly used in scd are hydroxyurea (hu) and chronic prbc transfusions. both interventions lead to increase of hemoglobin, decrease of hbs fraction, leading to decreased hemolysis. reports of effect of hu on pulmonary function are conflicting with some suggesting no effect and others proposing a slower decline of pulmonary function. the goal of our study is to evaluate effect of disease modifying therapies, like hu and chronic prbc on change of pulmonary function in pediatric patients with sickle cell disease. design/method: this study utilized a retrospective chart review of children with scd who had multiple pfts. we analyzed pfts from patients done during clinic visits. scd patients were divided into three treatment groups: hydroxyurea, chronic transfusions or neither. data was analyzed with linear correlations and analysis of variance (anova). comparison were made between the three groups specifically observing the changes in absolute numbers on pfts over time using the first and last pft the patient had. results: there were a total of patients with multiple pfts (ranging from - ); control ( ), hydroxyurea ( ) and chronic transfusion ( ). the mean changes of the control, and hydroxyurea for the pft parameters fev (- . the chronic transfusion group demonstrated a small improvement in pfts over time for fev ( . ), fvc ( . ), fef - ( . ), however there was a decline in fev /fvc (- . ). however, there was no statistically significant (p-value < . ) in the difference in any pfts parameters between any of the groups. in children with scd there is a decline of pf parameters over time. although no significant differences were seen between the three groups it appears chronic transfusion may improve or limit the decline in pfts. larger studies need to be done to evaluate difference in pf decline in patients with scd patients. background: the use of mobile technology in health care has been a growing trend. patients with chronic diseases such as sickle cell disease (scd) require close monitoring to provide appropriate treatment recommendations and avoid complications. we conducted a feasibility study for patients with scd hospitalized for pain using our self-developed mobile application (tru-pain: technology resources to better understand pain) and a wearable activity tracker. subjective symptoms such as pain and objective data such as heart rate (hr) were measured. we aimed to ) correlate nursing recordings with mobile technology recordings; ) get feedback from patients about usability. design/method: we enrolled patients with scd > years old and < hours from admission for uncomplicated vasoocclusive crisis, excluding patients admitted to icu. patients were given an ipad and a wearable device. they were instructed to record in the application at least once per day and to keep the wearable on, removing only to charge. prior to discharge, patients completed a feasibility questionnaire. we enrolled patients, % females, median age . (range to ) who were admitted for a median days (range to ) for uncomplicated pain crisis. patients used the application throughout hospitalization and made one entry/day (range to ). pain scores recorded via tru-pain correlated well (r = . , p< . ) with pain scores recorded in emr. there was an average of , data points recorded per day, by the wearable, with a maximum of , data points/day. the median amount of hours of wearable data per day was . (maximum of . ). the hr recorded via the wearable correlated significantly with the hr recorded in emr (r = . , p-value < . ). as for usability, % of patients indicated never having a problem with the technology, % found tru-pain 'very easy' or 'somewhat easy' to use, and % were 'very satisfied' with their participation in the study, indicating that it helped them track their pain. our pilot study during hospitalization shows strong potential for using tru-pain for patients with scd. pain data from application and hr from wearable correlated well to the emr data. according to the feedback received, our application was easy to use and helped patients track their pain. despite limitations of battery life, the use of wearable technology is feasible, providing additional data such as activity. we are optimistic that we can continue to improve our tru-pain system to help improve care in patients with scd. background: hydroxyurea, chronic blood transfusion, and bone marrow transplantation can reduce complications, and improve survival in sickle cell disease (scd), but are associated with a significant decisional dilemma because of the inherent risk-benefit tradeoffs, and the lack of comparative studies. these treatments are underutilized leading to avoidable morbidity and premature mortality. there is a need for tools to provide patients high-quality information about their treatment options, the associated risks, and benefits, help them clarify their values, and allow them to share in the process of informed medical decision making. objectives: to develop a health literacy sensitive, web-based, decision aid (ptda) to help patients with scd make informed choices about treatments, and to estimate in a randomized clinical trial the acceptability and effectiveness of the ptda in improving patient knowledge, involvement in decisionmaking and decision-making quality. design/method: we conducted qualitative interviews of scd patients, caregivers, stakeholders, and healthcare providers for a decisional needs assessment to identify decisional conflict, knowledge, expectations, values, support, resources, decision types, timing, stages, and learning, and personal clinical characteristics, and to guide the development of a ptda. transcripts were coded using qsr nvivo . stakeholders completed alpha and beta testing of ptda. we conducted a randomized clinical trial of adults, and of caregivers of pediatric patients to evaluate the comparative efficacy of the ptda, vs. standard of care. results: ptda (www.sickleoptions.org) was developed per decisional needs described by stakeholders and finalized following alpha testing, and beta testing by and stakeholders respectively. in a randomized trial of subjects considering various treatment options, qualitative interviews revealed a high level of usability, acceptability, and utility in education, values clarification, and preparedness for decision making of the ptda. a median % rated the acceptability of ptda as good or excellent and provided narrative comments endorsing the acceptability, ease of use, and utility in preparation for decision making. the ptda met international standards for content, development process, and efficacy with the exception of having a full range of positive and negative experiences in patient stories. compared to baseline ptda group had statistically significant improvement in preparedness for decision making (p = . ) and informed subscale of decisional conflict (p = . ) but not for decisional self-efficacy, knowledge, choice predisposition, or stages of decision-making. a ptda for patients with scd developed following extensive engagement of key stakeholders was found to be acceptable, useful, easy to use, to improve preparedness for decision making, and decrease decisional conflict. background: painful vaso-occlusive crisis (voc) accounts for the majority of emergency department (ed) visits and hos-pitalizations in sickle cell disease (scd). we are interested in studying mental stress and associated autonomic nervous system (ans) imbalance that cause vaso-constriction as possible triggers of scd pain. to this end, we developed a mobile phone application (app) to record daily pain frequency and intensity as clinical endpoints that might be predicted by ans parameters measured in the laboratory. in particular, we think that the aura may represent ans instability that precedes or even triggers change in blood flow and voc. objectives: to assess the feasibility of using an app to evaluate frequency and severity of voc and its potential association with mental stress and presence of aura. design/method: an app was developed for both ios and android systems to allow patients to track pain, stress, and aura. the idea was to create an app that was easy to use with the intent to only capture pain episodes, rather than detailed description of the pain. all scd patients were eligible and a parent version was available for younger children. de-identified data was automatically transferred to a hipaa compliant database via a cloud-based server interfaced to the main research project database. a feedback questionnaire was implemented after at least a month of utilization to assess usability. of the scd patients enrolled, participants utilized the app and of the participants that provided feedback indicated the app was easy to navigate. the mean pain scale was out of (standard deviation . ) for those that entered they had pain that day. although the mean stress level was out of , there was a statistically significant correlation between increasing stress levels and increasing pain scores (p < . ). aura was reported by patients, with patients reporting more than episodes. moreover, on days aura was present there was greater incidence that pain was present as well (p < . ). however, there was no statistically significant association between pain intensity and presence of an aura (p = . ). conclusion: consistent with prior research, reported pain intensity is significantly associated with reported stress intensity. although there was an association between presence of aura and pain, it did not seem to correlate with pain intensity. this uniquely designed app can monitor scd pain clinically and help understand the role of sickle dysautonomia in the genesis of scd pain. university of florida college of medicine, gainesville, florida, united states background: evidenced-based guidelines recommend the emergent evaluation of fever in children with sickle cell disease (scd). as the prevalence of bacteremia has decreased, outpatient management has become more common. however, fever can sometimes herald other complications of scd, such as acute chest syndrome, vaso-occlusive pain crisis, splenic sequestration, or aplastic crisis. institutional practices regarding fever management in scd remain variable, and little is known about the clinical outcomes of children hospitalized for uncomplicated fever. objectives: the primary objective was to determine the rate of bacteremia or scd-related complications per febrile episode in children with scd admitted to a single institution between january and june for uncomplicated fever. this was a retrospective cohort study of febrile patients up to years of age with scd, any genotype, admitted to the university of florida during the defined study period. eligible patients were identified by a database search using admitting diagnosis codes for scd and fever based on the international classification of diseases th and th revisions. encounters were manually reviewed to confirm eligibility. patients were excluded if they had other indications for hospitalization apparent at the time of admission, such as an acute vaso-occlusive episode requiring parental narcotics, asthma exacerbation, or additional complications of scd. the database search identified encounters, of which were excluded based on confounding indications for hospitalization. sixty-three eligible patients accounted for hospitalizations. the median age was years (range weeks- years); . % were male. mean duration of hospitalization was . days (range - days). eight positive blood cultures were identified; six of these were classified as contaminants. bacteremia or the development of a scd-related complication was identified in ( . %) admissions. these included acute chest syndrome (n = ), bacteremia (n = ), splenic sequestration (n = ), and red cell transfusion (n = ). exploratory analyses of potential predictors of bacteremia or scd-related complications showed no association with the presenting white blood cell count or degree of fever (p = . ). of the patients classified as having a scd-related complication, % had hemoglobin ss disease and % had at least one prior documented complication. % of the patients transfused had at least one prior transfusion. conclusion: while improvements in preventative care have substantially lowered rates of bacteremia in children with scd, fever warrants careful evaluation for other acute scdrelated complications. providers should consider inpatient observation in select cases. additional studies are warranted to define subsets of patients suitable for outpatient fever management. background: children with sickle cell disease (scd) exhibit lower neurocognitive functioning than healthy peers, even in the absence of stroke. among the domains commonly affected, working memory (wm) seems particularly affected by disease processes and wm deficits have significant implications for academic achievement and disease selfmanagement. few interventions to improve working memory in pediatric scd have been evaluated. to determine the effects of cogmed, a homebased computerized wm training intervention, in children with scd using a randomized controlled trial design. design/method: participants (ages - ) with scd completed a baseline neuropsychological assessment and those with wm deficits were randomized to either begin cogmed immediately or enter an -week waitlist. cogmed is a homebased intervention completed on an ipad that consists of increasingly challenging exercises targeting visual-spatial and verbal wm, practiced over sessions. at the end of training, participants completed a post-intervention neuropsychological assessment, including tests of visual-spatial and verbal wm from the wechsler intelligence scale for children-fifth edition (wisc-v). results: ninety-one participants (m age = . , sd = . ; % female; % hbss) enrolled in the study; % (n = ) exhibited wm deficits and were randomized to either begin cogmed immediately or wait - weeks before starting cogmed. among those that have received the intervention and reached the end of their training period (n = ), participants ( %) completed at least cogmed sessions, ( %) finished at least sessions, and finished at least sessions ( %). the mean number of completed cogmed sessions was . (sd = . ). paired samples t-tests revealed significant improvements on the working memory index (t[ ] = - . , p = . ) and on the digit span (t[ ] = - . , p = . ), and spatial span-backward (t[ ] = - . , p = . ) subtests. improvements were especially pronounced for participants completing at least sessions. partial correlations controlling for respective baseline scores indicated that the number of cogmed sessions completed was positively correlated with post-test scores on digit span (r = . , p = . ) and spatial span-backward (r = . , p = . ) subtests. among participants who completed at least cogmed sessions, % scored in the average range or higher on the working memory index at the post-intervention assessment, compared to % at baseline. results support the efficacy of cogmed in producing significant improvements in wm. a dose-effect was observed such that participants who completed more cogmed sessions had greater improvements in wm. home-based cognitive training programs may ameliorate scd-related wm deficits but methods for motivating and supporting patients as they complete home-based interventions are needed to enhance adherence and effectiveness. background: sickle cell disease is associated with myriad complications that lead to significant morbidity and early mortality. hydroxyurea has been used successfully to reduce the incidence of these complications and has led to significant improvements in quality and duration of life. at children's minnesota we recommend hydroxyurea in all patients with hb ss/s thalassemia as early as months of age with a goal of starting all patients before months of age. objectives: the purpose of this study was to evaluate the use of hydroxyurea therapy in young patients with sickle cell disease, with particular attention to those children less than one year of age. design/method: a retrospective chart review was conducted on patients less than years of age with sickle cell disease who began hydroxyurea therapy between january , and december , . the study population was divided into three cohorts based upon age at hydroxyurea initiation: cohort ( - year), cohort ( - years), and cohort ( - years). outcomes included laboratory data, clinical events (hospitalization, dactylitis, pain crisis, transfusion, splenic sequestration, acute chest syndrome), and toxicity occurring in the first years of life. results: a total of patients were included in cohorts (n = , mean age . months), (n = , mean age . months), and (n = , mean age . months). patients in cohort had higher hemoglobin (p = . ) and mcv (p = . ) and lower absolute reticulocyte count (p = . ) when compared to cohort . the wbc (p = . , < . ) and anc (p = . , . ) were significantly lower compared to both older cohorts. however, no patient had therapy held because of neutropenia. the mean baseline hemoglobin f in cohort was . % compared to . % and . % in cohorts and respectively (p = . , p< . ). the mean duration of therapy in cohort was . months, compared to . months in cohort (p = . ) and . months in cohort (p = . ). during this time, hb f levels remained higher in cohort (mean . %) compared to cohorts and (mean . %, p = . and mean . %, p = . ). patients in cohort experienced fewer hospitalizations (p = . ), pain crises (p = . ), and transfusions (p = . ). there was no difference in toxicity between groups. hydroxyurea was used safely in infants to months of age and resulted in more robust hematologic responses and a decrease in sickle-related complications when compared with patients starting hydroxyurea later in life. children's national health system, washington, district of columbia, united states background: children with sickle cell disease (scd) have a significantly greater risk of silent or overt cerebral infarction than the general population. infarcts are associated with declines in cognitive functioning and academic achievement. while infarcts are reliably identified using mri, scans are expensive and occasionally necessitate sedation. moreover, mri's are not recommended for routine monitoring of cerebral infarcts. additional tools are needed for discriminating the presence of a cerebral infarct that are brief, noninvasive, inexpensive, and repeatable. objectives: to evaluate differences in performance on cogstate, a computerized neurocognitive assessment, in patients with scd with and without history of cerebral infarct. design/method: participants included children with scd ages - (m = . , sd = . ; % female; % s of s hbss) enrolled in a cognitive intervention trial. participants completed the cogstate pediatric battery, which measures processing speed, sustained attention, verbal learning, working memory, and executive functioning. history of silent or overt infarct was determined via health record review. participants also completed measures of intelligence (iq) and math fluency. results: participants' standard scores across most neurocognitive measures were lower than expected compared to the standardization sample (mean iq = . , sd = . ). thirty percent of participants (n = ) had a documented history of cerebral infarct. participants with a history of cerebral infarct scored lower on cogstate tasks measuring sustained attention (t[ ] = . , p = . ) and executive functioning (t[ ] = . , p = . ), as well as on a measure of math fluency (t[ ] = . , p = . ). receiver operating characteristic (roc) analyses demonstrated that the cogstate task measuring sustained attention was a fair discriminant of patients with and without a history of infarct (auc = . , ci = . - . , p = . ), whereas iq score was not (auc = . , ci = . - . , p = . ). cogstate processing speed and sustained attention tasks fairly discriminated between patients with at least average or below average intelligence (auc = . , ci = . - . , p = . and auc = . , ci = . - . , p = . , respectively). finally, the cogstate processing speed task was good at discriminating between at least average or below average math fluency (auc = . , ci = . - . , p< . ). multiple tasks in the cogstate pediatric battery appear to adequately identify patients with a history of cerebral infarcts. in addition, cogstate tasks appear to be fair predictors of impairments in iq and academic achievement outcomes. cogstate is inexpensive and can be easily administered in a medical setting with minimal training in approximately minutes. results support the potential for cogstate to be used as a screening tool for medical and neuropsychological abnormalities in children with scd. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: cardiovascular disease contributes to the morbidity and mortality of patients with sickle cell disease (scd). hydroxyurea therapy in scd has known clinical efficacy including improving anemia, decreasing episodes of vasoocclusive crisis and acute chest syndrome, and decreasing mortality. effect of hydroxyurea on cardiac function in children with scd is not well studied. an earlier study suggested the protective effect of hydroxyurea on left ventricular (lv) hypertrophy in scd. we hypothesized that hydroxyurea use would be associated with decreased lv remodeling and improved cardiac function. we aimed to evaluate the association between hydroxyurea use and lv remodeling and cardiac dysfunction in children with scd. design/method: we completed a retrospective study of patients with scd who were to years old, followed at st. christopher's hospital for children and had an echocardiogram completed in the past months. data collected included gender, bmi, scd genotype, hydroxyurea use, chronic transfusion use, and d and doppler echocardiographic parameters. cardiac structure, geometry, systolic function, and diastolic function echocardiogram parameters were included. analysis of variance (anova) tests were performed to assess for statistical significance of differences in cardiac parameters between patients with and without hydroxyurea use. analysis of covariance (ancova) tests were performed to control for age. results: demographic and echocardiogram data was collected on all patients who met inclusion criteria. of the patients included, ( %) were on hydroxyurea therapy. patients on hydroxyurea had significantly lower mean relative wall thickness (p = . ) and significantly higher mean peak early lv filling velocities (p = . ) and peak early lv filling/septal annuli early peak (e/ea) velocities (p = . ); however, only the e/ea velocities remained significant when controlling for age (p = . ). mean peak early lv filling velocities approached significance when controlling for age (p = . ). hydroxyurea therapy resulted in a significantly higher e/ea velocity, suggesting that these patients had worse diastolic function. it is possible that the patients initiated on hydroxyurea already had worse disease manifestations than those not on hydroxyurea, possibly accounting for the decreased diastolic function. when controlling for age, hydroxyurea use did not result in significant differences in cardiac structure parameters, systolic function parameters or cardiac geometry. prospective studies and larger sample size are needed to validate our findings, examine for additional statistically significant differences, and develop preventive strategies for cardiovascular disease in children with scd. background: acute chest syndrome (acs) is now the leading cause of death in children with sickle cell disease; mortality in the u.s. is reported to be - % and is mostly due to respiratory failure. early transfusion improves clinical outcomes. although patients with concurrent asthma are considered at increased risk for poor outcomes, risk factors for respiratory failure in pediatric acs have not been well-defined. to determine whether specific epidemiological and clinical features of children hospitalized with acs are predictive of the need for mechanical ventilation. design/method: data from the kids' inpatient database were reviewed to identify patients age < years with a discharge diagnosis of acs for the years , , , and . outcomes were defined by the international classification of diseases, ninth revision, clinical modification code. data were weighted to estimate total annual hospitalizations according to hospital characteristics in the united states. trends in healthcare costs, length of hospital stay, transfusion, and mechanical ventilation use were analyzed using multivariable linear regression. in addition, multivariable logistic regression was used to ascertain specific clinical or epidemiologic factors associated with mechanical ventilation use after adjusting for patient and hospital characteristics. the total hospitalizations for acs were , in ; , in ; , in ; and , in . reported use of mechanical ventilation ranged from . % to . % and was associated with non-black compared to black children (or, . ; %ci, . to . ) and the fall season (or, . ; %ci, . to . ), but not with age, preexisting asthma or hb-genotype. comorbidities of obesity (or, . ; %ci, . to . ), obstructive sleep apnea (or, . ; %ci, . to . ) and heart disease (or, . ; %ci, . to . ) were associated with mechanical ventilation use. the use of simple and exchange transfusion during all acs admissions ranged from . % to . % and . % to . %, respectively. among pediatric acs patients, those with obesity, obstructive sleep apnea or heart disease were at increased risk for respiratory failure and might benefit from early intervention (e.g., transfusion). surprisingly, asthma in children with acs does not appear to be a distinct risk factor for respiratory failure, and further studies are needed to clarify whether differences in treatment approach (e.g., addition of corticosteroids, bronchodilators) might impact on acs progression and/or severity even in high risk patients without asthma. objectives: to compare pulmonary functions between aa and k children with scd and to assess if a high hb f level contributes to better function. design/method: a cross sectional study was done on children with scd (hb ss disease) followed in comprehensive sickle cell programs. aa patients were followed at brookdale hospital, ny and k patients were followed in mubarak hospital, kuwait. children between the ages of and years who had pulmonary function tests (pft) done as a routine screening were enrolled. pft was done using spirometer and plethysmography. patients with congenital or anatomical lung abnormality, heart disease, pulmonary disease such as acute chest syndrome, acute asthma or pneumonia within weeks were excluded. results: there were children ( in each group) with scd,. restrictive pattern on pft was seen in / ( %) of aa vs. / ( %) of k (p> . ). obstructive pattern was seen in / ( %) of aa vs. / ( %) of the k group (p> . ). in both groups, children ( %) had normal pft. three/ ( %) in the aa group had a hb f> % as compared to / ( %) in the k group (p< . ). abnormal pft was noted in / children ( %) in each group. hbf was > % in / ( %) in the aa group vs. / ( %) in the s of s k group (p< . ). in patients with abnormal pft, mean hbf was . ± . in aa group, compared to . ± in k group (p< . ). conclusion: abnormal pft is highly prevalent among children with scd in both groups. aa children are more likely to have restrictive disease and k to have an obstructive pattern. level of hbf did not seem to protect k patients from abnormalities on pft. this finding should emphasize the importance of performing pft as part of the initial evaluation of all children with scd. background: sickle cell disease (scd) is a life-threatening disease with varied clinical spectrum and severity leading to premature death. there is a lack of validated prognostic marker in scd. recent evidence suggests that inflammation and platelet adhesion plays a critical role in the pathophysiology of vaso-occlusion in scd. elevated mean platelet volume(mpv) values are associated with a higher degree of inflammation in many disease states but it's effect on sickle cell disease or it's severity is unknown. objectives: to analyze the role of mpv in predicting disease severity/mortality in pediatric patients with scd. design/method: this is a single center retrospective study and included patients with sickle cell disease between months and years of age during a -year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . demographic information, lab data and clinical information including acute chest syndrome (acs), priapism, transfusions, sepsis, pain crisis, avascular necrosis were collected. all laboratory data were collected in steady state with no crisis in the recent past months. the disease severity score/probability of death was calculated using a validated model to predict risk of death in sickle cell disease (sebastiani et al. blood ) . pearson test was used to analyze correlation between mpv and probability of death. results: total no. of patients = ; male ( . %); female ( . %). median age is . years. all patients were of african-american origin. disease severity, hb ss - ( %); hb sc - ( . %) and sickle-beta thalassemia ( . %). patients on hydroxyurea has significantly lower mpv, p = . and this is independent of hb f levels. mpv has a significant positive correlation with the probability of death, p = . and correlation coefficient, r = . . on subgroup analysis, the correlation is even more significant in the age group between and years, p = . , r = . . using linear regression model, with probability of death as a dependent variable and hydroxyurea, mpv as independent variables, mpv maintains a significant association with probability of death (p = . ). conclusion: mpv is an independent biomarker predicting disease severity and probability of death in pediatric patients with sickle cell disease. hydroxyurea a known disease ameliorating agent is associated with lower mpv values. this effect is independent of the levels of fetal hemoglobin and may be due to anti-inflammatory effect of hydroxyurea or effect on the platelets. background: major success with initial qi projects by the sickle cell care team at children's hospital has precipitated ongoing inclusion of the qi approach to many other aspects of patient care. objectives: to optimize scd patient care utilizing qi processes. design/method: success of the scd qi team's initial project on transcranial doppler studies (tcds) and a second more complex project on hydroxyurea (hu) adherence, led to additional projects on completion of key immunizations, rbc phenotyping, and vitamin d level testing. using similar processes and principles from the hu adherence project, plan-do-study-act (pdsa) cycles were used to conduct smallscale tests of change. patient chart prep sheets, created for bi-monthly pre-appointment chart prep meetings, were significantly modified to include these focused care qi objectives. because of difficulty with emr database capability, data collected from the emr was tracked in excel spreadsheets or other unique tracking vehicles for the various parameters. for example, due to the clinic's diffuse, geographically scattered population, many separate non-shared primary care emrs, and lack of a mandatory state immunization registry; immunization records needed to be retrieved from pcps, outlying hospitals, public health departments, and fqhcs, and added to the emr and excel database. starting in / , all such data was collected and updated monthly. in one year's time ( - ) , the average immunization completion rate for seven key immunizations (pcv , pcv , hepatitis a, hepatitis b, meningococcal a, meningococcal b, and hpv) has increased by %. the biggest improvements were a % and % increase in completion for meningococcal a and meningococcal b, respectively. completion rate for rbc phenotyping rose from . % to . %. patients with at least one vitamin d lab test increased from . % to . %. since starting the tcd project in , the percent of patients who have completed their annual tcd has gone from a baseline of % to a sustained value of > %. conclusion: these qi projects have not only increased adherence to national recommendations for care of scd patients, they have helped establish a scd clinic methodology to create and implement sustainable processes. having the focused care initiatives prominently displayed on the patients' chart prep sheet serve as a reminder to medical team members to check the status of that item. this methodology is currently being used to formulate additional qi projects on annual renal function parameters and specialty visits, such as annual eye and dental exams. background: dominican republic has a high burden of sickle cell disease, and - % of children with homozygous hbss (sickle cell anemia, sca) will develop primary stroke. transcranial doppler (tcd) ultrasonography is an effective screening tool for primary stroke risk, but is not routinely available in dominican republic. hydroxyurea and blood transfusions are available, but no prospective screening and treatment program for stroke prevention has been implemented to date. ( ) to screen a large cohort of children with sca living in dominican republic, using tcd to identify elevated stroke risk; ( ) to determine the effects of treatments for stroke prevention (hydroxyurea for conditional velocities and transfusions for abnormal velocities). we hypothesized that both hydroxyurea and blood transfusions will decrease elevated tcd velocities and help prevent primary stroke. design/method: stroke avoidance for children with república dominicana (sacred, nct ) features a research partnership between cincinnati children's hospital and robert reid cabral children's hospital in dominican republic. the protocol, consent forms, and redcap database were prepared collaboratively and translated into spanish, and then irb approval was obtained at both institutions. in the initial prospective phase, children receive tcd screening over a -month period; those with conditional tcd velocities (maximum time-averaged velocity - cm/sec) receive fixed-dose hydroxyurea at mg/kg/day, followed by dose escalation to maximum tolerated dose, while those with abnormal tcd velocities (≥ cm/sec) receive monthly transfusions for stroke prevention. results: a total of children were enrolled in sacred, with an average age of . ± . years. initial tcd screening revealed ( . %) normal, ( . %) conditional, ( . %) abnormal, and ( . %) inadequate velocities. among children ( males, females, average age . ± . years) who initiated hydroxyurea at mg/kg/day for conditional tcd velocities, completed six months of treatment with expected hematological benefits including significant increases in hemoglobin concentration ( . to . g/dl) and fetal hemoglobin ( . to . %). no clinical strokes have occurred in the treatment group. repeat tcd examination after -months of hydroxyurea treatment revealed % ( / ) with previous conditional velocities had normal tcd velocities. the prevalence of conditional tcd velocities in the dominican republic is high, indicating an elevated stroke risk among children with sca. hydroxyurea treatment is associated with improved hematological parameters, lower tcd velocities, and probable decreased stroke risk. sacred is an important prospective and collaborative research trial providing epidemiological data regarding tcd screening, stroke risk, and hydroxyurea effects among children with sca. background: red blood cell aggregation is a rheologic property that explains the shear-thinning behavior of blood. at lower shear rate blood flow, red cells tend to aggregate, s of s whereas in higher shear rate blood flow, these aggregates are dispersed. this property is especially important in the venous system, where low shear rate blood flow predominates. there is inconsistent data in the literature concerning aggregation and aggregability in sickle cell disease (scd). objectives: because the lorrca and myrenne instruments have been shown to be similarly effective methodologies in red cell aggregation measurements, we aimed to determine whether the measurement of aggregation indices in scd, by myrenne and by lorrca, is consistent in our lab. design/method: we measured aggregation in blood samples corrected to % hematocrit. aggregability was measured using kda dextran in the myrenne but not the lor-rca. aggregation index using lorrca was measured in patients with scd and healthy subjects enrolled in a study of blood flow between and . aggregation and aggregability using the myrenne was measured in patients with scd and healthy subjects enrolled in a separate study of blood flow between and . results: using lorrca, we found that aggregation index in patients with scd was less than that of healthy subjects (p< . ). in the myrenne, aggregation at stasis was slightly higher in patients with scd compared to healthy subjects (p = . ) but aggregation at low shear rotation was not different. aggregability was higher in the patients with scd compared to healthy subjects at both stasis and low shear rotation (p< . ). red cell aggregation is an important determinant of low shear blood flow. deoxygenated venous blood is particularly important to low shear blood flow in patients with sickle cell disease. we found that two different aggregometers predict different aggregation results for scd. it is unclear why there is a systematic difference between the two methods, but there are some possibilities. first, the syllectogram in the lorrca is generated by the backscatter of light from the laser, while the myrenne measures transmitted light. second, the distance between the bob and cup in the lorrca is microns, while the gap between plates in the myrenne is microns, which might affect the disaggregation of red cells. further work is needed to understand the differences in red cell aggregation and aggregability when using these instruments, particularly when using aggregation as a predictor of blood flow and tissue perfusion. background: children with sickle cell disease (scd) are at risk of acute splenic sequestration crisis (assc). assc is a life-threatening complication characterized by splenomegaly, pain and severe anemia. assc most often occurs in young children with the most severe forms of scd and one-third of patients will have more than one episode. treatment is based primarily on expert opinion and includes blood transfusion and surgical splenectomy. objectives: we plan to assess the clinical practice patterns of physicians treating children with assc. design/method: a survey study was performed. the survey included six scenarios of severe scd with variation in age, hydroxyurea-use, and episode number of assc; questions focused on the acute and chronic management of assc. the survey was disseminated on three occasions over a six-month period, using an online survey tool, surveymonkey, to pediatric hematologist-oncologists participating in the american society of pediatric hematology-oncology hemoglobinopathy special interest group. the survey had a response rate of % ( / ). most respondents were recent graduates ( %; / ) practicing in academic urban centers with greater than sickle-cell patients. seventy-nine percent ( / ) recommended hydroxyurea initiation in - m/o with severe scd. prophylactic penicillin after surgical splenectomy was continued by % ( / ) after years. for the acute management of assc results did not vary despite patient age, hydroxyurea use, and the number of previous assc episodes. simple transfusion was preferred by % ( / ), with % ( / ) recommending slow transfusion and % ( / ) recommending routine simple transfusion. for the chronic management of assc, results varied based on patient age and the number of previous assc episodes. for a m/o after the first episode, % ( / ) recommended observation and % ( / ) hydroxyurea initiation. for a m/o with any prior episode of assc, % ( / ) recommended chronic transfusion therapy and % ( / ) surgical referral for splenectomy. for a y/o after the first episode, % ( / ) recommended surgical splenectomy and % ( / ) increasing hydroxyurea dose. for a y/o with any prior assc episode, % ( / ) recommended referral for surgical splenectomy. in this survey, we found most providers continue to recommend simple transfusions for assc and surgical splenectomy after two episodes. the majority of providers continue to delay referral for surgical splenectomy until age two, but earlier referral in children under two and use of chronic transfusion therapy were also reported. variability in chronic management highlights the need for further research of splenic sequestration. background: developing therapies for sickle cell disease (scd) is challenging in part because the accepted endpoint, vaso-occlusive crisis (voc), occurs infrequently, does not measure full disease burden, and is a measure of healthcare utilization. in phase / studies of patients with scd, voxelotor (gbt ) has demonstrated increased hemoglobin (hb) levels and reduced hemolysis and has been safe and welltolerated. voxelotor is being evaluated in the ongoing hope phase trial. objectives: to report the innovative phase / hope trial design with novel primary and secondary outcomes to accelerate drug development. design/method: hope (nct ) is a phase , randomized, placebo-controlled, multicenter study of oral voxelotor in patients with scd (aged - years) with baseline hb . - . g/dl and - episodes of voc in the prior year. to accelerate clinical trials to support drug development, the study combines a phase exploratory, dose-selection phase (group ) with a pivotal phase (groups / ). patients in group will be randomized : : to voxelotor or mg/day or placebo. analysis for dose selection will occur when the final patient has received weeks of treatment. group will continue enrollment with randomization : : until dose selection based on analysis of the group cohort. group will allow for a seamless transition into group , which will randomize patients : to the selected dose or placebo. the final data analysis set will include group patients who received placebo or the selected dose and all group patients. the primary endpoint is an objective laboratory measure and surrogate of clinical benefit, increase in hb > g/dl, from baseline to weeks based on voxelotor mechanism of action (inhibition of hb polymerization). this trial is the first to use a patient-reported outcome (pro), the -item sickle cell disease severity measure, as a secondary endpoint. this novel electronic pro, developed specifically for the hope study following fda guidance, will evaluate changes in scd symptom exacerbation and total symptom score from baseline to weeks. additional secondary endpoints include measures of hemolysis, rates of voc, transfusions, and opioid use. the study was designed to enable selection of pro-defined symptom exacerbations or traditionally defined voc as the key secondary endpoint after the group analysis. results: this study is ongoing. the hope trial, expected to complete enrollment by late , will evaluate the efficacy and safety of voxelotor compared with placebo in patients with scd. supported by global blood therapeutics. background: inflammation, coagulation activation, oxidative stress and blood cell adhesion are elements of sickle cell disease (scd) pathophysiology. patients with scd have low levels of the omega- fatty docosahexaenoic acid (dha) and eicosatetraenoic acid (epa) in plasma and blood cell membranes. dha is a bioactive fatty acid with anti-inflammatory, anti-blood cell adhesion and anti-oxidant properties. altemi-atm is a novel dha ethyl ester formulation with a proprietary delivery platform (advanced lipid technology® (alt®)) that enhances oral dha bioavailability. the scot trial investigated the effects of altemiatm in children with scd. objectives: to demonstrate the effects of altemiatm on blood cell membrane omega- index and selected biomarkers of inflammation, coagulation, adhesion and haemolysis associated with scd. s of s design/method: children with scd, aged - years (n = ), were enrolled. subjects were randomized to receive either placebo or one of three daily oral doses of altemiatm ( - , - or - mg/kg/day dha) for two months. the effects of altemiatm on red blood cell (rbc), white blood cell and platelet membrane omega- fatty acids index (total dha + epa levels) were assessed after four weeks of treatment. the effects of altemiatm on markers of inflammation, adhesion, coagulation, and hemolysis were assessed after eight weeks of treatment. cell membrane dha and epa concentration was determined by using lc-ms/ms method. the percent changes from baseline on blood cell membrane omega- index and select scd biomarkers were compared between the three dose groups and placebo using a mixed-model repeatedmeasures (mmrm) analysis with baseline blood cell membrane omega- index, hydroxyurea use, and treatment as fixed effects and patient as a random effect. after four weeks of treatment, blood cell membrane dha and epa levels were significantly increased in all altemiatm doses (p< . ). after eight weeks of treatment, significant reductions were observed in se-selectin (p = . ), and d-dimer (p = . ) in patients exposed to altemiatm dose level vs. placebo. hemoglobin was significantly increased at altemiatm dose level versus placebo. plasma high-sensitivity c-reactive protein, lactate dehydrogenase, soluble vascular cell adhesion molecule- and white blood cell count showed improvement after weeks of treatment in all three altemiatm doses levels but did not reach significance. conclusion: treatment with altemiatm enriches dha and epa in blood cell membranes of patients with scd and improves select sickle cell disease biomarkers of blood cell adhesion and thrombin generation. these findings provide insight into the mechanisms of action of altemiatm in sickle cell disease. brown university -hasbro children's hospital, providence, rhode island, united states background: despite clinical advances in the treatment of sickle cell disease (scd) in pediatric and young adult patients, pain remains a significant source of disease-related morbidity. physical therapy has been shown to be useful for the treatment of pain in children and young adults with various chronic illnesses of which pain is a significant component, however no data exists regarding potential benefits of physical therapy in pediatric and young adult patients with scd. objectives: to query healthcare providers and others involved in the care of pediatric and young adult scd patients regarding possible benefits of and barriers to physical therapy as a potential treatment modality. design/method: we conducted a web-based survey of healthcare providers within the new england pediatric sickle cell consortium (nepscc) in an attempt to identify potential benefits of and barriers to outpatient physical therapy in this patient population. results: nearly % of survey participants felt that physical therapy had the potential to be "somewhat beneficial" or "very beneficial" in pediatric and young adult patients with scd. a majority of physicians reported having referred patients with scd for physical therapy in the past. the most frequently identified perceived potential benefits included improved functional mobility, improvement of chronic pain symptoms, decreased use of opiates, improved mood symptoms, improved acute pain symptoms, and improved adherence with medications and clinic visits. significant perceived barriers identified included lack of transportation, time constraints, patient lack of understanding, and difficulty with insurance coverage. our study indicates that healthcare providers have an overwhelmingly positive view of the use of physical therapy in the management of pediatric and young adult patients with scd. significant barriers exist which need to be addressed. future research should focus on patient and parent perspectives regarding physical therapy, as well as a randomized controlled trial of a physical therapy intervention in this patient population. background: vitamin-d deficiency is fast becoming increasingly recognized in patients with sickle cell disease (scd). while it is estimated that these patients are five times more likely to develop vitamin-d deficiency, the exact clinical significance of this is largely unknown. given that this deficiency can be inexpensively and easily treated, our study sought to establish the prevalence of vitamin-d deficiency in our patient population and its relationship with disease severity. objectives: to estimate the prevalence of vitamin-d deficiency in patients with scd in our institution and to analyze their disease severity in relation to their vitamin-d level. design/method: through retrospective chart review we analyzed subjects that represent a cohort of patients followed at the adult and pediatric hematology services at university of miami with known diagnosis of scd that had a vitamin-d level drawn between january st, and august st, . we conducted a cross-sectional study and recorded the first vitamin-d level during this period. patient demographics, medical and social history information were collected along with laboratory data. the number of admissions for vaso-occlusive crisis (voc) and acute chest syndrome within one year preceding the collection the vitamin-d level was also recorded. results: a total of charts were reviewed, adult charts and pediatric charts. after exclusion, patients were enrolled. subclinical vitamin-d deficiency is only evident on laboratory blood testing of vitamin-d ( -hydroxy) and according to this laboratory result patients were classified as sufficient (≥ ng/ml), insufficient (< to ng/ml) and deficient (< ng/ml). out of the cases, . % ( / ) were deficient, . % ( / ) were insufficient and . % ( / ) were optimal. after statistical analysis two negative correlations were identified, increasing vitamin-d levels with decreasing white blood cell count (ci %- . (- . , - . )and decreasing incidence voc (ci %- . (- . , - . ). conclusion: this study confirms that there is a significant prevalence of vitamin-d deficiency in patients with scd. furthermore, the results of this investigation proved that vitamin-d deficiency is associated with acute pain and leukocytosis in patients with scd. given the multitude of confounding factors that affect vitamin-d absorption and intake, multivariate analyses are required to truly further investigate this relationship. texas children's hospital, houston, texas, united states background: hemophagocytic lymphohistiocytosis (hlh) is a rare but life-threatening condition of hyper-inflammation that is characterized by splenomegaly, cytopenias, hyperferritinemia, hypertriglyceridemia, hemophagocytosis and coagulopathy. although timely diagnosis is imperative, it is often challenging as these individual signs and symptoms may occur in a variety of clinical conditions. to report a case of undiagnosed sickle cell anemia presenting with severe ebv viremia and associated hemophagocytic lymphohistiocytosis results: a -month-old previously healthy male presented with respiratory distress, increased fatigue, and a focal seizure following a two-week history of cough and lowgrade fevers. physical exam was consistent with hypovolemic shock and revealed significant splenomegaly. laboratory testing revealed severe hypoglycemia, acidosis and electrolyte disturbances including hyperkalemia, hyperphosphatemia, and hyperuricemia. labs showed a leukocytosis (wbc , ), severely low hemoglobin ( . ), and platelets of , . coagulation testing revealed prolonged pt/inr and ptt, hypofibrinogenemia and a highly elevated d-dimer. additional workup was completed to determine etiology of acute presentation, given broad differential diagnosis. infectious studies were consistent with an acute ebv infection (plasma ebv pcr > , ). elevated levels of soluble il- and ferritin completed / criteria for the diagnosis of hlh. bone marrow evaluation showed trilineage hematopoiesis with no abnormal blast population or hemophagocytosis. results from hemoglobin electrophoresis sent from the initial cbc sample were notable for hbs . %, hbf . %, and hba of %, confirming the diagnosis of sickle cell disease. the patient was started on hydroxyurea and penicillin and splenomegaly resolved. with supportive care, he demonstrated gradual improvement in symptoms and laboratory abnormalities, including normalization of soluble il- , ferritin, cd , il- levels, immunoglobulins, and declining ebv titers. nk cell function has remained abnormally low, not eliminating the possibility of acquired hlh despite spontaneous improvement. conclusion: splenic sequestration associated with sickle cell disease in combination with acute infectious mononucleosis could have explained many of the presenting symptoms including anemia, thrombocytopenia, and splenomegaly. however, it does not explain the unusually high ebv titer and degree of inflammation meeting diagnostic criteria for hlh, which raises concern for an underlying immunologic abnormality such as x-linked lymphoproliferative disorder (xlp). although testing for xlp was negative, he will require s of s continued monitoring in the future for signs of relapse. this case illustrates the complexity of diagnosing lymphohistiocytic disorders and the significant overlap in presentation between these disorders and other medical conditions. background: vaso-occlusive crisis (voc) is one of the most distressing occurrences in patients with sickle cell disease (scd). patient controlled analgesia (pca) is recommended by nih and expert opinions favor its early use. we aim to review the use of pca in patients with voc and to evaluate if its early use is associated with faster pain control and reduced length of stay (los). design/method: this retrospective single center study included all pediatric patients admitted and treated with pca for a severe voc from to . "early" use was defined as start of pca within hours of arrival in the emergency department (ed) and "late" use after hours. time to reach adequate analgesia was defined as oucher, verbal scale or faces pain scale < / obtained twice consecutively in a -hours interval. time to reach adequate analgesia and los were compared between early-pca and late-pca groups. results: a total of patients presented episodes of voc treated with pca during the study. sixty-one episodes ( %) were treated with early-pca and ( %) with late-pca. both groups were comparable in terms of age ( . vs . years old), gender ( . % female vs . %), hemoglobin phenotype ( . % hbss vs . %), but median pain score at admission was higher in early-pca than in late-pca ( / vs / , median difference ( % ci , ). early-pca was associated with a median reduction in los of . days ( % ci . , . ) (median early-pca los . vs late-pca . days). time to reach analgesia could be evaluated only in a subset of patients ( in early-pca and in late-pca group). although time to reach adequate analgesia tended to be shorter in the early-pca group, it was not statistically different: median . hours vs . hours, difference of . ( % ci - . , . ). side effects were observed during ( . %) pca treatments ( / ( . %) episodes in early-pca, / ( . %) in late-pca group) among which ( . %) were significant adverse events. these were observed in patients who required interventions: desaturations requiring oxygen without intubation, neurologic abnormalities (hallucinations, visual abnormalities, no stroke), urinary retentions. conclusion: early use of pca for severe voc was associated with a reduced length of hospital stay despite that these patients had higher pain score on admission. prospective studies are needed to support these positive outcomes. background: acute chest syndrome is one of the leading causes of death in children with sickle cell disease - . while the cause of acute chest syndrome most commonly is not identified, fat embolism and infectious causes are believed to be most common. with an extremely high mortality rate, rapid identification and initiation of therapy is essential for survival. case presentation: we describe the case of an -year-old female with sickle cell sc disease who was admitted for vasoocclusive pain crisis and quickly progressed to multi-system organ failure due to fat embolism syndrome and parvovirus b infection objectives: the case highlights the presentation and diagnosis so other providers can optimize outcomes for those with this under-recognized syndrome design/method: her parvovirus studies returned after days which showed: parvovirus b dna pcr detected; parvo igg . (positive > . ); and igm . (positive > . ). the patient experienced an approximately . g/dl drop in hemoglobin( . to . g/dl/ hrs) with progressive thrombocytopenia (from , to , /ul) and a peripheral smear showed microcytic,normochromic red cells with nucleated rbcs and occasional nuclear budding, slight polychromasia, schistocytes, and polymorphic cells with toxic granules that suggested leukoerythroblastosis. she was emergently transferred to the regional quaternary care hospital for ongoing ecmo therapy where she experienced a change in her pupillary exam prompting a stat ct scan that showed severe, diffuse cerebral edema with transtentorial herniation. the decision was made to withdraw life-sustaining therapies and her family refused a post-mortem autopsy examination. fat embolism syndrome is a severe and uncommonly recognized complication of sickle cell disease, seen most commonly in those with a non-ss phenotype and previous mild disease course who present with severe, unrelenting vaso-occlusive pain episode and/or acute chest syndrome that progresses to respiratory distress with altered mental status and cutaneous changes. rapid identification and initiation of exchange transfusion therapy should be initiated with clinical suspicion because of the extremely high mortality rate. although previously considered rare, it needs to be considered in the differential diagnosis of more commonly encountered complications of sickle cell disease. background: patients with sickle cell disease (scd) experience vaso-occlusive crisis (voc), which results in extreme pain, often requiring opioids and admission. genetic and environmental factors affect the frequency and severity of these episodes. previous research has born conflicting evidence on whether environmental temperature is contributory. edmonton, alberta is the northern most city with a population over a million in north america. there is an increasing sickle cell population which is exposed to extreme winter conditions. this provides a suitable population and atmosphere to study the influence on cold external temperatures in scd. this study sought to identify if pediatric patients with scd, experience greater morbidity in cold external temperatures. board approved retrospective case control series. patients were identified through a clinical database, and emergency visit, phone call and admission data was collected over a fiveyear period. the average, minimum and change in temperature on day of presentation, and hours prior, was collected from the government of alberta, and was statistically analyzed using descriptive statistics, to determine the relation to vaso-occlusive events. results: one-hundred and eighteen patients were identified, and voc events reviewed. the mean patient age was . years of age with a range from . - years old. the female to male ratio was equivalent with female ( . %) and male ( . %) voc events. eight records ( %) had docu-mented cold exposures. the analysis between the temperature and the frequency of events did not yield significant correlation. average and minimum temperature on day of admission had the largest percentage of voc events occur at mild temperatures, from - . to • c and - . to respectively. change in temperature on day of admission, and hours had the largest percentage of voc events at a mild to moderate change in temperature of - degrees. data at & hours prior to admission showed similar results. secondary data analysis accounting for the lower proportion of extreme weather days in comparison to moderate temperate days showed no significant impact. there was no correlation of average, minimum or change in temperature on day of admission, or hours prior. multiple cofounding factors likely contribute to these results. as it was a retrospective study many confounding and precipitant factors may not be recorded or identified. a prospective study to better record specific cold exposure is warranted. children's national health system, washington, district of columbia, united states background: achieving optimal anticoagulation with unfractionated heparin (ufh) in pediatric patients receiving extracorporeal membrane oxygenation (ecmo) is often challenging due to antithrombin (at)-mediated heparin resistance (hr). intermittent at dosing during pediatric ecmo support does not maintain adequate at levels. continuous at infusion (cati) presents an alternative strategy to achieving consistent goal at levels and optimizing heparinization. however, cati during pediatric ecmo has not been adequately studied. objectives: to describe our center's experience with an ecmo cati protocol. design/method: in , we modified our ecmo anticoagulation protocols to include ufh titration according to anti-factor xa (anti-fxa) levels and cati in patients with at-mediated hr. the cati rate was calculated using baseline and goal at levels while accounting for the circuit volume. cati was administered with ufh into the circuit via a s of s y-infusion set. at and anti-fxa levels were monitored every hours. recombinant at (r-at) concentrate was used at our center until with subsequent transition to a plasmaderived at (pd-at) concentrate. due to the longer half-life of pd-at concentrate, the protocol was modified so cati is stopped once target at and anti-fxa levels are achieved. we conducted a retrospective study of all patients who received cati during ecmo support at our center. data are reported as median and interquartile range and compared using the mann-whitney u test. two-tailed p-value < . was considered statistically significant. since , patients [ males, age month ( . - )] on ecmo support received catis ( rat, pd-at) per our protocol ( patients received pd-at infusions during one ecmo run). the duration of cati was hours ( - ). cati administration led to significant increases in at and anti-fxa levels from baseline of % ( - ) and . units/ml ( . - . ) to the first level within goal of % ( - ) and . units/ml ( . - . ), respectively (p< . ). the respective times to achieve goal at and anti-fxa levels were hours ( - ) and hours ( - ). the respective peak at and anti-fxa levels were % ( - ) and . units/ml ( . - . ). during cati, no patient required circuit change, patient developed cannula thrombosis and patients experienced non-fatal major bleeding. conclusion: cati in pediatric patients receiving ecmo support with close monitoring of at and anti-fxa levels was associated with significant rapid increase in at, optimization of heparin effect, and reduction in thrombotic complications without increase in major bleeding compared to prior reports. a prospective study of this at dosing strategy is warranted. children's hospital of orange county, orange, california, united states background: inherited factor xiii (f ) deficiency is a rare bleeding disorder with wide heterogeneity in clinical manifestations ranging from mild bruising, and mucosal and umbilical stump bleeding to spontaneous, severe intracranial bleeding. the bleeding phenotype is influenced not just by zygosity of the fxiii mutation alone, but also by co-inheritance of variants in other clotting protein genes that also play a major role in clot formation and stability. we present a series of three siblings found with f a gene variant and platelet dysfunction linked to bleeding phenotype. design/method: retrospective chart review of the index case, coagulation studies and whole gene sequencing. the index patient presented at two years of age with a subdural hematoma after a fall, requiring emergent craniotomy. a week after initial evacuation, she re-bled, prompting an extensive work-up for potential bleeding disorders, including f activity, von willebrand profile, comprehensive fibrinolysis panel, pai- antigen level, platelet mapping thromboelastogram (plt-teg), and f genetic analysis. the patient's identical twin and older sibling, who had symptoms of bruising, underwent a similar evaluation. the index patient demonstrated consistently low f activity ( - %), and platelet function testing revealed decreased response to adp agonists. the twin and older sibling had normal f levels, and only slightly decreased response to adp in platelet studies. whole gene analysis of f and other genes on our next generation panel, revealed several intronic deletions in the index patient that were not shared by her siblings, which likely account for her decrease in circulating f levels. her symptoms have responded well to monthly treatment with factor concentrate. all three children shared the f variant, pro leu, previously described as a risk factor for intracranial hemorrhage. the f mutation, pro leu, has been associated with intracranial hemorrhage in young women, but the presence of the variant alone may not be enough to cause a severe bleeding phenotype. family studies identified novel deletions in the index patient which may account for her decreased f levels, which would have been overlooked with standard sequencing. future studies, including evaluation of 'platelet' f levels, should be performed when platelet dysfunction is detected. further laboratory and clinical evaluation is required to delineate the long term implications of the interaction of even mild f deficiency if present with additional clotting disorders such as the platelet function defect in these siblings. background: acquired hemolytic anemia can occur due to mechanical shearing of red blood cells and is classically seen in patients with prosthetic heart valves. there are reports of this same traumatic effect with other repairs, including annuloplasty. following valvular procedures flow disturbances can exist across the valve that lead to shear stress and hemolysis. although von willebrand disease (vwd) is typically seen due to an inherited disorder in the pediatric population, flow disturbances in the setting of valve abnormalities can lead to acquired von willebrand syndrome (avws). von willebrand factor multimers become unfolded and elongated in the setting of shear stress resulting in increased susceptibility to cleavage by adamsts- . specifically, loss of high molecular weight multimers (hmwms) can lead to a syndrome akin to type a vwd. objectives: to describe a case of mechanical hemolysis with acquired type a vwd design/method: a -month-old girl with history of hypoplastic left heart syndrome and severe tricuspid valve insufficiency underwent norwood procedure, blalok-taussig shunt placement and subsequently a bidirectional glenn and tricuspid valve annuloplasty. during the following month she requires weekly red blood cell (rbc) transfusions due to intermittent anemia. she also experienced bloody stools and dark urine. laboratory evaluation was notable for normocytic anemia, reticulocytosis, elevated lactate dehydrogenase, and low haptoglobin consistent with hemolytic process. immune-mediated hemolysis from transfusion reaction or presence of autoimmune or alloimmune antibodies testing was negative. to investigate gi bleeding, work up for vwd revealed normal vw activity and antigen but with loss of high molecular weight multimers consistent with acquired type a vwd. in consultation with cardiology, it was felt her tricuspid valve insufficiency jet could be leading to mechanical hemolysis and avws. a repeat echo showed persistent moderate tricuspid insufficiency but no other significant changes. due to the patient's continued need for weekly rbc transfusions she was subsequently trialed on pentoxifylline which is used in adult patients to decrease blood viscosity and increase erythrocyte flexibility in patients with mechanical hemolysis. her transfusion needs remained the same and the medication was discontinued after two weeks. she required one transfusion a week later but no transfusions since that time. although not commonly seen in pediatric patients, the diagnosis of mechanical hemolysis accompanied by avws should be pursued in a patient with congenital heart disease with significant anemia and/or bleeding. the work up in these patients is difficult as echocardiograms can be inconclusive thus an extensive hematologic evaluation is usually necessary. objectives: our aim was to assess incidence of and potential risk factors for central line-related dvt at our institution between - . additionally, our goal was to analyze if that incidence differed between the three central line types and identification of line-specific risks. design/method: a retrospective chart review of central line placements in pediatric patients at cleveland clinic between - was conducted. data included demographics, potential risk factors, line characteristics and any related thrombotic events. the study cohort consisted of lines in pediatric patients aged - years of age. there were . thrombi ( % ci . - . ) per , line days. statistically significant risk factors for thrombus include diagnosis group (liquid tumor highest rate of %, solid tumor lowest at %), type of line (picc %, broviac %, and mediport %), location of line, greater number of lines per patient, peg asparaginase ( % vs %), sepsis, and history of procoagulant state. line characteristics such as lumen size and number of lumens were not identified as a significant risk. there was a significantly higher rate of thrombus in than in the previous years when pooled ( % in vs . % from - , p = . ). the incidence of dvt in pediatric patients at our institution was highest with broviac lines, and significant risk factors in our patient population included liquid tumor, femoral vein location, peg asparaginase, sepsis, and history of a procoagulant state. the incidence of thrombi was highest in , and therefore highlights the urgent need for improvement in nationwide hospital practices to minimize risk of thrombi formation and early detection in the higher-risk s of s populations. there is still much to be learned regarding the characteristics specific to different central lines, which would influence thrombi formation. nyu winthrop hospital, mineola, new york, united states background: pediatric immune thrombocytopenic purpura (itp) is an autoimmune disorder with platelet counts < causing increased risk for significant hemorrhage. there is increased immunologic platelet destruction due to production of specific autoantibodies along with inhibition of platelet production. few randomized trials exist to guide management and ultimately each patient requires an individualized treatment plan. itp may be acute (diagnosis to m) or chronic (> months). one of the treatments of chronic itp is laparoscopic splenectomy (ls), which is very well tolerated. a rare complication of ls is splenosis, an autotransplantation or implantation of ectopic splenic tissue within the abdominal cavity or in any other unusual body compartment. splenosis is sometimes associated with relapsed itp due to preserved immune activity. the usual management of symptomatic splenosis is surgical resection. objectives: to describe medical management in a young patient with itp relapsed due to extensive unresectable splenosis following ls design/method: our patient was originally diagnosed at years with itp and was treated with ls at years of age for chronic severe thrombocytopenia and persistent bleeding not responding to first line therapies. she tolerated it well and had a complete response (cr) defined as a platelet count of > measured on occasions > days apart and absence of bleeding. she maintained a normal platelet count for twelve years after which she relapsed (loss of response after cr) with severe thrombocytopenia and hematuria necessitating high dose steroids. ct scans showed multiple wellcircumscribed soft tissue masses in the left lower quadrant adjacent to uterus and left ovary, involving left omentum and the anterior abdominal wall partly. findings were confirmed by damaged rbc nuclear scan to be splenosis. during laparoscopy the splenosis lesions were deemed too extensive and were not resected completely to avoid postoperative morbidity. she was started on sirolimus around the same time for treatment of her relapsed itp and steroids were weaned off. results: eight months since beginning sirolimus with therapeutic levels she remains in cr with no bleeding and has not required any steroids, immunoglobulins or anti d immunoglobulin. conclusion: sirolimus is a safe and effective steroid-sparing agent in treatment of chronic itp. this is the first instance of a patient with poorly resectable splenosis responding well to medications for itp. more data is needed regarding the longterm efficacy of such an intervention and whether it will eliminate the need for a second surgery in relapsed itp patients with extensive splenosis. background: storage pool disorders affecting platelets result in bleeding symptoms related to a deficiency or defect in alpha granules or delta granules. in delta-storage pool disorders (dspd,) there is a deficiency of the delta granules and their constituents, which results in the inability of platelets to properly activate as well as lack of proper constriction of blood vessels during bleeding episodes. amongst patients with dspd, females most commonly present with menorrhagia, while males tend to present with epistaxis and easy bruising. the international society on thrombosis and hemostasis (isth) developed a screening bleeding assessment tool (bat) for mild bleeding disorders, shown to be a validated tool in children. diagnosis of dspd is classically made with a platelet electron microscopy (pem) value < . delta granules per platelet (dg/pl), but recently lower diagnostic thresholds of dg/pl or even . dg/pl have been suggested. objectives: evaluate the correlation between pem and bleeding scores, and also examine various cut-off values used to diagnose and risk stratify patients with dspd. design/method: retrospective chart review of pediatric patients followed by hematology with a diagnosis of dspd was performed. clinicians obtained bleeding scores for each patient as standard of care in the hemostasis clinic. quartile ranges were established to appropriate three stages of severity based upon bleeding scores. statistical analysis was performed using software r and exploratory data analysis to evaluate for a correlation. results: amongst all patients, the average bat score was . and pem was . dg/pl. the average bleeding score for pem between . dg/pl and dg/pl was . , while the average bleeding score for pem below dg/pl was . . the correlation coefficient between pem and bleeding scores is . . using a threshold of dg/pl, % of patients would have met diagnostic criteria. quartile ranges for the bleeding scores are as follows: st quartile was - , nd quartile was - , and rd quartile was > . conclusion: patients with a more marked granule deficiency do not exhibit a more severe bleeding phenotype, suggesting proper platelet function is not solely determined by granule quantity in these patients. bleeding severity may be more appropriately assessed with bleeding scores rather than pem values, and using quartile ranges may aide in risk stratification and therapeutic interventions for dspd patients. further work remains to determine the optimal diagnostic threshold of pem dspd in pediatric populations. texas children's hospital, houston, texas, united states background: warfarin management has many challenging aspects including pharmacogenomics, food and drug interactions, lack of standardized dosing, patient compliance, tracking lab results from multiple lab locations, and the potential for significant bleeding or thrombotic complications. a literature review revealed limited data highlighting anticoagulation monitoring workflow and emr documentation and specifically, no data in the pediatric population. historically, the texas children's hospital cardiology and hematology centers were each documenting anticoagulation data within the epic tm system differently. epic's tm original design for anticoagulation documenting resulted in the necessity to duplicate documentation in order to see at-a-glance critical anticoagulation monitoring information. objectives: the objective of this project was to standardize inr documentation across departments to reduce the risk of patient safety events and improve workflow. design/method: a workgroup assembled consisting of nurses from the cardiology and hematology departments, along with staff members from the epic tm is support group. the workgroup identified current documentation practices, available epic tm tools, and brainstormed ideas to streamline and improve both documentation with the current epic tm tools. physician partners were identified in cardiology, hematology and coagulation laboratory to gain their input. a new anti-coag (ac) encounter was developed and first made available in an epic tm practice environment, then once approved, epic tm written education and training session were completed by both departments' staff. results: surveys were sent to health care providers in the cardiology and hematology centers prior to the new ac encounter, and also to health care providers six months after implementing the ac encounter. six responses were received for each survey. the pre-implementation survey showed the most problematic part of the documentation system for anticoagulation was no single place in the emr to find a complete anticoagulation picture. post ac encounter implementation survey results revealed more health care providers using the epic tm inr reminder pool, less time needed to compile a report of three months of anticoagulation information, less time needed to document individual encounters, less locations needed to document ac information and decreased amount of types of documentation used. standardized ac encounters improves workflow with less time needed to document and compile information, less types of documentation utilized and easier access to patients ac information. next steps include retrospective review of patients' inr time in therapeutic range to determine if there was an impact on patient compliance and continue to evaluate and modify the ac encounter to enhance user friendliness. caitlin tydings, jennifer meldau, christine guelcher, carole hennessey, eena kapoor, michael guerrera, yaser diab s of s children's national health system, washington, district of columbia, united states background: venous anatomic abnormalities (vaas) are considered a risk factor for developing deep vein thromboses (dvts) that occur as a result of significant alterations in venous blood flow. identification of predisposing vaas can be challenging. hence, diagnosis can be delayed or overlooked especially in pediatric patients. dvts in children or adolescents with predisposing vaas have been only described in sporadic case reports and small case series. objectives: to describe characteristics and outcomes of dvts in pediatric patients with underlying vaa treated at our center. design/method: we conducted a retrospective chart review of all pediatric patients with objectively confirmed extremity dvt treated at our institution over a -year period from to and identified all patients with underlying vaas. patients were managed according to standardized institutional protocols based on published guidelines. post-thrombotic syndrome (pts) was assessed at our center using the manco-johnson instrument. relevant data were collected and summarized using descriptive statistics. during the study period, of pediatric patients ( %) [ females, median age years (range - )] diagnosed with extremity dvt at our center were found to have an underlying vaa. vaas included may-thurner anomaly ( patients), venous thoracic outlet obstruction ( patients) and inferior vena cava (ivc) atresia ( patients). additional provoking factors were identified in patients at time of presentation. dvt locations included upper extremity veins ( patients), lower extremity veins ( patients) and lower extremity veins and ivc ( patients). the majority of dvts [ patients, ( %)] were completely occlusive. high risk thrombophilia (defined as inherited deficiency of antithrombin, protein c, or protein s, or antiphospholipid antibody syndrome) was present in patients ( %). all patients were treated with therapeutic anticoagulation with patients continuing indefinite anticoagulation. endovascular interventions were performed in patients and included percutaneous pharmacomechanical thrombectomy and/or catheter-directed thrombolysis ( patients), balloon angioplasty ( patients) and stent angioplasty ( patients). surgical interventions included thoracic decompressive surgery ( patients) and surgical thrombectomy ( patient vvas represent an important risk factor for developing extensive extremity dvt in adolescents. this special population is at risk for short-term and long-term com-plications. early identification and correction of vaas may improve outcomes. however, multicenter, prospective studies are needed for developing optimal evidence-based treatment approaches. alexander glaros, roland chu, sureyya savasan, meera chitlur, madhvi rajpurkar, yaddanapudi ravindranath children's hospital of michigan, detroit, michigan, united states background: acute budd-chiari syndrome (bcs) is a rare thrombotic emergency in children, and etiologies/treatment are less well-defined than in adults. in adults, a systematic approach including anticoagulation, relief of venous obstruction, and treatment of the underlying cause has proven successful. more recently treatment has tilted towards aggressive surgical interventions, which carry significant risk and are often not feasible. objectives: review our experience with three different patients with bcs and suggest a mechanistic based approach to treatment. the records of three patients with bcs were reviewed and their presentations, etiologies, treatment, and outcomes were reported. results: patient a was a -year-old female with paroxysmal nocturnal hemoglobinuria who presented with recurrent worsening abdominal pain over several months. narrowing of inferior vena cava (ivc) and hepatic veins was noted on imaging. liver transplant was not considered surgically feasible. she was treated with eculizumab, steroids, and anticoagulation with restoration of hepatic venous flow in weeks. patient b was a -year-old male with several weeks of right upper quadrant pain, fatigue, and pre-syncopal episodes, with a history of blunt abdominal trauma from football scrimmage weeks earlier. he was found to have near complete occlusion of the ivc and hepatic veins. liver transplant was not considered feasible. he was successfully treated with anticoagulation alone. patient c was a -yearold male with acute myeloid leukemia in induction cycle who developed severe pancytopenia; typhlitis was diagnosed and managed medically. days later he acutely decompensated, arrested, and was placed on extra corporeal membrane oxygenation, and imaging showed complete occlusion of the portal vein, hepatic veins, and ivc to the level of the atrium, with bilateral pulmonary emboli. emergency liver transplant or catheter based interventions was deemed not feasible. treatment with eculizumab was considered for presumed inflammation induced complement activation (c mg/dl [normal - ]; ch was u/ml [normal - ]) as a trigger for thrombosis, but the patient progressed quickly and died before it could be initiated. our experience with bcs shows that invasive interventional options and liver transplant may not be feasible in most patients for multiple reasons. rapid diagnosis and aggressive etiology-based medical management are paramount to successful treatment of this rare complication. eculizumab may be considered in treating bcs with complement activation not only due to innate disorders, but also secondary to acute inflammation when proper laboratory evidence is present. background: platelet aggregation studies are the gold standard for the diagnosis of platelet function defects during the evaluation of a patient with bleeding problems. the platelet aggregation test measures how well platelets clot in response to different concentrations of epinephrine, adenosine diphosphate (adp), collagen, arachidonic acid and ristocetin. because platelet function defects are often under-recognized and under-diagnosed in the pediatric patient, the true incidence is unknown. we report our experience in the diagnosis of platelet defects at our institution over a -year period in order to add some clarity to the limited pediatric data available. objectives: our primary objective is to document correlations/trends between less well-known platelet function abnormalities and clinically significant bleeding at our institution over a -year period. design/method: after appropriate irb approval obtained, we performed a retrospective chart review of all children who had platelet aggregation testing done from to . data collected included demographics (age, sex, race), personal and family history of bleeding, screening for coagulation defects and platelet aggregation test results. symptoms examined in our data were limited to epistaxis and heavy menstrual periods. for each of these symptoms, results were further analyzed to those with abnormal responses to adp and epinephrine. patients with existing bleeding diagnoses and those with incomplete medical records were excluded. we identified patients. of the patients with epistaxis, % had abnormal platelet aggregation testing while only % of those with heavy menstrual periods had abnormal results. within our population, abnormal platelet function assay (pfa- ) results or race did not appear to correlate with abnormal platelet aggregation testing. in the cases of epistaxis, sex was also noncontributory. our preliminary results suggest that platelet aggregation testing was more useful in predicting platelet defects in those with a clinical bleeding history of epistaxis as opposed to heavy menstrual periods. for other presenting symptoms, platelet aggregation testing did not offer diagnostic benefit. abnormal response to adp in the platelet aggregation test was the most common finding in our population; the clinical significance of which is not well understood. going forward, we plan to document whether abnormal results correlated significantly with the subsequent final diagnoses of our patients. background: decision making for severe hemophilia a in previously untreated patients (pups) has recently become a significant ethical debate. recombinant factor viii (rfviii) products previously were recommended to avoid transmission of blood borne pathogens associated with plasma-derived fviii (pdfviii) products. however, the increased incidence of fviii alloantibody inhibitors with rfviii products compared to pdfviii products has challenged this former standard of care. despite the support of the medical and scientific advisory council, recommendations considering pdfviii products for a pup remains controversial. design/method: we used a modified utilitarian approach involving clinical, public health, and research ethics. shared decision making permeates the framework to maximize understanding, minimize bias, respect informed consent or dissent, and provide care that aligns with patient and family values when medically and practically feasible. the framework has three tiers. first, it evaluates whether resources are scarce or abundant for equitable resource allocation. if fviii products are scarce, we s of s recommend developing a central supply for emergency use and then evaluating the needs of the severe hemophilia a patients. prioritization of who receives the factor products would be decided by a designated team based on the availability of the factor products and clinical scenarios, with no preference given to those on research trials. however, if resources are abundant, treatment for acute bleeding and standard of care prophylaxis measures, including primary prophylaxis, could continue. the second tier accounts for whether there is a new infectious epidemic or concern where a pathogen cannot be eliminated. if there is, healthcare and public health workers may limit the use of pdfviii products. if not, pdfviii and rfviii products are to be equally considered. the third tier evaluates whether the clinical scenario is emergent or not. if there is acute, emergent bleeding, the immediately available resource should be used, along with bypassing and/or adjuvant resources as needed until the bleeding has resolved or improved. to align with patient and family preferences, attempts to have both pdfviii and rfviii products available at similar costs in institutions would be ideal. this ethical framework endeavors to balance autonomy, beneficence, nonmaleficence and justice in helping guide discussions among providers, pups with severe hemophilia a, and their families. disclaimer: findings and conclusions are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention, emory university, or children's healthcare of atlanta. background: von willebrand disease (vwd) is a common bleeding disorder which affects up to % of the population without gender predilection. bleeding associated with this condition results from a deficiency or abnormality in von willebrand factor interfering with formation of primary hemostasis. ehlers-danlos syndrome (eds) is a group of rare inherited connective tissue disorders which may have an associated bleeding manifestation without abnormalities in coagulation testing. bleeding symptoms reported in eds result from capillary and tissue fragility. joint hypermobility syndrome (jhs) is an inherited condition which is nearly indistinguishable from eds iii. reports of coinheritance of vwd and eds or jhs are infrequent. the objective of this retrospective study was to review patients with coexisting vwd and eds or jhs at the indiana hemophilia and thrombosis center in order to describe the type and severity of bleeding symptoms, physical examination findings, and pertinent laboratory data. design/method: the electronic medical record database of the indiana hemophilia and thrombosis center was queried for patients with a diagnosis of vwd and one of the following descriptors: hypermobility syndrome, hypermobility, hypermobile joints, or ehlers-danlos syndrome. the records of identified patients were reviewed for demographics, type and severity of bleeding symptoms, beighton scores (bs), vwd antigen, ristocetin cofactor, factor viii levels, vwd multimer pattern, vwd subtype, genetic testing for eds, and family history of eds. results: a total of patients with dual diagnoses of vwd and eds and patients with vwd and hypermobility were identified with this query. two patients had completed genetic testing for eds, and one had a col a gene mutation identified. significant bleeding symptoms in the vwd and eds group included hematuria and postoperative hemorrhage. two of these patients had delayed wound healing postoperatively. seven of the patients identified to have type i vwd and jhs had moderately severe and somewhat unusual bleeding episodes reported including hematuria, hematemesis, and hemoptysis; of these patients had significant perioperative bleeding. females composed % of the vwd and eds group and % of the vwd and jhs group. conclusion: coinheritance of vwd and eds is an uncommon phenomenon. patients with vwd and eds or jhs may have atypical and moderately severe bleeding, especially with procedural intervention. incorporation of bs into the assessment of patients with bleeding disorders is useful to identify potential inherited collagen disorders, as diagnosis of these conditions may impact clinical management. in the year-long phase ii study (ro fd ), / khe patients responded. patients were followed for years after study completion, collecting data on growth and development, complications of therapy, unexpected toxicities, and need for continuing sirolimus. objectives: after study therapy treatment of one year, objectives include: . assess long term toxicity over the - year period after study therapy completion . assess unexpected toxicity . assess overall condition of the patient . assess need for restart or continuation of sirolimus therapy design/method: prospective follow-up of patients with a diagnosis of khe from institutions. inclusion criteria: follow-up for - years post-study. results: follow-up included data at year (n = ) and - . year (n = ) time points. average age at the start of treatment was months. of patients were available for follow up. four patients are no longer on sirolimus: one patient completed study therapy and remains off treatment (ot) ( years), required years of treatment and is now . years ot and required an additional treatment course prior to successful discontinuation now and months ot. of the patients still on sirolimus, all restarted medication for symptoms of pain, swelling and/or edema interfering with quality of life and have made an average of . attempts to discontinue sirolimus. no patient had reoccurrence of kmp. all patients had improvement in clinical and radiologic appearance of khe but all have residual lesions noted on imaging and/or clinical exam. no unexpected toxicity, growth delay, developmental issues or other long term toxicity of sirolimus was noted. conclusion: this is the first prospective data on long-term follow up of khe patients treated with sirolimus. although numbers are small, sirolimus is well tolerated; however, over half the patients were still on medication at - year follow up. this stresses the need for continued long term follow up in these young patients and investigation of the mechanism of sirolimus effect. nationwide children's hospital, columbus, ohio, united states background: recent studies have identified that adult persons with hemophilia (pwh) have a higher prevalence of hypertension and renal disease than the general population. while hematuria is a known complication of hemophilia a and b (ha, hb), its long-term impact on pwh is not currently known. by annually screening our patients with urinalysis, our pediatric center identified that just under half of our patients demonstrated hematuria over a four-year period. motivated by a desire to identify early markers of hypertension and renal disease, we sought to determine if this finding is reflected in the pediatric hemophilia population as a whole. objectives: establish the population-wide prevalence of hematuria in pediatric pwh. design/method: we used the pediatric health information system (phis) database, which contains clinical and resource utilization data for inpatients from hospitals nationwide, to analyze the prevalence of hematuria, hypertension, renal disease and related diagnosis codes in pediatric pwh who were admitted from january to september . results: during the five-year period, , unique pediatric pwh accounted for , admissions. while the majority of admissions were for bleeding or infectious concerns, ( . %) patients had an affiliated admission code for hematuria. for admissions as a whole, the median age was years with % of those admitted being infants, % toddlers, % children, % adolescents, % older than . we identified % of admissions were for ha with the remaining % were for hb. there were ( %) admits in which a bypassing agent was administered. the median length of stay for persons with hematuria was days compared to days for nonhematuria/other bleeding. there were ( . %) admissions with hypertension reported; though, only patients received an antihypertensive medication during that admission. additionally, only ( . %) admissions reported a diagnosis code of renal disease. our study demonstrated that pediatric pwh are experiencing hematuria. in general, only patients with persistent hematuria require hospital admission so we suspect this data underrepresents the numbers of pwh experiencing hematuria that is managed in the outpatient setting. we also suspect that hypertension is grossly underreported and undertreated in pediatric pwh. additionally, there are a low number of patients experiencing renal disease requiring hospital admission among this cohort. given that there is little research into the long-term impact of hematuria in hemophilia, we feel these findings support the need for further vigilance of our pediatric pwh. background: gla and gsd can aggressively destroy bone, with significant impact on morbidity and mortality. the mtor inhibitor, sirolimus has been shown to be effective in the treatment of these diseases. based on the addition of mtor inhibition to bisphosphonate therapy in metastatic cancer therapy, regimens have been used for refractory or high risk gla and gsd but there is heterogeneity of diagnosis, and variability of drug regimens and assessment of effectiveness. objectives: . assess the variability of clinical features of gla and gsd . assess the heterogeneity of diagnosis . assess drug regimens and response assessment across multiple institutions design/method: we conducted a retrospective review from institutions of cases of gla and gsd treated with sirolimus and a bisphosphonate for at least months with assessment of clinical features, treatment protocols, response regimens and side effects. results: patients included gla (n = ) and gsd (n = ). the average age at diagnosis was years. clinical features included effusions: gla (n = ), soft tissue lymphatic malformations: gla (n = ), gsd (n = ), multiple splenic lesions: gla (n = ), and soft tissue swelling at the site of bony lesion: gsd (n = ). the presenting symptom in patients was pain with patients (gla) presenting with shortness of breath. fracture was noted in patients: gla ( ), gsd ( ). diagnostic and/or response imaging included mri, ct, bone scan, skeletal survey and dexa scan. treatment consisted of: initial sirolimus use with the addition of bisphosphonate secondary to worsening disease (n = ), initial therapy with other agents (interferon, chemotherapeutic agents, radiation) and change to sirolimus and bisphosphonate secondary to toxicity (n = ), sirolimus and bisphosphonates (n = ) and sirolimus, bisphosphonates and interferon (n = ). seventeen patients had stable disease and patients had improvement of disease. sirolimus protocol was standard; however, bisphosphonate protocol varied in dosing and frequency. side effects were tolerable and expected with no grade iii or iv toxicity. sirolimus and bisphosphonates are a safe and effective therapy for gsd and gla. a consistent medication regimen, redefined response and an improved radiologic classification will be important for the development of a prospective clinical trial. background: hemophilia a is a bleeding disorder from the deficiency of clotting factor viii. the most significant sequelae of hemophilia a is the tendency to develop hemarthrosis that incites joint destruction. the prevalence of overweight and obesity has been increasing in the general and hemophilia population and leads to several morbidities including arthropathy. this is a particular concern for hemophilia a as arthropathy is a consequence of joint bleeding. objectives: the purpose of this study was to detect the relation between body mass index (bmi) and joint health endpoints in a pediatric hemophilia population. design/method: participants in this study included patients from the hemostasis and thrombosis center at children's hospital los angeles. participants were pre-screened and approached for this study during routine follow-up appointments. patients aged - years old who have been diagnosed with hemophilia a, including mild, moderate, and severe, qualified for the study. informed consent was obtained from the patients or parents before enrollment. joint health was objectively measured by physical therapists from children's hospital los angeles using the hemophilia joint health score (hjhs). an hjhs total score is calculated by assessing: swelling, duration of swelling, muscle atrophy, crepitus on motion, flexion loss, extension loss, joint pain, and muscle strength in major joints. subjective data was also obtained by patients recording their annual bleed rate within the past year. of the patients, ( %) were normal weight, ( %) overweight, and ( %) obese. we used chi-square analysis to compare joint scores across bmi classifications (chi square = . , df = , p-value = . ). although, this did not approach statistical significance, the average hjhs score in patients who had a hjhs > shows an increasing trend among bmi classifications: . in normal bmi patients, . in overweight bmi patients, and . in obese bmi patients. the average number of annual bleeds in those with positive values show: in normal bmi patients, in overweight bmi patients, and in obese bmi patients. although a positive effect of adiposity was found in the joints of hemophilia a pediatric patients, the effect shows there was not enough evidence to conclude a difference. future studies are needed to address whether obesity has an effect on hemophilia and to determine whether overweight/obesity can lead to further complications in hemophilic joints. background: stagnant blood flow in slow-flow vascular malformations (vm), particularly in their venous components, can lead to localized intravascular coagulation (lic) that is characterized by elevated d-dimer levels, low fibrinogen and decreased platelet count this coagulation derangement can lead to localized thrombosis or bleeding which can result in pain, functional limitations, and possible progression to disseminated intravascular coagulopathy (dic). the treatment of vm and their associated coagulopathy has proven difficult. patients with complex vm are frequently managed with sirolimus, an mtor inhibitor, and have clinical benefits, including reduction of pain and improvement in functional impairment. it is possible that some of these improvements from sirolimus could be secondary to improvement in the coexisting lic. objectives: this study assessed the use of sirolimus to manage the coagulopathy seen in slow-flow vm. design/method: we reviewed charts of patients with vm who are followed in the vascular anomalies center at arkansas children's hospital and were started on sirolimus. efficacy was objectively assessed through improvement of ddimer, fibrinogen and platelet count. three sets of lab values (pre-sirolimus, - months post-sirolimus, and most recent) were obtained for each patient when available. we identified a total of patients who had been prescribed sirolimus. eighteen were excluded based on underlying condition other than slow-flow vascular malformation and for inadequate medical records. a total of patients ( combined vascular, venous) were included in the study. all had elevated d-dimer levels (mean . mcg/ml feu, median . mcg/ml feu, range ( . - . )) prior to treatment. two patients had an associated low fibrinogen (below mg/dl), indicating severe lic. with treatment, ( . %) patients showed an overall decrease in d-dimer levels with an average decrease of . mcg/ml feu between pre-and post-sirolimus labs, and an average decrease of . mcg/ml feu between pre-sirolimus and most recent values. the two patients with low fibrinogen prior to treatment showed a decrease in d-dimer levels (mean decrease of . mcg/ml feu) and an increase and normalization in fibrinogen (mean increase . mg/dl) after beginning sirolimus. no patient had thrombocytopenia. we report that treatment with sirolimus was effective in improving coagulopathy associated with slowflow vm as evidenced by decreased d-dimer levels and increased fibrinogen and/or platelets. long-term use of this medication in this population may decrease the bleeding and thrombotic complications that these patients experience, especially following invasive vascular procedures. background: safety and efficacy of bay - , a sitespecifically pegylated b-domain-deleted recombinant factor viii, in previously treated adolescents and adults aged - years with severe hemophilia a was demonstrated in the phase / protect viii study and ongoing extension. objectives: this subanalysis examines the efficacy and safety of bay - in adolescents in protect viii and the ongoing extension study (data cutoff, january ). design/method: in protect viii, patients (including adolescents) received bay - on demand or as prophylaxis for weeks. prophylaxis regimens for weeks - were twice-weekly ( - iu/kg), every- -days ( - iu/kg), or once-weekly ( iu/kg) infusions based on bleeding during a -week run-in period of iu/kg twice-weekly prophylaxis. patients continued their prophylaxis regimens in the extension or changed regimens at any time. results: twelve patients aged - years were included in the protect viii intent-to-treat population; s of s additional patient discontinued after dose (included in safety population). for patients receiving prophylaxis before study enrollment, median (range) number of total and joint bleeds in the months before study entry was . ( - ) and . ( - ), respectively. ten patients ( . %) had target joints at baseline (median [range], [ - ] per patient). during weeks - of protect viii for the entire time patients remained on their designated prophylaxis dosing frequency, the median (quartile [q] ; q ) annualized bleeding rate (abr) for patients receiving twice-weekly (n = ), every- -days (n = ), and once-weekly prophylaxis (n = ) was ( ; . ), . ( ; . ), and . ( ; . ), respectively (overall prophylaxis [n = ], . [ . ; . ]). two patients switched from once-weekly to twice-weekly (n = ) or every- -days prophylaxis (n = ), and number of bleeds decreased from to in one patient and to in the other. all patients from the main study continued in the extension; mean abr in the extension was . and varied by dosing regimen (twice weekly [n = ], . ; every days [n = ], . ; once weekly [n = ], . ). two patients changed from every- -days to once-weekly prophylaxis during extension (mean abr, . ). one patient had a nonneutralizing antibody to bay - at baseline; end-of-study titers were negative. no patient developed anti-peg antibodies or factor viii inhibitors or experienced a serious adverse event related to bay - during the main study or extension. in previously treated adolescents with severe hemophilia a, bay - prophylaxis was effective in prevention of bleeds, with less bleeding overall versus prestudy, and was generally well tolerated. funded by bayer. cincinnati children's hospital medical center, cincinnati, ohio, united states background: vascular malformations (vms) consist of a heterogeneous group of congenital disorders characterized by the abnormal development of blood and/or lymphatic vessels, which cause a broad spectrum of clinical manifestations. although considered benign, vms are frequently associated with cutaneous complications that can cause significant morbidity such as nodular overgrowth, skin thickening, pruritus, oozing or bleeding of lymphatic blebs and secondary infection. oral sirolimus has shown to be effective in the treatment of complicated vascular malformations but has known side effects and need for frequent laboratory monitoring. currently, there are limited studies on the use of topical sirolimus for the treatment of cutaneous manifestations of vascular malformations. objectives: to evaluate the efficacy and safety of topical sirolimus in vms with cutaneous complications and propose indications for use. design/method: this is a retrospective review of medical records of patients with vascular malformations treated with topical sirolimus from january to december . response was determined by subjective and objective improvement. results: twenty-four patients, ( %) females and ( %) males, with vascular malformations and cutaneous manifestations were treated with topical sirolimus. age ranged from - years. indications for treatment were: blebs ( %, n = ) causing either leaking, bleeding, pain, pruritus, swelling or recurrent infection; nodular overgrowth % (n = ); pyogenic granuloma % (n = ); bleeding % (n = ) and cosmetic % (n = ). treatment course ranged from - months. no major side effects were reported. one patient reported burning and itching sensation. regarding clinical response: % (n = ) patients had improvement in cutaneous lesions; % (n = ) had a stable lesions; and % (n = ) stopped treatment due to side effects. for prior/concomitant treatment: % (n = ) had prior surgery, laser or sclerotherapy; % (n = ) had concomitant oral sirolimus. of the patients not receiving concomitant systemic sirolimus, only % (n = / ) had been on oral sirolimus. of these patients, % (n = / ) had a very good response to topical treatment. : topical sirolimus appears to be beneficial and well-tolerated with a minimal side effect profile for the treatment of cutaneous manifestations of vascular malformations as a single agent or as adjuvant therapy with systemic sirolimus when symptoms are not adequately controlled. further studies are needed to prospectively analyze efficacy and safety of topical sirolimus in this patient population. objectives: to evaluate the safety and efficacy of long-term romiplostim in children with itp. design/method: all patients received weekly sc romiplostim from - g/kg to target platelet counts of - × ( )/l. median (min-max) treatment for the patients was ( - ) weeks for a total of patient-years, or . years per patient. at baseline, median (min-max) age was ( - ) years; % were female; . % had prior splenectomy. median (min-max) average weekly dose was . ( . - . ) g/kg, including escalation to a stable dose; patients started on g/kg. reasons for discontinuing romiplostim (n = , %) included consent withdrawn (n = ), required other therapy (n = ), and ae (n = ) (asthenia, headache, dehydration, and vomiting in one patient and anxiety in the other; none treatment related). fifty four serious aes occurred in patients but were treatment related in one (concurrent grade thrombocytopenia, grade epistaxis, and grade anemia). anti-romiplostim neutralizing antibodies were detected in one patient who discontinued to receive other therapy; antibodies were absent on retesting. from week on, median platelet counts remained > × ( )/l; median platelet counts were > × ( )/l from weeks - . nearly all ( %, / ) patients had ≥ platelet response (platelet counts ≥ × ( )/l, excluding ≤ weeks after rescue medication). most ( %, / ) patients had a platelet response ≥ % of the time and % ( / ) did ≥ % of the time. sixty ( %) patients (or caregivers) self-administered romiplostim. fifteen ( %) patients had treatment-free periods of platelet counts ≥ × ( )/l for ≥ weeks (ie, remission); these patients ( girls, boys) had had itp for a median (min-max) of . ( . - ) years, none had prior splenectomy, and had received romiplostim for . ( . - ) years. all had platelet counts > × ( )/l for ≥ months and / for ≥ months; the median (min-max) duration of being ≥ × ( )/l was ( - ) weeks. of baseline characteristics such as sex, platelet counts, itp duration, and number of past itp treatments ( , , , > ), only age < years was predictive of developing treatment-free periods ≥ weeks (p = . ). in this seven-year open-label extension, > % of children with itp achieved a platelet response and romiplostim was well tolerated. importantly, % of patients were able to discontinue all itp medications for ≥ months. funded by amgen inc. background: sirolimus is an immunosuppressive drug that is widely used in solid organ and bone marrow transplantation, and more recently for the treatment of vascular and lymphatic anomalies. sirolimus has been associated with decreased immunity in the transplant setting in patients that have received other immunosuppressive drugs or were immunosuppressed from previous chemotherapy. the effects of sirolimus on the immune system in chemotherapy naïve children who have not received other immunosuppressive agents are not well understood, and there is variability in the approach to fever and pcp prophylaxis. to understand the effects of sirolimus on the immune system of patients with non-complicated vascular or lymphatic anomalies by evaluating anc, alc prior to and after sirolimus therapy. design/method: multi-institutional retrospective review was done to include patients with non-complicated vascular or lymphatic anomalies. those with effusions/ascites, multiorgan involvement, or history of vascular-anomaly-related infections prior to treatment were excluded. results: twenty patients with kaposiform hemangioendothelioma (n = ), generalized lymphatic anomaly (n = ), cloves syndrome ( ), and simple vascular malformation (n = ) were included. age at initiation of sirolimus treatment ranged from . - years. male to female ratio was : . sirolimus was initiated due to extensive disease, lack of response to steroids or bisphosphonates, pain, dment, lymphatic drainage, and prevention of ongoing overgrowth. prior to the start of sirolimus (sir- ) the mean anc was and alc was . the target level of sirolimus varied by indication and patient, and ranged from to . after the st steady state level, month after sirolimus (sir- ) the mean anc decreased to and alc was . at months after sirolimus (sir- ) the mean anc was and alc was . the first sirolimus levels (sir- ) mean was . ; and sir- level was . . nine patients were placed on pcp prophylaxis at the start of sirolimus. none of these patients had an infectious complication while on sirolimus at a median f/u of months. one patient had mild neutropenia (anc > ) which normalized after discontinuation of pjp prophylaxis. conclusion: in this small cohort of patients we found that the anc and alc level in patients with non-complicated vascular or lymphatic anomalies at sir- was not different from the sir- or sir- . prospective studies that specifically track anc, alc, igg, and lymphocyte function should be conducted to better understand the effects of sirolimus in the immune system. this data will allow for uniform recommendations regarding prophylaxis and management of febrile episodes. background: acute infections and the associated systemic inflammation can increase the risk of venous thromboembolism (vte) and in certain well-defined clinical scenarios may be the primary trigger of vte in pediatric patients. pediatric data on vte in the setting of acute infection are sparse. objectives: to describe characteristics and outcomes of vte in pediatric patients with acute infections. we conducted a retrospective chart review of all pediatric patients with objectively confirmed vte treated at our institution since and identified all patients in whom an acute infection was identified as a vte trigger. patients were managed according to standardized institutional protocols based on published guidelines. relevant demographic, clinical and laboratory data were collected and summarized using descriptive statistics. since , acute infection was identified as a trigger in of vtes ( %) diagnosed at our center. the median age at time of vte diagnosis in this group was . years (interquartile range . - ). males were more commonly affected than females, representing % of cases. neonatal vte events accounted for % of cases. sepsis was the most common acute infection to be identified as a vte trigger [ / cases ( %)]. most vte events ( %) associated with acute infections were considered hospital-associated vtes. at time of vte diagnosis, % of patients were critically ill. extensive vte (defined as completely occlusive thrombosis involving > venous segment) occurred in % of patients. acute infection was deemed to be the primary trigger for vte in / patients ( %). infection-associated vtes in this cohort included cerebral sinus venous thrombosis due to sinus or cns infection ( patients, %), septic throm-bophlebitis ( patients, %), lemierre's or lemierre's-like syndrome ( patients, %) and osteomyelitis-associated deep vein thrombosis ( patients, %). systemic anticoagulation was prescribed in / patients ( %). anticoagulationrelated major bleeding occurred in / patients ( %). vte complications included vte recurrence ( patients, %), vte progression ( patient), acute pulmonary embolism ( patients) and arterial ischemic stroke ( patients). our study indicates that acute infection is a common risk factor for pediatric vte, especially in critically ill children, and can be the primary trigger in a significant proportion of vte cases associated with acute infections. anticoagulation appeared to be overall safe in this population and was associated with low rates of serious vte-related acute complications. however, our study also suggests that this population may be at increased risk for vte recurrence and anticoagulation-related major bleeding. background: epithelioid hemangiomas (eh) are rare benign vascular tumors that occur in soft tissues and bone and present between the third and sixth decades of life. a subset ( %) of eh harbor fos rearrangement. eh has been described in children, but little is known about the long-term outcomes of pediatric eh. the main objective is to obtain data to be used for improved understanding of this rare disease in order to provide standardization of care and development of future research studies. board-approved retrospective review of clinical, pathologic, and radiographic characteristics, and treatment outcomes in patients diagnosed with eh between and . results: eight patients were male; mean age at diagnosis was . years (range: - ). lesions involved the lower extremities (n = ), cranium (n = ), pelvis (n = ), and spine (n = ). multifocal disease was identified in five patients. the most common presentations involved significant localized pain and neurologic symptoms: headache, cranial nerve injury, loss of consciousness. radiographic studies identified variable features, such as multifocal lytic bony lesions with sclerotic margins, enhancing soft tissue component, and surrounding inflammatory edema. histologically, all specimens were composed of vascular channels lined by epithelioid endothelial cells without significant cytologic atypia; solid cellular areas (n = ). endothelial cells were positive for cd and egr, and negative for camta . fos rearrangement was assessed in only one specimen and detected. mean follow-up time was days (range: - ). patients were treated with surgical resection, intravascular embolization, bisphosphonates, propranolol, interferon, and sirolimus. one patient treated with interferon and one with sirolimus exhibited partial response for mean follow-up of . days. although eh is a benign neoplasm, it is difficult to manage without standard protocols and portends considerable morbidity. our findings suggest medical management, particularly sirolimus, may benefit these patients; however, long-term follow-up is needed in treated children. novel fos inhibitors are in development and may benefit patients with fos rearrangement. penn state health children's hospital, hershey, pennsylvania, united states background: central venous catheters (cvc) are often required in critical care settings in order to provide a secure point of access for life sustaining care. clinical studies identify cvc presence as the single most important risk factor for deep vein thrombosis (dvt) in children. venous thromboembolic event (vte) incidence rates in critically ill children with a cvc range from . - % and . - . per catheter days depending on the population studied. per institutional protocol, the penn state health children's hospital picu (hershey, pa) utilizes a low dose continuous infusion of unfractionated heparin (ldufh) at units/kg/hr as prophylaxis against cvc-related vte and to maintain line patency. the efficacy of this approach has never been evaluated. to determine if ldufh for prophylaxis results in lower incidence of cvc-related vte, catheter dysfunction and central line associated blood stream infection (clabsi) without increasing morbidities. to determine if the incidence of catheter related vte is lower than historical published data, a retrospective chart review was conducted utilizing the institutional electronic medical record for all patients in , aged - . years, who had a cvc during a picu admission. secondary objectives such as the incidence of catheter dysfunction, clabsi, and any associated bleeding complications are also being analyzed. results: interim data analysis revealed cvcs ( nontunneled cvc, totally implantable devices, tunneled lines, peripherally inserted central catheters [picc] ) in total patients with a median age of . years. overall vte incidence was . % ( / ) with vtes associated with non-tunneled cvc and with piccs. sixty one percent of non-tunneled cvcs received ldufh and % ( / ) of the patients with vtes associated with non-tunneled cvcs did receive ldufh prophylaxis. vte incidence rate of nontunneled cvcs with ldufh was . % ( / ) and . per picu catheter days. the only other vte events identified within our study cohort were in the picc group where two patients experienced vte, one of which was receiving ldufh. clabsi incidence was . % ( non-tunneled cvc, tunnel cvc, picc). no major bleeding complications were associated with ldufh. preliminary data demonstrates ldufh is efficacious in preventing cvc-related vte in comparison to published reports. further analysis will compare another similar sized and acuity level picu which does not practice the same method. background: fibroadipose vascular anomaly (fava) is a rare, challenging disorder associated with pik ca mutations. fava often causes painful replacement of muscle and soft tissues with fibrotic and adipose tissue and is associated with ectatic draining veins. treatments for focal lesions are surgical excision, cryoablation or sclerotherapy and the role of medical therapy is unclear. some fava lesions are too extensive or directly involve neurovascular structure, resulting in refractory pain. objectives: to retrospectively evaluate the efficacy of sirolimus in patient with residual symptoms after procedural therapies for fava design/method: retrospective review of individual cases from institutions of fava refractory to other therapies treated with sirolimus for at least months. cases were s of s identified by polling member of the aspho vascular anomalies special interest group. results: all seven patients report improvement on sirolimus therapy. all patients had received prior procedures, including sclerotherapy ( patients), cryoablation ( patients) and/or resection ( patients). mean age at sirolimus initiation was y (range - y). mean length of therapy is . months (range - months). six patients were treated with bid dosing and one adult received daily dosing. goals of sirolimus were improvement in pain or musculoskeletal dysfunction. pain and function improved in all patients, including discontinuation of narcotic use and resumption of participation in sports. time to symptom improvement ranged from - weeks. in four patients for whom dose was lowered, pain recurred in all four and responded to restarting or increasing sirolimus dose. while all patients do not have pre-and postsirolimus imaging, decrease in fava lesion size is seen in cases with available imaging. sirolimus side effects are similar to prior reports, most commonly mouth sores, elevated lipids and acne. we report the first known data supporting a role of sirolimus in refractory fava cases. sirolimus is welltolerated and initial improvement is rapid, within weeks of initiation. whether sirolimus has a role in upfront therapy to reduce lesion size prior to procedures deserves further study. objectives: to assess platelet responses in children with itp receiving romiplostim. design/method: eligible children had itp for ≥ months, ≥ prior therapy, and screening platelet counts ≤ × ( )/l or uncontrolled bleeding. weekly dosing was from - g/kg to target platelet counts of - × ( )/l. bone marrow biopsies were evaluated in europe at baseline and after or years (cohorts and ). as of mar , patients received ≥ dose. at baseline, median (min-max) age was ( - ) years, itp duration was . ( . - . ) years, and platelet count was ( - ) × ( )/l; patients ( %) had had prior splenectomy. the median (q , q ) % time with a platelet response (platelet count ≥ × ( )/l, no rescue medications past weeks) in months - was % ( %, %) (primary endpoint). over the course of the study, % ( / ) of patients had a platelet response. four patients maintained platelet counts ≥ × ( )/l with no itp medications for ≥ weeks. median (min-max) treatment duration was ( - ) weeks for patient-years in total. median (min-max) average weekly romiplostim dose over the course of the study was . ( . - . ) g/kg; the median dose was g/kg at year (n = ) and g/kg at years (n = ). most ( %) patients initiated self-administration. sixty-four patients ( %) discontinued treatment, most frequently for lack of efficacy (n = ), patient request (n = ), and adverse event (ae) (n = ). fortyone ( %) patients had serious aes (saes) including epistaxis ( %) and decreased platelet count ( %). five patients had treatment-related saes: headaches, abdominal pain, and each of presyncope and neutralizing antibodies (ab). there were cases of neutralizing ab to romiplostim (of patients tested), but none to tpo; / had continued elevated platelet counts and in / cases ab were not found on retesting. for cohort , of patients with baseline bone marrow biopsies, had evaluable on-study biopsies scheduled for year; patient had an increase from grade to . there were no findings of collagen or abnormalities. in this interim datacut of a romiplostim openlabel study in children with itp, % of children had a platelet response. overall, the median dose was . g/kg; the median romiplostim dose over time reached g/kg. no new safety signals were observed over patient-years. funded by amgen inc. background: hepatic hemangiomas are benign vascular tumors without a medical home, managed by multiple specialties. the diagnosis has been assigned historically to various vascular lesions affecting the liver with completely different clinical presentations, resulting in difficult standardized management. objectives: the consensus steering committee identified an acute need of clear definitions and evaluation guidelines using the updated international society for the study of vascular anomalies (issva) classification. the goal was to formulate recommendations that will be adopted by all specialties involved in the care of children with hepatic hemangiomas. design/method: we used a rigorous, transparent consensus protocol, with input from multiple pediatric experts in vascular anomalies from hematology-oncology, surgery, pathology, radiology and gastroenterology. in the first section, we precisely define the subtypes of hepatic hemangiomas seen in children (congenital and infantile) using clinical course, histology and radiologic characteristics. inclusion and exclusion limits to the diagnosis are noted. the following two sections describe these subtypes in further detail, including complications to be considered during monitoring and respectively recommended screening evaluations. conclusion: while institutional variations may exist for specific clinical details, a clear understanding of the diagnosis of hepatic hemangiomas affecting the pediatric population and the possible complications that require screening during the monitoring period should be standard. as patients with hepatic hemangiomas are managed by different medical and surgical specialties, a multidisciplinary consensus based on current literature, on the data extracted from the liver hemangioma registry and on expert opinion was required and was accomplished by this manuscript. objectives: to investigate the association between routine prophylaxis with bay - and bleeding outcomes after adjusting for key patient and pharmacokinetic (pk) characteristics. design/method: the leopold kids study evaluated safety and efficacy of bay - prophylaxis in previously treated boys aged ≤ years with severe hemophilia a. patients received bay - - iu/kg x/wk (n = ) or > x/wk (n = ) and were followed up for - months. prophylaxis dose and frequency were assigned by investigators. pk parameters, including area under the curve (auc), half-life, and clearance, were derived from a population pk model and reflect predicted pk values with a -iu/kg dose. patient characteristics were compared between the x/wk and > x/wk groups using wilcoxon rank sum or chi-square tests. negative binomial regression was used to model the association between prophylaxis frequency and annualized bleeding rate (abr) for total bleeds, first without adjustment and then adjusting for age, pk parameters, and bleed history. results: mean ± sd age for patients in this analysis was . ± . years. patients receiving prophylaxis x/wk had more bleeding episodes in the months before study entry (mean ± sd, . ± . [median, . ] for x/wk vs . ± . [ . ] for > x/wk; p = . ) and were more likely to have been treated on demand ( % vs %; p = . ). pk parameters were similar between the x/wk and > x/wk groups. without adjustments, abr during the study was % higher in the x/wk group compared with the > x/wk group (rate ratio [rr], . ; % ci, . - . ; p = . ). abr was % lower in the x/wk group (rr, . ; % ci, . - . ; p = . ) after adjusting for age, auc, and number of bleeds in the prior months. conclusion: abr was numerically lower but not significantly different between the x/wk and > x/wk groups after adjusting for age and pk parameters. these findings suggest that even among patient groups that are homogeneous with respect to age, pk, and bleed history, further individualization of bay - prophylaxis based on other characteristics may help reduce bleeding episodes even at a lower treatment frequency. larger real-world studies are needed to verify these findings. funded by bayer. stanford, palo alto, california, united states s of s background: vascular malformations may be of lymphatic, arterial, venous or capillary endothelial origin. they may be simple or complex, with complex malformations being a combination soft tissue and skeletal overgrowth. although likely present at birth, these malformations often become symptomatic with puberty or infection, and range from little or no clinical impact to life threatening symptoms. in malformations primarily of venous origin, pain may be significant and hypothesized to be caused by phlebolith development (intra-malformation thrombi), inflammation, consumptive coagulopathy, vascular engorgement, and endothelial proliferation. anti-angiogenic and anti-platelet therapies have been reported to relieve pain. however, the use of anticoagulation for pain is not well described. objectives: to report clinical features and outcomes of patients with vascular malformations of venous origin treated with anticoagulation for pain. we performed a retrospective review of patients with vascular malformations followed by the hematology service between january and december who were treated for pain with anticoagulation. pain relief was determined both by wong-baker pain scales and patient report. clinical data were extracted from electronic medical records. we identified five patients with venous malformations (vm) who had received anticoagulation for pain. four patients were female and median age was years old (range to years old) at time of initiation of anticoagulation. all five patients had vm of the extremity, two with vm of the lower extremity, and three patients had vm of the upper extremity. two patients had concomitant coagulopathy and demonstrated decreased d-dimer after initiation of anticoagulation. four patients received enoxaparin, and one adult patient received rivaroxaban. all patients reported improvement in pain after administration of anticoagulation. one patient exhibited mild epistaxis and bruising at the injection site. there was no significant bleeding or other complications. pain is a significant complication in patients with venous malformations. our case series suggests that anticoagulation is a safe and effective therapy for pain relief in this population. further investigation is indicated to compare the effect of anticoagulation to other therapeutic interventions such sclerotherapy, surgery, and sirolimus in the treatment of pain associated with venous malformation. maria ahmad-nabi, christine knoll, sanjay shah, lucia mirea phoenix children's hospital, phoenix, arizona, united states background: estimates of the incidence of dvt in patients with osteomyelitis range widely from %- %, however risk factors and outcomes of dvt in this cohort have not been thoroughly established. objectives: this study aims to estimate the incidence of dvt in patients with osteomyelitis, and to assess risk factors and outcomes of dvt in this cohort. design/method: after irb approval, a retrospective chart review was conducted for patients aged - years seen at phoenix children's hospital between - with icd / codes for osteomyelitis. exclusion criteria included chronic recurrent multifocal osteomyelitis, and chronic dvt. demographics, clinical factors and outcomes were compared between osteomyelitis patients with and without dvt using the fisher-exact and wilcoxon-rank sum tests, as appropriate for the data distribution. results: a total of study subjects with osteomyelitis had a mean (standard deviation) age of . ( . ) years. dvt was present in ( % of ) patients, and ( %), ( %) and ( %) patients received anticoagulation for < , - and ≥ weeks, respectively. patients with vs without dvt were more likely to be male ( % vs %; p-value = . ), and had significantly higher rates of bacteremia ( % vs %; p-value = . ). rates of central lines were comparable between dvt and non-dvt patients ( % vs %; p-value = . ); however patients with dvt vs without dvt had significantly longer mean length of stay ( vs days; p-value < . ) and higher rates of icu admission ( % vs %; p-value < . ). the incidence of dvt among osteomyelitis pediatric patients was estimated at %, with risk increased by male sex and bacteremia. patients with dvt had significantly higher rates of icu admission and longer length of hospital stay. many of these patients had standard practice management of their dvt with - weeks of anticoagulation. our data highlights the need for recognition of high risk patients, and the need for future efforts targeting dvt prophylaxis. baylor college of medicine, houston, texas, united states background: lymphatic malformations (lm) frequently occur in the head and neck and can often be disfiguring and even life-threatening. management options include observation, surgery, sclerotherapy, and sirolimus. the optimal sequence of therapeutic interventions has not been determined due to the lack of comparative clinical trials or established guidelines. thus, prenatal planning with a multidisciplinary team is beneficial. we present a case series of ten children with head and neck lms evaluated in at our multidisciplinary vascular anomalies center. a chart review was performed to assess treatment modalities and recent trends. results: seven of patients ( %) with head and neck lms were diagnosed prenatally. six patients required an ex utero intrapartum treatment procedure. all patients were started on sirolimus at a median age of . months (range days - years). four patients most recently started on sirolimus were less than months of age at the time of initiation. six patients underwent partial excision of lm during the first year of life; none of whom received sirolimus prior to surgery. sirolimus was discontinued in one patient given chronic clostridium difficile infections, and non-compliance in another patient. five patients received sclerotherapy. tracheostomy was necessary in six patients; one patient was de-cannulated after months on sirolimus. all patients have had radiographic and clinical improvement of lm with varying treatment modalities. current clinical observations show improved response with sirolimus and demonstrate tolerability of sirolimus at a young age. conclusion: treatment of pediatric head and neck lms is challenging and a multidisciplinary approach is necessary. as the majority of patients are diagnosed prenatally, prenatal planning and discussion of potential use of sirolimus is beneficial. availability of vascular anomalies experts in the prenatal/neonatal period offers the best management results, and early initiation of sirolimus should be considered for complex lesions. long-term follow up is warranted to investigate the efficacy and timing of treatment options. yale school of medicine, new haven, connecticut, united states background: to mitigate transfusion of pathogencontaminated platelets, amotosalen, a synthetic psoralen compound, is added to sdp components. exposure to uv-a light activates amotosalen and crosslinks dna/rna base pairs, preventing replication of a broad spectrum of viral, bacterial, and other pathogens that may contaminate platelets. pr-sdps were fda approved for clinical use with no age restrictions in . we initiated use of pr-sdps in november of for all patients. we retrospectively analyzed usage of pr-sdp vs conventional (non-pr) platelets (cp) in neonatal and pediatric patients with thrombocytopenia to compare hemostatic efficacy and the incidence of transfusion reactions (tr) for these products, after one year of a dual platelet inventory. design/method: since pr-sdp were fda-licensed, no irb approval was required; pr-sdp and cp were both considered standard of care. we evaluated transfusions for all pediatric patients age - years who received any platelet transfusion between november and november . we determined the volume (mean ml ± sd) of each type of platelet component transfused, the number of platelet transfusion episodes, and reported trs based on cdc hemovigilance guidelines. a subgroup analysis was performed for thrombocytopenic neonates ( - months). results: patients - years who received only cps (n = ) received a total of , ml of platelets ( ± ml/patient) over transfusions ( . ± . episodes/patient). for comparison, in patients who received only pr-sdp, a total of , ml of platelets ( ± ml/patient, p = . ) were infused over transfusions ( . ± . episodes/patient, p = . ). for neonates ( - months, n = ) who received only cps, , ml of cps ( ± ml/ patient) were transfused over episodes ( . ± . episodes/patient). for comparison, those who received only pr-sdp (n = ), received , ml of pr-sdp ( ± ml/patient, p = . ), transfused over episodes ( . ± . episodes/patient, p = . ). for all recipients - years (n = ), including additional patients who received both cp and pr-sdp, there were three reported allergic trs over transfusion episodes, while no allergic reactions were reported with pr-sdp transfusions. one febrile tr was reported to cp transfusion, while three were reported for pr-sdp. in conclusion, pr-sdps, in our pediatric population age - years, were comparable to cp products in regards to volume and episodes of platelet transfusions, and incidence/type of transfusion reactions. pr-sdp were safe and effective for use in this pediatric patient population. background: vascular anomalies are classified as either vascular tumors or vascular malformations. fibro-adipose vascular anomaly (fava) is a newly described entity which presents with distinct clinical, radiographic and histopathologic findings. we present a case in which the diagnosis of fava was complicated by a persistent low platelet count secondary to immune thrombocytopenia (itp). to describe a challenging diagnosis of a novel vascular anomaly (fava) complicated by severe thrombocytopenia. a year old male presented to hospital with bruising and left thigh pain related to a remote sports injury. blood work revealed a platelet count of × /l, but with an otherwise normal complete blood count. the following were also normal: aptt and fibrinogen; d dimer levels were slightly increased. he was treated with one dose of ivig ( . mg/kg) for presumed itp and responded well with his platelet count increasing to × /l. he returned to hospital weeks later with recurrent thrombocytopenia and worsening leg pain. an ultrasound of the left thigh revealed a . cm x . cm x . cm lesion within the vastus medialis. the diagnosis of an intramuscular hematoma secondary to persistent thrombocytopenia was made. the patient presented with multiple episodes of thrombocytopenia over the next several months. his itp did not respond to oral prednisone ( mg/day for days). he continued to have short-lived responses to ivig requiring infusions every other week as his platelet count would fall below × /l. his leg pain progressed, restricting him to a wheelchair. further imaging by mri brought into question the diagnosis of a hematoma and a biopsy of the thigh lesion was performed. the results were consistent with a diagnosis of fava; this was subsequently excised. conclusion: this is a unique case where a vascular anomaly was misdiagnosed as a hematoma due to a patient's persistent thrombocytopenia and history of an injury. fava is a newer entity which, unlike other vascular anomalies, has not been linked to thrombocytopenia or a localized consumptive coagulopathy. after excision of the fava, the patient's chronic pain, and mobility resolved, though his itp persisted. objectives: this preliminary, exploratory analysis of realworld administrative data was conducted to determine units dispensed and factor replacement product-related direct expenditures associated with a currently marketed shl or ehl rfix product. design/method: de-identified claims data from the commercially available truven health marketscan® research u.s. claims database were used to identify direct expenditures and number of international units (ius) dispensed for all patients aged - years with a diagnosis code of icd- . /icd- d who used nonacog alfa or eftrenonacog alfa during the study period (june , to july , ). reference weight measurements from the centers for disease control and prevention national center for health statistics' (cdc nchs) anthropometric data were used to estimate product dispensation on an iu per kg basis. the nonacog alfa and eftrenonacog groups comprised and patients, respectively. the median [iqr] age in the two groups was . [ . ] and . [ . ] years, respectively. while of the patients in the eftrenonacog alfa group had > calendar quarter of available data, only of the patients in the nonacog alfa group had > available quarter. the median rfix product dispensation per quarter was , ius (iqr, , ius) in the nonacog alfa group and , ius (iqr, , ius) in the eftrenonacog alfa group. incorporating attributed weight values, the median rfix product iu dispensation per kg per week was . iu/kg/wk (iqr, . iu/kg/wk- . iu/kg/wk) in the nonacog alfa group, and . iu/kg/wk (iqr, . - . iu/kg/wk) in the eftrenonacog alfa group. applying wac prices (eftrenonacog alfa = $ . /iu; nonacog alfa = $ . /iu), the calculated estimates of $/kg/week were $ and $ in the nonacog alfa and eftranonacog alfa groups, respectively. conclusion: preliminary real-world data derived from a large u.s. claims database revealed differences in product dispensation and factor product-related expenditures among pediatric patients with any severity of hemophilia b to whom an shl or ehl rfix product was prescribed. refinements of these data, potentially to exclude instances of sporadic usage, may shed light on real-world dispensation of rfix products among pediatric hemophilia b patients. background: vascular malformations can be classified as simple (including capillary, venous, lymphatic, arteriovenous), combined, malformations of major named vessels or associated with other anomalies. multiple modalities including laser treatments, sclerotherapy, embolization, surgery and pharmacological intervention (with mtor inhibitors like sirolimus) have been used for treatment of vascular malformations. these interventions have been used alone or in combination with varied outcomes. we present our institution's experience with a multimodal approach to simple and combined vascular malformations. design/method: we performed a retrospective chart review of patients with vascular malformations who were referred to our center for an interventional radiology evaluation from june -july . we included patients (age at presentation: months - years), referred initially for interventional radiology procedures (irp) for vascular malformations. all patients had symptoms of pain and/or swelling/deformity. diagnosis of was based on vascular imaging (doppler ultrasound, mri/a/v). nine patients had venous malformations (vm), five had macrocystic lymphatic malformations (lm), six had lymphatic-venous malformations (lvm), and two arteriovenous malformations (avm). patients initially underwent interventional radiology procedures. all the vm patients responded to sclerotherapy alone. three patients with lm responded to sclerotherapy alone, remainder required surgical intervention. one avm patient responded well to embolization, the other needed surgical resection after embolization. four lvm patients underwent irp with minimal improvement in symptoms ( - procedures attempted), surgical resection was attempted in patients with poor response and patients were started on sirolimus ( . mg/m /dose twice a day). all lvm patients started on sirolimus have responded well (decreased pain and swelling); time to initial symptom response ranged from weeks - month from starting medication. in this case series, patients with simple vm responded well to sclerotherapy alone, avm and lm patients needed irp and/or surgery for complete response. complex lvm did not respond well to surgery or irp; . % had improvement in clinical symptoms with addition of sirolimus to the treatment regimen. response to various modalities of treatment varied based on the type of vascular malformation. a multidisciplinary approach to management of vascular malformations is essential to provide multimodal therapeutic options for rapid symptom relief and improve the quality of life of these fragile patients, especially those with complex malformations. background: von willebrand disease (vwd) is the most common bleeding disorder in humans, affecting ∼ % of the united states' population. desmopressin (ddavp) is a longacting vasopressin analog that induces vasoconstriction and release of vwf. ddavp is used in patients with vwd and as a surgical prophylaxis, but carries anti-diuretic properties. to avoid electrolyte imbalance and hyponatremia, fluid restrictions are recommended in the hours post-ddavp administration. objectives: this study sought to examine perioperative practices and outcomes following ddavp administration and a fluid restriction protocol in a population of pediatric patients with von willebrand disease. design/method: a retrospective chart review was conducted for patients with von willebrand disease who underwent surgical procedures at children's hospital of pittsburgh of upmc between january , and december , . patient age, sex, weight, diagnosis, surgical procedure, total fluids administered, and post-operative sodium level were recorded. the primary outcomes noted were the proportion of patients exceeding % of the recommended fluid consumption for the -and -hour periods post-ddavp s of s administration, as defined by local guidelines. secondary outcomes were the presence of any bleeding requiring an er visit or readmission or hyponatremic seizures within hours of ddavp administration. results: data was compiled for patients ( females, males). the mean age was . years (sd . years), median age was years (range to years). procedures included dental ( ), otolaryngology ( ), orthopedics ( ), gastrointestinal ( ), plastics ( ), neurosurgery ( ), ophthalmology ( ), dermatology ( ), general surgery ( ) and gynecology ( ). % of patients exceeded % of the fluid volume recommended for the first -hour period post-ddavp administration while still in the surgical setting. no patients exceeded % of the fluid volume recommended for the total -hour period post-ddavp administration. post-operative sodium levels were obtained in only of patients. no patients returned to the er or were admitted for bleeding in the hours post-ddavp administration. no patients returned to the er or were admitted for hyponatremia or seizures in the hours post-ddavp administration. maintenance of a fluid restriction protocol effectively deterred negative outcomes in this cohort. however, a significant fluid volume was administered in nearly a third of patients despite the restrictions. given the risk of hyponatremia, and limited compliance with fluid restrictions, postoperative sodium levels should be recorded in following ddavp administration to assess the possibility of a hyponatremia and to reinforce the importance of fluid restrictions and their communication. results: a male fetus required in utero insertion of a pleuroamniotic shunt for bilateral pleural effusions diagnosed antenatally by ultrasound. shortly after delivery at term, he developed respiratory distress and was found to have reaccumulation of the pleural effusions. blood work on day of life showed a platelet count of , / l, which then decreased precipitously. he demonstrated schistocytes on blood-smear, signs of consumptive coagulopathy with hypofibrinogenemia and high d-dimers, and compensatory reticulocytosis. he required multiple transfusions and admissions to the intensive care unit for respiratory support. investigations ruled out congenital ttp, neonatal alloimmune thrombocytopenia, and noonan syndrome. given high clinical suspicion for an underlying vascular lesion causing kmp, a full body mri without contrast was undertaken. this showed a focal area of suspicious signal intensity in the upper paraspinal musculature. an ultrasound and mri with contrast demonstrated an extensive infiltrative vascular lesion involving the paraspinal musculature, prevertebral space, posterior extrapleural space, mediastinum, and neck. the child was commenced on prednisone ( mg/kg/day) and rapamycin ( . mg/m twice/day). there was no clinical or laboratory improvement after one month. a biopsy was performed which confirmed khe. in the second month of rapamycin therapy, the platelet count gradually normalized and the patient was discharged from hospital at . -months of life. prednisone was weaned off at . months of life. a repeat mri at months showed significant reduction in the khe. he is now almost years into therapy and doing well. conclusion: this is a unique case of khe with kmp that initially presented with extensive and recurrent pleural and pericardial effusions. this case demonstrates the importance of suspecting an underlying vascular malformation in the presence of kmp. our patient had a delayed but overall good response to rapamycin. further studies investigating duration of rapamycin therapy is key for the optimal management of these patients. rosa diaz, donald mahoney, lakshmi srivaths, donald yee texas children's hospital, houston, texas, united states background: since von willebrand disease (vwd) is the most common inherited bleeding disorder, it must co-exist with other less common bleeding disorders in some dually affected patients. however, reports of combined deficiencies in factor viii (fviii) and von willebrand factor (vwf) are rare. objectives: to study the prevalence and bleeding phenotype of combined deficiencies of fviii and vwf in males with hemophilia a in a hemophilia treatment center. design/method: we retrospectively reviewed the electronic medical records of males with hemophilia a followed at our institution during the past years. the primary and secondary outcomes for the study were ( ) the prevalence of combined fviii and vwf deficiencies and ( ) the bleeding phenotype of these patients. we identified vwf deficiencies in % (n = ) of the patients with hemophilia a. most (n = , %) patients were tested for vwf deficiency as part of the initial hemostatic evaluation, but one-third were tested due to clinical concern for inadequate response to fviii concentrate. the median duration of follow up was . years (range . to . ). patients were referred to our clinic at a median age of months (range to years) for evaluation of easy bruising (n = , %), mucosal (n = , %) and surgical bleeding (n = , %). primary diagnoses included with severe, moderate and mild discrepant hemophilia a. secondary diagnoses included with low vwf activity, type vwd and with type unclassified. patients experienced episodes of musculoskeletal (n = , %), mucocutaneous (n = , %) and cns bleeding (n = , %). a total of patients received factor prophylaxis. half of the patients were initially treated with fviii concentrates but subsequently changed to combined fviii/vwf products due to the frequency of breakthrough bleeding despite good compliance. all patients are on combined fviii/vwf products at the time of this review. a total of ( %) of this cohort developed chronic joint disease manifest as decreased range of motion and/or abnormal mri findings. combined deficiencies of fviii and vwf were present in % of our center's hemophilia patients. these patients exhibited a severe bleeding phenotype as evidenced by the high frequency of hemarthrosis, need for prophylaxis and high prevalence of chronic joint disease. while the optimal treatment strategy remains to be elucidated, early recognition of a combined deficiency may have important clinical implications, particularly in patients who demonstrate a suboptimal response to fviii concentrate alone. background: childhood neutropenia is heterogeneous and may be congenital or acquired. cerebral cavernous malformation (ccm ) is a neurovascular malformation disorder where lesions consist of low flow, dilated capillary endothelial channels with increased permeability, predisposing to hemorrhage and thrombosis. programmed cell death protein (pdcd ) activity has been implicated in glia and neuron migration, and recently linked to the dysregulation of the actin and microtubule cytoskeleton, thereby affecting cellular morphology and migration. variants of pdcd encoding pdcd have been associated with ccm . ccm causes a greater and earlier disease burden than other ccms, with % presenting younger than years. some patients have associated extra-neuronal manifestations, suggesting that pdcd plays a role in other tissues. we describe a patient with significant blood cytopenias associated with ccm . design/method: retrospective chart review to obtain patient data. results: an -month old female presented with seizure and was found to have multiple intracranial cystic lesions and abscesses due to s. pneumonia serotype f. during her treatment, she developed anemia (hemoglobin . - . g/dl), thrombocytopenia (platelets , - , cells/l), and profound neutropenia (absolute neutrophil counts of zero). initial bone marrow evaluation revealed a normocellular marrow but with marked granulocytic hypoplasia and % hematogones on flow cytometry. florescent in situ hybridization excluded cytogenetic changes characteristic of myelodysplastic syndrome. further evaluation included testing for neutrophil antibodies, chromosome breakage, and telomere length and results were normal. whole exome sequencing excluded mutations affecting congenital neutropenia genes, but detected a de novo pdcd variant (c. + g>a), thereby diagnosing ccm . the neutropenia has responded well to granulocyte colony stimulation factor (gcsf), which is still needed at months of age. moreover, the thrombocytopenia has progressed, requiring periodic platelet transfusions. over time, the bone marrow hematogone population has decreased to % at months of age, though the granulocytic hypoplasia persists. conclusion: our case describes the first patient with neutropenia and thrombocytopenia associated with ccm . we hypothesize the pdcd variant is the etiology of bone marrow dysfunction due to its role in actin and microtubule cytoskeleton formation, akin to the pathophysiology of xlinked neutropenia. supportive features of an underlying genetic cause of marrow dysfunction include the persistence of cytopenias beyond infection resolution as well as presence of hematogones. hematogones were previously reported to occur in patients with other congenital neutropenia disorders, indicating they could be a feature of congenital neutropenia and may be reactive to surrounding cell apoptosis. further testing of pdcd role in hematopoiesis should be explored. background: - % of adult women will suffer from heavy menstrual bleeding (hmb) during their lifetime. % of women with inherited bleeding disorders suffer from hmb. there is a paucity of data about hmb among adolescents and young adults (aya), a population in which hmb may have large social and educational effects. objectives: to study the social and academic implications of hmb in an aya population. design/method: this is a questionnaire based survey conducted in a medium-sized city in california. we recruited females - years of age from one high school and from local university. the questionnaire was set up in research electronic data capture (redcap) at our institute which allowed us to obtain objective data about the respondents' menstrual cycles. a link was sent to the high school students via their online portal schoolloop and to the university students via social media and word of mouth. data was collected over weeks from may to august . we received replies, some were not complete. using regression analysis, data was analyzed from respondents in the age group of - (with a mean age of ) years. we developed a composite score for hmb based on factors including saturation levels, number of pads, duration of bleeding, soaking of a pad within two hours, passage of clots, size and number of clots, and gushing sensation. we conducted statistical analysis of the drivers and implications of hmb based on the composite score. results indicate that having a relative with hmb, having other bleeding problems, and having anemia are drivers of higher hmb score. the results also indicate that hmb adversely affects quality of life as measured by participation in sports, social activities, after-school activities, tiredness, absenteeism, and gpa. hmb is also associated with increased rates of anemia and use of anti-depressants. hmb-driven anemia further adversely affects gpa. under-represented minorities are more likely to have a higher hmb score, as well as an increased adverse effect of hmb on gpa. the results suggest that the social costs of hmb are pervasive in the aya population, and especially pronounced among minorities. a relative with hmb is a significant driver of heavy menstrual bleeding. a hemostatic screen should be included when assessing the aya population with hmb. johns hopkins all children 's hospital, st. petersburg, florida, united states background: propranolol is a non-cardioselective beta blocker medication frequently prescribed for hemangiomas and hyperthyroidism. propranolol inhibits types i and ii iodothyronine deiodinases, enzymes that convert bioinactive thyroxine (t ) into bioactive triiodothyronine (t ). hypothyroidism is a well-recognized complication of diffuse hepatic hemangiomas that produce type iii deiodinase, an enzyme that converts t into bioinactive reverse t and t into diiodothyronine. thyroxine is typically selected for replacement in this population, even though doses up to % above physiologic may be necessary. we hypothesized that low dose, nearly physiologic t would be safer and equally effective because it bypasses propranolol's impact on the pituitarythyroid axis. we report an infant with diffuse hepatic hemangiomatosis and acquired hypothyroidism successfully treated with propranolol, prednisone, and triiodothyronine. design/method: a mo healthy female presented with abdominal distension, poor oral intake, and hepatomegaly. mri confirmed diffuse hepatic hemangiomatosis, the largest lesion measuring . cm by . cm. thyrotropin (tsh) was elevated at . (reference range* . - mcgiu/ml), total t # (rr - ng/dl), and total t ^ . (rr - mcg/dl). treatment was started with prednisone ( mg/kg/day) for three weeks, propranolol ( mg/kg/day) and t ( . mcg/kg/day). the t dose was slowly titrated to a maximum of . mcg/kg/day. thyroid hormone levels rapidly improved on t replacement. after two weeks, the tsh was . , tt , and tt . . after eight months, the tsh was . , tt , and tt . . at twelve months, the tsh dropped to . , tt , and tt . , suggesting decreased tumor production of type iii iodothyronine deiodinase. liver mri confirmed fewer hemangiomas, largest being . cm by . cm. the patient's t dose was reduced. both propranolol and t were discontinued after twenty-four months of treatment. one year off all therapy, this child has normal growth and development, only two < . cm hepatic hemangiomas and no evidence of hypothyroidism (tsh . ; tt ; tt . ). conclusion: t at near physiologic doses corrects the consumptive hypothyroidism associated with diffuse hepatic hemangiomas. t replacement is preferable to thyroxine due to its lower risk of rebound hyperthyroidism as the hemangiomas involute and type iii deiodinase production declines. there are two prior case reports describing t use without t , one employing propranolol and the other utilizing steroids for hemangioma management. this is the first case report with long term follow-up of a child treated with multimodal therapy including propranolol, prednisone, and triiodothyronine. *rr = reference range; #tt = total t ;^tt = total t background: multifocal lymphangioendotheliomatosis with thrombocytopenia (mlt) is a rare congenital disorder first described in that is characterized by multiple vascular abnormalities commonly involving the skin and gastrointestinal tract as well as consumptive coagulopathy often resulting in gi bleeding in infancy( ). to describe an unusual presentation and successful management of mlt in a neonate. design/method: baby h was born at full term after a pregnancy complicated by maternal sinus venous thrombosis requiring anticoagulation beginning at weeks. at birth, she was diagnosed with multiple hemangiomas based on clinical exam. at two weeks of age, she developed melena and hematemesis. cbc revealed platelet count of and she was referred to the ed. abdominal ultrasound was concerning for abnormal hepatic waveform; cxr showed multiple pulmonary nodules. workup revealed no other lesions and no further hematologic abnormalities. biopsy of presumed hemangioma ultimately revealed a smooth muscle-lined vascular proliferation without glut- immunoreactivity, consistent with mlt. her early course was complicated by an acute hemodynamically significant gi bleed; esophagogastroduodenoscopy identified six bleeding vascular malformations within the stomach that were injected with epinephrine and sclerosed with successful hemostasis. she received multiple prbc and platelet transfusions. central access was obtained and she was started on oral sirolimus based on previous reports of successful use in management of vascular malformations given its antiangiogenic and immunosuppressive effects ( ). she has tolerated it well with no evidence of toxicity and has achieved a partial response with stable of hemoglobin > and platelet count > . cutaneous lesions have diminished in intensity and she has had no further signs of gi bleeding. she receives pentamidine for pcp prophylaxis. she continues to have appropriate growth and development. we describe here an unusual presentation of an already rare disease. while cutaneous and gi lesions are typical of mlt, pulmonary involvement is not well-described in the literature. early identification of tissue-based diagnosis enabled timely stabilization and treatment of the patient. five months later, she continues to tolerate sirolimus and has shown significant response with diminished coloration of cutaneous lesions, stable blood counts, and no further bleeding. mlt is a relatively newly-recognized disorder with significant phenotypic variability. given that bleeding secondary to a kasabach-merritt-type consumptive thrombocytopenia is the major cause of morbidity and mortality in the first year of life in children with mlt, it is essential to recognize the diagnosis and initiate appropriate treatment as early as possible. north, arch background: patients with generalized joint hypermobility (jhm) may experience easy bruising or bleeding given the association between these symptoms and abnormalities in collagen, a required component of primary hemostasis. heavy menstrual bleeding (hmb) is a common initial presentation for females with underlying hemostatic defects and may be the sole manifestation of a bleeding disorder. however, limited reports describe jhm as a cause of hmb, leading to under recognition. objectives: to describe the clinical characteristics and management of young women presenting with hmb in the setting of jhm. design/method: this study utilized our hmb research registry. we included subjects - years, seen in the nationwide children's young women's hematology clinic between february and november with both hmb and jhm. medical records were retrospectively reviewed for history of presentation, menorrhagia impact questionnaire (miq): a validated quality-of-life tool for females with hmb, medication profiles and relevant laboratory studies. results: twenty-five patients met inclusion criteria (median age years, range - ) with an average beighton score of . (range to ). participants presented an average of . years (range months to years) after menarche despite % of patients reporting heavy to very heavy menses since menarche. according to the miq responses, most participants expressed hmb-associated limitations in physical activities ( %), social activities ( %), and work or school activities ( %). of the participants, % reported bleeding symptoms in addition to hmb, most commonly easy bruising ( %), epistaxis ( %) and cutaneous bleeding ( %). forty percent of young women presented with anemia due to chronic blood loss. results of hemostatic testing were unremarkable, with the exception of one patient who was also found to have type von willebrand disease. additionally, % of females reported arthralgia, with knees and ankles the most commonly affected joints. at time of presentation, % of participants reported failure of initial therapies and most patients ( %) were managed long-term with oral hormone therapy. in a small population of young women found to have jhm who initially presented with hmb, patients were likely to have prior bleeding symptoms as well as substantial delays from menarche to timing of presentation at our young women's hematology clinic despite limitations in activities of daily life. greater awareness of the associations between bleeding symptoms and jhm, despite typically normal hemostatic laboratory results, is necessary so that patients can more easily be identified and receive appropriate therapy. the objective is to determine the impact of cl care practices involving the home environment on ambulatory clabsi rates. design/method: information for the pi was collected through a comprehensive survey that was completed annually by the ccbdn member hospitals. responses to the questions about cl care practices involving the home environment were selected from the pi for . ambulatory clabsi rates and ambulatory total bloodstream infection (bsi) rates were obtained from another ccbdn database. the proportion of hospitals that did or did not employ a particular cl care practice was tallied. the mean ambulatory clabsi rate and mean ambulatory total bsi rate of the hospitals that did or did not employ a particular cl care practice were compared using generalized linear model techniques assuming an underlying negative binomial distribution. results: twenty-five hospitals submitted responses to the questions about cl care practices involving the home environment. one hospital was excluded for lack of bsi data. sixty-three percent of the hospitals programmatically educated parents about all aspects of the cl care bundle. the mean ambulatory clabsi rate for the hospitals that educated parents was significantly lower than that of the hospitals that did not ( . infections/ cl days vs. . infections/ cl days; p = . ). the mean ambulatory total bsi rate was also significantly lower ( . infections/ cl days vs. . infections/ cl days; p = . ). the mean ambulatory clabsi rates and mean ambulatory total bsi rates were not significantly different for the other cl care practices. conclusion: an analysis of cl care practices involving the home environment reveals that parental education of all aspects of the cl care bundle is associated with a lower ambulatory clabsi rate and lower ambulatory total bsi rate. this finding highlights the importance of systematically teaching family members the proper method of handling cl. background: children undergoing chemotherapy are at a high risk for developing nausea. dr. amy baxter in collaboration with pediatric oncology patients and nurses, developed and validated a pictorial nausea rating scale for children aged - years, called the baxter retching faces (barf) nausea scale. staff nurses at a large, academic, pediatric hospital located within washington, d.c., have identified variability in nursing assessment and documentation of chemotherapy induced nausea and vomiting (cinv) in pediatric oncology patients. the purpose of this quality improvement project was to utilize the barf scale to standardize assessment and documentation of nausea in pediatric oncology patients receiving chemotherapy. the primary aims of this project were to: assess feasibility of the barf scale in clinical practice; increase nursing knowledge about cinv through education sessions; increase documentation of nausea assessments through the use of the scale. the secondary aim of this project was to: increase the recognition of nausea through the use of a standardized assessment tool. design/method: the pdsa model was used to guide the design and implementation plan. in the first phase of the project data was collected to identify the prevalence of nausea in patients admitted for chemotherapy in the prior three months. education sessions discussing cinv and the utilization of the barf scale were conducted. pre and post assessment of nurses' knowledge of cinv and documentation were assessed. in the second phase the barf scale was implemented into practice. nurses were asked to utilize the barf scale to assess and document nausea scores in patients, aged to years, receiving chemotherapy. at the end of the implementation period nurses were surveyed about the feasibility of the scale. post data was collected to identify the prevalence of nausea documented in the electronic health record. this project was undertaken as a quality improvement initiative at children's national and it does not constitute as human subjects research. as such it was not under the oversight of the institutional review board. results: all data has been collected; however complete data analysis will be conducted in the upcoming weeks. background: sickle cell disease (scd) is the most common inherited blood disorder in the united states (us); however, there are few quality measurements to evaluate scd practice. in , the nhlbi published guidelines that include two key interventions for children with sickle cell anemia (sca): the use of transcranial doppler (tcd) screening for stroke prevention and hydroxyurea (hu) to prevent scd pain crisis. we conducted a national survey of scd management sent to providers in over institutions in the us to better assess knowledge of the guidelines and barriers to hu counseling and tcd screening guideline implementation. it was hypothesized that the barriers to tcd screening are different than barriers to hu counseling and prescribing. a -question anonymous survey was sent to providers by mail (follow-up by email). survey themes included nhlbi guidelines knowledge and comfort with understanding and implementing both tcd screening and hu use. the response rate was % ( / ) however one survey was incomplete. thus, were analyzed in the final data set. all of the respondents are in active practice, % s of s in academics and all care for children with scd. the majority of providers ( %) felt "very" or "extremely" confident in their knowledge of tcd screening and interpretation. similarly, % of providers felt "very" or "extremely" familiar with hu dosing and management. for tcd screening, % of providers estimated their screening rates were > % and % providers felt their annual screening rates were - %. the two biggest barriers to tcd screening noted by providers (of moderate to extreme significance) included: lack of support staff ( %) and lack of time during a patient visit ( %). regarding hu prescribing practices, % of providers offered hu to at least % of children with sca over nine months of age. the biggest barrier to hu prescribing noted by % of providers was concerns about patient adherence or access to the medication. only % providers felt that lack of support staff was a moderately significant barrier to hu prescribing. the pediatric scd providers surveyed all have access to the nhlbi guidelines. despite widespread guideline knowledge, there are different barriers for tcd screening versus hu prescribing, which prevent optimal implementation. as a result, although both recommendations are from the same nhlbi guideline, they likely will require different implementation strategies (systems-based interventions for tcd screening; interventions to improve patient adherence for hu counseling) to improve outcomes. background: invasive fungal disease (ifd) is a major cause of mortality and morbidity among pediatric immunocompromised patients such as those who receive chemotherapy or hematopoietic stem cell transplantation. the current diagnostic 'gold standard' of ifd remains culture of infected tissue obtained by biopsy. noninvasive biomarker testing for galactomannan or , -beta-d-glucan (bg) can have low sensitivity and does not provide species-level identification. nextgeneration sequencing (ngs) of cell-free plasma is a promis-ing noninvasive approach to providing species-level identification of ifd via a blood test and can further guide specific treatment. objectives: describe the incidence of positivity for fungal specific pathogens on ngs analysis in a high-risk immunocompromised pediatric population and correlate results with other 'standard' infectious studies if performed. design/method: immunocompromised pediatric patients with suspected ifd were enrolled and plasma was collected at time of enrollment. ngs was performed on extracted dna in cell-free plasma (karius, redwood city, ca). after removing human reads, remaining sequences were aligned to a curated database including pathogens. organisms present at a significance-level above a predefined threshold were reported. results: twenty-seven samples from enrolled patients have been processed thus far. of these subjects, were enrolled for prolonged febrile neutropenia (≥ hours) despite broad-spectrum antibiotics, for recrudescent febrile neutropenia, for abnormal imaging, and with other findings. after evaluation of routine studies performed, patients met criteria for proven ifd, for probable ifd, and for possible ifd using eortc/msg guidelines. the ngs plasma test identified the same pathogen as cultured from infected tissue or blood in % ( / ) of the proven cases. in the probable cases, pneumocystis jirovecii was identified in a patient with a positive bg ( pg/ml) and pneumonia. among the possible cases, toxoplasma gondii was detected in a patient with prolonged febrile neutropenia and lung imaging suggestive of ifd. additionally, candida glabrata was isolated in a patient with prolonged febrile neutropenia but no other criteria for ifd. numerous pathogens were also identified that could explain the above clinical parameters, including hsv , cmv, vzv, hhv , ebv, bk polyoma virus, and ureaplasma parvum. the cell-free plasma ngs test can detect invasive fungal infections from blood. the test identified fungi from proven ifd, detected pathogens in both probable and possible ifd cases, and is a useful diagnostic tool in the evaluation of ifd. supplies and sample shipment and processing supported by karius, inc. baylor college of medicine, texas children's hospital, houston, texas, united states background: practicing medicine is a lifelong learning process. as noted in the institute of medicine's seminal report, 'to err is human,' adverse outcomes do not typically result from individual recklessness; rather, they result from faulty systems, processes, or conditions that provide an environment conducive to making a mistake, or failing to prevent one. learning to systematically review errors and translate lessons learned into quality improvement (qi) initiatives is a critical component of practice-based learning and improvement for practitioners at all career levels. objectives: to develop a methodical, self-reflective and nonthreatening approach to incident analysis and translation of lessons learned into qi initiatives. design/method: we used a validated, structured case audit approach, modified from szostek et al: ) review all documentation relating to the case and identify all health care providers involved; ) interview stakeholders, including those who directly provided and supported care; ) use a qi tool to conduct a root-cause analysis; ) identify a systems issue that contributed to the outcome; and ) propose systems-level interventions and prioritize initiatives based on effort-yield projections. results: pdsa cycle : plan: establish a committee to ) identify potential cases, ) triage cases for conference presentation, ) determine timing and frequency of conferences, ) develop a training manual, ) record identified qi initiatives. do: we established a quarterly section-wide meeting to which all members of the pediatric hematology/oncology service are invited, including administrative and nursing leadership. we developed a training manual and structured presentation template. prioritized cases were discussed in advance during multidisciplinary case review sessions, and presented by senior fellows who were instructed to focus discussion on potential opportunities for qi. study: we identified cases, meeting criteria for mmi presentation. qi initiatives identified from this conference resulted in a number of systemic practice changes; however, we encountered challenges to sustaining these changes over time. act: objectives for the next pdsa cycle are to ) establish a method for tracking the adherence to recommended changes in practice, ) maximize sustainability by integrating qi initiatives into institutional qi leadership and practice standardization committees. we have successfully implemented an mmi conference that meets out of institute of medicine quality domains: safety, effectiveness, patient-centeredness, timeliness, and efficiency. a standardized, consistent approach to mmi presentations that includes identification of contributing factors and specific qi implications has the potential for improving both provider education and patient care/safety. johns hopkins university, baltimore, maryland, united states background: receiving a cancer diagnosis is a life-changing event for patients and caregivers, although little is known about the experience. while some oncologists receive dedicated training in delivering this bad news, the initial conversation is often with a primary pediatrician, and these providers often feel they do not receive adequate training in the communication of a cancer diagnosis. objectives: our objectives were two-fold: first, to better define the experiences of caregivers/patients when told of a cancer diagnosis, and to query how caregivers/patients believe providers can improve the disclosure of this bad news. secondly, to assess what, if any, training primary pediatricians received in this skill, and to assess how comfortable providers in various settings and stages of training are with communicating cancer diagnoses. design/method: from november - , semistructured, in-depth interviews were conducted with pediatric oncology patients and caregivers of patients (n = ) diagnosed in the past year regarding their experiences receiving the diagnosis at our institution. in addition, pediatric residents (n = ), outpatient pediatric primary care physicians and pediatric emergency medicine physicians (n = ) were interviewed regarding their experiences delivering cancer diagnoses. interviews were analyzed following principles of thematic analysis. interviewers with patients and caregivers had two common themes: ) all emphasized their wish for direct and thorough information; ) both patients and caregivers emphasized the gratitude they felt for physicians who gave them hope by emphasizing the good prognosis of their child's cancer. lack of training in this area, as well as lack of comfort delivering this news was common will all providers. additionally, providers report variable approaches to giving bad news, including ) whether to tell caregivers separately or tell the child and parents together, and ) whether to give favorable prognostic information. additionally, attending physicians also differed significantly in their approaches to teaching residents. while some believed residents should give the news to gain experience, others felt that this is not appropriate if residents are inexperienced. only one resident reported ever receiving feedback on his communication skills in this type of discussion. conclusion: we plan to build on these interviews to develop a national survey of patients, caregivers, and providers to better understand the issues surrounding this discussion. we will use the findings to develop a communication curriculum for pediatric residents, focusing on the discussions that occur in the outpatient setting by primary pediatricians. background: human papilloma virus (hpv), common in both females and males, is responsible for pathologies ranging from benign genital warts to cervical and penile cancer. hpv strains and are responsible for , malignancies each year in the united states, and one third of them arise in men. pharmaceutical companies have now developed a vaccine that will help prevent the virus-associated malignancies. the cdc initially recommended that females ages - years receive the vaccine series, then starting in they expanded the eligibility to males ages - years. despite being widely available and highly publicized, only % of eligible females receive the full vaccine series. objectives: this study aims to assess the knowledge of hpv, the attitudes towards the hpv vaccine, and identify barriers preventing its full utilization. once identified, we aim to overcome the barrier(s) in order to improve vaccination rates in eligible adolescents. we distributed a standardized questionnaire to the parents of eligible female and male patients in our pediatric hematology-oncology clinic. it assessed the parents' knowledge of hpv and the vaccine, their views of the vaccine, and reasons why they may oppose it. results: approximately % of parents claim they have been educated about hpv, mostly by their primary care physician. however, % did not know what disorders hpv caused; % felt the vaccine should not be added to the typical vaccine schedule; % of parents do not intend to vaccinate their child. of those that opposed the vaccine, one-third were concerned about potential side effects and nearly % feel they do not have enough information. additionally, % of parents are not aware that the vaccine is available at their child's doctor and only % of parents have discussed the hpv vaccine with their child's doctor. the largest barrier to the utilization of the hpv vaccine that we have identified appears to be lack of educa-tion. as a result, we have begun distributing the cdc's hpv and vaccine patient guide to our patients' families as an intervention. we are currently in the process of re-administering our survey to these families after implementing the intervention to assess its success in increasing both knowledge and utilization of the hpv vaccine. cancer institute, chennai, chennai, tamilnadu, india background: rasburicase is a recombinant urate oxidase enzyme approved for use in tumor lysis syndrome (tls) and it acts by reducing serum uric acid levels. using rasburicase at the recommended dose of . mg/kg/day for days is expensive and it is not known whether this extended schedule is clinically beneficial compared to a single fixed dose of . mg. the aim of the present study was to evaluate the efficacy of single dose rasburicase . mg in prevention and management of tls. design/method: rasburicase is available as single use . mg vial. at our institution a single dose of rasburicase . mg irrespective of bodyweight has been used in adults and in children a dose of . mg/kg (maximum . mg) has been used since for prevention and management of tls and subsequent doses are given based on biochemical response and clinical condition. we retrospectively analysed the case records of patients who had received rasburicase from january to january . the study included patients with hematological malignancies who received rasburicase. children accounted for . % (n = ) patients and males comprised % (n = ). rasburicase was used prophylactically in ( . %) patients, for laboratory tls in patients ( . %) and for clinical tls in ( . %) patients. single fixed dose rasburicase prevented laboratory/clinical tls in % of the prophylactic group and prevented clinical tls in % of the laboratory tls group. none of the patients in prophylactic and laboratory tls group developed clinical tls. however, majority of the patients with clinical tls required more than one dose rasburicase. single dose of . mg ( vial) rasburicase is efficient in preventing and managing laboratory tls and is economically viable in resource constrained settings. nicole wood, lauren amos, nicholas clark, chris klockau, karen lewing, alan gamis children's mercy kansas city, kansas city, missouri, united states background: medication reconciliation for newly diagnosed oncology patients is complicated and cumbersome. these patients are often admitted on no medications, and leave on multiple. chemotherapy and supportive medications are crucial. despite numerous individuals overseeing this process, prescribing errors or omissions still occur. when reviewing the literature, improvement occurs when there is an interprofessional and standardized process to medication reconciliation. objectives: this project's aim was to improve the accuracy of the discharge medication reconciliation process from % to % from february -august . the process measure was the percentage of patients discharged with an accurate checklist. additional time for staff spent in completing the checklist and avoiding an increased error rate by changing the prescribing process were followed as balancing measures. we created a discharge medication checklist which included a list of required home medications prescribed by the resident, ideally hours prior to discharge. it required fellow or attending review and pharmacy to review the list and educate the family. checklists were collected monthly and reviewed against the electronic medical record (emr) for accuracy. results: six pdsa cycles were completed. there were errors during the data collection time frame. in pdsa cycle , a patient received acetaminophen for pain control which is avoided at home. in addition, this patient received diphenhydramine instead of ondansetron, which is preferred as an antiemetic. in pdsa cycle , a patient with a pending diagnosis was sent home with acetaminophen. of note, this patient did not have a checklist completed upon discharge. this project provides a novel and important method to standardize the discharge medication reconciliation process in a complex patient population. it clarifies which types of medications these patients need, provides pharmacy teaching to families which was not done previously, and prescribes discharge medications to families sooner. after the first medication reconciliation error, the checklist was revised. no further errors were made following revision, with the exception of one patient without a completed checklist at dis-charge. our accuracy rate increased from % at baseline to % following implementation. we are in the process of making the checklist electronic and accessible in the emr. in the interim between the end of data collection and implementation into the emr, a leukemia patient was sent home without an epinephrine pen, further demonstrating the importance of this standardized discharge process. for this reason, we have re-instituted the checklist until the electronic version is available. background: survivors of pediatric cancer are at risk of losing pre-existing protective antibodies to vaccine preventable diseases. in a prior study, % of children < years lost humoral immunity to measles as a result of chemotherapy induced alterations in immune system. measles in recipients of immunosuppressive chemotherapy has mortality rates up to %. because of volitional vaccine refusal, there has been a dramatic increase in measles infection from cases in to in , including several statewide outbreaks. small pediatric oncology practices frequently share floor/clinic space with the general pediatric patients putting them at risk for measles since virulence starts hours prior to symptoms. there is no standard protocol for revaccinating post-chemotherapy patients. to assess measles risk based on serial humoral immune status in a cohort of pediatric oncology patients receiving intensive chemotherapy design/method: patients < years age with known vaccination status receiving intensive chemotherapy between july -june at our institution's pediatric oncology practice were included in this prospective study. serial measles igg antibodies were measured at diagnosis, months and months after initiation of chemotherapy using elisa. measles immunity was defined per lab standards. a comparison of pre-chemotherapy and serial post-chemotherapy immunization titers was made for all patients by diagnosis. the study population consisted of children ( male); patients had all, non-hodgkin lymphoma, sarcoma and other solid tumors. two patients ( . %), both unvaccinated had non-protective measles antibody levels at s of s baseline. of the remaining patients, . % patients ( leukemia, lymphoma and sarcoma) lost protective antibody titers at months after initiation of chemotherapy and . % ( leukemia, lymphoma and sarcoma) at months after initiation of therapy. % of the remaining patients who retained measles antibody titers within protective range at months also demonstrated a steady decline in antibody titers at and months from therapy initiation. the loss of protective measles humoral immunity occurred significantly more often in patients with leukemia compared to other malignancies. oncology patients in our practice undergoing intensive chemotherapy demonstrated progressive waning of protective measles igg titers. our data suggests that it should be standard practice to check all patients for measles humoral immunity prior to starting chemotherapy and at completion. larger studies need to be performed to establish guidelines for revaccinating post-chemotherapy pediatric patients, an intervention that is easily applicable and of low cost. background: the accurate determination of glomerular filtration rate (gfr) is important to screen for acute kidney injury, to dose chemo-therapy, and to identify risk for chronic kidney disease.being correlated with inulin clearance, measured gfr by iohexol plasma disappearance (igfr) is a new gold standard for measurement of gfr in pediatric cohort studies. igfr is based on the clearance of an exogenous marker and is unaffected by endogenous compounds or a patient's muscle mass. we compared igfr with -hour urine creatinine clearance ( crcl) and gfr estimating equations using serum creatinine (scr) and serum cystatin c (cystc) in pediatric patients with cancer. we recruited participants who were ages to yrs, continent of urine, and diagnosed with a malignancy in the past years. eligible subjects had stable kidney function for at least two weeks prior to the assessment of igfr. consented subjects had baseline assessments including height, weight and vital signs. blood samples were obtained for serum chemistry, and time zero iohexol. igfr determined by ml iohexol solution infused over - minutes followed by ml of sterile saline. blood was drawn at , , and minutes.at the same time of igfr, the crcl was collected. igfr was calculated using a two-compartment model and area under the curve. we compared igfr to published gfr equations (schwartz et al, kidney int ). results: ten subjects ( female/ male) agreed to participate. the distribution of diagnoses for the subjects: all = , lymphoma = , brain tumors = and hepatocellular carcinoma = . six patients were off therapy. the lower gfrs are noted in patients who had malignancies other than leukemia, likely due to the use of cisplatin based therapy. the average igfr was ml/min/ . m^ whereas crcl was . ml/min/ . m^ ; demonstrating the crcl overestimates gfr compared to igfr. comparing igfr to univariate equations using scr, cystc, and the multivariate equation with both, the univariate cystc equation correlated well with igfr; the others overestimated igfr. we found that crcl overestimated igfr. the univariate cystc equation better correlated to igfr than equations with scr. the poor performance of scr based methods to assess gfr might be due to decreased muscle mass and inadequate nutritional status. creatinine-based determinations of gfr alone, may not be accurate in this population. further study is needed to determine if igfr should be a standard of care to assess gfr in children with cancer particularly who are receiving nephrotoxic medications and incontinent of urine. background: pediatric oncology patients undergoing chemotherapy through indwelling venous catheters are at increased risk for severe sepsis especially when neutropenic due to chemotherapy. rapid triage and early recognition are essential because delayed initiation of antibiotics and fluids in these patients or delayed transfer to higher level of care after initial stabilization is associated with poor clinical outcome. our pediatric oncology out-patient clinic is designated as an article unit whereby the providers can initiate and give treatment such as intravenous fluid, antibiotics, chemotherapy and blood products. objectives: global aim-optimize management of early sepsis and decreased morbidity, mortality and hospital length of stay in the high risk pediatric oncology patients. smart aim-improve timely management with initiation of fluids and antibiotics and transfer of septic patients to higher levels of care by % in months in above patients design/method: multidisciplinary team with physicians and nurses was created. retropective chart review of sepsis patients treated at the clinic from april to october was done using an audit sheet to identify the barriers in the delivery of care. three patients were identified and data analyzed prior to intervention; two were analyzed post interventions. a key driver diagram was created by the group to drive intervention. a process map was designed to identify the different steps in the care of these patients to pinpoint areas needing improvement. different timed data points were used starting from time of arrival to clinic, time to antibiotics and fluids and time to transfer to higher level of care. rapid pdsa cycles were done to improve the processes and delivery of care. run charts were created. there was an improvement close to the goal of % for all data points used. pdsa cycles for improvement included conducting frequent mock codes with appropriate feedback real time coaching and process planning with nursing staff. we partnered with pharmacy for close loop communication with clinic staff and we improved communication between physicans at different levels. conclusion: sepsis in neutropenic pediatric oncology patients is deadly and can be reversed with timely management at different levels. given the promising results of the above project, we want re-inforcement of the processes to be a part of the daily practice of first line clinical staff. eventually we will extend the principles learnt in management and triage of sepsis to other outpatient emergencies chemotherapy related anaphylaxis background: chemotherapy-induced nausea and vomiting (cinv) is a common side effect in children receiving antineoplastic chemotherapy. recommended prophylactic antiemetic medications are based on the classification of chemotherapy emetogenicity. however, despite appropriate use of these antiemetic agents, some patients will still experience nausea and/or vomiting. children's oncology group clinical practice guidelines recommend the addition of olanzapine to prophylactic regimens for management of breakthrough cinv. objectives: our pediatric hematology oncology center implemented a quality improvement (qi) project aimed to increase the use of olanzapine in pediatric cancer patients years of age and older receiving moderately or highly emetogenic chemotherapy and experiencing breakthrough cinv over a month period. design/method: this qi project was conducted utilizing plan-do-study-act (pdsa) cycles. for the first pdsa cycle, baseline data was collected through chart review to determine the rate of olanzapine use for breakthrough cinv over a month period from july to december . breakthrough cinv was defined as use of or more doses of antiemetic agents other than those given for cinv prophylaxis. guidelines for treatment of breakthrough cinv were reviewed with pediatric hematology/oncology attending physicians and fellows. flyers were created that listed chemotherapy regimens considered moderately and highly emetogenic. if a patient experienced breakthrough cinv, a flyer was to be placed in the patient's roadmap binder to signal olanzapine should be added to the next chemotherapy block. data was collected over a month period in september following this first intervention. the second pdsa cycle consisted of didactic education and training of pediatric oncology nurses as well as pediatric residents regarding the addition of olanzapine for breakthrough cinv. rates of olanzapine use were then collected from october through november . results: olanzapine use increased from . % at baseline to . % after the first pdsa cycle ( = . , p = . ). after the second pdsa cycle, olanzapine use increased another . % to . % ( = . , p = . ). the administration of olanzapine was successfully increased by modifying patients' roadmaps after patients experienced breakthrough cinv as well as with education and training of pediatric oncology staff, fellows, residents, and nurses. background: venous thromboembolism (vte) is increasingly affecting children. according to an administrative database study, there was a % increase in the incidence of vte among children admitted to free-standing children's hospitals in the united states from to . risk factors for hospital-acquired vte are well-known and well-studied in adults, with evidence-based preventative measures available. similar guidelines are lacking for children. objectives: there is an ongoing national-initiative to develop and institute methods for screening and preventing hospitalacquired vte in children. in / , nationwide children's hospital instituted an electronic screening form required for all patients admitted ≥ hours. patients were scored and riskstratified based on eight risk-categories. a summated score was used to determine the vte risk level, and used to make prophylaxis recommendations for patients ≥ years; as well as patients ≥ years who were admitted to an intensive care (icu), surgical, or trauma unit. the purpose of this irb exempt, quality improvement initiative was to retrospectively review our experience with this risk-stratification tool. results: hospital-acquired vte events occurred in unique subjects. median age at vte diagnosis was years. only ( %) vte occurred in children ≥ years of age. ( %) vte were deep vein thrombosis (dvt), and ( . %) involved pulmonary embolism. vte was most common in subspecialty units including the pediatric and cardiac icus ( . %); neonatal icu, ( . %); and hematologyoncology, ( . %). ( %) vte were associated with central venous catheters (cvc) and events ( %) were associated with altered mobility. congenital heart disease/heart failure was the most common chronic medical condition associated with vte ( ( . %) events); whereas infection and trauma/surgery were the most common acute medical conditions associated with vte ( ( . %) and ( %) events, respectively). during ( %) events, subjects scored a summated score ≥ . in summary, in this single institution, prospectively maintained database, cvc remains the most common risk factor for vte, followed by cardiac disease, infection and trauma/surgery. most subjects who developed vte scored high (score ≥ ) on our screening tool. only a small proportion of vte occurred in patients older than years and thus eligible for thromboprophylaxis. our results indicate that future vte prevention endeavors should include these age groups in addition to exploring more aggressive prophylactic modalities including pharmacological prophylaxis. background: pediatric fellows are required to have active engagement in quality improvement (qi) activities, and yet a national acgme review found most trainees had "limited knowledge of qi methods" and "limited participation in interprofessional qi teams". the twenty fellows in our pediatric hematology/oncology training program identified blood culture utilization as their qi priority. our institution recently introduced a hospital-wide decision algorithm to guide providers regarding when to obtain blood cultures. there is often a low threshold to obtain blood cultures in immunocompromised pediatric oncology patients, but these are often low-yield or result in falsepositives. our fellows spearheaded a project to implement the algorithm in the inpatient pediatric oncology population and improve the proportion of appropriately drawn blood cultures. we investigated how appropriately the algorithm was being utilized on the inpatient pediatric oncology floor prior to and after several educational steps aimed at disseminating the algorithm to members of the care team. our primary endpoint was to quantify the proportion of culture episodes drawn "inappropriately", with a goal of reducing inappropriate episodes to ≤ %. the algorithm was initially introduced to the nursing staff and residents covering the twenty-bed inpatient unit in september . qi project planning took place with upper level fellows in january . fellows and faculty received intensive training on the algorithm in july-august . we then conducted a retrospective chart review of blood culture episodes drawn between august and november . upper level fellows scored ∼ culture episodes as to whether the decision to culture and number of cultures drawn were "appropriate" or "inappropriate", and catalogued the indications for culture episodes and if applicable, why the episode was found to be inappropriate. additionally, fellows discussed inappropriate culture episodes with the team onservice, to provide direct feedback on where the algorithm failed. results: between august -december on average cultures/ patient-days were drawn. forty-nine percent of culture episodes were inappropriate. from january -october , following targeted education on the algorithm, the rate of blood cultures drawn decreased to cultures/ patient-days. the average proportion of inappropriate culture episodes fell to . %, representing a % decrease in inappropriate culture utilization. correct application of a decision algorithm for blood culture utilization can reduce total cultures drawn on an inpatient pediatric oncology unit. fellow-led education of the multi-disciplinary team decreases the rate of inappropriate culture episodes as well as provides active engagement in qi. background: inadequate understanding of sickle cell disease (scd) is common and can affect patients' compliance and therefore their morbidity and mortality, especially after transition to adult care. optimal clinical care for scd includes disease education, which can be difficult given the breadth of possible topics and limited time in clinic. it is unclear how best to provide personalized, efficient education for adolescents with scd. this quality improvement (qi) study aimed to implement a questionnaire-based system to improve patients' knowledge of their scd and documentation of education by the nurse or physician. the study objective was to improve provider documentation and patient knowledge about their scd by identifying patients' gaps in comprehension. by january , the study aimed to increase education documentation from % to %. by april , the study aimed to increase use of a smart phrase for education documentation from % to %. by june , the study aimed to increase patients' knowledge about their disease by %. design/method: twenty-one scd patients enrolled on an irb approved qi study, with twenty active patients. our comprehensive team generated a questionnaire with knowledgebased questions for two age groups: - and - years old. at each comprehensive visit, a questionnaire was distributed, with at least -month intervals. the provider scored questionnaires and reviewed two educational topics, with wrong answers taking priority. plan-do-study-act (pdsa) cycles included pdsa# : patients completed questionnaire. pdsa# : a smart phrase addressing questionnaire topics was created and shared with providers. pdsa# : patients received education handouts during clinic education. documentation in clinic notes was the process measure and questionnaire scores was the outcome measure. results: pdsa# is complete, pdsa# has four patients remaining, and pdsa# is ongoing. due to variable visit frequency, there are multiple concurrent cycles. after pdsa# , free text documentation was completed an average of % over the course of months. after pdsa # documentation increased to % within months and questionnaire scores increased from an average of % to %. of the questions that patients got wrong on their first visit, they were significantly more likely to improve on retesting if the topic was taught to them than if it was not addressed ( % vs. %, p = . ). we are currently completing pdsa# and collection of post pdsa# data. questionnaire-based scd education coupled with standardized smart phrases improves patients' scd knowledge and documentation by providers. further improvement in knowledge is expected with the addition of handouts. background: exposure to suffering can have a profound impact on the wellness of caregivers, often referred to as the "cost of caring". this cost is especially high in pediatric hematology/oncology. repeated exposure to suffering has the potential to negatively impact resilience and increases the risk of burnout, thus impacting quality of care and patient satisfaction. we have developed a peer support team utilizing the critical incident stress management (cism) model. this model has been successfully used in other professions that frequently face traumatic events such as fire fighters, police and emergency medical technicians. the h.o.p.e.s. team (helping our peers endure stress) consists of volunteer multidisciplinary staff members who have received training to provide both group and peer support following any 'critical incident' that may impact one or more staff members. we hypothesize that implementation of the h.o.p.e.s. team will improve staff resilience, decrease overall rates of burnout and improve compassion satisfaction. s of s design/method: we are using both empiric metrics and anecdotal reports to assess the impact of the h.o.p.e.s. team. prior to the activation of the team, all pediatric hematology/oncology clinical staff members were surveyed using validated tools to assess their levels of resilience, burnout, secondary trauma and compassion satisfaction (proqolv and brief resilience scale). they were also asked to rate the number of times they had experienced critical incidents, as well as their perceived level of distress after dealing with traumatic events. after the h.o.p.e.s. team has been functional for months, we will send the same survey to staff members to measure changes, paying special attention to resilience and rates of burnout and compassion satisfaction. results: enthusiasm for development of the team has been high. of people approached to volunteer their time to participate in the multidisciplinary team agreed, including attending physicians, fellows, nurses, nurse practitioners, child life specialists, social workers, clergy and psychologists. all volunteers participated in a -day training conducted by an instructor from the international critical incident stress foundation. engagement in the first staff survey has been high, with of responding to date. data collection is ongoing. clinical staff in pediatric hematology/oncology may be particularly vulnerable to burnout and decreased resilience by repeatedly witnessing suffering and trauma. peer support interventions following critical incidents may lead to increased resilience and compassion satisfaction while decreasing rates of burnout. enthusiasm for the development of a peer support team has been high. background: monthly blood transfusions are an indicated therapy for pediatric patients with sickle cell disease with certain complications. maximizing transfusion efficiency in a busy infusion clinic requires: ensuring that appropriate blood units are available in the hospital blood bank; laboratory specimens are obtained from patients in advance; and coordination of clinic appointment and nursing availability. we sought to improve clinic efficiency through identifying ways to better communicate with patients/families regarding upcoming laboratory and transfusion appointments, and to assess the efficacy of implementing a web-based personalized text reminder (pinger.com). we measured the baseline frequency with which transfusion appointments were missed by families, moved to later within the week, or delayed due to late labs. a convenience sample of patients receiving monthly transfusions received a questionnaire about patient/parent preferences for appointment reminders and barriers to keeping appointments. those patients/parents who did not opt-out of an additional text reminder received personalized texts from their care team reminding them of lab and transfusion appointments. rates of missed/moved/delayed appointments were compared between the group receiving the additional text messages and the group only receiving standard, hospitalgenerated appointment reminders (telephone call). results: forty-one families ( patients) responded to the survey, capturing information on % of patients receiving chronic transfusion therapy. thirteen families ( %) declined the additional text reminders. families reported a preference for text reminders ( %), more often than email ( %) or telephone ( %), and % of families wanted to receive reminders for both transfusion and laboratory appointments. the majority ( %) of families reported competing work/life priorities as the reason for missed/late appointments. other families noted transportation/travel ( %), fear/illness/pain ( %), and lack of reminders ( %) as the reason for missed appointments. at baseline (twelve weeks), . % of appointments were missed on a weekly basis (range - of available per week), . % were moved, and % of appointments were delayed. during our intervention period (twelve weeks), % were missed, . % were moved, and . % were delayed (combined, both groups). there was no difference in missed ( . % texted vs . % standard), moved ( . % texted vs . % standard) or delayed ( . % text vs . % standard) appointments. though families at our center reported a preference for a text-based reminder, personalized text reminders for appointments did not improve clinic efficiency as measured by missed, moved or delayed transfusion appointments. there was no improvement in appointment adherence in the group receiving personalized texts in addition to standard hospital reminders. university of utah, salt lake city, utah, united states background: childhood cancer outcomes have improved significantly, in large part due to multi-institution collaborative clinical trials run by the children's oncology group (cog). approximately half of eligible children with cancer will enroll on a therapeutic trial, but little is known about the factors affecting caregiver decision-making regarding enrollment or how well the required elements of informed consent are conveyed during the consent process. objectives: . assess coverage of ten of the required elements of informed consent for cog therapeutic trials. . describe factors affecting caregiver decision-making regarding therapeutic trial enrollment. we surveyed families of children who were offered enrollment onto a phase cog therapeutic study for an initial cancer diagnosis in the previous months. fisher's exact or wilcoxon rank-sum tests were utilized to compare demographic and other motivating factors related to enrollment decision-making. results: seventy participants were surveyed. regarding of the basic required elements of informed consent, % knew the trial involved research, % knew consent was required, % knew the enrollment length for the trial, % knew they could continue care independent of enrollment, % knew who to contact with questions, % knew there were options besides enrollment, % knew they could withdraw at any time, % knew the information was confidential, % knew there were risks associated with the trial, and % knew there were benefits. of all participants, % (n = / ) enrolled onto a therapeutic study. among enrollees, % (n = / ) of the primary caregivers had completed college compared to % (n = / ) of those not enrolled (p = . ). when asked about factors impacting their decision, % (n = / ) of those enrolled said they felt there were no risks or did not know if there were risks associated with the study compared to % (n = / ) of those choosing not to enroll (p = . ). of those enrolled, % (n = / ) reported the physician recommendation "somewhat" or "strongly" affected their decision to enroll compared to % (n = / ) of those not enrolling (p = . ). of those who enrolled, % (n = / ) reported feeling pressured to enroll while % (n = / ) of those not enrolled reported pressure (p = . ). of enrollees, % (n = / ) reported they did not have enough time to decide compared to % (n = / ) of those not enrolled (p = . ). failure to convey all required elements of informed consent highlights possible deficiencies in the consent process for cog therapeutic trials. caregivers' perception of being pressured and lack of time to make an informed decision may impact clinical trial enrollment. background: abnormal uterine bleeding (aub) is a frequent adolescent gynecologic complaint. however, limited research exists to guide management, and acute care varies. we sought to improve emergency care for adolescents with aub by developing a clinical effectiveness guideline (ceg) and assessing its impact on quality of care. design/method: a stakeholder engagement group consisting of members from the departments of hematology/oncology, adolescent medicine, general pediatrics, and emergency medicine designed a ceg algorithm for emergency aub management. pediatric residents received ceg training and their knowledge and attitudes were assessed using pre and post intervention surveys. icd- and codes identified electronic health record data for patients presenting to the pediatric emergency department (ed) for aub months before and after ceg implementation. pre-pubertal patients and those with vaginal bleeding from trauma were excluded. a weighted, -point scoring system consisting of prioritized aspects of history, laboratory studies and management was developed to quantify the quality of care provided. t-test, chi square test, wilcoxon rank sum test, and a run chart were used for analysis. of the patients identified, met inclusion criteria. there were % of patients currently using some form of contraception, while . % had bleeding related to a current or recent pregnancy. median aub quality care scores were pre-and post-intervention (p = . ). run chart data showed no shifts or trends (overall median score, -points). both pre and post-implementation, points were deducted most frequently for not assessing personal/family clotting disorder history and inappropriate use/dosing of oral contraceptives. we successfully designed and implemented a ceg and educational intervention for aub management in a pediatric ed. these data suggest our ceg may be an effective tool to improve emergency aub care for adolescents, though additional cycles are needed. background: high-dose methotrexate (hd-mtx) is a common chemotherapy administered inpatient at most centers. its administration is particularly susceptible to error due to the need for frequent drug levels with resulting changes in supportive care. errors can prolong patient stay and cause patient harm. objectives: global aim-to reduce the length of stay (los) of hd-mtx admissions. smart aims-to increase the percentage of patients whose pre-hydration fluids are started by am from % to % by / / , and to increase the percentage of patients who receive hd-mtx by pm from % to % by / / . we used rapid process improvement methods to target earlier methotrexate administration. a key driver of prolonged los was hypothesized to be drug levels returning overnight rather than in the day time due to delayed hd-mtx start. changes implemented have included scheduling hd-mtx patients as the first patients of the day for their exam in clinic and scheduling labs to pass for hd-mtx on the day prior to admission. there are ongoing pdsa cycles to change the location of pre-hydration start from the inpatient room to the clinic exam room in order to meet hd-mtx administration time goals. we are piloting two different education materials to improve patient experience. one explains hd-mtx levels in a red/yellow/green stoplight format and the other reminds patients how to prepare for the admission. other interventions regarding how we test urine ph and safety checks in the ordering process for history of delayed clearance are in the planning stage. the project is ongoing, but as of / / , we start methotrexate by pm % of the time which is improved from a baseline of %. when the project was started, pre-hydration was never started before am. now, fluids are started by am % of the time. pdsa cycles are ongoing and we have yet to sustain reductions in los, but some months have shown decreased los by as much as hours from baseline measurements. rapid cycle improvement can be utilized to decrease los hd-mtx admissions. this has important financial implications as well as the potential to reduce secondary harm from unnecessary time in the hospital. pediatric cancer centers should schedule hd-mtx admissions first thing in the morning so that data regarding kidney injury and drug clearance can be interpreted by the day team and children are not cleared for discharge in the middle of the night. background: education and training for interdisciplinary pediatric oncology providers requires training in principles of palliative and end-of-life (eol) care. the experiences of bereaved parents can inform and enhance palliative care educational curricula in uniquely powerful and valuable ways. the objective of this study is to present an innovative palliative care educational program for oncology providers facilitated by trained bereaved parents who serve as volunteer educators in local and national palliative care educational forums and to describe how incorporation of bereaved parents in these educational forums affects participant comfort with communication and management of children at the eol. design/method: survey tools were adapted to determine how bereaved parent educators affected participant experiences in different educational forums: institutional seminars on pediatric palliative and eol care, role-play based communication training sessions, and an international symposium on pediatric palliative oncology. pre-and post-session surveys with incorporation of retrospective pre-program assessment item to control for response shift were used in the evaluation of institutional seminars and communication training sessions. results from feedback surveys sent to all attendees were used to appraise the participants experience in the international oncology symposium. results: involvement of trained parent educators across diverse, interdisciplinary educational forums improved attendee comfort in communicating with, and caring for, patients and families with serious illness. importantly, parent educators also derive benefit from educational with interdisciplinary clinicians. integration of bereaved parents into palliative and eol care education is an innovative and effective model that benefits both interdisciplinary clinicians and bereaved parents. background: poorly controlled chemotherapy-induced nausea and vomiting (cinv) significantly impairs patients' quality of life and contributes to ongoing medical costs through increased length of stay in the hospital or readmissions and outpatient visits for control of nausea, vomiting or dehydration. lack of adherence to national evidenced-based guidelines that dictate antiemetic prescribing for variably emetogenic chemotherapy leaves patients vulnerable to increased cinv and its ensuing complications. objectives: to review our institution's antiemetic prescribing practices and their consistency with the antiemesis guidelines from the national comprehensive cancer network (nccn) and children's oncology group (cog)-endorsed supportive care guidelines and to further develop tools to increase adherence to these national-based guidelines to improve control of cinv. we performed a retrospective chart review of inpatient chemotherapy encounters. we evaluated emetogenicty of chemotherapy (high, medium, low), initial antiemetic regimen ordered, number of as needed medications required and adherence to national evidenced based guidelines tailored to each level of emetogenicity in the prescription of antiemetics. results: fifty-five total inpatient chemotherapy encounters were reviewed over months. eighteen of these encounters were considered to have been highly emetogenic chemotherapy (hec) with the remaining of these considered to be moderately emetogenic. only out of hec encounters completely included all guideline-recommended agents. there was a demonstrable lack of consistency across providers with dosing of aprepitant and most as needed medications. there was significant variation in order of first, second and third line anti-emetics ordered -with lorazepam and promethazine being used most frequently. with an aim of improving antiemetic prescribing practices for our patients, we are currently rebuilding chemotherapy treatment plans in our electronic medical record to incorporate antiemetic drug order sets that follow evidenced-based guidelines for variably emetogenic chemotherapy. this will be used in conjunction with an education initiative about best practices in supportive care for all prescribers of antiemetics. review of our department's recent inpatient chemotherapy encounters show we are falling short in following nationally recommended standards for appropriate antiemetic coverage during chemotherapy. identification of these deficiencies allows for implementation of quality initiatives to improve prescriber adherence to evidenced-based guidelines for better control of cinv. background: there are currently no consensus guidelines for the management of pediatric oncology patients presenting with fever without neutropenia. historically, these patients had been treated similarly to neutropenic patients with empiric antibiotics. while there has been a shift towards reducing unnecessary empiric treatment, there has been limited research into the outcomes associated with withholding empiric iv antibiotics in this patient population. we assessed the safety and efficacy of our institution's current protocol of observing well-appearing patients who present with fever without neutropenia and compared the outcomes of the patients who did and did not receive empiric iv antibiotics. design/method: this was a prospective, single-institution cohort study. patients were included if they were currently undergoing chemotherapy for an oncologic diagnosis and presented initially as an outpatient with fever and nonneutropenia (defined as anc ≥ cells/mm ). for each episode we recorded lab and blood culture results, signs and symptoms of initial presentation, and clinical outcomes, including antibiotic administration and hospital admission. results: a total of episodes of well-appearing patients with fever without neutropenia were identified. compliance with the institutional protocol was high; . % of patients were observed without receiving empiric iv antibiotics. the majority of patients were discharged home and there were no serious complications or infectious deaths. the incidence of positive blood cultures was low ( . % including several likely contaminants), despite the presence of central venous catheters in the majority ( . %) of patients. there were no significant differences in age, oncologic diagnosis, central s of s line access, anc value, or incidence of bacteremia between patients who did and did not receive empiric iv antibiotics. patients who were admitted to the hospital were significantly more likely to have received iv antibiotics (p < . ) despite documentation of a reassuring exam. however, admitted patients who initially received iv antibiotics were just as likely to discharge within hours compared to patients who were observed. we propose that empiric iv antibiotic administration in febrile, non-neutropenic, otherwise well-appearing patients is unnecessary. our study demonstrated no adverse consequences of observation and no significant differences in clinical outcomes between patients who did and did not receive iv antibiotics aside from rate of hospitalization. this supports the practice of observation without empiric antibiotics for such patients. background: children with hepatoblastoma (hb) undergo repetitive computed tomography (ct) scans to determine response to treatment and assess for relapse. this imaging exposes children to radiation, anesthesia, and imposes financial and emotional burden. objectives: review our institutional experience to determine if afp measurements are sufficient to assess response to treatment and detect relapse. we conducted a retrospective chart review of all patients diagnosed with hb at our institution between - . data collected included serum afp, total number and type of imaging studies during and post treatment, and how relapse or progressive disease was detected. results: thirty-one patients were diagnosed with afp positive hb. during therapy, ct scans were performed: to assess for response to therapy or surgical planning (average scans/patient) and due to concern for progression with rising afp. off therapy, surveillance ct scans were performed (average of . scans/patient) and ( %) included the chest in patients with no lung metastasis at diagnosis. relapsed patients averaged . surveillance scans, . of which were done before relapse was noted on imaging. there were no cases of radiographic evidence of relapse without a prior increase in afp. during treatment, response to therapy based on imaging correlated with a decline in afp in all patients, arguing that repetitive scans are not needed in this setting unless required for surgical planning. only of scans performed during off therapy surveillance displayed evidence of relapse, all of which were preceded by rise in afp. our study represents the largest cohort of hb patients. prior studies suggest similar results, but included fewer patients, lower stage of disease and less than years of surveillance monitoring. at our institution, the cost of a ct c/a/p is $ , with reimbursement varying from - %. in comparison, the cost of an afp measurement is $ . . many scans also require anesthesia and result in emotional toil for families concerned about this procedure as well as the results. thus, afp demonstrates greater sensitivity, with significant cost savings and decreased emotional burden, and should be used for monitoring both during and off therapy, replacing routine serial imaging. background: we observed that our practice of drawing daily blood cultures in hospitalized patients with fever and neutropenia was wasteful; it resulted in excessive negative cultures that did not add to patient care. the smart aim of this quality improvement project was to reduce the number of negative blood cultures drawn on hospitalized patients with fever and neutropenia by % in months. design/method: after reviewing published evidence suggesting drawing daily blood cultures in febrile neutropenic patients was unnecessary, a new blood culture guideline was implemented: cultures were drawn at presentation for fever with neutropenia and, if negative at hours, repeat cultures were not drawn except for clinical change, new fever after being afebrile > hours, or antimicrobials were being changed/broadened. to impact key drivers, we educated staff and changed blood culture order sets to require providers to select a reason for ordering the culture and to eliminate a nursing order to draw daily cultures with fever. we compared the number of blood cultures drawn per central linedays (/ -cld) and the proportion of positive versus negative cultures pre-guideline (july -may ) and postguideline (june -december ). we calculated the cost savings from reducing cultures. to assess patient safety, potential septic events without a corresponding positive blood culture were reviewed. data were analyzed by service (oncology and stem cell transplant). a chi-square test was used to compare rates. in stem cell transplant patients, pre vs. postguideline, there were vs. total cultures drawn/ -cld; vs. positive ( % decrease, p = . ) and vs. negative cultures/ -cld ( % decrease, p< . ). in oncology patients, pre vs. post-guideline, there were vs. total cultures drawn/ -cld; vs. positive ( % decrease, p = . ) and vs. negative cultures/ -cld ( % decrease, p< . ). the decreased positive culture rate among oncology patients may be due to decreased culture contaminants and/or the effect of a concurrent initiative to decrease clabsi in that group. there were safety concerns; however, chart review concluded that the guideline did not lead to missed infections in these patients. for the first months of the guideline, the total cost savings in blood cultures was $ , . . the implementation of our new blood culture guideline successfully led to a substantial reduction in the collection of negative cultures and a cost savings without compromising the detection of bacteremia in hospitalized pediatric patients with fever and neutropenia. background: there are various evidence-based guidelines for treatment of adult cancers, such as the nccn guidelines. previously, care was standardized for most new diagnosis pediatric cancer patients through enrollment on a clinical trial. with decreasing clinical trial availability and enrollment and few, if any, evidence-based guidelines for pediatric cancer, care standardization is challenging for pediatric cancers. objectives: to assess consistency of care, as determined by plan of treatment by diagnosis, for pediatric patients receiving chemotherapy for newly diagnosed cancer at a single center. design/method: patients with a new cancer diagnosis at a large, tertiary care pediatric oncology center in calendar year were identified through reports from the chemotherapy order entry (coe) system. reports included diagnosis (recorded through standardized options) and the plan of treatment. chart review was used to exclude patients who started treatment elsewhere and patients being treated for relapse, to clarify diagnosis if the standardized options in coe were unclear, and to clarify treatment plan if needed. data was entered and analyzed in a redcap database. specific diagnoses were clustered into higher level disease groups and the distribution of treatment plans for patients within each was determined. this project was deemed exempt from irb approval for human subject research as a qualifying quality improvement project. of the patients with a first chemotherapy order in , were excluded due to one or more reasons: stem cell transplant ( ), transfer of care ( ), relapse ( ), and other ( ). an additional patients were excluded because < patients/year/diagnosis. there was no cns tumor disease group with > patients. thus, patients with hematologic malignancies or non-cns solid tumors are the focus of this analysis. for patients with intermediate risk rhabdomyosarcoma, the plan of treatment was the standard arm of a cog protocol, arst for patients and arst for subsequent patient after protocol activation. for all other diseases including lymphoblastic leukemia/lymphoma (excluding infants), classical hodgkin lymphoma, aml (excluding trisomy and apml), stage iii/iv burkitt lymphoma/diffuse large b-cell lymphoma, posttransplant lymphoproliferative disease, wilms tumor, rhabdomyosarcoma, ewings sarcoma, osteosarcoma, neuroblastoma, and retinoblastoma, only one treatment plan per risk category was used. conclusion: this analysis demonstrates highly consistent chemotherapy treatment at a single center for patients with hematologic malignancies and non-cns solid tumors. next steps include exploring strategies to group diagnoses for cns tumors and assessing the quality of evidence supporting the treatments given. background: rapid initiation of empiric antibiotics in patients with fever and neutropenia has been shown to reduce morbidity and mortality. current practice guidelines call for the initiation of antibiotics in these patients within sixty minutes and time-to-antibiotic (tta) has been suggested as a quality-of-care measure. many institutions, including our own, face barriers to meeting this time limit. objectives: utilizing a quality improvement model, determine barriers and implement an intervention to reduce the time-to-antibiotics for pediatric febrile patients with suspected neutropenia who present to the emergency department (ed) at our institution. we have identified and implemented an intervention utilizing the plan-do-study-act model for quality improvement. a twelve-month retrospective review was conducted to evaluate the efficacy of the current practice algorithm at our large, academic tertiary-care hospital. subjects identified were pediatric oncology patients undergoing active chemotherapy who presented to the ed with febrile neutropenia. we identified two specific barriers, triage level assignments and delay in ordering antibiotics. to address these barriers, we have created a wallet sized "fever card" that patients were instructed to show upon arrive to the ed. in collaboration with the ed staff, efforts were also made to educate all pediatric staff on the use of the fever card. post-intervention data collection is currently underway and pre-and post-intervention antibiotic delivery times will be compared. the pre-intervention cohort consisted of thirty-three encounters with a mean time-to-antibiotic delivery of minutes, or seventy-five minutes greater than the accepted standard of care. only one patient received antibiotics within sixty minutes of arrival. post-intervention data collection is currently underway. since identifying two barriers to meeting the standard of care at our institution, we have implemented a quality improvement measure that empowers patient families to direct appropriate triage in the ed as well as simplifying the treatment protocol for ed providers. we expect to identify an improvement in time-to-antibiotics from the pre-intervention to the post-intervention period. background: sickle cell disease (scd) is a genetic disorder in which sickle hemoglobin (hbs) triggers multiple downstream effects, including red cell sickling, hemolysis, vaso-occlusion, and inflammation. scd, a lifelong disease initiated at birth with injury that accumulates over time, causes significant end-organ damage and clinical complications that are undertreated and associated with early death. homozygous mutation (hbss) causes the severe form of scd. individuals with scd are at increased risk of infection, stroke, and retinopathy. clinical guidelines for pediatric patients with scd recommend prophylactic penicillin use (ages - ), annual screening for stroke with transcranial doppler (tcd) imaging (ages - ), and annual ophthalmology exams to assess for retinopathy (ages ≥ ). there are limited real-world data on implementation of these nhlbi-based recommendations. objectives: to describe utilization of penicillin, tcd screening, and ophthalmology care in children with hbss disease. medicaid administrative claims databases were used to identify us patients aged - years at first indication of hbss recorded in each calendar year from to . patients were required to have medical and pharmacy benefits for the calendar year in which they were identified and for months prior to their first recorded hbss indication. prior year utilization of penicillin, tcds, and ophthalmologist visits was measured for each annual cohort. annual cohorts included - commercial (mean age . years, % female) and - medicaid (mean age . years, % female) patients with hbss disease. fewer than half of all patients had received a tcd scan in the previous year, with similar rates seen across all age groups for both payers. ophthalmologist visits increased as patients aged, and while patients aged - years had the highest proportion with an ophthalmologist visit in both payer populations, the overall implementation remained low. in contrast to the low use of tcd and ophthalmology visits, penicillin use was highest in the - year age group: > % use in any given year for both payers. conclusion: although our data demonstrated high penicillin use in the - year age group, consistent with guidelines there is an opportunity to improve implementation of other guidelines-based recommended screening. for example, tcd screening can identify children at risk of scd-related stroke in order to initiate preventive therapies. further research to understand potential barriers to proper screening and to evaluate strategies to improve awareness, adherence, and implementation of recommended screenings in children with scd is warranted. supported by global blood therapeutics. background: childhood cancer therapy has improved where there are many long-term survivors. while psychosocial difficulties in pediatric cancer survivors are recognized, the prevalence of these problems at initial survivorship presentation is unclear. objectives: to examine the prevalence of overall internalizing symptoms (e.g., depression/anxiety) in pediatric cancer survivors presenting to a survivorship clinic and to examine how this is mitigated by receiving psychological services and by evidence of parental depression/anxiety. design/method: pediatric cancer survivors attending their first visit at the reach for survivorship clinic at vanderbilt (ages - ) were included. survivors' parents ( % female) completed the child behavior checklist (cbcl), beck depression inventory-ii, and beck anxiety inventory. survivors > years completed a self-report. the wilcoxon rank-sum and pearson's test were used for univariate analyses. the effect size and % confidence intervals (ci) estimated from the multivariable linear regressions were reported. results: childhood cancer survivors a median of years old and . years off therapy were included. thirty one survivors ( %) showed at least borderline clinical internalizing problems (t score > ) on the cbcl, but only of these patients ( %) reported receiving psychological services. nine other survivors with normal t score ≤ also reported receiving psychological services. parental depressive and anxiety symptoms were correlated to the parental report of survivor overall internalizing symptoms (spearman = . , p = < . and = . , p = < . respectively), however they were not correlated to survivor selfreports. furthermore, parents with mild to severe depressive symptoms or mild to severe anxiety symptoms were more likely to rate their child as having higher overall internalizing symptoms (p = . ; p = . , respectively). multivariable linear regression showed that when adjusted for age, gender, cancer diagnosis and time off treatment, reported utilization of psychological services ( = . , % ci [ . , . ],p = . ), and parent depressive symptoms ( = . , [. , . ],p< . ) were significantly associated with child overall internalizing symptoms. in an otherwise identical alternate model substituting parental anxiety for parental depression, parental anxiety was also a significant risk factor ( = . , [. , . ], p< . ). alternatively, parent anxiety/depressive symptoms were not significantly associated with child self-report of internalizing symptoms. childhood cancer survivors have an elevated prevalence of experiencing internalizing symptoms but seldom report receiving psychological services. childhood cancer survivors' parents with anxious/depressed symptoms are more likely to rate their children as having more internalizing problems, compared to patient self-reports. ongoing longitudinal analyses will help clarify the best timing for potential interventions. background: life expectancy for adults with sickle cell disease (scd) has remained unchanged over the past years despite improvements in pediatric scd survival. at greatest risk are the adolescents and young adults (ayas) transitioning from pediatric to adult care. allen county ranks rd in scd incidence among the counties in indiana, and has board certified pediatric hematologist-oncologists. when children "age out" of the pediatric system, there are few providers knowledgeable about managing adults with scd in the region. a novel partnership between hematologists and the family medicine residency program in allen county was initiated to educate family medicine residents (fps) about scd, hydroxyurea (hu), and management of scd-related complications with the goal to increase the number of knowledgeable providers to care for adults with scd. to determine the effectiveness of online learning modules in educating fps about hu, best practices for aya scd care and transition. three online learning modules about scd (comprehensive care of ayas with scd, hu, best practices in aya transition) were developed and cme-accredited. electronic pre-and post-tests were distributed to fps with five questions for each module covering: contraception; screening tests; hu indications, dosing and monitoring; developmental milestones and scd knowledge assessments. the st vincent irb reviewed the protocol and granted a waiver of consent. results: twenty-six fps ( %) completed the pre-and posttests. over two-thirds correctly identified the clinical benefits of hu on both assessments. knowledge about the rationale for hu therapy increased after the completion of the hu module ( % correct on pre-test vs. % on post-test, p = . ). the proportion of correct responses increased for all comprehensive aya scd care post-test questions, but only the leading cause of death and the priapism-related questions reached statistical significance ( % vs. %, p = . ; % vs. %, p = . , respectively). the proportion of correct responses for of the transition-focused questions was unchanged ( % for both), while the proportion of correct post-test responses on the self-care assessment question significantly increased ( % vs. %, p = . ). after module completion, fps were able to correctly identify common scd complications and why hu is an effective treatment for individuals with scd. the best practices of transition clinic module may need modification to improve physician understanding of the intricacies in establishing and maintaining a scd transition clinic. overall, online training is effective at educating fps and could be used to increase the number of providers knowledgeable about scd care. background: survival rates for pediatric hodgkin lymphoma (hl) exceed % with contemporary therapy. studies of pediatric hl survivors treated in the s- s have shown increased risk for treatment-related chronic health conditions. risk-adapted therapy, including tailored radiotherapy, has been developed to reduce long-term morbidity while maintaining excellent survival. little is known about chronic conditions associated with contemporary therapy presenting during the first years from therapy completion (early outcomes). objectives: to analyze survival and early outcomes of pediatric hl patients treated with contemporary therapy. we conducted a retrospective review of hl patients diagnosed < years of age at our institution from - . three-year overall (os) and event-free (efs) survival were calculated with kaplan meier statistics using sas . . results of standardized screening for targeted toxicities that developed between - years from therapy completion were identified and graded per ctcae criteria. censoring occurred at date of death, years from therapy completion, or december , . data from the last collection point were used for prevalence calculations in cases with multiple evaluations. we identified patients ( % male; % non-hispanic white; mean age at diagnosis . ± . years) with a median time since therapy completion of . years (range . - . ). initial treatment included: ( %) chemotherapy only and ( %) multimodality treatment. all patients received anthracyclines (median dose mg/m ) and % received alkylating agents (median cyclophosphamide equivalent dose [ced] mg/m ). the -year os was % with an efs of % ( % chemotherapy only, % multimodality treatment; p = . ). patients with relapsed/refractory disease received salvage treatment including chemotherapy only (n = ), multimodality therapy (n = ), or multimodality treatment including stem cell transplant (autologous n = ; autologous+allogeneic n = ). no patients developed thyroid dysfunction, cardiac dysfunction, subsequent neoplasm, or male gonadal dysfunction during the study period. pulmonary dysfunction was limited to ctcae grade . anti-mullerian hormone (amh) below the normal range was found in / pubertal females who received ced ≥ mg/m compared to / females who received ced < mg/m . two of the females with low amh also had follicle stimulating hormone > iu/ml. this study is the first to evaluate early outcomes in pediatric hl survivors. the results indicate contemporary chemotherapy and a lower rate of radiotherapy utilization lead to excellent -year survival rates with minimal early toxicities. females exposed to ced ≥ mg/m are at increased risk for gonadal dysfunction and should be prioritized for fertility preservation approaches prior to initiation of cancer therapy. background: cancer is one of the leading disease-related causes of death among individuals aged < years in the united states. recent evaluations of national trends of pediatric cancer used data from before , or covered ≤ % of the us population. objectives: this study describes pediatric cancer incidence rates and trends by using the most recent and comprehensive cancer registry data available in the us. design/method: data from us cancer statistics were used to evaluate cancer incidence rates and trends among individuals aged < years during - . data were from states and covered % of the us population. we assessed trends by calculating average annual percent change (aapc) in rates using joinpoint regression. rates and trends were stratified by sex, age, race/ethnicity, us census region, county-based economic status, and county-based rural/urban classification, and cancer type, as grouped by the international classification of childhood cancer (iccc). we identified , cases of pediatric cancer during - . the overall cancer incidence rate was . per million; incidence rates were highest for leukemia ( . ), brain tumors ( . ), and lymphoma ( . ). rates were highest among males, aged - years, non-hispanic whites, the northeast us census region, the top % of counties by economic status, and metropolitan counties. the overall pediatric cancer incidence rate increased (aapc = . , % ci, . - . ) during - and contained no joinpoints. rates increased in each stratum of sex, age, race/ethnicity (except non-hispanic american indian/alaska native), region, economic status, and rural/urban classification. rates were stable for most individual cancer types, but increased for non-hodgkin lymphomas except burkitt lymphoma (iccc group ii(b), aapc = . , % ci, . - . ), central nervous system neoplasms (group iii, aapc = . , % ci, . - . ), renal tumors (group vi, aapc = . , % ci, . - . ), hepatic tumors (group vii, aapc = . , % ci, . - . ), and thyroid carcinomas (group xi(b), aapc = . , % ci, . - . ). rates of malignant melanoma decreased (group xi(d), aapc = - . , % ci, - . -- . ). this study documents increased rates of pediatric cancer during - , in each of the demographic variables examined. increased overall rates of hepatic cancer and decreased rates of melanoma are novel findings using data since . next steps in addressing changing rates could include investigation of diagnostic and reporting standards, host biologic factors, environmental exposures, or potential interventions for reducing cancer risk. increasing pediatric cancer incidence rates may necessitate changes related to treatment and survivorship care capacity. background: while childhood cancer treatment modalities have improved, the delayed effects of cancer treatment continue to compromise the quality of life in survivors. metabolic syndrome (ms) is diagnosed based on the presence of three of the following findings -obesity, dyslipidemia, hypertension and insulin resistance per the world health organization (who) criteria. the increased risk of ms among childhood cancer survivors was first reported in the 's and is known to increase the incidence of cardiovascular disease in these individuals. objectives: assess the frequency of ms in childhood cancer survivors at our institution. . we conducted a retrospective chart review on pediatric cancer survivors, - years of age, who had been treated at sri ramachandra medical institute and research foundation between august and august . patients who received at least one year of treatment with s of s chemotherapy and/or radiation and surgery were included. medical history, family history of diabetes, cardiovascular diseases, and hypercholesterolemia, tanner staging, weight for height (< y per who criteria), bmi (> y per indian academy of pediatrics iap), blood pressure (nhlbi criteria), fasting blood sugar levels and lipid profile were obtained from the charts. statistical analysis of the data was done using ibm spss statistical software (version ). results: patients were studied, . % were male. . % were under years of age, . % between - years and . % above years. leukemia survivors comprised . % of the sample and non-leukemic's were . %. . % were treated with chemotherapy alone, . % with radiotherapy and chemotherapy, and . % underwent surgery with radiotherapy and chemotherapy. hypertension was found in . % of the study group, dyslipidemia in %, impaired fasting blood glucose in . % and . % were found to be obese. % of the study group was diagnosed with ms based on who criteria. conclusion: % of our study population was found to have ms per who criteria. individual metabolic complications were detected in % of the population. acute lymphoblastic leukemia (all) survivors appeared to be at high risk in our population. ms has been known to increase cardiovascular complications in cancer survivors. a multidisciplinary team approach to management of these patients is important to closely monitor and manage the long-term complications related to ms such as type diabetes and atherosclerosis. such an approach is essential to decrease long term morbidity and mortality from ms in this vulnerable population. background: the -year survival rate for childhood cancer exceeds %. however, up to % of these children require admission to the pediatric intensive care unit (picu) within three years of diagnosis. these children account for approximately % of all picu deaths, with mortality being higher for those post-hematopoietic stem cell transplant (hsct). national guidelines recommend that providers share informa-tion regarding prognosis and treatment options within the first hours of icu admission. these prognostic goals of care conversations (pgocc) are critical to the care of children with malignancies, a subpopulation at risk for increased mortality. to determine the frequency of pgocc as well as describe differences in patient characteristics and critical care therapies by pgocc status. design/method: a retrospective cohort study was conducted using the university of michigan virtual picu system database. picu admissions lasting longer than hours for patients ages to years between july , and june , with an oncologic diagnosis and/or hsct were identified. data on pgocc, patient demographics, diagnoses, picu interventions, and outcomes were recorded and compared between children with pgocc and those without using chi square test for categorical variables and kruskal-wallis test for continuous data. of picu admissions, % were male; the mean age was . years. the leading diagnoses were acute lymphoblastic leukemia ( %), acute myeloid leukemia ( %), lymphoma ( %), neuroblastoma ( %), and brain tumors ( %), and % of patients were post-hsct. pgocc was documented in ( %) patients. in comparison with patients who did not have a pgocc, children with a pgocc were more likely to be readmitted to the picu ( % vs. %, p < . ) and more likely to have had relapse of disease ( % vs. %, p< . ). patients with a pgocc had higher severity of illness scores (p = . ), higher use of non-invasive ( . % vs. . %, p = . ) and invasive conventional ventilation ( . % vs. . %, p< . ), and high frequency ventilation ( . % vs. . %, p < . ). also, patients with pgocc were more likely to receive continuous renal replacement therapy ( . % vs. . %, p< . ), arterial catheterization ( . % vs. . %, p< . ), and cardiopulmonary resuscitation ( . % vs. . %, p< . ). in only in critically ill children with hematologic-oncologic disease is pgocc held. children with pgocc were sicker and received more critical care interventions. future research is needed to evaluate the content of pgocc. background: central nervous system (cns) tumors and autism spectrum disorder (asd) represent significant disease cohorts in the pediatric population. asd diagnoses in children have a prevalence of %, in every children in the united states. additionally, more than , cns tumors are reported in children age to years in the united states with brain tumors being the most common solid tumor and the leading cause of death among all childhood cancers. the genetic etiology of autism and cns tumors is complex. specific gene alterations present in certain cancers have similarly been described and suspected to play a role in asd subtypes. targeted therapy panels, like foundation one (fo), have been beneficial in guiding treatment for some cancers based on distinct gene alterations. given the genetic overlap, the potential for therapeutic benefit and crossover from such actionable gene target panels merit further exploration in asd and cns tumors. we aim to identify and describe genetic alterations with known actionable targets in cancer therapy from fo as potential diagnostic, therapeutic and research targets for neurodevelopmental diseases. we plan to discuss the common genetic alterations between our cancers and neurodevelopmental diseases described in the literature. fo data was extracted and compared to the literature. each reported gene alteration from fo plus the keywords "autism", "psych" were used on pubmed to search for a suspected association if any with a neurodevelopmental disorder. results: twenty-one patients representing a cohort of six unique (astrocytoma-five, ependymoma-six, gbm-four, glioma-three, nerve sheath tumor-one, etmr-two) cns tumors were investigated. fo produced eighty total with sixty unique gene alterations. thirty-one ( %) of these yielded at least one published, suspected association to a neurodevelopmental disorder. the most common gene alterations were tp -four, cdkn a/b-five and braf-four. the main functional categories were cellular: proliferation, structure, differentiation and degradation; chromatin modeling; histone transcriptional modification; dna methylation and repair; strna; and neural signaling. sixty unique gene alterations were found in our cns tumor set using foundation one. thirty-one ( %) of these discrete alterations paired with at least one description in the literature as having been similarly altered in an asd subtype. many of these alterations have actionable targeted therapies presented through foundation one for our cns tumors and may be a relevant guide in the future of targeted therapy and research in asd subtypes. monoclonal antibody therapy usage is associated with significantly improved survival in b-cell nhl aya patients. although the usage has increased in the aya population from to , the magnitude of the increase is low. factors that affect the use of mab include race and insurance s of s type. further research is warranted to identify why privately insured patients are less likely to receive these drugs. background: prevention of chemotherapy-induced nausea and vomiting (cinv) remains a challenge despite advances in pharmacotherapy and the development of cinv clinical practice guidelines by the pediatric oncology group of ontario (pogo) that have been endorsed by the children's oncology group. achieving control of cinv in pediatrics further is complicated by the difficulty young children have vocalizing their symptoms. use of a validated nausea-assessment tool in conjunction with improved adherence to evidence-based guidelines may result in better quantification of symptoms and reduction of both nausea severity and vomiting frequency for pediatric patients undergoing chemotherapy. the pediatric nausea assessment tool (penat) has been validated for children ages - , and its integration into clinical practice may help optimize cinv control. objectives: this single-institution study sought to improve control of cinv in patients admitted for chemotherapy by standardizing the antiemetic regimens prescribed by all providers according to an institutional cinv algorithm developed from the pogo guidelines. we hypothesized that treatment using a standardized guideline would improve cinv control in patients admitted for chemotherapy. a baseline cohort of admissions for chemotherapy completed penat assessments and cinv diaries prior to receiving chemotherapy, four times daily during each admission, and daily for days following completion of chemotherapy from may , to january , . providers then were provided an institutional cinv treatment algorithm based on the pogo guidelines and received education at departmental meetings on appropriate implementation of this algorithm. a second cohort of admissions completed penat assessments and cinv diaries in a similar fashion from july , to december , . results: complete control of vomiting markedly improved following cinv guideline implementation ( % vs %, p <. ) with treatment failure also significantly reduced ( % vs %, p <. ). after controlling for the degree of emetogenicity of chemotherapy received, a patient was . times more likely to vomit prior to guideline implementation (or . , ci . - . ). there was no difference in nausea control, even after adjusting for the emetogenicity of chemotherapy. conclusion: control of chemotherapy-induced vomiting (civ) improved following widespread implementation of an institutional cinv treatment algorithm at a single institution. the severity of nausea reported remained unchanged which may reflect the difficulty of assessing nausea or an inadequate sample size. future research may focus on cinv treatment management through the use of guidelines specifically for breakthrough cinv and delayed cinv. background: aspho's professional development committee (pdc) recognized pediatric hematologists-oncologists (phos) serving in the united states (us) military have unique professional development needs that may not be addressed by aspho or a similar professional society. these individuals may also encounter challenges when transitioning to a civilian career. however, barriers to professional development have not been systematically characterized. the objectives were to characterize the number of phos with current or prior military service (mphos) and to identify any unmet professional development needs. design/method: a working group consisting of pdc members and both senior and early career mphos was formed. initial comments were solicited by email from known mphos regarding potential gaps in professional development and interest in working with aspho to improve support of mphos. a survey was developed and piloted with four members of the advisory group, questions were revised based on their feedback, and a final version was distributed via the aspho website and online community forum. targeted emails were sent to mphos identified through aspho and military databases. eligibility to complete the survey included ) completion of a fellowship in pediatric hematologyoncology, and ) current or prior service as an active duty military provider. quantitative and qualitative information were collected, including demographic data and perceived barriers to professional development. responses were summarized using descriptive statistics. results: sixty-five mphos were identified and surveys were completed for a % response rate. respondents were engaged in a variety of professional activities; % were male, % were serving active duty commitments, and % felt there were professional development gaps. areas of concern were categorized into nine themes with the most concerning being ) limited civilian knowledge of mpho practices ( % of participants), ) inability to attend professional society meetings ( %), and possibility of deployment ( %). participants expressed a desire for educational products to meet their specific needs and for networking opportunities with civilian colleagues. qualitative analyses identified concerns about low patient numbers and practice size. a subset of mphos perceive significant gaps in professional development. additional research is needed to better define areas for intervention, but many of the concerns align with those of similarly sized civilian programs and may be addressed through professional society networking opportunities, such as an aspho special interest group. background: infertility is an established cause of distress and has a negative impact on quality of life among childhood cancer survivors. the american society of clinical oncology has established guidelines on fertility counseling for individuals of reproductive age diagnosed with cancer, with the goal of improving reproductive and psychosocial outcomes. studies have shown that instituting a fertility team that can provide counseling and discuss fertility preservation (fp) options results in improved patient satisfaction in patients with cancer. objectives: the goal of this study was to examine predictors of referrals to the multidisciplinary fertility team, and documented fp interventions among these patients. design/method: an irb-approved retrospective medical record review was performed at a large pediatric academic center. all patients with new cancer diagnoses receiving chemotherapy were included from january (when the fertility team was established) to present. a standardized abstraction form was used to collect information about: age at diagnosis, gender, cancer type, whether a fertility consult was placed, and documented fp interventions. data were summarized descriptively and comparisons were made using nonparametric statistical methods. results: patients met inclusion criteria, of which ( %) were male. cancer types were as follows: leukemia/lymphoma, cns tumors, sarcomas, embryonal tumors, and langerhan's cell histiocytosis (lch). the mean age was . years, (range < - years). overall, % of all patients had a consultation with the fertility team. patients were significantly less likely to have a fertility consult if they were younger (p< . ). further, there were differences in the consultation rate between diagnoses, with % of sarcoma patients completing a consult, compared to % of those with cns tumors, % of those with embryonal tumor, % of those with leukemia/lymphoma and none of the patients with lch. our findings show that many children, adolescents, and young adults newly diagnosed with cancer are still not receiving fertility counseling despite: ) an expanding body of literature supporting the need to provide this counseling, ) guidelines published by several organizations recommending discussions about infertility risk and fp options, and ) presence of a multidisciplinary fertility team. specific strategies need to be developed to improve access for younger children, and for disease groups in whom fertility consults are underutilized, such as youth with cns tumors, embryonal tumors, and leukemia/lymphoma. background: socioeconomic status (ses) has on impact on overall survival in the pediatric oncology population. unfortunately, data are insufficiently detailed to explain the mechanism behind this phenomenon. how parents handle the health management demands placed on them at the time of a child's cancer diagnosis may represent a point of differentiation in health outcomes. objectives: determine the association between socioeconomic factors, cancer literacy, and parents' understanding of home emergency management and their responses to instances of pain, nausea, and fever. in a prospective observational study of parents whose children were newly diagnosed with cancer, we obtained demographic information and, using a validated instrument, (dumenci, ) we evaluated cancer literacy. we tested understanding of the education parents received about home emergency management with a -item multiple-choice vignette-based questionnaire focused on actions needed in home scenarios. we then followed parents' actual behavior through periodic phone calls assessing instances of nausea, pain, and fever and their responses to these episodes. results: preliminary analysis of participants showed an average score of on the -item parental understanding questionnaire (range - ). variables associated with increased score were college-level education by . points ( % ci [. to . ]), private insurance by . points [. to . ] and adequate cancer literacy by . points [. to . ]. actual behavior reported by families indicated that married parents and those with income above $ , were less likely to treat instances of pain by % ( % ci [ to ]) and % [ . to ], respectively. white parents, those with college-level education, and those with adequate cancer literacy were less likely to treat instances of nausea by % [ to ], % [ to ] and % [ to ], respectively. no associations were found between socioeconomic markers and parental responses to instances of fever. our findings suggest an association between demographic and socioeconomic markers and improved parental understanding of home emergency management. paradoxically, the same markers show a decrease in treatment response to pain and nausea. larger prospective studies are needed to link this behavior pattern to health outcomes, and help inform the extent of ses impact on home emergency management. emory university/children's heathcare of atlanta, atlanta, georgia, united states background: cardiovascular disease is a leading cause of morbidity and mortality in childhood cancer survivors (ccs). previous research showed wide practice variation in referral patterns to cardiology from the survivor clinic and in recommendations from cardiologists about the need for further testing or exercise restrictions. to develop a cardio-oncology algorithm in order to standardize referrals to cardiology and provide guidelines for cardiologists evaluating pediatric ccs. design/method: survivorship and cardiology experts developed a weighted scoring system for pediatric ccs who received cardiotoxic therapy based on time since treatment and risk factors identified by the children's oncology group (cog) and american heart association (aha). the cardiooncology algorithm assigned a score of - . the score range was categorized to guide cardiology referral: screening echo only ( - ), consider cardiology referral ( - ), recommend cardiology referral ( - ), and regular cardiology follow-up (≥ ). the algorithm also provides recommendations to cardiologists for screening and exercise modifications based on the score. after establishment of the algorithm, a convenience sample of institutional survivor clinic patient charts were retrospectively reviewed from the first month of each quarter from april -march to validate the algorithm, evaluate referral patterns to cardiology, and assess cardiology recommendations. the retrospective chart review evaluated patients ( % male; % non-hispanic white; % leukemia survivors; median age at diagnosis . years [range - . ]; median time off-therapy . years [range . - . ]). patients ( %) received anthracyclines (median dose mg/m , range - ) and ( %) received cardiac radiation. assigned cardio-oncology scores resulted in: % echo only, % consider cardiology referral, % recommend cardiology referral, and % regular cardiology followup. when evaluating detection rates of late effects by cardiooncology score, survivors ( %) had an abnormal echo: / echo only, / consider referral, / recommend referral, and / regular cardiology follow-up. assessing referral patterns prior to initiation of the algorithm revealed forty-two survivors ( %) referred to cardiology: / echo only, / consider referral, / recommend referral, and / regular cardiology follow-up. of the patients seen by a cardiologist at our institution, had further diagnostic testing ordered (i.e., stress test) and received exercise restrictions. a cardio-oncology algorithm and guidelines will standardize cardiac care for survivors by assigning a score to guide referral and cardiology practice after referral. prospective clinical use has begun and review will occur in one year to determine changes in detection rates of cardiac late effects, referrals, and recommendations from cardiologists. oregon health and science university, portland, oregon, united states background: delirium affects - % of patients (pts) in pediatric intensive care units (picu) and is associated with increased length of stay, decreased attention in school, and post-traumatic stress disorder. the diagnostic and statistical manual of mental disorders (dsm v) defines delirium as a "disturbance of consciousness […] with reduced ability to focus, sustain or shift attention" due to an underlying medical condition. despite the medical complexity of the hospitalized pho population, there are no published prospective studies looking at delirium in these pts. hypothesizing that delirium is under recognized in the pho population, we designed a year-long prospective study using a validated screening tool to determine the frequency of delirium in hospitalized pho pts and to identify associated clinical factors. design/method: baseline frequency of pts with symptoms suggestive of delirium was determined through retrospective chart review using a data mining program of electronic medical records (emr). for the prospective study, pho and picu nurses were trained to use the cornell assessment for pediatric delirium and to record scores within the emr on all pho pts once every -hour shift. predetermined demographic and clinical variables were entered daily into a red-cap database on all hospitalized pho pts. results: baseline frequency of delirium, without active screening, was determined to be . % of hospitalized pho pts. in the first months of the prospective study, consecutive admissions occurred among unique pho pts: oncology, hematology, and stem cell transplant pts. pts had at least positive delirium screen, for a prevalence per admission of . %. statistically significant variables associated with delirium, at p < . by univariate logistical regression, included prolonged length of stay, pt location (picu vs pho unit), and fever. adjusting for length of stay, administration of benzodiazepines and opiates were also significantly associated with delirium, p = . and . , respectively. on average, nurses completed delirium screening in % of each pts' -hour shifts. study accrual ends in jan and final data analyses will be reported in the abstract presentation. conclusion: delirium does occur in the pho hospitalized population and screening by trained nursing staff is feasible. pts at highest risk appear to be pts with prolonged hospital stays, picu admissions, or frequent use of benzodiazepines/opioids. routine screening should improve our recognition of delirium and allow us to promptly intervene, or prevent delirium in an effort to avoid potential acute and long term consequences. background: with high survival rates for children and adolescents with hodgkin lymphoma (hl), treatment regimens are now designed to maximize cure while decreasing risk of long-term health outcomes associated with chemotherapy and radiation therapy. within contemporary treatment regimens, the comparison of toxicities experienced by patients receiving chemotherapy plus radiotherapy (crt) versus only chemotherapy (co) has not been studied extensively. objectives: this study examines select self-reported adverse health outcomes in survivors of contemporarily-treated pediatric hl to better understand the balance between efficacy and toxicity associated with chemotherapy and radiation therapy. (cog) ahod that evaluated a response-based treatment paradigm in pediatric hl. patient who received initial chemotherapy were randomized based on early response to continued chemotherapy, chemotherapy plus radiotherapy or augmented chemotherapy plus radiotherapy. patients completed self-report questionnaires on health problems at , , , and years following therapy. we examined selected patient-reported pulmonary, gastrointestinal (gi), cardiac and endocrine outcomes. kaplan-meier survival curves were used to determine probability of survival without the selected adverse health outcome. log-rank tests were used to compare the co versus the crt group. results: a total of , enrolled patients, patients in the co group and patients in the crt group, completed , questionnaires at a median of . years after s of s completion of therapy (q , q : . , . ) which were analyzed. the cumulative -year incidence of endocrine dysfunction was significantly greater in the crt group versus those in the co group ( % versus %; p< . ), driven by the incidence of hypothyroidism ( % versus %; p< . ). there were no significant differences in cardiac ( % versus %; p = . ), pulmonary ( % versus % p = . ), and gastrointestinal dysfunction ( % versus %; p = . ) between the co and crt patients. conclusion: this study demonstrates low cumulative incidence overall of organ dysfunction early post completion of contemporary therapy for hl. the addition of radiation therapy significantly increased risk for hypothyroidism, but with no higher risk noted for cardiac, pulmonary or gi dysfunction. limitations include self-report status, potential selection bias, and relatively short latency period following end of therapy. longer follow-up is needed to determine more delayed risks for organ dysfunction in order to best define the balance between therapeutic efficacy and long-term adverse health outcomes related to chemotherapy and/or radiation therapy. background: identification of an organism via bronchoalveolar lavage (bal) or respiratory tract biopsy (rtb) has historically been considered the gold standard for diagnosis of invasive fungal infection (ifi); however, data previously published by our group showed that these procedures infrequently lead to a change in management in children with an oncological diagnosis or undergoing hematopoietic stem cell transplant (hsct). there is also a paucity of data on the cost of ifi in this population. to compare the costs of work-up and management of pulmonary ifi diagnosed based on ct scan alone versus ct scan or chest x-ray prompting a bal or rtb. design/method: we collected cost data on patients at ann & robert h. lurie children's hospital of chicago undergoing chemotherapy or within months of hsct who were suspected of having an ifi between and . in order to include sufficient time to account for post-procedure compli-cations but avoid including costs unrelated to ifi, data were included for days from the day of their diagnostic scan or procedure. cost data was available for of the patients previously studied. thirty-six of these patients were diagnosed with suspected ifi based on ct only and patients underwent bal or rtb. when evaluating specific costs, inpatient beds costs were higher in the bal and rtb group (median $ , versus $ , , p = . ), yet there was only a trend towards higher costs for antifungal agents (median $ , versus $ , , p = . ) and respiratory support (median $ versus $ , p = . ). many of the initial ct scans were not captured in the -day evaluation period for the bal or rtb group based on the study design; however, even when accounting for ct scans up to a week prior these procedures, the total cost of ct scans was higher in the ct only group (median $ versus $ , p = . ), as they had more scans. despite this, total costs were significantly higher for patients who underwent bal or rtb versus ct scan only (median $ , versus $ , , p < . ). combined with our previous data that bal and rtb infrequently leads to a change in management in children with an oncological diagnosis or undergoing hsct suspected to have an ifi, the significantly higher costs associated with these procedures makes these invasive diagnostic techniques even less desirable. batra, pediatr blood cancer, . background: while infants > months of age with acute lymphoblastic leukemia (all) have a poor prognosis, infants with acute myeloid leukemia (aml) fare better despite more intensive therapy. there are limited data on this difference, particularly differences in supportive care requirements during induction therapy for infants. objectives: to compare induction mortality and resource utilization in infants relative to non-infants aged < years, separately for all and aml. design/method: we used previously established cohorts of children treated for new onset all or aml at children's hospitals in the us contributing to the pediatric health information system. patients with down syndrome were excluded. follow-up started on the first day of induction chemotherapy and continued until the earliest of: days after commencement of chemotherapy, start of the subsequent course, or death. high acuity of presentation, defined as icu requirements involving or more organ systems within the first hours following initial admission were compared using log binomial regression. -day inpatient mortality was compared using cox regression. resource utilization rates (days of use per inpatient days) were compared using poisson regression. results: a total of all ( infants, non-infants) and aml ( infants, non-infants) were included in the analyses. infants were more likely to present with high acuity compared to non-infants for both all ( % and %, rr = . , % ci: . , . ; p< . ) and aml ( % vs %; rr = . , % ci: . , . ; p = . ). infants with all had higher inpatient mortality compared to non-infants even after accounting for differences in acuity of presentation ( . % vs . %, adjusted hr = . % ci: . , . ; p = . ). in contrast, inpatient mortality was more similar for infants and noninfants with aml ( . % vs . %, adjusted hr = . % ci: . , . ; p = . ) and comparable to rates among infants with all. infants with all and aml had higher rates of utilization of fresh frozen plasma, cryoprecipitate, diuretics, supplemental oxygen, and ventilation relative to non-infants. infants with all also had higher rates of total parenteral nutrition, ecmo, and patient controlled analgesics compared to noninfants. infants with all experienced significantly higher induction mortality compared to noninfants, a difference not entirely explained by acuity at presentation. differences in ru among infants may reflect higher presentation acuity and greater treatment related toxicity. further work is needed to elucidate the contribution of treatment related toxicity to early mortality in infants with all. background: fever in a child with cancer is a medical emergency due to the significant risk of a serious bacterial infection. many attempts have been made to risk stratify these patients. the respiratory pathogen panel (rpp) is a panel of polymerase chain reaction tests that identify seventeen common respiratory viruses and three bacterial infections. samples are taken via nasopharyngeal swab. rpps are frequently sent, but we do not have data to determine whether a positive result can lead to stratification to a lower risk of bacterial infection. ( ) to determine the epidemiology of respiratory virus-associated fever in pediatric oncology patients ( ) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all pediatric oncology patients seen in our emergency department (ed) with fever from the introduction of the rpp in april to september , . we reviewed the results of blood cultures, rpp, chest radiographs, and discharge summaries to identify sources of infection. we also identified the patients' cancer diagnosis, age, absolute neutrophil count (anc), and absolute lymphocyte count (alc). results: positive rpps were found among pediatric oncology patients who presented to the ed with fever. the most common positive rpp findings were rhinovirus/enterovirus (rev) ( %), parainfluenza ( %), influenza ( %), coronavirus ( %), and polyviral ( %). among patients with a positive rpp, % had bacteremia compared to % bacteremia among all pediatric oncology patients with fever (or . [ . - . ], p . ). all cases of bacteremia were associated with rev. there was no bacteremia identified in patients with rpps positive for other viruses (or . [ . - . ], p . ). rev positivity did not confer a lower risk of bacteremia than rpp negative patients ], p . ). anc (p = . ) and alc (p = . ) less than , and number of patients with severe neutropenia (p = . ) were not statistically different between the rev and non-rev positive rpp groups. rpps positive for viruses other than rev reduced the likelihood of bacteremia in febrile pediatric oncology patients in the ed setting. patients with bacteremia may have concurrent infection with rev. a larger study is warranted to determine if positive rpp results can inform clinical management of a child with febrile neutropenia. emily mueller, anneli cochrane, seethal jacob, aaron carroll s of s background: the usage of mobile health (mhealth), which refers to the application of mobile or wireless communication technologies to health and healthcare, has grown exponentially in recent years. mhealth tools have been used by caregivers of other vulnerable populations, but little has been focused on caregivers of children with cancer. objectives: to conduct a survey to understand the mobile technology usage, barriers, and desired mhealth tools by caregivers of children with cancer. we conducted a mailed cross-sectional paper survey of caregivers of all children who were diagnosed with cancer at riley hospital for children between june, and june, . the survey contained questions, both fixed and open-ended, in both english and spanish. up to three rounds of surveys were sent to those who did not respond. of the respondents, they were primarily parents ( . %), median age was . years (range - ), and most were white ( . %) and non-hispanic/latino ( . %). the top three annual household income brackets included $ , to $ , ( . %), $ , to $ , ( . %) and under $ , ( . %). the majority had an education: . % college graduates, % graduate degree, and . % high school education or ged. nearly all respondents owned a smart phone ( . %) and . % owned a tablet. the majority used an ios operating system ( . %), while . % reported use of a device with an android operating system. all caregivers reported use of at least one mobile website/app regularly for their personal use. while . % of respondents reported no barriers to mobile technology use, the top barrier selected was "data limitations" ( . %). overall, . % wanted at least one medical managementrelated website/app: medical knowledge ( . %), healthcare symptom tracking/management ( . %), and medication reminders ( %). healthcare system-related desires were high, as . % wanted access to their child's medical record and . % wanted a website/app to facilitate better communication with medical providers. there were no significant associations between socioeconomic status (income or education) with barriers or types of websites/apps desired by caregivers. since the vast majority of caregivers use mobile technology with minimal barriers, future research should focus on designing an mhealth tool to address the medical management needs by caregivers of children with cancer. by supporting caregivers through this type of mhealth tool, it could positively impact patient clinical outcomes through greater adherence to medications and treatment protocols. background: in children with fever and neutropenia, early initiation of targeted antibiotic therapy improves outcomes, yet there are no standards for choice of empiric antibiotics. in our institution implemented an early empiric ceftriaxone (eec) protocol to reduce time to antibiotic administration in febrile hematology-oncology patients who are potentially neutropenic when the absolute neutrophil count is not yet know. ceftriaxone is given immediately after obtaining blood for culture and lab studies. in patients found to be neutropenic, ceftriaxone is discontinued and cefepime is initiated. the purpose of this retrospective study was to evaluate our eec protocol in neutropenic patients by assessing ceftriaxone sensitivity of positive blood cultures and comparing rates of adverse outcomes with a cohort of patients treated prior to implementation of the protocol. we are now conducting a prospective study to more thoroughly investigate antibiotic sensitivities of organisms isolated from blood cultures of neutropenic patients. design/method: hematology-oncology patients with at least one positive blood culture between january and december were identified. patient demographics, neutrophil count, antibiotic treatment, isolated organisms and sensitivities, and adverse outcomes (increased respiratory support, hypotension requiring intervention, and icu admission) were obtained by retrospective chart review. fisher exact test was used to compare dichotomous variables between patient groups. we are now prospectively identifying febrile neutropenic patients with positive blood cultures and performing antibiotic sensitivity testing to several antibiotics commonly used as empiric therapy for febrile neutropenia. results: retrospectively, we identified neutropenic patients with a total of bacterial isolates from blood cultures. of organisms isolated, were tested for sensitivity to ceftriaxone and ( %) were not sensitive, / ( %) of gram-positive cultures and / ( %) of gram-negative cultures. ten of ( %) eec patients had an adverse outcome versus / ( %) of non-eec patients (p = . ). notably, % of eec patients required icu admission versus % of non-eec patients (p = . ). thus far our data obtained prospectively is revealing similar rates of ceftriaxone resistance with / cultures not sensitive to ceftriaxone ( %, ci . %- . %). in our retrospective study, no statistically significant difference was seen in overall adverse outcome rate between the two cohorts, though icu admission rates were significantly higher in eec patients. ceftriaxone resistance rates were high in tested isolates, which is further supported by preliminary data from our ongoing prospective study. given these data, eec may not be effective at improving outcomes in febrile neutropenic pediatric hematology-oncology patients. background: approximately in children diagnosed with cancer will die of their disease, despite advances in treatment. results: two focus groups of six parents each met in june . the parents were predominantly female ( female, male) and had lost their children an average of . years prior (range - . years). two parents were in the same family. nearly all patients were offered palliative care ( / ), all were offered hospice and most died at home ( at home, in the icu). parent discussion uncovered six broad themes: beneficial provider qualities, optimal communication, helpful systematic supports, struggles to feel like a good parent, struggles with a loss of control and unmet needs. parents appreciated providers who were consistent, reliable and honest. parents desired communication that was sensitive to the needs of the patient and family with a balance of hope and realism. parents appreciated the tangible supports pro-vided by social work and the emotional support of child life both for the patient and their siblings. some parents struggled to define and advocate for their child's quality of life, especially when it led to disagreeing with the medical team. several parents expressed frustration with unfamiliar caregivers in the hospital, especially trainees. they expressed a strong desire for more anticipatory guidance about the end of life including how to discuss it with their children. they also wished for a cancer-specific support group for bereaved parents. conclusion: bereaved parents of pediatric oncology patients in our focus groups appreciated consistent, reliable providers who communicated with a balance of realism and hope. they appreciated the tangible and emotional support they received and wanted more anticipatory guidance at the end of their child's life. these results can help guide clinical care, especially in communities without strong palliative care support. further research is needed to develop interventions to improve end of life care. background: clinical trials involving human subjects depend on informed consent (ic) to ensure ethical protections for participants. parents of children with cancer often lack full understanding of the basic elements of ic for clinical trials. additionally, the stress of their child's cancer diagnosis may affect their decision-making capabilities. this is especially problematic as these children rely on parents to fully comprehend clinical trials and weigh their benefits and risks. physician communication is critical for effective family-centered care. the acgme mandates that training programs teach and assess trainees' communication skills. however, there are currently no published curricula aimed at training pediatric hematology/oncology fellows to deliver ic effectively for cancer clinical trials. to develop and pilot-test a simulation-based curriculum to enhance communication skills of pediatric s of s hematology/oncology fellows in the delivery of ic for cancer clinical trials. we developed, tested, and implemented the curriculum from to in two phases. in phase- , we reviewed literature on simulation-based curricula and completed a needs assessment to create a clinical scenario and full curriculum using standardized patients. using miller's pyramid model, fellows' assessments included: immediate de-brief, surveys to assess pre/post confidence and knowledge of the basic ic elements ("knows" and "knows how"), and -degree summative assessments compiled from fellow self-assessments, faculty, and standardized patients ("shows how"). after initial testing and refinements done with fellow, in phase- , we implemented the curriculum with our fellows. likert scale ( strongly disagree- strongly agree) and basic p values are reported. results: fellows gave high mean ratings for training relevance ( . ) and standardized patients' preparedness ( ). almost all ( . ) reported they have used the knowledge gained in their clinical practice. increase in self-reported confidence (pre/post) was noted in all domains: general -describing possible benefits of the clinical trial . / vs. . / (p = . ), risks and potential side effects . / vs. . / (p = . ), and explaining alternatives . / vs. . / (p = . ); research -discussing purpose of the clinical trial . / vs. . / (p = . ), and randomization . / vs. . / (p = . ); and family-centered -addressing emotions during ic . / vs. . / (p = . ), and delivering bad news . / vs. . / (p = . ). summative evaluation mean ratings for all fellows were . (range . - . ). our novel simulated-based ic curriculum, significantly increased fellows' self-reported confidence and skills during ic delivery. importantly, our ic curriculum addressed not just research-related content but also management of parental emotional needs during the ic discussion. next phase includes kirkpatrick model program evaluation and dissemination across other training programs in our institution. national kaohsiung normal university, kaohsiung, taiwan, province of china background: taiwan's childhood cancer foundation reported in that the -year survival rate of childhood cancer was %. as a result, many childhood cancer survivors were back in school after treatment. however, childhood cancer survivors' educational outcomes suffered because of their long-term absence from school and late effects of cancer and cancer treatment. a few school reentry protocols have been developed by the nursing professionals in taiwan to facilitate students' return to school but remained experimental in nature and hardly accessible. parents, students, and teachers were left to their own devices to make individual school reentry plans. objectives: this study aimed to examine and uncover the commonalities among three middle school students' successful school reentry experiences from their teachers' perspectives and to analyze the factors contributing to their success. design/method: this is a qualitative interview study. indepth semi-structured interviews were conducted with three middle school teachers in december about their perceptions, observations, and experiences working with adolescent childhood cancer survivors. the students were two boys with leukemia and one girl with bone cancer. they were diagnosed in the first year of middle school when they were - years old and returned to school for the third and the final year. these students met the following criteria for successful school reentry: regular school attendance, average/above average academic performance, friendship maintenance, and high school diploma. the theme -bring the class to the hospital was found to be the key to the adolescents' successful return to school. without a prescribed school reentry protocol and in the face of limited bedside education services, the homeroom teachers, as links between school, home, and hospital, brought the class to their hospitalized students. they doubled as bedside teachers conducting lessons at the hospital or students' homes, became friends with the parents, witnessed firsthand the students' pain and triumph during treatment, brought the students back to school for visits and celebrations, delivered the classmates' wishes and news to the students, encouraged and welcomed classmates' visits to the hospital, and, together with parents and other teachers, developed flexible school reentry schedules for the students. this on-going study demonstrated the critical roles and functions of homeroom teachers in successfully bringing the students back to school during and/or after cancer treatment. further analysis will be focused on how and why these three homeroom teachers were able to carry out this unexpected task on top of their already full workload. jennifer kesselheim, shicheng weng, victoria allen, collaborative group fellowship program directors dana-farber/boston children's cancer and blood disorders center, boston, massachusetts, united states background: a novel, -module, case-based curriculum entitled "humanism and professionalism for pediatric hematology-oncology" (hp-pho) aims to foster pho fellows' reflection on grief and loss, competing demands of fellowship, difficult relationships with patients and families, and physician well-being and burnout. in small group facilitated sessions, fellows work to identify coping strategies and explore how the challenges of fellowship influence both their own doctoring and the patient experience. objectives: to administer the hp-pho curriculum in a prospective, cluster-randomized trial, measuring whether exposure to this educational intervention, compared to standard conditions, fosters humanism and professionalism and improves satisfaction with training. design/method: pho fellowship programs (n = ) were cluster-randomized to deliver usual training in humanism and professionalism (control) or the novel curriculum (intervention) during the - academic year. the primary outcome measure was the pediatric hematology-oncology self-assessment in humanism (phosah). secondary measures included a -point satisfaction scale, the maslach burnout inventory (mbi), the patient-provider orientation scale, and the empowerment at work scale. participating fellows were pre-tested in summer and post-tested in spring . a change score was calculated for each study instrument. we compared each outcome between arms using mixed effect models adjusted for pre-test score as a fixed effect and site as a random effect. results: randomization yielded intervention and control fellows. the two arms did not significantly differ in distribution of fellow age, gender, or post-graduate year. the intervention sites successfully administered of ( %) modules. change scores on the phosah were not significantly different between the control and intervention arms (adjusted mean difference = . ; % confidence interval [ci] - . , . ; p = . ). compared to the control arm, fellows' exposed to the curriculum gave significantly higher ratings on several items within the satisfaction scale including satisfaction with their training on "physician burnout" (adjusted mean difference = . ; % ci . , . ; p< . ), "physician depression" (adjusted mean difference = . ; % ci . , . ; p< . ), "balancing professional duties and personal life" (adjusted mean difference = . ; % ci . , . ; p = . ), and "humanism overall" (adjusted mean difference = . ; % ci . , . ; p = . ). change scores on other secondary measures were not significantly different between study arms. conclusion: exposure to the hp-pho curriculum did not alter fellows' self-assessed humanism and professionalism. however, the curriculum proved feasible to administer and intervention fellows expressed higher levels of satisfaction in their humanism training, indicating the curriculum's positive impact both for fellows and their learning environment. background: recent work has documented significant levels of unmet needs among adolescents and young adults with cancer, particularly psychosocial challenges during the transition to adulthood, (e.g., abrupt disruption to school and social life, and social isolation). given that adolescents and young adults drive mobile app use, a mobile-phone may be an ideal way to deliver a psychosocial intervention to adolescents and young adults with cancer. to use a patient-centered approach to inform a mobile-based mindfulness and social support intervention for adolescent and young adult patients with cancer. design/method: participants were ten aya with sarcoma ( % female; % adolescents); parents of the five adolescents, and six healthcare providers (n = ). formative research involved three steps: ( ) in-depth interviews were conducted with ten aya with sarcoma; parents of the five adolescents, and six healthcare providers (n = ). ( ) adaptations were made to an existing mindfulness app which offers a program for youth. modifications included creating a -week "mindfulness for resilience in illness" program, with relaxation exercises, and the addition of videos featuring two sarcoma survivors as program hosts. content was informed by the mindfulness curriculum for adolescents, learning to breathe. ( ) a private facebook usability group was organized to (i) elicit beliefs about the mindfulness app and potential future enhancements, and (ii) promote social support. results of the in-depth interviews revealed themes around adolescents' functioning and coping, including body image concerns; recurrence-related anxiety; anger over loss; and being overwhelmed by medical information. themes from the interviews were incorporated into a demonstration version of the mobile app. a patient-centered approach is widely recommended in the development of mobile-based health behavior change interventions and may be a useful way to inform development of a mobile-based mindfulness and social support intervention for adolescents and young adults with cancer. background: medical trainees consistently report suboptimal instruction and poor self-confidence in communication skills. despite these deficits, few training programs provide comprehensive pediatric-specific communication education, particularly in the provision of "bad news." an in-depth survey to examine the historical experience and communication needs of pediatric fellows was conducted at a large academic pediatric center as the first step towards the development of a comprehensive communication curriculum. to determine the previous educational and clinical experiences of pediatric subspecialty fellows, assess their levels of comfort in the context of various communication topics, and query potential modalities and topics for future communication training. design/method: the needs assessment survey was developed using previously developed and validated questions and review of the literature. the survey was reviewed by internal and external pediatric oncology and palliative experts and pre-tested with a subset of trainees to enhance content validity. results: thirty-two out of a total of fellows completed the survey ( % completion rate), of which % were pediatric hematology-oncology or subspecialty fellows. most fellows had participated in previous teaching sessions ( %), including those involving role play or simulation ( %). however, few fellows had received feedback from senior clinicians on their communication skills ( % of fellows had received feedback ≤ times). on a scale of -x, with indicating "not well prepared," the mean score for of communication items was < . fellows felt least prepared to lead discussions around informed consent for experimental therapies, end of life care, and autopsy. fellows indicated that didactic educational sessions and additional coursework were less useful strategies for improving their communication skills, whereas small group role play sessions with faculty and/or bereaved parent educators were most useful. fellows' overall communication preparedness score was not correlated with post-graduate year but was positively associated with the number of times they previously had delivered bad news to patients and families. fellows requested additional training on many topics, with greatest interest in learning skills to optimize communication with an angry patient or family. additional topic requests included placing limitations on resuscitation, withdrawing/withholding further therapy, and ageappropriate inclusion of patients in difficult discussions. despite self-report of prior communication skills training, pediatric subspecialty fellows felt underprepared to participate in difficult discussions with patients and families. learners identified role-playing and coaching with real-time feedback from other physicians and bereaved parents as more useful training strategies as compared to didactic sessions. background: when children die of cancer, parents must adjust to their child's absence amidst the lingering turmoil of what preceded their death: witnessing their child undergo painful treatments, making difficult decisions, and anticipating a devastating loss, all the while hoping for a recovery. adjustment to a child's death, as depicted by current bereavement literature, necessitates making meaning of one's loss. professional care staff can help parents make sense of their child's illness, and in turn, of their own parental experience during treatment. however, the extent to which relationships with professional care team members influence parents' ability to make sense of, and successfully cope with, their loss has not been examined. objectives: to examine how bereaved parents' interactions with their deceased child's pediatric oncology professional care team have impacted their grief symptoms design/method: to better understand how interactions with professional care staff relate to parents' grief outcomes, we conducted a mixed-methods study examining staff impact on parental grief. thirty participants whose children died of cancer one to three years ago completed an in-depth interview and psychometrically validated surveys measuring meaningmaking, depression, and grief symptoms. results: correlational analyses of the measures found that an increase in meaning making was associated with lower depressive and grief symptoms. a content analysis of the interviews found that many participants regarded staff "like family," had on-going relationships with staff after their child died, and described various ways staff interactions during treatment and after the child's death helped them make sense of their loss. in particular, participants described how interactions with staff have helped them find benefits in their loss and learn to create a new relationship with their child despite their physical absence. quantifying the interview data and statistically analyzing it along with the measures found that participants' increased frequency of describing staff's positive impact on their grief correlated with higher meaning-making scores and lower grief symptom scores. our study found that bereaved parents who lost their children to cancer were articulate in sharing their experiences of staff engagement and communication during treatment, offering numerous examples of how staff aided them in making meaning of their loss that were reliably associated with their subsequent grief. we hope the results of this mixed methods research encourage further study of the importance of staff interaction with families during the critical period of their children's care, and the lasting impact this can have regardless of the treatment outcome. memorial sloan kettering cancer center, new york, new york, united states background: although resiliency has been recognized as necessary for healthcare professionals, trainees feel unprepared for the emotional challenges inherent in caring for sick and dying patients. compounded by long hours, challenging work environments, and lack of formal training on handling emotionally difficult situations, many institutions are recognizing the need for interventions to reduce trainee distress. the goals of this fellow-led quality improvement initiative were: ) to determine whether there is a need for emotional support amongst pediatric hematology and oncology fellows, ) to provide formal resiliency and debriefing sessions, and ) to measure feasibility, acceptability and effectiveness of implemented curriculum. design/method: an anonymous survey to determine need for resiliency and debriefing sessions following a traumatic event was distributed to active pediatric hematology & oncology fellows at memorial sloan kettering cancer center in january . once need was established, an intervention consisting of a formal curriculum was developed and initiated in june , involving: ) scheduled and ad hoc debriefing sessions in response to traumatic events (including patient death, codes, interpersonal conflicts, end-of-life care); led by a psychiatrist and social worker with fellows and a pediatric oncologist mentor in attendance, and ) a resiliency didactic curriculum, led by a palliative medicine specialist, focused on skills such as contesting cognitive distortions and mindfulness. the effectiveness of these sessions will be measured using follow-up anonymous surveys at months (currently underway) and months post-initiation of intervention. the initial survey demonstrated most trainees ( / ) were present at or more deaths during their training, while less than half of respondents had attended a post-event debriefing session. % of respondents felt there was not sufficient emotional support from the institution for physicians caring for dying patients. a separate pre-intervention survey found all respondents ( / ) expressed a need for regular debriefings, and nearly all anticipated that they would benefit from such debriefings. concerns identified by trainees that would preclude participation in the curriculum included preference to deal with emotional situations privately and time constraints. trainees identified a need for formal debriefings and resiliency skill development. the program was easily implemented, and is both feasible and acceptable with good attendance. feedback received at the -month mark will determine deficits and possible improvements to the curriculum. the -month survey will measure effectiveness of the program and whether it should be continued. background: acute kidney injury (aki) is a common but under-recognized complication among patients with leukemia. it is associated with prolonged hospital stays, increased mortality, progression to chronic kidney disease, and delays or changes in cancer therapy which may affect a patient's prognosis. however, data on aki in pediatric patients with cancer is still lacking overall. we investigated the incidence of aki in patients who were newly diagnosed with all at our center from january to september . we performed a retrospective chart review of all patients who were newly diagnosed with all from neonate to years in our facility. we determined the incidence of aki in our population using the kidney disease: improving global outcomes (kdigo) diagnostic criteria. we also assessed for nephrotoxic exposures, nci all risk stratification and risk of aki, and tumor lysis syndrome (tls). we identified patients diagnosed during the study period who met inclusion criteria. median follow-up time was . months (range . - . ). the cohort was predominantly male ( . %) and hispanic ( . %). our analysis showed . % had aki by kdigo criteria ( % grade , . % grade , and % grade ), . % had aki on presentation, and % had multiple aki episodes during the study period. older age and longer length of hospitalization were associated with aki (p = . and p = . , respectively). there was no association between aki and nci all risk classification, contrast exposure, hyponatremia, elevated white blood cell count, uric acid levels, antimicrobial therapy, or diuretic use in this study. conclusion: aki was a common finding in our study population. the majority had grade aki by kdigo criteria. however, aki was associated with older age and a longer length of stay. further study is needed to determine the short-and long-term impact of aki on pediatric patients with all. st. jude children's research hospital, memphis, tennessee, united states background: in some regions, the availability of trained pediatric oncologists is a limiting barrier for the care of children with cancer. in , the unidad nacional de oncología pediátrica (unop) and the universidad francisco marroquín school of medicine in guatemala established a pediatric hematology/oncology fellowship program sponsored by st jude children's research hospital to provide central america and the caribbean with well-trained specialists. a systematic analysis of the impact of fellowship programs in pediatric oncology has never been done, especially in the context of a regional education program. objectives: this study sought to analyze the impact of the unop fellowship program based on the regional number of providers, pediatric cancer centers and patient volume. in addition, it sought to characterize the jobs and scientific output of the graduates. the impact will be evaluated in the context of a cost analysis. to define the volume of providers, pediatric cancer centers and patients, the directors of pediatric cancer centers in central america were sent an online survey to obtain these data. all the centers contacted maintain an updated hospital-based patient registry. in addition, the graduates of the fellowship program were also sent an online survey, asking about their job at graduation, current role and scientific productivity. the cost analysis will include assessment of direct costs including salaries and stipends for away rotations, as well as the indirect costs of faculty time spent teaching. since the establishment of the unop fellowship program, the region has more providers for pediatric cancer (p< . ) and centers treat a larger volume of patients (p< . ). two new centers have opened with graduates of the program. all but one graduate practice pediatric oncology ( / ) and the majority do it in their country of origin ( / ). no graduate practices outside of this region. almost half of the graduates ( %) hold a leadership role at their institution. the majority of their time is spent in the public sector (> %). the majority of graduates participate in clinical research ( %) and have participated in the creation or implementation of therapeutic protocols ( %). on average, the graduates have published peer-reviewed articles since completion of training. the unop fellowship program has had a favorable impact on pediatric cancer care in the region, contributing to the capacity to treat a larger volume of patients. graduates practice pediatric oncology in the region in the public sector, frequently hold leadership roles and are scientifically productive. background: abandonment of treatment is a major cause of treatment failure and poor survival in children with cancer in low-and middle-income countries. the incidence of abandonment in peru has not been reported. objectives: the aim of this study was to examine the prevalence and associated factors of treatment abandonment in pediatric patients with cancer of peru. we retrospectively reviewed the sociodemographic and clinical data of children referred between january and december to the two main tertiary centers for childhood cancer, located in lima, peru. definition of treatment abandonment was used from the siop (international society of paediatric oncology) podc (paediatric oncology in developing countries) abandonment of treatment working group recommendation. results: data of children diagnosed with malignant solid tumors and lymphomas were analyzed, of which ( . %) abandoned treatment. univariate logistic regression analysis showed significant higher abandonment rates in children living outside the capital city, lima (p< . ); prolonged travel time to a tertiary center (> hours; or . , p = . ); living in a rural setting (or . ; p< . ) and lack of parental formal job (or . ; p = . ). according to cancer diagnosis, children with retinoblastoma were more likely to abandon compared with other solid tumors. in multivariate regression analyses, rural origin and lack of formal parental employment were independently predictive of abandonment. conclusion: treatment abandonment prevalence in our country is high and closely related to socio-demographical factors. treatment outcomes could be substantially improved by strategies that help prevent abandonment of therapy based on these results. st. jude children's research hospital, memphis, tennessee, united states background: to improve the quality of a pediatric hematology/oncology fellowship program, a systematic assessment must be performed that can evaluate its current state and identify areas of opportunity, as well as modifications over time. unfortunately, widely agreed-upon metrics of quality for pediatric hematology/oncology fellowship programs currently do not exist. this is particularly important in this field due to the global shortage of specialists. for this reason, an assessment instrument that is applicable throughout the world must be created. objectives: the st. jude global education program assessment tool (epat) is a novel instrument that seeks to evaluate pediatric hematology/oncology fellowship programs around the world in systematic and objective way. epat will help determine key performance indexes that are relevant for quality education in pediatric hematology/oncology fellowship programs and establish the framework for improvement. design/method: firstly, key domains to be evaluated for program assessment were identified a priori based on the continuum of pediatric hematology/oncology fellowship programs in the context of geography and educational structure. subsequently, questions were formulated to evaluate these key domains, seeking to assess elements involved in ensuring competence in clinical practice, academic productivity and regional impact. due to the novelty of this tool and the lack of defined metrics of quality, epat relies on expert opinion in a two-step process: internally in the department of global pediatric medicine at st. jude children's research hospital and, subsequently, from a panel of experts in global pediatric oncology and medical education from around the world. ten key domains were identified to evaluate all aspects relevant to training programs around the world, regardless of educational and geographic context. questions have been created to assess these domains and, to make epat quantitative, these have assigned weights with a value reflective of their relative importance. this grading system allows for a score in each key domain, permitting monitoring of changes over time. epat is currently at the stage of external expert review, and subsequently will be piloted in five fellowship programs around the world to provide different geographical and patient care contexts for its validation. once epat is finalized, it will be distributed to pediatric hematology/oncology fellowship programs around the world to be applied. epat proposes a novel strategy to assess training programs in a systematic way that includes all aspects relevant for a training program in a global context. this tool will help guide improvements in pediatric hematology/oncology fellowship programs and assure a well-trained workforce. background: with the improvement in pediatric oncology patient survival and outcomes in the past several decades, monitoring for recurrence and long-term effects of therapy has become even more important. the utilization of personalized treatment summaries and survivorship care plans (scps) is one way to communicate this information with patients and families. the american college of surgeons commission on cancer (coc) created a standard regarding provision of scps to % of eligible patients by december , as a metric for accreditation of all cancer centers. the standard applies to all patients with stage i, ii, and iii cancer diagnoses and requires creation of the scp within one year of diagnosis or six months of completing treatment. during implementation at our pediatric cancer center, we identified barriers to use of the guidelines in the childhood cancer setting. objectives: define eligibility for an scp for pediatric oncology patients to include all patients with curative intent and to deliver scps within six months of finishing therapy. design/method: using chart review and a cancer center registry query, we identified childhood cancer patients potentially eligible for an scp by collecting stage, goal of therapy, and dates of treatment. all patients with curative intent were deemed eligible for an scp regardless of stage i-iv. patients being followed in the oncology clinic for posttreatment surveillance and care were included even if they had received an scp in the survivorship program or were greater than six months off therapy at time of implementation. as expected in the pediatric oncology population, acute lymphoblastic leukemia (all) was the most common diagnosis comprising . % of patients. all is stratified into risk groups instead of surgical staging categories, and treatment duration is greater than one year, unlike many adult-onset malignancies. these differences required interpretation of the guidelines to apply to our pediatric population for all and other pediatric diagnoses with non-surgically based staging. our pediatric oncology clinic has to date provided scps to of eligible patients by adapting the guidelines to focus on patients with curative intent to receive an scp by six months off therapy. cancer staging guidelines and goals for curative intent as well as lengths of treatment vary between the pediatric and adult populations. the coc guidelines require adaptation for optimal applicability to the pediatric oncology population. background: education in communication for fellows in fields that require difficult discussions with families are few in nature. adult learning pedagogies such as role play are under-utilized in medical education, and have been shown to be as effective as traditional teaching methods such as lecture. an -module course for fellows in hematology/oncology, hospice and palliative medicine, radiation oncology, and pediatric hematology/oncology was implemented in january/february . fellows participated in the program. topics covered including fundamentals of communication, coping and spirituality, delivery of bad news, communicating with families, sexual dysfunction during treatment, palliative care/death and dying, and burnout. objectives: overall goal of this course is to foster holistic physicians who views their patients as people with cancer, not cancer patients, and physicians that can communicate effectively with their patients throughout the disease continuum. by the end of the course, learners should be able to practice the fundamental principles of good communication. design/method: fellows initially participated in a pre-course osce to establish baseline skills. osce was facilitated by the center for learning and innovation at northwell, and included actors portraying a pediatric patient and family member to whom the fellow had to break bad news. two months later, the course was carried out over the span of eight weeks and included didactic sessions followed by minutes of role play scenarios. five of the eight modules included role play, with faculty members serving as simulated patients. after the course, a second breaking bad news osce was held. both osces were filmed, and feedback was given by the on-site actors. additionally, faculty members were given access to the videos in an on-line format and were given an evaluation tool to assess the fellows' performance pre-and post-intervention. fellows were given subjective surveys pre-and post-course as well. results: subjective data from participants showed a noticeable increase in comfort level in all areas on the pre-and post-course survey. data obtained from osce videos showed improvement in communication skills as assessed by sps and faculty members using a new evaluation tool developed by faculty. initial first-run data shows that this course is successful in improving communication skills as well as increasing fellows' comfort level across several domains of communication. future directions for our course include improving and validating our assessment tool, expanding our topic base to include more aya and pediatric scenarios, faculty development for improved role play, and investigating impact on practice after course completion. background: acute lymphoblastic leukemia (all) is the most common form of childhood cancer with approximately children diagnosed each year. survival rates have improved significantly over the past several years. children with all are at risk for developing musculoskeletal complications during and after completion of treatment, which can contribute to impaired activity, elevated body mass index (bmi), and risk for complications. interventions involving physical activity could improve musculoskeletal strength as well as overall health in these children. the aims of this study are to examine the feasibility of a directed physical activity program for children with newly diagnosed all during the initial intensive phase of therapy and to evaluate the overall health and quality of life of children participating in the directed physical activity program. design/method: all subjects will receive education materials about the importance and safety of physical activity and a nutrition handout. all subjects will also participate in the directed physical activity program under the supervision of a trained physical therapist for at least minutes every week for weeks. the program will entail four stations including a cardiovascular, balance/proprioception, strength and flexibility, and coordination and cardio. feasibility will be assessed by tracking the participation rate throughout the study period. other assessments will be made at study entry, at the end of weeks of physical activity initiative and months after completion of the intervention. assessments include overall strength and flexibility, weight, height, bmi, blood pressure and performance scores. descriptive statistics will be used for this study. results: a total of patients, male and female, enrolled in the study over a . month period. patient ages ranged from - years. half of the patients enrolled have completed the week program and all patients had stability or improvement of their physical functioning scores. further data collection and analysis is ongoing. patients in the early intensive phase of all therapy are at risk for complications that can affect their physical functioning. a directed physical activity protocol may improve their overall physical functioning. patients may not need specific physical therapy; however a directed physical activity program appears to be beneficial for these patients. the main roadblocks to successful completion of the program were difficulty with scheduling, strain on the parents and patient from treatment, unplanned admissions for fever, as well as nausea and fatigue at time of visit. albany medical center, albany, new york, united states background: communication skills are a core competency highlighted by the acgme. increasing resident confidence in delivering difficult news has been shown to lead to more s of s effective communication. currently, the majority of residency programs lack formal training in communication skills. our objective was to demonstrate feasibility and efficacy of integrating a standardized-patient based training program for communication skills into the curriculum of pediatric residents design/method: to date, pediatric and medicine/pediatric residents have participated in the program during the intern year. the program consists of three, two-hour long sessions, in which each resident is given several opportunities to act out case scenarios with a standardized patient. scenarios included informing a parent of their child's new cancer diagnosis and disclosure of a positive hiv test to a teenager. residents received post hoc peer to peer, and preceptor to learner feedback. pre and post-program surveys were completed by residents. results: following course completion residents reported an increase in confidence in multiple areas of communication including giving a difficult diagnosis (p< . ), discussing a poor prognosis (p< . ), responding to different patient/family member emotional responses i.e. crying or anger (p< . ), and organizing vital information to be relayed (p< . ). in conclusion, communication skills training of pediatric residents is feasible and provides a platform for developing valuable skills not taught elsewhere within the curriculum. background: for children with cancer, transitioning back to school during or after treatment can be challenging. literature supports the need for school re-entry programs to ease this transition. however, these programs vary widely among pediatric cancer institutions with little data addressing their program components. data from this study provides information on current school re-entry programs across these institutions. objectives: one objective of this study was to assess for correlation between the presence of a school re-entry program and other factors, such as geographic location and institution size. a second objective was to establish a list of differences between institutions' school re-entry program components. finally, we aimed to describe current school reentry practices, as well as program benefits and perceived areas for improvement. states with membership in the children's oncology group were offered enrollment in this study. a member of each institution was invited to participate in a survey established by the research team. this person was closely associated with the institution's school re-entry practices. each interview queried institution demographics, as well as program components (e.g., participants, target audience, resources). comment was also collected on program benefits and potential for improvements. analysis of transcripts was performed using pearson's correlation to assess for relationships between institution size, geographic location, and program presence. grounded theory was used for analysis of benefits and improvements. results: thirty-nine of forty-one pediatric institutions who were offered enrollment participated in this study. twentynine institutions ( %) indicated the presence of a school reentry program, and ten ( %) stated they had none. no correlation was found between institution size and the presence of a school re-entry program (p = . , ns). there was also no correlation found between institution location and the presence of a school re-entry program (p = . , ns). a major theme surrounding the benefits of having a program included education for the returning student's peers. for those with programs, perceived improvements included increasing staffing and the ability to offer more services. the results do not support the hypothesis that the presence of a school re-entry program is influenced by the size and geographic location of the treating institution. however, data seem to suggest that available staffing may influence the presence of a program. future studies are needed to address other potential influences, as well as to take an evidence-based approach to determine the effectiveness of the interventions present in these programs. cohen children's medical center/ zucker school of medicine at hofstra-northwell, new hyde park, new york, united states background: genetics/genomics is evolving at an extremely rapid pace. current advances lead to individual algorithms toward disease treatment for each disease with multiple branch points. fellows learn only a fraction of the knowledge and there is no formal approach to teaching critical analysis of information and application algorithms toward disease. additionally, as knowledge evolves extremely rapidly, any approach must teach self-acquisition and application of evolving discoveries. objectives: to create, implement and evaluate a novel curriculum for genetics/genomics targeted toward pediatric hematology/oncology fellows design/method: the curriculum includes four components: ) genetic and genomic medical knowledge, with one initial team-based learning session and weekly online multiple choice questions; ) essential pathways, which will teach molecular pathways common in oncogenesis and relevant to targeted therapy in microteaching sessions with using auditory, visual and tactile learning; ) knowledge acquisition and clinical judgment, to allow learners to gain experience into researching data available, then developing and prioritizing potential treatment plans using problem-based learning sessions in which they will stage a patient, research treatment options, prioritize and present findings; and ) synthesis to demonstrate independent ability to research and recommend therapy through an independent project in which the learner, given a case, will present the case and research findings, genetics/genomics, molecular pathways and make recommendations for therapy in molecular tumor board for faculty and fellows. to evaluate, we plan to recruit to institutions, match for size of programs and implement in half and evaluate nd and rd year fellows in both groups by mcq exam and satisfaction surveys. the creation of a multi-module, adult-learning based curriculum for genetics and genomics in pediatric oncology is feasible. implementation and evaluation are necessary to demonstrate efficacy. background: neuroblastoma is the most common extracranial solid tumor in children. chimeric anti-gd antibody ch . (dinutuximab) therapy has improved the survival of children with newly diagnosed high-risk, neuroblastoma patients as well at the time of first relapse/progression. acute neuropathic pain is a well-documented side effect of dinutuximab administration. however, additional adverse effects including sensorimotor neuropathy, ocular symptoms, and behavioral changes have been described. the incidence and severity of these effects are currently not well-documented in pediatric patients. with improved long term survival of patients receiving this modality, it is important to look for the potential late effects of dinutuximab. objectives: to determine the incidence and severity of neurologic, ophthalmologic, or behavioral changes after dinutuximab administration at our institution. we performed a retrospective chart review using our electronic medical record. we included all patients with high-risk neuroblastoma between the ages of and years at our institution diagnosed between and who received dinutuximab. patients with history of opsoclonus-myoclonus syndrome or gross sensorimotor neuropathy prior to receiving dinutuximab were excluded. we examined clinical documentation for subjective reports and objective exam findings of neurologic, ophthalmologic, or behavioral changes. we also looked for referrals made to neurology, ophthalmology, physical medicine & rehabilitation (pm&r), and psychology. : twenty-two patients met inclusion criteria. at the time of chart review, patients were alive and were deceased. eighteen patients received dinutuximab per anbl ; patients received dinutuximab per anbl . of these patients, patients reported symptoms of interest and reported multiple symptoms. six patients reported symptoms that began at least months after completing dinutuximab. nine patients had objective findings on exam, including decreased deep tendon reflexes, abnormal pupils, and nearsightedness. for patients, referrals were made to ophthalmology, pm&r for neuropsychologic testing, or neurology. two patients who reported symptoms of interest were not referred to a specialist. conclusion: neurologic, ophthalmologic, and behavioral symptoms were commonly reported and demonstrated on exam among pediatric patients with high-risk neuroblastoma who received dinutuximab. it is important to identify these effects so that appropriate specialist referrals can be placed for adequate management of these changes. we recognize that these symptoms may not be solely due to dinutuximab as these patients receive other agents including opioids, so a prospective trial is needed to further evaluate the long-term effects of dinutuximab and to determine how best to screen for these effects. akron children's hospital, akron, ohio, united states background: pediatric cancer is the leading cause of diseaserelated death in children in the united states (u.s.). in , over fifteen thousand children were diagnosed with cancer in the u.s. this population is at high risk for malnutrition due to the multimodal therapies they receive: surgery, chemotherapy, radiation therapy, antibody therapy, and/or bone marrow transplant. adverse effects of these therapies include taste changes, loss of appetite, diarrhea, vomiting, and/or mucositis, making it difficult for the children to be able to consume adequate amounts of nutrition during therapy. there is no "gold standard" measurement tool for identifying patients at risk for malnutrition. nutritional status is not frequently evaluated as a component of clinical trials. assessment of anthropometric measurements (weight, height, z-scores) at diagnosis, as well as over the duration of treatment, can assist in the early identification of malnutrition. the incidence and prevalence of malnutrition in this population is unknown at akron children's hospital. the purpose of this study is to describe the nutritional status and provision of nutritional support therapies in pediatric patients during their first year post new oncologic diagnosis. objectives: identify the incidence and prevalence of malnutrition across oncologic diagnostic categories over the first twelve months post diagnosis. we performed a retrospective records review of all patients newly diagnosed with cancer in at akron children's hospital. demographic and anthropometric data was collected at time of diagnosis and nutritional status categorized by z score. anthropometric and nutrition support data was then collected every two months for the first year after diagnosis along with incidence of unplanned inpatient admissions. results: a total of patients were included in the analysis, with . % malnourished at time of diagnosis; . % developed malnutrition the first year. patients with solid tumors represented % of patients with pre-existing or acquired malnutrition. overall, % of patients received at least one nutritional support modality. patients with pre-existing or acquired malnutrition had a non-significant increase in unplanned admissions (p = . ). our study demonstrated that patients with solid tumors were found to be at increased risk of pre-existing and acquired malnutrition, followed by leukemias, and experienced higher incidence of unplanned admissions in the time period observed. prospective, multi-center replication of this study, including detailed collection of nutrition therapies is recommended to guide development of diagnosis specific nutrition support guidelines. background: pediatric and young adult oncology patients treated with intense chemotherapy have a high incidence of transfusional iron overload. iron deposition can lead to heart failure/arrhythmias, liver abnormalities, endocrine dysfunction, ineffective erythropoiesis, and increased cancer and mortality risk. however, there is a paucity of data regarding recommendations for management of transfusional iron overload in these cancer survivors. consequently, long-term complications of transfusional iron overload specific to these patients have not been assessed. objectives: to assess screening and phlebotomy-based treatment algorithms for this population. design/method: a retrospective chart review of pediatric and young adults who completed oncology management, had iron overload, and initiated phlebotomy treatment was conducted. tiered screening occurred in patients that received at least packed red blood cell (prbc) transfusions. patients were recommended for evaluation and possible phlebotomy if: ( ) liver iron concentration (lic) > mg of iron/gram dry weight liver tissue by ferriscan and/or ( ) cardiac mri t * < ms. during phlebotomy, iron status was assessed quarterly and phlebotomy discontinued with lic < or normalization of ferritin/imaging lic verification. descriptive statistics were employed to report the characteristics of the study population. spearman correlations were utilized to describe associations between transfusions, lic, ferritin, iron saturation and number of phlebotomy sessions. results: twenty five survivors underwent phlebotomy. the mean age was . years (sd . ) and ( %) were female. oncologic diagnoses: all ( %), aml ( %), nhl ( %), ewing sarcoma ( %), osteosarcoma ( %), neuroblastoma ( %) and cns ( %). patients received a median of . (iqr - ) transfusions. median number of phlebotomy sessions was (iqr - ) over . years (iqr . - . ). prior to phlebotomy, median lic was . mg/g (iqr . - . ) and ferritin was . ng/ml (iqr - ) . no patients demonstrated abnormal cardiac t * mri (n = ). ( %) patients completed phlebotomy. one discontinued due to poor vascular access. no patients developed iron deficiency. lic was reduced by a median of . mg/g (iqr . - . ) and ferritin by ng/ml . correlation between number of transfusions and phlebotomy sessions was poor (r = . ). conclusion: management guidelines are lacking for transfusional iron overload in pediatric and young adult survivors of cancer. we demonstrate a phlebotomy algorithm that is effective and tolerated. correlation between number of transfusions received and phlebotomy treatments was poor, necessitating serial assessments. using this management algorithm, prospective studies can evaluate the effect of iron removal on iron overload complications in this patient population. penn state children's hospital, hershey, pennsylvania, united states background: cancer therapy leads to an impaired immune system that takes time to recover. it is important to ensure that these survivors have adequate immunity to prevent common yet potentially severe childhood illnesses. no validated guidelines currently exist for surveillance testing or re-immunization in this population. retrospective analysis involving a small cohort of pediatric cancer patients treated at penn state children's hospital showed % of patients screened for varicella immunity after therapy completion did not have adequate disease titers. to determine the proportion of pediatric cancer survivors who have lost humoral immunity to previously received vaccines; to determine the rate of response to single dose boosters or full vaccine series in seronegative subjects after one booster. design/method: pediatric cancer survivors treated at the children's hospital who are at least months from completion of cancer therapy are prospectively tested for antibody levels to hepatitis b, tetanus, varicella, measles, and strains of pneumococcus ( , b, v, c, f, and f). samples are analyzed by the cdc for measles and varicella avidity. seronegative subjects by commercial studies, are eligible to receive booster vaccines. titers are rechecked at least weeks after boosters to re-evaluate immunity; if still seronegative, subjects will receive the entire vaccine series. titers are finally tested at least weeks after the final dose of the vaccine series. immunity analyzed after therapy, after boosters, and after vaccine series. results: of pediatric cancers survivors who completed therapy, % were non-immune to hepatitis b, % nonimmune to > % of pneumococcal strains tested, % nonimmune to measles, % non-immune to varicella, and % non-immune to tetanus. of subjects who received mmr vaccine after therapy and prior to study enrollment did not have protective antibodies to measles. of the subjects who received varicella vaccine after end of therapy and prior to study enrollment, did not maintain protective antibody levels. cdc results for measles and varicella are pending, as well as repeat studies after vaccine boosters and series. conclusion: a significant percentage of pediatric cancer survivors do not retain immunity to hepatitis b, pneumococcus, measles, and varicella. after one booster, a high percentage of subjects did not develop protective immunity to varicella. only subject did not have immunity to tetanus, which is consistent with the high immunogenicity of tetanus toxoid. formal guidelines are needed to protect this population from vaccine-preventable illness post-therapy. children's hospital of richmond at virginia commonwealth university health system, richmond, virginia, united states background: childhood cancer survivors are at risk for being overweight. diet and physical exercise are important in maintaining a healthy lifestyle and weight; however, it has been reported that cancer survivors are less active than their peers. one reason for this may be that there are no clearly established risk-based exercise recommendations for cancer survivors. another reason may be that providers tend to focus s of s recommendations for exercise more towards patients who are overweight. objectives: to describe changes in physical fitness of childhood cancer survivors who exercise. design/method: 'moving forward' is a wellness and physical fitness program that the center for care beyond the cure at chor offers in partnership with the ask childhood cancer foundation and the ymca. the program is available for any childhood cancer survivor between y and y age, being seen at our center. survivors define their fitness or wellness goals and then work with a trainer once a week (at least) for min sessions throughout the year to achieve these goals. baseline and ongoing measurements for core strength, endurance, overall strength and balance were collected. the average of each of the parameters of all participants were compared from the beginning to the end of the program. over the year, there was a % increase in endurance as measured by the average of the miles walked in minutes, % increase in core strength as measured by the average number of sit-ups in secs, an % and % increase in overall strength as measured by the average weight lifted by leg press and the average weight lifted by chest press, and a % increase in balance as measured by the average number of seconds balancing on a single leg. in addition, each child had actually gained weight in the process with an approximately % increase in the average of the weights of all children. there are benefits to regular exercise beyond weight control, and improvements in physical fitness can be seen even without weight loss. regular physical exercise results in improved physical fitness and should be universally advocated to all patients. determining insulin resistance, measuring changes in fatigue and wellness perception following exercise are future directions that we intend to explore. dana-farber cancer institute, boston, massachusetts, united states background: improvements in adolescent and young adult cancer patient (aya) survival rates and quality of life outcomes have lagged behind those of children and older adults, highlighting a need for research targeting this unique population. current literature supports the value of strong ayaclinician communication, notably in facilitating therapeutic alliance, however little is known about aya communication priorities during cancer care and barriers to optimal ayaclinician communication. objectives: to explore aya and oncology clinician communication priorities and to identify barriers and facilitators to aya-oncology clinician communication. design/method: semi-structured interviews were held with aya cancer patients and survivors (ages - years) from a single large academic institution and oncology clinicians (physicians and nurse practitioners) from academic institutions in the northeastern united states. interviews were conducted in english by phone or in person. all interviews were audio-recorded and transcribed verbatim. analyses were aided by nvivo software. ayas identified a wide range of topics as important to discuss with clinicians. the most frequently identified topics were ) side effects of treatment (with an emphasis on physical appearance and function, n = ), ) social issues (including friendship, family, and school, n = ), ) looking ahead to the future (n = ), and ) sexual & reproductive health (including future fertility, contraception, and romantic relationships, n = ). clinicians prioritized ) cancer treatment and side effects (n = ), ) emotional and psychological health (n = ), and ) sexual and reproductive health with a focus on fertility risk and fertility preservation (n = ). aya reported facilitators to good communication including an open and long-established relationship with the clinician (n = ) and clinician engagement in age-appropriate and patient-directed conversations (n = ). barriers included parental presence during visits (n = ). clinicians reported barriers including ) clinician discomfort (not feeling wellequipped to discuss psychosocial topics such as sexual health, spirituality, and relationships with peers, n = ), ) presence of parents/family (n = ), and ) perceived patient discomfort discussing specific topics (such as sexual health, n = ). clinicians acknowledged the need for collaborative efforts with additional team members (i.e. nurses, psychosocial providers) to assist in meeting aya communication needs. conclusion: aya and clinician-reported communication priorities are largely aligned. however, ayas emphasize some topics, such as social function, appearance, and sexual health that are not highly prioritized by clinicians, which may result in gaps in care for ayas in treatment and in survivorship. these data identify opportunities for intervention, including clinician education, patient and family education, clinic-based intervention, and systems-based changes that can be developed and tested. background: primary care physicians (pcps) cite lack of knowledge and inadequate communication with the oncology team as major barriers to providing recommended surveillance for late effects of treatment to childhood cancer survivors. a standardized telephone handoff to pcps posttherapy is a potential strategy to increase survivorship care by pcps through interactive communication. to determine the feasibility of a structured telephone communication using the situation, background, assessment, and recommendation (sbar) communication tool delivered by a trained oncology nurse to increase pcp knowledge and willingness to provide survivorship care. design/method: from / / to / / , a registered nurse expert in childhood cancer survivorship attempted to contact by telephone the pcps of the most recent patients attending yale's childhood cancer survivorship clinic that were < years old, english-speaking, and ≥ years posttreatment. all pcps had been previously sent an individualized survivorship care plan (scp) that listed the patient's previous treatment history and recommended surveillance tests. upon successful contact and after confirming receipt of the scp, the nurse explained the definition of late effects, description of patient's diagnosis and treatment history, and associated potential late complications and schedule of recommended surveillance tests. the pcp was also asked about his/her ability and willingness to provide needed surveillance for late effects in the future. overall, of pcps were successfully contacted with a median of phone call (range: - ) that lasted a median of minutes (range: - ) after a median of business day (range: - ). no pcps ended the call mid-conversation. all pcps were receptive and expressed appreciation for the call. twenty-five of ( %) pcps expressed an understand-ing of the material discussed and endorsed belief in their ability and willingness to provide late effects surveillance for their patients. no pcps questioned discussing their patient's care with a nurse versus a physician. interactive, structured communications between nurses and pcps by telephone are feasible and are associated with high-levels of pcp confidence in providing survivorship care. background: childhood cancer (cc) admissions account for % of non-newborn pediatric hospitalizations. these hospitalizations are longer and more expensive than other hospitalizations. admission payer (medicaid or commercial) reflects both health policy and sociodemographic status. the objective of this study was to determine if length of stay (los) or cost of cc admissions differed by payer. we used the kids inpatient database, a sampling of all pediatric hospital discharges in the united states. analysis for this study was limited to admissions containing a cancer diagnosis in any discharge icd- codes. admissions were further subcategorized by discharge codes according to diagnosis (leukemia, lymphoma, solid tumor and brain tumor) and reason for admission (chemotherapy, procedure, infection, non-infectious toxicity or "other"). charges were converted to costs using cost-to-charge ratios. multivariable linear regression models were performed to control for age, gender, race, reason for admission, and diagnosis. results: there were , weighted admissions for children with a cancer diagnosis in . of these admissions, . % had medicaid, . % had commercial insurance, and less than % had other payers. the mean los for medicaid admissions was . days ( % ci . - . ), compared with . days ( % ci . - . ) for commercial insurance. surgical admissions accounted for the largest difference in length of stay with medicaid admissions being . days longer than those covered by commercial insurance ( . days vs . days), however, the difference was significantly different for all reasons for admission. in multivariable analysis admissions associated with commercial insurance were % shorter s of s (p< . ), accounting for approximately one hospital day, than admissions associated with medicaid after controlling for other variables including race. the mean overall cost for medicaid admissions was $ , ( % ci - ), compared with $ , ( % ci - ) for commercial insurance. in the multivariable model, cost was collinear with race. conclusion: los and cost of admissions associated with medicaid differed from those associate with commercial payers. medicaid admissions were % longer on average than commercial insurance, accounting for a difference in length of stay of approximately one day although the difference varied with the reason for hospitalization (chemotherapy, surgical procedure, infection, other toxicity, other). costs of admissions were not independent of race. further investigation into potential explanations for this difference including differential access to home care needs, outpatient reimbursement differences, social indications for prolonged hospitalization, and provider biases, is warranted. background: pediatric cancer is a major cause of morbidity and mortality among children surpassed only by accidents. despite improved outcomes in high income countries (hic) survival rates remain poor in the developing word. there are various diagnostic and therapeutic limitations contributing significantly for the survival gap. the main objective of the study is to to evaluate the outcomes of pediatric cancer in armenia and identify diagnostic and therapeutic limitations in the country. we conducted a retrospective study among (≤ years old) children with cancer (solid tumors and hematological malignancies), who were diagnosed and treated at the clinic of chemotherapy of muratsan hospital complex of yerevan state medical university between and . those patients, who didn't receive chemotherapy for any reason were not included in the study cohort. epidemiological, social, medical information was collected through the patient charts review. this included patient age at diagnosis, sex, place of residence (city vs village), the educational level and employment status of parents, type of cancer, stage, presentation of symptoms, first medical specialty consulted and the time consulted, initial work-up, the type of treatment received, information on the diagnosis/treatment received abroad. results: at our clinic during the mentioned period of time the majority of patients presented with hematologic malignancies- %. ( . %) patients had information on diagnosis delay. average delay in diagnosis was about days. in % of cases the first contact with "healthcare system" was through pediatrician, and in % with surgeon. out of relapsed patients received salvage treatment in armenia and abroad. from those who stayed for treatment in armenia patients survived. majority of relapsed patients had acute lymphoblastic leukemia. from leukemia patients immunophenotyping and cytogenetics were available for ( . %) patients; the majority of missing cases were between and , when these diagnostic modalities were not available or affordable in the country. ( %) patients received part of diagnosis and/or treatment abroad. the most frequent reason for going abroad was bone marrow transplantation, otherwise none available in armenia. out of patients were lost to follow-up, patients had a fatal outcome. patients were in remission at a median follow up of . years. conclusion: unavailability of cancer registry and several essential diagnostic/treatment modalities, luck of multidisciplinary care and palliative support, high rate of out-of-pocket expenses were among the main challenges of pediatric cancer care in armenia. background: adverse drug reactions (adrs) are increasingly recognized as important and sometimes irreversible complications of cancer treatment. anthracyclines and cisplatin are effective chemotherapeutic agents, but their use can be limited by cardiotoxicity (anthracyclines) and ototoxicity (cisplatin) in up to % of patients. genetic variants that can be used to predict who is most at risk of developing these adrs have been discovered and replicated. objectives: to create pharmacogenetic risk prediction models for anthracycline and cisplatin toxicities and discuss results with oncologists to facilitate incorporation into treatment decision-making when appropriate. design/method: risk prediction models were developed from the linear regression of strongly-predictive genomic variants (odds ratios ≥ ) discovered and replicated in at least three patient populations. these models were used to assess an individual patient's genomic risk of developing cardiotoxicity from anthracyclines or hearing loss from cisplatin. risk results were returned to oncologists showing where the specific patient's genetic risk of toxicity lies on a continuum between the lowest and highest risk groups across all studied patients using a multi-gene model. interviews were conducted with patients, families, and oncologists to determine how results were valued and utilized. results: patients have been genotyped and had their genetic risk results returned to their oncologists. the first patients have been characterized to determine the impact these test results have had on their clinical care. results were described as being useful in decision-making by patients and/or oncologists in % of cases. additionally, for patients in the most extreme risk groups (highest and lowest risk), a change in treatment plan was ordered % of the time for cisplatin patients and % of the time for anthracycline patients. this included increased cardiac and audiological monitoring, the addition of a protective agent, or choosing an alternative treatment protocol if the risk outweighed the benefits of remaining on the current treatment plan. in interviews, patients indicated that they felt more involved in decision making, and felt reassured by understanding their genetic risk of toxicities. genetic risk prediction models for anthracycline cardiotoxicity and cisplatin ototoxicity were highly utilized by patients and oncologists in decision-making. results were found to be an important tool for informing patients of the risk of adrs during cancer treatment, and resulted in patients and their families feeling more involved in decision-making. background: childhood cancer survivors are at increased risk of developing executive dysfunction, and low socioe-conomic status (ses) has been identified as one of the mediators of executive functioning. previous studies have used traditional measures of ses, such as parents' education level, family annual income and occupation. but more recently, area based socioeconomic measures like block group poverty status are deemed to be more useful in monitoring of social inequalities in health in the united states. block groups are statistical divisions of census tracts and generally contain between and , people. the current study aims to understand the association of block group poverty status (percentage of households in family's block group of residence living below the federal poverty level) with executive functioning among cancer survivor children. design/method: we used a retrospective cohort of childhood cancer survivors. relevant information was collected from the medical record, administrative data sets and parent-filled surveys. address information was geocoded using arcgis . to obtain data on the block group poverty status. a priori cut-points were set to represent block groups with families living below poverty level at %, . % to . %, and ≥ . %. executive functioning were assessed through a parent-rated instrument, the behavior rating inventory of executive functions (brief). multiple linear regressions were used to determine the relationship between block group poverty status and the brief scores. results: data was examined from families of childhood cancer survivors, ranging in age from to years. in this sample, . % families reported an annual income <$ , , . % reported income between $ , and $ , while . % reported annual income ≥$ , . primary care giver of . % of cancer survivors had more than more high school education, and . %, . % and . %, of families were living in a block groups with %, . - . % and ≥ % poor households respectively. block group poverty level was not significantly associated with annual income levels (spearman's rho = . , p = . ), or parental education level (spearman's rho = - . , p = . ). in a step-wise multiple linear regression, there was no statistically significant association seen between block group poverty status and executive functioning after adjusting for co-variables in the final model. future prospective study with a bigger sample size, longer follow up period and more robust measures of the executive functioning like a clinician administered test are needed to understand the effect of block group poverty status on executive functioning. to d completion was . days (range - ). all parents strongly agreed/agreed that d was helpful and would recommend d participation to another family. ten parents ( %) reported time spent on d was "just right." no parent felt more worried due to the intervention, though parent found d participation stressful. this interim analysis suggests that parents have a favorable d experience and recommend the intervention. to date, < % of enrolled parents fail to participate. d shows promise as an acceptable interdisciplinary communication intervention targeted to the early treatment period for childhood cancer. children 's hospital and research center oakland, oakland, california, united states background: screening echocardiograms are recommended by children's oncology group (cog) guidelines to assess for anthracycline-induced left ventricular (lv) systolic dysfunction. the yield of screening echocardiograms during chemotherapy and in the immediate post-therapy period is uncertain. objectives: to assess the incidence of lv dysfunction detected by screening echocardiograms during chemotherapy and in the immediate post-therapy period, defined as - months off-therapy. design/method: children diagnosed with cancer between january -march who received anthracycline chemotherapy were identified. echocardiograms were performed as per protocol, institutional and cog guidelines, and were reviewed retrospectively. lv dysfunction was defined as fractional shortening (fs) < % or ejection fraction (ef) < % ( ) results: in this cohort (n = , median age years), the most common diagnosis was all ( . %), followed by aml ( . %). of echocardiograms, ( . %) were performed during treatment and in the immediate posttreatment period. thirty-eight ( . %) patients had a > % decrease in fs compared to their pre-treatment echocardiograms. none of these patients required any treatment modification or cardiac medications. only patient ( . %) had echocardiogram-proven lv dysfunction discovered on a screening echocardiogram during her treatment course. she eventually died due to multi-organ failure following septic shock. this patient was receiving treatment for aml and had received mg/m of doxorubicin-equivalent anthracyclines at the time of the abnormal echocardiogram. one patient with metastatic ewing sarcoma had borderline lv dysfunction with a fs of % detected a month before completion of therapy. she had received mg/m of doxorubicin equivalent anthracyclines at the time of the abnormal echocardiogram. she did not require any therapy modification or additional cardiac medications. serial echocardiograms done on this patient have shown stable ventricular function. no off-therapy screening echocardiograms identified lv dysfunction. in our experience, the yield of echocardiograms to detect anthracycline-related cardiac dysfunction during treatment and in the immediate post-therapy period is very low. one patient developed lv dysfunction during treatment and one had borderline fs, while no lv dysfunction was identified within months of completing chemotherapy. though fs decreased in % of patients, none required intervention. further study is needed to optimize the use of echocardiography screening in children treated with anthracyclines. references: . landier w et al. jco . background: platinum-based chemotherapy increases the risk of sensorineural hearing loss in children with cancer. little is known about the impact of hearing loss on cognitive and emotional functioning in survivors. to determine the association of severe/profound hearing loss after platinum-based chemotherapy with ) cognitive impairment and ) emotional distress (i.e. anxiety and/or depression). cross-sectional study of all patients attending yale's childhood cancer survivorship clinic ≥ years off therapy for cancer diagnosed at < years and treated with cisplatin and/or carboplatin, but with no history of cns tumor, cranial radiation, congenital hearing loss, or developmental delay. hearing loss severity and hearing aid data were abstracted from audiograms and detailed clinical history. cognitive impairment was defined as behavior rating inventory of executive function t score ≥ , assessment by neuropsychologist, and/or history of special education. emotional distress was determined by brief symptom inventory t score ≥ (global or two subscales) or behavioral and emotional screening system t score ≥ , psychologist interview, and/or history of psychotropic medication/psychotherapy. the most recent available patient data were used. logistic regression with sas software, version . was performed. results: overall, patients ( % female, % white) met eligibility criteria with a median age of . years (iqr = . ) at diagnosis and . years at evaluation (iqr = . ) after a diagnosis of sarcoma ( %), neuroblastoma ( %), or other ( %) for which % received cisplatin and % received carboplatin. fifteen patients ( %) had severe/profound hearing loss in at least one ear. patients with severe/profound hearing loss had a significantly increased risk of cognitive impairment (or = . ; % ci = . - . ), but not emotional distress, compared to patients without severe/profound hearing loss. there was no significant association between age at diagnosis, current age, time since diagnosis, sex, race, ethnicity, or diagnosis with either cognitive impairment or emotional distress. similarly, there was no significant interaction between ) age at diagnosis and hearing loss or ) sex and hearing loss with either cognitive impairment or emotional distress. ten of the ( %) patients with severe/profound hearing loss in at least one ear were recommended hearing aids, of which ( %) reported compliance most of the time. we conclude that severe/profound hearing loss is significantly associated with cognitive impairment, but not emotional distress, in childhood cancer survivors. our data supports the need for interventions to improve hearing in these patients, including compliance with hearing aids. background: who grade anaplastic astrocytoma is a high grade glioma dependent on vascular endothelial s of s growth factor (vegf) mediated angiogenesis for its growth and infiltration. bevacizumab is a recombinant humanized monoclonal antibody which binds vegf-a and inhibits angiogenesis. common adverse effects of bevacizumab are hypertension, proteinuria, thrombosis and bleeding. while animal model based studies have shown that bevacizumab may impair ovarian function the effects of bevacizumab therapy on human fertility are not clear. since the physiology of pregnancy involves neovascularization/angiogenesis it is recommended that conception be avoided for at least months following exposure to bevacizumab. to describe the course of a young adult who became pregnant after receiving bevacizumab and radiation therapy for treatment of an anaplastic astrocytoma. a year old woman diagnosed with a localized hemispheric who anaplastic astrocytoma was treated with chemotherapy and radiation (temozolomide/ . gy) followed by cycles of bi-weekly bevacizumab/temozolomide. patient opted not to pursue fertility preservation prior to initiation treatment. she experienced bevacizumab-associated proteinuria and hypertension during treatment but received all protocol mandated doses (cumulative doses: bevacizumab = mg/kg; temozolomide = . gm/m ). she had a spontaneous unassisted pregnancy months after completing treatment. her pregnancy was uneventful and she was normotensive throughout. fetal ultrasonography at , , , weeks revealed no abnormality of the brain, heart, great vessels, kidney, extremities, placenta and umbilical cord. at weeks she delivered a female infant via cesarean section (birth weight: grams, apgars: and ) excessive post-partum hemorrhage was not reported. placenta was bi-lobed and weighed g. histological analysis revealed normal placental villous development and maturation and two small infarcts. conclusion: exposure to bevacizumab in our patient had no detrimental effect on fertility and on placental/fetal vascular development. we hope this report will add to the existing data on the effects of bevacizumab therapy on fertility. children's healthcare of atlanta, emory university school of medicine, atlanta, georgia, united states background: reports of malnutrition incidence and prevalence in young cancer patients are variable and not well established. previous research suggests children, especially less than years old, treated with intensive cancer-directed therapy are at higher risk for malnutrition. however, no standardized assessment has been used to evaluate risk in this population. objectives: we aim to assess the trends of weight-for-age for patients following cancer diagnosis. this study will be the first to use a standardized measure of treatment intensity (intensity treatment rating scale, itr- ) and will assist in targeting interventions for identification and treatment of malnutrition. design/method: this observational, retrospective study obtained data through the center's pediatric cancer registry and electronic medical record. patients were classified by tumor type (brain or non-brain tumor) and treatment intensity (itr- ). itr- incorporates diagnosis, chemotherapy, radiation, and surgery, beginning with lowest intensity ( ) to highest intensity ( ). inclusion criteria included new cancer diagnosis - at less than years old, with weight obtained and available within days of therapy start date. incomplete data, alternate growth charts, or treatment intensity of , were excluded. weight was obtained at start of therapy and through years after treatment initiation (approximately days) and converted to z-scores adjusted for age and sex. weight trajectories were modeled using generalized linear mixed models with subject-specific random intercepts and spline functions. separate functions were constructed for subgroups of interest (tumor type and itr). results: there were patients included: patients with brain tumors ( . %) and with non-brain tumors ( . %). of included patients, had treatment intensity of ( . %), of ( . %) and of ( . %). over the observation period, , valid weights were recorded. at initiation of treatment, no difference existed between z-score by tumor type (p = . ) or by intensity ( vs. , p = . ; vs. , p = . ; vs. , p = . ). tumor type did not affect z-score through the follow up period. z-scores were higher for intensity rating vs. and vs. (p = < . and p = . respectively) at days after the start of treatment and persisted through days (p = . and p< . respectively). higher treatment intensity is associated with decline in z-score and failure to return to baseline. future directions include further analysis on specific risk factors and timing of weight loss, longer-term follow-up of weight trends, and targeted interventions for identification, prevention, and treatment of malnutrition. objectives: asses the pt requirements for bleeding episodes in a prospective cohort of pcp using a < × e threshold compared to a < × e /l threshold in a historical cohort. we collected pt data in all pcps treated at our center between january/ through december/ . diagnosis, prescription for pt (prophylaxis vs bleeding disorder), plt count and transfused units were assessed for each pt. pcps treated from january/ through june received prophylactic pt with a < × e threshold (cohort a), and pts treated from july/ through december/ received prophylactic pt with a < × e threshold. pts done for procedures and pts with concomitant hemorrhagic pathology were excluded. we compared the number of pts prescribed as prophylaxis vs bleeding episode between cohorts. data analyzed: graphpad prims . ®. statistical analysis: percentages with confidence interval (ci); t-student test (parametric variables) and mann-whitney test (nonparametric variables). statistical significance: p< . . we reviewed pts ( in cohort a, cohort b) in patients. % had acute leukemia, % received and auto or allo hsct. diagnoses and the proportion of patients undergoing hsct was comparable in both cohorts. the average number of pts per patient was , in cohort a and , in cohort b (p = ns), but a significant difference was found when hsct patients were excluded from this comparison ( , pt per patient in cohort a vs , in cohort b, p = , ), which resulted in an estimated , % reduction in pts prescription. furthermore ( , %) pts were prescribed for bleeding episodes in cohort a versus ( , %) in cohort b (p = ns). patients receiving hsct in the entire group ver-sus those not receiving hsct had similar pt requirements for bleeding episodes ( % vs , % p = ns) conclusion: a < × e plt count threshold for prophylactic pts is safe in pcp in chemotherapy and hsct. it can result in a significant reduction in pt usage. key words: platelets, transfusions, prophylaxis, cancer, childhood. ucsf benioff children's hospital oakland, oakland, california, united states background: transition of care for adolescent and young adult (aya) survivors of childhood cancer from pediatric to adult-oriented long-term follow-up (ltfu) is complex. loss to follow-up is common, and little is known about the success rates among different models. the survivors of childhood cancer program (sccp) at ucsf benioff children's hospital oakland employs a community-based model for transitional care. our multidisciplinary team provides aya survivors a comprehensive treatment summary and recommendations, then facilitates transition to primary care or adult oncology ltfu programs. evaluate the success rate for transition of care among aya survivors of childhood cancer in our ltfu program, and identify barriers to successful transition. design/method: aya patients seen from november to august in the sccp with intent to transition were asked by email or telephone if they had followed up with their designated provider. the primary outcome was successful transition, defined as establishing care within months of their visit. patients were also asked about barriers to transition and to rate the new provider's familiarity with their cancer history and ltfu needs. results: transition was intended for patients. eightyseven were contacted and responded. of these, ( %) successfully transitioned, while ( %) were lost to followup. ages ranged from to years, at to years since completion of therapy. ten ( %) transitioned to a primary care provider, ( %) to an adult oncology ltfu program, and ( %) to a pediatrician. patients rated their new provider's knowledge above average ( . ) on a -point scale from poor ( ) to excellent ( ). survivors lost to follow up indicated the following barriers to transition: loss/change of insurance ( ), inability to find a provider ( ), too busy/forgot ( ), problems with transportation ( ), concerns about cost/copay ( ), and s of s other ( ). twelve patients requested further assistance with transition. conclusion: two-thirds of responding patients successfully transitioned. more work is needed to overcome various barriers to transition for one third of aya survivors. albany medical center, albany, new york, united states background: the transition from active treatment, to offtherapy follow-up, is a stressful event for parents of children with cancer. the psychosocial needs of parents after therapy have received limited attention in the united states with only published quantitative studies, the largest with parents. we have secured funding for and recruited a transition care coordinator (tcc) to investigate this further. objectives: our objective is to assess and screen parents at the end of their child's treatment, and to develop interventions to support parents during this time and thereafter. design/method: after informed consent, a standardized questionnaire, the psychosocial assessment tool (pat . ), was administered to parents at end of therapy (t ), months later (t ) and year later (t ). the tcc provided "universal" intervention to all families with an end of therapy binder containing a treatment summary, follow-up roadmaps, information on late effects, and survivor scholarships. based on their pat . scores, some parents were provided intervention specific to symptoms (targeted intervention for scores - . ) or referred to a behavioral health specialist through the clinic social worker for counseling (for scores > ). results: analysis of pat data showed that % of parents (n = ) scored in the targeted or clinical ranges; % of parents scored in those ranges at pat . significant gender differences were revealed with the mean score for men of . and for women of . . this was confirmed by showing statistical significance (p = . ) when analysis was conducted for only a subgroup of data composed of couples (n = ). analysis of pat data by couples (n = ) showed the mean score for men was . and for women was . (p = . ). gender differences were most apparent in caregiver stress reaction questions that focused on ptsd symptoms. when the subgroup of couples' scores (n = ) for caregiver stress reaction at pat was analyzed, there was a significant difference (p = . ) in caregiver stress reaction with a mean of . for men versus . for women. [note: subcategory scores range from to ]. this study was initiated in october using a tcc and the pat . screening tool. the results suggest greater stress on mothers after therapy, with a substantial proportion of parents having symptoms of ptsd after therapy. background: hodgkin lymphoma (hl) is a common childhood cancer characterized by an inflammatory microenvironment. chemotherapy and radiation may exacerbate this inflammation and contribute to the development of late effects (pneumonitis or pulmonary fibrosis). in a heterogeneous cohort of childhood cancer survivors exposed to pulmonarytoxic therapy, no association between pro-inflammatory cytokines and late pulmonary dysfunction was observed. our objective was to test this association in a relatively uniform cohort of survivors of hl, given the well-recognized proinflammatory background of this disease. objectives: to characterize off-therapy pulmonary function in survivors of hl treated with contemporary therapy, and to investigate its association with persistent systemic inflammation. design/method: blood samples, clinical data, and pulmonary function tests were obtained from survivors of hl ≥ months off therapy. lung function score (lfs), a validated method for assessing degree of pulmonary dysfunction on a scale of i to iv, was determined from diffusion capacity and forced expiratory volume in one second (fev ). for a control group, blood samples from patients with benign, noninflammatory hematologic conditions were used. plasma concentrations of inflammatory cytokines were measured on a luminex platform (emd millipore). associations between clinical features or cytokine levels and lfs i (normal) vs. ii-iv were evaluated using logistic regression or wilcoxon rank sum tests, respectively. results: of survivors (mean age at diagnosis: years, range: - ; mean time off therapy: . years, range: . - ), % were categorized as lfs ii (mild dysfunction), % as lfs iii (moderate dysfunction), and no survivors as lfs iv (severe dysfunction). higher lfs was associated with female sex (p = . ) but not other demographic, disease, or treatment factors. forty-eight survivors had blood samples collected at a mean age of . years (range: - ) with a mean time since treatment completion of . years (range: . - . ). of controls, the mean age at time of blood collection was years (range: - ). survivors did not have significantly elevated cytokine levels compared to controls. female survivors of hl ≥ months off therapy are at increased risk of pulmonary dysfunction. neither evidence for pulmonary dysfunction, as measured by lfs, nor duration of time off therapy were related to systemic inflammation in this study. pulmonary function deterioration and clinical pulmonary symptoms are rarely observed immediately following therapy but increase over time. future studies may consider exploring the contribution of systemic inflammation to pulmonary late effects in survivors farther off therapy, when risk for this late effect is greater. background: thyroid carcinoma is a very rare tumor in pediatrics, accounting for . - % of childhood carcinomas in the united states and europe. we aim to detect the risk of second malignancies among pediatric thyroid cancer survivors. the cohort analysis consisted of pediatric cancer patients aged less than years diagnosed with a primary thyroid cancer and identified by site code icd- - : c , reported to a seer database between and . they were followed up by death or the end of the study period (december , ) . out of patients diagnosed primarily with thyroid carcinoma, there were patients who had incidences of subsequent malignancies. the mean age of patients at initial diagnosis of thyroid cancer was years. females ( . %) had significantly higher incidence of second malignancies (sm) than males ( . %). the overall standardized incidence ratio (sir) of sm in thyroid pediatric patients was higher than expected (sir = . ). some specific sites showed significantly higher incidences: salivary gland (sir = . ), gum and other mouth (sir = . ) and kidney (sir = . ). the overall risk of sm in patients received radioactive iodine was higher than expected (sir = . ). the cumulative inci-dence of sms from the initial diagnosis of thyroid cancer was calculated with the survival methodology of competing risk, death treated as a competing event. cumulative incidence of sm was . % [ % ci ( . , . %)] at years and substantially expanded after years, reaching . % [ % ci ( . , . %)] at years. the cumulative incidence of each tumor type at years was . % [ % ci ( . , . %)] for breast cancer, . % [ % ci ( . , . %)] for salivary gland, . % [ % ci ( . , . %)] for each one of kidney and cervix uteri and . % [ % ci ( , . %)] for each one of ovary and melanoma of the skin. cumulative incidence of sm was stratified based on race, gender and radiotherapy exposure, but there was no statistical difference in each of them. conclusion: race, gender, histological subtypes, and radioactive iodine may play an important role as prognostic factors for developing sm among pediatric thyroid cancer survivors. identification of underlying mechanisms that raise the risk of sm is important for both treatment and follow-up strategy. background: the ethical practice of informed consent requires it be both voluntary and understood by the research participant. in pediatric oncology, parents must undergo informed consent to enroll their child with cancer into clinical trials, but often it can be difficult to understand especially for parents with low english proficiency. previous research has shown that parents of children with cancer have difficulty understanding voluntariness, and that parental satisfaction with informed consent does not always correlate with adequate comprehension. objectives: to examine socio-demographic and contextual correlates of comprehension of informed consent, voluntariness, and satisfaction in parents who consented to participation of their child in a cancer clinical trial. we focused on characterizing differences between non-hispanics and hispanics, the fastest growing ethnic group in the u.s. design/method: parents/guardians (n = ) of children aged - years with newly diagnosed cancer, who had consented to participation of their child in a clinical trial for cancer treatment at rady children's hospital-san diego were s of s prospectively recruited. parents completed questionnaires assessing comprehension, voluntariness, satisfaction, health literacy, socio-demographics, and acculturation level, if hispanic. comprehension was surveyed at baseline and longitudinally at months. comprehension, voluntariness and satisfaction outcomes were analyzed by socio-demographics, health literacy, and acculturation level using logistic regression. results: of the participants surveyed, ( . %) were hispanic and ( . %) were non-hispanic. we found that higher health literacy was associated with greater objective comprehension (p< . ), voluntariness (p< . ), socioeconomic status (p< . ), and acculturation (p< . ). hispanics reported lower objective comprehension (p = . ), voluntariness (p = . ), health literacy (p< . ) and ses (p = . ) compared to non-hispanics. spanish-speakers reported lower voluntariness (p = . ), health literacy (p< . ), and acculturation (p< . ) compared to englishspeakers. at the -month follow-up, comprehension in hispanics significantly improved (p = . ) compared to their baseline comprehension. satisfaction was moderately high across all subgroups and was not significantly impacted by socio-demographics, health literacy, or acculturation. in this study, with equivalent numbers of hispanic and non-hispanic participants, we found that hispanic and spanish-speaking parents of children with newly diagnosed cancer had inadequate informed consent comprehension, voluntariness and health literacy despite high satisfaction. our study suggests that hispanics and individuals with limited english proficiency are not making truly informed decisions for their child with cancer. to ensure the ethical practice of research in pediatric oncology, the informed consent and decision-making process must be improved with culturally and linguistically interventions for these underserved populations. memorial sloan kettering cancer center, new york, new york, united states background: pediatric oncology patients undergo repeated bone marrow aspirations and biopsies (bma/bx). these potentially painful procedures can exacerbate anxiety and distress. standard practice at memorial sloan kettering (msk) department of pediatrics is to use propofol, which has amnestic but no analgesic properties. we sought to evaluate whether the addition of local anesthetic would improve patient experience with bma/bx. the purpose of reppair: reducing procedural pain and improving recovery of quality of life (qol) (nct ) is to evaluate the efficacy of local anesthesia with ropivacaine in reducing procedural pain and improving post-procedure qol in pediatric neuroblastoma patients undergoing bma/bx with general anesthesia. reppair is a prospective, randomized, crossover clinical trial that opened for enrollment october . eligible patients were - years old with neuroblastoma. participants were observed on trial for two sequential bm procedures; one procedure with intervention a: propofol alone (pa), and the other with intervention b: propofol plus ropivacaine (p+r). participants were randomized to intervention sequence ab or ba and were blinded to the order of interventions. participants and recovery room (rr) nurses, who were also blinded, followed a standardized postprocedure pain management algorithm. the primary endpoint was percentage of participants requiring opioid analgesia in the hours post-procedure. secondary endpoints included total opioid in hours, non-opioid analgesia use, pain scores, time to first opioid, and short-term qol. qol was assessed by a parent-proxy metric that evaluated pain interference with sleep, physical, emotional, and social recovery. as of january , patients were assessed for eligibility and patients were randomized ( have completed both procedures). for the primary endpoint, a slightly higher proportion of participants required opioid for pa than p+r ( % versus %, p = . ). pain scores in the rr were significantly higher for pa than p+r (median [ th, th percentile]: [ , ] versus [ , ], p = . ). there were no statistically significant differences in total opioid or non-opioid analgesia, -and -hour pain scores, median time to first opioid, or pain interference scores. there were no adverse events. conclusion: preliminary findings of the reppair trial suggest that local anesthesia does not reduce the need for opioid analgesia or improve short-term qol in pediatric patients undergoing bma/bx with general anesthesia. local anesthesia did improve pain scores in the immediate recovery period. final results of this study will help establish evidence-based guidelines and optimize the experience of pediatric patients with bone marrow procedures at our center. background: children with advanced cancer experience a range of symptoms throughout treatment or at end of life, some of which are poorly controlled. minimizing suffering, including effective symptom management, in children with advanced cancer is a central value for pediatric oncology clinicians. patient-reported outcomes have been used in symptomrelated research in pediatric oncology patients; however the majority of literature specific to symptoms during palliative care and end of life for children and adolescents with advanced cancer is based primarily upon medical record reviews and to a lesser extent, patient self-report. the purpose of this study was to prospectively describe symptom frequency, severity, and level of distress in children/adolescents with advanced cancer using patient selfreport and parent proxy. design/method: a prospective cohort design was used for this study. five pediatric oncology institutions from across the united states participated. children and adolescents were eligible to participate if they were - years of age, englishspeaking, and had a diagnosis of advanced cancer, defined as a -week history of progressive, recurrent, or non-responsive disease or a decision not to pursue curative-focused therapy. a modified version of the memorial symptom assessment scale (msas) was used to measure symptom frequency, severity, and level of distress and was administered to child/parent dyads electronically via smartphones every two weeks. information regarding disease status and cancer treatment was collected concurrently. data was analyzed using descriptive statistics and univariate logistic regression analysis. results: a total of children and adolescents and parents participated in the study. the median age of child participants was years, with half being male. the median age of parents was years. the child participants had a variety of primary diagnosis, including: leukemia/lymphoma (n = , %), solid tumor (n = , %), and brain tumor (n = , %). the most frequently reported symptoms by children with advanced cancer and parents were pain (n = / , . %), lack of energy (n = / , . %), and nausea (n = / , . %). presence of disease (p = < . ), recent disease progression (p = . ), and receiving cancer therapy (p = . ) were significant factors on the presence of pain. high intensity cancer therapy was a significant factor on pain frequency (p = . ) and level of distress (p = . ). it is feasible to collect data prospectively in children with advanced cancer regarding symptom frequency, severity, and level distress. clinicians' increased understanding of the symptom experience may promote communication with children and adolescents and timely intervention. more research is needed to understand symptom clusters in children with advanced cancer. vanderbilt children's hospital, nashville, tennessee, united states background: febrile neutropenia (fn) is a frequent occurrence in children undergoing chemotherapy. though guidelines recommend adding a second antibiotic to broad-spectrum antipseudomonal coverage in specific scenarios, augmenting empiric therapy with a second antibiotic is common practice. additional empiric antibiotic (aea) use increases the risk of antibiotic toxicity and future antimicrobial resistance. data clarifying the indications for aea are limited in pediatric patients. objectives: to identify risk factors for gram-positive (gp) and gram-negative (gn) bacteremia in patients presenting with fn to determine situations in which aea use is warranted. design/method: a retrospective chart review was conducted of pediatric severe fn with absolute neutrophil count < / l occurring at a single institution between and . potential a priori risk factors based on clinical reasons for antibiotic expansion were chills, hypotension, mucositis, skin or soft tissue infections (sstis), recent administration of highdose cytarabine (hdac), and a diagnosis of acute myeloid leukemia (aml). potential factors for gn bacteremia were chills, hypotension, mucositis, and abdominal pain. the association between each potential risk factor and gp or gn s of s bacteremia was identified. logistic regression was used for multi-variable analysis. the review yielded episodes. gp bacteremia was isolated in cases ( . %) and gn bacteremia in episodes ( . %). in multivariable analysis, hypotension (or . ( % ci . , . ), p = . ) and sstis (or . ( . , . ) , p = . ) were independently associated with increased risk of gp bacteremia, while mucositis (p = . ), recent administration of hdac (p = . ) and chills (p = . ) were not. ten patients with aml didn't receive hdac, thus the association between aml and gp bacteremia could not be reliably estimated. hypotension (or . ( . , . ), p< . ) and chills (or . ( . , . ), p< . ) were independently associated with a higher risk of gn bacteremia, while mucositis (p = . ) and abdominal pain (p = . ) were not. of the gn infections, ( %) were resistant to cefepime, the empiric agent of choice at our institution. patients with fn with sstis, hypotension, or recent hdac had increased risk of gp bacteremia indicating potential benefit of empiric vancomycin in these settings, while mucositis and chills were not associated with gp bacteremia. hypotension and chills were associated with gn bacteremia, potentially warranting empiric antibiotic expansion, while mucositis and abdominal pain were not. identifying specific indications for aea use in pediatric severe fn use may improve antimicrobial utilization, decrease unnecessary antibiotic use, and improve patient outcomes. background: for children/young adults with incurable high grade gliomas (hggs), like diffuse intrinsic pontine glioma (dipg) or glioblastoma multiforme (gbm), oncologists endeavor to align therapy with patient/family goals of care, but may be influenced by providers' preferences or limited resources. ethical challenges can arise around the perceived purpose, risks and benefits of therapy options, provider conflicts of interest, access to care, deciding decisional priority between patients and families, and conflicts around end-oflife care. objectives: evaluate factors that play into longitudinal decision making for children and young adults with hggs, their families and oncologists using a qualitative approach with ethnographic elements. design/method: eligible patients were aged - with dipg, gbm, or secondary hgg. patient exclusions included: non-english speaking, in state custody, death prior to diagnosis, seen by oncology once, or an oncologist declined participation. key decision making visits (e.g. mri reviews) were serially audio-recorded, along with subsequent : semistructured interviews with patients and/or parents about the decision making process. field notes from clinician meetings, chart notes, and oncologist questionnaires were obtained. discussions and interviews were transcribed and independently coded by three investigators. inter-rater reliability was assessed during code book development. discrepancies were discussed until consensus met. constant comparison analysis with maxqda software continued until thematic saturation. results: twenty-two of eligible patients were approached; agreed to participate. one withdrew upon transferring care. mean age was . years (sd . ); % male, % caucasian, % african american, % hispanic, and % asian. four encounters, ( . hours), were recorded on average per patient. parent/patient interview themes included: ) hope (for a cure, prolonged life, and quality of life), ) importance of physician recommendations, ) importance of support systems (family, community, social media), ) food (as cancer etiology, intervention) ) finances (personal, research funding), ) communication (with medical providers, family, community), ) death, and ) god (beliefs, prayer, existential questions). oncologists desired prolonged quality of life, while patients/families transitioned to that hope from hope for a cure. decisions made in the setting of hggs are multi-factorial, ultimately reflecting the competing values of decision makers. optimism about treatment efficacy is held in tension with poor prognosis, allowing for functional hope. acknowledging patients' and families' shifting hopes allows for changes in goals of care and shared decision making. future work is needed to ) develop preference tools for pediatric patients and families to inform medical providers and ) provide training in communication and shared decision making with oncologists. emory university, atlanta, georgia, united states background: bone marrow transplantation (bmt) is a potentially curative but underutilized treatment for scd. our previous work has shown that there is variation in physician philosophy and practice in considering bmt as a treatment option for patients with scd, and physicians may not discuss this with patients and families as a potential treatment option. in a randomized clinical trial to test the effectiveness of a decision aid for disease modifying therapies for sickle cell disease, adult patients with scd as well as caregivers of adult/pediatric patients were interviewed about how they seek or have sought information related to scd, made decisions about treatments for scd, and identified a treatment option they were interested in learning more about using the decision aid tool. we performed a secondary analysis of these baseline data to understand patient information needs and attitudes regarding bmt as a treatment option for scd. the goals of this analyses was to understand patient and caregivers' attitudes and perceived information needs regarding bmt as a treatment option for scd. we performed an analysis of baseline interviews from caregivers of patients with scd or adult patients from a randomized control trial for a decision aid tool for scd. of the interviews belonged to caregivers of patients with scd. in addition to reviewing interviews for discussion of bmt, we interrogated for mention of terms such as 'bone marrow transplant' or 'cure' or 'stem cell transplant'. interviews were coded using nvivo and analyzed for emerging themes. results: of the baseline interviews, interviews met selection criteria. thirteen of the interviews were with caregivers of pediatric patients, and the remainder were with adult patients, including young adult patients with scd. the majority of participants want to learn about bmt or curative options. in many participants, this was expressed despite knowledge that they were not a likely candidate for transplant. desired information about bmt included eligibility, benefits, risks, long-term effects, quality of life and financial aspects related to bmt. of the patients who discussed how they learnt about bmt, approximately half mentioned that their healthcare provider had not previously mentioned this to them. we then examined knowledge of bmt and attitudes with demographic and clinical variables. patients and caregivers of pediatric patients with scd want to learn about bmt as a treatment option. healthcare providers should consider discussing bmt with their patients with scd. natasha frederick, anna revette, alexis michaud, jennifer mack, sharon bober dana-farber cancer institute, boston, massachusetts, united states background: adolescents and young adults (ayas) consistently identify the need for improved patient-clinician communication on sexual and reproductive health (srh) issues. however, oncology clinicians do not routinely integrate srh conversations with ayas through disease treatment and survivorship. little is known about why these conversations do not take place. objectives: explore aya perceptions of and receptiveness to srh communication with oncology clinicians and to identify barriers and facilitators to these conversations. design/method: semi-structured interviews were held with aya cancer patients and survivors (ages - years, men, women). twelve participants were on active treatment and were within years of treatment completion. interviews were conducted in english by phone or in person. the interview transcript underwent pre-testing with ayas. all interviews were audio-recorded and transcribed verbatim. transcripts were analyzed and summarized by two trained qualitative researchers according to standard comprehensive thematic qualitative analysis methods. analyses were aided by nvivo software. results: ayas perceived existing srh communication between ayas and oncology providers as inadequate. all ayas reported a need for improved srh communication with oncology providers, and three key areas of need emerged: ) general education; ) addressing specific srh issues experienced during treatment and survivorship; and ) understanding the long-term impact of cancer and treatment on srh. ayas felt that current srh discussions are limited and too narrow in scope and scale. ayas reported that most srh conversations focus exclusively on fertility (n = ), usually taking place at the start of treatment. other additional yet limited communication reported was about sexual activity (n = ), contraception (n = ), sexual function (n = ). no ayas reported conversations about potential treatment complications related to sexuality other than infertility. key barriers to srh conversations include patient discomfort initiating conversation (n = ) and presence of family members (n = ), with additional reported barriers including perceived provider discomfort (n = ), lack of rapport with provider (n = ), and age/gender differences (n = ). ayas felt that s of s communication tools such as handouts, brochures, and websites would be helpful facilitators to direct communication from the oncology clinician, and wanted conversations to start before treatment initiation and to continue through treatment and survivorship conclusion: ayas identify a key role for pediatric oncology providers in srh care from diagnosis through survivorship, however multiple barriers interfere with discussions about srh on a regular basis. identified barriers suggest that future efforts should focus on provider education and training in srh and srh-related communication in order to optimize care provided to this unique patient population. background: peripherally inserted central venous catheters (picc) provide secure vascular access in pediatric patients for the delivery of necessary therapies. the ease of placement in the inpatient and outpatient settings has expanded their utilization. however, recent data analyses show a significant increase in venous thromboembolism (vte) risk with the use of picc lines. with its rising use, modifiable risk factors need to be understood for preventative measures. objectives: in this study we aim to understand patient and catheter specific characteristics in relation to the development of vte. design/method: with irb approval, a retrospective interrogation of the electronic medical record and a picc database, at rainbow babies and children's hospital, was completed. the study cohort contained patients < years of age who had a picc line placed between january of and december of . data collected included indication for line placement, line dwell time, location of insertion including blood vessel and extremity, number of attempts at line placement, lumen size and indwelling line length. in addition, we collected number of days to vte formation, associated symptoms and location of vte. chi-squared analyses and fischer's exact test were used where appropriate for statistical analysis. we analyzed ( neonatal) newly placed picc lines. fifty line-associated vte events were found, for an incidence of . %. all vte occurred with the placement of the first picc line. intravenous therapies were the most common reason for line placement. no statistical significance was found between various indications for placement. the most common symptom of vte manifestation was extremity swelling, follow by extremity pain. right extremity picc was found to have a higher incidence of vte. larger catheter lumen sizes (> french) had a higher incidence of vte. we found a mean time of . days to vte detection. we were unable to find any clinical, patient or line specific factors leading to increased vte formation after statistical analysis. special consideration should be given to the duration of picc line use as this may reduce the incidence and comorbities associated with vte. there is still much to be understood about catheter associated vte formation as our analyses indicates the need for prospective data collection on a larger scale in hopes to create guidelines related to catheter use in pediatrics. background: the decision to transfuse a patient is a complex one and is never based solely on a number; however, certain hemoglobin or platelet count thresholds have been proposed in aiding physicians make transfusion decisions. in our hospital, the thresholds for packed red blood cell (prbc) and platelet transfusion in pediatric oncology patients are hemoglobin levels below . g/dl and platelet counts below , /mm (< , for brain tumors), respectively. recently, these thresholds have been questioned and we were asked whether we could safely lower the thresholds to < . g/dl of hemoglobin and < , /mm platelet count objectives: to investigate platelet and hemoglobin transfusion thresholds for oncology patients at children hospital of michigan design/method: retrospective chart review over a -month period, examining platelet and hemoglobin pretransfusion levels for each prbc and platelet transfusion given to oncology patients results: over the course of months, eligible oncology patients (median age years) received transfusions ( prbc transfusions and platelet transfusions). the mean pretransfusion hemoglobin level was . ± . g/dl (range . - . ) (n = ) for total prbc transfusions and this was not different among disease categories (p = . ). patients who had anemia symptoms and signs (n = ) had a slightly lower hemoglobin level compared to those who did not (n = ): . ± . vs . ± . g/dl (p = . ). the mean pretransfusion platelet count was , ± , /mm (range , - , ) for total platelet transfusions (n = ); , ± , /mm in patients with brain tumors (n = ); , ± , in patients with leukemia (n = ); and , ± , in patients with solid tumors (n = ). the mean pretransfusion platelet count was significantly higher in transfusions for brain tumors compared to that in the other disease groups (p< . for both). the mean pretransfusion platelet count was not different among those patients who had bleeding/bruising symptoms ( , ± , , n = ) versus those who did not ( , ± , , n = ) (p = . ). the bleeding/bruising rate was slightly but insignificantly higher in those who had platelet counts < , vs those who had ≥ , ( . % vs . %, p = . ). since most patients develop symptoms of anemia at hemoglobin above g/dl and about / of patients develop bleeding/bruising symptoms at platelet counts above , /mm , our current policy so far reflects a safe threshold for transfusion, and further lowering of the thresholds should be investigated in prospective studies. background: renal impairment is an important complication of childhood cancer and its treatment. serum creatinine level is frequently used as a screening test to monitor renal function; however, patients can have significantly decreased glomerular filtration rate (gfr) with normal serum creatinine. to determine the prevalence of chronic kidney disease (ckd) among children with cancer diagnosis, based on calculated gfr. to compare the difference between using serum creatinine value alone versus gfr in detecting ckd. design/method: retrospective review of medical records of patients, age - years, diagnosed between / - / with solid tumors were analyzed. serum creatinine and calculated gfr using schwartz formula were recorded. ckd as classified by the foundation of kidney disease and outcome quality initiative was used: ckd stage : gfr ( to ml/min per . m ) ckd stage : gfr ( to ml/min per . m ) statistical analysis using spss software v. . chi-squared test for proportions within group, and pearson chi-squared and fisher exact tests for statistical differences between groups. p-value < . was considered to indicate significance results: out of the records reviewed, ( %) were males and ( %) females, with mean age of . ± . years. ( . %) patients received one or more of nephrotoxic chemotherapy drugs; cisplatinum, carboplatinum, or ifosphamide mainly in the non-wilms solid tumors group ( . %) compared to ( . %) in the wilms tumor (wt) group. based on calculated gfr (by schwartz formula) ckd stage /or was diagnosed in ( %) patients with overwhelming majority ( %) were in the mild stage ckd, only ( . %) of those patients had abnormally high serum creatinine levels (p = . ). . % of patients who received nephrotoxic chemotherapy developed ckd, compared to . % in those who did not receive it, (p = . ). despite that only / ( %) of wt group patients received nephrotoxic chemotherapy, yet this group had higher percentage of ckd ( . %) compared to non-wt group ( . %) p = . . significantly lower mean gfr . ± was noticed in the wt group compared to . ± in non-wt group (p = . ) conclusion: high prevalence of mild ckd was found among solid tumor patients. using serum creatinine alone as measure of renal function significantly under estimates renal impairment in those patients. early identification of ckd is easily achieved by using calculated gfr, which can helps providers and care givers to avoid potential nephrotoxic antibiotics, contrast media, nsaids and dehydration that may further deteriorate renal function the university of texas southwestern medical center, dallas, texas, united states background: children with down syndrome (ds) have increased risk of developing leukemia. pediatric patients with ds-associated acute lymphoblastic leukemia (ds-all) are known to have significant toxicities with reinduction chemotherapy and historically poor outcomes with stem cell transplant (sct). anti-cd chimeric antigen receptor (car) t-cell therapy, tisagenlecleucel, demonstrated high rates of durable complete remission (cr) and a manageable safety profile in children with r/r b-cell acute lymphoblastic leukemia (b-all). objectives: characterize the efficacy and safety of tisagenlecleucel in pediatric/young adults with ds-all. design/method: pooled data from single-arm, multicenter, phase trials of tisagenlecleucel in pediatric/young-adult patients with r/r b-all (eliana, nct ; ensign, nct ) were analyzed. eight patients with ds-all were enrolled (data cutoff: eliana, november ; ensign, february ). seven were infused with tisagenlecleucel; patient died from all progression and intracranial hemorrhage before infusion. no manufacturing issues occurred during production. / infused patients were male, / had prior sct (age range, - years). / patients achieved cr or cr with incomplete blood count recovery (cri) by day (d) (cr+cri, %); died before d and was not evaluable. analysis of minimal residual disease was negative in bone marrow in responding patients. two patients had cd negative relapses at and months. ongoing remissions in patients without relapse ranged from to months. the safety profile (n = ) appears similar to that in patients without ds in the same trials (n = ). grade (g) / cytokine release syndrome occurred in % ( / ) of patients with ds and in % without ds. rates of other g / adverse events of special interest did not appear to favor a consistent trend between patients with/without ds (febrile neutropenia: % vs %; neurological events: % vs %; tumor lysis syndrome: % vs %). g / infections were not observed in patients with ds ( % vs %). one patient died after infusion due to intracranial parenchymal hemorrhage on d associated with ongoing coagulopathy. time and extent of tisagenlecleucel expansion and long-term persistence were similar between groups. conclusion: this is the first analysis of car t-cell therapy in pediatric patients with r/r b-all and ds. these data suggest that toxicities appear similar to those in patients with b-all without ds, remission rates in ds-all are high, and longterm outcomes with sustained persistence appear promising. further exploration of tisagenlecleucel as an alternative to sct in children with r/r ds-all is warranted. sponsored by novartis. background: hispanic adolescence and young adults are twice as likely to develop acute lymphoblastic leukemia (all) with high risk features as non-hispanic whites. they also have poor prognosis and % higher death rate. b-all with crlf overexpression caused by genetic alteration of the cytokine receptor, crlf is five times more common in this subgroup. approximately % of crlf b-all cases also have ikzf genetic alterations. ikaros is involved in transcriptional regulation of several important genes involved in leukemogenesis. overexpressed casein kinase ii (ck ) impairs functions of ikaros. objectives: understand the molecular mechanisms that regulate crlf expression in crlf b-all. here we present evidence that ikaros-mediated repression of crlf transcription in b-all in hispanic children is regulated by ck . design/method: primary b-all patient samples from hispanic children were used. ikaros retroviral transduction, ikaros shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: ikaros binding to promoter of crlf was confirmed using quantitative chip. functional experiments such as overexpression of ikaros in b-all primary cells results in transcriptional repression of crlf whereas ikaros silencing using shrna resulted in increased transcription. these results suggest that ikaros negatively regulates crlf expression. molecular inhibition of ck with shrna targeting the ck catalytic subunit, as well as pharmacological targeting of ck with cx resulted in transcriptional repression of crlf . ck inhibition was associated with increased ikaros dnabinding to the promoter of crlf . however, the ability of cx to repress crlf is lost or severely reduced, in cells with shrna silencing of ikaros, as compared to cells with intact ikaros. moreover, similar results were noted following treatment with cx in leukemia cells obtained from high risk b-all patients with deletion of one ikzf allele. ikaros binds poorly to promoters of crlf gene in these cells. treatment with cx restores ikaros dnabinding to the promoters of crlf , which is associated with its strong repression. serial qchip analysis of the epigenetic signature at the crlf promoter showed that increased ikaros binding to the crlf promoter, following ck inhibition, is associated with enrichment for the h k me histone modification, which is a marker of repressive chromatin. results demonstrate that crlf expression is epigenetically regulated by the ck -ikaros axis .cx show antileukemic effect via restoration of ikaros tumor suppressor function, resulting in crlf repression suggesting advantage of using ck inhibitors as potential therapeutic approach in crlf altered b-all. results: hypodiploid all (modal chromosome number < and/or di < . ) was identified in patients ( . % of all patients; . % of nci standard risk (sr) and . % of nci high risk (hr)), who were removed from frontline protocol therapy post-induction. overall -year efs and os were . %± . % and . %± . %. transplant status was retrospectively available for / ( %), of whom underwent hsct in cr . five-year efs with hsct was . %± . % vs. . %± . % without (p = . ). -year os with and without hsct was . %± . % vs. . %± . % (p = . ). when corrected for the median time to hsct ( days), there were no significant differences in -year efs or os rates with and without hsct: . %± . % and . %± . % vs. . %± . % and . %± . %. no nci risk group or mrd subset benefitted significantly from cr hsct. sr patients (n = ) had -year efs and os of . ± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. hr patients (n = ) had -year efs and os of . %± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. for those with end-induction mrd < . % (n = ), -year efs and os were . %± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. end-induction mrd-positive patients (n = ) fared poorly with both year efs and os of . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. multivariate regression analysis including nci risk group, mrd, and cr hsct, showed only mrd negativity was significantly associated with efs (hr . , p< . ) and os (hr . , p< . ). patients with hypodiploid all fare poorly, particularly those with end-induction mrd ≥ . %. while cr hsct is a standard treatment approach, it does not confer significant benefit. we were unable to assess bridging therapy prior to hsct, and comparator groups are small. taken together, however, new strategies are urgently needed for these patients. background: ras-pathway mutations are known to play a pivotal role in a significant proportion of myeloid malignancies, including upwards of % of pediatric aml cases. ras-pathway mutations in myeloid malignancy commonly co-occur with mutations of epigenetic regulators, suggesting cooperative leukemogenesis. among the epigenetic modifiers most frequently mutated in myeloid malignancy are regulators of dna methylation. this indicates that the alteration of dna methylation contributes to leukemogenesis. the ten-eleven translocation (tet ) is an epigenetic regulator that plays an important role in regulation of dna methylation through its action of hydroxylation of -methylcytosine, which ultimately leads to passive de-methylation of dna cytosines. in myeloid malignancy, loss of function tet mutation is one of the most frequently co-occurring lesions in ras mutated malignancy. how specifically the altered methylation patterns in ras-pathway driven diseases promotes leukemogenesis is unclear. objectives: we hypothesize in mice with a ras-pathway mutation, that when an epigenetic modifier co-occurs, such as loss of function of tet , this primes stem cells and/or early differentiating progenitors for transformation by preventing the repression of stem cell self-renewal genes, inhibiting differentiation, enhancing ras signaling and leading to leukemogenesis. we have generated a novel murine model with constitutive deletion of tet (tet -/-) combined with an inducible activating krasg d mutation (krasg d/wt). mice have been tracked for evidence of hematologic malignancies and compared to mice with corresponding single genetic lesions. cooperative leukemogenesis will be demonstrated by decreased latency to disease onset, impact on malignancy lineage, in addition to investigating mechanistically through which pathways leukemogenesis may be promoted. results: krasg d/wt/ tet -/-mice demonstrate statistically significant differences in peripheral white blood cell count, hemoglobin, and platelet levels as early as -weeks post ras-pathway activation. peripheral cell lineage analysis demonstrates early skewing toward myeloid differentiation and marked splenomegaly in mice harboring both genetic lesions compared to wild type or mice with single genetic lesions. phospho-flow cytometric analysis reveals increased perk and ps activation in krasg d/wt/ tet -/-sca- enriched bone marrow cells compared to either genetic lesion alone. our study utilizing a murine model to examine how in ras-pathway mutations the addition of a co-occurring epigenetic lesion demonstrates that these lesions appear to cooperate to promote early myeloid differentiation with attendant changes in signaling pathways. this exploration to elucidate the mechanics of ras-pathway mediated disease lay the foundation for identification of patients who may benefit from existing therapies, such as dmtis, or identify new signaling targets for therapeutic exploration. background: the humoral immunogenicity of car , a chimeric antigen receptor (car) with a murine scfv domain developed for treatment with tisagenlecleucel in relapsed/refractory (r/r) pediatric/young-adult acute lymphoblastic leukemia (all), was evaluated in studies. little is known about the presence/impact of preexisting/treatmentinduced anti-murine car (mcar ) antibodies in patients treated with car therapy. objectives: patients from eliana (nct ; n = ) and ensign (nct ; n = ) were evaluated before and after tisagenlecleucel infusion to determine the impact of anti-mcar antibodies on cellular kinetics, efficacy, and safety. design/method: anti-mcar antibodies were determined by flow cytometry and reported as median fluorescence intensity. assay validation included evaluation of the interferences of intravenous immunoglobulin (ivig) treatment with the anti-mcar antibody assay. impact of preexisting and treatment-induced immunogenicity on cellular kinetics, efficacy, and safety was determined. treatment-induced immunogenicity was defined by a positive increase in anti-mcar antibody levels over baseline and was assessed by calculating the fold-change between preexisting (ie, baseline) and postinfusion levels. results: % of patients displayed preexisting anti-mcar antibodies; a similar incidence was detected in healthy volunteer samples during method validation. % of patients developed treatment-induced anti-mcar antibodies. no relationship was identified between tisagenlecleucel expansion (auc - d) and preexisting/treatment-induced anti-mcar antibodies (r < . and r = . , respectively); similar results were seen for cmax. presence of treatment-induced anti-mcar antibodies did not appear to impact transgene persistence or response. kaplan-meier estimates showed that preexisting/treatment-induced anti-mcar antibodies did not appear to impact duration of response or event-free survival. strip plots showed consistent levels of preexisting/treatment-induced anti-mcar antibodies across patients with safety events, including cytokine release syndrome, neutropenia, thrombocytopenia, and neurological events. there was no apparent relationship between treatment-induced anti-mcar antibodies and b-cell recovery categories (≤ months, > and ≤ months, > months, and ongoing sustained aplasia). no association existed between time of b-cell recovery and presence of treatment-induced anti-mcar antibodies. b-cell aplasia requiring ivig occurred following tisagenlecleucel in the majority of patients. the tisagenlecleucel concentration-time profiles in patients with treatment-induced anti-mcar antibodies were categorized by time following ivig administration. time of ivig administration had no impact on in vivo transgene expansion and persistence. we report the first comprehensive assessment of the impact of anti-mcar antibodies on clinical endpoints with car therapy. pediatric/young-adult patients with r/r all had a high frequency of baseline anti-mcar antibodies, and preexisting/treatment-induced anti-mcar antibodies did not impact the cellular kinetics, safety, and efficacy of tisagenlecleucel. cell-mediated immunity studies are ongoing. sponsored by novartis. background: adoptive immunotherapy, using cd engager (cd -eng) t-cells, has shown success in preclinical studies, recognizing and killing acute myeloid leukemia (aml) blasts in vitro and in vivo. cd -eng t-cells secrete bispecific molecules that recognize cd (t-cells) and cd (aml blasts), and are able to direct transduced t-cells and recruit bystander t-cells to kill cd -positive blasts. however, cd -engs do not provide costimulation and have not shown the capability for sequential killing of targets in vitro. we are seeking to improve the expansion, persistence and sequential killing capabilities of cd -engs by genetically modifying these cells with an inducible costimulatory molecule, which can be activated by a chemical inducer of dimerization (cid). we generated a retroviral vector encoding cd -eng and the inducible costimulatory molecule myd .cd linked by a a sequence (cd -eng. a.imc). cd -eng and cd -eng.imc t-cells were generated by retroviral transduction, and their effector function was compared with and without cid. we used flow cytometric analysis to assess transduction efficiency, chromium release assays to evaluate cytolytic activity, and elisa to determine cytokine production. we successfully generated cd -eng.imc tcells and achieved a mean initial transduction efficiency of % that was maintained above % throughout our study period. cd -eng.imc t-cells +/-cid and cd -eng t-cells readily killed cd -positive aml blasts (molm and kg a) in cytotoxicity assays when compared to the cd -negative control (k ). in co-culture assays, cd -eng.imc t-cells secreted increased il- and ifn-gamma in the presence of cid and cd -positive targets (kg a and molm ) when compared to co-culture with cd -positive targets in the absence of cid. in addition, cd -eng.imc t-cells displayed enhanced sequential killing capabilities and ifn-gamma secretion when stimulated weekly with cid and tumor cells at a : ratio when compared to cd -eng t-cells. conclusion: cd -eng.imc t-cells are able to recognize and kill cd -positive aml blasts in an antigen dependent manner. cd -eng.imc t-cells have improved effector function in the presence of cid as judged by cytokine production and their ability to sequentially kill cd -positive target cells. thus, inducible myd and cd costimulation is a promising strategy to improve the effector function of cd -eng t-cells, and warrants further active exploration in preclinical studies. background: eliana (nct ; n = ) is a pivotal multicenter study testing the efficacy of tisagenlecleucel, anti-cd car-t, in children/young adults with r/r b-all. tocilizumab (toci) has been used for management of moderate/severe (grade / ) crs in ≈ % of patients treated with tisagenlecleucel at equivalent doses used in approved nononcological pediatric indications (< kg received mg/kg; ≥ kg received mg/kg [ mg max dose]).( ) crs onset, as graded by the penn grading scale, generally occurred at a median of days (range, - ) after infusion, requiring administration of - toci doses in some patients via a protocol-specific treatment algorithm. toci is a humanized monoclonal antibody that inhibits il- receptor (il- r) signaling. the pharmacokinetics (pk) and pharmacodynamics (pd) of toci in pediatric patients with b-all with carassociated crs have not previously been described. objectives: characterize toci pk/pd for crs management following tisagenlecleucel infusion and describe its impact on cellular kinetics. design/method: toci pk and levels of soluble il- r (sil- r) were determined from serum and quantified using validated assays. maximum toci concentration (cmax) was derived using noncompartmental methods. sil- r, proinflammatory cytokines, and crs resolution time were characterized to describe toci pd. summary statistics and graphical analyses of tisagenlecleucel exposure by number of doses were performed to describe the impact of toci on tisagenlecleucel kinetics in patients responding to tisagenlecleucel infusion. : / patients with crs received the first toci dose at a median of days (range, - ) after crs onset. seventeen patients received dose (range, . - mg/kg); received doses ( - mg/kg); received doses ( - mg/kg), per the crs treatment algorithm. first-dose mean cmax (sd) was ≈ ( . ) g/ml; second dose, ≈ ( ) g/ml. individual patient pd concentration-time profiles showed increased sil- r levels after the first toci dose which remained elevated following the second dose. following toci administration, median time to crs resolution (including fever resolution) was days (range, - ). crs onset coincided with tisagenlecleucel expansion, followed by a peak in serum cytokines, including il- . the geometric mean auc - day and cmax of tisagenlecleucel transgene (by pcr) were % and % higher in tisagenlecleucel-responding toci-treated patients. conclusion: crs symptoms resolved within a median of days after toci administration. toci levels achieved in patients with b-all were similar to reported pediatric nononcological indications (tocilizumab label) and resulted in concentration/time-dependent sil- r increases. transgene continued to expand and persist following toci administration. these data support treatment with toci for crs management. ( ) buechner, eha, . sponsored by novartis. background: in acute myeloid leukemia (aml), mesenchymal stem and stromal cells (mscs) in the bone marrow microenvironment contribute to extrinsically mediated chemo-resistance and are therefore important potential therapeutic targets. the study of patient-derived mscs is at a competitive disadvantage, however, because traditional means of isolating mscs from a bone marrow aspirate interferes with isolating the more highly prioritized leukemic cells. many opportunities to study mscs are therefore missed. objectives: to develop a novel method of isolating mscs using the otherwise discarded portion of a bone marrow aspirate, thereby de-coupling the isolation of primary mscs from the isolation of leukemia cells. design/method: aml patient bone marrow aspirates were obtained prospectively from the children's oncology group. healthy patient marrow was purchased. experimental mscs were isolated from the bottom-most layer (rbc-layer) produced by density-gradient separation of a bone marrow aspirate, which is typically discarded. control mscs were isolated from the buffy coat (mnc layer). non-adherent cells were removed after hours, and adherent cells were cultured at % co with mem-alpha containing % fbs. growth curves were obtained by seeding -well plates with , cells per well. cells were stained using oil red o to observe adipocyte differentiation. results: rbc-layer mscs grow successfully following overnight shipment of the aspirate. identical to mnc-layer mscs, rbc-layer mscs exhibit a fibroblastic morphology and are adherent to plastic. rbc-layer mscs persist in culture up to passages before senescence. they exhibit a slower growth curve relative to mnc-layer mscs, but their overall doubling time is similar at approximately hours. surprisingly, mscs from the rbc-layer exhibit adipocyte differentiation on stimulation, revealing their stem-cell like qualities. we present a method of isolating mscs from the discarded portion of a bone marrow aspirate that does not interfere with the isolation of leukemia cells from the same patient. this portion of the aspirate can be shipped, or can sit for at least hours, without sacrificing its mscs. rbclayer mscs are nearly identical to mscs obtained conventionally. perhaps most importantly, rbc-layer mscs retain a stem-cell like capacity, showing them to be a highly valuable cell population in aml research. future plans include investigating potential selective enrichment of stem-cell mscs in the rbc-layer, which could explain the unexpected difference in growth kinetics. aml researchers now have the opportunity to study this exciting component of the bone marrow microenvironment without sacrificing valuable leukemic cells in the process. background: neutropenia is one of the most frequent side effect of chemotherapy associated with an increase in the risk of infection, especially in the cases when the depth and duration of neutropenia are extended. some genes, as variations of darc, gsdma and cxcl are known to influence white blood cell and neutrophil counts. our previous study conducted in children with acute lymphoblastic leukemia (all), showed that polymorphisms in these genes might play a role in the onset of chemotherapy complications during consolidation and maintenance treatment. objectives: in order to support our previous finding, we have expanded the study to the induction period in a cohort of all children treated at the sainte-justine university health center between july and july . design/method: previous associated single nucleotide polymorphisms (snps) in darc, gsdma and cxcl genes were analyzed for an association with the complications occurring during induction including the duration of low neutrophil count (pnn) and low absolute phagocyte count (apc), proven infections and delay between induction and consolidation phases. results: significant effect was found for all studied polymorphims. minor alleles of darc rs , cxcl rs and gsdma rs were all associated with higher risk of complications during induction treatment, whereas that of darc rs (particularly gg genotype) had a protective effect. the gg genotype of rs was associated with a lower risk of post-induction delay (p = . or = . , %ci . - . ), less frequent febrile episodes (p = . ) and lower number of days with apc/pnn count reduction (p = . for apc< . and p = . for pnn< . ). in contrast, the minor t allele of another darc polymorphism (rs ), was associated with longer apc/pnn count reduction (p = . for apc< . and p = . for pnn < . ), as it was the tt genotype of gsdma rs (p = . for apc< . and p = . for pnn< . ). the patients with the gsdma rs had also a higher risk of documented febrile episodes (p = . or = . %ci - . ). the aa genotype of rs cxcl was associated with a higher risk of post-induction delay due to infection (p = . , or = . , % ci . - . ). conclusion: this complementary study confirmed our previous results, showing overall that variations in darc, gsdma and cxcl genes influence the onset of chemotherapy complications in pediatric all, regardless of treatment phases. these polymorphisms might be useful pharmacogenetics markers possibly guiding an adjustment of chemotherapy intensity. background: pediatric acute myeloid leukemia (aml) has a poor survival rate of about % and there is an urgent need for newer targeted therapies. car t-cell based therapies are effective against all but similar therapies against aml are still under development. recent clinical trials have highlighted the concerns about toxicity and therapy related deaths from car t-cells. antigen selection is the key factor determining the specificity, efficacy and toxicity of car t-cells. while contemporary adoptive t-cell therapies use monoclonal antibodies against tumor associated antigens we employed the naturally occurring flt ligand (fl) to target aml cells expressing flt receptors. flt receptor is expressed on multipotent and myelomonocytic progenitors as well as myeloid leukemia cells. to generate fl containing chimeric tlymphocytes designated flcar t-cells and to evaluate their efficacy against aml cells. design/method: flcar was constructed by fusing the coding sequences of the human fl, cd costimulatory domain, and cd -zeta chain (intracellular region) in series. it was then cloned into the phiv-egfp lentiviral vector for expression in cell lines and primary t cells obtained from healthy donors. the empty phiv-egfp vector was used as a negative control. flcar was expressed on both cd + and cd + t-lymphocytes, confirmed by western blot. cell cytoxicity was evaluated by co-culturing flcar t-cells and aml cells followed by flow cytometric analyses. cytokine production was assessed by analyzing expression of interleukin- using quantitative rt-pcr. results: flcar t-cells were generated from cd + jurkat and cd + tk- cell lines with up to % lentiviral transduction efficiency. the efficiency for primary t cells was lower ( - %). flcar was expressed as a ∼ kda protein in cells and was partially phosphorylated on tyrosine. the expression of flcar on lymphocytes lead to increased basal il- expression in the cells. this was further augmented (by > folds) upon co-incubating flcar t-cells with flt expressing target cells. jurkat cells, tk- cells and primary human t cells expressing flcar suppressed the growth of flt -expressing aml cell lines and primary aml cells in vitro. notably, flcar t-cells generated from healthy donors caused strong inhibition of aml cells even at a lower transduction efficiency. in vivo experiments using nsg-sgm mice xenografted with human aml cells are underway. our data demonstrate that flcar can be effectively expressed on t-lymphocytes and mediate potent cytotoxicity against flt -expressing aml cells in vitro. being a completely human derived chimeric protein, it represents a promising candidate for further therapeutic development. holly pacenta, kelly sullivan, ahwan pandey, kelly maloney, joaquin espinosa children's hospital colorado, denver, colorado, united states background: individuals with down syndrome (ds) have a -fold higher risk of developing acute lymphoblastic leukemia (all) than the typical population. there are several important differences between all in individuals with ds (ds-all) and all in individuals without ds (nds-all): first, patients with ds-all have a lower percentage of favorable cytogenetic features compared to nds-all. second, patients with ds-all are more likely to have activating mutations in jak , crlf overexpression, and ikzf deletions. despite these clear genotypic differences, this knowledge has not yet been exploited for therapeutic purposes in ds-all. when outcomes for ds-all are compared to nds-all with similar cytogenetic features, the survival rates are similar. however, individuals with ds-all have an increased risk of treatment-related mortality (trm). current therapy for ds-all is similar to that for nds-all, with the exception of small changes to decrease toxicities that are more prevalent in ds-all. it was recently identified that interferon signaling is constitutively activated in healthy individuals with t . we hypothesize that aberrant interferon signaling could play a role in the unique leukemias observed in ds patients. objectives: to identify differences in gene expression and intracellular signaling cascades that are unique to individuals with ds-all, relative to both nds-all and healthy individuals with ds that can be exploited for therapeutic use. design/method: bone marrow samples were obtained from ds-all patients and matched nds-all patients based on clinical characteristics and genetic features. rna sequencing of these samples was performed and a total of samples were used for the transcriptome analysis ( ds-all vs. nds-all). the differential expression data was generated by deseq and analyzed using ingenuity pathway analysis. the analysis revealed that the chromosome genes that have been implicated in leukemogenesis are not differentially expressed in the ds-all samples, relative to nds-all. an inflammatory signature was identified, which included interferon gamma as an upstream regulator with predicted activation in ds-all. this finding is consistent with prior observations from healthy individuals with ds. other examples of results with potentially actionable targets include the upregulation of several genes in the ras pathway and genes involved in histone methylation. the increased interferon signaling seen in healthy individuals with ds was also identified in ds-all. this may contribute to the development of mutations in inflammatory pathways such as jak and crlf in ds-all. targeting these common pathways with small molecule inhibitors may have a therapeutic benefit in ds-all. cincinnati children's hospital medical center, cincinnati, ohio, united states background: next-generation sequencing (ngs) guides precision medicine approaches in oncology using therapies targeting molecular alterations found within an individual cancer. increased availability of ngs coupled with a proliferation of targeted drugs in development heightens the need for reliable pre-clinical animal models. here we report a patientderived xenograft (pdx) system with integrated molecular profiling for pre-clinical testing of conventional cytotoxic and novel targeted agents. objectives: to utilize ngs from patients with pediatric leukemia to guide rational pre-clinical trials in pdx leukemia avatars, and to determine pdx mice tolerance of and response to cytotoxic and targeted therapies. pediatric acute lymphoblastic leukemia (all) samples were obtained in adherence to an irb-approved protocol and xenografted into nod/rag/interleukin- (il- )rg (nrg) mice. ngs was performed clinically using the foundationone® heme panel. a de novo all sample bearing mutations involving jak , crlf , ntrk , cdkn a/b, ptpn and wt was used for pre-clinical testing. thirty-seven nrg mice were transplanted with million patient cells/mouse via iv injection. standard -drug induction chemotherapy was administered consisting of vincristine, dexamethasone, pegaspargase, and daunorubicin [vxpd, n = mice], in comparison to vehicle control [n = ]. parallel pdx cohorts were treated with single agent targeted therapies based on ngs findings, including ruxolitinib [n = ], crizotinib [n = ] and loxo- [n = ]. the four-week treatment period began on day + from transplant after confirmation of engraftment. following completion of therapy, residual disease burden was analyzed by flow cytometry (hcd +, mcd -cells) in the bone marrow [bm] . to date, pdx models have been established using over thirty ngs-profiled pediatric all samples, including six samples bearing philadelphia (ph) chromosome or phlike mutations. pre-clinical testing was performed in a repre- conclusion: ngs reveals concomitant mutations in ph-like all that may represent additional targets for therapy, or predict tyrosine kinase inhibitor (tki) resistance. we show that all xenograft nrg mice can tolerate a -week multi-agent cytotoxic chemotherapy induction regimen, as well as rational targeted agents, and serve as a robust pre-clinical model for precision medicine trials. background: osteonecrosis is a well-characterized all therapeutic toxicity attributed to glucocorticoids, asparaginase, and methotrexate that disproportionately affects adolescents. in ccg- , alternate-week dexamethasone during double delayed intensification (di) reduced osteonecrosis vs continuous dexamethasone with single di in rapid early responders (rer) ≥ y. to compare efs and os between hr-all patients with vs without osteonecrosis. design/method: hr-all patients - y on aall ( - ) received cog augmented therapy with a × randomization to: ( ) induction dexamethasone ( mg/m d - ) vs prednisone ( mg/m d - ), and ( ) interim maintenance (im) high-dose methotrexate (hdm) vs escalating-dose methotrexate/pegaspargase (ema). rer received single, and slow early responders (ser) double, im/di. initially, all received monthly dexamethasone maintenance pulses, patients ≥ y received di alternate-week dexamethasone, and patients ≤ y received di continuous s of s dexamethasone. there were osteonecrosis-related amendments: after / all patients ≥ y received di alternateweek dexamethasone; after / all patients ≥ y were assigned to induction prednisone, and all patients received di alternate-week dexamethasone and maintenance prednisone pulses. results: osteonecrosis was confirmed in / patients. the y cumulative incidence (ci) was . % overall and increased with age: - y . %, - y . % (alternateweek dexamethasone . % vs continuous dexamethasone . %; p< . ), ≥ y . % (p< . ). among randomized rer patients ≥ y, ci differed by glucocorticoid (dexamethasone . % vs prednisone . %; p = . ) but not methotrexate assignment (hdm . % vs ema . %; p = . ). among randomized ser patients ≥ y, ci was . % with no difference by regimen. results were similar for patients ≥ y. in the entire study population, patients with osteonecrosis had superior y efs ( . % vs . %; p< . ) and os ( . % vs . %; p< . ) than those without osteonecrosis. y efs was significantly higher among randomized patients ≥ y with vs without osteonecrosis ( . % vs . %; p< . ); this finding was present in different age ranges (≥ y, ≥ y, ≥ y) and rer/ser subsets within each, especially in the ≥ y rer ( . % vs . %; p = . ) and ser ( . % vs . %; p< . ) cohorts. across groups, asparaginase allergy was significantly associated with reduced osteonecrosis risk (≥ y: hr . ; p = . ). patients who develop osteonecrosis have significantly increased efs and os, suggesting host differences that increase sensitivity to develop osteonecrosis and render all cells more chemo-responsive. pennsylvania state university, hershey, pennsylvania, united states background: cdc (cell division cycle protein ) belongs to rho family of small gtpases in ras-oncogene superfamily. pro-oncogenic role of overexpressed cdc in ras driven solid tumors are well known. however, role of cdc in leukemia is yet to be established. ikzf encodes ikaros protein which has important role in regulation of lymphoid development and tumor suppression in leukemia. casein kinase ii (ck ) oncogene is overexpressed in leukemia. ck impairs ikaros function which can be restored by using ck inhibitors. objectives: to investigate role of cdc in leukemia and regulation of cdc by ikaros and ck in b-cell acute lymphoblastic leukemia (b-all). shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: cdc is identified as one of the ikaros target genes by analysis of genome-wide dna binding of ikaros using chip-seq and qchip in b-all primary cells. expression of cdc was also noted to be higher in all patient samples compared to normal bone marrow. functional experiments showed that ikaros overexpression via retroviral transduction results in transcriptional repression of cdc . ikaros silencing using shrna resulted in increased expression of cdc . these data suggest that ikaros negatively regulates transcription and expression of cdc . ck directly phosphorylates ikaros and impairs its function as transcription factor. we noted that molecular inhibition of ck via sirna as well as treatment with specific ck inhibitor, cx also decreases expression of cdc . treatment with cx of primary b-all with ikaros haploinsufficiency restores ikaros binding to cdc promoter and represses cdc expression. however, this effect is evident only in presence of ikaros. treatment with cx in ikaros silenced (ikaros shrna) cells showed no change in expression of cdc . these results emphasizes the importance of ikaros in regulating cdc expression. furthermore, we analyzed the changes in epigenetic signature at the cdc promoter following treatment with cx . results show that loss of histone marker of open chromatin (h k ac) and increased histone marker for repressive chromatin (h k me ), at the cdc promoter. these data suggest that ikaros transcriptionally represses cdc via chromatin remodeling. a specific cdc inhibitor, ml showed cytotoxic effects on primary b-all cells. conclusion: cdc may have important role in hematologic malignancies. expression of cdc in b cell all is regulated by ikaros and ck . these results suggest that targeting cdc could be a potential therapeutic strategy in leukemia. caitlyn duffy, laura hall, justin godown, koyama tatsuki, scott borinstein monroe carell jr. children's hospital at vanderbilt, nashville, tennessee, united states background: systemic corticosteroids are widely used as treatment of acute lymphoblastic leukemia (all) and lymphoblastic lymphoma. there are anecdotal reports of bradycardia in pediatric patients receiving corticosteroids, but a more extensive analysis of this effect is needed. objectives: the aim of this study was to describe the incidence, severity, and timing of steroid-induced bradycardia and document any adverse events associated with bradycardia. design/method: we performed a retrospective review of all newly diagnosed patients at our center ( - ) with all/lymphoblastic lymphoma who received corticosteroids (dexamethasone - mg/m /dose or prednisone mg/m /dose) during induction chemotherapy. patients were excluded if they had a pre-existing cardiac abnormality or if they received prior corticosteroids. the average hour heart rate (hr) was assessed for the period prior to initiating steroid therapy and for the hour period surrounding the nadir following steroid administration. the degree and time of steroid induced bradycardia was assessed. adverse patient events and concomitant medication use was documented to identify other contributing factors to bradycardia. a total of children ( females, males, months- years) were included in the analysis with demonstrating a decrease in mean hr following steroid administration. median hr decrease was . beats per minute (quartiles . - ) from prior to initiating steroids to surrounding nadir. sixty one percent developed bradycardia less than or equal to the st percentile for their age range. nadir occurred doses (range - ) into treatment, which corresponded to hours ( - ) after initiation of therapy. of patients who experienced bradycardia, % were associated with dexamethasone rather than prednisone. hr nadir was not associated with other vital sign abnormalities. after completion of induction chemotherapy, % of patients had documented resolution of bradycardia with hr greater than the th percentile for age. it was observed that the children who continued to have relatively low hr were often younger ( months- years old). examination of nadir hr during subsequent hospitalizations in which steroids were not being administered (excluding hr during procedural sedation) did not demonstrate a significant incidence of bradycardia. concomitant opioid, beta-blocker, or other medication exposure did not contribute to the incidence of bradycardia. corticosteroid-induced bradycardia is extremely common in children, teenagers, and young adults with all receiving induction chemotherapy. bradycardia was not associated with clinical adverse events and resolved after completion of corticosteroid treatment. therefore, further cardiac assessment may not be warranted in the presence of bradycardia suspected to be secondary to steroid administration. baylor college of medicine, houston, texas, united states background: survival in newly diagnosed pediatric acute myeloid leukemia (aml) is approximately %; however survival falls dramatically if a patient relapses. currently, approximately one-third of patients with pediatric aml relapse on standard chemotherapy regimens. aml cells are exposed to proteotoxic stress at baseline due to their rapid and inefficient metabolism; proteotoxic stress increases after chemotherapy due to accumulation of reactive oxygen species resulting in misfolded proteins. this leads to activation of cell stress pathways, such as the unfolded protein response (upr) in the endoplasmic reticulum. because an activated upr can make cells more sensitive to proteotoxic stress, we hypothesize that upr activation correlates with response to chemotherapy. objectives: determine the status of upr in pediatric aml and its correlation with chemosensitivity; design/method: peripheral blood samples from pediatric patients with aml were collected at the start of induction chemotherapy, - hours (h) and h post initiation of systemic chemotherapy. tumor cells were sorted from peripheral blood mononuclear cells. expression of upr proteins was determined by chemiluminescence using an automated capillary electrophoresis system. clinical correlations were performed using an annotated database. we measured five upr proteins: grp (glucose regulated protein kda), phospho-eif , inositol-requiring enzyme (ire ) and activating transcription factor (atf ). patients with aml had - times higher expression of upr proteins (except atf ) at baseline than normal controls. grp -the key upr driver-had the highest level of protein expression in myeloid blasts. there was a wide variability in the level of baseline upr expression. eight out of samples expressed > fold increase in grp above those with the lowest grp levels. similarly, and patients respectively, had a > fold increase in peif and ire , compared to patients with low basal expression of these upr proteins. in our limited sample set, there was a trend towards lower overall survival (os) and event-free survival in patients with low baseline grp and ire . conclusion: upr has a variable expression at baseline in pediatric aml, with a trend towards lower os in patients with a low basal grp and low ire expression, suggesting less chemosensitivity in this subgroup. conversely, it is possible that blasts with an upregulated upr prior to chemotherapy manage proteotoxic stress less effectively, having faster apoptosis and hence a better response to chemotherapy in patients with a high basal upr. we are currently expanding our findings in a larger cohort of patients enrolled in the children's oncology group aaml protocol. background: children with newly diagnosed acute lymphoblastic leukemia (all) undergo chest x-ray (cxr) evaluation during initial diagnostic workup to ensure safe airway management. however, to our knowledge, no systematic assessment of cxr findings has been reported. objectives: to evaluate cxr findings at diagnosis of all and their associations with clinical characteristics. we reviewed the cxr findings at diagnosis of all in patients treated on the total xv and xvi protocols at st. jude children's research hospital. findings were evaluated for associations with clinical characteristics at presentation, and the clinical management of mediastinal masses was reviewed. mediastinal masses were seen in ( . %) of patients evaluated and were more common in older patients (mean age, . years) than in younger patients (mean age, . years) (p = . ), in males than in females (p = . ), and in patients with t-all than in those with b-all (p< . ). also associated with mediastinal masses were a higher white blood cell count (wbc) at diagnosis (mean, . × /l) (vs. a lower wbc; mean, . × /l) (p< . ), cns involvement (vs. no involvement) (p = . ), and standard/high-risk disease (vs. low-risk disease) (p< . ). other cxr findings included pulmonary opacity ( patients [ . %]), bronchial/perihilar thickening ( patients [ . %]), cardiomegaly ( patients [ . %]), and osteopenia/fracture/periosteal lesions ( patients [ . %]). pulmonary opacity was more common in younger patients (mean age, . years) than in older patients (mean age, . years) (p = . ) and in those with t-all (vs. b-all) (p = . ). bronchial/perihilar thickening, cardiomegaly, and osteopenia/fracture/periosteal lesions were also more common in younger patients than in older ones (p< . , p = . , and p< . , respectively) and in those with low-risk disease (versus standard/high-risk disease) (p< . , p = . , and p = . , respectively). of the patients with a mediastinal mass on cxr, underwent a confirmatory chest ct scan, and ( . %) were confirmed to have a mediastinal mass. notably, patients ( . %) had airway compression, and compression of venous structures was identified in of patients ( . %) who received iv contrast. the clinical course was evaluated for patients with mediastinal masses detected by cxr. fifty patients ( . %) required icu admission (mean stay, . days). general anesthesia was used for only patients ( . %), and patients ( . %) had a less invasive peripherally inserted central catheter. no deaths occurred in the acute phase. conclusion: cxr at the time of all diagnosis can detect various intrathoracic lesions and is helpful in planning initial diagnostic workup and management. background: mertk is a receptor tyrosine kinase that is aberrantly expressed in % of pediatric primary aml samples. mertk inhibition with the small molecule tyrosine kinase inhibitor (tki) mrx- decreases tumor burden and prolongs survival in aml xenografts. while treatment with mrx- reduces leukemia in the peripheral blood, it is less effective in the bone marrow, suggesting a role for the marrow microenvironment in therapeutic resistance. the jak/stat pathway has been implicated as a mediator of bone marrow derived resistance to tkis and inhibitors of this pathway are in clinical development for the treatment of aml. to determine the role of the bone marrow stromal niche in mediating resistance to mertk inhibition and to evaluate the efficacy of combined mertk and jak/stat inhibition. design/method: aml cell lines were cultured with or without the hs stromal cell line or hs conditioned medium, then treated with mrx- +/-the jak/stat inhibitor ruxolitinib, or control. induction of apoptosis and cell cycle arrest in aml cells was measured by flow cytometry. expression of h ax and total and phosphorylated stat were determined by immunoblot. results: co-culture with stromal cells significantly reduced aml cell death and g /m phase arrest in response to treatment with nm mrx- compared to no co-culture (cell death: . % versus . %, p< . ; g /m arrest: . % versus . %, p< . ). g /m arrest was accompanied by an increase in h ax expression which was similarly abrogated in co-culture. conditioned medium did not provide protection from mrx- induced apoptosis, g /m arrest, or h ax induction. mrx- inhibited stat phosphorylation but direct co-culture and conditioned medium potently increased basal stat phosphorylation which was not inhibited by mrx- . to determine whether the observed induction of stat phosphorylation was functionally relevant, cocultures were treated with both mrx- and ruxolitinib. while ruxolitinib potently inhibited the phosphorylation of stat in the presence of co-culture, combination treatment did not overcome stromal mediated protection from mrx- induced apoptosis. similarly, the addition of exogenous gm-csf induced stat phosphorylation but did not yield protection from mrx- functional effects in the absence of co-culture. together these data support a model whereby direct cell-cell contact with stromal cells in the bone marrow niche protects leukemia cells from mrx- induced apoptosis, cell cycle alterations, and dna damage. while co-culture potently induces phosphorylation of stat in leukemia cells, this is neither necessary nor sufficient for stromal-cell mediated protection from mertk inhibition and combined treatment with jak/stat inhibitors is unlikely to be therapeutically efficacious. background: mercaptopurine ( -mp) is an immunosuppressive thiopurine drug that is a key component of acute lymphoblastic leukemia (all) treatment. -mp is metabolized into -thioguanine ( -tgn), which is responsible for anti-leukemic effects, as well as -methylmercaptopurine nucleotides ( -mmpn/ -mmp), which are associated with hepatotoxicity. some patients preferentially metabolize -mp to -mmpn/ -mmp, increasing their risk for hepatotoxicity and potentially reducing anti-leukemic effects. hepatotoxicity can cause interruptions or delays in therapy that may jeopardize cure rates. allopurinol has been increasingly used in patients with inflammatory bowel disease (ibd) to shunt -mp metabolism toward -tgn and away from -mmpn to minimize hepatotoxicity and preserve therapeutic effects. objectives: this retrospective chart review expands upon our previously published case series of three patients with all in whom allopurinol was successfully used to redirect -mp metabolism. twelve additional patients have subsequently received allopurinol and -mp combination therapy at texas children's hospital. data from this larger patient sample, with longer follow up, is being analyzed to increase knowledge of the effectiveness and longitudinal effects of adding allopurinol to -mp to reduce risk of hepatotoxicity. design/method: data were abstracted from the electronic medical records of patients with all treated at texas children's hospital from to present, who had been found to have evidence of altered -mp metabolism and in whom allopurinol was added to -mp therapy due to concern for risk or recurrence of hepatotoxicity. metabolite levels, -mp dose, and alanine transaminase (alt) prior to initiation of allopurinol and approximately weeks later were compared. wilcoxon signed-rank test was applied for statistical analysis. : after the addition of allopurinol, patients experienced a significant decrease in mean levels of -mmpn (p = . ), correlating with a significant decrease in mean alt (p = . ). with the initiation of allopurinol, the mean -mp dose was decreased from to mg/m /day over an -week period. mean -tgn levels increased (p = . ). in follow up beyond weeks, no patients had further holds in -mp due to hepatotoxicity. addition of allopurinol appears to shift metabolism from -mmpn toward -tgn, with increases in mean -tgn levels despite a decrease in mean -mp dose. this may limit negative side effects, thus resulting in fewer gaps in therapy and possible improved outcomes. further analysis of -mp dose titration and effects on anc over time as well as effects on overall survival is ongoing. prospective background: alterations in epigenetic patterning are a fundamental feature in acute myeloid leukemia (aml). treatment with dna methyltransferase inhibitors (dnmti) yields responses in aml, but the molecular mechanisms underlying this effect are poorly understood. in prior work, we demonstrated induction of genes involved in the pirna rna (piwi) silencing pathway as a common gene feature of aml cell lines treated with decitabine. the piwi pathway is an rna silencing system, distinct from classical small rna transcriptional silencing, responsible for transposon-silencing in gametogenesis; emerging data suggest a role for this system in somatic cells. based on these data, we postulate that piwi induction plays a crucial role in aml recovery following demethylation and that disruption of this pathway would modulate response and/or recovery from decitabine treatment. to assess the effect contribution of the pirna pathway response following dnmti treatment in aml. design/method: to choose target genes in the pirna pathway for disruption, molm cells were first treated with escalating doses of decitabine. using quantitative rt-pcr, the dose-dependent expression of several pirna-associated genes were analyzed. two genes, mael and piwil , were selected for disruption experiments based on preliminary data suggesting decitabine dose-dependent responses. molm cells were transduced with shrna targeting these genes using a lentivirus delivery system with selection in puromycin. knockdown efficiency was assessed by rt-qpcr. to determine how gene disruption affected cell growth, knockdown cells were treated with decitabine nm. proliferation was assessed by celltiter glo assay following decitabine treatment. clonogenic potential was assessed by colony forming assays of transduced cells after treatment with decitabine at nm and nm. results: following decitabine exposure in molm , there was a markedly increased expression of mael and piwil compared to untreated cells ( : and : , respectively) . thus, these were the candidate genes chosen for disruption. of mael shrna constructs, two resulted in a % relative expression of mael compared to controls. of the piwil shrna constructs, the best knockdown showed % relative expression. there were no significant differences in proliferation or clonogenicity of stably selected mael or piwil knock-down molm cells following decitabine treatment. using gene knockdown procedures, mael and piwl do not appear to have a marked effect on growth and response to decitabine treatment in molm . however, these results may be limited by inefficient knockdown using shrna targeting methods. further work using a cas /crispr based inactivation of these genes is ongoing. children cancer hospital cairo, egypt background: hypodiploidy < chromosomes is very uncommon and have particularly poor outcomes in childhood acute lymphoblastic leukemia (all). it is subdivided into: near-haploid ( - chromosomes), lowhypodiploid ( - chromosomes) and high-hypodiploid ( - chromosomes). to determine if minimal residual disease (mrd) can identify a group of patients with better prognosis in the hypodploid population who can be treated with intensive chemotherapy alone. design/method: a retrospective study that included all patients under age of diagnosed as hypodiploid b-precursor all during the period between january -december and treated at children's cancer hospital egypt on sjcrh total study-xv for ir/hr all. sixteen patients had < chromosomes ( nearhaploid and low-hypodiploid), constituting % of all pediatric patients with b-precursor all during the study period. patients with near-haploid all had a median age of years (range - ), initial leukocyte count (wbc) median of . × /l (range . - . ), ( . %) were males and / ( . %) had hr-nci criteria. four patients ( . %) are alive in complete remission(cr) (range - months, median ), one died in induction and ( . %) had hematological relapse (range . - months, median ). patients with low-hypodiploid all had significantly older age (median years, range - ), median wbc . × /l (range . - . ), / ( . %) were males. one patient ( . %) is alive in cr, one died in induction, one failed to achieve cr post-induction and patients( %) had hematological relapse (range . - . months, median . ). mrd< . % by flow-cytometry on day- and end of induction was achieved in / ( . %) and / patients( %) with near-haploid, compared with / ( . %) and / patients( %) with low-hypodiploid; respectively (p = . , p = . ;respectively). allogeneic transplantation was performed during initial remission only in mrd negative patients (one relapsed and one is in cr) and in the patient with induction failure (relapsed post-transplant). five of the total six patients who had negative mrd on day- and end of induction are alive in cr ( / with chemotherapy alone). all patients with negative mrd at end of induction but with mrd levels≥ . % on day- (range . - . %) relapsed as well as all patients with detectable mrd at the end of induction. the difference in relapse was statistically significant in relation to negative-mrd on day- (p = . ), but not at end of induction(p = . ). conclusion: children with hypodiploid all and negative mrd on day- of induction are highly curable with intensive chemotherapy alone, while patients with negative mrd at the end of induction and detectable mrd on day- had dismal outcome. background: overall survival in pediatric acute myeloid leukemia (aml) has plateaued between - %, with death during induction chemotherapy seen in - % of patients. respiratory complications contribute to morbidity and mortality in pediatric aml induction, however the incidence, patterns, and predictors of respiratory adverse events (aes) during this period are unknown. to estimate the incidence of respiratory aes during induction therapy for de novo pediatric aml, to characterize and grade these respiratory aes, and to identify predictors of respiratory ae development. we conducted a retrospective longitudinal study from presentation to day in institutional de novo pediatric aml patients (≤ years) between march and december . outcomes included any nci ctcae grade - respiratory ae or death from another cause. demographic, disease, and treatment-related data were abstracted. the most specific, best-fitting ctcae category and grade for each ae was determined. descriptive statistics, survival analysis, multivariable logistic regression analysis, and time-toevent distributions were performed (sas v . , cary, nc) . among eligible patients, . % (n = ) experienced discrete respiratory aes. incidence of grade - aes was . % (n = ). a bimodal time-to-event distribution demonstrated peaks at treatment days and . induction death occurred in . % (n = ) including deaths from respiratory failure associated with disseminated fungal disease. in univariate analysis, those experiencing aes differed significantly in regards to older age at diagnosis (p< . ), higher initial wbc (p = . ), higher initial peripheral blast percentage (p = . ), coagulopathy at diagnosis (pt (p = . ), d-dimer (p = . )), fluid overload status (p< . ), occurrence of infection (p = . ), and occurrence of tumor lysis syndrome (tls) (p = . ). patients with hyperleukocytosis (p = . ), fluid overload (p< . ), and fab m morphology (p = . ) each had a significantly decreased probability of completing the follow up period without experiencing a respiratory ae. on multivariable analysis, fluid overload (aor . [ % ci: . - . ) and older age (aor . [ % ci: . - . ) were significantly associated with ae occurrence when gender, hyperleukocytosis, tls, and infection status were held constant. we describe a high incidence of respiratory aes during pediatric aml induction. fluid overload and older age at diagnosis are independently associated with ae development when controlling for other proposed risk factors. interventions focused on conservative fluid management and offset of fluid overload should be explored in newly diagnosed pediatric aml in an effort to reduce respiratory complications during induction. overall, all survival rates are outstanding and have continued to improve with risk-adapted therapy. the most striking improvement occurred in t-all where -year os rates now exceed % and parallel b-all. survival improvements, however, have not been observed uniformly across all subgroups. while the gap in outcome differences narrowed among blacks, outcomes for hispanics have remained static. further, no improvements in survival were observed in infants or ayas and new treatment approaches have been implemented for these populations. background: acute myeloid leukemia (aml) accounts for approximately % of new childhood leukemia cases. chest x-ray (cxr) is performed in all newly diagnosed aml cases to evaluate the safety of airway management for anesthesia during diagnostic procedures; however, cxr results in pediatric patients with aml have not been described. objectives: the primary objective was to evaluate cxr findings at diagnosis in patients with aml. the secondary objectives included assessing associations between cxr findings and clinical characteristics, with the overall goal of aiding in the evaluation of the use of cxrs as an initial diagnostic study in pediatric patients with aml. design/method: cxr findings and clinical characteristics were evaluated in patients with newly diagnosed aml who were enrolled in one of three protocols at st. jude children's research hospital (aml , aml , and aml ). the findings were categorized based on radiologic reports. further, the associations of these findings and clinical characteristics were evaluated. we evaluated cxr findings in a total of patients: from aml ; from aml ; and from aml . common cxr findings were pulmonary opacity (n = , . %), bronchial/perihilar thickening (n = , . %), splenomegaly (n = , . %), mediastinal mass and lymph nodes (n = , . %), pleural effusion/thickening (n = , . %), demineralization/fracture/periosteal lesions (n = , . %), scoliosis (n = , . %), and granulomatous disease (n = , . %). three cxr findings were associated with younger age at diagnosis: pulmonary opacity (median age, . years in patients with positive findings vs. . years in those with negative findings, p< . ), bronchial/perihilar thickening (median age, . years vs. . years, p< . ), and demineralization/fracture/periosteal lesions (median age; . years vs. . years, p = . ). two cxr findings were associated with older age at diagnosis: scoliosis (median age, . years vs. . years, p< . ) and granulomatous disease (median age, . years vs. . years, p = . ). higher white blood cell counts (wbcs) at diagnosis were associated with cxrs showing pulmonary opacity (median wbc; . × ^ /l vs. . × ^ /l, p = . ) or splenomegaly (median wbc; . × ^ /l vs. . × ^ /l, p = . ). french-american-british (fab) m /m subtypes were more frequently associated with pulmonary opacity compared with others (p< . ). we did not find significant differences between female and male patients. conclusion: cxr in patients with newly diagnosed aml showed a variety of thoracic, abdominal, and bony lesions that are important for the initial evaluation and management. pulmonary opacity was the most common finding and was frequently seen in patients who were younger or had higher wbcs at diagnosis or fab m /m . background: children diagnosed with acute lymphoblastic leukemia (all) require a central venous catheter (cvc) to administer chemotherapy safely. both external and internal cvcs carry risks of complications including thrombosis, infection, and possible replacement. internal catheters, such as a port, are generally used for the majority of patients for the duration of treatment since therapy lasts for several years. many institutions place a port at the time of diagnosis. other institutions prefer to start induction therapy via placement of a peripherally inserted central catheter (picc) and defer port placement until the completion of induction therapy due to concerns of increased risk of infectious complications with port placement. objectives: to compare rates of common cvc associated complications by type of cvc placed at start of induction therapy in children treated for newly diagnosed all at the jimmy everest center (jec) at the university of oklahoma health sciences center. design/method: a retrospective chart review analyzed data from newly diagnosed all patients treated at the jec between - . data was collected on complications including thrombosis, bacteremia, insertion site infection, cvc malfunction and need for removal. data collection began at the start of induction and was completed at the end of induction therapy. statistical analysis used a univariate and multivariate logistic regression model to compare complication rates between those who had a port versus those who had a picc placed at start of induction. results: data was collected on patients. fifty-six patients had a port placed at start of therapy while had a picc placed. fourteen percent of patients had a cvc associated complication. univariate analysis showed no statistically significant difference in rates of cvc associated complications between the groups (port %, picc . % p = . ). the rates of hospitalization for cvc associated complications were similar between both groups (port %, picc % p = . ). rates of cvc removal were also similar between both groups (port %, picc % p = . ). multivariate model that included baseline patient characteristics including type of all, patient body surface area, gender, ethnicity and age continued to demonstrate no significant difference in cvc associated complications between both groups. conclusion: this single institution study showed that there was no significant difference in cvc associated complications between port and picc line placement at the start of childhood all induction therapy. port placement can be considered as a safe option at the start of induction therapy. complete remission [cr] or cr with incomplete blood count recovery [cri]) within treatment cycles - . interim data are reported (nct ). results: seventeen patients were enrolled and received ≥ dose of lenalidomide; median age was years (range - ); patients were female. patients received median prior regimens (range - ). nine patients had previously undergone bone marrow transplantation (bmt). four patients had relapsed aml and were refractory to immediate prior treatment. median duration of study treatment was weeks (range - ); patients completed a median of treatment cycle (range - ). all patients were evaluable for primary outcome; achieved morphologic cri after cycles (no patients achieved cr). the responder was a -year-old male with history of r/r aml after first-and second-line treatment, bmt, and salvage chemotherapy. at baseline, he had a complex cytogenetic karyotype (monoallelic − q . , − q, − q . , − p ) with no identifiable molecular mutation; he was also positive for del( q) (− q , − q ). his post-treatment karyotype showed no abnormalities. sixteen patients experienced treatment failure; due to resistant disease, of indeterminate cause, and had treatment failure before a post-baseline assessment was performed. all patients experienced ≥ grade - treatment-emergent adverse event (teae). the most commonly reported were thrombocytopenia (n = ), anemia (n = ), febrile neutropenia (n = ), and hypokalemia (n = ). fifteen patients experienced ≥ teae related to lenalidomide. all patients discontinued treatment; remain in follow-up. the study is now closed to enrollment. ten patients died on study: during treatment, during follow-up. all deaths were attributed to aml or complications due to aml. conclusion: third-line lenalidomide monotherapy was associated with clinical response in of pediatric patients with r/r aml; however, treatment exposure was limited. safety data are consistent with the known profile of lenalidomide. lenalidomide was not an efficacious treatment for r/r pediatric aml. funding: celgene corporation, summit, nj, usa. cook children's medical center, fort worth, texas, united states background: it is well documented that pediatric patients with acute lymphoblastic leukemia (all) often experience significant weight gain during induction therapy and later struggle with obesity. however, some patients experience unintended weight loss during induction therapy; since this issue is not well reported, it often goes undertreated. although malnutrition is reported to be associated with decreased survival, increased risk of infection, and loss of lean body mass, there remains a scarcity of in-depth analysis of prevalence and risk factors that contribute to this problem. our study attempts to address this critical yet unmet need. objectives: our aim was to identify the clinical risk factors and outcomes associated with weight loss during induction therapy for pediatric all. design/method: this was a retrospective chart review of patients between and years of age diagnosed with all at cook children's medical center from / / to / / . for each patient, we collected height, weight, age, body mass index (bmi) z-scores at diagnosis and end of induction therapy, risk stratification, and whether consolidation was delayed. patients with a bmi > th percentile at diagnosis were categorized as being overweight or obese. using logistic regression analyses, we examined which variables predicted whether the patient had an increase or decrease in bmi z-score throughout induction. a critical alpha level of . indicated statistical significance. results: ninety-six patients met our inclusion criteria. of these, % experienced a decrease in bmi during induction therapy. compared to patients whose bmi increased during induction, patients with a decrease in bmi were more likely to be overweight or obese at diagnosis ( % vs. %; p< . ), to be ≥ years of age ( % vs. %; p< . ), to have a high-or very-high-risk stratification ( % vs. %; p< . ), and to experience a delay in the start of consolidation therapy ( % vs. %; p< . ). conclusion: this research highlights a risk not previously identified in the literature that may impact outcomes. patients treated on high-or very-high-risk protocols, who are overweight or obese at diagnosis, and who are ≥ years of age at diagnosis should be monitored closely for weight loss during induction therapy. patients who experience weight loss should receive prompt intervention. it is our hope that this information can be used for future prospective studies and to help develop evidence-based guidelines. background: p abnormalities have been observed in some patients with hematologic malignancies. loss of p function as a tumor suppressor gene in the chromosome plays an important role for development of leukemia. these patients usually have poor outcome due to the chemotherapy and are associated with poor prognosis. objectives: this study aimed to identify frequency of p abnormalities between iranian children and adult patients with aml (acute myeloid leukemia) malignancy. design/method: the p abnormalities were analyzed via bone marrow karyotyping and fish method in acute myeloid leukemia patients. in this study, p abnormalities were observed in ( %) patients out of diagnosed cases. a significant strong correlation between p abnormalities and other high risk factors (poor risk cytogenetic) were observed. from patients with aml malignancy ( p abnormalities), ( %) patients have complex karyotype, ( %) patients monosomal karyotype and ( %) patients have monosomal karyotype accompanied with a complex karyotype. overall, p abnormalities are independent risk factor in acute myeloid leukemia and evaluation of these abnormalities by fish or other complementary techniques prior to treatment, might help for better risk stratification of high risk aml patients. background: hepatotoxicity in treatment of acute lymphoblastic leukemia (all) is well studied and transiently affects most patients receiving antimetabolite therapy. rarely, patients develop liver injury severe or prolonged enough to undergo a liver biopsy. little is known about how these patients differ from patients that develop transient hepatotoxicity. we sought to describe disease and treatment characteristics for all patients that developed hepatotoxicity severe enough to undergo liver biopsy. we also looked for pre-dictive factors for liver biopsy, including signs of early hepatic injury from the initial treatment protocol. design/method: pathology reports of all patients from the liver biopsy database at children's healthcare of atlanta were collected. controls were matched : for age, all subtype, and treatment protocol. demographics, treatment protocols, and overall outcomes were collected through the electronic health record. hepatic lab results for transaminases, coagulation, and albumin were collected for induction, consolidation, interim maintenance, delayed intensification, and maintenance. results: sixteen patients diagnosed between - (median age at diagnosis years, range - ; % male; % pre-b all) were included in the case series. the median time from diagnosis to liver biopsy was . years (range - ). eight patients ( %) were in maintenance at the time of biopsy; none had active disease. eight ( %) were postbone marrow transplant. biopsy results included: steatosis ( ), acute inflammatory/infectious ( ), liver infiltration ( ), fibrosis ( ) and graft-vs-host disease (gvhd) ( ). six patients were deceased; -year all-cause mortality from diagnosis was %. thiopurine methyltransferase (tpmt) status was known in % cases and % controls. all cases had intermediate or wildtype status, which did not differ from controls (p > . ). patients requiring liver biopsy did not have evidence of acute hepatotoxicity (ast/alt > × normal values) during their initial treatment protocol. hepatotoxicity requiring liver biopsy is a rare outcome of all treatment. these patients had elevated rates of relapse, bmt, and -year all-cause mortality, suggestive of a more severe disease process. however, it is difficult to sort out the temporality of relapse, bmt, and hepatoxicity requiring biopsy in this limited sample. additionally, patients with bmt preceding liver biopsy have other confounding factors that makes them difficult to include in the analysis. finally, our limited descriptive data show no notable correlation between early hepatotoxicity and later indication for liver biopsy. future cohort or case-control studies with larger sample sizes are required to further explore early predictive factors for severe hepatotoxicity requiring liver biopsy. nathan gossai, joanna perkins, michael richards, yoav messinger, bruce bostrom background: the majority of chemotherapeutic agents used to treat hodgkin lymphoma are teratogenic. pregnancy screening prior to the start of chemotherapy is supported by clinical guidelines and baseline testing is a standard component in therapeutic trials. there is limited data available on the incidence of pregnancy screening prior to the start of hodgkin therapy but previous studies suggest that pregnancy screening, especially at pediatric institutions, is not consistently completed. objectives: the objective of this study is to evaluate the incidence of pregnancy screening and contraceptive counseling prior to the start of therapy in females diagnosed with hodgkin lymphoma. design/method: a retrospective chart review was performed for all female patients newly diagnosed with hodgkin lymphoma from to at the hospital for sick children in toronto, ontario. all patients who were intended to receive multi-agent chemotherapy were included, regardless of age. data collected included demographic and disease information, chemotherapy regimen and enrollment on clinical trial. all pregnancy testing within two weeks prior to the start of therapy was captured, as well as type of pregnancy test performed, documentation of menstrual status, contraceptive counseling and contraceptive provision. univariate and multivariate analyses were used to describe factors influencing the incidence of pregnancy testing. results: a total of female patients with newly diagnosed hodgkin lymphoma between the ages of and years were identified. sixty patients ( %) had pregnancy testing done prior to the start of therapy. testing modalities included serum and urine screens as well as quantitative beta-hcg measures. older age (p = . ), documentation of menstrual status at diagnosis (p = . ) and diagnosis between and (p = . ) were associated with higher incidence of screening. enrollment on a therapeutic trial was not associated with a higher incidence of screening (p = . ). contraceptive counseling was documented for patients ( %) and patients ( %) were prescribed contraceptive medications during therapy. pre-chemotherapy pregnancy testing was completed on % of females with newly diagnosed hodgkin lymphoma. improvement is required and interventions, including clarification of institutional standards, modification of chemotherapy order sets and staff education, are planned. (rao et al., cancer, ) . university of louisville, louisville, kentucky, united states background: granulocytic sarcomas (also known as chloromas or leukemia cutis) were first described by a. burns in . they are solid tumors comprised of immature granulocytic cells and represent extramedullary manifestations of underlying leukemia. chloromas are most commonly associated with acute myeloid leukemia. they may arise in other myeloproliferative disorders but are rarely seen in b or t cell acute lymphoblastic leukemia (all). objectives: although patients with all rarely have chloromas, it should remain on the differential for patients with unusual swelling or masses. design/method: we present a case series of two patients from our institution diagnosed with b cell all who had a chloroma as the presenting symptom. the first patient is a yo who presented to his primary provider with nasal congestion and a one-week history of bilateral eye swelling and was referred to an allergist when the symptoms did not resolve with anti-histamines. his review of systems was otherwise negative. he was referred urgently to ent two months later for a × cm mass palpated along the medial border of the left eye. an mri showed a left facial mass surrounding the zygoma and extending into the anterior inferior left orbit. biopsy revealed b cell acute lymphoblastic lymphoma, and bone marrow aspirate and biopsy confirmed the diagnosis as b cell all. the second patient is a yo who presented to his primary doctor for rapid growth of a scalp nodule that had been present for about months. he was referred to dermatology and treated for a supposed kerion from tinea capitis. the lesion continued to grow and became more irritated with this treatment. punch biopsy revealed a complicated phenotype of lymphoblastic lymphoma. however, after a lymph node biopsy and bone marrow aspirate and biopsy, the diagnosis was confirmed as b all. his only other positive point on review of systems was a questionably pathologic -pound weight loss and an area of matted cervical lymph nodes. for both of our patients, the chloromas completely disappeared during induction therapy. it is worth noting that both of these patients presented with the chloroma as the only symptom of the underlying leukemia. this led to initial misdiagnosis and delay in identifying their leukemia. therefore, while it is very rare for a patient with b all to present with a chloroma, our experience shows that all should be on the differential for patients presenting with unusual swelling or masses. background: hodgkin lymphoma (hl) is a lymphoproliferative neoplasm that commonly presents with history of adenopathy and a predictable pattern of disease involvement with or without systemic symptoms of fever and/or weight loss. in the hands of an experienced oncologist the diagnosis of hl is usually not a challenge. occasionally a diagnostic challenge is presented by a patient who has an atypical presentation which is suggestive of an alternative diagnosis. we describe a case series of patients diagnosed with hl whose initial clinical presentations lead to a diagnosis different form hl. honduras, nicaragua and the united states. results: six pediatric oncology centers from the american continent conducted a retrospective review of patients diagnosed with hl since . patients that had an initial presentation not suggestive of hl or who were initially diagnosed with a disease other that hl were included for a total of patients. argentina n = , guatemala n = , honduras n = , nicaragua n = , united states n = . five patients were female and male. patient's ages ranged from to years. most patients (n = ) were older than years. three patients ( %) presented with non-immune cytopenias without overt lymphadenopathy, of those one had active hemophagocytic syndrome. five patients ( %) were suspected to have localized solid tumors: ewing sarcoma n = , rhabdomyosarcoma n = , hepatocellular carcinoma n = , and soft tissue tumor of the cheek n = . two ( %) metastatic solid malignancy as they presented with disseminated pulmonary nodules. five ( %) with autoimmune disorders: hashimoto thyroiditis n = , autoimmune hemolytic anemia n = , nephrotic syndrome n = . ten ( %) with chronic infectious processes: brucella n = , tonsillar abscess n = , splenic abscess n = , and tuberculosis (tb) n = . patients with suspected tuberculosis were diagnosed outside of the united states. six of patients were ultimately diagnosed as having both tb and hl. seventeen patients had ann-arbor stage iii or iv, seven patient had stage ii with either b symptoms or bulky disease. patients were treated with various chemotherapy regimens according to the treating center: abvd, abve-pc oepa-copdac, avpc, beacopp. two patients had recurrent disease, one died of disease progression and one died from causes not related to hl conclusion: a small proportion of hl patients have atypical or unusual presentations. hl should be included in the differential diagnosis of solid tumors, autoimmune disorders, infections or cytopenias. the most common atypical presentation is an infectious process. background: acute lymphoblastic leukemia (all) represents the largest group of pediatric malignancies. the high cure rate of childhood all represents one of the most remarkable success stories in the war on cancer. in a lower middle income country (lmic) like the philippines, we reviewed the five year survival in a tertiary referral center. objectives: this retrospective cohort study aims to determine the survival of children - years old with all treated at a tertiary referral center for childhood cancer in the philippines from january to december . design/method: this is a retrospective cohort study that reviewed medical charts of newly diagnosed all ages to years old from january to december . a total of subjects were included in the study. the year overall survival (os) and event free survival (efs) were . % and . %, respectively. the year os for standard risk all was . % and for high risk patients was %. the year os for the patients on remission was . % and for those who relapsed was . %. univariate and multivariate by cox proportional hazards regression revealed wbc count at diagnosis, risk classification, immunophenotyping, and development of relapse showed significant prognostic impact for mortality. age and gender were reported with no prognostic significance. the -year os and efs were lower compared to developed countries but are comparable with other lmics. the prognostic factors for relapse and mortality were compatible with the literature. overall, the adopted treatment protocols for childhood all in this institution showed acceptable results. relapse has a significant prognostic impact for mortality. development of accessibility to care, increase awareness, early detection and resources at hand should be achieved. improvement in the follow up protocol to prevent delays in the treatment, patient education to prevent non-compliance and psychosocial support, to developed better supportive care, and expand facilities should be given emphasis to further improve survival and prevent relapse. objectives: here, we seek to further characterize this entity by describing the pathologic and clinical features of pediatric cases of burkitt-like lymphoma with q aberration. we collected pathologic and clinical data from the medical record on all pediatric high grade b-cell lymphoma (hgbcl) cases diagnosed at our institution over a -year period ( - ) . for those cases classified as neither burkitt lymphoma nor diffuse large b-cell lymphoma (dlbcl), fish for myc, bcl- and bcl- , as well as array comparative genomic hybridization (acgh), were performed. we identified cases of hgbcl, including cases of burkitt lymphoma presenting as purely leukemic phase. of the hgbcl cases, had burkitt lymphoma as defined by myc rearrangements, and had dlbcl. collectively, the majority of these patients had primary disease outside of the head/neck, and most patients presented with advanced stage (iii-iv) disease. of the remaining cases, q aberration was identified in cases using acgh. all cases histologically and immunophenotypically resembled burkitt lymphoma but lacked myc rearrangement, instead showing proximal gains in q -q and telomeric losses in q . qter. all cases involved primary disease in the cervical lymph node and/or tonsil. three of these cases were localized (stage ii), and the fourth case involved a few metabolically active but non-enlarged lymph nodes in the chest and abdomen (stage iii). all patients achieved complete remission with standard therapy for mature b-cell lymphoma, and were alive with no clinical evidence of disease at a median follow-up of months. although the number is small, our results suggest that the majority of non-burkitt, non-dlbcl cases of pediatric hgbcl carry q aberrations. in addition, patients with q aberrations appear to be more likely to present with lower stage disease, thus requiring less intensive therapy, and also tend to have primary disease in the head/neck. these findings further support the classification of burkitt-like lymphoma with q aberration as a distinct pathologic and clinical entity, and we propose that all pediatric non-burkitt, non-dlbcl cases of hgbcl regularly undergo further workup for possible q aberrations. marie claire milady auguste, joseph bernard st damien hospital, port-au-prince, port-au-prince, haiti background: hodgkin lymphoma (hl) and non-hodgkin lymphoma (nhl) account for % of cancers in the united states pediatric population ( , ). in central america and the caribbean, they are in second position among all types of pediatric cancers ( ). a previous study on pediatric cancers in haiti showed that the lymphomas were in fifth place after the leukemias, wilms tumor, retinoblastoma and the sarcomas ( ). the main objective of this study is to present the epidemiological profile of lymphomas managed at a haitian pediatric hospital. design/method: this is a retrospective study conducted on the cases of lymphoma diagnosed and managed at st damien hospital from january to december . key variables such as age, gender, stage at diagnosis, histopathological types and outcome were collected to present the characteristics of this retrospective cohort. of the cases of cancer diagnosed during the study period, ( . %) had the diagnosis of lymphoma. the sex ratio was . ( males for females) and the average age was . years [ - years]. there were cases of hl ( . %) and cases of nhl ( . %). . % of the patients were diagnosed at stages iii and iv. among the hl cases, ( . %) were nodular sclerosis lymphoma, ( . %) with mixed cellularity and ( . %) with lymphocytic predominance. for the nhl cases, ( . %) were burkitt's lymphoma and ( . %) lymphoblastic t-cell lymphoma. among the patients for who immunohistochemistry was found, the cases of hl were cd -positive and out of cases of nhl were cd -negative. only patient was hiv-positive, and patients had a confirmed exposure to epstein-barr virus. patients ( . %) were lost to follow-up, ( . %) were in remission, ( %) relapsed, ( . %) were still in treatment and ( %) were deceased. university of chicago, chicago, illinois, united states background: due to the adoption of risk-adapted therapy, pediatric and adolescent acute lymphoblastic leukemia (all) is associated with high cure rates. despite excellent outcomes in most children, patients with certain blast cytogenetic features do not fare as well. furthermore, african american, native american, and hispanic patients have worse outcomes than caucasian patients. while the outcome discrepancies are certainly multifactorial, and blast cytogenetics are related to age, it remains unclear whether ethnicity and blast cytogenetics correlate. the diverse patient population at the university of chicago provides an opportunity to evaluate for such a correlation. objectives: to describe cytogenetic findings in a racially and ethnically diverse population of patients of all age groups diagnosed with all at university of chicago from to and determine if there is a correlation between race/ethnicity and blast cytogenetics. results: a total of newly diagnosed patients with all between the ages of - from - were included in this study. of those, patients ( . %) had b-all, had t-all ( . %), one had early t-cell precursor all and one had mixed phenotype all (b/t). caucasians accounted for % of patients, african americans (aa) %, hispanics . %, asians . %, and % were of other races. age distribution had a bimodal pattern, with a peak in incidence at and another at years of age, consistent with published data. cytogenetic categories included: t( ; )(p ;q ), q rearrangements (kmt a), iamp , t( ; )(q ;p . ), t( ; ) (q ;q ), hypodiploidy, hyperdiploidy and double trisomy of chromosomes and . aa and hispanic patients with b-all presented more frequently between the ages of - years compared to caucasians (p = . and . , respectively). in aa patients, t( ; ) (q ;p . ) was overrepresented (p = . when compared to caucasians), and was mainly observed in patients between - years. caucasian patients were more likely than non-caucasians to have hyperdiploidy (p = . ), especially in patients aged - years. the rate of t( ; )(q ;p . ) was significantly higher in aa patients in our cohort, in particular in patients between the ages of - years. hyperdiploidy was more likely in caucasians aged - years. these findings may suggest that varying blast cytogenetics could contribute to outcome differences between races. ahmed elgammal, yasser elborai, mohamed fawzy, asmaa salama, eman d el-desouky, lobna shalaby national cancer institute, cairo, cairo, egypt background: hodgkin lymphoma (hl) in children is one of the malignancies that have a high chance of cure. stage iv hl remains a challenge for getting good clinical outcome as in other stages. many treatment protocols used to give combination chemotherapy while combined modality treatment is the mainstay in other treatment protocols. objectives: we aimed in to assess the outcome using consolidation radiotherapy to chemotherapy (combined modality treatment) versus combination chemotherapy alone in treatment of stage iv hl. design/method: we included patients with stage iv hl and whose data were retrieved from the medical records of the pediatric oncology department, national cancer institute, cairo university, egypt from till june and were followed till august . treatment was either to give cycles of abvd (adriamycin, bleomycin, vinblastine, dacarbazine) only or to give cycles of abvd followed by consolidation radiotherapy. the study included cases; were males and were females. mean age was . years ranging from to years. the histopathology subtype was nodular sclerosis in the majority of cases ( cases) followed by mixed cellularity ( cases) then only one case of lymphocyte rich. nine cases were initially bulky while cases were not. constitutional manifestations were present in cases while it was absent in cases. bone marrow was involved in only cases. radiotherapy was given after completion of chemotherapy to cases while cases received chemotherapy only. the -year overall survival for patients who received radiotherapy was superior to those who received chemotherapy alone; % versus . % respectively with statistical significance (p = . ). the -year progression free survival was also higher with radiotherapy than others; % versus . % (p = . ). patients with stage iv hl who received consolidation radiotherapy apparently had a better outcome than those who received chemotherapy only. this suggests that radiotherapy contributes significantly with chemotherapy to the cure rate for those patients. the feinstein institute for medical research, manhasset, new york, united states background: microrna (mirnas) are short non-coding rnas that play a decisive role in cancer biology, including leukemia. exosomes are microvesicles ( - nm) produced by most cells in biological fluids. exosomes represent the fingerprint of the parental tumor and are loaded with bioactive markers such as mirnas, which may regulate tumor growth. exosomal cargo can be transferred into target cells changing their biological properties. our study investigates a functional role for exosomal mir- a in pediatric acute lymphoid leukemia (p-all). objectives: / to demonstrate that p-all exosomes induce cell proliferation / to confirm that exosome-induced cell proliferation is disease-stage specific / to analyze exosomal mir- a expression profiles in p-all / to authenticate that inhibition of exosomal mir- a reduces leukemia proliferation design/method: exosomes were isolated by ultracentrifugation from healthy donors (hd) & p-all serum and conditioned medium (cm) of sup-b , jm , and cl- (control) human cell lines. cell lines were exposed to different sources of leukemia-derived exosomes in a paracrine or autocrine fashion for hrs in triplicates. proliferation was assessed by microscopic cell counting and confirmed by gene expression for proliferation, pro-survival and pro-apoptotic genes. mirna profiling was performed with the human cancer pathway finder microarray (qiagen). silencing of exosomal mir a was carried out by a mir- a inhibitor (qiagen), utilizing exo-fecttm exosome transfection reagent (sbi, system biosciences). further, exosomal mir- a silencing was confirmed by q-pcr. cellular uptake of texred-sirna (sbi, system biosciences) was confirmed by flow cytometry. transfer of exosomal mir a to the target cells was evaluated by q-pcr. we elucidated that cm-derived exosomes from sup-b and jm cell lines induce cell proliferation in sup-b , jm (autocrine and paracrine) and cl- cells (paracrine) (p< . ). serum p-all exosomes promote paracrine cell proliferation in all cell lines compared to hdderived exosomes (p< . ). heatmap analysis of mirna profiles of leukemia exosomes (all cell lines and p-all) identified mir- a significantly upregulated in leukemia exosomes compared to controls. mir- a was also upregulated in all cell lines after exposure to leukemia exosomes that induced proliferation. moreover, exosomal mir- a inhibition reduces leukemic proliferation in pediatric all. our data suggest that all exosomes induce cell proliferation of leukemic cell lines in both paracrine and autocrine fashion. exosomes regulate these phenomena in a highly orchestrated way, by transfer of functional exosomal mirnas such as mir- a. the results of this study suggest s of s that exosomal mir- a inhibition can act as a novel way for growth-suppression of pediatric leukemia. results: a total of disease sites were detected at pet/ct, while sites were detected at contrast-enhanced ct and bone marrow biopsy (bmb). pet/ct showed improved detection of nodal lesions (p < . ) (kappa value = . ), extranodal lesions (p < . ) (kappa value = . ) and bone marrow (p < . ) (kappa value = . ) compared to contrast enhanced ct and bmb. pet/ct had upstaged cases ( %) and down-staged cases ( . %) (p < . ) (kappa value = . ). among the upstaged cases, patients ( . %) were upstaged from stage ii to iii, based on residual in pet/ct not seen in contrast enhanced ct after abdominal mass excision. four patients ( . %) were upstaged from stage iii to iv based on bone marrow uptake in fdg-pet without positivity in bma or bmb.regarding response assessment, sensitivity was % for pet and % for contrastenhanced ct (p = . ). specificity was % for pet and % for ct (p< . ). positive predictive value for pet was %, while was % for ct scan (p< . ). negative predictive value for both pet and ct was % (p = . ). five patients had nd biopsy to confirm viability of the residual lesions, lesions were negative in pathological examination (all of them were metabolic negative in pet/ct; deauville score below ). one lesion was positive in pathological examination (was positive in pet/ct; deauville score of ). conclusion: pet/ct detected additional sites compared with contrast-enhanced ct and resulted in changing stage of disease. pet scan is significantly more specific than ct in the management of children with burkitt lymphoma. background: deep sequencing of the immunoglobulin heavy chain (igh) locus indicates that each b all is composed of innumerable subclones. in many cases, subclones exhibit differing phenotypic qualities. however, it remains unclear whether subclones demonstrate distinct tissue distribution within a patient. objectives: . to quantify the extent of clonal heterogeneity in diagnostic b all specimens; . to identify variability in clonal composition between bone marrow (bm) and peripheral blood (pb) disease sites. design/method: igh sequencing was performed on purified dna from pairs of matched bm and pb patient specimens. multiplex pcr was used to globally amplify the igh locus; next generation sequencing (ngs) was performed using illu-mina® miseq. index clones (defined as ≥ % of all sequence reads in a specimen) and their subclone progeny (defined by shared nucleotide bases immediately upstream of a common jh, or n_jx) were identified using igblast-determined vh and jh alignments (http://www.ncbi.nlm.nih.gov/igblast/) and an established in-house computational pipeline. results: up to index clones per specimen were discovered in of the samples. in the remaining ( bm/pb pairs), pair did not reveal a clonal igh and was eliminated from analysis; in the other, clone frequency did not reach the % index threshold, but predominant clonal precursors were inferred by the prevalence of their subclone progeny. subclone counts ranged from to , per index clone. a combined , subclones derived from pb index clones were observed; in contrast, bm index clones gave rise to only subclones. subclone heterogeneity was observed between all paired specimens. in bm/pb pairs, index clones existed in equivalent proportions between disease sites. in contrast, bm/pb pair demonstrated high-frequency index clones in the bm ( . % & . %) with limited representation of these clones in the pb ( . % & . %, respectively); in this case, the most prevalent clone in the pb ( . %) matched the least frequent index clone in the bm ( . %). similarly, another pair showed a predominant index clone in the pb ( . %) which was below index threshold ( . %) in the bm. in paired patient specimens, index clone predominance was discovered to be overtly distinct between bm and pb. among all pairs, the extent of subclone progeny derived from each index clone showed marked variability, with far higher subclone frequency in the pb than in the bm. our data indicate that b all clonal composition differs between disease sites. valley children's healthcare, madera, california, united states background: tuberculosis (tb) presenting with hodgkin lymphoma (hl) is rare. their coexistence could lead to delay in diagnosis of both tb and hodgkin lymphoma due to the similarities in signs and symptoms of presentation. most cases have been reported in the adult literature. we describe a case series of children that were suspected to have tb and were found to have coexisting tb and hl. results: a retrospective review of hl patients in guatemala and argentina over six years, uncovered patients with simultaneous diagnosis of tb and hl. eight patients were from guatemala (incidence of . %) and from argentina (incidence of . %). there were females and males. age ranged from - years (mean . years, media years). nine patients were suspected to have tb at presentation by the referring physician. two patients were found to have tb at the time of relapse through routine tissue culture. initial systemic symptoms included fever (n = ), weight loss (n = ), and night sweats (n = ). six patients had a second systemic symptom in addition to fever. time for referral to oncology center ranged from weeks to months. nine patients were diagnosed with tb and hl through a tissue cultures and with serum quantiferon. one patient was found to have hl without tb. two patients had no systemic symptoms and the diagnosis of tb came to light through routine tissue culture. five patients had stage iiib and ivb, two stage iia and one iib at diagnosis. hl treatment was given according to the insti-tutional standards depending on stage and risk with abvd, oepa/copdac +/-radiation therapy, and ice for relapse. five patients started anti tb treatment (isoniazid, rifampin, pyrazinamide +/-ethambutol for months followed by isoniazid and rifampin for - weeks) simultaneously with chemotherapy, and three others after completing cycles. the two relapsed patients started tb treatment after cycles of chemotherapy. seven patients are alive and have been followed for months - years. one patient died during therapy, another died for causes not related to tb or hl and one is currently receiving treatment. conclusion: tuberculosis can coexist with hl. in areas were the prevalence of tb is high, microbiology investigations of biopsy specimen should be strongly considered. therapy for tb can be given simultaneously with chemotherapy. coexistence of tb and hl does not appear to affect outcomes. the children's hospital affiliated to the capital institute of pediatrics, united states background: the pi k/akt signaling pathway plays a central role in cell growth, proliferation and survival in physiological conditions. this signal pathway is considered to be an innovative targeted therapy of cancer, and its abnormal activation has been proved to be related to t-cell acute lymphoblastic leukemia (t-all). despite improved treatment strategies, such as multi-drug combination, high-dose chemotherapy and all kinds of application and popularization of hematopoietic stem cell transplantation, children with drug resistance or relapse t-all are still rather worse and its overall outcome and prognosis are much poorer than the more common b-lineage all. objectives: to explore the relationship between the pi k/akt pathway and the pediatric t-all, so as to probe the exact molecular mechanisms of t-all and provide more directions for its treatment. design/method: cases of new or recurrent acute t lymphocyte leukemia children with clinical information were collected in the children's hospital affiliated to the capital institute of pediatrics from dec. to oct. , with age and gender matched healty children as control (all was informed consent). the expressions of key genes in pi k pathway were s of s analyzed by western blot rt-pcr analysis, the pi k enzyme activities were detected by elisa,and the ccrf -cem's proliferation and its apoptosis were tested by mtt and flow cytometry technology on t-all cell lines ccrf-cem in different treatment group. the results of t-all children in clinical showed that pi k protein and gene expression level were higher apparent than the control group (p< . ), and pi k enzyme activity increased as well (p< . ); pi k inhibitor ly made a significant inhibition of cell proliferation and promoted cell apoptosis. ly also enhanced the effectiveness of clinical commonly used chemotherapeutic drug dnr. in combination ly and dnr treatment group cell viability dramatically declined, apoptosis and the apoptosis relation protein casepase expression in t-all patients was obviously higher than the control and the single drug group; pi k/akt signaling pathway related proteins and gene expression level, pi k, akt, gsk transcription in ccrf-cem were significantly higher than the control (p< . ), while pten transcription was significantly lower than the control (p< . ). the abnormal activation of pi k/akt signaling pathway might play an important role in pediatric t-all patients, especially in the cell proliferation or apoptosis. the results might provide new train of thought and direction in targeted suppress this signal pathway or in combination with other chemotherapy drugs therapy in looking for the more effective and less cytotoxic treatment of pediatric t-all. cleveland clinic children's hospital, cleveland, ohio, united states background: non-hodgkin lymphomas (nhls) are a heterogeneous group of lymphoproliferative diseases which comprise % of all childhood malignancies. nhls can be divided in to b cell lymphomas and t cell/natural killer (nk) cell lymphomas depending on immunophenotype, molecular biology, and clinical response to treatment. although nk/t cell lymphomas occurring in childhood and adolescence comprise a small portion of all lymphomas, they present many diagnostic and therapeutic challenges. the role of angiogenesis in lymphoma pathogenesis is becoming more evident. high molecular weight kininogen (hk) is a central compo-nent of the kallikrein-kinin system. it has been previously reported that cleaved hk (hka) induces apoptosis of proliferating endothelial cells and inhibits angiogenesis in matrigel plug and corneal angiogenesis models. however, the role of endogenous kininogen in regulation of angiogenesis is in tumor microenvironment is unknown. objectives: to elaborate the role of hk in lymphoma angiogenesis, we used a murine t-cell lymphoma model and compared angiogenesis and tumor growth between wild-type and kininogen deficient (mkng -/-) mice. we also evaluated the effect of hka on lymphoma cell proliferation. design/method: el- murine t-cell lymphoma cells ( × ^ ) were implanted into wild-type and mkng -/-mice. tumor size was measured using calipers and tumor volume was calculated using the formula volume = length × width^ × . . seventeen days after cell implantation, tumors were harvested and processed by immunoblotting and immunofluorescent staining. cell proliferation assays (mts) were performed to investigate any possible inhibitory effect of hka on el- cell growth, with human umbilical vein endothelial cells (huvec) were used as a positive control. results: el- lymphomas grew more rapidly and to larger sizes in mkng -/-mice compared to wild-type mice, with significant differences apparent by day after tumor implantation (p< . ). by day , the volume of tumors in mkng -/-mice was approximately . -fold larger than in wild-type mice (mean volume ± standard deviation; ± vs. ± mm , respectively, p< . ). mts assays showed that hka does not directly inhibit the proliferation of el- cells in vitro, though it does significantly impair the viability of ecs studied simultaneously. conclusion: these findings suggest that hk is an important endogenous regulator of angiogenesis and tumor growth in this t-cell lymphoma model, and suggests that hka specifically modulates endothelial proliferation in tumor microenvironment. further work is needed to understand the mechanisms underlying these findings and provide future anti-angiogenic approaches to increase the therapeutic options for patients with nhl. bruce bostrom, jack knudson, nathan gossai, joanna perkins, michael richards, jawhar rawwas, susan sencer, julie chu, nancy mcallister, yoav messinger children's minnesota, minneapolis, minnesota, united states background: osteonecrosis causes significant pain and morbidity in older patients treated for acute lymphoblastic leukemia. besides altering the schedule of dexamethasone in delayed intensification there is no other intervention known to reduce the incidence of symptomatic osteonecrosis. pamidronate has been shown to reduce bone pain from osteonecrosis but not to prevent joint collapse when advanced. objectives: to compare the incidence of symptomatic osteonecrosis in patients who received prophylactic pamidronate compared with concurrent controls. to describe any increase in side effects from the use of pamidronate. design/method: patients age to years at time of all diagnosis were given intravenous pamidronate monthly for one year at the discretion of the primary oncologist starting in the first year of therapy. concurrent controls were patients age to who did not receive pamidronate. all patients were treated according to the concurrent cog protocols and received intermittent dexamethasone during delayed intensification. patients with bcr-abl all were excluded as the use of imatinib may increase the risk of osteonecrosis. imaging was only done if osteonecrosis was suspect based on clinical symptoms. patients were censored at the time of relapse. data were analyzed as of / / . this retrospective study was approved by the children's minnesota irb. of the patients evaluated % were male and % female, % had b-cell and % t-cell. the median followup is . years with a range of . to years. pamidronate was given to patients with developing symptomatic osteonecrosis. there were concurrent controls with developing osteonecrosis. there was no significant difference in the leukemia lineage, gender distribution or body mass index (bmi) at diagnosis between groups. for all patients the median bmi was with a range of to . the age at diagnosis was significantly higher in the pamidronate group with a median of . years vs. . in the controls (p = . ). by kaplan-meier analyses the incidence of symptomatic osteonecrosis was significantly lower in the pamidronate group at % vs. % in controls. the log-rank p-value was . and the breslow p-value, which is more sensitive to early events, was . . there were no untoward side-effects from pamidronate. pamidronate infusions significantly reduced the incidence of symptomatic osteonecrosis in patients over the age of compared to concurrent controls who did not receive pamidronate. arahana awasthi, dina edani, janet ayello, christian klein, mitchell cairo new york medical college, valhalla, new york, united states background: mature b-nhl, including bl and pmbl express cd +/cd b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ % yr. efs, cairo et al. blood. ; gerrard/cairo et al., blood, , goldman/cairo et al. leukemia, . pv has been demonstrated to possess significant preclinical activity against indolent cd b+nhl (polson et al. can. res. ). we previously observed that obinutuzumab (anti-cd mab) significantly enhanced cell death and increased overall survival against bl (awasthi/cairo et al., bjh ) in xenografted nsg mice. however, additive/synergistic effects of pv with obinutuzumab against mature pmbl/bl are unknown. to determine the efficacy of the pv or obinutuzumab/rtx alone or in combination against pmbl and rituximab (rtx) sensitive/resistant bl cell lines. design/method: raji rh (provided by m. barth, md, roswell park cancer institute) and raji/ karpas p (atcc, usa) were cultured in rpmi. tumor cells were incubated with pv, and/or anti-cd b, mmae (generously supplied by genentech inc.) with obinutuzumab /rituximab ( ug/ml) for hr with nk cells at : e: t ratio and cytotoxicity was determined by delfia cytotoxicity assay. six to week old female nsg (nod.cg-prkdcscid il rgtm wjl/szj), were divided into groups: pbs, isotype control, pv, anticd b mab and mmae ( mg/kg). mice were xenografted with intravenous injections of luc+ bl and pmbl cells and tumor burden was monitored by ivis spectrum system. results: os of mice receiving pv alone was significantly increased compared to anticd b ab or isotype control in raji ( . vs. vs. . our preliminary data indicates that pv significantly increased survival in bl and pmbl nsg xenografts compared to anti-cd b ab alone. furthermore, pv in combination with obinutuzumab significantly enhances in-vitro cytotoxicity in bl and pmbl compared to obinutuzumab or pv alone. results: maximal grades (g) / , , and crs occurred in , , and patients, respectively. median lowest fibrinogen levels were . , . , and . g/l in patients with maximal g - , , and crs, respectively. %, %, and % of patients with maximal g - , , and crs had lowest reported fibrinogen levels of ≥ to < . g/l. eight patients (all with g crs) had very low fibrinogen levels (< g/l), which occurred before (n = ) or during (n = ) maximal crs grade or at time of improvement (n = ). no patients with maximal g - crs had < g/l fibrinogen levels. at the onset of < g/l fibrinogen levels, patient had concurrent g , and had g - increased international normalized ratio and activated partial thromboplastin. cryoprecipitate was the primary treatment in the us, and fibrinogen concentrate (fc) guidelines for tisagenlecleucel-associated coagulopathy were developed for other countries because administration of fresh frozen plasma can be problematic. fc was available at / sites for infused patients: / (g crs) and / (g - crs). cryoprecipitate was available at / sites for infused patients: / (g crs), / (g crs), and / (g - crs). risk of bleeding increases in pediatric patients with comorbid thrombocytopenia and anticoagulant treatments. / patients had g / decreased platelets within day of < g/l fibrinogen levels. fatal case of intraparenchymal cranial hemorrhage occurred during resolving crs with g hypofibrinogenemia, ongoing thrombocytopenia, and continuous veno-venous hemofiltration with citrate. hypofibrinogenemia was observed more frequently in patients with higher crs grades during/when crs was improving or resolving. fc and cryoprecipitate treatment guidelines were developed. frequent monitoring and fibrinogen replacement are needed in patients with g / crs. sponsored by novartis. its prolonged cns half-life, may allow a reduction in the number of intrathecal injections. objectives: to safely reduce the burden of therapy by reducing the number of it injections and reducing the total dose of doxorubicin with the addition of liposomal cytarabine and rituximab. design/method: patients ( - years) with cd + b-nhl with fab group b good risk (=stage i/ii and stage iii with ldh < xuln), fab group b intermediate risk (=stage iii ldh ≥ xuln and stage iv {bm blasts < %}) and fab group c high risk were eligible. patients received fab backbone therapy with the addition of six rituximab ( mg/m ) doses; two doses prior to each of two induction courses and one dose prior to each of two consolidation courses. cumulative doxorubicin was reduced from to mg/m in gr patients. after systemic methotrexate clearance, patients received age based dosing of it liposomal cytarabine. it injections were reduced from nine to five. the primary outcome is safety and toxic deaths among evaluable patients with an estimated -year survival above %, monitored by an independent dsmb. results: to date, evaluable patients, fab group b and group c ( cns positive), median age years (range - ), males, burkitt/ dlbcl with gr, ir and hr have enrolled. there has been one grade anaphylactic reaction to rituximab and one grade facial nerve palsy. no other serious adverse events were attributable to protocol therapy. there has been death from progressive disease and relapse at a median follow up of months. efs and os are % and %, respectively. our initial results show excellent efs and os, consistent with published standard of care outcomes, with the addition of rituximab and intrathecal liposomal cytarabine despite the reductions in therapy. further enrollment is ongoing and continued long term outcomes are needed to confirm early results. future randomized studies are needed to examine both short term (mucositis, infections, hospitalization days) and long term (late cardiac toxicity) endpoints. . goldman etal, leukemia, . cairo etal, jco st. jude children's research hospital, memphis, tennessee, united states background: bereaved parents identify significant spiritual needs around time of death and throughout their bereavement journeys. spirituality has been identified as a primary means by which bereaved parents can find meaning in their losses, and this ability to find meaning is associated with lower maladaptive grief symptoms. the use of spiritual coping strategies has been associated with improved coping and mental health outcomes among bereaved parents. objectives: to better understand how bereaved parents' experiences with spirituality throughout bereavement effects objective measures of grief, depression, and meaning-making. design/method: thirty participants whose children died of progressive cancer or related complications one to three years prior to participation completed an in-depth semi-structured telephone interview about their experiences with grief. participants were prompted to describe the impact of their spirituality on their bereavement processes. additionally, participants completed surveys related to grief (prolonged grief disorder questionnaire, pg- ), depression (beck depression inventory, bdi), and meaning-making (integration of stressful life experiences scale, isles). results were analyzed using a mixed methods approach including semantic content analysis of qualitative content and kruskal-wallis h test and post-hoc analyses of quantitative data. results: correlation analyses demonstrated significant differences between participants with positive and negative spiritual experiences of bereavement. participants with negative experiences of bereavement had a statistically significant increase in scores on the pg- compared to those with positive spiritual experiences signifying greater symptoms of prolonged grief. participants with negative spiritual experiences with grief had significantly lower scores on the isles, suggesting a lesser degree of adaptive integration of their losses. there were no significant differences in depression scores between groups. conclusion: bereaved parents that have a negative spiritual experience of bereavement are at increased risk for prolonged grief symptoms and are less likely to find meaning in their children's deaths than bereaved parents that describe a positive spiritual experience of bereavement. providers should consider exploration of spiritual beliefs and provision of spiritual care for parents of children facing life-limiting illnesses during treatment and bereavement. background: langerhans cell histiocytosis (lch) is an inflammatory myeloid neoplasia characterized by frequent relapse, with treatment failure associated with higher risk of death and neurodegenerative disease (lch-nd). activating somatic mutations in mapk pathway genes have been identified in almost all cases, with braf-v e in approximately % of lesions. targeted therapies have been successful in treating other refractory cancers with braf v e mutations (such as melanoma). given the central role of mapk pathway activation in lch, mapk pathway inhibition may be an effective therapeutic strategy for children with lch. objectives: the purpose of this study was to report the efficacy and toxicity profile of a retrospective cohort of patients with lch treated with mapk pathway inhibitors. design/method: medical records from pediatric patients with lch (systemic and/or lch-associated neurodegeneration) who were treated with a mapk pathway inhibitor were retrospectively reviewed from five institutions. all patients had failed at least one prior systemic therapy and had a proven mapk pathway mutation. results: all patients in this series were less than years old (median = . years; range: - years) with a median of three prior treatments (range: - ). at the time of initial mapk inhibitor use, nine of the patients had lch-nd diagnosed clinically and/or by radiographic imaging; the remaining three patients had systemic disease. patients were treated for a median of months (range: - months) with various reasons for discontinuation. three patients received combination mapk inhibitor therapies and three patients received other concurrent lch-directed therapies. four of the twelve patients had a grade or toxicity reported and three of these patients required dose reduction in order to be able to successfully resume therapy. overall survival was % with median month follow-up (range: - months) with only one patient achieving transient complete response. the remaining ten patients had partial response or stable disease and four of these patients developed progressive disease while on therapy. conclusion: mapk pathway inhibitors may be a relatively safe salvage therapy for refractory systemic lch and lch-nd but the efficacy and durability of this strategy remains to be defined. combination with cytotoxic chemotherapies may be required in order to eradicate the disease-causing cell. future prospective trials of mapk pathway inhibitors for patients with refractory lch are needed in order to directly compare their efficacy and toxicity relative to other current salvage strategies. cincinnati children's hospital medical center, cincinnati, ohio, united states background: medication adherence during maintenance therapy has been shown to have a direct relationship with disease relapse in pediatric leukemia. previous research determined that patients who are ≤ % adherent to mercaptopurine ( mp) have a greater risk for relapse. the primary aim of the present study is to examine the relationship between metabolite profiles of mp with behavioral adherence rates obtained via electronic monitoring at , , and days. it is hypothesized that patients demonstrating low levels of thioguanine (tgn) and methylated mercaptopurine (mmp) will have lower behavioral adherence rates prior to the blood draw. design/method: in a multisite, prospective study of patients ages - years diagnosed with acute lymphoblastic leukemia (all) or lymphoblastic lymphoma (lbl), mp adherence was measured across months of maintenance therapy using behavioral adherence (electronic monitoring) and pharmacological (metabolites) measures of mp. mp is metabolized into mmp and tgn. cluster analysis was used to generate three mutually-exclusive profiles of mp adherence. behavioral adherence rates were calculated for , , and days prior to the blood draw. results: this study identified three metabolite profiles of mp across months. previous research indicated that low levels of both metabolites suggest nonadherence to medication. low levels of one metabolite with high levels of another metabolite indicate adherence to mp. in this study, . % of the low tgn-low mmp group had -day behavioral adherence rates ≥ % (mean = %); . % had adherence rates < % (mean = . %). in the high tgn-low mmp group, . % had a mean -day adherence of %; . % had adherence rates < % (mean = . %). the low tgn-high mmp group had % of patients with a mean -day adherence level of %; % had adherence rates < % (m = . %). at and -days, to % of patients in the low tgn-low mmp group had adherence rates < %. conclusion: these findings suggest that electronic monitoring and metabolite concentrations can be used to monitor mp medication adherence during maintenance therapy. it is notable that there is a sub-sample of pediatric patients who are identified as being nonadherent to mp based on electronic monitoring, however, metabolite levels indicate adherence to mp. similarly, a sub-sample of patients were identified as being adherent based on electronic monitoring, but metabolite profiles indicated sub-therapeutic levels of mp. our findings underscore the clinical significance of using both objective measures of medication adherence to inform clinical decision making. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hemophagocytic lymphohistiocytosis (hlh) is a life-threatening hyperinflammatory syndrome characterized by non-remitting fevers, rash, hepatosplenomegaly, cytopenias, liver dysfunction and coagulopathy, and can include central nervous system involvement. several genetic diseases cause hlh by impairing normal lymphocyte or macrophage function. the hlh panel at the cincinnati children's genetics laboratories includes genes associated with hlh and other lymphoproliferative diseases, including the genes that cause primary hlh (prf , unc d, stxbp , stx , rab a), x-linked lymphoproliferative diseases (sh d a, xiap), itk deficiency (itk), hermansky-pudlak syndrome types and (ap b and bloc s ), chediak-higashi syndrome (lyst), cd deficiency (cd ), xmen syndrome (magt ) and lysinuric protein intolerance (slc a ). deletion/duplication analysis is available as a reflex test for all genes, as copy number variations (cnvs) are not directly assessed by sequencing. objectives: the prevalence of cnvs among large groups of patients with hlh in north america is unknown. we assessed the frequency of cnvs in the genes on the hlh panel through a retrospective review of orders for deletion/duplication analysis performed after next-generation or sanger sequencing: orders for all genes on the panel, and orders of - genes from the panel. deletion/duplication analysis was performed on a custom × k microarray annotated against ncbi build (ucsc hg , march ). deletion/duplication analysis resulted in a confirmatory diagnosis in of cases ( . %). pathogenic or likely pathogenic cnvs were most common in the three x-linked genes: sh d a ( deletions), xiap ( deletions, duplication), and magt ( deletions). hemizygous deletions in xlinked genes in male patients were typically suspected after amplification failure during previous sequencing. of the autosomal recessive genes, pathogenic cnvs were observed once in each of three genes: rab a (heterozygous), lyst (heterozygous), and stxbp (homozygous). in the two heterozygous cases, a second change was not identified by sequencing, so deletion/duplication analysis did not offer a confirmatory diagnosis. in patients, deletion/duplication analysis was performed after a pathogenic or likely pathogenic variant was identified in an autosomal recessive gene during sequencing; however, in no case was a second mutation uncovered by cnv analysis. we recommend that deletion/duplication analysis be routinely performed in all male patients with hlh who lack a genetic diagnosis after sequencing of hlh-associated genes, especially if any regions failed to amplify. deletion/duplication analysis may be performed in female patients after sequencing if a genetic form of hlh is highly suspected, but the yield is expected to be low. cleveland clinic children's hospital, cleveland, ohio, united states background: the development of post-transplant neoplasia, typically from lymphoproliferative disease (ptld), is a severe complication in transplant recipients and affects approximately % of pediatric solid organ recipients. rates of lymphoma in adult heart transplantation patients are comparatively low, at less two percent at ten years. there are few published reports of the long-term outcomes of neoplasia after pediatric heart transplantation. we aimed to identify the subsequent malignancies that occurred in pediatric heart transplantation patients in a large single institution, and describe their treatment and subsequent clinical course. we performed a retrospective chart review of all pediatric heart transplant recipients followed at the cleveland clinic children's hospital from january to october . we excluded patients who died within days of heart transplantation. we reviewed in depth the history and clinical course of subjects who developed neoplasms. results: between and , patients underwent heart transplantation and survived at least days post transplantation. nine patients ( . %) developed a subsequent malignancy. in this case series, the median age at heart transplant was years old and the median time to develop neoplasia was . months. primary neoplasia included monomorphic ptld ( ), polymorphic ptld ( ), burkitt lymphoma ( ), hodgkin's lymphoma ( ), plasmacytoma-like lymphoma ( ) and epstein-barr virus-associated smooth muscle tumor (ebv-smt) ( ). one patient with hodgkin lymphoma subsequently developed monomorphic ptld, one patient with polymorphic ptld subsequently developed ebv-smt and later, an undifferentiated gastric cancer. one patient with monomorphic ptld developed an ebv-smt. evidence of epstein-barr virus was present in six of nine patients at diagnosis of first malignancy. four of nine patients received reduction in immunosuppression as a primary intervention for the initial malignancy, with two complete responses (cr), one partial response, and one with progressive disease. five patients were treated with chemotherapy, with four cr and one with progressive disease. three patients died of malignancy (recurrent ebv-smt, undifferentiated gastric cancer, and monomorphic ptld post-hodgkin disease) and two patients died of other transplant related complications. conclusion: secondary malignancies represent a significant disease burden to survivors of cardiac transplantation. as expected, much of the malignancy burden is driven by ebv. despite aggressive histology, many malignancies can be successfully cured in this setting with a multidisciplinary approach. stanford university school of medicine, palo alto, california, united states background: current treatment of langerhans cell histiocytosis (lch) is based on extent of organ system involvement and if high risk systems are affected. gastrointestinal (gi) involvement is diagnosed in about % of lch patients, and classically presents in children under years of age with malabsorption, failure to thrive, bloody diarrhea and anemia. although the gi system is considered standard risk, a mortality rate over % occurring within years of diagnosis has been reported. this study was performed due to this discrepancy and the limited number of published cases. objectives: to review the clinical course and outcomes of patients diagnosed with gi lch. design/method: a retrospective chart review of patients with histologically confirmed gi lch diagnosed in the last years identified from the bass center histiocytosis clinical database was performed. two other pediatric hematology/oncology centers (ucsf benioff children's hospital oakland and san francisco) were queried for additional cases. results: four patients with biopsy proven gi lch [ subjects ( . %) from database records and l from center queries] were identified. failure to thrive, hypoalbuminemia, bloody diarrhea and rash were the most common presenting symptoms. lch of the skin was found in all patients. risk organ systems were involved in patients. of note, subjects were of african racial background. the median age at diagnosis was . months ( . months to years), mean albumin . g/dl ( . - . g/dl), mean esr of mm/hr ( - mm/hr). all patients initially received combination therapy per lchiii protocol (vinblastine, prednisone, and mercaptopurine). two patients had recurrent disease and received second line therapy (cytarabine, cda, and local radiation therapy). all patients are alive without active disease at last follow-up ( to months after completion of therapy). a systematic approach to evaluate gi involvement should be performed in children diagnosed with lch. from our experience, combination chemotherapy for patients with lch involving the gi tract is an effective intervention for active disease. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bhatia indicated that rates of mp adherence ≥ % have better clinical outcomes. those with adherence rates ≤ % have an increased risk for disease relapse. the present study investigated patterns of mp medication adherence using group-based trajectory modeling in a large sample of pediatric patients. to describe patterns of behavioral adherence during the maintenance phase of therapy for a cohort of pediatric patients ages - years who were diagnosed with acute lymphoblastic leukemia or lymphoblastic lymphoma (n = ). previous research has documented the relationship between optimal levels of medication adherence with positive health outcomes. it was hypothesized that three groups would be identified: optimal adherence, deteriorating adherence, and chronic nonadherence. it was hypothesized that patients in the optimal adherence group would have adherence rates ≥ %. those with poor adherence would have adherence rates ≤ %. design/method: the present study was a longitudinal, multisite study investigating adherence to -mercaptopurine in a pediatric cohort of patients using electronic monitoring devices. daily adherence rates (electronic monitoring of mp) were examined across -months. health outcomes were measured at quarterly intervals through medical chart reviews. results: unconditional growth curve modeling indicated that the mean percentage of behavioral adherence was . % at baseline and declined to . % at -months. three trajectories of mp behavioral adherence were identified: ) optimal adherence ( % of patients): averaging % behavioral adherence across months; ) moderate adherence ( %): relatively stable nonadherence with rates of % across months; and, ) chronically nonadherent ( %): adherence decreased from % to %. with respect to patterns of medication adherence and relationship to clinically-relevant health outcomes, there were no significant differences in health outcomes between patients in the adherent versus nonadherent trajectories, including mean absolute neutrophil counts (anc), risk for infection as measured by anc, healthcare utilization, or risk for disease relapse. although longitudinal patterns of mp behavioral adherence were not related to health outcomes, it is notable that only % of the current sample had adherence rates ≥ %. in fact, % of the current sample demonstrated adherence rates ≤ %. our findings are important for development of future adherence promotion studies in pediatric cancer. our findings underscore the relative significance of tailoring adherence promotion interventions to subgroups of patients, including those with problematic patterns of adherence. patients who demonstrate adequate levels of adherence could still benefit from less intensive, preventative interventions to sustain and improve adherence. sophie gatineau-sailliant, pascale grimard, marie-claude miron, guy grimard, anne-sophie carret, jean-marie leclerc chu sainte-justine, montreal, quebec, canada background: vertebral involvement in langerhans cell histiocytosis (lch) is still a subject of interest, due to its low frequency and the absence of management's guidelines. objectives: to provide additional information on presentation, treatment and morbidity of pediatric lch vertebral lesions, we report cases of children with vertebral lesion of biopsy-proven lch, between january st and december st , at sainte-justine university health center (montreal, quebec, canada). we conducted a retrospective study by reviewing charts and imaging of vertebral lch in a population of children (median age of . years at lch diagnosis), followed for a median duration of months. symptoms at presentation, treatment modalities and morbidities were collected. results: vertebral lesions were present at lch diagnosis in of cases. they were usually diagnosed secondary to back pain in of cases and were asymptomatic in only one case. despite an epidural extension in of cases, no child developed neurological symptoms. lesions frequently involved vertebral body ( of cases) and were rarely unstable ( of cases). out of vertebral lesions, most of them had a dorsal localization ( of lesions) and of patients had lch in multiple vertebrae. at diagnosis, median vertebral height loss was . % compared to % at last imaging control. most used imaging modalities were pet-scan and plain x-rays. treatments were diverse and consisted in chemotherapy in all children but three and bisphosphonates in only cases. radiation therapy was not used in any patient. six out patients did benefit of an orthosis. a lch recurrence was observed in patients and involved vertebrae in cases. one patient with treatment-resistant lch disease had relapses, and required multiple lines of treatment. all children were alive and disease-free at their last follow-up, patients having radiological vertebral sequelae and only had clinical sequelae. our study is consistent with the epidemiological data described in larger cohorts of children with vertebral lesions of lch and the favorable prognosis associated with such lesions. nevertheless, aggressive treatment and long term follow-up seemed to be essential as recurrences are s of s not rare and spontaneous bone regeneration often incomplete. plain x-rays appears to be a good follow-up tool for vertebral lesions as it allows reliable measures, less exposure to radiation at lower cost. national cancer institue, giza, giza, egypt background: acute lymphoblastic leukemia (all) is the most common type of childhood cancer and also the most complicated in the treatment, so it requires many interventions for both treatment and to alleviate suffer form side effects. pancreatitis is one of the toxicities, which is more common in all as it appears in about % of the patients. it occurs in many drug combinations which induce pre-pancreatitis and even direct destruction of pancreatic tissues. pancreatitis can be induced by many drugs used in the treatment such as chemotherapeutic agents or supportive treatment. lasparaginase is the backbone drug of the treatment of all in which to doses are required to achieve complete remission status in the induction phase of treatment and to doses in the maintenance phase.it is an enzyme that destructs the l-asparagine amino acid into aspartic acid and ammonia thus deplete the asparagine from the extracellular matrix . many drugs are investigated for their effect on treatment of induced pancreatitis such as interleukin- , nsaid as antiinflammatory, glycerin tri nitrates as improvement of microcirculation, tnf-alpha antibody, paf inhibitor as specific anti-inflammatory and low molecular weight heparin .none of the drugs was investigated for their ability to prevent the occurrence of pancreatitis. objectives: this study was designed to evaluate the protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis design/method: acute pancreatitis was induced in rats by intra-muscular injection of l-asparaginase ( i.u/kg) given daily for five days. enoxaparin was given subcutaneous ( i.u/kg) and diclofenac was given intra-peritoneal ( mg/kg) daily for five days. then, markers of pancreatic injury, lipids, immune cell infiltration and oxidative stress were analyzed with histo-pathological examination of the pancreatic tissue results: during acute pancreatitis, oxidative stress markers were significantly changed as indicated by reduced tis-sue glutathione and increased malondialdehyde levels. this was accompanied with significant increase in immune cells infiltration as indicated by high levels of myeloperoxidase and pro-inflammatory cytokine tnf-alpha. triglyceride only showed increase level. treatment with enoxaparin and/or diclofenac restored levels of biochemical markers including serum alpha-amylase, reduced glutathione, malondialdehyde, pro-inflammatory cytokine tnf-alpha, myeloperoxidase and triglyceride. histological injuries of pancreatic tissues as vacuolation and necrosis of epithelial lining pancreatic acini, inflammatory cells infiltration and focal pancreatic hemorrhage were also reduced by treatment with enoxaparin and/or diclofenac. the present study emphasizes the potential protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis background: rosai dorfman disease (rdd), or sinus histiocytosis with massive lymphadenopathy (shml), is a rare condition of immune dysregulation of unknown etiology arising from the massive accumulation of non-langerhans type histiocytic cells inside lymph nodes. the disease classically presents as bulky, painless lymphadenopathy often associated with infection showing distension of lymph node sinuses by abundant histiocytic cells (cd a(-), s- (+)/cd (+)). in some cases, the disease can be self-limiting, but in cases with a prolonged chronic course of exacerbations and remissions, those with extranodal involvement, or disease that threatens vitals structures, treatment may be necessary. there is no treatment consensus. to describe a case of life-threatening, unresectable, recurrent rdd successfully treated with langerhans cell histiocytosis (lch) -inspired therapy. design/method: we compared this case to the current literature on chemotherapeutic treatments for rdd. we searched pubmed, ovid, and google scholar for similar cases. we believe this to be the first reported case of using lch therapy to successfully treat rdd. an -year-old male presented to an outside hospital with two years of massive neck swelling causing torticollis. biopsy confirmed rdd. he was intermittently treated with courses of antibiotics with partial response. surgical removal of the affected lymph nodes was unsuccessful due to proximity to the spinal cord. two years later, the patient presented to our institution. he was initially treated with prednisone with a fast tapering dose, but after a second relapse the decision was made to try chemotherapy following the lch- protocol of weekly vinblastine ( mg/m ), -mp ( mg/m ), and high dose steroid bursts. he experienced two additional relapses off therapy at ages and years old, including cmv(+) associated septic shock and cytokine storm requiring rapid response, picu admission, and ionotropic support. this last episode was treated with a more prolonged induction and maintenance therapy. an extended and slowly tapered maintenance therapy regimen of . years of daily -mp, monthly vinblastine and steroids with a slowly tapered dose during his fourth remission has resulted in -months of continuous complete remission-the longest stretch of his life. no similar cases were found. literature search demonstrated no consensus regarding the most effective treatment of rdd, with no previous cases being successfully treated following lch chemotherapy protocols. we hypothesize that the multi-agent relatively mild lch- therapy mitigates the immune dysregulation of rdd. this case suggests that lch- therapy can be used to treat cases of rdd that is not amendable to surgery or observation. nicklaus children's hospital, miami, florida, united states background: central venous catheters (cvc) are necessary in the management of patients with malignancies, especially children. patients with acute leukemia (al) have higher rates of central line associated complications such as bloodstream infections compared with other malignancies. objectives: to examine the choice of placement of cvc and the differences in outcome between peripherally inserted central catheters (picc) and ports in patients with leukemia during induction. design/method: retrospective chart review of patients with newly diagnosed leukemia at nicklaus children's hospital between and . results: ninety four patients with a new diagnosis of leukemia undergoing induction chemotherapy were identified. the average age was . years. overall, ( . %) patients had a port placed and ( . %) had a picc placed. the decision for picc or port was subjective and physician based. the main outcome measures were local inflammation/infection, bacteremia, thrombophlebitis, blocked catheter and premature removal. the most common complication was bacteremia ( . %). in a multiple logistic regression analysis for predicting whether patients had at least one complication, results showed that having at least one complication is . times the odds in patients with aml compared to patients with all (p = . ). when comparing picc vs. ports, patients with picc had more frequent episodes of blocked catheters ( . %) and premature removal ( . %) compared to the patients with ports ( . % and . %) (p = . and p = . respectively) during induction. local inflammation, bacteremia and thrombophlebitis were not statistically different (p = . , p = . and p = . respectively). the most common place for port placement was the right subclavian vein ( %). there was no significant association between port location and having at least one complication (p = . ). acute lymphocytic leukemia subgroup analysis: fourteen patients ( %) in the picc group had at least one complication and ( %) in the port group but that was not statistically significant (p = . ). our series showed a higher incidence of blocked catheters and premature removals with picc compared to ports in patients with leukemia during induction. the choice of placement of picc vs port was subjective and physician based. patients with all, despite receiving steroids and asparaginase during induction, did not show a statistically significant increase risk in thrombosis or infection but larger numbers may be needed in future studies. university of california, san francisco, san francisco, california, united states background: hemophagocytic lymphohistiocytosis (hlh) is classically a disorder of young children meeting systemic hyperinflammation criteria. presentation in late adolescence is uncommon. furthermore, though cns signs occur in - % of cases, initial isolated neurologic presentation is rare, frequently resembling encephalitis or demyelinating disorders. these cns signs can be isolated or precede systemic disease, delaying hlh diagnosis. hlh declaring in adolescence with predominant psychiatric features has not been well documented. objectives: to describe a case of cns hlh presenting with neuropsychiatric features in absence of classic hlh criteria. design/method: retrospective review of clinical, radiologic, histologic, immunophenotypic, and molecular features of a patient with cns hlh. a -year-old female presented with acute-onset headaches following nine months of progressive anxiety, short-term memory loss, emotional lability, perceptual disturbances, and hypomania. brain mri demonstrated numerous enhancing t hyperintense supratentorial and infratentorial white matter lesions in the left thalamus and caudate head. brain biopsy showed histiocyte-rich inflammation and associated demyelination. extensive evaluation including universal microbial pcr failed to reveal underlying infection or malignancy. past medical history was notable for presumptive pulmonary sarcoidosis diagnosed months prior with progressive respiratory failure with associated granulomatous pulmonary nodules which responded to systemic immunosuppression. at presentation of her neuropsychiatric symptoms, she had normal sil- r, ferritin, fibrinogen, and triglycerides. there was no pancytopenia, coagulopathy, bone marrow hemophagocytosis, fevers, or splenomegaly. given the possibility of partial immune suppression of systemic symptoms and the prominent neurologic symptoms, hlh screening labs were sent and notable for decreased natural killer and cytotoxic t lymphocyte function, normal granzyme expression and cd a mobilization, and absent perforin expression. genetic testing confirmed compound heterozygous mutations in prf (c. g>a, c. a>c) and familial hlh type . she was treated with low-dose dexamethasone and intrathecal chemotherapy per hlh- . due to lack of evidence of systemic inflammation, vp- and high-dose steroids were held. within one week of initiating therapy, she had decreased anxiety and improved cognition, with sustained, incremental neuropsychiatric improvement with additional intrathecal treatments. she tolerated dexamethasone tapering without symptom flare. mri also demonstrated parenchymal lesion improvement. for definitive treatment, she underwent unrelated allogeneic hematopoietic cell transplantation and remains at neurologic baseline as of eight months post-transplant with ongoing imaging improvement. conclusion: this case of familial hlh with compound heterozygous perforin mutations in an adolescent with isolated neuropsychiatric symptoms illustrates that cns hlh may be an underrecognized phenomenon in absence of systemic signs. standard hlh therapy may effectively reverse these symptoms with associated radiologic responses. rush university children's hospital, chicago, illinois, united states background: posterior reversible encephalopathy syndrome (pres), a recognized complication of pediatric leukemia treatment has been reported in up to % patients in various series. hypertension, chemotherapy and cortical spreading depression have been implicated in the pathophysiology. due to the combinations used, it is difficult to identify the offending drug, several have been implicated. since delay of chemotherapeutic treatment in children with high risk leukemia is unfavorable, it is important to recognize the characteristic radiologic findings, manage appropriately and reintroduce the treatment as soon as possible. pharmacoethnicity is now recognized as an important factor for variation in neurotoxicity in children with all. ethnic differences in reported pres events in pediatric patients with all has not been well described in literature. to describe the factors associated with pres in a cohort of high risk pediatric all patients at a single institution. design/method: a total of children with an average age of years ( - years) diagnosed with all between - were retrospectively reviewed for the occurrence of pres. various demographic factors, therapy received, clinical features, radiology related findings and management were reviewed. a search for all published articles on pres in leukemia was conducted using pubmed databases. results: five ( %) children (average age . years) developed pres during days - of induction. % of the patients that developed and % of those that did not develop pres were hispanic. all the patients that developed pres and % of those that did not were diagnosed with high risk all. all patients received vincristine, % received daunomycin and intrathecal methotrexate and % received asparaginase in the week prior to the event. mri findings confirmed pres in all patients with no evidence of methotrexate related leukoencephalopathy or leukemia. at the time of pres all patients were in remission based on mrd and spinal fluid cytology. two-thirds of the patients had seizures and hypertension at the time of the event with no prior history of either. all patients had complete recovery of normal mental status after resolution of pres. a higher incidence of pres than previously reported was noted in our series. hispanic ethnicity, high-risk all and exposure to vincristine, daunomycin and intrathecal methotrexate in induction were associated with pres in our cohort. a new association that emerged was that of hispanic ethnicity with pres .larger studies to understand the importance of pharmacoethnicity in pres may help in individualization of chemotherapy based on ethnic differences. children's hospital of illinois, peoria, illinois, united states background: hyper ige syndrome is a primary immunodeficiency characterized by susceptibility to skin and lung infections as well as increased propensity for malignancy. hemophagocytic lymphohistiocytosis (hlh) is a syndrome characterized by overwhelming activation of t lymphocytes and macrophages occurring as either primary hlh caused by genetic abnormalities or secondary hlh associated with infectious, malignant, metabolic, or immunodeficiency causes. we describe the first case to our knowledge of hlh in a patient with hyper ige syndrome. to describe a case of hlh in a pediatric patient with hyper ige syndrome. results: a -year old caucasian male with known autosomal dominant hyper ige syndrome (stat mutation) was transferred to the pediatric intensive care unit secondary to concern for septic shock. the patient had persistent slow bleeding from oral lesions and central catheter sites despite the addition of aminocaproic acid and recombinant factor viia. he also required numerous blood product transfusions sec-ondary to anemia and thrombocytopenia. clinical suspicion was high for hlh and the patient met criteria for diagnosis of hlh with the following: ferritin > , ng/ml, triglycerides mg/dl, decreased nk cell function with the sample only containing % nk cells, elevated soluble il- receptor at u/ml, splenomegaly, and fever. infectious workup was remarkable for a positive ebv qpcr with , copies/ml suggestive of ebv driven secondary hlh. familial hlh testing was unable to be completed. therapy was initiated based upon the hlh- study. the addition of ruxolitinib and anakinra were considered but the patient declined rapidly prior to treatment. ct of the head was concerning for a stroke with signs of edema and increased intracranial pressure likely leading to the development of symptoms consistent with brain stem herniation. the decision was then made to withdraw care. conclusion: to our knowledge, this is the first report of hlh in a patient with hyper ige syndrome. diagnosing hlh requires a high index of suspicion in critically ill patients, and prompt initiation of therapy is essential. this challenging case of hlh in a patient with hyper ige syndrome highlights the diagnostic challenge, variable presentation, and need for effective therapy in this vulnerable patient population. background: adolescents and young adults (ayas) with cancer are at risk for psycho-social as well as physical symptom burden during cancer therapy. the purpose of this study is to explore psychological and physical symptoms endorsed by aya while receiving therapy for cancer design/method: surveys were given in both inpatient and outpatient settings during cancer therapy. symptom screening in pediatrics tool (sspedi) and memorial symptom assessment scale (msas). symptoms severity was rated by teens on a point likert scale. spss , used for statistical analysis. results: : a total of aya on cancer therapy (age range - . years) % female, % male, . % acute leukemia, . % solid tumors, and . % diagnosis was not reported. % of aya on cancer therapy reported at least or more symptoms, % reported > symptoms cluster. of the physical symptoms that were reported as most distressing to the teens, mouth sores and headaches were the top causes. of the physical symptoms that were most frequently endorsed; fatigue was on the top ( %), followed by change in appetite %, vomiting %, and pain %., the least was bowel habit changes. aya rated sadness as the most frequent psychological symptom %, followed by feeling angry %, and scared %. statistically significant difference was noticed based on gender difference with more females reported symptoms (p = . ), while type of cancer (acute leukemia versus solid tumors) was not statistically different. conclusion: aya with cancer reported multiple physical and psychological symptoms with significant distress. females seem to report more symptoms compared to males. screening aya for cancer therapy related symptoms is feasible during routine visits and adds important information about the aya well-being. background: sinus histiocytosis with massive lymphadenopathy (shml), also known as rosai-dorfman disease, is a rare histiocytic proliferative disorder of unknown etiology. many treatment modalities have been employed; however, no uniform guidelines exist. objectives: literature review of treatment options for shml. design/method: chart review was performed on pediatric patients diagnosed with shml at the children's hospital at montefiore between and after irb approval. inclusion criteria included children between the ages of and years with shml. exclusion criteria included children with cutaneous shml. four cases of shml seen at montefiore are described. a comprehensive review of the literature identified additional cases published between and . manuscripts that did not include the treatment modality or outcome were excluded. results: many of the patients with shml responded to observation alone. of patients, patients were observed, with ( %) having resolution of disease, five having stable disease, and five being lost to follow-up. one patient received subsequent systemic therapy. surgical management was con-ducted upfront in patients. of those, ( %) had resolution of disease, one had stable disease, and one had recurrence with no further therapy noted. of the remaining nine patients, % were successfully treated with systemic therapy, consisting of either steroids ( ) or steroids and chemotherapy ( ). systemic therapy was used as first-line therapy in patients. steroids alone or in conjunction with chemotherapy resulted in resolution of disease in / and / patients ( / , %), respectively, with four patients having stable and three with progressive disease. chemotherapy without steroids resulted in resolution of or stable disease in / patients. radiation was ineffective. conclusion: shml is a rare disease with no published guidelines for treatment. from the results of the cases and a detailed review of the literature, it can be suggested that observation may be considered as first line management in patients providing there are no significant symptoms. for patients who are symptomatic or have significant progression, surgery may be considered. in patients with recurrence or refractory disease, steroids and/or chemotherapy may be used. the presence of nodal or extra-nodal disease did not seem to have a significant impact on the course of treatment. given the rarity of the disease, it is difficult to conduct a randomized control trial. further work, involving collaboration between centers and cooperation with the international rare histiocytic disorders registry would be helpful. boston children's hospital, boston, massechusettes, united states background: increasing census and intensified work compression on the inpatient oncology service at our institution was identified as leading to resident dissatisfaction, impaired resident learning and decreased perceived quality of patient care. objectives: to evaluate the impact of a redesign of a pediatric inpatient hematologic malignancy (ihm) service on resident perceptions of the educational value of the rotation and safety of patient care. design/method: during the - academic year, we initiated a bundled intervention on the ihm service. modifications included ) decreased patient volume: the ihm service was divided into two teams, utilizing an extra attending -a teaching service consisting of residents and fellows and a team comprised of nurse practitioners. ) intentional patient team assignment: patients were deliberately assigned to a care team based on educational opportunities and provider skill sets. ) intentional attending faculty selection: attending faculty with deeper clinical and teaching experience were selected to supervise on the teaching team. ) increased weekend staffing. after completing the service, junior residents completed an electronic survey to evaluate their perceptions of the educational value of the rotation, as well as their ability to deliver safe care while on the rotation. fisher's exact tests were used to compare responses from residents in who experienced the redesign to residents in , whose experience results: survey completion rates were % ( / ) in and % ( / ) in . intervention residents were significantly more likely than comparison group residents to choose the answers "very good" or "excellent" to describe both the overall quality of the rotation ( % intervention vs. % comparison, p< . ) and the educational experience on rounds ( % intervention vs. % comparison, p< . ). intervention residents also reported caring for fewer average primary patients daily on weekdays as compared to comparison residents ( . vs . patients, p< . , % ci - . to - . ). furthermore, intervention residents were more likely than comparison residents to "agree" or "strongly agree" that they could provide safe patient care on weekend days ( % intervention vs. % comparison, p< . ) and on nights ( % intervention vs. % comparison, p< . ) while on the oncology service. a redesign initiative of an oncology service with the development of a new teaching service led to improved resident perceptions of the educational value of the rotation and ability to provide safe care to patients. this approach could be useful to other services and institutions to promote similar outcomes in resident education and patient care. background: alk-positive histiocytosis is a rare histiocytic proliferative disorder that has been reported in three infants presenting primarily with hepatosplenomegaly, anemia, and thrombocytopenia. given the rarity of this disease, there are no standard treatment algorithms for this diagnosis and the disease course and outcomes remain largely unknown. the published series describes treatment ranging from monitoring alone to multi-drug chemotherapy regimens. there was ulti-mately resolution of presenting symptoms in all three cases despite varying treatment strategies. objectives: to report a newly diagnosed case of alkpositive histiocytosis that was treated with a novel approach using cytarabine monotherapy. results: a full term male infant presented at birth with difficulty feeding and hyperbilirubinemia. over the first few weeks of his life, he subsequently developed thrombocytopenia, transaminitis, and profound hypoalbuminemia. by six weeks of life, he was experiencing significant abdominal ascites requiring repeat paracenteses, massive hepatosplenomegaly, respiratory distress secondary to abdominal distension, anemia, and coagulopathy. he underwent numerous diagnostic tests, including a liver biopsy followed by a bone marrow biopsy that showed alk-positive histiocytic infiltrates in both sites. treatment was initiated with cytarabine mg/kg/day x days, repeating every weeks. throughout his course of five cycles of treatment, he experienced intermittent fevers and mild nausea with no other adverse events. by the end of five cycles, his hepatosplenomegaly resolved, his blood counts normalized, he demonstrated weight gain on oral feeds, and his liver enzymes normalized. he is currently months post completion of therapy and remains well with a normal physical exam and laboratory values. conclusion: treatment of alk-positive histiocytosis with lose dose cytarabine resulted in complete resolution of our patient's symptoms with minimal treatment related adverse effects, and few long-term treatment related risks. given the rarity of the diagnosis, the reporting of effective novel treatment options is important for future patient care. background: adult patients with melanoma or lung cancer harboring braf v e have benefitted from the development and subsequent approval of specific braf inhibitors. as such, delineating the subset of similarly targetable pediatric oncology patients may spur development and rational use of these inhibitors in children. importantly, other point mutations and fusions of braf may also be targetable in s of s children analogous to recent emerging data in adult cancer patients. objectives: to define the genomic landscape of known and novel braf alterations and raf fusions in pediatric malignancies and report index cases with clinical response to braf or mek inhibitors. design/method: dna was extracted from microns of ffpe sections of , tumors from pediatric (< years of age) oncology patients, and cgp was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of x for up to cancer-related genes plus introns from genes frequently rearranged in cancer. genomic alterations (ga) included base substitutions, indels, copy number alterations and fusions/rearrangements. a total of ( . %) braf-altered pediatric malignancies were identified. ( . %) harbored a single kinaseactivating braf short variant, indel, or fusion. an alteration resulting in reduced braf kinase activity was identified in ( . %) tumors while ( . %) tumors harbored multiple braf alterations, of which contained at least a single activating short variant. the remaining tumors ( . %) contained functionally uncharacterized variants. kinaseactivating braf alterations were identified in diverse tumor spectra comprised of brain tumors ( . %; subtypes), carcinomas ( . %; subtypes, with melanoma constituting % of cases), hematological malignancies ( . %; subtypes), sarcomas ( . %; subtypes), and extracranial embryonal tumors ( . %; subtypes). seventy-two ( . % of braf-altered cases) braf fusions were identified, ( . %) of which were kiaa -braf; involved the novel fusion partners: stard nl and khdrbs . seven ( . %) raf fusionpositive cases, predominantly brain tumors ( ), were identified; involved the novel fusion partners: tmf and sox . index cases of response to therapy of intracranial tumors will be presented. we describe a population of pediatric patients with targetable braf alterations predominantly enriched in primary intracranial tumors, but spanning diverse solid tumor types and hematologic malignancies. we additionally report a cohort of raf fusion-positive patients. an index case and multiple previous reports suggest raf or mek inhibitors may benefit pediatric patients with either intracranial or extracranial disease, and development of such drugs in pediatric indications is strongly warranted. background: diffuse midline gliomas (dmg) with h k m mutation, including diffuse intrinsic pontine glioma (dipg), are the leading cause of brain tumor-related deaths in children. there are no effective therapeutic strategies and the median survival remains dismal. genomic studies have identified a recurrent mutation in the majority of dmgs involving a lysine to methionine substitution (k m) in histones . and . , resulting in changes in the epigenetic landscape that dysregulate gene expression and promote gliomagenesis. panobinostat, a multiple histone deacetylase (hdac) inhibitor, was found to be one of the most effective agents against dipg patient-derived cell cultures and xenograft models in previous studies and is presently in clinical trial for dipg. hdac inhibition with panobinostat may also exhibit activity against h k m+ diffuse midline gliomas of the thalamus and spinal cord. to evaluate the effect of panobinostat as a single agent against patient-derived thalamic and spinal cord h k m+ diffuse midline glioma cell cultures and in an orthotopic xenograft murine model of h k m+ spinal cord glioma. design/method: patient-derived thalamic and spinal cord h k m+ diffuse midline glioma cell cultures were treated with single agent panobinostat at a range of concentrations. cell viability was evaluated using the celltiter-glo assay. panobinostat was systemically administered to orthotopic xenograft murine models of luciferase-expressing spinal cord h k m+ diffuse midline glioma. response to panobinostat was evaluated with ivis in vivo imaging. results: hdac inhibition with panobinostat significantly decreases cell proliferation with an ic of nm and nm in the spinal cord and thalamic glioma patient-derived cell cultures respectively. panobinostat slowed tumor growth in murine models of spinal cord glioma by . -fold in the brain (p = . , n = ) and -fold in the spinal cord (p = . , n = ) when compared to vehicle controls after week of administration. panobinostat is in clinical trials for dipg. this study suggests that hdac inhibition with panobinostat may also be beneficial for patients with thalamic and spinal cord diffuse midline glioma h k m mutants. background: brain tumors are the most common solid tumor of childhood and the leading cause of childhood cancer deaths. while medulloblastoma is the most common malignant brain tumor of childhood with a -year survival - %, children with high-grade gliomas (hggs) such as glioblastoma multiforme (gbm) fare much worse with a -year survival of - %. implicated in this poor outcome is the presence of treatment resistant brain tumor stem-like cells. gbm stem-like cells (gscs) have been implicated in tumor growth, treatment resistance and patient relapse, making them a key therapeutic priority. antipsychotic drugs (apds) have been used for decades in various psychiatric clinical settings and are associated with a lower incidence of cancer, including malignant brain tumors. currently, atypical apds are being evaluated for their potential to alleviate cancer and treatment induced side effects. furthermore these drugs may have direct anti-tumor effects, potentially via inhibition of dopamine d receptors (drd ). objectives: determine the anti-cancer effects of atypical apds on gbm stem-like cells design/method: the anti-cancer effects of apds (quetiapine and risperidone) were evaluated on gbm stem-like cell lines developed in our laboratory (glio and ) and the group medulloblastoma cell line hdmbo . cell proliferation/viability was determined using trypan blue exclusion and mts assays. the effect of apds on cancer stem cell self-renewal was determined by neurosphere assay. receptor expression and apds effect on cell cycle proteins were examined by western blot analysis. results: western blot analysis of gscs and hdmbo demonstrated robust drd expression indicating a viable therapeutic target. both apds induced dose dependent cell death of all cell lines tested. treatment with only um of either apd for days significantly reduced cell proliferation by % (hdmbo ) and - % (gscs). consistent with these findings, we observed an increase in cell cycle inhibitors p and p . furthermore at day both apds induced a robust increase in gsc death, approximately % compared to only % in non-treated controls. lastly, um apds significantly reduced gsc neurosphere formation compared to untreated controls by up to % suggesting inhibition of gbm stem cell self-renewal. our data indicates that clinically relevant concentrations (low micromolar) of these apds induce anticancer effects in both gscs, which are enriched with tumor initiation/propagation properties, and in the group (myc amplified) medulloblastoma cell line. these apds represent strong candidates as potential adjuvant therapies for the treatment of these brain tumors. background: while the poor prognosis for high risk neuroblastoma (hrnb) underscores the need for new treatment strategies, the elucidation of specific biologic subsets of neuroblastoma suggests a way to improve disease management. the identification of agents that target specific molecular pathways associated with the development or progression of diseases holds promise. dfmo, an inhibitor of odc, has been shown to decrease lin and mycn and target cancer stem cells in preclinical studies. currently % of patients undergoing immunotherapy relapse. dfmo is in studies to prevent relapse after immunotherapy and may be helpful during immunotherapy as well. the hypotheses for this study were that: ) the incorporation of a targeted therapy, selected based upon upfront tumor genomic interrogation, into standard induction chemotherapy for hrnb is safe, feasible and may increase the pr/cr/vgpr response rate at the end of induction therapy; and ) the addition of dfmo as maintenance during immunotherapy is safe and feasible and may decrease the relapse rate for hrnb. a multicenter feasibility pilot trial in subjects with newly diagnosed hrnb within the beat childhood cancer consortium. at diagnosis, patients' tumors underwent dna exome and rna sequencing which were analyzed within a molecular tumor board to identify the single best drug of targeted agents to be added to cycles - of induction chemotherapy. after consolidation with asct and radiation, the patients received dfmo along with standard dinutuximab and retinoic acid and dfmo for years after immunotherapy. patients were evaluated for additional toxicities with the addition of targeted agents and dfmo in addition to induction response. results: the pilot study of eligible patients has shown this process to be feasible. all patients have completed induction portions of the study. the combination of targeted agent with chemotherapy was shown to be safe without any unexpected toxicities. delays between induction cycles were < weeks and related to surgery, infection, or thrombocytopenia. the induction response demonstrated % cr/vgpr/pr rate, which suggests improvement over historical %. in addition, patients were eligible for the combination of dfmo with dinutuximab and retinoic acid was well tolerated and safe without additional toxicities due to dfmo. the pilot study of patients has shown the process of genomic sequencing and addition of a targeted agent to upfront chemotherapy and addition of dfmo to dinutuximab and retinoic acid maintenance therapy in newly diagnosed hrnb patients and is feasible and safe without any unexpected toxicities. background: identifying sub-populations of medulloblastoma tumors with stem cell-like properties holds promise for reducing disease recurrence, but there is no known unifying marker of medulloblastoma cancer stem cells. the granulocyte stimulating factor receptor (gcsf-r or cd ) is well understood in the context of hematopoiesis, but its role in solid tumor pathogenesis is less clear. neuroblastoma and melanoma subpopulations expressing gcsf-r have cancer stem cell properties of chemoresistance and increased tumorigenicity, and are enriched in tumors after chemotherapy. gcsf-r activation leads to signaling through the jak-stat pathway, suggesting a potential therapeutic target. we hypothesized that a subpopulation of medulloblastoma cells would express the gcsf-r and that this subpopulation would demonstrate chemoresistance and response to inhibitors of the jak/stat pathway. objectives: our objective was to identify a subpopulation of medulloblastoma cells expressing the gcsf-r and determine their relative growth rates, tumorigenicity, and responses to chemotherapy and jak/stat inhibition. design/method: medulloblastoma cell lines were sorted via flow cytometry for gcsf-r surface expression. subpopulations of gcsf-r-positive and -negative medulloblastoma cells were then monitored for growth by continuous live cell imaging. responses to chemotherapy were measured in subpopulations of gcsf-r-positive and -negative medulloblastoma cells using continuous live cell imaging to measure percent cell confluence and cell viability assays. ic values were calculated for each cell line and each agent. parental medulloblastoma cell lines and isolated gcsf-r-positive and -negative subpopulations were also treated with the jak / inhibitor ruxolitinib and growth rates, viability, and ic values were calculated. results: gcsf-r surface expression was identified on . - . % of medulloblastoma cell lines. isolated gcsf-r positive cells demonstrate a slower growth rate compared to gcsf-rnegative or parental unsorted medulloblastoma cells. gcsf-r positive cells are more resistant in vitro to vincristine, etoposide, and carboplatin, when compared to the gcsf-r negative population and an unsorted population of the same cell line. ruxolitinib is cytotoxic to medulloblastoma cells in vitro, with higher ic values noted in gcsf-r positive cells compared to unsorted and gcsf-r negative cells. we show that a subpopulation of gcsf-r positive cells are present in multiple medulloblastoma cell lines via flow cytometry, and that isolated gcsf-r-positive cells have a slower growth rate than gcsf-r-negative or unsorted populations. we also show that ruxolitinib has in vitro activity against medulloblastoma cell lines. we propose that jak inhibition may represent an adjunct therapy targeting overall tumor burden and specifically targeting the gcsf-r-positive subpopulation of medulloblastoma cells that may drive tumor recurrence. we investigated the efficacy of intensified adjuvant chemotherapy in osteosarcoma patients. design/method: we retrospectively analyzed the medical records of children with osteosarcoma treated at asan medical center between and . all patients received a -drug induction consisting of cycles of cisplatin and doxorubicin along with cycles of methotrexate (map), and proceeded to surgical resection. adjuvant ct was map or map with the additional ifosfamide and etoposide (mapie), and mapie was mainly considered for poor responders (tumor necrosis below %) or patients with metastases. results: among patients, patients had metastases at diagnosis. surgery was conducted in patients who responded to induction ct, and showed over % tumor necrosis. among patients who proceeded to adjuvant ct, and patients received to map and mapie protocols. with a median follow-up of months, the -year overall survival (os) and event-free survival (efs) rates of all patients were % and . %. of those patients, patients recurred, and of them died of disease progression. relapsed patients received salvage ct and/or surgery, and were rescued after autologous stem cell transplantation (sct). three patients developed treatment-related acute myeloid leukemia, and they are alive after allogeneic sct. according to the response to neoadjuvant ct, the os rates of good responders (n = ) and poor responders (n = ) were % and . % (p = . ), and efs rates were . % and . % (p = . ). of the poor responders, patients received map as adjuvant ct, and the other received mapie. the os rates of map and mapie group were . % and . % (p = . ), and efs rates were . % and . % (p = . ), respectively. when patients were classified into three groups: . localized disease & necrosis ≥ % (n = ), . localized disease & necrosis < % (n = ), . metastatic disease (n = ), survival rates were in the order of group > > (os = %: . %: . %, efs = . %: . %: %). in each group, intensified adjuvant ct by mapie did not improve survival outcomes. conclusion: initial metastatic disease and poor histological response to neoadjuvant ct were major risk factors for poor survival in osteosarcoma patients. we found that adding ifosfamide and etoposide to map did not improve survival outcomes of patients with adverse risk factors. more effective adjuvant therapy for these patients is needed. background: circulating cell-free dna (cfdna) that shed from tumors into circulation have been used for noninvasive molecular profiling in adult cancers but little is known about its utility in pediatric cancers. pediatric patients with metastatic and refractory solid tumors are known to have poor survival rates, and a key challenge in their management is obtaining biopsy samples especially at times when disease is widely spread or the patient is physically unfit for sampling. the development of a noninvasive profiling strategy is critical for optimizing molecularly guided therapy and assessing response to treatment. in this study, we want to determine the utility of cfdna to noninvasively analyze the molecular profiles of pediatric solid tumors such as neuroblastoma (nb), osteosarcoma (os), and wilms tumor (wt). design/method: tumor, plasma, and matched controls were collected from patients with nb, wt, and os, at diagnosis or time of disease progression. cfdna was extracted from the plasma and analyzed through multiple methodologies including a targeted next generation sequencing panels and shallow whole genome sequencing (swgs). results: fifteen nb patients, os patients, and wt patients had tumor molecular profiles known from different targeted next-generation sequencing platforms. in the cfdna of / nb patients, somatic mutations and copy number alterations previously reported in the tumors were detected, including recurrent nb drivers such as mycn amplification, alk, and atrx mutations. mutations not detected in the original tumor were also found in / nb patients including nras, mll , arid b, some of which are potentially actionable. in os, mutations known from the tumor were found in the cfdna of of patients, including atrx and notch mutations, as well as copy number alterations such as cdk amplification, which has targetable therapeutics available. of the two wt patients analyzed, cfdna revealed the same mutations as tumor in one patient, however in a cohort of patients where tumor was not available, cfdna revealed recurrent driver mutations such as amer , dicer . it is feasible to noninvasively identify somatic mutations and copy number alterations in cfdna of patients with pediatric solid tumors. establishing a platform using cfdna to identify molecular profiles of these tumors can serve as a powerful tool for guiding treatment and monitoring response to treatment. background: despite multi-modality therapy, the prognosis for patients with metastatic osteosarcoma remains poor necessitating development of novel targeted therapies. immunotherapy can be exploited to target osteosarcoma with exquisite specificity but remains limited by insufficient tumor specific targets. objectives: to overcome the dearth in tumor specific antigens, we have explored the use of tumor derived mrna (representing a tumor specific transcriptome) for development of personalized nanoparticle vaccines. design/method: rna-nanoparticles (rna-nps) can be amplified from limited amounts of biopsied tissue for induction of tumor specific t cells against osteosarcoma. since local vaccination strategies are mired by poor overall immunogenicity, we assessed the feasibility, immunogenicity and antitumor activity of intravenously administered rna-nps (tumor mrna complexed to dotap nanoliposomes) in pre-clinical murine and canine tumor models. we identified a clinically translatable np formulation for the delivery of rna to antigen presenting cells (apcs) that induces in vivo gene expression and preserves rna stability over time. tumor derived rna-nps induced antigen specific t cell immunity and mediated anti-tumor efficacy in several pre-clinical solid tumor models (i.e. b f , kr b). when administered intravenously, rna-nps increased expression of co-stimulatory molecules (i.e. cd , cd , cd , ccr ) and pd-l on cd c+ cells throughout reticuloendothelial organs (i.e. spleen, liver, bone marrow) and within the tumor microenvironment; this phenotype was strictly dependent on type i interferon. targeted inhibition of type i interferon signaling (via infar mabs) abrogated anti-tumor efficacy mediated by rna-nps. we enhanced the immunogenicity of this platform by simply combining mrnas encoding for immunomodulatory molecules (i.e. hcv-pamps, gm-csf) or by combining rna-nps with immune checkpoint inhibitors. addition of checkpoint inhibitors (pd-l mabs) to rna-nps increased tumor infiltrating lymphocytes, and intratumoral mhc class i/ii expression, and mediated synergistic anti-tumor activity in settings where pd- or pd-l inhibition alone did not confer therapeutic benefit. we then explored the feasibility of rna-nps in a large animal osteosarcoma model. in ongoing studies for canines with osteosarcomas, we have shown that sufficient amounts of rna can be extracted, amplified, and manufactured into personalized rna-np vaccines. conclusion: rna-nps reprogram systemic immunity and mediate anti-tumor activity providing near immediate immune induction without the complexity of cellular immunotherapy. the immune correlate of preclinical response to rna-nps is hallmarked by interferon dependent pd-l expression on activated apcs (cd c+ mhcii+ cd + cells). based on these findings, we are exploring the preclinical safety, efficacy and immunologic effects of rna-nps targeting canine osteosarcoma before first in-human evaluation. background: ewing sarcoma is an aggressive bone tumor affecting mainly adolescent and young adults. treatments are based on compressed schedule chemotherapy combined with local control (surgery and/or radiation). prognosis is poorer for patients with metastatic disease, older age and central primaries. survival when disease recurs within two years of diagnosis is < %. the ews-fli fusion gene t( ; ) (q ; q ) has been well characterized as a dominant ews driver-gene. the most common variation is ews exon with fli exon ( % of fusion positive patients). we designed a novel pbi-shrna tm ews/fli type lpx which has demonstrated, safety and efficacy in animal model (rao et all). the pbi-shrna strategy silences target gene expression by concurrently inducing translational repression and p-body sequestration as well as post-transcriptional mrna cleavage. to determine the safety and maximum tolerated dose of intravenous administration of pbi-shrna tm ews/fli type lipoplex in patients advanced ews. design/method: phase i study × escalation cohort. testing pbi-shrna tm ews/fli type lpx (starting iv dose of . mg/kg) on patients (≥ age ) with advanced ewing's sarcoma, all with a type translocation. intravenous infusion was given twice a week for weeks with the following escalation schema: % → % → % → % → %. required kps > % and adequate organ function. cytokines induction pre and post-infusion was analyzed (il- , il- , tnf-alpha, il ra). first cohort of patients has been enrolled (ages between - years). three relapsed patients had > lines of therapy and patient had refractory disease, patients received a complete cycle of pbi-shrna tm ews/fli type lpx with twice a week infusions. a total of doses were given. the most prominent related toxicity has been hematological, patient developed transient g neutropenia, another patient developed g anemia that required prbc transfusion, and of note this patient had significant bone and bone marrow involvement. one patient only received two lpx infusions; she developed a fatal rsv pneumonia. other reported grade toxicity includes fatigue and headache. evaluable patients (n ) had stable disease between and months before progression. one patient had sustained response for month before progression, two patients are still alive. our preliminary experience supports the safety and potential efficacy of pbi-shrna tm ews/fli type lpx as novel treatment for advanced ews with limited toxicity. il- increase correlates with higher bi-shrnai ews/fli lpx infusion rate and clinical symptoms. further clinical testing is indicated. background: as more children with cns malignancies (bt) are surviving, the late effects of the therapies they receive are better described. studies show that radiation therapy is particularly harmful to neurocognitive functioning, specifically processing speed, working memory, and attention span. these deficits have negative effects on quality of life, especially in academic and professional settings. a large proportion of s of s adult survivors of bt are unable to reach adult milestones such as living on their own, holding a steady job, and getting married. proton beam radiation therapy (pbrt), is touted for the potential to have fewer and less severe side effects than traditional photon radiation therapy (xrt). because of the properties of protons, the amount of damaging energy released in non-target healthy tissue is reduced when compared to xrt. although a study comparing iq testing between pbrt and xrt found no difference between the two therapies, no studies have compared the specific neurocognitive domains. it would be valuable to evaluate full neurocognitive testing scores (nct) since the specific domains, particularly processing speed (psi), appear to be most vulnerable to radiation therapy. objectives: our primary aim was to assess differences in psi for patients with bt who underwent pbrt versus xrt. a secondary aim was to assess differences in iq (fsiq) and working memory (wmi). we retrospectively evaluated all patients treated for bt at the jimmy everest cancer center within the past years who received rt and had nct post radiation. we examined the full nct results for both subsets of participants to evaluate differences in the specific domains of processing speed, working memory, and iq by measuring percentiles scored in these domains. objectives: we report our experience on imaging children with mm treated uniformly on an institutional melanoma trial. we retrospectively reviewed the clinical and imaging findings of patients with ajcc stage iic-iv cutaneous mm treated on our institutional mel protocol. brain mri/ct, pet/ct, ct chest, abdomen, and pelvis (ctcap) were performed at diagnosis in all patients. on treatment, stratum a patients (peg-interferon; ajcc iic, iiia, iiib) (n = ) had the same imaging repeated every months; stratum b (peg-interferon and temozolomide; unresectable measurable disease metastatic, or recurrent) (n = ) had pet scans every months and brain imaging every months; those in stratum b (peg-interferon and temozolomide; unresectable non-measurable, metastatic, or recurrent) (n = ) had the same imaging performed every months. off therapy all patients continued same imaging every months for years. results: there were patients ( female; median age years). eleven had spitzoid and conventional melanoma. primary sites included head/neck (n = ), trunk (n = ), and extremities (n = ). patients with spitzoid melanoma had imaging studies ( pet, ctcap, ct chest, ct brain, and mri brain) with a median of , , , and studies/patient respectively. median cost per patient was $ , . thirteen studies ( . %) showed suspicious lesions with additional scans and diagnostic biopsies of which one only was positive stratum a with tert promoter mutation and died from disease). for conventional mm, studies ( pet, ctcap, ct chest, ct brain, and mri brain) were performed with a median of , . , , , studies/patient respectively. median cost per patient was $ , . twenty ( %) showed suspicious lesions with additional scans and diagnostic biopsies; four were positive (two at diagnosis); both died of disease; the other two recurred locoregionally and were detected clinically; both are alive and disease free; one patient had diffuse metastases and died shortly after enrollment. after a median follow up of . years (range . - . ) patients are alive and disease free. children with spitzoid melanoma should have minimal imaging at diagnosis and follow-up given the low risk of recurrence and low yield and high cost of aggressive imaging protocols. patients with conventional mm should be imaged according to the adult guidelines. nationwide children's hospital, columbus, ohio, united states background: the role of infections in the long term outcome of patients with bone tumors is controversial. two retrospective studies have shown increased survival in osteosarcoma patients who had a post-operative wound infection, while another showed no changes in overall survival. to determine the relationship between wound infections and/or bloodstream infection (bsi) on survival in pediatric and young adult patients with osteosarcoma and ewing sarcoma treated at a tertiary children's hospital. design/method: a retrospective chart review was performed for patients with diagnosis of osteosarcoma or ewing sarcoma from - . patients received standard chemotherapy regimens for their disease type and stage. local control included surgical resection and/or radiation therapy. presence of infection was determined by bsi or wound cultures while receiving treatment for primary tumor. the median age of patients was (range - years) at diagnosis. % had a diagnosis of osteosarcoma and % had ewing sarcoma. of these, % of patients developed an infection during treatment; % had bsi, % had wound infections, and % had both. patients with bsi had a year os of . %, compared to % in those without bsi (p = . ). those with both bsi and wound infections had the poorest overall survival of %, compared to . % for patients without any infection. patients with wound infections alone had a year os of . %, compared to % of patients without a wound infection. our analysis revealed decreased os in patients with bsi; however, this could be due to other confounding factors in the presence of bsi. those with bsi or bsi and wound infections had the poorest survival. wound infections without bsi were associated with a slight increase in survival; however, this study was limited by the number of patients that had local wound infections. with the use of newer surgical techniques, availability of antimicrobials and routine use of prophylactic antibiotics, the incidence of infections while undergoing treatment is low. however, the importance of this clinical observation indicates a likely enhanced immune system associated with infection, supporting the role of immunotherapy for treatment of these aggressive tumors. background: hypoalbuminemia is a well-recognized effect of cancer and other chronic illnesses and is often regarded as a marker of malnutrition. in adults, hypoalbuminemia has been associated with adverse outcomes in patients with cancers of the lung, pelvis, head and neck, gastrointestinal tract, and bone marrow, as well as in some pediatric patients with ewing sarcoma and hodgkin lymphoma. hypoalbuminemia has not been well studied in children with cancer. to determine the incidence of hypoalbuminemia (using age-specific references) in children with cancer receiving chemotherapy at baseline (prior to starting chemotherapy) and to determine whether hypoalbuminemia is associated with inferior -year overall survival. design/method: we performed a single institution, irbapproved, retrospective review of pediatric oncology patients diagnosed between and . five-year survival was estimated using the kaplan-meier method; groups were compared using cox regression. we identified pediatric patients with a first diagnosis of cancer, brain tumor, or other condition possibly requiring chemotherapy. of these patients, were excluded for reasons including not receiving chemotherapy and missing data, leaving patients who had a serum albumin level within days prior to starting chemotherapy. the mean age was . years (sd . years); % were male; % were non-hispanic. the most common diagnosis was acute lymphoblastic leukemia ( of ; %). one hundred thirty nine of ( %) had hypoalbuminemia prior to starting chemotherapy. there was no statistically significant difference in -year overall survival between those with and without hypoalbuminemia ( % vs. %, respectively; hazard ratio . , % c.i. . - . ). conclusion: hypoalbuminemia at baseline in pediatric oncology patients requiring chemotherapy is common (one in five), and was not associated with inferior -year overall survival in this cohort. leptomeningeal metastases at diagnosis. standard treatment for completely resected, non-anaplastic supratentorial ependymomas is close observation. treatment for anaplastic or incompletely resected non-anaplastic ependymomas is maximal safe surgical resection followed by focal radiation. however, up to % of localized ependymomas recur. the role of chemotherapy in treating ependymomas is under investigation. extraneural metastases of anaplastic ependymomas have rarely been reported and the outcome is dismal. objectives: to report extraneural cervical node metastases of a non-anaplastic ependymoma and successful treatment with surgical resection, radiation, and systemic chemotherapy. design/method: retrospective review of patient medical records, including radiographic imaging and tumor tissue pathology, and comprehensive literature review. results: a previously healthy -year-old girl underwent gross total resection (gtr) of an isolated right parietal lobe ependymoma (who grade ii). at age years, magnetic resonance imaging (mri) revealed an isolated localized recurrence. she underwent gtr followed by observation. at age years, she again experienced isolated localized recurrence and underwent gtr followed by . gy focal conformal photon radiation. at each recurrence, pathology revealed a non-anaplastic ependymoma, and cerebral spinal fluid (csf) cytopathology and spine mri were negative. at age years, she developed an enlarged right posterior cervical chain lymph node. subsequent mri revealed a large rim-enhancing, t hyperintense lymph node and multiple abnormally enhancing regional nodes consistent with metastases. biopsy revealed a non-anaplastic ependymoma. mri of the brain and spine, computed tomography of the chest, abdomen, and pelvis, and csf and marrow evaluations were unremarkable. chemotherapy according to acns was initiated. mri after course demonstrated significant node size reduction. she underwent right neck node dissection. only one right level ii lymph node showed metastases. she was treated with . gy irradiation to the neck and additional courses of chemotherapy. she remains in remission months and months after diagnosis of metastatic disease and end of therapy, respectively. literature review reveals rare reports of extraneural metastatic disease of anaplastic ependymomas to bone, lung, or liver, and only involving lymph nodes, all associated with a poor outcome despite multimodal therapy. to our knowledge, this is the first report of extraneural metastases of a non-anaplastic ependymoma. extraneural metastases should be considered in children previously treated for non-anaplastic ependymomas who experience systemic symptoms, even in absence of cns relapse. multimodal treatment offers potential long-term disease control with acceptable toxicity. arun gurunathan, joel sorger, andrew trout, joseph pressey, rajaram nagarajan, brian turpin cincinnati children's hospital medical center, cincinnati, ohio, united states background: pigmented villonodular synovitis (pvns) is a benign neoplasm of the synovium. standard treatment is surgery, but post-operative recurrence rate is as high as %. radiation therapy can be used for local control, but is associated with late effects. while pvns is rarely fatal, aggressive disease and/or extensive surgery can result in substantial functional impairment. colony stimulating factor- (csf ) overexpression, often due to chromosomal translocation involving csf , drives pvns through recruitment of synovial-like mononuclear cells expressing the csf -receptor. tyrosine kinase inhibitors such as imatinib are active against the csf -receptor, and have shown benefit in the post-surgical relapse setting. however, questions remain regarding the broader application of imatinib and regarding optimal response assessment. to present three patients with pvns, each with different clinical scenarios, who demonstrate clinical response to imatinib monitored by changes in metabolic activity (maximum suv) on pet/ct. results: three patients with pvns demonstrate pet/ct response to imatinib, guiding management of their challenging clinical scenarios. patient is a year-old female with left hip pvns and high grade articular cartilage loss, with decrease in metabolic activity (suvmax . to . in months) on neoadjuvant imatinib, enabling total hip replacement surgery planning. patient is a year-old female with left knee pvns with recurrences after synovectomies, spared subsequent surgical control attempts after clinical improvement correlating with pet/ct response to imatinib (suvmax . to . in months). patient is a year-old male with right knee pvns that recurred after total knee replacement, now with clinical improvement correlating with pet/ct response to imatinib (suvmax . to . in months). all patients would have been characterized as stable disease by response evaluation criteria in solid tumors (recist). in each of these patients, imatinib has been tolerated well, with no therapy interruptions and absent or easily managed side effects (one patient takes dronabinol for decreased appetite, one patient takes prn immodium for diarrhea). all patients are currently still taking imatinib, with therapy length ranging from five to eleven months. in our series of three patients with pvns, imatinib shows promise for disease management in neoadjuvant and adjuvant settings with a tolerable side effect profile. imatinib should be considered in the treatment of pvns to spare surgical and radiotherapy related morbidity, and treatment effect can be monitored by pet/ct. background: metastatic rhabdomyosarcoma (rms) carries a poor prognosis with three-year event free survival rates ranging between %- % (depending on oberlin risk factors) due to the lack of significantly effective breakthroughs in the recent past. there is an urgent and unmet need for new treatment strategies against this disease. metastatic rms cell lines exhibit increased expression of the erm family membrane-cytoskeleton linker protein ezrin. knockdown of ezrin expression using sirnas decreases the metastatic potential of these cells, whereas forced expression of ezrin results in increased degree of metastasis. the activity of ezrin is controlled by its phosphorylation at the threonine (thr ) residue at the c-terminus of the protein, suggesting that alteration of ezrin phosphorylation may control rms growth and metastasis. our goal was to determine if pharmacological inhibition of thr phosphorylation in ezrin affects the growth, survival and metastasis in rms in vitro as well as in vivo. design/method: rms cell lines representative of the alveolar and embryonal histological subtypes were used. rms cells were treated with a small molecule inhibitor of ezrin, nsc , which specifically dephosphorylates ezrin at the thr residue. baseline expression of ezrin and perm levels as well as the effect of nsc on perm levels in the rms cell lines was determined by western blotting of cell lysates. viability of cells was assessed by trypan blue exclusion, and morphology visualized by bright field microscopy. the extent of apoptosis was detected by imaging caspase / activation using fluorescent microscopy. motility of rms cells was examined by performing a wound-healing assay. subcutaneous and orthotopic xenografts were established in nsg mice using rd cells (embryonal rms). mice harbor-ing xenografts were treated with intraperitoneal injections of nsc or dmso. results: ezrin is constitutively phosphorylated at the thr residue in a majority of the rms cell lines examined. nsc dephosphorylates ezrin at the thr residue in these cell lines. treatment with nsc inhibits growth, induces apoptosis and inhibits the migration of rms cell lines in vitro. further, treatment of nsg mice bearing subcutaneous or orthotopic embryonal rhabdomyosarcoma xenografts with nsc significantly impedes tumor progression without any obvious adverse effects. our findings suggest that dephosphorylation of ezrin at the threonine residue may have the potential to be a novel therapeutic strategy for rms patients. all india institute of medical sciences, new delhi, new delhi, delhi, india background: the role of laparoscopy in the management of pediatric intra-abdominal solid tumors is yet to be established. the safety of laparoscopic management of pediatric intra-abdominal tumors is still questionable. we study the results of the initial case series of pediatric intraabdominal tumors managed laparoscopically at our institute from july onwards. design/method: total children ( -males, females) who presented to us with pediatric intra-abdominal tumors were included. the tumors included wilms tumor (n = ), neuroblastoma(n = ), adrenal cortical tumor(n = ), ovarian teratoma(n = ).children were between months - years and received neo-adjuvant chemotherapy. a -port laparoscopic nephrectomy and lymph node sampling for wilms tumor and adrenalectomy for adrenal tumors was performed. the tumors were removed in-toto with no rupture (except in one). specimens were retrieved through a lumbar incision (n = ) or an inguinal incision(n = ). all the children are under regular follow up. two children with wilms tumor had recurrence. the neuroblastoma child underwent open surgery for recurrence later. conclusion: laparoscopy/laparoscopic assisted removal of pediatric intra abdominal tumor is a feasible and safe option. it has the advantage of less postoperative pain, shorter hospital stay and a better cosmetic result. proper patient selection, port placement and laparoscopic experience are contributory. background: targeting of proteins and cell surface antigens specific to cancer cells with monoclonal antibodies has proven to be an effective form of treatment in many forms of cancer. gd is a cell surface disialoganglioside that is expressed on the cell surface of some normal tissues including nerve cells, melanocytes, and mesenchymal stromal cells and is overexpressed in some pediatric cancers like neuroblastoma and osteosarcoma. dinutuiximab is a chimeric monoclonal antibody that is fda approved for the treatment of patients with high risk neuroblastoma and under investigation for the treatment of relapsed osteosarcoma. little is known about the patterns of gd expression in other pediatric malignancies. objectives: we sought to describe the patterns of gd expression in the following pediatric sarcomas: synovial sarcoma, rhabdomyosarcoma and ewing sarcoma. design/method: synovial sarcoma (n = ), rhabdomyosarcoma (n = ) and ewing's sarcomas (n = ) formalin fixed, paraffin embedded cores were obtained from the seattle children's research institute tissue microarray (tma) biorepository. tma blocks consisting of melanoma cores stained with and without gd antibody were used as positive and negative controls, respectively. slides were incubated with anti-ganglioside gd antibody clone q (ab from abcam) diluted : in % normal goat serum and % bsa in tbs overnight at ˚c. the negative control of human melanoma section was incubated in % normal goat serum and % bsa in tbs without primary antibody. the expression of gd was indicated by characteristic brown diaminobenzidine staining. the intensity and location of tissue staining were assessed and compared to positive and negative controls. staining was considered positive (+++) if the intensity of the staining was consistent with that of the positive control with - % of cells staining positive. classification of intermediate gd expression (++) was assigned to slides in which - % of cells stained positive. slides were classified as sporadic staining (+) if - % of cells stained positive. tissue was considered (-) if there was complete absence of staining, similar to the negative control. objectives: to evaluate the clinical presentation, management and treatment outcomes of children with malignant germ cell tumor at our institute design/method: a prospective study was conducted from june to dec in the department of pediatric surgery in a tertiary care institute in a developing country. all patients were evaluated for local disease and metastatic disease by imaging and tumor markers. risk stratified chemotherapy was used with low risk tumor receiving no chemotherapy, intermediate risk: courses of peb chemotherapy and high risk: courses of peb + courses of pe. upfront resection of the primary or the residual disease after neoadjuvant chemotherapy if feasible was performed. follow up was done with monthly tumor markers for months and imaging studies every - months for initial years. five year overall survival and disease free survival was calculated. results: during the study we treated children who formed the study group. of these ( %) were gonadal ( ; % testicular and ; % ovarian) and the remaining ( %) were extragonadal with sacrococcygeal (sct) being the most common site ( %). one hundred and thirteen children ( %) presented to us primarily while the remaining had received treatment elsewhere. stage or stage disease at presentation was present in ( %) children. recurrence was noted in ( %) patients. respectively. patients with testicular mgct and children with age - years and males had significantly poor rfs rates. conclusion: patients with mgct should be staged correctly and adjuvant chemotherapy is advisable to all patients except stage i endermal sinus tumor of testis. awareness regarding the same is still lacking in our country. meticulous follow up is needed as more than % of will recur. cure rates are dismal in children with recurrent mgct especially those who are not chemotherapy naïve. nemours children's specialty care, jacksonville, florida, united states background: radiotherapy for pediatric head and neck tumors often results in mucositis, limiting oral intake and compromising patients' nutritional status. this may be reduced through the improved conformality offered by proton therapy. despite widespread use of enteral tube feeding through a percutaneous gastrostomy (peg) or nasogastric tube (ngt), there is little data available regarding overall incidence of ngt/peg placement and perspectives of pediatric patients and caregivers. objectives: to (a) estimate the need for ngt/peg support and (b) characterize patient and caregiver perceptions surrounding enteral feeding in children with head and neck tumors undergoing proton therapy. design/method: dependent on development stage, patient (n = ) or parents (n = ) filled out a series of customized surveys according to a prospective irb approved study. seventythree percent of patients also received concurrent chemotherapy. questions addressed their current feeding route and perception, for example, "what aspect(s) of tube feedings are beneficial to you?" and "what aspect(s) of tube feeding worry or scare you?" fifty-five surveys were distributed before and after radiation, and with any change in feeding route. results: at the start of proton therapy, patient had a ngt and patients had peg. of these, patients ( %) had a ngt/peg in place exclusively for the administration of medication; only patient ( %) needed a ngt/peg for nutrition. in those patients without ngt/peg, % would "consider" enteral feeds. in patients without ngt/peg, the most commonly cited benefit was "maximizing my nutrition" ( %) and the most common negative aspect was "fear" of tube placement ( % of patients). all sub-populations ( % of patients) cited change in appearance as a negative aspect. in patients without ngt/peg at the start of proton therapy, % of patients/caregivers felt enteral feeding to be "unnecessary," and % of these patients would not "consider" ngt/peg even if their "physician advised it." over the course of proton therapy, the patients/caregivers who deemed enteral feeding "unnecessary" decreased from % to %. at completion of treatment, patients ( %) were using a ngt/peg tube for nutritional support but only one ( %) patient relied exclusively on their enteral feeds. two patients (without ngt/peg) ( %) required parenteral support. our data does not support prophylactic placement of ngt/peg in of children with head and neck tumors undergoing proton therapy. ongoing research is needed to identify which patients will need ngt or peg to supplement their diet. in this cohort, anticipatory counseling should focus on pain, cosmesis, and utility. children's national medical center, washington, district of columbia, united states background: ovarian sex cord-stromal tumors (osct) are rare neoplasms that typically present with signs/symptoms of an adnexal mass and signs of hormonal production approximately % of ovarian sex cord-stromal tumors in children are sertoli-leydig cell tumors (slct) with median age of presentation years overall. to our knowledge the youngest reported case in the literature describes a -month old female in china with a slct that was treated with oophorectomy alone. some studies have found an association in families between pleuopulmonary blastoma and osct with a germline mutation leading to dicer syndrome, which has been associated with a younger age at diagnosis. , objectives: to describe an unusual case presentation of slct in an infant results: -month old, twin female, ex- week premature infant presented to the emergency department on multiple occasions for abdominal distention and feeding intolerance initially thought to be related to previous omphalocele repair and umbilical hernia. an ultrasound demonstrated an × cm mass arising from the right ovary with large volume ascites. she required admission to the intensive care unit due to s of s respiratory distress from her significant ascites. serum tumor marker including hcg, afp and ldh were negative. patient underwent right oophorectomy with tumor capsule noted to be open at time of surgery. further imaging post operatively demonstrated no other sites of disease. the patient was classified as figo stage ic due to the presence of her significant abdominal ascites that was presumed to be malignant pre-operative tumor rupture. the pathological diagnosis was challenging and eventually resulted as a mixed germ cell sex cord stromal tumor with pattern of sertoli cell tumor with neuroendocrine differentiation. based on the staging of figo ic with pre-operative rupture, the decision was made to treat with a standard platinum based regimen as there is a higher incidence of relapse in stage ic patients when compared to ia treated with observation alone. our patient tolerated four cycles of chemotherapy well and end of therapy scans showed no evidence of disease. interestingly, her dicer mutation genetics performed by ion torrent tm next generation sequencing was negative in germline and tumor studies. to our knowledge, our patient is the youngest described with slct. she will continue to be followed with serial imaging alone as she had no evidence of elevated tumor markers at diagnosis. , due to young age and unusual diagnosis, she was referred to cancer genetics team. background: approximately % of patients with wilms tumor (wt) have metastatic disease at diagnosis and often have a grave prognosis. limited cell lines are available for the study of metastatic wt and long-term passaged cell lines do not always recapitulate the human condition. focal adhesion kinase (fak) is a non-receptor tyrosine kinase that controls cellular pathways involved in the tumorigenesis of pediatric renal tumors. using a novel patient-derived xenograft (pdx) model from a patient's primary wt (coa ) and matched isogenic metastatic wt (coa ), we previously demonstrated that fak is expressed and its inhibition led to decreased tumorigenicity of both the primary and metastatic pdxs. kinomic profiling is an innovative, high-throughput method used to investigate kinase signaling to identify potential therapeutic targets. to date, the kinomic profile of primary and metastatic wt has not been examined. objectives: investigate baseline kinomic differences between primary and metastatic wt and evaluate kinases upstream and downstream of fak as potential targetable therapies. design/method: cells from coa and coa were treated with pf- , (pf), a small molecule fak inhibitor. protein from cell lysates of treated and untreated coa and coa were combined with kinase buffer, atp, and fluorescently labeled antibodies and loaded into a phosphotyrosine kinase or serine-threonine kinase pam-chip® per the uab kinome core protocol. phosphopeptide substrate analysis with the pamstation® kinomics workstation (pamgene® international), pamchip® protocol using evolve software, and bionavigator v. . were used to analyze kinases upstream and downstream of fak. the primary wt had increased epha , ror sgk and decreased pdgfrb relative to the paired metastatic wt at baseline. treatment with pf increased ron, pdgfrb, p s kb, mak, camk g, vacamkl, camk d, ck a and pskh in the primary wt. treatment with pf decreased tnk , lmr , cck , epha , pdk , sgk , lkb and increased pskh in the paired metastatic wt. primary wt displayed a different kinomic profile compared to metastatic wt in a matched isogenic pdx model. these data reveal that alternative therapies to specifically target metastases are needed. furthermore, fak inhibition resulted in diverse kinomic alterations between primary and metastatic wt. inhibitors targeting many of these pathways, such as pdgfrb inhibitors, are currently available and potentially could be combined with fak inhibitors in the treatment of wt. the results of the current study indicate that kinases upstream and downstream of fak in primary and metastatic wt warrant further investigation. background: use of high-dose methotrexate (hd-mtx, g/m^ ) is a mainstay of standard therapy for pediatric osteosarcoma (os) in north america. in pediatric os, there is a narrow therapeutic window for hd-mtx, with decreased tumor response rate with mtx concentrations < m and decreased survival due to severe toxicity with concentrations > m. risk factors for hd-mtx toxicity have been defined in adults, including body mass index (bmi) and male gender, but such studies have not been conducted in children. we sought to examine the relationship between mtx levels and toxicities during hd-mtx infusion for pedi-atric os, thereby identifying risk factors for increased toxicity and providing a framework for therapeutic drug monitoring. design/method: this retrospective chart review included patients treated at texas children's hospital with hd-mtx as first-line therapy for os from - . data abstracted from electronic records included patient characteristics, bmi and body surface area (bsa), baseline and post-treatment laboratory values, mtx levels and hours after dose given ( h, h), hour mtx cleared (mtx < . um), grade / mucositis, myleosuppression, persistent lft elevation (ctace v . ), and % tumor necrosis. correlation between h mtx level and other covariates was summarized using descriptive statistics. we reviewed hd-mtx infusions corresponding to patients. bmi was found to significantly impact h mtx level (p< . ). female gender was also significantly associated with higher h mtx level (p< . ). percent necrosis (available in patients) was associated with h mtx levels at near-statistical significance (p = . ). h mtx level was not found to contribute to toxicities or associate significantly with mtx clearance. analysis in a larger cohort is ongoing. we have identified at least one patient factor (bmi) that significantly impacts h mtx levels and is of potential use for future modeling, as current models incorporate bsa only. our findings concord with studies in adult os in that bmi significantly impacts h mtx level but diverge in that female gender is associated with higher h levels. importantly, these data support targeting h mtx levels to ensure that minimum concentration for adequate tumor necrosis is reached. these results do not suggest that monitoring h levels would prevent toxicities, thus necessitating further characterization of any intrinsic patient factors that associate with toxicity. overall, our definition of the clinical factors that associate with h mtx levels contributes to a framework for therapeutic drug monitoring in pediatric os. children 's mercy hospital kansas city, kansas city, missouri, united states background: post consolidation immunotherapy with dinutuximab, aldesleukin (il- ), granulocyte macrophage colony stimulating factor (gmcsf) and isotretinoin is standard of care for children with high risk neuroblastoma. dinutuximab is combined in alternating cycles with s of s gmcsf or il , followed by a th cycle with isotretinoin alone. il- is administered as a hour continuous infusion on days - at miu/m /day followed by a higher infusion dose, . miu/m /day, in combination with dinutuximab on days - of cycles and . the miu/m /day dose may be administered inpatient or in the ambulatory setting. objectives: to retrospectively compare the incidence of inpatient and outpatient side effects and complications associated with low dose ( miu) il to provide the tolerability data necessary to evaluate these venues for future administration options. design/method: this study was a descriptive, singlecentered definitive study utilizing a retrospective convenience sample population of children with high risk neuroblastoma who received low dose il either as an inpatient or an outpatient without exclusion from may to june . subjects were identified by a tumor registry query post irb approval. electronic and paper medical records were reviewed for the dates and location of the infusions, the home health company used if applicable and all documentation regarding clinical status, side effects and toxicity. demographics was limited to age and gender. results: infusion venue was chosen by provider preference. twenty-six infusions, inpatient and outpatient via separate home health companies were all administered in entirety and without interruption. there were males and females ranging from - years of age. two children received a single outpatient infusion due to intolerance of il when combined with dinutuximab and received therapy in both settings. fever, inpatient and outpatient was the only common side effect. no source of infection was ever identified. there was one incidence of diarrhea and one patient with pruritus in both the outpatient and inpatient settings respectively. no planned outpatient infusions required subsequent admission however the outpatient fever did necessitate an er evaluation. conclusion: low dose il can successfully be administered outpatient. the medication has minimal side effects with fever occurring in %, none of which were associated with infection. no outpatient infusion required a subsequent admission. no patients who received cycle infusions outpatient opted to receive the next cycle inpatient. baylor college of medicine, houston, texas, united states background: metastatic ewing sarcoma (es) has an extremely poor overall survival, necessitating investigations into molecular mechanisms to identify novel targets and develop new therapies. we previously performed an in vivo study, using our mouse model, designed to provide insights into transcriptomic and proteomic signatures for metastatic es to identify potential therapeutic targets. comparing profiles of primary tumors to corresponding metastatic lesions, we identified aberrant expression of integrin ß (itgb ) and downstream activation of integrin-linked kinase (ilk) in metastatic lesions compared to primary tumors, implicating this pathway as a key regulator in the ability of es to establish and enhance metastasis. our hypothesis is that upregulation of itgb and its downstream signaling events play a key role in es metastasis and are viable therapeutic targets. objectives: to investigate the role of itgb and its downstream signaling pathways in driving the establishment and enhancement of metastasis in es and to investigate this pathway as a potential therapeutic target. to investigate the role of itgb and ilk in es metastasis, we used sirna to knock down itgb and ilk expression in established es cell lines and then performed functional assays in vitro, including cell proliferation and invasion/migration assays. we also tested inhibition of this itgb signaling pathway using available small molecule inhibitors targeting itgb , ilk and the downstream target ap- , using cilengitide, compound and sr , respectively. we are currently using these small molecule inhibitors as treatment in vivo and assessing rates of metastatic tumor formation. we generated stable itgb and ilk overexpression and knockdown cell lines, which we are using for similar in vitro and in vivo investigations. knockdown of itgb and ilk in our sirna cell lines resulted in decreased cell proliferation and decreased invasion and migration compared to controls. we also found significantly decreased cell proliferation using each of the small molecule inhibitors in vitro. our preliminary studies using compound in vivo established a safety profile and dose escalation is underway to assess the effectiveness of inhibiting es metastasis. these results support our hypothesis that itgb and its downstream signaling events play a key role in the ability of es to establish metastatic foci and may serve as a potential therapeutic target. we continue to investigate this pathway in vitro. we are also using our small molecule inhibitors and itgb and ilk overexpression and knockdown approaches to study these effects on metastatic tumor development in vivo using our mouse model. background: neuroblastoma (nbl) is characterized by phenotypic heterogeneity. outcome is excellent for patients with low-(lr) and intermediate-risk (ir) disease, whereas only % of high-risk (hr) patients will survive. -hydroxymethylcytosine ( hmc) is an epigenetic marker of active gene transcription, and hmc profiles are prognostic in many types of adult cancers. we hypothesized that hmc profiles will serve as robust biomarkers in children with nbl tumors, refining current risk stratification. objectives: analyze genome-wide hmc in nbl tumors and correlate hmc deposition with chromosomal copy number and gene expression. design/method: hmc was quantified by nano-hmc-seal-seq from the dna extracted from hr, ir and lr nbl tumors. read counts and clinical data were analyzed with deseq to identify genes with differential hmc patterns between risk groups. chromosomal copy number was assessed by chromosomal microarray analysis (cma) in a subset of samples ( lr and hr). expression of genes located on chromosome p was evaluated using publically available microarrays (e-mtab- ) of hr nbl tumors with known p loh status. results: globally, lr tumors had more hmc peaks ( , ) than ir ( , , p = . ) tumors, or hr tumors ( , , p = . ). , genes had different patterns of hmc deposition in hr versus lr tumors. ( %) of these genes mapped to chromosome p and had decreased hmc in hr versus lr tumors (padj < . ). in the cma analysis p deletion was detected in of the tumors tested. in the tumors with p loss, genes that map to p showed decreased hmc deposition compared to the hr tumors without p loss (p< . ). further, compared to the tumors without p loss, the expression of of the p genes was decreased (p< × - ), including chd , camta , and arid a, known and proposed tumor suppressor genes in nbl. conclusion: different patterns of hmc accumulation are associated with neuroblastoma risk classification. nano-hmc-seal-seq is sensitive to copy number variations and has the potential to identify these changes in patient tumors. our results suggest that hmc deposition contributes to the silencing of tumor suppressor genes in p and may also regulate the transcription of other genes that drive tumor phenotype. background: metastatic osteosarcoma has a -year survival rate of - %. pulmonary metastases remain a major treatment challenge in osteosarcoma. current treatment with conventional chemotherapy shows inadequate activity towards metastases and has toxic systemic side effects. chloroquine is a widely used anti-malarial drug and has been shown to have promising anti-cancer and anti-metastatic activity. polymeric drugs have been shown to have multiple advantages over their small molecular parent drugs, including enhancing the therapeutic efficacy, an improved pharmacokinetics profile and decreased systemic toxicity. we hypothesized that by developing chloroquine into a polymeric drug and combining it with conventional chemotherapy it will improve the treatment of metastatic osteosarcoma. objectives: to identify the optimal combination of polymeric chloroquine (pcq) with conventional chemotherapy active in osteosarcoma as a new means of treating metastatic disease in a murine osteosarcoma model. we synthesized and developed pcq and evaluated its anti-invasive activity using an osteosarcoma cell migration and invasion assay. we evaluated the efficacy of cell killing using combination drug therapies with pcq and a panel of conventional chemotherapy agents (doxorubicin, docetaxel, cisplatin and paclitaxel) using celltiter blue cell viability assay. to develop the murine osteosarcoma model, we intravenously injected luciferase-expressing human osteosarcoma cells b into nsg mice. we administered the drug combination that showed the strongest in vitro synergy to the mice and evaluated their anti-cancer and anti-metastatic effects in vivo. tumor growth and suppression were evaluated using whole body bioluminescence imaging. results: we successfully synthesized pcq that contains . % chloroquine with a molecular weight of . kd. pcq was also found to decrease the toxicity of the parent chloroquine. pcq showed strong inhibition of osteosarcoma cell migration with % inhibition compared to % by chloroquine. we screened the combination drug therapies and found the combination of pcq and doxorubicin to show the strongest synergism. the pcq/doxorubicin combination is currently being evaluated in the murine model. combination drug therapy using pcq and doxorubicin showed synergistic cell killing and inhibition of cell migration in vitro. the combination represents a promising treatment strategy for pulmonary metastatic osteosarcoma. emory university/children's healthcare of atlanta, atlanta, georgia, united states background: survival for relapsed high-risk neuroblastoma (rnb) is < %, underscoring the critical need for novel therapies. rnbs have increased ras/raf/mapk mutations and increased yes-associated protein (yap) transcriptional activity. yap is a transcriptional co-activator that binds with tea-domain (tead) transcription factors to regulate cellular proliferation, self-renewal, and survival. we found that shrna inhibition of yap decreases nb cell proliferation and sensitizes ras-mutated nbs to mek inhibitors, supporting yap as a tractable therapeutic target. verteporfin (vp), a photodynamic drug used for macular degeneration, is the only drug found to inhibit yap expression or yap:tead binding to kill tumor-derived cells. peptide is a mer yap peptidomimetic that also disrupts yap:tead interactions. we sought to determine whether these compounds are potent in nb via yap direct effects. design/method: yap expressing (nlf, sk-n-as) or yap null (ngp, lan , sk-n-as-shyap) human-derived nbs were incubated with vp, with and without direct light exposure, or with peptide . celltiter-glo and immunoblots were used to assess for cell death and yap-downstream protein expression, respectively. results: without direct light exposure, vp inhibits yap expression at nm dosing, yet no nb cell death was observed at equal or higher concentrations. egfr and erk / were inhibited along with yap, confirming yap/ras pathway coregulation. when vp was exposed to direct incandescent light for minutes, > % nb cell death occurred in all nbs tested, even those lacking yap. peptide caused no cell death or yap inhibition up to um. neuroblastomas are resistant to vp at doses sufficient to inhibit yap expression. in macular degeneration, light-activated vp produces reactive oxygen species, which we hypothesize is the off target mechanism killing nbs independent of yap. given the off target effects and the need for light activation, vp is not an ideal preclinical or clinical yap inhibitor. accordingly, peptide has poor cell permeability and low tead affinity, leading to its lack of efficacy. given the relevance of yap in rnb and other cancers, we are chemically optimizing a yap peptidomimetic with enhanced permeability, nuclear localization, and tead affinity to create a bonafide yap inhibitor for preclinical and clinical application. kayeleigh higgerson, aaron sugalski, rajiv rajani, josefine heim-hall, jaclyn hung, anne-marie langevin ut health san antonio, san antonio, texas, united states background: osteosarcoma is the most common bone malignancy in children, adolescents, and young adults. most study cohorts have to % hispanic patients that encompass many different hispanic backgrounds. the university of texas health science center at san antonio (uthscsa) sarcoma team serves a latino population that is predominantly mexican american, thus providing a unique opportunity for evaluation this population. this study expands on previous data collected from january to december from the same institution, providing increased insight into outcomes of mexican american children, adolescents, and young adults with osteosarcoma. objectives: to further understanding of osteosarcoma in latino children, adolescents and young adults. design/method: a retrospective analysis of demographics, tumor characteristics, response to treatment, and survival outcome of all localized osteosarcoma of the extremity patients below years of age diagnosed and treated by the uthscsa sarcoma team between january and june was performed. results: in our original cohort from january to december , we observed a significantly decreased -year eventfree survival (efs) in patients diagnosed before age (preadolescent) relative to patients diagnosed between ages and ( % vs. %, p< . ). patients had a -year overall survival (os) and event-free survival of % and % respectively. in our expanded cohort from january to june we evaluated sixty-six patients with a median age of (range, to y) with localized high-grade osteosarcoma of the extremity. the expanded cohort was % mexican american, with a median follow-up of months (range, to ). the analysis of our expanded cohort is ongoing and we postulate that the findings will hold true, as we increase the cohort size and length of follow-up. conclusion: analysis of our previous cohort, predominantly of mexican american ethnicity, showed that preadolescent patients had an increased rate of relapse when compared with previous large studies. we also showed a trend towards decreased efs for the entire cohort. we hypothesize that we will further validate these findings with this expanded cohort and this will support further investigation into potential causes of poor outcome in this vulnerable latino population. background: neuroblastoma in infants has the potential to regress or mature spontaneously. growing literature showed that some cases subjected to initial observation didn't show inferior outcome compared to actively treated similar categories. objectives: we investigated whether early active treatment can be safely avoided/deferred in selected favorable cases at the children's cancer hospital-egypt (cche). design/method: patients enrolled on the watch and see strategy (w&s) at cche had small primary tumor; inss stage - , uncomplicated stage s or stage infants (< days). tissue biopsy was not mandatory for infants below months of age with localized adrenal mass (stage - ). on progression, immediate intervention took place according to stage and risk of disease after biological characterization. results: thirty four nbl patients were enrolled on w&s strategy; m/f: . / . eighteen patients had stage s disease, patients had stage - and were stage . primary adrenal site was reported in patients ( . %), patients ( . %) had small mass measuring ≤ cm in its largest diameter. the -year os & efs were . ± . % and . ± %, respectively, with months median follow-up (range: - months). spontaneous total/near total resolution of mass occurred in / patients ( %). median time to eliciting regression was . months (range: . - . months), and . months (range: - months) till complete resolution. only / patients ( . %) witnessed progression ( local, distant and combined local and distant progression); median time to progression was months (range: - months) with / deaths after starting chemotherapy. watch and see strategy is a safe approach in localized and uncomplicated stage s neuroblastoma. progressive cases could be rescued. baylor college of medicine, houston, texas, united states background: ga- dotatate binds to somatostatin receptor expressed in neuroendocrine tumors (nets). it was approved by fda in for use with pet/ct scan for localization of somatostatin receptor positive nets in adult and pediatric patients. pediatric approval was based mainly on extrapolation of data from adults. objectives: to describe the use of ga- dotatate pet/ct scan in children with neuroendocrine tumors and compare with other imaging modalities. design/method: patients with nets enrolled in texas children's rare tumor registry between february and october were reviewed and those patients who underwent ga- scan were included. results: four patients with nets underwent ga- scans without any adverse reactions. first patient was a -yearold female with small bowel net with multiple liver metastases. mri abdomen and fdg pet at diagnosis showed s of s multiple liver metastases but could not identify the primary lesion. ga- scan was able to accurately identify the enlarged lymph nodes in the small bowel and was better than fdg pet in delineating the liver metastases. second patient was a -year-old female with recurrent small bowel net with liver, lung and paraspinal metastases. the lesions were initially detected by ct scan. octreotide scan failed to show any uptake in the identified lesions while ga- was taken up by the liver lesions, lung lesions > cm in size and the paraspinal lesion. third patient is an year-old male with pancreatic net with peripancreatic lymphadenopathy, multiple liver metastases and cardiophrenic lymph node involvement. the primary lesion in the pancreas could not be identified by ct scan, ct angiogram, mibg scan, or octreotide scan. in addition, there was uncertainty about involvement of the enlarged cardiophrenic lymph node. in addition to clearly identifying the primary lesion, ga- scan was able to detect multiple peripancreatic lymph nodes not detected by other scans and revealed uptake in the cardiophrenic lymph node confirming its involvement by the tumor. fourth patient is a -year-old female with malignant abdominal paraganglioma with solitary lung metastasis. both mibg scan and ga- scan were able to identify the primary lesion. ga- scan was performed after the lung metastasis was removed and thus its ability to detect it could not be confirmed. background: neuroblastoma is the most common extracranial solid tumor of childhood, with overall survival for high-risk patients (hrnbl) near %. the outcomes of hrnbl have improved with high dose chemotherapy followed by autologous stem cell rescue (abmt). data about factors influencing the rate of hematopoietic recovery following abmt in hrnbl is lacking in the literature. our objective was to identify factors influencing the rate of hematopoietic recovery following abmt in hrnbl. design/method: this was a retrospective chart review of patients with hrnbl treated at texas children's hospital from to . neutrophil engraftment was considered the first of three consecutive days with post-transplant neutrophil count greater than cells/ul. red blood cell and platelet engraftment were considered at a hemoglobin greater than g/dl and platelets greater than , /ul three days after the last transfusion. race and conditioning regimen were analyzed using one-way anova; amount of infused cells was analyzed using pearson correlation coefficients; chemotherapy delay and bone marrow (bm) involvement after cycle of induction chemotherapy were analyzed using independent sample t-tests. the study included males and females with a median age at diagnosis of . years. thirtyeight patients were caucasian, african-american, hispanic, asian, and did not have race documented. the mean dose of infused cd + cells was . × ^ cells/kg. forty-five patients received conditioning therapy with carboplatin/etoposide/melphalan (cem), received busulfan/melphalan (bu/mel), and received thiotepa/cyclophosphamide (thiotepa/cpm). the conditioning regimen administered was significant (p = . ) for time to engraftment of neutrophils, with bu/mel at . days, cem at . days, and thiotepa/cpm at days. a delay of chemotherapy during induction (n = ) was significant (p = . ) for time to platelet engraftment of greater than , /ul and trended towards significance (p = . ) for time to neutrophil engraftment. bm involvement at diagnosis and after cycle of induction was not significant for time to engraftment. dose of stem cells infused was the only variable significant for hemoglobin engraftment. background: osteosarcoma (os) is the most prevalent aggressive primary malignancy of the bone affecting children and young adults. approximately % to % of patients have metastatic disease at initial presentation, and % of those patients have isolated pulmonary metastases. although overall survival in patients with os has improved with advances in therapy, there have been no significant improvements in survival outcome in patients with metastatic disease. recent studies suggest that tumor-associated vascular cell adhesion molecule (tvcam- or cd ) plays a critical role in the metastatic progression of various tumors. indirect evidence from these studies suggest that vcam- / integrin signaling promotes tumor survival and metastatic progression by changing the tumor niche and associated immune response. to determine if interfering vcam- / signaling between pulmonary metastatic osteosarcoma (pos) and macrophages (macs) by down-regulating vcam- , depleting macs or blocking vcam- / signaling will reduce pos and improve overall disease-free survival. design/method: we used a pair of spontaneous, high-grade murine os cell lines from balb/c mouse (h- d), k and k m (derived from in vivo k metastasis). we used lentiviral shrnas to knockdown vcam- mrna and protein expression in k m (vcam- kd). we introduced luciferase into k , k m and various k m shrna cell lines to follow lung metastasis by bioluminescence (bli). we depleted macs by intranasal administration of liposomal clodronate formulation. we tested the ability of k and k m supernatants to polarize m macs into m or m phenotype in vitro. we also administered anti- monoclonal antibody (anti- mab) intranasally to assess the outcome of functional blockade of vcam- / signaling. results: k m over-expressed vcam- compared to k . mac depletion in k m -bearing animals exhibited reduced pos. weekly administration of anti- mab resulted in % tumor-free rescue among mice with established k m pos. interestingly, supernatant from k m but not k preferentially induced m -like macs, suggesting a novel integrin-mediated mechanism of m differentiation. validation data with additional os cell lines will be presented. despite aggressive multimodal therapy, overall outcome for patients with pos remains dismal at - %. for this reason, novel and directed therapy approaches are desperately needed. molecular targeted approaches for therapy are challenging, due to the complex genetic heterogeneity of os. immune-modifying therapy is a promising new alternative approach for pos. university of chicago, chicago, illinois, united states background: only half of all patients diagnosed with high-risk neuroblastoma achieve long-term survival. imetaiodobenzylguanidine (mibg) scans are routinely used to evaluate disease at diagnosis and following treatment, and the extent of disease is quantified using the curie scoring system. a previous study by yanik et al., has shown that for high-risk patients with mycn non-ampliified tumors, scores less than versus greater than following cycles chemotherapy are associated of superior survival, whereas scores less than versus greater than were prognostic in patients with mycn-amplified tumors. however, the prognostic significance of specific sites of metastatic disease at diagnosis is not known. to determine if site of metastatic disease determined by i-metaiodobenzylguanidine (mibg) imaging in high-risk patients at the time of diagnosis was associated with outcome design/method: we performed a retrospective chart review of high-risk neuroblastoma patients treated at comer children's hospital and lurie children's hospital in chicago between and with positive mibg scans at the time of diagnosis. we collected imaging data as well as other clinical data including bone marrow status. sites of disease were defined as curie regions with any positive value. kaplan-meier analysis was performed to evaluate the association with disease sites and survival. pearson correlation coefficients were calculated to compare bone marrow disease to sites of positivity on mibg scan. the cohort consisted of high-risk patients. had skull disease, and had pelvic disease. the presence of mibg positive disease in the skull and in the pelvis trended toward worse efs. efs at years for patients with disease in the skull at diagnosis was ± % and for patients without skull disease was ± % (p = . ). efs at years for patients with and without pelvic disease was ± % and ± % (p = . ). consistent with prior data, we found that the presence of bone marrow disease was associated with worse survival with year efs of ± % and ± % with and without marrow disease at diagnosis (p = . ). there is the highest correlation between pelvic disease on mibg scan and bone marrow disease with pearson coefficient . . pelvic disease noted on mibg scan likely reflects underlying bone marrow disease. in patients with high-risk neuroblastoma, skull disease and pelvic disease on mibg scan at diagnosis may predict worse event free survival. background: osteosarcoma is one of the deadliest cancers in the pediatric population with little progress in morbidity and recurrence rates since the 's. oncolytic herpes simplex- virus (ohsv) is an attenuated virus that has shown encouraging results against certain solid tumors. programmed cell death protein (pd)- -mediated t cell suppression via engagement of its ligand, pd-l , is also of particular interest due to recent successes in selected cancers, especially those with high genetic mutational loads. most pediatric cancers do not have a wide variety of mutations; however, osteosarcoma has a chaotic genome, prone to genetic mutations. it has been shown through numerous other studies that pd- inhibition alone is not sufficient to result in statistically significant tumor growth delays in osteosarcoma models and patients. we hypothesize the addition of ohsv therapy as an immunologic stimulus to pd- inhibition is efficacious for osteosarcoma. ( ) to determine whether ohsv therapy enhances response to pd- inhibition in immunocompetent murine models of osteosarcoma and ( ) to quantify and characterize the anti-tumor t-cells infiltration after treatment with ohsv and pd- inhibition individually and in combination. we utilized an immunocompetent transplantable murine model using a cell line derived from a spontaneous metastatic osteosarcoma (k m , balb/c background). we transplanted established tumor wedges subcutaneously and monitored tumor volume by caliper measurement. once tumors reached - mm , we administered intratumoral injections of hsv ( × plaque-forming units) every other day for a total of injections. we then gave intraperitoneal injections of ug anti-pd- or control antibody twice weekly, up to weeks, starting from the last dose of virus treatment. we monitored tumor growth via calipers twice weekly until tumors reached mm or cm diameter. we quantified and characterized innate and adaptive immune cell infiltrates in tumors using flow analysis. we found significantly prolonged survival with our combination therapy group compared to all other groups. we found that anti-pd- by itself had little impact on t cell recruitment while the combination group had higher influx of cd + cells with a reduced amount of t-regulatory cells (cd +foxp +cd +). we also found an increase in cd + effector memory cells. osteosarcoma is a deadly cancer with therapeutics remaining unchanged for the last years. here, we describe prolonged murine survival after treatment with combination of pd- inhibition and ohsv injection. the combination treatment changed the microenvironment to be more inflammatory. our data support further preclinical and clinical studies. background: neuroblastoma is the second most common cause of cancer related death in children. treatment for high-risk neuroblastoma has improved significantly over the past twenty years, however cure rates remain below %. immunotherapy has emerged as an effective therapy for neuroblastoma, however new modalities and targets are needed to improve outcomes. objectives: our lab has developed a chimeric antigen receptor (car) that targets b -h (cd ), an immune checkpoint molecule overexpressed on many cancers, including neuroblastoma. we hypothesized that b -h would be a good target for car based immunotherapy for neuroblastoma. design/method: neuroblastoma tissue microarrays of primary patient samples were screened for b -h expression by immunohistochemistry and cell lines were screened using flow cytometry. b -h car t cells were tested in vitro by measuring tumor cell killing and cytokine production after coculture with tumor cell lines and in vivo in an orthotopic model of neuroblastoma. results: b -h expression was detected by ihc on % of the screened neuroblastoma patient samples. b -h was expressed at high levels ( + or +) in more than half of these samples ( %). almost all cell lines screened were homogeneously positive for b -h by flow cytometry. retrovirally transduced b -h . - bb. car t cells were cocultured with three b -h positive neuroblastoma cell lines (sk-n-be , kcnr, and chla ) and robust tumor cell killing was demonstrated using an incucyte assay. supernatant from the co-cultures was harvested after hours and both interferon gamma and il- production were detected by elisa.in an orthotopic subrenal capsule xenograft model of neuroblastoma, mice treated with b -h car t cells show significant reductions in tumor growth and prolonged survival compared to those treated with untransduced control t cells. however, the treatment is not always curative.b -h car t cells express high levels of exhaustion markers (pd , tim , and lag ) when compared to cd car controls. in order to overcome inhibition from exhaustion, b -h car t cells were co-cultured with neuroblastoma cell lines and pd- blocking antibody. nivolumab significantly increased the production of il- and interferon-gamma by b -h car t cells. further studies are underway to determine if b -h car t cell activity is enhanced in vivo by treating animals with pd- blockade along with car t cells. conclusion: b -h is expressed on a majority of neuroblastoma samples and appears to be a promising candidate for car t cell therapy. b -h car t cells demonstrate activity against neuroblastoma xenografts that may be enhanced by the addition of pd inhibitors. helen devos children's hospital, michigan state university, grand rapids, michigan, united states background: osteosarcoma is the most common bone tumor in children. it is often metastatic at diagnosis and in this scenario less than % of children survive. polyamines, small molecules found in all cells, are involved in many cell processes including cell cycle regulation, immune modulation, cell signaling and apoptosis. they are also involved in tumor development, invasion and metastasis. in neuroblastoma, inhibition of the polyamine biosynthesis pathway with odc inhibitor alpha-difluoromethylornithine (dfmo) results in decreased cell proliferation and differentiation. these finding have led to multiple phase i and phase ii multicenter clinical trials in pediatric neuroblastoma patients. dfmo is an attractive drug as it is oral, well-tolerated, can be given for prolonged periods and is already used in pediatric patients. the polyamine pathway has not been evaluated in osteosarcoma. objectives: evaluate effect of inhibition of polyamine biosynthesis with dfmo on osteosarcoma proliferation and cell differentiation. design/method: up to three osteosarcoma cell lines were used: mg- , u- os and saos- . cells were exposed to mm dfmo for days with replacement of media and dfmo on day . intracellular polyamine levels were measured by high performance liquid chromatography (hplc). cell numbers were obtained with a hemocytometer using trypan blue. flow cytometry cell cycle distribution (facs) and propidium iodide were used to evaluate for cell cycle arrest. the protein expression of several osteosarcoma differentiation markers was measured by sds-page and western blot using differentiation specific antibodies. a bioluminescent cell viability assay was used to measure cell recovery over several days after dfmo was removed and replaced with standard media. results: dfmo exposure resulted in significantly decreased cell proliferation in all cell lines. after treatment, intracellular spermidine levels were nearly eliminated in all cells. cell cycle arrest at g was observed in u- os. cell differentiation was most pronounced in mg- and u- os cells as determined by increased osteopontin levels. remarkably, cell proliferation continued to be suppressed for several days after removal of dfmo. conclusion: based on our findings dfmo is a promising new adjunct to the current osteosarcoma therapy for high risk patients. it is a well-tolerated oral drug that is currently in phase ii clinical trials in pediatric neuroblastoma patients as a maintenance therapy. the same type of regimen may also improve outcomes in metastatic or recurrent osteosarcoma patients for whom there have been essentially no medical advances in the last years. background: recent studies demonstrate that lower levels of the ews-fli fusion oncoprotein are associated with enhanced metastatic capability in ewing sarcoma. the nf-kb transcription factor is a critical mediator of cxcr and cxcr -driven metastasis in multiple cancers, and increased cxcr and cxcr expression have each been associated with increased metastasis and poor prognosis in ewing sarcoma. we thus sought to investigate the impact of ews-fli on cxcr /cxcr -dependent nf-kb signaling in ewing sarcoma. objectives: the goals of this study are ) to determine the impact of cxcr /cxcr signaling on metastasis-associated nf-kb target gene expression in ewing sarcoma and then ) to investigate how the ews-fli fusion oncoprotein modulates this response . design/method: we utilized multiple ewing sarcoma cells lines including a , chla , chla , tc and tc . cxcr /cxcr cell surface expression was determined by flow cytometry. ews-fli level was modulated using sirna and expression levels were confirmed by western blot and rt-pcr. p dna binding was measured via elisa. nf-kb target gene expression was assessed via rt-pcr. results: consistent with ihc analysis of primary and metastatic patient tumor samples, the paired primary and metastatic ewing sarcoma cell lines chla and chla showed dramatic differences in cxcr and cxcr expression, with the metastatic chla line demonstrating much higher expression of both receptors. other cell lines (nonpaired) showed variable cxcr /cxcr expression. genetic knock-out of cxcr lead to significant decrease in expression of both cxcl /sdf- and il- , two nf-kb transcriptional targets known to play a key role in tumor metastasis. knock-out of cxcr did not alter endogenous ews-fli mrna levels. conversely, lowering the level of ews-fli using sirna lead to enhanced nf-kb signaling, indicated by an increase in p dna binding. consistent with this observation, treating ewing cell lines with ews-fli sirna also resulted in significantly increased nf-kb target gene expression compared to control cells and target gene expression was then further enhanced upon cxcr /cxcr receptor stimulation with the receptor ligand cxcl /sdf- . our findings indicate that the ews-fli oncoprotein negatively modulates cxcr /cxcr -dependent nf-kb signaling. this suggests that ews-fli low, cxcr /cxcr high cells, which are associated with enhanced metastasis and poor prognosis, would be anticipated to exhibit enhanced expression of key nf-kb target genes. importantly, the nf-kb pathway is a druggable target that could potentially serve as an "achilles heel" in this subset of high risk tumors. current work is evaluating nf-kb inhibition as an approach to treating metastatic and refractory ewing sarcoma. background: acute graft versus host disease (agvhd) is a major cause of morbidity and mortality following allogeneic bone marrow transplant (bmt) in pediatric patients. gastrointestinal (gi) agvhd is the most serious manifestation. recently, decreased paneth cell (pc) in a predominantly adult cohort was shown to correlate with agvhd clinical grading and response to treatment. we aim to demonstrate the relationship between pc counts and gi agvhd stage and response to therapy. design/method: charts of patients who underwent endoscopy following bmt between - were reviewed. for repeated biopsies during the course of agvhd, only the first was included for analysis. one pathologist retrospectively reviewed the biopsies and counted pcs in high powered fields; the average pc count was analyzed. twenty-six percent of biopsies were reviewed by a second blinded pathologist. statistical associations between pc counts and day (d ) response, agvhd stage, and other study covariates of interest were gauged using general linear regression. agreement in pathologist pc counts was quantified by intraclass correlation (icc). the research was approved by the children's healthcare of atlanta irb. results: seventy-eight biopsies were included in the analysis. mean age at transplant was . years ± . (range: months - years). most patients underwent transplant for hematologic malignancies ( , %). the majority of transplants used a matched unrelated donor graft -including cords ( , %) and myeloablative conditioning regimens ( , %) - % received total body irradiation. of these, % were diagnosed clinically with gi agvhd (stage , %; stage , %; stage , %; stage , %). icc showed good agreement ( . ) between the pathologists. mean pc was . for patients with no gut agvhd, . for stage , . for stage , . for stage and . for stage (p = . ). on multivariate analysis pc was strongly associated with gi agvhd stage (p< . ) after controlling for age, preparative regimen intensity, and diagnosis (malignant vs. non-malignant). mean pc counts were significantly lower in patients with no response to steroid therapy at d (complete response (mean . ) vs. persistent disease ( . ) vs. partial response ( . ) (p = . )). patients diagnosed with gi agvhd with pc counts less than had a higher risk of mortality (hr . , % ci: . , . ; p = . ). lower pc count correlated with stage gi agvhd, refractory disease at d , and mortality. incorporating pc count in pathology review during gi agvhd work-up may help in agvhd risk stratification. background: there have been increasing discussions pressuring health care teams and institutions for potentially bearing the cost of clostridium difficile infections (cdi) as a health care-associated infection in the recent years. the pediatric oncology patient population, though small, accounts for significant portion of all cdi with - -fold increased risk. hematopoietic stem cell transplant (hsct) recipients constitute a unique subset with distinct risk factors, such as severe immune deficiency state and graft versus host disease (gvhd). although there is ample data on cdi in adult hsct recipients, reports on pediatric experience are limited. objectives: to evaluate the incidence and patterns of cdi among pediatric hematology, oncology and hsct inpatients at our institution. a retrospective review of all clostridium difficile (cd) stool tests performed using toxin enzyme immunoassay and later, polymerase chain reaction targeting toxin genes between and in a large, urban academic children's hospital was performed. the data were analyzed for hematology, oncology, hsct inpatient population and all the other cases separately and statistical comparisons were performed. results: a total of samples were submitted to the microbiology laboratory for cd testing during the study period. while hematology patients constituted . %, oncology . %, hsct . % and others . % of the cases on whom cd testing was done; per patient average test number was . , . , . , and . , respectively. of all the cd tests per-formed, . % were positive. test positivity was higher in hsct ( . %) and oncology ( . %) cases tested compared with hematology ( . %) and other cases ( . %) with statistical significance (p< . ). overall recurrence rate was . %; hsct patients had the highest recurrence with a rate of % followed by oncology ( . %), hematology ( . %) and other ( . %) cases, again reaching statistical significance (p< . ). again, hsct patients had the highest average number of recurrences at . ( - ) followed by oncology . ( - ), general . ( - ) and hematology . ( - ) groups. there was no seasonal variability in the incidence of cdi among populations analyzed. prolonged hospital stay/antibiotic use and persistent diarrhea due to gvhd are the likely reasons for higher rate of cd testing in hsct as a result of increased monitoring and thus might have even caused underrepresentation of positive cd test frequency. higher incidence and frequencies of recurrence underscores the inevitable nature of cdi in hsct population as a consequence of the current therapies and may lead to future radical treatment approaches like fecal implantation. background: viral infections remain a challenge to treat post hct in children, and significantly contribute to morbidity and mortality. virus specific t cells (vsts) have shown tremendous clinical efficacy in treating viral infections post-hct, with minimal toxicity and long term efficacy. we have used donor-derived vsts in individual patients, however not all donors are agreeable to the process, and numerous patients may benefit from vsts who do not have an identified donor/have other disease indications objectives: we sought to actively build a third-party vst bank, for "off the shelf" use in eligible patients. design/method: vsts targeting cmv, adenovirus and ebv were manufactured using one of techniques. initially ebv transformed b cells were genetically modified with an ad f pp vector and used as antigen presenting cells (apc) to stimulate and expand ebv, ad and cmvpp specific t cells. more recently, vsts were expanded using s of s apc pulsed with commercially available peptide pools (pep-mixes) to expand ebv/cmv/ad specific t cells. products were entered into the "bank" via two mechanisms: a) left over products from our "donor-derived" protocol when patients no longer required vsts or were not at risk of developing viral infections, or b) by targeting regular blood donors based on their hla typing to ensure an appropriate mix of high frequency hla types for optimal patient matching and antigen presentation based on current knowledge of antigen presentation. results: a total of products are currently in the thirdparty vst bank ready for use. twenty seven of these are from our donor derived protocol, and three from targeted donors. all vst products met safety and in vitro efficacy testing. thirteen vst infusions have been given to patients. eleven infusions have been given for cmv and two for adenovirus. five out of seven patients responded to thirdparty vst infusions, with a median of vst infusions per patient (range - ). the median hla matching was out of per patient (range to ) no patients experienced adverse reactions, gvhd or other toxicity related to the vst infusion. a third-party vst bank is feasible and produces clinically appropriate vsts for use in patients with viral infections. hla typing and matching of vst products is essential to reduce toxicity and promote appropriate antigen presentation and expansion of vsts in vivo. further work is underway to further characterize the vsts using epitope mapping to better define the hla restriction and immunogenicity of each vst product. akron children's hospital, akron, ohio, united states background: acute graft-versus-host disease (agvhd) is a well-known complication of hematopoietic stem cell transplant (hsct) and a major cause of post-transplant related morbidity and mortality. first line therapy of agvhd involves corticosteroids and calcineurin inhibition. in patients with severe refractory gvhd, mortality can reach up to %. currently, there is no standard of care for the treatment of steroid refractory agvhd. many centers have looked at the use of antibody mediated control of agvhd to competitively inhibit the inflammatory cascade. basiliximab, a chimeric monoclonal antibody against the t-cell il- receptor, has been used in adults with steroid refractory agvhd. patients receiving this medication have demonstrated complete and partial responses to therapy with minimal toxicities. objectives: report the successful use of basiliximab in the treatment of agvhd in a -year-old following matched unrelated (mud) hsct. design/method: a -year-old male underwent mud transplant for high risk aml with monosomy . conditioning regimen included busulfan, fludarabine and equine atg. his clinical course was complicated by fever, mucositis and agvhd (stage skin; stage gi-biopsy proven). gvhd prophylaxis included tacrolimus and methotrexate, however with progressive skin rash, diarrhea, and early satiety, gvhd treatment with corticosteroids was initiated. as the patient continued to have worsening symptoms, basiliximab therapy was started. the patient received doses ( mg) iv basiliximab on two consecutive days and then received weekly therapy for a total of doses leading to initial improvement. the patient further developed acute on chronic gvhd on day + , and subsequently received a second course of basiliximab. after initial administration of basiliximab, the patient had near complete resolution of symptoms. however, with a small wean in his tacrolimus dose, the patient experienced another skin gvhd flare prompting the second basiliximab course. the patient was subsequently weaned off all immunosuppression by day + . the only acute complication the patient experienced while receiving basiliximab was right toe paronychia and asymptomatic low ebv titer. the patient is currently off all immunosuppression at the time of report without evidence of cgvhd. conclusion: this single case report, in a young pediatric patient, demonstrates the use of basiliximab may be a safe and efficacious treatment for pediatric patients with agvhd. university of california, san diego, la jolla, california, united states background: clinical outcomes after allogeneic hematopoietic stem cell transplantation (hsct) depend on restoration of t lymphocyte populations. association between recovery of cd +foxp + regulatory t cells (tregs) and protection from chronic graft versus host disease (cgvhd) has been described in adult hsct. in adults, t cell recovery is driven by expansion of donor t cells and treg reconstitution is hypothesized to result from peripheral conversion. restoration of t cells in pediatric patients has a larger contribution from thymopoiesis, however, the relationship between thymopoiesis and treg recovery is undefined. objectives: we hypothesized that effective thymopoiesis is important for restoration of treg populations and protection from cgvhd in pediatric hsct patients. design/method: we performed longitudinal flow cytometry of peripheral blood t cells from pediatric hsct patients and age-matched healthy donors. laboratory data were correlated with clinical outcomes to evaluate impact. recovery of tregs occurred in / ( . %) patients by post-transplant day . day treg frequency in patients that developed cgvhd ( . ± . % of cd + t cells) was reduced compared to cgvhd-free patients ( . ± . %). failure to restore tregs to > . % of cd + cells by day was associated with increased risk of cgvhd in the first year post-hsct (rr = . , p = . ). a majority ( . ± . %) of tregs from patients recovering the peripheral treg compartment expressed helios, a marker of thymic-derived tregs; only . ± . % of tregs expressed helios in patients failing to restore adequate tregs. this prompted examining the relationship between defects in thymopoiesis and inability to restore tregs. we evaluated thymic function by flow cytometry quantification of cd ra+cd +ptk + recent thymic emigrant (rte) cd + cells (confirmed by qpcr for trec content). most ( / , . %) hsct patients had detectable rtes by day post-hsct. thymic production of rtes was persistently absent in patients that developed cgvhd (< / ^ cd + cells in / patients), compared to cgvhd-free patients ( / patients > rte/ ^ cd + cells by day , average . ± . / ^ cd + cells). post-hsct thymic activity as measured by rte enumeration correlated with treg restoration; / ( %) rte+ patients restored tregs, compared to / ( %) of rte-patients. conclusion: failure to restore tregs after allogeneic hsct results in increased risk for cgvhd. in pediatric patients thymic generation of new t cells is an important contributor to restoration of the treg compartment. this data supports further investigation into mechanisms impairing post-hsct thymopoiesis and suggests peripheral blood tregs may be a prognostic biomarker for cgvhd. background: haploidentical stem cell transplantation (haplo sct) is riddled with unique challenges. objectives: we present our experience in the use of haplo sct with post-transplant cyclophosphamide (ptcy) and the adaptations required for each disorder for optimal outcome. design/method: we performed a retrospective study at the pediatric blood and marrow transplant unit, apollo cancer institutes, chennai, india. children up to years of age, diagnosed to have benign disorders and underwent haplo sct with ptcy from to july were included. results: ptcy was used in i.e. % haplo transplants for children with benign disorders. the underlying conditions included fanconi anemia , severe aplastic anemia , mds , jmml , hemoglobinopathy , prca , xld and primary immunedeficiency disorders (pid) . source of stem cells was peripheral blood in %, bone marrow in %. conditioning included fludarabine with treosulphan or cyclophosphamide for pids and aplastic anemia respectively. neutrophil engraftment by day+ - with a durable graft was noted in % transplants with graft versus host disease in %, cmv reactivation in %. mortality rate was % with infants less than months of age developing severe fatal cytokine release syndrome. the median follow up is year with years being the longest. no significant late effects have been noted with chronic skin gvhd in children. survival rate was superior among children with pids with survival of % in this group. haplo sct with ptcy is a feasible and costeffective option for cure in children with life-threatening benign disorders with no compatible family or matched unrelated donor. careful patient selection, reducing cyclophosphamide related free radical toxicity with the use of n acetylcysteine, limiting t cell numbers by capping cd at × /kg, post-transplant viral monitoring protocols are required to reduce morbidity and mortality. we have been working on universal access to care for children from s of s all socioeconomic background and incorporating innovations to reduce the cost of hsct without compromising outcomes. haploidentical hsct using tcr / depletion costs usd as compared to ptcy priced at usd. children with severe aplastic anemia and pids can be transplanted using reduced intensity conditioning and ptcy. in hemoglobinopathies, pretransplant immunosuppression is required to prevent graft rejection. graft versus host disease remains the main cause of mortality in children with fanconi anemia. mortality in infants less than months after ptcy has been high, tcr / depletion would be superior in this cohort. cincinnati children's hospital medical center, cincinnati, ohio, united states background: fanconi anemia (fa) is a congenital bone marrow failure syndrome with hsct the only curative option for associated bone marrow failure. patients with fa undergoing hsct may experience increased toxicity related to either their underlying disease, or the effects of medications, resulting in the inability to tolerate prophylactic medications or sideeffects from anti-microbial therapy. objectives: we postulated that increased cd cell dose would be associated with a rapid immune reconstitution and therefore early withdrawal of anti-infective prophylactic medications. design/method: patients with fa transplanted at cchmc from an unrelated donor had peripheral blood stem cell grafts collected and cd selection performed. where possible, patients had serial measurements of their immune system performed at varying intervals post hsct. we defined immune reconstitution as normalization of lymphocyte subsets-cd , cd , cd and cd cells, as well as a normal response to mitogen stimulation including phytohemagglutinin, concanavalin a and pokeweed. the first measurement of either normal cell number or mitogen response was recorded for each patient. results: a total of patients underwent hsct for fa at cchmc between and . patient demographics included a median age of years at hsct, the vast majority of patients having a fully matched or one anti-gen mismatched donor, and the majority of patients transplanted for bone marrow failure. there was a statistically significantly decreased time post-transplant to immune cell recovery in patients receiving > × /kg cd cells (median . ) compared to those receiving < × /kg cd cells (median . ). the median time to normalization of cd count was days (cd count > /kg) versus days (cd count < /kg), cd count days (cd count > /kg) versus days (cd count < /kg), cd count days (cd count > /kg) versus days (cd count < /kg) and cd count days (cd count > /kg) versus days (cd count < /kg). time to normalization of mitogen response was decreased posttransplant in those patients receiving increased cd cell dose at time of transplant, though this was not significant, reflecting low number of patients with evaluable responses. no patients in either group experienced gvhd or graft failure. patients with fa who are transplanted with higher cd cell doses have quicker immune reconstitution than those who receive lower cell doses. along with benefit to patients including less risk of infection and early termination of immune-prophylaxis medications, this supports the use of high dose cd selected grafts in this vulnerable population. background: parvovirus b (pvb ) infection after transplantation was first reported in . since then, numerous cases of pvb infections after hematopoietic stem cell transplantation (hsct) and solid organ transplantation (sot) have been reported. most report anemia as the predominant clinical manifestation. however, pvb has been associated with pancytopenia, hepatitis, myocarditis, and allograft rejection. we present a patient with acute lymphoblastic leukemia who developed bone pain and pancytopenia following hsct in the setting of pvb infection. to describe an unusual presentation of pvb in a patient with acute lymphoblastic leukemia following hsct. design/method: a search of the english-language medical literature was performed using pubmed and medline databases. a review of the patient's medical history was performed. a year old male with relapsed b-cell all and history of "fifth disease" in infancy presented four months after hsct with focal left arm pain and difficulties fully extending the arm. bone mri showed enhancement of the medullary space centered within incomplete transverse cortical fracture interpreted as pathologic fracture due to neoplastic involvement of the ulna with no history of inciting injury. subsequently, peripheral blood counts decreased from low normal values to wbc . k/microl, anc /microl, plt k/microl, and hemoglobin . g/dl. the patient's chimerism remained % donor. a bone marrow biopsy and aspirate were performed to assess for recurrent leukemia given persistence of bone pain and developing pancytopenia. marrow findings included morphologic cytopathic effects with erythroid precursors and strong parvovirus staining with no signs of red cell aplasia or recurrent b-cell disease by morphology or flow cytometry. pvb was detected in blood by pcr and immunoglobulins with resolution of cytopenia and bone pain. this case highlights an unusual constellation of symptoms following hsct in a child with all. unexplained bone pain and medullary infiltrates with pancytopenia suggestive of recurrent leukemia were likely triggered by pvb infection. the question remains if he had reactivation of pvb , a primary infection by a new strain, or the virus was aquired through stem cells. bone biopsy could not be justified in light of clinical improvement. so far, bone lesions have only been described with congenital pvb infection. pvb appears to be uncommon after hsct, with a review of literature yielding pediatric cases. however, it may be underestimated due to lack of routine screening. our patient's presentation supports that evaluating for pvb may be warranted in hsct patients presenting with symptoms suggestive of relapsed leukemia. background: cardiac injury may occur during hematopoietic stem cell transplant (hsct) in pediatric patients and can be asymptomatic for many years. recommendations for screening are available for patients who received anthracyclines or chest irradiation, but no guidelines exist for unexposed longterm survivors. we sought to define the prevalence of echocardiographic abnormalities in long-term survivors of pediatric hsct and determine the need for screening in asymptomatic patients. design/method: we analyzed echocardiograms performed on long-term survivors (≥ five years) who underwent hsct at cincinnati children's hospital between and . we analyzed echocardiograms for left ventricular ejection fraction (ef), end-diastolic dimension (lvedd), septal thickness, posterior wall thickness, and global longitudinal strain (gls). we normalized linear measurements for age and patient body surface area. we included for further analysis patients who had echocardiogram obtained for routine surveillance. results: a total of patients underwent hsct and were alive more than years after transplant in , with having an echocardiogram obtained ≥ five years postinfusion. those with an echocardiogram were transplanted more recently (median vs. ). however, no difference between screened and unscreened individuals was noted for age at transplant, sex, transplant indication, anthracycline exposure, chest irradiation, or cyclophosphamide based preparative regimen. indications for echocardiograms included: cardiac symptoms ( . %), congenital cardiac anomalies ( . %), hypertension ( . %), known cardiac or pulmonary disease ( . %), routine post-hsct surveillance ( . %), and unknown ( . %). the mean time post-hsct was . years. among routine surveillance echocardiograms, the mean ef z-score was - . . mean lvedd zscore was - . , mean septal thickness z-score - . , mean posterior wall thickness z-score - . , and mean gls - . %. for patients that had echocardiogram performed for routine surveillance, / patients ( . %) had ef measured, and / ( . %) had ef z-scores ≤ - . (abnormally low). patients exposed to anthracyclines had a mean z-score ef of - . vs. unexposed patients - . (p = . ). among individuals who received neither anthracyclines nor tbi only / ( . %) was found to have an abnormal ef, . % (z-score - . ) or gls (- . %). only one patient who had a normal ejection fraction (z-score - . , ef . %) had an abnormal gls, - . % (normal ≤ - . ). long-term survivors of pediatric hsct who are asymptomatic and did not receive radiation or anthracyclines likely do not require surveillance echocardiograms, unless indicated by clinical symptoms. patients exposed to anthracyclines or tbi require close echocardiographic s of s screening and clinical monitoring for the development of cardiac complications. duke children's hospital, durham, north carolina, united states background: children undergoing pediatric blood and marrow transplants (pbmt) experience significant symptom distress. mobile health (mhealth) technologies can be leveraged to collect and monitor patient generated health data, and subsequently enhance our understanding of pbmt symptom clusters, patterns, and trajectories. better understanding of symptom complexity can foster development of precision health strategies to improve patient outcomes. however, limited research exists in integrating mhealth technology into pbmt management. we aimed to explore the feasibility, acceptability, and usability of using a pbmt specific mobile application to collect and monitor symptoms and wearable technology (apple watch) to measure objective data such as heart rate (hr) and activity. design/method: an exploratory mixed method design began in october to monitor pbmt symptoms for patients using real-time data from: ) a self-developed mhealth application (app) to collect subjective symptom data; and ) apple watch to collect physiologic measures such as heart rate and number of daily steps. data is collected pre-transplant through days. acceptability will be assessed through satisfaction surveys at study completion. we have enrolled patients to date who are all currently using the app and watch. patients' average frequency of daily charting in the app %. the wearable average daily recorded measurements are for hr and for step count. most common symptoms recorded within the app include fatigue and pain. we have noted trends in data including a decrease in activity following transplant and gvhd and an increase following engraftment. patients have stated "the app is helpful to keep track of how my pain is doing day to day" and "i try to take more steps each day than the day before". patients often remove the watch for charging, then forget to put it back on, but consistently put it on upon reminder. finally, parents often were required to make app entries with patients too sick to record. we continue to enroll patients with enthusiasm from both patients and parents to use mhealth during pbmt. preliminary findings suggest feasibility of using the mhealth devices is strongly correlated to the patient's post-transplant stage and is facilitated by caregiver participation with device management (charging devices, reminders to wear watch and record in app). patients reported satisfaction and ease of use with devices, but found it difficult to keep up with charging and charting. these findings indicate using mobile devices may be useful methods to collect patient generated health data. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bacterial bloodstream infections (bsi) are a common complication following hematopoietic stem cell transplantation (hsct) in both pediatric and adult populations, and are associated with poor outcomes. there is limited data describing the outcomes and characteristics of patients who develop three or more bsi after hsct. objectives: to describe the characteristics and outcomes of pediatric patients who develop three or more blood stream infections in the first-year post hsct. design/method: we performed a retrospective chart review of consecutive patients who underwent hsct at our institution from through to compile this case series. data were collected through the first year post-hsct including: patient demographics, underlying disease and therapy characteristics; and transplant complications such as thrombotic microangiopathy (tma), graft versus host disease (gvhd) and overall survival. bsis were classified according to current center of disease control guidelines. results: of patients, ( %) developed or more bsi in the first-year post transplant (total bsi cases = including all patients). of the cases, the majority underwent allogeneic hsct (n = / ; %). most cases were from unrelated donor (n = / , %). more than half of patients had grade - gvhd (n = / , %). sixteen ( %) had tma. of these cases, tma preceded the first bsi in n = / ( %). the majority of bsis were classified as central line-associated bloodstream infections (clabsis, n = / , %), followed by mucosal barrier injury laboratory-confirmed bloodstream infections (n = / , %) and secondary bsi (n = / , %). the majority of isolated organisms ( %) were associated with mucosal barrier injury pathogens. one-year overall survival in the cohort was % (n = / ). pediatric patients undergoing hsct who develop or more bsis in the first-year post transplant demonstrated an increased rate of tma compared to the overall institutional incidence of roughly %. tma diagnosis preceded the first bsi in over half of patients, suggesting that tma may predispose to recurrent bsi. improved strategies for early detection and treatment of tma as well as prevention of clabsis may help reduce the number of bsis ultimately leading to decreased morbidity and mortality in this patient population. background: in neutropenic pediatric patients, infection remains a significant cause of morbidity and mortality. while granulocyte transfusions have been utilized for decades to treat infections, including in the pediatric population, the efficacy of this intervention remains poorly described. previous guidelines have primarily utilized information from adult populations. furthermore, recruitment of donors typically involves friends or relatives of the patient with periodic involvement of community donors. the use of a readily available local donor population to improve availability has yet to be well described. as the immunocompromised population is particularly susceptible to worsening infection and clinical deterioration, the ability to rapidly harvest and deliver granulocytes warrants further investigation. to investigate the efficacy, safety, and outcomes of severely immunocompromised patients receiving granulocyte transfusions from a local altruistic granulocyte program in a pediatric tertiary care center. design/method: a retrospective review was performed to evaluate the context for receiving a transfusion as well as primary outcomes including infection clearance, survival to discharge, and overall mortality. the indiana blood bank assisted with timing the interval from initial order placement to onset of first granulocyte infusion. results: among the patient population reviewed, patients received separate granulocyte regimens. ages ranged from - years with a mean neutrophil count of at time of first transfusion. indications for transfusions included bacteremia (n = ), fungal pneumonia (n = ), and fungemia (n = ). primary outcomes included clearing infection ( %) and surviving to discharge ( %). the median time from initial order placement to infusion was hours, although there was no significant difference between responders who cleared the infection and non-responders who did not. however, additional investigation found that ward patients had a % chance of surviving to discharge while patients in the icu at time of initial transfusion had a % chance of survival to discharge. the readily available granulocyte transfusion program allows patients to quickly receive therapy in neutropenic settings. this is beneficial for patients as transfusion prior to clinical decompensation correlates with increased likelihood of infection clearance, and subsequently improved mortality. further investigation is needed, likely as a prospective study, to better explore circumstances that are beneficial for granulocyte transfusions. background: donor lymphocyte infusions (dli) are composed of immune cells to treat relapse after hematopoietic cell transplantation (hct). to date, data regarding its efficacy is limited in pediatric populations. furthermore, while outcomes related to cd content have been characterized, to our knowledge, the relationship between outcomes and other cellular content in dli has never been reported. objectives: determine whether the primary hematological malignancy, presence/absence of graft-versus-host disease s of s (gvhd), and unique phenotypic content of each dli impact overall survival (os) in pediatric patients with hematological malignancies. design/method: irb-approved, retrospective study investigating all consecutive dlis given to patients at the children's hospital of wisconsin. analyses were conducted using mann-whitney, fisher's exact, and chi-square. from from - patients ≤ years old with hematologic malignancies [myeloid (aml/ mds/cml/jmml),n = ; lymphoid (all),n = ] underwent dlis ( %% ≥ dlis). the median time between hct and dli was . (range, . - . ) years. there were significant differences between the lymphoid and myeloid groups, respectively, in regard to median age at hct ( . vs . yrs, p = . ) and at first dli ( vs years, p = . ). ultimately, there were no statistically significant differences in gvhd or os in products with either higher or lower cd , cd , cd , cd , or cd cellular content. however, the median cd /kg content was more than double in the patients who developed gvhd as compared to patients who exhibited no gvhd after dli ( . × vs . × , p = . ). patients receiving one dli had a -year os of ± % vs those receiving + dli of ± % (p = . ). with a median follow-up of . (range, . - . ) years, the year estimated os of patients in the lymphoid group was higher at ± % vs ± % in the myeloid group, although not significant (p = . ). our results indicate a survival benefit when using dli in a subset of patients who relapse after hct. unlike adult studies demonstrating little effect of dli in lymphoid diseases, many children with all achieved durable remission. while our analysis did not demonstrate that dli cellular content had a statistically significant effect on gvhd or os, it is possible that differences could be found if a larger population and more targeted cell doses were studied. more data will be needed to further define these relationships and identify patients who stand to benefit most. cincinnati children's hospital medical center, cincinnati, ohio, united states background: many arabic speaking muslim parents of children requiring bone marrow transplantation (bmt) receive medical care in the united states. providers may not understand the impact of islamic parents' religious beliefs and practices on their health care experience. objectives: to explore how islamic parents used religion in decision making and to understand the impact of their religious beliefs and practices on their overall health care experience. design/method: we used grounded theory, an inductive method gathering data from interviews and analyzing text, to identify core themes. ten caregivers of bmt children from middle eastern countries were interviewed by an arabicspeaking provider; interviews were coded by an interdisciplinary team. we identified key themes: . patience is a core belief in islam. patience results from the acceptance of allah's will. behaviors showing patience include praying rather than questioning and crying. . al qur'an provides comfort, healing, and protection. families listen to recitations of al qur'an in the patient's room because they feel that this practice not only comforts them but promotes healing as well. for some, certain portions of the qur'an were especially meaningful such as surat al-baqara, which explains that while we may think something is bad for us, allah will know it is good for us. . religious care in the medical center helped families feel respected. religious care in the medical center included interactions with chaplains, who were understood to be "religion experts," and provision of space for prayer and religious resources. . seeking religious consultation. religious consultation from imams or religious scholars (muftis or sheikhs) provides interpretations of the qur'an applied to the family's specific situation helps families make difficult decisions and follow allah's plan. . muslim beliefs guided decision making; muslim practices brought comfort, strength, and peace. drawn from the parents' understanding of islam. parents who addressed this topic said they would only do what islam allowed. they did indicate that most aspects of healthcare were understood to be allowed within islam. additionally, muslim practices of prayer, reading/listening to qur'an, and giving alms all provided comfort, strength and peace. we identified several recurring themes through our interviews that allowed us to understand how families use their muslim faith to deal with their children's illnesses and how it influences their decision making. we believe this better understanding will allow for more informed conversations about patients' health care and decision making, and shows respect for religious beliefs and practices. nemours/dupont hospital for children, wilmington, delaware, united states background: virtually all children will be infected with human herpesvirus (hhv- ) by the age of two. hhv- reactivation after stem cell transplantation causes multiorgan toxicities, including encephalitis, with inflammation and destruction of the temporal lobes and hippocampi, memory loss, and seizures. catatonia is characterized by posturing, immobility, mutism, and autonomic instability, and it's associated with various psychiatric and medical conditions. we describe a patient with hhv- encephalitis and unusual neurologic sequelae, including cognitive and neurobehavioral dysfunction and catatonia, which may impact our understanding of the pathophysiology of hhv- reactivation encephalitis. objectives: describe a case of hhv encephalitis with practice implications for stem cell transplantation. results: our patient was diagnosed with acute myeloid leukemia at age . within years, he relapsed and received two stem cell transplants. on the th day after his second transplant, he developed hyponatremia and refractory seizures. brain mri showed edema in the medial right temporal lobe with linear ischemic change. eeg showed diffuse encephalopathy. cerebrospinal fluid (csf) demonstrated white blood cells, red blood cells, and hhv- by pcr. his prophylactic antiviral was switched to foscarnet and ganciclovir. repeat mri showed abnormal signals in bilateral medial temporal lobes and the right insula. three months later he developed episodes of diaphoresis, hypothermia, agitation, mutism, and unusual posturing, recurring almost daily, recognized as catatonia. mri showed improvement of the abnormalities in the bilateral medial temporal lobes and hippocampi. eegs showed diffuse slowing. after months of antiviral therapy, csf was negative for hhv- . over the ensuing years, he had numerous episodes of diaphoresis, hypertension, hypothermia, pruritis, confusion, agitation, cogwheel rigidity, and bizarre posturing. dopamine blocking agents did not help. clonazepam helped reduce their frequency, and hot showers helped break acute episodes. further mris showed generalized cortical volume loss. he suffered from depression and severely impaired sleep and cognitive function. we describe a novel, debilitating outcome of hhv encephalitis which may provide diagnostic considerations as we continue to improve our understanding of the breadth of possible neurologic sequelae in transplant patients. hhv- is understood to infect and destroy the temporal lobes and hippocampi, but our patient's autonomic dysfunction indicate involvement of the hypothalamus and basal ganglia. antidopaminergic agents may worsen catatonia, and they were not effective for our patient. treatment of catatonia includes benzodiazepines; electroconvulsive therapy was not attempted in this case but may also be useful. background: epstein-barr virus (ebv)-related posttransplant lymphoproliferative disorder (ptld) is a lifethreatening complication in patients following hematopoietic stem cell transplantation, with a frequency estimated at . % and a cumulative incidence of mortality estimated as high as %. studies of ebv have hypothesized that the tonsils are critical for propagating this infection, as tonsillar epithelial cells have been shown to be the site of primary viral infection and continued viral shedding; however, to date no studies have been performed assessing the role of tonsillectomy in patients with ebv ptld. objectives: identify patients with localized ebv ptld treated with tonsillectomy to identify prognostic factors that may be able to help guide future treatment decisions. design/method: patients treated at memorial sloan kettering cancer center who had received hematopoietic stem cell transplantation and had billing codes for both ebv and tonsillectomy were eligible for inclusion in this study. a retrospective chart review was performed, assessing patient demographics, transplant characteristics, laboratory values, tonsillar pathology, and clinical course. any patient who did not have unilateral or bilateral tonsillectomy performed or who had non-localized disease (defined as disease involvement outside of the oropharynx and neck) was subsequently immunodeficiency; % (n = / ) fanconi anemia (fa); % (n = / ) hemoglobinopathy; % (n = / ) non-fa marrow failure and % (n = / ) a metabolic disorder. seventy one percent (n = / ) had normal amh for age pre-transplant, % (n = / ) had low amh for age pre-transplant; of these, % (n = / ) had an oncologic diagnosis; % (n = / ) had fa; % (n = / ) had previously treated hlh; % (n = / ) had non-fa marrow failure; one had a metabolic disorder and one a hemoglobinopathy. of the patients with post-transplant amh measurement % (n = / ) had low levels. of the patients with previously normal pre-transplant amh % (n = / ) underwent myeloablative conditioning (mac) regimen with a % (n = / ) having low amh levels post-transplant compared to %(n = / ) who underwent reduced intensity conditioning (ric) regimen with % (n = / ) having low amh levels post-transplant (p . ). fifteen percent (n = / ) had low levels pre-transplant and underwent mac regimen with % (n = / ) remaining low; % of these patients (n = / ) had fa. nine percent (n = / ) had low levels and underwent a ric regimen with % (n = / ) of amh levels remaining low; % (n = / ) of these patients had hlh treated prior to transplant. conclusion: amh levels can be used for detection of premature ovarian failure and fertility counseling. there is a higher risk of premature ovarian failure with mac regimens and prior chemotherapy vs ric regimens. follow up of this cohort will provide more information to understand the effects of hsct in ovarian function and the usefulness of amh as a predictor of fertility potential. background: there are no proven strategies to prevent blood stream infections (bsi) secondary to oral mucosal barrier injury after hematopoietic stem cell transplant (hsct). additionally, we recently reported progressive gingivitis and dental plaque accumulation in hsct recipients despite our current oral standard of care (three times daily oral rinse). xylitol is a non-fermentable sugar alcohol that reduces dental caries, plaque accumulation, and oral disease progression by inhibiting bacterial growth. we hypothesized that the addition of xylitol to standard oral care will decrease dental plaque accumulation, gingivitis and bacteremia from oral flora. objectives: identify a clinically effective strategy to improve oral health and prevent bsi secondary to bacterial translocation through the oral mucosa in patients undergoing hsct. we are conducting a prospective randomized control study to test our hypothesis. those in the intervention arm receive our current standard of care (three times daily oral rinse) in addition to daily xylitol wipes; controls receive oral standard of care alone. oral exams are performed at baseline and weekly for the first days post hsct. metagenomic shotgun sequencing (mss) of gingival samples is performed at all time points to evaluate microbiome diversity and pathogenic bacterial load. finally, we performed whole genome sequencing of pathogenic bacterial isolates causing bacteremia to assess for genetic relatedness to corresponding strains present within the patient's oral microbiome preceding the infection. : preliminary interim analysis of patients demonstrates improved oral health in patients receiving xylitol (n = ) over those receiving standard of care (n = ), measured by the oral hygiene index (p = . ) and gingivitis index (p = . ). in the nine patients having complete oral mss analysis, xylitol appeared to be associated with decreased streptococcus mitis/oralis domination in the oral microbiome. finally, patients receiving xylitol had no incidence of streptococcus mitis/oralis bacteremia through the first days compared to three patients ( %) in standard of care arm. interestingly, streptococcus mitis/oralis comprised % of the oral microbiome in one child who subsequently developed a streptococcus mitis/oralis bsi. we expect to complete this study in the next months (n = ). the addition of xylitol to oral standard care appears to decrease dental plaque and gingivitis in patients undergoing hsct. xylitol may also impede streptococcus mitis/oralis dominance in the oral microbiome with potential reduction in blood stream infections. (range: - days). twenty-one mdli ( %) were administered because of lymphopenia, fourteen of them ( %) in patients with concomitant viral/opportunistic infections. mixed chimerism/graft failure was the motive of % of the mdli (n = ) and six ( %) were administered to accelerate immune reconstitution. all infusions were well tolerated without appearance or worsening of gvhd. an increase in t-cell counts was observed following six mdli ( . %), although it was a transitory response ( - weeks) in five cases. viral/opportunistic infections were controlled in five cases ( . %), requiring a median of mdli to achieve this response. none of the mdli administered in cases of mixed chimerism/graft failure were effective in reverting this situation. our preliminary data suggests that mdli, is a safe adoptive immunotherapy strategy even with high dose of t-cells without infusion side effects or gvhd complications. some efficacy has been observed in patients with lymphopenia and opportunistic infections, with no positive results in patients with mixed chimerism/graft failure, up to date. however, to determine the real efficacy of this strategy, prospective studies are required. jun zhao, kristen beebe, lucia mirea, alexandra walsh, shane lipskind, alexander, ngwube phoenix children's hospital, phoenix, arizona, united states background: male adolescents undergoing myeloablative hematopoietic stem cell transplantation (hsct) develop infertility with impaired spermatogenesis with reported rates ranging from % to %. in nonmalignant diseases, myeloablative regimens have been replaced with reduced intensity conditioning (ric) with the hopes of better survival rate, less organ toxicity and improved quality of life. despite the increased use of ric regimens for hsct, the effects of ric on fertility remain unknown. objectives: to assess fertility following ric hsct in young adult males. we assessed gonadal function and semen characteristics in adolescent males (> years) who received a single ric hsct at phoenix children's hospital for nonmalignant diseases during - . male patients who were a minimum of year from ric hsct and had postpubertal development at tanner stage iii or above were eligible for this study. gonadal status was assessed by measuring fsh, lh, testosterone, and inhibin b levels, and semen anal-yses assessed fertility indicators (semen volume, sperm concentration, motility, viability, forward progression, morphology, and total count). results: hormone levels and semen analysis have been obtained for patients thus far. the median time between transplant and semen analysis was years. post hsct, ( %) patients showed abnormally elevated lh levels, but fsh, testosterone (total and free), and inhibin b levels were within normal range for all patients. sperm morphology and viability testing were not able to be performed due to low concentrations and volumes. as a result, the total motile sperm count, the most useful estimate for fertile potential, is essentially for all patients. conclusion: recruitment is ongoing, but so far our limited results suggest that ric hsct may have detrimental longterm effects on male fertility. a multi-institutional trial may be appropriate due to small patient numbers at each institution. we are currently exploring options to expand to other centers. further consideration is warranted regarding decisions made by providers, ways to improve anticipatory counseling provided to patients and their families prior to transplant, and how to augment the preventive care of these patients in longterm follow-up. currently all male patients being considered for ric transplant should be counseled to sperm bank prior to transplant. background: a previous systematic literature review identified all published studies of defibrotide treatment for patients of all ages with vod/sos. to assess day+ survival for defibrotidetreated pediatric patients (≤ or ≤ years, per study) all patients exhibited infectious complications with at least viral infection. four patients also had bacterial infections. of note, no patient developed evidence of fungal infections. conclusion: early institution of ecp in patients with high risk acute gvhd (grade - ) was very effective at treating agvhd, allowed for an aggressive steroid taper and contributed to excellent overall survival rates ( %). infectious complications were primarily viral and bacterial, with no fungal infections in this very high risk population. background: vod/sos is a life-threatening complication of hsct conditioning. vod/sos with multi-organ dysfunction (mod) may be associated with > % mortality. defibrotide is approved to treat hepatic vod/sos with renal/pulmonary dysfunction post-hsct in the us and severe hepatic vod/sos post-hsct patients aged > month in the eu. there are few published data on survival of neuroblastoma patients with vod/sos post-hsct. objectives: to report day+ survival and safety post hoc for patients with neuroblastoma and vod/sos post-hsct in the defibrotide t-ind trial. design/method: vod/sos was diagnosed by baltimore or modified seattle criteria or biopsy, with/without mod, after hsct or chemotherapy. defibrotide treatment ( mg/kg/day) was recommended for ≥ days. this post hoc analysis is based on adult and pediatric patients receiving ≥ dose of defibrotide, including with mod. results: among patients with neuroblastoma, developed vod/sos after hsct. for these post-hsct patients, . % were male and . % were female, median age was years (range - years): . % aged - months, . % - years, . % - years, and patient > years. day+ survival data were available for / of these neuroblastoma patients ( with mod and without mod); had autologous and had allogeneic transplants. kaplan-meier estimated day+ survival for the neuroblastoma group was . % ( % confidence interval [ci] , . %- . %). for the mod and no mod subgroups, kaplan-meier estimated day+ survival was . % ( % ci, . %- . %) and . % ( % ci, . %- . %), respectively. in the overall t-ind hsct population aged ≤ years (n = ) and pediatric autologous hsct subgroup (n = ), kaplan-meier estimated day+ survival was . % and . %, respectively. treatment emergent adverse events (teaes) occurred in . % (n = / ), with serious teaes in . % ( / ; most common: multi-organ failure, . % [ / ]). teaes lead to treatment discontinuation in . % (n = ; most common: pulmonary hemorrhage, n = ); death occurred in . % (n = ; > %: multi-organ failure, . %; vod/sos, . %). treatment-related adverse events, as assessed by investigators, occurred in . % (n = ; most common: pulmonary hemorrhage, . %). this post hoc analysis found kaplan-meier estimated day+ survival of . % in patients with neuroblastoma and vod/sos post-hsct, which was consistent with outcomes in pediatric patients after autologous hsct. the safety profile of defibrotide in neuroblastoma patients was consistent with the overall hsct population in this study and other defibrotide studies in pediatric patients. cincinnati children's hospital medical center, cincinnati, ohio, united states background: blood stream infections occur in nearly % of patients undergoing hematopoietic stem cell transplant (hsct) and fever is often the first symptom. timely administration of antibiotics is associated with improved outcomes, thus, early recognition of fever is paramount. current standard of care (soc) includes episodic monitoring of temperature in hospitalized patients, which may delay fever detection. therefore, continuous real-time body temperature measurement may detect fever prior to the current soc. temptraq is a food and drug administration cleared class ii medical device and consists of a soft, comfortable, disposable patch that results: of patients, were started on a pca in the days post hct. % were male with median age of y. % had all, and % aml. matched related donors were used in % and % received tbi. pca was initiated median d+ . oral mucositis alone was the most common indication ( %). a majority of patients were started on hydromorphone ( %); % started on morphine and % started on fentanyl. % started on continuous infusion. pca was used for a median of days (range - days). median pain score was highest d+ of pca use, however, there was inconsistency in charting of numerical pain scores. on d+ , patients had insufficient data to determine efficacy of pain control; of the remaining patients, % had good pain control while % had moderate and % had poor pain control using our devised scale. the most common toxicity observed was respiratory depression (∼ %), however, etiology was often multifactorial and not due to opiates alone. analysis is ongoing to assess variables predicting pca use as well as efficacy of pain control and correlation between current reporting scales and patient perception. conclusion: pca use is common in pediatric hct yet pain control remains inadequate. there's a need for better evaluation of pca management, especially uniform assessment of pain, thereby improving quality of life post hct. children's national health system, washington, district of columbia, united states background: actinomycosis is a rare invasive anaerobic gram-positive bacterial disease caused by actinomyces spp. that may colonize the oropathynx, gastrointestinal tract and urogenitial tract and can lead to abscesses. respiratory tract actinomycosis is characterized by pulmonary cavities, nodules, consolidations and pleural effusions. although actinomyces are nearly always sensitive to penicillin they are frequently resistant to cephalosporins and variable sensitives to fluoroquinolones. although rare in children, immunosuppressed patients are at increased risk for actinomycosis. to describe a case of next-generation sequencing identification of actinomycosis. a -year-old male with a history of very high risk b-cell acute lymphoblastic leukemia who was months status post a / matched unrelated donor bone marrow transplant complicated by prolonged fevers, persistent weight loss, and splenic lesions, treated with posaconazole and levofloxacin developed fever and cough in the setting of neutropenia. blood cultures demonstrated staphylococcus epidermidis. ct showed micronodules and effusion not consistent with s. epi, prompting bronchoscopy. all bacterial cultures were negative. patient was prescribed a three-week course of vancomycin with rapid improvement. design/method: s next generation sequencing (ngs) from bronchoalveolar levage sample was performed at the university of washington laboratory results: ngs assay from bronchoalveolar lavage showed major abundance of actinomyces most closely related to meyeri or oodontolyticus. demonstrated actinomyces. the patient was started on a six month course of amoxicillin with continued clinical improvement. in retrospect, the splenic nodules that were presumed fungal disease were likely actinomycosis, partially treated with levofloxacin. this case highlights the potential utility of ngs in the diagnosis of rare diseases in immunocompromised patients. actinomycosis was only demonstrated through ngs and led to a change in treatment regimen and durable clinical improvement. because actinomyces often mimics malignancy, tuberculosis or nocardiosis, the use of this novel test both targeted appropriate therapy and reduced the exposure to unnecessary medications to treat the differential diagnosis. finally, we highlight that actinomyces should be considered in patients who present with unexplained fevers, weight loss, and night sweats. haneen shalabi, cynthia delbrook, maryalice stetler-stevenson, constance yuan, bonnie yates, terry j. fry, nirali n. shah center for cancer research, national cancer institute, national institute of health, bethesda, maryland, united states background: car-t therapy, while effective, may not be durable for all, and antigen negative escape is a growing problem. hct, in relapsed/refractory all, can be curative, particularly for those in an mrd negative remission. we demonstrated that cd directed car-t therapy effectively rendered patients into mrd negative remissions (by flow cytometry) and the leukemia free survival post-hct was high . in pastorek, jesssica bruce, michael a. pulsipher, chloe anthias, peter bader, andre willasch, jennifer sees, jennifer hoag, wendy pelletier, brent logan, pintip chitphakdithai, lori wiener university of pittsburgh, pittsburgh, pennsylvania, united states background: more than , pediatric hscts are performed in north american and europe each year. the ethics of exposing a healthy child to donation procedures which have some risks and no direct medical benefits continue to be a topic of debate. pediatric donors may experience psychological distress and poorer quality-of-life during and after donation compared to healthy controls. although there are fact/jacie requirements related to the management of pediatric donors, it is unclear what standardized practices exist for psychosocial assessment/management of this group. objectives: to describe transplant center practices for psychosocial evaluation/ management of pediatric donors (< years) and to examine differences in practices by location (cibmtr/ebmt) and number of harvests (volume). design/method: data were collected via a single crosssectional survey distributed electronically to cibmtr and ebmt centers between / / and / / . : / ( %) of cibmtr and / ( %) of ebmt centers completed the survey. most centers had written eligibility guidelines for pediatric donors ( %). most also had a process for ensuring that donors were freely assenting to donate ( %), managed by a transplant physician ( %). a single physician often jointly managed donor/recipient care ( %). half of centers had a pediatric donor advocate ( %), who was most often a physician ( %) or social worker ( %). cost was the largest barrier to having a donor advocate ( %). most centers performed psychosocial screening of donors ( %) but rarely declined donors based on psychosocial concerns ( %). less than half of centers provided post-donation psychosocial follow-up ( %). comparisons by center location indicated that ebmt centers were more likely to have a physician doing joint donor/recipient care ( % vs. %; p = . ), less likely to have a psychosocial assessment policy ( % vs. %; p = . ), less likely to have a donor advocate ( % vs. %; p = . ), but marginally more likely to do post-donation psychosocial follow-up ( % vs. %; p = . ). large volume centers were more likely to have a psychosocial assessment policy than their medium/smaller counterparts ( % vs. %, %; p = . ) â€"there were no other differences on key psychosocial management variables by volume. although most centers have written guidelines for pediatric donor eligibility and mechanisms for ensuring assent, substantial numbers of donors do not undergo psychosocial assessment, are jointly managed with the recipient by a single physician without an assigned donor advocate, and do not receive psychosocial follow-up. the field would benefit from guideline development for the psychosocial management of pediatric donors. background: germline mutations in samd and samd l genes cause mirage (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy) and ataxia-pancytopenia syndromes, respectively, and are associated with chromosome deletions, mds and bone marrow failure (bmf). there are limited data on outcomes of hct in these patients. to describe outcomes of allogeneic hct in patients with hematologic disorders associated with samd /samd l mutations. results: seven patients underwent allogeneic hct for primary mds (n = ), congenital amegakaryocytic thrombocytopenia (camt)(n = ), and dyskeratosis congenita (n = ). retrospective exome sequencing revealed gain-of-function mutations in samd (n = ) or samd l (n = ) genes. constitutional mosaic monosomy was present in cases. two samd patients had features of mirage syndrome. unusual findings of panhypopituitarism, laryngeal cleft, and glomerulosclerosis were noted in one case. in another case with a samd mutation hypospadias & bifid scrotum were the only findings. the remaining patients had no phenotypic abnormalities. median age at hct was y (range: . - . ). patients received transplants from bone marrow (matched unrelated (n = ) & hla identical sibling (n = )), or unrelated cord blood (ucb) (n = ). five mds patients received myeloablative s of s conditioning (busulfan-based (n = ) or tbi-based (n = )); patients (mds (n = ); camt (n = )) received reducedintensity conditioning (ric) (fludarabine, cyclophosphamide, with ratg or alemtuzumab). syndrome-related comorbidities (diarrhea, infections, malnutrition, electrolyte imbalance, lung disease and hypoxia) were present in both patients with mirage syndrome. one patient with a familial samd l mutation, mds and morbid obesity failed to engraft following ric double ucbt. she died one year later from refractory aml. all other patients achieved neutrophil and platelet engraftment, at a median (range) of ( - ) and ( - ) days, respectively. posttransplant complications included severe hypertension (n = ), pericardial effusions (n = ), veno-occlusive disease of liver (n = ), and recurrent aspiration pneumonias (n = ). one patient developed grade iii agvhd which resolved with treatment. one patient developed mild skin cgvhd and suffers from chronic lung disease. all surviving patients had resolution of hematological disorder and sustained peripheral blood donor chimerism ( - %). overall survival was % with a median follow-up of years (range: . - . y). patients with hematological disorders associated with germline samd /samd l mutations tolerated transplant conditioning without unusual, or unexpectedly severe toxicities. allogeneic hct led to successful resolution of mds or bmf, with excellent overall survival. more data is needed to refine transplant approaches in samd /samd l patients with significant comorbidities, and develop guidelines for their long-term follow-up. shyamli singla, tiffany simms-waldrip, andrew y. koh, victor m. aquino background: steroid-refractory acute graft versus host disease (agvhd) is a potentially fatal complication of allogeneic hematopoietic stem cell transplantation (hsct). basiliximab (anti-il -r monoclonal antibody) as a single agent or in combination infliximab (anti-tnf-monoclonal antibody) has demonstrated efficacy in adult cohorts with steroid-refractory agvhd, but has not been well studied in the pediatric population. we adopted the use of basiliximab and infliximab as our institutional standard of care for steroid-refractory agvhd in pediatric hsct patients. to determine the response and survival of hsct children who received basiliximab and infliximab for the treatment of steroid-refractory agvhd. design/method: we retrospectively reviewed children who received basiliximab and infliximab for steroid-refractory agvhd refractory between september and december . complete response (cr) was defined as resolution of all clinical signs of agvhd. partial response (pr) was defined as at least one grade reduction in one target organ (e.g. skin, gut or liver) without increased grade in another target organ. no response was defined as either no improvement or progressive worsening of agvhd in at least one organ. baseline demographics, transplant details, laboratory findings, and treatment outcomes were also evaluated. results: of the evaluable hsct patients, children (median age yrs, range mo- yrs) with steroid-refractory agvhd received combination monoclonal antibody (mab) therapy. the median time from the start of steroid therapy to initiation of mab was days. the overall glucksberg grade of agvhd at the time of initiating mab therapy was grade i (n = , . %) ii (n = ; %), iii (n = ; %) or iv (n = ; %). the overall response rate was %, with ( %) patients achieving cr, ( . %) patients achieving pr, and ( . %) patients with no response at days following the start of mab therapy. the median overall survival was , , and days for patients who exhibited cr, pr, and no response, respectively. the overall survival at year following start of mab therapy was %. background: the role of high dose chemotherapy (hdc) and autologous stem cell rescue (ascr) in patients with high risk (advanced metastatic or relapsed) soft tissue sarcomas is controversial. despite multimodal chemotherapy, radiotherapy, and local control measure advancements, prognosis of patients with advanced metastatic or unresectable and relapsed sarcomas remains poor, with less than % years disease free survival. objectives: to determine if consolidation with myeloablative hdc and ascr improves relapse free (rfs) and overall survival (os) outcomes in a high risk patient subgroup. we performed retrospective review of all high risk soft tissue sarcoma patients who underwent hdc and ascr at the children's hospital at montefiore, bronx, ny between october and january . the protocol was approved by albert einstein college of medicine institutional review board. results: patients ( primary metastatic high risk disease, relapsed or recurrent disease) received hdc with ascr. primary diagnoses were rhabdomyosarcoma (rms) (n = , alveolar histology), primary site nasopharynx (n = ) and lower extremity (n = ). ewing's sarcoma (ews) (n = ), axial site (pelvic) in patients ( %). median age years (range - years), ( %) were male. all patients were in complete metabolic remission before transplant. median pre transplant comorbidity index was (range - ). patients ( rms and ews) received conditioning with carboplatin, etoposide and melphalan. remaining patients with ews received conditioning with busulfan, melphalan and topotecan. all patients received peripheral blood mobilized hematopoietic stem cell transplantation. stem cell mobilization achieved with high dose filgrastim in all patients except one who required addition of plerixafor. median cd +/kg s of s recipient body weight cell dose infused was . × ^ (range . - . × ^ ). median times to neutrophil and platelet (> , / l) engraftment were (range - ) and ( - ) days respectively. patients ( %) developed bk viuria (one with grade iii hemorrhagic cystitis); ( %) developed cmv viremia; and one patient ( %) had asymptomatic ebv viremia. there was no graft failure, sinusoidal obstruction syndrome or transplant related mortality. median follow up post-transplant was days (range - days). year probability of os and rfs were % and % respectively. hdc with ascr is a promising therapeutic strategy to consolidate remission and improve survival in select high risk soft tissue sarcoma patient subgroups. prospective clinical trials will inform the impact of disease status prior to hdc and ascr on outcome, optimal conditioning and long term relapse free and overall survival. background: absence of minimal residual disease is paramount for cure of pediatric acute lymphoblastic leukemia (all). the testis may harbor occult leukemia and this disease may result in treatment failure. objectives: the purpose of this study was to assess the longterm outcomes of boys with or without testicular leukemia pre-hematopoietic stem cell transplantation (hsct). design/method: retrospective analysis of boys with high-risk de novo ( with hypodiploidy all) or recurrent/refractory all was conducted. flow cytometry of bone marrow mononuclear cells was used to determine remission status. testicular evaluations were performed by physical examination and wedge biopsy pre-hsct. the median age at time of transplant was . years. all patients were in remission by flow cytometry of bone marrow mononuclear cells at the time of transplant and none had evidence of clinically apparent testicular disease. testicular leukemia was detected in patient and he underwent bilateral orchiectomy. he developed acute graft versus host disease (gvhd) of the duodenum and sigmoid colon which resolved, and the leukemia remains in second complete remission and he is free of hsct-related morbidity . months post-hsct. of the patients without testicular leukemia died a median of . months (range, . to . ) post-hsct ( with adenovirus infection and each with thrombotic microangiopathy and aspergillus pneumonia); experienced infection (staphylococcus species, corynebacterium, enterococcus, klebsiella, citrobacter, e. coli, epstein barr virus, adenovirus, bk virus, human herpesvirus- , candida albicans, fusarium, aspergillus, yeast, and other fungus); experienced gvhd ( of the gi tract, of the skin, of the liver, of the eyes, of the mouth, and of the lungs); and developed a second neoplasia (right lower leg leiomyosarcoma). one patient developed bone marrow minimal residual disease ( . % phenotypically abnormal cells detected months after / matched sibling hsct). reinduction therapy comprised weekly doses of rituxan, courses of blinatumomab and donor lymphocyte infusions with il- . two subsequent bone marrow evaluations were minimal residual disease negative. thirteen months post-hsct residual disease recurred ( . %) and he will receive inotumumab. overall median survival post-transplant of the boys is . months (range, . to . ) and of the surviving boys is . months (range, to . ). conclusion: testicular biopsy can detect occult leukemia pre-hsct. testicular leukemia pre-hsct does not appear to increase the risk of subsequent relapse or other hsct-related adverse events compared to those without it. yaya chu, nang kham su, sarah alter, emily k. jeng, peter r. rhode, mathew barth, dean a. lee, hing c. wong, mitchell s. cairo new york medical college, valhalla, new york, united states background: rituximab has been widely used in frontline treatment of b-nhl including burkitt lymphoma (bl), however, some patients retreated with rituximab relapse, which limit patient treatment options. novel therapies are desperately needed for relapsed/refractory b-nhl patients. several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering immune cells with chimeric antigen receptors (car), as well as second-generation anti-cd antibodies. nature killer (nk) cells play important roles in the rejection of tumors. however, nk therapy is limited by small numbers of active nk cells in unmodified peripheral blood, lack of tumor targeting specificity, and multiple mechanisms of tumor escape of nk cell immunosurveillance. our group has successfully expanded functional and active peripheral blood nk cells (expbnk). b t m was generated by fusing alt- , an il- superagonist, to four single-chains of rituximab. b t m displayed tri-specific binding activity through its recognition of the cd molecule on tumor cells, activated nk cells to enhance adcc, and induced apoptosis of b-lymphoma cells. objectives: to examine if b t m significantly enhances the cytotoxicity of expbnk against rituximab-sensitive and -resistant bl cells. design/method: expbnks were expanded with lethally irradiated k -mbil - bbl and isolated using miltenyi nk cell isolation kit. alt- and b t m were generously provided by altor bioscience. nk receptors expression and cytotoxicity were examined as we previous described. ifng and granzyme b levels were examined by elisa assays. equal doses of rituximab, alt- , rituximab+alt- , obinutuzumab (obinu) were used for comparison. igg was used as controls. anti-cd car expbnk cells were generated as we previously described by mrna electroporation. rituximab-sensitive raji andresistant bl cells raji- r and raji- rh, were used as target cells. results: b t m significantly enhanced expbnk cytotoxicity against rituximab-sensitive raji cells, rituximab-resistant raji- r cells and resistant raji- rh cells compared to the controls igg, rituximab, alt- , rituximab+alt- , obinu (p< . , e:t = : ). furthermore, we confirmed the enhanced cytotoxicity by measuring ifn-g and granzyme b production. b t m significantly enhanced ifn-g and granzyme b production from expbnk against raji, raji- r and raji- rh compared to igg (p< . ), rituximab (p< . ), alt- (p< . ), rituximab+alt- (p< . ), and obinutuzumab (p< . ). when compared to anti-cd car expbnk cells, b t m + expbnk had the similar cytotoxicity against raji, raji- r and raji- rh as anti-cd car expbnk cells did (p> . ). conclusion: b t m significantly enhanced expbnk activating receptor expression and in vitro cytotoxicity against rituximab-sensitive and -resistant bl cells. the in vivo functions of b t m with expbnk against rituximab-sensitive and -resistant bl cells using humanized nsg models are under investigation. background: cardiac dysfunction, including left ventricular systolic dysfunction (lvsd), is a known complication in stem cell transplant (sct) survivors. while detection of lvsd by echocardiography is important in this population, there has been minimal research to determine if subclinical cardiac dysfunction exists in sct patients. cardiopulmonary exercise testing (cpet) is a valuable tool to assess cardiac function, and to determine how the heart responds to the stress of exercise. no studies have been performed to determine if sct patients with normal lvsd on standard echocardiography may have abnormal cpet. to determine the feasibility of cpet, as well as additional echocardiographic parameters, to detect dysfunction in sct patients with a normal ejection fraction on echocardiogram. design/method: we performed a cross-sectional analysis of sct survivors who were at least years post sct, years of age or older and with an ejection fraction > % (low end of normal range) on echocardiogram. we assessed the exercise capacity of all patients with cpet, and sub-clinical cardiac dysfunction through tissue doppler and strain analysis from the echocardiogram. results: seven patients ( male) have qualified and completed this study so far with an average age of . ± . years. the median time from transplant is . ± . years. all seven patients had a normal ejection fraction, however four patients had abnormalities on their cpet. these abnormalities included abnormal predicted peak oxygen consumption (vo ) ( %± . , normal > %) (the best predictor of functional capacity), predicted oxygen pulse ( %± . , normal > %) (measure of cardiac stroke volume) and ventilatory efficiency (ve/vco slope) ( ± . , normal < ). submaximal exercise data, used when patients are unable to complete a maximal effort test, demonstrated low-normal predicted vo at anaerobic threshold ( . %± . %, normal > % of was . days while patients who received autologous infusions had a mean number of days to engraftment of . . engraftment after hsct needs to be prompt to minimize duration of neutropenia and maximize survival rates . our data demonstrates that the infusion of hematopoietic stem cell products with a syringe or iv pump is an effective method of delivery for stem cell products and does not delay the time to engraftment. the median days to neutrophil engraftment was . days. this is comparable to data from the nmdp, which reports engraftment occurs within - days. the main limitation to this study was its small sample size due to the number of transplants done at our center. however, it does provide evidence to support that infusion of stem cell products via pump mechanism is a safe alternative to the infusion by gravity method in the process of the hematopoietic stem cell administration. johns hopkins all children 's hospital, st. petersburg, florida, united states background: leukemic relapse remains the most common cause of treatment failure after allogeneic hematopoietic cell transplant (allohct) for myeloid malignancies. most children who relapse post-allohct will die of their disease, making interventions to minimize this risk a high priority. objectives: to evaluate the safety and efficacy of posttransplant azacitidine for relapse prevention in children undergoing allohct for myeloid malignancy. design/method: we retrospectively reviewed the charts of children undergoing allohct for myeloid malignancies between february and november at johns hopkins all children's hospital. results: during the study period, children (ages to years, median ) underwent allohct for myeloid malignancies: de novo acute myeloid leukemia (aml), ; mixed phenotype acute leukemia, ; treatment-related aml, ; juvenile myelomonocytic leukemia with aml transformation, ; and myelodysplasia/aml, . thirteen were in first complete remission, were in cr or greater. most patients ( / ) received fludarabine/melphalan/thiotepa conditioning; received hla-identical related or unrelated donors, and received haploidentical bone marrow grafts with post-transplant cyclophosphamide. three patients never received planned azacitidine ( early relapse; early trm), leaving evaluable patients. azacitidine ( mg/m /dose for days, in -day cycles for up to cycles) was started at a median of days post-transplant (range - ). two-thirds ( / ) of patients received eight or more cycles. of five patients who stopped therapy early, only one was due to toxicity; other reasons included severe gvhd ( ), parental preference ( ), and relapse ( ). cycle delays occurred in patients, with a median cycles delayed per patient, mostly for mild myelosuppression with early cycles. no patient required blood product transfusion during therapy, but g-csf was used in three patients to maintain anc> / l. dose-modifications were made in patients (renal tubular acidosis, acute kidney injury, and myelosuppression). there were relapses ( %), two of which occurred in patients in cr , for a relapse incidence of % in patients in cr , with a median follow-up of months (range . to ). no patients who received azacitidine died of transplant-related mortality. conclusion: administration of azacitidine in children undergoing allohct for myeloid malignancies is safe and feasible, with most patients successfully receiving all planned cycles. toxicity was acceptable and there was no trm or secondary graft failure. despite the limitations of a small cohort, relapse incidence-particularly in patients transplanted in cr suggests a potential benefit in disease control that warrants investigation in follow-up studies. background: despite significant improvements in the success rate of hematopoietic cell transplantation (hct), graft failure remains an important complication in patients transplanted for severe aplastic anemia (saa). second allogeneic hct can salvage patients, but -year overall survival (os) rates have been reported as low as % . objectives: identify patients who developed dropping donor chimerism, graft rejection, and/or graft failure after first hct for saa, necessitating additional hcts or cellular boosts (defined as stem cell products infused without preceding chemotherapy), and evaluate treatment-related complications and os. with vod/sos with and without multi-organ dysfunction (mod) pubmed and embase databases were searched for "defibrotide and retrospective chart reviews; excluded publication types were: case reports (< cases); meta-analyses; reviews; animal, modeling, pharmacokinetic, chromatography, and adult-only studies; guidelines; articles; and letters. resulting reports were screened for exclusion criteria. full-text articles were then reviewed for eligibility. study characteristics of selected publications were summarized, and publications were categorized by patients' mod status. when necessary, additional data tables were requested. a random effects model was used for pooling data for efficacy. interstudy heterogeneity was assessed with cochran's q-test. percentage of total variation across studies due to heterogeneity (i ) was evaluated we quantified ∼ proteins in each sample. reproducibility for one donor at different time points children 's minnesota, minneapolis, minnesota, united states background: pediatric and young adult hodgkin lymphoma (hl) has five-year survival rates > %. chemotherapy required to achieve this rate is associated with a lifetime risk of cardiac deaths, second malignancies, pulmonary disease and infertility. as effective salvage therapy exists, outcomes may be improved by de-intensifying initial therapy to lessen toxicity.objectives: we piloted a regimen in low and intermediate risk hl patients using agents without known association to significant late effects. this retrospective chart review was approved by children's minnesota irb.design/method: the bvg(p) regimen incorporated bortezomib ( . mg/m day , , , ); vinorelbine ( mg/m day , ); gemcitabine ( mg/m day , ) every days and prednisone ( mg/m /dose bid x days). we treated newly diagnosed patients, ages - years, with non-bulk stage iia (n = ) or iib (n = ) hl. two patients received bvg and received bvgp with the addition of prednisone.results: newly diagnosed patients were all pet negative after the first or second cycle and remained pet negative at end of therapy, cycles. nausea was well controlled with -ht antagonists and scopolamine. pegfilgrastim was not necessary due to the high absolute neutrophil count nadir [median . and minimum . × /l]. there were no episodes of febrile neutropenia, infection or transfusion need. no patients experienced alopecia. one patient developed sensory neuropathy after the eighth dose of bortezomib that was controlled with gabapentin and a switch to subcutaneous bortezomib administration. of the five newly diagnosed patients, four remain in remission at , , , days; relapsed at previous disease sites at days and subsequently achieved remission with bvgp with the addition of brentuximab. this series provides early evidence to stimulate expansion of this pilot experience and subsequent multiinstitutional study leading to a randomized trial of bvgp and current chemotherapy for low and intermediate hl. st jude affiliate clinic at st francis hospital, tulsa, oklahoma, united states background: symptoms suggestive of morning hypoglycemia has been noticed in children receiving all chemotherapy. only few small studies looked at this therapy related complication. factors increase risk of hypoglycemia in all patients include accelerated starvation, steroid induced adrenal suppression, mercaptopurine therapy and prolonged fasting for procedures.objectives: to study the prevalence and risk factors for hypoglycemia during all therapy design/method: medical records of of children (up to years old) treated for all between - ( patients) were studied for evidence of morning hypoglycemia defined as blood sugar (bs) < mg/dl. statistical mean differences between the subgroups were analyzed with spss using a nonparametric mann-whitney u test.results: fifty two percent ( %) of patients developed hypoglycemia during all treatment, with an average of . episodes/patient. % were males and % females. almost / ( %) of patients with hypoglycemia were in maintenance phase of therapy. % of hypoglycemic episodes occurred in % of patients. majority of hypoglycemic episodes ( . %) occurred on the day of procedure when patients were fasting overnight. . % of hypoglycemic episodes occurred in children ≤ years, with . % in ≤ years. patients who developed hypoglycemia were significantly younger (mean age at time of diagnosis of all was . ± . at the hypoglycemia group versus the non-hypoglycemia ( . ± . ) p< . . no statistically significant difference was found regarding sex, or tpmt genotype. % of hypoglycemic children-all < years of age-presented with life threatening hypoglycemia symptoms including seizure and loss of consciousness. this study showed high prevalence of hypoglycemia during childhood all therapy. younger age, especially ≤ years, is associated with higher risk of hypoglycemia as well as life-threatening episodes. to decrease fasting hypoglycemia during therapy for childhood all, we recommend that children under the age of years receive bed time snack high in proteins and complex carbohydrates, and to get them up early the day of procedure to take clear sugary drink. hospital for sick children, toronto, ontario, canada ann & robert h. lurie children's hospital of chicago, chicago, illinois, united states background: childhood brain tumors are the most common solid malignancy and the leading cause of cancer-related mortality in children. the most aggressive type of pediatric central nervous system (cns) tumors is diffuse intrinsic pontine glioma (dipg). despite decades of clinical trials, there has been no substantial improvement with respect to therapeutic outcomes with most children eventually succumbing to the disease. research on adult high-grade gliomas has shown a targetable pathway through the inflammationinduced expression of indoleamine , dioxygenase (ido ) and its recognized ability to suppress the anti-tumor immune response. a limited understanding into the role of ido in pediatric central nervous system tumors serves as the foundation of this research project. furthermore, the integration of nanotechnology is a fundamental step for the investigation and targeting of ido . spherical nucleic acids (snas) composed of nanoparticles have been shown to transverse cellular membranes, exhibit stability in physiological environments, escape from degradation, and create precise targeting in brain tumors.objectives: the purpose of our project is to delineate the role of ido in pediatric dipg, and develop small inhibitory (si)rna oligonucleotides and snas aimed at therapeutically inhibiting the gene expression of immunosuppressive ido . our specific aims are to: ( ) confirm the gene expression ido in different human dipg cell lines; ( ) generate and characterize sirna oligonucleotides targeting human ido in vitro; and ( ) generate and characterize gold nanoparticles for targeted inhibition of ido .design/method: unique patient-derived dipg cell lines were grown in culture, stimulated with increasing concentrations of the proinflammatory cytokine, ifn , and analyzed for mrna levels. sirna specific to ido was transfected into cells. sna generation is in progress.results: ido is expressed in multiple human pediatric dipg cell lines. sirna targeting ido among exons and results in a significant decrease in overall ido expression by dipg cells. sna generation for targeting ido with improved penetration & stability is ongoing, with preliminary results demonstrating a robust ability to inhibit ido expression. the grim prognosis of children with dipg, the lack of effective therapies, and the expression of ido by human dipg cells emphasize the importance of developing the treatment capability to inhibit ido gene expression, as a excluded from this study. the remaining patients were analyzed using descriptive statistics.results: a total of patients meeting inclusion criteria were identified. of these, patients ( . %) received tonsillectomy alone, ( . %) underwent tonsillectomy and decreased immunosuppression, ( . %) received tonsillectomy and rituximab, and another ( . %) received tonsillectomy with additional therapy (including ebv-specific cytotoxic tlymphocytes, donor leukocyte infusion, and chemotherapy). of the patients who received tonsillectomy with or without a decrease in immunosuppression, all were diagnosed with high-grade lymphoma and achieved clinical remission following tonsillectomy with no evidence of relapse to date. on further analysis looking at ptld risk factors, all patients were under years of age, all received t-cell depleted grafts, and none had significant graft-versus-host disease (gvhd) at the time of ptld diagnosis. we have identified a population of patients with localized ebv ptld that achieved clinical remission with no evidence of recurrence following tonsillectomy, suggesting that tonsillectomy alone may be an adequate treatment for localized ebv ptld in a specific subgroup of patients. further analysis is needed to identify characteristics of this subgroup to determine which patients would be most likely to respond to this treatment. university of rochester, rochester, new york, united states background: malignant central nervous system (cns) tumors in young children have a poor prognosis and pose a significant therapeutic challenge. consolidation therapy with carboplatin and thiotepa was piloted in ccg- , cog acns , and cog acns with the goals of intensifying therapy and omitting or delaying radiation.objectives: to document outcomes for patients undergoing carboplatin/thiotepa consolidation with autologous stem cell rescue (ascr) and to demonstrate the feasibility and toxicity of this regimen.design/method: patients up to years old (median age: months) with malignant cns tumors treated at the university of rochester from - with at least one cycle of carboplatin ( mg/kg/day x days) and thiotepa ( mg/kg x days) followed by peripheral blood ascr were included in retrospective analysis. data were recorded on time to engraftment (defined by absolute neutrophil count (anc) recovery to > . × ^ /l), length of hospitalization, toxicity with each consolidation cycle, progression free survival (pfs) and overall survival (os). stem cell harvest data were also collected.results: eleven patients with malignant cns tumors ( atypical teratoid/rhabdoid tumor, primitive neuroectodermal tumor, glioblastoma multiforme, and pineoblastoma) received a total of cycles of carboplatin/thiotepa. of these, underwent stem cell harvest at our institution, with complications limited to procedure-related hypotension for patient with known autonomic instability, and catheter-associated deep vein thrombosis (dvt) for patient. four patients were in complete remission (cr) /status-post gross total resection, was in cr , and had residual tumor at the time of consolidation. nine patients received planned consolidation cycles, patient (of ) planned cycles, and patient of an anticipated cycles thus far. average time to engraftment for these cycles was . (+/- . ) days, with a mean hospital length of stay of (+/- . ) days. fever occurred in of cycles ( %); infectious toxicity included documented bacterial infection in cases (enterococcus faecalis bacteremia in , klebsiella pneumoniae in ). there were no regimenrelated deaths. with a mean follow-up of months, survivors have not yet completed all therapies, and patients have relapsed ( have died of disease). of the survivors, have been disease-free for > months. background: autologous hematopoietic stem cell transplantation (auto-hsct) has resulted in improved survival for patients with high-risk neuroblastoma. treatment intensification is however associated with greater complications. data on early infectious complications in low-and-middle income countries are limited.objectives: to review the early infectious complications following auto-hsct in patients with high-risk neuroblastoma.design/method: a retrospective chart review of pediatric patients with high-risk neuroblastoma who underwent auto-hsct at the american university of beirut medical center between and was conducted. infectious complications during the first days post-transplant were reviewed.results: forty-three patients ( males and females) with a median age at diagnosis of . years [range: . - . ] years underwent auto-hsct during the above-mentioned period. conditioning regimen consisted of melphalan, etoposide and carboplatin. all patients received antiviral and antifungal prophylaxis. median time for neutrophil engraftment was days [range: - ]. bacteremia and clostridium difficile infections occurred in ( %) and ( %) patients respectively. seven ( %) patients developed enterocolitis diagnosed by imaging, were adenovirus induced. cmv viremia was diagnosed in ( %) patients, of whom required treatment. varicella zoster reactivation, parvovirus viremia, toxoplasmosis encephalitis, bk virus cystitis ( patients) and central nervous system ebv related post-transplant lymphoproliferative disorder were diagnosed in different patients. there was no invasive fungal infection. sixteen ( %) patients have died, of whom died in the early post-transplant period, due to disease progression and ( . %) due to infectious complications. among the patients who died due to infection, developed toxoplasmosis encephalitis, developed severe enterocolitis, of which were adenovirus related. the mean igg level within one week post-transplant was lower in patients with clinically significant viral infection compared to others ( vs . mg/dl, p: . ). the mean igg level at the time of clinically significant bacterial infection was lower in infected patients compared to others ( . vs . mg/dl, p: . ). neither absolute lymphocyte count nor absolute neutrophil count at day post-transplant affected the incidence of clinically significant infections. our results show that the rate of infections during the early post auto-hsct period is higher than what has been described in developed countries and has a significant impact on mortality. prevention, early detection and improvement in the treatment is required to improve outcome. university of miami, miami, florida, united states background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative treatment for many malignant and non-malignant (bone marrow failure, immunodeficiency, or metabolic diseases) in pediatrics. despite advances in medicine, graft-versus-host-disease (gvhd) remains a significant cause of non-relapsed morbidity and mortality, specifically in those with malignant diseases.objectives: to highlight the complexity to acute gvhd management and seldom-described treatment approach. a year male with a history of high risk acute myeloid leukemia (aml) due to failed induction therapy. he received a matched ( / ) unrelated donor hsctmarrow product-conditioned with busulfan, fludarabine, and anti-thymoglobulin (atg). his post-transplant course was complicated by hhv- viremia, pres (prompting a change from prograf to cyclosporine), mucositis, and grade iii acute gvhd (skin s , gut s , liver s ) around post transplant day , which later morphed to ocular involvement by d+ . he was started on mg/kg steroids with good response but flared up with each attempt to taper steroid dose. a course of rituximab and later atg were tried without success in weaning off steroids. switching cyclosporine to sirolimus did not provide any additional benefit either. extracorporeal photopheresis (ecp) was started times a week. he initially responded well, yet was not able to wean off steroids. in addition, he developed a flare when ecp session was reduced to days per week. ecp was therefore increased to days per week, which appeared to stabilize skin lesions. a trial of weekly methotrexate was attempted to wean off steroids and photopheresis, which provided no response. finally, a trial of bortezomib on days , , , and of a day cycle as published in a case series of multiple myeloma patients who developed post hsct gvhd. skin lesions improved remarkably however dose had to be reduced due to related pancytopenia. given the response to therapy, he was continued on a weekly dose of bortezomib, receiving a total doses, which has permitted the slow taper of prednisone that has since been discontinued without a major flare. he however is currently maintained on ecp times per week, which is now been slowly withdrawn.conclusion: management of acute gvhd in pediatric patients after hsct can be challenging with no definite options for those who fail steroids or become steroid dependent after initial response. in these situations, bortezomib could be a valid therapeutic option. background: neuroblastoma (nbl) is the second most common solid tumor in children and despite recent treatment advances, overall survival for high risk nbl remains < %. the addition of immunotherapy has improved survival and includes anti-gd antibody therapy. the success of antibody therapy in neuroblastoma is primarily due to natural killer (nk) cell mediated antibody dependent cellular cytotoxicity. we previously demonstrated that nk cells from patients with high risk nbl can be successfully isolated and expanded to large numbers and exhibit potent anti-tumor effects against nbl ( ). thus, infusions of autologous expanded nk cells in high risk nbl in combination with anti-gd antibody are being studied in clinical trials. toll-like receptors (tlr) present on the surface of leukocytes are responsible for pathogen recognition, and activation of these receptors stimulate the production of cytokines that critically link innate and adaptive immune responses. the tlr agonist, poly(ic) is a synthetic analog of dsrna that has previously been shown to directly stimulate cytokine production and improve cytotoxicity in primary nk cells through activation of genes regulated by interferon-response elements (ire) ( ). we hypothesized that ex vivo activation of tlr pathways in nk cells during our normal -day expansion using k feeder cells expressing membrane bound il- would enhance their function.design/method: nk cells were isolated from peripheral blood mononuclear cells and expanded with our previously described expansion protocol in media containing il- and ug/ml poly(ic) ( ). at the end of the -day expansion, nk cells expanded with poly(ic) were compared to controls using a calcein cytotoxicity assay to measure cytotoxicity against high risk neuroblastoma and cytometric bead array to measure cytokine production. : surprisingly, the addition of poly(ic) during nk cell expansion did not improve proliferation, cytokine production or cytotoxicity compared to our standard expansion method. rnaseq demonstrated that our standard expansion method results in a modest decrease in tlr expression at the transcriptional level, but significant upregulation of several ireregulated genes. we conclude that either our standard approach interferes with tlr signaling or saturates the innate immune response pathway such that co-stimulation with poly ic does not produce an additive effect. we are performing expression analysis on nk cells receiving poly(ic) during expansion to further explore this hypothesis. background: gonadal dysfunction leading to infertility is a complication after hematopoietic stem cell transplant (hsct). anti-müllerian hormone (amh) is a marker of ovarian reserve; it is not controlled by gonadotropins and has minimal inter-cycle variations, therefore, it can be used as a marker of ovarian reserve and aid in fertility counseling.objectives: assess ovarian reserve in hsct patients utilizing amh levels. background: tgf beta is an immune suppressive cytokine frequently elevated in the tumor microenvironment causing tumor immune evasion. acute tgf beta treatment potently inhibits nk cell cytotoxicity, cytokine secretion, and proliferation. however, tumor infiltrating nk cells receive chronic inhibitory tgf beta signals in conjunction with activating signals from tumor cells. objectives: to this end, we hypothesized that long-term tgf beta-cultured nk cells would induce functional and phenotypical changes on nk cells that differ from short-term tgf beta treatment.design/method: to explore this, primary human nk cells were cultured with the leukemia cell line, k , alone or with exogenous tgf beta for weeks. : surprisingly, nk cells cultured in tgf beta proliferated faster, and upon challenge with a variety of cell line targets they secreted much greater quantities of ifnÎ ( -to -fold increase against / cell lines) and tnf ( -to -fold increase against / cell lines). further, the high cytokine secretion induced in these nk cells was no longer inhibited by adding additional tgf beta. degranulation was also increased ( / cell lines), however cytotoxicity was not enhanced in a -hour cytotoxicity assay. after resting in il- , the cytokine hypersecretion of tgf betacultured nk cells was maintained for several weeks suggesting this functional change might involve cellular reprogramming. we investigated the mechanism behind these functional changes and profiled genes involved in tgf beta signaling. we found significant reduction of smad transcription which corresponded to a striking decrease in smad chromatin accessibility. we also found significantly increased smad and decreased tgfbr expression. phenotypic analysis revealed that tgf beta also induced remodeling of the nk receptor repertoire with decreased nkp , cd , and klrg and upregulation of trail. the functional consequences of these tgf beta-induced changes on in vitro and in vivo nk cell function are currently under investigation. background: the use of t-cell depleted grafts in haploidentical stem cell transplantation (hsct) has been associated with a delay in early t-cell recovery which increases the risk of viral infections, relapse or graft rejection. conventional donor lymphocyte infusion (dli) after hsct transplantation is effective but conditioned because of a high prevalence of gvhd. the infusion of selected lymphocyte subpopulations with low aloreactivity is emerging as an effective strategy to rectify this issue. the depletion of cd ra+ naive lymphocytes, preserving cd ro+ memory t-cells, could provide a safe source of functional lymphocytes with anti-infection, antileukemic and anti-rejection properties, and lower rates of adverse effects. our objective is to present data of patients that have received cd ro+ memory t-cells dli (mdli) and assess its safety and outcome. we present data of mdli performed after hsct in cases of mixed chimerism, persistent lymphopenia, viral/opportunistic infections or as a strategy to accelerate immune reconstitution.results: fifteen patients with diagnosis of all (n = ), aml (n = ), mds (n = ), saa (n = ), sideroblastic anemia (n = ) and cgd (n = ), received mdli after hsct. a total of forty-three mdli were infused. the median dose of cd ro+ memory t-cells infused was . × /kg (range: . × - . × /kg), with a median dose of cd ra+ naive t-cells of . × /kg (range: - . × /kg). the mdli were infused at a median of seventy-seven days after hsct (range: - days), with a median interval between mdli of thirty-four days results: eight published studies reported survival outcomes for pediatric vod/sos patients (n = ), across all defibrotide doses. estimated day+ survival ( % confidence interval) was % ( %- %). for vod/sos with mod, studies were identified (n = ) with pooled estimated day+ survival of % ( %- %). only one openlabel expanded-access study, the treatment-ind, reported outcomes separately for pediatric vod/sos patients without mod (n = patients aged ≤ years). the day+ kaplan-meier estimated survival for those patients was % ( %- %). safety results were not pooled due to differences in reporting methodology; however, study results were consistent with the safety profile of the phase historicallycontrolled trial in vod/sos patients with mod ( % pediatric), in which / defibrotide-treated patients and all controls experienced ≥ ae. hypotension was the most frequent ae ( %, defibrotide; %, controls); common hemorrhagic aes (ie, pulmonary alveolar and gastrointestinal hemorrhage) occurred in % of defibrotide-treated patients and % of controls. in this pooled analysis of studies with defibrotide-treated pediatric patients with vod/sos, estimated day + survival was % (without mod, %; with mod, %). safety results in individual studies were generally consistent with the known safety profile of defibrotide. taken together, these results show a largely consistent defibrotide treatment effect in pediatric patients treated with defibrotide for vod/sos, with or without mod. results: six patients met inclusion/exclusion criteria. all patients were started on ecp while concurrently receiving . to mg/kg steroid therapy for agvhd plus a calcineurin inhibitor. patients had initiation of ecp within a maximum of weeks from initial diagnosis of agvhd (range - days). patients had grade - agvhd ( / patients with grade ) with skin, liver, and gi gvhd represented. patients received ei-ecp - times per week for the first weeks and then had ei-ecp frequency tapered based on initial response.after weeks of therapy patient had a decrease in overall gvhd grade by grade. all patients were able to have steroids tapered, with doses decreased by an average of % ( % - % decrease).at weeks of therapy, one patient with grade agvhd died of mof associated with infections. three patients had complete resolution of agvhd and patients decreased by grade. steroid doses were decreased by an average of % ( % - % decrease). continuously measures axillary temperature and wirelessly transmits real time-time data. the primary aim of the study was to evaluate the feasibility, safety and tolerability of continuous temperature monitoring in hsct patients using temptraq. we are performing a prospective observational study of pediatric patients ( - years of age) undergoing hsct at cincinnati children's hospital in cincinnati, ohio. enrolled patients wore a temptraq patch for days. a - rating scale survey was completed by the parent/guardian at the end of the study to determine tolerability, ease of use, satisfaction and desire for future use in the inpatient and outpatient setting. temperature data from the temptraq patch was compared to the standard episodic temperature monitoring to determine detection of febrile episodes. seven of ten patients have completed screening. we anticipate completion of the study in early february. the temptraq patch was well tolerated by study subjects (mean tolerability rating of . / ). one patient developed skin breakdown at the site of the temptraq patch attributed to recent thiotepa. the patch was easy to apply with an easy of application rating of . / . parents were overall satisfied (rating . / ) and would like to use the temptraq patches in future hospitalizations (rating . / ) and at home (rating . / ). temptraq patch identified fever (≥ . • f) in patients. the fever was never detected by episodic monitoring (soc) in patients and significantly delayed in the other patients (> hours). temptraq was well tolerated in pediatric hsct patients. timely fever detection was improved in temptraq over the current soc. background: serotherapy is commonly used in patients undergoing hematopoietic stem cell transplant (hsct) to reduce the incidences of engraftment failure and graft versus host disease. however, one well-known side effect is fever. as children undergoing hsct have compromised immune defenses, fever may also be an early indicator of bloodstream infection, which would warrant prompt use of broad-spectrum antibiotics. in a subset of patients with serotherapy-associated fever, antibiotics, which may induce antibiotic resistance and increase costs, may be unnecessary. we aimed to determine the incidence and characteristics of serotherapy-related fever, as well as the likelihood of concomitant bacteremia, in our institutional experience. a -year retrospective chart review was conducted of pediatric patients who received serotherapy as part of hsct conditioning at the university of minnesota. one-hundred sixty eight consecutive hsct patients who received serotherapy -either atg (n = ) or alentuzumab (n = ) -were identified. the median age at hsct was -years (range, . - years). a total of patients ( %) developed fever while on serotherapy (atg = , alentuzumab = ). one-hundred sixteen patients presented fever following the first infusion, and the median onset of fever was hours after commencing infusion (range, . - hours). fever resolved at a median hours (range, - hours). one hundred and fourteen patients ( %) underwent blood cultures. only seven patient were not started on ( %) empiric antibiotics, while % (n = ) were on antibiotic treatment prior to serotherapy for previously known or suspected infections. nine patients ( % of febrile patients, % of all patients) had positive blood cultures (atg = ; alentuzumab = ). no infection-associated deaths were observed.conclusion: while fever is common during serotherapy conditioning in children undergoing sct, episodes of concomitant bloodstream infection are rare. ongoing analysis identified potential risk factors for bacteremia as recent history of infection, first episode of fever following second or subsequent infusions, and previous central line placement. further analysis is being conducted to identify subgroups of patients for whom close monitoring alone may be safe. background: hsct is potentially curative for caya with high-risk leukemias; however, most lack an hla-matched aspho abstracts related donor. the risk of gvhd is increased with unrelated (urd) or partially matched related (pmrd) donors. selective t-cell depletion based on the elimination of t cells carrying and chains of the t-cell receptor may greatly reduce the gvhd risks, while allowing the maintenance of mature donor-derived alloreactive nk cells and / (+) t cells, which may augment the anti-leukemia effect.objectives: this is a prospective study of caya with acute leukemia who underwent hsct with mmrd or urds and tcr / /cd depletion. outcomes included engraftment, toxicities, viral reactivation, and relapse.design/method: this study included caya with acute leukemia transplanted between october and may . all received a myeloablative preparative regimen with targeted busulfan (n = ) or tbi ( cgy/ fractions) (n = ), with thiotepa ( mg/kg) and cyclophosphamide ( mg/kg). atg ( mg/kg x ) was given to those receiving haploidentical grafts and to the first who received urd grafts. immune suppression was not given post-hsct. the stem cell source was mobilized peripheral blood stem cells (pscs), which then underwent tcr / /cd depletion utilizing the clinimacs device under gmp conditions in the chop cellular immunotherapy lab.results: median age was (range . - . ). diagnoses included all ( -b-cell, -t-cell) and aml ( ; -secondary aml). urd were used for ; were / allele matched and were / matched. haploidentical donors were used for . median cd (+) dose - . × , / (+) cd (+) cells - . × , and b cells - . × . all patients achieved an anc at a median of d+ ( - ), and % had platelet engraftment at median d+ ( - ). nine patients ( %) developed acute gvhd (all skin, grades i-iv). five developed chronic gvhd (skin, gut, lung): limited in , extensive in . viral reactivations included: adenovirus ( , %), bk virus ( , %), cmv ( , %), and hhv ( , %). nine ( %) patients relapsed at a median of days (range - ) post-hsct, including aml patients ( . %) and all patients ( . %). transplant-related mortality was %; causes included sepsis ( ) and ards ( ). os was %; efs was % (gvhd-free efs %, lfs %). hsct with tcr / /cd depletion demonstrates excellent engraftment kinetics with limited gvhd without immune suppression. elimination of post-hsct immunosuppression may offer an excellent platform to augment anti-leukemic immune therapy or to enhance immune reconstitution. background: hematopoietic cell transplantation (hct) is the only curative treatment available for patients with sickle cell disease (scd). low bone mineral density (bmd) has been described in scd, but little is known about the impact of curative hct on this outcome. to determine the prevalence of low bmd and variables associated with low bmd in scd patients after hct. we conducted a retrospective chart review of scd patients who underwent hct at children's healthcare of atlanta (choa) between / and / and survived ≥ year post-hct. transplant characteristics, post-hct dual-energy x-ray absorptiometry (dexa) scan results, vitamin d levels, graft-versus-host-disease (gvhd) status, and fsh levels were reviewed. for patients - years of age, height corrected z-scores were calculated using a nihvalidated calculator, with t-scores used for older patients. bmd was categorized as low if between - and - sd below the mean and clinically significantly low if >- sd, in accordance with the children's oncology group long-term follow-up guidelines. vitamin d levels < ng/mol were considered deficient, and fsh levels > miu/ml suggestive of premature ovarian failure. fisher's exact test was used to compare variables in those with normal versus abnormal dexa scan results, with p< . considered significant.results: hct was performed on patients with scd, with surviving ≥ year post-hct. dexa scans were obtained in patients ( % female), with mean time from hct to dexa scan being years ( . - . years) and mean age at time of dexa . years ( . - . years). patients with and without dexa scans did not differ by sex, donor source, age at transplant, or vitamin d status. low bmd was noted in patients ( . %), with these patients more likely to be > years (pubertal; . versus . %, p = . ). acute gvhd was more common in patients with low bmd ( . versus . %), but not statistically significant (p = . ). clinically significant low bmd was noted in patients ( . % of those with dexa scans). these patients were older ( . years at testing), were more likely to be male ( . %), and all had acute and chronic gvhd, while none had evidence of gonadal failure.conclusion: clinically significant low bmd is uncommon after hsct for scd. patients at risk for low bmd include older patients and likely those with gvhd. this preliminary data suggests routine dexas may not be indicated for all patients who undergo hct for scd, but further data is needed. background: causes of renal dysfunction after hematopoietic cell transplantation (hct) include damage from radiation, nephrotoxic medications, graft vs. host disease (gvhd), hepatorenal syndrome, viral infections, or transplant associated microangiopathy. we sought to investigate the incidence of, and risk factors for, acute kidney injury in pediatric hct patients and associated risk with mortality.design/method: data from patients who underwent hct between and at a single institution were sequentially retrospectively captured on irb approved protocol. acute kidney injury (aki) was defined at multiple time points post-hct using the standardized criteria: kidney disease: improving global outcomes (kdigo). interval differences between values were analyzed using wilcoxon rank sum testing and categorical variables were analyzed using chi-square analysis.results: ninety-eight patients were included in the study: allogeneic (n = ) and autologous (n = ), mean age . years, of whom % were african american, % asian, % caucasian, % latino, and % mixed race. forty-seven percent of patients developed aki within the first years of hct. increased risk for aki was associated with a lower pre-transplant creatinine level (p = . ), abnormal pretransplant bun (p = . ) and an unrelated donor (p = . ) while preparative regimen intensity, race, or primary disease were not. twenty-six percent of patients developed aki within days of hct. of those with aki, % were exposed to either cidofovir, aminoglycosides, and/or ambisome for at least days versus % without aki and % were exposed to vancomycin compared to % without aki. evaluating outcomes at year after hct, of those with stage aki: % had reduced gfr and % died, while % had reduced gfr and % had died for patients with aki stage or . the absence of aki by day was associated with % reduced gfr and % death at -year after hct. overall, those with aki at any time in the first year post-hct had a . fold increased risk of death compared to those without. for patients who required renal replacement therapy (rrt, n = ), the risk of death was . fold greater compared to those who did not. in the % of patients who survived rrt, both recovered renal function within years.conclusion: acute kidney injury is common after pediatric hct, and may be associated with low creatinine, abnormal bun, unrelated donor pre-hct, and renal toxic medications. early-onset aki post hct is associated with an increased risk of mortality. these data should be validated in a larger prospective study but may offer opportunities to intervene and enhance outcomes. background: myeloablative hematopoietic stem cell transplant (hct) for pediatric malignant disease is associated with significant morbidity with % patients experiencing mucositis. patient controlled analgesia (pca) utilizing opioids is an effective strategy for pain management. we sought to describe and analyze pca use in d+ days post myeloablative hct for malignancies at lurie children's hospital of chicago from - .design/method: utilizing retrospective chart review, pca details were collected: indication, initiation day, pca duration, team managing pca (anesthesia or palliative), medication and dose in morphine equivalents, and pca toxicities. efficacy of pca was evaluated on pca day + , + , + , + using demands %, maximum pain score (rflacc, faces, vas) and subjective patient, parent and/or pain team perception of pain control. we devised a scale based on the above to designate pain control as good, moderate or poor. variables being analyzed include recipient age, sex, donor type, source, diagnosis, tbi use, gvhd/trm. this analysis, we analyze the depth of remission, car-t persistence, and post-transplant gvhd on our phase i anti-cd car-t protocol (nct ) to better understand the role of car-t in the peri-hct setting.design/method: children and young adults with relapsed/refractory cd + all treated on our phase i anti-cd car-t protocol were analyzed. mrd was assessed by flow cytometry (fc) in all, with pcr-based mrd analysis using igh or tcr testing assessed in select patients. hcts were performed at each patient's local institution based on standard of care and included varying conditioning regimens, donor types, stem cell source, and gvhd prophylaxis.results: on our cd car trial, patients were treated, the majority of patients (n = ) having relapsed following a prior hct. / patients ( %) attained a cr, of whom were mrd negative by fc. concurrent pcr based mrd analysis available in patients demonstrated that all patients achieved pcr based negativity. in , this was simultaneous with the month mrd negative fc, and in , pcr negativity was achieved over time (fc remained negative). patients proceeded to hct at a median time of days (range: - days) post-car-t, which was a first hct in . these two patients remain in an mrd negative cr, year post-car-t. no patients developed acute or chronic gvhd. car persistence was seen in patients who had detectable car-t cells on the pre-hct marrow suggesting the possibility of ongoing anti-leukemia surveillance prior to initiation of the conditioning regimen.conclusion: by inducing pcr negativity, car-t therapy may have a synergistic role with hct to improve leukemia free survival, prior to emergence of antigen negative leukemia, without an increased risk of gvhd. while the sample size is small, car-t therapy may offer an effective bridge to hct, particularly for those who are pcr negative, and those who have not had a previous transplant. given the underlying risk of hct related trm, pre-hct car may potentially allow for hct conditioning de-intensification as it may not be needed to eradicate residual disease. lee dw, ash abstract , background: post-transplant lymphoproliferative disease (ptld) is a complication after solid organ transplantation (sot) that is frequently due to epstein -barr virus (ebv) as a decrease in ebv-specific t cell immunity due to immune suppression allows for uncontrolled proliferation of ebv-infected b cells. outcomes for ptld are suboptimal with relapse rates approaching %. however, ebv-infected b cells in ptld express the ebv antigens lmp and lmp that can be targeted with immune therapy.objectives: we hypothesize that third party "off the shelf" lmp-specific t cell products may improve outcomes and decrease associated co-morbidities for patients with ptld by not only target the lymphoproliferating ebv-infected b cells but also restoring ebv-specific immunity.design/method: lmp-specific t cells (lmp-tcs) are manufactured from eligible donors with a broad range of hla types in our gmp facility to be used in a children's oncology group (cog) trial (anhl ) for patients with ptld after sot. lmp-tc products are manufactured from healthy donors using autologous monocytes and lymphoblastoid cell lines (lcl) transduced with an adenoviral vector expressing Δlmp and lmp as antigen presenting cells. lmp-tc products undergo comprehensive characterization by ifn-elispot assay to determine lmpspecific epitopes, class i and/or ii response, and hla restriction to guide selection of lmp-tc product for each patient.results: thus far, lmp-tc products have been manufactured. lmp-tcs were active against lmp (mean: sfu/ × ^ cells; range: - ), lmp ( ; - ), and lcl ( ; - ) as determined by ifn-elispot assay. at the time of cryopreservation, the lmp-tc products comprised a mean of % cd + t-cells, % cd + t-cells, and % nk cells. no b cells or monocytes were detected in the final products. thus far, we have identified novel lmp epitopes (lmp specific: n = ; lmp specific: n = ). approximately % of the lmp-tc products have lmp-specific activity through multiple hla alleles, and % have a mixed class i and class ii response. conclusion: thus, lmp-specific t cell products can be expanded from healthy donors to creat a third party bank, and identifying epitopes and hla alleles with lmp activity will facilitate selecting the most appropriate product for patients. while lmp-specific t cells have previously demonstrated safety and efficacy in phase i studies, anhl is the first trial using cellular therapy within a cooperative group setting. children's cancer hospital at the university of texas md anderson cancer center, houston, texas, united states background: in , the united states food and drug administration (fda) approved the first chimeric antigen receptor t cell (car-t) therapy; tisagenlecleucel. this cd -directed genetically modified autologous t cell immunotherapy has shown response rates of almost % among children and young adults with b-cell precursor acute lymphoblastic leukemia (all) that are refractory or in second or later relapse. cytokine release syndrome (crs) and car-t cell related encephalopathy syndrome (cres) are well described toxicities associated with car-t therapy. crs is a systemic inflammatory response and is typically characterized by fever, hypoxia, tachycardia, hypotension and multi-organ toxicity. cres may occur concurrently or following crs, or without any associated crs symptoms and is characterized by encephalopathy, delirium, seizures and rarely cerebral edema. almost half of patients who receive tisagenlecleucel may require pediatric intensive care unit (picu) support. crs and cres are generally reversible but may be associated with fatal outcomes. pediatric specific management guidelines, comprehensive training of multidisciplinary staff, effective communication and phased infrastructure ensure that adequate resources are available to facilitate early diagnosis and appropriate management of pediatric patients with crs and cres and allow for optimal patient outcomes and accreditation by the foundation for accreditation of cellular therapy (fact).objectives: develop a comprehensive program to ensure safe administration of immune effector cell (iec) therapy to pediatric patients.design/method: an inter-disciplinary pediatric cartox (car t cell therapy associated toxicity) committee consisting of cell therapy and picu physicians, neurologists, fellows, nursing leadership, advanced practice practitioners, pharmacists, registered nurses and social workers was created to monitor patient toxicity and establish specific clinical guidelines and diagnostic and treatments algorithms for pediatric patients receiving iec therapy. educational modules were developed as (i) live in-services and (ii) an online module with a competency based assessment. electronic medical record (emr) order sets and documentation and warning systems were also developed by the committee. the pediatric cartox committee developed a diagnostic and treatment algorithm for patients receiving iec therapy. emr orders and flowsheets were developed to support adherence to the algorithm. inter-disciplinary staff training and competency assessments were closely tracked. almost % of identified staff have completed training and achieved competency including, pediatric cell therapy staff, emergency center, picu, outpatient clinic/triage, neurology and sub-specialty staff and nocturnalists.conclusion: an inter-disciplinary approach can assist in institutional readiness for an iec program, promote quality assurance and perhaps fact iec accreditation. future directions include a program for ongoing staff competency assessments. predicted peak vo ) and abnormal oxygen uptake efficiency slope at the anaerobic threshold ( . ± . . , normal ± ). additionally, on echocardiogram three patients had evidence of diastolic dysfunction as evidenced by an elevated e/a ratio ( . ± . ) on tissue doppler. three patients demonstrated depressed longitudinal peak systolic strain (- . ± . ), indicating dysfunction not captured by ejection fraction. in this feasibility study, sct patients without evidence of lvsd on standard measures by resting echocardiogram can demonstrate abnormal exercise capacity. additionally, they can demonstrate systolic and diastolic dysfunction by measures not always included in standard echocardiography. these data suggest the need for a more thorough screening of survivors, and will be further validated as additional patients are recruited for this study. background: in hematopoietic transplantation, the t lymphocytes of the inoculum play a determining role in promoting hematopoiesis, transferring immunity to pathogens and acting as mediators of the graft-versus-leukemia effect (gvl). however, they are also responsible for graft-versus-host disease (gvhd), the main cause of post-transplant morbidity and mortality. the depletion of cd ra lymphocytes, by eliminating naive t lymphocytes from the inoculum, aims to conserve the gvl without producing gvhd.design/method: since april , patients ( boys and girls), with a median age of years, have undergone an allogeneic hematopoietic transplant from an hla donor identical with cd ra/cd depletion. the indication for transplant was: acute lymphoblastic leukemia ( ), acute myeloblastic leukemia ( ), myelodysplasia ( ) and medullary aplasia ( ). the donor was familiar in cases and unrelated in . the conditioning regimen was with fludarabine, busulfan and thiotepa. the median of cd + cells infused was . × / kg. on the day , + and + a programmed infusion of × / kg lymphocytes cd ra-was performed.results: all the patients grafted with a median leukocyte (> . × / l) and platelet (> × / l) engraftment time of and days, respectively. only one patient has developed acute gvhd grade i and no patient has developed chronic gvhd. immune reconstitution was early and rapid in all t cell subsets no patient has relapsed so far and only patient with myelodysplasia has developed an aml. she has received a nd transplant and has died of relapse. there was no case of toxic mortality. the event-free survival (sle) was ± % with a median follow-up of months. at present, patients are alive, out of immunosuppressive treatment and doing well. allogeneic transplantation with cd lymphocytes ra depletion resulted on very encouraging results, with a very low incidence of acute and chronic gvhd, but preserving the gvl effect by infusing cd ra-donor lymphocytes. miami children's health system, miami, florida, united states background: hematopoietic stem cell transplantation (hsct) using autologous or allogeneic progenitor cells is a potentially curative treatment for patients with high-risk malignancies and nonmalignant conditions. the american society for blood and marrow transplantation developed a task force to establish consensus guidelines for defining patient care in hsct and advocated for further studies to delineate safe procedural steps as an increasing amount of hsct are being offered to patients. there is limited evidence to support engraftment in recipients who receive their infusions via iv or syringe pump. we present novel data from patients who achieved neutrophil engraftment following hsct by a pump mechanism.objectives: to provide evidence supporting the use of pump (intravenous or syringe) infusion method in hematopoietic stem cell transplantations.design/method: a retrospective review was completed for patients who underwent hsct between and . inclusion criteria included patients who had received hematopoietic stem cell transplants between and and who were ages months to years old. the main outcome measure was days to neutrophil engraftment (defined as the first of three consecutive days with an anc > × /l).results: among patients who received infusion of hematopoietic stem cell products via pump mechanism, patients ( . %) received autologous products and ( . %) received stem cells from allogeneic donors. neutrophil engraftment (anc > × /l) occurred in a median of . days after stem cell infusion. the mean number of days to engraftment for patients who received allogeneic infusions s of s design/method: a retrospective chart review was performed at the children's hospital of wisconsin. statistical analyses included kaplan-meier estimate for os, mann-whitney test for comparing outcomes between subjects, and descriptive analyses.results: from - , patients with a median age of . ( . - . ) years at st hct were identified. patients were conditioned with cy/atg (n = ), cy/flu/atg (n = ), or cy/flu/atg/tbi (n = ) and received marrow (n = ) or cord blood (n = ) with median cd /kg dose of . ( . - . ) × . two patients developed grade i acute graftversus-host disease (gvhd); none developed chronic gvhd. due to dropping chimerism, graft rejection, or graft failure, nd hct (n = ) or boost (n = ) was offered. the median cd chimerism prior to hct/boost was ( - )%. median time between st hct and nd hct or boost was days ( days- . years). in patients receiving nd hct, used the same donor, of which used the same stem cell source (marrow) and switched to peripheral blood stem cells (pbsc). in patients who switched donors, used pbsc and used cord blood. most patients receiving nd hct underwent a uniform conditioning regimen of cy /flu /equine atg/ gy tli (n = ) or cy /flu /rabbit atg/ gy tbi (n = ); one received cy/atg. acute and chronic gvhd (limited seen in %) developed in % and % of patients, respectively. four patients required additional boosts and additional hct. after final intervention, cd and whole-blood chimerism at last follow-up was between - % (n = ) and - % (n = ), respectively. with a median follow-up of . ( . - . ) years, of patients are alive with an estimated -year os of . ± . %, having performance status ≥ % (n = ) or % (n = ). one patient developed chronic extensive gvhd and died of fungal infection . years after nd hct. our single-center experience demonstrates excellent ability to salvage patients who develop graft failure after initial hct. transplant-related complications such as gvhd and infections remain significant concerns. key: cord- -uj fe y authors: nan title: scientific abstracts date: - - journal: reprod sci doi: . / sha: doc_id: cord_uid: uj fe y nan by reduced placental oxygenation, hypoxia-induced oxidative stress is a predominant mechanism. we investigated the effect of hypoxic pregnancy, with and without antioxidant treatment, on placental and maternal circulatory indices of oxidative stress in rats. methods: on pregnancy day , wistar rats were randomised into: normoxia ( % o litters), hypoxia ( % o litters) and hypoxia + vit c ( % o + mg. ml - vit c in water, litters). on day , dams were anaesthetised, maternal blood taken, pups measured and weighed, placentae weighed and frozen. only placentae from two male pups from any one litter were investigated. blood was processed for ascorbate, urate, l-cysteine and glutathione (gsh) measurement. placental protein was analysed for heat shock protein (hsp ; western). results: hypoxia + vit c did not affect maternal food or water intake. vit c elevated maternal ascorbate by % of baseline; a similar increment to human trials (poston et al. ) . hypoxia elevated placental hsp and maternal plasma urate and l-cysteine, but decreased gsh. vit c in hypoxic pregnancies prevented all stimulated effects, but the reduction in gsh persisted. hypoxic pups had a reduced ponderal index and elevated head diameter: body weight ratio; effects also prevented by vit c. fetal oxygen uptake in normal and gdm pregnancies. emanuela taricco, tatjana radaelli, veronica cozzi, gabriele rossi, danila puglia, giorgio pardi, irene cetin. department of obstetrics gynecology "l. mangiagalli", irccs policlinico, mangiagalli e regina elena, milan, italy. background. diabetes in pregnancy has been associated with alterations of fetal growth probably due to increased nutrient availability and placental transport. fetal hypoxia and acidemia have been reported in pregestational diabetic pregnancies with poor glicemic control but this is still uncertain in well controlled patients. the role of placental function and the relationship between maternal and fetal circulation is crucial for efficient exchanges of oxygen and nutrients. since umbilical blood flow can be obtained by us in utero, we studied normal and gdm pregnancies in order to evaluate fetal oxygen uptake. methods. normal (n) and gdm pregnancies were studied at term, at the time of elective caesarean section. umbilical vein volume flow (qumb) was measured by us before caesarean section and blood samples from umbilical vein (uv) and artery (ua) were obtained. blood gases and acid-base balance were evaluated. results. average fetal weights were similar in both groups (n= ± ; gdm= ± g) while placental weights were significantly different (n= ± ; gdm= ± g). n and gdm pregnancies showed similar values of qumb and qumb/kg of fetal weight. (qumb: . ± . in n and . ± . ml/min in gdm; qumb/kg: . ± . in n and . ± . ml/min/kg in gdm). in fetuses from gdm pregnancies a significant reduction in o sat, o cont and po and a significant increased lactate conc was found in both uv and ua compared to n (table ) . o umb uptake (n= . ± . ; gdm= . ± . mmo/l/min) and o umb uptake/kg (n= . ± . ; gdm= . ± . mmo/l/ min/kg) were significantly lower in gdm compared to n fetuses. conclusions. our data indicate that fetuses from gdm pregnancies show a significant reduction in oxygen supply despite a normal blood flow/kg of fetal weight. these data may suggest that a good maternal metabolic control is not sufficient to ensure normal placental oxygen supply and/or utilization by the gdm fetus. background: synthetic glucocorticoids (sgc) are given to mothers at risk of preterm delivery to promote fetal lung maturation. evidence is emerging indicating long-term effects of such treatment on endocrine function and behavior in offspring. however, virtually nothing is known concerning potential transgenerational influences on growth, endocrine function and behaviour. we hypothesize that repeated treatment of grandmothers (f ) with sgc will alter hypothalamic-pituitary-adrenal (hpa) function in f offspring with no manipulation of the f pregnancy. methods: pregnant guinea pigs (f ) were subcutaneously injected with betamethasone (beta; mg/kg) or vehicle (veh) on gestational days / , / / . adult f female offspring from each group were mated with control males. hpa function was assessed in adult f offspring by non-invasive measurement of salivary cortisol concentrations: ) under basal conditions, ) during and following exposure to psychological stress (high frequency strobe light) or psychological/physical stress (forced swim) and, ) following dexamethasone suppression. results: there was no effect of beta (f ) on bodyweight from birth to adulthood in f offspring. basal salivary cortisol in the betaf females was lower than in the vehf group in the morning but not the afternoon; there were no differences in male f offspring. in contrast, both male and female betaf failed to mount adrenocortical responses to psychological stress compared to vehf offspring that mounted robust responses (p< . ). swim stress induced robust adrenocortical responses in all groups (p< . ), however, the response was consistently lower in betaf offspring. beta exposure also led to a significant difference (p< . ) in the cortisol response to dexamethasone suppression in female (f ) offspring, with a similar trend in males. conclusion: prenatal exposure to sgc (f ) causes transgenerational programming of adrenocortical function in adult f offspring. grand maternal exposure to beta results reduced basal adrenocortical activity in betaf female offspring, and caused stress hypo-responsiveness in both males and females. dexamethasone suppression tests indicate altered central glucocorticoid feedback. these findings have important ramifications for the management of human preterm labor. support: canadian institutes of health research. in the uterine and umbilical vasculatures, we hypothesized that their remodeling would also be blunted in pregnant enos -/-mice, leading to an elevated vascular resistance and decreased blood flow to the placenta contributing to fetal growth restriction. methods: utero-and umbilical-placental blood velocity waveforms and umbilical arterial diameters were measured using mhz ultrasound biomicroscopy in control (c bl/ j) and enos -/-mice at . days of pregnancy (n= mothers). spiral artery and fetal capillary morphologies, and uterine arterial diameters were evaluated from vascular corrosion casts. tissues were collected for hydroxyprobe- and actin immunohistochemistry to identify hypoxic and smooth muscle regions. we calculated resistance index ((s-d)/s) from systolic (s) and diastolic (d) velocities, and blood flow from mean velocity and vessel area. results: calculated uterine blood flow normalized to the weight of the uterus and its contents was % lower (p< . ) in pregnant enos -/-mothers due to large reductions in uterine artery diameter (- %, p< . ) and mean velocity (- %, p< . ). uterine arterial resistance index was % higher (p< . ), and the spiral arteries were less coiled and contained more smooth muscle actin in enos -/-mice than controls. more intense hypoxic immunoreactivity was detected in the spongiotrophoblast and trophoblast giant cell layers of the junctional zone of enos -/-placentas, whereas fainter staining was only detected in the spongiotrophoblast cell layer in controls. in the umbilical circulation, flow normalized to fetal weight was not significantly changed although the resistance index was slightly elevated ( %, p< . ) and capillary lobule length was reduced by (- %, p< . ). fetal organs showed increased hypoxic immunoreactivity suggesting reduced organ oxygen delivery in enos -/-fetuses. conclusions: results suggest that enos plays an important role in uterine and spiral artery remodeling and in augmenting utero-placental blood flow during pregnancy. it also appears to enhance oxygen delivery to the placenta and fetus. enos may contribute to normal fetal growth by these mechanisms. integrin methods: hpmcs isolated from term placentas were assessed for their phenotype markers, mutilineage capacity, and the expression of integrin molecules. the hpmcs were induced to endothelial cell differentiation in the presence of endothelial cell growth medium with % of fcs and ng/ml vegf for to days. the angiogenesis ability of these cells was demonstrated by using an in vitro angiogenesis kit and in vivo chick chorioallantoic membrane assay from ten-day-old embryos. blocking antibodies specific to integrin , associated with gdm. this study was adequately powered to detect association in the caucasian and asian group. the absence of association suggests that gdm and t dm may have more divergent molecular pathophysiology than previously suspected. background: uterine endometrium has a unique cycle of physiological angiogenesis. in mice, endothelial progenitor cells (epc) contribute to endometrial angiogenesis being incorporated after oestradiol administration. objective: to determine whether circulating © epc number and function vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: ten healthy, nulliparous, pre-menopausal, non-smoking women (mean age years) with regular menses ( - days) were studied. venous blood was collected during menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - , - ) . cepcs, serum oestradiol (e) and progesterone (p) were measured at each phase. cepcs were quantified by phenotype using flow cytometry (leukocytes co-expressing cd , kdr and cd ) and function by the epc-colony forming unit (cfu) assay. epc-cfus were stained for endothelial markers including uptake of acetylated low-density lipoprotein, binding of lectin (ulex europaeus) and endoglin. results: luteal p was > nmol/l in all women. cepc numbers increased in the menstrual and follicular phases being -fold higher in the follicular compared to periovulatory phase (p< . ). there was no significant variation in cepc function over the menstrual cycle. there was no correlation between serum e or p levels and cepc number or function. conclusion: cepcs number but not function (epc-cfu assay) vary during the menstrual cycle with numbers increasing during the menstrual and follicular phase and falling in the periovulatory and luteal phase of the menstrual cycle. this may represent mobilisation of cepcs from the bone marrow and subsequent incorporation into the endometrium. neither function nor cepc number correlate with serum p or e. our previous studies demonstrated that tissue factor (tf), which binds factor vii to act as a potent pro-coagulant and angiogenic factor, is over-expressed in endometriotic lesions. thus, we determined whether tf could serve as a target for the elimination of pre-established ectopic human endometrial growth in a mouse model. icon is composed of a mutated factor vii (fvii) domain targeting tf and an igg fc (fvii/igg fc) effector domain that activates antibody-dependent immune responses against tf bearing endothelial cells. methods: athymic, ovariectomized and estrogen-treated mice received intraperitoneal (i.p.) injections of . mg of proliferative phase human endometrial tissue derived from normal (disease-free) women. twelve days after inoculation to establish lesions, icon protein ( ug) was delivered i.p. once a week for weeks. after sacrifice, animals were subject to gross inspection. residual endometriotic tissue was formalin fixed, paraffin embedded and immunostained for von willebrand's factor (vwf) and tf. results: compared to control mice, treatment with ug of icon abolished all lesions in of mice and reduced both size ( . to . mm) and number of lesions ( . to per diseased mouse) in the remaining mice. moreover, residual lesions from icon treated mice were atrophic and displayed significant reductions in vessel areas of % +/- % (p= . , mean +/-sem, n= ) as determined by vwf immunostaining. no hemorrhagic or thrombotic sequelae were observed in icon treated mice. conclusions: unlike other treatments that target developing angiogenesis, icon can target both developing and established human endometriotic lesions in athymic mice. the gross and microscopic vessel analysis suggests that icon directly or indirectly destroys endometriotic vessels. thus, icon presents a novel, non-toxic therapy for endometriosis. compared to the miscarriage and top groups. ihc for crisp demonstrated increased secretion of crisp in the glandular epithelium and expression in the leucocytes of the tubal uterine decidua. conclusions: there are differences in decidual gene expression in tubal compared to iu pregnancies. we believe that potential biomarkers of tubal pregnancy can be discovered by focusing on secreted proteins associated with uterine decidualization. one of these proteins, crisp , is significantly increased in decidua of tubal ectopic pregnancies and we are currently investigating its expression pattern in sera from women with tubal compared to iu pregnancies. progesterone and hoxa regulate gaba-a pi receptor expression, membrane translocation and activation. homayoun sadeghi, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale school of medicine, new haven, ct, usa. objective the expression of the gaba-a pi receptor has been previously described in the human endometrium in both luminal epithelium and stroma. it is upregulated during stromal decidualization in the rat and in the implantation window of human endometrium. the gaba receptor is modulated by progesterone metabolites, with the resultant opening of the receptor ion channels which allow the water flux necessary for trophoblast attachment. here we identified regulators of pi subunit receptor gene expression and activity. the well-differentiated human endometrial adenocarcinoma cell line (ishikawa) and human endometrial stromal cells (hesc) were transfected with hoxa , treated with progesterone, or treated with vehicle or empty plasmid controls. gaba-a pi receptor mrna upregulation was evaluated by real time rt-pcr. protein expression was evaluated using immunohistochemistry. results gaba-a pi receptor mrna expression was increased with either progesterone treatment ( %, p= . ) or hoxa transfection ( %, p= . ). coadministration of progesterone along with increased hoxa transfection had no additive effect on the expression of gaba-a pi receptor mrna (p= . ). gaba-a pi receptor protein expression was similarly increased by each treatment. either hoxa or progesterone independently caused translocation of the gaba receptor from the cytoplasm to the cell membrane in ishikawa cells. conclusion gaba-a pi receptor expression is increased in the human luminal epithelium and stroma in the window of implantation. activation of the pi subunit leads to opening of ion channels, likely allowing flux of water into the epithelial cells and out of the uterine lumen. progesterone and hoxa each increase both pi subunit receptor expression and membrane translocation. the lack of additive effect suggests progesterone induced pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of hoxa expression. finally, after receptor expression and translocation, progesterone mediated gaba receptor ion channel activation mediates water resorption necessary for implantation. cancer. suzy davies, donghai dai, kimberly k leslie. obstetrics and gynecology, university of new mexico, albuquerque, nm, usa. objectives: endometrial cancer is the most frequent gynecologic cancer in women. it affects an estimated , women in the us every year, and long term outcomes for patients with advanced stage or recurrent disease are poor. targeted molecular therapy against the vascular endothelial growth factor (vegf) and its receptors constitute a new therapeutic option for patients that is now under study by the gynecologic oncology group in a phase trial, gog e (now in second stage accrual). the goal of our work was to assess the potential effectiveness of vegf/vegfr blockade in preclinical endometrial cancer models. methods: these studies employed two agents, bevacizumab (avastin, a vegfa blocking antibody) and vandetanib (zactima, a tyrosine kinase inhibitor of egfr and vegfr ). ic experiments were performed on endometrial cancer cells in culture using four established cell line models. xenografted athymic mice were also employed to test the ability of compounds to inhibit tumor growth in vivo. tumors were isolated from controls and treated animals, mrna was extracted, and affymetrix gene expression arrays were performed to determine the genes consistently modulated by treatment. results: compared to vandetanib with an ic of . m, bevacizumab showed little activity on cell proliferation in vitro, and cell numbers were not reduced by % using concentrations up to . m. however, bevacizumab demonstrated robust activity in the athymic mouse model, resulting in a significant decrease in tumor formation and growth compared to vehicle treated animals when dosed bi-weekly in a concentration of . mg/mouse ip. tumors from this model demonstrated that eighteen genes were consistently up or down regulated in the presence of bevacizumab. among the regulated transcripts was microrna , which was significantly down-regulated. microrna is an anti-apoptotic factor, and its inhibition by bevacizumab predicts for increased expression of the tumor suppressor pten, decreased cell proliferation, and a reduced capacity for metastasis. conclusions: these studies confirm that the vegf pathway is a good target for new therapies against endometrial cancer. blocking this pathway not only inhibits angiogenesis, but also results in changes in gene expression that enhance apoptosis and reduce cellular proliferation and tumor invasion. we collected fibroid and adjacent normal tissues following hysterectomy with patient consent and institutional irbs. to date, data points for each gene from arrays have been collected from patients. the fluorescence readings were log-transformed and normalized with the robust quartile normalization method. quality control of the normalized data was performed to remove arrays that deviated from twice the inter-quartile range calculated from the array signals. results: the custom microarray profiling identified genes that were differentially expressed between normal and fibroid tissues (p < . ; fdr< . ). among them, genes encode receptor tks, genes encode tk ligands, and genes encode cell cycle and apoptosis proteins. clustering analysis of the mean log ratios of these genes has led to the division of the patients into two major groups. thirty four ( %) belong to a group characterized by the significant downregulation of the cyr (ccn ) gene in fibroid tissues. the cyr -down group can be further divided into two sub-groups based on the expression of another tk ligand, efn a. other receptor differentially expressed tk did not segregate with the three defined sub-groups. finally, there was no sub-group segregation based on age of the patient, menstrual phase, or weight of the fibroid. conclusion: our results have demonstrated that tyrosine kinases and their ligands are uniquely differentially expressed between normal and fibroid tissues. unique tyrosine kinase ligands in our population were cyr and efn a, and these markers created three molecular classification groups based on their differential expression. these results support the hypothesis that tyrosine kinase ligands are involved in fibroid growth, and may offer targets for a strategy to avoid hysterectomy. microvascular perfusion sonographic imaging to detect early stage ovarian cancer. joanie mayer hope, arthur c fleischer, brian day, stephanie v blank, bhavana pothuri, robert wallach, john p curtin, david a fishman. gynecologic oncology, new york university school of medicine, new york, ny, usa; radiology, vanderbilt university medical center, nashville, tn, usa. objective: epithelial ovarian cancer (eoc) is the th leading cause of death in us women due to the inability to detect early stage disease. recent sonographic developments involving harmonics, pulse inversion, and the use of contrast agents justify the hope that depiction of aberrant tumor microvascularity associated with early disease can occur. this study utilizes these new techniques to assess the unique microvascularity associated with early stage eoc. methods: we used pulse inversion harmonic microvascular imaging (mvi) technology to depict differences between benign and malignant ovarian lesions. contrast enhanced harmonic transvaginal (tv) sonography was performed using the philips iu scanner after intravenous injection of g of definity (bristol-myer-squibb). split screen real-time images were acquired displaying conventional sonographic views adjacent to harmonic, low mechanical index images. morphologic features (thickened wall, papillary excrescence, calcifications) and aberrant vascularity were noted. q-lab quantification of wash-in, peak enhancement, and wash-out times as well as area-under-thecurve (corresponding to microvessel perfusion) were compared using student's t-tests. results: to date, contrast enhancement patterns of ovaries have been analyzed, benign and malignant. of the malignancies ( fallopian tube, ovarian: stage i, stage iii), / women were correctly identified using conventional tv imaging and others were identified using mvi / . all benign lesions were correctly identified by mvi / while tv detected normals ( false negatives). the lesions detected as malignant by mvi were -stage i fallopian tube and -stage i eoc. contrast enhancement kinetics of malignant lesions demonstrated similar wash-in ( . ± . vs . ± . , p = . ), greater peak enhancement ( . ± . vs . ± . , p < . ), longer wash-out ( . ± . vs . ± . (p < . ), and greater perfusion ( . ± . vs . ± , p < . ) when compared to benign lesions. conclusion: contrast enhancement patterns are significantly different in benign vs. malignant ovarian masses. this technique has clear potential in differentiating benign from malignant lesions and for detecting occult stage i disease. identification and characterization of mir- a as regulator of ikk expression and its function in ovarian cancer cells. rui chen, ayesha b alvero, thomas rutherford, gil mor. department of obstetrics and gynecology, yale university school of medicine, new haven, ct, usa. introduction: the proinflammatory environment associated with tumor growth and chemoresistance is produced by both immune cells and cancer cells. the nf-b pathway plays a critical role mediating the capacity of cancer cells to produce pro-inflammatory cytokines. recently, we described a group of epithelial ovarian cancer (eoc) cells characterized by ikk expression as the main factor promoting nf-b activation and cytokine production. in this study we evaluated the regulation of ikk in eoc cells. we describe the identification and characterization of mir- a as a regulator of ikk expression, thus indirectly of nf-b activity and function. materials and methods: human eoc cell lines were established from malignant ovarian cancer ascites. protein expression were determined by western blotting. ikk mrna was measured by rt-pcr. cytokines were profiled by the luminex system. ikk transfection was done with roche fugene transfection reagent. mirna microarray was done with invitrogen ncode multi-species mirna microarray kit. mir- a qrt-pcr was performed with invitrogen ncode sybr greener mirna qrt-pcr analysis kit. mirna transfection was done with ambion siport neofx agent. the ikk '-utr luciferase reporter plasmid was established based on ambion pmir-report mirna expression reporter vector. results: ikk expression was associated with nf-b cyclic activity and the ability of type i eoc cells (but not type ii) to produce inflammatory cytokines. transfection of ikk into type ii eoc cells reversed their phenotype. mirna microarray identified mirnas differentially expressed in type i versus type ii cells, one of which, mir- a, had putative binding sites in the '-utr of ikk mrna. mir- a introduction into type i cells inhibited ikk expression, and direct inhibition through ikk 's '-utr was confirmed by luciferase assay. conclusion: we describe for the first time the identification of ikk as a potential key switch between chemo-resistant and chemo-sensitive phenotypes, by regulating nf-b activity in eoc cells. furthermore, we identified mir- a as a direct regulator of ikk expression. ikk expression may represent an adaptational stage in tumor progression allowing cancer cells to create their own inflammatory environment. these findings may provide novel molecular targets and potential markers for individual therapy selection. regulation of cd in the rat uterus by nitric oxide and the involvement of p kinase pathway. uma yallampalli, rebakah elkins, pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cd is expressed in many cell types including uterine cells and has been shown to play an important role in protecting against compliment attack. we have previously shown in endometrial cell lines that cd levels were down regulated by nitric oxide (no) . in this study we extend our previous observations to determine if no down regulates cd in rat uterine tissues and assess its mechanisms of action. methods. non pregnant rats ( g; b wt) were bilaterally ovariectomized under ketamine anesthesia. groups of ovariectomized rats were implanted with alzet mini pumps to deliver nitro-l-arginine melthylester (l-name) at or /mg/rat/day in saline or saline alone. uteri were obtained from these rats at or hours after infusion. in another set of experiments uteri were obtained from ovariectomized rats and cut in to small pieces and incubated in vitro with either l-name ( mm), diethylenetriamine-no mm) or worthmanin ( . m) in mem without phenol red for hours. uterine tissues were homogenized in trizol and mrna levels for cd were measured using rt-pcr and expressed as a ratio to s. results. results show that in vivo treatment with l-name to ovariectomized rats caused elevations in uterine cd mrna levels in a time and dose dependent manner with maximal responses seen with mg l-name and at hours. in vitro studies show that deta-no suppressed cd mrna levels in the rat uterus. both l-name and pi kinase inhibitor, worthmanin caused increases in cd levels in these tissues and the effects of worthmanin are reversed by deta-no. these results suggest that cd levels in the rat uterus are down regulated by no and are upregulated when no synthesis is inhibited by l-name. further, pi kinase appears to be involved in cd regulation in the rat uterus and no donor appears to modulate this response. lung. lakeitha r foster, daniel b hardy, carole r mendelson. dept of pediatrics; depts of biochemistry and ob/gyn, ut southwestern medical center, dallas, tx, usa. during % of human pregnancy, the maternal uterus is maintained in a state of almost complete quiescence by elevated circulating levels of progesterone (p ). we previously observed that p acting through the progesterone receptor (pr) serves an anti-inflammatory role and inhibits uterine contractility by antagonizing nuclear factor b activation and cyclooxygenase- (cox- ) expression (hardy et al., ) . our previous findings also suggest that the fetus provides an important signal for the initiation of labor near term through augmented secretion of the major lung surfactant protein, sp-a, into amniotic fluid (condon et al., ) . secreted sp-a, in turn, activates fetal macrophages which migrate to the maternal uterus where they release cytokines and promote an inflammatory response, leading to labor. in the present study, we tested the hypothesis that maternal p also maintains uterine quiescence by inhibiting sp-a production by the fetal lung. age matched icr mice were injected s.c. once daily either with sesame oil (control) or p ( mg/ml) from to days post-coitum (dpc). as expected, treatment with p delayed parturition by - h. this also was associated with a decrease in uterine cox- mrna expression, as compared to the vehicle-injected controls. interestingly, maternal p treatment caused a marked decrease in the levels of immunoreactive sp-a protein secreted by the fetal lungs into in amniotic fluid at dpc. furthermore, sp-a protein and mrna levels were reduced in the fetal lungs of p -injected mothers, as compared to controls. this was associated with an inhibitory effect of p treatment on cox- protein and mrna levels in the fetal lungs. these findings were of interest, since cox- expression is markedly upregulated during differentiation of human fetal lung (hfl) explants in culture (hardy et al., ) and endogenous and exogenous prostaglandins increase sp-a expression in hfl (acarregui et al., ) . collectively, these findings suggest that maternal p treatment prevents increased uterine contractility, in part, by inhibiting inflammatory response pathways within the fetal lung. this, in turn, blocks the developmental induction of sp-a expression and its secretion into amniotic fluid. in this manner, maternal p inhibits an important fetal signal leading to labor. supported by nih p hd ; nih r hl . recent evidence suggests that leukocytes infiltrate uterine tissues at the time of parturition implicating inflammation as a key mechanism of human labor. ccl- is a pro-inflammatory cytokine that may contribute to the development of inflammatory reaction in the myometrium. previously we showed upregulation of rat ccl- gene expression in myometrium during term and ru -induced preterm labor. also ccl- was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium. the objective of this study was to investigate the role of mechanical stretch as a possible regulator of myometrial leukocyte infiltration and ccl- as a mediator of this stretch response. we also studied the effect of progesterone (p ) on the myometrial secretion of ccl- . methods. we used primary culture of rat myometrium smooth muscle cells (smcs) to study in vitro ccl- gene and protein induction by static mechanical stretch. ccl- gene expression analysis was performed by real-time rt-pcr and immunoreactive (ir) protein content was measured by elisa assay. we used primary rat monocytes to access whether stretch-induced ccl- production by myometrial smcs resulted in enhanced monocyte chemotactic activity. results. myometrial cells were stretched for - hours and the supernatants collected. analysis of media conditioned by primary myometrial smcs revealed that static mechanical stretch ( % elongation for hours) caused a significant accumulation in ir ccl- which was repressed by pretreatment with p ( um). the rise in ccl- protein levels was preceded by a transient increase on ccl- mrna. the migration of primary rat monocytes in response to conditioned medium from stretched myometrial smcs was much greater than that of conditioned medium from control non-stretched cells. co-incubation with a neutralizing antibody to ccl- significantly reduced the chemotaxis of monocytes in response to the stretch-conditioned medium. conclusion: uterine smcs play an active role in uterine inflammation by producing chemokines and promoting the chemotaxis of immune cells into the myometrium. the blockade of this effect by p offers a potential explanation for the therapeutic actions of this hormone in the prevention of preterm birth. membranes during human labor. nardhy gomez-lopez, , guadalupe estrada-gutierrez, lourdes vadillo-perez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico; escuela nacional de ciencias biologicas, ipn, mexico city, df, mexico. introduction. leukocytes arriving to the choriodecidua (chd) during labor are capable to secrete cytokines and matrix metalloproteinases that may play a role in the fetal membranes (fm) extracellular matrix degradation. objective. the aim of this work was to identify changes in the leukocyte subpopulations in the chd and fm during human labor. methods: fm were obtained from two groups of women: ) term without labor (n= ) and ) term with spontaneous labor (n= ). chd cells were isolated and analyzed by flow cytometry. explants of fm were embedded in paraffin and analyzed by confocal microscopy. in both techniques, cd , cd , cd , cd and cd subpopulations of leukocytes were identified. intracellular mmp- was also identified in these cells. results: major changes in leukocytes subpopulations during labor involved a higher amount of cd +, cd+ and cd + cells, both in the chd and inside the fm. mmp- was associated to cd + cells. cd + exhibited a more widespread localization in the fm and cd + cells were localized in the contact with the trophoblast layer during labor. conclusions: leukocyte populations changes both in the chd and fm during labor and are characterized by arrival and infiltration of specific subpopulation of lymphocytes and monocytes. nk cells are enriched in mmp- , which may be related to a role in extracellular matrix degradation leading to the rupture of fetal membranes. women with a history of a pregnancy complicated by preeclampsia or intrauterine growth restriction (iugr) have an increased risk of future cardiovascular disease. excessive weight, particularly abdominal fat mass, is associated with cardiovascular morbidity and mortality. objectives: the aim of this study was to investigate differences in body composition and fat distribution between women with a history of preeclampsia or iugr and uncomplicated pregnancies. methods: from a genetically isolated population in the southwest of the netherlands, non-pregnant women with a history of preeclampsia (n= ), iugr (n= ) and uncomplicated pregnancies (n= ) were recruited at a mean follow up time of . years after pregnancy. body composition and fat distribution were assessed by dual energy-x-ray absorptiometry (dxa) and anthropometric measurements. results: women with a history of preeclampsia compared to controls had higher mean total-, fat-and lean mass (p < . ) as well as higher mean indices of body mass, fat mass and lean mass (p< . ). no significant differences were found for these variables between women with a history of iugr and controls. women with a history of preeclampsia had higher waist circumferences and waistto-hip ratios (p< . ) as well as excess of android fat mass and increased android-to-fat ratios (p< . ). women after pregnancies complicated by iugr had higher waist-to-hip ratios (p < . ). after controlling for body mass index, both women with a history of preeclampsia or iugr had higher waist circumferences (p < . ) and waist-to-hip ratios (p < . ) as well as smaller hip circumferences (p < . ). conclusion: despite differences in body mass index, both women with a history of preeclampsia and women after pregnancies complicated by iugr have a metabolically adverse fat distribution, marked by an excess of fat deposition in the abdominal region relatively to the hip region. these findings may explain, at least partly, their increased cardiovascular risk. women with a history of preeclampsia. marc ea spaanderman, timo h ekhart, robert aardenburg, louis lh peeters. obstetrics and gynecology, radboud university medical center, nijmegen, netherlands; obstetrics and gynecology, university medical center maastricht, maastricht, netherlands. background: a history of preeclampsia is associated with persistent short-term alterations in circulatory function and remote cardiovascular disease. in this study we tested the hypothesis at least years after preeclamptic pregnancy renal and central hemodynamic function is impaired as compared to women with uncomplicated pregnancy. methods: in formerly preeclamptic women (pe) and healthy parous controls (control) who were normotensive at weeks post-partum follow up, we assessed at least years after delivery blood pressure (mmhg), cardiac output (co, doppler ultrasonography, l/min) and effective renal plasma flow (erpf, pah clearance, ml/min/ . m ) after which we calculated total peripheral vascular resistance (. dyne.s/cm ) and renal vascular resistance (. dyne.s/cm ). data were analyzed parametrically (p< . ). results: age and bmi were comparable between groups. blood pressure was higher in formerly pe. moreover, % of formerly pe women and % of control were hypertensive (p< . ). although cardiac out was comparable between groups, total peripheral and renal vascular resistance were about % higher and erpf % lower in formerly pe women as compared to control. conclusion: at least years after gestational hypertensive disease, women who were normotensive at direct follow up have impaired renal and central hemodynamic function and developed more often chronic hypertension. long-term follow up may also be warranted in apparently healthy formerly pe women who are normotensive at post-partum follow up. circulatory function in formerly preeclamptic women and healthy parous controls co erpf tpvr rvr formerly pe . ± . ± * ± * ± * control . ± . ± ± ± * = p< . pregnancy increases blood-brain barrier permeability coefficient (l p ) to lucifer yellow: role of estrogen. marchien j wiegman, , marilyn j cipolla. neurology, ob/gyn and pharmacology, university of vermont, burlington, vt, usa; ob/gyn, university medical center groningen, groningen, netherlands. background: eclampsia is similar to posterior reversible encephalopathy syndrome in which an acute rise in blood pressure causes breakthrough of autoregulation, blood-brain barrier (bbb) disruption, and cerebral edema formation. we previously showed that late-pregnant (lp) animals developed cerebral edema during breakthrough, a response that was absent in nonpregnant (np) animals. in the current study we hypothesized that pregnancy predisposes the brain to edema during acute hypertension by enhanced bbb permeability. we further hypothesized that the underlying effect of pregnancy on the bbb permeability is due to elevated estrogen levels. methods: permeability coefficients (l p ) to lucifer yellow (ly), a polar compound that does not pass through tight junctions, were compared in posterior cerebral arteries (pca) from groups of sprague dawley rats: np (n= ), lp (d ; n= ), ovariectomized and implanted with -estradiol ( . mg, -day release) and estriol ( . mg, -day release) pellets for days (ovx+e; n= ), and ovariectomized and implanted with placebo pellets for days (ovx; n= ). pcas were isolated, pressurized in an arteriograph, and perfused with . mg/ml ly in saline. concentration changes of ly outside the vessel wall were determined at pressures from - mmhg. the slope of the pressure vs. permeability curve is the rate of flux, or l p for ly. results: l p for ly was significantly increased in pcas from lp and ovx animals vs. np (p< . ; figure ). estrogen was protective of the bbb only in ovariectomized animals, decreasing l p % in ovx+e vs. ovx. however, pregnancy did not afford protection and had a l p that was % greater than np. conclusions: pregnancy significantly increases bbb permeability to ly, an effect that may predispose the brain to edema formation during acute hypertension. these data also show that estrogen modulates l p in ovariectomized animals differentially than pregnancy, suggesting that the increased bbb permeability in pregnancy is caused by a mechanism other than elevated estrogen levels. in both pre-and early pregnancy, the sympathoinhibitory response to volume expansion is blunted in formerly preeclamptic women with low plasma volume. ineke krabbendam, marc ea spaanderman, dorette a courtar, robert aardenburg, ben j janssen, fred k lotgering, louis lh peeters. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; obstetrics and gynecology, university hospital maastricht, maastricht, netherlands; pharmacology and toxicology, university of maastricht, maastricht, netherlands. background: the circulation of formerly preeclamptic women with a low plasma volume (lpv) is characterized by sympathetic dominance. these women respond to a new pregnancy with an aberrant rise in atrial natriuretic peptide (anp) and a times higher chance to develop recurrent gestational hypertensive disease compared to their counterparts with normal plasma volume (npv). anp has sympathicomimetic capacity. we postulate that the sympathetic overdrive in lpv-women is associated with a reduced venous capacitance. to this end, we compared the response to volume expansion (ve) in women with lpv and npv, both before and in pregnancy. method: in non-pregnant normotensive formerly preeclamptic women, we measured pv (hsa i indicator dilution method) at least months post partum. we intravenously infused ml of iso-oncotic fluid over minutes. during the infusion, we recorded changes in heart rate (hr, bpm), blood pressure (bp, mmhg), cardiac output (co, l/min), sympathetic activity (lfsys, mmhg , low frequency component of spontaneous fluctuations in systolic bp, portapress) and anp (nmol/l). eight women became pregnant within year and were evaluated at weeks gestation. changes in circulatory and autonomic function between and within groups were analyzed non-parametrically (p< . ). results: before pregnancy, ve leads to comparable changes in hr, bp and co in women with lpv ( / ) and npv ( / ). in npv, lfsys decreased %, but only % in lpv (p< . ). anp remained unaltered in npv, but increased in lpv. in the pregnant group, women had lpv and had npv. in both groups, pregnancy did not alter the response to ve. conclusion: irrespective of pregnancy, the sympathoinhibitory response to ve is diminished in lpv. these data suggest that in these women ve leads to venous overfill, giving rise to anp-release and consequently sympathetic activation, flattening the normal baroreceptor-mediated sympathoinhibitory response. we speculate that this mechanism contribute to circulatory maladaptation to pregnancy, sympathetic dominance and subsequent gestational hypertensive disease. in both pe and ht, serum sflt- was increased, and plgf reduced at all gestations (p< . ). seng levels were also increased in pe. after weeks (but not before) antihypertensive treatment was associated with a significant fall in serum sflt- and seng, in pe only. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). only sflt- was significantly reduced in the placenta in women who received antihypertensive therapy. conclusion in pe, antihypertensive therapy after weeks' gestation is associated with a significant fall in serum sflt- and seng, and in placental sflt- . these findings raise the possibility that these drugs may have an effect on the pathophysiology of pe other than their known antihypertensive action. the synergistic effect of soluble vegf receptor pre-treatment and small doses of tnf-on endothelial cells. tereza cindrova-davies, debbie a sanders, olivera spasic-boskovic, graham j burton, d stephen charnock-jones. dept of pdn, university of cambridge, united kingdom; dept of obstetrics and gynaecology, university of cambridge, united kingdom. introduction: preeclampsia is marked by an enhanced endothelial inflammatory response manifested by maternal endothelial activation. soluble fms-like tyrosine kinase- (sflt- , svegf-r ), a naturally occurring circulating antagonist of vegf-a and plgf, is one of the secreted factors implicated in the pathogenesis of preeclampsia. in women who develop preeclampsia, sflt rises sharply, preceding the onset of the clinical disease. the aim of this study was to examine the effect of a combined treatment with recombinant sflt- and tnf-on the activation of human umbilical cord endothelial cells (huvec) by examining leukocyte adhesion, and the expression of icam, vcam, endothelin and vwf. methods: huvec were seeded, grown overnight and pre-treated with recombinant sflt ( - ng/ml) in a basic % fbs-dmem medium for hr. on day , low doses of tnf-( . ng/ml) were applied to pre-treated cells for hr. antagonism of the vegf-a action was mimicked at the protein level by pre-incubating huvec with anti-flt antibody ( - g/ml), anti-kdr antibody ( g/ml), anti-vegf antibody ( g/ml), or vegf receptor inhibitor su ( m). at the rna level, the effect of sflt was mimicked by sirna transfection of huvec with siflt or sikdr. at the end of each experiment cells were either harvested for western blotting, fixed in % pfa for immunofluorescence or incubated with labelled hl leukocyte cells, followed by fluorescent detection of adhesion. results: pre-incubation of huvec with sflt and subsequent treatment with low doses of tnf-increased the adhesion of hl leukocytes and increased icam- , vcam- , endothelin and vwf, compared to tnf-treatment alone. similar results were obtained when cells were pre-treated with su , anti-flt, anti-kdr or anti-vegf. transfection knock-down of flt or kdr gene also significantly increased leukocyte adhesion when small doses of tnfwere added. conclusions: pre-incubation with recombinant sflt, anti-flt, anti-kdr, anti-vegf, su or knocking-down flt or kdr transcripts all antagonised the autocrine actions of vegf and/or plgf. this predisposes huvecs to be more sensitive to the effect of tnf-. our study shows that sflt and tnfcombine to induce an enhanced synergistic effect, activating endothelial cells. maternal obesity is associated with increased production of inflammatory cytokines and risk of poor perinatal outcome. inflammatory cytokines can stimulate production of reactive oxygen (ros) and nitrogen (rns) species, which can covalently modify protein function. placental oxidative and nitrative stress are increased in pathological pregnancies and associated with altered placental function. objectives: determine the effect of increasing body mass index (bmi) on placental nitrative stress, measured by the expression and localization of nitrated (nitrotyrosine) and oxidized proteins. methods: placental tissue was collected at term ( - wks) from lean kg/m ), overweight ( - . kg/m ) and obese ( - kg/m ) patients (n= or /group). tissue was sectioned for immunostaining with nitrotyrosine antibody. protein samples were either dot blotted onto nitrocellulose membrane, probed with nitrotyrosine ab, and nitrated proteins detected using ecl, or derivatized using , -dinitrophenylhydrazine (dnph) (oxyblot® kit), separated on sds-page, probed with anti-dnph ab ( : ) and oxidized proteins detected using ecl. protein band intensity was measured by densitometry. oxidized proteins were selected for maldi mass spectrometry analysis. results: nitrotyrosine residues were immunolocalized primarily in the fetal capillary endothelial cells and the villous stroma, but were almost absent in the syncytiotrophoblast. by dot blot, nitrotyrosine expression differed across the three groups (p< . , anova) with expression in tissue from obese women being significantly increased compared to lean (p< . ) and overweight (p< . , tukey test). several oxidized proteins were detected with significantly greater expression seen in the lean versus overweight groups (p< . , mann-whitney u). one oxidized protein was identified by maldi-ms with four peptide matches and . % coverage as -beta hydroxysteroid dehydrogenase ( bhsd). discussion: with increasing bmi, an increase in nitrative stress appears to occur in parallel with a decrease in oxidative stress. oxidative stress is apparently reduced as ros are consumed by the interaction with rns to give nitrative stress. bhsd is involved in the biosynthesis of steroid hormones and glucocorticoids. its activity and hence steroid metabolism in the placenta may be regulated by oxidation with implications for fetal development. background teenagers are susceptible to delivering small-for-gestationalage (sga) infants. previous studies implicate continued maternal growth as a causal factor . growing adolescent sheep have reduced fetal birthweight due to impaired placental development and nutrient transfer . we hypothesized that placental function is impaired in human teenage pregnancy if there is maternal growth. methods placentas were collected from teenagers ( - years) and adults. activity of the amino acid transporter, system a, was quantified by the sodium-dependent uptake of c-methylaminoisobutyric acid into placental fragments. teenagers were defined as growing (> mm increase in kneeheight per days) or non-growing. system a activity was analysed in relation to individualised birthweight centile and maternal growth. results placental system a activity was significantly lower in teenage compared to adult pregnancies (p< . ). this was unrelated to birthweight; teenagers who delivered infants appropriate-for-gestational age (aga) had significantly lower placental system a activity compared to aga infants delivered to adults (p< . ). growing teenagers did not deliver lower birthweight infants than non-growing teenagers (median birthweight g and g respectively). furthermore, system a activity in placentas from growing teenagers was significantly elevated compared to that in non-growing teenagers (p< . ), and was similar to placental activity in adults. conclusions system a activity was reduced in placentas from teenagers compared to adults. this suggests that inherently lower placental function predisposes teenagers to sga, but that other factors (e.g. adequate nutrition) can compensate in those teenagers delivering aga infants. in contrast to our hypothesis, placental system a was elevated in growing teenagers and mimicked that of adults. this may be related to a hormonal-milieu in growing teenagers that is conducive to fetal growth, in part through stimulating placental transport. homocysteine inhibition of system a amino acid transport activity in human placenta. eleni tsitsiou, susan l greenwood, colin p sibley, stephen w d'souza, jocelyn d glazier. maternal and fetal health research group, st. mary's hospital, university of manchester, manchester, united kingdom. background: elevated plasma levels of the amino acid homocysteine (hcy) during pregnancy are associated with vascular-related complications and adverse neonatal outcomes including a reduced birthweight. fetal and maternal plasma hcy concentrations are positively correlated suggesting placental transport of hcy may be an important determinant of fetal plasma hcy. the mechanisms involved in placental hcy transport are uncharacterised. evidence that the system a amino acid transporter, which transports neutral amino acids in a na + -dependent manner, is important in promoting fetal growth and that a reduced system a activity is associated with intrauterine growth restriction (iugr), led to our hypothesis that system a provides one mechanism for placental hcy transport. this hypothesis was tested by measuring the ability of hcy to inhibit system a activity in isolated microvillous plasma membrane (mvm) vesicles and placental fragments. materials and methods: mvm vesicles and placental fragments were isolated from placentas of normal pregnancies at term. system a activity was measured at initial rate ( s and min respectively) as na + -dependent cmethylaminoisobutyric acid (meaib) uptake into mvm vesicles ( . mm) or fragments ( . mm) in the absence (control) or presence of l-hcy and dl-hcy or model substrates. results: mm l-hcy (custom-synthesised) and dl-hcy (commercial source) significantly (p< . ) inhibited na + -dependent c-meaib uptake into mvm vesicles compared to control; comparable in magnitude to other model substrates (meaib, l-ala, l-ser, l-met; n= , kruskal-wallis with dunn's multiple comparison test). l-hcy, l-met and meaib ( . - mm) caused a dose-dependent inhibition of na + -dependent c-meaib uptake into mvm with ec values (in mm; mean ± se) of . ± . , . ± . , and . ± . respectively, n= ). na + -dependent c-meaib uptake into fragments was reduced substantially in the presence of mm l-hcy or dl-hcy causing a ± and ± % reduction (mean ± sd, n= - ) of control respectively. conclusion: these observations suggest that hcy is a relatively high affinity substrate for system a in the human placenta. we speculate that inhibition of placental amino acid uptake by hcy could impact on fetal growth and development. supported by the mrc and action research endowment fund. glucose regulates placental mtor activity and glucose deprivation down-regulates placental system l activity in an mtor-dependent manner. sara roos, theresa l powell, thomas jansson. department of physiology, institute of neuroscience and physiology, gothenburg, sweden; department of obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. placental amino acid transporters are down-regulated in intrauterine growth restriction (iugr). we have previously shown that mammalian target of rapamycin (mtor) regulates placental system l transporter activity and that placental mtor activity is decreased in iugr. however, the upstream regulators of placental mtor are unknown. in iugr, fetal hypoglycemia and reduced maternal glucose levels are common and the placenta may therefore be exposed to low glucose levels. hypothesis: we hypothesized that glucose availability regulates placental amino acid transporter activity mediated by changes in mtor signaling. methods: cytotrophoblast cells were isolated and cultured until syncytialization at hours. cells were cultured for an additional hours in culture media containing . mm, . mm, or mm glucose (control), which corresponds to standard culture media. at hrs, the activity of the mtor signaling pathway was assessed by measuring the protein expression of s k phosphorylated at thr- , the primary site of mtor phosphorylation. in another set of cells, system l activity was assessed by measuring the bchinhibitable uptake of h-leucine. results: as compared to control, phospho-thr- -s k expression was reduced by % in cells incubated in . mm results: fgr pregnancies were delivered earlier ( ± . v ± . w; p< . ), weighed less ( . ± . v . ± . kg; p< . ) and had higher s/d ratios ( . ± . v . ± . ; p< . ). eaa concentrations were similar between groups, but there were differences (non-parametric testing; p< . ) in transport rates between groups with his crossing considerably faster and ile crossing slower in fgr . the table shows fetal vein/maternal (fv/m) ratios standardized to the leu fv/m ratio for all eaa for both groups. amino acids fell into groups for fgr pregnancies: ) his (ratio . ), ) leu, phe, met (ratios ), and ) val, thr, ile, trp, lys with intermediate ratios ( . ns conclusion: this is the st study to compare the relative rates of in vivo placental transport for all eaa between normal and fgr human pregnancies showing striking differences in the transport rates of two eaa. in the absence of eaa concentration differences between groups, the higher his transport rate in fgr suggests higher utilization by the placenta. vasculature of women who received exogenous estrogen compared to those who received placebo. the purpose of this investigation was to identify the gene expression in response to the estrogen, equilin, as the major component of conjugated equine estrogen and genistein, a phytoestrogen in human coronary artery endothelial cells. human coronary artery endothelial cells from a -year old female were used. cells were treated with estradiol [e ], equilin [eq] ( . nm) or genistein [gen] ( micromolar) for hours. focused oligomicroarrays for cardiovascular disease and endothelial cell function were used to study gene expression. rt-pcr was used to confirm the transcription of genes analyzed in oligoarrays. vascular adhesion molecules and genes involved in the inflammatory response were most affected with the three estrogen sources. significant reduction of integrin alphae was seen with all three . , . and . for e , eq and gen respectively. similarly nfkb was also reduced . , . and . fold compared to controls. significant reduction of ccl was demonstrated with eq ( . fold) and gen ( . fold). tnfreceptor a and b were only significantly decreased by gen. vcam- which is regulated by proatherogenic factors and upregulated mainly at atherosclerosis -prone sites, was significantly reduced ( . -fold) with genistein, and a slight reduction was seen with e and no change was observed with eq. our data support the clinical findings that estrogen has favorable effects on the genes involved in atherosclerotic disease. while e has been shown to be slightly more effective than equilin in the modulation of gene expression, genistein was significantly more effective in the favorable expression of genes involved in particularly the inflammatory response. olga n lekontseva, sandra t davidge. physiology and obstetrics/gynecology, university of alberta, edmonton, ab, canada. introduction: the prevalence of cardiovascular disease in women dramatically rises in the postmenopausal period. although deficiency of estrogen has been implicated in the pathophysiology of systemic vascular dysfunction, the effects of estrogen on vasculature are complex and not completely understood. we have previously shown that estrogen exerts a beneficial effect on the aging vascular system by reducing circulating levels of the inflammatory cytokine tnf . tnf is a known regulator of matrix metalloproteinases (mmps), proteolytic enzymes that may modulate vascular tone through cleavage of vasoactive peptides such as big endothelin- (et- ). the role of estrogen in this pathway is unknown. we tested the hypothesis that in aging/estrogen deficiency, tnf -induced mmp activity mediates greater vasoconstriction, in part, through the et- pathway. we further hypothesized that estrogen replacement reduces vascular sensitivity to the constriction by preventing mmp activation. methods: aged ( month old) female sprague dawley rats were ovariectomized and treated with either placebo [ovx] , -estradiol [ovx+e ], or the tnf inhibitor etanercept [ovx+etan] . after four weeks, resistance mesenteric arteries were isolated and studied on the pressure arteriograph. concentrationresponse to exogenous big in the absence or presence of mmp inhibitor (gm , μm) was assessed in the vessels. results: treatment of "menopausal" [ovx] rats with either estrogen or the tnf inhibitor reduced sensitivity of arteries to big et- (ec = . ± . μm in ovx versus . ± . μm in ovx+e or . ± . μm in ovx+etan groups; p< . ). although mmp inhibition attenuated maximal constriction in all of the arteries, there was a significantly greater (p< . ) role of mmps in big et- -induced vasoconstriction in the ovx+e group (reduction in max constriction= . ± . %) compared to ovx ( . ± . %) and ovx+etan groups ( . ± . %). conclusions: both estrogen and tnf inhibition reduced big et- vasoconstriction. however, contrary to our hypothesis, tnf is not contributing to mmp modulation of et- vasoconstriction. interestingly, our study demonstrates a novel role for estrogen to increase mmp contribution to big et- vasoactivity with the net effect being less vasoconstrictive. understanding this unique pathway of regulation by estrogen in the aged vasculature will allow for development of new therapeutic options for women. mo, usa. introduction: the etiology of pelvic organ prolapse is multifactorial, with both inherited and acquired components. the molecular mechanisms of prolapse have not been established yet. we have previously shown that lysyl oxidase (lox) expression is suppressed in uterosacral ligaments of women with pelvic organ prolapse. it has also been shown that lox is a tumor suppressor gene inactivated by methylation in human gastric cancers. hypothesis: the aim of this study was to analyze the dna sequence of the promoter region of the lysyl oxidase gene in tissues from women with pelvic organ prolapse and identify whether methylation is present. our hypothesis is that the promoters of the lox gene in women with pelvic organ prolapse have significantly more methylation sites than women without prolapse. materials and methods: genomic dna was isolated from the uterosacral ligaments of eight women with pelvic organ prolapse and women without prolapse (controls). genomic dna samples were treated with ez dna methylation kit (zemo research, orange, ca). the lox gene promoter region of - to + was amplified by pcr and then cloned into pcr . -topo (invitrogen, carlsbad, ca) and transformed into an e. coli dh a strain. amplified plasmid dna samples containing the lox gene promoter region from each woman were sequenced and methylated cpg islands were identified by sequence comparison. results: a total of methylated cpg sites were found in the patient group with pelvic organ prolapse while only methylated cpg site was found in the non-prolapse control group. conclusion: these findings suggest that methylation in the promoter region suppresses lox gene expression in women with pelvic organ prolapse. background: reports from case-control and cohort studies have suggested an inverse association between lactation and breast cancer risk, but findings have been inconsistent. methods: we conducted a prospective observational cohort study of , parous women participating in the nurses' health study ii from to . our primary outcome was incident premenopausal breast cancer. results: during the study period, cases of premenopausal breast cancer were diagnosed during , person-years of follow-up. women who had ever breastfed had a % lower incidence of premenopausal breast cancer ( % confidence interval [ci] - %) compared with women who never breastfed, adjusting for parity, age at first birth, year of first birth, height, body mass index (bmi), bmi at age , family history, personal history of benign breast disease, participant birth weight, preterm birth, age at menarche, oral contraceptive use, physical activity, and alcohol consumption. no trend was observed with duration of lactation (p= . ). the association between ever-breastfeeding and premenopausal breast cancer was modified by use of medication to suppress lactation (p= . ); in analyses restricted to women who had never used suppressive medication, ever-breastfeeding was associated with a % ( %ci - %) reduction in incident disease. the association between lactation and premenopausal breast cancer was further modified by family history of breast cancer (p= . ). among women who had never used suppressive medication and reported a family history, those who had breastfed had a % lower covariate-adjusted risk of premenopausal breast cancer ( % ci - %) than women who had never breastfed. among women without a family history, ever-breastfeeding was not associated with breast cancer incidence (hazard ratio . , % ci . - . ). among women who had ever breastfed, we observed no association between breast cancer risk and duration of lactation amenorrhea or exclusive breastfeeding. conclusion: in a large, prospective cohort study, ever-breastfeeding was inversely associated with risk for premenopausal breast cancer. at the durations observed in our cohort, we observed no trend with duration of breastfeeding or lactation amenorrhea. the inverse association with ever-breastfeeding was stronger among women with a family history of breast cancer. regulatory and nkt cells at the maternal-fetal interface. thomas f mcelrath, rachael a clark. brigham women's hospital, boston, ma, usa; harvard skin disease research center, brigham women's hospital, boston, ma, usa. introduction: pregnancy represents an immunologically challenging event requiring maternal tolerance of the fetal semi-allograft. an increase in decidual cd + cd + t cells has been documented but it is unclear if these represent foxp + t cells (tregs) . the possibility also exists that other cd + lineages with potential regulatory function exist within the decidua parientalis. we examined if cd + cd + cells are true foxp + tregs and evaluated the frequency of other t cell subsets with possible regulatory potential. methods: we extracted t cells from term deciduas of planned cesarean deliveries. cells were stained with directly conjugated monoclonal antibodies and were analyzed on a six color flow cytometer. results: we found that only a subset (median %) of cd + t cells were true foxp + regulatory t cells. from donors, foxp + tregs accounted for a median of . % of all cd + cells. these cells were memory cd ro + t cells lacking ccr , and l-selectin but expressing cd , ctla- , gitr, and hla-dr. additionally we found that a median % of cd + t cells expressed the nkt marker cd . these cells were a mixture of cd and cd -cd -t cells, with variable numbers of cd t cells, suggesting they do not represent merely recently activated t cells. there was enrichment for t cells with nkt markers after culture on hela cells expressing cd d, suggesting that these cell represent true nkt cells. nkt cells were of the non-classical type, with a diverse t cell repertoire ( . % iv jq) and also expressed cd , hla class ii, cd ro but were ccr and l-selectin low. comments: we find that only a minority of cd -expressing t cell in the decidua are true foxp + tregs. because much of the work on treg in pregnancy has used cd as a treg marker, this suggests additional studies are needed to confirm the role of these cells in pregnancy. we find that a novel population of non-classical nkt cells exists in the human decidua. nkt cells can either promote or antagonize tolerance, depending on the immunologic context. the large number of these cells in the decidua suggests they may play a role equal or exceeding that of regulatory t cells. prolapse. marsha k guess, kathleen a connell, richard bercik, lloyd g cantley. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa; internal medicine/neprhology, yale university school of medicine, new haven, ct, usa. objectives: thy- is a cell surface glycoprotien expressed in human fibroblasts, neurons, hematopoetic stems cells and endothelial cells. thy- expression affects fibroblast proliferation and migration, cell-cell, as well as, cell-matrix interactions. moreover, thy- expression has been shown to play a critical role in fibroblast dedifferentiation into myofibroblasts, as well as in extracellular matrix (ecm) production and fibrosis. women with pelvic organ prolapse (pop) have alterations in vaginal ecm protein expression and metabolism, as well as decreases in smooth muscle fractional content. in the current study, we evaluated thy- as well as the smooth muscle markers alpha-smooth muscle actin (asma) and desmin expression in women with pelvic organ prolapse compared to women with normal pelvic support (controls). methods: anterior apical vaginal wall specimens from women with pop and controls were collected at the time of hysterectomy. messenger rna and protein expression of thy- , asma and desmin were evaluated using semiquantitative rt-pcr, real-time pcr and western blot analysis. gapdh and beta actin were used as internal controls. results: rt-pcr demonstrated the presence of thy- , asma and desmin in vaginal tissue from women with pop and controls. further, thy- mrna expression was downregulated % in women with pop compared to controls (p = . ). a parallel decrease in thy- protein was seen in women with pop compared to controls (p= . ). although a % and a % decease were seen in asma and desmin mrna, these differences were not statistically significant. similarly, no differences were seen in asma and desmin protein expression. conclusion: we demonstrate that there is significantly less thy- expression in vaginal tissue from women with pop compared to controls. differential expression of thy- in prolapsed vaginal tissues suggests that thy- may have a functional role in mediating ecm metabolism in the female genitourinary tract. hur expression is altered in ectopic endometrium. s karipcin, t altun, ua kayisli, e seli. ob gyn, yale u., new haven, ct, usa. introduction: cytokines and growth factors contribute to cyclic turnover of the normal endomterium and to the pathogenesis of endometriosis. cytokine and growth factor messenger rnas (mrnas) undergo rapid turnover that is primarily mediated by au-rich elements (are) that consist of multiple stretches of adenylate and uridylate residues located in the ' untranslated region ( '-utr) of their mrnas. hur is a ubiquitously expressed rna-binding protein that stabilizes are containing mrnas and prolongs their expression. we hypothesized that hur may play a role in the regulation of cytokine expression during normal menstrual cycle and in endometriosis. methods: tissue sections obtained from normal (n= ) and ectopic (n= ) endometrium were immunostained for hur. staining intensity was evaluated by hscore and grouped according to menstrual cycle phase. statistical analysis was done with one-way anova. cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (e ; - m), or progesterone (p, - m) for and h, and hur expression was determined using western analysis and normalized to ß-actin. results: hur immunostaining was nuclear in endometrial cells. hur immunoreactivity was significantly lower in the early proliferative and late secretory phases ( . ± . and . ± . , respectively), compared to the mid-late proliferative ( . ± . ) and early-mid secretory phases ( . ± . )(p< . ). moreover, hur expression was significantly lower in ectopic endometrial cells when compared to normal endometrium in midlate proliferative and early and mid-secretory phases (p< . ). progesterone suppressed hur levels significantly in cultured endometrial stromal cells at both and h compared to control (p< . ) while estrogen did not cause a significant change. discussion: decreased hur levels in the late secretory and early proliferative phases are likely to contribute to degradation of cytokines and result in lower cytokine levels observed mid-cycle. late secretory decrease in hur levels may be mediated by progesterone as suggested by in vitro findings. in ectopic endometrium, persistent low expression of hur compared to normal endometrium most probably results from elevated cytokine levels associated with endometriosis. the effect of lower hur expression in ectopic endometrium on other are-containing transcripts, and on the pathogenesis of endometriosis remains to be elucidated. tissue. hong zhao, joy innes, scott reierstad, mehmet bertan yilmaz, serdar e bulun. department of obstetrics and gynecology, northwestern university, chicago, il, usa. background: aromatase is the key enzyme for estrogen biosythesis, and is encoded by the cyp a gene. thus far, only three unique untranslated first exons associated with distinct promoters in the mouse cyp a gene were described (brain-, ovary and testis-specific exon i). however, it remains unknown whether aromatase is expressed in other mouse tissues via previously unknown tissue-specific promoters activating new exon is. methods: real-time pcr was used to examine the aromatase expression levels in various c bl mouse tissues. '-rapid amplification of cdna ends ( '-race) was used to determine the transcriptional start sites of cyp a transcripts. promoter activity was measured using serial deletion mutants of dna fused to the luciferase reporter gene. results: real-time pcr results showed that aromatase was expressed in male gonadal fat and the expression level is lower than that in testis. the adipose tissue-specific untranslated exon i of cyp a transcript was isolated using '-race and this novel gonadal fat-specific exon i of cyp a mrna did not show sequence similarities to previously reported ones. this new adiposespecific exon i was mapped to kb upstream of the translation start site in the coding exon ii. the genomic region upstream of the adipose-specific exon i was cloned into luciferase plasmids. transfection of murine t -l cells with these plasmids showed that promoter activity was conferred by the sequence located at - to - bp upstream of the transcriptional start site. dexamethasone significantly induced activity of the adipose-specific promoter region. conclusion: taken together, our results suggest that a novel cyp a transcript is regulated by a tissue-specific promoter in male murine gonadal fat. these sacrificed; reproductive and selected other organs were removed, weighed and evaluated histologically by an observer blinded to treatment. results: the table below shows that tcc augmented effects of tp on weights of accessory sex organs. histological assessment revealed that tcc induced greater glandular distention with more secretions compared to the effects of tp alone; furthermore, mitotic figures were seen only in prostates from rats exposed to tp+tcc. conclusions: tcc is a newly identified endocrine disruptor with unique and novel actions resulting in potentiation of androgen effects on sex organs. these observations underscore possible environmental risks related to exposure to tcc. background: blastocyst implantation is dependent on the differentiation of human endometrial stromal cells (hesc) into decidual cells. transforming growth factor family members have well defined roles in cell differentiation and proliferation. activin a, a tgf superfamily member, enhances hesc decidualization and localizes to decidualized cells in human endometrium. other tgf superfamily members, including bmp , bmp , bmp , gdf , gdf (myostatin), gdf and nodal, may also be present in decidual cells and therefore may also play a role during this important process. this study aimed to determine whether activin is the major family member driving decidualization or whether other family members contribute to the process. methods: broad ranging activin inhibitors (activin-m a and sb ) that effect receptor-ligand interactions of other tgf superfamily members were used in hesc decidualization. protein localization was examined in secretory phase endometrium and first trimester decidua by immunohistochemistry and mrna expression was examined in an ex vivo model. the secretion of candidate proteins was measured during hesc decidualization and certain recombinant proteins added during decidualization to examine their effect. results: m a ( nm) and sb ( m) significantly reduced decidualization ( % and % respectively) demonstrating that activin and possibly other tgf family members are involved in decidualization in vitro. bmp , gdf and tgf protein were detected in decidual cells of mid-late secretory endometrium and first trimester decidua whilst all ligands except nodal, were expressed by hesc. both bmp and tgf secretion increased during hesc decidualization and administration of both these proteins significantly enhanced decidualization in vitro. conclusions: these data support a role for activin a, bmp and tgf in hesc decidualization. this is important as the elucidation of factors involved during decidualization will aid in better understanding implantation and fertility. abnormal chromatin remodeling in diabetic murine oocytes. laura lawrence, ann ratchford, cybill esguerra, qiang wang, kelle moley. ob/ gyn, washington university, st. louis, mo, usa. background: diabetic women experience increased miscarriages and adverse pregnancy outcomes. previous studies suggest adverse diabetic outcomes may occur earlier than the preimplantation period, particularly during oogenesis. we hypothesize that diabetes affects chromatin remodeling and chromosomal condensation in murine oocytes. methods: mii oocytes from diabetic and control mice were fixed with % pfa, permeabilized with . % triton x- for min, and immunostained against a-tubulin and the nucleus for hr at rt. images were obtained with laser confocal scanning microscope. protein expression levels of chromatin with dimethyl h k modifications were measured in nondiabetic and diabetic denuded murine oocytes at hours post pmsg ( hour) and at six hours post hcg ( hour) via western immunoblots. mature nondiabetic denuded oocytes were fixed in %pfa and permeabilized with . % triton x- . they were stained via immunohistochemistry against histone protein h at lysine (h k me ) and heterochromatin. results: immunohistochemistry reveals that diabetic mii oocytes have aberrant spindle formations and metaphase chromosome alignment. approximately / ( %) diabetic oocytes examined had abnormal spindles and metaphase alignments compared with only about / normal oocytes ( . %). western blot demonstrated times higher expression of dimethylated chromatin in diabetic oocytes at time compared with nondiabetic oocytes. at time hours, diabetic oocytes had significantly fewer h k modifications than the controls. when staining mature murine nondiabetic oocytes for dimethylation of h k me by immunohistochemistry, we demonstrated h k me expression in a condensed heterochromatin ring surrounding the nucleolus, consistent with transcriptional silencing. conclusions: diabetic mii oocytes have a significant increase in abnormal spindle formation and metaphase chromosome alignment. they also have increased dimethylation compared with normal oocytes at a time point when they should be transcriptionally active, storing maternal mrna in preparation for the silencing period. in addition, after hcg injection to trigger maturation and gene silencing, the diabetic oocytes had decreased dimethylated chromatin changes. our findings suggest that diabetic oocytes may be exiting the transcriptionally active period prematurely and may ultimately experience decreased, partial, and incomplete gene silencing. and xenopus epab genes and proteins were performed. expression of human epab and pabpc mrna was tested in ten different somatic tissues, testes, and ovaries by rt-pcr. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. epab and pabpc expression in human prophase i (pi) and metaphase ii (mii) oocytes, -cell embryos and blastocysts was evaluated using quantitative real time pcr. results: human epab is a aa protein with % identity and % similarity to mouse epab and contains rna recognition motifs and a pabp domain. human epab mrna is detected in ovaries and to a lesser extent in testes and several somatic tissues including kidney, liver, and muscle. similar to its mouse orthologue, human epab mrna is expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. pabpc mrna is ubiquitously present in all tissues as well as -cell, and blastocyst stage embryos. however, its levels are significantly lower than that of epab in oocytes. conclusions: in this study we report the identification of human epab. similar to that observed in xenopus and mouse, human epab is the predominant poly(a) binding protein in oocytes and it is replaced by pabpc following zga, which occurs at -to -cell stage in human. our findings suggest that the unique translational regulatory pathways that control gene expression during oogenesis and early embryo development may be common between model organisms and humans. objective: c-jun nh -terminal kinase (jnk), a member of mitogen-activated protein (map) kinase family, is involved in cell proliferation, differentiation, and survival. fsh is required for granulosa cell proliferation and antral follicle growth but its mechanism of action in preantral stages is not well defined. we previously showed that pharmacological inhibition of jnk pathway halts invitro growth of murine preantral follicles in serum free media supplemented with fsh ( miu/ml). sigcs (spontaneously immortalized rat granulosa cell line) have characteristics similar to preantral granulosa cells and hence they were used in this study to determine whether the jnk pathway plays a key role in preantral granulosa cell proliferation. our specific aims were to determine whether: a) fsh activates jnk pathway in granulosa cells; b) the inhibition of jnk pathway blocks cell cycle progression. material and methods: sigcs were treated with miu/ml recombinant fsh in serum free media two days after serum starvation. activation of jnk pathway was analyzed with if and wb using phospho-jnk and phospho-cjun expression, respectively. fsh receptor expression (fshr) was analyzed with if. the inhibition of jnk pathway on cell cycle progression was analyzed by facs using a jnk inhibitor sp . results: fshr protein was expressed in sigc indicating that they can respond to fsh (fig a) . likewise by if, phospho-jnk expression was significantly increased in sigc hour post fsh exposure (fig- a) . similarly on wb, phospho-cjun expression increased as early as min after fsh exposure and peaked at hrs. cjun phosphorylation was abolished hr after treatment with sp ( mm) (fig b) . facs analysis showed that the inhibition of jnk by sp resulted in cell cycle arrest at g /m transition in a dose dependent fashion (fig c) . conclusion: these results strongly suggest that the proliferative effect of fsh on immature granulosa cells is mediated through the activation of jnk pathway. this is the first experimental observation implicating jnk signaling in granulosa cell cycle control. (nichd - ). the induction of {alpha}-hydroxylase (cyp ) expression in granulosa cells. satin s patel, victor e beshay, william e rainey, bruce r carr. reproductive endocrinology and infertility, university of texas at southwestern medical center at dallas, dallas, tx, usa; physiology, medical college of georgia, augusta, ga, usa. according to the traditional two-cell two-gonadotropin hypothesis of the ovary, androgen production arises exclusively from theca cells. the granulosa cells, in turn, utilize androstenedione, which is aromatized eventually to estradiol. studies involving immunohistochemical analysis of normal ovaries have shown that granulosa cells express significantly higher levels of the activator protein- (ap- ) transcription factor, cfos compared to theca cells, where cfos expression is virtually absent. we hypothesize that cfos functions to inhibit the expression of cyp in granulosa cells, thereby suppressing androgen production. hence, the inhibition of cfos activity might result in cyp expression in the granulosa cell. our objective was to define the role of cfos, in the regulation of cyp expression in granulosa cells. transformed human luteinized granulosa (hgl ) cells were utilized for all experiments. hgl cells were cultured in monolayer for h. cells were treated for h with and without pd (pd), a mapkk inhibitor, which also blocks cfos expression. rna was isolated and real time rt-pcr was performed for cyp . cfos rna interference experiments were carried out using rnaifect, cfos smartpool sirna and scrambled sirna for h. rna was isolated and rt-pcr was also performed for cyp . immunochistochemical studies were performed on normal ovaries, staining for cfos and cyp . treatment of hgl cells with the mapkk inhibitor pd for h, resulted in a -fold increase in cyp mrna expression compared to basal conditions. in cfos gene silenced cells, cyp mrna expression also increased by -fold compared to control sirna conditions. immunohistochemical staining for cfos and cyp showed significant staining of cfos in the granulosa cell layer, but absent staining for cyp . conversely, the theca cell layer did not stain for cfos, but staining was evident for cyp . these results suggest that the ap- transcription factor, cfos, may play a role in the inhibition of cyp expression in granulosa cells. this may provide an explanation for the lack of cyp expression in granulosa cells. the g /m stages of the granulosa cell cycle. as clip- has been identified as an mtor substrate, we hypothesized that its function at the mitotic spindle would be positively regulated by mtor during the late g and m phases of the cell cycle in granulosa cells. during periods of stress (e.g., mtor inhibition), mtor would fail to phosphorylate clip- , leading to spindle checkpoint failure and follicle undergrowth. objectives: the expression of clip- and mtor were evaluated. computational analysis of potential clip- phosphorylation sites and comparison with residues on known mtor targets were performed. clip- threonine and serine were chosen and evaluated as bona fide mtor phosphorylation sites. a preliminary assessment of the effects of mtor inhibition upon clip- function was performed. methods: for protein expression analyses, western blots, immunostaining of tissues and primary granulosa cells in culture were performed. computational analysis of potential clip- phosphorylation sites was followed by in vitro assessment of mtor kinase activity upon clip- and a peptide substrate. results: clip- was expressed in the ovarian stroma, blood vessels (including the endothelial cells of both arteries and veins), granulosa cells, and in the oocytes of primordial and growing follicles. overlapping expression was found between clip- , mtor, and the mtor cofactors raptor and rictor in granulosa cells. this expression was conserved between the mouse and the human. evaluation of clip- phosphorylation supported thr as a bona fide mtor target. conclusions: clip- was supported as an mtor substrate protein during granulosa cell mitosis. the mechanism of mtor action during granulosa cell growth and survival is likely to include the phosphorylation of clip- and subsequent positive regulation of mitotic spindle function. the effect of a selective oxytocin antagonist (barusiban) in threatened preterm labour: a randomised, double-blind, placebo-controlled trial. steven thornton, thomas m goodwin, gorm greisen, morten hedegaard, joan-carles arce. warwick medical school, university of warwick, coventry, united kingdom; maternal-fetal medicine, university of southern california, los angeles, usa; dept of neonatology, rigshospitalet, copenhagen, denmark; dept of obstetrics, rigshospitalet, copenhagen, denmark; clinical research development, ferring pharmaceuticals, copenhagen, denmark. objective: a mixed oxytocin/vasopressin v a antagonist, atosiban, has been shown to reduce uterine contractions in placebo-controlled clinical trials and is useful in the management of preterm labour. the objective of this study was to determine the effect of a selective oxytocin antagonist, barusiban, in delaying delivery and reducing uterine contractions in women with threatened preterm labour at a late gestational age and relatively high risk of delivery. methods: this was a randomised, double-blind, placebo-controlled multicentre study in countries. a total of women between + and + weeks gestation, and with uterine contractions of sec duration during min, cervical length mm, and cervical dilatation > and < cm were randomised to receive a single intravenous bolus dose of either barusiban . mg, mg, mg, mg or placebo. rescue tocolytics were prohibited. the primary end-point was percentage of women who did not deliver within h. uterine contractions were monitored by cardiotocography. obstetrical and neonatal outcomes were determined. results: there were no significant differences between the placebo and any barusiban group in percentage of women who did not deliver within h ( % in the placebo group and % to % in the barusiban groups). there was no dose-effect relationship nor an effect at or h. none of the barusiban groups were associated with a significant reduction in number of uterine contractions compared to placebo at any time point up to h post-dosing. postpartum blood loss and time to established lactation were not significantly increased with barusiban. barusiban was well tolerated and was not associated with safety concerns for the women, fetus or neonates. conclusion: a single intravenous bolus of a selective oxytocin antagonist, barusiban (dose range . - mg), did not delay delivery or reduce uterine contractions compared to placebo in women with preterm labour at late gestational age and with short cervical length. the results contrast those of the mixed oxytocin/vasopressin v a antagonist, atosiban. prolonged delivery intervals in triplet gestations. tracy a manuck, heather l mertz, leah passmore, david c merrill. obstetrics and gynecology, wake forest university health sciences, winston-salem, nc, usa. objective: delayed interval delivery is one management strategy for previable preterm labor affecting multiple gestations. prior reports of asynchronous deliveries have examined twins and higher-order multiples as a group. this study was conducted to analyze the unique situation of asynchronous triplet deliveries. study design: cases of asynchronous triplet deliveries resulting in an ongoing twin gestation were ascertained through medline. data were abstracted and combined with two similar previously unpublished cases. patients were grouped by management with and without rescue cerclage. variables compared included use of tocolytics, antibiotic administration, gestational age at delivery of each fetus, interdelivery interval, delivery mode, birthweights, and short and long term outcomes. chi-square or t-test analyses were used where appropriate. results: fifty-one cases of asynchronous triplet deliveries met inclusion criteria and were analyzed. twenty-three patients ( . %) underwent placement of a rescue cerclage following delivery of the first infant. these patients delivered the first fetus at a significantly earlier gestational age as compared to those patients without a cerclage ( . +/- . weeks vs. . +/- . weeks, p= . ). patients with a rescue cerclage had a significantly longer prolongation of the remaining twin gestation ( . +/- . days vs. . +/- . days, p= . ). no significant differences in use of tocolytics or antibiotics, gestational age at delivery of triplets "b" and "c," mode of delivery, short term outcome (alive at hours), or long term outcome (alive at discharge) were noted, despite delivery of triplet "a" at a significantly younger gestational age. conclusion: rescue cerclage, particularly when placed following previable delivery of a first triplet, may significantly prolong the delivery interval for the remaining twin gestation. mean pregnancy prolongation (days). objective the aim of our study was to evaluate the role of amnioinfusion in pregnancies complicated by pprom. we studied singleton pregnancies with pprom at < weeks gestation. all patients were managed conservatively with bed-rest, prophilactic antibiotics, tocolytics and steroids. only patients without vaginal bleeding and/or contractions were included: patients showed an amniotic fluid pocket (afp) persistently cm and did not undergo amnioinfusion (group b) whereas had a maximum afp < cm and were offered amnioinfusion to restore an adequate amount of amniotic fluid (group a). in patients of group a amnioinfusion was successful (afp cm for hours following the procedure: group a ) whilst in it was unsuccessful (afp< for hours: group a ) and repeated. results were analized with the student t test for unpaired samples and with the when appropriate. p values < . were considered significant. results the group where amnioinfusion was not successful (group a ) showed the worst outcome (see table) . there were intrauterine deaths, all in this group. pulmonary hypoplasia was present in / ( . %) newborns (both survived and deceased) newborns, / in group a . no maternal complications were recorded. conclusions our data confirm that a conservative-active management with amnioinfusion can be considered a reasonable option in women with pprom. in our series it was effective in preventing both neonatal death and pulmonary hypoplasia. group a (infusion successful) background. synthetic progestogens are effective in reducing the risk of spontaneous preterm birth in high risk singleton, but not multiple, pregnancy. we hypothesized that myometrial stretch may inhibit the response of human myometrium to progestogens. methods. myometrial strips obtained with written consent at the time of term planned cesarean section were studied using a modification of the method of young and zhang (jsgi ; : - ) . strips were maintained in individual tubes in tissue culture media in an incubator for a period of three days. the effect of prolonged stretch was assessed by comparing strips connected to a . g weight with those connected to a . g weight. the effect of prolonged exposure to progestogen was studied by adding medroxyprogesterone acetate (mpa, nm or nm). following the day incubation, myometrial strips were transferred to an organ bath containing kreb's solution. all were placed under g tension and responses obtained to mm potassium then oxytocin. contractility was expressed as the ratio of the maximum response to potassium or oxytocin to the wet weight of the tissue (units = g.tension per gram), summarized as the mean (sem) and compared using student's paired t test. prolonged stretch increased the maximum response to both potassium ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ) and oxytocin ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days reduced the maximum response to potassium (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ) and there was a trend towards a reduced maximum response to oxytocin (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days had no effect on either the maximum response to potassium . prolonged stretch increases human myometrial contractility in vitro. . prolonged exposure to a progestogen inhibits the contractility of human myometrium but this effect is blocked by prolonged stretch. these properties of human myometrium may explain the failure of oh progesterone caproate to reduce the incidence of spontaneous preterm birth in multiple gestations. sangeeta jain, william l maner, janet l brandon, gary dv hankins, robert e garfield. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine if transabdominal uterine electromyography (emg) correlates with parturition factors such as measurement-to-delivery time (mtdt), cervical dilation (cd), cervical effacement (ce), and station (s) for preterm labor patients with and without tocolysis. materials and methods: pregnant preterm labor women were included. uterine electromyography (emg) was measured for minutes. cd, ce, and s were assessed at or near the time of uterine emg measurement. the power density spectrum peak frequency (pdspf) was calculated on emg. patients were grouped (g : tocolysis, n= ; g : no tocolysis, n= ). pearson-product-moment test was used for correlation. significant differences were sought between groups using student-t test. p< . significant. results: there was a significantly higher uterine emg activity (pdspf: . ± . vs. . ± . ), but no difference in cd ( . ± . vs. . ± . ), for patients delivering within days of emg recording compared to those who delivered later, regardless of tocolysis. there was no apparent difference in uterine emg in tocolytic vs. non-tocolytic patients, regardless of mtdt (table ) . conclusions: uterine emg activity is significantly greater in patients in preterm labor who delivered within days of measurement, making it a viable alternative diagnostic parameter for assessing the state of parturition. tocolytics may not affect uterine emg, but this should be further verified with larger studies. supported by grant nih r -hd . objective: calcium sensitizers are a novel class of drugs with unique molecular and phsiological actions. levosimendan, the best characterized of these compounds and is used in the treatment of acute and chronic heart failure. levosimendan can exert an inotropic effect via sensitization of myofilaments to calcium. it also exerts a relaxant effect on vascular smooth muscle through the opening of atp-dependent potassium channels and has been shown to be a potent inhibitor of human uterine contractions in vitro. for these reasons we investigated the effects of levosimendan on uterine contractions, both spontaneous and agonist induced, in the presence of glibenclamide, a k-atp channel blocker. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to glibenclmide ( mmol) followed by cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. control experiments were performed simultaneously. results: levosimendan exerted an inhibitory effect on spontaneous and oxytocin induced contractions in human m yometrium in vitro, in comparison to control experiments. the effect of levosimendan was significantly antagonized by glibenclamide with the mean maximal inhibition seen due to levosimendan greatly reduced (n= , p< . ). conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on human uterine contractility in vitro. this action was antagonized by glibanclamide and this study demonstrates that the effect of levosimendan on uterine smooth muscle is mediated at least in part through the k-atp channel. introduction: the determination of the beginning and ending points for "bursts" of electrical activity occurring during uterine contractions is sometimes difficult. if bursts cannot be discerned, the preferred burst-by-burst analysis cannot be performed. one solution to is to analyze any given electrical recording in its entirety. but this approach has often lead to meaningless results when traditional analytic methods are applied. chaos analysis, using lyapunov exponents, may provide an answer. materials and methods: term patients were included in the analysis: were in labor (group ), while were non-labor (group ). minute recordings were analyzed using "lyapunov exponent." for each pair of subsequent trajectories in phase space, only the most positive lyapunov exponent was calculated. the mean largest exponent was found by averaging over all of the trajectories in the recording. the lyapunov exponent is given in units of bits per data sample. thus a value of + means that the separation of nearby orbits doubles on the average in the time interval between data samples. the mean largest exponent was found for each patient recording. these values were compared using t-test (p < . considered significant). results: the mean and sd of the lyapunov exponent for all the patients was . ± . . moreover, the lyapunov exponent calculated for each patient was positive. comparing lyapunov exponents of the two groups showed a statistically low value (low chaos) for the laboring group, compared to the non-laboring group (figure) . conclusions: there is a chaotic component associated with uterine emg traces, since small but non-negative lyapunov exponents were found in all the traces observed. the lyapunov exponent indicated significantly lower chaotic behavior in the whole emg traces of patients who were in labor than found in those who were not in labor, implying that this measure could be a good diagnostic parameter for labor, possibly eliminating the need for tedious burst-by-burst analysis. supported by grant nih r -hd . comparing uterine emg to tocodynamometer for monitoring contractions. robert e garfield, lynette b mackay, sangeeta jain, william l maner. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine whether uterine electromyography (emg) plots contractions similarly to tocodynamometer (toco). study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. uterine emg signals were sampled at hz and band-pass filtered in the . to . hz range. root-mean-square (rms) function was calculated from the uterine emg signals in order to produce a "toco-like" trace from the original emg trace. emg-generated rms contraction plots were then compared to toco contraction plots using the following criteria: contractions were assigned a marker value of " ." in-between contraction periods were assigned a " ." from these marker values, contraction rates were established. correlation was found between the contraction rates of rms and toco. temporal overlap of contractions plotted by the two methods was used to find overall percent agreement (opa), positive percent agreement (ppa), and negative percent agreement (npa). these parameters were corrected for within-patient variation using a bootstrap method. results: uterine rms contraction plots were seen to correspond with toco contraction plots (fig. ) . corrected opa, ppa, and npa were high at . %, . %, and . %, respectively. there was a large, statistically significant correlation between uterine emg and toco contraction frequency (fig. ) . conclusions: the similarity between toco and uterine emg contraction plots (specifically, using rms to convert) will allow emg to be used interchangeably with toco in the clinic. supported by grant nih r -hd . introduction -this is a secondary analysis of women participating in a center randomized placebo controlled trial (rct) evaluating the impact of -ohpc in preterm birth (ptb) prevention among women with twins. objective -to evaluate the relationship between plasma -ohpc concentrations and gestational age (ga) at delivery in women with twins receiving weekly injections of -ohpc. methods -women with twins were randomized between - weeks to receive weekly im injections of either -ohpc ( mg) or placebo until weeks. after a minimum of five consecutive injections had been administered to assure steady state concentrations a plasma sample was collected between - weeks. the sample drawn just prior to the next scheduled injection was analyzed for -ohpc by hplc-ms in a blinded manner. the lower limit of quantification of -ohpc was ng/ml. we conducted univariate analyses to assess the association of -ohpc concentration and ga at delivery. we also conducted a proportional hazards model to evaluate the time from sample draw to spontaneous delivery (censoring indicated preterm deliveries), and a logistic regression to evaluate ptb< weeks; in both analyses we adjusted for bmi, race and ga at sample draw. results - women assigned to -ohpc were included; all received all of their scheduled injections. the concentration of -ohpc was significantly higher in women delivering < weeks compared with those women delivering > weeks (p= . , table) . concentration of -ohpc was significantly correlated with ga at delivery (r = - . , p= . ). each unit increase of -ohpc was associated with a % increased odds of delivering < weeks (odds ratio . , % ci, . - . , p= . ) and a % increase in hazard of spontaneous delivery (hazard ratio . , % ci, . - . , p= . ) after adjusting for confounders. gestational age at delivery mean (sd) -ng/ml < weeks (n= ) . ( . ) > weeks (n= ) . ( . ) conclusion plasma -ohpc concentrations after weekly injections were inversely related to ga at delivery in women with twins. since -ohpc induces its own metabolism it is possible that higher concentrations during initial treatment are associated with lower plasma concentrations and reduced efficacy in later pregnancy . clearly more studies are needed. objective: in many non-human species, maternal circulating progesterone levels fall prior to delivery, leading to the theory that in humans progesterone withdrawal occurs on a local and/or functional level. our objective was to characterize maternal and fetal progesterone in human preterm and term labor. methods: women between . and . weeks' gestation (cases) or term controls ( - weeks) with either labor with intact membranes or premature rupture of the membranes prior to labor (prom) were enrolled in a prospective case-control study. progesterone was measured by immulite assay in maternal serum collected upon enrollment and again within minutes after delivery and in umbilical cord serum obtained at delivery. maternal progesterone treatment was not used in any subjects. results: cases and controls were studied (see table for comparisons). controls p value ga at enrollment, weeks . ± . . ± . < . interval to delivery, days (median, range) ( - ) ( - . ) < . maternal progesterone at enrollment, ng/ml ± ± < . maternal progesterone after delivery, ng/ml ± ± . cord progesterone, ng/ml ± ± . among cases, fetal but not maternal progesterone was significantly lower in preterm labor with intact membranes ( ± ng/ml, n = ), as compared to prom ( ± , n = ), p< . . this difference increased further when cases of clinical chorioamnionitis were excluded. conclusions: serum progesterone in laboring patients prior to delivery is higher at term than in the preterm period, which may be attributable to increased placental mass in late pregnancy. this disparity disappears shortly after delivery of the fetus and placenta. fetal progesterone levels are several-fold higher than peripartum maternal levels. preterm labor with intact membranes is associated with diminished fetal progesterone, a phenomenon unrelated to clinical infection. these findings suggest the possibility of fetally regulated progesterone withdrawal as a mechanism underlying preterm labor with intact membranes. [snps] and ptb. however, many of these studies are inconclusive and non reproducible. the challenge of identifying robust associations between genetic variation and either susceptibility or protection from ptb is enormous. a systematic review of literature followed by metaanalysis was performed to understand true associations between snps and ptb. methods: for systematic review, articles were chosen based on medline and embase searches ( ( -april and relevant articles were chosen based on stringent inclusion criteria. primarily, studies reporting genetic associations between snps in maternal dna in singleton pregnancies and spontaneous ptb were included. other criteria included, but not limited to, provided genetic data in a complete enough format so that it could be evaluated in meta-analysis and defined the clinical outcome clearly. meta-analysis was performed wherever > replication data sets were available results: a total of abstracts were reviewed and were selected for full text review. data were extracted from articles. over associations were reported between snps on various candidate genes and ptb; however only had replication dataset. meta-analysis documented significant association between pon a g (odds ratio [or]= . ( %ci . - . ), pon (rs# )(or= . ; ci- . - . ), tnfrsf - a/g (fas) (or= . ; ci- . - . ) and ptb. two snps pon s c (or= . ; ci- . - . ) and ifn gamma (rs ; or- . ; ci- . - . ) documented protective effect. conclusions: systematic review concludes significant heterogeneities leading to lack of reproducible data in genetic association studies of ptb. heterogeneities are contributed predominantly by lack of adequate power, poor phenotype selection, and population admixture. the functional relevance of the risk and protective alleles needs to be verified. jignesh parvadia, mounira habli, jeff livingstone, william polzin, foong lim, timothy crombleholme. pediatric and thoracic surgery, cincinnati children's hospital medical center, cincinnati, oh, usa; obstetric and gynecology, university of cincinnati, cincinnati, oh, usa; obstetric and gynecology, good samaritan hospital, cincinnati, oh, usa. objective little is known about the response of ttts to treatment either by amnioreduction or selective fetospopic laser in triplet pregnancy, particularly the survival of the bystander fetuses. in order to define the response of triplet pregnancies to treatment for ttts we reviewed our experience with higher order multifetal gestations complicated by ttts. study design retrospective chart review of patients diagnosed with in high order gestation from - was performed. results among cases of ttts / ( . %) patients with high order gestations were identified (n= ) with a mean ga at diagnosis of . ± . weeks. pregnancies ( . %) were dichorionic triamniotic and ( . %) were monochorionic triamniotic. cincinnati modification of quinterro staging was utilized to characterize recipient cardiomyopathy as mild (stage iiia, n= ), moderate (stage iiib, n= ) and severe (stage iiic or iv, n= ) categories. / ( . %) were treated with amnioreduction alone (ar), / ( . %) with selective fetoscopic laser photocoagulation (sflp) alone, / ( . %) with ar followed by sflp and / ( . %) with ar followed by intrafetal radio frequency ablation (rfa). / ( . %) patient had a cervical cerclage. / ( . %) patients were treated but remain undelivered. mean ga at delivery was . ± . weeks. overall survival was / ( . %) with bystander survival was / ( %), donor survival / ( . %), recipient survival was / ( . %). conclusion triplet pregnancies treated for ttts have % survival rate for bystander fetuses and have . % survival rates for donor and recipients comparable to twins treated for ttts. ga at diagnosis . ± . weeks cincinnati modification of quinterro (i), (ii), (iii), (iiia), (iiib), (iv) ga at delivery . ± . weeks live birth -donor / ( . %)* # -recipient / ( . %)*# -bystander / ( %) # birth weight -donor to assess the effect of breast feeding (bf) on perinatal outcome in relation to maternal antenatal methadone dose. study design a retrospective chart review study of methadone dependent mother and infant pairs. patients were categorized into groups based on maternal dose at time of delivery: group : dose mg, group : dose - mg, group : dose > mg. the finnegan's scoring system was used to monitor neonatal abstinence syndrome(nas). treatment for nas was initiated if there were scores of . neonatal outcome data included:% nicu admission, % of babies discharged(d/c) at time of maternal d/c, % nas, % treated for nas and total hospital stay. data were analyzed by t-test and fisher's exact test. maternal characteristics between the groups were similar. regardless of maternal methadone dose, bf infants have shorter hospital stays and higher rates of d/c at time of maternal d/c, lower incidence of nas and fewer nicu admission (table) . in all three groups, breast feeding did not impact the severity of nas as reflected in finnegan's score(fs). (table) conclusion regardless of maternal methadone dose, breast feeding improves perinatal objective: to evaluate the effects of preventive collagen plugging of the fetoscopic access port at the time of balloon removal on pregnancy outcome in fetoscopic endoluminal tracheal occlusion pregnancies. study design: fifty-one pregnancies involving fetuses with severe congenital diaphragmatic hernia (cdh) were studied. all patients underwent feto between - weeks gestational age (ga) and fetoscopic balloon removal around weeks ga. at the time of balloon removal, a purified dried collagen plug was inserted through the fetoscopic access port in consecutive pregnancies but not in the first pregnancies considered as controls. all patients underwent post-plugging ultrasound and magnetic resonance imaging studies to evaluate for membrane dehiscence, amniotic fluid volume and fetal well being. ga at delivery, incidence of premature rupture of the membranes (pprom), bleeding at port retrieval and adverse fetal effects were compared in both groups. results: mean (sd) ga at feto [ . ( . ) vs. . ( . ) weeks; p= ns] and balloon removal [ . ( . ) vs. . ( . ) weeks; p= ns] was similar in the treatment and in the control group. incidence of pprom following the second fetoscopy was / in the study group compared to / in the control group (p< . ). mean (sd) ga at delivery was . ( . ) weeks in the study group, compared to . ( . ) in the control group (p= . ). bleeding from the trocar insertion site occurred in cases in both groups, but clinically significant bleeding occurred only in one of the controls. membrane dehiscence was noted in patient in the treatment group compared to in the control group (p=ns). conclusion: preventive collagen plugging of the fetal membrane defect created by the fetoscopic access resulted in a significant reduction in pprom rates and a trend towards increased ga at birth without adverse fetal effects in feto pregnancies. wider application of this technique should be considered, but needs evaluation in larger, randomized trials. hydrops fetalis is an uncommon fetal condition characterised by the abnormal accumulation of fluid in two or more body cavities, traditionally associated with a poor prognosis. the relative rarity of this presentation has meant that published case series have consisted of small numbers. a retrospective review of case notes of all cases managed at kemh between and was performed. in western australia, kemh is the only tertiary maternity hospital incorporating a maternal-fetal medicine unit. cases were obtained from the mfm database. in the period to there was a total of pregnancies affected by hydrops (incidence . per births). the average maternal age was years. in cases a fetal abnormality had occurred in a previous pregnancy. the median gestational age at diagnosis was weeks (range - weeks). in just over half ( %) of cases, the diagnosis was confirmed prior to delivery. a post-mortem was performed on all but of the babies not born alive. edema was present in at least cavities in over half of cases (n= ). chromosomal anomalies included trisomy , trisomy and turners syndrome. in all cases of infection, parvovirus b was implicated. cardiac arrythmias included svt and atrial flutter. cases classified as other included alpha thalassemia and syndromic disorders. in cases an interruption of pregnancy was performed at a mean gestational age of weeks. of those who did not elect to terminate the pregnancy, there were fetal deaths in utero, live borns with neonatal death. for the live borns, the median gestational at delivery was . weeks (range to . weeks). the causes of hydrops in live birth cases included cardiac arrythmia (n= ), infection (n= ), chromosomal abnormality (n= ), unknown (n= ) and other (n= (dmv) have been noted to play a role in the development of hemorrhagic and periventricular leukomalacic lesions in premature babies. since deep vein drainage system is relatively more prominent in the developmental brain than adult brain, we investigated if dmv anomalies could be associated with clastic lesions in-utero. methods two senior neuroradiologists reviewed fetal brain exam performed between and , seeking for unequivocal anomalies in dmv, such as periventricular venular engorgement. all mr scanning is performed at . tesla, using surface abdominal coils and single-shot fast spin-echo t -weighted - mm thick sections, with . - . mm in planar spatial resolution. we found cases with dmv anomalies (tab. ). most of the dmv engorgement is located at frontal lobes level. from this limited preliminary series it appears that dmv involvement plays a role in the development of periventricular leukomalacia and periventricular hemorrhagic necrosis. the observation that these lesions are mostly located at frontal level may suggest that some of the term neonates carrying sequelae of atypically located leukomalacia (i.e. deep frontal lobe) might have developed these lesions in-utero. it is of interest to notice that most of our cases were related to heart failure. therefore, central venous hypertension affecting immature deep cerebral venous system has to be taken into account. in our center, patients with an estimated risk for chromosomal abnormalities at term greater that in after st trimester combined screening test (ft) are offered non-directive genetic counseling. the aim of this study was to evaluate the responses of women younger than attending this genetic counseling session. material and methods: data from patients referred for a positive ft from september , to july , was retrieved from our database. information concerning women younger than years of age at the estimated date of delivery was extracted and tabulated. results: during the study period women had genetic counseling for positive ft. thirteen patients were excluded from further analysis ( had incomplete clinical documentation and had spontaneous miscarriages prior to weeks gestation). four hundred and twenty-five patients were older than and were younger than at the estimated date of delivery. table depicts summary statistics for studied variables in this younger group of women. conclusions: overall this data suggests that approximately % of this younger group of women opted for chorionic villous sampling (cvs), % for amniocentesis and more than % declined prenatal genetic testing. moreover, this data also suggests that: ) these women opted for cvs when the ft risk (mean = in ) almost doubled the cvs procedure related risk quoted at % and, ) when the ft risk is between in and in almost half ( out of ) declined not only the st trimester cvs but also the nd trimester amniocentesis. we believe that understanding our patient population is important to optimize both the efficiency and efficacy of the alternative prenatal screening programs. acceptance for prenatal genetic testing after a positive first trimester combined screening test in women of less than to determine the optimal diagnostic test using prenatal ultrasonography and mri for predicting pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. methods: relevant papers were identified by searching medline ( medline ( - , embase ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the cochrane library ( issue ). in addition, the specialist literature on the topic and reference lists were hand searched for relevant articles. studies were selected if they examined diagnostic tests for the prenatal prediction of pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. the primary outcome measure was perinatal survival. study selection, quality assessment and data abstraction were performed independently and in duplicate by separate observers. results: of a total number of articles (published studies), there were eighteen studies that fulfilled the entry criteria. six examined entirely unique heterogeneous parameters. of the remainder, all examined the ultrasound measurement of lung to head ratios (lhr) at a 'cut-off of greater than or less than the thresholds of . , . , . , . . in addition, the presence of liver in the fetal thorax was included (if present) as a co-variable (liver "up"). a lhr > . compared to < . provided the strongest association with perinatal survival (peto or . , % ci . - . ). the finding of "liver up"in the fetal thorax had a negative association with survival (peto or . , % ci . - . ). only three studies provided data for lhr in conjunction with the presence of liver in the fetal thorax (peto or survival for lhr > . compared to < . was or . , % ci . - . ). data was also available for liver up and lhr > . which had a peto or of . ( % ci . - . ). discussion: the data supports the view that lhr may be a useful prognostic indicator of perinatal survival in fetuses with congenital diaphragmatic hernia. however, heterogeneity still exists regarding the timing of ultrasound measurement and the use of mri. the majority of studies have small sample sizes. objective: to estimate, in fetal anaemia, the diagnostic value of fetal ultrasonography and doppler blood flow in the evaluation of fetal anaemia methods: literature from to was identified using medline and embase, the cochrane library and relevant specialist register of the cochrane collaboration, and by checking reference lists of known primary studies and review articles. studies were selected if the accuracy of the fetal ultrasound parameters or doppler studies of blood flow in the fetal vessels was estimated compared with a reference standard. diagnostic tests evaluated were ultrasound measurement of the fetal spleen and liver length and doppler studies from the umbilical vein and middle cerebral artery. data from the selected studies were abstracted as x tables comparing the diagnostic test result with the reference standard. results were pooled where appropriate. diagnostic accuracy was expressed as sensitivity and specificity. twenty-nine primary studies were identified containing suitable data. twentyone of these gave data on middle cerebral artery doppler peak systolic velocity (mca-psv) and could be pooled in the meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases in detecting severe anaemia. four studies gave data on spleen perimeter and it was possible to pool three of these giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases. three studies had data for liver length measurements and two were pooled. the sensitivity was . ( . - . ) and the specificity was . ( . - . ) but only cases were used in the analysis. there were three studies on umbilical vein maximum velocity doppler studies and all were suitable for meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) with cases analysed. two studies gave data on middle cerebral artery time-averaged mean velocity score giving cases. the sensitivity was . ( . - . ) and specificity was . ( . - . ). discussion: middle cerebral artery peak systolic velocity dopplers remain the gold standard for non-invasive screening of fetal anaemia. middle cerebral artery time-averaged mean velocity scores require further investigation. other tests perform poorly when diagnostic accuracy is assessed. cochrane review of treatment in twin to twin transfusion syndrome. twin-twin transfusion syndrome is a condition affecting monochorionic twin pregnancies associated with a high risk of perinatal mortality and morbidity. the objective of this review was to evaluate the impact (maternal,fetal and pediatric)of treatment modalities in twin-twin transfusion syndrome. register and cochrane controlled trials register. we also searched conference proceedings and made personal contact with experts active in the area of the review. randomised and quasi-randomised studies of amnioreduction versus laser coagulation, septostomy versus laser coagulation or septostomy versus amnioreduction. eligibility was assessed by one reviewer. study authors were contacted for additional information. two studies were included. this review shows that laser coagulation of anastomotic vessels results in less fetal (rr . ; % ci . , . ) and neonatal deaths (rr . ; % ci . , . ) than amnioreduction ( figure ). there is no difference in perinatal outcome between amnioreduction and septostomy. more babies in the laser arm are alive without neurological abnormality at six months of age (developmental delay at < years rr . , % ci . , . )( figure ). conclusions: endoscopic laser coagulation of anastomotic vessels should be considered in the treatment of all stages of twin twin transfusion syndrome to improve perinatal outcome. further research on the effect of treatment on milder forms of twin twin transfusion syndrome (quintero stage and ) are required. (produced in collaration with the cochrane collaboration, uk). (cvs) concluded that although the risks of pregnancy loss are relatively low, lack of adequate controls tends to underestimate the true added risk of prenatal invasive procedures (obstet gynecol. ; ( ): - ). the west midlands is a large region within the uk containing approximately % of the total uk population. the congenital anomaly register for this region is able to monitor pregnancy outcomes with accurate deminator data. results: there were first trimester cvs performed, by ten operators, in the west midlands (uk) in . this equates to . procedures per births. significantly higher rates were noted in areas of high socioeconomic status. the median number of procedures performed per operator was (range - ). all operators were performing other invasive tests such as amniocentesis or cordocentesis,etc. the most common indication for cvs was: i) fetal anomaly on dating scan ( . %) ii) abnormal (> in ) risk on combined first trimester screening ( . %) iii) molecular genetic diagnosis ( . %) and iv) maternal request ( . %). using a combination of first trimester scanning and cvs, % had abnormal karyotype/structural anomaly.the corrected loss rate (background and procedure-related) following cvs in normally formed, singleton pregnancies was . % ( thci . - . %) up to days postnatal (perinatal loss) and . % ( thci . - . %) with days of procedure. the proportion of cvs in which an adequate sample was not obtained was . % ( th ci . - . %). conclusions: this epidemiological study using accurate demoninator data provide interesting statistics relating to the uptake and prenatal risks of first trimester cvs. with the increasing prevalence of obesity in the last two decades, we have seen a tremendous increase in bariatric procedures in reproductive aged women. malnourishment and vitamin deficiency are common complications after gastric bypass which may impact on fetal development. we present the case of a yo who underwent a duodenal switch procedure in . she was discovered to be pregnant during evaluation for persistent malnutrition in . multiple prenatal ultrasounds were performed; the first at weeks gestation was unremarkable. the week sono revealed a male fetus with shortened femurs and humeri bilaterally, nasal bridge hypoplasia, macroglossia, poorly defined hands, and possible clubbed feet. amniocentesis revealed a normal karyotype. d ultrasound redemonstrated the abnormal facial findings. a fetal echocardiogram was normal. the lagging long bone measurements continued to worsen, ultimately with femurs weeks behind. the fetal thoracic circumference was two standard deviations below the mean, giving rise to concern for pulmonary sequelae. growth restriction was noted at the wk sonogram. delivery by cesarean section was at / weeks secondary to nonreasuring fetal status. the birthweight was gm; apgars were , , and . postnatal radiographs confirmed antenatal ultrasonographic findings and demonstrated evidence of epiphyseal stippling. the infant remained intubated until weeks of life, after which it died secondary to respiratory complications associated with pulmonary hypoplasia. gene mapping studies have not found any point mutations on the recessive gene as an etiology of this disorder. rhizomelic chondrodysplasia punctata refers to a heterogeneous group of bone dysplasias with a familiar radiographic phenotype involving punctate calcifications and epiphyseal stippling. the etiology of this may be secondary to chromosomal abnormalities, mendelian gene disorders, or teratogens, notably warfarin. this case may be explained by vitamin k deficiency of the embryo due to maternal malabsorption after bariatric surgery. the maternal vit k level was <. ng/dl at time of delivery(normal > . ). the teratogenic effects of vitamin k deficiency in this instance highlight the need for strict counseling and screening for vitamin deficiency in those women undergoing bariatric surgery since previous obesity-related anovulation is reversed as patients lose weight, resulting in unexpected pregnancies and potentially preventable fetal abnormalities. term preeclampsia: any risk for the neonate? sindhu k srinivas, jamie bastek, christina m andrela, emmanuelle pare, michal a elovitz. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: preeclampsia continues to be a major contributor to maternal morbidity and mortality worldwide. preeclampsia at term is not associated with the same risk of neonatal morbidty and mortality as preterm preeclampsia . however, neonatal outcomes in term women with preeclampsia have not been adequately studied. we sought to compare short term neonatal outcomes in term infants born to women with and without preeclampsia. methods: this study was part of a large case control study. women with preeclampsia (n= ) and term controls (n= ) were prospectively identified. infants with congenital anomalies were excluded. hospital length of stay (los), admission or transfer to the nicu, and use of mechanical ventilation or cpap within first week of life were assessed. associations between neonatal outcomes and preeclampsia were evaluated using chi-square analysis and wilcoxon rank sum test. significant confounders were controlled for using multivariable logistic regression. results: discharge day of life was significantly different between neonates born to women with preeclampsia (median = ; mean = . ) versus those born to women with uncomplicated term deliveries (median = ; mean = . , p< . ). this difference persisted even when neonates with iugr and those born to diabetic mothers were excluded (p< . ). term infants born to women with preeclampsia have a higher odds of being admitted or transferred to the nicu (aor= . [ . - . ], p= . ) after controlling for iugr, delivery mode, race, and gestational age at delivery. these infants also have a higher odds of mechanical ventilation (aor= [ . - . ], p= . ) and cpap use (aor= . [ . - . ], p= . ) after controlling for the same confounders. there was no difference in ivh or nec between the two groups. conclusion: neonates born to women with preeclampsia have differences in short-term morbidity when compared to neonates born to women without preeclampsia, despite being born at term. whether this increase in neonatal morbidity is attributable to medications used in preeclampsia, such as magnesium sulfate, is unclear. these findings may have implications for patient counseling as well as hospital resource allocation. further investigation correlating these findings with long-term morbidity is warranted. could confined placental mosaicism account for adverse perinatal outcomes in ivf pregnancies? benoit c jacod, gh schuring-blom, kd lichtenbelt, jse laven, d van opstal, mjc eijkemans, nick s macklon. reproductive medicine gynaecology, university medical centre, utrecht, netherlands; clinical genetics, university medical centre, utrecht, netherlands; obstetrics gynaecology, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background: ivf singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. this may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. aim: to compare the incidence of confined placental mosaicism (cpm) in ivf/icsi pregnancies and spontaneous conceptions. methods: multicentre retrospective analysis of karyotype results obtained by chorionic villus sampling (cvs) performed because of advanced maternal age ( years at weeks of gestation) in the netherlands between and . results: from a total of pregnancies, cvs results were analysed: in the ivf/icsi group and in the control group. the mean age of women in both groups was . years (mean difference - . , % ci - . - . ). foetal karyotype was missing in cases of possible cpm, all in the control group. when taking into account missing data, the incidence of cpm was lower in the ivf-icsi group than in the control group, . % vs. . % (odds ratio . , % ci . - . ) whereas the incidence of foetal chromosomal anomalies was increased . % vs. . % (odds ratio . , % ci . - . ) although both differences are not significant. conclusions: the incidence of confined placental mosaicism is not increased in ivf/icsi pregnancies compared to spontaneous conceptions. cpm probably does not account for the adverse perinatal outcomes following ivf/icsi. fetal rh d, cc and ee genotyping using fetal dna from maternal blood is not impaired by the presence of maternal alloimmunization. chad a grotegut, stacey l jeronis, john p gaughan, enrique hernandez, ossie geifman-holtzman. obstetrics and gynecology, duke university, durham, nc, usa; obstetrics, gynecology and reproductive sciences, temple university, philadelphia, pa, usa; biostatistics, temple university, philadelphia, pa, usa. this study was conducted to assess the impact of maternal alloimmunization on the accuracy of fetal rh d, cc and ee genotyping from maternal blood. we performed a literature search of english-written articles describing fetal rh d, cc and ee determination from maternal blood (am j obstet gynecol ; : - ) . using this database, we determined the accuracy of rh d, cc and ee genotyping in the presence of maternal alloimmunization. for each subgroup, % confidence intervals for a proportion were calculated and compared between groups. we found english-written publications reporting non-invasive rhd genotyping and reporting non-invasive rh ce genotyping from maternal blood. fourteen ( . %) of the rh d articles and three ( %) of the rh ce articles provided accuracy results in the setting of alloimmunization. the accuracy results are reported as follows: . % of the samples were determined correctly in the presence of alloimmunization.* this accuracy was significantly lower than the accuracy reported for all rh cc samples. when only studies utilizing free fetal dna for rh cc genotyping were used (vs fetal cells), fetal cc genotype was determined correctly in / ( %, % ci . , ), which was similar to the overall success rate for rh cc determination. overall, there were no differences in the success of rh d, cc or ee determination in the setting of alloimmunization compared to the overall accuracy seen when free fetal dna was used. the presence of maternal alloimmunization does not reduce the accuracy of fetal rh d, cc or ee non-invasive genotyping from maternal blood utilizing free fetal dna. further research into structure and rearrangements of the rh d, cc and ee genes may further improve diagnostic accuracy of free fetal dna from maternal blood. fetal dna, most likely of trophoblast origin, is present in both the plasma of pregnant women and provides a potential basis for non-invasive fetal diagnostic tests. however, fetal dna in maternal plasma is highly contaminated with maternal dna, and this contamination is the main technical challenge in trying to accomplish non-invasive detection of fetal chromosome abnormalities. existing methods for the selective amplification of fetal dna have generally relied on specific sequence differences between the mother and fetus. as an alternative, we have developed a method for selective amplification of fetal dna that makes use of observation that trophoblast dna is globally hypomethylated in comparison with dna from other sources. in this method, a dna mixture is first digested with a methylation sensitive restriction enzyme and then amplified by linker-mediated pcr. after an initial amplification, a second isothermal rolling-circle amplification is performed. this procedure results in the differential amplification of short, relatively hypomethylated fragments. after amplification, the resulting "representations" are comparatively hybridized to a microarray consisting of oligonucleotides that correspond to restriction fragments generated by the initial digest. copy number differences are then detected through statistical analysis of array addresses that show significant amplification. to test the feasibility of this method for detecting aneuploidy, we have prepared mixtures of peripheral blood dna and first trimester trophoblast dna from either normal or from samples with trisomy and trisomy . we present data showing that aneuploidy can be detected even when % of the starting dna sample was derived from a euploid source and only % was from an aneuploid trophoblast sample. two different approaches to data analysis are presented. one relies on prior analyses of trophoblast methylation and the second is independent of any prior knowledge or analysis. both methods provide similar ability to detect aneuploidy. future work will focus on testing whether this approach can be used for non-invasive prenatal diagnosis. chromatin immunoprecipitation and emsa analysis of nf-b and c/ ebp synergism on the otr promoter. shirin khanjani, yun s lee, vasso terzidou, mark r johnson, phillip r bennett. institute of reproductive developmental biology, imperial college, london, united kingdom. we have shown, the transient transfections of the transcription factors nf-bp and c/ebp leads to a synergistic increase in otr promoter activity in human myocytes. this effect is mediated through a bp region of the promoter between - to - from tss. we now report that this sequence binds both nf-bp and c/ebp in vitro and chip studies show binding of both transcription factors to be increased by il- in vivo. materials and methods: emsa studies were performed using a bp oligonocleotide sequence (- to - ) , containing the bp region responsible for the synergistic activation of the otr promoter and nuclear extracts from primary human myocytes treated with il- ng/ml for hours. for chip analysis, dna protein complexes were crosslinked and antibodies recognizing p , c/ebp and h (positive control) and igg (negative control) were used for immunoprecipitation. primers were designed to amplify the region - to - , which includes the bp response sequence. to further confirm the interaction between nf-bp with c/ebp a xnf-b consensus/luc reporter construct was cotransfected with an expression vector for either nf-bp or c/ebp . results: specific nf-bp and c/ebp binding was seen in the emsa study. preincubation with antibodies to nf-bp and c/ebp led, not to supershift, but to elimination of dna binding for both nf-kb p and c/ebp . chip analysis confirmed increased in vivo binding of nf-b p and c/ebp to this region of the otr promoter following stimulation with il- . transfection of the nf-b/luc reporter construct with an expression vector for c/ebp caused a significant reduction in the basal promoter activity suggesting that c/ebp is binding to nf-kb. this interaction was further confirmed using a tf-tf interaction array. conclusion: these data support the role of nf-b and c/ebp in regulation of otr. the bp region contains a c/ebp but not a nf-b dna binding site suggesting that c/ebp primarily binds to this part of the otr promoter but also interacts with nf-b. the emsa data shows that the bp region binds both nf-b and c/ebp . the loss of the supershift observed in previous emsa studies suggests that the antibodies inhibit the interaction between c/ebp and nf-b, therefore inhibiting dna binding. chip analysis supports the concept that il- leads to binding of nf-b and c/ebp to the bp region. regulation of pro-labour genes by c/ebp, nf-b and ap- in human uterine myocytes. suren r sooranna, shirin khanjani, yun s lee, phillip r bennett, mark r johnson. imperial college parturition research group, imperial college, london, united kingdom; irdb, imperial college, london. introduction: the transcription factors c/ebp (lap), nf-b (p ) and ap- (c-fos and c-jun) are implicated in inflammatory processes such as parturition. the promoter regions of the pro-labour genes il- , pghs- and otr contain putative transcription factor-binding sites for these transcription factors. our aim was to determine the effect of transfecting these transcription factors either alone or in combination into uterine myocytes and to determine their effects on the expression of pro-labour genes including il- , pghs- , otr, connexin- and fp. methods: primary cultures of human uterine myocytes (n= ) were grown from myometrial samples obtained at the time of elective lscs. cells were cultured in well plates to % confluence at which point expression constructs for c/ ebp (lap), nf-b(p ), ap- (c-fos and c-jun) were transfected either alone or in different combinations. the empty expression vector psg was used as a filler construct. cells were cultured for and h after which culture medium was collected for elisa and cells frozen at - °c prior to rna extraction. copy numbers of il- , pghs- , otr, fp, connexin- and gapdh were measured by qpcr using a rotor-genetm (corbett research). results: h post transfection with c/ebp (lap), nf-b (p ), c-fos and c-jun alone or in combination showed no significant changes in pghs - , otr and connexin- expression. over expression of p alone or together with either c-fos or c-jun increased fp expression by , and % respectively (p= . ). nf-bp consistently increased il- expression either alone (by %; p= . ) or in combination with lap, c-fos or c-jun (by , and % respectively; n= ; p= , ). rel a, lap, c-fos and c-jun together also increased il- expression (by %; p= . ) and a small but significant increase was seen with a combination c-fos and c-jun (by %; p= . ). the changes observed in il- expression were reflected in the medium il- concentration at h post transfection. in the presence of rela and c-fos il- increased from . ± . to . ± . pg/ml of culture medium (mean ± sem; n= ). conclusions: nf-b (p ) consistently increased fp and il- expression in human myometrium. the data suggest that pghs- activation has greater dependence upon other transcription factor(s) in addition to p . true identity of myometrial pr-c: fact or fiction? yun s lee, suren r soorana, mark r johnson, jan brosens, phillip r bennett. imperial college parturition research group, hammersmith campus, london, united kingdom. progesterone is thought to be central to maintenance of pregnancy. multiple progesterone receptor (pr) isoforms underlie complex and diverse biological action of progestins. previously two human pr isoforms have been identified: pr-b and pr-a. pr-a is n-terminally truncated form of pr-b (initiation site methionine ). in some cells pr-a inhibits pr-b. it has been proposed that increased expression of pr-a in myometrium underlies a 'functional progesterone withdrawal'. the breast cancer cell t d contains a kda progestin-specific binding protein that is not found in pr negative cells. it was proposed that there is a downstream methionine (met ) which serves as a translation initiation site for the generation of a pr isoform of kda. based on such findings condon et al (mol endo ) have focused on the possible role of kda pr-c in human parturition. they found that expression of "pr-c" using pr antibody (sc- santa cruz, sc) is increased in upper segment myometrium with labour and that overexpression of this protein inhibits pr-b function. we cloned the same human pr cdna into psg expression vector. in vivo translation produced a protein of only kda. furthermore overexpression of pr-c did not significantly decrease pr-b activity in human myocytes. we examined other downstream initiation sites, which may produce a kda protein. we constructed potential pr isoforms in psg vector with initiation sites at met and . in vivo translation produced proteins of approximately and kda respectively. to determine the effect of pr isoforms on pr-b function, myocytes were co-transfected with pr-a, pr-c , pr-c and pr-c with constant amounts of pr-b. the progesterone dependent pre/luc was used as reporter. unlike pr-c both pr-c and pr-c significantly inhibited ligand dependent pr-b mediated transcriptional activity. we found in western analysis that the antibodies pgr- (nc) and the sc- (sc) detected both endogenous and overexpressed pr-a and pr-b but none of the pr-c isoforms. the sc- antibody detected only pr-a and pr-b very poorly. our data suggests that the sc- antibody would not detect any pr-c protein and that none of the commercially available antibodies in the uk do so. great care needs to be taken when over-expressing pr isoforms to ensure that proteins are of the expected size. if pr-c does exist in vivo then the kda but not the kda isoform might inhibit pr-b function. tnf receptor antibody (tnf ri ab), nf-b inhibitor (nf-b activation inhibitor) and erk inhibitor (u ) (p< . ), but not by tnf receptor antibody (tnf rii ab), p mapk inhibitor (sb ) and jnk inhibitor (sp ). by western blot analysis, we found that the protein level of tnf receptor associate factor (traf ) was higher than that of tnf receptor associate factor (traf ) (traf >traf ) in untreated ct cells. however, after tnf treatment for h to h, traf protein level was increased, but traf protein level was reduced (traf >traf ). the increase of traf and decrease of traf were blocked by tnf ri ab, but not by tnf rii ab. we also found that tnf rapidly (within - min) and significantly increased phosphorylation of ikk , erk / and jnk / / and the phosphorylation of these protein kinases by tnf was reduced significantly by tnf ri ab, but not by tnf rii ab. moreover, we found that the changes of increased traf and decreased traf in ct cells (traf >traf ) resulted in a dramatic deficiency in phosphorylation of the above protein kinases induced by tnf compared with the normal ct cells (traf >traf ). nuclear localization of nf-b p in tnf treated cells was increased compared to untreated controls. conclusion: we have demonstrated for the first time that tnf stimulates mmp- production in ct cells through tnf ri-trafs-ikk /erk-nf-b signaling pathways, but not through the jnk/p -ap- pathway. these studies reveal steps within this pathway as possible therapeutic targets to inhibit mmp- expression potentially attenuating tnf -induced degradation of extracellular matrix and pre-term rupture of the fetal membranes. objective: women with preterm birth are at elevated risk of cardiovascular disease, but mechanisms that might relate these conditions are not understood. we hypothesized that women with spontaneous preterm vs. term births may have early gestation evidence of activation of the fibrinolytic cascade, as measured by the thrombin-antithrombin iii (tat) complex. we also tested if this relation may be associated with inflammation. methods: tat was measured in plasma collected < weeks gestation (mean . weeks, sd . ) among women without chronic medical conditions, preeclampsia or growth restriction who delivered singleton liveborn infants. inflammation was assessed by c-reactive protein (crp) measured in serum from the same samples. women with spontaneous preterm birth (sptb) < weeks (n= ) and -< weeks (n= ) were compared to women with term births >= weeks (n= ). high tat was defined as > . ng/ml (highest quartile among women with term births) and high crp was defined as >= ug/ml. multinomial logistic regression was utilized to relate elevated tat and inflammation to risk of sptb subtypes. results: women with sptb were more likely to have tat concentrations in the highest quartile compared to women with term births (< weeks, . %; -< weeks, . %; >= weeks . %, p< . ). women with high tat concentrations had a . -fold ( % ci: . - . ) increased risk for sptb < weeks, after adjustment for body mass index, race, age and gestational age at sampling. there was no relation between high tat and sptb -< weeks (or . , % ci . - . ). additional adjustment for elevated crp (>= ug/ ml) did not effect the estimates associated with tat, and elevated crp was independently related to risk for both sptb subtypes (< weeks, or . [ . - . ]; -< weeks, or . [ . - . ]). thus, women with high tat and elevated crp appeared to be at particularly elevated risk of sptb < weeks (or . , % ci . - . ). conclusions: the thrombin-antithrombin iii complex was elevated early in gestation among women with sptb < weeks, perhaps secondary to microvascular injury. this effect was independent of inflammation, suggesting that the elevated fibrinolytic cascade may function independently from inflammation among women with sptb. plasma cortico-releasing hormone and cortisol concentrations and psychological stress among pregnant women. katherine p himes, hyagriv n simhan. obstetrics and gynecology, university of pittsburgh medical center, magee womens hospital, pittsburgh, pa, usa. objective: many studies have found an association between psychological stress and preterm birth. we sought to determine if women with greater psychological stress during pregnancy had higher concentrations of plasma cortico-releasing hormone (crh) or cortisol. study design: this is a secondary analysis of a multicenter case-control study, nested within an observational cohort. of , participants, plasma crh and cortisol concentrations at and weeks gestation were available in controls who delivered after weeks and cases who delivered before weeks. the abbreviated scale for the assessment of psychosocial status in pregnancy (asaps) was available for all women. concentrations of crh and cortisol were compared between women above and below the lowest quartile score on the asaps among cases and controls. the same analysis was done for the portion of the scale related to psychological stress. concentrations of crh and cortisol and psychological stress were also compared between black and non-black cases and controls. univariate analysis was performed with kruskal wallis or chi-square. results: there was no difference in crh or cortisol concentrations at or weeks among women above or below the lowest quartile on the asaps (controls:p= . - . cases:p= . - . ). greater psychological stress was not associated with higher concentrations of crh or cortisol at or weeks(controls:p= . - . cases:p= . - . ). crh concentrations were not different between blacks and non-blacks. among both cases and controls, cortisol concentrations at and weeks were lower in black women than non-black women (controls:p< . cases:p< . ). the median cortisol concentration among control black women was . g/ml at weeks compared to . g/ml among non-black women and . g/ml compared to . g/ml at weeks. black women reported less psychological stress than non-black women (p= . ) conclusion: we found no relationship between psychological stress and plasma crh or cortisol. furthermore, while stress is hypothesized to play a role in the racial disparity of preterm birth, black women reported less psychological stress and had lower cortisol concentrations than non-black women. improved assessments of psychological stress and additional biomarkers involved in the stress response may broaden our understanding of how stress contributes to preterm birth. expression, tissular traffic and activation of mmp- in human fetal membranes during labor. rodrigo vega-sanchez, arturo flores, marisol castillo, nardhy gomez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico. introduction. rupture of the fetal membranes (fm) during human labor occurs as a consequence of extracellular matrix degradation. this process is controlled by increased secretion and activity of matrix metalloproteinases, particularly mmp- .several evidences suggest that mmp- is mainly produced by infiltrating choriodecidual leukocytes that could arrive from placental circulation. characterization of the synthesis, transport and activation of mmp- within the fm is critical to understand the process of membrane rupture during human labor. objectives. expression and secretion of mmp- in placental leukocytes, trafficking of the enzyme through the fm and one possible mechanism for its activation were analyzed. methods. leukocytes were isolated from placental blood of women after active labor. maternal leukocytes were used as controls. cells were cultured for h. relative expression of mmp- by rt-pcr and enzyme secretion by elisa and zymography were followed at different times. to analyze the traffic of mmp- through the fm, fluorescein-conjugated prommp- was added to the choriodecidual side of the fm in an in vitro system that allows the separation of amnion and chorion. labeled mmp- was localized at distinct times by confocal microscopy. the protease responsible for the activation of mmp- was identified using neutralizing antibodies and specific inhibitors. results. no difference in the relative expression of mmp- in leukocytes throughout the culture was found. however, secretion of the enzyme significantly increased since h (p< . ). experiments using labeled mmp- , repeatedly showed that after h in culture, the enzyme was mainly localized within the amniotic epithelium. specific inhibition of mmp- significantly decreased the activation of pro-mmp- . conclusions. our results demonstrate that the increased secretion of mmp- by placental leukocytes is not associated to increased gene expression, suggesting that homing of a specific leukocyte subpopulation to the choriodecidua is occurring during labor. mmp- can be trafficked from the choriodecidua to the amnion, suggesting a transmembranal pathway that may regulate the tissular localization of the enzyme to the area of the fm with high content of connective tissue. once secreted by the placental leukocytes, activation of mmp- depends mainly on mmp- , which seems to be derived from the same leukocytes. objective: preterm labour is a major problem in terms of perinatal morbidity and mortality. the histone-deacetylase inhibitor (hdaci), trichostatin a (tsa) has been shown to have an inhibitory effect on myometrial contractility. the aim of this study was to evaluate the effect of the hdaci's suberic bishydroxymate (sbha) and valproic acid (vpa) on human uterine contractions and hence their potential role as tocolytic agents. methods: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of sbha in the concentration range of nmol/l to mmol/l and vpa ( nmol/l- mmol/l). control experiments were run simultaneously. results: sbha and vpa exerted a potent and cumulative inhibitory effect on spontaneous and oxytocin-induced contractions, compared to control strips. the mean maximal inhibition (mmi) values for sbha were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). the mmi values for vpa were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). conclusion: these results raise the possibility that hdaci's may have tocolytic potential, in addition to their current clinical indications. the inhibitory effect observed may be linked to the ability of hdac inhibitors to induce the expression of genes involved in the maintenance of myometrial quiescence via epigenetic mechanisms but may potentially also involve non-epigenetic pathways. progestin suppresses thrombin-enhanced interleukin- expression in term decidual cells: implications for abruption-induced preterm delivery. edward kuczynski, lynn f buchwalder, frederick schatz, charles j lockwood. obstetrics/gynecology reprod. sciences, yale university school of medicine, new haven, ct, usa. background and objective: decidual hemorrhage (abruption) promotes binding of factor vii to decidual cell (dc)-expressed tissue factor to generate thrombin. thrombin in turn induces several biological effects leading to preterm delivery via activation of cell surface protease-activated receptors (pars). interleukin- (il- ) is a pleiotropic proinflammatory cytokine induced by pars. this study assessed the separate and interactive effects of thrombin and medroxyprogesterone acetate (mpa) on il- expression in term dcs. methods: term decidua from stripped fetal membranes were isolated and the dcs were purified on a percoll gradient, grown to confluence and passaged until leukocyte-free. confluent dcs were primed in - m estradiol (e ) of e + - m mpa for days, then incubated in a defined medium (dm) with corresponding steroids ± thrombin. after hours, il- levels in conditioned dm were measured by elisa and western blotting. in parallel -hour incubations, il- mrna levels were assessed by quantitative rt-pcr and normalized to -actin mrna. results: secreted il- levels were similar in cultures maintained in e alone ( . ± . ) and e + mpa ( . ± . pg/ml/ug protein; mean ± sem; n = ). the addition of thrombin ( . u/ml) enhanced secreted il- levels by . ± . fold (p< . ) in incubations with e and by . ± . -fold (p< . ) in incubations with e + mpa. the inhibitory effect of mpa was statistically significant (p< . ). in confluent dcs incubated with e + mpa, exogenous thrombin ( . - . u/ml) elicited a concentration-dependent increase in secreted il- levels. hirudin acted as a pure thrombin antagonist, exerting no agonist effects alone, but counteracting thrombin-enhanced il- secretion. western blotting confirmed the elisa results. quantitative rt-pcr confirmed that il- m-rna levels corresponded to protein changes. conclusion: thrombin enhances il- mrna and protein expression in term dcs and progestin blunts these effects. since thrombin-generating abruption is closely associated with preterm delivery, anti-inflammatory effects induced by progestin inhibition of dc-derived il- may contribute to the protection against ptd, and may explain the reported protective effects of administration of -oh-progesterone in recent clinical trials. introduction: catechol-o-methyltransferase (comt) catalyzes the methylation of the phenolic hydroxyl groups in a variety of catechols. during estrogen metabolism, this enzyme converts the catechol estrogen, -hydroxyestrogen ( ohe ), to -ethoxyestrogen ( meohe ). the comt substrate, -ohe , can exhibit an anti-estrogenic effect in multiple biologic assays while the methoxyestrogen ( -meoe ) can exhibit an estrogenic effect. the biologic activities of these estrogen metabolites ( ohe meoe) depend upon their concentrations and tissue type. since comt activity ultimately controls levels of these metabolites, it appears to be a key factor in regulating the cellular estrogenic milieu. we have recently reported that amnion layers of human fetal membranes from laboring women exhibited folds higher comt mrna expression when compared to non-laboring women (wentz et. al. sgi ) . objective: to investigate the impact of comt inhibition on prostaglandin e (pge ) production by human amniotic membrane explants. study design: explants consisting of -cm circular sections of the amnion layer (obtained from term pregnant women who underwent elective repeat cesarean section) were prepared and placed in tissue culture explants media at ºc. after a -hour incubation, explants were treated with the selective comt inhibitor ro - , at and m concentrations. the incubation media was harvested after and -hour intervals. the levels of prostaglandin e (pge ) in the media were measured by sensitive elisa and were normalized against total protein concentration. results: ro - comt inhibitor induced major reductions in pge production in media collected from amnion explants of human fetal membranes. in the group treated with m of ro - for hours there was %± % reduction of pge after hours compared to untreated control (p< . ). in the amnion explants treated with m of ro - , there was %± % after hours and %± % after hours of treatment compared to untreated control (p< . ). conclusions: this finding indicates that comt activity in the amnion layer of human fetal membranes affects pge production. by facilitating a pro-estrogenic milieu in human fetal membranes in late gestation, increased comt activity may indirectly increases production of factors associated with labor such as pge . hypothesis: an extensive remodeling of the human cervical connective tissue takes place throughout pregnancy with a decrease in the total concentration of collagen and proteoglycans due to an altered higher metabolic turnover. we hypothesize that the profound changes in proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. we also hypothesize that proteoglycan production in cervical fibroblasts from preterm partal women are simmilar to the production in fibroblasts from partal women. method: cervical biopsies were obtained from non-pregnant women, women during elective cesarean section, woman after spontaneous parturition and after a preterm vaginal delivery. by explant technique fibroblasts were cultured from the biopsies. produced proteoglycans were metabolically labeled with s during hours and then purified by ion-exchange chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. results: the total proteoglycan production decreases with approximately % in partal and preterm partal cell cultures. the reduction of proteoglycans in preterm partal and partal cell cultures is significant compared to non pregnant fibroblast cultures. the distribution of biglycan and perlecan are similar in partal and preterm partal cells. biglycan are significantly reduced by around % and perlecan are significantly increased by around % compared to non pregnant cultures. preterm partal cervical fibroblasts secreates significantly more heparan sulfate proteoglycans compared to non pregnant cultures. the changes in total proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. both partal and preterm partal cell cultures differ in their proteoglycan production compared to their non pregnant counterpart, suggesting a role for proteoglycans in cervical ripening. the role of the oxytocin/oxytocin receptor system is not well defined in human amnion. previous studies in rabbit amnion have demonstrated an up-regulation of oxytocin receptors in the end of pregnancy and have shown that there is a large increase in the ability of ot to stimulate pge production. we and others have previously shown a role for nf-b in otr regulation. in whole genome array analysis of human amnion we found that otr was the gene with the second highest increase associated with activation of nf-b (the highest being cox- ). the present work was directed towards further understanding of otr expression and function in human amnion at term. we have shown that pge release by pre-labour primary human amnion cells is significantly increased after oxytocin treatment for hrs (ot: - m; n= ; triplicate samples; fold increase p< . ). the expression of otr in labour (+) and labour (-) primary amnion cells was measured with real time rt-pcr. we found a significantly higher level of expression in the labour (+) cells (n= ; duplicate samples; p< . ). western blot analysis confirmed the upregulation of otr in labouring amnion. treatment with il- b resulted in a significant upregulation of otr which peaked with a fold induction after hours (p< . ). il- caused a fold increase in otr mrna levels in labour (-) cells, bringing the expression level up to that found in labour (+) cells. our findings provide further evidence for a role of otr in human amnion. expression of otr in human amnion is significantly increased after the onset of labour. term non laboured amnion can be stimulated with il- to increase otr expression to levels of laboured amnion. one of the functions of otr in amnion cells is stimulation of prostaglandin synthesis. expression of antioxidant defence proteins in human myometrium before and after the onset of labour. vasso terzidou, mandeep s kandola, shirin khanjani, jan j brosens, phillip r bennett. parturition reserach group, irdb, imperial college, london, united kingdom. background oxidative stress is a result of an imbalance between the production of reactive oxygen species (ros) and the antioxidant defence mechanisms present in biological systems. parturition and infection-induced preterm labour resemble inflammatory processes that are linked to the production of ros including super-oxide (o -), hydrogen peroxide (h o ) and peroxynitrite. low concentrations of ros can act as second messengers in the regulation of several cellular functions. in an attempt to maintain redox homeostasis cells are equipped with machineries to both produce and scavenge ros. enzymatic scavengers include superoxide dismutase (sod), glutathione peroxidise and catalase. sod is the first enzymatic step in the defence system against oxidative stress. nuclear factor-kappa b (nf-b), a transcription factor family classically associated with inflammation, is activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. labour is associated with an increase in nf-b activity in human myometrium and in fetal membranes. nf-b is known to regulate a range of genes associated with the onset of labour. in a study using affymetrix whole genome arrays analysis nf-b overexpression was associated with increased expression of sod- ( . fold). to determine changes in ros scavenging potential upon onset of labour, lower segment myometrial biopsies were taken at term before and after the onset of labour (n= ; each group). cdna was extracted from the tissues and real time rt-pcr was performed for sod- , serum/glucocorticoidinduced protein kinase- (sgk- ) and the dna repair enzyme gadd a. we found that labour onset is associated with a two fold increase in the levels of expression of each. oxidative damage at the fetomaternal interphase has been extensively studied in the placenta as part of investigations for first trimester pregnancy losses, iugr and pre-eclampsia. the role of ros is less well defined in human decidua and the underlying myometrium. our results suggest a role for oxidative stress and redox homeostasis in the maintenance of myometrial quiescene during pregnancy and onset of labour. plasma anandamide levels increase during labour induction and appear to delay labour progression. vijianitha nallendran, anthony h taylor, patricia mw lam, stephen c bell, david j taylor, justin c konje. cancer studies and molecular medicine, university of leicester, leicester, united kingdom. background: the evidence for a role of the endocannabinoid, anandamide (aea) in labour is conflicting. we previously showed elevated aea plasma levels in labouring women whilst another group showed that activation of functional receptors for anandamide actually inhibited uterotonin-induced contractions through an adenylate cyclase pathway . the aim of this study was to explore further the relationship between labour and plasma aea levels. methods: plasma aea levels in women undergoing induction of labour for various indications at term, were measured by a sensitive isotope dilution method using hplc-ms/ms. each volunteer had an assessment of her cervix prior to the start of the induction when the first sample for aea was collected. once active labour was established (cervix cm dilated; contractions every - minutes), a second sample was collected. results: seventeen ( %) of the subjects were multigravida. the inductions were for postdates(n= ); decreased fetal movements(n= ), fetal growth restriction(n= ), symphysis pubis dysfunction(n= ), diabetes mellitus(n= ), pre-eclampsia(n= ), fetal cardiac complication(n= ), spontaneous rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes(n= ). plasma aea levels increased significantly once labour was established (figure) and demonstrated in ( %) of the cases. the median (interquartile range) plasma aea level increased from . nm ( . - . ) at induction to . nm ( . - . ); *p= . ; mann-whitney u-test) at active labour. there was a positive correlation between plasma anandamide levels taken before induction and the time taken for the women to enter into active labour (r = . , p= . pearson correlation). plasma aea levels were higher in labouring women compared to non-labouring women after the induction of labour confirming our previous observations and suggesting a direct role for this endocannabinoid in labour. further studies are required to elucidate this role. nuclear factor-kappa b (nf-b) is a transcription factor family classically associated with inflammation, activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. as a cytokineinducible transcription factor it plays a key role in the expression of a variety of genes involved in inflammatory responses and cell survival. nf-b dna binding and transcriptional activity plays an important role in labour associated gene expression in myometrium. in this study we examined the range of genes regulated by nf-b in myometrium using transient transfections and wholegenome array analysis. myometrial cells were extracted from myometrial biopsies taken at the time of elective caesarean sections at term. transient transfections of primary myocytes were performed using expression vectors for nf-b p .the amount of dna was constant and psg was used as a control construct. cdna was made from myocytes transfected with either nf-b or psg . affymetrix genechip u microarray was performed (n= , each group). we found that genes were significantly differentially expressed between control and overexpressed nf-b samples. twenty eight of these genes were upregulated with nf-b and were down regulated. several chemokines and cytokines were identified in the upregulated group. interleukin demonstrated the highest, fold, induction in the upregulated group, followed by tumor necrosis factor, a-induced protein (tnfaip ), chemokine ligand (ccl ), chemokine ligand (ccl ), pentaxin related gene (ptx ), interleukin (il ) and superoxide dismutase (sod- ). nine genes were present in the nf-b group that were absent in the control group. these included chemokine ligand (ccl ), chemokine ligand (ccl ), chemokine ligand (cxcl ) and il . ingenuity pathway analysis demonstrated that immune response, inflammatory, cell growth and proliferation and cell death were the main pathways involved. standardisation experiments have been performed, and the microarray results were confirmed with real time rt-pcr in several candidate genes. our results provide further support in the role of nf-b in human labour and suggest its direct link in upregulation of inflammatory genes, cytokines and chemokines, consistent with the imflammatory nature of the biochemistry of labour. inflammation is widely accepted to be a key feature of human labor. secretory leukocyte protease inhibitor (slpi), an innate immune molecule, has been shown to be an antimicrobial and anti-inflammatory protein. the aims of this study were to verify its expression and localization in human myometrium methods: specimens were obtained at time of cesarean delivery with or without labor. expression and localization of slpi was detected using immunohistochemistry. slpi expression pattern relative to nf-b p subunit was compared between not in labor and in labor subjects, between different tissue sections as well as in in vitro model systems including myometrial explants, uterine smooth muscle cells (usmc) and ishikawa endometrial adenocarcinoma cells. slpi was predominantly localized to the nuclei of myocytes. the observed nuclear immunoreactivity of myocytes was increased during the labor relative to not in labor, paralleled with p nuclear translocation. the nuclear pattern of slpi is specific to myometrium since slpi immunostaining was present exclusively in the cytoplasm of all other tissues examined, including amnion, chorion, decidua and endometrium. slpi staining was also positive in macrophages, indicated by co-localization of slpi with cd positive cells. treatment with il- or tnf-induced nuclear translocation of p in myometrium explants and usmc, but not in ishikawa cells. in human myometrium, slpi is predominantly localized in the nuclei of myocytes and in macrophages. the nuclear expression pattern of slpi is myometrium-specific and increased following the onset of labor and correlated with nf-b activation. further understanding of its physiological significance may suggest new strategies aimed at preventing preterm birth. application of a new proteomic technology on amniotic fluid in prom. sara consonni, niccolo bosso, marianna andreani, agnese pizzardi, fulvio magni, anna locatelli. obstetrics and gynaecology, university of milano-bicocca, monza, italy; experimental medicine, university of milano-bicocca, monza, italy. objective: mass spectrometry (ms) is the obligatory tool for proteomics studies. biological samples must be purified before ms analysis due to the matrix complexity. a recent approach combines active surface prepurification with maldi-tof (matrix assisted laser desorption) analysis. clinprot technology provides the prepurification of the sample through the use of magnetic beads (mb) with activated surface. this technique can be carried out by robot in an automated way on a large number of samples. a unique example of the use of mb before maldi-tof analysis to determine proteomic profiles of amniotic fluid (af) is reported for rapid detection of fetal aneuploidies (wang ) . the objective of our study was to verify the applicability of clinprot prepurification before maldi-tof analysis on amniotic fluid collected noninvasively in premature rupture of membranes (prom). methods: we sampled af from vaginal posterior fornix of women with preterm prom (group , n= ) and term prom (group , n= ). samples were prepared with mb and analyzed with ms maldi-tof in order to generate proteomic profiles. results: it was possible to generate average proteomic profiles in the two study groups and the observed profiles were different. samples of af non invasively collected in prom can be analyzed by ms maldi-tof after preparation with mb. this technique allows to retain part of the eluted sample for characterization of protein peaks of interest. due to the less laborious characteristics of this method in comparison with techniques based on bidimensional electrophoresis its application can be useful in clinical proteomics. progestins accentuate the maternal but not fetal inflammatory response of women with intra-amniotic inflammation. sonya abdel-razeq, irina a buhimschi, michael cackovic, guoyang luo, antonette dulay, victor rosenberg, mert bahtiyar, errol norwitz, edmund funai, catalin buhimschi. ob./gyn. reprod.sci., yale university, new haven, ct, usa. introduction: data from animal research suggests that progestins have a marked pro-inflammatory capacity. recent studies support the administration of -hydroxyprogesterone caproate in women at risk for preterm birth. we sought to determine the impact of progestins during gestation on the extent of maternal and fetal inflammatory responses in pregnant women with intraamniotic inflammation. methods: amniotic fluid, placenta and cord blood were obtained from women who delivered preterm (median[range], ga: [ - ] wks). an amniocentesis was done to rule out infection. women exposed to progestins (n= ) within one week prior to amniocentesis were matched to controls (crl) by age, parity, history of preterm birth ga, membrane status and interval to delivery. proteomic profiling of amniotic fluid [mass restricted (mr) score] identified the presence or absence of intra-amniotic inflammation. an mr score of or confirmed intra-amniotic inflammation. amniotic fluid and umbilical cord interleukin- (il- ) levels were measured by elisa. histological chorioamnionitis was graded based on recognized criteria. results: overall, women and their fetuses exposed to progestin did not exhibit an increased inflammatory response (table) . however, sub-analysis restricted to women with mr or (n= ) showed that in the context of intraamniotic inflammation, progestins were associated with significantly elevated amniotic fluid il- levels compared to unexposed women (progestins (n= ): vs. crl (n= ): [ . - ] ng/ml, p= . ). these relationships were maintained after correction for steroid and antibiotic exposure. such significance was not found for amniotic fluid glucose, ldh, wbc count or cord blood il- . conclusion: our results suggest that progestins may amplify the maternal, but not the fetal inflammatory response of women with intraamniotic inflammation. objective: recently progestins have been shown to reduce the incidence of recurrent preterm labor in women. progestins have long been known to inhibit preterm labor in some species including rats and are also known to delay term labor. nitric oxide (no) donors, including ng, inhibit uterine contractility and have been used as tocolytics. the aim of this study was to examine the inhibitory effects of r on preterm labor in rats induced with a progesterone antagonist (onapristone, zk) with and without ng. materials and methods: charles river s-d timed pregnant rats (n= /group) were treated with zk ( mg/rat, s.c.) alone or vehicle (controls) on day of gestation. other groups of rats were treated with zk in combination with various doses of r ( . , or mg/rat s.c) with and without ng ( mg s.c. pellet) from days to of gestation. all rats were sacrificed on day of gestation and the number of fetuses and implantation sites were counted to determine the preterm delivery rate. one way anova was used for statistical analysis. p< . was considered significantly different. results: rats treated with zk alone delivered all their fetuses prematurely compared with controls (p< . ) treated with vehicle only (ca. % fetuses delivered). ng treatment alone did not affect the delivery rate (p> . ) compared to controls. similarly zk + ng did not reduce the preterm delivery rate compared to zk alone (p> . ). however r dose dependently reduced (p< . at all doses) the number of fetuses delivered prematurely in response to zk and the premature delivery rate was further reduced when treatment included the combination of r plus ng (p< . ). conclusions: zk effectively induces premature delivery. premature delivery produced by zk can be effectively reduced with a r , a progestin known to bind with high affinity to nuclear progesterone receptors. ng by itself, at the dosage used, does not reduce the prematurity rate caused by zk. however, r and ng act synergistically to reduce the preterm delivery rate. this study indicates that combinations of a progestin with an no donor may be an effective treatment for preterm labor and delivery. monkeys. pl grigsby, jp rasanen, , dw sadowsky, m bertolino, m carbonatto, e gillio tos, s canali, j lacy, a chollet, mj novy. reprod sci, oregon primate res ctr, beaverton, or; ob/gyn, oregon health sci univ, or, usa; rbm, merck serono, italy; merck serono, switzerland. objective: to investigate the pharmacokinetics (pk) and pharmacodynamics (pd) of as , a novel orally active non-peptide oxytocin (ot) antagonist in rhesus monkeys. study design: dose finding and pk/pd studies were done in chronically instrumented non-pregnant (n= ) and pregnant ( - dga, term d; n= ) monkeys. maternal and amniotic fluid compartments were serially sampled during dosing and washout. treatment phases included: ot infusion control, ot infusion + as , and ot rescue therapy. ot infusions ( - mu/kg/hr, iv) were given to determine the lowest dose required to produce stable, sub-maximal uterine contractions in each animal. as was administered p.o. at , , mg/kg and the effects on inhibition of uterine activity (hca, mmhg.sec/hr) were compared. to verify antagonist reversibility, objectives: infection such as chorioamnionitis is thought to be the cause of premature rupture of the membrane and induce uterine contractions leading to preterm delivery. however, the mechanism for enhancement of uterine contractility is not well understood. we have reported that non-selective cation channels (nsccs) regulate pacemaker potentials to generate rhythmical contractions and should be targets of magnesium ions used for tocolysis. the purpose of this study is to investigate the changes of the expression of nsccs during normal pregnancy and the effect of inflammation in preterm. methods; atp receptors (p x) and transient receptor potential canonical (trpc) channels were examined as uterine nsccs. the mrna was extracted from the rat myometrium of non-pregnant and pregnant rats at days , , and . the expression of each subtype of p x and trpc channels was measured by real time rt-pcr (abi ) with taqman probes (abi). as an inflammatory model lipopolysaccharide (lps; . mg/kg) was injected into intraperitoneal cavity at day and the tissue was sampled after six hours. results; p x and p x were determined to be dominant subtypes of p x channel. the expression of p x was increased by % at day , compared with day and that of p x was enhanced by %. on the other hands, trpc and trpc were detected dominantly. the expression of trpc was increased three times in the late stages of gestation. however, trpc was suppressed by %. in the lps treated rat myometrium cox- mrna expression was measured to be fold higher than that of the control rat, showing inflammatory effects in the myometrium. in this model the expressions of p x , p x and trpc were enhanced by . , . and . times, but trpc was not changed. the mrna expression of p x , p x and trpc channels in rat myometrium was increased in the late stages of pregnancy. these channels are suggested to be concerned with onset of labor. in the inflammatory model the expression of these channels was accelerated dramatically and these values were much higher than those in normal pregnancy. this finding supposed inflammation may enhance some types of nsccs to accelerate uterine contractility and induce preterm delivery. anandamide ( to determine the mechanism of rho protein activation during oxytocin and carbachol induced contraction, freshly prepared myometrial strips in krebs henseleit buffer were treated with oxytocin ( nm) and carbachol ( μm) under isometric and tension free conditions. control strips were exposed to buffer only. treated myometrial strips were solubilised and separated into membrane and cytosolic extracts and equal aliquots were immunoblotted with rhoa and rhob antibodies. rhoa translocated to the membrane after oxytocin and carbachol stimulation under both isometric and tension free conditions (p< . ). there were no significant changes in rhob membrane to cytosol ratios relative to control. agonist induced contraction in human myometrium is associated with rhoa but not rhob membrane translocation. during pregnancy components of the intracellular camp signalling pathway show increased gene expression resulting in the maintenance of myometrial quiescence until term where a substantial decrease in expression of these genes is observed. protein kinase a regulatory subunit riia (rii ) is upregulated at both the mrna and protein levels in the human myometrium during pregnancy. this particular subunit is membrane-bound and by directing phosphorylation to myometrial cytoskeletal proteins may affect contractile machinery thus playing a role in maintaining uterine relaxation. acetylation of histones promotes a favourable chromatin environment for transcriptional activity of many genes. this process is largely inhibited by histone deacetylases (hdacs), whereby its activity leads to transcriptional repression. hdacs can be recruited to the promoter region of a gene by other transcription factors such as sp proteins. since the rii promoter contains three sp - consensus binding sequences in its proximal part, we investigated whether this gene is a target for transcriptional regulation by hdacs. using dna precipitation assays we found that sp , and as well as hdac and form complexes with biotin-labelled fragments relating to sp - elements in the promoter region of rii . additionally, treatment of myometrial primary cell cultures with hdac inhibitor trichostatin a (tsa), or with the methyltransferase inhibitor -azac resulted in increased mrna and proteins levels. further studies with full and truncated luciferase constructs of the promoter region of the rii gene in transiently transfected myometrial cells confirmed that all three sp - elements are involved in the transcriptional regulation of the gene. this process involves hdacs, as h treatment with tsa significantly increased the luciferase signal. changes in the binding of sp proteins and hdacs to the promoter after tsa and azac treatment were investigated employing chromatin immunoprecipitation assays. alterations in the methylation status of the promoter after treatment were examined by bisulfite modification and dna sequencing. together, this study highlights the importance of chromatin modifications in the maintenance of uterine quiescence during pregnancy as well as identifying a potential mechanistic target for drugs that may reduce the incidence of preterm labour. objective: progesterone maintains pregnancy by promoting myometrial quiescence. typically progesterone effects are thought to be mediated through the classic genomic pathway. there is evidence, however, that progesterone also acts via a non-genomic pathway by interacting with specific membrane progesterone receptors (mprs) and in particular progesterone receptor membrane component - (pgrmc- ). the role of non-genomic progesterone actions in human pregnancy and parturition is not clearly understood. the goal of this study was to measure the extent of mpr expression in biopsy specimens of human myometrium obtained at cesarean delivery, and to determine whether expression changes with advancing gestation or the onset of labor. study design: lower uterine segment myometrial biopsies were obtained at the time of delivery from consenting women who were at term and not in labor (n= ), preterm and not in labor (n= ) and term and in labor (n= ). protein extracts were prepared and subjected to polyacrylamide gel electrophoresis and immunoblot analyses for pgrmc- and gapdh. abundance of pgrmc- protein relative to gapdh was determined by digital densitometry. we also performed immunohistochemistry (ihc) to determine the cellular localization of pgrmc- in the human pregnancy myometrium. results: pgrmc- protein was identified in each biopsy specimen. there was a -fold increase in pgrmc- protein in term compared with preterm biopsies (relative to gapdh p< . ). the relative level of pgrmc- protein was not different between biopsy specimens from laboring and non-laboring women at term (compared to gapdh, p= . ). pgrmc- immunoreactivity was localized to granular cytoplasmic staining. conclusion: this is the first description of the presence of pgrmc- protein in the human myometrium during pregnancy. its presence suggests that progesterone may influence contractility non-genomically via these receptors. the functional significance of the gestational age associated two fold increase in pgrmc- is unclear. changes in expression of this receptor during pregnancy may be important for the hormonal control of parturition and can be the focus of future studies. (ruddock et. al., abstract smfm, ) . the aim was to examine the mechanism of p inhibition. methods: uterine tissues from women (n= ) at term with cesarean section, were suspended in organ chambers and exposed to various agents or solvents. contractility was registered, stored, analyzed and compared before and after addition of agents or kcl. tissues were treated with p alone ( - to - m) or p bound to bovine serum albumin (bsa/p , - to - m p ), a progestin with low affinity to mpr (r , - - - m), or a non-sex steroid (cholesterol, - to - m). other tissues were pretreated with selective inhibitors of adenylate cyclase (sq , - m), guanylate cylase (odq, - m), phosphodiesterase (rolipram, - m), nitric oxide (no) synthases (l-name, - m) or a nuclear p receptor antagonist (mifepristone, mif, - m), followed by p . data were analyzed by anova for statistical differences (p< . ). results: p rapidly (< hour), effectively ( %) and dose-dependently inhibits spontaneous and kclinduced contractility (ed of < - m incubation of strips with zd- , at concentrations up to m for one hour prior to the experiment, led to no significant reduction in the total work done. however, incubation of strips with l- with a concentration of nm for one hour prior to the experiment, led to a statistically significant reduction in the total work done after one and a half hours. furthermore, when increasing concentrations of pge ( - to - ) were added to l- ( nm) pre-treated strips, total work done per contraction was comparable to that of non-treated controls. similar effects were not observed in zd- ( nm) pre-treated strips. since the reduction in contractility caused by l- was greater than that caused by zd- , it is likely that the stimulating effect of pge acts predominantly via ep receptors. taken together with our previous data showing an increase in ep at labour, this data shows that targetting an ep receptor may be a useful strategy in managaing pre-term labour. recently a truncated kda isoform of the er-has been described in human endothelial and testicular cells. we describe the presence of this kda erisoform in pregnant and immortalized non-pregnant human myometrial cells. methods: myometrial tissue obtained from non-laboring pregnant women undergoing cesarean section at term (n= ) was dissected prior to being finely minced. a portion of the tissue was placed in pbs and sonicated, while the remainder of the tissue was dissociated with collagenase prior to filtration and placement on culture plates. cells were then cultured in mem w/ % fetal bovine serum (fbs) until confluence. cultured cells were then dissociated with . % typsin/edta, subsequently re-plated and grown to confluence. immunohistochemistry directed toward smooth muscle protein was used to verify myometrial phenotype.) protein was extracted and quantified. western blot was performed with mouse monoclonal anti-er-receptor antibody (santa cruz biotechnology) to the f- domain of the human er-receptor. an immortalized non-pregnant human myometrial cell line provided by ann word, htert, was cultured and isolated as described above. results: htert cells expressed both the truncated ( kda) and the full length ( kda) er-isoforms. fresh and cultured myometrial cells from non-labored term pregnant patients also expressed both isoforms of er-. subsequent subcultures of myometrial tissue continued to express the kda erisoform, yet the expression of the kda isoform was lost. a representative blot is below. conclusions: we demonstrate, for the first time, the presence of a kda erisoform in cultured and non-cultured pregnant and cultured nonpregnant human myometrial tissue. discovery of this isoform of er-in myometrial tissue could provide insight into the molecular mechanisms involved in parturition. the action of prostaglandin f (pgf ), a potent uterotonic stimulant that is associated with labour at term and preterm, is mediated by its receptor, ptgfr. myometrial ptgfr mrna levels fall during pregnancy and this likely plays a role in uterine quiescence. however, the mechanisms by which this occurs are poorly understood. we previously reported that pgf downregulates fp mrna expression in cultured human myometrial ultr cells in a protein kinase c (pkc) dependent manner. in addition to the downregulation of mrna levels, receptor desensitization may also represent another mechanism of decreasing ptgfr activity because ligand binding to g protein coupled receptors often results in receptor internalization. we therefore hypothesized that pgf treatment of ultr cells also results in pkc dependent ptgfr internalization. methods: near confluent cultured human myometrial ultr cells were treated +/- - m or - m pgf for , , or hr. cells were fixed with formaldehyde and visualized for localization of ptgfr by immunofluorescence. to examine the potential involvement of pkc in the process, ultr cells were treated +/- - m pgf and +/- m myristoylated pkc inhibitor ( - ) and examined for ptgfr cellular localization as described above. results: pgf treatment resulted in a dose dependent decrease in ptgfr membrane signal at , , and h. this decrease was dependent on pkc as cotreatment with the myristoylated pkc inhibitor ( - ) prevented the pgf induced decrease in membrane ptgfr at h treatment. there was no visible effect of the pkc inhibitor on ptgfr membrane signal on its own. conclusion: we conclude that pgf decreases membrane levels of ptgfr protein in human myometrial ultr cells in a pkc dependent manner. these results suggest that pkc may be required for both the pgf induced internalization and desensitization of ptgfr protein and downregulation of ptgfr mrna in human myometrial ultr cells. therefore pkc may play a crucial role in downregulating ptgfr expression and activity and maintaining uterine quiescence during pregnancy. rhoa is a small gtpase that acts as a molecular switch to control a variety of signalling pathways in smooth muscle, including contractility. it is thought that increases in rhoa-gtp levels facilitates phosphorylation of target proteins such as cpi- , promoting contractility at pre-term and term labour in humans. however, in situations of acute or chronic hypoxia in the uterus, it is important that myometrial contractility and subsequent labour is not facilitated prematurely. given the importance of rhoa to a cell's response to hypoxia in other cell types, it was hypothesised that rhoa plays a central role in the mechanism controlling smooth muscle contraction in the uterus too. following acute hypoxia ( . % o ) for one to six hours, rhoa mrna, total protein and activation (rhoa-gtp) levels were analysed, using semi-quantitative pcrs and western blot, and compared to normoxic non-pregnant human uterine smooth muscle control cells. next, we investigated whether reduced oxygen conditions affected oxytocin induced activation of rhoa, following a two hour treatment of nm oxytocin. firstly, our results demonstrate that the rhoa itself is significantly activated under low oxygen conditions, resulting in phosphorylation of myosin phosphatase, myosin light chain and cofilin, three proteins known to be central in contraction and actin filament organisation. secondly, hypoxia significantly reduced the coupling of oxytocin to rhoa activation under the conditions examined. we observed a significantly reduced level of rhoa expression and activation which correlated with an increase in the level of another rhogtpase protein, rhoe. we propose that rhoa inactivation occurs through a rhoe-mediated mechanism, suggesting a balance in the activity of these two antagonistic rhogtpases in oxytocin-induced hypoxic human uterine smooth muscle cells. these results provide a possible explanation for the reduced coupling of oxytocin as a stimulant of myometrial contractions during slowly progressing labours. oxytocin-induced release of calcium ions (ca + ) from sarcoplasmic reticulum (sr) and sensitisation of contractile proteins to ca + have been suggested to mediate the oxytocin-induced potentiation of myometrial contractions. objective: we investigated the effects of oxytocin in the presence of nifedipine, a known inhibitor of the l-type calcium channel (ltcc). method: samples of myometrium were obtained from women undergoing term caesarean section with the approval of the local ethics committee. a standard organ bath system (ad instruments, uk) was employed to analyse contractile activity. stable spontaneous contractions were recorded for - minutes before addition of nifedipine. results: in agreement with our previous findings, application of oxytocin to spontaneously active strips produced a two-component effect: a transient tetanus-like contraction, followed by prolonged augmentation of phasic contractions. nifedipine ( um) rapidly abolishes spontaneous contractions, subsequent addition of nm oxytocin produced an initial, transient rise in force, approxiamately % compared to oxytocin alone, followed by high frequency oscillations in > % of strips. calcium-free solutions were used to confirm that oscillations were due to ca + entry. disabling the sr store using thapsigargin ( um) had no effect on oscillations, confirming the sr not to be involved. the t-type calcium channel blocker, mibefradil ( um ) showed no inhibition of oscillations. an ip receptor and store-operated calcium channel inhibitor, -aminoethyldiphenylborate ( -apb) um also had no effect on oxytocin-induced oscillations. the store-operated calcium channel inhibitor skf- ( um) showed partial inhibition of oscillations. conclusions: based on these results, we propose that the most likely mechanism of ca + entry producing oxytocin-induced oscillations in the presence of nifedipine is the transient receptor channel-c (trpc) channel, known to be present in the human myometrium. further work needs to be completed to clarify this further. identification of stim and orai in human myometrium. a new paradigm in store-operated calcium signaling. evonne c chin-smith, mark r johnson, rachel m tribe. division of reproduction and endocrinology, king's college london, london, united kingdom; department of maternal fetal medicine, imperial college school of medicine, chelsea and wesminster hospital, london, united kingdom. background: recent reports have suggested that two novel proteins, stim and orai, are involved in the regulation of store-operated calcium entry. we have previously reported that members of the trpc family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labour associated stimuli il- beta and mechanical stretch. although stim and orai isoforms have been reported in other smooth muscle cell types, there are no published reports of stim and orai expression in human myometrium. the aim of this study was to identify mrna expression of stim , stim , orai and orai in human myometrium. methods: human myometrial biopsies were obtained from women undergoing elective caesarean section at term (prior to labour) with informed written consent and institutional ethics committee approval. whole myometrial tissue was either snap frozen and stored at - o c or used for cell culture. rna was extracted from whole tissue (n= ), primary cultured myometrial cells (n= ) and passaged (p ) myometrial cells (n= ) and stim , stim , orai and orai mrna expression was assesed by quantitative real-time pcr. results: all four genes were expressed in whole myometrial tissue and cells. stim and stim mrna expression in cultured myometrial smooth muscle cells (primary and passaged) was significantly reduced compared to myometrial tissue expression (p< . ). however, there was no significant difference in either orai or orai expression in whole tissue versus cultured myometrial smooth muscle cells. conclusion: to our knowledge this is the first report of stim / and orai / mrna expression in human myometrium. these genes may contribute to the regulation of calcium signalling in human myometrium, but the functional significance of their expression remains to be determined. funded by the bbsrc. obstetrics and gynecology, national university of ireland, galway, galway, ireland. objective: the ability of uterine smooth muscle cells to stimulate collagen contraction has been well established as an in vitro model of myometrial contractility. devost and zingg ( ) reported that the contractility of human myometrial cell lines layered onto collagen matrices was increased by oxytocin while another group described the stimulation of human uterine smooth muscle cell contractility, cultured within collagen lattices, with endothelin- (dallot et al., ) . our study investigated the response of human primary uterine smooth muscle cells cultured within collagen lattices, to various compounds, including the non-specific depolarizing agent potassium chloride (kcl), the inflammatory cytokine tnf , the rock- inhibitor y- , oxytocin, and oxytocin plus its clinically used antagonist, atosiban. methods: human primary uterine smooth muscle cells (utsmc) (lonza) were maintained in dmem high glucose media. cells ( , per well) passage - , were embedded in . mg/ml collagen, in . ml aliquots, in dmem-f media on well plates (invitrogen) (dallot et al., ) . effects on contraction were studied by monitoring changes in gel area (alpha innotech imager, image j software (nih)). statistical significance was determined by the student t test. results: the utsmcs displayed basal contraction of the collagen gels while the non-contractile cell line hek cells, did not. kcl ( nm) stimulated an % increase in contractility (n= , p= . ) while nm tnf resulted in an . % increase (n= , p= . ), in comparison to unstimulated cells embedded in gel. the rock inhibitor y- ( m) inhibited contractility, with a . % decrease in collagen gel contractility (n= , p= . ). a % increase (n= , p= . ) in utsmc embedded collagen contractility was observed with nm oxytocin, which was antagonized by atosiban ( m) (n= ). conclusion: this study highlights the importance of the development and optimisation of a reproducible human in vitro myometrial contractility model, to evaluate the effect of various known labor-associated, and also unknown compounds. this should aid in our understanding of the many complex biochemical pathways involved in myometrial contractility at labor, and ultimately contribute to the prevention of preterm labor. changes in intra-mural myometrial blood flow during spontaneous labour using d power doppler angiography (pda). nw jones, , nj raine-fenning, h mousa, mj taggart, k jayaprakasan, gj bugg. nottingham university hospitals nhs trust, united kingdom; nottingham university, united kingdom; university of newcastle upon tyne, united kingdom. methods d pda was used to measure the percentage (%) change in the vascularization index (vi), the flow index (fi) and the vascularization flow index (vfi) in a volume of myometrium at the uterine fundus of nulliparous women at term, in the first stage of uncomplicated spontaneous labour. d data sets were obtained during a single cycle of uterine relaxation (r ), contraction and subsequent relaxation (r ). measurements were made independantly by two authors (nwj and gjb) using vocal® (ge kretz) and the mean value was used for analysis. the results from each woman are presented as a % change of r . data is presented as medians [interquartile range (iqr)] and analysed using non-parametric tests. the median volume (cm ) of interest for r was . cm ( . - . cm ), for the contraction was . (fig. ) . the % change for all three indices between r and r was not significantly different. however, there was a significant difference between the contraction and r (p< . ). the mean intra-class correlation coefficient and % confidence interval (ci) of vi, fi and vfi for the authors (nwj and gjb) were . ( . - . ), . ( . - . ) and . ( . - . ), indicative of good inter-observer reliability. conclusion d pda is a useful and reliable tool in the assessment of changes in intramural myometrial blood flow, which was found to reduce significantly during a contraction but increase again during the following uterine relaxation. . raine-fenning nj, et al. the inter-observer reliability of d pda acquisition within the female pelvis. ultrasound obstet gynecol. ; : - . the role of pgf on myometrial contractility; studied with a selective fp antagonist. shankari arulkumaran, andre chollet, phillip r bennet. imperial college parturition research group, institute of reproductive and developmental biology, hammersmith hospital campus, london, united kingdom; merck serono, geneva, switzerland. objectives: human myometrial strips established in culture will usually begin contracting after one hour. we have previously shown that stretch upregulates prostaglandin synthesis and have therefore hypothesized that spontaneous contractions occur because of stretch-related prostaglandin synthesis. methods: experiments were performed using x mm human pre-labour, lower uterine segment myometrial strips in a dmt myograph ms in oxygenated kreb's solution, with adi powerlab software. spontaneous contractions required stretch force and initial experiments determined that the maximum number of strips attaining spontaneous contractions was greatest at a force of - g. a novel antagonist to pgf (fpa) was studied in this model. the fpa has a ki of nm for fp and is - fold selective for fp compared with other prostanoid receptors. results: addition of pge and pgf after the commencement of spontaneous contractions caused a statistically significant increase in the total work done by the strips. the effect of pge was being greater than that of pgf . pre-incubation of the baths with a novel and selective fp antagonist (fpa) with concentrations up to m ( - ) did not affect the total work done by spontaneous contractions compared to non-treated controls. however, increasing concentrations of the fpa ( - to - ) decreased the total work done by -fold on strips treated with pgf beforehand in comparison the pgf -treated strips alone. conclusion: these data suggest that stretch and synthesis of prostaglandins is essential for spontaneous contractility in human myometrial strips. since fpa is able to block pgf induced but not spontaneous contractions, it is likely that pgf does not play a role in spontaneous myometrial contractility in vitro, although it may do so in vivo. because other factors may combine to increase contractility, the combination of an fp antagonist with other inhibitors may therefore, be a more effective strategy in reducing pre-term deliveries. objectives: brain natriuretic peptide (bnp) is synthesized in fetal membranes and inhibits oxytocin-induced contraction of preterm human myometrium. we hypothesized that bnp may be a paracrine mediator of human myometrial quiescence. we showed bnp content is higher in membranes from preterm pregnancies absent labor and significantly decreased with idiopathic preterm labor. while bnp activates natriuretic peptides receptors a (npr-a), b (npr-b), and its clearance receptor (npr-c), we have shown bnp does not inhibit myometrial contraction via npr-a or npr-b. herein, we test in part the hypothesis that bnp inhibits myometrial contractions by activating npr-c by quantitating npr-c in human myometrium at different gestations and labor status. methods: myometrial samples were obtained at the time of cesarean section after informed consent from groups of patients: preterm not in labor (pt-nl), preterm in labor (pt-l), term not in labor (t-nl) and term in labor (t-l). myometrial samples were obtained from women - weeks' gestation, and term between and weeks. mrna for npr-a, b and c were semiquantitated by realtime pcr (normalized by s mrna), and npr-c protein by western blot. results: natriuretic peptide receptors a, b and c mrnas were identified in all groups. while there were no differences in mrna levels among groups, npr-c protein was increased in samples from term laboring women. conclusion: since bnp inhibits the contraction of human preterm but not term myometrium independent of npr-a and npr-b, we have previously speculated that bnp activates another "unknown" receptor. herein we find that myometrial npr-c protein but not mrna increases during human labor at term. the increase in npr-c at term could compete with the unknown quiescent receptor to functionally reduce the availability of bnp and thus permit or promote myometrial activation/contraction. (supported by a grant from chilean government fondecyt ). objective: bnp is synthesized within human chorion and amnion and inhibits oxytocin-induced contraction of human myometrium. bnp activates guanylate cyclase (gc) natriuretic peptides receptors a (npr-a) and b (npr-b). bnp also stimulates the clearance receptor (npr-c) whose action is not mediated by gc. the intracellular pathway of bnp/npr-c inhibition is not known. we determined that bnp does not inhibit myometrial contraction via npr-a or -b. we hypothesized that bnp inhibits myometrium by npr-c activation. we test aspects of our hypothesis by determining whether bnp/npr-c pathway inhibits myometrial contractions via myometrial nos pathway. methods: bnp and canp (specific npr-c agonist) were added to primary human myometrial cell cultures and enos/inos activity/expression determined. cell cultures (n= ) were prepared from myometrial samples of nonlaboring, term pregnant women. after confluence ( d), the cells were incubated ( h) with nm bnp and/or canp. nos activity was measured ( l-citruline assay) and transcription/translation semi quantitated (realtime pcr and western blotting). results: bnp had no effect on either nos expression or activity. canp significantly reduced inos but not enos mrna level. canp did not alter the protein level of nos but significantly reduced nos activity. co-incubation with bnp and canp reduced both mrna levels (p< . ) and significantly decreased enos, but not inos, protein without change in overall nos activity. within the female pelvis. ultrasound obstet gynecol. ; : - . conclusion: the activation of npr-c by canp reduces nos activity and expression. the same effect was not observed by bnp. the difference may be explained because bnp (but not canp) activates all natriuretic peptide receptors, and they may have opposite actions on nos pathway. we conclude unlikely bnp inhibits human myometrial contraction by npr-c activation via the nos pathway. ( introduction: ultr is a retroviral immortalized human uterine smooth muscle cell line which we use as a model for uterine hypertrophy studies. however, this cell line has limited usage due to a reduced rate of cell division and eventually replicative senescence in culture. one of the mechanisms of human somatic cellular senescence is un-compensated shortening of telomeres, the specialized dna structures located at the ends of eukaryotic chromosomes. introduction of human telomere reverse transcriptase (htert) has been shown to induce telomerase activity and telomere elongation, and extend life-span of normal human cells. objective: to recover ultr cell division without altering the phenotypic characteristics of smooth muscle cells by introducing htert, therefore improving ultr cell line. methods: ultr cells were transfected with a modified htert expression vector at a relatively early stage (passage ) in the presence of selective antibiotic. ultr cell growth rate, with (ultr-ht) or without transfection, was determined by plating cells in multiple plates at a fixed density and counting cell numbers after days. single cell clones of stably transfected cells were further isolated by plating in well plates. expression of htert, smooth muscle specific genes (smc-sgs), and target genes was identified by rt-pcr of total rna extracted from ultr and ultr-ht cells. results: rt-pcr demonstrated successful introduction of htert into ultr cells. the growth rate of ultr-ht cells was increased and cell morphology was improved (free of the typical aneupoid appearance). at passage , ultr-ht cells grew . fold (p< . ) and . fold (p< . ) faster than ultr cells at passages and , respectively. currently single cell clones have been selected. ultr-ht cells express a set of smc-sgs, including -actin, caldesmon, calponin, myosin heavy chain, sm , and smoothelin, confirming that the smooth muscle phenotype is preserved. a panel of genes involved in angiotensin ii signalling, including angiotensin ii receptors (at / ), nox family (nox , , and duox ), nox-associated genes (p phox, p phox, p phox, and rac / ), was also preserved. conclusion: this is the first report of rescuing uterine smooth muscle cell replicative senescence via activation of telomerase. we have established a human uterine smooth muscle cell line which will provide an improved in vitro model for studying human myometrium. at term, myometrium is characterized by spontaneous contractions which vary in the amplitude, frequency and duration depending on specie and hormonal status. repetitive depolarization of plasma membrane followed by transient elevation in [ca + ] i is thought to underlie the contractions. however, the detailed pathways which regulate the spontaneous contractility remain unclear. objective: this study was designed to elucidate the effect of protein kinase c (pkc) on the spontaneous contractions and [ca + ] i transients in the myometria from term pregnant mice and women. methods: the human samples were obtained from women undergoing cesarean section with the approval from the institutional review board committee while mice myometria were dissected from the days pregnant mice sacrificed according to the animal study protocol. the isometric force and [ca + ] i were measured simultaneously in fura- loaded myometrial strips using spectrofluorometer equipped with force transducer. results: the pkc activator phorbol , -dibutyrate (pdbu) applied at - m first stimulated the amplitude of spontaneous contractions in mice and human myometria followed by their inhibition. under the pdbu treatment the frequency of the contractions was first increased and then decreased in both species. at the same time, pdbu didn't initially increase the amplitude of [ca + ] i transients but attenuated it over time. however, the frequency of the transients was first increased and later decreased upon the pdbu exposure. in addition, pkc activation with pdbu resulted in the elevation of the uterine basal tone without corresponding changes in the basal level of [ca + ] i in human myometrium. in mice myometrium, on the contrary, pkc activation didn't result in an increase of the muscle tone and the basal level of [ca + ] i . conclusions: we propose that pkc causes bi-phasic effect on uterine spontaneous contraction in mice and human first to potentiate the amplitudes and the frequencies and later decreases them. the amplitude of [ca + ] i was not initially potentiated by pdbu suggestive of the dissociation of the contractile and [ca + ] i response. the stimulatory effect of pdbu on the basal muscle tone in human myometrium and the absence of the effect in mouse suggest different mode of pkc action on uterine contraction in human and mice. introduction: mechanical stretch of uterine myocytes is detected through integrins on the cell surface which form part of the focal adhesion complex, this signals through mapk, ultimately leading to the up-regulation of various pro-labour genes including pghs- and il- . on integrin activation fak is recruited to the focal adhesion complex and activated by autophosphorylation at tyr- , creating a src binding site. this promotes further fak phosphorylation at tyr- , and , enhancing fak catalytic activity. however fak can also act as a scaffold protein and its exact role in the expression of pro-labour genes is uncertain. in this study we used inhibitors for the kinase activity of fak and mek / in order to test the affect of fak kinase activity on expression on pghs- mrna. methods: primary human myometrial cell cultures were grown from myometrial biopsies taken from women undergoing elective caesarean section. cells were plated onto -well flexible bottom plates coated in type i collagen. cells were subjected to % static stretch for up to minutes. cells were also incubated with either the rho kinase inhibitor y or the mek / inhibitor u prior to being stretched. western blots were performed using antibodies to fak phospho- , fak phospho- and erk / phospho- / , -actin was used as a loading control. rna was also extracted and levels of pghs- measured using q-pcr. results: stretch increased levels of phosphorylation at both tyr- and tyr- with the greatest increases occurring at and minutes. however the increase at tyr- appeared to be greater than at tyr- . incubation with the rho kinase inhibitor reduced phosphorylation of fak- , however this did not affect phosphorylation of erk / or stretch induced up regulation of pghs- mrna. in contrast incubation with the mek / inhibitor reduced erk / phosphorylation and expression of pghs- mrna, whilst also reducing fak phosphorylation (n= ; p= . ). conclusions: fak has previously been shown to be important in activation of the stretch induced mapk cascade and pghs- expression. however these data suggest that while stretch causes fak phosphorylation fak kinase activity is not essential to pghs- expression. this suggests fak may be acting as a protein scaffold. . our aim was to examine the effects of both natural progesterone and hp on spontaneous myometrial contractions. myometrial biopsies were taken with informed consent and ethics approval from non-labouring women at elective caesarean section weeks gestation. strips of myometrium mm long , mm wide were cut and suspended under a resting tension of mn in organ baths of krebs gassed with % o / % co within hours of collection. progesterone or hp were added in a cumulative manner at -minute intervals. changes in amplitude were recorded. results were compared using anova. following equilibration for hours, myometrial strips contracted in a rhythmic manner (amplitude . ± . mn, n= pairs). progesterone ( nm- m) produced a concentration dependent inhibitory effect on myometrial contractions (fig ), which was greater than that of vehicle (p< . ). maximum inhibition measured . ± . % and . ± . % for progesterone and vehicle, respectively. hp exerted an inhibitory effect, this was not significantly different from the vehicle (p> . ). progesterone exerts an inhibitory effect on myometrial contractility in vitro. this is apparent within minutes suggesting a nongenomic action. our data are in agreement with some reports in the literature but conflict with others[ ]. this acute inhibition of myometrial contractility may contribute to the mechanism by which progesterone prevents preterm birth. in contrast, we were unable to demonstrate an inhibitory effect of hp on contractility despite its demonstrated ability to reduce the incidence of preterm delivery [ ] . our data suggest that natural progesterone may be a more effective tocolytic agent in the acute setting than hp. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: chemokines has been shown to play an important role in regulating uterine function. recent evidence demonstrates that monocyte chemotactic protein (mcp) level in amniotic fluid increases during spontaneous labor. the aim of this study was to examine the expression of mcp in human myometrium. methods: myometrial biopsies were taken from nonpregnant women undergoing hysterectomy and term pregnant women undergoing cesarean section followed written consent and local ethics committee approval. elective cesarean section was performed before the onset of labor while emegency section was done after the onset of labor. immunolocalization (n = each) was performed on paraffin sections by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . reverse transcriptionpolymerase chain reaction (n = each) using gene specific primer against mcp and mcp receptor was performed to identify mcp messenger(m) rna in human myometrium . results: immunohistochemical findings demonstrated mcp in human myometrial cells from nonpregnant, term pregnant women before and after the onset of labor. mcp was labelled on plasma membrane and cytoplasm of myocytes from these three groups of women. similarly, mcp and mcp receptor mrna were found in nonpregnant and term pregnant women before and after the onset of labor. in this prospective study a group of fetuses was consecutively enrolled. one ultrasound examination was performed to each patient within days from delivery. we considered fetal macrosomia a birthweight g and big babies a birthweight g. cut-off points for identifying the best value of fetal abdominal circumference for fetal macrosomia prediction were chosen by receiving operator characteristics' curve (roc) analysis. using the best cut-off indicated by roc analysis, specificity and sensitivity were calculated. mean gestational age at delivery was + weeks ( + sd). neonates weighted less than g, had a weight range between and g and weighted more than g. the fetal ac measurement was the selected criterium to evaluate the risk of fetal macrosomia. to identify macrosomic fetuses the roc curve analysis identified a cut off of ac > mm which allowed to select fetuses. analysing this population we found true negative cases, false negatives, false positives and true positives, with a sensitivity of . % and a specificity of %. to detect big babies the roc curve analysis identified a cut off of ac > mm (n. fetuses), reaching a sensitivity of % and a specificity of %, without false negative cases and with false positives ( true positives, true negatives). the cases that weighted between and g, were included in the cases considered false positives by this cut-off. conclusions. these results clearly indicated that ultrasounds alone can not be used to manage a pregnancy suspected for fetal macrosomia or big babies. in fact, to avoid shoulder dystocia and all other complications strictly related to macrosomic fetuses, clinicians should perform a large number of useless elective cesarean sections. . pathway analysis of differentially expressed genes (pa; z-score . ) showed up-regulation of axon guidance, folate biosynthesis, nitrogen metabolism and down-regulation of steroid biosynthesis, insulin signaling, oxidative phosphorylation, tgf-beta signaling, and ubiquinone biosynthesis pathways in cm vs cf. comparison of mnr vs c in f showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation steroid biosynthesis, fatty acid metabolism, glycolysis/gluconeogenesis, phosphatidylinositol signaling, ketone body metabolism, and ubiquinone biosynthesis pathways and down-regulation of bile acid biosynthesis, cell adhesion molecules, dna polymerase, notch signaling and ti diabetes mellitus pathways in mnr vs c. comparison of mnr vs c in m showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation of jak-stat signaling, autophagy, renin-angiotensin system (ras), and ubiquinone biosynthesis pathways and down-regulation of apoptosis, basal transcription, folate biosynthesis, nitrogen metabolism, protein export, and snare interaction pathways in mnr vs c. only the ubiquinone biosynthesis pathway up-regulation of mnr vs. c was common to both m and f. conclusions: ta and pa demonstrate sex-specific transcriptome expression in fetal kidneys at . g. in addition, these results show sexspecificity in response to mnr. we have seen similar effects of mnr on gene expression for autophagy, apoptosis, ras, ubiquinone and cell adhesion pathways at . g, suggesting these pathways may contribute to persistent affects of mnr. finally, we postulate that stress in utero may contribute to sex differences in risk of hypertension in adult life. leptin and neuropeptied y protein expression paradoxally increased in gestationall food restricted dams. louiza belkacemi, chun-hung chen, andrea jelks, michael g ross, mina desai. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: placental insufficiency is associated with marked increase in placental leptin production. this results in a rise in maternal leptin levels that serves as an early index of placental dysfunction. further increased placental leptin and suppressed neuropeptide y (npy) are associated with preeclampsia. leptin, an anorexigenic hormone and npy, an orexigenic peptide regulate food intake. importantly, leptin also serves as a placental and fetal growth factor. we have shown that maternal food restriction (mfr) results in intrauterine growth ... sf ()to:!)) df(,..)l) ...._ bf(jtolo) ... -m bf(,..lj) =- ( . ) <(u.)) ( . ) )( . )"" ' ~. ) !( . )' d.'cbom< ( ) f( ))""" s( l) background teenagers are more likely to deliver small-for-gestational age (sga) infants than adults, even after adjustment for socioeconomic factors , . previous studies of mostly black and hispanic subjects in the usa have suggested that maternal growth may contribute to reduced infant birthweight, due to preferential nutrient partitioning to the mother . the impact of maternal growth on birthweight and nutrient partitioning in pregnant teenagers in the uk has not been examined. methods skeletal growth (change in knee-height from st to rd trimester), weight gain and skinfold thicknesses were measured in pregnant teenagers (n= , % non-white) in london and manchester. key mediators of nutrient partitioning and metabolism: insulin-like growth factor(igf)- , igf binding protein(bp)- and leptin, were measured in maternal plasma ( weeks gestation). results maternal growth (defined as increase in knee-height > mm/ days) was detected in % of pregnant teenagers. this growth was not associated with sga birth; in fact these mothers were more likely to deliver large-forgestational age (lga) infants (p< . ). maternal weight gain and fat accrual at peripheral and central sites were greater in growers (p< . ). these parameters correlated positively with maternal igf- and leptin but negatively with the igf inhibitor, igfbp- (p< . for all). subjects delivering sga infants gained significantly less weight (p< . ) and had lower igf- levels (p< . ) than those delivering non-sga infants. conclusion maternal growth in teenage pregnancy was not associated with reduced birthweight. indeed the increased weight gain and fat accrual observed in growing teenagers may protect against sga birth and promote fetal growth. igf- and leptin promote fetal growth, primarily through effects on maternal metabolism and nutrient partitioning to the fetoplacental unit. these data suggest that higher maternal igf- and leptin in growing teenagers may provide an anabolic drive for both maternal and fetal growth. background. umbilical oxygen uptake (o umb uptake) has been estimated in human pregnancies only in acute experiments at the time of caesarean section. the recently developed possibility to measure umbilical blood flow by ultrasound in utero, prompted us to study normal and iugr pregnancies in order to evaluate fetal oxygen uptake utilizing the fick principle, i.e. uptake equals umbilical blood flow times (a-v) differences. methods. thirty-six iugr pregnancies were studied at the time of elective caesarean section and compared to twenty-one controls (c) (gestational age: c= . ± . and iugr= . ± . wks). an ultrasound examination was performed within hours from the caesarean section in all the recruited patients. umbilical vein absolute volume flow (qumb) was measured as the result between umbilical vein area and the time-averaged peak velocity * . . blood samples from umbilical vein (uv) and artery (ua) were obtained after the delivery and blood gases and acid-base balance were evaluated. umbilical oxygen uptake was calculated as o umb uptake = qumb*(uv-ua) o content. results. as expected, average fetal and placental weights were significantly different in the studied groups ( ± and ± g in iugr vs ± and ± g in n) . iugr pregnancies showed a significant reduction in qumb ( . ± . vs . ± . ml/min; p< . ) but no differences in the qumb/kg of fetal weight. iugr fetuses showed a significant reduction in o sat, o cont and po in both uv and ua compared to n. (uv-ua)o content ( . ± . vs . ± . mmol/l; p< . ) and o umb uptake/kg ( . ± . vs . ± . mmol/min/kg; p< . ) were significantly reduced in iugr. conclusions. we here report an evaluation of fetal oxygen uptake that proved surprisingly similar to the values reported in chronically catheterized animals. however, iugr fetuses showed a significant reduction in both blood and oxygen supply: this latter was reduced more that % on a per kg basis. iugr fetuses therefore utilize less oxygen than normally grown fetuses. circulating levels of vitamin d and il- in pregnancies with iugr. calvin j hobel, chander p arora, adegoke adeniji, priya arora, susan e jackman, olga miadel, baldjyan lilit. ob-gyn, cedars-sinai medical center, burns ans allen research institute, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: any condition resulting in under exposure to sunlight, including the use of sun block or poor nutrition may result in insufficiency ( . - nmol/l) or even deficiency of vitamin d (< . nmol/l).vitamin d regulates placental development and function. vitamin d deficiency has been linked to increased risk of serious chronic and inflammatory diseases. objective: to determine if the circulating levels of vitamin d and interleukin - (il- ) in maternal plasma correlates to pregnancies resulting in fetus with intrauterine growth restriction (iugr). hypothesis: the metabolism of vitamin d initiates the biochemical cascade of events leading to the expression of il- and the inflammatory response in iugr births. study design: in a behavior in pregnancy study, plasma samples at all three time points were analyzed in a cohort of women for (oh)d using elisa. the samples were also analyzed for il- at three stages of pregnancy: t ( - weeks), t ( - weeks) and t ( - weeks). iugr was defined as birth weight below the tenth percentile for gestational age. none of the iugr cases had spontaneous preterm birth. results: iugr was diagnosed in of women with available samples from a behavior in pregnancy study (bips) . out of these subjects, were selected as matched case controls. circulating levels of vitamin d ( (oh)d) were significantly lower in iugr cases at each visit (p<. ). the levels indicated deficient ( . ± . nmol/l) vitamin d in iugr group at t but sufficient vitamin d levels ( . ± . nmol/l) in controls. subsequent visits also showed lower levels in the iugr cases compared to the control group (t : ± . nmol/l vs ± . nmol /l; t : ± . nmol/l vs ± . nmol /l). at all three time intervals, significantly (p<. ) higher levels of il- were associated with the iugr cases ( pg/ml, pg/ml and pg/ml respectively) as compared to the controls ( pg/ml, pg/ml and pg/ml respectively). conclusions: vitamin d deficiency or even insufficiency may be an unrecognized cause of iugr. it is possible that a primary non-infectious inflammatory process is activated by vitamin d deficiency. combined assessment of vitamin d deficiency and il- expression during different stages of pregnancy may facilitate the resognition of the risk of developing iugr. response to global % maternal nutrient restriction (mnr). nathan drever, thomas j mcdonald, peter w nathanielsz, cun li. obstetrics and gynecology, university of texas health science center at san antonio, san antonio, tx, usa. background: igf-ii is a major growth factor in the developing pancreas and studies in the human fetus demonstrate that the peptide localizes to b cells of the islets (j endocrinology : ). rodent studies show decreased fetal pancreatic growth and igf-ii and ins abundance and increased apoptosis with mnr (j endocrinology : ). we previously demonstrated a fall in most components of the placental and fetal baboon liver igf systems with mnr. here we have evaluated fetal pancreatic igf-ii and ins changes in response to mnr. methods: pregnant baboons were fed ad lib (ctr, n= ) or % of wt adjusted ctr diet (mnr, n= ) from . gestation (g) and fetuses were recovered at c-section under general anesthesia at . (n= ; ctr and mnr) and . g (n= ; ctr and mnr). igf-ii and ins expression were determined by immunohistochemistry (ihc) and quantified by image analysis for fraction (area immunostained/area of the field x %) and density. data are expressed as mean + sem; ctr data are expressed] first; comparison made with two tailed t-test. results: at . g there was no difference in igf-ii or insulin fraction or density between groups. at . g, igf-ii fraction ( . ± . vs . ± . ,p< . ) and density ( . x ± . x vs . x ± . x , p< . ) and insulin fraction ( . ± . vs . ± . , p= . ) were reduced. conclusion: moderate mnr decreases abundance of fetal pancreatic igf-ii and ins at . g. in the fetal baboon and supports the extant evidence for impaired pancreatic development with mnr seen in rodents. maintenance of liver growth in the hypoxic growth restricted fetal sheep: a role for intrahepatic glut ? sheridan gentili, janna l morrison, i caroline mcmillen. sansom institute, unisa, adelaide, south australia, australia. objective: we have previously demonstrated that there is a differential tissue response to chronic placental and fetal growth restriction. growth of fetal tissues such as the brain and the adrenal are consistently spared in the face of chronic substrate restriction, whilst we have demonstrated that the growth of the fetal liver may be either maintained or reduced. it is unclear whether the growth response of the fetal liver to hypoxia and hypoglycemia are determined by intrahepatic metabolic adaptations. hypothesis: we hypothesize that there will be a differential profile of hepatic expression of glut , hsd , the gluconeogenic and glycolytic enzymes pepck and g pdh and the transcription factors pgc and ppar in animals in which liver growth is maintained or reduced. methods: carunclectomy was performed in non-pregnant ewes to induce placental restriction (pr). vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. mean gestation po < mmhg was defined as hypoxic (h; normoxia, n). post mortem was performed at - d. hepatic mrna expression of glut , hsd , pepck, g pdh, pgc and ppar was determined using qrt-pcr. results: four experimental groups were defined by fetal po and liver growth (c-n, pr-n, pr-h and pr-h-reduced liver growth). fetal weight correlated with mean gestational po (r = . , y= . x+ . , p< . ). liver weight was significantly lower in a cohort of pr-h fetuses (c, . ± . ; pr-n . ± . ; pr-h . ± . ; pr-h-rlg . ± . g:kg; p< . ). glut expression was highest in those pr-h fetuses in which liver growth was maintained, whilst the expression of hsd , pgc , ppar and pepck was highest in the pr-h fetuses in which liver growth was reduced (p< . ). conclusions: in the pr-h fetuses in which liver growth was reduced, the increase in hsd expression may be associated with an increase in hepatic exposure to cortisol and an associated increase in pepck, pgc and ppar . interestingly the compensatory increase in hepatic glut expression did not occur in fetuses in which liver growth was reduced. predicting the trajectory of fetal growth. racine n edwards-silva, jeffrey gornbein, calvin j hobel. obstetrics gynecology, los angeles, ca, usa; biomathematics, david geffen school of medicine at university of california, los angeles, ca, usa; obstetrics gynecology, david geffen school of medicine at university of california, los angeles, ca, usa. objective: to evaluate twelve potential predictors of fetal growth trajectory defined as the rate of fetal weight change over time. study design: a longitudinal prospective study of singleton fetal growth trajectory. the twelve potential predictors considered were: fetal gender, gestational age, parity, race, bmi, age, weight at st clinical exam, cumulative weight gain at each exam, smoking, and alcohol use. estimated fetal weight was computed using the hadlock formula and sonographic fetal biometric parameters at - weeks, - weeks, and - weeks. the rate of change in fetal weight was defined as x (fetal wt at exam j -fetal wt at exam i )(j > i)/ (gestational age at exam j -gestational age at exam i ). bivariate statistical analysis included the non-parametric spearman rank correlation and wilcoxon rank sum test. all factors were assessed multivariately using multiple linear regression. results: there were multi-ethnic women included in the study, after were excluded. they underwent a total of , exams. fetal gender (p= . ), maternal weight at st exam (p= . ), and cumulative maternal weight gain at exam (p < . ) were significant predictors of fetal growth trajectory. in this model, male fetuses had an average rate of fetal weight change of . grams per days higher than females. the rate of fetal weight change increased by an average of . grams per days for each lb increase in maternal weight at the st exam. the fetal growth rate increased . grams per days for each lb of cumulative weight gain at the rd exam. conclusions: in this study, maternal weight at the st exam and cumulative maternal weight gain were the significant determinants of fetal growth trajectory. adequate initial maternal weight and cumulative gestational weight gains probably ensure sufficient nourishment for normal placental growth, uteroplacental blood flow, and fetal nutrient uptake. this supports the emphasis on periconceptual nutritional counseling for all pregnant women. the implication of this study is that similar to fetal programming of adult diseases, there is nutritional programming of fetal growth trajectory. high risk patients. other predictors include the known risk factors of age < and > , single, tobacco/alcohol use, and african american race. interestingly, hispanic and native american patients have a lower lbw rate compared to other groups. introduction: catecholamines released by the sympathetic nervous system and adrenal medulla act via b-ars to regulate glucose and insulin function in liver, pancreas, adipose and muscle tissue. b-ar knock out mice show increased fat mass and glucose intolerance (asenslo et al.,diabetes, ) . mnr animal models have increased sympathetic activity. we, therefore, evaluated effects of mnr on baboon fetal liver b -ar. methods: baboons were fed as ad lib controls (ctr) or % of wt adjusted ctr diet (mnr) from . gestation(g) with fetuses retrieved at c-section under general anesthesia at . or . g. protein expression determined by immunohistochemistry for b -ar in the central liver lobule was quantified by image anaysis and expressed as fraction = area immuno-stained/area of the field x %. all data are expressed as mean + sem with ctr data presented first. liver glycogen expression was determined by the periodic accid schiiff (pas) method. comparisions were made with student's t-test with alpha level set at . . results: fetal body and liver wts were not changed by mnr at either age. pas stained liver glycogen at . g = . ± . vs. . ± . %, p< . . b -ar fraction was lower following mnr at . g and at . g (p< . ; fig ) conclusions: mnr decreased b -ar over % at . g and % at . g. decreased fetal liver b -ar in mnr alters glucose metabolisam and may result in reduced lipolysis predisposing to fatty liver. infection with noncytopathic bovine viral diarrhea virus (ncpbvdv) during early bovine pregnancy (< d gestation) results in fetal immunotolerance, persistent infection (pi) and intrauterine growth restriction (iugr). in contrast, infection after the development of adaptive immune competence (> d gestation or postnatal) results in a transient infection (ti). we have previously reported an iugr in pi fetuses presenting as decreased body weight and ponderal index. a growth defect can be the result of many factors, including placental insufficiency and nutrient restriction, however it was hypothesized that the iugr seen in bvdv pi fetuses may be an immunopathological effect caused by the persisting virus. our two part experimental design examined the relationship between bvdv and its pi host. in experiment (exp) , blood cell mrna was collected from pi steers (n= ; confirmed by virus isolation), or uninfected control steers (n= ) and used to identify differentially expressed genes using microarray (affymetrix) and qtrt-pcr approaches. in exp , bvdv naïve pregnant heifers (n= per group) were not infected (control) or infected with ncpbvdv on d. or d. of pregnancy creating pi and ti fetuses, respectively. fetuses were collected by c-section on d. ; infection was confirmed by elisa and qtrt-pcr. histology of placental tissue revealed no placentitis or pathology, and glucose and lactate levels in fetal serum were normal. microarray analysis revealed genes that were differentially regulated in pi vs. controls (p< . , > . fold). qtrt-pcr of steer and fetal blood revealed a significant upregulation of activators and products of the antiviral type-i interferon (ifn-i) pathway. as ifn-i can act as a growthsuppressive cytokine, a long-term upregulation may contribute to the iugr seen in persistent bvdv infection and in other viral infections observed during pregnancy. nricg - from the csrees. patients with a short cervix are at an increased risk for spontaneous preterm delivery. therefore, it is possible that women with a short cervix during pregnancy are also at risk to deliver an sga neonate. this study was conducted to address this question. study design: patients > weeks of gestation were prospectively enrolled into an observational study ( / to / ). transvaginal sonographic examinations were performed every weeks until delivery. the shortest cervical length between - weeks was used for analysis. sga was defined as less than the tenth percentile of birth weight. results: asymptomatic patients were studied. . % of patients delivered an sga neonate ( / ). the median cervical length was mm ( to mm); patients had a cervical length < mm. of these, % ( / ) delivered an sga neonate. similarly, % ( / ) patients with a cervical length < mm had an sga neonate. no relationship was found between a short cervix and sga (short cervix was defined as either < mm and < mm). the frequency of sga was not significantly different between women with a short cervix and those with a long cervix [ % ( / ) vs. % ( / ); p= . ]. newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced background folate is an essential micronutrient for cellular growth. recommendations on periconceptional folic acid use are mainly focussed on prevention of neural tube defects, despite growing evidence that folic acid use may have positive effects on birth weight. objective to examine associations between folic acid use, intrauterine fetal growth and birth weight. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods information on folic acid use was obtained by questionnaires and categorized into three groups: ) preconception start of folic acid use; ) start of folic acid use in first ten weeks of gestation; ) no folic acid use at all. fetal growth measurements included head circumference, abdominal circumference and femur length measured in mid-and late pregnancy, i.e., gestational age - and > weeks, respectively, and birth weight. fetal weight in mid-and late pregnancy was estimated using the haddock method. results data from , pregnant women were available. overall, folic acid use was positively associated with fetal growth. preconceptional folic acid use resulted in an increased growth of grams ( %ci . - . , p< . ) per week from late pregnancy to birth, compared to no folic acid use. similarly, start of folic acid use in the first ten weeks of gestation resulted in an increased growth of grams ( %ci . - . , p= . ) per week from late pregnancy to birth. both preconceptional folic acid use and folic acid use started in the first ten weeks of gestation resulted in higher birth weights of grams ( %ci - ) and grams ( %ci - ), respectively, compared to no folic acid use. a tendency was found for an increased risk of birth weight less than grams when folic acid was not started preconceptionally (or . , %ci . - . ). conclusion periconceptional folic acid use is significantly associated with increased fetal growth resulting in a higher birth weight. ductus venosus isovolumetric relaxation in severely premature growth-restricted fetuses. jason l picconi, katherine drennan, farhan hanif, michael kruger, giancarlo mari. obstetrics and gynecology, wayne state university/dmc, detroit, mi, usa. objective: ductus venosus (dv) doppler waveforms are characterized by two periods in which blood velocity decreases. the first represents the isovolumetric relaxation (ir) at the end of ventricular systole and the second represents atrial contraction at the end of ventricular diastole (a). ductus venosus reversed flow (dvrf) occurring at the time of the a-wave is considered a risk factor for intrauterine fetal demise (iufd). we have previously reported that absent or reversed a-wave flow can be present for weeks before iufd occurs or delivery is performed for non-reassuring fetal testing. the guiding hypothesis for this study is that decreased flow at the time of ir in combination with absent or reversed a-wave flow allows a more accurate prediction of fetal outcome than a-wave absent or reversed flow alone. material and methods: ductus venosus doppler was serially studied in severely premature iugr fetuses (estimated fetal weight < th percentile and umbilical artery pulsatility index > th percentile) from diagnosis until demise or delivery. ductus venosus waveforms were assessed quantitatively for peak systolic velocity (psv), isovolumetric relaxation velocity (irv), and end diastolic velocity (edv). the psv/irv + edv were compared to fetal and neonatal outcome. a kruskal-wallis one way anova, mann whitney u post-hoc test, and a roc, were used for statistical analysis. a p < . was considered statistically significant. results: all fetuses were delivered at < weeks. six cases resulted in iufd, five cases resulted in neonatal demise (nd), and six cases resulted in neonatal survival (ns) at the time of discharge from the hospital. the psv/irv+edv correlated better than a-wave reversal of flow with perinatal outcome. a psv/irv+edv score less than - . resulted in iufd, whereas a score greater than - . resulted in live birth. live births segregated based on estimated gestational age, where those fetuses at less than weeks resulted in nd and those fetuses at or greater than weeks resulted in ns. all results were statistically significant. conclusions: the isovolumetric relaxation velocity is a novel doppler parameter in the assessment of severely premature iugr fetuses. these data indicate that assessment of irv should be considered part of the evaluation of severely iugr fetuses. background: fetal growth restriction has been linked to an increased incidence of chronic hypertension, which may be the result of extracellular matrix changes (ecm) within the vascular tree. in previous studies, ecm changes were observed in the umbilical arteries of preterm growth restricted infants. objective: to determine if there are alterations in collagen subtypes within the umbilical cords from growth restricted fetal rat pups after a period of maternal nutrient restriction. methods: timed pregnant sprague-dawley rats were fed either a % food restricted diet (mfr; n= ) or were fed ad libidum (control; n= ) from d until d of gestation. litter size, fetal weights and placental weights were then noted and umbilical cords from randomly selected pups in each litter were snap frozen. gene expression for collagens i, iii, xiv and decorin was evaluated by real-time rt-pcr with normalization to the gadph housekeeping gene. data were analyzed from fitting general linear regression models with estimation based on the quasi-likelihood estimation (generalized estimating equations) to account for clustering of responses within litters. data are shown as cycles to amplification (ct), which is inversely proportional to mrna levels. results: no difference in median litter size was detected. however, fetal and placental weights in the mfr group were significantly less than those from control dams. no significant differences in umbilical cord gene expression for collagens i, iii, xiv or decorin were detected between mfr and control pups. conclusions: maternal food restriction does not result in any detectable alterations in collagen or decorin expression within the intact umbilical cord, despite causing significant reductions in fetal growth. control (n= ) p-value litter size (median, range) ( , ) ( , ) . fetal weight (mean ± sd) g . ± . . ± . < . placental weight (mean ± sd) g . ± . . ± . . collagen i (mean ± sem) ct . ± . . we have previously demonstrated that the restriction of placental and fetal growth results in fetal hypoxia and fetal brain sparing suggesting a redistribution of cardiac output. furthermore, placentally restricted (pr) hypoxic fetuses are more dependent on their sympathetic nervous system for the maintenance of blood pressure during late gestation. nerve growth factor (ngf) plays a significant role in sympathetic innervation. hypothesis: we hypothesize that the expression of ngf will be higher in the aorta and femoral artery of the pr hypoxic compared to the control fetus. method: carunclectomy was performed in non-pregnant ewes to induce pr. vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. all pr fetuses introduction elevated sflt- levels have been shown to be a feature of pre-eclampsia and is considered to play a significant role in the pathogenesis of the condition. the role of angiogenic factors in fetal growth restriction has not been as well established. this study evaluated the levels of circulating vascular endothelial growth factor (vegf) and its soluble receptor sflt- , in normal pregnancies and isolated placental vascular disease without evidence of pre-eclampsia. method maternal peripheral venous samples were collected antenatally from two groups of pregnant women between - weeks of gestation. group a: uncomplicated normal pregnancies (n = ) and group b: pregnancies complicated by isolated placental vascular disease( n = ) as defined by birth weight less than th centile for gestation and umbilical artery doppler s:d ratio above the th centile for gestation, with no evidence of maternal preeclampsia or pre-existing hypertension. plasma vegf and sflt- levels were measured using standard eliza techniques. comparison between groups were performed by using one way analysis of variance. the maternal plasma sflt- levels (figure ) in group a: normal pregnancies increased with gestation (p < . ). the sflt- levels in group b: isolated placental vascular disease were significantly higher throughout all gestations (p = . ) and did not show a significant variation with gestation ( p = . ). the plasma vegf levels were below the detectable levels of the assay in all samples except normal pregnancies. the significantly increased maternal plasma sflt- levels in established isolated placental vascular disease without evidence of pre-eclampsia suggest a disease of placental origin. the dysregulation of angiogenic factors may be part of a repair and regeneration process in the placenta. objectives: production of -reduced neurosteroids is critical for reducing fetal vulnerability to stressors in pregnancy and inhibition of -reductases ( r) increase acute hypoxia-induced apoptosis in the fetal brain (yawno et al neurosci : . we have developed a model of placental insufficiency that results in fetal growth restriction (gr) in the guinea pig (palliser et al repro sci # ). the aim of the present study was to determine the effects of suppression of -reduced steroid synthesis on apoptosis in vulnerable regions of the fetal brain and on neurosteroid synthetic enzymes in pregnancies compromised by chronic placental insufficiency. methods: placental insufficiency was induced in guinea pig dams by surgical ablation of uterine artery branches at mid gestation (term d). sham operated or gr dams received finasteride (a r inhibitor; mg/kg/day) during late gestation ( d until term). activated caspase- , a marker of apoptotic cell death, was measured by immunohistochemistry and steroidogenic enzymes, r and cytochrome p side chain cleavage (p scc) were measured by real time pcr and western blotting in fetuses ( d) and neonates h after birth. results: placental insufficiency significantly reduced fetal body and organ weight by % whilst sparing brain weight. the number of activated caspase- positive cells was significantly increased in the fetal hippocampus of gr fetuses and further increased in the cortex of gr fetuses receiving finasteride. the neonatal brain also exhibited changes in caspase- activation following gr and finasteride treatment. the fetal adrenal and the placenta responded to the compromise with increased expression of p scc mrna and r protein, respectively. conclusion: the combination of placental insufficiency and suppressed neurosteroid system leads to markedly increased apoptotic cell death in the fetal brain which continues to affect the neonatal brain. the fetus responds to these conditions by increasing steroid synthetic enzyme expression in the placenta and adrenal glands suggestive of a possible neuroprotective feedback process. to study the effect of smoking by mothers on the fetal and neonatal brain using non-invasive magnetoencephalography technique (meg). materials and methods: using fetal magnetoencephalography, cortical auditory evoked responses (aer) were measured from fetuses ranging from to weeks gestational age for a total of recordings. measurements were taken from mothers with a history of smoking (sm) and from mothers with no smoking history (ns). after delivery, five sm and five ns newborns had meg aer measurements twice for a total of recordings. aer was quantified by cross-correlation analysis and its significance was assessed by boot-strap technique. results: aers were detectable in out of fetal recordings in the ns group and of in the sm group. the neonatal response rate was % for each group. the latencies were divided into three components: c ( - ms), c ( - ms) and c ( - ms). in both fetuses and neonates as well, there was a statistically significant difference (p< . ) between the two groups in the c -component and the sm group showed faster aer compared to the lr group. conclusion: meg technique provides a non-invasive approach to study the effects of smoking on developing fetal and neonatal brain. the observed decrease in the latency of fetuses and neonates of the smoking mothers could indicate a hypersensitive cortical response to auditory tone. adenosine (ado) modulates metabolism in adult mammals through multiple mechanisms that involve ado a and a a receptors. objective: this study was designed to test the hypothesis that ado a and a a receptors participate in fetal metabolic homeostasis. methods: experiments were performed in chronically catheterized fetal sheep (> . term). intravascular infusion for h of dpcpx (a receptor antagonist) or zm (zm, a a receptor antagonist) was performed alone or in concert with ado administration. the highly selective ado receptor antagonists were also infused in fetuses in which hypoxia was induced for h by having the ewe breathe a hypoxic gas mixture (fio = . ). data were analyzed by two-way repeated measures of anova. results: blockade of ado a receptors (n= ) increased significantly (p < . ) fetal concentrations of glucose [control (c): . ± . (se)]; experiment (e): . ± . mg/dl] and lactate (c: . ± . ; e: . ± mg/dl) without significantly altering insulin levels and arterial blood gases or ph. antagonism of ado a a receptors (n= ) did not affect plasma levels of glucose, lactate, or insulin. intravenous infusion of ado (n= ), which did not alter pao or paco , increased concentrations of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . ). zm (n= ), but not dpcpx (n= ), abolished ado-induced rise in glucose and lactate concentrations. isocapnic hypoxia (pao torr), which increases ( - fold) fetal plasma ado levels to those similar to ado infusion, decreased arterial ph (c: . ± . ; e: . ± . ), and increased fetal levels of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . mg/dl) without altering changing insulin concentrations. these effects of hypoxia were not altered by dpcpx or zm. conclusions: ) a receptors modulate plasma levels of glucose and lactate in normoxic fetuses; ) a a receptors mediate the adoinduced rise in plasma glucose and lactate; and ) a and a a receptors are not significant modulators of hypoxia-induced changes in plasma levels of glucose and lactate. supported by usphs hd- . objective: to investigate the dynamic changes of the relationship between leptin, adiponectin and resistin in maternal and fetal circulation during pregnancy and in the early post-natal period. materials and methods: thirty pregnant women with uncomplicated singleton pregnancy delivered at term. maternal and fetal/neonatal venous blood samples were obtained at delivery and at hours from birth. leptin, adiponectin and resistin were measured by specific elisa assays. neonatal anthropometric measurements, glucose metabolism and lipid profile, blood pressure information were obtained. statistical analysis was performed by anova followed by student t test or duncan's test whenever appropriate. correlations were calculated by using the pearson coefficient. results: adipokines concentration at birth and at h from birth was showed in table. multivariate regression analysis showed that fetal leptin levels were positively associated with female gender and adiponectin levels, but not with anthropometric characteristics. fetal leptin and adiponectin levels were not correlated with maternal concentration, whereas fetal and maternal resistin levels were. fetal and maternal resistin concentration was positively associated with gestational age and birth weight and fetal resistin levels correlated negatively with lipid profile. after birth leptin concentration in maternal and neonatal circulation decreased dramatically and the correlation between leptin and adiponectin levels was lost. a positive correlation between resistin and leptin concentration in neonatal circulation was found at h from birth. conclusions: in contrast to adult life a positive correlation between leptin and adiponectin is present in fetal life. placental secretion of leptin, but not of adiponectin and resistin, contributes significantly to maternal and fetal circulating levels. significant changes in the relationship among adipokines occurred immediately after birth and may affect growth and development in early post-natal period. adipokines concentration in maternal and fetal circulation ) antagonism has been shown to normalize placental perfusion and fetal growth in several rat models of fetal growth restriction. however, direct administration of et a antagonists to newborn rats within hours of delivery has been consistently associated with neonatal demise due to failure of the ductus arteriosus to close, raising concerns about the safety of their use late in pregnancy. perinatal exposure to et a antagonists (maternal administration in late gestation) and its impact on rat pup survival and oxygen saturation has not been investigated. objective: to determine the impact of a maternally administered et a antagonist on oxygen saturation in newborn and -day-old rat pups. methods: timed pregnant sprague-dawley rats were treated with fr ( mg/kg/day; et a antagonist) or . % nahco vehicle, by subcutaneous osmotic pump connected to an intravenous catheter, from gestational day (term= days) through parturition. all five pregnant rats in each group delivered spontaneously and nursed their pups through postpartum day . oxygen saturation of each rat pup was measured by pulse oximeter on postpartum days and . results are presented as means ± se. newborn -day neonate vehicle . ± . . ± . eta antagonist . ± . . ± . there were no statistically significant differences between the treatment groups. maternal administration of an et a antagonist from gestational day through parturition, at a dose sufficient to ameliorate fetal growth restriction, has no adverse impact on oxygen saturation in neonatal rat pups. helen l torrance, jan b derks, martijn a oudijk, avnesh s thakor, tereza cindrova-davies, frank van bel, gerard ha visser, graham j burton, dino a giussani. perinatal center, university medical center utrecht, netherlands; department of physiology, development neuroscience, university of cambridge, united kingdom. introduction: the management of perinatal asphyxia remains a major concerns in obstetrics. umbilical cord compressions (ucc) induce fetal asphyxia and ischaemia-reperfusion (i/r). i/r increases reactive oxygen species, for instance via activation of the xanthine oxidase (xo) pathway, which may promote oxidative stress in the fetal circulation. while treatment with allopurinol of asphyxic human neonates reduced free radicals and improved cardiovascular status, treatment started postnatally was deemed too late to prevent oxidative damage (benders et al. arch dis child : , ) . consequently, in complicated pregnancy, recommendations to treat the fetus via the mother, rather than the neonate, with allopurinol are being entertained today. we investigated the effects of maternal allopurinol treatment on indices of oxidative stress in the fetal heart following repeated ucc in sheep. methods: at . of gestation, surgically instrumented sheep fetuses were submitted to i/r ( x min repeated ucc) under maternal allopurinol (n= ) or saline vehicle (n= ) infusion. fetal hearts were collected h after i/r and snap frozen for measurement of (anti)oxidant proteins by western blot. hearts from uninstrumented fetal sheep at . gestation served as controls. statistical comparisons were made using one-way anova. results: i/r episodes led to increased expression of cox- , enos, hsp and decreased expression of sod and gluthatione peroxidase (gpx) in the fetal heart, findings consistent with cardiac oxidative stress. maternal treatment with allopurinol ameliorated these effects (fig. objectives: hypoxic-ischemic fetal brain injury (hie) is a major cause of neonatal death and morbidity. evidence suggests that the brain cell injury associated with chronic hpx (in contrast to an acute ischemic reperfusion injury) is a complex process reflecting a series of adaptive intracellular events that are duration and gestational age dependent. we applied advanced proteomic tools as a next step toward ascertaining a more complete understanding of the impact of hpx on the fetal brain proteome. methods: time-mated guinea pigs were housed in a chamber beginning on day for d, breathing either room air (nmx), or . % or % o ( % o hpx or . % o hpx). on day (term), the fetal brains were removed and preserved for study. total protein was extracted, and the proteome first characterized by d gel electrophoresis. the density of the resulting protein spots were acquired and analyzed using the gs densitometer and pdquest software. identified spots of interest were trypsin digested and subject to maldi mass spectrometry using the proteomics analyzer mass spectrometer for peptide mapping or sequencing. the hpx-induced protein spots were identified based on a minimum of a x change from nmx. results: hpx had a clear effect on the fetal brain proteome. superoxide dismutase (sod), heat shock protein (hsp ), actin (actg ), interleukin- (il- ), and glutamine synthetase (gs) were each up regulated, while cofilin- , brain-type creatine kinase (bb-ck), and - gtp binding protein were each down regulated. all protein changes were proportional to the hpx ( % o hpx vs nmx, % o hpx vs . % o hpx, p< . ). conclusions: this initial application of proteomic techniques confirms that fetal brain damage secondary to chronic hpx (in contrast to acute hpx) is a complex processes characterized by fetal adaptations mediated by multiple protein activations and inactivations. while sod, il- , and gs have each been previously investigated, the potential roles of actg , bb-ck, beta- gtp binding protein, and cofilin- in the fetal response to hpx and the associated brain damage are unclear and represent strong candidates for future investigation. acknowlegement: this study was supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). intermittent umbilical cord occlusion occurs in % of human pregnancies.given that elastogenesis within the vascular wall is in part mediated by hemodynamic conditions during development, the blood pressure response to acute hypoxic insults such as cord occlusion may alter arterial composition. elastin content of a central and peripheral artery and blood pressure responses in fetal sheep exposed to varying degrees of cord occlusion were determined. methods: over a day period, near term fetal sheep received total umbilical cord occlusion (uco) lasting min/ hour (mild group; n= ), min/hour (moderate group; n= ), min/hour (severe group; n = ) or no occlusion (control group; n= ). fetal arterial blood samples were drawn min prior to and at the end of cord occlusions. mean arterial pressure (map) was monitored continuously. the carotid and superior mesenteric arteries were excised and a colorimetric assay (biocolor) performed for determination of elastin content. results are presented as mean ± sem. results:umbilical cord occlusions produced decreases in fetal arterial oxygen pressure and oxygen saturation that were progressively more pronounced across mild, moderate and severe uco groups (p < . ). lactate concentration rose during occlusions in the moderate and severe groups, but not the mild group (p < . ).elastin content of the superior mesenteric artery did not differ between the experimental groups and the control group. elastin content of the carotid artery and map response elastin content (μg/mg tissue) max ∆ in map duration of rise in map (min) control . ± . . ± . -mild . ± . . ± . ** . ± . moderate . ± . . ± . ** † . ± . † severe . ± . * . ± . ** † † . ± . † † * p < . ; ** p < . : experimental groups compared to control † p < . ; † † p < . : compared to mild group the max in map was positively correlated with elastin content (r = . , p < . ). conclusions:the transient rise in blood pressure and preferential blood flow to the brain that occur in response to acute hypoxemia during severe intermittent umbilical cord occlusion induce an increase in elastin synthesis in the carotid artery.this may give rise to adaptive programming of postnatal central arterial compliance. electrocortical activity during repetitive umbilical cord occlusions (uco) with worsening acidemia in the ovine fetus near term. martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. obgyn, chri, univ of western ontario, london, canada; obgyn, harbor-ucla med ctr, torrance, ca. objective: uterine contractions during labour can restrict umbilical blood flow compromising fetal oxygenation and leading to adverse neonatal outcome including newborn encephalopathy/subsequent cerebral palsy. while electronic fetal heart rate (fhr) monitoring is widely used for assessment during labour, abnormal fhr patterns as used clinically have a poor positive predictive value for concerning/significant acidosis at birth. there is limited study of fetal electrocortical activity (ecog) in animal models with induced hypoxia/ acidemia as might be seen during labour. thus, we aimed to induce repetitive ucos in fetal sheep leading to worsening acidemia to determine the predictive value of ecog activity for fetal compromise. methods: near-term fetal sheep (n= ) underwent chronic preparation with artery catheters, ecg/ecog electrodes and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) ucos with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood samples for blood gases and ph were taken at selected time points during the baseline, ucos, and recovery periods. arterial blood pressure (abp) and fhr were continuously monitored and spectral edge frequency (sef) was calculated from ecog. correlation of sef with fhr change was calculated for time intervals using sef maxima and fhr minima during uco series. data are presented as means±sem. results: repetitive ucos led to development of a marked acidosis (ph . ± . to . ± . , p< . ). ± min prior to fetal ph drop < . , the sef of ecog began to increase abruptly during each fhr deceleration from ± hz up to ± hz (p< . ) and was correlated to both fhr change and a decrease in fetal abp during each fhr deceleration at this time (p< . ). conclusion: our findings suggest that in the animal model studied ( ) fetal ecog activity is impaired with progressive acidemia accompanied by fhr decelerations and pathological abp decreases; ( ) there is a consistent temporal relationship between the occurrence of ecog alterations and the subsequent critical drop of ph < . . these findings could contribute to improvement in the clinical ability to predict fetal compromise during labour using ecog/fhr monitoring. background: the ductus arteriosus (da) plays a pivotal role in fetal development and circulation. during gestation, patency of the fetal da is maintained by nitric oxide (no) and prostaglandins (pg). no and pgs are downstream effectors of estrogen (e ) and progesterone (p ). however, the roles of e and p in da regulation have not been studied. objective: we hypothesized that: e and p have opposite effects in the da; that rising e and falling p levels help trigger da closure via specific hormone receptors; and that no and/or pgs mediate da responses to e and p . methods: expression of er , er , pra, prb, and the putative membrane receptors mpr-, -, -, pgrmc- , - , and serbp- was examined by rt-pcr and qpcr on days , , (term), and p . the effects of e and p ( - - - m) on the d fetal da were examined in a cannulated microvessel myography system. e and p effects were also studied in the presence of receptor antagonists (ici , ru ) and no and pg inhibitors (l-name, indomethacin). results: er and pr receptors were expressed at low levels; pgrmc- expression was stronger than other membrane prs, and increased with advancing gestation. under fetal o conditions, e induced rapid, concentrationdependent dilation at - - - m (n= ); p induced progressive vasodilation at all doses (n= ). e and p -induced dilation occurred within - seconds of exposure. while e -dilated das did not respond to ici, ici-pretreated das failed to dilate to the same dose of e (n= ) suggesting antagonism of e effects. the vasodilatory effects of e were partially inhibited by pretreatment with l-name. p -dilated das did not respond to ru , whereas ru pretreated das showed a small, significant response to p (n= ), suggesting partial antagonism or signaling via alternative, ru -independent pathways. pre-treatment with indocin did not block the vasodilatory effects of p . conclusions: contrary to our expectation, both e and p have dilating effects on the fetal da, via no, pgs and non-no, non-pg pathways. expression studies and the rapid response of ex vivo das are consistent with non-genomic actions via membrane receptors. hormone shifts in parturition may have longterm effects on da preparation for postnatal closure, but strategies to maintain fetal da patency or treat newborns with pda will require better understanding of this process. background. for the fetus, arterial blood gases are critical in the regulation of cerebral blood flow (cbf) and cerebral oxygenation. however, the relation of cbf, cortical tissue po (tpo ), and electrocorticographic (ecog) activity to arterial o tension (pao ) are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that in the near-term fetus, acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with ecog state. methods. by use of a laser doppler flowmeter with a fluorescent tissue o probe, and with fluorescent-labeled microspheres, and with ecog electrodes, in near-term fetal sheep (n = ) we measured laser doppler cbf (ld-cbf), tpo , ecog (root mean square (rms) voltage with high voltage low frequency, hvlf versus low voltage high frequency, lvhf) and spectral edge frequency- % (sef ) in response to min moderate isocapnic hypoxia. results. ld-cbf, cerebral o delivery, tpo , and several other variables correlated highly with ecog state. in the normoxic control fetus, in association with a shift from hvlf to lvhf ecog activity, tpo decreased briefly to ± from a control value of ± torr; however, as ld-cbf increased ± %, and sef increased to ± from ± %, tpo returned to near normal value. with acute hypoxia (pao = ± torr) when in the lvhf state ld-cbf increased only ± %, as opposed to a ± % increase when in hvlf ecog state. with this degree of hypoxia, tpo decreased to ± torr, sef remained at ± %, and cerebral metabolic rate for o (cmro ) decreased ± % (p< . ). conclusions. for the near-term fetus, normoxia with changes in ecog state was associated with brief periods of decrease in tpo , which were restored quickly by increased ld-cbf. in contrast, acute hypoxia was associated with a significant depression of cortical tpo , cmro , and ecog state, with increased ld-cbf failing to restore cortical tpo . thus, we reject the null hypothesis that in such fetuses, hypoxia demonstrates no compromise in cerebral oxygenation. (supported by usphs hd- ). releasing hormone and arginine vasopressin in the ovine fetus. charles a ducsay, kanchan m kaushal, malgorzata mlynarczyk, kimberly hyatt, dean a myers. ctr. for perinatal biol., loma linda univ., loma linda, ca; ob/gyn, univ. oklahoma hlth. sci. ctr., oklahoma city, ok. background: long term hypoxia (lth) profoundly affects the hypothalamicpituitary-adrenal axis of the fetal sheep. we previously showed that lth causes augmented corticotrope function. the present study was designed to test the hypothesis that lth enhances sensitivity to the acth secretagogues; cortiocotrophic releasing hormone (crh) and arginine vasopressin (avp) resulting in increased anterior pituitary corticotrope secretion of acth. methods: pregnant ewes were maintained at high altitude ( , m) from day to - of gestation (dg), when they were returned to the lab and a maternal tracheal catheter was implanted. maternal po was maintained at a level comparable to that observed at altitude ( mmhg) by nitrogen infusion. on dg, lth (n = ) and age-matched, normoxic control (n = ) fetuses were implantated with vascular catheters. each fetus received a min infusion of either saline vehicle, ng/kg of ovine crh or ng/kg of avp (estimated body weight/min) in a randomized order over consecutive days ( - dg). blood samples were collected at min (baseline prior to infusion), , , and min following the start of the infusion and analyzed for acth, as well as the acth precursors pro-opiomelanocortin and the major processing intermediate kda proacth. results: vehicle had no effect on any of the measured parameters. with crh infusion, acth (pg/ml) increased in both groups over the course of the study. however, peak concentrations (at min) were significantly higher in the lth group compared to control ( ± vs. ± , respectively; p< . ). acth precursor secretion (pm) was greater in lth fetuses compared to controls during the experiment (p< . ). in response to avp, peak acth concentrations were also higher in the lth fetuses compared to control ( ± vs. ± respectively; p< . ), however peak levels were reached at between and min after start of infusion with levels in both groups returning to pre-infusion values. a similar pattern was observed with precursor levels ( . ± . vs. . ± . , p< . , lth vs. control). conclusions: lth significantly increases pituitary sensitivity to both crh and avp. this enhanced sensitivity may be mechanism of our previously observed enhanced corticotrope function. (supported my nih grants hd and hd ). martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. ob/gyn, chri, university of western ontario, london, canada; ob/gyn, harbor-ucla med. ctr., torrance, ca. objective: repetitive uco leading to worsening fetal acidosis with fetal heart rate (fhr) decelerations (fhr dec ) are accompanied by pathological decreases of fetal arterial blood pressure (bp). we hypothesized this bp change may be caused by the bjr, a vagally mediated reflex with bradycardia and hypotension to reduce cardiac workload, via stimulation of cardiac chemoreceptors during systemic acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. after a control period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco each lasting h or until ph decreased to < . . fetal arterial blood samples were taken at selected time points during the control and uco periods. bp and fhr were continuously monitored. individual fhr nadir during each fhr dec and accompanying bp change ( bp = [bp at the time of a fhr nadir] -[bp at baseline preceding a uco series]) were determined during all uco. data are presented as means±sem. results: control period ph ( . ± . ), fhr ( ± bpm) and bp ( ± mmhg) were within the physiological range. average depth of fhr dec for all uco was ± bpm and increased with higher lactate concentrations (r = . , p < . ) and lower ph (r = . , p = . ). bp during all uco demonstrated an initial hypertensive response to fhr dec , which decreased with lower ph (r = . , p < . ). bp increased ± mmhg (p < . ) during mild uco (ph to . ± . ), ± mmhg during moderate uco (ph to . ± . , p < . ) and ± mmhg during severe uco (ph to . ± . , p < . ). conclusion: these results suggest that bjr, a short-acting, ph dependent depressor reflex, blunts the physiologic hypertensive response to cord occlusion insults leading to worsening acidemia as might be seen in the human fetus during labour with repetitive variable fhr dec . the failure to increase bp may prevent optimal blood flow distribution responses necessary for preservation of vital organs. role of nos and pde in placental dysfunction following fetal bypass. mitali basu, r scott baker, christopher t lam, kenneth e clark, pirooz eghtesady. cardiothoracic surgery, cincinnati children's hospital, cincinnati, oh, usa; ob/gyn, university of cincinnati, cincinnati, oh, usa. introduction rising placental vascular resistance following fetal cardiopulmonary bypass (bypass) remains the achilles' heel of fetal cardiac surgery. we have previously shown in real-time that nitric oxide (no) production rises during bypass and falls post-bypass, while cgmp levels rise throughout. using immunohistochemical and western analysis of placenta, we examined the involvement no pathway components in this placental vascular pathophysiology. ovine fetuses at - days gestation were placed on bypass for minutes and followed post-bypass for hours. placental samples were collected immediately prior to bypass and at and min post-bypass (n= ) and compared to a group of similarly instrumented controls, (n= ). placental enos, inos and pde protein expression was measured using standard methods and relative expression normalized to beta-actin. statistical analysis utilized students t-test, and anova for trend and group-wise analysis, (significance at p= . ). pre-bypass protein expression did not differ between groups. pde protein levels and phosphorylated pde- expression were both elevated min post bypass, and reduced min post bypass compared to sham, (p= . ). enos levels in the bypass group increased linearly from pre-bypass to min postbypass (p< . ), and were also elevated compared with shams (p< . ), while shams had declined significantly by min post bypass, (p< . ). similarly, phosphorylated enos expression in the bypass group increased linearly from pre-bypass to min post-bypass, (p= . ), while shams trended towards decline by min post-bypass. simultaneously, placental inos expression remained stable within groups, but was lower in the bypass group at and min post-bypass, (p< . ). the preceding data correlated with observed immunohistological changes in the same placental cotyledons. fetal bypass leads to significant increases in placental protein levels of pde , phosphorylated pde- , enos and ostensibly phosphorylated enos. increased pde expression may be a response to increased no and the generated cgmp. this data suggests a compensatory upregulation of pde and enos that eventually fails, leading to increasing placental vascular resistance and subsequent lethal placental dysfunction. angiotensin receptors (rat-ii) in neuronal nuclei of the brainstem have been implicated in integration of baroreceptor responses. in near term fetal sheep, we have previously shown that days of mild chronic hypoxemia increases heart rate (hr) and blood pressure (bp). the aim of the present study was to investigate the effects of chronic mild hypoxemia on the expression of rat-ii in the medulla oblongata and on baroreflex control of the circulation. methods: at days of gestational age, pregnant sheep were submitted to days of hypoxemia ( % of fetal arterial po ; at day fetus ± vs ± . ; mother ± vs ± mmhg). hr and arterial bp were continuously recorded from mother and fetus in control (n= ) and hypoxemic (n= ) animals. baroreflex sensitivity (brs) as well as hr and diastolic bp variability were analyzed (nevrokard software). brainstem was collected and rat-ii expression in the nucleus tractus solitarius (nts) and dorsal motor nucleus of the vagus (dmnx) was measured by autoradiography using i-sarthran labeling. data are shown as mean±sem and were analyzed by t-test. results: hypoxemia induced a greater total binding of i-sarthran in nts and dmnx resulting from an increased expression of at receptors. in the time-domain analysis of brs hypoxemia induces lower baroreflex sensitivity in the fetus (brs in ms/mmhg; . ± . vs. . ± . , p< . ). in the frequency domain, hypoxemia changes the relative power of low frequencies (lf: . - . hz) and high frequencies (hf: . - . hz) of rri and dbp with an increased value of the lf/hf ratio (rri lf/hf . ± . vs ± . , p< . ; dbp lf/hf . ± . vs . ± . , p< . ). also the alpha index for baroreflex sensitivity is decreased in hypoxemic animals (alpha lf . ± vs . ± . , p< . ; alpha hf . ± . vs . ± . , p< . ). no differences were observed in maternal variables. conclusions: our results suggest a link between a prenatal insult, alterations in cns receptors and functional alterations of baroreflex responses. a higher sympathetic outflow, suggested by a greater lf/hf ratio, and impaired reflex gain, both possibly mediated by increases in nts rat , may have potential long-term consequences for the development of hypertension. hd ;hd ;hl . objective : we evaluated velocity profiles, relative wall distension rate (rwdr), and wall shear rate (wsr) of fetal descending aorta (fda) in uncomplicated singleton gestations. study design: ninety seven uncomplicated singleton fetuses were studied throughout gestation using multigate spectral doppler analysis (msda) working with gasp software. this consists of a personal computer (pc) add-on board including a single high-speed digital signal processor. the analysis of echo-signals backscattered from range cells located along the axis of the interrogating ultrasound (us) beam. post-processing was accomplished using gasp software. statistical analysis consisted of spearman correlation and chi-square test. results: velocity profiles, wall distension, wall shear rate were obtained from fetal descending aorta throughout gestation establishing gestational age specific norms. wdr[%] is highly correlated with gestational age in appropriate growth fetuses ( . ± . , rs= . p< . ) with linear regression with standardized coefficient of . (p< . ). in contrast, wsr ( ± ) is unchanged during the first , second and third trimesters (p= . ).conclusion: we speculate that the relative wdr changes observed during gestation, in normally grown fetuses, may be secondary to adaptive vascular and autonomic responses and the evolving composition of the vessel wall, particularly with respect to elastin. conversely, the mean wsr for the study group was independent and constant throughout the gestation. these findings suggest that there is an increase in the diameter of the fetal aorta, which provides adaptation to the progressive flow demands, while preserving other key hemodynamic parameters. in this study, we have established normative values of rwdr and wsr, which are new rheological parameters that may be useful in distinguishing normal and pathologic hemodynamic states. objective: placental dysfunction is a key barrier to successful fetal cardiopulmonary bypass (cpb) for repair of congenital heart defects in utero. endothelial cells regulate vascular tone during fetal cpb through interactions of vasodilation by nitric oxide (no) and endothelin- (et- )-mediated vasoconstriction. the objective was to determine the time during fetal cpb when endothelial cell-mediated changes occur. methods: human umbilical vein endothelial cells (huvec) were cultured in media containing % serum collected from ovine fetuses (n= ) that underwent min of cpb, then were maintained for min. serum was collected before cpb, from pump prime before initiation of cpb, min on cpb, or and min after fetal cpb. control cells were cultured in normal fetal serum. cells were harvested and hr after addition of fetal serum. no production was measured in real time with an electrochemical detection system (inno-t, harvard apparatus). et- was measured in the culture media by elisa. results: no production by huvec after and hr was stimulated above control levels by fetal serum collected during and up to min after fetal cpb (p< . ). serum collected from fetuses that were surgically instrumented, but not yet subjected to cpb, decreased no levels below controls (p<. ). stimulation of et- after and hr of huvec culture peaked with serum collected at min after fetal cpb (p<. compared with control), but was elevated above control levels at each collection time point (p<. , table ). conclusions: fetal cpb releases serum proteins that elevate endothelial cell no and et- production during and for at least min after cpb. although the specific regulatory proteins remain to be identified, the no and et- pathways share circulating mediators and participate in a feedback loop to modulate vascular tone. to test the hypothesis that hypoxia-induced upregulation of no is linked to cardiac inos expression, a selective inos inhibitor, l-nil (l-n -( -iminoethyl)-lysine), was administered in vivo to fetal guinea pigs and no levels measured in fetal hearts. methods: pregnant guinea pigs were exposed to either normal room air (normoxia; %o ) or . %o (hypoxia) in a hypoxic chamber for days prior to term (term= d). l-nil was administered to pregnant normoxic and hypoxic guinea pigs via their drinking water at a dose of - mg/kg/d for days. at d gestation, pregnant sows were anesthetized and near-term fetuses removed via hysterotomy. the fetal hearts were excised, weighed, and normalized to their respective fetal body weights. left cardiac ventricles were obtained and frozen in liquid n and stored at - o c until ready for analysis. the effect of total no product (no and no -, nox) of left ventricles of fetuses exposed to normoxia (n= ), hypoxia (n= ) and hypoxia plus l-nil (n= ) was quantified by a commercial fluorometric no assay kit. results: intrauterine hypoxia significantly reduced fetal body weight by % and increased placenta/fetal body wt by % as expected for hypoxic stress. hypoxia induced a slight increase in heart/fetal body wt by %. fetal cardiac nox levels (pmoles/mg) were increased by hypoxia ( . + . ) by . fold compared to normoxic controls ( . + . ). l-nil significantly decreased (p< . ) nox levels in hypoxic hearts by % ( . + . vs . + . ; hypoxic vs hypoxic+l-nil, respectively). conclusion: l-nil inhibits inos-derived no generation in the hypoxic fetal guinea pig heart. since previous study in our lab showed a significant increase in inos expression but a decrease in enos and no change in nnos expression, we hypothesize that hypoxia upregulates cardiac no generation via the inos pathway. further study is needed to identify the important role of cardiac inos in the adaptive response of fetal hearts to chronic intrauterine hypoxia. objective: fetal asphyxia-mediated metabolic acidosis results in a ph decrease and an increase of lactate and base deficit in blood (bd blood ) and extracellular fluid (bd ecf ). it is not clear whether bd blood and bd ecf are similarly altered with worsening fetal acidosis. in the present study we sought to study the dynamic relations of bd blood and bd ecf to lactate in the ovine fetus subjected to repetitive uco with worsening acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood was sampled at baseline, immediately before and during the first mild, moderate and severe uco and at min intervals during the moderate and severe uco. bd gap (bd blood -bd ecf ) was studied in relation to lactate. presented as means±sem. results: lactate correlated to bd blood (r= . , p< . ). bd ecf correlated strongly with bd blood (r= , p< . ). bd ecf increased by . ± . mmol/l more than bd blood from baseline to ph nadir, with bd gap therefore decreasing with increasing lactic acidemia ( fig. ) . lactate, bd blood , bd ecf and bd gap correlated to ph (r= . , r= . , r= . and r= , respectively, all p< . ) and ph could therefore be predicted with any of the four acid-base parameters. conclusion: the increases of bd blood and bd ecf and the decrease of bd gap with increasing lactic acidemia suggest a relatively more rapid accumulation of [h+] in ecf during uco of increasing severity. this may be because the metabolic build-up of acidosis occurs primarily in the tissues and the endothelial permeability for [h+] is impeded during increasing acidosis with atp depletion thus decreasing [h+] movement from ecf to plasma. previous studies have shown that intraperitoneal transplantation of human umbilical cord blood (hucb)-derived mononuclear cells led to the specific 'homing' of these cells to a hypoxic-ischemic brain lesion in perinatal rats. motor deficits resulting from the lesion were alleviated upon transplantation. thus, the presence of hucb cells at the lesion site seems to be a major prerequisite for their potential beneficial effect. however, the mechanisms of cell 'homing' are still unclear. in this study, we focused on elucidating mechanisms underlying the specific migration of hucb-derived mononuclear cells to the brain lesion. one possibility to induce cell 'homing' are chemotactic signals present at the lesion site. the cxc chemokine stromal derived factor- (sdf- ), which was previously shown to be a potent chemoattractant for directed migration of other stem and progenitor cells, is a putative candidate in our lesion paradigm. therefore we investigated the spatial and temporal expression of sdf- in brain hemispheres with or without hypoxic-ischemic lesion. sdf- expression was substantially increased at the lesion site during the investigated period of fourteen days after the insult. furthermore, hla-positive hucb cells were mainly detected in sdf- expressing brain regions and we were able to show that these cells express the sdf- receptor cxcr on their surface. the functional implication of sdf- in directing hucb cell migration was determined by application of neutralizing sdf- antibodies in vivo, resulting in a reduced number of hucb-derived mononuclear cells at the lesion site. with these functional effects, together with the observed timing and location of its expression, the involvement of the chemokine sdf- in hucb cell 'homing' seems conceivable. novel pathways in inflammation-induced fetal brain injury. michal a elovitz, jinghua chai. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. intro: while survival of extremely preterm infants continues to improve, the number of children with cognitive impairment and/or cerebral palsy is increasing. if preterm birth (ptb) cannot be prevented, then strategies to identify and treat fetal brain injury in the setting of a ptb must be investigated. these studies were performed to explore novel pathways involved in fetal brain injury in the setting of inflammation-induced ptb. methods: cd- mice on e receive intrauterine injection of lipopolysaccharide (lps) or saline. hours after injection, fetal brains from the left upper horn were harvested. fetal brains were removed from each dam with dams per treatment group. separate rna samples were prepared and used for microarray (ma) analysis. all protocols were conducted as described in the affymetrix genechip expression analysis technical manual in the ma core facility using the moe av chip. data analysis was performed using significance analysis for microarray (sam). the brain samples from the same dam were considered as dependent samples. pathway analysis and functional annotation clustering tools were used with david. validations studies using qpcr with fetal brain samples (n= - per group) were performed. results: while there was significant differences in gene expression between lps and saline exposed fetal brains, variability existed even between pups from the same dam. genes were significantly differentially expressed between lps and saline brains (p< . ). with a p value of < . , genes were differentially expressed. pathway analysis revealed significant involvement of ) the cadeherin, cadherin-like, cell fraction and calcium binding (enrichment score . ) and ) ribosomal processing, rna metabolism, pyrimidine metabolism (enrichment score . ). specifically, genes involved in neurogenesis, synaptic function, and neuronal and glial metabolism were most differentially regulated. qpcr confirmed observed fold changes in / genes analyzed. inflammatory pathways were not differentially regulated. conclusions: current theories regarding fetal brain injury in ptb focus on activation of inflammatory processes as essential events. this data suggests that long-term neurological injury in a ptb may be secondary to altered neuronal function, metabolism and/or communication. disruptions in these pathways should be explored as key mechanisms to adverse neurological outcomes in preterm neonates and should be targets for future investigations. regulation of microglial activation in the developing murine brain. mariya hristova, virginia zbarsky, daniel cuthill, adam wallace, donald peebles, gennadij raivich. institute for women's health, university college london, london, united kingdom. introduction: the aim of this study was to assess the process of microglial differentiation in developing white matter, an area of the brain that is particularly vulnerable to damage pre-myelination (approx weeks gestation). microglia form a distinctive non-neuronal component. although related to peripheral macrophages they undergo highly specific processes of regional maturation and differentiation inside the brain with a slimming of the cell body, development of very elaborate crenulated arborised branches and downregulation of most macrophage activation markers. this process is relatively rapid in most grey matter brain regions, but is retarded in and around the subcortical white matter (swm) giving rise to the phagocytic fountains of microglia (fom). methods:we examined the process of deactivation and morphological differentiation in the cortex and swm of mice - days after birth (p -p ) using confocal microscopy for monoclonal antibodies against alpha and beta integrin subunits and the costimulatory factor b . , colocalised with standard microglial marker iba . results: strikingly, only the fom macrophages, but not cortical microglia, strongly expressed typical activation markers alpha- , alpha- , alpha-m, alpha-x, beta- and b . . the data for alpha-x are shown as an example in the figure; cortical microglia are shown in the grey bars and swm in black. fom activation was maximal at p , decreased linearly over p and p and disappeared at p . this process followed the presence of ingested phagocytic material but correlated only moderately with ramification, demonstrated by non-ramified but inactive p cortical microglia and formation of stubby processes in p fom. conclusion: these data describe strong and selective biochemical activation of fom phagocytes in p -p swm, roughly equivalent to early rd trimester human foetal development. this presence of highly active phagocytes in the neighbourhood of vulnerable wm could play an important role in the genesis of axonal damage in the foetus and premature neonate. , pp. - ) . mbp is expressed in mature oligodendrocytes (jakovcevski and zecevic, glia ) . although myelination in the fetal sheep brain, a model extensively used to evaluate fetal development, has been described using conventional staining techniques (barlow, j comp neurol ) the use of specific mbp staining allows a more precise determination of the onset of myelination (antonow-schlorke et al., reprod sci . , , a) . aim: to use mbp expression to determine the trajectory of development of different white matter tracts of fetal sheep brain. methods: the ontogenetic profile of mbp expression was estimated in healthy fetuses at . (n= ), . (n= ), . (n= ), . (n= ), . (n= ), . (n= ) and . (n= ) of gestation (term days). after brain fixation and embedding in wax, sections at the level of the optic chiasm were stained with a monoclonal anti-mbp antibody using the abc-technique. immunohistochemical distribution of mbp was morphometrically assessed in the dorsal internal capsule and cortical white matter tracts, i.e. the lateral centrum semiovale, superficial white matter and median corpus callosum using an image analysis system (scion image . , nih, usa). results: mbp expression of various fetal white matter structures started at different time points; initially in the internal capsule at . of gestation followed by the cortical white matter structures at . of gestation. cortical myelination advanced from the cortical deep white matter via superficial white matter to the corpus callosum reflecting different rates of progression. conclusions: the onset of mbp expression estimated here explicitly antedates previous observations in sheep (barlow, ) the way the embryonic heart functions before cardiac morphogenesis is completed is still subject of many studies. to gain more insight into the early cardiac structure-function relationship, doppler blood flow velocity waveforms at four different locations in the embryonic chicken heart during cardiovascular development were assessed. we collected waveforms using high frequency ultrasound biomicroscopy with a -mhz transducer at hh stages , and which are comparable to humans at to weeks of gestation. waveforms were obtained at the inflow tract, the primitive left ventricle, the primitive right ventricle, and at the outflow tract in ten different embryos per stage. by exploring the time relation between the waveforms, cardiac cycle events were outlined. our results demonstrate that stage and location dependent, intracardiac blood flow velocity waveforms can be obtained in the chicken embryo which reflect stage dependent pumping mechanisms. the blood flow profiles assessed at the four locations in the embryonic heart demonstrated a developmental related increase in velocity. in the primitive ventricle the passive filling wave decreased, whereas the active filling wave increased resulting in a decreased p to a ratio in the course of time. high frequency derived cardiac blood flow velocity characteristics support previous findings that the embryonic heart functions like a suction pump at early development and transforms towards another pumping mechanism at later developmental stages. these findings are of importance for the interpretation of human first trimester cardiac velocimetry studies. figure a . changes in the perimeter of the thymus with gestational age by fetal gender are depicted in figure b . background. for the fetus, the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (cbf) and cerebral metabolic rate for oxygen (cmro ), cortical tissue po (tpo ), and electrocorticographic (ecog) activity. in addition, metabolites such as adenosine (ado) are important in this regard. nonetheless, the relation of adenosine and its metabolites to these indices of cerebral oxygenation are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with adenosine. the most common cause of perinatal brain injury is chronic hypoxia/ischemia. the mechanisms are complex and poorly understood. applying proteomics tools, we identified a set of hypoxia-related proteins in the fetal guinea pig (gp) brain (companion abstract). one protein, cofilin- , which regulates the rapid cycling of actin assembly and disassembly, was dramatically altered by chronic brain hpx. herein, we test the hypothesis that confilin- modifications during hpx contributes to brain damage via apoptosis and is an example of an adaptive response that becomes maladaptive. methods: time-mated gps were housed in a chamber from d to d (term) with either room air (nmx) or . % or % o . fetal brains were removed and sections prepared for double staining specific to bax and dephosporylated cofilin- . total protein was isolated and equal amounts loaded on a sds gel, separated by electrophoresis, and transfered to pvdf membranes probed with primary antibodies to dephosphorylated and phosphorylated cofilin- , bax and bcl-xl, and incubated with second antibody. protein signals were detected, quantified by densitometry, and normalized to -actin. total rna was isolated, reverse-transcribed, and quantified by real-time pcr using sybr green i labeling. expression was calculated by the ct method using s for control. melt analysis was performed to confirm specificity of the amplification, and efficiency determined by the slope of the standard curve. the mitogen activated protein kinase (mapk) signalling pathway is upregulated in perinatal hypoxic-ischaemic brain. the role of the extracellular signal-regulated kinase (erk) cascade, a pivotal component of mapk signalling, remains unclear with reported actions ranging from mitogenic and trophic effects to a neuronal death-promoting role. the aim of this study was to assess the role of neuronal erk activity using selective neuronal inhibition in a perinatal model of hypoxic-ischemic brain injury. methods: we explored the effects of selective neuronal inhibition of erk activation using transgenic mice expressing dominant-negative (dn) mek , the upstream activator of erk / , which was under the control of the pan-neuronal talpha alpha-tubulin promoter. p-erk immunoreactivity was quantified following a hypoxic inschemic (hi) insult in p c bl/ mice, caused by unilateral occlusion of the carotid artery, followed by hypoxia with % oxygen for mins at °c. the volume of surviving brain on the affected hemisphere was expressed as a % relative to the control side. results: expression of the mek dn was associated with a reduction in erk / activation following hypoxia ischaemia, as assessed by p-erk immunoreativity. compared with the wild type, littermate controls, mek dn animals exhibited significantly decreased volume of forebrain damage brain following unilateral, min hi insult (*p< . , **p< . , anova). similar protective effects of mek dn were observed in cerebral cortex, hippocampus, thalamus and striatum when compared to wild type littermate controls and correlated with a significant reduction in microglial activation in all brain areas. conclusion: overall, these results suggest that neuronal mek and its downstream signals have an important death-inducing role in this model of perinatal brain injury and could serve as potential targets for therapeutic intervention. oup, ) . however, whether melatonin protects placento-fetal development during hypoxic or undernourished pregnancy is unknown. we investigated in rats the effects on placental and fetal growth of maternal treatment with melatonin in hypoxic and undernourished pregnancy. methods: on pregnancy day , wistar rats were divided: control ( % o ) + melatonin ( g.ml - drinking water), hypoxia ( % o ) + melatonin, and undernutrition ( % reduction in food intake) + melatonin (n= per group). on day , dams were anaesthetised, the pups, placentae and fetal brain were weighed. results: relative to controls (fetal weight: . ± . g; brain/body weight ratio: . ± . mg/g; n= ), both hypoxic ( . ± . g; . ± . mg/g, n= ) and undernourished pregnancy ( . ± . g; . ± . mg/g, n= ) promoted asymmetric iugr (p< . ), without affecting placental weight. melatonin treatment had no effect on iugr, but it decreased placental weight in normoxic ( . + . , n= ), hypoxic ( . ± . , n= ) and undernourished ( . ± . , n= ) pregnancies relative to untreated controls ( . ± . , n= ; p< . ). when fetal body weight was expressed relative to placental weight, a measure of placental efficiency, melatonin prevented the fall in the ratio in undernourished, but not hypoxic pregnancies (fig. objective: midkine (mk) is a -kda heparin-binding growth factor with various functions including cell proliferation, migration, differentiation and angiogenesis. mk expression is strictly regulated in temporal sequence; it is highly expressed during midgestation. we recently studied mk expression in the midgestation human fetus, and showed that the highest mk expression were observed in the adrenal gland, brain, lung and kidney. in the present study, we investigated the profile of mk in human amniotic fluid (af) and amnion (am). methods: amniotic fluid and amniotic membranes were collected at diagnostic amniocentesis, preterm no labor, and term no labor. mk protein levels were analysed by western blot. expression of transcripts encoding mk and putative mk receptors were examined by rt-pcr and real-time quantitative rt-pcr (qpcr). results: ) western blot analysis demonstrated abundant mk protein in the human am; mk levels were higher at midgestation than at term ( -fold: wks vs. wks). ) tissue transglutaminase known to polymerize mk was abundant in af. ) qpcr revealed that mk mrna was not expressed in am whereas it was highly expressed in the fetal adrenal, kidney and lung (positive controls). ) among the receptors implicated in mk signaling, lowdensity lipoprotein receptor-related protein and syndecan- were expressed in am while protein-tyrosine phosphatase, anaplastic lymphoma kinase, and syndecan- were not. conclusions: abundant mk protein in the midgestation af is likely to be derived from the fetus. mk in af may play a role in feto-amniotic communications and/or development of fetal organs exposed to af. short term hfix expression. we hypothesized that long term hfix expression could be achieved in fetal sheep using an aav vector, without stimulating an immune response to the transgenic hfix protein. we injected aav hfix vector ( - x p/kg) into the peritoneal cavity of fetal sheep under ultrasound guidance in early (n = ) or late (n = ) gestation. fetal blood was retrieved by ultrasound guided sampling from the intra-hepatic umbilical vein. fetal and lamb blood was tested for hfix expression using elisa, antibody responses using functional assays, and for liver damage up to a year after birth. vector spread was detected in maternal and fetal tissues by quantitative pcr analysis. results: the highest level hfix was detected days after late ip injection ( % and % normal human levels). early gestation ip injection gave . % and . % at and days after injection. hfix levels dropped rapidly correlating with the increase in size of the fetal liver and lamb. however, hfix was detectable at a low levels ( . %) year after birth in early and months after birth in late gestation injected lambs. up to year after birth, liver function tests and bile acid levels were normal, showing no evidence of liver pathology. no functional antibodies to the hfix protein or aav vector were detectable. high vector levels were detected in the fetal liver, and other peritoneal organs; no vector was present in fetal gonadal tissue. conclusion: hfix expression is detectable up to year after delivery of aav vector to the fetal sheep using a clinically applicable method. this is the first study to show long term hfix expression after fetal gene therapy in a large animal. further work will include testing for immune tolerance to exogenous hfix protein in these animals. objective: placental estrogens play a pivotal role in the endocrine control of pregnancy and may be involved in the key changes occuring during parturition. it has been established that crh interacting with crh receptor has a positive effect on estrogen production throughout pregnancy. urocortin (ucn ), a novel peptide of the crh family binding exclusively crh receptor , is expressed by human placenta and the aim of the present study was to evaluate the influence of ucn on estrogen biosynthesis in cultured trophoblast cells. methods: cultured term placental cells were treated with various concentrations of ucn in the presence of the estrogens precursors dehydroepiandrosterone sulphate (dhea-s), androstenedione or testosterone. estradiol secretion was measured in the culture medium using a specific elisa, p arom mrna and protein expression were evaluated by real time pcr and western blot analysis respectively. results: the addition of ucn , in presence of dheas significantly increased e levels at , and hours treatment, while in presence of androstenedione and testosterone an increase in e secretion was detected only at , and hours (at different ucn doses). both p arom mrna and protein were up-regulated in presence of each estrogen precursor and the addition of ucn caused a synergistic increase. anti-sauvagine (a crh type receptor antagonist) resulted in a significantly attenuated ucn effects on e secretion and on p arom mrna and protein expression. conclusions: the present study supports a possible role of ucn on placental e biosynthesis: e secretion and p arom transcript and protein expression were significantly increased after ucn treatment. in conclusion, the crf family may play a major role on placental steroidogenesis, stimulating dheas secretion in fetal adrenals by crh and controlling placental estrogen biosynthesis through ucn . a possible influence on the mechanisms of parturition may be hypothesized. increased expression of kiss- gene in preterm placenta. letizia galleri, michela torricelli, chiara voltolini, fernando m reis, felice petraglia. department of pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. introduction placenta expresses a large number of peptides involved in delivery. neuropeptides, cytokines, are expressed and secreted by human placenta at the time of preterm delivery. human placenta is a major source of kiss- , a -amino-acid peptide encoded by a putative metastasis suppressor gene. kiss- acts on its placental g protein-coupled receptors (kiss- r); this peptide stimulates release of oxytocin in rats, the most potent known uterine stimulant, suggesting a possible role of kiss- in the mechanisms of labor. aim of study the aim of this study was to evaluate placental expression of kiss- at preterm labor. material and methods placental tissue and plasma samples (both maternal and fetal) were collected at term in the absence of labor (tnl), at term spontaneous vaginal delivery (tl), and at preterm labor (ptl). changes in placental mrna expression were determined by real-time quantitative rt-pcr analysis. kiss- protein levels were measured by specific immunoenzymatic assay (elisa). results placental kiss- mrna expression was significantly higher (p< . ) in ptl than in tnl and in tl. however, maternal and fetal plasma kiss- levels did not differ among tnl, tl, and ptl samples. conclusion the present study showed that placental kiss- mrna expression is increased in preterm delivery. further studies are needed to better understand the role of kiss- on cascade leading term and preterm labor. placental angiogenesis is essential for placental development, which occurs under a hypoxic environment ( - % o ) during normal pregnancy. it has been reported that in transformed human dermal microvascular endothelial cells, hypoxia activates erk / , which stabilizes hypoxia-inducible transcription factor- (hif- ). however, signaling mechanisms governing placental angiogenesis under hypoxia is largely unknown. herein, we tested whether hypoxia affected fgf -and vegf-stimulated cell proliferation partly via activating erk / and stabilizing hif- protein levels in human placental artery endothelial (hpae) and transformed human placental microvascular endothelial (hpme) cells. methods: cells cultured under normoxia ( % o ) or hypoxia ( % o ) for days were treated with fgf and vegf. after days, the number of cells was determined. activation of erk / and levels of hif- protein were determined by western analysis. results: under normoxia, fgf and vegf stimulated (p < . ) cell proliferation and induced ( min, p < . ) erk / phosphorylation in hpae and hpme cells. hypoxia promoted (p < . ) fgf -and vegf-stimulated cell proliferation in hpae cells, whereas hypoxia blocked (p < . ) such actions in hpme cells. hypoxia enhanced fgf -, but not vegf-induced erk / phosphorylation in hpae cells. in contrast, hypoxia promoted (p < . ) vegf-, but not fgf -induced erk / phosphorylation in hpme cells. hypoxia increased (p < . ) hif- levels in the hpae cells: first detected at . hr and maintained up to hr. hpme cells had high basal levels of hif- ( folds; p < . ) compared with hpae cells. hypoxia did not alter hif- levels up to hr and decreased (p < . ) hif- levels at hr in hpme cells, conclusions: in hpae cells, hypoxia enhances fgf -and vegf-stimulated cell proliferation possibly partially via promoting activation of different protein kinases. this stimulatory effect is associated with increased hif- protein levels. however, inhibition by hypoxia on fgf -and vegf-stimulated hpme cell proliferation is associated with relatively high hif- levels in the first hr and decreased hif- levels at hr. thus, these data suggest that different signaling mechanisms are involved in hypoxia-modulated growth factor-induced placental angiogenesis in which an increase in hif- levels plays a critical role. objective: corticotrophin releasing factor (crf) is a well established neurohormone involved in the initiation of the stress response. we recently documented that rat placenta is analogous to human placenta in the expression of crf-mrna and protein, and that placental crf release may contribute to in utero meconium passage. time-of-day variation in crf expression is a highly recognized phenomenon at the brain cellular sites of crf synthesis. we sought to determine whether crf expression in rat placenta is subject to time-of-day variation. method: time-dated pregnant rats were obtained on day of gestation (term= days), housed in under h- h light-dark conditions (lights on ). lab chow and water were continuously available. on day , pregnant rats were quickly anesthetized by exposure to isoflurane, abdomen opened and fetuses and placenta exteriorized either at - hr (zeitgeiber time, zt ) or - hr (zt ). individual placentas were processed either for rna extraction or immunohistochemical investigation. the levels of crf-mrna were assessed by pcr using rat specific pcr primers. pcr bands were subsequently cloned and sequenced. bouin's solution fixed paraffin sections of placenta were subjected to crf immunohistochemistry with antibodies specific to rat species and intensity of immunostaining was analyzed using image pro-plus software and expressed in arbitrary units (au). results: one specific pcr band of bp size was consistently identifiable in placenta harvested at zt but not at zt . nucleotide sequence of the pcr band confirmed its identity as crf-mrna. intensity of crf-immunostaining was significantly greater at zt in giant trophoblast (gt) cells (gt-crf: zt = . ± . au, zt = . ± . au, p= . ) but not in spongiotrophoblast cells (stc) (stc-crf: zt = . ± . au, zt = . ± . au, p=ns) or labyrinth cells (lbc) (lbc-crf: zt = . ± . , zt = . ± . au, p=ns) . conclusion: similar to adult brain, rat placenta expresses crf mrna and protein. time-of-day variation of crf expression originally seen in central nervous system neurons is also identifiable in giant trophoblasts cells at zt . these findings suggest that stress-mediated placental crf release, and potentially fetal meconium passage, may be dependent upon time-of-day. objectve: transplacental water flow is essential for the provision and maintenance of fetal body water and amniotic fluid. water flow across membranes is regulated by aquaporin (aqp) water channels in many tissues. thus aqp , expressed in the placenta, is a candidate to regulate maternal to fetal fluid exchange. maternal beta-mimetics have been hypothesized to augment fetal growth by increasing the availability of nutrients and perhaps water. as camp acts as a second messenger to increase expression of selected aqps in other tissues, we sought to determine if betamimetics acting through camp could modulate placental aqp , and potentially influence placental water transfer. methods: trophoblastic cell cultures were established in first trimester-derived extravillous htr- /svneo cells and term placenta-like trophoblast carcinoma cell line jeg- . cultures were treated with sp-camp, a membrane-permeable and phosphodiesterase resistant camp, and forskolin, an adenylate cyclase stimulator, in doses of . , and m for hrs (aqp mrna expression) and hrs (aqp protein expression). for time course experiments, cells were incubated with μm of either sp-camp or forskolin for , and hrs at °c, % co . after cell harvest, mrna and protein expression were assayed using real time pcr and western blotting. results: sp-camp and forskolin increased aqp mrna expression in both cell lines after hrs (p< . ) in a dose-dependent manner. protein expression paralleled the increase seen in the mrna. m of sp-camp and forskolin stimulated aqp mrna expression after hrs in htr- /svneo cells and after hrs in jeg- cells (p< . ). m of either sp-camp or forskolin stimulated aqp protein expression in both cell lines after hrs (p< . ) and the expression remained high at hrs. conclusion: aqp gene expression in trophoblast cells is up-regulated by camp agonists. these results suggest that maternal beta-adrenergic agonists or antagonists may modulate maternal-fetal water flux via modulation of aqp water channels. rat placenta expresses corticotrophin releasing factor-binding protein and mrna. jayaraman lakshmanan, thomas r magee, bindu cherian, sharon k sugano, hanalise huff, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor-binding protein (crf-bp), a kda secreted glycoprotein, was originally isolated from human plasma. it binds crf and urocortin i with an equal or greater affinity than does the crf receptor. crf-bp expression has been reported in target tissues such as brain and placenta. based on its ability to inhibit crf actions in vitro, it is speculated to function as a "gate keeper" for crf-initiated stress responses. we recently documented expression of crf and urocortin- in rat placenta. in the present study we sought to establish the expression of crf-bp in rat placenta by immunohistochemical and pcr-analyses. methods: placental tissues (n= ) collected from sprague-dawley pregnant rats on day of gestation (term= ) were either fixed in % paraformaldehyde solution (ph . ) and paraffin embedded or processed for total rna extraction. paraffin sections of five micron thickness were subjected to immunohistochemical analysis with goat polyclonal antibodies to human crf-bp precursor (sc- santa cruz biotechnology, ca) by avidinbiotin-peroxidase complex technique. the structure of cloned human crf-bp precursor exhibit significant amino acid sequence homology among all species studied. immunoreactivities on the sections were quantified using image pro . software and staining intensity (od/area) expressed as arbitrary units (au). all values are expressed as mean±sem. for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, clone into plasmid vector and dna sequenced. results: the crf-bp antibody elicited strong positive staining in decidua, giant trophoblasts, spongiotrophoblasts and labyrinth cells. the results of image analyses revealed (au): decidua: . ± . , giant trophoblast cells: . ± . , spongiotrophoblasts: . ± . , fetal membranes: . ± . . pcr analyses identified a single bp band, consistent with crf-bp. conclusion: our study establishes for the first time that rat placenta is analogous to humans in that both express crf-bp mrna and protein. immunohistochemical findings reveal that crf-bp protein expression occurs at multiple sites within the placenta. expression of per clock protein in rat placenta: an internal timer for placental functions. jayaraman lakshmanan, reuben lakshmanan, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor (crf) expression time-of-day variation occurs in giant trophoblast cells on the maternal side of the rat placenta. this temporal change in crf expression pattern is very similar to that of central nervous system neurons, suggesting that placental cells may use a similar mechanism to read time of the day. the self-sustaining rhythm generating capacity of the suprachiasmatic nuclei is believed to be derived from cell-autonomous, transcriptional feed-back loops dependent on a number of canonical clock genes. in the present study we sought to determine whether rat placenta expresses period gene (per ), one of the clock/cycle related genes. methods: time-dated pregnant sprague-dawley rats were received on day of gestation and housed in a controlled environment ( - h lights on) with free assess to food and water. placentas collected on days and of gestation (term= days) between and . am were fixed in % paraformaldehyde and paraffin embedded. for each gestational ages a total of placentas from different pregnant rats were used. paraffin sections ( sections per placenta) were subjected to immunohistochemical analysis with antibody to per ( : , santa cruz biotechnology, ca) by abc technique and , 'diaminobenzidine as a chromagen. sections were examined under microscope, imunostaining quantified by image pro . software and expressed in arbitrary units (au). data are presented as mean ± sem. statistical significance was analyzed by anova with a p value < . as significant. conclusion: the present results indicate that rat placental cells, devoid of any neural innervations, express per clock protein, similar to central nervous system neurons. based on the differences in the relative intensities between trophoblasts and labyrinth cells on day of gestation, we conclude that fetalmaternal interactions in per regulation disappear at term (or at the time of birth.). our findings imply that per may function as internal physiological modulator in placenta. to determine the mechanism(s) underlying placental time-of-day crf variation, we examined the effect of maternal administration of betamethasone (a synthetic glucocorticoid) on nuclear gc receptor expression in placental cells. method: time-dated pregnant rats (n= ) on day of gestation were given a single subcutaneous injection (at : am) of betamethasone ( μg/kg body weight) while control pregnant rats (n= ) received saline. dams were exposed to isoflurane anesthesia at : am, and placentas harvested, fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analysis with gc-receptor antibody (sc- , santa cruz biotechnology), with immunoreactive material identified by abc technique using , ' diaminobenzidine as a chromagen. percentages of gc-nuclear receptor (gc-nr) positive cells and their intensities (od/area) were quantified using image pro . plus software. all values are expressed as mean ± sem. statistical analysis was by anova with p< . considered significant. results: in placentas of saline exposed pregnant rats, isolated labyrinth (lb) cells (< %) were positive to gc-nr. no positive staining for gc-nrs was seen either in giant trophoblasts (gt) or in spongiotrophoblasts (st). betamethasone administration was associated with a significant and marked increase in gc-nr staining in all three placental cell types (p< . ). among the cells, gt ( ± %) demonstrated a greater percentage of cells expressing gc-nr than did st ( ± %) or (lb= ± %) (p< . ), though there was similar immunoreactive intensities (gt: . ± . , st: . ± . , lb: ± . au; p=ns) conclusion: our findings indicate that all placental cells respond to gc with upregulation of gc-nr with an enhanced response among gt cells. these results suggest that endogenous or exogenous maternal stress-induced gc exposure may influence signaling responses within both maternal and fetal placental compartments. ovine placenta at very-preterm gestation expresses corticotrophin releasing factor (crf), crf-binding protein (crf-bp) and glucocorticoid receptors (gr). jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: in humans, the placenta is the major source of maternal and fetal plasma crf. based on its critical functions, crf is considered as a "placental clock" of parturition. we recently reported that ovine fetal as well as maternal plasma contain measurable amounts of crf at near-term but not at very preterm gestation. we interpret the absence of crf in plasma at very preterm gestation is either due to lack of placental crf expression and/or placental crf release. here, we examined the expression status of crf, crf-bp (a known specific binding protein for crf and a known regulator of crf functions in human placenta) and gr (a known positive regulator of crf expression in human placenta) in ovine placenta collected at very-preterm gestation. method: placenta harvested from time-dated pregnant ewes on ± days gestation were fixed in bouins's solution and processed for paraffin embedding. paraffin sections were subjected to immunohistochemical analysis with polyclonal antibodies specific to ovine crf( : - : , phoenix pharmaceuticals, ca,) crf binding protein ( : - : , sc ) and gr ( : - : , sc: , santacruz biotechnology, ca). immunoreactive material on the sections were identified as brown staining by abc technique using diaminobenzidine as chormagen. immunoreactive material was quantified by image analysis using image pro . plus software and the immuoreactive intensity (od/area) expressed as arbitrary units (au). results: strong positive staining for crf ( . ± . au), crf-bp ( . ± . au) and nuclear-gr ( . ± . au) were noticed in syncytiotrophoblast cells in all placental sections obtained from pregnant ewes at days gestation. control sections exhibited no positive staining. conclusion: our findings indicated that ovine placenta at very-preterm gestation expresses crf, crf-bp and gr. these results suggest that absence of measurable crf in maternal and fetal plasma at very-preterm gestation is not due to lack of placental crf expression but rather due to the absence of regulated crf secretory mechanisms. rat placenta expresses urocortin i protein and mrna. thomas r magee, michael g ross, john d richard, sharon k sugano, jayaraman lakshmanan. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: urocortin i (ucn- ), a amino acid neuropeptide belongs to corticotrophin releasing factor (crf) stress hormone family. in humans, the placenta and several gestational tissues have been reported to express ucn- protein and ucn- mrna. a number of published studies indicate that ucn- is similar to crf in expression pattern and biological functions. we recently reported that rat placenta is a site of crf protein and crf mrna expression. in the present study we sought to determine whether rat placenta expresses ucn- protein and ucn- mrna. methods: placenta (n= ) collected from pregnant rats at day gestation were either fixed in bouin's solution and processed for paraffin embedding or placed in rna later preservative and frozen for rna extraction. for immunohistochemical localization, five micron thickness paraffin sections were cut and immunostained with rabbit polyclonal antibodies to ucn- ( : to : , sigma) by standard abc technique. control sections were incubated with omission of ucn- antibody. immunoreactive materials on the sections were identified using , ' daminobenzidine as chromagen. immunostaining intensity (od/area) was quantified by image-pro plus software and expressed in arbitrary units (au). for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, cloned into a plasmid vector and dna sequenced. all values are given as mean ± sem. results: ucn- polyclonal antibody elicited strong immunostaining in placental trophoblast cells with variable intensity. the pattern of immunostaining is as follows: giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au. the relative intensity in labyrinth cells was significantly lower than the other two cell types (p< . ). pcr analyses revealed the presence of a single band of bp, consistent with ucn- mrna. conclusion: similar to human placenta, rat placenta expresses ucn- protein and ucn- mrna, with most expression localized to the maternal side trophoblast cells. these results support our hypothesis that rat placenta can be used as a model to understand the role of this peptide in feto-maternal stress. the role of the nuclear hormone receptors fxr, pxr and car in placental bile acid homeostasis in normal and pathological pregnancy. victoria geenes, peter dixon, selina raguz, jenny chambers, kishore bhakoo, catherine williamson. imperial college, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder characterised by raised maternal serum bile acid levels and associated with adverse fetal outcome. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from an accumulation of bile acids in the fetal circulation. bile acids are the toxic end products of hepatic cholesterol metabolism and are synthesised from weeks gestation. in common with other waste products, accumulation in the fetal compartment is prevented by excretion across the placenta into the maternal compartment. however, studies of maternal and cord serum from normal and oc pregnancies have suggested that bidirectional transfer of bile acids is possible. hepatic bile acid transport and metabolism is regulated by members of the nuclear hormone receptor family, namely fxr, pxr and car, but the mechanisms for regulating placental transfer are unknown. objectives: this study used placenta from normal and cholestatic pregnancies to investigate the expression of genes involved in hepatic bile acid homeostasis. methods: villous trophoblast samples from oc and normal pregnancies (np), and human livers were collected and preserved in rnalater. explant cultures were prepared from a further np placentas and cultured for days at % oxygen. on day they were treated with chenodeoxycholic acid, lithocholic acid or vehicle for hours prior to fixing in rnalater. total rna was extracted using trizol, and reverse transcribed to cdna. quantitative real-time pcr was performed using sybr green. target gene mrna abundance was calculated from a standard curve and normalised to l . results: the expression of fxr, pxr and car, the nuclear hormone receptors responsible for hepatic bile acid homeostasis and several fxr target genes (shp, mdr and bsep) was found to be very low in np, and unaffected by the presence of maternal cholestasis. furthermore, the expression of these genes could not be induced by bile acid treatment in an in vitro model. here we have shown that the nuclear hormone receptors (fxr, pxr and car) involved in hepatic bile acid homeostasis are expressed at very low levels in normal and pathological pregnancy and are thus likely to be of limited importance in placental bile acid transfer. a placental phenotype for obstetric cholestasis. victoria geenes, jenny chambers, jo wyatt-ashmead, kishore bhakoo, catherine williamson. imperial college, london, united kingdom; hammersmith hospital, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder that affects . % of pregnant women in the uk. biochemically, oc is characterised by liver dysfunction with elevated maternal serum bile acids (sba), and clinically by an increased risk of fetal complications including fetal distress, meconium staining of the amniotic fluid, preterm labour and sudden intrauterine death. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from the toxic effects of bile acids, which can cause vasoconstriction in the placenta and fetal cardiac dysrrhythmias. furthermore, in a rodent model of oc, bile acids cause oxidative stress in the placenta and a reduction in litter size. these changes are absent in animals treated with ursodeoxycholic acid (udca), a drug used to treat oc. however human data are lacking. objectives: this study used whole placenta and explant cultures to investigate the effects of bile acids on human placental architecture and to establish whether udca protects the placenta against bile acid induced damage. methods: villous trophoblast samples were collected from oc and normal pregnancies (np) and fixed in formalin. explant cultures were prepared from a further np placentas, and cultured for days at % oxygen. on day a subset were treated with udca overnight, and on day the explants were treated with taurocholic acid, taurochenodeoxycholic acid or vehicle for hours prior to fixing. m sections were stained with haematoxyllin and eosin. slides were reviewed by a perinatal pathologist and syncytial knots (sk) counted. results: oc tissues showed marked alterations in morphology, including congestion of the terminal villi, and loosening of the stroma and fibrotic change of the membranes of the stem villi. there were significantly more sk the oc samples (mann-whitney u test p= . ). furthermore, sk were increased in explants treated with bile acids, but not those treated with udca prior to the addition of bile acids. conclusions: here we describe several morphological abnormalities of the placenta associated with maternal cholestasis. these changes were confirmed using a placental explant model, which also showed that udca protects the placenta against bile acid induced damage. in summary, this study indicates that udca is likely to protect the fetus in oc. the placenta has complex metabolic and endocrine activities and is essential for the growth and survival of the fetus in utero. ultrasound is the most sensitive and less invasive method to evaluate placental size, morphology and function. the three-dimensional approach allows to calculate placental volume in the i and ii trimester of pregnancy. pregnancies from assisted reproduction technologies (art) show an increased rate of pathologies potentially related to placental insufficiency such as intrauterine growth restriction and preterm delivery. aim. to determine placental volume in art pregnancies in a cross sectional study in the first trimester of gestation. design. using three-dimensional ultrasound machine (ge ) placental volume measurement from art pregnancies (n= ; - wks) were compared with data from normal controls (n= ) matched for gestational age. data were analysed with software stata . results. mean placental volume was . ml (sd± . ) in art pregnancies and . ml (sd± . ) in normal controls. mean difference resulted in . ml (- - . ) and was statistically different (p= . ). multiple linear regression analysis showed a statistically significant interaction (p= . ) between gestational age and case status, i.e. differences in placental volume increase significantly with advancing gestational age between cases and normal controls. mean gestational age at birth was not essentially different between the two subsets ( . weeks ± . ) however a statistically significantly lower mean fetal birthweight was found in art pregnancies: g (sd± ) vs .(sd± . ) (p= . ). conclusions. placental volume is slightly decreased in art pregnancies. measurements of placenta volume by d ultrasound may play a role in identifying the degree of placental growth early in gestation in a risk population. in vitro effects of adenovirus-mediated gene transfer of insulin like growth factor- (ad-igf- ) in trophoblast cells. mounira habli, datis alae, foong yen lim, jignesh parvadia, timothy crombleholme. obstetrics and gynecology; pediatric surgery, university of cincinnati/children's hospital, usa. objective:recently, ad-igf- corrected both fetal and placental weights in rat model of fetal growth restriction (fgr) . to elucidate the mechanism of action of ad-igf- ;we examined the effect of ad-igf- on trophoblast proliferation, differentiation and invasion. methods: rcho- trophoblast cell line derived from rat choricarcinoma was used. ad-igf- and galactosidase transgene (ad-lacz) were given at moi of : and : in all the experiments.transduction efficiency was assessed hr after infection with ad-lacz by galactosidase enzyme activity. the invasiveness of rcho- was measured by using bdmatrigel invasion chamber kit. rcho- proliferation cell density was assessed by crystal violet assay. morphologic analysis of rcho- differentiation in response to treatment (differentiated cells are multinucleated giant cells) was assessed byimunocytochemistry staining using placental lactogen- antibodies(specific hormone produced by giant cells). results: % efficiency of gene transfer of ad-lacz to rcho- cells was observed at both moi's.there was no significant difference in proliferation between treated control group regardless of the dose at and hrs. ad-igf- induced morphologic differentiation of trophoblast cells compared to control (fig ) at hrs.ad-igf- induced higher rate invasion in a dose dependent fashion as compared to control(ad-lacz) group( % at moi vs . % in control p< . )(fig ). conclusion: ad-igf- gene transfer induces differentiation and invasiveness of trophoblast cells. these may be the mechansim of correction of fgr in rat model of placental insufficiency. placental gene transfer may be an effective treatment strategy for placental insufficiency. types of human placenta. martina dieber-rotheneder, ursula hiden, gernot desoye, mila cervar-zivkovic. department of obstetrics and gynaecology, medical university graz, graz, austria. background and hypothesis: endothelin- is a polypeptide with a wide range of functions. in the placenta it acts as a potent regulator of vasotonus on endothelial and smooth muscle cells, whereas on the trophoblast it regulates cell proliferation, invasion and apoptosis. endothelin- action is differently signaled through two endothelin receptor (etr) subtypes, etr-a and etr-b. several alternative splice variants of etr were identified. here we hypothesize that the etr-a splicing varies with gestational age and is different in trophoblast vs endothelial cells of the human term placenta. methods: mrna of placental tissue from first trimester (pregnancy terminations, missed abortions) and term of gestation, first trimester and term trophoblasts, arterial and venous placental endothelial cells as well as cell lines representing first trimester trophoblast (ach p) and term placental endothelial cells (hpec) were analyzed for full length etr-a and known as well as unknown splice variants by sqrt-pcr using primers spanning the exons - . the predominant dna bands in agarose gel were excised and sequenced. results: all tissues and cells expressed full length etr-a. in all tissues an additional -deletion variant was identified which was not found in the isolated cells. trophoblasts expressed another yet unidentified splice variant. the endothelial cells expressed a -deletion variant and a novel splice variant, which was identified as partial -partial deletion. this novel splice variant was found regardless of the arterial or venous origin of the cells as well as in the cell line (sv -transformed). in tissues und trophoblast no difference in splicing was found between first trimester and term. conclusion: gestational age does not alter splicing of endothelin receptor-a. splicing is different between trophoblasts and endothelial cells. the endothelial cells contain a novel splice variant. the differential splicing may allow maternal and fetal endothelin- to induce different effects on the two major cell types of the placenta. (supported by grant , jubilee fund, austrian national bank, vienna). adrenomedullin production and secretion by human trophoblast cells are regulated by glucocorticoids and hypoxia. francesca ciardo, katia pacioni, emanuela marinoni, giovanna corona, massimo moscarini, alfredo patella, romolo di iorio. department of gynecology, perinatology and child health, university "la sapienza", rome, italy; department of obstetetrics and gynecology, university of ferrara, ferrara, italy. objectives: adrenomedullin (am) is produced by intrauterine tissues and is involved in the regulation of implantation, placental hemodynamics and endocrine function. circulating am is increased in pregnancy complications such as preeclampsia and intrauterine growth retardation. we investigated whether am output by human trophoblast cells is regulated by hypoxia and/or glucocorticoids. study design: trophoblast cells obtained by human placentas at term (n= ) were cultured in presence or absence of hypoxia ( % o ) and treated with or without betamethasone at the dose of - , - , - m. media and cells were collected at h and at , and h from syncytiotrophoblast cultures. am was measured in cultured media by specific ria kit. protein expression in trophoblast cells was evaluated with immunohistochemistry and western blot. results: hypoxia stimulated am output and protein expression by cytotrophoblast and syncytiotrophoblast cells. betamethasone induced an increase in am production and secretion in a time-and dose-dependent manner (figure). effects of hypoxia were partially reversed by betamethasone in a dose and time-dependent fashion. conclusions: am production in trophoblast cells is up-regulated by hypoxia and glucocorticoids, independently. increased am levels in pregnancy complications characterized by placental insufficiency might derived by an increase in am placental secretion stimulated directly by hypoxia or indirectly by an increase in fetal cortisol levels. preeclampsia complicates - % of pregnancies, and while associated with significant maternal and fetal morbidity and mortality, the mechanisms responsible for preeclampsia are not completely understood. recent advances suggest there are imbalances of pro-and antiangiogenic factors, present from the time of implantation affecting vascular responsiveness of the fetal placental unit and maternal vasculature. in this study, we investigate vasomotor responsiveness of placental arteries from normal and preeclamptic (pet) pregnancies using an in vitro muscle bath contractility assay. transverse rings of placental arteries were cut and equilibrated in the muscle bath containing a bicarbonate buffer aerated with % o / % co at °c. viability was demonstrated by contraction to mm kcl prior to all experiments. cumulative logarithmic dose dependent responses to four different contractile agents: pgf , serotonin, carbochol, and norepinephrine were compared. placental arteries precontracted with pgf at half maximal concentration were relaxed with three vasorelaxants: sodium nitroprusside (snp), forskolin (fsk) and lactate (lct). agonist studies revealed contraction to both pgf and serotonin but not to carbochol or norepinephrine. there were no statistically significant differences between the responses of normal and pet arteries. both normal and pet arteries demonstrated heightened responsiveness to sodium nitroprusside compared with forskolin and lactate. this study reveals that the placental vessels are more sensitive to sodium nitroprusside, that mediates relaxation through nitric oxide -cyclic gmp signal transduction pathway, than forskolin that induces relaxation through the cyclic amp pathway. cancer complicates approximately one in , pregnancies. depending on the type of cancer and trimester, patients can terminate the pregnancy or choose treatment such as chemotherapy. since there is limited knowledge on the safety of chemotherapy on fetal tissues in utero, clinicians cannot efficiently analyze the risks of particular anticancer agents when deciding on a course of therapy. since carboplatin is among the most common anticancer agents used for treatment of cancer during pregnancy the primary objective of this study was to evaluate if carboplatin will readily cross to placental barrier and determine total potential platinum fetal exposure. this project utilizes an ex vivo placenta perfusion model to determine the concentration of carboplatin that crosses the human placental barrier. placentas are obtained within minutes of spontaneous delivery and re-perfused. two carboplatin concentrations were selected of ng/ml and ng/ml to represent clinically relevant maternal plasma concentrations. antipyrine was used as the internal control. serial samples were collected every minutes for total minutes in open-open model then experiment repeated with closed circuit model. antipyrine concentrations were determined by hplc methods. platinum concentrations in media samples were determined with a validated atomic absorption assay. a cell culture-based approach will be used to determine whether cell cycle or apoptotic proteins are altered (p , bax, bcl- , aif, and pro-caspase- ) using western blotting as a detection method. a total of two placentas have been completed at the low carboplatin concentration and one at the high concentration. the mean carboplatin clearance was . +/- . ml/min and . +/- . ml/min at the low and high carboplatin concentrations, respectively. an estimated % increase in the transport fraction was observed in the high concentration experiments compared to the low concentrations model. fetal carboplatin exposure ranged from to ng/ml. the placental perfusion experiments conducted at carboplatin and ng/ml indicate that carboplatin crosses the placenta through simple diffusion. the toxicology assays are ongoing to determine potential effect on fetal tissues. preterm delivery represents one of the predominant causes of perinatal mortality and morbidity, and is one of the most unpredictable gestational disturbances. urocortin is a peptide expressed by trophoblast and gestational tissues (amnion and chorion), whose maternal levels correlate with gestational length, since they are increased at preterm delivery and decreased in post-term pregnancy, when compared to term pregnancy. the addiction of urocortin enhances contractility of human myometrial strips, suggesting a possible role on uterine contractility. high maternal urocortin levels in threatened preterm delivery correlates with the timing of delivery, suggesting a possible role in the predictivity pf preterm delivery. in the present study urocortin concentrations in amniotic fluid collected at mid gestation for amniocentesis were correlated with gestational age at delivery. a case-control study with amniotic fluid obtained from healthy women undergoing amniocentesis for genetic indications was performed; urocortin concentrations were measured by a specific elisa. amniotic fluid urocortin concentrations resulted significantly lower (p= . ) in women delivering preterm (n= ; ucn= . ± . pg/ml) (m ± se) than in those delivering at term (n= ; ucn= . ± . pg/ml). in conclusion, the present preliminary data showed that amniotic fluid urocortin concentrations at mid gestation may represent predictive marker of preterm delivery. human placentas throughout pregnancy. annunziata mastrogiacomo, elisabetta federico, francesca caprio, maria teresa schettino, gabriele coppola, antonio de luca, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; department of medicine and public health, section of anatomy, second university of naples, nales, italy. hypothesis apoptosis is intimately involved in placental homeostasis, growth and remodeling and the apoptotic rates increase progressively during normal pregnancy as part of normal placental development. moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction, diabetes. in the present study, we describe differences in the expression of pro-apoptotic protein bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. material and methods human placental samples were obtained with informed consent from patients undergoing surgery such as first trimester voluntary termination of pregnancy (n= ), first trimester abortion (miscarriage) (n= ), delivery section (n= ), spontaneous birth (n= ), preeclampsia (n= ) and diabetes (n= ). the gestation period ranged from to weeks. the specimens were immediately fixed in formalin for immunohistochemistry. we first observed a strong increase of bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. secondly, we showed a more intense immunopositivity for bax in the third trimester spontaneous birth respect to the third trimester caesarean birth. thirdly, we observed an increase of bax expression in preeclamptic placentas compared to the normal full-term placentas. on the contrary, we observed a moderate bax expression in diabetic placentas only slightly decreased compared to the normal full-term placentas. our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies. objectives: maternal endothelial activation in preeclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. the application of proteomic technologies such as mass spectrometry promises the reward of identifying and characterising these factors. using a placental explantconditioned culture media model system we have developed a proteomics workflow to obtain relative quantification of proteins released into placental explant culture media. methods: term villous explants were cultured in serum-free conditions and exposed to differing oxygen concentrations ( % %) to mimic physiological and non-physiological intervillous o tensions. the d media was concentrated, immunodepleted to remove fibrinogen (using beckman igy- columns) and proteins labelled using an itraq (applied biosystems) kit following the manufacturer's protocol. labelled peptides were fractionated by strong cation exchange and then analysed using lc-maldi on a maldi-tof/tof (applied biosystems) mass spectrometer. data was analysed using protein pilot . (applied biosystems). results: when matched pooled (n= ) media samples were subjected to our proteomics workflow over proteins were identified with a calculated false positive identification rate of < %. of these proteins a total of display a statistically significant difference in protein levels between the % % o samples (p=< . ). from a list of proteins of interest we selected proteins for validation using elisa/western blotting to measure their relative abundance in both pooled and individual explant media samples. interleukin is an example of a low-abundance differentially expressed protein identified in our proteomic workflow and subsequently validated by elisa; il- (pg/ml) in % o : . , range . to . ; % o : . , range . to . ; values are median with interquartile range. *** p< . , mann-whitney test (n= ) conclusions: we demonstrate a successful reduction to practice of a relative quantitative proteomics strategy applied to placental explant-conditioned culture media. having optimised and validated this proteomic workflow we will apply the same methods to compare proteins released by normal and preeclamptic placentas. a proteomics screen of human placental microvillous syncytiotrophoblast (stb) revealed the expression of dysferlin (dysf), a membrane repair protein associated with certain muscular dystrophies (vandré et al., ) . a second ferlin protein, myoferlin (myof), was also discovered which has not yet been characterized in the placenta. in human c c myoblasts, dysf and myof are reciprocally expressed as a function of differentiation into multinucleate syncytial myotubes, with dysf predominating in differentiated cells. we hypothesized that dysf and myof would show a similar reciprocal expression pattern in human trophoblasts as a function of syncytialization. methods term placentas from uncomplicated pregnancies were obtained with informed consent (n= ) and either fixed for immunofluorescence (if) labeling or flash frozen for subsequent immunoblotting (ib). human trophoblastic (bewo, jar, and jeg- ) and other cell lines (mrc- , hl- , huvec) were cultured in the absence or presence of μm forskolin or solvent control for - days. dysf and myof expression was examined by rt-pcr, ib, and if. results ib validated proteomics data showing myof expression in term placenta. by if, myof labeling was predominant in apical and basal stb plasma membranes. myof was expressed in trophoblastic cell lines (bewo, jar, and jeg- ), cultured endothelium (huvec), and a fibroblast cell line (mrc- ), but was not detected in a leukemic cell line (hl- ). dysf was constitutively expressed in jar, but minimally expressed in unstimulated bewo. following forskolin-induced syncytialization, we observed a time-dependent increase in dysf expression in bewo concomitant with increasing syncytin- levels. by if, dysf expression was restricted to bewo cells in syncytial structures. in contrast, myof expression was robust in mononuclear bewo cells and not down-regulated over the course of days of differentiation. conclusions two ferlin-family genes are expressed in trophoblasts. fusion-competent bewo cells behave similarly to cultured myoblasts and ctbs with regard to dysf expression, which is restricted to syncytializing cells. myof, in contrast, is expressed constitutively in bewo and other models. while both proteins are likely to function in stb plasma membrane repair (e.g., following syncytial sprouting), the relative contributions of each to this process awaits clarification. features of chronic placental insufficiency are significantly more common in small for gestational age placentas from infants with intrauterine growth retardation. emily king, violetta kolesnikova, carri tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: there is a strong association between intrauterine growth restriction (iugr) and small for gestational age (sga) placentas (heinonen et al. placenta ( ), : - ) . the cause is likely multifactorial, but we hypothesize that chronic uteroplacental insufficiency may play a role. our objective was to test whether sga placentas from human neonates with iugr show significantly more pathologic features of placental insufficiency compared to controls. design: we performed a retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ), excluding elective terminations and spontaneous abortions before weeks gestation. clinical records were reviewed and pregnancy outcomes recorded, including: ) neonatal sex; ) weight (iugr calculated by routine methods), ) trimmed weight of the placenta (sga calculated by routine methods), ) gross placental infarctions, ) gestational age at delivery, and ) maternal features (e.g. race, gravida). controls were defined as cases within the series without sga or iugr (e.g. submitted for meconium, infection, etc). histologic sections were scored by two pathologists while blinded to clinical diagnoses as positive or negative for features of placental insufficiency, including accelerated villous maturation (avm), chorangiosis, and microscopic infarctions. significant associations were tested by analysis and logistic regression for multiple variables. results: similar to prior reports, we observed a strong association between iugr and sga placentas ( . ; p < . ). this relationship was independent of maternal race, fetal sex, and parity, although it was more common in primigravidas. avm and placental infarctions were significantly more frequent in sga placentas with superimposed iugr. controls ( introduction: messenger rna expression peripheral blood cells (pbc) has been recently used as biomarkers of environmental exposures (ionizing radiation or tobacco), physiological conditions (stress) and diseases (hypertension, neurological disorders). pbc evaluation is a useful diagnostic tool in an era of individualized medicine. since there is an urgent need for non-invasive methods for determination of fetal (f) and placental (p) function, this study was designed to evaluate the genes differently and commonly expressed in p tissue and leukocytes in maternal (m) and f circulation.material and methods. p (n= ), f (n= ) and m blood (n= ) were obtained during cesarean section in pregnant baboons at term. total rna from a buffy coat pellet was isolated using a modified procedure of the qiagen rneasy mini kit (qiagen). anti-sense rna (arna) was synthesized and purified using the illumina rna amplification kit (ambion, usa). hybridization of arna to illumina sentrix human whole genome (wg- )beadchips and subsequent washing, blocking and detection was performed using illumina's beadchip ´ protocol. samples were scanned on the illumina beadarrayer gx reader using illumina beadscan image data acquisition software (ver. . . . ). differential gene expression data analysis was performed using illumina beadstudio software (ver. . . . ). results. the detection level of gene transcripts using illumina methodology with control human rna was - at p< . . gene transcripts were detected in f blood and in maternal blood. transcripts were uniquely expressed in fetal blood. transcripts were found in m, but not in f leukocytes. the number of gene transcripts expressed in p tissue was and of these genes were not expressed in m leukocytes. conclusion. despite white blood cells trafficking through the p barrier there is a set of unique genes expressed only in p or in the m or f circulation. the application of these genes as the biomarkers of p barrier function still need to be evaluated. objective: to evaluate if a relationship exists between duration of placental exposure to meconium in vivo and histologic evidence of severity and extent of meconium uptake by macrophages. study design: from a cohort of / ( %) consecutive singleton liveborn infants delivered at term with thick meconium-stained fluid, ( %) had placental histologic examination performed, and in / the timing of meconium appearance after membrane rupture was documented. placental histologic examination quantitatively evaluated the intensity of meconium uptake by resident macrophages based on the number of macrophages/field, and the extent of uptake based on histologic location, graded in a score to . results: mean interval between meconium appearance and delivery was . ± . min (range - ). after exclusion of cases in which severe placental inflammation interfered with analysis, meconium uptake by macrophages was documented in / cases at the level of amniochorionic membranes, in / cases at the placenta, and in / cases at the umbilical cord. there was no correlation between the interval meconium appearance-to-delivery in relation to presence of meconium in the membranes (p= . ), in the placenta (p= . ), in the cord (p= . ), or score of severity of meconium uptake (p= . ). the results did not change after correcting for gestational age, oligohydramnios, presence of placental acute inflammatory or vascular lesions. conclusion: there is no relationship between duration of placental exposure to meconium and the extent and intensity of its uptake by macrophages in cases with exposure up to . hours. transfer of bisphenol a across the human placenta. biju balakrishnan, , kimiora henare, , eric thorstensen, , murray d mitchell. , the liggins institute, university of auckland; national research centre for growth and development, auckland, new zealand. introduction: there are growing concerns over the effects of developmental exposure to the xenoestrogen bisphenol a (bpa). animal studies have shown that bpa is transferred through the placenta and can cause deleterious effects to the fetus. the presence of bpa in feto-placental tissues in humans has also been reported. however, a detailed study of the time-course of bpa transfer across the human placenta has not been performed. the aim of this research was to study the transfer of bpa in ex-vivo perfused human placental tissues. methods: a dual recirculating single cotyledon perfusion was used to monitor the placental transfer of bpa. bpa ( ng/ml), antipyrine (ap) ( μg/ml), and fitc dextran (fitc-dx) ( . μg/ml) were added to the maternal perfusate, and perfusion was continued for hours. perfusate samples were collected from both reservoirs at timed intervals and analysed. bpa, ap, and fitc-dx were determined by hplc with fluorescent detection, hplc with uv detection, and spectrofluorometry respectively. the viability and metabolic activity of the placentae were assessed by measuring -hcg (elisa), glucose utilization, and lactate production (autoanalyzer). fetal pressure, ph, flow rates and fluid shifts were monitored continuously. results: the biochemical validation parameters (glucose consumption, lactate production and -hcg secretion) indicated that the placental tissue was metabolically active and viable throughout the -hour perfusion period. the physical parameters observed (fetal pressure < mmhg, ph range . - . ) were in concordance with other published works for placental perfusion. membrane integrity was confirmed by fluid shifts from either circuit of < ml/hr, and by < % materno-fetal transfer of fitc-dx. an observed ap transfer of - % further validated our model. bpa first appeared in the fetal compartment within minutes of perfusion and reached a peak of about - % of maternal concentration within hours of perfusion. this figure is likely to be an underestimate since it does not include conjugated bpas. conclusion: this first study of bpa transfer in ex-vivo perfused human placental tissue shows that our model can serve as a useful tool to study the transfer kinetics and metabolism of bpa in human term placentae. we found that bpa rapidly crosses the human placenta at environmentally-relevant doses, with potentially harmful effects on the human fetus. angiogenic growth factor secretion by uterine natural killer cells in co-culture with extravillous trophoblast. gendie e lash, katsu naruse, , barbara a innes, stephen c robson, judith n bulmer. institue of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom; obstetrics and gynaecology, nara medical university, nara, japan. objectives. uterine natural killer (unk) and extravillous trophoblast (evt) cells have been proposed to play roles in remodeling of uterine spiral arteries through secretion of various angiogenic growth factors. we have previously demonstrated that unk cells are a significant source of angiogenic growth factors within the decidua, many of which alter with gestational age. however whether the secretion of these proteins is altered by interactions of unk cells with evt is unclear. hypothesis. unk cell angiogenic growth factor secretion is regulated by evt in early pregnant decidua. methods. placental and decidual samples were collected from women undergoing termination of pregnancy with written informed consent ( - and - weeks gestation, n= each group). . × cd + unk cells were positively selected from decidua and co-cultured with evt ( . × ) or cytotrophoblast (ctb; . × ) purified from the same placenta for h in direct or indirect contact (n= each group). angiogenin, ang , pdgf-bb, fgf-basic, timp , icam and vegf-a were measured by fast quant ® angiogenesis multiplex assay system, and vegf-c, ang and plgf by elisa. results of unk co-culture with evt or ctb (negative control) at each gestational age were analyzed with wilcoxon rank test. in addition, the effect of direct and indirect co-culture of unk cells with evt at each gestational age was compared with mann whitney u test. results. at - weeks gestation unk secretion of ang (p= . ), icam (p= . ) and plgf (p= . ) was increased in the presence of evt compared with ctb. no differences were observed at - weeks gestation. in addition, at - weeks gestation unk secretion of ang (p= . ), vegf-c (p= . ) and ang (p= . ) was increased in direct co-culture with evt compared with indirect co-culture. at - weeks gestation unk secretion of timp- (p= . ) was reduced in direct co-culture with evt compared with indirect co-culture. conclusions. unk cell secretion of several key angiogenic growth factors was altered by direct culture with evt. these data suggest that a membrane bound molecule (such as hla-g) mediates this modulation of unk cell activity. abnormalities in placentation and impaired placental circulation can lead to fetal growth restriction. -d ultrasound can be used to evaluate this through the quantification of the power doppler signal that may be expressed as a percentage of colour voxels within a user-defined volume (vi: vascularisation index). we aimed to test the hypothesis that increased fetoplacental blood flow correlates with an increased vi, using the in vitro dual perfusion model of the human placental lobule. three term lobules were dually perfused through both circulations with earles bicarbonate buffer (ebb), and supplemented on the fetal-side only with adult erythrocytes, prepared to a % haematocrit. following initial equilibration perfusion at normal flow values, fetal-side flow was varied between and ml/min, whilst maternal-side flow was held at ml/min. images were obtained with a 'voluson i' ultrasound machine and a neonatal transducer (pulse repetition frequency = . hz, wall motion filter = low , and gain = . ). three -d datasets were acquired at each flow rate from each placental lobule and these were measured in triplicate using vocal. a sphere was centred on a visibly perfused cotyledon along the chorionic-decidual axis, with a diameter corresponding to placental thickness. linear regression analysis was used to assess the relationship between the total fetal-side flow and mean vi. the mean vi showed a high degree of correlation with total fetal-side flow for each lobule ( figure ) suggesting increased vascular perfusion and the inclusion of perfused vessels that cross the detection threshold with increased flow. this data provides qualifying information for translation to a clinical application, where early gestational fetoplacental blood flow will be assessed to predict the onset of fetal growth restriction. anthony n imudia, brian a kilburn, anelia petkova, samuel s edwin, roberto romero, d randall armant. objective survival of first trimester cytotrophoblast cells depends on their upregulation of heparin-binding egf-like growth factor (hbegf), which is downregulated in placental tissues diagnosed with preeclampsia. we have examined the expression and cytoprotective activity of hbegf in term villous explants subjected to hypoxic stress in vitro. non-pathological placentas were collected by cesarean section at term (n= ). chorionic villous explants were prepared and cultured at either % or % o and treated with the hbegf antagonist crm or recombinant hbegf. paraffin sections were assayed for trophoblast cell death by the tunel assay, proliferation by immunohistochemical labeling of nuclear ki and hbegf expression by semi-quantitative immunohistochemistry. data were compared using anova and the student-newman-keuls posthoc test. results crm ( mg/ml) increased trophoblast cell death after culturing villous explants h at % o (p< . ), but only slightly affected proliferative capacity. culture at % o increased trophoblast cell death % above explants incubated at % o (p< . ). trophoblast cell proliferation decreased after h in explants cultured at either % or % o (p< . ). exogenous hbegf ( nm) prevented the elevation of cell death during hypoxia (p< . ) and maintained nuclear ki expression at %, but not %, o . contrary to first trimester trophoblast, hbegf was not upregulated by hypoxia in term trophoblast. the failure of term trophoblast to elevate hbegf expression in response to hypoxia could contribute to their decreased survival at low o compared to early gestation. endogenous hbegf signaling appears to facilitate survival of term trophoblast during villous explants culture. exogenous hbegf supplementation prevented cell death due to hypoxia and maintained trophoblast proliferation rates under in vitro conditions. therefore, hbegf, which is downregulated in preeclampsia, could have significant impact on trophoblast survival during late gestation. supported by the intramural research program of nichd. conspicuous meconium-laden macrophages in the chorionic plate are an independent predictor of clinically significant fetal distress. violetta kolesnikova, emily king, carrie tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: meconium is a common indication for placental examination, present in approximately % of placentas routinely submitted to pathologists (beebe et al. obstet gynecol ; : - ) . prolonged meconium exposure leads to accumulation of meconium-laden macrophages in the chorionic plate (estimated to require at least hours). our objective was to test whether prolonged meconium exposure is associated with clinically significant fetal distress. design: retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ) was performed, excluding abortions before weeks gestation, and placentas without representative sections of the chorionic plate. clinical records were reviewed and pregnancy outcomes recorded, including: ) evidence of fetal distress prompting c-section (non-reassuring fetal heart rate), ) gestational age, ) maternal diagnosis, ) apgar scores, and ) neonatal length of hospital stay. routine histologic sections were independently scored by two pathologists while blinded to clinical diagnoses and outcomes. cases were scored as positive or negative for: ) diffusely conspicuous meconium-laden macrophages in the chorionic plate (at least / hpf), ) chorioamnionitis, and ) features of placental insufficiency. significant associations were tested by the mann-whitney u test for paired comparisons, x analysis, and logistic regression for multiple variables. results: conspicuous meconium staining of the chorionic plate was common ( / , %) and was significantly more frequent in c-sections performed for fetal distress ( / cases, %) (x . ; p-value < . ). logistic regression modeling showed that this association was independent of chorioamnionitis and features of placental insufficiency. there was no association between meconium and neonatal outcome, including no difference in apgar scores or length of hospital stay. conclusions: our data support the hypothesis that meconium-laden macrophages in the chorionic plate are associated with fetal distress prompting c-section. whether meconium is the cause or consequence of this distress is uncertain. however, given the acute time frame between clinical diagnosis and c-section, we suspect prolonged meconium exposure may be a significant cause of non-reassuring fetal heart rate changes. apoptotic index in normal and intrauterine growth-restricted rat placentas. elissa scotland, tri nguyen, s chiang, radmila runic. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: intra-uterine stress caused by maternal food-restriction may have adverse fetal and placental effects. we sought to assess the effect of maternal food restriction during pregnancy on placental apoptosis. methods: rat placentas were analyzed from control dams fed ad libitum and dams % food-restricted from day of gestation. placentas were harvested at embryonic days and (n= ). placentas were fixed in % pfa and two methods of analysis were utilized to determine the proportion of apoptotic to non-apoptotic cells within maternal food restricted and control rat placentas: immunohistochemistry (ih) using fas-ligand and in situ tunel (terminal deoxynucleotidyl transferase biotin-dutp nick end labeling). tunel: after rehydration, slides were subjected to tdt enzyme. dab was used to visualize brown apoptotic nuclei. dna-se treated slide was used as a positive control. ih: primary rabbit polyclonal fas-ligand antibody was used at : dilution, after which secondary antibody linked to peroxidase and dab staining was performed. results: food-restricted placentas demonstrated . % relative apoptotic index when compared to . % in the control group. the iugr iod (integrated optical density) per unit area in the placental membrane was higher than in the control group. fasl demonstrates an iod of . + . in food restricted placentas as compared to . + . per μm surface area in controls. apoptosis was seen in the amnion as well as trophoblast (syncytialtrophoblasts and some cytotrophoblasts). conclusion: the increased apoptotic index in maternal food restricted placentas suggests that the accompanying iugr may be a result of both maternal/fetal nutrient restriction and increased fetal stress. this study found that maternal food restriction during pregnancy affects placental apoptosis. there is more apoptosis in the iugr placental bed at e than at e , with the reverse being true for the placental membrane. more research is required to statistically validate the data. the suggested next step is to evaluate fas and fas-l and to address possible mechanisms of placental apoptosis. cord. jayaraman lakshmanan, avish arora, lilit baldjyan, sharon k sugano, olga miadel, adegoke adeniji, michael g ross, calvin j hobel. ob-gyn, harbor-ucla medical center, torrance, ca, usa; ob-gyn, cedars-sinai medical center, los angeles, ca, usa. background: crf-bp is a kda protein, that specifically binds corticotrophin releasing factor (crf). the structure of cloned crf cdnas in all species examined predicts that the precursor is larger than the kda in size and contains one n-glycosylation site. marked reductions in plasma crf-bp levels seen in pregnant women prior to both preterm and term delivery led to the notion that crf-bp is a "gatekeeper" of crf responses. recently we identified crf-bp expression in term human umbilical cord (uc) by immunohistochemical analyses. objective: to characterized the expression of crf-bp by immunohistochemical and biochemical analyses in human uc. methods: freshly obtained human preterm ( to < weeks, n= ) umbilical cord ( pieces of mm thickness taken at - cm intervals close to placenta) were fixed in bouin's solution and paraffin embedded. also, pieces of ucs were dissected, arteries and vein separated, weighed and frozen at - c. for immunohistochemical localization, uc sections were subjected to immunostaining with polyclonal antibodies to human crf-bp precursors. for western blot analyses, whole uc as well as isolated arteries and vein were homogenized in a buffer containing detergents and protease inhibitors. the homogenate supernatant proteins were subjected to western analyses by standard protocol. immunoreactive protein bands were identified by chemiluminescent reagent. results: both goat and rabbit crf-bp polyclonal antibodies elicited weak to moderate positive staining in uc epithelial layers, vascular musculature and barely endothelial cells. they identified a strong kda band in whole uc as well as in isolated artery and venous preparations. in addition minor immunoreactive protein bands of , , kda in size were noticed in all three preparations. conclusion: we conclude that preterm uc expresses crf-bp. we postulate that the kda major band is either glycosylated crf-bp precursor or glycosylated kda mature protein. the low molecular weight immunoreactive proteins likely represent proteolytically processed, glycosylated crf-bp precursor or a proetolytically processed mature da crf-bp. uc obtained at delivery could be useful as a tool to understand the critical functions of crf-bp in feto-placental unit. objective: adenosine, known to be released from inflammatory sites and tisse ischemia, has many important biologic roles. four specific adenosine receptors have been cloned to date, termed a , a a, a b, and a . recently our study has shown that increased a receptor in the trophoblast of preeclamptic pregnancy was noted and non-vascular and trophoblast-mediated a receptor may play an important role in the pathogenesis of preeclampsia. there are evidences of impaired trophoblast invasion related to matrix metalloproteinase (mmp) in preeclampsia and the relationship between adenosine receptor and mmp in other fields. the objective of this study is to evaluate the effect of mmp expression by adenosine a receptor in preeclamptic villous explants at different oxygen conditions. methods: placental villous explants from normal (n= ) and preeclamptic (n= ) pregnancies were cultured at high ( %) and low ( %) oxygen levels for days. explants were analyzed for mmp- /- and timp- /- by rt-pcr and western blot. preeclamptic villous explants in hypoxic culture condition were treated with a receptor agonist, cl-ib-meca and a receptor antagonist, mre. mmp- / - expression was determined in a time-and dose-dependent manner by rt-pcr, western blot. also mmp- /- activity was evaluated by zymogram assay. results: there were significantly increased a receptor intensity and reduced mmp- /- and timp- /- expression at low oxygen level in normal and preeclamptic villous explants. interestingly, in preeclamptic villous explants, after high oxygen culture mmp- /- and timp- /- expression were recovered to almost same level compared to those in normal villous explants. treatment of preeclamptic villous explants with cl-ib-meca in low oxygen level resulted in a time-and dose-dependent enhanced expression of mmp- /- . this cl-ib-meca-induced expression of mmp- /- was inhibited by pretreatment with mre. conclusion: to our knowledge, this study is the first to evaluate modulation of mmp secretion by adenosine a receptor in preeclamptic villous explant. our results provide evidence for the existence of functional adenosine a receptors in the trophoblast and suggest that adenosine a receptor will be investigated as a therapeutic target in preeclampsia. murray d mitchell, timothy a sato, anderson wang, jeffrey a keelan, anna p ponnampalam, michelle glass. liggins institute; department of pharmacology and clinical pharmacology, university of auckland, auckland, new zealand. introduction: it is well established that prostaglandins play critical roles in multiple aspects of pregnancy and that the fetal membranes are an important site of intrauterine prostaglandin production. endocannabinoids have been implicated in the maintenance of pregnancy and parturition in women and are a source of arachidonic acid which is a substrate for the production of prostaglandins. the aim of the present study was to determine the effects of endocannabinoids on the production of prostaglandins in extraplacental membranes -amnion, chorion and decidua. methods: explants of term amnion and choriodecidua were established and treated with endogenous endocannabinoids -arachidonoyl glycerol ( ag) and anandamide (aea) and with the synthetic cannabinoid cp , , to determine the ability of these substances to modulate prostaglandin e (pge ) production. pge was measured by radioimmunoassay. the explants were also treated with cp , in the presence of either sr a (a selective antagonist of the cannabinoid receptor cb ) or ns (a cox- inhibitor), to determine whether any observed stimulation of pge production was mediated through cox- activity and/or the cb receptor. cox- , cox- , cpla and pgdh protein levels were measured by western blotting. results: all three cannabinoids caused a significant increase in pge production in amnion but not in choriodecidua. however, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. the enhanced pge production caused by cp , was abrogated by co-treatment with either sr a or ns , illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by cb agonism and cox- . preliminary data from western blotting show that cannabinoid treatment results in the up-regulation of cox- expression. however, there was no change in cox- expression and no evidence either for up-regulation of cpla or for down-regulation of pgdh expression. conclusion: this study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labour and membrane rupture. inactivation of vegf receptor- , but not vegfr- or vegfr- , during the peri-implantation period prevents normal pregnancy development in the rodent through disruption of uterine angiogenesis. nataki c douglas, hongyan tang, raul gomez, bronislaw pytowski, daniel j hicklin, jan kitajewski, mark v sauer, ralf c zimmermann. division of reproductive endocrinology and infertility, columbia university, new york, ny, usa; imclone systems, inc., new york. objective: vegf is involved in the regulation of uterine angiogenesis and implantation in both rodents and non-human primates. vegfr- , r- , and r- are expressed in the uterine decidua and are involved in the regulation of vessel formation in many systems. to determine if these receptors have a functional role in the regulation of post-implantation angiogenesis and pregnancy development, we examined the effects of blocking vegfr- , r- , and r- function. design: prospective animal laboratory material and methods: to avoid effects of vegf receptor neutralization on ovarian function, we utilized a progesterone replaced, ovariectomized mouse model. vegf receptor blocking antibodies were administered on ed . , prior to embryonic expression of these receptors. embryonic development was evaluated on ed . , blood vessel density and apoptosis on ed . , and cellular proliferation on ed . (n= per time point in each group). anova with bonferroni correction was used to compare sample means. results: see tables. conclusions: neutralization of vegfr- and vegfr- , but not vegfr- resulted in a significant reduction in cellular proliferation and decidual angiogenesis. vegfr- mediates decidual angiogenesis, but is not required for normal pregnancy development. in contrast, an intact vegf/vegfr- pathway is required for the decidual angiogenesis that mediates early pregnancy development. effect of vegfr neutralization on ed . control anti r- anti r- anti r- no. of implantation sites . +/- . +/- . . +/- . . +/- . hoxa encodes a transcription factor required for endometrial receptivity and embryo implantation. our objective was to identify and to characterize those molecular markers regulated by hoxa in multiple cellular model-systems. using microarray technologies, we identified putative hoxa target genes involved in early implantation. liposome-mediated transfection delivering either empty vector or the same plasmid constitutively expressing hoxa was introduced into newly impregnated mice during laparotomy or layered onto cultured human endometrial stromal-cells (hescs). rna products from these in vivo and in vitro transfections were used to identify targets and to validate the microarray screen employing semi-quantitative real-time pcr (qrt-pcr). we identified statistically-significant genes regulated by hoxa overexpression of which genes were down-regulated greater than -fold when compared to controls. cellular ontogenies of differentially-expressed genes include: cell adhesion molecules, signal transduction factors as well as metabolic regulators. furthermore, we identified the -phosphoglycerate dehydrogenase gene, (pgdh) whose products are regulated by hoxa during implantation in both murine model systems in and cell culture. this genes codes for an enzyme critical to de novo l-serine biosynthesis via a phosphorylation-dependent pathway. microarray analysis demonstrated a fold expression decrease when hoxa is overexpressed. this diminution in pgdh expression was noted in the validation experiments using qrt-pcr and corroborated in hesc cells where the mrna levels decreased to % when compared to controls. the repression of pgdh during the implantation window may represent a conservation of activity as secretory-phase protein synthesis may be suppressed in order to promote cellular differentiation and resultant implantation. these regulatory relationships identified in mouse implantation likely function to enhance uterine receptivity and may have a role in human implantation. objective: hoxa is expressed in endometrium, where it is regulated by sex steroids and is necessary for endometrial receptivity and implantation. hoxa is also expressed in leukocytes. here we hypothesized that hoxa would be regulated by sex steroids in both cell types. we further hypothesized a correlation between expression of hoxa in peripheral blood cell (pbcs) and endometrium in both mice and in humans. methods: real-time pcr was used to determine differential expression of hoxa mrna in u cells, a monomyelocytic cell line, in response to increasing concentrations of estradiol. to determine if hoxa is expressed in leukocytes in vivo, peripheral leukocyte hoxa mrna expression was measured over sequential estrus cycles in mature cd nulliparous mice and correlated to vaginal smear-cytology. additionally, peripheral leukocytes were isolated either from pregnant or normally-cycling women to assess hoxa mrna expression. results: there was a direct, dose-responsive correlation between exposure to increasing estradiol and hoxa mrna expression levels in u cells. in a murine model, we demonstrated that hoxa mrna expression-levels varies throughout the estrus cycle with a marked increase in expression following vaginal plug detection. the nadir of hoxa expression is prior to proestrus and increased up to -fold during the receptive phase. this increased expression continues throughout gestation. the heightened expression in murine leukocytederived hoxa mrna also is demonstrated across species. our preliminary data suggests that the greatest fold-increase of expression occurs during the window of implantation of the secretory phase in normal, cycling women. this level was sustained in pregnancy. there appears to be a trend with the highest levels of expression associated with viable gestations. women with attenuated expression profiles had non-viable gestations. conclusions: hoxa expression is regulated by sex steroids in both leukocytes and endometrium. the temporal pattern of peripheral hoxa transcript expression demonstrated in mice and humans mimics the differential rna expression documented within the uterus. leukocyte hoxa expression during the reproductive cycle in mice and humans is a marker of endometrial receptivity. peripheral leukocyte expression of hoxa mrna may correlate with implantation success. carolien m boomsma, annemieke kavelaars, marinus jc eijkemans, gijs teklenburg, bart cjm fauser, cobi heijnen, nick s macklon. reproductive medicine and gynaecology and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands. the purpose of this study is to assess the association between the intra-uterine cytokine expression profile at the time of embryo transfer and successful embryo implantation following ivf/ icsi treatment. materials and methods women undergoing ivf/ icsi underwent endometrial secretion aspiration prior to embryo transfer. known soluble mediators of implantation were measured using a multiplex immunoassay, namely il ß, il , il , il , il , il , il , il , tnf , vegf, ifn , eotaxin, mcp- , ip- , dkk- and hbegf. mif was determined using an elisa. the total protein concentration was measured for normalization purposes. data were log transformed to obtain normal distribution. multivariable logistic regression analysis with a backward elimination procedure (p< . ) was used, potential confounders (age, blood contamination, embryo quality) were included in a forward stepwise model. ten mediators of the analysed were detectable in - % of the samples. il was detectable in % of samples, dkk- %, il- %, il- %, il- % and hbegf was detectable in % of samples. ifn-was not detectable in any of the samples. multivariable logistic regression showed only logmif concentrations to have a significant correlation with achieving clinical pregnancy (p = . ). higher mif concentrations were correlated with a higher chance of conceiving. endometrial secretion analysis represents a novel means of assessing the intra-uterine milieu encountered by the embryo and offers new perspectives in the study of endometrial receptivity. in this large prospective study assessing an array of cytokines, mif was found to be significantly correlated with pregnancy. mif, macrophage migration inhibitory factor, is a cytokine with numerous proinflammatory, immunomodulatory, angiogenic and tissue remodelling properties. mif induces the synthesis and secretion of matrix metalloproteinases by endometrial cells, which may contribute to embryo invasion. its expression is particularly increased during the secretory phase, suggesting a role in reproductive processes. analysis of aspirated endometrial secretions offers a direct clinical test of endometrial receptivity which can be applied during treatment cycles without disrupting implantation. endometrial receptivity and secretory differentiation require progesterone (p). it has been hypothesized that low p levels result in delayed endometrial differentiation and infertility due to reduced receptivity. objective: test the effects of low luteal p on histologic and molecular markers of differentiation and function. methods: normal cycling women (n= ) were treated with daily leuprolide ( . mg/d) beginning in the midluteal phase and continuing through the protocol. after menses, subjects received transdermal estradiol (e, . mg/d) for days. after day of e, subjects also received daily i.m. injections of p, randomized to mg/d (sub-physiological) or mg/d (physiological). endometrial biopsy was performed after days of combined e and p treatment. additional untreated women had biopsies performed days after spontaneous lh surge. endometrial histologic dating was performed by two individuals according to the criteria of noyes et al. mrna levels were assessed using real-time rt-pcr. results: mean(s.d.) of peak and trough p serum concentrations in the mg/d p group were . ( . ) and . ( . ) ng/dl, respectively, while those in the mg/d group were . ( . ) and . ( . ) ng/dl. there were no differences between treatment groups for histologic dating; the mean(s.d.) histologic date was . ( . ), . ( . ), and . ( . ) for the mg, mg, and spontaneous cycle groups, respectively. there also were no differences among the three groups in mrna levels of ten functional markers (er , pr, integrin subunit, osteopontin, cyr , egr- , fkb , c-fos, and cd ), although variability of gene expression was greater in those who received mg/d p than in those who received mg/d p. there was also no correlation between serum progesterone level and gene expression or histologic date. conclusions: sub-physiological levels of progesterone, in the range seen in ovulatory women, do not induce detectable changes in expression of marker genes or histological dating, although low p levels were associated with greater variability of gene expression. these data suggest that abnormalities in endometrial histologic development and function likely result from intrinsic abnormalities rather than from low levels of p secretion. (supported by unc nova carta fund and nih u hd- ). endometrial ip attracts trophectoderm through cxcr interaction. simcha yagel, caryn greenfield, hen sela, jacob hanna, irit manaster, orna singer, ronit haimov-kochman, shira natanson-yaron, diana prus, benjamin reubinoff, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical centers, jerusalem, israel; lautenberg center for immunology, jerusalem, israel; human embryonic stem cell research center, jerusalem, israel. introduction: implantation is initiated in part by attraction of the blastocyst to the endometrium lining the uterus. we hypothesized that this process is partly accomplished by chemokines expressed by the endometrium interacting with chemokine receptors on the blastocyst, suggesting that they play an important role in implantation. materials and methods: chemokine receptors were characterized in jeg cells, placental villi, primary trophoblast cell culture, trophectoderm cells derived from human es cells, blastocyst trophectoderm, and st trimester placental tissue sections. expression of chemokines was tested in decidua, endometrium, and ishikawa and hec- cell lines. immunohistochemistry, intracellular staining, elisa, facs analysis, and rt-pcr were employed to characterize chemokine receptor and ligand expression. functional testing was performed using transwell migration assays and in a nude mouse model using a matrigel gel plug cell attraction assay followed by facs analysis. results: trophoblasts demonstrated expression of various chemokine receptors, most prominently cxcr and cxcr . immunohistochemistry of trophoblast from placental villi plated on matrigel expressed cxcr and cxcr as well as hla-g. noteworthy is that trophectoderm cells derived from hes cells treated with bmp- and jeg cells, and blastocyst trophectoderm expressed principally cxcr . elisa and immunohistochemistry showed that decidua and endometrium expressed chemokines ip and il . migration assays demonstrated that ip significantly attracted various trophoblast and trophectoderm cells in vitro, and in the mouse model in vivo. taken together these results demonstrate the interaction between trophoblasts and endometrial cells is mediated by cxcr and cxcr , and il and ip . these interactions are important in the attraction of trophoblasts at the feto-maternal interface. however, ip -cxcr is the most relevant to early implantation as only cxcr is expressed consistently by trophectoderm. the endometrial proteome: changes from proliferative to secretory phase. lois a salamonsen, jenny i-c chen, xian mak, peter j stanton, david m robertson, andrew n stephens. prince henry's institute of medical research, melbourne, vic, australia. global gene analyses have demonstrated major changes across the menstrual cycle, but which of these are reflected in the proteome is not known. this study aimed to globally assess proteins differentially expressed in the endometrium between the proliferative and. secretory phases. d page analysis with dige minimal dye labelling was conducted across the pi range - on endometrial tissue from either the mid-proliferative or midsecretory phases (n= /group). profiles were assessed using samespots software. differentially expressed proteins were identified using maldi-tof ms and the interrelationship of proteins examined using ingenuity software. a total of spots were detected: were differentially expressed (p < . ) with spots having an overall false discovery rate < % (q < . ). hierarchical clustering analysis revealed that these proteins lay within three main branches in the protein dendrogram. one cluster had proteins upregulated in the proliferative phase and two contained proteins up-regulated in the secretory phase. the unique protein profiles were also revealed using principle component analysis (pca): proteins clustered into two main groups, according to cycle phase. pca thus indicated similar unique protein signatures as suggested by hierarchical clustering. thirty one of the differentially expressed proteins were identified using maldi-tof ms. these proteins could be grouped into seven categories, which included structural ( ), transport ( ), regulatory ( ), membrane ( ), enzyme ( ), motor ( ) and others ( ). proteins involved in matrix assembly and those needed for subsequent establishment of secretory endometrium were up-regulated in proliferative endometrium. proteins important for cellular organisation and communication as well as products responding to environmental stress and the immune system were highly up-regulated during the secretory phase. biological pathways were constructed based on the proteins identified. the top network for secretory endometrium clustered around tgf-b. others related to inhibition of cell death / cell viability and leukocyte extravasation. these studies provide a global approach to the cyclic changes of the endometrium and highlight the complex dynamics of protein expression in human endometrium. hormonal regulation of prokineticins in the human fallopian tube: potential regulators of embryo transport. aw horne, hn jabbour, p lourenco, s wright, s battersby, arw williams, hod critchley. reproductive and developmental sciences, university of edinburgh, edinburgh, united kingdom; mrc human reproductive sciences unit, queen's medical research institute, edinburgh, united kingdom. background: understanding the factors regulating embryo transport in the fallopian tube (ft) has important clinical implications. embryo retention in the tube due to ft dysfunction is thought to lead to tubal pregnancy, a considerable cause of morbidity and occasional mortality. transport of the embryo through the ft is, for the greater part, accomplished by smooth muscle contraction. a group of multi-functional proteins and their receptors, called prokineticins, have been shown to affect smooth muscle function in other tissues, such as the intestine. the expression pattern of prokineticins, and their receptors, was examined in normal human ft obtained throughout the menstrual cycle, and the effect of the sex steroids on prokineticin expression was examined in an in-vitro model of the ft. methods: ft biopsies (n= ) and sera (for measurement of oestradiol and progesterone for endocrine staging) were collected from women undergoing gynaecological procedures for benign conditions. using a combination of quantitative taqman rt-pcr and immunohistochemistry, the mrna and protein expression pattern of prokineticins, and their receptors, were examined in the ft throughout the menstrual cycle. tubal explant culture was established using surgical tissue from the biopsies and exposed to varying concentrations, and time courses, of oestrogen and progesterone. results: prokineticin (pk ) and prokineticin receptor (pkr ) mrna are up-regulated in the progesterone-dominant mid-secretory phase. pk and pkr protein are expressed in the epithelium, smooth muscle and around the blood vessels of the ft. stimulation of tubal explant cultures with a physiological concentration of progesterone showed an up-regulation of pk and pkr . conclusions: prokineticins show temporal variation in expression in human ft and appear to be regulated by progesterone. their role in embryo transport needs to be investigated to further understanding of pregnancy complications, such as tubal pregnancy. translating mouse to human: a dynamic model of xenografted human endometrium. alex j polotsky, liyin zhu, nanette santoro, jeffrey w pollard. albert einstein college of medicine, bronx, ny, usa. in the mouse endometrium, the hormonal environment controls cellular proliferation and cell cycle activity. estradiol (e ) inhibits glycogen synthase kinase beta (gsk- ), resulting in nuclear accumulation of cyclin d and progression of the cell cycle, as well as dna replication licensing. in utero administration of the gsk- inhibitor, licl, results in epithelial cell proliferation in the absence of e (zhu, pollard. pnas. ; : ) . in this study, we derived a functional model of xenografted human endometrium to perform mechanistic studies of human endometrial proliferation. methods: human endometrial samples were obtained from volunteers aged - . immuno-compromised mice were transplanted with disaggregated/ recombined human epithelial glands and stroma under the kidney capsule. after weeks of out-growth, mice were ovariectomized, and replaced with e or licl. xenografts were harvested and processed for immunohistochemistry (ihc) and glandular labeling index (li). t test was used to compare group means. results: - % of engraftments were successful, resulting in a vascularized endometrium with characteristic architecture (a, b). hoechst staining confirmed that xenografts were made up of human cells ©. e (e) induced significantly greater proliferation compared to control (d) as assessed by ihc for ki , with li of . ± . and . ± . , respectively, p = . ). minichromosome maintenance- , a protein involved in dna replication licensing, was more sensitive for e -treated cells synthesizing dna than was ki staining (i). estrogen (g) and progesterone receptors (h) were expressed in xenotransplant tissue, the latter being up-regulated by e . licl (f) induced proliferation similar to e and greater than control (li = . ± . , p= . for e vs. licl). conclusion: xenografted human endometrium provides a dynamic model of endometrial proliferation that is well suited for translational studies. administration of licl in the absence of e induced glandular proliferation, supporting the notion that similar mechanisms are operative in human proliferation as in the mouse. gnrh analogs have been extensively used in assisted reproduction. although the main effects of gnrh analogs are via gnrh receptors on the pituitary gonadotrope, gnrh and gnrh receptors have been identified in many reproductive tissues including human endometrium, suggesting their potential action at endometrial level. in the present study, we examined the potential regulatory action of gnrha, la on sex steroid mediated gene expression in human endometrium. human endometrial surface epithelial (hes) cells and isolated endometrial stromal (esc) cells were used as in vitro models. all experiments lasted for h. the cells were treated with estradiol (e , nm, h), progesterone (p , nm, h), gnrha (la μm, h), e ( h) followed by p (last h) and gnrha plus e plus p where, gnrha added either first, second or last in order at h intervals. total rna was extracted, reversed-transcribed and subjected to real-time pcr simultaneously, measuring the expressions of il- , il- , il- , il- , il- , il- , il- , il- p , il- p , il- , ifn-and tnf . both hes and esc expressed majority of these cytokines with the exception of low to undetectable levels of il- , il- , il- and ifn-. treatment with e and p , either alone, or in combination significantly upregulated the expression of many of these cytokines at varying extend as compared to controls. however, gnrha either alone or in combination with e and p significantly diminished e , p or e plus p induced mrna expression of cytokines. the suppressive effects of gnrha on some of the cytokines varied significantly by the order which gnrha was introduced into the culture medium. we conclude that gnrha by acting directly on the endometrial cells effectively suppresses the mrna expression of several key proinflammatory cytokines upregulated by ovarian steroids. our results imply that gnrha therapy during assisted reproduction may modify endometrial receptivity via downregulation of proinflammatory cytokines induced by estradiol and progesterone. supported by nih grant hd . introduction: endometrial angiogenesis is characterized by a rapid increase during the early proliferative phase that peaks midcycle, followed by a gradual decrease in the secretory phase, while menses involves generalized endometrial inflammation, necrosis, and vascular thrombosis. vascular endothelial growth factor (vegf), whose expression is regulated by sex steroids, is a mediator of endometrial angiogenesis. recently, myoferlin, a kd transmembrane protein, was identified in endothelial cells where it mediates vegf-dependent endothelial cell proliferation, migration, and nitric oxide synthesis by affecting vegf receptor- function and stability. myoferlin also appears to play a role in vesicle trafficking and membrane repair. objective: to characterize myoferlin protein expression in endometrium. methods: western analysis of cultured human endometrial stromal cells (hesc) and human endometrial endothelial cells (heec) treated with physiologic concentrations of estradiol and progesterone was performed using a polyclonal rabbit antibody against myoferlin. subsequently, immunohistochemistry (ihc) was performed on human endometrium samples obtained from various stages of the menstrual cycle. results: myoferlin protein was expressed in hescs and heecs, but expression was not affected by sex steroids. ihc staining for myoferlin was specific, intense, and localized to the apical membrane of glandular epithelial cells and endothelial cells, with less intense staining in the stroma. h-score quantification showed that in endometrial endothelial cells, myoferlin protein expression was highest during the early proliferative and early secretory phases, while glandular and stromal myoferlin expression peaked during the late proliferative/early secretory phase. conclusion: myoferlin expression in human endometrium correlates with periods of greatest endometrial angiogenesis, and expression is not limited to endothelial cells, but also includes glandular and stromal cells. given the involvement of myoferlin in vegf signaling as well as membrane repair, understanding its role in human endometrium may further elucidate an understanding of endometrial development both under physiologic and pathologic conditions. the human embryo is the primary regulator of embryo-endometrial molecular cross talk during early implantation. gijs teklenburg, , cobi heijnen, esther baart, karima amarouchi, carolien boomsma, janet carver, helen mardon, annemieke kavelaars, nick macklon. reproductive medicine and gynaecology, and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands; nuffield department of obstetrics and gynaecology, university of oxford, women's centre, john radcliffe hospital, oxford, united kingdom. introduction: uterine receptivity and implantation are controlled by locally acting trophic factors and cytokines. in humans, the regulation of the embryo-endometrial dialogue beyond the early blastocyst stage is poorly understood. we hypothesized that the interaction between a healthy conceptus and the receptive endometrium is associated with a distinct local regulation of cytokine production favouring implantation. methods: human embryos, cryopreserved at day after fertilization and donated for research, were thawed and cultured under standard conditions until day five. forty-two embryos from donors developed to the blastocyst stage. following removal of the zona pellucida, they were placed in individual coculture on a confluent monolayer of endometrial stromal cells. on day , the developmental potential of each embryo was assessed as early arrested, late arrested or developing. culture supernatants were analysed for concentrations of il- , il- , il- , il- , il- , il- , il- , il- , tnf-, mcp- , ip- , eotaxin and hb-egf using a multiplex immunoassay. day supernatants from culture systems in which no embryo had been placed were also analysed as controls. results: out of ( %) embryos continued to develop and were able to attach and invade into the stromal cell compartment of the co-culture environment. twelve late arrested embryos showed signs of degradation on day and the totally disintegrated embryos were assigned to the early arrested group. supernatants from both early and late arrested embryo cultures contained significantly lower levels of a number of cytokines and growth factors in comparison to developing embryo cultures. moreover, the levels of these mediators in co-cultures were significantly lower than those in non-embryo control stromal cell culture supernatants. conclusion: these data suggest a pivotal role of the embryo in embryoendometrial cross talk. whether reduced mediator expression in co-cultures reflects a selective down regulation of stromal cell cytokine and growth factor production is now being investigated. epithelial cell dynamics during human implantation. hiroshi uchida, tetsuo maruyama, toru arase, masanori ono, takashi kajitani, maki kagami, hideyuku oda, sayaka nishikawa, yasunori yoshimura. department of obstetrics and gynecology, keio university school of medicine, shinjuku, tokyo, japan. epithelio-mesenchymal transition (emt) is thought to play a role in functional differentiation of endometrial epithelial cells during human implantation. molecular mechanism of epithelial sheet remodeling caused by embryo invasion remains elusive. to address this, we investigated cellular dynamics of n-cadherin and vimentin, the two representative major markers of emt, during implantation. in in vitro implantation assay using a human endometrial epithelial cell line, ishikawa, and a human choriocarcinoma cell line, jar (uchida et al., hum reprod ), we pre-treated human ishikawa cells with or without ovarian steroid hormones ( -estradiol + progesterone; ep), fa- (n-cadherin blocking ab), or suberoylanilide hydroxamic acid (saha), one of histone deacetylase inhibitors, which has a potential to improve in vitro implantation (ibid). implantation or treatment with or without ep or saha enhanced the expression of n-cadherin and vimentin but down-regulated ecadherin. furthermore, treatment with ep or saha accelerated ishikawa cell motility and increased the number and spreading area of jar spheroids. in vitro implantation assay, the most prominent staining intensity of n-cadherin was observed just around the adhered spheroid from which its intensity decreased away. functional blockade of n-cadherin by fa- resulted in the complete suppression of ishikawa cell motility, the unique distribution of n-cadherin around jar spheroids, and the spreading area of jar spheroids, while it did not affect the number of the adhered spheroids. human implantation consists of the multiple steps, including apposition, adhesion and penetration. thus, these results collectively indicate that emt may take place after the apposition and that n-cadherin may be required for the remodeling and emt of the epithelial sheet during embryo invasion. n-cadherin may enhance the recruitment of spheroid-neighboring cells, suggesting its role in the covering-up of the invading embryo through acceleration of epithelial cell motility. endocannabinoid regulation in human endometrium. jessica g scotchie, marc a fritz, steven l young. obstetrics and gynecology, university of north carolina, chapel hill, nc, usa. background: research in mouse models demonstrates two cyclically regulated endocannabinoids produced in murine endometrium, anandamide and -arachidonoyl glycerol ( -ag); both play critical roles in murine embryonic implantation. no studies of endocannabinoids in human endometrium have been performed. objectives: determine menstrual cycle expression and localization of synthetic and degradative enzymes for anandamide and ag in human endometrium. methods: human endometrium was collected from volunteers across the menstrual cycle (n= ). quantitative rt-pcr was performed analyzing the expression of: n-acylphosphatidylethanolamine (nape) and fatty acid amide hydrolase (faah), the synthetic and degradative enzymes for anandamide, respectively; sn- -diacylglycerol lipase-a (dagla), and b (daglb), the synthesis enzymes for ag; and monoacylglycerol lipase (magl) and cyclooxygenase- (cox ), the degradative enzymes for ag. the constitutive gene ppia was used for comparison. immunohistochemical localization of nape protein was performed using nape-pld polyclonal antibody (cayman chemical, # ). anova and student's t-test analysis performed on samples grouped by proliferative (pro), early, mid-, and late secretory (es, ms, ls) phases. results: mrna expression of all enzymes responsible for synthesis and degradation of anandamide and ag was detected throughout the cycle. no significant cyclic change in nape, faah, or daglb gene expression was seen. a decrease in dagla gene expression in the ms and ls phases compared to the pro and es phases (p= . ) was seen. magl gene expression was higher in the secretory phase than the pro phase (p= . ). cox gene expression was detected at low levels in the pro, es and ms phases, with marked increase in the ls phase (p< . for all comparisons). protein localization of nape showed a cytoplasmic epithelial location, with increased staining on pro and es samples compared to ms and ls samples. immunolocalization of remaining proteins is ongoing. conclusion: this is the first report documenting the presence of endocannabinoid synthetic and degradative enzymes in human endometrium. genes controlling anandamide expression do not fluctuate significantly across the cycle. however, ag's degradative enzymes increase in the secretory phase, suggesting that lower ag levels may be advantageous for embryo implantation. our findings suggest that human endometrial endocannabinoid regulation differs from murine regulation. interleukin- beta (il- ) regulates il- signaling in decidua-implication in the pathophysiology of preeclampsia (pe). sj huang, cf yen, cp chen, f schatz, cj lockwood. obstetrics, gynecology and reproductive sciences, yale university, new haven, ct, usa; ob/gyn, chang gung memorial hospital, tao-yuan, taiwan; ob/gyn, mackay memorial hospital, taipei, taiwan. objective: previously, we found much higher cytoplasmic immunoreactive il- levels in the preeclamptic decidual cells than in adjacent interstitial trophoblasts. such decidual cell-derived il- contributes to the systemic endothelial cell dysfunction that elicits the proteinuria and hypertension of the maternal syndrome. il- promotes the transition from innate to adaptive immunity. moreover, by skewing monocyte differentiation from a dendritic to a macrophage phenotype, decidual il- may promote the macrophage excess observed in the preeclamptic decidua. macrophages impair trophoblast decidual invasion to foster incomplete spiral artery remodeling that elicits placental ischemia and hypoxia. the current study: ) localized il- mrna levels in preeclamptic versus normal decidual sections; ) evaluated mechanisms regulating il- synthesis by targeting intracellular signaling pathways with specific inhibitors; ) identified potential il- targets by immunolocalizing the il- receptor (il- r) to specific cell types in placental bed biopsies. methods: in situ hybridization localized il- mrna in normal versus preeclamptic decidua. il- r was immunolocalized in placental bed biopsies. leukocyte-free first trimester decidual cells were incubated with e and mpa ± il- ( ng/ml) ± an inhibitor of p mapk (sb ) or protein kinase c (calphostin c) or nf b (activation inhibitor iii) for hrs. an elisa measured secreted il- levels. results: il- mrna was present primarily in decidual cells with increased il- mrna levels observed in pe. preferential expression of the il- r was observed on decidual cells in placental bed biopsies. compared with basal il- levels ( . ± . pg/ml/ g cell protein) by decidual cells, il- enhanced il- output by decidual cells ( . ± . pg/ml/ g cell protein). only the p mapk inhibitor significantly reduced this output to . ± . pg/ml/ g cell protein (n= , p< . ). our results indicate that inflammatory cytokine enhances il- synthesis in decidual cells of the preeclamptic decidua by a mechanism involving p mapk. such il- is likely to act as an autocrine/paracrine effector via decidual cell-expressed il- r to contribute to the macrophage excess observed in the preeclamptic decidua. heparanase is up-regulated by estrogen and during the secretory phase of the human endometrium. ronit haimov-kochman, shira natanson-yaron, caryn greenfield, achinoam lev-sagie, lichtenstein michal, haya lorberboum-galsky, israel vlodavsky, simcha yagel, arye hurwitz. ob/ gyn, jerusalem, israel; cellular biochemistry human genetics, hadassah hebrew university medical centers, jerusalem, israel; cancer and vascular biology research center, technion school of medicine, haifa, israel. introduction: heparanase is an endoglycosidase that cleaves heparan sulfate (hs) proteoglycan of the extracellular matrix. the full-length proheparanase is activated by cleavage into an active isoenzyme, resulting in the release of hsbound cell-differentiation factors, such as hb-egf. the cycling endometrium involves remarkable steroid hormone-induced tissue remodeling. in vivo, increasing exposure to unopposed estrogen may lead to endometrial malignant transformation. aim: to investigate heparanase expression and regulation in the cycling endometrium. materials and methods: heparanase mrna levels were measured by quatitative rt-pcr in naturally menstruating women and in hec a, estrogen receptor (er)-negative and ishikawa, er-positive endometrial carcinoma cell lines exposed to increasing doses of estradiol. heparanase isoenzymes were localized by immunohistochemistry using specific anitbodies in murine endometrium and human normal, hyperplastic and malignant endometrium. results: heparanase mrna level increased fold in secretory phase (d ) compared to proliferative phase (d ) endometrium. heparanase transcript levels increased fold during hr culture in er positive adenocarcinoma cell line exposed to increasing doses of estradiol, but not in hec a, er negative cell line. both heparanase isoforms were localized to murine glandular endometrium. human glandular endometrium at both proliferative and secretory phases was immunoreactive with the active isoform of heparanase. proheparanase was detected in basal membrane of endometrial glands and endometrial stroma during secretory phase. along with malignant transformation of the endometrium the presence of proheparanase increased dramatically from none in stroma of normal and hyperplastic endometrium to abundance in malignant tumors. conclusions: heparanase gene expression is higher during the window of implantation and up-regulated with estrogen in endometrial cells via er in vitro and vivo. heparanase is differentially localized in the secretory phase of the endometrium compared to the proliferative phase, suggesting a role for this molecule during the window of implantation in man. interleukin- (il- ) system mrna and protein expression in the human fallopian tube with ectopic implantation. hong-yuan huang, , tien-hung huang, chin-jung li, chyi-long lee, , hsin-shih wang, , yung-kuei soong. , obstetrics and gynecology, chang gung memorial hospital, kwei-shan, tao-yuan, taiwan; obstetrics and gynecology, chang gung university and school of medicine, kwei-shan, tao-yuan, taiwan. objective: ectopic pregnancy, an abnormal implantation of a fertilized ovum outside the uterine cavity, has been increasing in number at a staggering pace of all pregnancies. il- system is one of the major cytokines involved in human endometrium during embryo implantation and might perform a defensive role against maternal immune response. very little information is available regarding the expression and synthesis of cytokines in the pathogenesis of fallopian tube with ectopic gestation. the purpose of this study is to investigate il- system expression in human fallopian tubes with ectopic pregnancy. methods: paired segments of human fallopian tubes with ectopic implantation site and side portion close to ectopic gestation (n= ) were collected from women undergoing laparoscopic salpingectomy after informed consent and irb approval. segments of fallopian tubes from women undergoing tubal ligation (n= ) were used as control groups. total extracted rna was reverse transcribed and amplified by pcr using specific primers for gapdh ( bp), il- ( bp), il- bp ( bp) and il- r ( bp). quantitative il- and il- bp mrna expression in human fallopian tube was determined by real-time pcr. to determine the presence of il- system proteins, tissues were fixed and processed for immunohistochemical study. data analysis was done with anova and pearson's correlation. results: il- and il- bp as well as il- r mrna were all expressed in tubal ectopic implantation and normal tubes. according to real-time pcr with c t value quantification and -ct method, a significantly higher il- expression in tubal ectopic implantation and lower ratio of il- antagonist to agonist in portion close to ectopic implantation is demonstrated in comparison to normal tubes (p< . ). immunoreactive il- system at the protein levels was also present in human fallopian tubes with ectopic implantation and normal tubes. conclusions: these results suggest that fallopian tube il- system expression may play a crucial role during the process of early embryonic implantation. the expression and ratio of antagonist to agonist in fallopian tubes may indicate an earlier "dialogue " in human fallopian tubal gestation prior to uterine implantation. replacement. marcia c ferreira, ines kd cavallo, fernando m reis. gynecology, ufmg, belo horizonte, mg, brazil. activin a is a growth factor expressed in the endometrium, where it modulates tissue remodelling and enhances decidualization. the effects of activin a are counteracted by two binding proteins, namely follistatin and follistatin-like (fstl ). while the endometrial expression of activin a increases during the secretory phase of menstrual cycle, the effects of ovarian steroids on these proteins and their mrnas has not been assessed yet in postmenopausal women or in ovariectomized animals. we have evaluated the effects of estrogen alone or estrogen plus progestin on the endometrial expression of activin beta-a subunit, follistatin and fstl in ovariectomized rats. adult female wistar rats (n= ) were ovariectomized and received one week later a single dose of estradiol benzoate ( . mg/kg body weight, i.m. injection), either alone (n= ) or associated with depot medroxyprogesterone acetate ( . mg/kg body weight, i.m. injection, n= ), or oil vehicle (control group, n= ). one week after the hormone or placebo treatment, the animals were sacrificed and their uteri were removed and processed by immunohistochemistry and real-time pcr. data were normalized to the expression of ribosomal phosphoprotein p (rpp ) and analyzed with the delta-delta ct method, anova and newman-keuls test. activin beta-a subunit mrna levels increased significantly in the uteri of rats treated with estradiol alone ( . fold increase over controls, p< . ) and to the same extent in rats receiving estradiol plus medroxyprogesterone ( . fold increase over controls, p< . ). this was accompanied by increase of beta-a subunit immunostaining in estradiol and estroprogestin-treated rats, which was noted only in the surface endometrial epithelium. follistatin mrna expression, conversely, showed a significant decrease in the groups treated with estrogen alone ( . fold compared to controls, p< . ) and estrogen plus progestin ( . fold compared to controls, p< . ), while follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprotestin-treated rats compared to controls. fstl expression was similar in the groups. in conclusion, the expression of activin beta-a subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin. endometrial nk cells are a unique inert nk subset until pregnancy. simcha yagel, irit manaster, jacob hanna, ronit haimov-kochman, miri godin, yuval bdolach, caryn greenfield, shira natanson-yaron, arye hurwitz, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical center, jerusalem, israel; lautenberg center for tumor immunology, hadassah-hebrew university medical center, jerusalem, israel. introduction: we recently demonstrated that nk (natural killer) cells play a critical role in trophoblast migration and angiogenesis at the fetal maternal interface. nk cells populate the endometrium at the secretory phase of the menstrual cycle, the time of anticipated blastocyst implantation. peripheral blood (pb) nk cells and decidual nk (dnk) cells express a variety of activating receptors, including nkp , nkp and nkp , collectively known as natural cytotoxicity receptors (ncrs), and nkg d which regulate nk cell killing and growth factor production. to compare endometrial nk cell (enk) activating receptor expression and function to pbnk and dnk cells and endometrial ligand expression, with a focus on their roles in blastocyst implantation. patients and methods: subjects were ivf patients undergoing natural menstrual cycles. endometrium samples were collected on treatment days and . a lymphocyte profile of the endometrial cells and pb was performed. facs analysis was performed on isolated endometrial nk cells, pbnk cells and dnk cells for cd , cd , nkp , nkp , nkp and nkg d. ncr ligand expression was characterized on adherent endometrial cells using ncr-ig fusion proteins and nkgd -ig and nkg d specific ligands as well as control ccmi-ig. redirected killing assays and cytokine secretion assays of ifn , vegf, plgf, and il- with and without il- were performed. results: endometrial lymphocytes of day and in these women are mostly cd bright cd -nk cells, with a significant amount of t cells, similar to pbnk cells and in marked contrast to dnk cells. unlike pbnk and dnk cells, enk receptors do not express nkp , nkp . nkp and nkg d are the only activating enk receptor expressed. like decidual cells, adherent stromal endometrial cells expressed the ligands for nkp , nkp and nkg d, suggesting that these nk cells have potential for activation. finally enk cells could not kill or secrete cytokines. conclusions: these findings of a unique activating receptor profile on endometrial nk cells, unlike that of dnk and pbnk, suggest that enk cells are a special local population of nk cells that change dramatically and are activated at the onset of pregnancy. variation in platelet activation throughout the menstrual cycle. fiona c denison, amy o robb, imogen b smith, nicholas l mills, hilary od critchley, david e newby. centre for reproductive biology; centre for cardiovascular sciences, the university of edinburgh, united kingdom. background: platelet-monocyte aggregation (pma) is a sensitive and novel measure of platelet activation with important proinflammatory consequences including release of cytokines and chemokines. previous studies using less sensitive techniques suggest that platelet activation alters during the menstrual cycle in response to circulating concentrations of sex steroids. the effect of sex steroids on circulating (c) pmas during a single menstrual cycle is not known. objective: to determine whether cpmas, platelet surface (ps) p-selectin and plasma (p) p-selectin vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: healthy, nulliparous, pre-menopausal, non-smoking women (mean age years), with regular menses ( - days) were studied. subjects gave written informed consent and the study had ethical approval. serial venous blood samples were taken at menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - and - ). cpmas (monocytes positive for the platelet marker cd a) were measured by flow-cytometry. psp-selectin expression was calculated on cd a positive cells. isotype-matched controls were used. serum oestradiol (e) and progesterone (p), plasma and pp-selectin were measured by elisas. data were analysed by one-way anova with repeated measures and bonferroni's post-tests for multiple comparisons. results: luteal phase p was > nmol/l in all women. numbers of cpmas and expression of psp-selectin were both significantly higher during menstrual compared with periovulatory phase of the menstrual cycle ( . ± . vs. . ± . %, p= . and . ± . vs . ± . %, p< . , respectively). there was no significant difference in pp-selectin concentration during the menstrual cycle (p= . ). there was no correlation between levels of serum e or p and numbers of cpmas, expression of psp-selectin or pp-selectin concentration. conclusions: numbers of cpmas and expression of pspselectin are maximal at menstruation with neither numbers of cpmas nor expression of psp-selectin correlating with serum e or p levels. this study suggests that activated platelets may potentially contribute to the inflammatory response at menstruation by releasing inflammatory mediators. by angiogenic factors and peri-cellular proteases in decidual secretory endometrium (dse), decidua parietalis (dp), and basalis (db) of miscarriage patients and matched controls. comparison of these parameters between the two groups enabled hypothesizing about their correlation with the occurrence of miscarriages. methods: decidua was obtained during st trimester termination of pregnancy (control group) and vacuum aspiration of missed abortions (case group). vascularization was studied by cd -immunohistochemistry. the expression of vascular endothelial growth factor-a, placental growth factor, flt- , kdr, angiopoietin- , angiopoietin- , tie- and the membrane-type matrix metalloproteinases mt -, mt -, mt -and mt -mmp were determined at mrna and antigen level and cd -positive unk cells, cd -positive macrophages, proliferation (ki ) and apoptosis (activated caspase- ) were evaluated by immunohistochemistry in consecutive serial sections. results: the decidual vascularization pattern showed differences between cases and controls: i.e. fewer vessels with larger circumference in cases, and this correlated with the differential expression of various angiogenic factors and proteases at mrna and antigen level. moreover, the endothelial protein expression of flt , kdr, mt -and mt -mmp was increased at the implantation site of cases. ki and active caspase- showed similar levels in the two groups and also the immune cells, both unk cells and macrophages, showed no differences at the implantation site between both groups. conclusion: differences between cases and controls appeared not to be based on altered proliferation, apoptosis, and/or inflammation. the differences in vascularization pattern and in the expression of angiogenic factors and proteases between both study groups suggest a correlation between decidual vascularization and the occurrence of miscarriages. respond to adrenomedullin. yaun-lin dong, hong y wen, janice endsley, alison hogg, hui-qun wang, manubai nagamani, chandra yallampalli. background: natural killer (nk) cells are the predominant lymphocytes present in human implantation site. decidual nk cells express perforin, an essential molecule required for lysis. formation of the placenta involves cooperation between maternal nk cells and fetal trophoblast cells that remodels the blood supply; however, the interaction between trophoblasts and decidual nk cells is largely unknown. adrenomedullin (adm) has been implicated in regulating early placental function and fetal growth. objective: to determine the role of multifunctional peptide adm in the decidual nk cells and fetal trophoblast cells interactions. methods: decidual and placental tissues were obtained from normal firsttrimester pregnancies terminated for social reasons. ethical approval to use these tissues was obtained from the irb of university of texas medical branch. cell preparations containing all decidual mononuclear cells were isolated by collagenase enzymatic disaggregation. cd decidual nk cells were purified by magnetic bead isolation. results: ) immunohistochemical analysis showed that adm is expressed primarily in decidual cells and trophoblast cells at the human implantation site; ) confocal imaging analysis demonstrated that decidual nk cells, which were identified by anti-cd staining, express adm receptor components crlr/ramp /ramp and their mrna expressions were futher confirmed by rt-pcr; ) k target cell killing assay indicates that adm inhibits cytokine il- /il- -induced decidual nk cell cytotoxicity; and ) immunofluorescent labeling and flow cytometric analysis revealed that adm suppresses perforin expression by decidual nk cells. conclusion: trophoblast-derived adm inhibits decidual nk cell cytotoxicity via suppressing perforin expression, thus, our results provide evidence for a new paradigm of embryonic-maternal communication involving a adm mediated interaction between decidual nk cells and fetal trophoblasts. leandro g oliveira, gendie e lash, judith n bulmer, barbara a innes, roger f searle, stephen c robson. institute of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom. background: we have previously demonstrated that co-culture with extravillous trophoblast cells (evt) (expressing hla-g) alters cytokine secretion by uterine natural killer (unk) cells, particularly at - weeks gestation. we have also reported that unk cells can stimulate evt invasion, but only at - weeks gestation (not at - weeks gestation). in addition, unk cell cytokine profiles alter with increasing gestational age. other reports have suggested that evt or hla-g expressing cells may alter the expression of cytokines and angiogenic growth factors by unk cells. hypothesis: hla-g expressing cells alters unk secretion of cytokines. methods: cd + unk cells were isolated from early pregnancy decidua ( - and - weeks gestation, n= each group) using enzyme digestion and positive immunomagnetic bead separation. the human b lymphoblastoid . transfected with either hla-g ( g) or a mock cdna ( cdna) were obtained as a kind gift from mr r apps (university of cambridge, uk). isolated unk cells were cultured in the presence or absence of the two cell lines in either direct or indirect contact (n= each group and each gestational age) for hours. cell supernatants were analysed for cytokines using a fastquant® th /th multiplex protein assay (il- , il- , il- , il- , il- , tnf-, il- , il- , ifn-) or by standard elisa (tgf- ). the effect of direct co-culture of unk cells with g compared with co-culture with cdna at each gestational age was tested using mann whitney u test. the effect of co-culture of unk cells with g in both direct and indirect contact was also tested using mann whitney u test. results: there was no difference in the level of cytokines secreted by the g or cdna cells. cytokine secretion by unk cells was not altered after direct co-culture with either g or cdna cells at either gestational age. in addition, direct or indirect co-culture of g or cdna with unk did not alter cytokine secretion at either gestional age. conclusions: hla-g does not alter the secretion of cytokines by unk cells from either - or - weeks gestation. other evt or decidua derived factors (including hla-e) may be responsible for the alteration in secretion of cytokines by unks with increasing gestational age. introduction: natural killer (nk) lymphocytes are central to innate immunity and contribute to tissue homeostasis by eliminating altered cells. their nkg d receptor pathway plays a fundamental role in target elimination through binding nkg d ligands on the cell surface. reduction in the nkg d ligand, ulbp , expression is associated with immune resistance in neoplastic processes. we have previously shown that fibroblasts from adhesion tissue (at) are characterized by increased extracellular matrix molecules and inflammatory cytokines compared with normal peritoneal (np) fibroblasts. objective: to determine if there is a difference in nk lymphocyte-mediated elimination between np and at fibroblasts and to investigate potential role of nkg d pathway in this process. material and methods: expression of nkg d ligands; ulbp , ulbp , mica, and micb was evaluated by flow cytometry and western blot in primary cell cultures of fibroblasts from np and at, established from two patients. peripheral blood nk lymphocytes (cd +cd -) from three healthy volunteers were isolated using macs system with purity greater than % and kept in interleukin overnight. fibroblast elimination with and without ulbp blocking was investigated following -hour co-incubation with allogeneic nk lymphocytes using our established flow cytometric cell mediated cytotoxicity assay. paired t test was used in statistical analysis. results: the flow cytometry studies showed that nkg d ligands (ulbp , ulbp , mica and micb) were lower in at compared to np fibroblasts, reaching a statistical significance in ulbp expression (p = . ). western blot analysis also revealed a lower ulbp protein level in at than np fibroblasts. furthermore, nk lymphocyte-mediated elimination was % lower in at in comparison with np fibroblasts. blocking ulbp expression resulted in decreased nk lymphocyte-mediated np fibroblast elimination by %, supporting the role of nkg d receptor pathway in the process. conclusions: our results demonstrate that nkg d pathway is operational in at fibroblast resistance to immune elimination, and extends our prior observations of the potential role of immunological mechanisms in the pathogenesis of adhesion development. objective: galectin- is an anti-inflammatory lectin that has pleiotropic regulatory functions at the crossroad of innate and adaptive immunity. human galectin- is expressed in the placenta and immune privileged sites and it has been implicated in establishing immune tolerance. the aim of this study was to examine the evolution and placental expression of the lgals gene in primates. methods: seven primate nucleotide sequences were generated, aligned to vertebrate orthologs from all classes and subjected to phylogenetic analysis. deduced amino acid sequences were analyzed for functionally important substitutions. placental galectin- expression was studied by immunohistochemistry and western blot. results: ) the lgals gene had high sequence identity among all investigated species. ) phylogenetic analysis revealed that intense purifying selection had been acting on the lgals gene in placental mammals (dn/ds= . ); ) residues responsible for sugar binding or molecule stabilizing were highly conserved in primates. ) immunostaining showed a uniformly abundant and ubiquitous galectin- expression pattern in human, old and new world monkey and prosimian placentas, regardless the type of placentation. the lgals gene has conserved sequence and placental expression pattern in primates that may suggest its important function in maternal-fetal immune interactions. these results support the view that immune interactions at the maternal-fetal interface evolved in concert with invasive placentation and that these interactions have been maintained regardless of the degree of placental invasion in primates and other mammals. expression of interleukin- in human endometrium throughout the menstrual cycle and early pregnancy. yesim h uz, , william murk, umit a kayisli, aydin arici. department of obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa; department of histology and embryology, trakya university school of medicine, edirne, turkey. background: interleukin- (il- ) is a recently discovered heterodimeric cytokine, comprised by a novel p subunit and a p subunit shared by il- . it has biological activities that are similar to but distinct from il- , and is known to be involved in th /th cell class switching and the regulation of cytokines such as ifn-gamma, il- , tnf-alpha, and il- . early pregnancy is associated with alterations in the maternal immune response, such as changes in cytokine expression, and leukocyte recruitment and subtype switching. we hypothesized that expression of il- in the human endometrium is menstrual cycle-and pregnancy-dependent. materials and methods: endometrial samples from women (n= ) undergoing surgery for benign gynecologic conditions, and decidual tissues from women (n= ) with clinically normal pregnancies terminated voluntarily in the first trimester, were obtained after receiving informed consent. endometrial samples were grouped according to menstrual phase. paraffin sections were stained with il- p antibodies and evaluated semi-quantitatively with hscore. statistical analysis of the data was done using anova, with p< . considered significant. results: il- immunoreactivity was predominantly located in the cytoplasm of both endometrial stromal (esc) and glandular (egc) cells. escs showed mild il- immunoreactivity without significant changes in intensity throughout the menstrual cycle. on the other hand, first trimester decidual cells showed significantly stronger il- staining compared to escs from non-pregnant endometrium (p< . ). il- immunoreactivity in egcs was high in the late proliferative phase, as compared to other cycle phases and first trimester tissues (p< . ). moreover, egcs from the early secretory phase (p< . ) and first trimester tissues (p< . ) showed higher il- immunoreactivity compared to the early proliferative and late secretory phases. conclusions: this is the first study describing il- expression in the human endometrium and decidua. these results suggest that il- has a cycledependent expression in endometrial cells and may be involved in regulating cytokine expression and immune cell modulation during the menstrual cycle and early pregnancy. gercel-taylor, douglas d taylor. obstetrics, gynecology, and women's health, university of louisville, louisville, ky, usa. objective: estrogen appears appear to be a critical regulator of the immune system. since hypoestrogenism is present in the postmenopausal woman, our objective was to determine whether t cell activation and function, defined as il- production and signaling molecule expressions at the transcriptional and translational levels, were affected by a low estrogen environment. design: prospective study in a university research laboratory. materials and methods: jurkat . t cells, initially grown in estrogen free media, were incubated in pm (representing postmenopausal levels) or pm (premenopausal levels) of estradiol (e ) for hours. cells were either resting or activated with a phorbol ester, -phorbol -myristate -acetate (pma), and ionomycin. enzyme-linked immunosorbent spot assay (elispot) was performed to analyze production of il- . expression of signaling protein components, cd and jak, were determined by western immunoblotting. real time-polymerase chain reaction was performed to quantify cd , jak , and jak gene expression. a p value of < . was considered significant. results: jurkat cells exposed to pm e and activated exhibited significantly diminished numbers of il- producing colonies compared to t cells exposed to pm ( . ± . vs. . ± . colonies, p< . ). analysis of cellular cd and jak protein demonstrated that jurkat cells incubated in pm e expressed a . -fold decrease in cd and . -fold decrease in jak compared with cells incubated in pm e (p< . ). these diminished protein levels appeared to be the consequence of suppressed transcription, as the mrna levels of cd , jak and jak were significantly decreased in jurkat cells incubated in low levels of estrogen ( . , . , and . fold, respectively, compared to pm). conclusions: jurkat cells exposed to low postmenopausal estrogen levels produce significantly less il- following activation, which was associated with a significant decrease in signaling proteins. the diminished levels of signaling proteins appear to result from decreased cd , jak and jak gene expression in the presence of low estrogen. these findings support the observation of decreased cellular immune response in postmenopausal women and may provide a basis for the increased risk of infections and cancer proliferation associated with aging. support: dept. of ob/gyn research seed fund. the expression pattern of novel cytokines (il- and ) in human fetal membranes. judith eckardt, , stephen j fortunato, holger maul, ramkumar menon. the perinatal research center, nashville, tn, usa; womens' hospital, university of heidelberg, heidelberg, baden-wuerttemberg, germany. objective: interleukin (il) and are novel cytokines produced by various immunological cells in response to microbial antigens. the functions of these cytokines in reproductive system is unknown. this study examines the expression pattern of il- and il- in human fetal membranes from preterm and term births and in in vitro in normal term membranes in response to bacterial endotoxin (lipopolysaccharide-lps). methods: fetal membranes collected (n= ) from cesareans at term (normal, not in labor) were placed in an organ explant system for hours and were stimulated with lps for an additional hrs. fetal membranes were also collected (n= ) either at preterm or term after vaginal deliveries. in a case -control study (preterm birth vs. normal term deliveries) amniotic fluids (af) (n= ) were collected to document the role of il- and il- in ptb. tissue expressions of il- and il- were studied by rt-pcr using specific primers. elisa documented culture media and af cytokine concentrations. statistical analysis was performed using non-parametric mann-whitney u test. results: both il- and il - expressions were seen in fetal membranes in culture (in vitro) regardless of stimulation. in vivo in membranes from preterm and term deliveries and membranes at term not in labor also documented the expression of these cytokines. culture media analysis documented higher concentration of il- after lps stimulation (lps- . vs. . pg/ml; p= . ) whereas no difference was noticed in il- concentration (lps- . control- . pg/ml; p= . ) between the two groups. af analysis, regardless of the status, did not document detectable concentrations of either of the cytokines (lower limit . pg/ml for both). conclusion: this is the first study to document the expression of two novel cytokines in laboring and non laboring human fetal membranes and also in membranes from preterm deliveries. il- production was stimulated by lps whereas il- was not affected. these cytokines are not physiological components of af and their role in fetal membranes is unclear. higher il- concentration in response to lps but lack of its presence in term or preterm af is suggestive of an autocrine immune response during pregnancy in response to a microbial antigen. evidence for a selective migration of fetus specific cd + cd bright regulatory t cells from the peripheral blood to the decidua in human pregnancy. tamara tilburg, , dave l roelen, barbara j van der mast, godelieve m de groot, carin kleijburg, sicco a scherjon, frans hj claas. department of obstetrics, leiden university medical centre, leiden, netherlands; department of immunohematologie and bloodbank, leiden university medical centre, leiden, netherlands. during pregnancy the maternal immune system has to tolerate the persistence of fetal alloantigens. many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. murine studies suggest that cd + cd + t cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. previous studies by our group demonstrate that a significantly higher percentage of activated t cells and cd + cd bright t cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy ( ) . in this study we examined the phenotypic and functional properties of cd + cd bright t cells derived from maternal peripheral blood and decidual tissue. depletion of cd + cd bright t cells from maternal peripheral blood demonstrates regulation to a rd party umbilical cord blood cells comparable to non-pregnant controls, whereas the suppression capacity to umbilical cord blood cells of her own child is absent. furthermore, maternal peripheral blood shows a reduced percentage of cd + cd bright foxp + and cd + cd bright hla-dr + cells compared to peripheral blood of non-pregnant controls. in contrast, decidual lymphocyte isolates contain high percentages of cd + cd bright t cells with a regulatory phenotype that are able to down regulate fetus-specific and non-specific immune responses. these data suggest a preferential recruitment of fetus-specific regulatory t cells from maternal peripheral blood to the fetal-maternal interface where they may contribute to the local regulation of fetus specific responses. ( ) tilburgs t, roelen dl, van der mast bj, van schip jj, kleijburg c, de groot-swings gm, kanhai hh, claas fh, scherjon sa. differential distribution of cd (+) cd (bright) and cd (+) cd (-) t-cells in decidua and maternal blood during human pregnancy. placenta. apr; suppl a:s - . alicia del toro-arreola, lourdes nunez-atahualpa, juan velazquez-rodriguez, laura gonzalez-lopez, jorge i gamez-nava, adrian daneri-navarro. there is evidence that the maternal immune system is influence by changes in the hormonal levels during the menstrual cycle (mc). so far, the information related to the levels of t, treg, nk cells and receptors of activation and inhibitors is scarce. aim: to analyze the populations of t, treg, nk cells and their receptors of peripheral blood of healthy women and their correlation with hormones during mc. material and methods: we studied to women not using hormonal contraceptives in the day th of the follicular phase and st of the luteal phase of the mc. pbmc subsets and their receptors were determined by flow cytometry and hormone levels by chemiluminescence method. we found that the progesterone and prolactin were positive correlated (rho= . , p< . and rho= . , p< . , respectively) with cd /nkg in t cells and negative correlated (rho= - . , p< . and rho= - . , p< . , respectively) with nk cells. meanwhile cortisol was positive correlated (rho= . , p< . ) with the receptor nkg d expressed in nk cells. the results observed in this study in the luteal phase of mc on the expression of cd /nkg inhibitor receptor and nkg d activator receptor were related to a particular hormone (progesterone, prolactin and cortisol) might contribute to understanding the physiological role of the neuroendocrine axis on the expression of some receptors of the immune system in order to keep the homeostasis milieu of the mc. objective: sp-d, a key component of the innate immune system, is detected in amniotic fluid (af) and believed to originate in the fetal lung. however, sp-d is produced by other cells and therefore extra-pulmonary sources must be considered. the objective of this study was to determine the maternal and fetal plasma and af concentrations of sp-d to gain insight into the behavior of this natural antimicrobial peptide in pregnancy. moreover, we studied sp-d expression in maternal and fetal peripheral leukocytes. methods: maternal and fetal plasma and af samples were obtained from patients in the following groups: ) term not in labor (tnl; n= ); ) term in labor (til; n= ); ) preterm labor without histologic chorioamnionitis (ptl; n= ) and ) preterm labor with histologic chorioamnionitis (ptl-hc; n= ). sp-d concentration was measured by elisa. sp-d mrna expression in maternal and fetal leukocytes was evaluated by real-time qrt-pcr. flow cytometry and confocal microscopy were used to study the localization of sp-d in leukocytes. results: ) the af sp-d concentration increased as a function of gestational age (mean, til: , . ng/ml vs. ptl: , . ng/ml; p< . ); ) in contrast, the maternal and fetal plasma sp-d concentrations decreased with advancing gestational age (mean, til: . ng/ml vs. ptl: . ng/ml; p< . , and til: . ng/ml vs. ptl: . ng/ml; p< . , respectively); ) the maternal plasma sp-d concentration was lower than that of fetal plasma (mean: . ng/ml vs. . ng/ml; p< . ); ) however, sp-d mrna expression in maternal leukocytes was . fold higher than that of fetal leukocytes (p< . ); ) neutrophils (both maternal and fetal) expressed sp-d as demonstrated by flow cytometry and confocal microscopy. conclusion: ) the concentrations of sp-d in the maternal and fetal circulation decreased with gestational age while the af concentration increased; ) the expression of sp-d mrna is higher in maternal leukocytes than in fetal leukocytes; ) we report for the first time that maternal and fetal neutrophils are a source of sp-d and propose that this molecule plays a role in host defense against infection and in the modulation of the maternal and fetal immune response. introduction intrauterine insemination (iui) is a fertility technique that allows for couples to have intercourse after the procedure is performed. it has been postulated that intercourse after iui may increase the pregnancy rate by either endometrial stimulation or because it may represent a second spermatic flow in the periovulatory period. in the present study we evaluate the effect of intercourse on the pregnancy rate of patients undergoing iui. material and methods: from to patients were enrolled in the study. every couple undergoing iui was instructed at the moment of insemination to decide whether to have or not intercourse on the same day of the procedure. all couples were abstinent three to seven days before iui. the information regarding intercourse was recorded the day after treatment. ovulatory, insulin resistant, cervical, male, tubal and endometrial factors as well as parity and time of infertility were compared between the two groups. all these factors were analyzed based on number of follicles that ovulated in each group. our principal outcome was to determine the pregnancy rate. intercourse was practiced by . % of the couples. the global pregnancy rate was . %. the pregnancy rate for the couples who had intercourse was . % and . % for those who did not have intercourse (p< . ). even though age, parity, time of infertility and stimulation protocols were similarly distributed in both groups, the proportion of tubal and endometrial factors were higher among those who had intercourse (p< . ). when subjects with tubal and endometrial factors were excluded, the pregnancy rate between both groups (n= ) was similar ( . % vs . % for positive and negative intercourse, respectively). the average number of ovulatory follicles was . + . for the group that had intercourse and . + . for those who did not. according to our results, intercourse after iui does not improve pregnancy rate after this procedure is performed. furthermore our study indicates that iui does not interfere with sexual intimacy since almost % of the couples decided to have intercourse on the same day of the procedure. . we sought to investigate the effects of gravidity on mmc and fmc in healthy, parous women. methods: mc was assayed in dna extracted from peripheral blood mononuclear cells (pbmc). hla-genotyping was first conducted and mc quantified employing a q-pcr assay targeting a non-shared maternal-or fetalspecific hla polymorphism. gravidity was dichotomized as a history of one pregnancy compared with two or more, and the prevalence of mc was analyzed using logistic regression. possible confounders were included as appropriate, including subject age and time since last pregnancy. adjustment for possible correlation between values was also made when there were repeated measures for the same subject. results: for the mmc analysis, there were subjects with observations. for the fmc analysis, there were subjects with observations. table provides a summary of mmc and fmc by gravidity. mmc was significantly decreased with higher gravidity. fmc was not affected by gravidity. gravida gravida or more adjusted* or ( %ci) mmc / ( %) / ( %) . ( . - . ) fmc / ( %) / ( %) . ( . - . ) *adjusted for possible correlation between values within a subject, subject age, and time from last pregnancy, as appropriate. increasing gravidity is significantly associated with a decreased prevalence of mmc. despite additional sources of fmc, there does not appear to be an increase in fmc prevalence with increasing gravidity. the biology of mc is incompletely understood, and the nature of mmc and fmc are likely to be different given that acquisition of the former, but not the latter, occurs within a nascent immune system. these data raise interesting questions when considered as interactions of acquired grafts within a host, including whether emergence and persistence of one dominant source of mc may be most advantageous for an individual. anti-igd antibody treatment as a novel immunosuppressive agent for autoimmune diseases and its effects on th /th gene expression. tommie g nguyen, eileen d gallery, , jonathan m morris. elevated t-helper cell type- (th ) and type- (th ) cytokine expression have a role in autoimmune diseases, allograft rejection and pregnancy-related complications. thus, molecules that can shift the immunity away from th /th responses toward a th response represent a novel therapeutic treatment for these conditions. we have previously demonstrated that pregnancy is associated with a suppression of t-bet in peripheral t cells. in this study, we examined a novel effect of anti-igd antibody on t-bet expression, th /th gene expression in human peripheral blood mononuclear cells (pbmc) and its therapeutic effects in an animal model of collagen-induced arthritis. methods: human pbmc were isolated and then cultured in the presence of anti-igd antibody at various time points followed by stimulation with pma/ionomycin (p/i) for hrs. gene expression was examined by rt-pcr, western blotting and elisa. for in vivo study, arthritis-prone dba/j mice were induced to undergo joint inflammation by intradermal injections with bovine type-ii collagen. these mice were then given daily doses of mg/kg of intravenous injection with anti-igd antibodies as preventive or therapeutic treatments (n = per group). results: treatment with anti-igd antibodies significantly suppressed p/iinduced expression of t-bet (a master regulator of th immunity), tnf-(a classical pro-inflammatory th cytokine), and il- (a critical proinflammatory th cytokine) in human pbmc. this suppression is highly specific to these genes because anti-igd antibodies have no effects on the expression of ifn-g and il- (two classical th cytokines). in vivo experiment showed that anti-igd antibody treatment markedly reduced clinical severity of joint inflammation when comparing the clinical score of control mice group ( . ± . , mean ± s.e.m), preventive group ( . ± . ) and therapeutic group ( . ± . ) . conclusions: our study has demonstrated that suppression of t-bet by anti-igd antibodies, similar to the changes seen in human pregnancy is a novel in vivo anti-inflammatory effect. given the essential role of t-bet, tnf-and il- in the pathogenesis of human autoimmune diseases, anti-igd antibodies may represent a novel immunosuppressive treatment that needs further studies and evaluation. objective: women with circulating anti-phospholipid antibodies (apl) are at risk for recurrent miscarriage, preeclampsia and preterm labor. apl antibodies directly target the placenta by binding to phospholipids or phospholipid-binding proteins expressed on the surface of viable trophoblasts. the objective of this study was to determine the effects of apl antibodies on first trimester trophoblast cells. methods: two mouse igg anti-human beta -glycoprotein i monoclonal antibodies (mabs), designated id and iic , were used in these studies. the first trimester trophoblast cell line, htr , was incubated with either medium, a mouse igg control, id or iic ( - g/ml), in the presence or absence of unfractionated heparin ( ng/ml). trophoblast cell death and apoptosis was determined using a viability assay, hoechst staining and a caspase activity assay. cytokine production was evaluated by multiplex analysis. results: following a hour incubation, significant trophoblast cell death was induced by iic ( . ± . %) and id ( . ± . %) at the high dose of g/ml, when compared to the medium and mouse igg controls (p< . ). hoechst staining showed that id -and iic -induced trophoblast cell death was a result of apoptosis. moreover, id and iic induced a significant increase in caspase- , - and - activity (p< . ). treatment of trophoblasts with heparin significantly inhibited the effects of iic and id on cell death by . ± . % and . ± . % %, respectively (p< . ). following a hour incubation at lower concentrations ( g/ml), treatment of trophoblast cells with id or iic resulted in a significant upregulation of il- , mcp- , gro production (p< . ), and a significant reduction in il- secretion (p< . ). conclusion: this study demonstrates that at low levels apl antibodies can modulate trophoblast cytokine production, while at higher levels, the same antibodies induce trophoblast apoptosis in a caspase-dependent manner. these findings shed new light on the mechanisms by which apl antibodies may impact placental survival and function. antigenic targets for the diagnosis of premature ovarian failure. hc bohler, c gercel-taylor, lt ku, st nakajima, dd taylor. obstetrics, gynecology, women's health, university of louisville, louisville, ky, usa. objective: premature ovarian failure (pof) is a premature depletion of ovarian follicles before the age of , affecting approximately % of women < years. the involvement of autoimmune mechanisms in pof has been suggested and similar mechanisms have been postulated for other ovarian pathologies, including idiopathic infertility, polycystic ovary syndrome (pcos), or endometriosis. while the association of autoantibodies has been demonstrated for these ovarian pathologies, variation in specificity and frequency of false positivity have limited the diagnostic use of autoantibodies. the objective of this study was to develop an antigen array to differentiate antibody recognition patterns of pof from other infertility pathologies. design: prospective study in a university research laboratory. materials and methods: patients diagnosed with infertility were included in this pilot study: endometriosis (n= ), pcos (n= ) and pof (n= ). autoantibodies were assayed by dot immunoblotting using an antigen array derived from endometrial and ovarian cells. for the cellular antigen preparations, solubilized total proteins were separated by reverse phase-hplcquid chromatography and the individual proteins were blotted onto nitrocellulose membranes and reactivity visualized by peroxidase-labeled antihuman igg. results: patients with pof, endometriosis, and pcos all exhibited autoantibodies reactive with these cellular proteins. while some antigenic reactivities were shared by all infertility patients, the pattern of antigen recognition was distinct for patients with pof. patients with pof all recognized a common antigenic proteins (row , antigens a,b,d-g). conclusions: alterations in autoreactivity are observed in patients with the diagnosis of infertility; however, distinct patterns of autoantibody recognition can be demonstrated for patients with different pathologies. while this study needs to be expanded to reliably establish the specificity, sensitivity and positive and negative predictive values, patients with pof clearly exhibit a shared recognition pattern that may be useful a diagnostic marker. cicek gercel-taylor. ob/gyn, university of louisville, louisville, ky, usa. objective: americans' consumption of nutraceuticals is one of the most rapidly expanding health markets, growing at a rate of % annually. multiple nutraceuticals containing phytoestrogens have been marketed as "immune boosters" despite suboptimal evidence-based medicine to support such statements. as immunomodulatory therapies should affect downstream cytokine expression, the relative effects of estradiol and genistein in regulating expression of cd -and jak were tested. these markers were chosen since they are central to t cell signaling. cd -is a critical transducer of tcr activation and regulates t cell proliferation and cytokine production. jak upregulation is a specific marker for hematopoietic cell stimulation. methods: to test the immunomodulatory effects of phytoestrogens and estrogen, jurkat . (t cell leukemia) cells were grown in estrogen-free, phenol red-free media for hours. these cells were then exposed for hours to pm, pm (postmenopausal), or pm (premenopausual) of estradiol in the presence of increasing concentrations of genistein ( , . , . , . , , and m). cells were then solubilized and cellular protein quantitated. protein concentrations were standardized and western blots for each set of culturing conditions were run in triplicate. cd -and jak expression were quantitated following visualization with chemiluminescence by digital pixel quantification. results: our findings show that in the absence of estradiol and at postmenopausal levels of estradiol, genistein induced a dose dependent increase in cd -reaching a maximum of fold. although cultivation of t cells in pm of estradiol significantly increased the levels of cd -and jak relative to hypoestrogenic conditions, the genistein mediated dose response was not observed. conclusions: these in vitro results indicate that genistein can at least partially reverse suppression of signaling molecules observed in postmenopausal estrogen environments. clinically, this suggests that phytoestrogens may have greater immunomodulatory properties for postmenopausal females than those that are premenopausal. maternal serum il- as a biomarker of acute immunologic rejection of pregnancy. joaquin santolaya-forgas, juan deleon-luis, isabel galan. obstetrics and gynecology, brigham and women's hospital, boston, ma, usa; amarillo women's health research institute, texas tech university health science center, amarillo, tx, usa. objective: markers of acute rejection of pregnancy are very scarce. in this study we aimed at determining if rapid changes in maternal serum concetration of a variety of biomarkers could be used for this purpose. we used an established baboon model for in utero stem cell therapy to introduce at - days from conception and via ultrasound-guided celocentesis, human hematopoietic stem cells with different proportions of natural killer t-cells (nk). maternal blood was collected before and hours after celocentesis for quantification of hormones and il- using solid phase, enzyme labeled, chemiluminescent sequential immunometric assays. pearson correlation analysis was used for determination of significant changes from baseline (p< . ). results: all animals survived their pregnancies. seven animals receiving < % concentration of nk delivered at term ( days gestation) while animal receiving more than % concentration of nk had dead fetuses on ultrasound evaluation hours after celocentesis. table depicts mean maternal serum concentration of the biomarkers investigated (all n.s.). figure shows mean il- changes from baseline in continuing (n.s.) and rejected pregnancies (p< . ). conclusions: we have described a model in which in utero graft vs host disease can be studied. these preliminary results suggest that of all the biomarkers investigated, il- might be the most sensitive for detection of an acute rejection of pregnancy. biomarkers of acute immunologic rejection of pregnancy biomarker unit pre-celocentesis ( ) the activity of cytotoxic cd + t cells during pregnancy protects the mother and fetus from infection. however, pregnancy's effect on the proliferation and apoptosis of cd + t cells has not been clearly defined. objective: to determine if normal pregnancy changes the number of proliferating or apoptotic splenic cd +t cells. methods: female c bl/ mice were used unmated (um) or underwent timed mating. one day prior to harvest, mice were i.p. injected with bromodeoxyuridine (brdu), which is incorporated into replicating dna. harvested spleens were homogenized, enumerated, and stained for cell surface expression of cd and t cell receptor beta chain (tcr ). apoptotic cells were detected by treatment with terminal transferase and fitc-dutp (tunel). the numbers of cd +tcr + cells that were brdu+ or tunel+ were calculated from the percentage of positive cells obtained by flow cytometry and the absolute number of cells counted. for each experiment, the ratio of the number of positive cells in pregnant to um mice was compared by anova with dunnett's post-test. results: at day of pregnancy (n= ), the number of brdu positive cd + t cells was two fold higher than that found in um (n= , p< . ). the number of proliferating cd + t cells continued to be non-significantly elevated at day ( . x, n= ), day ( . x, n= ), and day of pregnancy ( . x, n= ) compared to um. by day of pregnancy (n= ) the number of proliferating cd + t cells returned to the um level, however by this time the total number of splenic cd + t cells was . fold higher than um (n= p< . ). on gestational day , the number of proliferating cd + t cells declined further ( . x, n= ), and the number of splenic cd +t cells returned to the um level (n= , p> . ). compared to um mice, there was no significant difference in the number of cd + t cells undergoing apoptosis at any gestational day examined ( . - . x, p> . ). in normal murine pregnancy, the number of cd + t cells is increased in late gestation, and then returns to baseline at the end of pregnancy. this is due to an early increase then gradual decline in cd + t cell proliferation, accompanied by a steady rate of apoptosis. this argues that the maternal immune system undergoes dynamic homeostatic changes, and is not globally suppressed. supported by nihro hd - niht ai . arturo cerbulo-vazquez, cun li, , gene hubbard, natalia e schlabritz-loutsevitch, , peter w nathanielsz. objectives: early thymocyte (t) maturation occurs in the cortex while later stages occur in the medulla. thymic epithelial cells (tec) synthesize gc and t express gr. tec may influence t cell maturation by regulating apoptosisinduced gc-gr interactions. igf- (also synthesized by tec) may support thymocyte prolifetarion. human fetuses of mothers in premature labor are exposed to gc. gc administered to pregnant baboons at . , . , and . gestation (g) alters fetal lymphocyte populations at . (g) (j repro immunol, , : ) . we determined if fetal gc exposure alters thymic ) structure; ) gr and igf-i protein. methods: pregnant baboons received saline (control ctr; n= ) or betamethasone i.m. ( μg/kg daily for two days at . , . and . g; gc group; n= ), c-sectioned at at . g under general anesthesia and thymic gr and igf-i evaluated by immunohistochemistry. results: gr localized to medulla and a few cortical cells. igf-i localized to cortex with little medullary expression. medullary necrosis was greater in ctr than gc fetuses. t gr was located in cytoplasm. no gross differences were observed between ctr or gc fetuses for either igf-i or gr. conclusions: a) early thymocyte maturation may be supported by igf- , b) later differentiation involves gr, and c) after exposure to gc doses equivalent to human therapy, no gross effects were detected on gr or igf, but d) natural thymic necrosis was inhibited. lindsay s christensen, peyman bizargity, elizabeth a bonney. ob/gyn, university of vermont, burlington, vt, usa. background: the exact mechanism by which bacterial products trigger preterm delivery and the immune cellular circuits involved remain unclear. our recent data in normal c bl/ (b ) or recombinase deficient c bl/ mice (rag-ko) indicates that t and b cells are not critical for lps-induced preterm delivery and stresses the importance of related innate mechanisms. rag-ko mice are more susceptible to lps, suggesting that t or b cells may control the innate response. macrophages are vital to innate immunity and produce proinflammatory cytokines that can activate prostaglandin synthesis and myometrial contraction. we questioned whether differences in susceptibility between the strains are due to differences in the uterine macrophage response to lps and thus examined macrophages at the maternal-fetal interface early after injection. objective: to compare uterine macrophages levels at and hours after lps injection in pregnant b and rag-ko mice. methods: b and rag-ko mice were mated and on gestation day , females were injected intraperitoneally with μg lps in l saline (pbs) or l pbs alone. euthanasia and uterine harvest occurred or hours after injection. frozen uterine sections were stained with the macrophage marker f / or an isotype-matched control followed by an alexafluor -conjugated secondary and a nuclear stain (dapi). sections were visualized by fluorescence microscopy. for each mouse, f / + dapi+ and total dapi + cells were counted in areas of representative section and the percentage of f / + dapi+ cells was calculated. the mean percentage for at least representative areas per experimental group was analyzed by anova. results: two hours post-injection, macrophages levels were similar in b and rag-ko mice injected with pbs (b , n= , . ± . ; rag-ko, n= , . ± . % + per area). lps injection increased macrophages (p< . ) in both strains (b , n= , . ± . ; rag-ko, n= , . ± . ,); no difference was evident between strains. the percentage of f / + cells was similar hours post-injection (b , n= , . ± . v. rag-ko, n= , . ± . ), and not elevated relative to the hour time point. objective. decay-accelerating factor (cd ), is expressed in the plasma membranes and protects mammalian cells against the lytic action of serum complement. phosphoinositide -kinases (pi ks) are involved in the regulation of cell functions by synthesizing a second messenger molecule ptdins ( , , ) p . akt, a serine-threonine kinase acts downstream of pi k and regulates cell survival, growth and proliferation. the pi k-akt activity is controlled by tumor suppressor gene pten. in this study we assessed whether the pi k-akt activity affects the expression of cd in human endometrial and cervical cells. methods. endometrial and cervical cell lines which differ in the constitutive pi k activity were used in this study. ishikawa and rl - endometrial cell lines harbor pten mutation and have high levels of phosphorylated akt (p-akt). hec- -a and kle endometrial cell lines and hela cells express wild-type pten and have minimal or no demonstrable levels of p-akt. the expression of cd was evaluated by rt-pcr, immunoblotting and flow cytometry. the pi k activity was assessed by immunoblotting with anti-p-akt antibodies. the effect of inhibition of pi k-akt pathway on cd expression was evaluated in cells treated with wortmannin ( nm), ly ( mm), or with akt inhibitor sh ( mm). results. immunoblotting densitometry and measurements of mean fluorescence intensities showed that the level of cd expression correlates with the status of pi k-akt pathway. the cd expression was lowest in hec- -a, ishikawa and rl - cells which constitutively express p-akt. higher cd expression was found in hela cells and kle cells which express wild-type pten product and has no detectable phospho-akt. mean fluorescence intensities were . -fold higher for kle cells and -fold higher for hela cells compared to hec- -a cells. treatment of cells with akt inhibitor led to . - . -fold increase in cd expression. the . - . -fold increase following treatment with pi k inhibitor wortmannin was found in ishikawa cells, rl - and kle cells. conclusions. human endometrial cell lines with elevated pi k-akt activity express lower level of cd compared to cell lines with intact pten gene function. these findings may indicate that structural alteration at the dna level and resultant overexpression of pi k-akt pathway are involved in the downregulation of cd . endometrial and cervical cells. pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cell shape is determined by the cytoskeleton, which provides the mechanical support and is involved in cellular signaling. apoptotic cells undergo major morphological changes such as rounding and contraction, a process regulated by caspases, the cysteine proteases responsible for events controlling the cell disassembly. the motifs in certain cytokeratins make them substrates for caspase degradation. anti-apoptotic serine/threonine kinase akt provides a survival signal by phosphorylating downstream effector molecules including caspase- . while studying the akt distribution in human endometrial cell line we found that akt shows filamentous pattern of staining resembling cytoskeleton organization. in this study we evaluated whether akt staining correlates with the microfilaments (mf), microtubules (mt) or intermediate filaments. endometrial ishikawa, rl - , hec- -a, kle and cervical hela cell lines were used in the study. incubation with cytochalasin d, ( ug/ml) or nocodazole ( mg/ml) and labeling with bodipy fl phallacidin, anti -tubulin or anti-akt antibody were used to assess whether cytoskeleton disruptors affect akt distribution and mf and mt organization. for colocalization, cells were stained with anti-cytokeratin mouse antibody followed by anti-mouse alexa conjugate, and then stained with anti-akt rabbit antibody and anti-rabbit alexa conjugate. the scans collected with laser scanning confocal microscope from channels filtered for alexa and alexa were combined digitally and evaluated with imaris colocalization analysis software. filamentous pattern of akt staining was most pronounced in ishikawa and less obvious in hec- -a cells. treatment with cytochalasin d or nocodazole resulted in disruption of mf and mt but had no effect on cytokeratin organization and akt distribution. double staining with anti-cytokeratin- and anti-akt antibody showed overlapping staining for cytokeratin and akt. analysis of digitally acquired images showed highest correlation for colocalized channels in rl - cells ( . ) followed by ishikawa ( . ) and hela cells ( . ). lowest correlation was found for kle ( . ) and hec- -a cells ( . ) conclusions. this study indicates a strong colocalization pattern of serine/ threonine kinase akt with cytokeratins, and suggests a mechanism by which cytokeratins might be protected against cleavage by caspase- and caspase- in the early apoptotic stages. background: cd + cd + t regulatory cells (t-reg), express foxp ,suppress antigen-specific immune responses and are important for allograft tolerance. during pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus), requiring substantial changes in immune regulation over a programmed period of time. the presence of t-reg cells (cd + cd highfoxp +) was assessed in the peripheral venous blood of non-pregnant, pregnant and seven postnatal healthy women by flow cytometry. human decidua was obtained by surgical termination of pregnancy in the first (n= ), second (n= ) and third (n= ) trimester of human pregnancy. paraffin sections were immunostained for foxp and cd . foxp +cells were quantified in x fields and results compared between first, second and third trimester samples and according to the presence of extravillous trophoblast. results: fluorometric studies of blood samples indicate an increase % of circulating cd + cd highfoxp t-cells in pregnant ( . % [range . - . %]) vs. non-pregnant controls ( . % [range . - . %]; p< . ). a progression from st, nd and rd trimesters indicated the % of cd + cd highfoxp t-cells was . %, . % and . %, respectively. low numbers of foxp + cells were detected in all decidua samples and their distribution mirrored that of cd + cells. in st trimester samples, foxp + cells were often detected in lymphoid aggregates adjacent to endometrial glands. increased numbers of foxp + cells were detected in st ( . ± . ) compared with nd trimester decidua ( . ± . ; p< . ) but there was no difference between st and rd trimester ( . ± . ), nor between nd and rd trimester decidua. in st trimester decidua, numbers of foxp + cells were increased in areas without extravillous trophoblast. conclusion: normal human pregnancy is associated with an increase in the number of circulating cd + cd highfoxp t-cells. the presence of foxp + cells in early gestation human decidua may be important in the initiation of materno-fetal tolerance at an autocrine level. (supported by mrc). aims: -defensins are small cationic peptides with antibiotic and antimicotic activity. hyaluronan and its degradation products have been described as endogenous ligands for tlr and tlr , whose involvement in -defensin expression has been reported in different epithelial tissues and cell lines. we aim to investigate weather low molecular weight hyaluronic acid induces -defensin release by keratinocytes, via tlr and tlr . methods: the induction of -defensin production following in vitro treatment of human keratinocytes with a low molecular weight hyaluronic acid solution was evaluated by pcr-analysis and elisa techniques. studies on the involvement of tlr and tlr in -defensin production have been performed using specific blocking antibodies. results: pcr and elisa revealed an intense -defensin production following hyaluronic acid treatment in human keratinocytes. the -defensin production induced by hyaluronan was abolished following block of tlr and tlr by specific antibodies, demonstrating the involvement of these receptors. the same hyaluronic acid treatment did not induce activation of inflammatory genes, such as il- , tnf-, il- and il- . conclusion: our data show that hyaluronic acid is an efficient inducer ofdefensin production in keratinocytes, via tlr and tlr . this observation might be important to open new perspectives in the development of possible topical products containing hyaluronic acid, to improve the release ofdefensins by keratinocytes, ameliorating the self-defence of the skin in case of skin infections. therefore, one of the possible applications for this kind of topical products might be the treatment of infective vulvitis, one of the most distressing gynaecological diseases for adult women. pregnancy outcome? kiera von besser, serena wu, mary d stephenson. , obstetrics and gynecology, university of chicago, chicago, il, usa; obstetrics and gynaecology, university of british columbia, vancouver, bc, canada. objective: to investigate whether gender of an ongoing pregnancy impacts the probability of a successful outcome, and, to ascertain whether the gender of prior live birth(s) impacts subsequent pregnancy outcome, among women with rm/aps. materials and method: cohort-control study. rm subjects were evaluated by mds between - (stephenson, . couples who met aps criteria (wilson et al, ) , restricted to rm only, were followed prospectively. cohort data was compared to live birth data from the vital statistics agency of british columbia from - . secondary sex ratios (ssrs) among successful pregnancy outcomes were calculated by dividing the number of male live births by female. sex ratios were calculated for all pregnancies weeks, regardless if they ended in success or demise. pearsons test with yates continuity correction was applied. results: subjects were identified. subjects had prior live births of known gender ( male/ female), giving a ssr of . . there were also prior fetal demises wks of known gender ( / ) giving a sex ratio for all prior pregnancies at wks of . . subjects delivered subsequent live births of known gender ( / ), giving a ssr of . . there were also subsequent fetal demises ( / ) giving a sex ratio for all subsequent pregnancies of . . subjects delivered both prior and subsequent live births. the ssr was . ( / ) among their prior and . ( / ) among their subsequent live births. including fetal demises, subjects had ongoing prior and subsequent pregnancies. the sex ratio was . among their prior pregnancies and . among their subsequent. as the control, a ssr of . ( , / , ) was calculated from vital statistics data. when the prior and subsequent ssrs of the cohort were compared to each other, as well as to the control, there were no statistically significant differences. conclusions: our findings, from the largest study of its kind to date, suggest that, in patients with rm/aps, the gender of an ongoing pregnancy does not significantly affect the probability of a successful outcome, to any greater degree than it does in the general population. also, the gender of a prior ongoing pregnancy does not significantly impact the likelihood of developing rm/aps. oocyte maturation. jk friend, fb bezirci, e seli. ob gyn, yale u., new haven, ct, usa. introduction: oocyte maturation is associated with repression of transcription. during oocyte maturation, fertilization, and early embryo development until the onset of zygotic gene expression, proteins are synthesized from maternallyderived mrnas. the regulation of protein expression from these maternal mrnas is post-transcriptional, and occurs mainly via poly(a)-tail elongation. the embryonic poly(a)-binding protein (epab) is the predominant poly(a)binding protein before the activation of the zygotic genome, and plays a critical role in the activation of certain maternal mrnas, those bound by cpeb and probably pumilio. we are characterizing additional functions of epab during the process of oocyte maturation. methods and results: our model system is the xenopus laevis oocyte where oocyte maturation is induced by the addition of progesterone. our preliminary findings indicate that epab is phosphorylated, and that levels of phosphorylated epab increase upon progesterone-induced oocyte maturation. moreover, glycerol gradient centrifugation revealed that nonphosphorylated and phosphorylated epab are contained in distinct complexes that change mobility upon oocyte maturation. furthermore, oligo-dt selection for poly(a)-containing mrnas strongly suggests that these mrnas are bound exclusively by phosphorylated epab. using affinity purification, we have determined that nonphosphorylated epab exists primarily in a large protein complex prior to oocyte maturation that is later disassembled after the addition of progesterone. conclusions: based on these preliminary findings, we conclude that prior to oocyte maturation, the bulk of epab is nonphosphorylated and is found in a protein-rich complex separate from poly(a)-containing mrnas. upon oocyte maturation (when certain maternally-derived mrnas are activated for translation), the majority of epab becomes phosphorylated, and this phosphorylated form of epab is likely bound to translationally-active mrnas. we are currently investigating what kinase phosphorylates epab and whether this phosphorylation plays a role in translational up-regulation of epab-bound mrnas. introduction: reactive oxygen species (ros) play important roles in all aspects of cellular fate. nadph oxidase isoforms, a family of genetically preserved enzyme complexes, have been shown to be the main sources of ros in various cell types. however, the role of nadph oxidase isoforms in human myometrium proliferation and differentiation has not been defined. in the myometrium, different smooth muscle phenotypes maybe associated with specific physiologic functions. we have shown that angiotensin ii (angii) stimulates hypertrophy but not cell proliferation in ultr cells, an in vitro model of human myometrium. ultr cells at greater than passages display a replicative senescent phenotype. by introducing human telomerase reverse transcriptase (htert) gene, we have obtained a stable cell line (ultr-ht) which has a significantly increased division rate and distinct cellular morphology than the original ultr cells. objective: to determine the relationship of expression of nadph oxidase to ultr cellular fate. methods: early and late passages (p - ) of ultr and ultr-ht (p - ) cells were grown on either plastic or collagen iv (cn )-coated surfaces. ultr-ht cells were further stimulated with angii ( . um) for hrs. expression of nadph oxidase core (nox - and duox / ) and associated subunits (p phox, p phox, noxo , p phox, noxa , p phox, and rac / ), and angii receptors at / was identified by rt-pcr from cellular total rna. fluorescent immunohistochemistry (ihc) was employed to determine protein expression and localization. results: the mrna level of house keeping gene -actin was unchanged by any cellular manipulation. the senescent phenotype of ultr cells was accompanied by an apparent down-regulation of nox , p phox, and noxa genes, and an up-regulation of at / . overexpression of htert did not reverse nox , p phox and noxa expression while cell division rate was increased. however, there was a down-regulation of nox , at / and rac . plating ultr-ht cells on cn induced nox / down-regulation and up-regulation of duox / , with no apparent change of at / . however, exposure to cn re-directed cellular response to angii such that only nox was induced by angii stimulation. conclusion: expression of nadph oxidase isoforms nox , , , and duox / are correlated with ultr cell differentiation and cell fate control. data also suggests that ang ii-induced myometrial hypertrophy involves nox mediated ros generation. fluids from reproductive women -the influence of aging. eriko y fujii, masahiro nakayama. women's health, national center for child health and development, tokyo, japan; aska reproductive clinic, nara, japan. [introduction] receptor for advanced glycation end products (rage) is a multiligand type glycoprotein, and is characterized based on its ability to bind ages, adducts formed by non-enzymatic glycation and oxidation of protein and lipids. this process occurs during normal course of aging. ages/rage interaction regulates various physiological function, such as inflammation, angiogenesis through vegf inducement. a soluble form of rage (srage) works as a decoy in the body and inhibits intracellular signaling. [objectives] the balance of these factors may contribute to reproductive dysfunction by aging. we aimed to measure the ages, srage and vegf concentrations in plasma and follicular fluids from reproductive women, and examined the differences of those factors between young group and old group. [material and methods] patients' plasma and follicular fluid were collected with consent based on regulations of the ethical committee, and we measured ages (pentosidine, cml), srage and vegf in duplicate using commercially available elisa kits (fushimi co, r d and cyelex). concentrations were calculated from each standard curve, and compared between the young group under years old, and the old group over y.o. data were evaluated for the difference in two groups by student's t test , and the significance was determined by p< . . [results] ) srage in plasma, ± pg/ml (mean±sd), n= in the young group was significantly higher than ± pg/ml, n= in the old group. there was no significant difference in plasma vegf. ) vegf in follicular fluid was ± pg /mg protein, n= in the young, and ± pg /mg protein, n= in the old was increased significantly. ) we could not see statistical difference of pentosidine nor cml concentrations between two groups in plasma and follicular fluid samples. [conclusions] it has been reported that higher concentration of vegf in follicular fluid may relate to worse pregnancy rate in art. there was a significant decrease of plasma srage in older group in our result, and because of this decrease of 'decoy', focal ages-rage-vegf signaling might be activated in older women. our results showed the possibility that ages/rage and vegf regulation may contribute to the reproductive dysfunction by aging. and leiomyosarcoma cells. qun pan, xiaoping luo, nasser chegini. ob/ gyn, university of florida, gainesville, fl, usa. as a part of a novel endogenous rna silencing machinery, a noncoding short rna strand referred to as "microrna" (mirna) has been identified to regulate the stability of the target gene expression through mrna degradation and repression. we have identified the expression of many of these mirnas in leiomyoma, myometrium, their isolated smooth muscle cells (lsmc and msmc), transformed lsmc (t-lsmc) and sklm (leiomyosarcoma cell line), including mir- which is predicted to target the expression of many genes, including tgf-b and tgf-b type ii receptor (tgf-brii). however, the biological significance of these mirnas in various cellular processes remains to be established. as such in the present study we examined the expression, regulation and function of mir- in lsmc, msmc, t-lsmc and sklm. we found that mir- is expressed and regulated by b-estridiol and medroxyprogesterone acetate ( - m) in these cells (p< . ). we further assessed the regulatory function of gain of and loss of function of mir- on the expression of tgf-brii. transfection of lsmc, msmc, t-lsmc and sklm with pre-mir-and anti-mir- oligonueclotides resulted in a significant increase and/or inhibition of mir- expression in these cells, respectively as determined by realtime pcr (p< . ). over-expression of mir- resulted in a significant reduction, while transfection with antimir- increased the expression of tgfbrii mrna and protein in these cells as compared to controls (p< . ). we concluded that mir- is expressed in leiomyoma and myometrial cells, its expression is regulated by the ovarian steroids and it functions by targeting the expression of tgfbrii and possibly other genes with key regulatory action on cell growth, angiogenesis, transcription regulation, ecm turnover and apoptosis that results in leiomyomas growth and regression. (supported by nih grant hd ). objective: placenta and a number of gestational tissues are well recognized to express corticotrophin releasing factor (crf), urocortin (ucn ), ucn , ucn and crf -r and crf-r receptor subtypes together with crfbinding proteins locally. ucn and ucn are implicated in the reversal of stress responses initiated by crf. in the present investigation, we evaluated functions of crf and ucns by quantifying their contents in venous smooth muscle layers using image pro . in human umbilical cords collected at preterm and term gestation methods: umbilical cord specimens ( - mm thickness, pieces per umbilical cord) collected at preterm and term (n= each) at delivery were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analyses with polyclonal antibodies to crf ( : ), ucn ( : ), ucn ( : ) and ucn ( : ) (peninsula laboratory, pa and sigma aldridge, ms) by standard abc technique. immunoreactive materials on the sections were identified using , '-diaminobenzidine as a chromagen. immunostaining intensities (od/area) on uc-sections were quantified by image pro . software and expressed as arbitrary units (au). all values were expressed as mean ± sem. differences between groups were evaluated by anova, followed by the post-hoc tukey test for multiple comparison. results: antibodies to crf and ucns elicited positive immunostaining of variable intensity in venous smooth muscle layers in uc-sections of preterm and term gestations. immunostaining intensity (au) of venous smooth muscle layers at preterm (pt) and term (t) are as follows: crf-pt= . ± . vs crf-t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p=ns); ucn -pt= . ± . vs . ± . (p< . ). conclusion: crf content in venous smooth muscle layer markedly increased at term, while ucn and ucn contents significantly decreased and no significant change occurred in ucn content. based on the opposing changes in crf vs ucn and ucn immunostaining, we speculate that crf, but not ucn or ucn , is the major key player of vasodilation and function locally at the level of venous smooth muscle cells at term. ucn and ucn may perform cytoprotective functions at preterm. physiol. ; : - , am j physiol. ; : - ) . these studies showed that when ad libitum (al) feeding was resumed in cr females, fertility was sustained well beyond the age at which al-fed females became infertile. however, much of what is known on the effects of cr on fertility derives from models in which cr was initiated at weaning. further, there is large variation in how the experiments were conducted. objective: herein we tested if adult-onset cr could delay age-related infertility in females. methods: cr ( %, nia protocol as described in j geront. a:b -b ) was initiated in c bl/ female mice at months and continued until . months of age, at which time al feeding was resumed. matings were initiated at months of age. for mating during cr, a male mouse was housed overnight in a cage with a female and removed the next morning, so that the female mice could be fed their dietary food ration. al-fed and cr females followed the same mating regimen. the number of offspring born and that survived to weaning (day ) were recorded. results: fertility was lost in of al-fed females by months of age and continued to decline through . months of age. age-matched females maintained on cr during the same period exhibited poor fertility, with a total of pregnancies achieved out of females. although cr females showed poor fertility while on cr, their fertility improved dramatically after the reinitiation of al feeding at . months of age. while only out of , al-fed females achieved a pregnancy between . - . months of age, out of age-matched cr-then-al-fed females achieved a total of pregnancies in this -month time. notably, % of the pups born to cr-then-al-fed females between . - . months of age survived and developed to weaning without complications. conclusions: adult-onset cr allows maintenance of female fertility into advanced maternal age after the reinitiation of al feeding. how long fertility can be maintained and the minimum time needed for the beneficial effects of cr to be realized remain to be investigated. nonetheless, these observations suggest that there may be ways to safely extend fertility in females at ages when reproductive function is suboptimal (nih r -ag ). bile acids in human ovarian follicular fluid. laura p smith, , kaila deiorio-haggar, jason reindollar, alan s penzias, , anny usheva-simidjiyska. ob/gyn reproductive endocrinology infertility, bidmc, boston, ma, usa; boston ivf, boston, ma, usa; endocrinology, bidmc, boston, ma, usa. introduction: bile acids are known to serve important functions in the hepatobiliary and gastrointestinal systems. the presence of bile acids in the human ovary and relation with fertility potential have never been previously evaluated. methods: human follicular fluid (ff) from large follicles and small follicles was obtained at vaginal oocyte retrieval. human serum was obtained before and hours after human chorionic gonadotropin (h-cg). follicular fluid and serum samples were analyzed for total bile acids by spectrophotometry. bile acid concentrations were correlated with age, number of retrieved and mature oocytes, number of fertilized oocytes, and pregnancy. results: bile acid concentrations were analyzed and compared to the normal human serum bile acid concentration which ranges from to micromoles/l. bile acids are present in follicular fluid with a mean concentration of . micromoles/l in large follicles and . micromoles/l in small follicles (p= . ). pre and post h-cg serum bile acid concentrations differed significantly ( . micromoles/l vs. . micromoles/l, p= . ). there was a trend toward higher bile acid concentration in large follicles of young patients < years old compared to older patients years old ( . ± . vs. . ± . , p= . ). there was also a trend toward higher pre h-cg serum bile acid concentrations in older patients ( . ± . vs. . ± . , p= . ). there was no correlation between serum and follicular fluid bile acid concentrations and number of oocytes retrieved or number of mature oocytes, but there did appear to be a positive correlation between pre h-cg serum bile acid concentration and number of fertilized oocytes (spearman's correlation coefficient . , p= . ). conclusions: this is the first demonstration of the presence of bile acids within human ovarian follicular fluid. there may be a relationship between bile acid concentration and fertility potential. the precise function of bile acids in human ovarian follicular fluid is under investigation. objective to investigate the impact of ovarian hyperstimulation treatment on the biomarkers of the homocysteine pathway in blood and follicular fluid, and their association with the follicle diameter as a measure of follicular maturation. methods in women undergoing ivf/icsi treatment blood samples were collected on cycle day and the day of hcg administration. during oocyte retrieval in each woman the diameter of two follicles was measured and the corresponding follicular fluids were collected. in blood and follicular fluid total homocysteine (thcy), folate, cobalamin and pyridoxal' 'phosphate (plp) concentrations were determined. women with a serum folate . nmol/l were classified as folic acid supplemented. results ovarian hyperstimulation significantly decreased thcy and cobalamin blood levels (both p . ). the blood and follicular fluid concentrations of thcy, folate, cobalamin and plp were significantly correlated (all p . ). in the total group, a two-fold increase of thcy in follicular fluid resulted in a . mm decrease of the follicular diameter (p . ). in non-supplemented women this decrease was . mm (p . ). in supplemented women a twofold increase of follicular fluid folate resulted in a . mm decrease of the follicular diameter (p . ). conclusions ovarian hyperstimulation reduces thcy blood levels independent of folic acid supplementation. however, high follicular fluid thcy and folic acid supplementation may have detrimental effects on the maturation of the follicle. ), post-fixed for rain in % w/v osmium tetroxide in the same buffer, quickly dehydrated in a series of ethanol solutions, and embedded in epon. thin sections were stained with uranyl acetate followed by lead citrate and were observed at electron microscope. results: ten blastocysts from each group were collected and analyzed. compared to the in vivo embryos, blastocysts generated in vitro exhibited significant differences. the surface of their trophectoderm (te) layer had a reduced number of microvilli, the number of the apoptotic cells in the inner cell mass (icm) was higher and the presence of non functional mitochondria was elevated. in this study we have, for the first time, compared the ultrastructure of the in vivo and in vitro conceived blastocysts. taken together, these results suggest that both, the higher rate of apoptosis and the morphological alterations in the mitochondrial structure in ivf embryos, are associated with stress during in vitro embryo culture. therefore, these parameters can be used, in the future, as markers for the assessment of the embryo wellbeing in the ivf settings. deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age.here, we explore a newly discovered role of nitric oxide (no) in the sustenance of oocyte quality. methods: sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (icsi) with cauda-epididymal spermatozoa following exposure to either the no donor, s-nitroso n-acetyl penicillamine (snap, . m/min); a soluble guanylyl cyclase inhibitor, h-[ , , ] oxadiazolo [ , -a] quinoxalin- -one (odq, m) or an no synthase inhibitor, n w -nitro-l-arginine methyl ester (l-name, mm). their sibling oocytes were subjected to icsi either before (young) or after culture for the corresponding period (old). outcomes of fertilization, cleavage and development to the morula and blastocyst stages were compared. some embryos from each subgroup were also subjected to tunel assay for apoptosis. results: oocytes deteriorated in their ability to undergo normal fertilization and development to morulae/blastocysts after aging in culture, as compared to their sibling cohorts subjected to icsi immediately after ovulation (p< . ). this deterioration was prevented after oocyte exposure to snap. while, exposure to l-name or odq resulted in a significant compromise in fertilization and development to the morulae/blastocysts (p< . ) with detection of apoptosis, which was also noted in embryos derived from aged oocytes but not in those from young or snap exposed oocytes. conclusions: no is essential to sustain oocyte fertilizability and developmental ability, and to prevent blastomere apoptosis. objective to investigate the effects of the levels of the biomarkers of the homocysteine pathway on ivf outcome. methods from women undergoing an ivf or icsi procedures, two blood samples and two mono follicular fluid samples were collected for determination of folate, cobalamin, and total homocysteine (thcy). total protein was determined in follicular fluid to adjust the biomarker concentrations for follicular maturation. primary endpoint of the study was oocyte quality measured by fertilization and embryo quality (range - ; with being best quality). secondary endpoint was the occurrence of pregnancy. results % of the included women used a folic acid supplement (serum folate . nmol/l). in non-supplemented women higher cobalamin levels in follicular fluid correlated with a better embryo quality (estimate - . ; p . ) and higher thcy levels (median . mol/l, range . - . ) correlated with a worse embryo quality (estimate . ; p . ). in supplemented women higher follicular fluid thcy (median . mol/l, range . - . ) correlated with better embryo quality (estimate - . ; p . ). the follicular fluid folate level of oocytes that did not fertilize was . -fold higher than in the fluids of a fertilized oocyte ( % ci . - . ; p . ). a two-fold increase of follicular folate corresponded with a . higher chance to achieve pregnancy ( % ci . - . ). conclusions cobalamin levels in follicular fluid are correlated with embryo quality. folic acid supplementation modifies the thcy and folate levels in follicular fluid and thereby affects oocyte quality. the level of folate in follicular fluid is important in the fertilization process. we recently reported that increasing relative oocyte immaturity is associated with worsening outcome, and that cycles with many immature oocytes are more common in younger women. (moore et al, asrm annual meeting, ) to further investigate this trend, we conducted a case/control analysis of patients with repeated cycles of high-level oocyte immaturity (hloi). methods: oocyte maturity data was collected on all icsi cycles starting in . we defined a cycle with hloi as having > % immature oocytes (> sd's above the median). a case was defined as a patient with hloi on more than one cycle. control subjects were age-matched and defined as having </= % ( sd above median) immature oocytes on all cycles. results: from subjects, we identified cases of recurrent hloi (comprising icsi cycles) and control subjects. at baseline, cases had more oocytes retrieved per cycle than controls ( . vs. . , p<. ). all other baseline characteristics were similar. all case subjects were younger than . outcomes are shown in table . discussion: clinical pregnancy and live births were reduced in the case group, but more than expected based on % immaturity. during cycles, there were no live births among the case group. patients with recurrent hloi represent a previously undescribed group of young patients with a very poor prognosis during icsi treatment. this study may suggest that the problem underlying the immaturity may relate to overproduction of oocytes during stimulation, a factor which may be modifiable. however, the additional trend (though not significant) that even the mature oocytes from the case patients tend to have poor outcomes suggests the possibility of a maturation defect and warrants further consideration. data on response to gonadotropins, fert rates, and implantation rates will be presented. amongst those, were performed for an initial diagnosis of premature ovarian aging (poa), for the diagnosis of premature ovarian failure, and for repeated pregnancy loss. in women under age , b-fsh levels over miu/ml and up to < miu/ml denoted a diagnosis of poa, while levels of miu/ml were considered diagnostic for pof. all patients signed consent permitting review of medical records for clinical research purposes. results: day fsh levels ranged from . to . miu/ml (mean . ± . miu/ml). amh levels ranged from < . to . ng/ml (mean . ± . ) and allele repeats ranged from to , (mean for allele- , . ± . ; allele- , . ± . ). mixed model analysis of variance for all patients, adjusted for age, race, and allele- revealed a statistically significant correlation between b-fsh levels and number of repeats in allele- ( figure ; p < . ). we did not observe a significant linear relationship between serum amh levels and cgg repeat counts, however cgg repeat counts above were significantly associated with serum amh levels less than ng/ml (chi square = . ). conclusions: triple repeats above , a level currently considered well within normal, are associated with evidence of premature decline in ovarian function. the evaluation of triple repeats in frm gene offers a first objective assessment of ovarian function and should be predictive of autologous reproductive life span in most women. objective: the levels of mis in the serum and in the ovary have been demonstrated to decrease in response to increasing doses of cisplatin. we hypothesize that ovarian stimulation also will reveal dose related decreases in ovarian and serum mis levels after cisplatin exposure, further demonstrating the follicular damage from chemotherapy. methods: adult female sprague-dawley rats were treated with saline, . mg/kg cisplatin or . mg/kg cisplatin as two weekly injections. five days following the last cisplatin injection, the rats were injected with pregnant mare serum gonadotropin (pmsg) and euthanized hours following the pmsg injection. serum was collected and both ovaries were obtained for protein analysis. serum and ovarian levels of mis were determined using an elisa kit. western blot analysis, immunohistochemical analysis, and tunel analysis were also performed on the ovarian proteins. results: stimulated mis levels declined in a linear fashion in response to increasing cisplatin doses (p< . ). ovarian protein levels measured by elisa and western blot both indicated that stimulated ovarian mis levels also decrease in a linear fashion (p= . and p< . respectively.) immunohistochemical analysis revealed that while the total number of follicles remains the same, both the total number of mis positive follicles, and the percentage of mis positive follicles declines in a linear fashion (p= . for both). tunel analysis reveals that there is no change in the incidence of apoptosis in the stimulated ovaries from any treatment group. conclusions: the administration of pmsg after treatment with cisplatin causes a dose dependent decrease in the levels of mis in both the serum and in the ovary. serum mis levels following ovarian stimulation may be useful as a serum biomarker in this animal model to assess ovarian damage following the administration of cisplatin. correlations of micro-rnas a, b, - pp, and - p with inflammatory and steroidogenic factors in human granulosa/cumulus cells. tannaz toloubeydokhti, orhan bukulmez, kenneth c drury, r stan williams, nasser chegini. obstetrics and gynecology, university of florida college of medicine, gaineville, fl, usa. as part of the novel endogenous rna silencing machinery, noncoding short rna strands, referred to as micrornas (mirnas), have been identified to regulate the stability of the target gene expression through degradation and repression. the expression of many of these mirnas has been identified in human tissues however, their biological significance in various cellular processes remains to be elucidated. in this cross-sectional study of human granulosa cells, we aimed to study the potential correlations between five selected mirnas, mir- a, mir- b, mir- - p, mir- and mir- - p, and mrna expressions of some of their predicted target genes namely, cyclooxygenase (cox)- , il- , steroidogenic acute regulatory protein (star), aromatase and estrogen receptor (er) . granulosa/cumulus cells were obtained from women (age range - ; mean age . ± . ) undergoing oocyte retrieval for assisted reproduction. total rna was extracted from these cells and reversed-transcribed and real-time pcr was performed to measure steady-state levels of mirnas and mrna transcripts. we observed that the expression of mir- a, mir- - p, mir- and mir- - p displayed a significant and positive correlation with the expression of cox- . moreover, mir- b significantly correlated with star mrna expression, but not with other genes tested (p< . ). with advancing age, the expression of mir- - p and cox- was significantly decreased, with cox- expression displaying a positive correlation with aromatase, star and er mrna expressions (p< . ). none of the factors tested showed any significant correlations with peak estradiol levels and number of oocytes retrieved. receiver operating characteristic curve analysis revealed that female age cut-off value of . y best predicted whether mir- - p was below its mean arbitrary value. in conclusion, given the importance of mirnas' regulatory function in gene expression, our results suggest that mir- b may play an important role in gene expression related to granulosa cell luteinization, while variation in mir- - p expression with female age may suggest its potential role as a predictor of oocyte quality and granulosa cell function. given the importance of mirnas in gene regulation, the role of mirnas in ovarian physiology and aging requires further investigation. support: nih grant . introduction: a symbiotic relationship between ovarian granulosa cells (gc) and the enclosed developing oocyte is critical to reproductive efficiency. genetic modulations in gc's can lead to reproductive insufficiency, highlighting the critical role of gc's in reproductive competence. defects in the oocyte-gc repertoire in women with dor include lower e levels, luteal deficiency, poor fertilization rates, higher incidence of aneuploidy, lower pregnancy and higher miscarriage rates. utilizing global gene expression analyses in cumulus gc's, we attempt to enhance our understanding of biological mechanisms that may contribute to poor reproductive capacity in young women with dor. methods: infertile women (< years old) undergoing ivf were prospectively enrolled into two groups based on ovarian reserve (normal reserve, fsh< dor, fsh > miu/ml). at egg retrieval, cumulus gc's were isolated, rna extracted transcribed into cdna. microarray targets were generated cdna hybridized to affymetrix human genome u plus . genechips. microarray data were analyzed (array assist) and normalized (robust multichip analysis). a difference in gene expression of > fold was considered biologically relevant. results: of the , genes identified to be differentially expressed in young women with dor compared to normal reserve, genes demonstrated consistency of expression across five different normalization schema; were down regulated and up-regulated. expression of gremlin, a member of the dan family of genes known for its highly regulated expression pattern during folliculogenesis, was noted to be down-regulated -fold over two probe sets (- . ) in women with dor versus normal reserve; this down-regulation was confirmed by real-time pcr (- . ) . conclusions: this is the first demonstration linking differential expression of gremlin with etiology of infertility in women. gremlin is a downstream effector of oocyte-derived gdf- which facilitates cumulus cell expansion, a critical event in reproductive physiology. our finding of a significant downregulation of gremlin expression in cumulus gc's associated with dor may partly explain the physiology of poor reproductive performance. nih k cd . nih k rr . ferring pharmaceuticals. the objective of the present study was to investigate the effects of the gnrh antagonist ca on expression of mis and aromatase (cyp ) using luteinized human granulosa cells obtained during in-vitro fertilization cycles and an immortalized human granulosa cell line (hgl ). the granulosa cells were cultured +/-dibutyryl camp ( mm) and incubated +/-the gnrh antagonist, ca, for - h. rna was isolated, reversed-transcribed and real-time pcr was performed to measure mrna transcripts for ovary-specific cypiia and mis. we observed that camp markedly induced aromatase mrna in both the primary and immortalized human granulosa cells. interestingly, camp treatment of these cells also caused an upregulation of mis mrna. objectives: bisphenol a (bpa), a known endocrine disruptor, is a chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins and is present in multitude products, including the interior coating of food cans, milk containers, and baby formula bottles. bpa can leach into foods during heat processing and is known to exert a variety of endocrine-like effects on different cell types acting as an estrogen because it contains two hydroxyl groups in its diphenyl structure. in this study we focused on the effects of bpa on aromatase expression and estradiol production in the human granulosa kgn cell line. we also evaluated its effects on several transcription factors crucial in cyp expression. materials and methods: kgn cells were cultured in f- dmem and were starved for h before experiments. subsequently they were treated for h with vehicle (control), fsh ( ng/ml), and/or bpa ( , , , , um) . messenger rna expression was quantified by real time pcr and estradiol secrection was measured in supernatants by elisa. results: fsh induced a -fold increase in aromatase expression. bpa induced a dose-dependent decrease in cyp production, with the greatest effect at um (p< . ), resulting in +/- % (mean+/-sem) inhibition, compared to aromatase expression induced by fsh alone. bpa also reduced levels of estradiol secretion in a dose-dependent manner, with the greatest inhibition at um (p< . ) resulting in +/- % decrease. we also evaluated expression of transcription factors known to be involved in regulating the activity of the ovary-specific aromatase proximal pii promoter. interestingly factors known for induction of aromatase such us steroidogenic factor- and gata- , mimic the pattern of cyp expression after bpa treatment, whereas, other receptors previously reported to act as aromatase inhibitor, such as ppar gamma were up-regulated by the addition of bpa. moreover, expression of creb remained virtually intact, suggesting that most likely mechanisms governing endocrine disruption by bpa are highly selective. conclusions: bpa inhibited fsh stimulated aromatase expression and downstream estradiol secretion. we propose that constant exposure to this chemical may result in endocrine disruption which may contribute to reduced fertility and early ovarian senescence. oocyte maturation occurs during folliculogenesis as a result of complex cell-tocell communications between the granulosa cells and the oocyte. maintaining the granulosa cells' spherical structure and network of gap junctions surrounding the oocyte is critical. this project tests the ability of a novel substrate-free threedimensional culture system to form viable granulosa cell spheroids. methods: after irb approval, freshly obtained follicular fluid from in vitro fertilization was obtained and granulosa cells were purified by percol gradient. nonadhesive agarose hydrogels, containing cylindrical round bottom recesses m in diameter, were cast from micro-molds designed using computer-assisted rapid prototyping. granulosa cells seeded at a density of , cells per gel were incubated for up to days. cellular viability was assessed with live:dead assay. results: after three days in culture, granulosa cells formed spheroids of densely packed cells that were difficult to disperse with multiple pipettings. the cells remained viable for at least days. conclusions: granulosa cells can be cultured in a novel substrate-free threedimensional culture system. the cells form tightly adherent spheroids that remain viable for extended culture. the cohesiveness of the cells suggests the formation of gap junctions. this is under investigation with immunohistochemistry and electron microscopy. these experiments suggest that a substrate-free threedimensional hydrogel culture system may be ideal to reassemble follicular structure important for future in vitro evidence testing and oocyte maturation. known to function as responders to pathogens, inflammation, and tissue injury. previous studies in our laboratory demonstrated that saa was produced in mouse granulosa and production was regulated by cytokines. ovulation has long been considered an inflammatory reaction and patients with chronic inflammatory conditions often experience infertility. the present study was undertaken to explore the role of saa in human ovarian function. methods: ovarian granulosa and luteal cells were obtained from surgically removed specimens and mural and cumulus granulosa-luteal cells were obtained from ivf aspirates. rna was extracted from fresh or cultured cells. some cells were treated in vitro with tnf or other cytokines for h. expression of saa was assessed by quantitative rt-pcr. in addition, serum levels of saa were determined using a commercial elisa in women undergoing ovulation induction (oi) and art. saa levels were measured at baseline, during oi, on the day of hcg administration and at the time of the pregnancy test. results: saa mrna was expressed in theca, granulosa, and granulosa-luteal cells. in granulosa-luteal cells both saa and saa mrnas were expressed at higher levels in cumulus compared to mural granulosa. expression of saa and saa in theca was increased following treatment with tnf in vitro. serum levels indicated that patients with ovulatory dysfunction had increased levels of saa at the time of hcg injection while patients without ovulatory dysfunction had lower saa levels as compared to the baseline level. in addition, patients undergoing oi who achieved pregnancy exhibited increased levels of saa at the time of the pregnancy test compared to baseline levels, whereas patients who did not become pregnant had lower post-cycle levels of saa. interestingly, saa levels did not change in art patients that became pregnant without undergoing oi (donor egg or frozen embryo transfer) conclusions: human ovarian cells express saa mrnas which can be altered in vitro. serum levels of saa may correlate with the responsiveness of the ovary to gonadotropins as evidenced by altered levels in women with ovulatory dysfunction, and by an increase in pregnant patients following ovarian stimulation. yeh, beom su kim, felicia hercules, jennifer peresie, armando arroyo. gynecology-obstetrics, university at buffalo, buffalo, ny, usa. objective: cisplatin is a common chemotherapeutic agent given to women for treatment for a wide variety of cancers. we hypothesize that one mechanism by which cisplatin may cause damage to ovarian structures is by decreasing the amount of anti-oxidant activity in the ovary. we examined super-oxide dismutase (sod ), a critical anti-oxidant enzyme that has been shown to be affected by cisplatin in other tissues, in the ovaries of cisplatin treated animals. methods: adult female sprague dawley rats were injected with saline, cisplatin . mg/kg or cisplatin . mg/kg as weekly doses. five days following the last injection, the rats were euthanized and both ovaries were excised. one ovary was processed for immunohistochemistry and the other was processed for protein analysis using western blot techniques for sod . the anti-sod antibody was purchased from santa cruz. the immunohistochemical sections were scored using a semiquantitative h scoring method. results: immunohistochemistry analysis of the expression pattern of sod following cisplatin administration revealed that there was a significant linear decrease in a dose response pattern in the expression of sod in antral follicles and in corpora lutea (p< . for both). no change was found in the h score of sod in other ovarian structures. western blot analysis of sod in the ovaries following increasing doses of cisplatin revealed no changes in the overall protein levels of sod in the ovary. conclusions: this is the first report that administration of cisplatin causes changes in the expression pattern of sod in antral follicles and in copora lutea. cisplatin decreases the amount of sod available in these structures, possibly leading to increased oxidative stress and free radical damage, thereby leading to ovarian damage found after cisplatin administration. is there evidence for aromatase activity in the stroma of postmenopausal ovaries? mf landay, rh fogle, rb allen, s patel, fz stanczyk, rj paulson. ob/gyn, usc, keck school of medicine, los angeles, ca, usa. background: following menopause, the ovaries continue to secrete androgens and estrogens. we have recently confirmed the production of androstenedione, testosterone and estradiol (e ) up to ten years after menopause by measuring gradients from ovarian venous effluent and peripheral blood. anti-müllerian hormone (amh) and inhibin b have been shown to be markers of follicular activity. peripheral levels of these hormones have previously been found to be undetectable in menopause, suggesting the absence of follicular activity in the postmenopausal ovary. objective: to investigate if the postmenopausal ovary continues to demonstrate evidence of follicular activity as the source of steroid production. design: observational study materials and methods: eight subjects aged ± . yr (range - ) were enrolled. postmenopausal status was confirmed by preoperative fsh levels of more than u/l and/or amenorrhea greater than months. serum was collected from the ovarian veins during total abdominal hysterectomy and bilateral oophorectomy. peripheral blood was also collected pre-operatively, intraoperatively and postoperatively. all samples were analyzed for amh and inhibin b using elisas with sensitivities of . ng/ml and pg/ml, respectively. androgen and estrogen levels in these samples have previously been reported, and documented a gradient between ovarian venous effluent and peripheral serum in all cases. results: ) six patients had no detectable follicular activity by amh and inhibin b levels. ) one patient demonstrated detectable inhibin b levels with an -fold gradient between ovarian venous effluent ( pg/ml) and peripheral blood ( pg/ml), however no amh was detected. ) in one patient, aged and months postmenopause, both amh and inhibin b were detected. peripheral inhibin b levels were high at pg/ml. amh was detectable at levels of . ng/ml. conclusions: ) in the majority of patients, continued e and androgen production in the ovary occurs in the absence of follicular activity as detected by amh and inhibin b production. ) some patients have evidence of follicular function up to ten years after menopause. ) since e is produced in post-menopausal ovaries in the absence of follicular activity, these data provide evidence for aromatase activity in the stroma of post-menopausal ovaries. to elucidate the process by which prostaglandin f (pgf ) mediates luteal regression, this study examined the role that the transcription factor yin yang (yy ) and histone deacetylase (hdac ) play in altering luteal cholesterol uptake by the scavenger receptor class b type i (sr-bi), intracellular cholesterol transport by steroidogenic acute regulatory protein (star), and cholesterol processing by p side chain cleavage enzyme (p scc) expression. rat luteal cells ( days post-ovulation) were treated with pgf ( hr) and trichostatin a (tsa; hr), a potent hdac inhibitor. protein expression was measured post-treatment by western blot and cholesterol was localized via filipin staining. star and sr-bi promoter activation was also assessed in hek t cells treated with yy , myy , a deletion mutant lacking an essential region required for transcriptional repression, and tsa. pgf caused a significant -fold decline in star (p< . ), and smaller declines in sr-bi and p scc which occurred concomitantly with an increase in yy ( -fold, p< . ) and intracellular lipid staining ( % increase). in contrast, nm tsa treatment caused a dose dependent increase in sr-bi, star, and p scc protein levels, . -fold (p< . ), . -fold (p< . ), . -fold (p< . ), respectively, and a . -fold decline (p< . ) in intracellular lipid levels. tsa prevented the pgf -induced decline in sr-bi, star, and p scc expression. promoter analysis demonstrated that wildtype yy , but not myy , repressed sr-bi and star activation while the addition of tsa overcame the repressive effects. this study shows the critical role that yy plays in pgf induced luteal regression by recruiting a histone deacetylase to block three essential processes in steroid production. in luteal cells yy -mediated global repressive action prevents cholesterol metabolism by inhibiting cholesterol uptake, intracellular transport, and processing thus leading to functional and structural luteal demise. supported by nih hd and nih hl . regulation of intermedin expression in cycling rat ovary. madhu chauhan, rebekah elkins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd) is a ct/cgrp family peptide involved in a variety of physiological functions, including vasodilatation and fetoplacental growth. this peptide is expressed in a variety of tissues such as stomach, placenta, uterus, pituitary and ovary suggesting its different functions including in reproduction. imd gene has an estrogen response element and we have shown that the plasma concentration of immuno-reactive imd is elevated in rats with pregnancy. however, expression of imd in the ovary and its regulation during estrous cycle is not known. we hypothesize that imd is expressed in the ovary and its expression is hormonally regulated throughout the estrous cycle in rat. objective: ) to assess expression of imd mrna and its receptors components calcitonin receptor like receptor (crlr), and receptor activity modifying proteins, ramp and ramp mrna in rat ovary; on diestrus, proestrus and estrus stages of rat estrus cycle and ; ) to demonstrate immunohistolocalization of imd, crlr, ramp , ramp and ramp in rat ovary. methods: groups of sprague-dawley non-pregnant and pregnant rats on day of gestation were used in this study. non-pregnant rats were sacrificed on diestrus, proestrus and estrus stage. ovaries were collected and total rna was extracted using trizol method. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). immunohistolocalization of imd, ramp , ramp and ramp proteins were assessed in tissue sections of ovaries from pregnant rats sacrificed on day . results: ) imd mrna is regulated in rat estrus cycle and its expression is significantly downregulated in estrus stage compared to the diestrus and proestrus; )expression of imd receptor crlr is highest in the diestrus stage, followed by a decline in proestrus which further declined during estrus; ) expression ramp mrna is higher in proestrus compared to diestrus and estrus (p< . ) but there is no significant change in the ramp expression during the estrus cycle and ; ) imd, ramp , ramp and ramp are immunolocalized in rat ovary in granulose cells of all follicles and granulosa cells of the corpus luteum. conclusion: imd mrna and protein are expressed in rat ovary suggesting a possible role for imd in ovarian function. defining and defying deterioration in oocyte quality with advancing chronological age: role of nitric oxide. pt goud, ap goud, mp diamond, b gonik, hm abu-soud. div reprod endocrinology, dept ob/gyn, wayne state university, detroit, mi, usa; div maternal and fetal medicine, sinai grace hospital, wayne state university, detroit, mi, usa. nitric oxide (no) is a ubiquitous free radical essential for oocyte maturation, function and sustenance of oocyte quality post-ovulation. the current study investigates the role of no insufficiency in the causation of oocyte quality deterioration with advancing chronological age. methods: in set , oocytes were retrieved from the following superovulated b d f mice: (a) young breeders (yb, n= ); (b) retired breeders (rb, n= ), and © old animals (oa, n= ), aged - , - , and - weeks respectively; and studied for zona pellucida dissolution time (zpdt), spindle ( -tubulin fluorescence immunocytochemistry, sigma) and chromosome morphology (dapi, vector), ooplasmic microtubule (mt) dynamics (omd) in response to taxol [ ], and cortical granule (cg) status (rhodamine conjugated lectin, vector). in set (n= ), oocytes from retired breeders were studied after exposure in vitro to an no-donor, s-nitroso acetyl penicillamine (snap in m- , . m no/min , n= , h, °c, % co ), while their sibling control oocytes were cultured for corresponding period under identical conditions without snap. [ ]. zpdt, spindle and chromosome morphology, omd and cg status were assessed with a confocal microscope and compared between the subgroups using anova, chi square and/or fisher's exacts test and appropriate post-hoc tests. results: in set , a significantly fewer oocytes were retrieved per animal (mean) in rb ( ) and oa ( ) compared to yb ( . , p< . ). advancing age was also associated with a significant increase in zpdt, omd and cg loss in rb compared to yb (p< . ). furthermore, significantly fewer oocytes from rb than yb had normal spindle and chromosome morphology (p< . ). oocytes from oa had significant spindle and chromosome disarray, a near total cg loss and significantly harder zp (p< . ). these oocytes also exhibited diminished omd in response to taxol although, metaphases were exquisitely sensitive to disruption with taxol. in set , exposure to snap in oocytes from rb resulted in a significantly lowerzpdt, omd and cg loss, and significantly higher incidence of normal spindles ( the gamma-aminobutyric acid (gaba), its biosnthetic enzyme gad and gaba-a receptors have been found in the oviduct and ovary. objective: this study examined the expression of gaba-a receptor subunit genes and gad and whether the gaba-a receptor could alter cytosolic ca + in gc. methods: for qrt-pcr, both human cumulus and mural gc were obtained at the time of oocyte retrieval for ivf and cultured separately. total rna was isolated separately from each gc type and from human brain ( positive control). the total rna from patients was pooled for each gc type and all rna was treated with dnase i. two-step qrt -pcr was performed using gene-specific lux™ primers for all gaba-a receptor subunits, gad and gad plus gapdh. single and specific qrt-pcr products were verified by melting curve analysis, gel electrophoresis, and dna sequencing. for ca + study, gc were cultured on coverslips. gc were loaded with fura- -am and changes in ca + concentration of gc were studied using a dynamic digital ca + imaging system. gaba-a agonist, muscimol, was used to study any dose-dependent effects on gc. gaba-a antagonist, m bicuculline, were perfused min. prior to and during application of muscimol. the responses were represented as changes in the / nm fluorescence ratio over time. m atp was used as positive control. results: the qrt-pcr results indicated that all gaba-a receptor subunits were expressed to various degrees in both types of gc, with the expressed highest in both cell types. gaba-a receptor subunits showing the next highest expression in both cell types were , and . gad isoenzyme was more abundantly expressed in cumulus and mural gc than gad . ca + imaging showed that muscimol, had the ability to increase ca ++ in gc, about % gc (n= ) cells responded to muscimol. muscimol increased intracellular ca + in a dose-dependent manner. the muscimol responsive cells was reduced by bicuculline, from % to % (n= , p< . ). conclusion: qrt-pcr indicates that gaba-a receptor subunit gene and gad expression occurs in both cumulus and mural gc. the ability of bicuculline to inhibit the ca + response to muscimol suggests the activation of gaba-a receptor. the current study confirms the presence of functional gaba-a in gc for the first time, and suggests that gaba may exert trophic effects in the ovary via gaba-a receptor. the present study test the hypothesis that administration of l-nitro-l-arginine methyl ester (l-name), a nos inhibitor, prior to hypoxia prevents the hypoxiainduced activation of p mapk, erk and jnk in the cerebral cortex of the guinea pig fetus during gestation. to test this hypothesis guinea pig fetuses at and days gestation were assigned to normoxic (nx, n= ), hypoxic (hx, n= ) and hypoxic pretreated with nos inhibitor (hx+l-name, mg/kg i.p., n= ) groups. hypoxia in the fetuses was induced by exposing the pregnant guinea pigs at both gestational ages to an fio of . for min. cerebral tissue hypoxia was documented biochemically by determining the tissue levels of atp and phosphocreatine (pcr). neuronal nuclei were isolated, purified and proteins separated by sds-page, and then probed with specific phosporylated erk, jnk and p antibodies. in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx) . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx) . ± . (hx+l-name). the data show that administration of l-name prior to hypoxia decreased the expression of phosporylated p , erk and jnk at both gestation ages however expression of phosporylation was higher at term as compared to preterm. since a nos inhibitor prevented the hypoxia-induced phosphorylation of p , erk and jnk in both gestational ages, we conclude that the hypoxia-induced activation of p , erk and jnk in the cerebral cortical nuclei of preterm and term guinea pig fetus is no-mediated. we speculate that no-mediated modification of cysteine residue leading to inhibition of map kinase phosphatases results in increased activation of p , erk and jnk in the guinea pig fetus. (nih-hd , nih-hd and st. christophers foundation for children). the the endocannabinoid, anandamide (aea), is involved in the hormone-cytokine network that regulates implantation and early pregnancy maintenance with levels at the endometrial level considered a major checkpoint . high levels ( nm) in culture are associated with embryo death while plasma levels (> nm) at weeks in women undergoing ivf-et are associated with failed pregnancy . what is uncertain is whether systemic aea levels after spontaneous conception in women presenting with threatened miscarriage are predictive of pregnancy outcome. our aim was therefore to measure plasma aea levels in women presenting with threatened miscarriage and to relate these to outcomes. methods: plasma aea levels were measured using a sensitive and previously validated hplc-ms method at - weeks gestation in women (nonsmokers, bmi < kg/m ) presenting with threatened miscarriage and in whom a viable pregnancy was confirmed by ultrasound scan. results: nine of the women subsequently had a miscarriage. the plasma aea levels in those women who had live births was . -fold lower than that in those who subsequently miscarried ( . ± . nm versus . ± . nm, mean ± sem; respectively; p< . unpaired student's t-test). the roc analysis revealed an area under the curve of . ± . with a sensitivity of % ( %ci of . % to . %) and a specificity of . % ( % ci of . % to . %). using an aea level of . nm as the optimal cut-off point, a single plasma aea measurement provided a sensitivity of % and a specificity of % with a negative predictive value of % and a positive predictive value of % for subsequent miscarriage. conclusion: these findings suggest a possible predictive role for plasma aea in women presenting with threatened miscarriage. the data also indicate that systemic aea levels may reflect local endometrial levels and therefore the local hormonal milieu in the early stages of normal pregnancies and in those complicated by threatened miscarriage. introduction: follicle stimulating hormone (fsh) mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation. the ovarian response of different women to fsh is variable, ranging from poor response to ovarian hyperstimulation and has been partly attributed to two common variants of the fsh receptor (fshr). we have previously identified four abnormal fshr splicing products ( exon deletions and intron insertion) in women with low and high response to fsh. two of the splice variants, deletion of exon and deletion of exon , showed a correlation with low and high response to fsh, respectively. in the current study, we evaluated the functional competence of the mutant fshrs in vitro. methods: we established stable hek cell lines expressing wild-type (wt) and splice-variant (del) fshr under the control of the inducible tet on/off system. the cells were transfected with a camp-responsive luciferase reporter plasmid and stimulated with fsh. results: the subcellular distribution of all splicing variants was the same as the controls and the protein localized mainly on the cell surface. all four splicing variants showed markedly decreased camp activation compared to controls when stimulated with fsh. however, all variants were able to form functional heterodimers with the wt receptor when co-expressed. interestingly, the heterodimer containing the form of fshr lacking exon , found in patients with decreased response to fsh, resulted in lower intracellular camp compared with the wild-type homodimer. conclusion: our findings suggest that fshr variants can contribute to abnormal response to stimulation in certain women undergoing ivf treatment. the variants require the presence of wild-type receptor in order to initiate signaling in response to fsh. further analysis of this signaling cascade in granulosa cells is underway to estimate the final production of estrogen from these heterodimeric receptors. objective: to compare the proteome of human endometrium in the prereceptive versus the receptive phases of the menstrual cycle. m m: endometrial biopsies were collected and days after urinary lh surge in the same menstrual cycle from three fertile women (n= ). proteins were extracted using sample grinding kit (ge healthcare) and interfering substances removed using -d clean-up kit (ge healthcare). labelling was performed with cydye dige fluors (ge healthcare) and proteins were separated using difference gel electrophoresis (dige). for the isoelectric focusing, cm ipg-strips in the nonlinear range of ph - were used. the second dimension was carried out using sds-page. differentially expressed proteins were detected by image analysis using decyder v . and the statistical module eda (ge healthcare). the spots of interest were subject to protein identification based on in-gel digestion, maldi-tof/tof mass spectrometry and database searching. western blot analysis were performed in the same biopsies in order to validate some candidate proteins. results: table displays the differentially protein abundance between days lh+ and lh+ (> -fold change). of these proteins, were increased at lh+ in comparison with lh+ , whereas only proteins were decreased. stathmin was found more than -fold decrease in lh+ compared to lh+ in the three patients studied in the validation studies performed by western blot. conclusions: this study shows that the human endometrium has a differential protein repertoire during the window of implantation compared to the prereceptive phase. the role of these proteins in the molecular events directed to embryo implantation is under research. differential protein abundance in human endometrium ( the extracellular signal-regulated kinases and (erk / ) are a mitogen-activated protein kinase (mapk) subfamily that act as key links in eukaryotic intracellular signaling transduction. activated by phosphorylation in response to specific stimuli, erk / is known to play a role in the regulation of cellular proliferation and survival. the human myometrium is a tissue known to undergo cycle-dependent proliferative and apoptotic changes in response to sex steroids. we hypothesized that erk / activity is involved in mediating menstrual cycle-dependent changes in the myometrium. materials and methods: immunostaining for phospho-erk and total-erk was performed on myometrial tissues obtained from normal women (n= ) at different phases of the menstrual cycle. staining intensities were evaluated by hscore. myometrial smooth muscle cells were isolated and cultured from normal women and treated with vehicle, estrogen ( - m), and progesterone ( - m) for and minutes, and then subjected to western blot analysis for p-and t-erk. statistical analysis was performed using one-way anova. results: tissue staining revealed that p-erk was mostly nuclear in all tissues, while t-erk was cytoplasmic and nuclear. p-erk staining was significantly stronger in the secretory phase and strongest at the early secretory phase, compared to other phases (p< . ). t-erk staining intensity was moderatehigh without variation across the menstrual cycle. in cultured myometrial cells, progesterone significantly increased p-erk levels within and minutes (p< . ) when compared to control. our results suggest that erk activity in the human myometrium is regulated in a menstrual cycle-dependent manner. the increased phosphorylation in the secretory phase suggests the involvement of progesterone in erk activation, as supported by our in vitro results. this increased erk activity may play a role in regulating myometrial proliferation. measures of insulin resistance (ir) including: fasting serum insulin, gir and homa. measures of plasma levels of endothelin- (et- ), soluble intercellular adhesion molecule- (sicam- ), soluble vascular cell adhesion molecule- (svcam- ) and high sensitivity c-reactive proteins (hscrp). results: women with pcos exhibited significantly higher levels of et- (p < . ), sicamp- (p < . ), svcam- (p < . ) and hscrp (p < . ) as compared with age-matched controls, respectively. positive correlations were evident between et- and fai (r = . ; p < . ) but et- negatively correlated with shbg (r = - . ; p < . ). svcam- positively correlated with total t (r = . ; . ), hscrp correlated with: bmi (r = . ; p < . ), and homa (r = - . ; p < . ), respectively. conclusions: women with pcos exhibited abnormal levels of endothelial and inflammatory markers, which appear to be inter-related to hyperandrogenaemia. ( ), and a higher expression of fatty acid amide hydrolase (faah) in peripheral lymphocytes post-ovulation ( ), suggest an involvement of the endocannabinoid system in menstrual cycle control. our aims were to investigate changes in plasma aea levels and in endometrial faah expression throughout the menstrual cycle. methods: plasma aea levels were measured using a hplc ms/ms method from women, median age yrs (range - ) and bmi kg/m (range - ), with regular menstrual cycles, with no medical problem and not on any medication for the preceding months. the menstrual cycle was divided to early follicular d - (n= ), late follicular d - (n= ), ovulatory d - (n= ), early luteal d - (n- ) and late luteal phases d - (n= ). uterine biopsies were taken from hysterectomy specimens taken for benign conditions and subjected to immunohistochemistry for faah with polyclonal antibodies. results: aea levels were significantly higher around ovulation in comparison to the pre-ovulatory or post-ovulatory phases as shown in the figure. faah expression in the endometrial stroma was unchanged throughout the follicular phase but increased during the mid to late luteal phase reaching a maximum in the late luteal phase. a high aea level in the early follicular phase and during ovulation suggests a role for aea in ovulation. the lower levels of aea in the luteal phase, essential for successful implantation, may be regulated by increased faah expression at the uterine level. the modulation of plasma aea levels during the menstrual cycle strongly suggests that it is regulated by gonadal steroid hormones. ( , , , , , , and , as well as at pregnancy. binding activities of igfbp and their protein levels (igfbp , , , , , ) were assessed by western ligand blot (wlb) and western blot (wb), respectively. the glyscosylation and phosphorylation status of igfbps were examined by deglycosylation treatment. our results showed that both glycosylated and unglycosylated igfbp elevate in fetal and newborn rat and graduately decline to a lower level at day and kept lower constant level since then. interestingly, biding activity of glycosylated igfbp was not detected by wlb assay. the igfbp- cleaved products were observed after rat day age day , which associated with a decrease in full length of igfbp- , suggesting endoproteolytic processing may involved in decreasing igfbp content. igfbp- , with heterogeneous glycosylation, were appeared after age day and disappeared during pregnancy, recurrence again postpartum. glycosylation of igfbp has no effect on its binding activity. unglycosylated and glycosylated igfbp were constant in life time. physiologically constant igfbp were detected by wb in protein, but not by wlb in binging activity. conclusion: highly elevated circulation igfbp suggest physiological role in new born and early postnatal development. its binding activity are well regulated by its posttranslation modification, such as glycosylation of igfbp in inactivating binding activity and possible involvement of active endoproteolytic processing in maintaining binding active igfbp- at low background: cigarette smoking affects hormone biosynthesis, storage, release, binding, transport and clearance, resulting in changes in circulating hormone levels. we used metabolomics to analyze the effects of cigarette smoking and second hand smoke (shs) on changes in hormone levels in women of childbearing age. methods: this is a three arm study; women aged - years who are active smokers, exposed to shs (passive smokers), or non-exposed were recruited from the washington d.c. area. all women completed a detailed staffadministered questionnaire probing their medical history, occupational, lifestyle factors and diet. blood and urine samples were collected at the follicular phase. hormone profiles were determined using metabolomics for serum thyroxine and triiodothyronine and steroid hormones, as well as for cotinine using isotopedilution tandem mass spectrometry (lc/ms/ms-api ). in addition, all samples were analyzed for serum lh, fsh, tsh and creatinine. results: the relationships of cigarette smoking, age, relative weight, and dietary intake to serum thyroxine, triiodothyronine, estradiol (e ), estrone (e ), progesterone (p), -hydroxyprogesterone ( ohp),dehydroepiandrosterone (dhea), dehydroepiandrosterone sulfate (dheas), androstenedione, cortisol, -deoxycortisol, corticosterone, testosterone, aldosterone and vitamin d were analyzed using lc/ms/ms. mean tsh in non-exposed, shs-exposed and smokers: . , . , and . miu/ml respectively. similarly, mean t . , . ( % increase) (p= . ), . μg/dl (- %) in active smokers; (active vs. exposed p= . ). shs increased dhea levels ( % higher, p = . ), dheas ( % higher, p = . ), cortisol ( % lower, p = . ), aldosterone ( % lower, p = . ) and androstenedione ( % higher, p = . ). these data suggest that active smoking and shs can have a profound effect on serum t , adrenal steroid and sex hormone concentrations in women of childbearing age. objective: to determine the prevalence and predictors of the metabolic syndrome (mbs) among in saudi women with polycystic ovary syndrome (pcos) in comparison to women without pcos and to assess the role of androgens and insulin resistance (ir) in mbs development. design: a prospective case control study. setting: tertiary referral university hospital. subjects: six hundreds saudi women living in the jeddah area were classified as follows: with pcos and age-matched women without pcos. interventions: blood samples were collected from all women with or without pcos between : - : , after an overnight fast. main outcome measures: measures of abdominal obesity, blood pressure and that of serum levels of lh, fsh, tsh, ft , -ohp, -a, dheas, total t, free t, shbg, insulin, hdl-c, triglycerides and plasma levels of glucose. measures ir including: fasting serum insulin, gir and homa. results: age-adjusted prevalence of mbs was higher in women with pcos ( . %, % ci: . - . %) as compared with women without pcos ( . %, % ci: . - . %) (p< . ). in the same age group, the risk of mbs in women with pcos was greater than that for women without pcos (p< . ). markers of ir were significantly abnormal in women with both pcos and mbs in comparison to those without mbs (p< . ). the most common abnormal components of mbs in women with both pcos and mbs (after adjustment for age) were: decreased hdl-c ( . ± . %); increased triglycerides ( . ± . %); and increased bmi ( . ± . %), respectively. the prevalence of mbs from lowest to highest tertile of free t level was . , . and . %, respectively; in women with both pcos and mbs. in women with pcos, % exhibited all components of mbs; . % had components, and . % had components. conclusions: women with pcos exhibited significantly higher prevalence of mbs ( . -fold) as compared with age-matched control without pcos. ir is a possible common pathogenetic factor for both mbs and the pcos. it is suggested that more intensive screening and/or therapy monitoring of mbs among women with pcos should be part of the therapeutic modalities of the condition. case of sisters with complete androgen insensitivity syndrome and discordant mullerian remnants. jennifer l nichols, jennifer j gell, eric j bieber. reproductive endocrinology and infertility, geisinger medical center, danville, pa, usa. complete androgen insensitivity is an x-linked recessive disorder resulting in the abnormal expression of the androgen receptor. affected individuals are most commonly phenotypically female but genotypically male. the prevalence of this disorder is in , live male births. we present a case of complete androgen insensitivity in members of the same family with differing residual mullerian tissue. sister a presented at age for evaluation of primary amenorrhea. a karyotype revealed ,xy. an mri of the pelvis showed a hypoplastic uterus but no ovaries. this patient underwent laparoscopic bilateral gonadectomy and hemihysterectomy. on examination under anesthesia, she was noted to have a normal vagina with no cervix noted. at laparoscopic evaluation, she was noted to have bilateral elongated gonads and what appeared to be a remnant of uterine tissue. pathology revealed portions of immature testicles and fragments of smooth muscle in what grossly appeared to be the uterine remnant. the patient's total testosterone following surgery was noted to be elevated at . ng/ml. other laboratory evaluation showed fsh . miu/ml, lh . miu/ml, free testosterone . pg/ml, and estradiol . pg/ml. approximately two years later sister b presented at age for evaluation of primary amenorrhea. no uterus or ovaries were visualized on pelvic ultrasound. again a karyotype revealed ,xy. laboratory evaluation demonstrated fsh . miu/ml, lh . miu/ml, estradiol . pg/ml, total testosterone . ng/ml, and free testosterone . pg/ml. she underwent a laparoscopic bilateral gonadectomy. no uterus, cervix or pelvic masses were identified on exam under anesthesia. at laparoscopy, both gonads were visualized and removed without difficulty but no uterus was visualized. pathology reported two testicular and epididymal-like structures. this case demonstrates the presentation and laparoscopic results of complete androgen insensitivity syndrome discovered in two siblings. although both girls are genotypically male, they differ in the presence of vestigial mullerian tissue. the case shows with complete androgen insensitivity, as an x-linked defect, one should consider apparent sisters of affected individuals, as well as offspring of unaffected individuals with a family member diagnosed. background: anandamide (n-arachidonoylethanolamine, aea) is an endocannabinoid that binds to and activates the cannabinoid receptors, cb and cb and may have important roles in the regulation of human reproduction. the traditional lipid extraction methods used for aea are cumbersome, slow and of low efficiency. the aim of this study was to determine whether the use of a solid phase (spe) method of aea extraction from human plasma would offer any advantages over the traditional liquid phase (lpe) method. methods: pooled human plasma was obtained from the local blood transfusion unit and aliquots stored at - °c prior to extraction. an internal standard of . pmol of deuterated aea (aea-d ) was added to each plasma sample and aea extracted from aliquots on each occasion over three days using the lpe and spe methods. spe was performed with waters oasis hlb cc/ mg cartridges on a waters vacuum manifold. cartridges were activated with methanol and water, the samples applied and washed with % methanol at ml/min. aea was eluted with ml acetonitrile and the eluents dried under a stream of nitrogen before reconstitution in ml acetonitrile. aea levels were measured using uplc-ms/ms against authentic standards. results: these are shown in the table. conclusion: the spe method was -fold more efficient at extracting aea compared to the traditional lpe method. the intra-day and inter-day assay variability were similar for both techniques, although the spe method was quicker, cheaper and required less plasma to generate data similar to that from the traditional lpe method, suggesting that the spe method may be more efficient than the lpe method, and thus making it more suitable for routine analysis of multiple plasma aea samples. background: the precise role of the endocannabinoid, anandamide (narachidonoylethanolamine, aea) in reproduction has been hampered by difficulties in its accurate measurement. aea levels have previously been measured by tlc, gc-ms and hplc-ms but these are laborious. our aim was to improve the analysis of aea using uplc and tandem ms/ms with a standard isotope-dilution method , . methods: authentic non-labelled and deuterium-labelled aea (aea-d ) diluted in acetonitrile were maintained at °c before analysis and separation by uplc on a c ( . x mm) column maintained at °c using a gradient of % a, . min: % a, . min: % a, . min: % a, . min: % a with a flow rate of . ml/min. the mobile phases were a ( mm ammonium acetate, . % formic acid) and b (acetonitrile, . % formic acid). the analytes were quantified using multiple-reaction monitoring in positive ion mode with a quattro premier mass spectrometer. entry, collision and exit energies were - , and - ev, respectively. calibration curves were performed in triplicate with . pmol aea-d as the internal standard. transitions employed were . > . and . > . for aea and aea-d , respectively. results: calibration curves ( . to fmol aea on column; n= ) were linear, producing a mean (±sd) gradient of y = . ± . x, crossing the y-axis very close to the origin ( . ± . units). variability was limited, with an r = . . measurements were precise, fmol aea produced a cv of only . %, and the retention time was consistent at . ± . min (n= ). the limit of quantification (signal/noise> ) was . fmol on column and the limit of detection (lod) was . fmol on column (signal/noise= ). accuracy for . fmol, . fmol and fmol aea was . ± . %, . ± . % and . ± . %, respectively. conclusions: the method described is an improvement over other lc-ms/ms methods , with a lower lod [ . fmol v. fmol or fmol ] and shorter run time [ min v. min or . min ]. thus, an improvement in terms of speed, increased sensitivity and better reproducibility will allow for a more accurate assessment of aea concentrations in a number of biological samples. objectives: the gata family of transcription factors consists of six zinc-finger proteins with a critical role in tissue-specific and developmentally-regulated gene expression. gata factors exert their effects alone and through interactions with cofactors, such as friend of gata (fog), as well as with nuclear hormone receptors, including steroidogenic factor- (sf- ), a well-described activator of gonadotropin gene expression. the objective of these studies was to define the role of gata family members in the gonadotrope. methods: ) total rna was extracted from the gonadotrope cell line, l t , and analyzed by standard rt-pcr analysis. ) the cv- fibroblast cell line was transiently transfected by the calcium phosphate precipitation method with a rat - /+ lh promoterreporter vector as well as cmv-driven expression vectors for gata- , gata- , dngata- , fog- and/or sf- . results: gonadotrope l t cells were found to express transcripts encoding gata- , gata- , and gata- as well as fog- and fog- . gata- and gata- stimulated lh gene promoter activity by approximately -fold (p< . ) and synergistically increased the sf- response ( -fold versus -fold for sf- alone; p< . ). the gata-mediated increase in lh gene expression was nearly eliminated with co-transfection of fog- . similarly, co-transfection with a gata- dominant negative construct blunted wild-type gata- effects in a dose-dependent fashion. conclusions: these data demonstrate expression of both gata and the functionally related fog proteins in a well-characterized gonadotrope cell line. furthermore, they demonstrate a functional role for these factors in regulation of gonadotrope function, specifically expression of the lh gene. low-dose dexamethasone (dex) therapy early in pregnancy is used in fetuses with suspected risk of congenital adrenal hyperplasia. several adverse neurological events in prenatally treated children have been reported and the fetal hypothalamic-pituitary-adrenal (hpa) axis may be involved. aim: to investigate the immediate and long-term effects of early maternal dex administration on fetal growth and pituitary-adrenal activity in sheep. method: pregnant ewes carrying singleton fetuses (total n= ) were randomized to control ( ml saline/ewe) or dex treatment ( . mg/kg ewe weight) consisting of four intramuscular injections at -hourly intervals over hours on - days of gestation (dg). at , , , and dg fetal weights were recorded. i -ria, qrt-pcr and in-situ hybridisation were used to measure fetal plasma cortisol and acth levels and to analyse adrenal and pituitary mrna expression. results: dex-exposed fetuses were lighter than control animals at dg*, but not at other times; in general fetal organ weights were similar between treatment groups. fetal plasma acth was unaffected by dex and did not differ between genders. similarly, pomc and pc- mrna in pars distalis were unaltered after dex. however, fetal plasma cortisol was reduced after dex in both male and females at dg*, was similar at and dg, then elevated at dg*. plasma cortisol in female fetuses in control and after dex was significantly higher than in males. the increases in cortisol after dex at dg* were associated with increased fetal adrenal expression of p c and bhsd mrna in females, reduced expression of mc r in males, but no difference in star mrna. conclusion: we conclude that in sheep, early dex programs the developmental trajectory of the fetal hpa with increased activation directly of the adrenal, but not pars distalis function. in females this effect may be attributed to increased fetal adrenal steroidogenic activity. the effect of dex in increasing cortisol in males, albeit at a significantly lower level than in females, appears to be independent of the enzymes that we have measured.*p< . . synaptophysin and gonadotropin-releasing hormone (gnrh) are colocalized in rat hypothalamus. armando arroyo, beom su kim, amanda biehl, blenna cl bett, , john yeh. gynecology-obstetrics, university of buffalo, buffalo, ny, usa; physiology and biophysics, university at buffalo, buffalo, ny, usa. the cellular and molecular mechanisms that control gonadotropin-releasing hormone (gnrh) release are not completely understood. gnrh is stored in synaptic vesicles and released by exocytosis at gnrh nerve terminals. there are currently nine families of synaptic vesicle proteins that are involved in neurotransmitter release by exocytosis. synaptophysin is one of the most common synaptic vesicle proteins present in synaptic vesicles in neurons. the hypothesis of this study is that synaptophysin is expressed in gnrh neurons. we obtained sections from the hypothalamus of female sprague dawley rats. double-label fluorescence immunohistochemistry was performed on free-floating sections. sections were incubated with a mixture of mouse monoclonal antibody against gnrh ( : -chemicon international) and with a rabbit polyclonal antibody against synaptophysin ( : -santa cruz biotechnology) for h. after incubation the sections were washed and incubated with a mixture of alexa conjugated goat antimouse and alexa conjugated goat antirabbit ( : ; molecular probes) for h. slices were visualized with confocal microscopy (zeiss lsm- ). fifteen out of a total of fifteen gnrh cell bodies in the medial preoptic area and most gnrh neuron terminals in the median eminence showed intense immunostaining for synaptophysin. this is the first study to demonstrate that synaptophysin is expressed in rat gnrh neuron terminals. this suggests that synaptophysin is present in gnrh neuron vesicles. thus, gnrh release by exocytosis may be mediated by synaptic vesicle proteins. objective: our aim was to identify the effects of early gestation gc exposure on fetal and postnatal hpa axis development and function in postnatal life. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dex groups ( . mg/kg), consisting of four at h intervals on days - of pregnancy. at months postnatal age, catheters were implanted; a bolus injection of crh ( . mg/body weight) and avp ( . mg/body weight) were administered and arterial blood samples were taken at - , - , , , , , , , and min. levels of hepatic cbg mrna were determined by qpcr, expressed relative to s rrna. plasma cortisol and cbg levels were measured by radioimmunoassay. results: both total and free cortisol levels in the dex females (n= ) were lower than in dex males (n= ) from to min (p . ) and lower than in control females (n= ) at minutes (p . ), also in the dex-m group were higher than in control males (n= ) at min (p . ). plasma cbg levels and cbg mrna expression were not altered by dexamethasone exposure or sex. conclusions: these findings suggest that prenatal glucocorticoid exposure alters the development of the hpa axis differentially according to the sex of the exposed fetus. to determine maternal injections of synthetic glucocorticoids in early gestation can alter expression of hippocampal corticosteroid receptors at months postnatal age. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dexamethasone treatment ( . mg/kg) consisting of four injections at h intervals on days - . hippocampal was collected from the offspring at months postnatal age. levels of mrna of gr and mr were determined using qpcr and levels relatived to s rrna. results: dexamethasone-treated male animals (n= ) had significantly higher levels of mr gene expression than both control males (n= ; p= . ) and females (n= ; p= . ). gr gene expression levels were higher in treated vs. control males (p= . ), but in females levels in treated and control animals were similar. total body, brain and hippocampus weights were similar. conclusions: maternal dexamethasone administration in early pregnancy resulted in gender-dependent changes in hippocampal gene expression when measured in the offspring seven months after birth. objective: diminished ovarian reserve (dor) affects many younger women. we analyzed superovulation with intrauterine insemination (so) cycles in vitro fertilization (ivf) cycles of women with dor to determine if women < with dor differ from their older counterparts. method: irb approved retrospective review of so ivf cycles from / - / with follicle stimulating hormone (fsh) levels based on age < or . results: so cycles were performed in women. no differences were noted in clinical pregnancy. women < were more likely to have inseminates with a lower mean tms/ins, median tms/ins was . among pregnancy cycles compared to for unsuccessful cycles. for ivf, mean total gonadotropin dosage was significantly lower in women < , mean number of follicles mm peak e were significantly higher in women < . so ivf measures are in tables , respectively. conclusions: similar clinical pregnancy outcomes were seen despite age. in ivf, women < required less gonadotropins and generated more follicles but with no significant difference in number of mature oocytes or clinical pregnancies. of note, pregnancy rates for so in women < with dor are substantially lower than expected in our clinical practice. as ivf yielded a substantially higher chance of pregnancy, consideration should be made to expedite progression to ivf. to assess the effects of intraovarian injection of ad-fshr on the reproductive system of fshr (-/-) mice. methods: about l containing x pfu of ad-hfshr were injected directly into each ovary of treated group and same amount of ad-lacz were injected into the ovaries of control animals. vaginal smears were collected and body weight was measured daily. four weeks after the injection animals were sacrificed and all organs were weighted and evaluated by h e. fsh, e and p measured before and after treatment. results: ad-hfshrtreated mice showed obvious estrogenic changes in vaginal smear while in control animals vaginal smear remained at diestrus stage. significant increase in total body weight and estrogen dependent organs weight (uterus, ovary, vagina) was observed in treated animals compare to control group (p< . ). no significant weight changes were observed in other organs. h e evaluation of the ovaries showed significant increases in both the total number of follicles and the collective diameter of the follicles in treated animals compare to controls. on average follicles/ovary were observed in ad-hfshr-treated group of which follicles were at the antral stage while only follicles observed in ad-lacz control group, with zero follicles at antral stage. a . to folds increase in e and about % decrease of fsh observed in treated animals compared to control mice. there was no significant change in serum progesterone level between treated and control groups. conclusion: intraovarian injection of an adenovirus expressing human fshr gene is able to restore folliculogenesis and resume estrogen hormone production in female forko mice. objective: the sentinel issue surrounding multiple gestations following ivf is the inability to precisely estimate the reproductive potential of individual embryos with the currently used embryo grading systems based on embryo cleavage rate and morphology. recently, metabolomic profiling of spent culture media using raman and near-infrared spectroscopy have been reported to predict reproductive potential of embryos. in this study we applied proton nuclear magnetic resonance (¹h nmr) spectroscopy to analyze metabolomic profile of embryo culture media and to identify components of the media that correlate with reproductive potential. methods: eighteen spent media samples from embryos that failed to implant, and samples from embryos that resulted in pregnancy and delivery were individually collected after embryo transfer on day , and evaluated using ¹h nmr. the spectra obtained were analyzed using a selective genetic algorithm (ga) to determine regions predictive of pregnancy outcome as determined by logistic regression. to avoid random correlations, a leave-one out crossvalidation was used. sensitivity and specificity of predicting pregnancy (described as implantation and delivery) were calculated. results: using the ga, two areas in the ¹h nmr spectral region were identified as most discriminatory between the two groups. viability indices calculated by ¹h nmr using these regions were significantly higher in culture media of embryos with proven reproductive potential ( . ± . ) compared to those that failed to implant ( . ± . ) (p< . ). compiled outcomes from the leave-one-out cross-validation of the logistic regression using the ¹h nmr measurements resulted in a sensitivity of % and a specificity of . %. quantification by integration showed significantly decreased glutamate levels (p= . ) and a trend toward an increase in pyruvate levels (p= . ) in culture media of embryos that did not cause pregnancy. conclusion: metabolomic profile of spent embryo culture media using ¹h nmr correlates with the reproductive potential of embryos. the lower glutamate levels detected in culture media of embryos that failed to implant could potentially be due to the toxicity associated with increased embryonic glutamate uptake. additional studies using complementary approaches are needed to further delineate molecular components associated with reproductive potential. objective: beta-carotene and other carotenoids are known antioxidants previously identified in human follicular fluid (ff). in addition to their inherent antioxidant properties, carotenoids have been identified as precursors of the antioxidant retinol in bovine ff. high retinol levels in bovine ff are associated with non-atretic follicles. this would suggest a possible role for retinol and its carotenoid precursors in follicular heath and the general oxidative state of the follicle. we sought to measure carotenoids and retinol in the ff of women undergoing ivf and correlate these levels with normal fertilization as a marker of follicular/oocyte health. design: prospective cohort study materials and methods: ff from a single - mm follicle was obtained from women (age - ) undergoing ivf and intracytoplasmic sperm injection (icsi). serum was also obtained at the time of oocyte retrieval. retinol, vitamin e ( , , and tocopherol) and carotenoids ( -carotene,cryptoxanthin, lycopene and lutein/zeaxanthin) were measured using hplc. we correlated ff carotenoid and retinol levels with oocyte fertilization status following icsi. results: as previously reported, retinol, vitamin e and carotenoids were all identified in the ff. each fat-soluble vitamin level was significantly lower in ff compared to serum (p< . for all analytes) and the levels were strongly correlated (r > . , p< . for all analytes). mean levels of ff -carotene were significantly higher in those follicles that resulted in a fertilized oocyte ( . +/- . g/ml vs. . +/- . g/ml, p= . ). other carotenoids, retinol, and vitamin e levels did not correlate with fertilization outcomes. conclusions: our finding of a strong association between ff -carotene concentration and subsequent normal fertilization of the oocytes suggests an important antioxidant role for -carotene in the health of the human ovarian follicle/oocyte. the lack of correlation with other carotenoids, retinol and vitamin e suggests that the antioxidant properties of -carotene act by preventing singlet oxygen and scavenging the peroxyl radical and may directly influence oocyte competence. mature oocytes obtained during egg retrieval have been shown to undergo spindle depolymerization as a result of cooling. for this reason, ivf programs strive to maintain constant temperature during follicle aspiration. we sought to elucidate if there was a difference in temperature of fluid aspirated into a hand held (hh) or heating block (hb) encased collection tube. the experiment was performed in an ambient temperature of °c. thermistors, pinhead sized sensors with an accuracy of %, were used to monitor temperature differences between control fluid (cf) (sterile water ºc) and aspirated fluid. data was recorded using an -channel data acquisition system from dataq instruments. one thermistor was placed into the cf, the other was placed into an empty collection tube set in the heating block or held in a gloved hand. a cm, gauge, single lumen needle connected to cm of tubing was used to aspirate into the empty collection tube. temperature was recorded x/second, each experiment was repeated times. baseline empty collection tube temperature was significantly cooler in the hh vs the hb group ( . ± . °c vs . ±. °c, p=. ). two seconds after aspiration, the lowest aspirate temperature was observed, (hh . ±. °c, hb . ±. °c, p>. ) no difference between groups. in both groups, temperature quickly increased as aspiration progressed (hh . ±. °c, hb . ±. °c, p>. ). the hb group took an average of . min to return to baseline ( °c), the hh group never returned to baseline. substantial cooling of aspirated fluid occurs during oocyte retrieval, with a mean temperature decrease of . ±. °c corresponding to . °c. considering this dramatic decrease, the difference between temperatures in the hh vs. the hb group is negligible. current aspiration systems poorly regulate temperature, thus the choice of aspirating into a test tube warmed by hand or by heating block is inconsequential. clinical management of twin gestations with recurrent preterm labor symptoms. lesley de la torre, luis roca, niki b istwan, debbie j rhea, gary j stanziano, victor hugo gonzalez-quintero. obstetrics and gynecology, university of miami medical center, miami, fl, usa; clinical research, matria healthcare, marietta, ga, usa. objective to examine pregnancy outcomes in women with twin pregnancies receiving nifedipine tocolysis (nt) who experienced recurrent preterm labor symptoms (rptlsx). study design twin pregnancies enrolled for outpatient preterm labor (ptl) surveillance services prescribed nt following an initial episode of ptl were identified from a database (n= ). eligible for inclusion were patients later hospitalized with acute rptlsx (n= ). included were those < weeks' gestational age (ga), with intact membranes, remaining undelivered for > hours after rptlsx . pregnancy outcomes of women resuming nt (rnt group, n= ) following hospitalization were compared to those having an alteration in treatment (alttx group, n= ) to continuous subcutaneous terbutaline. per normality assumptions, either independent student´s t or mann-whitney u test statistics were used for continuous variables; pearson´s chi-square for categoric. all p-values presented as two-sided, significant at < . . results overall, ( . %) of twin pregnancies prescribed nt experienced rptlsx; ( . %) were not eligible for continued tocolysis. pregnancy outcomes are presented in table. conclusion rptlsx are common. in twin pregnancies receiving nt, alteration of tocolytic treatment following rptlsx had a positive impact on pregnancy prolongation and neonatal outcomes. routine cervical dilatation during elective cesarean section -is it really necessary? arie koifman, avi harlev, eyal sheiner, fernanda press, arnon wiznitzer. obstetrics and gynecology, soroka university medical center, ben-gurion university of the negev, beer-sheva, israel. objective: the purpose of this study was to examine the necessity of routine cervical dilatation during elective cesarean section. material and methods a retrospective cohort study was performed, including all cases of elective cesarean sections performed at a tertiary medical center during . stratified analysis, using the mantel-haenszel technique, was done to control for confounders. results out of a total of elective cesarean deliveries (cd), underwent routine cervical dilatation. the overall rate of febrile morbidity was . %. no significant differences in postpartum febrile morbidity were noted between the groups ( . and . %; p= . ). in addition, hospitalization duration did not differ between the groups ( . ± . and . ± . days p= . ). about % of all elective operations were repeated cd. there was no difference in febrile morbidity between the groups even in that subgroup of the elective cd. likewise, there was no difference in anemia rate between the two groups (hemoglobin . ± . mg and . ± . mg p= . ). controlling for a previous vaginal delivery, using the mantel-haenszel technique, no significant association was noted between cervical dilatation and fever (weighted or= . ; % ci . - . ; p= . ). nevertheless, in a subgroup of patients following a previous vaginal delivery, cervical dilatation was significantly associated with post-operative fever (or= . the majority of women with an unfavorable cervix undergoing iol at term deliver vaginally, even with a prolonged first stage of labor. this is important information to discuss with women prior to iol when establishing labor expectations. providers should consider the ongoing success of labor induction when contemplating a diagnosis of "failed induction". objective: this study was performed to investigate and compare changes of the lipid peroxide levels and the protein carbonyls formation in the maternal venous plasma of preterm premature rupture of membrane (pprom) during antibiotics administration. materials and methods: thirty-six pregnant women with pprom between and weeks of gestation were chosen for this study. eighteen patients (group ) were treated with amoxicillin and erythromycin for day period while the other patients (group ) were treated with rd generation cephalosporin and erythromycin for the same period. samples of maternal blood were obtained from the two groups at before the antibiotics administration, day , and day after the antibiotics administration. lipid peroxide levels were measured by thiobarbituric acid reaction and protein carbonyl contents were determined by the , -dinitrophenylhydrazine method. results: . the lipid peroxide levels and protein carbonyls formation in the maternal venous plasma of pprom was significantly higher than that of normal pregnancy ( . ± . vs . ± . nmol/mg protein, p< . ), ( . ± . vs . ± . nmol/mg protein, p< . ). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the maternal venous plasma with pprom mixed and incubated by amoxicillin, cefodizime, and erythromycin (in vitro). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the venous plasma of group among before the antibiotics administration, day , and day after the antibiotics administration. . the protein carbonyls formation in the venous plasma of group was significantly decreased at day and day after the antibiotics administration than that of before the antibiotics administration ( . ± . , . ± . , . ± . nmol/mg protein, p< . ). conclusion: in the maternal venous plasma of pprom, the lipid peroxide levels and protein carbonyls formation were increased. the results suggest that reactive oxygen species formation by inflammatory reaction is suppressed by combined treatment of rd generation cephalosporin and erythromycin. objective: the aim of the present prospective cohort study was to evaluate the correlation between blood flow pulsatility index in fetal middle cerebral artery (mca) and the onset of spontaneous labor. study design: doppler evaluation of fetal mca were performed between and weeks gestation in consecutive pregnant women with a singleton pregnancy and known gestational age. the study population was divided according to the mca pi (< . or >/= . mom) and, using survival analysis and cox regression, the two subgroups obtained were compared. in these analyses, the event was delivery (following spontaneous labor) and the time variable was time elapsed since doppler exam. results: the median time elapsed between doppler evaluation and spontaneous labor was significantly shorter in the women with mca pi lower than . mom ( . days, interquartile range - ) in comparison with the women with mca pi higher or equal than . mom ( . days, interquartile - . days (p< . , mann-whitney test). after correction for birth weight and umbilical artery pi, survival analysis and cox regression confirmed that mca pi was independently associated with the number of days elapsed from doppler to spontaneous labor and delivery (p< . , exp(b) . , ci % . - . ). conclusions: the present data show that, at term of pregnancy, fetal cerebral resistance reduction anticipates the onset of spontaneous labor. novel calcium sensitizers and human uterine contractility. mark p hehir, audrey t moynihan, terry j smith, john j morrison. department of obstetrics and gynaecology, national university of ireland, galway, ireland. objective: the factors regulating contractility of uterine smooth muscle are central to the occurrence of preterm labour and delivery. calcium sensitizers are a novel class of drugs with unique molecular actions. levosimendan, the best characterized of these compounds, is used in the treatment of acute and chronic heart failure and is a compound which exerts a number of effects on smooth muscle. it can exert an inotropic effect via sensitization of myofilaments to calcium and also exerts a relaxant effect by opening atp-dependent potassium channels. for these reasons we hypothesized that levosimendan may have an effect on myometrial contractility and investigated its action on both spontaneous and agonist induced contractions. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. two sets of control experiments were performed simultaneously as follows: . strips exposed to either physiological salt solution (pss) only, for spontaneous contractions, or . nmol/l oxytocin; . strips exposed to pss/oxytocin and vehicle for levosimendan. results: levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, compared to control strips. the mean maximal inhibition values were as follows: . ± . % for spontaneous contractions (n= ; p< . ) and . ± . % for oxytocin-induced contractions (n= ; p< . ). no significant difference was found between control and control for both spontaneous and oxytocin induced contractions. conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on uterine contractility in vitro. this action was seen in both spontaneous and agonist induced contractions. the results from this study raise the possibility of calcium sensitizers holding potential tocolytic properties in vivo and further studies are required to investigate the potential benefits of this novel class of drugs. to determine to what degree extensive patient movement affects uterine emg signals and toco signals. study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. baseline recordings were obtained when patients were still, and these were used as control records. test recordings for both devices were obtained from all patients by asking the patients to perform movements. area under the rectified-voltage amplitude curve of the uterine emg signals and area under the curve for the toco signals were found. mean %-increase was calculated for the uterine emg and toco devices (each device %-increase values were averaged over all patients). mann-whitney rank-sum test was used to look for any statistical differences in %-increase in area for uterine emg vs. toco methods (p < . considered significant). results: there was a large increase in activity (artifact) on both devices' signals during patient movement (figure ) . both devices' traces eventually returned to baseline after the patient movement stopped. toco movement artifact was significantly higher than emg movement artifact ( table ) . conclusions: both uterine emg and toco signals experience artifact when patients move the uterine emg electrodes and toco pressure transducer, respectively. toco seems to be more adversely affected by such movements. uterine emg may be a preferred method for monitoring contractions of laboring patients in the clinic. supported by grant nih r -hd . results: % of obstetricians completed the questionnaire. none would counsel patients against labour unless there were contraindications. the majority would recommend labour for all indications for previous caesarean section investigated although in all instances, personal preferences were lower (p< . ); after a failed instrumental delivery, % obstetricians would recommend labour but only % would choose that option for themselves (p< . ). overall, female obstetricians would contemplate and recommend labour more readily than male obstetricians. labour augmentation and induction were recommended to patients more frequently ( % and % respectively) than chosen for personal care ( % and %). reluctance for labour augmentation and induction was greatest among younger consultants. conclusion: consultants have responded to consumerism and aim to meet the requests of their patients. they more readily recommend labour than they would choose for personal care, and a majority would recommend labour induction when necessary. informed patient choice is paramount rather than attaining a target figure for women attempting to labour, and the views of those requesting delivery by caesarean section should be respected. (table) . cd increased in nullipara singleton, cephalic, term pregnancy in spontaneous labor, mostly due to dystocia; nullipara singleton, cephalic term pregnancy with induced labor, mostly due to non-reassuring fetal status and dystocia; singleton, cephalic term pregnancy with previous cd, mostly due to elective cd; singleton cephalic preterm. no changes in neonatal outcome were observed. conclusion: clinical audit was useful to keep under control cd rate in our institution in comparison with italian reality, but it was not sufficient to maintain stable cd rate suggesting the need of other, multifaceted strategies. elective delivery at weeks' gestation is a common obstetric intervention. the purpose of this study is to estimate the maternal mortality rates (mmr) associated with this acog -approved intervention. there are no reliable us data describing mmr by mode of delivery. therefore, we base our estimates on british data indicating a procedure related mmr of . / , , . / , and . / , for vaginal, elective cesarean section (c/s) and emergency-unplanned c/s deliveries, respectively. a decision tree model was constructed assuming that all eligible patients (approx. , , /annually in the u.s.) would be delivered at weeks by either an elective c/s or an induction of labor. we further assumed that % of inductions would result in a vaginal delivery. our estimates show that the annual, delivery-related maternal mortality associated with an elective delivery of all patients at weeks would be and for elective c/s and induction of labor, respectively. because vaginal delivery results in the lowest mmr, we performed a oneway sensitivity analysis to identify the impact of changing the success rate of induction on the estimated mmr. the results indicate that once the success rate exceeds %, this intervention would be associated with a lower mmr as compared to elective c/s. our estimates indicate that although the overall mmr associated with elective delivery at weeks is relatively low (approximately . / , deliveries), it is certainly not negligible. in addition, the mmr is highly dependent on the likelihood of a successful vaginal delivery following induction of labor. when the success rate for induction of labor falls below %, an elective c/s appears to be the safer delivery method. background: the first obstetric visit is an opportunity to provide the pregnant patient information regarding substances that can cause potential harm to the pregnancy. little is known about how obstetric care providers handle these topics. objective: to examine patient-provider communication about substance use during the first prenatal visit. methods: we audiotaped first prenatal visits and qualitatively analyzed those tapes in which patients disclosed substance use. we invited patient participants to return for a semi structured interview during which they reviewed their audiotaped conversations and described their reactions to the providers' communication styles. results: providers ( residents, midwives, nurse practitioners) and patients participated. providers asked about smoking, alcohol and drug use in all ( %) visits. patients reported being smokers, reported alcohol use, and reported drug use. provider responses to smoking disclosures included brief discussions of smoking effects on pregnancy, encouragement to quit/cut down, and referral to smoking cessation programs. provider responses to alcohol or drug disclosures included only general statements regarding effects on pregnancy (e.g., "we find that this is bad for babies.") and referral to ultrasound/genetics for reassurance. few alcohol or drug discussions assessed whether the patient had intentions or concerns regarding continued use during the pregnancy. few discussions addressed strategies for behavioral change. none included assessment for motivations, readiness, or barriers to change. in follow up interviews, patient participants said they expected to be asked about substance use but advised providers to ask non-judgmentally. those who used alcohol/drugs wanted more information on potential effects of these substances on the pregnancy/fetus and appreciated the reassurance from referrals to ultrasound/genetics. discussion: counseling for risky behaviors in the first obstetric visits contained only limited discussion of the effects of the risky behaviors and primarily focused on referral-which may be a proxy for avoiding a difficult and time consuming conversations. background. there are conflicting results regarding the association of maternal psychiatric disorders or psychological distress due to stressful life events with pre-term birth and low birth weight. aims. to investigate the association between maternal psychiatric disorders and/or stressful life events and intrauterine growth abnormality, low birth weight or preterm birth. method. three mutually exclusive and homogeneous groups of pregnant women ( with actual psychiatric disorder, with stressful life events, and healthy comparisons) underwent serial fetal ultrasound examinations and uterine and umbilical artery doppler velocimetry at (+ ), (+ ) and (+ ) weeks of gestational age. subjects were recruited from all consecutive women attending two antenatal clinics. the presence of any maternal medical illness, drug treatments, fetal chromosomal and/or structural malformations, were exclusion criteria. all women recruited underwent a structured interview at - weeks for the psychiatric diagnosis (mini international neuropsychiatric interview -mini) (sheehan et al, ) ; moreover, the -item hamilton rating scale for depression and the hamilton rating scale for anxiety were included in the assessment. the person who obtained obstetrical clinical data was blind to the results of psychological evaluations. results. the three groups were comparable for: age, parity, socioeconomic status, smoking, alcohol consumption, body mass index. gestational age at birth was not different in the three groups. infants of women with psychiatric disorders had significantly lower birthweight ( + g) and higher percentage of birth weight below the th centile for gestational age ( %) than infants of healthy mothers ( + g and %, respectively). there was also a trend towards lower mean birth weight ( + g) and higher incidence of birth weight below the th centile ( %) in the stressful life event group. there was no significant difference among groups in the percentage of abnormal uterine or umbilical doppler results. conclusions. maternal psychiatric disorders are associated with a lower birth-weight, but the effect is unlikely to be due to abnormal utero-placental or feto-placental vascularisation. objective: the purpose of this study was to evaluate antenatal ultrasound as a tool for the detection of intrauterine growth restriction (iugr) and small for gestational age (sga) infants among subjects with elevated human chorionic gonadotropin (hcg) levels on second trimester serum screening. although iugr has been linked to elevated hcg levels, the optimal screening regimen for antenatal sonographic surveillance has not been previously established. methods: a retrospective cohort study was performed at saddleback memorial medical center where serial ultrasounds from weeks-delivery are generally recommended for patients with hcg levels > . mom. all pregnancies were dated by second trimester ultrasound ± last menstrual period. subjects with an hcg > . mom, who had at least one antenatal ultrasound evaluation for iugr, were identified from an electronic ultrasound database used for clinical report generation. ultrasound data were then linked to an obstetrical birth outcomes database for relevant demographic/delivery information using unique identifiers. iugr was defined as a sonographic estimated fetal weight (efw) < %ile for the estimated gestational age (ega). sga was defined as an actual birthweight < %ile for the ega at the time of delivery. results: from - , there were subjects with elevated hcg levels who underwent antenatal ultrasound surveillance for iugr and who had known delivery information. a total of ultrasound examinations were performed. the median number of examinations per subject was with a range from - examinations per subject. the incidence of iugr and of sga were . % (n= / ) and . % (n= / ), respectively. no fetus with iugr demonstrated absent or reverse end diastolic umbilical artery doppler flow. antenatal ultrasound examinations only identified . % (n= / ) of sga infants. however, the sensitivity for the detection of sga was % when an efw cut-off of % was used. conclusions: although the majority of sga infants did not demonstrate growth restriction on antenatal ultrasound, a sonographic efw > %ile appears to be a safe cut-off to rule out fetuses at risk for sga. neonatal dry lung syndrome after pprom: reason to change intrauterine referral practice in the netherlands? ellen s hoogakker, christiaan v hulzebos, gerda g zeeman. obstetrics and gynecology, university medical center groningen, groningen, netherlands; pediatrics, division of neonatology, university medical center groningen, groningen, netherlands. background: neonatal dry lung syndrome (dls) is a distinct clinical entity following pprom, mimicking pulmonary hypoplasia but with dramatic respiratory improvement during the first - h . its pathogenesis implies complete collapse of small airways to a degree that capillary forces impede distension by ordinary ventilatory pressures. in the netherlands, women with pprom remain admitted at a level iii nicu perinatal center until they reach wks. when they are referred back to their community hospital, unless they live in the neighborhood of the tertiary center. we question this referral pattern because we still see severe respiratory problems occur > wks. we sought to determine the prevalence of such morbidity, in particular dls, in women with pprom who deliver > wks. methods: retrospective descriptive study of neonatal outcome data of singleton pregnancies complicated by pprom between - wks, who deliver > wks (latency > h) , during the -yr period of - at a single academic center. data were extracted from medical records and electronic department databases. results: pprom pregnancies were identified. all but received at least full course of steroids. ( %) delivered > wks. newborns born at the community hospital needed emergency transportation to a level iii nicu for respiratory morbidity. neonatal outcome data (means ± sd) are listed in the table. there were no cases of late onset sepsis, nec or perinatal mortality. conclusions: respiratory morbidity still occurs after pprom with delivery > wks. further investigation of pregnancy-related characteristics, such as the presence of anhydramnios and the latency period, with regards to dls is needed. modification of current referral practice depends upon complete data derived from all dutch level iii perinatal care centers (n = ). tertiary care center (n = ) to determine if an association exists between macrosomia (birthweight > g) and perinatal outcomes in women with and without gestational diabetes mellitus (gdm). study design: this is a retrospective cohort study of , singleton pregnancies. the study cohort was stratified by the diagnosis of gdm, with the presence or absence of macrosomia as the dependent variable. perinatal outcomes examined included neonatal hyperbilirubinemia, hypoglycemia, respiratory distress syndrome (rds), shoulder dystocia and erb's palsy. chisquare tests were performed as well as multivariable analyses controlling for confounders, using p< . to indicate statistical significance. results: in women diagnosed with gdm, macrosomia is associated with a higher frequency of hypoglycemia, respiratory distress syndrome, shoulder dystocia and erb's palsy. though the prevalence of these outcomes is relatively decreased in patients without gdm, they are still more prevalent in macrosomic patients. macrosomia is associated with a higher prevalence of adverse perinatal outcomes in women with and without gdm. therefore, it is important to evaluate neonates with birthweights greater than grams for hypoglycemia and unrecognized erb's palsy. conclusion a significant proportion of our obstetrical patients are obese and many do not perceive themselves to be obese. while our finding of an inverse correlation between level of education and bmi may be confounded by socioeconomic status, our results suggest that in order to address the problems of obesity in our population an important first step will be improving the education of our reproductive age women regarding normal weight gain and nutrition in pregnancy. this may have a significant impact on improving pregnancy outcomes of today's obstetrical population. objective: the aim of the study was to evaluate the impact of a "flat" oral gct upon the outcome of pregnancy. the gct was considered flat when the difference between the basal value and the after load value was %. methods: we prospectively analyzed the outcomes of pregnancies who delivered at our department. inclusion criteria were singleton pregnancy; bmi < kg/m , absence of major risk factors for diabetes and of pregestational diabetes. g -hour oral gct was performed at - weeks of gestation. women were subdivided into groups according to the result of the gct: group = negative (load glucose > % and < mg/dl) women ( . %); group =flat (load glucose % than basal and < mg/dl) women ( . %); group =positive gct/negative ogtt, women ( . %). data are mean ± sd. differences were calculated with the student t test for unpaired samples and test. regression analysis were performed by the least squared method. p-values were considered significant at p< . . results: the characteristics and obstetric outcomes in the three groups are presented in the table . in all patients there was a significant linear relationship between the load and basal glucose values (load value= . + . basal value; r= . ; p< . ) and between birthweight and load values (birthweight= . + . load value; r= . ; p< . ). the relationships were significant also in group and separately but not in group . in this preliminary study we found no major outcome differences in women with flat gct compared to women with normal and gct positive/ ogtt negative results. it seems important, however, to futher investigate the meaning of a flat curve in a bigger population and/or by means of metabolic studies with the use of stable isotopes. results characteristic of the study population and obstetric outcomes are presented in table . the probability to be primiparous and to deliver babies < ° centile decreased significantly with bmi whereas the risk of cesarean section, of post partum hemorrhage at vaginal delivery and to deliver babies > ° centile increased significantly. also the risk to develop preeclampsia and gestational diabetes was increased, although not significantly, with bmi. compared to lean and normal, obese were more likely to be hypertensive and diabetic before pregnancy (p< . ) and to start pregnancy without any medical and obstetrical risk but obesity ( . % vs . and . %; p< . ). african women exhibited the highest bmi and the highest rate of obesity (table ). conclusions we confirm that obese women have an increased risk of prepregnancy and pregnancy complications: less than % have an uncomplicated pregnancy. at delivery there is an increased risk of cesarean section and post partum haemorrhage. objective: overweight and obese women often give birth to larger babies, which is associated with traumatic birth injuries and an increased risk to develop obesity, diabetes and hypertension in childhood and later in life. the mechanisms underlying fetal overgrowth in obese pregnant women are largely unknown. the aim of this study was to establish a mouse model of obesity/high fat diet in pregnancy. we hypothesized that a moderately high fat diet prior to and during pregnancy would result in increased maternal adiposity, fetal overgrowth and a metabolic profile similar to that of obese newborn offspring with persistent pulmonary hypertension, despite enhanced pregnant women. method: c bl/ j female mice were fed control (c, % of energy from fat) or isocaloric high fat (hf, % of energy from fat) diets ad libitum for weeks prior to mating and during gestation. at gestational day . maternal blood samples were obtained to measure adiponectin, leptin and cytokine levels and maternal fat pads were isolated and weighed. in a sub set of animals measurements of transplacental transport of neutral amino acids and glucose were performed in vivo under ketamine anesthesia using h-meaib, and c-glucose. results: no significant differences were observed in maternal pre-pregnancy bodyweight, total caloric intake, weight of the dam at e . or litter size between treatment groups. fetal weight was increased in the hf group by % (p< . ). maternal adiponectin levels were significantly (p< . , n= ) decreased (hf ± , c ± g/ml) and leptin levels increased by % in animals fed a high fat diet, but this difference did not reach statistical significance (n= ). adiposity (fat pad weight) was increased by % (p< . , n= in the dams fed high fat diet, however no difference was observed in maternal il- levels and neither group had measurable levels of tnf-. maternal red blood cell lipid profiles were altered in high fat animals with an increase in stearic and linoleic acids but decreased oleic acid levels. preliminary data showed that placental uptake and transfer to the fetus of glucose and meaib were increased by at least % in dams fed high fat diet. conclusion: this murine high fat diet model has several features consistent with human obesity in pregnancy and the maternal metabolic environment is similar to that seen in the human. the increase in placental uptake and transfer of nutrients constitute a key mechanism underlying fetal overgrowth in overweight/obesity in pregnancy. objective: epidemiological studies have demonstrated a positive relationship between maternal overnutrition and the development of the metabolic syndrome in the offspring. we previously reported that lambs of well fed ewes have increased plasma glucose levels in early life, this may be a consequence of altered hepatic glucose production. increased hsd expression is associated with an increase in intracellular cortisol, promotion of hepatic insulin resistance and a consequential increase in gluconeogenic activity. hypothesis: we hypothesised that maternal overnutrition in late gestation in the sheep results in increased hepatic expression of hsd in the lamb before and after birth. methods: ewes were provided with either % (control,c) or % (well fed, wf) of maintenance energy requirements from d gestation until delivery. post-mortem was performed on either ± d gestation (c= ,wf= ) or and postnatal day (c= ,wf= ). plasma glucose and leptin concentrations in the fetuses and lambs were determined. the relative hepatic mrna expression of hsd and reference gene rpp were determined by qrt-pcr. results: relative liver weight was significantly higher in lambs of wf ewes compared to c at d (p< . ). the expression of hsd mrna was significantly higher in the postnatal compared to the fetal lamb (p< . ) independent of maternal nutritional treatment. however, there was no effect of maternal overnutrition on the hepatic expression of hsd mrna before or after birth. there was no effect of prenatal nutrition on fetal or postnatal plasma cortisol concentrations. the expression of hsd in the liver of lambs of wf, but not c ewes, was inversely related to plasma glucose concentrations in the first hrs after birth (r =- . , p= . ). we have therefore demonstrated that exposure to prenatal overnutrition results in an inverse relationship between hsd mrna expression in the liver at d and plasma glucose concentrations on the first day of life. this suggests that exposure to high glucose levels before and immediately after birth results in a reduced expression of hepatic hsd . we would expect a reduction, rather than promotion of intra-hepatic cortisol production, and therefore it is unlikely to explain the hyperglycemia present in lambs of wf ewes in early life. ninety-seven ( %) of mothers were classified as obese (bmi> ) based on their first pregnancy weight. glycemic control at weeks was superior in the non-obese group (table ) . there were no differences in glycemic control during the last week of pregnancy. obesity was significantly associated with increased maternal weight gain during pregnancy. mean birth weights, ponderal indices and rates of macrosomia were significantly higher in infants born to obese women when compared to non-obese (table ) . primary cesarean deliveries rates were comparable. the rate of neonatal hypoglycemia, hyperbilirubinemia, phototherapy and neonatal icu admissions did not differ between obese and non-obese diabetic women (table ) . although not statistically significant, there was a trend towards an increased rate of birth injuries in the obese group. a similar comparison between obese and non-obese women treated with medication (glyburide or insulin) demonstrated a higher mean birth weight ( g+ vs. g+ , p=. ) and higher rate of macrosomia ( vs. %, p= . ). there were no differences in glycemic control, cesarean delivery rates and other neonatal outcomes between the obese and non-obese treated with medication. conclusion: obese women with type ii and gdm give birth to larger infants than their non-obese counterparts and have a higher incidence of fetal macrosomia. there was a trend towards increased rate of birth injuries in the obese group. despite these differences other maternal and neonatal outcomes were similar which may be a reflection of glycemic control. introduction: tlrs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. the placenta, and more specifically the trophoblast, may play an important role in the response to infection. previously, we described the expression of tlr- by human trophoblast and their ability to respond to polyinosinic-polycytidylic acid (polyi:c), a synthetic double strand rna which mimics viral rna. in the present study we evaluate the effect of polyi:c in mouse pregnancy and characterize the local and systemic response. material and methods: human first trimester trophoblast cell line, htr , was treated with polyi:c. c b/ wild type and tlr- knock out mice were injected intraperitoneally with polyi:c at . gestation day. cytokine and chemokine level were determined in supernatant and lysates using the bio-rad multiplex assay and analysis was done using the bio-plex is. results: polyi:c induced cytokine (il , il and il ) and chemokine (il , rantes, gro , mcp and mip ) secretion and production by human cultured trophoblast in a time ( - h) and a dose ( - g/ml) dependent manner. injection of polyi:c to c b/ wild type mice induced preterm delivery within h at a dose of mg/kg body weight. no effect was observed in tlr- knock out mice. a robust systemic (spleen and serum) and local (placenta and amniotic fluid) inflammatory response was observed and h following polyi:c treatment. trophoblast cell cultures from tlr- ko mice confirmed that the response to polyi:c is tlr- dependent. conclusion: we demonstrate that viral infection may trigger an immune response leading to preterm labor. furthermore we show that the trophoblast is able to recognize and respond to viruses through the expression of tlr- . our findings provide a novel mechanism of pathogenesis of preterm labor associated with tlr- mediated inflammatory response. introduction: adverse neurological outcome is a major cause of neonatal morbidity after a preterm birth (ptb). a growing body of evidence demonstrates the involvement of inflammatory pathways in ptb and implicates these pathways as a causative factor in fetal brain injury. however, activations of cytokine pathways in normal labor (whether iatrogenic preterm or at term) has been observed. what remains understudied is the effect of labor on the preterm fetal brain and whether an inflammatory stimulus is essential for fetal brain injury. methods: mouse models were utilized: ( ) a model of intrauterine inflammation (lps into uterine horn) (n= ); controls received intrauterine saline (n= ) and ( ) autism is a neurodevelopmental disorder with a strong genetic component and several known environmental risk factors, such as infection. in addition, its onset of etiology is likely to occur during prenatal development. we propose that subjecting fetal sheep via amniocentesis to the bacterial endotoxin lipopolysaccharide (lps) injected to the amniotic fluid at gestational day (gd) will result in morphological alterations in the offsprings' cerebellum resembling alterations found in the cerebellum of patients with autism. using high precision design-based stereology, we investigated mean total-and layer-specific volume and mean total granule and purkinje cell (pc) number in the cerebellum of lps infected animals and controls. the results of the present study showed preserved volumes of the total cerebellum as well as of the molecular layer, outer and inner granular cell layers and white matter. interestingly, compared to controls, the lps infected brains showed a statistically significant increase (+ . %) in the mean total number of granule cells, whereas the pcs did not show any difference between the groups. these seemingly paradoxical results might be explained by ( ) the so-called time of origin of these neurons, i.e. the pcs develop prenatally whereas the granule cells develop postnatally or ( ) the direct correlation between pcs and granule cell number in the cerebellum. these results might contribute, as an animal model, to our understanding of the biological basis for interindividual differences in morphological alterations found in the brains of patients with autism. the previous studies have shown that there is a higher incidence of spontaneous preterm birth and poorer neonatal outcome in pregnancies with a male fetus. in vitro studies also report a sex dependent pattern in different placental enzymatic systems. we have shown previously that lactobacillus rhamnosus gr- supernatant is able to antagonize the actions of lps on cytokines and ptgs in placental trophoblasts. we hypothesize that fetal sex will influence the production of cytokines and prostaglandin regulating enzymes in lps and lactobacilli treated placental trophoblast cells. methods: term placentae were collected from women undergoing elective caesarean section. placental trophoblasts were isolated using established primary culture protocols. cells were pretreated with lactobacilli supernatant and subsequently treated with lps. pgdh and ptgs expression levels were measured by western blot analysis and tnf-, and il- concentrations measured by elisa. results: lps stimulation caused a marked increase in production of tnf-( . pg/ml to . pg/ml, n= , p< . ), an effect that was greater in placentae of the male fetuses ( . pg/ml, n= ) compared to female fetuses ( . pg/ml, n= ). lactobacilli supernatant abolished this response in both sexes. lps-activated trophoblasts from placentae of the male fetuses showed an increase in il- production (n= , p< . ) and ptgs expression (n= , p< . ). however, there was no response to lps in placentae of the female fetuses. lactobacilli supernatant up-regulated pgdh (n= ) by %, and this effect was greater in placentae of the female fetuses (n= ). conclusion: we conclude that human placentae from pregnancies carrying male fetuses are more responsive to lps by producing more pro-and anti-inflammatory cytokines, as well as ptgs . conversely, placentae of the female fetuses upregulate pgdh with lactobacilli treatment. these findings may explain the underlying mechanism for the higher incidence of preterm birth and adverse pregnancy outcomes seen with male fetuses in the clinical setting. , ) . this study investigates whether iai is associated also with altered expression of neuropilin- . methods: (i) immunohistochemistry (ihc) was performed on tissue sections of term decidua with or without clinical / histologic evidence of iai (n= for each). neuropilin- expression was scored by an investigator blinded to the identity of the samples. (ii) cultured term dscs were retrieved from elective cesarean (n= ), purified, and depleted of leukocytes. after treatment with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days, dscs were stimulated with il- b ( - ng/ml), tnfa ( ng/ ml), or thrombin ( . iu/ml) for h. since no elisa exists for neuropilin- , protein expression was determined by immunocytochemistry (icc). (iii) total rna was extracted and the effect of il- b on neuropilin- mrna expression measured by real-time rt-qpcr and corrected for b-actin mrna. results: neuropilin- expression in term decidua was increased in tissues with iai vs controls (p< . ), and localized primarily to dscs. using icc, an increase in neuropilin- was noted after stimulation with il- b and tnfa, but not thrombin. il- b increased neuropilin- mrna expression in dscs by . ± . -fold (from . ± . to . ± . neuropilin- mrna/b-actin mrna; p= . ). conclusions: iai is associated with increased expression of the vegf receptor, neuropilin- , in term decidual tissues. il- b and tnfa (but not thrombin) stimulated neuropilin- expression in term dscs, and this effect appears to be mediated at the level of gene transcription. since aberrant vegf function alters vascular permeability, these data provide a mechanism by which iai can promote 'decidual activation' and preterm labor. inflammation, university of glasgow, glasgow, united kingdom. objective: asthma is associated with inappropriate activation of airway smooth muscle, chemokine expression and accumulation of mast cells which drive smooth muscle reactivity. labour is similarly associated with smooth muscle activation and expression of cxcr and cxcr ligands. the role of mast cells in human parturition is unknown; however, mast cell products can stimulate myometrial contractions and preterm labour in animal models. we have quantified mast cells in association with human labour and determined whether they express cxcr and cxcr . methods: lower segment myometrial and cervical biopsies were taken at term caesarean section from women not in labour (nil) (myometrium n= ; cervix n= ) and in labour (il) (myometrium n= ; cervix n= ). mast cells were localised in myometrial and cervical sections by icc with a primary antibody against c-kit. the number of cell transects in randomly selected high-powered fields ( x) was quantified blindly by two observers for each specimen, with median density and interquartile range (iqr) calculated. backto-back icc was performed to determine whether c-kit co-localised with the chemokine receptors cxcr and cxcr . results: mast cells were in close association with myometrial smooth muscle in non-labouring lower segment myometrium. labour was associated with a significant influx and increase in mast cells numbers (nil median . , iqr . - . ; il median . , iqr . - . , p= . ). in contrast no significant increase in mast cells was observed in cervical tissue in association with labour (nil median . , iqr . - . ; il median . iqr . - . , p= . ). analysis of chemokine receptor expression demonstrated co-localisation of cxcr to c-kit positive cells present within the myometrium. conclusions: human labour at term is associated with an increase in mast cells within the myometrium, with close approximation to smooth muscle bundles. these mast cells express the chemokine receptor cxcr , the ligands of which we have previously shown to be up-regulated in labouring myometrium. mast cells are not accumulated in cervix in association with labour suggesting a less critical role in cervical ripening. further analysis of the role of mast cells in modulating myometrial smooth muscle physiology is warranted. progestins and the glucocorticoid receptor in human myometrial and amnion-derived wish cells. alison j tyson-capper, stephen c robson. reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. background and objectives: progesterone (p) can reduce the risk of preterm birth in some high risk women. in this context there is accumulating evidence that this, at least in part, may be due to anti-inflammatory and immunoregulatory properties of p. target tissue responsiveness to p is considered to be determined by the progesterone receptors (pr) and nuclear co-factors that directly interact with pr. pr and glucocorticoid receptors (gr) share several structural and functional characteristics, including similarities in dna sequence recognition by binding to the same hormone response elements. pr and gr interact with similar chaperones in the absence of ligand and with a similar group of co-activators in the presence of hormones; both can display comparable anti-inflammatory activities under specific physiological conditions. in this study we aimed to investigate whether the anti-inflammatory properties of progestins may be mediated by pr and gr signalling. methods: primary cultures of non-pregnant and term pregnant human myometrial (passage - ) and wish cells were serum starved for hrs and treated with -hydroxyprogesterone ( -hp), progesterone (p), dexamethasone (dex) and immunofluorescent staining and immunoblotting analyses performed. in some experiments cells were pre-treated with ru (a pr/gr antagonist) or org (a pure pr antagonist). results: in the absence of hormone gr appeared to be predominantly cytoplasmic, whereas, upon treatment with -hp and p ( and m) and dex ( nm) gr was abundant within the nuclei of myometrial cells. immunoblotting analyses demonstrated that levels of gr progressively increased within the nuclear fractions of both pregnant myometrial and wish cells in response to increasing concentrations of p ( nm to m), and decreased sequentially within cytoplasmic fractions. in the presence of org gr protein levels remained constant within the cytoplasm. there also appeared to be a slight increase in gr expression, though not statistically significant (p> . ) within both cells types in response to p. conclusion: in this study we show that gr is activated by -hp and p and translocates to the nuclei of human myometrial and amnion-derived cells. in addition, levels of gr increase in response to p. whether p and -hp act as agonists or antagonists for gr in the regulation of hormone response genes associated with the onset of term and preterm labour remains to be elucidated. objective: using a mouse model of inflammation-induced preterm birth (ptb), we have demonstrated dramatic cytokine elevations in the uterus and placenta with concomitant, though less dramatic, cytokine elevations in the fetal liver and brain, associated with neuronal injury. because precise mechanisms of fetal injury in ptb remain unclear, we sought to examine inflammatory cell trafficking, and target organ damage by histopathologic assessment of the placenta, fetus, and fetal brain. study design: hours after intrauterine infusion of saline or lps into the right lower uterine horn of cd- mice, the left upper horn, with the gestational sacs(gs) in situ, was removed en bloc(n= per group) each with - gs with fetuses/treatment group. specimens were fixed, bisected and processed for histology and ihc. inflammatory and hematopoietic cells were quantified using pas, gata- (erythroid precursors), cd , and bm (macrophage-mp) within the placenta, liver, extremity mesenchyme, brain and leptomeninges. the presence of hemorrhage, necrosis, and apoptosis (h ax stain) was assessed. erythopoietin (epo) levels were measured in brain and liver by elisa. results: more neutrophils were present in maternal decidual vessels in lps compared to saline (p= . ). in lps-exposed, fetal mp were increased in the placenta (p= . ), fetal extremity mesenchyme (p= . ), fetal liver (p= . ) and leptomeninges (p= . ) but not in the brain or spinal cord compared to saline. no necrosis, hemorrhage or increased apoptosis was noted in the fetal brains. % of lps-exposed fetuses and % of saline-exposed had liver hemorrhages (p< . ). increases in nucleated erythrocytes and erythroid precursors were found in fetal vasculature of the placenta in lps-exposed (p= . ). epo levels were not elevated in either group. conclusion: intrauterine lps infusion induces acute inflammation predominantly in the maternal circulation of the placenta. in the fetus, there is widespread mp activation, liver hemorrhage and increased erythroid precursors seen in the fetal circulation of the placenta. although histologic evidence of cns damage was not evident, the increased mps present in the leptomeninges may play an important role in inflammatory-mediated cns damage. non-toll-like innate immune proteins: do they change during pregnancy? juan m gonzalez, hua xu, ella ofori, michal a elovitz. obgyn; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: trem- (trigger receptors expressed on myeloid cells) is an important regulator of innate immunity. the natural ligand for trem has not been identified. activation of trem- in the presence of toll-like receptors results in substantial amplification of the host inflammatory response (klesney-tait et al nature immunology ). since inflammatory pathways are implicated in adverse pregnancy outcomes, this novel mediator of inflammation may play a critical role in preterm birth (ptb). therefore, we sought to determine trem- expression in the uterus, cervix, and placenta across gestation and to determine if trem- levels are altered by intrauterine inflammation. methods: in cd- mice, trem- was investigated in non-pregnant (np) and throughout gestation e , e (n= - per group). uterine, cervical, and placental tissues were harvested. using an established mouse model of inflammation-induced ptb, uterine tissue was collected hours after intrauterine infusion of saline (n= ) or lipopolysaccharide (lps) (n= ). for a non-pregnant model, using cd- mice, lps (n= ) or saline (n= ) was injected into the uterine horn following same procedures as with pregnant mice. uteri were harvested hrs later. quantikine ® mouse trem- immunoassay was utilized for these studies. statistical analysis was performed using oneway anova followed by pair-wise comparison if statistical significance was reached (p< . ) results: trem levels are significantly different between np and pregnant uterine tissues (p= . ). e and e trem expression is significantly increased . and . -fold compared to np (p= . and . respectively). trem- levels in the placenta and cervix were not significantly different between e and e . trem levels increased about -fold in the uterus after intrauterine infusion of saline or lps compared to e controls. in nonpregnant, trem levels were significantly different (p= . ) with a -fold increase in trem expression in uteri exposed to lps or saline compared to controls. conclusions: non-toll-like innate immune proteins are differentially regulated during pregnancy compared to the non-pregnant state. the role of trem- in inflammation-induced ptb requires further study. research is warranted to determine if uterine up-regulation of trem in gestation is associated with an increased likelihood of responding to pathogens or severe as a protective mechanism. reduced plasma levels of vitamin d in caucasian women at term are associated with increased rate of infection. chander p arora, adegoke adeniji, susan e jackman, babak forooghi, isaac mostadim, phillip yadegari, calvin j hobel. og-gyn, cedars-siani medical center, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: vitamin d plays an important role in human pregnancy by acting as a regulator of immunity at the fetal-maternal interface. inflammatory changes associated with pro-inflammatory cytokines were reduced by vitamin d while anti-inflammatory cytokines were increased in t lymphocytes. vitamin d status has been defined as deficiency (< . nmol/l), insufficiency ( . to nmol/l) and sufficiency (> nmol/l) . objective: to assess the involvement of vitamin d in the occurrence of maternal infection during pregnacy in women with term deliveries. hypothesis: vitamin d metabolism could affect the rate of infection during pregnancy. study design plasma levels of (oh)d were determined by elisa and the rate of infection was recorded in a behavior in pregnancy study. in this study, ethnically diverse women were evaluated at - weeks (t ), - weeks (t ) and - weeks (t ). maternal infections were documented at each stage as well as at baseline visit with history of infection in current pregnancy. of these subjects who delivered at term two groups ( with no infection during pregnancy, with infection or history of infection) were matched further for non-smoking status, non-diabetics, ethnicity and maternal age. plasma from these were assayed for (oh)d and analyzed for the rate of maternal infection using fisher´s exact test or chi-square test. results although the women delivered at term, the levels of (oh)d in caucasians were significantly lower in the subjects with infection than the ones without (p<. ). women with vitamin d insufficiecy in the first trimester were more likely to develop infection during pregnancy ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ) but not subjects with sufficient vitamin d at t ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ). conclusion the results reveal a positive association between (oh) d concentrations and greater risk of infection. vitamin d deficiency or even insufficiency may, therefore be involved in the pathogenesis of maternal infection during pregnancy. it is probable that vitamin d deficiency or even insufficiency could modulate the maternal susceptibility to infection during pregnancy by a proinflammatory mechanism. in vitro and in vivo observational data suggest that infection leads to caspase activation and apoptosis in the placenta and membranes, however currently there are no data on the role of apoptosis in the pathogenesis of infection associated preterm delivery. here we used group b streptococcus (gbs) as a model pathogen and examined the role of caspase dependent and independent apoptosis in preterm delivery. methods: we injected ( . x ) heat killed group b streptococcus organisms (hk-gbs) intraperitoneally (i.p.) in . day pregnant c b/l mice. the mice were euthanized at hr (n= ) and hr (n= ), the placentas and membranes were removed and assessed for apoptosis by tunel staining. caspase activation and expression were determined by immuno-histochemistry (ih) and western blotting. the effect of apoptosis on preterm delivery was assessed by i.p. treating the pregnant mice with pbs (n= ), dmso (n= ) or pancaspase inhibitor z-vad-fmk (n= ) prior to hk-gbs and observing the animal for delivery for hrs. results: there was a time dependent, gbs-induced increase in apoptosis by tunel assay and caspase activation in the placenta and membranes. in addition hk-gbs-induced the expression of caspase and caspase independent m-calpain in the placenta. z-vad-fmk ( mg/kg), at the maximum concentration that did not induce maternal illness, did not prevent hk-gbsinduced preterm delivery. conclusions: caspase dependent and independent mechanisms are activated in the placenta upon exposure to gbs. systemic adminstration of a pan-caspase inhibitor did not impact upon the occurance or timing of bacterially induced preterm delivery. further studies are needed to assess the role of apoptosis in the pathogenesis of infection associated preterm delivery. early and ( . %) had a late sptb. the mean + sd gestational age at blood draw was + weeks. the median level of bb was higher in women with early as compared with late sptb or term births (p= . for trend). women with bb in the top quartile were . times more likely to have an early sptb as compared with women who had lower levels of bb ( % ci . to , p = . ). there was no association between bb and late sptb (rr= . , % ci = . to ). the adjusted or of an elevated bb for early sptb was . ( % ci = . to , p= . ). when the analysis was restricted to the women with sptb the rr of an elevated bb for early sptb was . ( % ci . to . , p = . ). conclusions: a significant relationship was found between an elevated bb in early pregnancy and early sptb suggesting inflammatory events in early pregnancy, perhaps infection-related, are part of the pathogenic mechanisms. objective: genital tract infection and/or inflammation appears to contribute to the majority of ptds preceding weeks of gestation. ptd in humans has been associated with colonization and/or infection with a variety of different organisms including gram positive and negative bacteria, mycoplasma, ureaplasma, trichomonads, malaria parasites and viruses. the innate immune response to these pathogens is produced by a family of pattern-recognition cell membrane receptors known as the toll-like receptors (tlrs). these studies sought to characterize the tlr isoforms expressed in the preterm mouse uterus, and their modulation during lipopolysaccharide (lps)-induced ptd. methods: using sterile surgical technique, day- pregnant cd- mice underwent intrauterine injection of g lps. subsequently, the mice were euthanized at , , , , and hours after lps injection. uterine tissue was harvested and placed in rnalater; subsequently total rna was isolated using the trizol reagent and genomic dna was removed using turbo dnafree. cdna was made using iscript cdna synthesis kit. pcr primers were designed using published mouse tlr gene sequences. real-time quantitative rt-pcr was performed using the power sybr green master mix and an abi prism multicycler. to confirm tlr amplicon sizes, the rt-pcr products were visualized on a % agarose/tbe gel stained with gelred. results: these studies have confirmed mrna expression of all of the reported mouse tlr isoforms. these tlr amplicons range in size from to bp as expected; amplicon sequences are pending. quantitative rt-pcr studies performed using uterine tissues from five mice at each time point demonstrated that at hours after lps injection, tlr increased -fold and tlr increased -fold (both p< . ). in contrast, the expression of tlr (the ligand for lps) remained stable during the hours after lps; the expression of tlr , , , , , and also remained stable. tlr expression trended upward and tlr trended downward, although neither was statistically significant. conclusions: these studies have confirmed expression of all tlrs within the preterm pregnant mouse uterus, along with characterization of their modulation during lps-induced ptd. these observations are important because they contribute to our understanding of the immunologic signaling events leading to ptd. (funded by nih hd ). udp-glucose and its receptor p y as a new innate immune system in the female reproductive tract. toru arase, tetsuo maruyama, hiroshi uchida, takashi kajitani, masanori ono, maki kagami, hironori asada, yasunori yoshimura. obstetrics and gynecology, keio university, shinjukuku, tokyo, japan. objective: innate immune system involving toll-like receptors has recently emerged in the female reproductive tract (frt). we hypothesize that there may exist new other mucosal immunity in frt. recently, it has been reported that p y , a g protein-coupled p y receptor for udp-glucose (udp-g), is involved in the lung epithelial immune system. the aim of this study is to investigate whether udp-g and p y have a potential as the defense immune system in frt, in particular endometrium. materials and methods: we obtained human endometrial tissues from consenting reproductive-aged patients. the spatiotemporal expression of p y in human and mouse endometrial tissues was analyzed using rt-pcr and ihc. isolated human endometrial cells and a human endometrial epithelial cell line, ishikawa, were cultured, treated with udp-g, and subjected to rt-pcr analysis for il- mrna expression. we also measured the il- secretion using elisa. small interfering rna was used to knock down p y expression. the chemotactic activity of udp-g on neutrophils was tested using transwell assay with ishikawa cells. lastly, mouse uterine tissues were incubated with udp-g and subjected to rt-pcr analysis for mrna expressions of kc and mip- , the il- homologues in mice. results: p y was exclusively expressed in the glandular and luminal epithelium both in human and mouse uteri. treatment with udp-g induced the secretion of il- in ishikawa and human endometrial glandular cells, but not stromal cells, in a dose-and a time-dependent manner. p y knockdown abrogated udp-g-induced il- production. treatment with udp-g also significantly increased the chemotaxis of neutrophils, which was attenuated by co-addition of anti-human il- neutralizing antibody. in the mouse uterus stimulation of udp-g significantly up-regulated the expressions of kc and mip- mrna. conclusions: udp-g is an endogenous molecule and released into the extracellular environment in a lytic manner after cell damage. taken together, our results collectively substantiate a model in which udp-g released from endometrial cells damaged by infection stimulates il- production via p y in endometrial glandular epithelium, which, in turn, recruits neutrophils thereby preventing the progression of infection. thus, udp-g and its receptor p y may be involved in the trans-species mucosal immune system in frt. (jsgi ; , (suppl) , abst # ) but in utero effects on fetal lung remain to be established. we have examined the relationship between duration of iai and subsequent azi treatment on the severity of fetal lung histopathology. we hypothesized that early treatment would prevent the development of advanced lesions, while late treatment may reduce the severity of lung damage. study design. thirteen chronically instrumented rhesus monkeys received intraamniotic inoculation of u. parvum (serovar ; - x cfu) at ± . days gest. age (dga, mean ± sem, term= d). six of these animals received maternal i.v. azi ( . mg/kg q h or q h for d) either alone (n= ) or in combination (n= ) with dexamethasone (dex; mg/kg/d i.v. for d) and indomethacin (indo; mg/d p.o for d). tissues were obtained at cesarean section for histopathologic assessment. leukocytic infiltration of aveolar spaces and septal walls, type ii pneumocyte hyperplasia and peribronchiolar lymphocytic aggregates were scored as absent ( ), minimal ( ), moderate ( ) and severe ( ). results. inoculation-to-delivery interval was - d for combined treatment groups and was similar to long duration infection without treatment ( - d). treatment effects were tabulated as mean scores and compared as follows: control (n= ), score ; short duration infection ( - d; n= ), score ; long duration infection ( - d; n= ), score ; short duration infection + treatment (n= ), score ; long duration infection + treatment (n= ), score . conclusions. histopathologic findings of fetal pneumonia progressively worsen with duration of u. parvum iai. early maternal azi treatment prevents development of advanced lung lesions, while later treatment may reduce the severity of fetal lung damage. our results suggest that in utero treatment of iai may lower the risk of neonatal bronchopulmonary dysplasia. support: nih hd , rr . brain associated with adverse neurodevelopmental outcome. lps triggers proinflammatory responses through toll-like receptor- (tlr ). mitogen activated protein kinases including c-jun-n-terminal kinase (jnk) have been reported to be implicated in tlr signalling pathways and play important role in both onset of labor and brain injury. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of specific inhibitor of jnk signaling, d-jnk inhibitory peptide (d-jnki) can (i) inhibit jnk activity in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr- specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. in vitro kinase assay demonstrated the activation of jnk in response to lps in the maternal uterus and fetal brain. furthermore, the brain specific jnk was found to be the major jnk isoform activated by lps in the fetal brain. co-administration of d-jnki with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and reduced pup mortality up to %. this was associated with inhibition of jnk activity in both maternal uterus and fetal brain. in addition, we have found that treatment with lps significantly up-regulated cox- , cxcl (il- equivalent) and ccl in myometrium and this is significantly suppressed after co-administration of d-jnki. we conclude that specific inhibition of jnk signaling may have a potential of controlling preterm labor and preventing fetal brain damage as a result of infection/inflammation. the prostaglandin -deoxy- , -prostaglandin j delays lps-induced preterm delivery and reduces mortality in the mouse. intrauterine infection is a common trigger for preterm birth, and is also a risk factor for the development of neurodevelopmental abnormalities in the neonate. bacterial lipopolysaccharide (lps) binds to toll-like receptor- (tlr ) to activate pro-inflammatory signaling pathways. the transcription factor nuclear factor kappa b (nf-kb) is a key player in the orchestration of the inflammatory response and has a central role in parturition. we have previously shown that exposure to the anti-inflammatory cyclopentenone prostaglandin -deoxy- , -prostaglandin j ( d-pgj ) inhibits il- b-induced nf-kb activity and cox- expression in human myometrial and amnion epithelial cells in vitro. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of d-pgj can (i) inhibit nf-kb in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. co-administration of d-pgj with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and conferred protection from lps-induced fetal mortality up to %. we have looked at the expression profile of several labor associated genes in myometrium hours after lps administration. (otr, connexin and , cox- , cox- , cxcl (il- equivalent) and ccl ). we have found that treatment with lps significantly up-regulated cox- , cxcl and ccl and this is significantly suppressed after with co-administration of d-pgj . western analysis for ser -phosphorylated p and ikkb in-vitro kinase assay has demonstrated that lps induced activation of nf-kb at both h and h. co-administration of d-pgj was associated with inhibition of nf-kb activation in both the maternal uterus and the fetal brain. conclusion d-pgj may have potential as a therapeutic agent in the management of preterm labor and, by targeting the player nf-kb, may have the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation. synergistic macrophage response to co-activation of tlr- and tlr- : mechanisms and implications for bacterial/viral co-infection. vladimir ilievski, emmet hirsch. , department of ob/gyn, evanston northwestern healthcare, evanston, il; department of ob/gyn, feinberg school of medicine, northwestern university, chicago, il. background: toll-like receptors (tlrs) recognize structural components of pathogens and initiate host defenses. tlr- responds to gram-positive organisms and peptidoglycan (pgn), a gram-positive cell wall constituent. tlr- is activated by viral infection in response to double-stranded rna. polyinosinic-cytidylic acid (poly(i:c)) is a tlr- ligand. we have shown that pgn and poly(i:c) have a synergistic effect on the expression of downstream genes for both tlr- and tlr- . here we identify mechanisms underlying this synergy. methods: mouse peritoneal macrophages or a mouse macrophage cell line (raw . ) were treated in tissue culture with either pgn ( g/ml), poly(i: c) ( g/ml) or both pgn and poly(i:c) either simultaneously or sequentially for - hours. total rna was extracted and duplex rt-pcr was performed for inducible nitric oxide synthase (inos), interleukin (il- ), tumor necrosis factor (tnf), the chemokine rantes and tlr- , normalized to the housekeeping gene gapdh. results: compared to stimulation with either pgn or poly(i:c) alone, costimulation of raw . cells with both pgn and poly(i:c) resulted in synergistic expression of inos, il- , tnf and rantes (p< . for all) at and hours . sequential stimulation with either pgn or poly(i:c) for h followed by incubation for an additional h with the alternate ligand also induced synergistic expression of the same rnas, albeit at lower levels than were elicited by simultaneous stimulation. in contrast, incubation with either pgn or poly(i:c) for h followed by medium for h induced minimal to no gene expression. both pgn and poly(i:c) induced tlr- mrna after h but not h. tlr- mrna was not detectable by rt-pcr. in primary peritoneal macrophages, similar synergy due to pgn and poly(i:c) was seen. conclusions: simultaneous or sequential exposure to pgn and poly(i:c) exerts a synergistic effect on the expression of inflammatory mediators in macrophages. interestingly, either one of these two tlr pathways can prime cells for activation of the other pathway. a possible mechanism for this effect may be induction of tlr- by either tlr- or tlr- activation. these observations have implications for bacterial/viral co-infection. this is a secondary analysis of a prospective cohort study. after irb approval, daily blood samples were obtained from pprom subjects and analyzed for il- by elisa. paired maternal serum il- levels from subjects were divided into groups: il- levels obtained - hours prior to completion of antibiotics and those obtained - hours after completion of antibiotics. the wilcoxon signed rank test was used to perform the data comparison on the analyze-it statistical software program. statistical significance was defined as p< . . of the pprom subjects, the maternal age was . yrs; gestational age at admission was . weeks; latency was . days; gestational age at delivery was weeks; and infant birth weight was grams. median maternal serum il- levels obtained off antibiotics were significantly higher when compared to those on antibiotics ( . vs. . pg/ml, p< . ). the results of this investigation suggest that maternal serum il- levels rise after discontinuation of antibiotics. the optimal duration of antibiotics administration in the setting of pprom is unknown. this data suggests a role for continuation of antibiotics in women with pprom in order to prolong the latency period and potentially decrease neonatal morbidity. to identify clinical and pathologic factors associated with fetal inflammatory responses in the placenta from term parturients. methods: retrospective cohort study of consecutive term parturients with submitted placentas in . placentas with histologic chorioamnionitis were divided into two cohorts: group -maternal inflammatory responses only, and group -maternal and fetal inflammatory responses. maternal and fetal inflammatory responses in the placenta were classified according to guidelines established by the amniotic fluid infection nosology committee of the perinatal section of the society of pediatric pathologists. selected demographic, intrapartum, newborn and placental characteristics were analyzed with t-tests and chi-square tests as appropriate. multiple logistic regression was used to identify independent predictors of fetal inflammatory responses in the placenta. results: of consecutively submitted placentas, had histologic chorioamnionitis: with maternal inflammatory responses only (group ) and with both maternal and fetal inflammatory responses (group ). fetal inflammatory responses observed in group were associated with higher stages of maternal inflammatory responses (p<. ). % of fetal inflammatory responses were stage i (acute chorionic vasculitis or phlebitis).group patients were more likely to have had amnioinfusion ( % v %, p=. ) and less likely induction of labor ( % v %, p=. ). group was more likely to have had intrapartum fever ( % v %, p=. ) and maternal tachycardia ( % v %, p=. ). newborns from group were more likely to have been observed for sepsis ( % v %, p <. ) and have an apgar score at minutes ( % v %, p=. ). a logistic regression model showed that stage ii or greater maternal inflammatory responses (or . ) and amnioinfusion (or . ) were independent predictors of fetal inflammatory responses. conclusion: higher stages of maternal inflammatory responses in the placenta and amnioinfusion were independent predictors of fetal inflammatory responses in term parturients with histologic chorioamnionitis. objective: previous studies have shown that sulfasalazaine (sasp) inhibits lipopolysaccharide (lps)-induced nf-b activation and decreases lpsstimulated interleukin- (il- ) production by cultured explants of placenta, amnion and choriodecidua with no effect on cell viability. bacteria-induced il- production in the cervix is a potential mechanism for premature cervical ripening that may lead to preterm birth. it is unclear, however, whether sasp can inhibit il- production by endocervical cells. therefore, we performed a series of in vitro studies to determine if sasp inhibits il- production by endocervical epithelial cells stimulated with bacterial pathogens associated with preterm birth. methods: human endocervical epithelial cells were incubated with - . mm sasp overnight and then stimulated with ccu/ml ureaplasma parvum, cfu/ml escherichia coli, or x cfu/ml gardnerella vaginalis for another overnight incubation in -well cultures. conditioned medium was then harvested and production of il- was quantified by elisa. viability of the cells was ascertained at the end of the experiment with the mtt-assay. each treatment was applied in quadruplicate wells and experiments were repeated times using different batches of cells for each pathogen. results were evaluated using the general linear models procedure of sas for a randomized block design. results: sasp had no detectible effect on il- production by endocervical cells not treated with bacteria. at the highest concentration tested ( . mm), sasp significantly inhibited il- production by cultures stimulated with e. coli (p< . ), u. parvum (p< . ), and g. vaginalis (p< . ). viability of the cells, however, was significantly reduced by sasp at . and . mm in the both the presence and absence of bacteria for all experiments. conclusion: although high concentrations of sasp inhibit il- production by cultures of endocervical cells stimulated with pathogens associated with preterm birth, this effect may be due to toxicity of the antibiotic on the cells. the effect of -hydroxyprogesterone caproate on lipopolysaccharide stimulated peripheral blood mononuclear cells from pregnant women. richard h lee, aimin li, frank z stanczyk, jinwen i lin, t murphy goodwin. obstetrics and gynecology, university of southern california, los angeles, ca, usa. introduction: -hydroxyprogesterone caproate ( -ohpc) reduces the rate of recurrent preterm birth in high-risk women. however, there are lines of evidence to suggest that -ohpc alters inflammatory response in the setting of gram-negative infection. in a mouse model, -ohpc decreased the rate of preterm birth but was associated with significantly increased maternal morbidity when mice were exposed to lipopolysaccharide (lps). in non-pregnant women, -ohpc pre-treatment of whole blood exposed to lps significantly increased the production of tnf-. objective: to determine if -ohpc has an effect on the production of proinflammatory cytokines from peripheral blood mononuclear cells (pbmc) in pregnant women. methods: pbmc were isolated from fresh whole blood samples of pregnant women between and weeks. pregnant women had no prior history of preterm birth. cells were treated with -ohpc ( m) and escherichia coli lipopolysaccharide (lps, g/ml) alone or in combination. after and hours of culture, supernatants were collected and tested for tnf-and il- levels by enzyme-linked immunosorbent assay (elisa). statistical analysis was performed using non-parametric tests. p < . was considered significant. results: pbmc exposed to lps significantly increased tnf-and il- secretion compared to untreated controls (p= . and p= . ). tnf-concentrations were not significantly different between lps and lps+ -ohpc treated cells at both and hours (p= . and p= . ). il- production was significantly increased after -hour treatment with lps+ -ohpc compared to lps alone ( , [ , - , ] background. recent clinical trials indicate that progestins reduce the incidence of preterm birth in some high risk pregnancies. it has been proposed that progesterone promotes uterine quiescence, in part, via its antiinflammatory properties with inhibition of pro-inflammatory gene expression. it is intriguing that progestins are clinically effective given the considerably increased background circulating levels of the hormone during pregnancy. we hypothesised that non-classical progesterone signalling pathways contribute towards mediation of the anti inflammatory effects of progestins. to the endotoxin lipopolysaccharide (lps) by activation of inflammatory pathways. myometrial cell cultures were treated with lps ( g/ml)+/progestin ( nm). two progestin compounds were investigated. natural progesterone (p) is known to have a strong affinity with progesterone receptor (pr) analogues in contrast to -hydroxyprogesterone ( -hp) which, in the absence of esterification with caproate or acetate, has been reported to have no progestogenic activity at pr. the effect of p and -hp on the activation of two inflammatory genes known to be associated with labour (cycloxygenase [cox- ], toll-like receptor [tlr- ]) was evaluated. cox- and tlr- were detected at the protein and mrna levels using sds-page and rt-pcr. results. lps-induced expression of cox- and tlr- proteins were significantly inhibited by both p (p< . and p< . , respectively) and -hp (p< . and p< . , respectively). furthermore, preliminary results indicate that co-incubation with the anti-progesterone mifepristone, fail to abrogate the anti inflammatory effect associated with progestin treatment. conclusion. non-classical progesterone signalling pathways have a role in mediating the anti-inflammatory properties of progestins. further elucidation of the molecular actions of progestins may allow novel approaches to ameliorate the inflammatory response associated with preterm labour. detection and transcriptional expression of tlr- , tlr- and tlr- at the maternal-fetal interface. jacqueline p tilak, karen zakharian, alexandra tungol, gabriel o schubiner, david m svinarich. patient care research, providence hospital, southfield, mi, usa. preterm labor and delivery remains a leading cause of neonatal morbidity and mortality and bacterial infection is considered to be the most common etiology. toll-like receptors (tlr's) and the associated components of the innate immune system may represent a first line of defense against these pathogens. tlr's belong to a family of pattern-recognition receptors that bind to highly conserved pathogen-associated molecular patterns (pamps) including lipopolysaccharide (lps), lipotechoic acid (lta) and cpg dna, and are a key component of the innate immune system. this study was undertaken to characterize the transcriptional expression and regulation of tlr- , tlr- and tlr- within gynecologic and gestational tissues. human first trimester trophoblasts, endometrial mesoderm, ectocervix, choriocarcinoma and neonatal epithelium, were cultured in media alone or in the presence of either lps ( mg/ml) or lta ( mg/ml) for , , , , and hours. total rna was isolated and semi-quantitative rt-pcr was performed using intron-spanning amplimers to tlr- , tlr- , tlr- and gapdh. following gel electrophoresis, the integrated optical densities were determined for the corresponding pcr products and normalized with respect to gapdh levels. inducible transcription of tlr- with lta was observed in choriocarcinoma cells ( -fold, h), and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in ectocervical cells ( -fold, h), choriocarcinoma cells ( -fold, h) and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in choriocarcinoma cells ( fold, h) and neonatal epithelial cells ( -fold, h). all cell lines showed at least constitutive levels of transcription for tlr- , tlr- and tlr- . these data demonstrate that tlr- , tlr- and tlr- are transcriptionally expressed either constitutively or inducibly in both gynecologic (endometrial mesoderm, ectocervix) and gestational (chorion, trophoblast), tissues. translation of these receptors suggests that the innate immune system may function at the maternal-fetal interface to help protect the fetus against infection and prevent or diminish the likelihood of prematurity. objective: further to our development of a sheep model of intrauterine ureaplasma spp infection, we aimed to examine the capability of ureaplasma colonization in the amniotic fluid to infect the fetus and alter lung and brain development. methods: at days of gestation (d, term= d) ewes bearing single fetuses were given a single ultrasound-guided intra-amniotic injection of (a) x colony forming units (cfu) of u parvum (serovar , n= ; serovar , n= ), (b) x cfu of u parvum (serovar , n= ; serovar , n= ) or (c) media control (n= ). at d, fetuses were delivered by cesarean section. amniotic fluid and umbilical arterial blood samples were collected. fetal body weight was recorded, fetal cerebrospinal fluid (csf) collected and a descending pressurevolume curve constructed after inflation of the lungs to cmh o. fetal membranes were immersion fixed and the fetal brain was perfusion fixed with % paraformaldehyde. the fetal brain and membranes were blocked, paraffin embedded, stained and viewed under the light microscope. results: chronic intra-amniotic colonisation with u parvum serovar or (ureaplasmas) did not result in fetal abortion or death. amniotic fluid ureaplasma titers at delivery were not dose-dependent. chronic ureaplasma exposure did not affect fetal body or brain weights, or result in a fetal systemic inflammatory response. umbilical arterial blood gases at delivery were similar between ureaplasma-and media-exposed fetuses. chronic intra-amniotic exposure to ureaplasmas resulted in higher inflammatory cell scores in the fetal membranes compared to media controls (p< . ). lung compliance was increased in ureaplasma-exposed fetuses compared to controls (p< . ). there were no gross anatomical changes observed in the white or grey matter in the cerebral hemispheres of fetuses that had been exposed to ureaplasmas; even in animals (n= ) that had csf ureaplasma colonisation. conclusion: chronic ureaplasma colonisation enhances fetal lung compliance without gross anatomical changes in the fetal brain. background: an important source of vitamin d is its synthesis by skin from uv solar radiations. the skin pigment melanin absorbs uv photons thus reducing the vitamin d synthesis by more than % making african americans at high risk for vitamin d deficiency. low maternal vitamin d status during pregnancy has been linked to molecular pathways for adverse outcomes. objective: the nf b transcription factor regulates genes involved in inflammation and immune processes, and is proposed to play a major role in the successful outcome of pregnancy and labor. prior immunohistochemical (ihc) and biochemical studies have been conflicting regarding nf b activation in human intrauterine tissues. the aim of this study was to quantify nuclear localization of p , the major tranactivating nf b subunit, in full-thickness fetal membranes (fm) and myometrium using ihc. methods: a retrospective analysis of paired fm, decidua, and myometrial samples was conducted using tissues collected from women in cohorts: preterm no labor (pnl, n= ), preterm labor (ptl, n= ), spontaneous term labor (stl, n= ), and term no labor (tnl, n= ). subjects were delivered between gestational ages - weeks (preterm) and - weeks (term) by cesarean. paraffin sections were stained with polyclonal (rabbit) anti-human p and microscopically examined. myometrial samples were categorized in a blinded fashion to groups of staining: no nuclear p (-), rare nuclear p (+), and > % nuclear p (++). a p nuclear labeling index (nli; % nuclear p /total cells) was calculated for each histological layer in full-thickness fm specimens using a blinded targeted randomization scheme consisting of non-overlapping low-magnification fields. results: nuclear p labeling was rare in amnion and inconsistently present in chorion. in decidua, p nuclear labeling was observed in all cases; however, decidual nli did not vary significantly across cohorts [pnl- % ( - %); ptl- % ( - %); tnl- % ( - %); stl- % ( - %); all values are median (iqr)]. in myometrium, ++ p nuclear labeling was significantly associated with labor, but present only in a portion of cases (ptl- %; stl- %). despite a trend, decidual nli was not significantly correlated with myometrial nuclear p labeling: myometrial specimens classified as -, +, and ++ corresponded with decidual nli of % ( - %) [median (iqr)], % ( - %), and % ( - %), respectively. conclusions: in a comprehensive ihc analysis, significant nuclear p immunolabeling was observed in myometrial cells following labor. nuclear p labeling in decidua was present in all cases, and did not vary significantly among the cohorts. the inflammatory cytokines interleukin- and tnf-increase g-csf expression in term decidual cells. objectives: chorioamnionitis (cam) elicits premature rupture of the membranes and pre-term delivery. previously, we found that the decidua from cam patients contains much higher neutrophil numbers than control decidua, but macrophage numbers are similar in both groups. granulocyte colony-stimulating factor (g-csf) enhances granulocyte colony formation chemoattraction and activation. the amniotic fluid during cam contains elevated tnf-and il- levels. to account for the marked influx of neutrophils infiltration in cam-complicated decidua, tnf-and il-effects were assessed on g-csf expression in term decidual cell (dc) monolayers. methods: confluent leukocyte-free term dcs from normal term deliveries were primed with - m estradiol (e ) + - m medroxyprogesterone acetate (mpa) for days, then switched to serum-free defined medium (dm) with steroids ± tnf-or il- . after h, aliquots of conditioned dm supernatants, cell lysates and extracted rna from h parallel incubations were frozen. secreted g-csf was measured by elisa in conditioned dm and normalized to cell protein and mrna was assessed by quantitative real time rt-pcr and normalized to -actin mrna. results: in cultured first trimester dcs, basal g-csf levels in conditioned dm were . ± . pg/ml/ug cell protein [mean ± sem, n= ] and did not differ from e +mpa basal levels. the addition of ng/ml of tnf-or il- significantly elevated g-csf output to . ± . and ± respectively (p< . ), representing more than a -fold and , -fold change; respectively. western blotting confirmed the elisa results. quantitative rt-pcr demonstrated corresponding changes in g-csf mrna levels as found for the elisa measurements. concentration-dependent effects for both tnfand il- from . to . ng/ml were observed. conclusions: when extrapolated to the placental milieu of cam, the marked increase in g-csf elicited in term dcs by tnf-and il- may provide a mechanism to account for the selective increase in decidual neutrophils versus macrophages. background. the immunological mechanisms of the female reproductive tract are unclear. toll-like receptors (tlrs), innate immune receptors that combat microbial infections and establish adaptive immunity, may play a role. infection in pregnancy has been associated with preterm birth and tlrs may modulate the host response to such infections. we hypothesised that the distribution of tlr and tlr in cervical epithelial cells may differ with pregnancy, and that oestrogen may contribute to the modulation of these receptors. aims and objectives. . to compare tlr and tlr protein expression, using immunocytochemistry, in the cervical epithelium of pre-menopausal non-pregnant women with pregnant women at term. methods. fresh non-pregnant (n= ) and pregnant (n= ) human cervical cells were obtained by smear and tlr and tlr expression investigated by immunocytochemistry. cervical epithelial cells from nonpregnant women obtained by smears were then coincubated with varying concentrations of estradiol, and tlr and tlr protein expression quantified by immunocytochemistry. results. using pixel-based image analysis software, we demonstrated a reduction in tlr expression in late pregnant compared with non-pregnant cervical epithelium (p= . ), whilst tlr did not appear to differ. there was an apparent up-regulation of tlr protein expression in response to beta-oestradiol (n= ) objective: to evaluate in vitro antimicrobial activity of cranberry-exposed urine against common urinary pathogens. subjects were pregnant women enrolled in a clinical trial evaluating the effect of daily cranberry juice cocktail or matching placebo on asymptomatic bacteriuria and other urinary tract infections. methods: -hour urine samples from pregnant women who were randomized to cranberry or placebo in three treatment arms: a. cranberry two times daily with meals (c, c; n= ), b: cranberry in the am, then placebo at dinner (c, p; n= ), c: placebo two times daily with meals (p, p; n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment groups and collected -hour urine specimens from them. the ph of all urine specimens was adjusted to ph= and filtered. aliquots of each were independently inoculated with overnight culture of - cell/ml each of single strains of e. coli with fimbriae type i and type ii, k. pneumoniae, and c. albicans. after hours of incubation for e. coli and k. pneumoniae, and hours for c. albicans cfu/ml of each specimen were enumerated by subculture with quantitative plate counts in duplicate. results: there were no differences for any of the antimicrobial activities between pregnant and non-pregnant groups, or based on treatment allocation. we were able to perform antimicrobial assays on the urine of women exposed to cranberry juice or placebo, but unable to demonstrate differences based on treatment allocation or pregnancy. this may be due to beta-error. further studies are planned. supported by r dk - ; clinical trials registration nct . ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • objective: to measure compliance, we sought to evaluate the use of a bioassay for cranberry in the urine of women enrolled in a clinical trial designed to determine the effect of daily dosing of cranberry juice cocktail or matching placebo on the incidence of asymptomatic bacteriuria (asb) and other urinary tract infections (uti) during pregnancy. methods: we collected -hour urine specimens from pregnant women who were randomized to ingest cranberry or placebo in three treatment arms: a: cranberry two times daily with meals (n= ), b: cranberry in the am, then placebo at dinner (n= ), c. placebo two times daily with meals (n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment regimens (group a: n= , group b: n= , group c: n= ), and collected -hour urine specimens from them. bacterial anti-adhesion effects of the cranberry-exposed urine were evaluated utilizing a human red blood cell hemagglutination assay specific for uropathogenic p-fimbriated e. coli. activity was quantified as , , and %. results: when combining all subjects and dosing regimens, the sensitivity of the assay was %, range % in the pregnant group assigned single daily dose of cranberry to % in the non-pregnant group assigned to multiple daily doses. the specificity ranged from % to %. conclusions: these data indicate that the bioassay can be applied to the pregnant patient population, although the sensitivity of the assay is variable. higher daily dosing appears to confer greater sensitivity. further studies are needed to evaluate the utility of this bioassay for compliance. supported by r dk - . clinical trials registration nct . objective: increasing evidence suggests that inflammation plays a crucial role in initiation of labour both at term and preterm. we have previously shown upregulation of pro-inflammatory cytokines in myometrium, cervix and membranes at term labour. we have also shown that myometrium and cervix is invaded by leukocytes at the time of labour, and these leukocytes express pro-inflammatory cytokines. in this study, we aimed to test the hypothesis that pro-inflammatory cytokines and toll-like receptor and (tlr and ) mrna expression are up-regulated in circulating leukocytes during term and preterm labour. methods: peripheral blood samples were taken from pregnant women: - weeks gestation (w) preterm not in labour (ptnl) n= ; - w preterm in labour (ptl), n= ; - w term not in labour (tnl) n= ; and - w term in labour (tl) n= . leukocytes were isolated using dextran sedimentation. total rna was isolated and reverse transcribed using high capacity cdna archive kit, and mrna expression determined by real-time pcr (abi, taqman). student's t-test was used to compare outcomes between groups. results: messenger rna expression of il- , icam- , mcp- , tlr and tlr was significantly increased in tl compared to gestation matched tnl. the expression levels of il- b, il- and tlr- were significantly greater in ptl compared with gestation matched ptnl. (table i) . conclusions: we show up-regulation of pro-inflammatory cytokine production in circulating leukocytes in both term and preterm labour. thus, the pathophysiology of labour seems to involve the upregulation of proinflammatory cytokine production in peripheral blood leukocytes, followed by their invasion into the myometrium and cervix. this work further contributes to our understanding of the pathophysiology of parturition, and suggests that peripheral blood leukocytes may be potential targets for therapeutic agents aimed at modifying the time course of parturition. introduction. the mechanisms of innate immunity and tolerance in the female reproductive tract (frt) are ill-understood but involve the pattern recognition toll-like receptors (tlrs). we have previously demonstrated high expression levels of tlr gene and protein in fresh human cervical epithelium. aims. in order to gain insight into the immunological role of cervical epithelial cells (cecs), we sought to determine cec responses to the following tlr- agonists: macrophage-activating lipopeptide (malp- ), pam csk , and peptidoglycan. methods. cecs were isolated by smears and compared between pregnant ( rd trimester) and nonpregnant women. following cell preparation, flow cytometry was performed using a direct staining procedure with mouse anti-human tlr primary antibody and its igg , isotype control, to determine tlr- protein expression. a further nonpregnant smear samples were each prepared, and seeded at a concentration of , cervical epithelial cells/ml into -well cell plates. cells were incubated at °c in % co overnight with the tlr agonists malp- and pam csk (at concentrations of and ng/ml), peptidoglycan( ng/ml), il- ( ng/ml, positive control) and rpmi + -glutamine media only (vehicle). following centrifugation, all resulting supernatants were stored at - °c until the concentration of il- was determined by elisa and an array of cytokines by bead assays. results. both pregnant and nonpregnant cecs expressed tlr (specific mean fluorescence . vs . respectively) but did not differ (p = . ). unstimulated cells incubated with buffer alone demonstrated high il- levels ( pg/ml), which did not differ following stimulation with malp- ( pg/ml), pam csk ( pg/ml) or peptidoglycan ( pg/ml). results of an array of pro-and anti-inflammatory cytokines following incubation of cells stimulated with tlr agonists are pending. conclusion. high basal il- levels suggest constitutive expression by cecs but the physiological relevance of this intriguing observation is unclear. that cec stimulation with tlr- agonists did not lead to further release of il- may epitomise the resistance of these cells to antigenic challenge by the vaginal commensal flora. cecs may play a pivotal role in modulating the immunological tolerance necessary to minimise inappropriate inflammation in the cervix. there are several epidemiological and clinical studies that omega- fatty acids and related fish oils can decrease the premature labor and birth. however, the scientific mechanisms are not well elucidated. this study was carried out to investigate the effects of omega- fatty acids on producing pge and il- , in human umbilical vascular endothelial cell(huvec) media with artificial inflammation as an infection-induced premature labor tissue model. also, if there are some significant effects with omega- fatty acids, we want to investigate the possible mechanisms. materials and methods; human umbilical vascular vascular cell primary culture was done. in the adequate cell confluence in each cell dish, pretreatment with various concentrations of epa, dha and troglitazone (another ppar-ligand) and incubation were done for hours in °c, co incubator. after that, g/ml conc. of lipopolysaccharide(lps) was treated to the each cell dishes and incubated for hours. the cell media were collected, and pge and il- concentration were checked by elisa. the each cell lysates were collected and western blot anaysis for cyclooxygenase(cox)- were done. on the other hand, nuclear factor kappa b (nf b) luciferase vector was transfected and then did the same pretreatment with epa, dha and troglitazone and lps treatment to each cell dishes. after that, nf b luciferase activity was checked with luminometer. statistical analysis was done by student t-test. results: epa, dha and troglitazone decreased the pge (p< . ) and il- (p< . ) significantly in elisa. cox- expression revealed the significant reduction with pretreatment of epa, dha and troglitazone in higher concentration ( , m) in the western blotting (p< . ). nf b luciferase activity were also significantly decresed with pretreatment of epa, dha and troglitazone in higher concentration (p< . ). conclusion; this study offers some scientific mechanisms, by which omega- fatty acids (epa, dha) and troglitazone may be one kind of the preventive medicine for infection-induced preterm labor and delivery. also, these effects may come from the common mechanism of epa, dha and troglitazone, ppar-ligands. the next study would be how the cox- , nf b and pparare related. ( mg/ml). cytokine expression was measured in medium using the bio-plex suspension array system (bio-rad). mean expression was compared between the two groups using t test. results: -aminopurine significantly inhibited lps stimulated cytokine production by human myometrium. in contrast, there were no significant differences in expression after mpa treatment, although a trend towards inhibition of proinflammatory cytokine and an increase in il- production was noted. introduction: intrauterine infection is now recognised as a major antecedent of white matter injury (wmi) in the preterm infant brain, which can manifest later as cerebral palsy or as varying degrees of cognitive dysfunction. wmi in these infants is characterized by damage to immature oligodendrocytes, and has been linked both to microglial activation and to elevated levels of tnfa, il- b and il- . we have developed a fetal sheep model for diffuse and focal wmi, based on repeated administration of e. coli lipopolysaccharide (lps) as the stimulus for an inflammatory response. methods: surgery to implant fetal vascular catheters was performed on pregnant ewes carrying single fetuses at d - of gestation. fetuses received repeated iv injections of lps ( ng/kg estimated fetal weight/day) between d and d . plasma levels of pro-inflammatory cytokines were determined in fetal arterial blood samples taken between d and d . at d ewes and fetuses were euthanized and fetal brain tissue samples collected for histological analysis. results: five days after final administration of lps to fetuses we observed a pattern of wmi similar to that seen clinically, ranging from focal to diffuse injury within the periventricular region, impairment of white matter (cnpase; marker for immature/mature oligodendrocytes) tracts, and thinning of the corpus callosum, characterised by oligodendrocyte disruption and microglial proliferation in the surrounding and sub-cortical white matter. we also found a progressive rise in fetal plasma tnf levels between days and (day two of treatment to third day following final dose of lps). background: plasma fatty acid (fa) levels are associated with altered host inflammatory responses, blood pressure regulation, and insulin resistance, characteristic features of pregnancy-induced hypertension (pih). most studies compare the n- and n- polyunsaturated fatty acids (pufas). in addition, recent data demonstrate that saturated and trans-fas promote inflammation. based on the immunomodulatory activity of various fas, we explored their effects on placenta inflammatory responses ex vivo. methods: placenta cells were isolated from fresh human (anonymous), term placentas and treated with/without lipopolysaccharide (lps) with various fas, including saturated fas, trans-fas, and n- and n- pufas (at physiological concentrations). after an overnight treatment, tnf-alpha (tnf) production was determined. data were analyzed by analysis of variance (anova) followed by the dunnett's test. results: oleic acid ( : n- ), a cis-monosaturated fa common in the mediterranean diet, did not induce significant placenta tnf production (fig. a) . by contrast, elaidic acid ( : n- ), a trans-monosaturated fa, induced tnf production by -fold compared to vehicle (fig. a) . likewise, palmitic acid ( : ), a saturated fa, induced placenta tnf production by -fold (fig. a) . to mimic inflammation, placenta cells were treated with lps ex vivo in the presence/absence of fas. docosahexanoic acid ( : n- , dha) reduced placenta tnf production by up to % following stimulation (fig. b) . similarly, treatment of placenta cells with linoleic acid ( : n- , la) or arachidonic acid (n : n- , aa) suppressed tnf production by up to % and %, respectively (fig. b) . conclusions: both saturated fas and trans-fas, which positively correlate with hypertension, induce placental tnf production. the n- fa precursors to prostaglandins, aa and linoleic acid, reduce placental tnf production following stimulation. likewise, dha, a product of n- fa metabolism commonly consumed in fish oil and associated with improved blood pressure, suppresses tnf production by stimulated placenta cells. vegf and flk mediated glomerulogenesis in offspring exposed to maternal hypernatremia. roy z mansano, mina desai, ahmed abdel-hakeem, thomas r magee, tazmia q henry, cynthia nast, john s torday, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: growth restricted human and animal offspring, resulting from maternal nutrient restriction or uteroplacental insufficiency, consistently demonstrate reduced glomerular number and a predisposition to adult systemic hypertension and renal compromise. in contrast, maternal water restriction (wr) produces newborns with a unique programmed phenotype of increased glomerular number. glomerulogenesis is dependent, in part, on appropriately timed and quantified vascular development. as vegf and its receptor flk have crucial stimulatory effects on renal vasculogenesis, we hypothesized that maternal wr-induced morphologic renal changes are secondary to vegfmediated vasculogenic effects. methods: from day to term gestation, pregnant rats received either ad libitum food and water (control, n= ), or wr to produce an increment of meq/l in plasma sodium (n= ). following delivery, all dams received ad libitum food and water. at d after birth, offspring kidneys were extracted for mrna. vegf and its receptor, flk , mrna levels were determined using real-time rt-pcr (presented as fold difference normalized to s). at d after birth, offspring glomerular number were determined in formalin fixed m sections using histomorphometric analysis. values are means±se. results: wr offspring (day ) had higher glomerular number than control (wr ± , control ± per mm , p< . ). flk mrna expression was increased in wr offspring kidneys (wr . ± . , control . ± . , p< . ) as compared to controls. in contrast, vegf mrna expression in wr offspring kidney was comparable to control (wr . ± . , control . ± . , p= . ). conclusion:prenatally wr offspring demonstrate significantly increased glomerular number. as vegf recruits angioblasts to the developing glomerulus via its receptor flk , increased receptors (flk ) with the same level of the ligand (vegf) suggest that enhanced vasculogenesis may represent a putative mechanism for increased nephrogenesis in wr offspring. modulation of newborn vasculogenesis via vegf and flk expression may represent a therapeutic option for growth restricted offspring with decreased glomerular number. objective: maternal food restriction (mfr) during gestation (embryonic day to birth) reduces rat kidney glomerular number by % at weeks of age. undernutrition during human gestation leads to similar impaired nephrogenesis and increased hypertension in adults. renal development may be delineated into stages including ureteric bud branching, mesenchymal to epithelial transformation and glomerulogenesis. previously, we detected dysregulation of genes controlling nephrogenesis at gestational ages, e -e , suggesting that mfr has significant effects on ureteric bud branching. in the present study we evaluated the effect of this dysregulation on early nephron development in e fetal kidney explants from dams with mfr. methods: e fetal kidneys were collected from % mfr pregnant rats and ad libitum control rats (n= per group and time point), incubated in dmem/f k medium for , , , , and days, and fixed with % paraformaldehyde. kidneys were stained with fluorescein-labeled dolichos biflorus agglutinin (dba), images captured and terminal ureteric buds quantitated. all values are presented as mean ± sem. differences were considered significant at p< . . results: mfr ex-vivo kidneys at day demonstrated an initial moderate reduction in terminal branches versus control (c) samples (mfr: ± vs c: ± terminal branch ends per kidney). both mfr and control (c) kidneys demonstrated significant (p< . ) increases in branching in explant culture to maximum values at days (mfr: ± vs c: ± ). with increasing culture days, the percent reduction in mfr branching increased with terminal branch point decreases of % at day , % at day , % at day , % at day , and % at day (p< . , mfr vs c at day ). conclusion: kidney explant cultures from mfr treated fetuses display basal and culture-based decreased branching compared to controls. this decrease in terminal ureteric buds, in combination with our previous findings of dysregulation of branching-associated kidney transcription and growth factors, suggests mfr induces early (e ) dysregulation of branching as a major mechanism of the associated nephropenia. these results indicate that early programming events in kidney development induce nephropenia and renal disease in adults. intrauterine growth restriction (iugr) has important implications for the neonate not only at the time of birth, but also as an adult. in humans and animals with iugr, the elevated risk of developing hypertension is thought to involve an upregulation of the renin-angiotensin system (ras). however, the link between the intrauterine insult and enhanced ras is not known. a novel mechanism leading to hypertension has emerged recently involving two orphan g-protein coupled receptors (gcr and gcr ) and their endogenous ligands, succinate and -ketoglutarate. infusion of succinate into adult mice induces hypertension, an effect which was eliminated in gcr knockout mice or by pretreatment with ace inhibitors. interestingly, succinate levels have been shown to increase in the circulation under conditions of oxidative stress, one of the hypothesized mediators of developmental programming of hypertension. thus, we hypothesized that gcr and gcr are upregulated in a rat model of iugr and may contribute to in utero programming of hypertension. timedpregnant rats were fed either control (c, % casein) or low protein (lp, % casein) diet throughout gestation. kidneys were collected on embryonic day (e ), post-natal day (d ) or (d ), n . real-time pcr was used to compare gcr , gcr and renin expression. data were standardized to a housekeeping gene (s ) and are expressed as fold increase/ decrease compared to control. a student's t-test was used to determine significance between groups (p< . ). offspring from lp dams were significantly smaller at birth and did not display catch-up growth. in kidneys from lp fetuses, both gcr and gcr were significantly elevated compared with controls ( . fold and . fold respectively; p= . ), whereas renin was . fold lower. on post-natal d , there was a trend towards increased expression of both gcr ( . fold; p= . ) and renin ( . fold; p= . ) in offspring from lp dams. at d , there were no significant differences between groups. in conclusion, these preliminary data suggest that in iugr offspring from lp rats, the gcr / pathway may be enhanced, indicating a novel mechanism for the programming of hypertension. objective: in addition to excess adipose tissue, obesity is accompanied by increased fat storage in organs such as the liver. peroxisome proliferatoractivated receptors (ppars) are ligand-activated transcription factors involved in the regulation of lipid metabolism, and lipid-associated inflammatory response. obesity represents a state of chronic low-level inflammation, with ppar and ppar implicated in this process. we have previously shown that nutrient restriction in pregnancy results in intrauterine growth restricted (iugr) newborns which develop adult obesity with elevated c-reactive protein (crp) levels. as crp is derived from the liver, we hypothesized that iugr-induced obesity inhibition of hepatic ppar and ppar is associated with an increased inflammatory response. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to to produce iugr newborns. all pups were nursed by control dams and weaned at weeks to ad libitum feed. at day and months of age, male offspring were analyzed for hepatic ppar , ppar and crp mrna (real time rt-pcr) and protein (western blot) expression. data was normalized to -actin and presented as fold change for protein levels. at months, hepatic triglyceride content was determined enzymatically. results: at d of age, iugr pups showed significant downregulation of ppar ( . -fold) and ppar ( . -fold) expression, though crp expression was significantly upregulated ( -fold). findings persisted at months of age, with continued downregulation of ppar and ppar ( . -fold) and upregulation of crp expression ( -fold). furthermore, iugr adults had significantly increased hepatic triglyceride content ( ± vs. ± mg/g liver, p< . ). conclusions: reduced expression of hepatic ppar and ppar in iugr offspring may contribute to elevated hepatic crp levels and triglyceride content. thus, developmental hepatic dysregulation leads to programmed obesity-induced inflammation in iugr offspring. offspring. mina desai, ederlen casillas, guang han, darran n tosh, , michael g ross. dept. of ob/gyn, labiomed at harbor-ucla med. ctr., torrance, ca, usa; dept. of physiology, univ. of adelaide, australia. objective: leptin and insulin mediate central anorexigenic signaling responses via different receptor molecules: leptin binds to the obrb receptor activating jak-stat and pi k pathways, whereas insulin activates pi k pathway by binding to its receptor (ir) and substrate (irs ). maternal food restriction in pregnancy results in iugr newborns that develop hyperphagia and adult obesity. the iugr newborns have significantly decreased plasma leptin levels with increased hypothalamic expression of basal obrb, stat and decreased expression of ir and irs , suggesting altered anorexigenic pathways. we studied the response of hypothalamic leptin/insulin signal molecules to peripheral leptin in iugr newborns. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to . day old male offspring were given saline or leptin ( g/g, i.p). hypothalamus was dissected at , and minutes and protein expression of total stat , phosphorylated stat (p-stat ), inhibitor of leptin signal (socs ), ir, irs , total akt and phosphorylated akt was determined by western blot (normalized to -actin). data is compared between leptin and saline treatments in iugr and controls. results: in response to peripheral leptin, iugr newborns showed marked dysfunction in stimulated hypothalamic leptin and insulin signaling responses. ( ) jak-stat : leptin-treated controls show progressively increased pstat ( -fold) with initial suppression of socs ( . -fold) as compared to salinetreated controls. conversely in leptin-treated iugr, the pattern is reversed such that there is sustained decline in pstat expression ( . -fold) with failure to downregulate socs . ( ) pi k pathway: leptin-treated controls showed a significant reduction in irs ( . -fold) and pakt ( . -fold) as compared to saline-treated controls. however, leptin-treated iugr newborns exhibited a paradoxical increase expression of irs ( -fold) and pakt ( -fold). conclusion:the iugr offspring demonstrate persistent upregulation of leptin receptor, a reduced phosphorylated stat (p-stat ) response in conjunction with an enhanced socs- response. the persistent increase in insulin responses indicates a dysfunction in dynamic signaling, leading to altered anorexigenic response and development of programmed obesity. we have previously demonstrated that maternal food restriction (mfr) in rats induces a marked increase in the expression of vegf protein in the aorta and mesenteric arterioles, accompanied by an increase in tgf-and collagen in both vessel types in adult rat offspring (am j physiol, ) . the aim of this study was to determine if this in vivo finding could be reproduced in an in vitro preparation. methods: two types of preparations were used in this study. we isolated endothelial cells from week old male control rat aortas. these cells were used after the third passage. staining with von willerbrand factor demonstrated that these cells were pure endothelial cells. the second type of preparation was aortic explants from week old male control rats. endothelial cells and aortic explants were transfected with a vegf adenovirus ( - viral infective particles) or a -galactosidase-adenovirus as a control. after hours of culture protein was isolated from cells and explants for western blot analysis using rat specific antibodies. culture media was assayed for vegf by elisa. results: transfection of vegf adenovirus induced a dose-dependant increase in the expression of vegf protein in primary endothelial cells and aortic explants. the transfection of vegf into the endothelial cells showed a bell shaped curve, and was accompanied by an increase in media levels of vegf protein. maximal secretion of vegf was found with viral infective particles. vegf adenovirus transfection induced a dose-dependant increase in c-reactive protein (crp) (inflammatory marker), and tgf-protein in both aortic explants and primary endothelial cells. these results indicate that upregulation of vegf in blood vessels induces inflammation and tgf-expression which in turn can induce collagen synthesis. thus the increased collagen expression and reduced compliance previously reported by us in vessels of mfr offspring can be explained by the over-expression of vegf which we reported. therapeutic intervention aimed at prevention of the increase in vascular vegf expression in mfr offspring could potentially prevent programmed hypertension. maternal regulation of high fat nourishment during lactation period reduce a hypertension of male offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med.univ., fukushima, fukushima, japan. objective: exposure to undernutrition or high fat nourishment during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of maternal feeding regulation during lactation period on blood pressure of offspring is unclear. our objective was to investigate the effects of either high-fat diet (hfd) during gestation to lactation period or restrictive fed a hfd during lactation period on blood pressure in male rat offspring. we use types pregnant wistar rats as fed with normal nutrition (group a), with a high fat diet (hfd) during gestation to lactation period (group b) and with hfd nutritionally restricted by feeding with % of the normal lactationmatched dietary intake from the day of delivery to the end of lactation period (group c). the male offspring was measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using oneway annova. results: body weight was significantly reduced in c offspring compared to a and b in male offspring at day after delivery (p< . ). at weeks old, the body weight of c offspring was no difference to catch up compared to a and b offspring. systolic and diastolic blood pressures were significantly elevated at all , and weeks in offspring of b > c >a. (p< . , vs. a) conclusions: under high-fat nutrition during gestation to lactation period induced hypertension in male rat offspring. maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. background: uterine artery (uta) doppler velocimetry has been validated in populations of heterogeneous parity in the second trimester for prediction of obstetric outcomes requiring preterm delivery to include: fetal growth restriction, fetal demise, hypertensive disorders of pregnancy, abruption, and indicated preterm delivery. understanding that parity may affect uta doppler indices in subsequent pregnancies, we sought to validate these predictive values in the first trimester in a homogeneous population of multiparous women. study design: multiparous women undergoing first trimester screening of singleton pregnancies were enrolled and followed prospectively until delivery (n= ). these women were divided into controls, ri < . (n= ) and cases, ri . (n= ), based on prior studies. demographic, clinical, and sonographic data (including uta indices and assessment of notching) were obtained. statistical analysis included student's t test and chi square. results: cases were not significantly different from controls in terms of maternal age, ethnicity, bmi, or medical history. uta doppler indices were significantly different between the two cohorts in terms of the presence of unilateral or bilateral notching ( % vs, % p< . and % vs. %, p< . , respectively). in contrast to that observed in patients of heterogenous parity previously, ri . was not associated with adverse pregnancy outcomes despite an average ri of . , significantly above this threshold. conclusions: in this multiparous cohort ri was not predictive of adverse obstetrical outcome, in contrast to that observed in cohorts including nulliparous patient. parity may affect uta vasculature and obscure the ability of doppler velocimetry to predict adverse obstetric outcome in multiparous women. presence of uta doppler notching in the first trimester remained a robust predictor of adverse obstetric outcomes in multiparous patients. objective: epidemiological studies have shown that offspring exposed to preeclampsia during fetal development are more susceptible to airway disease later in life. we have shown previously that gender, but not sflt- over-expression during pregnancy determines higher reactivity in the offspring airways at months of age. the objective of this study was to examine the effect of preeclampsia on the trachea from female and male offspring in our model of sflt- induced preeclampsia at year of age and compare responses between the two age groups. methods: cd- mice at day of gestation were injected via the tail vein with adenovirus carrying sflt (adsflt , pfu/ l) or mfc (admfc, pfu/ l). mice were allowed to deliver. tracheas were isolated from female and male offspring at months and year of age, and rings were mounted in organ chambers for isometric tension recording. responses to potassium chloride (kcl, mm), the mast cell degranulating agent compound / ( / , g/ml), and concentration-responses curve to acetylcholine ( - - - m) were obtained. results: there was no significant difference in responses to acetylcholine, kcl, or compound / between year old offspring born to the sflt and mfc groups. when comparing offspring within the same pregnancy exposure groups, responses to acetylcholine in adsflt -treated group were significantly higher in year old females than males. comparison between age groups by pregnancy exposure revealed that in the mfc group, year old male offspring had higher responses to compound / and acetylcholine than months old males. responses to kcl were significantly higher in months old males than year olds independent of maternal treatment during pregnancy. in females, the only difference between age groups was observed in the mfc group, where months old offspring demonstrated significantly higher responses to acetylcholine compared to year old offspring. conclusions: our findings did not show that airways of year old offspring born to mice with a preeclampsia-like syndrome induced by sflt- over-expression have airway hyperreactivity. however, sex and age differences in airway responses dependent on maternal exposure during pregnancy were observed, and needs to be explored further to elucidate underlying mechanisms. objective: maternal food restriction (fr) results in iugr newborns that when normally nursed exhibit rapid catch-up growth and adult obesity. continued fr during nursing delays catch-up growth and prevents adult obesity. igf- , which modulates growth and is secreted by the liver, may contribute to these morbidities. igf- is epigenetically regulated involving two promoters, alternative exon splicing and multiple transcription termination sites. we determined if hepatic igf- mrna levels correlate with obesity, and whether these changes are due to programmed epigenetic modification. methods: control pregnant rats received ad libitum food from gestation day to and lactation, whereas study dams were % fr. fr pups were nursed by either control (fr/adlib) or fr dams (fr/fr) and weaned to ad libitum feed. at day and months, male livers were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k trimethyl, and associated levels of each igf- species were measured by pcr. results: at months, obese fr/adlib males showed increased mrna levels of igf- a, igf- b, igf- exon , and igf- exon as compared to controls ( ± , ± , ± , and ± %). comparing fr/adlib month to newborn offspring, h /k was increased at igf -promoter , promoter , exon , utr# and utr# ( ± , ± , ± , ± , and ± %), though there was no differences between control month and newborns. in contrast, month fr/fr males had comparable mrna levels to the controls except for igf- b (% of control: ± ). further, fr/fr month h /k was only different from newborns at utr# (% of newborn: ± ). conclusion: iugr newborns with rapid catch-up growth and adult obesity have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. conversely, iugr with delayed catch-up growth and absence of adult obesity have levels similar to that of controls. thus, modulation of the rate of iugr newborn catch-up growth may protect against igf- epigenetic modifications. introduction: igf-ii is synthesized as a pro-hormone (proigf-ii; -amino acid peptide) which is then processed into its active forms: "big" igf-ii ( - ) and mature igf-ii ( - ). these active forms are essential for placental and fetal development and have also been shown to persist into postnatal life. since maternal smoking is known to adversely affect feto-placental growth and postnatal development, we postulated that these effects might be mediated through nicotine-induced alterations in igf-ii processing. methods: in the present study, nulliparous female wistar rats ( - g) were given nicotine ( mg/kg/day) or saline for days prior to mating, during pregnancy, and throughout lactation. at gestational day , and , dams were euthanized and we collected serum (fetal and maternal), amniotic fluid and recorded fetal body weight. a subset of dams were allowed to deliver at term. following parturition, serum samples from the offspring were collected at birth (pnd ) and weaning (pnd ). body weight was recorded weekly from birth to weaning. pro, "big" and mature igf-ii levels were determined by western blot analysis. results: maternal nicotine exposure during pregnancy resulted in a significant reduction in fetal body weight by gestational day . however, there was no effect of nicotine on fetal serum or amniotic fluid igf-ii levels at any gestational age examined. in maternal serum, mature igf-ii in control animals decreased with advancing gestational age such that igf-ii levels were lowest at gestational day . nicotine administration prevented this decline, which resulted in significantly higher mature igf-ii levels in nicotine-exposed mothers at gestational day . in postnatal life, nicotine exposed offspring had significantly lower levels of "big" igf-ii expression at weaning (pnd ). conclusions: these data demonstrate that nicotine can alter the amount of the active forms of igf-ii in the mother and the newborn. dysregulation of maternal igf-ii occurs concomitantly with suboptimal fetal growth. results from this study suggest a mechanism by which maternal smoking causes impaired fetal growth and adverse postnatal health outcomes. objective: maternal food restriction in pregnancy results in iugr newborns which develop adult metabolic syndrome. programming of both increased appetite-mediated hyperphagia and enhanced adipogenesis contribute to the development of obesity. transcription factors, peroxisome-proliferatoractivated-receptor (ppar ), ccaat/enhancer binding-protein (c/ebp ), and sterol regulatory element binding-protein (srebp c) regulate adipogenesis and lipogenesis. although iugr offspring exhibit acute upregulation of the adipogenesis signaling cascade prior to the development of obesity, we determined if this increased adipogenic potential was an intrinsic cellular response, and thus maintained in cell culture. we further examined the responses to adipocyte stimulators (ppar activator-ligand rosiglitazone) and inhibitors (ppar repressor-ligand badge). methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to term. adipocytes from day old iugr and controls were isolated and cell proliferation rate was determined (mtt). primary adipocyte cell cultures were established and following % confluence, iugr and control adipocytes were treated to two doses ( and m) of either rosiglitazone or badge for h. mrna and protein was extracted for expression of ppar , c/ebp , srebp c. data was normalized to -actin and compared to the respective untreated cells. results: iugr adipocytes had significantly increased protein expression of ppar ( . -fold) and c/ebp ( -fold) as compared to control adipocytes, though srebp c levels were unchanged. mrna levels showed similar changes in iugr newborns. importantly, iugr adipocytes exhibited increased cell proliferation ( % of control, p< . ) and showed greater response to rosiglitazone ( . -fold), though similar response to badge, as the control adipocytes conclusion: iugr primary adipocytes cell culture exhibit basal phenotypic characteristic of programmed upregulation of adipogenic transcription factors which promote adipose cell proliferation. the enhanced response to the adipogenic stimulant is further evidence of the predisposition to obesity. in contrast, the normal suppressive response to the inhibitor suggests that iugr adipocytes may respond to pharmacologic approaches to prevent obesity during this period. objective: maternal nutrient restriction results in intrauterine growth restricted (iugr) newborns which develop programmed obesity despite a normal post-weaning diet. the epidemic of obesity has been attributed in part to programmed "thrifty phenotype" and exposure to "western" diets. hepatic igf- is epigenetically regulated involving two promoters, alternative exon splicing, and multiple transcription termination sites. iugr offspring with normal post-weaning diet have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. we hypothesized that iugr newborns that develop programmed obesity would demonstrate discernable hepatic igf- changes which are distinct from diet-induced obesity. we determined igf- hepatic mrna levels and epigenetic characteristics in programmed (iugr) and dietinduced (dio) offspring. methods:: control pregnant rats received ad libitum food whereas study dams were % maternal food restricted from day to . all pups were nursed on ad libitum fed dams. controls were weaned to high-fat (fat, %) diet whereas iugr were weaned to normal ad libitum diet (fat, %) to produce diet-induced (dio) and programmed obese groups, respectively. at months, male hepatic igf- were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k dimethyl and h k trimethyl, and associated levels of each igf- species were measured by pcr. result: relative to dio control males, iugr had increased mrna of igf- a, exon and exon ( ± , ± , ± %). chip with h k dimethyl showed increased igf- exon ( ± %) and with h k trimethyl, increased igf- promoter and promoter ( ± , ± %) as compared to dio controls. conclusion: adult obese iugr males exposed to normal postweaning diet have increased hepatic igf- a mrna and h k dimethylation and trimethylation of igf- than dio controls. changes in igf- in adulthood from a prenatal insult thus suggest that igf- is programmed during the fetal period and may be associated with programmed adult obesity. rebekah elkins, pandu gangula, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa; internal medicine, university of texas medical branch, galveston, tx, usa. objectives: we previously reported that the offspring of rats fed % protein during gestation develop hypertension at two to four months and that the hypertension is exacerbated in males. this study is to evaluate: ) changes in estrogen receptor (er) angiotensin ii subtype receptor (at -r) and endothelial nitric oxide synthase (enos) in the mesenteric artery and aorta of offspring and assess if these changes, if any, are gender specific. methods: pregnant sprague dawley rats were fed either % protein (ctrl) or % protein (lpd) from day of gestation. the offspring were evaluated for hypertension by means of systolic blood pressure measurements. at four months for the males and nine months for the females, mesenteric artery and aorta were collected in rnalater. expression of estrogen receptor a (er-a) and b (er-b), at -r, and enos were analyzed by western immunoblotting and rt-pcr and expressed relative to b-actin or s. results: mesenteric artery shows no differences between ctrl and lpd female offspring in at -r, enos, er-a or er-b. similarly mesenteric artery shows no diet exposure related changes in at -r, er-a or er-b in male offspring. however, enos expression was lower in mesenteric artery of lpd male offspring. on the other hand, in the aorta both er-a and er-b levels are lower in lpd female offspring while there were no changes in at -r or enos. no changes in at -r, er-a or er-b were observed in male offspring aorta of ctrl and lpd rats. conclusion: the in utero exposure to lpd results in adult hypertension in both male and female offspring. some mechanisms for hypertension include the decrease in er-a and er-b but not at -r or enos in females, and the decrease in enos but not at -r or er in males indicating gender related differences. offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. our objective was to investigate the effects of either severe undernutrition during late gestation or lactation period on blood pressure and the development of vascular function in male rat offspring. we use normal pregnant wistar rats (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b) and % restricted after delivery to the end of lactation period (group c). the offspring was measured blood pressure at and weeks by using indirect tail-cuff method. rings of thoracic aorta with intact endothelium from the male offspring of a and b at weeks, were equilibrated at g passive tension in organ chambers filled with krebs-henseleit solution continuously bubbled with %co in air ( °, ph . ) for isometric tension recording. concentration-response relationships to norepinephrine (ne) and angiotensinii(atii) were obtained in the absence or presence of n(omega)-nitro-l-arginine methyl ester (l-name) or a selective atii type- receptor blocker (valsartan). responses to cumulative concentrations of sodium nitroprusside (snp) and to - m oxyhemoglobin (hb, nitric oxide scavenger) were also determined. contractions were expressed as a percent of the reference contraction induced by potassium chloride ( mm). statically analysis was performed using one-way annova. results: body weight was significantly reduced in b offspring compared to a and c in male offspring at day (p< . ). at weeks the body weight of offspring of b was no difference to catch up compared to a and c offspring. systolic and diastolic blood pressures were significantly elevated at both and weeks in offspring of b > c >a. ne concentration-dependently stimulated tension of aortic rings from in a and b offspring, which was not significantly (n= ). maximal contractions to ne were significantly stimulated by l-name in a (p< . ), but not b offspring. valsartan significantly inhibited aortic contractions by ne in r (p< . ), but not a offspring. there was no significant difference on responses of aortic rings by atii, snp and hb in a and b offspring. conclusions: severe under nutrition during not only late gestation but also lactation period induced hypertension in male rat offspring in adulthood. fetal origin of adult hypertension might be vascular endothelial dysfunction. fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. objective: exposure to undernutrition during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of either high fat nourishment or undernutrition during lactation period on blood pressure is unclear. our objective was to investigate the most effective maternal nourishment and feeding period for offspring induced adulthood hypertension in using high-fat diet (hfd). study design: we use types pregnant wistar rats as fed with normal nutrition (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b), % restricted after delivery to the end of lactation period (group c), with a high fat diet (hfd) during gestation to lactation period (group d) and with hfd nutritionally % restricted from the day of delivery to the end of lactation period (group e). the offspring was measured body weight (bw) and measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using one-way annova. results: bw was significantly reduced in b offspring compared to another (a, c, d, e) male offspring at day (p< . ). at day after delivery, bw was significantly reduced in c, e offspring compared to a, d in male offspring (p< . ). at weeks old, bw of all type offspring was no difference. systolic and diastolic blood pressures were significantly elevated at weeks in offspring of d> b > e > c >a. (p< . , vs. a). at weeks, hypertensive offspring as b>d>e>c>a (p< . , vs. a). at weeks, d> e > b > c >a (p< . , vs. a). conclusions: maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. in case with normal food, restrictive feeding during late gestation is more effective than lactation period for inducing hypertensive male offspring. regulation of maternal feeding not only during late gestation but also lactation period may control adulthood hypertension. the strongest epigenetical factor of maternal nutrition is high fat feeding during pregnancy to lactation period for f blood pressure, respectively. or residence at high altitude, impacts fetal growth and development. in a preliminary study, we observed a significant decrease in birth weight, subsequent compensatory postnatal growth, and an increase in relative right ventricular (rv) weight at postnatal (pn) day in female offspring of rats exposed to hypoxia ( , ft; . % po ) from days thru of gestation (dga). thus, our objective was to further elucidate the impact of prenatal hypoxia on fetal growth and postnatal development. methods: pregnant dams (hx, n= ) were hypoxic from - dga with additional control dams either fed ad libitum (al, n= ), pair-fed with the hx dams throughout gestation (pg, n= ), or only pair-fed during the window of hypoxia (ph, n= ). female offspring from hx, pg, and ph dams were cross-fostered onto additional al dams (n= /litter) by h after birth. results: at birth, there was no difference in litter size; however, body weight (bw) of the hx, pg, and ph pups was lower (p< . ) than that of al pups, and hx pups were lighter (p< . ) than ph pups. weight of hx offspring remained lower (p< . ) than al pups until the termination of the study at pn , while the pg and ph pups reached weights comparable to the al offspring by pn . relative to bw, heart weight and left ventricular/septal (lvs) weight was not different among groups; however, right ventricular weight (rv/bw) was greater (p< . ) in the hx offspring at pn as was rv/lvs (p< . ). cardiac function was evaluated by echocardiography at pn . rv wall thickness was % greater (p< . ) in hx pups as compared to al pups, confirming the significantly higher relative rv weight observed at necropsy. pep, pep/at, and pep/et were %, %, and % higher respectively in the hx offspring relative to the al offspring. lv end diastolic and end systolic diameters were smaller (p< . ) in hx and ph offspring relative to the al group. myosin heavy chain (mhc) and mrna concentrations in the rv were evaluated by qrt-pcr, and the mhc / mrna ratio was greater (p< . ) in the hx pups. conclusion: prenatal hypoxia from - dga impacted both fetal and postnatal growth, altered postnatal heart development and function, with the primary impact being on the rv. supported by nih hd . reproductive sciences; pharmacology experimental therapeutics, university of maryland, baltimore, md, usa. background: exposure to nicotine (nic) is a significant risk to normal fetal development. fetal nic, which readily crosses the placenta, can be acquired from pregnant mothers by smoking or nicotine replacement therapy. the impact of nic on fetal organs may be mediated directly and/or via intrauterine hypoxia (hpx) via constriction of the uterine circulation. in adult hearts, both nic and hypoxia stimulate gene expression of matrix metalloproteinases (mmp), although the study of nic and hypoxia on gene expression in fetal organs remains incomplete. because mmps are involved in the regulation of extracellular matrix turnover and cardiac remodeling, we tested the hypothesis that prenatal nic and intrauterine hpx upregulate protein expression and activity of mmp in the fetal guinea pig heart. methods: pregnant guinea pigs were placed in either normoxia (nmx) or hpx ( . %o in chamber) for d prior to term ( d). in two separate groups, nic was also added to the drinking water ( mg/kg/d) for d at a dose that generates fetal nic levels ( ng/ml cotinine) equivalent to a moderate smoker. anesthetized near-term fetuses ( d) were excised and weighed. left ventricles of hearts were obtained and frozen at - c for storage. mmp protein levels and enzymatic activity were measured by western analysis and gel zymography, respectively. results: nic alone (nmx+nic) decreased (p< . ) fetal body wt by %, increased (p< . ) the relative fetal brain wt (brain wt/fetal body wt ratio) by . % and had no effect on relative placental or fetal heart wts. hpx alone decreased (p< . ) fetal body wt by . %, increased the relative fetal brain wt by % but, in contrast to nic alone, increased relative placental wt by . %. both mmp protein levels (mmp /a-actin density values) and activity (clear band density) were increased (p< . ) by nic alone (by . and . fold, respectively) and hpx alone (by . and . fold, respectively). in addition, both protein and activity levels of hpx hearts were further increased by nic (by . and . fold) compared to hpx alone. conclusion: prenatal nic upregulates mmp expression in nmx fetal hearts and is potentiated by hpx. this suggests that under conditions of intrauterine stress cardiac remodeling by mmp activation may be an important mechanism by which nic and hpx affect fetal heart function. objective: in addition to peripheral hypoglycemic effects, insulin induces central anorexigenic responses via stimulation of the phosphoinositide- kinase (pi k) pathway and cellular growth by mitogen activated protein kinase (mapk) pathway. the pi k signaling cascade is activated by insulin binding to its receptor (ir), recruiting ir substrate (irs- ), and phosphorylating pi k. activated pi k in turn causes phosphorylation of protein kinase b/akt which subsequently modulates hypothalamic anorexigenic responses. in contrast, the mapk (erk /erk ) signaling pathway likely involves irs- . further, insulin signaling is inhibited by the lipid phosphatase pten. we have previously shown that maternal food restriction (mfr) during pregnancy results in iugr newborns that develop hyperphagia, obesity and insulin resistance as adults. we sought to determine if altered hypothalamic basal insulin signaling expression of pi k and mapk pathways contribute to reduced satiety responses and thus enhanced growth in iugr newborns methods: pregnant control dams received ad libitum (n= ) food, whereas study dams were % mfr (n= ) from pregnancy day to to produce iugr newborns. at day , hypothalamic region was dissected and analyzed for mrna levels (real time rt-pcr) of insulin signaling components via pi k (ir, irs , pi k and akt) and mapk (irs , erk , erk ) pathways, and pten. data is presented as fold difference normalized to beta- -microglobulin. results: at d of age, iugr pups exhibited downregulation of the entire pi k pathway with significantly decreased (p< . ) mrna levels of ir ( . -fold), irs- ( . -fold), pi k ( . -fold) and akt ( . -fold). further, iugr pups showed similar decreased mrna expression of erk ( . -fold) and erk ( . -fold). however pten expression was similar to the controls. conclusion: reduced insulin-mediated pi k signaling likely contributes to the suppressed anorexigenic responses and development of obesity in iugr offspring. reduction of central mapk signaling suggests a potential maldevelopment of additional neuronal pathways in iugr offspring. objectives: in the rat, uteroplacental insufficiency restricts fetal growth and impairs mammary development further compromising postnatal growth. both male and female growth-restricted offspring have a reduced nephron endowment but only males develop hypertension with glomerular hypertrophy, which can be reversed by improving the lactational environment. this study used cross-fostering to assess the influence of the prenatal and postnatal environments on renal development and nephrogenesis. methods: bilateral uterine vessel ligation (restricted, r) or sham surgery (control, c) was performed on day of gestation in wky rats. control and restricted pups were cross-fostered onto c or r mothers on postnatal day (pn ). post mortem was carried out on pn (c and r) and pn (c-on-c, c-on-r, r-on-c, r-on-r). results: body and kidney weights were decreased in r and r-on-r pups on pn and pn (p< . ). there was some evidence of accelerated pup growth for r-on-c relative to r-on-r on pn . male, but not female, relative bmp mrna expression on pn was higher in r than c (p< . ) while gdnf, tgf and at receptor were not different. on pn , wnt (but not at r, vegf-a) mrna expression (males only) was relatively higher in r-on-r (p< . ) when compared to c-on-c (p< . ). this and the histological analyses suggests an up-regulation of nephrogenic activity with more immature nephrons (males and females) in r-on-r (p< . ) when compared to c-on-c, while r-on-c remained intermediate. intrauterine growth-restricted pups were born lighter and with smaller kidneys. this was partially rescued by improving lactational nutrition (r-on-c) at pn . higher bmp mrna expression indicates impaired branching morphogenesis in pn r male, but not female kidneys, suggesting the timing and/or molecular mechanisms underlying the nephron deficit may be sex specific. at pn there was evidence of extended and increased nephrogenic activity in r-on-r, however, this was unable to restore the later nephron deficit. improved lactation for r-on-c, which prevented the adult nephron deficit and hypertension, increased and extended nephrogenesis to a lesser degree than r-on-r suggesting that the restoration of nephron endowment was likely to have occurred prior to pn . amy m tetrault, sarah b lieber, marya shanabrough, tamas l horvath, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa. objective: classically recognized for its role in energy balance, body weight and appetite, ghrelin has also been implicated in reproduction. ghrelin (-/-) mice are infertile while administration of ghrelin to wt mice results in decreased litter size and constrained embryonic growth. here we investigate the effect of maternal ghrelin deficiency on in utero developmental programming of the female reproductive tract. hox genes determine developmental identity of the paramesonephric duct. we determined that hoxa is regulated by ghrelin in vitro and that in utero ghrelin deficency alters f hoxa gene expression and reproductive success. methods: wild-type females mice parented by ghrelin +/-b d f (ghrellin deficient) mice were analyzed for litter size, oocyte, and corpus luteum number. rna was extracted from the uterus of mice exposed to ghrelin deficiency in utero. ishikawa cells were treated with ghrelin with/without receptor (ghsr) antagonist, or saline and rna extracted. in both hoxa expression was analysed by real time rt-pcr normalized to -actin and also determined by ihc. experiments were repeated in triplicate and mrna expression compared by student's t-test. results: wild-type female offspring of ghrelin deficient dams had smaller litter sizes than controls (n= , . ± . pups; n= , . ± . pups, respectively; p< . ). no differences were seen in oocyte or corpus luteum number suggesting a uterine defect. hoxa mrna and protein expression were decreased in the uterus of the f females. ghsr was expressed in uterine endometrium. treatment of ishikawa cells with nm to nm ghrelin resulted in a to % increase (p< . ) in hoxa expression. treatment with ghrelin and ghsr antagonist resulted in similar increases in hoxa expression indicating a non-receptor mediated mechanism. conclusion: ghrelin contributes to reproductive tract developmental programming; in utero ghrelin deficiency compromises reproduction in female offspring. the developmental effects of ghrelin were mediated by alteration in hox gene expression and not through the classic ghsr receptor. obesity and decreased ghrelin may lead to defects in developmental programming of the reproductive tract. these findings demonstrate the importance of nutrition, energy utilization and appropriate ghrelin levels on normal uterine development. we have previously studied the deleterious effects of lack of the endothelial nitric oxid synthase (nos ) in mouse dams and their offspring. our laboratory demonstrated that adaptive responses in subsequent pregnancies may offset the harmful effects of the genetic deficiency of nos . in this study we aimed to determine hepatic and renal histopathologic damage in nos deficient pregnant mice comparing animals carrying their first versus their second pregnancy. study design: gravid nos +/+wt and nos -/-ko mice during their first (p ) or second (p ) pregnancy were sacrificed at day of gestation. livers and kidneys were stained and analyzed for the presence and extent of histopathologic lesions. results: nos +/+wt dams displayed a low incidence of significant renal or hepatic lesions in either the first or the second pregnancy. in nos -/-ko mice the incidence of liver necrosis and inflammation during the first pregnancy was % and %, respectively. in nos -/-ko dams sacrificed during the second gestation the incidence rates for the same lesions were % and %, respectively (p< . ). this correlation persisted when we analyzed the relative severity of hepatic lesions between p and p animals. although a similar trend was observed, the difference between p and p animals with regards to kidney lesions did not reach the level of statistical significance in our study. conclusions: a second pregnancy in this animal model of hypertension was associated with a significantly improved hepatic histopathology compared with the first pregnancy. this observation is consistent with our previous studies showing a decrease in systemic vascular resistance in p versus p nos -/-ko mice. the beneficial effects of a prior pregnancy may partially underlie the phenomenon of a decreased risk of preeclampsia in multiparous versus nulliparous women. further studies are required to delineate the counterregulatory mechanisms leading to improved cardiovascular function in subsequent pregnancies in these genetically modified animals. maternal hypomethylation is associated with congenital heart defects in down syndrome. lmjw van driel, , r de jonge, wa helbing, bd van zelst, j lindemans, eap steegers, rpm steegers-theunissen. , , , obstetrics gynecology; pediatrics; clinical chemistry; epidemiolog y biostatistics; clinical genetics; erasmus university mc, rotterdam, netherlands. background: maternal age and hyperhomocysteinemia are risk factors for having a child with down syndrome (ds) and congenital heart defects (chds), respectively. evidence is rising that ageing is associated with a state of hypomethylation. objectives: to investigate whether the risk of a child with ds and chd is associated with maternal hypomethylation. methods: we conducted a case-control triad study at months after the index-pregnancy. case-children (n= ) were included if they had ds and chd. children (n= ) without a major congenital malformation served as controls. the concentrations of s-adenosyl methionine (sam), s-adenosyl homocysteine (sah), sam/sah ratio, and homocysteine in maternal blood were measured as biomarkers for methylation. the data were analyzed using the mann-whitney u test and a logistic regression model. results: maternal age was included in the model as potential confounder. the levels and the crude and adjusted or( %ci) of the biomarkers are shown in table . an increase of the sam/sah ratio with unit decreases the risk of a child with ds and chd with percent. moreover, every increase of mol/l of homocysteine . fold increases this risk. conclusions: maternal hypomethylation is significantly associated with an increased risk of having a child with ds and chd. since, the effects are confounded by maternal age, hypomethylation can be considered as feature of ageing. the developmental origins hypothesis postulates that during critical ontogenetic periods, transient environmental stimuli perturb developmental pathways and induce permanent changes in gene expression, metabolism, and chronic disease susceptibility. one likely mechanism is via early nutritional influences on epigenetic gene modification consisting of the presence of a methyl group on the carbon of a cytosine residue. this modification is responsible for an important form of gene regulation in eukaryotes. in the present study, we have tested the hypothesis that maternal low-protein diet altered epigenetic regulation of specific gene of the offspring. c bl/ female mice were mated and on the day the plug was detected, these females were then randomly allocated to be fed isocaloric diets consisting % protein or % protein. at delivery, offspring were killed and the livers were removed immediately, frozen in liquid nitrogen and stored at - c. genomic sequencing after bisulfite modification is used to study site-specific dna methylation. dna methylation status of oct- and sphk- gene upstream regions in the mouse liver was analyzed. hepatic oct- or sphk- promoter methylation was not significantly different between both groups. however, dna methylation pattern of the genomic dna is specific in low-protein diet group. aberrant oct- and sphk- gene expression may cause perturbations in cell differentiation. we suggest that the epigenetic mechanism consisting of dna methylation underlies the fetal programming theory. primary human cytomegalovirus (hcmv) infection during pregnancy can have devastating consequences for both the mother and fetus. hcmv infection has been implicated in the development of pre-eclampsia and intrauterine growth retardation (iugr), as well as congenital cmv syndrome in newborns exposed in utero. previously, we have shown that hcmv infection of placental cytotrophoblasts inhibits their normal invasion, proliferation, and migration. however, the mechanisms occurring during early establishment of placental infection are largely unknown. we assessed the impact of hcmv infection on cytotrophoblasts by performing immuno-based assays for various cytokines and cellular growth factors. we detected significant cytokine dysregulation at both and hours after in vitro hcmv infection of cytotrophoblast cells. soluble cytokines involved in recruitment of monocytes and macrophages (gro-a, mcp- ) were downregulated at both and hours after infection. sdf- , which is chemotactic for lymphocytes during early inflammation, was also decreased. these results suggest that recruitment of cells involved in the anti-viral immune response is being interrupted early in the course of infection by hcmv. additionally, a large decrease in the amount of soluble hgf was seen. hgf normally induces migration of cytotrophoblasts along the invasive pathway, and downregulation of this factor could severely affect these processes. finally, we saw increased amounts of soluble icam- , contrasted by decreased amounts of vcam- , indicating dysregulation of adhesion molecules that are necessary for successful placental invasion to occur. all together, these data indicate significant alterations in cytokine profiles as early as hours after hcmv infection, which could provide important clues to the pathogenesis of hcmv in placental invasion and inflammation. additional studies will further elucidate this dysregulation, and determine whether these effects are due to alterations in pre-existing cellular factors or if transcriptional alterations are involved. method: studies were performed in pregnant ewes (n= in each group) with twin fetuses at - days (very preterm) and - days (near-term). neither mother nor fetuses were instrumented prior to the time of blood collection. maternal blood was collected from the jugular vein before sedation. anesthesia was then induced with isofluorane, the abdomen was opened, fetuses exteriorized and blood collected from umbilical cords. blood samples were transferred to plastic tubes containing ethylene-diamino tetraacetic acid and reduced glutathione, plasma separated and stored at - c. commercial radioimmunoassay kits for ovine-crf (phoenix pharmaceuticals, b : . ± . pg/ml) and cortisol (diagnostic laboratory, b : . ± . ng/ml) were used according to the manufacturer's instructions. results: in very-preterm gestations, maternal and fetal plasma crf levels were undetectable. maternal, but not fetal, plasma cortisol levels were measurable ( ± ng/ml). in near-term gestations, both cortisol and crf were measurable in maternal (crf: ± pg/ml; cortisol: ± ng/m) and fetal plasma (crf: ± pg/ml; cortisol: ± ng/ml). plasma crf levels were higher in nearterm fetuses than in their maternal ewes (p < . ). conclusion: ovine plasma crf levels are measurable in maternal and fetal plasma in near-term but not very-preterm gestations. the absence of crf in preterm plasma, perhaps due to reduced placental expression and/or placental crf release, may contribute to the rarity of in utero meconium passage in preterm gestations. interleukin - objectives: interleukin- (il- ) is a pro-inflammatory cytokine produced in adipose cells. recent studies suggest il- may be a marker of maternal obesity and in utero fetal programming. our hypotheses were ) il- correlates with maternal obesity and ) il- mediates the effect of maternal obesity on infant birth weight. methods: the parity, inflammation, and diabetes (pid) study is a longitudinal study of adipokine levels in a diverse sample of pregnant women. we present a cross-sectional analysis of first trimester il- levels from non-diabetic women who underwent a live birth. the independent variable was il- (pg/ml), measured with monoclonal antibody elisa assays. the dependent variable was infant birth weight (gms). maternal bmi categories were: normal/underweight (< kg/m ); overweight ( - kg/m ); and obese (> kg/m ). data on demographic and clinical factors, nutrition and physical activity were collected at baseline. average il- levels were compared across bmi categories using anova. the association of il- levels with infant birth weight was estimated using multiple linear regression, adjusting for covariates. results: average il- levels were significantly higher in obese women ( . ± . ) compared to overweight ( . ± . ) and normal/underweight women ( . ± . ) [p< . ]. after adjustment, il- levels was positively correlated with pre-pregnancy bmi [regression coefficient (rc) . ; % ci: ( . , . )]. as shown in the table below, elevated levels of il- were statistically significantly associated with a . gm higher infant birth weight after full adjustment. each unit increase in pre-pregnancy bmi was associated with a gm higher infant birth weight. table . association of il- with infant birth weight characteristic regression coefficient % confidence interval interleukin- . . , . pre-pregnancy bmi . , gestational weight gain - - , bmi = body mass index; coefficients adjusted for demographics, clinical factors, pre-pregnancy bmi, gestational weight gain, non-fasting first trimester glucose levels, gestational age, nutritional intake and physical activity conclusion: il- and pre-pregnancy bmi were associated with infant birth weight after adjustment for covariates. our findings suggest that the effect of maternal obesity on infant birth weight may be mediated through il- or an alternative independent pathway. we have demonstrated that -tetrahydrocannabinol (thc), in physiologically relevant concentrations, inhibits the growth and tight transcriptional control of the bewo trophoblast cell line . the mechanism involved the decreased expression of the transcriptional regulator histone deacetylase (hdac ). in these experiments we sought to answer the question, 'does anandamide (aea) work in the same manner as thc?' methods: the first trimester human trophoblast cell, bewo were plated at or x cells /well to -well and -well plates for growth and rna experiments, respectively. after growing to - % confluence, cultures were treated with varying concentrations of aea up to a maximum of m for hr. cell numbers were determined using the xtt apoptosis/proliferation assay. total cellular rna was prepared and the relative levels of hdac and gapdh determined by end-point rt-pcr. aea exhibited an inhibitory effect on the bewo cell cultures, but only at concentrations in excess of m where confluency was significantly reduced from % at m to - % at m and m (*p< . ; one-way anova with tukey's hsd test; n= ). cultures treated with m and m aea did not exhibit increased cell death or failure to attach to the substratum, as evidenced by the lack of increase in the shedding of cells into the spent medium. bewo cells treated with aea showed a dose-dependent decrease in hdac mrna expression with a significant effect at . m (*p< . ; oneway anova with tukey's hsd test; n= ). at this dose, the effect of aea had reached an effective maximum decrease in hdac mrna levels ( %) because hdac mrna levels were not decreased further by either m aea ( %) or m aea ( %). the alteration of hdac gene expression by aea in bewo cells with its associated decrease in cell number suggest that the trophoblast cell may be an important target for circulating endocannabinoids during the st trimester of pregnancy. the data indicates that although exocannabinoids and endocannabinoids both inhibit bewo cell growth, they do so using different transcriptional mechanisms. further understanding of the mechanism(s) by which aea alters placental physiology may lead to new strategies for the prevention of pregnancy complications such as st trimester miscarriages. ( ) taylor background: anandamide (aea) exerts its effects by acting on two cannabinoid receptors, cb and cb with the main regulator for aea levels being the metabolising enzyme, fatty acid amide hydrolase (faah). aea, cb , cb and faah constitute the endocannabinoid system and previous studies have shown that faah and cb are expressed in term human placenta( ) suggesting that the endocannabinoid system might be present earlier in gestation. this study aimed to document changes in faah, cb and cb expression in the placenta during the first trimester of pregnancy. methods: first trimester samples ( to weeks gestation) were fixed in % neutral-buffered formalin for days before embedding into paraffin wax or frozen in liquid nitrogen for rna analysis. for immunohistochemistry, faah polyclonal antibodies were used at an optimal dilution of : in pbs, cb at : and cb at : . transcripts were measured using q-pcr with gene-specific primers. results: immunoreactive faah, cb and cb were detected in all samples. faah immunoreactivity in the syncytiotrophoblast increased between the th and th gestational week and by week faah was barely detectable within large parts of the placenta. simultaneously, faah immunoreactivity increased in the mesenchymal core of the developing villi. immunoreactive cb and cb localised to the syncytiotrophoblast, cytotrophoblast and mesenchymal core with cb immunoreactivity showing diminished intensity after week , although this did not reach significance at the transcript level. cb immunoreactivity was absent from fetal blood cells and infiltrating maternal plasma cells, whereas cb and cb immunoreactivity was detected in endothelial cells but not in the vascular smooth muscle cells of blood vessels. the intensity of cb immunoreactivity in the syncytiotrophoblast differed from that of cb and faah in that it remained constant throughout. conclusion: the data suggest that placental faah and cb levels do not alter significantly during the first trimester, but alter their cellular distribution from the syncytiotrophoblast to the mesenchymal core. the significant loss of cb expression from the syncytiotrophoblast after the th week of gestation, a point of critical alteration in the developing placenta, suggests that its retention may be detrimental to normal placental development. reference: park, b. et al., ( ) hankins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: numerous angiogenic proteins synthesized in the placenta are thought to be involved in placental vascularization and development; however, the molecular mechanism modeling the angiogenic process in early pregnancy remains elusive. calatonin gene-related peptide (cgrp) is a multifunctional peptide expressed at the human implantation site; but its influence on in vitro angiogenesis by human micro vascular endothelial cells is not known. objective: the present study was designed to determine the influence of cgrp on angiogenesis by human dermal microvascular endothelial cell (hdmvec) in vitro. methods: hdmvecs (vec technologies) were cultured in mcdb- complete solution containing cgrp ( - m), cgrp plus its antagonist cgrp - ( - m), in well plates with x cells per well. the existence of cgrp receptor components calcitonin receptor-like receptor (crlr) and receptor activity modifying protein (ramp ) was determined using immunofluorescent staining. cell proliferation was examined using methylthiazoltetrazolium (mtt) assay. the pro-angiogenic bioactivity of cgrp was evaluated using cell migration and capillary like tube formation on the matrigel. results: ) immunofluorescent staining showed that cgrp receptor components crlr and ramp are abundantly expressed by hdmvecs. replacement of the primary antibodies with preimmune serum resulted in a negative staining; ) cgrp dose-dependently ( - to - m) stimulated hdmvec proliferation, and this effect was totally blocked by cgrp antagonist, cgrp - ; ) quantitative analysis for cell migration revealed that cgrp increases hdmvec migration in a dose and time-dependant manner; and ) cgrp promotes hdmvec capillary like tube formation, and the length of capillary tube induced by cgrp ( - m) was significantly increased over that of the untreated controls. this increase was observed at hours of treatment and further increase was noted at hours of culture. conclusion: cgrp induces in vitro angiogenesis by promoting microvascular endothelial cell proliferation, migration and capillary like tube formation. therefore, trophoblast derived cgrp at the implantation site may play a role in placental angiogenesis and fetal growth. over-expression of socs- gene promotes il- production by jeg- trophoblast cells. qin dong, , ruping fan, yang gu, david f lewis, yuping wang. biochemistry, harbin medical university, harbin, heilongjiang, china; obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: suppressor of cytokine signaling- (socs- ) plays an important role in negative regulation of inflammatory response in biological cells. evidence has shown anti-inflammatory cytokine il- expression was significantly reduced in trophoblasts of preeclamptic (pe) placentas. we sought to determine if over-expression of socs- in placental trophoblasts could promote il- production. methods: full-length socs- open reading frame (socs- cdna) was generated by rt-pcr from total rna samples isolated from human leukocytes and cloned into a pzsgreen -n vector, which encodes a green fluorescent protein zsgreen . successful socs- cloning was confirmed by sequencing. for transfection, jeg- cells were placed into well/cluster plates at a concentration of . x /well. the tranfection was carried out with . g of socs- /zsgreen plasmid (psocs- /zsgreen ) for hours when cell reached - % confluence. siport lipid were used. jeg- cells transfected with zsgreen plasmid (pzsgreen ) only was used as control. after approximately hours of transfection, cells were treated with il- at and ng/ml. medium was then collected and measured for il- by elisa. il- production was calculated as the percentage of increase by psocs- /zsgreen transfected cells compared to the cells transfected with pzsgreen only. result: il- production was increased by psocs- /zsgreen transfected jeg- cells compared to the cells transfected with pzsgreen only when stimulated by il- , control: . % increase; ng/ml il- : . % increase and ng/ml il- : % increase, p< . , respectively. data are means from three independent experiments. conclusion: over-expression of socs- gene could promote il- production by placental trophoblast cells. reduced sil- r release and increased ratio of sgp /sil- r production by placental tissues from women with preeclampsia. shuang zhao, ruping fan, jingxia sun, yang gu, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa; obstetrics and gynecology, first hospital, harbin medical university, harbin, heilongjiang, china. objective: placental tissue/trophoblasts release more inflammatory cytokines (il- , il- and tnf ) in preeclampsia (pe) than in normal pregnancies. however, the reason for increased inflammatory cytokines released by pe placentas is not clear. soluble il- receptor (sil- r) and membrane receptor il- /gp complex play an important role in the negative regulation of cytokine signaling in suppressor of cytokine signaling (socs) pathway. in contrast, soluble gp (sgp ) is an antagonist for il- /il- r trans-signaling. this study was undertaken to determine sil- r and sgp production by villous tissues from normal and pe placentas. methods: placentas delivered by normal and pe pregnant women were used in this study. placental explants were incubated with dmem for h. the culture medium was collected. placental villous tissue productions of sil- r and sgp were measured by elisa. all samples were assayed in duplicate. data are expressed as mean ± se and analyzed by mann whitney test. a p level < . was considered statistically different. results: placental tissues from pe produced significantly less sil- r than tissues from normal pregnancies, . ± . vs. . ± . pg/mg of wet tissue, p< . . soluble gp production was relatively compatible between pe and normal placental tissues: . ± . vs. . ± . pg/mg of wet tissue. the ratio of sgp /sil- r release was significantly higher in pe than in normal placentas, . ± . vs. . ± . , p< . . conclusion: reduced sil- r production and/or increased ratio of sgp /sil- r production by pe placentas suggest less cytokine inhibitory activity in pe placentas, which may contribute to the increased toxic cytokine production in placentas from pe. using chemiluminescence immunoassays. peter s uzelac, jing dai, frank z stanczyk, daniel r mishell, jr. obstetrics and gynecology, university of louisville, louisville, ky, usa; obstetrics and gynecology, university of southern california, los angeles, ca, usa. objective: characterizing human chorionic gonadotropin (hcg) levels throughout normal pregnancy is critical to its use as a bio-marker for abnormal gestations. there is a paucity of data describing hcg trends during pregnancy and most of the relevant studies use older, less specific assays. our first objective was to characterize hcg levels throughout normal gestation using two different contemporary chemiluminescence immunoassays. our second objective was to compare hcg patterns in healthy gestations with pregnancies affected by diabetes, a common obstetrical complication. methods: a single blood sample was collected from healthy pregnant women and diabetic pregnancies. gestational age was confirmed by ultrasound. serum hcg levels were quantified by chemiluminescence immunoassays using the acs- and immulite systems. data was grouped in -week intervals until weeks of gestation and -week intervals thereafter, with to samples in each interval for healthy women and to samples in each interval for diabetic women. paired t-test and wilcoxon rank sum test were used for statistical analysis. results: using the acs- system, mean hcg levels (miu/ml) for healthy pregnant women were , at - weeks, , at - weeks, , at - weeks, , at - weeks, , at - weeks, and , at - weeks. mean hcg levels obtained from the immulite system were similar. for diabetic pregnancies, mean hcg levels (miu/ml) were , , , , , , , , , and , , respectively. between - weeks of gestation, the hcg levels were significantly lower in diabetic pregnancies (p= . ) compared to healthy controls. ) compared to healthy pregnancies, diabetic gestations have significantly lower peak hcg levels. the cause of this difference is an area deserving further investigation. background: mouse and human placentae share several cellular and molecular features, including a haemochorial interface, allowing the murine labyrinth to be compared with the human fetal placenta. there is an autonomous embryonic ras from at least the time of implantation. ace converts angiotensin i to the active angiotensin ii (angii). angii's actions as a pro-inflammatory agent, in promoting cell migration, angiogenesis and cellular growth and apoptosis, strongly suggest a rôle in placentation. hypothesis: there would be counterregulation of the placental ras if the effect of ace were removed. this study investigated for the first time whether the knockout of somatic ace affected the expression and localisation of various components of the ras in the placentae of wild-type (wt/wt), heterozygous (wt/ ) and ace knockout ( / ) mice. methods: immunohistochemistry (dako envision plus) was used to localise and semiquantify ang type receptor (at r), ang type receptor (at r), ace, and ace- in the three genotypes (n= /group). placental sections were blinded to genotype; a score range of - was used. the test was used (spss version . ) to analyse the difference in staining score by genotype. results: immunoreactivity of all antigens increased in the placental labyrinth of / mice compared to wt/wt and wt/ mice (at r p< . ; at r p<< . ; ace p<< . ; ace- p<< . ). at r and ace- displayed increased staining in the fetal vascular endothelium of / mice (at r p<< . ; ace- p= . ), and in the cells lining the maternal central artery (at r p<< . ; ace- p<< . ). ace- expression was very high in cytotrophoblast lining the maternal blood space in all genotypes. no gross structural differences were seen. comment: the antibody used did not differentiate between ace and the membrane-bound "testicular"-ace (tace). immunohistochemically-identified ace expression was upregulated in / placentae despite the loss of somatic ace, suggesting that t-ace can be expressed placentally. we believe this is the first demonstration of such expression. ace and t-ace are catalyticallysimilar in converting angi to angii. furthermore, angii acting via the at rs is vasodilatory and ace- catalyses production of the vasodilatory ang ( - ); placental blood flow was presumably well-maintained and pregnancy outcome is normal in / mice. it is generally accepted that prostaglandin production plays a crucial role in both term and preterm parturition, and recently the administration of alpha hydroxyprogesterone acetate has been shown useful in preventing preterm labor. what is not clear is the biochemical pathway of prostaglandin production during labor and what, if any, effect alpha hydroxyprogesterone acetate has on this pathway. in order to address this question, we used immunohistochemical staining techniques to evaluate the effect of treating human placental amnion and chorion decidua with alpha hydroxyprogesterone acetate. membranes from unlabored patients were obtained at cesarean section and immediately seperated into control and drug treated specimens. controls were from the same placenta and were subjected to all experimental procedures except for the addition of alpha hydroxyprogesterone acetate to the culture media. specimens were compared at zero, six, and twenty four hour intervals. at the appropriate time, each specimen was formalin fixed and then paraffin blocked. tissue sections were then mounted on slides which were immunohistochemically stained using appropriate primary and secondary antibodies and standard techniques. the slides were then analyzed via light microscopy for changes in staining of three enzymes involved in prostaglandin production--cyclooxygenase (cox- ), cyclooxygenase (cox- ), and -hydroxy prostaglandin dehydrogenase (pgdh). compared to control, the slides treated with alpha hydroxyprogesterone acetate had differing amounts of enzyme expression. cox- was relatively unchanged and pgdh was only slightly increased, but cox- was noticeably decreased in the treated slides. these results were time dependent. this data suggests that alpha hydroxyprogesterone acetate decreases prostaglandin production in fetal membranes primarily by downregulation of the cyclooxygenase enzyme. objectives: in the human placenta, proliferation, differentiation and fusion of cytotrophoblasts (ct) are essential events in the formation of the multinucleated syncytiotrophoblast, however the regulation of these processes is poorly understood. using an explant model of human first trimester placenta we have established that both igf-i and -ii enhance ct proliferation, differentiation and survival mediated via igf r signalling. we have also shown that non-specific inhibition of protein tyrosine phosphatases inhibits igf-mediated signalling in trophoblast; therefore, we have now used sirna-mediated knockdown to investigate the role of the tyrosine phosphatase shp- in this pathway. methods: amaxa nucleofector technology was used to deliver shp- or scrambled sirna ( nm) to bewo cells or first trimester villous tissue fragments. knockdown was confirmed by q-pcr and western blotting. transfected cells and tissue were maintained in culture for hours, then treated with igf-i or igf-ii ( nm) for a further hours before immunohistochemical (ihc) analysis for cell proliferation (ki , brdu) or apoptosis (m ). results: sirna-mediated knockdown of shp- in bewo cells ( % reduction on western blot) demonstrated that igf-induced proliferation was reduced from . ± . % to . ± . % (p< . , n= ). ihc analysis of tissue demonstrated that shp- is localised to ct. following knockdown ( % decrease by q-pcr), igf-i-and igf-ii-induced ct proliferation was decreased by . ± . % and . ± . % respectively (p< . , n= ). furthermore, the ability of igf-i-and igf-ii to prevent ct apoptosis (m staining) was reduced by . ± . % and . ± . % respectively (p< . , n= ) after shp- knockdown. conclusions: igf stimulation of cytotrophoblast proliferation is mediated by shp- . exogenous igf rescues cytotrophoblast from apoptosis, and this pathway is also shp- -dependent. villous endothelial cells. emily j su, zhi-hong lin, ping yin, scott reierstad, joy innes, serdar e bulun. obstetrics and gynecology, northwestern university feinberg school of medicine, chicago, il, usa. background: within the human vascular system, estrogens have been shown to enhance vasodilatation in both normal and abnormal endothelium. estrogenic function occurs by activation of one or both of two estrogen receptors, estrogen receptor-alpha (esr ) and estrogen receptor-beta (esr ). these estrogen receptors are expressed in a wide variety of tissues. within the vasculature, estrogen receptors regulate the expression of multiple vasodilator and vasoconstrictor proteins. specifically, esr has been shown to be critical in maintaining normal vascular physiology in a murine model, where esr knock-out mice demonstrate significant systolic and diastolic hypertension. we hypothesize that within placental endothelium, estrogen plays an important role in maintaining normal vascular function that is critical for normal fetal growth and development. methods: term placentas from uncomplicated pregnancies were obtained, and the decidua was removed. an iv cannula was inserted into the umbilical vein, which was perfused with a collagenase/dispase solution. the perfusate was collected and subjected to further purification. these cells were cultured in complete medium, and after the initial passage of these cells, purity was confirmed via immunofluorescence and flow cytometric studies. estrogen receptor expression was determined in these cells via western blotting. additionally, these endothelial cells underwent treatment with varying doses of estradiol ( - m to - m), and quantitative real-time pcr was performed thereafter for mrna levels of various genes important in prostanoid production. results: western blotting demonstrated that esr is the only estrogen receptor expressed within villous placental endothelial cells. estradiol induced cyclooxygenase- (cox- ) mrna levels -to -fold, as quantified by real-time pcr (p< . ). conversely, there was no effect of estradiol on cyclooxgenase- (cox- ). conclusion: these results suggest that estradiol and esr are important in mediating the balance of prostanoid production that is essential in maintaining placental vascular health. future studies will further delineate estrogenic effects on prostanoid production within placental endothelial cells in health and disease. supported by the smfm/aaogf scholarship award and the nih grant u -hd . previously we established that proteins secreted by the decidua promote the differentiation of extravillous trophoblasts (evt) from a proliferative phenotype (characterized by cx , her- and alpha integrin protein expression) to an invasive phenotype (characterized by her- and alpha protein expression). the ability of decidua-conditioned media (dcm) to induce trophoblast differentiation was inhibited in the presence of the her- receptor antagonist, ag . furthermore, dcm-induced jar cell migration was also attenuated in the presence of ag . thus, the purpose of this study is to define the role of her signaling in evt differentiation and invasion. methods: evt differentiation was assessed in placental villous explant outgrowths and jar cells using antibodies against markers of the proliferative and invasive phenotypes. trophoblast migration was assessed using jar cells in transwell migration assays. results: treatment of placental villous explants with egf, a her- ligand, resulted in the downregulation of her- and an upregulation of her- expression, as well as an induction of alpha integrin expression. pre-treatment of placental villous explants with ag blocked this effect. in the jar cell line, egf treatment mimicked the differentiation-promoting effects of dcm by downregulating her- and upregulating her- expression, effects that were both blocked when jar cells were pre-incubated with ag . in contrast to dcm however, egf stimulation did not induce jar migration. stimulation of jar cells with hb-egf, a her- /her- heterodimer ligand, induced jar migration in a dose-dependent manner. analysis of dcm using antibody arrays confirmed the presence of many members of the egf family including hb-egf. immunohistochemical assessments of placental villous explants verified the expression of her- in evt outgrowths and in jar cells; her- expression was not affected by stimulation with either egf or hb-egf. conclusions: her signaling is an important and necessary component of the invasive evt differentiation cascade. our data supports a role for her- signaling in the induction of the invasive evt phenotype. chandrasekhar thota, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: parathyroid hormone related peptide (pthrp) is expressed in trophoblast cells and may play a role in placental growth and function. studies conducted in pregnant rats using pthrp antagonist showed decreases in fetoplacental growth during mid gestation. objective: to assess the effects of pthrp silencing on the expression of growth factors in immortalized first trimester trophoblast cells (htr- /svneo cells). methods: htr- /svneo cells cultured at º c and %co in rpmi- medium supplemented with % fbs were transiently transfected with nm of three different sirna sequences of pthrp, si , auaccuaacucaggaaacuuu; s i , g a g c u g u g u c u g a a c a u c a u u ; a n d s i , caagauuuacggcgacgauuu. for control, a scrambled sirna sequence was used. at % confluency, cells from each well were split and transfected again in triplicates with respective sirna sequences. total rna was isolated using trizol reagent h after transfection, and protein was extracted using lysis buffer h after transfection. the isolated rna and protein were subjected to reverse transcription and polymerase chain reaction (rt-pcr) and western analysis, respectively, using primers and antibodies specific for pthrp, plgf, vegf and lif. the results are expressed relative to either s for changes in mrna expression or -actin for changes in protein expression. results: rt-pcr of total rna obtained from htr cells subjected to double transfection with sirnas for pthrp showed a significant decrease in pthrp expression. expression of growth factors plgf, vegf and lif showed decreases with all the three sirna sequences used compared to the scrambled sequence. western analysis of cell lysates obtained from htr cells subjected to transfection with sirnas for pthrp showed a significant decrease in protein expression of pthrp and vegf. however the protein expression for plgf, and lif decreased in cells transfected with only si sequence of pthrp. conclusions: our studies showed that transient transfection of htr cells with sirna for pthrp caused decreases in both mrna and protein expression of pthrp. our results further suggests that decrease in pthrp peptide in transfected cells resulted in a decrease in vegf, plgf and lif suggesting that pthrp may play role in regulating trophoblast cell functions. objective. maternal obesity poses an increased risk to the fetus during pregnancy, and has long term consequences for the progeny. we tested the hypothesis that maternal caloric excess effects growth-related gene expression changes in the murine placenta. methods. female c bl/ mice were fed a hypercaloric diet ( % fat, % sugar) or standard chow for six weeks prior to mating and throughout pregnancy. near-term (day gestation) the dams ( controls, overfed) were sacrificed. following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on regulated genes. results. maternal overfeeding was associated with a two-fold increase in body fat mass. probe sets, corresponding to genes showed differential expression (p < . ); twenty-seven of which were up-regulated, and ten down-regulated, as compared to the placenta of control fed dams. of note, several genes related to obesity, diabetes, dna methylation, and the tgf beta pathway were differentially expressed. conclusions. diet-induced obesity in mice was associated with altered placental gene expression, including genes involved in tgf beta signaling and dna methylation pathways. these findings may have important implications for placental growth and epigenetic regulation. (supported by usphs hd- , earnest eu framework , tommy's the baby charity, uk). objective. maternal dietary protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. to comprehend more completely stress responses to maternal protein restriction, we measured gene expression changes in the mouse placenta. methods. pregnant fvb/nj mice were fed an isocaloric diet containing % less protein than normal chow ( % vs. % protein content) from embryonic day . (e . ) to e . . following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on the regulated genes, and used both qrt-pcr and immunohistochemistry to verify the results. results. the weights of the e . pups were decreased % (p< . ). probe sets, corresponding to genes, were regulated by protein restriction (p< . ); ninety-one being up-regulated and down-regulated. of particular note, several genes related to the p pathway were up-regulated. along with p itself, positive regulators of p (zmiz , jmy, hipk ) and genes activated by p (inpp d, cebpa) were induced. for selected genes we confirmed these results using qrt-pcr and immunohistochemistry. conclusions. by microarray analysis, we have described the genetic response to maternal protein deprivation in the mouse placenta. we observed that pups were growth restricted, and genes related to the p pathway were regulated. we propose a model through which intrauterine growth restriction is triggered, in part, by activation of the p pathway. (supported by usphs hd- and the sgi medical student grant to cpg). purpose: human placental villous tissue cultures have been underused in the study of placental drug disposition. thus we assessed the utility of this model by studying the effect of time in culture on the viability and integrity of the tissue and the, expression and function of proteins involved in the formation and efflux of -chloro- , -dinitrobenzene (cdnb) conjugate , -dinitrophenyl-s-glutathione (dnp-sg) as a model system for phase ii metabolism and cellular efflux. methods: placental tissue samples were obtained within minutes of cesarean deliveries following normal pregnancies in three patients. villous tissue was cultured in m medium to hr. at , , , , , and hr post culture, villous tissue was preincubated without or with atpase inhibitor sodium orthovanadate, exposed to μm cdnb, rinsed and incubated in buffer at °c to determine formation and efflux of dnp-sg, which was assayed by hplc. changes in expression of gstp - , abc transporter isoforms b , c and g (abcb , abcc , and abcg , resp.) were assessed by immunoblotting. lactate dehydrogenase (ldh) release, methyl tetrazolium thiazolyl blue (mtt) incorporation, and total tissue glutathione content were monitored up to hr. villous tissue morphology was assessed by immunohistochemistry. results: villous tissue structure and protein expression of glutathione-stransferase isoform p - (gstp - ) and abcg remained unchanged over hr in culture. expression of abcb and abcc , and total tissue glutathione decreased with culture time. ldh release was unchanged up to hr and increased at hr, while mtt incorporation remained constant to hr and decreased at and hr suggesting a decline in tissue integrity and viability at hr. however, dnp-sg formation, dnp-sg buffer/tissue ratio, and the extent of inhibition of dnp-sg efflux by sodium orthovanadate remained unchanged through hr. sodium orthovanadate decreased the dnp-sg buffer/tissue ratio by . ± . % (p< . ), consistent with inhibition of apical abc transporters. conclusions: these results support the use of this model to study the coordinated function of metabolizing enzyme gstp - and apical abc transporters in the formation and efflux of the model substrate dnp-sg. the model may be useful to study metabolism and transport of other compounds. syncytiotrophoblast. shauna f williams, ewa fik-rymarkiewicz, stacy zamudio, nicholas p illsley. obstetrics, gynecology and women's health, umd-new jersey medical school, newark, nj, usa. introduction: multiple inputs influence placental protein synthesis. nutritional, endocrine and metabolic factors have been implicated but its regulation has not been investigated. one of the factors shown to be associated with the inhibition of protein synthesis is hypoxia. the goal of this study was to determine the effects of hypoxia on a marker of placental protein synthesis. eukaryotic initiation factor (eif ) is a subunit of eif which is required for initiation of translation however when phosphorylated, eif is unable to participate in the assembly of the initiation complex. hypoxia has been shown previously to cause increased phosphorylation of eif . we hypothesized that hypoxia would increase the levels of phosphorylated eif in term syncytiotrophoblast, thus inhibiting protein synthesis. methods: primary syncytiotrophoblast cultured from term cytotrophoblast were incubated for hr in atmospheres of , , or % o or in the presence of the hypoxia-mimetic, dimethyloxalylglycine (dmog, . - . mm) in % o . cell extracts were analyzed by western blotting to determine the degree of eif phosphorylation. results: incubation in , , or % o did not increase eif phosphorylation relative to the % o control (n= , separate placental preparations). incubation in dmog concentrations up to . mm did not affect eif phosphorylation however incubation in . mm dmog increased eif phosphorylation by ± % (p < . , n= ). conclusions: contrary to our expectations, inhibition of protein synthesis via the eif regulatory pathway was not apparent except when induced by the highest concentration of dmog, consistent with severe hypoxia. thus while eif phosphorylation does occur, we did not observe changes at dissolved oxygen levels of %. these data suggest that a reduction in syncytial protein synthesis via the eif pathway takes place only under severe hypoxic stress. (supported by nih hd ). the increasing prevalence of overweight and obese women of childbearing age is a growing public health concern. the impact of maternal obesity on placental aa transport, which is essential for normal fetal development, remains poorly defined. there are three sub-types of the placental na-dependent system a transporter, snat , and which mediate neutral aa transport. snat is ubiquitously expressed in mammalian tissues and is likely responsible for the majority of placental system a activity. objective: to examine the impact of maternal obesity and over-nutrition on the fetal: maternal (f:m) aa ratio and placental protein abundance for snat . methods: nonpregnant ewes were randomly assigned to a control (c, % of nrc recommendations) or obesogenic (ob, % of nrc) diet from - to days of gestation (dg). under isofluorane anesthesia, maternal and fetal blood samples were collected for aa analysis by hplc from five twin bearing ewes in each dietary group. after euthanasia, placental cotyledonary (cot) tissue was separated from caruncular tissue, frozen in liquid nitrogen and stored at - c for western blot analysis. results: fetuses from ob ewes were % heavier (p< . ) than those from c ewes at dg ( ± vs. ± g). blood concentrations of asn, thr, cit, arg, tau, tyr, trp, val, phe, leu and orn were higher (p< . ), or tended to be higher (met and lys, p< . ) in ob than c ewes. in contrast, f:m ratios, for asn, ser, gln, his, gly, thr, cit, arg, b-ala, tau, ala, tyr, trp, met, val, phe, leu, orn and lys were reduced (p< . ) in ob compared to c ewes. snat content in cot tissue was reduced in ob when compared to c ewes ( . ± . vs. . ± . arbitrary units; p< . ). conclusions: maternal obesity in pregnancy reduced expression of placental snat protein and efficiency of placental aa transport in ewes, providing a mechanism whereby fetuses may mitigate excessive delivery of aa under conditions of maternal obesity and over-nutrition. decreased aa transport to the fetus may play a role in altered cellular structure and function. nih inbre p rr . early gestation utero-placental hemodynamics in an ovine model of fetal growth restriction. lucia dohnal, james s barry, henry l galan, randall b wilkening, russell v anthony. perinatal research center, university of colorado health sciences center, aurora, co. objective: fetal growth restricted (fgr) pregnancies, during late gestation, exhibit altered placental hemodynamics, and reduced capacity for o and nutrient transfer. it was our objective to examine utero-placental hemodynamics and o uptake during early gestation in an ovine model of fgr. methods: singleton-bearing ewes were instrumented with uterine artery flow probes, uterine venous and femoral artery catheters before being placed into a highambient temperature (fgr; n= ) or normothermic (con; n= ) environment at days of gestation (dga). maternal arterial and venous blood, uterine artery flow, heart rate, arterial pressure and respiration rate was collected until dga, at which time umbilical venous blood, fetal weight, placental weight and tissue were harvested. data reported here were analyzed by students t-test. results: maternal respiration rate ( . ± . vs . ± . breaths/min) and arterial po ( . ± . vs . ± . mmhg) were increased (p . ), whereas maternal heart rate ( . ± . vs . ± . beats/min), blood pressure ( . ± . vs . ± . mmhg) and arterial pco ( . ± . vs . ± . mmhg) were reduced (p . ) in fgr pregnancies. at dga, fetal weight was not different (p . ), but placental (total placentome) weight ( . ± . vs . ± . g) was reduced (p . ) in fgr pregnancies. while uterine artery (pregnant horn) flow ( . ± . vs . ± . ml/min) tended (p= . ) to be reduced in fgr pregnancies, relative uterine artery flow ( . ± . vs . ± . ml/min/g fetus; . ± . vs . ± . ml/min/ g placenta) was not different (p . ). uterine o uptake (mmol/min), relative uterine o uptake (ml/min/g fetus or ml/min/ g placenta) and uterine o extraction (%) were not different (p . ) between fgr and con pregnancies. at dga, umbilical vein po (mmhg), o content (mm) and o capacity (mm) were also not different between fgr and con pregnancies. conclusions: reduction in absolute uterine artery flow (ml/min) did not impact utero-placental o uptake or transfer to the umbilical vein, and may have resulted from reductions in maternal cardiac output. relative uterine artery flow was not reduced, suggesting that uterine blood flow and delivery of o to the conceptus does not mediate the ongoing placental growth restriction initiated during early gestation. supported by nih hd . barton c staat, anna maria marconi, cinzia paolini, alex cheung, henry l galan, frederick c battaglia. obstetrics gynecology and pediatrics, univ of colorado at denver health sciences center, aurora, co, usa; dept of obstetrics and gynecology, san paolo institute of biomedical sciences, university of milano, milano, italy. objective: to determine relative contributions of transplacental flux vs fetal production for myo-inositol and mannose in normal term pregnancies using stable isotopic methodolgy. background: myo-inositol and mannose are important in biologic functions. an external supply of mannose may be required for glycoprotein synthesis. low maternal myo-inositol is associated with spina bifida. mannose concentrations are known to be higher in the mother than the fetus. in contrast, myo-inositol concentrations are higher in the fetus than the mother. what remains unknown is whether fetal levels of these polyols are a result of direct maternal transport or from conversion of glucose. design: four term uncomplicated pregnancies undergoing an elective ceasaran section were infused with c labeled isotopes of glucose, myo-inositol and mannose over hours prior to delivery. maternal samples were obtained prior to infusate being administered, and at hour (h), . h and h. fetal concentrations were measured from umbilical artery and vein plasma. the concentrations of labeled and unlabeled glucose, mannose and myo-inositol were measured using high pressure anion exchange chromatogrpahy permitting detection of polyols and sugars at concentrations in the μm range. the feto-maternal molar percent enrichment (mpe) ratio was calculated for each glucose, mannose, and myo-inositol as the ratio between fetal plasma enrichment and the maternal plasma enrichment at steady state. steady state was calculated as the mean of the three maternal samples taken during infusion. results: the feto-maternal mpe ratios of mannose ( . ± . , p= . ) and glucose ( . ± . , p= . ) were not significantly different from . , consistent with transplacental supply. the feto-maternal ratio for myo-inositol ( . ± . , p= . ) indicates little transplacental flux ( % of fetal inositol derived from maternal plasma). conclusion: in normal term pregnancies, fetal mannose and glucose concentrations are dependent upon maternal transplacental supply. in contrast, fetal myo-inositol concentration is not dependent upon transplacental supply, but fetal demands are met by placental conversion, likely from glucose. christian wadsack, manuela augsten, christian guelly, ursula hiden, ingrid lang, manfred moertl, uwe lang, gernot desoye. clinic ob/gyn; center of med res; inst cell biol, histol embryol, med univ graz, austria. background placenta and fetus need lipids for growth and synthesis functions. part of the lipids is supplied from maternal sources by transplacental transfer. recently, we identified in human placenta the high density lipoprotein (hdl) receptor scavenger receptor class b type i (sr-bi). among other functions it mediates hdl-induced ser -phosphorylation of endothelial nitric oxide synthase (enos) resulting in enos activation. this mechanism allows hdl to contribute to regulation of vasotonus in arteries. we hypothesized that term placental endothelial cells (ec) express sr-bi at levels different between arteries and veins. methods sr-bi was localized by ihc and quantified by qrt-pcr in rna isolated from arterial and venous vessels. arterial (eca) and venous (ecv) placental ec were rigorously characterized. sr-bi levels were measured by qrt-pcr and immunoblotting. hdl binding and uptake was measured in eca and ecv with i-labelled hdl. hdl from human donors was used to stimulate ser enos phosphorylation. epigenetic regulation was studied by methylationspecific pcrs for cpg-rich promoter regions of sr-bi. pdzk a key adaptor for sr-bi mediated enos activation was measured by sqrt-pcr. in situ analyses (ihc, qrt-pcr) showed more sr-bi in arteries than in the vein. the differential expression persisted in vitro in isolated eca and ecv even after passages and culture under same conditions suggesting epigenetic mechanisms regulating sr-bi. however, no methylation was found in eca or ecv. sr-bi was functional since hdl cell association was -fold higher in eca than in ecv ( ± . vs ± . ng hdl/mg prot). hdl did not induce ser enos phosporylation in eca or ecv, which was stimulated by ionomycin about -fold in both cell types. pdzk was undetectable in eca and ecv, whereas it was expressed in placental tissue. conclusion more sr-bi is expressed in ec from arteries than from veins in situ and in vitro. this is not the result of different methylation of sr-bi promoter and, hence, unlikely an epigenetic phenomenon. mechanism of differential expression and its functional consequences for vasotonus regulation is yet unknown. the lack of pdzk may account for the failure of hdl to activate enos. (grants , , oenb). cells. juan a arroyo, brad ziebell, mi-hye park, henry l galan. obstetrics and gynecology, university of colorado andgealth sciences center, aurora, co, usa. introduction: mtor is a protein that regulates cell growth in response to nutrients and growth factors. downstream effectors of the mtor pathway include the p and the ebp proteins. activation by phosphorylation of these proteins increases protein synthesis. given that various signaling pathways are regulated by hypoxia in human trophoblast and that mtor is expressed in human trophoblast, our objective was to determine the effects of hypoxia in the activation of mtor, p and ebp in cultured human trophoblast. study designs: trophoblast cell were isolated from term uncomplicated placentas using a trypsin, dnase and disapase solution. cytokeratin immunocytochemistry confirmed trophoblast cells culture purity. trophoblast cells were treated with hypoxia ( % o ) or normoxia ( % o ) for and hours. western blot for p-mtor, mtor, p-p , p , p- ebp , and ebp were done for each time studied. results: trophoblast cells demonstrated: ) positive staining for cytokeratin, ) non significant differences for mtor at either ( . -fold; p= . ) or hours ( . -fold, p= . ), ) no differences in p protein at ( . fold; p= . ) or hours ( . -fold, p= . . ), ) no differences for ebp at either or hour. conclusion: we conclude that the mtor pathway is not regulated under hypoxic conditions in cultured trophoblast, which suggests that hypoxia does not affect protein synthesis in cultured human trophoblast. however, this may not reflect what happens in vivo in iugr. (supported by nih grant r hl - a ). increased expression of phospho-mtor, phospho-p , phospho-akt and phospho-erk in an ovine model of fetal growth restriction. juan a arroyo, brad ziebell, henry l galan. obstetrics and gynecology, university of colorado and health sciences center, aurora, co, usa. objective: both phosphorylated (p) mtor and p are known to be involved in protein synthesis and are regulated by physiological conditions such as fetal growth restriction (fgr). in a hyperthermic (ht) ovine model of fgr we hypothesize that mtor, p , ebp , erk and akt will be phosphorylated (activated) in the placentae of age (dga) animals. study design: ewes were exposed to ht conditions for days to induce iugr and were placed in ambient conditions. at necropsy ( dga), placentomes were separated into the maternal (caruncle) and fetal (cotyledon) components and frozen for western blot analysis with antibodies against (p) mtor, mtor, (p) p , p , (p) ebp , ebp , (p) erk, erk, (p)akt and akt. results: compared to control animals, fgr animals had smaller fetuses ( ± g v. ± g; p= . ) and smaller placentae ( ± g v. ± g; p= . ) at dga. fgr cotyledon showed an increase in p-mtor ( . -fold; p= . ), p-p ( . -fold; p< . ), p-erk ( . -fold; p< . ) and p-akt ( . -fold; p< . ). in contrast, caruncle (maternal) did not show any changes for the mtor pathway. conclusion: in fgr ovine pregnancies, the fetal placental tissues (cotyledons) showed upregulation of the mtor pathway for protein synthesis via phosphorylation of the p but not ebp while this was not seen in the maternal (caruncle) tissues. in addition neither the cotyledon or caruncle tissues at mid-gestation ( dga) showed changes in these endpoints, which is prior to the exponential fetal growth that starts at mid-gestation. (supported by nih grant r hl - a ). hyperuricemia has long been recognized as a common clinical finding in preeclamptic (pe) women. to date, elevated uric acid concentrations in these women have been considered a marker of disease severity. however preeclamptic pregnancies with hyperuricemia, are associated with an increased frequency of preterm birth and fetal growth restriction. over the past decade several pathogenic roles for uric acid have become evident, raising the possibility of a role(s) for uric acid in the altered vascular and placental functions associated with pe. objective: examine the effects of syncytial uric acid uptake on system a amino acid transport across the human placenta using a primary placental villous explant model. methods: placental villous explants from placentae of healthy, term pregnancies were incubated for hours with uric acid ( . mg/dl), corresponding to concentrations of uric acid observed in pe women. these experiments were conducted in the presence or absence of probenecid ( m), a uric acid cellular uptake inhibitor. system a amino acid transport was subsequently assayed using a radiochemical assay in which na+-dependant uptake of radio-labeled system a substrate, [ c] methyl-amino-isobutyric acid, was measured over minutes. data were analyzed using a paired student's t-test and presented as mean ± sem. results: uric acid attenuated system a amino acid placental transport by % (± . %, p< . ). this inhibitory effect of uric acid on system a activity was prevented by probenecid. conclusions: uric acid reduces placental amino acid transport at concentrations observed in pe women. this inhibitory effect of uric acid is dependant upon syncytial uptake of uric acid, being inhibited by the uric acid transporter inhibitor probenecid. these results may be relevant to the increased frequency of fetal growth restriction observed in hyperuricemic pe. additionally the results of this study, indicating a detrimental effect of hyperuricemia on placental function, also suggest a role for uric acid in the pathophysiology of pe. hyperuricemia, a well-documented clinical finding in preeclamptic women, is associated with pre-term birth and intrauterine growth restriction. uric acid is higher in women destined to develop preeclampsia as early as weeks of gestation at a time when cytotrophoblast are invading decidua and myometrium and remodeling uterine spiral arterioles. we propose that elevated concentrations of uric acid may have detrimental effects on placental development in part through inhibition of trophoblast invasion through the decidua. objective: examine the effects of increasing concentrations of uric acid on trophoblast invasion through a reconstituted extracellular matrix. methods: using the in-vitro matrigel invasion assay, the effects of increasing concentrations of dissolved uric acid ( . mg/dl, . mg/dl and . mg/dl) on the ability of immortalized first trimester extravillous trophoblast cells (htr -svneo) to invade through a reconstituted extracellular membrane were assessed. the concentrations of uric acid used were comparable to those measured in healthy pregnant women and preeclamptic women with an increase in uric acid of two or four standard deviations above normal. cells that successfully invaded through the matrigel membrane within hours were fixed with methanol, stained with hematoxylin and counted. data were analyzed using a one-way analysis of variance with fisher's post-hoc analysis. results: uric acid attenuated trophoblast invasion in a dose-dependent fashion (p< . ), with decreases of % (± . %), % (± . %) and % (± . %) respectively compared to untreated controls. conclusions: exogenous uric acid, at physiological and pathological concentrations, is capable of attenuating trophoblast invasion through a reconstituted extracellular membrane in a dose dependent fashion. these results suggest uric acid is a potential contributor to the pathophysiology of altered placental perfusion in preeclamptic pregnancies. previous studies have shown that transforming growth factor (tgf)-is a key inhibitory factors in the invasion of early trophoblast cells, suggesting therefore that overcoming tgf-beta signaling may be necessary for successful implantation. smad ubiquitin regulatory factor (smurf ), a hect type e ubiquitin ligase, is a key regulator of tgf-signaling pathway, targeting tgf-receptors and various smads for proteasome-mediated degradation. in this context, smurf has been shown to play important roles in embryonic development, cell senescence and tumor formation. as a key regulator of tgfbeta signaling, we wished to determine whether smurf has a physiological role during embryo implantation, especially in trophoblast invasion. we have examined the spatio-temporal expression of smurf in human placental villi during pregnancy. we have also investigated the possible function of smurf in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, htr /svneo. our results showed that expression of smurf in placental villi was the highest during the first trimester and the expression decreased in the nd trimester. expression of smurf was lowest in placental villi at parturition. overexpression of smurf in htr /svneo cells reduced tgf beta type i receptor levels and attenuated the inhibitory effect of tgf-on cell migration and invasion. conversely, rnai-mediated down-regulation of smurf resulted in significant increase of tgf-type i receptor protein levels. in contrast, the levels of smad , another potential target of smurf , was unchanged. in conclusion, the present study suggests that smurf participates in trophoblast cell migration and invasion by down-regulating the expression of tgf-type i receptor. our previous data demonstrated the extensive expression of mmp- in various kinds of trophoblast cells in human placenta at the early pregnancy. however, the modulation of the enzyme in trophoblasts is largely unclear. in the present study, the effects of the two types of gonadotropin releasing hormone (gnrh) on mmp- expression were examined in an immortalized human cytotrophoblast cell line, b tert- that has been established in this lab. real-time quantitative pcr and western blot analysis revealed that both types of gnrh (gnrh i and gnrh ii) could increase mmp- mrna and protein levels in b tert- cells in time-dependent manners. in particular, regulatory effect of gnrh i on mmp- expression was concentration-independent, whereas that of gnrh ii was dose-dependent. moreover, both gnrh i and gnrh ii could evidently activate jnk kinase, and sp , an inhibitor of a jnk kinase, reversed the up-regulation of mmp- induced by either gnrh i or gnrh ii. on the other hand, it is not likely that erk / pathway participates in the signaling of gnrh i or gnrh ii. collectively, our observations suggest that gnrh i and gnrh ii elicit their modulation effects in human trophoblastic cells through jnk pathway leading to up-regulation of mmp- . during the first trimester of pregnancy, the oxygen tension of the developing trophoblast cells is less than %. however, the majority of studies on primary trophoblast cell development have been performed at % oxygen. primary third-trimester trophoblast cells are believed to be nonproliferative syncytiotrophoblast cells. we have previously demonstrated that low oxygen tension dramatically affects the differentiation pathway of these cells. we now hypothesize that cell culture in low oxygen tension will improve cell growth and restore proliferation. methods: primary trophoblast cells were purified from third-trimester placenta by enzymatic dispersion and cd- negative selection and cultured at %, % or . % oxygen tension for up to days. the number of cells in culture was assessed by cell counting and by measuring genomic dna. live:dead and mtt assays were used to determine viability. proliferation was assayed with brdu and immunohistochemistry for proliferating cell nuclear antigen. to assess cellular activity, radioactivity of protein precipitated from cells cultured in the presence of tritiated leucine was measured. results: there were no obvious morphologic changes in the cells cultured in different oxygen tensions. the amount of cell loss was directly proportional to oxygen tension: at % oxygen % of the cells remain in culture; at . % oxygen tension % of the cells remained. the cells at . % oxygen tension were proliferating and had a five-fold increased metabolic activity. conclusions: it was previously believed that third-trimester trophoblast cells are non-proliferative. we have demonstrated that low oxygen tension increases the survival of primary third-trimester trophoblast cells. this may reflect the change in the differentiation pathway of these cells. however, the cells also begin to proliferate and increase their metabolic activity. trophoblast cells in vivo form a three dimensional structure which promotes critical complex cell-to-cell interactions that cannot be studied with traditional monolayer cell culture. we developed a substrate-free three-dimensional trophoblast culture system capable of studying cellular interactions without a confounding artificial matrix. methods: nonadhesive agarose hydrogels containing cylindrical recesses m in diameter were cast from molds designed using computer-assisted prototyping. tcl trophoblast cells were seeded into the gels ( , cells per) for up to days. viability and cellular stress were assessed and the threedimensional structures of the spheroids were analyzed. results: tcl trophoblast cells formed uniform spheroids within three days of seeding. the spheroids remained intact after being removed from the mold. when placed in traditional cell culture dishes the cells adhered to the plate within one hour and rapidly proliferated into a monolayer. repetitive reseeding allowed easy transition between monolayer and spheroid without affecting cellular morphology. serial sectioning on days , and revealed central vacuolization forming a trophoblast vesicle with an outer rim . m (+/- m) thick. this rim size remained constant for at least days. live:dead assay demonstrated that the outer cells remained viable and staining against proliferating cell nuclear antigen demonstrated that the cells were proliferating. the inner cells undergo apoptosis as demonstrated by caspase- staining. there is an abundance of vegf staining in the cells remaining in the on the inside of the sphere suggesting a gradient of nutrient or oxidative stress. the formation of a vesicle has been confirmed with confocal imaging. em imaging revealed the structure of the rim. conclusions: trophoblast cells cultured in a novel substrate-free three dimensional system form trophoblast vesicles within days of seeding. these vesicles remain viable after long-term culture and can be repeatedly reformed with repetitive seeding. this new cell culture technique allows us to better study placental cell-cell interactions with the potential of forming microtissues. the transcription factor glial cell missing- (gcm ) mediates cell cycle arrest and differentiation of human trophoblast progenitors into villous syncytiotrophoblast and invasive extravillous cytotrophoblast (evt). micro-array analysis of total rna extracted from cultured bewo cells, in which gcm mrna and protein were repressed using sirna, identified tissue inhibitor of metalloproteinase- (timp- ) in the highest ( -fold) upregulated group of genes. confirmatory rtpcr demonstrated a -fold mrna induction. in placental villi, gcm acts as a transcription factor promoting expression of the fusogenic protein syncytin that mediates syncytial fusion into the overlying syncytiotrophoblast. by contrast, syncytial fusion is uncommon in evt. rather these cells invade several millimeters into the distal myometrium where they transform spiral arterioles. to investigate the role of timp and gcm in the trophoblast we assessed its mrna by qrt-pcr and protein by western blot in cellular extracts from both bewo cells grown under standard cultivation conditions (synchronized by prior thymidine exposure) and floating cultured first trimester villous explants cultured in % oxygen with prior exposure to either gcm sirna or anti-sense oligo-nucleotides to gcm . gcm inhibition in the bewo system was associated with a - % increase in timp- protein expression and alteration of cell proliferation and differentiation in both models. we are presently utilizing the explant model of evt invasion (explant tips cultured on matrigel in % oxygen) to test the hypothesis that gcm mediates metallo-proteinase expression and evt invasion via timp- . presently we conclude that gcm -mediated evt differentiation involves more than an arrest of mitosis and may include promotion of invasion via repression of timp- . funding: cihr. scott h purcell, jeremy d cantlon, virginia d winn, russell v anthony. , colorado state university, fort collins, co; university of colorado health sciences center, aurora, co. background: periattachment factor (pf) is a nuclear protein first described in the bovine conceptus. our research in sheep has shown pf mrna concentration peaks when the conceptus is undergoing elongation and initial apposition to the endometrium, and that pf is a nuclear protein localized to the trophoblast. in silico analysis identified a human homolog, hprr . objective: the objective of this experiment was to determine if pf was expressed in the human placenta, and to develop short-hairpin (sh) rnas for hpf to begin investigating its function. materials and methods: immunohistochemistry was performed on paraffin embedded first and second trimester human placental samples. placental sections were immuno-stained using rabbit polyclonal antiovine pf or anti-human cytokeratin- . cytotrophoblasts from first trimester pregnancies (n= ) were subjected to an in vitro invasion assay and rna was harvested following , , and h. quantitative rt-pcr was performed on these samples with intron-spanning primers for hpf, and normalized on hs mrna concentrations. based on the human pf sequence, four putative shrna constructs were generated and cloned into a lentiviral expression vector. bewo human choriocarcinoma cells were treated with one of four shrna contructs or an empty vector for h and then rna was harvested from cells for analysis by quantitative rt-pcr. results: periattachment factor was present in the nuclei of both first and second trimester cytotrophoblasts. hpf mrna concentration increased as invasion occurred from , , to h in all samples; while hypoxia decreased expression at h of invasion compared to h under normoxic conditions. the four lentiviral vectors expressing shrna against hpf resulted in hpf mrna concentrations at , , and % of hpf mrna concentration with the control vector. conclusion: the presence of pf in the human placenta and the increase in pf mrna during cytotrophoblast invasion may indicate this gene plays a role during implantation. we have developed shrnas against pf that result in greater than % mrna knockdown and will be using these to begin to elucidate the function of pf in the human placenta, specifically during the invasion process. recently we demonstrated that infusion of imd antagonist (imd - ) in rat caused distorted labyrinth indicative of a deficient vasculature in placenta. we hypothesize that imd has a role in migration of first trimester trophoblast cell (htr- /svneo) via regulating human leukocyte antigen (hla-g) and stimulating mek / / erk / phosphorylation. objectives: ) to asses the effect of imd on migrating capacity of htr- sv/neo cells using scratch assay in presence or absence of mek and ras/raf inhibitor, u and manumycin a, respectively ; ) to assess the effect of imd peptide on phosphorylation of mek / and erk / in first trimester htr cells ) to analyze the effects of imd on the expression of human leukocyte antigen, hla-g, a critical factor involved in the invasion and vascular remodeling of spiral uterine arteries and subsequent pregnancy in human. methods: htr- sv/neo cells were used to assess the effect of imd ( - m) on the expression of hla-g mrna and phosphorylation of erk / and mek / protein by reverse transcriptase polymeration chain reaction (rt-pcr) and western blot analysis respectively. scratch wound assay was used to determine the migration capacity of htr cells. total rna was isolated from cells using trizol reagent and processed for rt-pcr and results are expressed relative to s mrna. trichloroacetic acid was used for the extraction of total protein for western blot analyses. results: our data demonstrates that, ) imd enhances the migrating capacity of htr cells (compared to the untreated cells) and these effects are inhibited by mek and ras/raf inhibitors, u and manumycin a, respectively; ) imd ( - m ) stimulates phosphorylation of erk / and mek / proteins in htr cells, ) imd increases the expression of hla-g mrna in htr cells. conclusion: imd promotes migration of first trimester htr cells through mek/ erk signaling pathway and modulates the expression of immunoregulatory molecule, hla-g in these cells. chymotrypsin-like protease promotes the placenta tissue release of sflt- . yang gu, shuang zhao, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: the placenta is a major source of soluble vegf receptor- (sflt- ) in the maternal circulation during pregnancy. increased placental release of sflt- is believed to play an important role in the pathophysiology and pathogenesis in pe. however, the mechanism of increased placental sflt- release in pe is unknown. we recently reported increased chymotrypsin-like protease (clp) activity and expression in placental trophoblasts from pe. in this study, we tested if proteolytic effects of chymotrypsin may play a role in promoting sflt- release by placental trophoblasts. methods: placentas delivered by normal pregnant women (n = ) were used. we tested if chymotrypsin could promote sflt- release by placental tissue, in which villous explants were cultured with dmem containing chymotrypsin at . , . , and . g/ml for hours. the culture medium was then collected for measuring sflt- . we then determined the specificity of chymotrypsin induced sflt- release. villous tissues were cultured with or without chymotrypsin inhibitor (ci) in culture and then the medium was collected and measured for sflt- . soluble flt- was measured by elisa. all samples were assayed in duplicate. data are presented as mean ± se and analyzed by anova. a p level < . is considered as statistically different. results: ) sflt- concentrations in the medium were increased when chymotrypsin was present in culture and the increased sflt- release induced by chymotrypsin was in a concentration-dependent manner: control: . ± . ; . g/ml: . ± . ; . g/ml: . ± . ; and . g/ml: . ± . (p< . ) pg/mg tissue/hour. ) ci could attenuate sflt- release. this inhibitory effect was also revealed in a concentration-dependent manner: control: . ± . ; ci . g/ml: . ± . ; and ci . g/ml: . ± . (p< . ) pg/mg tissue/hour. conclusions: increased placental sflt- release stimulated by chymotrypsin and decreased placental sflt- release inhibited by chymotrypsin inhibitor suggest that the proteolytic effect of clp may play a role in sflt- generation. therefore, increased clp activity in placental trophoblasts may contribute to the increased placental sflt- production in pe. (supported nih grants hl and hd ). the change of autophagy-related proteins, lc and beclin- , by tnfa stimulation in cultured primary trophoblasts. soo-young oh, kyung hee kim, suk-joo choi, jong-hwa kim, cheong-rae roh. department of obstetrics and gynecology, samsung medical center, sungkyunkwan university school of medicine, seoul, korea. objective: our previous work have demonstrated that the expression of lc , but not beclin- , was increased in placentas from pregnancies complicated by severe preeclampsia (sgi abstract # ) . to understand the regulatory mechanism of these autophagy-related proteins in trophoblast cells, we investigated the changes in these proteins in response to cytokine or hypoxic stimulation in cultured primary trophoblast. material and methods: primary human cytotrophoblasts obtained from normal term placenta were cultured with stimulation of tnf-a or cocl for a given time and the changes of beclin- and lc were assessed using immunoblot analysis. paired t test was used for statistic analysis. results: tnf-a stimulation induced a significant increase of the expression of lc -ii in cultured primary trophoblasts while decreasing the expression of beclin- (p< . for each). however, cocl stimulation did not induce a significant change of both lc -ii and beclin- . conclusions: our data suggests that tnf-a stimulation in cultured primary trophoblasts is associated with increased autophagic activity. background: thyroid hormones play vital roles in the development of the fetal brain. mutations in mct , recently recognised as a specific thyroid hormone transporter, define a novel syndrome of severe x-linked psychomotor retardation accompanied by elevated serum t . we previously reported that mct expression in n-tera- (nt ) cells (a human embryonal cell line with characteristics of cns precursors), as well as mct -null jeg- choriocarcinoma cells, resulted in markedly reduced cell proliferation. further, the s x mct mutation, as reported in males affected by severe psychomotor impairment, resulted in a similar repression of proliferation to wild type, whereas the l p mutant failed to influence cell turnover compared with control. methods: we now examine the effect of "knocking down" mct via sirna and evaluate the effects of cell proliferation (mtt and [ h]-thymidine assays) and tri-iodothyronine (t ) uptake. results: repression of endogenous mct expression in nt cells by % caused a significant increase in proliferation compared to matched-dose nonspecific sirna treatment, independent of t concentration ( . %, . % and . % induction at , and nm t , n= , p< . ). we also sought to examine the role of mct in t uptake. in jeg- cells, wild type mct induced a . -fold increase in the uptake of i-labelled t . by contrast, mutants s x and l p failed to significantly augment t uptake, though r h caused a mild but significant . fold induction in uptake, hence retaining approximately % of wt activity. in parallel experiments, co-transfection of mu-crystallin, a t binding protein, resulted in a similar increase in t uptake compared with control ( . -fold; n= ; p< . ), implying that mct plays only a minor role in thyroid hormone efflux in jeg- cells. mutants s x, l p and r h showed analogous responses to those in the absence of mu-crystallin. conclusion: these results further extend the evidence of a potential role for mct in the modulation of cell proliferation, independent of t transport. to determine predictors of failure for labor induction in women with preeclampsia. we conducted a retrospective cohort study to examine cesarean delivery rates in all the preeclamptic women at a single institution undergoing labor induction between - with a singleton pregnancy >= weeks gestational age (ga). bivariate analyses informed the creation of multivariable logistic models to predict the risk of cesarean delivery using multiple predictors (maternal age, race/ethnicity, unfavorable cervix, gestational diabetes, diabetes, and gestational age). analyses were stratified by parity. our study population included , preeclamptic women undergoing labor induction. in the bivariate analyses, the risk of cesarean delivery ranged from as low as . % (p= . ) among multiparous women - weeks ga to as high as . % (p< . ) among nulliparous women with diabetes. a total of , women had adequate data to be included in the multivariable analyses. odds ratios of the predictors are presented in the objective: preeclampsia and cardiovascular disease share many risk factors, and women with preeclampsia are at increased risk of cardiovascular mortality later in life. we investigated whether risk factors associated with cardiovascular disease and preeclampsia remain elevated months postpartum. methods: we measured plasma sflt , endoglin, plgf, cellular fibronectin (cfn), uric acid, homocysteine, and asymmetric dimethylarginine (adma) in women with uncomplicated normotensive pregnancies compared to women with preeclampsia in samples collected at pre-delivery and again several months postpartum (average . ± . months). data are mean±sd or median (interquartile range). statistical analysis was by wilcoxin rank-sum or students unpaired t-tests with statistical significance accepted at p< . . results: the mean concentration of sflt , endoglin, plgf, homocysteine, adma, cfn, and uric acid were all significantly different in samples collected pre-delivery in subjects with preeclampsia compared to controls (table). adma, cfn and uric acid remained significantly higher postpartum in subjects with previous preeclampsia compared to postpartum controls (table) . conclusions: biological markers associated with altered vascular function or cardiovascular risk are elevated in women with preeclampsia, and some remain significantly higher in postpartum preeclamptic women. these data suggest that vascular dysfunction persists in women with previous preeclampsia, and may contribute to the increased risk of future cardiovascular disease. funded in part by national institutes of health nih- mo -rr and nih- po -hd . (ajp, ) . as leptin is a known potent angiogenic factor we hypothesized that leptin deficiency and/or resistance to leptin-induced vegf expression might be a mechanism for reduced angiogenesis in mfr offspring. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. mfr and control offspring were sacrificed on day of life (p ). some tissues were used to determine the expression of leptin by western blot analysis. for culture experiments, thoracic aortas were dissected, cut into - mm explants and incubated with leptin ( - ng/ml) in dmem ( % fbs). after hours of culture, rna was extracted from the tissues and subjected to real time rt-pcr using specific rat primers for vegf, vegfr and r , and ob-ra, stat and socs ( s mrna as control). culture media was analyzed for vegf protein by elisa. results: expression of leptin mrna and protein in p mfr aortas was significantly reduced. in culture, leptin significantly increased expression of vegf, vegfr and vegfr mrna in explants of aortas obtained from the control but not mfr tissues. as expected, control but not mfr aortic explants secreted significantly more vegf in vitro. to determine the mechanism for resistance to leptin-induced vegf in mfr offspring, we assessed expression of leptin receptor (ob-ra) in explants treated with leptin. leptin was found to induce the expression of ob-ra in aortas from both dietary groups. this upregulation of leptin receptor was accompanied by significant upregulation of stat and socs mrna in the control tissues. in contrast, in mfr explants only the ng/ml concentration of leptin induced an increase in stat mrna, and the magnitude of socs mrna increase by both concentrations of leptin was significantly less in the mfr explants. conclusion: these results indicate that reduced angiogenesis in mfr vessels is in part due to reduced leptin expression and ability of leptin to stimulate vegf expression. although in vitro leptin induced the expression of its receptor in both groups, it was only in the mfr group in which leptin up-regulated vegf and its receptors. our results suggest that this defect in leptin receptor function in mfr vessels is due, in part, to defects in jak/stat signaling. objective: women with a history of early-onset preeclampsia are at increased risk of developing major cardiovascular disease (cvd) related events, that have a detrimental effect on their long-term health and life expectancy. in this follow-up study, we measured established risk factors predictive of first cvd events after early-onset preeclampsia. study design: over a -year interval, primiparous women with a history of early-onset preeclampsia (delivery < weeks gestation) were included and tested for major cardiovascular risk factors at least six months after delivery, in addition to a population-based control group of healthy non-pregnant women. women with chronic hypertension were excluded. results: mean age was . years for cases compared to . years for controls (p<. ). after adjustment for age, we observed significantly increased mean values for weight (p=. ), body-mass index (p<. ), systolic blood pressure (p<. ), diastolic blood pressure (p<. ), total cholesterol (p=. ), ldl cholesterol (p<. ), triglycerides (p=. ), fasting blood glucose (p<. ), and lower hdl cholesterol (p<. ) in women with previous early-onset preeclampsia. no difference was found for height, smoking, diabetes, and ethnicity. estimated -year risk of first cvd events by framingham risk scores remained < % for all women (low-risk). nonetheless, at mean (sd) . ( . ) years after early-onset preeclampsia, % of women met the criteria for metabolic syndrome, % of women exhibited >= , % of women >= and % of women >= major cvd risk factors. conclusion: the majority of women with a history of early-onset preeclampsia exhibit at least one modifiable risk factor for future cvd. although most of these women are classified as low-risk according to the current aha guidelines, this is mainly due to their young age masking other, mostly modifiable, major risk factors. our data thus support life-style intervention programs aimed at primary prevention of cvd in women with a history of early-onset preeclampsia. it has recently been shown that antihypertensive drugs can stimulate cytokine release in normal and hypertensive pregnancy. there is evidence that these cytokines alter the secretion of inhibin a. inhibin a and activin a levels are increased in pre-eclampsia (pe), but it is not known if antihypertensive therapy can affect their secretion. patients and methods we recruited women with hypertensive disorders in pregnancy ( pe and non-proteinuric hypertension [ht]) and matched normotensive controls. inhibin a and activin a levels, before and - hours after initiating antihypertensives, were measured in serum and urine, using an elisa. the same markers were measured using validated assays in placentas delivered at cesarean section at similar gestational age ( pe, ht and controls). analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. marker levels before and after antihypertensive therapy were compared using paired t-test. we compared placental concentrations between the group which received antihypertensive therapy and the group which did not, using an independent t-test. data were analysed using spss®. in pe, both serum and urine levels of inibin a and activin a were increased at all gestations (p< . ). activin a (but not inhibin a) level was also increased at all gestations in ht (p< . ). after weeks' gestation (but not before), antihypertensive treatment was associated with a significant fall in both inhibin a and activin a serum levels, and urinary inhibin a, in both pe and ht. the placental concentration of inhibin a, but not activin a, was significantly higher in women with pe compared with controls (p< . ). there was no significant difference in either marker between controls and women with ht. the fall in serum levels of inhibin a and activin a following antihypertensive treatment after weeks' gestation may indicate that these drugs have an effect on the pathophysiology of pe other than their known antihypertensive action. pre-eclampsia (pe) is a placental disease of unknown etiology. anti-angiogenic factors, such as soluble fms-like tyrosine kinase (sflt- ) and soluble endoglin (seng), and pro-angiogenic factors, such as vascular endothelial growth factor (vegf) and placental growth factor (plgf), are believed to play an important role in its pathophysiology. maternal plasma concentrations of these markers are altered in pe, even weeks before the clinical manifestations. the aim of this study was to compare the concentration of these markers in placental extracts of normotensive pregnant women, and women with pe and non-proteinuric hypertension (ht). patients and methods placental samples were collected at cesarean section from women with pe (n = ), ht (n = ) and normotensive pregnancies of similar gestational age (n = ). these samples were stored at - ºc. the frozen tissue samples were homogenised and these four markers measured by specific, validated enzymelinked immunosorbent assays. analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. data were analysed using spss®. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). there was no significant difference in plgf concentration between controls and women with pe or ht. placental vegf concentrations in both pe and ht were higher than in controls (p< . ); there was no significant difference between the levels in pe and ht (p= . ). the fact that placental concentrations of sflt- and seng mirrored the known rise in serum levels in pe suggests that the placenta is the main source of these circulating factors. although sflt- was significantly raised in pe, plgf was not reduced. this suggests that the lower levels of free plgf found in the serum of women with pe are not the result of impaired placental production or secretion, but are due to increased binding by (the increased levels of) sflt- in the serum. objective. the purpose of this study was to evaluate whether systematic screening with uterine artery doppler (utad) and serum biochemical markers of oxidative stress, endothelial dysfunction and vasculogenesis performed during the first trimester predict efficiently pre-eclampsia (pet), specifically early-onset pet, in an unselected chilean population. methods. this nested case-control study involved asymptomatic pregnancies scanned at + - + week of gestation. the subjects for biochemical testing were women who were delivered due to pet (n= ) and normotensive controls (n= ) that were enrolled during the first trimester scan. mean pulsatility index (pi) of the utad was calculated. blood samples were obtained and stored at - o c until biochemical analysis of oxidative stress, endothelial dysfunction and vasculogenesis were performed. normally distributed data were analysed by the unpaired t test, and non-normally distributed data by the mann-whitney rank sum test. chi-square tests were used for the comparison of categorical variables. a probability level of p< . was considered significant. multiple logistic regressions were used to develop a combined predictive index. results. there was % and % significantly increased of the mean pi utad in women who later developed pet or early-onset pet compared to control pregnancies during the first trimester scan. although oxidative stress and endothelial dysfunction biochemical markers were not different between all pet pregnancies and control groups, plasma levels of sflt ( . ± . vs. . ± . pg/ml, p< . ) and placenta growth factor ( . ± . vs. . ± . pg/ml, p< . ) were significantly higher in women who subsequently developed early-onset pet compared to controls. multivariate logistic regression showed that a combination between abnormal utad and both biochemical markers of abnormal vasculogenesis were the best predictor test for early-onset pet, being its detection rate % with % false positive rate. conclusion. this study has shown early and selective changes in markers of impaired placentation and angiogenic state in women who later developed earlyonset pet, without alteration in oxidative stress and endothelial dysfunction. supported by fondecyt . currently it is unknown whether maternal inflammatory changes are specific to pregnancy or reflect an innate susceptibility to inflammation. c-reactive protein (crp) and interleukin- (il- ) are markers of the acute-phase inflammatory response and predictive of future cardiovascular events. we compared crp and il- levels after influenza vaccination, as an in vivo model for lowgrade inflammation, in non-pregnant women with a history of early-onset preeclampsia and controls with only uneventful pregnancies. methods: forty-four women with a history of early-onset preeclampsia (delivery < weeks' gestation) and twenty-nine controls with at least one uneventful pregnancy received an influenza vaccination. we then compared plasma levels of crp and il- at baseline, . days and . days after vaccination. results: median baseline crp and il- levels of women with a history of early-onset preeclampsia were comparable to controls ( . versus . mg/l; p= . and . versus . pg/l; p= . , respectively). however, high crp and il- responses to vaccination were more common in cases (ors for response > th, > th, > th, > th and > th percentile based on the distribution of control values of . , . , . , . and for crp [p for trend . ] and of . , . , . , . and . for il- [p for trend . ], respectively). the relationship between high il- responses and early-onset preeclampsia persisted after adjustment for body-mass index (p for trend . ). conclusion: women with a history of early-onset preeclampsia more frequently exhibit an innate pro-inflammatory phenotype not specific to pregnancy. background altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (hellp) syndrome. we examined whether allelic variants of the innate immune receptors toll-like receptor (tlr ) and nucleotide-binding oligomerization domain (nod ), that impair the inflammatory response to endotoxin, are related to preeclampsia and hellp syndrome. we determined five common mutations in tlr (d g and t i) and nod (r w, g r and l fs) in primiparous women with a history of early-onset preeclampsia, of whom women developed hellp syndrome and in women with a history of only uneventful pregnancies as controls. in addition, we assessed plasma levels of pro-inflammatory biomarkers c-rp, il- , sicam- , fibrinogen and von willebrand factor in a subset of women included at least six months after delivery. after adjustment for maternal age and chronic hypertension, attenuating allelic variants of tlr were more common in women with a history of early-onset preeclampsia than in controls (or . [ % ci . - . ]). highest frequencies for tlr variants were observed in women who developed hellp syndrome (adjusted or . [ % ci . - . ]). in addition, high levels of il- and fibrinogen were associated with a history of early-onset preeclampsia. combined positivity for any of the tlr and nod allelic variants and high levels of il- was . -fold more common in women with a history of early-onset preeclampsia ( % ci . - . ) compared to controls. we observed an association of common tlr and nod gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and hellp syndrome, that suggests involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy. thus, we sought changes in pbef in the serum of patients with mild and severe pe, compared with matched controls. immunodistribution of pbef in fetal membranes and placentas from similar patients was also studied. methods: serum samples ( ) were collected with clinical data including; gestational age, medications, ethnicity and recognized complications. patients in labor or infection were excluded. the standard bp and proteinuria criteria was utilized to classify cases for pe grouping; no pe (n= ), mild pe (n= bp; / - / , proteinuria; trace to + or - mg/ hr urine) and severe pe (n= bp; > / , proteinuria; > + or > g/ hr, or other associated symptoms). pbef concentration was determined by eia (phoenix pharmaceuticals) in accordance with the manufacturers instructions. fetal membranes and placentas of additional patients, no pe (n= ) and with pe (n= ) were fixed and embedded in paraffin. sections ( um) were immunostained with pbef antibody / (pheonix) and treated with abc reagent (vector labs) followed by dab ( . % mg/ml), washed, counterstained, mounted and viewed under brightfield microscopy. results: the concentrations of pbef in serum were between - ng/ml and were significantly higher in those patients with mild pe (p= . ) and further significantly elevated in those with severe pe (p= . ) compared with the matched controls. pbef was detected by immunocytochemistry in the placental syncytiotrophoblast and in the amnion and choriodecidua of the fetal membranes. conclusions: pbef was elevated in the serum of patients according to the degree of pe severity and may be derived from the placenta and/or fetal membranes. (supported by nih #u rr - to the pacific research center for early human development, university of hawaii). background: platelet-monocyte aggregation (pma) is a novel sensitive measure of platelet activation and indicates a proinflammatory state (cytokine release). less sensitive techniques demonstrate platelet activation during pregnancy and pre-eclampsia (pe) but platelet activation has not been assessed by pma.objective: longitudinal study of pma in normal pregnancy and pe. methods: healthy, non-smoking primigravida with an uncomplicated pregnancy and primigravida women with pe were studied. pe was defined by standard definitions. informed consent was obtained and the study had ethical approval. serial venous blood collected at , , , wks in controls, at time of diagnosis in pe cases and wks post-natal (pn) in all. pma, platelet surface p-selectin (psp-sel) and monocyte cd expression (mcd ) were analysed by flow-cytometry and plasma (p) p-sel by elisa. results: groups were matched for mean age and bmi. in controls, pmas, psp-sel and mcd expression and pp-sel increased with gestation and decreased post-natally (table ) . for pe analysis, data was divided into pre-term (sampled at mean wks), and term (mean wks). pp-sel was lower in pre-term pe than control (normal pregnancy wks; p= . ) there was no significant difference in other measures between pe and control ( objective: much effort has been put into the evaluation of novel markers to identify pregnant women at risk for the development of pre-eclampsia. soluble endoglin (seng) and soluble fms-like tyrosine kinase (sflt ), two antiangiogenic agents, appear to be involved in the pathogenesis of pre-eclampsia. despite several studies describing higher midtrimester serum concentrations of these markers in women with subsequent pre-eclampsia, information on first trimester serum levels is scarce. the aim of this study was to assess seng and sflt as first trimester serum markers for the prediction of pre-eclampsia. methods: sera were obtained between + and + weeks of gestation from women who later developed late-onset pre-eclampsia and from controls matched for gestational age, maternal age, maternal pre-pregnancy weight, and storage. using commercially available microplate enzyme immunoanalytical methods, seng and sflt were determined and the results analyzed using non-parametric statistical tests. results: the serum concentration of seng was found to be increased in women with subsequent pre-eclampsia when compared to controls (mean ± sd, . ± . ng/ml versus . ± . ng/ml, p = . , unpaired mann-whitney test). similarly, the serum levels of sflt were higher in women later developing late-onset pre-eclampsia ( ± ng/ml) when compared to controls ( ± ng/ml, p = . ). sensitivities and specificities for predicting pre-eclampsia were % and % for seng and % and % for sflt- , respectively. the combination of the two markers by multiplication yielded a sensitivity of % and a specificity of %. conclusion: seng and sflt , showing increased first trimester serum levels in women with subsequent pre-eclampsia, might both fulfill the characteristics of first trimester markers to predict pre-eclampsia. the combination of the two, however, did not improve the sensitivity nor the specificity compared to their individual determinations. moderate sensitivities and specifities, however, limit the clinical use of these molecules as single markers. pregnancy is associated with increased monocyte/platelet aggregate formation in whole blood. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is associated with increased rates of platelet clearance, changes in platelet function and platelet activation. the goal of the current study was to examine basal levels of platelet activation through pregnancy beginning prior to conception, and to examine the association of platelet activation with the development of hypertensive complications during pregnancy. methods two indices of platelet activation, platelet cd expression (%cd ) and monocyte/platelet aggregate (%mp) formation, were measured in whole blood by flow cytometry using specific, fluorescentlylabeled monoclonal antibodies in healthy, nonsmoking women during the follicular phase of their menstrual cycle (pp, cycle day . + . ). all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - wks) and late pregnancy (lp, - wks). five of these women were diagnosed with hypertensive complications ( hypertension, preeclampsia) at term although hypertension was not observed at any study time point. data are expressed as mean±sem. p< . was accepted for significance. results subjects were . + . years old with a bmi of . + . kg/m at the time of prepregnancy studies. a significant increase in the %mp formed over time of pregnancy was observed (p= . ). there was little change in the %mp formed between pp and ep (pp, . ± . % and ep, . ± . %, p= . ). however, the %mp increased significantly in lp ( . ± . %) as compared to pp (p= . ) and ep (p= . ). this increase occurred independent of the development of hypertensive complications (p= . ) and independent of pp platelet activation status. although statistically significant increases in cd expression were not observed, the change in cd expression over pregnancy correlated with the change in %mp over pregnancy (r= . , p= . ) and cd expression correlated with %mp in lp (r= . , p= . ). conclusion these combined observations suggest that pregnancy is associated with increases in levels of unstimulated platelet activation and that these increases occur in the presence or absence of subsequent hypertensive complications. furthermore, we observed a correlation between the changes in two distinct platelet activation events, %mp and cd expression, during pregnancy. supported by nih hl . backgound sildenafil citrate (sc) has been proposed as a therapy to improve uterine perfusion in pregnancies complicated by iugr or preeclampsia. we sought to determine the effects of sc on uterine vascular resistance, uterine blood flow and cardiac output in young healthy nulliparous women. methods eleven young healthy nulligravid women were studied during the luteal phase (cycle day + ) of the menstrual cycle. women were randomized in a double-blind fashion to receive placebo (pl), or sc at a dose of or mg. uterine artery vascular resistance, uterine artery volumetric flow, brachial artery volumetric flow and cardiac output were measured at baseline and at and hours post dosing employing color doppler ultrasound. comparisons were made by anova between those randomized to pl (n= ) versus sc (n= ). p< . was accepted for significance. data are expressed as mean + s.e.m. results there were no significant differences in subject age, cycle day, body mass index, uterine blood flow, brachial blood flow or cardiac output at baseline comparing the two groups. there was a tendency towards increased uterine blood flow in subjects randomized to receive sc ( % increase) compared to pl (no change), changes in uterine blood flow, brachial blood flow and cardiac output are outlined in the objective reduced maternal plasma volume in the third trimester has been associated with both fetal growth restriction and preeclampisa. we sought to determine the degree to which third trimester plasma volume is dependent on plasma volume prior to pregnancy. methods sixteen young ( . + . years) healthy nulligravid women had their plasma volume measured during the follicular phase (cycle day . + . ) of the menstrual cycle and subsequently conceived. subjects were predominantly caucasian ( / ) with a mean prepregnancy bmi of . + . kg/m . plasma volume was re-estimated at - weeks gestation. all patients were placed on sodium and total calorie balanced diets for days prior to each plasma volume determination. plasma volume was determined employing evans blue dilution with multiple post injection sampling time points. data are expressed as mean + s.d. results baseline prepregnant plasma volume was , + ml or + ml/unit bmi. third trimester plasma volume was , + ml representing a % increase (p< . ). the range of plasma volume expansion was - % dependent upon prepregnant plasma volume. plasma volume in the third trimester of pregnancy was strongly correlated to prepregnant plasma volume r= . (p= . ). plasma volume expansion was consistent across the range of prepregnancy plasma volume. conclusions pre-pregnancy plasma volume contributes approximately % of the variance in third trimester plasma volume. the observed increase is plasma volume is independent of prepregnancy volume resulting in a greater percentage increase for those starting at the lower end of the plasma volume range. as third trimester plasma volume is strongly associated with pregnancy outcome the correlation of prepregnancy plasma volume to third trimester plasma volume suggests that prepregnancy status contributes to these adverse reproductive events.this work was supported by nih hl . background: hydatidiform mole is a rare disorder of pregnancy and may predispose the mother to severe morbidity. molar pregnancies are known to be associated with high risk for the development of early onset preeclampsia. in recent years, the expression of sflt- (soluble vegfr- ) was found to be increased in preeclampsia, and contributes to the pathogenesis of the maternal systemic disease. the objective of the present study was to examine the expression of sflt- in placentae from molar pregnancies. methods: placental samples from unique cases of twin pregnancies with complete molar pregnancy in one sac and developing fetus in the other sac were prospectively collected (n= ). the first set delivered at weeks due to excessive bleeding. the second set delivered at weeks due to severe iugr and elevated blood pressure. mrna level of sflt- was measured by quantitative real-time pcr using specific taqman primers and probe. protein expression of sflt- in placental tissue lysates were measured by western blot analysis using a polyclonal antibody against flt- . immunohistochemistry of paraffin embedded samples was performed using specific antibody for sftl- . results: mrna level of sflt- was increased by . fold in the molar placentae compared to matched controls. the placentae of the developing fetuses which were growth restricted exhibited . fold increase compared to controls. sflt- protein expression in the molar placentae was increased by . fold compared to controls, while the co-twin placentae exhibited a . fold increase compared to controls. immunohistochemistry revealed strong positive immunoreactivity for sflt- in the trophoblast layer of both molar pregnancies and iugr co-twin relative to controls. conclusion: our data suggest that sflt- expression is increased in placentae from molar pregnancies and thus may explain the increased risk for developing early onset preeclampsia. the expression of sflt- in the growth restricted twin placenta is also increased compared to controls and support our previous observation (supported by cihr and owh/igh). (n= ); ) neonates of patients with pe (n= ); and ) sga neonates (n= ). cord blood was collected immediately after delivery and pz plasma concentrations were measured by elisa. pz deficiency was defined as a cord plasma concentration < th percentile of the normal pregnancy group. non-parametric statistics were used for analysis. results: ) cord plasma pz concentration differed significantly among the study groups (kruskal wallis, p< . ); ) neonates of patients with pe and sga neonates had a significantly lower median cord plasma pz concentration than those delivered after normal pregnancy (pe: median . g/ml, range . - . , p< . ; sga: median . g/ml, range . - . , p= . ; normal pregnancy: median . g/ml, range . - . ); ) there were no differences in the rate of pz deficiency among the groups; and ) there was no relationship between placental histologic findings and median cord plasma pz concentrations between and among the sga and pe groups. conclusions: ) at the time of delivery, the median cord plasma pz concentration was lower in sga neonates and those born to women with pe than in neonates born to normal pregnancies; ) there was no difference in the rate of pz deficiency among the study groups, suggesting that the lower median pz cord blood concentrations in pe and sga groups may result from activation of the coagulation cascade rather than an inherited pz deficiency. objective: periconceptional multivitamin (mv) use may be related preeclampsia risk. we examined the relation between timing and frequency of periconceptional multivitamin use and the risk of preeclampsia. methods: women in the danish national birth cohort who delivered singleton liveborn infants (n= , ) reported upon enrollment at . weeks (sd . ) the number of weeks of regular multivitamin use during a week periconceptional period (lmp- to lmp+ ). preeclampsia cases were identified using icd- codes (n= , . %). logistic regression was used to estimate the effect of frequency (number of weeks of use) and timing of use to lmp+ ] and post-conception [lmp+ to lmp+ ]). results were stratified a priori by overweight status. results: overall, , women ( %) reported mv use in the periconceptional period. after adjustment for bmi, smoking, parity and chronic hypertension, infrequent mv use (< weeks of use) had no relation to risk (or . ; % ci . , . ) but regular use (>= weeks) was associated with modestly reduced risk ( . ( . , . ). similarly, when mv use was modeled as a continuous variable, each additional week of use was related to reduced risk for preeclampsia (or . , % ci . - . ). this potential dose effect of periconceptional mv use appeared to be limited to normal weight women (bmi < kg/m², or . ; % ci . - . ), with no apparent effect among overweight women (bmi kg/m², or . ; % ci . - . ). a total of , women reported regular mv use in both the preconception and post-conception periods, and , women reported regular use only in the post-conception period. among normal weight women, regular use in the preconception period had no effect on preeclampsia risk (or . , % ci . - . ). in contrast, use in the post conception period was associated with reduced risk for preeclampsia (or . , % ci . - . ). conclusions: regular periconceptional mv use was associated with a modestly reduced risk for preeclampsia among normal weight women. if causal, mv use immediately after conception appeared to be the critical exposure window. background: pre-eclampsia (pe), a disorder of pregnancy characterized by maternal inflammation, results in immune, cardiovascular and metabolic dysfunction. in non-pregnant persons, inflammatory disorders are treated with and prevented by pharmaceuticals and lifestyle methods such as physical activity (pa). while most pharmaceuticals are contraindicated for pregnant women, pa during pregnancy has been found safe, healthy and beneficial for both mother and baby. clinical evidence has found pa can beneficially affect pregnancy outcome, decrease excessive inflammation and decrease the risk of pe. epidemiological studies indicate that pa may be useful in preventing pe. unfortunately, previous studies have quantified pa based on recall of postpartum women and have not controlled for differences in women's interpretations of amount, type or intensity of pa. however, investigating pa utilizing a laboratory-based exercise intervention to control these variables inflicts difficulties translating the intervention into a community-based program that attracts and retains pregnant women in order to enhance public health. method: a retrospective study was performed to determine the rates of pe among the , women who gave birth at yale-new haven hospital (ynhh) during and , and a person subset of this group who performed prenatal pa in a community-based program that is evidence-based and standardized, thereby controlling for type and intensity of pa. additionally, the program is established in the community, has been offered to the public for years and is internationally known. results: during during - , the pe rate for the general population at ynhh was . %. for the pa group, the rate was . %. two women in the pa group were diagnosed with pe in the last month of pregnancy and delivered normal infants at term. no pe was observed in this group (pa group) during the second or early third trimesters nor was there any prematurity in this group. significance: these findings support the hypothesis that adequate physical activity provided in a standardized community-based group setting may provide a non-pharmacological approach for preventing pe. growth restriction. margreet plaisier, esther streefland, pieter koolwijk, frans m helmerhorst, jan jaap hm erwich. department of gynecology and reproductive medicine, leiden university medical center, leiden, netherlands; department of obstetrics and gynecology, university medical center groningen, groningen, netherlands; department of physiology, vu univerity medical center, amsterdam, netherlands. objective: disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications, like pre-eclampsia (pe) or fetal growth restriction (fgr). whether the regulation of decidual vascular adaptation to implantation is altered in these illnesses, is not elucidated yet. the present study focused on the role of first-trimester angiogenic factors in the pathogenesis of pe and or fgr. methods: first-trimester decidua samples were obtained during routine chorionic villous sampling. the expression of vascular endothelial growth factor (vegf-a), placental growth factor (plgf), flt- , kdr, angiopoietin- (ang- ), angiopoietin- (ang- ) and tie- mrna was determined by rt-pcr. the expression of the angiogenic factors was related to the pregnancy outcome, i.e. uncomplicated, pe or fgr. results: the first-trimester decidual tissues expressed all angiogenic factors. mrna levels of vegf-a, plgf, kdr, ang- , ang- and tie- appeared increased in fgr cases compared to matched controls. in addition, plgf, ang- and tie- mrna appeared increased in pe cases compared to matched controls. the differential expression of angiogenic factors was more pronounced in cases with fgr than pe. the large inter-individual variation disallowed a significant outcome. conclusions: various angiogenic factors showed differential mrna expression in st trimester decidua of patients developing pe or fgr in later pregnancy compared to their matched controls. the first-trimester decidual samples provided a unique opportunity to obtain information regarding the onset of pe and fgr. early st trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. in turn, this may set the stage for increased non-branching angiogenesis and altered decidual and placental vascular adaptation, which may be part of the pathogenesis of pe and/or fgr. complications. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is characterized by endothelial dysfunction. the goal of the current study was to prospectively measure plasma levels of the soluble endothelial cell adhesion molecules, sicam- and svcam- , beginning prior to pregnancy and determine if subjects destined to develop hypertension complicating pregnancy had differences in the concentrations of these molecules. methods serum levels of sicam- and svcam- were measured in healthy, nonsmoking women (cycle day . + . , prepregnancy) by elisa. all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - weeks) and late pregnancy (lp, - weeks). five of these women developed hypertensive complications ( gestational hypertension, pre-eclampsia) near term. all subjects were normotensive at all study time points. data are expressed as mean ± sem. p< . was accepted as significant. results subjects were . + . years old with a mean bmi of . + . kg/m at the time of prepregnancy studies. significant differences in sicam- levels as a function of pregnancy were observed (p= . ) and are outlined in table p= . differences were dependent upon the stage of pregnancy in those women who were not diagnosed with hypertensive complications with a decrease in sicam- levels in ep (p= . ) followed by an increase in sicam- levels in lp (p= . ). in women with hypertension in pregnancy, these differences in sicam- levels were not evident (p= . ). there were no differences in sicam- levels comparing women with or women without hypertensive complications prior to pregnancy (p= . ). in contrast to sicam- , we observed no significant differences in svcam- levels over pregnancy or between those with and without hypertension. conclusions these combined observations suggest that levels of the soluble adhesion molecule sicam- change significantly over time in normal pregnancies. subjects destined to develop hypertension did not demonstrate the early pregnancy reduction in sicam- . supported by nih hl . yuval bdolah, uriel elchalal, shira natanson-yaron, hadas caspi, tali bdolah-abram, angelika bord, caryn greenfield, debra goldman-wohl, ariel milwidsky, franklin h epstein, s ananth karumanchi, simcha yagel, drorith hochner-celnikier. ob/ gyn, jerusalem, israel; ob/gyn medicine, beth israel deaconess medical center, harvard medical school, boston, ma, usa. objectives: nulliparity is a risk factor for preeclampsia (pe) with a reported incidence of up to - times higher than multiparous pregnancies. soluble fms-like tyrosine kinase- (sflt ), a circulating anti-angiogenic molecule of placental origin plays a pivotal role in pe by antagonizing placental growth factor (plgf). increased sflt and sflt /plgf have been shown to antedate clinical signs in pe. we therefore hypothesized, that the higher risk of pe in nulliparous pregnancies is associated with high sflt (or sflt /plgf). methods: maternal serum samples from nulliparous (n= ) and multiparous (n= ) term singleton pregnancies without pe, at the time of admission to delivery room, were used. serum samples were analyzed for levels of sflt and plgf by elisa. statistical analysis was performed applying t-test and the kruskall-wallis test and using spss software. results: for nulliparous and multiparous pregnancies, the mean serum sflt levels were , ± and , ± , (p= . ), the mean serum plgf levels were ± and ± (p= . ), and the mean ratios of sflt- /plgf were ± and ± (p= . ), accordingly. in a subgroup of multigravidous nulliparous pregnancies, sflt levels were , ± . correcting for maternal age did not alter the results. moreover, results did not differ between multiparous pregnancies with a - years interpregnancy interval compared with a - years interval. conclusions: in nulliparous pregnancies, circulating sflt levels and sflt / plgf ratios are significantly higher than in multiparous pregnancies. these findings suggest that the increased risk of pe in nulliparous pregnancies may involve anti-angiogenic imbalance. nulliparity may be more substantial than primigravity, as a risk factor for pe, suggesting that first semester abortions in primigravidas may not protect from pe in a subsequent term pregnancy. nevertheless, even - years intervals from the previous gestation do not increase the risk for pe. different normograms of angiogenesis should be used, when assessing the risk for pe in multiparous versus nulliparous pregnancies. objective: we determined whether maternal serum levels of angiogenic proteins namely soluble fms like tyrosine kinase (sflt- ), soluble endoglin (seng), and placental growth factor (plgf) -measured during the first trimester are associated with the subsequent development of placental abruption. methods: we performed a prospective, nested case-control study of women enrolled in the massachusetts general hospital obstetric maternal study (moms). first trimester serum samples from placental abruption cases and normal pregnancies were measured for angiogenic factors. cases and controls were matched by body mass index and age. placenta abruption was diagnosed by standard clinical findings and pathological examination of the placenta. women with confirmed preeclampsia or chronic hypertension were excluded. results: compared to controls, cases had more pregnancies, delivered infants at an earlier gestational age and with lower birth weight. first trimester levels of seng were significantly increased in cases compared to controls: . ± . ng/ml vs. . ± . ng/ml, p < . . there were no significant difference in serum levels of plgf, . ± . ng/ml versus . ± . ng/ml, p=ns, although sflt levels were lower in cases: . ± . ng/ml vs. . ± . ng/ml, p=ns. in logistic regression analysis adjusted for age, race, smoking, number of pregnancies, gestational age at delivery, gestational age of blood sampling, and blood pressure at first prenatal, seng levels remained independently associated with subsequent risk (odds ratio . , % ci . - . ) of placental abruption. examining this relationship by tertiles of seng, in the unadjusted model, women in the second (or . , % ci . - . ) and third (or . , % ci . - . ) tertiles were at increased risk of developing placental abruption compared with women in the lowest tertile. after adjusting for known risk factors of placental abruption, women in the second (or . , % ci . - . ) and third (or . , % ci . . ) tertiles remained at increased risk for placental abruption. conclusion: increased first trimester maternal serum levels of seng are associated with increased risk of subsequent placental abruption. ob/gyn, univ of vermont. shear stress is the most potent physiologic stimulus for elevating endothelial no production for flow-mediated vasodilatation. we measured in vivo shear stress during the proliferative and secretory phases and at and weeks of gestation hypothesizing that uterine blood flow (ubf) elevations in turn increase shear stress in early and late gestation. methods: during proliferative, secretory phases, and at and - weeks of pregnancy ua blood velocity and internal radius were measured bilaterally using color doppler ultrasound. blood viscosity was measured at shear rates in excess of /sec. results: compared to the proliferative phase viscosity was decreased at weeks and more so at weeks gestation (p< . ). . ± . . ± . nsd . ± . *** . ± . *** ua velocity (cm/sec) . ± . . ± . *** . ± . *** . ± . *** ubf (ml/min) . ± . ± *** . ± . *** . ± . *** shear stress (dynes/cm ) . ± . . ± . *** . ± . *** . ± . *** internal ua radius was not altered by the menstrual cycle, and was greater at weeks (+ %) and weeks (+ %). compared to proliferative, secretory phase showed significant rises in unilateral ubf and velocity that rose progressively during gestation. in contrast, shear stress increased in secretory ( %) and did not rise further in early pregnancy but by weeks shear stresses was further elevated %. conclusions: equivalent rises in shear stress during the secretory phase and weeks gestation demonstrate increases in radius and profound remodeling of uas that reflect the physiologic process of "normalization of shear". by late gestation, continued but modest rises in radius illustrate that further increases in shear stress occur almost solely due to rises in ubf via falls in down stream impedance. continued rises in shear stress into late gestation provide progressive stimuli for no production by ua endothelium. nih hl , hd , hl background: hypertension during pregnancy is associated with altered uterine vascular reactivity and blood flow, although its effects on arterial myogenic behavior have not been explored. the purpose of this study was to evaluate the effects of hypertension and no inhibition on myogenic tone in pregnancy, as the ability of a vessel to constrict and dilate in response to pressure plays a key role in regulating blood flow to the uterus. methods: three groups of sprague dawley late pregnant (day ) rats were used: control (n= ), hypertensive ( . g/l l-name in the drinking water, n= ), and treated with l-name and hydralazine (also in the drinking water, . g/l, n= ) to prevent the blood pressure increase, yet maintain no inhibition. resistance-sized radial arteries (< m) were mounted in a pressure arteriograph and equilibrated at mmhg (in pss containing l-nna and indomethacin) to induce a myogenic response. vessels were then subjected to pressure steps from to mmhg. tone (%) was calculated by comparing the vessel diameter at each pressure with the passive diameter at the same pressure (determined by incubation with . mm papaverine and m diltiazem). results: myogenic tone developed in controls ( ± % maximal), and was maintained over a broad range of transmural pressures . arteries from the l-name group did not develop tone at any pressure. co-treatment with hydralazine reinstated tone ( ± % maximal) over the same range of pressures as in the control group. the reduction in average placental weights in the l-name group ( vs. mg, p< . ) was restored by hydralazine ( mg, p< . vs. l-name). average fetal weights were also reduced in the l-name group ( . vs. . g, p< . ), but only partially restored by hydralazine co-treatment ( . g, p< . vs. control and l-name groups). conclusions: surprisingly, radial uterine arteries from the l-name group did not develop tone over any range of pressures, despite the fact that matched arteries from late-pregnant controls developed significant myogenic tone. this abolishment of tone was reversed by hydralazine, which also had beneficial effects on fetal and placental growth. these results implicate hypertension rather than no inhibition as the key factor in the suppression of myogenicitiy and dysregulation of uterine blood flow. charles r rosenfeld, timothy roy. division of neonatal-perinatal medicine, ut southwestern medical center at dallas, dallas, tx, usa. background: upbf b rises -fold in ovine pregnancy; but the mechanisms responsible for the rise and maintenance are unclear. we (jsgi ) reported that uterine vascular smooth muscle bk ca k + channels contribute to uterine vasodilation and upbf b maintenance; but up-stream activators are unclear. uterine vascular prostacyclin synthesis increases in pregnancy, but cyclooxygenase inhibition does not alter upbf b (ajp ) . vascular nitric oxide synthase (nos) also increases; but acute inhibition with l-name decreases upbf b only % (jci ) . it is unclear if l-name doses were insufficient or if prolonged nos inhibition has a greater effect. objective: to determine if local nos inhibition with l-name dose-dependently decreases upbf b and if prolonged inhibition exerts a greater effect on upbf b . methods: pregnant ewes were studied at - d gestation age (ga). had doseresponse studies with uterine artery l-name infusions to achieve . - . mg/ml over min. had h arterial l-name infusions to achieve and maintain levels of . mg/ml at (n= ), (n= ) and d (n= ) ga, while measuring arterial pressure (map), heart rate (hr) and upbf b before, during ( , , , , , , and h) and after ( , h) infusions. uterine arterial and venous cgmp were measured. data were analyzed by anova. results: acute nos inhibition decreased upbf b - %, but was not doserelated. h arterial l-name infusions decreased upbf b by - h at each ga (p . , anova) and values returned to baseline by h postinfusion. sensitivity did not differ between ga (p= . , anova), upbf b falling - % at each ga. contralateral upbf b was unaffected at all ga. map rose and hr fell during infusions at and d ga, p . ; but were unaffected at d ga. venous-arterial cgmp concentration differences were seen at d and absent at h of l-name infusion, p= . . conclusions: uterine vascular nos increases in ovine pregnancy, but its inhibition decreases upbf b %, suggesting study doses were insufficient to fully inhibit vascular nos activity. alternatively, nos contributes to the maintenance of upbf b , but other mediators, not yet identified, are more important in activating bk ca and regulating upbf b . notably, l-name reached the systemic circulation, and although further diluted, map rose - %, suggesting the systemic vasculature may be more sensitive to nos inhibition than upbf b . charles r rosenfeld, xiao-tie liu. division of neonatal-perinatal medicine, university of texas southwestern medical school, dallas, tx, usa. background: uteroplacental blood flow (upbf) rises -fold in ovine pregnancy, reflecting increases during pre-implantation, placentation and finally, in the last third of gestation. we reported that bk ca density in uterine vascular smooth muscle (uvsm) increases in ovine pregnancy (sgi ) and inhibition with tetraethylammonium ( . mm) dose-dependently decreases basal upbf % in the last third of pregnancy (jsgi ) . it is unclear how bk ca subunit expression changes and activity is regulated in pregnancy; since uterine vascular nitric oxide is increased, signaling via cgmp-dependent protein kinase g (pkg) is one potential pathway. objective: to determine simultaneous changes in uvsm bk ca density and regulatory -subunit expression and the cgmp signaling pathway for activation in ovine pregnancy. methods: endothelium-denuded segments of nd generation uterine arteries obtained from nonpregnant (n= ), pregnant (n= , - d gestation; term d) and postpartum (n= , - d) sheep were used to measure expression of bk ca -and regulatory -subunits and signaling proteins in uvsm by immunoblot analysis and immunohistochemistry. results: uvsm bk ca density, reflected as a change in the kda -subunit, rose % with placentation (p< . ) and was unchanged thereafter. expression of the kda regulatory -subunit paralleled the rise in bk ca density during placentation, increasing % (p< . ), but increased another -fold and exponentially in the last third of pregnancy (p< . , r = . , n= ). changes in subunit immunostaining in uvsm paralleled increases in protein. although uvsm soluble guanylyl cyclase was unchanged in pregnancy (p= . , r= . , n= ), pkg expression rose -fold (p= . , r= . , n= ) and gradually returned to nonpregnant levels by d postpartum (p= . , r= . , n= ). uvsm cgmp is being measured. conclusions: these are the st data suggesting that increases in ovine upbf during placentation involve vascular growth and bk camediated vasodilation. the rise in upbf in the last third of pregnancy reflects bk ca -mediated vasodilation due to enhanced channel activation via increases in uvsm pkg, bk ca phosphorylation and changes in subunit stoichiometry due to an exponential rise in the regulatory -subunit. it is unclear what initiates and directs these changes in bk ca expression and sensitivity. relaxin (rlx) is a hormone traditionally associated with changes in the female reproductive tract during pregnacy. recent evidence suggests that rlx may play a pivotal role in regulating cardiovascular function during gestation. analogous to pregnancy, administration of recombinant human relaxin (rhrlx) to nonpregnant female rats reduces systemic vascular resistance, as well as increases global arterial compliance. additionally, we demonstrated that, concurrent with rlx´s influence on overall cardiovascular function, small renal arteries (sra) from rhrlx-treated mice and rats are characterized by reduced passive stiffness and increased arterial wall area whereas external iliac arteries (eia) are not. we hypothesized that rlx regulates arterial passive mechanical properties by altering the cellular and biochemical composition of the arterial wall. nonpregnant female mice were administered rhrlx for days after which sra and eia were isolated. we measured arterial collagen, elastin, and total protein using the sircol collagen assay, the fastin elastin assay, and the pierce bca protein assay, respectively. additionally, we quantified arterial smooth muscle cell (smc) density using immunofluorescent techniques. sra isolated from rhrlx-treated mice were characterized by a significant reduction in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein; mean±sem; p< . ) as well as a significant increase in smc density ( . ± . vs . ± . cells/ μm ; p< . ) compared to control mice with no significant change in elastin content ( ± vs ± μg elastin/mg dry weight). in contrast, there were no significant changes in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein), smc density ( . ± . vs . ± . cells/ μm ) or elastin content ( ± vs ± μg elastin/mg dry weight) in eia from the rhrlx-treated mice compared to control mice. of note, comparable results were observed for rlx knock-out (rlx -/-) and wild-type mice with rlx -/mice exhibiting increased arterial collagen and decreased smc density. we conclude that the rlx-induced decrease in passive stiffness of sra that we previously reported is, at least in part, due to rlx-induced alterations in arterial wall cellular and biochemical composition. further, our findings suggest that these vessel wall remodeling effects of rlx are artery-type specific. relaxin is a peptide hormone that emanates from the corpus luteum of the ovary and circulates during pregnancy. this hormone plays an important role in renal vasodilation and hyperfiltration, two fundamental maternal adaptations in pregnancy. chronic administration of recombinant human relaxin (rhrlx) to both virgin rats and mice inhibits myogenic reactivity and increases compliance of small renal arteries, thus further mimicking pregnancy. we hypothesize that these arterial responses to rhrlx are mediated by the lgr , and not the lgr receptor. both lgr and lgr receptor-deficient, and wild-type virgin mice were investigated. the mice were chronically infused with rhrlx or vehicle (veh) for days. small renal arteries were isolated and mounted in a pressure arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a mmhg step increase in intraluminal pressure). small renal arteries from rhrlx-infused lgr wild-type mice showed inhibited myogenic reactivity with a . ± . % increase in diameter whereas the arteries from rhrlx-infused lgr knock-out mice exhibited robust myogenic reactivity with only a . ± . % change in diameter (p= . ). in contrast, myogenic reactivity of small renal arteries was inhibited in both the rhrlx-infused lgr knock-out and wild-type mice. the veh-treated lgr and lgr mice, regardless of genotype, exhibited robust myogenic reactivity. arterial compliance was also assessed for each genotype/treatment group. rhrlx infusion increased arterial compliance of small renal arteries from lgr wild-type, but not from lgr knock-out mice (p= . ). in contrast, the arteries from rhrlx-infused lgr wild-type and knock-out mice showed increased compliance relative to veh-infused animals. conclusion: relaxin-induced inhibition of myogenic reacitivity and increase in compliance of small renal arteries is mediated by the lgr , and not the lgr receptor. smoking is associated with adverse pregnancy outcomes including fetal growth restriction. pathologic effects of smoking on maternal vasculature is a potential mechanism leading to fetal growth restriction. the objective of this study was to determine whether cigarette smoke exposure during pregnancy affects the functional properties of uterine and peripheral arteries using a gravid murine model. study design: pregnant and virgin c bl/cj mice were exposed to whole body side stream smoke using an inhalational chamber for hours/day. smoke exposure was increased from day of gestation until late pregnancy (day - ) with mean total suspended particle levels of mg/m , representative of moderate to heavy smoking in humans. control animals were exposed to ambient room air. late pregnant and virgin mice were sacrificed and uterine, mesenteric, and renal arteries were isolated and studied in a pressure arteriograph system (n= - in each group). plasma cotinine was measured by elisa. means were compared using t-test or analysis of variance. results: fetal weights were significantly reduced in mice exposed to smoke compared to control fetuses ( . ± . g vs . ± . g, p= . ), while litter sizes were not different. cotinine levels in smoking mice were significantly elevated compared to control mice ( . ± . vs . ± . ng/ml for virgin mice and . ± vs . ± . ng/ml for pregnant mice). there was no significant difference in phenylephrine responses between groups. endothelial mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. no difference in endothelial-mediated relaxation was seen in isolated renal arteries in pregnant mice exposed to cigarette smoke, however relaxation was significantly reduced in renal arteries from smokeexposed virgin mice. conclusions: passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in a gravid murine model. functional vascular perturbations during pregnancy, specifically reduced uterine blood flow and impaired peripheral vasodilation, may be a mechanism by which smoking results in fetal growth restriction. human chorionic gonadotropin (hcg) is essential during early human gestation for "rescue" of the corpus luteum. however, its potential contribution to the widespread maternal vasodilation of pregnancy that occurs at this stage of pregnancy remains uncertain. our objective was to use the renal circulation of conscious rats as an experimental model in which to test the vasodilatory potential of hcg. in addition, we investigated both myogenic reactivity and relaxation responses in small renal and mesenteric arteries isolated from rats, as well as in small human subcutaneous arteries using a pressure arteriograph. we chronically instrumented rats for measurement of renal function. five were ovariectomized, and sham-ovariectomized. after days of surgical recovery, baseline glomerular filtration (gfr) and effective renal plasma flow (erpf) were measured on two separate days and the values averaged. then, an osmotic minipump containing hcg ( iu/min) was implanted s.c. and renal function was again assessed and days thereafter. gfr and erpf significantly increased and calculated effective renal vascular resistance decreased from baseline in the intact (p< . vs. baseline), but not ovariectomized (p=ns) rats on both days and of hcg administration. in the intact, but not ovariectomized rats, plasma osmolality declined and progesterone increased (both p< . vs baseline). plasma hcg concentrations were , miu/ml and comparable in both groups of rats. incubation of small renal arteries from rats with recombinant human relaxin (rhrlx, - ng/ml), but not hcg ( , - , miu/ml) in vitro inhibited myogenic reactivity and relaxed phenylephrine (pe)-constricted arteries. in contrast, both rhrlx and hcg inhibited myogenic reactivity and relaxed pe-constricted small mesenteric arteries from rats and small human subcutaneously arteries. in conclusion, consistent with our earlier work showing a virtually exclusive role for relaxin in mediating the renal circulatory changes of pregnancy, hcg is likely to play little or no role. in contrast, hcg and relaxin are both likely to contribute to the vasodilation of other organ circulations during pregnancy. pierre-andre scott, , michele brochu, jean st-louis. , research centre, chu ste-justine, montréal, qc, canada; pharmacology, université de montréal, montréal, qc, canada. uterine vasculature undergoes major structural and functional changes during pregnancy. estrogens have been shown to induce increased uterine blood flow in this circulation. we have reported that estradiol ( -e ) induced direct vasorelaxant response on smooth muscle of the uterine arteries that, for it major part, is not mediated through tissue nitric oxide. to investigate the cellular effectors mediating this vasorelaxant effect of -e , we set-up uterine arteries of non-pregnant rats in wire myograph systems for microvessels. -e and -e were equipotent in relaxing phe ( μmol/l)-preconstricted uterine arteries, although the later produced significant smaller relaxations. genistein, a phytoestrogen presumed to inhibit phosphatases, also produced uterine artery relaxation with significant lower potency. to try interfere with the vasorelaxant effect of -e , tissues were preincubated with different substances. cycloheximide (protein biosysthesis inhibitor), ici , (estrogen receptor modulator), and kt (pka inhibitor) did not significantly influenced the response to -e . rp- -pcpt-cgmps (pkg inhibitor) slightly displaced the concentration-relaxation curve to -e to the right. inhibitors of potassium channels, penitrem a (bkca) and glybenclamide (katp), showed opposite effects for -e concentration-response curve; the former producing a right shift and the latter a small not significant left shift. these data indicated that the direct acute effect of -e in uterine artery is the result of complex interactions within smooth muscle cells, involving potassium channels, and protein kinases and phosphatases. the renin-angiotensin-aldosterone system is paradoxically activated during pregnancy, since blood pressure decreased. earlier data showed that the high levels of aldosterone present during pregnancy may be involved in cardiovascular adaptation to pregnancy. in order to delineate this effect of aldosterone on vascular tone, potassium canrenoate, an antagonist of mineralocorticoid receptors (mr), was administered ( mg/kg/day) to pregnant rats from the day to of gestation. rats were sacrificed at day , and of gestation together with untreated and day pregnant rats. the thoracic aorta was quickly removed and set up in tissue baths as ring ( - mm) preparation. as observed previously, canrenoate enhanced responsiveness to phe for all time of treatment tested, but only at day ( days treatment) for kcl responsiveness. aortic contractile responses to tea (tetraethylammonium) gradually decreased during pregnancy to almost disappear at the end of gestation. in the present results, canrenoate treatment made the response statistically different by day of pregnancy. finally, the activity of na/k-atpase was measured by the relaxant effect of kcl added to physiological solution without potassium. the activity of the pump was decreased when approaching parturition compare to day of pregnancy. canrenoate treatment abolished this effect. the present data show that vascular changes that occurred during pregnancy are markedly modified by treatment of rats with an antagonist of mineralocorticoid receptors, suggesting that aldosterone may be involved in vascular adaptation to pregnancy. background impaired endothelium-dependent vasodilatation has previously been demonstrated in small myometrial arteries from women with gestational diabetes. this impairment may play a role in mediating the complications observed in diabetic pregnancies. it is not known which mechanisms of endothelium-dependent vasodilatation are affected in myometrial arteries by gestational diabetes. aim to investigate mechanisms of endothelium-dependent vasodilatation in uterine arteries using a mouse model of pregnancy complicated by diabetes. methods diabetes was induced in female c bl /j mice (streptozotocin; mg/kg) prior to mating. mice were culled at day of pregnancy (term) and uterine arteries dissected, mounted on a wire myograph, normalised to mmhg and equilibrated ( °c; %co /air). arteries were constricted with phenylephrine ( m) and a concentration-response curve to the endotheliumdependent vasodilator acetylcholine (ach; . nm- m) constructed in the presence and absence of a nitric oxide synthase inhibitor (l-nna; m), a cyclooxygenase inhibitor (indomethacin; m) or a combination of the two to determine the contribution of nitric oxide (no), prostacyclin and endothelium derived hyperpolarising factor (edhf) to vasodilatation. results sensitivity to ach was comparable between diabetic and vehicle treated mice (ec . ± . nm vs . ± . nm). the contribution of individual endothelium-dependent vasodilators was significantly altered in arteries from diabetic mice. at m ach, edhf-mediated relaxation was significantly reduced (p= . , one-way anova) compared with controls ( . ± . vs . ± ). in comparison, no-mediated relaxation was significantly increased (p= . , one-way anova) compared with controls ( . ± . vs . ± . %). conclusions endothelium-dependent relaxation was not reduced in uterine arteries of diabetic mice compared with controls. however, there was a profound change in the contribution of endothelium-dependent vasodilators in arteries of diabetic mice. this may alter compensatory capacity as disease progresses. supported by mfn training grant (cihr). raf- serine/threonine protein kinase has been extensively studied as the upstream kinase linking ras activation to the mek/erk module. mek/erk has been shown to play a role in the modulation of vascular contraction. however, the role of raf kinase in vascular contraction and its possible involvement in alteration of maternal vascular function during pregnancy is not known. objectives: to determine ( ) if raf kinase contributes to phenylephrine (pe)induced contractile response, ( ) the role of raf kinase inhibitor (gw ) in regulating vascular tone during pregnancy, and ( ) mechanism by which gw produces vasodilatation in rat mesenteric arteries. methods and results: conscious non pregnant (np) and pregnant (p) sprague dawley rats received increasing doses of pe in the absence or presence of gw . pe induced a dose-dependent increase in map in both np and p rats but responses were substantially depressed in pregnancy. gw shifted the pe-induced dose response to the right in both np and p rats. gw itself did not affect basal blood pressure. isometric tension studies in mesenteric arteries showed that gw did not change the kcl-evoked contraction but significantly inhibited the contraction to pe in both np and p arteries. interestingly, at a given concentration, gw produced greater inhibition of pe-induced contractile response in p than in np arteries. also, in p arteries the inhibitory effects of gw were greater as the pregnancy progressed from day to day . raf kinase expression and activity was significantly decreased in arteries of p compared to np rats. in mesenteric vascular smooth muscle cells (vsmcs), pe stimulated activation of raf kinase as indicated by phosphorylation of its immediate target, mek / in a time-and dose-dependent manner. measurement of [ca + ] i with fura- showed that gw -mediated inhibition of pe-induced contraction was not associated with decrease in [ca + ] i . vsmcs treated with pe exhibited higher levels of the contractile proteins, p-mypt and p-mlc which was inhibited by gw . conclusion: to our knowledge, for the first time we show that raf kinase plays an important role in the regulation of vascular contractility. decreased expression and/or activity of raf kinase during pregnancy may explain in part, for decreased systemic vascular reactivity during late gestation. the mechanism(s) that contribute to reduced vascular sensitivity to phenyleperine (pe) during pregnancy is not well understood. pe, in addition to activating the classical contractile pathways, also stimulates growth factor pathways that results in activation of ras/mitogen-activated protein kinases. it is not clear whether these pathways play a role in the modulation of vascular contraction. hence, the present study was designed to determine ) if ras is involved in mediating the pressor response to pe in non pregnant (np) and pregnant (p) rats, ) if so, the mechanism by which ras protein contributes to pe-induced vascular contraction, and ) any differential expression and/or activity of ras during pregnancy that might contribute to altered vascular response. methods: ) mean arterial pressure (map) was measured in conscious np and p rats. ) isometric tension was measured in mesenteric arteries of np and p rats. ) expression of contractile proteins, p-mlc and p-mypt were studied in cultured vascular smooth muscle cells (vsmcs). ) expression and activity of ras in mesenteric arteries of np and p rats were measured by western blot analysis. results: ( ) intravenous administration of pe resulted in a dose-dependent increase in map but responses were substantially depressed in pregnancy. inhibiting ras activation with manumycin, decreased pe-induced increase in map in both np and p rats. manumycin by itself had no effects on basal map. ( ) isometric contraction studies in myograph with mesenteric arteries showed that manumycin shifted pe-induced dose response curve to the right with a decrease in e max in np and p rats. higher concentration of manumycin was required to inhibit pe-induced contraction in np ( μm) compared to p ( - μm) rats. ( ) pretreatment with manumycin inhibited pe-induced increase in the extent of phosphorylation of both mlc and mypt in mvsmcs. ( ) compared to p rats, mesenteric arteries of np rats revealed increased basal and pe-induced expression and activity of ras. reduced expression of this contractile ras pathway might contribute to decreased vascular reactivity during pregnancy. conclusion: ras protein plays a role in regulation of vascular contraction. the decreased amount and activity of vascular ras may be a novel mechanism that explains the reduced vascular resistance during pregnancy. overweight affects the relaxin-induced response of mesenteric arteries. joris van drongelen, arianne van koppen, marc ea spaanderman, paul abm smits, frederik k lotgering. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; pharmacology and toxicology, radboud university nijmegen medical centre, nijmegen, netherlands. objective: overweight affects pregnancy-induced vascular adaptation and predisposes to gestational hypertensive disease. relaxin plays a key role in normal gestational vascular adaptation by increasing endotheliumdependent vasodilation and compliance, and reducing myogenic reactivity. we hypothesized that overweight blunts the vascular response to relaxin. the vascular responses to flow and pressure of mesenteric arteries after pre-treatment to human recombinant relaxin (rlx) or placebo (plac) were examined in overweight (ow) and normal weight (nw) rats in a pressure-perfusion myograph. overweight was established by a high-fat diet. the endothelium-dependent vasodilatation was measured in response to flow: e (flow inducing % dilatation) and e max (maximal dilative effect). active contractile (myogenic reactivity) and passive dilative (compliance) vascular responses to pressure were determined. all vascular responses were calculated as the proportional change in diameter to % precontraction with u . results: in nw rats rlx decreased e and increased e max to flow and attenuated myogenic reactivity without affecting vascular compliance. in ow plac-treated rats, compared to nw rats, an upregulated vasodilative state was present (decreased e and increased e max , and lower myogenic reactivity). in ow rats rlx increased e to flow and unaffected e max . rlx increased myogenic reactivity mildly in absence of changes in vascular compliance. values are presented as mean±sem; * p< . between nw/ow, # p< . within nw/ow. whereas rlx stimulates endothelium-dependent vasodilation to flow and myogenic reactivity in nw rats, ow overturns the flow-induced response and decreases myogenic reactivity to a lesser extent than present in nw rats. we speculate that these overweight-induced adverse effects of rlx prelude to vascular maladaptation and gestational hypertensive disease. compared to the luteal (lut) phase, uterine blood flow is increased in vivo during the follicular (fol) phase and more so during pregnancy (preg). both of these are physiologic states of high estrogen and shear stress. endothelial cells express enos and produce greater amounts of nitric oxide (no) in response to elevations of shear stress. phosphorylation of enos is a signaling marker of activation. we have recently validated lut, fol and preg uaec culture models for evaluating enos phosphorylation responses to shear stress and vascular mediators. we hypothesized that uaecs derived from fol and preg sheep will show greater enos phosphorylation than lut phase uaecs, and with more robust responses in the presence of estrogen (e ). methods: uaecs were cultured until % confluence, and then subjected to (static control), or dynes/cm for hrs in the absence or presence of e ( nm). western analysis was used to compare optical densities of ser -penos (penos) normalized to total-enos (mean + sem). results: in lut uaec penos was equally increased two fold by and dynes/cm (from . + . to . + . and . + . , respectively). compared to lut uaecs, the static control fol uaecs appeared to have higher penos levels ( . + . ) and this was further increased . - . fold with dynes/cm ( . + . ) and dynes/cm ( . + . ). as seen with fol uaecs, preg uaec static penos ( . + . ) appeared higher than lut uaec, but unexpectedly, neither nor dynes/cm significantly raised these levels of penos ( . + . and . + . ). regardless of shear stress level, e replacement in the culture media only increased the penos levels in the nonpregnant, but not the pregnant derived uaecs. conclusions: increasing amounts of shear stress have a corresponding increase in the ratio of enos that is phosphorylated at serine in lut and fol uaecs. pregnant uaecs do not appear to increase the constitutive ratio of serine phosphorylation of enos at either or dynes/cm . in contrast to our hypothesis, chronic treatment with e for hrs did not augment the ratio of enos phosphorylation in pregnancy. nih hl , hd , hl . cells. honghai zhang, wu xiang liao, dong-bao chen. reproductive medicine, university of california san diego, la jolla, ca, usa. s-nitrosylation (sno) is a rapid, reversible, and nitric oxide (no) dependent post-translational protein modification critical for signal transduction. estrogen stimulates endothelial cell (ec) no production but yet to be determined is if this leads to increased formation of sno-proteins in ec. hypothesis: we hypothesize that estrogen stimulates the formation of sno via a receptor and endogenous no dependent pathway in huvec or ovine uterine artery ec. methods: cells on glass coverslips were treated with mm of no donor gsno or dea nonoate, estradiol- ( nm) in the presence of l-name ( mm) for min. the cells were fixed with methanol. in situ sno-proteins were detected by a rapid biotin derivatization method by blocking thiols by methylmethanethiosulfonate (mmts), followed by ascorbate reduction and labeling with texas red-labeled ethylmethanethiosulfonate (mtsea) and examined by fluorescence microscopy. cells ( x /group) were lysed in hen buffer, and then similarly prepared but labeled with biotin-mtsea. a portion of the samples were separated on sds-page and blotted with anti-biotin antibody for total sno-protein patterns. the biotin-labeled sno-proteins were captured by avidin, followed by immunoblotting with specific antibodies. results: basal sno-protein labeling was apparent in both ec types. exogenous no donated by gsno or dea nonoate significantly increased red fluorescence labeling of sno-proteins in the cytosol of both ec types. treatment with estradiol- also increased total sno-protein labeling in both ec types. pretreatment with l-name significantly reduced sno-protein labeling. sds-page and immunoblotting with anti-biotin antibody analyses identified multiple bands of sno-proteins. in avidin captured biotinylated samples, multiple proteins were indentified. conclusion: exogenous no donated by gsno or dea nonoate and endogenous no upon estrogen stimulation increased s-nitrosylated proteins in ec (supported by nih ro hl and ). background and objective: the renin-angiotensin system (ras) functions both systemically and locally as a primary regulator of blood pressure and fluid volume and is therefore involved in the etiopathogenesis of essential hypertension as well as preeclampsia, a pregnancy-induced hypertensive disorder in pregnant women. while much progress has been made in the development of strategies to block the systemic ras and thus treat essential hypertension, relatively less advance has been achieved in the development of effective local ras inhibitors to treat preeclampsia, primarily due to the fact the conventional systemic ras inhibitors generate potential teratogenic risks to pregnant women during critical stages of their pregnancies. ginger has been widely and effectively used in pregnant women to treat pregnancyinduced nausea and vomiting with minimal adverse effects. the present study was designed to investigate whether ginger could down-regulate renin secretion by human decidual cells (the major source of renin in the tissue-based uteroplacental ras), as an initial step toward a long-term goal to develop potential safe and effective ras inhibitors for use in obstetric patients. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were treated with ginger juice at various doses in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. the ginger juice used was freshly prepared from the ginger roots purchased from a local grocery store and different batches of juice preparations were standardized based on their optical density values at a wavelength of μm on a spectrophotometer. results: a dominant band of renin at approximately kd was detected in all samples. when compared with the control (i.e. %), ginger juice decreased the renin protein contents in the culture supernatants in a dose-dependent manner. no significant difference in the cell morphology was observed between the control and treatment groups. conclusion: ginger root juice down-regulates renin secretion in human decidua, showing a great potential of a novel ras inhibitor, particularly, in obstetric patients. noninvasive quantification of the autonomic nervous system throughout pregnancy. dietmar schlembach, karoline pickel, daniela ulrich, philipp klaritsch, isa alkan, uwe lang, manfred g moertl. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. introduction: the analysis of heart rate variability (hrv), blood pressure variability (bpv), and baroreflex sensitivity (brs) has become a powerful tool for the assessment of autonomic control. although hrv analysis was initially developed for risk stratification in cardiology, the field of clinical application has broadened in recent years. the aim of our study was to investigate the adaptation of autonomic control during pregnancy based on analysis of heart rate variability and baroreceptor sensitivity. methods: patients with uncomplicated pregnancy were measured with the taskforce® monitor i made by cnsystems. all measurements were performed in supine position under standardized resting conditions. autonomic parameters were recorded at rest and within the "deep breathing method" as a "cardiovascular challenge test". results: throughout pregnancy a shift to higher sympathetic activity respectively a lower parasympathetic activity was observed. , and . ± . [> wks]) (figure ). during the deep breathing maneuver we could show an increase of the sympathetic / parasympathetic ratio (figure ). the noninvasive determination of autonomic parameters throughout pregnancy is possible. these results can be used as basic parameters for classifying and assessing autonomic changes in pathological conditions in pregnancy such as hypertensive disorders. how far the characterization and challenge of these autonomic functions can be utilized for diagnosis and prediction of preeclampsia has to be shown in future studies. hemodynamic parameters measured noninvasively throughout pregnancy. daniela ulrich, manfred g moertl, karoline pickel, philipp klaritsch, isa alkan, uwe lang, dietmar schlembach. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. background: hemodynamic changes throughout pregnancy have been measured predominantly by invasive techniques. the discussion on the valence und the low acceptance of these invasive procedures by pregnant women demands a noninvasive method for evaluating and distinguishing cardiovascular adaptative mechanisms throughout normal and especially complicated pregnancy. method: healthy patients with uncomplicated pregnancy were measured with the taskforce® monitor i (cnsystems, austria) at different time points throughout pregnancy. cardiovascular parameters were recorded in supine and left lateral position under standardized conditions. results: throughout pregnancy an increase of global parameters such as heart rate (hr), blood pressure (bp), total peripheral resistance (tpr) and total peripheral resistance index (tpri) were observed. stroke volume (sv), stroke index (si), cardiac output (co), contractility index (ci), acceleration index (aci), and left ventricular ejection time (lvet) decreased throughout pregnancy (table). discussion: the noninvasive determination of cardiovascular parameters throughout pregnancy is possible and the results of this pilot study can serve as basic parameters for classifying and assessing cardiovascular changes in pathological conditions in pregnancy such as hypertensive disorders. assigning pregnant hypertensive women into hyper-and hypodynamic groups may aid in planning individual therapeutic strategies. objective: both gnrh i and gnrh ii are expressed in humans. gnrh i is produced by the hypothalamus under the regulation of gonadal steroids and stimulates pituitary gonadotropins. during pregnancy gnrh i is also produced by the placenta and affects hcg. gnrh ii, the ancient isoform, is produced by numerous human tissues including immune and reproductive tissues and circulates in blood in quantifiable levels. we have demonstrated that gnrh ii analogs directly affect fertilization and uterine function, and propose that it acts via immune regulation. during pregnancy it is known to regulate numerous hormone productions, yet the levels of gnrh ii has not been reported. in these studies we have determined the circulating levels of gnrh ii throughout pregnancy and in early pregnancy loss. study design: thirty-three women having normal pregnancies were followed prospectively. plasma samples were drawn at , , , , , , weeks gestation and during labor. plasma was also collected from patients have spontaneous abortions (n= ). circulating gnrh ii and crh and gnrh i were measured by specific radioimmunoassays. results: circulating gnrh ii increased from non-pregnant levels ( +/- pg/ ml, mean+/-sem) to +/- pg/ml by -week lmp. gnrh ii concentrations continued to increase through weeks gestation over the -week levels, although a significant increase was only attained by weeks. gnrh ii continued to increase in late term, attaining levels of +/- pg/ml which was significantly higher than that of early gestation. during labor and delivery gnrh ii in maternal plasma was further increased to +/- pg/ml, i.e., . times that of -week gestation ( +/- pg/ml). circulating gnrh ii did not parallel gnrh i in early pregnancy but did in late gestation. gnrh ii did correlate with crh in early pregnancy but not in late gestation. patients having spontaneous abortion had increased circulating gnrh ii at -weeks lmp ( +/- pg/ml) as compared to normal pregnancies ( +/- pg/ml). conclusion: gnrh ii increased throughout pregnancy attaining highest concentrations during labor and delivery. patient with early pregnancy loss had increased gnrh ii expression. the function of gnrh ii or factors affecting this increased gnrh ii in normal or abnormal human pregnancy should be investigated. introduction: plasma levels of the endocannabinoid, anandamide (aea) decrease during the luteal phase of the menstrual cycle and early pregnancy and increase during parturition. high plasma aea levels at weeks in women undergoing ivf-et was associated with a failure to achieve an ongoing pregnancy . what is uncertain is what happens to aea levels when the pregnancy has already failed. we aimed to quantify aea levels in women presenting in early pregnancy with a diagnosed non-viable pregnancy and to compare them to those of a viable pregnancy. methods: plasma aea was measured by a sensitive isotope dilution hplc-ms/ms method from women in early pregnancy ( - weeks) of whom had a viable pregnancy and had a non-viable pregnancy. serum hcg and progesterone were measured in blood samples by standard elisa methods. results: the ages and bmi of both groups were similar ( ± . versus ± . years; mean ± sd; p= . ; student's unpaired t-test and ± . versus ± . kg/m ; p= . ) respectively. plasma aea levels in women with non-viable pregnancies were significantly higher than those in women with viable pregnancies ( . ± . versus . ± . nm; p= . ). there was no correlation found between aea levels and hcg or progesterone levels p= . and r= . ; p= . , respectively) , despite progesterone being significantly lower in the non-viable group ( . ± . versus . ± . ng/ml; p= . ). conclusion: higher plasma aea levels were associated with early pregnancy failure, and this association appeared to be independent of serum hcg or progesterone concentrations. these data suggest that plasma aea levels are linked with pregnancy failure through a mechanism that does not involve hcg or progesterone production. the precise involvement of anandamide in early pregnancy needs to be investigated further. n- ) and arachidonic (aa, : n- ) acids, the major brain long-chain polyunsaturated fatty acids (lc-pufa) are generated by elongation-desaturation of dietary essential fatty acids (efa). the maternal liver is principally responsible for efa elongation-desaturation. objetive. we determined effects of protein restriction in pregnancy on expression of d, d and elongases and (elov and ) in the maternal liver rat. methods. pregnant rats were fed control ( % casein; c) or restricted ( % casein; r) isocaloric diets. at day gestation maternal blood and livers were collected. serum triglycerides, cholesterol and glucose were determinate with the synchron cx autoanalyser and leptin and insulin by ria. liver fat determination was performed by the soxhlet method. liver ara and dha were calculated by gas chromatography based upon retention times from methyl ester standards. liver d, d, elov and mrna were measured by rt-pcr and northern blot. data are m ± sem; analysis by t-test. results. liver weights did not differ in c and r. total liver fat ( ± vs ± mg) serum leptin ( ± . vs ± . ng/ml) and insulin ( . ± . vs . ± . ng/ml) differed in c and r respectively (p< . ). serum triglycerides, cholesterol, and glucose did not differ. desaturase and elongase mrna and % of maternal liver aa and dha were lower in r (fig ) . conclusion. low liver fat content and desaturase and elongase mrna in r indicate impaired lc-pufa synthesis which may adversely impact fetal development, especially the brain. objective: to test the hypothesis that obstetrical dic results from an excessive leak of fetal material into the maternal circulation. a secondary objective was to assess maternal morbidity. methods: all cesarean hysterectomy cases for hemorrhage at our hospital from to were included. intravascular presence of fetal material was determined by two pathologists, blinded to each other and any clinical information. the percentage of those with any fetal material in the maternal circulation was calculated for each diagnosis for hemorrhage. for a given diagnosis, the percentage of intravascular fetal material in those with the given diagnosis was compared to those without that diagnosis using fisher's exact test. most patients had multiple hemorrhage diagnoses. a two sample t-test was used to evaluate the difference in mean blood loss between those with and without intravascular fetal material. results: primary outcome* % of patients received blood products and % received clotting agents. the average blood loss was cc. there were no maternal deaths and injuries to adjacent organs, all to the bladder. conclusions: there was no association between the presence of intravascular fetal material and any specific hemorrhage diagnosis or amount of blood loss. although the power to detect a relationship was low, the excessive leakage of fetal material as a specific and exclusive mechanism of obstetrical dic, as postulated, seems unlikely. while % of the population was transfused, there were few intraoperative complications and no maternal deaths. hypertension, tel-aviv university, sheba medical center, ramat-gan, israel. objective: the joint national committee on high blood pressure (jnc ) determined blood pressure of - / - in adults to be prehypertension that requires health promoting life style modifications to prevent cardiovascular disease. similarly, we hypothesize that gestational prehypertension in pregnancy is associated with increased risk of pregnancy complications and its management would improve pregnancy outcome. methods: prospective and retrospective recruitment resulted in a study group consisting of patients who were diagnosed in the first and early second trimester (< weeks) with blood pressure of - / , and a control group consisting of patients with blood pressure < / at similar gestational age. comparisons between the two groups were accomplished for demographic characteristics and outcome measures using the chi-square test statistic and two-sample t-tests for categorical and continuous variables, respectively. results: all outcome measures analyzed resulted in poorer results being associated with the study group compared with control as follows: % versus % of the study and control group pregnancies, respectively, experienced preeclampsia (p= . ); and % versus % of the study and control group, respectively, were associated with gestational hypertension (p= . ). % of the control group deliveries were associated with admission to the nicu, compared to only % in the control group (p= . ); % versus % of the study and control group deliveries, respectively, were preterm (p= . ); twenty-nine percent of the study group were cesarean deliveries, compared to % in the control group (p= . ). on admission mean sbp and dbp was and mmhg, respectively, in the study group, compared to and mmhg in the control group (p= . for sbp and p= . for dbp). conclusions: "gestational pre-hypertension" may be a real pregnancy condition that when is recognized, physician attention, patient close monitoring during prenatal care and delivery and early intervention in patients at risk, may all promote improved pregnancy outcome. larger scale study is in progress to evaluate and confirm these findings in the larger pregnant population. are contractions at - weeks gestation less painful than those at full term? kristy a ruis, karin blakemore, abimbola aina-mumuney, valerie jones. gynecology and obstetrics, johns hopkins hospital, baltimore, md, usa. objective to determine if gestational age plays a role in severity of pain associated with uterine contractions. study design self-reported pain from women in labor, on a scale of - , is assessed and recorded in an obstetrical database by nurses at regular intervals at two hospitals within our medical system and was retrospectively reviewed from - . pain at various dilatations ( - cm) of women who were laboring and then delivered at - weeks' gestation was compared to women in labor at term. maternal demographics of age, race, education level, bmi, presence of support person, request for analgesia at any point in labor, and epidural placement were abstracted from maternal records. categorical data were analyzed using chi square or fisher exact test; continuous variables using student t-test. results laboring patients between - weeks gestation and at term were identified. term and preterm patients did not differ with respect to maternal demographics. pain reported by preterm patients was significantly less at - cm dilatation ( . vs. . ‚ p= . ) and significantly greater at - cm ( . vs. . ‚ p= . ). pain reported at - cm and - cm was not statistically different between the groups. of note, fewer of the preterm patients received an epidural despite the request for analgesia. conclusion preterm laboring patients between - weeks gestation may perceive less pain at - cm dilatation compared to term laboring patients; however, their pain perception at advanced dilatation was comparable to those at term despite the fact that they were less likely to have an epidural in place at the time of pain evaluation. in light of these findings, the widely accepted etiology of labor pain as the result of cervical dilatation may need to be re-examined. also, factoring in the patient's discomfort level into the evaluation for onset of early active labor may not be valid in the preterm patient. background and objective: asthma is a risk factor for adverse pregnancy outcome. this has been attributed to the effect of allergy in pregnancy. mast cell mediators can induce uterine contractility. the objective of the study was to test the hypothesis that pregnant women with asthma have different uterine electrical signals from those generated by normal pregnant women. materials and methods: uterine electromyography (emg) recordings from gestational age matched pregnant women at term were analyzed. the cases consisted of patients with asthma and the controls, those women without asthma. emg was recorded for approximately minutes from surface electrodes placed upon the maternal abdomen, with the electrical signals filtered in the uterine-specific range of . to . hz to remove noise components. the recordings were analyzed in their entirety first by power spectrum analysis and then by lyapunov analysis. the power-spectrum-largest-peak frequency and the largest lyapunov exponent were calculated for each patient, and then the mean and standard deviation of these parameters was compared using student's t-test. results: patients with asthma had significant lower power spectrum frequency and higher lyapunov exponent than women without asthma. see ) the power spectrum frequency and the lyapunov exponent used to analyze uterine emg signals were different in women with and without asthma; ) the results suggest that uterine electrical activity is altered in women affected by asthma; ) these findings may explain the observed increased frequency of preterm birth in women with asthma. further studies are required to dissect the mechanisms. fetal microchimeric cells trafficked into the maternal circulation persist in blood and tissues for years after pregnancy. increasing data suggest that microchimerism occurs after every pregnancy, but the biological role of fetal microchimerism or the cell types involved is unclear. while persistent fetal cells were initially implicated in autoimmune disease, animal studies suggest these fetal cells play a broader role in response to tissue injury. methods: appendix specimens were acquired from women undergoing appendicectomy during pregnancy. detailed reproductive histories were obtained. fluorescence in situ hybridisation (fish) with two different probes allowed investigation of the presence of male presumed-fetal cells and nested pcr amplification of sry gene confirmed male dna in the appendix. immunostaining was used to determine the fetal cell phenotype. results: male cells were identified in appendix tissues from women with known male pregnancies (n= ) and also from a woman with no sons and a previous miscarriage of undetermined gender (n= ). no male cells were observed in the control (n= ), a woman with daughters. male cells of presumed fetal origin were evenly distributed in the muscle, mucosa and submucosa layers of the appendix. morphology and co-localisation analysis suggested the identified male cells had differentiated primarily into muscle cells or lymphocytes. combined immunostaining and y-fish demonstrated male desmin+ muscle cells and cd + and cd + lymphocytes. finally, the presence of male dna in the appendix specimens was confirmed by nested pcr. male cells of presumed fetal origin were identified in the appendix of pregnant women. microchimerism frequency varied according to the reproductive history and the degree of inflammation. microchimerism rates were higher in the appendix from women with current male pregnancies than in those with previous male pregnancies and were higher in those with histological acute inflammation, when compared to milder cases. male microchimeric cells identified were of haematopoietic and mesenchymal origin. this study suggests that fetal cells are present in sites of tissue injury and may participate in tissue repair during pregnancy. collagen i and collagen-binding integrins mrna expression in rat cervix during gestation. huiling ji, tanya l dailey, vit long, edward ks chien. obstetrics and gynecology, maternal fetal medicine, women and infants' hospital of rhode island, providence, ri, usa. objective cervical remodeling is associated with cell proliferation and extracellular matrix (ecm) remodeling. integrins are a family of multifunctional cell adhesion receptors which mediate ecm-cell interactions including binding collagen. of the integrin (i) heterodimers, , , and are primary receptors for collagen. the predominant cervical collagen is collagen type . the purpose of this study is to investigate collagen and integrin expression in the pregnant rat cervix. the cervix was harvested from timed pregnant sprague-dawley rats. non pregnant (np)and timed pregnant day , , , , and animals were euthanized using a protocol approved by the iacuc. four animals were sacrificed on each day of gestation. quantitative rt-pcr was used to evaluate mrna expression and normalized to -actin. a standard curve was generated from a single sample. data was analyzed using anova and multiple comparisons testing. collagen i mrna expression increased through mid-gestation but decreased to np levels on day . a similar pattern was seen with the integrin beta subunit. the pattern of integrin alpha subunit expression was different for each subunit during pregnancy. the changes in collagen, integrin alpha , , and integrin beta were found to be statistically significant. (figure) . the pattern of collagen binding integrin alpha subunit expression during the rat gestation appears to vary independently of each other. the expression of the subunit paralleled col expression. collagen binding integrins may play independent roles in signaling and biomechanics during gestation. funding: women infants' hospital research fund; phs nih-ncrr p rr p rr . background. the effects of chemotherapy on human fetal development are poorly studied, mainly due to the lack of adequate animal models in which placentation and embryological development resembles humans and pharmacokinetic studies, prenatal sonography as well as histological studies can be performed. aim of the study. to test the baboon as model for studying pharmacokinetics and fetal effects of chemotherapy administered during pregnancy. methods. experiments were performed in pregnant baboons at a mean gestational age of (+/- ) days. detailed ultrasound examination (biometry, doppler, screening) was performed days before, at and day after the drug administration. the administration of different schemes of chemotherapy and the fetal samplings occurred under endotracheal anaesthesia. maternal blood samplings, as well as percutaneous ultrasound guided fetal blood ( ml) and amniotic fluid ( ml) samplings were performed at least once immediately after drug administration. in case of fetal demise, the fetus underwent detailed macro-and microscopic examination. in the other animals mother and fetus were euthanized h after the experiments, with collection of blood, amniotic fluid and tissues for further analysis. results. all mothers survived the experiments, one mother developed paralitic ileus. none of the fetuses died during the acute phase of the experiment but / ( %) fetuses died within h following the experiment. none of the animals showed significant clinical or histological signs of infection or anaemia. a total of ultrasound examinations were performed in fetuses, allowing the creation of sonographic growth charts in this model. at the time of drug administration the mean maternal weight was . kg (range . - . ) and the estimated fetal weight was gr (range - ). sixteen out of cordocenteses ( %) and all amniocenteses ( %) were successful in obtaining the required samples for analysis. conclusion. the pregnant baboon can be used as a model for studies on pharmacokinetics and fetal effects of chemotherapy administered during gestation. prenatal ultrasound is comparable to the human situation, and amniocentesis and cordocentesis can be performed with a high success rate and short term survival. therapy to prevent preterm birth is limited". moreover, -hydroxyprogesterone ( ohp) caproate is a category d drug according to the fda (evidence of fetal harm). p is produced in the adrenal glands, gonads and brain. after weeks gestation p is secreted from the placenta independently to the mother and the fetus. p is the precursor of aldosterone and after conversion to ohp, of cortisol and androstenedione. baboons have similar reproductive system and development to humans and are used to study mechanisms of labor and prevention of preterm labor. currently, there are no normative data on the concentration of p, ohp, cortisol ©, estradiol (e ) or inhibin (i) throughout their pregnancy. therefore, a doseresponse or the teratogenic effects of p or ohp caproate cannot be studied in a controlled manner in this animal model. the aim of this study was to generate reference values for these hormones during baboon gestation and early postpartum life. material and methods: hormonal quantitative measurements were performed (immulite analyzer) results: the mean concentrations of these hormones are depicted in figure (maternal serum, from conception to term days) and figure (newborn serum) conclusion: this study provides reference values for hormones quantified during the baboon gestation and early postpartum life. this may be useful for future research concerning the effects of p and ohp administration during pregnancy. ( )non-pregnant women eligible for ivf treatment and ( )pregnant women receiving prenatal care. the pre- / samples were drawn in the year prior to / / ; the post- / samples were drawn within months after. the pre- / and post- / normal pregnant samples were drawn at + weeks' gestation. the non-pregnant samples were from ivf candidates with serum e levels< newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced pg/ml and fsh serum levels< . miu/ml. the samples were assayed for acth and cortisol by a commercially available immulite™ system. hsp and hsp were measured by commercially available elisa. these results were compared in the patient populations before and after / . t-tests were used for analysis. statistical significance was set at p< . . results: in the pregnant subjects, there were no statistical differences in the mean levels of acth, cortisol, and hsp pre-and post- / (table ) . only serum hsp levels were significantly increased in the pregnant women post- / .* mean serum acth, cortisol, hsp , and hsp levels were all significantly different in non-pregnant subjects pre-and post- / . conclusions: except for hsp , serum markers for stress were unchanged in pregnant subjects after the stress of / / in comparison to non-pregnant women whose serum hsp , hsp , cortisol, and acth levels were significantly different. the lack of change in serum markers of stress in pregnant women suggests that the hormonal milieu of pregnancy may buffer against acute environmental stressors. objective: in human pregnancy, maternal body composition provides an indicator of maternal nutritional status and metabolic capacity. previously, we reported that ß hsd- activity was significantly reduced in term placentas from thin women and women with a smaller mid-upper arm circumference before conception. this suggests that these fetuses may have been exposed to inappropriate levels of maternal cortisol, which is a mediator of gestation length. in this study, we hypothesize that the duration of gestation will be shorter in women who tend to be thin and have a lower mid-upper arm circumference prior to conception. methods: within the longitudinal, population-based southampton women's survey (sws), analyses were performed on women whose estimated date of conception was set using an algorithm that combined menstrual and early ultrasound scan data and who had a spontaneous onset of labour and delivered after weeks of gestation. linear regression was used to examine the relationships between maternal body composition and the duration of gestation. results: within the women surveyed, lower maternal body mass index, mid-upper arm circumference and arm muscle area before conception were all associated with shorter duration of gestation at delivery (r= . , p= . ; r= . , p= . ; and r= . , p= . , respectively). a lower subscapular skinfold thickness, sum of skinfolds and height were also associated with shorter duration of gestation (r= . , p= . ; r= . , p= . and r= . , p= . , respectively). mother's age, own birthweight and ratio of subscapular/triceps skinfold thickness were not related to the duration of gestation. conclusions: in this study, we found that thinner women with a lower midupper arm circumference and arm muscle area tended to have a shorter duration of gestation. our findings of shorter gestation length in thinner women are in keeping with our previous observation of lower ß hsd- activity in term placentas from thinner women. we conclude that metabolic capacity prior to conception could influence duration of gestation through mechanisms that include alteration in placental metabolism and fetal cortisol exposure. fecal incontinence (fi) is a debilitating condition affecting - % of the us. our prior studies found that % of women report new onset fi after childbirth. the goal of our study was to examine the impact of fi on postpartum quality of life (qol). women reporting fi on a statewide survey who agreed to participate in a -year study of qol were included in the analysis. women were considered to have fi based upon the nih definition of fi. the quality of life survey was based upon the uebersax incontinence impact questionnaire and was administered every months for years. qol in women with fi was examined using chi square, and impact of severe fi (stool incontinence) was determined by multivariate logistic regression. results women with fi were surveyed and of those, ( %) returned at least survey during the study period, completed all survey questions, and were included in the final analysis. among women with fi, % felt frustrated due to fi, % reported fi impacted their emotional health, . % reported fi impacted child-caring abilities, and . % reported a negative impact on social activities. qol was similar across survey periods. one out of three women with fi reported severe symptoms (incontinence of stool). women with severe symptoms were - times more likely to report negative impacts on qol compared to milder (e.g. flatus) fi after adjusting for age, parity and urinary incontinence. . % felt their stool was stored elsewhere before bowel movements, . % reported using digital defecation and more than half ( %) reported symptoms of urinary leakage. despite the substantial impact on postpartum quality of life, few women sought medical help with only % of women at months, . % at year, and . % at years ever reporting their symptoms to a medical provider. postpartum women report that fecal incontinence has a substantial negative impact on their qol after delivery including their emotional health and ability to care for their newborn. despite this profound impact, few women will discuss fi with medical providers. these data suggests there may be a benefit for providers to inquire about fi at postpartum visits. objective: the objective of this study was to determine what characteristics contribute to racial/ethnic differences in breastfeeding rates. study design: a retrospective cohort study of all women who delivered a viable infant (n= , ) was conducted. the primary outcome was breastfeeding upon discharge of the hospital. we first examined the association between race/ ethnicity and breastfeeding. next, we conducted stratified analyses examining a variety of predictors of breastfeeding within the racial/ethnic groups including maternal demographics, obstetric interventions, and perinatal complications. results: we found that both asians (or . , % ci . - . ) and blacks ( . , % ci . - . ) had statistically significant lower rates of breastfeeding, while latinas (or . , % ci . - . ) showed a trend towards higher rates of breastfeeding. in latinas, we found that rd and th degree lacerations were significantly associated with lower rates of breastfeeding, but were not predictive of breastfeeding in the other racial/ethnic groups. epidural use was predictive of lower rates of breastfeeding in caucasians and blacks, but was not predictive in latinos and asians. some of the other predictors which differed between the racial/ethnic groups were obesity and induction of labor (table ) . conclusion: race/ethnicity is significantly associated with breastfeeding, with blacks and asians having the lowest rates of breastfeeding at discharge. a variety of other factors are associated with breastfeeding and interestingly, their effect appears to differ between the racial/ethnic groups. future studies might elucidate the sociocultural and biomedical reasons that explain the differences. these results help focus our efforts during the peripartum period to advocate for mothers who have factors that might impede breast feeding. epidemiology, erasmus medical centre, rotterdam, netherlands; pediatrics, erasmus medical centre, rotterdam, netherlands; public health, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background recommendations on folic acid use to prevent neural tube defects are launched in several countries. however, the adequate use of folic acid supplements during the periconception period seems to be low. objective to assess the prevalence of adequate folic acid use, defined as the preconception start of supplements, and to identify its determinants in a multi-ethnic population. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods from all women in the cohort who delivered between april and january information on folic acid use and potential determinants was obtained by questionnaires and physical examination. logistic regression models were used to identify determinants of periconception folic acid use. results. data from , pregnant women were available. of all women % adequately used folic acid supplements. the most important risk factors for inadequate use were unplanned pregnancy (or . , p< . ), nonwestern ethnicity (or . , ci . - . , p < . ) and a low educational level (or . , ci . - . , p< . ). other risk factors were single marital status, smoking, multiparity (all p< . ) and alcohol use (p< . ). prior spontaneous abortion was associated with increased adequate folic acid use (p< . ). conclusion adequate periconceptional folic acid use is low. improved preconception care and public health education programs are necessary to improve the uptake of folic acid. although methamphetamine (ma) use has been front and center of the united states drug control policy since , little attention has been given to ma use in pregnancy, despite the fact that pregnant admissions to drug treatment for ma have been rising sharply. to determine trends in the prevalence of ma treatment admissions during pregnancy, we undertook a secondary analysis of the treatment episode data set (teds), an administrative data set that captures at least % of all known treatment admissions in the us. in particular, we investigated risk factors for ma use and how these characteristics have changed over time. demographic, geographic and substance use data were collected for the , pregnant admissions captured between and . logistic regression models were constructed by year. confounding was assessed via backwards elimination with a change-in-estimate criteria of . considered substantial. trend results were reported as adjusted proportions and represented graphically. overall ma prevalence, reported as the primary drug of use upon admission, rose from . % in to . % in . although white women had times the odds of using ma in (adjusted or ( %ci) . [ . , . ]), this had dropped to . [ . , . ] by , mostly due to an increase in latina ma use in pregnancy. pregnant women admitted for ma had fewer prior treatment admissions, were more likely to be unemployed, and less likely to use alcohol or marijuana. we found great geographic variability in use. ma was more common in the pacific region and least common in new england. there was little change in regional variation over the study time period. less is known about the perinatal effects of ma compared with other substances, yet it is the primary drug of choice for pregnant women admitted into treatment. as pregnant women occupy a unique place in drug treatment, analysis of national trend data is essential to guide both policy and research. background: epidemiologists have grouped the multiple disorders that lead to extremely preterm delivery in a variety of ways. we sought to identify characteristics that would support the combining or dividing of the disorders that lead to preterm delivery. methods: we enrolled , women who delivered a live born singleton infant between and completed weeks gestation at tertiary centers in the united states. each delivery was classified according to the complication that prompted presentation: preterm labor, preterm premature rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes (pprom), preeclampsia, placental abruption, cervical incompetence, and fetal indication/intrauterine growth restriction (iugr). we compared these entities on the frequency of characteristics identified by standardized interview, chart review, histological examination of the placenta, and culture of placenta parenchyma. results: the percents of women who presented with each antenatal complication were: preterm labor ( ), pprom ( ), preeclampsia ( ), placental abruption ( ), cervical insufficiency ( ), and fetal indication/iugr ( ). after considering antecedents and correlates of the processes leading to preterm delivery, we observed two overarching epidemiologic patterns. the first pattern, characterized by recovery of organisms from the placenta and by histologic chorioamnionitis, tended to be associated with preterm labor, pprom, placental abruption, and cervical insufficiency. the second pattern, characterized by a paucity of organisms and inflammation and the presence of histologic features of dysfunctional placentation, tended to be associated with preeclampsia and fetal indications/iugr. conclusions: disorders leading to preterm delivery can be categorized broadly into two groups: those associated with intrauterine inflammation and those with aberrations of placentation. predictors of compliance with tuberculosis screening in pregnancy. nadav schwartz, sarah wagner, sean keeler, may tam, julian mierlak, , aaron caughey. obstetrics and gynecology, nyu school of medicine, new york, ny; obstetrics and gynecology, ucsf, san francisco, ca; obstetrics and gynecology, gouverneur health care services, new york, ny. objective: poor compliance with tuberculosis screening and treatment is a major obstacle to the containment of this disease. we sought to identify predictors of ppd and cxr compliance during pregnancy. methods: a retrospective cohort study at a single institution which serves a largely immigrant population in nyc, from november , through june , . data on maternal age, ethnicity, country of origin, level of education, ppd and cxr status were collected. results: of the , pregnancies, asian and hispanic race/ethnicities accounted for . % and . % of the population, respectively, with . % being us-born. the mean age was . years and the overall ppd+ rate was . %. there was % non-compliance with ppd testing, and % of ppd+ patients were non-compliant with their chest x-rays. asian women were more likely to be ppd-compliant than hispanic or caucasian women. ppd+ asian women were also more likely to be compliant with cxr. us-born women were significantly less likely to be compliant with their ppd or with their cxr. women > years old were less likely to be compliant with their cxr, while women with an elementary school education or less were more likely to be compliant. (see table for odds ratios and %ci) conclusions: age, education, immigrant status and other cultural and ethnic factors appear to play a role in compliance with tuberculosis screening. further elucidation of these effects may help clinicians target at-risk subpopulations and improve overall compliance, working towards better control of this disease. background: in the us, differential outcomes in cervical cancer among medically underserved women are linked to multiple barriers impacting loss to follow-up and failure or delay in diagnostic resolution. prior studies have found risk factors for acquisition of hpv and for inability to obtain a pap smear. no study has explored health literacy and physician communication as a key factor. methods: this research represents the formative phase of a randomized controlled trial of african american (aa) and hispanic women aimed to improve communication and abnormal pap smear follow-up in chicago. semi-structured interviews and focus groups were conducted face to face in spanish or english. each interview lasted minutes, and each focus group lasted - minutes. we recruited patients ( hispanic, aa) and providers from a purposive sample representative of two large clinics that serve low-income women. results: all interviews were transcribed by two investigators. each interview was coded by two investigators separately and then a third investigator reviewed the transcripts and coding to achieve triangulation. codes for these themes were developed and the responses were tabulated using the coding scheme. atlas ti was utilized to analyze all qualitative data. from these data, we uncovered provider-patient challenges in cancer communication including: the providers' trade off between medical accuracy and/or literacy when communicating with their patients. for the patients, the word cancer was important to hear since they wanted the truth and needed to hear this word in order to encourage them to respond more quickly. however, many providers believed that the word cancer was too "scary" and to "extreme" of a word that may communicate too much exaggerated information. both the hispanic and aa patients did not seem to differ in their responses. conclusion: results exemplify not only the importance of health literacy and patient provider communication, but demonstrate the wealth of information gained from qualitative research-a method of data collection seldom utilized in ob/gyn investigations. funding: women's reproductive health research award: hd - ; r ca (spring). population. kristine beckers, isabelle guelinckx, greet vansant, roland devlieger. obstetrics and gynaecology, university hospital gasthuisberg, leuven, belgium; clinical nutrition, katholic university leuven, leuven, belgium. objective: to generate reference charts for weight gain during pregnancy for the different bmi-categories (underweight, normal weight, overweight, obesity), based on recent data in a homogeneous caucasian population. methods: in a retrospective study at the department of obstetrics of the leuven university hospital (belgium), weight gain and pre-pregnancy bmi were determined in belgian pregnant women with accurately dateable, uncomplicated singleton pregnancies. centile curves for the different bmicategories were constructed using of the linear mixed model, one set of charts based on the absolute weight gain, another set based on the relative (expressed as percentage) weight gain. the effect of parity on weight gain was examined. results: overall mean weight gain was . ± . kg ( . ± . lbs). mean weight gain was . ± . kg ( . ± . lbs) in the underweight population, . ± . kg ( . ± . lbs) in the population with normal weight, . ± . kg ( . ± . lbs) in the overweight population and . ± . kg ( . ± . lbs) in the obese population. weight gain (pattern and amount) of the underweight and normal weight patients differed significantly of the overweight and obese patients. parity had a statistical, but no clinical significant influence on amount and evolution of weight gain. conclusion: by using strict inclusion criteria, bmi-category-specific reference charts were generated representing the optimal gestational weight gain, rather than the mean weight gain. this enables the weight charts to be used as a clinical tool during the counselling of pregnant women. further studies are required to assess the effectiveness of this clinical tool. female fshr(-/-) mice are sterile, because of a block in folliculogenesis at the primary follicle stage, show decrease in e and elevated fsh. this animal model is an appropriate model for studying hypergonadotropic ovarian dysgenesis and infertility, caused by c t mutation in fshr gene. objective: to investigate the effects of bmt on serum hormonal levels, follicular maturation and fertility of fshr(-/-) mice. methods: fshr (-/-) mice at - weeks of age were randomized into treated versus control groups.bm from syngenic female donor was injected into the tails of recipients. control group received vehicle alone. vaginal smears were collected, body weight was measured daily. sample animals were sacrificed at , , , , and weeks post bmt. all organs were weighted and examined by h e. fsh, e , and p were measured before and after treatment. for donor cell tracking, dna was extracted from various organs. specific primer sets were designed for normal and mutant hfshr gene. pcr amplification was done and pcr products were analyzed in % agarose gel. results: total body weight significantly increased in treated animals(p< . ). significant increase in both the total number of follicles, and the collective diameter of the follicles in treated animals observed(p< . and p< . ). six out of treated animals showed estrogenic changes in daily vaginal smear. e level increased . - . times and fsh level dropped to % in treated animals. normal (donor allele) fshr gene was amplifiable in out of recipient mice, and was detected only in the ovaries and uterus but not in any other tested organs.control group did not show any changes in vaginal smear, hormonal level, and normal fshr allele. conclusion: intravenously injected syngeneic bone marrow cells were able to home to the ovaries of female fshr (-/-) mice and restore folliculogenesis and resume steroid hormone production. potential mechanisms for these observations will be discussed. conclusion: neural stem cells (nscs) give rise to progenitors, which expand by rapid proliferation until cell cycle arrest followed by differentiation along one of cns cell lineages: neurons, astrocytes and oligodendrocytes. increased differentiation of neuronal cells with ins and even more with leptin suggests that iugr-associated reduction of these growth factors may contribute to impaired hypothalamic pathway development. objective: to develop a technology of modulating hucb in order to achieve neuronal cells for future potential replacement of damaged neuronal tissue. design: we developed a two-dimensional ( d) tissue culture technology for isolation and differentiation of collagen-adherent hucb neural progenitors (hucbnps). we further used the extracellular matrix protein collagen, organized in a three-dimensional ( d) gel, supplemented with neuronal conditioning medium and nerve growth factor (ngf), to facilitate ex-vivo long term neuronal differentiation of hucbnps. we developed a stable green fluorescence protein (gfp)-pc cell model, to be used as a positive control for monitoring neuronal outgrowth for proper evaluation of the hucbnps growth in the d scaffold, mimicking a neural tissue organization. results: our experimental data indicate that d collagen environment is neuronal biocompatible, supporting attachment, long-term survival, proliferation and differentiation of both hucbnps and gfp-pc cells. conclusions: improvement of the d technology with cultures of hucbnps that is in progress in our laboratory might be the first step in validating the concept for the feasibility of generating a neuronal d tissue construct for future potential treatment of neurodegenerative disorders. piane, justin tsai, gnanaratnam giritharan, emin maltepe, paolo rinaudo. reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: ivf embryos are often used to generate stem cells. however, it is unknown if stem cell lines originated from in vitro generated embryos are different from stem cell originated from in vivo embryos. trophoblastic cells, due to their external position within the embryo, are most susceptible to environmental factors encountered in vitro. furthermore, our previous data showed that trophoblast transport functions may be impaired after culture in vitro and that the number of trophoblastic cells is reduced in the embryo after ivf. in this study, we assess the differentiation characteristics of ts cell lines obtained from in vivo and in vitro fertilized embryos. methods: oocytes were isolated from superovulated c bl/ j female mice and in vitro fertilized with sperm from male c bl/ j mice. the resulting late-cavitating blastocysts were harvested. in vivo controls were obtained by flushing the blastocysts from the uteri of superovulated pregnant mice days post hcg. ts cells from the two groups were allowed to develop and maintained in vitro in the presence of fgf and heparin, using a feeder layer of human placental fibroblasts. ts cells were then allowed to differentiate without fgf and heparin, fixed on day , stained with -tubulin and zo- antibodies and then observed under fluorescence microscopy. three ts cell lines per group were analyzed and their differentiative capacity was evaluated using morphological criteria. results: in vivo and ivf derived ts exhibit a similar differentiation pattern. in particular, the number and timing of trophoblast giant cells and spongiotrophoblasts derivation is similar in the two groups. there are no obvious abnormalities in the immunologic staining morphology of the different cell lines at different time points. conclusion: there are no apparent morphological alterations in ts cells lines derived from ivf embryos as compared to in vivo embryos. this finding in an animal model increases our confidence in the reliability of human stem cells derived from ivf. as a further investigation, markers of trophoblast giant cells, spongiotrophoblast, syncytiotrophoblast and chorionic trophoblast cells will be examined and compared in the two different groups by northern blot hybridization. genomewide high density snp-cgh reveals several new deletion copy number variants on the x chromosome in pof patients. erik ah knauff, cisca wijmenga, ruben van 't slot, lude franke, bart cjm fauser. reproduction gynecology, university medical centre, utrecht, netherlands; complex genetics group, university medical centre, utrecht, netherlands. introduction: around % of women have a post-menopausal hormonal profile before age , referred to as premature ovarian failure (pof). pof could act as a genetic model for accelerated follicle loss and may render useful information about the polygenetic background of major individual differences in menopausal age. macrodeletions on the x chromosome are associated with pof but karyotyping has a maximal resolution of mb. when using genotyping whole genome arrays it is now possible to detect submicroscopic deletions and duplications (copy number variants (cnv)) up to kb effective resolution. we have screened for microdeletions on the x chromosome in a well-phenotyped cohort of pof patients. methods: our study included caucasian, ,xx patients with spontaneous secondary amenorrhea before age , fsh > iu/l and absence of (low) x/ xx mosaicism and fmr premutations. dna analysis was performed using illumina k cnv arrays containing , probes. , probes were located on the x chromosome ( probe for every kb), of which , probes were specifically designed to detect known cnvs. ten samples with call rates < % were removed from the analysis and one sample gave inconsistent x probe intensities. we screened for deletions ( kb) using an algorithm detecting at least five consecutive probes with intensities (logr > - . ) below the mean probe intensity. we identified a total of x chromosomal microdeletions, divided in loci and varying between - kb in size. of the samples showed at least one deletion on the x chromosome. % of the identified deletions had already been recorded in the database of genomic variants. in the newly identified newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced loci, we found coding regions containing genes. eight of these are established or potential pof candidate genes, including five that are clustered on the terminal xq critical pof region. conclusions: we observed abundant variation in the cnv regions, a proof-of-principle for this specially designed cnv-chip. snp comparative genomic hybridization revealed possible new deletions specific to pof on the x chromosome. data on duplications, validation and whole genome cnv analysis, compared to a control cohort, will become available soon and will be presented at the sgi annual scientific meeting. unexplained intrauterine fetal death (iufd) is associated with long qt syndrome. irene cetin, patrizio antonazzo, sabrina cozzolino, stefania calabrese, lia crotti, francesca ferrari, roberto insolia, fabio facchinetti, peter j schwartz. dept. of obst/gynecol, univ. of milano, milano, italy; molec cardiol lab, policlinico san matteo, pavia, italy; mother-infant dept., univ. of modena/reggio emilia, modena, italy. introduction: in developed countries, nearly in every pregnancies ends in late fetal loss. many iufds can be attributed to maternal disorders, fetal pathology, placental pathology and fewer to complications of labor and delivery. however, - % of cases remain unexplained. we recently demonstrated that % of sudden infant death syndrome (sids) cases carry functionally relevant genetic variants in long qt syndrome (lqts) genes. aim of the study was to analyze whether lqts genes are associated with iufd. materials and methods: patients with iufd were enrolled in two years, as part of an italian multicentre study. iufd was defined as fetal death at weeks or more of gestation according to the definition of late fetal death of the who. out of cases were classified as "unexplained stillbirths" according to the wigglesworth and aberdeen classifications. at birth placental and/cord samples were collected and dna extracted. the main lqts genes kcnq , kcnh , scn a, kcne and kcne were screened through dhplc and sequence analysis. any amino-acid substitution identified in the samples was checked for in a control population of caucasian women with uneventful pregnancies. preliminary data on the first cases (gestational age of death: - weeks) are reported. results: a total of missense mutations were identified in of stillbirths ( %), two on scn a and one on kcnh . the two mutations on scn a (v l; p a) were observed in two iufd occurred at term; they had been previously associated to sids and shown to increase the late sodium current. the mutation on kcnh is a novel genetic variant absent in reference alleles, never described in any control populations; this mutations was present in a case of iufd diagnosed at weeks of gestation. we are currently performing the electrophysiological cellular studies to define its functional effect. conclusions: these preliminary data indicate that a potentially significant number of currently unexplained iufd might be caused by ion channel diseases such as lqts. the potential prevention of sids or iufd recurrence and the identification of other affected family members could have important implications for the affected families. alternative splicing of epab is regulated by exonic splicing enhancers. e seli, a yaba, o guzeloglu-kayisli, md lalioti. ob gyn, yale u., new haven, ct, usa. introduction: alternative splicing is an important mechanism by which the genome gives rise to the observed diversity of proteins. embryonic poly(a) binding protein (epab), expressed exclusively in oocytes and early embryos, mediates translation of maternal mrnas. we identified an alternatively spliced form of epab lacking exon (cex del), and investigated its regulation as a model for alternative splicing in early development. specifically, we evaluated: imprinting (expression from maternal or paternal allele only); rna editing (post-transcriptional single nucleotide substitution); and exonic splicing enhancers (eses, exonic sites that bind splicing proteins). methods: a single nucleotide polymorphism (snp) detected in exon (c a/g) served as a marker for the parental origin of the spliced form. snp genotyping was performed by pcr amplification of exon followed by restriction enzyme digestion. to evaluate imprinting, we characterized heterozygous mice (a/g) that inherited the snp from either the mother or the father. to test for rna-editing and exonic enhancer contribution we tested mice homozygous for the exon snp (a/a or g/g). efficiency of alternative splicing in different genetic backgrounds was tested using real-time pcr normalized to actin expression. results: in mice heterozygous (a/g) for the exon snp, cex del was only expressed from the (a) allele. however, this was independent of the parental origin of the allele, ruling out imprinting. in mice homozygous (g/g) for the exon snp, the cex del variant also contained (g). therefore, rna editing did not occur. further sequence analysis led to the identification of an additional snp in exon (c g/c) that co-segregated with the exon snp. presence of c g led to the formation of an ese that binds splicing regulatory protein srp , leading to efficient exclusion of exon . real time pcr revealed a five-fold increase in the expression of the cex del alternative splicing variant in animals carrying the enhancer (homozygous g/g) for the exon snp compared to those that did not (homozygous c/c) at the same locus (p < . ). conclusions: in this study, we found that eses mediate the alternative splicing of oocyte-specific transcripts. our findings suggest that single nucleotide polymorphisms may alter the ratio between alternative splicing variants of oocyte-specific proteins. the role that these subtle differences play in determining individual reproductive outcome remains to be identified. epigenetics and chromosomal abnormalities in human oocytes. ilse van den berg, , joop se laven, robert jan galjaard, j hikke van doorninck. , obstetrics gynaecology; clinical genetics, erasmus medical center, rotterdam, netherlands. humans have a low fertility rate compared to other mammalian species. moreover their fertility declines with increasing age. both phenomena are largely due to an increasing number of chromosomal abnormalities in human oocytes during life. identifying factors that cause aneuploidy in oocytes may offer possibilities to diminish these abnormalities in vitro or in vivo. chromosomal segregation errors can result from aberrant recombination but little is known about epigenetic factors that may cause aneuploidy. epigenetic modifications such as histone acetylation and subsequent de-acetylation in oocytes are necessary for a correct progress through meiosis. if disturbed it may lead to aberrant segregation of chromosomes or chromatids due to decreased kinetochore function and imperfect spindle figures resulting in aneuploidy. mice oocyte data have shown a correlation of abnormal histone de-acetylation and aneuploidy and a correlation between age, remaining histone acetylation and aneuploidy (akiyama et al., ) . our research focused on human oocytes and investigated whether a similar relationship between histone acetylation, age and aneuploidy is present. human oocytes were surplus from standard ivf/icsi treatments (ic+). human oocytes showed immunostaining for histone , lysine acetylation (h k ) at the germinal vesicle stage and complete deacetylation at the mii stage in % of the oocytes, while % keep high levels of h k acetylation. treatment of germinal vesicle oocytes with a histon deacetylase inhibitor (tsa) during in vitro maturation until mii stage resulted in high levels of acetylation. remaining acetylation in tsa treated oocytes was correlated significantly with abnormal spindle figures (tubulin staining), a hallmark of developing aneuploidy. similarly, % of oocytes with naturally remaining h k acetylation showed abnormal spindle figures. in contrast % of the normal de-acetylated oocytes showed normal spindles (p= . ). this suggests that defective de-acetylation of h k in human oocytes leads to abnormal spindle figures and subsequent aneuploidy. advanced maternal age is associated with a reduction in de-acetylation during in vitro maturation and an increase in spindle abnormalities. these results may stimulate the development of assays for histone modifications as biomarkers to follow oocyte quality in in vitro maturation studies or in optimizing general ivf treatments. objective: racial disparity in preterm birth (ptb) is unexplained and genetic risk factors are suspected as a major cause. this large scale candidate gene study examines differences association of single nucleotide polymorphisms [snps] in caucasians (c) and african americans (aa) to help elucidate racial disparity in preterm birth (ptb) methods: in this case (preterm birth < weeks) control study (term birth > weeks) maternal and fetal dna from ( cases and controls) c and ( ptb and term) aa were collected. a high-throughput candidate gene association study was performed examining snps in genes of selected from hypothesized ptb pathways. single locus association analyses were performed separately on maternal and fetal samples. results: snps in genes associated with ptb (p< . ) were common between races with both maternal and fetal dna analyses. however, snps in genes in c and in aa in both maternal and fetal dna differed in its association with ptb. in c maternal dna, the single strongest association between ptb and snp was in plasminogen activator tissue (plat) gene (c- t; rs ) at both allelic (p = . x - ) and genotypic (p= . x - ) level with an odds ratio (or) of . ]. the single strongest effect in c fetal dna was observed in a snp in the interleukin- receptor antagonist gene (a g; rs ) for both allele (p= . ) and genotype (p= . x - ), or . ). in aa, the strongest associations were in interluekin- (c t-rs , allele p= . x - , genotype p= . x - ) in maternal dna with an or= . [ci . - . ] and in fetal dna interleukin- receptor b (a g; rs , allele p= . x - , genotype p= . x - ) with an or . ]. conclusion: large scale high throughput analyses of snps in candidate genes of ptb pathway reveals significant differences between races at both maternal and fetal levels. we found that the strongest single locus associations differed in the two races in both maternal and fetal dna samples. these findings support the hypothesis that underlying genetic predispositions may differ between these populations, perhaps partly explaining racial disparity in preterm birth. candidate gene association study indicates differential etiologies in gdm and in t dm. johann urschitz, tarik sultan, kenneth ward. obgyn, jabsom, university of hawaii, honolulu, hi, usa. objective: recently several genome-wide association studies have associated multiple single nucleotide polymorphisms (snps) in multiple genes with increased risk of type diabetes. as risk factors are similar between t dm and gestational diabetes (gdm), we investigated a possible association of those snps with gdm. study design: blood was collected from caucasian ( cases, controls) women who met coustan-carpenter criteria for gdm and non diabetic controls. dna was extracted and a candidate gene association study was performed (taqman, abi). results: chi contingency tests were used to analyze genotype and allele frequencies in controls and gdm affected pregnancies (see table below). none of the snps showed a significant association after bonferroni correction. conclusion: several polymorphisms which displayed highly significant associations with type diabetes are not associated with gestational diabetes. these results suggest that gdm is not a simple unmasking of a t dm predisposition by the metabolic demands of pregnancy; rather it appears that different biological mechanisms are responsible for the respective diseases. introduction: histone acetylation/deacetylation plays an important role in the regulation of gene expression. histone deacetylase inhibitors (hdaci), in general, maintain gene expression, although in some cases they cause repression of specific genes. in this context we have previously shown that the hdaci tsa suppresses activation of the pro-contractile gene cox- in human myometrial and amnion-derived cells [reproductive sciences, vol , no (supplement, # )]. we have also shown that expression profiles for hdacs ( - , ) differ significantly within upper and lower regions of the myometrium during pregnancy and in labour. objectives: since changes in both acetylation and phosphoryation status can influence the activity of individual hdacs we aimed to define whether there are any changes in the levels of acetylation and phosphoryation of myometrial hdac , and during pregnancy and labour. methods: protein homogenates prepared from non-pregnant, term and labouring myometrium were treated +/-shrimp alkaline phosphatase and subjected to sds-page and western immunoblotting (wb) using antibodies to hdac , and . the acetylation status of the hdacs was assessed by a co-immunoprecipitation assay using anti-hdac and anti-acetylated lysine antibodies. results: distinct patterns of acetylation were observed for the individual hdacs; hdac and appeared to be more acetylated in non-pregnant and labouring lower tissues when compared to pregnant myometrium. in contrast, hdac appeared to be slightly more acetylated in lower uterine samples from pregnant and labouring myometrium. in experiments to evaluate changes in phosphorylation status we observed that ) myometrial hdac appeared to less phosphorylated in both upper and lower labouring samples when compared to non-pregnant and pregnant samples; ) hdac appeared to be more phosphorylated in labouring samples than non-pregnant and pregnant myometrium; ) no changes in phosphorylation levels were observed for hdac using this test system. conclusions: the difference in hdac acetylation and phosphorylation levels in the human myometrium may indicate differential regulation of the activity of the hdacs within the distinct myometrial regions, perhaps leading to the alteration of their epigenetic effect on genes related to myometrial quiescence and contraction and the subsequent onset of both term and preterm labour. objective: native hawaiians and pacific islanders experience a higher perinatal morbidity secondary to the increased incidence and earlier onset of heart failure. this increased incidence is likely multi-factorial and includes co-morbidities and genetic factors. mutations in the human adrenergic receptor gene may play a role in the determination of heart function. mutations in the b -adrenergic receptor (adrb ) located on chromosome have been identified as a cause progressive cardiomyocyte loss leading to a dilated cardiomyopathy. the minor allele frequencies for most of these polymorphisms have been determined for caucasian, japanese, african-american and chinese as part of the hapmap project. the frequencies have not been determined in the pacific islander groups. this study helped determine the hawaiian allele frequency and determine the allele frequency in the presence of cardiac or other vascular co-morbidities such as hypertension, preeclampsia, and gestational diabetes. study design: real time pcr technology (taqman genotyping assays, applied biosystems) was used to screen maternal dna (n= ) from the phenotyping sample core of the pacific center for early human development (prcehd) for the rs single nucleotide polymorphisms (snps). genotype frequencies were also determined in % complete ethnic controls as determined by a four grandparent descent. results: chi square was used to analyze allele and genotype frequencies differences between controls and affected pregnancies. the results are summarized in the following tables. conclusion: the rs snp was not significantly associated with hypertension, preeclampsia or gdm in our overall hawaiian population. the genotype frequency in the % pacific islander group was significantly different from the caucasians (p= . ) and asians (p= . ) in our overall hawaiian population. angiotensin-converting enzyme and -adducin polymorphisms in preeclamptic mothers and fetuses. c mando, p antonazzo, s tabano, f colleoni, a martinelli, s calabrese, c benedetto, l marozio, f facchinetti, m miozzo, i cetin. inst. obstetrics and gynecology, fondaz. irccs policlinico mangiagalli regina elena, univ. milan, milan, italy; dept. biology and genetics, univ. milan, italy; dept.obstetrics and gynecology, univ. torino, italy; univ. modena and reggio emilia, modena, italy. background the genes encoding angiotensin-converting enzyme (ace) and -adducin (add ) share the potential of influencing blood pressure. previous studies demonstrated in humans the association of hypertension with the combined effect of both ace insertion/deletion (i/d) polymorphism, which leads to a different activity of the enzyme, and add g w non-sense single nucleotide polymorphism (snp). ace i-i genotype has been associated with low serum ace activity. controversial studies concerning the association of ace polymorphism with preeclampsia (pe) were reported and the possible combined effect of both ace i/d and add g w polymorphisms yet remains to be investigated. population association study we genotyped polymorphisms (ace i/d and add g w) in women: with pe ( / with severe pe) and controls. moreover, we investigated a subset of their fetuses: from severe pe, from mild pe and controls, in order to identify specific maternal and/or fetal genotypes conferring a higher risk to develop pe. we both evaluated the single and the combined effects of ace and add genotypes on mother-fetus couples and singularly on mothers and fetuses. ace i/d genotype was analyzed using a pcr method; an allelic discrimination approach was performed to detect the add snp. in mother-fetus genotype couples, neither ace nor add polymorphisms are associated with pe, nor are the combination of their genotypes separately in mothers and in fetuses. nevertheless in mothers with mild pe ace i-i genotype is significantly less frequent ( % vs %; p< . ) and ace i-d is significantly more frequent ( % vs %; p< . ) compared to controls. ace i allele frequency is not significantly different in mild pe compared to controls ( % vs %). in women with mild pe the i allele seems to move from i-i to i-d genotype. it leads to the increase in mild pe women of those genotypes with a higher ace activity conferring a higher susceptibility to develop pe. this association with mild pe and not with severe pe could be due to the contribution in the latter of many other genetic and environmental factors. introduction: maternal mrnas stored in the oocyte are critical for early development as transcription ceases upon oocyte maturation, and gene expression until zygotic genome activation (zga) is mediated by translation of maternal transcripts. cytoplasmic polyadenylation element binding protein (cpeb) plays a central role in this process by stabilizing maternal mrnas and regulating their timely activation. this function has been well characterized in xenpous, and a similar role in mammals is suspected as the cpeb knockout mouse displays infertility as a result of arrested oogenesis. in order to investigate if translational regulation of maternal transcripts by cpeb is maintained in mammals, we characterized the spatial and temporal expression of cpeb- in the mouse and human. methods: ten different somatic tissues, testes, and ovaries were tested by rt-pcr for the expression of cpeb mrna in mouse and human. cpeb mrna expression in mouse was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. in human, pi and mii oocytes, -cell embryos and blastocysts were evaluated. sequencing of the pcr products was performed to confirm specific amplification of cpeb. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results and discussion: highest level of cpeb mrna expression was detected in ovaries and testis of both mouse and human. in addition, differential expression of cpeb in somatic tissues was also observed. among these, prominent expression was present in brain, where a role for cpeb in facilitating gene expression through cytoplasmic polyadenylation has been proposed. these data would suggest that translational control of mrna by cpeb may be a mechanism utilized by multiple somatic tissues to regulate gene expression. in the mouse, cpeb was expressed in pi and mii oocytes and -cell and -cell embryos, and became undetectable in -cell or more advanced embryos. human cpeb was expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. zga occurs at late -cell stage and -to- -cell stage, in mouse and human, respectively. the tightly controlled temporal expression of cpeb prior to zga in both mouse and human is consistent with a conserved role for cpeb in the regulation of maternal mrna expression during early development in mammals. background embryonic stem cells (esc) express specific transcription factors that are indicative of their ability to maintain pluripotency. these factors are activated during preimplantation embryo development. the interplay between the transcription factors pou f (oct / ) and caudal-homeobox domain (cdx ) is thought to regulate inner cell mass (icm) and trophectoderm (te) differentiation; however, the mechanism of cell fate determination in mammalian embryos is poorly understood. genetic ablation of oct / in mice prevents icm development, while cdx knockout embryos fail to implant. esc deficient in nanog, a second key regulator of pluripotency, fail to maintain pluripotency and undergo differentiation. expression of nanog in primate embryos has not been investigated, therefore the localization of oct / , nanog and cdx was determined in rhesus macaque blastocysts. methods oocytes were collected from rhesus macaque females, fertilized and cultured in vitro to the blastocyst stage. embryos were collected hours post insemination, then fixed prior to incubation with primary antibodies directed against oct / , nanog and cdx . following incubation with cy conjugated secondary antibodies, nuclei were counterstained with dapi and embryos visualized using an olympus bx fluorescence microscope. results nanog protein was restricted to the icm of monkey blastocysts. unlike the mouse embryo, oct / protein was detected in both the icm and te, based on two different antibodies. the expression of cdx was localized specifically to the te. conclusions the ubiquitous pattern of oct / expression is consistent with observations in human, cow and pig embryos. significantly, lack of restricted oct / protein, and icm localization of nanog in primate blastocysts, suggests that nanog more specifically determines cell fate in primate embryos. these results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of nanog. importantly, this difference may underlie observations that regulatory mechanisms in esc differ between mice and primates. further investigations will focus on determining the onset of marker expression, and the upstream regulators of nanog activation. supported by nih grants r hd r rr , and the intramural research program of nichd. in vitro-conceived mice tend to be smaller at birth and throughout their life. luisa delle piane, annemarie donjacour, francesca di sebastiano, gnanaratnam giritharan, paolo rinaudo. obstetrics, gynecology and reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: epidemiological evidence indicates that ivf is associated with an increased incidence of low birth weight. this phenomenon has not been studied in a mouse model; in addition, it is unknown if the resulting offspring show different growth pattern later in life. we therefore created an ivf mouse model to follow growth patterns till adulthood. methods: we generated experimental groups of b mice: one cohort of in vivo generated mice (in vivo group), one cohort of in vitro generated animals (ivf group) transferred to cd foster mothers and one cohort of animals fertilized in vivo and transferred to cd foster mothers (flushed blastocysts group, fb). the fb group was generated because our preliminary results showed that embryo transfer to b foster mothers was not successful. all pups were delivered at term, measured and weighed at birth and then weekly up to weeks. parametric tests (anova with bonferroni correction) were used as appropriate. a mixed model was used to compare growth curves. results: average litter size was . (in vivo), . (fb) and . (ivf). birth weight of male mice both ivf ( . mg) and fb ( . mg) was larger than male in vivo mice ( . mg) (p< . ). ivf mice tended to be smaller than fb mice in both sexes (p= . ). the bmi (body mass index) was not different among all the groups. male fb growth curves were different from in vivo mice (p< . ) and more importantly from ivf growth curves (p= . ) (fig. ) . conclusion: the method of conception and the maternal environment play a significant role in determining birth weight in this mouse model, emphasizing that the fb group is the best control for the effects of ivf in this model. these preliminary results confirmed for the first time an ivf effect on growth and interestingly the ivf males did not show a catch-up growth. the finding of smaller ivf mice when compared to fb is both noteworthy, because it confirms human data, and worrisome, because lower birth weight is associated with long term sequelae according to the barker hypothesis. the effect of betamethasone exposure in mid-gestation on renal sodium excretion in male sheep. lijun tang, luke carrey, nancy valego, philip deibel, james perrott, jorge figueroa, mark chappell, james c rose. obestetrics and gynecology, wake forest university, medical school, nc, usa; hypertension center, wake forest university, medical school, nc, usa. objective: whereas prenatal exposure of ovine fetuses to clinically relevant doses of glucocorticoids during the time of peak nephrogenesis results in a reduction in nephron number in adulthood, there is little information about its effect on sodium excretion. in the present study, we evaluated the effect of exposure to betamethasone on renal sodium excretion in adult male sheep. methods: we studied nineteen conscious adult rams at . years of age which were exposed to either vehicle or betamethasone at - days gestation. we implanted vascular and bladder catheters and then allowed the animals a - days recovery period prior to study. inulin and para-aminohippuric acid (pah) clearances were performed for estimating glomerular filtration rate (gfr) and renal plasma flow (rpf) respectively. following determination under basal conditions, an acute hypertonic sodium load was administrated intravenously by a continuous infusion of nacl ( . meq/kg/min at . ml/min) for minutes. urine was continuously collected for determination of na + excretion. results: basal gfr was decreased in steroid exposed adults ( . ± . ml/min/kg) compared with the vehicle animals ( . ± . ml/min/kg) (p= . ). rpf was similar in the vehicle and steroid exposed group ( . ± . ml/min/kg vs . ± . ml/min/kg). at basal conditions, na + excretion (u na v) was similar in vehicle and steroid exposed group ( . ± . μmol/h vs . ± . μmol/h). this similarity was also present after normalization by body weight ( . ± . μmol/h/kg vs . ± . μmol/h/kg). the vehicle group excreted . ± . % of the dose of na during the experiment while the betamethasone exposed group excreted only . ± . % (p< . ). gfr and prpf did not change during the experiments. these results suggest that prenatal exposure to glucocorticoids affects renal function in adult male sheep which results in a decreased basal gfr and an attenuated ability to excrete on acute sodium load. the elevated blood pressure previously observed associated with this prenatal steroid treatment may be related to the alteration in renal function. the study is supported by nih grants hd , hl and hd . characterisation of human fetal cardiac stem cells. marah alfakir, nicholas dawe, annette meeson, stephen c robson. school of surgical reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. objectives: for many patients with severe cardiac disease treatment is limited to surgical intervention and/or heart transplantation. the heart has a limited regenerative capacity but it is insufficient to repair extensive damage. cellular therapies might provide an alternative treatment. we have identified stem cells (sp cells) in the adult mouse and human heart and have shown that the mouse cardiac sp cells can differentiate along a cardiac lineage. sp cells can be identified using facs combined with hoechst dye efflux. this ability to efflux hoechst dye is due to expression of abcg (an abc transporter). we have hypothesized that the fetal heart would contain more cardiac stem cells, relative to the adult, and the aim of this project was to determine the spatial and temporal expression of known stem cells markers in the embryonic/fetal heart. methods: human fetal hearts were collected aged between - wk. we used rt-pcr, using primers for several stem cell (abcg , cd , cd ) and cardiac specific (mlc- v, mhc) markers, and ihc on frozen and paraffin sections, using a panel of antibodies (abcg , cd , cd , islet ). cdna was generated from the following regions of the heart at different developmental stages: right and left atrium, right and left ventricle, and outflow tract. results: abcg mrna was highly expressed in all regions of the fetal heart between - wk. expression was down regulated at - wk especially in the la and lv. a similar pattern of expression was observed for cd . cd showed low/moderate expression in all heart regions examined; however, this expression was absent in the lv at - wk. mrna and protein analysis showed similar results. whilst protein expression of abcg was robust at wk (approximately - %), it declined with increasing gestational age. cd was also strongly expressed at weeks of age. there was no co-localisation between abcg and islet . conclusion: abcg and cd are both expressed between - wk of age. based on this analysis, further studies are underway to isolate and characterise both the abcg and cd expressing fetal cardiac cells which might be a potential source of cardiac stem/progenitor cells. xanthine oxidase plays a significant role in the fetal cardiovascular defence to hypoxia. ja hansell, a kane, e herrera, da giussani. physiology, cambridge university, united kingdom. prenatal hypoxia remains a major concern in obstetrics. the fetal defence to hypoxia includes redistribution of the cardiac output, away from peripheral and towards essential ciculations, such as those perfusing the brain (cohn et al . ajog : , ) . the physiology underlying this response is well characterised and involves chemoreflex and endocrine responses (giussani et al. fet mat med rev : , ) . more recently, local factors such as nitric oxide (no) and reactive oxygen species (ros), and the interaction between them, have been shown to play a role. antioxidants, such as vitamin c, scavenge hypoxia-induced ros, maintain no high and thereby depress peripheral constriction (thakor et al., sgi ) . in this study, we address the source of hypoxia-induced ros production and investigated the effects on the in vivo fetal femoral constrictor response to hypoxia of maternal treatment with the xanthine oxidase inhibitor allopurinol in sheep. methods: under anaesthesia, sheep at . gestation, were instrumented with maternal and fetal catheters and a fetal femoral transonic probe. five days later, all animals were subjected to h normoxia, . h hypoxia (mat fio to reduce fetal pao to ca. mmhg) and h recovery, either following maternal i.v. treatment with vehicle or allopurinol in low ( mg.kg - over min) or high ( mg.kg - over min ) doses. treatments finished min prior to hypoxia. the low allopurinol dose was adopted from human studies (benders et al. arch dis child : , ) . the timing of the hypoxic challenge coincided with peak concentrations in fetal sheep plasma of oxypurinol (active metabolite). we evaluated the effect of bpa exposure on uterine gene expression in a nonhuman primate model. method: a total of nine vervet monkeys (cercopithecus aethiops sabaeus) were ovariectomized. three monkeys were treated with bpa via alzet pumps ( microgram/kg/day), two with estradiol benzoate and three received combined treatment with bpa and estradiol. the remaining monkey was untreated and served as a control. following days of treatment the monkeys were hysterectomized and immunohistochemistry performed to examine endometrial gene expression. we evaluated hoxa , proliferating cell nuclear antigen (pcna), and caspase iii gene expression as markers of differentiation, proliferation, and apoptosis respectively. differential expression was evaluated by h-score. results: exposure to a combination of estradiol and bpa resulted in increased expression of hoxa and pcna compared to control (p< . ). lower, but increased, levels of hoxa and pcna gene expression were seen in the specimens exposed solely to estradiol (p< . ). those specimens exposed to bpa alone demonstrated a small induction in hoxa expression (p< . ) with no change in caspase iii or pcna expression when compared to the control. conclusion: bpa induced the expression of hoxa in the uteri of a non-human primate. exposure in the presence of endogenous estrogen further augmented estrogenic stimulation confirming that bpa is a xenoestrogen. bpa's effect of developmental gene expression may alter uterine differentiation. bpa acts as an endocrine disruptor by altering estrogen regulation on a gene required for fertility. contractility and meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, ahmet karadag, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: endogenous glucocorticoids (gcs) increase with advancing fetal age suggesting that gcs function as a hormonal modulator of organ maturation. although fetal colonic motility and meconium passage primarily occur near term, the role of gc in colonic maturation is poorly understood. we sought to examine gc-nuclear receptor (gc-nr) expression in ovine fetal distal colon smooth muscle from very-preterm to term gestation to define the cascade of gc initiated biochemical changes as a prerequisite for maturation-associated meconium passage. methods: ovine fetal distal colonic segments removed at very-preterm (vpt: - d gestation), preterm (pt: - d), near term (nt: - d) and term (t: - d) (n= fetuses in each group) were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochmical analysis with gc-receptor antibody ( : , sc- , santacruz biotechnology, ca) using standard abc regimen. immunoreactive material identified by , 'diaminobenzidine as chromogen. the percentage of gc-nr staining in smooth muscle-enteric unit was analyzed by counting dark brown immunostained nuclei at different fields at x magnification. all values are expressed as mean ± sem. results: the gc-receptor antibody immunostained nuclei both in smooth muscle and enteric units and the observed pattern (expressed as percentage of total nuclei) are as follows: nuclear gc expression varies with gestational age with maximal expression occurring near-term in smooth muscle and enteric neurons, in a synchronized manner. a near total disappearance of gc-nr expression occurs at term. these results suggest that peak gastrointestinal gc-nr mediated effects may occur prior to term with secondary signaling effects resulting in distal colonic motility maturation and meconium passage. the rna-binding protein hud co-localizes with choline acetyl mechanisms of in utero meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, octavio balbuena, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: we have previously speculated that crf, acting through its receptor crf-r , increases gastrointestinal (gi) motility and potentiates in utero meconium passage. patients with paraneoplastic syndrome with auto-antibodies to hud, a neuronal rna binding protein, develop severe gi dysmotility, indicating a role for hud in gi motility. hud binds mrna for actylcholinesterase implying a role in the cholinergic neurotransmitter system. based on these known functions, we hypothesized that hud may regulate post-transcriptional activity in fetal colonic cholinergic neurons expressing crf-r . method: bouin's solution-fixed paraffin-embedded sections of distal colonic segments were prepared from very preterm (vpt: - days), preterm (pt: - days), near term (nt: - days) and term ( - days) ovine fetuses (n= at each age). sections were immunostained with anti-human neuronal protein huc/hud antibodies ( : to : ) with abc reagents and examined at x. neurons positive for hud staining were quantified. double immunofluorescence and laser confocal analyses evaluated co-expression of hud in neurons expressing peripheral choline acetyl transferase (pchat) (a marker for peripheral cholinergic neurons) and crf-r receptor. results: hud immunostaining was seen in either entire cytoplasm (c) or cytoplasm and nuclear regions (c+n) in both submucosal and myenteric neurons. a greater percentage of submucosal (vpt: ± , pt: ± , nt: ± , and t: ± %) than myenteric neurons (vpt: ± , pt: ± , nt: ± , t: ± %) exhibited positive staining at all ages. the percentages of neurons with c+n staining in submucosal neurons were significantly lower at very-preterm gestation. confocal studies co-localized the hud staining with pchat and crf-r receptor immunoreactivity both in submucosal and myenteric neurons. conclusion: in the fetal enteric nervous system hud may function both as a rna-binding protein and as a nuclear cytoplasmic shuttling protein. colocalizaion studies suggest that both pchat-mrna and crf-r mrna species are target transcripts for hud in myenteric neurons. we speculate upregulation of hud contributes to in utero meconium passage. meconium passage during mild stressors. jayaraman lakshmanan, guo l liu, john d richard, sharon k sugano, raina khan, octavio balbuena, kimberly chap, virender rehan, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: mr exhibit a greater affinity to glucocorticoids (gc) than do glucocorticoid receptors (gr). thus low circulating gc levels may preferentially bind mr during mild-stress periods, while the high gc levels may bind gc-receptors. mr antagonism increases gc-mediated responses, suggesting that mrs have a regulatory impact on gc-mediated stress responses. we recently documented that fetal in utero meconium passage is a neurovisceral stress response. to test our hypothesis that mrs play a primary role in mediating suppressive gc effects, we examined the expression patterns of mr in ovine fetal distal colon. methods: bouin's solution fixed paraffin sections of distal colonic segments collected from ovine fetuses (n= for each gestational ages) at very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - days) and term (t: - days) were subjected to immunohistochemistry with polyclonal antibodies to mr. digital photos ( field per colonic ring, rings each gestational age) taken at x were used to count the number of intensely stained neurons, referred to as "mr-capped neurons". differences over time were determined with anova. results: mr antibody elicited a punctate staining pattern in all layers of ovine fetal distal colon. significant immunoreactive intensity was observed in submucosal and myenteric ganglia at all ages: submucosal ganglia: vpt= . ± . , pt= . ± . , nt= . ± . , t= . ± . ; a subpopulation of enteric neurons exhibited dense staining ("mr-capped"). advancing gestation was associated with a significant decreased in the percentage of mrcapped myenteric (vpt = ± , pt = ± , nt = ± , t = ± %; p< . ), though not submucosal ganglia neurons. results indicate a significant decrease in the percentage of mr capped myentric neurons with advancing gestation. in view of the potential inhibitory effect of mr on gc-mediated stress responses, these results suggest that the decrease in mr expression may contribute to near term and term in utero meconium passage. the ambiguity of myometrial progesterone receptor expression in pregnancy and labour. alison j tyson-capper, elizabeth a shiells, stephen c robson. surgical reproductive sciences, institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom. background and aims: in contrast to many other species, human parturition is not due to a reduction in circulating levels of progesterone (p) but appears to be related to changes in expression, ratios or signalling events of p receptors (pr-a and pr-b). the literature on pr expression in human myometrium in pregnancy and labour remains conflicting. we hypothesise this is due, at least in part, to differing specificities of the various pr antibodies employed. we carried out a comprehensive analysis of pr expression using ten 'pr-specific' antibodies that recognise different amino acid epitopes within the pr proteins. two phosphorylation-specific antibodies for pr were also included to define whether phosphorylation status of myometrial pr changes in pregnancy and in labour. methods: western immunoblotting and semi quantitative rt-pcr were undertaken using protein lysates and rna prepared from myometrial tissue from: -first (n= ) and second (n= ) trimesters, paired upper and lower segment myometrium from preterm ( - wks, n = ), term not in labour (n = ), term spontaneous labour (n = ) and non pregnant (n = ). results: using the same set of myometrial lysates for each antibody, we found that the specificity of individual pr antibodies, in particular those raised against internal and c-terminal epitopes of the pr proteins, varied considerably resulting in different patterns of expression. there also appeared to be temporal and spatial differences in levels of myometrial pr proteins ( - kda/ - kda) in pregnancy and in labour with use of the phosphorylation-specific pr antibodies, however, it remains to be resolved which pr isoforms these may be. in contrast, data using four antibodies all of which react with the amino terminal domain of pr consistently indicated that expression of pr, in particular pr-b, decreased significantly at term (p < . ) and in labour (p < . ) when compared to non-pregnant levels. a decrease in levels of pr-b protein was also observed when comparing preterm levels to non-pregnant levels. rt-pcr using primers specific to pr-b consistently indicated that levels of pr-b mrna decreased at term and in labour. conclusion: interpretation of gestation-related changes in myometrial pr expression and phosphorylation status must take into account the pr antibodies used. further studies are now underway to validate the specificity of the pr antibodies. objective to test the hypothesis that expression of fshr in mural gl cells from ivf follicles varies with the infertility diagnosis and correlates with the outcome of ovulation induction (oi). materials and methods women undergoing ivf were classified as: . "no ovarian factor", (tubal or male factor and egg donors, nof;n= ); . endometriosis (endo; n= ); . poor responders (pr; n= ); . polycystic ovary syndrome (pcos; n= ). ovulation induction was carried out using a long or microflare or antagonist protocol based on clinical parameters and gonadotrophin doses were selected based on ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual response. after ultrasound guided egg retrieval, mural gl cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-cd immunobeads to eliminate wbcs, viability was assessed by trypan blue. fshr was measured by rt-pcr as relative expression compared to beta actin. statistical analysis was performed with the spss using pearson´s correlation, one way anova and student´s t-test. results fshr expression in nof ( ± . ) was statistically significantly higher than in pr ( ± . ) and lower than in pcos ( ± . ). both endo ( ± . ) and pr levels of fshr were statistically significantly lower than in pcos. analysis of all cycles showed that fshr expression correlates positively with the number of total (r= , ; p< , ) and mii (r= , ; p< , ) oocytes and negatively with the units of fsh (r=- , ; p< , ) and lh (r=- , ; p< , ) administered for oi. separate analysis of nof showed a positive correlation with the number of total and mii oocytes retrieved and with estradiol levels on day of hcg but not with the total dose of gonadotropins received during oi. fshr expression correlates negatively with day fsh levels in all patients except in pcos (r=- , ; p< , ). conclusions these results suggest that expression of fshr in the hyperstimulated ovarian follicle is "average" in normoresponders, high in high responders (pcos) and low in poor responders (pr and endo). knowledge of the level of expression of fshr might be useful to individualize gonadotrophin doses and oi protocols thus improving pregnancy rates. comparison of intracellular atp levels between non-vitrified and surviving post-vitrification/thawed human oocytes. somjate manipalviratn, zhi-bin tong, eric widra, , alan h decherney. reproductive biology and medicine branch, nichd/nih, bethesda, md, usa; department of obstetrics and gynecology, georgetown university hospital, washington, dc, usa; shady grove reproductive science center, washington, dc, usa. objective: to compare intracellular atp level in non-vitrified and surviving post-vitrification/thawed human oocytes using an atp bioanalysis assay. design: prospective match-controlled laboratory study material and methods: all oocytes were obtained from women undergoing controlled ovarian stimulation for ivf/icsi. oocytes were discarded due to nuclear immaturity at the time of planned icsi. all immature oocytes (gv and mi) were incubated overnight. oocytes from patients with or more discarded eggs which matured to mii stage were used in this study. oocytes from each women were randomly divided into groups. in group , the oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. in group , the oocytes were vitrified using % ethylene glycol, % dimethyl sulphoxide and . m sucrose as cryoprotectant. these oocytes were kept in liquid nitrogen for - days before thawing with a rapid thawing method. oocyte survival was determined by morphological assessment after minutes of incubation then oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. intracellular atp level was determined using luciferin-luciferase bioluminescent assay. result: ninety-one discarded human oocytes were obtained from women. oocytes were vitrified/thawed before measuring for atp content. the other oocytes were measured for their atp content without undergoing vitrification/thawing process. oocyte survival rate after vitrification/thawing is . % ( / ). mean oocyte atp levels in non-vitrified oocytes is significantly higher than surviving post-vitrification/thawed oocytes (table ). coefficient of variation of luciferin-luciferase bioluminescent assay is less than %. conclusion: oocyte atp level in surviving post-vitrification/thawed human oocytes is significantly lower than non-vitrified oocytes. objective to study the expression of genes involved in cell proliferation and steroidogenesis in the human follicle (kl , kl , fshr, papp, p ) and its relationship with ivf outcome. materials and methods patients with different infertility diagnosis underwent ovulation induction with either a long or microflare or antagonist protocol based on clinical parameters; the dose of gonadotrophin used for ovulation induction was selected based on the ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual patient response. ultrasound guided egg retrieval was performed hours after administration of . iu of hcg. mural granulosa-lutein cells (gl cells) were isolated from pooled follicular fluids (ff) from each patient using a percoll gradient and anti-cd immunbeads to eliminate wbcs, viability was assessed by trypan blue. the genes under study were measured by rt-pcr as relative expression compared to actin. statistical analysis was performed with the spss statistical software using pearson´s correlation and mann-whitney u. results kl expression correlates positively with kl levels (r= , ; p< , ) and with genes implicated in granulosa cell function such as fshr (r= , ; p< , ), papp (r= , ; p< , ) and p (r= , ; p< , ). the number of mii oocytes obtained is positively correlated with both fshr (r= , ; p< , ) and papp (r= , ; p< , ) expression, but not with the other genes. kl expression in women who became pregnant during the ivf cycle studied was higher ( , ± ) than in non pregnant women ( , ± , ).(n= , mann-whitney u p< , ). conclusions patients who become pregnant have increased expression of kl , which is associated with optimal granulosa cell proliferation and maturation. this is in accordance with the presence of lower apoptosis in granulosa cells in the same patients. syndrome during assisted reproduction treatment. k jayaprakasan, r jayaprakasan, h al-hasie, js clewes, bk campbell, ir johnson, nj raine-fenning. school of human development, university of nottingham, nottingham, united kingdom. objective: to test the hypothesis that an increased pre-treatment ovarian blood flow is associated with the development of ovarian hyperstimulation syndrome (ohss) and to evaluate ovarian vascularity as a predictor of ohss during invitro fertilization (ivf). methods: subjects undergoing first cycle of ivf had d transvaginal ultrasound in the early follicular phase of the menstrual cycle preceding ivf. of them developed ovarian hyper-response, defined as retrieval of oocytes and ohss. subjects had normal ovarian response with retrieval of to oocytes in the absence of ohss. antral follicle count (afc), ovarian volume (ov), and ovarian vascularity (vascularisation index, vi; flow index, fi and vascularisation flow index, vfi) were measured and an unpaired t-test was used to compare these parameters between the ohss and the control groups. multiple logistic regression analysis was used to assess the predictive value of these variables against age, bmi and basal fsh for the development of ohss. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both the groups. afc and ov were significantly higher (p< . ) in the ohss group ( . ± . and . ± . cm respectively) than in the control group ( . ± . and . ± . cm respectively). afc was the only significant (p= . ) predictor of ohss on multiple regression analysis (table ) . conclusion: women developing ohss during ivf do not demonstrate an increased pre-treatment ovarian blood flow as measured by d ultrasound but do have a significantly higher afc, which is the only significant predictor of ohss. compared to other species, little is known about the steroid biosynthetic capacity and gene expression profiles of human cumulus cells. objective: to examine the ability of human cumulus cells in primary and long-term culture to synthesize steroids and respond to gonadotropin or camp-dependent stimulation. methods: human cumulus cells were isolated from cumulusoocyte complexes during assisted reproductive technology (art) and placed in primary culture or propagated to third passage. at subconfluence, cells were transferred into serum-free conditions in the presence of vehicle, forskolin, fsh, or hcg. at h, media was collected and progesterone (p ) and estradiol (e ) levels were determined by eia. aromatase activity was measured by the tritiated water assay. aromatase (cyp ) and cholesterol side-chain cleavage (cyp a ) mrna abundance was determined by quantitative real-time pcr (qrt-pcr). transcriptional regulation of the cyp and cyp a promoters was investigated by transient transfection of cumulus cells with promoter luciferase constructs. results: substantial amounts of p and e were synthesized by cumulus cells in culture, which were further elevated by forskolin, hcg, or fsh treatment. the presence of cyp and cyp a mrna in cumulus cells was confirmed by qrt-pcr, and the relative mrna abundance of both transcripts was induced by forskolin, hcg, or fsh treatment. changes in cyp and cyp a gene expression in response to camp and gonadotropin stimulation were associated with increased transcriptional regulation of both the cyp and cyp a gene promoters. our studies demonstrate that human cumulus cells are sites of significant p and e biosynthesis and respond to camp-and gonadotropin-stimulation in vitro. the ability to examine human cumulus cells in primary and long-term culture provides a unique model system to investigate cumulus cell function, and the paracrine role of cumulus cells in oocyte development, maturation, and subsequent fertilization. background: there is increasing evidence suggesting that events occurring during fetal development may result in increased predisposition to adult conditions, such as diabetes. in vitro fertilization (ivf) is a new environmental stressor and the long-term effects of these manipulations are currently unknown. there is some evidence that lower number of insulin-secreting cells at birth signals predisposition to diabetes later in life. in this study, we compare areas of pancreatic insulin-secreting cells in newborn mice conceived in vivo versus in vitro. methods: oocytes were collected from super ovulated cf- mice and fertilized in vitro with cauda epididymal sperm from b d f /j mice. fertilized eggs were cultured in whitten medium under % co in humidified air at °c for h. blastocysts were transferred to the uteri of pseudo-pregnant recipients. control mice were allowed to conceive in vivo. the newborn animals were sacrificed within hours of birth. pancreases were sectioned, immunostained with anti-insulin, and total areas and stained areas were compared using t-test with two-tailed distribution. p value of < . was considered significant. results: there were total of newborn animals: females ( ivf, controls) and males ( ivf, controls). percentage of total pancreatic area occupied by insulin-secreting cells was lower in female ivf ( . mm , . %) animals compared to female controls ( . mm , . %) and higher in male ivf ( . mm , . %) animals compared to controls ( . mm , . %). the average of males and females was slightly lower for ivf ( . mm , . %) than controls ( . mm , . %). none of these differences were statistically significant. there was a trend in newborn female mice towards lower amount of insulin-secreting cells in the ivf offspring, suggesting possible predisposition to diabetes later in life. this effect was not observed in newborn males. while our study failed to demonstrate consistent and significant differences in the areas of insulin-secreting cells between newborn mice conceived in vitro and in vivo, the number of animals in the study may have been too small to show significant results. larger studies are needed to further investigate this question. peter s uzelac, phyllis risch, kassi shelton, steven t nakajima. obstetrics and gynecology, university of louisville, louisville, ky, usa. objective: several fertility preservation methods currently available may not be viable options to certain patients due to their high cost and limited number of centers offering these services. for women undergoing bilateral oophorectomy, in vitro maturation (ivm) of oocytes retrieved from unstimulated whole ovary specimens may represent a more practical solution. here we describe out initial experience in offering ivm as a fertility-preserving measure to two patients undergoing total abdominal hysterectomy and bilateral salpingo-oophoectomy (tah-bso). case one was a year old nulligravid with chronic pelvic inflammatory disease unresponsive to medical management. case two was a year old nulligravid with stage ia endometrial carcinoma. surgery was planned for the mid-follicular phase in order to collect prophase i oocytes before the onset of late-follicular phase atresia. upon surgical removal, suction aspiration of all identifiable follicles was performed. ovaries were then serially sectioned and all tissue was examined for the presence of prophase i oocytes. results: total number of prophase i oocytes retrieved was and , maturation rate after hours was % ( / ) and % ( / ), fertilization rate after hours was % ( / ) and % ( / ), maturation rate after hours was % ( / ) and % ( / ) and fertilization rates after hours % ( / ) and % ( / ), for case and case respectively. ivm of oocytes retrieved from unstimulated whole ovary specimens may be a simple and inexpensive approach to fertility preservation in women undergoing bilateral oophorectomy. by requiring minimal adjustments to in vitro fertilization protocols, this treatment could easily be implemented in centers which currently have no fertility preservation program. patient age at time of retrieval may be predictive of developmental potential. continued patient enrollment and follow-up studies on the developmental potential of embryos derived from this technique are necessary to fully evaluate its potential for a role in fertility preservation. the routine use of progesterone as luteal phase support in women with a diagnosis of polycystic ovary syndrome (pcos) undergoing ovulation induction cycles with oral agents has not been fully elucidated. we hypothesize that women with pcos utilizing either clomiphene citrate or letrozole, an aromatase inhibitor, should administer intravaginal progesterone in the luteal phase to increase pregnancy rates. design: retrospective chart review. materials and methods: cycle data from women with pcos undergoing ovulation induction with clomiphene citrate or letrozole at the university of cincinnati medical center from to were evaluated. diagnosis of pcos was based on rotterdam criteria and all other infertility diagnoses were excluded. clinical pregnancy rates (presence of fetal cardiac activity on ultrasound at - weeks gestation) in women who received intravaginal micronized progesterone ( mg bid) following ovulation induction (with and without intrauterine insemination) were compared to those who did not receive progesterone. results: no significant differences were noted in demographic parameters, including patient age or bmi. a total of cycles were evaluated in women treated with clomiphene citrate. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to . % ( / ) of the non-progesterone group (p < . ). forty-three cycles were evaluated in patients treated with letrozole. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to none ( / ) in the non-progesterone group (p < . ). conclusions: patients with pcos who used letrozole for ovulation induction had superior clinical pregnancy rates when using intravaginal micronized progesterone compared to women who did not receive luteal phase support. there was a trend toward increased clinical pregnancy rates in pcos patients utilizing luteal support following clomiphene citrate for ovulation induction. therefore, in women with pcos undergoing ovulation induction with oral agents, luteal supplementation with progesterone should be strongly considered, especially in those using letrozole. were measured and an unpaired t-test was used to compare these parameters between poor and normal responders. multiple logistic regression analysis was used to assess the predictive value of these variables against age and basal fsh for poor ovarian response. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both poor and normal responders. afc and ov were significantly lower (p< . ) in poor responders ( ± . and . ± . cm respectively) than in normal responders ( . ± . and . ± . cm respectively). afc was the best (p< . ) predictor of poor ovarian response on multiple regression analysis ( objective: gay male couples increasingly seek parenthood through in vitro fertilization using an oocyte donor and a gestational carrier, but no studies describe this unique experience. the purpose of this study was to determine medical and psychosocial issues unique to gay men using art. design: qualitative analysis of semi-structured interviews with gay male couples seeking parenthood through art. materials and methods: sixteen gay males (eight couples) entering an art program were assessed through the use of a semi-structured interview. characteristics evaluated included age, relationship status, duration of their relationship, psychological health and stability, how the decision was made concerning who would donate the sperm, the decision to use an anonymous or known oocyte donor, and whether their gestational carrier was someone previously known to them or not. results: the average age of the men in this study was years. all eight couples were in a committed relationship and had been together for an average of . years. five of the couples ( %) had been joined in a civil union which is legal in the state of connecticut. all subjects were psychologically stable and in good health. six couples ( %) were very clear about which partner would inseminate the oocytes. of those couples, two felt that the older partner should donate; two felt that the partner who cared more about a genetic connection to the child should donate; and two felt that the partner with "better genes" should donate. the remaining two couples chose to inseminate equal numbers of oocytes in order to transfer an embryo from each partner. all couples chose an anonymous oocyte donor, two couples chose relatives as their gestational carriers, while the others chose carriers recruited through an agency. conclusions: participants in this study were determined to become parents through assisted reproduction. they had given thoughtful consideration to the medical and psychosocial issues unique to this process including which partner would be the genetic parent, and why. clomiphene superovulation. rb allen, az steiner, rh fogle, mj kalan, rj paulson. ob/gyn, usc keck school of medicine, la, ca, usa; ob/gyn, unc, chapel hill, nc, usa. intro: micro-dose hcg has been demonstrated to increase ovulation and pregnancy rates following clomiphene administration in women resistant to clomiphene. since micro-dose hcg stimulates growth in follicles expressing the lh receptor, we hypothesized that its use following clomiphene in women with unexplained infertility would increase the number of follicles that ovulate and subsequently, pregnancy rates. m m: irb approval was obtained for this prospective pilot study of women with unexplained infertility. on day # a baseline ultrasound was performed and serum fsh and e levels were measured. subjects were given mg of clomiphene daily from days - and then returned for serial ultrasounds on day # . when at least follicles mm were present, iu of hcg im daily was initiated. cycles were monitored by ultrasound every days until a +lh surge occurred. all subjects had previously undergone a cycle using clomiphene alone for superovulation followed by iui and these cycles were used for comparison. results: subjects, aged . ± . yrs (mean±sem) and bmi . ± . kg/m with . ± . yrs of infertility were enrolled. the mean fsh and e levels were . ± . miu/ml and . ± . pg/ml, respectively. there was an average of . ± . days from starting clomiphene to the attainment of follicles mm. out of the subjects had follicles mm by day # . a mean of . ± . days of treatment were required until ovulation occurred. there were twice as many follicles in the micro-dose hcg cycles, although due to the small sample size this did not reach statistical significance ( . ± . follicles, measuring . ± . mm in the micro-dose hcg cycles, compared to . ± . (p= . ), measuring . ± . mm (p= . ) in the control cycles). there was no difference in the endometrial thickness at the time of ovulation between the micro-dose hcg cycles and the control cycles: . ± . mm vs. . ± . mm (p= . ), respectively. all subjects had at least ovulatory sized follicles in the study cycles. % of subjects had an iui performed, however there were no pregnancies resulting from this study. conclusions: ) the addition of micro-dose hcg to clomiphene may allow more follicles to reach an ovulatory size, which should theoretically increase pregnancy rates ) this pilot study demonstrates that adding micro-dose hcg may increase the effectiveness of clomiphene when given in a superovulatory protocol. jessica salas, donald maier, john nulsen, claudio benadiva, lawrence engmann. obstetrics gynecology, university of connecticut, farmington, ct, usa. objective: to evaluate the antepartum, intrapartum, and neonatal complications in patients undergoing ivf using a gnrh-antagonist protocol where a gnrhagonist was used to induce final oocyte maturation. materials and methods: a retrospective review of data from high responders undergoing their st or nd ivf cycle using a gnrh-antagonist protocol who were triggered with leuprolide acetate (study group) or hcg (control) and achieved at least a singleton pregnancy reaching the third trimester. patients younger than years old were included. both groups received luteal phase support with intramuscular progesterone. the study group received estrogen patch supplementation. outcomes measured were antenatal and intrapartum complications, order of gestation, gestational age at delivery, birth weight, neonatal adverse outcomes, and congenital anomalies. pearson chi square, fisher's exact test, or independent t-test were used as appropriate. results: the baseline characteristics were different (table ) . maternal antenatal and intrapartum complications were similar in both groups ( . % vs . %, p= . ). there were more singletons in the study group ( . % vs . %, p< . ). a subgroup analysis of gestational age at delivery, birth weight, neonatal complications and congenital anomalies in singletons showed no difference (table ) . conclusions: this is the first study reporting the maternal and perinatal outcomes after gnrh-agonist trigger. differences in response to ovarian stimulation may dictate use of gnrh-agonist triggering for prevention of ohss, which may explain the differences in baseline characteristics. maternal and neonatal complications remain unaffected. table lupron (n= ) objective: moderate to severe ovarian hyperstimulation syndrome (ohss) occurs in - % of assisted technology cycles and carries the potential for severe complications. traditional management consists of hospitalization with intravenous fluids (ivf), bedrest, and close monitoring. early and aggressive paracentesis is an alternative method of treatment for ohss in an outpatient setting, but the economic consequences of the two treatment regimens have not been compared. design: cost-effectiveness analysis materials and methods: two scenarios, outpatient management with transvaginal paracentesis and conservative therapy with hospitalization, were compared. potential initial outcomes were analyzed for the conservative group to include hospitalization either in a ward hospital bed or the intensive care unit (icu) for an average of seven days. costs included ivf administration, ultrasound, and daily bloodwork. initial outcomes for the outpatient management group included no further therapy beyond the initial transvaginal paracentesis, bloodwork, ivfs, and ultrasound versus admission for an average of three days to a ward bed with similar management. the probability of the negative outcome for the conservative group was set at % (icu admission) and % for the outpatient group (regular hospitalization). results: the cost of conservative therapy including first tier complications ranged from a low of $ , to a high of $ , . the cost of outpatient management with aggressive paracentesis and its first tier complications ranged from a low of $ to a high of $ , . this resulted in an estimated cost burden of $ , to $ , for conservative management with hospitalization. the main improvement factor was that patients in the outpatient management group had a much lower likelihood for prolonged hospitalization than the conservatively managed group. conclusions: aggressive outpatient treatment of moderate to severe ohss with early paracentesis appears to be a cost-effective strategy. induction. zeynep alpay, michael p diamond, michael l kruger, elizabeth e puscheck. obstetrics and gynecology, division of reproductive endocrinology and infertility, hutzel hospital, wayne state university, detroit, mi, usa. introduction: aromatase inhibitors (ai) are a new method of ovulation induction and is proposed to replace clomiphene citrate (cc) due to their reported advantages. their superior effect is thought to be due to up-regulation of the estrogen receptors and increase in the sensitivity of the endometrium, resulting in better proliferation despite low estrogen levels in the circulation. objective: to compare the effect of different ovulation induction methods, including ai, cc and gonadotropins (gt) on the endometrium in infertility patients. methods: we reviewed ovulation induction cycles performed in our institution in the last one-year period retrospectively. there were gt, cc and ai cycles. age, gravida, day (d et) and midcycle (mcet) endometrial thickness, endometrial pattern (ep) on day of hcg injection, endometrial growth during the induction cycle (d-et), which was calculated by the difference between mcet and d et, and the pregnancy rates (pr) were compared. the ep was categorized as type a (homogenous and hyperechogenic), type b (intermediate isoechogenic pattern and a poorly defined central echogenic line), and type c (multilayered triple-line). chi-square, anova, t test and pearson correlation were used for statistical analysis. results: in all cycles, ep was closely related to the d-et (p < . ). this correlation appeared to be more pronounced when observed ep was compared with d-et (r = . ; p < . ) rather than with mcet (r = . ; p = . ). there was a reverse relationship between the age of the patients and ep (r = - . ; p < . ). a negative correlation was also found between the gravida and ep (r = - . ; p < . ). pregnancy rate was significantly correlated with ep (r = . ; p < . ). pregnancy rate was % in type a ep, . % in b, % in c when all cycles were analyzed. aromatase inhibitors and gt treatments each resulted in % type c pattern in contrast to % with cc (p = . for ai vs. cc; p = . for gt vs. cc). the d-et was greater in gt cycles compared with ai or cc (p < . ). conclusion: these results show that ep has a strong correlation with pr in ovulation induction cycles. additionally, ai and gt treatments have similar effects on ep. both ep and the growth of the endometrium during the induction (d-et) are good prognostic variables for the successful pregnancy initiation. ovarian response in patients undergoing ovarian stimulation after myomectomy. hyacinth browne, , desiree mccarthy-keith, , barbara stegmann, , alicia armstrong. , reproductive biology and medicine branch, nichd, nih, bethesda, md, usa; reproductive endocrinology and infertility, walter reed army medical center, washington, dc, usa. objective: the impact of myomectomy on ovarian function has not been wellstudied. other surgical treatments of fibroids, such as hysterectomy and uterine artery embolization, have shown an increase of fsh into the peri-menopausal range. the objective of this study is to examine ovarian response in infertile women undergoing ovarian stimulation after abdominal myomectomy. design: retrospective analysis. materials and methods: a retrospective analysis of all infertile women with known fibroids who had a failed art cycle, from january to , followed by an abdominal myomectomy and a subsequent art cycle was performed. women served as their own controls. ovarian function pre and post-myomectomy was assessed by age, day and fsh levels, days of stimulation, total gonadotropins used, peak estradiol level, number of oocytes retrieved, embryos obtained, and high-grade embryos, and pregnancy outcome. quantitative results are presented as mean + sd. results: four women had a failed art cycle and underwent an abdominal myomectomy prior to a subsequent art cycle. the mean age was and pre-and post-myomectomy, respectively. all subjects had uterine factor infertility. two of these women also had tubal factor infertility, and one had endometriosis and male factor infertility. we found no difference in ovarian response pre and post-myomectomy. pre-myomectomy post-myomectomy age (years) + . + . day fsh (u/l) . + . . + . day fsh (u/l) . + . . + . objective: anti-mullerian hormone ( amh) is produced by developing primordial follicles. amh levels are stable through the menstrual cycle and therefore can be drawn randomly. the objective of this study was to assess the association of anti-mullerian hormone ( amh) and oocytes obtained at ivf retrieval. design: cross-sectional cohort study. materials and methods: the study cohort is comprised of thirty women in cycles of in vitro fertiization (ivf). serum levels antimullerian hormone (amh) were drawn before starting an ovulation induction cycle for ivf. standard ovulation induction was performed with leuprolide flare and miu/ml per day of follicle stimulating hormone. we performed linear regression of log convert d oocyte number against the log converted amh level. serum amh was measured using an enzymatically amplified two-site immunoassay dsl- - active mis/amh elisa. statistical analysis was performed using spss version . . continuous values are presented as mean std error. results: the log number of oocytes retrieved was directly related to the log value of amh (p = . ). conclusions: our data demonstrate an association between serum amh and oocytes produced in response to ovulation induction. using amh levels in addition to fsh levels in evaluating for ovarian reserve and pregnancy outcome may help improve predictions of reproductive performance. support: foundation for reproductive medicine. context: prediction of outcome after in vitro fertilization (ivf) can be difficult due multiple factors. human chorionic gonadotropin (hcg) levels correlate with pregnancy outcome, but data that can be used to easily counsel patients on their possible outcome is lacking. objective: to investigate the use of hcg levels along with other significant factors to predict the likelihood of an ivf pregnancy progressing to the point of detection of cardiac activity by ultrasound design: retrospective data analysis of ivf cycles performed from january to july resulting in pregnancies. multiple logistic regression analysis modeling was performed to determine the factors most predictive of an ongoing early pregnancy and to assess possible confounding variables. setting: an academic fertility center. patients: patients undergoing in vitro fertilization using autologous fresh embryos. intervention: none main outcome measure: pregnancy continuation to the documentation of cardiac activity by ultrasound. results: maternal age, day (post-oocyte aspiration) hcg level, and day hcg levels were significant in predicting pregnancy outcome. day and day hcg levels were highly correlated and can be considered proxies for each other. the most accurate predictive model used only a single day hcg level and maternal age. the type of fertilization method used, the cycle number for that patient, and the number of embryos transferred were not found to be significantly different. ongoing pregnancy rates were directly proportional to day hcg level, and inversely proportional to maternal age. the incidence of multiple pregnancies also increased proportionally to the initial hcg level. % of ongoing pregnancies had hcg level > miu/ml. conclusions: a single day post-oocyte aspiration hcg level and maternal age are the most predictive of an ongoing ivf pregnancy. there was no difference in outcome between fertilization methods or number of embryos transferred. there is no benefit to obtaining serial hcg levels after the initial one. erk activation with u ( m)) reduced fsh stimulated cyclin d mrna expression by %. fsh has also been shown to stimulate mtor, a regulator of growth and proliferation of many cell types. inhibiting mtor activation with nm rapamycin for min significantly reduced fsh-mediated cyclin d mrna expression. dht exposure for hours also inhibited fsh stimulated mtor signaling, as shown by a % reduction in the phosphorylation of its down stream target p s kinase. furthermore, pretreatment with dht resulted in significantly reduced fsh-mediated tsc- phosphorylation, which is an upstream regulator of mtor pathway. further studies revealed that fsh mediated tsc- phosphorylation and mtor signaling is regulated by erk, but independent of akt. based on these results we conclude that elevated reduced metabolites of androgens inhibit fsh-stimulated pka-erk pathway resulting in the inhibition of multiple mitogenic signaling pathways leading to defective follicle maturation culminating in anovulation. thus, rescuing the erk activation might serve as a potential therapeutic target to restore normal granulosa cell proliferation in hyperandrogenic states. (supported by nih grant hd- ). searching pcos-genes: results of a genome screen for pcos in a dutch founder population. olivier valkenburg, annemarie g mulders, aida bertoli-avella, ben a oostra, joop se laven. department of gynecology and obstetrics, erasmusmc, rotterdam, netherlands; department of internal medicine, erasmusmc, rotterdam, netherlands. context: although the etiology of pcos is not yet fully understood, evidence has accumulated for a complex genetic background i.e. a combination of multiple genetic and environmental factors. to reduce genetic heterogeneity, this study was conducted in a genetically isolated population in the netherlands . objective: in order to identify genomic loci that are associated with pcos, a whole genome screen using highly polymorphic microsatellite markers was performed in a founder population. subjects and methods: pcos patients ( rotterdam criteria) were identified in the founder population (rucphen, the netherlands) of ± . inhabitants. patients underwent a standardized screening procedure that included clinical, ultrasound and endocrine evaluation. all patients plus unaffected first-degree family members were genotyped using polymorphic markers on (microsatellites) from the abi prism ® linkage mapping set md- (average spacing cm). association-analysis was performed with the transmission disequilibrium test (tdt, genehunter software). linkage analysis was performed in clusters of or more closely related patients (simwalk ). results: there was an average number of . alleles per polymorphic marker. the tdt identified two non-adjacent markers on chrome (d s and d s ) that showed significant association with pcos (p , ). other markers that showed significant association were positioned on chromosomes (d s ), (d s ), (d s ) , (d s ), (d s ) and seventeen (d s ) (all p values . ). linkage analysis in sub-pedigrees revealed no significant linkage for these, or other, loci with pcos. moreover the results of a prior report of linkage of a marker on chromosome (d s ) could not be confirmed. conclusions: the lack of consistent results suggests the absence of a consistent genetic background in this select group of pcos patients. this supports the hypothesis of a complex genetic background for pcos that allows relatively small contributions of multiple risk-genes to be involved in the pathogenesis of this syndrome. in this founder-population the genetic heterogeneity was not sufficiently reduced to find risk-loci in a genome wide screen using highly polymorphic markers with an average spacing of cm. objectives: to objectively quantify uterine and endometrial blood flow in women with polycystic ovarian syndrome (pcos) and to examine if this was different in women with different phenotypic expressions of the disease. methods: transvaginal d and d ultrasound was performed in women with pcos, as defined by the rotterdam criteria, and sub-group analysis conducted based on the subjects' body mass index (bmi), ovulation status, and hirsutism score. anova was used to compare the mean values between the groups. results: pcos women with clinical hyperandrogenaemia had significantly lower endometrial and subendometrial blood flow than their anovulatory normoandrogenic counterparts (table ). there were no differences between lean and obese women or between anovulatory and ovulatory women with pcos. the pulsed wave doppler parameters were similar in all three phenotypic groups. conclusions: hirsute women with pcos have impaired endometrial perfusion compared to their normoandrogenic counterparts which is only evident with d ultrasound and not conventional pulsed wave doppler. nusayba a bagegni, jill blaine, anuja dokras. obstetrics gynecology, university of iowa hospitals clinics, iowa city, ia, usa; obstetrics gynecology, university of pennsylvania, philadelphia, pa, usa. background: there is conflicting evidence on the association between pcos and early and late obstetric complications. it is unclear if the reported risks are independent of bmi, preexisting hypertension and diabetes. we examined the risk of early and late obstetrical complications in a large group of women with pcos compared to controls. methods: we reviewed pregnancy records of women with pcos (rotterdam criteria, n= ) and controls (tubal infertility, n= ) after in vitro fertilization at university of iowa from - . the wilcoxon rank sum test and fisher's exact test were used to evaluate differences between variables and logistic regression analysis was used to determine the independent risk of pcos. results: subject demographics and medical history are shown in table. the first trimester miscarriage rate was % in women with pcos and % in controls. after logistic regression analysis pcos was not associated with miscarriage (p= . ). the prevalence of gestational dm (gdm) was similar in both groups % pcos vs % controls. pcos was not associated with gdm after adjusting for age and bmi (p= . ). however, bmi was significantly associated with gdm after adjusting for age and pcos (p= . ). risk of both pre-eclampsia and pih was % in pcos and % in controls, but not statistically significant after adjusting for age, bmi and twin gestation. preexisting htn showed a significant association with preeclampsia (p< . ). there was no significant difference in preterm delivery, cesarean section, twin gestation, intrauterine fetal death and intrauterine growth restriction in the groups. conclusion: despite adequate power, our study did not detect an increased risk of miscarriage in women with pcos. obesity was a significant contributor to late obstetric complications, namely gdm. these findings may warrant aggressive counseling of women with pcos on the potential benefits of weight loss prior to pregnancy. objective: to assess the prevalence of polycystic appearing ovaries (pcao) as defined by the rotterdam criteria; and to determine whether metabolic parameters differ between regularly cycling women with pcao and those with normal ovaries. studies have demonstrated a population frequency of pcao of - %, with - % among women with regular cycles. most were performed in a young reproductive age population; none examined the impact of age. all were performed prior to adoption of the rotterdam criteria. recent studies have shown an increased prevalence of metabolic syndrome among women diagnosed with polycystic ovarian syndrome (pcos). however studies focused on women in their s found no increase in bmi or fasting insulin with pcao but regular menses. participants: women aged - with regular cycles (every - days) enrolled in the ova study, a population-based study of ovarian aging. each subject underwent a transvaginal ultrasound for assessment of ovarian volume and antral follicle count (afc). outcomes collected included waist measurements and hdl, ldl, total cholesterol, triglycerides, fasting glucose and insulin. pcao were determined by the rotterdam criteria (afc of on one ovary or ovarian volume > cc). student's t-test and chi-square tests were used to assess differences between those with and without pcao for continuous and categorical variables, respectively. the prevalence of pcao decreased with increasing age (table ) . of the women with pcao, % met the afc criterion only, while % met the volume criterion only, and % met both. women with and without pcao did not differ in waist measurements, or in fasting lipids, insulin, or glucose. the rotterdam criteria, while less subjective than those described by adams in , have led to an increased prevalence of pcao among women with regular menses, over / of women in their s. women with pcao do not differ from those with normal ovaries in metabolic parameters associated with pcos. consideration should be given to adopting an age-adjusted criterion for afc, or a combination of afc and ovarian volume, for diagnosing pcos. background: pcos, especially accompanied by obesity, has been reported to be associated with a characteristic dyslipidemia comprising elevated triglycerides (tgs) and depressed hdl, especially the hdl- fraction. this is an atherogenic profile; in fact, studies suggest low hdl- may correlate most strongly with cardiovascular disease risk. weight loss is a mainstay of treatment and improves all manifestations of the disease, but the optimal diet to recommend remains undetermined. preliminary studies show a high protein diet may improve total hdl levels and insulin responses. however, the effect of weight loss and dietary composition on hdl- levels in pcos has not been investigated. objective: to evaluate the fasting lipid profile in newly diagnosed obese pcos patients and to determine the effects of a high-protein diet with or without metformin on weight loss, hdl- and other lipoproteins, and menstrual cyclicity. methods: in this pilot retrospective observational study, the fasting lipid profile of obese women newly diagnosed with pcos was determined. they were then placed on a high protein ( - g/day), low carbohydrate ( - g/day) diet with or without metformin ( and %, respectively) and followed monthly for an average of months (range - ). results: at diagnosis, % had low hdl and % had low hdl- ; only % had elevated tgs. on the diet, the patients demonstrated an average weight loss of . lbs ( . to . , p< . ) and decreased bmi of . kg/m ( . to . , p< . ). hdl levels increased significantly ( % increase from . to . , p< . ), especially the hdl- fraction ( % increase from . to . , p< . ). triglycerides decreased as well ( . to . , p< . ). ldl decreased but did not reach statistical significance. resumed menstrual cycles, were started on oral contraceptives, and had a hysterectomy. pregnancies occurred. no difference was seen with metformin use. conclusion: a majority demonstrated decreased hdl and hdl- at diagnosis. the high protein diet resulted in significant weight loss and improvement in hdl- levels, as well as improvements in total hdl, tgs and menstrual cyclicity. metformin produced no added benefit. prospective trials based on these data will help determine the optimal diet to reduce the significant short-and long-term morbidity in the large population of women with pcos. resistin is an adipokine that has been associated with obesity and insulin resistance in animal models. studies on the role of resistin on insulin resistance in humans have been controversial. recently resistin has been shown to exert atherosclerotic effects and elevated resistin levels have been observed in women with coronary heart disease (chd). women with polycystic ovary syndrome (pcos) are at high risk for chd. our present study investigates potential association of resistin and markers of inflammation, c-reactive protein (crp) and insulin resistance in women with pcos. methods: thirty two women with pcos participated in the study. all were hirsute and had irregular cycles. nineteen women with normal ovulatory cycles who matched the pcos patients in bmi served as controls. fasting glucose, insulin, resistin and crp levels were measured in all women. after a high carbohydrate diet for days, a standard oral glucose tolerance test (ogtt) was performed. blood samples were collected for glucose and insulin before and , and hrs after g oral glucose. the area under the curve (auc) for insulin and glucose was calculated. women with overt diabetes were excluded from the study. results: fasting insulin levels ( . + . μu [±se] /ml) and insulin response to oral glucose (auc) ( . + . μu/ml) were higher (p < . ) in women with pcos compared to controls. all were insulin resistant with homa-ir value > . mol x μu / l . resistin levels in women with pcos ( . ± . ng/ml) was significantly (p < . ) higher compared to control women ( . ± . ng/ml). crp levels in women with pcos ( . ± ng/ml) was also significantly (p < . ) higher than the controls ( . ± ng/ml). there was a significant positive correlation between resistin and crp levels (r = . , p < . ). there was no correlation between resistin levels and fasting insulin levels or insulin auc. there was also no correlation between resistin levels and fasting glucose or glucose auc. conclusions: our results indicate that ( )women with pcos and insulin resistance have increased resistin levels, ( ) there is a strong association between resistin and crp ( ) elevated resistin and crp levels may predict women who are at increased risk for chd ( ) ) it is unlikely that resistin plays a major role on insulin resistance in women with pcos. vuk p jovanovic, enrico carmina, prati vardhana, michel ferin, rogerio a lobo. department of obstetrics and gynecology, columbia university, new york, ny, usa; department of internal medicine, university of palermo, palermo, italy. current diagnostic criteria for pcos includes both ovulatory (ov) and anovulatory or "classic" (c) phenotypes. in an effort to further characterize differences and /or similarities between these phenotypes, we studied hyperandrogenic women with pcos (age . ± . , bmi . ± . ) and age matched controls (age . ± . , bmi . ± . ). women with pcos were divided into weight matched (ov) n= and (c) n= groups. fat and weight distribution were assessed by dexa (total fat, r fat, trunk fat) as well as fasting levels of lh, e , mis/amh, kisspeptin and testosterone, the adipocytokines (leptin, adiponectin, visfatin and retinol-binding-protein- rbp ) and serum glucose, insulin and crp. the hyperandrogenic pcos groups had characteristically altered hormone profiles compared to matched controls. although total fat mass was comparable, women with c-pcos had a significantly larger waist circumference ( . . vs. cm, p< . ) trunk fat, r fat and %trunk and %r fat compared to ov-pcos (p< . ). leptin, rbp and visfatin did not significantly differ among the pcos subgroups although adiponectin was lower in the c-pcos group (p< . ). quicki was significantly lower in c-pcos ( . ± . vs. . ± . , p< . ) and insulin was higher ( . ± . vs. . ± . , p< . ). serum lh was also higher ( . ± . vs. . ± . , p< . ) but kisspeptin, testosterone and estradiol were similar. mis/amh ( . ± . vs. . ± . ng/ml, not significant) and crp ( . ± . vs. . ± . , p< . ) were higher in c-pcos. significant correlations (p< . ) were noted among kisspeptin/rbp (r . ), mis/testosterone (r . ), insulin/ %trunk fat (r . , p< , ), total fat/leptin (r . , p< . ), %trunk fat/adiponectin (r - . ). in conclusion, women with c-pcos when compared to similarly hyperandrogenic women with ov-pcos with similar bmi, have increased abdominal fat, and appear to have differences in serum lh, crp and increased insulin resistance. the latter, as well as subtle differences in the adipocytokines may explain these differences in anthropometric findings. problems of normal oogenesis and folliculogenesis but also disturbed oogenesis and folliculogenesis in polycystic ovaries are not fully understood. oocyte specific genes play an essential role in oogenesis and folliculogenesis. there are suggestions about possible role of some oocyte specific genes in etiopathophysiology of pcos. zona pellucida gene (zp ) is recently identified gene, which belongs to zona pellucida genes such as zp , zp and zp . the role of zp , contrary to above-mentioned other zp genes is not well described. the aim of this study was to analyze zp coding sequence and expression in patients with polycystic ovary syndrome. material included blood received from patients (mean age , +/- , years; mean bmi , +/- , kg/m ) with polycystic ovary syndrome. all patients with pcos were diagnosed with the use of eshre/asrm criteria from . dna was isolated from blood cells( after separation of blood cells from serum) using a dna isolation kit (qiagen ). genomic dna was used for in vitro amplification by pcr with a specific set of primers complementary to the coding sequence of the zp gene. products from each pcr reaction were examined by sscp method. samples with changes detected by sscp in comparison to control probes were cloned into plasmid vector and then automatically sequenced from a total of patient samples with pcos, we identified nucleotide changes in the zp coding seguence : silent nucleotide changes in exons , , , , and nucleotide change in the exon ( position , t>g). the mutation in exon ( t>g ) results in substitution of cystein for glycin of amino acid in position of zp protein. in summary, our data demonstrate that zp nucleotide changes account for % of patients with pcos. relationship between serum mullerian inhibiting substance levels and insulin in women with polycystic ovary syndrome. rebecca a chilvers, shilla chakrabarthy, summer james, xin ma, manubai nagamani. ob/gyn, university of texas medical branch, galveston, tx, usa. müllerian-inhibiting substance (mis) is a member of the transforming growth factor-superfamily of growth factors. it is expressed exclusively in granulosa cells and is believed to play a role in the regulation of follicle selection and maturation. women with pcos have anovulation and most of them have hyperinsulinemia. the purpose of our study is to investigate possible association between insulin and mis in the dysregulation of folliculogenesis in pcos. methods: twenty one women with pcos who had anovulatory cycles and hyperandrogenism were recruited for the study. sixteen women with ovulatory cycles and matched the pcos patients in age and bmi served as controls. an oral glucose tolerance test (ogtt) was performed in all women. blood samples were obtained at fasting and , and hours after glucose ingestion for measurement of glucose and insulin. to investigate the effect of hyperinsulinemia on mis secretion, mis levels were measured in ten patients during the ogtt. fasting mis, testosterone, dheas, fsh, and lh levels were measured in all patients. results: fasting insulin levels ( . + . μu/ml) vs . + μ u/ml [+ se]) (p < . ) and area under the curve (auc) of insulin ( . + . vs . + . μu/ml) (p < . ) were significantly increased in women with pcos compared to control women, while the glucose levels were normal indicating insulin resistance. mis levels were significantly (p < . ) increased in women with pcos ( . + . ng/ml) compared to controls ( . + . ng/ml). there was no correlation between age and mis levels in pcos patients while there was a highly significant negative correlation between the age and mis levels in the control women ( r = - . , p< . ). there was significant negative correlation between mis and fasting insulin levels (r = - . , p < . ) and insulin auc during the ogtt (r = - . , p < . ). there were no changes in mis levels during ogtt. conclusions: results of our study indicate that in women with pcos, ( ) there is an increase in secretion of mis, ( ) higher insulin levels are associated with lower mis levels, ( ) acute increase in insulin levels has no effect on mis levels, ( ) lower mis levels in women with severe hyperinsulinemia could be due to associated increased follicular atresia and a decrease in the ovarian reserve. further studies are needed to investigate the role of insulin on mis secretion. hiroyuki asakura, noriko tanaka, kyoko nishio. ohgimachi ladies' clinic, osaka, japan. objective: elevation of serum anti-müllerian hormone (amh) levels among polycystic ovary syndrome (pcos) patients has been reported. however, the regulatory factors of amh in pcos remain unknown. we examined correlations between amh values and various indices of insulin resistance in pcos women. methods: infertile women compatible with rotterdam criteria for pcos were recruited under informed consent. the subjects underwent g oral glucose tolerance test ( ggtt, sampling at , , minutes), and -step elisa for amh (sensitivity . pmol/l, inter intra-assay c.v. : . %, . %, respectively). p< . was considered as statistical significance. results: average amh level of the subjects (age: . + . years, bmi: . + . kg/m , mean+s.d.) was . + . pmol/l, which was higher than normo-ovulatory women ( . + . pmol/l, n= ). amh levels had significant positive correlation with total ovarian volume by ultrasound ( . + . cc), but not with bmi, waist-hip ratio ( . + . ), and levels of serum lh ( . + . iu/l), total testosterone ( . + . ng/dl). although fasting glucose levels ( . + . mg/dl) were positively correlated with total ovarian volume (r= . ), amh levels had no significant correlation with fasting insulin ( . + . iu/l), fasting glucose/insulin ration ( . + . ), homa-ir ( . + . ), and the sum of insulin levels ( . + . iu/l)during ggtt. conclusions: increased serum amh level of pcos and its positive correlation with total ovarian volume implies that determination of serum amh level would aid in confirming diagnosis of the ovulatory disorder. absence of relationship between amh and various clinical indices of insulin resistance suggests alternative regulatory factors of amh gene expression in the follicular compartment. reproductive tissues. amisra a nikrodhanond, keeley l mui, helen h kim. ob/gyn, university of chicago, chicago, il, usa. background: although primarily a neuroendocrine hormone, gonadotropinreleasing hormone (gnrh) is also produced in reproductive tissues, where it acts locally. the study of gnrh regulation in these tissues is limited by low levels of expression. objective: to elucidate mechanisms that regulate gnrh expression in male tissues, our objective was to identify regions of the gnrh gene that target expression in vivo. methods: transgenic mice were generated with different fragments of the mouse gnrh gene promoter (- /+ and - /+ bps), fused to the luciferase reporter gene. in these mice, luciferase activity (detected as light) reflects gnrh promoter activity. using bioluminescent imaging, gnrh promoter activity was assayed in live gnrh-luc mice and in their reproductive tissues ex vivo. to confirm imaging results, luciferase activity was also measured as relative light units (rlu) in tissue homogenates. for each dna construct, male mice were examined. with whole-body imaging, bioluminescence was detected in the genital region of the gnrh-luc transgenic mice (fig.a) . in mice that incorporated the - luc transgene, examination of reproductive tissues ex vivo revealed bioluminescence in the prostate and penis, but not in the seminal vesicles, epididymis, or testes (fig.b) . in contrast, in the - luc mice, bioluminescence was detected in the testes, but not in other tissues (fig.c) . examination of luciferase activity in tissue homogenates from - luc mice confirmed gnrh promoter activity in the prostate ( ± rlu) and penis ( ± rlu) and absence in the testes ( ± rlu). in the - luc mice, however, luciferase activity was detected only in the testes ( ± rlu) and not in the prostate ( ± rlu) or penis ( ± rlu). conclusions: our studies demonstrate that gnrh is present in male reproductive tissues, but may be differentially regulated in the testes vs. prostate/penis. promoter elements, contained within the proximal - bp of the mouse gnrh gene, are sufficient to mediate testicular gnrh expression while - bp of the gene promoter are necessary to direct expression to the prostate and penis. characterization of mouse ringo/speedy homologues. z walton, s uckac, o guzeloglu-kayisli, md lalioti, d sakkas, e seli. ob gyn, yale u., new haven, ct, usa. introduction: ringo/speedy (ringo/spy) is a recently discovered cyclindependent kinase (cdk) activator that functions similar to cyclins in controlling the cell cycle. ringo/spy plays a crucial role during meiotic maturation in xenopus by inducing meiotic g /m progression and germinal vesicle breakdown (gvbd). more recently, padmanabhan and richter demonstrated that ringo/spy mrna is repressed in the xenopus oocyte cytoplasm by pumilio . upon meiotic reactivation, pumilio loses its interactions with ringo/spy permitting its translation. ringo/spy is then expressed, leading to activation of cpeb by phosphorylation, which in turn elicits polyadenylation and translation activation of the mrna for a critical oocyte maturation factor, the mos kinase. in this study, we investigated the expression of ringo/spy in mouse. methods: ten different somatic tissues, testes, and ovaries were tested by reverse transcription-polymerase chain reaction (rt-pcr) for the expression of ringo/spy homologues and their alternative splicing variants. ringo/spy mrna expression was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results: we analyzed the two previously identified homologues of ringo/spy (a and b). the two alternative splicing variants of ringo/spy a (a and a ) were separately studied for their expression profile. we also identified an additional alternative splicing variant of ringo/spy b and evaluated similarly. ringo/spy a ( and ) were expressed in testes, ovaries, and certain somatic tissues including brain, and spleen. ringo/spy b (both variants) were only expressed in testis. ringo/spy a or b were not present in mouse oocytes or early embryos. conclusions: our findings indicate that the previopusly described ringo/spy homologues a and b are not expressed in mouse oocytes or early embryos suggesting that either an alternative cdk-activator or a yet to be identified homologue of ringo/spy may be mediating meiotic g /m progression in mouse. testis specific expression of ringo/spy b, and previously described role of ringo/spy in meiosis suggests a role for this protein in male gametogenesis in mouse. treated with glyburide. jennifer aguayo, gladys a ramos, alethea hanley, carri r warshak, thomas r moore. reproductive medicine, university of california, san diego, san diego, ca, usa. objective: to determine the risk factors that may predict inadequate response to first-line glyburide monotherapy in pregnant women with type diabetes mellitus (dm) or gdm and to assess if non-responders are at increased risk of adverse pregnancy and neonatal outcomes. study design: this was a retrospective cohort of women diagnosed with type ii or gdm initially treated with glyburide at a single institution from - . maternal characteristics, adequate glycemic control defined as more than % of fasting glucose < mg/dl and one-hour post-prandials < mg/dl, and neonatal outcomes were assessed. non-responders were defined as failure to achieve adequate glycemic control on maximum daily doses of glyburide or intolerance due to side effects, necessitating switch to insulin therapy. statistical methods included bivariate analyses. results: of the women initially treated with glyburide, ( %) failed to achieve adequate glycemic control or did not tolerate glyburide. reasons for glyburide non-response were maximum dose ( mg/d) reached ( %), maternal hypoglycemia ( %) and other side effects ( %). there were no statistically significant differences between non-responders and responders with respect to family history of diabetes ( % vs. %, p= . ), prior history of gdm ( % vs. %, p= . ) and macrosomia (> g) ( % vs. %, p= . ) or hour glucose challenge test ( + vs. + mg/dl, p= . ). non-responders had a higher rate of obesity (bmi> ) ( % vs. %, p= . ) and earlier gestational age at initiation of therapy ( + . vs. + . wks, p= . ). there were no differences between the groups in the mean -week fasting ( + vs. + mg/dl, p= . ) and post-prandial glucose values ( + vs. + mg/dl, p= . ). no significant differences were observed in incidence of pre-eclampsia, primary cesarean delivery or birth weight. neonatal outcomes including ponderal index, neonatal hypoglycemia, nicu admission, and birth injuries also did not differ between the groups. conclusion: women who are obese and who require earlier initiation of glyburide therapy are at increased risk of non-response to glyburide monotherapy. however, despite non-response, these women can be managed with subsequent insulin therapy to achieve similar glycemic control and pregnancy and neonatal outcomes. a different diagnostic strategy using the gram, -hour glucose tolerance test for the diagnosis of gestational diabetes mellitus. yvonne w cheng, ingrid block-kurbisch, jennifer lydell, aaron b caughey. obstetrics, gynecology and reproductive sciences, university of california, san francisco, san francisco, ca, usa. objective: to examine whether a simplified strategy using the gm, -hour glucose tolerance test (gtt) may be useful for the diagnosis of gestational diabetes mellitus (gdm). methods: this is a retrospective cohort study of women with singleton pregnancy who received the -gm, -hour glucose challenging test (gct) for initial screening and the gm, -hour gtt as confirmatory tests for the diagnosis of gdm between and . various combinations of the gm, -hour gtt results were examined and compared to the diagnostic criteria for gdm established by the carpenter and coustan criteria using the receiver-operator characteristic (roc) curves. perinatal outcomes of women who would have had a false-positive or false-negative test results were compared to those who did not have gdm using the carpenter and coustan criteria. potential confounding factors were controlled for using multivariable regression models. results: , women had gm, -hour gtt results available for analysis during the study period. using gdm diagnosed by the carpenter and coustan criteria as reference, various diagnostic strategies was compared using roc curves (figure ) . summation of the -hour and -hour gtt results with a diagnostic threshold of mg/dl yielded the most optimal balance between sensitivity ( . %) and specificity ( . %). when compared to women without gdm, women who were diagnosed with gdm by c c criteria but not by summation of -hour and -hour gtts had higher odds of operative vaginal delivery (aor= . , % confidence interval [ci] . - . ) and neonatal birthweight > gm (aor= . , % ci . - . ). conclusion: using only the summation of only the -hour and -hour gtt results of the gm -hour gtt offers an alternative test strategy which may be more convenient and less costly for the diagnosis of gdm. however, women who would have gdm by the carpenter and coustan criteria but not by the summation method have higher odds of having an operative vaginal delivery and neonatal birthweight > gm. outcome of induction of labor indicated for term prom among women with or without a uterine scar. yifat ochshorn, avital skornick rapaport, adi reches, joseph b lessing, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. objective: we aimed at comparing the outcome of induced term deliveries presenting with prom with or without a previous uterine scar. methods: the computerized files of women delivered following induction of labor due to term prom, were reviewed. perinatal outcome parameters such as the mode of delivery, indication for cesarean section, rate of low ' apgar scores and nicu admissions were compared between women with and without a previous cesarean. results: during the study period women delivered in our institution following prom and induction of labor , of them had a previous uterine scar. parturients of both groups (with and without a scar) were similar with regard to age, gestational age at delivery and parity. cesarean section rate was higher for the previous scar group ( % vs %, p< . ). the most common indication for cesarean was arrest of dilatation and/or descent among women with previous scar accounting for % and % (p< . ) for women with and with no uterine scar, respectively. no differences were noted in neonatal outcome parameters such as rate of low ' apgar scores and nicu admission rate between the two groups. conclusion: induction of labor due to prom culminates in higher cesarean rate in women with one previous scar compared with parturients with no scar. perinatal outcome is similar between the two groups. acid base balance and immediate perinatal outcome of vertex compared with breech presentation in elective cesarean section. avital skornick rapaport, yifat ochshorn, joseph b lessing, yuval yaron, michael kupferminc, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. backround: apgar scores, umbilical blood ph and bicarbonate are generally lower and pco levels are higher in vaginally delivered breech neonates compared to cephalic deliveries. although cesarean delivery improved apgar scores there is a debate wether it improved the acid base status. this retroprospective study compared umbilical cord blood acid-base values and perinatal outcome of elective cesarean breech-delivery with those of elective cephalic cesarean delivery and to determine whether a different metabolic status and perinatal outcome should be expected in neonates in breech presentation. study design: the study group included singleton pregnancies delivered by elective cesarean section at term between january and march . computerized files of singleton breech presentation elective cesarean sections were compared to those of singleton vertex neonates delivered by elective cesarean section. demographic data included: maternal age, pregnancy week at delivery and parity. perinatal outcome measures checked were: birth weight, apgar scores at 'and ', umbilical cord venous and arterial ph and base excess. results: during the period between january and march there were singleton elective cesarean sections, of them were breech and vertex. the mean age, gravida and parity were significantly different between groups ( . vs. . , . vs. . . and . vs. . respectively, p< . ). the birth weight was significantly different - . gram for the vertex and . gram in the breech deliveries (p< . ) there were no differences in either apgar scores or umbilical ph between the breech and vertex neonates delivered by cesarean section at term ( - + w). venous and arterial po and pco levels were significantly different, though the differences were very small and we doubt if these differences have any clinical importance. conclusion: although vaginal breech deliveries are associated with increased risk of asphyxia during delivery, elective cesarean breech deliveries are not at increased risk for lower ph levels or apgar scores. the clinical significance of bleeding during the second trimester of pregnancy. arie koifman, amalia levi, yaron zaulan, avi harlev, eyal sheiner. obstetrics gynecology, soroka university medical center, ben gurion university of the negev, beer-sheva, israel; epidemiology and health services evaluation, faculty of health sciences, ben gurion university of the negev, beer-sheva, israel. objective: this study aimed at investigating clinical importance and pregnancy outcome in women suffering from bleeding during the second half of their pregnancies. methods: a population based study including all deliveries which took place in the soroka university medical center between the years - were examined. comparison was performed between patients with and without second trimester bleeding pregnancies terminated before weeks, multiple gestations and women lacking prenatal care were excluded . stratified analysis, using the mantel-haenszel technique, and a multiple logistic regression model were performed . results: during the study period, , singleton deliveries occurred in our institute. of these, ( . %) were complicated with bleeding upon admission during the second half of pregnancy. the cases were attributed to placental abruption ( . %; n= ) and placenta previa ( . %; n= ). independent risk factors associated with bleeding, were oligohydramnios, polyhydramnions, (odds ratio [or]= . ; % confidence interval [ci] . - . ; p= and . ; . - . ;p< . respectively ), suspected intra uterine growth restriction (iugr, . ; . - . ; p<. ), gestational age, previos abortions and maternal age. these patients subsequently were more likely to deliver by cesarean section (cs, . % vs. . %, or= . ; %ci . - . ; p< . ). perinatal mortality among patients admitted due to second half bleeding was significantly higher as compared to patients without bleeding (p<. ). conclusion: bleeding upon admission during the second half of pregnancy is an independent risk factor for perinatal mortality. careful surveillance, including fetal monitoring, is suggested in these cases in order to reduce the adverse perinatal outcome. crude and adjusted odds ratios for perinatal mortality among patients with vaginal bleeding. characteristics or % ci p crude or for perinatal mortality . . - . < . or adjusted for: < . iugr . . - . < . oligohydramnios . . - . < . premature rupture of membranes . . - . < . the predictors included neonatal pi, bmi, or birthweight while the outcomes included hypoglycemia, shoulder dystocia, acidemia and hyperbilirubenemia. roc curve analyses were utilized to evaluate the relationship between sensitivity and specificity for each of the predictors and outcomes, with an area under the curve (auc) significantly greater than . indicating a screening test that is better than chance. results: neonatal pi, bmi and birthweight are poor predictors of nicu admission, acidemia and hypoglycemia with all auc values not statistically significantly different than chance alone. they are a reasonable predictor of shoulder dystocia, with birthweight functioning the best (p< . ). in general, neonatal anthropometric measurements do not appear to be good predictors of short term neonatal outcomes. although they are a reasonable predictor of shoulder dystocia, they are not useful clinically for this outcome since they cannot be calculated until after birth. in future studies, new models of neonatal anthropometrics should be created to better predict adverse neonatal outcomes. neonatal anthropometric measurements and their relationship to perinatal outcomes expressed as auc and % confidence intervals history of at least one spontaneous preterm delivery were offered protocolbased prenatal care including first-or second trimester bacterial vaginosis screening, repeated ultrasound cervical length assessment, and supportive care by specialized nurses. women with a positive test for bacterial vaginosis were treated with oral metronidazol therapy, and women with a cervical length below . cm (before weeks of gestation) underwent a vaginal cerclage. progesterone administration was not provided. data on obstetrical and medical history, pregnancy, delivery and neonatal outcome were prospectively collected and evaluated. results: in total, pregnant women were prospectively followedup. eighty-seven women had experienced a preterm delivery in their last pregnancy, of whom had delivered before weeks. three women were diagnosed previously with a bicornual or septal uterus. twelve women received metronidazol therapy and women underwent a vaginal cerclage. eighty-four women ( . %) delivered at weeks or beyond. one-hundred and three women ( . %) delivered no earlier than weeks, and only one woman delivered at weeks. two women gave birth to a twin (at and weeks, respectively). two neonatal deaths due to pulmonary hypoplasia were recorded. conclusion: women at high risk of preterm delivery who were offered protocol-based prenatal care not including progesterone therapy had a better pregnancy outcome than would be expected from previous studies. our findings may question the reported beneficial effects of prenatal progesterone administration. premature rupture of membranes. tracy a manuck, alexandra g eller, m sean esplin, robert m silver. obstetrics gynecology, university of utah health sciences, salt lake city, ut, usa. objective: historically, outcomes following expectant management of midtrimester preterm premature rupture of membranes (pprom) have been uniformly poor. thus, many patients elected pregnancy termination. however, outcomes may be improved with recent advances in neonatal medicine. our purpose was to assess outcomes in expectantly managed early pprom in an era of improved maternal and neonatal care. study design: this is a retrospective cohort of patients from tertiary healthcare systems from - experiencing pprom . weeks gestation. patients electing immediate termination of pregnancy, carrying fetus(es) with lethal anomalies, or delivering within hours of pprom were excluded. survival without major morbidity was the primary outcome. data were analyzed using student t-test and chi-square as appropriate. results: a total of women carrying fetuses ( singleton, twin, triplet, quadruplet) met inclusion criteria. only the fetus in the "ruptured sac" was studied. pprom occurred at a mean of . (+/- . , range . - ) weeks. the average latency period was . (+/- . , range . - ) days, with a mean delivery gestational age of . (+/- . , range . - ) weeks. this communication provides the first report of fundal uterine necrosis following placement of multiple uterine compression sutures. only two previous published cases report occurrence of uterine necrosis following application of a uterine compression suture --both identified as the b-lynch technique --and neither of these cases report necrosis confined to the fundus. in our case, uterine atony was refractory to pharmacologic therapy and manual compression of the uterus appeared to decrease the amount of blood loss. therefore, we applied a traditional b-lynch which effectively contracted the majority of the uterus, while the fundus remained atonic. a second horizontal, square suture was then placed between the cornua and carried over the fundus (see figure one: red reflects b-lynch suture; blue represents second fundal, square stitch). the patient subsequently experienced significant, persistent fevers despite antibiotic therapy. a repeat laparotomy was performed, at which time we found uterine necrosis confined to the uterine fundus (see figure : photograph taken at the time of hysterectomy). the placenta showed no histologic evidence of chorioamniotis --hence, the patient's main risk factor for fundal necrosis was the compression sutures. physicians should be aware of the risk of uterine necrosis, especially after placement of multiple compression sutures and, more specifically, after placement of a compression suture confined to the uterine fundus. background: migraines are far more common in women than in men. the incidence of migraines increases in girls after puberty, reaching an incidence of % in women who experience migraine at least once a year around middle age. migraine has been postulated as one of the major risk factors for stroke during pregnancy and the puerperium.triptans are a class of serotonin receptor agonists used in the treatment of migraine headaches. triptans administered in combination with other drugs have been known to precipitate serotonin syndrome, a rare but potentially life-threatening condition clinically manifested by a triad consisting of mental-status changes, autonomic hyperactivity, and neuromuscular abnormalities. objective: to determine whether triptan monotherapy is associated with serotonin syndrome methods: using data mining techniques we analyzed the entire food and drug administration's (fda) adverse event reporting system (aers) database. results: after excluding reports of concomitant serotonergic medication or other potentially confounding medication, eleven reports remained of serotonin syndrome associated with triptan use without concomitant serotonergic medication. the mean age for these eleven cases was . years; nine cases occurred in females and two occurred in males. there were no apparent instances of overdose among these eleven cases. conclusions: serotonin syndrome is a rare but serious occurrence with the use of triptan monotherapy. such cases were seen with eletriptan, rizatriptan, sumatriptan, and zolmitriptan. because of the spontaneous nature of voluntary reporting to aers, the actual number of occurrences of serotonin syndrome in patients using triptans is probably higher and cannot be assessed from aers data. users of triptan in combination with an ssri or snri should be warned of this rare but serious adverse effect. during periods of drug initiation, dose escalation, or the addition of another serotonergic agent, patients should be particularly vigilant for symptoms of concern and seek urgent medical attention if any occur. the offending agents should be withdrawn and the patients closely monitored and treated with supportive measures as required. rising maternal mortality in the midst of modern technology. oormila p kovilam, jane khoury, padmini c sekar, ralph c buncher. obstetrics gynecology, university of cincinnati, cincinnati, oh, usa; center for epidemiology and biostatistics, cincinnati children's hospital medical center, cincinnati, oh, usa; environmental health, university of cincinnatic, cincinnati, oh, usa. introduction: maternal mortality rate (mmr) is an index of overall wellbeing of the community and safe motherhood should be given utmost priority in obstetric care. as per , who estimates % of maternal mortality occurs in developed countries . this rate has established without much decline in recent years. the mmr for ohio for to was reported by cdc to be . per , live births with % confidence interval . to . . objective: our objective was to audit the trend in maternal mortality in the state of ohio for the last years. methods: information from the death certificates completed by attending physicians, medical examiners, coroners, funeral directors filed with ohio state registration offices were analyzed. we only used women who were residents of the state of ohio at the time of death, and cause of death was coded as a "complication of pregnancy, childbirth and the puerperium", icd codes to and icd codes o to o . five year maternal mortality pattern and long term trend was assessed. results: the number of maternal deaths recorded as due to pregnancy complications varied over the years from a high of in to a low of in and . in general the five-year mortality rate was decreasing over time until the last four years to , when we may be seeing an upward trend compared to to (p= . ). the rates and associated % confidence intervals (ci) are shown in the table below. years mmr % ci ci - ci . . - . ci - . . - . - . . - . - . . - . - . . - . the rate of maternal mortality is on the rise after a period of downward trend. we should develop a multidisciplinary approach to analyze and target the root causes of maternal death. introduction. women with thrombophilia are at higher risk of vte during pregnancy due to the acquired hypercoagulable state. it is likely that not only maternal veins but also placental vessels are more prone to the development of thrombosis. our objective was to compare maternal and fetal placental circulation disorders in women with intra-uterine fetal death (iufd) and thrombophilia and women without thrombophilia. methods. in a dutch multi-centre study on iufd, during the period - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. factor v leiden and prothrombin g a were tested at induction of labour. panel classification of cause according to the tulip classification was performed by assessors after individual investigation of structured patient information. we studied the cause of death group "maternal and fetal placental circulation disorders": placenta bed pathology with abruption or infarction as origin of mechanism and placental parenchyma pathology with fetal thrombotic vasculopathy and massive perivillous fibrin deposition as origin of mechanism. results. of the women tested for factor v leiden, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of factor v leiden. of the deaths with another cause of death ( . %) mothers were carriers of factor v leiden (p= . ). of the women tested for prothrombin g a, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of prothrombin g a mutation. of the deaths with another cause of death ( . %) mothers were carrier of prothrombin g a mutation (p= . ). in women with iufd and factor v leiden or prothrombin g a mutation "maternal and fetal placental circulation disorders" did not seem to cause iufd more often than in non-carriers. , university medical center groningen, groningen, netherlands; pathology, university medical center groningen, groningen, netherlands; trial coordinating center, dept of epidemiology, university medical center groningen, groningen, netherlands; hematology, university medical center groningen, groningen, netherlands. introduction. growing evidence suggests that women with thrombophilic defects may be at higher risk of fetal loss. although some paternal components to the predisposition of preeclampsia have been demonstrated it is not known whether paternal components contribute to intra-uterine fetal death (iufd). our objective was to investigate the relation between paternal thrombophilic defects and iufd. methods. in a dutch multi-centre study on iufd, from - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. we tested male partners of women with iufd for antithrombin (at), protein c, protein s type i and iii, factor v leiden, prothrombin g a (factor ii) and factor viii: ag. standard tests were performed in one laboratory. normal ranges were determined in healthy male blood donors. we compared prevalence of thrombophilic defects to reference values from the literature. . of the men tested for factor v leiden, ( . %) were carrier versus ( . %) non-carriers. prevalence of factor v leiden in the normal population is % (p= . ). men were tested for prothrombin g a, ( . %) were carriers and ( . %) non-carriers. all were heterozygous. prevalence of prothrombin g a mutation in the normal population is % (p= . ). decreased levels of antithrombin, protein c, protein s type i and iii and increased levels of factor viii: ag were observed significantly more often in male partners of women with iufd compared to the normal population. conclusion. in our iufd group the prevalence of male factor v leiden carriers was comparable to the normal population, prevalence of prothrombin g a mutation was lower. the difference in other factors imply an as yet unexplained association of male thrombophilia with fetal death in their partner. objective fetal macrosomia, the most important complication of gdm, increases the risk of shoulder dystocia, erb's palsy and intrauterine hypoxia. we compared frequencies of fetal macrosomia and erb's palsy in two gdm cohorts with different severity of glycemic disturbance and in healthy controls. methods we studied consecutive gdm women with singleton childbirth and or abnormal values in -h ogtt with g of glucose. abnormal plasma glucose values of the ogtt were: fasting . , -h . and -h . mmol/l. insulin treatment was started when values were . preprandially or . mmol/l postprandially in the -h glucose profile done within days of diagnosis. if the same woman had more than childbirth during the study period, only the last pregnancy was included. the control group consisted of women from a nearby town with singleton childbirth in the same hospital. women with diabetes were excluded from controls. macrosomia was defined as birth weight (bw) > . sd above the mean of a standard population. erb's palsy was diagnosed by pediatric surgeons. results gdm women were older, more obese, had more childbirths and more often a previous child with bw > g than controls. insulin was started in gdm women ( . %). c/s rate was . % in controls, . % in diettreated and . % in insulin-treated gdm women. macrosomia rate was . % in controls, . % in diet-treated and . % in insulin-treated gdm women (p< . compared with controls or diet-treated gdm women). the frequency of macrosomia did not differ between the diet-treated gdm and control women. erb's palsy occurred in . % of controls, in . % of diet-treated and in . % of insulin-treated gdm women. the frequency of erb's palsy was significantly higher in both gdm groups than in controls (p= . ). by regression analysis, previous child with bw > g (p< . ), previous c/s (p= . ), mother's age (p= . ), bmi (p= . ), insulin treatment (p= . ) and fasting plasma glucose of the -h profile (p= . ) were significant independent predictors of fetal macrosomia. conclusions the -h glucose profile done shortly after the diagnosis of gdm clearly distinguishes between low-risk (diet-treated) and high-risk (insulintreated) gdm pregnancies for fetal macrosomia. unfavourable fetal body composition of diet-treated gdm women is the likely explanation for the high rate of erb's palsy in this group. . '-:·:tpartq,-, '-:·:tpartq,-, '-:·:tpartq,-, % p= . ). first trimester uta doppler indices were similar in the two cohorts in terms of the resistance and pulsatility indices (ri . vs. . , p= . ; pi . vs. . , p= . ) . however, bilateral notching was much more common in the cohort of prior adverse outcomes ( % vs. % p= . ) as well as in patients destined to have a subsequent poor outcome (p= . ). conclusions: not surprisingly, prior poor obstetric outcome was strongly associated with recurrent adverse obstetric outcome. uta notching was robustly associated with prior poor obstetric history as well as a recurrent poor outcome. this clinical history had no discernable influence on ri or pi. reassurance may not be offered based on first trimester uta ri and pi. anti-retroviral therapy is associated with increased blood loss and uterine atony for patients undergoing primary cesarean section. carey eppes, alice cootauco, melissa russo, jessica bienstock. gynecology and obstetrics, johns hopkins university school of medicine, baltimore, md, usa. objective: anti-retroviral therapy has been associated with gastrointestinal smooth muscle dysfunction. it has also been hypothesized that myopathies can occur secondary to anti-retroviral therapy due to mitchondrial alterations. in our experience, we have noticed an increased incidence of uterine atony in our hiv patients on anti-retroviral therapy. we sought to examine the incidence of postpartum hemorrhage and uterine atony in patients currently on anti-retroviral therapy. study design: a retrospective case-controlled study was conducted on all hiv positive pregnant women on anti-retroviral therapy undergoing a primary low segment transverse cesarean section from through . these patients were obtained from an irb approved database containing hiv positive patients within our institution. patients' medical records were abstracted for demographic data, use of uterotonics, preoperative and postoperative hematocrits, and incidence of blood transfusions. controls were matched for age, parity, gestational age and surgical indication. data was analyzed using the t-test, fischer's exact test and chi square. results: there were no differences in demographics, incidence of chorioamnionitis or magnesium sulfate use between groups. patients on anti-retroviral therapy had a statistically greater decrease in hematocrit and estimated blood loss compared to controls. they also had an increased need for uterotonics and blood transfusions. conclusion: anti-retroviral therapy may impact uterine smooth muscle contractility in pregnancy, as evident by the increased incidence of uterine atony and change in hematocrit. additional research is needed to elucidate the mechanism. clinicians should be aware of the potential for uterine atony and excessive blood loss in patients on anti-retroviral therapy. background: adolescent pregnancy is frequently associated with adverse outcomes, especially small-for-gestational age (sga) deliveries. some studies have implicated maternal nutritional status in these poor outcomes. methods: in a prospective longitudinal study, ethnically-diverse, pregnant adolescents were studied from booking to parturition, with collection of anthropometric and nutritional variables. blood samples ( - weeks' gestation; n= subjects) were assayed for a spectrum of nutritional biomarkers. logistic regression was used to determine significant associations between studied variables and pregnancy outcomes. results: median age at recruitment was . years (iqr: . - . ). outcome data was available for subjects. ( . %) had uncomplicated pregnancies. median birthweight was , g (iqr: , - , g) and median birthweight centile was . % (iqr: . - . %). ( . %) infants were born sga and ( . %) were preterm. spontaneous vaginal deliveries occurred in . % of cases. there were ( . %) cases of pre-eclampsia and ( . %) admissions to neonatal care. . % of subjects reported smoking at booking. iron deficiency anaemia was prevalent in . % of subjects by - weeks and was strongly associated with higher infant birthweight centile (p< . ). . % had serum -hydroxy vitamin d concentrations < ng/ml, although this was not associated with any outcomes. low folate status, as indicated by low red cell folate (p= . ), low serum folate (p= . ) and high serum homocysteine (p= . ) concentrations, was associated with higher rates of sga birth. conclusion: adolescent pregnancy in inner-city populations is associated with a high risk of sga and preterm birth, increasing the likelihood of health problems in later life and perpetuating social disparities in health. the association between anaemia and higher birthweight is unexplained. impaired maternal folate status may contribute to impaired fetal growth. we suggest that sga birth in this population could be reduced by the use of antenatal supplements or the mandatory fortification of flour with folic acid. amount of cigarette use pre-pregnancy and during pregnancy were self-reported and subgrouped into nonsmokers, quitting during pregnancy, and continued smoking throughout pregnancy. categorical outcomes were compared using chi-square test. multivariable logistic regression analyses were used to control for potential confounders (continued smoking compared to quitting during pregnancy). results: women who quit smoking during pregnancy had higher rates of pregnancy-associated hypertension and cesarean delivery but lower rates of preterm delivery and neonatal birthweight < gm compared to non-smokers. compared to women who quit smoking during pregnancy, those who continue to smoke have lower odds of pregnancy-associated hypertension and cesarean delivery, but higher odds of preterm delivery, neonatal birthweight < gm, and apgar score < at minutes (see table) conclusion: quitting cigarette smoking during pregnancy appears to reduce undesirable neonatal outcomes, though an increase in pregnancy associated hypertension. these findings should be emphasized to women who are smoking during pregnancy. to achieve effective tamponade the balloon was inflated initially up to - ml with incremental increases of volume by ml until bleeding stops. to measure intrauterine balloon pressures we used a standard pressure transducer (edwards lifesciences) connected to the balloon port and to a pressure monitor (ge dash ). the pressure transducer was mounted on the bedrail at uterine level, primed with sterile saline and then connected to the balloon port of the catheter. the system was zeroed to atmospheric pressure, the stopcock of the intrauterine balloon port was opened and the pressure was recorded (p : the total pressure on the fluid). to verify the accuracy of measurements we used another balloon catheter (reference), inflated with the same amount of saline, connected to the pressure transducer and zeroed to atmospheric pressure. the reference balloon measured p (the intrinsic balloon pressure caused by distension at that volume). the system was then zeroed to the reference balloon. the pressure transducer was then reconnected with the intrauterine balloon and the actual intrauterine pressure was recorded (p ). we calculated the correlation coefficient between p and systolic, diastolic and mean blood pressure using a linear regression (statsdirect statistical software there is a growing body of evidence to suggest that peripartum assessment of fetal or neonatal lactate levels are as good as or better than standard blood gas analysis in the prediction of neonatal outcome. in this study we have evaluated the ability of umbilical cord blood gases and lactate levels in the prediction of neonatal hypoxic-ischaemic encephalopathy (hie). department of neonatal paediatrics, king edward memorial hospital, perth, western australia, australia; women and infants research foundation, king edward memorial hospital, perth, western australia, australia. objective: current evidence suggests that umbilical cord ph at delivery provides the most sensitive reflection of birth asphyxia. paired umbilical artery/vein blood gases have been routinely collected at king edward memorial hospital over the last years. the objective of this study was to determine: local reference ranges; accuracy of sampling; and the rates of metabolic acidosis. of the , births ( ) ( ) ( ) ( ) , accurate paired results were available on % of births. over the study period there was a progressive improvements in accuracy rates of paired sampling (p< . ). the median ( . th , . th centile) values for cord arterial blood gases were: ph . ( . , . ); po . mmhg ( . , . ); pco . mmhg ( . , . ); base excess - . (- . , . ); and lactate . mmol/l ( . , . ). there was a progressive improvement in all blood gas measures over the years of this study (all p< . ). moreover, there were significant reductions in all measures of metabolic acidosis (see table) . the progressive improvement in the measures of metabolic acidosis remained significant after multivariate analysis including obstetric, fetal, and demographic factors associated with metabolic acidosis. the introduction of universal umbilical cord blood gas analysis to all births is associated with significant improvements in all markers of metabolic acidosis. together with other compelling evidence in the literature, these data support the routine use of cord arterial and venous gases at all births; however, improved accuracy rates on paired sampling requires an ongoing education program umbilical artery indicators of acidosis, percentage of total ph < . ph < th centile* objective: intrapartum pcn prophylaxis aims to prevent early-onset gbs sepsis by interrupting vertical transmission from colonized mothers to their newborns. however, despite its wide clinical use, systematic pharmacokinetic evidence in support of the current pcn dosage regimen is lacking. current cdc guidelines recommend intensified surveillance and testing of infants exposed to < h of prophylaxis. our goal was to examine the relationship between maternal time of exposure to pcn and fetal serum pcn levels among maternal-fetal dyads exposed to short durations of pcn prophylaxis (< h) compared to those exposed to longer durations. study design: ninety-eight laboring gbs positive women were administered million units (mu) of intravenous pcn to be followed by . mu every hours until delivery (cdc ) . subjects with renal disease, multiple gestation, and preterm delivery (< wks) were excluded. umbilical cord blood samples were collected at delivery and pcn levels measured by high-performance liquid chromatography. intra and inter-assay coefficients of variation were < %. results: the pcn concentrations (mean±sd) by duration of prophylaxis were: < h, . ± . g/ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ml (n= );> h . ± . g/ml (n= ); and for those without a second dose after h, . ± . g/ml (n= ). fetuses exposed to short duration (< h) had higher levels of pcn than those exposed to > h (p= . ). in multivariable linear regression analysis, fetal pcn levels were determined by total duration of exposure, time since last dose, dosage, and number of doses, but not maternal bmi. pcn levels in cord serum increased linearly until hour; thereafter, they decreased rapidly, but all groups were significantly above the minimum inhibitory concentration (mic) for gbs ( . g/ml)(p< . ). furthermore, every sample individually remained - fold above the mic. conclusion: in this study, even short durations of prophylaxis achieved levels above the mic, suggesting a benefit to prophylaxis even in precipitous labors. the data also suggests that the current cdc designation of infants exposed to < h of pcn prophylaxis as particularly at risk for gbs sepsis may be inaccurate from a pharmacokinetic standpoint. objective: -oh aa is a metabolite of tryptophan with pro-oxidant and proapoptotic properties and has been shown to increase in umbilical cord blood in pregnancies with intra-uterine infection. since labour-related events may also activate inflammatory pathways, we sought to determine the placental release of -oh aa into the umbilical circulation in labouring vs non-labouring patients at term. methods: twenty-six patients were studied (term labour n= , and term elective cesarean section n= ) with blood sampling from a clamped segment of umbilical cord after delivery of the fetus and from the cord at its insertion into the placenta after delivery of the placenta, with subsequent measurement of blood gases/ph and -oh aa (isocratic hplc using fluorometric detection with assay sensitivity at pmol). results: -oh aa measurements from respective umbilical and placental cord vessels were all variably higher in the labouring group vs the elective cesarean group patients (table ) . for labouring group patients, the -oh aa levels from the umbilical vein were significantly higher than those from the umbilical artery, indicating net release from the placenta into the fetal circulation. placental vein levels were also significantly higher than those from the umbilical vein, indicating continued placental release of -oh aa into the cord blood after delivery of the fetus. conclusion: labour at term is associated with changes in the placental metabolism of tryptophan resulting in the increased release of -oh aa into the fetal circulation with the potential for pro-oxidative and apoptotic effects in many tissues, including the brain. obstetrics, gynecology, and reproductive sciences, new brunswick, nj, usa; department of obstetrics and gynecology, mineola, ny, usa. objective: ischemic placental disease (preeclampsia, small for gestational age, sga and placental abruption) is a major contributor to pregnancy-related morbidity. although the placenta is considered a fetal organ, it is accepted that ischemic placental disease (ipd) can present clinically with either fetal or maternal manifestations. we hypothesized that the pattern of diagnosis (maternal versus fetal) varies by gestational age and would provide insights in to origins of indicated and spontaneous preterm birth. methods: this was a retrospective cohort study utilizing the maternallylinked reproductive history data for missouri residents , restricted to singleton live births. women who experienced spontaneous onset of labor and subsequently delivered preterm were classified as spontaneous preterm birth. medically indicated preterm birth included women who delivered preterm through a labor induction or (prelabor) cesarean delivery. ipd was classified as maternal (preeclampsia only), fetal (sga only) or both (preeclampsia with sga or abruption, and all conditions). results: among term births with ipd, . % presented as maternal disease only, . % as fetal disease, and the remainder ( . %) as both. among spontaneous preterm births with ipd, a greater proportion were of fetal presentation ( bethesda, md, usa; dept ob/gyn, wayne state univ, detroit, mi, usa; dept pathology, wayne state univ, detroit, mi, usa; dept ob/gyn, soroka univ medical center, israel. objective: hemoglobin (hg) and its catabolic products have been observed in cases of amniotic fluid (af) discoloration, which is a risk factor for intraamniotic infection/inflammation (iai). the study aimed to determine the association between af fetal hg concentration and gestational age, term and preterm labor and iai. study design: this cross-sectional study included: ) mid-trimester (n= ); ) term not in labor (tnl) (n= ); ) term in labor (tin) (n= ); ) preterm labor (ptl) who delivered at term (n= ); ) ptl without iai (n= ); ) ptl with iai (n= ); ) preterm prelabor rupture of membranes (pprom) with (n= ) and without iai (n= ). af fetal hg concentrations were determined by elisa. results: ) fetal hg was detected in . % of all af and . % of mid-trimester samples; ) women at tnl had a higher median af fetal hg concentration than patients at mid-trimester ( . ng/ml, iqr - vs . ng/ml, iqr . - . , p= . ); ) no differences were found in median af fetal hg concentration among patients with and without labor at term (til: . ng/ml, iqr - . ; p= . ); ) median af fetal hg concentration was not significantly different among the ptl subgroups [ptl with iai: . ng/ml, ptl who delivered preterm: . ng/ml, ptl without iai who delivered at term: . ng/ml, p= . (kruskal wallis) ]; ) in pprom, there were no differences among patients with and without iai ( . ng/ml, respectively; p= . ) ; ) median af fetal hg concentrations were significantly higher in ptl or pprom, with or without iai than in pregnant women at term, with and without labor (p< . for all comparisons). conclusions: ) immunoreactive af fetal hg increases with gestational age; ) among women with ptl or pprom, the median af fetal hg concentration is not associated with iai; ) the median af fetal hg concentration is higher in pregnancies complicated with ptl or pprom than in term pregnancies. the impact of latency time to delivery after preterm premature rupture of membranes (pprom) on neonatal outcome. dan nayot, deborah penava, barbra de vrijer, orlando da silva, bryan s richardson. obstetrics and gynaecology; pediatrics, university of western ontario, london, on, canada. objective: there continues to be controversy as to the management of pprom with conservative management to advance gestational age (ga) versus aggressive management with early induction to avoid chorioamnionitis. we have therefore used the perinatal and neonatal databases of a large regional patient population to determine the association of pregnancy variables with latency time to delivery after pprom and the impact of latency duration on adverse neonatal outcomes. methods: the perinatal/neonatal database of st. joseph's health care, london, ontario was used to obtain demographic and neonatal outcome information for all patients with pprom > and < weeks gestation, singleton and no major anomalies, delivering between january , and december , . patients were grouped according to ga at pprom stratified for latency time < hrs vs > hrs with incidences for those pregnancy related variables and neonatal outcomes available from the database then compared with the use of logistic regression analysis. results: there were patients who met the inclusion criteria of whom %, %, and % had pprom at - wks , at - wks, or at - wks, respectively, and with these pprom groupings showing a stepwise decrease in the percentage of patients with latency to delivery > hrs, at %, %, and %, respectively. pregnancy related variables and neonatal outcomes for these patient groupings are as shown in table . conclusion: despite a to fold increase in the incidence of chorioamnionitis with latency to delivery > hrs, a policy of conservative management to advance ga after pprom will result in decreased severe infant morbidity until weeks, and moderate infant morbidity until weeks. hospital. outcomes studied were prolongation of latency period with intact membranes and prom using magnesium sulfate (mgso ), nifedipine, terbutaline and tocolytic agents. secondary outcomes examined were maternal complications. statistical analysis performed were t-test and . comparisons were expressed as odds ratios ( % ci). results: in a -year period, twin gestations were admitted in ptl and administered tocolytic agent(s) with mean gestational age of . weeks. when tocolytic agents were used, maternal complications were: ( . %) with intact membranes and ( . %) with prom had pulmonary edema (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had chorioamnionitis (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had postpartum hemorrhage (or= . , ci . - . ). conclusion: there is no difference in prolongation of latency period achieved in twin gestations in ptl with intact membranes or prom using tocolytic agents. similarly, there is no difference between the groups with latency period hrs. there is an increased likelihood of pulmonary edema and chorioamnionitis with use of tocolytic agents. however, results are not significant due to type ii error. mean latency period and latency ≥ hrs using single or multiple tocolytic agent ( introduction: high sensitivity crp (hscrp) is a serum marker of inflammation and has proven clinical utlility in predicting cardiovascular disease (cvd). considering the hypothesized association between preeclampsia (pre) and inflammation and cvd, it is plausible that hscrp may have utility in predicting pre. prior to widespread utilization of this marker, the affect of labor on levels of crp needs to be clarified. we assessed the association between labor and hscrp levels in term deliveries and between elevated hscrp and adverse perinatal outcomes. a secondary analysis comparing hscrp in women with preeclampsia (pre) to those without was performed. methods: women presenting for term delivery or pre were prospectively identified as part of a case-control study. clinical data and serum were collected for all subjects. a standard immunoturbidimetric assay was used to measure hscrp levels. women presenting for induction of labor or planned cesarean delivery (non-labor) were compared to women presenting in labor. a secondary analysis comparing non labor women with and without pre was performed. serum was collected prior to labor induction in the non-labor group. nonparametric comparisons were made using wilcoxon rank sum tests. mvlr was used to evaluate dichotomous outcomes and control for confounders. results: women were included (non-labor group (n= ), labor group (n= ), non-labor pre (n= )). the median and mean hscrp levels were and and and in the non-labor and labor groups respectively (p= . ). elevated levels of hscrp in these term deliveries were not associated with chorioamnionitis (p= . ), maternal postpartum complications (endometritis, hemorrhage, transfusion) (p= . ), mode of delivery (p= . ), or admission to the nicu ( . ). levels of hscrp were significantly greater in the non-labor pre group compared to non labor without pre (mean . vs. . , median vs. , p< . ). conclusion: use of hscrp as a biomarker may improve clinical prediction of obstetrical complications such as pre. levels of hscrp are affected by labor and this should be taken into account when studying the utility of this biomarker. further, crp levels are elevated in women with pre even after excluding patients in labor. further investigations to determine if crp elevation in term labor is associated with adverse outcomes may be warranted. the role of hscrp as a valid and discriminating biomarker in pre should be assessed. review of the electronic labor record facilitated collection of maternal demographic, intrapartum, and newborn data. data analyzed with t-tests, chi-square tests, and calculated odds ratios with % cis as appropriate. a forward stepwise logistic regression analysis was used to identify predictors of histologic chorioamnionitis. results: of submitted placentas, had histologic chorioamnionitis (cases) and did not (controls). the groups were similar with respect to age, race, gbs status, and mode of delivery. gestational age, birthweight, duration of labor and ruptured membranes, and number of vaginal exams were greater in the cases (p . ). the cases were more likely to have had epidural anesthesia (or . ), internal monitoring (or . ), fever (or . ), maternal tachycardia (or . ) and fetal tachycardia (or . ) and less likely to have had induction of labor (or . ). newborns in the histologic chorioamnionitis group were more likely to have been observed for sepsis (or . severe sepsis defined as sepsis associated with acute respiratory distress syndrome (ards) or cardiovascular dysfunction(cvd) or with or more other organ dysfunction.all patients were resuscitated with fluids and treated with broad spectrum antibiotics and supportive care as needed. outcome data were: etiology, management, maternal complications, duration of icu stay and perinatal survival. results patients were young (mean age= . ± . years) with a mean gestational age at delivery . ± . weeks. etiologies were pyelonephritis(n= ), septic abortion(n= ), endomyometritis (n= ), chorioamnionitis (n= ), ruptured appendix(n= ), pneumonia( n= ) and one unknown. eighteen ( %) were diagnosed during antepartum and ( %) postpartum period.there were ( %) maternal deaths and high rate of major morbidities (table) . among the antepartum patients,there were, abortions, iufd, neonatal death for a perinatal survival rate of only %. conclusion pregnancies complicated with severe sepsis/septic shock are associated with substantial maternal and perinatal morbidities. the low maternal mortality rate in our study as compared to previous reports is attributed to early diagnosis and aggressive management of maternal complications. fetal loss rate, however continues to be high when septic shock develops antepartum. and tnfa levels are elevated. we developed a dynamic computer (in silico) model of pregnancy (uterine myometrial environment -infection/inflammation and endocrine crosstalk). mathematical differential equations were used to describe the interactions between molecules. infection was represented as increased levels of activated, nuclear transcription factor nf-kb. in the model ru inhibited both the glucocorticoid receptor and progesterone receptors. simulations were run adding different concentrations of ru ( . um= dose used in patients, . um, . um) at different time points during infection (before, at the time of or after nf-kb activation). ru degradation kinetics was also included. the effect of ru on nf-kb induced il- and tnfa levels was assessed. results: infection induced nf-kb activation led to increased il- and tnfa levels. there was a subsequent increase in cortisol that led to dampening of nf-kb activation, il- and tnfa levels. in the presence of ru , il- and tnfa levels continued to rise. the effect of ru on nf-kb induced il- and tnfa was dose dependent and was more prominent in slow metabolizers who had ru in the system for a longer time. addition of ru after the onset of nf-kb activation led to increased il- and tnfa levels above those observed without ru . the addition of misoprostol (prostaglandin e analogue), at the concentrations used together with ru for medical abortion, did not add to the effect of ru on nf-kb induced il- or tnf. conclusions: ru has dose dependent effects on infection induced immune activation and may contribute to the pathogenesis of c sordelii induced sepsis syndrome. the increased susceptibility of neonates to infection remains a major clinical problem. we previously found that after intraperitoneal (ip) listeria monocytogenes infection, neonatal mice have an ld that is orders of magnitude lower than adults. we also found that the inflammatory response of neonatal mouse macrophages, but not neutrophils, in the peritoneal fluid was deficient, and that this correlated with low levels of macrophage chemokines mcp- and rantes. given that the liver and spleen are important organs in listeria infection, we sought to characterize the innate immune response in these organs. a sublethal dose of listeria was injected intraperitoneally into balb/c - week old adult mice and - day old neonatal mice. liver and spleen was collected at , , and hours, sectioned serially for staining with hematoxylin-eosin and primary antibodies (rabbit anti-listeria monocytogenes igg; mhc class ii rat anti-mouse igg, a marker for activated macrophages; f / rat antimouse igg, a marker for macrophages) and secondary antibodies (cy- goat anti-rabbit igg; af goat anti-rat igg) were applied. negative controls used only secondary antibody. real time pcr was used to compare the levels of the chemokines mcp- and rantes in adult and neonatal liver. after ip infection, both adults and neonates showed similar influx of neutrophils to the sites of infection within the liver and spleen. at hours post-infection with listeria, adult liver and spleen showed increased staining for f / and class ii, which increased further and became confluent surrounding microabscesses at and hours. in contrast, the listeria infected neonatal mouse showed some increase in f / around microabscesses but no apparent increase in staining for mhc class ii. the neonates showed greater staining for listeria at each time point. mcp- and rantes levels were higher in infected neonatal liver compared to adults. in the neonatal mouse, the innate immune response in the liver and spleen was characterized by a deficiency of activated macrophages. this deficiency was not correlated with hepatic expression of macrophage chemokines. is there a seasonal pattern in the incidence of post-cesarean endometritis? tamula m patterson, alan tn tita, william w andrews. obstetrics and gynecology, the university of alabama at birmingham, birmingham, al, usa. objective: several theories, including one suggesting a peak in july coincident with resident turnover, postulate seasonality in post-cesarean infections. we assessed whether there is seasonal variation in endometritis. a retrospective cohort study of post-cesarean endometritis at our university-based institution using our obstetric computerized database to compare annual variation in monthly incidence patterns from to . prior to establishing an average aggregate seasonal pattern for all years, years were assessed separately for a recurrent pattern of peaks and nadirs in incidence. peak incidence (or nadir) was defined as any monthly incidence that differed from the mean incidence for the year by over %. results: a total of . % ( , ) of , deliveries from to were by cesarean. annual cesarean rates increased by an absolute rate of over %; while, post-cesarean endometritis rates decreased from % to %. monthly incidence of post-cesarean endometritis did not reveal a consistent recurrent pattern of peaks (figure ) or nadirs. the month of july accounted for only out of a total of peaks for all years. the adjacent months of june and august accounted for only each. the month with the highest number of peaks was april with only . these findings contraindicated the establishment of an average aggregate monthly pattern for all years. the incidence of post-cesarean endometritis did not follow a seasonal pattern. chi-square analyses were used to compare associations between race (black (bl) vs non-black (nbl)) and dichotomous characteristics. student´s t-test was used to compare continuous variables. results , patients were evaluated ( cases and controls). % and % of cases and % and % of the controls were bl and nbl respectively. the baseline prevalence of chtn in bl and nbl controls was . % and . % (p= . ). when comparing bl and nbl cases, bl women had a higher mean systolic blood pressure and screening bmi. bl women also had a trend toward being discharged on post partum blood pressure medicine when compared to nbl women. there was no difference in chtn, diabetes, severity of disease, iugr, or delivery < wks between the two groups (table) . to determine the leading causes of death in a case series of stillborn infants examined in a large hospital autopsy service, and to describe the most common post-mortem observations. study design: one hundred and sixty one stillborn infants were examined. gross pathology observations were recorded at autopsy and during the placental exam, and tissue sections were collected and examined microscopically. immediate and underlying cause of death (cod) were recorded, along with contributory cod, concomitant/significant cod, and incidental findings. statistical analysis was conducted using the software spss v. . . results: the immediate anatomic cod could be determined in . % of all infants examined. in over % of these cases, cod was attributable to placental or umbilical cord findings affecting the maternal-fetal blood supply. the most prevalent among these findings were placental lesions (maternal floor infarction, placental abruption, fetal thrombotic vasculopathy), umbilical cord lesions (entanglement, true knot, compression, excessive length/twisting), and infectious/inflammatory processes (chorioamnionitis, chronic villitis objective: advanced maternal age (ama) is associated with increased risk of intrauterine fetal demise (iufd). antenatal testing (at) is widely used in clinical practice to prevent iufd due to uteroplacental insufficiency and has been suggested to reduce the risk of iufd in ama women. we sought to assess the impact of at on obstetrical interventions and compliance with new practice recommendations. methods: retrospective cohort of ama women ( and older at their due date) who delivered at or after weeks. non-exposed women delivered from july to december (when at for ama was not routinely recommended); exposed women delivered from july to december (after at for ama was introduced at our institution). subjects were identified through the perinatal database; records were abstracted for demographics, medical history, and labor/delivery variables. outcomes included rates of at and induction of labor (iol) and mode of delivery. associations between at for ama and outcomes were tested using t test and chi square. assuming a baseline rate of iol of %, we had % power to detect an increase to % after the introduction of at. results: women met the inclusion criteria: delivered before the introduction of at (non-exposed=before at) and delivered after the introduction of at (exposed=after at). baseline clinical characteristics were similar in both groups. as anticipated, at was more common in the after at group than in the before at group ( % vs %; p< . ). women were not eligible for or declined trial of labor, thus not "at risk" for iol and not included in all analyses. ob intervention rates were increased after at compared to before at (table ) . the corrected iufd rates were similar in both groups ( / vs / ). conclusion: at an academic center, compliance with new practice recommendations was excellent. introducing at testing for a new indication seemed to increase iol and cs rates. these findings should be considered when assessing the risks:benefits ratio of antenatal testing. . we stratified indications for cesarean into the following: failed induction (cervix < cm dilated), arrest of dilation, arrest of descent, failed operative delivery, fetal intolerance of labor (fil), and "other" reasons (e.g., pre-eclampsia, abruption, chorioamnionitis, malpresentation). both fil and "other" reasons were more likely to occur among the medically indicated group (rr . , . physicians in solo practice had higher rates of elective inductions (p< . ), but there was no association between cesarean and practice type. conclusions: inductions accounted for . % of cesareans. these results suggest an increased risk for cs for patients undergoing medically indicated inductions at our institution. there was no association between cesarean and type of practice, whether solo or group, suggesting institutional clinical policies may be more important than practice type in determining delivery outcome after induction. further research is needed to understand how age, race/ethnicity, or other unmeasured patient factors may impact these findings. given rising rates of both cesarean delivery and inductions, this information may be pertinent to women considering elective induction prior to weeks. objective: fetal demise can lead to a consumptive coagulophathy ("fetal death syndrome") traditionally attributed to the release of "tissue thromboplastin", now known as "tissue factor" (tf). tf is the most potent activator of coagulation. despite the appeal and acceptance of this proposed pathophysiology, there is no evidence supporting this view. this study was undertaken to determine if fetal death prior to development of fetal death syndrome is associated with changes in maternal plasma concentration of cd l (a marker of platelet activation), tf and its soluble inhibitor (tfpi). methods: a cross-sectional study included the following groups: ) women with normal pregnancy (n= ) and ) patients with fetal demise without disseminated intravascular coagulation (n= ). plasma concentrations of scd l, tf and tfpi were measured by elisa. standard coagulation tests were performed. non-parametric statistics were used for analysis. results: ) patients with fetal demise had a higher median maternal plasma scd l concentration than women with normal pregnancy (median . pg/ml, range - vs. median . pg/ml, range . - . , p< . ); ) there was no significant difference between the groups in the median maternal plasma tf concentration and ) in contrast, the median maternal plasma tfpi concentration was significantly lower in patients with fetal demise than in women with normal pregnancy (median . ng/ml, range . - . vs. median . ng/ml, range . - . , p< . ). conclusions: ) a change in the plasma concentration of tf was not demonstrated; ) a change in the ratio of tf/tf inhibitor pathway may predispose to thrombin generation and activation of the coagulation cascade; ) however, maternal platelet activation is present in patients with a fetal demise without fetal death syndrome and ) the role of tf in fetal death syndrome remains to be proven. lipoic acid inhibits matrix metalloproteinase production, activity and prostaglandin e secretion by cultured amnion epithelial and mesenchymal cells. r moore, j novak, d kumar, j moore. case western reserve university, cleveland, oh, usa. introduction: cytokines, free radicals, matrix metalloproteinases (mmp) and prostaglandins (pg) have been implicated in processes of fetal membrane rupture and labor. dietary anti-oxidant supplementation has been suggested as a possible therapy for high risk patients, however, clincal evidence supporting the efficacy of agents such as vitamin c or n-acetylcysteine remains controversial. in fact, we have previously shown that vitamin c increases matrix metalloproteinase (mmp) activity in isolated fetal membrane fragments and fails to inhibit tumor necrosis factor (tnf) induced fetal membrane weakening in vitro. in this study, we examine the effect of the naturally occurring anti-oxidant, -lipoic acid, on tnf induced mmp activity/protein and pge secretion in isolated amnion epithelial and mesenchymal cells. methods: amnion epithelial and mesenchymal cells were pre-treated with increasing doses of -lipoic acid ( - mm/ h), then with increasing doses of tnf ( - ng/ml/ h). medium and cells were analyzed by gelatin zymography/western blotting for mmp /mmp activities/protein. pge output was determined by immunoassay. results: tnf induced a dose dependent increase in mmp production, secretion and activity in amnion epithelial cells. tnf ( ng/ml) induced an fold increase in cellular active mmp production and fold increase in secreted mmp enzyme activity by amnion epithelial cells. these increases were reduced - % following h pre-treatment with . - . mm -lipoic acid. mmp protein/activity and pge secretion by amnion epithelial cells were barely detectable and unaffected by tnf and/or -lipoic acid treatment. in striking contrast, mesenchymal cells exhibited little basal or tnf induced mmp protein/activity. mmp protein/activity in mesenchymal cells were unaffected by either tnf and/or -lipoic acid. however, tnf treated mesemchymal cells exhibited a dose dependent increase in pge production ( fold increase/ ng/ml tnf/ h) that was inhibited by %- % following . objective: nearly fifty years after the discovery of microphthalmia-associated transcription factor (mitf), its gene was identified as a specialized transcription factor that dictates cell-specific differentiation. unique mitf isoforms are generated from alternative promoter usage. an isoform of mitf (mitf-cx) is down-regulated in cervical stromal cells of the ripened cervix. further, mitf-cx inhibits il- gene expression and thereby suppresses signaling of the final pathway in cervical ripening. since mitf binds to canonical eboxes (canntg) in promoter regions of target genes, we sought to determine if mitf regulated its own promoter through ebox motifs. methods: the kb genomic dna sequence upstream of the mitf-cx transcription start site was cloned into pgl luciferase reporter vectors which were co-transfected with wild type or mutmitf-cx (impaired dna binding) into cervical stromal cells or hek cells. at h, promoter activity was determined and normalized for transfection efficiency. results: gel-shift assays conducted with oligonucleotides corresponding to eboxes in the mitf-cx promoter revealed two strong binding sites (eboxes - to - and - to - ). specific binding was established using oligonucleotides with or without mutated ebox, antibody supershift experiments, competition with cold probe, and absence of binding to mutmitf-cx. binding specificities were confirmed in nuclear extracts from cells that overexpressed mitf-cx, but not control or mutmitf-cx. reporter gene studies indicated that mitf-cx, but not mitf-m, increased mitf-cx promoter activity -to -fold. whereas mutations in ebox or resulted in significant decreases in mitf-stimulated promoter activity, mitfinduced increases in promoter activity were abolished by mutations in both eboxes. conclusions: collectively these experiments indicate that mitf-cx is a vital regulator of its own promoter activity and acts in a positive feedforward loop through two specific binding sites in its promoter. moreover, isoform-specific amino acids are important to mediate mitf-induced mitf-cx promoter activity. decreasing mitf protein or mutating its promoter would interrupt this loop resulting in rapid reduction of mitf synthesis. since mitf-cx suppresses il- gene expression in cervical stromal cells, we suggest that preservation of mitf-cx-induced mitf gene expression is an important mechanism to maintain the "brake" on cervical ripening and ensure cervical competency during pregnancy. the role of cd in cervical remodeling. denisse sanchez, brenda timmons, mala mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa. objective: prior to the onset of parturition, the uterine cervix undergoes a remodeling process from a closed, rigid structure, to one that is soft and dilatable. many changes occur, including increases in hyaluronan (ha), a glycosaminoglycan that facilitates loosening of the collagen matrix. in the postpartum period, the concentration of ha is reduced to that of the nonpregnant state. cd , a transmembrane glycoprotein expressed in hematopoietic and epithelial cells, is a receptor for ha. cd expression by immune cells is important in extravasation of leukocytes into tissue. cd may also be required for ha catabolism through the action of hyaluronidases and . in the cervix, cd is expressed in the endo-cervical epithelia as well as in immune cells localized in the stromal matrix. to study the importance of cd in cervical remodeling, mice with a null mutation for cd (cd -/-) were evaluated during pregnancy, parturition and postpartum. methods: changes in ha amount and size distribution were assessed using ha molecular weight gels and fluorophore assisted carbohydrate electrophoresis. to identify defects in cervical remodeling in the cd -/mice, differences in expression for genes regulated in the cervix were studied by quantitative real time pcr . to determine whether cd plays a role in the recruitment of immune cells during cervical ripening and postpartum repair, immunohistochemistry with antibodies against leukocytes was done. in the postpartum period, there is a higher ratio of high molecular weight ha relative to low molecular weight ha in the cd -/mice. this suggests that postpartum breakdown of ha in cd -/cervices is delayed. as compared to wt cervix, there was a significant increase in hyaluronidase (hyal- ) mrna in the postpartum period. the distribution and relative numbers of immune cells in the cd -/cervix was similar to wt. conclusion: these studies provide evidence that cd may play a role in remodeling of the postpartum cervix back to the nonpregnant state. our current data suggests that the catabolism of ha after birth is delayed in the mutant mice and upregulation of hyal may compensate to allow ha removal. furthermore, the activity of hyal- may be dependent on cd . little difference in the recruitment of immune cells between cd and wt animals suggest that cd expression is not required for this process. these experiments provide a greater understanding for the role of cd and ha in cervical remodeling. in background: during in vitro experiments we have shown that separation of amnion from choriodecidua occurs as an integral part of the process of fetal membrane (fm) rupture. although spontaneous amnion and choriodecidual separation is seen in fm after both svd and elective c/s deliveries, its etiology is uncertain. biochemical degradation at the amnion-choriodecidua interface may be a key contributing factor. our previous biomechanical studies have demonstrated that separated fm require less physical work to rupture than intact membranes. the purpose of this study was to determine whether fm separation was associated with clinical differences in the birth process. hypothesis: during term, normal labor, spontaneous separation of fm is associated with differences in the clinical parameters of labor and delivery. study design: fm from consecutive term deliveries were cut off the placental disk. separated areas of fm were cut from the intact areas. both were weighed and their weight ratios determined. maternal medical, pregnancy, and delivery data were collected and analyzed. results: term fm had the following characteristics: maternal age ± . yr, gravida . ± . , gestation ± . wks, elec. cs . %, duration of rom ± min, duration of contractions ± min, african american %. % of the fm had < % separation; % had more than % separation. srom fm with > % separation (vs. < %) had significantly shorter duration of rom (p= . ) and admission to birth (p= . ) times. srom fm with > % separation (vs. < %) had even shorter rom (p= . ), duration of contractions (p= . ) and admission to birth (p= . ). the > % group (vs. < %) was further along, gestationally (p= . ). srom fm (vs. arom) had shorter admission to birth (p= . ), but longer rom to birth (p< . ) times. absence of epidural (p= . ), srom mode of rupture (p= . ), svd (vs. elec. c/s) (p= . ), and the presence of meconium (p= . ), were all associated with increased fm separation. conclusion: spontaneous separation of fetal membranes is nearly universal and is associated with increased gestation, spontaneous rupture of membranes, shorter duration of contractions, and svd. speculation: we speculate that programmed biochemical changes initiate fm separation which then facilitates rom and childbirth. whole genome array and si-rna investigation of the function of nfkb in human amnion epithelial cells. sheri e lim, shirin khanjani, yun s lee, tg teoh, , philip r bennett. institute of reproductive and developmental biology, imperial college, london, united kingdom; maternal fetal medicine, st. mary's hospital, london, united kingdom. introduction: labour is associated with activation of nfkappab in the amnion. nfkappab increases prostaglandin synthesis through the upregulation of cyclooxygenase- (cox- ), which is essential to the labour process. cox- mrna expression increases with gestation in the amnion. primary amnion epithelial cells cultivated from tissue collected prior to the onset of labour display a spectrum of nfkappab activation, similar to the spectrum of cox- expression presumably relating to the nearness of labour. our aim was to investigate the full range of genes under nfkappab control in amnion epithelial cells by using whole genome arrays. methods: amnion from women undergoing elective caesarean section was collected and primary cell cultures established. total rna and protein were extracted from each culture. nuclear localization of nfkappab is required for its activation. western analysis of nuclear p was therefore performed to identify the samples displaying the lowest and highest nfkappab activity. the corresponding rna samples displaying the three lowest and three highest nuclear p protein concentrations were used for whole genome analysis using affymetrix u arrays. results and conclusions: we identified significantly regulated genes. the gene with the highest fold change was cox- (x . ) followed by oxytocin receptor (x . ), ch orf (x . ), integrina (x . ), and connective tissue growth factor (x . ). other significant genes included interleukin- (il- ) (x . ). pathway analysis revealed the majority of other nfkappab associated genes were involved in cell signaling, turnover and proliferation. we used real-time pcr (rtq-pcr) to validate cox- and il- expression. to prove that cox- is directly regulated by nfkappab, primary amnion epithelial cells were then transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna pool. western blot analysis confirmed knockdown of nfkappab p associated with inhibition of cox- demonstrating that nfkappab is essential for cox- expression. objective: premature birth is a major public problem accounting for over , deaths and , surviving infants with life-long morbidity yearly. in order to develop a rational basis for treatment and prevention of premature fetal membrane (fm) failure, we first need to understand the sub-failure fm structural and mechanical behavior at near full term. methods: we utilized planar biaxial mechanical testing, which approximates the physiologic loading state, for mechanical evaluation of the fm, and a structural constitutive model approach was used to offer insight into the structure-strength of the fm by integrating information on tissue composition and structure. small angle light scattering (sals) was used to nondestructively quantify the collagen fiber architecture of both intact and separated fm layers. results: in the stress free state, the gross collagen fiber architecture of the fm and separated layers were not homogenously align but exhibited small regions of fiber alignment. the amnion layer displayed the greatest alignment. the model fit the equi-biaxial strain data well (r = . ) and indicated that fm collagen fibers were rapidly recruited and straightened well below failure stress levels. collagen fibers were gradually recruited followed by a drastic increase in fiber recruitment. conclusion: this study provided the first data on the effective collagen fiber stiffness in the intact fm under physiologic biaxial loading, which was related to quantitative collagen fiber architectural measures. modeling results indicated that the collagen fibers became fully loaded and straighten well below physiological loading levels. failure did not occur during physiological loading, indicating that fibers do not begin to fail until all collagen fibers are fully straightened and bearing load. this result suggested modest structural reserve in the fm collagen architecture, and may be an important aspect of its failure properties. previously, we demonstrated that a physically "weak zone" exists overlying the cervix in the fm, evident of collagen remodeling and cellular apoptosis. we are currently extending the present study to include the "weak zone" tissues, allowing us to elucidate the micro-mechanical mechanisms that facilitate failure in this newly identified fm zone. supported by nih . the extracellular matrix of the cervix undergoes extensive remodeling during parturition. hyaluronan (ha) is a major constituent of the extracellular matrix of the term pregnant cervix. the onset of labor is preceded by an increase in ha and after delivery, the concentration of cervical ha gradually decreases to that of the non-pregnant state. these dramatic changes suggest that ha plays an important role during parturition. hyaluronan synthase (has ) is one of three known ha synthases and the most abundant isoform in the pregnant cervix. transcripts for has are regulated by two alternative promoters, one upstream of the first coding exon (proximal promoter) and another upstream on an untranslated exon (distal promoter). the focus of the current study is to further our understanding of the transcriptional regulation of has during cervical ripening. methods: rna blotting was carried out using transcript specific probes corresponding to the distal and proximal promoter of the mouse has gene. the regulation of has was evaluated in a cervical epithelial cancer cell line (caski cells) which we have previously shown to express endogenous has . regulation of has expression by epidermal growth factor was assessed in the caski cells by western blotting and quantitative real time pcr assessment of transcripts. results: has mrnas in the nonpregnant (np) and pregnant cervix are transcribed from the distal promoter upstream of exon . two transcripts of approximately . kb and . kb were detected that arise from use of polyadenylation sequences. as compared to np, the expression of has is increased , , and fold on gestation days , and shortly postpartum respectively. has mrna expression is increased upon treatment of caski cells with epidermal growth factor ( ng/ml) and is suppressed in cells treated with ag ( m), an inhibitor of egf receptor phosphorylation. maximal stimulation was observed at and hours of treatment. conclusion: has is the major ha synthase expressed during cervical ripening and the majority of transcripts are driven by the distal promoter in the has gene. in vitro studies using caski cells suggest has is regulated in part by the egf signaling pathway resulting in a several fold increase in has expression. these results provide an understanding of has gene regulation at the time of cervical ripening which will ultimately enhance our understanding of the molecular mechanisms important to cervical ripening. chorion and decidua cells. chad a grotegut, bernard j canzoneri, liping feng, phil heine, amy p murtha. obstetrics and gynecology, duke university, durham, nc, usa. objective: preterm premature rupture of the fetal membranes accounts for approximately % of all preterm deliveries. cigarette smoking independently carries a fourfold increase risk for pprom. our laboratory has previously demonstrated that the chorion layer undergoes apoptosis in women with pprom. this study was conducted to determine if extract of cigarette smoke causes cell death in specific cells of the fetal membrane. fetal membranes were collected at the time of elective cesarean section from women without labor and at term. the chorion and decidua layers were separated and purified on a gradient spin column and then plated near confluence. cigarette smoke extract (cse) was collected in cell media and used to treat chorion and decidua cells in culture at concentrations ranging from % to % in -well plates. cell viability was determined at , and hours following treatment with a non-radioactive cell viability assay. data were analyzed using paired t test (analyse-it, leeds, uk). chorion and decidua cells underwent cell death when exposed to cse in a dose dependent fashion. increasing concentrations of cse resulted in increased cell death at hours in both cell types (figure ). at hours, chorion exhibited greater percent cell death compared to decidua at concentrations of , and % cse (p= . , . , and . , respectively). for any given concentration of cse, the degree of cell death increased with increasing length of exposure ( , and hours) for each cell type. chorion cells routinely exhibited greater percentage of cell death following treatment with cse at concentrations ranging from - % compared to decidua cells. human chorion and decidua cells in primary cell culture exhibit a dose-response and time dependent cell death in the presence of cse. human chorion cells show greater sensitivity to cell death when compared to decidua cells. further studies are needed to determine the mechanisms through which these cell types undergo cell death and the implications for the differential sensitivity to cigarette smoke. yoon ha kim, tae-bok song, cheol hong kim, jong woon kim, moon kyoung cho, sung yeul yang, bong whan ahn. obstetrics gynecology, chonnam national university medical school, gwangju, korea; biochemistry, chonnam national university medical school, gwangju, korea. objective: to investigate the lipid peroxide levels and protein carbonyls levels in the amniotic fluid of pregnant women with preterm premature rupture of membranes (pprom). the lipid peroxide levels in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced measured by thiobarbituric acid reaction. the protein carbonyl contents in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were determined by the , -dinitrophenylhydrazine method. after amniotic fluid of them were mixed and incubated up to hours with . ml of mm moxalactam, cefodizime, amoxacillin, erythromycin, the lipid peroxide levels and protein carbonyl contents in them were measured. results: . the lipid peroxide levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein, p< . ). . the protein carbonyl levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein p< . ). . the lipid peroxide levels and protein carbonyls formation by moxalactam in the amniotic fluid of pregnant women with pprom was significantly higher than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . the lipid peroxide levels and protein carbonyls formation by cefodizime in the amniotic fluid of pregnant women with pprom was significantly lower than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . there were no significant differences in the levels of lipid peroxide and protein carbonyls by amoxacillin and erythromycin in the amniotic fluid of pregnant women with pprom between antibiotics-induced and basal levels. background: chorioamnionitis (cam) is a major antecedent of preterm delivery (ptd) associated with elevated amniotic fluid tnf and il . we hypothesized that these cytokines enhance the term decidual cell (dc) expression of the matrix metalloproteinases (mmp) and , which can then promote ptd by degrading the extracellular matrix of the decidua, fetal membranes, and cervix. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on cam-complicated (n= ) and gestational age-matched control decidua (n= ), and staining intensities were evaluated by hscore. confluent, leukocyte-free term dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ), and quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in cam-complicated decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in control decidua ( ± , and ± respectively; p< . ). in cultured term dcs incubated with e , tnf and il significantly increased secreted levels of mmp- compared to e alone (pg/ml/ g protein: . ± . and . ± . , respectively, vs. . ± . ; p< . ). in parallel incubations with e +mpa, basal mmp- output was lowered by %, and tnf -and il -elicited mmp levels were blunted by % and %, respectively. rt-pcr confirmed that tnf and il increased mmp mrna levels (p< . ), although mrna levels in e +mpa incubations were not different from those of e alone. mmp levels in all treatments were similar. conclusions: mmp- and mmp- are elevated in cam decidua compared to controls. our in vitro results suggest that mmp- expression is enhanced by the high levels of il and tnf associated with cam, and that mpa may be able to blunt this effect. we have previously found a similar regulatory mechanism of mmp- and mmp- and their over-expression in cam-complicated tissues. synergy among these mmps may represent a potent pathogenic mechanism of cam that can be targeted through the therapeutic use of progestins in preventing cam-induced ptd. domerudee preechapornprasert, patama promsonthi, wasun chantratita, mana rochanawutanon, patcharee karnsombut, chutatip srichunrusami, stephen j lye, boonsri chanrachakul. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: cervical ripening is an inflammatory process involving chemokines, cytokines and various mediators. recent study has shown that the level of monocyte chemotactic protein (mcp) , a chemokine, increases in amniotic fluid during spontaneous labor. the aim of this study was to examine the localization and expression of mcp in human cervix before pregnancy, during pregnancy and after the onset of labor. methods: this study was approved by the local ethics committee and written informed consent was obtained from each participant. cervical biopsies were taken from groups of women; nonpregnant women, first trimester pregnant women, term pregnant women with and without labor. tissue samples were fixed in % formal saline for paraffin section. immunohistochemistry (n = each) was performed by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . the mcp messenger(m) rna was identified by reverse transcription-polymerase chain reaction using gene specific primer against mcp and mcp receptor (n = each). results: immunohistochemistry demonstrated mcp in cervical tissues from all four groups of women. mcp was localized on plasma membrane and cytoplasm of both squamous epithelial and columnar cell lining of endocervical gland. mcp and mcp receptor mrna were identified in nonpregnant, first trimester and term with and without labor human cervix. conclusion: mcp and mcp receptor were located in cervical tissues of nonpregnant and pregnant women at different gestation both before and after the onset of labor.ongoing studies are investigating the role of this chemokine during pregnancy and labor. whether preterm cervical ripening is just an aberrant regulation in timing or whether divergent mechanisms and pathways are involved in preterm versus term cervical ripening remains to be elucidated. methods: cervical tissue was collected from groups of cd- mice. group : mouse model of ptb that utilizes intrauterine infusion of lipopolysaccharide (lps). group : e dams representing preterm controls. group : e . - dams selected from a timed pregnant batch where half of the dams had delivered representing term cervical ripening. n= dams/treatment group. separate rna samples were used for microarray analysis (ma). significance analysis for ma and partek software was used for biostatistical analysis. pathway analysis was performed using david. quantitative pcr was performed to confirm the most differentially regulated genes. results: using a cut-off of -fold change with p value of < . , genes in the cervix were differently regulated between the groups. principal component analysis revealed three distinct groups (see graph). functional annotation clustering demonstrated the following pathways: ) in preterm cervical ripening (e lps vs e ): immune and inflammation response, defense response ) in term cervical ripening compared to preterm controls: negative regulation of cellular process and biological process, ecm, cell-cell communication. qprc confirmed the highly significant differences found in ma (see table) . conclusions: the molecular mechanisms and pathways governing preterm and term cervical ripening are distinctly different. elucidating these unique pathways can lead to improved therapeutics for prevention of ptb as well as for postdate pregnancies. cervical ripening at term involves activation of apoptotic enzymes. maria kb sennstrom, valentina ciani, gunvor e ekman. obstetrics and gynecology, women and child health, karolinska institute, stockholm, sweden; obstetric and gynecology, university of siena, siena, italy. aim: to investigate if the human cervical ripening at term involves programmed cell death. during the final cervical ripening the extracellular matrix dominated cervix undergoes an extensive remodelling of the tissue. inflammatory mediators such as the cytokines increase in ripening cervical tissue at term. programmed cell death, apoptosis, has been suggested as important in this process. apoptosis can be induced by inflammatory mediators such as cytokines. we also looked upon the distribution of inflammatory cells in cervical tissue. materials and methods: cervical biopsies from pregnant women at term and post partum women with fully ripened cervix were studied. biopsies from non-pregnant women served as controls. immunohistochemical analysis of the apoptotic enzyme caspase- and the inflammatory cell marker cd was performed on paraffin embedded sections of cervical tissue. double staining was performed. mann-whitney u-test was used for statistical analysis. results: there was a significantly higher frequency of caspase- staining in the post partal sections from ripened cervical tissue compared to tissue from term pregnant (p= . ) with unripe cervix and from non-pregnant patients (p= . ). the inflammatory cells staining for cd increased in post partal and term pregnant sections compared to non-pregnant (p= . ). there was a higher frequency of caspase- positive cells in the post partal tissue than of cd positive cells (p= . ). the localization of cd- positive staining was highest in the epithelia and basal lamina while caspase- staining was most pronounced in stromal tissue and around vessels. conclusion: our data show apoptotic activity in stromal tissue in fully ripened human cervix at term of pregnancy, suggesting that apoptotic mechanisms are involved in the extracellular matrix remodelling at term. the apoptotic activity is not co-localized with inflammatory cells suggesting non-infectous inflammation with apoptosis as important for cervical ripening at term. objective: cervical biomechanical responses are important for accommodating the increased stress induced by an enlarging uterus. a mechanical testing system was modified to evaluate the stress relaxation response in the pregnant cervix. methods: tissue harvested from the non pregnant and timed pregnant (days , , , , and ) sprague-dawley rats underwent tensile testing using an instron material testing system. the testing regimen consisted of tissue extension to near maximal strain over seconds followed by a period of constant strain for minutes, then return to rest over seconds. this cycle was repeated additional times with a minute rest period between cycles. strain and force measurements were recorded at second intervals. - animals were used for each time point. in addition, stress and strain at the yield point was also determined for each gestational time point. results: the pregnant and non pregnant samples exhibit marked differences in response in both stress and strain. the timed pregnant tissue demonstrated progressively increased compliance and lower nominal stress compared to non pregnant tissue. peak nominal stress declined with each successive cycle. this was demonstrated in the peak nominal stress and strain values at the yield point. conclusion: the cervix becomes more distensible (compliant) but less resistant to force with increasing gestational age. ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • introduction: a growing body of evidence supports that inflammatory processes are implicated in spontaneous preterm birth (ptb). using an inflammatory and non-inflammatory mouse model of ptb, we sought to determine if activation of these inflammatory pathways are essential for ptb and/or cervical ripening to occur. methods: timed pregnant cd- mice were used in these two models of ptb: ) a model of intrauterine inflammation where lipopolysaccharide (lps) is injected into the uterine horn (n= ); controls for this model received intrauterine saline (n= ) and ) a non-infectious model of ptb using ru sq ( ugrams/dam) (n= ); controls for this model received no intervention (n= ) were used for these studies. for both models, hours later uterine and cervical tissues were harvested. the tissues were processed for protein and rna studies. elisas were performed to assess il- and tnf-alpha in the uterine tissue. mrna expression of ifn , il- , il- , il- , tnf-alpha were assessed in cervical tissue from both models by quantitative pcr. results: in uterine tissues, both il- and tnf were significantly elevated in the lps-induced ptb when compared to control, saline, and non-infectiousinduced preterm birth (p< . ) (figure ). in cervical tissue, an increase in il- , il- beta and tnf mrnawas observed in both models, while ifn-gamma and il- were only increased in the lps model. (figure ). conclusions: up-regulation of pro-inflammatory cytokines in the uterus do not appear to be essential for ptb. cytokine expression in the cervix is greater in an inflammatory model of ptb but is also present in a non-infectious model. these studies suggest that targeting a cytokine response in the cervix may hold the most promise in prevention of ptb. the initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (af) and infiltration of the myometrium by neutrophils and macrophages (m ). whereas, in preterm labor, intra-amniotic infection may provide the stimulus for increased af interleukins and inflammatory cell migration, the stimulus for these events at term has remained uncertain. in studies using pregnant mice, we observed that the m that invade the maternal uterus near term arise from the fetus. furthermore, we obtained compelling evidence that surfactant protein-a (sp-a), a developmentally regulated c-type lectin secreted by the fetal lung into af near term, activates af m , which migrate to the uterus where they promote an inflammatory response culminating in labor. we propose that interactions of m surface receptors with sp-a, at term, or bacterial lipopolysaccharide at preterm, initiate changes in m phenotypic properties, resulting in the enhanced expression of genes that promote their migration to the uterus. the objectives of the present study were to analyze the numbers and phenotypic properties of mouse af m during late gestation and to identify their putative tissue source(s) of origin. to assess changes in the number of m in af during late gestation, af cells were isolated and stained for the m marker f / . the density of adherent f / + cells greatly increased in equivalent volumes of af between . and . days postcoitum (dpc) ( . dpc = term). interestingly, the f / + cells at . dpc were highly similar in morphology to those present in . dpc fetal lung, but distinctly different from those in fetal liver, suggesting their pulmonary origin. the af m were foam cell-like, suggesting the presence of lipid inclusions, a property shared by adult alveolar m . to further analyze gestational changes in the m population(s) in mouse af, we used flow cytometric analysis. in our initial studies, cells isolated from af were stained for f / and for cd , a pan-leukocyte marker. we observed that af from . and . dpc mice contained two sub-populations of cd + f / + cells. studies are in progress to analyze these af m populations for expression of cell surface antigens indicative of their maturity, activation state and chemotactic properties in association with the developmental induction of sp-a synthesis and secretion by the fetal lung. april bleich, patrick keller, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. the role of prs, proinflammatory cytokines, cell adhesion molecules, cox- , and toll-like receptors in mediating cervical ripening prior to labor is not clear. the objective of this study was to quantify pr isoforms and determine the relative expression of certain inflammation-related genes in cervical stroma from nonpregnant and pregnant women in early gestation (eg), term before and after cervical ripening, and during labor. methods: standard curves of pr-b, -a, pra+b and qpcr were used to quantify total and pr-b in cervical stroma from nonpregnant (proliferative, n = ; progestin treatment, n = ) and pregnant women undergoing hysterectomy (eg, n = ; term before ripening, n = ; after ripening, n = ; in labor, n = ). cervical status was determined by modified bishop scoring. results: total pr expression was maximal in nonpregnant women in the proliferative phase ( . ± . pg/ug cdna) and decreased % by progestins ( . ± . pg/ug cdna). this level was maintained in stroma from pregnant women before labor ( . ± . , eg.; . ± . before ripening; . ± . after ripening pg/ug cdna). in contrast, total pr was decreased significantly in the dilated cervix ( . ± . pg/ug, p < . ). interestingly, whereas pr-b was ± % that of total pr in the nonpregnant cervix, pr-b mrna levels were ± % to ± % in cervical tissues from all pregnant women and did not vary with labor status. using immunoblot analysis and pr-specific antibodies (pgr ), pr-b immunoreactivity was % that of total pr in all samples from pregnant women, and both pr-a and -b were downregulated significantly in the dilated cervix (from ± to ± units/atub). decreased expression of pr in the dilated cervix was accompanied by significant increases in il- , mcp- , tlr- , cd l, cox- , and s a mrna (all p < . , anova) but not cd b or tlr- . pgdh mrna was decreased significantly in the dilated cervix. with the exception of cox- , expression of these genes was similar before labor regardless of cervical ripening. conclusions: both pr and pr-b are decreased proportionately in the dilated cervix, but not during cervical ripening. further, a number of inflammatory gene products are increased dramatically in the cervix during labor, but not before. taken together, the results suggest that cervical ripening is distinct from cervical dilation and involves upregulation of cox- but not il- , mcp- , or toll-like receptors. ifn-y il- il- , il- tnf- "p value < . lps/saline ru /controls . "" . " . " . * " " . * . . " association between interleukin- (il- ) and il- to examine association of amniotic fluid interleukin (il- ) concentration with il- and its receptor il -r haplotypes in term and preterm caucasians (c) and african americans (aa) samples. methods: in this study case (preterm birth -ptb [< weeks]) and control (term [> weeks]) amniotic fluid (af) il- concentrations were analyzed for association with haplotypes of the il- and il r genes in aa and c separately. in il- , eight, and in il- r, single nucleotide polymorphisms (snps) were examined. aa and c maternal and fetal genotypes were assessed (aa: maternal:cases- controls- fetal: cases- fetal controls-; c: maternal cases- , controls- , fetal:cases- ; controls= ). haplotype associations were performed by using a sliding window with outcome il- concentration. analyses were performed separately on maternal and fetal dna. results: the strongest haplotype associations were observed in il- r rather than in il- . in c fetal dna il- r haplotypes defined by markers - bp from the transcription start site associated most strongly with af il- concentrations (global p= . x - ) and in aa maternal il- r haplotype markers at - - - (global p= . x - associated with il- concentrations. in the c fetal cases the - haplotype with the highest concentration was a-g (log(cytokine) = . pg/ml). in aa maternal samples the highest concentration was observed for haplotype t-t-g-c at - - - .this was seen in both cases and controls at (case log (cytokine) = . ; control log(cytokine) = . pg/ml). significant associations from haplotype analyses converged on three regions of the il- r in both races. no strong differences were observed between the haplotypes of cases with and without microbial invasion of the amniotic cavity (miac), with the exception of aa fetal samples that showed two overlapping haplotypes in il- that associated in cases with miac but not in cases without miac (- and - ; - and - )(both with p < x - ) conclusion: differences in the af il- concentration in ptb do not result from single snp effect on il- but are a result of complex relationships between il- and il- r haplotypes. these associations exhibit racial disparity. the role of tnf-in parturition. helen alexander, amanda tattersall, mark tattersall, suren sooranna, peta grigsby, leslie myatt, mark johnson. obstetrics gynaecology, imperial college, london, united kingdom; obstetrics gynaecology, university of cincinnati college of medcine, cincinnati, oh, usa. introduction: tumour necrosis factor-alpha (tnf-) is thought to play a role in inflammation-induced preterm labour since the decidua produces tnfin response to bacterial products and amniotic fluid tnf-concentrations are increased in the presence of intra-amniotic infection. the aims of this study were to (i) investigate the expression of myometrial tnf-and its receptors in relation to the onset of preterm and term labour; (ii) to identify which intracellular pathways are activated by tnf-; and to investigate the effect of tnf-alone and in combination with il- or il- on gene expression in uterine myocytes. methods: biopsies of human myometrium were taken at caesarean section from women before and after the onset of preterm and term labour and analysed for tnf-and its receptor mrna expression. a further samples were obtained before the onset of labour (n= ) from which myocytes were isolated and cultured in -well plates. when cells were - % confluent either tnf-at a concentration of ng/ml was added to the cells for , , , and min and the cells analysed by western blotting or tnf-at concentrations of , . and ng/ml was added either alone or in combination with similar concentrations of il- or il- to cells for hours and mrna was extracted and converted to cdna to determine il- and gapdh gene expression by qpcr. results: there was no change in tnf-mrna expression in relation to the onset of labour, but the expression of tnfr and tnfr mrna levels were significantly increased with gestation and further increased with the onset of labour. incubation of uterine myocytes with tnf-( ng/ml) activated all three mapk substypes: erk, jnk, and p , activation peaked between - minutes. preliminary data suggest that tnf-induces il- mrna expression but exposure to il- or il- itself did not enhance this response. conclusions: although there is no significant increase in tnf-concentration from baseline during labour we have shown tnf receptor mrna levels do increase with labour at term. exposure of isolated uterine myocytes to tnfcauses activation of all mapk subtypes and an increase in il- mrna expression. this enhanced mapk-dependent il- expression at term may be mediated via increased myometrial sensitivity to tnf-through increased tnf receptor expression. remodeling. brenda c timmons, anna-marie fairhurst, mala s mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa; immunology, ut southwestern medical center, dallas, tx, usa. objective: the molecular mechanisms involved in cervical ripening are not well understood. immunohistochemical studies from our lab report a recruitment of inflammatory cells to the cervical stroma one day before birth in the mouse using a neutrophil/monocyte marker (neutrophil / ). in this study, we sought to identify and quantitate inflammatory cells migrating into the mouse cervix and to determine if this recruitment was affected by changes in progesterone levels. peripheral blood was also evaluated to see if changes in the cervix was paralleled in blood. methods: flow cytometric analysis was performed using cervical cells and peripheral blood obtained before and during cervical ripening along with - h postpartum. dispersion of cervical cells was optimized. these cells were stained with a panel of fluorescent conjugated antibodies directed against leukocyte antigens and analyzed on an lsrii flow cytometer. cells were also sorted and stained to visualize cell morphologies. to determine the effect of progesterone on the migration of leukocytes, gestation d mice were treated for h with a progesterone receptor (pr) antagonist prior to tissue collection. results: neutrophils do not appear to increase in the cervix until after birth. monocyte (mo) numbers do increase during cervical ripening (late day , d . ) and remain high through postpartum (pp). macrophages (mØ) are present prior to cervical ripening and steady state levels are maintained during labor and pp. pr antagonist treatment on d resulted in a premature increase in mo but not neutrophils or mØ. in contrast to the cervix, mo and neutrophil numbers do not significantly increase in the peripheral blood until pp. results from lymphocyte studies suggest a low level of b and t cells in the cervix. in the peripheral blood, b cells remain consistent through parturition and the t cells decrease by d . and continue to decrease pp. conclusion: tissue mo are increased in the cervix during ripening. this recruitment is dependant on loss of pr function. in contrast, neutrophils are increased in the pp cervix while mØ numbers appear constant. timing of changes in mo and neutrophil numbers in the peripheral blood differed from that observed in the cervix suggesting quantitation of these cell types in blood is not reflective of what is occurring in the cervix during ripening, dilation and pp repair. novel interactions between nf-b and other labour-associated transcription factors identified by a tf-tf array. shirin khanjani, yun s lee, suren r sooranna, mark r johnson, phillip r bennett. irdb, imperial college, london, united kingdom. introduction: external stimuli lead to changes in cellular gene expression through activation of inducible transcription factors. nf-b is a ubiquitous transcription factor classically associated with inflammation, which is activated in response to infection and proinflammatory cytokines such as those prevalent during the labour. the tf-tf interaction array uses a novel technology for detecting interactions between transcription factors based on binding of tfs to their own consensus dna binding sequence. materials and methods: primary myometrial cells were grown until - % confluent and stimulated with ng/ml il- prior to nuclear protein extraction. the nuclear extracts were incubated with the provided set of biotin-labeled, double-stranded oligonucleotide probes, which represent a known library of cis-elements. during the incubation step, these tf probes bind to their specific tfs in the nuclear extract. next, immunoprecipitation was performed using an antibody against nf-bp , which pulled out nf-bp and any tfs bound to it, bound to corresponding cis-elements. normal igg was used in a parallel experiment to represent a negative control. free cis-elements and nonspecific binding proteins were washed away and the cis-elements were finally eluted and hybridized to the array membrane, which is spotted with different tf consensus sequences. results: table shows the different tfs interacting with nf-bp based on the degree of binding stimulated by il- compared to no-il- control. conclusion: these data show that il- stimulation causes nf-b to bind to a wide variety of other treansciption factors. of particular interest in the area of parturition is binding to the other pro-inlammatory tfs such as c/ebp and ap- , which are known to regulate labour-associated genes in synergy with nf-b. the lack of association between nf-b and pr without il- stimulation supports the concept that with the onset of labour inflammation leads to functional progesterone withdrawal, rather than pr acting to inhibit inflammation. table high ap- , c/ebp, cbf, creb , c-myb, e f- , ets, ets- /pea ,fast- , gas/isre, hse, mef- , mef- , myc-max, nf- , nfatc, nf-e , nf-e , pax- , objective: pre-partum cervical ripening involves remodeling of collagen structure and inflammatory immune cell activity (jsgi : , ; reprod biol endo : , ) . although parturition is associated with systemic or local progesterone withdrawal (ajog : , ) , effects of a decline in progesterone on cervical ripening is not known. the present study tested the hypothesis that progesterone withdrawal promotes collagen degradation, innervation, and immune cell trafficking in the cervix of nonpregnant mice. methods: adult virgin female c bl mice received capsules (sc) with oil vehicle (v) or estradiol (e) and progesterone (p) to simulate concentrations in pregnancy (hum reprod : , ) . after days, mice in the v and e+p groups were euthanized. the p capsule was removed from some mice on day (e-p) and groups killed on days and . cervix sections were stained for collagen, nerve fibers, macrophages, or neutrophils (n= /group/day; sections/ cervix; biol reprod : , ; jsgi : , ) . stained macrophages and neutrophils were counted (image pro-plus , media cybernetics). results: e+p treatment for days promoted hypertrophy of the cervix compared to v controls, i.e., collagen content and structure diminished, cell nuclei density declined, and nerve fibers increased. removal of p did not affect these endpoints. for immune cells, e+p for , , or days decreased immune cell numbers. by contrast, p removal increased macrophages and neutrophils in the cervix on days and (p< . , e-p vs e+p groups, respectively). the census of resident immune cells in e-p groups at and h after p removal equaled that in the v group. conclusions: mimicking gonadal steroid concentrations in circulation during pregnancy promotes hypertrophy and suppresses immigration of immune cells in the cervix. in this non-pregnant murine model for parturition, progesterone withdrawal recruits immune cells, but fails to promote further remodeling or hyperplasia of nerve fibers in the cervix. the findings raise the possibility that ripening of the cervix requires not only recruitment, but also activation of immune cells. whether proinflammatory activities by specific immune cells affect nerve fiber hypertrophy or neural activity, as part of the mechanism for ripening of the cervix, remains to be determined. we have previously identified and characterized a truncated pr (pr-m), that localizes to the mitochondrion by multiple experimental techniques, including confocal imaging of a recombinant gfp fusion protein, western blot analysis after cellular fractionation of nuclear pr negative t d-y breast cancer cells and western blot analysis of purified human heart mitochondrial proteins. initial studies with nuclear pr negative mcf- a breast epithelial cells shown to express pr-m demonstrated an increase in mitochondrial membrane potential (mmp) with progesterone/progestin treatment. these studies led to the hypothesis that progesterone modulates cellular respiration via the mitochondrial receptor, pr-m. objectives: the present studies sought to further localize pr-m to the outer, inner or matrix portion of the mitochondrion and to correlate the increase in mmp with total cellular atp production. additionally, the potency of progesterone and synthetic progestins on the change in mmp was evaluated. methods: the location of pr-m was determined by western blot analysis after fractionation of human heart mitochondria with digitonin treatment and differential centrifugation. mmp was determined in mcf- a breast epithelial cells and a rhabdomyosarcoma cells by the change in fluorescent emission of jc- . total atp was determined by a bioluminescent assay. results: western blot analysis after mitochondrial fractionation showed pr-m localization exclusively in the outer membrane. a dose-dependent increase in mmp was seen in cell lines with - min treatment with progesterone and r which were inhibited by a specific pr antagonist, rti- - b, and not affected by the translational inhibitor, cycloheximide. similar changes in mmp were seen with the same concentration of progesterone, mpa and r . progesterone/progestin treatment for min led to an increase in total cellular atp without a change in cell number. conclusions: progesterone/progestin treatment results in an increase in cellular respiration in cells expressing an outer mitochondrial membrane pr and known to lack nuclear pr expression. this may represent a mechanism whereby progesterone enhances cellular energy production to meet the demands of pregnancy. introduction pge is a major product of the fetal membranes, decidua and myometrium and plays an important role in cervical ripening and myometrial contractions. there are four pge receptors, ep- and ep- mediate contractions whilst ep- and ep- mediate quiescence. ep- contains multiple consensus sequences for transcription factors known to be of importance in labour, in particular nfkappab. we therefore performed experiments to determine the effect of activation and inhibition of nfkappab upon ep- expression. myocytes plated in well plates were treated with il- ( ng/ml), to activate nfkappab. myometrial cells were also transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna to knock down nfkappab p . rna was extracted for amplifying ep- using quantitative rt-pcr with amplification of l as a control to normalise data. il- caused an increase in expression of ep- . knock down of nfkappab using si-rna resulted in a further increase in ep- . this data suggests that although il- stimulates expression of ep- it does so through an nfkappab independent mechanism. the upregulation of ep- with sirna knock down of p suggests that activation of nfkappab would inhibit ep- expression consistent with the concept that activation of nfkappab at term causes the myometrium to adopt a more contractile phenotype. introduction: a role for the pro-inflammatory cytokine il- is suggested in preterm and term birth, independent of the presence of infection. we previously showed that mice with a null mutation in the il- gene (ko) delivered one day later than wild type (wt) mice due at least in part to altered timing of uterine expression of prostaglandin (pg) f receptor, ptgfr, mrna. we also observed differences in mrna expression of other uterine activation proteins (uaps), pg h synthase (pghs)- , oxytocin receptor (otr) and connexin- (cx- ), suggesting multiple physiological effects for il- in term delivery. objective: to examine the effect of il- deficiency on the peri-partal uterine mrna expression of the pge receptors (ep) , and ; the post-partum changes of all uaps; and the relationship of these to serum progesterone (p ) concentrations. methods: gestational length was observed in pregnant c bl/ wt (n= ) and ko (n= ) mice. uap mrna levels were measured by real time rt-pcr in wt and ko dams sacrificed from d through delivery and up to h postdelivery. serum p was determined by ria. data were analyzed by one-way and two-way anova using the holm-sidak test to differentiate treatment effects at p< . . results: birth was delayed in the il- ko mice ( . ± . d vs. . ± . d), and this affected the timing of peri-partal changes for all uaps similarly. both ep and ep (relaxatory receptors) were elevated at d in ko dams, but returned to low levels before delivery and were elevated at delivery (ep ) or afterwards (ep ). ep levels did not change. otr increased several hours before delivery in all dams. cx- increased at delivery, then fell, while pghs- increased at delivery and remained elevated afterwards. ptgfr mrna increased - -fold at delivery and a further - -fold after delivery, suggesting loss of pgf permitted enhanced ptgfr expression. p serum concentrations fell pre-partum in both groups. conclusions: il- ko alters the expression pattern of several pregnancy and parturition-related genes and may delay the pre-partum p fall, suggesting a potential ovarian effect. a uterine role for il- in regulating the timing of normal term parturition cannot be ruled out. objective: recent studies have highlighted the prevalence of vitamin d deficiency in pregnant women, particularly in those from ethnic groups with darker skin who require higher levels of uv light to make parental vitamin d. as the active form of vitamin d, , -dihydroxyvitamin d ( , (oh) d ) is a potent immunomodulator, we postulated that vitamin d deficiency may lead to dysregulated placental immunity. both trophoblast and decidua express the enzyme -hydroxylase (cyp b ) which catalyzes synthesis of , (oh) d from the inactive pro-hormone -hydroxyvitamin d ( ohd ). in view of the role of trophoblast as a barrier site protecting the fetus against infection, we investigated the impact of cyp b , ohd and , (oh) d on innate immune responses in trophoblastic cells. methods: a trophoblast cell line was used to assess the effect of vitamin d on innate immune responses. the cells were treated for hrs with various concentrations of , (oh) d ( - nm) and antimicrobial cathelicidin expression was assessed by real time pcr. to assess whether expression of cyp b was affected by pathogenic stimuli, a cells were treated with ligands for toll-like receptor (tlr) - , cyp b expression (real time pcr) and ohd utilization were assessed. results: a trophoblast cell line; which shows temperature-sensitive differentiation, revealed expression of cyp b with higher levels of enzyme activity under conditions of syncytiotrophoblast development. cells treated for hrs with , (oh) d showed dose-dependent induction of the antimicrobial defensin cathelicidin expression ( . - fold induction), whilst cells treated pro-hormone ohd ( nm) showed . -fold induction of cathelicidin expression. activation of tlr (poly i:c) and tlr (lipopolysaccharide) enhanced the expression of cyp b ( -and . -fold) and increased the sensitivity to ohd as a consequence. conclusion: these data show that autocrine synthesis of , (oh) d from ohd can stimulate trophoblast immune responses in a similar fashion to macrophages. as ohd is the major circulating form of vitamin d, we hypothesize that trophoblast innate immunity may be significantly compromised under conditions of vitamin d deficiency. introduction: secretory leukocyte protease inhibitor (slpi) is a potent -kda protein inhibitor of neutrophil elastase and it is a mediator of mucosal immunity and an inhibitor of nfkb regulated inflammatory responses. however, its source, function and regulation within the uterus during pregnancy and at parturition are not well defined. it has previously been shown to be present in fetal membranes and cervical mucus and in amniotic fluid, where its levels is increased from second trimester to term and with a further increase at parturition. slpi has also been shown to be responsive to progesterone in human epithelial cells. our aim was to determine the effects of il- on slpi gene expression in human myometrium. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates and when cells were - % confluent they were serum starved overnight and incubated with m methyl- -hydroxy-progesterone acetate for h and with or without ng/ml il- for a further hours. at the end of incubations rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of slpi, gapdh and beta-actin were measured by qpcr. results: h incubation of uterine myocytes with ng/ml il- caused a marked increase in slpi by % (n= ; p< . ). incubation of uterine myocytes with m progesterone for h also increased slpi by % (n= ; p< . ). incubation with il- for h in the presence of h with progesterone increased slpi: gapdh mrna ratio from . ± . to . ± . (mean ± sem; p< . ). slpi expression was similar in the upper and lower segment myometrium in preterm patients. in term myometrium the slpi: beta-actin mrna ratio was increased by -and -fold in term labour versus term non labour samples in the lower and upper segment respectively (mean ± sem; p< . ). these data show slpi is present in human myometrium and that it is increased by il- . progesterone also increases its expression. its expression is increased in term labour where its anti-bacterial, anti-fungal and anti-viral properties could allow it to act as an endogenous block to infections. objective: pre-b cell colony-enhancing factor (pbef) downstream mapk signaling and transcription factor activation. introduction: pbef is expressed in all layers of the human fetal membranes and the myometrium and is upregulated by labor, infection, nf-kb, ap- and stretching. pbef has the ability to protect a variety of cells from apoptosis, however it also appears to act as an insulin mimetic via the insulin receptor (fukuhara et al. science : ; - ) . its levels are elevated in obese patients, those with type diabetes and in gestational diabetes. because pbef has poorly understood biological activities an investigation of mapk signaling and transcription factor activation induced by pbef has been undertaken in order to gain insights into its mechanisms of action. methods: primary aec were isolated from fetal membranes and treated with rhpbef ( ng/ml) for h, lysed and the resultant proteins labeled with cy or cy (amersham). there were used on a protein microarray containing constituents of the mapk signaling pathways (sigma). isolated aec were transfected with luciferase constructs for nf-kb, ap- , cre, hse and gre response elements using the exgen reagent. the cells were treated with rhpbef ( . , . , , ng/ml) hrs, lysed and ul of sample was analyzed for luciferase activity with dual-luciferase reporter assay system (promega). co-transfection with gfp and sv luciferase constructs to control for transfection efficiency and cell number (respectively) was also performed for each experiment. results: pbef significantly upregulated mapk signaling components including functioning as part of the erk pathways and belonging to p signaling. it also activated nf-kb and camp response elements. however, it significantly down regulated signaling molecules belonging to the jnk pathway that resulted in decreased c-jun phosphorylation. conclusions: pbef signaling in aec is consistent with its anti-apoptotic ability and its upregulation of the pro-inflammatory cytokines. although some mapk components responsive to pbef could be associated with insulin receptor signaling, some may not. therefore, it appears likely that pbef interacts with another potentially unique, but currently unidentified receptor. was the first effector to be characterised, but camp is now known to have other effectors, including camp receptor protein, cyclic nucleotide-gated channels and camp-guanine nucleotide exchange factor/exchange protein directly activated by camp (camp-gef/epac). two epac's have been identified and they consist of functional domains: camp-binding domains, a dep domain, a ras exchange motif and a gef domain. our aim was to determine the presence of epac's in human myometrium and to study the effect of camp responses on prolabour genes such as il- , pghs- , otr and fp. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates until - % confluent. cells were serum starved overnight and incubated with μm methyl progesterone, . mm sodium -bromo-camp, . mm forskolin, μm kt , ng/ml brefeldin a and . mm -pmeopt- 'o-me-camp either alone or in combination for h. rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of epac , epac , il- , pghs- , otr, fp, gapdh and -actin were measured by qpcr. results: epac and epac were present in the upper and lower segment of myometrium with epac levels being some -fold higher than those of epac . there was no change with the onset of labour at or before term. treatment with il- for h significantly increased epac (n= ; p< . ) but had no effect on epac . when conditions mimicked pregnancy, (the presence of forskolin and progesterone), brefeldin a, an epac antagonist, increased basal pghs- and fp mrna expression (n= ; p< . ), but had no effect on il- and tended to reduce otr. the il- -induced increase in pghs- and il- , but not fp, was greater with the epac antagonist. conclusions: these data show epac's are present in human myometrium and that camp acts via epacs to reduce pghs- and fp expression during pregnancy. background: inflammatory events have been implicated in the process of labour. glucocorticoids mediate strong anti-inflammatory effects through binding to the glucocorticoid receptor (gr), which on activation translocates to the nucleus and either increases or decreases the expression of responsive genes thereby suppressing inflammation. objective: to characterise the expression profile for gr protein and mrna in human myometrium during fetal maturation and parturition. methods: western immunoblotting (wb) was employed to characterise gr protein expression in first (n= ) and second trimester myometrium (n= ), and in paired upper and lower segment pregnant (non-labouring, p, n= ) and labouring (l, n= ) myometrium; as compared to non-pregnant (np, n= ) control samples. immunofluorescence staining with confocal microscopy and rt-pcr were also undertaken. results: detection of gr protein by wb revealed two bands at - kda, representing the alternatively spliced isoforms, gr-and gr-. densitometric analysis showed that gr levels decreased significantly during pregnancy and remained at very low levels at term and in labour when compared with np samples (p< . ); this decrease was seen for gr-and gr-. no significant temporal variations were observed in gr protein levels at term or during labour. gr protein was localised by immunofluorescence staining to the nuclei and cytoplasm of cells. less intense staining was apparent in p compared to np tissues; consistent with wb data. rt-pcr showed a consistent predominance of gr-to gr-mrna in all the tissues used. the observed decrease in protein expression was also mirrored at the mrna level: gr-mrna levels appeared to gradually decrease throughout gestation (p< . ). differences between the upper and lower myometrial regions were only observed in the labouring samples, where gr-mrna levels were significantly decreased in the lower segment (p< . ). nested pcr was additionally used to amplify gr-, but no consistent pattern of mrna expression was obtained. conclusions: these data are the first to characterise gr expression in human myometrium. spatial and temporal variations have been found with expression evolving through the three trimesters of pregnancy and in labour. further studies are now underway to evaluate whether gr contributes to the sequence of inflammatory events implicated in triggering term and preterm labour. these studies sought to determine the mechanism by which pas modulate the immune response. methods: ) an in vitro co-culture model mixed with human cervical epithelial hela cells and pma-induced human macrophage u cells at epithelial/macrophage cell ratio of : was employed. ) the co-culture was pre-treated with medroxyprogesterone acetate (mpa), progesterone (prog) and dexamethasone (dex) at concentrations of , , and nm for hrs followed by day stimulation of g/ml lipopolysaccharide (lps). these experiments were repeated using hela cells transfected with sirna for glucocorticoid receptor (gr). the production of cytokines il- , il- and il- , il- and tnf were determined using elisas. ) the co-culture was pre-treated with nm of each pas for hours prior to lps stimulation at g/ml for . , . , , , and hours. the phosphorylation of p mapk and gr and the expression of mapk phosphatase (mkp ) were determined by western-blotting. results: il- , il- and il- , il- and tnf were elevated by . fold, . fold, fold, . fold and . fold in the co-culture model in response to lps(p< . for all). pretreatment of mpa and dex, but not prog, inhibited the lps-induced cytokine production. the anti-inflammatory effect of mpa occurs in a dose-dependent manner. in the absence of gr, the inhibitory effect of mpa and dex on il- , but not on il- , was lost. mpa and dex, but not prog, induced the phosphorylation of gr and expression of mkp . p mapk was activated by lps for up to hrs and pretreatment of mpa and dex attenuated this response. the ability of mpa and dex to suppress the inflammatory response in cervical tissues appears to be mediated through a gr-dependent pathway, specifically though p mapk. our studies suggest that prog is not a significant immunomodulator in these tissues. background: progesterone (p ) has been shown to play a critical role in maintaining pregnancy and preventing preterm birth. these effects are likely related to its immunomodulatory properties. we investigated whether camp plays a role in the immunosuppressive action of progesterone on fetal mononuclear cells. methods: umbilical cord blood mononuclear cells were isolated using density gradient centrifugation. to establish a p effect and optimize concentrations, cells were pretreated with p ( - m- - m), dexamethasone ( um) or vehicle for hour prior to overnight lps ( ng/ml) stimulation. supernatants were assayed for tnf-using elisa. ldh assays confirmed the absence of a cytotoxic effect. next, cells were pre-incubated with forskolin (adenylate cyclase activator) or db-camp (camp agonist) prior to lps to determine the effects of camp on tnf production. finally, cells were pretreated with rp-camp (camp antagonist) prior to p incubation and lps stimulation to determine whether the p effect was reversed by a camp antagonist. results: p significantly inhibited lps-induced tnf production in a dose dependent manner, with maximum suppression observed at - m. both forskolin and db-camp suppressed lps-induced tnf production in a doserelated manner (fig ) . finally, a dose-dependent partial reversal of tnf production was observed when cells were pretreated with rp-camp. (fig ) conclusions: progesterone suppresses the inflammatory response in fetal mononuclear cells as measured by tnf expression. this effect is mimicked by adenylate cyclase activators and camp agonists and partially reversed by camp antagonists. this implicates the camp pathway in mediating the immunomodulatory actions of p . objectives estrogen improves endothelial function after vascular injury via largely unknown mechanisms. endothelial progenitor cells (epcs) are known to be implicated in various vascular events requiring endothelialization. we hypothesized that estrogen and progesterone could influence the function and the change of epcs in menstrual cycle. material and methods peripheral blood mononuclear cells (pbmcs) were isolated from peripheral blood of healthy young women in each menstrual period by density gradient centrifugation with ficoll separating solution. pbmcs were seeded in endothelial basal medium with or without estrogen or progesterone. on the th day in culture, nonadherent cells were removed. on the th day in culture, adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. the number of ldl-and lectin-positive cells was measured as epcs using flowcytometry. the expression of estrogen and progesterone receptor mrna in epcs were measured by real time pcr in menstrual and luteal period. the number of epcs was significantly increased in the menstrual and luteal period compared with the follicular phase. estrogen and progesterone significantly increased the number of adherent epcs dose dependently in menstrual period, but not in luteal period. the expression of estrogen-alpha receptor in menstrual period was higher than luteal period. also, estrogen-beta receptor in luteal period was strongly expressed compared with menstrual period. these results suggest the expression of estrogen receptor and progesterone receptor play important roles to regulate epcs' proliferation during menstrual cycle. objective: elevated levels of fetal plasma avp are associated with the development of oligohydramnios, in part a result of avp-mediated reduction in fetal urine production. as avp urinary concentration effects are mediated via upregulation of renal tubular aqp water channels, we propose that avp modulates placental aqp channels, influencing bidirectional maternal-fetal water flow. we sought to study the effect of avp on trophoblast aqp gene expression using the first trimester-derived extravillous htr- /svneo cells and the term placenta-like trophoblast carcinoma cells jeg- . methods: cultures of both cell lines were treated with a physiological concentration of avp ( . nm) to determine aqp mrna and protein expression. negative controls consisted of cells incubated in medium supplemented with % fbs without avp. to determine whether avp regulation of aqp occurs by a camp signaling pathway, jeg- cells were preincubated with μm -(tetrahydro- '-furyl) adenine (sq ), a cell-permeable camp inhibitor, before being treated with avp ( . nm). cells were incubated for hrs for aqp mrna expression and for hrs for protein extraction at °c. after harvest, real time pcr and western blotting analysis were used to detect the aqp mrna and protein expression levels, respectively. results: avp increased aqp mrna expression in both cell lines by . and . fold after hrs. aqp protein expression paralleled the increase seen in the mrna (p< . ). pretreatment of jeg- cells with sq inhibitor completely blocked the stimulatory effect of avp. conclusion: aqp gene expression is up-regulated by avp in first trimester and term trophoblast cells, with a higher induction in the later. avp activation of aqp gene expression occurs via a camp mediated pathway, as the adenyl cyclase inhibitor blocked avp effects on aqp gene expression. these results suggest that increased fetal plasma avp may contribute to oligohydramnios by an increase in aqp-mediated fetal to maternal water flow. objective: changes in expression, ratio and activity of progesterone receptors within human myometrium have been proposed as potential contributory mechanisms for the functional progesterone withdrawal effect which precedes labour. progesterone receptor c (pr-c) is an n terminally truncated isoform of the full length progesterone receptor; pr-c has been shown to be abundant in fetal membranes, placenta and upper segment of laboring myometrium ( , ). the function and regulation of pr-c is at present unclear. in this study we aimed to investigate the regulation of pr-c in cultured human myometrial and amnion-derived wish cells. methods: myometrial primary cell cultures were prepared from non pregnant and term pregnant uteri. both myometrial and wish cell cultures were treated with natural progesterone ( , nm, mm, mm, mm) for , and hrs. western immunoblotting was employed using nuclear and cytoplasmic extracts prepared from treated cells to observe the effect of progesterone on a) expression and b) subcellular localisation of pr-c within both cell types. immunofluorescent staining and confocal microscopy were also employed. experiments were also undertaken whereby nuclear and cytoplasmic extracts were subjected to shrimp alkaline phosphatase treatment to evaluate the phosphorylation status of pr-c in response to hormone. results: data indicates that a kda cytoplasmic protein, representing pr-c is abundant in myometrial and amnion cell cultures. we show that expression of pr-c increases in both cytoplasmic and nuclear fractions within both cell types in response to progesterone. evidence also suggests that pr-c is phosphorylated in a progesterone-independent manner. concurrent treatment with progesterone and the pr-antagonists ru and organon in amnion-derived cells still led to an increased abundance of pr-c but seemed to alter the phosphorylation status of pr-c. conclusion: pr-c expression and subcellular localisation within myometrial and wish cells alters in response to progesterone. speculation is generated about potential role of pr-c in reproductive tissues and its contribution to functional progesterone withdrawal. . taylor smooth muscle responses have been studied, data on regional blood flow (bf)distribution are scarce. we have studied the effect of a single dex course on relative bf distribution within the brain, skeletal muscle, heart, omental fat, placenta and adrenal in fetal sheep at . g, the equivalent of weeks. methods: fetal sheep received amniotic, jugular, carotid and femoral artery and vein catheters at days g (dg). experiments started at dg in dex ( mg) and saline treated controls (ctr), each receiving injections h apart. fetal blood pressure was recorded continuously (windaq pro+). microspheres ( . x total; sterispheres, biopal) were injected at : am into fetal jugular and femoral veins before maternal i.m. injection (t ) and h after the rd injection (t ) in both groups. tissues were collected hours after the th injection for assessment of bf (biopal). results: fetal bp increased above baseline after dex ( . + . mmhg; p= . ) with no change in ctr (- . + . mmhg). regional bf distribution is presented as a percent of the total counts per g tissue (table ) and after normalization within each animal to placentomal flow, shown previously to be unchanged by dex, in table . no significant differences in flow were found. discussion: although dex altered bp, regional distribution of bf among key organs was unaffected. whether gc does not affect regional bf, or the rise in bp is the result of a non-specific, system-wide vasoconstriction, or the impact of gc on bf is similar in all organs, will be subject of further investigation that will include determination of regional vascular resistance and absolute flow. stimulator while crf-r is an inhibitor of gi motility at times of stress. we recently hypothesized that stress-induced in utero meconium passage in fetuses is analogous to stress-induced defecation in adult rats and likely mediated by crf pathway. in support, we demonstrated hypoxia-induced meconium passage in conjunction with marked increases in fetal plasma crf levels. as the mouse is a common experimental model, with known genome, we sought to determine the presence of crf-r and crf-r receptors in mouse fetuses. methods: time-dated cd- pregnant mouse were anaesthetized and fetuses (n= ) were collected at e (term = e ). whole gi tracts were dissected, fixed in bouin's solution, paraffin embedded and sectioned at five micron thickness. sections were immunostained with rabbit polyclonal antibodies to crf-r and crf-r by abc technique with vector abc reagents. immunoreactivity was identified as brown staining using ', diaminobenzamidine as chromagen. slides were counter stained with mayer's hematoxylin and examined under the microscope. immunoreactive signals in the smooth muscle layers of gi tracts were quantified by image analysis using image pro . plus software. results: immunostaining for both crf-r and crf-r was seen in the smooth muscle layers of all lumens examined ( lumens per section). the immunostaining intensity expressed as intensity over density (iod) in arbitrary units for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) and for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) greatly varied between lumens. the mouse fetal gi tract expressed both crf-r and crf-r receptors, suggesting that the mouse is an appropriate model for fetal-stress induced neurovisceral motor responses. the non-uniform distribution of crf receptors in the fetal gi tract suggests the existence of regional differences in the expression patterns of crf-receptors of both types. we speculate that mouse devoid of crf and crf-r or r receptors will be useful to confirm the participation of crf pathway in stress-induced in utero meconium passage. antenatal exposure to glucocorticoids (gc) is associated with a reduction in nephron number and hypertension in adult life. one of the mechanisms for the development of hypertension is thought to be the long term effect of single nephron hyperfiltration. however, in most studies there is only a - % reduction in nephron number with no change in gfr. thus, although a reduction in nephron number may be a contributing factor it is unlikely the only variable. the aim of the present study was to evaluate nephron mass, renal function and blood pressure in a cohort of adult sheep exposed antenatally to betamethasone. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hs apart at days gestational age and allowed to deliver at term. at . yr of age in female (f) and male (m) offspring, glomerular filtration rate (gfr) was measured as inulin clearance and effective renal plasma flow (erpf) as pah clearance. blood pressure was measured though an indwelling catheter in the femoral artery over a -hour period. nephron number was measured by the acid maceration technique. data mean±sem were analyzed by anova and/or two sample t test. results: antenatal bm was associated with an elevation in arterial blood pressure and a reduction in nephron number in both f and m offspring. in contrast, a reduction in gfr and erpf was present only in males. plasma and urinary electrolytes as well as urinary protein excretion were not different from ctr. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on blood pressure regulation. interestingly, while the decrease in nephron number was of similar magnitude in m and f, evidence of alterations in renal function was present only in males. these suggest that the decrease in renal function observed in males is not solely a consequence of the decrease in nephron number. furthermore, it is possible that different mechanisms are responsible for the elevation in arterial blood pressure observed in m and f. hl ; hd p hd . clr l>t-x to evaluate the effects on blood pressure, urine output, sodium excretion and gfr of the intrarenal infusion of angiotensin - in the male sheep with or without prenatal exposure to b. we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation and were born at term. catheters were placed in the femoral artery, femoral vein, left renal artery and the bladder. the right kidney was removed. after days recovery the sheep received an infusion of ang - ( ng/kg/min) with or without its antagonist, [d-ala ]-ang-( - )(d-ala, ng/kg/min), into the renal artery for hours. blood pressure, gfr, urine output and sodium excretion were measured before and after the infusion. two-way analysis of variance (anova) was used to test mean values between the groups. with or without d-ala, map and urine output didn't change significantly during ang - infusion. however, the infusion of ang - resulted in a decrease (change from baseline) (p= . ) in na + excretion of . ± . meq/kg/ h in the vehicle animals and . ± . meq/kg/ h in the b animals. there was no significant change in gfr during ang - infusion. gfr was lower in the b group during the combined ang - and d-ala infusion ( . ± . ml/min vs . ± in vehicle group, p= . ). intrarenal infusion of ang - at ng/kg/min is followed by a significant decrease in sodium excretion but no significant change in urine output and gfr. the decrease tended to be greater in the vehicle animals and was not markedly attenuated by the antagonist d-ala. this suggests the effect of ang - may be mediated by a receptor other than the mas receptor for ang - and may be of lesser magnitude in animals exposed to b before birth. and norepinephrine ( . - . g/kg/min) and to an l-name ( mg/kg) bolus were studied. vascular reactivity was analyzed by curve fitting of either the blood pressure or hr responses to obtain the maximal response. data are expressed as mean±sem of change from baseline. statistical significance was established using t test. results: bm-exposed animals displayed a higher blood pressure response to at-ii (atii max ± vs ± % of baseline, p< . , figure left panel). no differences in blood pressure or heart rate responses to norepinephrine infusion were detected in bm when compared to control animals (figure right panel) . maximal response to l-name was also elevated in bm-exposed animals but did not reach statistical significance with the current sample size. conclusion: we have shown that antenatal gc treatment significantly increases blood pressure in sheep. here we show an enhanced in vivo response to at-ii. this response may contribute to the increased blood pressure observed. the greater response to l-name most likely represent an increased no production as a compensatory mechanism to the higher blood pressure observed. hl . women at risk of delivering preterm are frequently treated with glucocorticoids to facilitate fetal lung maturation. animal studies have revealed that prenatal exposure to glucocorticoids may alter aspects of hypothalamic-pituitary adrenal functioning in later life. in this study we examined the effects of clinically relevant prenatal betamethasone treatment on the responsiveness of pituitary cells (in terms of adrenocorticotropin, acth, secretion) isolated from adult sheep. time-dated pregnant sheep were injected with betamethasone ( . mg/kg) or vehicle on days and of gestation. thereafter pregnancy was allowed to continue unimpeded and offspring were born. pituitaries were isolated from adult sheep aged between and months and cells were dispersed and plated at a density × cells per well in well plates. after h, cells were stimulated with normal medium, nm arginine vasopressin (avp), nm avp, nm corticotropin releasing factor (crf) or nm crf for h. medium was collected and analyzed for acth using a commercially available kit. acth secretion (given as % increase from untreated cells) was similar between the control and betamethasone groups following nm avp ( . ± . vs. . ± . ) and nm crf ( . ± . vs. . ± . ). at higher stimulatory concentrations of both avp and crf, acth secretion remained similar in control cells ( . ± . and . ± . respectively), but was significantly decreased in betamethasone cells ( . ± . and . ± . respectively). these findings suggest that prenatal exposure to clinically relevant doses of betamethasone can alter pituitary responsiveness to both avp and crf in adulthood. this research was supported by nih grants hd and hd . lcc is supported by an rjr-leon golberg fellowship in pharmacology and toxicology. objective: newborn meconium (mec) passage normally occurs within the first - h after birth. in contrast, more than half a million infants born annually in the us pass mec in utero. neither the physiological mechanism(s) nor causes for in utero mec passage are well understood, though both fetal maturation and stress are associated. we recently reported in utero mec passage and marked increases in plasma crf levels in term fetal rats exposed to maternal hypoxic stress. we hypothesize that stress-induced in utero mec passage is mediated by the crf pathway in a manner analogous to stress-induced defecation in adult rats. in the present investigation we examined placental crf content prior to and following maternal hypoxia, to determine the source of increased plasma crf. methods: time-dated pregnant rats (n= ) were exposed to a paradigm of graded, stepwise hypoxia on day (term= ) (pediatr. res. : - , ) . control pregnant rats were exposed to % oxygen for a similar duration as experimental animals. at the end of the study, placentas were harvested, fixed in % paraformaldehyde and paraffin embedded. sections (n= per placenta) were subjected to immunohistochemical analyses with rat/human crf antibody (peninsula laboratory) by abc technique. immunoreactivities on placental sections were quantified using the image pro . software and intensity expressed as arbitrary unit (au). all values are mean± sem. results: in control maternal rats exposed to normoxia, positive staining for crf was seen in decidua ( . ± . au) and in all three major placental cells types (giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au.) in contrast, in animals exposed to hypoxia, there was a total absence of crf staining in the placental cells, with only positive crf staining seen only in the decidua ( . ± . au). conclusion: the total absence of crf staining in both the basal and labyrinth zones in placentas of pregnant rats subjected to hypoxic stress suggests that placental cells rapidly release crf into the maternal and fetal circulation in response to hypoxic-stress. our findings support our hypothesis that the peripheral crf pathway mediates in utero mec passage. offspring. angela g massmann, jie zhang, jorge p figueroa. department of obstetrics and gynecology, wake forest university school of medicine, winston-salem, nc, usa. objective: in rats and sheep, exposure to glucocorticoids (gc) in the perinatal period induces hypertension in adult life. recent evidence suggests that endothelin b (etb) receptor plays an important role in the regulation of sodium balance and blood pressure. renal medulla is an important site of expression and action of etb receptor. furthermore, in kidney it is the medulla (km) where the highest concentrations of immunoreactive et- and etb receptor are found. the aim of this study was to measure eta and etb expression in adult sheep kidney exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (v) hours apart at days of gestational age and allowed to deliver at term. at . yr of age, male and female offspring exposed to either vehicle or bm were euthanized and the kidneys harvested. kidney medulla (km) was obtained by sharp dissection. eta and etb protein and mrna expression were evaluated by western blot and rnaase protection assay. data are expressed as mean±sem and were analyzed by two way anova. results: a significant decrease in etb (f= . , p= . ) but not eta protein was observed in km of bm exposed male and female adult sheep. at the mrna level, bm exposure also affected etb, but not, eta expression. however, bm treated animals had higher mrna levels than control sheep (f= . ; p= . ). conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects. the activation of etb receptor by et- inhibits sodium transport function in the collecting duct and the selective gene deletion of the etb in medulla results in hypertension.therefore, our finding of a significant reduction in etb protein suggests that there may be an impairment in the kidney's ability to regulate sodium reabsorption. the functional relevance of the alterations in etb protein expression as well as the discrepancies in mrna regulation need to be established. hl ; hd p hd . in the placenta, -hydroxysteroid dehydrogenase type ( hsd ) regulates the transfer of cortisol. maternal glucocorticoid (gc) administration has been shown to affect the ovine fetal growth trajectory and regulate hsd expression in mid and late gestation. however, little is know about the effects of gc administration in early gestation. we hypothesized that maternally administered low-dose dexamethasone (dex) in early gestation would affect hsd expression, which will subsequently alter fetal birth weight. pregnant ewes were randomized and received intramuscular injections consisting of either saline ( ml saline/ewe) or dex ( . mg/kg) given hours apart starting on days of gestation (dg). the animals were sacrificed at , , , and dg and fetal weights were recorded and placental tissue was collected. qrt-pcr was used to measure hsd placental mrna expression. statistical analysis was done by anova with student's t-test posthoc. overall, there was no significant effect of dex treatment on placental hsd mrna expression across gestation. however at dg, there was a significant increase in hsd expression after dex (p= . ). there was an increase in placental hsd mrna progressively from dg (mean = . ) to dg (mean = . ), independent of treatment. overall, a positive correlation between hsd and fetal weight (r = . ) was apparent at dg and at dg (p= . ) in both control (p= . ) and dex (p= . ). significant positive correlations between placental hsd and fetal weight (r = . ) were found in females (p= . ) and a similar trend was found in male fetuses (p= . ). we conclude that in the sheep placenta, hsd expression increases throughout gestation and may respond, at least transiently in early gestation, to elevations in maternal gc. the positive correlation between hsd and fetal weight is consistent with the thesis that the fetal growth trajectory may be influenced by maternal cortisol levels and that placental hsd is an important mediator of these effects. antenatal gc exposure induces hypertension in the young adult rat (neuroendocrinol; ; : ) and increases femoral vascular resistance in the lamb (jphysiol; ; : ) . unpublished own examinations in preparation of this study have shown age dependency of the vascular reactivity. vascular contractility of the middle cerebral artery (mca) decreased in response to k + and noradrenaline (na) between mo and , y of age (p< . ). vascular relaxation to acetylcholine (ach) but not to pge decreased during the same time (p< . ). vascular contractility of the renal artery (ra) increased in response to k + (p< . ). aims: to examine if antenatal gc alter vascular reactivity in the aged individual when cardiovascular diseases are predominant. we studied the ra because its vascular tone is involved in modulation of arterial pressure control (amjphysiol; ; :r ) and the mca because depressive disorders that are associated with dysregulation of the hpa axis (jcem; ; : ) increase stroke mortality for unknown reasons (amjpsychiatry; ; : ) . to be clinically relevant, we administered dexamethasone at the dose used to enhance lung maturation in babies threaten premature labor. methods: pregnant dams received saline (n= ) or μg/kg dexamethasone (n= ) i.p. at day e / equivalent to x mg dexamethasone administered to a kg pregnant woman. at . years of age, vascular response of the ra and mca to endothelium-dependent and independent mediators was measured using wire myography. vessels were inspected histologically for intact endothelium. results: basal vasoconstrictory and dilatory responses to all mediators were less pronounced in the mca than in the ra probably reflecting autoregulatory properties of cerebral vessels (p< . ). after prenatal dexamethasone exposure, contractility but not sensitivity to k + and na was enhanced in the mca and even more pronounced in the ra (p< . ). relaxation to ach and pge was similarly diminished in both vessels after precontraction with na (p< . ). conclusions: prenatal dexamethasone exposure at the dose used clinically increases renal and cerebral vascular tone in the aged rat by endotheliumdependent and independent mechanisms. this effect may be a potential mechanism of fetal programming of cardiovascular diseases in later life. betamethasone (bmz) therapy during pregnancy to improve fetal lung maturity may result in long term side effects, including hypertension, during adulthood. the kidney is an important organ which regulates systemic blood pressure via the local renin angiotensin system (ras). the major enzymes of the intrarenal ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the hypothesis of this study is that prenatal bmz exposure will result in alterations of the enzymes regulating the intrarenal ras. specifically, sheep that are treated with bmz in utero will have higher amounts of ace activity in kidney cortex compared to control animals. pregnant sheep of known mating date were either treated with vehicle (n= ) or with two doses of bmz (n= ), . mg/kg, hours apart at days of gestation. renal cortex from the male offspring was harvested at - months of age. cortical membranes were then solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were separated by reversephase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results: bmz treatment resulted in a significant increase in ace activity compared to control animals (p= . ). ace and neprilysin levels were not significantly different between treatment groups. when expressed as a ratio of ace/ace activity, bmz treated animals exhibited a . fold greater proportion of ace activity versus control animals (p= . ). this study demonstrates that bmz exposure during fetal life results in a significant increase in renal ace activity during adult life. this increase in enzyme activity would be expected to be associated with increased levels of ang ii in the kidney and na + retention, possibly underlying the development of hypertension. hd , hd . we recently hypothesized that stress-induced in utero meconium passage in term fetuses utilizes peripheral and/or central crf pathways in a manner analogous to stress-induced defecation in adult rats. as participation of central crf pathway requires the crf circuitry system in brain-gut axis, we exploited immunohistochemical techniques to address whether term fetal colon is wired by crf-nerve fibers. methods: fetal distal colon segments (n= ) were dissected from term ovine fetuses ( - d gestation). in addition, lumbosacral spinal cord with spinal roots and dorsal root ganglia (drg) (n= ) were dissected from ovine fetuses, and vagal nerve trunk with nodose ganglia (n= ) were dissected from mouse fetuses. paraffin sections fixed either in bouin's solution or zamboni's were subjected to immunohistochemistry with rabbit polyclonal antibodies specific to ovine-crf (ocrf). immunoreactivity on the sections was identified by standard abc technique, immunostaining quantified by image-pro plus software and expressed (intensity over area) as arbitrary units (au). results: ocrf antibody elicited strong positive staining on the serosal surface (port of entry for extrinsic nerve fibers) in distal colonic segments objective: we hypothesize that stress-induced in utero meconium passage is mediated by the crf pathway. ucn-i, similar to crf, is a potent contractility inhibitor of preterm ovine fetal distal colon, which has abundant crf-r receptors. both ucn- and crf thus may inhibit colonic motility and prevent preterm in utero meconium passage via crf-r receptors. however, ucn-i is reported to stimulate contractility of adult rat colonic smooth muscle strips more strongly than crf. we sought to study the pattern of ucn-i innervation in distal colon of ovine fetuses to delineate whether ucn- functions as a facilitator or inhibitor of colonic propulsive motility. methods: bouin's solution fixed, paraffin sections of very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - ) and term (t: - days) ovine fetal distal colon rings were subjected to immunohistochemistry with polyclonal antibodies to human ucn- ( : sigma) by abc system. intensity of ucn- immunostaining in smooth muscle layers and myenteric neurons were quantified by image-pro plus software and expressed (intensity/area) in arbitrary units (au). differences over time were assessed with anova. in all groups very preterm was significantly greater than term (p< . ). conclusion: ucn- immunoreactivity in all three muscle layers, as well as myenteric and submucosal neurons, is highest at very preterm as compared to more mature gestations. these results suggest that the reduction of ucn- inhibitory function may facilitate stress-induced meconium passage at term. to evaluate the effect on blood pressure (bp), urinary output (uop), sodium excretion (nae) and gfr of the intrarenal infusion of ang ii in the male sheep after prenatal exposure to b. methods: we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation. catheters were placed in the femoral artery and vein, left renal artery and in the bladder. a right side nephrectomy was performed. after days of recovery, the sheep were infused with ang ii ( ng/kg/min) with or without candesartan ( ng/kg/day) or pd (pd g loading dose and ng/kg/min infusion) into the renal artery over hours. bp, gfr, uop and nae were measured before and after the infusion. two-way analysis of variance was used to test mean values between the groups. results: bp and uop did not change with ang ii infusion with or without candesartan. the infusion of pd did not affect uop but did increase bp in the b sheep (p= . ). ang ii decreased the nae in both groups(p= . ). candesartan increased nae among controls(controls . and b . meq/kg/h, p= . ). pd infusion decreased nae among controls but this was not significant. baseline gfr was higher among vehicle compared to b animals (p= . ). during ang ii infusion there was a decrease in gfr among control compared to b sheep (- . vs. - . ml/min, p= . ). this difference was not seen during candesartan or pd infusion. conclusion: ang ii infusion led to a decrease in nae in both groups and gfr among controls but not b animals. infusion of ang ii with candesartan increased nae while pd decreased nae among controls but not b animals. this suggests that prenatal steroid exposure can alter at receptor mediated response in renal function by decreasing the effect of ang ii on renal sodium excretion. the differences in gfr suggest that this may be due to an effect on tubular sodium reabsorption rather than glomerular perfusion. antalarmin antagonism of acth-induced cortisol secretion by ovine adrenal cells probably not at acth receptor. nancy k valego, james c rose. center of research for ob/gyn, wake forest u. school of medicine, winston-salem, nc, usa. in addition to regulating the hpa axis, crh and its type receptor (crhr- ) occur peripherally and in the female reproductive system. late in human pregnancy, a surge in placental crh probably stimulates adrenal secretion of cortisol and dheas requisite to parturition. we previously reported that, in dispersed fetal or adult ovine adrenal cortical cells, hour incubation with the specific crh-r antagonist, antalarmin (ant), significantly reduced both cyclicamp and cortisol responses to acth, suggesting an interaction with acth-r (like crh-r , a g-protein-coupled membrane receptor). however, forskolin (fsk; direct stimulant of adenylyl cyclase)-stimulated cortisol secretion was also attenuated by hour co-incubation with ant. our objective was to clarify the effect of ant on binding of acth to its receptor after a short ( . hours) treatment. method: acth binding: the adrenals from adult sheep were obtained at necropsy and the cortex cells dispersed and plated @ cells/well. after hours in culture, wells were rinsed x and refilled with serum-free dmem/ f containing vehicle (dmso) with or without ant. after . hours, wells were washed very gently and the binding assay (adapted from rainey et al; j biol chem, : , ) completed. triplicate vehicle or ant wells were treated with i -tyrosine human acth ( - ) with or without - m acth (for non-specific binding). after hour, wells were chilled, washed, and the cells lysed and counted on a gamma counter. fsk treatment: cells were treated as above except that fsk ( - m) was added to the wells. after . hours, medium was removed and stored @ - ºc for camp eia and cortisol ria. results (mean±se) of . hour incubation on acth binding and fskinduced secretion. vehicle antalarmin acth binding (net cpm) n= ± ± cortisol (ng/ml/ . hr) n= ± ± * (p=. ) camp (pmol/ml/ . hr) n= . ± . . ± . * (p=. ) * indicates significant difference from vehicle alone. conclusion: the crh-r antagonist, antalarmin, attenuates acthstimulated cortisol secretion but not by preventing acth receptor binding. however, its effect is early in the secretory process and is associated with a reduced camp response. objective glucocorticoids are often administered to pregnant women to prevent neonatal respiratory distress syndrome. prenatal steroid treatment increases blood pressure in adult sheep. exposure to excess corticosteroids before birth is hypothesized to be a key mechanism underlying the fetal origins of adult disease hypothesis and effects on the renin-angiotension system (ras) may modulate the steroid-induced increases in blood pressure. we therefore sought to determine if renin processing and secretion were altered in adult female sheep exposed antenatally to betamethasone ( ) and to compare them with data from males studied previously. methods pregnant sheep were randomized to receive doses of . mg/kg of or vehicle, at and days of gestation; the offspring were studied at and months of age. in female offspring, active renin concentration (arc) and total renin concentration in plasma were measured by ria of angiotensin i generated by incubation with excess substrate. prorenin concentration (prc) is the difference between total and active renin. nine or control exposed female animals born at term ( - days of gestation) were brought from the farm at - months of age, and had vascular and bladder catheters placed. five days after surgery, a sodium load of hypertonic nacl ( . meq/kg/min at . ml/min) was given for min. blood samples were obtained. data are expressed as mean sem and were analyzed by t test. the prc was significantly higher in the females than in the males ( . ± . vs . ± . p< . ) but there was no effect of prenatal steroid treatment. arc was similar in both genders. however, arc was a significantly greater percent of the total plasma renin concentration in the males ( . ± . vs . ± . p< . ) during the na infusion experiment, the exposed females had lower arc than did the control females ( . ± . vs . ± . p< . ). conclusion the data suggest that prenatal exposure to didn't alter the processing and secretion of renin in adult female sheep. prenatal steroid treatment does not appear to alter the effect of gender on plasma renin levels in adult sheep.it seems unlikely that the elevated blood pressure seen in adult ewes after prenatal exposure is the result of increased secretion or processing of renin. supported by hd and hd . background: mrap is a recently discovered protein with alpha and beta isoforms, a common amino terminal region and divergent c-terminal sequences generated by alternative splicing. in human and mouse mrap plays an essential role in the generation of a functional, g-protein coupled acth receptor (melanocortin- receptor; mc r) but has not been described for ruminants. we previously reported that lth fetal sheep exhibited elevated basal acth - yet decreased expression of key steroidogenic enzymes and the mc r in the adrenal cortex while basal cortisol levels were not different from control. we hypothesized that mrap could play a key role in mediating the effect of lth as well as developmental regulation of fetal adrenal cortisol synthesis in fetal sheep. the goals of the present study were to determine the ontogenic pattern of mrap expression in the sheep fetus and to determine if lth alters mrap expression. methods: we searched the bovine genomic sequence database (www.ncbi.nlm. nih.gov/genome/guide/cow/) and found a sequence with > % homology to the human mrap n-terminal residues, with partial homology to the beta isoform in the carboxyl region ( %). using primers based on the bovine sequence, we confirmed the presence of mrap in the ovine fetal adrenal cortex. adrenal cortical tissue was collected from sheep fetuses from - (n= ) and near term ( - ; n= ) days of gestation (dg) as well as from lth (n= ) fetuses (exposed to high altitude hypoxia from to - dg) and age-matched normoxic controls (n= ). cyclophilin was used as a housekeeping mrna. data are expressed in fg mrna/ ng total rna. results: the expression of mrap, based on quantitative rt-pcr, was low between - dg ( . ± . ) and increased (p< . ) approx. -fold near term ( - dg; . ± . ). levels of mrna for mrap were highly correlated to cyp mrna in individual samples. mrap expression in control ( . ± . ) and lth ( . ± . ) did not differ between groups. gender differences in hypertension are well described and there is growing evidence that the regulation of the renin angiotensin system (ras) is influenced by sex hormones. the major enzymes of the ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the peptide products of these enzymes have opposing actions. angiotensin ii (ang ii), the product of ace, is a potent vasoconstrictor. on the other hand, the peptide products of ace and neprilysin, ang ( - ) and ang ( - ), exhibit vasodilatory properties. thus, modifications in the relative proportions of these enzymes and their peptide products can result in alterations of systemic blood pressure. the purpose of this study was to describe the gender differences in angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin (nep) enzyme activities in adult sheep kidney cortex. renal cortex from male (n= ) and female (n= ) sheep were harvested at - months of age. the tissue membranes were solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were then separated by reverse-phase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was then quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results ace activity was over times greater ( ± . vs. . ± . fmol/mg/min, p< . ), ace activity was . times greater ( ± . vs. . ± . fmol/mg/min, p= . ) and nep activity was nearly times greater ( . ± . vs. . ± . fmol/mg/min, p= . ) in female kidney cortex. in adult sheep, key enzymes of the intrarenal ras have signficantly greater activity in female kidney cortex. these findings suggest that there are fundamental, gender specific, differences in the regulation of the enzymes of the intrarenal ras. the physiologic significance of these findings remain to be elucidated. hd , hd . in rats and sheep exposure to glucocorticoids (gc) in the perinatal period is associated with a reduction in nephron number and hypertension in adult life. furthermore, antenatal exposure to gc alters glucose tolerance in animals and in people. the aim of the present study was to determine ) if insulin resistance is a contributing factor for the development of hypertension in adult sheep exposed antenatally to gc and ) if diet-induced obesity has a more pronounced effect in sheep exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hours apart at days gestational age and allowed to deliver at term. at mo of age, female sheep were randomly allocated to be fed at either % of recommended nutritional allowance or ad libitum for three months. sheep were chronically instrumented under general anesthesia to place intravascular catheters. insulin sensitivity was evaluated both by iv glucose tolerance test (ivgtt) and euglycemic clamp (hec)techniques. for the ivgtt a . g/kg glucose bolus was used and for the hec mu/kg human insulin was used. data mean±sem were analyzed by anova and/or two sample t test. results: ad lib fed sheep gain > % of the original weight. antenatal bm was associated with an elevation in basal and ivgtt plasma insulin values. as shown on the figure, diet-induced obesity significantly increased insulin plasma levels during ivgtt in bm-exposed adult female sheep (f= . ;p< . ). insulin sensitivity derived from hec was significantly decreased by obesity in bm-exposed adult female sheep. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on glucose metabolism regulation. bm-exposed sheep exhibit alterations in glucose tolerance, hyperinsulinemia and insulin resistance. these abnormalities are exagertated by diet-induced obesity. hl . syngergistic induction of -hydroxysteroid deyhdrogenase type expression by cortisol and interleukin- in human fetal lung fibroblasts. z yang, p zhu, cm guo, l myatt, k sun. school of life sciences, fudan university, shanghai, china; obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. objectives: glucocorticoids acting via glucocorticoid receptor (gr), serve as crucial hormones in fetal lung maturation. glucocorticoids and proinflammatory cytokines to induce b-hydroxysteroid dehydrogenase type ( b-hsd ) which converts inactive cortisone to active cortisol, but their effect on b-hsd expression has not been addressed in human fetal lung. we examined the interactions and mechanism of cortisol and interleukin- b (il- b) effect on b-hsd in human fetal lung fibroblasts (hfl- cells). methods: the expression of b-hsd in hfl- was examined with immunocytochemistry and pcr. b-hsd , prostaglandin h synthase- (pghs- ) and cytosolic phospholipase a a (cpla ) mrna levels in cultured human fetal lung fibroblasts treated with cortisol and il- b were measured with real time pcr. the roles of gr and c/ebps in the effect of cortisol and il- b were studied using a gr antagonist (ru ) and transfection of plasmid carrying c/ebp-specific dominant-negative gene (cmv -a/cebp). results: hfl- cells expressed a high level of b-hsd . both cortisol ( - - - m) and il- b ( . - ng/ml) induced b-hsd mrna expression in a concentration-dependent manner, an effect blocked by the mrna transcription inhibitor , -dichlorobenzimidazole riboside ( μm). ru ( - m) blocked the induction of b-hsd by cortisol. induction of b-hsd mrna expression by cortisol ( - m) was synergistically increased by co-treatment with il- b ( . - ng/ml) in a concentration-dependent manner. in contrast, the induction of cpla and pghs- expression by il- b was inhibited by cortisol, suggesting a different mechanism of interaction. transfection of the cells with c/ebp-specific dominant-negative plasmid attenuated induction of b-hsd mrna expression by either cortisol or il- b. these data suggest induction of b-hsd expression by cortisol is a gr dependent process involving c/ebps, which also mediate induction of b-hsd expression by il- b. conclusions: cortisol and il- b synergistically induce b-hsd expression in human fetal lung fibroblasts. this would lead to greater local cortisol production, perhaps providing either a self-attenuating mechanism for control of inflammation or a mechanism for enhancing fetal lung maturation when the fetus is exposed to cytokines e.g. with infection-induced preterm labor. do alterations in placental -hydroxysteroid dehydrogenase ( hsd) activities explain differences in fetal hypothalamic pituitary adrenal function following periconceptional undernutrition or twinning in sheep? kl connor, pl van zijl, cw rumball, , al jaquiery, , je harding, , mh oliver, , fh bloomfield, , jrg challis. medicine, university of toronto. periconceptional undernutrition (pcun) leads to activation of fetal hypothalamic pituitary adrenal (hpa) function, whereas twinning results in delayed fetal hpa activation. we hypothesized that these differences in fetal hpa activity were the result of altered patterns of expression of placental of hsd isozymes and hence of the maternal glucocorticoid (gc) effect on the fetus. we developed a mass spectrometric assay for the measurement of hsd- and - activities and validated this method against a widely used thin layer chromatography method. sheep were randomly assigned to ad libitum (n control) concentrates throughout gestation or were undernourished (un) from days before until days after mating to reduce maternal body weight by %, with ad libitum feeding thereafter. placentomes were collected on days , , , and of gestation (term, d) and hsd- and - activities were determined by measuring the rate of interconversion between cortisone to cortisol. with un, there was a trend towards lower hsd- activity at d (p= . ), a significant reduction at d (p< . ), but no difference at d or d . hsd- activity was not different between n and un animals at any time. there was no effect of twinning on hsd- or - at d . however, with twins both hsd- (p= . ) and - (p< . ) activities increased at d . hsd- activity was reduced in twins (p= . ) at d but was higher than any singleton pregnancies at d (p= . ). hsd- was not different between singletons and twins at either d or d . overall, hsd- was lower in placentae of male compared to female fetuses in late gestation; hsd- was higher in male than female fetuses. there was no interaction with un in either sex. we conclude that pcun and twinning result in alterations of placental hsd activities in sheep. modifications in these enzymes during critical periods of fetal development may affect transplacental transfer or placental generation of gcs reaching the fetus potentially influencing the timing of activation of the fetal hpa axis, fetal maturation and later life development. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. control animals had ad libitum food. offspring were sacrificed on day of life (p ) and at months (n= - per group). adrenals were dissected and snap frozen in liquid nitrogen for later extraction of rna. real time rt-pcr using specific rat primers was used to quantify mrna levels ( s as control). we evaluated the expression of -beta hydroxylase (cyp b ), aldosterone synthase (cyp b ), acth receptor (mcr ), p side chain cleavage enzyme (cyp a ), star protein, -hydroxysteroid dehydrogenase type (hsd ) and type (hsd ), glucocorticoid receptor (gr), and mineralocorticoid receptor (nr c ). fold changes in mrna expression in controls and mfr offspring was compared by student's t-test. results: there was a marked downregulation in expression of cyp b (p=. ), cyp b (p=. ), hsd (p=. ), p (p=. ), acth receptor (p=. ), star (p=. ) and nr c (p=. ) mrna in p mfr offspring, with no changes in hsd and gr. gender specific differences were found in the adult mfr offspring. in the male mfr offspring the expression of hsd and gr were significantly upregulated with a trend towards an increase in acth receptor (p=. ) whereas in the female mfr the expression of acth receptor (p=. ) was increased and nr c (p=. ) and cyp b were decreased (p=. ). in combined data for adult male and female offspring the expression of acth receptor was significantly (p=. ) increased. conclusion: these results indicate that mfr has a suppressive effect on steroidogenic enzymes of the newborn offspring regardless of gender. this may be an adaptive mechanism in the fetus/newborn to offset the high circulating maternal glucocorticoids in response to undernutrition. in adult male mfr offspring, increased hsd indicates an increase in gc synthesis whereas in the females there were no changes in gc synthesizing enzymes with a suppression of mc synthesizing pathway. the common finding of an increased acth receptor expression in both genders would suggest an increased sensitivity of adult mfr offspring adrenals to the effects of stress. objective: during gestation the fetal adrenal undergoes phases of active growth ( - d), quiescence ( - d) followed by reactivation (> d) before birth. insulin like growth factors play an important role in stimulating adrenal growth throughout late gestation. interestingly the prepartum activation of the adrenal is delayed in the female compared with the male fetus, but the mechanisms underlying this delay are unknown. hypothesis: we hypothesize that there are gender specific differences in the gestational profile of adrenal igf mrna expression between male and female fetuses. methods: a total of twin fetuses were used in this study. post mortem was performed at either - d, - d or - d gestation. adrenal mrna expression of igf , igf , igf r, igf r and cyp was determined by qrt-pcr. results: fetal weight was not different between males and females, and increased (p< . ) with increasing gestational age. the relative adrenal weight was lower however after d when compared to the earlier gestation age group (p< . ). adrenal igf mrna expression was lower (p< . ) at - d when compared to the earlier gestation age groups. interestingly, igf r expression was highest at - d (p< . ). there was an interaction between the effects of age and gender on adrenal igf and igf r mrna expression such that the expression was higher in males compared to females at - d (p< . ), but was not different to either the earlier or later gestational ages. there was no effect of either gender or gestational age on adrenal cyp mrna expression. conclusions: it has been speculated that a delay in prepartum activation of the adrenal in female fetuses may be due to gender specific differences in the intra-adrenal bioavailability of igf . in this study we have demonstrated there is an increase in both igf and igf r mrna expression in males compared to female fetuses. this may be evidence that adrenal igf expression plays a role in the earlier activation of the adrenal gland in male fetuses. we have previously shown that in response to a secondary stressor, in vivo cortisol secretion is elevated in long term hypoxic (lth) ovine fetus despite lower acth receptor mrna expression, and no differences in plasma adrenocorticotropic hormone (acth) levels when compared to normoxic controls. the present study was designed to determine the potential mechanism(s) of this enhanced cortisol secretion. specifically we tested the hypothesis that post receptor signaling events including camp production and expression of steroidogenic acute regulatory protein (star) are enhanced following lth. methods: for the lth group, pregnant sheep were maintained at high altitude ( , m) from day to near term. on days - (term = days), fetal adrenal glands were collected from lth (n= ) and age-matched, normoxic signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. the roles of extracellular signal-regulated kinase / (erk / ) and p map kinase in the differentiation and invasion of human trophoblasts have been studied. however, the in vivo expression and activation of erk / and p at the placental bed has not been elucidated. the study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n= ) and with preeclampsia (n= ) between and weeks of gestation. we evaluated the expressions and phosphorylations of erk / and p map kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry. results: p and phospho-p map kinase were not detected in invasive trophoblasts in cases or controls. erk / and phospho-erk / were positive in invasive trophoblasts albeit with variable staining. phosphorylation of erk / was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls. conclusion: these findings suggest that preeclampsia is associated with decreased activation of erk / in invasive trophoblasts in vivo. objective: placentae from preeclamptic pregnancies are characterized by an excess of immature hyperproliferative trophoblast cells, however the molecular mechanisms regulating cell cycle progression in this pathology are unclear. our aim was to examine the expression of g phase cell cycle regulators in normal and preeclamptic placentae and to establish whether the hyperproliferative state of trophoblast cells in preeclampsia may result from a developmental delay. methods: human placental samples were collected from normal pregnancies throughout gestation (n= ) and from severe early onset preeclamptic (n= ), and age-matched control placentae (n= ). protein expression of cyclin e , cyclin d , cyclin d and cell cycle inhibitors, p , p , p , and p was assessed by western blot analysis. spatial and temporal localization of cyclins e , d , d , p and p was determined by fluorescence immunohistochemistry. expression of cyclin e and cyclin d mrna was evaluated by qpcr analysis. results: immunohistochemical analysis showed cyclin e and cyclin d to be localized to cytotrophoblast (ct) cells of the chorionic villi; additionally cyclin e was also expressed in the extravillous trophoblast cells of the anchoring columns. in contrast, cyclin d expression was predominantly restricted to the villous stroma. cell cycle inhibitor p was expressed in both ct and syncytiotrophoblast (st) cells whereas p was restricted to the st. during normal placentation, levels of both cyclin e and cyclin d were high in the first trimester and decreased with advancing gestation. the expression of cyclin d , p and p showed an inverse correlation to cyclins e and d whereby their expression increased towards term. levels of p and p remained constant throughout pregnancy. preeclamptic placentae showed a significant increase in both cyclin e and p and a decrease cyclin d and p expression, as compared to age-matched control tissues. interestingly, preeclampsia was associated with an increased number of cyclin e positive progenitor ct cells in the floating villi and an increased expression of p in the endothelial cells lining the villous vessels. conclusion: preeclampsia displays an altered expression of g phase cell cycle regulatory molecules portraying an expression profile that closely resembles that of first trimester placentae. (supported by cihr, owh/igh). we have also demonstrated elevated, hif- -mediated placental and circulating sflt- at high altitude. we sought to correlate circulating levels of plgf, free vegf and sflt- with maternal and fetal oxygen tensions. we hypothesized that circulating sflt- and free vegf would be increased at m, and that plgf, known to be up-regulated by higher oxygen tension, would be decreased. methods: we collected both serum and plasma samples, the latter treated with inhibitors of platelet activation. maternal and umbilical samples were obtained from and healthy mother-infant pairs living at m and m respectively. plgf, free vegf and sflt- were measured in both serum and plasma using commercially available elisa kits (r d). data were log-transformed and analyzed by unpaired student's t test or anova as appropriate. results: plgf did not differ between altitudes. free vegf ( ± pg/ml vs. ± pg/ml, p<. ) and sflt- ( . ± . vs. . ± . ng/ml) were increased at m. however concentrations of all the angiogenic growth factors were reduced when platelet activation was prevented (- ± % plgf; - ± % free vegf; - ± % sflt- , p<. ). cord blood free vegf was -fold greater than in the mothers, but inhibition of peripheral cell activation abolished detectable levels in % of the babies. similar results were obtained for sflt- . thus, while free and total maternal circulating vegf and sflt- are elevated at high altitude, these increases are due to activation of peripheral blood cells. moreover, free vegf does not exist in detectable amounts in human pregnancy. there was no relationship between variation in the angiogenic growth factors and maternal or fetal oxygen tensions. the objective of this study is to identify candidate genes responsible for the variance between severe preeclampsia (spe) and hellp syndrome (hs). placental biopsies from spe (n= ) and hs (n= ) were collected. diagnosis of spe was confirmed by blood pressure and protein criteria. hs was diagnosed in preeclamptic patients who developed characteristic laboratory abnormalities. placental tissues were embedded in oct for sectioning, h e staining and rna isolation (invitrogen, carlsbad). gene expression data was obtained by hybridizing fluorescently labeled reverse transcription products to spotted cdna microarrays. the microarrays were produced by the laboratory of microarray technology at the van andel institute (grand rapids, mi) using a custom microarrayer. microarrays were scanned using an agilent g b scanner. images were analyzed using genepix . (axon). limmagui was used to generate lists of discriminating genes for these data, while david provided functional annotations. there were differentially expressed genes between spe and hs (p<. ). among these candidate genes, were up-regulated and were downregulated. the most up-regulated genes are ( )-deoxyribonucleotidase (dnt- ), superoxide dismutase , hydroxy-delta- -steroid dehydrogenase, caspase , and general transcription factor iih. further analysis of functional groups revealed the most enriched gene categories are related to cellular energy (mitochondria), cell cycle regulation, and protein metabolism. the underlying role of the placenta in the development of hs is currently unknown. this study shows that there is a relatively mdest number of genes that are differentially expressed between hs versus spe placentals. thes data provide the molecular context for placental changes seen in this variant of preeclampsia and provides an opportunity to study the role of the effected genes in disease variance. ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and control group ( patients) uncomplicated pregnancies. total rna was isolated and reversely transcribed to c-dna. the mrna level of tert was detected with a probe-specific real-time quantitative pcr assay using ß-actin as the reference gene. crossing point (cp) values were obtained during the pcr amplification and the relative expression level of htert equals to (cp htert -cp ß-actin) . statistical analysis was performed using the student's t test. immunohistochemistry (ihc) staining was employed to localize htert protein on placenta tissue sections using abc method, incubation with rabbit anti-htert antibody followed by application of a goat anti-rabbit antibody with results evaluation using microscope. objective tgf s are involved in the regulation of trophoblast differentiation and invasion, and we have previously reported that tgf- is over expressed in pre-eclamptic placentae. tgf s signal via a receptor complex composed of type ii (t r-ii) and type i (t r-i) receptor. to date, seven type i receptors, designated as activin receptor-like kinase (alk - ) have been identified. our aim was to investigate the expression pattern of alk and alk receptors, known to exert tgf signaling, in preeclamptic and iugr placentae. methods human placental tissue throughout gestation was used in order to determine the development profile of the receptors. in addition, placental tissue from preeclamptic and iugr pregnancies and from age matched controls was collected. all iugr pregnancies were characterized by absence of end diastolic velocity in the umbilical artery and had no evidence of preeclampsia. expression of alk and alk mrna was measured by real-time pcr analysis, and protein by western blot analysis using alk and alk antibodies. immunoblot analysis demonstrated a unique developmental profile whereby alk expression increased with advancing gestation. in preeclamptic placentae alk expression was significantly increased compared to preterm and term controls, whereas alk expression was significantly decreased. preeclamptic placentae also exhibited decreased phosphorylation of smad , a tgf signaling molecule, which is activated by alk and increased phosphorylation of smad , which is triggered by alk . the expression of alk and alk in iugr placentae differed from that of preeclampsia as both alk and alk mrna levels were significantly increased in iugr compared to preterm and term controls. however, only alk expression was significantly increased in iugr placentae at the protein level, while no differences in alk protein levels were noted between iugr and controls. conclusions imbalance between alk and alk signaling pathways might play a role in the pathogenesis of preeclampsia and iugr. as tgf signaling via either alk or alk has been found to differentially regulate vasculogenesis, changes in these signaling pathways may contribute to the altered vasculogenesis found in these pregnancy-related disorders. (supported by cihr and owh/igh). ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and patients, the control group who had uncomplicated pregnancies. total protein was isolated from placental tissue. gadd a and its downstream signal proteins--phospo-p , phospho-mkk /mkk were assessed by western blot. immunohistochemistry (ihc) staining was employed to localize the expression of gadd a and sflt- proteins in placenta tissue sections using abc method. huvec cells were cultured to - % confluence and were divided into groups: control, stress induction (sorbitol, . m, h), p inhibition (sb- , ug/ml, ug/ml and ug/ml, hour) + sorbitol ( . m, h). total protein was isolated from cells and the supernatant of huvec was collected. western blot was processed to detect the induction of gadd a and phospho-p . supernatant sflt- was measured with an eia kit and the results were read at nm wavelength. results: gadd a protein was elevated in the preeclamptic placentas with its downstream proteins (mkk and p ) activation compared with control. overexpression of gadd a and sflt- in preeclamptic placentas was observed with ihc staining. in huvec cells, gadd a was induced by sorbitol, triggering the activation of the downstream p- pathway and the accumulation of sflt- in the supernatant. the up-regulation of sflt- secretion by inducing gadd a was depleted when treated with p- inhibitor. conclusions: our study reveals that gadd a and its down stream p- pathway were activated in preeclamptic placentas and this stress inducible signal pathway regulates the secretion of sflt- , which is a key player in preeclampsia. it provides novel evidence that links placental stress to sflt- secretion via the gadd . trophoblast adipose triglyceride lipase (atgl) expression is upregulated in preeclampsia. beth a plunkett, jennifer a doll, emily j su, serdar e bulun, mona cornwell, susan e crawford. obstetrics and gynecology, northwestern university, chicago, il, usa; pathology, northwestern university, chicago, il, usa. objective: preeclampsia is characterized by placental endothelial cell dysfunction and elevated maternal triglyceridemia (tg). tg traverse the placenta in a process of uptake followed by lipolysis with subsequent release of fatty acids to the fetus. although three placental lipases (hormone sensitive lipase, endothelial lipase and lipoprotein lipase) have been identified, they do not account for all lipolytic activity. here, we introduce a new lipase, adipose triglyceride lipase (atgl), which is responsible for the hydrolysis of triglycerides to diglycerides in adipocytes and was recently identified as a receptor for endothelial cell modulator pigment epithelium-derived factor. the purpose of this study is to determine if expression of atgl, a potential modulator of both lipid metabolism and vasculature, is upregulated in preeclampsia. methods: immunohistochemical studies were performed on placental tissues from normal pregnancies (n= ) and those complicated by severe preeclampsia (n= ) with anti-atgl antibodies. the degree of positivity in the trophoblasts and endothelial cell was scored ( =none, =spotty, light, =consistent, dark). to determine if atgl is a product of placental endothelial cells (plec), microvascular cells were isolated from normal placental tissue. purity of the sample was confirmed using flowcytometry (> / positivity for factor antigen). atgl was detected immunohistochemically and via western blot using anti-atgl antibodies. results: mean atgl expression in preeclamptic trophoblast was significantly higher than normal placentas ( . + standard deviation versus . + . , p = . ). endothelial expression was not significantly different in preeclamptic ( . + . ) versus normal placentas ( . + . , p> . ). atgl stained intensely and demonstrated a beaded pattern in the endothelial cells, suggestive of a lipid droplet pattern. immunohistochemistry of plec and western blot analysis of cell lysates revealed strong immunopositivity for atgl, although at a smaller size than anticipated. conclusion: these findings demonstrate that a novel lipase, atgl, is produced by trophoblasts and is upregulated in severe preeclampsia. plec express high levels of atgl, suggesting that atgl could prove to be important in the vascular dysfunction and lipid abnormalities characteristic of preeclampsia. increased endothelial chymotrypsin-like protease (chymase) expression is responsible for endothelial activation in preeclampsia. yuping wang, yang gu, yanping zhang, david f lewis. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: endothelial (ec) activation is an important component of inflammatory phenotypic changes in preeclampsia (pe). our previous study showed enhanced chymotrypsin-like protease (clp)/chymase expression in the maternal vessel endothelium in women with pe. in this study, specific effect of placental-derived clp on ec activation was examined. methods: human uterine microvascular endothelial cells (utmvecs) were used. placental conditioned medium (cm) was prepared by culturing villous explants from normal and pe placentas. confluent utmvecs were treated with placental cm with or without depletion of chymotrypsin. ec adhesion molecule expressions for icam, vcam, p-selectin and e-selectin were determined by a colorimetric assay at od nm. depletion of chymotrypsin from cm was performed by immunoprecipitation. to further determine if activation of endogenous clp/chymase in ecs is responsible for up-regulation of p-selectin and e-selectin expression, chymase sirna was applied to ec culture before the cells were treated with normal or pe cm and then ec adhesion molecule expressions were examined. data was expressed as mean ± se and analyzed by anova. a p level < . was set for statistically different. results: ) expressions of vcam, p-selectin and e-selectin, but not icam, were significantly increased in pe-cm treated utmvecs compared to those of normal-cm treated cells and untreated controls, p< . ; ) there was no difference for adhesion molecule expression in utmvecs between normal-cm treated with untreated controls; ) utmvecs transfected with chymase sirna significantly reduced p-selectin and e-selectin expressions when exposed to pe-cm, p< . . conclusion: placental-derived clp/chymase is responsible for activating ecs and inducing ec adhesion molecule expression. activation of ec chymase may be directly related to the inflammatory phenotypic changes that occur in ecs in pe. (supported nih grants hd and hl ). corin is a transmembrane serine protease that is important in processing natriuretic peptides (nps) and maintaining normal blood pressure. genetically modified mice without corin function develop a syndrome during pregnancy similar to preeclampsia. corin is present in the pregnant uterus and a deficiency in the enzyme may lead to hypertension and preeclampsia. we tested the hypothesis that corin expression was increased in human myometrium from women with preeclampsia. methods: myometrium was obtained from groups of women at the time of primary cesarean section: (i) preterm no labor with preeclampsia (n= , ptspe, . weeks); (ii) preterm labor (n= , ptl, . weeks); (iv) term no labor (n= , tnl, . weeks); (v) term labor (n= , tl, . weeks). microarray gene profiling was performed using affymetrix human genome u plus . arrays. women who were not in active labor at the time of delivery (tnl) served as the control group. conventional and real time pcr was performed to verify corin expression in the arrays was directionally accurate. corin protein expression was examined by western blot. results: compared to tnl control, corin levels decreased nearly -fold in myometrium from women in term labor (tl). there was a small increase in corin gene expression of preeclamptic women (ptspe) and little-to-no change in myometrium from women in preterm labor (ptl). these results were confirmed by pcr analysis. conclusion: blood volume surges during pregnancy, increasing the potential for hypertension and preeclampsia in the mother. the corin-np control system could contribute to this condition. however, there are no reports on corin message or protein levels in women with preeclampsia. our preliminary results suggest that preeclampsia is not a consequence of a corin deficiency (decreased corin preeclampsia). acknowlegement: supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). the expression of gp at implantation site: implication for the pathogenesis of preeclampsia. objective: gp is a common shard signal transducing subunit of the il- cytokine family which is critical for implantation, and viewed as marker of endometrial blastocyst receptivity. preeclampsia (pe) is highly related to the restricted trophoblast invasion, which leads to impaired spiral artery remodeling. our hypothesis was that the poor placentation of pe is associated with the altered expression of gp at the implantation site. methods: human decidua from patients with pe and uncomplicated term deliveries (n = , respectively) were immunostained for gp . the intensity and distribution of immunostaining on decidual cells and extravillous traphoblasts were evaluated with hscore. statistical analysis of the data was performed using student's t-test and kruskal-wallis one way anova on ranks followed by post hoc test. results: immunostaining of gp was significantly higher in decidual cells of patients with pe compared with normal specimens, with hscore (median, [interquartile range]) value [ . - . ] and , respectively (p< . ). in pe specimen, the hscore of decidual cells was also significantly higher than that of trophoblast ( [ - ]; p< . ). there were no difference in the comparison of hscore of trophoblasts between the pe and normal specimens, and in normal specimens between decidal cells and trophoblasts. conclusions: the increase of gp expression in the decidual cells of preeclamptic placenta may be implied to the pathogenesis of poor placentation in preeclampsia. we have previously demonstrated that decidual cells are the major source of renin at the human uteroplacental interface, but little is known regarding the human decidual renin expression in hypoxic conditions. therefore, the present study was undertaken to determine the effect of hypoxia on renin secretion by human decidual cells in vitro. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were exposed to normoxia or hypoxia ( % or % oxygen) in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. a dominant band of renin at approximately kd was detected in all samples. when compared with the cells cultured in the normoxic condition, the cells cultured in both hypoxic conditions (i.e. % and % oxygen) had significantly lower renin protein contents in their culture supernatants. conclusion: our data for the first time show that hypoxia down-regulates renin secretion in human decidua, suggesting a link between the uteroplacental ras and oxygen tension during human gestation. the effect of nucleated fetal red blood cells derived from preeclamptic patients on endothelial progenitor cell proliferation. keiichi matsubara, emiko abe, yuko matsubara, shinji hyodo, masaharu ito. obstetrics and gynecology, ehime university school of medicine, toon, ehime, japan. objectives inadequate uteroplacental circulation results in placental ischemia and the development of preeclampsia (pe). endothelial progenitor cells (epcs) are thought to be a key player in the fetal angiogenesis. vascular endothelial growth factor (vegf), which is up regulated in pe, is involved in epcs proliferation. recently, it was reported that fetal nucleated red blood cells (nrbcs) have the capability to generate vegf. we hypothesized that nrbcs could influence epcs proliferation in the placenta and may be involved in the pathogenesis of pe. material and methods mononuclear cells (mncs) were isolated from the umbilical venous blood of normal pregnant women and preeclamptic patients by density gradient centrifugation. mncs were incubated with anti-cd antibody conjugated with microbeads. nrbcs were collected using a mini macs separator. nrbcs were incubated for hours with or without angiotensin ii (ang ii) and erythropoietin (epo). vegf and placental growth factor (plgf) concentrations in the supernatant were measured using elisa. also, pbmcs without nrbcs were seeded in endothelial basal medium with or without nrbcs using a boyden chamber. these samples were incubated for days with or without ang ii and epo. the adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. di-ldl and lectin positive cells was considered to represent epcs and the number was measured using flowcytometry. the number of nrbcs derived from umbilical venous blood was significantly increased in pe. both ang ii and epo significantly increased vegf concentration in the supernatant of nrbcs derived from normal pregnant women. however, ang ii and epo did not influence the nrbcs' vegf production in pe patients. plgf was not detectable in the supernatant. the number of epcs in the umbilical venous blood was significantly decreased in pe and the number was not changed by nrbcs. on the other hand, the number of epcs was significantly decreased in the culture with nrbcs. epo significantly increased the number of epcs in pe at the lower concentration of epo compared with normal pregnant women. conclusions it appears that fetal nrbcs may inhibit fetal epcs proliferation in pe. since epo reduced the inhibitory reaction of nrbcs without vegf production epo may affect epcs proliferation independently of nrbcs. relationship between the hypoxia-inducible factor- (hif- ) and the receptor svegf-r /sflt- : implication for phatophysiology of preeclampsia. julio e valdivia-silva, , juan c gonzalez-altamirano, keisy lopez- the trophoblast invasion is critical for the establishment of the uteroplacental circulation. at early phases of this process local oxygen pressure in the placenta is lower, that pathologically in preeclampsia remain constant. because of this, is important to understand the response of placental cells against these stimuli. in the present work, we use primary cultures of trophoblast cells, fibroblasts of the villous, and human umbilical endothelial cells, isolated of preterm and term placentas (with and without preeclampsia), to explore the effect of the oxygen pressure in the expression and synthesis of vegf, svegfr- /sflt- , hif- and- . our results show that the low pressure of oxygen resulted in a significant increase of the mrna and the protein of the receptor svegf-r selectively in the cts. the vegf's expression and synthesis was raised in three cellular types, but the free protein (not bounded to svegf-r ) of the cts was diminished. on the other hand, the expression of the arnm of hif- or - in cells was comparable in all the types of placentas, nevertheless, the protein hif- was more increased in the cts of preeclamptic placentas. to evaluate the relation of hif- and the increase of the receptor svegfr- , we used sirna-hif . in response to the inhibition, the expression of the receptor svegf-r diminished dramatically. the blockade of hif- did not alter vegf's expression. our data are the first that propose that the protein of the factor of transcription hif- is one of the molecules involved in the selective expression of the receptor svegf-r in trophoblast cells during hypoxia. figure: reduction of the expression to soluble receptor flt- /svegfr- after transfection with sirna-hif in trophoblast cells. sirna= interference rna, lu=luciferase. luc-sirna was used as control. effects of estradiol on synthesis, secretion, and activation of von willebrand factor in endometrial endothelial cell. shumei zhao, chainarong choksuchat, michael s scholfield, todd d deutch, thomas d kimble, david f archer. obstetrics and gynecology, eastern virginia medical school, norfolk, va, usa. objectives: heavy menstrual bleeding (hmb) is a serious clinical condition affecting % of women. due to inefficient and ineffective medical interventions, women with hmb often elect endometrial ablation or hysterectomy to eliminate hmb symptoms. morbidity and loss of fertility linked to these surgical treatments support the search for more effective medical remedies. von willebrand factor (vwf), a principle initiator of blood clotting produced by endothelial cells. women with von willebrand disease (vwd), have a high incidence of hmb indicating poor clotting. these findings suggest vwf heavily impacts the amount of blood loss during menstruation. estrogen stops/reduces hmb and has been used to treat hmb in women with vwd, although the mechanism(s) is unknown. this proposal addresses the hypothesis that estradiol (e ) increases the synthesis and activation of vwf, promoting clotting. materials and methods: immortalized human endometrial endothelia cells (heecs) were used for the studies. to determine if e increases synthesis of vwf, we treated heecs with e at . μm, . μm and . μm for hours. vwf protein and mrna levels were determined by western blotting and real time pcr, respectively. to establish if e can convert vwf from an inactive to an active conformation, the release of activated vwf by cells into culture medium will be assessed by elisa. decreased adamts (a disintegrin and metalloproteinase with thrombospondin type motif) activity results in increased amounts of active vwf. rrelease of activated adamts will be determined by fret. to ascertain if e regulated vwf secretion is by genomic pathway, heecs will be exposed to the estrogen receptor antagonist ici , . elisa and fret will assess the release of active vwf and adamts , respectively. results: western blotting demonstrated e increases vwf mrna and protein levels in a dose-dependent manner in heecs.conclusion: the project will determine if e acts at the heecs to increase synthesis, secretion and activation of vwf. preliminary results show e increases intracellular vwf protein and mrna levels in heecs, supporting our hypothesis that e increases synthesis of vwf in heecs in vitro. if e increases activated vwf, it could reduce/stop hmb by increasing activated vwf, providing justification for a clinical trial of e to treat hmb. over-expression of vegf-d does not induce lymphangiogenesis in the mouse endometrium. peter a rogers, jacqui f donoghue, marc g achen, steven a stacker, jane e girling. obstetrics gynaecology, monash university, melbourne, victoria, australia; ludwig institute for cancer research, melbourne, victoria, australia. background: the human endometrial functionalis has reduced lymphatics compared to the basalis and myometrium . this study examines the distribution of lymphatics in mouse uterus and investigates if over-expression of the lymphangiogenic growth factor vascular endothelial growth factor-d (vegf-d) stimulates growth of new endometrial lymphatic vessels. methods: the distribution of uterine lymphatics was examined in c bl/ jxcba mice collected during the oestrus cycle, early pregnancy and following oestrogen and progesterone treatment. human ebna cells with/without stable transfection of vegf-d were injected into the uterine horn of nod/scid mice. uteri were collected after weeks. serial sections were immunostained with lyve- and/or vegfr (lymphatic endothelial cell markers), cd (blood endothelial cell marker), mab (human vegf-d), mab (human mitochondria) and pcna (proliferative cell nuclear antigen). results: lymphatic vessel profiles were mostly found in the connective tissue between the longitudinal and circular muscle layers of the myometrium. they were rare in the endometrium and only observed in % of the sections. when present in endometrium, lymphatic vessel profiles were usually situated adjacent to the endometrial/myometrial border. ebna tumours formed inside and outside the uterine horn of both the control (n= of ) and vegf-d group (n= of ). localization of ebna cells within the mouse uterus was confirmed by anti-human mitochondrial expression. vegf-d immunostaining confirmed that transfected ebna cells expressed vegf-d in vivo. over-expression of vegf-d did not stimulate endometrial lymphangiogenesis, although there was an increase in vessel diameter of lymphatics in the myometrium adjacent to tumours. initial analysis shows no significant effect of vegf-d ebna cells on endometrial blood vascular density or endothelial cell proliferation. conclusions: minimal lymphatics are present in the mouse endometrium, as is the case for lymphatic vessels in the human endometrial functionalis. the lack of endometrial lymphangiogenesis in response to vegf-d suggests the presence of an inhibitory factor limiting lymphatic growth in this tissue. in early pregnancy, decidual-derived vegf mediates angiogenesis and is required for implantation and placentation. the role of decidual vegf in later pregnancy is poorly understood. decidual hemorrhage (placental abruption) generates excess thrombin and is a major risk factor for pprom and preterm birth. this study compares immunohistochemical (ihc) localization of vegf in decidual tissue sections from term pregnancies complicated by abruption and gestational age-matched controls, and investigates the effect of thrombin on vegf expression by cultured human term decidual stromal cells (dscs). study design: ihc was performed on serial sections of term placental tissues (no labor) with (n= ) and without (n= ) abruption. purified term dscs were passaged until > % free of cd + cells by facs. confluent dscs were primed with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days. after h incubation in defined medium with corresponding steroids ± thrombin ( . - . iu/ml), conditioned supernatants were analyzed for vegf by elisa. extracted total rna was used to assess vegf mrna levels by quantitative rt-pcr using established primers. results: vegf expression was localized by ihc primarily to dscs in placental tissue sections, and was increased in tissues from placental abruption vs controls. in term dscs, thrombin increased vegf secretion in a dosedependent fashion irrespective of the hormonal milieu (eg, . -fold stimulation by . iu/ml thrombin from . ± . to . ± . pg/ml per mcg protein for e +mpa; p= . ). this effect was abrogated by the thrombin inactivator, hirudin. vegf mrna were similarly increased by thrombin with or without steroid hormones (eg, . -fold for e +mpa; p< . ). conclusions: placental abruption is associated with increased vegf expression in term decidual tissues in vivo with thrombin enhancing vegf mrna and protein expression in term dscs in vitro. excess thrombinmediated vegf expression in term decidua aberrantly increases endothelial cell permeability to further generate thrombin by continuous exposure of tissue factor-expressing decidual cells to circulating factor vii. thrombin-enhanced matrix metalloproteinase expression in term dscs would degrade decidual and fetal membrane extracellular matrix to induce pprom and preterm birth. basal directional release of angiotensin ii by endothelial cells stimulated by chymotrypsin-like protease (clp)/chymase. yuping wang, david f lewis, yang gu. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: chymotrypsin-like protease (clp)/chymase is a serine protease which plays a major role in angiotensin ii (ang ii) generation in the human heart. our previous study showed a higher clp activity in the maternal plasma in women with pe than in normal pregnancies. we also found enhanced chymase expression in the maternal vessel endothelium in women with pe. in this study, we determined if clp could promote endothelial cell (ec) generation of ang ii. methods: we specifically examined basal directional release of ang ii by cultured ecs. ecs were grown on cell culture insert ( well/plate, micron pore size). when ecs reached confluence, chymotrypsin (chy) at concentrations of . , . , . , . , and . g/ml were added to the upper chamber of the cell insert. after hours of culture, medium in the lower chamber was collected. medium concentrations of ang ii were measured by enzymelinked immunoassay (eia). all samples were measured in duplicate. data are expressed as mean ± se and analyzed by anova. a p level < . was considered statistically different. results: chymotrypsin produced a concentration-dependent increase in basal directional release of ang ii by cultured ecs, control: . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml (p< . ); chy . : . ± . g/ml (p< . ), respectively. data are means from independent experiments. conclusion: apical exposure of ecs with chymotrypsin-like protease could promote basal directional release of ang ii. our result implicates that in pe, elevated clp levels in the maternal circulation are very likely to affect ec generation of ang ii. basal directional released ang ii may bind to its receptor on underlying vascular smooth muscle cells and contribute to the increased vasoconstriction in pe. (supported nih grants hd and hl ). vegf stimulates angiogenesis and vasodilation critical for dramatic rises in materno-feto interface blood flows directly linked to fetal growth/survival. extracellular signal-regulated kinase (erk / ) pathway mediates partially vegf-induced angiogenic and vasodilatory responses in placental endothelial cells (ec). it is, however, unknown how this vegf-induced signaling is organized in placental ec. objectives: ovine fetoplacental artery ec (ofpaec) and its transformed counterpart, sv -of to test whether: ) vegf-activated erk / signaling is compartmentalized in the caveolae and disruption of caveolae interferes vegf-induced erk / activation and; ) caveolin- , the structure protein of caveolae, regulates vegf-stimulated erk / phosphorylation. methods: ofpaec or sv -of cells were cultured in mcdb- / % fbs/antibiotics. serum-starved subconfluent ( %) cells were treated with rhvegf ( to ng/ml) for various times. caveolae were disrupted by -cyclodextrin ( -cd, mm, min) or caveolin- scaffolding domain (cav-sd, m, hr). sv -of cells were used for fractionation of caveolae membranes by discontinuous sucrose gradient ( %/ %/ %) ultracentrifugation. activation of erk / signaling pathway were analyzed by western-blotting with specific antibodies. results: in total cell extracts, vegf stimulates erk / phosphorylation in a time-and dose-dependent manner. erk / phosphorylation maximized by vegf ( ng/ml) at - min, which was abrogated by -cd or cav-sd. all the molecules for compromising the erk / signaling module, plc , pkc , src, ras, raf- , mek / and erk / , were detectable in purified caveolae membranes positive for various markers including caveolin- , enos, flotillin- , and -adaptin. in caveolae, vegf dramatically increased phosphorylated erk / without altering total erk / in a time-dependent manner similar to that in total cell extracts, which also maximized at - min. pretreatment with -cd or cav-sd blocked vegf stimulation of erk / phosphorylation in caveolae. conclusion: vegf activates the erk / signaling pathway in caveolae and caveolae integrity is essential for vegf-activated erk / signaling pathway. we conclude that caveolae/caveolin- serves as a platform for compartmentalizing the vegf-induced erk / signaling pathway in placental ec (hl and hl ). hypoxia upregulates gcm in human placenta. david mccaig, fiona lyall. institute of medical genetics, university of glasgow, glasgow, united kingdom. introduction: studies in transgenic mice have shown that a variety of genes regulate the differentiation of trophoblast cells. these genes include gcm . gcm is also expressed in the human placenta. placental gcm- protein has been reported to be reduced in pre-eclampsia, in view of the close link between hypoxia, hypoxia-reoxygneation, pre-eclampsia, placental development and the reported reduction in gcm we hypothesised that gcm expression would be affected by hypoxia. aim: the aim of this study was to determine the effects of hypoxia on gcm expression in the human placenta. two model systems were used; ( ) free floating villous explants and ( ) cultured primary cytotrophoblast and syncytiotrophoblast cells as described previously*. methods: explants or cell cultures were exposed to either hypoxia or hypoxia followed by re-oxygenation. western blot analysis was used to assess gcm protein levels. bands on the gels were quantified using scanning densitometry. statistical differences (n= experiments for both models used) were calulated by anova and turkey's post-hoc test. results: gcm protein was detectable at a low level in villous explants maintained for h in % o . a striking increase in gcm protein was observed when villous explants were incubated for h in % o (p< . ). incubation of villous explants for h in % o followed by re-oxygenation for h in % o resulted in a marked decline in gcm protein (p< . ). expression of gcm was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in % o or reduced oxygen ( % o ) conditions. gcm protein was not detected in any of the experimental conditions used. discussion: the present study has shown that acute hypoxia increases gcm- protein in villous explants. the experiments with purified trophoblast do not support a role for hypoxia increasing gcm- in these cells under the experimental conditions used. the present findings are in keeping with the complex effects of oxygen depending on the conditions used. the observed hypoxic effects on gcm warrant further investigation. the effect of acute alcohol exposure on histone -lys modification in the mid-gestation embryonic lung. xiangyuan wang, debra wolgemuth, , , laxmi baxi. ob/gyn; genetics development; human nutrition, columbia university medical center, new york, ny, usa. objective maternal alcohol abuse during pregnancy produces an array of birth defects comprising fetal alcohol syndrome. lung development depends on a balance between cell proliferation and apoptosis. we have previously shown that the acute alcohol exposure in the mid-gestation embryo can delay lung development and induce apoptosis. acute exposure to ethanol of selected tissues in mouse embryos has been reported by others to initiate apoptosis within hours after exposure and result in histone modifications. specifically, histone acetylation and deacetylation are involved in transcriptional activation and repression, respectively, but can also involve apoptosis. in the present study, we have investigated the effect of alcohol on acetylation of histone at lysine (ach lys ) in the mid-gestation embryonic lung. pregnant c bl/ j mice at day . of gestation (e . ) were injected intraperitoneally with doses of % ethanol ( . g/kg ), hr apart (alcoholexposed: ae) or with ringers solution (controls: c). ae and five c fetuses were retrieved and hr later and the lungs were fixed and processed for morphological evaluation and staining with rabbit polyclonal anti-ach ly antibody. the entire lung tissue field was evaluated for the levels of ach lys staining and scored as (-) to (++++). three areas were selected randomly from each sample and the total number of cells and staining positive cells were counted in the bronchial epithelium and in the mesenchyme. twelve hr after alcohol exposure at e . , the morphology of ae embryonic lungs was normal. however, high levels of ach lys were detected in % of the bronchial epithelial cells and % of the mesenchymal cells. the expression level in both lineages decreased hr after alcohol exposure. in the controls, the expression of ach lys was virtually undetectable in both the bronchial epithelium and the mesenchymal cells. our previous study showed that ae e . lungs significantly increased apoptotic cell in both bronchial epithelium and mesenchyme hours after alcohol treatment. we now observe that elevated expression of ach lys in the embryonic lung preceded the observation of apoptosis, suggesting that alteration in the acetylation of h could be one of the molecular mechanisms involved in the induction of apoptosis following acute alcohol exposure. objective: our purpose was to evaluate the effect of vitamin c and e supplementation on lipid peroxide levels, total antioxidant ability, and antioxidant levels in the umbilical venous plasma. materials and methods: women at risk for preeclampsia (nullipara, previous preeclampsia, chronic hypertension) were recruited at to weeksgestation and randomly assigned to receive either mg of vitamin c and iu of vitamin e (study group, n= ) or placebo (control group, n= ) daily until delivery. umbilical venous blood were collected after full term delivery. lipid peroxide levels, oxygen-radical absorbance capacity (orac) values, antioxidant levels were measured by each method (thiobarbituric acid reaction, cao's method, and high performance liquid chromatography). results: . the lipid peroxide levels in the umbilical venous plasma of study group were significantly lower than that of control group ( . ± . vs. . ± . nmol/mg protein, p< . ). . the orac values in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . u/ml, p< . ). . the -tocopherol levels in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . nmol/ml, p< . ). . there were no significant differences in the ascorbic acid, uric acid, -carotene, retinol, and -tocopherol levels in the umbilical venous plasma between control and study group. conclusion: this suggested that maternal vitamin c and e supplementation may affect the oxidant-antioxidants balance of the utero-placenta unit and fetus. placental tnf-related apoptosis-inducing ligand (trail) in normal pregnancy and pre-eclampsia. xilian bai, jenny e myers, philip n baker, john d aplin, ian p crocker. maternal and fetal health research group, the university of manchester. introduction: enhanced placental trophoblast apoptosis is well known to occur in pre-eclampsia. however, the potential role of membrane associated or soluble trail, an apoptosis-inducing ligand, and its death receptor, dr , is not known. we tested the hypotheses that the trail/trail-receptor system is compromised in pre-eclampsia and that soluble trail is a circulating factor which triggers the vascular complications of pre-eclampsia. method: this study was conducted on placental samples and plasma (edta) from women with uncomplicated pregnancies (n= ) and with pre-eclampsia (n= ) at - weeks gestation. protein expression levels and tissue localisation of trail and dr were defined in villous tissue by western blotting and immunohistochemistry. soluble trail (strail) was measured in maternal plasma from both groups using a commercial elisa (diaclone). results: placental villous trail and dr protein was unaltered in preeclampsia compared to normal pregnancy. whilst there was differential distribution of trail and dr within the component cells, this also was unaffected in pre-eclampsia. within the villi, trail was mainly confined to the cytoplasm and perinuclear regions of cytotrophoblast, syncytiotrophoblast, stromal cells and the fetal capillary endothelium. conversely, dr was restricted to trophoblast only, distributed evenly between cytoplasm, plasma membranes and nucleus. strail was present in plasma from non-pregnant women of childbearing age [ . ( . - . ) , median (interquartile range), pg/ml, n= ]. however, there was no significant increase either in pregnancy [ . ( . - . ) pg/ml, n= ] or pre-eclampsia [ . ( . - . ) pg/ ml, n= ] (kruskal-wallis test, p> . ). conclusions: these results suggest that placental villous trail is not adversely regulated in pre-eclampsia. the absence of dr in stromal and endothelial cells may increase resistance to apoptotic stimuli in cells of the villous core. the co-localisation of trail and dr in trophoblast suggests a role in autocrine regulation of cell turnover in this cell type. introduction: preeclampsia is associated with apoptosis of the syncytial layer of placenta and the release of particulate and soluble factors which are deleterious to maternal endothelial function. the goal of the current study was to determine whether laser capture microdissection (lcmd) and western blotting could be used to assess levels of syncytial fas ligand (fasl), a key protein in the apoptotic cascade. methods: frozen sections of term placenta delivered from uncomplicated pregnancies at term were used for study (n= ). following staining with mayer's hematoxylin (n= ), lcmd (leica instruments) of an intact terminal villus was carried out using a focused laser pulse directed to the area of interest using a microscope. in the first round of microdissection the placental villus core consisting of fibroblast, macrophages, fetal vessels, and connective tissue, were removed. in the second round of lcmd, the syncytial layer of that same villus was removed and collected in lysis buffer containing detergent and protease inhibitors, and the number of nuclei per sample was recorded. this procedure was repeated until syncytial tissue from - villi were collected. electrophoretic separation of lysate proteins was then carried out, and western blotting and immunodetection using an anti-fasl antibody was performed. results: we observed that fasl was detected in microdissected syncytial specimens at a molecular weight of approximately kda ( figure) , consistent with our previous reports. it is of note, that western blotting of samples containing approximately (lane ), (lane ), (lane ), and (lane ) nuclei revealed that fasl could be reliably detected in specimens containing as few as nuclei. conclusions: since fasl is a cytokine of low to moderate abundance in placenta, this suggests that lcmd coupled with western blotting will be a valuable methodology to elucidate pathways of syncytial apoptosis and pathophysiology in pregnancies complicated by preeclampsia. supported in part by nih grant hd . the placenta of preeclamptic pregnancies shows oxidative and nitrative stress. we have shown low protein abundance but paradoxically high activity of inducible nitric oxide synthase (inos) in the placenta with severe preeclampsia compared with normal pregnancies. protein nitration and phosphorylation are post-translational modifications possibly involved in nos activity. objectives: examine inos localization and tyrosine phosphorylation in the preeclamptic placenta and the effect of a peroxynitrite generator (sin- ) on inos expression in primary human placental microvascular endothelial cell (hpmec) cultures. methods: placental lysates from normal (n= ), mild (n= ) and severe (n= ) preeclamptic pregnancies were western blotted for inos and what are the roles played by peroxynitrite in preeclamptic women? yoshikatsu suzuki, tamao yamamoto, yoshimasa watanabe, takeo itoh, hidetaka izumi. obstetrics and gynecology, nagoya city university, nagoya, japan; pharmacology, nagoya city university, nagoya, japan; obstetrics and gynecology, izumi women's hospital, fukuoka, japan. aim preeclampsia is characterized by hypertension plus proteinuria. it is hypothesized that the endothelial cell function might be activated by placental faculty in early pregnancy and the activation might cause the vascular disease in preeclampsia. peroxynitrite, which is produced by combination with superoxide (o -) and nitric oxide (no), is strong oxidant as well as o -.we investigated whether or not the localization of peroxynitrite in both placenta and resistance artery might play important roles of developing preeclampsia. omental arteries or placentas were obtained from severe preeclamptic and term-normotensive pregnant women at cesarean section. they were stained by ant-nitrotyrosine (nt, a marker of peroxynitrite) antibody. furthermore, the concentration of nt was measured in omental arteries. in formed consent was obtained from all patients in written. preeclampsia was diagnosed according to the criteria of japan society of obstetrics and gynecology. the localization of nt was seen in placentas obtained from sever preeclamptic women ( / ), although it was not seen from normotensive pregnant women ( / ). the localization was also seen in placentas from of severe preeclamptic women with intrauterine fetal restriction. in omental arteries from both groups, the localization of nt was seen to same degree, and the concentration of nt was similar ( . ± . for sever preeclampsia and . ± . for normotensive pregnant women). from these results, it was suggested that an increase in o production, a decrease in no as well as peroxynitrite production in the placentas might cause the vascular dysfunction and subsequently damage uteroplacental blood circulation in preeclampsia. however, the role played by peroxynitrite in resistance artery might be different and more complicated. background: preeclampsia (pe) is associated with shallow cytotrophoblast (ct) invasion of the decidua, leading to impaired vascular transformation and poor uteroplacental perfusion. ct invasion requires the selective proteolysis of the peri-decidual cell (dc) extracellular matrix. we hypothesized that il and tnf , cytokines that have been linked to pe, may induce aberrant expression of the matrix metalloproteinases (mmp) and in dcs, thereby preferentially degrading the decidual ecm and interfering with sequential ct invasion. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on decidua from normal (n= ) and preeclamptic (n= ) women, and staining intensities were evaluated by hscore. confluent, leukocyte-free first trimester dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ) and confirmed by western blotting. quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in preeclamptic decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in normal decidua ( ± and ± , respectively; p< . ). in cultured first trimester dcs incubated with e , tnf increased secreted mmp- and mmp- levels by ± and ± -fold, respectively, while il increased them by ± and ± fold, respectively (p< . ). in parallel incubations with e +mpa, basal mmp- and mmp- output were lowered by approximately % and %, respectively, while tnf -and il elicited mmp- and mmp- levels were blunted by - %. western blotting confirmed the elisa results, and mrna levels corresponded to changes in mmp- and mmp- secreted protein levels. conclusions: over-expression of mmp- and mmp- in decidual cells may promote pe by disrupting decidual ecm and impairing normal ct invasion. the high levels of il and tnf associated with pe may be contributing to this over-expression. our in vitro observations that mpa blunts tnf -and il -elicited mmp expression suggests that exogenous progestin may offer a novel therapeutic approach in preventing pe. to produce -methoxyestradiol ( -me), a compound with diverse biological activities including inhibition of hif- , a transcription factor that mediates cellular response to hypoxia. circulating levels of -me and placental comt activity are significantly reduced in preeclampsia, raising the possibility that reduced production of -me contributes to the pathophysiology of preeclampsia by altering placental response to hypoxia. genetic variation in the comt gene is linked to comt activity and has been associated with intrauterine fetal growth restriction. we determined if a snp in exon of the comt gene (rs ), which does not change amino acid sequence ( leu ), but reduces comt mrna translation, was associated with preeclampsia. we analyzed comt genotypes in paired dna samples extracted from maternal and cord blood from normal pregnancies and pregnancies complicated by preeclampsia by allele discrimination. the study population was predominantly (> %) african-american. the frequency of the minor rs "g" allele, which is associated with low comt activity, was similar in maternal cases and controls (cases: %; controls: %, p= . ), but was significantly greater in fetal dna from pregnancies complicated by preeclampsia compared to control pregnancies (g allele frequency cases: %; control: %; p< . ). likewise, fetal carriage of the rs "g" allele conferred a significantly greater risk of preeclampsia (odds ratio: . ; % c.i.: . , . , p< . ). there was also a significant discordance between paired maternal and fetal rs genotypes with significantly greater discordance for the "g" allele in fetuses hosted in preeclamptic pregnancies (odds ratio: . ; % c.i.: . , . ). we conclude that genetic variation in the comt gene is associated with risk of preeclampsia, possibly through a mechanism involving reduced production of -me. supported by nih p md . smoking is associated with elevated adma in preeclampsia. michael p frank, robert w powers. , magee-womens research institute, pittsburgh, pa, usa; obstetrics gynecology, university of pittsburgh, pittsburgh, pa, usa. objective: cigarette smoke exposure paradoxically reduces the risk of preeclampsia. asymmetric dimethylarginine (adma) is an endogenous competitive inhibitor of nitric oxide synthase (nos), an independent risk factor for cardiovascular mortality, adma is elevated in women who develop preeclampsia, and adma has been reported to be both higher and lower in smokers. the objective of this study was to investigate the concentration of adma in pregnant smokers and nonsmokers with and without preeclampsia. study design: case-control study of women with uncomplicated pregnancy (controls), and women with preeclampsia matched for gestational age at sample collection. adma was measured by hplc. cigarette smoke exposure was determined by questionnaire and confirmed by plasma cotinine. data are mean±sd. analysis was by two factor anova with fishers post-hoc testing, significance accepted at p< . results: as previously reported, maternal plasma adma concentrations were higher in women with preeclampsia compared to controls (p< . ). in addition, the concentration of adma was significantly higher in preeclampsia smokers compared to controls and preeclampsia nonsmokers (p< . ). in contrast, there was no difference in adma concentration between control smokers and nonsmokers. conclusion: these data may suggest a differential effect of cigarette smoke exposure on circulating adma concentrations between women who do and do not develop preeclampsia. previous data has suggested that cigarette smoking is associated with lower adma in low risk elderly patients, and higher adma in high-risk subjects with diabetes. therefore, the data in preeclamptic and non-preeclamptic subjects may be consistent with these studies, however, the underlying biological explanation for this differential effect has yet to be determined. objective: eclampsia is similar to hypertensive encephalopathy in which an acute elevation in blood pressure causes autoregulatory breakthrough, hyperperfusion and edema formation. we previously reported that the pressure of breakthrough was similar between nonpregnant (np) and late-pregnant (lp) rats, but only lp animals developed edema. this study tested the hypothesis that lp animals have decreased in cerebrovascular resistance (cvr) and hyperperfusion in response to breakthrough vs. np. we further hypothesized that acute hypertension would cause greater blood-brain barrier (bbb) permeability in lp rats due to elevated hydrostatic pressure. methods: in vivo models of bbb permeability and cerebral blood flow (cbf) were used in np (n= ) and lp (d - ; n= ) rats that were either normotensive or hypertensive (np-htn, lp-htn) by infusion of phenylephrine to raise mean arterial pressure. permeability was determined in anterior and posterior brain regions by calculating the flux of kd dextran into the brain tissue, measured by a fluorescent spectrophotometer after flushing the vasculature with saline. cbf and cvr were measured by infusion of m fluorescent microspheres and determined based on the flow rate and fluorescence intensity of a reference sample for each animal. animals were ventilated to maintain blood gases within normal ranges (po > mmhg, pco = - mmhg). results: although the pressure change was similar between np and lp ( and mm hg), lp animals responded to acute hypertension with hyperperfusion. cbf increased from ± to ± ml/ g/min in np ( %) and ± to ± ml/ g/min in lp ( %; p< . vs. np). hyperperfusion in lp animals was associate with decreased cvr vs. np ( . ± . vs. . ± . mm hg/(ml/ g/min); p< . ). bbb permeability was significantly increased in lp animals at breakthrough vs. np in both anterior and posterior brain regions. the flux of dextran in anterior and posterior brain regions for np vs. lp animals was: ± vs. ± for anterior (p< . ) and ± vs. ± for posterior (p< . ). conclusions: these data demonstrate that pregnancy decreases cvr and causes hyperperfusion of the brain during acute hypertension. because increases in cvr is a protective function in the brain, impairment of these mechanisms during pregnancy may predispose the brain to edema when blood pressure is elevated, as in eclampsia. introduction: this study used the in vitro dually perfused human placental lobule to test the hypothesese that placental release of vegf and the fetoplacental vasodilatory response to exogenous vegf- are altered by tissue oxygenations that mimic healthy and preeclamptic pregancies. methods: lobules were dually perfused for six hours under one of two oxygenation conditions, representing 'normoxia' and 'hypoxia' (n = each): delivering maternal side inflow perfusate at oxygen concentrations of . % and %, respectively, distributed via cannulae; and fetal side inflow perfusate oxygen concentration of - % in both systems. venous perfusates were sampled and assayed, appropriate to side of release, for erythropoietin (epo), macrophage inflammatory protein- alpha (mip- alpha) (reference oxygen sensitive hormones), free vegf, svegfr- and plgf. in separate perfusions, fetoplacental vasodilation in response to pm vegf was investigated, following preconstriction of the fetoplacental vasculature to steady state fetal-side inflow hydrostatic pressure (fihp) with the thromboxane mimetic, u , (n = each). results: maternal-side mip- alpha release was higher in the 'hypoxic' than the 'normoxic' system ( ± and ± pg/ml, respectively, at hours, mean ± se; -way anova: p< . ). maternal-side epo and fetal-side soluble free vegf and svegfr- release were not different between groups. fetal-side release of plgf was higher in the 'hypoxic' group than the 'normoxic' group ( . ± . and . ± . pm, respectively, at hours, mean ± se; -way anova: p < . ). there was no difference in the vasodilatory response to vegf- in the fetoplacental vasculature between the groups ( . ± . and . ± . % change in fihp, mean ± se). discussion: differences in mip- alpha and plgf release provide evidence for metabolic separation of the adapted systems, caused by a changed oxygen environment. our failure to observe differences in epo, vegf and svegfr- release may be explained by longer lag-times for up regulation of their gene expression. vegf associated endothelial signalling appears to be unaffected by 'hypoxia' in the placenta over the time course studied here. background: there is a placental renin-angiotensin system (ras) from very early pregnancy. ang iv mediates various effects by binding to its specific receptor, the at r, the active site of which is an insulin-regulated aminopeptidase (irap). there is at r expression in both endothelial and smooth muscle cells. ang iv at low concentrations is vasodilatory, increasing blood flow via the at rs; it also stimulates nf-kappa beta and modulates glut- . to date at r expression has not been investigated in the placenta. we propose that at r plays a part in the placental vascular development necessary for successful pregnancy, and that reduced at r expression may be associated with inadequate vascular adaptation contributing to pre-eclampsia (pe). aim: to identify and locate at r expression in both np and pe placentae. methods: the study had hospital ethical approval; written informed consent was obtained from all women. placental samples were obtained from np and pe at delivery (gestational ages: and weeks respectively). samples were taken from areas, near cord, middle and outer edge of the placenta. paraffin embedded sections were immunostained for irap reactivity using a rabbit polyclonal antibody (gift from professor david james, garvan institute, australia). immunoreactivity of trophoblast and uterine cell populations was assessed using a semi-quantitative grading system. grade = no positive labelling, = - %, = - %, = - % and = - % of cells positively labelled. median (max, min) are shown. results: ) at r immunostaining was prominent in the syncytiotrophoblast and hofbauer cells of all placental villi examined, with no differences in expression between sampling sites. ) at r positivity was reduced in near cord pe samples ( . ( . , . )) compared to np ( . ( . , . ) p= . ). conclusion: we have shown for the first time, dense at rs in syncytiotrophoblast and hofbauer cells in np placentae, and their down-regulation in pe. reduced ang iv/at r binding may contribute to increased placental vasoconstriction resulting in increased ischemia/reperfusion. this in turn may stimulate xanthine oxidase, which is itself stimulated by angii, leading to increased superoxide production. further work is needed to clearly define the role of this newly identified component of the renin-angiotensin system in normal pregnancy and pe. pre-eclampsia is associated with lower percentages of regulatory t cells in maternal blood. jr prins, hm boelens, jj hm erwich, j heimweg, s van der heide, ajm van oosterhout, aej dubois, jg aarnoudse. obstetrics, university medical center groningen, groningen, netherlands; laboratory of allergology and pulmonary disease, university medical center groningen; pediatric allergology, beatrix children's clinic, university medical center groningen. pre-eclampsia is a serious disease of human pregnancy and immunological mechanisms play a role in its pathophysiology. normal pregnancy is associated with an increase in regulatory t (treg) cells and with a predominant th immune profile. treg cells are a subpopulation of cd + lymphocytes and are specifically characterized by the lineage specific transcription factor foxp . treg cells seem to induce immunological changes that have a protective role in maintaining normal pregnancy. we hypothesised that percentages of treg cells are decreased in pregnancies complicated by pre-eclampsia. methods in total, women with pregnancies complicated by pre-eclampsia and healthy pregnant controls were enrolled. to obtain control umbilical cord blood as well, control group i consisted of eighteen healthy pregnant women at term. in addition, since women with pre-eclampsia delivered preterm, control group ii (peripheral blood only) consisted of women during normal pregnancy with a gestational age matched for the preterm pre-eclamptic group. treg cells were measured from whole blood using four-color flow-cytometry. women with a pregnancy complicated by pre-eclampsia had a significantly lower percentage of cd + foxp + treg cells ( . vs . %; p< . ). in the pre-term group the pregnancies complicated by pre-eclampsia showed a significantly lower percentage of cd + foxp + cells in the peripheral blood as compared to the healthy pregnant controls. at term this percentage was also lower but not significantly so. between pre-term and term pregnancies both complicated by pre-eclampsia no significant difference was found in the percentage of cd + foxp + treg cells. no difference was found in umbilical cord blood ( . vs . %). conclusions our data suggest that pre-eclampsia is associated with a diminished percentage of treg cells in peripheral blood. we conclude that a deficiency of regulatory t cells may play a role in the pathophysiology of pre-eclampsia. background: preeclampsia and eclampsia are significant causes of maternal and fetal death. however, the pathophysiology of these conditions is unclear. we and others have reported that inhibition of endogenous nitric oxide (no) synthesis produces symptoms similar to preeclampsia in pregnant rats. several studies demonstrate that fetoplacental weights are altered in pregnancies of spontaneous hypertensive (shr) rats. in addition, impaired synthesis of tetrahydrobiopterin (bh ), a major co-factor for endothelial nitric oxide synthase (enos) activity and enhanced expression of enos has been observed in the pathogenesis of hypertension. in the current study, we examined whether supplementation of bh and sepiapterin (sep, a precursor for bh biosynthesis in the salvage pathway) reduces increased blood pressure and improves fetoplacental weights in shr pregnant rats. methods: groups ( - ) of shr pregnant rats were either treated with bh , sep ( mg/k.g body weight/day/rat, oral tablets) or vehicle (normal diet) beginning from day of pregnancy until day of gestation. animals were sacrificed on day of gestation and fetoplacental weights were recorded immediately. western blot analysis was performed to determine vascular enos expression (enos/gapdh). results: significant (p< . ) elevations in blood pressure (bp, mmhg) were observed in shr (shr, ± . vs. wky, ± . mmhg) compared to wistar-kyoto (wky) group. supplementation of either bh or sep ( ± . ), significantly (p< . ) reduced elevated bp beginning from day of pregnancy. fetal but not placental weights were significantly (p< . ) reduced in shr ( . ± . grams) compared to wky ( . ± . ) rats. bh ( . ± . ) treatment partially (p< . ) increased fetal weights compared to the shr group. vascular enos expression is significantly (p< . ) elevated in shr ( . ± . ) compared to wky rats ( . ± . ). further, treatment with bh ( . ± . ) but not sep ( . ± . ) significantly (p< . ) reduced elevated enos protein expression in shr rats. conclusions: bh may be beneficial treatment of preeclampsia to reduce blood pressure and improve fetal perfusion to increase fetal weights. genetic risk factor for severe preeclampsia: significance of endothelial nitric oxide synthase gene t- ->c and missense glu asp variants. toshihiro yoshimura, michihiro yoshimura, masafumi nakayama. obstetrics and gynecology, kumamoto university school of medicine, kumamoto, japan; cardiovascular medicine, jikei university school of medicine, tokyo, japan; cardiovascular medicine, kumamoto university school of medicine, kumamoto, japan. introduction: we recently identified two endothelial nitric oxide synthase (enos) gene polymorphisms, a glu asp missense variant in exon and a t- -->c variant in the '-franking region, which are associated with coronary spasm and myocardial infarction in japanese population. and we also identified a missense glu asp variant is associated with severe preeclampsia and placental abruption. our objective was to analyze the association between the t- -->c and severe preeclampsia. materials and methods: the study participants included patients with histories of severe preeclampsia. this is a preliminary study, therefore, the comparisons were made with the general normal population. results: the analyses revealed that the frequency of the missense glu asp variant (n= / , %) was significantly higher than the general population (n= / , %), as we previously published. however, the frequency of the t- -->c variant (n= / , %) was not different from the general population (n= / , %). interestingly, only one patient had both t- -->c and missense glu asp variants, and she developed placental abruption. conclusion: although our sample size is small, it is very unlikely that the t- -->c variant is associated with severe preeclampsia. the t- -->c variant may not be a genetic susceptibility factor to severe preeclampsia. the t- -->c may have some reproductive significance in combination with missense glu asp variant, however huge number of patients would be needed to analyze such rare ( . %) combination of the variance. the association between the development of preeclampsia and methylenetetrahydrofolate reductase, angiotensinogen, vascular endothelial growth factor single nucleotide polymorphism genotype combinations. hyun soo park, jong kwan jun, chan-wook park, joong shin park, bo hyun yoon, hee chul syn. obstetrics and gynecology, dongguk university international hospital, goyang-si, gyeonggi-do, republic of korea; obstetrics and gynecology, seoul national university college of medicine, seoul, republic of korea. objective this study was conducted to investigate if there exists any genotype combination of multiple single nucleotide polymorphism (snp)s which is frequently found in preeclampsia patients. study design one hundred sixty two preeclampsia patients and normotensive pregnant women were included in this study between jan and jul . diagnosis of preeclampsia and assignment of severity were made according to the criteria by national high blood pressure education working group and american college of obstetricians and gynecologists. the patients were reclassified as early ( weeks or before) and late-onset ( weeks or beyond) disease. genotypes were measured with pcr-rflp for methylenetetrahydrofolate reductase (mthfr) c t, angiotensinogen (agt) m t, vascular endothelial growth factor (vegf) c t with the dna extracted from maternal blood. case-control study for each snp was done and the frequencies of genotype combination were compared. anova, t-test, chi-square test, fisher's exact test and logistic regression analysis were used for statistical analysis. a p value of < . was considered statistically significant. results genotypes of mthfr polymorphism showed significant difference between late onset preeclampsia and control (cc+ct/tt, or: . , p< . ) but agt and vegf polymorphism did not show statistical difference between any case-control combination. only out of possible genotype combinations were found and there was no statistical difference in the frequencies of genotype combination between case and control group. conclusion mthfr polymorphism might be associated with the development of preeclampsia, but there was no combination of mthfr, agt and vegf polymorphisms which is associated with the development of preeclampsia. diffuse staining and vascular smooth muscle (vsm) staining. resistance-sized vessels ( - μm) were evaluated. results: for mpo, the intensity of staining (fig) and the % vessels with neutrophil, diffuse and vsm staining was significantly greater for obese than for overweight or normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . , p< . ); % vsm staining ( . ± . vs. . ± . vs. . ± . , p< . ). for mmp , obese and overweight patients had a greater (p< . ) % vessels with neutrophil, diffuse and vsm staining than normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . ); % vsm staining ( . ± . vs. . ± . vs. . ± . ). conclusions: neutrophils infiltrate the systemic vasculature of obese women and release mpo and mmp . speculation: obesity may put women at risk for pe because their vasculature may already be dysfunctional due to neutrophil infiltration and release of mpo and mmp . hl . collagen is an important protein that maintains the structural integrity of tissues. disruption of vascular smooth muscle collagen could result in vascular dysfunction in women with preeclampsia. recently, neutrophil infiltration of the systemic vasculature was demonstrated in preeclamptic women. neutrophils produce inflammatory mediators, such as reactive oxygen species (ros) and tnf-. we hypothesized that neutrophils, ros and tnf-would alter expression of collagen regulating genes. methods: primary cultures of human vascular smooth muscle cells (vsmc) were seeded into t- flasks ( , cells/flask) and grown for days to % confluence. the cells were treated for hours with medium control, ros (hx, . mm + xo . u/ml), tnf-( ng/ml); and neutrophils ( , ) activated with arachidonic acid, μm, ( : ratio of neutrophils to vsmc). rna was extracted from cell homogenates and analyzed for gene expression with an rt profiler pcr array system for human extracellular matrix genes (superarray). to determine the fold-change of gene expression, the results were first normalized to a housekeeping gene and then ct was calculated across two rt-pcr arrays where group was the control and group was the experimental treatment. results: table . conclusions: neutrophils, ros and tnf increased mmp expression. interestingly, genes involved in collagen synthesis (col a ) or inhibition of mmp- activity (timp ) were either not affected or down-regulated. these data suggest that neutrophil infiltration in preeclamptic women could cause vascular dysfunction by creating an imbalance between collagen synthesis and collagen breakdown favoring breakdown. hl , fogarty d tw , p md . objective: hypoxia increases membrane attack complex (mac) binding to cultured human trophoblasts, and mac enhances apoptosis in trophoblasts exposed to low compared to normal fio . trophoblast microparticles and cellular fragments released into the maternal circulation in vivo may contribute to the systemic pathophysiology of preeclampsia. we tested the hypothesis that hypoxia induced mac deposition on cultured human trophoblasts yields microparticles and fragments coated with mac. study design: primary cytotrophoblasts from term human placentas (n= ) were cultured h in % and % oxygen in dmem with % human serum with active mac or heat inactivated serum (control). media were centrifuged to obtain pellets of microparticles and cell debris which were immunostained for mac or exposed - h to confluent, phorbol myristate acetate differentiated u macrophages. the percentage of macrophages that ingested trophoblast debris was quantified by counting the number of macrophages with immunofluorescence for trophoblast cytokeratin filament staining, as assessed by confocal microscopy. results: cultures exposed to normal human serum, but not heat inactivated control serum, showed mac immunofluorescence on microparticles and fragments in medium, with the highest level of mac in cultures exposed to extreme hypoxia. the maximal percentage of macrophages that ingested the trophoblast debris coated with mac from cultures with % oxygen was . %, not different from the . % from cultures exposed a % fio and control serum. conclusion: trophoblasts exposed to hypoxia and active complement release microparticles and cellular fragments coated with mac into the extracellular environment. mac coating does not influence phagocytic removal of the debris by macrophages suggesting that placental derived, membrane bound mac could circulate to yield systemic affects on maternal endothelium. supported by nih hd and hd . is there a role for fatty acids in the pathogenesis of pre-eclampsia? nicola j robinson, laura j minchell, jenny e myers, philip n baker, carl a hubel, ian p crocker. maternal and fetal health research group, the university of manchester; obstetrics, gynecology and reproductive sciences, university of pittsburgh. objectives: women with pre-eclampsia (pe) display altered lipid metabolism as characterized by elevated circulating triglycerides and non-esterified fatty acids (nefa) and these changes are evident before the disease is clinically apparent. we have tested the hypothesis that the increased circulating levels of nefa contribute to endothelial dysfunction in pe. methods: human umbilical vein endothelial cells were incubated for h with pooled plasma ( %) from normal or pe pregnancies, or with palmitic, oleic and linoleic acid in culture media at the concentrations and molar ratios to albumin identified in normal ( , , m, ratio . ) and pe pregnancies ( , , m, ratio . ) , . lipid droplet accumulation was determined using an oil red o absorbance assay. endothelial metabolism was measured using the mtt test and mitochondrial membrane potential determined by jc- assay as a marker of early apoptosis. results: plasma from pe pregnancies increased endothelial cell lipid droplet accumulation compared to normal plasma (p< . , wilcoxon signed ranks, n= ). this change was replicated following exposure to nefa at the combined concentrations found in pe compared to normal pregnant controls (p< . , n= ). plasma from women with pe caused a significant decrease in mitochondrial dehydrogenase activity (mtt test; p< . , n= ) and a reduction in jc- fluorescence (p< . , n= ), compared to normal plasma, suggestive of mitochondrial membrane depolarization and increased cellular apoptosis. again these effects were replicated using nefa in culture medium at the levels found in pe compared to normal pregnancies (mtt test: p< . , n= ; jc- assay: p< . , n= ). conclusions: in endothelial cell cultures, plasma from women with pe caused increased lipid droplet accumulation, decreased cellular metabolism and increased apoptosis. these changes to cellular function were mirrored using nefa in culture medium at the concentrations and molar ratios to albumin previously reported in pe. these findings provide evidence that the changes in endothelial cell function induced by plasma from women with pe may be due to the increased nefa circulating levels and that increased palmitic, oleic and linoleic acid, in combination, could play a role in pathogenesis of pe. lorentzen ( ) bjog; endresen ( ) ajog. background: skewing of the maternal endothelial phenotype in pre-eclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. we hypothesise that factors secreted from pe placental tissue will impair endothelial cell function. we have tested the effect of soluble factors by menopausal status, there was a significant increase in bmi in pre-but not in postmenopausal women. in postmenopausal women there was insufficient power to note a statistically significant change in bmi. results are summarized with the follow-up for each group represented by "n" in the graph below. premenopausal patients were divided into hysterectomy with oophorectomy (pbso) versus hysterectomy alone (ph). the ph group showed an increase in bmi that plateaus at time . in the pbso group the bmi continued to increase over time. subgroup analysis comparing ph to pbso demonstrates initial weight loss in pbso but a significant increase in bmi from baseline at time compared to ph. conclusions: hysterectomy appears to be associated with an increase in bmi over time. subgroup analysis suggests that, in premenopausal women, oophorectomy is more strongly associated with continuing weight gain than hysterectomy alone. purpose: obesity is implicated as a key risk factor in chronic disease, but no studies have associated central obesity to the presence of chronic abdominal and/or pelvic pain. we set out to identify the prevalence of chronic abdominal/ pelvic pain in an underserved, primarily latina population by a cross-sectional study in the olive view-ucla outpatient gynecology clinic. we sought to identify an association between the presence and severity of abdominal/pelvic pain and central obesity. methods: nonpregnant women presenting to the gynecology clinic were prospectively evaluated and grouped according to the presence of abdominal/ pelvic pain ('none-mild' or 'moderate-severe' pain). body mass index (bmi) and abdominal circumference (ac) were measured. patients with 'moderate-severe' pain completed standardized questionnaires for pelvic pain and global health scores. results: / ( %) of patients has 'none-mild' pain, and ( %) had 'moderate-severe' pain. pain prevalence was not significantly associated with bmi (mean: 'none-mild' . + . kg/m ; 'moderate-severe' . + . kg/m , p= . ), nor was pain severity (p= . ). pain prevalence was significantly associated with ac (mean: 'none-mild' . + . inches; 'moderate-severe' . + . , p= . ). a borderline positive association exists between ac and pain severity (p= . ). conclusions: we demonstrate an association between both the presence and severity of chronic abdominal/pelvic pain and central obesity, independent of bmi. ac appears to be a more relevant factor than other traditional measures of habitus in patients with this chronic malady. to improve preventative care in women's health management, further evaluation of the role of central obesity in the pathogenesis of chronic pain is necessary. aims: vulvitis is one of the most frequently diagnosed gynaecological infections. we aim to assess the efficacy against infective vulvitis of a new topical medical device containing low molecular weight hyaluronic acid (lmw-ha). the ability of this molecule to stimulate -defensin release in keratinocytes has been recently shown. methods: we report preliminary data regarding women suffering from infective vulvitis, as assessed by a gyneacologist: patients were randomly selected to receive sphg ( patients), a cream containing low-molecular weigh hyaluronic acid, or vehicle ( patients). patients were asked to apply the cream to the vulva twice-daily for days. at the end of treatment, evaluation of efficacy, tolerability and acceptability of the cream was assessed by a specific questionnaire. results: preliminary results show that patients receiving sphg report a significative improvement of vulvitis symptoms, in terms of itch, redness of the skin and burning, in comparison to vehicle. sphg also showed a good tolerability, cosmetic acceptability and symptomatic relief perceived by patients. conclusion: sphg seems to be efficacious in ameliorating the symptoms of infective vulvitis. this activity may be probably related to the lmw-ha presents in this formulation: in fact, low molecular weight hyaluronic acid has been recently shown to induce -defensin production by human keratinocytes. since this peptide exerts antimicrobial and antimicotic activity, the improvement of symptoms assessed in patients receiving sphg might be linked to a reduced infective charge, attributable to the activity of -defensin . background: allogeneic hematopoietic stem cell transplant (hsct) is a treatment used for many malignant and nonmalignant diseases of the bone marrow and immune system. hsct may be complicated by chronic graft-versus-hostdisease (cgvhd) in to % from matched unrelated donors. genital cgvhd complicates about % of hsct and may uncommonly result in labial fusion. case: year old woman with a history of ewing's sarcoma and acute myelogenous leukemia, had received chemotherapy and total body irradiation (tbi) followed by a matched unrelated donor hsct. menarche occurred at years of age after normal pubertal development. she menstruated regularly until cancer diagnosis. premature ovarian failure resulted after chemotherapy and tbi and oral contraceptive pills were used for hormone replacement. after transplant, she developed chronic gvhd involving the skin, eyes, mouth and joints, and concomitantly complained of vulvar pruritus. she was presumed to have a yeast infection which was treated with fluconazole without a pelvic exam. she was evaluated by a gynecologist when she was unable to insert a tampon. pelvic exam revealed dense labia minora adhesions from the clitoris to urethral meatus and posteriorly leaving a cm opening at the urethra. pelvic mri revealed a normal uterus and ovaries. after weeks of topical estrogen cream, the adhesions remained dense and were lysed under general anesthesia. vaginal examination revealed pale, minimally rugated, normal mucosa. cervical cytology was normal. post-operatively she used daily topical estrogen and hydrocortisone creams. at months after surgery, her urinary stream was stronger. on pelvic exam, the labial opening was cm, but a small posterior forchette adhesion elicited severe pain. after using dilators coated with topical steroids and estrogen, she was able to insert a tampon. conclusion: genital gvhd should be considered in women with genital tract complaints after hsct. labial fusion secondary to chronic gvhd may be treated successfully with surgery and medical therapy. support: rbmb/nichd/nih. dana r ambler, mazen e abdallah, rahi victory, michael p diamond, elizabeth e puscheck, jay m berman. obstetrics and gynecology, wayne state university/detroit medical center, detroit, mi, usa. objective: to determine which factors are predictive of a ruptured ectopic pregnancy, and whether endometrial stripe thickness can be used as an alternative to such criteria in the diagnosis of an ectopic pregnancy. design: retrospectively collected ectopic pregnancy database. setting: detroit medical center, detroit, michigan. patients or participants: women with a diagnosis of an ectopic pregnancy were studied, with surgically confirmed tubal rupture cases. interventions: abstracted data included hcg(iu), gestational age (days), presenting symptoms of pain and/or bleeding, hemoglobin (hgb), hematocrit (hct), historical risk factors, ultrasound-determined ectopic size, endometrial stripe thickness (mm), amount of cul de sac fluid (cds), tubal rupture at time of surgery, and estimated blood loss (ebl)(ml). covariates included demographics. results were significant when p< . . results: chi square analysis revealed that there is a relationship between endometrial stripe thickness and hcg levels. logistic regression models demonstrated that endometrial stripe thickness was not predictive of ectopic rupture, (or . , p= . ) . however, logistic regression, both forced and forward stepwise analysis, demonstrated that hcg (or= . , p< . ), a large volume of cul-de-sac fluid (or= . , p< . ), and increasing pain (or= . , p= . ) were associated with increased risks of rupture. gestational age, and ectopic related risk factors were also not predictive of rupture. conclusions: endometrial stripe thickness is not a useful predictor for the diagnosis of a ruptured ectopic pregnancy. serum hcg measurement, cds fluid volume and the presence of pain are much stronger diagnostic indicators of ectopic pregnancies. clinical profile of migraineurs in a university hospital gynecology department in japan. [objectives] the changes in hormonal milieu associated with menarche, pregnancy, menopause, and post-menopause are frequently accompanied by changes in the patterns and frequency of migraine. little is known on the relationship of women's issues of migraine though the balance between estrogen and progesterone is critical in the elimination of migraine. our aim was to investigate the relations among the prevalence of migraine, the reproductive stage, and gynecologic diseases. [materials and methods] female patients (average age: . years old) who consulted a physician and agreed to answer about the questionnaires during september, -june, . migraineurs were diagnosed with the migraine screener by the japanese headache medical treatment promotion committee in . and the patients answered questionnaires that screened about menopausal disorder and abnormal menstruation at once. they were conducted a survey in the form of a questionnaire and sometimes were taken blood samples. [results] in patients had migraine ( . % average age: . years old). prevalence were . % in one's twenties, higher than another ages. most patients with migraine was complicated by menstruation disorders (premenstrual syndrome %, dysmenorrhea . %), sterility ( . %), and severe menopausal disorder ( %). when trh and the lh-rh test were examined for the sterility patient, migraineurs had higher prolactin basal level and lh level after lord but lower fsh level before and after lord than nonmigraineurs. on the other hand, the prevalence of migraine for postmenopausal women and women who had gynecology cancer treatment was low, and there was no relation between migraine and pregnancy history. [conclusions] this study provides migraine headache is influenced by reproductive stage and that women with migraine are frequently complicated by menstruation disorder. it is thought that migraine account for abnormality of hormone milieu including abnormality of the hypothalamus-pituitary system. objective: this study evaluated the efficacy of doses of estradiol (e ) gel . % (divigel ® ), a novel formulation of e consisting of mg e per g transdermal gel, to reduce frequency and severity of vasomotor symptoms and signs of vulvar and vaginal atrophy (vva) associated with menopause. design: postmenopausal women were evaluated in a -week study comparing placebo to e gel . % at doses of . g/day, . g/day, and . g/day with estimated nominal daily deliveries of . mg, . mg, and . mg of e respectively. endpoints included mean change from baseline in daily frequency and severity of moderate to severe hot flashes (msvs). vaginal ph and % superficial cells were collected at baseline and end of study. results: e gel . % showed statistically significant improvements from placebo gel as early as week (table) that were maintained throughout treatment. signs of vva (vaginal ph and % of superficial cells) showed statistically significant improvements from baseline with all doses of e gel . % compared to placebo. conclusion: e gel . % significantly decreased the frequency and severity of msvs at all doses evaluated in this trial. e gel . % offers multiple dosing options to individualize patient therapy, including the lowest effective dose that was studied ( . mg e , delivering . mg e /day), to treat vasomotor symptoms associated with menopause. estradiol (e ) gel . % (divigel ® ) is a novel formulation of e consisting of mg e per g transdermal gel for the treatment of vasomotor symptoms associated with menopause. safety and tolerability of e gel . % were evaluated in a large placebo-controlled trial. design: postmenopausal women participated in a -week study comparing placebo to . g/day, . g/day, and . g/day of e gel . %. circulating e and estrone (e ) concentrations were measured. safety analyses included the incidence of adverse events (aes) and clinical laboratory evaluations, including plasma levels of sex hormone binding globulin (shbg). application site tolerability was assessed using the draize scale. results: all doses of e gel . % produced physiologic e :e ratios similar to those seen in premenopausal women. e :e increased from a baseline mean of . to . , . , and . with, respectively, the . , . , and . g/day doses of e gel . %. the most frequently reported aes were breast tenderness and postmenopausal bleeding that appeared to be dose-related and would be expected with increased circulating estrogen concentrations. there were no remarkable changes in hematology, blood chemistry, urinalysis, lipid, coagulation, and carbohydrate values following treatment with e gel . %. shbg levels remained unchanged after weeks of treatment at all doses. the vast majority of patients had no evidence of skin irritation throughout the treatment period. mean draise scale scores after , , and weeks of treatment were . for all treatment groups except for a mean value of . ± . for the . g dose group after weeks of treatment. conclusion: e gel . % is a safe and well-tolerated therapy for the treatment of menopausal symptoms. design: pharmacokinetic parameters, dosing, efficacy, and safety information for divigel ® , elestrin ™ , evamist ™ , estrogel ® , and estrasorb ® were obtained from current prescribing information (obtained from manufacturer websites) and the data were compared. results: together, these new transdermal therapies offer multiple dosing/ delivery options and contain a wide range of e ( . mg to . mg), with the lowest systemic daily delivery of e attained by the divigel ® . g dose. following weeks of treatment, across the dosing options, each treatment significantly reduced both the frequency and severity of moderate to severe vasomotor symptoms (msvms) compared to placebo. change in frequency of msvms ranged from - . (divigel ® . g) to - . (estrasorb ® ); change in severity scores for msvms ranged from - . (divigel ® . g) to - . (divigel ® . g). all treatments are safe and well tolerated. breast tenderness was the only adverse event reported in % of subjects, occurring with all therapies. conclusions: current treatment guidelines recommend using the lowest effective dose of estrogen for the treatment of menopausal symptoms. this side-by-side comparison of the recently available e non-patch transdermal options to the standard transdermal therapies is meant to assist physicians in individualizing treatment for their patients. introduction: cdb- (cdb) is a relatively new progesterone receptor modulator being clinically evaluated for contraception and treatment of fibromas. its use in an intrauterine device/system (ius) has not been reported. in this study we prepared cdb-filled intrauterine devices (cdb-ius) and evaluated their effects on endometrial growth and bleeding patterns in rhesus macaques. methods: short ( . - . cm) lengths of silastic tubing (od . mm), either empty (n= ),or filled with silicone rubber matrix containing % of micronized cdb (n= ), were inserted into the uterine lumens of ovariectomized rhesus macaques. animals were induced to cycle by sequential treatment with systemic estradiol and progesterone (p) as reported (brenner et al, ann ny acad, ) . after . cycles, at the end of the follicular phase, the uterus was removed and processed. results: when systemic p treatment was withdrawn at the end of each cycle, animals with empty ius menstruated normally, while animals with cdb-ius bled little or not at all. over the whole . cycles, animals bearing a blank ius bled for an average of . ± . days while cdb treated animals bled for an average of only ± . days. at the end of treatment, animals exposed to blank ius had mean endometrial wet weights of ± mg while the cdb-treated endometria weighed only ± mg. the proliferation markers ki- and phospho-h were substantially lower in the cdb-ius treated than in the blank treated animals. histologically, the cdb exposed endometria were atrophied with evidence of glandular degeneration while the blank controls were proliferative and normal. summary: in cycling rhesus macaques, a cdb-ius prevented progestational development, blocked menstruation after p withdrawal, and suppressed endometrial proliferation. if these effects are confirmed in women, the cdb-ius could provide estrogen-free and bleed-free contraception, and could help control heavy menstrual bleeding. supported by rr , hd and the population council. introduction: irregular uterine bleeding is a major side effect and cause for discontinuation of ltpoc use. while endometria of ltpoc-exposed women display abnormally enlarged, fragile blood vessels (bv), decreased blood flow and evidence of oxidative stress, the mechanisms by which structural and vasomotor endometrial dysfunction occurs remains unknown, in part by the difficulty of manipulating hormone levels in women. the aims of this study were ) to validate the guinea pig (gp) as a model to study uterine effects of ltpoc and ) to investigate ltpoc-effects on endometrial histology and oxidative stress markers. methods: oophorectomized gps were implanted s.c. with time release pellets containing either placebo (crl,n= ); estradiol (e ,n= ); medroxyprogesterone acetate (mpa,n= ) or e +mpa (n= ). after days, uterine horns were weighed and frozen or paraffin embedded. angiogenesis was assessed by quantitative image analysis of vonwillebrand factor staining and included bv density and size. oxidative stress was detected by isoprostane and -oh-deoxyguanosine ( oxog). apoptosis was investigated by the tunel method. statistical analysis was by -way and -way anova. results: gp uteri were enlarged by both e (p< . ) and mpa (p= . ). effects of mpa on uterine weight differed significantly depending on e levels (p< . ), where mpa opposed the e effect in combined treatments. angiogenesis parameters were similarly impacted upon. thus, mpa alone increased bv density (p= . ) and bv average area (p= . ). the presence of e significantly decreased these parameters (bv density mean± sem: crl: . ± . %, e : . ± . %, mpa: . ± . %, e +mpa: . ± . %, p= . ). these changes were associated with highly elevated -isoprostane content in e +mpa-treated uteri compared to all other groups (p< . ). abnormalities in the e +mpa group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased nuclear oxog staining and a marked increase in tunel labeling. conclusions: ltpoc exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. the gp is an excellent model for the study of ltpoc effects on the uterus. physiologic and psychological symptoms associated with injectable and oral contraceptive use. abbey b berenson, susan odom, carmen r breitkopf, mahbubur rahman. ob/gyn, utmb, galveston, tx, usa. objective: to compare physiologic and psychological symptoms over mo among users of depomedroxyprogesterone acetate (dmpa), an oral contraceptive with micrograms ethinyl estradiol (oc), and non-hormonal (nh) contraception. methods: a total of women reported the presence of symptoms prior to initiating contraception ( dmpa, oc, nh) and every mo thereafter for mo. longitudinal relationships between symptoms and contraceptives (reference: nh), as well as race/ethnicity (non-hispanic black, non-hispanic white, and hispanic) were assessed by gee-analysis after adjusting for age, visits and baseline status of symptoms. persistence, resolution, and new development of symptoms were noted by method in mo increments for mo and compared with that of nh controls. the gee analyses showed that oc was protective against mastalgia (or= . ), cramping (or= . ), hair loss (or= . ), acne (or= . ), nervousness (or= . ) and mood swings (or= . ). when race was considered, oc was protective for all women against acne, for whites against mastalgia and cramping, and for non-whites against nervousness. for whites, it was a risk factor for bleeding between menses. oc use resulted in resolution of acne within mo in nearly % of those with it at baseline, but no significant resolution after mo. also mastalgia ( % at baseline) was less likely to persist at and mo. bleeding between menses was reported for the first time at mo by % of oc users. dmpa users of all races had an increased risk of missed periods (or= . ), bleeding between menses (or= . ), bleeding > d (or= . ) and loss of libido (or= . ) relative to nh users. it reduced cramping (after mo) and bloating (all mo). racial differences were observed with dmpa protective against mastalgia and mood swings in whites, cramping in whites and hispanics, and bloating in non-whites. it was a risk factor for loss of energy in whites only. neither method affected depressive symptoms. conclusion: side effects of these two methods are mostly related to abnormal bleeding. very low dose pills can be protective against symptoms (mastalgia, cramping, hair loss, acne, nervousness and mood swings) commonly associated with pills while dmpa protects against cramping and bloating. knowledge about racial differences will allow physicians to individualize therapy. counseling should include that some symptoms can develop after mo while resolution often occurs within mo. methods: twenty-four women were enrolled in irb approved prospective, randomized, cross-over trial comparing months of oc (ortho cyclen) vs. tc ortho evra). the daily oc administers micrograms of ee; the weekly tc contains . milligrams of ee. each treatment was followed by months of washout and months of the alternative contraceptive. blood was drawn at baseline and final week of treatment for each arm of the study. ee was quantified by ria, with preceding organic solvent extraction and celite column partition chromatography. data were analyzed by t test with boferroni's correction, p < . . results: after two months of treatment the mean (+/-sem) ee levels for tc = . pg/ml (+/- . ) and oc = . pg/ml (+/- . ). the ee level is not significant different for the two medication (p = . ). conclusions: there is no difference in ee levels with the use of these oral and transdermal contraceptives. this suggests the transdermal contraceptive, despite the lack of the hepatic first pass effect, has similar levels of ethinyl estradiol compared to oc. the continuous elevation in ee seen with the tc route of administration, versus the episodic increases seen with the oc route, raises concerns of constant exposure to ee. this may explain the increased risk of estrogen-induced thrombotic events with this tc route of administration. impact of paracervical block, in combination with general anesthesia, on post-abortion pain. gweneth b lazenby, tod aeby. obstetrics and gynecology, university of hawaii, honolulu, hi, usa. objective to evaluate the impact of paracervical block with a long-acting local anesthetic, in conjunction with general anesthesia, on post-operative pain. methods a power analysis determined patients per arm were needed to demonstrate a significant difference of in mean pain scores. seventy-two patients were allocated to one of two arms using urn randomization. all patients received standardized anesthesia; intravenous sedation for gestational age under weeks and general anesthesia for over weeks. thirty-nine patients were randomized to receive a paracervical block with . % bupivacaine and thirtythree were randomized to no local anesthesia prior to surgical abortion. patients completed visual analog scales for pain and anxiety prior to the procedure, upon awaking, and minutes post-operatively, and prior to discharge. data were analyzed using an anova and students t-test the experimental and control groups were equivelent in age, ethnicity, gravidity, parity, prior abortions, prior vaginal deliveries, prior c-sections, gestational age, number of laminaria, pre-operative and intra-operative dilation, operative time, estimated blood loss, and reported complications. pain and anxiety were not significantly affected by placement of a paracervical block. these data do not support the hypothesized benefit of local anesthesia, prior to surgical abortion under general anesthesia, to reduce post-operative pain. we do not recommend the routine use of a paracervical block to decrease post-operative pain. age, parity, history of abortion and contraceptive choices affect the risk of repeated abortion. oskari heikinheimo, mika gissler, satu suhonen. ob gyn, university of helsinki, helsinki, finland; national research and development centre for welfare and health, helsinki, finland. objective the rate of repeat abortion varies from to % in northern europe. however, risk factors for repeat abortion are poorly understood. we characterized risk factors (demographic, as well as those related to abortion and postabortal contraception) of repeat abortion. design a prospective cohort study of women undergoing medical abortion between august and december . the subjects were followed by means of finnish registry on induced abortions until december ; the follow-up time (mean ± sd) was . ± . months. results altogether ( . %) of the subjects requested repeat abortion within the follow-up time. in univariate analysis previous abortion, parity, young age, smoking and failure to attend the follow-up visit were associated with increased risk of repeat abortion. immediate -in contrast to postponed -initiation of any contraceptive method was linked to lower risk of repeat abortion. in comparison to combined oral contraceptives, use of intrauterine contraception was most efficacious in reducing the risk of another pregnancy termination. in multivariate analysis the effects of young age, parity, previous abortion and type of contraception on the risk of another abortion persisted. conclusions increased focus on young, parous and those with the history of an abortion may be efficacious in decreasing repeat abortion. contraceptive choices made at the time of abortion have an important effect on the rate of reabortion. postabortal use of intrauterine contraception, specifically that of lng-ius, might decrease the rate repeat abortion. objective. to determine the rate of failure and to analyze factors associated with failure for the essure permanent birth control device at the detroit medical center (dmc). methods. a chart review was conducted on patients who underwent essure placement at the dmc from january through june . patient demographics, past medical and surgical history, anesthesia type, procedure time, intraoperative complications, and procedure failures were noted. data were analyzed for statistical significance using spss. results. there were essure procedures attempted at the dmc from january through june . of the attempted procedures, there were failures ( . %). of the failures were attributed to difficulty visualizing the ostia ( %). other causes of failure included expulsion of the device ( ), tubal spasm ( ), uterine perforation ( ), and tubal ostia too large for the device ( ). there were cases of failed placement for undocumented reasons, one case requiring a laparoscopic tubal ligation secondary to postprocedure tubal patency, and post-procedure pregnancies. age, race, body mass index, gavidity, parity, history of sexually transmitted infections, medical history, history of cesarean section, tobacco or illicit drug use, anesthesia type, and physician experience with the procedure were not significantly associated with placement failure or difficulty visualizing the ostia. a longer procedure time was significantly associated with failure ( . vs . min, p = . ), and history of ectopic pregnancy was significantly associated with difficulty visualizing the ostia ( . % vs . %, p = . ). conclusion. the failure rate for placement of the essure permanent birth control device at the dmc is . % with a pregnancy rate of . %. the majority of failures may be attributed to difficulty visualizing the ostia. a history of ectopic gestation was significantly associated with difficulty visualizing the ostia; thus, it may be reasonable to advise these women that success in essure placement may be reduced. introduction. the essure permanent birth control device is a relatively new form of minimally invasive sterilization for women. under hysteroscopic guidance, a dynamically expanding micro-insert is introduced into the proximal portion of the fallopian tube. the micro-insert induces local fibrosis and ultimately occlusion of the tubal lumen. a hysterosalpingogram (hsg) is performed three months after the procedure to confirm bilateral tubal occlusion. objective. to determine the follow-up rate for the post-essure hsg for a clinic population. methods. a retrospective chart review was conducted on university health center (uhc) patients who underwent placement of the essure permanent birth control device at the detroit medical center from january through june . follow-up for the post-essure hsg as well as the result of the hsg were noted for each patient. results. placement of the essure permanent birth control device was attempted in uhc patients of which were successfully completed. of the patients, ten underwent a post-essure hsg ( . %). the hsg was performed three to six months after placement of the essure permanent birth control device. bilateral tubal occlusion was documented in all ten patients. conclusion. despite counseling patients prior to their procedure that a hsg is needed and providing an information sheet, the follow-up rate for the post-essure hsg for this clinic population is only . %. for those in whom a hsg was performed three to six months after essure placement, bilateral tubal occlusion was confirmed in all. steps and or approaches to improve compliance with post-procedure confirmation of tubal occlusion should be employed to increase follow-up in the future. towards fibroids gene therapy: adenovirus mediated delivery of herpes simplex virus thymidine kinase gene/ganciclovir shrinks uterine leiomyoma in the eker rat model. memy hassan, dong zhang, salama salama, cheryl walker, hala el-mazar, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. aim: assessment of the efficacy of gene therapy of uterine leiomyoma in the immune-competent eker rat model using adenovirus mediated delivery of herpes simplex- -thymidine kinase gene followed by ganciclivir treatment (ad-tk/gcv). method: female eker rats with mri-confirmed uterine fibroid lesions were randomized to a single treatment with direct intratumor injection of ad-tk/gcv, ad-lacz/gcv, or medium.the tumor volume was evaluated by serial mri scanning and confirmed with caliper measurement at time of euthanasia. sample rats were selected randomly and killed at the following time points; , and days post treatment. samples were collected from tumors, other body organs and blood to assess the safety and efficacy of the treatment. results: ad-tk/gcv treatment produced dramatic shrinkage of the total uterine fibroid volume by % ± , % ± and %± of pretreatment volume at days , and respectively. the tumor size in negative control animals receiving ad-lacz/gcv continued to grow by + %± , + %± , + % ± while receiving media continue to grow by + % ± , + % ± and + % ± at same time points. ad-tk/gcv induced significant increase in caspase activity, bax expression, decrease in bcl and parp proteins expression and increased tunnel apoptosis index. additionally ad-tk/gcv treatment decreased cyclin d and pcna expressions. ad-tk/gcv did not produce any significant change in liver function tests or relative uterine horns weight to total body weight. the adenovirus transfection did not disseminated significantly to other distal organs except to liver and myometrium in limited number of animals. h e staining of non targeted organs did not revealed any sign of tissue damage. ad-transfection increased local cd and cd expressions as well as serum anti-ad antibodies. conclusion: ad-mediated delivery of hsv tk gene by direct intra-tumor inoculation followed by sc treatment of gcv for ten days effectively shrinks uterine leiomyoma lesions in eker rats. this effect is mediated via induction of apoptosis and decreasing the proliferation. the treatment regimen is well tolerated. these studies provide essential preclinical data for the development of gene therapy as an alternative non-surgical treatment option for women with symptomatic uterine fibroids. inhibitors of catechol o-methyl transferase shrinks uterine fibroids in the eker rat model. memy hassan, dong zhang, hala el-mazar, cheryl walker, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. background :the sex hormone dependent pattern of uterine leiomyomas and their high content of catechole -o-methy transferase (comt) raise the possibility for the development of novel treatment option using comt inhibitors.aim : to assess the potential therapeutic utility of a synthetic comt inhibitor (ro - ) in the eker rat model of uterine leiomyoma. methods female eker rat were evaluated by mri to confirm the prescence uterine fibroid lesion, then randomized for sc treatment with ro - mg /kg ,twice/ day for days versus vehicle injection.fibroid tumor burden was evaluated by serial mri measurement and confirmed by direct caliper measurement at time of euthanasia. sample animals were euthanized at and weeks. at that time tumor tissue, blood and most of animal organs including long bones were collected and subjected for further evaluations. hours urine samples were collected for evaluation of estrogen (e ) metabolites and bone resorption marker. results:animals treated with ro - exhibited significantly lower uterine fibroid tumor burden ( % and %) of pretreatment volume at and weeks post treatment respectively. conversely, the tumor size in control animals continued to grow and reached %, and % of pretreatment size at the same time points. ro - treatment resulted in an increase in urinary / hydroxy e metabolite ratio. in addition ro - increased bax expression and decreased parp , pcna and cyclind experssions . all ro - treated animals tolerated the treatment protocol with no signs of toxicity. h e staining of different body organs did not reveal any signs of tissue damage. furthermore, there was no significant change in both liver function tests (alt, ast, billirubin) and bone resorption marker , deoxypyridinoline croslinks, between treated and control rats. conclusion ro - , a synthetic selective comt inhibitor, caused immediate arrest of the growth of eker rat uterine leiomyoma. this effect might be in part due to modulation of various estrogen dependent genes regulating leiomyoma apoptosis (parp, bax) and proliferation (pcna, cyclin d ). this anti-estrogenic effect is due to the accumulation of the the antiestrogenic metabolite hydroxy estrogen secondary to comt inhibition.comt inhibitors might present an alternative non-surgical option for the treatment of women with symptomatic uterine fibroids. background development of uterine leiomyomas (fibroids) is the most common pathological feature in the female reproductive tract. they negatively impact patients of virtually every gynecologist. despite such morbidity, leiomyoma development is poorly understood. we have recently demonstrated that leiomyomas have a genomic expression pattern that limits retinoic acid (ra) exposure. our group and others have demonstrated that tgf-beta regulation is altered as well. these two pathways likely play central roles in leiomyoma development. the central feature of uterine leiomyomas, the extracellular matrix (ecm), is regulated by both all-trans retinoic acid and tgf- , focusing on versican as a critical ecm component. human uterine leiomyoma and patient-matched myometrium were obtained from surgical specimens under an irb-approved protocol. these tissues were immortalized and treated with either all-trans retinoic acid, tgf- , or anti-tgf- antibody. rna was isolated for qrt-pcr. human immortalized leiomyoma cells demonstrated the same increased template expression of tgf- ( . ± . fold), retinoic acid metabolizing protein (cyp a ; . ± . ), and versican variant v ( . ± . fold) as was found in the progenitor tissue. when treated with all-trans ra, expression of versican variant v decreased to levels found in myometrial cells ( . ± . fold). conversely, when treated with tgf- , expression of versican variant v increased . ± . fold. to confirm that tgf- was central to the overexpression of v , we treated leiomyoma cells with anti-tgf- antibody, and found that baseline over-expression of v template was decreased to expression levels similar to untreated myometrial cells. finally, we elucidated a link between the ra and tgf-pathways by assessing the impact of ra treatment of tgf- expression, demonstrating that tgf- template decreased to levels comparable to myometrial cell expression ( . ± . fold). the disrupted leiomyoma ecm, of which versican is a central component, defines the leiomyoma phenotype. in this study, the leiomyoma fibrotic phenotype regressed when treated with ra and increased when treated with tgf- , providing the basis for novel therapeutic interventions directed at cell differentiation and ecm formation. objectives: uterine fibroids are the leading cause for hysterectomies in the us. the lack of an appropriate in vitro cell model for the initiation of fibroid growth has hindered advancement in understanding the cellular and molecular basis for the development and progression of uterine fibroids. fibrosis is the underlying mechanism of uterine fibroid formation and myofibroblasts cells are the principal fibrogenic cell type in the uterus. we sought to develop a myofibroblast in vitro cell model for analyzing the initiating molecular events of uterine fibrosis. methods: smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and cultured in the presence of % serum until % confluent. for the next h cells were cultured in serum-free media followed by replacement with serum containing media. cells were fixed at , , , , m later. cell fine structure and cytoskeletal organization were evaluated by transmission electron microscopy. smooth muscle specific alpha-actin ( -sma) and progesterone receptors (pr) were detected by western blot. results: we observed smc differentiation into myofibroblasts, marked by the presence of notched nuclei ( figure) and the increased expression of -sma m after serum replacement. pr-a and pr-b were detectable at , and m. conclusions: the development of myofibroblasts is important in wound healing and fibrosis. we show for the first time that uterine myofibroblasts can be derived in culture from myometrial smcs. thus, these cells will be utilized as a model for developing "in vitro fibroids". this model will enable the study of myofibroblast activation, cytokine signaling, intracellular regulation of uterine fibrogenesis, production of extracellular matrix proteins and development of antifibrotic drugs. the presence of prs in our model enables us to evaluate pr mediated events in fibroid pathogenesis and treatment. this model will be more useful in determining the molecular biology of fibroid initiation than cell models derived from established fibroids that are already well past their initial stages of development. novel approach to genome-wide expression profiling analysis. liping feng, morgan walls, insuk sohn, millie behera, sin-ho jung, phyllis leppert. obgyn; biostatistics and bioinformatics, duke university medical center, durham, nc, usa. background: microarray studies have examined the differential gene expression between uterine fibroid and normal myometrium. all previous studies considered the fibroid as a whole and analyzed only fold changes. we have developed a novel statistical approach to genome-wide expression analysis comparing two fibroid tissue sites to myometrium. methods: using affymetrix tm u a genechip, we have compared the gene expression between c and e and matched adjacent m. data has been analyzed by considering the specimens per subject and subjects as individuals. we used a block one-way anova method to test if each gene was differentially expressed among the three sites. the p-value is calculated using a permutation method accounting for possible dependency among three lesions. the multiple testing issue was addressed by controlling the false discovery rate. expression values were calculated using the robust multichip average (rma) method. rma estimates are based upon a robust average of background corrected perfect/mismatch (pm) intensities. normalization was done using quantile normalization. expression values were then transformed by taking logarithm base . confirmatory rt-pcr was performed. results: we applied a hierarchical clustering analysis to all raw data sets and then displayed a dendrogram, where the height of each branch point indicates the similarity level at each generated cluster. identical gene expression among sites clusters together. due to a strong site effect, m tissues clustered separately from e and c combined. genes were differentially expressed when we used a . q-value cut off. expression data revealed concordant changes in genes regulating cholesterol biosynthesis, gene transcription, estrogen and extracellular matrix formation when both e and c were examined. cyp was detected and we report for the first time that scc- (a cell cycle-regulated molecule) folliculin and l-selectin are differentially expressed suggesting that they may be involved in the regulation of cell growth and proliferation of uterine fibroids. conclusions: our novel robust analysis of gene expression provides new clues to the relevant pathways of fibroid development. this new statistical approach that can be used in clinical and/or translational studies to identify differentially expressed genes comparing treatment regimens, cells or tissues. objectives: thrombospondin- (tsp- ) is a large matricellular glycoprotein secreted by many cell types. matricellular proteins modulate interactions between cells and their environment, regulate cell adhesion and are expressed during tissue formative processes. they are especially important in fibrosis. tsp- plays an important role in angiogenesis and is an activator of tgf - . in a previous study, we found that differential expression of tsp- in uterine fibroids may contribute to an altered healing process leading to fibrosis. this alteration in tissue response to injury initiates the development of abundant, nonaligned collagen fibrils and changes in other components of the ecm. methods: we measureed the pattern of mrna and protein expression of tsp- by rt-pcr and western blot in an in vitro serum-deprived differentiated myometrial cell (myofibroblast) model. specifically, smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and grown in primary culture to % confluence. then smc were serum deprived for h and treated back with % serum for , , , and m. cells were collected for rna and protein, and tsp- expression was evaluated. in addition, cells were stained using the combination of anti-cd and anti-smooth muscle -actin or combination of anti-cd d and anti-smooth muscle -actin as well as the appropriate single and double negative controls. stained sections were analyzed using zeiss axio imager widefield fluorescence confocal microscopy. results: tsp- mrna and protein was present in cells in this serumstarvation model after the addition of serum and the expression level remained elevated for m following the addition of serum. fluorescence staining analysis indicated that these cells were positive for human smooth muscle -actin, but negative for leukocyte antigen cd and platelet marker cd d suggesting that the myofibroblasts cells themselves were the source of the tsp- . conclusions: unlike skin wounds, where tsp- is derived from the blood macrophages, monocytes and platelets, differentiated myometrial cells appear to produce tsp- . elucidating the roles of tsp- in myometrium physiology and pathobiology will increase our understanding of the etiology of uterine fibroids and may lead to improved therapies. further studies utilizing this cell model to determine the role of tsp- in the activation of tgf - are indicated. phospholipid s p via gi, rac and rho pathways. yoel smicun, armando wu pimentel, jennifer gilman, david a fishman. obstetrics gynecology, new york university school of medicine, new york, ny, usa. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of ovarian cancer patients. we have demonstrated that low concentration s p enhances while high dose s p inhibits invasion of epithelial ovarian carcinoma (eoc) cells in a dose and attachment mode dependent manner. we sought to further dissect the pathways by which s p affects invasion, using specific inhibitors. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi, p -mapk, rac and rock, thereafter cells were detached and tested for invasion towards m lpa chemoattractant in matrigel-coated chambers. conditioned media from pretreated cells and invading cells were quantified for upa activity using colorimetric assays. the significance of results was calculated by student's t-test. results: inhibition of gi mildly increased invasion of both control ( p= . ) and m s p treated cells ( p= . ). inhibition of both p -mapk and rac did not affect m s p treated cells, in contrast invasion of control cells was mildly increased (p= . , . ). inhibition of rock, a protein effector downstream of rho, highly elevated invasion of both control and m s p treated cells ( fold, p= . , fold, p= . ). both upa and gelatinase activities were higher in conditioned media of invading cells than of attached cells. gelatinase activity was enhanced by both concentrations of s p (p= . , . ). ptx fully inhibited gelatinase activity of control and . m s p treated cells, and partially of the m s p treated cells (p< . ). . m s p significantly increased upa activity of attached (p= . ) but not of invading cells. this increase was sensitive to ptx and rac inhibitor. m s p inhibited upa in both attached and invading cells (p= . ), this inhibition was rock dependent. conclusions: these findings suggest a strong inhibition of invasion and upa by the rho pathway, of both control and m s p treated cells. this inhibition is induced partially by upstream gi protein. increased invasion by . m s p is associated with elevation of gelatinase activity through gi, rac and rock pathways. this suggests that attached cells and invading cells affect upa activity through different pathways. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of epithelial ovarian cancer (eoc) patients. we have demonstrated that invasion of attached eoc cells differentially react to s p as compared to invading cells. we examined the impact of the inhibitors for gi and rac on attached and invading eoc. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi (pertussis toxin (ptx)), and rac (nsc , (rac-i)), and cells were detached and assayed for invasion towards m lpa in matrigel-coated chambers. to distinguish the response of attached from invading cells, inhibition of cells pretreated with inhibitors was either continued or not in the invasion chambers. conditioned media (cm) of invading cells were quantified for upa and gelatinase activity by fluorometric and colorimetric assays. significance of results was calculated by student's t-test. results: the significant (p= . ) increase of invasion by . m s p was inhibited by both ptx and rac-i, either by pretreatment alone or by continuous treatment (p= . - . ). however, the invasion was higher in cells inhibited continuously than cells inhibited only in dishes. both inhibitors did not affect cells treated with m s p. control cells invasion was increased ( . fold, p= . ) by continuous rac-i treatment. . m s p increased gelatinolysis in cm of invading cells. this and control cells activity was inhibited by ptx pretreatment. continuous treatment with ptx or rac-i elevated and fold gelatinase activity of control and m s p treated cells. in contrast upa activity was inhibited by both . m and m s p. activity of control cells was inhibited by both pretreatment and continuous treatment with both ptx and raci. upa activity of cells treated with . m s p was increased only by pretreatment with ptx and raci. in contrast, in cells treated with m s p upa was inhibited only by continuous inhibition with ptx and raci. conclusions: invasion of s p induced eoc cells correlated with the gelatinase and upa activities in their microenvironment. ptx and rac-i affect attached and invading cells in different manner, inhibition of invasion and gelatinolysis of attached and increased invasion of invading cells. this suggests a dual effect of the gi-rac pathway, inhibiting attached and stimulating invasion via gelatinolysis of invading cells. lysophosphatidic acid (lpa) levels are elevated in the ascites and plasma of early-and late-stage ovarian cancer patients, underscoring the unique role this bioactive lipid plays in the pathobiology of epithelial ovarian cancer (eoc). lpa binding to its cognate g-protein-coupled receptor can transactivate the receptor tyrosine kinase, egfr, which is often overexpressed in ovarian tumors. in the current study, we investigated the role that lpa activation of egfr plays in the processing of the metalloproteinase, mmp- , and eoc dissemination. lpa stimulates tyrosine phosphorylation of egfr in ovarian cancer cells, and egfr kinase activity is required for optimal lpa induction of pro-mmp- activation. lpa-dependent pro-mmp- activation does not require the egfr ligands, amphiregulin, b-cellulin, egf, hb-egf, and tgf-a. we previously reported that when cells are cultured at high density, lpa represses rhoa activity to induce loss of stress fibers, and these changes in actin microfilament organization contribute to pro-mmp- activation. in the current study, inhibition of rho-kinase/rock with y- reversed the repression of lpa-stimulated pro-mmp- processing observed with treatment of the egfr kinase inhibitor, ag . correspondingly, lpa induced the loss of stress fibers, while inhibition of egfr kinase restored stress fiber formation in lpa-treated cells. this suggests that lpa acts through egfr to modulate microfilament organization and pro-mmp- processing. finally, lpa-induced cellular haptotactic migration and invasion are abrogated when egfr kinase activity is blocked. taken together, these results suggest that egfr signaling plays a critical role in lpa regulation of metastatic pathways by mediating changes in the cytoskeleton which impact protease activity. objectives: membrane-derived vesicles are active modulators of tumor dissemination; they promote and contribute to extracellular matrix degradation and tumor cell invasion. we have isolated vesicles from ascites of ovarian cancer patients and from ovarian cancer cells in vitro. here we analyzed the functional consequences of exposure of cancer cells to vesicles derived from a different type of malignancy in order to evaluate their proinvasive properties. methods: ovarian cancer (dov ), breast cancer (mda mb ) and mesothelioma (hp- ) cells were grown in media supplemented with % fbs. after serum deprivation, % vesicle-free fbs was added for hr to induce vesicle release. vesicles were isolated from media by differential centrifugation and quantified with a bradford assay. fusion to cells was followed by fluorescence of the lipophilic tracer dii. each cell line was stimulated with and g/ml of each type of vesicles and tested in a matrigel invasion assay. changes in proliferation were evaluated in an mts assay. gelatin zymography was used to assess matrix metalloproteinase activity of vesicles. results: zymographic analysis showed that vesicles contained mmp- and . fusion experiments showed that all vesicles fused to all cell types. all vesicles induced the invasion of their respective cell types in a dose-dependent manner. when vesicles were used at g/ml they induced significantly high levels of invasion in all cell types tested. at g/ml they significantly inhibited invasion in different cell types. both concentrations of vesicles stimulated cell proliferation in all cell types tested. conclusions: membrane derived vesicles are potent mediators of invasion for mesothelioma, breast and ovarian cancer cells in vitro. this effect occurs in a dose-dependent manner when vesicles induce invasion of the same cell type from which they were isolated. when vesicles are used to induce a different cell type, low concentrations induce invasion while high concentrations inhibit invasion, this effect was independent of cellular proliferation. these results suggest that a novel mechanism may be at play where activation of recognition of self and non-self specific pathways may determine the invasive potential of the tumor cell upon fusion to microvesicles. the identification of signaling pathways responsible for this heterotypic signaling is a current effort. vegfr- as a potential target to inhibit lpa-induced epithelial ovarian cancer (eoc) invasion. sonia dutta, fengqiang wang, elaine barfield, david a fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objective: vegf and vegf receptors (vegfrs) play important roles in ovarian cancer metastasis. in this study, we examined the expression profiles of vegf and vegfrs (vegfr , vegfr , co-receptor nrp and nrp ) in established eoc cells lines (dov , r , ovca , skov ) and an immortalized normal ovarian epithelium (iose- ). the effect of lysophosphatidic acid (lpa) on vegf and vegfrs expression and the effect of vegfr- silencing by rnai on lpa-induced invasion were also evaluated. methods: vegf and vegfrs expression in ovarian cancer cell lines and normal ovarian epithelium was quantified by real time pcr. cell invasion differentiate into either a pro-tumor or anti-tumor phenotype depending on the specific tumor microenvironment. previously, we described a subgroup of epithelial ovarian cancer (eoc) cells with a functional tlr- -myd -nf-b pathway (type i eoc cells). these cells have constitutive nf-b activity and constitutively secrete il- , il- , mcp- , and gro . we hypothesize that type i eoc cells, but not type ii, can promote macrophage differentiation into a tumor-supporting immune cell. methods: eoc cell conditioned media (cm) was prepared by incubating eoc cells in log-phase growth in optimem for h. migration assay was done using an in vitro cell culture insert with m-size pore and pkh red fluorescentlabeled thp- . cytokine profile of thp- cells co-cultured with eoc cell cm was determined using xmap technology. modulation of response to apoptotic bodies was determined by "pre-educating" thp- cells with eoc cell cm for h prior to exposure to apoptotic bodies ( : ratio with thp- cells). results: monocytes migrate toward eoc cells. however, migration is significantly higher towards type i eoc cells. type i, but not type ii, eoc cells alter monocytes' cytokine profile by inducing the secretion of high levels of progrowth and angiogenic cytokines il- , il- , mcp- , and gro . furthermore, type i eoc cells modify monocytes response to apoptotic bodies by inducing a significant increase in the secretion of il- , il- , mip- , mip- , and gro ( -fold, . -fold, -fold, -fold, and -fold respectively). conclusion: we demonstrate for the first time a differential interaction between two subtypes of eoc cells and monocytes. we showed that the microenvironment created by type i eoc cells is able to modify the function and differentiation of immune cells towards a tumor supporting phenotype. understanding the molecular mechanisms mediating this tumor-immune interaction will help to design appropriate immune therapies that will take into consideration the tumor microenvironment. objectives: the standard of treatment for patients with ovarian cancer (oc) is intravenous combination chemotherapy (ct) after primary cytoreductive surgery. although initial response is above %, most of these patients experience recurrence. the only approach for these patients is salvage ct which may prolong their lives for months. early detection of patients who are not responding to current therapy or are at risk of experiencing an early relapse of disease might improve response rates and survival if alternative therapy is possible. no single predictive marker has yet been proven sufficiently sensitive or specific to find a place in the daily clinic. in the present study we use a newly described biomarker test for the detection of oc (visintin et al clinical cancer research) and evaluated the ability of the test to monitor ct response methods: patients with oc, figo stage i-iv, were included in this study. all patients recieved postsurgery first line combination ct (paclitaxel/carboplatin). samples were evaluated at ) baseline (mean days after surgery), prior to the first cycle of ct, and ) after cycles of ct. μl serum samples were analyzed by multiplex assay (beadlyte® cancer biomarker panel kit) for six markers. changes during ct and differences in markers between patients with short time to progression (ttp) and patients with long ttp were determined. results: positive test for oc was observed in % of the patients evaluated at baseline. all patients had residual disease after surgery. from patients with long ttp, / patients (specificity %) had a negative test after cycles. from the patients with short ttp, / had a positive test (sensitivity %) p= . ( ²). the risk of experiencing a ttp shorter than months when having a positive test after cycles of ct was %. conclusion: we describe for the first time the use of a panel of biomarkers for oc that might have an application for monitoring ct response and risk of relapse. the test detects the presence of residual disease following debulking surgery, and differentiates between long term and short term progression. a longitudinal study is performed to determine how early during ct the test can identify responder versus non responder. ksp inhibitor (arry- ) as an alternative for paclitaxel in myd -positive epithelial ovarian cancer cells. ki h kim, ayesha b alvero, yanhua xie, david trollinger, gil mor. obstetrics, gynecology, and reproductive sciences, yale university school of medicine, new haven, ct, usa; array biopharma inc., boulder, co, usa. background: we previously described that epithelial ovarian cancer (eoc) cells ubiquitously express tlr- , but not the adaptor protein myd . when treated with paclitaxel, a known tlr- ligand, myd -positive eoc cells exhibited nf-b activation, increased secretion of il- , il- , mcp- , and gro , and increased p-erk levels . since majority of eoc patients are given paclitaxel in combination with a platinum drug, it is not only important to distinguish those patients that should not be given paclitaxel; it is also important to identify alternative chemotherapy agents that would benefit this sub-group of patients. the objective of this study is to determine if the ksp inhibitor, arry- , can be an alternative for paclitaxel in myd -positive ovarian cancer patients. methods: myd -positive and myd -negative eoc cell lines isolated from either ascites or tumor tissue were treated with increasing concentrations of arry- ( to nm) or paclitaxel ( . to m) for , , and hours and cell viability was determined using the celltiter aqueous one solution cell proliferation assay. cytokine profiling was performed from supernatants using xmap technology. nf-b activity was determined using a luciferase reporter system. p-erk levels were measured by western blot analysis. results: arry- and paclitaxel exhibited the same cytotoxic effect on myd -negative and positive eoc cells. the ic at h for myd -negative eoc cells was . m and . um for arry- and paclitaxel, respectively. for myd -positive eoc cells, the ic at h was m and m for arry- and paclitaxel, respectively. however, unlike paclitaxel, arry- did not induce nf-b activation or enhance the secretion of il- , il- , mcp- , and gro , and did not induce erk phosphorylation on myd positive cells. conclusions: administration of paclitaxel to patients with a myd -positive tumor could have detrimental effects due to the paclitaxel-induced enhancement of cytokine production which promotes chemoresistance and tumor growth. arry- has similar anti-tumor activity in eoc cells as that of paclitaxel. however, unlike paclitaxel, it does not induce cytokine production in myd positive eoc cells, and therefore, the ksp inhibitor arry- may represent an alternative to paclitaxel in this subgroup of eoc patients. introduction: nf-b activation has been associated with cell proliferation, angiogenesis, metastasis and suppression of apoptosis in ovarian cancer. in addition, nf-b activity induces the production of pro-inflammatory cytokines which may contribute to chemoresistance. inhibition of nf-b may represent a new approach to prevent tumor growth and reverse chemoresistance. in the present study we described the characterization of a novel nf-b inhibitor, eriocalyxin b (erib) that is able to re-establish the apoptotic cascade in chemoresistant ovarian cancer cells by suppressing pro-inflammatory cytokines and antiapoptotic proteins. materials and methods: eoc cell lines were isolated from malignant ovarian cancer ascites. caspase activity was determined by caspase-glotm assay. cytokine production and secretion were determined using multiplex assay. erib effect on cancer cells was evaluated in a time and dose dependent manner using celltiter cell proliferation assay. protein expression was determined by western blot analysis. combination studies were done with paclitaxel and carboplatin in addition to erib treatment. nf-b activity was determined by monitoring the expression of a nf-b luciferase reporter. results: erib decreased cell viability of ovarian cancer cells with an ic of . - μm in hours and was associated to increasing levels of caspases , and activity. intracellular changes induced by erib included: ) inhibition of nf-b activity; ) decrease in cytokine production; ) down regulation of anti-apoptotic proteins xiap and flip, and reversal of resistance to tnf-and fasl-mediated cell death; and ) chemosensitization to carboplatin and paclitaxel. at present, evidence is accumulating regarding the existence of unique populations of specialized tumor-initiating, stem-like cells within various tumor types of distinct origins. these cancer stem cells (csc), with characteristics reminiscent of normal stem cells, are thought to be responsible for driving tumor growth. we propose that ovarian cancers arise from csc, and are using microarray-based technology to identify specific genes/cell surface markers associated with ovarian csc that will permit distinction of these rare cells from the remaining tumor bulk. to identify unique gene signatures associated with an ovarian tumor-initiating cell population, we have utilized an in vivo serial transplantation model. this model selects for primary human ovarian tumor cells with increased tumorigenic capacity, given that time to tumor formation decreases with successive serial transplant despite fewer cells injected. our initial studies used cells derived from a human ovarian clear cell carcinoma, serially transplanted for three passages in nod/scid mice. rna derived from these transplanted tumors was analyzed on human genome microarrays. from these analyses, several differentially expressed genes were identified. the differential expression noted for potential genes of interest is currently being validated by rt-pcr. of particular interest, expression of the transmembrane glycoprotein muc was found to increase both at the rna and protein level with successive transplant of this clear cell carcinoma. further studies are ongoing to determine the functional significance of muc and other identified differentially expressed genes in ovarian clear cell cancer. in addition, we are carrying out parallel microarray analyses in other ovarian tumor subtypes. background recent observations suggest a decreased incidence of neoplastic lesions in hiv infected individuals treated with highly active anti-retroviral therapy comprised of protease inhibitors, such as ritonavir. the polycomb group protein ezh is associated with aggressive human malignancies via transcriptional suppression of dna repair proteins. objective objective of the present study was to assess the antineoplastic impact of ritonavir on epithelial ovarian cancer (eoc) cell lines. methods eoc cell lines (mdah and skov- ) were treated with serial dilutions of ritonavir ( - mm) dissolved in dmso. normal diploid human fibroblasts served as controls. growth curves, apoptosis and cell cycle analysis were performed with cell counting kit- , annexin v and flow cytometry. signal transduction was studied with western blotting. dna double strand breaks (dsb) were induced with . mm etoposide treatment for hours. homologous recombination (hr) repair of dna damage was measured by assessment of rad foci formation in the nucleus of the cells as visualized by fluorescence microscopy according to previously published criteria. untreated eoc cells expressed higher levels of ezh and lower levels of rad and xrcc as compared to controls and in turn, had lower prevalence of rad repair foci formation in response to dsb. serial treatments of eoc cells with ritonavir resulted in a decrease in expression of ezh and an increase in expression of rad and xrcc when compared to untreated eoc cells. after induction of dsb, the rad repair foci formation was significantly more prevalent in eoc cells treated with ritonavir as compared to untreated eoc cells. in addition, ritonavir induced apoptosis in ovarian cancer cell lines by down-regulation of akt pathway and caused g cell cycle arrest mediated by down modulating levels of prb phosphorylation and depleting the cyclindependent kinase and . ritonavir effectively induces apoptosis, cell cycle arrest and improves repair of dna damage by hr in ovarian cancer cell lines. as impaired hr is a key event in causation and progression of neoplastic lesions, ritonavir may have a role in chemoprophylaxis and treatment of human malignancies. a rare case of ovarian cystic lymphangioma. tomer singer, tamer seckin, noa feldman, susan jormark, michael divon. department of obstetrics and gynecology, lenox hill hospital, n.y., ny, usa; department of pathology, lenox hill hospital, n.y., ny, usa. cystic lymphangioma (cl) is a rare, benign malformation of the lymphatic system whose exact etiology remains uncertain. cl may arise in different sites: the spleen, the mediastinum, the axillary region, the retroperitoneum, and the mesentery. retroperitoneal cl is extremely rare and its true incidence is unknown. the majority of cases are symptomatic during childhood. clinical presentation of adult cl is variable and may be misleading. typically, this is a slow-growing tumor and it remains asymptomatic for a long period of time. it is most often found incidentally during abdominal or pelvic imaging studies, surgeries or autopsies. total surgical removal of the lesions with microscopically clear margins is the best approach when it is possible. we report, for the first time, a case of cystic lymphangioma arising from the ovary in a post-menopausal woman and present the feasibility and the advantages of laparoscopic surgery, allowing accurate diagnosis, optimal treatment and minimal risk for the patient. lgsc) is a chemoresistant ovarian neoplasm that has been molecularly linked to low malignant potential tumor (lmp), which often behaves in a non-invasive fashion. micropapillary features within lmp (lmp-mp) are associated with increased invasive behavior. the aim of this study was to determine the differential gene expression of lmp, lmp-mp, and lgsc to identify genes involved in malignant transformation and carcinogenesis. methods: laser capture microdissection was used to isolate epithelial cells from snap-frozen lmp (n= ), lmp-mp (n= ) and lgsc (n= ). rna was extracted, amplified, reverse transcribed to cdna and hybridized to affymetrix u plus arrays. data was analyzed by significance analysis methods: medical records were reviewed for patients who underwent hysterectomy for all indications at wsu hospitals from / / - / / . pathology reports were reviewed to identify the incidence of adenomyosis. data obtained from medial records included: age, race, insurance, bmi, social history, medical history, and presenting symptoms. the correlation between adenomyosis and all the above factors was tested using the appropriate statistical methods. results: patients were included. adenomyosis was confirmed by pathology in patients, an incidence of . %. incidence was not significantly different among races after controlling for parity. . % in african americans, . % in caucasians, and . % in others. incidence was not statistically different between uninsured( . %), privately insured ( . %), and medicaid ( . %) patients. p=. . incidence of adenomyosis was not different between smokers ( . %) and nonsmokers ( . %). p= . . table i shows the correlation between adenomyosis and different risk factors. conclusion: adenomyosis was diagnosed in . % of hysterectomy specimens. race, socioeconomic status or social habits didn't affect its incidence. diabetes and endometrial cancer were negative risk factors for adenomyosis, whereas htn, hypothyroid, breast cancer, fibroids, polyps and endometriosis didn't affect its incidence. menorrhagia, dysmenorrhea, dyspareunia, and chronic pelvic pain but not metrorrhagia had a positive correlation with adenomyosis. there is a protective adipocytokine profile. we hypothesize that this finding may precede some ill-defined threshold of fat mass and/or insulin resistance after which adiponectin decreases. the objective: to correlate reproductive hormone production with menstrual bleeding patterns among women in the menopause transition. methods: a sub-cohort of the swan study consisting of women age - was studied. each woman collected daily first void urine samples for one complete menstrual cycle or days (whichever came first) once a year for years. urine was assayed for excreted levels of fsh, lh, estrogen metabolites and progesterone metabolites which were normalized for creatinine concentration. ovulation was detected by a validated algorithm. menstrual bleeding parameters were derived from daily calendars. correlations between bleeding characteristics, hormone concentrations, and other potential clinical predictors were analyzed using multivariate logistic regression models. results: the cohort was ethnically diverse with a median age of and with % in the early perimenopause at the start of the study. % of all cycles were anovulatory. short cycle intervals (< days) were associated with the early perimenopause (or . , ci . , . ) and with anovulation (or . , ci . , . ). long cycle intervals ( + days) were associated with late perimenopause (or . , ci . , . )and with anovulatory cycles (or . , ci . , . ). both short ( - days) and long ( + days) duration of menstrual bleeding were significantly associated with anovulation (or . and . , respectively). women with anovulatory cycles were less likely to report heavy menstrual bleeding than women with ovulatory cycles. menorrhagia was not associated with steroid hormone concentrations but was associated with obesity (or . , ci . , . ) and with the self-reported presence of fibroids (or . , ci . , . ). conclusions: among women in the menopause transition, abnormalities in timing of menstrual bleeding (cycle intervals or bleeding duration) have a hormonal basis and are frequently associated with anovulation. in contrast, abnormally heavy periods do not have a hormonal basis and are less likely following anovulatory cycles. heavy periods are associated with obesity and fibroids. to undetectable levels. the use is vaginal e is contraindicated, although these women have even higher rates of av. topical testosterone (tt) has successfully treated vulvar atrophy; testosterone receptors are also present in the vaginal epithelium. objective: assess the effect of tt on vaginal maturation index (mi) and relief of av symptoms. methods: postmenopausal women on aromatase inhibitor with symptomatic atrophic vaginitis were enrolled in prospective, irb approved study. estradiol (e) and testosterone (t) levels, av questionnaires (score - ; none to most severe symptoms of dryness, irritation, and dyspareunia), gynecologic exam, and vaginal smears (for ph and vaginal maturation index, mi, by meisels criteria) were performed at baseline and after month of daily treatment with mcg of tt. data was assessed by t test and fischer's exact test; significant p < . . results: e levels were undetectable at baseline and following treatment. t levels increased (mean +/-sem) from baseline ( +/- . ) to treatment ( +/- . ); the difference was not significant; p = . , although one patient had an appreciable rise in serum t level. two av symptoms improved significantly with tt use; comparing baseline to treatment scores: vaginal dryness ( . vs. . ; p = . ) and dyspareunia ( . vs. ; p = . saliva analysis is a convenient, non-invasive and rapid method for assessing estradiol (e ) levels. however, particularly in postmenopausal women, the low salivary e levels are often near or below the sensitivity of available assays, compromising both accuracy and precision. we present results using an extraction step prior to e assay, which concentrates the sample to increase sensitivity and removes potentially interfering substances. morning saliva samples were obtained from premenopausal (mid-luteal phase, n= , ) and postmenopausal women (n= , ) not taking hormones, and from postmenopausal women receiving oral conjugated equine estrogens (cenestin, n= ; premarin, n= ), oral micronized e (estrace, n= ; compounded e , n= ), transdermal e patches (climara, n= ; vivelle, n= ); or topical e cream (compounded e , n= ). e levels were determined by an automated enzyme immunoassay (eia) after solid phase extraction. the functional sensitivity of the assay was determined to be . pg/ml, compared with > pg/ml without extraction. . . . . . oral conjugated equine estrogens; oral e ; e patch; topical cream salivary e levels corresponded with the hormone dosage, suggesting a reliable assessment of unbound e levels with each formulation, dosage and type of estrogen therapy. extraction prior to eia in an automated assay dramatically increased precision and accuracy at low concentrations. omitting the extraction step may have contributed to poor serum versus saliva correlations in other studies. this method may therefore allow reliable monitoring of estrogen therapy without the need for expensive and inconvenient blood tests. the role of fibulin- in pelvic organ prolapse. david d rahn, jesus f acevedo, patrick w keller, lihua marmorstein, r ann word. ob-gyn, ut southwestern, dallas, tx, usa; visual sciences, univ arizona, tuscon, az, usa. objectives: fibulin- (fib- ) is crucial for normal elastic fibers in the protein showed a statistically significant reduction in its expression (p= . ). lox, loxl , and proteins were detected by immunohistochemistry in all three layers of vaginal skin biopsies: ( ) stratified squamous epithelium; ( ) the lamina propria and ( ) the muscularis layer from both patients with pop and controls. significantly, in both groups we detected a numerous macrophages scattered throughout the vaginal thickness which displayed a very strong immunostaining to loxl . patients with severe pop showed reduced expression of proteins regulating collagen and elastin biogenesis. our finding raises the possibility that failure of ecm homeostasis could underlie the etiology of pop in women. fibroblast proliferation is regulated by hoxa : molecular implications for pelvic organ prolapse. kathleen a connell, marsha k guess, richard bercik, hugh s taylor. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa. objectives: the integrity of the extracellular matrix (ecm) is maintained by a delicate balance between collagen synthesis and degradation. previously, we have demonstrated that hoxa is essential for the development of the uterosacral ligament in mice and regulates the expression of collagen type iii and mmp . we have also shown that hoxa is deficient in the uterosacral ligament of women with pelvic organ prolapse (pop) compared to women with normal support. the exact mechanisms by which hoxa regulates pelvic organ integrity and repair remains to be elucidated. the aim of this study was to determine the effect of hoxa expression on fibroblast proliferation, and its potential role in pop. methods: nih t cells, a murine fibroblast cell line, were seeded onto a six well plate ( x cells/well) and transfected with either a vector carrying a hoxa cdna or empty vector alone as a control. immunohistochemistry using bromodeoxyuridine (brdu) was performed to evaluate cell proliferation. cell division in the uterosacral ligament (usl) was also compared in women with and without pop. usl specimens were obtained at the time of hysterectomy for benign disease. immunohistochemistry was performed on paraffin embedded sections of the usl to evaluate expression of two mitotic markers, cyclin b and phospho-histone . results: constitutive expression of hoxa in murine fibroblasts resulted in significantly higher proliferation. cells transfected with hoxa had a mean brdu incorporation of . + . cells/ cells compared with . + . cells/ cells in controls (p= . ). in the usl obtained from women with and without pop, cell proliferation as determined by cyclin b and phospho-histone expression was not significantly different. cyclin b and phosphor-histone were expressed in comparable numbers of cells in both groups. conclusion: hoxa is necessary for usl development and promotes proliferation of adult fibroblasts. hoxa may have a similar function in vivo in usl, and may regulate fibroblast proliferation during growth and the acute phase response following trauma when fibroblasts are activated to proliferate and remodel the ecm. it is likely that hoxa mediated proliferation of usl fibroblasts contributes to the tensile strength and resilience of these structures and thereby prevents pop. supported by nichd wrhr: k hd - . connective tissue composition, in term of collagen i, iii and proteoglycans content, in support and nonsupport structures of women with uterine prolapse. elisabetta trabucco, gennaro acone, sara d'avino, marco torella, gennaro trezza, annamaria marenna, nicola colacurci, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; loreto mare hospital, naples, italy. objective. connective tissue consists mainly of collagen and elastic fibers, glycoproteins and proteoglycans (pgs) and is considered an important factor of the support and nonsupport structures of the genitourinary region. it has been already demonstrated altered morphologic features in the pelvic support connective tissue in women with genital prolapse. however, analysis of nonsupport tissue may provide a more accurate reflection of body collagen. the objective of our study was compare the expression of collagen i, iii and four pgs (decorin, fibromodulin, lumican, biglycan), essential for synthesis and regular assembly and diameter of the collagen fibrils, in uterosacral ligaments and in a nonsupport tissue, the uterine cervix, between premenopausal women with uterine prolapse respect to age matched controls. we characterized uterosacral ligaments and uterine cervix of premenopausal women with uterine prolapse and controls. this immunohistochemical study was performed on paraffin-embedded sections. results. uterosacral ligaments of the prolapsed uteri are characterized by a higher expression of collagen iii, decorin, fibromodulin and byglican and a lower quantity of collagen i. no differences in the immunoreactivity of lumican between the two patients groups. the abnormalities of support connective tissue composition are not observed in the uterine cervix of patients with prolapse. conclusions. our results suggest an altered remodeling of connective tissue in the ligaments of premenopausal patients with prolapse, with a significant decrease in collagen i content and an increase in collagen iii and pgs expression . in the prolapse patients this abnormal collagen metabolism and organization, mainly related to the observed change in pgs expression, might affect significantly the tensile strength of the connective tissue and consequently the support that is provided by the suspensory apparatus to the uterus. the alterd remodeling of support tissues, not detectable in nonsupport tissues, may suggest a predominant role of local biomechanical stresses (childbirth, chronic straining) in the pathogenesis of prolapse respect to systemic connective tissue deficiency. objective: to achieve vaginal delivery with minimal maternal injury, vaginal smooth muscle (sm) contractility likely decreases. the objective of this study was to characterize changes in rat vaginal sm contractility in pregnancy that affords circumferential vaginal distension. methods: a tubular segment of the vagina of virgin, late pregnant , immediate and late post vaginal delivery rats were mounted in an organ bath between a force transducer and an adjustable support block. dose response curves to norepinephrine (ne) and potassium (k+) were used to assess contractility. relaxation capacity was determined in ne preconstricted vagina, using cumulative doses of sodium nitroprusside (snp). differences in active vs. passive length-tension curves were used to measure sm contractility relative to the vaginal wall forces. sm -actin levels were measured using quantitative confocal immunofluorescence. results: cumulative doses of ne induced a maximum constriction force of . ± mn/g in virgin vagina while no measurable force was generated in response to ne in late pregnant or postpartum vaginas. virgin vagina relaxed with cumulative doses of snp; no measurable relaxation response was observed from pregnant or postpartum animals. in the presence of the high dose k+ ( mm), virgin vagina generated the greatest contractile force ( . ± mn/g) vs. late pregnant vagina ( . ± . mn/g, p< . ) or immediate post partum vagina ( . ± mn/g, p= . ). four week postpartum k+ induced forces were similar to virgin levels ( . ± mn/g). sm force generation (difference in active vs. passive length tension curves at mm of displacement) was decreased in late pregnancy ( . ± mn/g) compared with virgin ( ± mn/g, p < . ) and week postpartum vagina ( ± mn/g, p < . ). the expression of sm -actin was lowest in late pregnancy ( ± . , p < . ) and immediate postpartum vagina ( ± . , p < . ) relative to virgin ( ± ) with a return to virgin levels week postpartum ( ± ). conclusions: vaginal sm contractility diminishes in pregnancy. the altered contractility is mirrored by a decrease in sm -actin protein expression. near complete recovery to pre-pregnancy levels occurs by weeks post partum. these functional and biochemical changes likely represent maternal adaptations designed to minimize trauma during passage of the fetus(es). background: diagnosis or exclusion of endometriosis usually requires laparoscopy and peritoneal biopsy. we have described a novel and consistent observation of small nerve fibers in the functional layer of eutopic endometrium in women with endometriosis (tokushige et al, ) . these nerve fibers are not present in women without endometriosis. this finding allows the possibility endometriosis in the nude-mouse and inhibited mmp- expression in human endometrial stroma. the present findings may lead to the development of novel treatments of endometriosis involving statins. supported by nih u hd . k-ras actived immunocompetent retrograde menstruation model of endometriosis. ching-wen cheng, , di licence, , cristin g print, , d stephen charnock-jones. , pathology, university of cambridge, cambridge, united kingdom; obstetric gynaecology, university of cambridge, cambridge; department of molecular medicine pathology, university of auckland, auckland, new zealand. objective: to establish a new murine model of endometriosis that mimics the retrograde menstruation theory using immuno-competent mice. method: ovariectomised donor female k-ras v +/-/cre +/+ /rosa r-lacz +/+ transgenic mice were treated sequentially with steroid hormones. to induce decidualization and activation and k-ras transgene, mg/kg b-nf dissolved in maize oil was injected into the uterine lumen. tissue degeneration mimicking menstruation was induced by hormone withdrawal. menstruating endometrial tissue was collected from donor mice hours after last hormone treatment, re-suspended in matrigel, and implanted subcutaneously in female c bl/ recipients. immunohistochemical and morphometric methods were used to characterize the endometriosis-like lesions. microarray analysis (illumina, n= in each group), was used to study the molecular changes in "menstruating" uteri following k-ras activation. result: simple transplantation of decidualised endometrial tissue into immunocompetent animals does not lead to endometriotic lesion development but using tissue with the genetic modification described here overcomes this. viable endometriosis-like lesions are visible days after implantation. these lesions are histologically similar to those seen in man with intact glands, functional blood vessels, leukocyte infiltration and collagen deposition. statistical analysis revealed that transcripts were differentially regulated in the ras activated and control tissue. gene ontology (go) analysis indicated that transcripts associated with epithelial cell function and differentiation were over represented within this gene set (chloride transport and epidermis development) as were those associated with the acute inflammatory response and neutrophil chemotaxis. we developed a new model of endometriosis using immunocompetent mice to mimic retrograde menstruation induced endometriosis. this permits for the first time, the ready use of transgenic and knock-out tools to investigate the cellular and molecular mechanisms underlying endometriosis. since the animals have an intact immune system it also allows the testing of therapeutic agents that modify the inflammatory response. stra is expressed and induced by progesterone in the endometrium and is absent in endometriosis. mary ellen pavone, serdar e bulun, scott s reierstad, joy innes, magdy p milad, you-hong cheng. obstetrics and gynecology, northwestern univeristy, chicago, il, usa. objective: retinoic acid (ra) plays important roles in development, growth and differentiation by regulating the expression of target genes. in the mouse endometrium, ra deficiency leads to hyperkeratinization, while high concentrations of retinoids promote secretory characteristics. this leads many to believe that ra mediates important actions of progesterone in the endometrium and may account for progesterone resistance in endometriosis. the mechanism for regulation of ra production by progesterone is unknown. moreover, the conversion from retinol to retinoic acid is not different between normal endometrium and endometriotic tissue. we hypothesize that retinol intake into cells rather than conversion of retinol to ra is the critical step that determines ra activity in the endometrium and endometriosis. stra , a widely expressed multitransmembrane domain protein and member of a group of "stimulated by retinoic acid" genes, has been identified as the retinol binding protein receptor. it is strongly expressed in adult mice in several tissues including the female genital tract. we hypothesize that ra activity in the endometrium may be regulated by stra . here we investigate the differential expression of stra in the endometrium and endometriosis. design: we studied primary stromal cells isolated from the eutopic endometrium of disease free women and walls of ovarian endometriomas from women with endometriosis with respect to stra expression and regulation by progesterone. materials and methods primary culture of eutopic endometrial and endometriotic stromal cells were treated with - m estradiol (e ), - m r , a progesterone agonist, or vehicle for hours. real-time pcr was employed to quantify stra mrna levels. we treated cells for hours and quantified protein levels by immunoblotting. results basal mrna of stra levels in endometrial cells (n= ), were > , fold higher than those in vehicle-treated endometriotic cells (n= , p<. ). in endometrial stromal cells (n= ), r stimulated stra mrna levels by . - fold. r induced stra protein levels in endometrial stromal cells (n= ). conclusions stra is highly expressed and regulated by progesterone in endometrial stromal cells, whereas it is practically undetectable in endometriotic cells. stra may mediate important actions of progesterone in endometrium. upregulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity (pc) may influence their survival and persistence via local estrogen synthesis. the inducing factors for the upregulation of aromatase in endometriosis are not well defined, but increased expression of sf- has been suggested to play an important role. given that the aromatase substrate androstenedione (a ) is the predominant steroid in peritoneal fluid, we aimed to determine whether a has a role in the regulation of aromatase expression in human endometrium. we found that culture of primary endometrial stromal cells and explants with a ( - nm) for h significantly upregulated expression of aromatase mrna transcripts containing exon iia at their '-ends. moreover, in human endometrial surface epithelial cells (hes), dose-response studies with a ( - nm) revealed that nm a maximally upregulated expression of both aromatase and sf- . when tissue samples were evaluated from women with endometriosis and control endometrium, expression of aromatase mrna mirrored sf- . treatment of hes cells ( h) with tritiated a demonstrated its metabolism to estradiol (e ), testosterone (t), dihydrotestosterone (dht) and androstanediol (a-diol). although equimolar concentrations of a , t and e upregulated aromatase and sf- mrna expression in hes cells, the nonaromatizable androgens, dht and a-diol, had no effect. a positive feedback role of estrogen in aromatase upregulation was suggested by the finding that the estrogen receptor antagonist, ici , , markedly diminished aromatase and sf- mrna expression induced by a . finally, chromatin immunoprecipitation revealed that a significantly enhanced recruitment of sf- to its response element (- bp) upstream of cyp exon iia. collectively, our findings strongly suggest that exposure of endometrial cells within the pc to c -steroids may cause an acute upregulation of cyp gene expression through their aromatization to estrogens. thus, estrogen may play a critical positive feedback role in the pathogenesis of endometriosis. supported by nih r -dk . the hpa axis and depression/anxiety scores in chronic pelvic pain patients. b stegmann, b frank, j gemmill, g chrousos, m ballweg, p stratton. rbmb, nichd/nih, bethesda, md; som, wake forest university, winston-salem, nc; endometriosis association, milwaukee, wi. stress, pain, anxiety and depression adversely affect the hypothalamic-pituitaryadrenal (hpa) axis. we examined the influence of depression and anxiety on the hpa axis response to corticotropin-releasing hormone (crh) testing in women with and without chronic pelvic pain (cpp). methods: healthy women, aged - , with without cpp, with regular menses and off hormonal contraception were studied. none had pelvic infections, untreated depression, manic depression, fibromyalgia, or chronic fatigue syndrome. after ovine crh injection ( mcg/kg), serial blood samples (- , , + , + , + minutes) were obtained for acth and cortisol measurements. depression and anxiety were scored using the duke quality of life questionnaire. hpa response was abnormal if peak acth levels were > pg/ml without a rise in cortisol levels. subject groups were: no pain and normal acth response (npnr), pain and normal acth response (pnr), no pain with an abnormal acth response (npar) and pain with an abnormal acth response (par). student t-test was used for comparisons. results: women ( cpp, controls) had a mean age of . ± . years (range: - ). women responded normally ( pnr, npnr) and women responded abnormally ( par, npar). peak and absolute rise in acth were significantly higher in abnormal (a) vs normal (n) responders; (peak: . a vs . pg/ml n, p< . ; absolute: . a vs . pg/ml n, p= . ). there was a significant difference within groups: peak acth: . par vs . pg/ml pnr, p= . ; . npar vs . pg/ml npnr, p< . ; absolute acth: . par vs . pg/ml pnr, p= . , . npar vs . pg/ml npnr, p< . ). however, total cortisol level was not different between or within the groups ( . a vs . mcg/dl n, p= . ). mean depression score did not differ among cpp patients ( . par vs . pnr, p= . ), but differed among controls ( npar vs . npnr, p= . ). anxiety scores did not differ between or within groups ( . npar vs . npnr p = . ; par vs . pnr, p= . ). conclusions: chronic pelvic pain is associated with an abnormal hpa response, regardless of anxiety or depression symptoms. abnormal hpa responses in control women, however, appear to be influenced by depression. the mechanism and clinical significance of these findings should be explored. support: rbmb/nichd/nih and endometriosis association. objective: the eutopic endometrium in women with endometriosis demonstrates altered endometrial receptivity and altered gene expression. it is unknow if the endometrium is defective giving rise to a predisposition toward endometriosis or alternativly if the endometriosis causes the altered endometrial receptivity. here we created experimental endometriosis in a mouse model through allotransplantation of the uterus to the peritoneal cavity in immunocompetent mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. materials and methods: the uterus of -week-old cd female mice was transected at cervicovaginal junction and each horn divided and transplantated into the abdomidnal cavity of eight cd mice. seven controls received sham surgery only. after weeks the uterus was removed, snap-frozen in trizol. total rna was extracted and cdna generated. quantitative real time rt-pcr using sybrgreen was performed and normalized to -actin. fold changes in normalized hoxa , hoxa , bteb , emx , igfbp , integrin - , total progesterone receptor (pr-ab) and progesterone receptor-b (pr-b) were assessed. all experiments were conducted in duplicate, repeated at least three times and compared using mann-whitney rank sum test. results: hoxa , hoxa , bteb , emx , and igfbp mrna expression showed . -fold (p= . ), . -fold (p< . ), . -fold (p= . ), . fold (p= . ), and . -fold (p< . ) decrease in the uterus of mice with experimental endometriosis compared with controls. pr-ab and pr-b mrna showed . -fold (p= . ) and . -fold (p= . ) increase in endometriosis group compared to controls, respectively, however the ratio of pr-b to pr-ab (b/ab) showed no significant change in both groups ( . vs. . , endometriosis vs. control, p> . ). there was no significant change in integrin - mrna expression ( . -fold, p> . ). conclusion: we demonstrate significant changes in multiple markers of endometrial receptivity in eutopic endometrium after induction of endometriosis. these findings suggest that origionally normal endometrium can develop defects with the creation of endometriosis; an abnormal endometrium is not a prerequisite for the development of endometriosis or associated abnormalities. this data also suggest the existance of a signal conduction pathway from endometriosis that alteres endometrial gene expression. endometrial expression patterns of relaxin and relaxin receptor mrna suggest involvement of relaxin in endometriosis. sara s morelli, felice petraglia, gerson weiss, stefano luisi, pasquale florio, jeff gardner, andrea wojtczuk, laura t goldsmith. obstetrics, gynecology and women's health, new jersey medical school, newark, nj, usa; pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. objective: in normal endometrium, relaxin is a potent inhibitor of matrix metalloproteinases, which have been implicated in the invasive process of endometriosis. we tested the hypothesis that relaxin plays a role in endometriosis by comparing expression of relaxin and its lgr receptor in normal human endometrium to levels in samples from patients with endometriosis. materials and methods: total rnas, extracted from ectopic (n= ) and eutopic (n= ) endometrium of patients with endometriosis and from endometrium of normal controls (n= ), were reverse transcribed into cdnas. real-time pcr was performed using primers previously shown to identify human lgr relaxin receptor mrna, h relaxin mrna, and beta-actin mrna, with sybr-green detection of double stranded dna products. the comparative c t method ( -ct ) determined relative lgr and relaxin mrna expression (normalized to beta-actin mrna expression). relaxin mrna was detectable in normal endometrium from of ( %) control patients. in contrast, relaxin mrna was detectable in a lower proportion of samples [ of ( . %)] from patients with endometriosis, among whom relaxin mrna was detectable in a lower proportion of ectopic samples [ of ( %)] than in eutopic samples [ of ( . %)]. lgr relaxin receptor mrna was detectable in all samples, with lower expression in endometriosis samples than in endometrium from normal controls. relaxin receptor lgr mrna levels vary with cycle phase, with greater expression in the secretory phase (sp) than in proliferative phase (pp): in normal controls . -fold higher levels in the sp than pp; and in endometriosis patients . -fold higher levels in the sp than pp. in both phases, lgr mrna levels were lower in ectopic samples than in either eutopic samples ( . -fold lower in pp and . -fold lower in sp) or endometrium from normal controls ( . -fold lower in pp and . -fold lower in sp). eutopic endometrium had similar lgr mrna expression to controls throughout the cycle. decreased local expression of relaxin and relaxin receptor mrna in ectopic endometrium from patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective against endometriosis. ovarian reserve after laparoscopic cystectomy of endometriotic ovarian cysts. shinichi hayasaka, takashi murakami. obsterics and gynecology, tohoku university, sendai, japan. objective laparoscopic ovarian cystectomy is generally recommended for endometriotic ovarian cysts because it has been associated with a higher pregnancy rate and a lower recurrent rate. however, residual ovarian function after laparoscopic cystectomy of endometriotic ovarian cysts has been questioned. in this study, we retrospectively evaluated ovarian response to hyperstimulation in women selected for ivf and icsi cycles who previously underwent laparoscopic cystectomy of a monolateral endometriotic ovarian cyst. methods a retrospective review of the patients between january and september was performed. the operated ovary and contralateral intact ovary were compared in terms of number of oocytes retrieved, number of follicles with a mean diameter > mm at the time of hcg administration. the patients who had recurrent endometriotic ovarian cysts were excluded. in total, ten patients were identified. the mean(±sd)number of oocytes retrieved was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the mean(±sd)number of follicles with a mean diameter > mm was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the age of patients and diameter of the operated ovary had little influence on the difference between the response of the control ovary and one of the previously operated ovary. laparoscopic cystectomy of endometriotic ovarian cysts is associated with a significant reduction in ovarian reserve. background pre-eclampsia (pe) is associated with systemic maternal endothelial dysfunction, which is thought to result from the presence of circulating factors released following placental damage. it is hypothesised that this occurs as a result of reduced oxygen (o ) delivery. some features of placental pathology seen in pe, such as increased apoptosis, can be reproduced by culture of placental explants in % o . metabolomics operates to study 'all' metabolites within a biological system. this strategy has previously identified differences in maternal plasma between normal pregnancies and those complicated by pe. in these experiments we used metabolomics to investigate differences in the metabolic profile of explants cultured in different o tensions. hypothesis the metabolic profile of placental villous explants is altered by culture in different o tensions. methods placental villous explants were cultured with either % serum (n= ) or in serum-free conditions (n= ) for h in %, % and % o . after h, conditioned culture medium and tissue were collected and snap frozen. tissue was homogenised in % ice cold methanol prior to analysis. samples were analysed using gas chromatography-mass spectroscopy (gc-ms). samples cultured at %, % and % o were compared to identify concentration differences in metabolites in conditioned cultured medium and tissue lysate. kruskal-wallis test was used for statistical analysis. due to the large number of metabolites identified, a p value of . was considered significant. results the mean intra-assay variability was . %. there were no differences in the metabolic profile of conditioned culture medium from % and % o . several metabolites differed in culture medium from % compared to % and % o including: deoxyribose, glycerol and threionic acid. these changes were present in both serum and serum-free conditions. using these metabolites alone, culture in % o could be completely discriminated from % and % o . deoxyribose was also elevated in tissue lysate from % o compared to % o . conclusion this metabolomic strategy can identify differences in metabolic profile in placental tissue cultured in % o . these novel compounds may provide further insight into pathophysiology of pe. objective a strategy of metabolic footprinting (the study of extracellular metabolites, which are related to intracellular metabolism) was used to detect a wide array of low molecular weight metabolites. metabolic profiles were employed to differentiate between placental explants cultured at different oxygen tensions. two separate studies were combined to validate initial observations. methods metabolic footprints of placental villous explants, obtained from uncomplicated pregnancies, (n= ) were cultured for h in %, % and % oxygen. conditioned culture medium was then collected and frozen, prior to analysis by uplc/ltq-orbitrap mass spectrometry in both negative and positive ion mode. samples cultured at %, % and % were compared to identify differences in the metabolic profiles. this procedure was repeated for different explants and the results compared across the studies in order to validate initial findings. approximately unique peaks were detected in both sample sets in negative ion mode and peaks in positive ion mode. a number of metabolites were identified as being significantly different using kruskal-wallis (pval< . ) under different oxygen tensions. some differences were seen between the results obtained from both runs due to separate batch preparation of the medium but good reproducibility was obtained for many metabolites between batches. metabolites of biological interest included amongst others -deoxyribose, valine, tyrosine and aspartic acid. the largest differences were those seen between o % and o %. comprehensive metabolic profiles were detected and employed to identify differences in the metabolic footprints of explants cultured under differing oxygen conditions. a number of biologically interesting metabolites were characterized. objective: brain weight and dna content were reduced in leptin deficient (lep ob /lep ob ) mice postnatally. leptin is detected in the sera and its receptors are expressed in the brain of the mouse fetus. we examined the role of leptin in the proliferation and differentiation of neural lineage cells in the mouse fetus. methods: the number of total cells in the cerebral cortex was compared between (lep ob /lep ob ) and wild type fetuses from embryonic day (e) to . the number of brdu positive cells was also compared on e and e . brdu uptake, the ratio of viable colony number to plated cell number, the proportion of multipotent, neuronal or glial progenitor colonies, and the expression levels of hes mrna were compared between leptin-and non-treated neurosphere cells. moreover, we examined stat phosphorylation by leptin stimulation with elisa to investigate whether or not jak/stat pathway transduces the leptin signal in the prenatal period as in the adult. results: lep ob /lep ob fetuses had reduced total cell numbers in the ventricular zone (vz) on e and e , and in the cortical plate on e . the number of brdu-positive cells was reduced in vz of e and e lep ob /lep ob fetuses. brdu uptake and the number of viable colonies were increased by days-leptin treatment in the neurosphere culture. the proportions of glial-restricted and multipotent precursor colonies were increased by leptin, whereas that of oligodendrocyte precursor colonies were decreased. hes mrna expression was enhanced in neurosphere cells by leptin. neither the amount of phosphorylated stat nor that of stat was increased in neurosphere cells by leptin stimulation. conclusion: our study suggests that leptin maintains the neural stem and glial-restricted precursor cells through upregulation of hes expression and increases the number of cerebrocortical cells in the mouse fetus, however, jak/stat pathway does not mediate the leptin signal in undifferentiated neural lineage cells. a vaginal wall, and > % of fib- knockout (ko) mice develop pelvic organ prolapse by weeks of age. in contrast, fib- and - deficiencies do not result in prolapse, and fib- kos die shortly after birth. herein, we evaluated the role of fib- in pelvic organ support. methods: two observers serially measured the degree of vaginal, perineal, and anal prolapse in fib- ko (fib -/-) and in fib -/-mice severity of prolapse was significantly related to age, but not parity, and the average age at diagnosis was wks. fib -/-animals with advanced prolapse had attenuated uterosacral ligaments and descent of the bladder and uterus caudal to the symphysis. pro-mmp ( -fold, p= . ), active mmp ( -fold, p= . ), and prommp ( -fold, p= . ) were increased in vaginal tissues from mice with gross prolapse compared with age-, strain-, and cycle-matched controls. this increase in protease activity, however, was also accompanied by increased expression of fib- and tropoelastin ( . -fold, p< . ). regardless of prolapse maternal morbidtties tf ards-n(%) ( ) acute ren~m f lure-n (%) ( ) car~tovascular ~ysfunrtion-n ("/o) ( ) hepatc fa..lure-n (%) dt sst'dlm atl:d inlfavascul..-coagul tipalhy-n(%) ( ) durallon of !cu suy (days. mean+sd) :t . hospital my (days.. me +sd) . condon, j. c., hardy, d. b., kovaric, k., and mendelson, c. r. ( ) mol endocrinol ( ), - . objective: innervation of the uterine cervix is important for the process of parturition (physiol beh : , ; j histochem cytochem : , jsgi : , ) . the hypogastric nerve is a major pathway that innervates the uterine cervix, yet its contribution to processes associated with cervical ripening and parturition is not known. the objective of this study was to determine the effect of hypogastric nerve transection on processes associated with cervical remodeling and parturition. methods: time-dated pregnant rats were sham-operated or the hypogastric nerve bilaterally transected just anterior to the inferior mesenteric ganglion on day post-breeding. live pups were born spontaneously by all rats at term on days - post-breeding. on the day of birth, the cervix was excised, postfixed overnight, sectioned, and processed to evaluate collagen content and structure (nih image j). sections were also processed by immunohistochemistry to assess cell nuclei density, the census of resident macrophages, and area of tissue that contained nerve fibers. results: hypogastric neurectomy did not affect cell nuclei density, the number of macrophages, or density of nerve fibers in the cervix. the failure of hypogastric nerve transection to affect indices of cervical remodeling is consistent with the finding that duration of pregnancy and timing of birth were not different in sham-operated and hypogastrectomized rats. conclusions: nerve fibers in the hypogastric nerve that innervate the lower uterus and cervix, including sympathetic and neuropeptidergic projections, are thus not essential for birth. these novel findings provide support for the contention that innervation of the uterine cervix other than through the hypogastric nerve contributes to processes associated with cervical ripening and parturition. receptor system in prostaglandin synthesis in pregnancy. eugenie r lumbers, , della m yates, carolyn m mitchell, jonathan j hirst, , tamas zakar. , school of health sciences, university of newcastle, newcastle, nsw, australia; mothers and babies research centre, hunter medical research institute, newcastle, nsw, australia; obstetrics and gynaecology, john hunter hospital, school of medical practice and public health, university of newcastle, newcastle, nsw, australia. the fetal membranes and decidua contain prorenin, which requires proteolytic activation. ngyuen (j clin invest. : ) described a renin/prorenin receptor that conformationally activates prorenin, so that it can form ang i from aogen. in addition, receptor-bound prorenin can stimulate intracellular pathways directly. prostaglandins (pgs) participate in the control of term and preterm labour, and renin can stimulate pg production by amnion and decidua when angiotensin's action is blocked mitchell placenta : ) . the prorenin receptor may mediate these effects. our aim was to find out if the prorenin receptor gene is expressed and colocalised with prorenin and prostaglandin h synthase- (pghs- ) in human placenta, amnion, chorion and decidua. we have also determined if there is any change in the expression of the prorenin and prorenin receptor genes with labor. placentae with attached membranes were collected after term birth either by elective caesarian section or by spontaneous labor, and prorenin, prorenin receptor and pghs- mrna levels were quantitated relative to beta-actin mrna by real-time rt-pcr in amnion, chorion, decidua and placenta. before labor, mean prorenin mrna levels were . times higher in decidua and . times higher in chorion and placenta than in amnion (p< . ). there were no significant changes with labor. proenin receptor mrna was expressed in all tissues. proenin receptor mrna levels in prelabor amnion, chorion and placenta were similar, while levels in decidua were % of those in amnion (p= . ). this low level of prorenin receptor mrna in decidua persisted after labor (p< . ). pghs- mrna expression was highest in amnion; in decidua and placenta it was only and % of amnion. similar differences were present after labor. we conclude that the decidua is the principal source of prorenin within the pregnant uterus, and all gestational tissues are targets for prorenin. decidual prorenin may affect pghs- expression in amnion through the prorenin receptor forming a maternal-fetal paracrine system that stimulates pg production at labor. the immuno-suppressive effects of progesterone on umbilical cord blood mononuclear cells and the effect of culture media estradiol content. nadav schwartz, xiangying xue, christine n metz. obstetrics and gynecology, nyu school of medicine, new york, ny, usa; feinstein institute for medical research, manhasset, ny, usa. objective: progesterone (p ) is known to supress the maternal immune response and similar effects have been seen with fetal cells. we sought to ascertain whether p action was modulated by the phenol red and/or estrogen content of the culture media. methods: umbilical cord blood mononuclear cells were isolated using a density gradient centrifugation. the cells were incubated with p ( - m) hour prior to overnight stimulation with lps. supernatants were assayed for tnf-using elisa. the following culture media were used: a)'red' media: rpmi+ % fbs, b)'yellow' media: phenol red-free rpmi+ % fbs, and c)'clear' media: phenol-free rpmi+ % dextran/charcoal treated fbs. finally, the 'clear' media experiments were repeated with estradiol add-back. results: p suppressed tnf production in all medias. tnf levels were suppressed to . % (p< . ) of controls using 'red' media and to . % (p< . ) in 'yellow' media. the degree of suppression was not as substantial using 'clear' media where tnf levels were . % (p< . ) of control. (fig ) adding estradiol to the 'clear' media had little or no effect on the degree of tnf suppression. (fig ) conclusions: progesterone exerts an immuno-suppressive effect on lpsstimulated fetal mononuclear cells which is more pronounced when using media containing untreated fetal bovine serum. the phenol-red/estradiol content of the culture media did not modulated the p effect. dextran/charcoal treatment of fbs appears to deplete the media of factors other than estradiol that are necessary for p to exert its full effects. culture conditions should be optimized when studying progesterone's effects on immune response. ovine fetal regional blood flow following prenatal dexamethasone (dex) at . gestation (g). antonine d van heesewijk, jan g nijhuis, susan l jenkins, peter w nathanielsz, mark j nijland. ob/gyn, academisch ziekenhuis maastricht, maastricht, netherlands; ob/gyn, cpnr, uthscsa, san antonio, tx, usa. background: pregnant women at risk for premature labor receive antenatal glucocorticoids (gc) to reduce neonatal morbidity and mortality. while fetal blood pressure (bp), heart rate and vascular endothelial and control fetuses (n= ). dispersed adrenal cortical cells ( . x cells/tube; in duplicate) were challenged with - m acth. samples were collected at time (baseline), , , , and min after acth treatment, and tissue and media samples were frozen for determination of cortisol, camp, and star. results: cortisol output (ng/ . x cells) was higher in the lth group compared to the control, (p< . ) at min ( . ± . vs. . ± . ), min ( . ± . vs. . ± . ), and min ( . ± . vs. . ± . ). peak camp levels (fmol/ . x cells) were observed at min but did not differ between control ( . ± . ) and lth ( . ± . ) adrenal cells. western analysis demonstrated that star protein expression was higher in the lth adrenal cortex compared to control (p< . ) at min ( . ± . vs. . ± . ), and at min ( . ± . vs. . ± . ), relative optical density units. conclusions: results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in lth fetuses is the result of increased adrenal acth sensitivity. this enhanced sensitivity is not due to differences in camp generation. star, which is a key element involved in cholesterol transfer at the level of the mitochondrial membrane appears to play a key role in the enhanced cortisol response to acth in the lth group. (nih grants hd , hd and llu-nih imsd r gm - ). expression. hiroaki itoh, makoto kawamura, shigeo yura, haruta mogami, tsuyoshhi fujii, norimasa sagawa. obstetrics and gynecology, national hospital organization osaka national hospital, osaka, japan; gynecology and obstetrics, kyoto university graduate school of medicine, kyoto, japan; obstetrics and gynecology, mie university school of medicine, tsu, japan. objective: epidemiological evidences suggested that undernutrition in utero develops risk factors for adult cardiovascular disorders, such as blood pressure increase. the present study was designed to prove the hypothesis that maternal iso-caloric high protein diet alleviates blood pressure increase in the adult offspring by affecting placental beta-hydroxysteroid dehydrogenase type ( beta-hsd ) expression. methods: the % calorie restriction was applied to pregnant mice by using either standard protein diet (spd; % casein protein; spd-un) or high protein diet (hpd; % casein protein; spd-un). in some groups, these pregnant mice were sacrificed at . d.p.c.; then maternal and fetal plasma as well as placental tissues were corrected. while in other groups, systolic blood pressure was measured in the offspring at wks. fetal and maternal corticosterone levels were measured by elisa. the placental beta-hsd gene expression was measured by quantitative taqman pcr. results: systolic blood pressure in the spd-un offspring at weeks ( mmhg) was higher than that in spd-nn offspring ( . mmhg). by contrast, systolic blood pressure in the hpd-un offspring ( . mmhg) was similar to that in spd-nn offspring. maternal calorie restriction with spd and hpd caused similar elevation of maternal plasma corticosterone concentrations. by contrast, fetal plasma corticosterone levels in hpd-un offspring ( ng/ml) was lower than those in spd-un offspring ( ng/ml), indicating the amelioration of fetal exposure to high corticosterone by maternal hpd. the placental beta-hsd gene expression in the hpd-un was % higher than that in spd-un, suggesting that maternal hpd augmented placental beta-hsd gene expression in the placenta and probably facilitated the inactivation of maternal cortocsterone in the process of placental transfer to fetal circulation. conclusion: the preset study suggests that maternal high protein ingestion may elevate placental beta-hsd gene expression and ameliorate blood pressure increase in the adult offspring via modification of fetal exposure to maternal corticosterone. elevated cortisol levels at birth exert critical maturational effects through action at glucocorticoid receptors, gr. in contrast, the low cortisol concentrations in the preterm fetus, before the time of increased fetal cortisol secretion, are near to the kd for cortisol at the higher affinity mineralocorticoid receptor, mr. we hypothesized that endogenous cortisol in the fetus exerts physiologic actions at mr in tissues without appreciable cortisol-inactivating enzyme, hsd , including the fetal lung and brain. we therefore tested the effect of infusion of a mr-antagonist, ru (mra, . mg/h over h) into - day fetal sheep. we tested for effects on lung liquid production rate, lung liquid and plasma electrolytes, plasma acth and cortisol, and hematocrit at - h after start of the infusion. although there was no significant difference in the liquid production rate in fetuses infused with ru , the ratio of na + to k + in lung liquid was significantly greater in mra-treated fetuses than in the control fetuses ( . ± . vs . ± . ). plasma acth was significantly greater in the mra-treated fetuses than in control fetuses at h ( ± vs ± pg/ml); in the mra-treated fetuses, acth concentrations at h were greater than in the same fetuses at h ( ± pg/ml), wheras there was no increase in plasma acth in the control fetuses ( h: ± pg/ml). similarly, plasma cortisol concentrations at and h were greater in the mra infused fetuses than in control fetuses ( h: . ± . vs . ± . ng/ml), and cortisol increased significantly over time in the fetuses infused with mra, but not in control fetuses ( h, mra: . ± . , control: . ± . ng/ml). infusion of mra did not alter fetal plasma electrolyte, fetal blood pressure or fetal heart rate. however, fetal packed cell volume was significantly increased at - h of infusion of mra ( h: ± vs ± %) and fetal pco was significantly greater at h of infusion of mra as compared to control fetuses ( . ± . vs . ± . ). these results suggest that endogenous cortisol concentrations in the preterm fetus exert negative feedback control of acth via action at mr in the fetal brain, and play a role in regulation of fetal lung liquid composition and in fetal fluid balance. preparturient increases in fetal acth secretion are cyclooxygenase- (cox- ) dependent. charles e wood. dept. physiology and functional genomics, university of florida college of medicine, gainesville, fl, usa. maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. in sheep, increased preparturient activity of the fetal hpa axis appears to be responsible for triggering parturition itself. this study was designed to test the hypothesis that prostaglandin generation, mediated by cox- in the fetal brain, is critically important for stimulation of the preparturient increase in fetal acth secretion. singleton fetal sheep were chronically catheterized with vascular, amniotic, and lateral cerebral ventricular catheters. nimesulide, a selective cox- inhibitor, was infused ( mg/day, n= ), and vehicle ( % dimethylsulfoxide, n= ) was infused. arterial blood samples were drawn from fetuses at hour intervals for measurement of plasma hormone concentrations and arterial blood gases. in the vehicle-treated fetuses, fetal plasma acth, pomc, and cortisol concentrations increased exponentially (p< . by anova) before spontaneous parturition at ± . days. in the nimesulide-treated fetuses, fetal plasma acth and pomc were constant and did not increase prior to parturition (p=ns by anova). in contrast, fetal plasma cortisol increased independent of acth (p< . by anova) and the fetuses were born spontaneously on ± . days gestation. the slight delay in spontaneous parturition in the nimesulide-treated fetuses was statistically significant (p< . by mantel-cox test). intracerebroventricular nimesulide infusion did not decrease fetal plasma concentrations of prostaglandin e , demonstrating that the action of the nimesulide was restricted to the fetal brain. fetal blood gases were normal in all of the fetuses, and there were no differences in blood gases between groups. we conclude that the spontaneous increase in fetal acth and pomc prior to parturition is cox- dependent. however, we also conclude that the increase in fetal plasma cortisol concentration can occur independent of increases in fetal acth, suggesting that the increase in cortisol can result from increases in adrenal sensitivity to acth. the results of this study demonstrate that the timing of parturition in the sheep is not dependent upon increased acth secretion, and the results suggest that parturition is regulated primarily by changes in adrenal sensitivity. expression of extracellular signal-regulated kinase / and p mitogen-antiphosphotyrosine. immunolocalization of inos was performed in the same tissues. hpmec were used to determine effect of sin- ( mm) on inos protein expression ( min, and h). results: inos protein was detected in microvascular endothelium, smooth muscle and syncytiotrophoblast of the placenta. inducible nos levels in placentas from severe preeclampsia were significantly decreased compared to normal (p< . ) but were not different from mild preeclampsia. aligning western blots of anti-phosphotyrosine and inos revealed a phosphorylated band corresponding to the molecular weight of inos. the proportion of tyrosine phosphorylated inos was reduced by % in severe preeclampsia compared with normotensive. preliminary data with ip for phosphotyrosine and crossblotting with inos confirmed this finding. sin- treatment decreased inos protein abundance at and h in hpmec. conclusions: our results demonstrate decreased tyrosine phosphorylation of inos in preeclamptic placenta. phosphorylation of tyrosine in inos has been reported to negatively regulate its activity. we therefore postulate that decreased phosphorylation of inos may be responsible for the increased catalytic activity of the enzyme that we have previously observed. the decreased levels of inos observed on sin- treatment may be due to nitration, an effect analogous to preeclampsia, where the presence of peroxynitrite has been well established. it remains to be seen if protein nitration has an effect on enzyme stability. objective: -isoprostane ( -iso), a prostaglandin-like compound formed in vivo, is a reliable measure of oxidative stress which occurs in response to many different stimuli, including infection. periodontal disease is a chronic oral infection associated with fetal growth restriction and preeclampsia. our objective was to determine if maternal periodontal disease is associated with oxidative stress as measured by -iso. methods: a secondary analysis was conducted using prospective data from the oral conditions and pregnancy study. a cohort of healthy women enrolled < weeks underwent oral health examination and serum sampling. maternal periodontal disease was categorized as healthy, mild, or moderate-severe by clinical criteria. maternal serum was analyzed for -iso by ultra-sensitive elisa. elevated -iso was defined by a value th %tile. maternal factors associated with elevated -iso were determined using chi-square or t-tests as appropriate. a logistic regression model was created to determine adjusted odds ratios ( th %ci) for elevated -iso. results: women had complete data for analysis. the median -iso level (iqr) was , ( - , pg/dl). using bivariate analysis, moderate-severe periodontal disease, non-white race, use of wic/food stamps, unmarried status, obesity, and lack of insurance were associated with elevated -iso. the logistic regression model for elevated -iso is shown below. adjusted or ( th ci)* mild periodontal disease . maternal periodontal disease and utilization of public assistance are associated with oxidative stress in pregnancy. the relationship between periodontal disease, social hardship, oxidative stress, and adverse pregnancy outcome remains to be determined. antioxidant therapy could represent novel therapy for the prevention of adverse pregnancy outcome. severe transient immunological thrombocytopenia in a preeclampsia patient whose bone marrow production of platelets had been restricted. toshihiro yoshimura. obstetrics and gynecology, ntt-west kyushu general hospital, kumamoto-city, kumamoto, japan. introduction: idiopathic myelofibrosis is a chronic myeloproliferative disorder in which clonal haemopoetic stem cell proliferation is accompanied by reactive fibrosis. case report: a -year-old primiparous woman was referred to our hospital for prenatal care after weeks of gestation. her medical history was significant for idiopathic myelofibrosis, and congenital agenesis of the spleen. the laboratory workup showed the patient's white blood cell count to be , / l; hemoglobin, . gm/dl; and platelet count, , / l. her bleeding time was normal ( min). until the th week, the patient's pregnancy was uneventful. during the th week, however, the patient's platelet count declined to , / l, when proteinuria became evident. her bleeding time was prolonged to min. the coagulation system was normal. by the th week, the patient's blood pressure was elevated to / mmhg, and her urinary protein excretion exceeded g/day. our initial diagnosis was thrombocytopenia due to bone marrow suppression. the patient had no history of platelet transfusion, but we expected it would increase the platelet count, particularly since in the absence of destruction by the spleen, the lifespan of the platelets would be prolonged. this was not the case, however. elective induction of labor was carried out after weeks. at the same time, the patient was transfused with units of platelets, but her platelet count remained unchanged ( , / l). it was then that we realized that immunological thrombocytopenia may be involved, and massive doses ( mg/kg) of gamma globulin were given intravenously for one day. thereafter, platelets ( units) were again transfused, this time raising the platelet count to , / l. cesarean section was promptly carried out under general anesthesia. we later found that the patient's serum platelet associated immunoglobulin (paigg) level was positive, though antiplatelet and antinuclear antibodies were negative. the postoperative course was uneventful. the maternal platelet count declined to the pretransfusion level within days after delivery, but gradually increased to the prepregnancy level by weeks. comment: this idiopathic myelofibrosis patient in whom bone marrow production of platelets had been severely restricted demonstrates that immunological destruction may play a major role in the development of thrombocytopenia in preeclampsia. early l-arginine therapy improves notably the fetal growth in preeclamptic women. a randomized controlled trial. keisy lopez-molina, juan c gonzalez-altamirano, julio e valdivia-silva. , oncoinmunología y biología vascular, facultad de medicina, universidad nacional san agustín, arequipa, peru; cardiología y cirugía de tórax, hospital nacional case essalud, arequipa, peru; inmunología, instituto de investigaciones biomédicas, méxico, distrito federal, mexico. objective: to assess the benefit of early administration to l-arginine, the precursor to nitric oxide, on fetal growth. methods: one hundred women with preeclampsia were randomized to receive either l-arginine or placebo until day postpartum. live singleton infants were born after preeclamptic pregnancies and compared those with other control infants. birth size was expressed as the ratio between observed and expected birth weights, and infants smaller than two standard deviations from expected birth weights were classified as small for gestational age (sga). all the women had neither previous precedents of the preeclampsia nor other factors that they cause sga. results: no significant differences existed between the groups with preeclampsia before randomization. preeclampsia was associated with a % ( % confidence interval [ci] %, %) reduction in birth weight. women with hellp syndrome had to leave the study. the risk of sga was three times higher (relative risk [rr] = . ; % ci . , . ) in infants born after preeclampsia without l-arginine therapy than in control pregnancies, and two times higher (relative risk [rr] = . ; % ci . , . ). in infants born after preeclampsia with l-arginine therapy. conclusion: fetal growth improve markedly with l-arginine therapy in women with preeclampsia. introduction: although the pathogenesis of severe preeclampsia (pe) and intrauterine growth restriction (iugr) is poorly defined, inadequate remodeling of uterine spiral arties may promote reperfusion injury leading to focal infarction and reduced nutrient flow between mother and fetus. our goal was to determine whether an intravillous inflammatory cytokine cascade was associated with pe and iugr. methods: immunohistochemistry (ihc) examined il- and il- expression in preterm placentas with severe pe+iugr, and idiopathic preterm controls (ptc) with no evidence of clinical or histological chorioamnionitis. cultures of fibroblasts (fibs) and syncytiotrophoblasts (scts) from term placentas (n= ) were treated with il- and cytokine levels in conditioned media were determined using elisa or multiplex array. results were analyzed by student's t test or anova. results: the gestational age at delivery was not significantly different in pe+iugr and ptc groups ( . ± . vs . ± . wks). ihc of pe+iugr samples revealed intense villus core staining of il- adjacent to infarcts. villi distal to infarcts stained with a lower intensity. in contrast, staining was virtually undetectable in ptc. the pattern of il- staining was nearly identical in pe+iugr and ptc groups, was localized to the syncytium, and did not differ with respect to distance from infarct. to identify cellular targets of il- action, primary cultures of fibs and scts were incubated for h in serum-free medium ± ng/ml il- . by elisa we observed that il- treatment of fibs increased il- levels from ± to ± pg/ g protein (p< . ). similarly, multiplex array revealed that levels of il- , il- , and il- were induced -, -, and -fold, respectively in fibs. the presence of ng/ml il- receptor antagonist reduced the il- -mediated increase in il- levels ± % (p< . ), indicating that this response was mediated through il- receptor. in marked contrast, il- treatment did not significantly affect levels of il- , il- , il- or il- in scts, indicating that this response was cell type-specific. conclusions: these results indicate that increased expression of il- in the placental villus core in pe and iugr may promote an inflammatory cascade in fibs at this site leading to focal infarction and reduced flow of nutrients to the fetus. introduction: in human pregnancy, decreased responsiveness to angiotensin ii (angii) starts in week , promoting an expanded vascular bed. at the same time levels of the vasodilatory hemopexin increases . during pre-eclampsia the vascular bed is contracted and the responsiveness upon angii persists. this may be due to the increased levels of extra cellular atp, a natural inhibitor of hemopexin . in the present study we tested whether extra cellular atp is toxic in the pregnant condition. methods: pregnant rats were infused with either atp ( g/kg bw; n= ) or saline (n= ) on day of pregnancy (permanent jugular vein cannula/ hr infusion). non-pregnant rats (n= ) were infused with atp identically. one day before and , , days after infusion, hr urine and blood samples (wbc count and hemopexin activity) were collected. seven days after the infusion, rats were sacrificed and kidney tissue processed for immunohistology. results: urinary albumin excretion was increased in pregnant atp infused rats only (fig ) . wbc were also increased only in pregnant rats infused with atp (days and vs pre-infusion value). in pregnant atp infused rats intraglomerular influx of monocytes was increased, which correlated with urinary albumin excretion on the same day (r = . ). staining of glomeruli for the angii receptor (at- r) showed decreased at- r expression in control pregnant rats as compared to non-pregnant rats, while at- r expression in atp infused pregnant rats was increased as compared to control pregnant rats. hemopexin activity was increased on days and in control pregnant rats as compared to all other groups. discussion: these data support the notion that atp is toxic exclusively in the pregnant condition. it may be suggested that atp induced an inflammatory response, although the exact mechanism by which atp induced its effects needs further investigation. background: hepatocyte growth factor (hgf) is a multifunctional cytokine that is known to promote division, motility, invasion and morphogenesis of a wide range of cell types and to inhibit apoptosis. the effects of hgf are mediated through its interaction with the tyrosine kinase receptor c-met. in pregnancy hgf/met system is involved in the physiologic growth and development of the feto-placental unit. hgf/met effects are counteracted by transforming growth factor- (tgf- ), that is known to promote progressive fibrosis in human tissues. moreover tgf- plays a major role in trophoblast growth and differentiation. tgf- inhibits hgf expression as well as hgf inhibits tgf- expression. an understanding of the mechanisms regulating placental balance of these growth factors may provide insights into the processes that occur in complications of pregnancy, such as preeclampsia (pe) and fetal growth restriction (fgr). objective: to verify the hypothesis that hgf, c-met and tgf- are differently expressed in tissues of normal and pe placentas. methods: we studied placentas from pregnancies complicated by pe ( with normal fetal growth and with fgr) and placentas from normal pregnancies. from each placenta random samples were excised and rna was extracted; quantitative real-time rt-pcr analysis was used to investigate mrna expression of hgf, c-met and tgf- in pe (with or without fgr) and in normal placentas. results: gestational age, neonatal weight and placental weight were, as expected, lower in pe groups. the mrna expressions of the three molecules are higher in pe than in normal placentas, but the difference is statistically significant only for c-met. the higher values are mainly due to the increase in the pe group without fgr for c-met and tgf- and the increase in the pe-fgr group for hgf. conclusion: increased levels of expression of hgf/met system in pathological placentas could be explained as an attempt to repair placental damages; nevertheless placental regeneration could be inhibited by the increase of tgf- , that promote fibrosis. placental expression of hgf, c-met and tgf- is increased in all pe placentas, but particularly in placentas of pe with normal fetal growth, where placental tissue is probably less compromised. obesity is a risk factor for preeclampsia (pe), but the reason for this risk is unknown. previously, we found that neutrophils infiltrated into the vasculature of pe women released myeloperoxidase (mpo) and matrix metalloproteinase (mmp ), products that can cause oxidative stress and vascular dysfunction. if neutrophils infiltrate the vasculature of obese women and release mpo and/or mmp , this may help explain why they are at risk of developing pe. hypothesis: systemic vascular tissue of obese women will have a significant presence of mpo and mmp as a result of neutrophil infiltration. methods: subcutaneous fat, which is highly vascularized, was obtained at abdominal surgery from normal weight, overweight and obese women. formalin fixed, paraffin embedded μm sections of fat biopsies were stained using immunohistochemistry with specific antibodies for mpo and mmp . data were evaluated for intensity of vessel staining by visual score ( - ), density of staining using image analysis software, and % vessels with neutrophil staining, liberated from serum-free placental villous explant cultures from normal and pe pregnancies on human endothelial cells, and identified candidate mediators of their differential effects on metabolism and behaviour. methods: term placental villous tissue from normal (n= ) or pe (n= ) pregnancies were explanted for days at % oxygen. conditioned h medium (day - ) was applied to human umbilical vein endothelial cells (huvecs). an angiogenesis assay was conducted in which tubule length and number were measured by morphometric imaging following seeding on % matrigel. the effect of explant conditioned medium on endothelial cell metabolism was determined by mtt and bioluminescent atp assay. the release of vasoactive metabolites (nitrite, endothelin- , prostacyclin) from huvecs exposed to this medium was also measured. finally, a luminex bead array was used to screen the explant media for a panel of chemokines/cytokines. results: in the angiogenesis assay, tubule length (p< . , mann-whitney u test) and number (p< . ) were significantly decreased in pe compared to normal pregnancy medium. there was no change in mtt reduction, but endothelial cellular atp levels were also significantly reduced following exposure to pe explant medium (p< . ). both normal and pe-derived medium stimulated huvecs to produce vasoactive metabolites. the following cytokines were detectable in the explant media: interleukin (il)- , il- , gro-, monocyte chemotactic protein- and macrophage inflammatory protein- (mip- ). higher levels of both il- and gro-were present in the normal medium compared to pe (both p< . ). mip- was present in pe conditioned media but undetectable in media generated from normal placental explants. conclusions: these results suggest that pre-eclampsia stimulates the release of soluble factors from the placenta which have adverse effects on endothelial cell angiogenic potential and metabolism. altered levels of several cytokines were detected in the explant medium, and the effects of these on endothelial cell function are currently being addressed. yang gu, davis f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: placentas from women with preeclampsia (pe) release more chymotrypsin-like protease (clp). the purpose of this study was to determine if placenta-derived clp was responsible for altering endothelial (ec) barrier function in pe. approaches: endothelial junctional protein complex ( -catenin/ve-cadherin/ p ) expression and junctional protein ve-cadherin distribution were examined in ecs treated with pe placental conditioned medium. methods: ) confluent ecs were treated with pe placental conditioned medium (cm). the association of ec junctional protein complex ve-cadherin/catenin/p was examined by a combined immuno-precipitation (ip) and immuno-blotting (ib) assay, in which total cellular protein was immunoprecipitated with monoclonal antibody against -catenin and then immunoblotted with antibodies against ve-cadherin or p- . ) confluent ecs grown on cover slips were exposed to pe placental cm with or without depletion of chymotrypsin. ec junction protein ve-cadherin distribution was examined by fluorescent microscopy. results: ) ve-cadherin and p are expressed in control ecs but not in ecs exposed to pe cm, which indicate that the junctional protein complex vecadherin/ -catenin/ p is lost in ecs exposed to pe cm. ) ecs exposed to pe placental cm showed a discontinuous distribution and reduced expression for ve-cadherin at cell contact areas. the zipper like structure was lost and cleft was formed at cell-cell contacts. these observations indicate that the homotypic cell-cell adhesion and junction protein intracellular partner complex are disrupted. these disruptive phenomena in cells treated with pe conditioned medium were not present in control cells and in cells treated with cm after depletion of chymotrypsin. conclusion: clp released by the placenta could be a candidate agent that is responsible for disrupting ec integrity and inducing endothelial permeability in pe. (supported by nih grants hl and hd ). introduction: chronic pelvic pain (cpp) syndromes such as endometriosis, irritable bowel syndrome, and interstitial cystitis are associated with visceral hyperalgesia, and often coexist in the same patient. one possible explanation for this phenomenon is viscero-visceral cross-sensitization in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input from an unaffected organ to the same dorsal root ganglion (drg). nociception induces upregulation of perk and substance p. the purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of perk-and sp-positive neurons in sensory ganglia innervating both uterus and colon. methods: colonic and uterine drgs were retrogradely labeled with fluorescent tracer dyes micro-injected into the colon and uterus. ganglia were harvested, cryoprotected and cut for fluorescent microscopy. results:approximately % neurons were colon-specific and % were uterus-specific. among these uterus-or colon-specific neurons, up to % of labeled drg neurons in the l -s levels innervated both visceral organs. uterine inflammation increased the number of perk and sp-immunoreactive neurons in drg neurons innervating colon, uterus, and those innervating both organs. furthermore, this effect was specific as non-retrograde labeled drg neurons did not manifest a significant increase in perk or sp immunoreactive cells. conclusions: localized uterine inflammation leads to increased expression of sp and perk in uterine afferents as well as dichotomizing afferents innervating both uterus and colon, suggesting that viscero-visceral convergence is present at the level of drg primary afferent cell bodies. this visceral sensory integration may underlie the co-morbidity of female pelvic pain disorders and may provide basic information regarding the etiology of cpp syndromes. purpose: success rates of medical and surgical modalities for the treatment of severe fecal incontinence due to obstetric trauma are modest. we tested whether the intrasphincteric injection of autologous myoblasts is clinically safe and feasible for postobstetric fecal incontinence. methods: using ultrasound guidance a suspension of autologous myoblasts was injected in the anal sphincter muscle complex in three women with severe postobstetric fecal incontinence. main outcome measures were safety, feasibility and the wexner grading score. secondary outcome measures were incontinence episodes, rockwood fecal incontinenece quality of life scale, external anal sphincter morphology and anal pressure values. results: all procurement procedures and injections were performed without complications; there were no clinically or laboratory signs of infection or inflammation. three months post myoblast injection, wexner continence grading scores and rockwood incontinence scales were markedly improved and incontinence episodes reduced in all three patients. no change could be observed in sonographic anal sphincter morphology. mean anal squeeze pressure improved. conclusion: autologous myoblast injection for fecal incontinence is clinically feasible and safe; further studies will evaluate the efficacy of this approach. objective: to evaluate whether hysterectomy is associated with a change in postoperative bmi. methods: a retrospective cohort study was conducted of hysterectomies performed for benign indications from june to june . institutional review board approval was obtained. the data were collected by a medical student blinded to the hypothesis. basic demographic data were recorded. body mass index (bmi) was determined at time points: time (immediately postoperatively), time ( weeks to months postoperatively), time ( months to year postoperatively) and time ( to . years postoperatively). one-way anova was performed using ncss statistical software. a bonferroni multiple comparison test (p< . ) was used to compare median changes in bmi from baseline. the starting bmi served as the control group. results: there was a statistically significant increase in bmi at time compared to baseline in all women ( . to . ). when sorted was measured by matrigel invasion assay. small interference rna targeting vegfr- were predesigned by ambion inc. and cells were transfected using ambion siport neofx according to the optimized protocol. results: of the four receptors (vegfr- , vegfr- , nrp- and nrp- ), vegfr- was the predominant receptor that expressed in the more invasive cell lines (dov , skov and r ). lpa, at - m, significantly induced vegfr- expression in dov and skov cells (p< . ), without significantly affecting vegfr- expression. lpa ( - m) also significantly induced the expression of vegf and vegf (p< . ) in dov and skov cells. by small interference rna (sirna) transfection, we demonstrated that inhibition of vegfr- expression could significantly decrease dov cells' invasiveness (p< . ) and moderately decrease skov cells' invasiveness. moreover, silencing of vegfr- by sirna significantly suppressed lpa-induced dov and skov invasion. conclusion: these results suggest that knocking down vegfr- expression by rnai may be an effective strategy to inhibit lpa-induced ovarian cancer metastasis. cancer. fengqiang wang, david fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objectives: expression of matrix metalloproteinases, such as mmp- and mmp- , has implicated in epithelial ovarian cancer (eoc) invasion and metastasis. osteopontin (opn) was also expressed in various human cancers and associated with tumor progression, invasion and metastasis. in the present study, we examined the correlation of mmp- , mmp- and osteopontin expression with tumor stage in ovarian tumor tissues and normal ovaries using a commercial tissue scan array. methods: complimentary dna (cdna) from normal ovaries (n = ) and ovarian tumors (n = ) of different stages (stage i, n = ; stage ii, n = ; stage iii, n = ; stage iv, n = ) was amplified by real time pcr using gene specific primer pairs for mmp- , mmp- , and osteopontin. gapdh was also amplified as a reference control. the expression level of mmp- , mmp- and osteopontin was calculated as relative expression normalized to that of gapdh in each tissue sample, and the expression in sample that has the lowest target gene expression was arbitrarily set as . the average expression of mmp- in ovarian tumor tissues ( ± ) is fold of that in normal ovaries ( ± , p = . ). using an arbitrarily set standard, of normal ovaries ( . %) has elevated mmp- expression; in ovarian tumor tissues, the percentage is . % ( of ), significantly higher than in normal tissues (p = . ). in early stage tumors (stage i/ii, n = ), of them have elevated mmp- expression ( . %, p = . vs. normal ovaries), whereh the average expression of mmp- is fold of that in normal ovaries (p = . ) and . fold of that in late stage tumors (stage iii/iv, n = ). the mmp- expression in ovarian tumors (n = , ± ) is fold of that in normal ovaries (n = ), however, the difference is not significant (p > . ) due to the extremely high expression of mmp- in two tumor tissues. osteopontin expression in ovarian tumor tissues is . fold of that in normal ovarian tissues ( ± . , n= vs. . ± . , n = , p< . ). in early stage (i/ii) ovarian tumors, osteopontin expression is . fold of that in normal ovaries, while in late stage (iii/iv) ovarian tumors, its expression is . fold of that in normal ovaries (p< . ), suggesting an increase trend associated with disease stages. conclusions: our results suggest that mmp- , mmp- and osteopontin alone or combined may have clinical value for ovarian cancer detection. objectives: -tubulin acetylation has been proposed to control the dynamic nature of microtubule stability. acetylated -tubulin plays a role in regulating microtubule functions in mitosis and cell migration. here, we sought to identify the relationship between post-translational -tubulin acetylation and the expression of epithelial cell adhesion molecules (ep-cam) after exposure to microtubule interacting agents in ovarian cancer cells. methods: epithelial ovarian cancer cell line, hey was treated with microtubule stabilizing agents (taxol, epothilone b and discodermolide), microtubule-destabilizing agent (vinblastine), hdac inhibitor trichostatin a (tsa), anti-metabolite fluorouracil ( fu), or alkalyting agents (cisplatin and carboplatin). cells were separately treated with either ic or -fold ic of each agent, and incubated at °c for h, h and h. acetylation oftubulin and pan--tubulin were evaluated by western blot analysis followed by protein quantification. cell surface ep-cam expression was examined by flow cytometry. results: increased acetylation of -tubulin was seen with taxol, epothilone b, discodermolide, vinblastine and tsa. -tubulin acetylation was time and dose dependent. the highest level of -tubulin acetylation ( . -fold) was observed with vinblastine at -fold ic after h. exposure to microtubule interacting agents and tsa resulted in increased cell surface expression of ep-cam in a time and dose dependent manner. the highest level of cell surface ep-cam expression ( . fold) was observed with -fold ic of vinblastine at h. the increase in acetylated -tubulin and ep-cam expression was clearly detectable after h treatment. this data reveals a similar dose and time dependent increases between the acetylation of -tubulin and the increase of ep-cam expression. conclusions: these data demonstrate the promotion of -tubulin acetylation and cell surface ep-cam expression by a microtubule destabilizing agent and by microtubule stabilizing agents. interestingly, vinblastine induces the highest -tubulin acetylation and ep-cam expression. acetylation of alpha-tubulin may be associated with redistribution of cell surface antigens in ovarian cancer cells. objective: epothilone b (epob) is similar to taxol in its ability to enhance tubulin polymerization and to stabilize microtubules. epob is currently being evaluated as an antitumor agent and is in phase iii clinical trials. the tumor suppressor gene, p plays an important role in the induction of apoptosis by a variety of anticancer drugs. p mitogen-activated protein kinase is activated by a wide array of stress stimuli including chemotherapeutic agents and promotes apoptosis. since both p and p activation induces apoptosis, we hoped to evaluate the relationship between p , p-p and parp cleavage, an early indicator of apoptosis, in ovarian cancer cells after treatment with epob. methods: hey cells were treated with epob ( to nm) for h. parp cleavage product p as well as p-p , p , phospho-p (ser- ), p and survivin were determined by western blot analysis. a wild type ovarian cancer cell line hey, was treated with or without m sb , a specific inhibitor of p-p , followed by treatment with epob ( or nm) for h. lysates were prepared and western blot analysis was performed with polyclonal phospho-p and anti-phospho-p antibodies. results: epob induces p activation and apoptosis demonstrated by increased parp cleavage product. time course studies indicated that phosphorylation of p precedes phosphorylation of p in hey cells. the expression of p targeted gene, p (survivin) were differentially expressed depending on the dose of epob and the duration of drug exposure. pretreatment with specific inhibitor of p markedly inhibited the p phosphorylation at serine . conclusions: nm epob (a concentration that triggered mitotic arrest) causes a decrease in p expression and an increase in survivin expression. epob induces parp cleavage. p inhibitor sb inhibits epo-b -induced p phosphorylation. these results suggest that phosphorylation of p may lead to p activation and these signaling events may be related to epo-b induced cell death in ovarian cells. conclusions: nf-b has been shown to control the expression and function of anti-apoptotic proteins and pro-inflammatory cytokines. in the present study we demonstrated that specific inhibition of nf-b by erib reversed the anti-apoptotic state of chemoresistant ovarian cancer cells, therefore may provide a new potential venue for the treatment of ovarian cancer patients. expression in cervical cancer. madhu chauhan, chandra yallampalli. gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd)/adrenomedullin is a ct/cgrp family peptide. imd is expressed in a variety of tissues such as pituitary, stomach, placenta, uterus, and ovary .we have shown that imd plays a role in a variety of physiological functions, including vasodilatation and fetoplacental growth regulation. further we have data indicating an angiogenic activity of imd in first trimester trophoblast cells. we hypothesize that imd is expressed in cervix and may have a role in the pathology of cervical cancer objective: ) to analyze expression of imd mrna in human cervix, rat cervix from non-pregnant and pregnant rats; ) to assess the differences in the expression of imd in normal human cervix and cervical carcinoma tissues and ) to analyze the effect of imd on the expression of tnf-alpha and il- beta in human epithelial cervical carcinoma cells (hela). methods: groups of sprague-dawley, non-pregnant and pregnant rats on day of gestation were used in this study. cervical tissues were collected from the non-pregnant and pregnant rats, women undergoing hysterectomy and from women diagnosed with cervical carcinoma. total rna was extracted using trizol method. hela cells were grown to % confluency in rpmi medium supplemented with % fbs. the cells were starved in %fbs for hrs followed by treatment with imd ( - m) in presence or absence of imd antagonist, imda ( - m). cells were further incubated for hrs and total rna was extracted using trizol reagent. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). the rt-pcr data was normalized to that of s. results: ) imd mrna is expressed in rat and human cervix and in hela cells. ) expression of imd is elevated in pregnant rat cervix as compared to the non-pregnant. ) imd has no effect on tnf-alpha expression in hela cells treated with imd but caused a decline in the expression of il- beta mrna and, ) expression of imd mrna is significantly elevated (p< . ) in cervical carcinoma as compared to the normal cervix. conclusion: imd is expressed in both rat and human cervix and is elevated in pregnant rat suggesting that it may have a role in cervical function during pregnancy in rat. in addition we demonstrate that imd is involved in the pathology of cervical carcinoma and thus may have a clinical significance in the pathology of cervical cancer. objective: there is minimal information about the impact of treatment delays or dose reductions on chemotherapeutic treatment responses and overall outcomes in ovarian cancer patients. the primary objective of this study was to quantify the impact of treatment delays and dose reductions in an in vivo xenograft ovarian cancer model and to evaluate if the growth factors could improve overall survival. methods: heya- and skov -ip xenograft mouse models to determine the effect of treatment delays or dose reductions on tumor response. results: the results indicated that full dose of paclitaxel ( mg/kg) and carboplatin ( mg/kg) delivered on timeevery daysachieved better tumor response in both aggressive (heya- ) and metastatic (skov -ip ) human ovarian tumors compared to the non-treatment and vehicle groups. in heya- mice, paclitaxel/carboplatin alone and paclitaxel/carboplatin followed by growth factor support agents including pegfilgrastim ( μg/kg) and darbepoetin ( μg/kg) improved overall survival rate. growth factors also improved the tolerability in heya- model. for skov -ip mice, the treatment delays resulted in a significant reduce in overall survival time compared to full dose, on time treatment (p< . ). for the dose reduction group, there was a significant different survival comparing to full dose, on time treatment (p< . ). conclusion: treatment delays had a negative impact on tumor response and overall survival compare with treatment controls. in addition, use of growth factor agents also improved treatment response and tolerability of chemotherapy and ultimately overall survival. - ) . median age at recurrence was ( - ), and median interval to recurrence . months ( - ). ( %) patients died of recurrent disease and ( %) of other causes. site of recurrence and was local in , groin in and distant in . of the local recurrences were managed with wide local excision (wle) radical surgery ( radical excisions with skin flaps, posterior exenterations, anterior exenteration, total exenteration), wle and radiotherapy (rt)/chemort, chemo/chemort/rt, and palliation. groin recurrences were managed surgically ( adjuvant rt), rt alone, chemotherapy alone, and palliation. patients with distant metastases were managed surgically, with rt/chemo/chemort, and palliation. overall median survival after recurrence was months. median survival (months) by site of recurrence was: local , groin , distant , groin or distant . there was a significant difference in survival in local vs groin node recurrence (p< . ), local vs groin and distant (p< . ), and groin node vs distant (p . ). and years after recurrence survival rates were: local % and %, groin % and % distant , . conclusion patients with vscc remain at risk of recurrent disease therefore long term follow up these patients is essential. patients with local recurrence often have a good prognosis. outcomes for groin and distant recurrences are poor, but a proportion of those with groin recurrences can be salvaged. therefore early identification of local and groin recurrences is essential for improving outcomes in recurrent vscc particularly in cases with more conservative management of primary disease. introduction: endocrine disrupting chemicals (edcs) are environmental agents possessing hormone-like activity. exposure to edcs during differentiation can interfere with hormonal signaling, resulting in predisposition to some cancers. it has been suggested that some of these effects may be epigenetic, mediated by changes in dna and histone methylation. we hypothesized that edcs, diethylstilbestrol (des), and bisphenol-a (bpa), may act by altering the expression of dna and histone methyltransferases (dnmts and hmts). methods: ishikawa (endometrial) and mcf (breast) cells were cultured in steroid-free, phenol-free media with bpa ( m), des ( x - m) or vehicle for hours. pregnant cd- mice were treated with intraperitoneal injections of des ( mg/kg) or vehicle on days - of gestation. weeks after birth, offspring were sacrificed and tissue obtained. mrna was extracted, cdna was generated and quantitative real time rt-pcr was performed and normalized to -actin. all experiments were conducted in triplicate, repeated three times and compared using anova. results: dnmts (dnmt , a, and b) and hmts (mll and ezh ) were screened after in vitro exposure to edcs (table ) . ezh mrna expression was significantly increased in ishikawa cells after treatment with des or bpa. a similar response in ezh expression was seen in mcf cells treated with des or bpa. due to the consistent induction of ezh in all cells with either treatment, we examined the effect of des exposure on ezh expression in vivo. in adult mice exposed to des in utero, ezh expression was persistently increased ( . ± . fold, p< . ). conclusions: breast and uterine cell lines show increased expression of ezh in response to bpa or des exposure. this differential expression persists in the adult offspring of mice exposed to des in utero. ezh has been identified as a risk for neoplastic progression in the breast and for increased proliferation in uterine cancers. des induced ezh expression may be a mechanism for the increased incidence of breast and reproductive tract cancers seen in des exposed women. defects in cellular programs that control apoptosis lead to an imbalance of cell proliferation and cell death in ovarian cancer. recent evidence suggests that the use of some anti-inflammatory drugs decreases risk of ovarian cancer. natural curcumin-based anti-inflammatory therapies were shown to be beneficial in preclinical models of ovarian cancer (lin et al., ) . in this study, we examined the effects of plant derived curcumin on cell proliferation, apoptosis, and vegf expression in cultivated ovarian cancer cells. ovarian cancer igrov cells were cultured under standard conditions to study the effects of curcumin on cell kinetics and on vegf expression. cell proliferation was measured by srb and mtt assays. apoptosis was determined by measuring cytoplasmic histone-associated-dna-fragments (cell death detection elisa, roche, germany). vegf gene promoter-reporter activation and real-time quantitative reverse transcription polymerase chain reaction were used. conditioned media concentrations of vegf were measured with a commercially available enzyme-linked immunosorbent assay (quantikine, r d systems, minneapolis, mn). we observed that curcumin dose-dependently suppressed cell growth in igrov cancer cells (ic = um). treated cells showed a - fold increase in dna fragmentation compared to controls. curcumin also resulted in a significant decrease of vegfmrna expression and vegf protein secretion into the conditioned media in a dose-dependent manner. in this study, we have demonstrated that curcumin induces apoptosis, suppresses growth, and inhibits vegf gene and protein expression in an ovarian cancer cell line. experiments are underway to identify specific mechanism of curcumin action. curcumin may act as a chemosensitizing drug by potentiating the antitumor effects of standard treatments including taxols and platins in ovarian cancer. supported by the caldwell family foundation and the vesa w. and william j. hardman, jr. charitable foundation inc. daniel r christie, faheem m shaikh, john a lucas iii, susan l bellis. obsterics and gynecology, university of alabama at birmingham, birmingham, al, usa; physiology and biophysics, university of alabama at birmingham, birmingham, al, usa. introduction: previous work has shown that an upregulation of the enzyme st gal i, which is responsible for the , sialylation of integrins, confers a more tumorigenic/metastatic phenotype to colon carcinoma cell lines. it is not known, however, if this is unique to colon cancer, or if it is more broadly applicable to other forms of cancers. quantitatively increased expression of st gal i has been reported in ovarian cancers versus controls, but no biochemical or functional assays have been described to date. objective: because integrins are involved in cell adhesion and migration, and because metastasis of epithelial ovarian cancers is largely an intraperitoneal dissemination, we hypothesized that upregulation in the expression of st gal i in an ovarian cancer cell line would enhance binding with the extracellular matrix, increase invasiveness, and alter migration. methods: in the present study we forced a stable transduction of st gal i into the ov ovarian tumor cell line, which we found to be lacking the st gal i enzyme, establishing a parental (par), an empty lentiviral vector (ev), and an st gal i expressing (st ) cell line. a collagen i cell adhesion assay was performed and quantified by staining adherent cells and measuring absorbance. a haptotactic collagen i cell migration assay was performed by seeding cells in boyden chambers lined with collagen i concentration gradient, and quantified with cell staining and absorbance measurement. cell invasion through a reconstituted basement membrane (matrigel) was quantified as previously described for the cell migration assay. results: we were able to demonstrate successful creation of the ov -st gal i cell line by western blot analysis. functional assays demonstrated increased adhesion to collagen i (p < . ), increased haptotactic collagen i cell migration (p < . ), and increased invasiveness (p < . ) in the st cell line as compared to par and ev when analyzed by one-way anova. conclusion: this initial study into st gal i in ovarian cancer may have future therapeutic implications, and, in addition, lend greater insight into the intraperitoneal dissemination of disease.of microarrays (sam), arrayassist and binary tree structured vector quantization. differentially expressed genes were integrated with a database of known predicted protein-protein interactions (ophid) and key genes are being validated with real-time pcr and immunohistochemistry. results: unsupervised hierarchical clustering revealed collective clustering of all tumors, irrespective of their pathological classification. sam analysis has highlighted probe sets as differentially expressed between lmp and lmp-mp, probes between lmp and lgsc, and probes between lmp and lmp-mp+lgsc. no differential gene expression was detected between lmp-mp and lgsc. ophid analysis demonstrated gene members of the egfr and mapk / pathways to be differentially regulated between the non-invasive and the invasive tumors. to date, we have successfully validated members of the mapk / pathway-poly adp ribose polymerase (ppol) and traf family associated nf kappa b activator (tank)-using real-time pcr. conclusion: our data demonstrate that although the tumors have related genetic profiles, lmp-mp and lgsc are similar to each other and different from lmp, in keeping with their clinical behavior. members of the mapk / and egfr pathways appear to play a key role in low grade serous cancer. identification of novel genes associated with malignant transformation, may lead to development of more effective targeted therapy for lgsc. background: approximately % of pap smears with the ambiguous diagnosis of atypical squamous cells, cannot exclude high grade (asc-h), are negative for dysplasia in follow up colposcopic examination and biopsy. although hpv testing provides excellent negative predictive value (npv) ( %), the prevalence of high risk hpv infection is high in young women and the positive predictive value (ppv) in asc-h pap smears is no better than cytologic diagnosis alone ( %). recent studies have shown that immunostaining for p ink a , or proex tm c, supports a diagnosis of dysplasia in surgical biopsies. our objective was to determine whether staining for p or proexc provides sufficient predictive value to reliably distinguish high grade dysplasia in asc-h pap smears. design: we retrospectively collected samples from liquid based pap smears diagnosed as asc-h at either ohsu or portland oregon kaiser permanente ( - ). known hsil and negative pap cases were included as immunostaining controls. asc-h cases with followup cervical biopsies (n= ) were included for subsequent immunostaining according to manufacture's instructions. samples were also sequestered for hpv testing (pending). immunostained slides were scored as positive or negative by two independent cytopathologists (as and tm) while blinded to pap diagnoses and surgical biopsy outcomes. results: we observed excellent agreement between pathologists' scores (pairwise kappa statistic . ). the correlation between p and proexc scores was moderate (kappa . ). chi-square analysis comparing staining to biopsy outcome revealed a significant association between proex c positivity and cervical dysplasia (*** p< . jing lin, zhenmin lei, ch v rao. ob/gyn women health, university of louisville health sciences center, louisville, ky, usa. introduction: matrix metalloproteinases (mmps) are a family of highly homologous zinc-dependent endopeptidases, which degrade all kinds of extracellular matrix proteins. the degradation process is required for tissue growth and remodelling. these enzymes are probably involved in uterine growth and devolopment and its differentiated functions. ovarian steroid hormones, estradiol (e ) and progesterone (p ), are known to regulate some of the uterine mmps. since it is now known that lh/hcg can directly regulate uterine growth and devolopment and its functions, we questioned whether these gonadotropins could also regulate mmps in the uterus. methods: sixty day old wild type (wt) and lh receptor knockout (lhrko) mice and -day e and p treated -day old animals were used. in addition, primary cultures of uterine epithelial cells from wt animals were treated for hrs either with no hormone or with a single or a combination of pg/ml of e or p , ng/ml of hcg. then mmp- , - and - mrna levels were quantified by the semiquantitative rt-pcr. results: while the uterine mmp- mrna levels were unaffected, mmp- mrna levels significantly decreased and mmp- mrna levels significantly increased in lhrko animals as compared with wt siblings. e and p treatment reversed mmp- and mmp- changes in lhrko animals. treatment of wt type primary endometrial epithelial cell cultures with hcg had no effect on mmp- mrna levels, but it did increase mmp- mrna levels. this increase was synergistic with both e or p . conclusion: while lh/hcg do not regulate uterine mmp- and mmp- , they seem to co-regulate mmp- with ovarian steroid hormones. cancer. radhika gogoi, christine ardalan, dorota popiolek, leslie gold, john curtin, stephanie v blank, bhavana pothuri. new york university, new york, ny, usa. objective: megesterol, a synthetic progestin with strong androgenic properties, is used in the medical management of patients (pts) with atypical endometrial hyperplasia (aeh) or endometrial carcinoma (emca). our hypothesis was that androgen receptor (ar) expression in the endometrium of aeh and emca pts would correlate with degree of response to treatment. methods: pre-and post-treatment endometrial biopsy specimens were obtained from pts treated with megesterol for aeh or emca. ar expression was investigated by immunohistochemical (ihc) analysis with appropriate positive and negative controls. ihc staining was scored for intensity ( - ) and percentage of positive cells ( - ) in the glandular and stromal compartments by a pathologist, blinded to clinical response data. a composite score utilizing both intensity and percentage of positive cells was calculated. we evaluated pre-and post-treatment ar expression in responders and nonresponders as well as ar expression in emca, aeh and normal endometrium. the mann whitney u and the wilxocon signed rank tests were utilized for statistical analysis. results: eight pts' pre-and post-treatment samples were obtained; with emca and with aeh. three pts had no response, , a partial response and , complete responses. ar expression in emca samples when compared to the normal endometrium was significantly lower in both the glands (mean . vs. ; p< . ) and the stroma (mean . vs. ; p< . ). although there was no statistically significant difference in glandular ar expression in the pretreatment biopsies of responders compared to nonresponders (mean . vs. . ; p= . ), there was a significantly higher level of glandular ar expression in the post treatment biopsies of responders compared to non-responders (mean . vs. ; p< . ). furthermore, we noted a trend towards higher levels of glandular ar expression in the post-treatment versus the pre-treatment biopsies in the responders (mean . vs. . ; p= . ). we noted a significant decrease in ar expression in emca compared to the normal endometrium. increased ar expression after treatment in responders suggests an important role of the ar in pts treated conservatively with progestational therapy, and needs further prospective validation as a means to predict treatment response in these pts. objectives: study the correlation between adenomyosis and some potential non-surgical risk factors. objective: o -methylguanine-dna methyltransferase (mgmt) acts to repair dna damaged by alkylation of guanine residues. mgmt promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes. the loss of mgmt activity is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas. we sought to determine if mgmt promoter methylation plays a role in endometrial cancer. methods: one hundred and twenty primary endometrial cancers were analyzed for mgmt promoter methylation by combined bisulfite restriction analysis (cobra). the cohort included endometrioid endometrial cancers, endometrial tumors of adverse histologic type, and endometrial cancer cell lines. twenty one endometrioid and mixed endometrioid ovarian cancers were also analyzed. a subset of the primary tumors was analyzed for mgmt expression by immunohistochemistry. results: no mgmt promoter methylation was seen in the endometrial cancers evaluated or the endometrial cancer cell lines. one of the ovarian cancers showed methylation. immunohistochemistry for mgmt expression is ongoing. conclusion: mgmt promoter methylation is an infrequent event in endometrial cancer. mgmt expression in tumor cells and repair of alkylguanine residues could explain in part the limited response of endometrial tumors to alkylating chemotherapy. objective: uterine sarcomas ( % of gynecological malignancies) originate from uterine mesenchyma. patients with high-risk disease (i.e. high grade) or at advanced stages have poor -years overall survival (< - %). pelvic radiation and/or chemotherapy have not demonstrated to improve survival. identification of new therapies for this malignancy is a major goal. few in vitro models have been established to test therapeutic agents for uterine sarcoma. here we sought to establish a new human uterine sarcoma cell line and to test the effects of chemotherapeutic drugs: tnf-related apoptosis inducing ligand (trail) used alone or in combination. methods: tissue sample was obtained from a woman with uterine sarcoma undergoing hysterectomy. sarcoma cell lines were established using a published protocol for endometrial cancer. phenotypic characterization was made through the different passages ( to ) by western blot. levels of estrogen (er), progesterone (pr) and trail receptors were also studied (rt-pcr, w-b). cells were incubated with chemotherapy agents (cisplatin, paclitaxel and doxorubicin and trail) and cytotoxicity (mts assays) and apoptosis (flow cytometry, parp cleavage by w-b, and dna laddering) measured. results: human uterine sarcoma cell line was established from a highgrade uterine sarcoma. through the different passages the cell line remains expressing cytokeratin, vimentin, tissue factor, caveolin- and -actin. this cell line expresses low er and pr levels. trail receptors (r and r ) were also detectable (rt-pcr, w-b). cisplatin, paclitaxel and doxorubicin ( um for h) produced low cell cytotoxicity (< %). trail ( ng/ml for h) induced about % cell cytotoxicity. apoptosis was confirmed by parp cleavage. doxorubicin significantly enhances trail mediated cytotoxicity (up to %), this was demonstrated by a significant increase in the sub g /g region in the dna histogram. conclusions: we establish a human uterine sarcoma cell lines using protocols for endometrial cancer. more importantly, we demonstrated that doxorubicin enhances trail effect on this uterine sarcomas cell line. thus, this combination might be considered as a treatment for high-risk uterine sarcomas. (financial support fondecyt ). defining the tumor initiating capacity of cd + human endometrial cancer cells. anne m friel, petra a sergent, christine l cummings, rosemary foster, current data suggest rare subpopulations of cells with tumor initiating capabilities are a common feature of solid tumors. several investigators have recently identified cd , a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages, as a potential tumor initiating cell (tic) marker in solid tumors of the brain, colon and pancreas. in our efforts to investigate such a population in human endometrial cancers (enca), we developed an in vivo model that is based on serial transplants of tics from endometrial tumor explants. serial transplant experiments were initiated in nod/scid mice that were injected with primary human enca cells. a subset of cells derived from the generated tumor was subsequently transplanted into new recipients. tumors formed following serial transplantation retained the original histological phenotype of the primary enca, and the number of cells required to initiate tumor formation was reduced at each successive transplant stage suggesting an increase in the ratio of tics to non-tics. we exploited this model in our initial efforts to investigate the tumor initiating potential of cd -expressing enca cells. to evaluate the tumorigenic potential of cd + cells, the tumor initiation capacity of xenograft derived cd + and cd cells were compared following subcutaneous injection of each population into nod/scid mice. only the cd + fraction was tumorigenic consistent with the hypothesis that tumors are generated and maintained by a subpopulation of cells with phenotypically distinct profiles. we are further investigating the functional significance and characteristics of this fraction in vitro and in vivo. objectives. central issues in tumor biology are the understanding of factors that control tumor cell proliferation and the identification of extracellular matrix cues controlling the signaling transducing repertoire that make cancer cells proliferate and invade the host tissues. among these factors, small leucine-rich proteoglycans (srlps):decorin, fibromodulin, lumican, biglycan, are emerging as powerful modulators of angiogenesis and cell growth, by affecting several key elements including matrix assembly, growth factor binding, and receptor tyrosine kinase activity. recently has been demonstrated that srlps can act as a pan-erbb ligand and, in doing so, down-regulate the activity of one of the most potent oncogenic proteins, erbb , whose overexpression is linked to poor prognosis and increased cancer mortality in breast, ovary, and prostate. since srlps have not been previously investigated in the endometrial cancer biology, we investigated their role in the benign and malignant endometrial neoplasia. method. the sampling has been obtained in women (n= ; mean age ± . ) with endometrial hyperplasia (n= ), polyps(n= ), and cancer(n= ), during therapeutic and diagnostic procedures (hysterectomy, colpohysterectomy, hysteroscopy). physiological endometrial samples (n= ) were obtained from menopausal women (n= ; mean age ± . ), during procedures for others gynaecologic indications. immunohistochemestry was the biology technique used for the detection of srlps.results. in the physiological endometrial samples immunoreactivity for decorin, fibromodulin and biglycan was intense (+++), while it was weak in polyps and hyperplasia (+) and absent (-) in cancer. no significant difference in the staining of lumican between the physiological and pathological samples. conclusions. these results could provide a mechanism by which naturally occurring proteins normally synthesized by fibroblasts and smooth muscle cells, the two key components of the tumor stroma, may play a protective role in the genesis and progression of endometrial neoplasia counteracting the growth of neoplastic cells and suppressing tumor cell-mediated angiogenesis. although further studies are necessary to understand mechanisms whereby srlps might influence endometrial cell growth and survival, these molecules may represent potential target for pharmacological cancer therapy. myostatin is a member of the tgf-beta superfamily of protein and a wellknown inhibitor of skeletal muscle proliferation. the muscular component of the uterus is the myometrium, a tissue that regulates its mass in response to different physiologic conditions under the influence of steroids. we determined the expression of myostatin mrna in immortalized pregnant human myometrial (phm ) cells and we verified its biological activity. functional assays showed myostatin induced phosphorylation of smad- and reduction of proliferation of phm cells in a time and dose-dependent manner. to investigate the physiological relevance of our in vitro findings, the expression of myostatin in rat uterus was examined at various phases of the estrous cycle. for the first time we report that myostatin is expressed in rat uterus and that levels of myostatin mrna change during distinct phases of the estrus cycle. uterine levels of myostatin peaked during late estrous and were the lowest at pro-estrous. to further examine the role of steroids in myostatin regulation, we examined the effects of gonadal steroid administration in ovariectomized (ovx) rats. ovaryectomy increased myostatin expression compared to normal cycling controls. estrogen treatment strong decreased myostatin levels while progesterone produced less robust effects on myostatin expression. these findings taken together suggest an important role for myostatin in the regulation of myometrial functionality. her neu over-expression and pi kinase/akt pathway activation in paget's disease of the vulva. amy stenson, bita behjatnia, jaime shamonki, jianyu rao, amer karam, jonathan berek, oliver dorigo. ob/gyn and pathology, ucla, los angeles, ca, usa; ob/gyn, stanford university, palo alto, ca, usa. background: paget's disease of the vulva is rare with high recurrence rates. treatment of recurrent disease is challenging due to its extent and location. non-surgical approaches have limited clinical efficacy, obviating the need for novel therapies. in contrast to paget's of the breast with well-described overexpression of her neu, molecular features of vulvar paget's are poorly characterized. our objective was to study therapeutic targets in vulvar paget's, including the her neu protein and the phosphorylated oncoprotein akt (pakt). in addition, detailed clinical characteristics were correlated with molecular expression. methods: specimens with vulvar paget's disease were retrieved from ucla's department of pathology. protein expression was evaluated by immunohistochemistry on slides from paraffin embedded tissue using the hercep test (dako) for her neu expression and a standard protocol to assess expression of activated pakt. slides were scored by two independent pathologists based on a nominal scale of (negative) to (strongly positive). clinical data was retrieved via chart review. results: between and , patients with vulvar paget's were identified. median age was yrs ( - yrs). a family history of cancer was found in / ( %), / ( %) were smokers and / ( %) had a history of hormone use. intraepithelial lesions accounted for the majority ( / , %), while / ( %) demonstrated invasion and / ( %) were associated with underlying gi malignancy. / ( %) had at least one recurrence, with median time to recurrence months. so far, specimens were stained for her neu and pakt. overexpression of her neu was found in / ( %.) positive staining for pakt was evident in / ( %.) statistical analysis suggested a correlation between her neu and pakt expression. conclusions: our study demonstrates that overexpression of her neu and activation of the pi kinase/pakt pathway are important features of vulvar paget's disease. to the best of our knowledge, this is the largest series evaluating these molecular pathways in vulvar paget's. our data suggest that her neu and pakt may be important molecular targets for novel therapies using for example the monoclonal antibody trastuzumab directed against her neu, or a pi kinase pathway inhibitor like rapamycin. introduction: uterine leiomyomas are the most frequent tumor of the female reproductive tract and are the primary indication for hysterectomy in women worldwide. these tumors occur in up to % of adult women, and their prevalence is especially high in africa-american women. currently there is no effective and safe medical treatment for uterine fibroids and surgery is the main stay. epigallocatechin gallate (egcg) constitutes the main solid extract of green tea and is believed to contributes most of the antioxidant and antiinflammation capacity of green tea. egcg has been shown to have beneficial effects on prostate cancer and breast cancer by inducing apoptosis and inhibiting proliferation of cancer cells. in this study, we investigated the ability of egcg to inhibit proliferation of human leiomyoma cells (hlm) in vitro. methods: human immortalized leiomyoma cells were cultured at ºc in a humidified atmosphere of % co - % air in smbm medium supplied with %fbs, . % insulin, . % hfgf-b, . % ga- and . % hegf(lonza). the cells were plated at density of × cells/well in -well plates and grown under the same conditions above. the monolayer cultures at approximately % confluence were treated with various concentrations ( , . , . , , , and μm) of egcg (sigma) for days. a fluorometric assay using hoechst dye (sigma) for dna quantitation was conducted at the following designed time points, day , , , and post egcg treatment. the cells were lysed and dna content was determined using hoechst dye solution ( μg/ml). fluorescence was measured after excitation at nm and emission at nm. results: egcg exhibited marked anti-proliferation effect on the growth of hlm cells. compared with untreated control, the inhibitory effect of egcg on hlm cells was observed at μm and peaked at μm concentration. the difference was statistically significant (p = . ). evaluation of the mechanism of action of egcg is cuurently under investigation in the lab.conclusion: egcg significantly inhibited the proliferation of human leiomyoma cells in a dose-depended pattern. egcg may present a potential effective and safe medicinal treatment for uterine fibroids. objective: choriocarcinoma is an aggressive form of germ cell tumor that exhibits rapid growth with early metastases. choriocarcinomas autonomously secrete hcg which acts as an autocrine/paracrine growth factor in these cancers. we hypothesize that a novel hcg antagonist (hcg-ant) can limit tumor expansion by blocking hcg-induced expression of pro-invasive genes. we investigated if hcg-ant could alter rna expression of matrix metalloproteinases (mmp- and mmp- ), which facilitate basement membrane degradation and hence invasion, and metastin (kisspeptin), a strong suppressant of metastasis, in the choriocarcinoma cell line jeg- . design: in vitro experiments using the jeg- cell line. materials and methods: after plating jeg- cells in a well tray overnight in rpmi media containing % fbs, cells were washed twice with pbs and then cultured in ul of serum-free rpmi media containing one of four treatments: ) saline; ) hcg ( iu); ) hcg ( iu) plus hcg-ant ( iu); or ) hcg-ant ( iu). rna was extracted from each well using trizol and reverse transcribed using sensiscript (qiagen). the relative expression of mmp- , mmp- , and metastin mrna was quantified using sybr-green based real time pcr. the expression of the housekeeping gene hprt was used to normalize expression data. results: treatment of jeg- cells with hcg-ant vs. hcg alone reduced expression of mmp- ( . ± . vs. . ± . ) and . hcg-ant reduced mmp- and mmp- expression by % and %, respectively (p< . ). treatment with hcg-ant vs. hcg increased metastin expression ( . ± . vs. . ± . ). metastin expression was increased by % following hcg-ant treatment. conclusion: treatment of the jeg- choriocarcinoma cell line with hcg-ant reversed the increased expression of mmp- and mmp- following treatment with hcg. metastin expression was increased by hcg-ant. this data suggests that hcg antagonism is capable of altering gene expression thereby inhibiting invasion in a choriocarcinoma cell line. the role of hcg-ant as an adjuvant therapy in hcg sensitive tumors offers promise. retinoids, but not progesterone, directly induce differentiation and apoptosis of endometrial cancer cells. you-hong cheng, serdar e bulun. ob/gyn, northwestern university feinberg school of medicine, chicago, il, usa. objectives: the opposing actions of estrogen and progesterone during the menstrual cycle regulate endometrial proliferation, differentiation and secretion. the unopposed action of estrogen contributes to the development of type i endometrial cancer. however, the mechanisms for progesterone protection of estrogen-induced carcinogenesis in endometrium remain unclear. methods and results: in the current study, we demonstrated that retinoids ( -cis retinoic acid (ra) or all-trans ra (atra)) significantly inhibited basal and hormone-stimulated ishikawa cell proliferation by over % using mtt assay. the same experiment indicated that estrogen had no significant effect, whereas progesterone slightly induced, on cell proliferation. cell cycle analysis indicated that atra significantly increased the g /g cell population by % and decreased s phase cells by %, suggesting that ra induces cell cycle arrest at the s phase. knock-down of rar alone or both rar and rxr significantly increased proliferating cell nuclear antigen (pcna) levels in epithelial ishikawa cells, suggesting that ra signaling via rar/rxr activation is critical for normal endometrial growth and differentiation. to determine whether retinoids are naturally secreted by the endometrial stromal cells, we cultured primary stromal cells and analyzed the culture media using hplc. we found that retinol is the predominant retinoid form secreted by stromal cells. the average concentration of retinol in the cultured media of eutopic endometrial stromal cells was approximately to ng/ml/ cells (n= ). although there was less than . ng/ml/ cells of all-trans retinal or atra in the cultured media, we did find a small peak for all-trans retinal and atra in the media using hplc analysis. furthermore, progesterone significantly increased secreted retinol levels from eutopic endometrial stromal cells, but decreased retinol levels secreted from endometriotic stromal cells. retinol is a precursor for ra that is converted to retinal and then to ra. conclusions: we demonstrated that progesterone signaling via pr induces endometrial stromal cells to secrete paracrine retionids that in turn control the phenotype of adjacent epithelial cells. conversely, this interaction is disrupted in diseased endometrial tissues, such as endometrial cancer and endometriosis. the effects of hormonal contraceptives on antimullerian hormone by obesity status. anne z steiner, frank z stanczyk, stan patel, alison edelman. ob/gyn, university of north carolina, chapel hill, nc, usa; ob/gyn, usc keck school of medicine, los angeles, ca, usa; ob/gyn, oregon health and sciences university, portland, or, usa. background: antimullerian hormone (amh) is emerging as a predictor of reproductive potential. serum levels of fsh, a commonly used measure of ovarian reserve, are suppressed with the use of oral contraceptives (ocs) thereby limiting its use. the impact of ocs and on serum amh levels in normal and obese women is unknown. objective: to examine the impact of ocs on serum amh levels by obesity status in reproductive-age women. materials and methods: ovulatory women, ages - years, of normal (< kg/m ; n = ) and obese (> kg/m ; n = ) body mass index (bmi) received a low dose oc ( mcg ethinyl estradiol/ mcg levonorgestrel) for two cycles. serum was obtained at three time points: after days of active pills (cycle , day ), at the end of the -day hormone-free interval (cycle , day ), and during the first week of active pills in cycle (cycle , day , , or ). amh levels were quantified by elisa; fsh and lh levels were determined by chemiluminescent immunoassay. amh at the three time points was compared using repeated measures anova. models were used to assess the relationship between amh and cycle day by obesity status. amh and gonadotropin levels were compared using spearman's correlation. results: amh levels did not differ by oc cycle day (p anova = . ) or by active versus placebo pill use ( . ng/ml ± . vs. . ng/ml ± . , p= . ) among normal bmi women. among obese women, amh levels differed by oc cycle day (p anova = . ), but not by use of active or placebo pill ( . ng/ml ± . vs. . ng/ml ± . , p = . ). across the cycle, cv(standard deviation/mean) averaged . %± . in the obese and . %± . in the normal bmi women ( p= . ). modeling to determine differences in amh throughout the cycles based on obesity status showed a significant interaction (p = . ) and lower amh levels in the obese group (p< . ). among both bmi groups, serum amh and fsh levels did not correlate during active pills or after days of placebo pills. conclusions: in young, normal bmi women serum amh levels do not appear to fluctuate during oc use. among obese women, amh levels are lower and fluctuate significantly. these fluctuations do not appear to mirror changes in gonadotropins and may complicate clinical interpretation of amh. background: menopausal hormone therapy (ht) is a confusing topic for many clinicians and patients. objective: to assess comprehension of basic clinical trial features among prospective participants for the keeps trial, designed to study the effects of ht initiated within years of menopause on chd markers. methods: screening materials were provided giving an overview of study purpose, duration, medications, likelihood of receiving drug vs. placebo, ht related risks and side effects. at a subsequent interview, a -item questionnaire assessed the participant's level of comprehension. a score of % was adequate to complete the informed consent process. those scoring < % were re-counseled and retested. demographic variables determining the likelihood of an initial score > % were evaluated by univariate and multivariable analyses. results: % ( / ) scored > % on initial testing. all women scored > % after re-counseling and retesting. likelihood of an initial response > % correct was unrelated to age or time since menopause. ability to correctly respond was influenced by highest educational level attained. none of women whose furthest educational level was high school scored > % on initial testing, significantly less than those with a college education (p= . ). a higher proportion of college graduates ( / ) scored > % compared to those attaining further education ( / ) (p= . ). comprehension was greatest for study purpose and duration ( / and / correct responses respectively) and least for questions related to results of the whi hormone trial breast cancer and chd. that e alone was not associated with an increased risk of chd ( %) or breast cancer ( %) was poorly understood. conclusion: comprehension of the risks and benefits of ht by potential research subjects is low despite provision of reading materials prior to the informed consent process. (supported by the montefiore medical center/albert einstein college of medicine site for the kronos longevity research institute and k -hd to ns). variation in menopausal symptoms within a sample of hispanic women: swan, the study of women's health across the nation. robin r green, alex j polotsky, aileen p mcginn, carol a derby, rachel p wildman, lhasa ray, kavitha t ram, gerson weiss, nanette f santoro. albert einstein coll of med, bronx, ny; univ of med and dent of new jersey, newark, nj. background: menopausal symptoms are experienced by over % of women. purpose: to describe symptom frequency in a sample of midlife hispanic women from different countries of origin. methods: the study of women's health across the nation (swan) recruited women at baseline who self-identified as hispanic. their baseline responses to validated questionnaires regarding common menopausal symptomatology were examined. symptoms were reported over the previous two weeks and scored on a frequency scale ranging from (not at all) to (every day). for all analyses, symptoms were dichotomized into "absent" vs. "present" variables. responses were correlated with acculturation ( -item scale: marin, hisp j behav sci : , ) and analyzed by sub-ethnicities: central/south american (c/s am), puerto rican (pr), dominican (dr), and cuban (cu). associations between symptoms and sub-ethnicity were tested by chi-square. logistic regression was used to test for associations with acculturation and being us-born. there was significant variation in several menopausal symptoms. while puerto-rican women had the highest likelihood of reporting trouble sleeping (or= . , %ci: . - . ) and headaches (or= . , %ci: . - . ), dominican women were most likely to report vaginal dryness (or= . , %ci: . - . ) acculturation and being us-born did not explain the variation between subethnicities in any of the models tested conclusion: there appear to be significant differences among hispanic women with respect to menopausal symptomatology. these differences were not readily explained by measures of acculturation. (supported by the study of women's health across the nation (swan) has grant support national institutes of health, dhhs, through the national institute on aging, from the national institute of nursing research and the nih office of research on women's health (grants nr ; ag , ag , ag , ag , ag , ag , ag , ag and cd ). purpose: to compare the relative effects of conjugated equine estrogen, raloxifene, and tamoxifen therapies on cognition among women aged years or older. participants and methods: annual modified mini mental state ( ms) examinations were used to assess global cognitive function among the , women enrolled in the two randomized placebo-controlled clinical trials of the women s health initiative memory study (whims) and women enrolled in co-star, the cognitive substudy of the nsabp's study of tamoxifen and raloxifen (star) trial who had baseline ms testing. associations between baseline cognitive risk factors common to both trials and baseline ms scores were assessed and interactions used to examine whether risk factor relationships differed between cohorts. factors for which relationships were similar were used as covariates in analyses comparing ontrial ms scores. factors for which relationships did not appear to be similar were used to stratify analyses. results: compared to placebo, each of the active therapies was associated with a small mean relative deficit in ms scores of . units or less, which was fairly consistent between women with and without prior hysterectomy. overall, relative deficits appeared to be most marked for tamoxifen (unadjusted p= . ) but were also evident for raloxifene (p= . ) and cee (p= . ). conclusions: while unmeasured differences between trials may have confounded our analysis, these findings suggest that both tamoxifen and raloxifene may adversely affect cognitive function in older women. weight gain and increased abdominal fat have been found in women after menopause and is associated with higher levels of leptin, and decreased levels of the cardioprotective adipocytokine adiponectin. at the same time, bmd characteristically decreases. in an effort to determine the evolution and correlates of these changes, we studied postmenopausal women (pm) within years of menopause (age . ± yrs) of normal weight (bmi . ± ) and compared them to weight matched (bmi . ± ) premenopausal (pre) controls (age . ± yrs.) all subjects had bmd and body composition studies by dexa and measurements of leptin, adiponectin, visfatin and retinol-binding protein (rbp .) while total fat was similar in the groups, pm had more trunk and abdominal fat ( ± ; ± g) compared to pre ( ± g p< . ; ± g p< . ) pm also had greater %trunk fat and %central abdominal fat compared to pre, p< . .serum leptin ( . ± . vs ± pg/ml) and visfatin ( . ± vs. . ± . ng/ml) were similar but adiponectin ( ± vs. . ± g/ml) and rbp ( . ± vs. . ± ng/ml) were higher, p< . in pm. while in pre, abdominal fat correlated negatively with adiponectin (p< . ) in pm only leptin correlated with various parameters of fat mass, p< . , and adiponectin did not correlate but correlated positively with age (r . , p< . .) as expected, pm had reduced bmd at the lumbar spine and hip ( . ± . vs. . ± . g/cm ; . ± . vs. . ± . g/cm , p< . ) but there was a correlation between total and trunk fat in pm and lumbar bmd (r . , . , p< . ) but not with hip bmd; or any correlations in pre. there was a correlation between leptin and lumbar bmd in pm (r . , p< . ) but not in pre. in summary, in normal weight early pm, abdominal fat is increased, but only adiponectin and rbp are altered with an increase in the former correlating with age. lumbar bmd correlated with fat mass in pm and is partially explained by increases in leptin. it is suggested that in spite of increasing abdominal fat in normal weight early pm, (which correlates with a higher lumbar bmd) david d rahn, jesus f acevedo, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. objectives: matrix metalloprotease (mmp) activity is increased in the postpartum vagina of wild type (wt) animals, and this activity is accompanied by a burst in elastic fiber synthesis. the mechanisms that precipitate these changes are unclear. the goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. methods: nonpregnant ( ) and pregnant (d ) wt mice underwent either vaginal distention with a μl balloon x min (to simulate parturition) or sham procedure. tissues were obtained at and h for immunoblot analysis, zymography, and histology. distention was also performed in young fbln -/-, fbln +/-, and wts, and prolapse was quantified for weeks. results: distention resulted in marked increases in mmp activity in nonpregnant animals (prommp , . -fold; active mmp , . -fold; prommp , . -fold; active mmp , . -fold; all p < . compared with sham) which was accompanied by fragmented and disrupted elastic fibers in the vaginal wall. abundant amounts of tropoelastin and fibulin- in the nonpregnant vagina were not increased further by distention. in pregnant animals (which normally have suppressed vaginal wall fibulin- and tropoelastin), however, distention resulted in -fold upregulation of both proteins (p< . ). distention also induced increased mmps in pregnant animals (mmp- , . -fold; prommp- , . -fold; active mmp- , . -fold; all p < . ). thirteen young fbln -/-( - wks prior to age of prolapse development), het siblings, and age-matched wts underwent serial exams after ballooning. distention induced rapid increases in perineal bulge and vaginal diameter (within d) in fbln -/mice which never recovered. conclusions: in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. the finding that distention results in accelerated pelvic organ prolapse in fbln -/animals, but not wt, indicates that distention results in loss of pelvic organ support if elastic fiber synthesis is compromised. further, the data suggest that elastic fiber synthesis is crucial for recovery of the vaginal wall from parturition/distention-induced increases in vaginal protease activity. the molecular etiology of pelvic organ prolapse (pop) is complex and not yet well understood. defects in the connective tissue, such as a decrease in extracellular matrix (ecm) protein content may predispose women to pop. our objective was to study the expression and the enzymatic activity of matrix metalloproteinases (mmps) and their tissue inhibitor (timps) in vaginal tissue of patients with advanced pop and controls. after informed consent, pre-menopausal caucasian patients affected by pop ( grade by pop-q), and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer. western immunoblot analysis was performed (patients: n= , controls: n= ) to study the protein expression of mmp- , - , timp- , - , - . gelatin zymography was used to quantify the activity of mmp- and - . immunohistochemical analysis for timp- and - was performed on pfa-fixed vaginal biopsy tissue (n= ) in each group. both patient and control vaginal biopsy samples expressed latent and active forms of mmp- , and mmp- . the protein expression of the kda active form of mmp- was significantly increased in patients with pop compared to controls (p= . ). zymography detected the enzymatic activity of the proform and active form of both mmp- and mmp- . we found a significant increase in gelatinolytic activity of both kda pro-form and kda active forms of mmp- (p= . and p< . respectively) as well as kda active form of mmp- (p< . ) in patients with pop compared to controls. timp- protein expression in vaginal tissue showed a significant reduction in pop patients compared to controls (p= . ). timp- and - immunostaining were mainly localized in the smooth muscle cells at the muscularis layer of the vaginal biopsies. in vaginal tissue of patients with pop, we have shown a decrease in timp protein expression paralleled by an increase in mmp protein expression and activity. these findings reflect an active ecm remodelling that may compromise the structural integrity of the pelvic floor leading to pop. aberrant elastin and collagen synthesis may play a role in the pathogenesis of pelvic organ prolapse (pop). the lysyl oxidase (lox) family of enzymes direct cross linking of collagen and elastin polymers, however to-date no information is available on the expression and localization of these proteins in human vaginal tissue. our objectives were to study the expression and in situ localization of lox, loxl , loxl and loxl proteins in the vaginal tissue of patients with advanced pop and asymptomatic controls. pre-menopausal caucasian patients affected by pop ( grade by pop-q) and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer and western immunoblot analysis was performed (patients: n= , controls: n= ). pfa-fixed vaginal biopsy tissues (n= for each group) were used for immunohistochemical study. vaginal biopsy samples from both patient and control groups expressed all four members of lox family proteins: lox ( kda pro-form and kda active form), loxl ( kda pro-form and kda active form), loxl ( kda) and loxl ( kda). the expression of all lox family proteins was reduced in patients with pop compared to controls; however only the pro-form of lox of making the diagnosis of endometriosis from an endometrial biopsy. objective: to evaluate the diagnostic value of examining endometrial biopsy specimens for small nerve fibers in women with pelvic pain or infertility in a double-blinded prospective comparison with laparoscopy. methodology: we undertook to compare the detection of endometrial nerve fibers with laparoscopy and peritoneal biopsy for the diagnosis of endometriosis in women (aged . years; range - years) who presented with chronic pelvic pain or infertility. small nerve fibers were detected in the functional layer of endometrium using immuno-histochemical staining with the highly specific pan-neuronal marker pgp . , using a carefully validated technique and blinded assessment of nerve fiber density. laparoscopic assessment of the presence of endometriosis and peritoneal biopsies was undertaken by three experienced gynecologic endoscopic surgeons. data from these assessments were recorded independently of endometrial findings and maintained under blinded coding until the codes were broken. results: small nerve fibers were detected in all of the women in whom endometriosis was surgically diagnosed and in none of the women in whom endometriosis was excluded at laparoscopy, giving the specificity and sensitivity of %. the density of nerve fibers in the endometriosis cases was . per mm²± . (mean ± sd). conclusions: endometrial biopsy provided a reliability of detection or exclusion of endometriosis which was equal to that of diagnostic laparoscopy carried out by experienced gynecologic laparoscopists. background: erk / are mapk intracellular signaling proteins involved in cell survival and differentiation. endometriosis requires angiogenesis for ectopic implant growth. we hypothesized that the endometriotic peritoneal environment, known to be high in estrogen (e ), vegf, and cytokines such as il- and mcp- , stimulates angiogenesis in human endometrial endothelial cells (heec) through erk signaling. methods: serial sections from normal (n= ) and ectopic (n= ) endometrium were immunostained for total-(t-) and phospho-(p-) erk / and cd (an endothelial progenitor cell marker); results were quantified by computer densitometry and grouped by menstrual phase. cultured normal heec were treated with control, e ( - m), il- , mcp- , and vegf (all ng/ml) for min, and western blotted for p-/t-erk (n= ). heec were treated with peritoneal fluid (pf; diluted : in basal media) from normal (n= ) and endometriotic (n= ) women, with or without pd erk / inhibitor ( m) for h, and cell viability was analyzed by mtt assay. statistical analysis was done with one-way anova. results: tissue staining revealed that ectopic cd + endothelial progenitor cells undergoing angiogenesis (vessel sprouting and/or angiogenic cell cord formation) exhibited the strongest levels of p-erk. heec of ectopic foci showed moderate-high p-erk staining, while surrounding mesothelial capillaries were weak for p-erk. in normal endometrium, p-erk was cycledependent, with low levels in the late secretory phase vs. other phases (p< . ). p-erk of ectopic heec showed no cycle dependence, with moderate-high levels in all phases. t-erk in all tissues was high and invariable. in cultured heec, treatment with pf from endometriotic women significantly increased heec viability after h compared to normal pf (p< . ). this effect was abrogated by erk / inhibitor. among factors known to be high in endometriotic pf, vegf increased p-erk in cultured heec in and min (p< . ), while e , il- , and mcp- had no effect. conclusions: high p-erk found specifically in sprouting endothelial progenitor cells and focal ectopic capillaries suggests that erk / is involved in endometriotic angiogenesis. the peritoneal microenvironment of endometriosis may be persistently stimulating erk-mediated endometrial angiogenesis through vegf. further investigation into erk / inhibitors may offer a novel treatment of endometriosis. the events leading to the development of post operative adhesions are unknown, though recent observations emphasize the crucial role played by the superoxide ion generated by hypoxia. exposure of normal peritoneal fibroblasts to hypoxia caused the development of the adhesion phenotype, which is characterized by increased extracellular matrix molecules and inflammatory cytokines as well as high protein nitration and low nitric oxide. superoxide dismutases (sod) are a family of metalloenzymes that eliminates superoxide by converting it to hydrogen peroxide, protecting mammalian organisms against superoxide. objective: to determine whether sod is differentially expressed between normal peritoneal and adhesion fibroblasts and whether its expression is modulated by hypoxia. methods: fibroblasts from normal peritoneal and adhesion tissues were cultured under normal ( % o ) and hypoxia ( % o ) conditions for and hours. real time rt/pcr was utilized to measure the absolute mrna levels for sod in both cell lines before and after hypoxia exposure. results: normal peritoneal fibroblasts exhibited significantly higher basal mrna levels for sod ( . pg/ grna, p< . ) as compared to adhesion fibroblasts ( . pg/ grna, p< . ). short exposure to hypoxia resulted in a significant increase in sod mrna levels to . and pg/ grna in normal and adhesion fibroblasts respectively, p< . . in contrast, long exposure to hypoxia resulted in a significant decrease in sod mrna levels to . and . pg/ grna in normal peritoneal and adhesion fibroblasts respectively, p< . . conclusion: sod mrna levels are lower in adhesion as compared to normal fibroblasts, both basally and following short and longer exposure to hypoxia. reduced sod expression creates an milieu with greater free radical levels, which leads to the development of the adhesion phenotype. enhancement of sod levels and/or function are likely to be of benefit for reduction of postoperative adhesion development. objectives: development of endometriosis requires ectopic attachment and proliferation of endometrial tissue. the invasive process required to establish endometriosis may involve matrix metalloproteinases (mmps), including mmp- . recently, we have demonstrated that statins inhibit proliferation of endometrial stroma. this study evaluated the effects of simvastatin on a nude mouse model of endometriosis and on modulation of mmp- . methods: proliferative phase human endometrial biopsies were established as organ cultures or utilized for stromal cell isolation. to establish endometriosis in the nude mouse, endometrial tissues were maintained in nm estradiol (e) for hrs and subsequently injected intraperitoneally into ovariectomized nude mice. mice were treated with e ( μg, silastic capsule implants) and simvastatin ( - mg/kg/day) by gavage for days beginning day after tissue injection. control mice received e+vehicle. subsequently, animals were sacrificed and endometrial implants were evaluated. studies of endometrial stroma involved plating the cells in the presence of e ( nm) or e+medroxyprogesterone acetate (mpa; pm) with and without simvastatin ( - μm). some cultures additionally received interleukin- (il- , ng/ml). conditioned media was subjected to western analysis for expression of mmp- . results: in vivo studies demonstrated a dose-dependent inhibitory effect of simvastatin on number and volume of endometrial implants in mice. at the highest dose of simvastatin ( mg/kg/day), the number of endometrial implants was decreased by % and the volume by % in comparison to the control group. isolated stromal cells expressed abundant levels of mmp- following treatment with e, but minimal levels in cultures additionally receiving mpa or simvastatin. although il- induced a dramatic increase in mmp- secretion from cells pretreated with e alone, treatment with either mpa or simvastatin prevented this induction. cultures receiving e+mpa+simvastatin were the most resistant to mmp- induction by il- . objective: to increase awareness of potential presentations of adenomyosis and the differential of a uterine mass. material and method: in a tertiary medical center, a patient was being evaluated for a uterine cystic mass and cyclic dysmenorrhea. the patient is a year old nulliparous woman who has been complaining of cyclic dysmenorrhea for several years. the pain starts with the onset of menses and lasts around weeks. the patient did not improve on contraceptives. the patient had prior laparoscopy and imaging studies which misdiagnosed the mass as either a leiomyoma or an adnexal hemorrhagic cyst. the patient underwent exploratory laparotomy and resection of the mass. results: on ultrasound, the mass appeared as an echodense cyst within the uterus. intraoperatively, a cm thick-walled well circumscribed uterine chocolate cyst was identified. the resection of the cyst was performed in similarly to an ovarian cystectomy. tissue examination confirmed the diagnosis of cystic adenomyosis. following surgical intervention, the patient reported significant improvement of symptoms. conclusion: this case highlights an unusual presentation and treatment of adenomyosis and cyclic dysmenorrhea. cystic adenomyosis should be on the differential of uterine cystic mass, particularly in young women with cyclic dysmenorrhea and menorrhagia. the earlier misdiagnosis probably resulted from the unfamiliarity of physicians with this condition. similar clinical presentations may occur with congenital uterine anomalies (with an obstructed uterine segment) and cystic degeneration of a leiomyoma. the incidence and pathogenesis of cystic adenomyosis are unknown. oral contraceptives may be helpful as a first line therapy. however, resection of the adenomyotic cyst appears to be more effective, particularly in women seeking fertility. objective: it is hypothesized that abnormalities within the eutopic endometrium of females with endometriosis can cause the ectopic growth of the endometrial tissue at extrauterine sites. previous studies have shown that gene expression in eutopic endometrium of women with endometriosis is markedly different from disease-free females. inflammatory cytokines and receptor-dependent kinase pathways are widely recognized as therapeutic targets for immune disorders, which is believed to be the underlying pathogenesis of endometriosis. in this study we asked whether responses of primary eutopic endometrial cell cultures are dysregulated between females with or without endometriosis. methods: a total of biopsies of endometriotic eutopic endometrium (eee) and disease-free endometrium (dfe) were obtained from proliferative phase females. the primary cell cultures established from these biopsies were treated with tnfa to induce expression of inflammatory mediators. parallel cultures were also treated with kinase inhibitors of p , mek, pi k and ikk. after a period of or hours, concentrations of il- , mcp- , and gm-csf in cell culture supernatants were analyzed by elisa. results: in eee, basal concentrations of mcp- and gm-csf were - times higher, while il- was times higher compared to dfe. as expected, tnfa treatment stimulated higher levels of cytokine secretion in dfe mimicking disease state, however, the same treatment had almost no effect on eee. kinase inhibitors were very effective in lowering the cytokine levels of dfe, however, their effect on eee were less dramatic. conclusion: eee expresses higher levels of inflammatory cytokines under basal conditions, which is in concert with the current literature. our results validate that high il- levels in endometrium are diagnostic for females with disease. the increase of gm-csf, il- and mcp- following tnfa treatment was expected in dfe however; tnfa's effect was blunted in eee, which implies that eee are already highly activated. the effect of kinase inhibitors on cytokine production from eee was unaltered relative to dfe, which implies that tnfa stimulated kinase pathways are modified even in eee. endometrial cancer is the fourth leading cause of cancer in women, and hyperplasia and adenocarcinoma are more commonly seen in women with polycystic ovary syndrome (pcos). mig- , a negative inhibitor of egf signaling, is expressed in normal secretory phase endometrium and associated with hyperplasia in mig- knockout mice. objective: we examined and compared endometrium from normal and pcos women for mig- expression and characterized its regulation using in vivo and in vitro models. methods: immunohistochemistry (ihc) and real-time pcr were performed in endometrium from normal (n= ) women and pcos (n= ) women. regulation of mig- was studied in ishikawa and ecc- endometrial cell lines, treated with sex steroids or egf. endometrial xenografts from normal and pcos women were implanted in ovxd rag -(c) immunocompromised mice, treated with e or e + p pellets to mimic a natural cycle. results: mig- protein expression was low in the functionalis of the proliferative phase and high in the secretory phase; this pattern was reversed in the basalis layer. pcos secretory phase endometrium had significantly less mig- protein and mrna than normal endometrium (p < . ). xenografts using pcos samples had paradoxically elevated expression of mig- compared to normal, and appeared unresponsive to steroid treatments. mig- expression in endometrial cell lines was regulated by egf but not by ovarian steroids, e or e + p. conclusions: mig- expression is low in proliferating endometrium and regulated by egf. risk of hyperplasia or cancer in pcos women can be explained by altered expression of mig- . reduced expression mig- provides insight into endometrial function and may lead to better treatment options for disorders of endometrial proliferation including endometriosis, adenomyosis, endometrial hyperplasia and cancer. supported in part by nih-u -hd (sly), u hd (lcg) and u hd (fjd). could androgens influence human luteal cells function? anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. in pcos patients reproductive dysfunctions are frequently observed even if ovulation occurs. an impaired luteal function could partially explain this subfertility, since luteal steroidogenesis deficiency and premature luteal degeneration have been described in pcos patients. based on the frequent observation of high plasmatic levels of androgens in pcos, we investigated whether hyperandrogenism could negatively affect luteal function.to this aim, we tested the in vitro effects of androgens on progesterone (p) and on vascular endothelial growth factor (vegf) production by human luteal cells. indeed, vegf is essential for normal luteal development and function being an important regulator of angiogenesis and vascular permeability. since prostaglandins (pgs) play a central role in modulating luteal function, the influence of androgens on pge and pgf a secretion was also investigated. in order to investigate whether testosterone and androstenedione act directly or after local aromatisation, we tested the in vitro effects of estriol, estrone and --estradiol on p, vegf, pge and pgf secretion by human luteal cells. moreover, we tested the effects of testosterone and androstenedione in presence of exemestane -an aromatase inactivator. highly purified human luteal cells were cultured for h with medium alone (control) or in presence of increasing concentrations of testosterone or dihydrotestosterone or androstenedione (from - to - m) or in presence of increasing concentrations of estriol, estrone and --estradiol (from . to ng/ml). moreover, testosterone and androstenedione - m were tested in presence of exemestane (from to nm). in the culture medium vegf, p, pge and pgf secretion was assayed by commercially available elisa kits. in order to evaluate the influence of androgens and estrogens on vegf mrna expression on luteal cells real-time rt-pcr has been performed. our results demonstrated that testosterone, androstenedione and dihydrotestosterone were all able to significantly reduce vegf secretion in human luteal cells, while no effect was seen on vegf mrna expression. androgens were also able to significantly decrease p and pgf secretion, while an increase was observed on pge production. moreover our preliminary results demonstrated that in human isolated luteal cells estriol, estrone and -estradiol at all tested doses are able to mimic androgens effects on p and pge production. on the contrary estrogens were able to increased vegf release. estrogens seemed to have no effect on pgf  released. data regarding exemestane inhibition of testosterone and androstenedione are still in progress. increased levels of androgens were able to decrease luteal vegf, p and pgf release and might be involved in pcos-luteal phase defect. nevertheless, the observed effects could probably be attributed -at least in part -to estrogens locally produced via the aromatase enzyme, rather than be directly mediated by testosterone and androstenedione. the in vitro effect of proghrelin-derived peptides on purified human luteal cells. anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. ghrelin, well known mediator of neuroendocrine effects, has been demonstrated to have a role as signal for energy insufficiency. several evidences suggested that ghrelin might also operate as regulator of reproductive function. indeed, we recently demonstrated that both p and vegf released were significantly decreased by ghrelin in isolated human luteal cells. moreover ghrelin was also able to reduce prostaglandin (pg) e and increase pgf luteal cells release. in the present work we investigated whether two ghrelin-related peptides derived by the same ghrelin precursor (unacylated ghrelin and obestatin) might affect human isolated luteal cells function. conventionally regarded as an inert form of ghrelin, unacylated ghrelin has been recently proven as biologically active in specific cellular contexts. obestatin has been suggested to directly control porcine ovarian cell functions. in these assumptions, we investigated whether unacylated ghrelin and obestatin could directly affect luteal progesterone (p) release. moreover, since vascular endothelial growth factor (vegf) is known to play a critical role in luteal development and function, the influence of unacylated ghrelin and obestatin on luteal vegf, pgf and pge production was also analysed. highly purified human luteal cells were incubated for h with medium alone (control) or with hcg ( ng/ml) or with cocl ( μm) or in presence of increasing concentrations of unacylated ghrelin ( - nm) and obestatin ( - nm). in the culture medium vegf, pgf , pge and p secretion was assayed by commercially available elisa kits. moreover real-time rt-pcr has been performed in order to evaluate whether unacylated ghrelin and obestatin could affect vegf mrna in human luteal cells. our preliminary results demonstrated that either unacylated ghrelin or obestatin are able to negatively affect luteal steroidogenesis. moreover, both peptides seemed to increase the release of two luteotropic factors -vegf and pge and to reduce pgf release -a luteolytic prostanoid -from isolated human luteal cells. finally data regarding the expression of vegf mrna are still in progress. our results suggest that unacylated ghrelin and obestatin -two ghrelin-related peptides -could play a role in regulating luteal function affecting both luteal steroidogenesis and luteotropic/luteolytic imbalance. the mechanisms responsible for labor progression have yet to be fully elucidated. in a previous study, over-expression of small conductance calcium-activated k + channel isoform (sk ) in transgenic mice compromised parturition, suggesting a role for sk channels in this process. based on these findings, we hypothesized that sk channel expression is reduced late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. we investigated the effects of sk channel expression on gestation and parturition by comparing transgenic mice over-expressing mice sk (sk t/t ) mice to wild-type (wt). in wt mice, sk transcript and protein are significantly reduced during pregnancy. the force produced by uterine strips from sk t/t mice on the day of delivery was significantly less than wt or sk knockout control (sk dox ), and the contractile force reached wt levels by application of the sk channel inhibitor, apamin. moreover, lipopolysaccharide and ru , which induce pre-term labor in wt mice, failed to result in completion of delivery in sk t/t mice. thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when sk channel activity is in excess. the mechanism(s) down-regulating this channel in the uterus during pregnancy is unknown. the sk gene promoter region contains two specificity protein (sp) binding sites; ) sp , is a transcription factor known to enhance the transcription of genes in response to estrogen, and ) sp , which competes for the same binding motif as sp , reduces gene expression. sp protein expression in mice uteri dramatically decreases in late pregnancy, while sp protein level remains consistent. our data indicate that sk channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice. the changes in sk channel expression may be transcriptionally regulated by sp and sp . key: cord- -t x gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober ‐ , date: - - journal: transfusion doi: . /trf. sha: doc_id: cord_uid: t x gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks , , , and following index donations from donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by months. urine and saliva detection decreased significantly after weeks and was undetectable by months. of donors who were enrolled in the acute pre-seroconversion stage of infection % ( / ) developed multiple zikv related symptoms week post index donation, compared to only % ( / ) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* , yuelong guo , ritchard g. cable , joseph e. kiss , michael paul busch , grier page , stacy endres-dighe , steve kleinman , simone glynn , alan mast and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) . american red cross, rti international, american red cross blood services, blood systems inc., blood systems research institute, university of british columbia, nih/ nhlbi, blood research institute, nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over , donors were enrolled from us blood centers for ferritin testing. the study population was enriched for racial minorities [ african-american (aa), asian (as), hispanic (hisp)] and for "super donors" ( , who had completed donations in two years without low hemoglobin deferral). the minority donors and the remaining non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml). results/findings: across all subjects, % had ais and % had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed % % decreased risk for ais compared to nhw, while hisp donors had % higher risk. daily use of exogenous iron reduced risk for lf and ais by to %, respectively, while the estimated benefit from less-than-daily use was lower ( to % protection). regular use of antacids was associated with a % or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by % - %, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean ( sd) *p < . compared to batf / , ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to % of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d and d of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration ( % on d vs % on d of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized ( %) and macrophage-depleted ( %) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than % at h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at h posttransfusion. at h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* , amanda l richards and krystalyn e hudson . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod otii mice are predisposed to have autoreactive cd t cells. study design/method: four cohorts of hodxotii f mice ( - mice/ cohort) were bled monthly for months to assess for autoab production. peripheral rbcs were stained with anti-complement (c ) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd and ter to assess for the presence of rbc progenitors. statistical analysis between hod otii autoab vs. hod otii autoabvs. hod -otii was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd t cells were not deleted in the thymus of hod otii mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod otii . however, as they aged, - % of hod otii were positive for rbc autoab by months. thereafter, $ % of the autoab mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in of the cohorts, - % of autoab mice were female. hod otii autoab mice also had enlarged spleens compared to hod otii autoaband hod -otii mice ( . g vs. . g and . g, resp., p< . ). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd hi ter inter ) were observed in the spleens of hod otii autoab mice but not in hod otii autoaband hod -otii ( . % vs. . % and . % resp., p< . ). moreover, autoab and c deposition were found ( . - % and - %, resp.) on ter rbcs in all of the hod otii autoab mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b -a a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards , christopher a tormey and krystalyn e hudson* . background/case studies: red blood cell (rbc) alloimmunization occurs in up to % of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c bl/ (b ) mice were treated with pbs, or anti-ly g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at - hours post-transfusion. anti-hod alloantibody generation was assessed days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n ), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n ). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls ( / experiments, p< . ). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l ), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p< . ); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b -a b cxcr pd and ccr expressions characterize responders to rbc immunization benoît vingert* , , , marie tamagne , , , sadaf pakdaman , , , anoosha habibi , , , philippe bierling , , , , , rachid djoudi and france pirenne , , , . efs ile de france, laboratory of excellence gr-ex, imrb u -eq , ap-hp, universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd t cells have a th profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd t cells with a cxcr pd phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr pd hi profile, with a differentiated expression of ccr . ccr is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr and cxcr can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr pd lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr pd cells were compared in groups of transfused sickle cell patients : alloimmunized (n ) and non-alloimmunized patients (n ). the analysis was also performed in non-transfused healthy controls (n ). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr pd hi subpopulation expression was identical between transfused groups and controls. ccr and cxcr expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr expression was very strong independently of the expression of pd . in the aim to determine the help of the circulating cxcr pd cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr pd subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr pd cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr on circulating cxcr pd cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr pd profile and the ccr expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* , ashley bennett , kathryn girard-pierce , connie arthur , amanda mener , patty zerra , christopher a tormey , jeanne hendrickson and sean stowell . emory university, yale-new haven hospital, yale university, emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod kel). to examine the role of cd t cells, pic/kel primed b recipients were cd t cell depleted prior to transfusion. in addition, b recipients were adoptively transferred with cd t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < . ); pic/kel primed recipients transfused with (hod kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < . ) and transfer of cd t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < . ). conclusion: these results demonstrate that cd t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c bl/ ) mice or oti mice, whose cd t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after , , , , and hours and on days - . results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after hours and peaking at hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is - % versus > % in wt recipients at hours (p< . ), whereas transfusion of wt platelets into either oti or wt recipients is approximately % at hours after transfusion. adoptive transfer of oti cd t cells into wt mice recapitulates the effect, with significant mova platelet clearance at hours compared to wt platelet clearance (p< . ). conclusion: this work extends the ability of cd t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c bl/ mouse blood and treated with uv r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv r treated wbc-rich prp, or uv r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c bl/ donor blood. a second transfusion of untreated wbc-rich c bl/ prp was given weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p< . ) or necrotic (p< . ) wbcs, but not those given uv r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c bl/ wbcs were reduced in recipients of either uv r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so ) of venous blood is generally assumed to be around - % as measured from a central venous line. however, a recent investigation of so levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so distribution (mean . % . % [yoshida et al. ; blood transfusion , ] . the present study was undertaken to determine the distribution of so in lr-rcc produced at a medium-size blood center using a novel non-invasive so probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so levels. study design/method: the so from units of lr-rcc were examined on five consecutive days representing % of the collected units during the period at a regional blood center where all the units were processed at room temperature within hours of blood collection. so was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a u ; pendar technologies, cambridge ma). in addition to so , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n ) from human volunteers were stored in as- under normoxic, hyperoxic, or hypoxic conditions for up to days (so ranging from < to > %) prior to uhplc-ms metabolomics analyses in presence of c, n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so carried out non-invasively at a blood center yielded a similar wide distribution as previous study from units of lr-rcc procured and sampled invasively within hours after blood collection [yoshida ibid]. the shape of the so distribution appeared near normal with the mean of . % . %, median . %, range < % to > % and inter-quartile range (iqr) of . %- . %. male donors showed higher so compared to female donors (p< . ). no correlations were observed between so levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so levels ameliorate the energy and oxidative metabolic lesion. lower so levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so levels was observed from lr-rcc manufactured at a blood center using -hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a -fold increase in the absolute lymphocyte count (ko . . x /l vs. wt . . x /l, p . ), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko . . fl vs. wt . . fl, p . ). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd foxp regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd foxp tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd foxp tregs derived exosomes and their functions involved in cd tregs mediated immune-modulation were seldom reported. study design/method: cd t cells were freshly purified from pbmcs, cultured with anti-cd /cd antibody packaged beads and il- , and then polarized with tgf-b and rapamycin into cd foxp treg cells. the harvest cells were co-cultured with cd /cd beads stimulating cd cd effector cells in the transwell plate. the supernatant derived from cd tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd , cd , tsg and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir- , let- b, let- d were measured by qpcr. the precipitated exosomes were further purified by cd immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd treg cells could suppress the proliferation of effector cells with a small decline (p> . ), which means some non-contact factors involved in the cd treg mediated immune modulation. a total number of . . / cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle - nm in diameter ( . . nm by nta). cd and cd were expressed on these background/case studies: regulatory t cells (tregs), containing cd and cd subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd foxp regulatory t cells (ntregs) in inflammation conditions (including instability of foxp , conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd regulatory t cells in inflammation and transfusion. study design/method: human cd tregs were induced with tgf-b and rapamycin from cd t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd tregs when encountering with inflammation were test by foxp expression, th and th cells conversion in inflammations conditions (il tgf-b il il and il tgf-b il b il ) on day , day and day . in vivo, cd tregs were transfused into cia mice and then their survival in mice and foxp express were evaluated to reveal the stability of cd tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd tregs when induced factor tgf-b and rapamycin were removed by testing the foxp expression on day , day and day . results/finding: ex vivo induced human cd treg were foxp ( . . %) and did not secret il a (both in supernatant and % of cells). foxp express in cd tregs were maintained after induced factor tgf-b and rapamycin were removed on day , day and day . in vitro, foxp , il and ifn-c expression has no significant difference when compared with controlled tregs on day , day and day and did not secret il a when encounter with inflammation conditions (il tgf-b il il and il tgf-b il b il ). in vivo, cd treg cells were transfused into cia mice on the peak of disease onset ( days after the first collagen immunization, has inflammation condition in vivo) to test cd tregs survival. cd tregs were found in cia mice foot ( . . %), blood ( . . %) and spleen cells ( . . %) hours after transfusion and their % of foxp were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd tregs are stable in inflammation and transfusion and can maintain foxp expression when induced factor were removed, these make cd treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd /cd /cd expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p s k and its downstreanm proteins, especially the protein s , which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s related protein translation inhibition. xiaoyun fu* , , mikayla anderson and james c zimring , . bloodworksnw research institute, university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day (one day past their expiration). bioactive lipids including common fatty acids, oxylipins, and lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in stored rbc units. for example, arachidonic acid (aa) ranges from . - . mm, linoleic acid (la) ( . - mm), dihomo-c-linolenic acid (dgla) ( . - . mm), eicosapentaenoic acid (epa) ( . - . mm), docosahexaenoic acid (dha) ( . - . mm), and alpha-linolenic acid (ala) ( . - . mm). ten oxylipins including hetes, hodes, and dihomes, and lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of analytes quantified, showed a significant difference in concentration among different blood types by one-way anova testing (fdr< . ). the ab rh blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh ). the fold increase of o rh-/o rh among pufas ranges from . to . , suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna- was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna- targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps ( nm) surrounded by a thick fluorescent silica shell ( nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r rnaprobe . and r dnaprobe . ) with mirna- concentration, down to a -nm limit of detection. hybridization assays in % human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in % human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. . . a . . . . a . . . . a . . total atp,lm/ghb . . a . . . . a . . . . a . . extracellular lactate,mm a extracellular glucose,mm a a a extracellular na ,mm a extracellular k ,mm a a a a p< . , paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n ) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by % ( / , ) compared to the previous year without pathogen inactivation ( / , , p . , chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased ( / , vs / , ). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count ( . x ) than unaffected controls ( . x ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above . x . in vitro quality of single dose amotosalen/uva treated platelets in % plasma/ % pas- after days of storage crystal stanley , marguerite kelher , nero evero , melissa vongoetz , betsy donnelly and anna erickson* . belle bonfils memorial blood center, university of colorado, cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas- to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct ) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas- and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in % plasma/ % pas- , collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, . . platelets in ml, were collected on the trima apheresis platform in % plasma/ % pas- . a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n , were ml (t) and ml (c) with doses of . . (t) and . . (c). all pc were stored under the same conditions and evaluated on day and day for physical/metabolic characteristics. results/finding: on days and all t and c pc had ph c ! . . the dose recovery for t was % %. on day , t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table ) . conclusion: trima pc in % plasma/ % pas- treated with the inter-cept blood system for platelets using the sv set and stored for days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan , , yang yu , li-ping sun , shu-fang wang , rui wang , lei-ying zhang and deqing wang* . the department of blood transfusion, the pla general hospital, the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d , d and d . we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in % final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. levels of k and lactic acid (la) were tested using automatic biochemical analyzer. k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. treated hips-cms with d ssrbcs, d k and cell culture media for h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: d ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d ssrbcs stop beating, but beating patterns restored at h. hips-cms treated with d ssrbcs stop beating, and beating patterns did not restored at h. levels of k and la in ssrbcs changed most obviously. only d k solution made hips-cms stop beating and can restore in h; d k, d k and la solution did not influence the beating pattern in at the end of the treatment for h, hips-cms treated with d ssrbcs show obvious shrinkage. at the end of the treatments for h, cells treated with d k and d ssrbcs both show obvious shrinkage, the shrinkage in d ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. gene expression array results show a total of genes were differentially expressed in d ssrbcs group compared with naive group. there was no consistent separation within the d k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. further study should be applied to signal pathways on ssrbcs induced cytotoxicity. large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as- as compared to sagm. the presence of citrate in as- seems to be necessary to prevent hemolysis of thawed cells. during storage in as- , atp and , -dpg levels rapidly decline. recently developed additive solutions like pag m and as- have shown to better maintain , -dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag c in which the mannitol of pag m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at - c in pag c. study design/method: leukoreduced rcc (n ) in pag c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at - c. on day , rccs were glycerolized using acp (haemonetics v r , braintree, ma) to a final concentration of % (w/v), frozen and stored for at least two weeks at - c. after thawing and deglycerolization using acp , rcc were resuspended in pag c. during storage at - c, stability (hemolysis), atp and , -dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n ) resuspended in or sagm (n ). results/finding: pre-freeze storage in pag c resulted in increased , -dpg levels at day as compared to storage in sagm, resp. . . mmol/g hb and . . mmol/g hb. hemolysis during post-thaw storage in pag c remained below . % for days and was comparable with storage in as- . in sagm, hemolysis remained below . % for days. during the first weeks of post-thaw storage in pag c, both atp and , -dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag c showed significantly higher atp and , -dpg levels compared to as- or sagm. while in sagm and as- , , -dpg levels were undetectable after days post-thaw storage, in pag c, , -dpg levels only decreased to . lmol/g hb after days of storage. conclusion: pre-freeze storage in pag c resulted in increased , -dpg levels. as compared to as- , post-thaw storage in pag c showed comparable hemolysis while atp and , -dpg levels were much better maintained. based on a maximum allowed hemolysis of . % and an atp content of > . mmol/g hb, thawed rcc can be stored at - c for days in pag c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold ( c, c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n - ) in % iso using a trima or in % int using an amicus and stored for days at rt and c. samples were tested on day (baseline, bl), , , and of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at % hct. labeled blood was perfused through microfluidic channels (fluxion) coated with ug/ml type- collagen at s - shear rate. images were acquired every sec for min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p< . . results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day of storage compared to bl (bl: . . %, rt: . . %; p< . ). c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day (p . ) and compared to c-int by day (p< . ). conclusion: our work suggests that c storage of plts collected with a trima ap system in iso for up to days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at c. these results are surprising since both c-int and c-iso have been shown to express similar levels of cd p, pac- , and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table . comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after -week storage between unirradiated and irradiated groups (n ) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone ( mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin ( mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th cells and increase in cd regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th ,th ,tfh and tfr cd subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* , sarah kloss , sara crew and sandra j nance . american red cross, american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from plasma and serum clinical specimens. group contained a single hpa alloantibody specificity with or without hla antibodies (n ). group included specimens with hla antibodies alone and group consisted of patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa- , hpa- , hpa- , hpa- , hpa- , gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is % concordance observed for hpa- a, hpa- b and hpa- b antibodies. the pak-plus assay had difficulty discriminating hpa- b from hpa- a antibody when hpa- a antibody was present ( false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa- a when compared to hpa- b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within % of the cut-off for pakplus and < . adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut overexpression on cell motility. results/findings: fut overexpression increases both ley expression and cell migration, while fut knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac . as tumor promoter, hdac becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n ) and stored at c for up to days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means sem, and paired student's t-tests were used to determine statistical significance (p< . ). results/finding: on day , p-selectin levels were significantly higher in pre than bl (p . ). mirasol treatment caused a significant increase in pac- expression compared to pre (pre: . . %, post: . . %; p . ), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post- samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after day storage at c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day c-stored pas plts followed by incubation ( minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* , arthur p. bode , anne s hale , michael stanton , mark johnson and g. michael fitzpatrick . cellphire, bodevet, inc, cellphire, background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to , , and . % of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control ( -day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to % of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of % and % of the tcpc produced a significant decrease in blood loss. the lcps at % and % tcpc were as effective in mitigating blood loss as -day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to % of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of % and % of the tcpc reduced blood loss. these results suggest a starting dose above . % tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as % tcpc had similar efficacy signals as % tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso c. the study on pcr-ssp technique for the genotyping of cd - del.ac mutation and the genetic polymorphism of cd - del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd (platelet glycoprotein iv, scarb ) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd deficiency in china. cd gene mutation is the main reason that leads to cd deficiency. cd - del.ac (frameshift at aa ) mutation is one of the cd mutations that causes cd deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in pediatric patients who received both doses of the mmr vaccine at and months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are ! % for all mmr components. results/finding: table shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged . years ( . to . years). thirteen patients ( %) were chronically transfused at the time of serology. twenty-three patients ( %) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper , , franklyn cladis , richard saladino , darrell triulzi , barbara a gaines and mark yazer* . university of pittsburgh, children's hospital of pittsburgh of upmc, institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients ! years old and ! kg with evidence of hemorrhagic shock were eligible to receive up to cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (< ) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately months, trauma patients received wb: group o and group a recipients, % male, median (iqr) age was ( . - ) and % blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of ( - ) and % mortality rate. the median (iqr) quantity of wb transfused to group o recipients was . ( . - . ) ml/kg versus . ( - ) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean standard deviation haptoglobin concentrations for non-group o recipients was . . mg/dl on day , . . mg/dl on day , and . . mg/dl on day ; the corresponding haptoglobin concentrations for group o recipients were . . mg/dl, . . mg/dl, and . . mg/dl, respectively (p> . for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n ) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n ). the mean standard deviation platelet volume administered was cc for whole blood recipients versus cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) with anti-human igg only, and a positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a minute c incubation, followed by automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer and the breast milk showed anti-d with a titer between and . the patient had a consistent plasma anti-d titer of . the patient's mother chose to stop breast feeding after weeks, and the patient's hemoglobin was improved at and weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that % of c scd patients from the west indies and west and central africa are partial c and at ( %) risk for alloimmunization to the c antigen through transfusion of c rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce( g, t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce( - )-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of patients with genotype/rh phenotype data available, ( . %) were c antigen positive serologically. the allele frequency of rhce*ce( g, t) was . . in total, ( . %) patients possessed rhce*ce( g, t) in the absence of conventional c gene in trans. of the c antigen positive patients, individuals ( . %) were predicted to be partial c based on four molecular profiles [rhce*ce( g, t)/rhce*ce: ; rhce*ce( g, t)/rh*ce: ; rhce*ce( g, t)/rh*ce( g): ; rhce*ce( g, t)/rh*ce( g, t): ]. in these partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after transfusion exposures ( c-antigen negative units; mean: , range: - ), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce( g, t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* , megan l nguyen , melanie c proctor , david e krysztof , gregory a foster , erin k sash , sandy s dickson , joua yang , jeffrey m linnen , kui gao , jaye p brodsky and susan l stramer . american red cross, grifols diagnostic solutions inc., grifols diagnostic solutions, inc, quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on / / (fl, ga, sc, ms, al). following revised guidance on / / , testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on / / . travel history questions were discontinued on / / . confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of / / , , , donations were tested including , ( %) in , mps. no reactive donations were identified by mp-nat. of the , , id-nat donations, were initial reactive (ir) of which ( %) confirmed positive (cp) by subsequent testing (cp rate of : , ; positive predictive value of %; specificity of . %). five ( %) cp donations were id-nat repeat reactive (rr); ( %) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and in fl, of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from to days prior to donation. two donors with a travel risk reported clinical symptoms; cp donors ( %) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than copies (c)/ml to about Ê c/ml. at the time of writing, the longest period of detection in rbcs was days vs. days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from ir and all rr donors, ranging from to c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of were prepared by diluting nat plasma : and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n ) were sorted into categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm /high vl; igm /low vl. results/finding: of , donations collected april -december , were reactive for zikv rna. igm-index donations had higher vls (mean . x vs . x iu/ml) and higher proportions of simulated mp-detectable results ( % vs %) than igm donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm donations increased (table ) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r / systems lisa lee pate* , phillip c williamson , michael paul busch , susan rossmann , scott jones , ann butcher , john duncan , jean stanley and susan a galel . roche molecular systems, inc., creative testing solutions, blood systems research institute, gulf coast regional blood center -sugar land, qualtex laboratories background/case studies: in february , the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march , and testing of puerto rico donations began on april , . as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august , the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april , -february , using the investigational cobasv r zika for use on the cobas v r / systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a : dilution to simulate mini-pool testing. results/findings: a total of , , blood donations were screened using cobasv r zika. of ir donations, were repeat reactive (rr), non-rr and had no repeat testing. of the rr donations, were positive by altnat; of these were igm positive. all altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the rr donors that were not igm positive on index, enrolled in follow-up and all seroconverted. of non-rr donations, were altnat negative and is pending supplemental testing. / donors were igm positive on index. donors were igm negative on index; / enrolled in follow-up; remained igm negative and was gm inconclusive. of donations without repeat testing results, met criteria for positive ( was altnat positive, igm negative and altnat negative, igm positive). donation is pending additional testing. altogether, / ir donations met the criteria for true positive on the index donation. / ( %) true positive donations were reactive when retested in a simulated minipool. / were igm positive. conclusion: . % of the , , donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* , marc germain , gilles delage , maria esther lopes and yves gr egoire . hemorio, hemaquebec, h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia ( ) , and in brazil ( / ), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since % of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january st , through november, th , , from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: days, with % of the values lower than days); % of infected donors with symptoms lasting days; . donation/donor/year for wb and . for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x ( -proportion of refused donors) x ( proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain : , , for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from donations). the residual volume of mp plasma, . - . ml, is routinely discarded. beginning in april each blood center saved $ mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april - ) mp were combined into pools of donations; thereafter mp were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and % confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity ( % limit of detection < copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first months of samples is complete for , mp, comprised of , donations collected from april to october , . a total of pools were positive, with detected between april-june . the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over . % of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over . % of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in , zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* , sze sze chua , mars stone , michael paul busch and ai leen ang . health sciences authority, blood services group, blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on august . the numbers rose rapidly to cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since october . zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using blinded frozen samples consisting of replicates of half log dilutions of the who international standard for zikv and replicates of negative controls prepared by bsri. probit analysis was performed to determine the % and % limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a member blinded zikv reference panel from the usa-fda. results/finding: a total of , donations were screened from october to march , with false positive case and zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of . x copies/ml. zika igm was negative in the index donation sample but present in the -day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be . copies/ml at % lod and . copies/ml at % lod. the procleix zikv assay detected rna in out of patient samples and provided . % agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of in , donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types , and express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to reactivity on initial gel testing. if genotyping demonstrated weak d types , or , the intent was to manage the patient as rhd-positive. if weak d types , or were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in to weeks. results/finding: rhdgenotyping was performed on patient samples over months. of these patient samples, ( %) were weak d types or . the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types and patients have not received transfusions at this institution since they were genotyped. four of obstetric weak d types and patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this month study period serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using fda approved anti-d reagents. when reactivity with all reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of months we tested rhd-negative blood donors. there were ( . %) partial-d, weak d ( . %), and ( . %) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs - _ deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these donors showed that rhd-negative recipients received rbcs from of these donors. five of these recipients underwent antibody screening after an average follow-up period of months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which % of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d " with reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october to march , we performed routine d typing (neo, immucor) on obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of using at least antibody. solid phase and manual testing used the series and series reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh ) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all samples. two of ( . %) were d with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs - c, rs ; ivs c, rs ; and ivs a, rs ). two ( . %) were d and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four ( . %) patients had rhd alleles with known potential to make anti-d (rhd*dol , rhd*dar . , and with weak d type . ). one had weak d type , which has uncertain susceptibility to alloimmunization and one was weak d type , which has not yet been associated with anti-d. interestingly, two ( . %) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type is a variant of the rhd protein that comprises an amino acid substitution located in the th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c. g>c which is the first nucleotide of the exon of the rhd gene and thus could be implicated in exon skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v (agilent) and the nextseq platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of patients previously characterized by beadchip technology. interestingly, out of carry the c. - c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last patients, one has been previously characterized as rhd weak type carrying the c. g>c (p.gly ala). independently, sanger sequencing on unrelated rhd weak type samples pinpoint to a linkage disequilibrium between c. g>c (exac, maf . ) and the c. - c>t (exac, maf . ). in silico analysis of both mutation located close to the splice acceptor site of the exon does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon , mutant rhd c. g>c, mutant rhd c. - c>t and double rhd mutants c. g>c plus c. - c>t, we showed no influence on skipping of exon due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type between ala (transmembrane helix ) and val (transmembrane helix ) hampering membrane insertion. conclusion: the c. - c>t variation is always associated in cis with the missense mutation c. g>c on the allele rhd weak type . the c. - c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type red blood cells is due to the substitution of alanine at amino acid position to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has transmembrane (tm) and tilted ureapore a-helices, a long extracellular connector segment, and cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p. . we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and - aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, ) . results/finding: seven snmvs located within amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all at the exofacial ends (p.a t, p.w r, p.v d) are jk-weak; the two jkneg exceptions p.g e and p.g e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v m, p.e k, p.v i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the jk-neg variants are within aa (p. -p. ) of jk a/b at p. . none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n s and p.s p are adjacent to p. f and p. l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc a gene, which encodes the urea transporter ut-b . the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in . in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc a gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within aa from tm a-helix end v i, a t, w r, w r, g e, g e, v d* cytoplasmic n-terminal v m, g s, e k, l p in membrane tm and urea-pore a-helices r w, r q, g d, i t, a v, l r, a t ‡, a a §, l f, n s, s p, t m / * second nucleotide variant in this allele is synonymous (p.p p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a mbp region in q . - . with an lod score of . . using deep sequencing, we identified a potential deleterious mutation in the znf gene, which deletes bp resulting in loss of an entire zing finger domain. the identical del -znf mutation is present in all affected individuals, and is absent from all controls tested (n> ). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf del . none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc a gene, is a urea transporter that has been associated with renal function, we found that people with the znf del in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf , prevalent in southern spain due to a founder mutation, leads to ut-b dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-( -ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis( -ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at days and year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the pools included group a, group o and group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than - c within hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day (pool), day , and year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day , day , and year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp . ppm; mehp . ppm; deht . ppm; and meht . ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than % of its initial value. plasma stored in deht bags had an average plasticizer content % lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at g to separate rbcs from platelet-rich plasma (prp). prp was diluted -fold in pipes-saline with . mm pge and centrifuged at g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of - % and , - , platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted : (spdp %) with plasma from a patient with type vw disease (t vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of s - for seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was % (spdp/ffp > . ). results/finding: six batches of spdp/ffp were evaluated using subjects. there was no statistical difference between the spdp/ffp pairs (p . ). the mean ratio of spdp/ffp was . with a % ci of . - . . comparing spdp vs. spdp %, there was no difference (median ratio . , range: . - . ) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was % greater than in samples reconstituted with ffp. the lower limit of the th % ci is a difference of %, which is less than the a priori determined margin of noninferiority of %. even with % dilution with t vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* , qiyong peter liu , grantham c. peltier , ryan c. carney , ashley s. taylor , colby s. mcintosh , james a. bynum and andrew p cap . u.s. army institute of surgical research, velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: ( ) ffp; ( ) ffp with mm glycine; ( ) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; ( ) spdp pretreated with glycine-hcl ( mm); and ( ) spdp pretreated with glycine-hcl:glycine ( mm: mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood ( % hct with platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < . ). fibrin polymerization density was slightly diminished in rspdp vs. ffp ( . vs. . o.d., p < . ), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < . ). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < . ) and an additional twofold in pretreated spdps vs. rspdp (p < . ). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < . ). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples ( . % surface coverage vs. . - . %, p < . ). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* , anita tuip-de boer , ruqayyah almizraq , jason p. acker , philip j. norris , jennifer a muszynski and nicole juffermans . academic medical center, university of alberta, canadian blood services, blood systems research institute, nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from donors (blood type a or b). supernatants were prepared after - (fresh) and - days of storage (stored) for measurement of thrombin generation and ev analysis. a type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to % stretch using a cellstretcher. control cells were not stretched. after hours, il- and il- production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il- and il- production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p< . ) . incubation of stretched cells with stored wbf products resulted in higher il- production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by different methods from individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after - days (fresh) and - days (expiry). monocytes were co-cultured in media plus % rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table ) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin- production was higher after exposure to fresh wbf ( % control, p . ) or wbd at expiry ( % control, p . ). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* , alessandro tocchio , anita howell , kaushik sridhar , jason p. acker and utkan demirci . stanford university, canadian blood services, centre for innovation, background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of - g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at , , , , and days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from volunteers with four different age and sex categories (male, - years; male, > years; female, - years; female, > years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young ( . g/ml) and older female donors ( . g/ml) (p < . ). moreover, rbcs from young males ( . g/ml) were significantly less dense compared to rbcs profiled from older female donors ( . g/ml) (p < . ). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il- (pg/ml) il- (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p< . ). substantial correlations were also found between orp and free hemoglobin (p< . ) and orp and free heme (p< . ). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections vs (p< . ). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than days compared to rbcs stored for days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of to days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to days storage duration -reference group), medium age (at least rbc of - days storage), and oldest (at least rbc greater than days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every days, and b) a finer partition using cut-points every days. results/finding: , patients receiving , rbcs were included in the analysis. exposure to rbcs stored for more than days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for days or less) after adjusting for several fixed and time-dependent potential confounders (hr . ; % ci: . , . ; p . ). exposure to blood stored for at most - days yielded a similar hazard ratio (hr . ; % ci: . , . ; p . ). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than days compared to exclusive exposure to rbcs stored days or less was not significant (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . similar findings were obtained with partitioning exposure data into day intervals where exposure to rbcs stored for - days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for - days (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . conclusion: individuals exposed to rbcs stored for more than days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* , cynthia walser , tatsuro yoshida , andrew dunham and pedro cabrales . university of california san diego, new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o saturation < %) stored rbcs, or anaerobic/hypercapnic (o saturation < % and pco (@ c) $ mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as- after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either ) conventional; ) anaerobic; or ) anaerobic/hypercapnic conditions. rats ( - g) were hemorrhaged to % of blood volume, held in hypovolemia for minutes, and resuscitated to recover blood pressure to % pre-hemorrhage with prbc stored for either or weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. ( %) neg patient showed short term response and ( %) patients showed progressive disease. at the neg group standard eval ( %) patient showed response and ( %) had progressive disease. ( %) neg patient had long term response compared to ( %) pos patients. at the pos short term eval ( %) patients showed response and ( %) patients had progressive disease. at the pos group standard eval, ( %) patients showed response and ( %) patients had progressive disease. overall, ( %) pos patients responded compared to ( %) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd neutralizing substance could play a role in treatment response. alternatively, reduced cd expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a -hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate % replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of . m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* , laurie sutor , , germ an leparc , marjorie doty and william crews . carter bloodcare, ut southwestern medical center, oneblood background/case studies: anti-cd drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with . m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and . m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual ( th edition). each of the plasma aliquots was further separated into aliquots and stored at - c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. ( ) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than with the untreated or dtt-treated cells during the study. conclusion: long term storage of . m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque- ), pooled, suspended in cryopreservation media ( % dmso; : ) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips ( c, % co , h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o ) rbcs sensitized with either anti-d (positive control), anti-scianna- (sc ) or anti-anwj or lipopolysaccharide stimulated for h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/ monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed . % viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il- b, il- , il- , mip-a (p < . ), mip-b and gro (p < . ) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc -and anwj-sensitised rbcs resulted in a pi of . % and . . % respectively vs anti-d sensitized rbcs (pi: . %). a weak ( ) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi> %). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in patients, involving antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all patients had - positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients and typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c by hea precise-type. ega-treated rbcs gave reactions with the same anti-c reagent. patient rbcs gave variable reactivity (vw- ) with bio-rad seraclone and ortho bioclone anti-c. patient rbcs gave reactivity with all anti-c reagents when incubated for the maximum incubation time allowed. patient rbcs were jk(a ) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients and tested s with bio-rad seraclone anti-s ( - ), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat rbcs but not all manufacturers include reagent limitations regarding testing of dat rbcs. we describe cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and cases with false positive tests with anti-s (n ) and anti-jk a (n ) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with - positive dat and supports testing to dissociate igg from rbcs strongly dat before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/ opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r : prob(ab|al-loexp), so that prob(ab) prob(ab|alloexp)*prob(alloexp) and r ; rewriting prob(alloexp) prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar months before ( / - / ) and months after ( / - / ) was selected; for state b, a similar months before ( / - / ) and months after ( / - / ) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (< months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a -and -fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from to (state a) and to , (state b), which annualized, represents a potential gain of , (state a) and , (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from - , there were donors identified who had changed their gender from their birth gender; female donors changed their gender to male and male donors changed their gender to female. there were ( %) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to . ml/kg or % of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the % limit. variable volume scales [vvs] can be programmed to vary unit volume (up to ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by ml at ebvs < . l in donors ! yo, but increase by - ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the mos. before a mo. phased implementation of the vvs, and the subsequent mos. multivariable analysis [mva] by -mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods & , continued during impl and post-impl periods & , returning to the baseline rate in post-impl periods & (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods & . the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of . ml during post-impl periods & from the temporally matched baseline & pre-impl period . conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* and yves gr egoire . hema-quebec, h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, . the vaccine is produced with live and attenuated yfv, which can circulate for at least weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received , blood donors candidates; from those, , were accepted as a blood donor, after medical interview. the deferral rate was . %. at the same period of the year , there were , prospective donors, and blood donations. the deferral rate was . %. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, , additional donors, compared to same dates. that represents a . % increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from . % in to . % in . conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of ! . x is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: , apheresis collections from centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of . x for single (s), . x for double (d), and . x for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or % plasma) assuming i) a minimum dose (allowing for production loss) of . x for s and . x for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as %) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc rbc plt/p plt plt/rbc/p plt/rbc plt plt/rbc plt/p #donations citrate exposure (mls) - study design/method: a randomized ( : ), placebo-controlled, single blind, subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into cohorts, receiving increasing doses, ranging from / , - / of the lowest effective dose found in the above rabbit model. cohorts and received the / th dose, but cohort received two / th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for hrs post infusion and followed for up to days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of aes: were treatment emergent (teae), of which were treatment-related ( thrombosomes and control). all teaes were mild or moderate in severity. in cohorts and , / thrombosomes subjects had treatment related adverse events. one cohort subject developed an upper respiratory infection and elevated wbcs within hours post infusion, which resolved by hours, and an elevated d-dimer at hours post infusion, which resolved by day . this subject also had an elevation of prothrombin fragment at baseline, which increased post transfusion and peaked at hours with resolution by day . one cohort subject developed non-specific t-wave changes at and hours following her nd infusion that resolved by day without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort subject developed an igg platelet autoantibody on days - , which was undetectable on days - ; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days - , and negative on days - . background/case studies: cryopreservation of platelets (plts) could extend the shelf life from - days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with % dmso and stored at c. after thawing, the unit was reconstituted in thawed ffp spiked with either lm puromycin (pm) or nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after , and hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x- containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by -fold during hour storage. immunoblot analyses of the plts showed a -and -fold increase in pm incorporation after and hours of storage, respectively. massspectrometry revealed unique proteins that were synthesized after hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac , rap and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac , rap and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in , the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r - cooler with units of ffp, units of rbcs, and unit of whole blood. three to ml of platelets were collected via syringe from each unit at min (before storage in cooler or refrigerator) and after . , , , , and hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac- binding) were measured by coulter counter, channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p< . deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c -a h molecular sieving: beyond genotyping ghazala hashmi , reinhard klemm and michael seul* , . biomolecular analytics, immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi http://bit.ly/ ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, * * samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for sensitized sickle cell anemia ("sca") patients (tb in cas-tro , http://bit.ly/ oplxhr, excluding le and e(variant) and assuming request per patient), presenting with up to allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only = plate holding * candidate units from actual black donors, followed by profiling of samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for of requests ( . %), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining pooled samples produced additional assignments to a second set of requests, for a total of assignments from only wells. in another scenario, sieving of a full plate of * samples, produced $ assignments for two successive batches of requests from sca patients, a yield exceeding . x. sieving alone typically fills - % of requests of moderate complexity ( ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez , monica kalvelage , ghazala hashmi* and michael seul . biomolecular analytics, lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou http://bit. ly/ ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including at the rhce locus) that encode mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag " (e.g. e ,c ,e ,c ) to specific combinations of "ag " (e.g. c e k fya and c e jsa ) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the c samples, that were also v and vs and, among the e samples, that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select specific "ee" pools of which were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b ( pools), co a|b ( ) and others. conclusion: molecularsieving of a single -well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c , e and jsa . these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam (landsteiner-wiener) and ackr (duffy). for longer genes, such as abo of > kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of > kb each was used for all physically confirmed ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v . . was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least clades representing clusters of to alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors ) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons - , and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors . a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos (b galnt ) cells, and cell-surface expression of fors antigen was immunologically monitored with a monoclonal anti-fors antibody. results/findings: we found that met thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon or of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met thr/ ser or exon / deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt genes is reminiscent of common ancestral origin of alpha , -gal(nac) transferase genes. the finding that at can synthesize fors implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of , cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period / / - / / . abo genotyping targeting specific snps for groups a, a , b, o , and o and, if needed, gene sequencing was conducted in cases with indeterminate results, and in cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two ( . %) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in % the predicted abo phenotype was a rh neg (table a ). the predominant donor race was caucasian ( %). four cbu with abo discrepancy were also evaluated by genotyping (table b) . in of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r / systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: , fresh and , frozen edta plasma samples from american red cross donors, collected from february - , were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of , valid results, a total of donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a -year old male in indiana, a -year old male in california, and a -year old female in kentucky. all donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna ( iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as a, the california donation genotype b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was % ( % exact ci: . % to %). conclusion: based on the confirmed-positive donations of , tested, the hev prevalence was . % ( % exact ci: . % to . %) with a detection rate of : , ( % ci, : - : , ). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than % of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using % confidence intervals. this analysis contains data from / / - / / . results/findings: among , , donations reported ( . % from firsttime and . % from repeat donors), there were respectively , and cp results for hbv, hcv and hiv with corresponding rates of . , . and . per , (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of : , : and . : for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv . vs . ; hcv . vs . ; hiv . vs . ). in general, higher rates for all markers were seen among minority donors, those in the - -year age group (also - year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when -month periods were compared. conclusion: data from major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* , melanie c proctor , deanna self , monique portugal , adrian gurrola , laura tonnetti , sonia bakkour , cheryl lobo , michael paul busch , susan l stramer and jeffrey m linnen . grifols diagnostic solutions inc., american red cross, blood systems research institute, new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening , unlinked whole blood donations collected from august th to april th in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of . results/finding: the procleix babesia assay detected all four babesia species with a % lod ranging from . - . copies/ml. the preliminary % lod in parasites/ml ranged from . - . p/ml for b. microti (n ), from . - . p/ml for b. duncani (n ), and from . - . p/ml for b. divergens (n ). of the , donations screened, initial reactive and confirmed positive donations were identified for specificity of . % ( %ci: . - . %). of the confirmed positive specimens, were reactive by both ifa and pcr, by ifa only and by pcr only. all confirmed positive samples were reactive in lysate pools of . donors of reactive donations resided in ct ( ), nj ( ), nh ( ) and me ( ) for an overall incidence of : , , and : , in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* , whitney r steele , ed p notari , james haynes , roger y dodd and susan l stramer . american red cross, american red cross (retired) background/case studies: from - , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from - . study design/methods: prevalence was calculated in -year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of . , . and . days for hbv, hcv and hiv, respectively. linear regressions were calculated with p< . (*) as significant. results/findings: from / / - / / , there were more than million donations from , , donors ( . % female, % first-time (ft), . % caucasian). there were significant decreases in donation prevalence for hbv and hcv (p . and . ), but no significant decrease in hiv during the years (see table for f and r values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p . and . ). prevalent ft donors were significantly more likely to be male ( . % -hbv, . % -hcv, . % -hiv; p< . ). incidence for all agents declined (significant only for hbv; p . ). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last -year period ( in - vs. in - ) . hcv incident donors in - were more likely to be male ( . % vs . % in - , p< . ) and were younger ( . % vs. . % in - < years, p . ). overall, incident donors were more likely to be caucasian males (p< . ). rrs for all agents decreased over time with rrs in - of in , , ; in , , ; and in , , for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the -year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in , mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as- rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with lm amustaline, and incubated for hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, > . log , or > . log pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was > . log , or > . log pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts- inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts activity of < % and high inhibitor ( . - ). mean age of cohort . years (range - ). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean . x /l, range - x /l) and low a-ipc (mean . x /l, range . - . x /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab mg/m ( patients) and cyclophosphamide mg/m (one patient). tpe continued until platelet count reached x /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of . days [range - days]) when they achieved a three-fold increase in a-ipc from baseline (mean . x /l, range . - . x /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean . x /l, range - x /l) and a-ipc (mean . x /l, range - . x /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of . days (range - days) mean platelet count was . x /l (range x /l) and mean a-ipc . x /l (range . - . x /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of . days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* , michelle n stram , joan sevcik , alesia kaplan , and joseph e. kiss , . department of pathology, university of pittsburgh medical center, blood systems inc., university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within - hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january , to november , was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts- activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the ttp patients identified, were excluded due to missing temporal data for important variables. the majority ( %) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients ( %) had a prior history of ttp and % had severe adamts deficiency on admission. the median time from tpe request to initiation was . hours (interquartile range: . - . hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table ) . treatment was not started within an -hour window in patients; the median time to cv access was significantly longer in these patients ( . vs . hours, p< . ). two of these patients had a prior history of ttp and only four patients had severe adamts- deficiency. the majority (more than %) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table ) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus - hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi- a transfusion vol. supplement s hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, through january, , we performed cytapheresis (cy) treatments (txs) for pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. pts ( %) had cml-at and received leukapheresis (lp) txs; pts ( %) had et and received thrombocytapheresis (tc) txs. cml-at pts presented with median wbc x /l (range - x /l), of which % had blast percent > % or blast count > x /l. median age was years ( - years); % were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. % of cml-at pts had no sxs of lks; % pts had sxs of either cns or pulm lks ( sxs), and % pts had sxs of both cns and pulm lks ( sxs). et pts presented with median platelet (plt) count of: x /l ( - x /l)and % pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was years ( - years); % pts were male. results/finding: all pts received a course of cy tx with following objectives: ) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and ) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) < x /l for cml-at pts, and plt ct < x /l for symptomatic et pts and < x /l for asymptomatic et pts. cml-at pts received median of lp txs (mean . txs/pt; range - txs). et pts underwent median of tc txs (mean . txs/pt; - txs). outcomes were evaluated by percentage of pts who: ) reached wbc (or plt ct) tx goal, and ) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved > % reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, % pts improved, % pts stabilized; and % pts worsened. in et cohort, % improved, % stabilized, and % were unchanged. for cml-at pts, median final wbc ct x /l (range - x /l); % pts received ind chemo. for et pts, median final plt ct x /l ( - x /l); % pts had resolution of thrombotic a transfusion vol. supplement s symptoms. % of cml-at pts and % of et pts expired within - days after course of cy tx. of expired pts, pts had both blast crisis and sxs of cns/ pulm lks; pt had intracranial hemorrhage or cva; and pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median - txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used % albumin or % albumin- % normal saline ( / ) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered ( % albumin vs / ), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where % albumin was used versus those that used / . covariates included were fluid types, age and gender. odds ratios (or) and % confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < . . results/finding: during the study period, procedures were documented for subjects ( % female), age range - years, of which , ( . %) received / . the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with % albumin had a significantly lower risk of having either event than by using / , [p . , or (ci): . ( . , . )] , and also had a significantly lower risk of causing hypotension [p . , or (ci): . ( . , . )] in addition to a lower risk of causing citrate toxicity [p . , or (ci): . ( . , . )]. age had a significant effect on having a hypotensive event [p . , or (ci): . ( . , . )] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a year period and compared it to published literature. study design/method: we conducted a -year retrospective study of ta procedures performed and aes were classified according to criteria described in table . during the study period, ta were performed using cobe spectra (software versions . and . ) and since the spectra optia apheresis system (version . ). literature search was conducted for data published on aes associated with ta. four studies from us and non-us studies (canada, europe and japan) were analyzed. trend for ae rates from - was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was . % ( of , procedures) during year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher ( . %, p< . ) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho . , p . ) over the years and significant down trend of moderate and severe aes with a spearman rho of - . (p . ) and - . (p . ) respectively. there were no fatalities during the study period. majority of aes were grade i ( %) and grade ii ( %): / ( . %) procedures were not completed due to aes. comparison of aes [ . % ( / , )] to both european [ . % (n , , / , ) ] and other us studies [ . % (n , / , )] showed a statistically significant difference (p< . ). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table ) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether platelet donors with a donation activity of up to platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* , jordy jurgens , jacoline buchner-doeven , joris roelofs , philip spinella , jennifer a muszynski , carel goslings and nicole juffermans . academic medical center, washington university school of medicine, nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells ( days old) and platelets ( days old) by washing. plasma was filtered through a . um filter. rats ($ grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $ % of their estimated blood volume, which was calculated to be ml/kg. hemorrhage continued until a mean arterial pressure of mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of ml/kg of blood products in a : : ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april , which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and -day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january and july (n ). because of missing data on patient characteristics, patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january to march , n ) and after (april to july , n ) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of baseline variable, generating pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common ( . % vs . %, p< . ) and ventilation time was longer ( h vs h, p . ) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group ( ml vs ml, p . ; and ml vs ml, p< . , respectively). however, -day mortality was not statistically different between the groups ( . % vs . %, p . ). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april , www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over , joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy . length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of -factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: -factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in . marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p . ) and ptt (p . ) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p . ) and plasma (p . ) after off-label use was significantly greater than on-label use. cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were . times (p . ) more with cell saver or anh, and . (p . ) times more with cpb. post-pcc thromboses were identified in cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to days. the transfusion service medical director reviews the case and gives final approval. we observed only patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight ( ) patients were in-patients continuously until delivery. five patients were discharged prior to delivery- moved to another state, was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was days (range - ). six ( ) patients delivered within days of approval. after approval, the mean number of additional specimens per patient was . (range, - ). no patient required transfusion prior to delivery. five patients received transfusion of at least rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only patients delivering within days of approval for extended specimens, patients avoided collection of at least specimen each, and patients avoided at least collections each. since new antibodies are not detectable for at least days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* , , jan m. binnekade , benjamin nota , pieter r tuinman , kirsten van de groep , olaf l cremer , janneke horn , marcus j schultz , robin van bruggen and nicole p juffermans . academic medical center, sanquin research and landsteiner laboratory, vu university medical center, university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in tertiary icus in the netherlands comparing patients who developed ai during icu stay with control groups: non-anemic patients with sepsis, non-anemic patients without sepsis, and patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron ( . vs. . mmol/l, p< . ) and transferrin saturation ( vs. %, p< . ), and low ferritin ( vs. mg/l, p< . ). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately . x cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to %, whilst leaving % of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than % of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately % of the cd fraction post separation and freeze thaw (table ) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table ) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd cd ) and early projenitor cells expressing oct and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: . routine recovery of the wcf at levels higher than current methods, independent of volume. . higher percentage recoveries of all cell types tested than can be achieved with existing methods. . markedly higher post-thaw recovery of viable nucleated cells than any current methodology. . almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving > . x lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd cell target of . x /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf plerixafor (g pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< . considered significant. results/finding: no alc and alc collections occurred among the patients. fenwal amicus was used for % of the no alc and % of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was hodgkin's and non-hodgkin's lymphoma (no alc); hodgkin's and non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc . , lymph . x /l) than alc (wbc . , lymph . x /l). equivalent whole blood (corrected for ac) was processed for no alc ( . l) and alc ( . l). for alc group, extra collections beyond cd target were: days: %, day: %, days: %, days: %, and days: %. significantly more patients were mobilized with g pl in no alc group (n ) than alc group (n ) and collections in alc group had mobilization discontinued after cd cell target reached. there was no significant difference in g ( . x lymph) compared to g pl mobilized collections ( . x lymph); both were significantly higher than the collections where mobilization had been discontinued ( . x lymph). days to wbc engraftment ( . no alc vs . alc) and platelet engraftment ( . no alc vs . alc) were not significantly different. median number of collections for no alc ( ) and alc ( ) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the . x lymph/kg or even the . x lymph/kg targets. implementation of a lymph target increased patients obtaining . x lymph/kg from % to %. only % had < . x lymph/ kg. discontinuation of mobilization once cd cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* , and nicolas pineault , . canadian blood services, university ottawa, canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost hours at room temperature (rt) as long as units are cryopreserved by -hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n ) were split with one half processed immediately (baseline - hours) and the second after hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd cells and cd cell (n ). primary nsg mice were transplanted with a ucb cell dose that contained a total of , annexinv neg viable cd cells. the latter was done to avoid any bias towards one group or another. short term platelets ( vs. hplt/ml, p . ) and leucocytes ( . % vs. . % hcd , p< . ) engraftment at -weeks were significantly reduced in stored mice vs. baseline (n ), and similar results were observed long-term at -weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated months post-transplant. strikingly, the frequency of human cd bm cells was -fold greater in baseline vs. stored mice (p< . , n ). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured -weeks post-transplants were reduced by % in unit , and by % in unit . conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($ mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to %, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of sec). total lymphocyte recovery was % and monocyte concentration was reduced %. furthermore, in a two-pass process platelets were reduced by %. in a -fold parallel system we tested rbc separation from plasma and achieved % separation at ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from clinical protocols from - . an infusion reaction was defined as any symptom from the time of nk cell infusion up to hours afterwards. a severe reaction was defined as any symptom with grade or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r . . . two major endpoints of interest were: ) infusion reaction with any symptom and ) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of nk cell infusions. there were ( %) patients with an infusion reaction of any symptom and there were ( %) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . ). infusion rate (ml/min/kg) was also similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . respectively). incubation of nk cell product overnight in il- vs il- had similar reaction rates for those with any symptom ( % had reaction with il- , % had reaction with il- , p . ) and those with severe reaction ( % had severe reaction with il- , % had severe reaction with il- , p . ). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median . x ) versus those without (median . x , p . ). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan , maryanne c herzig* , barbara a christy , james a. bynum and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at - . mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at - , cells/ well and cultured in well plates for - h in their respective medias. on day , mscs were washed, resuspended in pbmc media and incubated with or without , freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, - lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by h, with > fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was . %. inter-assay variation of msc preps run under identical conditions was . %. inhibition of pbmc proliferation was graded from - % over the range msc concentrations therefore an ec of msc cell number resulting in % suppression of pbmc could be determined for each msc prep. this ec however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute % or more of the us blood supply. differences between donors - years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged - were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the / academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml) were estimated for , , and - yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of donors contributed donations. donors were evenly split by gender, % were ft donors, and % were - yo. ft and rpt - yo donors had on average lower ferritin values at enrollment (p<. ), and a greater percentage were iron-depleted than donors - yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged - have sharply higher risk for iron depletion than donors - yo. odds for lf were to times greater in the younger donors, and for ais were -to fold higher. preliminary statistical models indicate yo donors may have greater risk for lf than or yo by to percentage points, controlling for other factors (p . ). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in - yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on / / by a large blood collector. testing was performed on successful - y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < ng/ml in females (f) and < ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations ( months for f and months for m) and counseled to take - mg of elemental iron daily for days. for m and f, a ferritin < ng/ml indicated absent iron stores (ais) and < ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! ng/ml in f and ! ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior months. an appreciable number of donors with no rbc donations in the prior months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* , joan williams , michelle humphries , nancy haubert , ben reynolds , michael phillips , randall spizman , ralph r vassallo , hany kamel , sally caglioti , german leparc , and phillip c williamson . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. study design/methods: over , serum samples from donors aged , and years were analyzed for ferritin levels using the beckman coulter au instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among , adolescents (ages - ; . % female) who donated blood from to . study design/method: abnormal risk factor levels were defined as hba c ! . %, sbp/dbp ! / mm hg and tc ! mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, , ( . %) adolescents had at least one abnormal risk factor ( . % of males, . % of females). of these, , adolescents had isolated abnormal risk factors, and , adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as ! . g/dl for men and ! . g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from blood services worldwide and complete data were available for blood services. deferral percentages for low hb varied from . % to . % among male donors and . % to . % among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with % lower hb deferral rates in men ( % confidence interval [ci] % to %) and % lower rates in women ( %ci % to %). iron supplementation was associated to % lower hb deferral rates among women ( %ci % to %) but there was no evidence of such an effect among men (p . ). each one-week increase in minimum donation intervals resulted in % lower hb deferral rates among women ( %ci % to %) but not among men (p . ). at the % level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: . % of all candidates for wb donation were deferred in continental france in . deferral was significantly more frequent in women ( . %) than in men ( . %), due to anemia in . % of deferred women and . % of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from to weeks. analysis (table) identified main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the previous years. conclusion: the main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, days stored or days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, ng/kg). blood was sampled every hours up to hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from . e (iqr . e - . e ) /ml in the fresh product to . e (iqr . e - . e /ml; p< . ) in the stored product (p < . ), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: cases of taco and matched controls were enrolled from , transfused patients who received , blood components from may until july . taco incidence was case per patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation ( % vs. %; p < . ), experienced longer intensive care ( vs. days; p . ) and hospital length of stay following transfusion ( vs. days; p< . ), and had higher mortality ( % vs. %; p . ). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from to days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july and to routinely extend ap outdate to day since february . this study reports a month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july -january , ap underwent rt on day . day and units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day had a second rt performed. from february -january , ap underwent rt on day with routine outdate extension to days by performing a second rt on day and a third rt on day , as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type ) or repeat rt positive with negative confirmatory culture (type ). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july , , ap were entered into inventory. of these, , ( %) were transfused prior to rt testing. the remaining ( %) underwent rt on day or day . of these ( . %) were rt positive ( type fp, returned to inventory; type fp, discarded), leaving a total available inventory of units tested by rt. of these, ( % of original inventory) were transfused before the end of day and the remaining ( % of original inventory) reached a day outdate. a total of ( % of original inventory) were transfused on day or day . of these, underwent a second rt on day ( rt positives; fp type one and fp type ) and underwent a third rt on day (no positive results). a total of ( % of original inventory) outdated on day . of these, underwent a second rt on day (no positive results). conclusion: to date we have performed rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted % to only %. a total of ap have been tested twice by rt ( on day and ; on day and ) with ( . %) positive results, both of which were deemed fp by repeat testing or culture. a total of units have been tested times (day , day and day ) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with to million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in . contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between - , parasites/ml. each parasite concentration in wb was tested x . an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at c for up to days; platelets were stored at c (rt) under agitation for days and plasma was frozen at - c. aliquots for culture were removed weekly from rbcs, daily from platelets and after days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at c for detection of live parasites for up to weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at c, rbcs from all units spiked with , parasites/ml were positive for up to days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with parasites/ml were positive for up to days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with , and parasites/ml were positive up to days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at c for up to weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* , marion lanteri and larry corash . cerus corporation, scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom ( - ), french ( - , swiss ( - ), and belgium( - hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately . million dlvbc-screened were issued with a day outdate after release into inventory days after collection, and $ . million amotosalen/uva-treated pc were released into inventory on day or , with a to day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored > . million conventional, non-dlvbc-screened pc and recorded str and fatalities. concurrently, zero definite and possible str were reported with , amotosalen/uva-treated pc, significantly fewer than with conventional pc (table ) ( . str per million vs. . per million, p< . ). one definite, possible, undetermined/indeterminate non-fatal str and contaminated "near miss" pc were reported with . million dlvbc-screened pc between and , for a reduced falsenegative rate compared with the prior five years ( . str per million vs. . per million, p < . ). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october for all platelets received at our institution. at time of receipt at the blood bank (day post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at c for three days. results/finding: a total of / , ( . %) platelet products were successfully cultured ( / [ . %] and / [ . %] in october and march respectively). over the -month period, two true positive cultures were obtained (incidence of in platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us . per product tested. the cost per averted case was $us , . conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january and december . the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the -year study period, a total of transfusions reactions were reported, of which were bcptr ( . % of transfusion reactions). of the bcptr, ( %) were associated with apheresis platelets, ( %) with red blood cells, and ( %) with plasma. recipient diagnoses spanned hematologic/oncology (n ), renal (n ), cardiac (n ), autoimmune (n ), and obstetrics (n ). an organism was identified in both the blood product and recipient in ( %) cases; in ( %) cases an organism was grown in the blood product but not the recipient; and in ( %) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in of the cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever ( %), chills ( %), nausea and vomiting ( %), pain ( %) and dyspnea ( %). blood pressure (bp) decreased in %, increased in %; % of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie , jenna lebedev , linda kapp , xiaohong wang , meghan delaney , lay see er and james c zimring* . bloodworksnw research institute, bloodworks nw, university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg -igg ), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all known variants. study design/methods: the heavy and light chain variable regions of an anti-k monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg , igg , and igg had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table ). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg or for any of the monoclonal ahgs tested. monoclonal anti-igg had a blindspot for igg - , due to the shorter hinge region on igg - . no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* , yves dominique pastore and maryse st-louis . chu sainte-justine, hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since , our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of , scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september , ( %) patients had been transfused and had antibodies with known blood group antigen specificity: anti-c, anti-e ( ), anti-hrb, anti-fya, anti-jka, anti-jkb ( ), anti-s, anti-m, anti-sc , anti-leb ( ). seventeen patients ( . %) were either d or partial d. rhce results showed that patients expressed a normal c antigen and expressed partial c. as for e antigen, had a normal antigen, bore a partial antigen and were weakly expressed. fy(a b ) phenotype was found in ( %) patients. a total of genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos , emilia sippert , mayra dorigan de macedo , sheila fatima perecin menegati and lilian castilho* , . hemocentro unicamp, university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa- a, il b- t cytokine polymorphisms, rhag g>a and hla-drb * alleles may predict a good responder phenotype (sippert et al, transfusion ) and that rhag a and hla-drb* alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included non-alloimmunized patients with scd, homozygous for hbs, receiving a range of - rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa- g>a, il b- c>t) and the rhag g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among non-alloimmunized patients, were homozygous or compound heterozygous for rh variant alleles. from those, had rhag a and/or hla-drb* alleles and at least one cytokine polymorphism (tnfa- a or ilb - t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and were confirmed by sequencing. the third sample was found to be rhce*cevs. ,rhce*cebi on sequencing (predicted phenotype v ,vs ). the samples were typed as v (or ce s ) and vs (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[ g]in samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising % of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies ( % of patients) bound young and old rbcs with no apparent prejudice. band- is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band- is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band- to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band- aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band- tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing type i and type ii patients, we found that type i is characterized by percollv r fractions (similar to healthy storage-matched controls) but increased band- tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by - percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band- tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band- suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* , burak bahar , jeanne hendrickson , krystalyn e hudson and christopher a tormey . yale-new haven hospital, yale university, background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam abstract algorithm, e value x - , word size , gap costs: existence exten-sion ). search results were restricted to bacteria and fungi, with a selective threshold of > % identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b - . , r . & p . ); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of alloimmunized patients reviewed, were culture-positive. of these, % of the anti-c/c group ( of patients) and % of the anti-k group ( of patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from - . %. overall, . % ( of patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of > % sequence identity. while . % ( of ) patients reviewed had positive cultures for klebsiella species, . % of these ( of patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed tegs performed on patients undergoing cv surgeries at our institution from jan to dec , . no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at minutes (min) all within reference range), "hypocoagulable" (r> min, k> min, a< degrees, ma< mm) and "hypercoagulable" (r< min, k< min, a> degrees, ma> mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of tegs analyzed, patients ( %) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients ( % vs. %, p . ). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused ( % vs. %, p . ). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused ( % versus %, p . ). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from liver transplants, performed from to in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, olts were performed. a total of patients was submitted to cs. the median age was years (range - yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in , % of the patients. the average meld score was , , and it was slightly higher in the cs group ( , vs , , p< , ) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was ml and mean reinfused blood volume was ml. allogeneic blood transfusion was required in , % patients in the cs group, compared to , % patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group ( , units vs , units, p< , background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july -december was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp ( and products, respectively) however, obp wastage occurred more frequently in the month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations ( versus products). this is skewed by one month when products were wasted due to expiration of product on the floor. cooler-related issues ( ) and products dwelling too long out of a controlled environment ( ) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were . %, . %, and . %, respectively, with a total exsanguination protocol waste rate of . %. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p . ). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d ) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d- (table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* , edward smith , thomas brown , foeks jeremy , metcalf suzanne , james johnson , peter davis , karafa sw badjie and abba zubair . department of laboratory medicine and pathology, transfusion medicine, mayo clinic, department of anesthesia, mayo clinic background/case studies: our institution performs an average of solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused o( ) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the o( ) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in % of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a y.o. female patient should not have received o( ) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with admissions during the hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight ( kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o( ) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o( ) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately , surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from - %, whereas transfusion rates for vaginal and robotic pfd surgeries range from . - . % and . - . %, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately % of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may -may in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p< . . results/finding: we identified patients for analysis, of whom ( . %) had a preoperative t&s ordered. two ( . %) of these patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine ( . %) of the patients required a second abo/rh specimen per hospital protocol; ( . %) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table ) . no abo/rh discrepancies were identified. one patient received unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution ( . % vs. %, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n ) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of . g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs- i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), units were subjected to the volume reduction while recording the time needed to process all units. this was performed twice for a total of units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of % (range %- %). in units between and days (n ), the estimated mean residual k was . meq (range . to . ). in the two mock mtp trials, the time to complete the procedure was approximately minutes and we estimate an additional - minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal ( ) and caesarian ( ) births. uc collections were divided into segments to test conditions. segment explants were placed on . % gelatin-coated gridded tissue culture plates ( explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the remaining tissue segments were soaked in (ab/am) saline solution for hr and hrs at c, respectively. tissue segments were frozen in cryo bags with a proprietary % dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process or times the total blood volume (bv) of the patient, up to a maximum of liters (l) per procedure, to obtain peripheral blood cd stem cells. as a consequence, a patient often would need to spend hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd pre-count and cd yield, normalized per liter of blood processed, was derived utilizing the patient's cd pre-count, the patient's weight in kilograms (kg), and the target cd dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd stem cells. the initial equation was modified to add an additional % to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in patients, representing both allogeneic and autologous donors, the average blood volume processed was . l. the range was . l - . l. the target dose was achieved in all patients. our previous practice for these patients would have required, assuming a standard bv procedure, processing an average of up to l per patient, with a range of - l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd yield. the result was a high correlation between these two ratios (r . ), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r . , confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses ( - ) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june and march . patients (n ) evaluated were diagnosed with malignant lymphoma (n ), multiple myeloma (n ) and primary amyloidosis (n ) and were mobilized according to standard protocols. collection cd cellularity target was established ! x e /kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by > years old, previous fludarabine, lenalidomide, or bendamustine treatments or ! previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v . . results/finding: the media (range) general collection parameters were: cd (day ) . /ml ( . - . /ml), blood volume processed ml ( - ml) and . ( - . ) exchanged volemias. seventeen patients were considered bad mobilizers, needed plerixafor and had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p . ]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n ), fever (n ) and flu syndrome; all grade ]. two patients could not undergo hematopoietic stem cell transplantation due low cd cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd ) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with % fetal bovine serum. bmsc and amsc at passage - were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses ( . /ml, /ml and . /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par ) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within min and hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: seconds, versus low, medium and high doses of amsc ( , , and seconds), and bmsc ( , , . seconds), p< . ), clot formation time (cft, p< . ) and increased alpha angle (p< . ) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par . no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* , neil bagamasbad , reynold dilag , melissa nasser , nicole bauer , jennifer wheeler and mary berg . department of pathology, university of colorado -anschutz medical campus, department of medicine, division of hematology, university of colorado hospital, scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from collection procedures using the mnc protocol and collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd )-positive (cd ) throughput, cd collection efficiency (ce%), platelet loss a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included and allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd throughput was significantly higher in the cmnc group than the mnc group. the cd ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient # , originally typed as an a , had bone marrow donor and cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient # is now typing as type o. patient # was originally typed as a and received a bone marrow transplant from a type b donor. patient # is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient # and patient # indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with % fetal bovine serum under either normoxia ( % o ) or hypoxia ( . % o ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd /cd and cd were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc ( . /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by %, but depressed the growth of amsc by % at day in comparison to normoxia. both bmsc and amsc equally expressed cd and cd but not cd under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: (control), versus (bmsc), and (amsc) seconds) by natem. hypoxia also significantly shortened ct ( (bmsc), (amsc) seconds, p< . as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd cells. study design/method: cryopreserved cd cells from healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef -alpha-yfp lentivirus ( . % concentration) and media (x-vivo- , human serum albumin(hsa), ng/ml each of cytokines (scf, tpo and flt -l) over days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of % dmso, % pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd cd -cd ra -cd cd f cells) phenotyping and cfu assays were done following first thaw (pt ), post-transduction (ptxn) and second cryopreservation-thaw (pt ). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd %, cfus were similar before and after pt . hscs ranged from to cells/ cd cells in the pt -tr arm compared to a range of to / cd cells after pt . viability, % cd and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt (table) . conclusion: dec of mpb human cd cells decreases tnc recovery, but has minimal effects on cd cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd % in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of % hydroxyethyl starch, % human serum albumin and % dmso at final concentration. pbsc were cryopreserved by direct immersion on - c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of consecutive days of neutrophil count > . x /l and platelet count > x /l after days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd was below x cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd on the day of the collection versus collected cd per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd was calculated. final laboratory count of cd per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss software. results/findings: among patients collecting hpc for autologous transplantation, , % needed only one day of hpc harvesting, while , % needed two days and , % needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was - , %. after comparing predicted values with cd collected in the final product, we found a very strong correlation of . (p< . ) for patients and a strong correlation . for healthy donors (p< . ). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; ! % cd , cd , cd and % cd , cd , cd , cd , hla-dr) study design/method: umbilical cord tissue (n ) was washed, blood vessels removed, cut into . - mm pieces, and washed twice in saline. fresh tissue was immersed in . % saline for same day culture, while frozen tissue was cryopreserved for at least hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a cm tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures > % confluence. all cells were tested on an msc flow panel at passage just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of days (fresh . , frozen . ), and days (total) for the msc's to reach passage (fresh . , frozen . ). all cells were ready for flow analysis in approximately weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p . ), or their growth rates (p > . for all). flow cytometry showed average ! % for positive markers and % negative markers. there was no statistical difference between fresh and frozen flow result (p > . ). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of up to years ( to ) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with % concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a c water bath and . ml aliquots were diluted at a : proportion with % human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using -aad marker through flow cytometric analysis. results/finding: ucb storage period was . years (mean) and cell recovery was . % (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p . ). post-thaw cell viability of . % (mean) showed no statistically significant correlation with unit storage period (p . ). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln vapor in a dmso-based cryoprotectant for yrs. ( . . ; n ). units were rapidly thawed and rinsed in dpbs, then pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a x grid pattern in msc-supportive medium and incubated for days, after which the tissue was discarded and media exchanged. cells were isolated on the th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a % success rate. cells were positive for the msc markers cd , cd , and cd ( . . %, . . %, and . . %, respectively) and negative for the hematopoietic markers cd / ( . . %). passage and passage doubling times were . . days and . . days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical a transfusion vol. supplement s research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd yield prediction algorithm ines bojanic* , nelly besson , ivana vidovic and branka golubic cepulic . department of transfusion medicine and transplantation biology, university hospital centre zagreb, terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version ) in adult and pediatric lvl. a prediction algorithm for cd cell yield was also tested. study design/method: we evaluated retrospectively lvl performed in adult patients, and lvl in pediatric patients treated in uhc zagreb from march till september . mobilization regimen combined chemotherapy and filgrastim; poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio : ). in patients weighting kg (n ), a rbc prime was performed. cd , lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd cell count and cd cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd values to real cd yield. results are presented as median (iqr). results/finding: in both groups, cd , ly and mo ces were high. target cd dose was successfully reached in procedure in ( , %)adults and in ( . %) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in ( . %) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd cells and cd cells collected/ blood volume was observed in both groups (r . and . in adults and children respectively, p< . ) suggesting cd yield could be predicted based on precd cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd yield and observed cd yield (r . and . in adults and children respectively, p< . ) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of . ( . - . )l of blood in adult procedures, and . ( . - . )l in pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd , ly and mo ce were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* , aniko barta , arpad batai , zoltan csukly , zita farkas , laszlo gopcsa , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) times per case weekly at a dose of million cells/kg. clinical response was assessed days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all patients had received cycles of msctreatment ( dose per cycle). the median age was years old ( - ) with a male/female ratio of : . distribution of the original malignancies (n): acute myeloid leukemia: ; acute lymphoblastic leukemia: ; myelofibrosis: ; myelodysplastic syndrome: ; multiple myeloma: ; t-cell lymphoma: . nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median rd day ( - ). the involved organs were skin ( ), gut ( ), skin and gut combined ( ) and even lung in cases. the median time of msc's first infusion was days after the stem cell transplantation (hsct) and ( - ) days after the first episode of gvhd. of the cycles of msc-treatment led to complete remission ( . %) and resulted inpartial remission ( . %). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with % overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is million cd cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the million cells/kg goal. the ideal minimum cd cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from patients to evaluate the predictive value of the cd cells/ml level. data was collected over months from every patient who underwent a stem cell collection. four patients were allogenic donors and were autologous donors. the patients' weight, diagnosis, and pre-procedure cd cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd cells collected were recorded. the collection efficiency and the cd cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd cells/ml and post-procedure cd cells/kg (r . ). any patient who had a pre-procedure cd cells/ml count of or greater had a collection of at least million cells/kg. any patient who had a pre-procedure cd cells/ml count of or less collected less than conclusion: the pre-procedure cd cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd cells/kg level. to confidently know that a patient will be able to produce the desired million cells/kg, a pre-procedure cd cells/ml count of at least should be obtained. for any patient with a count below , they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between and cd cells/ml should be conducted. heidi elmoazzen , antonio giulivi , michael halpenny* , lisa martin , donna perron , chris bredeson , lin yang , locksley mcgann , paul birch and jason p. acker . canadian blood services, ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso ( % final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd , viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in % dmso and . % hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of . days for anc with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current % dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using % dmso and . % hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from % to %. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is . mg/kg, therefore patients weighing > kg would require a second vial, thus doubling the drug cost. in we implemented a policy of capping plerixafor at mg for patients weighing > kg. this retrospective study compares the mobilization of patients > kg who received capped doses ( ) ( ) ( ) ( ) , with historical control patients ( - ) who received full or uncapped doses. study design/method: patients weighing > kg with crcl > ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of and consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd /cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at mg for patients > kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (< yrs) have a higher pre-apheresis %cd level than any of the other groups, reaching statistical significance when comparing the %cd pre-apheresis between the youngest group (< yrs) and the oldest group (> yrs). hispanic donors show statistically similar %cd pre-apheresis levels over all age groups. moreover, the hispanic older age group (> yrs) had a statistically higher %cd pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last years was analyzed to determine the th percentile, median and th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc ( x cell / ml) and three low wbc ( x cells / ml) concentrations, each at high ( ml), low ( ml) and median ( ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax (pericell protocol, cs. . kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and -aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting -aad viability of % [range - ]% and a hematocrit of % [ - ] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was % [ - ] with a % [- - ] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of [ , ] % and a change in -aad viability of [ , ] % from the input product. the method was found to have a cv of . %. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating a transfusion clinical assessment. in the first phase, cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd -aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to hours post thaw. the second in vivo phase included use of an infusion pump for consecutive autologous patients, with comparison of infusion and transplant outcomes to previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than % within hour, while cd cell viability remained stable up to hours post thaw. small aggregates appeared after hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were . . and . . , respectively (p-value . ). platelet days to engraftment for pump and drip were . . and . . , respectively (p-value . ). infusion rates were slightly higher for the pump group. for control patients, required transfer of products to syringes due to slow infusion rate and others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the zikv ineligible cbus was: caucasian %, asian %, black/aa %, and multi-race %. racial distribution of all clinical cbu donors was caucasian %, asian %, black/aa %, and multi-race %, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: % of all ineligible cbus and % of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete , -diphosphoglycerate ( , -dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring , -dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n ), which were stored in sagm for days, to act as untreated controls. the remainder of each unit ($ ml) underwent treatment with the rejuvenation solution ( ml, minutes at o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros . pc were centrifuged at g in a sorvall rc c centrifuge (sorvall, usa) for min. the combination cryoprotectant dmso dextran (cryosure dex , germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at degrees c (barkey plasmatherm) for min. cpc osmolality was measured with an osmomat osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru u ). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso dextran (cryosure dex ) , as a cryoprotectant, to obtain a final concentration of % dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m were instrumental in automating that phase. pc to be frozen had an osmolality of no less than mosm/l. prp and ppp were frozen at a cooling rate of - c/min and stored at - in the chest freezer for up to months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru u ). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to mosm/l. freeze-thaw recovery of platelets was % or more of the original population. defrosted pc were stored at - with continuous gentle stirring from a helmer platelet agitator for no longer than hours before transfusion. it took no more than min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day and day evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day . a total of lrap units were tested over a -month period: were cultured and rapid tested on day ; were rapid tested on day . the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were true negatives (tn) and false positive (fp) on day when tested by bact/alert, with tns on day . bactx testing results showed tns on day and tns on day . testing using the pgd kit showed tns on day ; and tns and fps on day . fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in % plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day and day during the night shift to be accomplished without additional staffing and allows to extend outdate to day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for year jos lorinser , pieter f van der meer , hans van der heiden and dirk de korte* . department of product and process development, sanquin blood bank, mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers ( ml) is unknown. if these products can be stored at - c it will be feasible to store this product in -star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at - c or <- to - c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <- c for - months and controlled thawing, six different sera were used to fill a large number of mini ( ll) containers, which were refrozen and stored at either - c or <- c. during storage at months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <- c. growth factors tested were pdgf-aa&ab/bb, tgf-ß / / , vegf, a transfusion vol. supplement s egf, fgf . the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß were the most abundant growth factors, on average , resp. ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average ng/ml. tgf-ß , egf and vegf were detected at relatively low values, resp. ng/ml, . ng/ml and . ng/ml. average levels of fgf and tgf-ß were close to detection limit (< . ng/ml). the controls stored at <- c showed for all growth factors close to % of the initial values in samples at t (moment of filling mini containers). for serum stored at <- c for up to months, most factors showed less than % decrease, except for pdgf-aa and tgf-ß , showing % resp. % lower values. for serum stored at - c the values for tgf-ß , egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß showed a decrease of resp. , and %. conclusion: human serum eye drops can be stored in the new micro dose device at - c ( -star household freezers) or <- c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at - c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond year. ruqayyah almizraq* , heather inglis , phillip norris , , jennifer a muszynski , nicole juffermans , jelena holovati and jason p. acker , . university of alberta, blood systems research institute, university of california, san francisco, nationwide children's hospital, academic medical center, canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n per method). residual platelets and white blood cells (wbcs) were measured on day using flow cytometer (fc). on storage day and , number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day , apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p< . , wbd p< . ) and wbf (vs: apheresis p< . , wbd p< . ) methods. while rcf units yielded the lowest count of platelet-evs (cd a ) on day and , the highest number of platelet-evs were in apheresis (day ) and in wbd (day ). similarly, there was significant difference among methods in the number of wbc-evs (cd , cd , cd , cd , cd b ) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day vs day in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< nm) was greater than large evs (! nm) in all of the products on day and , and the highest level of evs < nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p< . ). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at centigrade maryanne c herzig* , crystal lafleur , chriselda g fedyk , sherrill j. slichter and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research, university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least weeks of storage at c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for , , , or days after collection. units were stored for days without agitation. units stored for , or days were agitated during storage with a model hybridization incubator at c set for end over end rotation at - rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at - c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai- ) as another fibrinolytic measure; and complement activation markers c a, c d, c a and c b- . data was analyzed by one way repeated measure anova. results/finding: only % of the platelets were recovered in units stored for days without agitation. these levels did not meet fda requirements of . x platelets per wb unit. subsequently, wb was agitated and platelet recovery was - %. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t (day of collection) and t , , , or measurements. significant elevations of pai- and scd l indicate activation of platelets and inhibition of fibrinolysis (p< . ). activated complement peptides c a, c a, and c d were all elevated over time (p< . ) while sc d- was not. however, only c a and c d levels at t were above normal reference ranges at . and . times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at c for - days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc d- reported, wb showed elevation of c a, a and c d and not sc d- . complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* and christian todd . cerus corporation, community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly . x . x . x . x rcf . x . x . x . x apheresis . x . x . x . x wbd . x . x . x . x platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following collection targets: . x in ml, . x in ml, . x in ml, and . x in ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of . x or . x was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: % of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was . . conclusion: it is possible to treat % of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward % while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* , andrey skripchenko , fei xu , ying li , stephen j wagner , pamela h whitley and jaroslav g vostal . fda/cber/ obrr/dbcd/lch, american red cross holland laboratory, american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature ( - o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts ( hrs ct: hr o c [tc]). autologous apheresis plts stored for -days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n ) and the same non-labeled plts were also infused into mice (n ). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts < % were considered background. results/finding: the mean recoveries of infused plts were . . % for rt, . . % for tc and . . % for ct in humans. in mice, mean recoveries of the same plts were . . % for rt, . . % for ct and . . for ct (mean sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $ % and ct was $ % of rt. in mice tc was $ % and ct was $ % of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to % for rt plts. human tc plts had % auc while ct plts had % auc compared to rt plts in humans. in comparison, the same tc plts had % auc and ct plts had % auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are . and . , respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* and geeta paranjape , . coastal bend blood center, carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g with the compomaster net software for data management. implementation was planned for a november go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g s and compomaster in june . training and validations were successfully completed and a full launch occurred mid-march . device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december . validation was completed and signed off in march of . manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g system. data points were collected from units bi and units ai. results/finding: upon initial implementation, staff training and use, the compomat g was found to be easy. plt weight spread was reduced from an average of gm to an average of gm. actual plt weights were reduced from an average of gm to gm, resulting in an average increase in recovered plasma of . ml per unit. plt count on average increased from a count of to ( /mm ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by . % after implementation of the compomat g and our plt concentrations increased on average by %. we were able to consistently produce a smaller volume plt (average gm), which gave us . ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell , angela hill , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: abo/rh matched lr sagm rccs were pooled and split to produce large ( ml) and small ( ml) rccs. the rccs were stored to d and glycerolized manually by mixing ml of glycerol with the rcc in a ml freezing bag. units were frozen at - c for ! h before being removed from frozen storage and thawed in a c water bath. large rccs and small rccs were deglycerolized using the organization's current procedure on the cobe cell processor prior to re-suspension in . % saline, . % dextrose. the remaining rccs were transferred into a l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of %, and deglycerolized in a ml centrifuge bowl on the acp- with re-suspension in as- . rbc quality was tested at h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p . , acp : p . ) and lower cell recovery (cobe: p . , acp : p< . ) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe had higher hemolysis (p< . ) and supernatant potassium (p . ) than did small volume rccs. large cobe rccs had higher hematocrits (p . ), hemoglobin (p . ), and recovery (p . ) than did large acp- rccs. however, all cobe rccs had higher (p< . ) hemolysis ( . . %) levels than did acp- rccs ( . . %). cobe rccs failed to meet regulatory hemolysis standards of . %. conclusion: addition of a ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as- and storage for h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp- cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using . % saline and centrifugation and the semi-automated washing method (sam) using the cobe blood cell processor. study design/method: in this study, units of single donor platelets were evaluated ( washed using the mm and washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas . results/finding: table shows that the average platelet recovery for the sam ( %) was significantly higher compared to the mm ( %). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took - minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas- ) for input platelet doses of . to . platelets in to ml of to % plasma and - % pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas- containing doses of . to . platelets in a volume of to ml. study design/methods: apheresis pcs (amicus v r ) were collected in % plasma and % pas- . one study was performed at the nominal dose ( . - . x platelets), volume ( - ml) in % pas/ % plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition ( . - . x platelets in - ml) using either single or pooled donations. input pcs (n ) were treated with the intercept ts set by the end of day post collection; the incubation time in the compound adsorption device (cad) container ranged from to hours and the intercept treated pcs were stored in containers (n ). day and post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas- treated in the intercept ts set demonstrated acceptable in vitro function (table ). all intercept treated pcs had ph( c) ! . . platelet dose and volume recovery post-treatment ranged from % to % and % to %, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing % plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n ). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n ). numerous in-vitro quality markers (plt concentration, atp, po , pco , ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days , , and for apheresis pcs, and on days , , and for wb-derived pcs. two flow cytometry assays were used to evaluate cd p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion vol. supplement s results/finding: platelet recovery was % and % for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells ( % (test), vs. . % . (ctl) on day ) and a higher rate of cd p expression than control pc units ( % (test), vs. % (ctl)) on day ). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* , lorraine blagg , christi e marshall , herman woodson , sean erony , krishna patel and eric gehrie . the johns hopkins hospital, johns hopkins hospital transfusion medicine dept, johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day . as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day . the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of lrap were tested. lraps initially tested negative by bactx, while lraps initially tested positive by bactx. all initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was . %. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only platelet units. the expected rate of bacterial contamination of platelets is less than per units. the . % initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as- and cp d/as- rbc alan d. gray* , matt landrigan , pamela whitley , michael wellington , sherrie sawyer , shalene hanley , emily rondeau , louise herschel , neeta rugg , patricia a.r. brunker , shawnagay nestheide , jose cancelas-perez , larry dumont and zbigniew m. szczepiorkowski . and , -dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for > hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood ( - ml) was collected and processed at sites into leukocyte-reduced rbc (a total of n cpd/ as- and n cp d/as- ). ml of rejuvenation solution (citra labs) was added to each rbc on day (d- ), incubated for minutes with agitation at c water bath (helmer dh ), washed (haemonetics acp ), and stored in as- at - oc for days (d- through d- ). in vitro recovery (%) was calculated and hemolysis, atp, and , -dpg were determined on day , d- , d- after rejuvenation and washing (postrjv), d- , d- , d- , and d- . all units were cultured on d- postrjv and on d- , and then concentrated by centrifugation on d- . results/finding: in vitro rbc recoveries were . % and . % (as- and as- , respectively) and no bacterial growth was observed. hemolysis on d- was maintained < % in / ( %) as- units and / ( . %) as- units. all as- and as- units ( %) had hemolysis < % following concentration by centrifugation. morphology score was reduced to % (as- ) and % (as- ) by d- , restored after rejuvenation ( %, %, respectively) and maintained through d- (> %). atp was restored and maintained above fresh levels after rejuvenation. , -dpg was restored above fresh levels and was maintained ! % of fresh levels through d- . all values were significantly different compared to d- except as noted (p< . , paired ttest) ( table ) . conclusion: rejuvenation of stored rbc restores atp and , -dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d- when compared to nonrejuvenated rbc on d- . this study is funded by zimmer biomet. storage > hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante , jason p. acker* , and jelena holovati . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during -day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and , -dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome rejuvesol-treated (l r). the prbcs were incubated for h at c with hepes-nacl (sham), liposomes (dopc:chol, : mol%, mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and , -dpg at day hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control ( . . %): l ( . . %, p . ), r ( . . %, p . ), l r ( . . %, p . ). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r ( . . , p . ) and l r ( . . , p . ) treatments compared to s ( . . ) but not l ( . . , p . ). rbc rigidity (kei) increased in all treatments compared to sham ( . . ): l ( . . , p . ), r ( . . , p . ) and r l ( . . , p . ). aggregation amplitude was significantly increased by r treatment only ( . . au vs. . . au, p . ). atp levels were significantly higher in all treatments compared to sham ( . . mmol/g hb): l ( . . mmol/g hb, p . ), r ( . . mmol/g hb, p . ), l r ( . . mmol/g hb, p . ). the levels of , -dpg were no longer detectable in s and l treatments at day . the combined treatment was comparable to r ( . . mmol/g hb vs. . . mmol/g hb, p . ). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer , eric ducas , patricia landry , nathalie dussault , jacques bernier , danny brouard* and anne maltais . h ema-qu ebec, institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the -ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < c) of one to six -ml whole blood units (wbu) within h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between c and c for h under extreme external conditions (- c to c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for h at - c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, -ml wb bags were filled with ml saline . % at t c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (- c) and summer ( c) conditions. shipping boxes were filled with either one or six bags (n ). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under c in . . h and maintain their internal temperature between c and c for h with final values ranging between . c and . c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the c threshold value in . . h and the bags' internal temperatures were within the acceptable range for h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than - c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* , christopher c. c silliman , beth shaz , marguerite kelher and claudia s. cohn . new york blood center, bonfils blood center, department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either % donor plasma (n ) or % pas- / % donor plasma (n ). within hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd ligand (scd l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c ( / products) compared to plasma platelets ( / products); however, the hla-antibody screen-positive supernatants of pas- a transfusion vol. supplement s abstract c platelets had fewer hla specificities ( specificities) compared to those of the plasma platelets ( specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd l levels were increased in the supernatant of pas-c compared to plasma platelets (table ) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd l and not bioactive lipids. although scd l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below x wbc in us and x wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n ) and leukoreduced (lr) rbc units (n ) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of wbc/ul. the spiked samples of , . , , , and wbc/ul were prepared from the source sample of wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations ( , . , , , , wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples ( , , , wbc/ul). tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of samples (plt and rbc) were run on both analyzers, repeated for days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt . , slope . ), (r-rbc . , slope . ). the %diff-plt at , , wbc/ul were . , . and , respectively. the %diff-rbc at , , wbc/ul were . , . and . , respectively. the average total testing time was similar on both instruments; min for the facsvia and min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated % ( of min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* and mickey koh . department of laboratory medicine and pathology, university of minnesota, st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of completed and partially completed surveys were received. results/findings: responses came from countries, but the majority of responses came from the united states (us). of the respondents, % reported aprp use in their hospital. aprp was used predominantly for outpatients, though > % of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by - mds; however, hospitals had > mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures ( %); however, respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, ) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n ) under blood bank conditions at day (d ), at day (d ), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from . - . % at d to . - . % at d . rejuvenation markedly reduced this storage-induced spherocytic shift ( . - . %) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell , angela hill , tracey turner , april xu , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units ( ) than in rcf units ( ), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as- additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: small rcf ( - ml) and large wbf ( - ml) rccs were stored for d before being glycerolized and frozen at - c for ! h. large rccs whose red cell mass exceeded the capacity of the ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a c water bath, deglycerolized and re-suspended in as- . rccs were stored d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p< . ) hematocrit, specific gravity, hemoglobin per unit, supernatant k and na concentration, deformability (ei max ), and higher (p< . ) recovery than did large wbf units. no significant differences in hemolysis, atp, , -dpg, p , rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table ) , however of large wbf units had rbc recoveries < % due to pre-glycerolization volume reduction, and of the small rcf units had hemoglobin values < g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above % and the hemoglobin failure rate would be below % of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. . . . . . . . . elongation index ( pa) . . . . . . . . this study is funded by zimmer biomet. (hasan ) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two ( ) rbc units (leukocyte-reduced), cpd/as- or cp d/as- , on day , day , and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o /g hb) and total releasable oxygen (tro) of the unit (ml o ). orc was determined by assessing the change in % o saturation from mm hg po (e.g., lung) to mm hg po (e.g., venous blood) multiplied by . ml o /g hb (li ). a simulated baseline pretransfusion vo of ml o /min was estimated using the day orc and assuming a g/dl transfusion trigger with a cardiac output of l/min and l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day restored orc and tro to levels greater than day ( table ) . orc of the rejuvenated unit was . . times and . . times greater than rbc on day and day , respectively (p< . ). vo increased after a simulated single unit transfusion of rbc (day , day , and pw) by . %, . %, and . % over the pre transfusion vo , respectively (p< . ). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release . times the volume of o compared to standard, untreated rbcs stored for days. inferior oxygen delivery to tissues (vo max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for days vs days which seem dependent on genetic variability and storage time (bennett-guerrero ). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* , jay srinivasan , gustaaf de ridder , alan d. gray , matt landrigan , keaton charles stoner , angela crabtree , jessica poisson and ian welsby . duke university school of medicine, duke health pathology, citra labs, a zimmer biomet company, zimmer biomet, duke university, department of pathology, durham veterans affairs medical center, duke university hospital, duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted , -diphosphoglycerate ( , -dpg). the loss of , -dpg increases the oxygen affinity of hemoglobin, resulting in lower p (partial pressure of oxygen at % hemoglobin saturation). decreased p may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and , -dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a , leukoreduced prbc stored in as- were obtained from our local blood center. after days of storage, units were divided into separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution ( ml) was added to the cr group, and all groups were then stored for another days at - c. on day of storage, the sr group was incubated for hour at c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps ) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a . . results/finding: significant differences in p were noticed between all groups (table ) . ei, ps , and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p ) seen over days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc ( ml) units stored in as- , obtained from a regional blood donor center at expiration ( days), were passed by gravity through sorbent-devices containing ml of multifunctional polymer bead, at a flow rate of ml/min. supernatants were analyzed for k removal as well as free hb, antibodies and cytokines ( -plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k ] from . . to . . meq/l; equivalent to an . % reduction. free hb was reduced by . % from . . to . . mg/ml. antibodies, specifically igg, iga, and igm decreased from . . to . . mg/ml ( . %), . . to . . mg/ml ( . %), and . . to . . mg/ml ( . %), respectively. inflammatory cytokines were significantly reduced, specifically: ip- from . . to . . pg/ml ( . %), mip- b from . . to . . pg/ml ( . %), and pdgf from . . to . pg/ml ( . %). filtration had no significant impact on cell surface markers of rbc viability (< . % decrease) or sensitivity to osmotic changes. values listed represent mean sem (p < . for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency ( . %) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in apheresis and whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and , -dpg profiles to fresh levels. the objective was to compare % hemoglobin-oxygen saturation (p ) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was . . %. hemolysis (%) was similar on day before and after dry-air incubation with the rejuvenation solution ( . . % vs . . %). percent hemolysis (%) decreased after washing ( . . %) and was maintained below < % for all units during storage for hr ( . . %). average atp and , -dpg were restored above the average fresh values. the morphology score decreased $ % by day , which was restored to near fresh values following rejuvenation and washing and storage hr ( . % and . %, respectively). rbc oxygen affinity, as assessed by p , was restored above fresh values. all values were significantly different compared to day (p< . , paired t-test) ( table ) . conclusion: rbc morphology was restored to near fresh and average atp, , -dpg, and p were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and , -dpg were maintained during storage hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d tetrahydrocannabinol (thc) and -oh-d -tetrahydrocannabinol ( -oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and -oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and -oh-thc to produce samples for lc-ms/ms analysis. lc used a c column. post-column detection by ms/ms used positive ion electrospray with q :q ion pairs of m/z . : . (internal standard (is), d -thc), m/ z . : . (thc), and m/z . : . ( -oh-thc). quantitative results for thc and -oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from - ng/ ml for both thc and -oh-thc. limits of quantitation, defined as standard deviations above background, were . ng/ml for thc and ng/ml for -oh-thc. results/finding: a total of donor plasma samples were tested for thc and -oh-thc. no samples tested positive for either thc or -oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a % probability of one or more positives at a prevalence of . % positive samples, and a % probability of one or more positives at a prevalence of . % positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than % among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than hours for post-exposure detection of thc and/or -oh-thc in plasma. conclusion: testing of donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than %. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* , ruqayyah almizraq , daniel millar and jason p. acker . university of british columbia, university of alberta, lightintegra technology inc., canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days , , and of storage. one rcf rcc was tested on days , , , , and and six ml aliquots were stored in parallel and tested on days , , and . all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days , and (p< . ) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the aliquots were consistent at each time point but statistically higher than in the original rcc on and after day of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow % screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n ) and paired ffp aliquots were stored for - days at - c and - c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! % levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c esterase and alpha -proteniase inhibitors). the level of factor xiii in odp was slightly lower, about % of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i ii and ddimer) and complement (c a and c a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion vol. supplement s abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to % in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to minutes. in addition, donations with collection times between and minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from - minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n ) were prepared from one - minutes bc and ml of autologous plasma in a ml pvc-dehp container. as a reference, spc from donations with collection times of < minutes were prepared (n ). in addition, pc were prepared from bc, of which at least bc were from - minutes donations (n ). after pooling of the bc, ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for days at c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume ( vs. ml) and platelet content ( vs. x ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day , ph( c): . . vs . . , other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day and/or in / pc (possibly because sometimes ab incompatibility was accepted). on day , plt showed low cd p expression ( . . %) and phosphatidylserine exposure (annexin v binding, . . %). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from - minutes whole blood donations had a normal composition and showed good in vitro quality during day storage. to substantiate that the exclusion of - minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* , jessie miller , ranee marie wannarka-farlinger , sandra bryant , scott a hammel , sherry stern and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as- rbcs, irradiated and non-irradiated, were selected for the study. the units varied in age, ranging from to days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< . ) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p . ). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after days of storage pei lun karen lim* , erma sofia sumardi , isamar eduardo ancheta , susan lim , christina yip , lip kun tan and shir ying lee . national university hospital singapore, national university hospital, national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within hours of processing and stored at - c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for days and kept at to c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than iu/dl. study design/method: randomly selected units of ffp (n ) were measured for fviii concentration based on clotting assay (stav r -deficient a transfusion vol. supplement s viii diagnostica stago). fviii levels were measured at five time points: prefreezing, , , and hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at to c for minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration ( hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at to c for days for subsequent testing. results/finding: results obtained were listed in table . units to were not tested for fviii at post thaw- hour due to operational issues. the overall fviii concentration decreased at an average of % from pre-freezing to post thaw hour. after further storage of tp post thaw- hour and - hour, residual fviii level remain to be above iu/dl except unit which had a lower initial fviii concentration. at post thaw- hour, out of units tested had residual fviii activity within the pre-set standard of iu/ dl. the average decline from -hour post-thaw to -hour, -hour and hour post-thaw was . %, . % and . % respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least iu/dl of fviii. typically patients with factor levels below iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi , ayman mohamad sabri , ali abdullah alajeafi , ashwaq hasan alhekri , saleem bin mahfouz , ali hasan alkhodari , rawya saeed shealy , marcus picard-maureau* and hussain bana almalki . king abdulaziz hospital and oncology center, cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in % plasma over a day storage period and the new "test" pathogen-reduced, pooled (pools of ) prp pc in % plasma over a day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of leucoreduced test pcs were assessed at day of storage and compared to leucoreduced control pc at day of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day ; the process was completed by day post-collection. samples were taken daily for quality analysis from test and control pc until day and day , respectively. for bacterial spiking, additional pc were spiked with each receiving ml of mcfarland ($ . x cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was . % . , the total average platelet loss at day was . % . . the average platelet loss in the control units at day was . % . . the average ph of the test units at day was . . and in the same range as the control pc, ph . . . glucose concentration in test pc at day ( . . mmol/l) was lower than in the day control units ( . . mmol/l). lactate levels increased during the course of storage; lactate levels at days and were outside the range of the assay (> mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* , jay srinivasan , jessica poisson and ian welsby . duke university, duke university school of medicine, duke university hospital, duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified proteins in cryo; of the most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf ( . mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial ( Á lm) filtration. cryoprecipitate mini-pools ( ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di( -ethylhexyl) phthalate (dehp)] adsorption device and a Á lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table ) . kit ensured bacterial sterility (table ) and most importantly, final product was free of hbv, hcv and hiv (table ) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase- , thereby blocking synthesis of thromboxane a from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n ) were prepared from a nsaid-bc and ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for days at c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n ) were investigated as a reference. values are expressed as mean sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume ( vs. ml) and plt content ( vs. x ) were similar in both groups. on day , both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day was significant higher in a subset of donors who had used ibuprofen (n ). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin ( , - , p< . ), diclofenac ( , - ) and naproxen ( , - , p< . ), compared to normal controls ( , . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (< hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, ] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type diabetes (t d). because of the strong rise of people with t d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t d, but accepted as donor. study design/method: twelve whole blood donors with t d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for days at c and sampled on day , or and . the diabetic marker hba c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 'good' (ph day > . ) and 'poor' (ph day < . ) storing spc were selected and analysed in more detail. results/finding: donors were of age year and primarily men ( %). donors with t d had a higher mean bmi ( . . vs. . . kg/m ) and higher hba c than controls. the spc of both groups had the same volume ( vs ml) and plt content ( vs x ) but on day glucose concentration was higher in the diabetic group ( . . vs . . mm, p< . ). on day , the average in vitro quality was comparable in both groups (data not shown). when combining a transfusion vol. supplement s the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed ( . . vs . . mmol/ day/ plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc- ) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day ('poor': . . vs 'good': . . , p< . ). conclusion: bc from donors with t d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table ). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* , , katharine a downes , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy month old unvaccinated girl presented with history of diarrhea for days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: x /l, platelets: x /l, bun: mg/dl, creatinine: . mg/dl, lactate dehydrogenase u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to x /l, adamts sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation ( x /l and a-ipc of . x /l). two consecutive tpe were completed which resulted in a platelet count decrease to x /l and a-ipc of . x /l. a-ipc ratio was . below the ratio of which has been reported for ttp patients. similarly a-ipc count was not below x /l threshold reported in setting of ttp with severe adamts deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o :h toxin. testing of c , c , factor h, factor h autoantibody, factor i and factor b were normal. adamts activity was %. patient was treated for the infection and platelet count improved within days to x /l, with resolution of her renal failure: bun: mg/dl, creatinine: . mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts activity. many patients with severe autoantibody-mediated adamts deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts activity < %) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value - days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column ), increased peak a-ipc value (results shown as percent increase, column ), delayed a-ipc peak, and delayed plt recovery (table ) . moreover, recurrent episodes required more procedures compared to initial presentation (table ) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february to march on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version . after taking consent from the donors. the target collection of each procedure was a dose of x platelets in - ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at consecutive donations within days were considered. data was analyzed by stata . within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the donors, repeated the plateletpheresis within a week (group i) and underwent nd plateletpheresis within - days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the groups (p . ). though above the eligibility cutoff of . lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at nd plateletpheresis in group ii donors. there were donors who presented to us for the rd time for plateletpheresis with a mean gap between st and rd plateletpheresis being days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p . ). plateletpheresis through all the cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type- receptor antibody (at rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at r ab are reviewed. results/findings: case : the patient is a currently -year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age , and a second deceased donor transplant due to a rejection of the transplanted kidney at age . three years post-transplant, her creatinine (cr) started to rise from . to . mg/dl and a biopsy showed banff criteria grade amr, grade a t-cell mediated rejection (tcmr) and grade interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at rab was identified at > u/ ml (high: > u/ml, intermediate: - u/ml, negative: < u/ml). she received tpe treatments every other day and started losartan. after a course of tpe, at rab decreased to u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to . ml/dl. in one month, her at rab increased again to > u/ml, therefore, she received more tpe treatments with a decrease in her at rab to u/ml. although at rab level increased slightly to u/ml after months, her cr has been stable at . - . ml/dl. case : the patient is a -year-old mean /-se - . /- . % * . % /- . %* * p< . a female with malignant hypertension who received a deceased donor kidney transplant at age . her cr started to rise weeks post-transplant from . to . mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at rab level at u/ml. she received tpe procedures every other day and at rab decreased to u/ml with a decrease of cr to . mg/dl and improved arteriopathy in histology. because her at rab level slightly increased to u/ml over the next weeks, she started weekly tpe treatment. after weekly tpe, tpe treatment was stopped because her at rab level remained relatively unchanged. her cr has been stable at around . ml/dl to date. conclusion: we present kidney transplant recipients who received tpe treatments for high at rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at rab levels; however, weekly tpe had no effect on reducing at rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < %], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a year old man (patient b) with a medical history of hypothyroidism (on synthroid for years), end stage renal disease and non-ischemic cardiomyopathy (ef of - %) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a - . plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within hours of the procedure completion. their total t , t and free t levels trended to normal or near normal range within hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy - weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* , laura martínez molina , cristina muniesa montserrat , octavio servitje bedate , silvia cosano navarro and maria isabel gonz alez medina . banc de sang i teixits, dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) , as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category . since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of patients diagnosed with ss and compare them in their first evaluation once the th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( -methoxypsoralen, -mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from . to total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in / patients) and with online system (therakos) just in . main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is ' % (partial remission . % and complete remission . % with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : u/l) requiring transfusions, mild thrombocytopenia ( x /l), acute kidney injury (bun mg/dl, creatinine . mg/dl). by the third hospitalization day hgb improved to g/dl, however with worsening thrombocytopenia ( x /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days - ). platelet count and a-ipc improved to x /l and . x /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count ( x /l) and a-ipc . x /l. these dynamics did not resemble those which had been described for ttp patients with adamts deficiency. adamts obtained prior to tpe initiation was resulted at this time and was %. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc ( . x /l) that preceded platelet count increase to x /l three days later when patient was discharged. other laboratory values at this time were ldh of u/l, hgb: . g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts deficiency eiman hussein* and jun teruya . department of clinical pathology, cairo university, texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts . since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts , the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts . the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january through march were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts activity of less than %. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts deficiency. eight patients ( %) were associated with suspected bacterial infection. four of the patients ( %) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than kilograms using a single apheresis procedure. study design/method: in october and june , two children with possible leukemia were submitted to tl procedure. they were and months old, and weighted , and , kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, % hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin ( ml of acd-a and , units of heparin), at a blood to anticoagulant ratio of : . a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were . and . / mm . the formula "collection pump flow , x inlet flow x preapheresis wbc count" was used with the goal of removing up to x leukocytes/ml. a single leukapheresis procedure was performed with total blood volume processed per patient. immediately after the -hour procedures, wbc count were . and . wbc/mm , and -hour post tl, wbc count were respectively . and . /mm . net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since . we report the data from the year . study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of , apheresis procedures were performed at hospitals. therapeutic plasmapheresis was the most frequent procedure ( . %) followed by autologous peripheral blood stem cell (pbsc) collection ( . %), allogeneic pbsc collection ( . %), donor leukapheresis ( . %), and therapeutic leukapheresis ( . %). cobe spectra ( . %) and amicus ( . %) were the most widely distributed instruments. centrifugation was the dominant technique ( . %) for therapeutic plasmapheresis. detailed information was given for , therapeutic plasmapheresis procedures performed on patients (some items were not completely filled out). spectra optia ( . %) and cobe spectra ( . %) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently ( . %) as the replacement fluid followed by % albumin ( . %), % albumin ( . %), and % albumin ffp ( . %). most of the procedures were performed for plasma volume ( . %). acd ( . %) and heparin ( . %) were used for anticoagulation. central venous catheter ( . %) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation ( . %), antibody mediated rejection in renal transplantation ( . %), thrombotic microangiopathy ( . %), desensitization for abo compatible renal transplantation ( . %), neuromyelitis optica spectrum disorders ( . %), and hyperviscosity in monoclonal gammopathies ( . %). adverse reactions were observed in . % of the procedures. allergic reaction ( . %), hypocalcemic symptom ( . %), and hypotension ( . %) were frequently reported. therapeutic effect was achieved in . % of the patients. our apheresis registry has been well run for years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride > - mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $ deaths/ , cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of pts who were diagnosed with hp from january, through january, , and referred for immunotherapy evaluation. / ( %) pts received conventional therapy (ct) and pe (pe group), and / ( %) pts received ct alone (ct group). mean age was years (range - ), and % were female. baseline mean triglyceride level (normal < mg/dl) for pe group was , mg/dl ( , - , ) versus , mg/dl ( , - , ) for ct group. baseline mean lipase level (normal < u/l) for pe group was , u/l ( - , ) versus u/l ( - , ) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving - medications. / ( %) of pe group and / ( %) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. / ( %) of pe group and / ( %) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of . pe treatments (txs) (median of , range - daily txs) using % albumin; / ( %) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were < - mg/dl and lipase < - u/l ( . - . x upper limit of normal). mean triglyceride levels after pe txs were , mg/dl ( - , ) for pe group (mean decrease %); mean triglyceride levels after additional hours of ongoing ct were , mg/dl ( - , ) for ct group (mean decrease %). while the pe group achieved a greater mean decrease in triglyceride levels after pe txs (compared to the ct group after hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p> . ). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a -year-old man with a chronic history of hypertriglyceridemia > mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa guidelines, in a patient unresponsive to optimal medical management. asfa guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely % albumin for exchange fluid ( % albumin procedures) or partial plasma replacement ( - units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while were performed with % albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* , , metha apiwattanakul , sompis santipong , jutaluk jaipian , jettawan siriaksorn and ponlapat rojnuckarin . chulalongkorn university, king chulalongkorn memorial hospital, prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june through february were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a % solution. before using, it was diluted to a % albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of tpes in patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was , (range , - , ) ml. although the corrected calcium level was low (< mg/dl) in . % ( / ) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in patients. the first patient had events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was . % ( / ). in , the incidence of tpe adverse effects was . % ( / ) when commercial albumin was used. the difference was not statistically different (p . ). median serum albumin levels pre-tpe and post-tpe were . ( . - . ) and . ( . - . ) g/dl. the increase in serum albumin after tpe was statistically significant (p< . ). eighty-two percent of pre-tpe serum albumin levels were lower than . g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether outpatients who underwent a total of la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer ( %) and ovarian cancer ( %). based on differences in the study protocols la was performed either one-time ( %), two-times ( %) or three-times ( %), with an interval of at least weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, out of patients ( %) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in % of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was . x wbc consisting of . x mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was % lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg -related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to , iu/mls (ref. range < ) and anti-cyclic citrulline peptide antibody was elevated to , units (ref. range < ) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were , and mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was . . peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be . centipoise (cp) at admission (ref. range . - . ). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated - total plasma volume; replacement fluid - % albumin and normal saline in a : ratio; replacement fluid volume: % of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to . cp. serum igg, igm and iga levels decreased to , and mg/dl respectively. her rf had decreased to , iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan -dec ). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with % human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between to years age (m: f; : ) underwent tpe procedures with an average of . per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three ( %) patients had only visual symptoms, ( %) had both visual as well as muscular symptoms whereas ( %) patients had muscular symptoms only. three ( %) out of the seven tested, were positive for aqp -igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in patients with grade- , in patient, and by grade- , in seven. adverse events were observed in % ( / ) of the procedures with allergic reactions to replacement fluid as most common event (n- ) followed by hypotension (n- ). follow up was available in % ( / ) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from . to . for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version . as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version . . the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to / / , plt collections were performed on nine trima accel machines operating with version . . upgrading and validating all nine machines to version . occurred from / / to / / . the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version . ( / / to / / ) was compared to version . ( / / to / / ). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version . and . , adjusting for multiple visits per donor, with significance defined as p-value < . . results/findings: following the upgrade to version . , staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version . of the trima accel showed a statistically significant increase in possible leukocyte contamination from % to % of collections as compared with version . . both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version . . conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version . software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version . currently does not provide added value over version . for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of ml % albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with acute episodes requiring prolong hospital admission of approximately months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of mg/dl, lipase u/l, glucose mg/dl, bicarbonate mmol/l, anion gap . ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day after admission. results/finding: the patient tg decreased by % ( mg/dl) with medical therapy, followed by additional % ( mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below mg/dl at days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from to , a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected , cases whose ferritin levels have been monitored more than twice with an interval of detection in - days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over lg/l. and the upper limit was set to be lg/ l in male and lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: times, to times, to times, to times and more than times. the high frequency (more than times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were , donors included in the study, of which , were male ( . %) and were female ( . %). the mean ferritin was . lg/l in male ( % ci: . - . lg/l) and . lg/l in female ( % ci: . - . lg/l). the result of anova indicates that the group with the highest frequency (more than times) has the significant lowest ferritin level (p< . ). the average change of ferritin if donation over times would up to . and . lg/l in younger and elder y/o male and and lg/l in female. and then for high frequency (half a year more than times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over times in $ days) was reduced from . lg/l in the first period to . lg/l in the third period ( period $ days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* , mary townsend and lizabeth rosenbaum . university of new mexico hospital, blood systems, inc., blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* , william korzun , teresa nadder , susan roseff and elizabeth ripley . virginia commonwealth university, virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for % of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a -hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from to could be a result of a change in the blood drive timing of the training schedule of that location. in , basic trainees at site a were scheduled at day of . in january , the blood drive date changed to day of . the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from to of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in . these observations support the hypothesis that the increase in hemoglobin deferrals in resulted from the implementation of the male hemoglobin standard change from . to . g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional % of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* , eva alonso , oscar bascuñana , monica romero , teresa vich , elena castaño , laura carbonell , eva palomas , saray almerge , francesc carpio and xavier curia . banc de sang i teixits, institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january to december we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, donors came to give blood, of these, ( %) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t but his hemoglobin levels were lower than our selection criteria. of the donors selected for donation ( . %) had sci lower than t and t . adverse reactions to donation ( . %) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of , automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an month period from january to november . the same information was assembled for the automated bp process for the month period of january to november . the automated bp process implemented in mid-december ; so the december data for both and has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < . . results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p . ). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < . ) with the automated bp while and reactions remained non-significantly lower (p . ). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below . g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < . . results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under . . statistically more visits with hgb less than . g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under . was . % higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from january to march . after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of donors who has donated blood in the blood bank's main branch were used as the baseline for this study. % of donors (n ) accepted automatic appointment booking, whereas some donors (n ) were not comfortable with it. % of those who declined still preferred walk-ins (n ) based on their own time schedule, the rest decided that variable situations (n ), donation frequency (n ) and choice of preferred donation locations (n ) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was %. a comparison was made and found that this study shown a significant increase of acceptance rate by %. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. ) , . . poisson distribution, normal distribution, logistic distribution, lognormal distribution a transfusion (p> . ) in donor and reference populations except in younger ( - yrs) male donors (p< . ; donor . %, reference . %). mean donor sbp, dbp, and pulse were . mmhg, . . mmhg, and . . bpm, respectively. screening blood pressure levels consistent with hypertension ( . % male; . % female) in the - year donor group, significantly (p< . ) higher than the reference population ( . % male; . % female). no differences were observed in the - year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in - year old females. developing blood donor educational materials gay wehrli* , susan rossmann , louis m. katz and dan a waxman . university of virginia health system, gulf coast regional blood center -sugar land, americas blood centers, indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, . page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table . results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level ( th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from to after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from . % to . % in and , respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from . % in to % in . moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately . million doses of ap transfused within . billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since . firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than times and had not donated for more than days or less than times with an interval of more than days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by . times from to and the doses of ap increased by . times from to within years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into groups: those who donated ap once, those who donated - times, - times, - times, and those who donated more than times, respectively. it was found that the number of permanent ap donors who donated ap more than times was only ( . %), but they denoted a total of doses of ap ( . %) from to . conclusion: aps increased at a rapid and steady pace in wuhan blood center from to , which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at sites on consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer < ng/ml and zpp levels > umol/mol heme) at hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp . % (r . ) at first and . % (r . ) at second donations. at first donation when compared to fs hb, only . % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. at second donation, when compared to fs hb, only % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p< . ) suggesting strong evidence against correlation. % ( ) responded to the survey of which % ( ) reported not feeling well after donation. it should be noted that noted that % ( ) female study participants reported feeling unwell after the first donation and had ferritin levels below ng/ml but the zpp levels were less than umol/mol heme. of the % ( ) male participants that reported not feeling well none had ferritin levels below ng/ml nor ven or fs zpp levels above umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from . to . gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from blood centers over two intervals, july-dec. and july-dec. (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male m, female f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab . , chicago il). p <. was considered significant. results/findings: data were provided by of centers invited, representing , , and , , wb donations and , and , ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from . % to . % in the two intervals among aggregated donation attempts (p<. ), and for m ap from . to . % (p<. ). the mean "by center" deferral rates (table) were similar to that and significant (p<. ). mean by center hb deferral rates among f donations during the two intervals were . and . % (p . ) for wb, . and . % (p . ) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only centers could provide specific high vs. low vs. irregular pulse deferrals; provided only a summary (i.e total pulse deferrals), and could provide none. for bp, provided detail (high vs. low), summary and none. p deferrals increased in the successive intervals among f wb donors from a center mean of . to . % (p . ) and for m wb donors from . to . % (p . ). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba c levels among those with rbs > mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from st march to st march . total of , blood donors were tested for rbs. those with rbs > mg/dl were further tested for hba c by gold standard hplc method using variant ii biorad. blood donors with > mg/dl rbs and hba c > . % were advised to consult a physician for further evaluation. results/findings: of the , donors tested, ( . %) donors showed a rbs of > mg/dl. forty two ( . %) were males and ( . %) females with a mean age of . years ( - years). of these, ( . %) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining , ( . %) of them had a family history of dm. of these donors, donors did not give a consent for testing for hba c. among the donors tested for hba c levels, ( . %) had hba c > . %. all the donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is . % ( of donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* , hwee huang tan and ai leen ang . health sciences authority blood services group, health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from . to . g/dl last may . the current minimum acceptable hemoglobin for male donors in singapore is . g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels ( . - . ) and in donors with hemoglobin g/dl and above. study design/method: during a month period, serum ferritin testing was performed on regular male whole blood and regular male apheresis donors who made at least donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin . - . ) group b (whole blood with hemoglobin ! , group c (apheresis with hemoglobin . - . ) and group d (apheresis with hemoglobin ! ). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below ug/l is considered low and levels below < ug/l are considered having absent iron stores. results/findings: . % of donors in the study have ferritin levels below ug/l. there were more donors with low ferritin in group a compared to group b, % and % respectively (p< . ). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, % and % respectively (p . ). ferritin results for the groups can be seen in table . conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or . % have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin . - . g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to . g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of % male and % female students in the age group of - years. only % of the students have heard about voluntary blood donation and % of the students have given blood once in their lifetime and among them % are blood donors at the moment. % of the participants believed that there is a specific reason why they don't donate blood and % believed that there is a risk involved for the donors, when donating blood. % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. ( ); miscellaneous effects were reported in courses. side effects led to interruption of supplementation in instances. ferritin levels (mgt sd) at entry into the program and at the last visit were . and . . mg/l in participants, vs . . and . . mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took ! % of the tablets. ferritin levels< mg/l were found in , % of participants and . % of controls. deferral for low hemoglobin was below % in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only % of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under cfr . and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § . to make a collection under this provision if the requirements set forth in § . (a)( ) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in , an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of - ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached , of whom ( %) are c y homozygotes. without active recruitment, accrual rate is about per quarter, with % of subjects qualifying as allogeneic donors. the mean current age is . years, % male, % caucasian. the majority of hh donors ( of an active cohort of ) are in the maintenance phase of therapy with an average of . donations/year and a % deferral rate. over the last years, hh donors contributed approximately - % of the hospital's allogeneic blood supply, averaging whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided - % of blood for in vitro research at our institution with an average of wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over years. since / / , with an increase in male hgb deferral threshold to g/dl, there has been only hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm- non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm- non-invasive occlusion spectroscopy device. over a span of days, eligible blood donors, both male and female, were first screened by the nbm- non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the blood donors for the performance of hb measurement on the sysmex hematology analyzer within - hours of collecting the venous samples. results/finding: the sd of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed . g/dl. the hb measurements obtained from the nbm- and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be . g/dl. the precision of the nbm- yielded a co-efficient of variation of . g/dl and a standard deviation of . g/dl. conclusion: the operators found the nbm- easy to install, maintain, and operate with minimal training. the nbm- non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder , ravi reddy , dhuly chowdhury , don brambilla and edward l. murphy* . sanbs, rti international, ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in and followed them for one year. within days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used -point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. ) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included , first-time black donors with median age and female predominance ( %). within one year, , donors ( %) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable ( strongly agree to strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) . , % ci . - . ), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or . , % ci . - . ). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or . , % ci . - . ) and "i wasn't treated well by the <blood center> staff" (or . , % ci . - . ). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or . , % ci . - . ). a secondary analysis treating the likert scales as -level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard , ramya ghantasala , obhijit d hazarika , nicole leonard , cori a polonski , zachary b wunrow , michelle heleba , jan k carney and mark k fung* . university of vermont larner college of medicine, american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" ( . %) and "traveling is a time for me to relax." ( . %). of the respondents who travel in the summer, very few reported donating while traveling ( . %). summer donation rates between summertime travelers ( . %) and non-travelers ( . %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website ( . %) and phone ( . %). willingness to use a regional blood donation smartphone app was highest among respondents ages of to ( - %) and lowest among ages and older ( - %). of respondents with no prior knowledge of summer seasonal shortages ( %), / rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv , and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin- (il- ) and il . others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent hard spins at rpm for minutes with separation after each spin on a compomatev r g . plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers ( m [ %]: [ %] f); median age years (range - ) donated a unit ( ml) blood from which buffy coats (average volume ml) were processed. the buffy coat process was previously validated on wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures and . all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by % within months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for days. results/findings: there were completed responses, of which ( %) indicated that their hospital had an msbos and ( %) did not. the majority of hospitals without an msbos were academic centers ( / , %) from oceania ( / , %) or europe ( / , %), had between - beds ( / , %); the majority of these hospitals transfused between , - , rbcs ( / , %) per year. / ( %) are going to implement an msbos in . of those with an msbos, the majority / ( %) were from north america. the majority were academic hospitals ( / , %) with - beds ( / , %) that transfused ! , rbc units per year ( / , %) offering a wide range of surgical services. on average there were procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of % of the procedures listed, a pre-operative type and screen for %, crossmatching rbc units for %, and for % of procedures a different recommendation was made. most ( / , %) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only / ( %) of msbos' were created solely by using procedure-specific data, and most ( / , %) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually ( / , %), and the hospital transfusion committee is often ( / , %) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents ( %) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, % of respondents felt that it was regularly used by all surgeons and anesthesiologists; % felt that it was not used at all at their hospital, % did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, % of the hospitals currently without one indicated that it would be implemented in suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a -bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see , hospital admissions and nearly , emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in was approximately $ . m. in nov. , an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $ . m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns ( and ), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee . development of evidence based transfusion triggers. . education on evidence based transfusion triggers across multiple campuses, specialties and resident programs . clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to " " unit instead of " " units. . updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in , we were still able to reduced blood product expenditures by $ , when compared to . conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* , anna w rains and christopher t clark . university of tennessee graduate school of medicine, univeristy of tennessee medical center, univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for week post-delivery, with cost of $ . per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc system. by using the blood volume values, and assigning a value of . ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in , there was a total of , infants delivered at our facility. out of all the deliveries, ( %) infants were transferred to the nicu. of those infants, % received at least one red blood cell transfusion and % received at least one platelet transfusion. of the infants transferred to the nicu, ( %) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to % (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from . % all the way up to . %. in those infants, the birth weight ranged from - grams, and the gestational age ranged from weeks to weeks and days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* , shannon davis , suzan new , vaishali patel and oren guttman . university of texas southwestern medical center, ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable : communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to % of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july ; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of randomized controlled trials ( , conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* and stephanie rogers . dignity health st joseph's medical center, dignity health background/case studies: over million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels > . g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of . g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of . g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to hospitals including cme presentations, online physician and nursing educational videos, communication tools including infographics and " is the new " buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb > . g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy to fytd , there was a % reduction in prbc units transfused to patients with hgb > . g/dl, starting at a baseline of % down to %. this represents an fy annualized savings of $ . m, from a baseline of units per , patients days down to an average units and approximately , fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels > . g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm - . ) reduced rbc units and two studies decreased the percentage of patients transfused (or . ). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or . ) and rbc units transfused (sdm - . ). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a- method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was g/dl/ % or g/dl/ %. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately % of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was units/ month. after the first months of buc activation the mean number of units was units/month a reduction of units/month or % of nonsurgical blood use (p . by t-test). non-surgical rbc use now represents approximately % of the total rbc use hospital wide a % reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital - month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital ( beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance ( q vs. q ): present and completed consents ( vs. %), present and completed nursing flow sheets ( vs. %), transfusion thresholds supported ( vs. %), discharge instructions provided ( vs. %); ( q vs. q ) vital sign compliance ( % vs. %). jwp increased from to ( / - / ). cost savings were realized by decreased utilization and implementation of bpa. (table - q ) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately - trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only percent of coolers fully used. it also consumed valuable staff time as technologists typically made - trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated - hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains - units of group o rhd negative, units of o rhd positive, and units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in , the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june -october were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a -month period, , cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table below. overall, ( %) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." ( ), "active bleeding" ( ), "platelet count of . . ." ( ), and "downtrend" ( ), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, ( %) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years - . study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of , g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey ( questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of complete responses ( %). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, % with physicians, and of these, % reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only % responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of - g/dl ( %), platelet count of - , ( %), and inr of greater than . ( %). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per , inhabitants may vary folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies ( . % and . %), for the remaining strategies weights varied between . % and . %. we estimate that , patients would be eligible for pbm strategies in one year time horizon, resulting in premature death avoided ( . % reduction) corresponding to a gain of approximately , life years and a reduction of , ( . %) disability adjusted life years (daly) relative to the current clinical practice. a decrease of , in-hospital days is expected mainly due to a . % reduction in hospital length of stay and a . % reduction in -day readmission rate. in this population the overall transfusion rate could decrease to . % from the current . % ( . % reduction) implying , blood transfusion avoided and , red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted < ml of rbcs, all cases with ml transfused were captured and only . % of the time > ml were used. if - ml rbcs were predicted to be transfused, > ml were used % of the time. if predicted usage was > ml, % of the time it exceeded ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for > ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than . is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a month period (jan to mar ). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the month period, there were patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of units of blood were requested. units were crossmatched, of which units were sent to the operating theatre (ot). only . % of blood issued to ot were transfused (n ) while the rest were unutilized. the observed ct ratio was . . conclusion: although only % of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of ! . , with almost % of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study patients who received tranexamic acid (txa) (study group) and patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < . g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were unique alert encounters. of these, ( . %) led to a crossmatch being ordered while ( . %) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds ( . g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds ( . or . g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at academic hospitals with data-derived msbos. study design/method: the hospitals were in groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if % of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if - % of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if ! % of the patients had been transfused. data were collected at each center over a month period between january to march and included a maximum of cases per hospital during that one month to ensure equal representation between centers results/finding: between these centers there were a total of cases analyzed. some of the more frequently performed surgeries included orthopedics ( % of cases), general surgery ( %) and cardiac surgery ( %). there were t&s ordered for these cases, of which were positive for antibodies on the day of surgery. of all the t&s ordered, % were ordered in accord with the msbos recommendation, % were ordered when the msbos did not recommend one, and in . % a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio . for prenatal hemoglobin (hgb) - . ). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of % among obstetric patients. study design/method: we studied a sample of anemic (hgb< . g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of women were enrolled, with median age (interquartile range - ) years, median gravida / para and median gestational age weeks. mean hgb before referral was . g/dl and most were already taking oral iron therapy. a total of women were hiv positive with mean cd lymphocytes counts of cells/ul; ( %) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and ( %) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in ( %) of women. there was concurrent chronic disease (n ), infection (n ), vitamin b deficiency (n ) and antenatal hemorrhage (n ); had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a month old boy presented to our institution after a month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige ( ku/l; rr: - . ). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp , while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp cd lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $ . %. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of . % over the last years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from to , with a peak of . % (range . %- . %). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for mtps. results/finding: in , mtp was activated times. in cases the patient did not receive any blood product and in cases plasma was already available at the time of rbc allocation/issue. this left cases to evaluate. the median time to plasma availability was minutes (range minutes - minutes). the mean plasma wastage for mtp activations was % (range - %). of the cha replies, were using thawed plasma and their wastage was </ %. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of minutes. our implementation began with thawing a single unit of plasma for "level neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are . %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated cases of gtx in pediatric patients with age from months to years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of granulocytes donations from to were collected from health donors ( male/ female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, mcg/kg/dose (donors up to kg, maximum of mcg) and oral dexamethasone ( mg), h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with gy. results/finding s: granulocytes number increased times from basal levels and the median of cells collected were . x (range . - . ). in general donors reported no significant adverse reactions. from donations, only in was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table . adverse reactions for patients were minor and not frequent ( in gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* , elena kurnikova and pavel trakhtman . russian institute for paediatric haematology, oncology and immunology, instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period / - / . donors underwent granulocyte mobilization with g-csf in a dose - mg/kg s.c. - hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed granulocytes collections in donors ( f, m), ages to . the mean increment of wbc after stimulation was , x /l ( , - , x /l; f- , x /l; m- . x /l); in the age groups - years (n ) and over years (n ), the mean increment of wbc was respectively , x /l ( , - , x /l) and , x /l ( , - x /l). the mean volume of treated blood was , a , ) . the mean number of total wbc in the product was , x /l ( . - x / l; f- , x /l, m- , x /l). only three donors ( , %) had apheresisrelated adverse effects the analysis included granulocyte transfusions in cases of infectious complications in patients. the mean number of transfusions was . ( - ). each patient received transfusions from mean donors ( - ); the granulocyte transfusions started at the mean days ( - ) after infection; the mean dose of wbc on the transfusion was . x /kg ( - . x /kg). wbc increment was able to estimate after transfusions. wbc increment was recorded after transfusions ( . %); the mean increment was , x /l ( , - , x /l). efficacy was determined by the number of patients who survived an infection episode and amounted to . %. in the cases of severe sepsis, the efficacy of transfusions was . %. the frequency of post-transfusion reactions was %, (n ): febrile nonhemolytic reactions , % (n ); pulmonary complications , % (n ); the anti-hla antibodies formation , % (n ) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp hemolytic disease of the newborn due to anti-rh keegan barry-holson* and jennifer andrews . stanford university, dept of pathology, stanford university, dept of pathology & pediatrics background/case studies: anti-rh is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g p > mother with a negative st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at hours of life because he was found to have anemia (hemoglobin . g/dl), severe hyperbilirubinemia (total bilirubin (t bili) . mg/dl), reticulocytosis ( %) and a positive direct antiglobulin test (igg ). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of . mg/dl on day of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin . g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* , cyril jacquot , valli criss , philippe p pary , jay greenberg , naomi lc luban and edward cc wong . children's national medical center, quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as - %. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of . g/dl/ . %. wbc counts ( x /l) were mildly elevated and platelet counts ( x /l) were within normal limits. her history was notable for upper respiratory tract infection days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total . mg/dl, direct . mg/dl), normal ldh ( u/l), and undetectable haptoglobin (< mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin ( - reactivity) with positive autocontrol. dat was positive for anti-igg and negative for c despite a positive cold antibody screen. the patient weighed . kg with an estimated total blood volume of ml. she initially received simple transfusions totaling ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of %, utilizing the central venous catheter. no adverse events took place over the course of the hour exchange. her one hour post-exchange hb was . g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin ( ). after initiation of steroid therapy (methylprednisolone, mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels ( mg/dl). at a subsequent follow-up visit months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a or gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, . /- . ml/ second) or with a mechanical syringe pump (slowly, ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the g catheter, the mean change in hct was - . /- . % with the one-way valve and . /- . % without (p< . ). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p< . ). during rapid manual transfusion with a g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse . ml when using a one-way valve (change in hct versus time: r - . , p< . ) compared to a significantly different (p . ) slight increase in hemolysis for samples that took less time to transfuse . ml when not using a one-way valve (change in hct versus time: r . , p . ). correlations between time and hemolysis were similar, but insignificant using g with washed rbcs and the g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in . % of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge . study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at weeks gestation with passive anti-d and an anti-ge titer of . she was d-and ge:- ,- , by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge . the birth hemoglobin (hgb) was . g/dl, reticulocyte (retic) was . %, bilirubin (bili) was . mg/dl; the infant was discharged. on day of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb . g/dl, retic . %, and bili . mg/dl. ge -blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh . obstetrics had to authorize maternal blood donation due to her hgb of . g/dl. maternal blood collection and rbc washing was expedited and the infant received ml of maternal rbcs within hours, at which time his hgb was . g/dl. post-transfusion hgb was . g/dl. one week later, the infant was symptomatic with hgb . g/dl, retic . %, bili . mg/dl. a nd aliquot of ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb . g/dl, retic . %, anti-ge titer , and needed another transfusion. the maternal blood stored for just weeks had hemolyzed necessitating a nd maternal donation for baby's rd transfusion. at weeks, the infant's anti-ge titer was , hgb . g/dl, retic . %; no transfusion was necessary. at weeks of life, hgb was . g/dl, retic was . %, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge hdfn. molecular analysis revealed that the mother was homozygous ge -negative ge* .- , the father had homozygous wild type ge* , and the infant was heterozygous ge* /ge* .- . conclusion: the infant had hdfn due to antibodies to the high prevalence ge antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge . hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results ( ) supporting a hemoglobin trigger of g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in , , rbc orders occurred and the top three patient groups were: % in congenital heart disease patients, % in hematology/oncology patients and % in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was . g/dl as measured in the hours prior to rbc order placement. in , ffp orders occurred and the top three patient groups were: % in neonates in the nicu, % in congenital heart disease patients and % in pediatric intensive care patients. average inr of every patient was . as measured in the hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates ( - months of age), infants (> - months of age) and children (> months- years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after months, pr-plt represented % of our platelet inventory (average daily plt inventory: units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among transfused neonates ( - m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased , dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of pediatric patients receiving prbc transfusion over a month period were retrospectively reviewed. a total of patients were identified as receiving allogeneic prbc transfusion. patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was . g/dl with post-transfusion hgb rising to . g/dl. the mean prbc volume transfused was . ml using a dose of ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of - ml/kg in a kg patient, for instance, would translate into full prbc units (about ml) in an average size adult. the current standard dose of - ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a month-old, previously healthy female infant who presented to the hospital with a -week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of . g/dl and . %, respectively, platelets of , , and a reticulocyte count of . %. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of . mg/dl with a direct fraction of . mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens ( total) were non-detectable. the patient was started on a -day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c . an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her -day hospital course, the patient received rbc transfusions on the day of admission and several rbc transfusions thereafter (see table ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day . her hemoglobin rose to . g/dl on hospital day and increased to . g/dl on hospital day . at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to . g/dl on day after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is or . platelet and leukocyte immunohematology, testing and genetics table . of pairs, pairs were complete match ( / ), pairs were partial match ( / ), pairs were complete mismatch ( / ). the matching rate of hla-dpb in our study is %. conclusion: the matching rate of hla-dpb in / hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china ( ) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts . therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a year old male with a -year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts < % and inhibitor of . . on day of admission platelet count was x /l which decreased within five days to x /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were x /l and x /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to . x /l achieving the ratio of previously shown to be diagnostic of ttp. on day his a-ipc and platelet counts had improved to . x /l and x /l respectively. absence of anti-pf antibodies ruled out heparin-induced thrombocytopenia at this time. on day he had an unexpected decrease in both a-ipc and platelet count to . x /l and x /l respectively, worsening by day to . x /l and x /l respectively despite daily tpe. patient received additional tpes that failed to improve a-ipc or platelets which on day were . x /l and x /l respectively. a-ipc had remained at this level for days suggesting that the observed decrease was irreversible. adamts activity remained < % low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa- a, hpa- a, and hpa- a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa- a, hpa- a, and hpa- a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p and gi , specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa- aa, hpa- ab and hpa- aa) were collected and reacted with anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex . the hpa- a serum was diluted to serial dilutions (from neat to / ) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa- a, hpa- a, hpa- a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( . vs . ), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa- a, anti-hpa- a, anti-hpa- a. furthermore, because the platelet was hpa- aa, the hpa- b serum did not react with the coupled beads with mfi was comparable to negative control ( . vs . ). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa- a was / ( . iu/ml) and / ( . iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa- a, hpa- a and hpa- a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in % of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from january to december . clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c , c bp, thbd, dgke, cfhr , cfhr , cfhr and cfhr ) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of patients tested, pathogenic mutations were detected in % ( / ) and vus in % ( / ). % ( / ) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and ( %) had vus. % ( / ) of patients had primary ahus; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. % ( / ) of patients had secondary tma; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. in patients with pathogenic mutations, % ( / ) were children, . % ( / ) had a positive family history of ahus and % ( / ) had recurrent disease. patients with primary ahus had a significantly lower age at presentation ( vs. yrs; p-value: . ) and a higher proportion of pathogenic mutations ( % vs. % p-value: . ) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl and sysmex xn compared with a flow cytometric method farshid ezligini , kjersti roen eriksen , annette vetlesen , thomas larsen titze and geir hetland* , . oslo university hospital, university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion ). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl (horiba abx, montpelier, france) and xn sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x /l sd were , (<) , (<) and for counting by sysmex toa, pentraxl , and the gallios flow cytometer, respectively. sysmex count was the very lowest a transfusion vol. supplement s abstract ( . % less than for flow cytometry), but all plt counts were significantly different (p< . ), although least so ( . %) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing , arishma lata , roland russnak , zachary antovich , heather dunckley and thierry viard* . new zealand blood service, linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of reactions that identify both variants of relevant snps located within hpa genes (hpa- through hpa- , and hpa ). genomic dna purified from blood samples, previously genotyped for hpa- ,- ,- ,- ,- and - by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were % concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late and to date has tested dna samples from blood donors ( donors were tested in duplicate). concordance between the sample replicates was %. there were occasions where the assay had to be repeated, giving a repeat rate of . %. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa- ( . %) and hpa- ( . %) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd antigen deficiency expression in jiangsu chinese han population qing chen* , jianyu xiao and chengyin huang . jiangsu province blood center, jiangsu province blood center background/case studies: cd has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd deficiency varies widely among different ethnic populations, with the frequency of - % in asians and . % of african americans, respectively. however, there is little information on the molecular basis of individuals with cd deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd expression levels and to determine the molecular basis of cd deficiency on the platelet surface of the han population in jiangsu region. cd expression levels on platelets were detected by flow cytometry among blood donors in jiangsu region. donors without cd antigen expression on their platelet surface were further to be determined the expression of cd antigen on their peripheral blood monocyte cells. the coding exons of cd gene and adjacent introns were amplified and sequenced in cd deficient individuals. results/finding: among these blood donors, cd -deficient and cd -expression individuals were . % ( / ) and . % ( / ), respectively. the frequencies of type i and type ii cd deficiency among the study population were . % ( / ) and . % ( / ), respectively. among individual with platelet cd expression, according to mean fluorescence intensity (mfi) value, , and individuals showed low, moderate and high expression levels of cd , respectively, and their mfis were . . , . . and . . (p< . ), respectively. the type i cd deficiency individual were heterozygous for - a>g and - c>g, respectively. among type ii cd deficiency individuals, two harbored a t insertion at position in exon which caused frameshift at codon ; one has a t>c exchange at position in exon which resulted in a tryptophan to arginine substitution at codon ; one has a a insertion before the th bp of the start codon atg in the promoter region; one were heterozygous for t>c and t>g, respectively. conclusion: platelet cd surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd deficiency was higher than that in type i. the study findings indicated that the frequency of cd deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd -deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd -deficient individuals widely varies among ethnic groups, with % to % in japanese, % in sub-saharan africans, . % in african americans, and . % in caucasians. although some studies of cd deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from unrelated platelet-apheresis donors in the eastern china. the expression of cd antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd and peanti-cd ). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd -nagtive phenotype. for those donors with cd -negative platelets, cd antigen expression on monocytes was analyzed further to distinguish between cd type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china ( ) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd in all samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd phenotypes using the (mean sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd deficiency on platelet, in which one sample was cd negative both on platelet and monocyte. the frequency of cd type i and type ii deficiency in the eastern chinese donors was . % and . %, respectively. the average mfi of cd deficiency samples was significantly lower than cd positive samples ( . . vs . . , p< . ). conclusion: the frequency of platelet cd deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd -deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd antibody. background/case studies: cd (gpiv, chromosome q . ) is an kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd deficiency (cd -n) is observed in - % of africans (t g) & is classified as either type i (cd -n plt, cd -n mono) or type ii (cd -n plt, cd mono). an acquired type ii cd -n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type cd -n individuals can develop anti-cd alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an year-old, group o african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin . g/dl & plt count k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, - % blasts & a complex karyotype with del( )(q q ) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < . hla antibody testing was negative (class i panel reactive antibody (pra) %). the patient was plt-xm-incompatible with most donors ( / ). a trial of group o, plt-xm-compatible plts was unsuccessful (cci ). subsequent testing for plt-specific alloantibodies identified anti-cd . fc-phenotyping showed no cd on patient's mono or plt, consistent with type i cd -n. preliminary dna results show that the patient is heterozygous for t g. because cd -n apheresis plt were unavailable from blood suppliers, the patient's children & grandson were screened as possible donors: all showed normal cd expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day ) demonstrated new class i alloantibodies (pra %) in response to transfusion ( apheresis plts, rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd -n & severe plt refractoriness in the setting of new mds, and q-chromosomal abnormalities. the absence of cd on plt & mono support congenital type cd -n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel , kristopher fernandez* , eric senaldi , pascal george , michael seul and ghazala hashmi . biomolecular analytics, central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur http://bit.ly/ q heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $ adult donors who had made ! donations in the previous months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for , , , , , , , , , and for hla class (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/ pdplf ) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a * : chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are , hla alleles documented according to the imgt / hla sequence database in janury , and more than % of them were identified in the last years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a * : allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a * : : : , but nucleotide substitution in exon , by nt c-a (codon acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a year-old pregnant woman typed as ab , who delivered a baby affected by severe hdfn. the newborn was typed as b and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted with the four monoclonal anti-d used (igm clones p x and rum and the blends clones th ms and d d ) and were typed as c-c e-e . an anti-d was identified in her serum. molecular analysis showed the c>t and a>c in exon , the snp t>c changes in exon and the t>g nucleotide change in exon . the set of snps found is similar to the molecular background of dol , except for a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of immuno hematological tests ( abo/d grouping (including newborn samples), extended erythrocytic phenotype, antibody screening, antibody identification, dat) and crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in . % of the abo/d tests (n ), , % of the antibody screening tests (n ), , % of the antibody identification tests (n ) and % of the dat tests (n ). there were discrepancies ( abo/d for the same patient, for antibody screening and antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than min). detection threshold of the d antibody was assessed at . ng/ml ( . ui/ml) whereas the french recommendations are ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a year old g p presented during her fifth pregnancy with anti-kpb with an initial titer measured of . by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams ( . moms) peaking at weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was . mg/dl with . g/dl hemoglobin. the baby typed as o positive, kp (b ) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by weeks of age. his hemoglobin recovered to . g/dl with an indirect bilirubin of . mg/dl at weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c d-specific dat may be too insensitive to detect low, but significant levels of c d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a " " reaction corresponds to about molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c d have been published. however, these are mainly designed to quantify the fraction of rbcs with c d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c d. study design/method: ten microliters (ul) of : (after documenting experimentally that this amount ensured maximum binding of anti-c d) mouse monoclonal anti-human anti-c d (abcam, clone c ) were added to ul of a . % rbc suspension. after incubation for minutes at c, samples were washed x , and ul of : diluted anti-mouse-f(ab) -pe (ro , dako) were added. after incubation at c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a rbcs stained with levels ( fold dilution, : - : ) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c d, edta-stabilized samples from healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c d (values ranging from to , abc) with level of -serum dilution (used to sensitize a rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r . , p < . ). compared with dc-screening , the sensitivity of the flow cytometric assay was superior. it detected c d sensitization at least dilution steps further. the median normal level of rbc-bound c d was abc (range - abc, n ). the assay enabled demonstration of specific c d-sensitization in the patient; the level of rbc-bound c d in the sample was significantly elevated ( , abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt gene. in humans, the abo and gbgt genetic loci are located on chromosome q , and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors ) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt genes have been constructed of vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion vol. supplement s the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c , e-, e and k -. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d , c-, e-, partial c , partial e . the probable rhce genotype, rhce*ce-jal/rhce*ce g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant woman and her newborn carolina bonet bub* , maria giselda aravechia , thiago costa , marilia sirianni , eduardo bastos , leandro santos , lilian castilho and jos e kutner . hospital israelita albert einstein, hemocentro unicamp background/case studies: rhd*weak d type is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion ) the c. g>c change (p.g a), which characterizes the rhd*weak d type allele is a splicing variant that induces skipping of the whole rhd exon . we report an altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- /ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d w c-c e e . the samples showed weak hemagglutination reactions ( / ) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon in both dna samples. sequencing showed the c. g>c change and the intronic c. - t>a and c. - t>c substitutions, which are associated to the rhd*weak d type allele. conclusion: our results showed that c. g>c associated with c. - t>a and c. - t>c variations had probably a functional impact on splicing inducing exclusion of exon in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type . background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than % of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with , patients and studies from other regions (brazil, egypt and france) with patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was to years and for the other countries to years. available data from us studies included a total of alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens ( . %, . %, . %, . %, . % and . % respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of . % ( . to . , % ci) vs. . % for non-us studies ( . - . , % ci) (p . ) and a transfusion vol. supplement s more alloantibodies per transfused patient ( . vs. . , p . ). similarly, the number of alloantibodies per transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort ( Á vs. . , p Á ). average number of rbc units transfused per patient in the us was also higher compared to data from france ( vs. , p . ). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* , melissa grohotolsky , lisa deblass , bala carver and kip kuttner . health network laboratories, miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: year old white mennonite female g ,p presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at . although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g (r g r) cells. both rhce*c and rhd genes encode ser which determines g expression. rare rhd variant antigens lacking ser are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e c e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r r , r'r, r g r and rr cells. this patient is predicted to be r r (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is a transfusion vol. supplement s excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* , christa voliva , kathy fletcher , heather vaught and tracie ingle . indiana university health, indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology ; : - ) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were > dat positive, were weakly dat positive and were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman . - . ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: bb, bg and bp. the ph of all eluates ranged from . - . with the highest percentage of eluates at a ph of . ( %). sixteen of the eluates tested yielded the same results in both automation platforms (concordance of %). four eluates with different results are summarized in table . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results ( ) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in led to the discovery of human blood groups. in the abo system > alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor ( units over years) who is actually a w . study design/methods: donations were tested with the pk instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns , and and exons and . specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table . tests with anti-a, -a , -b anti-a,b were negative as were the a cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table . the significant changes were found in exons and . in exon there was a nucleotide (nt) deletion of g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly ala. mutations in exon included a nt substitution causing a pro -leu change and a nt deletion c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o. . /abo*aw. ] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a and b cells. this abo discrepancy was caused by the presence of anti-a in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer > : . (schmidt, nacarrow et al. ) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. a transfusion vol. supplement s extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: edta blood tubes collected from random blood donors were used to extract dna from microliters of whole blood on day , and days post collection. blood samples were stored at - c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a /a using a nanodrop (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result ) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the samples, samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from . to . ng/ul. all readings with the exception of ( . ng/ ul) had concentrations > ng/ul. interestingly, the one that was < ng/ ul on day , yielded > ng/ul on day and post collection. over the next months, sets of samples were extracted and tested by hea. eighty-three ( . %) failed extraction and ( . %) failed hea. none of the samples that failed extraction were or days post collection; none of those that failed hea were days post collection; . % were > < days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas , carlos villa , rachel davis-rauser* , helen carpenter and vrunda patel . university of pennsylvania, hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in . communications suggest all patients receiving therapy would have a positive antibody screen because cd is a common antigen expressed on red blood cells. currently, patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at : dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for negative patients was observed up to a : dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a y/o g p at approximately weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [ , ] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m n-s s phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency > . ), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [ , ] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [ ] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays a transfusion vol. supplement s that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in dna samples from chronically transfused patients with scd, in patients with thalassemia and in dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in levels: ( ) rh and k matching; ( ) extended matching and ( ) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for % of our thalassemic and scd patients at level , % for scd patients and % for patients with thalassemia at level and % for patients with scd and % for patients with thalassemia at level . the patients were transfused with a median of . rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to - % with c e k matching and < % with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube ( drops plasma, minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to minutes and specimen volume to drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of clinically significant antibodies were detected using sprca technique, as well as warm autoantibodies and nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being % specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions ( versus ), it identified more clinically significant antibodies ( ) than liss ( ). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd on rbcs patricia a arndt* , anthony salazar and regina m. leger . american red cross blood services, long beach memorial medical center background/case studies: monoclonal anti-cd , e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or aminoethylisothiouronium bromide (aet). chapuy et al described ( ) and validated ( ) ), and % aet (ph . ) as per the aabb technical manual, th ed. these treated and untreated rbcs were stored in alsevers at c and tested on days , , , and by two methods: ) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from total dara patients were tested with reactivity - ), and ) flow cytometry using phycoerythrin (pe)labeled anti-cd . rbcs were also tested on days and or with a serum containing anti-k by peg iat. results/findings: the . m dtt in ph . pbs had a final ph of . and the ph of the commercial . m dtt was . . results are in table ; flow cytometry results from days , and (data not shown) were similar to those from days and . rbcs treated with . m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd by flow cytometry for up to days after treatment. rbcs treated with . m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening ( - %) of reactivity with pe anti-cd . background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon and ) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr ) in gestational week . the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of pregnant women were included. nip rhd was positive in / ( %), negative in / ( %) and inconclusive in / ( . %). compared to the postnatal rhd type, / ( . %) of nip rhd results were false positive (fp) and / ( . %) were false negative. in / ( %) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n ) at gestational week was . ( -and -percentiles: . - . ). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p , ). the fraction of ccfdna was calculated for randomly selected nip rhd true positive cases. median ccfdna ratio was . (the distribution had a highly positive skew, -and -percentiles: . - . ). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r , ; p . ). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of , suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only in , donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, lan-rbc units were transfused over days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day , the patient had symptomatic anemia with hemoglobin (hb) . g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of . g/dl was maintained for days. the antibody screen was negative on day post-transfusion, but strongly panreactive on day , with a positive dat (igg , c ) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from . g/dl on day to . g/dl by day with no bleeding identified, and increase in total bilirubin and ldh (peak . mg/dl and u/l on day ) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day with good response (hb . g/dl). the patient remained stable and was discharged to a skilled nursing facility days later. conclusion: transfusion of lan rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* , andrea gerner , lynne stewart , carol sostok , mollie bell and gregory r halverson . st. elizabeth healthcare, hoxworth blood center background/case studies: anti-f was first described in by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the th antigen assigned to the rh blood group system (isbt rh ). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused unit o-rbcs. two weeks later the patient received an additional o-rbc. within days the hgb had decreased from . to . g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused r r (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c e f phenotype. the rh phenotype and as was repeated on a sample collected days later. the c typing was micro positive, mixed field only after minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post hour hgb increment from the receipt of a standard unit of blood should be near g/dl (or % hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a . g/dl increase, and the second unit was only . g/dl. the last transfusion of units increased by only . g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of r r units was nearly complete. in a case from , ohto and kariyone (transf. ; vol , no. ) reported a cr Ásurvival study of f rbcs in a patient with anti-f. they showed that the initital survival of f cells was fairly normal, however, after days, there was a sudden increase of red cell destruction, and by day all f cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with . m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to days following the dtt treatment of rrbc. reactions were graded using standard serological grading of (negative) to (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to days. this suggests that dtttreated reagent red cells can be stored for at least days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with . m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract - c, and observed for hemolysis (none was seen) for up to days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted - with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* , monique scott , garcia curtis , ellice wong , alexa j siddon and christopher a tormey . yale-new haven hospital, va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately % to % of persons of african descent is characterized by neutrophil count of < . x /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p< . . results/finding: subjects who were clinically identified as having probable ben included patients (mean age . ; all self-identified as african-american; / were male) and controls included patients (mean age . ; self-identified as african american; ( / male). all of the cases ( %) diagnosed with ben had fy(a-b-) phenotype. mean anc ( . x /ul) and wbc counts ( . x /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p . and . , respectively) compared with controls (mean anc . x /ul ; mean wbc count . x /ul). there was no significant difference in mean platelet counts ( x /ul vs x /ul; p . ) or mean hemoglobin levels ( . g/dl vs . g/dl; p . ) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih- had % concordance for all blood grouping assays. for ahg assays, the ih- detected an anti-jka e, anti-fya warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih- identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih- with anti-igg,-c d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih- analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih- is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of tests were performed on , adult patient samples and donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in . % of samples tested. there were discrepancies, all antibody screening ( false positives, failure to detect a very weak prophylactic anti d and positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing - group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* , kathleen bensing , michael schanen , cindy piefer , randall w. velliquette , christine lomas-francis and connie m. westhoff . immunohematology reference laboratory, versiti/bloodcenter of wisconsin, immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new / / / cecf / / / rhce*ce or rhce*ce compound heterozygotes ce g ce g or ce c, g or ces or ceti / / / ce g ce c, g or c, g / / / ce g ces or cemo or ceek or ceek(var) or cern / / / ce c, g ce c, g or cemo or ceti / / / ce c, g/ce g/ g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi / / / total / / / a transfusion vol. supplement s reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd / and rd / , and a licensed comparator anti-e (p gd ms ), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n ) or edta blood from donors (n ) and were tested using a manual tube method or on a pk automated platform. a score ( ) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table ) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r r , r r , r r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: were rhce*ce that were in trans to rhce*ce; were various rhce*ce plus rhce*ce c compound heterozygotes; were rhce*ce or rhce*ce homozygotes; were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with rhce*cear/rhce*ce c compound heterozygote, and with rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and of rhce*cecf homozygotes were detected using the comparator reagent. rd / and rd / failed with and e variants, respectively (table ) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd / clone. none of the reagents detected e antigen variant expressed on example of rhce*cehar/rhce*ce. conclusion: rd / and rd / anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of blood group genes associated with the expression of blood group antigens from blood group systems. we used the illumina's hiseq / system to perform next generation sequencing first on sureselect-enriched genes from dna reference samples with average target design coverage of . %, and then on haloplex-enriched genes from dna reference samples with average target design coverage above . %. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for blood group genetic variants in these genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads ( . %) were mapped to the target regions relative to the sureselect reads ( . %). the mean sequence coverage depth of the targeted bases was around x for sureselect method and x for haloplex method. some exons, such as rhd exons and , , rhce exon , ermap exons and , cd exons and , cr gene (most exons) and gypb exon , are consistently covered with less than x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than % concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than % blood group genetic variants in selected genes. evidence rhce*cehar does not encode for rh (hr b ) antigen debra j bailey* , trina horn , paul mansfield , najmi qazi , pamela nickle , jessica keller , margaret a keller and jan r hamilton . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in and has a phenotype of c e c e w f w , g , hr w , hr , hr s , rh: , rh: with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh: (hr b ) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c. c>g and rhd c. c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d c e c e . her plasma contained an alloanti-s and an antibody that reacted strongly with all random e k s reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d and dc red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e s k red cells homozygous for the rhd*diiia-ce( - )-d, rhce*-ce c, g, t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce c, g, t/ rhd*diiia-ce( - )-d, rhce*ce c, g, t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr ( of sources) and hr b ( of sources) phenotype. conclusion: the rhd*diiia-ce( - )-d, rhce*ce c, g, t haplotype is one of the rh haplotypes expressed by the original hr b individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between / / and / / . all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately years, patients had htla established at least once by titration studies. serological investigations on a total of samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on samples was successful in rule out in % of cases. in an additional patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only % of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for % of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c. c>t (p.thr met). the dau allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion , : ) recently summarized serologic characteristics and associated anti-d alloimmunization for dau family alleles. we investigated two samples with the c. c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample was from a yo multiracial female. her rbcs reacted s at immediate spin (is) and in iat with immucor gammaclone and series and , and mi at is and in iat with ortho bioclone anti-d. rbcs did not react with of (lhm / & / ) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c. c>t characteristic of dau. rhd sequencing confirmed c. c>t and identified two adjacent changes, c. g>t and c. g>t (c. _ delinstt), in exon encoding p.gly leu. sample rbcs reacted w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series and , and quotient albaclone blend and alpha anti-d. papain treated rbcs were s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c. c>t. sequencing confirmed c. c>t and found a new c. c>t change (p.ser -leu) in exon . the c. t has not been reported, but c. g encodes a stop codon (p.ser stop) in japanese (vox sang , : ). conclusion: we report two new alleles: rhd with c. _ delinstt (p.gly leu) and rhd with c. c>t (p.ser leu), both also carrying the c. c>t (p.thr met) characteristic of the african dau cluster. d antigen associated with p. leu is a partial d antigen with a novel epitope pattern. the p. leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by / commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to . the number and diversity of alleles in the dau cluster supports that the c. c>t change is a major ancestral african background allele (wagner et al, blood , : ). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september to present day we got samples of repeated blood donors who are known to be d negative, c positive and/or e negative from blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon , exon and exon in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon to confirm the existence of c. g>a and c. t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the sample, we identified cases ( . %) of total rhd deletion, cases ( . %) of rhd-ce-d hybrid, and cases ( . %) of rhd variant. of rhd variant were determined to be asian type del with c. g>a variation. cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n ) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon and exon of the abo gene were amplified and sequenced. the abo*b . allele was confirmed by pcr-rflp analysis. results/finding: among subjects with b or ab phenotypes, were genotyped as abo*b . . the abo*b . group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage ( . . ) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other subjects with b or ab subjects, genotyped as abo*b . (n ), abo*bw. (n ), abo*bw. (n ), abo*bw. (n ) and abo*bw. (n ), displayed flow patterns differed from the abo*b . group. the abo*bw. , abo*bw. and abo*b . subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (< % in abo*bw. and abo*bw. subjects and < % in abo*b . subject). both abo*bw. and abo*bw. displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b . genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout , the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in % (n ) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in % (n ), % (n ), and % (n ) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n ), anti-jka (n ), anti-k (n ), anti-jkb (n ), both anti-e and anti-c (n ) (see table ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly in cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study volunteers in the gambela region (nct ). the whole ackr gene was amplified in one reaction covering , base pairs (bp). this primary amplicon was re-amplified using nested primers covering nucleotides. nucleotide sequence was obtained by sequencing reactions and manually annotated using ncbi refseq ng_ . . the sequencing covered bp of both exons, bp of intron, bp of '-flanking region, bp of '-utr, bp of '-utr and bp of '-flanking region and encompassed all the variations present in dbsnp and nhlbi esp databases. results/finding: among the samples, a total of snps, including one novel snp in '-utr were observed. snps occurred in the exons, in 'and 'flanking region, in '-utr and in the intron. all individuals carried the snp indicative of the common fy: phenotype; while individuals were homozygous and was heterozygous for the gata box mutation. no splice site mutation was detected. as individuals were observed as being homozygous or heterozygous for snp, we could unambiguously assign distinct alleles. in the remaining individuals with or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than . kb of the ackr gene and identified at least different alleles. the present study found that the vast majority of alleles ( / ) in the gambela population as defined by snps, were similar to the clinically relevant fy* n. allele, which in turn is defined by only snps at positions c. - t>c and c. g>a. out of the remaining alleles, were similar to fy* with the fy(b ) phenotype and was similar to fy* w. with the fy x phenotype. the high frequency of fy* n. ( %) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique ( %- %). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce( - )-d is the most common hybrid and is found in african blacks. it arose by conversion of exons - of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample (male) and sample (multiracial female), both c c e e (presumed r r ), presented with weaker than expected d typing; is and / at iat. rhd beadchip identified the common african rhd*diiia-ce( - )-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c. c>g and c. g>t (heterozygous) was also detected. as rhce*ce with g and t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons and replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce( - )-d were found in both samples. sample (scd male), d c e c e , by rh beadchip had one conventional rhd and rhd*diii type , and rhce*ce g/ces. as rhd*diiia type has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce( c) exons and had replaced those exons in the common hybrid rhd*diiia-ce( - )-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky and ky . we report two different and novel complex rh rearrangements: two samples thought to be r r had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia( - )-ce. in kind, a sample genotyped as diii type rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce c( - )-diiia( )-ces( - )-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples and have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a -year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at g/l. her pregnancy history was not provided. she had received units of packed red blood cells (rbcs) in the past including unit within the last months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r r , r r , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse polymorphisms which determine antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a ) and lu(b ). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of antigens encoded by the kel gene, is organized into exons. there are approximately kel alleles associated with a kell null phenotype (k ) in which no kell antigens are expressed, and alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons , , , and and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k kp(a-b ) js(a-b ). kel-cdna sequence analysis was performed and detected a single transcript species with c. c, c. c, t, and missing the sequences corresponding to exons , and . amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel* allele. this donor was presumed to have a k phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel* m. . here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a and a cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c. deleted g, characteristic of o alleles, c. t, characteristic of a and some uncommon o alleles, and c. a and c. a, characteristic of b alleles. genomic sequencing of exons and confirmed the presence of an o allele, abo*o del g, t, t), and the presence of a b allele ( g, g, t, a, a, c, and a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* , tatiane vendrame , janaína muniz , diana gazito , afonso cortez , lilian castilho and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the -bp deletion in smim in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting and in samples with reactivity of . dna was isolated from peripheral blood and smim was sequenced. results/finding: from donor samples studied, were serologically vel negative by gel-iat but positive by adsorption-elution, presented a reaction and the remained samples showed a reactivity of . genotyping results showed that the samples with negative results and of samples that presented reaction were heterozygous for the bp deletion and presented the a allele rs in homozygous status. from the of remaining samples with reactivity of , ( %) had the a allele of rs and ( . %) had the a allele of rs . in contrast, in the samples with stronger reactions we found the a allele of rs in ( . %) samples and the a allele of rs in ( . %) samples. conclusion: the molecular changes rs and rs are located in intron distancing nucleotides. this study reinforces the association of the a allele of rs with reduction of vel expression and suggests the involvement of a new rs change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as (rh negative), or ! on the neo or ! on the echo (rh positive) for both series and series anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series and series anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in of ( . %) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in of ( %) of samples. of ( . %) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. of ( %) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining of ( . %) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but < on the galileoneo or positive but < on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* , tatiane vendrame , janaína muniz , rosangela person , lilian castilho , afonso cortez and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of samples were included in the study, being previously genotyped as rhd*dar . , rhd*dar . and rhd*dau . the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from - corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon . rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that of samples ( dar . , dar . and dau ) had rhd genes, were phenotyped as c e-c e and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in dar . samples showed the rhce*-cear/ce s genotype, in dar . samples the rhce*cevs. /ce s genotype and in the dau sample the rhce*ce s /ce genotype. table describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized f antibody (hu f -g ) that binds human cd has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu f -g (anti-cd ) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a year old female with progressive follicular lymphoma who was enrolled in phase b/ trial of hu f g in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a year old male with refractory diffuse large b cell lymphoma enrolled in hu f -g clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing ( with anti-a, non-reactive with anti-b) and the reverse typing ( with both a cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase ( to ), at liss- c ( to ), at liss-polyspecific ahg (m ), and at peg-anti-igg (m to ). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at o c ahg phase. the abo typing of the second case performed after anti-cd administration showed a discrepancy between the forward ( with anti-a) and the reverse ( with both a and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is ( ), at c in liss ( ), and liss-polyspecific ahg (m ). the dat and autocontrol were negative. his genotype was determined to be a /o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o. . / o. . , consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n ) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt and a galt. papain-treated patient rbcs were used to screen donor plasmas (n ) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with polyclonal anti-a,b and a monoclonal anti-b (clone g / ) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors or nor antigens. the patient was le(a-b ) and thus a secretor. a positive crossmatch was seen with % of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type or ) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $ years earlier. the reactions are likely due to uptake of recipient-derived ble b (type ) antigen (isbt no. ), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b ). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of . shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive ( ) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from / / to / / . reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of , columns run as part of antibody screens, , ( . %) columns generated "?" results. assuming seconds of technologist time per "?", we estimate that . hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table ) . in cases, all three columns were visually negative but the analyzer reported reactivity with of cells. all cases had mts-gel tm antibody identification panels performed, of also had a mts-gel tm ficin panel. the yield for the panels performed was two routine panels with weak reactivity against hla cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; were negative. one patient newly demonstrated anti-jka. fifty percent ( / ) of visually negative but analyzer positive samples were tested with gel card lot number , % ( / ) with lot , and % ( / ) with lot . conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from patients ( cross-match samples, units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in / patients. further testing was performed in / . eight were tested for the presence of antibodies at c and confirmed in / . rouleaux formation was observed in / patients, / had reactivity detectable at c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* , sandra nance , david moolten and p. dayand borge . ( ): - ). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this . year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from random allogeneic and autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing . % bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* , anna burgos , virginia lew , sunitha vege , susan veneman , christopher j gresens , jonathan hughes and connie m. westhoff . immunohematology and genomics laboratory, new york blood center, blood centers of the pacific, bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons - , and long range sequencing of exon - ( . kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- / ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m n-individuals (meyer et al. br j haematol. ; : - ) . the st a allele, also described as gyp* , is a hybrid gypb-gypatranscript with the crossover in intron . we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a years old pregnant, african american female g p was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a like phenotype. genetic testing did not support the serological findings of a subgroup and a new abo allele, abo* c that has never been reported in correlation with an a like subgroup was detected study design/method: the patient rbcs were typed with anti-a (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a antibody work up was performed using three different lots of a cells and three lots of a cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and c for min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo* c found in the mother. the previously reported abo* a allele encoded an aspartic acid to asparagine change at position p. consistent with an a weak phenotype. also, at least five other alleles encoding an a phenoytpe consisted of polymorphisms at positions c. through c. , giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a / aweak phenotype is encoded by the variant allele abo* c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since ; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n ) were processed with the automated biorobot m robot using the magattract dna mini m extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of . ng/ml and . respectively. in the second phase of the study (n ), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was - ng/ll and the recommended purity was an absorbance ratio of . - . (a / ) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was % concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also % concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano , izaskun apraiz , maría azcarate , miguel angel vesga , montserrat rubia , mercedes piedrabuena , fernando puente , barbera veldhuisen , ellen van der schoot and m onica l opez* . progenika biopharma, a grifols company, centro vasco de transfusi on y tejidos humanos, banco de sangre y tejidos de arag on, sanquin blood supply research background/case studies: it is well established that weak d , and phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type , rhd*weak d type , rhd*weak d type , rhd deletion, rhd*pseudogene and rhd*diiia-ce( - )-d and itgb gene: hpa a and hpa b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa- blood group typing. study design/method: a cohort of previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications / /ce for a ivd product of list a (! % clinical samples, > % neonatal specimens and ! % weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n , %). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa- predicted phenotype were used for comparison. transfusion results/finding: no system failure, % call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a % concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was %. the following id rhd xt predicted phenotype results were obtained: d negative (n ), no amplification variant (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d types , or not detected (n ). regarding hpa- blood group, the predicted phenotype results obtained by id rhd xt were % concordant with bds results: hpa- a positive (n ) and hpa- a negative (n ), hpa- b positive (n ) and hpa- b negative (n ). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types ( % specificity and % sensitivity for d antigen, hpa- a and hpa- b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value < . (clinical analysis) or < x - (gwa) was considered statistically significant. results/finding: of the cohort patients, ( . %) transfused subjects were included with alloimmunized children < years ( . % of ) and alloimmunized adults ( . % of ). in multivariable logistic regression models, age (or . , p . , for age compared to - ), gender (or . , p . , for female compared to male), transfusion history (or . , p< . , for transfusions compared to - ), site, hemolysis (or . , p . , for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or . , p< . ) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from newborns less than , gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on sample pairs. dat test was negative on sample pairs and two were positive. there was % concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on placental blood samples and heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < , g birth weight newborns. o-( . %), ab ( . %), b-( . %), and a-( . %). among the tested donors, . % were d positive with r r being the most common rh phenotype. in the kell blood group system, . % of the donors were k positive, while the k antigen was found to be . %. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a b ) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a b ) and m n-s s at % and . % respectively. the le a and le b alleles were seen in . % and . % of donors respectively, while lu b -phenotype was found in . % of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a b ) and lu(a-b-) were . % , . % and . % respectively, while the m n-s-s-and m-n s-s-phenotypes were not found. the frequency of the p antigen was found to be at . % similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph . and the supernatant was discharged. a dilution buffer containing % human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with ll of washed red cells. the cell suspensions were incubated for min at c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional min at c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for - nm. events were recorded at a frequency of cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* , mark yazer , nancy m. dunbar and biomedical excellence for safer transfusion (best) collaborative . university of vermont medical center, university of pittsburgh, dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of . study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a : dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a -year and -year period, respectively. one center provided plasma and wb testing data for a -year period. results/findings: in total there were group a plasma units tested and ( . %) had a high titer anti-b. the range of high titer group a plasma units between these three centers was . %- . %. of the wb units tested, ( . %) units had a high titer; / ( . %) of the units had a high titer anti-a, / ( . %) had a high titer anti-b, and / ( %) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with . m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd on reagent rbcs and render them free from plasma anti-cd drug interference. procedures for the preparation of . m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on . m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of . m dtt, was adjusted to ph . , ph . , ph . using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n ) were treated with the . m dtt solutions in parallel by mixing : ratio of packed rbcs to . m dtt solution followed by incubation at c. for up to minutes during treatment, the expression of k antigens was measured every minutes by tube method using different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every minutes for each ph level. the reduction in average scores between different phs were also calculated at every minutes to measure the impact of . m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by ! ) after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . complete loss of k expression was seen after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . the reactivity patterns of k antigen tested with sources of anti-k correlate with each other. the reductions in average scores were seen between to minutes range of dtt treatment time when ph . was raised to ph . ; to minutes range when ph . was raised to ph . ; and to minutes range when ph . was raised to ph . . conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to minutes and/or beyond minutes of incubation. the ph of the . m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of . m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n , ) drawn between july , and june , . these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, , patients were found to possess clinically significant red cell antibodies for an overall incidence of . percent. the three most commonly encountered antibodies were anti-d (n ) . %, anti-m (n ) . %, and anti-e (n ) with a frequency of . %. a total of ( . %) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with instances ( . %) followed by anti-e and anti-c with ( . %), and anti-c, anti-e with ( . %). of the multiple antibodies identified, ( . %) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with or . % of the total. the west had ( . %), the midwest ( . %) and the northeast with ( . %). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of . and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the . thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p . ). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* , lindsy rich , sherry stern , sharon wangen and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was to days. results/finding: of the ntd specimens from the immucor neo, resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all results, it was determined that there was no reactivity and a valid result was present. the other samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the absc specimens that were resulted out as positive on the immucor neo, specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: - with ms (n ), - s with ms (n ), no reaction with ms , dgc , p x (n ). samples tested with a polyclonal anti-c showed a - reactivity. d c e c e cases tested with a polyclonal and monoclonal anti-e (ms , ms , ms , ms ) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c. g>a mutation in exon , predicted to encode the p.gly ser substitution. for apparent r r donors, a f-negative type allowed the prediction of a rhce*ce a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce a allele (c and e in cis) in all samples. d c e c e individuals were reactive s with the original source of anti-rh , slightly weaker when compared to rhce*ce a/rhce*ce rbc samples available from our cryobank ( ). conclusion: our results confirm that the c. g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh ). the locr reactivity appears to be rather similar when coded by rhce*ce a or rhce*ce a alleles. this was quite an unexpected finding, since the p.gly ser substitution is close to the critical amino-acid for c/c expression (p.pro ser). none of our cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce a was reported to code for a partial c (rh:- ), we consider that rhce*ce a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes , or . the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d , , or genotypes. study design/methods: between / and / samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types , , or genotypes, but a transfusion vol. supplement s had evidence of rhd genetic sequences in exon and/or intron in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons - to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients ( %) followed by transfusion patients ( %); % had no clinical indication provided. samples ( %) were found to be weak d type , , or ( , , and samples, respectively). samples ( %) appear to be genetically rhd negative. genetic sequencing was performed on samples; had rhd genetic variants that were not weak d types , , or (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples ( %) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types - . of the samples that had evidence of an rhd gene and did not carry the known weak d types - polymorphisms, ( %) of were found to have other rhd variants, and ( %) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types - variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel , curr opin hematol : ) , that individuals with weak d types , , and are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report months experience with rhd genotyping on samples referred with discrepant or weak d typing investigated from january to april . study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for samples ( . % caucasian, . % african american/african, . % multiracial, . % hispanic, % asian, and . % other). results/finding: rhd genotyping identified weak d types , , and in / ( %) and alleles known to encode partial d phenotypes in / ( . %) (table) . uncommon or rare weak d alleles including types , , , , , , (n ), , , , , and were found in ( . %). the partial d alleles found were diverse, but the largest number included partial rhd*d . (n ) and *dar ( conclusion: in a multiracial cohort of individuals with weaker than expected d typing % were due to weak d types , , or and would not be considered at risk of clinical significant anti-d, but for % there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp rhd* . allele causes discrepant genotyping results for rhce small c sabine scholz* , sandra schneider , sabrina k€ onig , susanne helmig and vicky van sandt . inno-train diagnostik gmbh, rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c ( c) and c ( t) is caused by the snp on position on the rhce gene. the rhd* . allele (also known as rhd cat vii type ) carries the snp t>c on the rhd gene and additionally the snp t>c. this rhd* . allele has been described to partially express rhc on the d polypeptide (faas, transfusion, ) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, ) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles ( t>c, t>c) confirming a rhd* . and one rhd* allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series and anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series and series , and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c. a>g change in exon encoding an amino acid change p.met val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c. a>g (p.met val) change in exon . several snps, deletions, and insertions have been reported with changes in exon . phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c. a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* , juan merayo-rodriguez , christopher lough and nancy eckert . lifesouth community blood centers, life south community blood centers, lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is % in most populations and greater than % in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is . / . and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal (k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to . / . the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient was a yo female, c e c e , whose rbcs reacted by echo and by neo with anti-d , and '?' with anti-d . testing with d and d by tube gave and w on initial spin (is) respectively and by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and w with immucor gammaclone anti-d, and all were at iat. rbcs did not react with two (lhm / & / ) of anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c. c>a change encoding p.his gln. patient was a yo pregnant female, c e c e , whose rbc were w at is and at iat with immucor series and and gammaclone, and moderately reactive, is and iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm / & / ) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon from c. to c. encoding amino acid changes p.ile leu and ser asn. conclusion: we found two previously reported rare alleles: rhd with a c. c>a (p.his gln), previously found in france (lefloch et al. genbank ku ), and rhd*dar with part of exon replaced by rhce, reported in sub-sahara africa (granier et al. transfusion : ) designated rhd*dar(ce :v v-s n) with an allele frequency of . to . . blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu f -g is a human monoclonal igg antibody recognizing cd that is in clinical trials to treat hematologic or solid malignancies. cd is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd is thought to enhance phagocytosis and promote anti-tumor responses. cd is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n ) from patients were tested over the course of month treatment. plasma was tested at immediate spin (is) and by iat with r r , rr, d--, rh mod and rh null rbcs, as cd expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg ) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd was observed in plasma as soon as hour post infusion. plasma reacted to at is and with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker ( and ) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was . in contrast, iat reactivity using gamma-clone anti-igg was only w to , and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd titer was at is and peg iat with gamma-clone anti-igg, but was ! with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n ) were reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after x allo-adsorption with papain treated rr cells, but in some samples low level (micro- ) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu f -g anti-cd therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu f -g is igg . reactivity was observed in all phases and with all test methods. cd is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg , can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in . in august , she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was with untreated and with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:- etc. had been excluded, k-phenotyping revealed a k -phenotype. a total of silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel* n. with c. t encoding p. ter (reported in an individual from austria in ). there are two known k -patients in our country, both homozygous for c. t. the daughter was a c. t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k -donors are available in our country. with the help of the isbt rare donor working party, a k o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* , kshitij srivastava , houda romdhane , saloua jemni yacoub and willy albert flegel . nih, dtm/cc/nih, regional blood transfusion center sousse, regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, a transfusion vol. supplement s since . the tunisian population has the largest known prevalence of weak d type . alleles, occurring in of rh haplotypes, compared to in , or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type . in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of , random blood donors were serologically screened for the d antigen using routine techniques. samples with weak reactivity were tested with a panel of monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of discrepant samples ( . %) were observed and expressed the serologic weak d phenotype. among them, carried an allele of the weak d type cluster ( . %), of which samples ( . %) showed the weak d type . allele. only sample each was found for the weak d types , and and the dvii, while samples showed the consensus rhd sequence. no mutation in any of the rhd exons was detected in another samples. the molecular analysis of the rhce gene showed that out of samples with serologic weak d phenotype ( . %) had a variant rhce allele and the most common associations were: weak d type . linked to rhce*cevs. . ; weak d type . . with cear; and weak d type . to rhce*cevs. , while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost % of the weak d phenotypes in tunisia were caused by alleles of the weak d type cluster, of which % represented the weak d type . allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type . in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type . phenotype. there is a possibility that the rhce*cevs. . allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* , heather simmons , christine lomas-francis , gayane shakarian , sunitha vege , lauren hutelmyer , sandra nance , jessica poisson , nicholas bandarenko and connie m. westhoff . duke university hospital, immunohematology and genomics laboratory, new york blood center, arc pennjersey, american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a , year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with . m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c , k , fy(a ),s ]. reactivity was detected to a titer of ; it was not removed by prewarm technique or by x peg alloadsorption. the adsorbed plasma reacted with . m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k , k , js(b ), kp(a b ) and sc: , . her plasma reacted with k o , mcleod, sc: , rbc samples and dtt-treated sc: rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused aliquots of crossmatch incompatible kp(b ), s rbcs. her post-transfusion dat was with anti-igg, with anti-c d. the eluate reacted with all rbc samples except kp(b ) sample. she tolerated additional aliquots from phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k , k , kp(a b ), js(a b ) and sc: , , discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c. a>t (p.glu val) (kel* . ) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a ). sc sequencing found heterozygosity for a '- g>a change (rs , to % prevalence) and conventional sc* , predicting sc: , , . kel and sc results on the mother were kel* /kel* . , heterozygous for the sc change '- g>a, and her rbcs typed k k kp(a b ), sc , ula , consistent with dna predictions. plasma collected months later was nonreactive at rt and in peg iat. her rbcs were dat and now typed k , kp(a b ), ul(a ) sc and sc ,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel* . homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc: , rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b ). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a -year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs - a)genotype, associated with a jkb null phenotype. anti jk was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a ) and jk(b ) . genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk . however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms a transfusion vol. supplement s (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a b-); kp(a b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon revealed a g>a mutation, fy *n. , known to silence fya. sequencing of kel exons - exposed a silent polymorphism in exon , g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy *n. mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly % of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have genetic variants not previously reported in those of african descent. only was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient . study design/methods: patient was a year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at years of age. anti-b changed from undetectable/weak to strong at the age of years. patient was a month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was / . both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient with - reactions up to years of age. thereafter, abo typing showed mainly strong anti-b. patient had / anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for children on long-term tpn. patient had absent/weak anti-b since birth up to years of age, then developed strong anti-b with no change in feeding regiment and medications. patient had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* and elizabeth hart . brigham and women's faulkner hospital, university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title , cfr part . (a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in . a total of samples were evaluated. each abid was subcategorized; ( ) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and ( ) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of abids were performed on new patient samples. of the new abid samples, ( %) had microscopic absc results. for the previously known antibody patients, there were which accounted for % of the total abids performed. when reviewing the total abid workups, a total of ( %) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the new antibody samples were: conclusion: a total of % of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: ( ) discontinue the use of the microscope, ( ) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or ( ) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than % in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a -yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a -year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only ( - ), and negative with anti-c b, c d reagent. the antibody showed a peak gel-igg iat titer of . results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w ), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from to %, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody anti-p anti-m anti-sd a anti-le b anti-jk a anti-k anti-e anti-c results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of % (type ), % (type ), % (type ), % (type ) and % other than , , or . further investigation was conducted to determine the molecular identity of the «others». out of samples, ( %) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon or both exons and . a surprising amount of samples were discovered to be normal rhd. conclusion: along with sandler et al. ( ) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types , , and obtained in serological weak d, years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was , %. no trali happened in the period. prophylaxis were used in % of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p < . ). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) ( . % vs . %, p < . ) or aes with a non-allergic type inflammation etiology ( . % vs . %, p < . ) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes ( . % vs . %, p . ) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for , , , and weeks were . %, . %, . % and . %, respectively (p . , logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week ( . %) and longer than a week ( . %) (p < . ). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found patients from - , who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these cases, only of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as out of transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was . g/dl but declined to . g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days and with poor responses. on day , routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from . mg/dl to . mg/dl (reference . - . mg/dl), and lactate dehydrogenase was above reportable linearity, > u/l (reference - u/l). testing revealed additional anti-e, anti-jkb, dat c , plasma free hb . mg/dl (reference - . mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b ), one of which was also e . one volume tpe was performed to remove free hb on days , , and using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at . mg/dl on day , decreased to . mg/dl before discharge on day results/findings: twenty three cases were identified, of which had medical records available for analysis. ten ( %) patients were male, the mean age was . years (range - years), ( %) had an underlying hematologic malignancy or bone marrow disorder, and ( %) had a history of coronary artery disease (cad). the implicated units included ( %) red blood cells and ( %) platelets; ( %) patients received a single unit, and ( %) received two or more within the previous hours; the mean volume transfused was . ml (range - ml). the mean time to onset of chest pain was . minutes (sd minutes), with % of patients presenting within . hours and % within hours of starting the transfusion. chest pain was present as the only symptom in % of the cases, and for the other cases the accompanying symptoms included dyspnea ( %), fever ( %), back pain ( %), and hypo-and hypertension ( %). a post-transfusion chest x-ray was performed in % of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in % of cases and showed no findings to suggest acute ischemia. three ( %) patients had a minimal increase in their troponin levels, although had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen ( %) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than minutes in the majority of patients ( %). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts . based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, degree, or anti-human globulin phase. check cells were found to be . these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o to a ) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a -year-old o man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha- antitrypsin deficiency who presented for olt (donor o ). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving units of o rbcs and units of o plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a plasma were transfused to wash out anti-a antibody prior to transfusion of a rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for hours post-transplant. the patient's total estimated blood loss was > l. he received a total of units of rbcs (including a ), units of plasma (including a ), units of cryoprecipitate, and units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o . on postoperative day (pod) , a ts showed predominantly a rbcs with trace o rbcs, as well as very low anti-a igm and igg titers (table ) . he received two additional o rbc units ( each on pod and pod ) with increasing o rbcs on ts and rising anti-a titers. his blood type was unequivocally o by pod . the patient showed recovery of liver synthetic function on pod (factor activity %) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod , the patient had reverted to o with recovery of anti-a titers. at months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* , alfred mingo , maria isabel gonzalez , antoni mena and juan pedro benitez . bst, at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h ) and an oncology center (h ). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of transfusion activity in both h and h shows pse and bca, out of and respectively, since the tss deployment in . retrospective analysis and classification of security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h , nm related to pse accounted for . % of all, being the mistake in concordance between patient identification and prescription order the most frequent ( . %). the nm detected in bcas were . % of all and mostly ( . %) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are . % of all and mostly ( %) the systems detects a not assigned bracelet. for h , nm related to pse accounted for the . % of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent ( . %). the nm detected in bcas accounts for . % of all and in . % occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are . % of all and in . % of them the blood components were assigned to another patient. ( , , , , , , , , , , , , , ) were analyzed via a commercially available elisa. comparison of adequate response to ppv , defined as ! mcg/ml for > serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in patients (alloimmunized, ); pre-and post-vaccine titers were available for patients (alloimmunized, ). of the patients, were on chronic transfusions, were on hydroxyurea, were surgical splenectomized, patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv in the previous years; / also reported previous history -valent sp conjugate vaccine within the last years. baseline pre-vaccination titers (n ) showed no difference between alloimmunized and non-alloimmunized patients (all p-values > . ). in the group with pre-and post-vaccination (n ) titers available, out of ( %) non alloimmunized patients had an adequate response versus out of in the alloimmunized group ( %, p ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from to ). patient harm events recorded within the veritas system from january to july were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of tres per month and per year were found over five years. % of tres are associated with pre-bb activities, % occur within bb, and % are post-bb events. sample collection and handling represent % of total tres. most tres ( %) were reported by bb staff, % were reported by non-bb staff. patient harm analysis revealed an average of four level (near miss), three level (no known harm), and . level (patient harm) per month. no deaths related to tre were detected over the seven month january to july period. patient harm was associated with tres occurring in the bb ( %) and post-bb ( %). these events were reported externally ( %) and by bb staff ( %). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january , and march , was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by and added to the number of individual whole blood plts; apheresis plasma units were multiplied by and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was . the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the -hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions ( [ /l vs. [ /l, p . ). there were no significant differences in the frequency of effective therapeutic ( % vs. %, p . ) and prophylactic ( % vs. %, p . ) transfusions between the prcs and gypcs. we did not find significant differences between prcs and gypcs in cci after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . . ) transfusions, in cc after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . .) transfusions. there were no significant differences between prcs and gypcs also in ma after prophylactic ( . . vs. . , p . ) and therapeutic ( . . vs. . . , p . ) pc transfusions. reduction of the severity of bleeds was obtained in ( %) of the cases after prpc transfusions and in ( %) of cases after gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, and cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in . it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were units administered using the emergency transfusion process in the months before the change was implemented. it was found that / ( %) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month ( components administered), / ( %) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: ) a stable anemic inpatient, ) a stable anemic inpatient to be discharged, and ) an asymptomatic post-operative inpatient. results/finding: we identified canadian tm experts: ( . %) provided a response and most had a primary place of practice in a laboratory setting ( / ; . %). for a stable, non-bleeding, anemic inpatient, . % of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with . % generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period ( - hours), a repeat hemoglobin > hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse ( . %) compared to an inpatient potentially symptomatic due to anemia ( . %). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium- ( cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n ) were divided into two ml aliquots, which were labeled with selected concentrations of s-nhsbiotin ( and lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of . . %). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that nm excitation of phycoerythrin (pe)-sa and high laser power ( mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with lg/ml of biotin resulted in $ , mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, % ci) of in , ( . %). the lld for rbc labeled with biotin at lg/ ml was $ in million ( . %). biotinylation was not associated with increased levels of hemolysis ( . . % before labeling versus . . % after labeling; p . ) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes ( ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer- (hboc- ) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: ( ) wb, ( ) wb % hboc volume (model of two units in an adult), ( ) wb % fdp, and ( ) wb % hboc % fdp. samples ( )-( ) simulated autoresuscitation by adding % plasmalyte to - . susceptibility to lysis was tested with ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of %, %, %, and % volume replacement with hboc and/or fdp, with or without prior % plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb hboc, and wb hboc fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb hboc in autodilution simulation (mean lysis . % vs. . %; p<. ). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc ( %) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even % hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc- , there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in / and / . we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of patients, were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery ( %). pcc was given for warfarin reversal in % of cases. a subset of patients received plasma within hours prior to pcc ( %) or hours after ( %). pcc was most frequently ordered in the or/perioperative service ( %). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in , several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; ) simplify and expedite the process; ) improve communication and expectations to decrease tat; ) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood ( units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of er episodes were received. the average tat from order to delivery at the bedside was reduced by % ( . minutes compared to minutes previously), while the compliance rate for er orders and physician documentation was % ( / ), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february -february . mt was defined as the transfusion of ! rbc units in a -hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at days. results/findings: thirty mts occurred during a one year period. a total of , blood products were transfused during that time period. gender distribution was / ( %) males, and the average age of all patients was with a range of to years of age. surgical patients accounted for / ( . %) mts, and / ( . %) were critical care patients. tumor categories included carcinomas ( / ), sarcomas ( / ), leukemias ( / ) and lymphoma ( / ). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer ( / ) was the most common disease seen followed by sacral chordoma ( / ). mtps were activated in only / ( . %) cases. thirty-day survival was seen in / ( . %) patients. only of mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage ( / ) or perisplenic hematoma ( / ). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a -year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a -year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a -year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* , , rebecca ross , christopher a tormey , , and amit gokhale , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: daratumumab (dara) is a igg monoclonal antibody therapy that specifically targets cd , a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, subjects were identified for analysis. their mean age was . years, with male and female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of % ( / ) prior to administration of dara. of these patients, were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these ( %; / ) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september , through april , were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in , , unique women receiving a total of , units of red cells (rc) in , transfusion episodes were identified. their median age was (range - ). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in ( . %) and ( . %). ( . %) transfusion episodes were associated with the use of units or more rc. as a result, ( . %) episodes resulted in a post transfusion hb ! g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given (< . %) and ( . %) women respectively. upon discharge, ( . %), ( . %) and , ( . %) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to ( . %) women. conclusion: in the present study, it is observed that . % transfusion episodes were given at hb ! g/dl. a substantial number of episodes ( . %) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! g/dl). for iron replenishment and bleeding control, up to . % transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes ( . - . x per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with gy and transfused over - hours within hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* , lee grabner , brenda herdman , robert fallis , amin kabani and charles musuka . canadian blood services, kenora rainy-river regional laboratory program, diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between and before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and % were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels ( . g/dl; p . ); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units ( . vs . g/dl; . vs. . g/dl; both p . ). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients ( . g/dl vs. . g/dl; p< . ). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p . ). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p . and p . , respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than % of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered times in total, affecting patients and users. stratified by location, the majority of triggers occurred in the perioperative areas ( times) and the liver icu ( times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin % iv solution, human albumin % iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* , , mahmut akgul , , hollie m reeves , , robert w maitta , , marcie pokorny , anne capetillo and katharine a downes , . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a -month retrospective study (january-july ) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was - mg/dl with critical value of mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were adult (> years) orders reviewed by tms out of which were approved. of the approved orders, ( . %) were in agreement with tms's estimated dose. of ( . %) orders that were not in agreement with the tms's estimate, ( %) were underestimated and ( %) were overestimated. seventeen of orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of . mg/dl (range . to mg/dl) and a median excess of . mg/dl (range . to mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was mg/dl above target, which is significantly higher with intervention than without (which could have been mg/dl below the target; p< . ). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention ( vs. . mg/dl, p . ). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention ( vs. - . mg/dl, p< . ). seven of ( ) ( ) orders were for critically low fibrinogen (< mg/dl) and of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were and units ( . % and %) i.e. and pools and the most frequent orders in the disagreement group were , , and units ( %, %, % and %). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was . and posttransfusion was . . only % of patients had their inr corrected to . , while % had no change, or had increased inr. (table) . the majority ( %) of patients received units of plasma. the mean plasma dose was ml/kg. there were transfusion reactions reported, non-hemolytic and transfusion associated circulatory overload reactions in which required admission to the icu. two patients experienced bleeding during ir procedures (tips) and developed a hematoma (tunneled central line). the median of inr correction in this study was . with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is . . randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. of the patients experienced bleeding complications indicating that inr of . may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately % of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population ( %) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately % of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july , as follows: units of group a plasma and units of group ab plasma is provided for the massive transfusion protocol (mtp) along with units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed mtps at our institution between july and march . twenty patients ( . %) were transfused with incompatible group a plasma ( group ab and group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april , our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in , bonfils immunohematology reference lab (irl) sent out approximately special platelets for patients with hla antibodies. by , hla platelet orders increased dramatically and the irl sent out over special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over , donors in the database with historical hla typing. however, only approximately of those donors actively donate. in the denver area, one of the most common hla types is a a b b . only of the , donors have this type ( . %). therefore, to fill an hla platelet order request for a common hla type, only donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a a b b , there is only out of , donors ( . %) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a for example, all of the a positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in , approximately % of these special order platelets were pra matched and the remaining % were hla matched by donor recruitment. by , approximately % of special platelets sent are pra matched. this change resulted in a . fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains packed red blood cells (prbcs), fresh frozen plasma (ffp) units, and plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august to march mtps were activated at ucm, of which orders could be traced to the origin: on inpatient floor (including icus), in the operating rooms, in the emergency department, in labor and delivery, and in other procedure rooms. of the prbcs that were issued, were transfused ( % utilized); of the units of ffp that were issued, were transfused ( % utilized); of the platelet packs that were issued, were transfused ( % utilized); of the units of cryoprecipitate that were issued, were transfused ( % utilized). since march , the time of first product issue after the initiation of an mtp has also been tracked. of the events that fall within this time period, ( %), had the first product issued in minutes or less. another ( %) were issued between - minutes, resulting in over % of patients being issued their first blood product within the first minutes. only of ( %) events had an initial time greater than minutes and none were greater than minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($ - %). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* , shailesh macwan , arline stein , jane fischman , nancy nikolis , matthew bank , lennart logdberg , alexander indrikovs , sherry shariatmadar and vishesh chhibber . north shore university hospital, northwell health background/case studies: massive bleeding is generally defined as any patient who requires blood volume replacement within hours and/or receives transfusion of greater than or equal to units in one hour with a transfusion vol. supplement s ongoing bleeding. our mtp was officially implemented in in preparation for an initial verification as a level trauma center by acs. our mtp has the following packages: st pack has a ratio of : : (rbcs, plasma & platelets) and subsequent packs a ratio of : : . our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march to add cryo and pcc at a defined point in the mtp (cryo is included in the rd pack and pcc in the th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received > rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had patients who received > rbc in and . mtp had been activated for all patients and all patients received between . to unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt > seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of mg/dl (table ) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of > rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's -day and -day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective -year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of published cases ( reports) of ta-pls, ( reports) were stem cell and ( reports) were organ transplants. all ( %) stem cell transplants and ( %) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the reports of stem cell ta-pls, actively screened for antibodies in the immediate post-transplant period, and of the reports of organ ta-pls, actively screened for antibodies. these screens detected cases of stem cell ta-pls before hemolysis became apparent and cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* , rosario mallari , marc de asis , elaine shu , jonathan hughes and tho pham , . stanford university, stanford health care, stanford blood center, bloodsource background/case studies: mur antigen is present in - % of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received - rbc units every - weeks to maintain a hemoglobin (hb) level of g/dl. the patient remained stable for months when his hb level acutely dropped to . g/dl. the antibody screen remained negative for an additional months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for of the rbc units he received. units were from an asian donor, and a unit transfused days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of , donors at a hospital-associated blood center located in a region where asians comprise approximately % of the population. results/finding: . % of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for of ( . %) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over % of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* , princess maynie , carol chandler , shelia garret and pampee young . vanderbilt, vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [ ] [ ] [ ] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of samples have been analyzed (table) , rh and non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being . (range - ) times higher. the average fold change for rhd/c/e antibody titers were . , whereas the average fold change for non rh titers was . (range - ). the range for anti d titers was particularly variable, - , whereas for c/e, it was - . the overall reproducibility/precision of the automated analyzer was $ %. to correlate the a transfusion vol. supplement s increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $ times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of . units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to % of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately % of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix . % suspension of a and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, group a, and group b. results/finding: of the whole blood edta samples tested, ( group o and group b) exceeded a high titer threshold of . when the pas samples of these donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of , a % decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the ( %) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these hospitals had a lower overall rate of wastage including their own donations than the other hospitals that did not collect in-house plt. the other hospitals had wastage rates ranging between - %. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* , , christopher j gresens , anne capetillo , hollie m reeves , and katharine a downes , . case western reserve university school of medicine, university hospitals cleveland medical center, bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of < . %. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a -year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure ( / mm hg to / mm hg) followed by hematuria ( ml). chills and rigors resolved; blood pressure stabilized after min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of (igm) and , (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* , leila patricia de sousa fontenele , isabel nagle reis , carolina bonet bub , araci sakashita , raffael zamper , cristiane nakazawa , tatiane almeida omura paula , patricia silva batista , marcio dias almeida , fernanda loureiro de andrade orsi and jose mauro kutner . hospital israelita albert einstein, hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of cases of olt performed between and in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of consecutive patients submitted to liver transplantation between and were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or , - % ci: , - , , p: , ), absence of hcc (or , - % ci: , - , , p: , ), cirrhosis of any cause (or , - % ci , - , -p: , ), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or , %ic , - , ) and retransplantation due to primary non function of the graft (or , %ci , - , , , p: , ) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a /b in group o platelet products. charles k. childers* , mark destree , ashley rose and theresa nester , . madigan army medical center, bloodworks northwest, bloodworks nw, dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a /b in group o platelet products is presented from a large regional blood center collected over - months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in ml edta sample tubes. a single : dilution of plasma was prepared using a hamilton microlab series dilutor using . ml saline diluent and . ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at rpm for seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of is used, approximately % of group o apheresis platelets will have a high titer, most commonly with anti-a . less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their day outdate. after months of testing pspp units and verifying that the products rarely had a high titer ( . %), the blood center stopped performing this testing for pspp units. rh ) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to % (from % on the previous day). his hematocrit did not increase ( %), and over the ensuing hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of % while reducing the number of a rbcs in his circulation by approximately %. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes - days for antibodies to develop and they are short-lived ( - weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the calendar year from hospitals. an additional hospital* provided data for august-december . rbc transfusions in patients < year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/ years was also determined. results/findings: see table . the fraction of all transfused rbcs that were oneg ranged from - % (row f). the percentage of oneg rbcs transfused to oneg patients ranged from - % (row g); thus, non-oneg patients received - % of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients ( %- %; row i). overall use of oneg rbcs could have been reduced by %- % if opos units had been given to all oneg patients >/ years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* , nehad mohammed , marwa aly and nashwa fahmy . national blood transfusion services, nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on multitransfused patients who received blood transfusion chronically at our central blood center. they were thalassemia patients ( bthalassemia patients, one patients with a thalassemia), sickle cell anemia patients and immune hemolytic anemia patients ( auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). oncology patients, chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: out of ( %) alloimmunized patients and out of ( %) non alloimunized patients(p< . ) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june . screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified articles for data abstraction, where articles were transfusion guidelines. there were guidelines ( %) that made a recommendation, for a single unit and for multiple unit transfusion strategy (table ) . review b identified retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or . , % ci . - . ), although heterogeneity was high (i %). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december and february was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the months, units of platelets were transfused to recipients. over the months, a median of units was given to each patient with a range of to . the overall distribution of products used was % plasma, % pr, % pas f and % pas c. thirty percent of patients (n ) received all of their products on a single day. single units were given to patients while , and received , , and units respectively. the distribution by product type was % plasma, % pr, % pas c and % pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the month period (p . ). the distribution by service was different for the groups receiving multiple units. for single units the distribution was % hematologic malignancy, % infusion clinic (nos), % solid tumor medicine, % surgery, and % pediatrics. for those receiving multiple units the distribution was % surgery and % each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of . . conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* , , yvemarie b.o. somsen , maike e. van hezel , marleen straat , robin van bruggen and nicole p juffermans . sanquin research and landsteiner laboratory, academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains mg of iron and % of the rbcs are cleared by macrophages within hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in icu patients who received one rbc transfusion, different iron parameters were measured before and hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il- levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion ( . vs. . mmol/l, p . ). also, the transfusion had no effect on transferrin saturation ( vs. %, p . ), ferritin ( . vs. . mg/l, p . ) and il- levels ( . vs. . pg/ml, p . ). hepcidin levels increased in these icu patients after rbc transfusion ( vs ng/ml, p . ). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (- . vs. . % change, p . ). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il- or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since and pathogen reduced platelets have been available since . in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between and . during this time pas c, pas f and pr went from % to % of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in patients received an average of . units/recipient/month and in the average was . units/recipient/month. the intervening data points for , , and were . , . , and . respectively. the year average was . . the slope of the graph for all points was y - . . . the two sample t-test showed that the plt/recipient/month from to was not statistically different with a p value of . . conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that % to % develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least k/ul. the analysis revealed that the mean of . k/ul (n ) had a percent lower bound confidence interval platelet increment of k/ul (p< . ) results/findings: (median range [ - ]) hla matched leucoreduced irradiated sda platelets were transfused to ( m/ f) patients, median age years (range - ). / ( %) patients showing broad alloimmunization to hla class i/class ii antigens. / ( %) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority / ( %) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); / ( %) female patients had prior exposure via pregnancy and / ( %) had a history of hsct. ( %) platelets were abo identical-platelet increment median k/ul (range - to ), ( %) were abo compatible -platelet increment median of k/ul (range - to ) and ( %) were abo incompatible with platelet increments median k/ul ( range - to ). platelet counts were performed within hours in ( %) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* , renee leblanc , dongfu xie , alice cabe and yanyun wu . overlake hospital, bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about for years (from to ) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than . % of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a -year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of . g/dl (baseline - g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was . g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin ( . to . mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to . g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october to march . patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were rbc products transfused to waiha patients. twenty-three ( . %) patients received at least incompatible unit. the mean age was . years (range - yrs) with % women. ethnic composition was % african-american, % caucasian, and % patients of mixed/other ethnicity. one hundred fourteen ( %) of these products were released as li products and ( %) were compatible. ninetythree ( . %) of the li product transfusions had a post-tfn hct change of < % whereas only ( %) of the compatible product transfusions resulted in a post-tfn hct change of < % (p . , v ( ), exact methods). the mean hct increase in the compatible group was . % per unit vs. a slightly lesser per-unit increase of . % in the li group (p . , t-test, -tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for units. units that were incompatible had a lower mean post-tfn hct rise compared to all other li units ( . % vs. . %); however, this difference was not statistically significant (p . ). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected % per unit more frequently than it was for waiha patients who received compatible products ( . % vs. %). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first -hours from mt onset) was calculated with % and . % control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were mt cases from hospitals ( tertiarylevel, smaller/medium sized acute-care and specialist women's). number of mt cases per hospital ranged from to . patient median age was years (iqr , ), % were male and % required admission to intensive care. the most common clinical groups were cardiac surgery ( % cases), trauma ( %) and gastrointestinal hemorrhage ( %); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the tertiary-level hospitals was % (range % to %) and / ( %) were within the % control limit. cb that required ! rbcs within -hours of mt onset occurred in % of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals ( % versus %; p . ). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of bcs and ml of platelet additive solution iii (pasiii) were used to produce pcs (n ). pcs were stored on a flatbed agitator ( cycles/min) in a temperature-controlled cabinet at c for - days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of mm hg was applied. using clamps, a flow velocity of - ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted - %, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration ( . - . x /l) and number (> x /unit). simulated transfusion had no effect on the percentages of cd p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* , , joan sevcik and joseph e. kiss , . university of pittsburgh, blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution : is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient and and (control). results/finding: all patients were ab blood type. patient , a year old female with recurrent adamts deficient ttp, received courses of tpe (total tpe procedures) for relapse and exacerbation. ten out of procedures were performed with ab and a plasma (average ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of tpe procedures. four out of procedures were performed with ab and a plasma (average . ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with adamts deficient ttp who served as a control, received a total of procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between patients. the trends of hemolysis laboratory data for patient and were comparable with patient . all patients had negative dat. only patient received rbc transfusions. all patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to % of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd negative platelet unit is not available for a patient with anti-cd antibodies sameer khatri* , charles harmon , brian r curtis and chisa yamada . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd ) is one of the identified plt surface ags and deficiency is rare, but found in asians ( - %), sub-saharan africans ( - %) and also in some people from mediterranean descent. two types of cd deficiency have been described. type deficiency is the complete lack of cd on both plts and monocyte-macrophages whereas type deficiency lacks cd on plts with variable expression ( - %) on monocytemacrophages. transfusing plts in a patient with cd deficiency is challenging given the rarity of cd negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than units of apheresis plt units over a week period without any significant increase in plt count. cross-match compatible plt unit found in of units and hla matched units were tried without success. at that point, a cd ab was identified in the serum and the patient's type cd deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was % due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd- negative (but blood type different and hla a transfusion vol. supplement s unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd antibody positive reactivity in serial dilutions has reduced from : to : dilutions and his hla class i pra has decreased to %. he is currently receiving apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to . k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec , , blood donations were screened for b microti by immunetics elisa. of those, ( . %) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between . % and . %. no patient babesia transmission has been reported since implementing this test, but we only had documented babesia ttd cases from - . donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of positive test results, lookback investigations were initiated representing % of positive donations. lookbacks were only performed when there was a donation within months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to % were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented . % loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only . % of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in > % of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for blood donors, were positive with an average load of . x copies/ml of plasma, a median value of . copies/ml of plasma, ranging from to . x copies/ml. pre-transplant viral loads were similar. for transplant candidates, were positive with an average of . x copies/ml of plasma, a median value of copies/ml of plasma, ranging from to . x copies/ml. post-transplant viral loads were remarkably different. for transplant recipients, all were positive with an average of . x copies/ml of plasma, a median value of . x copies/ml of plasma, ranging from to . x copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around - copies were present in > % of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt ( ) . post-ivig: all plt ( ) . post-ivig: cd -negative plt from relative ( ) . post-ivig: single donor apheresis ( ) . post-ivig: cross-match compatible ( ) . post-ivig: flow cross-match compatible plt ( ) . a transfusion vol. supplement s detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv ( / / / ), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows % of specificity, with no false positive results on the control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of tcid /ml for denv- , denv- and chikv and of tcid /ml for denv- , denv- and zikv. finally, the first results obtained on denv( ), zikv( ) and chikv( ) clinical samples show %, % and % correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of , , and were prepared from plasma or serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from : to : ( -fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of or higher and was not eliminated by the addition of a blocking step. pools of or samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of or samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $ cfu/ml of organisms associated with platelet contamination and incubated at room temperature for - hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher ( log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc , were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta d and virtuo the organism was recovered % . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc . however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of ml lrap demonstrated % recovery when loaded onto the virtuo and d ( table ) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus - , and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in , a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as- rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and hrs after amustaline addition, respectively, for titration by plaque assay on vero cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, > . log or log /ml of rrv was achieved, with > . log or > . log /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; - ml), large volume (lv; - ml) and dual storage containers (ds; - ml) designed to treat platelet doses between . and . x . the new triple storage (ts) set was designed to expand the dose range to . x and the maximum volume to ml, generating either or doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or % plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in % plasma/ % pas or % plasma with a final volume of $ ml and a dose of - platelets. these conditions represent inactivation using the lowest amotosalen concentration ( mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of - logs in respiratory secretions of mers patients, and with lower genomic titers of - logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log reduction/ml (log /ml) %plasma/ % pas e .coli . <- . > . e. cloacae . <- . > . k. pneumoniae . < . > . s. aureus . <- . > . blue tongue virus . <- . > . bovine viral diarrhea virus . <- . > . adenovirus- . <- . > . %plasma k. pneumoniae . - . > . s. aureus . <- . > . adenovirus- . <- . > . n a transfusion vol. supplement s abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged times up to days, assessing the infectious titer and genomic titer every rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of ! . log infectious titer. no viral replication was observed after days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* , jen-wei chen , chi-ling chen , sheng-nan lu and pei-jer chen . department of research, head office, taiwan blood services foundation, graduate institute of clinical medicine, college of medicine, national taiwan university, division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by , and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since ) and -sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since ) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during - and - , respectively. age-standardized prevalence and its % confidence interval ( % ci) were calculated with adjustment of who world standard population - . for the incidence study, donors who have donated blood two or more times during - and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its % confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ) during - , and the agestandardized prevalence was also decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ). the agestandardized prevalence of anti-hcv was generally higher in female donors before , but it was significantly higher in male donors at (p-value . ). a total of , hcv rna positive cases, . % of them were anti-hcv negative, identified from , first-time donors during - , and the crude and age-standardized prevalence of hcv rna was . per , ( % ci: . - . ) and . per , ( % ci: . - . ), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value < . ), but no significant difference was found after age standardization (p value . ). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend< . ). in the incidence study, a total of new hcv rna positive cases, . % of them were anti-hcv negative, found from , , donors followed for , , person-years. the incidence of hcv rna was . per , person-years ( % ci: . - . ), and no significant difference was observed between both genders (p-value . ) and between age groups (p for trend . ). conclusion: the prevalence of hcv infection has been dramatically decreased by . % during - . it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* , sahar muhmmad and dalia el dewi . national blood transfusion services, azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is - days, hiv from to days, and hbv from - days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from to , the total number of donor samples to be screened is , the age of the donors ranged from to years, and they were of both sexes (m: f : ).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v ) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of nat yield donations among ( . %) seronegative donors. among these nat yields cases, ( . %) were reactive for hbv, ( . %) were reactive for hcv and ( . %) were reactive for hiv- . we stratified the age of the donors into groups; group a ( - years), group b ( - years) and group c ( - years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p . ; with % confidence interval (ci) . - . & p . ; with % ci . - . respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p . ; with % ci . - . ). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p . ; with % ci . - . ). nat-hcv; did not differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups a and c & p . ; with % ci - . to . between groups b and c). nat-hiv; did not also differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p . ; with % ci . to . ). nat hbv was significantly higher in males (p . ; with % ci . - . ), but the prevalence of either hcv or hiv did not differ significantly between males and females (p . ; with % ci - . - . & p . ; with % ci - . - . ; respectively). conclusion: in this study the nat yield of in assumes more significance when one considers the fact that single donation is used for generating components that can be used by recipients. hence, in effect the nat yield becomes times that is, in . saving recipients from tti out of ( . %) is indeed very significant. results/finding: of the , donors who were tested by our donor center, , ( . %) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at : titer. the screening elia s/co of this donor was . . both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were - transfusion transmitted babesia cases per year from - (table ). in the months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed ( in , donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of years of additional nat testing at blood bank, dmch, ludhiana from september to december . results/finding: results . % ( of ) units were initially nat reactive. these units were further tested, of which . % were discriminated ( hiv, hcv, hbv and co-infections). the remaining . % ( ) were repeat non-reactive and . % ( ) could not be discriminated. overall, nat yield rate was one in , whereas virus-specific nat yield rates were one in , for hiv, one in for hcv, one in for hbv and one in , for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past years has increased the screening sensitivities to check viral load and prevented transmission of probable transfusion transmitted viral infections. assuming % component preparation it saved transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu , , wei mao , tao he , yashan yang , , zhan gao , , chunhong zhang , hongmei liao , jingxing wang , and miao he* , . institute of blood transfusion, chinese academy of medical sciences & peking union medical college, sichuan blood safety and blood substitute international science and technology cooperation base, chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from , voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as e - . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: . gb raw data with , , reads were generated in the dna library. meanwhile, . gb raw data with , , reads were generated in the rna library. after cleaning the human background, reads from bacteria, reads from viruses, and reads from parasites were identified (table ) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table ) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of february to february . thrombocyte concentrates are prepared out of single donation units by the buffycoat method. results/finding: over the period platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* , germ an leparc , phillip c williamson , lani palmer , ben reynolds , maria noedel and lindsey houghton . creative testing solutions, oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february , recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march , . with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february , and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february , , the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in , who reported $ million new cases worldwide, resulting in > , deaths. malaria prevalence is highest in sub-saharan africa, home to % of all infections accounting for % of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of . mm amustaline and mm glutathione (gsh) and a h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with % fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching > % parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at > . log or > . log /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from to days using an fda cleared rapid test (rt). in february , our hospital based transfusion service implemented the use of rt on day , and to routinely extend ap shelf life to days. prior to this, we tested aps by rt on day and transfused day or day units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of day platelets. study design/methods: data were obtained for two -month study periods: october -september (pre-implementation) and february -january (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table . the number of ap transfusions increased by % post-implementation, comparable to a % increase in inpatient admissions and an % increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different ( . pre; . post, p . ). the number of rts performed increased by %. the percentage of transfused units tested at least once by rt prior to transfusion increased by % (p< . ). the outdate rate decreased from % to % (p< . ). ad-hoc ordering decreased from % to % (p< . ). conclusion: use of an approved rt for routine ap outdate extension to day was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* , vito scalia , carla osiowy , michael carpenter , anton andonov and margaret fearon . canadian blood services, public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low ( . and . per , donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of units. hcv nat was in place since (using minipools of ) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at - o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the ' ntr-e and ns b regions. sanger sequencing of these regions represents approximately % of the genome. results/findings: all confirmed positive donations were whole blood donations. there were hbv positive donations. of these, had tested hbv nat positive. genotypes were type a, b, c, d and e. there were samples hbv nat negative but hbsag positive ( were anti-hbc reactive). of these, could not be sequenced and one was genotype a (also anti-hbc reactive). there were samples considered hcv positive. of these, samples were hcv nat positive. genotypes were type a, b, c, b and a. there were also samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert d microbial detection system (bta d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert d (bta d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels ( - cfu/ml) of bacterial species commonly associated with platelet contamination, and replicates ( per instrument) of ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta d and the other into virtuo and incubated until signaled positive by the instruments or for up to days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august . this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a % voluntary donor program since and is the only center in the country that has achieved this goal. results/findings: a total of , blood donations were studied from august to december . in the rbdc, where only voluntary blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of : , for hbv; : , for hiv and : , for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s system. hbv s region was also sequenced. results/finding: obi were found in the , donations. in the viral loads assay, samples were negative and samples' viral loads were lower iu/ml. the mean viral loads was . . (log ) iu/ml in other samples,while the mean viral loads with hbsag /hbv dna samples was . . (log ) iu/ml. samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype ( . %) and the other was hbv c genotype( . %). compared the samples with hbsag /hbv dna ,we found two obi samples carrying with t>c mutation, which could cause an amino acid s f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag /hbv dna , and some unique variation was identified in the obi individuals. a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat th days, month, months& months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may , we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may to dec was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s platform using pools of serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, seronegative donations were screened by nat and a total of hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group - years old showed a large proportion, who accounted for % of reported infections. most of the hbv dna cases (about . %) reached senior high school education. the average hbsag dna positive rate was . % ( / ). incidence among apheresis donors in this period for hbsag dna were . / . these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf with day stability at c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control ( ml) and test components ( ml ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within hr and wbd pf within hr. cryo was manufactured according to site sops and frozen at - c (test ml, control ml ). test and control cryos were thawed at c, and characterized immediately post thaw (t ), and after d storage at c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over d at c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through d storage at c. conclusion: pr cryo can be processed from plasma sources, including pf , and stored at rt for days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf with stability over days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , lynne fleischmann , mirjana sarac , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics, abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects - million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over days using a panel of positive and negative samples. sensitivity was evaluated on presumed antibody positive specimens and specificity was evaluated on random blood donor samples. results/finding: precision was % cv or less for positive samples over days. the overall specificity in a blood donor population was . % ( / ). sensitivity was . % for presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , lynn martin , daniela kaleve , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over days. sensitivity was evaluated using known positive samples, commercially available seroconversion panels, the who standard, hbsag mutants, and hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed positive samples. sensitivity was % for all genotypes. % of the mutants were detected vs % for the comparator assay. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. analytical sensitivity ranged from . to . iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , kevin callear , susan sullivan , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types and (anti-hiv- / ). blood centers require very high throughput anti-hiv- / assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv- / antibodies and hiv- p antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv- / antibodies and hiv- p antigen was evaluated on the abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv- , hiv- and hiv group o antibodies and hiv- p antigen. seroconversion sensitivity was evaluated with commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed antibody positive samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. also, sensitivity was % for antigen positive viral isolate samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , joyce siregar , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who st international standard. seroconversion sensitivity was evaluated using commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from . to . iu/ml. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* , anton vanweert , ed bakker , mark paradowski , jane bryant , tuan bui , joyce siregar , george chen , george schlauder and gregg williams . abbott laboratories, sanguin diagnostics, abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over days using htlv i and htlv ii positive samples. specificity was evaluated using , blood donor specimens from europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot . . results/finding: imprecision was less than . % for positive samples over days. clinical sensitivity was . % ( / ) on preselected htlv i and htlv ii positive samples. the specificity was . % ( , / , ) on a blood donor population and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez , michel garcia* , fernando palomino and guillermo orjuela-falla . national blood bank colombian red cross, universidad del rosario, fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than . ; abbott architect i sr) underwent supplemental testing by immunoblot (either chiron riba hcv . sia or hcv blot . test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: - . , - . , > . band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in . % ( / ) of samples, indeterminate in . % ( / ) and were positive in . % ( / ). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group - . ( . %) compared with the - . ( . %). in samples with indeterminate results, ns _ was the most frequent band detected ( , %). in contrast, the most frequent band in the group of positive results was core ( , %). only one sample from the indeterminate group ( . %) had a strong band intensity ( ), compared with samples from the positive group ( . %). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group ( - . ) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (> ). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns _ and ns _ cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* , ivanka mihaljevic , manuela miletic , miljana stojic vidovic , irena jukic , jane bryant , mark paradowski , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, croatian institute of transfusion medicine, abbott gmbh & co. kg, abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over days using positive samples. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than . % cv for positive samples over days. clinical sensitivity was . % ( / ) on preselected syphilis positive samples. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* , ed bakker , anton vanweert , jane bryant , mark paradowski , tuan bui , lynn martin , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over days evaluating positive samples. sensitivity was evaluated using preselected positive samples and seroconversion panels. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than . % cv for positive samples over days. overall clinical sensitivity was % on preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying more bleeds than the comparator assay. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of , was declared as the public health emergency of international concern by who in feb . in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where . % of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, . % of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately : to : . thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of lm and assayed after illumination with visible light from both sides for , , and min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was . log % tissue culture infectious dose (tcid )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for min, min or min and the losses of the infectivity were further demonstrated by repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial min of treatment whereby ct-value jumped from . (control) to . (mbpt for min) (table ) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* , margaret fearon , susan l stramer , megan l nguyen , france bernier , sheila o'brien , vito scalia , sakina smith , yves gr egoire and boris hogema . h ema-qu ebec, canadian blood services, american red cross, sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in , canadian blood donors in . in a subset of , donor samples the seroprevalence was . %. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately , canadian whole blood donors including , from canadian blood services (cbs) and , from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all a transfusion vol. supplement s donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test ( % lod . iu/ml, % ci . - . ) for use on the cobas v r / system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous months are destroyed. recipients will be traced in the event of any products transfused in the previous months. results/finding: as of april , , of , ( , cbs, , hq) tested samples with valid results have been found hev-nat reactive: donors have been confirmed by further testing to date. confirmation is pending in donor. of the donors, were from quebec, and one each from nova scotia and alberta ( male, female). ages ranged from to years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: ate pork (including who ate pork liver), ate shellfish, ate venison, and drank well water. one donor had no identifiable risk factor. viral loads ranged from to iu/ml, of which were < , were - , and were > iu/ml; were anti-hev igm positive and anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around / . the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about to million cases and to million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august . both thin and thick glass stained blood smears of blood samples with giemsa was examined using microscope. results/finding: of the donated blood samples, ( . %) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p> . ), majority of the blood donors that tested positive belonged to blood group o ( . %). the prevalence of malaria parasite in the study was . %. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert d (bta d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life ( , and days after collection). study design/method: pooled lrap were seeded with low levels of organisms commonly associated with platelet contamination at , and days post collection. the seeded lrap were inoculated into bpa and bpn bottles on different days (not consecutive) alternating between teams of people each. seeded bottles were loaded into a virtuo and a bta d and incubated until declared positive or negative (up to days). additionally, bpa and bpn bottles inoculated with ml of unseeded lrap were tested on the virtuo and the bta d ( and bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of . % for the virtuo and . % for the bta d. the virtuo demonstrated an average improved ttd of . hours, when compared to the bta d in the presence of ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for days barring continued zikv testing and nonreactive results. a total of , donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low ( . %). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn , felicia santa maria , yvette girard , peter bringmann , marion lanteri* and adonis stassinopoulos . microbiology department, cerus corporation, scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in . the rapidly increasing number of infections in brazil, with hundreds of fatalities since december , is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate d-yfv using amotosalen (s- ) and uva light prt of platelet components (pc). study design/method: pc in %pas (n ) or % plasma (n ) were spiked with high titers of d-yfv and treated with s- /uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were . . log pfu/ml for pc in % plasma and . log pfu/ml for pc in % plasma while titers in post-prt samples were <- . . log pfu/ml for pc in % plasma and <- . log pfu/ml for pc in % plasma. inactivation to the limit of detection of > . . log or inactivation of > . . log pfu/ml was achieved for pc in % plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples ( hiv- antibody and hiv- p antigen, and htlv-i antibody and htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were . for hcv, hbc, syphilis, cmv igg, and chagas, . for hiv ag/ab and htlv i/ii, and . for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were . % (antibody sample) and . % (antigen sample) for hiv ag/ab combo; . % (htlv i antibody sample) and . % (htlv ii antibody sample); - . % for anti-hcv, - . % for chagas, - . % for hbsag, - . % for anti-hbc, - . % for syphilis, and - . % for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of ng/ml. robustness of the abbott prism methods to biotin interference c fischer , r schneider , w leonard , m cobb , g schlauder , g williams , m zuske m janulis* . transfusion medicine, abbott diagnostics, wiesbaden, germany, add diagnostics, transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from to colony forming units (cfus)/bag (i.e. . to . cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of - cfu/ml over the days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, hours (day ) after collection a sampling volume of spiked platelets ( . - cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate ( . %). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann , frank tolksdorf , wiebke handke , thomas h. m€ uller and axel seltsam* . german red cross blood service nstob, maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from buffy coats using the additive solution ssp (macopharma) with a residual plasma content of %. for inactivation kinetics, pcs (n ) were spiked with bacteria to a final concentration of approx. colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately . cfu/ml. bacteria were allowed to grow for h in the pcs at c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log reduction factors ranged from to for enterobacter cloacae ( . . , pei-b-p- ), pseudomonas fluorescens ( . . , pei-b-p- ), staphylococcus aureus ( . . , pei-b-p- ), and streptococcus bovis ( . . , pei-b-p- ). pcs (n for each species) spiked with these different bacteria species were efficiently sterilized ( out of ). treated pcs remained sterile during storage for days, while bacteria in non-treated pcs grew to high titers of - cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july , to july , was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than . were considered significant. results/finding: a total of , consecutive blood donors were screened between and . the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was . %, . %, . % and . % respectively. the hiv-hbv co-infection was higher among blood donors ( . %) followed by hbv-hcv co-infection whish accounts about ( . %). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of - . in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was , . ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a month old infant patricia davenport* , geeta paranjape and laurie sutor , . carter bloodcare, ut southwestern medical center background/case studies: in at a large pediatric hospital, a month old infant was supported for days by extracorporeal membrane oxygenation (ecmo). over this time blood products were transfused. about days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of units of red blood cells. approximately weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of : . the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* , andrea j linscott and donny dumani . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from - , % of transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a -year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day of hospitalization showed no growth after five days. on day , the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the ml unit had been aliquoted via sterile connecting device days prior for a pediatric patient. all ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was . c. within minutes of starting the transfusion, the patient's temperature increased to . c and subsequently reached a maximum of . c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at - c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of determinations of a low positive control in run. inter-assay variability was determined by testing at least representative negative production pool samples, at least low positive sample (about s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of . and were . and . respectively. the hbsag assay detection limit was . iu/ ml for source plasma and . iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at - c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between : , to : , , depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than % for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than %. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on donors ( - days after the index donation) - donors in the follow up study and tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all donors. denv antibodies were negative in donations and equivocal in . our initial reactive rate is higher than that reported to date for the procleix zikv tma of per , [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since . over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late , for which the program consisted of types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june , bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with ml of plasma, vs. ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, ; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation # : updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation # : implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over , donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on - year olds and premenopausal women (ages - ) donors. on average, - year olds donate . times a year and premenopausal women donate . times a year. if both of these groups were limited to donating once a year, a total of , donations from - year olds and , donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin # - , the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for - year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a . hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: % ( / ) of the simulation group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of hybrid group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of online only students improved their post-test scores and had an average likert scale rating of . (good). the average changes in scores were statistically significant within all training groups (p value < . ). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p< . ) and the hybrid group (p< . ). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in . all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july through january were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a -month period, cases of complex antibody identification workups ( %), transfusion reactions ( %), consultations for blood component utilization ( %), and deviations from standard operating procedures and massive transfusion protocols ( %) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $ , of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* , david lancaster , dianne geary , robert scott , anh thu nguyen , adam garcia , raina shankar , leslie buchanan and tho pham . stanford health care, stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to ( ) streamline the ordering process to accurately reflect inventory status and transfusion practices and ( ) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a -month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a -month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: ( ) the ratio of units transfused per week to the number stocked (t:s), ( ) the number of products ordered as stat, and ( ) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. lines of code were written for both programs, including class modules and distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were . , . , and . before the pilot period compared with . , . , and . during the pilot period. these differences did not reach statistical significance (p . ). we also monitored the number of stat ordered products before and during the pilot period, which were and stat units per week, respectively (p . ). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were and units, respectively (p . ). an estimated hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to . fte and $ , per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* , yulia lin , troy thompson , allison collins and sheena scheuermann . ontario regional blood coordinating network, sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that of the province's hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in indicated that % plan to implement or already have implemented the ptqip and % of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in . publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* , rana hajjeh and cees th. smit sibinga . world health organization regional office for the eastern mediterranean, international quality management (iqm) consulting background/case studies: more than million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between and ; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may in tunisia. results/finding: we found publications on disaster from five countries in the region and publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries ( . %) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated - % of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. ( , , and for - ) . the number of collections per registered trt donor varied significantly, ranging from to therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from . to . between and . conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from through . it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of . after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a % response rate (n ). of these, . % have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. . % of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although . % of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only % of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate % ( units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in , apheresis red cells (arc) represented . % of total red cell collections at our center. hae mcs ln was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs operators first and then operators new to apheresis with a training goal of % of mobile staff. validation of the alyx began / / and took approximately days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs machines were removed from service / / . alyx go-live occurred / / . additional operator training continued through september . results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $ . each providing an estimated annual savings of $ , . conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately . % and decrease our kit costs by %. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october -march . background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! g/l) and for females (minimum interdonation interval increased from to days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october , changes in rebooking of donation appointments in december and culminating with eprogesa criteria changes on march , . both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by . percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from . % in the weeks pre-implementation to . % in the weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were . % in september, . % in october/november, and . % from december to march, . conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past years, , blood products, derived from , procedures, were distributed to different investigators in over laboratories. whole blood was the most common product ( . %), followed by unmanipulated mononuclear cell collections ( . %), and elutriated monocytes or lymphocytes ( . %). less common requests included platelets ( . %), plasma ( . %) and granulocytes ( . %). adverse donor reactions were infrequent ( . % of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable % collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in % plasma. the corresponding pathogen reduction system used for the study has kits with different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than . x , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than x /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of x / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of . x and platelet concentration of x /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of . x and . x at the volume of mls and platelet yield of . x , . x , and . x at the volume of mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* and steve cihura . bonfils blood center, bbc / bsi background/case studies: in , the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated devices with the following criteria in mind: ) device disposable costs, ) reagents/controls/quality control, ) objective hgb/hct measurement, ) portability and durability for a mobile environment, ) ease of use, ) donor experience, ) battery life, ) validation requirements plans, ) blood center suitability, and ) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february with a targeted implementation date of july . after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately %. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to . % in and . % in . during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in , the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may . conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* , ayumi araki , hiromi sanyoshi , hiromi kanai , hiroya kikuchi , katsushi tsukada and kazuhide mure . hokkaido red cross blood center, japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from individuals who donated platelets during the -month period between february and august , and data from the donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the men ( . %) and of the women ( . %) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the donors in the vvr group was . . %, which was significantly higher than the . . % in the non-vvr group. at a maximum dbf threshold of %, sensitivity for discriminating between vvr and non-vvr donors was . % and accuracy was . %. when % dbf was used as the alert level, alerts were issued for donors, including in the vvr group. therefore sensitivity for predicting vvr was . % and specificity was . %. mean time from alert to diagnosis in the vvr group was . . minutes, and accuracy of the alert was . %. some of the vvr could not be predicted even the value of maximum dbf exceeded %. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* , , salam abdus and shabrina shah . inova blood donor services, inova fairfax medical campus, inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every to weeks from december to march . blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of patients, females and males, who underwent a total of rbcx from october to march , using an average number of rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of healthy volunteers were collected before blood donation and after blood donation immediately, day, week, weeks, and weeks among men and weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component ( c ) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at weeks among men and weeks among women, while c significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover week after blood donated and reached their levels before blood donated within weeks among men and weeks among women. conclusion: the biomarkers mutually changed over the course of weeks among men and weeks among women. donating ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low % split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate percent and increased concurrent plasma collections by percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october . after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in and , a total number , rns and rns completed the questionnaires, giving a response rate of . % and . % respectively. the overall mean score in was . points (range to ). the mean score in was . points (range to ). the percentage of rns having perfect scores of increased from . % in to . % in . table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; purposively selected blood prescribing clinicians and nurses from hospitals in countries of the human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at . level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r . ; p . ; df ) and accessibility (r . ; p . ; df ) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f( , ) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, junior faculty co-investigators from west coast institutions each had months to create a minute powerpoint presentation on a fundamental tm topic, after which other members had months to review and edit. therefore, each member created and reviewed presentations (three total steps). during each step, members wrote multiple-choice questions for those particular topics. in the end, each topic would have quiz questions to assess learning. at completion, evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: ) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; ) pre and post-lecture question validated examination (best collaborative) to assess learning; ) resident in-service examination trends specific to tm. results/finding: six presentations were developed as of the abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables and . abo leaders pre-test data could not be obtained for institution b, and trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsÕ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the minute vignette performance and the minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean %) when compared to the pre-test scores (mean %) ttest p< . . during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and % reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between . to . (in the range of - ). nps varies between and . according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased - from . to . . conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* , anne eder , beth a. dy and mary o'neill . american red cross, georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about in , apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. , a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to , hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the months before and after launching the educational outreach. results/finding: the web based course was completed by more than participants; were physicians. based on a review of the evaluations, the course was highly valued with % of participants rating it excellent or very good. the blood center physicians gave over presentations to hospital customers. reporting of suspected strs in increased by % compared to the prior year. the increased reporting came from specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required - minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, .) subscription-based e-learning utilizes - minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june , with a new equipment module assigned each month for the following five months. the series concluded in december with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of . on a -point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with positive and negative comment. level : learning the average score of users increased % from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of . and a t-stat value of . . level : results while equipment-related errors decreased by % after training, there is not enough data to demonstrate a statistical significance. conclusion: our level and evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october , a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n ) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was %. of those, % endorse that fbc creates a climate of respect within our transfusion practice, % believe it has improved communication between work units, and % feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only % responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: students of undergraduate semester and students of semester participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with % of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, / / from - am. there were attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, / / from - am. there were attendees, including regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) % as patients with fcr % may benefit from delaying the procedure for performance in the future. validation process included ( ) a deming regression to globally assess the predicted vs. actual results and ( ) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| %. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during days ( academic hours) the trainees can attend lectures, discuss the methodical materials, participate in seminars, interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, with the group capacity a transfusion vol. supplement s of up to people the number of medical specialists who have attended the training is nearly . results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of - ; being least satisfied/comfortable and being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of total technologists, technologists took the pre esd survey and technologists took the one year post esd implementation survey. table shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, ( . %) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were unplanned sop deviations; in the year after esd implementation there were only deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from to . the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in % plasma is broken up in to two days. on day platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for - hours. on day products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day and day . in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: of employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was . for the two month training period the daily average rose to . conclusion: our "flip the switch" training plan for implementing prt platelets in % plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly % of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as % of an activity. in we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of we performed a supply inventory on all departments to plan future purchases and control residual stocks. in , we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter (q / ) with last quarter (q / ). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q / blood bags were used compared to in q / , demonstrating an activity " . %. price negotiation resulted in . % readjustment. both indicated an estimated cost " . % with a possible impact of over us$ , . we have identified a real cost # . % in q / , representing an overall # . % and us$ , . (r$ , . ) savings. conclusion: economy had deteriorated in our country in with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih- tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v , statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in . in the - nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on % individual and % for the other four levels ( % for each). the bonus (%) is calculated based on the iis as follows; category a: % (iis > %), category b: % (iis: -< %), category c: % (iis: -< %) and category d: % (iis < %). on the pilot implementation, the individual scores for staff ranged from % to %. the iis were % to %. the number of staff in each bonus categories were , % (category a) and , % (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* , deborah r fludd and sandra j nance . background/case studies: rare donors are defined as a blood type occurring in less than in people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in , there were , active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the ( . % of total active donors) returned contact cards alerting ardp of changes in calendar year , ( . %) were donors moving from one ardp facility to another, ( . %) were donors no longer eligible to donate, and an additional ( . %) were address changes. other changes were ( . %) reactivated donors and ( . %) donors who we were notified were deceased, or did not want to be listed in the ardp. in , new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in was ( ), which would be . % of the new donors submitted. conclusion: with nearly a % response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in , donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* , barbara j bachman , mike leamy , susan olson and candace williams . university of florida college of medicine, bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih- (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two ( ) runs of one to six ( ) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing , types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. regardless of quality or speed metrics evaluated, the ih- demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < . ). ih- process steps and time studies addressed in the table below did not account for the ih- reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih- ( % reduction, a difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* , kimberly monnin , barbara j bachman , kyla warren , susan olson and candace williams . clinical pathology labs, bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih- tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of separate test runs of or samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately , type & screens (t&s). the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih- demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < . ). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih- (difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih- tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih- and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih- with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v . statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/findings: using automated result verification, only . % out of , samples evaluated for abo/rh testing would require visual verification, resulting in a % reduction in operator touchpoints (p < . ) and a labor saving of minutes ( : hh:mm) for abo/rh testing. for , antibody screens, automatic validation of results would result in . % reduction in operator touchpoints (p < . ) and a labor savings of minutes ( : hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by % annually and peaked at % in mid . to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an week period in late . hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than day shelf life remaining. units with tie tags attached were in hospital inventories for up to months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was % of the hospitals surveyed. mean percent o neg rbc gross sales for a month period before, during, and after the survey was . %, . % and . %, respectively. mean percent o neg net sales during the -month survey fell to . % compared to an average of . % in the months prior. during the -month survey period o neg rbc monthly return rate increased to an average of . % compared to an average of . % in the months prior. for the months after the survey the average o neg rbc return rate further increased to . % while mean percent o neg rbc net sales trended slightly upward to . %. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* , jeremie gachelin , veronique ollivier , thibaut mutin , xavier telot , benoit ho tin noe and sandra sanfilippo . aenitis technologies, hôpital bichat, inserm u background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac ) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than % (p< . ) and a purity of platelets close to . %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over . blood donations for an area with more than million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in , orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in , when we moved from this equipment to atreus c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in we changed to atreus c (terumo bct) and finally in , we moved to reveos system (terumo bct). since the changes in , our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in , bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during and . conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of - patients and an average, round-trip travel time of approximately -minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about % of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november , the hb has significantly improved the turnaround time of rbc issue -from -minutes to less than -seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately rbc per month out of the window for non-surgical patients. this has been reduced to approximately rbc per month, a % average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in , they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the st rescue squadron ( rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the rqs master sergeant in january . we asked what rqs's order and delivery expectations were. he said sporadic use and the blood order would be rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the rqs needs. staff was trained based on data from january meeting. we contacted the rqs in september to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the rqs association with a civilian blood center. based on his field experiences, he changed the blood order from to rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and rqs performed a mock run on october , and we felt prepared for any future events. results/finding: on november , , the rqs was deployed to a civilian aeromedical evacuation. we anticipated a rbc order. the actual order was rbcs and ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at : am by the rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november th , we established a maximum blood order of rbc and liquid plasma, noting future orders may request fewer products, yet meet the preferred rbc; plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table ). the highest mean fib concentration was mg/donor unit; lowest mean fib concentration was mg/donor unit. all sites had a mean fib concentration at least mg/donor unit above the fda minimum requirement of mg/donor unit. fifteen of blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; manufactured pooled donor cryoprecipitate. most froze plasma in a - c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every minutes for a hour period or until the temperature exceeded c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached > c in minutes as shown in table . the control thermometer recorded temperatures maintained at - c for the entire hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at - c for more than minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: % conventional (c-pc), % pr-pc, and mix of % c-pc/ % pr-pc. the model predicts a modest ($ %) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january . a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august , inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january . a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from to (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in , zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within weeks (sept. phase ) and nationwide within weeks (nov. phase ). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools ( -donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab had % and lab had % of clients requiring universal zkv testing. we evaluated a -month test result upload performance period to determine the impact of zkv test implementation. results/finding: during , lab upload time performance ranged from % to . % from january to july; upload time performance fell between august through november, returning to . % performance in december. lab upload time performance ranged from . % to . % january to august. performance fell september through december . % - . %. lab experienced a low of % upload time performance during phase when there was a rapid implementation; % clients required zkv nat. improved performance was observed during phase , with a % increase in zkv clients. for lab : phase experienced a modest decline of upload performance ranging from . % to . % with . % of clients implementing zkv nat. performance was . % in phase , when an additional . % of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* and colleen a. aronson . advocate lutheran general hospital, acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in of the hospital transfusion services. the sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the sites which implemented the verax pgd test perform testing on all day and day platelets to be issued for transfusion. this abstract summarizes the data collected for the first weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day , day , and those that were tested twice. inventory reports were reviewed for platelets issued on day or day that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february ( week of performing the test), . % of all platelets issued by the participating transfusion services were day or day platelets. in march that number dropped to . %. it is expected that this number will level off at some percentage at or below . % with further data collection. in february . % of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day or day ) will likely level off at some number at or below . % due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, of the sites performing testing are level trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed %. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n ). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days , , and of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was ( . . )%, relative change rate of hsr was ( . . )%, the residual leukocytes were ( . . ) . the ph, hsr, and the cd p expression of pooled platelet concentrates before and after filtering were ( . . ) vs ( . . ), ( . . )% vs ( . . )% and ( . . ) % vs ( . . )%. there is significant change for wbc after filtering (p< . ). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table . conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb - ) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of ml and ml. two users then evaluated the ss tubing segment types with ml or ml samples for a total of data points. samples were collected into the attached ml or ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january , a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by %. the platelet outdate rates dropped after implementing the platelet inventory tool from % ( units) to % ( units); a % decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of fte to . fte, lowering fte by %. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* , jaclyn mckay , jennifer curnes and rowena punzalan , . bloodcenter of wisconsin, children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: ( ) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, ( ) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within -hours after collection. this tight hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight -hour completion time constraint for cryo production (capacity expansion). in particular, during the th -quarter of , a blood processing region was able to process about more cryo units/month (an increase of %) at a slightly lower collection cost (cost avoidance), resulting in an approximately % reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the -the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after -day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from to days in the usa using an fda cleared rapid test (rt). in august , our hospital based transfusion service began using a rt on day and to routinely extend ap shelf life to days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of -day ap. study design/methods: data were obtained for two study periods: september -february (pre-implementation) and september -february (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from -day to -day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by % post-implementation while inpatient admissions and surgical volume increased by % and %, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by % post-implementation and the outdate rate decreased from % to % (p< . ). ad-hoc ordering was not statistically different between study periods (p . ). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different ( . and . , respectively, p . ). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years ( ) ( ) ( ) ( ) ( ) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was , , and units respectively. the mean number of discarded rbc units of the five years of the study exceeds % of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below mg/dl, and so the rejection threshold for lipaemia is level equal to or more than mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including rbcs, plasma products and apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from mg/dl to mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* , , rebecca ross , debra mraz , anne baker , zenna neal , melanie champion and edward l. snyder , . johns hopkins university school of medicine, yale university, yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, pr-sdp were transfused at our hospital (out of a total of platelets transfused). after months of scale-up, pr-sdp were approximately % of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($ mg per g) to the content in pr-sdp (< ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september , the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from donors was collected in k edta tubes. plt concentrations were determined at the qc department using the coulter act diff hematology analyzer (beckman coulter). sample tubes were stored at - c and measured at , and hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd ( ml; n ) or dpd units ( ml; n ). plt pools were stored at - c under mild agitation for seven days except for dpd, which were split in two -ml bags after h. samples were taken on days and . ph, po and pco , hypotonic shock response (hsr), extent of shape change (esc), cd p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at h ( pltx /l), h ( pltx /l) and h ( pltx /l) postdonation. dpd can be stored in the same collection bag for h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits ( - pltx / l) before splitting. on day , lactate and pco concentrations increased, and po decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march . there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted % increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* , colleen vincent and patricia kopko . university of california -san diego, american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only - % of double platelet collections meet requirements for pathogen reduction treatment. blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: , apheresis platelets annually), which includes two hospitals ( inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week ) and the bone marrow transplant (bmt) ward (week ). an e-mail communication explained the change to all physicians and nurses. in phase , we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase , we expanded usage to include the inpatient bmt ward. in phase , we lifted all restrictions so prp could be used throughout the health system, with the goal to reach % prp within months. results/finding: in phase (weeks - ), we requested prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the cancer center. in week , excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase . in phase (weeks - ), we formally expanded issuing of prp to include the inpatient bmt ward and requested prp products weekly. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the phased-in areas. in phase (weeks - ), we began issuing prp throughout the health system. our supplier provided an average of prp weekly (range - ), and prp constituted % of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table ) resulting in a cost savings of $ k. an additional cost savings of approximately $ k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as a transfusion vol. supplement s we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* , james r stubbs , scott a hammel and manish gandhi . mayo clinic, mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing % pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of . , . , and . x may end up below a . in the final storage bag and would need a post-processing sample to ensure the product met criteria at ! . x platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a . yield but ended with a yield below . . these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe- d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that of results were . or . x / mcl with the exception being the wbc failure with a count of . . further monitoring of the wbc counts discovered a result of . which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of . will be tested on the adam to confirm a leukoreduced product. we also discovered of ( %) of the . preprocessing yields products ended with a post processing yield < . . we decided to increase the yields requiring post processing samples to include the . . conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of ! . x platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from / - / were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need ) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in ; the revised prediction method (estimated clinical need ) was then evaluated retrospectively using the same data sources covering / - / and then compared to the prior method. results/finding: the average number of platelets transfused from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted amount and the number of units used was - . u/d. % days ( d/month) were under-predicted (average: u/d). % of days ( ) were under-predicted by ! u (average: u; max: u ( x)). the average number of platelets used from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted and units used was a . u/d. % days ( d/ month) were under-predicted (average: . u/d). one day ( %) over this period was under-predicted ! u ( u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages ( % a %), reduced the number of days under-predicted ( % a %), and decreased the discrepancy on those under-predicted days ( . u a . u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* , maria noedel , nancy haubert , kenneth hudson , larry morgan , robert shaw , tracy fickett , jamie jue , valerie winkelman , sally caglioti , german leparc , and phillip c williamson . background/case studies: on / / , fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within weeks; nationwide in weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on of manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for clients within weeks, and an additional clients within weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the -week period but overcome during the -week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* , hala samuel boules , fatemah saleh al matroud , rabab hussien ali dashti , hanan alawadhi and reem al radwan . kuwait central blood bank, kuwait central blood bank, kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of rbcs baby units were randomly chosen to be traced to their final deposition from the year till . half of them ( units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb ( units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is % efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (< days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march . results/findings: a total of products were tested. fifteen units ( %) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all by the vendor and none were true positives by re-culture. of the units that were successfully tested, were tested again on day for use on day ( %). there were platelets transfused ( %) and expired after day ( %). the cost to test the products including controls was $ , and our calculated cost to produce products would be $ , . if we had needed to import products to meet needs, the cost would be roughly $ , without shipping costs which are estimated at $ , . . we averaged expired platelet products per month (range - ) before verax testing and (range - ) after implementation. conclusion: using verax point-of-care testing saved platelet products from discard. the cost savings were $ , . from importing and $ , from producing a replacement for those products. the average discard rate per month went from to after verax implementation. extending platelet shelf life to days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for data, units were investigated without the use of secure texting. of these, units were identified as preventable wastage, and wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: ) product not released after procedure/ or when patient stabilized ( ) ) product returned outside of appropriate temperature range ( ) ) clinician unaware product was assigned ( ). thus far in , wastage records have identified units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, responses thanked the transfusion service for the information, and in instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: . % dmso, plas-a, % hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) . %dmso: plas-a : %hsa : : . plas-a and hsa are kept at room temperature ( - c, rt) and refrigerated at c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point . c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total tests. at least minutes cooling after dmso, before adding the next reagent. see table: ( ) after directly adding . % dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. ( ) in tube, autologous plasma first, dmso next, powder-like precipitates. ( ) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. ( ) & ( ) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at c. ( )&( ) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the c group is slightly milder/slower than rt group. so hsa should not be added first. ( )&( ) trace of hsa(< ml) was mixed into the plas-a bag ( ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive -month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from . % to . %. with an annual platelet usage of approximately , units, this reduction equates to approximately units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $ , to $ , , per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* , russell thorsen , rosaline ma , antonio g insigne , amy decourten , florence panganiban , patricia mckean , cyril jacquot , sara bakhtary and ashok nambiar . ucsf health, children's national medical center background/case studies: plasma (ffp, pf , pf rt ) stored at - c outdates hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (< months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited ( hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk and richard gammon* . oneblood, oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average - minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average - minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from % to %). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp- using the low glycerol ( %) freezing method and frozen at - c within six days of collection. thawing occurred in a c water bath in the following order: o positive and o negative on january ; o positive and o negative on february ; and o negative on february . deglycerolization occurred on site using the acp- with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $ - and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $ per unit to produce and distribute. drbcs have a shorter shelf life, days versus the days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp- 's and deglycerolize four units at a time. in january and february , it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c izekial butler* , karen leighton , scott jones and rachel beddard . qualtex laboratories, biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c instrument. precision of the new assay parameters was determined by testing replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from iu/ml to iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of . to . iu/ml. stability of samples was determined by testing samples stored at - c and - c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from . to . . so, precision was acceptable since the %cv for all samples tested was %. the mean values for the samples tested in the accuracy study were all % of the expected value which was much lower than the acceptance criteria which was % of the expected value. the linearity of the assay was acceptable with a r ! . %. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to days at - c and up to month at - c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for days at - c and stored up to one month at - c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, ) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april , to march , . local research ethics board approval was obtained. results/finding: patients received ivig for itp at smh over the study period for a total of unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding ( , %), pre-operative or antepartum care ( , %), a platelet count of less than and contraindication to corticosteroids ( , %). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between - x /l. patients received ivig for a likely diagnosis itp while a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. / ( %) of infusions consisted of g/kg over days; the remainder of infusions consisted of g/kg. of those who received g/kg, of patients ( %) had evidence of partial remission after a first g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to staff members in early february of . twenty-two technologists responded for a % response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, % indicated that workspace size was insufficient and % that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* , neil bangs and kimberly sanford . vcu health system, virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from / / to / / to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (< hours, > hours < hours), and if a second sample was required. results/finding: a total of t&s orders were initiated from the ed in this time period. ( . %) patients were not subsequently transfused any type of blood product within hours of tsd and ( . %) patients were not subsequently transfused any type of blood product within hours of tsd. a total of ( . %) patients required a second sample. of these patients requiring a second sample, ( . %) were not subsequently transfused any type of blood product within hours of tsd and ( %) were not subsequently transfused any type of blood product within hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within hours of tsd amounted to an estimated $ , in unnecessary patient charges and approximately . nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* , debra berry , yunchuan delores mo and gay wehrli . university of virginia health system, children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* , elizabeth halperin , sharon breining and mona papari . acl laboratories/ advocate hospitals, advocate health care, itxm background/case studies: a large midwest hospital system with level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and months of data were evaluated that contained events. results/finding: there was an equal number of events that were initiated in the ed and the or ( ). male patients were involved % of the time and % of time the patients expired. trauma of some type was the majority of the cause but . % of the cases involved gi bleed and only . % were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average . with the post hgb average of . . ratios of : for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of : . it was found that the rbc: plasma was . : , rbc: plt was . : and rbc to cryo was . : . use of txa was only . % and cacl was utilized in . % of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* , jody a barna , donald e ulinski and nancy m. dunbar . dartmouth hitchcock medical center, dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july , we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august and november (pre-implementation) , newly identified clinically significant antibodies were resulted for patients compared to patients between the months of august and november (post-implementation). the tat for allergy alert entry for both periods is shown in table . we observed that % of allergy comments were performed within hours in the post-implementation period versus only % pre-intervention (p . ). using the new process, nearly all of the alerts were entered into the emr within hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* , shailesh macwan , nancy nikolis , arline stein , janelle richardson , manju bagu , lennart logdberg , alexander indrikovs , vishesh chhibber and sherry shariatmadar . north shore university hospital, northwell health background/case studies: our institution is a tertiary care facility performing over cardiovascular surgeries (cvs) in , an increase of % after the healthcare system cvs integration in . transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december , the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after reported events in q that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: . open discussions and collaboration between blood bank and cvs nursing teams . mapping the process using flowcharts for additional blood orders from cvs. . identify bottlenecks and brainstorm solutions. . a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. . the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. . follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period / / - / / the blood bank has received verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* , tracie ingle and heather vaught . indiana university health, indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter - and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with - o, d negative red blood cells over days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series , anti-d series , a cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion ; : - who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for % and % of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february -june as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june -october . results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table ) . this led to a reduction in observed overall sli ( . . days vs . . days, p< . ) and odr ( . % vs . %). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. ( )) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january and december thirty-five ( ) notifications were sent to physicians using epic letters and of those, fourteen ( ) responded to the epic notification and five ( ) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* , denden benabdessadek , annu george and alexandra jimenez . westchester medical center, new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of orders were reviewed. approximately, % of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases ( %), but of the issued products, all were returned to the blood bank in % of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> type and screens (t&s) per day] blood banks , respectively. our laboratory which serves a large -bed multispecialty academic hospital and receives - t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of . . and . . minutes , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was % longer and had a larger standard deviation (s.d.) than the published trial result of . . . transfusion vol. supplement s abstract during phase i visionv r performed panels. during phase ii visionv r performed of the visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* , john roback , ronald arkin , michael bartlett , robert geiger and jaxk reeves . university of georgia, emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over , emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a % target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of participants responded to the survey ( . % response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n- - df) of the null hypothesis that the mean vs. the alternative that the true mean is> . overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on run cycles. ct was comprised of metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc ( : ), xm ( : ), cb ( : ) and dat ( : ). by implementing the future state, an average $ . min. lt and vt is saved on each sample loaded for ts equating to a % labor reduction over the current state. a % improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a % lt reduction. on average, a min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $ type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing -column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on patient samples for abo/d typing and antibody screening; of which at least had a positive antibody screen. out of the , had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which were d(-) and were d( ). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the patient and donor samples tested ( % concordance). all samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave % sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for samples at different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* , craig fletcher and peter millward . beaumont hospital, beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short -day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october and quality data was reviewed from august to december . the collected data was then analyzed using descriptive statistical methods. results/finding: data from indicates platelet wastage comprised % of total received platelets and % of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units months preimplementation of the report was units, compared to units months post-implementation and units months post-implementation. wastage rates improved from % (wasted yearly platelets/total received yearly platelet units) in , the year of report implementation, to post-implementation rates of % in and % in (see table) . importantly, this occurred despite a greater than % increase in platelet inventory between and and resulted in cost savings of over $ , in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* , lisa marie button , lori scanlan-hanson , karen koch , janet finley , deepi goyal and camille van buskirk . mayo clinic-rochester, mayo clinic, mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in and were / (events/ed transfusions - ). study design/method: electronic ordering for the ed was implemented march st . any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients < kg and pediatric patients > kg. a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for years (april -march ), and during that time there was instance of blood being ordered for an unintended patient . % ( / ). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in , / ( . %) units were transfused in the ed but not charted in the patient's medical record. in , / ( . %) transfusions were not charted. however, in , the first full year of electronic transfusion order capability, only / ( . %) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level trauma center, with over inpatient beds and over operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option (verbal) for verbal orders and coolers set up; and option (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by % with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* , steven zibrat , geoffrey wool and angela treml . university of chicago medicine, university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in , blood bank accounted for % of all rejected clinical laboratory samples, yet comprised only % of total laboratory volume; % of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was . %. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september . results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of . %, three times the national average of . %. under the new system, rejected blood bank samples decreased from an average of % to % of all rejected laboratory samples, a % decrease. implementation of the new process produced a net savings of $ . k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ). in the postimplementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ; p . ). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* , marcus holme , johnathan bakst , gunta musa and angela treml . university of chicago medicine, university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an minute turnaround time (tat). in april of , the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased %. tat analysis of a representative one week sampling per month showed an increase in outliers from per month to per month. average monthly tys samples performed is . these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was / / . results/finding: the average number of outliers decreased % from per month to . further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $ , for fiscal year . conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from - days with a mean of . days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of - days with a mean of days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* and randy levine . northwell health, lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and only of those units ( %) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, additional irradiated units were issued ( / ; %). post-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and all of those units ( %) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus of the ( %) total units were ordered as irradiated. again, additional irradiated units were issued ( / ; %) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* , charlotte van dyke , dina garza van hoose and rachel beddard . biobridge global, south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january , to april , , , total collections were flagged for additional qc by our trima accels and haemonetics instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in , the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: ) risk assessment, ) quality control plan and ) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* , rachel jug , kimberly ingersoll , nicholas bandarenko , nicole guinn and jessica poisson . duke health pathology, duke university hospital, duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced : : transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july -december . data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the : : ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* , nancy nikolis , linda benison , ruthmire thelusca , renee liberty , sherry shariatmadar , alexander indrikovs and vishesh chhibber . north shore university hospital, northwell health background/case studies: our blood bank (bb) processes approximately , specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june , a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed / . the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of . the implementation of this new process has led to a decrease in the number of unacceptable specimens up to % quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from % to %, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* , , morgan rockwell , joseph hagan , jun teruya , and shiu-ki hui . texas children's hospital, baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within hours pre and post transfusion were evaluated. patients ( - years) receiving prophylactic ptx from july to december admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < ml/kg for patients < kg and one apheresis unit (au) for patients > kg; therefore, patients were separated into groups: < kg and > kg. a significant proportion of orders for both < kg and > kg did not meet patient platelet threshold criteria (p< . ). conclusion: ptx threshold above ir for both groups were ( kg) and % (> kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than % of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p> . ) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only . % of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated ( x ) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately patients ( x ) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts ( . %) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by % with the communication category average rpn having the greatest reduction of %. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* , karen king and joseph sweeney . rhode island hospital, lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different ( minutes for the provuev r and for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over minutes are shown in the table. the results show a reduction in tat by minutes with a % reduction of tat greater than minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* , , lorna orengo , monique scott and christopher a tormey , , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as in , blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as % prior to interventions, but may potentially be reduced to as little as . %. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of < %, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged . %. reasons for specimen rejection were divided into groups: ) hemolysis, ) blood bank witness collection form errors, ) quantity not sufficient, abstract ) duplicate sample, and ) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table ) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a . % rejection rate with only rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s system christopher shahan* , christina dejesus , mosi mccall , fallon hampton , tangi herring , judy davis , anjali patel , sonya gomillion and bonnie maltby . qualtex laboratories, qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual - hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently % of whole blood donor testing turnaround time delays are due to issues and failed runs on the s system and we have technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s system. study design/method: the number of technician related failed runs on the s system were tracked from / / thru / / . a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s system. after counter measures were implemented, the number of technician related failed runs decreased from to . failures per week, which was a % decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by %. patients who were transfused for pre-transfusion hgb > g/dl with resulting post-transfusion hgb > g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: patient charts were reviewed. were excluded for bleeding and cardiovascular instability. / ( . %) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors ( . % vs . %, p . ) and anemia of chronic disease ( . % vs . %, p . ) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb . g/dl vs . g/dl, p< . ). inappropriately transfused patients also had higher median post-transfusion hemoglobin ( . g/dl vs . g/dl, p< . ). moreover, lab evalutions revealed association with lower folate levels (median . nmol/l vs . nmol/l, p . ). / ( . %) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. / providers were responsible for . % of all inappropriate transfusions. / appropriately-transfused patients experienced an fnhtr. deaths unrelated to transfusion occurred ( in appropriate, in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* , willem martin smid and ashley john duits . red cross blood bank foundation, sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* , dee dee cassidy , jed b gorlin , and nancy l van buren , . hennepin county medical center, innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant or more minutes may be required for transit of units often released in less than minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july . data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january -june , and post change included august -december . july data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of rbc/month in coolers. post change this dropped to rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of rbc/month were returned ( %). post change, the average rbc/ month returned was ( %), this represents an absolute % reduction in number of returned products. each rbc dispensed and returned takes approximately minutes to complete paperwork and transport, therefore this change saved an average of minutes per month. it was also noted that the average rbc/month transfused was for baseline and post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of - minutes (estimated) was reduced to - minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over patent-applications have been filed related to "transfusion medicine" and over related to "transfusion alarm", during the last years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an / inch color cmos sensor, providing effectively . mp, a . mm lens, ir-cut, day/night minimum illumination . lux/f and viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood a transfusion vol. supplement s abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h. , video frame rate (fps) - /s, refresh rate hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/ . /b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p p is provided. typical v power-supply, sized x x mm and weighing g. client software is required. the ir range is - m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a gb sd-card. pan/tilt-horizontal o and pan/tilt-vertical o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot ( cfu), multishot cfu or highdose k organism preparations at a low level (< cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < days and the fungal organisms in < days. the overall agreement was . % in bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* , diane schafer , debra brown , jesse cox , scott koepsell and sara shunkwiler . nebraska medicine, the nebraska medical center, university of nebraska medical center background/case studies: anticipating the implementation of the new ( th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september , ), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling in months post implementation compared to in the months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late ' s and in early , intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january , to june , . data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: % were female and % were less than weeks of gestational age. none had co-existing g pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn ( %) and rhesus-hdn ( %). antibodies most often implicated in rh-hdn were anti-d ( / ), anti-d and anti-c ( / ) and anti-c ( / ). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was g/kg (range from , g/kg to , g/kg). neonates received one to four ivig administrations. table shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, required an et for rh-hdn and for abo-hdn. forty-five ( %) patients needed top-up transfusions during hospitalisation and until three months of age: with abo-hdn and with rh-hdn. the mean number of transfusions was three (range: to ). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over % case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a -part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate ( each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, georgia institute of technology ap reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to a) all adult males (am), b) women of non-childbearing age (wncba), and c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to a) women of childbearing age (wcba) and b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed %) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap saving apheresis platelets through use of verax point of care testing jennifer rhamy* and rebecca wride . st. mary's regional blood donor center, st. mary's regional medical center background/case studies: our rural hospital-based blood center serves hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between and per day in ), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson and jun teruya , . texas children's hospital, baylor college of medicine ap vision titers --easier or problematic? (table ) . results/findings: post intercept, t had volumes of - ml, with % hemoglobin (hb) recovery. t had -fold less extracellular protein than c. after days of storage t had higher atp and na than c while lactate and hemolysis were lower. hct, ph, k and glucose were equivalent between t and c on d . d hemolysis for t was . - . %, while for c it was . - . %. t and c atp was > mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table ) . hematocrit (hct, %) . . * . . . . . . hemoglobin (g/unit) not measured hemolysis (%) . . * . . . . * . . ph ( c) . . * . . . . . . total atp (mmol/g hb) . . * . . . . * . . k (mm) . . * . . . total tested total plts issued feb mar totals table: . resident reports to the intranet "drop box" increased from . % to . % to %, each over month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from . per day or , dpmo to . per day or , dpmo. this is a statistically significant difference since the p-value calculated was . . conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately , specimens per month. since , the requirement of having a second blood type on record was met by: . utilizing the historical blood type and the current specimen, or . having second type performed on same specimen by different technologist, and . each type and screen specimen signed by staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards th edition, # . . . a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april , the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding: . there was a minor adjustment period with more phone calls made to blood bank to explain the process. . there was minimal impact on turn around times for release of components. . aborh retype workload decreased from to ( % to % of t&s volume) per month. . unnecessary blood draws minimized, improving patient experience. . no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as to %. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our bed hospital, a retrospective chart review was performed ( / / - / / ) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< grade" or "> grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples ( %) were > titer results higher, while the majority was titer results different ( %). the cost analysis is summarized in table . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated % decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc. samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another samples were measured with fc in ucc maribor. results/finding: samples ( rbc, plt, ffp-all leucocyte depleted and non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from , - , ; plt from , - , ; ffp from , - , ; and for non-leucocyte depleted ffp from , - , (table ) . samples ( rbc, plt, ffp -all leucocyte depleted and nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from , ; plt from , , ffp from , ; and for non-leucocyte depleted ffp from , - , (table ) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than x /unit for leucocyte depleted or x / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control.